Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4.

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity.

Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance has been reported against all clinically available antimalarials, threatening our ability to control the disease and therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identified the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity relationship of the identified class and the optimisation of asexual stage activity while maintaining selectivity against the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed in in vitro ADME profiling. A significant challenge remains to correct these liabilities for further advancement of the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a curative or prophylactic antimalarial treatment.

Structural Basis of Small-Molecule Aggregate Induced Inhibition of a Protein-Protein Interaction.

A prevalent observation in high-throughput screening and drug discovery programs is the inhibition of protein function by small-molecule compound aggregation. Here, we present the X-ray structural description of aggregation-based inhibition of a protein-protein interaction involving tumor necrosis factor α (TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor (JNJ525) induces a quaternary structure switch of TNFα that inhibits the protein-protein interaction between TNFα and TNFα receptors. SPD-304 may employ a similar mechanism of inhibition.

JNJ-40255293, a novel adenosine A2A/A1 antagonist with efficacy in preclinical models of Parkinson's disease.

Adenosine A2A antagonists are believed to have therapeutic potential in the treatment of Parkinson's disease (PD). We have characterized the dual adenosine A2A/A1 receptor antagonist JNJ-40255293 (2-amino-8-[2-(4-morpholinyl)ethoxy]-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one). JNJ-40255293 was a high-affinity (7.5 nM) antagonist at the human A2A receptor with 7-fold in vitro selectivity versus the human A1 receptor. A similar A2A:A1 selectivity was seen in vivo (ED50's of 0.21 and 2.1 mg/kg p.o. for occupancy of rat brain A2A and A1 receptors, respectively). The plasma EC50 for occupancy of rat brain A2A receptors was 13 ng/mL. In sleep-wake encephalographic (EEG) studies, JNJ-40255293 dose-dependently enhanced a consolidated waking associated with a subsequent delayed compensatory sleep (minimum effective dose: 0.63 mg/kg p.o.). As measured by microdialysis, JNJ-40255293 did not affect dopamine and noradrenaline release in the prefrontal cortex and the striatum. However, it was able to reverse effects (catalepsy, hypolocomotion, and conditioned avoidance impairment in rats; hypolocomotion in mice) produced by the dopamine D2 antagonist haloperidol. The compound also potentiated the agitation induced by the dopamine agonist apomorphine. JNJ-40255293 also reversed hypolocomotion produced by the dopamine-depleting agent reserpine and potentiated the effects of l-dihydroxyphenylalanine (L-DOPA) in rats with unilateral 6-hydroxydopamine-induced lesions of the nigro-striatal pathway, an animal model of Parkinson's disease. Extrapolating from the rat receptor occupancy dose-response curve, the occupancy required to produce these various effects in rats was generally in the range of 60-90%. The findings support the continued research and development of A2A antagonists as potential treatments for PD.

Substituted thieno[2,3-d]pyrimidines as adenosine A2A receptor antagonists.

A novel series of benzyl substituted thieno[2,3-d]pyrimidines were identified as potent A2A receptor antagonists. Several five- and six-membered heterocyclic replacements for the optimized methylfuran were explored. Select compounds effectively reverse catalepsy in mice when dosed orally.

Design and characterization of optimized adenosine A2A/A1 receptor antagonists for the treatment of Parkinson's disease.

The design and characterization of two, dual adenosine A(2A)/A(1) receptor antagonists in several animal models of Parkinson's disease is described. Compound 1 was previously reported as a potential treatment for Parkinson's disease. Further characterization of 1 revealed that it was metabolized to reactive intermediates that caused the genotoxicity of 1 in the Ames and mouse lymphoma L51784 assays. The identification of the metabolites enabled the preparation of two optimized compounds 13 and 14 that were devoid of the metabolic liabilities associated with 1. Compounds 13 and 14 are potent dual A(2A)/A(1) receptor antagonists that have excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse and rat models of reserpine-induced akinesia, and the rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation.

Overcoming the genotoxicity of a pyrrolidine substituted arylindenopyrimidine as a potent dual adenosine A(2A)/A(1) antagonist by minimizing bioactivation to an iminium ion reactive intermediate.

2-Amino-4-phenyl-8-pyrrolidin-1-ylmethyl-indeno[1,2-d]pyrimidin-5-one (1) is a novel and potent selective dual A(2A)/A(1) adenosine receptor antagonist from the arylindenopyrimidine series that was determined to be genotoxic in both the Ames and Mouse Lymphoma L5178Y assays only following metabolic activation. Compound 1 was identified as a frame-shift mutagen in Salmonella typhimurium tester strain TA1537 as indicated by a significant dose-dependent increase in revertant colonies as compared to the vehicle control. The metabolic activation-dependent irreversible covalent binding of radioactivity to DNA, recovery of 1 and its enamine metabolite from acid hydrolysis of covalently modified DNA, and protection of covalent binding to DNA by both cyanide ion and methoxylamine suggest that the frame-shift mutation in TA1537 strain involved covalent binding instead of simple intercalation to DNA. Compound 1 was bioactivated to endocyclic iminium ion, aldehyde, epoxide, and α,ß-unsaturated keto reactive intermediates from the detection of cyano, oxime, and glutathione conjugates by data-dependent high resolution accurate mass measurements. Collision-induced dissociation of these conjugates provided evidence for bioactivation of the pyrrolidine ring of 1. The epoxide and α,ß-unsaturated keto reactive intermediates were unlikely to cause the genotoxicity of 1 because the formation of their glutathione adducts did not ameliorate the binding of compound related material to DNA. Instead, the endocyclic iminium ions and amino aldehydes were likely candidates responsible for genotoxicity based on, first, the protection afforded by both cyanide ion and methoxylamine, which reduced the potential to form covalent adducts with DNA, and, second, analogues of 1 designed with low probability to form these reactive intermediates were not genotoxic. It was concluded that 1 also had the potential to be mutagenic in humans based on observing the endocyclic iminium ion following incubation with a human liver S9 preparation and the commensurate detection of DNA adducts. An understanding of this genotoxicity mechanism supported an evidence-based approach to selectively modify the structure of 1 which resulted in analogues being synthesized that were devoid of a genotoxic liability. In addition, potency and selectivity against both adenosine A(2A) and A(1) receptors were maintained.

Adenosine A(2A) Receptor Antagonists and Parkinson's Disease.

This Review summarizes and updates the work on adenosine A(2A) receptor antagonists for Parkinson's disease from 2006 to the present. There have been numerous publications, patent applications, and press releases within this time frame that highlight new medicinal chemistry approaches to this attractive and promising target to treat Parkinson's disease. The Review is broken down by scaffold type and will discuss the efforts to optimize particular scaffolds for activity, pharmacokinetics, and other drug discovery parameters. The majority of approaches focus on preparing selective A(2A) antagonists, but a few approaches to dual A(2A)/A(1) antagonists will also be highlighted. The in vivo profiles of compounds will be highlighted and discussed to compare activities across different chemical series. A clinical report and update will be given on compounds that have entered clinical trials.

In vivo characterization of a dual adenosine A2A/A1 receptor antagonist in animal models of Parkinson's disease.

The in vivo characterization of a dual adenosine A(2A)/A(1) receptor antagonist in several animal models of Parkinson's disease is described. Discovery and scale-up syntheses of compound 1 are described in detail, highlighting optimization steps that increased the overall yield of 1 from 10.0% to 30.5%. Compound 1 is a potent A(2A)/A(1) receptor antagonist in vitro (A(2A) K(i) = 4.1 nM; A(1) K(i) = 17.0 nM) that has excellent activity, after oral administration, across a number of animal models of Parkinson's disease including mouse and rat models of haloperidol-induced catalepsy, mouse model of reserpine-induced akinesia, rat 6-hydroxydopamine (6-OHDA) lesion model of drug-induced rotation, and MPTP-treated non-human primate model.

Optimization of arylindenopyrimidines as potent adenosine A(2A)/A(1) antagonists.

Two reactive metabolites were identified in vivo for the dual A(2A)/A(1) receptor antagonist 1. Two strategies were implemented to successfully mitigate the metabolic liabilities associated with 1. Optimization of the arylindenopyrimidines led to a number of amide, ether, and amino analogs having comparable in vitro and in vivo activity.

Methylene amine substituted arylindenopyrimidines as potent adenosine A(2A)/A(1) antagonists.

A novel series of arylindenopyrimidines were identified as A(2A) and A(1) receptor antagonists. The series was optimized for in vitro activity by substituting the 8- and 9-positions with methylene amine substituents. The compounds show excellent activity in mouse models of Parkinson's disease when dosed orally.

Syntheses and in vitro evaluation of arylsulfone-based MMP inhibitors with heterocycle-derived zinc-binding groups (ZBGs).

Several classes of arylsulfone-based MMP-2/-9 inhibitors utilizing 6- to 8-membered heterocyclic rings as zinc-binding groups (ZBGs) have been synthesized and their enzyme inhibitory activities were evaluated. Although a number of 6- and 7-membered heterocycles were effective, the most potent arylsulfone inhibitors are based on the rigid 1- or 3-hydroxypyridone ZBG.

1-Hydroxy-2-pyridinone-based MMP inhibitors: synthesis and biological evaluation for the treatment of ischemic stroke.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the breakdown of the blood-brain barrier during cerebral ischemia. As a result, inhibition of MMP-9 may have utility as a therapeutic intervention in stroke. Towards this end, we have synthesized a series of 1-hydroxy-2-pyridinones that have excellent in vitro potency in inhibiting MMP-9 in addition to MMP-2. Representative compounds also demonstrate good efficacy in the mouse transient mid-cerebral artery occlusion (tMCAO) model of cerebral ischemia.

Synthesis and biological evaluation of thiol-based inhibitors of glutamate carboxypeptidase II: discovery of an orally active GCP II inhibitor.

A series of 2-(thioalkyl)pentanedioic acids were synthesized and evaluated as inhibitors of glutamate carboxypeptidase II (GCP II, EC 3.4.17.21). The inhibitory potency of these thiol-based compounds against GCP II was found to be dependent on the number of methylene units between the thiol group and pentanedioic acid. A comparison of the SAR of the thiol-based inhibitors to that of the phosphonate-based inhibitors provides insight into the role of each of the two zinc-binding groups in GCP II inhibition. The most potent thiol-based inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (IC(50) = 90 nM), was found to be orally bioavailable in rats and exhibited efficacy in an animal model of neuropathic pain following oral administration.

Regioselective preparation of 2-substituted 3,4-diaryl pyrroles: a concise total synthesis of ningalin B.

Methylisocyanoacetate undergoes a 2 + 3 cycloaddition with alpha,beta-unsaturated nitriles to provide a regioselective synthesis of 2-substituted 3,4-diaryl pyrroles. The ease of preparation of alpha,beta-unsaturated nitriles allows the rapid synthesis of pyrroles with varied substituents. Using this method, a key intermediate (1) for the synthesis of the marine natural products lukianol A, lamellarin O, and lamellarin Q was prepared in two steps. A total synthesis of ningalin B (11) was also accomplished utilizing this methodology.

Pyridinylimidazole based p38 MAP kinase inhibitors.

The p38 MAP kinase is thought to be involved in a variety of inflammatory and immunological disorders such as rheumatoid arthritis. The pyridinylimidazole class of compounds was the first to potently inhibit this kinase. Since the original reports of their efficacy, they have become the most widely studied series of inhibitors of this kinase. This framework has served as a starting point for further synthetic work and several compounds have entered clinical trials. These compounds have also been utilized to elucidate the role of p38 kinase in the immune system, and more recently have been used to examine the role of this kinase in central nervous system disorders.