Effect of isouronium/guanidinium substitution on the efficacy of a series of novel anti-cancer agents

Abstract

Considering our hypothesis that the guanidinium moiety in the protein kinase type III inhibitor 1 interacts with a phosphate of ATP within the hinge region, the nature of the interactions established between a model isouronium and the phosphate groups of ATP was computationally analysed indicating that an isouronium derivative of 1 will interact in a similar manner with ATP. Thus, a number of compounds were prepared to assess the effect of the guanidinium/isouronium substitution on cancer cell growth; additionally, the molecular shortening and conformational change induced by replacing the di-substituted guanidine-linker of 1 by an amide was explored. The effect of these compounds on cell viability was tested in human leukaemia, breast cancer and cervical cancer cell lines and the resulting IC₅₀ values were compared with those of the lead compound 1. Replacement of the di-substituted guanidine-linker by an amide results in the loss of cytotoxicity; however, substitution of the mono-substituted guanidinium by an isouronium cation seems to be beneficial for cell growth inhibition. Additionally, the effect of these compounds on the MAPK/ERK pathway was studied by means of Western blotting and the results indicate that the isouronium derivative 2 decreases the levels of phosphorylated, and thus activated, ERK (pERK) both in leukaemia and breast cancer cells, whereas lead compound 1 only shows an effect on pERK levels in breast cancer cells. This confirms that both compounds could interfere with the MAPK/ERK pathway although other targets cannot be ruled out.

Fig. 1. Structure of compounds 1, 2, 3 and 4, highlighting the comparison of the two cationic moieties (guanidinium in the green box and isouronium in the blue box). The guanidine and amide linkers are indicated by the red boxes.

Scheme 1. Reagents and conditions: (a) PPTS, 3,4-dihydro-2H-pyran, CH₂Cl₂, rt, 2 h, 82%; (b) CuI, picolinic acid, K₃PO₄, DMSO, 80 °C, 24 h, 86%; (c) NaH, TFAA, THF, 0 °C, 5 h, 61%; (d) HgCl₂, Et₃N, CH₂Cl₂, rt, overnight, 58%; (e) montmorillonite KSF, MeOH, 50 °C, 4 h, 65%; (f) HgCl₂, Et₃N, CH₂Cl₂, rt, overnight, 55%.

Scheme 2. Reagents and conditions: (a) HgCl₂, Et₃N, CH₂Cl₂, 25 °C, 12 h, 51%; (b) Et₃N, CH₂Cl₂, overnight, 51%.

Scheme 3. Reagents and conditions: (a) Et₃N, CH₂Cl₂, overnight, 43%; (b) montmorillonite KSF, MeOH, 50 °C, 4 h, 74%; (c) HgCl₂, Et₃N, CH₂Cl₂, 25 °C, 12 h, 97%.

Fig. 2. Effect of compounds 1, 2, 3 and 4 on the viability of HL-60, MCF-7 and HeLa cells determined by the AlamarBlue® assay. Cells were seeded at a density of 4 × 10⁴ (HL-60), 5 × 10³ (MCF-7) and 5 × 10³ (HeLa) cells per well on a 96-well plate and treated with vehicle alone [1% (v/v) EtOH or 0.1% (v/v) DMSO], or the compounds at 0.1–100 μM. Sorafenib was used as control. Cells were incubated for 72 h at 37 °C after which they were treated with AlamarBlue® and left in darkness in an incubator for 4–5 h. The resulting fluorescence was read using a plate reader from which percentage viability was calculated. The points on the three graphs represent mean values ± S.E.M. from at least three independent experiments performed in triplicate.

Fig. 3. Effect of compounds 1, 2 and sorafenib (as a control) on the level of expression of phosphorylated (activated) and total ERK. HL-60 and MCF-7 cells were treated with either vehicle (0.1% EtOH), sorafenib (10 μM), compound 1 (10 μM) or compound 2 (10 μM) for 16 hours. Cells were lysed and 20 μg protein was loaded, separated on 12% SDS-page gel and transferred to PVDF membrane. The membrane was then blocked and probed with anti-ERK and anti-pERK [1 : 1000] and anti-rabbit secondary antibodies. Anti-GAPDH [1 : 2500] was used as a loading control and probed with anti-mouse secondary. HL-60 and MCF-7 results are representative of 4 and 2 independent experiments respectively.