Discovery of a class of highly potent Janus Kinase 1/2 (JAK1/2) inhibitors demonstrating effective cell-based blockade of IL-13 signaling.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

A restricted role for TYK2 catalytic activity in human cytokine responses revealed by novel TYK2-selective inhibitors.

TYK2 is a JAK family protein tyrosine kinase activated in response to multiple cytokines, including type I IFNs, IL-6, IL-10, IL-12, and IL-23. Extensive studies of mice that lack TYK2 expression indicate that the IFN-α, IL-12, and IL-23 pathways, but not the IL-6 or IL-10 pathways, are compromised. In contrast, there have been few studies of the role of TYK2 in primary human cells. A genetic mutation at the tyk2 locus that results in a lack of TYK2 protein in a single human patient has been linked to defects in the IFN-α, IL-6, IL-10, IL-12, and IL-23 pathways, suggesting a broad role for TYK2 protein in human cytokine responses. In this article, we have used a panel of novel potent TYK2 small-molecule inhibitors with varying degrees of selectivity against other JAK kinases to address the requirement for TYK2 catalytic activity in cytokine pathways in primary human cells. Our results indicate that the biological processes that require TYK2 catalytic function in humans are restricted to the IL-12 and IL-23 pathways, and suggest that inhibition of TYK2 catalytic activity may be an efficacious approach for the treatment of select autoimmune diseases without broad immunosuppression.

Novel triazolo-pyrrolopyridines as inhibitors of Janus kinase 1.

The identification of a novel fused triazolo-pyrrolopyridine scaffold, optimized derivatives of which display nanomolar inhibition of Janus kinase 1, is described. Prototypical example 3 demonstrated lower cell potency shift, better permeability in cells and higher oral exposure in rat than the corresponding, previously reported, imidazo-pyrrolopyridine analogue 2. Examples 6, 7 and 18 were subsequently identified from an optimization campaign and demonstrated modest selectivity over JAK2, moderate to good oral bioavailability in rat with overall pharmacokinetic profiles comparable to that reported for an approved pan-JAK inhibitor (tofacitinib).

2-Amino-[1,2,4]triazolo[1,5-a]pyridines as JAK2 inhibitors.

The advancement of a series of ligand efficient 2-amino-[1,2,4]triazolo[1,5-a]pyridines, initially identified from high-throughput screening, to a JAK2 inhibitor with pharmacodynamic activity in a mouse xenograft model is disclosed.

Structure-based discovery of C-2 substituted imidazo-pyrrolopyridine JAK1 inhibitors with improved selectivity over JAK2.

Herein we describe our successful efforts in obtaining C-2 substituted imidazo-pyrrolopyridines with improved JAK1 selectivity relative to JAK2 by targeting an amino acid residue that differs between the two isoforms (JAK1: E966; JAK2: D939). Efforts to improve cellular potency by reducing the polarity of the inhibitors are also detailed. The X-ray crystal structure of a representative inhibitor in complex with the JAK1 enzyme is also disclosed.

Azepines and piperidines with dual norepinephrine dopamine uptake inhibition and antidepressant activity.

Herein, we describe the discovery of inhibitors of norepinephrine (NET) and dopamine (DAT) transporters with reduced activity relative to serotonin transporters (SERT). Two compounds, 8b and 21a, along with nomifensine were tested in a rodent receptor occupancy study and demonstrated dose-dependent displacement of radiolabeled NET and DAT ligands. These compounds were efficacious in a rat forced swim assay (model of depression) and also had activity in rat spontaneous locomotion assay.

Identification of C-2 hydroxyethyl imidazopyrrolopyridines as potent JAK1 inhibitors with favorable physicochemical properties and high selectivity over JAK2.

Herein we report on the structure-based discovery of a C-2 hydroxyethyl moiety which provided consistently high levels of selectivity for JAK1 over JAK2 to the imidazopyrrolopyridine series of JAK1 inhibitors. X-ray structures of a C-2 hydroxyethyl analogue in complex with both JAK1 and JAK2 revealed differential ligand/protein interactions between the two isoforms and offered an explanation for the observed selectivity. Analysis of historical data from related molecules was used to develop a set of physicochemical compound design parameters to impart desirable properties such as acceptable membrane permeability, potent whole blood activity, and a high degree of metabolic stability. This work culminated in the identification of a highly JAK1 selective compound (31) exhibiting favorable oral bioavailability across a range of preclinical species and robust efficacy in a rat CIA model.

Discovery of potent and selective pyrazolopyrimidine janus kinase 2 inhibitors.

The discovery of somatic Jak2 mutations in patients with chronic myeloproliferative neoplasms has led to significant interest in discovering selective Jak2 inhibitors for use in treating these disorders. A high-throughput screening effort identified the pyrazolo[1,5-a]pyrimidine scaffold as a potent inhibitor of Jak2. Optimization of lead compounds 7a-b and 8 in this chemical series for activity against Jak2, selectivity against other Jak family kinases, and good in vivo pharmacokinetic properties led to the discovery of 7j. In a SET2 xenograft model that is dependent on Jak2 for growth, 7j demonstrated a time-dependent knock-down of pSTAT5, a downstream target of Jak2.

Identification of imidazo-pyrrolopyridines as novel and potent JAK1 inhibitors.

A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as rheumatoid arthritis (RA), by specific targeting of the JAK1 pathway. Examination of the preferred binding conformation of clinically effective, pan-JAK inhibitor 1 led to identification of a novel, tricyclic hinge binding scaffold 3. Exploration of SAR through a series of cycloamino and cycloalkylamino analogues demonstrated this template to be highly tolerant of substitution, with a predisposition to moderate selectivity for the JAK1 isoform over JAK2. This study culminated in the identification of subnanomolar JAK1 inhibitors such as 22 and 49, having excellent cell potency, good rat pharmacokinetic characteristics, and excellent kinase selectivity. Determination of the binding modes of the series in JAK1 and JAK2 by X-ray crystallography supported the design of analogues to enhance affinity and selectivity.

Discovery and optimization of C-2 methyl imidazopyrrolopyridines as potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2.

Herein we report the discovery of the C-2 methyl substituted imidazopyrrolopyridine series and its optimization to provide potent and orally bioavailable JAK1 inhibitors with selectivity over JAK2. The C-2 methyl substituted inhibitor 4 exhibited not only improved JAK1 potency relative to unsubstituted compound 3 but also notable JAK1 vs JAK2 selectivity (20-fold and >33-fold in biochemical and cell-based assays, respectively). Features of the X-ray structures of 4 in complex with both JAK1 and JAK2 are delineated. Efforts to improve the in vitro and in vivo ADME properties of 4 while maintaining JAK1 selectivity are described, culminating in the discovery of a highly optimized and balanced inhibitor (20). Details of the biological characterization of 20 are disclosed including JAK1 vs JAK2 selectivity levels, preclinical in vivo PK profiles, performance in an in vivo JAK1-mediated PK/PD model, and attributes of an X-ray structure in complex with JAK1.

Mono- and dicationic short PEG and methylene dioxyalkylglycerols for use in synthetic gene delivery systems.

A range of monocationic and dicationic dioxyalkylglycerol cytofectins have been synthesised possessing methylene and short n-ethylene glycol spacers. The monocationic compounds were found to be effective in transfections when formulated as lipopolyplexes with peptide and DNA components, in particular with shorter PEG head groups which may have less effect on peptide targeting in the ternary complex.

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector.

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

Mannose-pepstatin conjugates as targeted inhibitors of antigen processing.

The molecular details of antigen processing, including the identity of the enzymes involved, their intracellular location and their substrate specificity, are still incompletely understood. Selective inhibition of proteolytic antigen processing enzymes such as cathepsins D and E, using small molecular inhibitors such as pepstatin, has proven to be a valuable tool in investigating these pathways. However, pepstatin is poorly soluble in water and has limited access to the antigen processing compartment in antigen presenting cells. We have synthesised mannose-pepstatin conjugates, and neomannosylated BSA-pepstatin conjugates, as tools for the in vivo study of the antigen processing pathway. Conjugation to mannose and to neomannosylated BSA substantially improved the solubility of the conjugates relative to pepstatin. The mannose-pepstatin conjugates showed no reduction in inhibition of cathepsin E, whereas the neomannosylated BSA-pepstatin conjugates showed some loss of inhibition, probably due to steric factors. However, a neomannosylated BSA-pepstatin conjugate incorporating a cleavable disulfide linkage between the pepstatin and the BSA showed the best uptake to dendritic cells and the best inhibition of antigen processing.

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo.

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.

Asymmetric synthesis of dialkyloxy-3-alkylammonium cationic lipids.

The cationic diether-linked cytofectin DOTMA (available commercially as a mixture, Lipofectin comprised of DOTMA:DOPE, 1:1) and analogues including DIMRIE and DORIE are frequently used for in vitro and in vivo transfections. Despite this wide usage direct synthetic routes to the optical isomers have received little attention to date. Here we describe strategies to synthesize enantiomers of DOTMA and analogues, including an extremely concise procedure to the trimethylammonium salts. One strategy utilized N-protection, as the imine, with concomitant ether formation and deprotection during the workup. Methylation of the 1-amino-2,3-dialkyloxypropane then generated the trimethylammonium cationic lipids directly. This methodology was extended to synthesize a novel headgroup functionalized lipid. A second route was also developed using an alternative chiral synthon.