Considering our hypothesis that the guanidinium moiety in the protein kinase type III inhibitor <b>1</b> 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 <b>1</b> 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 <b>1</b> 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 IC<sub>50</sub> values were compared with those of the lead compound <b>1</b>. 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 <b>2</b> decreases the levels of phosphorylated, and thus activated, ERK (pERK) both in leukaemia and breast cancer cells, whereas lead compound <b>1</b> 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.