In vivo activity of benzoyl ester clerodane diterpenoid derivatives from Dodonaea polyandra.

Four new benzoyl ester clerodane diterpenoids, 15,16-epoxy-8α-(benzoyloxy)methylcleroda-3,13(16),14-trien-18-oic acid (1), 15,16-epoxy-8α-(benzoyloxy)methyl-2α-hydroxycleroda-3,13(16),14-trien-18-oic acid (2), 15,16-epoxy-8α-(benzoyloxy)methyl-2-oxocleroda-3,13(16),14-trien-18-oic acid (3), and 15,16-epoxy-2α-benzoyloxycleroda-3,13(16),14-trien-18-oic acid (4), have been isolated from the leaves and stems of Dodonaea polyandra. The anti-inflammatory activities of compounds 1, 2, and 4 were evaluated by means of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema. Compounds 2 and 4 exhibited maximum inhibition of inflammation (70-76%) at doses of 0.22 and 0.9 µmol/ear, respectively. Modest activity (~45% inhibition) was maintained at nanomole/ear doses.

Selective biocatalytic hydroxylation of unactivated methylene C-H bonds in cyclic alkyl substrates.

The cytochrome P450 monooxygenase CYP101B1 from Novosphingobium aromaticivorans selectively hydroxylated methylene C-H bonds in cycloalkyl rings. Cycloketones and cycloalkyl esters containing C6, C8, C10 and C12 rings were oxidised with high selectively on the opposite side of the ring to the carbonyl substituent. Cyclodecanone was oxidised to oxabicycloundecanol derivatives in equilibrium with the hydroxycyclodecanones.

Sequential and selective Buchwald-Hartwig amination reactions for the controlled functionalization of 6-bromo-2-chloroquinoline: synthesis of ligands for the Tec Src homology 3 domain.

Src homology 3 (SH3) domains are highly conserved protein-protein interaction domains that mediate important biological processes and are considered valuable targets for the development of therapeutic agents. In this paper, we report the preparation of a range of new 6-heterocyclic substituted 2-aminoquinolines using Buchwald-Hartwig chemistry. 6-Heterocyclic substitution of the 2-aminoquinoline has provided ligands with increased binding affinity for the SH3 domain relative to the lead compound, 2-aminoquinoline, that are the highest affinity ligands prepared to date. The key step in the synthesis of these compounds required a selective Buchwald-Hartwig amination of an aryl bromide in the presence of an activated heteroaryl chloride. The optimization of reaction conditions to achieve the selective amination is discussed and has allowed for cross-coupling with a range of cyclic amines. Introduction of the amino functionality of the 6-heterocyclic 2-aminoquinolines involved additional Buchwald-Hartwig chemistry utilizing lithium bis(trimethylsilyl)amide as an ammonia equivalent.

Antimicrobial compounds from Eremophila serrulata.

We report a search for antimicrobial compounds in the Australian plant Eremophila serrulata. Bioassay directed fractionation of a diethyl ether extract prepared from the leaves of E. serrulata led to the isolation of two compounds, an omicron-naphthoquinone, 9-methyl-3-(4-methyl-3-pentenyl)-2,3-dihydronaphtho[1,8-bc]pyran-7,8-dione (2), and a serrulatane diterpenoid, 20-acetoxy-8-hydroxyserrulat-14-en-19-oic acid (3). Two other known serrulatane-type diterpenoids, 8,20-dihydroxyserrulat-14-en-19-oic acid (4) and 8,20-diacetoxyserrulat-14-en-19-oic acid (5) were also isolated. None of these compounds had previously been tested for antimicrobial activity. Compounds 2-5 showed antimicrobial activity against Staphylococcus aureus (ATCC 29213) with minimum inhibitory concentrations (MICs) ranging from 15.6 to 250mug/mL. Compound 2 was the most active with an MIC of 15.6mug/mL and a minimum bactericidal concentration (MBC) of 125mug/mL. This compound also showed antimicrobial activity against other Gram-positive bacteria including Streptococcus pyogenes, and Streptococcus pneumoniae. No activity was observed for this compound against all Gram-negative bacteria tested.

Enantioselective assay for the determination of perhexiline enantiomers in human plasma by liquid chromatography.

Effective use of the antianginal agent perhexiline is difficult because saturable metabolism by the polymorphic cytochrome P450 2D6 (CYP2D6) isoform produces elevated plasma perhexiline concentrations that have been associated with serious hepatic and neurological toxicity. Perhexiline is marketed for therapeutic use as a racemate and there is evidence for differences in the disposition of its enantiomers. The current study describes an enantioselective HPLC-fluorescent method utilising pre-column derivatization with (R)-(-)-1-(1-napthyl)ethyl isocyanate. Following derivatization, the enantiomers are resolved on a C18 column with gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of (+)- and (-)-perhexiline in human plasma following clinical doses and demonstrates sufficient sensitivity, accuracy and precision between 0.01 and 2.00 mg/l for each enantiomer, with intra-assay coefficients of variation and bias <20% at 0.01 mg/l and <10% at 2.00 mg/l, and inter-assay coefficients of variation and biases <15% at 0.03 mg/l and <10% at 0.40 and 0.75 mg/l. The application of this method to plasma samples collected from a patient treated with perhexiline revealed that (+)-perhexiline concentrations were higher than (-)-perhexiline concentrations, confirming the stereoselective disposition of perhexiline. The current study describes an enantioselective method that utilises pre-column formation of fluorescent diastereomers that are resolved on a C18 HPLC column using a gradient of methanol and water.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

Aldehyde-sequestering drugs: tools for studying protein damage by lipid peroxidation products.

Elevated levels of reactive alpha,beta-unsaturated aldehydes (e.g. malondialdehyde, 4-hydroxynonenal and acrolein) in the affected tissues of various degenerative conditions suggest these substances are active propagators of the disease process. One experimental approach to attenuating damage by these intermediates employs 'aldehyde-sequestering drugs' as sacrificial nucleophiles, thereby sparing cell macromolecules and perhaps slowing disease progression. Drugs with demonstrated trapping activity toward lipid-derived aldehydes include various amine compounds such as aminoguanidine, carnosine and pyridoxamine. We have focused on identifying scavengers of acrolein, perhaps the most toxic aldehyde formed during lipid peroxidation cascades. Various phthalazine compounds (hydralazine and dihydralazine) were found to trap acrolein readily, forming hydrazone derivatives in a rapid Schiff-type reaction. These compounds strongly protect against acrolein-mediated toxicity in isolated hepatocytes.

Michael addition of acrolein to lysinyl and N-terminal residues of a model peptide: targets for cytoprotective hydrazino drugs.

The antihypertensive drug hydralazine blocks acrolein-mediated toxicity by trapping both free aldehyde- and acrolein-adducted proteins, with the latter property more closely related to cytoprotection in cellular models. Here we report the identification of products from 'protein adduct-trapping' reactions using electrospray ionisation mass spectrometry (ESI-MS). Reaction of a 13-residue peptide containing a single lysine with acrolein for 30 min generated ions corresponding to mono- and bis-Michael-adducted peptides. An ion corresponding to a cyclic species formed from bis-adducted lysine was conspicuous at later times (60, 180 min). Tandem mass spectrometric (MS/MS) analysis revealed Michael adduction also occurred on the N-terminus, with a novel N-terminal (3-formyl-3,4-dehydropiperidino) species formed on this residue. Addition of hydralazine to acrolein-adducted peptides generated a diverse range of hydrazones that were also characterised by MS/MS analysis. The results confirm that mass spectrometry is a powerful tool for characterising the reactions of noxious electrophiles with biological macromolecules.

Antimicrobial compounds from the Australian desert plant Eremophila neglecta.

A crude extract from the Australian desert plant Eremophila neglecta has recently been shown to possess antibacterial activity in a survey of candidate plants that may bear novel antimicrobial compounds. Bioassay-directed fractionation of the Et(2)O extract of E. neglecta using a broth microdilution assay led to the isolation of three new serrulatane-type diterpenoids, 2,19-diacetoxy-8-hydroxyserrulat-14-ene (2), 8,19-dihydroxyserrulat-14-ene (3), and 8-hydroxyserrulat-14-en-19-oic acid (4), and a known o-naphthoquinone commonly referred to as biflorin (5). The structures of 2-5 were determined using 1D and 2D NMR, FTIR, and high-resolution mass spectrometry. Compounds 3-5 showed antimicrobial activity against Gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, and S. pneumoniae. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 6.5 to 101.6 microM and 12.7 to 202.9 microM, respectively. No activity was observed for these compounds against Gram-negative bacteria.

Hydralazine inhibits rapid acrolein-induced protein oligomerization: role of aldehyde scavenging and adduct trapping in cross-link blocking and cytoprotection.

Hydralazine strongly suppresses the toxicity of acrolein, a reactive aldehyde that contributes to numerous health disorders. At least two mechanisms may underlie the cytoprotection, both of which involve the nucleophilic hydrazine possessed by hydralazine. Under the simplest scenario, hydralazine directly scavenges free acrolein, decreasing intracellular acrolein availability and thereby suppressing macromolecular adduction. In a second "adduct-trapping" mechanism, the drug forms hydrazones with acrolein-derived Michael adducts in cell proteins, preventing secondary reactions of adducted proteins that may trigger cell death. To identify the most important mechanism, we explored these two pathways in mouse hepatocytes poisoned with the acrolein precursor allyl alcohol. Intense concentration-dependent adduct-trapping in cell proteins accompanied the suppression of toxicity by hydralazine. However, protective concentrations of hydralazine did not alter extracellular free acrolein levels, cellular glutathione loss, or protein carbonylation, suggesting that the cytoprotection is not due to minimization of intracellular acrolein availability. To explore ways whereby adduct-trapping might confer cytoprotection, the effect of hydralazine on acrolein-induced protein cross-linking was examined. Using bovine pancreas ribonuclease A as a model protein, acrolein caused rapid time- and concentration-dependent cross-linking, with dimerized protein detectable within 45 min of commencing protein modification. Lysine adduction in monomeric protein preceded the appearance of oligomers, whereas reductive methylation of protein amine groups abolished both adduction and oligomerization. Hydralazine inhibited cross-linking if added 30 min after commencing acrolein exposure but was ineffective if added after a 90-min delay. Adduct-trapping closely accompanied the inhibition of cross-linking by hydralazine. These findings suggest that cross-link blocking may contribute to hydralazine cytoprotection.

Synthesis of N-benzylated-2-aminoquinolines as ligands for the Tec SH3 domain.

In recent work, we have been developing 2-aminoquinolines as ligands for Src Homology 3 (SH3) domains, so far the only reported examples of small-molecule ligands for these domains. In this paper, we report the synthesis of a series of N-benzylated-2-aminoquinolines by reductive amination of aryl aldehydes with 2-aminoquinoline. These ligands bound the SH3 domain with ca. one and a half to twofold reduced affinity relative to 2-aminoquinoline; however, some evidence was found to suggest that the benzylic substituents made new contacts with the SH3 domain surface. These results provide useful SAR information that may assist in future ligand design.

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.

Reactivity of hydrazinophthalazine drugs with the lipid peroxidation products acrolein and crotonaldehyde.

The nucleophilic drug hydralazine strongly inhibits cell toxicity mediated by acrolein, a short chain 2-alkenal formed during lipid peroxidation. We here report the chemistry of acrolein-trapping by hydralazine, and show that together with its structural analogue dihydralazine, it also readily traps crotonaldehyde. Isolable reaction products included (1E)-acrylaldehyde phthalazin-1-ylhydrazone (E-APH), (1Z)-acrylaldehyde phthalazin-1-ylhydrazone (Z-APH), (1E,2E)-but-2-enal phthalazin-1-ylhydrazone (E-BPH) and (1Z,2E)-but-2-enal phthalazin-1-ylhydrazone (Z-BPH). Concentration-dependent formation of (1E)-acrylaldehyde phthalazin-1-ylhydrazone was observed in the culture media of cells co-exposed to hydralazine and the acrolein precursor allyl alcohol. These aldehyde-sequestering properties of hydrazinophthalazine drugs may contribute to the protection they provide against 2-alkenal-mediated toxicity.

Strong protein adduct trapping accompanies abolition of acrolein-mediated hepatotoxicity by hydralazine in mice.

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde that readily alkylates nucleophilic centers in cell macromolecules. Typically, such reactions proceed via Michael addition chemistry, forming adducts that retain an electrophilic carbonyl group. Since these species participate in secondary deleterious reactions, we hypothesize that inactivation of carbonyl adducts may attenuate acrolein toxicity. Indeed, we recently established that the nucleophilic antihypertensive drug hydralazine readily "traps" acrolein adducts in cell proteins and strongly suppresses acrolein-mediated toxicity in isolated hepatocytes. This work sought to determine whether hydralazine prevents the in vivo hepatotoxicity of the acrolein precursor allyl alcohol in whole mice and whether adduct trapping accompanies any such hepatoprotection. Mice received allyl alcohol alone or in conjunction with several doses of hydralazine. Four hours later, mice were sacrificed to allow for the determination of liver enzymes in plasma as markers of hepatic injury, whereas livers were assessed for glutathione and hydralazine-stabilized protein adducts. Hydralazine afforded strong, dose-dependent protection against the increases in plasma marker enzymes but not the hepatic glutathione depletion produced by allyl alcohol. Western blotting revealed intense, dose-dependent adduct trapping by hydralazine in numerous liver proteins over a broad 26- to 200-kDA mass range. In keeping with these findings, immunohistochemical analysis of liver slices indicated diffuse, extranuclear adduct trapping by hydralazine that was uniformly distributed across the liver lobule, with partial localization in parenchymal cell membranes. These findings concur with our hypothesis that hydralazine readily inactivates reactive carbonyl-retaining protein adducts formed by acrolein, thereby preventing secondary reactions that trigger cellular death.

Reactivity with Tris(hydroxymethyl)aminomethane confounds immunodetection of acrolein-adducted proteins.

The toxic alpha,beta-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (> or =60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.

Protein adduct-trapping by hydrazinophthalazine drugs: mechanisms of cytoprotection against acrolein-mediated toxicity.

Acrolein is a highly toxic aldehyde involved in a number of diseases as well as drug-induced toxicities. Its pronounced toxicity reflects the readiness with which it forms adducts in proteins and DNA. As a bifunctional electrophile, initial reactions between acrolein and protein generate adducts containing an electrophilic center that can participate in secondary deleterious reactions (e.g., cross-linking). We hypothesize that inactivation of these reactive protein adducts with nucleophilic drugs may counteract acrolein toxicity. Because we previously observed that 1-hydrazinophthalazine (hydralazine) strongly diminishes the toxicity of the acrolein precursor allyl alcohol, we explored the possibility that hydralazine targets reactive acrolein adducts in proteins. We report that hydralazine abolished the immunoreactivity of an acrolein-modified model protein (bovine serum albumin), but only if the drug was added to the protein within 30 min of commencing modification by acrolein. The ability of a range of carbonyl-trapping drugs to interfere with "early" events in protein modification strongly correlated with their protective potencies against allyl alcohol toxicity in hepatocytes. In mass spectrometry studies using a model lysine-containing peptide, hydralazine rapidly formed hydrazones with Michael adducts generated by acrolein. Using an antibody raised against such ternary drug-acrolein-protein complexes in Western blotting experiments, clear adduct-trapping was evident in acrolein-preloaded hepatocytes exposed to cytoprotective concentrations of hydralazine ranging from 2 to 50 microM. These novel findings begin to reveal the molecular mechanisms whereby hydralazine functions as an efficient "protein adduct-trapping" drug.