Triethylamine-Promoted Oxidative Cyclodimerization of 2H-Azirine-2-carboxylates to Pyrimidine-4,6-dicarboxylates: Experimental and DFT Study.

An unprecedented oxidative cyclodimerization reaction of 2H-azirine-2-carboxylates to pyrimidine-4,6-dicarboxylates under heating with triethylamine in air is described. In this reaction, one azirine molecule undergoes formal cleavage across the C-C bond and another across the C=N bond. According to the experimental study and DFT calculations, the key steps of the reaction mechanism include nucleophilic addition of N,N-diethylhydroxylamine to an azirine to form an (aminooxy)aziridine, generation of an azomethine ylide, and its 1,3-dipolar cycloaddition to the second azirine molecule. The crucial condition for the synthesis of pyrimidines is generation of N,N-diethylhydroxylamine in the reaction mixture in a very low concentration, which is ensured by the slow oxidation of triethylamine with air oxygen. Addition of a radical initiator accelerated the reaction and resulted in higher yields of the pyrimidines. Under these conditions, the scope of the pyrimidine formation was elucidated, and a series of pyrimidines was synthesized.

One-Pot Synthesis of Multifunctionalized 1-Pyrrolines from 2-Alkyl-2H-azirines and Diazocarbonyl Compounds.

A novel strategy for the synthesis of 1-pyrrolines based on formal [4 + 1] annulation of 2-alkyl-2H-azirines with diazocarbonyl compounds has been developed. This one-pot approach includes the Rh(II)-catalyzed formation of 4-alkyl-2-azabuta-1,3-dienes, followed by the DBU-promoted cyclization, and features a good substrate tolerance. The 1-pyrrolines containing an ester group at the C3 were prepared in a three-step one-pot procedure starting from 5-alkoxyisoxazoles. The cyclization of 2-azabutadienes to 1-pyrrolines most likely proceeds via the 6π electrocyclization of a conjugated NH-azomethine ylide.

Non-Aziridination Approaches to 3-Arylaziridine-2-carboxylic Acid Derivatives and 3-Aryl-(aziridin-2-yl)ketones.

Highly functionalized aziridines, including compounds with aromatic moieties, are attractive substrates both in synthetic and medical areas of chemistry. There is a broad and interesting set of synthetic methods for reaching these compounds. Aziridination represents the most explored tool, but there are several other more specific, less well-known, but highly promising approaches. Therefore, the current review focuses on recently described or updated ways to obtain 3-arylated aziridines via different non-aziridination-based synthetic methods, reported mainly since 2000. The presented methods belong to two main directions of synthesis, namely, cyclization of open-chain substrates and rearrangement of other heterocycles. Cyclization of open-chain substrates includes the classic Gabriel-Cromwell type cyclization of halogenated substrates with amines, base-promoted cyclization of activated aminoalcohols (or its analogues), and the oxidative cyclization of ß-dicarbonyls. Rearrangements of other heterocycles are presented as the Baldwin rearrangement of 4-isoxazolines, the cycloaddition of 1.3-dipoles or dienes to 2H-azirines, and the addition of C- and N-nucleophiles to the double bond of azirines.

Copper(II)-Catalyzed (3+2) Cycloaddition of 2H-Azirines to Six-Membered Cyclic Enols as a Route to Pyrrolo[3,2-c]quinolone, Chromeno[3,4-b]pyrrole, and Naphtho[1,8-ef]indole Scaffolds.

A method for the [2+3] pyrroline annulation to the six-membered non-aromatic enols using 3-aryl-2H-azirines as annulation agents is developed in the current study. The reaction proceeds as a formal (3+2) cycloaddition via the N1-C2 azirine bond cleavage and is catalyzed by both Cu(II) and Cu(I) compounds. The new annulation method can be applied to prepare pyrrolo[3,2-c]quinoline, chromeno[3,4-b]pyrrole, and naphtho[1,8-ef]indole derivatives in good to excellent yields from enols of the quinolin-2-one, 2H-chromen-2-one, and 1H-phenalen-1-one series.

Carbon Atom Insertion into Pyrroles and Indoles Promoted by Chlorodiazirines.

Herein, we report a reaction that selectively generates 3-arylpyridine and quinoline motifs by inserting aryl carbynyl cation equivalents into pyrrole and indole cores, respectively. By employing α-chlorodiazirines as thermal precursors to the corresponding chlorocarbenes, the traditional haloform-based protocol central to the parent Ciamician-Dennstedt rearrangement can be modified to directly afford 3-(hetero)arylpyridines and quinolines. Chlorodiazirines are conveniently prepared in a single step by oxidation of commercially available amidinium salts. Selectivity as a function of pyrrole substitution pattern was examined, and a predictive model based on steric effects is put forward, with DFT calculations supporting a selectivity-determining cyclopropanation step. Computations surprisingly indicate that the stereochemistry of cyclopropanation is of little consequence to the subsequent electrocyclic ring opening that forges the pyridine core, due to a compensatory homoaromatic stabilization that counterbalances orbital-controlled torquoselectivity effects. The utility of this skeletal transform is further demonstrated through the preparation of quinolinophanes and the skeletal editing of pharmaceutically relevant pyrroles.

2H-Azirine-Based Reagents for Chemoselective Bioconjugation at Carboxyl Residues Inside Live Cells.

Protein modification by chemical reagents has played an essential role in the treatment of human diseases. However, the reagents currently used are limited to the covalent modification of cysteine and lysine residues. It is thus desirable to develop novel methods that can covalently modify other residues. Despite the fact that the carboxyl residues are crucial for maintaining the protein function, few selective labeling reactions are currently available. Here, we describe a novel reactive probe, 3-phenyl-2H-azirine, that enables chemoselective modification of carboxyl groups in proteins under both in vitro and in situ conditions with excellent efficiency. Furthermore, proteome-wide profiling of reactive carboxyl residues was performed with a quantitative chemoproteomic platform.

Synthesis of α-Aminophosphonic Acid Derivatives Through the Addition of O- and S-Nucleophiles to 2H-Azirines and Their Antiproliferative Effect on A549 Human Lung Adenocarcinoma Cells.

This work reports a straightforward regioselective synthetic methodology to prepare α-aminophosphine oxides and phosphonates through the addition of oxygen and sulfur nucleophiles to the C-N double bond of 2H-azirine derivatives. Determined by the nature of the nucleophile, different α-aminophosphorus compounds may be obtained. For instance, aliphatic alcohols such as methanol or ethanol afford α-aminophosphine oxide and phosphonate acetals after N-C3 ring opening of the intermediate aziridine. However, addition of 2,2,2-trifluoroethanol, phenols, substituted benzenthiols or ethanethiol to 2H-azirine phosphine oxides or phosphonates yields allylic α-aminophosphine oxides and phosphonates in good to high general yields. In some cases, the intermediate aziridine attained by the nucleophilic addition of O- or S-nucleophiles to the starting 2H-azirine may be isolated and characterized before ring opening. Additionally, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and non-malignant cells (MCR-5) was also screened. Some α-aminophosphorus derivatives exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over non-malignant cells (MCR-5) has been detected in almost all compounds tested.

Terminal Diazirines Enable Reverse Polarization Transfer from 15 N2 Singlets.

Diazirine moieties are chemically stable and have been incorporated into biomolecules without impediment of biological activity. The 15 N2 labeled diazirines are appealing motifs for hyperpolarization supporting relaxation protected states with long-lived lifetimes. The (-CH15 N2 ) diazirine groups investigated here are analogues to methyl groups, which provides the opportunity to transfer polarization stored on a relaxation protected (-CH15 N2 ) moiety to 1 H, thus combining the advantages of long lifetimes of 15 N polarization with superior sensitivity of 1 H detection. Despite the proximity of 1 H to 15 N nuclei in the diazirine moiety, 15 N T1 times of up to (4.6±0.4) min and singlet lifetimes Ts of up to (17.5±3.8) min are observed. Furthermore, we found terminal diazirines to support hyperpolarized 1 H2 singlet states in CH2 groups of chiral molecules. The singlet lifetime of 1 H singlets is up to (9.2±1.8) min, thus exceeding 1 H T1 relaxation time (at 8.45 T) by a factor of ≈100.

Traceless Immobilization of Analytes for High-Throughput Experiments with SAMDI Mass Spectrometry.

Label-free assays, and particularly those based on the combination of mass spectroscopy with surface chemistries, enable high-throughput experiments of a broad range of reactions. However, these methods can still require the incorporation of functional groups that allow immobilization of reactants and products to surfaces prior to analysis. In this paper, we report a traceless method for attaching molecules to a self-assembled monolayer for matrix-assisted laser desorption and ionization (SAMDI) mass spectrometry. This method uses monolayers that are functionalized with a 3-trifluoromethyl-3-phenyl-diazirine group that liberates nitrogen when irradiated and gives a carbene that inserts into a wide range of bonds to covalently immobilize molecules. Analysis of the monolayer with SAMDI then reveals peaks for each of the adducts formed from molecules in the sample. This method is applied to characterize a P450 drug metabolizing enzyme and to monitor a Suzuki-Miyaura coupling chemical reaction and is important because modification of the substrates with a functional group would alter their activities. This method will be important for high-throughput experiments in many areas, including reaction discovery and optimization.

Molecular Targets of the Hydrophobic Block of Pluronics in Cells: a Photo Affinity Labelling Approach.

PURPOSE: Pluronics are known as inhibitors of multidrug resistance thus making tumor cells sensitive to therapeutic doses of drugs. The purpose of our study consists in revealing molecular targets of the hydrophobic poly(propylene oxide) block of pluronics in living cells and the dependence of the polymers chemosensitizing efficiency upon targeting. METHODS: A photo sensitive tracer was attached to the hydrophobic poly(propylene oxide) block of 3H-labeled tert-Bu-EO-PO copolymer. The conjugate was used for treatment cells in culture. We searched for its complexes with cellular lipids or proteins using RP TLC and SDS-electrophoresis, respectively. The chemosensitizing efficiency of pluronics was evaluated by their least concentrations sufficient for MDR reversion (CMDR). RESULTS: The poly(propylene oxide) block inserts in the lipid core of plasma membrane. No preferential binding of the conjugate with any cellular protein, particularly P-gp, has been detected. FITC-labeled pluronic L61 bound to alcohol insoluble cellular targets did not participate in MDR reversion. CMDR values of 13 block copolymers have been determined. These values inversely correlated with the polymers affinity toward lipids and the ability to accelerate flip-flop. CONCLUSION: Insertion of the hydrophobic poly(propylene oxide) block of amphiphiles in the lipid core of plasma membrane and acceleration of flip-flop of lipids underlie the mechanism of MDR reversion.

Cationic Amphiphilic Tris-Cyclometalated Iridium(III) Complexes Induce Cancer Cell Death via Interaction with Ca2+-Calmodulin Complex.

In our previous paper, we reported on the preparation of some cationic amphiphilic Ir complexes (2c, 2d) containing KKGG peptides that induce and detect cell death of Jurkat cells. Mechanistic studies suggest that 2c interacts with anionic molecules and/or membrane receptors on the cell surface to trigger an intracellular Ca2+ response, resulting in the induction of cell death, accompanied by membrane disruption. We have continued the studies of cell death of Jurkat cells induced by 2c and found that xestospongin C, a selective inhibitor of an inositol 1,4,5-trisphosphate receptor located on the endoplasmic reticulum (ER), reduces the cytotoxicity of 2c, suggesting that 2c triggers the release of Ca2+ from the ER, leading to an increase in the concentration of cytosolic Ca2+, thus inducing cell death. Moreover, we synthesized a series of new amphiphilic cationic Ir complexes 5a-c containing photoreactive 3-trifluoromethyl-3-phenyldiazirine (TFPD) groups, in an attempt to identify the target molecules of 2c. Interestingly, it was discovered that a TFPD group functions as a triplet quencher of Ir complexes. It was also found that 5b is useful as a turn-on phosphorescent probe of acidic proteins such as bovine serum albumin (BSA) (pI = 4.7) and their complexation was confirmed by luminescence titrations and SDS-PAGE of photochemical products between them. These successful results allowed us to carry out photoaffinity labeling of the target biomolecules of 5b (2c and analogues thereof) in Jurkat cells. A proteomic analysis of the products obtained by the photoirradiation of 5b with Jurkat cells suggests that the Ca2+-binding protein "calmodulin (CaM)" is one of target proteins of the Ir complexes. Indeed, 5b was found to interact with the Ca2+-CaM complex, as evidenced by luminescence titrations and the results of photochemical reactions of 5b with CaM in the presence of Ca2+ (SDS-PAGE). A plausible mechanism for cell death induced by a cationic amphiphilic Ir complex is discussed on the basis of our results.

Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new candidate PET radioligands for P2X4 receptor.

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer's disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 30-50% decay corrected radiochemical yields with 370-1110GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD, [18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]FE-5-BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5-25% decay corrected radiochemical yields with 111-740GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD.

Photoaffinity electrophoretic mobility shift assay using photoreactive DNA bearing 3-trifluoromethyl-3-phenyldiazirine in its phosphate backbone.

Photoaffinity cross-linking enables the analysis of interactions between DNA and proteins even under denaturing conditions. We present a photoaffinity electrophoretic mobility shift assay (EMSA) in which two heterogeneous techniques-photoaffinity cross-linking using DNA bearing 3-trifluoromethyl-3-phenyldiazirine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis-are combined. To prepare the photoreactive DNA, which is an essential tool for photoaffinity EMSA, we first determined the optimal conditions for the integration of 4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl bromide to the specific site of oligonucleotide where phosphodiester linkage was replaced with phosphorothioate linkage. The photoaffinity EMSA was developed using the POU (initial letters of three genes: Pit-l, Oct-1,2, and unc-86) domain region of Oct-1 protein, which specifically bound to octamer DNA motif (ATGCAAAT). The affinity-purified recombinant POU domain proteins conjugated with glutathione-S-transferase (GST) contained three distinct proteins with molecular weights of 34, 36, and 45 kDa. The photoaffinity EMSA could clearly distinguish the individual binding abilities of three proteins on a single lane and showed that the whole POU domain protein specifically bound to octamer DNA motif by competition experiments. Using the nuclear extract of HeLa cells, the photoaffinity EMSA revealed that at least five specific proteins could bind to the octamer DNA motif. These results show that photoaffinity EMSA using 3-trifluoromethyl-3-phenyldiazirine can provide high-performance analysis of DNA-binding proteins.

The organocatalytic asymmetric Neber reaction for the enantioselective synthesis of spirooxindole 2H-azirines.

The asymmetric Neber reaction of 3-O-sulfonyl ketoxime, in situ generated from isatin ketoxime and sulfonyl chloride, for the synthesis of chiral spirocyclic oxindole compounds is reported. With the developed protocol, a range of chiral spirooxindole 2H-azirines could be obtained in good to excellent yields and up to a 92 : 8 enantiomeric ratio by using (DHQD)2PHAL as the catalyst. This methodology is the only example of the catalytic asymmetric construction of spirooxindole 2H-azirine compounds.

Selective inhibition of BET bromodomains.

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Rh(II)-catalyzed [3+2] cycloaddition of 2 H-azirines with N-sulfonyl-1,2,3-triazoles.

Rh(II)-catalyzed intermolecular [3+2] cycloaddition of 2 H-azirines with N-sulfonyl-1,2,3-triazoles is disclosed, in which a series of fully functionalized pyrroles is produced via rhodium azavinyl carbene intermediates. A distinct feature of this reaction is that the azavinyl carbene serves as a [2 C] equivalent, instead of as [1 C] or aza-[3 C] synthons, which have been reported previously in cyclopropanations and [3+n] cycloadditions. Moreover, this methodology has also been successfully applied in the total synthesis of URB447 as well as the formal synthesis of Atorvastatin (Lipitor).

Synthesis of pyrazines from rhodium-catalyzed reaction of 2H-Azirines with N-sulfonyl 1,2,3-triazoles.

An efficient synthetic route to a wide range of trisubstituted pyrazines is developed from Rh-catalyzed reaction of 2H-azirines with N-sulfonyl-1,2,3-triazoles through the elimination of nitrogen molecule and arylsulfinic acid. The present reaction proceeds through formation of in situ generated dihydropyrazines.

Theoretical insight into the mechanism of gold(I)-catalyzed rearrangement of 2-propargyl 2H-azirines to pyridines.

The title reaction is investigated in detail theoretically using density functional theory. After 5-endo-dig cyclization by nucleophilic attack, five possible pathways are taken into account in this work: direct ring expansion followed or accompanied by proton-transfer (paths A and B, respectively), 1,3-cationic migration (path C), proton-transfer before ring expansion (path D), and processing via a gold-nitrene (path E). Results indicate that the reaction would undergo the favored sequential pathway (path A) rather than other pathways. Moreover, the concerted mechanism (path B), which is designed to account for the selectivity of product in the experiment, would be unlikely in the reaction. The selectivity of product could be explained by the hindrance of ligand (t-BuXPhos) and the stability of the carbocation. Moreover, the binding energy of product complexes could account for the observed reaction rate.

Synthesis of New 2-Halo-2-(1H-tetrazol-5-yl)-2H-azirines via a Non-Classical Wittig Reaction.

The synthesis and reactivity of tetrazol-5-yl-phosphorus ylides towards N-halosuccinimide/TMSN3 reagent systems was explored, opening the way to new haloazidoalkenes bearing a tetrazol-5-yl substituent. These compounds were obtained as single isomers, except in one case. X-ray crystal structures were determined for three derivatives, establishing that the non-classical Wittig reaction leads to the selective synthesis of haloazidoalkenes with (Z)-configuration. The thermolysis of the haloazidoalkenes afforded new 2-halo-2-(tetrazol-5-yl)-2H-azirines in high yields. Thus, the reported synthetic methodologies gave access to important building blocks in organic synthesis, vinyl tetrazoles and 2-halo-2-(tetrazol-5-yl)-2H-azirine derivatives.

Copper-Catalyzed Trifluoromethylazidation of Alkynes: Efficient Access to CF3-Substituted Azirines and Aziridines.

A novel method for convenient access to CF3-containing azirines has been developed, and involves a copper-catalyzed trifluoromethylazidation of alkynes and a photocatalyzed rearrangement. Both terminal and internal alkynes are compatible with the mild reaction conditions, thus delivering the CF3-containing azirines in moderate to good yields. The azirines can be converted into various CF3-substituted aziridines.