John R. Harding

Synthesis of [1,2-13C2, 15N]-L-homoserine and its incorporation by the PKS-NRPS system of Fusarium moniliforme into the mycotoxin fusarin C.

The fusarins, for example, fusarin C (1) are toxic metabolites isolated from a number of fungi including Fusarium moniliforme and Fusarium venenatum. They are representative of a wider class of substituted 2-pyrrolidinone metabolites that have been isolated from a number of micro-organisms, for example, fuligorubin A (2) from the slime mould Fuligo septica and pramanicin (3) and equisetin (4) from terrestrial fungi Staganospora sp. and Fusarium heterosporum, respectively. In addition 2pyridones such as tenellin (5 ; Beauveria bassiana) are probably biosynthesised from the related 2-pyrrolidinones, such as militarinone (6 ; Paecilomyces militaris) by ring expansion. Feeding C-labelled acetates and methionine to cultures of F. moniliforme confirmed the formation of the fusarins via a reduced and highly methylated heptaketide intermediate, with the remainder of the carbon skeleton (carbons 14, 15, 18 and 19) being derived from a C4 Krebs cycle-derived intermediate. It was suggested that oxaloacetate could be the immediate precursor of these carbon atoms. This biosynthetic hypothesis would require an aminotransferase step to provide the required nitrogen atom. More recently, a parallel hypothesis emerged when it was shown that a 12 kb open reading frame (ORF), isolated from the genomes of F. venenatum and F. moniliforme, was involved in the biosynthesis of fusarin C. This ORF was shown to encode an unusual polyketide synthase (PKS) fused to a non-ribosomal peptide synthetase (NRPS) module (Figure 1) to form a megasynthetase know as FUSS (fusarin synthetase). Such PKS-NRPS systems are now known to be a rather common feature in fungi. In the majority of genome sequences, however, the products of these PKS-NRPS systems are not known, although a similar system has been shown to be responsible for equisetin biosynthesis. Studies of bacterial NRPS modules have shown that analysis of signature sequences of noncontiguous residues in the binding pocket of the adenylation (A) domain allows prediction of the amino acid substrate. This analysis has not been extended to fungal NRPS A domains because the signature sequences are very poorly conserved. Thus, no inference about the substrate selectivity of the FUSS A domain can be made. Therefore it is conceivable that the substrate for the FUSS A domain could be either an intact amino acid or an amino acid precursor, such as oxaloacetate, that would require later transamination. In the case of fusarin C, however, structural analysis indicates that if the A-domain substrate were an amino acid, it would be homoserine. In order to discriminate between these possibilities, we report herein an efficient route for the enantioselective synthesis of [1,2-C2, N]-l-homoserine and its use in feeding experiments in cultures of F. moniliforme to determine the origin of the nitrogen atom and carbons 14, 15, 18 and 19 in fusarin C. Triply labelled [1,2-C2, N]-l-homoserine (15) was ideally ACHTUNGTRENNUNGrequired for the feeding study as it could be used not only to establish if homoserine (14) is indeed a biosynthetic precursor of fusarin C but also to investigate if incorporation occurred intact or by degradation. A number of approaches have been described for the preparation of amino acids incorporating this labelling pattern, for example, with either glycinate equivalents or chiral auxiliaries. Recently a chiral phase-transfer catalyst has been used to prepare protected homoserine in 90% ee, and this strategy may be adapted for the introduction of isotopic labels. However, to the best of our knowledge the synthesis of [1,2-C2, N]-l-homoserine has not been reported. It was envisaged that the target could be prepared readily from protected [1,2-C2, N]-l-allylglycine, which in turn was to be accessed from commercially available [1,2-C2, N]-glycine. We [a] D. O. Rees, Dr. R. J. Cox, Prof. Dr. T. J. Simpson, Prof. Dr. C. L. Willis School of Chemistry, University of Bristol Cantock’s Close, Bristol, BS8 1TS (UK) Fax: (+44)117-9298611 E-mail : chris.willis@bristol.ac.uk [b] Dr. N. Bushby, Dr. J. R. Harding AstraZeneca UK Ltd, Mereside Alderley Park, Macclesfield, SK10 4TG (UK)

Identification of the Thiophene Ring of Methapyrilene as a Novel Bioactivation-Dependent Hepatic Toxicophore

Methapyrilene (MP), a 2-thiophene H1-receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse ≈ human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [3H]MP intravenously. MP produced concentration- and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes.

Development of a microwave-enhanced isotopic labelling procedure based on the Eschweiler–Clarke methylation reaction

A number of primary and secondary amines have been rapidly methylated under microwave-enhanced conditions using formic acid-formaldehyde mixtures, providing a route to 2H(D)-containing compounds and the potential for 3H(T), 11C, 13C and 14C labelling.

Glucuronidation of steroidal alcohols using iodosugar and imidate donors

We report a study of the glucuronidation of a number of important steroidal secondary alcohols. The alcohols studied are androsterone 7, epiandrosterone 8, 17-acetoxy-androstane-3alpha,17beta-diol 9, 11alpha-hydroxyprogesterone 10, and 3-benzoylestradiol 11. These were first glucuronidated using the Schmidt trichloroacetimidate method with variations in acyl substituent (viz. derivatives 2 and 3), Lewis acid catalyst and order of addition. The results are contrasted with those obtained using our recently described glycosyl iodide donor 4, catalysed either by N-iodosuccinimide (NIS) or various metal salts.

Glucuronide and sulfate conjugates of ICI 182,780, a pure anti-estrogenic steroid. Order of addition, catalysis and substitution effects in glucuronidation

Abstract The 3-sulfate 4 and 3- and 17-glucuronide conjugates 5 and 6 of the pure anti-estrogenic steroid ICI 182,780 1 , which is expected to be an effective agent for the treatment of breast cancer, have been prepared. The synthesis of 6 could only be satisfactorily achieved using an inverse addition technique, not previously employed in the glucuronic acid series: the value of this technique for some other aglycones is discussed.

Integrated HPLC-MS and 1H-NMR spectroscopic studies on acyl migration reaction kinetics of model drug ester glucuronides

Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-β-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an α,α-dimethylated ibuprofen analogue. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37°C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and 1H-NMR spectroscopy and the results compared. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, 1H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of 1H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than 1H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-β-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

Stereocontrolled synthesis of 2,4,5-trisubstituted tetrahydropyrans

Cyclisation of homoallylic acetals under acidic conditions leads to the formation of 2,4,5-trisubstituted tetrahydropyrans with the creation of two new asymmetric centres with excellent stereocontrol. By varying the acid and the nucleophile, the reaction may be adapted for the preparation of 2,4,5-trisubstituted tetrahydropyrans with either a halide, alcohol, acetate or amide at C-4.

High-performance liquid chromatography/mass spectrometric and proton nuclear magnetic resonance spectroscopic studies of the transacylation and hydrolysis of the acyl glucuronides of a series of phenylacetic acids in buffer and human plasma

The use of high-performance liquid chromatography/mass spectrometry (HPLC/MS) and proton nuclear magnetic resonance ((1)H NMR) spectroscopy for the kinetic analysis of acyl glucuronide (AG) isomerisation and hydrolysis of the 1-β-O-acyl glucuronides (1-β-O-AG) of phenylacetic acid, (R)- and (S)-α-methylphenylacetic acid and α,α-dimethylphenylacetic acid is described and compared. Each AG was incubated in both aqueous buffer, at pH 7.4, and control human plasma at 37°C. Aliquots of these incubations, taken throughout the reaction time-course, were analysed by HPLC/MS and (1)H NMR spectroscopy. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the calculated rates of reaction were much faster than for buffer and, in contrast to the observations in buffer, hydrolysis to the free aglycone was a significant contributor to the overall reaction.A diagnostic analytical methodology based on differential mass spectrometric fragmentation of 1-β-O-AGs compared to the 2-, 3- and 4-positional isomers, which enables selective determination of the former, was confirmed and applied. These findings show that HPLC/MS offers a viable alternative to the more commonly used NMR spectroscopic approach for the determination of the transacylation and hydrolysis reactions of these AGs, with the major advantage of having the capability to do so in a complex biological matrix such as plasma.

Microwave-enhanced hydrogen isotope exchange studies of heterocyclic compounds

The microwave-enhanced hydrogen–deuterium exchange reaction of a number of nitrogen-containing heterocyclic compounds has been achieved extremely rapidly (<20 min) via their hydrochloride salts, whilst the corresponding Raney-nickel catalysed reactions of indole and N-methylindole are also complete in a similar time interval. Furthermore, the pattern of labelling in the latter reaction depends critically on the choice of solvent used.

Probing the mechanism of Prins cyclisations and application to the synthesis of 4-hydroxytetrahydropyrans

Trapping intermediates on the Prins cyclisation pathway with carbon-based nucleophiles has given further insight into factors affecting the acid-mediated reactions of homoallylic alcohols with aldehydes, enabling the design of efficient syntheses of 4-hydroxy-2,6-disubstituted tetrahydropyrans.