Characterization of 4-hydroxy-2-nonenal metabolism in stellate cell lines derived from normal and cirrhotic rat liver

During oxidative stress, reactive aldehydes, including trans-4-hydroxy-2-nonenal (4-HNE), are generated by peroxidation of membrane lipids and purportedly stimulate hepatic stellate cells to produce excessive extracellular matrix, including type I collagen. An important question concerning the ability of 4-HNE to modulate collagen production by stellate cells is the potential of these specialized cells to detoxify 4-HNE. The objective of the present study was to characterize the ability of stellate cell lines, derived from normal (NFSC) and cirrhotic (CFSC) rat livers, to metabolize 4-HNE by oxidative, reductive and conjugative pathways. These two stellate cell lines were noted to have differing susceptibilities to the cytotoxic effect of 4-HNE. Treatment of both stellate cell lines with a range of 4-HNE doses demonstrated that the concentration which was cytotoxic to 50% of CFSC (TD(50)) was 25% greater than that for NFSC (967.57+/-9.26 nmol/10(6) cells vs. 769.90+/-5.32 nmol/10(6) cells respectively). The capacity of these cell lines to metabolizes 4-HNE was determined by incubating them in suspension with 50 microM 4-HNE (10 nmol/10(6) cell); 4-HNE elimination and metabolite formation were quantified over a 20 min time course. Both stellate cell lines rapidly metabolized 4-HNE, with the CFSC line eliminating 4-HNE at a rate that was approx. 2-fold greater than the NFSC line. The rate of 4-HNE metabolism attributable to glutathione S-transferase (GST) was similar in both cell lines, though differential cell specific expressions of GST isoforms GSTP1-1 and GSTA4-4 were observed. The greater rate of 4-HNE elimination by CFSC was attributable to its aldehyde dehydrogenase (ALDH) activity which accounted for approx. 50% of 4-HNE metabolism in CFSC but was insignificant in NFSC. Neither cell line had detectable alcohol dehydrogenase activity or protein levels. Measurement of cellular GSH concentrations revealed that NFSC contain approx. 2-fold greater concentrations of GSH when compared to CFSC and that following 4-HNE treatment, GSH levels were rapidly depleted from both cell lines. Concomitant with 4-HNE mediated GSH depletion, a corresponding increase in the 4-HNE-glutathione adduct formation was observed with the NFSC line forming greater amounts of the glutathione adduct than did the CFSC line. Taken together, these data demonstrate that both stellate cell lines have the capacity to metabolize 4-HNE but that CFSC have a greater rate of metabolism which is attributable to their greater ALDH activity, suggesting that the stellate cells isolated from cirrhotic liver may be differentially responsive to the biologic effects of 4-HNE.     

High-fat diet induces changes in adipose tissue trans-4-oxo-2-nonenal and trans-4-hydroxy-2-nonenal levels in a depot-specific manner

Protein carbonylation is the covalent modification of proteins by α,β-unsaturated aldehydes produced by nonenzymatic lipid peroxidation of polyunsaturated fatty acids. The most widely studied aldehyde product of lipid peroxidation, trans-4-hydroxy-2-nonenal (4-HNE), is associated with obesity-induced metabolic dysfunction and has demonstrated reactivity toward key proteins involved in cellular function. However, 4-HNE is only one of many lipid peroxidation products and the lipid aldehyde profile in adipose tissue has not been characterized. To further understand the role of oxidative stress in obesity-induced metabolic dysfunction, a novel LC–MS/MS method was developed to evaluate aldehyde products of lipid peroxidation and applied to the analysis of adipose tissue. 4-HNE and trans-4-oxo-2-nonenal (4-ONE) were the most abundant aldehydes present in adipose tissue. In high fat-fed C57Bl/6J and ob/ob mice the levels of lipid peroxidation products were increased 5- to 11-fold in epididymal adipose, unchanged in brown adipose, but decreased in subcutaneous adipose tissue. Epididymal adipose tissue of high fat-fed mice also exhibited increased levels of proteins modified by 4-HNE and 4-ONE, whereas subcutaneous adipose tissue levels of these modifications were decreased. High fat feeding of C57Bl/6J mice resulted in decreased expression of a number of genes linked to antioxidant biology selectively in epididymal adipose tissue. Moreover, TNFα treatment of 3T3-L1 adipocytes resulted in decreased expression of GSTA4, GPx4, and Prdx3 while upregulating the expression of SOD2. These results suggest that inflammatory cytokines selectively downregulate antioxidant gene expression in visceral adipose tissue, resulting in elevated lipid aldehydes and increased protein carbonylation.     

Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal : A Route to 4S-Hydroperoxy-2E-Nonenal and Related Products

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O₂ uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.     

Ethanol withdrawal increases glutathione adducts of 4-hydroxy-2-hexenal but not 4-hydroxyl-2-nonenal in the rat cerebral cortex

Ethanol withdrawal increases lipid peroxidation of the polyunsaturated fatty acid (PUFA) docosahexaenoate (22:6; n-3) in the CNS. To further define the role of oxidative damage of PUFAs during ethanol withdrawal, we measured the levels of glutathione adducts of 4-hydroxy-2-hexenal (GSHHE) and 4-hydroxy-2-nonenal (GSHNE) as biomarkers of brain lipid peroxidation of n-3 and n-6 PUFAs, respectively. In this study rats received an ethanol-containing diet for 6 weeks followed by withdrawal ranging from 0 to 7 days. GSHHE content was elevated (> 350%) in the cerebral cortex after 2 days of withdrawal with no change in GSHNE. The levels of GSHHE were significantly greater (2- to 20-fold) than those of GSHNE in multiple brain regions. Experiments demonstrated that intoxication and withdrawal did not alter the enzymatic rate of formation of GSHHE or GSHNE, but the rate of formation of GSHHE was higher (～ 50%) than that of GSHNE. These results indicate that selective oxidative damage to n-3 PUFAs occurs in the cerebral cortex as a result of ethanol withdrawal and that 4-hydroxy-2-hexenal is metabolized to the GSH adduct more efficiently than HNE.     

Convergence of the 5-LOX and COX-2 pathways: heme-catalyzed cleavage of the 5S-HETE-derived di-endoperoxide into aldehyde fragments

Oxygenation of the 5-lipoxygenase product 5S-hydroxyeicosatetraenoic acid by cyclooxygenase-2 yields a bicyclic di-endoperoxide. The di-endoperoxide contains two peroxides spanning from carbons 9 to 11 and 8 to 12, and two hydroxyls at carbons 5 and 15 of arachidonic acid (Schneider C., et al. 2006. Convergent oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2. J. Am. Chem. Soc. 128: 720). Here, we report that treatment of the di-endoperoxide with hematin or ferrous chloride results in cleavage of both peroxide O-O bonds and of the bonds between the carbons that carry the peroxide groups, producing the aldehydes 4-hydroxy-2E-nonenal (4-HNE), 8-oxo-5S-hydroxy-6E-octenoic acid, and malondialdehyde (MDA). The hematin- and ferrous iron-catalyzed transformation of the di-endoperoxide proceeded with a similar yield of products as the cleavage of the prostaglandin endoperoxide PGH₂ to 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid and MDA. Chiral phase HPLC analysis of the 4-HNE cleavage product showed greater than 98% 4S and thus established the S configuration of the 15-carbon of the di-endoperoxide that had not previously been assigned. This transformation of the 5-lipoxygenase/cyclooxygenase-2 derived di-endoperoxide invokes the possibility of a novel pathway to formation of the classic lipid peroxidation products 4-HNE and MDA.     

Marked expression of glutathione S-transferase A4-4 detoxifying 4-hydroxy-2(E)-nonenal in the skin of rats irradiated by ultraviolet B-band light (UVB).

Enzyme, Western blot, and immunohistochemical analyses indicated that rat skin cytosol contained no detectable level of the homodimeric, alpha-class glutathione S-transferase (rGST) A4-4 which catalyzes the GSH conjugation of the toxic product, 4-hydroxy-2(E)-nonenal (HNE), nonenzymatically formed from n-6 polyunsaturated fatty acid residues of lipids by lipid peroxidation. Rats irradiated by single doses (4000-24,000 mJ/cm(2)) of ultraviolet B-band light (UVB, 200 mJ/cm(2)/min) markedly expressed rGSTA4-4 in the skin at a level one-fifth that of the liver in apparent specific activity toward HNE at a single dose of 24,000 mJ/cm(2). Skin rGSTA4-4 was isolated, purified to homogeneity, and identified with hepatic rGSTA4-4 by reverse-phase partition HPLC and by amino acid sequence analysis of its CNBr fission peptides. Immunohistochemistry with polyclonal antibody raised against rGSTA4-4 demonstrated the selective expression of rGSTA4-4 in epidermis and sebaceous glands localized in dermis after UVB irradiation.     

Synthesis,structure elucidation and in vitro anticancer activities of novel derivatives of diethyl (2E)-2-[(2E)-(1-arylimidazolidin-2-ylidene)hydrazono]succinate and ethyl (4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)acetate

The worked out and optimized synthesis routes and remarkable antitumour activities in vitro of novel polynitrogenated derivatives of diethyl (2E)-2-[(2E)-(1-arylimidazolidin-2-ylidene)hydrazono]succinate (7–10) and ethyl (4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)acetate (11–16) are presented. Small molecules based on the privileged 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one scaffold (11–16) were obtained with fairly modest to good overall yields by very facile addition reactions of the nucleophilic centred 1-aryl-2-hydrazonoimidazolidine hydroiodides to diethyl acetylenedicarboxylate (DEAD) in the presence of triethylamine (TEA) and a subsequent cyclocondensation of the putative intermediate chain hydrazones. Heterobicyclic products 12 and 14–16 could also be prepared in high overall yields by an effective intramolecular cyclocondensation of the isolated stable and antiproliferative active heterocyclic hydrazones, namely, diethyl (2E)-2-[(2E)-(1-arylimidazolidin-2-ylidene)hydrazono]succinates (7–10), performed in refluxing DMF. These intermediates are the first products to be formed in the result of an addition of the nucleophilic reactants, namely, 1-aryl-2-hydrazonoimidazolidines of the 1–6 type, bearing the basic nitrogen atom of the hydrazono moiety (N–NH₂), to the carbon–carbon triple bond of the highly electrophilic alkyne, that is, DEAD. Molecular structures of the synthesized compounds (7–16) in the DMSO-d₆ solutions were verified by ¹H NMR and ¹³C NMR spectral data. These were finally confirmed based on the advanced 2D HMBC and HMQC NMR experiments, which were performed for the two representatives (8 and 11) of the two synthesized sets of the bioactive substances. Among the majority of antiproliferative active molecules, the disclosed herein ethyl [4-oxo-8-(3-chlorophenyl)-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl]acetate (14) is proposed as a promising lead structure for the design of novel highly selective antitumour agents because of the distinctly marked lower cytotoxicity towards the primary cell line of normal HSF cells and several-fold higher against cancer cells used. A double fluorochrome mix-staining was performed in order to find out about the possible mode of action by which this novel small heterobicycle reveals remarkable antiproliferative effects in vitro. Taking into account the obtained double staining results, this small molecule was identified as capable of inducing significantly higher levels of necrotic cells in human cancer cell lines (T47D and HeLa) than in normal HSF cells. Furthermore, its cytotoxicity against cells was found to be connected to the predominant induction of necrosis over apoptosis.     

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by β oxidation of fatty acids in the isolated rat heart

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on β-oxidation pathways. The hypothesis driving this report is that perturbations of β oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1 mM octanoate). During ischemia, HNE concentrations doubled and the glutathione–HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through β oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to β oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.     

2(S),3(S)-3-hydroxy-4-methyleneglutamic Acid from Gleditsia caspica

A new natural product, 2(S),3(S)-3-hydroxy-4-methyleneglutamic acid (G3) has been isolated from seeds of Gleditsia caspica. The structure has been established by chemical and spectroscopic methods. Catalytic reduction of G3 yields 2(S),4(S)-4-methylglutamic acid and a new amino acid, 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid. Ozonolysis of G3 followed by oxidation gives 2(S),3(R)-3-hydroxyaspartic acid. The S- (or l-) configurations at C₂ in G3 and in 2(S),3(S),4(S)-3-hydroxy-4-methyglutamic acid and the S-configurations at C₃ for G3 and 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid and at C₄ for 2(S),3(S),4(S)-3-hydroxy-4-methylglutamic acid are inferred from the configurations at C₂ in 2(_S),4(_S)-4-methylglutamic acid and at C₂ and C₃ in 2(S),3(R)-3-hydroxyaspartic acid. The seeds also contain appreciable quantities of 2(S),3(S),4(R)-3-hydroxy-4-methylglutami c acid (G1) and 2(S),4(R)-4-methylglutamic acid.     

Metabolism of 4-hydroxynonenal by rat Kupffer cells

Kupffer cells are known to participate in the early events of liver injury involving lipid peroxidation. 4-Hydroxy-2,3-(E)-nonenal (4-HNE), a major aldehydic product of lipid peroxidation, has been shown to modulate numerous cellular systems and is implicated in the pathogenesis of chemically induced liver damage. The purpose of this study was to characterize the metabolic ability of Kupffer cells to detoxify 4-HNE through oxidative (aldehyde dehydrogenase; ALDH), reductive (alcohol dehydrogenase; ADH), and conjugative (glutathione S-transferase; GST) pathways. Aldehyde dehydrogenase and GST activity was observed, while ADH activity was not detectable in isolated Kupffer cells. Additionally, immunoblots demonstrated that Kupffer cells contain ALDH 1 and ALDH 2 isoforms as well as GST A4-4, P1-1, Ya, and Yb. The cytotoxicity of 4-HNE on Kupffer cells was assessed and the TD50 value of 32.5+/-2.2 microM for 4-HNE was determined. HPLC measurement of 4-HNE metabolism using suspensions of Kupffer cells incubated with 25 microLM 4-HNE indicated a loss of 4-HNE over the 30-min time period. Subsequent production of 4-hydroxy-2-nonenoic acid (HNA) suggested the involvement of the ALDH enzyme system and formation of the 4-HNE-glutathione conjugate implicated GST-mediated catalysis. The basal level of glutathione in Kupffer cells (1.33+/-0.3 nmol of glutathione per 10(6) cells) decreased significantly during incubation with 4-HNE concurrent with formation of the 4-HNE-glutathione conjugate. These data demonstrate that oxidative and conjugative pathways are primarily responsible for the metabolism of 4-HNE in Kupffer cells. However, this cell type is characterized by a relatively low capacity to metabolize 4-HNE in comparison to other liver cell types. Collectively, these data suggest that Kupffer cells are potentially vulnerable to the increased concentrations of 4-HNE occurring during oxidative stress.     

Synthesis of (S)-13-Hydroxy (2E,4E,8E)- and (2E,4E,8Z)-Tetradecatrienoic Acids

The syntheses of (S)-13-hydroxy-(2E,4E,8E)-tetradecatrienoic acid (1) and (2E,4E,8Z)-tetradecatrienoic acid (2) were carried out by using the Wittig reaction as the key step. The asymmetric center at C-13 and the double bond between C-8 and C-9 for natural compound 1 were reconfirmed as being of (S) configuration and E, respectively.

The relationship between the structure of the unsaturated hydroxy fatty acids and their inhibitory effect on the growth of lettuce was investigated.     

(E)-2-Styrylchromones as potential anti-norovirus agents

Human noroviruses (NoV) are now recognized as the most frequent cause of outbreaks and sporadic cases of acute gastroenteritis. Despite the significant economic impact and considerable morbidity of norovirus disease, no drug or vaccine is currently available to treat or prevent this disease, therefore the discovery of anti-norovirus drugs is urgent.In the present work, a total of 12 structure related chromone and (E)-2-styrylchromones were evaluated for their potential anti-norovirus activity using the murine norovirus (MNV) as a surrogate model for human NoV.From the 12 compounds studied, six (E)-2-styrylchromones were found to have with interesting anti-norovirus activity. The best compounds of the series were (E)-5-hydroxy-2-styrylchromone and (E)-4′-methoxy-2-styrylchromone with an IC₅₀ ≈ 7 μM. A first insight into the mechanism of action of these compounds was possible. An interesting relationship between the anti-norovirus activity and the chemical structure was observed. The present study points out that the (E)-2-styrylchromones skeleton is an important one which deserves to be developed and further explored as new antiviral drugs against NoV.     

A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues

A mouse glutathione S-transferase (GST) isozyme designated as GST 5.7 or mGSTA4-4 belongs to a distinct subclass of the α-class isozymes of GST. It is characterized by kinetic properties intermediate between the α- and π-classes of GSTs. We have recently cloned and expressed this isozyme (rec-mGSTA4-4) in E. coli and have reported its complete primary sequence (Zimniak, P. et al. (1992) FEBS Lett., 313, 173–176). Using antibodies raised against the homogenous rec-mGSTA4-4 expressed in E. coli, we now demonstrate that an ortholog of this isozyme was selectively expressed in various human tissues. The human ortholog of mGST A4-4 purified from liver had a pI value of 5.8 and constituted approx. 1.7% of total GST protein of human liver. Similar to other α-class GSTs, the N-terminus of this isozyme (GST 5.8) was also blocked. CNBr digestion of the enzyme yielded two major fragments with M_r values of 12 kDa and 6 kDa. The sequences of these two fragments showed identities in 16 out of 20 residues and 17 out of 20 residues with the corresponding sequences of its mouse ortholog (mGSTA4-4), and showed significant homologies with the rat and chicken orthologs, GST 8-8 and GST CL3. Human liver GST 5.8 showed more than an order of magnitude higher activity towards t-4-hydroxy-2-nonenal as compared to 1-chloro-2,4-dinitrobenzene. This isozyme also expressed glutathione-peroxidase activity towards fatty acid, as well as phospholipid hydroperoxidase suggesting its role in protection mechanisms against the toxicants generated during lipid peroxidation. Western blot analysis of human tissues revealed that this GST isozyme was selectively expressed in human liver, pancreas, heart, brain and bladder tissues, but absent in lung, skeletal muscle, spleen and colon.     

A 4-oxo-2(E)-nonenal-derived glutathione adduct from 15-lipoxygenase-1-mediated oxidation of cytosolic and esterified arachidonic acid

15(S)-Hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE) undergoes homolytic decomposition to bifunctional electrophiles such as 4-oxo-2(E)-nonenal. 4-Oxo-2(E)-nonenal reacts with glutathione to form a thiadiazabicyclo-4-oxo-2(E)-nonenal–glutathione adduct (TOG). Therefore, this endogenous glutathione adduct can serve as a specific biomarker of lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation. A monocyte/macrophage cell line was generated to constitutively express human 15-lipoxygenase-1. In these cells, TOG was formed from 15(S)-HpETE-derived 4-oxo-2(E)-nonenal in a nonlinear dose-dependent manner upon arachidonic acid treatment. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate abolished arachidonic acid-mediated TOG formation. The calcium ionophore A23187 was also used to induce the formation of 15(S)-HpETE from esterified arachidonic acid present in the membrane lipids. In the 15-lipoxygenase-1-expressing cells, the calcium ionophore A23187 significantly increased TOG levels compared with mock-transfected cells. This was due to the 15-lipoxygenase-mediated formation of 15(S)-HpETE in the forms of free fatty acid and esterified lipids, which was subsequently converted to 4-oxo-2(E)-nonenal. The increase in TOG formation was again abrogated by pretreatment with cinnamyl-3,4-dihydroxy-α-cyanocinnamate. Only 8.7% 15(S)-HETE (both the free fatty acid and its esterified form in the cell membrane) was formed after ionophore A23187 stimulation compared with that formed after the addition of arachidonic acid. In contrast, the TOG levels after treatment with ionophore A23187 or arachidonic acid were comparable. Thus, it is likely that esterified 15(S)-HpETE underwent homolytic decomposition to 4-oxo-2(E)-nonenal more efficiently than the free 15(S)-HpETE that was formed in the cytosol.     

(2E)-4-hydroxy-2-nonenal--an active component of a new natural antimicrobial substance

A mixture of water-soluble oxidation products of Sardinops melanosticta sardine oil was found to contain (2E)-4-hydroxy-2-nonenal. This was isolated by column chromatography on silica gel and reversed-phase HPLC. Its structure was elucidated by physicochemical methods. The activity of (2E)-4-hydroxy-2-nonenal against test cultures of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was about 20% of the total antimicrobial activity of the preparation.     

New 4H-chromen-4-one and 2H-chromene derivatives as anti-picornavirus capsid-binders

Substituted (E)-3-styryl-4H-chromen-4-ones 1a–d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a–d, (E)-3-styryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a–d were designed and synthesized to improve the anti-picornavirus activity of previously tested analogues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14 and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although considerable differences were observed in the sensitivity of viruses to each compound. Generally, both HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as the most effective inhibitor of both HRVs showing IC₅₀ values of 0.20 μM and 1.38 μM towards serotype 1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high therapeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structurally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus infection, probably at the adsorption and/or uncoating level.     

Enantioselective metabolism of trans-4-hydroxy-2-nonenal by brain mitochondria

Trans-4-hydroxy-2-nonenal (HNE) is a product of lipid peroxidation with many cellular effects. HNE possesses a stereogenic center at the C4 carbon that influences the metabolism and alkylation targets of HNE. We tested the hypothesis that rat brain mitochondria metabolize HNE in an enantioselective manner after exposure to racemic HNE. The study of HNE chirality, however, is hindered by the lack of facile methods to chromatographically resolve (R)-HNE and (S)-HNE. We used a chiral hydrazine, (S)-carbidopa, as a derivatization reagent to form diastereomers with (R)-HNE and (S)-HNE that were separated by reverse-phase HPLC. After exposure to racemic HNE, rat brain mitochondria metabolized HNE enantioselectively with a higher rate of (R)-HNE metabolism. By using the purified enantiomers of HNE, we found that this enantioselective metabolism of HNE was the result of higher rates of enzymatic oxidation of (R)-HNE by aldehyde dehydrogenases compared to (S)-HNE. Conjugation of HNE to glutathione was a minor metabolic pathway and was not enantioselective. These studies demonstrate that the chirality of HNE affects its mitochondrial metabolism and potentially other processes in the central nervous system.     

Enantioselective metabolism of (R)- and (S)-4-hydroxy-2-nonenal in rat

4-Hydroxy-2-nonenal (HNE) is an endogenous product of lipid peroxidation, which is believed to play a biological role in the pathogenesis of various diseases. HNE is formed as a racemic mixture of (R)- and (S)- enantiomers. These enantiomers differ in their biological properties. The aim of this study was to investigate separately the in vivo metabolism of the two HNE enantiomers in male rats after intravenous administration of the corresponding radiolabeled compounds and to compare the results with those obtained with the racemic mixture. Although the difference in the excretion rates was not statistically significant, the HPLC profiles of urinary metabolites showed qualitative and quantitative differences between the two enantiomers. The level of 3-mercapturic acid-1,4-dihydroxynonane, which is considered as the major urinary metabolite of HNE, was significantly lower in the case of (S)-HNE injected rats. In vitro studies using rat liver cytosolic incubations and HNE-glutathione conjugate as substrate were performed to clarify the intermediate pathways involved in their metabolism. Large differences were obtained in the reduction and retro-Michael conversion steps of the metabolism between the conjugates originating from the two enantiomers.     

Synthesis and HMG-CoA reductase inhibition of 2-cyclopropyl-4-thiophenyl-quinoline mevalonolactones

A novel series of 2-cyclopropyl-4-thiophenyl quinoline-based mevalonolactones were synthesized from the substituted anilines by several reactions. Among them, (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(4-fluoro-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1d), (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1f) and (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4,7-di(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1q) showed potent HMG-CoA reductase inhibitory activity comparable with pitavastatin.     

Preparation of novel (Z)-4-ylidenebenzo[b]furo[3,2-d][1,3]oxazines and their biological activity

A reaction of 2-acetyl-3-acylaminobenzo[b]furans (9d–o) with Vilsmeier (VM) reagent afforded a mixture of (E)- and (Z)-{(E)-2-aralkenylbenzo[b]furo[3,2-d][1,3]oxazin-4-ylidene}acetaldehydes (5) with a characteristic exo-formylmethylene group on the oxazine ring. The Z-isomer was more stable than the E-isomer. The Z-isomers ((Z)-5) were reacted with phosphonate reagents under two different conditions to obtain various butadiene derivatives (12) containing benzo[b]furo[3,2-d][1,3]oxazine skeleton. Typical compounds (5 and 12) were evaluated for their anti-osteoclastic bone resorption activity, antagonistic activity for the cysLT1 receptor and growth inhibitory activity for MIA PaCa-2 and MCF-7. Compounds 12f and 12j showed potent anti-osteoclastic bone resorption activity comparable to E₂ (17β-estradiol).