New alkaloids from Banisteriopsis caapi

Three new alkaloids isolated from Banisteriopsis caapi, were identified as harmic amide (1-carbamoyl-7-methoxy β-carboline), acetyl norharmine (1-acetyl-7-methoxy β-carboline) and ketotetrahydronorharmine (7-methoxy-1,2,3,4-tetrahydro-1-oxo-β-carboline)     

Microradioisotopic assay for indoleamine N-methyltransferase and analysis of products by liquid chromatography

A rapid method for the separation of tryptamine, 5-hydroxytryptamine, and their N-methylated derivatives is described. The method involves liquid chromatography using a cation exchange column with the eluant monitored either by ultraviolet or fluorescence spectroscopy. The latter technique permits the detection of picogram quantities of indoleamines. Using normal-phase liquid chromatography a complete separation of tryptamine, its N-methylated derivatives, and their β-carboline analogs was also achieved. A radioisotopic assay with the potential to detect indoleamine N-methyltransferase activity in milligram quantities of rabbit lung tissue was developed. The radioisotopically labeled products formed from a number of substrates in such assays were characterized by liquid chromatography.     

Methylene-beta-phenylethylimine formation from 5-methyltetrahydrofolic acid and beta-phenylethylamine.

We have identified, by TLC, the product from the reaction between 5-methyltetrahydrofolic acid (5-MTHF) and β-phenylethylamine (βφEA) in rat brain extracts as methylene-β-phenylethylimine (MβφEI), a Schiff-base compound produced when formaldehyde, enzymatically formed from 5-MTHF, condenses with the amine. The formation of MβφEI in various brain regions, ranging from 517 ± 56 pmol formed per mg protein per hour in corpus striatum to 118 ± 9 pmol formed in hippocampus, is significantly correlated with that of 1,2,3,4-tetrahydro-β-carboline formed from 5-MTHF and tryptamine (r = 0.88; p < 0.01), which we reported elsewhere (1). In corpus striatum MβφEI formation is found nearly exclusively in cytosol, as was the β-carboline formation. We suspect that the enzyme involved in both reactions is methylenetetrahydrofolate reductase.     

Synthesis, in vitro anti-inflammatory and cytotoxic evaluation, and mechanism of action studies of 1-benzoyl-β-carboline and 1-benzoyl-3-carboxy-β-carboline derivatives

In the present study, various 1-substituted and 1,3-disubstituted β-carboline derivatives were synthesized by a modified single-step Pictet-Spengler reaction. The compounds were examined for cytotoxicity and anti-inflammatory activity, as measured by the inhibition of prostaglandin E(2) (PGE(2)) production and nitric oxide (NO) production. While only two compounds (28 and 31) showed marginal cytotoxicity against four human cancer cell lines, most of the tested compounds exhibited potent inhibitory activity of both NO and PGE(2) production. Moreover, compounds 6 and 16 significantly reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2), suggesting that β-carboline analogs can inhibit NO and PGE(2) production at the translational level. In addition, several of the β-carboline derivatives (1, 2, 4-8, 11, 13, 22, 25, 27, 31, and 41-43) displayed significant inhibitory activity of superoxide anion (O(2)(·-)) generation or elastase release compared to the reference compound, with 6 being the most potent. N-Formyl-L-methionyl-phenylalanine (FMLP)-induced phosphorylation of c-JunN-terminal kinase (JNK) and protein kinase B (AKT) were also inhibited by 6, suggesting that it suppresses human neutrophil functions by inhibiting the activation of JNK and AKT signaling pathways. Therefore, the synthetic 1-benzoyl-3-carboxy β-carboline analogs may have great potential to be developed as anti-inflammatory agents.     

β-carboline compounds, including harmine, inhibit DYRK1A and tau phosphorylation at multiple Alzheimer's disease-related sites

Harmine, a β-carboline alkaloid, is a high affinity inhibitor of the dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) protein. The DYRK1A gene is located within the Down Syndrome Critical Region (DSCR) on chromosome 21. We and others have implicated DYRK1A in the phosphorylation of tau protein on multiple sites associated with tau pathology in Down Syndrome and in Alzheimer's disease (AD). Pharmacological inhibition of this kinase may provide an opportunity to intervene therapeutically to alter the onset or progression of tau pathology in AD. Here we test the ability of harmine, and numerous additional β-carboline compounds, to inhibit the DYRK1A dependent phosphorylation of tau protein on serine 396, serine 262/serine 356 (12E8 epitope), and threonine 231 in cell culture assays and in vitro phosphorylation assays. Results demonstrate that the β-carboline compounds (1) potently reduce the expression of all three phosphorylated forms of tau protein, and (2) inhibit the DYRK1A catalyzed direct phosphorylation of tau protein on serine 396. By assaying several β-carboline compounds, we define certain chemical groups that modulate the affinity of this class of compounds for inhibition of tau phosphorylation.     

Synthesis and antitumor activity of β-carboline 3-(substituted-carbohydrazide) derivatives

A series of β-carboline derivatives bearing a substituted-carbohydrazide moiety at C-3 were synthesized and evaluated for their antitumor activity against eight human cancer cell lines. The β-carboline N-(substituted-benzylidene)carbohydrazides showed, in general, a greater antitumor activity than their N-(alkylidene)carbohydrazide analogues. The N(9)-methylation of β-carboline N-(substituted-benzylidene) carbohydrazides resulted in a decrease of antitumor activity. Among compounds tested, the benzylidene-carbohydrazides 3, 4, 11, 13, 16, 21 and 22 were the most active, possessing IC(50) less than 10 μM for six of the eight tumor cell lines assayed. The derivative 4 displayed the most significant activity toward all tested cell lines, with a remarkable cytotoxicity against renal (786-0) cell lines (IC(50)=0.04 μM). Compound 4 was assayed for its in vivo antineoplastic activity in the Ehrlich solid carcinoma assay.     

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.     

Pharmacokinetic and pharmacodynamic factors contributing to the convulsant action of β-carboline-3-carboxylic acid esters

Both the methyl ester of β-carboline-3-carboxylic acid and the 6, 7-dimethoxy-4-ethyl derivative of this compound are potent convulsants in rodents, while the ethyl ester of β-carboline-3-carboxylic acid does not cause convulsions, even when administered at very high doses. The rate of degradation of these compounds by rat plasma ($\text{in vitro}$) parallels their potencies as convulsants. In contrast, 3-carboethoxy-β-carboline was found to potently elicit tonic and clonic convulsions in the squirrel monkey ($\text{Saimiri sciureus}$). Furthermore, the rate of degradation of 3-carboethoxy-β-carboline in monkey plasma ($\text{in vitro}$) is negligible compared with rats. No significant differences were observed in either the potency or efficacy of GABA to inhibit [³H] β-carboethoxy-β-carboline binding in rat and monkey brain. These data strongly suggest that pharmacokinetic, as well as pharmacodynamic, factors may determine the pharmacologic profile of these β-carboline-3-carboxylic acid esters.     

β-Carboline alkaloids from the leaves of Trigonostemon lii Y.T. Chang

A phytochemical work on the alkaloid constituents from Trigonostemon lii Y.T. Chang was conducted to give six new β-carboline alkaloids, trigonostemines A-F (1-6) and eight known β-carboline alkaloids (7-14). Their structures were elucidated by extensive spectroscopic techniques including 2D NMR experiments and mass spectrometry. All of the compounds were evaluated for their cytotoxic activities against the HL-60, SMMC-7721, A-549, MCF-7, and SW480 human cancer cell lines. Trigonostemines A and B (1 and 2) exhibited stronger inhibitory activities than the positive control (cisplatin) in some cell lines.     

Characterization of enzyme-catalysed endogenous β-hydroxylation of phenylethanoid glycosides in Euphrasia rostkoviana Hayne at the molecular level

Phenylethanoid glycosides, the main constituents of the aerial part of eyebright (Euphrasia rostkoviana Hayne) were treated by the endogenous hydroxylase enzyme and the concomitant biotransformation was characterized by applying high-performance liquid chromatography with UV and MS detections and NMR spectroscopy. In the extracts of the untreated (intact) samples, acteoside and eukovoside were determined as main compounds. The enzymatic treatment resulted in the quantitative transformation of these phenylethanoid glycosides into their corresponding hydroxyl derivatives identified as two epimers of β-hydroxyacteoside and β-hydroxyeukovoside. As to the importance of this hydroxylation β-hydroxyeukovoside was identified as a new compound and β-hydroxyacteoside was described for the first time in eyebright. We proved for first time that a β-hydroxylase enzyme is active in eyebright tissues which can transform phenylethanoid glycosides into their β-hydroxyl derivatives. Our new enzymatic method combined with a preparative HPLC facilitates the isolation of β-hydroxyl phenylethanoid glycosides from the aerial part of eyebright.     

Digitization of sedimentation equilibrium and velocity data for analysis by minicomputer

A procedure is described whereby phosphorylated seryl residues may be unequivocally identified during the sequential degradation of a polypeptide chain by the Edman technique. The phosphoseryl residue, Ser(P), was first converted by treatment with methylamine in dilute alkali to a β-methylaminoalanyl residue which was split from the polypeptide by the degradative procedure as the derived phenylthiohydantoin. This was identified by high-performance liquid chromatography. The procedure was highly effective when the Ser(P) occupied an isolated position in a polypeptide chain but was less so when grouped consecutively with other Ser(P).     

Selective determination of phosphopeptide β-CN(1–25) in a β-casein digest by adding iron: characterization by liquid chromatography with on-line electrospray-ionization mass spectrometric detection

A β-casein tryptic digest has been analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) with on-line electrospray-ionization mass spectrometry (ESI-MS). Analyses of peptides were carried out before and after addition of iron(II) to the peptides in solution. In both cases, the majority of peptides were identified by the determination of molecular masses by ESI-MS and by prior knowledge of the amino acid sequence of β-casein, and thus of its corresponding tryptic peptides. In the presence of iron(II), only phosphopeptide β-CN(1–25) was able to bind iron to form different complexes that have increased retention times on the RP-HPLC column and that also absorbed at 280 nm. The method presented here appears to be selective for peptides containing phosphoseryl cluster(s).     

Synthesis and investigations into the anticancer and antibacterial activity studies of β-carboline chalcones and their bromide salts

A series of sixteen β-carbolines, bearing chalcone moiety at C-1 position, were prepared from easily accessible 1-acetyl-β-carboline and various aldehydes under basic conditions followed by N²-alkylation using different alkyl bromides. The prepared compounds were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. N²-Alkylated-β-carboline chalcones 13a-i represented the interesting anticancer activities compared to N²-unsubstituted β-carboline chalcones 12a-g. Off the prepared β-carbolines, 13g exhibited broad spectrum of activity with IC₅₀ values lower than 22.5?µM against all the tested cancer cell lines. Further, the N²-alkylated-β-carboline chalcone 13g markedly induced cell death in MDA-MB-231 cells by AO/EB staining assay. The most cytotoxic compound 13g possessed a relatively high drug score of 0.48. Additionally, the prepared β-carboline chalcones displayed moderate antibacterial activities against tested bacterial strains.     

The occurrence of 2-methyl-1,2,3,4-tetrahydro-β-carboline and variation in alkaloids in Phalaris arundinacea

2-Methyl-1,2,3,4-tetrahydro-β-carboline was isolated from reed canarygrass (Phalaris arundinacea L.) and the occurrence of 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline confirmed. Clones of reed canarygrass that contained N,N-dimethyltryptamine or 2-methyl-1,2,3,4-tetrahydro-β-carboline did not contain their respective methoxy or hydroxy derivatives. Five of the 12 clones tested contained either or both of 5-methoxy-N,N-dimethyltryptamine and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline. The data suggest that clones that contain gramine are not likely to contain N,N-dimethyltryptamine and/or β-carbolines. Thus, an inverse biosynthetic relationship between gramine and the tryptamine and β-carboline alkaloids seems to exist. However, further work is needed to firmly establish any such relationship between these alkaloids.     

LC-MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang)

A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35°C and 100°C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35°C and 100°C, respectively. From the targeted metabolites identification, it was found that 3,4?-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100°C.     

Influence of production media on Cinchona cell cultures; spontaneous formation of β-carbolines from L-tryptophan

Evidence is presented that the β-carboline alkaloids norharman and harman are artifacts formed from L-tryptophan. Transfer of Cinchona ledgeriana suspension cultured cells to Zenk's alkaloid production medium (ZAP medium) resulted in cell death. No quinoline or terpenoid indole alkaloids were formed. However, the L-tryptophan present in the production medium was transformed to the β-carboline alkaloids, harman and norharman. It was demonstrated that norharman was also formed in ZAP medium without cells and in ZAP medium containing frost-killed cell material.     

Eudistomidins H-K, new β-carboline alkaloids from the Okinawan marine tunicate Eudistoma glaucus

Four new β-carboline alkaloids, eudistomidins H-K (1-4), were isolated from an Okinawan marine tunicate Eudistoma glaucus and the structures of 1-4 were elucidated on the basis of spectroscopic data. Eudistomidins H (1) and I (2) were new β-carboline alkaloids possessing a unique fused-tetracyclic ring system consisting of a tetrahydro β-carboline ring and a hexahydropyrimidine ring. Eudistomidin J (3) showed relatively potent cytotoxicity against murine leukemia cells P388 and L1210, and human epidermoid carcinoma cells KB in vitro.     

Structure-activity relationships for the reserpine-like actions of derivatives of beta-carboline in vitro

The abilities of 10 derivatives of β-carboline to competitively inhibit the ATP-Mg²⁺ stimulated uptake of epinephrine (0.1 mM) were studied in isolated rat adrenal medullary storage vesicles. Uptake was inhibited 72% by harmine (0.03 mM), 64% by harmaline, 59% by 2-methylharmine, 43% by harmol and 25% by harman. Norharman, 6-methoxyharman, 6-methoxyindole, 6-methoxytetrahydroharman and yohimbine did not inhibit epinephrine uptake, nor did any of the 10 derivatives affect metaraminol uptake. The potencies of epinephrine uptake inhibitors were unrelated to the lipid solubility or pK of the drugs, suggesting that differences in activity reflected differences in affinity for the catecholamine transport site. Harmol, 2-methylharmine and harmaline were themselves incorporated into the vesicles, but the temperature dependence was much smaller than that of epinephrine or metaraminol (Q₃₀ of 1.5–2 vs. 4.5–7). These data suggest that β-carboline derivatives interact with a catecholamine carrier on the outside surface of the vesicle membrane and that the attachment involves at least 3 portions of the molecule. The different structure- activity relationships for inhibition of epinephrine uptake vs. uptake of the β-carboline derivatives themselves indicate that two separate processes, inward transport and subsequent intravesicular binding, contribute to the measured uptake.     

Rat brain aryl acylamidase: Further characterization of multiple forms

• 1.1. Two fractions of aryl acylamidase (EC 3.5.1.13) were further separated from rat brain extracts at pH 7.5 by ammonium sulfate precipitation and Bio-Gel chromatography.
• 2.2. 1,2,3,4-Tetrahydro-β-carboline competitively inhibited (67%) fraction-1 but slightly inhibited (13%) fraction-2. Tetrahydroharman, 6-hydroxy-tetrahydroharman and harminic acid slightly inhibited both fractions. Harmalol inhibited fraction-1 but enhanced fraction-2. 6-Methoxy-harman, 6-methoxy-harmalan and harmaline enhanced both fractions.
• 3.3. Pargyline did not affect either fraction. Methiothepin, cyproheptadine and chlorimipramine inhibited fraction-1 but stimulated fraction-2.
• 4.4. Neostigmine moderately (30%) inhibited AAA-2 but did not have any significant effect on AAA-1.
• 5.5. These results indicate that the β-carboline compounds might play a role in regulating activity of AAA-1 and 2 in brain.
• 6.6. Both fractions might be related to serotonergic neurons but only AAA-2 might be associated with acetylcholinesterase.

     

Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography–mass spectrometry

We first detected glutathionyl hemoglobin (Hb) β-chain in hemodialysis patients and healthy subjects using electrospray ionization liquid chromatography–mass spectrometry. The ratio of glutathionyl Hb β-chain to total β-chain was markedly increased in the hemodialysis patients as compared with healthy subjects. Glutathionyl Hb will be used as a new clinical marker of oxidative stress.