Synthesis and purification of iodoaziridines involving quantitative selection of the optimal stationary phase for chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 °C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 °C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control. Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by (1)H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by (1)H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

Aziridine alkaloids as potential therapeutic agents.

The present review describes research on natural aziridine alkaloids isolated from both terrestrial and marine species, as well as their lipophilic semi-synthetic, and/or synthetic analogs. Over 130 biologically active aziridine-containing compounds demonstrate confirmed pharmacological activity including antitumor, antimicrobial, antibacterial effects. The structures, origin, and biological activities of aziridine alkaloids are reviewed. Consequently this review emphasizes the role of aziridine alkaloids as an important source of drug prototypes and leads for drug discovery.

N-acylaziridines as potential proinsecticides of carboxylic acids Part VI. Direct HPLC monitoring of the metabolism in insect tissues.

To determine the reversible masking potential of carboxylic acids afforded by the N-acyl structure in a proinsecticide perspective, the hydrolysis of monosubstituted N-acylaziridines and unsubstituted N-acylpyrrolidine was studied by reversed-phase high-performance liquid chromatography (HPLC) during in vitro assays conducted in the presence of insect tissues or of alpha-chymotrypsin. Chromatographic analysis of unextracted biological samples so-called "the direct injection approach" was simpler and more accurate than the "extraction approach" because it avoids problems associated with extraction. Thus, periodical injections of samples of biological insect tissues or of alpha-chymotrypsin incubated with N-acyl substrates were performed on packings allowing direct injection: a wide-pore column or a monolithic column. Moreover, to allow the simultaneous monitoring of the carboxylic acids and of the parent substrates, ion-pairing was used. In these conditions, it was shown that N-acylpyrrolidine is not hydrolyzed whatever the enzymatic conditions or the pH. On the other hand, the unmasking of the carboxylic acid is the preponderant mode of hydrolysis of N-acylaziridines in the presence of alpha-chymotrypsine and the exclusive one in the presence of locust fat-body, which establishes the convenience of this structure in our proinsecticide perspective. Due to the enzymatic character of the unmasking of the carboxylic acid during biological hydrolysis of N-acylaziridines, the research of possible chiral recognitions was undertaken. Thus, the enantiomeric composition of these substrates was analysed at the stage of their approximative half hydrolysis using a chiral alpha-AGP column. It appeared that locust fat-body hydrolyses preferentially the (R)-isomers of N-acylaziridines while the reverse is observed when alpha-chymotrypsine is used.

Comparison of the separation of aziridine isomers applying heptakis(2,3-di-O-methyl-6-sulfato)beta-CD and heptakis(2,3-di-O-acetyl-6-sulfato)beta-CD in aqueous and nonaqueous systems.

Aziridines are attracting interest as protease inhibitors, which might be used, e.g., for treatment of parasitic diseases. Within the framework of greater projects dealing with the search of new selective protease inhibitors, a huge number of aziridines with two stereogenic centers will be synthesized. Thus, a fast and reliable screening method for the evaluation of the isomeric composition is needed. Robust baseline separations were obtained using heptakis(2,3-di-O-acetyl-6-sulfato)beta-CD (HDAS) in acidic methanol and sulfated beta-CD in acidic phosphate buffer. With HDAS the resolutions were higher and migration times shorter. Thus, the method will be used as a screening method for further isomeric mixtures of aziridines.

Development and validation of a separation method for the diastereomers and enantiomers of aziridine-type protease inhibitors.

Aziridine derivatives are attracting pharmacological interest as protease inhibitors. Due to their two centers of chirality, the aziridines studied here are mixtures of two diastereomers and corresponding enantiomers. Applying cyclodextrin-modified capillary electrophoresis resulted in a baseline separation of the four isomers. The most robust separation was obtained by means of 2 mM sulfated beta-cyclodextrin in 50 mM phosphate buffer of pH 2.5. Using this method, 0.25% of the trans-diastereomers aziridine could be precisely and accurately quantified in the presence of 99.75% of the cis-isomers. The corrected peak-area ratios, migration times, and resolutions were found to be robust with respect to small variations of voltage, buffer concentrations, pH, temperature, chiral selector concentration, and different lots.

Miraziridine A: natures blueprint towards protease class-spanning inhibitors.

The natural product miraziridine A isolated from the marine sponge Theonella aff. mirabilis unifies within one molecule three structurally privileged elements: (i) (2R,3R)-aziridine-2,3-dicarboxylic acid, (ii) (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine), and (iii) (E)-(S)-4-amino-7-guanidino-hept-2-enoic acid (vinylogous arginine). The alignment of them realized in the tetrapetide allows for a simultaneous inhibition of the proteolytic activity of trypsin-like serine proteases, papain-like cysteine proteases, and pepsin-like aspartyl proteases. Therefore, this unique compound represents a blueprint for the design of protease class-spanning inhibitors.

Purity determinations in the bulk drug of the novel indoloquinone antitumor agent EO9.

The pharmaceutical development of the investigational cytotoxic drug EO9 included the structural characterization of the bulk drug by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and infrared (IR) spectroscopy, and analytical characterization by high-performance liquid chromatography and ultraviolet/visible spectrophotometry. The presence of impurities in the bulk drug was investigated. The intermediates in the synthesis of EO9 were structurally characterized by NMR spectroscopy and MS, and analytically characterized by HPLC analysis with photodiode array (PDA) detection. All of the intermediates were below their limits of detection in EO9 bulk drug. The amounts of residual organic solvents were determined by gas chromatography. Methanol and ethanol were detected, but the amounts present did not exceed the limits as set in the United States Pharmacopeia XXII.

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).