Design, Synthesis and Bioactivity Evaluation of Heterocycle-Containing Mono- and Bisphosphonic Acid Compounds

Abstract

Fosmidomycin (FOS) is a naturally occurring compound active against the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, and using it as a template for lead structure design is an effective strategy to develop new active compounds. In this work, by replacing the hydroxamate unit of FOS with pyrazole, isoxazole and the related heterocycles that also have metal ion binding affinity, while retaining the monophosphonic acid in FOS or replacing it with a bisphosphonic acid group, heterocycle-containing mono- and bisphosphonic acid compounds as FOS analogs were designed. The key steps involved in the facile synthesis of these FOS analogs included the Michael addition of diethyl vinylphosphonate or tetraethyl vinylidenebisphosphonate to β-dicarbonyl compounds and the subsequent cyclic condensation with hydrazine or hydroxylamine. Two additional isoxazolinone-bearing FOS analogs were synthesized via the Michaelis-Becker reaction with diethyl phosphite as a key step. The bioactivity evaluation on model plants demonstrated that several compounds have better herbicidal activities compared to FOS, with the most active compound showing a 3.7-fold inhibitory activity on Arabidopsis thaliana, while on the roots and stalks of Brassica napus L. and Echinochloa crus-galli in a pre-emergence inhibitory activity test, the activities of this compound were found to be 3.2- and 14.3-fold and 5.4- and 9.4-fold, respectively, and in a post-emergency activity test on Amaranthus retroflexus and Echinochloa crus-galli, 2.2- and 2.0-fold inhibition activities were displayed. Despite the significant herbicidal activity, this compound exhibited a DXR inhibitory activity lower than that of FOS but comparable to that of other non-hydroxamate DXR inhibitors, and the dimethylallyl pyrophosphate rescue assay gave no statistical significance, suggesting that a different target might be involved in the inhibiting process. This work demonstrates that using bioisosteric replacement can be considered as a valuable strategy to discover new FOS analogs that may have high herbicidal activities.

Keywords: DXR; bisphosphonic acid; herbicidal activity; heterocycle; monophosphonic acid.

Figure 1

The MEP pathway for the synthesis of the isoprenoid precursors, IPP and DMAPP, that lead to two essential isoprenoid products, phytol and β-carotene, as examples in plants, with the function of DXR enzyme in converting DXP to MEP highlighted.

Figure 2

Structures of two naturally occurring DXR inhibitors, FOS and FR, and reported non-hydroxamate DXR inhibitors (A), exemplified nitrogen-containing heterocycles in commercial herbicides (B), and the design strategy for heterocycle-containing mono- and bisphosphonic acids in this work (C).

Scheme 1

Synthesis of the monophosphonic acid series 5 and 6. Reagents and conditions: (a) RCOCH₂COOEt or RCOCH₂COR, K₂CO₃, TEBAC, CH₃CN, 80 °C; (b) N₂H₄·H₂O, EtOH, 75 °C; (c) NH₂OH·HCl, K₂CO₃, EtOH, 75 °C; (d) (i) TMSBr, CH₂Cl₂, 0 °C to r.t.; (ii) THF/H₂O, r.t.

Scheme 2

Synthesis of the bisphosphonic acid series 12 and 13. Reagents and conditions: (a) (i) (CHO)_n, Et₂NH, MeOH, 65 °C; (ii) TsOH, toluene, 110 °C; (b) RCOCH₂COOEt or RCOCH₂COR, LiHMDS, THF, 0 °C to r.t.; (c) N₂H₄·H₂O, EtOH, 75 °C; (d) NH₂OH·HCl, K₂CO₃, EtOH, 75 °C; (e) (i) TMSBr, CH₂Cl₂, 0 °C to r.t.; (ii) THF/H₂O, r.t.

Scheme 3

Synthesis of the target compounds 19 and 20. (a) (i) NH₂OH·HCl, NaOH, H₂O, 0 °C to r.t.; (ii) NaOH, Na₂CO₃, H₂O, 45 °C; (b) Br(CH₂)₃Br, NaH, DMF, 0 °C to r.t.; (c) HPO(OEt)₂ or 7, Cs₂CO₃, TBAI, DMF, r.t.; (d) (i) TMSBr, CH₂Cl₂, 0 °C to r.t.; (ii) THF/H₂O, r.t.

Figure 3

Inhibition and bleaching effects of the 10 active compounds on Arabidopsis.

Figure 4

Relative activities of the 10 active compounds, plus compounds 19 and 20, on the root and stalk of BN and EC. The red/blue bars represent relative activities higher/lower than that (yellow) of FOS, respectively.

Figure 5

Rescue of bleaching and developmental arrest in Arabidopsis by adding exogenous DMAPP. (A) Images of 13e- and FOS-treated Arabidopsis before and after rescue with DMAPP, along with the control check (CK). The concentrations of 13e and FOS were 20 mg/L and 40 mg/L, respectively, and the DMAPP concentration was 150 mg/L. (B) Green channel pixel values of Arabidopsis images after different treatments. ** p < 0.01; ns: no significance.

Figure 6

The docked conformation of 13e with DXR enzyme and the interactions with the surrounding residues and (A) the conformational superimposition of 13e with CBQ and FOS in the DXR active site (B). Key residues are shown as slate sticks, the hydrogen bonds and coordinate bonds are highlighted in yellow dashed lines and the Mg²⁺ ion is presented as a wheat sphere.