sp3 -Rich Heterocycle Synthesis on DNA: Application to DNA-Encoded Library Production.

DNA encoded library (DEL) synthesis represents a convenient means to produce, annotate and store large collections of compounds in a small volume. While DELs are well suited for drug discovery campaigns, the chemistry used in their production must be compatible with the DNA tag, which can limit compound class accessibility. As a result, most DELs are heavily populated with peptidomimetic and sp2 -rich molecules. Herein, we show that sp3 -rich mono- and bicyclic heterocycles can be made on DNA from ketochlorohydrin aldol products through a reductive amination and cyclization process. The resulting hydroxypyrrolidines possess structural features that are desirable for DELs and target a distinct region of pharmaceutically relevant chemical space.

Enabling late-stage drug diversification by high-throughput experimentation with geometric deep learning.

Late-stage functionalization is an economical approach to optimize the properties of drug candidates. However, the chemical complexity of drug molecules often makes late-stage diversification challenging. To address this problem, a late-stage functionalization platform based on geometric deep learning and high-throughput reaction screening was developed. Considering borylation as a critical step in late-stage functionalization, the computational model predicted reaction yields for diverse reaction conditions with a mean absolute error margin of 4-5%, while the reactivity of novel reactions with known and unknown substrates was classified with a balanced accuracy of 92% and 67%, respectively. The regioselectivity of the major products was accurately captured with a classifier F-score of 67%. When applied to 23 diverse commercial drug molecules, the platform successfully identified numerous opportunities for structural diversification. The influence of steric and electronic information on model performance was quantified, and a comprehensive simple user-friendly reaction format was introduced that proved to be a key enabler for seamlessly integrating deep learning and high-throughput experimentation for late-stage functionalization.

Investigations into mRNA Lipid Nanoparticles Shelf-Life Stability under Nonfrozen Conditions.

mRNA LNPs can experience a decline in activity over short periods (ranging from weeks to months). As a result, they require frozen storage and transportation conditions to maintain their full functionality when utilized. Currently approved commercially available mRNA LNP vaccines also necessitate frozen storage and supply chain management. Overcoming this significant inconvenience in the future is crucial to reducing unnecessary costs and challenges associated with storage and transport. In this study, our objective was to illuminate the potential time frame for nonfrozen storage and transportation conditions of mRNA LNPs without compromising their activity. To achieve this goal, we conducted a stability assessment and an in vitro cell culture delivery study involving five mRNA LNPs. These LNPs were constructed by using a standard formulation similar to that employed in the three commercially available LNP formulations. Among these formulations, we selected five structurally diverse ionizable lipids─C12-200, CKK-E12, MC3, SM-102, and lipid 23─from the existing literature. We incorporated these lipids into a standard LNP formulation, keeping all other components identical. The LNPs, carrying mRNA payloads, were synthesized by using microfluidic mixing technology. We evaluated the shelf life stability of these LNPs over a span of 9 weeks at temperatures of 2-8, 25, and 40 °C, utilizing an array of analytical techniques. Our findings indicated minimal impact on the hydrodynamic diameter, zeta potential, encapsulation efficiency, and polydispersity of all LNPs across the various temperatures over the studied period. The RiboGreen assay analysis of LNPs showed consistent mRNA contents over several weeks at various nonfrozen storage temperatures, leading to the incorrect assumption of intact and functional LNPs. This misunderstanding was rectified by the significant differences observed in EGFP protein expression in an in vitro cell culture (using HEK293 cells) across the five LNPs. Specifically, only LNP 1 (C12-200) and LNP 4 (SM-102) exhibited high levels of EGFP expression at the start (T0), with over 90% of HEK293 cells transfected and mean fluorescence intensity (MFI) levels exceeding 1. Interestingly, LNP 1 (C12-200) maintained largely unchanged levels of in vitro activity over 11 weeks when stored at both 2-8 and 25 °C. In contrast, LNP 4 (SM-102) retained its functionality when stored at 2-8 °C over 11 weeks but experienced a gradual decline of in vitro activity when stored at room temperature over the same period. Importantly, we observed distinct LNP architectures for the five formulations through cryo-EM imaging. This highlights the necessity for a deeper comprehension of structure-activity relationships within these complex nanoparticle structures. Enhancing our understanding in this regard is vital for overcoming storage and stability limitations, ultimately facilitating the broader application of this technology beyond vaccines.

Identifying opportunities for late-stage C-H alkylation with high-throughput experimentation and in silico reaction screening.

Enhancing the properties of advanced drug candidates is aided by the direct incorporation of specific chemical groups, avoiding the need to construct the entire compound from the ground up. Nevertheless, their chemical intricacy often poses challenges in predicting reactivity for C-H activation reactions and planning their synthesis. We adopted a reaction screening approach that combines high-throughput experimentation (HTE) at a nanomolar scale with computational graph neural networks (GNNs). This approach aims to identify suitable substrates for late-stage C-H alkylation using Minisci-type chemistry. GNNs were trained using experimentally generated reactions derived from in-house HTE and literature data. These trained models were then used to predict, in a forward-looking manner, the coupling of 3180 advanced heterocyclic building blocks with a diverse set of sp3-rich carboxylic acids. This predictive approach aimed to explore the substrate landscape for Minisci-type alkylations. Promising candidates were chosen, their production was scaled up, and they were subsequently isolated and characterized. This process led to the creation of 30 novel, functionally modified molecules that hold potential for further refinement. These results positively advocate the application of HTE-based machine learning to virtual reaction screening.

Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties.

Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.

Rapid 18 F- and 19 F-Difluoromethylation through Desulfurative Fluorination of Transient N-, O-, and C-Linked Dithioles.

The difluoromethyl group plays an important role in modern medicinal and agrochemistry. While several difluoromethylation reagents have been reported, these typically rely on difluoromethyl carbenes or anions, or target specific processes. Here, we describe a conceptually unique and general process for O-H, N-H and C-H difluoromethylation that involves the formation of a transient dithiole followed by facile desulfurative fluorination using silver(I) fluoride. We also introduce the 5,6-dimethoxy-1,3-benzodithiole (DMBDT) function, which undergoes sufficiently rapid desulfurative fluorination to additionally support 18 F-difluoromethylation. This new process is compatible with the wide range of functional groups typically encountered in medicinal chemistry campaigns, and the use of Ag18 F is demonstrated in the production of 18 F-labeled derivatives of testosterone, perphenazine, and melatonin, 58.0±2.2, 20.4±0.3 and 32.2±3.6 MBq µmol-1 , respectively. We expect that the DMBDT group and this 18 F/19 F-difluoromethylation process will inspire and support new efforts in medicinal chemistry, agrochemistry and radiotracer production.

Antagonizing somatostatin receptor subtype 2 and 5 reduces blood glucose in a gut- and GLP-1R-dependent manner.

Somatostatin (SS) inhibits glucagon-like peptide-1 (GLP-1) secretion in a paracrine manner. We hypothesized that blocking somatostatin subtype receptor 2 (SSTR2) and 5 (SSTR5) would improve glycemia by enhancing GLP-1 secretion. In the perfused mouse small intestine, the selective SSTR5 antagonist (SSTR5a) stimulated glucose-induced GLP-1 secretion to a larger degree than the SSTR2 antagonist (SSTR2a). In parallel, mice lacking the SSTR5R showed increased glucose-induced GLP-1 secretion. Both antagonists improved glycemia in vivo in a GLP-1 receptor-dependent (GLP-1R-dependent) manner, as the glycemic improvements were absent in mice with impaired GLP-1R signaling and in mice treated with a GLP-1R-specific antagonist. SSTR5a had no direct effect on insulin secretion in the perfused pancreas, whereas SSTR2a increased insulin secretion in a GLP-1R-independent manner. Adding a dipeptidyl peptidase 4 inhibitor (DPP-4i) in vivo resulted in additive effects on glycemia. However, when glucose was administered intraperitoneally, the antagonist was incapable of lowering blood glucose. Oral administration of SSTR5a, but not SSTR2a, lowered blood glucose in diet-induced obese mice. In summary, we demonstrate that selective SSTR antagonists can improve glucose control primarily through the intestinal GLP-1 system in mice.

Quaternary Ammonium Trifluoromethoxide Salts as Stable Sources of Nucleophilic OCF3.

The reaction of nucleophilic tertiary amines with trifluoromethyl and pentafluoroethyl methyl ethers provides quaternary ammonium trifluoromethoxide (NR4OCF3) and pentafluoroethoxide (NR4OCF2CF3) salts, respectively, in good yields. The new trifluoromethoxide salts disclosed herein are uniquely stable for extended periods of time in both the solid state and in solution, which complements contemporary reagents. Here we describe the preparation of a range of NR4OCF3 salts, their long-term stability, and utility in substitution reactions.

Modulation of Pharmacologically Relevant Properties of Piperidine Derivatives by Functional Groups in an Equatorial or Axial ß-Position to the Amino Group.

Thirteen epimeric pairs of 5-substituted N-piperonyl-3-phenylpiperidine derivatives were synthesized in order to explore the stereospecific modulation of basicity, lipophilicity, aqueous solubility, and membrane permeation by functional groups in equatorial or axial positions beta to the amine unit. While this comprehensive data set provides enhanced insight into multiple factors that affect basicity and lipophilicity, it fills an important knowledge gap, providing a frame of reference for the property-based design of bioactive compounds. Impacts on amine basicity are very pronounced for the ß-equatorial functional groups and parallel basicity-lowering effects known for acyclic amine derivatives. For ß-axial functional groups, the basicity-lowering effects are generally decreased, with the nitrile group as the only exception. Basicity and lipophilicity modulations observed for ß-axial functional groups are quite diverse and rationalized in terms of intramolecular hydrogen bonding, dipolar interactions, and special solvation effects. Aqueous solubility and (artificial) membrane permeability are discussed with reference to lipophilicity.

A Convenient Synthesis of Difluoroalkyl Ethers from Thionoesters Using Silver(I) Fluoride.

Herein we report the mild and rapid fluorodesulfurization of thionoesters using only silver(I) fluoride. This reaction demonstrates excellent functional group tolerance and complements existing strategies for difluoroalkyl ether synthesis, which rely on toxic and often dangerous reagents that demonstrate limited functional group compatibility. We additionally report the translation of this finding to the production of 18 F-labelled difluoroalkyl ethers using fluoride-derived [18 F]AgF. This new process should enable the synthesis of a wide range of difluoroalkyl ethers with applications in medicinal and materials chemistry, and radiotracer production.

Paracrine crosstalk between intestinal L- and D-cells controls secretion of glucagon-like peptide-1 in mice.

DPP-4 inhibitors, used for treatment of type 2 diabetes, act by increasing the concentrations of intact glucagon-like peptide-1 (GLP-1), but at the same time, they inhibit secretion of GLP-1, perhaps by a negative feedback mechanism. We hypothesized that GLP-1 secretion is feedback regulated by somatostatin (SS) from neighboring D-cells, and blocking this feedback circuit results in increased GLP-1 secretion. We used a wide range of experimental techniques, including gene expression analysis, immunohistochemical approaches, and the perfused mouse intestine to characterize the paracrine circuit controlling GLP-1 and SS. We show that 1) antagonizing the SS receptor (SSTr) 2 and SSTr5 led to increased GLP-1 and SS secretion in the mouse, 2) SS exhibits strong tonic inhibition of GLP-1 secretion preferentially through SSTr5, and 3) the secretion of S was GLP-1 receptor dependent. We conclude that SS is a tonic inhibitor of GLP-1 secretion, and interventions in the somatostain-GLP-1 paracrine loop lead to increased GLP-1 secretion.

18F-Branched-Chain Amino Acids: Structure-Activity Relationships and PET Imaging Potential.

The large, neutral L-type amino acid transporters (LAT1-LAT4) are sodium-independent transporters that are widely distributed throughout the body. LAT expression levels are increased in many types of cancer, and their expression increases as cancers progress, leading to high expression levels in high-grade tumors and metastases. Because of the key role and overexpression of LAT in many types of cancer, radiolabeled LAT substrates are promising candidates for nuclear imaging of malignancies that are not well revealed by conventional radiotracers. The goal of this study was to examine the structure-activity relationships of a series of 18F-labeled amino acids that were predicted to be substrates of the LAT transport system. Methods: Using a photocatalytic radical fluorination, we prepared a series of 11 fluorinated branched-chain amino acids and evaluated them and their nonfluorinated parents in a cell-based LAT affinity assay. We radiofluorinated selected branched-chain amino acids via the same radical fluorination reaction and evaluated tumor uptake in U-87 glioma xenograft-bearing mice. Results: Structure-activity relationship trends observed in a LAT affinity assay were maintained in further in vitro studies, as well as in vivo using a U-87 xenograft model. LAT1 uptake was tolerant of fluorinated amino acid stereochemistry and chain length. PET imaging and biodistribution studies showed that the tracer (S)-5-18F-fluorohomoleucine had rapid tumor uptake, favorable in vivo kinetics, and good stability. Conclusion: By using an in vitro affinity assay, we could predict LAT-mediated cancer cell uptake in a panel of fluorinated amino acids. These predictions were consistent when applied to different cell lines and murine tumor models, and several new tracers may be suitable for further development as oncologic PET imaging agents.

Direct heterobenzylic fluorination, difluorination and trifluoromethylthiolation with dibenzenesulfonamide derivatives.

Functionalization of heterocyclic scaffolds with mono- or difluoroalkyl groups provides unique opportunities to modulate drug pKa, influence potency and membrane permeability, and attenuate metabolism. While advances in the addition of fluoroalkyl radicals to heterocycles have been made, direct C(sp3)-H heterobenzylic fluorination is comparatively unexplored. Here we demonstrate both mono- and difluorination of a range of alkyl heterocycles using a convenient process that relies on transient sulfonylation by the electrophilic fluorinating agent N-fluorobenzenesulfonimide. We also report heterobenzylic trifluoromethylthiolation and 18F-fluorination, providing a suite of reactions for late-stage C(sp3)-H functionalization of drug leads and radiotracer discovery.

Synthesis of acyl fluorides via photocatalytic fluorination of aldehydic C-H bonds.

Acyl fluorides are versatile acylating agents owing to their unique stability. Their synthesis, however, can present challenges and is typically accomplished through deoxyfluorination of carboxylic acids. Here, we demonstrate that acyl fluorides can be prepared directly from aldehydes via a C(sp2)-H fluorination reaction involving the inexpensive photocatalyst sodium decatungstate and electrophilic fluorinating agent N-fluorobenzenesulfonimide. This convenient fluorination strategy enables direct conversion of aliphatic and aromatic aldehydes into acylating agents.

Site-Selective, Late-Stage C-H 18 F-Fluorination on Unprotected Peptides for Positron Emission Tomography Imaging.

Peptides are often ideal ligands for diagnostic molecular imaging due to their ease of synthesis and tuneable targeting properties. However, labelling unmodified peptides with 18 F for positron emission tomography (PET) imaging presents a number of challenges. Here we show the combination of photoactivated sodium decatungstate and [18 F]-N-fluorobenzenesulfonimide effects site-selective 18 F-fluorination at the branched position in leucine residues in unprotected and unaltered peptides. This streamlined process provides a means to directly convert native peptides into PET imaging agents under mild aqueous conditions, enabling rapid discovery and development of peptide-based molecular imaging tools.

2 H-1,2,3-Triazole-Based Dipeptidyl Nitriles: Potent, Selective, and Trypanocidal Rhodesain Inhibitors by Structure-Based Design.

Macrocyclic inhibitors of rhodesain (RD), a parasitic cysteine protease and drug target for the treatment of human African trypanosomiasis, have shown low metabolic stability at the macrocyclic ether bridge. A series of acyclic dipeptidyl nitriles was developed using structure-based design (PDB ID: 6EX8 ). The selectivity against the closely related cysteine protease human cathepsin L (hCatL) was substantially improved, up to 507-fold. In the S2 pocket, 3,4-dichlorophenylalanine residues provided high trypanocidal activities. In the S3 pocket, aromatic residues provided enhanced selectivity against hCatL. RD inhibition ( Ki values) and in vitro cell-growth of Trypanosoma brucei rhodesiense (IC50 values) were measured in the nanomolar range. Triazole-based ligands, obtained by a safe, gram-scale flow production of ethyl 1 H-1,2,3-triazole-4-carboxylate, showed excellent metabolic stability in human liver microsomes and in vivo half-lives of up to 1.53 h in mice. When orally administered to infected mice, parasitaemia was reduced but without complete removal of the parasites.

Prospective Evaluation of Free Energy Calculations for the Prioritization of Cathepsin L Inhibitors.

Improving the binding affinity of a chemical series by systematically probing one of its exit vectors is a medicinal chemistry activity that can benefit from molecular modeling input. Herein, we compare the effectiveness of four approaches in prioritizing building blocks with better potency: selection by a medicinal chemist, manual modeling, docking followed by manual filtering, and free energy calculations (FEP). Our study focused on identifying novel substituents for the apolar S2 pocket of cathepsin L and was conducted entirely in a prospective manner with synthesis and activity determination of 36 novel compounds. We found that FEP selected compounds with improved affinity for 8 out of 10 picks compared to 1 out of 10 for the other approaches. From this result and other additional analyses, we conclude that FEP can be a useful approach to guide this type of medicinal chemistry optimization once it has been validated for the system under consideration.

18F-Fluorination of Unactivated C-H Bonds in Branched Aliphatic Amino Acids: Direct Synthesis of Oncological Positron Emission Tomography Imaging Agents.

A mild and selective photocatalytic C-H 18F-fluorination reaction has been developed that provides direct access to 18F-fluorinated amino acids. The biodistribution and uptake of three 18F-labeled leucine analogues via LAT1 mediated transport in several cancer cell lines is reported. Positron emission tomography imaging of mice bearing PC3 (prostate) or U87 (glioma) xenografts using 5-[18F]-fluorohomoleucine showed high tumor uptake and excellent tumor visualization, highlighting the utility of this strategy for rapid tracer discovery for oncology.

Intramolecular Inverse Electron-Demand [4 + 2] Cycloadditions of Ynamides with Pyrimidines: Scope and Density Functional Theory Insights.

4-Aminopyridines are valuable scaffolds for the chemical industry in general, from life sciences to catalysis. We report herein a collection of structurally diverse polycyclic fused and spiro-4-aminopyridines that are prepared in only three steps from commercially available pyrimidines. The key step of this short sequence is a [4 + 2]/retro-[4 + 2] cycloaddition between a pyrimidine and an ynamide, which constitutes the first examples of ynamides behaving as electron-rich dienophiles in [4 + 2] cycloaddition reactions. In addition, running the ihDA/rDA reaction in continuous mode in superheated toluene, to overcome the limited scalability of MW reactions, results in a notable production increase compared to batch mode. Finally, density functional theory investigations shed light on the energetic and geometric requirements of the different steps of the ihDA/rDA sequence.

A Convenient Late-Stage Fluorination of Pyridylic C-H Bonds with N-Fluorobenzenesulfonimide.

Pyridine features prominently in pharmaceuticals and drug leads, and methods to selectively manipulate pyridine basicity or metabolic stability are highly sought after. A robust, metal-free direct fluorination of unactivated pyridylic C-H bonds was developed. This convenient reaction shows high functional-group tolerance and offers complimentary selectivity to existing C-H fluorination strategies. Importantly, this late-stage pyridylic C-H fluorination provides opportunities to rationally modulate the basicity, lipophilicity, and metabolic stability of alkylpyridine drugs.