A series of novel vindoline–piperazine conjugates were synthesized by coupling 6 N-substituted piperazine pharmacophores at positions 10 and 17 of Vinca alkaloid monomer vindoline through different types of linkers. The in vitro antiproliferative activity of the 17 new conjugates was investigated on 60 human tumor cell lines (NCI60). Nine compounds presented significant antiproliferative effects. The most potent derivatives showed low micromolar growth inhibition (GI₅₀) values against most of the cell lines. Among them, conjugates containing [4-(trifluoromethyl)benzyl]piperazine (23) and 1-bis(4-fluorophenyl)methyl piperazine (25) in position 17 of vindoline were outstanding. The first one was the most effective on the breast cancer MDA-MB-468 cell line (GI₅₀ = 1.00 μM), while the second one was the most effective on the non-small cell lung cancer cell line HOP-92 (GI₅₀ = 1.35 μM). The CellTiter-Glo Luminescent Cell Viability Assay was performed with conjugates 20, 23, and 25 on non-tumor Chinese hamster ovary (CHO) cells to determine the selectivity of the conjugates for cancer cells. These compounds exhibited promising selectivity with estimated half-maximal inhibitory concentration (IC₅₀) values of 2.54 μM, 10.8 μM, and 6.64 μM, respectively. The obtained results may have an impact on the design of novel vindoline-based anticancer compounds.

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca²⁺ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.

The Vinca alkaloid vindoline was coupled at position 17 with several trisubstituted phosphine derivatives and their in vitro anticancer activities on 60 human tumor cell lines (NCI60) were investigated. This phosphonium-type ionic side chain is beneficial because it allows therapeutic molecules to pass through the cell membrane. Thus, the candidates coupled to it can exert their activities in the mitochondria. The coupling of vindoline with the trisubstituted phosphines was achieved through flexible or rigid linkers. Instead of the ionic phosphonium structural part, a neutral moiety, namely the triphenylmethyl group, was also added to the side chain, being sterically similar but without a charge and phosphorus atom. In addition, the triphenylphosphine element was also built at position 10 of vindoline. Most of the derivatives showed low micromolar growth inhibition (GI₅₀) values against most cell lines. Among them, conjugate 9e was outstanding: it exhibited nanomolar anticancer activity on the RPMI-8226 leukemia cell line (GI₅₀ = 20.0 nM). Compound 9g elicited cell cycle disturbance and apoptosis on A2780 ovary cancer cells and inhibited their migration at subantiproliferative concentrations. The selectivity of the conjugates was determined by their effects on non-tumor Chinese hamster ovary (CHO) cells in the CellTiter-Glo Luminescent Cell Viability Assay. Compound 9e showed an estimated half-maximal inhibitory concentration (IC₅₀) value of 1.36 µM, suggesting good selectivity on cancer cells. These results open new perspectives of novel phosphonium-based vindoline derivatives as anticancer compounds.

One of the best-known flavonoid chrysin was coupled at position 7 with several trisubstituted phosphine derivatives with a flexible spacer, and their in vitro anticancer activities were investigated on 60 human tumor cell lines (NCI60) and on several gynecological cancer cells. The trisubstituted phosphines contained different substituents on the aromatic ring(s), e.g., methyl and methoxy groups or fluoro atoms. The phosphorus atom was substituted not only with aromatic rings but with cyclohexyl substituents. The ionic phosphonium building block is important because it allows the therapeutic agents to transfer across the cell membrane. Therefore, the pharmacophores linked to it can exert their effects in the mitochondria. Instead of the ionic phosphonium element, a neutral moiety, namely the triphenylmethyl group, was also added to the side chain, being sterically similar but without a charge and phosphorus atom. Most of the hybrids exhibited low micromolar growth inhibition (GI₅₀) values against the majority of the tested cell lines. Notably, conjugate 3f stood out, demonstrating nanomolar antitumor activity against the K-562 leukemia cell line (GI₅₀ = 34 nM). One selected compound (3i) with promising cancer selectivity elicited cell cycle disturbances and inhibited the migration of breast cancer. The tumor-selectivity of 3a and 3f was assessed based on their effects on non-tumor Chinese hamster ovary (CHO) cells using the CellTiter-Glo Luminescent Cell Viability Assay. Given their estimated half-maximal inhibitory concentration (IC₅₀) values on non-tumor CHO cells (2.65 µM and 1.15 µM, respectively), these conjugates demonstrate promising selectivity toward several cancer cell lines. The excellent results obtained may serve as good starting points for further optimization and the design of even more effective flavonoid- and/or phosphonium-based drugs.