Discovery of 7-tetrahydropyran-2-yl chromans: ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors that reduce amyloid ß-protein (Aß) in the central nervous system.

In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aß1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aß1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.

Spirocyclic ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors: from hit to lowering of cerebrospinal fluid (CSF) amyloid ß in a higher species.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aß), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aß is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aß in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aß lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aß in rodents and in monkey.

Single-agent inhibition of Chk1 is antiproliferative in human cancer cell lines in vitro and inhibits tumor xenograft growth in vivo.

Chk1 is a serine/threonine kinase that plays several important roles in the cellular response to genotoxic stress. Since many current standard-of-care therapies for human cancer directly damage DNA or inhibit DNA synthesis, there is interest in using small molecule inhibitors of Chk1 to potentiate their clinical activity. Additionally, Chk1 is known to be critically involved in cell cycle progression of unperturbed cells. Therefore, it is plausible that treatment with a Chkl inhibitor alone could also be an efficacious cancer therapy. Here we report that Chk1-A, a potent and highly selective small molecule inhibitor of Chk1, is antiproliferative as a single agent in a variety of human cancer cell lines in vitro. The inhibition of proliferation is associated with collapse of DNA replication and apoptosis. Rapid decreases in inhibitory phosphorylation of CDKs and a concomitant increase in CDK kinase activity and chromatin loading of Cdc45 suggest that the antiproliferative and proapoptotic activity of Chk1-A is at least in part due to deregulation of DNA synthesis. We extend these in vitro studies by demonstrating that Chk1-A inhibits the growth of tumor xenografts in vivo in a treatment regimen that is well tolerated. Together, these results suggest that single-agent inhibition of Chk1 may be an effective treatment strategy for selected human malignancies.

Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors.

Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

Non-charged thiamine analogs as inhibitors of enzyme transketolase.

Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.

Prodrug thiamine analogs as inhibitors of the enzyme transketolase.

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.