Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.

In search of compounds with improved specificity for targeting the important cancer-associated P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported glutathione S-transferase (GST)-activated latent alkylating agent gamma-glutamyl-alpha-amino-beta-[[[2-[[bis[bis(2-chloroethyl)amino]ph osp horyl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (3) have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained its specificity and was found to be more readily activated by GSTs than 3. The GST activation concept was further broadened through design, synthesis, and evaluation of a novel latent urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered to have varying levels of GST P1-1 isozyme. New analogs 4 and 5 exhibited increased toxicity to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The alkylating nature of the decomposition products was further demonstrated by trapping those transient species as relatively stable diethyldithiocarbamic acid adducts. These results substantially extend previous efforts to develop drugs targeting GST and provide a paradigm for development of other latent drugs.

Glutathione-S-transferase (GST) isozyme-selective inhibitors were designed by an empirically guided strategy. In the first phase, literature data were used to select C-terminal modifications which generated maximum variation in the catalytic efficiency (Vmax/Km) for glutathione (GSH) analogs used as substrates with different rat GSTs. Also, on the basis of literature data, the sulfhydryl group was functionalized with a selection of alkyl and aryl groups to maximize potential isozyme specificity. Affinity chromatography sorbents were prepared from these which showed isozyme selectivity for both rat tissue and recombinant human GST isozymes. Some of these compounds also showed selective inhibition of GST activity in catalysis of the reaction of 1-chloro-2,4-dinitrobenzene with GSH. In the second phase, electronic effects were explored through synthesis of an isostructural series of S-benzyl GSH ligands with different substituents on the aromatic ring. GST isozyme specificity for these ligands, measured by binding to derivatized sorbents, varied substantially, with hydrophobic substituents favoring the human GST M1a isozyme and electronegative moieties favoring GST P1. In the third phase, information obtained from testing both series of compounds was combined and used to prepare GSH analogs with chemical features responsible for isozyme specificity at both the C-terminus and the sulfur. This approach gave two new compounds which showed improved potency while still maintaining selectivity in the inhibition of GSTs. A detailed discussion of the logic used in the selection of functional groups for maximum potency and selectivity is included.

No abstract

Tumor-associated macrophages (TAMs) are thought to be regulators of solid tumor development based on their capacity to enhance metastatic, invasive, and angiogenic programming of neoplastic tissue. Colony stimulating factor-1 (CSF-1) is a key cytokine involved in recruitment and activation of tissue macrophages, exerting these effects through binding to a high-affinity receptor tyrosine kinase, the CSF-1 receptor. We have developed a small molecule CSF1R inhibitor AC708 in an attempt to impact the TAM-related progression of human tumors. Here we demonstrate that AC708 possesses significant specificity for CSF1R relative to the rest of the kinome, and to the closely related PDGFR family receptors PDGFRα and β, FLT3, and KIT. In cell based assays, AC708 potently inhibited CSF1R phosphorylation mediated by CSF-1 (IC50 = 26 nM) and by IL-34 (IC50 = 33 nM). It also inhibited the viability of growth-factor dependent cells cultured in CSF-1 (IC50 = 38 nM) or IL-34 (IC50 = 40 nM), and inhibited the CSF-1-mediated differentiation and survival of primary human osteoclast with an IC50 of 15 nM. In cytokine release experiments where enriched human monocytes were stimulated with either CSF-1 or IL-34, AC708 inhibited MCP-1 release with nearly identical IC50 regardless of which cytokine was used (CSF-1 (93 nM), IL-34 (88 nM)), and with a lower IC50 than that obtained with the benchmark compound GW-2580 (CSF-1 (148 nM), IL-34 (140 nM)). In vivo, AC708 was assessed for its ability to inhibit the intraperitoneal growth of M-NFS-60 cells in mice. In these experiments, AC708 inhibited M-NFS-60 growth in a dose-dependent manner, with a greater than 80% reduction in cell number at 100 mg/kg, similar to that achieved with the benchmark compound Ki-20227. Two assays were employed to assess the ability of AC708 to modulate endogenous CSF1R. In the first model, AC708 inhibited CSF-1-mediated MCP-1 release in vivo by 60% when dosed at 100 mg/kg. In the second model, plasma levels of TRAP5b were determined following injection of recombinant Parathyroid hormone-related protein (PTHrP) with or without co-administration of AC708. PTHrP-induced increases of plasma TRAP5b were reduced by AC708 in a dose-dependent manner, with levels falling to below baseline in the 100 mg/kg dose group. Lastly to assess the ability of AC708 to modulate TAMs we utilized the 4T-1 breast cancer line implanted orthotopically. Although primary tumor growth was relatively unchanged by AC708 treatment in this model, administration of drug for two weeks resulted in a dose-dependent reduction of tumor resident macrophages, with a 70% reduction at the 100 mg/kg dose relative to vehicle control. AC708 impact on tumor angiogenesis and metastatic potential is currently under investigation and will be reported. These studies further validate CSF1R as a potential target in cancer, and support the development of AC708 as a therapeutic in oncology. Citation Format: Robert C. Armstrong, Barbara Belli, Martin W. Rowbottom, Ron R. Nepomuceno, Alan Q. Dao, Allison M. Rooks, Dan Brigham, Craig W. McMannus, Michael D. Hocker, Mark W. Holladay, Gang Liu. AC708 is a potent and selective inhibitor of CSF1R and reduces tumor associated macrophage infiltration in a breast tumor model . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 903. doi:10.1158/1538-7445.AM2013-903