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  • A previous report showed that the transfer


    A previous report showed that the transfer of the CSF1 gene into tumor Z-YVAD-FMK induced TAM infiltration into syngeneic BALB/c mice. High expressions of CSF-1 and CSF-1R in tumor and stromal cells and a high TAM density are known to be correlated with poor prognoses in many cancer types. CSF-1 promotes metastates via the regulation of TAMs in the tumor microenvironment. In human metastatic breast cancer, elevated CSF-1 levels were correlated with marked CSF-1R-positive macrophage infiltration into tumors and were associated with poor outcomes. Another study also showed that macrophage-deficient/CSF-1-deficient mice rarely developed pulmonary metastases, despite the rapid growth of primary mammary tumors. Furthermore, some chemotherapies stimulate tumor cells to release CSF-1, which then recruits TAMs expressing CSF-1R. These TAMs in the microenvironment stimulate tumor progression by enhancing tumor resistance to chemotherapy. In combination therapy with paclitaxel and CSF-1R antagonists on primary and metastatic tumors of mammary tumor-bearing mice, inhibition of TAM infiltration via CSF-1R antagonists increased the anti-tumor efficacy of cytotoxic agents. For these reasons, modulation of the CSF-1 signaling pathway in the microenvironment has been of great interest in chemotherapy research. In this paper, Z-YVAD-FMK we describe the identification of the potent FMS inhibitor compound and evaluate its anti-tumor activities against breast cancer. By computational screening compounds from the internal small molecule compound bank of Hanmi Pharmaceutical, we identified 1-indazol-4-yl)thieno[3,2-]pyrimidine derivative () as a highly potent (IC = 13 nM) FMS inhibitor. To improve its enzyme activity and cellular activities on NFS-60 cells that express endogenous FMS,, structure-activity relationship (SAR) studies probed substituent effects at the N-1 position (R) and C-3 position of the indazole moiety (R). Initially, analogues were synthesized with substitutions at R (). The replacement of the phenyl moiety with methoxy phenyl moiety () led to an almost two-fold loss of enzyme and cellular activity. In contrast, the replacement of the phenyl moiety with a pyridine moiety (–) showed good enzyme activity (IC < 10 nM). To further improve enzyme and cellular activity, we added an adopted substituent to the pyridine moiety. Halogen substituents (–) in the pyridine moiety had similar enzyme activities compared to regular pyridine moieties (–), and cellular activity did not dramatically improve. In contrast, alkyl substituents (–) on the pyridine moiety also showed improved cellular activity. In particular, a methyl substituent () on the pyridine moiety showed good cellular activity (GI = 27.5 nM) in an NFS-60 cell line whose growth depends on FMS., Next, we tried to attach an alicyclic, heterocyclic, or alkynyl substituent (–) to the indazole moiety. Although all the resultant compounds showed good enzyme activities, they did not show sufficiently strong cellular activity. The synthetic route of R-changed derivatives is shown in . Compounds – were prepared from commercially available 4-nitro-1-indazole (). Bromination of was performed using bromine gas, and gave an excellent yield (94%). The 1-substituted indazole of was easily derived from an SN2 reaction using an alkyl-halide. Reduction of was performed using Pd/C, and the amide coupling of and using commercially available HATU. Each step was isolated via column chromatography. Compound was the most potent compound of the R changed-derivatives, based on cellular activity. This compound also showed good oral bioavailability (37%) (). Next, we fixed R as 2-methylpyridine and replaced R with the C-3 position of an indazole moiety (compound Br). We then tried halogen (–) and alkyl (–) substituents at R (). Compound with a halogen moiety showed more potent cellular activity (3.4 fold) than compound , and compound with an alkyl moiety had the most potent enzyme activity (IC = 2 nM). Compound showed more stable liver metabolism than compound in microsomal stability test using human hepatocytes. Remaining value of parent molecule at 60 min of compound and were 87% and 66%, respectively.