Discovery of Novel 3-Piperidinyl Pyridine Derivatives as Highly Potent and Selective Cholesterol 24-Hydroxylase (CH24H) Inhibitors.

Cholesterol 24-hydroxylase (CH24H or CYP46A1) is a brain-specific cytochrome P450 enzyme that metabolizes cholesterol into 24S-hydroxycholesterol (24HC) for regulating brain cholesterol homeostasis. For the development of a novel and potent CH24H inhibitor, we designed and synthesized 3,4-disubstituted pyridine derivatives using a structure-based drug design approach starting from compounds 1 (soticlestat) and 2 (thioperamide). Optimization of this series by focusing on ligand-lipophilicity efficiency value resulted in the discovery of 4-(4-methyl-1-pyrazolyl)pyridine derivative 17 (IC50 = 8.5 nM) as a potent and highly selective CH24H inhibitor. The X-ray crystal structure of CH24H in complex with compound 17 revealed a unique binding mode. Both blood-brain barrier penetration and reduction of 24HC levels (26% reduction) in the mouse brain were confirmed by oral administration of 17 at 30 mg/kg, indicating that 17 is a promising tool for the novel and selective inhibition of CH24H.

Invention of MK-8262, a Cholesteryl Ester Transfer Protein (CETP) Inhibitor Backup to Anacetrapib with Best-in-Class Properties.

Cholesteryl ester transfer protein (CETP) represents one of the key regulators of the homeostasis of lipid particles, including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) particles. Epidemiological evidence correlates increased HDL and decreased LDL to coronary heart disease (CHD) risk reduction. This relationship is consistent with a clinical outcomes trial of a CETP inhibitor (anacetrapib) combined with standard of care (statin), which led to a 9% additional risk reduction compared to standard of care alone. We discuss here the discovery of MK-8262, a CETP inhibitor with the potential for being the best-in-class molecule. Novel in vitro and in vivo paradigms were integrated to drug discovery to guide optimization informed by a critical understanding of key clinical adverse effect profiles. We present preclinical and clinical evidence of MK-8262 safety and efficacy by means of HDL increase and LDL reduction as biomarkers for reduced CHD risk.

Synthesis and Modeling of Ezetimibe Analogues.

Ezetimibe is a well-known drug that lowers blood cholesterol levels by reducing its absorption in the small intestine when joining to Niemann-Pick C1-like protein (NPC1L1). A ligand-based study on ezetimibe analogues is reported, together with one-hit synthesis, highlighted in the study. A convenient asymmetric synthesis of (2S,3S)-N-α-(R)-methylbenzyl-3-methoxycarbonylethyl-4-methoxyphenyl ß-lactam is described starting from Baylis-Hillman adducts. The route involves a domino process: allylic acetate rearrangement, stereoselective Ireland-Claisen rearrangement and asymmetric Michael addition, which provides a δ-amino acid derivative with full stereochemical control. A subsequent inversion of ester and acid functionality paves the way to the lactam core after monodebenzylation and lactam formation. It also shows interesting results when it comes to a pharmacophore study based on ezetimibe as the main ligand in lowering blood cholesterol levels, revealing which substituents on the azetidine-2-one ring are more similar to the ezetimibe skeleton and will more likely bind to NPC1L1 than ezetimibe.

Anti-hyperlipidemic potential of natural product based labdane-pyrroles via inhibition of cholesterol and triglycerides synthesis.

Hyperlipidemia is the clinical condition where blood has an increased level of lipids, such as cholesterol and triglycerides. Therefore controlling hyperlipidemia is considered to be a protective strategy to treat many associated diseases. Thus, a novel natural product derived pyrrole, and pyrazole-(E)-Labda-8(17),12-diene-15,16-dial conjugates with cholesterol and triglycerides synthesis inhibition potential was designed through scaffold hopping approach and synthesized via one-pot selective cycloaddition. Amongst the tested hybrids, 3i exhibited excellent activity against triglyceride and cholesterol synthesis with the percentage inhibition of 71.73 ± 0.78 and 68.61 ± 1.19, which is comparable to the positive controls fenofibrate and atorvastatin, respectively. Compounds 3j and 3k also exhibited the considerable potential of promising leads. The HMG CoA reductase inhibitory activity of the compounds was consistent with that of inhibitory activity of cholesterol synthesis. Compound 3i showed the highest inhibitory potential (78.61 ± 2.80) percentage of suppression, which was comparable to that of the positive control pravastatin (78.05 ± 5.4). Favourably, none of the compounds showed cytotoxicity (HepG2) in the concentration ranging from 0.5 to 100 µM.

Cholesteryl ester transfer protein (CETP) inhibitors based on cyclic urea, bicyclic urea and bicyclic sulfamide cores.

Cholesteryl ester transfer protein (CETP) inhibitors reduce the transfer of cholesteryl esters from the high-density lipoprotein (HDL-C) to apolipoprotein such as VLDL/LDL, with exchange of triglycerides. Thus, this inhibition increases the HDL-C levels, which is believed to lower the risk for heart disease and stroke. We report here a series of CETP inhibitors based on the cyclic, bicyclic urea and sulfamide cores. These CETP inhibitors exemplified by 15, 31, and 45 demonstrated in vitro potency in inhibiting the CETP transfer activity, and 15, 31 showing in vivo efficacy to increase HDL-C levels in cynomolgus-CETP transgenic mice. The synthesis and biological evaluations of these CETP inhibitors are described.

The Role of Structure and Biophysical Properties in the Pleiotropic Effects of Statins.

Statins are a class of drugs used to lower low-density lipoprotein cholesterol and are amongst the most prescribed medications worldwide. Most statins work as a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), but statin intolerance from pleiotropic effects have been proposed to arise from non-specific binding due to poor enzyme-ligand sensitivity. Yet, research into the physicochemical properties of statins, and their interactions with off-target sites, has not progressed much over the past few decades. Here, we present a concise perspective on the role of statins in lowering serum cholesterol levels, and how their reported interactions with phospholipid membranes offer a crucial insight into the mechanism of some of the more commonly observed pleiotropic effects of statin administration. Lipophilicity, which governs hepatoselectivity, is directly related to the molecular structure of statins, which dictates interaction with and transport through membranes. The structure of statins is therefore a clinically important consideration in the treatment of hypercholesterolaemia. This review integrates the recent biophysical studies of statins with the literature on the physiological effects and provides new insights into the mechanistic cause of statin pleiotropy, and prospective means of understanding the cholesterol-independent effects of statins.

Preparation of phytosteryl ornithine ester hydrochloride and improvement of its bioaccessibility and cholesterol-reducing activity in vitro.

In this work, synthesis of phytosteryl ornithine ester hydrochloride was studied for the first time using an intermediate phytosteryl N,N'-bis[tert-butoloxycarbonyl(BOC)]-ornithine ester. This method also involved esterification of phytosterols with N,N'-bis(BOC)-ornithine and deprotection. The maximum yield was 90% and deprotection of BOC group was more than 99% using the HCl/ethyl acetate method. As a result, thermal stability and water solubility as well as emulsifying activity and stability of phytosterols were improved through coupling with ornithine, which is favorable for their application in water-based food systems. We also observed increased bioaccessibility of phytosteryl ornithine hydrochloride (4.5%) and 65% of phytosteryl ornithine hydrochloride was hydrolyzed in vitro. These results indicated that ornithine phytosteryl ester hydrochloride can reduce dissolution capacity of cholesterol in vitro, representing improved cholesterol-reducing activity, which will further expand the applications of phytosteryl ornithine ester hydrochloride.

Synthesis and Evaluation of 2-Azetidinone and 1H-Pyrrole-2,5-dione Derivatives as Cholesterol Absorption Inhibitors for Reducing Inflammation Response and Oxidative Stress.

Excess lipid accumulation can initiate the development and progression of atherosclerotic lesions, thus eventually leading to cardiovascular disease. Lipid-lowering medication therapy is one of the cornerstones of cardiovascular disease therapy. On the basis of the cholesterol absorption inhibitor ezetimibe, we successfully synthesized seven 2-azetidinone derivatives and eighteen 1H-pyrrole-2,5-dione derivatives. Most of the new compounds significantly inhibited cholesterol uptake in vitro. In addition, one of the most active inhibitors, 3-(4-fluorophenyl)-1-[(3S)-3-hydroxy-3-(4-hydroxyphenyl)propyl]-4-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione (14q), showed no cytotoxicity in L02 and HEK293T cell lines. Further evaluation indicated that 14q inhibited considerably the amount of TNF-α, ROS, MDA, and LDH in vitro. Therefore, 14q might be a novel cholesterol absorption inhibitor.

Cyclodextrin-Based Macromolecular Systems as Cholesterol-Mopping Therapeutic Agents in Niemann-Pick Disease Type C.

Over the last decade, cyclodextrins (CDs) have gained considerable attention as a potential therapeutic intervention in the treatment of the rare genetic condition Niemann-Pick type C disease (NPC). However, the oligosaccharide in its monomeric form suffers from serious side effects, especially from a pharmacokinetic and biodistribution standpoint. CD-based macromolecular systems hold great promise to overcome such limitations and might provide an improved therapeutic approach in reducing cholesterol accumulation in NPC. In the present article, the latest developments and synthetic strategies in the preparation of CD-containing polymers as cholesterol-mopping therapeutic agents in NPC are summarized.

Synthetic Approaches Towards Antihypercholesterolemic Drug Simvastatin.

Cardiovascular diseases are among the most threatening problems being faced by twenty-first century humans. The core cause of these diseases is high cholesterol level. Simvastatin (1: Synvinolin) is a well-known cholesterol-lowering drug marketed under the trade name Zocor®, which significantly reduces the risk of cardiovascular diseases related to hypercholesterolemia and is effective in lowering the total plasma cholesterol, low-density and very low-density lipoprotein cholesterol. It also enhances the high-density lipoprotein cholesterol. This review article aims to provide an overview of several chemical and biological methods utilized for the production of simvastatin in high yields and purity. Many robust and scalable methods have been described using lovastatin (2: Mevinolin) as a starting material, produced by the fungal strain of Aspergelius terreus. Enzymatic synthesis of simvastatin is also highlighted in this review. In addition, detailed experimental conditions, as well as the compatibility for industrial-scale preparations of simvastatin are also discussed.

Phytosterol and γ-Oryzanol Conjugates: Synthesis and Evaluation of their Antioxidant, Antiproliferative, and Anticholesterol Activities.

Fifteen new multifunctional conjugates were designed and synthesized by chemically linking the steroidal framework of natural occurring γ-oryzanol and γ-oryzanol-derived phytosterols to a wide range of bioactive natural compounds (fatty acids, phenolic acids, amino acids, lipoic acid, retinoic acid, curcumin, and resveratrol). Starting from γ-oryzanol, which is the main component of rice bran oil, this study was aimed at assessing if the conjugation strategy might enhance some γ-oryzanol bioactivities. The antioxidant activity was evaluated through three different mechanisms, namely, DPPH-scavenging activity, metal-chelating activity, and ß-carotene-bleaching inhibition. Measurement of the in vitro cell growth inhibitory effects on three different human cancer cellular lines was also carried out, and the potential hypocholesterolemic effect was studied. Compounds 10 and 15 displayed an improved antioxidant activity, with respect to that of γ-oryzanol. Compounds 2, 6, and 12 exerted an antiproliferative activity in the low micromolar range against HeLa and DAOY cells (GI50 < 10 µM). As for the claimed hypocholesterolemic effect of γ-oryzanol, none of the synthesized compounds inhibited the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis.

Lipid reducing activity of novel cholic acid (CA) analogs: Design, synthesis and preliminary mechanism study.

Bile acids, initially discovered as endogenous ligands of farnesoid X receptor (FXR), play a central role in the regulation of triglyceride and cholesterol metabolism and have recently emerged as a privileged structure for interacting with nuclear receptors relevant to a large array of metabolic processes. In this paper, phenoxy containing cholic acid derivatives with excellent drug-likeness have been designed, synthesized, and assayed as agents against cholesterol accumulation in Raw264.7 macrophages. The most active compound 14b reduced total cholesterol accumulation in Raw264.7 cells up to 30.5% at non-toxic 10 µM and dosage-dependently attenuated oxLDL-induced foam cell formation. Western blotting and qPCR results demonstrate that 14b reduced both cholesterol and lipid in Raw264.7 cells through (1) increasing the expression of cholesterol transporters ABCA1 and ABCG1, (2) accelerating ApoA1-mediated cholesterol efflux. Through a cell-based luciferase reporter assay and molecular docking analysis, LXR was identified as the potential target for 14b. Interestingly, unlike conventional LXR agonist, 14b did not increase lipogenesis gene SREBP-1c expression. Overall, these diverse properties disclosed herein highlight the potential of 14b as a promising lead for further development of multifunctional agents in the therapy of cardiovascular disease.

Molecular Diversity by Olefin Cross-Metathesis on Solid Support. Generation of Libraries of Biologically Promising ß-Lactam Derivatives.

The application of the reagent-based diversification strategy for generation of libraries of biologically promising ß-lactam derivatives is described. Key features are the versatility of the linker used and the cross-metathesis functionalization at the cleavage step. From an immobilized primary library, diversity was expanded by applying different cleavage conditions, leading to a series of cholesterol absorption inhibitor analogues together with interesting hybrid compounds through incorporation of a chalcone moiety.

Synthesis and evaluation of 1H-pyrrole-2,5-dione derivatives as cholesterol absorption inhibitors for suppressing the formation of foam cells and inflammatory response.

Excess lipid accumulation in the arterial intima and formation of macrophage-derived foam cells in the plaque could cause atherosclerotic lesion. Cholesterol absorption inhibitors could suppress the lipid accumulation of human macrophage, inflammatory response and the development of atherosclerosis. In this research, a series of 1H-pyrrole-2,5-dione derivatives were synthesized as cholesterol absorption inhibitor and tested in in vitro experiments. One of the most active inhibitors, compound 20 exhibited stronger in vitro cholesterol absorption activity than ezetimibe, no cytotoxicity in HEK293 and RAW264.7 cell lines and satisfied lipophilicity. The further study indicated that 20 could inhibit lipid accumulation of macrophage and reduce the secretion of LDH, MDA, TNF-α and ROS in a concentration-dependent manner. In conclusion, as a novel and potent cholesterol absorption inhibitor, compound 20 could suppress the formation of foam cells and inflammatory response.

Polymeric bile acid sequestrants: Review of design, in vitro binding activities, and hypocholesterolemic effects.

Polymeric bile acid sequestrants (BAS) have recently attracted much attention as lipid-lowering agents. These non-absorbable materials specifically bind bile acids (BAs) in the intestine, preventing bile acid (BA) reabsorption into the blood through enterohepatic circulation. Therefore, it is important to understand the structure-property relationships between the polymer sequestrant and its ability to bind specific BAs molecules. In this review, we describe pleiotropic effects of bile acids, and we focus on BAS with various molecular architectures that result in different mechanisms of BA sequestration. Here, we present 1) amphiphilic polymers based on poly(meth)acrylates, poly(meth)acrylamides, polyalkylamines and polyallylamines containing quaternary ammonium groups, 2) cyclodextrins, and 3) BAS prepared via molecular imprinting methods. The synthetic approaches leading to individual BAS preparation, as well as results of their in vitro BA binding activities and in vivo lipid-lowering activities, are discussed.

Synthesis, Biological Evaluation, and Molecular Modeling Study of Substituted Benzyl Benzamides as CETP Inhibitors.

Cardiovascular disease is the most common cause for mortality and morbidity in the developed world; its risk is inversely related to the high-density lipoprotein (HDL) cholesterol levels. Therefore, there is a great interest in developing new cholesteryl ester transfer protein (CETP) inhibitors capable of raising HDL as a novel approach for the prevention of cardiovascular disease. Herein, the synthesis and characterization of ten benzyl benzamides 8a-j that aim at CETP inhibition was performed. The in vitro CETP inhibition bioassay revealed that benzamide 8j had the best activity, with a percent inhibition of 82.2% at 10 µM concentration and an IC50 value of 1.3 µM. The docking study shows that the verified compounds accommodate the binding cleft of CETP and are enclosed by a hydrophobic lining. Furthermore, the scaffold of 8a-j matches the pharmacophoric points of CETP inhibitors, particularly in its hydrophobic and aromatic functionalities.

Dissolution and precipitation behavior of ternary solid dispersions of ezetimibe in biorelevant media.

The effects of different formulations and processes on inducing and maintaining the supersaturation of ternary solid dispersions of ezetimibe (EZ) in two biorelevant media fasted-state simulated intestinal fluid (FaSSIF) and fasted-state simulated gastric fluid (FaSSGF) at different temperatures (25 °C and 37 °C) were investigated in this work. Ternary solid dispersions of EZ were prepared by adding polymer PVP-K30 and surfactant poloxamer 188 using melt-quenching and spray-drying methods. The resulting solid dispersions were characterized using scanning electron microscopy, differential scanning calorimetry (DSC), modulated DSC, powder X-ray diffraction and Fourier transformation infrared spectroscopy. The dissolution of all the ternary solid dispersions was tested in vitro under non-sink conditions. All the prepared solid dispersions were amorphous in nature. In FaSSIF at 25 °C, the melt-quenched (MQ) solid dispersions of EZ were more soluble than the spray-dried (SD) solid dispersions and supersaturation was maintained. However, at 37 °C, rapid and variable precipitation behavior was observed for all the MQ and SD formulations. In FaSSGF, the melting method resulted in better solubility than the spray-drying method at both temperatures. Ternary solid dispersions show potential for improving solubility and supersaturation. However, powder dissolution experiments of these solid dispersions of EZ at 25 °C may not predict the supersaturation behavior at physiologically relevant temperatures.

From PK/PD to QSP: Understanding the Dynamic Effect of Cholesterol-Lowering Drugs on Atherosclerosis Progression and Stratified Medicine.

Current computational and mathematical tools are demonstrating the high value of using systems modeling approaches (e.g. Quantitative Systems Pharmacology) to understand the effect of a given compound on the biological and physiological mechanisms related to a specific disease. This review provides a short survey of the evolution of the mathematical approaches used to understand the effect of particular cholesterol-lowering drugs, from pharmaco-kinetic (PK) / pharmaco-dynamic (PD) models, through physiologically base pharmacokinetic models (PBPK) to QSP. These mathematical models introduce more mechanistic information related to the effect of these drugs on atherosclerosis progression and demonstrate how QSP could open new ways for stratified medicine in this field.

Stereoselective Synthesis of Ezetimibe via Cross-Metathesis of Homoallylalcohols and α-Methylidene-ß-Lactams.

Ru-catalyzed cross-metathesis (CM) reaction between ß-arylated α-methylidene-ß-lactams and terminal olefins was developed. The CM reaction is effectively catalyzed with Hoveyda-Grubbs second-generation catalyst affording corresponding α-alkylidene-ß-aryl-ß-lactams in good isolated yields (41-83%) with exclusive Z-selectivity. The developed protocol was successfully applied for stereoselective preparation of Ezetimibe, the commercial cholesterol absorption inhibitor.

Biosynthetic Study on Antihypercholesterolemic Agent Phomoidride: General Biogenesis of Fungal Dimeric Anhydrides.

To elucidate the general biosynthetic pathway of fungal dimeric anhydrides, a gene cluster for the biosynthesis of the antihy-percholesterolemic agent phomoidride was identified by heterologous expression of candidate genes encoding the highly reducing polyketide synthase, alkylcitrate synthase (ACS), and alkylcitrate dehydratase (ACDH). An in vitro analysis of ACS and ACDH revealed that they give rise to anhydride monomers. Based on the established monomer biosynthesis, we propose a general biogenesis of dimeric anhydrides involving a single donor unit and four acceptor units.