Discovery of novel ketoxime ether derivatives with potent FXR agonistic activity, oral effectiveness and high liver/blood ratio.

The farnesoid X receptor (FXR) is a promising therapeutic target for nonalcoholic steatohepatitis (NASH) and other bile acid related diseases because it plays a critical role in fibrosis, inflammation and bile acid homeostasis. Obeticholic acid (OCA), a FXR agonist which was synthesized from chenodeoxycholic acid, showed desirable curative effects in clinical trials. However, the pruritus which was the main side effect of OCA limited its further applications in NASH. Although pruritus was also observed in the clinical trials of non-steroidal FXR agonists, the proportion of patients with pruritus was much smaller than that of OCA. Thus, we decided to develop non-steroidal FXR agonists and discovered a series of novel FXR agonists which were synthesized from GW4064 by replacing the stilbene group with ketoxime ether. Encouragingly, in the following biological tests, our target compounds 13j and 13z not only showed potent FXR agonistic activities in vitro, but also effectively promoted the expression of target genes in vivo. More importantly, in the pharmacokinetic experiments, compounds 13j and 13z displayed high liver/blood ratio characteristics which were helpful to reduce the potential side effects which were caused by prolonged systemic activation of FXR. In summary, our compounds were good choices for the development of non-steroidal FXR agonists and were deserved further investigation.

Dipeptidyl peptidase 4 inhibitor sitagliptin protected against dextran sulfate sodium-induced experimental colitis by potentiating the action of GLP-2.

Dipeptidyl peptidase 4 (DPP4), a ubiquitously expressed protease that cleaves off the N-terminal dipeptide from proline and alanine on the penultimate position, has important roles in many physiological processes. In the present study, experimental colitis was induced in mice receiving 3% dextran sulfate sodium (DSS) in drinking water. We found that mice with DSS-induced colitis had significantly increased intestinal DPP activity and decreased serum DPP activity, suggesting a probable correlation of DPP4 with experimental colitis. Then, we investigated whether sitagliptin, a specific DPP4 inhibitor could protect against DSS-induced colitis. We showed that oral administration of single dose of sitagliptin (30 mg/kg) on D7 remarkably inhibited DPP enzyme activity in both serum and intestine of DSS-induced colitic mice. Repeated administration of sitagliptin (10, 30 mg/kg, bid, from D0 to D8) significantly ameliorated DSS-induced colitis, including reduction of disease activity index (DAI) and body weight loss, improvement of histological score and colon length. Sitagliptin administration dose-dependently increased plasma concentrations of active form of GLP-1 and colonic expression of GLP-2R. Co-administration of GLP-2R antagonist GLP-23-33 (500 µg/kg, bid, sc) abolished the protective effects of sitagliptin in DSS-induced colitic mice. Moreover, sitagliptin administration significantly decreased the ratio of apoptotic cells and increased the ratio of proliferative cells in colon epithelium of DSS-induced colitic mice, and this effect was also blocked by GLP-23-33. Taken together, our results demonstrate that sitagliptin could attenuate DSS-induced experimental colitis and the effects can be attributed to the enhancement of GLP-2 action and the subsequent protective effects on intestinal barrier by inhibiting epithelial cells apoptosis and promoting their proliferation. These findings suggest sitagliptin as a novel therapeutic approach for the treatment of ulcerative colitis.

OL3, a novel low-absorbed TGR5 agonist with reduced side effects, lowered blood glucose via dual actions on TGR5 activation and DPP-4 inhibition.

AIM: TGR5 agonists stimulate intestinal glucagon-like peptide-1 (GLP-1) release, but systemic exposure causes unwanted side effects, such as gallbladder filling. In the present study, linagliptin, a DPP-4 inhibitor with a large molecular weight and polarity, and MN6, a previously described TGR5 agonist, were linked to produce OL3, a novel low-absorbed TGR5 agonist with reduced side-effects and dual function in lowering blood glucose by activation of TGR5 and inhibition of DPP-4. METHODS: TGR5 activation was assayed in HEK293 cells stably expressing human or mouse TGR5 and a CRE-driven luciferase gene. DPP-4 inhibition was assessed based on the rate of hydrolysis of a surrogate substrate. GLP-1 secretion was measured in human enteroendocrine NCI-H716 cells. OL3 permeability was tested in Caco-2 cells. Acute glucose-lowering effects of OL3 were evaluated in ICR and diabetic ob/ob mice. RESULTS: OL3 activated human and mouse TGR5 with an EC50 of 86.24 and 17.36 nmol/L, respectively, and stimulated GLP-1 secretion in human enteroendocrine NCI-H716 cells (3-30 µmol/L). OL3 inhibited human and mouse DPP-4 with IC50 values of 18.44 and 69.98 µmol/L, respectively. Low permeability of OL3 was observed in Caco-2 cells. In ICR mice treated orally with OL3 (150 mg/kg), the serum OL3 concentration was 101.10 ng/mL at 1 h, and decreased to 13.38 ng/mL at 5.5 h post dose, confirming the low absorption of OL3 in vivo. In ICR mice and ob/ob mice, oral administration of OL3 significantly lowered the blood glucose levels, which was a synergic effect of activating TGR5 that stimulated GLP-1 secretion in the intestine and inhibiting DPP-4 that cleaved GLP-1 in the plasma. In ICR mice, oral administration of OL3 did not cause gallbladder filling. CONCLUSION: OL3 is a low-absorbed TGR5 agonist that lowers blood glucose without inducing gallbladder filling. This study presents a new strategy in the development of potent TGR5 agonists in treating type 2 diabetes, which target to the intestine to avoid systemic side effects.

Discovery and Structure-Activity Relationship Study of 4-Phenoxythiazol-5-carboxamides as Highly Potent TGR5 Agonists.

A novel therapy that stimulates endogenous glucagon-like peptide-1 (GLP-1) secretion by Takeda G-protein-coupled receptor 5 (TGR5) agonists might be a superior alternative for the treatment of type 2 diabetes mellitus. A series of 4-phenoxythiazol-5-carboxamides were developed as highly potent TGR5 agonists using a bioisosteric replacement strategy based on the scaffold of 4-phenoxynicotinamides. The structure-activity relationship on the bottom phenyl ring and the thiazole ring was extensively studied, and the 2-methyl-thiazole derivatives 30c and e displayed the best in vitro potency toward human TGR5, with EC50 values of approximately 1 nM. While endowed with excellent in vitro potency, the 2-methyl-thiazoles were flawed with high microsomal clearance.

Yhhu4488, a novel GPR40 agonist, promotes GLP-1 secretion and exerts anti-diabetic effect in rodent models.

G protein-coupled receptor 40 (GPR40) is predominantly expressed in pancreatic ß-cells and activated by long-chain fatty acids. GPR40 has drawn considerable interest as a potential therapeutic target for type 2 diabetes mellitus (T2DM) due to its important role in enhancing glucose-stimulated insulin secretion (GSIS). Encouragingly, GPR40 is also proven to be highly expressed in glucagon-like peptide-1 (GLP-1)-producing enteroendocrine cells afterwards, which opens a potential role of GPR40 in enhancing GLP-1 secretion to exert additional anti-diabetic efficacy. In the present study, we discovered a novel GPR40 agonist, yhhu4488, which is structurally different from other reported GPR40 agonists. Yhhu4488 showed potent agonist activity with EC50 of 49.96 nM, 70.83 nM and 58.68 nM in HEK293 cells stably expressing human, rat and mouse GPR40, respectively. Yhhu4488 stimulated GLP-1 secretion from fetal rat intestinal cells (FRIC) via triggering endogenous calcium store mobilization and extracellular calcium influx. The effect of yhhu4488 on GLP-1 secretion was further confirmed in type 2 diabetic db/db mice. Yhhu4488 exhibited satisfactory potency in in vivo studies. Single administration of yhhu4488 improved glucose tolerance in SD rats. Chronic administration of yhhu4488 effectively decreased fasting blood glucose level, improved ß-cell function and lipid homeostasis in type 2 diabetic ob/ob mice. Taken together, yhhu4488 is a novel GPR40 agonist that enhances GLP-1 secretion, improves metabolic control and ß-cell function, suggesting its promising potential for the treatment of type 2 diabetes.

MiR-146a alleviates inflammatory bowel disease in mice through systematic regulation of multiple genetic networks.

Introduction: Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy. Whether there is a single gene that systematically regulates IBD is not yet known. MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed. Methods: RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background. The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates. Bulk RNA-sequencing, RT-qPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon. Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice. Results: We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD. DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice. The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a-/- and WT mice. Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes. Quantitative RT-PCR or immunostaining confirmed that Ccl3, Saa3, Csf3, Lcn2, Serpine1, Serpine2, MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, p-P65, and IRAK1 were regulated by miR-146a in DSS induced IBD. Among them, MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A were involved in the active stage of IBD in humans. Discussion: Our date demonstrated that miR-146a acts as a top regulator in C57/BL6N mice to systematically repress multiple genetic regulatory networks involved in immune response of intestine to environment factors, and combinatory treatment with miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through down-regulating multiple genetic regulatory networks which were increased in colon tissue from IBD patients. Our findings suggests that miR-146a is a top inhibitor of IBD, and that miR-146a-5p and miR-146a-3p mimics might be potential drug for IBD.

Discovery of Highly Potent and Selective Thyroid Hormone Receptor ß Agonists for the Treatment of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive stage of nonalcoholic fatty liver disease (NAFLD) and is characterized by steatosis, inflammation, hepatocyte ballooning, and fibrosis. While there are currently no approved therapies for NASH, the thyroid hormone receptor ß (THR-ß), primarily expressed in the liver, is emerging as an effective molecular target for the treatment of NASH. However, the adverse cardiac and bone effects mediated by thyroid hormone receptor α (THR-α) need to be minimized. Herein, we reported the discovery of a series of novel THR-ß agonists featuring pyrrolo[3,2-b]pyridin-5-one skeletons based on structure-based drug design. Further optimization led to compound 15, which exhibited higher potency and selectivity for THR-ß over THR-α compared to clinical drug MGL-3196. More significantly, an excellent liver-to-serum ratio of 93:1 was observed for compound 15. We believe that the high hepatic concentration of compound 15 may result in no cardiotoxicity.

Synthesis and evaluation of piperidine urea derivatives as efficacious 11ß-hydroxysteroid dehydrogenase type 1 inhibitors in diabetic ob/ob mice.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) has attracted considerable attention as a potential target for the treatment of diabetes and metabolic syndrome. Herein we report the design, synthesis and efficacy evaluation of novel amide and urea 11ß-HSD1 inhibitors. Structure-activity relationship studies led to the identification of 10c, which was efficacious in a diabetic ob/ob mouse model and reduced fasting and non-fasting blood glucose levels after ip dosing.

Design, synthesis, and pharmacological evaluation of benzamide derivatives as glucokinase activators.

A series of benzamide derivatives were assembled by using the privileged-fragment-merging (PFM) strategy and their SAR studies as glucokinase activators were described. Compounds 5 and 16b were identified having a suitable balance of potency and activation profile. They showed EC(50) values of 28.3 and 44.8 nM, and activation folds of 2.4 and 2.2, respectively. However, both compounds displayed a minor reduction in plasma glucose levels on imprinting control region (ICR) mice. Unfavorable pharmacokinetic profiles (PK) were also observed on these two compounds.

Emodin, an 11ß-hydroxysteroid dehydrogenase type 1 inhibitor, regulates adipocyte function in vitro and exerts anti-diabetic effect in ob/ob mice.

AIM: Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a potent and selective inhibitor of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) with the ability to ameliorate metabolic disorders in diet-induced obese mice. In the present study, we investigated the effects of emodin on adipocyte function and the underlying mechanisms in vitro, and its anti-diabetic effects in ob/ob mice. METHODS: 3T3-L1 adipocytes were used for in vitro studies. 11ß-HSD1A activity was evaluated with a scintillation proximity assay. The adipogenesis, glucose uptake, lipolysis and adiponectin secretion were investigated in 3T3-L1 adipocytes treated with emodin in the presence of active (corticosterone) or inactive glucocorticoid (11-dehydrocorticosterone). For in vivo studies, ob/ob mice were administered emodin (25 and 50 mg·kg⁻¹·d⁻¹, ip) for 26 d. On the last day of administration, the serum was collected and the mesenteric and perirenal fat were dissected for analyses. RESULTS: Emodin inhibited the 11ß-HSD1 activity in 3T3-L1 adipocytes in concentration- and time-dependent manners (the IC50 values were 7.237 and 4.204 µmol/L, respectively, after 1 and 24 h treatment. In 3T3-L1 adipocytes, emodin (30 µmol/L) suppressed 11-dehydrocorticosterone-induced adipogenesis without affecting corticosterone-induced adipogenesis; emodin (3 µmol/L) reduced 11-dehydrocorticosterone-stimulated lipolysis, but had no effect on corticosterone-induced lipolysis. Moreover, emodin (3 µmol/L) partly reversed the impaired insulin-stimulated glucose uptake and adiponectin secretion induced by 11-dehydrocorticosterone but not those induced by corticosterone. In ob/ob mice, long-term emodin administration decreased 11ß-HSD1 activity in mesenteric adipose tissues, lowered non-fasting and fasting blood glucose levels, and improved glucose tolerance. CONCLUSION: Emodin improves the inactive glucocorticoid-induced adipose tissue dysfunction by selective inhibition on 11ß-HSD1 in adipocyte in vitro and improves glycemic control in ob/ob mice.

Design and exploration of gut-restricted bifunctional molecule with TGR5 agonistic and DPP4 inhibitory effects for treating ulcerative colitis.

Ulcerative colitis (UC) is a gastrointestinal disease with complex etiology, and the shortage of the treatment further intensifies the need to discover new therapies based on novel mechanisms and strategies. TGR5 and DPP4 are beneficial to treat UC through multiple mechanisms, notably increasing GLP-2 levels by promoting secretion and inhibiting degradation respectively. However, some unwanted systemic effects caused by systemic exposure hinder development, especially the gallbladder-filling effects. Herein, we firstly reported a series of high-potency gut-restricted TGR5-DPP4 bifunctional molecules by gut-restriction and multitarget strategies to utilize the positive impacts of TGR5 and DPP4 on UC and avoid unwanted systemic effects. In particularly, racemic compound 15, a high-potency TGR5-DPP4 bifunctional molecule, showed favorable intestinal distribution, preferable efficacy in mice colitis model and good gallbladder safety. Therefore, the feasibility of gut-restricted TGR5-DPP4 bifunctional molecule was confirmed for the treatment UC, providing a new insight into the development of anti-UC drugs.

Alisol B Alleviates Hepatocyte Lipid Accumulation and Lipotoxicity via Regulating RARα-PPARγ-CD36 Cascade and Attenuates Non-Alcoholic Steatohepatitis in Mice.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease worldwide, with no effective therapies available. Discovering lead compounds from herb medicine might be a valuable strategy for the treatment of NASH. Here, we discovered Alisol B, a natural compound isolated from Alisma orientalis (Sam.), that attenuated hepatic steatosis, inflammation, and fibrosis in high-fat diet plus carbon tetrachloride (DIO+CCl4)-induced and choline-deficient and amino acid-defined (CDA)-diet-induced NASH mice. RNA-seq showed Alisol B significantly suppressed CD36 expression and regulated retinol metabolism in NASH mice. In mouse primary hepatocytes, Alisol B decreased palmitate-induced lipid accumulation and lipotoxicity, which were dependent on CD36 suppression. Further study revealed that Alisol B enhanced the gene expression of RARα with no direct RARα agonistic activity. The upregulation of RARα by Alisol B reduced HNF4α and PPARγ expression and further decreased CD36 expression. This effect was fully abrogated after RARα knockdown, suggesting Alisol B suppressed CD36 via regulating RARα-HNF4α-PPARγ cascade. Moreover, the hepatic gene expression of RARα was obviously decreased in murine NASH models, whereas Alisol B significantly increased RARα expression and decreased CD36 expression, along with the downregulation of HNF4α and PPARγ. Therefore, this study showed the unrecognized therapeutic effects of Alisol B against NASH with a novel mechanism by regulating RARα-PPARγ-CD36 cascade and highlighted Alisol B as a promising lead compound for the treatment of NASH.

Metabolomic Study of High-Fat Diet-Induced Obese (DIO) and DIO Plus CCl4-Induced NASH Mice and the Effect of Obeticholic Acid.

The pathophysiology of nonalcoholic fatty liver disease (NAFLD) is a complex process involving metabolic and inflammatory changes in livers and other organs, but the pathogenesis is still not well clarified. Two mouse models were established to study metabolic alteration of nonalcoholic fatty liver and nonalcoholic steatohepatitis, respectively. The concentrations of metabolites in serum, liver and intestine content were measured by the AbsoluteIDQ® p180 Kit (Biocrates Life Sciences, Innsbruck, Austria). Multivariate statistical methods, pathway analysis, enrichment analysis and correlation analysis were performed to analyze metabolomic data. The metabolic characteristics of liver, serum and intestine content could be distinctly distinguished from each group, indicating the occurrence of metabolic disturbance. Among them, metabolic alteration of liver and intestine content was more significant. Based on the metabolic data of liver, 19 differential metabolites were discovered between DIO and control, 12 between DIO-CCl4 and DIO, and 47 between DIO-CCl4 and normal. These metabolites were mainly associated with aminoacyl-tRNA biosynthesis, nitrogen metabolism, lipid metabolism, glyoxylate and dicarboxylate metabolism, and amino metabolism. Further study revealed that the intervention of obeticholic acid (OCA) could partly reverse the damage of CCl4. The correlation analysis of metabolite levels and clinical parameters showed that phosphatidylcholines were negatively associated with serum alanine aminotransferase, aspartate aminotransferase, NAFLD activity score, and fibrosis score, while lysophosphatidylcholines, sphingomyelins, amino acids, and acylcarnitines shared the reverse pattern. Our study investigated metabolic alteration among control, NAFLD model, and OCA treatment groups, providing preclinical information to understand the mechanism of NAFLD and amelioration of OCA.

Design of G-protein-coupled bile acid receptor 1 (GPBAR1, TGR5) soft drugs with reduced gallbladder-filling effects.

The G-protein-coupled bile acid receptor TGR5 agonists were widely developed in type 2 diabetes and gastrointestinal disorders, but were also full of challenges, due to the systemic on-targeted side effects, especially the gallbladder-filling effects. Here, to circumvent these risks, several TGR5 agonists with soft-drug designation had been designed and synthesized with the properties of rapid metabolized after drug effect. Among them, compound 19 showed negligible systemic exposure and favorable gallbladder safety on a 3-day continuous administration, providing a novel strategy for developing TGR5 agonists.

Discovery of Highly Polar ß-Homophenylalanine Derivatives as Nonsystemic Intestine-Targeted Dipeptidyl Peptidase IV Inhibitors.

Although intensively expressed within intestine, the precise roles of intestinal dipeptidyl peptidase IV (DPPIV) in numerous pathologies remain incompletely understood. Here, we first reported a nonsystemic intestine-targeted (NSIT) DPPIV inhibitor with ß-homophenylalanine scaffold, compound 7, which selectively inhibited the intestinal rather than plasmatic DPPIV at an oral dosage as high as 30 mg/kg. We expect that compound 7 could serve as a qualified tissue-selective tool to determine undetected physiological or pathological roles of intestinal DPPIV.

TGR5 agonist ameliorates insulin resistance in the skeletal muscles and improves glucose homeostasis in diabetic mice.

BACKGROUND AND PURPOSE: TGR5 plays an important role in many physiological processes. However, the functions of TGR5 in the regulation of the glucose metabolism and insulin sensitivity in the skeletal muscles have not been fully elucidated. We synthesized MN6 as a potent and selective TGR5 agonist. Here, the effect of MN6 on insulin resistance in skeletal muscles was evaluated in diet-induced obese (DIO) mice and C2C12 myotubes, and the underlying mechanisms were explored. METHODS: The activation of MN6 on human and mouse TGR5 was evaluated by a cAMP assay in HEK293 cell lines stable expressing hTGR5/CRE or mTGR5/CRE cells. GLP-1 secretion was measured in NCI-H716 cells and CD1 mice. The acute and chronic effects of MN6 on regulating metabolic abnormalities were observed in ob/ob and DIO mice. 2-deoxyglucose uptake was examined in isolated skeletal muscles. Akt phosphorylation, glucose uptake and glycogen synthesis were examined to assess the effects of MN6 on palmitate-induced insulin resistance in C2C12 myotubes. RESULTS: MN6 potently activated human and mouse TGR5 with EC50 values of 15.9 and 17.9 nmol/L, respectively, and stimulated GLP-1 secretion in NCI-H716 cells and CD1 mice. A single oral dose of MN6 significantly decreased the blood glucose levels in ob/ob mice. Treatment with MN6 for 15 days reduced the fasting blood glucose and HbA1c levels in ob/ob mice. MN6 improved glucose and insulin tolerance and enhanced the insulin-stimulated glucose uptake of skeletal muscles in DIO mice. The palmitate-induced impairment of insulin-stimulated Akt phosphorylation, glucose uptake and glycogen synthesis in C2C12 myotubes could be prevented by MN6. The effect of MN6 on palmitate-impaired insulin-stimulated Akt phosphorylation was abolished by siRNA-mediated knockdown of TGR5 or by the inhibition of adenylate cyclase or protein kinase A, suggesting that this effect is dependent on the activation of TGR5 and the cAMP/PKA pathway. CONCLUSIONS: Our study identified that a TGR5 agonist could ameliorate insulin resistance by the cAMP/PKA pathway in skeletal muscles; this uncovered a new effect of the TGR5 agonist on regulating the glucose metabolism and insulin sensitivity in skeletal muscles and further strengthened its potential value for the treatment of type 2 diabetes.

Design, synthesis and structure-activity relationship studies of GPR40 agonists containing amide linker.

Free fatty acid receptor 1 (FFAR1/GPR40) attracted significant attention as a potential target for developing novel antidiabetic drugs because of its unique mechanism in glucose homeostasis. Several reports have expressed concerns about central nervous system (CNS) penetration of GPR40 agonists, which is possibly attributed to their high lipophilicity and low total polar surface area. Herein, we report our efforts to improve the physicochemical properties and pharmacokinetic profiles of LY2881835, a GPR40 agonist that had undergone Phase I clinical trial, through a series of structural optimizations. We identified an orally efficacious compound, 15k, which possessed increased plasma exposure, prolonged half-life and reduced CNS exposure and liver to plasma distribution ratio compared with LY2881835. 15k is a potentially valuable lead compound in the development of safe and efficacious GPR40-targeted drugs to treat type 2 diabetes mellitus.

Intestinally-targeted TGR5 agonists equipped with quaternary ammonium have an improved hypoglycemic effect and reduced gallbladder filling effect.

TGR5 activation of enteroendocrine cells increases glucagon-like peptide 1 (GLP-1) release, which maintains glycemic homeostasis. However, TGR5 activation in the gallbladder and heart is associated with severe side effects. Therefore, intestinally-targeted TGR5 agonists were suggested as potential hypoglycemic agents with minimal side effects. However, until now no such compounds with robust glucose-lowering effects were reported, especially in diabetic animal models. Herein, we identify a TGR5 agonist, 26a, which was proven to be intestinally-targeted through pharmacokinetic studies. 26a was used as a tool drug to verify the intestinally-targeted strategy. 26a displayed a robust and long-lasting hypoglycemic effect in ob/ob mice (once a day dosing (QD) and 18-day treatment) owing to sustained stimulation of GLP-1 secretion, which suggested that robust hypoglycemic effect could be achieved with activation of TGR5 in intestine alone. However, the gallbladder filling effect of 26a was rather complicated. Although the gallbladder filling effect of 26a was decreased in mice after once a day dosing, this side effect was still not eliminated. To solve the problem above, several research strategies were raised for further optimization.

Discovery of Intestinal Targeted TGR5 Agonists for the Treatment of Type 2 Diabetes.

Activation of TGR5 stimulates intestinal glucagon-like peptide-1 (GLP-1) release, but activation of the receptors in gallbladder and heart has been shown to cause severe on-target side effects. A series of low-absorbed TGR5 agonists was prepared by modifying compound 2 with polar functional groups to limit systemic exposure and specifically activate TGR5 in the intestine. Compound 15c, with a molecular weight of 1401, a PSA value of 223 Å(2), and low permeability on Caco-2 cells, exhibited satisfactory potency both in vitro and in vivo. Low levels of 15c were detected in blood, bile, and gallbladder tissue, and gallbladder-related side effects were substantially decreased compared to the absorbed small-molecule TGR5 agonist 2.

4-Benzofuranyloxynicotinamide derivatives are novel potent and orally available TGR5 agonists.

A series of 4-benzofuranyloxynicotinamide derivatives were identified to be novel, potent, and orally available TGR5 agonists. Among them, compound 9r had the highest potency in vitro (hTGR5 EC50 = 0.28 nM, mTGR5 EC50 = 0.92 nM). Further in vivo studies disclosed that 9r could effectively lower the blood glucose, but meantime caused an increase in the gallbladder volume of mice. Subsequent research toward eliminating the gallbladder toxicity resulted in compound 19 with low permeability. Although the EC50 of mTGR5 of 19 was larger one order than that of 9r, it still had good glucose-lowing activity. Nevertheless, 19 also caused the adverse effects to the gallbladder. The drug levels detection disclosed that the concentration of 19 was only lower than that of 9r in plasma but was higher in bile and gallbladder tissue. This result indicated that low exposure in plasma could not guarantee low exposure in bile and gallbladder tissue, and thus resulting in the gallbladder toxicity of 19.