Michael Addition Adducts Research Articles

A Chiral Functionalized Salt‐Catalyzed Asymmetric Michael Addition of Ketones to Nitroolefins

AbstractThe chiral functionalized salt catalysis, which differs from the known enzyme‐ and transition metal‐based methods, has been successfully developed to carry out the Michael addition of ketones to nitroolefins. Chiral anion salt (type I), chiral cation salt (type II), or chiral anion‐chiral cation salt (type III) could be expected to be remarkably effective catalysts and afford the corresponding chemically and optically pure Michael addition adducts. A primary amine group activating the ketone during salt catalysis was their obvious and common property. Based on preliminary experimental investigations and previous reports on primary amine catalysis, a reaction pathway via imine, enamine, iminium ion to imine was proposed.

Differences in Lysine Adduction by Acrolein and Methyl Vinyl Ketone: Implications for Cytotoxicity in Cultured Hepatocytes

Acrolein is a highly toxic environmental pollutant that readily alkylates the epsilon-amino group of lysine residues in proteins. In model systems, such chemistry involves sequential addition of two acrolein molecules to a given nitrogen, forming bis-Michael-adducted species that undergo aldol condensation and dehydration to form Nepsilon-(3-formyl-3,4-dehydropiperidino)lysine. Whether this ability to form cyclic adducts participates in the toxicity of acrolein is unknown. To address this issue, we compared the chemistry of protein adduction by acrolein to that of its close structural analogue methyl vinyl ketone, expecting that the alpha-methyl group would hinder the intramolecular cyclization of any bis-adducted species formed by methyl vinyl ketone. Both acrolein and methyl vinyl ketone displayed comparable protein carbonylating activity during in vitro studies with the model protein bovine serum albumin, confirming the alpha,beta,-unsaturated bond of both compounds is an efficient Michael acceptor for protein nucleophiles. However, differences in adduction chemistry became apparent during the use of electrospray ionization-MS to monitor reaction products in a lysine-containing peptide after modification by each compound. For example, although a Schiff base adduct was detected following reaction of the peptide with acrolein, an analogous species was not formed by methyl vinyl ketone. Furthermore, while ions corresponding to mono- and bis-Michael adducts were detected at the N-terminus and lysine residues following peptide modification by both carbonyls, only acrolein modification generated ions attributable to cyclic adducts. Despite these differences in adduction chemistry, in mouse hepatocytes, the two compounds exhibited very comparable abilities to induce rapid, concentration-dependent cell death as well as protein carbonylation. These findings suggest that the acute toxicity of short-chain alpha,beta-unsaturated carbonyl compounds involves their ability to form acyclic Michael addition adducts rather than Schiff conjugates or heterocyclic adducts.

Novel Imidazolidine-Tetrazole Organocatalyst for Asymmetric Conjugate Addition of Nitroalkanes

The Michael addition of nitroalkanes to alpha,beta-unsaturated enones catalyzed by a novel chiral imidazolidine-2-yltetrazole organocatalyst has been investigated. The new more soluble organocatalyst decreases reaction times and improves enantioselectivities compared to other catalysts. The Michael addition adducts were obtained with up to 92% ee. [reaction: see text]

Estimation of daily exposure to 4-hydroxy-2-alkenals in Korean foods containing n-3 and n-6 polyunsaturated fatty acids

4-Hydroxy-2-alkenals are cytotoxic aldehydes generated from the oxidation of n-3 and n-6 polyunsaturated fatty acids. The compounds have shown various biological effects via Schiff base adducts or Michael addition adducts at levels higher than physiological ones. To assess human exposure to 4-hydroxy-2-alkenals in the diet, 4-hydroxy-2-alkenals in vegetable oils, fish and shellfish were monitored using GC/MS/SIM. 2001 National Health and Nutrition Survey (2002) data were employed for the dietary intake pattern. The Korean daily exposure to 4-hydroxy-2-alkenals, excluding a possible one from fried food, was 4.3 µg day−1, constituted of 1.6 µg 4-hydroxy-2-hexenal (HHE) and 2.7 µg 4-hydroxy-2-nonenal (HNE). It was calculated that Koreans could be additionally exposed to more than 11.8 µg day−1 4-hydroxy-2-alkenal from fried foods. Thus, the combined exposure would be 16.1 µg day−1, which corresponds to 0.3 µg kg−1 body weight day−1 for a 60 kg Korean adult. In spite of the biological toxicity of 4-hydroxy-2-alkenals, the risk for human could not be quantified due to the lack of a virtually safe dose of the compounds. However, considering the basal level of 4-hydroxy-2-alkenals in many tissues, the present value from the diet may not pose a significant risk for human health.

New Aspects on the Selective Synthesis of 7‐Arylpyrido[2,3‐d]pyrimidines.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Oxidative bioactivation of crotyl alcohol to the toxic endogenous aldehyde crotonaldehyde: association of protein carbonylation with toxicity in mouse hepatocytes.

Recent confirmation that the toxic unsaturated aldehyde crotonaldehyde (CA) contributes to protein damage during lipid peroxidation confers interest on the molecular actions of this substance. However, since a plethora of structurally related aldehydes form during membrane oxidation, clarifying the toxicological significance of individual products (e.g., CA) is challenging. To facilitate study of the mechanisms underlying CA toxicity, we explored the possibility that it can be formed enzymatically from an unsaturated precursor, crotyl alcohol. This is analogous to the way allyl alcohol is converted in vivo to its toxic oxidation product, acrolein. In kinetic studies, we found that crotyl alcohol was readily oxidized by equine liver alcohol dehydrogenase, with electrospray-mass spectrometry confirming that CA was the main product formed. Moreover, in mouse hepatocytes, crotyl alcohol produced marked time- and concentration-dependent cell killing as well as pronounced glutathione depletion. Both cytotoxicity and glutathione loss were abolished by the alcohol dehydrogenase inhibitor 4-methylpyrazole, indicating an oxidation product mediated these effects. In keeping with expectations that carbonyl-retaining Michael addition adducts would feature prominently during protein modification by CA, exposure to crotyl alcohol resulted in marked carbonylation of a wide range of cell proteins, an effect that was also abolished by 4-methylpyrazole. Damage to a subset of small proteins (e.g., 29, 32, 33 kDa) closely correlated with the severity of cell death. Collectively, these results demonstrate that crotyl alcohol is a useful tool for studying the biochemical and molecular events accompanying intracellular CA formation.

New aspects on the selective synthesis of 7-arylpyrido[2,3- d]pyrimidines

Pyrido[2,3- d]pyrimidines have been synthesized via a selective cyclocondensation reaction between 6-aminopyrimidines 1 and the Mannich bases, propiophenone hydrochlorides 2 . Intermediates for this reaction, such as the Michael addition adduct and the further hydrated pyrido[2,3- d]pyrimidine were isolated to prove the postulated mechanism. NMR analysis of bidimensional experiments allowed to determine unambiguously the process to obtain 7-arylpyrido[2,3- d]pyrimidines.

Extensive protein carbonylation precedes acrolein-mediated cell death in mouse hepatocytes.

Allyl alcohol hepatotoxicity is mediated by an alcohol dehydrogenase-derived biotranformation product, acrolein. This highly reactive alpha,beta-unsaturated aldehyde readily alkylates model proteins in vitro, forming, among other products, Michael addition adducts that possess a free carbonyl group. Whether such damage accompanies acrolein-mediated toxicity in cells is unknown. In this work we established that allyl alcohol toxicity in mouse hepatocytes involves extensive carbonylation of a wide range of proteins, and that the severity of such damage to a subset of 18-50 kDa proteins closely correlated with the degree of cell death. In addition to abolishing cytotoxicity and glutathione depletion, the alcohol dehydrogenase inhibitor 4-methyl pyrazole strongly attenuated protein carbonylation. Conversely, cyanamide, an aldehyde dehydrogenase inhibitor, enhanced cytotoxicity and protein carbonylation. Since protein carbonylation clearly preceded the loss of membrane integrity, it may be associated with the toxic process leading to cell death.

Efficient Baylis--Hillman reaction using stoichiometric base catalyst and an aqueous medium.

A practical and efficient set of conditions were developed using stoichiometric base catalyst, 1,4-diazabicyclo[2,2,2]octane (DABCO), and an aqueous medium to overcome problems commonly associated with the Baylis--Hillman reaction, such as low reaction yields and long reaction time. These simple modifications to the classical conditions, using more base catalyst and an aqueous medium, proved to be successful in converting a variety of aliphatic and aromatic aldehydes to their corresponding Baylis--Hillman products. The inclusion of environmentally friendly water in the reaction solvent was critical for achieving the high yield of Baylis--Hillman adducts. Our deuterium-exchange experiments suggest that the Michael addition adduct formed between DABCO and methyl acrylate is the active intermediate for the Baylis--Hillman reaction in aqueous conditions, and its hydrolysis, a nonproductive side reaction facilitated by the quaternary ammonium ion, leading to the formation of a stable betaine product, consumes both the catalyst and methyl acrylate, making it necessary to add more base catalyst and methyl acrylate.

A Schiff base is a major DNA adduct of crotonaldehyde.

Previous studies have demonstrated that the reaction of crotonaldehyde with DNA produces Michael addition products, and these have been detected in human tissues as well as tissues of untreated laboratory animals. A second class of crotonaldehyde-DNA adducts releases 2-(2-hydroxypropyl)-4-hydroxy-6-methyl-1,3-dioxane (paraldol, 12) upon hydrolysis, and these adducts are quantitatively more significant than the Michael addition adducts in vitro. In this study, we demonstrate that the major source of the paraldol-releasing DNA adducts of crotonaldehyde is a Schiff base. Reaction of crotonaldehyde with DNA, followed by treatment with NaBH(3)CN and enzyme hydrolysis, resulted in the formation of N(2)-(3-hydroxybutyl)dG (10), identified by its UV, MS, and proton NMR. Reactions of crotonaldehyde or paraldol with dG demonstrated that the Schiff base precursor to N(2)-(3-hydroxybutyl)dG is N(2)-(3-hydroxybutylidene)dG (7), identified by UV, LC-APCI-MS, and MS/MS. Four isomers of N(2)-(3-hydroxybutylidene)dG were observed. The (R)- and (S)-isomers were identified by reactions of chiral paraldol with dG; each existed as a pair of interconverting (E)- and (Z)-isomers. These data indicate that the structure of the major Schiff base DNA adduct in crotonaldehyde-treated DNA is N(2)-(3-hydroxybutylidene)dG (7). This adduct is unstable at the nucleoside level and accounts for more than 90% of the paraldol released from crotonaldehyde-treated DNA. However, the adduct is stable in DNA and therefore is a likely companion to the Michael addition adducts in human DNA.

Role of the C(6)-Hydroxy Group in Bicyclomycin: Synthesis, Structure, and Chemical, Biochemical, and Biological Properties

Bicyclomycin (1) is a commercial antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. A recent structure-activity relationship study of 1 showed that replacing the C(6)-hydroxy group with alkoxy and thioalkoxy substituents led to dramatic losses of inhibitory activity in rho biochemical assays. The origin for this structural specificity has been explored by the synthesis and chemical, biochemical, and biological evaluation of C(6)-amino- (13), C(6)-(hydroxylamino)-(14), and C(6)-mercaptobicyclomycin (15). These compounds, like 1, are capable of entering into hydrogen bond donor interactions with rho and are capable of undergoing C(6) ring opening to generate alpha, beta-unsaturated carbonyl, imine, or thione systems. The chemical reactivity of 13-15 was compared with that of 1. We observed that 1, upon treatment with EtSH under moderate and basic conditions, readily underwent C(6) hemiaminal bond cleavage followed by conjugate addition to beta-methylene-alpha-ketoamide 2 to give Michael addition adducts whereas 13-15 reacted by initial cleavage of the C(1)-O(2) bond. Biochemical and biological assays of 13-15 and related analogues demonstrated that the C(6) hydroxy group in 1 was essential for activity. We found that replacing the C(6)-hydroxy group in 1 with weaker hydrogen bond donors led to low inhibitory activities in the rho-dependent ATPase and transcription termination assays. None of the bicyclomycin derivatives exhibited antibiotic activity against E. coli W3350 cells at a 32 mg/mL concentration. The apparent specificity for the C(6)-hydroxy group in 1 suggests that an efficient hydrogen bond donor interaction from the C(6)-hydroxy group to rho or the C(6) hemiaminal bond cleavage to 2 or both is necessary for drug function.

Maximum entropy deconvolution of heterogeneity in protein modification: Protein adducts of 4‐hydroxy‐2‐nonenal

To explore the chemistry of the reactions of the cytotoxic aldehyde trans-4-hydroxy-2-nonenal (HNE) with proteins, we incubated this aldehyde in vitro with beta-lactoglobulin B, a model protein of molecular weight 18,277 Da. Direct characterization of reaction products using electrospray ionization mass spectrometry yielded spectra whose complexity suggested extensive product heterogeneity. Spectra were transformed to a true mass scale using both a conventional transform algorithm and a maximum entropy algorithm. Both transformations demonstrated the formation of aldehyde-protein adducts containing from three to nine aldehyde molecules per molecule of protein. Maximum entropy deconvolution resolved Schiff base adducts and/or dehydration products, differing from the Michael addition adducts by 18 Da. The dominant reaction pathway, however, was Michael addition of the aldehyde to nucleophilic functional groups on the protein. The large number of Michael adducts relative to the one available cysteine requires that other amino acids, such as histidine and lysine, also be modified. The data suggest that methods for analysis of HNE that involve displacement of Schiff base groups from proteins will only recover a small fraction of HNE.

Reaction of 5‐acetyl‐10,11‐didehydro‐5H‐dibenz[b, f]azepine with pyrrole, N‐methylpyrrole, imidazole and N‐methylimidazole: Cycloaddition versus michael addition

AbstractReaction of 5‐acetyl‐10‐bromo‐5H‐dibenz[b, f]azepine 1 with potassium t‐butoxide results in the reactive intermediate 5‐acetyl‐10,11‐didehydro‐5H‐dibenz[b, f]azepine (2). The intermediate 2 reacts with N‐methylpyrrole to give a mixture of the Michael addition adduct 10‐(2‐N‐methylpyrrolyl)‐5H‐dibenz[b, f]azepine (9) and the Diels‐Alder/retro Diels Alder adduct 8H‐(N‐methylpyrrolo[3,4‐d]dibenz[b, f]azepine (8a). Reaction of 2 with pyrrole gives a mixture of two Michael addition adducts 10‐(1‐pyrrolyl)‐5H‐dibenz[b, f]azepine (16) and 10‐(2‐pyrrolyl)‐5H‐dibenz[b, f]azepine (18). Reaction of 2 with imidazole results in the Michael addition adduct 10‐(1‐imidazolyl)‐5H‐dibenz[b, f]azepine (21).

A Study of the Hetero Diels-Alder Reaction of N-Alkyl-2-cyano-1-azadienes with 2-Vinylindole

The N-alkyl-2-cyano-1-azadienes (2a) and (2b) derived from ethanolamine react with 2-vinylindole under thermal conditions to give the Michael addition adducts (9) and (9') as the principle product. In the reacaon of 2a with vinylindole the DielsAlder product (11) was also isolated. In the reaction with 2b, formation of compounds (9) was accompanied by the cyclized product (10) and the Diels-Alder adduct (13)

Drug-Excipient Interactions of Seproxetine Maleate Hemi-Hydrate: Isothermal Stress Methods

Seproxetine maleate hemi-hydrate was originally formulated with pregelatinized starch, to provide 1 and 20 mg free base equivalent gelatin capsule dosage forms for storage at 25°C and 40°C. HPLC analysis after 3 months revealed the formation of a 1,4 Michael addition adduct in each case. No additional degradation products were detected. To pursue a less interactive formulation, 5 mg formulation equivalent mixtures of seproxetine maleate hemi-hydrate were prepared with pregelatinized starch, lactose, and talc; thus, three distinctly different excipient classifications. These were evaluated in additional isothermal stress experiments at 25°C, 40°C, and 50°C. The results indicated that each excipient interacted with the drug in a unique chemically and thermally dependent manner. Thus, the drug-pregelatinized starch data may be represented by an Arrhenius type relationship, with activation energy of 32 kcal/mol, and formation of the previously described adduct. However, the drug-lactose data suggest reaction ...

Differential effects of thiols on DNA modifications via alkylation and Michael addition by alpha-acetoxy-N-nitrosopyrrolidine.

The hepatocarcinogen NPYR is metabolically activated by alpha-hydroxylation mediated by cytochrome P-450 enzymes to yield a 4-oxobutylating agent and 2-butenal (crotonaldehyde). Both are reactive intermediates capable of modifying DNA with guanine either by simple alkylation or by Michael type addition, respectively. In order to assess the roles of these pathways in NPYR tumorigenesis, we are interested in identifying agents which can selectively modify one of these two pathways. In this study, we examined the effects of three thiols--(mesna), glutathione (Glu), and N-acetylcysteine (Nac)--on DNA adduct formation by alpha-acetoxyNPYR, a stable precursor of alpha-hydroxyNPYR. Calf thymus DNA isolated from incubation of alpha-acetoxyNPYR with or without thiol was hydrolyzed and analyzed for the adducts formed by alkylation (adducts 1 and 2) and Michael addition (adducts 3-5). The results showed that the addition of mesna completely blocked the formation of the crotonaldehyde-derived adducts 3-5, whereas it exerted little effect on the formation of the alkylated adducts 1 and 2. These results indicate the preferential conjugation of mesna with crotonaldehyde. In contrast, Nac had little selectivity on adduct formation; levels of adducts 1 to 5 were were reduced by 36-75%. These results suggest that Nac conjugated with both alkylating agent and crotonaldehyde. Similar to mesna, Glu blocked the formation of the crotonaldehyde-derived adducts (adducts 3-5) efficiently. However, unlike mesna, Glu inhibited the formation of adduct 1, while it did not inhibit the formation of adduct 2, although both adducts are presumably derived from the 4-oxobutylating agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Arene Hydrides, 10 Michael Additions of Anthracene Hydride. Selective Reduction of the ArCC Moiety by Fragmentation of the Michael Adduct

Anthracene hydride (AH−) in THF undergoes Michael addition onto the acrylic acid derivatives 1 and 2. Secondary reactions of the first formed anions 3 and 4 of the Michael adducts depend on the absence (3) or presence (4) of an aryl group in β position. The non-benzylic 3 or its protonated or deprotonated form can cyclize via ester condensation to yield the “dibenzo-bicyclo[3.2.2.]nonanones” 10b, c. Deprotonation of the anion 4 by excess AH− usually leads to fragmentation of the CCAr bond formed in the Michael addition. The overall result is a selective C = C reduction of cinnamonic acid derivatives and analogues: PhR′C = C → PhR′CH CH. This fragmentation is hampered in the Michael addition adduct 4h with dimethyl benzylidenemalonate 2h probably due to the necessity of a pronounced conformational reorganization during the fragmentation step.