Potential antiatherosclerotic agents. 4. [(Functionalized-alkyl)amino]benzoic acid analogues of cetaben.

The synthesis of a series of analogues in which the alkyl group of cetaben is substituted with various functional groups or replaced entirely by a functionalized alkanoyl moiety is described. Also reported are the syntheses of branched-chain (alkylamino)benzoic acids in which branching is specifically localized at the terminus of the alkyl chain. Structure-activity relationships of these compounds, both as hypolipidemic agents and as inhibitors of the enzyme fatty acyl-CoA:cholesterol acyltransferase (ACAT), are discussed. Certain compounds were specifically synthesized to test the hypothesis that groups located near the terminus of the alkyl chain of cetaben might retard metabolic degradation of the molecule and, thus, enhance biological activity. Some of these (48-50) were found to be the most active analogues synthesized.

Potential antiatherosclerotic agents. 6. Hypocholesterolemic trisubstituted urea analogues.

The discovery that a series of N,N-dialkyl-N'-arylureas were inhibitors of the ACAT enzyme has led to a structure-activity study involving the systematic modification of three sites of the urea backbone. This study culminated in the selection of N'-(2,4-dimethylphenyl)-N-benzyl-N-n-butylurea (115) for more extensive biological evaluation. ACAT inhibitors are seen as potentially beneficial agents against hypercholesterolemia and atherosclerosis.

Potential antiatherosclerotic agents. 3. Substituted benzoic and non benzoic acid analogues of cetaben.

The synthesis of a series of analogues in which the carboxylic acid group of cetaben is replaced by carboxylate ester, carboxamide, or a variety of other substituent groups is described. Also reported are the syntheses of analogues in which the phenyl ring of cetaben is either modified by the presence of additional substituents or replaced entirely by another moiety. Structure-activity relationships of these compounds both as hypolipidemic agents and as inhibitors of the enzyme fatty acyl-CoA:cholesterol acyltransferase (ACAT) are discussed. Analogue syntheses designed to produce compounds that would be better absorbed orally than cetaben failed to yield any congeners of enhanced biological activity. In contrast, analogue syntheses directed toward non carboxylic acids of similar acidity to cetaben produced a very active class of sulfonamides.

Potential antiatherosclerotic agents. 2. (Aralkylamino)- and (alkylamino) benzoic acid analogues of cetaben.

The syntheses of a series of (aralkylamino)- and (alkylamino)benzoic acids, as well as the corresponding esters and sodium salts, are described. The compounds were evaluated in vivo in rats for serum sterol and triglyceride lowering activity and in vitro for activity in inhibiting the principle cholesterol-esterifying enzyme of the arterial wall, fatty acyl-CoA:cholesterol acyltransferase (ACAT). Based on a combination of these two activities, cataben sodium (150) was selected for development as a hypolipidemic and potential antiatherosclerotic agent.

The effects of endothelial cell-conditioned media on the proliferation of aortic smooth muscle cells and 3T3 cells in culture.

Normal aortic endothelial cells cultured in serum-free medium elaborate a factor(s) which cause the proliferation of smooth muscle cells or 3T3 cells grown in medium containing plasma-derived serum. For smooth muscle cell growth plasma factors are required in addition to the endothelial cell-conditioned medium whereas 3T3 cells will grow in the presence of endothelial cell-conditioned medium alone. Injured, growing cultures of endothelial cells do not appear to elaborate any more of the mitogen than normal, quiescent endothelial cells. The mitogenic factor is as yet poorly characterized but is heat stable at 56 degree C for 30 minutes. These results suggest that endothelial cell-smooth muscle cell interactions may contribute to the intimal smooth muscle cell proliferation which is a characteristic of the atherosclerotic lesion.

CL 277,082: a novel inhibitor of ACAT-catalyzed cholesterol esterification and cholesterol absorption.

CL 277,082 (I) was found to be a potent inhibitor of acyl CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in microsomes from a variety of tissues with IC50 values of 0.14 microM for intestinal mucosal microsomes, 0.74 microM for liver, and 1.18 microM for rat adrenal. I was also shown to inhibit ACAT in cultured smooth muscle cells (IC50 = 0.8 microM) and was found to be specific in inhibiting cholesterol esterification since it did not inhibit fatty acid incorporation into triglycerides or phospholipids. Also, other cholesterol esterifying enzymes such as lecithin:cholesterol acyltransferase (LCAT) and pancreatic cholesterol esterase were not inhibited by I, nor was esterification of retinol by acyl CoA:retinol acyltransferase (ARAT) from intestinal mucosal microsomes inhibited. I was a potent inhibitor of cholesterol absorption in cholesterol-fed rats by markedly inhibiting increases in liver and serum cholesterol concentration (ED50 = 5.2 mg/kg per day) while increasing the excretion of neutral 14C-labeled sterol in the feces.

Perifused adipose cells, quantitation and kinetics of lipolysis.

The perifused fat cell system is a system with which lipolytic activity can be monitored on a minute-to-minute basis. Thus, the rate at which lipolysis changes following the addition and removal of hormones can be followed. Catecholamines and other lipolytic agents produced a time-dependent increase in lipolysis following addition of agents, and a time-dependent decrease in lipolysis occurred following removal of the agent. ACTH also produced an increase in lipolysis. However, on termination of ACTH infusion, the lipolytic rate did not return to basal level but remained elevated for at least an additional 30 min (persistent phase). The persistent phase could be terminated by removal of Ca2+. Readdition of Ca2+ in the absence of additional ACTH resulted in a rapid increase in glycerol release. No persistant phase occurred following ACTH if the adipocytes were perifused in a Ca2+-free buffer. However, if Ca2+ was added to the system 20 min after termination of ACTH infusion, lipolysis increased to a rate greater than that obtained initially by infusing ACTH in a Ca2+-free buffer. It is concluded that ACTH is bound to some component of the fat cell in a Ca2+ independent, tenacious manner, and the full manifestation of that binding is dependent on the presence of Ca2+.

Hypercholesterolemia in the rabbit induced by feeding graded amounts of low-level cholesterol. Methodological considerations regarding individual variability in response to dietary cholesterol and development of lesion type.

While a number of studies have presented detailed examinations of lesion development in the cholesterol-fed rabbit, individual variability in response to cholesterol feeding and type of lesion produced relative to the degree of cholesterol exposure is not well defined. This study analyzed such critical parameters in an attempt to further characterize the model and establish a baseline for future testing of treatments targeted at limiting atherosclerosis. For these experiments, male New Zealand White rabbits were fed atherogenic diets consisting of 0.05%, 0.10%, 0.15%, 0.20%, or 0.25% cholesterol dissolved in 6% peanut oil for 31 to 32 weeks. Raising dietary cholesterol from 0.05% to 0.15% resulted in a less than twofold stepwise increase in total plasma cholesterol (TPC) exposure (area under plasma cholesterol versus time curve), whereas further increases in cholesterol intake resulted in an exponential four- to fivefold increase in TPC exposure. Regression analysis of TPC exposure with aortic sudanophilia demonstrated a threshold of approximately 5000 cholesterol weeks; below this limit lesions were minimal, and above this value the degree of plaque correlated with TPC exposure. Furthermore, a wide biological variability occurred among rabbits with respect to individual responsiveness to dietary cholesterol. In the aorta, various types of plaques, from fatty streaks to atheromatous lesions, were observed, depending on the degree of cholesterol intake. Diets consisting of < 0.15% cholesterol resulted in the development of fatty streak lesions, while transitional lesions and atheromatous plaques were mostly found with higher cholesterol feeding. Coronary artery atherosclerosis was present in > 50% of animals fed diets > or = 0.15% cholesterol. Despite the level of TPC exposure, coronary lesions in epicardial vessels were generally the fibrous type, whereas intramyocardial arteries demonstrated predominantly intimal foam cells. In conclusion, by adjusting dietary cholesterol intake and selecting rabbits with a similar responsiveness to cholesterol, the overall cholesterol exposure can be more closely controlled to minimize the inherent individual variability among animals in this model. The nature of the target lesion must also be carefully considered, because the efficacy of some treatments may depend on the type of atherosclerotic plaque.

Beta-3 adrenoceptor selectivity of the dioxolane dicarboxylate phenethanolamines.

The beta-1, beta-2 and beta-3 adrenergic properties of several benzodioxole-containing phenethanolamines were determined in vitro in both functional and binding assays. In addition, two of the compounds were evaluated for their effects on radioligand binding and cyclic AMP (cAMP) production in stably transfected Chinese Hamster Ovary (CHO) cells expressing the cloned rat or human beta-3 adrenoceptor or the human beta-2 or beta-1 adrenoceptor. The (+/-)-R*,R*-racemate, CL 314,514, and the pure (-)-R,R enantiomer, CL 316,243, stimulated rat adipocyte lipolysis (beta-3 effect) with EC50 values in the low nanomolar range, while having no effect on the rate of contraction of guinea pig atria (beta-1 effect) and little or no ability to prevent the insulin-stimulated incorporation of [14C]glucose into rat soleus muscle glycogen (beta-2 effect) with concentrations as great as 100 microM. The lack of beta-1 and beta-2 adrenergic activity was confirmed by the low affinity of the compounds for beta-1 or beta-2 adrenoceptors in plasma membranes from rat heart or rat soleus muscle, respectively. In CHO cells expressing each human beta adrenoceptor subtype, CL 314,514 bound to beta-3-CHO cells with a Ki of 2 microM and stimulated cAMP production with an activation constant (Kact) of 1 microM, whereas it did not bind to either beta-1- or beta-2-CHO cells at 100 microM. CL 316,243 bound to membranes from rat beta-3-CHO cells with a Ki of 1 microM and stimulated cAMP production in beta-3-CHO cells with a Kact of 0.7 nM.(ABSTRACT TRUNCATED AT 250 WORDS)