Discovery of pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase

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Abstract

In an effort to identify hepatoselective inhibitors of HMG-CoA reductase, two series of pyrroles were synthesized and evaluated. Efforts were made to modify (3R,5R)-7-[3-(4-fluorophenyl)-1-isopropyl-4-phenyl-5-phenylcarbamoyl-1H-pyrrol-2-yl]-3,5-dihydroxy-heptanoic acid sodium salt 30 in order to reduce its lipophilicity and therefore increase hepatoselectivity. Two strategies that were explored were replacement of the lipophilic 3-phenyl substituent with either a polar function (pyridyl series) or with lower alkyl substituents (lower alkyl series) and attachment of additional polar moieties at the 2-position of the pyrrole ring. One compound was identified to be both highly hepatoselective and active in vivo. We report the discovery, synthesis, and optimization of substituted pyrrole-based hepatoselective ligands as potent inhibitors of HMG-CoA reductase for reducing low density lipoprotein cholesterol (LDL-c) in the treatment of hypercholesterolemia.

Graphical abstract

Novel substituted pyrroles containing lower alkyl groups and polar functionality were shown to be potent hepatoselective inhibitors of HMG-CoA reductase.

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Introduction

Coronary heart disease (CHD) is the leading cause of death in most industrialized countries and affects 12–15 million people in the USA alone.1 The major cause of CHD is hypercholesterolemia or elevated serum cholesterol levels, particularly non-high density lipoprotein cholesterol (non-HDL-c), which is most effectively lowered by the use of statin drugs such as; simvastatin, atorvastatin, and rosuvastatin.2 Statins work by inhibiting 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate limiting enzyme involved in the biosynthesis of cholesterol. When cholesterol biosynthesis is inhibited, the low density lipoprotein cholesterol (LDL-c) receptor is upregulated and LDL-c is rapidly cleared from the bloodstream.3 In addition to lowering LDL-c, statins have been shown to lower very low density lipoprotein cholesterol (VLDL-c) and triglycerides, and sometimes raise HDL-c. Furthermore, results from multiple clinical studies have indicated that statins may be beneficial for restoring endothelial function, stimulating bone formation, decreasing vascular inflammation, and enhancing the stability of plaques associated with atherosclerosis.4, 5

An adverse side effect occasionally associated with all statins is myalgia, mild muscle pain or weakness which generally increases with higher doses of the drug.6, 7 In rare cases, statins can cause rhabdomyolysis, a severe form of myopathy or muscle toxicity, which prompted cerivastatin’s removal from the market.8 In an effort to reduce the potential for myalgia, there has been increased emphasis on the design of more hepatoselective HMG-CoA reductase inhibitors. It is anticipated that such hepatoselectivity might reduce systemic exposure and therefore limit a compound’s effect on muscle cells. One strategy for obtaining hepatoselectivity is to lower the lipophilicity of the drug. As an affirmation of this strategy, rosuvastatin (log D = −0.33), the most potent and hydrophilic statin on the market, was shown in the clinic to be much more liver-selective than the more lipophilic statins, simvastatin and cerivastatin (log D > 1.5).9, 10 The basis for greater hepatoselectivity of hydrophilic statins is presumably due to decreased passive permeability into non-hepatic cells, while active uptake into hepatocytes via the organic anion transporting polypeptides is maintained. To obtain a more potent and hepatoselective statin, chemical modifications were implemented on atorvastatin because of its overall efficacy and safety profile compared to other statins currently on the market. Other coworkers in our group began making modifications on the core pyrole template by shifting the nitrogen atom over by one carbon unit while retaining the same functionality as in atorvastatin. This modification led to the preparation of 30, which was found to be quite potent as an inhibitor of cholesterol in rat hepatocytes (IC50 = 0.43 nM), but did not exhibit the hepatoselectivity profile that we were searching for11 (see Fig. 1).

To obtain a more hepatoselective statin, the lipophilicity of 30 was reduced by replacing the 4-phenyl substituent with either a polar pyridyl function or a lower alkyl moiety. Lipophilicity was further reduced by functionalizing the phenyl group in the amide position with polar substituents. Herein these changes in 30 will be the focus of our discussion. The synthesis and hepatoselectivity profile will also be described.

Section snippets

Chemistry

As outlined in Scheme 1, the preparation of the various pyridyl substituted pyrroles commenced with a Knoevenagel condensation of 2-pyridylacetonitrile 1 and 4-fluorobenzaldehyde to generate cyano-styrene 2. Pyrrole formation was accomplished by treatment of 2 with ethyl isocyanoacetate in the presence of potassium tert-butoxide to afford pyrrole 3.12 Subsequently, N-alkylation of 3 using isopropyl iodide and potassium hydroxide provided N-isopropyl pyrrole 4. Reduction of the ethyl ester of

Biology

All new analogs were evaluated in a microsomal HMG-CoA reductase assay as well as in both hepatocyte and myocyte cellular assays.21 The ratio of inhibition of cholesterol synthesis in heptaocytes versus myocytes was used to determine the hepatoselectivity of individual analogs. Compounds with sufficient in vitro potency and selectivity were then evaluated in an in vivo efficacy model for measuring inhibition of cholesterol synthesis. For the analogs 1113 and 15 containing the pyridyl function,

Conclusion

In summary, we have demonstrated that the hepatoselectivity of pyrrole-based HMG-CoA reductase inhibitors can in general be increased by decreasing their lipophilicity, consistent with literature precedent for other statins. Although most of the new compounds had in vivo activity inferior to the less hepatoselective lead 30, we were successful in identifying one compound 12 with hepatoselectivity 10-fold higher than 30 that maintained robust in vivo activity.

Experimental

Melting points were determined on an Electro thermal Melting Point Apparatus and are uncorrected. Samples were characterized on a 400 MHz Nuclear Magnetic Resonance Spectrometer using either deuterated dimethylsulfoxide or deuterated chloroform. Mass spectra were determined on an LC Platform Mass Spectrometer using Chemical Ionization. A Rotoray Evaporator was used to remove solvents under reduced pressure. The precursor to intermediate 7 was purchased from Kaneka. Compound 24 was purchased from

Acknowledgments

Thanks to the staff from our high pressure lab for conducting the catalytic hydrogenation experiments.

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