ß-Hairpin mimics containing a piperidine-pyrrolidine scaffold modulate the ß-amyloid aggregation process preserving the monomer species.

Alzheimer's disease is a neurodegenerative disorder linked to oligomerization and fibrillization of amyloid ß peptides, with Aß1-42 being the most aggregative and neurotoxic one. We report herein the synthesis and conformational analysis of Aß1-42-amyloid related ß-hairpin peptidomimetics, built on a piperidine-pyrrolidine semi rigid ß-turn inducer and bearing two small recognition peptide sequences, designed on oligomeric and fibril structures of Aß1-42. According to these peptide sequences, a stable ß-hairpin or a dynamic equilibrium between two possible architectures was observed. These original constructs are able to greatly delay the kinetics of Aß1-42 aggregation process as demonstrated by thioflavin-T fluorescence, and transmission electron microscopy. Capillary electrophoresis indicates their ability to preserve the monomer species, inhibiting the formation of toxic oligomers. Furthermore, compounds protect against toxic effects of Aß on neuroblastoma cells even at substoichiometric concentrations. This study is the first example of acyclic small ß-hairpin mimics possessing such a highly efficient anti-aggregation activity. The protective effect is more pronounced than that observed with molecules which have undergone clinical trials. The structural elements made in this study provide valuable insights in the understanding of the aggregation process and insights to explore the design of novel acyclic ß-hairpin targeting other types of amyloid-forming proteins.

Asymmetric synthesis of 2-amino-3-hydroxynorbornene-2-carboxylic acid derivatives.

The enantioselective synthesis of 2-amino-3-hydroxynorbornene-2-carboxylic acid derivatives (5) was studied using the Diels-Alder reaction between cyclopentadiene and different dienophiles, i.e., alkyl 5-oxo-2-phenyloxazol-4-methylenecarbonates (1) or 2-benzoylamino-3-alkoxycarbonyloxy-acrylates (12), operating with different Lewis acids and both with thermal and with ultrasound conditions. The enantioselective synthesis of the exo/endo compounds 5c,d and 5'c,d was achieved starting from the chiral menthyl acrylates 12b,c using Mg(ClO(4))(2) as the catalyst and ultrasound. The cycloadducts were obtained in very good yield, in mild conditions, in short time, and in good diastereomeric excess (exo, 80%; endo, 87%). Finally, the use of alkylidene-oxazolones or acrylates and EtAlCl(2) or Mg(ClO(4))(2) as the catalyst allowed control of the cycloaddition reaction in favor of the exo or endo products.

Conformationally constrained serine analogues: synthesis of new 2-amino-3-hydroxynorbornanecarboxylic acid derivatives.

A synthesis of the new oxazolone 2, functionalized with the ethoxycarbonyloxy group on methylenic carbon, is presented, starting from 4-hydroxymethylenoxazolone 1 and ethyl chlorocarbonate. Oxazolone 2 was reacted with cyclopentadiene in the presence of EtAlCl(2), giving the two diastereoisomeric cycloadducts exo-3 and endo-3 in a 70:30 ratio. Selective hydrolysis of the lactone ring (THF, HCl) gave the corresponding acids 5 and 6 which were transformed into hydroxyacid derivatives 7 and 8, respectively, operating in an ethanolic solution of Me(2)NH. The new 3-hydroxy-2-aminonorbornane-2-carboxylic acids 11 and 12, in which the serine skeleton is included, were obtained by reduction of acids 5 and 6 to derivatives 9 and 10 and a subsequent hydrolysis with HCl.

N-deacetyl-N-aminoacylthiocolchicine derivatives: synthesis and biological evaluation on MDR-positive and MDR-negative human cancer cell lines.

A new series of N-deacetyl-N-(N-trifluoroacetylaminoacyl)thiocolchicine derivatives 9-15 have been synthesized starting from the corresponding N-deacetylthiocolchicine (3) and the N-trifluoroacetylamino acids 5-8 which were used as a racemic mixture. The trifluoroacetyl protecting group has been removed easily, giving the corresponding N-deacetyl-N-aminoacylthiocolchicines 16-22. Optical pure compounds 9-22 were isolated from the diastereoisomeric mixture or were prepared starting from compound 3 and an optical pure amino acid derivative; the configuration of each compound was assigned unequivocally. The diastereoisomeric couples of amino acids synthesized were tested, and their antiproliferative activity on MDR-positive and MDR-negative human cancer cell lines was evaluated.

A Novel Class of Conformationally Constrained, Masked Cysteines: Synthesis of 2-Alkyl- and 2-Arylsulfanyl-1-aminocyclopropanecarboxylic Acids.

A convenient synthesis of 4-sulfanylmethylene-5(4H)-oxazolones 3 was realized starting from 4-(chloromethylene)oxazolone 1 and mercaptans 2. Oxazolones 3 were used as starting materials for the preparation of unknown 2-sulfanyl-1-aminocyclopropanecarboxylic acid derivatives 5 and7. Oxazolones 3 were cyclopropanated at the exocyclic double bond with diazomethane, giving a mixture of the two (Z)- and (E)-spirocyclopropane oxazolones 4 with good diastereoselectivity. These were then treated with ethanol and DMAP to produce the corresponding carboxylates 5. The trityl derivative 5d was converted into a mixture of diastereoisomeric disulfides 6 using iodine in ethanol solution. Disulfides 6 are convenient synthons for the preparation of 3-sulfanyl-substituted 2,3-methanoamino acids 7.

Antiproliferative activity of colchicine analogues on MDR-positive and MDR-negative human cancer cell lines.

In this study the in vitro antitumor activity of a series of 20 colchicine analogues was tested and compared with colchicine and thiocolchicine on three different human cancer cell lines, two of which express the multidrug-resistance (MDR) phenotype. At concentrations from 1 nM to 100 microM, all compounds tested inhibited cancer cell proliferation. The IC50 values indicate that the three fluorinated analogues were the most active compounds, with a similar decreasing order of potency (IDN 5005 > IDN 5079 > IDN 5080) on the two MDR-expressing cell lines, whereas thiocolchicine was the most effective compound on the MDR-negative MDA-MB 231 cells. A strong correlation (r = 0.94; P = 0.004) was found between IC50 values obtained using the two MDR-positive cell lines. Conversely, IC50 values obtained in MDA-MB 231 cells did not show a significant correlation with MDR-positive cell lines, thereby suggesting some difference in the antiproliferative mechanism(s) of colchicine analogues. Cell cycle analysis of the most active analogues in breast cancer cells showed a relationship between cell cycle blocking activity and growth inhibition. The most active agents on the MDR-positive MCF7 ADRr cell line, after 24 h of culture, in terms of cell cycle blocking activity were the three fluorinated analogues. Interestingly, after 72 h, when the cell cycle block subsided, a consistent amount of DNA fragmentation was evident. The extent of cell cycle block, measured as the G2/G1 ratio, was significantly correlated with the apoptosis rate expressed as a percentage of DNA fragmentation on both cell lines, thereby suggesting that a large number of blocked cells underwent the apoptotic pathway.