Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

BMS-196085: a potent and selective full agonist of the human beta(3) adrenergic receptor.

A series of 4-hydroxy-3-methylsulfonanilido-1,2-diarylethylamines were prepared and evaluated for their human beta(3) adrenergic receptor agonist activity. SAR studies led to the identification of BMS-196085 (25), a potent beta(3) full agonist (K(i)=21 nM, 95% activation) with partial agonist (45%) activity at the beta(1) receptor. Based on its desirable in vitro and in vivo properties, BMS-196085 was chosen for clinical evaluation.

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.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: oxime ether analogs of pravastatin.

Pravastatin, a potent anti-hypercholesteremic drug, has been developed by Bristol-Myers Squibb for treatment of hypercholesterolemia and other related diseases. Several structurally related compounds (SQ 31,554, SQ 31,879, SQ 31,947, SQ 32,391, SQ 32,770, SQ 32,390 and SQ 32,469) modified at the 3-position of the hexahydronaphthalene ring system of pravastatin were prepared in the course of developing the basic reagents for a radioimmunoassay of the parent drug. The biological activity of these analogs was comparable to pravastatin itself. Indeed, one member of this series was found to be several times more potent than pravastatin.

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).

Differential release of plasminogen activator and latent collagenase from mononuclear cell-stimulated synovial cells.

Independent studies have previously shown that mononuclear cell supernatants stimulate the release of plasminogen activator and latent collagenase from synovial cell monolayer cultures. Simultaneous secretion of these enzymes could be an important pathway for tissue destruction under inflammatory conditions, since plasminogen activator can cause activation of latent collagenase in the presence of plasminogen. We investigated the kinetics of release of the two enzymes from synovial cells in response to the addition of mononuclear cell supernatants and retinoic acid. Synovial cells derived from osteoarthritic and rheumatoid arthritic patients responded similarly. Plasminogen activator is released within a few hours of stimulation, and secretion usually stops when the stimulus is removed. In contrast, significant amounts of collagenase are secreted only after an initial lag period of 1--2 days, and secretion is sustained long after removal of mononuclear cell supernatant. Another difference in regulation of the secretion of these two neutral proteinases is that the addition of all-trans retinoic acid to the same synovial cell culture elevates plasminogen activator secretion while suppressing that of latent collagenase. Differential regulation of these enzymes under conditions of chronic inflammation may allow for continual accumulation of latent enzyme(s) which are activated during short periods of plasminogen activator release.

Rabbit synovial tissue in culture: influence of the inflammatory state in vivo on the secretion of collagenase.

Primary rabbit adherent synovial cell monolayer cultures were prepared from animals with antigen-induced synovitis. Primary cultures derived from chronically inflamed tissues released elevated levels of collagenase into the culture media compared to cultures derived from either normal or acute inflammatory tissues. No active of latent collagenase activity was detected in media of cultures derived from normal tissues grown under identical conditions. Cultures of acute inflammatory tissues secreted 1/4 to 1/10 the amount of collagenase when compared to cultures from chronically inflamed tissues. Collagenase secreted by cultures from inflammatory tissues was detected only in latent form. Fetal bovine serum enhanced the trypsin activation of latent collagenase produced by these cultures.

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.

Human synovial fibroblasts: the relationships between cyclic AMP, bradykinin, and prostaglandins.

Human synovial fibroblasts in culture respond to bradykinin (8 X 10(-9) M) with an increment in intracellular cyclic AMP concentration. These bradykinin (BK) concentrations are comparable to levels of the nonapeptide found in pathological synovial effusions. The cyclic AMP response to BK is enhanced by a heat stable factor(s) in fetal calf serum (FCS) and by the addition of arachidonic acid (AA) to monolayer cultures incubated in serum-free media. Synovial fibroblasts initially treated with BK are refractory to rechallenge with this agent as measured by the absence of an increment in cyclic AMP. These BK refractory cells do respond with significant increment in cyclic AMP to challenge with prostaglandin E1 (PGE1). Cells that have become refractory to PGE1 stimulation respond to BK. this suggests that a receptor or activator system different from the one for PGE1 and PGE2 exists for BK. When both BK and PGE1 are incubated together with synovial fibroblasts, the cyclic AMP response elicited is more than additive as compared to the response of each hormone separately. Indomethacin (IM) inhibits the BK evoked cyclic AMP response unless cell cultures are pretreated with PGE1. The PGE1 analog, 7-oxa-13-prostynoic acid, is a better inhibitor of the cyclic AMP response induced by BK than by PGE1. BK does not elicit a cyclic AMP response solely by elaborating PGE1, yet the prostaglandin pathway and its products seem to have a role in the degree of the cyclic AMP response to BK challenge.

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.

Human synoviocytes: activation and desensitization by prostaglandins and 1-epinephrine.

The human synoviocyte increases its intracellular adenosine 3':5'-cyclic monophosphate (cAMP) concentration significantly after incubation with prostaglandin E1, prostaglandin E2, or l-epinephrine. The cells can be desensitized to these same hormones. Hormone-induced desensitization is receptor site specific and associated with a significant increase in intracellular 3':5'-cyclic AMP phosphodiesterase (EC 3.1.4. 17; 3':5'-cyclic AMP 5'-nucleotidohydrolase) activity, whereas cAMP-induced desensitization is not hormone specific.

PGE1-mediated cyclic AMP refractoriness: effects of cycloheximide and indomethacin.

Human synoviocytes in culture respond to prostaglandin E1 (PGE1) by increasing their intracellular concentration of cyclic AMP. Readdition of PGE1 to cells previously treated with PGE1 elicits no change in the intracellular concentration of cyclic AMP. This refractory state is partially prevented by the inhibitors of protein synthesis, puromycin (PM) and cycloheximide (CH). Indomethacin (IM), which reduces angiotensin tachyphylaxis, does not prevent the occurrence of refractoriness to PGE1 with respect to accumulation of cyclic AMP. This agent does alter the release of cyclic AMP from human synovial cells. We postulate that other factors, independent of new protein synthesis, are necessary for the development of the complete PGE1 refractory state in these cells.