Verubulin (Azixa) Analogues with Increased Saturation: Synthesis, SAR and Encapsulation in Biocompatible Nanocontainers Based on Ca2+ or Mg2+ Cross-Linked Alginate

Abstract

Tubulin-targeting agents attract undiminished attention as promising compounds for the design of anti-cancer drugs. Verubulin is a potent tubulin polymerization inhibitor, binding to colchicine-binding sites. In the present work, a series of verubulin analogues containing a cyclohexane or cycloheptane ring 1,2-annulated with pyrimidine moiety and various substituents in positions 2 and 4 of pyrimidine were obtained and their cytotoxicity towards cancer and non-cancerous cell lines was estimated. The investigated compounds revealed activity against various cancer cell lines with IC₅₀ down to 1-4 nM. According to fluorescent microscopy data, compounds that showed cytotoxicity in the MTT test disrupt the normal cytoskeleton of the cell in a pattern similar to that for combretastatin A-4. The hit compound (N-(4-methoxyphenyl)-N,2-dimethyl-5,6,7,8-tetrahydroquinazolin-4-amine) was encapsulated in biocompatible nanocontainers based on Ca²⁺ or Mg²⁺ cross-linked alginate and it was demonstrated that its cytotoxic activity was preserved after encapsulation.

Keywords: apoptosis inducer; aromatic nucleophilic substitution; cross-linked alginate; nanocontainer; pyrimidine; tetrahydroquinazoline; tubulin polymerization inhibitor; verubulin.

Figure 1

Verubulin (1) [36], some of its previously described analogues [25,33,34] and compounds studied in the present work.

Scheme 1

Synthesis of 4-aminopyrimidines 2a–l.

Scheme 2

Synthesis of 4-aminopyrimidines 2m–r.

Figure 2

Immunofluorescence microscopy images of the microtubules (MTs) in A549 cells treated with tested compounds, combretastatin (CA-4) as positive control or DMSO as negative control. (A) 2b, 10 μM (depolymerization of MTs). (B) 2e, 10 μM (full depolymerization of MTs). (C) 2c, 10 μM (full depolymerization of MTs). (D) 2a, 100 μM (intact MTs). (E) 0.03 μM CA-4 (full depolymerization of MTs). (F) 0.5% DMSO (intact MTs). Scale bar 10 μm. Images illustrate representative examples of two to four independent experiments.

Figure 3

Influence of compounds 2c and 2e in comparison with verubulin (positive control) on Tb + MAP polymerization as a time-dependent increase in absorbance at 355 nm. Background-subtracted data were normalized to control probe. This figure shows the results of the representative experiment. Each curve is the average of three repeats; each point is mean ± SEM.

Figure 4

(A) Root mean square deviation of the protein, GDP, GTP and ligand (2c) non-hydrogen atoms during molecular dynamics simulation of the complex with the α,β-tubulin heterodimer. (B) Binding mode of compound 2c refined using molecular dynamics simulation (detailed view of the binding site in the β-subunit). Ligand 2c is represented by a green-colored stick model; the amino acid residues located within 3 Å of the ligand are represented by beige stick models. The position of verubulin (PDB ID: 5XKF, matched to the β-subunit) is shown by a mauve-colored stick model. The guanosine triphosphate molecule bound in the α-subunits is shown by stick models on the right in the background. The hydrogen atoms are omitted for clarity.

Figure 5

Apoptotic profile of HCT116 cells treated with selected compounds after 48 h. Concentration of compounds 2 × IC₅₀ (see Supplementary Materials).