The HDAC binding ligand was derived from our previously developed degrader molecule A6, except that the linker in the HDAC inhibitor pharmacophore was changed to a benzyl moiety to achieve better HDAC6 selectivity. The ZBG within the HDAC6 ligand was changed from a hydroxamate to an ethyl hydrazide group. To attach the HDAC6 ligand to the spacer of the final PROTACs, we chose two distinct connecting units. One is a carboxamide directly connected to the phenyl ring of the HDAC6 ligand and the other is the bioisosteric 1,2,3-triazole ring, which is attached to the HDAC6 ligand by an ether group. The linkers were derived from A6 as well, and thus a C8 alkyl linker was chosen for the compounds bearing the carboxamide as a connecting unit, while C6 alkyl linkers were selected for the triazole-containing compounds. This was done in order to achieve a similar total distance between the E3 ligase ligand and the HDAC6 ligand. To compare alkyl versus polyethylene glycol (PEG) linkers, we also included PEG2 for carboxamides and PEG1 for triazole-based PROTACs. To investigate the effect of different E3 ligases we chose two different E3 recruiters. Firstly, we selected a CRL4 CRBN engaging ligand like in A6 but changed it from the classical thalidomide to a 6-fluorothalidomide, which is connected in 5-position to the PROTAC spacer by an amine bond. Secondly, we used the CRL2 VHL binding ligand VH032-amine, which is connected to the spacer by an amide group. Figure . Design of ethyl hydrazide-based PROTACs derived from our previously developed HDAC6 degrader A6. The synthesis of the HDAC ligand was done using two different pathways, one for the synthesis of the amide-connected ligands and the other for the triazole connected compounds. The aromatic amine 5 was synthesized for the use in amide-connected PROTACs (Scheme 1). An amide coupling using TBTU as a coupling agent generated intermediate 3. The methyl ester was then saponified by aqueous NaOH in a THF/MeOH mixture. Subsequently, 1-Boc-1-ethylhydrazine was introduced by another coupling reaction using HATU as the coupling agent. Finally, the nitro group of 4 was reduced by catalytic hydration in MeOH with a palladium catalyst to afford the key intermediate 5. The terminal propargyl ether 10 was synthesized for the desired 1,2,3-triazole connected compounds (Scheme 2). Here, we synthesized ether 8 by Williamson ether synthesis of 4-hydroxybenzoic acid (6) and propargyl bromide (7) using potassium carbonate as a base. The carboxylic acid (8) was then subjected to an amide coupling using HATU in DMF and methyl 4-(aminomethyl) benzoate. Again, the resulting methyl ester (9) was hydrolyzed and coupled with 1-Boc-1-ethylhydrazine using HATU to obtain the key intermediate 10. . All compounds showed low single-digit micromolar inhibitory activity against HDAC1 and HDAC2, with the exception of 17a,b, which demonstrated high submicromolar inhibitory activity against these two isoforms, and 17d, which inhibited HDAC2 with a slightly submicromolar half maximal inhibitory concentration (IC50) value. All compounds showed submicromolar inhibition of HDAC3, making all tested compounds preferable HDAC3 inhibitors over HDAC1 and 2. 18a exhibited the lowest IC50 value of 0.126 µM against HDAC3. The class IIb isoform HDAC6, was preferably inhibited by all compounds except for 18a, which showed the highest activity against HDAC3. All other compounds inhibited HDAC6 with IC50 values ranging from 0.108 to 0.594 µM. These assays confirmed target inhibition and, consequently, binding. Although most compounds showed a preference for HDAC6 inhibition, the nuclear isoforms HDAC1-3 were also inhibited.