DNDI-6148: A Novel Benzoxaborole Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a parasitic disease endemic across multiple regions of the world and is fatal if untreated. Current therapies are unsuitable, and there is an urgent need for safe, short-course, and low-cost oral treatments to combat this neglected disease. The benzoxaborole chemotype has previously delivered clinical candidates for the treatment of other parasitic diseases. Here, we describe the development and optimization of this series, leading to the identification of compounds with potent in vitro and in vivo antileishmanial activity. The lead compound (DNDI-6148) combines impressive in vivo efficacy (>98% reduction in parasite burden) with pharmaceutical properties suitable for onward development and an acceptable safety profile. Detailed mode of action studies confirm that DNDI-6148 acts principally through the inhibition of Leishmania cleavage and polyadenylation specificity factor (CPSF3) endonuclease. As a result of these studies and its promising profile, DNDI-6148 has been declared a preclinical candidate for the treatment of VL.

Design and Synthesis of a Pan-Janus Kinase Inhibitor Clinical Candidate (PF-06263276) Suitable for Inhaled and Topical Delivery for the Treatment of Inflammatory Diseases of the Lungs and Skin.

By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.

Quantitative Chemical Proteomic Profiling of Ubiquitin Specific Proteases in Intact Cancer Cells.

Deubiquitinating enzymes play an important role in a plethora of therapeutically relevant processes and are emerging as pioneering drug targets. Herein, we present a novel probe, Ubiquitin Specific Protease (USP) inhibitor, alongside an alkyne-tagged activity-based probe analogue. Activity-based proteome profiling identified 12 USPs, including USP4, USP16, and USP33, as inhibitor targets using submicromolar probe concentrations. This represents the first intact cell activity-based profiling of deubiquitinating enzymes. Further analysis demonstrated functional inhibition of USP33 and identified a synergistic relationship in combination with ATR inhibition, consistent with USP4 inhibition.

Novel Amino-pyrazole Ureas with Potent In Vitro and In Vivo Antileishmanial Activity.

Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected tropical diseases. Treatment options are limited, and there is an urgent need for new therapeutic agents. Following an HTS campaign and hit optimization, a novel series of amino-pyrazole ureas has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26 shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating proof of concept for this series.

Multiparameter optimization in CNS drug discovery: design of pyrimido[4,5-d]azepines as potent 5-hydroxytryptamine 2C (5-HT2C) receptor agonists with exquisite functional selectivity over 5-HT2A and 5-HT2B receptors.

A series of 4-substituted pyrimido[4,5-d]azepines that are potent, selective 5-HT2C receptor partial agonists is described. A rational medicinal chemistry design strategy to deliver CNS penetration coupled with SAR-based optimization of selectivity and agonist potency provided compounds with the desired balance of preclinical properties. Lead compounds 17 (PF-4479745) and 18 (PF-4522654) displayed robust pharmacology in a preclinical canine model of stress urinary incontinence (SUI) and no measurable functional agonism at the key selectivity targets 5-HT2A and 5-HT2B in relevant tissue-based assay systems. Utilizing recent advances in the structural biology of GPCRs, homology modeling has been carried out to rationalize binding and agonist efficacy of these compounds.

Identification of type-II inhibitors using kinase structures.

Spleen tyrosine kinase is a non-receptor tyrosine kinase, overactivation of which is thought to contribute to autoimmune diseases as well as allergy and asthma. Protein kinases have a highly conserved ATP binding site, thus making challenging the design of selective small molecule inhibitors. It has been well documented that some protein kinases can be stabilized in their inactive conformations (Type-II inhibitors). Herein, we describe a protein structure/ligand-based approach to successfully identify ligands that bind to novel conformations of spleen tyrosine kinase. By utilizing kinase protein crystal structures both in the public domain (RCSB) and within Pfizer's protein crystal database, we report the discovery of the first spleen tyrosine kinase Type-II ligands. Compounds 1 and 3 were found to bind to the DFG-out conformation of spleen tyrosine kinase, while compound 2 binds to a DFG-in, C-Helix-out conformation. In this instance, the C-helix moved significantly to create a large hydrophobic pocket rarely seen in kinase protein crystal structures.

Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor agonists: design, synthesis, and evaluation of PF-3246799 as a treatment for urinary incontinence.

New pyrimido[4,5-d]azepines 7 are disclosed as potent 5-HT(2C) receptor agonists. A preferred example, 7b had minimal activation at either the 5-HT(2A) or 5-HT(2B) receptors combined with robust efficacy in a preclinical canine model of stress urinary incontinence (SUI) and attractive pharmacokinetic and safety properties. Based on this profile, 7b (PF-3246799) was identified as a candidate for clinical development for the treatment of SUI. In addition, it proved to be critical to build an understanding of the translation between recombinant cell-based systems, native tissue preparations and in vivo preclinical models. This was a significant undertaking and proved to be crucial in compound selection.

Second generation N-(1,2-diphenylethyl)piperazines as dual serotonin and noradrenaline reuptake inhibitors: improving metabolic stability and reducing ion channel activity.

New N-(1,2-diphenylethyl)piperazines 6 are disclosed as dual serotonin and noradrenaline reuptake inhibitors (SNRI) which may have potential in treating stress urinary incontinence (SUI). In this Letter, we present new data for SNRI PF-526014 (4) including performance in a canine in vivo model of SUI, cardiovascular assessment, pharmacokinetics in dog and determination of the primary routes of metabolism in vitro. Starting from 4, detailed structure activity relationships established that potent dual SNRIs could be achieved by appropriate substitution of the phenyl rings (6: R; R(1)) combined with a preferred stereochemistry. From this set of compounds, piperazine (-)-6a was identified as a potent and selective dual SNRI with improved metabolic stability and reduced ion channel activity when compared to 4. Based on this profile, (-)-6a was selected for further evaluation in a preclinical model of SUI.

Discovery of a novel azepine series of potent and selective 5-HT2C agonists as potential treatments for urinary incontinence.

A range of heterocycle fused azepines were synthesized in order to find a CNS penetrant, selective 5-HT(2C) agonist for the treatment of incontinence. The pyridazo-azepines such as compound 11 were shown to be potent 5-HT(2C) agonists and have potential for CNS penetration and good in vitro ADME properties but lacked selectivity against 5-HT(2B). Fusing a further heterocycle gave the selective triazolopyrimido-azepines. An example of this series, compound 36, was shown to be potent, selective, metabolically stable in vitro and efficacious in an in vivo model of stress urinary incontinence.

N-[(3S)-Pyrrolidin-3-yl]benzamides as novel dual serotonin and noradrenaline reuptake inhibitors: impact of small structural modifications on P-gp recognition and CNS penetration.

The structure-activity relationship and the synthesis of novel N-[(3S)-pyrrolidin-3-yl]benzamides as dual serotonin and noradrenaline monoamine reuptake inhibitors (SNRI) is described. Preferred compound 9 aka PF-184,298 is a potent SNRI with good selectivity over dopamine reuptake inhibition (DRI), good in vitro metabolic stability, weak CYP inhibition and drug-like physicochemical properties consistent with CNS target space. Evaluation in an in vivo preclinical model of stress urinary incontinence showed 9 significantly increased urethral tone at free plasma concentrations consistent with its in vitro primary pharmacology.

6,7-Dihydro-5H-pyrrolo[1,2-a] imidazoles as potent and selective alpha1A adrenoceptor partial agonists.

Novel pyrroloimidazoles have been identified as potent partial agonists of the alpha(1A) adrenergic receptor, with good selectivity over the alpha(1B), alpha(1D) and alpha(2A) receptor subtypes. Pyrimidine 19 possessed attractive CNS drug-like properties with good membrane permeability and no evidence for P-gp mediated efflux.

4-Piperidines and 3-pyrrolidines as dual serotonin and noradrenaline reuptake inhibitors: design, synthesis and structure-activity relationships.

Single enantiomer [(aryloxy)(pyridinyl)methyl]piperidine and pyrrolidine derivatives 5-9 are inhibitors of monoamine reuptake. Structure-activity relationships established that monoamine reuptake inhibition are functions of amine, pyridine isomer, aryloxy ring substitution and stereochemistry. Consequently, selective NRIs, selective SRIs, dual SNRIs and triple SNDRIs were all identified. Dual SNRIs 5l-a and 9c were evaluated in additional pharmacology and pharmacokinetic studies as representative examples from this series.

Potent and selective alpha1A adrenoceptor partial agonists--novel imidazole frameworks.

Novel imidazole frameworks have been identified as potent partial agonists of the alpha(1A) adrenergic receptor, with good selectivity over the alpha(1B), alpha(1D) and alpha(2A) receptor sub-types. Nitrile 28 possessed attractive CNS drug-like properties with good membrane permeability and no P-pg mediated efflux. 28 also possessed excellent solubility, metabolic stability and wide ligand selectivity.

Synthesis and evaluation of 2-pyridyl pyrimidines with in vitro antiplasmodial and antileishmanial activity.

A series of 2-pyridyl pyrimidines, reported inhibitors of Plasmodium falciparum methionine aminopeptidase 1b were synthesized and evaluated for their antiplasmodial activities. An analysis of physicochemical properties demonstrated a link between lipophilicity and antiparasitic activity. Cross screening of the library against cultured Leishmania donovani parasites revealed this class of compounds as potent inhibitors of parasite development in vitro.

Chloroquine-astemizole hybrids with potent in vitro and in vivo antiplasmodial activity.

A dual activity, conjugated approach has been taken to form hybrid molecules of two known antimalarial drugs, chloroquine (CQ) and the non-sedating H1 antagonist astemizole. A variety of linkers were investigated to conjugate the two agents into one molecule. Compounds 5-8 possessed improved in vitro activity against a CQ-resistant strain of Plasmodium falciparum, and examples 7 and 8 were active in vivo in mouse models of malaria.

Novel 2-imidazoles as potent, selective and CNS penetrant alpha1A adrenoceptor partial agonists.

A novel series of central nervous system (CNS) penetrant indane 2-imidazoles have been identified as potent, partial agonists of the alpha(1A) adrenergic receptor, having good selectivity over the alpha(1B), alpha(1D) and alpha(2) sub-types. A key structural motif to impart selectivity is a methylene spacer between the indane and a pendant substituent, which includes heterocycles, sulphones and ethers. Introduction of an ortho-halogen to this group led to a lowering of intrinsic efficacy (E(max)).

Derivatives of (3S)-N-(biphenyl-2-ylmethyl)pyrrolidin-3-amine as selective noradrenaline reuptake inhibitors: Reducing P-gp mediated efflux by modulation of H-bond acceptor capacity.

Derivatives of (3S)-N-(biphenyl-2-ylmethyl)pyrrolidin-3-amine are disclosed as a new series of noradrenaline reuptake inhibitors (NRI). Carboxamide 9e, carbamate 11b and sulfonamide 13a were identified as potent NRIs with excellent selectivity over SRI and DRI, good in vitro metabolic stability and weak CYP inhibition. Carbamate 11b demonstrated superior transit performance in MDCK-mdr1 cell lines with minimal P-gp efflux which was attributed to reduced HBA capacity of the carbamate group. Evaluation in vivo, in rat microdialysis experiments, showed 11b increased noradrenaline levels by 400% confirming good CNS penetration.

N-Benzyl-N-(pyrrolidin-3-yl)carboxamides as a new class of selective dual serotonin/noradrenaline reuptake inhibitors.

The structure-activity relationship and the synthesis of novel N-benzyl-N-(pyrrolidin-3-yl)carboxamides as dual serotonin (5-HT) and noradrenaline (NA) monoamine reuptake inhibitors are described. Compounds such as 18 exhibited dual 5-HT and NA reuptake inhibition, good selectivity over dopamine (DA) reuptake inhibition and drug-like physicochemical properties consistent with CNS target space. Compound 18 was selected for further preclinical evaluation.

Design and synthesis of morpholine derivatives. SAR for dual serotonin & noradrenaline reuptake inhibition.

Single enantiomer (SS) and (RR) 2-[(phenoxy)(phenyl)methyl]morpholine derivatives 5, 8-23 are inhibitors of monoamine reuptake. Target compounds were prepared using an enantioselective synthesis employing a highly specific enzyme-catalysed resolution of racemic n-butyl 4-benzylmorpholine-2-carboxylate (26) as the key step. Structure-activity relationships established that serotonin and noradrenaline reuptake inhibition are functions of stereochemistry and aryl/aryloxy ring substitution. Consequently, selective SRI, selective NRI and dual SNRIs were all identified. One of these compounds, a potent and selective dual SNRI, (SS)-5a was selected as a candidate for further pre-clinical evaluation.

Novel 2-imidazoles as potent and selective alpha1A adrenoceptor partial agonists.

Novel 2-imidazoles have been identified as potent partial agonists of the alpha(1A) adrenergic receptor, with good selectivity over the alpha(1B), alpha(1D) and alpha(2A) receptor sub-types. Sulfonamide 23 possessed attractive drug-like properties with respect to physicochemical and ADME properties and wide ligand selectivity.