Total synthesis and biochemical evaluation of azumamides A–E and analogs

Histone deacetylases (HDAC) are a family of enzymes, which serve as epigenetic modulators. Their biological function has been related to DNA transcription and regulation of various biochemical pathways. Development of isoform selective HDAC inhibitors could be useful for dissecting the individual biochemical pathways associated with each HDAC isoform and these compounds could potentially serve as anti-cancer drugs. Macrocyclic peptides and depsipeptides is an interesting class of HDAC inhibitors, which are found in Nature. These compounds are characterized by being highly potent and moderate selective HDAC inhibitors and we turned our attention to a class of cyclic tetrapeptides, known as azumamides. We developed a synthetic route, which allowed us to complete the total synthesis of azumamides A–E. This is the first reported total synthesis of azumamide B–D and our results validate the proposed structures. The key step in this route is a diastereoselective Mannich reaction, which enabled us to prepare two site-specifically edited epimeric azumamide analogs, where the stereochemistry in the unique -amino acid was inverted. The two epimeric homologs were screened together with azumamide A−E against the entire panel of recombinant HDAC isoforms. Thus, providing the first full profiling of the azumamides. The epimers were inactive against the full panel of HDAC enzymes and show that the -amino acid scaffold is highly sensitive to modifications in the stereochemistry. The profiling of the natural products showed that the azumamides are poor inhibitors of class IIa HDACs, but potent inhibitors of HDAC1–3, 10, and 11 (IC50 values between 14 to 67 nM). Furthermore we showed that carboxylic acid containing compounds (azumamide C and E) were more potent than their carboxyamide counterparts (azumamide A and B). Isoform selectivity was observed in class I and class IIb. In class I, azumamides C and E were 60–350-fold more potent towards HDAC1–3 over HDAC8 and in class IIb they were >200-fold more potent against HDAC10 over HDAC6. Finally, we found that azumamide C was ~2-fold more potent than azumamide E, which indicate having a tyrosine residue in the macrolactam ring increase the activity compared to the phenylalanine homolog. The synthetic route was elaborated to produce structurally edited azumamide analogs. A series of 2- desmethylated compounds were synthesized in parallel to a series of 2-dimethylated analogs. Having observed the importance of the aromatic amino acid in the azumamides, a tryptophan-series was also prepared. The synthesis of these compounds underline the broad perspective and flexibility of the developed Mannich strategy. The dimethylated analogs were found to be poor HDAC inhibitors and only the tryptophancontaining compound showed activity below 20 μM. The removal of the 2-methyl group induced a 1.5–18- fold loss in potency across the different isoforms. The methyl group was found to be less important for inhibition of HDAC 6 and 8 (1.5−3-fold decrease in activity). Based on NMR solution structures we hypothesize that the 2-methyl group, found in the natural products, guides the 3-side chain towards the active site. Judging from the biochemical data on the desmethylated series, this directing feature is important for the activity of this type of inhibitors. Furthermore, a 3-propyl azumamide C analog was developed in order to investigate the effect of the zinc-binding moiety. Preliminary testing showed that this compound was active against HDAC3 with an IC50 of 3 μM. The straight forward synthesis of the -amino acid required for this analog also illustrate the effectiveness of the developed Mannich reaction. On a different project, a promising Bsmoc-based scaffold was probed to serve as a linker in anti-body drug conjugates (ADC) and preliminary results encourage further investigations of this strategy.