(S)-α-methyl,α-amino acids: a new stereocontrolled synthesis

A new and convenient stereocontrolled synthesis of the optically pure (S)-α-methyl,α-amino acids 6(a–d) that exploits the chiral synthon 1,4-N,N-[(S)-1-phenylethyl]-piperazine-2,5-dione (1) is described. The (S)-1-phenylethyl group, bonded to each of the N-atoms of the 2,5-diketopiperazine, acts as a chiral inductor in the first alkylation, while the steric hindrance appears to be the determining factor of stereocontrol in third and forth alkylation.     

Rapid sexual maturity and short life span in the blue-legged frog and the rainbow frog from the arid Isalo Massif, southern-central Madagascar

Longevity and age at sexual maturity were estimated in two anurans from the arid Isalo Massif (southern-central Madagascar), the blue-legged frog (Mantella expectata) and the rainbow frog (Scaphiophryne gottlebei). Phalanges from 69 individuals of M. expectata and 38 individuals of S. gottlebei were analyzed, using the skeletochronological method, in samples collected during two periods: January-February and November-December 2004. The male gonads of both species were also analyzed in order to better correlate reproductive activity with phenology. The phalangeal diaphysis in adults of both species was composed of two concentric bone layers: an innermost endosteal bone, which was less developed or sometimes lacking in S. gottlebei, and an outermost and broader layer of periosteal bone. Lines of arrested growth (LAGs) were observed in both species, although their recognition was more problematic and their distinctiveness much less evident in S. gottlebei. The results presented here indicate that M. expectata and S. gottlebei have a short life span and attain sexual maturity within the first active season after metamorphosis. Maximum longevity was 3 years in M. expectata and 2 years in S. gottlebei. In S. gottlebei the adult body size is likely attained during the same season in which metamorphosis occurs, but then breeding occurs only after the first latency period. Thus, mature individuals have only one LAG, corresponding to 1 year. The low number of individuals with two LAGs suggests that most animals die before the second latency period.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopy

Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour‐suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down‐regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N‐terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein–protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti‐cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd.     

2-Methyl-3-furanyl-4H-1,2,4-triazol-3-ylthioamides: a new class of selective orexin 2 antagonists

A new class of selective orexin 2 antagonist was identified among commercial products. Initial SAR was obtained using commercial derivatives only prior to starting ad hoc medicinal chemistry activities.     

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results.     

Novel benzothiophene H1-antihistamines for the treatment of insomnia

SAR of lead benzothiophene H₁-antihistamine 2 was explored to identify backup candidates with suitable pharmacokinetic profiles for an insomnia program. Several potent and selective H₁-antihistamines with a range of projected half-lives in humans were identified. Compound 16d had a suitable human half-life as demonstrated in a human microdose study, but variability in pharmacokinetic profile, attributed to metabolic clearance, prevented further development of this compound. Compound 28b demonstrated lower predicted clearance in preclinical studies, and may represent a more suitable backup compound.     

Identification of a novel selective H1-antihistamine with optimized pharmacokinetic properties for clinical evaluation in the treatment of insomnia

Analogs of the known H₁-antihistamine R-dimethindene with suitable selectivity for key GPCRs, P450 enzymes and hERG channel were assessed for metabolism profile and in vivo properties. Several analogs were determined to exhibit diverse metabolism. One of these compounds, 10a, showed equivalent efficacy in a rat EEG/EMG model to a previously identified clinical candidate and a potentially superior pharmacokinetic profile as determined from a human microdose study.