Sophia S. Michaelidou

Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity.

The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the proteins Cys residue(s). A dose-response analysis of the most active compounds gave IC(50) values in the range of 3-30μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein.

Synthesis of [(4-chloro-5H-1,2,3-dithiazol-5-ylidene)amino]azines.

The reactions of 2-, 3- and 4-aminopyridines with 4,5-dichloro-1,2,3-dithiazol-ium chloride (Appel salt) 4 to give N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)pyridin-X-amines 1a (X = 2), 1g (X = 3) and 1k (X = 4) were optimized with respect to base, temperature and reaction time. Based on these conditions a total of thirteen [(dithiazol-ylidene)amino]azines 1a-m were prepared and fully characterized.

1,2,3-Dithiazoles – new reversible melanin synthesis inhibitors: a chemical genomics study

A chemical genomic screen of an in-house library of small molecule heterocycles was carried out using Xenopus laevis embryos. This led to the identification of N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-4-methoxyaniline (1c), which elicits loss of pigmentation in melanophores and the retinal pigment epithelium (RPE) of developing embryos, independent of the developmental stage of initial exposure. The phenotype was reversible, since pigmentation returned upon compound removal while analysis of neural crest cell markers (Pax7) and melanophore markers (Dct/Xtrp2) revealed that both neural crest precursors and fully differentiated melanophores were present in the dithiazole 1c treated embryos. A subsequent focused structure–activity relationship (SAR) study identified the more active dithiazole 4-benzyloxy-N-(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-aniline (1l) and the need for a chlorine substituent at the dithiazole C-4 position. Both the initial chemical genomic screen and the focused SAR study highlighted the toxicity of (dithiazolylidene)aminoazines, and also of methoxyaniline (anisidine) analogues that hosted strong electron-withdrawing or electronegative substituents or acidic hydroxyl groups on the anisidine moiety. This study suggests that 1,2,3-dithiazoles can act as reversible melanin synthesis inhibitors, revealing a new biological activity for this class of compounds. The inhibition of melanin synthesis is medically relevant as a potential treatment for pigmentation disorders such as melasma.

(Z)-2-(4-Chloro-5H-1,2,3-dithiazol-5-ylideneamino)-6-ethoxy-4- phenylpyridine-3,5-dicarbonitrile

2-Amino-6-ethoxy-4-phenylpyridine-3,5-dicarbonitrile 1 reacts with 4,5-dichloro-1,2,3-dithiazolium chloride 2 in the presence of pyridine (2 equiv.) to afford (Z)-2-(4-chloro-5H-1,2,3-dithiazol-5-ylideneamino)-6-ethoxy-4-phenylpyridine-3,5- dicarbonitrile 3 in 88% yield.

3-Amino-6-ethoxy-4-phenyl-1H-pyrrolo[2,3-b]pyridine-2,5-dicarbonitrile

(Z)-2-(4-Chloro-5H-1,2,3-dithiazol-5-ylideneamino)-6-ethoxy-4-phenylpyridine-3,5-dicarbonitrile 1, when treated with either triphenylphosphine (4 equiv.) or polymer bound triphenylphosphine (5 equiv.) in dichloromethane at room temperature for 3 days affords 3‑amino-6-ethoxy-4-phenyl-1H-pyrrolo[2,3-b]pyridine-2,5-dicarbonitrile 2 in 60–62% yields.