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.

Ring opening of photogenerated azetidinols as a strategy for the synthesis of aminodioxolanes.

α-Aminoacetophenones are identified as promising building blocks for the synthesis of highly substituted dioxolanes. The presented strategy is founded on the build and release of molecular strain and achieves a formal transposition of a methyl group. During light irradiation, 3-phenylazetidinols are forged as reaction intermediates, which readily undergo ring opening upon the addition of electron-deficient ketones or boronic acids. Key to the successful development of this two-step process is the identification of a benzhydryl-protecting group, which orchestrates the photochemical Norrish-Yang cyclization and facilitates the subsequent ring opening.

Substituent and Solvent Effect Studies of N-Alkenylnitrone 4π Electrocyclizations.

The roles of substituent and solvent effects in promoting the 4π electrocyclization of N-alkenylnitrones to give azetidine nitrones have been investigated by experimental examination of relative rates, activation energies, and linear free energy relationships. These transformations are synthetically important because they favor the formation of a strained heterocyclic ring with imbedded functionality and stereochemical information for versatile derivatization. Mechanistic investigations, including Hammett studies, solvent-dependent Eyring studies, and solvent isotope effects, provide insight into the steric and electronic factors that control these electrocyclizations and identify trends that can be used to advance this approach towards the rapid synthesis of complex azetidines.

Design, Synthesis, Antimicrobial Evaluation of Novel 2-Oxo-4-Substituted Aryl-Azetidine Benzotriazole Derivatives.

A series of compounds was synthesized and characterized to explore new antimicrobial agents. These compounds were evaluated by using the agar cup plate method. The most active compound exhibited a zone of inhibition 18±0.09 mm and 19±0.09 mm against E. Coli and S. aureus, respectively. To gain insights into the intermolecular interactions, molecular docking studies were performed at the active site of the glucosamine fructose 6 phosphate synthase (GlcN 6 p) enzyme (PDB Id: 1XFF). The results of the molecular docking studies are in agreement with the pharmacological evaluation with potent compounds, exhibiting docking scores of -11.2. However, deformability, B-factor and covariance computations showed a result that the most active compound favored molecular connections with the protein. Therefore, our research is important for the development of antimicrobial agents.

Synthesis and neuroprotective activity of 3-aryl-3-azetidinyl acetic acid methyl ester derivatives.

A library of 3-aryl-3-azetidinyl acetic acid methyl ester derivatives was prepared from N-Boc-3-azetidinone employing the Horner-Wadsworth-Emmons reaction, rhodium(I)-catalyzed conjugate addition of arylboronic acids, and subsequent elaborations to obtain N-unprotected hydrochlorides, N-alkylated and N-acylated azetidine derivatives. The compounds were evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity, revealing several derivatives to possess AChE inhibition comparable to that of the AChE inhibitor rivastigmine. The binding mode of the AChE inhibitor donepezil and selected active compounds 26 and 27 within the active site of AChE was studied using molecular docking. Furthermore, the neuroprotective activity of the prepared compounds was evaluated in models associated with Parkinson's disease (salsolinol-induced) and aspects of Alzheimer's disease (glutamate-induced oxidative damage). Compound 28 showed the highest neuroprotective effect in both salsolinol- and glutamate-induced neurodegeneration models, and its protective effect in the glutamate model was revealed to be driven by a reduction in oxidative stress and caspase-3/7 activity.

Length-Dependent Transition from Extended to Folded Shapes in Short Oligomers of an Azetidine-Based α-Amino Acid: The Critical Role of NH···N H-Bonds.

Hydrogen bonds (H-bonds) are ubiquitous in peptides and proteins and are central to the stabilization of their structures. Inter-residue H-bonds between non-adjacent backbone amide NH and C=O motifs lead to the well-known secondary structures of helices, turns and sheets, but it is recognized that other H-bonding modes may be significant, including the weak intra-residue H-bond (called a C5 H-bond) that implicates the NH and C=O motifs of the same amino acid residue. Peptide model compounds that adopt stable C5 H-bonds are not readily available and the so-called 2.05-helix, formed by successive C5 H-bonds, is an elusive secondary structure. Using a combination of theoretical chemistry and spectroscopic studies in both the gas phase and solution phase, we have demonstrated that derivatives of 3-amino-1-methylazetidine-3-carboxylic acid, Aatc(Me) can form sidechain-backbone N-H···N C6γ H-bonds that accompany-and thereby stabilize-C5 H-bonds. In the capped trimer of Aatc(Me), extended C5/C6γ motifs are sufficiently robust to challenge classical 310-helix formation in solution and the fully-extended 2.05-helix conformer has been characterized in the gas phase. Concurrent H-bonding support for successive C5 motifs is a new axiom for stabilizing the extended backbone secondary structure in short peptides.

L-Proline Prevents Endoplasmic Reticulum Stress in Microglial Cells Exposed to L-azetidine-2-carboxylic Acid.

L-Azetidine-2-carboxylic acid (AZE) is a non-protein amino acid that shares structural similarities with its proteogenic L-proline amino acid counterpart. For this reason, AZE can be misincorporated in place of L-proline, contributing to AZE toxicity. In previous work, we have shown that AZE induces both polarization and apoptosis in BV2 microglial cells. However, it is still unknown if these detrimental effects involve endoplasmic reticulum (ER) stress and whether L-proline co-administration prevents AZE-induced damage to microglia. Here, we investigated the gene expression of ER stress markers in BV2 microglial cells treated with AZE alone (1000 µM), or co-treated with L-proline (50 µM), for 6 or 24 h. AZE reduced cell viability, nitric oxide (NO) secretion and caused a robust activation of the unfolded protein response (UPR) genes (ATF4, ATF6, ERN1, PERK, XBP1, DDIT3, GADD34). These results were confirmed by immunofluorescence in BV2 and primary microglial cultures. AZE also altered the expression of microglial M1 phenotypic markers (increased IL-6, decreased CD206 and TREM2 expression). These effects were almost completely prevented upon L-proline co-administration. Finally, triple/quadrupole mass spectrometry demonstrated a robust increase in AZE-bound proteins after AZE treatment, which was reduced by 84% upon L-proline co-supplementation. This study identified ER stress as a pathogenic mechanism for AZE-induced microglial activation and death, which is reversed by co-administration of L-proline.

Synthesis of New Azetidine and Oxetane Amino Acid Derivatives through Aza-Michael Addition of NH-Heterocycles with Methyl 2-(Azetidin- or Oxetan-3-Ylidene)Acetates.

In this paper, a simple and efficient synthetic route for the preparation of new heterocyclic amino acid derivatives containing azetidine and oxetane rings was described. The starting (N-Boc-azetidin-3-ylidene)acetate was obtained from (N-Boc)azetidin-3-one by the DBU-catalysed Horner-Wadsworth-Emmons reaction, followed by aza-Michael addition with NH-heterocycles to yield the target functionalised 3-substituted 3-(acetoxymethyl)azetidines. Methyl 2-(oxetan-3-ylidene)acetate was obtained in a similar manner, which was further treated with various (N-Boc-cycloaminyl)amines to yield the target 3-substituted 3-(acetoxymethyl)oxetane compounds. The synthesis and diversification of novel heterocyclic amino acid derivatives were achieved through the Suzuki-Miyaura cross-coupling from the corresponding brominated pyrazole-azetidine hybrid with boronic acids. The structures of the novel heterocyclic compounds were confirmed via 1H-, 13C-, 15N-, and 19F-NMR spectroscopy, as well as HRMS investigations.

Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells.

L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0−2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1ß, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.

Asymmetric Synthesis of Spiro[Azetidine-3,3'-Indoline]-2,2'-Diones via Copper(I)-Catalyzed Kinugasa/C-C Coupling Cascade Reaction.

Spiro[azetidine-indolines] are important scaffolds in diverse bioactive compounds. Current efforts to synthesize spiro[azetidine-indolines] are limited to chiral spiro[azetidine-2,3'-indolines]. Asymmetric synthesis of structurally similar chiral spiro[azetidine-3,3'-indolines] remains unexplored. In this work, the first copper(I)-catalyzed asymmetric Kinugasa/aryl C-C coupling cascade reaction is described. This provides a straightforward access to densely functionalized chiral spiro[azetidine-3,3'-indoline]-2,2'-diones in good yields and with high enantioselectivity.

Ring-opening of azetidiniums by nucleophiles. Synthesis of polysubstituted linear amines.

This microreview focuses on the nucleophilic ring-opening of azetidiniums presenting various substitution patterns at C2, C3, and C4. In most cases, the nucleophilic ring-opening occurred in a stereoselective and regioselective fashion producing functionalized linear amines. Experimental selectivities associated with Density Functional Theory (DFT) calculations have allowed a better understanding of the parameters governing the regioselectivities.

Azetidines of pharmacological interest.

Azetidines are almost unexplored among nitrogen-containing saturated heterocycles due to difficulties associated with their synthesis. However, over the past few years, attempts have been made by scientists to advance their synthetic feasibility. Compounds with the azetidine moiety display an important and diverse range of pharmacological activities, such as anticancer, antibacterial, antimicrobial, antischizophrenic, antimalarial, antiobesity, anti-inflammatory, antidiabetic, antiviral, antioxidant, analgesic, and dopamine antagonist activities, and are also useful for the treatment of central nervous system disorders and so forth. Owing to its satisfactory stability, molecular rigidity, and chemical and biological properties, azetidine has emerged as a valuable scaffold and it has drawn the attention of medicinal researchers. The present review sheds light on the traditional method of synthesis of azetidine and advancements in synthetic methodology over the past few years, along with its application with various examples, and its biological significance.

Synthesis of 2-Oxazolines from Ring Opening Isomerization of 3-Amido-2-Phenyl Azetidines.

Chiral 2-oxazolines are valuable building blocks and famous ligands for asymmetric catalysis. The most common synthesis involves the reaction of an amino alcohol with a carboxylic acid. In this paper, an efficient synthesis of 2-oxazolines has been achieved via the stereospecific isomerization of 3-amido-2-phenyl azetidines. The reactions were studied in the presence of both Brønsted and Lewis acids, and Cu(OTf)2 was found to be the most effective.

Theoretical Study on the Photo-Oxidation and Photoreduction of an Azetidine Derivative as a Model of DNA Repair.

Photocycloreversion plays a central role in the study of the repair of DNA lesions, reverting them into the original pyrimidine nucleobases. Particularly, among the proposed mechanisms for the repair of DNA (6-4) photoproducts by photolyases, it has been suggested that it takes place through an intermediate characterized by a four-membered heterocyclic oxetane or azetidine ring, whose opening requires the reduction of the fused nucleobases. The specific role of this electron transfer step and its impact on the ring opening energetics remain to be understood. These processes are studied herein by means of quantum-chemical calculations on the two azetidine stereoisomers obtained from photocycloaddition between 6-azauracil and cyclohexene. First, we analyze the efficiency of the electron-transfer processes by computing the redox properties of the azetidine isomers as well as those of a series of aromatic photosensitizers acting as photoreductants and photo-oxidants. We find certain stereodifferentiation favoring oxidation of the cis-isomer, in agreement with previous experimental data. Second, we determine the reaction profiles of the ring-opening mechanism of the cationic, neutral, and anionic systems and assess their feasibility based on their energy barrier heights and the stability of the reactants and products. Results show that oxidation largely decreases the ring-opening energy barrier for both stereoisomers, even though the process is forecast as too slow to be competitive. Conversely, one-electron reduction dramatically facilitates the ring opening of the azetidine heterocycle. Considering the overall quantum-chemistry findings, N,N-dimethylaniline is proposed as an efficient photosensitizer to trigger the photoinduced cycloreversion of the DNA lesion model.

TORC1 regulates autophagy induction in response to proteotoxic stress in yeast and human cells.

Misfolded and aggregated proteins are eliminated to maintain protein homeostasis. Autophagy contributes to the removal of protein aggregates. However, if and how proteotoxic stress induces autophagy is poorly understood. Here we show that proteotoxic stress after treatment with azetidine-2-carboxylic acid (AZC), a toxic proline analog, induces autophagy in budding yeast. AZC treatment attenuated target of rapamycin complex 1 (TORC1) activity, resulting in the dephosphorylation of Atg13, a key factor of autophagy. By contrast, AZC treatment did not affect target of rapamycin complex 2 (TORC2). Proteotoxic stress also induced TORC1 inactivation and autophagy in fission yeast and human cells. This study suggested that TORC1 is a conserved key factor to cope with proteotoxic stress in eukaryotic cells.

Involvement of the stress-responsive transcription factor gene MSN2 in the control of amino acid uptake in Saccharomyces cerevisiae.

The transcriptional factor Msn2 plays a pivotal role in response to environmental stresses by activating the transcription of stress-responsive genes in Saccharomyces cerevisiae. Our previous studies demonstrate that intracellular proline acts as a key protectant against various stresses. It is unknown, however, whether Msn2 is involved in proline homeostasis in S. cerevisiae cells. We here found that MSN2-overexpressing (MSN2-OE) cells showed higher sensitivity to a toxic analogue of proline, l-azetidine-2-carboxylic acid (AZC), as well as to the other amino acid toxic analogues, than wild-type cells. Overexpression of MSN2 increased the intracellular content of AZC, suggesting that Msn2 positively regulates the uptake of proline. Among the known proline permease genes, GNP1 was shown to play a predominant role in the AZC toxicity. Based on quantitative real-time PCR and western blot analyses, the overexpression of MSN2 did not induce any increases in the transcript levels of GNP1 or the other proline permease genes, while the amount of the Gnp1 protein was markedly increased in MSN2-OE cells. Microscopic observation suggested that the endocytic degradation of Gnp1 was impaired in MSN2-OE cells. Thus, this study sheds light on a novel link between the Msn2-mediated global stress response and the amino acid homeostasis in S. cerevisiae.

Cell death and mitochondrial dysfunction induced by the dietary non-proteinogenic amino acid L-azetidine-2-carboxylic acid (Aze).

In addition to the 20 protein amino acids that are vital to human health, hundreds of naturally occurring amino acids, known as non-proteinogenic amino acids (NPAAs), exist and can enter the human food chain. Some NPAAs are toxic through their ability to mimic protein amino acids and this property is utilised by NPAA-containing plants to inhibit the growth of other plants or kill herbivores. The NPAA L-azetidine-2-carboxylic acid (Aze) enters the food chain through the use of sugar beet (Beta vulgaris) by-products as feed in the livestock industry and may also be found in sugar beet by-product fibre supplements. Aze mimics the protein amino acid L-proline and readily misincorporates into proteins. In light of this, we examined the toxicity of Aze to mammalian cells in vitro. We showed decreased viability in Aze-exposed cells with both apoptotic and necrotic cell death. This was accompanied by alterations in endosomal-lysosomal activity, changes to mitochondrial morphology and a significant decline in mitochondrial function. In summary, the results show that Aze exposure can lead to deleterious effects on human neuron-like cells and highlight the importance of monitoring human Aze consumption via the food chain.

Optimization of an azetidine series as inhibitors of colony stimulating factor-1 receptor (CSF-1R) Type II to lead to the clinical candidate JTE-952.

Optimization of novel azetidine compounds, which we had found as colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors, provided JTE-952 as a clinical candidate with high cellular activity (IC50 = 20 nM) and good pharmacokinetics profile. JTE-952 was also effective against a mouse collagen-induced model of arthritis (mouse CIA-model). Additionally, the X-ray co-crystal structure of JTE-952 with CSF-1R protein was shown to be a Type II inhibitor, and the kinase panel assay indicated that JTE-952 had high kinase selectivity.

Discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by the use of docking models.

We report the discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by using a structure-based drug design (SBDD) based on a docking model. The work leads to the representative compound 4a with high CSF-1R inhibitory activity (IC50 = 9.1 nM). The obtained crystal structure of an azetidine compound with CSF-1R, which matched our predicted docking model, demonstrates that the azetidine compounds bind to the DFG-out conformation of the protein as a Type II inhibitor.