Methylenedioxy Bridge Research Articles

Coumarins in helianthus tuberosus : characterization, induced accumulation and biosynthesis

The biosynthesis of 7-hydroxylated coumarin phytoalexins has been examined in the tuber of Helianthus tuberosus. Ayapin and scopoletin where characterized by TLC, HPLC and GC-MS. Both compounds were excreted from sliced tuber tissues. They accumulated in response to treatment with chemical elicitors like CuCl 2 or sucrose, but not in response to MnCl 2. Scopoletin accumulation preceded ayapin build-up in the medium. Highest and earliest accumulation of both coumarins was measured after copper treatment. The formation of methylenedioxy bridges in several secondary metabolites is catalyzed by cyt P450 oxygenases. The changes in cyt P450 content, trans-cinnamate 4-hydroxylase and NADPH-cytochrome c reductase activities in microsomes from tuber tissues were, therefore, monitored for three days after wounding and elicitation. Cyt P450 content and NADPH-Cyt c reductase activity were not significantly modified in copper treated tissues, but increased three to five fold in response to sucrose. Copper elicitation resulted in a decrease in cinnamate-4-hydroxylase activity, while sucrose was a very strong and specific inducer of this Cyt P450 enzyme. No formation of ayapin was observed when radiolabelled scopoletin was incubated with NADPH and elicited plant microsomes, or fed to induced tuber tissues in vivo. No formation of methylenedioxy metabolite was obtained in vitro or in vivo from radiolabelled ferulate or feruloyl CoA.

Furanofuran lignan metabolism as a function of seed maturation in sesamum indicum: methylenedioxy bridge formation

Sesamum indicum seeds accumulate the antioxidant lignans, (+)-sesamin and (+)-sesamolin. It was established that seeds of 8-week old S. indicum plants catalyzed distinct lignan transformations (methylenedioxy bridge formation in piperitol and sesamin, as well as oxygen insertion in sesamolin) depending upon the stage of seed maturity, i.e. lignan formation was developmentally regulated. The most mature seeds at the eight week developmental stage efficiently converted (+)-[3,3′-O14CH3] pinoresinol into (+)-piperitol and (+)-sesamin, whereas younger seeds had a higher conversion into (+)-sesamolin; the corresponding (−)-[3,3′-O14CH3] pinoresinol antipode was not metabolized. Microsomal preparations obtained from S. indicum seeds catalyzed methylenedioxy bridge formation via an O2\\NADPH\\cytochrome P-450 dependent transformation of (+)-pinoresinol into (+)-piperitol. Preliminary characterization of the enzyme established its cytochrome P-450 dependence.

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures.

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.

The characterization of some 3,4-methylenedioxycathinone (MDCATH) homologs

In the past 35 years, a wide variety of illicit drugs have appeared in the clandestine market. Many of these compounds are based on the structure of amphetamine (1-phenyl-2-aminopropane) to which various functional or structural groups have been added. Previous modifications to the amphetamine molecule include addition of a methylenedioxy bridge to give 3,4-methylenedioxyamphetamine, and attachment of a β-keto oxygen to yield cathinone. A chemical synthesis integrating the salient functional/structural groups of these two classes of amphetamine analogs results in manufacture of methylenedioxycathinone (MDCATH). In each instance, N-alkylation of these analogs provides a series of homologs. Furthermore, many of these analogs/homologs meet several criteria which typically support the clandestine laboratory synthesis of novel illicit drugs (`designer drugs'). The MDCATH analogs represent a potentially new series of `designer drugs' whose chemical characteristics have not previously been reported. Appropriate selection of analytical, chemical and physical tests will enable rapid identification of these analogs by a comparative analysis using the data provided.

Cytochrome P450-dependent methylenedioxy bridge formation in Cicer arietinum

Microsomal preparations from elicited heterotrophic cell cultures of chickpea in the presence of O 2 and NADPH catalyse methylenedioxy bridge formation from pratensein (3′-OH-biochanin A) and calycosin (3′-OH-formononetin). The enzymatic activities are inhibited by CO and various P450 inhibitors and are therefore characterized as cytochrome P450-dependent.

Canadine synthase from Thalictrum tuberosum cell cultures catalyses the formation of the methylenedioxy bridge in berberine synthesis

An enzyme system catalysing the formation of the methylenedioxy bridge at ring A of ( S)-canadine [ = ( S)-tetrahydroberberine] from ( S)-tetrahydrocolumbamine has been detected in microsomal preparations from different Ranunculaceae and Berberidaceae cell cultures. The cytochrome P-450 enzyme complex has been partly characterized from a protoberberine alkaloid producing Thalictrum tuberosum L. cell line. The enzyme complex consisting of a microsomal associated oxidase with a cytochrome P-450 reductase has a pH optimum at pH 8.5 and a temperature optimum of 40°. The apparent K m values are 33 μM for NADPH and 11.5 μM for tetrahydrocolumbamine.

A new model for coenzyme B12 based on a Costa‐type ligand

AbstractThe synthesis of a new Costa‐type ligand (H2mdo), in which the propylene bridge has been replaced by a methylenedioxy bridge has been accomplished: two molecules of butanedione monoxime are coupled via a CH2 group by reaction with CH2Cl2 and 50% aqueous NaOH in the presence of a phase‐transfer reagent. Dihalidocobalt and alkyl(halido)cobalt complexes of the deprotonated ligand have been prepared and structurally characterized by NMR spectroscopy.

Novel formation of 4-methylthiopyrene in a Hofmann elimination reaction directed toward the synthesis of 17,19-dioxa[2.2.3](1,2,3)cyclophanediene

The synthesis of 17,19-dioxa[2.2.3](1,2,3)cyclophanediene was attempted via a Stevens rearrangement–Hofmann elimination sequence on 19,21-dioxa-2,11-dithia[3.3.3](1,2,3)cyclophane. Pyrene and 4-methylthiopyrene were, however, the isolated products. The structure of the latter was fully characterized by UV, MS, 1D and 2D 1H NMR spectra. Formation of 4-methylthiopyrene was expected to involve a novel base-induced ring cyclization resulting in elimination of methanoate anion from the methylenedioxy bridge followed by a novel 1,4-hydride shift resulting in the elimination of methane.

Enzymatic Cleavage of the Methylenedioxy Bridge of Piperonylic Acid

Enzymatic Cleavage of the Methylenedioxy Bridge of Piperonylic Acid

Two methylenedioxy bridge forming cytochrome P-450 dependent enzymes are involved in ( S)-stylopine biosynthesis

The enzymatic synthesis of the alkaloids ( S)-[10-OC 3H 3]scoulerine and ( S)-[3-OC 3H 3]cheilanthifoline is described. A microsomal preparation from Eschscholtzia californica cell suspension cultures in the presence of NADPH and O 2 catalyses the formation of two methylenedioxy bridges in two consecutive steps: ( S)-cheilanthifoline from ( S)-scoulerine and ( S)-stylopine from ( S)-cheilanthifoline. Nandinine was not found to be an intermediate. The release of HO 3H into the aqueous incubation mixture from the radiolabelled substrates served as an assay for both enzyme activities. The enzymes were characterized as to their substrate specificity, cofactor requirements and inhibition by cytochrome P-450 specific inhibitors. Both enzymes were drastically induced (12- and 5-fold) 20 hr after challenging the cell suspension culture with elicitor. The mechanism of enzymic methylenedioxy bridge group formation from an o-methoxyphenol function is discussed.

Cornigerine, a potent antimitotic colchicum alkaloid of unusual structure : Interactions with tubulin

Cornigerine is a natural product analog of colchicine produced by Colchicum cornigerum in which the vicinal 2- and 3-methoxy groups are condensed into a methylenedioxy bridge. This produces a fourth ring and a structure which resembles a hybrid of colchicine, podophyllotoxin, and steganacin. Cornigerine was somewhat more toxic than colchicine with L1210 murine leukemia cells and caused them to accumulate in metaphase arrest. Cornigerine resembled colchicine in its interactions with tubulin in vitro, and it was also somewhat more potent than colchicine in these drug-tubulin interactions. Cornigerine inhibited tubulin polymerization both with and without microtubule-associated proteins, inhibited the binding of radiolabeled colchicine to tubulin, and stimulated tubulin-dependent GTP hydrolysis. Indirect evidence suggested that the binding of cornigerine to tubulin is relatively slow and temperature-dependent, like the binding of colchicine to the protein.

Alternative final steps in berberine biosynthesis in Coptis japonica cell cultures

In Coptis japonica cell cultures an alternative pathway has been discovered which leads from (S)-tetrahydrocolumbamine via (S)-canadine to berberine. The two enzymes involved have been partially purified. (S)-Tetrahydrocolumbamine is stereospecifically transformed into (S)-canadine under formation of the methylenedioxy bridge in ring A. This new enzyme was named (S)-canadine synthase. (S)-Canadine in turn is stereospecifically dehydrogenated to berberine by an oxidase, (S)-canadine oxidase (COX), which was partially purified (25-fold). This enzyme has many physical properties in common with the already known (S)-tetrahydroprotoberberine oxidase from Berberis but grossly differs from the latter enzyme in its cofactor requirement (Fe) and its substrate specificity. Neither (S)-norreticuline nor (S)-scoulerine serves as substrate for the Coptis enzyme, while both substrates are readily oxidized by the Berberis enzyme. The four terminal enzymes catalyzing the pathway from (S)-reticuline to berberine are housed in Berberis as well as in Coptis in smooth vesicles with a density of ρ=1.14 g/ml. These vesicles have been enriched and characterized by electron microscopy.

Occurrence of the Methylene-dioxy Bridge in the Phenolic Components of Plants

THE methylene-dioxy bridge is a common feature of the structure of several groups of plant alkaloids and occurs also, though less commonly, among natural phenolic substances. Geissman and Hinreiner1, in a recent review of natural phenols, list twenty-four different substances containing the methylene-dioxy bridge; fourteen of them are C6—C3 type and all are characterized by a generally high degree of methylation of hydroxyl groups in the molecule; indeed, none of them bears a free hydroxyl in the ring that bears the bridge. The alkaloids are similarly characterized by a high degree of methylation of phenolic hydroxyl groups2. Botanically, the methylene-dioxy phenolic substances are distributed widely but not haphazardly, for they occur in six families of Polypetalae (Anonaceae, Magnoliaceae, Rutaceae, Leguminosae, Rosaceae and Umbelliferae), three of Apetalae (Myristicaceae, Lauraceae and Piperaceae) and one of the monocotyledons (Orchidaceae). Of these families, Lauraceae and Piperaceae are much the most abundant sources, and it seems from this (as from the occurrences in Magnoliaceae, Rutaceae, Umbelliferae and Myristicaceae) that there may be some relationship between aromatic properties and the occurrence of the bridge. It may be significant, too, that of the eleven families of plants which have yielded methylene-dioxy alkaloids, no fewer than four occur in the above list of phenolic-bearing families (Anonaceae, Rutaceae, Leguminosae and Piperaceae2).