Zur Biogenese der Betacyane: Versuche mit [2-14C]-Dopaxanthin

On the biogenesis of betacyanins. Expaiments with [2-¹⁴C]-dopaxanthine Labelled dopaxanthine is prepared from betanin by a double exchange procedure replacing first its cyclodopa part by diethylamine and effecting the second exchange with 2-[¹⁴C]-dopa on the purified intermediate ‘DEA-betalain’; the specific activity delivered with the dopa allowed accurate determination of ? in the electronic spectrum of dopaxanthine (λ_max 488 nm, ? = 41.800). The C(2)-labelled dopaxanthine is incubated into the fruits of Opuntia bergeriana. The incorporation rates into betanin are found to correspond approximately to those obtained in earlier experiments with labelled dopa alone: Only 1--5% of the label showed up in the cyclodopa moiety of the betanin (which is isolated as cyclodopa-glucoside) whereas 99--95% of the radioactivity was associated with the betalamic acid part (recovered in form of indicaxanthine).     

Radiolabelled peptide ergot alkaloids.

Paspalic acid ( 11 ), labelled at different positions with ³H or ¹⁴C and at specific activities up to 1 Ci/mmol (³H) and 2mCi/mmol (¹⁴C), has been prepared biosynthetically in a scale of 2–5 mmol in submerged cultures of a selected strain of Claviceps paspali by incorporation of DL -[5-³H]-tryptophan, DL -[6-³H]-tryptophan, DL -[alanine-2,3-³H]-tryptophan, or DL -[alanine-3-¹⁴C]-tryptophan. Radioactive lysergic acid ( 12 ) was obtained from paspalic acid by base catalysed rearrangement. The procedures for labelling the precursors at high specific activity arc described. The ergolene carboxylic acids 11 and 12 were used as key intermediates for tlie chemical synthesis of radiolabelled peptide ergot alkaloids 14 required for pharinacokinetic and metabolic studies. Linking of the aminocyclols 13 (peptide parts) of the ergotarnine (R¹ = methyl), ergoxine (R¹ = ethyl) and ergotoxine (R¹ =isopropyl) series with a reactive derivative of either lysergic acid or its mixture with paspalic acid was accomplished by standard procedures. l-Mctliyl-[13-³H]-ergotarnine (MY 25) ( 16 ) and 2-brotno-α-ergocryptine (bromocriptine, C73 154) ( 17 ), labelled with ³H at position 12 and with ¹⁴C in position 4, were obtained by alkylating ³H-ergolamine and by ljrominating appropriately labelled α-ergocryptines. Radioactive peptide ergot alltaloitls labelled by tlic present nietliotl proved suitable for the use in biological tracer studies.     

Biosynthesis of cytochalasans. Part 4. The mode of incorporation of common naturally-occurring carboxylic acids into cytochalasin D1.

Utilization of sodium [1-¹⁴C]-, [2–¹⁴C]-, and [1,2-¹³C]-acetates, [1-¹⁴C]-, [1-¹³C]-, or [2-¹⁴C]-propionates, [1-¹⁴C]-or [2-¹⁴C]-malonates, of [1-¹⁴C]- or of [1-¹⁴C]-myristic acid, or of [1-¹⁴C]- and [1-¹⁴C]-palmitic acid in the biosynthesis of cytochalasin D ( 1 ) by Zygosporium masonii was determined by degradation studies or by carbon magnetic resonance spectroscopy. The precursors were incorporated primarily via the acetate-malonate pathway to generate 1 from nine intact acetate units, eight of which are coupled in a head to tail fashion to form the C₁₆-polyketide moiety.     

Biosynthesis of the Verrucarins and Roridins. Part 1. The transformation of mevalonic acid into verrucarinic acid. Evidence for a hydrogen 1,2-shift. Verrucarins and roridins, 27th communication

Incorporation experiments using sodium [2-¹⁴C]-, [2-³H]-, (3R)-[5-¹⁴C]- and [2-³H, 2-¹⁴C]-mevalonates and with mevalonates stereospecifically tritiated at C(2) demonstrate the transformation of mevalonic acid ( 8 ) into verrucarinic acid ( 5 ). Degradation experiments showed that this transformation occurs with a hydrogen 1, 2-shift of the ‘pro-2R’ hydrogen atom of mevalonate to C(3) of verrucarinate. A possible mechanistic pathway is discussed.     

The biosynthesis of the alkaloids of croton sparsiflorus morong

Incorporation of tyrosine, dopa, dopamine, 4-hydroxyphenylpyruvic acid, (±)-, norcoclaurine-1-carboxylic acid, -norcoclaurine, -coclaurine, and -N-methylcoclaurine into N-methylcrotsparine, N-methylcrotsparinine and N-methylsparsiflorine in Croton sparsiflorus Morong has been studied. The evidence supports the direct oxidative coupling of (+)-, and (-)-N-methylcoclaurines to give N-methylcrotsparine and N-methylcrotsparinine respectively. Tracer experiment show that N-methylcrotsparine undergoes dienone-phenol rearrangement to give N-methylsparsiflorine. A double labelling experiment with (±)-N[¹⁴C]methyl[1-³H]coclaurine demonstrated that the H atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion. The intermediacy of norcoclaurine-1-carboxylic acid and specific incorporation of dehydro-N-methylcoclaurinium salt into the bases have been demonstrated.     

A rapid microwave induced synthesis of [carboxyl-14C]-nicotinic acid (vitamin B3) and [carbonyl-14C]-nicotinamide using K14CN

Microwave assisted direct aromatic substitution of 3-bromopyridine with K¹⁴CN as the cyanide source and catalytic amount of tetrabutylammonium bromide afforded [3-¹⁴C]-cyanopyridine 3 in 90% yield. Microwave assisted hydrolysis of 3 with a mixture of concentrated hydrochloric acid and propionic acid afforded [carboxyl-¹⁴C]-nicotinic acid in 95% yield whereas microwave assisted hydrolysis of 3 with a mixture of concentrated sulfuric acid and propionic acid afforded [carbonyl-¹⁴C]-nicotinamide in 85% yield.     

Einbauversuche mit (3H und 14C)-doppelmarkiertem Farnesol in Cantharidin. 5. Mitteilung zur Biosynthese des Cantharidins

Incorporation experiments with (³H and ¹⁴C) doubly labelled farnesols into cantharidin After injection of 11′, 12-[³H]-7-[¹⁴C]-farnesol or 11′, 12-[³H]-5,6-[¹⁴C]-farnesol, the ³H-label is located specifically in the C(9)-methyl-group of cantharidin, whereas the ¹⁴C-labelling pattern follows an incorporation via acetic acid (Scheme 4). C-Atoms 5, 6 and 7 from the middle part of the farnesol molecule are utilized for cantharidin biosynthesis to an extent that is about 2.1–11% of the incorporation rate of the methyl groups C(11′) and C(12), depending on the position of the ¹⁴C-label in farnesol. These results confirm our earlier hypothesis [1] that the C₁₀-molecule cantharidin is biosynthesized from the C₁₅-precursor farnesol which is cleaved between C(1)–C(2), C(4)–C(5), and C(7)–C(8). The synthesis of 7-[¹⁴C]-farnesol and of 5,6-[¹⁴C]-farnesol is described.     

On the incorporation of geraniol and farnesol into cantharidin (author's transl)

On the incorporation of geraniol and farnesol into cantharidin Earlier investigations [1] have shown that cantharidin (1) is biosynthesized by the male Lytta vesicatoria L. (Meloidae, Coleoptera) from the common terpenoid precursors mevalonate and farnesol (3) . To prove if geraniol (2) is incorporated via farnesol (3) into cantharidin (1) the following geraniols have been synthesized and injected into either larvae or male adult Lytta vesicatoria, partly in a mixture with synthetic 11′, 12-[³H]-farnesol as an internal standard: 2-[¹⁴C]-, 7-[¹⁴C]-, 7′, 8-[¹⁴C]-, 7′, 8-[³H]-geraniol. Unexpectedly, geraniol (2) was not specifically incorporated into cantharidin (1) perhaps due to its higher toxicity or its faster degradation relative to the other precursors before incorporation. The incorporation of U-[¹⁴C]-leucine, U-[¹⁴C]-isoleucine and 1-[¹⁴C]-glucose into cantharidin (1) via their metabolites is evident by degradation studies, whereas 1-[¹⁴C]- and 2-[¹⁴C]-glycine do not serve as precursors for cantharidin (1) .     

Regioselectively [15NO2]-labeled N-methoxypicramide and DPPH prepared by using crown ether and solid sodium [15N]nitrite

The present paper reports the regioselective [¹⁵NO₂]-labeling of N-methoxy-2,4,6-trinitroaniline and 2,2-diphenyl-1-picrylhydrazine (reduced DPPH). Starting from N-methoxy-2,6-dinitroaniline, or N-methoxy-2,4-dinitroaniline, nitration in methylene chloride with solid sodium [¹⁵N]nitrite and 15-crown-5-ether afforded N-methoxy-2,6-dinitro-4-[¹⁵N]nitroaniline and N-methoxy-2,4-dinitro-6[¹⁵N]nitroaniline, respectively. The same compounds could be prepared in higher purity by nitrodecarboxylation (ipso-substitution) under the same conditions starting from N-methoxy-4-carboxy-2,6-dinitroaniline (4-methoxyamino-3,5-dinitrobenzoic acid) and N-methoxy-2-carboxy-4,6-dinitroaniline (2-methoxyamino-3,5-dinitrobenzoic acid). Similarly,ipso-substitution of 2,2-diphenyl-1-(4-carboxy-2,6-dinitrophenyl)-hydrazine afforded, under the same reaction conditions, 2,2-diphenyl-1-(2,6-dinitro-4-[¹⁵N]nitrophenyl)-hydrazine. By¹H-NMR and¹³C-NMR it was also observed that under these reaction conditions a¹⁴NO₂ group can be replaced by a¹⁵NO₂ group.     

Saturated heterocycles. 254 . synthesis and stereochemistry of saturated or partially saturated pyridazino-[6,1-b]- and phthalazino[1,2-b]quinazolinones

By the reaction of anthranilic hydrazide 1 with cis-2-(p-methylbenzoyl)-1-cyclohexanecarboxylic acid 2a or diendo-3-(p-methylbenzoyl)bicyclo[2.2.1]heptane-2-carboxylic acid 2b , fused tetra- and pentacyclic ring systems 3a, b were prepared, trans-2-Amino-1-cyclohexanecar-bohydrazide 4b was reacted with 3-(p-chlorobenzoyl)propionic acid 5 to yield the pyridazino[6,1-b]quinazolinone 6 . From the reaction of cis-2-amino-1-cyclohexanecarbohydrazide 4a with 2a , three isomeric partially saturated 8H-phthalazino[1,2-b]quinazolin-8-ones 7a-c were formed. The reaction of diexo-2-aminobicyclo[2.2.1]heptane-3-carbohydrazide 4c and 2a furnished the pentacyclic derivatives 8 and 9 containing a 3-aryl-4,5-dihydropyridazine or 3-arylhexahydropyridazine ring C with cis annelated C/D rings. The formation of 8 and 9 involving different ring systems can be rationalized by two reaction pathways: (i) in the bislactam 9 the carboxyl group acylates the hydrazide, while (ii) in 8 it forms a pyridazine ring with the cyclic amino group by cyclocondensation. The structures of the products were elucidated by ¹H and ¹³C nmr methods, including DEPT, DNOE and 2D-HSC measurements.     

Use of Dibutyl[14C]formamide as a Formylating Reagent in the Vilsmeier–Haack Reaction and Synthesis of a 14C‐Labeled Novel Phosphodiesterase‐4 (PDE‐4) Inhibitor

A simple, high‐yielding synthesis of dibutyl[¹⁴C]formamide ([¹⁴C]DBF; 1 ) from ¹⁴CO₂ was developed (Scheme 1): reaction of LiBEt₃H and ¹⁴CO₂ followed by aqueous workup gave H¹⁴CO₂H in high yield. Conversion of the [¹⁴C]formic acid to 1 was effected by a standard carbodiimide coupling procedure. The utility of 1 as an alternative to dimethyl[¹⁴C]formamide ([¹⁴C]DMF) in alkylation reactions and in the [¹⁴C]Vilsmeier–Haack reaction was demonstrated for several substrates (Table 2). A ¹⁴C‐labeled phosphodiesterase‐4 (PDE‐4) inhibitor, [¹⁴C]‐ 2 , was synthesized by application of this technology (Scheme 2).     

Biosynthesis of verrucarins and roridins. 2. Incorporation of acetate in cis-transmuconic acid and of mevalonate in roridinic acid]

Incorporation experiments using sodium [1-¹⁴C]-acetate and sodium [2-¹⁴C]- and [2-¹⁴C,2-³H₂]-mevalonate and degradations of the verrucarin A ( 1 ) and roridin A ( 2 ) so produced demonstrate that cis,trans-muconic acid ( 3 ) is formed from 3 acetate units. The cis,trans-muconic acid and the C₂-side-chain structural elements of roridinic acid ( 6 ) are built up from 4 acetate units; the cis-oricntcd C(11)-carboxyl group of 6 originates from C(1) of acctic acid. The structural moiety of roridinic acid ( 6 ) corresponding to verrucarinic, acid ( 7 ) originates from mevalonate, as does 7 . A new degradation scheme was devised for roridinic acid ( 6 ); the oxime 23 of its 13 dehydro-detrahydro derivative 21 underwent cleavage with SOCl₂ and subsequent hydrolysis to yield verrucarinolactone ( 8 ), acetonitrile ( 26 ) and methyl adipaldohydate ( 27 ) by a heterolytic Beckmann fragmentation reaction.     

Synthesis of 24-Methylidene[24-14C]- and 24-Methylidene[7-3H]cholesterol

The syntheses of 24-methylidene[24-¹⁴C]cholesterol ( 7a ) and of 24-methylidene[7-³H]cholesterol ( 7b ) from commercially available (20S)-3-oxopregn-4-ene-20-carbaldehyde ( 1 ) are described. The method also provides simple preparations of 3β-acetoxy[24-¹⁴C]chol-5-en-24-oic acid ( 4 ) and 24-oxocholest-5-en-3β-yl acetate ( 6b ).     

Zur Biosynthese der Rubratoxine

In order to check the hypothesis that rubratoxin B ( 2 ), a C₂₆-metabolite, is formed biogenetically by head-to-tail coupling of two identical C₁₃-precursors derived from decanoic acid and oxaloacetic acid, two labelled forms of the postulated C₁₃-intermediate 2-((E)-1'-octenyl)-3-[¹⁴C]methyl- and 2-((E)-1'-octenyl)-3-[¹³C]-methylmaleic anhydride ( 10 ), were synthesized. The labelled compounds 10 as well as a number of other ¹⁴C]- and [¹³C]-labelled potential precursors were administered to growing cultures of Penicillium rubrum STOLL . Significant incorporation rates of acetate (as intact units) and malonate were observed. Propionate was incorporated after decarboxylation. Succinate exhibited the highest rate of incorporation. The results are in agreement with the assumption that the C₁₀-chain is formed by the fatty acid pathway and the C₃-unit via the tricarboxylic acid cycle. After administration of 10 randomization of the label was observed. Thus the question whether compound 10 is a biogenetic intermediate remains unanswered.     

On the biosynthesis of dendrolasin, a body constituent of the ant Lasius fuliginosus Latr

By feeding the ant Lasius fuliginosus LATR . with [¹⁴C]-1-acetate, [¹⁴C]-2-acetate, [¹⁴C]-2-mevalonate, [¹⁴C]-2-mevalonate, [¹⁴C]-1-glucose and [¹⁴C]-U-glucose, incorporation ratios of 10^?4 – 0,15% were obtained in the sesquiterpenoid dendrolasin. It was shown by analysis of the labelling pattern in dendrolasin that the insertions were spread over the whole molecule in exactly the manner that would be expected from terpene biosynthesis.     

Angiotensin-II Analogues. I: Synthesis and incorporation of the halogenated amino acids 3-(4′-iodophenyl)alanine, 3-(3′,5′-dibromo-4′-chlorophenyl)alanine, 3-(3′,4′,5′-tribromophenyl)alanine,and 3-(2′,3′,4′,5′,6′-pentabromophenyl)alanine

The synthesis of the polyhalogenated phenylalanines Phe(3′,4′,5′-Br₃) ( 3 ), Phe(3′,5′-Br₂-4′-Cl) ( 4 ) and DL -Phe (2′,3′,4′,5′,6′-Br₅) ( 9 ) is described. The trihalogenated phenylalanines 3 and 4 are obtained stereospecifically from Phe(4′-NH₂) by electrophilic bromination followed by Sandmeyer reaction. The most hydrophobic amino acid 9 is synthesized from pentabromobenzyl bromide and a glycine analogue by phase-transfer catalysis. With the amino acids 4, 9 , Phe(4′-I) and D -Phe, analogues of [1-sarcosin]angiotensin II ([Sar¹]AT) are produced for structure-activity studies and tritium incorporation. The diastereomeric pentabromo peptides L - and D - 13 are separated by HPLC. and identified by catalytic dehalogenation and comparison to [Sar¹]AT ( 10 ) and [Sar¹, D -Phe⁸]AT ( 14 ).     

Novel Synthesis of Agaritine,a 4-Hydrazinobenzyl-Alcohol Derivative Occurring in Agaricaceae

The 4-hydrazinobenzyl alcohol ( 3 was prepared (58%)) by diiobutylaluminiumhydride reduction of methyl 4-hydrazinobenzoate ( 4 ), whereas LiA1H₄ or LiBh₄ reduction of 4 proceeded further to yield (via intermediate 3 ) (4-tolyl)hydrazine ( 5 ). The alcohol 3 was stable under O₂-free conditions and exhibited no tendency to eliminate H₂O, neither thermally nor with H⁺ catalysis. Oxidation of 3 with SeO₂ yielded 4-(hydroxymethyl)benzine-diazonium ion ( 8 ), identified by its azo coupling product 9 with 2-naphthol. Condensation of 3 with 1-benzyl 5-Hydrogen N-(benzyloxycarbonyl)-L-glutamate ( 10 ) in presence of dicyclohexylcarbodiimide afforded 81% of N²-(benzyloxycarbonyl)-L- glutamic acid 1-(benzyl-ester) 5-{2-[4-(hydroxymethyl)phenyl]hydrazide} ( 11 ) which upon controlled hydrogenolysis (quinoline-sulfur-poisoned Pd/C catalyst) gave 82% of L-Glutamic acid 5-{2-[4-(hydroxymethyl)phenyl] hydrazide} ( 1 ), i. e. agaritine, a metabolite of Agaricus bisporus. Without poisoning of the catalyst, hydrogenolysis of ( 11 ) yielded L-glutamic acid 5-[2-(4-tolyl)hydrazide] ( 12 ).     

A convenient method for <Superscript>14</Superscript>C-labeling of <Emphasis Type="Italic">N</Emphasis>-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-1H-pyrrole-2-carboxamide-[carboxy-<Superscript>14</Superscript>C] as CCK-A antagonist

N-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-1H-pyrrole-2-carboxamide-[carboxy-¹⁴C] was prepared as part of a five-step sequence from pyrrol-2-carbonitrile-[cyano-¹⁴C] as a key synthetic intermediate which has been synthesized from 2-bromopyrrole and zinc [¹⁴C]-cyanide in the presence of tetrakis (triphenylphosphine)palladium.     

Zur Biosynthese des Strychnins. 135. Mitteilung über Alkaloide

From the feeding of young plants of Strychnos nux-vomica with [¹⁴C]-1-and [¹⁴C]-2-acetate it could be deduced that the C-atoms 22 and 23 were derived from acetate. [¹⁴C]-2-mevalonate, [¹⁴C]-2-geraniol and [¹⁴C]-2-geranyl pyrophosphate were also incorporated into strychnine. The distribution of radioactivity in the «mevalonate-strychnine» was in agreement with the monoterpenoid hypothesis. Feeding experiments especially with [¹⁴C]-tryptophane showed that the main production centre of the alkaloid lay in the roots and that only a small part of it was carried to the leaves. Tritium labelled WIELAND GUMLICH aldehyde as well as N_(a)-[¹⁴C]-1-acetyl WIELAND GUMLICH aldehyde were not converted into strychnine by S. nux-vomica.     

Facile syntheses of isotope-labeled chiral octahydroindole-2-carboxylic acid and its <Emphasis Type="Italic">N</Emphasis>-methyl analog

We have synthesized deuterium and carbon-14 labeled enantiomerically pure octahydroindole-2-carboxylic acid (PD0140417), N-methyl octahydroindole-2-carboxylic acid (PD0348183) and their racemic analogs (PD0108405 and PD0338055). [ring-U-¹⁴C]PD0140417 was prepared from [ring-U-¹⁴C]benzoic acid in a seven-step synthesis in 6.2% overall radiochemical yield. [¹⁴C]PD0348183 was prepared from [¹⁴C]BaCO₃ in a five-step synthesis in 16% radiochemical yield. Additionally, [D]PD0108405 and [D]PD0338055 were synthesized by direct platinum-catalyzed hydrogenation with deuterium gas.