Applications of the 1,3-dipolar cycloaddition reaction towards the target guided synthesis of small molecule inhibitors and towards peptide stapling

Abstract

Based on the structure of the known MDM2 inhibitor Nutlin, a series of pyrazoline and pyrazole compounds were synthesized and tested in an ELISA test format, from which 1,5-Bis-( 4-chloro-phenyl)-3-(2-isopropoxy-4-methoxy-phenyl)-1 H -pyrazole-4-carboxylic acid showed improved water solubility and an IC 50 of 40.5 μM against the MDM2-p53 interaction. In situ assembly of potent inhibitors in the presence of the enzyme by means of click chemistry has been shown to be a viable strategy in target guided synthesis. We were intrigued to investigate if the photoactivatable 1,3-dipolar cycloaddition of tetrazoles and alkynes could be applied towards the MDM2-templated assembly of inhibitors using this method. Based on the lead structure of 1,5-Bis-( 4-chloro-phenyl)-3-(2-isopropoxy-4-methoxy-phenyl)-1 H -pyrazole-4-carboxylic acid, a group of carboxylic acid phenyl acetylenes and biphenyl substituted tetrazole building blocks were selected to test the ability of MDM2 to template the assembly of the best building blocks. Additionally, we investigated the presence of any template effect by the structurally similar to MDM2 protein MDMX. Neither MDM2 nor MDMX showed the ability to template the in situ assembly of a selected pair of a tetrazole and a carboxylic acid phenyl acetylene dipolarophile via the photoactivatable 1,3-dipolar cycloaddition reaction. The 1,3-dipolar cycloaddition reaction has also been employed in our laboratory towards the macrocyclization or "stapling" of linear peptides bearing and alkene (dipolarophile) and tetrazole moieties located at the side chains on the i and i + 4 or i + 7 positions. Peptide stapling via the 1,3 dipolar cycloaddition reaction allows the stabilization of the helical form of such peptides and is expected to reduce the barrier to membrane penetration and increase the resistance to protease cleavage due to the intramolecular hydrogen bonding associated with helix formation since this would reduce the exposure of the polar amide backbone. A series of stapled BIM BH3 peptides (residues 88-103, human numbering) by means of the 1,3-dipolar cycloaddition were proposed given the relevance of the BH3 domain of the BIM protein on the induction of mitochondrial apoptosis. Preliminary studies showed that the most suitable positions for side chain modification are E93 and E100, which is in agreement with structural data on the human BIMBH3: BCL-X L interaction.