Gary E. Aspnes

Discovery of (S)-6-(3-Cyclopentyl-2-(4-(trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic Acid as a Hepatoselective Glucokinase Activator Clinical Candidate for Treating Type 2 Diabetes Mellitus

Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk. Herein, we report structure-activity studies on a carboxylic acid containing series of glucokinase activators with preferential activity in hepatocytes versus pancreatic β-cells. These activators were designed to have low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.

Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.

Trifluoromethylpyrimidine-based inhibitors of proline-rich tyrosine kinase 2 (PYK2): structure-activity relationships and strategies for the elimination of reactive metabolite formation.

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.

Designing glucokinase activators with reduced hypoglycemia risk: discovery of N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)benzofuran-4-yloxy)pyrimidine-2-carboxamide as a clinical candidate for the treatment of type 2 diabetes mellitus

Glucokinase is a key regulator of glucose homeostasis and small molecule activators of this enzyme represent a promising opportunity for the treatment of Type 2 diabetes. Several glucokinase activators have advanced to clinical studies and demonstrated promising efficacy; however, many of these early candidates also revealed hypoglycemia as a key risk. In an effort to mitigate this hypoglycemia risk while maintaining the promising efficacy of this mechanism, we have investigated a series of substituted 2-methylbenzofurans as “partial activators” of the glucokinase enzyme leading to the identification of N,N-dimethyl-5-(2-methyl-6-((5-methylpyrazin-2-yl)-carbamoyl)benzofuran-4-yloxy)pyrimidine-2-carboxamide as an early development candidate.

A potentiator of orthosteric ligand activity at GLP-1R acts via covalent modification

We report that 4-(3-(benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), which behaves as a positive allosteric modulator at the glucagon-like peptide-1 receptor (GLP-1R), covalently modifies cysteines 347 and 438 in GLP-1R. C347, located in intracellular loop 3 of GLP-1R, is critical to the activity of BETP and a structurally distinct GLP-1R ago-allosteric modulator, N-(tert-butyl)-6,7-dichloro-3-(methylsulfonyl)quinoxalin-2-amine. We further show that substitution of cysteine for phenylalanine 345 in the glucagon receptor is sufficient to confer sensitivity to BETP.

Regioselective Synthesis of 2H-Indazoles Using a Mild, One-Pot Condensation–Cadogan Reductive Cyclization

An operationally simple and efficient one-pot synthesis of 2H-indazoles from commercially available reagents is reported. Ortho-imino-nitrobenzene substrates, generated via condensation, undergo reductive cyclization promoted by tri-n-butylphosophine to afford substituted 2H-indazoles under mild reaction conditions. A variety of electronically diverse ortho-nitrobenzaldehydes and anilines were examined. To further extend the scope of the transformation, aliphatic amines were also employed to form N2-alkyl indazoles selectively under the optimized reaction conditions.

The design and synthesis of a potent glucagon receptor antagonist with favorable physicochemical and pharmacokinetic properties as a candidate for the treatment of type 2 diabetes mellitus.

A novel and potent small molecule glucagon receptor antagonist for the treatment of diabetes mellitus is reported. This candidate, (S)-3-[4-(1-{3,5-dimethyl-4-[4-(trifluoromethyl)-1H-pyrazol-1-yl]phenoxy}butyl)benzamido]propanoic acid, has lower molecular weight and lipophilicity than historical glucagon receptor antagonists, resulting in excellent selectivity in broad-panel screening, lower cytotoxicity, and excellent overall in vivo safety in early pre-clinical testing. Additionally, it displays low in vivo clearance and excellent oral bioavailability in both rats and dogs. In a rat glucagon challenge model, it was shown to reduce the glucagon-elicited glucose excursion in a dose-dependent manner and at a concentration consistent with its rat in vitro potency. Its properties make it an excellent candidate for further investigation.

Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a dioxino[2,3-d]pyrimidine core

A novel series of potent DGAT-1 inhibitors was developed originating from the lactam-based clinical candidate PF-04620110. Incorporation of a dioxino[2,3-d]pyrimidine-based core afforded good alignment of pharmacophore features and resulted in improved passive permeability. Development of an efficient, homochiral synthesis of these targets facilitated confirmation of predictions regarding the stereochemical-dependence of DGAT-1 inhibition for this series. Compound 10 was shown to be a potent inhibitor of human DGAT-1 (10 nM) and to suppress triglyceride synthesis at oral doses of <3mg/kg.

Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5)

Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints.

Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2)

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.