Lead Optimization of Small Molecule ENL YEATS Inhibitors to Enable In Vivo Studies: Discovery of TDI-11055.

Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.

MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants.

HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

Development of potent macrocyclic inhibitors of genotype 3a HCV NS3/4A protease.

A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.

Development of macrocyclic inhibitors of HCV NS3/4A protease with cyclic constrained P2-P4 linkers.

A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Soluble Adenylyl Cyclase (sAC, ADCY10) Inhibitors with Slow Dissociation Rates.

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.

MK-7009, a potent and selective inhibitor of hepatitis C virus NS3/4A protease.

The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.

Discovery of triarylethanolamine inhibitors of the Kv1.5 potassium channel.

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure-activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.

Phosphorous acid analogs of novel P2-P4 macrocycles as inhibitors of HCV-NS3 protease.

HCV-NS3 protease is essential for viral replication and NS3 protease inhibitors have shown proof of concept in clinical trials. Novel P2-P4 macrocycle inhibitors of NS3/4A comprising a P1 C-terminal carboxylic acid have recently been disclosed. A series of analogs, in which the carboxylic residue is replaced by phosphorous acid functionalities were synthesized and found to be inhibitors of the NS3 protease. Among them the methylphosphinate analogue showed nanomolar level of enzyme inhibition and sub-micromolar potency in the replication assay.

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists.

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure-activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K(+) channel. Preferred compounds were subsequently evaluated for selectivity in an alpha(1)-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.

Molecular modeling based approach to potent P2-P4 macrocyclic inhibitors of hepatitis C NS3/4A protease.

Molecular modeling of inhibitor bound full length HCV NS3/4A protease structures proved to be a valuable tool in the design of a new series of potent NS3 protease inhibitors. Optimization of initial compounds provided 25a. The in vitro activity and selectivity as well as the rat pharmacokinetic profile of 25a compare favorably with the data for other NS3/4A protease inhibitors currently in clinical development for the treatment of HCV.

Atrial antifibrillatory effects of structurally distinct IKur blockers 3-[(dimethylamino)methyl]-6-methoxy-2-methyl-4-phenylisoquinolin-1(2H)-one and 2-phenyl-1,1-dipyridin-3-yl-2-pyrrolidin-1-yl-ethanol in dogs with underlying heart failure.

Drug discovery efforts have focused recently on atrial-selective targets, including the Kv1.5 channel, which underlies the ultrarapid delayed rectifier current, I(Kur), to develop novel treatments for atrial fibrillation (AF). Two structurally distinct compounds, a triarylethanolamine TAEA and an isoquinolinone 3-[(dimethylamino)-methyl]-6-methoxy-2-methyl-4-phenylisoquinolin-1(2H)-one (ISQ-1), blocked I(Kur) in Chinese hamster ovary cells expressing human Kv1.5 with IC(50) values of 238 and 324 nM, respectively. In anesthetized dogs, i.v. infusions of TAEA and ISQ-1 elicited comparable 16% increases in atrial refractory period, with no effect on ventricular refractory period or QTc interval. Plasma concentrations at end infusion for TAEA and ISQ-1 were 58.5 +/- 23.6 and 330.3 +/- 43.5 nM, respectively. The abilities of TAEA and ISQ-1 to terminate AF, with comparison to the rapidly activating component of delayed rectifier potassium current blocker (+)-N-[1'-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4'-piperidin)-6-yl]methanesulfonamide] monohydrochloride (MK-499) and the class IC 1-[2-[2-hydroxy-3-(propylamino)-propoxy]phenyl]-3-phenyl-1-propanone (propafenone), were assessed in conscious dogs with heart failure and inducible AF (entry criterion). All test agents administered in i.v. bolus regimens terminated AF in at least half of animals tested; conversely no agent was universally effective. MK-499, ISQ-1, TAEA, and propafenone terminated AF in five of six, four of seven, four of six, and five of six animals at plasma concentrations of 32.6 +/- 18.7, 817 +/- 274, 714 +/- 622, and 816 +/- 240 nM, respectively. Directed cardiac electrophysiologic studies in anesthetized dogs using i.v. bolus (consistent with AF studies) plus infusion regimens with TAEA and ISQ-1 demonstrated significant increases in atrial refractory period (12-15%), A-H and P-A intervals, but no effects on ventricular refractory period, H-V, and HEG intervals. The demonstration of AF termination with TAEA and ISQ-1 in the dog heart failure model extends the profile of antiarrhythmic efficacy of Kv1.5 blockade.

Identification and characterization of 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]piperidine-1-carboxylate, an orally bioavailable, brain penetrant NR2B selective N-methyl-D-aspartate receptor antagonist.

The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure-activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease.

Design and synthesis of novel isoquinoline-3-nitriles as orally bioavailable Kv1.5 antagonists for the treatment of atrial fibrillation.

Novel 3-cyanoisoquinoline Kv1.5 antagonists have been prepared and evaluated in in vitro and in vivo assays for inhibition of the Kv1.5 potassium channel and its associated cardiac potassium current, IKur. Structural modifications of isoquinolinone lead 1 afforded compounds with excellent potency, selectivity, and oral bioavailability.

Preclinical pharmacology and pharmacokinetics of CERC-301, a GluN2B-selective N-methyl-D-aspartate receptor antagonist.

The preclinical pharmacodynamic and pharmacokinetic properties of 4-methylbenzyl (3S, 4R)-3-fluoro-4-[(Pyrimidin-2-ylamino) methyl] piperidine-1-carboxylate (CERC-301), an orally bioavailable selective N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) antagonist, were characterized to develop a translational approach based on receptor occupancy (RO) to guide CERC-301 dose selection in clinical trials of major depressive disorder. CERC-301 demonstrated high-binding affinity (K i, 8.1 nmol L(-1)) specific to GluN2B with an IC 50 of 3.6 nmol L(-1) and no off-target activity. CERC-301 efficacy was demonstrated in the forced swim test with an efficacy dose (ED 50) of 0.3-0.7 mg kg(-1) (RO, 30-50%); increase in locomotor activity was observed at ED 50 of 2 mg kg(-1), corresponding to an RO of 75%. The predicted 50% RO concentration (Occ50) in humans was 400 nmol L(-1), similar to that predicted for rat, dog, and monkey (300, 200, and 400 nmol L(-1), respectively). Safety pharmacology and neurotoxicity studies raised no specific safety concerns. A first-in-human study in healthy males demonstrated a dose-proportional pharmacokinetic profile, with T max of ~1 h and t 1/2 of 12-17 h. Based on the preclinical and pharmacodynamic data, doses of ≥8 mg in humans are hypothesized to have an acceptable safety profile and result in clinically relevant peak plasma exposure.

P2-quinazolinones and bis-macrocycles as new templates for next-generation hepatitis C virus NS3/4a protease inhibitors: discovery of MK-2748 and MK-6325.

With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK-5172. Quinazolinone-containing macrocycles were identified as promising leads, and optimization for superior cross-genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK-2748. Additional investigation of a series of bis-macrocycles containing a fused 18- and 15-membered ring system were also optimized for the same properties, leading to the discovery of MK-6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next-generation HCV NS3/4a protease inhibitors.

NR2B-selective N-methyl-D-aspartate antagonists: synthesis and evaluation of 5-substituted benzimidazoles.

Two classes of 5-substituted benzimidazoles were identified as potent antagonists of the NR2B subtype of the N-methyl-d-aspartate (NMDA) receptor. Selected compounds show very good selectivity versus the NR2A, NR2C, and NR2D subtypes of the NMDA receptor as well as versus hERG-channel activity and alpha(1)-adrenergic binding. Benzimidazole 37a shows excellent activity in the carrageenan-induced mechanical hyperalgesia assay in rats as well as good pharmacokinetic behavior in dogs.

Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux.

To facilitate the discovery of novel N-methyl-d-aspartate (NMDA) receptor antagonists, we have developed a high-throughput functional assay based on fluorescence detection of free intracellular calcium concentrations. Mouse fibroblast L(tk-) cells expressing human NR1a/NR2B NMDA receptors were plated in 96-well plates and loaded with fluorescence calcium indicator fluo-3 AM. NR2B antagonists were added after stimulation of NMDA receptors with 10 microM glutamate and 10 microM glycine. Changes in fluorescence after the addition of the antagonists were fitted by a single exponential equation providing k(obs). The concentration dependence of k(obs) was linear for all NR2B antagonists at concentrations where k(obs) < 0.2 s(-1). The values of k(obs) for six structurally distinct NR2B antagonists were in the range of 1.1 to 7.5 x 10(5) M(-1)s(-1). These values were several orders of magnitude slower than that obtained for diffusion limited Mg(2+) channel block. The rate constants k(off) provided the values of t(1/2) for dissociation of NR2B antagonists in the range of 1.8 min for ifenprodil to 240 min for the slowest novel antagonist. The IC(50) values obtained from the end-point fluorescence measurements agree with K(d) values calculated from kinetic measurements. All kinetic constants, obtained using our fluorescence method, correlate well with data measured by voltage clamp.

Novel Quinoline-Based P2-P4 Macrocyclic Derivatives As Pan-Genotypic HCV NS3/4a Protease Inhibitors.

We have previously reported the discovery of our P2-P4 macrocyclic HCV NS3/4a protease inhibitor MK-5172, which in combination with the NS5a inhibitor MK-8742 recently received a breakthrough therapy designation from the US FDA for treatment of chronic HCV infection. Our goal for the next generation NS3/4a inhibitor was to achieve pan-genotypic activity while retaining the pharmacokinetic profile of MK-5172. One of the areas for follow-up investigation involved replacement of the quinoxaline moiety in MK-5172 with a quinoline and studying the effect of substitution at 4-position of the quinoline. The rationale for this effort was based on molecular modeling, which indicated that such modifications would improve interactions with the S2 subsite, in particular with D79. We wish to report herein the discovery of highly potent inhibitors with pan-genotypic activity and an improved profile over MK-5172, especially against gt-3a and A156 mutants.