Benjamin D. Stevens

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.

Discovery of a Clinical Candidate from the Structurally Unique Dioxa-bicyclo[3.2.1]octane Class of Sodium-Dependent Glucose Cotransporter 2 Inhibitors

Compound 4 (PF-04971729) belongs to a new class of potent and selective sodium-dependent glucose cotransporter 2 inhibitors incorporating a unique dioxa-bicyclo[3.2.1]octane (bridged ketal) ring system. In this paper we present the design, synthesis, preclinical evaluation, and human dose predictions related to 4. This compound demonstrated robust urinary glucose excretion in rats and an excellent preclinical safety profile. It is currently in phase 2 clinical trials and is being evaluated for the treatment of type 2 diabetes.

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.

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 evaluation of a 2-(2,3,6-trifluorophenyl)acetamide derivative as an agonist of the GPR119 receptor.

The design and synthesis of a GPR119 agonist bearing a 2-(2,3,6-trifluorophenyl)acetamide group is described. The design capitalized on the conformational restriction found in N-β-fluoroethylamide derivatives to help maintain good levels of potency while driving down both lipophilicity and oxidative metabolism in human liver microsomes. The chemical stability and bioactivation potential are discussed.

Optimization of triazole-based TGR5 agonists towards orally available agents

Reported herein is a medicinal chemistry effort towards the identification of orally available TGR5 agonist 12, which served as a dog tool compound for studies to increase confidence in this mechanism. With the challenge of striking the balance of TGR5 potency and desired clearance profile, the screening strategy as well as medicinal chemistry strategy are discussed in this article.

A novel series of glucagon receptor antagonists with reduced molecular weight and lipophilicity.

A novel series of glucagon receptor antagonists has been discovered. These pyrazole ethers and aminopyrazoles have lower molecular weight and increased polarity such that the molecules fall into better drug-like property space. This work has culminated in compounds 44 and 50 that were shown to have good pharmacokinetic attributes in dog, in contrast to rats, in which clearance was high; and compound 49, which demonstrated a dose-dependent reduction in glucose excursion in a rat glucagon challenge experiment.

In Vitro Metabolism of the Glucagon-Like Peptide-1 (GLP-1)-Derived Metabolites GLP-1(9-36)amide and GLP-1(28-36)amide in Mouse and Human Hepatocytes

Previous studies have revealed that the glucoincretin hormone glucagon-like peptide-1 (GLP-1)(7-36)amide is metabolized by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase 24.11 (NEP) to yield GLP-1(9-36)amide and GLP-1(28-36)amide, respectively, as the principal metabolites. Contrary to the previous notion that GLP-1(7-36)amide metabolites are pharmacologically inactive, recent studies have demonstrated cardioprotective and insulinomimetic effects with both GLP-1(9-36)amide and GLP-1(28-36)amide in animals and humans. In the present work, we examined the metabolic stability of the two GLP-1(7-36)amide metabolites in cryopreserved hepatocytes, which have been used to demonstrate the in vitro insulin-like effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis. To examine the metabolic stability of the GLP-1(7-36)amide metabolites, a liquid chromatography–tandem mass spectrometry assay was developed for the quantitation of the intact peptides in hepatocyte incubations. GLP-1(9-36)amide and GLP-1(28-36)amide were rapidly metabolized in mouse [GLP-1(9-36)amide: t1/2 = 52 minutes; GLP-1(28-36)amide: t1/2 = 13 minutes] and human hepatocytes [GLP-1(9-36)amide: t1/2 = 180 minutes; GLP-1(28-36)amide: t1/2 = 24 minutes), yielding a variety of N-terminal cleavage products that were characterized using mass spectrometry. Metabolism at the C terminus was not observed for either peptides. The DPP-IV and NEP inhibitors diprotin A and phosphoramidon, respectively, did not induce resistance in the two peptides toward proteolytic cleavage. Overall, our in vitro findings raise the intriguing possibility that the insulinomimetic effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis and oxidative stress might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from the enzymatic cleavage of the peptide backbone in the parent compounds.

Demonstration of the Innate Electrophilicity of 4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a Small-Molecule Positive Allosteric Modulator of the Glucagon-Like Peptide-1 Receptor

4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.

Intrinsic electrophilicity of the 4-methylsulfonyl-2-pyridone scaffold in glucokinase activators: Role of glutathione-S-transferases and in vivo quantitation of a glutathione conjugate in rats

Previous studies on the in vitro metabolism of 4-alkylsulfonyl-2-pyridone-based glucokinase activators revealed a facile, non-enzymatic displacement of the 4-alkylsulfonyl group by glutathione. In the present studies, a role for glutathione-S-transferases (GST) as catalysts in the desulfonylation reaction was demonstrated using a combination of human liver microsomes, human liver cytosol and human GSTs. The identification of a glutathione conjugate in circulation following intravenous administration of a candidate 4-methylsulfonyl-2-pyridone to rats confirmed the relevance of the in vitro findings.