Nature can serve as an excellent source of novel biologically active compounds. Unfortunately, a large number of known active compounds are often encountered during the search for novel chemistries. Therefore, early stage identification, and determination of novelty is of critical concern to natural product chemists. We have designed a screening process utilizing a single quadrupole LC/MS system to efficiently distinguish novel compounds from known compounds in crude biologically active extracts. This "dereplication" process is accomplished by chromatographing an extract on a LC column, diverting 95% of the eluent to a 96 well plate, and directing the remainder of the eluent to the MS for spectral analysis. Bioassay of the 96 well plate contents localizes the biologically active compounds, which are then structurally interrogated using MS, accurate MS, accurate MS/MS, and MSn. These data are often sufficient to allow identification of the active compounds from the initial small scale extract, avoiding costly sample recollection or refermentation. (C) 2003 American Chemical Society.

Two lactating goats were given a daily oral dose of either [UL-aniline-(14)C; AN] or [triazolopyrimidine-7,9-(14)C; TP]cloransulam-methyl for 5 consecutive days. Each animal received a dietary equivalent of approximately 10 mg/kg of test material, approximately 2225 times the realistic maximum dietary exposure for a dairy animal. Milk, urine, and feces samples were collected in the morning and afternoon for each animal. Each goat was sacrificed within 23 h of receiving the last dose, and the liver, kidneys, samples of blood, fat, muscle, and gastrointestinal tract contents, and urine from the bladder were collected. All of these samples were analyzed for (14)C content. Cloransulam-methyl (CM) was rapidly excreted by the animals, with 99.9% of the recovered radioactivity appearing in the urine and feces. Radiochemical analysis showed very low residues, with the highest being in the kidneys at 0.122 and 0. 128 mg equiv of CM/kg (AN and TP labeled compounds, respectively). Radioactive residues were extracted and fractionated from kidney, liver, and milk. Analysis showed approximately 0.066 mg/kg CM in the kidney but <0.003 mg/kg in the liver. Only one metabolite, cloransulam, was identified (in liver, 9.5% of total radioactive residue; 0.005 mg/kg). All other metabolites were present at lower levels. Sulfonanilide bridge cleavage was not a significant degradation route for cloransulam-methyl in ruminants. These data indicated a very low bioaccumulation potential for cloransulam-methyl and its metabolites in ruminants. For a ruminant exposed to anticipated levels of cloransulam-methyl in its diet, parent and metabolites, in total, would not be expected to exceed 50 ng/kg in the kidney and liver.

The leaf uptake, translocation, and metabolism of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) and its ethylene glycol butyl ether and 2,2-dimethyl-1,3-dioxolane-4-methyl (solketal) esters were investigated in a range of representative plant species, in relation to the species-selective phytotoxicity of the herbicide. Plant species used were: wheat (Triticum aestivum, cv Norman, tolerant), barley (Hordeum vulgare, cv Igri, moderately tolerant), and chickweed (Stellaria media, susceptible). Neither uptake nor translocation was found to contribute to the selective action of triclopyr. The hydrolysis of both esters was 94% complete in all species after 3 days, and the half-life of the ethylene glycol butyl ether ester was less than 12 hr in each species. Ester hydrolysis did not, therefore, contribute to selective action. Rates of metabolism of the ester hydrolysis product, triclopyr acid, varied between species and half-lives were qualitatively correlated with tolerance: wheat (12 hr), barley (24 hr), and chickweed (48 hr). Therefore, the rate of metabolism of triclopyr is probably a major contributing factor to the selectivity among these species. Metabolic profiles were similar in wheat and barley and, although no products were definitively identified, the major metabolites had the general properties of saccharide esters of the parent compound. 1-β-d-Glucopyranosyl 3,5,6-trichloro-2-pyridinyloxyacetate (triclopyr glucose ester) was not a metabolite in the cereals. The major metabolic product from chickweed was identified as triclopyr aspartate by combined gas chromatography-mass spectrometry of its dimethyl ester. The significance of these metabolites, in relation to the selective action of triclopyr, is discussed.

The uptake and metabolism of the herbicide triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) by cell suspension cultures of soybean, Glycine max. var. Harcor were investigated in comparison with those of 2,4-D. After 7 days two major metabolites of triclopyr were formed, in ca. 1.5:1 ratio. These were isolated by ion-exchange and C18-high-performance liquid chromatography. Combined gas chromatography-mass spectrometry of their methyl esters identified the metabolites as the aspartate (major) and glutamate (minor) amide conjugates. In contrast, 2,4-D was predominantly metabolized to two more polar products having the properties of glucosyl ethers of hydroxylated 2,4-D. The effect of both incubation period and growth medium upon the behavior of soybean cells with respect to these herbicides was also studied. Cells grown in Miller's medium supplemented with naphthyl-1-acetic acid took up slightly more [14C]triclopyr, but significantly more [14C]2,4-D, than those grown in Gamborg's B5 medium supplemented with 2,4-D. In either medium, active metabolism of absorbed herbicides continued until at least 21 days, with the qualitative nature of products being only substrate dependent.

Three new esters of 2-(indol-3-yl)ethanol (tryptophol) containing the acyl residues of dehydrocrepenynic (octadeca-9Z,14Z-dien-12-ynoic), oleic and linoleic acids have been identified in fruiting bodies of the Basidiomycete Craterellus cornucopioides supplied with exogenous tryptophol. The esters were formed reversibly in high yields and are possible storage forms of auxin precursors which may be deposited in lipophilic cell compartments.

The mode of action of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid), a broadleaf herbicide, is unknown. No data concerning the fate of triclopyr in plants have yet been published. This communication summarizes a comparison of the uptake and metabolism of triclopyr and 2,4-D (2,4dichlorophenoxyacetic acid) using cell suspension cultures of soybean, Glycine max, var. Harcor. Soybean cells were maintained in aseptic shake culture, on the following media: Gamborg’s B5 (Gamborg, 1975) supplemented with 2,4-D (2.3 PM), indole-3-acetic acid ( 2 . 9 p ~ ) , kinetin ( 4 . 6 ~ ~ ) and sucrose (2% w/v) at pH 6.5, or Miller’s medium (Miller, 1963) supplemented with naphthylI-acetic acid (1 1 PM), kinetin ( 2 . 6 ~ ~ ) and sucrose (2% w/v) at pH 5.8. Cells were subcultured (10ml into 40ml) at 14-16 day intervals. Seven days after subculturing, [2,6-’4CZ]triclopyr (0.82 pCi, 15.6 mCi/mmol) or 2,4-dichlorophenoxy[2-’4C]acetic acid (0.85 pCi, 13.4mCi/mmol, diluted from 55mCi/mmol, Amersham) was added in 50% aq. ethanol. After 6 h, 3 days, 7 days and 21 days, cultures were filtered under vacuum through nylon mesh. Cells were ground (liquid N,) and extracted (80% methanol, 3 x 15 ml), cell debris was sedimented by centrifugation and the supernatant solutions were taken. The I4C recovered from each culture (cells and medium) was quantified by liquid scintillation counting. Cell extracts and media were examined by t.1.c. Radioactive zones were located by radiography, cut from t.1.c. plates, suspended in scintillant and quantified by liquid scintillation counting. All incubations were performed in duplicate. In Miller’s medium: (i) 2,4-D was taken up slightly faster than triclopyr. (ii) Metabolites of both herbicides were largely retained in the cells, except the 2,4-D metabolites nos. 1 1 and 12 (see Fig. l), up to 30% of which appeared in the medium after 21 days. (iii) Metabolism of both herbicides continued until 21 days when no 2,4-D, and ca. 10% triclopyr, remained. (iv) Triclopyr was metabolized to two major compounds (Fig. l), having the following properties: (a) ion-exchange chromatographic behaviour and derivatization by diazomethane showed these metabolites to be acids; (b) acid hydrolysis (2 M-HCl, 80°C, 6 h) yielded triclopyr as sole product; (c) /-glucosidase treatment (citrate phosphate buffer, pH 4.5, 37”C, 18 h) left both metabolites unchanged; ( d ) the metabolites co-eluted on both silica gel t.1.c. and C,,-reverse phase h.p.1.c. with authentic triclopyraspartate (major) and triclopyr-glutamate (minor). Under the same conditions as above, 2,4-D was metabolized to several compounds (Fig. 1). By their chromatographic properties, the products of acid and P-glucosidase treatments, comparison with the triclopyr metabolites and standards described above, and precedents in the literature (Mumma & Hamilton, 1978), these metabolites were probably 2,4-D-glutamate (no. 8), 2,4-D-aspartate (no. 9), 4OH-2,5-D-O-glucoside (no. 10) and 4-OH-2,3-D-0glucoside (no. 11). The behaviour of cells grown on B5 differed significantly from that of cells grown on Miller’s medium in the following respects: (i) B5 cells rapidly released unchanged 2,4-D, but not triclopyr, after initial herbicide uptake. At 3 days and

An improved synthesis of 7-hydroxy-2-oxoindolin-3-ylacetic acid via the base-induced condensation reaction between oxalate esters and 7-benzyloxyindolin-2-one is described. 7-Benzyloxyindolin-2-one was prepared in four steps and 50% overall yield from 3-hydroxy-2-nitrotoluene. The yield of the title compound from 7-benzyloxyindolin-2-one was 56%. This route was used to prepare 7-hydroxy-2-oxoindolin-3-yl[13C2]acetic acid in 30% yield from [13C2]oxalic acid dihydrate. The method could not be extended to the preparation of the corresponding [14C2]-compound. However, an enzyme preparation from Zea mays roots catalysed the conversion of carrier-free [5-n-3H]indol-3-ylacetic acid with a specific activity of 16.7 Ci mmol-1 to a mixture of 7-hydroxy-2-oxo[5-n-3H]indolin-3-ylacetic acid and its [5-n-3H]-7-O-glucoside in ca. 3 and 40% radiochemical yield respectively. The glucoside was converted into the 7-hydroxy compound in 80% yield by means of beta-glucosidase.

7-Hydroxy-2-indolinone-3-acetic acid was identified as a catabolite of indole-3-acetic acid in germinating kernels of Zea mays and found to be present in amounts of ca 3.1 nmol/kernel. 7-Hydroxy-2-indolinone-3-acetic acid was shown to be a biosynthetic intermediate between 2-indolinone-3-acetic acid and 7-hydroxy-2-indolinone-3-acetic acid-7'-O-glucoside in both kernels and roots of Zea mays. Further metabolism of 7-hydroxy-2-[5-3H]-indolinone-3-acetic acid-7'-O-glucoside occurred to yield tritiated water plus, as yet, uncharacterized products.

Resuspension cultures of Gibberella fujikuroi, strain GF-1a, were shown to metabolise potassium [3′-13C] mevalonate to 13C-enriched C19-gibberellins, plus 13CO2 (derived from the loss of carbon-20). The formation of [13C]-gibberellins could be observed in vivo using 13C NMR; however that of 13CO2 could not. In contrast, removal of the mycelium and concentration of the filtrate at pH 12 enabled the 13CO2 produced to be observed using 13C NMR. During incubations of H14CO2Na with this fungus, complete conversion to other radioactive products was observed, and the significance of these results in the light of previous work is discussed.

The 13C n.m.r. spectra of eighteen derivatives of ent-kaur-16-en-19-oic acid are recorded and the additivity of substituent effects is investigated. Application of these additive effects to the prediction of the 13C-shifts in new polysubstituted derivatives is assessed.

Die Cyclisierung der Disäure (Ia) zu dem Acetat (III) verläuft über die Zwischenzustände (II) und (Ib).

An n.m.r. study of the reaction of 3-hydroxy-3-methylpentane-1,5-dioic acid (5) with excess of [2H6]-acetic anhydride is described. It has shown that 3-hydroxy-3-methylpentane-1,5-dioic acid anhydride (2), previously described by Scott and Shishido 1 as an intermediate in their synthesis of [3′-13C]mevalonolactone, is formed only transiently, along with 3-acetoxy-3-methylpentane-1,5-dioic acid (6). Both intermediates eventually give 3-acetoxy-3-methylpentane-1,5-dioic acid anhydride (3).To obtain (R,S)-mevalonolactone, sodium borohydride reduction of 3-hydroxy-3-methylpentane-1,5-dioic acid anhydride (2), prepared from the diacid (5) and N,N-dicyclohexylcarbodi-imide, is shown to be better than reduction of 3-acetoxy-3-methylpentane-1,5-dioic acid anhydride (3).

In Gibberella fujikuroi , strain GF-1a, the effect of the sodium salt of compactin on the incorporation of both radiolabelled acetate and mevalonate into gibberellic acid has been investigated. In each case, a concentration of 40 mg/1. caused a significant reduction in the incorporation.