β-Eliminative Releasable Linkers Adapted for Bioconjugation of Macromolecules to Phenols

Abstract

We recently reported a chemical approach for half-life extension that utilizes sets of releasable linkers to attach drugs to macromolecules via a cleavable carbamate group (Santi et al., Proc. Nat. Acad. Sci. U.S.A. 2012, 109, 6211-6216). The linkers undergo a β-elimination cleavage to release the free, native amine-containing drug. A limitation of the technology is the requirement for an amino group on the drug in order to form the carbamate bond, since most small molecules do not have an amine functional group. Here, we describe an approach to adapt these same β-elimination carbamate linkers so they can be used to connect other acidic heteroatoms, in particular, phenolic hydroxyl groups. The approach utilizes a methylene adaptor to connect the drug to the carbamate nitrogen, and an electron-withdrawing group attached to carbamate nitrogen to stabilize the system against a pH-independent spontaneous cleavage. Carbamate cleavage is driven by β-elimination to give a carboxylated aryl amino Mannich base which rapidly collapses to give the free drug, an aryl amine, and formaldehyde.

Macromolecular Prodrug That Provides the Irinotecan (CPT-11) Active-Metabolite SN-38 with Ultralong Half-Life, Low Cmax , and Low Glucuronide Formation

“…Table 1 shows that the rates of base-catalyzed β-elimination are directly related to the electron-withdrawing ability of the modulator, and at pH 7.4, they have half-lives ranging from ∼9 to 450 h. The rate of 14A is only slightly slower than that of the analogous p-nitrophenyl ether, indicating little dependence of the β-elimination reaction on the leaving group pK a of the ether. 7 They are, however, 6-to 8-fold faster than reported simple primary carbamates with the same modulators 6 because of the higher acidity of the initially formed carbamic acids. 7 Importantly, β-elimination rates dominate the significantly slower spontaneous S N 1 hydrolysis at physiological pH.…”

“…7 They are, however, 6-to 8-fold faster than reported simple primary carbamates with the same modulators 6 because of the higher acidity of the initially formed carbamic acids. 7 Importantly, β-elimination rates dominate the significantly slower spontaneous S N 1 hydrolysis at physiological pH. It is noteworthy that for 14D and 16D the solvolysis rates are relatively unaffected by the spatially remote DBCO or amide connectors to the PEG macromolecule.…”

“…Mechanism. The pH-independent spontaneous hydrolysis of 14E has precedence in the analogous reactions of Nsubstituted N-aryloxy- 7,15 and N-acyloxy-methyl 16 carbamates.…”

“…Recently, we described an approach to adapt such βeliminative linkers for use with phenol-containing drugs (Scheme 2, 4). 7 In addition to the pK a modulator, the modified linkers include a methylene adaptor that connects the carbamate nitrogen to the oxygen atom of the drug and an electron-withdrawing N-aryl stabilizer that inhibits undesirable cleavage of the adaptor−oxygen bond. Upon β-elimination (Scheme 2, path A), a carbamic acid is released, which rapidly disassembles to the free phenol-containing drug.…”

Releasable Conjugation of Polymers to Proteins

“…More recently, the authors expanded this linker design for the modification of phenol groups rather than amino groups, though this has not yet been tested for peptide/ protein−polymer conjugation. 50 …”