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.

Discovery of 3-hydroxymethyl-azetidine derivatives as potent polymerase theta inhibitors.

Inhibition of the low fidelity DNA polymerase Theta (Polθ) is emerging as an attractive, synthetic-lethal antitumor strategy in BRCA-deficient tumors. Here we report the AI-enabled development of 3-hydroxymethyl-azetidine derivatives as a novel class of Polθ inhibitors featuring central scaffolding rings. Structure-based drug design first identified A7 as a lead compound, which was further optimized to the more potent derivative B3 and the metabolically stable deuterated compound C1. C1 exhibited significant antiproliferative properties in DNA repair-compromised cells and demonstrated favorable pharmacokinetics, showcasing that 3-hydroxymethyl-azetidine is an effective bio-isostere of pyrrolidin-3-ol and emphasizing the potential of AI in medicinal chemistry for precise molecular modifications.

Local and Sustained Baricitinib Delivery to the Skin through Injectable Hydrogels Containing Reversible Thioimidate Adducts.

Janus kinase (JAK) inhibitors are approved for many dermatologic disorders, but their use is limited by systemic toxicities including serious cardiovascular events and malignancy. To overcome these limitations, injectable hydrogels are engineered for the local and sustained delivery of baricitinib, a representative JAK inhibitor. Hydrogels are formed via disulfide crosslinking of thiolated hyaluronic acid macromers. Dynamic thioimidate bonds are introduced between the thiolated hyaluronic acid and nitrile-containing baricitinib for drug tethering, which is confirmed with 1H and 13C nuclear magnetic resonance (NMR). Release of baricitinib is tunable over six weeks in vitro and active in inhibiting JAK signaling in a cell line containing a luciferase reporter reflecting interferon signaling. For in vivo activity, baricitinib hydrogels or controls are injected intradermally into an imiquimod-induced mouse model of psoriasis. Imiquimod increases epidermal thickness in mice, which is unaffected when treated with baricitinib or hydrogel alone. Treatment with baricitinib hydrogels suppresses the increased epidermal thickness in mice treated with imiquimod, suggesting that the sustained and local release of baricitinib is important for a therapeutic outcome. This study is the first to utilize a thioimidate chemistry to deliver JAK inhibitors to the skin through injectable hydrogels, which has translational potential for treating inflammatory disorders.

Four-Component Strain-Release-Driven Synthesis of Functionalized Azetidines.

Despite the favorable properties that azetidine rings can engender on drug-compounds, methods for the diversity-oriented synthesis of azetidine-based structures are significantly underdeveloped. Herein, we report the successful realization of a multicomponent [1,2]-Brook rearrangement/strain-release-driven anion relay sequence and its application to the modular synthesis of substituted azetidines. The rapidity of the reaction, as confirmed by in situ infra-red spectroscopy, leverages the strain-release ring-opening of azabicyclo[1.1.0]butane to drive the equilibrium of the Brook rearrangement. The three electrophilic coupling partners, added sequentially to azabicyclo[1.1.0]butyl-lithium, could be individually varied to access a diverse compound library. The utility of this methodology was demonstrated in a 4-step synthesis of the EP2 receptor antagonist PF-04418948.

The Anisotropic Chemical Reaction Mechanism of 1,3,3-trinitroazetidine (TNAZ) under Different Shock Wave Directions by ReaxFF Reactive Molecular Dynamics Simulations.

1,3,3-Trinitroazetidine (TNAZ) has good thermal stability and low shock sensitivity, among other properties, and it has broad prospects in insensitive ammunition applications. In this study, a molecular dynamics calculation based on the ReaxFF-lg force field and multiscale shock technique (MSST) was used to simulate the shock-induced chemical reaction of TNAZ with different shock wave directions. The results showed that the shock sensitivity of TNAZ was in the order of [100] > [010] > [001]. There were significant differences in molecular arrangements in different shock directions, which affected the reaction rate and reaction path in different directions. The molecular arrangement in the [010] and [001] directions formed a "buffer" effect. The formation and cleavage of bonds, formation of small molecules and growth of clusters were analyzed to show the effect of the "buffer". The polymerization reactions in the [010] and [001] directions appeared later than that in the [100] direction, and the cluster growth in the [010] and [001] directions was slower than that in the [100] direction. In different shock loading directions, the formation and cleavage mechanisms of the N-O bonds of the TNAZ molecules were different, which resulted in differences in the initial reaction path and reaction rate in the three directions

Intramolecular, Visible-Light-Mediated Aza Paternò-Büchi Reactions of Unactivated Alkenes.

Azetidines are of particular interest in medicinal chemistry for their favorable properties, including increased resistance to oxidative metabolism and lower lipophilicity. The recent development of [2 + 2] reactions has significantly expanded the limited repertoire of methods for azetidine synthesis, but access to more complex architectures still requires further development. Herein, we report a visible-light-enabled intramolecular [2 + 2] cycloaddition of unactivated alkenes that proved previously unreactive to access tricyclic azetidines with 3D complex structures and high levels of saturation.

Structural insight into the molecular mechanism of cilofexor binding to the farnesoid X receptor.

Farnesoid X receptor (FXR) is a bile acid-related nuclear receptor and is considered a promising target to treat several liver disorders. Cilofexor is a selective FXR agonist and has already entered phase III trials in primary sclerosing cholangitis (PSC) patients. Pruritis caused by cilofexor treatment is dose dependent. The binding characteristics of cilofexor with FXR and its pruritogenic mechanism remain unclear. In our research, the affinity of cilofexor bound to FXR was detected using an isothermal titration calorimetry (ITC) assay. The binding mechanism between cilofexor and FXR-LBD is explained by the cocrystal structure of the FXR/cilofexor complex. Structural models indicate the possibility that cilofexor activates Mas-related G protein-coupled receptor X4 (MRGPRX4) or G protein-coupled bile acid receptor 1 (GPBAR1), leading to pruritus. In summary, our analyses provide a molecular mechanism of cilofexor binding to FXR and provide a possible explanation for the dose-dependent pruritis of cilofexor.

Bicyclic azetidines target acute and chronic stages of Toxoplasma gondii by inhibiting parasite phenylalanyl t-RNA synthetase.

Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis.

Synthesis of PF-6870961, a major hydroxy metabolite of the novel ghrelin receptor inverse agonist PF-5190457.

Preclinical and human studies have indicated involvement of the ghrelin system in alcohol-related behaviors illuminating the possibility of using ghrelin receptor blockers as a pharmacological intervention for alcohol use disorder (AUD). Preliminary data from a recently conducted phase 1b human study with a ghrelin receptor inverse agonist, PF-5190457 (2-(2-methylimidazo[2,1-b][1,3thiazol-6-yl)-1-{2-(1R)-5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl]-2,7-diazaspiro[3.5]non-7-ylethanone), provided evidence on the safety and tolerability of this compound when co-administered with alcohol. Furthermore, the study revealed important information on the biotransformation pathways for this compound and prompted the discovery and then synthesis of a newly identified major metabolite, PF-6870961 ((R)-1-(2-(5-(2-hydroxy-6-methylpyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)-2-(2-methylimidazo[2,1-b]thiazol-6-yl)ethan-1-one). The metabolite was synthesized and fully characterized through a design that enabled it to be prepared in useful quantities. The synthesis provided direct access to the recently discovered PF-6870961 and is allowing researchers to conduct additional and deeper evaluation of its in vitro and in vivo properties.

Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition.

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.

Novel Reversible-Binding PET Ligands for Imaging Monoacylglycerol Lipase Based on the Piperazinyl Azetidine Scaffold.

Monoacylglycerol lipase (MAGL) is a 33 kDa serine protease primarily responsible for hydrolyzing 2-arachidonoylglycerol into the proinflammatory eicosanoid precursor arachidonic acid in the central nervous system. Inhibition of MAGL constitutes an attractive therapeutic concept for treating psychiatric disorders and neurodegenerative diseases. Herein, we present the design and synthesis of multiple reversible MAGL inhibitor candidates based on a piperazinyl azetidine scaffold. Compounds 10 and 15 were identified as the best-performing reversible MAGL inhibitors by pharmacological evaluations, thus channeling their radiolabeling with fluorine-18 in high radiochemical yields and favorable molar activity. Furthermore, evaluation of [18F]10 and [18F]15 ([18F]MAGL-2102) by autoradiography and positron emission tomography (PET) imaging in rodents and nonhuman primates demonstrated favorable brain uptakes, heterogeneous radioactivity distribution, good specific binding, and adequate brain kinetics, and [18F]15 demonstrated a better performance. In conclusion, [18F]15 was found to be a suitable PET radioligand for the visualization of MAGL, harboring potential for the successful translation into humans.

Medicinal applications of coumarins bearing azetidinone and thiazolidinone moieties.

Coumarins (2H-chromen-2-ones), also known as benzopyran-2-ones, are a family of naturally occurring heterocyclic ring systems that contain a lactone moiety. Coumarins exhibit a wide range of well-studied pharmacological properties. Over the last few decades, as a result of advances in diverse oriented synthetic routes, physicochemical properties and numerous biological activities, coumarins have become globally studied molecules from various synthetic and medicinal chemists. Recently, several bioactive coumarins bearing azetidinone and thiazolidinone moieties have been found to display a range of therapeutic characteristics, including antimicrobial, anticancer, antidiabetic and anti-inflammatory properties. This review offers a brief description of the synthetic methodologies, known bioactivity and structure-activity relationships of coumarins bearing azetidinones and thiazolidinones.

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections.

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3-H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC50 = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC50 = 12 µM by visual CPE score; EC50 = 8.3 µM by TMS score; MCC > 100 µM, CC50 = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC50 in the range 14.5-97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at ß-lactamase, thus protecting the antibiotic from undesirable biotransformation.

Discovery of new anticancer thiourea-azetidine hybrids: design, synthesis, in vitro antiproliferative, SAR, in silico molecular docking against VEGFR-2, ADMET, toxicity, and DFT studies.

With the aim to discover potent and novel antitumor agents, a series of thiourea compounds bearing 3-(4-methoxyphenyl)azetidine moiety were designed according to the essential pharmacophoric features of the reported VEGFR-2 inhibitors and synthesized. All the synthesized compounds were evaluated for their in vitro anticancer activity against various human cancer cell lines (lung (A549), prostate (PC3), breast (MCF-7), liver (HepG2), colon (HCT-116), ovarian (SKOV-3), skin (A431), brain (U251) and kidney (786-O)). 3-(4-Methoxy-3-(2-methoxypyridin-4-yl)phenyl)-N-(4-methoxyphenyl)azetidine-1-carbothioamide (3B) was found to be most potent member against PC3, U251, A431, and 786-O cancer cell lines with EC50 values 0.25, 0.6, 0.03, and 0.03 µM, respectively and showed more potency than Doxorubicin in PC3, A431, and 786-O cell lines. Compounds 1B to 7B showed EC50 values ranging from 0.03 to 12.55 µM in A431 cell line. Compound 3-(4-methoxy-3-(pyridin-4-yl)phenyl)-N-(4-methoxyphenyl)azetidine-1-carbothioamide (1B) was found to be highly efficient in A431 and 786-O cell line with EC50 values of 0.77 and 0.73 µM respectively. All the compounds exhibited good to moderate cytotoxic activity. The pharmacophoric features and molecular docking studies confirmed the potentialities of compounds 1B, 2B, 3B and 5B to be VEGFR-2 inhibitors. Moreover, in silico ADMET prediction indicated that most of the synthesized compounds have drug-like properties, possess low adverse effects and toxicity. In addition, the DFT studies for the most active compounds (1B and 3B) were carried out. In the end, our studies revealed that the compounds 1B and 3B represent promising anticancer potentialities through their VEGFR-2 inhibition.

Design, synthesis and antitumor evaluation of novel chiral diaryl substituted azetidin-2-one derivatives as tubulin polymerization inhibitors.

A novel class of diaryl substituted azetidin-2-one derivatives were designed, asymmetrically synthesized, and evaluated for antiproliferative activities. The in vitro antitumor assay revealed that among the 4-aryl-substituted 1-(3,4,5-trimethoxyphenyl)azetidin-2-ones (B series), most possessed moderate to strong activities, with compound B7c that bears a 2-naphthyl substituent being the most potent one (IC50 0.16-0.40 µM) against a panel of human cancer cell lines. In contrast, none of the 3-(arylmethylene)-substituted 1-(3,4,5-trimethoxyphenyl)azetidin-2-ones (L series) showed significant activities in the assay. Further studies indicated that B7c inhibited tubulin polymerization, disrupted in vitro vascularization, blocked cell cycle progression at G2/M phase, induced cell apoptosis, decreased mitochondrial membrane potential, and increased the intracellular reactive oxygen species level in a dose-dependent way. Compound B7c also inhibited significantly tumor growth in a xenograft mice model with no obvious drop in the mice body weights. Collectively, these results suggested that B7c and its analogues should merit further investigation as new promising antitumor agents.

Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues.

Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.

Densely Functionalized 2-Methylideneazetidines: Evaluation as Antibacterials.

Twenty-two novel, variously substituted nitroazetidines were designed as both sulfonamide and urethane vinylogs possibly endowed with antimicrobial activity. The compounds under study were obtained following a general procedure recently developed, starting from 4-nitropentadienoates deriving from a common ß-nitrothiophenic precursor. While being devoid of any activity against fungi and Gram-negative bacteria, most of the title compounds performed as potent antibacterial agents on Gram-positive bacteria (E. faecalis and three strains of S. aureus), with the most potent congener being the 1-(4-chlorobenzyl)-3-nitro-4-(p-tolyl)azetidine 22, which displayed potency close to that of norfloxacin, the reference antibiotic (minimum inhibitory concentration values 4 and 1-2 µg/mL, respectively). Since 22 combines a relatively efficient activity against Gram-positive bacteria and a cytotoxicity on eucharyotic cells only at 4-times higher concentrations (inhibiting concentration on 50% of the cultured eukaryotic cells: 36 ± 10 µM, MIC: 8.6 µM), it may be considered as a promising hit compound for the development of a new series of antibacterials selectively active on Gram-positive pathogens. The relatively concise synthetic route described herein, based on widely available starting materials, could feed further structure-activity relationship studies, thus allowing for the fine investigation and optimization of the toxico-pharmacological profile.

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.

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.

The targetable nanoparticle BAF312@cRGD-CaP-NP represses tumor growth and angiogenesis by downregulating the S1PR1/P-STAT3/VEGFA axis in triple-negative breast cancer.

BACKGROUND: Overexpressed vascular endothelial growth factor A (VEGFA) and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) cause unrestricted tumor growth and angiogenesis of breast cancer (BRCA), especially triple-negative breast cancer (TNBC). Hence, novel treatment strategy is urgently needed. RESULTS: We found sphingosine 1 phosphate receptor 1 (S1PR1) can regulate P-STAT3/VEGFA. Database showed S1PR1 is highly expressed in BRCA and causes the poor prognosis of patients. Interrupting the expression of S1PR1 could inhibit the growth of human breast cancer cells (MCF-7 and MDA-MB-231) and suppress the angiogenesis of human umbilical vein endothelial cells (HUVECs) via affecting S1PR1/P-STAT3/VEGFA axis. Siponimod (BAF312) is a selective antagonist of S1PR1, which inhibits tumor growth and angiogenesis in vitro by downregulating the S1PR1/P-STAT3/VEGFA axis. We prepared pH-sensitive and tumor-targeted shell-core structure nanoparticles, in which hydrophilic PEG2000 modified with the cyclic Arg-Gly-Asp (cRGD) formed the shell, hydrophobic DSPE formed the core, and CaP (calcium and phosphate ions) was adsorbed onto the shell; the nanoparticles were used to deliver BAF312 (BAF312@cRGD-CaP-NPs). The size and potential of the nanoparticles were 109.9 ± 1.002 nm and - 10.6 ± 0.056 mV. The incorporation efficacy for BAF312 was 81.4%. Results confirmed BAF312@cRGD-CaP-NP could dramatically inhibit tumor growth and angiogenesis in vitro and in MDA-MB-231 tumor-bearing mice via downregulating the S1PR1/P-STAT3/VEGFA axis. CONCLUSIONS: Our data suggest a potent role for BAF312@cRGD-CaP-NPs in treating BRCA, especially TNBC by downregulating the S1PR1/P-STAT3/VEGFA axis.