Through the application of TRAP® (Target-Related Affinity Profiling), we identified a novel class of heteroaroylphenylureas that inhibit human CCL2-induced chemotaxis of monocytes/macrophages both in vitro and in vivo. This inhibition was concentration-dependent and selective with regards to other chemokines. The compounds, however, did not antagonize the binding of 125I-labeled CCL2 to the CCR2 receptor, nor did they block CCR2-mediated signal transduction responses such as calcium mobilization. Optimization of early leads for potency and pharmacokinetic parameters resulted in the identification of 17, a potent inhibitor of chemotaxis (IC50 = 80 nM) with excellent oral bioavailability in rats (F = 60%). Compound 17 reduced swelling and joint destruction in two rat models of rheumatoid arthritis and delayed disease onset and produced near complete resolution of symptoms in a mouse model of multiple sclerosis.

Protease inhibitor (PI) therapy for the treatment of patients infected with human immunodeficiency virus is frequently associated with insulin resistance and diabetic complications. These adverse effects of PI treatment result to a large extent from their inhibition of insulin-stimulated glucose transport. Insulin receptor (IR) activators that enhance the insulin signaling pathway could be effective in treating this resistance. However, there are no agents reported that reverse inhibition of insulin action by PIs. Herein, we describe the effects of TLK19781. This compound is a non-peptide, small molecule, activator of the IR. We now report in cultured cells, made insulin resistant HIV by PI treatment, that TLK19781 both increased the content of insulin-stimulated GLUT4 at the plasma membrane, and enhanced insulin-stimulated glucose transport. In addition, oral administration of TLK19781 with the PI, indinavir improved glucose tolerance in rats made insulin resistant. These results suggest, therefore, that IR activators such as TLK19781 may be useful in treating the insulin resistance associated with PIs.

A novel series of symmetrical ureas of [(7-amino(2-naphthyl))sulfonyl]phenylamines were designed, synthesized, and tested for their ability to increase glucose transport in mouse 3T3-L1 adipocytes, a surrogate readout for activation of the insulin receptor (IR) tyrosine kinase (IRTK). A structure-activity relationship was established that indicated glucose transport activity was dependent on the presence of two acidic functionalities, two sulfonamide linkages, and a central urea or 2-imidazolidinone core. Compound 30 was identified as a potent and selective IRTK activator. At low concentrations, 30 was able to increase the tyrosine phosphorylation of the IR stimulated by submaximal insulin. At higher concentrations, 30 was able to increase tyrosine the phosphorylation levels of the IR in the absence of insulin. When administered intraperitoneally (ip) and orally (po), 30 improved glucose tolerance in hypoinsulinemic, streptozotocin-treated rats. These data provide pharmacological validation that small molecule IRTK activators represent a potential new class of antidiabetic agents.

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Ezatiostat hydrochloride (TLK199, Telintra®) is the first glutathione S-transferase P1-1 (GST P1-1) inhibitor prodrug to demonstrate a clinically significant improvement in the cytopenias affecting patients with myelodysplastic syndrome (MDS), thereby validating this enzyme as a candidate target for pharmacologic intervention (Blood 2009; 113(26):6533-6540). Structurally, ezatiostat is a tripeptide glutathione analog diethyl ester that is metabolized in vivo to the diacid, which is a potent and selective inhibitor of GST P1-1, an enzyme overexpressed in many human hematologic cancers. This inhibition induces a dissociation of GST P1-1 from its complex with c-jun N-terminal kinase (JNK)/c-Jun and activates signaling pathways that lead to cell proliferation and differentiation of normal hematopoietic cells and apoptosis of malignant cells. To improve the inhibitory potency and selectivity for the target enzyme GST P1-1, a series of diacid analogs of ezatiostat bearing different substituents on the cysteinyl sulfur were synthesized and evaluated in several biological assays. Replacement of the benzyl group by a 2- or 4-biphenylmethyl or a 1-naphthylethyl substituent resulted in a 30- to 130-fold improvement in IC50 against GST P1-1 and 3- to 10-fold higher selectivity relative to the GST A1-1 and GST M1-1 isoforms. The most active analogs were esterified and tested in cell-based and in vivo assays. The esters induced growth arrest and cellular apoptosis in human leukemia cells (HL-60) with lower or similar CC50 values as ezatiostat (6-17 μM range). In an HL-60 differentiation assay measuring induction of cell surface CD11b expression, the compounds increased CD11b expression by approximately 2-fold. Similarly to ezatiostat, the compounds also increased the production of reactive oxygen species in HL-60 and 293T cells by 2- to 3-fold after a 2 hour exposure, as measured by the generation of a fluorescent signal from a redox-sensitive dye. Select ezatiostat analogs were evaluated in rodents for toxicity as well as for acceleration of recovery of neutrophil levels in a standard model of 5-FU chemotherapy-induced neutropenia. The compounds were well tolerated when administered at doses up to 200 mg/kg and, as ezatiostat, led to a significant >1 day accelerated recovery in the 5-FU-induced neutropenia model in rats compared to growth factor treatment. Substitution at the cysteinyl sulfur was established as the most critical position in the structure-activity relationship analysis and is consistent with the unique mechanism of action of these tripeptide inhibitors. Further investigation of the most potent and selective analogs in biochemical and cellular assays is underway. The availability of more selective and potent GST P1-1 inhibitors may help elucidate the role of GST P1-1 in the development and progression of MDS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3246. doi:10.1158/1538-7445.AM2011-3246