WLB-87848, a Selective σ1 Receptor Agonist, with an Unusually Positioned NH Group as Positive Ionizable Moiety and Showing Neuroprotective Activity.

The synthesis and pharmacological activity of a new series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as sigma-1 receptor (σ1R) ligands are reported. A hit from a high-throughput screening program was evolved into a highly potent and selective σ1R agonist (14qR) that contains a free NH group as positive ionizable moiety, not fulfilling the usual pharmacophoric features of the σ1R. The compound shows good physicochemical and ADMET characteristics, displays an agonist profile in the binding immunoglobulin protein/σ1R association assay, induces neuron viability in an in vitro model of ß-amyloid peptide intoxication, and presents positive results against recognition memory impairment induced by hippocampal injection of Aß peptide in rats after oral treatment, altogether making 14qR (WLB-87848) an interesting candidate for neuroprotection.

Towards Multitargeted Ligands as Pain Therapeutics: Dual Ligands of the Cavα2δ-1 Subunit of Voltage-Gated Calcium Channel and the µ-Opioid Receptor.

The synthesis and pharmacological activity of a new series of dual ligands combining activities towards the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) and the µ-opioid receptor (MOR) as novel pain therapeutics are reported. A careful exploration of the pharmacophores related to both targets, which in principle had few common characteristics, led to the design of novel compounds exhibiting both activities. The construction of the dual ligands started from published Cavα2δ-1 ligands, onto which MOR ligand pharmacophoric elements were added. This exercise led to new amino-acidic substances with good affinities on both targets as well as good metabolic and physicochemical profiles and low potential for drug-drug interactions. A representative compound, (2S,4S)-4-(4-chloro-3-(((cis)-4-(dimethylamino)-4-phenylcyclohexyl)methyl)-5-fluorophenoxy)pyrrolidine-2-carboxylic acid, displayed promising analgesic activities in several in vivo pain models as well as a reduced side-effect profile in relation to morphine.

Discovery of WLB-89462, a New Drug-like and Highly Selective σ2 Receptor Ligand with Neuroprotective Properties.

The synthesis and pharmacological activity of a new series of isoxazolylpyrimidines as sigma-2 receptor (σ2R) ligands are reported. Modification of a new hit retrieved in an HTS campaign allowed the identification of the compound WLB-89462 (20c) with good σ2R affinity (Ki = 13 nM) and high selectivity vs both the σ1R (Ki = 1777 nM) and a general panel of 180 targets. It represents one of the first σ2R ligands with drug-like properties, linked to a good physicochemical and ADMET profile (good solubility, no CYP inhibition, good metabolic stability, high permeability, brain penetration, and high oral exposure in rodents). Compound 20c shows neuroprotective activity in vitro and improves short-term memory impairment induced by hippocampal injection of amyloid ß peptide in rats. Together with the promising effects in the chronic models where 20c is currently being evaluated, these results pave the way toward its clinical development as a neuroprotective agent.

EST79232 and EST79376, Two Novel Sigma-1 Receptor Ligands, Exert Neuroprotection on Models of Motoneuron Degeneration.

Motor neuron diseases (MNDs) include sporadic and hereditary neurological disorders characterized by progressive degeneration of motor neurons (MNs). Sigma-1 receptor (Sig-1R) is a protein enriched in MNs, and mutations on its gene lead to various types of MND. Previous studies have suggested that Sig-1R is a target to prevent MN degeneration. In this study, two novel synthesized Sig-1R ligands, coded EST79232 and EST79376, from the same chemical series, with the same scaffold and similar physicochemical properties but opposite functionality on Sig-1R, were evaluated as neuroprotective compounds to prevent MN degeneration. We used an in vitro model of spinal cord organotypic cultures under chronic excitotoxicity and two in vivo models, the spinal nerve injury and the superoxide dismutase 1 (SOD1)G93A mice, to characterize the effects of these Sig-1R ligands on MN survival and modulation of glial reactivity. The antagonist EST79376 preserved MNs in vitro and after spinal nerve injury but was not able to improve MN death in SOD1G93A mice. In contrast, the agonist EST79232 significantly increased MN survival in the three models of MN degeneration evaluated and had a mild beneficial effect on motor function in SOD1G93A mice. In vivo, Sig-1R ligand EST79232 had a more potent effect on preventing MN degeneration than EST79376. These data further support the interest in Sig-1R as a therapeutic target for neurodegeneration.

Piperazinyl Bicyclic Derivatives as Selective Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels.

The synthesis and pharmacological activities of a new series of piperazinyl quinazolin-4-(3H)-one derivatives acting toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) are reported. Different positions of a micromolar HTS hit were explored, and best activities were obtained for compounds containing a small alkyl group in position 3 of the quinazolin-4-(3H)-one scaffold and a 3-methyl-piperazin-1-yl- or 3,5-dimethyl-piperazin-1-yl-butyl group in position 2. The activity was shown to reside in the R enantiomer of the chain in position 2, and several eutomers reached single digit nanomolar affinities. Final modification of the central scaffold to reduce lipophilicity provided the pyrido[4,3-d]pyrimidin-4(3H)-one 16RR, which showed high selectivity for Cavα2δ-1 versus Cavα2δ-2, probably linked to its improved analgesic efficacy-safety ratio in mice over pregabalin.

Preliminary in vitro approach to evaluate the drug-drug interaction potential of EST73502, a dual µ-opioid receptor partial agonist and σ1 receptor antagonist.

The potential for drug-drug interactions (DDI) of EST73502 was preliminary explored in vitro. EST73502 is a new chemical entity intended for oral pain treatment with dual sigma-1 receptor (σ1R) antagonism and µ-opioid receptor (MOR) partial agonism, that presents a promising potent analgesic activity.Several enzymes were involved in EST73502 metabolism catalysing the formation of different metabolites, CYP3A4 and CYP2D6 being the main ones.Fraction unbound was determined due to its impact in interactions, a considerable proportion of EST73502 being available.EST73502 showed a low potential for CYP inhibition, except for CYP2D6 that showed time-dependent inhibition.No induction potential was found for CYP1A2 and 3A4, while CYP2B6 was induced at high concentration.EST73502 seemed to be a potential efflux transporter substrate (efflux ratio ≥ 2) but a negligible in vivo impact would be expected due to its high solubility and permeability in Caco-2 cells. P-gp inhibition was observed while no BCRP inhibition was detected.Preliminary in vitro interaction studies suggested that neither CYPs nor efflux transporters interactions would preclude further development of EST73502 to thoroughly assess the clinical relevance of these findings.

Bicyclic Diazepinones as Dual Ligands of the α2δ-1 Subunit of Voltage-Gated Calcium Channels and the Norepinephrine Transporter.

The synthesis and pharmacological activity of a new series of bicyclic diazepinones with dual activity toward the α2δ-1 subunit of voltage-gated calcium channels (Cavα2δ-1) and the norepinephrine transporter (NET) are reported. Exploration of the positions amenable for substitution on a nonaminoacidic Cavα2δ-1 scaffold allowed the identification of favorable positions for the attachment of NET pharmacophores. Among the patterns explored, attachment of the 2-ethylamino-9-methyl-6-phenyl-6,7,8,9-tetrahydro-5H-pyrimido[4,5-e][1,4]diazepin-5-one framework to the meta-position of the phenyl ring of the 3-methylamino-1-phenylpropoxy and 3-methylamino-1-thiophenylpropoxy moieties provided dual compounds with excellent NET functionality. Alternative bicyclic frameworks were also explored, and some lead molecules were identified, which showed a balanced dual profile and exhibited good ADMET properties.

Preliminary in vitro assessment of pharmacokinetic drug-drug interactions of EST64401 and EST64514, two sigma-1 receptor antagonists.

EST64401 and EST64514 are two selective sigma-1 receptor ligands that showed a good profile in a lead optimization process for oral pain treatment. Their potential for pharmacokinetic-based drug-drug interactions was assessed to anticipate clinical interactions.Both compounds showed a low potential for CYP inhibition with percentages of inhibition <50% at 1 µM in recombinant human CYPs (CYP1A2, 2C9, 2C19, 2D6 and 3A4) and IC50 ≥75 µM for CYP3A4 and 2D6 in human liver microsomes.No CYP induction was observed for CYP1A2, 2B6 and 3A4 at concentrations ≤25 µM (EST64401) or ≤50 µM (EST64514) in human hepatocytes using as endpoints CYP activities and mRNA levels.More than one enzyme participated in compound metabolism. The main enzymes involved were CYP3A4 for EST64401 and CYP2D6 besides CYP3A4 for EST64514.Neither EST64401 nor EST64514 seemed to be substrates of P-gp or BCRP in Caco-2 cells (efflux ratio ≤2). Transporter inhibition was observed at concentrations ≥20 µM; EST64401 only inhibiting P-gp at higher concentrations (≥125 µM).Preliminary in vitro interaction studies suggest a similar profile for EST64401 and EST64514. Therefore, other properties will have to be considered for compound differentiation and selection for further development.

Preliminary in Vitro Assessment of the Potential of EST64454, a Sigma-1 Receptor Antagonist, for Pharmacokinetic Drug-Drug Interactions.

EST64454 is a selective sigma-1 receptor ligand intended for orally administered pain treatment that showed a promising profile in the lead optimization process. As part of the preliminary compound profiling, the potential for future drug-drug interactions was explored in vitro. Both direct and time-dependent CYP inhibition for CYP1A2, 2C9, 2C19, 2D6 and 3A4 was studied in human liver microsomes. EST64454 showed a low potential for CYP inhibition (IC50 between 100 and 1000 µM) and as time-dependent inhibitor (IC50 shift mainly around 1). CYP induction studies with HepaRG™ cells revealed no CYP induction at concentrations ≤50 µM, as shown by the CYP1A2, 3A4 and 2B6 activities measured. Reaction phenotyping was assessed after incubation with recombinant human enzymes. Although a very low metabolism was observed, several enzymes catalyzed the formation of metabolites, including CYP3A4, 2C19 and flavin monooxygenases (FMO) 1 and 3. EST64454 was not a P-glycoprotein (P-gp) substrate and was highly permeable in Caco-2 cells. P-gp inhibition was only observed at 200 µM, the highest concentration studied. Preliminary studies suggest that neither CYP nor P-gp interaction of EST64454 would be of any concern for further development. At later stages, the interaction kinetics and the clinical relevance of these findings will be thoroughly evaluated.

Synthesis and pharmacological evaluation of fluorinated benzo[7]annulen-7-amines as GluN2B-selective NMDA receptor antagonists.

Because of their neuroprotective potential, GluN2B-selective ligands are of great interest for the treatment of various neurological and neurodegenerative disorders. Fluorinated benzo[7]annulen-7-amines, capable for PET, were synthesized by combining fluorinated phenylalkylamines with differently substituted ketones. Relationships between substitution pattern and GluN2B affinity as well as selectivity towards σ1 and σ2 receptors were investigated. Two promising ligands (18a and 20c) were selected for further pharmacological evaluation. Besides a slight serotonin transporter (SERT), norepinephrine transporter (NET), and hERG affinity, they did not show interaction with other targets. Furthermore, the pKa value of a set fluorinated ligands, bearing the fluorine atom in different positions, was determined.

Synthesis, Receptor Affinity, and Antiallodynic Activity of Spirocyclic σ Receptor Ligands with Exocyclic Amino Moiety.

In order to detect novel σ receptor ligands, the rigid spiro[[2]benzopyran-1,1'-cyclohexan]-4'-one was connected with amino moieties derived from σ2 receptor preferring lead compounds resulting in mixtures of trans- and cis-configured amines 6, 18, and 27. In a four step synthesis the methyl acetals 6 were converted into fluoroethyl derivatives 13 and 30. The most promising σ2 receptor ligand is the methyl acetal 6a bearing a 2,4-dimethylbenzylamino moiety. The fluoroethyl derivatives 13c and 13d reveal high σ1 affinity but moderate selectivity over the σ2 subtype. In mice 13c and 13d showed antiallodynic activity that is stronger than that of the reference σ1 antagonist BD-1063 (34). Since the antiallodynic activity of 13c could only be partially reversed by the σ1 agonist PRE-084 (35), it is postulated that a second mechanism contributes to its overall antiallodynic effect. In contrast, the antiallodynic effect of its diastereomer 13d can be totally explained by a σ1 antagonism.

Synthesis and biological evaluation of a new series of hexahydro-2H-pyrano[3,2-c]quinolines as novel selective σ1 receptor ligands.

The synthesis and pharmacological activity of a new series of hexahydro-2H-pyrano[3,2-c]quinoline derivatives as potent σ1 receptor (σ1R) ligands are reported. This family, which does not contain the highly basic amino group usually present in other σ1R ligands, showed high selectivity over the σ2 receptor (σ2R). The activity was shown to reside in only one of the four possible diastereoisomers, which exhibited a perfect match with known σ1R pharmacophores. A hit to lead program based on a high-throughput screening hit (8a) led to the identification of compound 32c, with substantially improved activity and physicochemical properties. Compound 32c also exhibited a good ADMET (absorption, distribution, metabolism, excretion, toxicity) profile and was identified as a σ1R antagonist on the basis of its analgesic activity in the mouse capsaicin and formalin models of neurogenic pain.

Biaryl analogues of teriflunomide as potent DHODH inhibitors.

The structure-activity relationships of a novel series of biaryl dihydroorotate dehydrogenase (DHODH) inhibitors related to teriflunomide are disclosed. These biaryl derivatives were the result of structure-based design and proved to be potent DHODH inhibitors which in addition showed good antiproliferative activities on peripheral blood mononuclear cells and good efficacies in vivo in the rat adjuvant-induced-arthritis model.

Discovery of potent and selective bicyclic A(2B) adenosine receptor antagonists via bioisosteric amide replacement.

Several new potent and selective A(2B) adenosine receptor antagonists have been prepared in which the aryl-amide moiety of the lead series, exemplified by 1a, has been replaced by bioisosteric bicyclic moieties. Although the majority of compounds had generally improved microsomal stability as compared to 1a, this was not translated into overall improvements in the pharmacokinetic profiles of a representative set of compounds.

Discovery and characterization of 4'-(2-furyl)-N-pyridin-3-yl-4,5'-bipyrimidin-2'-amine (LAS38096), a potent, selective, and efficacious A2B adenosine receptor antagonist.

A novel series of N-heteroaryl 4'-(2-furyl)-4,5'-bipyrimidin-2'-amines has been identified as potent and selective A(2B) adenosine receptor antagonists. In particular, compound 5 showed high affinity for the A(2B) receptor (Ki = 17 nM), good selectivity (IC(50): A(1) > 1000 nM, A(2A) > 2500 nM, A3 > 1000 nM), displayed a favorable pharmacokinetic profile in preclinical species, and showed efficacy in functional in vitro models.

Racemic and chiral sulfoxides as potential prodrugs of 4-pyrone COX-2 inhibitors.

The preparation of the sulfoxide analogues 7, 8, and 9 and their enantiomerically pure forms is discussed as well as their ability to act as prodrugs of the potent and selective sulfone-containing COX-2 inhibitors 1, 2, and 3. Sulfoxide derivatives 7 and 9 were shown to be rapidly transformed in vivo into the corresponding sulfone derivatives 1 and 3, after oral administration to rats.

Racemic and chiral sulfoxides as potential prodrugs of the COX-2 inhibitors Vioxx and Arcoxia.

The preparation of the sulfoxide analogues 2 and 4, and their enantiomeric pure forms is discussed as well as their potential to act as prodrugs to the potent and selective sulfone-containing COX-2 inhibitors rofecoxib and etoricoxib. Sulfoxides 2 and 4 were shown to be effectively transformed in vivo into rofecoxib and etoricoxib, respectively, after oral administration in rats. In the case of sulfoxide 2, both a slightly improved pharmacokinetic profile and a better pharmacological activity in an arthritis model were seen when compared with rofecoxib.