Bulky 1,4-benzoxazine derivatives with antifungal activity.

For many years the development of new azole antifungals has been quite empirically based. More recently, the publication of the crystal structure of CYP51 of Mycobacterium tuberculosis (MT-CYP51) provided new opportunities to rationalize the knowledge about antifungal action of this class of compounds. Recent studies reported that a 'channel 2 opened' conformation of the enzyme could better explain the interaction with ketoconazole (KTZ)-like drugs. Conformational changes were made on our model of Candida albicans CYP51 (CA-CYP51) previously reported and docking experiments were performed. The results allowed new KTZ analogues to be designed, by predicting that the 1,4-benzoxazine moiety could replace the KTZ aryl-piperazinyl chain. The synthesis of derivatives 12 and 13 was planned. The in vitro antifungal activity was evaluated against different Candida species and low and high capsulated strains of Cryptococcus neoformans. Since the in vitro activity do not necessarily correlate with the in vivo antifungal activity the newly synthesized compounds were also tested in a murine model of systemic C. albicans infection. The therapeutic effect was evaluated in terms of animal survival and of fungal growth in the kidneys, the target organ in systemic candidiasis.

Oxime and oxime ether derivatives of 1,4-benzothiazine related to oxiconazole.

The synthesis, in vitro antifungal activity, and molecular docking experiments of some oxime and oxime ether derivatives of azole 1,4-benzothiazine are reported herein, with the aim of evaluating the influence of a partially constrained scaffold that is structurally related to Oxiconazole and bearing the 1,4-benzothiazine moiety, on the inhibition of Candida albicans CYP51.

Insight into the inhibition of human choline kinase: homology modeling and molecular dynamics simulations.

A homology model of human choline kinase (CK-alpha) based on the X-ray crystallographic structure of C. elegans choline kinase (CKA-2) is presented. Molecular dynamics simulations performed on CK-alpha confirm the quality of the model, and also support the putative ATP and choline binding sites. A good correlation between the MD results and reported CKA-2 mutagenesis assays has been found for the main residues involved in catalytic activity. Preliminary docking studies performed on the CK-alpha model indicate that inhibitors can bind to the binding sites of both substrates (ATP and choline). A possible reason for inhibition of choline kinase by Ca(2+) ion is also proposed.

Novel ketoconazole analogues based on the replacement of 2,4-dichlorophenyl group with 1,4-benzothiazine moiety: design, synthesis, and microbiological evaluation.

As a part of a program to develop novel antifungal agents, new compounds which incorporate the 1,4-benzothiazine moiety into the structure of ketoconazole (KTZ) were prepared. These compounds were computationally investigated to assess whether the 1,4-benzothiazine moiety was a suitable bioisosteric replacement for the 2,4-dichlorophenyl group of KTZ in order to obtain a more potent inhibition of CYP51 enzyme of Candida albicans. Results of preliminary microbiological studies show that the racemic cis-7 analogue has a good in vivo activity, comparable to that of KTZ, but the best activity was observed in the racemic trans-7 analogue.

Design, synthesis, and microbiological evaluation of new Candida albicans CYP51 inhibitors.

In a program aimed at the design and synthesis of novel azole inhibitors of Candida albicans CYP51 (CA-CYP51), a series of azole 1,4-benzothiazines (BT) and 1,4-benzoxazines (BO) were recently synthesized. A morphological study of the enzyme active site highlighted a hydrophobic access channel, and a docking study pointed out that the C-2 position of the BT or BO nucleus was oriented toward the access channel. Here, we report the design, synthesis, and microbiological evaluation of C-2-alkyl BT and BO compounds. In both series, introduction of the alkyl chain maintained and in some cases improved the anti-Candida in vitro activity; however, there was not always a strict correlation between in vitro and in vivo activity for several compounds.

Synthesis and evaluation of anti-apoptotic activity of L-carnitine cyclic analogues and amino acid derivatives.

Two series of derivatives were synthesised. In one series (R)-4-hydroxy-2-pyrrolidinone was used as a mimic of cyclic L-carnitine analogue and in the second series 3-amino-2-piperidinone was used as a cyclic ornithine analogue. N-Benzyloxycarbonyl derivatives of some amino acids were also prepared. The newly synthesised compounds were tested for their ability to inhibit Fas-activated apoptosis of human Jurkatt T-cell line. The results confirm the previously described anti-apoptotic activity of carnitine and indicate new carnitine and amino acid analogues (1, 3, 6, 7, 20) that inhibit Fas-induced apoptosis.

1,4-benzothiazine analogues and apoptosis: structure-activity relationship.

We have previously shown 1,4-benzothiazine (1,4-B) derivatives induce thymocyte apoptosis in vitro and thymus cell loss in vivo. Apoptosis is mediated through a complex of biochemical events including phosphatidylcholine specific-phospholipase C (PC-PLC) activation, acidic sphingomyelinase (aSMase) activation and ceramide generation, caspase-8 and caspase-3 activation. As preliminary analysis of the structure-activity relationship (SAR) suggested some structural features were responsible for apoptosis, we synthesised several derivatives and tested for apoptosis activity at equimolar concentrations. In particular, we synthesised analogues that differed in the nature of skeleton (1,4-benzothiazine, 1,4-benzoxazine and 1,2,3,4-tetrahydroquinoline) and in the nature of side chain (imidazole, benzimidazole or piperazine as azole substituent; presence, absence or transformation of alcoholic group). Results of apoptosis induction indicate that transforming the 1,4-benzothiazine skeleton into 1,2,3,4-tetrahydroquinoline does not result in significant change. Transformation into 1,4-benzoxazine decreased activity. Replacing imidazole at the side chain with different piperazines also decreased activity while replacing it with benzimidazole does not change apoptotic activity. Finally, removal of the alcoholic group by dehydration to olefin, or by transforming it into ether, increased activity. Moreover, in an attempt to analyse further the SAR characteristics that are responsible for 1,4-B-activated apoptosis we tested the effect on caspase-8,-9 and-3 activation. 1,4-B analogues activate caspases and the structural requirements correlate with those responsible for apoptosis induction.