Ideas Behind the Tryptophan-Mediated Petasis Reaction (TMPR) Concept for Peptide Stapling.

This Concept short review offers an insightful analysis of pivotal research papers and explores the key synthetic ideas behind the intersection of two realms in peptide chemistry: using tryptophan and Petasis multicomponent reactions for macrocyclisation and labelling of peptides. The recently published tryptophan-mediated Petasis reaction (TMPR) concept represents a critical junction between these two worlds, highlighting how combining such methodologies leads to more effective and versatile synthetic strategies, setting a potentially new direction for future research in the field of peptide-drug conjugates.

Site-selective peptide functionalisation mediated via vinyl-triazine linchpins.

Herein we introduce 3-vinyl-1,2,4-triazines derivatives as dual-reactive linkers that exhibit selectivity towards cysteine and specific strained alkynes, enabling conjugate addition and inverse electron-demand Diels-Alder (IEDDA) reactions. This approach facilitates site-selective bioconjugation of biologically relevant peptides, followed by rapid and highly selective reactions with bicyclononyne (BCN) reagents.

A cell-active cyclic peptide targeting the Nrf2/Keap1 protein-protein interaction.

The disruption of the protein-protein interaction (PPI) between Nrf2 and Keap1 is an attractive strategy to counteract the oxidative stress that characterises a variety of severe diseases. Peptides represent a complementary approach to small molecules for the inhibition of this therapeutically important PPI. However, due to their polar nature and the negative net charge required for binding to Keap1, the peptides reported to date exhibit either mid-micromolar activity or are inactive in cells. Herein, we present a two-component peptide stapling strategy to rapidly access a variety of constrained and functionalised peptides that target the Nrf2/Keap1 PPI. The most promising peptide, P8-H containing a fatty acid tag, binds to Keap1 with nanomolar affinity and is effective at inducing transcription of ARE genes in a human lung epithelial cell line at sub-micromolar concentration. Furthermore, crystallography of the peptide in complex with Keap1 yielded a high resolution X-ray structure, adding to the toolbox of structures available to develop cell-permeable peptidomimetic inhibitors.

Disulfide re-bridging reagents for single-payload antibody-drug conjugates.

Numerous antibody-drug conjugate (ADC) linker technologies exist for the synthesis of ADCs with drug-to-antibody ratios (DARs) being an even integer (typically 2, 4 or 8). However, ADCs with odd-integer DARs are significantly harder to synthesise. Here, we report the synthesis of ADCs loaded with a single warhead, using TetraDVP linkers which simultaneously re-bridge all four interchain disulfides of an IgG1 antibody.

Tryptophan in Multicomponent Petasis Reactions for Peptide Stapling and Late-Stage Functionalisation.

Peptide stapling is a robust strategy for generating enzymatically stable, macrocyclic peptides. The incorporation of biologically relevant tags (such as cell-penetrating motifs or fluorescent dyes) into peptides, while preserving their binding interactions and enhancing their stability, is highly sought after. Despite the unique opportunities offered by tryptophan's indole scaffold for targeted functionalisation, its utilisation in peptide stapling has been limited as compared to other amino acids. Herein, we present an approach for peptide stapling using the tryptophan-mediated Petasis reaction. This method enables the synthesis of both stapled and labelled peptides and is applicable to both solution and solid-phase synthesis. Importantly, the use of the Petasis reaction in combination with tryptophan facilitates the formation of stapled peptides in a straightforward, multicomponent fashion, while circumventing the formation of undesired by-products. Furthermore, this approach allows for efficient and diverse late-stage peptide modifications, thereby enabling rapid production of numerous conjugates for biological and medicinal applications.

Modulation of FLT3-ITD and CDK9 in acute myeloid leukaemia cells by novel proteolysis targeting chimera (PROTAC).

Oncogenic mutations in gene encoding FLT3 kinase are often detected in acute myeloid leukaemia (AML) patients, and several potent kinase inhibitors have been developed. However, the FLT3 inhibitor treatment often leads to the resistance development and subsequent relapse. Targeted degradation of oncogenic protein kinases has emerged as a feasible pharmacological strategy, providing more robust effect over traditional competitive inhibitors. Based on previously developed competitive inhibitor of FLT3 and CDK9, we have designed and prepared a novel pomalidomide-based PROTAC. A series of biochemical and cellular experiments showed selectivity towards FLT3-ITD bearing AML cells and confirmed proteasome-dependent mechanism of action. Dual FLT3-ITD and CDK9 protein degradation resulted in the block of FLT3-ITD downstream signalling pathways, apoptosis activation and cell cycle arrest of FLT3-ITD AML cells. Moreover, transcriptional repression caused by CDK9 degradation significantly reduced expression of crucial genes involved in AML pathogenesis. The obtained results indicate the beneficial impact of simultaneous FLT3-ITD/CDK9 degradation for AML therapy.

[68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice.

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvß3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [68Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [68Ga]Ga-DFO-c(RGDyK) can be used for αvß3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.

1,4,6-Trisubstituted imidazo[4,5-c]pyridines as inhibitors of Bruton's tyrosine kinase.

Herein, we report an efficient synthetic approach towards trisubstituted imidazo [4,5-c]pyridines designed as inhibitors of Bruton's tyrosine kinase (BTK). Two alternative synthetic routes for the simple preparation of desired compounds with variable substitutions at the N1, C4, C6 positions were introduced with readily available building blocks. Further, the developed synthetic approach was feasible for isomeric compounds bearing imidazo [4,5-b]pyridine scaffolds. In contrast to expectations based on previous studies, the imidazo [4,5-c]pyridine inhibitor exhibited a significantly higher activity against BTK compared to its imidazo [4,5-b]pyridine isomer. An inherent SAR study in the series of imidazo [4,5-c]pyridine compounds revealed a remarkably high tolerance of C6 substitutions for both hydrophobic and hydrophilic substituents. Preliminary cellular experiments indicated selective BTK targeting in Burkitt lymphoma and mantle cell lymphoma cell lines. The inhibitors could thus serve as starting points for further development, eventually leading to BTK inhibitors that could be used after ibrutinib failure.

Polymer-supported synthesis of N-substituted anthranilates as the building blocks for preparation of N-arylated 3-hydroxyquinolin-4(1H)-ones.

Fast and simple access to N-arylated 3-hydroxyquinolin-4(1H)-ones starting from easily available 1-methyl-2-iodoterephthalate and variously substituted anilines is presented. N-Alkylated anthranilic acid derivatives represent important intermediates. They can be advantageously prepared by solid-phase synthesis, by Buchwald-Hartwig amination or reductive amination with wide substrate scope and with excellent crude purities.

Scaffold hopping of the SYK inhibitor entospletinib leads to broader targeting of the BCR signalosome.

Spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK) are attractive targets in human haematological malignancies with excessively activated B-cell receptor (BCR) signalling pathways. Entospletinib is a SYK inhibitor that has been evaluated as a clinical candidate. We designed and prepared five isosteres in which the imidazo[1,2-a]pyrazine scaffold of entospletinib was altered to pyrazolo[3,4-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, imidazo[4,5-b]pyridine, imidazo[4,5-c]pyridine and purine. The last two isosteres were the most potent SYK inhibitors, with IC50 values in the mid-nanomolar range. Importantly, three compounds also inhibited BTK more effectively than did entospletinib. Further experiments then showed that BCR signalling was suppressed in Ramos cells by the potent compounds. Preliminary kinase inhibition screening also revealed LCK and SRC as additional targets. Our results further support the hypothesis that multikinase targeting compounds could produce more robust responses in the treatment of B lymphoid neoplasms.

Configuration-Dependent Medium-Sized Ring Formation: Chiral Molecular Framework with Three-Dimensional Architecture.

This report describes a configuration-dependent [6 + 8 + 5] fused ring formation via a tandem cyclic N-acyliminium nucleophilic addition reaction. Cyclization of the acyclic precursor prepared on a solid phase using l-Ser and a racemic mixture of Fmoc- trans-2-aminocyclohexanecarboxylic acid predominantly yielded the cyclic diastereomer with the (1 R,2 R)-2-aminocyclohexane moiety rather than the tricyclic diastereomer from the (1 S,2 S)-enantiomer. In contrast, the model compound prepared with d-Ser predominantly cyclized with the (1 S,2 S)-2-aminocyclohexanecarboxylic acid substrate. The outcome of the cyclization was not influenced by the type of resin, the spacer, or the N-substituent. The analogous synthesis of the [6 + 7 + 5] fused ring system yielded inseparable diastereomers in a 1:0.6 ratio.

Triterpenic azines, a new class of compounds with selective cytotoxicity to leukemia cells CCRF-CEM.

AIM: From betulinic acid (1a), we synthesized 30-oxobetulinic acid (2a) that is highly cytotoxic against many cancer cell lines; however, its generic toxicity is the main obstacle in further development as cytostatic. Methodology & results: From 2a, we prepared a new class of compounds - nonsymmetrical azines and tested their in vitro cytotoxicity. All new azines with a free 28-COOH group (4a-4e) were highly and selectively cytotoxic against the T-lymphoblastic leukemia cell line CCRF-CEM and exhibited dose-dependent inhibition of RNA and DNA synthesis and other cell-cycle alterations, including the M-phase block. CONCLUSION: The potential use of azines (4a-4e) in drug development focused on hematological cancers is significantly higher than that of previously studied acids 1a and 2a.

A Synthetic Approach for the Rapid Preparation of BODIPY Conjugates and their use in Imaging of Cellular Drug Uptake and Distribution.

A solid-phase synthetic (SPS) method was developed for the preparation of BODIPY-labeled bioactive compounds that allows for fast and simple synthesis of conjugates for use in fluorescent microscopy. The approach was used to visualize cellular uptake and distribution of cytotoxic triterpenes in cancer cells.

A Stepwise Approach for the Synthesis of Folic Acid Conjugates with Protein Kinase Inhibitors.

Herein, we report an alternative synthetic approach for selected 2,6,9-trisubstituted purine CDK inhibitor conjugates with folic acid as a drug-delivery system targeting folate receptors. In contrast to the previously reported approaches, the desired conjugates were constructed stepwise using solid-phase synthesis starting from immobilized primary amines. The ability of the prepared conjugates to release the free drug was verified using dithiothreitol (DTT) and glutathione (GSH) as liberating agents. Finally, binding to the folate receptor (FOLR1) overexpressed in a cancer cell line was measured by flow cytometry using a fluorescent imaging probe.

Diastereoselective Flexible Synthesis of Carbocyclic C-Nucleosides.

Carbocyclic C-nucleosides are quite rare. Our route enables flexible preparation of three classes of these nucleoside analogs from common precursors-properly substituted cyclopentanones, which can be prepared racemic (in six steps) or optically pure (in ten steps) from inexpensive norbornadiene. The methodology allows flexible manipulation of individual positions around the cyclopentane ring, namely highly diastereoselective installation of carbo- and heterocyclic substituents at position 1', orthogonal functionalization of position 5', and efficient inversion of stereochemistry at position 2'. Newly prepared carbocyclic C-analog of tubercidine, profiled in MCF7 (breast cancer) and HFF1 (human foreskin fibroblasts) cell cultures, is less potent than tubercidine itself, but more selectively toxic toward the tumorigenic cells.

Solid-Phase Synthetic Strategies for the Preparation of Purine Derivatives.

This Review summarizes all of the currently described strategies applicable for the solid-phase synthesis of purine derivatives. The individual approaches are classified according to the immobilization procedure used resulting in a linkage of the final scaffold at various positions.