Identification of farnesol as an intermediate in the biosynthesis of cantharidin from mevalonolactone

Identification of farnesol as an intermediate in the biosynthesis of cantharidin from mevalonolactone Simultaneous injection of 2-[¹⁴C]-mevalonolactone (2-[¹⁴C]- 1 ) and (E,E)-11′,12-[³H]-farnesol (11′,12-[³H]- 2 ) into Lytta vesicatoria L . (Coleoptera, Meloidae) yields doubly labelled cantharidin ( 3 ). The remainder of the precursor farnesol, re-isolated from the insects after the incubation period, has incorporated ¹⁴C-radioactivity. The labelling pattern in this farnesol, as determined by two independent degradative reaction sequences, is in agreement with the isoprene rule. Since specific incorporation of farnesol ( 2 ) into cantharidin ( 3 ), and of mevalonolactone ( 1 ) into both, farnesol ( 2 ) and cantharidin ( 3 ) is observed, the sesquiterpene alcohol 2 acts as an intermediate in the biosynthesis of the C₁₀-compound 3 (Scheme 1).     

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.     

Experimente zum kompetitiven Einbau der Stereoisomeren des Farnesols in Cantharidin. 6. Mitteilung zur Biosynthese des Cantharidins

Experiments on the competitive incorporation of farnesol-stereoisomers into cantharidin Farnesol ( 2 ) has been demonstrated to be an efficient precursor for cantharidin ( 1 ), into which it is transformed by elimination of C(1), C(5), C(6), C(7) and C(7′) [1]. The following incorporation experiments with doubly labelled (³H and ¹⁴C) stereoisomers of farnesol present strong evidence that (E,E)- farnesol ((E,E)- 2 ) in fact is the precursor for cantharidin, whereas (2E, 6Z)- 2 and (Z,Z)- 2 are not utilized for the biosynthesis of cantharidin. A possible mechanism for the incorporation of (2Z,6E)-farnesol ((2Z,6E)- 2 ) to an extent of 56,8% relative to (E,E)- 2 is discussed.     

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.     

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.     

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 ).     

The synthesis of novel polycyclic heterocyclic ring systems. 3. Synthesis and NMR spectroscopy of 15-chloro[1]benzo-thieno[2″,3″:3′,4′]naphtho[1′,2′:4,5]thieno[2,3-c]quinoline

The polycyclic heterocyclic compound with a novel ring system, 15-chloro[1]benzothieno[2″,3″:3′,4′]-naphtho[1′,2′:4,5]thieno[2,3-c]quinoline was synthesized via photocyclization of 3-chloro-N-phenyl[1]-benzothieno[2′,3′:3,4]naphtho[2,1-b]fhiophene-2-carboxamide followed by chlorination with phosphorus oxychloride. The assignment of its ¹H and ¹³C nmr spectra was accomplished by utilizing two-dimensional nmr methods.     

Biosynthesis of cytochalasins. II Building blocks of cytochalasun D

A number of potential ¹⁴C- and ³H-labelled precursors were fed to growing cultures of Zygosporium masonii after the rate of formation of cytochalasin D had been measured. By chemical degradation of [¹⁴C]- and [³H, ¹⁴C]-cytochalasin D the distribution of the radioactivity originating from incorporated [4′-3H, U-¹⁴C]-L -phenylalanine and [CH₃-¹⁴C]-L -methionine was determinated. The results demonstrate that the building blocks of cytochalasin D are 1 unit of pheylalanine, 3 units of methionine and acetate units.     

The synthesis of novel polycyclic heterocyclic ring systems via photocyclization. 23 . Naphtho[2′,1′:4,5]thieno‐[2,3‐c]naphtho[2,1‐f]quinoline

A previously unknown heterocyclic ring system, naphtho[2′,1′:4,5]thieno[2,3‐c]naphtho[2,1‐f]quinoline ( 14 ), was synthesized via oxidative photocyclization of 3‐chloro‐N‐(2‐phenanthryl)naphtho[1,2‐b]‐thiophene‐2‐carboxamide ( 9 ). Further elaboration of the lactam 10 yielded the unsubstituted ring system 14 . Structural confirmation of compound 14 was accomplished by a total assignment of its ¹H and ¹³C nmr spectra utilizing the concerted two‐dimensional nmr spectroscopic methods.     

The Synthesis of Novel Polycyclic Heterocyclic Ring Systems via Photocyclization. 12. Benzo[h]naphtho-[2′,1′:4,5]thieno[2,3-c]quinoline and benzo[f]-naphtho[2′,1′:4,5]thieno[2,3-c]quinoline

The synthesis of two previously unknown heterocyclic ring systems, namely benzo[h]naphtho[2′,1′:4,5]thi-eno[2,3-c]quinoline (1) and benzo[f]naphtho[2′,1:4,5]thieno[2,3-c]quinoline (2) was accomplished via photocyclization of the appropriate amides followed by chlorination and catalytic dechlorination. The total assignment of ¹H and ¹³C nmr spectra of 2 was determined utilizing two-dimensional nmr methods, providing unequivocal structural proof of the two novel polycyclic ring systems.     

Late stages in the biosynthesis of abnormal erythrina alkaloids

The incorporation of (±)-, N-norprotosinomenine, N-nor-orientaline, N-nor-reticuline, norlaudanosoline, protosinomenine, and N-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-2-(4-hydroxyphenyl) ethylamine into coccuvine has been studied, and the specific utilisation of the (±)-norprotosinomenine demonstrated. A double labelling experiment with (±)-[1-³H,4'-methoxy-¹⁴C]-N-norprotosinomenine showed that the 4'-O-Me group of the precursor is retained in the bioconversion and the erythrinan ring system is not formed by addition of secondary amino function onto an ortho-quinone system. Feeding of (±)-[1-³H, 7-methoxy-¹⁴C]norprotosinomenine established that O-demethylation is the terminal step in the biosynthesis of coccuvine. Feeding of labelled abnormal Erythrina alkaloids revealed that isococculidine is converted into coccoline via coccuvinine and isococculine into coccolinine via coccuvine.     

The synthesis of novel polycyclic heterocyclic ring system via photocyclization. 18. Benzo[h]naphtho-[1′,2′:4,5]thieno[2,3-c]quinoline and benzo[h]naphtho-[1′,2′:4,5]thieno[2,3-c] [1,2,4]triazolo[4,3-a]quinoline

The synthesis of two previously unknown polycyclic ring systems, benzo[h]naphtho[1′2′:4,5]-thieno[2,3-c]quinoline ( 1 ) and benzo[h]naphtho[1′,2′:4,5]thieno[2,3-c][1,2,4]triazolo[4,3-a]quinoline ( 2 ), was achieved via oxidative photocyclization of 1-chloro-N-(1-naphthyl)naphtho[2,1-b]thiophene-2-carboxamide ( 5 ). The total assignment of their ¹H and ¹³C nmr spectra was determined by the concerted use of two-dimensional nmr methods.     

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.     

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.     

Polycyclic N-heterocyclic compounds. XLI. Synthesis of 4-substituted 6,7-dihydro-5H-pyrimido[5,4-d][1]benzazepines, 1,2,5,6-tetrahydro-4H-imidazo[1′,2′:1,6]pyrimido[5,4-d][1]benzazepines and their related compounds as a series of potential blood platelet aggregation inhibitors

As a series of polyheterocyclic compounds for exploitation as anti-platelet agents, tricyclic heterocyclic compounds, 4-substituted 6,7-dihydro-5H-pyrimido[5,4-d][1]benzazepines 3–6, 9, 12–14 , and 16–26 , having nitrogen, oxygen, or sulfur containing functional groups at the 4-position, were prepared. In addition, tetra-cyclic heterocyclic compounds, 3-methyl-1,2,5,6-tetrahydro-4H-imidazo[1′,2′:1,6]pyrimido[5,4-d][1]benzaze-pinium chloride ( 7 ), 1,2,5,6-tetrahydro-4H-imidazo[1′,2′:1,6]pyrimido[5,4-d][1]benzazepines 10a-e , 2,3,6,7-tetrahydro-1H 5H-pyrimido[1′,2′:1,6]pyrimido[5,4-d][1]benzazepine ( 11 ), and 1,2,5,6-tetrahydro-4H-thiazolo-[3′,2′:1,6]pyrimido[5,4-d][1]benzazepinium chloride ( 15 ) via ring closure of 4-(hydroxyalkylamino)- 6, 9a-e , and 3c , and 4-(2-hydroxyethylthio)-6,7-dihydro-5H-pyrimido[5,4-d][1]benzazepine ( 14 ) with phosphoryl chloride or thionyl chloride, respectively, were also prepared. Their inhibitory activities against collagen-induced aggregation of rabbit blood platelets in vitro were investigated. Among them, compound 5 having a morpholino group at the 4-position on the tricyclic nucleus, which enhanced the activity more than 14-fold as compared with aspirin, was found to have the most satisfactory in inhibitory activity.     

An Efficient Synthesis of Spiro‐oxindole Derivatives by Three‐Component Reactions in Water

An efficient synthesis of (3S)‐1,1′,2,2′,3′,4′,6′,7′‐octahydro‐9′‐nitro‐2,6′‐dioxospiro[3H‐indole‐3,8′‐[8H]pyrido[1,2‐a]pyrimidine]‐7′‐carbonitrile is achieved via a three‐component reaction of isatin, ethyl cyanoacetate, and 1,2,3,4,5,6‐hexahydro‐2‐(nitromethylidene)pyrimidine. The present method does not involve any hazardous organic solvents or catalysts. Also the synthesis of ethyl 6′‐amino‐1,1′,2,2′,3′,4′‐hexahydro‐9′‐nitro‐2‐oxospiro[3H‐indole‐3,8′‐[8H]pyrido[1,2‐a]pyrimidine]‐7′‐carboxylates in high yields, at reflux, using a catalytic amount of piperidine, is described. The structures were confirmed spectroscopically (IR, ¹H‐ and ¹³C‐NMR, and EI‐MS data) and by elemental analyses. A plausible mechanism for this reaction is proposed (Scheme 2).     

The synthesis of novel polycyclic heterocyclic ring systems via photocyclization. 6. Naphthothienonaphthyridines

Five novel polycyclic heterocyclic ring systems are reported via photocyclization. The specific final products in these ring systems are: naphtho[1′,2′:4,5]thieno[2,3-c][1,8]naphthyridin-6(5H)-one ( 5 ), naphtho-[1′,2′:4,5]thieno[2,3-c][1,6]naphthyridin-6(5H)-one ( 6 ), naphtho[1′,2′:4,5]thieno[2,3-c]-1,5-naphthyridine ( 9 ), naphtho[1′,2′:4,5]thieno[2,3-c][1,2,4]triazolo[4,3-a]-1,5-naphthyridine ( 12 ), and naphtho[2′,1′:4,5]thieno[2,3-c]-1,5-naphthyridine ( 17 ). The direction of photocyclization to produce 9 was established from a zero quantum two-dimensional nmr spectroscopy experiment (ZQCOSY) using 6-chloronaphtho[1′,2′:4,5]thieno[2,3-c]-1,5-naphthyridine ( 8 ) as the model compound.     

Synthesis of two novel ring systems via photocyclization: Thieno[2′,3′:4,5]thieno[2,3-c][1,10]phenanthroline and thieno[3′,2′:4,5]thieno[2,3-c][1,10]phenanthroline

The synthesis of thieno[2′,3′:4,5]thieno[2,3-c][1,10]phenanthroline ( 5 ) and thieno[3′,2′:4,5]thieno-[2,3-c][1,10]phenanthroline ( 10 ) are described. Each compound was obtained in four steps from known starting materials. The basic skeleton of the molecule and of the phenanthroline ring were formed via photocyclization. The total assignment of ¹H-nmr spectra was accomplished with the aid of two-dimensional nmr methods.     

Synthesis and debenzoylation products of two perbenzoylated 2-substituted 5-D-galactosyl-1,3,4-oxadiazoles

The synthesis of 2-(p-chlorophenyl)-5-[1′,2′,3′,4′,5′-penta-O-benzoyl-D-galactopentitol-1-yl]-1,3,4-oxadiazole is described. Its debenzoylation gave an equilibrium mixture of the 1,3,4-oxadiazole derivative without protection of the hydroxyl group and the N-benzoyl-D-galactono-1,4-lactonehydrazone. A similar equilibrium was observed by debenzoylation of 2-phenyl-5-[1′,2′,3′,4′,5′-penta-O-benzoyl-D-galactopentitol-1-yl]-1,3,4-oxadiazole. The ¹H, ¹³C nmr and ms spectra of these compounds are presented.     

Biogenesis of betalaine. Biotransformation of dopa and tyrosine in betalaminic acid part of the betanins

During studies on the biogenesis of betalains (I) in cactus fruits (Opuntia sp.). DL -dopa-1-[¹⁴C] and -2-[¹⁴C] were incorporated into betanin (III) which was obtained radiopure after crystallization. The specific activity remained constant after conversion to betanidin (IV) and to a neobetanidin derivative (IX). Reaction of radiobetanin with proline afforded indicaxanthin (V) carrying more than 90% of the radioactivity. Dopa (VI) is thus an efficient precursor for betalamic acid (VIII) but not for cyclodopa (VII). Decarboxylation of radiobetanidin and radioindicaxanthin showed that the carboxyl group of dopa remained a carboxyl group in the biotransformation to betalamic acid. It is concluded that the aromatic ring of dopa is cleaved and that re-cyclization involving the nitrogen generates the dihydropyridine moiety. Under the same conditions mevalonic acid, aspartic acid and phenylalanine showed low incorporations. Studies with beet seedlings and DL -dopa-1-[¹⁴C], -2-[¹⁴C] and DL -tyrosine-1-[¹⁴C] afforded similar results but with low incorporations.