Nucleophilic Attack of Azide at Electrophilic Azides: Formation of N6 Units in Hexazene and Aminopentazole Derivatives

With the help of selective ¹⁵N labeling experiments, it has been confirmed that nucleophilic attack of azide at iminium‐activated organic azides leads to short‐lived hexazene intermediates. Such species do not only tend to a cleavage reaction with formation of N‐azido compounds, but also undergo ring closure to generate unprecedented amidino‐functionalized pentazoles. Thus, treatment of the parent Vilsmeier reagent with two equivalents of sodium azide creates an aminopentazole derivative as the main product, which is easily characterized by NMR spectroscopy.     

Preparative isolation and purification of steroidal glycoalkaloid from the ripe berries of Solanum nigrum L. by preparative HPLC–MS and UHPLC–TOF‐MS/MS and its anti‐non‐small cell lung tumors effects in vitro and in vivo

Overcoming epidermal growth factor receptor resistance is a critical problem that needs to be solved in clinical practice. Drugs that downregulate the fatty acid synthase‐epidermal growth factor receptor will become novel treatments for non‐small cell lung cancer. Solanum nigrum, extracted with water at 4°C, shows strong cytotoxic activity and inhibits tumor growth in Lewis tumor bearing‐mice in a dose‐dependent manner. A novel active compound in S. nigrum, solaoiacid, was successfully separated and purified from S. nigrum by preparative high‐performance liquid chromatography with mass spectrometry and ultra high performance liquid chromatography with time‐of‐flight tandem mass spectrometry. The IC₅₀ of solaoiacid on lung cancer cells was 2.3 µmol/L, which was significantly lower than that of the known steroidal glycoalkaloid. Label‐free proteomics and STRING Network analysis were used to identify significantly deregulated proteins in lung cancer cells that were treated with the fresh ripe fruit extracts of S. nigrum. S. nigrum regulates multiple signal pathways, including the epidermal growth factor receptor pathway. S. nigrum downregulated 24 main proteins with direct roles in fatty acid biosynthesis. Both S. nigrum and solaoiacid showed strong downregulation of the fatty acid synthase‐epidermal growth factor receptor and anti‐non‐small cell lung cancer effects, and thus will become a novel drug for the treatment of non‐small cell lung cancer.     

Two Reaction Mechanisms via Iminium Ion Intermediates: The Different Reactivities of Diphenylprolinol Silyl Ether and Trifluoromethyl‐Substituted Diarylprolinol Silyl Ether

The reactions of α,β‐unsaturated aldehydes with cyclopentadiene in the presence of diarylprolinol silyl ethers as catalyst proceed via iminium cations as intermediates, and can be divided into two types; one involving a Michael‐type reaction (type A) and one involving a cycloaddition (type B). Diphenylprolinol silyl ethers and trifluoromethyl‐substituted diarylprolinol silyl ethers, which are widely used proline‐type organocatalysts, have been investigated in this study. As the LUMO of the iminium ion derived from trifluoromethyl‐substituted diarylprolinol silyl ether is lower in energy than that derived from diphenylprolinol silyl ether, as supported by ab initio calculations, the trifluoromethyl‐substituted catalyst is more reactive in a type B reaction. The iminium ion from an α,β‐unsaturated aldehyde is generated more quickly with diphenylprolinol silyl ether than with the trifluoromethyl‐substituted diarylprolinol silyl ether. When the generation of the iminium ion is the rate‐determining step, the diphenylprolinol silyl ether catalyst is the more reactive. Because acid accelerates the generation of iminium ions and reduces the generation of anionic nucleophiles in the Michael‐type reaction (type A), it is necessary to select the appropriate acid for specific reactions. In general, diphenylprolinol silyl ether is a superior catalyst for type A reactions, whereas the trifluoromethyl‐substituted diarylprolinol silyl ether catalyst is preferred for type B reactions.     

Trapping Reactive Intermediates by Mechanochemistry: Elusive Aryl N‐Thiocarbamoylbenzotriazoles as Bench‐Stable Reagents

Monitoring of mechanochemical thiocarbamoylation by in situ Raman spectroscopy revealed the formation of aryl N‐thiocarbamoylbenzotriazoles, reactive intermediates deemed unisolable in solution. The first‐time isolation and structural characterization of these elusive molecules demonstrates the ability of mechanochemistry to access otherwise unobtainable intermediates and offers a new range of masked isothiocyanate reagents.     

Profiling primaquine metabolites in primary human hepatocytes using UHPLC‐QTOF‐MS with 13C stable isotope labeling

Therapeutic efficiency and hemolytic toxicity of primaquine (PQ), the only drug available for radical cure of relapsing vivax malaria are believed to be mediated by its metabolites. However, identification of these metabolites has remained a major challenge apparently due to low quantities and their reactive nature. Drug candidates labeled with stable isotopes afford convenient tools for tracking drug‐derived metabolites in complex matrices by liquid chromatography‐tandem mass spectrometry (LC‐MS‐MS) and filtering for masses with twin peaks attributable to the label. This study was undertaken to identify metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of ¹³C₆‐PQ/PQ with primary human hepatocytes. Acquity ultra‐performance LC (UHPLC) was integrated with QTOF‐MS to combine the efficiency of separation with high sensitivity, selectivity of detection and accurate mass determination. UHPLC retention time, twin mass peaks with difference of 6 (originating from ¹³C₆‐PQ/PQ), and MS‐MS fragmentation pattern were used for phenotyping. Besides carboxy‐PQ (cPQ), formed by oxidative deamination of PQ to an aldehyde and subsequent oxidation, several other metabolites were identified: including PQ alcohol, predictably generated by oxidative deamination of PQ to an aldehyde and subsequent reduction, its acetate and the alcohol's glucuronide conjugate. Trace amounts of quinone‐imine metabolites of PQ and cPQ were also detected which may be generated by hydroxylation of the PQ/cPQ quinoline ring at the 5‐position and subsequent oxidation. These findings shed additional light on the human hepatic metabolism of PQ, and the method can be applied for identification of reactive PQ metabolites generated in vivo in preclinical and clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Intermediates Formed in the Reactions of Organocuprates with α,β‐Unsaturated Nitriles

Conjugate additions of organocuprates are of outstanding importance for organic synthesis. To improve our mechanistic understanding of these reactions, we have used electrospray ionization mass spectrometry for the identification of the ionic intermediates formed upon the treatment of LiCuR₂ ? LiCN (R=Me, Bu, Ph) with a series of α,β‐unsaturated nitriles. Acrylonitrile, the weakest Michael acceptor included, did not afford any detectable intermediates. Fumaronitrile (FN) yielded adducts of the type Li_n?1Cu_nR_2n(FN)_n^?, n=1–3. When subjected to fragmentation in the gas phase, these adducts were not converted into the conjugate addition products, but re‐dissociated into the reactants. In contrast, the reaction with 1,1‐dicyanoethylene furnished the products of the conjugate addition without any observable intermediates. Tri‐ and tetracyanoethylene proved to be quite reactive as well. The presence of several cyano groups in these substrates opened up reaction pathways different from simple conjugate additions, however, and led to dimerization and substitution reactions. Moreover, the gas‐phase fragmentation behavior of the species formed from these substrates indicated the occurrence of single‐electron transfer processes. Additional quantum‐chemical calculations provided insight into the structures and stabilities of the observed intermediates and their consecutive reactions.     

An improved cell membrane chromatography method for the simultaneous screening of two epidermal growth factor receptor antagonists from radix scutellariae

A high‐expression epidermal growth factor receptor cell membrane chromatography using the silica gel with the average particle size of 3 μm as the stationary phase carrier coupled with high‐performance liquid chromatography and mass spectrometry was established for the online screening of epidermal growth factor receptor antagonists from Radix Scutellariae (Huang Qin in Chinese), a traditional Chinese medicine. In this study, the growth factor receptor cell membrane chromatography model using the smaller particle size carrier showed a higher efficiency for simultaneous screening baicalein, another one of the potential epidermal growth factor receptor antagonists from Radix Scutellariae extract besides wogonin, which was found in our previous work. The molecular docking result showed the occupancy site and binding mode of baicalein and wogonin with epidermal growth factor receptor tyrosine kinase were similar to gefitinib. The result of the assay for the in vitro inhibitory activity showed that baicalein and wogonin inhibited the growth of the high‐expression epidermal growth factor receptor cell in a dose‐dependent manner and even achieved a better inhibition effect than gifitinib in the low‐dosage range.     

Asymmetric Multicomponent Reactions Based on Trapping of Active Intermediates

Metal carbenes derived from transition metal‐catalyzed decomposition of diazo compounds react with nucleophiles with heteroatoms, such as alcohols and amines, to generate highly active oxonium/ammonium ylides intermediates. These intermediates can be trapped by appropriate electrophiles to provide three‐component products. Based on this novel trapping process, we have developed novel multicomponent reactions (MCRs) of diazo compounds, alcohols/anilines, and electrophiles. The nucleophiles were also extended to electron‐rich heterocycles (indoles and pyrroles)/arenes, in which the resulting zwitterionic intermediates were also trapped by electrophiles. By employing efficient catalysis strategy, the reactions were realized with excellent stereocontrol and wide substrate scope. In this personal account, we introduce our breakthroughs in the development of novel asymmetric MCRs via trapping of the active ylides and zwitterionic intermediates with a number of electrophiles, such as imines, aldehyde, and Michael acceptors, under asymmetric catalysis. Transition metal/chiral Lewis acid catalysis, transition metal/Brønsted acid catalysis, and chiral transition‐metal catalysis, enable excellent stereocontrolled outcomes. The methodologies not only provide experimental evidence to support the existence of protic onium ylides intermediates/zwitterionic intermediates and the stepwise pathways of carbene‐induced O?H, N?H and C?H insertions, but also offer a novel approach for the efficient construction of chiral polyfunctional molecules.     

Tumor‐Targeting of EGFR Inhibitors by Hypoxia‐Mediated Activation

The development of receptor tyrosine‐kinase inhibitors (TKIs) was a major step forward in cancer treatment. However, the therapy with TKIs is limited by strong side effects and drug resistance. The aim of this study was the design of novel epidermal growth factor receptor (EGFR) inhibitors that are specifically activated in malignant tissue. Thus, a Co^III‐based prodrug strategy for the targeted release of an EGFR inhibitor triggered by hypoxia in the solid tumor was used. New inhibitors with chelating moieties were prepared and tested for their EGFR‐inhibitory potential. The most promising candidate was coupled to Co^III and the biological activity tested in cell culture. Indeed, hypoxic activation and subsequent EGFR inhibition was proven. Finally, the compound was tested in vivo, also revealing potent anticancer activity.     

NMR Spectroscopic Observation of a Metal‐Free Acetylide Anion

A metal‐free acetylide was observed by using NMR spectroscopy. Metal‐free acetylides are closely related to reactive intermediates (carbanions) in solution; therefore, they have been regarded as unobservable species. However, we generated this highly reactive and unstable species through the deprotonation of phenylacetylene by using the strong nonmetallic phosphazene base tBu‐P4. In the presence of tBu‐P4, the J coupling between the ethynyl carbon and hydrogen nuclei (¹J_C,H) of phenylacetylene disappeared; this indicates the deprotonation of the alkyne terminal. Furthermore, a large low‐field shift (approximately 90 ppm) of the alkyne carbon resonance was observed. We concluded that we have observed a metal‐free carbanion with a formal charge on an sp‐hybridized carbon atom for the first time.     

Breslow Intermediates from Aromatic N‐Heterocyclic Carbenes (Benzimidazolin‐2‐ylidenes,Thiazolin‐2‐ylidenes)

We report the first generation and characterization of elusive Breslow intermediates derived from aromatic N‐heterocyclic carbenes (NHCs), namely benzimidazolin‐2‐ylidenes (NMR, X‐ray analysis) and thiazolin‐2‐ylidenes (NMR). In the former case, the diamino enols were generated by reaction of the free N,N‐bis(2,6‐diisopropylphenyl)‐ and N,N‐bis(mesityl)‐substituted benzimidazolin‐2‐ylidenes with aldehydes while the dimer of 3,4,5‐trimethylthiazolin‐2‐ylidene served as the starting material in the latter case. The unambiguous NMR identification of the first thiazolin‐2‐ylidene‐based Breslow intermediate rests on double ¹³C labeling of both the NHC and the aldehyde component. The acyl anion reactivity was confirmed by benzoin formation with excess aldehyde.     

Integrated One‐Flow Synthesis of Heterocyclic Thioquinazolinones through Serial Microreactions with Two Organolithium Intermediates

The synthesis of pharmaceutical compounds via short‐lived intermediates in a microreactor is attractive, because of the fast flow and high throughput. Additionally, intermediates can be utilized sequentially to efficiently build up a library in a short time. Here we present an integrated microfluidic synthesis of biologically active thioquinazolinone libraries. Generation of o‐lithiophenyl isothiocyanate and subsequent reaction with aryl isocyanate is optimized by controlling the residence time in the microreactor to 16 ms at room temperature. Various S‐benzylic thioquinazolinone derivatives are synthesized within 10 s in high yields (75–98 %) at room temperature. These three‐step reactions involve two organolithium intermediates, an isothiocyanate‐functionalized aryllithium intermediate, and a subsequent lithium thiolate intermediate. We also demonstrate the gram‐scale synthesis of a multifunctionalized thioquinazolinone in the microfluidic device with a high yield (91 %) and productivity (1.25 g in 5 min).     

Asymmetric Organocatalysis: The Emerging Utility of α,β‐Unsaturated Acylammonium Salts

Although acylammonium salts are well‐studied, chiral α,β‐unsaturated acylammonium salts have received much less attention. While these intermediates are convenient synthons, which are readily available from several commodity unsaturated acids and acid chlorides, and possess three reactive sites, their application in organic synthesis has been limited because of the lack of appropriate chiral Lewis bases for their generation. In recent years, the utility of chiral, unsaturated acylammonium salts has expanded considerably, thus demonstrating the unique reactivity of this intermediate leading to the development of a diverse array of catalytic, asymmetric transformations including organocascade processes. This Minireview highlights the recent and growing interest in these intermediates which might spark further research into their untapped potential for asymmetric organocascade catalysis. A cursory comparison is made to related unsaturated iminium and acylazolium intermediates.     

Insight into Isothiourea‐Catalyzed Enantioselective Addition of Saturated Esters to Iminium Ions

The possible mechanisms and origin of the selectivities of isothiourea‐catalyzed addition of saturated esters to iminium ions have been investigated by density functional theory. The favorable reaction pathway includes three stages: formation of an ammonium enolate intermediate, enantioselective addition of the ammonium enolate intermediate to the iminium ion, and dissociation of the catalyst to form the product. The enantioselective addition process is the stereoselectivity‐determining step, while the chemoselectivity‐determining step is included in the formation of the final product. The calculated energy barriers show that the chemoselectivity is thermodynamically controlled, and it depends on the polarities of the products and the nucleophilicities of the N atoms of the enamine reactant moieties of the intermediates. The origin of the stereoselectivity was investigated by non‐covalent interaction analysis of the key transition states. Hydrogen bonding interactions were identified as the determining factor for controlling the stereoselectivity. The obtained insight will be valuable for rational design of novel Lewis base organocatalyst‐promoted enantioselective addition reactions with special chemoselectivities.     

Isolation and X‐Ray Structures of Reactive Intermediates of Organocatalysis with Diphenylprolinol Ethers and with Imidazolidinones

Reaction of 2‐phenylacetaldehyde with the Me₃Si ether of diphenyl‐prolinol, with removal of H₂O, gives a crystalline enamine ( 1 ). The HBF₄ salts of the MePh₂Si ether of diphenyl‐prolinol and of 2‐(tert‐butyl)‐3‐methyl‐ and 5‐benzyl‐2,2,3‐trimethyl‐1,3‐imidazolidin‐4‐one react with cinnamaldehyde to give crystalline iminium salts 2, 3 , and 4 . Single crystals of the enamine and of two iminium salts, 2 and 3 , were subjected to X‐ray structure analysis (Figs. 1, 2, and 6), and a 2D‐NMR spectrum of the third iminium salt was recorded (Fig. 7). The crystal and NMR structures confirm the commonly accepted, general structures of the two types of reactive intermediates in organocatalysis with the five‐membered heterocycles, i.e., D, E (Scheme 2). Fine details of the crystal structures are discussed in view of the observed stereoselectivities of the corresponding reactions with electrophiles and nucleophiles. The structures 1 and 2 are compared with those of other diphenyl‐prolinol derivatives (from the Cambridge File CSD; Table 1) and discussed in connection with other reagents and ligands, containing geminal diaryl groups and being used in enantioselective synthesis (Fig. 4). The iminium ions 3 and 4 are compared with N‐acylated imidazolidinones F and G (Figs. 9, 12, and 13, and Table 3), and common structural aspects such as minimalization of 1,5‐repulsion (the ‘A^1,3‐effect’), are discussed. The crystal structures of the simple diphenyl‐prolinol?HBF₄ salt (Fig. 3) and of Boc‐ and benzoyl‐(tert‐butyl)methyl‐imidazolidinone (Boc‐BMI and Bz‐BMI, resp.; Figs. 10 and 11) are also reported. Finally, the crystal structures are compared with previously published theoretical structures, which were obtained from high‐level‐of‐theory DFT calculations (Figs. 5 and 8, and Table 2). Delicate details including pyramidalization of trigonal N‐atoms, distortions around iminium C?N bonds, shielding of diastereotopic faces, and the π‐interaction between a benzene ring and a Me group match so well with, and were actually predicting the experimental results that the question may seem appropriate, whether one will soon start considering to carry out such calculations before going to the laboratory for experimental optimizations.     

Enantioselective organocatalytic amine conjugate addition

The first enantioselective organocatalytic amine conjugate addition has been successfully developed. The application of LUMO-lowering iminium catalysis has enabled the highly chemo- and enantioselective 1,4-addition of a rationally designed N-silyloxycarbamate nucleophile (HOMO-raised) to alpha,beta-unsaturated aldehydes. Imidazolidinone 2*pTSA was found to catalyze the addition of various orthogonally N-protected silyloxycarbamate nucleophiles to a range of alpha,beta-unsaturated aldehydes, affording synthetically useful beta-amino aldehyde intermediates. The synthetic utility of the protocol was demonstrated in the rapid synthesis of enantioenriched beta-amino acids in one operation and 1,3-amino alcohol derivatives in three operations.     

Coupling a Wireless Bipolar Ultramicroelectrode with Nano‐electrospray Ionization Mass Spectrometry: Insights into the Ultrafast Initial Step of Electrochemical Reactions

We report a new mass spectrometric method for detecting electrogenerated intermediates. This approach is based on simultaneous activation of electrospray ionization and redox reaction on a wireless bipolar ultramicroelectrode, which is fabricated in the tip of a quartz nanopipette. The hollow structure of the ultramicroelectrode permits rapid transferring the transient species from electrode–electrolyte interfaces into the gas phase for mass spectrometric identification on the time scale of microseconds. The long‐sought fleeting intermediates including TPrA^.+, whose lifetime in solution is only 200 μs, and catecholamine o‐semiquinone radicals, the second‐order rate constant of which is typically 10⁹ m ^?1 s^?1, were successfully identified, helping clarify the previously hidden reaction pathways. Accordingly, our method may have wide applicability in exploring the dynamics of many electrochemical reactions, especially their ultrafast initial steps.     

О,О‐Dialkyl‐S‐(1,1‐dimethyl‐2‐oxoethyl)dithiophosphates: Synthesis and Reactions with n‐Nucleophiles

Hydrolysis of the new types of iminium salts was used to synthesize О,О‐dialkyl‐S‐(1,1‐dimethyl‐2‐oxoethyl)dithiophosphates or 2‐dialkoxythiophosphorylthio‐substituted aldehydes with carbon isochain. Reactions of aldehydes with N‐, N,N‐, and O,N‐nucleophiles gave new phosphorylated imines containing an acetal group at different positions, perhydro‐1,3‐diazol and oxazol with the diisopropoxythiophosphorylthio group in a side chain, and hydrazone of this aldehyde and diphenylphosphinylacetic acid hydrazide.     

Mechanistic studies of hydrazide-catalyzed enantioselective Diels-Alder reactions

The mechanism of the enantioselective, hydrazide-catalyzed Diels-Alder cycloaddition was investigated in detail. Both the formation of the reactive iminium species and the hydrolysis of the product iminium intermediates were found to be extremely rapid, leaving the cycloaddition as the kinetically significant step. Mechanistic studies using NMR showed that a retro-Diels-Alder reaction occurred during the catalytic cycle, suggesting a thermodynamic component to the reaction.     

One‐Pot Organocatalytic Asymmetric Synthesis of 3‐Nitro‐1,2‐dihydroquinolines by a Dual‐Activation Protocol

Generally, amine‐catalyzed enantioselective transformations rely on chiral enamine or unsaturated iminium intermediates. Herein, we report a protocol involving dual activation by an aromatic iminium and hydrogen‐bonding. An enantioselective aza‐Michael–Henry domino reaction of 2‐aminobenzaldehydes with nitroolefins has been developed through this protocol using primary amine thiourea catalysts to provide a variety of 3‐nitro‐1,2‐dihydroquinolines in moderate yields and with up to 90 % ee. The mechanism for the catalytic enantioselective reaction was confirmed by ESI mass spectrometric detection of the reaction intermediates. The products formed are substructures found in skeletons of important biological and pharmaceutical molecules.