STAT3 offers an attractive target for cancer therapy but small molecule inhibitors with appealing pharmacologic properties have been elusive. Here we report hydroxamic acid-based and benzoic acid-based inhibitors (SH5-07 and SH4-54, respectively) with robust bioactivity. Both inhibitors blocked STAT3 DNA binding activity in vitro and in human glioma, breast, and prostate cancer cells and in v-Src-transformed murine fibroblasts. STAT3-dependent gene transcription was blocked along with Bcl-2, Bcl-xL, Mcl-1, Cyclin D1, c-Myc and Survivin expression. Nuclear magnetic resonance analysis of STAT3-inhibitor complexes defined interactions with the SH2 and DNA binding domains of STAT3. Ectopic expression of the SH2 domain in cells was sufficient to counter the STAT3 inhibitory effects of SH4-54. Neither compound appreciably affected STAT1 or STAT5 DNA binding activities, STAT3-independent gene transcription or activation of a panel of oncogenic kinases in malignant cells. Each compound decreased the proliferation and viability of glioma, breast and prostate cancer cells and v-Src-transformed murine fibroblasts harboring constitutively active STAT3. Further, in mouse xenograft models of glioma and breast cancer, administration of SH5-07 or SH4-54 effectively inhibited tumor growth. Our results offer preclinical proof of concept for SH5-07 and SH4-54 as candidates fof further development as cancer therapeutics.
The molecular determinants for the activities of the reported benzoic acid (SH4-54), salicylic acid (BP-1-102), and benzohydroxamic acid (SH5-07)-based STAT3 inhibitors were investigated to design optimized analogues. All three leads are based on an -methylglycinamide scaffold, with its two amine groups condensed with three different functionalities. The three functionalities and the CH group of the glycinamide scaffold were separately modified. The replacement of the pentafluorobenzene or cyclohexylbenzene, or replacing the benzene ring of the aromatic carboxylic or hydroxamic acid motif with heterocyclic components (containing nitrogen and oxygen elements) all decreased potency. Notably, the Ala-linker analogues, and, and the Pro-based derivative , all with ()-configuration at the chiral center, had improved inhibitory activity and selectivity against STAT3 DNA-binding activity , with IC of 3.0 ± 0.9, 1.80 ± 0.94, and 2.4 ± 0.2 μM, respectively. Compounds ,, , and other analogues inhibited constitutive STAT3 phosphorylation and activation in human breast cancer and melanoma lines, and blocked tumor cell viability, growth, colony formation, and migration. Pro-based analogue, , with a relatively polar tetrahydropyranyl (THP) ring, instead of the cyclohexyl, showed improved permeability. In general, the ()-configuration Pro-based analogs showed the overall best profile, including physicochemical properties (e.g., microsomal metabolic stability, Caco-2 permeability), and in particular, showed improved tumor-cell specificity.
We optimized our previously reported proline-based STAT3 inhibitors into an exciting new series of ( R )-azetidine-2-carboxamide analogues that have sub-micromolar potencies. 5a , 5o , and 8i have STAT3-inhibitory potencies (IC 50 ) of 0.55, 0.38, and 0.34 μM, respectively, compared to potencies greater than 18 μM against STAT1 or STAT5 activity. Further modifications derived analogues, including 7e , 7f , 7g , and 9k , that addressed cell membrane permeability and other physicochemical issues. Isothermal titration calorimetry analysis confirmed high-affinity binding to STAT3, with K D of 880 nM ( 7g ) and 960 nM ( 9k ). 7g and 9k inhibited constitutive STAT3 phosphorylation and DNA-binding activity in human breast cancer, MDA-MB-231 or MDA-MB-468 cells. Furthermore, treatment of breast cancer cells with 7e , 7f , 7g , or 9k inhibited viable cells, with an EC 50 of 0.9–1.9 μM, cell growth, and colony survival, and induced apoptosis while having relatively weaker effects on normal breast epithelial, MCF-10A or breast cancer, MCF-7 cells that do not harbor constitutively active STAT3.
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