Nick Bushby

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)

In Vitro Hepatic Metabolism of Cediranib, a Potent Vascular Endothelial Growth Factor Tyrosine Kinase Inhibitor: Interspecies Comparison and Human Enzymology

The in vitro metabolism of cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors in late-stage development for the treatment of colorectal cancer and recurrent glioblastoma was investigated in hepatic proteins from preclinical species and humans using radiolabeled material. In human hepatocyte cultures, oxidative and conjugative metabolic pathways were identified, with pyrrolidine N+-glucuronidation being the major route. The primary oxidative pathways were di-and trioxidations and pyrrolidine N-oxidation. All metabolites with the exception of the N+-glucuronide metabolite were observed in rat and cynomolgus monkey hepatocyte preparations. Additional metabolism studies in liver microsomes from these or other preclinical species (CD-1 mouse, Han Wistar rat, Dunkin Hartley guinea pig, Göttingen mini-pig, New Zealand White rabbit, beagle dog, and cynomolgus and rhesus monkey) indicated that the N+-glucuronide metabolite was not formed in these additional species. Incubations with recombinant flavin-containing monooxygenase (FMO) and UDP-glucuronosyltransferase (UGT) enzymes and inhibition studies using the nonselective cytochrome P450 (P450) chemical inhibitor 1-aminobenzotriazole in human hepatocytes indicated that FMO1 and FMO3 contributed to cediranib N-oxidation, whereas UGT1A4 had a major role in cediranib N+-glucuronidation. P450 enzymes had only a minor role in the metabolism of cediranib. In conclusion, species differences in the formation of the N+-glucuronide metabolite of cediranib were observed. All other metabolites of cediranib found in humans were also detected in rat and cynomolgus monkey. Non-P450 enzymes are predominantly involved in the metabolism of cediranib, and this suggests that clinical drug interactions involving other coadministered drugs are unlikely.

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.

The synthesis of tritium, carbon-14 and stable isotope labelled selective estrogen receptor degraders.

As part of a Medicinal Chemistry program aimed at developing an orally bioavailable selective estrogen receptor degrader, a number of tritium, carbon-14, and stable isotope labelled (E)-3-[4-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl]prop-2-enoic acids were required. This paper discusses 5 synthetic approaches to this compound class.

A modern approach to the synthesis of 2-(4-chlorophenyl)[2- 14C]thiazol-4-ylacetic acid ([14C] fenclozic acid) and its acyl glucuronide metabolite

An updated approach to the 1960s synthesis of [(14)C] fenclozic acid from labelled potassium cyanide is presented. By employing modern synthetic methodology and purification techniques, many of the inherent hazards in the original synthesis are avoided or significantly reduced. The concomitant labelled stereoselective synthesis of the key acyl glucuronide metabolite (the 1-β-O-acyl glucuronide) is also described.

Isotope Labelling by Reduction of Nitriles: Application to the Synthesis of Isotopologues of Tolmetin and Celecoxib

The aryl methyl group is found in many drug-like compounds, but there are limited ways of preparing compounds with an isotope label in this methyl position. The process of cyanation of an aryl halide followed by complete reduction of the nitrile to a methyl group was investigated as a route for preparing stable and radiolabelled isotopologues of drug-like compounds. Using this methodology, carbon-13, deuterium, carbon-14, and tritium labelled isotopologues of the nonsteroidal anti-inflammatory drug tolmetin were produced, as well as carbon-13, deuterium, and carbon-14 labelled isotopologues of another nonsteroidal anti-inflammatory drug, celecoxib. The radiolabelled compounds were produced at high specific activity and the stable isotope labelled compounds with high incorporation making them suitable for use as internal standards in mass spectrometry assays. This approach provides a common synthetic route to multiple isotopologues of compounds using inexpensive and readily available labelled starting materials.

[14C]‐AZD1152 drug substance manufacture: challenges of an IV‐infusion dosed human mass balance study in patients

[(14) C]-AZD1152 (barasertib) drug substance was prepared in order to support a hADME study that was to be dosed as an intravenous infusion to patients with acute myeloid leukaemia. A long patient recruitment time (1-2 years) was expected, and because of its limited stability several batches of [(14) C]-AZD1152 drug substance needed to be prepared in order to maintain a supply of [(14) C]-AZD1152 in the clinic over this period. A method was developed to purify the [(14) C]-AZD1152 to a GMP-like standard at high specific activity to provide material for each of these batches. This approach to the delivery of the drug substance was successful in supplying the study with radiolabelled drug for over 1 year until all patients had been recruited.

Synthesis of [2H6]ceftazidime as a stable isotopically labeled internal standard

Ceftazidime is a third generation cephalosporin antibiotic that has activity against a wide range of Gram-negative and Gram-positive bacterial pathogens, including Escherichia coli and Pseudomonas aeruginosa. Stable isotope-labeled ceftazidime was required for use as an internal standard in LC-MS/MS assays, and a route was developed to make [(2)H6]ceftazidime in eight steps from the commercially available labeled starting material [(2)H7]isobutyric acid.