Crafting Unnatural Peptide Macrocycles via Rh(III)-Catalyzed Carboamidation.

Contemporary developments in the field of peptide macrocyclization methodology are imperative for enabling the advance of drug design in medicinal chemistry. This report discloses a Rh(III)-catalyzed macrocyclization via carboamidation, reacting acryloyl-peptide-dioxazolone precursors and arylboronic acids to form complex cyclic peptides with concomitant incorporation of noncanonical α-amino acids. The diverse and modular technology allows for expedient access to a wide variety of cyclic peptides from 4 to 15 amino acids in size and features simultaneous formation of unnatural phenylalanine and tyrosine derivatives with up to >20:1 diastereoselectivity. The reaction showcases an expansive substrate scope with 45 examples and is compatible with the majority of standard protected amino acids used in Fmoc-solid phase peptide synthesis. The methodology is applied to the synthesis of multiple peptidomimetic macrocyclic analogs, including derivatives of cyclosomatostatin and gramicidin S.

Synthesis of Membrane-Permeable Macrocyclic Peptides via Imidazopyridinium Grafting.

Macrocyclic peptides (MPs) are a class of compounds that have been shown to be particularly well suited for engaging difficult protein targets. However, their utility is limited by their generally poor cell permeability and bioavailability. Here, we report an efficient solid-phase synthesis of novel MPs by trapping a reversible intramolecular imine linkage with a 2-formyl- or 2-keto-pyridine to create an imidazopyridinium (IP+)-linked ring. This chemistry is useful for the creation of macrocycles of different sizes and geometries, including head-to-side and side-to-side chain configurations. Many of the IP+-linked MPs exhibit far better passive membrane permeability than expected for "beyond Rule of 5" molecules, in some cases exceeding that of much lower molecular weight, traditional drug molecules. We demonstrate that this chemistry is suitable for the creation of libraries of IP+-linked MPs and show that these libraries can be mined for protein ligands.

Discovery of Thioether-Cyclized Macrocyclic Covalent Inhibitors by mRNA Display.

Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS). Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date. This study outlines the workflow for integrating chemical functionalization─installation of a covalent warhead─with mRNA display and showcases its application in targeted covalent ligand discovery.

Synthesis and Functionalization of Azetidine-Containing Small Macrocyclic Peptides.

Cyclic peptides are increasingly important structures in drugs but their development can be impeded by difficulties associated with their synthesis. Here, we introduce the 3-aminoazetidine (3-AAz) subunit as a new turn-inducing element for the efficient synthesis of small head-to-tail cyclic peptides. Greatly improved cyclizations of tetra-, penta- and hexapeptides (28 examples) under standard reaction conditions are achieved by introduction of this element within the linear peptide precursor. Post-cyclization deprotection of the amino acid side chains with strong acid is realized without degradation of the strained four-membered azetidine. A special feature of this chemistry is that further late-stage modification of the resultant macrocyclic peptides can be achieved via the 3-AAz unit. This is done by: (i) chemoselective deprotection and substitution at the azetidine nitrogen, or by (ii) a click-based approach employing a 2-propynyl carbamate on the azetidine nitrogen. In this way, a range of dye and biotin tagged macrocycles are readily produced. Structural insights gained by XRD analysis of a cyclic tetrapeptide indicate that the azetidine ring encourages access to the less stable, all-trans conformation. Moreover, introduction of a 3-AAz into a representative cyclohexapeptide improves stability towards proteases compared to the homodetic macrocycle.

Biocompatible Synthesis of Macrocyclic Thiazol(in)e Peptides.

Macrocyclic peptides containing a thiazole or thiazoline in the backbone are considered privileged structures in both natural compounds and drug discovery, owing to their enhanced bioactivity, stability, and permeability. Here, we present the biocompatible synthesis of macrocyclic peptides from N-terminal cysteine and C-terminal nitrile. While the N-terminal cysteine is incorporated during solid-phase peptide synthesis, the C-terminal nitrile is introduced during cleavage with aminoacetonitrile, utilizing a cleavable benzotriazole linker. This method directly yields the fully functionalized linear peptide precursor. The biocompatible cyclization reaction occurs in buffer at physiological pH and room temperature. The resulting thiazoline heterocycle remains stable in buffer but hydrolyzes under acidic conditions. While such hydrolysis enables access to macrocyclic peptides with a complete amide backbone, mild oxidation of the thiazoline leads to the stable thiazole macrocyclic peptide. While conventional oxidation strategies involve metals, we developed a protocol simply relying on alkaline salt and air. Therefore, we offer a rapid and metal-free pathway to macrocyclic thiazole peptides, featuring a biocompatible key cyclization step.

Challenges with the Synthesis of a Macrocyclic Thioether Peptide: From Milligram to Multigram Using Solid Phase Peptide Synthesis (SPPS).

We describe an optimization and scale-up of the 45-membered macrocyclic thioether peptide BMS-986189 utilizing solid-phase peptide synthesis (SPPS). Improvements to linear peptide isolation, macrocyclization, and peptide purification were demonstrated to increase the throughput and purification of material on scale and enabled the synthesis and purification of >60 g of target peptide. Taken together, not only these improvements resulted in a 28-fold yield increase from the original SPPS approach, but also the generality of this newly developed SPPS purification sequence has found application in the synthesis and purification of other macrocyclic thioether peptides.

Process Development of a Macrocyclic Peptide Inhibitor of PD-L1.

This article outlines the process development leading to the manufacture of 800 g of BMS-986189, a macrocyclic peptide active pharmaceutical ingredient. Multiple N-methylated unnatural amino acids posed challenges to manufacturing due to the lability of the peptide to cleavage during global side chain deprotection and precipitation steps. These issues were exacerbated upon scale-up, resulting in severe yield loss and necessitating careful impurity identification, understanding the root cause of impurity formation, and process optimization to deliver a scalable synthesis. A systematic study of macrocyclization with its dependence on concentration and pH is presented. In addition, a side chain protected peptide synthesis is discussed where the macrocyclic protected peptide is extremely labile to hydrolysis. A computational study explains the root cause of the increased lability of macrocyclic peptide over linear peptide to hydrolysis. A process solution involving the use of labile protecting groups is discussed. Overall, the article highlights the advancements achieved to enable scalable synthesis of an unusually labile macrocyclic peptide by solid-phase peptide synthesis. The sustainability metric indicates the final preparative chromatography drives a significant fraction of a high process mass intensity (PMI).

Total Synthesis of an Immunosuppressive C25 Macrocyclic Terpenoid Produced by Terpene Synthase (LcTPS2).

Macrocyclic natural products, particularly those with no functionalities except unsaturation, are recognized for their therapeutic potential but are notoriously challenging to synthesize. In this study, we report the first total synthesis of an unconventional 18-membered, C25 macrocyclic terpenoid, which has demonstrated substantial immunosuppressive activity. This synthesis was achieved through strategic modifications and innovative reaction engineering, utilizing α-terpineol and geraniol as starting materials, highlighting a novel approach in macrocyclic terpenoid synthesis.

Capturing Mercury-197m/g for Auger Electron Therapy and Cancer Theranostic with Sulfur-Containing Cyclen-Based Macrocycles.

The interest in mercury radioisotopes, 197mHg (t1/2 = 23.8 h) and 197gHg (t1/2 = 64.14 h), has recently been reignited by the dual diagnostic and therapeutic nature of their nuclear decays. These isotopes emit γ-rays suitable for single photon emission computed tomography imaging and Auger electrons which can be exploited for treating small and metastatic tumors. However, the clinical utilization of 197m/gHg radionuclides is obstructed by the lack of chelators capable of securely binding them to tumor-seeking vectors. This work aims to address this challenge by investigating a series of chemically tailored macrocyclic platforms with sulfur-containing side arms, namely, 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), and 1,7-bis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane-4,10-diacetic acid (DO2A2S). 1,4,7,10-Tetrazacyclododecane-1,4,7,10-tetracetic acid (DOTA), the widest explored chelator in nuclear medicine, and the nonfunctionalized backbone 1,4,7,10-tetrazacyclododecane (cyclen) were considered as well to shed light on the role of the sulfanyl arms in the metal coordination. To this purpose, a comprehensive experimental and theoretical study encompassing aqueous coordination chemistry investigations through potentiometry, nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and density functional theory (DFT) calculations, as well as concentration- and temperature-dependent [197m/gHg]Hg2+ radiolabeling and in vitro stability assays in human serum was conducted. The obtained results reveal that the investigated chelators rapidly complex Hg2+ in aqueous media, forming extremely thermodynamically stable 1:1 metal-to-ligand complexes with superior stabilities compared to those of DOTA or cyclen. These complexes exhibited 6- to 8-fold coordination environments, with donors statically bound to the metal center, as evidenced by the presence of 1H-199Hg spin-spin coupling via NMR. A similar octacoordinated environment was also found for DOTA in both solution and solid state, but in this case, multiple slowly exchanging conformers were detected at ambient temperature. The sulfur-rich ligands quantitatively incorporate cyclotron-produced [197m/gHg]Hg2+ under relatively mild reaction conditions (pH = 7 and T = 50 °C), with the resulting radioactive complexes exhibiting decent stability in human serum (up to 75% after 24 h). By developing viable chelators and understanding the impact of structural modifications, our research addresses the scarcity of suitable chelating agents for 197m/gHg, offering promise for its future in vivo application as a theranostic Auger-emitter radiometal.

Discovery of macrocyclic covalent inhibitors for severe acute respiratory syndrome coronavirus 2 3CL protease.

The coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spread worldwide for more than 3 years. Although the hospitalization rate and mortality have decreased dramatically due to wide vaccination effort and improved treatment options, the disease is still a global health issue due to constant viral mutations, causing negative impact on social and economic activities. In addition, long COVID and complications arising from COVID-19 weeks after infection have become a concern for public health experts. Therefore, better treatments for COVID-19 are still needed. Herein, we describe a class of macrocyclic peptidomimetic compounds that are potent inhibitors of SARS-Cov-2 3CL protease (3CLpro). Significantly, some of the compounds showed a higher stability against human liver microsomes (HLM t1/2 > 180 min) and may be suitable for oral administration without the need for a pharmacokinetic (PK) boosting agent such as ritonavir.

Macrocyclic peptides derived from AcPHF6* and AcPHF6 to selectively modulate the Tau aggregation.

Ten macrocyclic peptides, each comprising 14 amino acids, were designed and synthesized based on the Tau aggregation model hexapeptides AcPHF6* and AcPHF6. The design took into account the aggregation tendencies of each residue in AcPHF6* and AcPHF6, their aggregation models, while employing peptide-based structural design principles including N-methylation to promote turns and to block hydrogen bond propagation and elongation of the aggregation chain. NMR analysis supported that all these peptides adopted an antiparallel ß-sheet conformation. Self-aggregation studies characterized the aggregation properties of these peptides, identifying two peptides with the highest (P3) and lowest (P8) aggregation tendencies. In cross-aggregation studies with the parent peptides AcPHF6* and AcPHF6, P3 and P8 were found to promote and reduce aggregation, respectively. Furthermore, P3 and P8 demonstrated an enhancement and diminution effect on the aggregation of K18wt, indicating their capacity to modulate aggregation even at the macromolecular level. Thus, the two simple peptides, P3 and P8 selectively exhibit pro- or anti-aggregation effects on PHF peptides and Tau. This study, has thus developed structurally well-defined non-complex peptides, derived from AcPHF6* and AcPHF6, to modulate Tau aggregation as desired, offering applications in Tau model studies and the development of Tau aggregation inhibitors or promoters.

Synthesis, structural, multitargeted molecular docking analysis of anti-cancer, anti-tubercular, DNA interactions of benzotriazole based macrocyclic ligand.

Biologically important macromolecule 1, 1', 3, 3' Bis - [2,3,5,6-Tetramethyl-p-phenylenebis(methylene)] dibenzotriazlinium dibromide hydrate (BTD) was synthesized and characterized using FT-IR, NMR and single-crystal XRD (SCXRD). SCXRD revealed that the compound was crystallized as a monoclinic system and associated through weak intermolecular interactions like H-bonding and π- π stacking interactions. These weak intermolecular interactions in BTD were studied using Crystal Explorer and Gaussian. The calculated energies for the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) showed the stability and reactivity of the title compound. Molecular electrostatic potential (MEP) surface analysis was used to investigate the crystal's nucleophilic and electrophilic reactive sites. The molecular shape and intermolecular interactions in the crystal structure were determined using Hirshfeld surface analysis and fingerprint plots. Anticancer, anti-bacterial and DNA binding ability of BTD were investigated by experimental and theoretical techniques. The obtained results suggest that BTD possesses better anti-cancer, anti-bacterial and DNA binding abilities. The mode of action of antibiotic and anticancer approach was discussed. This provides promising therapeutic advantages for further development.

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B-NS3 protease.

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors.

Cobalt(III)-Macrocyclic Scaffolds with Anti-Cancer Stem Cell Activity.

Cobalt(III) compounds with tetradentate ligands have been widely employed to deliver cytotoxic and imaging agents into cells. A large body of work has focused on using cobalt(III)-cyclam scaffolds for this purpose. Here, we investigate the cytotoxic properties of cobalt(III) complexes containing 14-membered macrocycles related to cyclam. A breast cancer stem cell (CSC) in vitro model was used to gauge efficacy. Specifically, [Co(1,4,7,11-tetraazacyclotetradecane)Cl2]+ (1) and [Co(1-oxa-4,8,12-triazacyclotetradecane)Cl2]+ (2) were synthesised and characterised, and their breast CSC activity was determined. The cobalt(III) complexes 1 and 2 displayed micromolar potency towards bulk breast cancer cells and breast CSCs grown in monolayers. Notably, 1 and 2 displayed selective potency towards breast CSCs over bulk breast cancer cells (up to 4.5-fold), which was similar to salinomycin (an established breast CSC-selective agent). The cobalt(III) complexes 1 and 2 were also able to inhibit mammosphere formation at low micromolar doses (with respect to size and number). The mammopshere inhibitory effect of 2 was similar to that of salinomycin. Our studies show that cobalt(III) complexes with 1,4,7,11-tetraazacyclotetradecane and 1-oxa-4,8,12-triazacyclotetradecane macrocycles could be useful starting points for the development of new cobalt-based delivery systems that can transport cytotoxic and imaging agents into breast CSCs.

Ligand-Enabled Pd-Catalyzed sp3 C-H Macrocyclization: Synthesis and Evaluation of Macrocyclic Sulfonamide for the Treatment of Parkinson's Disease.

The development of simplified synthetic strategy to create structurally and functionally diverse pseudo-natural macrocyclic molecules is highly appealing but poses a marked challenge. Inspired by natural scaffolds, herein, we describe a practical and concise ligand-enabled Pd(II)-catalyzed sp3 C-H alkylation, olefination and arylation macrocyclization, which could offer a novel set of pseudo-natural macrocyclic sulfonamides. Interestingly, the potential of ligand acceleration in C-H activation is also demonstrated by an unprecedented enantioselective sp3 C-H alkylation macrocyclization. Moreover, a combination of in silico screening and biological evaluation led to the identification of a novel spiro-grafted macrocyclic sulfonamide 2 a, which showed a promising efficacy for the treatment of Parkinson's disease (PD) in a mouse model through the activation of silent information regulator sirtuin 3 (SIRT3).

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Recent Advances in Macrocyclic Drugs and Microwave-Assisted and/or Solid-Supported Synthesis of Macrocycles.

Macrocycles represent attractive candidates in organic synthesis and drug discovery. Since 2014, nineteen macrocyclic drugs, including three radiopharmaceuticals, have been approved by FDA for the treatment of bacterial and viral infections, cancer, obesity, immunosuppression, etc. As such, new synthetic methodologies and high throughput chemistry (e.g., microwave-assisted and/or solid-phase synthesis) to access various macrocycle entities have attracted great interest in this chemical space. This article serves as an update on our previous review related to macrocyclic drugs and new synthetic strategies toward macrocycles (Molecules, 2013, 18, 6230). In this work, I first reviewed recent FDA-approved macrocyclic drugs since 2014, followed by new advances in macrocycle synthesis using high throughput chemistry, including microwave-assisted and/or solid-supported macrocyclization strategies. Examples and highlights of macrocyclization include macrolactonization and macrolactamization, transition-metal catalyzed olefin ring-closure metathesis, intramolecular C-C and C-heteroatom cross-coupling, copper- or ruthenium-catalyzed azide-alkyne cycloaddition, intramolecular SNAr or SN2 nucleophilic substitution, condensation reaction, and multi-component reaction-mediated macrocyclization, and covering the literature since 2010.

Synthesis of 20-Membered Macrocyclic Pseudo-Natural Products Yields Inducers of LC3 Lipidation.

Design and synthesis of pseudo-natural products (PNPs) through recombination of natural product (NP) fragments in unprecedented arrangements enables the discovery of novel biologically relevant chemical matter. With a view to wider coverage of NP-inspired chemical and biological space, we describe the combination of this principle with macrocycle formation. PNP-macrocycles were synthesized efficiently in a stereoselective one-pot procedure including the 1,3-dipolar cycloadditions of different dipolarophiles with dimeric cinchona alkaloid-derived azomethine ylides formed in situ. The 20-membered bis-cycloadducts embody 18 stereocenters and an additional fragment-sized NP-structure. After further functionalization, a collection of 163 macrocyclic PNPs was obtained. Biological investigation revealed potent inducers of the lipidation of the microtubule associated protein 1 light chain 3 (LC3) protein, which plays a prominent role in various autophagy-related processes.

High-Yielding Flow Synthesis of a Macrocyclic Molecular Hinge.

Many molecular machines are built from modular components with well-defined motile capabilities, such as axles and wheels. Hinges are particularly useful, as they provide the minimum flexibility needed for a simple and pronounced conformational change. Compounds with multiple stable conformers are common, but molecular hinges almost exclusively operate via dihedral rotations rather than truly hinge-like clamping mechanisms. An ideal molecular hinge would better reproduce the behavior of hinged devices, such as gates and tweezers, while remaining soluble, scalable, and synthetically versatile. Herein, we describe two isomeric macrocycles with clamp-like open and closed geometries, which crystallize as separate polymorphs but interconvert freely in solution. An unusual one-pot addition cyclization reaction was used to produce the macrocycles on a multigram scale from inexpensive reagents, without supramolecular templating or high-dilution conditions. Using mechanistic information from NMR kinetic studies and at-line mass spectrometry, we developed a semicontinuous flow synthesis with maximum conversions of 85-93% and over 80% selectivity for a single isomer. The macrocycles feature voids that are sterically protected from guests, including reactive species such as fluoride ions, and could therefore serve as chemically inert hinges for adaptive supramolecular receptors and flexible porous materials.

Diverse Proton-Conducting Nanotubes via a Tandem Macrocyclization and Assembly Strategy.

Macrocycles that assemble into nanotubes exhibit emergent properties stemming from their low dimensionality, structural regularity, and distinct interior environments. We report a versatile strategy to synthesize diverse nanotube structures in a single, efficient reaction by using a conserved building block bearing a pyridine ring. Imine condensation of a 2,4,6-triphenylpyridine-based diamine with various aromatic dialdehydes yields chemically distinct pentagonal [5 + 5], hexagonal [3 + 3], and diamond-shaped [2 + 2] macrocycles depending on the substitution pattern of the aromatic dialdehyde monomer. Atomic force microscopy and in solvo X-ray diffraction demonstrate that protonation of the macrocycles under the mild conditions used for their synthesis drives assembly into high-aspect ratio nanotubes. Each of the pyridine-containing nanotube assemblies exhibited measurable proton conductivity by electrochemical impedance spectroscopy, with values as high as 10-3 S m-1 (90% R.H., 25 °C) that we attribute to differences in their internal pore sizes. This synthetic strategy represents a general method to access robust nanotube assemblies from a universal pyridine-containing monomer, which will enable systematic investigations of their emergent properties.