Unprecedented Rearrangement of β-Difluoroboryloxy Ethers: A Route to C-2 Alkyl-chromenones

Abstract

The addition of boron trifluoride etherate (BF₃·OEt₂) to allenic ketones has led to the isolation of the isolated boron difluoride enolates. The single-crystal structure of boron enolate has been solved. The unprecedented C₁–C₁₀ migration of (Z)-β-difluoroboryloxy ether derivatives is observed to deliver rearranged phenol derivatives which are functionalized to C-2 alkyl-chromenones. Interestingly the isolated boron enolates have exhibited significant anticancer properties.

Key words

enolate - lewis acid - migration - chromenone

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• 10 Cytotoxicity Assay in Cancer Cells Further, the antiproliferative activity of (Z)-ethyl 2-(2-{3-[(difluoroboryl)oxy]but-2-enoyl}phenoxy) acetate (4a, EDFB-EPA) was assessed in two different types of cancer cell, such as cervical cancer cells (HeLa cells) and bone cancer cells (MG 63) by using MTT assays. Moreover, to understand the realistic anticancer application of a new compound, observing its effect on healthy cells was also necessary. Thus, the cytotoxicity of our compound was also assessed in two normal cell lines: HaCaT (human keratinocytes) and 3T3 (murine fibroblast). Cells were cultured in DMEM media, supplemented with 10 % FBS, 2 mM glutamine and 0.1 mg/mL antibiotics (penicillin and streptomycin). Cells with a 1 × 10⁴ cells/mL density were seeded on each well plate with 100 μL of fresh media, and the cells were allowed to proliferate for 24 h. The cells were further administered EDFB-EPA, and the cytotoxicity was assessed by using a standard MTT assay at the end of the next 24 h. Six hours before the end point, the MTT reagent was added. At 24 h, media was removed, and DMSO was further added to dissolve formazan. The absorbance of the solution was measured @595 nm, and the percentage of cell viability was expressed. Live/Dead Cell Assay Performed Using the Annexin V/7AAD Method Further, a live/dead cell assay was performed via a double-staining assay method using the Annexin V/7AAD dye. The kit was purchased from BD Life Science (Catalog No. 550474) and applied to cancer cells (HeLa) as well as normal cells (HaCaT). Cells were seeded in 6-well culture plates with an approximate cell density of 1 × 10⁵ per well and cultured for 24 h at 37 °C. After 24 h of the culture, the cell media was replaced with fresh media containing various doses of EDFB-EPA. After a treatment of another 24 h, the standard assay was performed using FACS (10000 events/sample). The dye Annexin V usually binds to apoptotic cells with exposed phosphatidyl serine, while 7-AAD interacts with necrotic cells with a damaged membrane. The percentages of the various states of cells that were present, such as viable (Annexin V–ve/7-AAD–ve), early apoptotic (Annexin V+ve/7AAD–ve), necrotic (Annexin V–ve/7AAD+ve), and late apoptotic or secondary necrotic (Annexin V+ve/7AAD+ve) cells, were determined. Cytotoxicity Study The cytotoxicity of EDFB-EPA was assessed in two different types of cancer cell, namely HeLa and MG63 cells. EDFB-EPA noticeably demonstrated the dose-based cytotoxicity in both the cancer cells, in which the cell viability reduced to 21.7 % (MG63) and 15.2 % (HeLa) at a concentration of 200 μg/mL (Figure-S3A and S3B in the Supporting Information). Further, from the cell viability plot, the IC50 of EDFB-EPA was determined to be 105.21 and 88.42 μg/mL for MG-63 and HeLa cells, respectively. While the IC50 of EDFB-EPA showed its promise as a potential drug candidate for cancer treatment, its cytocompatibility toward healthy cells was also desired for practical applications. Hence, we evaluated its cytocompatibility in two normal cell lines, HaCaT and 3T3, and found that EDFB-EPA has no significant effect on the viability of normal cells. Hence, EDFB-EPA might be considered as a potential therapeutic compound for anticancer applications. Live/Dead Cell Assay (Annexin V/7AAD) Method Performed Using Flow Cytometry The cytotoxic effect of EDFB-EPA toward cancer cells (HeLa cells) as well as healthy (HaCaT) cells was also studied using a double staining technique (Annexin V/7AAD) using FACS. The study was performed using 200 μg /mL EDFB-EPA. From Figure-S4 in the Supporting Information, it was clear that EDFB-EPA showed selective cytotoxicity toward cancer cells by causing nearly 66 % cell death in HeLa cells after 24 h of post administration of EDFB-EPA, which was compared to an untreated control sample. However, in healthy cells (HaCaT), EDFB-EPA was observed to cause only a negligible effect compared to the untreated control sample after 24 h of postadministration. Thus, from the previous results and the live-death cell assay, we confirmed that EDFB-EPA possesses a specific cytotoxicity toward cancer cells and has no significant effect on the healthy cells.