H2S-releasing properties of new thioamide, iminothioether and isothiocyanate derivatives

V Calderone1, A Martelli1, L Testai1, A Marino1, C Pellegrini1, I Pugliesi2, S Taliani1, G Nesi2, S Rapposelli2, F Da Settimo2, MC Breschi1. 1University of Pisa, Dept. of Psychiatry, Neurobiology, Pharmacology and Biotechnologies, I-56126 Pisa, Italy, 2University of Pisa, Dept. of Pharmaceutical Sciences, I-56126 Pisa, Italy

Presently, hydrogen sulphide (H2S) is emerging as an endogenous modulator [1] with important implications in controlling the homeostasis of the cardiovascular system. Indeed, its physiological concentrations induce vasorelaxing and cardioprotective effects [1, 2], while its deficiency has been associated with cardiocirculatory pathologies, such as hypertension. Therefore, the development of original H2S releasing agents is viewed as a timely and challenging issue, for both experimental purposes and potential perspectives for clinical use. In this study, some original compounds showing potential H2S-releasing moieties, such as benzothioamides, heterocyclic thioamides, heterocyclic iminothioether, or isothiocyanates, were tested. These compounds were incubated (1milliM) in PBS buffer (pH 7,4; 35-37°C), in the presence or in the absence of Cysteine 4 milliM. The well-known H2S-releasing derivatives diallyldisulphide (DADS) and GYY4137 were selected as reference drugs. The eventual generation of H2S was amperometrically measured, by means of an Apollo-4000 (WPI) detector and H2S-selective mini-electrodes, allowing a “real-time” determination of the H2S-release and a qualitative/quantitative description of the process.

Many of the tested compounds exhibited an interesting behaviour of slow H2S-donors, leading to a long-lasting production of stable concentrations of H2S, ranging in the microM order of magnitude. These H2S-releasing agents could be divided in two groups: some of them showed Cysteine-independent (i.e. spontaneous) mechanism of H2S release, while other derivatives required the presence of Cysteine, suggesting a thiol-mediated mechanism. Noteworthy, such a different feature could be closely related with clear structure/activity relationships. In particular, the incubation of benzothioamides and isothiocyanate derivatives led to a Cysteine-dependent release of H2S, with levels of maximal concentration at the steady state (Cmax) ranging between 10 and 16 microM and t50 values (time required to reach a half-value of Cmax) spanning between 1.5 and 2.5 min. These parameters were almost comparable with those exhibited by the reference drugs DADS and GYY4137, both endowed with Cysteine-dependent H2S-releasing properties. In the absence of Cysteine, the H2S-release from the new compounds and the reference agents was negligible. By contrast, the heterocyclic thioamides and the heterocyclic iminothioethers showed a Cysteine-independent behavior of H2S-donors, exhibiting Cmax of 5-20 microM and a t50 parameters of 1,5-2 min (both in the presence and in the absence of Cysteine). Both the different levels of effectiveness and the different mechanisms of H2S release represent features potentially useful for a rational development of new pharmacological agents, allowing us a) to select well calibrated properties of H2S-donors, able to release physiological concentrations of this mediator; b) to use chemically stable agents whose H2S-release is triggered only in biological environments by the presence of endogenous thiols.

Acknowledgements

This work was supported by the “Regional Health Research Program 2009” of Regione Toscana, Italy.

References

[1] Martelli A, Testai L, Breschi MC, Blandizzi C, Virdis A, Taddei S, Calderone V. Hydrogen sulphide: novel opportunity for drug discovery. Med Res Rev 2011; in press (published on-line DOI: 10.1002/med.20234).

[2] Zhao W, Zhang J, Lu Y, Wang R. The vasorelaxant effect of H2S as a novel endogenous gaseous K(ATP) channel opener. EMBO J 2001; 20: 6008-6016.