Preparative Synthesis of an RP-Guanosine-3′,5′-Cyclic Phosphorothioate Analogue, a Drug Candidate for the Treatment of Retinal Degenerations

Post-printCyclic guanosine monophosphorothioate analogue 1a is currently showing potential as a drug for the treatment of inherited retinal neurodegenerations. To support ongoing preclinical and clinical work, we have developed a diastereoselective synthesis via cyclization and sulfurization of the nucleoside 5′-H-phosphonate monoester, which affords the desired RP-3′,5′-cyclic phosphorothioate in 9:1 ratio to the undesired SP-diastereomer. This route was made viable as a result of the silyl protection sequence used, which achieved >80% selectivity for 2′,5′-hydroxyls over 3′,5′-hydroxyls. Finally, the chromatography-free process allowed for a scale-up, as intermediates and the final product were isolated by crystallization to give 125 g of 1a (13.8% total yield) with over 99.9% HPLC purity.The authors would like to thank Professor Thorsteinn Loftsson at the University of Iceland for his continued support and guidance, particularly to O.P. as academic supervisor, and Professor Jacek Stawiński at Stockholm University for fruitful discussions. We also thank Dr. Frank Schwede at BIOLOG Life Science Institute (Bremen, Germany) for discussions about synthetic strategy as well as providing material for structural comparisons to our product. This work was supported by the European Commission (H2020-MSCA-765441; HEALTH-F2-2012-304963)Peer Reviewe

A Study on Synthesis and Upscaling of 2′-O-AECM-5-methyl Pyrimidine Phosphoramidites for Oligonucleotide Synthesis

2′-O-(N-(Aminoethyl)carbamoyl)methyl-modified 5-methyluridine (AECM-MeU) and 5-methylcytidine (AECM-MeC) phosphoramidites are reported for the first time and prepared in multigram quantities. The syntheses of AECM-MeU and AECM-MeC nucleosides are designed for larger scales (approx. 20 g up until phosphoramidite preparation steps) using low-cost reagents and minimizing chromatographic purifications. Several steps were screened for best conditions, focusing on the most crucial steps such as N3 and/or 2′-OH alkylations, which were improved for larger scale synthesis using phase transfer catalysis (PTC). Moreover, the need of chromatographic purifications was substantially reduced by employing one-pot synthesis and improved work-up strategies

A Study on Synthesis and Upscaling of 2′-<i>O</i>-AECM-5-methyl Pyrimidine Phosphoramidites for Oligonucleotide Synthesis

2′-O-(N-(Aminoethyl)carbamoyl)methyl-modified 5-methyluridine (AECM-MeU) and 5-methylcytidine (AECM-MeC) phosphoramidites are reported for the first time and prepared in multigram quantities. The syntheses of AECM-MeU and AECM-MeC nucleosides are designed for larger scales (approx. 20 g up until phosphoramidite preparation steps) using low-cost reagents and minimizing chromatographic purifications. Several steps were screened for best conditions, focusing on the most crucial steps such as N3 and/or 2′-OH alkylations, which were improved for larger scale synthesis using phase transfer catalysis (PTC). Moreover, the need of chromatographic purifications was substantially reduced by employing one-pot synthesis and improved work-up strategies

Synthesis and stability studies of bicyclo[6.1.0]nonyne scaffolds for automated solid-phase oligonucleotide synthesis

Two novel bicyclo[6.1.0]nonyne (BCN) linker derivatives, which can be directly incorporated into oligonucleotide sequences during standard automated solid-phase synthesis, are reported. Stabilities of BCN-carbinol and two BCN-oligonucleotides are evaluated under acidic conditions. In addition, derivatized BCN linkers (non-acidic and acid treated) are evaluated for strain-promoted alkyne-azide cycloaddition (SPAAC).</p

Crystal Modifications of a Cyclic Guanosine Phosphorothioate Analogue, a Drug Candidate for Retinal Neurodegenerations

Abstract In contribution to the pharmaceutical development of cyclic guanosine monophosphorothioate analogue cGMPSA as a potential active pharmaceutical ingredient (API) for the treatment of inherited retinal degenerations (IRDs), its neutral form (cGMPSA‐H) and salts of sodium (‐Na), calcium (‐Ca), ammonium (‐NH4), triethylammonium (‐TEA), tris(hydroxymethyl)aminomethane (‐Tris), benethamine (‐Bnet), and benzathine (‐BZ) were prepared. Their solid‐state properties were studied with differential scanning calorimetry, thermogravimetric analysis, hot‐stage microscopy, and dynamic vapor sorption, and their solubilities were measured in deionized H2O as well as aqueous HCl and NaOH buffers. A total of 21 crystal modifications of cGMPSA were found and characterized by X‐ray powder diffraction. Despite their crystalline character, no API forms featured any observable melting points during thermal analyses and instead underwent exothermic decomposition at ≥163 °C. Both the vapor sorption behavior and solubility were found to differ significantly across the API forms. cGMPSA‐BZ featured the lowest aqueous solubility and hygroscopicity, with 50 μg/mL and 5 % mass gain at maximum relative humidity. The synthesis and crystallization of some crystal modifications were upscaled to >10 g. Single crystal X‐ray diffraction was performed which resulted in the first crystal structure determination and absolute configuration of a cyclic guanosine monophosphorothioate, confirming the RP‐ conformation at the phosphorus atom

Development of a Multi-Kilogram-Scale Synthesis of AZD1283: A Selective and Reversible Antagonist of the P2Y₁₂ Receptor

Ethyl 6-chloro-5-cyano-2-methylnicotinate (<b>4</b>) was coupled with 4-piperidinecarboxylic acid (isonipecotic acid) in 81% yield to pyridine acid <b>10</b>. An amide coupling between <b>10</b> and benzylsulfonamide (<b>6</b>) afforded AZD1283 (<b>1</b>) in 79% yield using CDI as coupling reagent. The synthesis has been developed and scaled up to 20 kg batches of <b>1</b>, supporting preclinical and clinical studies. Development work towards 2-chloropyridine <b>4</b> and benzylsulfonamide (<b>6</b>) is included

A Scalable Route to 5‑Substituted 3‑Isoxazolol Fibrinolysis Inhibitor AZD6564

A practical and chromatography-free multikilogram synthesis of a 3-isoxazolol containing antifibrinolytic agent, AZD6564, has been developed in eight steps and 7% overall yield starting from methyl 2-chloroisonicotinate. Highlights in the synthesis are a Negishi coupling and an enzymatic resolution of a racemic ester