br Conflict of interest br Acknowledgements
Conflict of interest
Acknowledgements This study was supported by the National Natural Science Foundation of China (No.31571839), the Chinese Ministry Program for New Century Excellent Talents in University (NCET-12-0865), and Special Fund for Agro-scientific Research in the Public Interest (No.201203047).
Introduction Cardiovascular heart disease (CHD) is the leading cause of death worldwide . One of the major risk factors for CHD are elevated serum cholesterol concentrations . Lowering the level of serum cholesterol is an established clinical practice for CHD treatment, intervention, and prevention. Pharmacologically, circulating cholesterol concentrations are reduced by statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, affecting biosynthesis of endogenous cholesterol , . Another approach is to block the RHC 80267 of dietary cholesterol, which is the other major contributor to serum cholesterol concentrations in the small intestine. Ezetimibe 1 (Zetia, Ezetrol; approved in 2002) (Fig. 1) is the only cholesterol absorption inhibitor on the market today . It can be applied either as a monotherapy or in combination with statins . Ezetimibe 1 was originally discovered without a known molecular target through in vivo screening of cholesterol-fed hamsters . In 2004, researchers from the Schering-Plough Research Institute identified Niemann Pick C1-like1 (NPC1L1) protein as a molecular target of ezetimibe 1. Ezetimibe 1 acts by blocking the internalization of NPC1L1, thereby preventing cholesterol from entering the cytoplasm of enterocytes . A thorough structure–activity relationship (SAR) study  of the β-lactam cholesterol absorption inhibitors in cholesterol-fed hamsters revealed that the 2-azetidinone backbone as well as the aryl group at the N-1 and C-4 position of the β-lactam ring are required for activity. The aryl group at the C-4 position of the β-lactam ring is optimally substituted with alkoxy or hydroxy groups at the para-position. The side chain at the C-3 position of the β-lactam ring with three linking atoms bearing a pendent aryl group is optimal. Preferred configuration at the C-4 chiral center of the β-lactam ring is S and the C-3 atom tolerates S or R configurations . Introduction of a heteroatom at the 1′-position of the C-3 side chain can also contribute to the activity, whereas isosteric groups at the 3′-position of the side chain decrease the activity . In continuation of our research in the field of β-lactam chemistry , , , ,  and taking into consideration the requirements determined by SAR studies , we synthesized bioisosteres 5 and 6 (Fig. 2) of ezetimibe 1 bearing a –NH– group at the C-3 position of the β-lactam ring. Bioisosterism is a useful approach for lead compound modification that can result in improved pharmacological activity, decreased toxicity, and optimized pharmacokinetics. With the classical bioisosteric exchange of the –CH2– with a –NH– group we aimed at investigating whether the change in polarity of the side chain, the ability of additional H-bond, and ammonium salt formations would affect their cholesterol absorption inhibition and cytotoxicity. Here we show that our new ezetimibe analogs 5, 6 and their diastereoisomeric mixture 5/6 (70:30) are potent novel cholesterol absorption inhibitors.
Results and discussion
Conclusion This report demonstrates that we have successfully synthesized two novel ezetimibe bioisosteres 5, 6 and their diastereoisomeric mixtures 5/6 (70:30) and 6/5 (85:15) from enantiomerically pure trans-(3R,4R)-amino-β-lactam 2. Crystal structure of 6 was determined to establish unambiguously both absolute and relative configurations at the new stereogenic centre C17 and were assigned to be S. Both diastereoisomeres 5 and 6 as well as their diastereoisomeric mixture 5/6 showed significant cholesterol absorption inhibitory activity both in vitro and in vivo. Based on our data and the pKa value for 5 and 6 being 9.35, indicating that both compounds are present in the form of NH4+ in the blood and small intestine, other diastereoisomeric mixtures (e.g. 6/5 (85:15)) may exhibit similar in vivo results as 5/6 (70:30). Results from this study implicate a therapeutic potential of these novel compounds to reduce cholesterol plasma concentrations and improve CHD.