Christopher Fotsch

Mitigating Heterocycle Metabolism in Drug Discovery

■ INTRODUCTION In the past few decades, drug metabolism research has played an ever increasing role in the design of drugs. In vitro metabolism assays have become an integral part of the routine profiling of compounds made in drug discovery. The data from these assays have allowed medicinal chemists to focus their efforts on compounds with improved metabolic stability. Detailed metabolite identification studies are also done more routinely, which provide information on how to strategically replace or block metabolically labile sites. Additionally, in vivo PK studies are regularly conducted in drug discovery, which helps to build in vitro−in vivo PK relationships. The positive influence that these advances in PKDM sciences have had on drug discovery is reflected in the fact that fewer drug candidates fail in the clinic for PKDM related issues. This suggests that medicinal chemists are successfully integrating the data generated by their PKDM colleagues into the design of compounds with fewer metabolic liabilities. Extensive data from metabolism studies have allowed medicinal chemists to develop general principles for reducing compound metabolism. These methods include, but are not limited to, reducing lipophilicity, altering sterics and electronics, introducing a conformational constraint, and altering the stereochemistry of their compounds. While no single method is able to solve every metabolic problem, these principles do give medicinal chemists guidance on how to improve the metabolic liabilities of their compounds. If the specific site of metabolism is known, medicinal chemists block the site, typically with a fluorine, or replace the metabolically labile group with a bioisostere. While several authors have reviewed these techniques for reducing metabolism, there is no review that summarizes different approaches to improving the metabolic stability of heterocycles. In this review, we summarize examples where changes were made at or near the heterocycle to improve metabolic stability. By summarizing these examples, we hope to provide a useful guide to medicinal chemists as they attempt to improve the metabolic profile of their own heterocyclic compounds. The majority of the examples that are included in this review came from searching the online open access database CHEMBL. In addition to having pharmacology data on compounds from the medicinal chemistry literature, CHEMBL has over 120 000 points of data on the ADMET properties of compounds. With the help of the visualization software Spotfire, we were able to cull examples from the CHEMBL ADMET data that focused on heterocycles. We also identified examples from papers that cite leading reviews in the drug metabolism field and were present in other recent reviews on drug metabolism. The main criteria that we placed on the examples selected for this review was that the change made to improve metabolism had to occur at or near the heterocycle and nowhere else on the molecule. This allowed us to eliminate examples where a change made to a compound away from the heterocycle may have influenced the metabolism. The data that we included in this review is predominantly from in vitro microsomal stability studies. However, we have included some data from bioactivation studies and in vivo PK studies to provide additional information about the overall metabolic profile. In several instances, the compound with the improved metabolic profile also became the lead compound in the paper, so we felt that including the data on the intended target was informative even though this is not a discussion point for the review. Of course, in some examples when the heterocycle was modified to improve metabolic stability, the activity at the intended biological target diminished. However, we felt that these examples of improved metabolic stability would still be of value to the reader. In the discussion below, we have organized the review by first discussing saturated heterocycles and then heteroaromatic compounds. Within each section we have organized the discussion by ring size.

Calcium sensing receptor activators: calcimimetics.

The calcium sensing receptor (CaR) is a G protein-coupled receptor (GPCR) that plays a fundamental role in serum calcium homeostasis. The CaR is expressed on the chief cells of the parathyroid gland and is responsible for controlling the secretion of parathyroid hormone (PTH). PTH acts on several organs including the bone, kidney, and intestine to tightly regulate the concentration of serum calcium. Substances other than calcium that activate the CaR are referred to as calcimimetics. Calcimimetics that bind to the CaR as agonists are referred to as type I. Type II calcimimetics bind to a site that is distinct from the physiological ligand and function as positive allosteric modulators of the CaR. Type II calcimimetics amplify the sensitivity of the CaR to serum calcium and are thus able to lower the concentration of serum PTH. Calcimimetics are being pursued as therapeutics for the treatment of disorders that are characterized by elevated levels of PTH such as primary and secondary hyperparathyroidism (primary HPT and secondary HPT). In this review, we provide an overview of key results in the discovery of cinacalcet HCl (Sensipar in the US, Mimpara in Europe). In addition, other recently disclosed type II calcimimetics are discussed.

11β-Hydroxysteroid dehydrogenase-1 as a therapeutic target for metabolic diseases

11β-Hydroxysteroid dehydrogenase-1 (11βHSD1) is a therapeutic target for Type 2 diabetes that has stimulated the interest of many pharmaceutical companies. Mounting evidence obtained from both preclinical and clinical studies support the contention that inhibiting 11βHSD1 will have a therapeutic benefit by lowering glucose output and increasing insulin sensitivity. In just over two years, 21 applications containing 11βHSD1 inhibitors have been published. In this review, the target rationale and patent applications from Merck, Novo Nordisk, AstraZeneca, Sterix, Biovitrum, Janssen and Novartis will be discussed.

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK–GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Discovery of a Potent, Orally Active 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor for Clinical Study: Identification of (S)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221)

Thiazolones with an exo-norbornylamine at the 2-position and an isopropyl group on the 5-position are potent 11beta-HSD1 inhibitors. However, the C-5 center was prone to epimerization in vitro and in vivo, forming a less potent diastereomer. A methyl group was added to the C-5 position to eliminate epimerization, leading to the discovery of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221). This compound decreased fed blood glucose and insulin levels and reduced body weight in diet-induced obesity mice.

Blockade of Glucocorticoid Excess at the Tissue Level : Inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1 as a Therapy for Type 2 Diabetes

Glucocorticoids are stress hormones with regulatory effects on carbohydrate, protein, and lipid metabolism. There are two forms of glucocorticoids in humans: the active cortisol (corticosterone in rodents) and inactive cortisone (11-dehydrocorticosterone in rodents). The physiological actions of glucocorticoids are mediated by glucocorticoid receptor (GR) which, upon binding to its natural ligand cortisol, is activated and regulates diverse physiological events. GR is a nuclear receptor ubiquitously expressed in tissues and triggers biological effects through transcriptional activation or suppression of target genes (Figure 1). Cortisol is synthesized in the adrenal glands as part of adrenal steroidogenesis that also involves the production of mineralocorticoids and androgens. Cortisol is secreted in a relatively high level at 10-20 mg/day. Cortisol biosynthesis is tightly controlled by adrenocorticotropic hormone (ACTH), a peptide hormone secreted from the anterior pituitary and is itself regulated by the hypothalamic peptide corticotrophin-releasing hormone (CRH). Circulating cortisol regulates its own biosynthesis by sending negative feedback signals to the pituitary and hypothalamus. Together, this neuroendocrine feedback circuit constitutes the hypothalamic-pituitary-adrenal (HPA) axis. The HPA activity is stimulated by physical or psychological stress and varies throughout the 24 h cycle. As a result, the circulating cortisol undergoes circadian rhythm, reaching its peak concentration of ∼800 nmol/L in the morning and nadir of ∼200 nmol/L at midnight in humans. About 96% of the circulating cortisol is protein-bound with 6% to albumin and 90% to corticosteroid binding globulin (CBG). Circulating CBG levels are approximately 700 nmol/L and regulated by estrogens and disease conditions. Free cortisol dictates glucocorticoid action. It is thought that CBG may serve to restrict access of cortisol to target tissues and regulate its bioavailability and metabolic clearance. CBG may also serve as a carrier for cortisol facilitating transport of cortisol in blood to certain tissues. In contrast, the inactive cortisone is in a free unbound form and its plasma concentration remains steady at approximately 100 nmol/L throughout the day. The metabolism of both cortisol and cortisone occurs in liver involving the A-ring reductases and several other enzymes, and the principal metabolites are tetrahydrocortisone (THE) and 5Rand 5 -tetrahydrocortisol (5Rand 5 -THF) (Figure 2). Another aspect of the regulation of glucocorticoid production involves two 11 -hydroxysteroid dehydrogenase (11 -HSD) isozymes that interconvert cortisone and cortisol (Figure 2). 11 -HSD1 is a reductase in vivo converting cortisone to cortisol and amplifies glucocorticoid action in a tissue-specific manner. In contrast, its isozyme 11 HSD2 acts as a dehydrogenase and catalyzes the opposite reaction, converting cortisol to cortisone. 11 -HSD1 is predominantly expressed in liver, adipose, placenta, and brain. 11 -HSD2 is primarily expressed in kidney and functions as the main source of cortisone production. Together, glucocorticoid homeostasis is maintained by the HPA axis and the activities of the 11 -HSD enzymes. The metabolic syndrome is a cluster of metabolic abnormalities including central obesity, insulin resistance, atherogenic * To whom correspondence should be addressed. Telephone: 805-4475598. Fax: 805-499-0953. E-mail: mwang@amgen.com. † Department of Medicinal Chemistry. ‡ Department of Metabolic Disorders. a Abbreviations: 11 -HSD1 or -2, 11 -hydroxysteroid dehydrogenase type 1 or 2; CBG, corticosteroid binding globulin; CBX, carbenoxolone; CRH, corticotrophin-releasing hormone; GA, glycyrrhetinic acid; GR, glucocorticoid receptor; HPA axis, the hypothalamic-pituitary-adrenal axis; THE, tetrahydrocortisone; THF, tetrahydrocortisol.  Copyright 2008 by the American Chemical Society

2-amino-1,3-thiazol-4(5H)-ones as potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitors: enzyme-ligand co-crystal structure and demonstration of pharmacodynamic effects in C57Bl/6 mice.

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11beta-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5 H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11beta-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11beta-HSD1 with compound 6d (Ki=28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11beta-HSD1 (Ki=3 nM) and also inhibited 11beta-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.

Design of a new peptidomimetic agonist for the melanocortin receptors based on the solution structure of the peptide ligand, Ac-Nle-cyclo[Asp-Pro-dPhe-Arg-Trp-Lys]-NH2

The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional groups led to analogues with lower affinity for the MC receptors.

Time of the day for 11β‐HSD1 inhibition plays a role in improving glucose homeostasis in DIO mice

Aims:  The physiological effects of glucocorticoids in a given tissue are driven by the local level of the active glucocorticoid, which is determined by two sources: the plasma cortisol in human (or corticosterone in rodents) and the cortisol produced locally through 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) activity. Because of the circadian variation of plasma glucocorticoids, the pharmacological efficacy of 11β‐HSD1 inhibition may depend on the time of the day for inhibitor administration.

Structural characterization and pharmacodynamic effects of an orally active 11beta-hydroxysteroid dehydrogenase type 1 inhibitor.

11β‐Hydroxysteroid dehydrogenase type 1 regulates glucocorticoid action and inhibition of this enzyme is a viable therapeutic strategy for the treatment of type 2 diabetes and the metabolic syndrome. Here, we report a potent and selective 11β‐hydroxysteroid dehydrogenase type 1 inhibitor with a binding mode elucidated from the co‐crystal structure with the human 11β‐hydroxysteroid dehydrogenase type 1. The inhibitor is bound to the steroid‐binding pocket making contacts with the catalytic center and the solvent channel. The inhibitor binding is facilitated by two direct hydrogen bond interactions involving Tyrosine183 of the catalytic motif Tyr‐X‐X‐X‐Lys and Alanine172. In addition, the inhibitor makes many hydrophobic interactions with both the enzyme and the co‐factor nicotinamide adenine dinucleotide phosphate (reduced). In lean C57BL/6 mice, the compound inhibited both the in vivo and ex vivo 11β‐hydroxysteroid dehydrogenase type 1 activities in a dose‐dependent manner. The inhibitory effects correlate with the plasma compound concentrations, suggesting that there is a clear pharmacokinetic and pharmacodynamic relationship. Moreover, at the same doses used in the pharmacokinetic/pharmacodynamic studies, the inhibitor did not cause the activation of the hypothalamic–pituitary–adrenal axis in an acute mouse model, suggesting that this compound exhibits biological effects with minimal risk of activating the hypothalamic–pituitary–adrenal axis.