Reductive C(sp2)–C(sp3) Coupling Protocol to Enable Linker Exploration of Cereblon E3-Ligase BRD4 Proteolysis-Targeting Chimeras

May 14, 2025 06:30 CST Updated 06:30

Johnson & Johnson

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PROTACs

DOI: 10.1021/acs.jmedchem.4c03157

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Heterobifunctional Degraders(Targeted Protein Degradation Chimeras, i.e., PROTACs) As an emerging drug entity, they have become an alternative therapeutic approach for targeting pathogenic proteins that are difficult to modulate with conventional inhibitors, showing highly promising application prospects.

In current clinical research, almost all PROTACs hijack the ubiquitin-proteasome system using CRBN. Therefore, in the design and synthesis of PROTACs, the rational design and high-throughput reactions based on CRBN ligands conjugated with linkers are crucial.

Recently (May 8), fromJohnson & Johnson The scientific team, inJ. Med. Chem.A high-efficiency was disclosed onNickel-Catalyzed Cross-Coupling Scheme, using high-throughput assays to identify new methods for C(sp2)-C(sp3) coupling on CRBN ligand warheads, replacing the traditional amide linkage approach.

In this study, it was found that, compared with CRBN binders linked by amide bonds,Alkyl-linked CRBN bindersExhibitedBetter Cell PermeabilityAndLower new substrate activity (i.e., reduced off-target degradation activity)。

In addition, the research team also produced a heterobifunctional BRD4 degrader for comparison with the corresponding amide bond counterpart.Demonstrates Improved CRBN Neo-Substrate Selectivity, further demonstrating the application value of this alkyl coupling scheme and the alkylated linkage sites it can generate.

PROTACs Are Developing at the Right Time

1.1 The Importance and Development Status of PROTACs

PROTACs are heterobifunctional molecules that connect two different ligands via a linker, simultaneously binding to E3 ligases (such as CRBN, VHL) and target proteins (POI), inducing ubiquitination and degradation of the target protein, becoming an important strategy for addressing difficult-to-drug targets.

Among them, CRBN is the most frequently used E3 ligase in current clinical applications.

In CRBN-based PROTACs, the binding motif responsible for recruiting CRBN is derived fromImmunomodulatory Drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide. These IMiDs bind to CRBN through the interaction of the glutarimide moiety with a conserved hydrophobic pocket on the CRBN protein. This also contributes to their anti-myeloma activity.

These IMiD-derived CRBN binders exhibit excellent drug-like properties and stand out among the currently disclosed E3 ligase ligands.The smallest molecular weight, the fewest hydrogen bond donors.It is not hard to imagine that the PROTACs developed based on this have the advantage of: greater potential for oral administration!(See the end of the article for related past shares)

The structural factors affecting the oral bioavailability of PROTACs, a super "Rule of Five" (bRo5) entity molecule, are multifaceted:

(1) Derived from the POI binding motif;

(2) Derived from the binding motif of E3 ligase (such as CRBN);

(3) Derived from the linker motif connecting POI ligands and E3 ligase ligands.

Among these, the linker itself is also part of the binding motif for both POI and E3 ligase. Therefore, the composition of the linker and its coupling method (bonding mode, coupling output site), among others, will have a profound impact on the properties of PROTACs.

For example, previous studies have shown that differences in the binding site of CRBN conjugates can affect the logD and stability of PROTACs; the research structure shared yesterday by Shanghai Runjia also indicated that differences in the binding site (4-position vs 5-position) on CRBN conjugates can have a profound impact on binding affinity, PROTACs degradation efficacy, and more.

Therefore, the developmentHigh-efficiency Conjugation、Multi-type Conjugation、High-throughput batch conjugationThe method is crucial for the design and generation of PROTACs molecules.

1.2 Research Objectives, Challenges, and Innovative Approaches

In previous studies, the connection between the linker and CRBN ligand has been mostly limited to"Amino-alkyl", "Alkyl-ether", "Amino-methylacetamide", "Methylamino-acyl"Connection method.

These may lead to issues such as new substrate activity (off-target toxicity, such as degradation of IKZF1, CK1α, etc.) and insufficient cell permeability.

One of the solutions isDevelopment of "Carbon-Carbon" Connection Method, structurally affecting CRBN ligandsWarhead ActivityAndPhysical and Chemical Properties, andPROTACs Degradation Activity。

Therefore,The research objective of this article is: Develop an efficient C-C coupling method to directly modify CRBN ligands, optimizing the linkage to improve the physicochemical properties and new substrate selectivity (off-target toxicity) of PROTACs.

However, if you investigate the current heterobifunctional degraders, you will find that: There are limited primary alkyl attachment sites on CRBN. This is probably due toGlutarimide motifIn the presence of precursors, the development of C-C coupling chemistry is quite challenging.

Some CRBN warhead ligands, such as lenalidomide, are inherently complex to synthesize, so the methods for conjugating alkyl linkers often involve multi-step reactions. The lengthy synthesis greatly restricts rapid SAR exploration. As shown in Figures A and B below, these are the main approaches reported for constructing C-C bonds:

These methods are not only lengthy and cumbersome, but also not always feasible, and sometimes even limited toDiversity of Terminal Alkynes and Borate Building Blocks is Lacking, as well asStringent Requirements of Strong Reducing Conditions, it is very difficult to obtain a fully saturated carbon chain.

Johnson & Johnson's R&D team discovered,Direct C(sp3)-C(sp2) coupling based on glutarimide is possible., As shown in Figure C above, multiple research groups have reported direct nickel-catalyzed cross-coupling based on the phthalimide/isatin-glutarimide motif. These methods are also similarly limited by challenges in scalability and the scarcity of starting materials.

However, these reports gave Johnson & Johnson new inspiration,Development of Nickel-Catalyzed Cross-Coupling Reactions Using Alkyl Pentane Sulfonates as Raw Materials, as shown in the figure below.

Based on this, the researchers developed a universal and scalable cross-coupling scheme to enable alkylation coupling of various CRBN ligands in the presence of glutarimide fragments. This coupling scheme was applied to improve the performance of PROTACs and enhance the selectivity of new substrates, realizing its practical application value.

Optimization of Cross-Coupling Protocols and Substrate Applicability

2.1 Response Modulation Optimization

During the condition optimization phase, the research team screened catalysts and ligands. The optimal conditions were determined by high-throughput experimentation (HTE) screening of nickel catalysts and ligands, as shown in the figure below.

(1) For the short-chain p-toluenesulfonate ester (Compound 2), the use of a picolinamide ligand without other additives resulted in the highest yield, with an analytical yield of 50% (isolated yield 45%); further optimization revealed that usingThe use of free-base pyridine amide instead of pyridine amide hydrochloride ligand can further increase the separation yield to 59%.。

(2) For the long-chain p-toluenesulfonate ester (Compound 3), using dtbbpy ligand and 4-ethylpyridine, the highest yield was obtained with an analytical yield of 48%.

Therefore, the above two conditions will be used as the optimal optimization schemes respectively, depending on the differences in the substrate sulfonate.

2.2 Substrate Applicability

Based on the optimal reaction conditions obtained, the researchers explored the substrate suitability for different CRBN ligand warheads and successfully achieved conjugation.Isoindolinone, Phthalimide, PhenylVarious aryl bromide-linked glutarimide substrate scaffolds, as shown in the figure below.

Among them, not only the coupling efficiency of carbon chains of different lengths was explored, but also the coupling efficiency of different coupling sites on three types of aromatic rings, all achieving appropriate yields ranging from 23% to 59%.

Next, the research team also exploredRange of Available Alkyl Electrophiles (p-Toluenesulfonate), as shown in the figure below. The study found that a series of primary and secondary alkyl electrophiles are suitable for the optimized protocol, while also being compatible with some cyclic amines, Boc groups, tert-butyl esters, PEG chains, and silyl protecting groups, demonstrating broad substrate applicability.

These compatibilities are particularly crucial for the design of PROTACs linkers and the conjugation of POI ligands.

2.3 BRD4 Degrader Synthesis

To further verify the practicality of this conjugation scheme, researchers used I-BET726 as a starting material to prepare primary alkyl tosylate, and adopted the introduction of a 5-bromoisoindolinone CRBN ligand warhead in the final step to synthesize BRD4 PROTACs as a proof of concept (POC).

As shown in the figure below, BRD4 PROTACs, compound 22, were synthesized with an isolated yield of 25%.

Applications of Carbon-Carbon Cross Coupling

3.1 Impact on the Binding Affinity of CRBN Ligand Warheads

As is well known, the strong affinity of IMiDs mainly arises from the binding of the glutarimide ring to the tri-tryptophan pocket of the CRBN protein.

The modification of the carbon-carbon linkage is close to the glutarimide fragment, and the research team envisioned that thisChanges in the key chain approach may impact the binding affinity and selectivity of CRBN ligand warheads.。

Therefore, the researchers usedBased on Homogeneous Time-Resolved Fluorescence (HTRF)The CRBN biochemical probe displacement assay was applied to all 18 synthesized alkyl-linked CRBN ligand warheads.The relative binding affinity of (Compound 4-22) was tested, as shown in the table below.

Among them, these analogs all exhibit sub-μM affinity, and the vast majority of the analogs show higher binding potency than lenalidomide (Len) and pomalidomide (Pom). Compounds generated through C-C coupling clearly maintain CRBN binding affinity, confirming their potential as effective ligand warheads for PROTAC degradation.

In addition, the researchers also constructedCompounds 5, 22The corresponding amide bond coupling was tested, as shown in the table above/figure below,Compounds 23, 24As can be seen from the table above, C-C coupling has better binding affinity compared to amide bond coupling.

3.2 The impact on the selectivity of new substrates (i.e., off-target safety)

As we all know, IMiDs can also act as molecular glues, recruiting novel substrates such as IKZF1, CK1α, and GSPT1.

Therefore, developing PROTACs based on IMiDs analogs presents a significant challenge in the development process, as it is necessary to address off-target defects while exploring the specificity of POI targets.

The research team envisioned that, by varying the C-C connectivity and attachment sites, it might not only improve the physicochemical properties of CRBN ligand warheads but also potentially...To modulate or eliminate the new substrate activity, i.e., potential off-target activity, brought by these warheads through structural changes.。

Researchers adopted two approaches to verify the impact of alkyl coupling on CRBN neosubstrate selectivity: Method 1, cell-based high-throughput HiBit assay; Method 2, molecular dynamics simulation.

(1) Method 1: Cell-Based High-Throughput HiBit Assay

As shown in the table below, alkyl-linked CRBN binders (Compound4–21) hardly degrades IKZF1, CK1α, GSPT1, whileAmide Analogues23 Significantly induce off-target degradation.

(2) Method 2: Molecular Dynamics Simulation

Amide linkages stabilize the CRBN conformation through hydrogen bonding, promoting the recruitment of new substrates; alkyl linkages, on the other hand, offer conformational flexibility.Inhibition of Off-Target Binding。

POC Verification Based on BRD4 Degrader

4.1 Targeted BRD4 Proteolysis Chimeric Activity and Properties

To evaluate the impact of this carbon-carbon linkage on the intact PROTACs system, researchers compared the amide analog 24 with the alkyl-linked analog 22, as shown in the table below.

Comparison reveals that the carbon-carbon bonding methodCompound 22Bisamide-linkedCompound 24, withSuperior BRD4 Degradation Efficiency Activity（DC₅₀: 0.2 nM vs 2.5 nM) andDegradation Capability（D_max: 98% vs 90%), and higher cell permeability (MDCK P_app：0.84 vs 0.08）。

4.2 Formation of PROTACs Ternary Complexes

Alkyl Linker PROTAC22 Promote the formation of CRBN-BRD4-PROTAC ternary complex (AUC >100), while the amide bond24 Less active.

4.3 Omics Analysis

PROTACs 22 Selective degradation of BRD4 shows significantly lower off-target effects on CK1α and WIZ.24, which also verifies the optimization effect of carbon-carbon bonding.

Summary and Outlook

This study successfully optimized the linker strategy for CRBN-PROTACs through an innovative nickel-catalyzed C–C cross-coupling method, overcoming the limitations of traditional amide linkages, providing a significant tool for the development of highly efficient and selective protein degraders.

In the future, it may further expand to other E3 ligases and targets, promoting the clinical application of PROTAC technology. This C-C coupling has many advantages and application values:

(1) A one-step conjugation method simplifies the multi-step synthesis process, supports the rapid construction of PROTAC libraries, and accelerates Structure-Activity Relationship (SAR) studies;

(2) Alkyl linkers significantly improve the potential of PROTACs for cellular permeability and oral bioavailability;

(3) This connection method reduces off-target degradation and increases the therapeutic window, providing a new strategy for the development of highly selective CRBN-PROTACs.

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