Discovery of Lanraplenib (GS-9876): A Once-Daily Spleen Tyrosine Kinase Inhibitor for Autoimmune Diseases.

Spleen tyrosine kinase (SYK) is a critical regulator of signaling in a variety of immune cell types such as B-cells, monocytes, and macrophages. Accordingly, there have been numerous efforts to identify compounds that selectively inhibit SYK as a means to treat autoimmune and inflammatory diseases. We previously disclosed GS-9973 (entospletinib) as a selective SYK inhibitor that is under clinical evaluation in hematological malignancies. However, a BID dosing regimen and drug interaction with proton pump inhibitors (PPI) prevented development of entospletinib in inflammatory diseases. Herein, we report the discovery of a second-generation SYK inhibitor, GS-9876 (lanraplenib), which has human pharmacokinetic properties suitable for once-daily administration and is devoid of any interactions with PPI. Lanraplenib is currently under clinical evaluation in multiple autoimmune indications.

Discovery of Potent and Selective MTH1 Inhibitors for Oncology: Enabling Rapid Target (In)Validation.

We describe the discovery of three structurally differentiated potent and selective MTH1 inhibitors and their subsequent use to investigate MTH1 as an oncology target, culminating in target (in)validation. Tetrahydronaphthyridine 5 was rapidly identified as a highly potent MTH1 inhibitor (IC50 = 0.043 nM). Cocrystallization of 5 with MTH1 revealed the ligand in a Φ-cis-N-(pyridin-2-yl)acetamide conformation enabling a key intramolecular hydrogen bond and polar interactions with residues Gly34 and Asp120. Modification of literature compound TH287 with O- and N-linked aryl and alkyl aryl substituents led to the discovery of potent pyrimidine-2,4,6-triamine 25 (IC50 = 0.49 nM). Triazolopyridine 32 emerged as a highly selective lead compound with a suitable in vitro profile and desirable pharmacokinetic properties in rat. Elucidation of the DNA damage response, cell viability, and intracellular concentrations of oxo-NTPs (oxidized nucleoside triphosphates) as a function of MTH1 knockdown and/or small molecule inhibition was studied. Based on our findings, we were unable to provide evidence to further pursue MTH1 as an oncology target.

Discovery of Potent and Selective Tricyclic Inhibitors of Bruton's Tyrosine Kinase with Improved Druglike Properties.

In our continued effort to discover and develop best-in-class Bruton's tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.

Discovery of highly potent and selective Bruton's tyrosine kinase inhibitors: Pyridazinone analogs with improved metabolic stability.

BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.

Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834.

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.

Discovery of GS-9973, a selective and orally efficacious inhibitor of spleen tyrosine kinase.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis.

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1ß and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Imidazo[1,2-a]pyrazine diaryl ureas: inhibitors of the receptor tyrosine kinase EphB4.

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent and Nexavar, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.

Approach to the homoerythrina alkaloids using a tandem N-alkylation/azomethine ylide cycloaddition.

Synthetic efforts toward the homoerythrina alkaloids 1-3 are described. Two separate model systems guided the pivotal [3 + 2] azomethine ylide cycloaddition cascade to form the A-C rings of these alkaloids. The cycloaddition precursors 63 and 68, prepared in nine and ten steps, respectively, from alkyne 47, each contain an enolizable ketone, a tethered electrophile, and an electron-poor dipolarophile. Heating 63 and 68 with the stannyl amine 17 generated demethoxyschelhammeridine 65 and demethoxyschelhammericine 70, the products of intramolecular azomethine ylide cycloadditions. Subsequent attempts to install the C-3 methoxy group of 1-3 are also described.

Studies on a total synthesis of the microbial immunosuppresive agent FR901483.

A strategy is outlined for construction of the fungal immunosuppressant FR901483 (1). It was possible to convert 1,4-cyclohexanedione monoethylene ketal in five simple steps to iodoacetamide ketone 10, which was cyclized in good yield to the key bridged keto lactam 11 containing the A/B 2-azabicyclo[3.3.1]nonane ring system of the natural product. This intermediate could be transformed to N-Boc lactam 16, whose derived enolate underwent stereoselective hydroxylation with the Davis oxaziridine to produce alcohol 17 having the desired C-2 configuration. Compound 17 was then converted in three steps to alkoxy carbamate 20. The N-acyliminium ion derived from intermediate 20 could be alkylated in good overall yield with p-methoxybenzylmagnesium chloride to afford a 5:4 mixture of the desired PMB product 21 and the epimer 23. In an attempt to improve the stereoselectivity in this alkylation, the inverted C-4 protected alcohol N-Boc lactam 33 was prepared and its enolate was hydroxylated. Inexplicably, the product of this reaction was the undesired equatorial alcohol 34. Some model systems were investigated toward annulation of the C-ring of the natural product. It was found that homoallylic amine 40 could be cyclized with PhSCl in the presence of silica gel to generate the desired 5-endo tetracyclic product 42 in moderate yield. This cyclization protocol was also successfully applied to the actual FR901483 system 22, leading to the requisite tricycle 43.