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  • br Conflict of interest br Acknowledgements This study was s


    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 [1]. One of the major risk factors for CHD are elevated serum cholesterol concentrations [2]. 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 [3], [4]. Another approach is to block the Fmoc-Arg(Pbf)-OH 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 [5]. It can be applied either as a monotherapy or in combination with statins [6]. Ezetimibe 1 was originally discovered without a known molecular target through in vivo screening of cholesterol-fed hamsters [7]. In 2004, researchers from the Schering-Plough Research Institute identified Niemann Pick C1-like1 (NPC1L1) protein as a molecular target of ezetimibe 1[8]. Ezetimibe 1 acts by blocking the internalization of NPC1L1, thereby preventing cholesterol from entering the cytoplasm of enterocytes [9]. A thorough structure–activity relationship (SAR) study [5] 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 [5]. 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 [10]. In continuation of our research in the field of β-lactam chemistry [11], [12], [13], [14], [15] and taking into consideration the requirements determined by SAR studies [5], 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.