Direct chromatographic methods for enantioresolution of amino acids: recent developments.

α-Amino acids are present in two opposite configurations due to the presence of a central carbon atom which is a chiral center. While L-amino acids are present in large amount in nature, only tiny quantities of their D-enantiomers exist. For a long time, D-amino acids have been considered of bacterial origin only, but recently we realized that they are present in all living organisms: notably, D-amino acids play specific and relevant functions in the different organisms. Detection and quantification of D-amino acids are mandatory to shed light on their physiological roles, especially related to foods and human health. Chromatographic techniques are among the most commonly used analytical methods for the enantioseparation of amino acids. Here, we revised the latest improvements in chromatographic direct methodologies based on chiral selectors and aimed to improve analytical speed, sensitivity, robustness, and reproducibility. While current methods are well suited for D-amino acid analysis in foodstuffs and pharmaceuticals, further improvements seem required for their simultaneous, fast and sensitive detection in biological fluids, an emerging field since D-amino acids have been proposed as biomarkers of different and relevant human pathologic states.

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-γ when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.

Synthesis, biological characterization and molecular modeling insights of spirochromanes as potent HDAC inhibitors.

In the last decades, inhibitors of histone deacetylases (HDAC) have become an important class of anti-cancer agents. In a previous study we described the synthesis of spiro[chromane-2,4'-piperidine]hydroxamic acid derivatives able to inhibit histone deacetylase enzymes. Herein, we present our exploration for new derivatives by replacing the piperidine moiety with various cycloamines. The goal was to obtain highly potent compounds with a good in vitro ADME profile. In addition, molecular modeling studies unravelled the binding mode of these inhibitors.

Cinnamic anilides as new mitochondrial permeability transition pore inhibitors endowed with ischemia-reperfusion injury protective effect in vivo.

In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.

Synthesis and biological characterization of spiro[2H-(1,3)-benzoxazine-2,4'-piperidine] based histone deacetylase inhibitors.

Histone Deacetylases (HDACs) have become important targets for the treatment of cancer and other diseases. In previous studies we described the development of novel spirocyclic HDAC inhibitors based on the combination of privileged structures with hydroxamic acid moieties as zinc binding group. Herein, we report further explorations, which resulted in the discovery of a new class of spiro[2H-(1,3)-benzoxazine-2,4'-piperidine] derivatives. Several compounds showed good potency of around 100 nM and less in the HDAC inhibition assays, submicromolar IC50 values when tested against tumour cell lines and a remarkable stability in human and mouse microsomes. Two representative examples exhibited a good pharmacokinetic profile with an oral bioavailability equal or higher than 35% and one of them studied in an HCT116 murine xenograft model showing a robust tumour growth inhibition. In addition, the two benzoxazines were found to have a minor affinity for the hERG potassium channel compared to their corresponding ketone analogues.

Spiro[chromane-2,4'-piperidine]-based histone deacetylase inhibitors with improved in vivo activity.

A series of spiro[chromane-2,4'-piperidine] derivatives based on a previously published lead benzyl spirocycle 1 and bearing various N-aryl and N-alkylaryl substituents on the piperidine ring were prepared as novel histone deacetylase (HDAC) inhibitors. The compounds were evaluated for their abilities to inhibit nuclear HDACs, their in vitro antiproliferative activities, and in vitro ADME profiles. Based on these activities, 4-fluorobenzyl and 2-phenylethyl spirocycles were selected for further characterization. In vivo pharmacokinetic (PK) studies showed that both compounds exhibit an overall lower clearance rate, an increased half-life, and higher AUCs after intravenous and oral administration than spiropiperidine 1 under the conditions used. The improved PK behavior of these two compounds also correlated with superior in vivo antitumor activity in an HCT-116 xenograft model.

Discovery, synthesis, and pharmacological evaluation of spiropiperidine hydroxamic acid based derivatives as structurally novel histone deacetylase (HDAC) inhibitors.

New spiro[chromane-2,4'-piperidine] and spiro[benzofuran-2,4'-piperidine] hydroxamic acid derivatives as HDAC inhibitors have been identified by combining privileged structures with a hydroxamic acid moiety as zinc binding group. The compounds were evaluated for their ability to inhibit nuclear extract HDACs and for their in vitro antiproliferative activity on different tumor cell lines. This work resulted in the discovery of spirocycle 30d that shows good oral bioavailability and tumor growth inhibition in an HCT-116 murine xenograft model.

New taxane derivatives: synthesis of baccatin[14,1-d]furan-2-one nucleus and its condensation with the norstatine side chain.

New taxanes 15 and 18, containing the unsaturated and saturated baccatin[14,1-d]furan-2-one nucleus, respectively, were prepared starting from the readily available 13-oxo-7-Tes-baccatin III (3). Sequential formation of the enolate of 3 and reaction with ethyl glyoxylate gave the 13-oxo-7-Tes-baccatin[14,1-d]-3,4-dehydrofuran-2-one 4. The reduction of 4 can result in the formation of a mixture of compounds corresponding to 13-hydroxy alcohol 5 and 13-enol derivative 6. Both 5 and 6 were transformed into 13-oxo-7-Tes-baccatin[14,1-d]furan-2-one 8 by treatment with a base. Further reduction of 8 gave 13-hydroxy compound 9. Esterification of 6 and 9 with N,O-protected norstatine 12, followed by deprotection, gave the new promising anticancer taxanes 15 and 18, respectively.

Diastereoselective 14beta-hydroxylation of baccatin III derivatives.

14beta-Hydroxybaccatin III, a compound with limited availability by natural sources, is the starting material for the synthesis of the second-generation anticancer taxoid ortataxel. The 7-tert-butoxycarbonyl (1a) and 7-triethylsilyl (1b) derivatives of 14beta-hydroxybaccatin III 1,14-carbonate were synthesized from 10-deacetylbaccatin III (3). The crucial steps were (a) the C(14)beta hydroxylation of the corresponding 13-oxobaccatin III derivatives by oxaziridine-mediated electrophilic oxidation and (b) the reduction of the C(13) carbonyl group with sodium or alkylammonium borohydrides. This protocol provides a practical way for the semisynthesis of ortataxel from 10-deacetylbaccatin III, a compound readily available from various yews.