Phase 1 clinical trial of losmapimod in facioscapulohumeral dystrophy: Safety, tolerability, pharmacokinetics, and target engagement.

AIMS: Evaluate safety, tolerability, pharmacokinetics (PK) and target engagement (TE) of losmapimod in blood and muscle in facioscapulohumeral dystrophy (FSHD). METHODS: This study included Part A: 10 healthy volunteers randomized to single oral doses of losmapimod (7.5 mg then 15 mg; n = 8) or placebo (both periods; n = 2); Part B: 15 FSHD subjects randomized to placebo (n = 3), or losmapimod 7.5 mg (n = 6) or 15 mg (n = 6); and Part C: FSHD subjects received open-label losmapimod 15 mg (n = 5) twice daily for 14 days. Biopsies were performed in FSHD subjects at baseline and Day 14 in magnetic resonance imaging-normal appearing (Part B) and affected muscle identified by abnormal short-tau inversion recovery sequence + (Part C). PK and TE, based on pHSP27:total HSP27, were assessed in muscle and sorbitol-stimulated blood. RESULTS: PK profiles were similar between healthy volunteers and FSHD subjects, with mean Cmax and AUC0-12 for 15 mg in FSHD subjects (Part B) of 85.0 ± 16.7 ng*h/mL and 410 ± 50.3 ng*h/mL, respectively. Part B and Part C PK results were similar, and 7.5 mg results were approximately dose proportional to 15 mg results. Dose-dependent concentrations in muscle (42.1 ± 10.5 ng/g [7.5 mg] to 97.2 ± 22.4 ng/g [15 mg]) were observed, with plasma-to-muscle ratio from ~0.67 to ~1 at estimated tmax of 3.5 hours postdose. TE was observed in blood and muscle. Adverse events (AEs) were mild and self-limited. CONCLUSION: Losmapimod was well tolerated, with no serious AEs. Dose-dependent PK and TE were observed. This study supports advancing losmapimod into Phase 2 trials in FSHD. CLINICAL TRIAL REGISTRATION: Clinical trial identifier ToetsingOnline: NL68539.056.18 Nederlands Trials Register NL8000.

Synthesis of functionalized derivatives of the gamma-secretase modulator BMS-932481 and identification of its major metabolite.

In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo.

p38α Regulates Expression of DUX4 in a Model of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the loss of repression at the D4Z4 locus leading to aberrant double homeobox 4 (DUX4) expression in skeletal muscle. Activation of this early embryonic transcription factor results in the expression of its target genes causing muscle fiber death. Although progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent, and specific inhibitors of p38α/ß, we show a robust reduction of DUX4 expression, activity, and cell death across patient-derived FSHD1 and FSHD2 lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/ß inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38α in the aberrant activation of DUX4 in FSHD and support the potential of p38α/ß inhibitors as effective therapeutics to treat FSHD at its root cause. SIGNIFICANCE STATEMENT: Using patient-derived facioscapulohumeral muscular dystrophy (FSHD) myotubes, we characterize the pharmacological relationships between p38α/ß inhibition, double homeobox 4 (DUX4) expression, its downstream transcriptional program, and muscle cell death. p38α/ß inhibition results in potent and specific DUX4 downregulation across multiple genotypes without significant effects in the process of myogenesis in vitro. These findings highlight the potential of p38α/ß inhibitors for the treatment of FSHD, a condition that today has no approved therapies.

Identification and Preclinical Evaluation of the Bicyclic Pyrimidine γ-Secretase Modulator BMS-932481.

A triazine hit identified from a screen of the BMS compound collection was optimized for potency, in vivo activity, and off-target profile to produce the bicyclic pyrimidine γ-secretase modulator BMS-932481. The compound showed robust reductions of Aß1-42 and Aß1-40 in the plasma, brain, and cerebrospinal fluid of mice and rats. Consistent with the γ-secretase modulator mechanism, increases in Aß1-37 and Aß1-38 were observed, with no change in the total amount of Aß1-x produced. No Notch-based toxicity was observed, and the overall preclinical profile of BMS-932481 supported its further evaluation in human clinical trials.

Discovery of new indole-based acylsulfonamide Nav1.7 inhibitors.

Screening of 100 acylsulfonamides from the Bristol-Myers Squibb compound collection identified the C3-cyclohexyl indole 6 as a potent Nav1.7 inhibitor. Replacement of the C2 furanyl ring of 6 with a heteroaryl moiety or truncation of this group led to the identification of 4 analogs with hNav1.7 IC50 values under 50 nM. Fluorine substitution of the truncated compound 12 led to 34 with improved potency and isoform selectivity. The inverted indole 36 also maintained good activity. Both 34 and 36 exhibited favorable CYP inhibition profiles, good membrane permeability and a low efflux ratio and, therefore, represent new leads in the search for potent and selective Nav1.7 inhibitors to treat pain.

Discovery of Indole- and Indazole-acylsulfonamides as Potent and Selective NaV1.7 Inhibitors for the Treatment of Pain.

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Nav1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund's adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia and models of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The observed efficacy could be correlated with the mouse dorsal root ganglion exposure and NaV1.7 potency associated with 29.

BMS-986163, a Negative Allosteric Modulator of GluN2B with Potential Utility in Major Depressive Disorder.

There is a significant unmet medical need for more efficacious and rapidly acting antidepressants. Toward this end, negative allosteric modulators of the N-methyl-d-aspartate receptor subtype GluN2B have demonstrated encouraging therapeutic potential. We report herein the discovery and preclinical profile of a water-soluble intravenous prodrug BMS-986163 (6) and its active parent molecule BMS-986169 (5), which demonstrated high binding affinity for the GluN2B allosteric site (Ki = 4.0 nM) and selective inhibition of GluN2B receptor function (IC50 = 24 nM) in cells. The conversion of prodrug 6 to parent 5 was rapid in vitro and in vivo across preclinical species. After intravenous administration, compounds 5 and 6 have exhibited robust levels of ex vivo GluN2B target engagement in rodents and antidepressant-like activity in mice. No significant off-target activity was observed for 5, 6, or the major circulating metabolites met-1 and met-2. The prodrug BMS-986163 (6) has demonstrated an acceptable safety and toxicology profile and was selected as a preclinical candidate for further evaluation in major depressive disorder.

Discovery of morpholine-based aryl sulfonamides as Nav1.7 inhibitors.

Replacement of the piperidine ring in the lead benzenesulfonamide Nav1.7 inhibitor 1 with a weakly basic morpholine core resulted in a significant reduction in Nav1.7 inhibitory activity, but the activity was restored by shortening the linkage from methyleneoxy to oxygen. These efforts led to a series of morpholine-based aryl sulfonamides as isoform-selective Nav1.7 inhibitors. This report describes the synthesis and SAR of these analogs.

Preclinical Characterization of (R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169), a Novel, Intravenous, Glutamate N-Methyl-d-Aspartate 2B Receptor Negative Allosteric Modulator with Potential in Major Depressive Disorder.

(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169) and the phosphate prodrug 4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl dihydrogen phosphate (BMS-986163) were identified from a drug discovery effort focused on the development of novel, intravenous glutamate N-methyl-d-aspartate 2B receptor (GluN2B) negative allosteric modulators (NAMs) for treatment-resistant depression (TRD). BMS-986169 showed high binding affinity for the GluN2B subunit allosteric modulatory site (Ki = 4.03-6.3 nM) and selectively inhibited GluN2B receptor function in Xenopus oocytes expressing human N-methyl-d-aspartate receptor subtypes (IC50 = 24.1 nM). BMS-986169 weakly inhibited human ether-a-go-go-related gene channel activity (IC50 = 28.4 µM) and had negligible activity in an assay panel containing 40 additional pharmacological targets. Intravenous administration of BMS-986169 or BMS-986163 dose-dependently increased GluN2B receptor occupancy and inhibited in vivo [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding, confirming target engagement and effective cleavage of the prodrug. BMS-986169 reduced immobility in the mouse forced swim test, an effect similar to intravenous ketamine treatment. Decreased novelty suppressed feeding latency, and increased ex vivo hippocampal long-term potentiation was also seen 24 hours after acute BMS-986163 or BMS-986169 administration. BMS-986169 did not produce ketamine-like hyperlocomotion or abnormal behaviors in mice or cynomolgus monkeys but did produce a transient working memory impairment in monkeys that was closely related to plasma exposure. Finally, BMS-986163 produced robust changes in the quantitative electroencephalogram power band distribution, a translational measure that can be used to assess pharmacodynamic activity in healthy humans. Due to the poor aqueous solubility of BMS-986169, BMS-986163 was selected as the lead GluN2B NAM candidate for further evaluation as a novel intravenous agent for TRD.

Discovery of non-zwitterionic aryl sulfonamides as Nav1.7 inhibitors with efficacy in preclinical behavioral models and translational measures of nociceptive neuron activation.

Since zwitterionic benzenesulfonamide Nav1.7 inhibitors suffer from poor membrane permeability, we sought to eliminate this characteristic by replacing the basic moiety with non-basic bicyclic acetals and monocyclic ethers. These efforts led to the discovery of the non-zwitterionic aryl sulfonamide 49 as a selective Nav1.7 inhibitor with improved membrane permeability. Despite its moderate cellular activity, 49 exhibited robust efficacy in mouse models of neuropathic and inflammatory pain and modulated translational electromyogram measures associated with activation of nociceptive neurons.

Synergistic inhibition of Aß production by combinations of γ-secretase modulators.

Alzheimer's disease is associated with the accumulation of amyloid-ß (Aß) in the brain. In particular, the 42-amino acid form, Aß1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aß1-42 production without inhibiting the production of other forms of Aß. Here we describe the novel discovery of synergistic inhibition of Aß by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aß peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aß occurred only when one GSM was of the "acid GSM" structural class and the other was of the "non-acid GSM" class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aß production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aß. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target.

Development of New Benzenesulfonamides As Potent and Selective Nav1.7 Inhibitors for the Treatment of Pain.

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Discovery of furo[2,3-d][1,3]thiazinamines as beta amyloid cleaving enzyme-1 (BACE1) inhibitors.

This Letter describes the synthesis and structure-activity relationships of a series of furo[2,3-d][1,3]thiazinamine BACE1 inhibitors. The co-crystal structure of a representative thiazinamine 2e bound with the BACE1 active site displayed a binding mode driven by interactions with the catalytic aspartate dyad and engagement of the biaryl amide toward the S1 and S3 pockets. This work indicates that furo[2,3-d]thiazine can serve as a viable bioisostere of the known furo[3,4-d]thiazine.

Discovery of S3-Truncated, C-6 Heteroaryl Substituted Aminothiazine ß-Site APP Cleaving Enzyme-1 (BACE1) Inhibitors.

Truncation of the S3 substituent of the biaryl aminothiazine 2, a potent BACE1 inhibitor, led to a low molecular weight aminothiazine 5 with moderate activity. Despite its moderate activity, compound 5 demonstrated significant brain Aß reduction in rodents. The metabolic instability of 5 was overcome by the replacement of the 6-dimethylisoxazole, a metabolic soft spot, with a pyrimidine ring. Thus, truncation of the S3 substituent represents a viable approach to the discovery of orally bioavailable, brain-penetrant BACE1 inhibitors.

The γ-Secretase Modulator, BMS-932481, Modulates Aß Peptides in the Plasma and Cerebrospinal Fluid of Healthy Volunteers.

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aß39, Aß40, and Aß42 while increasing Aß37 and Aß38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aß40 and Aß42 were observed with no change in total Aß; in CSF, modest decreases in total Aß were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aß42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aß lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.

Robust Translation of γ-Secretase Modulator Pharmacology across Preclinical Species and Human Subjects.

The amyloid-ß peptide (Aß)-in particular, the 42-amino acid form, Aß1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aß synthesis or increase the clearance of Aß have entered clinical trials, including γ-secretase inhibitors, anti-Aß antibodies, and amyloid-ß precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aß1-42 production, and may also decrease Aß1-40 while simultaneously increasing one or more shorter Aß peptides, such as Aß1-38 and Aß1-37. GSMs are particularly attractive because they do not alter the total amount of Aß peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, ß-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aß1-42 and Aß1-40 levels while increasing Aß1-38 and Aß1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

Targeting the BACE1 Active Site Flap Leads to a Potent Inhibitor That Elicits Robust Brain Aß Reduction in Rodents.

By targeting the flap backbone of the BACE1 active site, we discovered 6-dimethylisoxazole-substituted biaryl aminothiazine 18 with 34-fold improved BACE1 inhibitory activity over the lead compound 1. The cocrystal structure of 18 bound to the active site indicated two hydrogen-bond interactions between the dimethylisoxazole and threonine 72 and glutamine 73 of the flap. Incorporation of the dimethylisoxazole substitution onto the related aminothiazine carboxamide series led to pyrazine-carboxamide 26 as a very potent BACE1 inhibitor (IC50 < 1 nM). This compound demonstrated robust brain Aß reduction in rat dose-response studies. Thus, compound 26 may be useful in testing the amyloid hypothesis of Alzheimer's disease.

Expedient Synthesis of Furo[2,3-d][1,3]thiazinamines and Pyrano[2,3-d][1,3]thiazinamines from Enones and Thiourea.

Michael addition of thiourea to enones with subsequent intramolecular aminal ether formation provided easy access to furo[2,3-d]thiazinamines and pyrano[2,3-d][1,3]thiazin-2-amines. These amines served as versatile intermediates to a variety of beta-amyloid cleaving enzyme-1 (BACE1) inhibitors.

Synthesis of pyrimido[4,5-c]azepine- and pyrimido[4,5-c]oxepine-based γ-secretase modulators.

This Letter describes an efficient ring-closing metathesis approach to 2-chloro-4-amino-pyrimido[4,5-c]azepines and 2-chloro-4-amino-pyrimido[4,5-c]oxepines. These chlorides were applied to the synthesis of several potent γ-secretase modulators (GSMs).