Synthesis, Optical Properties, and Antiproliferative Evaluation of NBD-Triterpene Fluorescent Probes.

A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize, in yields of 72-86%, 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids (Aa) of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, 12 NBD-Aa-triterpene conjugates have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these NBD-Aa-triterpene conjugates enabled their uptake and subcellular distribution to be followed in order to probe in detail their biological properties at the cellular and molecular level.

Synthesis and Biological Activity of Triterpene-Coumarin Conjugates.

A set of 12 maslinic acid-coumarin conjugates was synthesized, with 9 being maslinic acid-diamine-coumarin conjugates at the C-28 carboxylic acid group of triterpene acid and the other three being maslinic acid-coumarin conjugates at C-2/C-3 and/or C-28 of the triterpene skeleton. The cytotoxic effects of these 12 triterpene conjugates were evaluated in three cancer cell lines (B16-F10, HT29, and Hep G2) and compared, respectively, with three nontumor cell lines from the same or similar tissue (HPF, IEC-18, and WRL68). The most potent cytotoxic results were achieved by a conjugate with two molecules of coumarin-3-carboxylic acid coupled through the C-2 and C-3 hydroxy groups of maslinic acid. This conjugate showed submicromolar IC50 values in two of the three cancer cell lines tested (0.6, 1.1, and 0.9 µM), being between 110- and 30-fold more effective than its corresponding precursor. Furthermore, this conjugate (10) showed percentages of cell viability for the three nontumor lines of 90%. Four maslinic acid-coumarin conjugates displayed apoptotic effects in the treated cells, with total apoptosis rates of between 40 and 85%, relative to the control. Almost all the compounds assayed caused cell-cycle arrest in all cancer cell lines, increasing the number of these cells in the G0/G1 phase.

Novel Oleanolic and Maslinic Acid Derivatives as a Promising Treatment against Bacterial Biofilm in Nosocomial Infections: An in Vitro and in Vivo Study.

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm.