Kinuko Iwasa

Antibacterial activity and structure-activity relationships of berberine analogs

Summary Analogs of berberine 1 and related compounds were prepared to evaluate structure-activity relationships. Among the 13-alkyl-substituted and the 13-unsubstituted protoberberinium salts, the 13-ethyl-9-ethoxyl homolog 30, the 13-ethyl analog 29, and the 13-methyl derivative 3 showed an increase in antibacterial activity against Staphylococcus aureus by eight-, four- and twofold respectively over the parent base berberine 1; this is suggestive that steric effects play a significant role in the antibacterial activity. Reduction of the protoberberinium salts yielding the tetrahydro derivatives greatly reduced the antibacterial activity. Replacement of methoxyl groups at the C-2 and the C-3 of ring A by a methylenedioxy group resulted in increased antibacterial activity. These data strongly suggest that the quaternary nitrogen atom such as in protoberberinium salts, an alkylsubstituent at C-13, and a methylenedioxy function at C-2 and C-3 are required for enhanced activity. Tetrahydroprotoberberine α-N-metho salts showed higher activity than tetrahydroprotoberberine hydrochlorides, but appreciably lower activity than protoberberinium salts. The effects of substitution at C-13 and on ring A in the α-N-metho salt were similar to those in protoberberinium salts. Stereochemical changes of the B/C ring juncture from trans to cis, and of the methyl group at C-13 from α to β, had, respectively, marked and slight effects on the activity. The tested compounds were less active against Escherichia coli (Gram-negative bacterium) and Candida albicans (fungus) than S aureus (Gram-positive bacterium).

Simple isoquinoline and benzylisoquinoline alkaloids as potential antimicrobial, antimalarial, cytotoxic, and anti-HIV agents

Twenty-six simple isoquinolines and 21 benzylisoquinolines were tested for antimicrobial, antimalarial, cytotoxic, and anti-HIV activities. Some simple isoquinoline alkaloids were significantly active in each assay, and may be useful as lead compounds for developing potential chemotherapeutic agents. These compounds include 13 (antimicrobial), 25, 26, and 42 (antimalarial), 13 and 25 (cytotoxic), and 28 and 29 (anti-HIV). A quaternary nitrogen atom of isoquinolium or dihydroisoquinolinium type may contribute to enhanced potency in the first three types of activities. In contrast, anti-HIV activity was found with tetrahydroisoquinoline and 6,7-dihydroxyisoquinolium salts.

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe2+-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.

Molecular cloning and characterization of methylenedioxy bridge‐forming enzymes involved in stylopine biosynthesis in Eschscholzia californica

(S)‐Stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge‐forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557–38565], no information is available regarding the genes for methylenedioxy bridge‐forming enzymes in stylopine biosynthesis. Two cytochromeu2003P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C.u2003japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge‐formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)‐cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)‐cheilanthifoline, (S)‐scoulerine, and (S)‐tetrahydrocolumbamine. An expression analysis in E.u2003californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S‐adenosyl‐l‐methionineu2003:u20033′‐hydroxy‐N‐methylcoclaurine 4′‐O‐methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E.u2003californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed.

Molecular Cloning and Characterization of CYP80G2, a Cytochrome P450 That Catalyzes an Intramolecular C–C Phenol Coupling of (S)-Reticuline in Magnoflorine Biosynthesis, from Cultured Coptis japonica Cells

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C–C phenol-coupling and intermolecular C–O phenol-coupling reactions. We report here the isolation and characterization of a C–C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C–O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C–C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C–C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-l-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.

Knockdown of berberine bridge enzyme by RNAi accumulates ( S )-reticuline and activates a silent pathway in cultured California poppy cells

Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310xa0μg/g-fresh weight. In addition, 1xa0g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6xa0mg/20xa0mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.

CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in Eschscholzia californica

Eschscholzia californica produces various types of isoquinoline alkaloids. The structural diversity of these chemicals is often due to cytochrome P450 (P450) activities. Members of the CYP719A subfamily, which are found only in isoquinoline alkaloid-producing plant species, catalyze methylenedioxy bridge-forming reactions. In this study, we isolated four kinds of CYP719A genes from E. californica to characterize their functions. These four cDNAs encoded amino acid sequences that were highly homologous to Coptis japonica CYP719A1 and E. californica CYP719A2 and CYP719A3, which suggested that these gene products may be involved in isoquinoline alkaloid biosynthesis in E. californica, especially in methylenedioxy bridge-forming reactions. Expression analysis of these genes showed that two genes (CYP719A9 and CYP719A11) were preferentially expressed in plant leaf, where pavine-type alkaloids accumulate, whereas the other two showed higher expression in root than in other tissues. They were suggested to have distinct physiological functions in isoquinoline alkaloid biosynthesis. Enzyme assay analysis using recombinant proteins expressed in yeast showed that CYP719A5 had cheilanthifoline synthase activity, which was expected based on the similarity of its primary structure to that of Argemone mexicana cheilanthifoline synthase (deposited at DDBJ/GenBank™/EMBL). In addition, enzyme assay analysis of recombinant CYP719A9 suggested that it has methylenedioxy bridge-forming activity toward (R,S)-reticuline. CYP719A9 might be involved in the biosynthesis of pavine- and/or simple benzylisoquinoline-type alkaloids, which have a methylenedioxy bridge in an isoquinoline ring, in E. californica leaf.

Antimalarial activity and structure-activity relationships of protoberberine alkaloids

The thirty-nine protoberberine derivatives including berberine 1 and palmatine 2 were tested for antimalarial activity in vitro against Plasmodium falciparum and structure-activity relationships are proposed. The activity of the protoberberine alkaloids was influenced by the type of the quaternary nitrogen atom, the nature and the size of the substituents at the C-13 position, and the type of O-alkyl substituents on rings A and D. The activity of the quaternary protoberberinium salts with an aromatic ring C such as berberine was higher than that of the quaternary salts such as the N-metho salts or the N-oxides of tetrahydro and dihydro derivatives as well as tertiary tetrahydroprotoberberines. Of the 13-alkyl derivatives of 1 and 2, the activity did not always increase as the length of the aliphatic chain rose in the order methyl, ethyl, propyl, butyl, and hexyl group. 13-Butylberberine (1Bu) and 13-propylpalmatine (2Pr) were the most active compounds among the 13-alkylberberines and 13-alkylpalmatines, respectively. 13-Hydroxyberberine 3 possessed the same level of activity as 1. Of 1 and 2 with different substituents types on Ring A, the activity of 1 was significantly higher than that of 2. Among berberrubines 4 and 5 and their C-9-O-alkyl derivatives 6 and 7, the activity of 9-O-ethylberberrubine 6 was the highest. Of the potent protoberberinium salts, the activity decreased in the order: 1, 3 > 2Pr > 6 > 1Bu. A positive effect on the activity might be exerted by the introduction of a more hydrophilic function into the C-13 position of the protoberberinium salts.

N-Propargylamine protects SH-SY5Y cells from apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol, through stabilization of mitochondrial membrane and induction of anti-apoptotic Bcl-2

Summary.Propargylamine derivatives, rasagiline and (–)deprenyl, are anti-Parkinson agents and protect neurons from cell death as shown by in vivo and in vitro experiments. The studies on the chemical structure-activity relationship proved that the propargyl moiety is essentially required for the neuroprotective function. In this paper, neuroprotective activity of free N-propargylamine was studied using SH-SY5Y cells expressing only type A monoamine oxidase (MAO) against apoptosis induced by an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol. N-Propargylamine prevented apoptosis, whereas N-methylpropargylamine and propiolaldehyde did not. N-Propargylamine stabilized mitochondrial membrane potential and induced anti-apoptotic Bcl-2 at 1u2009µM–10u2009nM. N-Propargylamine inhibited MAO-A in competition to substrate with the apparent Ki value of 28u2009µM, which was significantly higher than the concentration required for neuroprotection. It indicates that MAO inhibition is not prerequisite for the protective function of N-propargylamine. The anti-apoptotic function of N-propargylamine is discussed in terms of neuroprotection by propargylamines in neurodegenerative diseases, including Parkinson’s disease.

Structure-activity relationships of quaternary protoberberine alkaloids having an antimalarial activity.

Abstract Seventeen quaternary protoberberine alkaloids related to berberine 1 were tested for antimalarial activity in vitro against Plasmodium falciparum and structure-activity relationships are proposed. The activity of the protoberberine alkaloids was influenced by the type of the oxygen substituents on rings A, C and D and the position of the oxygen functions on ring D. The position of the oxygen functions on ring D and the type of the oxygen substituents at the C-13 position (ring C) strongly influenced the activity. Shifting the oxygen functions at C-9 and C-10 to C-10 and C-11 on ring D resulted in a significant increase in the activity. Compounds bearing a methylenedioxy function at C-2 and C-3 (ring A) or C-9 and C-10 (ring D) showed higher activity than those which have methoxy groups at the same positions. Introduction of a methoxy group into the C-1 position (ring A) decreased the activity. Replacement of a hydroxy group at C-2 or C-3 (ring A) by a methoxy group led to a reduction in the activity. Displacement of a hydroxy function at C-13 (ring C) by the oxygen substituents such as OMe, OEt, OCOOEt, and OCON(Me) 2 reduced the activity. In the same replacement at C-9 (ring D), the activity depended upon the type of the oxygen function. Six protoberberines displayed more potent activity than berberine 1 . The activity decreased in the order: 10 , 11 , 17 and 18 > 7 and 8 > 1 .