A novel series of highly potent benzimidazole-based microsomal triglyceride transfer protein inhibitors.

A series of benzimidazole-based analogues of the potent MTP inhibitor BMS-201038 were discovered. Incorporation of an unsubstituted benzimidazole moiety in place of a piperidine group afforded potent inhibitors of MTP in vitro which were weakly active in vivo. Appropriate substitution on the benzimidazole ring, especially with small alkyl groups, led to dramatic increases in potency, both in a cellular assay of apoB secretion and especially in animal models of cholesterol lowering. The most potent in this series, 3g (BMS-212122), was significantly more potent than BMS-201038 in reducing plasma lipids (cholesterol, VLDL/LDL, TG) in both hamsters and cynomolgus monkeys.

An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

1,1-Bisphosphonate squalene synthase inhibitors: interplay between the isoprenoid subunit and the diphosphate surrogate.

Inhibitors of squalene synthase have the potential to be superior cholesterol-lowering agents. We previously disclosed that lipophilic 1,1-bisphosphonates I are potent squalene synthase inhibitors and orally active cholesterol-lowering agents in animal models (Ciosek, C. P., Jr.; et al. J. Biol. Chem. 1993, 268, 24832-24837). In this paper, we describe modifications to the bisphosphonate moiety, in an attempt to reduce the number of acidic functions contained in these inhibitors. Replacing one of the acidic groups with a methyl (II, R2 = CH3) results in potent inhibitors when paired with a close mimic of the naturally occurring farnesyl moiety (R1 = farnesylethyl) but not when paired with the shorter isoprene surrogates (R1 = geranylethyl or 4-biphenylpropyl). In contrast, all three corresponding bisphosphonates I are potent squalene synthase inhibitors. Inhibitory potency is recovered with the shorter isoprene surrogates when R2 is CH2OH or CH2OCH3. It is proposed that these R2 groups serve as hydrogen bond acceptors with the active site of the enzyme. The properties of these compounds as cholesterol biosynthesis inhibitors in rats are described, and synthetic routes to these and related compounds are detailed.

Lipophilic 1,1-bisphosphonates are potent squalene synthase inhibitors and orally active cholesterol lowering agents in vivo.

Squalene synthase catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to form squalene at the final branchpoint of the cholesterol biosynthetic pathway. We report herein that isoprenyl 1,1-bisphosphonates and related analogs are potent inhibitors of rat microsomal squalene synthase (I50 = 0.7-32 nM). In addition, members of this family are potent inhibitors of cholesterol biosynthesis in rats on intravenous and oral dosing, as well as cholesterol lowering agents in rats and hamsters. Significant inhibition of cholesterol biosynthesis in rats by lovastatin occurs with a concomitant inhibition of dolichol and coenzyme-Q9 synthesis. In contrast, bisphosphonate 4 has no effect on dolichol and coenzyme-Q9 biosynthesis in rats under conditions where cholesterol biosynthesis is > 90% inhibited.

Phosphorus-containing inhibitors of HMG-CoA reductase. 2. Synthesis and biological activities of a series of substituted pyridines containing a hydroxyphosphinyl moiety.

A series of 2,3,4,(5),6-substituted pyridines containing a hydroxyphosphinyl functionally have been prepared and were evaluated for their ability to inhibit the enzyme HMG-CoA reductase. Systematic substitution of both R1-R4 and X-Y led to compounds of type 3-6 with in vitro potency greater than that of mevinolin (Na salt).

Cartilage-associated collagenolytic activity in rabbits with antigen-induced chronic synovitis.

Rabbit articular cartilage fragments from knees with normal and antigen-induced chronic synovitis were assayed for active and latent collagenolytic activity. Significant levels of latent collagenolytic activity, as measured by hydroxyproline release, were associated with cartilage from chronic synovitis knee cartilage but not from normal knee cartilage. Neither normal nor chornic synovitis knee cartilage contained demonstrable levels of spontaneously active cartilage-associated collagenolytic activity. Cartilage-associated latent collagenolytic activity was demonstrated only after activation with either trypsin or p-aminophenylmercuric acetate and was inhibited by EDTA (10(-2)M) and 1,10-phenanthroline (10(-3) M). Cartilage-associated latent collagenolytic activity was demonstrated after incubation of activated cartilage at 37 degrees C but not 4 degrees C. The activity could not be removed or diminished by extensive prewashing of these cartilage fragments. Treatment of rabbits undergoing development of antigen-induced chronic synovitis, with methylprednisolone (1 mg/kg/day), significantly suppressed the level of cartilage-associated collagenolytic activity in antigen-challenged knees. The measurement of cartilage-associated collagenolytic activity may be useful for the evaluation of potential antirheumatic drugs.

Effects of antiinflammatory agents on the acute response of immune synovitis in rabbits.

Immune synovitis in rabbits was investigated as a potential in vivo model for evaluating new antiinflammatory agents. Antigen-induced increases in knee width as well as beta-glucuronidase and acid phosphatase activities in exudates were observed. Histologically, polymorphonuclear leukocytes appeared within hours in synovial tissues and reached maximum infiltration at about 24 hours. Subsequently, mononuclear cells, including plasma cells, appeared. The 6-hour Arthus-like phase of synovitis can be depressed by some antiinflammatory agents, colchicine and steroids being particularly effective. It is suggested that this model can be utilized to define potentially more effective antiinflammatory drugs.