Alkaloid synthesis: stereoselective approach towards fawcettimine-serratinine group of lycopodium alkaloids

The concept of carbocycle-heterocycle equivalency has been utilised to assemble the framework of fawcettimine-serratinine group of alkaloids from 1,5-cyclooctadiene through a common tricarbocyclic intermediate 3.

Synthesis of DEF ring skeleton of naulafine alkaloid

6, 8-Dichloro-4, 5-dihydro-2-methyl-[2, 7]acenaphthyridin-1-one (5), obtained from the. reaction of 5-cyano-2, 6-dihydroxy-3, 4-cyclopentenopyridine with Vilsmeier reagent, has been transformed to the key synthon 4a in two steps.

A novel structural derivative of natural alkaloid ellipticine, MDPSQ, induces necrosis in leukemic cells

DNA intercalating molecules are promising chemotherapeutic agents. In the present study, a novel DNA intercalating compound of pyrimido4',5':4,5]selenolo(2,3-b)quinoline series having 8-methyl-4-(3 diethylaminopropylamino) side chain is studied for its chemotherapeutic properties. Our results showed that 8-methyl-4-(3 diethylaminopropylamino) pyrimido 4',5':4,5] selenolo(2,3-b)quinoline (MDPSQ) induces cytotoxicity in a time- and concentration-dependent manner on leukemic cell lines. Both cell cycle analysis and tritiated thymidine assays revealed that MDPSQ affects DNA replication. Treatment with MDPSQ resulted in both elevated levels of DNA strand breaks and repair proteins, further indicating its cytotoxic effects. Besides, Annexin V/PI staining revealed that MDPSQ induces cell death by triggering necrosis rather than apoptosis.

Evaluation of the Genotoxic and Antigenotoxic Potential of the Alkaloid Punarnavine from Boerhaavia diffusa

Boerhaavia diffusa is a traditional herbal medicine extensively used in the Ayurveda and Unani forms of medicine in India and many parts of the world. Different parts of the plant are used as an appetizer, alexiteric, eye tonic, for flushing out the renal system, and to treat blood pressure. This study was conducted to evaluate the in vivo genotoxic and/or antigenotoxic potential of punarnavine, a separated alkaloid from the root of B. diffusa using toxicity studies (OECD guideline 474, 1997). The genotoxic and antigenotoxic potential of punarnavine was assayed using the comet assay on lymphocytes, liver, spleen, brain, and bone marrow as well as using the micronucleus test in bone marrow cells including the in vitro chromosomal aberration test. The results demonstrated that none of the tested doses of punarnavine showed genotoxic effects by the comet assay, or clastogenic effects in the micronucleus test. On the other hand, for all cells evaluated, the three tested doses of punarnavine promoted inhibition of DNA damage induced by cyclophosphamide. Based on these results, we concluded that punarnavine, an alkaloid from the Boerhaavia diffusa root, has no genotoxic or clastogenic effects in our experimental conditions. However, it caused a significant decrease in DNA damage induced by cyclophosphamide. It is suggested that the antigenotoxic properties of this alkaloid may be of great pharmacological importance and beneficial for cancer prevention.

Anticancer Effects of Brominated Indole Alkaloid Eudistomin H from Marine Ascidian Eudistoma viride Against Cervical Cancer Cells (HeLa)

Marine invertebrates called ascidians are prolific producers of bioactive substances. The ascidian Eudistoma viride, distributed along the Southeast coast of India, was investigated for its in vitro cytotoxic activity against human cervical carcinoma (HeLa) cells by the MTT assay. The crude methanolic extract of E. viride, with an IC50 of 53 mu g/ml, was dose-dependently cytotoxic. It was more potent at 100 mu g/ml than cyclohexamide (1 mu g/ml), reducing cell viability to 9.2%. Among nine fractions separated by chromatography, ECF-8 exhibited prominent cytoxic activity at 10 mu g/ml. The HPLC fraction EHF-21 of ECF-8 was remarkably dose- and time-dependently cytotoxic, with 39.8% viable cells at 1 mu g/ml compared to 51% in cyclohexamide-treated cells at the same concentration; the IC50 was 0.49,mu g/ml. Hoechst staining of HeLa cells treated with EHF-2I at 0.5 mu g/ml revealed apoptotic events such an cell shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies. Cell size and granularity study showed changes in light scatter, indicating the characteristic feature of cells dying by apoptosis. The cell-cycle analysis of HeLa cells treated with fraction EHF-21 at 1 mu g/ml showed the marked arrest of cells in G(0)/G(1), S and G(2)/M phases and an increase in the sub G(0)/G(1) population indicated an increase in the apoptotic cell population. The statistical analysis of the sub-G(1) region showed a dose-dependent induction of apoptosis. DNA fragmentation was also observed in HeLa cells treated with EHF-21. The active EHF-2I fraction, a brominated indole alkaloid Eudistomin H, led to apoptotic death of HeLa cells.

Indole oxygenase from the leaves of Tecoma stans

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and at 30°C. Oxygen (2 mol) is consumed and anthranilic acid (1 mol) is formed for every mole of indole oxidized. Neither sulfhydryl reagents nor sulfhydryl compounds inhibited the enzyme activity. The oxygenase also attacks, apart from indole, 5-hydroxyindole, 5-bromoindole and 5-methylindole. It is not inhibited by copper specific chelators or non-heme iron specific chelators. Atebrin did not inhibit the enzyme activity suggesting that it is not a flavoprotein, unlike other indole oxygenases and indole oxidases. Dialysis resulted in complete loss of enzyme activity. The inactive enzyme could not be reactivated by addition of various cofactors.

Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes

Background and Objective: Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular b asis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.Material and Methods:Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.Results:Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase.Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.

Expression of hyoscyamine 6 beta-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L.

Hyoscyamine 60-hydroxylase (H6H: EC 1.14.11.11), a key enzyme at the terminal step of tropane alkaloid biosynthesis, converts hyoscyamine to scopolamine. The accumulation of scopolamine in different organs, in particular the aerial parts for storage, is subject to the expression of hyoscyamine 6-phydroxylase as well as its transport from the site of synthesis. To understand the molecular basis of this regulation, we have analyzed, in parallel, the relative levels of hyoscyamine and scopolamine, and the accumulation of H6H (both protein and transcript) in leaves, stems and roots of D. metel. The root, stem and leaf tissues all contain about 0.51-0.65 mg g(-1) dry weight of scopolamine. Hyoscyamine content was extremely low in leaf and stem tissues and was about 0.28 mg g(-1) dry weight in the root tissue. H6H protein and its transcript were found only in roots but not in the aerial parts viz. stems and leaves. The immunolocalization studies performed on leaf, stem, root as well as hairy root tissues showed that H6H was present only in the pericycle cells of young lateral and hairy roots. These studies suggest that the conversion of hyoscyamine to scopolamine takes place in the root pericycle cells, and the alkaloid biosynthesized in the roots gets translocated to the aerial parts in D. metel. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

An alpha-aminomethyl carbanion equivalent via a novel Barbier reaction: (1H-naphtho[1,8-de]-1,2,3-triazin-2-yl)methyl anion

A novel sonication-promoted Barbier reaction putatively generated the titled species from the corresponding naphthotriazinylmethyl chloride and magnesium in THF: its formal addition to a variety of carbonyl compounds in situ occurred in excellent yields. Subsequent catalytic hydrogenolysis of the triazine moiety demasked the amine, thus defining a route to various phenylethylamines (including the alkaloid 'mescaline'), or ethanolamines (in two cases), in excellent overall yields. (C) 2000 Elsevier Science Ltd. All rights reserved.

Indole oxygenase from the leaves of Jasminum grandiflorum

An indole oxygenase from the leaves of Jasminum grandiflorum was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 4.8 and at 30°C. Apart from indole, the oxygenase also attacks 5-hydroxy indole and 5-bromoindole. Both sulfhydryl reagents and sulfhydryl compounds inhibited the enzyme activity. Copper specific metal chelators such as salicylaldoxime, diethyl dithiocarbamate and neocuproine, inhibited the enzyme activity drastically. Inhibition caused by atebrine, could be reversed by FAD. Dialysis resulted in complete loss of enzyme activity. Inactive enzyme could be reactivated only by the addition of both FAD and Cu2+, suggesting that indole oxygenase is a cuproflavoprotein.

Biochemical and structural characterization of recombinant hyoscyamine 6 beta-hydroxylase from Datura metel L.

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6 beta-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. mete! (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. mete! (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6 beta-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K-m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 mu M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of alpha-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M-1 and 0.56 M-1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Total Synthesis of (+)-Eburnamonine

The enantiospecific total synthesis of vinca alkaloid (+)-eburnamonine is accomplished from L-ethyl lactate. Key feature of the synthesis is the construction of the chiral quaternary center involving a Johnson-Claisen rearrangement and assembly of the pentacyclic core by the Pictet-Spengler reaction and ring-closing metathesis.

Activation of TGF-beta Pathway by Areca Nut Constituents: A Possible Cause of Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-beta signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-beta in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-beta. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-beta induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (T beta RI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-beta in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-beta downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-beta in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-beta signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-beta 2 and THBS1 (activator of latent TGF-beta) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-beta. These data suggest a major causative role for TGF-beta that is induced by areca nut in OSF progression.

An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp-CrP14, obtained from stem tissues, and Talaromyces radicus-CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 mu g/ml and 20 mu g/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus-CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus-CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 mu g/l) in modified M2 medium and of vinblastine (70 mu g/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns.