Discovery of Clinical Candidate 2-(4-(2-((1 H-Benzo[ d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)- N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide Hydrochloride [K-604], an Aqueous-Soluble Acyl-CoA:Cholesterol O-Acyltransferase-1 Inhibitor.

2-(4-(2-((1 H-Benzo[ d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)- N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide hydrochloride (K-604, 2) has been identified as an aqueous-soluble potent inhibitor of human acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also known as SOAT)-1 that exhibits 229-fold selectivity for human ACAT-1 over human ACAT-2. In our molecular design, the insertion of a piperazine unit in place of a 6-methylene chain in the linker between the head (pyridylacetamide) and tail (benzimidazole) moieties led to a marked enhancement of the aqueous solubility (up to 19 mg/mL at pH 1.2) and a significant improvement of the oral absorption (the Cmax of 2 was 1100-fold higher than that of 1 in fasted dogs) compared with those of the previously selected compound, 1. After ensuring the pharmacological effects and safety, we designated 2 as a clinical candidate, named K-604. Considering the therapeutic results of ACAT inhibitors in past clinical trials, we believe that K-604 will be useful for the treatment of incurable diseases involving ACAT-1 overexpression.

Design, synthesis and pharmacology of aortic-selective acyl-CoA: Cholesterol O-acyltransferase (ACAT/SOAT) inhibitors.

We describe our molecular design of aortic-selective acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also abbreviated as SOAT) inhibitors, their structure-activity relationships (SARs) and their pharmacokinetic (PK) and pharmacological profiles. The connection of two weak ligands-N-(2,6-diisopropylphenyl)acetamide (50% inhibitory concentration [IC50] = 8.6 µM) and 2-(methylthio)benzo[d]oxazole (IC50 = 31 µM)-via a linker comprising a 6 methylene group chains yielded a highly potent molecule, 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)nonanamide (3h) that exhibited high potency (IC50 = 0.004 µM) toward aortic ACAT. This head-to-tail design made it possible to markedly enhance the activity to 2150- to 7750-fold and to discriminate the isoform-selectivity based on the double-induced fit mechanism. At doses of 1 and 3 mg/kg, 3h significantly decreased the lipid-accumulation areas in the aortic arch to 74 and 69%, respectively without reducing the plasma total cholesterol level in high fat- and cholesterol-fed F1B hamsters. Here, we demonstrate the antiatherosclerotic effect of 3hin vivo via its direct action on aortic ACAT and its powerful modulator of cholesterol level. This molecule is a potential therapeutic agent for the treatment of diseases involving ACAT-1 overexpression.

A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels.

BACKGROUND: Acyl-coenzyme A:cholesterol O-acyltransferase-1 (ACAT-1), a major ACAT isozyme in macrophages, plays an essential role in foam cell formation in atherosclerotic lesions. However, whether pharmacological inhibition of macrophage ACAT-1 causes exacerbation or suppression of atherosclerosis is controversial. METHODS AND RESULTS: We developed and characterized a novel ACAT inhibitor, K-604. The IC(50) values of K-604 for human ACAT-1 and ACAT-2 were 0.45 and 102.85 micromol/L, respectively, indicating that K-604 is 229-fold more selective for ACAT-1. Kinetic analysis indicated that the inhibition was competitive with respect to oleoyl-coenzyme A with a K(i) value of 0.378 micromol/L. Exposure of human monocyte-derived macrophages to K-604 inhibited cholesterol esterification with IC(50) of 68.0 nmol/L. Furthermore, cholesterol efflux from THP-1 macrophages to HDL(3) or apolipoprotein A-I was enhanced by K-604. Interestingly, administration of K-604 to F1B hamsters on a high-fat diet at a dose of >or=1mg/kg suppressed fatty streak lesions without affecting plasma cholesterol levels. CONCLUSIONS: K-604, a potent and selective inhibitor of ACAT-1, suppressed the development of atherosclerosis in an animal model without affecting plasma cholesterol levels, providing direct evidence that pharmacological inhibition of ACAT-1 in the arterial walls leads to suppression of atherosclerosis.