Design,Synthesis And Biological Evaluation Of Intestine Targeted TGR5 Agonists

Intestine targeted drugs are orally administered compounds that exert their therapeutic effects locally in the intestinal tract.Such drugs reach high concentrations in the intestinal tract,with low absorption into the systemic circulation,thus could minimize potential side effects related to systemic exposure.Their therapeutic areas included diabetes,obesity,infectious diseases,and inflammatory disorders.TGR5(Takeda G-protein receptor 5),also known as GPBAR1,was identified as the first membrane receptor responsive to bile acids.It is widely expressed in sites including the gallbladder,intestine,brown adipose tissue and skeletal muscle.Functions of TGR5 included modulating glucose hemostasis,reducing inflammatory response,regulating liver,gallbladder and gastrointestinal tract.TGR5 activation of enteroendocrine cells promotes release of glucagon-like peptide 1(GLP-1),GLP-2 and PYY.GLP-1 maintains blood glucose homeostasis by increasing glucose-induced insulin secretion,promoting satiety,delaying gastric emptying,suppressing glucagon release,and increasing glucose disposal in peripheral tissues.GLP-2 is primarily involved in maintaining structural integrity of the intestinal tract including promoting intestinal growth and enhancing intestinal function.PYY could promote satiety.Therefore,TGR5 agonists would have potential therapeutic effects towards type 2 diabetes and gastrointestinal disorders such as Crohn's disease and short bowel syndrome.However,it was suggested that TGR5 activation in the gallbladder caused gallbladder filling effect.Therefore,localized TGR5 activation within the intestinal tract might maintain hypoglycemia with minimal side effects.We focused on soft drugs to develop intestinal targeted TGR5 agonists.Soft drugs are biologically active compounds designed to undergo predictable and controllable metabolic inactivation into predictable,nontoxic metabolites.We initially constructed our structure optimization based on B1,however no proper soft drug skeletons were found.Then we searched for corresponding skeletons in our compound library,and finally found ester B12 a,which could be cleaved in plasma to its inactive metabolite B13 a.In an effort to accelerate the plasma metabolism rate,we removed the chlorine of the benzofuran ring,optimized the ester moiety and adjusted the middle pyridine ring,and finally obtained C1 g and C1i with best in vitro activity and rapid plasma metabolism rate.The PK assay in ICR mice assured that the drug concentrations of C1 g and C1i in plasma,bile and gallbladder were extremely low.In consistent with these,the gallbladder filling effects of C1 g and C1i in ICR mice and db/db mice were not significant.From the tests above,we could assume that the gallbladder filling effect and drug exposure grew in inverse proportion to the plasma metabolism rate.In the OGTT assay in ICR mice,C1 g and C1i displayed significant hypoglycemic effects.Their stabilities in 0.25% CMC-Na solutions and pH 1.2 acid solutions were confirmed.All these date confirmed the strategy of soft drugs as intestine targeted compounds.C1g and C1i still have some shortcomings.First was their relatively high cell permeability(Papp of about 1×10-6 cm/s),second is their relatively low drug concentration in intestinal tissues.Therefore,we decided to introduce highly polar substituents to the ester position,tetrahydroquinoxaline moiety or the pyridine nitrogen position.Finally we alkylated the basic pyridine nitrogen to get the corresponding quaternary ammonium compounds,among which,D5 g showed moderate hTGR5 activity,high metabolism rate in plasma and extremely low Caco-2 cell permeability(Papp = 0.01×10-6 cm/s).D5g showed rather low in vitro activity towards mTGR5.This species difference was thought to partly attribute to the benzofuran ring.Thus,we cleaved the benzofuran ring of C1 g,C1i to yield D7 j,D7l with similar in vitro activity,but higher plasma metabolism rate.Ring opening of benzofuran ring of D5 g afforded the best compound D9 m.Its hTGR5 EC50 was 84 nM,and its mTGR5 EC50 was 499 nM.Apparently,its species difference of in vitro activity was greatly reduced.Decreasing aqueous solubility offers another approach to reducing drug absorption.Cyclization might increase the lattice energy of compound and thus decreasing its aqueous solubility.We performed cyclization at different positions of A21 a and B1,and synthesized 4 series of compounds.After initial SAR optimization,we finally found E5 c with the best in vitro activity.