Traceless Staudinger ligation enabled parallel synthesis of proteolysis targeting chimera linker variants

Abstract

A parallel, one-pot assembly approach to proteolysis targeting chimeras (PROTACs) is demonstrated utilizing activated esters generated in situ, and traceless Staudinger ligation chemistry. The method described allows for rapid structure-activity relationship studies of PROTAC linker variants. Two previously studied systems, cereblon and BRD4 degraders, are examined as test cases for the synthetic method. The two related strategies to assemble PROTAC linker variants discussed can accommodate the chromotographic separations capabilities of labs of many sizes and incorporates commercially available degrader building blocks, thereby easing synthetic entry into PROTAC chemical space.

Fig. 1.

Schematic representation where a heterobifunctional molecule or PROTAC (yellow) is used to target the degradation of a protein of interest (POI, green). In this process, the PROTAC contains motifs that bind to both POI and E3 ligase (blue), yielding a ternary complex. Ubiquitin (red) can then be transferred to the POI in a proximity dependent manner, leading to proteolysis of the POI.

Scheme 1.

The ‘one-pot’ PROTAC assembly approach begins with conversion of a carboxylic acid functional group 4 to its an acyl-imidazolate in situ followed by coupling to amines 3a–3c. The resulting azides 5a-5c are then coupled with thioester 6, yielding bifunctional molecules 1a-1c and 2a-2c. We chose to use N,N-carbonyldiimidizole (CDI) for our initial study due to the ease of by-product removal, however other coupling reagents may be used.

Scheme 2.

Application to homobifunctional PROTACs. Three PROTACs 1a–1c were assembled in a one-pot fashion beginning with thalidomide acid 7. Compound 10 was synthesized and isolated from thiol 9 prior to the one-pot procedure. Intermediates 8a–8c were formed in situ and were not isolated.

Scheme 3.

Application to heterobifunctional PROTACs. Three heterodimeric PROTACs 2a–2c were assembled in a one-pot fashion beginning with 7 (route A) or 8 (route B). The choice of coupling reagent should be optimized for each system in order to consume the linker amine 3 and avoid aberrant reactivity and consumption of thioester 6 (Scheme 1). This process can be conducted on analytical scales and evaluated prior to use. One-pot procedure beginning with 7 may require HPLC purification, however starting with a collection of degrader building blocks 8 (commercially available) enables purification via standard flash column chromatography. Both methods are conducive to parallel synthesis, thereby producing all linker variants in a concerted effort from a stock solution of thioester 6 (Scheme 1).