Ennio Bousquet

Improvement of water solubility and dissolution rate of ursodeoxycholic acid and chenodeoxycholic acid by complexation with natural and modified β-cyclodextrins

The inclusion complexes of ursodeoxycholic and chenodeoxycholic acid with β-cyclodextrin, heptakis-(2,6-di-O-methyl)-β-cyclodextrin and soluble polymerized β-cyclodextrin were investigated in solution (1H-NMR spectrometry) and solid state (FT-IR spectroscopy and differential scanning calorimetry). Stability constants were determined at pH 7.4 and different temperatures and consequently thermodynamic parameters were obtained. All cyclodextrins are able to increase water solubility of the bile acids, particularly polymerized β-cyclodextrin. All complexes show high dissolution rate at 37°C and pH 1.1 and in particular freeze-dried complexes.

Calix[4]arene Decorated with Four Tn Antigen Glycomimetic Units and P3CS Immunoadjuvant : Synthesis, Characterization, and Anticancer Immunological Evaluation

A novel anticancer vaccine candidate built on a nonpeptidic scaffold has been synthesized. Four S-Tn tumor-associated glycomimetic antigens have been clustered onto a calix[4]arene scaffold bearing an immunoadjuvant moiety (P3CS). The immunogenicity of the synthetic construct has been investigated by immunization of mice in vivo. ELISA assay has evidenced that the tetravalent construct stimulates a higher production of anti-Tn antigen IgG antibodies when compared to an analogous monovalent compound. This result is ascribable to an antigen cluster effect and makes the reported vaccine candidate a good mimic of the natural motifs present on the mucine surface.

1-Alkylazacycloalkan-2-one esters as prodrugs of indomethacin for improved delivery through human skin

Abstract 1-Alkylazacycloalkan-2-one esters of indomethacin were synthesized and assayed to determine their stability in aqueous media, their susceptibility to undergoing in vitro enzymatic hydrolysis and their flux through excised human skin. 1-Methylazacycloalkan-2-one esters of indomethacin (I–IV) proved unstable in aqueous media while 1-ethylazacycloalkan-2-one esters (V–VIII) were much more stable. Esters V–VIII were readily hydrolyzed in vitro by porcine esterase and penetrated excised human skin better than the parent drug. The lipophilic index was determined by means of HPLC for esters I–VIII. Plotting the flux through the skin of esters V–VIII against their lipophilic index resulted in a parabolic relationship, the maximum being ester VI.

Capsular polysaccharide of Streptococcus pneumoniae type 19F: synthesis of the repeating unit

A new and more versatile synthesis of beta-D-ManpNAc-(1-->4)-alpha-D-Glcp-(1-->2)-alpha-L-Rhap, the trisaccharide repeating unit of the Streptococcus pneumoniae type 19F capsular polysaccharide, is described. The present approach allows a simple access to different fragments containing the trisaccharide and the conjugation of the product(s) to a protein through the selective manipulation of the anomeric position at the reducing end and of the HO-4 function at the nonreducing end. The synthetic scheme shows an efficient application of the sulfoxide method for the stereoselective and high yielding formation of the glycosidic linkages.

Analysis of aliphatic amines in air samples by HPLC with electrochemical detection.

A method was developed for the analysis of primary aliphatic amines by high performance liquid chromatography coupled with electrochemical detector. The electrochemical oxidation of aliphatic amines derivatized with 2,5-dihydroxybenzaldehyde was investigated at porous graphite electrodes. The derivatization reactions were performed off-line, before the chromatographic separation. The compounds were separated on a reversed phase column with a methanol-acetonitrile-phosphate buffer and detected setting at an oxidation potential of +0.5 V. The influence of the mobile phase buffer concentration and pH on the detector response was also studied. The derivatization was shown to be quantitative and the response linear between 50 and 200 ng/ml. The method is sensitive, selective and could be applicable for the assay of volatile amines in the field of environmental toxicology and also for biological monitoring after occupational exposure.

Synthesis and Immunostimulating Activity of A Thioglycolipopeptide Glycomimetic As A Potential Anticancer Vaccine Derived From Tn Antigen

ABSTRACT The Tn epitope is one of the tumor associated O-linked cell surface glycopeptides. It is expressed in over 70% of human epithelial cancers such as lung, colon, stomach and breast carcinomas. The glycosidic linkage of the Tn antigen, between N-acetylgalactosamine and serine or threonine, can be cleaved either chemically or enzymatically in the presence of glycosidases. The latter case is particularly a problem in vivo. Therefore, it would be of great interest to obtain a metabolically stable analogue of the Tn antigen that maintains or improves the immunogenic activity of the latter. The purpose of this work was to synthesize a totally synthetic vaccine using a chemically and metabolically stable glycomimetic of the Tn antigen in which the interglycosidic oxygen was replaced by a sulphur atom (S-Tn). The S-Tn thioglycopeptide was linked to the P3CS immunoadjuvant to obtain the potential S-Tn vaccine. Moreover, we synthesized the natural Tn antigen and derivatized it similarly to obtain the Tn vaccine. Last, we evaluated the immunostimulating activity of the two synthetic potential vaccines in vitro using cultured mouse splenocytes. The S-Tn construct showed immunostimulating activity comparable, in terms of maximal response, to the Tn analogue. Moreover, due to its higher stability the S-Tn construct reached its maximal effect at lower doses compared to the Tn analogue.

4-Quinazolinones: synthesis and reduction of prostaglandin E2 production

We synthesized and evaluated the anti-inflammatory activity of a series of 4-quinazolinone derivatives. Two approaches were used to yield the title compounds. A first group of quinazolinone derivatives was obtained by the appropriate substituted anthranilates. A second group of quinazolinone compounds was prepared through the benzoxazin-4-ones intermediate. The pharmacological results reveal that the synthesized derivatives exhibit a significant anti-inflammatory effect in an experimental ocular inflammation model. In fact, all the tested compounds lowered the prostaglandin E2 (PGE2) production with respect to the control group (P < 0.05). The 3-cyclohexyl-6-chloro-quinazolin-4(3H)-one and 3-cyclohexyl-quinazolin-4(3H)-one derivatives were the most active compounds. These compounds significantly reduced PGE2 levels even more than the reference drug tolmetin and significantly lower protein concentration and polymorphonuclear leukocytes number compared to the control group (P < 0.05). Therefore, these compounds may be useful to prevent ocular inflammatory reactions.

Synthesis of (+)- and (−)-cis-2-[(1-adamantylamino)-methyl]-1-phenylcyclopropane derivatives as high affinity probes for σ1 and σ2 binding sites

Selective ligands for either sigma1 (σ1) or σ2 binding sites are potentially useful for gaining a better understanding of the physiological functions of these proteins. Moreover, potent and selective homochiral σ1 and σ2 binding site ligands represent leads to potential radioligands for tumour imaging with positron emission tomography (PET). On the basis of their structural similarity to previous leads, new (+)- and (−)-cis-2-[(1-adamantylamino)-methyl]-1-phenylcyclopropane derivatives were synthesised and their binding affinities for σ1 and σ2 binding sites were determined. Each enantiomer showed high affinity for both σ1 and σ2 binding sites, but only (−)-cis-methyl-2-{[1-adamantyl(methyl)amino]methyl}-1-phenylcyclopropane-carboxylate, (−)-4, showed appreciable selectivity for binding to σ1 versus σ2 sites. The enantiomers of cis-(2-{[1-adamantyl(methyl)amino]methyl}-1-phenylcyclopropyl)methanol, 6, expressed the highest affinity for σ1 and σ2 binding sites. Ligands (−)-4, (+)-6 and (−)-6 might be rapidly labelled in their N-methyl groups by methylation of the N-desmethyl analogues with [11C]iodomethane to provide prospective radioligands for PET. The N-desmethyl analogues, which are also high affinity ligands, were prepared and shown to undergo satisfactory methylation with iodomethane.

Synthesis and spasmolytic action of 2-substituted thienopyrimidin-4-one derivatives.

In the search for novel compounds to treat disorders of smooth muscle function, efforts have focused on some 2‐substituted thieno[2,3‐d]pyrimidin‐4‐onederivatives that show interesting spasmolytic action. Our laboratories have developed a new series of quaternary salts of 2‐substituted thieno[2,3‐d]pyrimidin‐4‐one and thieno[3,2‐d]pyrimidin‐4‐oneisomers with therapeutic potential. These substances were prepared starting from simple derivatives of thiophene. Their spasmolytic activity was evaluated on transmurally stimulated guinea‐pig ileum. The most active compounds (IC50 1.12‐2.71 μM) 7f‐7h, 12d and 12f had the terminal piperidino nucleus in the thioalkyl chain and lacked two methyl groups in the thiophene ring. Their relaxant activity on the isolated ileum was potentiated (approx. 20–25%) by phosphodiesterase inhibitors. Compounds 7f‐h, 12d and 12f were less effective in inhibiting contractions of the guinea‐pig ileum induced by acetylcholine (IC50 26.7–41.4 μM) or histamine (IC5041.5–63.4 μM) and had a moderate binding activity to muscarinic receptors in membrane homogenates from the rat heart (M2 sites; pKi values between 5.55 ± 0.08 and 5.14 ± 0.12; n = 3) and submaxillary gland (M3 sites; pKi values between 6.15 ± 0.07 and 5.76 ± 0.08; n = 3). Action involving soluble guanylyl cyclase or any potential binding to guinea‐pig ventricular L‐type calcium channels was not considered likely. It is concluded that at least two different mechanisms of action contribute to their spasmolytic activity.

Determination of Chenodeoxycholic Acid in Pharmaceutical Preparations of Ursodeoxycholic Acid by High Performance Liquid Chromatography with Coulometric Electrochemical Detection

1-(2,5-Dihydroxyphenyl)-2-bromoethanone (2,5-DBE) has been used as an electroactive labelling reagent for high performance liquid chromatographic (HPLC) analysis of chenodeoxycholic acid impurity in pharmaceutical formulations of ursodeoxycholic acid. The preparation of this reagent has been performed in a single step by bromination of 2,5-dihydroxyacetophenone (2,5-DAP), with remarkable improvement as regards the synthesis previously reported. Several experiments have been performed in order to give maximum conversion of the bile acids to their electroactive esters. The determination of the derivatized compounds has been carried out on an Adsorbosphere column with a methanol-acetonitrile-acetate buffer eluent and detected by a porous graphite electrode set at an oxidation potential of + 0.6 V. The high sensitivity and specificity shown by this novel method allows the determination of low concentrations of the cited impurity in capsule and tablet formulations containing the above therapeutic agent.