The inhibition of cellular factors that are involved in viral replication may be an important alternative to the commonly used strategy of targeting viral enzymes. The guanylhydrazone CNI‐1493, a potent inhibitor of the deoxyhypusine synthase (DHS), prevents the activation of the cellular factor eIF‐5A and thereby suppresses HIV replication and a number of other diseases. Here, we report on the design, synthesis and biological evaluation of a series of CNI‐1493 analogues. The sebacoyl linker in CNI‐1493 was replaced by different alkyl or aryl dicarboxylic acids. Most of the tested derivatives suppress HIV‐1 replication efficiently in a dose‐dependent manner without showing toxic side effects. The unexpected antiviral activity of the rigid derivatives point to a second binding mode as previously assumed for CNI‐1493. Moreover, the chemical stability of CNI‐1493 was analysed, showing a successive hydrolysis of the imino bonds. By molecular dynamics simulations, the behaviour of the parent CNI‐1493 in solution and its interactions with DHS were investigated.