Scheme 1.

Synthesis of MTX-conjugated G5 PAMAM dendrimers 2–4, each MTX molecule tethered to the dendrimer through an EG-based spacer. Reagents and conditions: i) Glutaric anhydride, Et₃N, MeOH, rt; ii) EDC, NHS, DMAP, DMF, rt; iii) H₂N(CH₂CH₂O)₂CH₂CH₂NHC(=O)(CH₂)₃CO₂H, Et₃N, rt, DMF; iv) (a) PyBOP, HOBt, DIPEA, DMF; (b) H₂N(CH₂CH₂O)₂CH₂CH₂NH₂; v) (a) EDC, NHS, DMAP, DMF, rt; (b) 7, Et₃N, rt; vi) (a) PyBOP, HOBt, DIPEA, DMF; (b) 7, rt.