Synthesis and hLDH Inhibitory Activity of Analogues to Natural Products with 2,8-Dioxabicyclo[3.3.1]nonane Scaffold.

Human lactate dehydrogenase (hLDH) is a tetrameric enzyme present in almost all tissues. Among its five different isoforms, hLDHA and hLDHB are the predominant ones. In the last few years, hLDHA has emerged as a therapeutic target for the treatment of several kinds of disorders, including cancer and primary hyperoxaluria. hLDHA inhibition has been clinically validated as a safe therapeutic method and clinical trials using biotechnological approaches are currently being evaluated. Despite the well-known advantages of pharmacological treatments based on small-molecule drugs, few compounds are currently in preclinical stage. We have recently reported the detection of some 2,8-dioxabicyclo[3.3.1]nonane core derivatives as new hLDHA inhibitors. Here, we extended our work synthesizing a large number of derivatives (42-70) by reaction between flavylium salts (27-35) and several nucleophiles (36-41). Nine 2,8-dioxabicyclo[3.3.1]nonane derivatives showed IC50 values lower than 10 µM against hLDHA and better activity than our previously reported compound 2. In order to know the selectivity of the synthesized compounds against hLDHA, their hLDHB inhibitory activities were also measured. In particular, compounds 58, 62a, 65b, and 68a have shown the lowest IC50 values against hLDHA (3.6-12.0 µM) and the highest selectivity rate (>25). Structure-activity relationships have been deduced. Kinetic studies using a Lineweaver-Burk double-reciprocal plot have indicated that both enantiomers of 68a and 68b behave as noncompetitive inhibitors on hLDHA enzyme.

Synthesis of Analogs to A-Type Proanthocyanidin Natural Products with Enhanced Antimicrobial Properties against Foodborne Microorganisms.

Developing new types of effective antimicrobial compounds derived from natural products is of interest for the food industry. Some analogs to A-type proanthocyanidins have shown promising antimicrobial and antibiofilm activities against foodborne bacteria. We report herein the synthesis of seven additional analogs with NO2 group at A-ring and their abilities for inhibiting the growth and the biofilm formation by twenty-one foodborne bacteria. Among them, analog 4 (one OH at B-ring; two OHs at D-ring) showed the highest antimicrobial activity. The best results with these new analogs were obtained in terms of their antibiofilm activities: analog 1 (two OHs at B-ring; one OH at D-ring) inhibited at least 75% of biofilm formation by six strains at all of the concentrations tested, analog 2 (two OHs at B-ring; two OHs at D-ring; one CH3 at C-ring) also showed antibiofilm activity on thirteen of the bacteria tested, and analog 5 (one OH at B-ring; one OH at D-ring) was able to disrupt preformed biofilms in eleven strains. The description of new and more active analogs of natural compounds and the elucidation of their structure-activity relationships may contribute to the active development of new food packaging for preventing biofilm formation and lengthening the food shelf life.

Lactate dehydrogenase A inhibitors with a 2,8-dioxabicyclo[3.3.1]nonane scaffold: A contribution to molecular therapies for primary hyperoxalurias.

Human lactate dehydrogenase A (hLDHA) is one of the main enzymes involved in the pathway of oxalate synthesis in human liver and seems to contribute to the pathogenesis of disorders with endogenous oxalate overproduction, such as primary hyperoxaluria (PH), a rare life-threatening genetic disease. Recent published results on the knockdown of LDHA gene expression as a safe strategy to ameliorate oxalate build-up in PH patients are encouraging for an approach of hLDHA inhibition by small molecules as a potential pharmacological treatment. Thus, we now report on the synthesis and hLDHA inhibitory activity of a new family of compounds with 2,8-dioxabicyclo[3.3.1]nonane core (23-42), a series of twenty analogues to A-type proanthocyanidin natural products. Nine of them (25-27, 29-34) have shown IC50 values in the range of 8.7-26.7 µM, based on a UV spectrophotometric assay, where the hLDHA inhibition is measured according to the decrease in absorbance of the cofactor ß-NADH (340 nm). Compounds 25, 29, and 31 were the most active hLDHA inhibitors. In addition, the inhibitory activities of those nine compounds against the hLDHB isoform were also evaluated, finding that all of them were more selective inhibitors of hLDHA versus hLDHB. Among them, compounds 32 and 34 showed the highest selectivity. Moreover, the most active hLDHA inhibitors (25, 29, 31) were evaluated for their ability to decrease the oxalate production by hyperoxaluric mouse hepatocytes (PH1, PH2 and PH3) in vitro, and the relative oxalate output at 24 h was 16% and 19 % for compounds 25 and 31, respectively, in Hoga1-/- mouse primary hepatocyte cells (a model for PH3). These values improve those of the reference compound used (stiripentol). Compounds 25 and 31 have in common the presence of two hydroxyl groups at rings B and D and an electron-withdrawing group (NO2 or Br) at ring A, pointing to the structural features to be taken into account in future structural optimization.

Recovery and Seasonal Variation of Cinnamtannin B-1 from Laurel (Laurus nobilis L.) Pruning Wood Wastes.

Cinnamtannin B-1 (C-B1) is a commercially-available trimeric A-type procyanidin with remarkable cellular actions mainly derived from its high radical scavenging activity. C-B1 is the main phenolic compound of laurel wood, which has previously been isolated by a combination of conventional chromatographic techniques. The first aim of this work was to find laurel trees containing as much C-B1 as possible, and learn about the influence of variables, such as gender and harvest time, on the production of C-B1 by the tree. It was found that all studied trees tend to give higher C-B1 percentages in the May-July period, from 6 % to 18 %, and lower ones around March (spring) and November (fall), from 1 % to 8 %. In a general way, it also seems that the female trees tend to produce a bit more C-B1 (from 2.8 % to 17.3 %) than male ones (from 1.7 % to 13.4 %). In addition, eight minor phenolic compounds [(-)-ent-catechin (1), (-)-ent-epicatechin-(4α→8)-ent-epicatechin (2), (epi)catechin-(4→8)-(epi)afzelechin-(4→8)-(epi)catechin (3), (+)-epiafzelechin-(4ß→8)-epicatechin (4), (-)-epicatechin (5), (-)-afzelechin-(4α→8)-epicatechin (6), (epi)afzelechin-(4→8)-(epi)afzelechin-(4→8)-(epi)catechin (7) and (+)-epicatechin-(4ß→8,2ß→O-7)-epicatechin-(4ß→8)-catechin (C-D1)] were found and quantified in the ethyl acetate extract of the wood samples. The second aim of this work was to improve the recovery of C-B1 from laurel wood. The use of the fast centrifugal partition chromatography (FCPC) technique has allowed for a recovery of 96 % of a technical-grade C-B1 (64 % in a previous protocol using conventional column chromatography on silica gel and size-exclusion chromatography).

Evolution of Flavylium-Based Color Systems in Plants: What Physical Chemistry Can Tell Us.

Anthocyanins are the basis of the color of angiosperms, 3-deoxyanthocyanins and sphagnorubin play the same role in mosses and ferns, and auronidins are responsible for the color in liverworts. In this study, the color system of cyanidin-3-O-glucoside (kuromanin) as a representative compound of simpler anthocyanins was fully characterized by stopped flow. This type of anthocyanin cannot confer significant color to plants without intra- or intermolecular interactions, complexation with metals or supramolecular structures as in Commelina communis. The anthocyanin's color system was compared with those of 3-deoxyanthocyanins and riccionidin A, the aglycone of auronidins. The three systems follow the same sequence of chemical reactions, but the respective thermodynamics and kinetics are dramatically different.

Light-Mediated Toxicity of Porphyrin-Like Pigments from a Marine Polychaeta.

Porphyrins and derivatives form one of the most abundant classes of biochromes. They result from the breakdown of heme and have crucial physiological functions. Bilins are well-known representatives of this group that, besides significant antioxidant and anti-mutagenic properties, are also photosensitizers for photodynamic therapies. Recently, we demonstrated that the Polychaeta Eulalia viridis, common in the Portuguese rocky intertidal, holds a high variety of novel greenish and yellowish porphyrinoid pigments, stored as granules in the chromocytes of several organs. On the follow-up of this study, we chemically characterized pigment extracts from the worm's skin and proboscis using HPLC and evaluated their light and dark toxicity in vivo and ex vivo using Daphnia and mussel gill tissue as models, respectively. The findings showed that the skin and proboscis have distinct patterns of hydrophilic or even amphiphilic porphyrinoids, with some substances in common. The combination of the two bioassays demonstrated that the extracts from the skin exert higher dark toxicity, whereas those from the proboscis rapidly exert light toxicity, then becoming exhausted. One particular yellow pigment that is highly abundant in the proboscis shows highly promising properties as a natural photosensitizer, revealing that porphyrinoids from marine invertebrates are important sources of these high-prized bioproducts.

Ground and excited state properties of furanoflavylium derivatives.

The comparison of the ground-state reactivity of anthocyanins and aurone model compounds (i.e. with and without the furano bridge) has shown that the kinetic paradigm does not depend on the bridge but only on the hydroxyl substituent pattern, independently of the presence of the bridge: (i) bell shaped kinetics for those with two hydroxyl substituents in position 4' and 7, and (ii) four distinct kinetic steps for the mono substituted compounds with a hydroxyl in position 4'. The excited state proton transfer (ESPT) properties of these compounds were also investigated using steady-state and time-resolved spectroscopic techniques. It was found that the ESPT efficiency is significantly higher for the bridged compounds. Interestingly, pH-dependent steady-state fluorescence emission experiments show that in 4',7-dihydroxyfuranoflavylium the hydroxyl group in position 7 is the more acidic one in the excited state, while 1H NMR titration curves indicate a higher acidity constant in the ground state for the proton at the hydroxyl group in position 4'. Differently, the fluorescence emission spectrum of the quinoidal base deprotonated at position 7 is only observed upon excitation of the flavylium cation while the one from the base deprotonated at 4' is observed upon direct excitation.

Thermodynamic Stability of Flavylium Salts as a Valuable Tool To Design the Synthesis of A-Type Proanthocyanidin Analogues.

A convenient method to synthesize A-type proanthocyanidin analogues from flavylium salts and π-nucleophiles has been developed. It was found that the thermodynamic stability of the starting flavylium salt, assessed by the measurement of the apparent acidity constant ( K'a), was the key parameter to design effective one-pot reactions between flavylium salts and nucleophiles such as phloroglucinol and (+)-catechin. When flavylium salts have a p K'a value of 1.7 or lower, the synthesis of the corresponding 2,8-dioxabyciclo[3.3.1]nonane derivative was properly achieved.

Effect of Methyl, Hydroxyl, and Chloro Substituents in Position 3 of 3',4',7-Trihydroxyflavylium: Stability, Kinetics, and Thermodynamics.

The effect of methyl, hydroxyl, and chloride substituents in position 3 of the 3',4',7-trihydroxyflavylium core structure was studied. The stability, relative energy of each of chemical species (thermodynamics), and their rates of interconversion (kinetics) are very dependent on these substituents. By comparing the mole fraction distribution at equilibrium of the three multistate systems with the parent 3',4',7-trihydroxyflavylium, introduction of a methyl substituent in position 3 increases the mole fraction of hemiketal at the expense of the trans-chalcone and increases the hydration rate very significantly; a hydroxyl substituent in position 3 gives rise to a degradation process, as observed in anthocyanidins. In the case of 3-chloro-3',4',7-trihydroxyflavylium, a dramatic increase of the flavylium cation acidity was observed and a photochromic system can be operated upon irradiation of the respective trans-chalcone in 1 m HCl. According to the photochromic response of 3,3',4',7-tetrahydroxyflavylium and 3',4',7-trihydroxyflavylium, some requirements for a good photochromic performance are discussed.

Small Molecule-Based Enzyme Inhibitors in the Treatment of Primary Hyperoxalurias.

Primary hyperoxalurias (PHs) are a group of inherited alterations of the hepatic glyoxylate metabolism. PHs classification based on gene mutations parallel a variety of enzymatic defects, and all involve the harmful accumulation of calcium oxalate crystals that produce systemic damage. These geographically widespread rare diseases have a deep impact in the life quality of the patients. Until recently, treatments were limited to palliative measures and kidney/liver transplants in the most severe forms. Efforts made to develop pharmacological treatments succeeded with the biotechnological agent lumasiran, a siRNA product against glycolate oxidase, which has become the first effective therapy to treat PH1. However, small molecule drugs have classically been preferred since they benefit from experience and have better pharmacological properties. The development of small molecule inhibitors designed against key enzymes of glyoxylate metabolism is on the focus of research. Enzyme inhibitors are successful and widely used in several diseases and their pharmacokinetic advantages are well known. In PHs, effective enzymatic targets have been determined and characterized for drug design and interesting inhibitory activities have been achieved both in vitro and in vivo. This review describes the most recent advances towards the development of small molecule enzyme inhibitors in the treatment of PHs, introducing the multi-target approach as a more effective and safe therapeutic option.

Synthesis of A-Type Proanthocyanidins and Their Analogues: A Comprehensive Review.

Proanthocyanidins (PACs) are oligomers or polymers composed of units of flavanols. A-type PACs are a subclass of PACs characterized by the presence of at least a double linkage between two consecutive monomers of flavanol. These A-type PACs are found in some fruits and spices and possess potential health benefits as a result of their interesting biological activities, and consequently, their isolation and synthesis have given rise to great interest in the past. This review summarizes the synthetic efforts made to obtain both naturally occurring A-type PACs and their structurally simplified analogues. Most of the synthetic protocols reported involve the addition of a π-nucleophilic molecule over a molecule with two electrophilic carbons, such as a chalcone, a flavylium salt, or a flavanol derivative, among others. Synthesis of A-type PACs remains an issue at a very early stage of development compared to that of PACs with single linkages between monomers (B-type PACs), but the advances that are taking place in the last few years point to a significant development of the subject in the near future.

Synthesis and Evaluation of Antimicrobial and Antibiofilm Properties of A-Type Procyanidin Analogues against Resistant Bacteria in Food.

Natural A-type procyanidins have shown very interesting biological activities, such as their proven antiadherence properties against pathogenic bacteria. In order to find the structural features responsible for their activities, we describe herein the design and synthesis of six A-type procyanidin analogues and the evaluation of their antimicrobial and antibiofilm properties against 12 resistant bacteria, both Gram positive and Gram negative, isolated from organic foods. The natural A-type procyanidin A-2, which had known antiadherence activity, was also tested as a reference compound for the comparative studies. Within the series, analogue 4, which had a NO2 group on ring A, showed the highest antimicrobial activity (MIC of 10 µg/mL) and was one of the best molecules at preventing biofilm formation (up to 40% decreases at 100 µg/mL) and disrupting preformed biofilms (up to 40% reductions at 0.1 µg/mL). Structure-activity relationships are also analyzed.

Phytochemicals and Biological Activities of Laurel Tree (Laurus nobilis).

This review summarizes the chemical composition reported up to date on Laurus nobilis L. (Lauraceae), an evergreen shrub or tree cultivated for its aromatic leaves and ornamental interest. It has been focused on non-volatile phytochemicals such as sesquiterpene lactones, flavonoids and proanthocyanidins, among others. Moreover, biological activities of laurel extracts and pure compounds have also been reviewed.