6-Azaspiro[2.5]octanes as small molecule agonists of the human glucagon-like peptide-1 receptor.

Activation of the glucagon-like peptide-1 (GLP-1) receptor stimulates insulin release, lowers plasma glucose levels, delays gastric emptying, increases satiety, suppresses food intake, and affords weight loss in humans. These beneficial attributes have made peptide-based agonists valuable tools for the treatment of type 2 diabetes mellitus and obesity. However, efficient, and consistent delivery of peptide agents generally requires subcutaneous injection, which can reduce patient utilization. Traditional orally absorbed small molecules for this target may offer improved patient compliance as well as the opportunity for co-formulation with other oral therapeutics. Herein, we describe an SAR investigation leading to small-molecule GLP-1 receptor agonists that represent a series that parallels the recently reported clinical candidate danuglipron. In the event, identification of a benzyloxypyrimidine lead, using a sensitized high-throughput GLP-1 agonist assay, was followed by optimization of the SAR using substituent modifications analogous to those discovered in the danuglipron series. A new series of 6-azaspiro[2.5]octane molecules was optimized into potent GLP-1 agonists. Information gleaned from cryogenic electron microscope structures was used to rationalize the SAR of the optimized compounds.

Small molecule inhibitors of PCSK9. SAR investigations of head and amine groups.

Our previous work on the optimization of a new class of small molecule PCSK9 mRNA translation inhibitors focused on empirical optimization of the amide tail region of the lead PF-06446846 (1). This work resulted in compound 3 that showed an improved safety profile. We hypothesized that this improvement was related to diminished binding of 3 to non-translating ribosomes and an apparent improvement in transcript selectivity. Herein, we describe our efforts to further optimize this series of inhibitors through modulation of the heterocyclic head group and the amine fragment. Some of the effort was guided by an emerging cryo electron microscopy structure of the binding mode of 1 in the ribosome. These efforts led to the identification of 15 that was deemed suitable for evaluation in a humanized PCSK9 mouse model and a rat toxicology study. Compound 15 demonstrated a dose dependent reduction of plasma PCSK9 levels. The rat toxicological profile was not improved over that of 1, which precluded 15 from further consideration as a clinical candidate.

Discovery of a Novel Potent and Selective HSD17B13 Inhibitor, BI-3231, a Well-Characterized Chemical Probe Available for Open Science.

Genome-wide association studies in patients revealed HSD17B13 as a potential new target for the treatment of nonalcoholic steatohepatitis (NASH) and other liver diseases. However, the physiological function and the disease-relevant substrate of HSD17B13 remain unknown. In addition, no suitable chemical probe for HSD17B13 has been published yet. Herein, we report the identification of the novel potent and selective HSD17B13 inhibitor BI-3231. Through high-throughput screening (HTS), using estradiol as substrate, compound 1 was identified and selected for subsequent optimization resulting in compound 45 (BI-3231). In addition to the characterization of compound 45 for its functional, physicochemical, and drug metabolism and pharmacokinetic (DMPK) properties, NAD+ dependency was investigated. To support Open Science, the chemical HSD17B13 probe BI-3231 will be available to the scientific community for free via the opnMe platform, and thus can help to elucidate the pharmacology of HSD17B13.

Discovery and evaluation of non-basic small molecule modulators of the atypical chemokine receptor CXCR7.

The atypical chemokine receptor C-X-C chemokine receptor type 7 (CXCR7) is an attractive therapeutic target for a variety of cardiac and immunological diseases. As a strategy to mitigate known risks associated with the development of higher molecular weight, basic compounds, a series of pyrrolidinyl-azolopyrazines were identified as promising small-molecule CXCR7 modulators. Using a highly enabled parallel medicinal chemistry strategy, structure-activity relationship studies geared towards a reduction in lipophilicity and incorporation of saturated heterocycles led to the identification of representative tool compound 20. Notably, compound 20 maintained good potency against CXCR7 with a suitable balance of physicochemical properties to support in vivo pharmacokinetic studies.

Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5'-Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

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.

Pyrimidone-based series of glucokinase activators with alternative donor-acceptor motif.

Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor-acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor-acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor-acceptor motifs may find utility in other glucokinase activator series or beyond.

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.

Identification of novel series of pyrazole and indole-urea based DFG-out PYK2 inhibitors.

Previous drug discovery efforts identified classical PYK2 kinase inhibitors such as 2 and 3 that possess selectivity for PYK2 over its intra-family isoform FAK. Efforts to identify more kinome-selective chemical matter that stabilize a DFG-out conformation of the enzyme are described herein. Two sub-series of PYK2 inhibitors, an indole carboxamide-urea and a pyrazole-urea have been identified and found to have different binding interactions with the hinge region of PYK2. These leads proved to be more selective than the original classical inhibitors.

The design and synthesis of indazole and pyrazolopyridine based glucokinase activators for the treatment of type 2 diabetes mellitus.

Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells.

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.

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.

Differences in CYP3A4 catalyzed bioactivation of 5-aminooxindole and 5-aminobenzsultam scaffolds in proline-rich tyrosine kinase 2 (PYK2) inhibitors: retrospective analysis by CYP3A4 molecular docking, quantum chemical calculations and glutathione adduct detection using linear ion trap/orbitrap mass spectrometry.

Previous studies have demonstrated the CYP3A4 mediated oxidation of the 5-aminooxindole motif, present in the trifluoromethylpyrimidine class of PYK-2 inhibitors, to a reactive bis-imine species, which can be trapped with glutathione (GSH) in human liver microsomal incubations. The corresponding 5-aminobenzsultam derivatives, which should possess a similar oxidative liability, do not form GSH conjugates in microsomal incubations. In the current study, we conducted a retrospective analysis on representative 5-aminooxindole and 5-aminobenzsultam PYK-2 inhibitors utilizing CYP3A4 molecular docking and quantum chemical calculations to rationalize the bioactivation differences. Our analysis revealed key differences in (a) active site binding and (b) two-electron oxidation rates, which correlate with GSH adduct formation with the two moieties. The value of linear ion/orbitrap mass spectrometry to detect GSH conjugates with greater sensitivity, compared with conventional triple quadrupole mass spectrometry approaches, was also demonstrated in the course of these studies.

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.