Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

The Combination of Mesyl-Phosphoramidate Inter-Nucleotide Linkages and 2'-O-Methyl in Selected Positions in the Antisense Oligonucleotide Enhances the Performance of RNaseH1 Active PS-ASOs.

Antisense oligonucleotides (ASOs) that mediate RNA target degradation by RNase H1 are used as drugs to treat various diseases. Previously we found that introduction of a single 2'-O-methyl (2'-OMe) modification in position 2 of the central deoxynucleotide region of a gapmer phosphorothioate (PS) ASO, in which several residues at the termini are 2'-methoxyethyl, 2' constrained ethyl, or locked nucleic acid, dramatically reduced cytotoxicity with only modest effects on potency. More recently, we demonstrated that replacement of the PS linkage at position 2 or 3 in the gap with a mesyl-phosphoramidate (MsPA) linkage also significantly reduced toxicity without meaningful loss of potency and increased the elimination half-life of the ASOs. In this study, we evaluated the effects of the combination of MsPA linkages and 2'-OMe nucleotides on PS ASO performance. We found that two MsPA modifications at the 5' end of the gap or in the 3'-wing of a Gap 2'-OMe PS ASO substantially increased the activity of ASOs with OMe at position 2 of the gap without altering the safety profile. Such effects were observed with multiple sequences in cells and animals. Thus, the MsPA modification improves the RNase H1 cleavage rate of PS ASOs with a 2'-OMe in the gap, significantly reduces binding of proteins involved in cytotoxicity, and prolongs elimination half-lives.

Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA.

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

Site-specific incorporation of 5'-methyl DNA enhances the therapeutic profile of gapmer ASOs.

We recently showed that site-specific incorporation of 2'-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5'-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5'-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2'-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5'-Me and R-5'-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5'-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.

Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

Design and Synthesis of Novel and Selective Phosphodiesterase 2 (PDE2a) Inhibitors for the Treatment of Memory Disorders.

A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2a inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, the increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2a active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2a inhibition.

New synthetic technology for the construction of N-hydroxyindoles and synthesis of nocathiacin I model systems.

A new synthetic method providing expedient access to a wide range of polyfunctionalized N-hydroxyindoles (IV) is reported. These unique constructs are assembled by nucleophilic additions to in situ generated α,ß-unsaturated nitrones (III) through carbon-carbon and carbon-heteroatom bond formation. The new synthetic technology was applied to the synthesis of nocathiacin I (1) model systems (2 and 3a-c) containing the N-hydroxyindole structural motif.

Enantioselective partial reduction of 2,5-disubstituted pyrroles via a chiral protonation approach.

[reaction: see text] The partial reduction of 2,5-pyrrole diester 1 followed by enantioselective protonation in situ to furnish synthetically useful building blocks is described. An enantiomeric excess of up to 74% was achieved using (-)-ephedrine and related analogues as chiral proton sources. The pyrroline product obtained could be recrystallized to give enantiomerically pure material.

Synthesis of enantiopure dihydropyranones: aldol-based ring expansion of dihydrofurans.

[reaction: see text] The stereoselective Birch reduction of 3-methyl-2-furoic acids using a readily available chiral auxilairy is described; by coupling this process to an oxidative cleavage/aldol ring closure sequence we were able to produce highly functionalized and enantiopure dihydropyranones in high yield. This sequence has ample flexibility built into it, either by the use of different electrophiles during reductive alkylation or by subsequent derivatization of the dihydropyranone after ring expansion.