Antituberculosis Macozinone Extended-Release Tablets To Enhance Bioavailability: a Pilot Pharmacokinetic Study in Beagle Dogs.

Macozinone (MCZ; PBTZ169) is a first-in-class antituberculosis clinical-stage benzothiazinone-based drug candidate. Although its efficacy and safety have been strongly proven in several preclinical and clinical studies, the physicochemical and pharmacokinetic properties specific to MCZ required further optimization. Accordingly, this study aimed to evaluate the pharmacokinetics of MCZ administered as extended-release (ER) tablets F2 and F6 compared to immediate-release (IR) dispersible tablets for oral suspension. Oral absorption of MCZ from ER tablets was significantly different from that of IR tablets after a single oral dose in Beagle dogs in both fasted and fed states. In addition, food directly affects the bioavailability of MCZ from ER tablets but does not affect it from IR tablets. The high values of relative bioavailability of the prolonged-release tablets F2 and F6 compared to the IR tablets may indicate an indirect confirmation of their gastroretentive properties. Taken together, pharmacokinetic parameters have demonstrated that these MCZ oral formulations not just enhance drug bioavailability but may also improve regimen adherence by reducing MCZ dose frequency and reducing the development of drug resistance. IMPORTANCE Macozinone (MCZ) is the newest first-in-class clinical-stage benzothiazinone-based drug candidate for the treatment of tuberculosis. Yet, the extremely low oral bioavailability of MCZ, a major problem in clinical trials, needed to be addressed, and we are pleased to present our attempts to solve this issue. We report that extended-release tablets of MCZ significantly increased key pharmacokinetic parameters in the preclinical setting. We suggest that these MCZ oral formulations not just enhance drug bioavailability but may also improve regimen adherence by reducing MCZ dose frequency and reducing the development of drug resistance.

Synthesis, inhibitory activity and oral dosing formulation of AV5124, the structural analogue of influenza virus endonuclease inhibitor baloxavir.

BACKGROUND: The development and clinical implementation of the cap-dependent endonuclease (CEN) inhibitor baloxavir marboxil was a breakthrough in influenza therapy, but it was associated with the emergence of drug-resistant variants. OBJECTIVES: To design and synthesize structural analogues of CEN inhibitors and evaluate their safety, pharmacokinetics and antiviral potency in vitro and in vivo. METHODS: The drug candidate AV5124 and its active metabolite AV5116 were synthesized based on pharmacophore modelling. Stability in plasma and microsomes, plasma protein binding, cytotoxicity and antiviral activities were assessed in vitro. Pharmacokinetics after IV or oral administration were analysed in CD-1 mice. Acute toxicity and protective efficacy against lethal A(H1N1)pdm09 influenza virus challenge were examined in BALB/c mice. RESULTS: Pharmacophore model-assisted, 3D molecular docking predicted key supramolecular interactions of the metal-binding group and bulky hydrophobic group of AV5116 with the CEN binding site (Protein Data Bank code: 6FS6) that are essential for high antiviral activity. AV5116 inhibited influenza virus polymerase complexes in cell-free assays and replication of oseltamivir-susceptible and -resistant influenza A and B viruses at nanomolar concentrations. Notably, AV5116 was equipotent or more potent than baloxavir acid (BXA) against WT (I38-WT) viruses and viruses with reduced BXA susceptibility carrying an I38T polymerase acidic (PA) substitution. AV5116 exhibited low cytotoxicity in Madin-Darby canine kidney cells and lacked mitochondrial toxicity, resulting in favourable selective indices. Treatment with 20 or 50 mg/kg AV5124 prevented death in 60% and 100% of animals, respectively. CONCLUSIONS: Overall, AV5124 and A5116 are promising inhibitors of the influenza virus CEN and warrant further development as potent anti-influenza agents.

Synthesis, biological evaluation and in silico modeling of novel pan-genotypic NS5A inhibitors.

A series of novel small-molecule pan-genotypic hepatitis C virus (HCV) NS5A inhibitors with picomolar activity containing 2-[(2S)-pyrrolidin-2-yl]-5-[4-(4-{2-[(2S)-pyrrolidin-2-yl]-1H-imidazol-5-yl}buta-1,3-diyn-1-yl)phenyl]-1H-imidazole core was designed based on molecular modeling study and SAR analysis. The constructed in silico model and docking study provide a deep insight into the binding mode of this type of NS5A inhibitors. Based on the predicted binding interface we have prioritized the most crucial diversity points responsible for improving antiviral activity. The synthesized molecules were tested in a cell-based assay, and compound 1.12 showed an EC50 value in the range of 2.9-34 pM against six genotypes of NS5A HCV, including gT3a, and demonstrated favorable pharmacokinetic profile in rats. This lead compound can be considered as an attractive candidate for further clinical evaluation.

Non-rigid Diarylmethyl Analogs of Baloxavir as Cap-Dependent Endonuclease Inhibitors of Influenza Viruses.

4-Substituted 2,4-dioxobutanoic acids inhibit influenza virus cap-dependent endonuclease (CEN) activity. Baloxavir marboxil, 4, is approved for treating influenza virus infections. We describe here the synthesis and biological evaluation of active compounds, 5a-5g, and their precursors (6a, 6b, 6d, and 6e) with flexible bulky hydrophobic groups instead of the rigid polyheterocyclic moieties. In silico docking confirmed the ability of 5a-5g to bind to the active site of influenza A CEN (PDB code: 6FS6) like baloxavir acid, 3. These novel compounds inhibited polymerase complex activity, inhibited virus replication in cells, prevented death in a lethal influenza A virus mouse challenge model, and dramatically lowered viral lung titers. 5a and 5e potently inhibited different influenza genera in vitro. Precursors 6a and 6d demonstrated impressive mouse oral bioavailability with 6a, providing effective in vivo protection. Thus, these novel compounds are potent CEN inhibitors with in vitro and in vivo activity comparable to baloxavir.

Synthesis, biological evaluation and in silico modeling of novel integrase strand transfer inhibitors (INSTIs).

Although a relatively wide range of therapeutic options is currently available for the treatment of HIV/AIDS, it is still among the most serious and virulent diseases and is associated with a high mortality rate. Integrase strand transfer inhibitors (INSTIs), e.g., FDA-approved dolutegravir (DTG), bictegravir (BIC) and cabotegravir (CAB), have recently been included in standard highly active antiretroviral therapy (HAART) schemes as one of the five major components responsible for the most beneficial clinical outcome. In this paper, we describe a combinatorial amide synthesis, biological evaluation and in silico modeling of new INSTIs containing heteroaromatic bioisosteric substitution instead of the well-studied halogen-substituted benzyl fragment. With the focus on the mentioned diversity point, a medium-sized library of compounds was selected for synthesis. A biological study revealed that many molecules were highly active INSTIs (EC50 < 10 nM). Two compounds 1{4} and 1{26} demonstrated picomolar antiviral activity that was comparable with CAB and were more active than DTG and BIC. Molecular docking study was performed to evaluate the binding mode of compounds in the active site of HIV-1 IN. In rats, lead compound 1{26} showed two-fold greater bioavailability than CAB and had a similar half-life. Compound 1{26} and its sodium salt were considerably more soluble in water than the parent drugs. Both molecules were very stable in human liver microsomes and plasma, demonstrated high affinity towards plasma proteins and did not show cytochrome (CYP) inhibition. This benefit profile indicates the great potential of these molecules as attractive candidates for subsequent evaluation as oral long-acting drugs and long-acting nanosuspension formulations for intramuscular injection.

AVN-492, A Novel Highly Selective 5-HT6R Antagonist: Preclinical Evaluation.

Discovery of 5-HT6 receptor subtype and its exclusive localization within the central nervous system led to extensive investigations of its role in Alzheimer's disease, schizophrenia, and obesity. In the present study, we present preclinical evaluation of a novel highly-potent and highly-selective 5-HT6R antagonist, AVN-492. The affinity of AVN-492 to bind to 5-HT6R (Ki = 91 pM) was more than three orders of magnitude higher than that to bind to the only other target, 5-HT2BR, (Ki = 170 nM). Thus, the compound displayed great 5-HT6R selectivity against all other serotonin receptor subtypes, and is extremely specific against any other receptors such as adrenergic, GABAergic, dopaminergic, histaminergic, etc. AVN-492 demonstrates good in vitro and in vivo ADME profile with high oral bioavailability and good brain permeability in rodents. In behavioral tests, AVN-492 shows anxiolytic effect in elevated plus-maze model, prevents an apomorphine-induced disruption of startle pre-pulse inhibition (the PPI model) and reverses a scopolamine- and MK-801-induced memory deficit in passive avoidance model. No anti-obesity effect of AVN-492 was found in a murine model. The data presented here strongly indicate that due to its high oral bioavailability, extremely high selectivity, and potency to block the 5-HT6 receptor, AVN-492 is a very promising tool for evaluating the role the 5-HT6 receptor might play in cognitive and neurodegenerative impairments. AVN-492 is an excellent drug candidate to be tested for treatment of such diseases, and is currently being tested in Phase I trials.

Design, synthesis and biological evaluation of novel 5-oxo-2-thioxoimidazolidine derivatives as potent androgen receptor antagonists.

A series of novel highly active androgen receptor (AR) antagonists containing spiro-4-(5-oxo-3-phenyl-2-thioxoimidazolidin-1-yl)-2-(trifluoromethyl)benzonitrile core was designed based on the SAR studies available from the reported AR antagonists and in silico modeling. Within the series, compound (R)-6 (ONC1-13B) and its related analogues, including its active N-dealkylated metabolite, were found to be the most potent molecules with the target activity (IC50, androgen-sensitive human PCa LNCaP cells) in the range of 59-80 nM (inhibition of PSA production). The disclosed hits were at least two times more active than bicalutamide, nilutamide and enzalutamide within the performed assay. Several compounds were classified as partial agonists. Hit-compounds demonstrated benefit pharmacokinetic profiles in rats. Comparative SAR and 3D molecular docking studies were performed for the hit compounds elucidating the observed differences in the binding potency.

Discovery of novel highly potent hepatitis C virus NS5A inhibitor (AV4025).

A series of next in class small-molecule hepatitis C virus (HCV) NS5A inhibitors with picomolar potency containing 2-pyrrolidin-2-yl-5-{4-[4-(2-pyrrolidin-2-yl-1H-imidazol-5-yl)buta-1,3-diynyl]phenyl}-1H-imidazole cores was designed based on the SAR studies available for the reported NS5A inhibitors. Compound 13a (AV4025), with (S,S,S,S)-stereochemistry (EC50 = 3.4 ± 0.2 pM, HCV replicon genotype 1b), was dramatically more active than were the compounds with two (S)- and two (R)-chiral centers. Human serum did not significantly reduce the antiviral activity (<4-fold). Relatively favorable pharmacokinetic features and good oral bioavailability were observed during animal studies. Compound 13a was well tolerated in rodents (in mice, LD50 = 2326 mg/kg or higher), providing a relatively high therapeutic index. During safety, pharmacology and subchronic toxicity studies in rats and dogs, it was not associated with any significant pathological or clinical findings. This compound is currently being evaluated in phase I/II clinical trials for the treatment of HCV infection.

2-Substituted 5,6-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines: new series of highly potent and specific serotonin 5-HT6 receptor antagonists.

Syntheses, biological evaluation, and structure-activity relationships for a series of novel 2-substituted 3-benzenesulfonyl-5,6-dimethyl-pyrazolo[1,5-a]pyrimidines are disclosed. In spite of a wide, four orders of magnitude, SAR range (K(i) varied from 260 pM to 2.96 µM), no significant correlation of 5-HT(6)R antagonistic potency was observed with major physiochemical characteristics, such as molecular weight, surface polar area, cLogP, or number of rotatable bonds. Statistically significant trend was only observed for size of substitute group, which was not enough to explain the deep SAR trend. Besides with the substitute group size, another factor that presumably plays a role in defining the compound potencies is a relative position of the heterocycle and sulfophenyl moieties. Among all synthesized derivatives, (3-benzenesulfonyl-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 18 is the most potent (K(i) = 260 pM) and extremely selective, 5000 to >50,000-fold relative to 55 therapeutic targets, antagonist of the 5-HT(6) receptor.

Synthesis and biological study of 3-(phenylsulfonyl)thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines as potent and selective serotonin 5-HT6 receptor antagonists.

A number of 3-(phenylsulfonyl)thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines were prepared and their 5-HT6 receptor binding affinity and ability to inhibit the functional cellular responses to serotonin were evaluated. 3-[(3-chlorophenyl)sulfonyl]-N-(tetrahydrofuran-2-ylmethyl)thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5-amine 2{5,26} appeared to be the most active in a functional assay (IC50=29.0 nM) and 3-(phenylsulfonyl)-N-(2-thienylmethyl) thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidin-5-amine 2{1,28} demonstrated the greatest affinity in a 5-HT6 receptor radioligand binding assay (Ki=1.7 nM). A screening of 5-HT2A and 5-HT2B receptor affinity revealed that 3-(phenylsulfonyl)thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines are highly selective 5-HT6 receptor ligands.

(3-Phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-2-yl)amines: potent and selective antagonists of the serotonin 5-HT6 receptor.

5-HT(6) receptors are exclusively localized in the CNS and have high affinity with many psychotropic agents. Though the role of this receptor in many CNS diseases is widely anticipated, lack of definite progress in the development of 5-HT(6) receptor-oriented drugs indicates a need for further discoveries of novel chemotypes with high potency and high selectivity to the receptor. Here we present preparations and biological evaluation of a series of (3-phenylsulfonylcycloalkano[e and d]pyrazolo[1,5-a]pyrimidin-2-yl)amines. Phenylsulfonylcyclopentapyrazolopyrimidine 7 was found to be a highly selective 5-HT(6) receptor antagonist with high affinity (low picomolar range) and potency. 7 and a few of its analogues were further tested for biological effect on 5-HT(2B) receptors and hERG potassium channels, potential liability targets. Such liability appears to be minimal, based on the in vitro data.

Solution phase parallel synthesis of substituted 3-phenylsulfonyl-[1,2,3]triazolo[1,5-a]quinazolines: selective serotonin 5-HT(6) receptor antagonists.

Here we present the solution phase parallel synthesis of a combinatorial library consisting of 776 new substituted 3-phenylsulfonyl-[1,2,3]triazolo[1,5-a]quinazolines and a study of the relation of their structure with a 5-HT(6) receptor antagonistic activity in a functional cell (HEK 293) analysis and radioligand competitive binding. We have found highly active and selective 5-HT(6)R antagonists. The most active 5-HT(6)R antagonists have IC(50) <100 nM in a functional assay, and K(i) <10 nM in a binding assay, which is 100 times higher than the activity with respect to other serotonin receptors.

Synthesis of cycloalkane-annelated 3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines and their evaluation as 5-HT6 receptor antagonists.

Synthesis and biological evaluation of 1 ('angular') and 2 ('linear') cycloalkane-annelated 3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines as novel ligands of the 5-HT(6) receptors are disclosed. The new compounds 1 and 2 are highly selective antagonists of the receptor with sub-nanomolar affinities (K(i)<1 nM). In its structure, this new chemotype lacks a basic ionizable side chain, which is considered as the characteristic feature of the 5-HT(6) receptor antagonists pharmacophore model.

Novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamides as selective GSK-3 inhibitors.

Synthesis, biological evaluation, and SAR dependencies for a series of novel aryl and heteroaryl substituted N-[3-(4-phenylpiperazin-1-yl)propyl]-1,2,4-oxadiazole-5-carboxamide inhibitors of GSK-3beta kinase are described. The inhibitory activity of the synthesized compounds is highly dependent on the character of substituents in the phenyl ring and the nature of terminal heterocyclic fragment of the core molecular scaffold. The most potent compounds from this series contain 3,4-di-methyl or 2-methoxy substituents within the phenyl ring and 3-pyridine fragment connected to the 1,2,4-oxadiazole heterocycle. These compounds selectively inhibit GSK-3beta kinase with IC(50) value of 0.35 and 0.41 microM, respectively.