Discovery of Pyrazolopyrazines as Selective, Potent, and Mutant-Active MET Inhibitors with Intracranial Efficacy.

Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that serves a critical function in numerous developmental, morphogenic, and proliferative signaling pathways. If dysregulated, MET has been shown to be involved in the development and survival of several cancers, including non-small cell lung cancer (NSCLC), renal cancer, and other epithelial tumors. Currently, the clinical efficacy of FDA approved MET inhibitors is limited by on-target acquired resistance, dose-limiting toxicities, and less than optimal efficacy against brain metastasis. Therefore, there is still an unmet medical need for the development of MET inhibitors to address these issues. Herein we report the application of structure-based design for the discovery and development of a novel class of brain-penetrant MET inhibitors with enhanced activity against clinically relevant mutations and improved selectivity. Compound 13 with a MET D1228N cell line IC50 value of 23 nM showed good efficacy in an intracranial tumor model and increased the median overall survival of the animals to 100% when dosed orally at 100 mg/kg daily for 21 days.

Progress on the Synthesis Pathways and Pharmacological Effects of Naturally Occurring Pyrazines.

As one of the most essential types of heterocyclic compounds, pyrazines have a characteristic smell and taste and have a wide range of commercial applications, especially in the food industry. With the development of the food industry, the demand for pyrazines has increased. Therefore, understanding the properties, functions, and synthetic pathways of pyrazines is one of the fundamental methods to produce, control, and apply pyrazines in food or medical systems. In this review, we provide an overview of the synthesis pathways and physiological or pharmacological functions of naturally occurring pyrazines. In particular, we focus on the biosynthesis and pharmacological effects of 2,3,5,6-Tetramethylpyrazine (TTMP), 2,5-Dimethylpyrazine (2,5-DMP), and 2,3,5-trimethylpyrazine (TMP). Furthermore, areas where further research on pyrazines is needed are discussed in this work.

Discovery of 6,7-Dihydropyrazolo[1,5-a]pyrazin-4(5H)-one Derivatives as mGluR2 Negative Allosteric Modulators with In Vivo Activity in a Rodent's Model of Cognition.

Allosteric modulators of the metabotropic group II receptors, mGluR2 and mGluR3, have been widely explored due to their ability to modulate cognitive and neurological functions in mood disorders, although none have been approved yet. In our search for new and selective mGluR2 negative allosteric modulators (NAMs), series of 6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were identified from our published series of 1,3,5-trisubstituted pyrazoles. SAR evolution of the initial hit resulted in 100-fold improvement in the mGluR2 NAM potency and subsequent selection of compound 11 based on its overall profile, including selectivity and ADMET properties. Further pharmacokinetic-pharmacodynamic (PK-PD) relationship built showed that compound 11 occupied the mGluR2 receptor in a dose-dependent manner. Additionally, the compound revealed in vivo activity in V-maze as a model of cognition from a dose of 0.32 mg/kg. Compound 11 was selected to be evaluated further.

Discovery of pyridazinone derivatives bearing tetrahydroimidazo[1,2-a]pyrazine scaffold as potent inhibitors of transient receptor potential canonical 5 to ameliorate hypertension-induced renal injury in rats.

Transient receptor potential canonical 5 (TRPC5) is a calcium-permeable non-selective cation channel involved in various pathophysiological processes, including renal injury. Recently, GFB-887, an investigational pyridazinone TRPC5 inhibitor, demonstrated significant therapeutic potential in a Phase II clinical trial for focal segmental glomerulosclerosis (FSGS), a rare and severe form of chronic kidney disease (CKD). In the current study, based on the structure of GFB-887, we conducted extensive structural modification to explore novel TRPC5 inhibitors with desirable drug-like properties and robust nephroprotective efficacy. A series of pyridazinone derivatives featuring a novel tetrahydroimidazo[1,2-a]pyrazine scaffold were synthesized and their activities were evaluated in HEK-293 cells stably expressing TRPC5 using a fluorescence-based Ca2+ mobilization assay. Among these compounds, compound 12 is turned out to be a potent TRPC5 inhibitor with apparent affinity comparable to the parent compound GBF-887. Compound 12 is highly selective on TRPC4/5 over TRPC3/6/7 and hERG channels, along with acceptable pharmacokinetic properties and a favorable safety profile. More importantly, in a rat model of hypertension-induced renal injury, oral administration of compound 12 (10 mg/kg, BID) efficaciously reduced mean blood pressure, inhibited proteinuria, and protected podocyte damage. These findings further confirmed the potential of TRPC5 inhibitors on the CKD treatment and provided compound 12 to be a valuable tool for exploring TRPC4/5 pathophysiology.

A hit expansion of 3-benzamidopyrazine-2-carboxamide: Toward inhibitors of prolyl-tRNA synthetase with antimycobacterial activity.

This study presents an exploration of the chemical space around derivatives of 3-benzamidopyrazine-2-carboxamides, previously identified as potent antimycobacterial compounds with predicted binding to mycobacterial prolyl-transfer RNA synthetase. New urea derivatives (Series-1) were generally inactive, probably due to their preference for cis-trans conformation (confirmed by density functional theory calculations and experimentally by nuclear overhauser effect spectroscopy NMR). Series-2 (3-benzamidopyrazine-2-carboxamides with disubstituted benzene ring) demonstrated that substituents larger than fluorine are not tolerated in the ortho position of the benzene ring. This series brought two new compounds (21: R = 2-F, 4-Cl and 22: R = 2-F, 4-Br) with in vitro activity against Mycobacterium tuberculosis H37Rv as well as multidrug-resistant clinical isolates, with minimum inhibitory concentration ranging from 6.25 to 25 µg/mL. The lactone-type derivatives 4H-pyrazino[2,3-d][1,3]oxazin-4-ones (Series-3) were inactive, but solvent stability studies of compound 29 indicated that they might be developed to usable lactone prodrugs of inhibitors of mycobacterial aspartate decarboxylase (PanD).

Discovery of a potent Gilteritinib-based FLT3-PROTAC degrader for the treatment of Acute myeloid leukemia.

Fms-like tyrosine receptor kinase 3 (FLT3) proteolysis targeting chimeras (PROTACs) emerge as a promising approach to overcome the limitations of FLT3 inhibitors, while the development of orally bioavailable FLT3-PROTACs faces great challenges. Here, we report the rational design and evaluation of a series of Gilteritinib-based FLT3-PROTACs. Among them, B3-2 exhibited the strongest antiproliferative activity against FLT3-ITD mutant AML cells, and significantly induced FLT3-ITD protein degradation. Mechanistic investigations demonstrated that B3-2 induced FLT3-ITD degradation in a ubiquitin-proteasome-dependent manner. More importantly, B3-2 exhibited an oral bioavailability of 5.65%, and oral administration of B3-2 showed good antitumor activity in MV-4-11 xenograft models. Furthermore, B3-2 showed strong antiproliferative activity against FLT3 resistant mutations, highlighting its potential in overcoming drug resistance.

Acipimox inhibits human carbonic anhydrases.

Acipimox, a nicotinic acid derivative in clinical use for the treatment of hyperlipidaemia, incorporates a free carboxylic acid and an N-oxide moiety, functionalities known to interact with the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and inhibit its activity. Herein we report that acipimox acts as a low micromolar CA inhibitor (CAI) against most human (h) isoforms possessing catalytic activity, hCA I - XIV. By using computational techniques (docking and molecular dynamics simulations), we propose that acipimox coordinates through its carboxylate group to the zinc ion from the enzyme active site cavity, whereas the N-oxide group is hydrogen-bonded to the proton shuttle His residue in some isoforms (hCA I) or to active site Thr or Gln residues in other isoforms (hCA II, III, IV, VII, etc). As some CA isoforms are involved in lipogenesis, these data may be useful for the design of more effective CAIs with antiobesity activity.

Design, synthesis and biological evaluation of substituted 3-amino-N-(thiazol-2-yl)pyrazine-2-carboxamides as inhibitors of mycobacterial methionine aminopeptidase 1.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the number one cause of deaths due to a single infectious agent worldwide. The treatment of TB is lengthy and often complicated by the increasing drug resistance. New compounds with new mechanisms of action are therefore needed. We present the design, synthesis, and biological evaluation of pyrazine-based inhibitors of a prominent antimycobacterial drug target - mycobacterial methionine aminopeptidase 1 (MtMetAP1). The inhibitory activities of the presented compounds were evaluated against the MtMetAP1a isoform, and all derivatives were tested against a broad spectrum of myco(bacteria) and fungi. The cytotoxicity of the compounds was also investigated using Hep G2 cell lines. Overall, high inhibition of the isolated enzyme was observed for 3-substituted N-(thiazol-2-yl)pyrazine-2-carboxamides, particularly when the substituent was represented by 2-substituted benzamide. The extent of inhibition was strongly dependent on the used metal cofactor. The highest inhibition was seen in the presence of Ni2+. Several compounds also showed mediocre in vitro potency against Mtb (both Mtb H37Ra and H37Rv). Despite the structural similarities of bacterial and fungal MetAP1 to mycobacterial MtMetAP1, title compounds did not exert antibacterial nor antifungal activity. The reasons behind the higher activity of 2-substituted benzamido derivatives, as well as the correlation of enzyme inhibition with the in vitro growth inhibition activity is discussed.

Identification of (6S)-cyclopropyl-6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxamines as new HBV capsid assembly modulators.

GYH2-18 is a type II HBV CAM with 6,7-dihydropyrazolo[1,5-a]pyrazine-5(4H)-carboxamine (DPPC) skeleton discovered by Roche INC. A series of GYH2-18 derivatives were designed, synthesized and evaluated for their anti-HBV activity. Two compounds 2f and 3k exhibited excellent anti-HBV activity, low cytotoxicity and accepted oral PK profiles. Chiral separation of 2f and 3k was conducted successfully, and (6S)-cyclopropyl DPPC isomers 2f-1, 2f-3, 3k-1 and 3k-3 were identified to be much more active than the corresponding (6R)-ones. The preliminary structure-activity relationship, particle gel assay and molecular modeling studies were also discussed, which provide useful indications for guiding the further rational design of new (6S)-cyclopropyl DPPC analogues.

Synthetic Attempts Towards Eminent Anti-viral Candidates of SARS-CoV.

Severe Acute Respiratory Syndrome (SARS) aka SARS-CoV spread over southern China for the first time in 2002-2003 and history repeated again since last year and took away lives of more than two million people so far. On March 11, 2020 COVID-19 outbreak was officially declared as pandemic by World Health Organization (WHO). The entire world united to fight back against this ultimate destruction. Around 90 vaccines are featured against SARS-CoV-2 and more than 300 active clinical trials are underway by several groups and individuals. So far, no drugs have been currently approved that can completely eliminate the deadly coronavirus. The promising SARS-CoV-2 antiviral drugs are favipiravir, remdesivir, lopinavir, ribavirin and avifavir. In this review, we have discussed the synthetic approaches elaborately made so far by different groups and chemical companies all around the world towards top three convincing anti-viral drugs against SARS-CoV-2, which are favipiravir, remdesivir and lopinavir.

Synthesis of Novel Methyl 7-[(Hetero)arylamino]thieno[2,3-b]pyrazine-6-carboxylates and Antitumor Activity Evaluation: Effects in Human Tumor Cells Growth, Cell Cycle Analysis, Apoptosis and Toxicity in Non-Tumor Cells.

Several novel methyl 7-[(hetero)arylamino]thieno[2,3-b]pyrazine-6-carboxylates were synthesized by Pd-catalyzed C-N Buchwald-Hartwig cross-coupling of either methyl 7-aminothieno[3,2-b]pyrazine-6-carboxylate with (hetero)arylhalides or 7-bromothieno[2,3-b]pyrazine-6-carboxylate with (hetero)arylamines in good-to-excellent yields (50% quantitative yield), using different reaction conditions, namely ligands and solvents, due to the different electronic character of the substrates. The antitumoral potential of these compounds was evaluated in four human tumor cell lines: gastric adenocarcinoma (AGS), colorectal adenocarcinoma (CaCo-2), breast carcinoma (MCF7), and non-small-cell lung carcinoma (NCI-H460) using the SRB assay, and it was possible to establish some structure-activity relationships. Furthermore, they did not show relevant toxicity against a non-tumor cell line culture from the African green monkey kidney (Vero). The most promising compounds (GI50 ≤ 11 µM), showed some selectivity either against AGS or CaCo-2 cell lines without toxicity at their GI50 values. The effects of the methoxylated compounds 2b (2-OMeC6H4), 2f and 2g (3,4- or 3,5-diOMeC6H3, respectively) on the cell cycle profile and induction of apoptosis were further studied in the AGS cell line. Nevertheless, even for the most active (GI50 = 7.8 µM) and selective compound (2g) against this cell line, it was observed that a huge number of dead cells gave rise to an atypical distribution on the cell cycle profile and that these cells were not apoptotic, which points to a different mechanism of action for the AGS cell growth inhibition.

Determination of the Absolute Configuration of Bioactive Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones and Study of Their In Vitro Metabolic Profile.

In recent decades, fungi-derived naturally occurring quinazolines have emerged as potential drug candidates. Nevertheless, most studies are conducted for bioactivity assays, and little is known about their absorption, distribution, metabolism, and elimination (ADME) properties. To perform metabolic studies, the synthesis of the naturally occurring quinazolinone, fiscalin B (1), and its chloro derivative, 4-((1H-indol-3-yl)methyl)-8,10-dichloro-1-isobutyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-dione (2), disclosed as an antibacterial agent, was performed in a gram scale using a microwave-assisted polycondensation reaction with 22% and 17% yields, respectively. The structure of the non-natural (+)-fiscalin B was established, for the first time, by X-ray crystallography as (1R,4S)-1, and the absolute configuration of the naturally occurring fiscalin B (-)-1 was confirmed by comparison of its calculated and experimental electronic circular dichroism (ECD) spectra as (1S,4R)-1. in vitro metabolic studies were monitored for this class of natural products for the first time by ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). The metabolic characteristics of 1 and 2 in human liver microsomes indicated hydration and hydroxylation mass changes introduced to the parent drugs.

Discovery and Toxicological Profiling of Aminopyridines as Orally Bioavailable Selective Inhibitors of PI3-Kinase γ.

Using a novel physiologically relevant in vitro human whole blood neutrophil shape change assay, an aminopyrazine series of selective PI3Kγ inhibitors was identified and prioritized for further optimization. Severe solubility limitations associated with the series leading to low oral bioavailability and poor exposures, especially at higher doses, were overcome by moving to an aminopyridine core. Compound 33, with the optimal balance of on-target activity, selectivity, and pharmacokinetic parameters, progressed into in vivo studies and demonstrated good efficacy (10 mg/kg) in a rat model of airway inflammation. Sufficient exposures were achieved at high doses to support toxicological studies, where unexpected inflammatory cell infiltrates in cardiovascular tissue prevented further compound development.

New Pyrazine Conjugates: Synthesis, Computational Studies, and Antiviral Properties against SARS-CoV-2.

Currently, limited therapeutic options are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have developed a set of pyrazine-based small molecules. A series of pyrazine conjugates was synthesized by microwave-assisted click chemistry and benzotriazole chemistry. All the synthesized conjugates were screened against the SAR-CoV-2 virus and their cytotoxicity was determined. Computational studies were carried out to validate the biological data. Some of the pyrazine-triazole conjugates (5 d-g) and (S)-N-(1-(benzo[d]thiazol-2-yl)-2-phenylethyl)pyrazine-2-carboxamide 12 i show significant potency against SARS-CoV-2 among the synthesized conjugates. The selectivity index (SI) of potent conjugates indicates significant efficacy compared to the reference drug (Favipiravir).

Favipiravir-Based Ionic Liquids as Potent Antiviral Drugs for Oral Delivery: Synthesis, Solubility, and Pharmacokinetic Evaluation.

Coronavirus disease 2019 (COVID-19) has spread across the world, and no specific antiviral drugs have yet been approved to combat this disease. Favipiravir (FAV) is an antiviral drug that is currently in clinical trials for use against COVID-19. However, the delivery of FAV is challenging because of its limited solubility, and its formulation is difficult with common organic solvents and water. To address these issues, four FAV ionic liquids (FAV-ILs) were synthesized as potent antiviral prodrugs and were fully characterized by nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FT-IR) spectrometry, powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), derivative thermogravimetry (DTG), and differential scanning calorimetry (DSC). The aqueous solubility and in vivo pharmacokinetic properties of the FAV-ILs were also evaluated. The FAV-ILs exhibited improved aqueous solubility by 78 to 125 orders of magnitude when compared with that of free FAV. Upon oral dosing in mice, the absolute bioavailability of the ß-alanine ethyl ester FAV formulation was increased 1.9-fold compared with that of the control FAV formulation. The peak blood concentration, elimination half-life, and mean absorption time of FAV were also increased by 1.5-, 2.0-, and 1.5-fold, respectively, compared with the control. Furthermore, the FAV in the FAV-ILs exhibited significantly different biodistribution compared with the control FAV formulation. Interestingly, drug accumulation in the lungs and liver was improved 1.5-fold and 1.3-fold, respectively, compared with the control FAV formulation. These results indicate that the use of ILs exhibits potential as a simple, scalable strategy to improve the solubility and oral absorption of hydrophobic drugs, such as FAV.

Fragment-Based Optimization of Dihydropyrazino-Benzimidazolones as Metabotropic Glutamate Receptor-2 Positive Allosteric Modulators against Migraine.

Our previous scaffold-hopping attempts resulted in dihydropyrazino-benzimidazoles as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs) with suboptimal drug-like profiles. Here, we report an alternative fragment-based optimization strategy applied on the new dihydropyrazino-benzimidazolone scaffold. Analyzing published high-affinity mGluR2 PAMs, we used a pharmacophore-guided approach to identify suitable growing vectors and optimize the scaffold in these directions. This strategy resulted in a new fragment like lead (34) with improved druglike properties that were translated to sufficient pharmacokinetics and validated proof-of-concept studies in migraine. Gratifyingly, compound 34 showed reasonable activity in the partial infraorbital nerve ligation, a migraine disease model that might open this indication for mGluR2 PAMs.

Discovery and Early Clinical Development of LY3202626, a Low-Dose, CNS-Penetrant BACE Inhibitor.

The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer's disease drug target owing to its critical role in the production of amyloid-beta. We have previously reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity. LY2886721 advanced to Phase II, but development was halted due to abnormally elevated liver enzymes. Herein, we report the discovery and clinical development of LY3202626, a highly potent, CNS-penetrant, and low-dose BACE inhibitor, which successfully addressed these key development challenges.

Application of cation-π interactions in enzyme-substrate binding: Design, synthesis, biological evaluation, and molecular dynamics insights of novel hydrophilic substrates for NQO1.

Cation-π interaction is a type of noncovalent interaction formed between the π-electron system and the positively charged ion or moieties. In this study, we designed a series of novel NQO1 substrates by introducing aliphatic nitrogen-containing side chains to fit with the L-shaped pocket of NQO1 by the formation of cation-π interactions. Molecular dynamics (MD) simulation indicated that the basic N atom in the side chain of NQO1 substrates, which is prone to be protonated under physiological conditions, can form cation-π interactions with the Phe232 and Phe236 residues of the NQO1 enzyme. Compound 4 with a methylpiperazinyl substituent was identified as the most efficient substrate for NQO1 with the reduction rate and catalytic efficiency of 1263 ± 61 µmol NADPH/min/µmol NQO1 and 2.8 ± 0.3 × 106 M-1s-1, respectively. Notably, compound 4 exhibited increased water solubility (110 µg/mL) compared to that of ß-lap (43 µg/mL), especially under acidic condition (pH = 3, solubility > 1000 µg/mL). Compound 4 (IC50/A549 = 2.4 ± 0.6 µM) showed potent antitumor activity against NQO1-rich cancer cells through ROS generation via NQO1-mediated redox cycling. These results emphasized that the application of cation-π interactions by introducing basic aliphatic amine moiety is beneficial for both the water solubility and the NQO1-substrate binding, leading to promising NQO1-targeting antitumor candidates with improved druglike properties.

Synthesis of New Triazolopyrazine Antimalarial Compounds.

A radical approach to late-stage functionalization using photoredox and Diversinate™ chemistry on the Open Source Malaria (OSM) triazolopyrazine scaffold (Series 4) resulted in the synthesis of 12 new analogues, which were characterized by NMR, UV, and MS data analysis. The structures of four triazolopyrazines were confirmed by X-ray crystal structure analysis. Several minor and unexpected side products were generated during these studies, including two resulting from a possible disproportionation reaction. All compounds were tested for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum (3D7 and Dd2 strains) and for cytotoxicity against a human embryonic kidney (HEK293) cell line. Moderate antimalarial activity was observed for some of the compounds, with IC50 values ranging from 0.3 to >20 µM; none of the compounds displayed any toxicity against HEK293 at 80 µM.

Structure-Activity Relationship Studies on Oxazolo[3,4-a]pyrazine Derivatives Leading to the Discovery of a Novel Neuropeptide S Receptor Antagonist with Potent In Vivo Activity.

Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure-activity relationships and provide an updated model of ligand/NPSR interactions.