# Carbazole Derivatives as STAT Inhibitors: An Overview
## 3 STATs Inhibitors
### Carbazoles as STAT Inhibitors
Although prior research substantiated that [31; 32] some carbazoles affected the STAT3-mediated transcription and/or STAT3-DNA binding and phospho-STAT3 at high doses (30-50 \(\upmu\)M), little evidence regards the molecular mechanisms involved in this process. [PERSON] et al. [107] have been previously fascinated by the possibility of targeting STAT3 pathways. They, indeed, tried to identify therapeutic agents against cancer. They focused on the development of fluorescent small molecules. The silver lining of these compounds was the capability to be observed directly as it interacts with receptor-positive cell lines [108; 109; 110]. However, a preceding survey assessed that the antiproliferative activity of the free carbazole on the cancer cell lines is lower than the corresponding scaffolds with the dansyl group. In view of the above, this research group synthesized a small series of carbazole derived compounds with fluorophore (Figure 10). Among them, compound **15** was the most effective compound. Figure 10: General structure of carbazoles fluorophore and structure of 7-hydroxy-1-methyl-9H-carbazol-2-yl 5- (dimethylamino)-naphthalene-1-sulfonate (**15**). Figure 9: Structure of: hydroxyisophthalate derivatives (**13**) and methyl salicate derivatives (**14**). It inhibited the STAT3-mediated transcription and IL-6-induced phosphorylation of STAT3 in triple negative breast cancer (TNBC) cells. Indeed, they examined the antiproliferative effects of **15** on a panel of breast cancer cells, finding the best results for the invasive ductal carcinoma cells SUM1315 MO2. Moreover, **15** suppressed cell proliferation in A431 (a squamous carcinoma cell line), A549 (a lung cancer cell line) and PC-3 (a prostate cancer cell line) with GI50 values 0.16 uM (for A431), 2.5 uM (for A549), and 3 and 7.9 uM (for PC-3), respectively. According to the inhibition of STAT-3, the cell treatment with compound **15** provoked a reduction of cyclin D1 levels, transcriptional target of STAT3. Besides, in vitro and in vivo studies demonstrated that the inhibitory effects of **15** on phospho-STAT3 were via the up-regulation of cytoplasmic protein\(-\)tyrosine phosphatase PTPN6. This was measured on the two lines of invasive ductal carcinoma HS578T, and SUM149 PT. Further analysis assessed a cytoplasmic fluorescence of compound **12** in MDA-MB-231 cells, evidencing that the compound enters human breast cancer cells. Compound **15**, in fact, triggered apoptosis in breast cancer cell lines in vitro and it was efficient at inhibiting the in vivo growth of human TNBC xenograft tumors (SUM149) with a 57% reduction of tumor volume on the fifth day without showing any toxicity. Furthermore, compound **12** also suppressed the growth of human lung tumor xenografts (A549) harboring aberrantly active STAT3. The maximum inhibitory effect was achieved on the ninth day (inhibition rate 73.76%) [107]. Subsequently, in 2017, the same research group focused their attention on this compound deepening the knowledge of the molecular mechanism [111]. Indeed, as just reported, in their preceding work they discovered that **15** was able to inhibit phospho-STAT3 (Y705) by induction of PTPN6/SHP-1 expression. However, previous studies underlined that PTPN6/SHP-1 is epigenetically repressed by STAT3- DNMT1 [112] and acetylation of STAT3 (K685) is critical to bind DNA (cytosine-5)-methyltransferasease 1 (DNMT1). They found that **15** not only decreased the phospho-STAT3 (Y705) but also the acetyl-STAT3 (K685) levels in HS578T cells, both in a dose-dependent manner. By using Western blot experiments, they highlighted that STAT3 binds to DNMT1 in HS578T cells. However, **15** inhibited the capability of forming this interaction at 0.3 uM. The disruption of STAT3-DNMT1 interaction by **15**, instead, happened at 0.1 uM. This effect was achieved also in SUM1315 MO2 cells. In contrast, the reference molecules used, the JAK2 inhibitor (AZD1480) or JAK1/2 inhibitor (CP690550) did not impact STAT3-DNMT1 interaction.