Peter J. Sinclair

Biochemical characterization of cholesteryl ester transfer protein inhibitors

Cholesteryl ester transfer protein (CETP) has been identified as a novel target for increasing HDL cholesterol levels. In this report, we describe the biochemical characterization of anacetrapib, a potent inhibitor of CETP. To better understand the mechanism by which anacetrapib inhibits CETP activity, its biochemical properties were compared with CETP inhibitors from distinct structural classes, including torcetrapib and dalcetrapib. Anacetrapib and torcetrapib inhibited CETP-mediated cholesteryl ester and triglyceride transfer with similar potencies, whereas dalcetrapib was a significantly less potent inhibitor. Inhibition of CETP by both anacetrapib and torcetrapib was not time dependent, whereas the potency of dalcetrapib significantly increased with extended preincubation. Anacetrapib, torcetrapib, and dalcetrapib compete with one another for binding CETP; however anacetrapib binds reversibly and dalcetrapib covalently to CETP. In addition, dalcetrapib was found to covalently label both human and mouse plasma proteins. Each CETP inhibitor induced tight binding of CETP to HDL, indicating that these inhibitors promote the formation of a complex between CETP and HDL, resulting in inhibition of CETP activity.

Biphenyl-Substituted Oxazolidinones as Cholesteryl Ester Transfer Protein Inhibitors: Modifications of the Oxazolidinone Ring Leading to the Discovery of Anacetrapib

The development of the structure-activity studies leading to the discovery of anacetrapib is described. These studies focused on varying the substitution of the oxazolidinone ring of the 5-aryloxazolidinone system. Specifically, it was found that substitution of the 4-position with a methyl group with the cis-stereochemistry relative to the 5-aryl group afforded compounds with increased cholesteryl ester transfer protein (CETP) inhibition potency and a robust in vivo effect on increasing HDL-C levels in transgenic mice expressing cynomolgus monkey CETP.

2-Arylbenzoxazoles as CETP inhibitors: Substitution of the benzoxazole moiety

A series of 2-arylbenzoxazole inhibitors of the cholesterol ester transfer protein (CETP) is described. Structure-activity studies focused on variation of the substitution of the benzoxazole moiety. Substitution at the 5- and 7-positions of the benzoxazole moiety was found to be beneficial for CETP inhibition. Compound 47 was found to be the most potent inhibitor in this series and inhibited CETP with an IC(50) of 28nM.

Benzamide derivatives as blockers of Kv1.3 ion channel

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.

[3H]paraherquamide binding to Caenorhabditis elegans : studies on a potent new anthelmintic agent

Paraherquamide was identified recently as a potent anthelmintic agent. In this paper we describe the identification and characterization of a specific, high-affinity paraherquamide binding site in a membrane preparation isolated from the free-living nematode, Caenorhabditis elegans. [3H] Paraherquamide bound specifically to C. elegans membranes with an apparent dissociation constant, Kd, of 263 nM. A series of paraherquamide analogs were examined, and their relative affinity for the paraherquamide binding site correlated with their nematocidal activity. Phenothiazines were the only other class of anthelmintics tested which inhibited specific [3H]paraherquamide binding. These results suggest that the anthelmintic activity of paraherquamide and phenothiazine is mediated via an interaction with a common binding site.

2-Arylbenzoxazoles as CETP inhibitors: raising HDL-C in cynoCETP transgenic mice.

We describe structure-activity studies leading to the discovery of 2-arylbenzoxazole 3, the first in a series to raise serum high-density lipoprotein cholesterol levels in transgenic mice. Replacement of the 4-piperidinyloxy moiety with piperazinyl provided a more synthetically tractable lead, which upon optimization resulted in compound 4, an excellent inhibitor of cholesteryl ester transfer protein function with good pharmacokinetic properties and in vivo efficacy.

Preparation and in vitro activities of naphthyl and indolyl ether derivatives of the FK-506 related immunosuppressive macrolide ascomycin

Abstract The synthesis of naphthyl- and indolyl-ethers of the immunosuppressive macrolide ascomycin using pentavalent bismuth reagents is described. The in vitro activities of the aryl ether analogs are reported. The indole ether analogs show increased immunosuppressive activity in vitro relative to the parent macrolide.

A tacrolimus-related immunosuppressant with reduced toxicity

BACKGROUND Tacrolimus (FK506) has potent immunosuppressive properties reflecting its ability to block the transcription of lymphokine genes in activated T cells through formation of a complex with FK506 binding protein-12, which inhibits the phosphatase activity of calcineurin. The clinical usefulness of tacrolimus is limited, however, by severe adverse effects, including neurotoxicity and nephrotoxicity. Although this toxicity, like immunosuppression, appears mechanistically related to the calcineurin inhibitory action of the drug, a large chemistry effort has been devoted to search for tacrolimus analogs with reduced toxicity but preserved immunosuppressive activity that might have enhanced therapeutic utility. METHODS Here, we report on the identification of such an analog, which was synthetically derived from ascomycin (ASC), the C21 ethyl analog of tacrolimus, by introducing an indole group at the C32 position. The profile of biological activity of indolyl-ASC was characterized in rodent models of immunosuppression and toxicity. RESULTS Indolyl-ASC was found to exhibit an immunosuppressive potency equivalent to that of tacrolimus in T-cell activation in vitro and in murine transplant models, even though indolyl-ASC bound about 10 times less to intracellular FK506 binding protein-12 than tacrolimus or ASC. Further evaluation of indolyl-ASC revealed that it is threefold less potent than tacrolimus in inducing hypothermia, a response that may reflect neurotoxicity, and in causing gastrointestinal transit alterations in mice. Moreover, indolyl-ASC was at least twofold less nephrotoxic than tacrolimus upon 3-week oral treatment in rats. CONCLUSIONS Altogether, these data indicate a modest but definite improvement in the therapeutic index for indolyl-ASC compared with tacrolimus in rodent models.

The medicinal chemistry of FK-506

Substantial strides have been made in the past decade in the discovery of potent immunosuppressants that are effective in the prevention of rejection in organ transplantation and in the treatment of autoimmune diseases. The past three years have witnessed stunning breakthroughs in the elucidation of the common mechanism of immunosuppressive action of FK-506 and cyclosporin A (CsA). FK-506 is 50- to 100-fold more potent than CsA in its immunosuppressive activity in vitro and in vivo, but retains the toxic side effects of CsA. This review describes the medicinal chemical evaluation of FK-506 with the objective of identifying positions on the macrolactam that can be altered without substantially reducing immunosuppressive activity. Suitable modification at such positions may reduce toxicity, thereby improving the overall therapeutic index and broadening the patient population that can be treated for autoimmune-related diseases.

A tacrolimus-related immunosuppressant with biochemical properties distinct from those of tacrolimus

BACKGROUND Tacrolimus (FK506) is an immunosuppressive drug 50-100 times more potent than cyclosporine (CsA), the current mainstay of organ transplant rejection therapy. Despite being chemically unrelated, CsA and tacrolimus exert their immunosuppressive effects through the inhibition of calcineurin (CaN), a critical signaling molecule during T-lymphocyte activation. Although numerous clinical studies have proven the therapeutic efficacy of drugs within this class, tacrolimus and CsA also have a strikingly similar profile of unwanted side effects. METHOD Our objective has been to identify a less toxic immunosuppressant through the modification of ascomycin (FK520). Quantitative in vitro immunosuppression and toxicity assays have demonstrated (see the accompanying article, p. 18) that we achieved our goal with L-732,531 (indolyl-ascomycin; indolyl-ASC), a 32-O-(1-hydroxyethylindol-5-yl) ascomycin derivative with an improved therapeutic index relative to tacrolimus. RESULTS We report that the attributes of indolyl-ASC may result from its distinctive biochemical properties. In contrast to tacrolimus, indolyl-ASC binds poorly to FK506 binding protein 12 (FKBP12), the major cytosolic receptor for tacrolimus and related compounds. However, the stability of the interaction between the FKBP12-indolyl-ASC complex and CaN is much greater than that of the FKBP12-tacrolimus complex. These distinguishing properties of indolyl-ASC result in the potent inhibition of CaN within T lymphocytes but may lower the accumulation of the drug at sites of toxicity. CONCLUSIONS Indolyl-ASC may define those properties needed to increase the therapeutic efficacy of a macrolactam immunoregulant for treating both human autoimmune disease and organ transplant rejection.