Potential of Vitamin B6 Dioxime Analogues to Act as Cholinesterase Ligands

Abstract

Seven pyridoxal dioxime quaternary salts (1-7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). All compounds, prepared in good yields (43-76%) and characterized by 1D and 2D NMR spectroscopy, were evaluated as reversible inhibitors of cholinesterase and/or reactivators of enzymes inhibited by toxic organophosphorus compounds. Their potency was compared with that of their monooxime analogues and medically approved oxime HI-6. The obtained pyridoxal dioximes were relatively weak inhibitors for both enzymes (K_i = 100-400 µM). The second oxime group in the structure did not improve the binding compared to the monooxime analogues. The same was observed for reactivation of VX-, tabun-, and paraoxon-inhibited AChE and BChE, where no significant efficiency burst was noted. In silico analysis and molecular docking studies connected the kinetic data to the structural features of the tested compound, showing that the low binding affinity and reactivation efficacy may be a consequence of a bulk structure hindering important reactive groups. The tested dioximes were non-toxic to human neuroblastoma cells (SH-SY5Y) and human embryonal kidney cells (HEK293).

Keywords: AChE; BChE; cytotoxicity; dioxime; molecular docking; pyridoxal oxime; reactivation; reversible inhibition.

Scheme 1

A general schematic representation of the preparation of pyridoxal dioximes.

Figure 1

The chemical structures of the tested pyridoxal dioximes 1–7 and medically approved oxime HI-6.

Figure 2

Schematic presentation of the S_N1 mechanism occurring in pyridoxal dioxime synthesis.

Figure 3

Enumeration scheme for the assignment of the NMR spectra when R=H, F, Cl, Br, NO₂, OCH₃, or phenyl.

Figure 4

Part of the ¹H-¹³C HMBC spectra (600 MHz, DMSO-d₆) of 3. The ovals indicate cross peaks, important for the assignment and confirmation of the synthesized compound. The part of the ¹H NMR spectrum is shown at the top and part of the ¹³C NMR spectrum is shown at the left edge.

Figure 5

Physicochemical characteristics (molecular weight (M), number of hydrogen bond donors and acceptors (HBDs and HBAs), number of rotating bonds (RBs), lipophilicity (logP), and topological polar surface area (TPSA)) of pyridoxal dioximes in relation to the reference literature values given by the red dashed line [30].

Figure 6

Close-up of the active site of the model complexes between AChE and compound 1 (A) or 7 (B) and BChE and compound 7 (C). Dashed lines represent different types of non-bonding interactions. Red spheres represent conserved water molecules. For clarity, only water molecules predicted to be engaged in non-bonding interactions are shown.