M. Antonia Martín

An Overview of Analytical Techniques Employed to Evidence Drug-DNA Interactions. Applications to the Design of Genosensors

The demonstration of the existence of non-covalent bonding interactions between different biomolecules (DNA, enzymes, proteins) and drugs or potentially mutagenic agents requires sensitive analytical techniques. Technological advances in key techniques, together with the miniaturization in fluorescence or surface plasmons resonance techniques, have allowed obtaining information in real time concerning the nature, the localization and the strength of drug-DNA interactions. The present chapter describes these analytical techniques and their application to achieve two fundamental goals, namely a deeper knowledge of the nature of the interactions and their application to the design of different devices (aptamers, molecular beacons, DNA-arrays, genosensors) consisting of short single stranded oligonucleotides produced in vitro by well-established methods (polymerase chain reaction, PCR; systematic evolution of ligands by exponential enrichment, SELEX,...) that are capable to detect genetic peculiarities and diseases, biological entities (micro-organisms, genetically modified seeds...) and also genotoxic agents in the environment and in foodstuffs.

Isolation and quantitative methods for analysis of non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs are widespread employed in both human and animal health care to reduce ongoing inflammation, pain and fever due to their anti-inflammatory, analgesic and, antipyretic actions. Apart from the well-known biological samples, nowadays these compounds are frequently found in the environment, leading to longterm exposure resulting in adverse effects on humans and wildlife. Therefore, it is important to develop analytical methodologies to detect and control the presence of these pharmaceuticals in very different kinds of samples, from urine, serum or plasma, to river and waste water, sediments or sewage sludge, most of them having very complex matrices. Other problems to solve are the low concentration of the target analytes, the presence of a great number of potential interferences and, sometimes, incompatibilities with the detection systems. Consequently, sample pre-treatment is a very important step to take into account in the non-steroidal anti-inflammatory drugs determination. Herein we reviewed the main extraction and clean-up procedures reported in the literature for these substances: ultrasonic extraction, Soxhlet extraction, pressurized- liquid extraction, supercritical fluid extraction, microwave-assisted extraction, dispersive liquid-liquid microextraction, hollow fiber liquid phase microextraction, pressurized hot water extraction, solid-phase extraction, molecularly imprinted solid-phase extraction and solid-phase microextraction. Several analytical methodologies have been developed to quantify non-steroidal anti-inflammatory drugs, including gas chromatography-mass spectrometry, liquid chromatography with ultraviolet detection, diode array detection, fluorescence detection and tandem mass spectrometry.

Eco-friendly liquid chromatographic separations based on the use of cyclodextrins as mobile phase additives

Acetonitrile and methanol are the most popular solvents employed in analytical HPLC, but they suffer from a number of drawbacks from the environmental point of view. Alternative, greener mobile phases employing methanol or the less toxic solvent ethanol as the sole organic solvent are proposed in this paper, and applied to the problem of the separation of β-carbolines on C18-stationary phases. The use of β-cyclodextrin (β-CD) and (2-hydroxypropyl)-β-cyclodextrin (HPβ-CD) as mobile phase additives allowed us to increase the proportion of water in the mobile phases without loss in the resolution or efficiency of the separations, leading initially to a considerable reduction of the proportion of methanol in the mobile phase (from 70% to 50%) and at a later stage, to the development of a mobile phase containing only 30% of ethanol. The β-carboline–cyclodextrin association constants were determined by HPLC, and the inclusion complexes were also characterized by 1H-NMR, 13C-NMR and 2D-ROESY experiments, and these studies were used to explain the chromatographic behaviour. The new chromatographic methodology developed was validated and applied to the quantitation of β-carboline derivatives in spiked human serum samples. For the extraction of β-carboline alkaloids from serum samples, liquid–liquid extraction (LLE) and solid-phase extraction (SPE) procedures were compared. It was concluded that the combination of a pre-treatment procedure (ionic exchange SPE) with a water-enriched chromatographic separation leads to a promising, environmentally friendly new methodology.

Study of non-covalent interactions of luotonin A derivatives and the DNA minor groove as a first step in the study of their analytical potential as DNA probes

The interaction between DNA and several newly synthesized derivatives of the natural anticancer compound luotonin A has been studied. The results from our work reveal an effective and selective alkaloid/double-stranded DNA (ds-DNA) interaction. In the presence of increasing amounts of ds-DNA, a noticeable fluorescence quenching of the luotonin A derivatives under study was observed. However, this effect did not take place when single-stranded DNA (ss-DNA) was employed. The association constant alkaloids/ds-DNA was calculated by quantitation of such a quenching effect. The influence of other quenchers, namely Co2+ and Br− on the native fluorescence of luotonin A and derivatives was also studied, and a remarkable quenching effect was observed for both ions. We have also investigated how by binding DNA the alkaloids could get protected from the external Co2+ and Br− quenchers. The Stern–Volmer constants (KSV) for Co2+ and Br− quenching effect on the studied alkaloids were considerably reduced (10–50%) after incubation of the compounds in the presence of DNA with regard to the KSV values in absence of DNA. An increase in the fluorescence anisotropy values of luotonins was also produced only in the presence of ds-DNA but not in the case of ss-DNA. To better characterize the nature of that interaction, viscosimetry assays and ethidium bromide displacement studies were conducted. With regard to DNA reference solutions, the viscosity of solutions containing DNA and luotonin A derivatives was reduced or not significantly increased. It was also observed that the studied compounds were unable to displace the intercalating agent ethidium bromide. All of these results, together with the obtained association constants values (Kass = 2.2 × 102 – 1.3 × 103), support that neither covalent nor intercalating interactions luotonin A derivatives/ds-DNA are produced, leading to the conclusion that these alkaloids bind ds-DNA through the minor groove. The specific changes in the fluorescence behavior of luotonin A and derivatives distinguishing between ss-DNA and ds-DNA binding, lead us to propose these compounds as attractive turn-off probes to detect DNA hybridization.

Influence of the presence of methyl cyclodextrins in high-performance liquid chromatography mobile phases on the separation of β-carboline alkaloids

The presence of cyclodextrins (CDs) in the mobile phase alters the chromatographic equilibria and induces a secondary chemical equilibrium associated to the chromatographic separation by HPLC. In this study the influence of the presence of CDs in the mobile phase as chemically modified beta-CDs, i.e. 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) on the separation of the alkaloids norharmane, harmane and harmine is described. These beta-carboline alkaloids are chemically and structurally related and their quantitation by RP-HPLC is of interest due to their biological and pharmacological properties. Two stationary phases (methyl-, C(1) and octadecyl-, C(18)) were employed, with methanol:buffered aqueous solution and ethanol:buffered aqueous solution as mobile phases. The role of tert-butyl alcohol as a mobile phase modifier and also as an inclusion complex stabiliser was also considered. The concentrations of DMbeta-CD and TMbeta-CD vary from 0 to 17 mM. The presence of increasing amounts of CDs in the mobile phase reduces the retention factor. The changes observed in the retention factor allow the determination of the alkaloid/CD apparent association constants, whose magnitude is influenced by the chemical and structural properties of the guest molecules but also by the composition of the mobile phase. Assuming a 1:1 stoichiometry for the inclusion complexes, the apparent association constants obtained were higher for norharmane and for both DMbeta-CD and TMbeta-CD. The strength of the complexes is higher for DMbeta-CD than for TMbeta-CD and this behaviour can be explained considering the steric problems associated to the permethylated-beta-CDs. Besides significant differences in the magnitude of the apparent association constants were observed for the two stationary phases employed and thus can be related to the adsorption of CDs on the stationary phase. A significant reduction in the proportion of organic solvent in the mobile phase (50%) without a decrease in the selectivity or resolution of the separation is a favourable consequence of the presence of the CDs in the mobile phase.

B-ring-aryl substituted luotonin A analogues with a new binding mode to the topoisomerase 1-DNA complex show enhanced cytotoxic activity.

Topoisomerase 1 inhibition is an important strategy in targeted cancer chemotherapy. The drugs currently in use acting on this enzyme belong to the family of the camptothecins, and suffer severe limitations because of their low stability, which is associated with the hydrolysis of the δ-lactone moiety in their E ring. Luotonin A is a natural camptothecin analogue that lacks this functional group and therefore shows a much-improved stability, but at the cost of a lower activity. Therefore, the development of luotonin A analogues with an increased potency is important for progress in this area. In the present paper, a small library of luotonin A analogues modified at their A and B rings was generated by cerium(IV) ammonium nitrate-catalyzed Friedländer reactions. All analogues showed an activity similar or higher than the natural luotonin A in terms of topoisomerase 1 inhibition and some compounds had an activity comparable to that of camptothecin. Furthermore, most compounds showed a better activity than luotonin A in cell cytotoxicity assays. In order to rationalize these results, the first docking studies of luotonin-topoisomerase 1-DNA ternary complexes were undertaken. Most compounds bound in a manner similar to luotonin A and to standard topoisomerase poisons such as topotecan but, interestingly, the two most promising analogues, bearing a 3,5-dimethylphenyl substituent at ring B, docked in a different orientation. This binding mode allows the hydrophobic moiety to be shielded from the aqueous environment by being buried between the deoxyribose belonging to the G(+1) guanine and Arg364 in the scissile strand and the surface of the protein and a hydrogen bond between the D-ring carbonyl and the basic amino acid. The discovery of this new binding mode and its associated higher inhibitory potency is a significant advance in the design of new topoisomerase 1 inhibitors.

Challenging core-shell stationary phases with the separation of closely related anti-cancer compounds: performance studies and application to drug quantitation in cell cultures with multi-well plate clean-up.

In the present paper, we compare the chromatographic behaviour of six different stationary phases (5 μm fully porous and 2.6 μm core-shell particles, featuring octadecyl-, pentafluorophenyl-propyl- and phenyl-hexyl- functionalizations) for the separation of camptothecin and a family of closely related analogues of the natural anti-cancer drug luotonin A under different experimental conditions (organic solvent nature and proportion, temperature and presence of modifiers). We found two differentiated behaviours for the phenyl-hexyl-functionalized supports depending on the temperature. The efficiency and kinetic performance of fully porous vs. core-shell particles was compared by means of van Deemter and kinetic plots, using an optimized conventional HPLC system. Columns packing core-shell particles afforded improved efficiencies, permeabilities, analysis times and consumption of solvents. Nevertheless, our optimized system was not able to release the full intrinsic efficiencies of these columns. The replacement of acetonitrile with methanol had a dramatic effect in the behaviour of the pentafluorophenyl-propyl stationary phase. The aggregate of these results led us to select the core-shell C-18 stationary phase along with a H2O:MeCN gradient for the rapid and sustainable quantitation of the anti-tumour agents in cell cultures. A simple and rapid clean-up step was optimized using 96-well deproteinization and delipidation plates. The validation of the proposed analytical method showed excellent figures of merit. The combination of this sample pre-treatment procedure with the separation on core-shell particles yielded a fast methodology able to process a large number of samples with minimal waste generation and environmental impact.

Tetrahydroisoquinoline-Derived Urea and 2,5-Diketopiperazine Derivatives as Selective Antagonists of the Transient Receptor Potential Melastatin 8 (TRPM8) Channel Receptor and Antiprostate Cancer Agents

Tetrahydroisoquinoline derivatives containing embedded urea functions were identified as selective TRPM8 channel receptor antagonists. Structure-activity relationships were investigated, with the following conclusions: (a) The urea function and the tetrahydroisoquinoline system are necessary for activity. (b) Bis(1-aryl-6,7dimethoxy-1,2,3,4-tetrahydroisoquinolyl)ureas are more active than compounds containing one tetrahydroisoquinoline ring and than an open phenetylamine ureide. (c) Trans compounds are more active than their cis isomers. (d) Aryl substituents are better than alkyls at the isoquinoline C-1 position. (e) Electron-withdrawing substituents lead to higher activities. The most potent compound is the 4-F derivative, with IC50 in the 10(-8) M range and selectivities around 1000:1 for most other TRP receptors. Selected compounds were found to be active in reducing the growth of LNCaP prostate cancer cells. TRPM8 inhibition reduces proliferation in the tumor cells tested but not in nontumor prostate cells, suggesting that the activity against prostate cancer is linked to TRPM8 inhibition.

A down-scaled fluorimetric determination of the solubility properties of drugs to minimize waste generation

A miniaturized fluorescence assay on multi-well plates has been developed to study the solubility enhancement effect of (2-hydroxypropyl)-β-cyclodextrin on three anti-tumor alkaloids. The measurement of the fluorescence emission on a multi-well plate format has been proved to be a rapid and efficient technique to evaluate the solubility of pharmaceutical formulations of new drugs that help save time, reagents and wastes in the search for greener analytical strategies. The proposed methodology was compared with a reference HPLC solubility study and was employed to examine the enhancement of the solubility of camptothecin, luotonin A, and a synthetic derivative of the latter in the presence of (2-hydroxypropyl)-β-cyclodextrin. Considerable reductions in the time of analysis (almost 50 times faster) and the volume of organic solvents employed (close to 25 times less acetonitrile needed) were achieved. The nature of the inclusion complexes was investigated by analysis of the phase-solubility diagrams obtained by the newly developed method and was complemented with spectrofluorimetry and ESI-MS experiments. The concentrations of solubilised compounds found by both methodologies were in good agreement (R2 > 0.98). The analytical figures of merit of both methodologies were compared and the adequacy of the proposed method for the development of drug solubilisation studies was discussed.

Three-component synthesis of a library of m-terphenyl derivatives with embedded β-aminoester moieties

The three-component reaction between alkyl- or arylamines, β-ketoesters and chalcones in refluxing ethanol containing a catalytic amount of Ce(IV) ammonium nitrate allowed the construction of a large library of highly substituted dihydro- m-terphenyl derivatives containing β-alkylamino- or β-arylamino ester moieties. This process generates three new bonds and one ring and proceeds in high atom economy, having two molecules of water as the only side product. Another domino process, in which the original MCR was telescoped with a subsequent aza Michael/retro-aza Michael sequence, allowed the one-pot preparation of a library of compounds with a N-unsubstituted β-aminoester fragment. Finally, to extend the structural diversity of these libraries, we also examined the aromatization of the central ring of our compounds in the presence of dichlorodicyanoquinone. This reaction sequence did not affect the integrity of a stereogenic center belonging to the amino component.