Aristolochic acid and its derivatives as inhibitors of snake venom L-amino acid oxidase.

Snake venom L-amino acid oxidase (LAAO) exerts toxicity by inducing hemorrhage, pneumorrhagia, pulmonary edema, cardiac edema, liver cell necrosis etc. Being well conserved, inhibitors of the enzyme may be synthesized using the template of the substrate, substrate binding site and features of the catalytic site of the enzyme. Previous findings showed that aristolochic acid (AA), a major constituent of Aristolochia indica, inhibits Russell's viper venom LAAO enzyme activity since, AA interacts with DNA and causes genotoxicity, derivatives of this compound were synthesized by replacing the nitro group to reduce toxicity while retaining the inhibitory potency. The interactions of AA and its derivatives with LAAO were followed by inhibition kinetics and surface plasmon resonance. Similar interactions with DNA were followed by absorption spectroscopy and atomic force microscopy. LAAO-induced cytotoxicity was evaluated by generation of reactive oxygen species (ROS), cell viability assays, confocal and epifluorescence microscopy. The hydroxyl (AA-OH) and chloro (AA-Cl) derivatives acted as inhibitors of LAAO but did not interact with DNA. The derivatives significantly reduced LAAO-induced ROS generation and cytotoxicity in human embryonic kidney (HEK 293) and hepatoma (HepG2) cell lines. Confocal images indicated that AA, AA-OH and AA-Cl interfered with the binding of LAAO to the cell membrane. AA-OH and AA-Cl significantly inhibited LAAO activity and reduced LAAO-induced cytotoxicity.

Total synthesis of the aristolochic acids, their major metabolites, and related compounds.

Plants from the Aristolochia genus have been recommended for the treatment of a variety of human ailments since the time of Hippocrates. However, many species produce the highly toxic aristolochic acids (AAs), which are both nephrotoxic and carcinogenic. For the purposes of extensive biological studies, a versatile approach to the synthesis of the AAs and their major metabolites was devised based primarily on a Suzuki-Miyaura coupling reaction. The key to success lies in the preparation of a common ring-A precursor, namely, the tetrahydropyranyl ether of 2-nitromethyl-3-iodo-4,5-methylendioxybenzyl alcohol (27), which was generated in excellent yield by oxidation of the aldoxime precursor 26. Suzuki-Miyaura coupling of 27 with a variety of benzaldehyde 2-boronates was accompanied by an aldol condensation/elimination reaction to give the desired phenanthrene intermediate directly. Deprotection of the benzyl alcohol followed by two sequential oxidation steps gave the desired phenanthrene nitrocarboxylic acids. This approach was used to synthesize AAs I-IV and several other related compounds, including AA I and AA II bearing an aminopropyloxy group at position-6, which were required for further conversion to fluorescent biological probes. Further successful application of the Suzuki-Miyaura coupling reaction to the synthesis of the N-hydroxyaristolactams of AA I and AA II then allowed the synthesis of the putative, but until now elusive, N-acetoxy- and N-sulfonyloxy-aristolactam metabolites.

A novel pre-column fluorescent derivatization method for the sensitive determination of aristolochic acids in medicinal herbs by high-performance liquid chromatography with fluorescence detection.

Aristolochic acids (AAs) are a family of structurally related nitrophenanthrene carboxylic acids existing in Aristolochia, Bragantia, and Asarum species. AAs have been proven to have nephrotoxic and carcinogenic toxicity. In this study, a novel pre-column fluorescence derivatization procedure followed by high-performance liquid chromatography-fluorescence detection (HPLC-FLD) is developed for the analysis of AAs in medicinal herbs. The nitro group in the phenanthrene ring of AAs was removed by NaBH(4) in water-THF (2:1, v/v), resulting in the corresponding aristolic acids. The analysis of AAs in medicinal herbs was based of the sensitive fluorescence detection of aristolic acids after the chemical derivatization. Because the produced aristolic acids are highly fluorescent the limit of detection (LOD) of AAI and AAII were lowered to 0.06 and 0.04 ng/mL, respectively, which is at least an order of magnitude lower than those in the reported HPLC and LC-MS methods. Good linearity with correlation coefficients higher than 0.997 were obtained for AAI and AII in the calibration ranges of 0.2-800 ng/mL. The derivatization conditions such as reaction temperature, time and the amount of NaBH(4) were optimized. The developed method provided satisfactory intra-day and inter-day precisions with RSDs less than 1.4% and 3.8%, respectively. The relative analytical error was less than 7% for the analysis of AAI and AAII in spiked matrix samples.

Semi-synthetic aristolactams--inhibitors of CDK2 enzyme.

Several analogs of aristolochic acids were isolated and derivatized into their lactam derivatives to study their inhibition in CDK2 assay. The study helped to derive some conclusions about the structure-activity relation around the phenanthrin moiety. Semi-synthetic aristolactam 21 showed good activity with inhibition IC50 of 35 nM in CDK2 assay. The activity of this compound was comparable to some of the most potent synthetic compounds reported in the literature.

DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells.

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In approximately 50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA-->T and AL-II-dG-->T transversions.

[Synthesis and mass spectrometric analysis of aristolochic acid-deoxyguanosine adducts].

To synthesize aristolochic acid (AA)-2'-deoxyguanosine 5'-monophosphate (dGp) adducts in vitro and develop a novel method for the characterization of the adducts using multiple mass spectrometric techniques. AA was incubated with dGp in vitro using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-dGp adducts, and the reaction conditions were optimized. Crude extracts were analyzed by techniques of liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-MS/MS) and high accuracy mass data and isotope pattern of super high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICRMS). The quasi-molecular ion peaks of the AA-dGp adducts were obtained in the negative ion mode. Analysis by electrospray ionization/tandem mass spectrometry (ESI-MS/MS) provided useful structural information about AA-dGp adducts. AA can bind covalently to the exocyclic amino group of deoxyguanosine to form AA-dGp adducts. MS analysis is a powerful tool to detect and identify AA-dGp adducts simply, rapidly and accurately.

Regio- and stereoselective synthesis of isoindolin-1-ones via electrophilic cyclization.

[reaction: see text] A variety of substituted isoindolin-1-ones are readily prepared in good to excellent yields under very mild reaction conditions by the reaction of o-(1-alkynyl)benzamides with ICl, I(2), and NBS. In a few cases, substituted isoquinolin-1-ones were obtained as the major product instead. This methodology accommodates various alkynyl amides and functional groups and has been successfully extended to heterocyclic starting materials. This chemistry has been successfully applied to the synthesis of a biologically interesting alkaloid, cepharanone B.

Synthesis and biological evaluation of aristolactams.

A variety of aristolactam derivatives were synthesized and evaluated for cytotoxicity. Modulations were carried out on the phenanthrene nucleus and the lactam moiety as well. N-(N-dialkylaminoalkyl) derivatives exhibited interesting cytotoxic activity against the L1210 leukemia cell line.