Benedetto Natalini

Discovery of 6α-ethyl-23(S)-methylcholic Acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity.

In the framework of the design and development of TGR5 agonists, we reported that the introduction of a C(23)(S)-methyl group in the side chain of bile acids such as chenodeoxycholic acid (CDCA) and 6-ethylchenodeoxycholic acid (6-ECDCA, INT-747) affords selectivity for TGR5. Herein we report further lead optimization efforts that have led to the discovery of 6alpha-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a novel potent and selective TGR5 agonist with remarkable in vivo activity.

D-3,4-‘cyclopropylglutamate’ isomers as nmda receptor ligands: Synthesis and enantioselective activity.

Abstract Dirhodium(II) tetraacetate catalyzed decomposition of ethyl diazoacetate in the presence of D-Cbz-vinylglycine methyl ester (11) afforded a mixture of the cyclopropyl esters D-CGA A-D (13) from which the corresponding 2R-acids 7-10 were obtained and their absolute configurations assigned. The (2R,3S,4R) α-(carboxycyclopropyl)glycine (D-CGA C, 9 resulted to be the most potent and selective among the NMDA receptor ligands yet reported.

Determination of bile salt critical micellization concentration on the road to drug discovery.

With the discovery of the bile acid (BA)-activated nuclear and membrane receptors, the role of BAs as signalling molecules in important paracrine and endocrine networks has been fully documented in the last decade. Besides regulating their own synthesis and transport, BAs have been demonstrated being involved in triggering the adaptive response to cholestasis and other insults to liver. More to the point, their recognized ability to control the general energy-related metabolism and inflammation processes has contributed to justify the renewed interest towards this class of amphiphilic steroidal compounds. All these evidences feed a continuing interest in the BA research aimed at designing and synthesizing new side chain- and body-modified derivatives endowed with improved biological and physico-chemical profiles, as well as with proper ADMET behaviour. In this context, the micellar aggregation of BAs, and the respective critical micellization concentration (CMC) value (determined on the BA sodium salt, BS), is considered a key parameter that needs to be determined in the preliminary phase of compound characterization, being implicated in cytotoxicity issues. An extraordinary variety of different analytical techniques and methods have been proposed along the years with the aim of better identifying the start of the self-aggregation process of BS monomers. The unicity of the physico-chemical nature of such class of compounds can be invoked to explain this unusual interest. Accordingly, a number of both invasive and non-invasive approaches have been developed along with a limited number of indirect chromatographic-based estimation strategies. Worth to be mentioned among the non-invasive determination methods are those based on potentiometry, freezing point depression, surface tension, nuclear magnetic resonance, viscosimetry, turbidimetry, microcalorimetry, refractometry, conductimetry, spectrophotometry, cholesterol solubilization, and monoglucuronide solubilization. Dye solubilization- and fluorescence-based methods deserve instead credit among the invasive methodological approaches. Indirect chromatographic methods based on capillary electrophoresis and high performance liquid chromatography analysis also demonstrated to be profitably exploited for the CMC estimation, especially when a small amount of sample is available. The collection of literature data reveals that the CMC value of a given BS is markedly related to the method selected for determining it as well as to the experimental conditions applied during the analysis.

S-Trityl-(R)-cysteine, a powerful chiral selector for the analytical and preparative ligand-exchange chromatography of amino acids

S-trityl-(R)-cysteine [(R)-STC] is the new selector of a dynamically coated, chiral ligand-exchange stationary phase which proved to be highly effective in both analytical and preparative-scale separation of enantiomers of some natural and unnatural underivatized amino acids, with good separation and resolution factors. With the aim of identifying the best chromatographic conditions suitable for the preparative-scale separations, some parameters controlling retention, separation and resolution factors (such as the type and amount of cupric salt and the eluent pH) were investigated. The relatively easy removal of the Cu(II) ions renders this technique suitable for obtaining small amounts of enantiomerically pure samples for preliminary biological evaluations.

The effect of the copper(II) salt anion in the Chiral Ligand-Exchange Chromatography of amino acids.

Due to the strong involvement in numerous biochemical processes, the separation and resolution of amino acids is a continuously challenging task. Among the experimental parameters affecting the performances in a Chiral Ligand-Exchange Chromatography (CLEC) environment, the effect of the copper(II) salt counter-ion has received very limited attention. Aimed at evaluating the Cu(II) counter-ion effect upon the overall chromatographic performances when the S-trityl-(R)-cysteine is adopted as the chiral selector for the coated stationary phase, a number of both organic (acetate, formate, trifluoromethane-sulfonate) and inorganic (bromide, chloride, nitrate, perchlorate, sulfate) salts has been engaged for the enantiomer separation of a selected set of aliphatic (Allo-Ile, Ile, Leu, Nor-Leu, Nor-Val, Val) and aromatic (AIDA, ATIDA, His, Phe, Phg, Tyr) amino acids. By varying the physico-chemical nature of the Cu(II) anion, a pronounced impact upon the resolution factor (R(S)) has been observed. Even if to a lesser extent, the enantioseparation factor values (alpha) underwent variation, as well. A molecular modelling investigation has also been carried out as a rationalization attempt of the observed chromatographic outcomes.

The effect of mobile phase composition in the enantioseparation of pharmaceutically relevant compounds with polysaccharide-based stationary phases

Mobile phase variables have a deep influence on the chromatographic behavior with polysaccharide-based chiral stationary phases. Basic additives are generally used to minimize peak broadening arising from unwanted interactions between polar solutes and underivatized silanols. However, basic additives can improve enantioselectivity through disruption of hydrogen bonds and modification of the polymer morphology. Acidic additives are incorporated into the mobile phase during the analysis of acidic compounds as efficiency enhancers. Acidic additives can also improve enantioselectivity by minimizing within the chiral recognition site nonenantioselective retention. Peak shape without acidic additive in the eluent could be severely distorted during the analysis of salified compounds. Concentration and type of alcohol modifier can have an effect on the morphology of the polymer. The different winding of the chiral selector, caused by alcohol modifiers of different size/shape, ultimately results in different stereo environment of the chiral cavities in the polymer chain. Trace amounts of water in normal-phase eluents can affect retention time, tailing, and resolution. Deliberate addition of water to the eluent can improve peak resolution and save analysis time and solvent needs. Immobilized-type polysaccharide-derived chiral stationary phases offer new selectivity profiles and often improved enantioselectivity.

Modulation of glutamate receptor pathways in the search for new neuroprotective agents

Excessive stimulation of excitatory amino acid (EAA) receptors is responsible for a wide variety of acute and chronic neurological impairments. A separate line of investigation has focused on oxidative stress as one of the main reasons for several of these degenerative disorders. Current evidence has confirmed that activation of both ionotropic and metabotropic glutamate receptors can also result in either neuroprotection or neurodegeneration according to the role played by oxidative stress mechanisms. An outline of this research, together with our recent results aimed at the discovery of new subtype selective modulators of the central nervous system pathways as well as new classes of free radical scavengers, is presented.

Direct enantioseparation of underivatized aliphatic 3-hydroxyalkanoic acids with a quinine-based zwitterionic chiral stationary phase.

While aliphatic 2-hydroxyalkanoic acids have been more or less successfully enantioseparated with various chiral stationary phases by HPLC and GC, analogous applications on underivatized aliphatic 3-hydroxyalkanoic acids are completely absent in the scientific literature. With the aim of closing this gap, the enantioseparation of 3-hydroxybutyric acid, 3-hydroxydecanoic acid and 3-hydroxymyristic acid has been performed with two ion-exchange type chiral stationary phases (CSPs): one containing the anion-exchange type tert-butyl carbamoyl quinine chiral selector motif (Chiralpak QN-AX), and the other carrying the new zwitterionic variant based on trans-(S,S)-2-aminocyclohexanesulfonic acid-derivatized quinine carbamate (Chiralpak ZWIX(+)) as the chiral selector and enantiodiscriminating element, respectively. The zwitterionic enantiorecognition material provided better results in terms of enantioselectivity and resolution compared to the anion-exchanger CSP at reduced retention times due to the intramolecular counterion effect imposed by the sulfonic acid moiety and its competition with the 3-hydroxyalkanoic acid analyte for ionic interaction at the quininium-anion exchanger site. It is thus recommended as the CSP of first choice for enantioseparations of the class of aliphatic 3-hydroxyalkanoic acids. With use of polar organic eluent composed of ACN/MeOH/AcOH - 95/5/0.05 (v/v/v), a good compromise in terms of analysis time and enantioresolution quality was accomplished. The major experimental variables have been investigated for optimization of the resolution and allowed to derive information on the enantiorecognition mechanism. Corresponding Chiralpak ZWIX(-), based on pseudo-enantiomeric selector derived from quinidine and trans-(R,R)-2-aminocyclohexanesulfonic acid with opposite configurations provided reversed enantiomer elution orders. It has further to be stressed that these separations can be obtained with mass spectrometry compatible mobile phases.

Pyrazole[3,4-e][1,4]thiazepin-7-one derivatives as a novel class of Farnesoid X Receptor (FXR) agonists

A virtual screening procedure was applied to the discovery of structurally diverse non-steroidal Farnesoid X Receptor (FXR) agonists. From 117 compounds selected by virtual screening, a total of 47 compounds were found to be FXR agonists, with 34 of them showing activity below a concentration of 20 μM. 1H-Pyrazole[3,4-e][1,4]thiazepin-7-one-based hit compound 7 was chosen for hit-to-lead optimization. A large number of 1H-pyrazole[3,4-e][1,4]thiazepin-7-one derivatives was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activity against FXR. Most of them exhibited low micromolar range of potency and very high efficacy.

Cysteine-based chiral selectors for the ligand-exchange separation of amino acids.

Three cysteine-based coated selectors, S-benzyl-(R)-cysteine, S-diphenylmethyl-(R)-cysteine and S-trityl-(R)-cysteine, have been used in the ligand-exchange separation of a selected set of natural and unnatural underivatized amino acids. With only few exceptions, a gain in enantiodiscrimination was obtained when the most lipophilic discriminating agent, characterized by the presence of a trityl moiety, was engaged. Moreover, a new descriptive structure-separation relationship study through molecular surface (Jurs) and shape (Shadow) descriptors provided evidence that specific physico-chemical features of the employed chiral selector result decisive in establishing which property of the analyte is responsible for the enantiorecognition accomplishment.