Resumen de Diseño sintesis y evaluacion biologica de nuevos derivados de fosforamida como inhibidores de ureasa
Maria Jose Dominguez Fernandez
"Design, synthesis and biological evaluation of new derivatives from phosphoramides as urease inhibitors". Mª José Domínguez Fernández. Science, University of Navarra, 2006.
Employment of nitrogen fertilizers in current agriculture is causing not only econòmic losses but also problems of environmental pollution and problems over human health. The most used nitrogen fertilizer is the urea because of its easy commercial availability, its high content of nitrogen per unit of product and its solubility in the soil solution. The urea is hydrolysed in soil by the urease enzyme and as a result ion ammonium will be real i sed which could be assimilated by plants. However, this is not a very stable form and could be transformed into ammonia that is loosed by volatilization or into ion nitrate that is loosed by lixiviation. This works is aimed at designing, synthesising and biologically evaluating new phosphoramides' derivates as urease inhibitors to reduce the losses of ammonium. Studies focusing on the active centre of the enzyme have used Molecular Modeling and subsequently the geomètric and electrònic properties that enable the interaction between an inhibitor compound and the enzyme have been defined and a bio specific affinity model has been proposed. 40 phosphorous derivate compounds were synthesized and were biologically evaluated against Jack Bean urease in Inabonos S.A. All the compounds were identified by the following techniques: infrared spectroscopy, nuclear magnètic resonance spectroscopy, mass spectrometry and elemental analysis.
Due to the difficulties found in the synthesis of phosphorous derivates it was necessary a meticulous adjustment on the reaction's conditions and the purification techniques in order to optimize the method. On some of the compounds, a Molecular Modeling study was carried out, describing the interaction between the enzyme active centre and the compounds and its characteristics related to the activity as urease inhibitors. On all the synthesized compounds, in vitro inhibitory activity tests were carried out. Twelve of the compounds showed IC50 vàlues inferior than 1 um and eight of them improved the activity of NBPT (100 nm), which is the only inhibitor marketed as Agrotain.
Based on the activity results and the conclusions of the Molecular Modeling study, a structural model for new potential inhibitors with the next structural requirements was defined :
- An oxygen atom substituted on the phosphorous (.P=0).
- Polar groups without substituent (NH2, OH) that interact with nickel àtoms in the active centre. - An heteroatom (NH, O), between phosphorous atom and the variable rest of the molecule, which allows an appropriate space distribution of the compounds in the active centre and favours the hydrolysis after the interaction with the nickel atoms.
- And, a variable aromatic or aliphatic fragment with electron-withdrawing groups replaced on the chain that polarize the P-Y bond (Y = the above-mentioned heteroatom) and allow the hydrolysis. Two of the compounds were evaluated in soil and showed higher activity than NBPT and better stability in soil at different pH.
