Smallest Maximum Intramolecular Distance: A Novel Method to Mitigate Pregnane Xenobiotic Receptor Activation.

Induction of cytochrome P450 isoform 3A4 via activation of the pregnane xenobiotic receptor (PXR) is a concern for pharmaceutical discovery and development, as it can lead to drug-drug interactions. We present a novel molecular descriptor, the smallest maximum intramolecular distance (SMID), which is correlated with PXR activation, and a method for using the SMID descriptor to guide discovery chemists in modifying lead compounds to decrease PXR activation.

Novel, highly potent systemic glucokinase activators for the treatment of Type 2 Diabetes Mellitus.

Glucokinase (GK, hexokinase IV) is a unique hexokinase that plays a central role in mammalian glucose homeostasis. Glucose phosphorylation by GK in the pancreatic ß-cell is the rate-limiting step that controls glucose-stimulated insulin secretion. Similarly, GK-mediated glucose phosphorylation in hepatocytes plays a major role in increasing hepatic glucose uptake and metabolism and possibly lowering hepatic glucose output. Small molecule GK activators (GKAs) have been identified that increase enzyme activity by binding to an allosteric site. GKAs offer a novel approach for the treatment of Type 2 Diabetes Mellitus (T2DM) and as such have garnered much attention. We now report the design, synthesis, and biological evaluation of a novel series of 2,5,6-trisubstituted indole derivatives that act as highly potent GKAs. Among them, Compound 1 was found to possess high in vitro potency, excellent physicochemical properties, and good pharmacokinetic profile in rodents. Oral administration of Compound 1 at doses as low as 0.03mg/kg led to robust blood glucose lowering efficacy in 3week high fat diet-fed mice.

Discovery of orally active hepatoselective glucokinase activators for treatment of Type II Diabetes Mellitus.

Systemically acting glucokinase activators (GKA) have been demonstrated in clinical trials to effectively lower blood glucose in patients with type II diabetes. However, mechanism-based hypoglycemia is a major adverse effect that limits the therapeutic potential of these agents. We hypothesized that the predominant mechanism leading to hypoglycemia is GKA-induced excessive insulin secretion from pancreatic ß-cells at (sub-)euglycemic levels. We further hypothesized that restricting GK activation to hepatocytes would maintain glucose-lowering efficacy while significantly reducing hypoglycemic risk. Here we report the discovery of a novel series of carboxylic acid substituted GKAs based on pyridine-2-carboxamide. These GKAs exhibit preferential distribution to the liver versus the pancreas in mice. SAR studies led to the identification of a potent and orally active hepatoselective GKA, compound 6. GKA 6 demonstrated robust glucose lowering efficacy in high fat diet-fed mice at doses ⩾10mpk, with ⩾70-fold liver:pancreas distribution, minimal effects on plasma insulin levels, and significantly reduced risk of hypoglycemia.

eCounterscreening: using QSAR predictions to prioritize testing for off-target activities and setting the balance between benefit and risk.

During drug development, compounds are tested against counterscreens, a panel of off-target activities that would be undesirable for a drug to have. Testing every compound against every counterscreen is generally too costly in terms of time and money, and we need to find a rational way of prioritizing counterscreen testing. Here we present the eCounterscreening paradigm, wherein predictions from QSAR models for counterscreen activity are used to generate a recommendation as to whether a specific compound in a specific project should be tested against a specific counterscreen. The rules behind the recommendations, which can be summarized in a risk-benefit plot specific for a counterscreen/project combination, are based on a previously assembled database of prospective QSAR predictions. The recommendations require two user-defined cutoffs: the level of activity in a specific counterscreen that is considered undesirable and the level of risk the chemist is willing to accept that an undesired counterscreen activity will go undetected. We demonstrate in a simulated prospective experiment that eCounterscreening can be used to postpone a large fraction of counterscreen testing and still have an acceptably low risk of undetected counterscreen activity.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes as thrombin inhibitors.

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.

Extracellular loop C of NPC1L1 is important for binding to ezetimibe.

Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.

Spiroimidazolidinone NPC1L1 inhibitors. Part 2: structure-activity studies and in vivo efficacy.

Ezetimibe (Zetia®), a cholesterol-absorption inhibitor (CAI) approved by the FDA for the treatment of hypercholesterolemia, is believed to target the intestine protein Niemann-Pick C1-Like 1 (NPC1L1) or its pathway. A spiroimidazolidinone NPC1L1 inhibitor identified by virtual screening showed moderate binding activity but was not efficacious in an in vivo rodent model of cholesterol absorption. Synthesis of analogs established the structure-activity relationships for binding activity, and resulted in compounds with in vivo efficacy, including 24, which inhibited plasma cholesterol absorption by 67% in the mouse, thereby providing proof-of-concept that non-ß-lactams can be effective CAIs.

Spiroimidazolidinone NPC1L1 inhibitors. 1: Discovery by 3D-similarity-based virtual screening.

A series of spiroimidazolidinone NPC1L1 inhibitors was discovered by virtual screening of the Merck corporate sample repository using 3D-similarity-based screening. Selection of 330 compounds for testing in an in vitro NPC1L1 binding assay yielded six hits in six distinct chemical series. Follow-up 2D similarity searching yielded several sub- to low-micromolar leads; among these was spiroimidazolidinone 10, with an IC(50) of 2.5 microM. Compound 10 provided a useful scaffold to initiate a medicinal chemistry campaign.

Discovery of isoxazole voltage gated sodium channel blockers for treatment of chronic pain.

A series of novel isoxazole voltage gated sodium channel blockers have been synthesized and evaluated. Substitutions on the benzylic position of benzamide were investigated to determine their effect on Na(v)1.7 inhibitory potency. The spirocyclobutyl substitution had the most significant enhancement on Na(v)1.7 inhibitory activity.

Discovery of a novel class of isoxazoline voltage gated sodium channel blockers.

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity, while non-aromatic heterocycle replacements were well tolerated.

Structure-based design of novel groups for use in the P1 position of thrombin inhibitor scaffolds. Part 2: N-acetamidoimidazoles.

Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.

Knowledge-Based Approaches to Off-Target Screening.

Selective binding of a drug for its target vs other proteins is an important consideration in designing compounds with minimal risk of toxicity and therefore a key aspect of lead optimization. Screening all compounds against all known off-target proteins would be prohibitively expensive, so project teams must decide which compounds to screen in which assays. This chapter describes informatics-based methods that help prioritize testing, including screening minipanels and prioritization using predictive models (e-counterscreening).

Use of density functional calculations to predict the regioselectivity of drugs and molecules metabolized by aldehyde oxidase.

Aldehyde oxidase is a molybdenum hydroxylase that catalyzes the oxidation of aldehydes and nitrogen-containing heterocycles. The enzyme plays a dual role in the metabolism of physiologically important endogenous compounds and the biotransformation of xenobiotics. Using density functional theory methods, geometry optimization of tetrahedral intermediates of drugs and druglike compounds was examined to predict the likely metabolites of aldehyde oxidase. The calculations suggest that the lowest energy tetrahedral intermediate resulting from the initial substrate corresponds to the observed metabolite >or=90% of the time. Additional calculations were performed on a series of heterocyclic compounds where the products resulting from metabolism by xanthine oxidase and aldehyde oxidase differ in many instances. Again, the lowest energy tetrahedral intermediate corresponded to the observed product of aldehyde oxidase metabolism >or=90% for the compounds examined, while the observed products of xanthine oxidase were not well predicted.

Inhibition of recombinant cytochrome P450 isoforms 2D6 and 2C9 by diverse drug-like molecules.

The affinities of a diverse set of 500 drug-like molecules to cytochrome P450 isoforms 2C9 and 2D6 were measured using recombinant expressed enzyme. The dose-response curve of each compound was fitted with a series of equations representing typical or various types of atypical kinetics. Atypical kinetics was identified where the Akaike Information Criterion, plus other criteria, suggested the kinetics was more complex than expected for a Michaelis-Menten model. Approximately 20% of the compounds were excluded due to poor solubility, and approximately 15% were excluded due to fluorescence interference. Of the remaining compounds, roughly half were observed to bind with an affinity of 200 microM or lower for each of the two isoforms. Atypical kinetics was observed in 18% of the compounds that bind to cytochrome 2C9, but less than 2% for 2D6. The resulting collection of competitive inhibitors and inactive compounds were analyzed for trends in binding affinity. For CYP2D6, a clear relationship between polar surface area and charge was observed, with the most potent inhibitors having a formal positive charge and a low percent polar surface area. For CYP2C9, no clear trend between activity and physicochemical properties could be seen for the group as a whole; however, certain classes of compounds have altered frequencies of activity and atypical kinetics.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.

Synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. Appropriately substituted imidazole acetic acids such as 10j were found to be potent inhibitors of activated TAFI and selective versus the related carboxypeptidases CPA, CPN, and CPM but not CPB. Further, 10j accelerated clot lysis in vitro and was shown to be efficacious in a primate model of thrombosis.

Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase.

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.

Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines.

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazinone acetamide template (e.g., 1) which resulted in the modification of each of the three principal components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral bioavailability and long plasma half-lives.

Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors.

In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.84 nM. A study of ortho-substituted five-membered-ring heterocycles was undertaken and subsequently demonstrated that the o-triazole and tetrazole rings were optimal. Combination of these potent P(1) aryl heterocycles with a variety of P(2)-P(3) groups produced a compound with an extraordinary thrombin inhibitory activity of 1.4 pM. It is hoped that this potency enhancement in P(1) will allow for more diversification in the P(2)-P(3) region to ultimately address additional pharmacological concerns.