Influence of Linker Attachment Points on the Stability and Neosubstrate Degradation of Cereblon Ligands.

pubs.acs.org/acsmedchemlett Letter Influence of Linker Attachment Points on the Stability and Neosubstrate Degradation of Cereblon Ligands Aleša Bricelj,‡ Yuen Lam Dora Ng,‡ Dominic Ferber, Robert Kuchta, Sina Müller, Marius Monschke, Karl G. Wagner, Jan Krönke, Izidor Sosic,̌ Michael Gütschow, and Christian Steinebach* Cite This: ACS Med. Chem. Lett. 2021, 12, 1733−1738 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Proteolysis targeting chimeras (PROTACs) hijacking the cereblon (CRBN) E3 ubiquitin ligase have emerged as a novel paradigm in drug development. Herein we found that linker attachment points of CRBN ligands highly affect their aqueous stability and neosubstrate degradation features. This work provides a blueprint for the assembly of future heterodimeric CRBN-based degraders with tailored properties. KEYWORDS: PROTACs, CRBN, Stability, Neosubstrates, Medicinal Chemistry T tives, a variety of other linker connections have been realized (Table S1). Despite being used frequently in degraders, one fundamental consideration of thalidomide-based compounds needs to be emphasized, i.e., their stability. Previous studies showed that IMiDs are hydrolytically20−22 and metabolically23−25 unstable under physiological conditions, rendering their chemical properties challenging. For instance, during previous studies our group and others noticed that certain CRBN-based PROTACs suffered from hydrolytic susceptibility, resulting in the less durable degradation of the desired target protein.26,27 Interestingly, such PROTACs mainly differed in their linker attachment points to the CRBN ligand. To thoroughly examine this observation, we sought to unravel the influence of the linker junction of phthalimide− linker conjugates on their hydrolytic stability and their capability to induce neosubstrate degradation. A broad set of related thalidomide- and lenalidomide-derived compounds was synthesized (Scheme 1). Thalidomide analogs were prepared from phthalic anhydrides by condensing them with 3-aminopiperidine-2,6-dione in acetic acid.28,29 Lenalidomide-based conjugates were synthesized from methyl 2-(bromomethyl)- he past decade has seen tremendous advancements in the field of proteolysis targeting chimeras (PROTACs). Several entities revealed promising activity in various diseases in the preclinical setting1 and recently entered clinical trials in human patients.2 As discussed in several of the latest reviews on the topic of chimeric degraders,3−5 very few E3 ligases are utilized with this technology. These include cullin-RING E3 ubiquitin ligases (CRL), cereblon (CRBN), and von Hippel− Lindau. Two ligases that do not belong to the CRL class, i.e., cellular inhibitor of apoptosis protein 1 and mouse double minute 2 homologue, are also frequently used. Of the former class, CRBN is the most commonly used E3 ligase for PROTAC design.6 Immunomodulatory drugs (IMiDs) such as thalidomide and related derivatives mediate their effects by interacting with CRBN.7,8 Upon binding, the newly established molecular interface promotes the recognition and ubiquitination of neosubstrates, namely, the transcription factors IKZF1 and IKZF3,9,10 as well as casein kinase 1A1.11 The IMiDs lenalidomide and pomalidomide are currently approved as treatments for multiple myeloma,12 other B-cell neoplasms, and myelodysplastic syndrome,13 and new IMiD derivatives are being studied in other cancers and autoimmune disorders.14 The widespread utilization of CRBN as the recruited E3 ligase in PROTACs encouraged the utilization of different linker types (Figure 1).15−17 The optimization of the linker length has been a central part of numerous studies.18,19 However, little attention was paid to the intercomparison of different linker attachment points. Although the majority of CRBNbased PROTACs contain N-alkylated pomalidomide deriva© 2021 American Chemical Society Received: July 3, 2021 Accepted: October 13, 2021 Published: October 18, 2021 1733 https://doi.org/10.1021/acsmedchemlett.1c00368 ACS Med. Chem. Lett. 2021, 12, 1733−1738 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Scheme 1. Synthesis of Tailored Phthalimide-Linker Conjugates in This Studya Figure 1. Selected examples of CRBN-based PROTACs with different linker attachments. benzoates. The so-obtained functionalized CRBN ligands were then coupled to a respective polyethylene glycol-based linker equipped with a terminal benzyl ether group, which was stable under all pH conditions applied in our stability experiments. The entire set spans 16 thalidomide derivatives and six lenalidomide derivatives potentially suitable for PROTAC design (Scheme 1). Next, ligand-linker conjugates were tested for their susceptibility toward hydrolysis under different conditions. Triplicates of compound solutions in an acetonitrile/buffer mixture were incubated for 0, 24, or 48 h at 37 °C, and analyte decomposition was monitored by HPLC. A first control compound (CC1, see the Supporting Information) was used as an internal standard due to its very high stability at all pH levels tested and its favorable long retention time on the HPLC system. The whole data set is presented in Tables S2 and S3. These compounds were only marginally affected at pH 1 and pH 6, but hydrolysis rates were more pronounced at pH 7.4, indicating the potential risk of hydroxide-promoted ringopening reactions. Hydrolysis was significantly influenced by the different linker junctions (Table 1). Whereas amino conjugates T01/T02 and T07/T08 represented very stable entities, alkynyl- (T13/T14) and carboxamide-derived conjugates (T15/T16) were almost completely degraded after 24 h at pH 7.4. In all cases, a linker attachment at position 4 resulted in more stable derivatives. To investigate whether the corresponding linker junctions increased or decreased the stability of the phthalimide ring system, a second control compound (CC2) was synthesized that carried the linker at the imide nitrogen and kept the aromatic positions unsubstituted (Figures S1A and S1B). Relative to this thalidomide derivative, only pairs T01/T02, T05/T06, and T07/T08 showed improved stabilities. As expected, hydrolysis occurred at both the phthalimide and glutarimide rings as judged by comparing T05 with simplified compounds CC3 and CC4 (Figures S1A and S1C). To shed light on potential determinants, stability data, 13C NMR resonances of the atom adjacent to the linker junction, and logD and TPSA values of all compounds were plotted in a correlation matrix (Figure S2). The 13C NMR shifts correlated convincingly with the stability data. Because neosubstrate degradation can be recognized either as a desired quality or an undesired off-target effect of PROTAC molecules, it is highly desirable to install an on−off switch for this feature. Thus, we also investigated whether a FG = functional group. compounds from our unique set of linker conjugates can induce the d (...truncated)