Jeffrey G. Varnes

Discovery of novel positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5)

Novel in vitro mGlu(5) positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC(50) 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC(50) 156.3 nM). Derivation of a second-generation of mGlu(5) PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC(50) 50.1 nM) as a potent and soluble mGlu(5) PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.

Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo, and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC(50)=551 nM), is 20-fold more potent against rat FAAH (IC(50)=27 nM), and is inactive at 10 μM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC(50)=360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB(1) activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.

Identification and optimisation of 7-azaindole PAK1 inhibitors with improved potency and kinase selectivity

A novel series of PAK1 inhibitors was discovered from a kinase directed screen. SAR exploration in the selectivity pocket and solvent tail regions was conducted to understand and optimise PAK1 potency and selectivity against targeted kinases. A liganded PAK1 crystal structure was utilised to guide compound design. Permeability and kinase selectivity impacted the translation of enzyme to cellular PAK1 potency. Compound 36 (AZ-PAK-36) demonstrated improved Gini coefficient, good PAK1 cellular potency and has utility as a tool compound for target validation studies.

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors.

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-xL in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the RS versus SS sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-xL.

Identification of N-(2-(azepan-1-yl)-2-phenylethyl)-benzenesulfonamides as novel inhibitors of GlyT1

A novel series of glycine transporter 1 (GlyT1) inhibitors is described. Scoping of the heterocycle moiety of hit 4-chlorobenzenesulfonamide 1 led to replacement of the piperidine with an azepane for a modest increase in potency. Phenyl sulfonamides proved superior to alkyl and non-phenyl aromatic sulfonamides, while subsequent ortho substitution of the 2-(azepan-1-yl)-2-phenylethanamine aromatic ring yielded 39 (IC(50) 37 nM, solubility 14 microM), the most potent GlyT1 inhibitor in this series. Favorable brain-plasma ratios were observed for select compounds in pharmacokinetic studies to evaluate CNS penetration.

Structure-Based Design of Selective Noncovalent CDK12 Inhibitors

Cyclin‐dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA‐damage‐response genes and sensitizes BRCA wild‐type cells to poly(ADP‐ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR‐3029, resulting in lead compound 5 [(S)‐2‐(1‐(6‐(((6,7‐difluoro‐1H‐benzo[d]imidazol‐2‐yl)methyl)amino)‐9‐ethyl‐9H‐purin‐2‐yl)piperidin‐2‐yl)ethan‐1‐ol]. Further structure‐guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)‐2‐(1‐(6‐(((6,7‐difluoro‐1H‐benzo[d]imidazol‐2‐yl)methyl)amino)‐9‐isopropyl‐9H‐purin‐2‐yl)piperidin‐2‐yl)ethan‐1‐ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single‐point kinase panel screening against 352 targets at 0.1 μm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C‐terminal repeat domain of RNA polymerase II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells.

Fragment-assisted hit investigation involving integrated HTS and fragment screening: Application to the identification of phosphodiesterase 10A (PDE10A) inhibitors

Fragment-based drug design (FBDD) relies on direct elaboration of fragment hits and typically requires high resolution structural information to guide optimization. In fragment-assisted drug discovery (FADD), fragments provide information to guide selection and design but do not serve as starting points for elaboration. We describe FADD and high-throughput screening (HTS) campaign strategies conducted in parallel against PDE10A where fragment hit co-crystallography was not available. The fragment screen led to prioritized fragment hits (IC50s ∼500μM), which were used to generate a hypothetical core scaffold. Application of this scaffold as a filter to HTS output afforded a 4μM hit, which, after preparation of a small number of analogs, was elaborated into a 16nM lead. This approach highlights the strength of FADD, as fragment methods were applied despite the absence of co-crystallographical information to efficiently identify a lead compound for further optimization.

Bicyclo((aryl)methyl)benzamides as inhibitors of GlyT1.

A series of isoquinuclidine benzamides as glycine uptake inhibitors for the treatment of schizophrenia are described. Potency, lipophilicity, and intrinsic human microsomal clearance were parameters for optimization. Potency correlated with the nature of the ortho substituents of the benzamide ring, and reductions in lipophilicity could be achieved through heteroatom incorporation in the benzamide and pendant phenyl moieties. Improvements in human CLint were achieved through changes in ring size and the N-alkyl group of the isoquinuclidine itself, with des-alkyl derivatives (40-41, 44) demonstrating the most robust microsomal stability. Dimethylbenzamide 9 was tested in a mouse MK801 LMA assay and had a statistically significant attenuation of locomotor activity at 3 and 10 μmol/kg compared to control.