N-oxide Formation Research Articles

Synthesis, Insecticidal Assay and Molecular Docking Study of Novel Neonicotinoids N‐Oxide Analogues

AbstractEight novel neonicotinoids N‐oxide analogues were designed and synthesized. All the compounds have been identified by 1H NMR and HRMS. The N‐oxide analogues exhibit high insecticidal activity against cowpea aphids (Aphis craccivora) at 250 mg·L−1. The influence of N‐oxide formation on the biological activity was elucidated by computational chemical study, and it indicated that the water bridge hydrogen bonding network was broken due to the influence of the O atom connected with the pyridine ring.

Synthesis of 1-Aroxyisoquinolines from Phenols and 2-Alkynylbenzaldoximes

Reported is the silver-catalyzed synthesis of 1-aroxyisoquinolines from 2-alkynylbenzaldoximes and phenols in the presence of PyBroP. This work is a direct extension of other ­recent work by the same authors (D. Zheng, Z. Chen, J. Liu, J. Wu Org. Biomol. Chem. 2011, 9, 4763), where amines were used instead of phenols to prepare 2-aminoisoquinolines. This previous work itself was based on the known silver-catalyzed formation of isoquinoline N-oxides from benzaldoximes (H.-S. Yeom, S. Kim, S. Shin Synlett 2008, 924), and the amination of isoquinoline N-oxides using PyBroP (A. T. Londregan, S. Jennings, L. Wei Org. Lett. 2010, 12, 5254). The proposed mechanism is directly analogous to the mechanism of the previously reported reaction, which combined the two known mechanisms of the individual steps. As in the previous work, there is no explanation for the requirement of two equivalents of PyBroP.

Study of the oxidation of 3-hydroxypyrroloindoles to pyrrolobenzoxazine alkaloids

This report describes a detailed study of the oxidation-Meisenheimer rearrangement of N-methyl-3-hydroxy-7-chloropyrroloindoline ethyl ester and the corresponding O-Boc and N-Boc derivatives. Experimental conditions were found, which allowed the selective Boc protection of either the tertiary alcohol substituent or the NH group of the aminal function. It was shown that both the parent compound and its O-Boc derivative yielded a mixture of oxazines and, in some cases, N-oxides upon treatment with m-CPBA. MS fragmentation (APCI) clearly differentiates formation of N-oxides and oxazines. The N-Boc derivatives exclusively yielded the N-oxides showing that the Meisenheimer rearrangement requires the presence of a high energy lone pair on the neighbouring nitrogen atom. Both the parent compound and the O-Boc derivative gave a mixture of rearranged products and N-oxide depending on the reaction conditions.

In vitro identification of the human cytochrome p450 enzymes involved in the oxidative metabolism of loxapine

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) enzymes responsible for the oxidative metabolism of loxapine to 8-hydroxyloxapine, 7-hydroxyloxapine, N-desmethylloxapine (amoxapine) and loxapine N-oxide. These studies included use of cDNA-expressed enzymes, correlation analysis with 12 phenotyped human liver microsomal samples, and use of selective inhibitors of cytochrome P450s. The resultant data indicated that loxapine was mainly metabolized by human liver microsomes to (i) 8-hydroxyloxapine by CYP1A2, (ii) 7-hydroxyloxapine by CYP2D6, (iii) N-desmethyloxapine by CYP3A4 and (iv) loxapine N-oxide by CYP3A4. The involvement of flavin-containing monooxygenase (FMO) in the formation of loxapine N-oxide was also observed.

The first synthesis of 3-nitro-4-[(s-tetrazin-3-yl)amino]furazans

Oxidation of 3-amino-4-[(s-tetrazin-3-yl)amino]furazans with peroxy acids or 30% H2O2/Na2WO4/H2SO4 system results in transformation of the amino group into the nitro one and is accompanied by formation of N-oxides at tetrazine moiety.

Complexation of Phenol and Thiophenol by Amine N‐Oxides: Isothermal Titration Calorimetry and ab Initio Calculations

To develop a new solvent-impregnated resin (SIR) system for removal of phenols from water, the complex formation of dimethyldodecylamine N-oxide (DMDAO), trioctylamine N-oxide (TOAO), and tris(2-ethylhexyl)amine N-oxide (TEHAO) with phenol (PhOH) and thiophenol (PhSH) is studied. To this end we use isothermal titration calorimetry (ITC) and quantum chemical modeling (on B3LYP/6-311G(d,p)-optimized geometries: B3LYP/6-311+G(d,p), B3LYP/6-311++G(2d,2p), MP2/6-311+G(d,p), and spin component scaled (SCS) MP2/6-311+G(d,p); M06-2X/6-311+G(d,p)//M06-2X/6-311G(d,p), MP2 with an extrapolation to the complete basis set limit (MP2/CBS), as well as CBS-Q). The complexes are analyzed in terms of structural (e.g., bond lengths) and electronic elements (e.g., charges). Furthermore, complexation and solvent effects (in benzene, toluene, and mesitylene) are investigated by ITC measurements, yielding binding constants K, enthalpies ΔH(0), Gibbs fre energies ΔG(0), and entropies ΔS(0) of complex formation, and stoichiometry N. The ITC measurements revealed strong 1:1 complex formation between both DMDAO-PhOH and TOAO-PhOH. The binding constant (K=1.7-5.7×10(4) M(-1)) drops markedly when water-saturated toluene was used (K=5.8×10(3) M(-1)), and π-π interaction with the solvent is shown to be relevant. Quantum mechanical modeling confirms formation of stable 1:1 complexes with linear hydrogen bonds that weaken on attachment of electron-withdrawing groups to the amine N-oxide moiety. Modeling also showed that complexes with PhSH are much weaker than those with PhOH, and in fact too weak for ITC determination. CBS-Q incorrectly predicts equal or even higher binding enthalpies for PhSH than for PhOH, which invalidates it as a benchmark for other calculations. Data from the straightforward SCS-MP2 method without counterpoise correction show very good agreement with the MP2/CBS values.

Thermally induced intramolecular oxygen migration of N‐oxides in atmospheric pressure chemical ionization mass spectrometry

N-Oxides are known to undergo three main thermal degradation reactions, namely deoxygenation, Cope elimination (for N-oxides containing a β-hydrogen) and Meisenheimer rearrangement, in atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The ions corresponding to these thermal degradants observed in the ensuing APCI mass spectra have been used to identify N-oxides as well as to determine the N-oxidation site when the analyte contains multiple tertiary amine groups. In this paper, we report a thermally induced oxygen migration from one N-oxide amine to another tert-amine group present in the same molecule through a six-membered ring transition state during APCI-MS analysis. The observed intramolecular oxygen migration resulted in the formation of a new isomeric N-oxide, rendering the results of the APCI-MS analysis more difficult to interpret and potentially misleading. In addition, we observed novel degradation behavior that happened after the Meisenheimer rearrangement of the newly formed N-oxide: a homolytic cleavage of the N-O bond instead of elimination of an aldehyde or a ketone that usually follows the rearrangement. Understanding of these unusual degradation pathways, which have not been reported previously, should facilitate structural elucidation of N-oxides using APCI-MS analysis.

Abstract 3474: Formation of myosmine under oxidation of nicotine

Abstract Nicotine and myosmine are alkaloids in tobacco products at which nicotine occurs in 200fold higher concentrations than myosmine. Nicotine is restricted on tobacco mainly, but myosmine is widespread in the biosphere and food materials additionally. Activation of nicotine showed amongst others N-nitrosation conversion to a compound class, the tobacco specific N-nitrosamines (TSNA). N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) represent nicotine derived substances. These are assumed to be responsible for tumorigenesis and cancer disease, especially from the lung after activation to highly reactive intermediate species pyridyloxobutanone (POB). POB is known in the context of CYP 450 dependent activation of TSNA and was detected in DNA of tissues from laboratory animals, after TSNA application. The POB-DNA adducts release under hydrolysis 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) which was observed also during myosmine metabolism in rats, under N-nitrosation and under peroxidation procedures of this compound. Partially, nicotine and myosmine seem to be metabolized on similar way and support assumption from employment of identical CYP450 isozymes. Oxidation processes of nicotine and myosmine employing non CYP450 dependent systems undergoe different pathways involving N or N’ ring positions of their pyridine and pyrrole structures. Peroxidation of nicotine is reported under formation of nicotine N-oxides mainly. First results of finding higher myosmine contents in cigarette filter tips from smoked in relation to unburned cigarettes gave rise to deliberation of possible conversion of nicotine to myosmine. Following, (−)-nicotine in concentrations equivalent to amount in cigarettes 4.9-5.5µmol was incubated with hydrogen peroxide at 37°C and analyzed. To simulate the assumed reaction on biological systems, (−)-nicotine was incubated with horse radish peroxidase (HRP) and occurence of the relevant compounds analyzed. The assays were directly measured with high pressure liquid chromatography (HPLC) instrumentation and confirmed by cochromatography with respective standard compounds. Our results showed formation of myosmine under peroxidation from (−)-nicotine. This would mean conversion of nicotine to myosmine releases activated methyl-species, which could undergo protein or DNA methylation. This possible effect would indicate ability of nicotine to become activated not only as nitrosated but also as peroxidated species. Additional experiments are demanded to elucidate possible carcinogenic risk of nicotine conversion to myosmine. Acknowledgement: This work was supported by DFG grant 59/2-2 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3474.

Distribution, metabolism, and excretion of toceranib phosphate (Palladia™, SU11654), a novel tyrosine kinase inhibitor, in dogs

Toceranib phosphate (Palladia, SU11654), a multireceptor tyrosine kinase inhibitor with anti-tumor and anti-angiogenic activity, has been developed for the treatment of mast cell tumors in dogs. An overview of the distribution, metabolism, and excretion of toceranib phosphate in dogs is presented. When [(14)C]-toceranib was orally administered to dogs, the majority of the radioactivity (92%) was excreted in feces and only a small portion (7%) was excreted in urine. Seven days after a single 3.25 mg/kg oral dose, radioactivity was the highest in bile and liver, with measurable concentrations in lymph nodes, colon, adrenals, bone marrow, kidneys, lungs, spleen, pancreas, and skin. Plasma protein binding of toceranib in fresh plasma ranged from 90.8% to 92.8% at concentrations between 20 ng/mL and 500 ng/mL and was independent of concentration. Microsomal and hepatocyte incubations resulted in the formation of a single metabolite. Spectrometric analysis of the metabolite was consistent with the formation of an alicyclic N-oxide of toceranib. The combination of the high rate of fecal excretion and the long elimination half-life of toceranib indicate enterohepatic recirculation of the parent compound and/or the N-oxide metabolite.

Synthesis and characterization of new heterocyclic Schiff base palladacycles: Ring activation through N-oxide formation

Reaction of the Schiff base ligand derived from 4-pyridinecarboxaldehyde NC 5H 4C(H) N[2′,4′,6′-(CH 3)C 6H 2], ( 1), with palladium(II) acetate in toluene at 60 °C for 24 h gave [Pd{NC 5H 4C(H) N[2′,4′,6′-(CH 3)C 6H 2]} 2(OCOCH 3) 2], ( 2), with two ligands coordinated through the pyridine nitrogen. Treatment of the Schiff base ligand derived from 4-pyridinecarboxaldehyde N-oxide, 4-(O)NC 5H 4C(H) N[2′,4′,6′-(CH 3)C 6H 2], ( 4), with palladium(II) acetate in toluene at 75 °C gave the dinuclear acetato-bridged complex [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}(OCOCH 3)] 2, ( 5) with metallation of an aromatic phenyl carbon. Reaction of complex 5 with sodium chloride or lithium bromide gave the dinuclear halogen-bridged complexes [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}(Cl)] 2, ( 6) and [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}(Br)] 2, ( 7), after the metathesis reaction. Reaction of 6 and 7 with triphenylphosphine gave the mononuclear species [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}(Cl)(PPh 3)], ( 8) and [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}-(Br)(PPh 3)], ( 9), as air stable solids. Treatment of 6 and 7 with Ph 2P(CH 2) 2PPh 2 (dppe) in a complex/diphosphine 1:2 molar ratio gave the mononuclear complexes [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}(PPh 2(CH 2) 2PPh 2)][Cl], ( 10), and [Pd{4-(O)NC 5H 3C(H) N[2′,4′,6′-(CH 3)C 6H 2]}(PPh 2(CH 2) 2PPh 2)][PF 6], ( 11), with a chelating diphosphine. The molecular structure of complex 9 was determined by X-ray single crystal diffraction analysis.

Polyvinyl pyridine metal complex as permanent antimicrobial finishing for viscose fabric

Viscose fabrics were treated with polyvinyl pyridine (PVP) using padding technique, followed by oxidation with hydrogen peroxide or peracetic acid, which was prepared by the reaction of tetra acetyl ethylene diamine (TAED) with hydrogen peroxide. Peracetic acid gives higher oxidation of PVP than hydrogen peroxide. FTIR study proved the formation of N-oxide as a result of oxidation. Incorporation of copper and silver ion onto oxidized PVP was also proved by FTIR. The antimicrobial study emphasise that Cu/oxidized PVP and Ag/oxidized PVP have retarded the growth of bacteria significantly, and Ag/oxidized PVP has a far better biocidal activity. The antibacterial activity of both metal ions survived after washing 10 times.

Iodine-mediated electrophilic cyclization of 2-alkynylbenzaldoximes leading to the formation of iodoisoquinoline N-oxides

Reaction of 2-alkynylbenzaldoximes 1 with 5 equiv of iodine in EtOH at room temperature gives the corresponding iodoisoquinoline N-oxides 2 in good to excellent yields. The cyclization proceeds very smoothly and quickly without any additives such as bases under very mild reaction conditions.

Change in the hybridization of the N→O group oxygen atom upon complex formation of pyridine and quinoline N-oxides with v-acceptors

It was shown on the basis of X-ray structural and NMR data that the (sp 2→sp 3) rehybridization of the oxygen atom of the N→O group can take place in reactions of heteroaromatic N-oxides with Lewis and Bronsted-Lowry acids. The hybridization type depends on the ligand and acceptor natures, composition of a complex, and spatial stresses arising during the complex formation.

Role of Flavin-Containing Monooxygenase in Oxidative Metabolism of Voriconazole by Human Liver Microsomes

Voriconazole is a potent second-generation triazole antifungal agent with broad-spectrum activity against clinically important fungi. It is cleared predominantly via metabolism in all species tested including humans. N-Oxidation of the fluoropyrimidine ring, its hydroxylation, and hydroxylation of the adjacent methyl group are the known pathways of voriconazole oxidative metabolism, with the N-oxide being the major circulating metabolite in human. In vitro studies have shown that CYP2C19, CYP3A4, and to a lesser extent CYP2C9 contribute to the oxidative metabolism of voriconazole. When cytochrome P450 (P450)-specific inhibitors and antibodies were used to evaluate the oxidative metabolism of voriconazole by human liver microsomes, the results suggested that P450-mediated metabolism accounted for approximately 75% of the total oxidative metabolism. The studies presented here provide evidence that the remaining approximately 25% of the metabolic transformations are catalyzed by flavin-containing monooxygenase (FMO). This conclusion was based on the evidence that the NADPH-dependent metabolism of voriconazole was sensitive to heat (45 degrees C for 5 min), a condition known to selectively inactivate FMO without affecting P450 activity. The role of FMO in the metabolic formation of voriconazole N-oxide was confirmed by the use of recombinant FMO enzymes. Kinetic analysis of voriconazole metabolism by FMO1 and FMO3 yielded K(m) values of 3.0 and 3.4 mM and V(max) values of 0.025 and 0.044 pmol/min/pmol, respectively. FMO5 did not metabolize voriconazole effectively. This is the first report of the role of FMO in the oxidative metabolism of voriconazole.

Molecular modeling optimization of anticoagulant pyridine derivatives

Intravascular clotting remains a major health problem in the United States, the most prominent being deep vein thrombosis, pulmonary embolism and thromboembolic stroke. Previous reports on the use of pyridine derivatives in cardiovascular drug development encourage us to pursue new types of compounds based on a pyridine scaffold. Eleven pyridine derivatives (oximes, semicarbazones, N-oxides) previously synthesized in our laboratories were tested as anticoagulants on pooled normal plasma using the prothrombin time (PT) protocol. The best anticoagulant within the oxime series was compound AF4, within the oxime N-oxide series was compound AF4-N-oxide, and within the semicarbazone series, compound MD1-30Y. We also used a molecular modeling approach to guide our efforts, and found that there was good correlation between coagulation data and computational energy scores. Molecular docking was performed to target the active site of thrombin with the DOCK v5.2 package. The results of molecular modeling indicate that improvement in anticoagulant activities can be expected by functionalization at the three-position of the pyridine ring and by N-oxide formation. Results reported here prove the suitability of DOCK in the lead optimization process.

Metabolism of substituted vs. unsubstituted pyridine analogs of NNRTIs

A new series of NNRTIs with substituted or unsubstituted pyridine has been developed, and members of this series have demonstrated potent activity against NNRTI-resistant virus. In this study, both in vitro microsomal stability assay and in vivo animal pharmacokinetics study were conducted for selected NNRTIs in order to establish the in vitro-in vivo correlations. There was a good correlation for the substituted pyridine analogs, but for the unsubstituted pyridine analogs, the in vivo clearance was under-predicted in vitro. We investigated the metabolic pathways of several substituted pyridine analogs and one unsubstituted pyridine analog. The hydroxylation on the ring and N-hydroxylation of the amino group were found to be the major metabolic pathways for the substituted pyridine analogs. However, for the unsubstituted pyridine analog, N-oxide formation was the single most important metabolic pathway. This pathway was under-represented in the liver microsomes, which indicates that the N-oxide formation was likely catalyzed by non-P450 enzyme systems. Further investigations are underway to identify the enzyme system that is responsible for the N-oxide formation on the unsubstituted pyridines. This research is supported by Gilead Sciences, Inc.

Lack of effect of ketoconazole-mediated CYP3A inhibition on sorafenib clinical pharmacokinetics

Sorafenib is a novel, small-molecule anticancer compound that inhibits tumor cell proliferation by targeting Raf in the Raf/MEK/ERK signalling pathway, and inhibits angiogenesis by targeting tyrosine kinases such as vascular-endothelial growth factor receptor (VEGFR-2 and VEGFR-3) and platelet-derived growth factor receptor (PDGFR). In vitro microsomal data indicate that sorafenib is metabolized by two pathways: phase I oxidation mediated by cytochrome P450 (CYP) 3A4; and phase II conjugation mediated by UGT1A9. Approximately 50% of an orally administered dose is recovered as unchanged drug in the feces, due to either biliary excretion or lack of absorption. The aim of this study was to evaluate the effect of CYP3A inhibition by ketoconazole on sorafenib pharmacokinetics. This was an open-label, non-randomized, 2-period, one-way crossover study in sixteen healthy male subjects. A single 50 mg dose of sorafenib was administered alone (period 1) and in combination with ketoconazole 400 mg once daily (period 2) (ketoconazole was given for 7 days, and a single 50 mg sorafenib dose was administered concomitantly on day 4). No clinically relevant change in pharmacokinetics of sorafenib and no clinically relevant adverse events or laboratory abnormalities were observed in this study upon co-administration of the two drugs. Plasma concentrations of the main CYP3A4 generated metabolite, sorafenib N-oxide, decreased considerably upon ketoconazole co-administration. This effect is in accordance with the in vitro finding that CYP3A4 is the primary enzyme for sorafenib N-oxide formation. Further, these data indicate that blocking sorafenib metabolism by the CYP3A4 pathway will not lead to an increase in sorafenib exposure. This is consistent with data from a clinical mass-balance study that showed 15% of the administered dose was eliminated by glucuronidation, compared to less than 5% eliminated as oxidative metabolites. Since there was no increase in sorafenib exposure following concomitant administration of the highly potent CYP3A4 inhibitor ketoconazole with low dose sorafenib, it is postulated that higher therapeutic doses of sorafenib may be safely co-administered with ketoconazole, as well as with other inhibitors of CYP3A.

Efficient Synthesis of Halohydroxypyridines by Hydroxydeboronation.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, retrorsine, leading to DNA adduct formation in vivo.

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. The high toxicity of many pyrrolizidine alkaloids has caused considerable loss of free-ranging livestock due to liver and pulmonary lesions. Chronic exposure of toxic pyrrolizidine alkaloids to laboratory animals induces cancer. This investigation studies the metabolic activation of retrorsine, a representative naturally occurring tumorigenic pyrrolizidine alkaloid, and shows that a genotoxic mechanism is correlated to the tumorigenicity of retrorsine. Metabolism of retrorsine by liver microsomes of F344 female rats produced two metabolites, 6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), at a rate of 4.8 +/- 0.1 nmol/mg/min, and retrorsine-N-oxide, at a rate of 17.6 +/- 0.5 nmol/mg/min. Metabolism was enhanced 1.7-fold by using liver microsomes prepared from dexamethasone-treated rats. DHP formation was inhibited 77% and retrorsine N-oxide formation was inhibited 29% by troleandomycin, a P450 3A enzyme inhibitor. Metabolism of retrorsine with lung, kidney, and spleen microsomes from dexamethasone-treated rats also generated DHP and the N-oxide derivative. When rat liver microsomal metabolism of retrorsine occurred in the presence of calf thymus DNA, a set of DHP-derived DNA adducts was formed; these adducts were detected and quantified by using a previously developed 32P-postlabeling/HPLC method. These same DNA adducts were also found in liver DNA of rats gavaged with retrorsine. Since DHP-derived DNA adducts are suggested to be potential biomarkers of riddelliine-induced tumorigenicity, our results indicate that (i) similar to the metabolic activation of riddelliine, the mechanism of retrorsine-induced carcinogenicity in rats is also through a genotoxic mechanism involving DHP; and (ii) the set of DHP-derived DNA adducts found in liver DNA of rats gavaged with retrorsine or riddelliine can serve as biomarkers for the tumorigenicity induced by retronecine-type pyrrolizidine alkaloids.

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, monocrotaline, leading to DNA adduct formation in vivo

Monocrotaline is a representative naturally occurring genotoxic pyrrolizidine alkaloid. Metabolism of monocrotaline by liver microsomes of F344 female rats generated (+/−)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5 H-pyrrolizine (DHP) and monocrotaline-N-oxide as major metabolites. Metabolism in the presence of triacetyleandomycin, a P450 3A enzyme inhibitor, reduced the formation of DHP by 52% and monocrotaline N-oxide formation by 59%. Dexamethasone significantly induced microsomal monocrotaline metabolizing enzyme activities in rat liver and lung. Previously, we have identified a set of DHP-derived DNA adducts from DHP-modified calf thymus DNA by 32P-post labeling/HPLC analysis. Metabolism of monocrotaline in the presence of calf thymus DNA resulted in a similar set of DHP–DNA adducts. These DHP–DNA adducts were also found in the liver DNA of rats treated with monocrotaline. The time course of the DHP-derived DNA adduct formation and removal in the liver of rats gavaged with a single dose (10 mg/kg) of monocrotaline was similar to that of rats treated with riddelliine. The levels of DHP–DNA adducts in liver DNA of rats treated with monocrotaline were much lower than that of riddelliine-treated rats. Results from this study indicate that (i) DHP is a common reactive metabolite for retronecine-type of pyrrolizidine alkaloids, (ii) the formation of DHP-derived DNA adducts in the liver DNA of rats treated with monocrotaline suggests that monocrotaline-induced tumorigenicity is through a genotoxic mechanism.