A Unified Platform for Nucleoside Analog Synthesis

1. Introduction

Nucleosides and nucleotides, the building blocks of genetic material, are ubiquitous across all known life forms. Nucleoside analogs (NAs), synthetic mimics of endogenous nucleosides, have emerged as a well-established class of therapeutics, with over 30 approved for clinical use. Primarily employed in treating viral infections like HIV/AIDS and hepatitis, NAs also play crucial roles in oncology. Functioning as antimetabolites, NAs mimic natural nucleosides, inhibiting key enzymes involved in nucleic acid metabolism or incorporating into nucleic acids to disrupt chain termination, epigenetic modification, or DNA repair processes.

Despite their therapeutic significance, the exploration of NA chemical space has been hindered by synthetic complexities. Nucleosides are highly polar small molecules susceptible to enzymatic degradation, necessitating designs that enhance potency, specificity, cellular uptake, and stability. Traditional synthetic strategies are laborious, often involving lengthy sequences of transformations and limiting structural diversification. Moreover, the Vorbrüggen reaction, commonly used for nucleobase attachment, is restricted to N-linked nucleosides and often results in mixtures of anomers.

2. Results

2.1 Platform Development

To address these challenges, we envisioned a synthetic strategy incorporating a "nucleobase-last" philosophy, leveraging the flexibility of de novo processes while enabling rapid enantio- and diastereoselective production of a universally flexible building block. Trifluoroborate salts, known for their stability and versatility in coupling processes, were selected as the functional handle of choice. We aimed to develop a unified platform capable of synthesizing all major NA classes from a single nucleoside synthon, prepared in near enantiomeric purity from cheap and achiral materials.

2.2 Building Block Synthesis

Our strategy began with the proline-mediated aldol reaction of α-chloro boroaldehyde 4 with dioxanone 5, yielding borylated chlorohydrin 6 in good yield and excellent diastereoselectivity (Fig.2.2A). This reaction proceeds via dynamic kinetic resolution, preferentially forming the major diastereomer through a more favorable transition structure. Notably, the racemic cocrystal of 6 could be easily removed by filtration, providing virtually enantiomerically pure material (>99% ee).

Fig. 2.2A. Large-scale preparation of a borylated aldol adduct and enantioenrichment.

Elaboration of key synthon 6 into modified ribose cores involved selective reduction and cyclization steps, yielding building blocks BB1-BB9 featuring various modifications at the C4’ position, including methyl, cyano, azido, and methoxy groups (Fig.2.2B). Additionally, reductive amination and cyclization of 6 efficiently delivered iminonucleoside scaffolds, while treatment with NaSH provided thionucleoside scaffolds in excellent yields.

Fig. 2.2B. Synthetic routes for the preparation of modified NA building blocks.

2.3 Photoredox Coupling Optimization

With building blocks BB1-9 in hand, we focused on the final photoredox coupling step to convert these intermediates into desired NA products. Initial optimization studies for C-C coupling reactions revealed aryl iodides as superior coupling partners, with rigorously degassed 1,4-dioxane serving as the optimal solvent. Among various photocatalysts evaluated,Ir[dF(CF3)ppy]2(dtbbpy)PF6 proved most effective, delivering protected pseudouridine derivative 13 in 44% yield from 5-iodouracil (Fig. 2.3A).

Fig. 2.3A. Optimization of C-C photoredox coupling.

For C-N coupling reactions, an acridinium organophotoredox catalyst emerged as the most effective after screening 11 candidates. High-throughput optimization identified Copper (II) acetate as the optimal copper source, with 4,4-di-tert-butyl-2,2’-bipyridine as the ligand. The addition of potassium acetate and tert-butyl hydroperoxide significantly improved reaction yields, providing protected N-linked NAs 17 and 18 in a combined yield of 68% (Fig. 2.3B).

Fig. 2.3B. High-throughput optimization of C-N photoredox coupling.

2.4 Library Generation and Scope

Leveraging this platform, we synthesized a diverse NA library comprising 71 members, including ProTides, C4’-functionalized NAs, iminonucleosides, and thionucleosides. Notably, all 45 ProTide derivatives generated were novel, substantially expanding this underexplored chemical space. The platform enabled efficient access to challenging iminonucleoside and thionucleoside scaffolds in significantly fewer steps than previously reported methods.

2.5 Cheminformatic and Biological Evaluation

To visualize the diversity of our library, we compared it with existing NA chemical space using clustering and dimensionality reduction techniques on molecular fingerprints. The resulting map indicated that our library spans the chemical space of known NA drugs, with representatives in three out of four identified clusters. Screening the library against HIV in a cell-based replicon assay revealed three hit compounds with nanomolar to micromolar antiviral activity, comparable to FDA-approved anti-HIV drugs.

3. Discussion

The developed platform represents a significant advancement in NA synthesis, addressing key challenges that have historically limited the exploration of NA chemical space. By unifying the synthesis of multiple NA classes from a single, scalable intermediate, this approach enables rapid generation of diverse NA libraries suitable for high-throughput screening. The platform's flexibility and efficiency are demonstrated through the synthesis of a 71-member library, encompassing structurally diverse NAs with antiviral activity.

The identification of hit compounds with anti-HIV-1 activity underscores the platform's potential for accelerating NA drug discovery. Future efforts will focus on further expanding the library's diversity, optimizing lead compounds, and exploring additional therapeutic applications. Additionally, the platform's modular nature allows for easy integration of new building blocks and coupling strategies, ensuring its continued relevance in the evolving landscape of NA therapeutics.

4. Conclusion

In conclusion, we have developed a scalable, high-throughput platform for the rapid and modular synthesis of diverse NA libraries. This approach overcomes key synthetic challenges, enabling efficient access to previously challenging NA scaffolds and significantly expanding accessible NA chemical space. The platform's utility in drug discovery is demonstrated through the synthesis of a 71-member library and the identification of hit compounds with antiviral activity, highlighting its potential for accelerating the development of next-generation NA therapeutics.