Using the structure of (3,5-dichlorophenyl)aminomethylenebisphosphonic acid as a lead compound, 25 new phosphonates were synthesized and evaluated as possible inhibitors of Arabidopsis thaliana delta1-pyrroline-5-carboxylate (P5C) reductase. Derivatives substituted in the phenyl ring retained the inhibitory potential, though to a different extent. On the contrary any variation in the scaffold, i.e., the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of biological activity. The availability of several structures capable of interfering with the catalytic mechanism in the micromolar to millimolar range allowed a proper structure-activity relationship analysis, leading us to hypothesize about the steric and electronic requirements for maintenance or enhancement of the inhibitory properties. Reversal experiments with suspension cultured cells provided evidence for the occurrence of enzyme inhibition in vivo. Because in higher plants the step catalyzed by P5C reductase is shared by all pathways leading to proline synthesis, these compounds may be exploited for the design of new substances endowed with herbicidal activity.

Analogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthesized and evaluated as inhibitors of Arabidopsis thaliana δ(1)-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness, two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 μM. Experimental evidence supporting the ability of these inhibitors to interfere with proline synthesis in vivo is also shown.

BACKGROUND: Aiming at the rational design of new herbicides, the availability of the threedimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry.

Alpha-cyclodextrin was shown to be convenient chemical shift reagent for determination of the enantiomeric composition of alpha-hydroxyphosphonic acids by means of 31P NMR. The developed methodology appeared to be reliable, repetitive, easy to perform and simple for interpretation. Enantiomeric discrimination in the 31P NMR spectra for 12 of 13 studied hydroxyphosphonates was achieved, with baseline separation of resonances obtained for eight compounds. In those cases, the chemical nonequivalence values ranged from 0.069 to 0.313 ppm. The studies showed that enantioselectivity is strongly influenced by the solution pD and the optimal condition was found at pD 2 or 10 depending on the guest structure. On the basis of the ROESY spectra the complexation modes of selected hydroxyphosphonates with alpha-cyclodextrin was postulated.