acyl-CoA:cholesterol Acyltransferase Inhibitory Activity Research Articles

Synthesis of a novel series of 2-alkylthio substituted naphthoquinones as potent acyl-CoA: Cholesterol acyltransferase (ACAT) inhibitors

We report a new series of naphthoquinone derivatives as potent ACAT inhibitors, which were obtained through structural variations of previously disclosed lead 1. Several analogs represented by 3i–l, 4k–m, 6a–n, 7a, and 7i demonstrated potent human macrophage ACAT inhibitory activity by a cell-based reporter assay with human HepG2 cell lines. In particular, compounds 4l and 6j emerged as highly potent inhibitors, exhibiting significantly high inhibitory potencies with IC50 values of 0.44 μM and 0.6 μM, respectively. Moreover, compound 4l significantly reduced the accumulation of cellular cholesterol in HepG2 cell lines.

2,3,22,23-Tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene, an Acyclic Triterpenoid Isolated from the Seeds of <i>Alpinia katsumadai</i>, Inhibits Acyl-CoA : Cholesterol Acyltransferase Activity

In order to isolate a cholesterol-lowering compound from Alpinia katsumadai, an inhibitor for acyl-CoA : cholesterol acyltransferase (ACAT), an enzyme responsible for the cholesterol ester formation in liver, was purified, its chemical structure was determined, and in vivo and in vitro inhibition activities were performed. In a high fat diet mouse model, we discovered that the ethanol extract of Alpinia katsumadai reduced plasma cholesterol, triglyceride, and low density lipoprotein (LDL) levels. An acyclic triterpenoid showing ACAT inhibitory activity was isolated from the extract of seeds of A. katsumadai. By NMR spectroscopic analysis of its (1)H-NMR, (13)C-NMR, (1)H-(1)H correlation spectroscopy, heteronuclear multiple bond connectivity (HMBC), hetero multiquantum coherence (HMQC) and nuclear Overhauser effect, chemical structure of 2,3,22,23-tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (1), were elucidated. The acyclic triterpenoid was found to be responsible for the ACAT inhibition activities of rat liver microsomes with IC(50) values of 47.9 µM. It also decreased cholesteryl ester formation with IC(50) values of 26 µM in human hepatocyte HepG2 cell. The experimental study revealed that the ethanol extract of A. katsumadai has a hypolipemic effect in high fat diet mice, and the isolated acyclic triterpenoid has ACAT inhibition activity, showing a potential novel therapeutic approach for the treatment of hyperlipidemia and atherosclerosis.

Discovery of a Potent and Orally Available Acyl-CoA: Cholesterol Acyltransferase Inhibitor as an Anti-atherosclerotic Agent: (4-Phenylcoumarin)acetanilide Derivatives

Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes cholesterol esterification. ACAT inhibitors are expected to be potent therapeutic agents for the treatment of atherosclerosis. A series of potent ACAT inhibitors based on an (4-phenylcoumarin)acetanilide scaffold was identified. Evaluation of the structure-activity relationships of a substituent on this scaffold, with an emphasis on improving the pharmacokinetic profile led to the discovery of 2-[7-chloro-4-(3-chlorophenyl)-6-methyl-2-oxo-2H-chromen-3-yl]-N-[4-chloro-2-(trifluoromethyl)phenyl]acetamide (23), which exhibited potent ACAT inhibitory activity (IC50=12 nM) and good pharmacokinetic profile in mice. Compound 23 also showed regressive effects on atherosclerotic plaques in apolipoprotein (apo)E knock out (KO) mice at a dose of 0.3 mg/kg per os (p.o.).

Novel Tetrahydroisoquinoline Derivatives with Inhibitory Activities against Acyl-CoA: Cholesterol Acyltransferase and Lipid Peroxidation

To find a novel acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor with anti-lipid peroxidative activity, a series of tetrahydroisoquinoline derivatives were synthesized and evaluated. A compound with a N-(4-hydroxy-2,3,5-trimethylphenyl)carbamoyl moiety at the 3-position and an octanoyl moiety at the 2-position (7) was demonstrated to show anti-foam cell formation activity stronger than and anti-lipid peroxidative activity comparable to those of Pactimibe, while it was hardly absorbed orally. To increase its bioavailability, the acyl chain at the 2-position was shortened and various polar or basic moieties were introduced at the 7-position of 7. Among the synthesized derivatives, (S)-7-dimethylamino-N-(4-hydroxy-2,3,5-trimethylphenyl)-2-isobutyryl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide hydrochloride (21) showed about 16-fold stronger anti-foam cell formation activity, 3-fold stronger hepatic ACAT inhibitory activity, similar anti-low density lipoprotein (LDL) oxidative activity and 2-fold more potent protective activity against macrophage cell death by oxidative stress in comparison with Pactimibe. Compound 21 was efficiently absorbed after oral administration at 10 mg/kg in rats and dogs and its C(max) values were higher than its IC(50) values for in vitro activities. In conclusion, a tetrahydroisoquinoline structure is a useful scaffold for designing a phenolic anti-oxidative ACAT inhibitor, and compound 21 is expected to effectively prevent atherosclerosis.

Novel indoline-based acyl-CoA: cholesterol acyltransferase inhibitor: Effects of introducing a methanesulfonamide group on physicochemical properties and biological activities

A novel series of indoline-based acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors with a methanesulfonamide group at the 5-position were synthesized and their lipophilicity and biological activities were evaluated. Hepatic ACAT inhibitory and anti-foam cell formation activity increased dependent on lipophilicity of derivatives with various alkyl chains at the 1-position. The log D 7.0–biological activity curve of the derivatives with a methanesulfonamide group shifted leftward compared to that of Pactimibe derivatives with a carboxymethyl group, and derivatives with no substituent, suggesting that a methanesulfonamide group plays an important role in the interaction with ACAT protein. Among derivatives, N-(1-ethyl-5-methanesulfonylamino-4,6-dimethylindolin-7-yl)-2,2-dimethylpropanamide ( 1b) had about twofold lower log D 7.0 than Pactimibe, while it showed twofold higher hepatic ACAT inhibition than and the same anti-foam cell formation as Pactimibe, respectively. The C max of 1b (10 mg/kg, po) was higher than that of Pactimibe in rats. The plasma protein binding ratio of 1b was lower than that of Pactimibe: 64.8% and 97.9%, respectively. Compound 1b showed greater inhibitory effects on hepatic cholesterol secretion in mice than Pactimibe. In conclusion, the introduction of a methanesulfonamide group is effective to design less lipophilic, more efficacious and more bioavailable indoline-based ACAT inhibitors than previous indoline-based inhibitors.

Synthesis of a Novel Series of Imidazo[1,2-α]pyridines as Acyl-CoA: Cholesterol Acyltransferase (ACAT) Inhibitors

A novel series of imidazo[1,2-<TEX>$\alpha$</TEX>]pyridines was designed, synthesized, and tested for their ability to inhibit acyl- CoA:cholesterol acyltransferase. Preliminary lead optimization efforts resulted in the identification of ACAT inhibitors represented by analogues 5b, 5c, 6a, 6c, 7b, and 7c. The ACAT inhibitory activity of these compounds was further established by potent inhibition of cholesteryl ester formation in HepG2 cells by a representative analogue 7b.

Preferential pharmacological inhibition of macrophage ACAT increases plaque formation in mouse and rabbit models of atherogenesis

The cholesteryl ester, foam cell-enriched vulnerable plaque is a principle pharmacological target for reducing athero-thrombosis. Acyl CoA:cholesterol Acyl Transferase (ACAT) catalyzes the esterification of free cholesterol in intestine, liver, adrenal and macrophages, leading in the latter cells to intracellular cholesteryl ester accumulation and foam cell formation in the arterial intima. Previous studies suggested the existence of several isoforms of ACAT with different tissue distribution and this has largely been confirmed by molecular cloning of ACAT-1 and ACAT-2. We developed a series of ACAT inhibitors that preferentially inhibited macrophage ACAT relative to hepatic or intestinal ACAT based on in vitro assays and ex vivo bioavailability studies. Four of these compounds were tested in three models of atherosclerosis at oral doses shown to give sufficient bioavailable monocyte/macrophage ACAT inhibitory activity. In fat-fed C57BL/6 mice, chow fed apo E−/− mice and KHC rabbits, the various ACAT inhibitors had either no effect or increased indices of atherosclerotic foam cell formation. Direct and indirect measurements suggest that the increase in plaque formation may have been related to inhibition of macrophage ACAT possibly leading to cytotoxic effects due to augmented free cholesterol. These results suggest that pharmacological inhibition of macrophage ACAT may not reduce, but actually aggravate, foam cell formation and progression.

Synthetic conversion of acat inhibitor to acetylcholinesterase inhibitor

Natural product acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor pyripyropene A was synthetically converted to acetylcholinesterase (AChE) inhibitor via heterolitic cleavage of the 2-pyrone ring, followed by γ-acylation/cyclization with several aroyl chlorides. The 4-pyridyl analogue selectively showed AChE inhibitory activity (IC 50=7.9 μM) and no ACAT inhibitory activity IC 50=>1000 μM.

Potent inhibitors of acyl-CoA:cholesterol acyltransferase. 2. Structure-activity relationships of novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amides.

Novel N-(2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives 1 were synthesized and tested for their ability to inhibit rabbit small intestinal ACAT (acyl-CoA:cholesterol acyltransferase) and lower serum total cholesterol in cholesterol-fed rats. Among the synthesized compounds, N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)amide derivatives showed potent ACAT inhibitory activity. The synthesis and structure-activity relationships of these compounds are described. A methyl group at position 6 of the 2,3-dihydrobenzofuran moiety was important for potent ACAT inhibitory activity. In the series of N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl) amides, lipophilicity of the acyl moiety was necessary for the potent ACAT inhibitory activity. The highly lipophilic acid amides N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl)-2,2- dimethyldodecanamide (10) and 6-(4-chlorophenoxy)-N-(2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-y l)-2,2-dimethyloctanamide (50) showed potent activity. Introduction of a dimethylamino group at position 5 of the 2,3-dihydrobenzofuran moiety resulted in highly potent activity. The most potent compound, N-[5-(dimethylamino)-2,2,4,6-tetramethyl-2,3-dihydrobenzofuran-7-yl ]-2,2-dimethyldodecanamide (13, TEI-6620), showed highly potent ACAT inhibitory activity (rabbit small intestine IC50 = 0.020 microM, rabbit liver IC50 = 0.009 microM), foam cell formation inhibitory activity (rat peritoneal macrophage IC50 = 0.030 microM), extremely potent serum cholesterol-lowering activity in cholesterol-fed rats (71% at a dose of 0.3 mg/kg/day po), and good bioavailability in fed dogs (Cmax = 2.68 microg/mL at 1 h, 10 mg/kg po).

Inhibitors of acyl-CoA: cholesterol acyltransferase. II. Preparation and hypocholesterolemic activity of optically active dibenz[b,e]oxepin-11-carboxanilides.

In a previous paper, we reported a novel inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT), 2-bromo-N-(2,6-diisopropylphenyl)-6,11- dihydrodibenz[b,e]oxepin-11-carboxamide (1). In this work, we prepared both enantiomers and tested them for ability to inhibit ACAT (liver microsomes from cholesterol-fed rabbits) in vitro and to decrease serum total cholesterol in cholesterol-fed golden hamsters in vivo. The precursor carboxylic acid 4 was optically resolved with cinchonidine. The obtained (-)- and (+)-4 were converted to (-)- and (+)-1 without racemization, respectively. The enantiomer (-)-1 showed potent ACAT inhibitory activity in vitro with an IC50 value of 8 nM and was approximately 10-fold more active than (+)-1. Furthermore, (-)-1 showed strong hypocholesterolemic activity in vivo, whereas (+)-1 was inactive. A molecular modeling study showed that the difference of ACAT inhibitory activity between the enantiomers was derived from the spatial alignment of the bromine. Compound (-)-1 was selected for further evaluation as KW-3033.

Chemical modification and structure-activity relationships of pyripyropenes. 1. Modification at the four hydroxyl groups.

Four hydroxyl groups of pyripyropenes have been modified and evaluated for their ability to inhibit microsomal acyl-CoA:cholesterol acyltransferase (ACAT) activity in vitro and to lower cholesterol absorption in vivo in a cholesterol-fed hamster. 7-O-n-Valeryl derivative (8c) improved the in vitro ACAT inhibitory activity (IC50 = 13 nM) about 7 times better than pyripyropene A. Introduction of methanesulfonyl group at 11-hydroxyl group (17a) increased both in vitro activity (IC50 = 19 nM) and in vivo efficacy (ED50 = 10 mg/kg).

Potent inhibitors of acyl-CoA:cholesterol acyltransferase. Structure-activity relationships of novel N-(4-oxochroman-8-yl)amides.

Novel N-(4-oxochroman-8-yl)amide derivatives 1 were synthesized and tested for their ability to inhibit rabbit small intestinal ACAT (acyl-CoA:cholesterol acyltransferase) in vitro and to lower serum total cholesterol in cholesterol-fed rats in vivo. Among the synthesized compounds, N-(7-alkoxy-4-oxochroman-8-yl)amide derivatives showed potent ACAT inhibitory activity both in vitro and in vivo. The structure-activity relationships of these N-(4-oxochroman-8-yl)amides and related compounds are discussed on the basis of these two assays. The carbonyl group at position 4 of the 4-chromanone was essential for potent ACAT inhibitory activity. N-(Chromon-8-yl) derivatives were less potent than N-(4-oxochroman-8-yl) derivatives. An alkoxy group at position 7 of the 4-chromanone moiety was important for potent ACAT inhibitory activity. In the N-(7-alkoxy-4-oxochroman-8-yl)amide derivatives, another necessary factor to elicit the potent ACAT inhibitory activity was lipophilicity of the molecules. The highly lipophilic acid amides N-(7-methoxy-4-oxochroman-8-yl)-2,2-dimethyldodecanamide (35) and 4-[[6-(4-chlorophenoxy)hexyl]oxy]-N-(7-methoxy-4-oxochroman-8- yl)benzamide (63) showed potent activity. Introduction of a highly lipophilic alkoxy group at position 7 of the 4-chromanone moiety instead of methoxy group also resulted in potent activity. In this case, highest inhibitory activity was obtained by N-[7-(decyloxy)-4-oxochroman-8-yl]-2,2-dimethylpropanamid e (65). The most potent compound, N-(7-methoxy-4-oxochroman-8-yl)-2,2-dimethyldodecanamide (35, TEI-6522), showed significant ACAT inhibitory activity (rabbit small intestine IC50 = 13 nM, rabbit liver IC50 = 16 nM), foam cell formation inhibitory activity (rat peritoneal macrophage IC50 = 160 nM), and extremely potent serum cholesterol-lowering activity in cholesterol-fed rats (61% at a dose of 0.1 mg/kg/day po).

N-(1-phenyl-2-benzimidazolyl)- N′-phenylurea derivatives as potent in hibitors of acylcoa:cholesterol acyltransferase (ACAT)

A novel series of N-(1-phenyl-2-benzimidazolyl)- N′-phenylurea derivatives were prepared as ACAT inhibitors. These compounds showed potent ACAT inhibitory activity in vitro (liver microsomes from cholesterol-fed rabbits) and hypocholesterolemic activity in vivo (cholesterol-fed golden hamsters).

Biochemical alterations in guinea pig adrenal cortex following administration of PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase.

To assess whether previously reported ultrastructural alterations of adrenocortical mitochondria induced by the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor PD 132301-2 are accompanied by functional deficits in tissue energy stores, phosphorylated adenine nucleotide levels in guinea pig adrenal cortex were quantitated. Adrenals of male guinea pigs were obtained at 1, 2, 6, or 24 hours after oral administration of 100 mg/kg PD 132301-2 or 0.5% methylcellulose vehicle. In treated animals, ATP levels and ATP/ADP ratios were decreased approximately 50% with concurrent increases in AMP. Calculated energy charge was also decreased; these decreases were maximal by 6 hours. Cholesterol esterification in adrenal cortex was inhibited, resulting in progressive accumulation of free cholesterol up to 3-fold over control by 24 hours, consistent with the ACAT inhibitory activity of the drug. These data suggest that PD 132301-2 distributes to the adrenal cortex where early alterations of tissue bioenergetics occur in a time frame consistent with ultrastructural alterations of mitochondria.

Terpendoles, novel ACAT inhibitors produced by Albophoma yamanashiensis. I. Production, isolation and biological properties.

A series of new acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors termed terpendoles were isolated from the culture broth of a fungal strain FO-2546 which was proposed to belong to a new genus designated as Albophoma yamanashiensis. Among four structurally related terpendoles, terpendole C showed the most potent ACAT inhibitory activity with an IC50 value of 2.1 microM in an in vitro enzyme assay, followed by terpendoles D (IC50: 3.2 microM), A (15.1 microM) and B (26.8 microM). Evaluation of their ACAT inhibition in the cell assay using J774 macrophages indicated that terpendole D exhibited the highest specificity (cytotoxicity vs. ACAT inhibition) among microbial ACAT inhibitors we discovered so far.

Inhibitors of acyl-CoA:cholesterol acyltransferase. 1. Synthesis and hypocholesterolemic activity of dibenz[b,e]oxepin-11-carboxanilides.

A series of N-phenyl-6,11-dihydrodibenz[b,e]oxepin-11-carboxamides and related derivatives were prepared on the basis of structures of the reported inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). These compounds were tested for their ability to inhibit ACAT (liver microsomes from cholesterol-fed rabbits) in vitro and to decrease serum total cholesterol in cholesterol-fed golden hamsters in vivo. The structure-activity relationships in vitro were as follows. Substitution at positions 2 and 6 in the anilide resulted in potent inhibitory activity, and the potency increased with increasing size of the substituents, with maximum potency being obtained with a 2,6-diisopropyl substitution. The position of the substituent on the dibenz[b,e]oxepin ring system influenced the activity, and substitution at position 2 was critical for potent activity. The electronic effect of the substituent at position 2 does not influence activity, but bulkiness seems to be a significant factor. The lipophilicity of the compounds also plays an important role in determining ACAT inhibitory activity. Among the compounds tested, 2-bromo-N-(2,6-diisopropylphenyl)-6,11-dihydrodibenz-[b,e]++ +oxepin-11- carboxamide (33, KF17828) showed significant in vitro activity (rabbit liver microsomes IC50 = 23 nM) and the most potent in vivo activity (complete reduction in elevated serum total cholesterol levels at a dose of 10 mg/kg in hamsters).

Pyripyropenes, novel inhibitors of acyl-CoA:cholesterol acyltransferase produced by Aspergillus fumigatus. I. Production, isolation, and biological properties.

Aspergillus fumigatus FO-1289, a soil isolate, was found to produce a series of novel inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Four active compounds, named pyripyropenes A, B, C and D, were isolated from the fermentation broth of the producing strain by solvent extraction, silica gel column chromatography, ODS column chromatography and preparative HPLC. Pyripyropenes A, B, C and D show very potent ACAT inhibitory activity in an enzyme assay system using rat liver microsomes with IC50 values of 58, 117, 53 and 268 nM, respectively.

Inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). 2. Modification of fatty acid anilide ACAT inhibitors: bioisosteric replacement of the amide bond.

In order to further define the structural features necessary for potent inhibition of acyl-coenzyme A:cholesterol acyltransferase (ACAT) in vitro and cholesterol lowering in vivo, systematic study of bioisosteric replacements for the amide bond in our previously identified series of fatty acid anilide ACAT inhibitors was undertaken. Only replacement of amide bonds with isosterases having both hydrogen bond donor and acceptor functionalities yielded compounds retaining ACAT inhibitory activity. Replacement of the amide bond with the urea bioisostere yielded compounds that were potent ACAT inhibitors in vitro and efficacious hypocholesterolemic agents in vivo. Examination of the structure activity relationships in the phenyl ring and alkyl portion of the N-phenyl-N'-alkylureas revealed that 2,6-diisopropyl substitution was optimal in the phenyl ring. When the 2,6-diisopropyl moiety was kept constant, potency in vitro and in vivo was maintained with straight and branched alkyl groups from 6 to 18 carbons in length.

Inhibition of acyl-CoA: cholesterol acyltransferase activity by cyclodepsipeptide antibiotics.

The effect was studied of the fungal cyclodepsipeptide antibiotics beauvericin and seven distinct enniatins on acyl-CoA: cholesterol acyltransferase (ACAT) activity. In an enzyme assay using rat liver microsomes, all the compounds were found to inhibit ACAT activity. The drug concentration that caused 50% inhibition (IC50 value) of the enzyme activity was determined to be 3.0 microM for beauvericin, indicating that the compound is one of the most potent ACAT inhibitors of microbial origin. Enniatins exhibited much higher IC50 values of 22 to 110 microM. More hydrophobic enniatins showed more potent inhibitory activity. Furthermore, the ACAT inhibitory activity was evaluated as inhibition of cholesteryl ester formation in a cell assay using J774 macrophages. Calculation of the ratio, CD50 value (the drug concentration causing 50% cell damage)/IC50 value of cholesteryl ester formation, indicated that beauvericin shows the highest specificity. These data indicate that beauvericin is one of the most potent and specific ACAT inhibitors of microbial origin.