Solid Phase Synthesis of Casimersen and Its 3ʹ-end Modification for Therapeutic Benefits

Phosphorodiamidate Morpholino Oligonucleotides (PMO) have been well established in the treatment of muscular dystrophies with five drugs approved so far on this scaffold. However, the literature data on synthetic methodology for PMOs is very limited in the public domain. In this report, micromole scale synthesis of PMO has been achieved with improved yield and HPLC purity. Casimersen (Amondys 45), an FDA approved drug for treatment of Duchenne Muscular Dystrophy (DMD) was selected as the standard sequence for screening various conditions. Optimization of different reaction conditions revealed that 1:1 salt of 4-cyanopyridine and methane sulphonic acid served as an efficient deblocking agent, Ramage Chemmatrix resin with sarcosine linker-loading was a suitable solid support and 1,3-Dimethyl-2-imidazolidinone (DMI) was the appropriate solvent. The compound was purified by reverse phase HPLC in Trityl ON mode and characterized by MALDI-TOF. Conjugation of PMO with stearic acid, docosanoic acid, and a phosphorothioate based linker has been shown to significantly enhance serum binding properties while avoiding adverse effects such as hemolysis or immunostimulation. These modifications not only improve the bioavailability of PMOs but also suggest potential strategies for optimizing their pharmacokinetic profiles. The minimal immune activation observed compared to traditional PMOs underscores their promising safety and efficacy in clinical applications. However, the varying rates of dialysis among different conjugates indicate a complex interaction with albumin, highlighting the need for further detailed investigation in this area.