Synthesis,Anti-Oomycete and Anti-fungal Activities of Novel Cinchona Alkaloid Derivatives Containing Sulfonate Moiety

Using cinchona alkaloid as the lead compound, twenty-four cinchona alkaloid sulfonate derivatives ( 1 a – l , 2 a – c , 3 a – c , 4 a – c , and 5 a – c ) were designed and prepared by modifying their C9 position, and structurally confirmed by ¹H-NMR, ¹³C-NMR, HR-MS and melting points. Moreover, the stereochemical configurations of compounds 1 f and 1 l were unambiguously confirmed by single-crystal X-ray diffraction. Furthermore, we determined the anti-oomycete and anti-fungal activities of these target compounds against Phytophthora capsici and Fusarium graminearum in vitro. The results showed that two compounds 4 b and 4 c exhibited prominent anti-oomycete activity, and the median effective concentration (EC₅₀) values of 4 b and 4 c against P. capsici were 22.55 and 16.32 mg/L, respectively. This study suggested that when the C9 position of cinchona alkaloid sulfonate derivatives is in the S configuration and the 6′-position methoxy group is not present, the anti-oomycete activity is superior. In addition, five compounds 1 e , 1 f , 1 k , 3 c and 4 c displayed significant anti-fungal activity, with EC₅₀ values of 43.64, 45.07, 80.18, 48.58 and 41.88 mg/L against F. graminearum, respectively. This result indicates that only when a specific substituent is introduced into the structural framework of the target compound, the corresponding compound exhibits significant inhibitory activity against fungi.     

The ergot alkaloid gene cluster: Functional analyses and evolutionary aspects

Ergot alkaloids and their derivatives have been traditionally used as therapeutic agents in migraine, blood pressure regulation and help in childbirth and abortion. Their production in submerse culture is a long established biotechnological process. Ergot alkaloids are produced mainly by members of the genus Claviceps, with Claviceps purpurea as best investigated species concerning the biochemistry of ergot alkaloid synthesis (EAS). Genes encoding enzymes involved in EAS have been shown to be clustered; functional analyses of EAS cluster genes have allowed to assign specific functions to several gene products. Various Claviceps species differ with respect to their host specificity and their alkaloid content; comparison of the ergot alkaloid clusters in these species (and of clavine alkaloid clusters in other genera) yields interesting insights into the evolution of cluster structure. This review focuses on recently published and also yet unpublished data on the structure and evolution of the EAS gene cluster and on the function and regulation of cluster genes. These analyses have also significant biotechnological implications: the characterization of non-ribosomal peptide synthetases (NRPS) involved in the synthesis of the peptide moiety of ergopeptines opened interesting perspectives for the synthesis of ergot alkaloids; on the other hand, defined mutants could be generated producing interesting intermediates or only single peptide alkaloids (instead of the alkaloid mixtures usually produced by industrial strains).     

Synthesis of constrained phenylalanine derivatives via a [2+2+2] cycloaddition strategy

A simple synthesis of dialkyne building blocks (6, 7, 8 and 9) embodying amino acid moiety is described. The dialkyne 6 participated in a [2+2+2] cycloaddition reaction with various monoalkynes in presence of Wilkinson's catalyst to give 5- and 5,6-disubstituted indan-based -amino acid derivatives. Cobalt catalyst [e.g., CpCo(CO)2] has also been employed in the synthesis of various 2-indanyl glycine derivatives via co-trimerization reaction of the diyne building blocks 6 and 7 with several monoalkynes.     

Synthesis of CMP-9'-modified-sialic acids as donor substrate analogues for mammalian and bacterial sialyltransferases

Cytidine-5'-monophospho-sialic acid (CMP-Neu5Ac) derivatives bearing a phenyl group in which the tether length between the phenyl group and the 9-position of Neu5Ac varied were synthesized and evaluated as substrates for sialyltransferases. In the synthesis of the compounds, a coupling reaction between methyl 5-acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-beta-D-glycero-D-galacto-2-nonulopyranosonate and 2-cyanoethyl 2',3'-O,N4, triacetylcytidine-5'-yl N,N-diisopropylphosphoramidite was carried out and the phosphite derivative thus obtained was oxidized and then deprotected to yield CMP-9'-azido-Neu5Ac. Modification of the 9-amino group prepared by reduction of the azido groups was performed by the use of several phenyl-substituted alkylcarboxylic acid derivatives. Using these CMP-9'-modified-Neu5Ac analogues bearing the phenyl-substituted alkyl-amide group, sialyltransferase assays were performed with both rat liver alpha-(2-->6)-sialyltransferase and Photobacterium alpha-(2-->6)-sialyltransferase. These 9-modified analogues could be transferred to disaccharide acceptors, and a practical enzymatic synthesis using CMP-9'-modified-Neu5Ac yielded sialoside analogues and sialylglycoproteins in good yield. These experiments demonstrate that the Photobacterium sialyltransferase can be used in the synthesis of sialoside analogues having a large substituent at the 9-position of Neu5Ac.     

Natural alkaloids and synthetic relatives as chiral templates of the Orito's reaction

The enantioselective hydrogenation of methyl or ethyl pyruvate over cinchona‐platinum catalyst system (Orito's reaction) is one of the most intensively studied heterogeneous catalytic asymmetric hydrogenation reactions. Studies aiming at systematic changes of the chiral template have played a crucial role in creating hypotheses for the mechanism of Orito's reaction. It is very important to clarify which structural unit of the alkaloid takes part in the enantiodifferentiation, and learn about the role of the different structural units of chiral templates. In this article, we made an attempt to describe the behavior of natural alkaloids, their synthetic derivatives, and analogues as chiral templates in the heterogeneous catalytic asymmetric hydrogenation of activated ketones. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Alkaloid diversity in Galanthus elwesii and Galanthus nivalis

Seventy alkaloids of galanthamine, lycorine, homolycorine, tazettine, haemanthamine, narciclasine, and tyramine types were detected by GC/MS in 25 Galanthus elwesii and seven Galanthus nivalis populations, collected from different locations in Bulgaria. Intraspecies diversity in the alkaloid profiles regarding the main alkaloid types (chemotypes) was observed. Tyramine-type protoalkaloids (namely, hordenine and its derivatives) were dominant in 19 populations of G. elwesii. In other populations of G. elwesii, the plants accumulated mainly homolycorine-, lycorine-, and galanthamine-type alkaloids. The alkaloid profiles of G. nivalis were dominated by narciclasine-, galanthamine-, lycorine-, haemanthamine-, or tazettine-type compounds. Geographical distribution of chemotypes indicated a relationship between populations, since adjacent populations often displayed similar alkaloid profiles. The results from year-to-year sampling and transplantation experiments imply genetic determination of alkaloid synthesis in the two studied species of Galanthus.     

Correlation between growth and ergot alkaloid biosynthesis in Claviceps purpurea batch fermentation

Summary Following the growth kinetics of a C. purpurea ergotoxine-producing strain it was observed that alkaloid synthesis and alkaloid distribution depend on fungal growth velocity during idiophase. Formation of extracellular alkaloids is favoured by higher fungal growth velocity, while intracellular alkaloids begin to accumulate at a lower rate of growth. Simple lysergic acid derivatives prevali among extracellular alkaloids, whereas ergotoxines predominate among intracellular alkaloids. By varying cultivation conditions, by feeding the culture, or by varying the inoculum size, alkaloid composition can be influenced within the limits of strain capabilities.     

Enhanced DNA binding of 9-ω-amino alkyl ether analogs from the plant alkaloid berberine

To understand the structure-activity relationship of isoquinoline alkaloids, absorption, fluorescence, circular dichroism, and thermodynamics were employed to study the interaction of five C-9-ω-amino alkyl ether analogs from the plant alkaloid berberine with double-stranded calf thymus DNA. The C-9 derivatization resulted in dramatic enhancements in the fluorescence emission of these compounds. The most remarkable changes in the spectral and binding properties were in the BC4 and BC5 derivatives. Interactions of these analogs, which have an additional recognition motif with DNA, were evaluated through different spectroscopic and calorimetric titration experiments. The analogs remarkably enhanced the DNA binding affinity and the same was directly dependent on the alkyl chain length. The analog with six alkyl chains enhanced the DNA binding affinity by about 33 times compared with berberine. The binding became more entropically driven with increasing chain length. These results may be of potential use in the design of berberine derivatives and understanding of the structure-activity relationship for improved therapeutic applications.     

Enantioselective Michael addition catalyzed by cinchona alkaloids.

Enantioselective Michael additions of cyclic beta-ketoesters to methyl vinyl ketone catalyzed by cinchona alkaloids were studied. The results revealed that the induced enantioselectivity was significantly influenced by both the structure of the catalyst and that of the substrate. Interesting differences in the effect of the structure of the alkaloid on the enantioselectivity of this reaction in the case of three beta-ketoesters were discovered. High enantioselectivities were obtained in the reaction of ethyl 2-oxocyclopentanecarboxylate and ethyl 2-oxocyclohexanecarboxylate (up to 83 and 80%, respectively) at a low cinchona:reactant ratio of 1:500. As the specific rotations of the product enantiomers were unknown, they were determined by optical rotation and chiral GC measurements and verified by NMR experiments.     

The analysis of 2-acetamido-2-deoxyaldose derivatives by gas-liquid chromatography and mass spectrometry

Syntheses are reported of 4-substituted, 4-deoxy analogs of methyl β-D-galactopyranoside (the 4-amino-4-deoxy, 4-azido-4-deoxy, 4-bromo-4-deoxy, 4-chloro-4-deoxy, 4-deoxy-4-fluoro, 4-deoxy-4-iodo, and 4-thio derivatives) as potential substrates of D-galactose oxidase. These syntheses involved nucleophilic displacement of the 4-(p-bromophenylsulfonyl)oxy group of appropriate D-glucose derivatives, although the more reactive (trifluoromethylsulfonyl)oxy group was also utilized as a novel leaving-group. Formation of the bromo and iodo derivatives was accompanied by appreciable halogen exchange and a resulting overall retention of configuration, and formation of the thio compound was attended by competing reactions. Optical rotatory characteristics of the halogeno compounds, their triacetates, and tribenzoates are described, and “anomalous” behavior of the last group is noted.     

Biochemical processing of plant acquired pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi

Leaf beetles of the genus Platyphora, feeding on plant species containing pyrrolizidine alkaloids of the lycopsamine type, not only sequester these alkaloids and concentrate them in their exocrine defensive secretions, but also specifically process the plant acquired alkaloids. Using P. boucardi as subject, three mechanisms were studied: (i). utilization of host plant alkaloids that are not sequestered per se; (ii). elucidation of the mechanism of the already documented C-7 epimerization of heliotridine O(9)-monoesters; (iii). the specificity of insect catalyzed necine base esterification. P. boucardi does not sequester the triester parsonsine, the principal alkaloid of its host plant Prestonia portobellensis (Apocynaceae). Beetles fed with a purified mixture of nor-derivatives of parsonsine, obtained from Parsonsia laevigata, did not sequester the triesters but transformed them by partial degradation into monoesters that are accumulated in the defensive secretions. The mechanism of the previously described transformation of rinderine into intermedine by C-7 epimerization was elucidated by feeding C-7 deuterated heliotrine (3'-methylrinderine). The transformation of heliotrine into epiheliotrine (3'-methylintermedine) catalyzed by P. boucardi is accompanied by complete loss of deuterium, indicating the same mechanism of an oxidation-reduction process via a ketone intermediate as recently demonstrated in a pyrrolizidine alkaloid sequestering lepidopteran. P. boucardi is able to form ester alkaloids from five different necine bases fed as radioactively labeled substrates. However, besides C-7 epimerization the beetles are not able to convert simple necine bases into retronecine. The functional importance of the various alkaloid transformations is discussed in comparison to striking parallels of analogous reactions known from pyrrolizidine alkaloid sequestering Lepidoptera.     

Correlation between Polyamines and Pyrrolidine Alkaloids in Developing Tobacco Callus

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) α-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) α-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.     

Structural determination of an exocellular mannan from Rhodotorula mucilaginosa YR-2 using ab initio assignment of proton and carbon NMR spectra

The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acetamido-2-deoxy-alpha,beta-D-mannose (N-acetyl-alpha,beta-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and 3-N-tert-butyloxycarbonyl compounds were further characterised as their peracetates. A preliminary study has found that these C-3 nitrogen-substituted derivatives of ManNAc not to be substrates for Neu5Ac aldolase.     

Enantioselective hydrogenation of α‐ketoesters catalyzed by cinchona alkaloid stabilized Rh nanoparticles in ionic liquid

The heterogeneous enantioselective hydrogenation of α‐ketoesters catalyzed by rhodium nanoparticles (Rh NPs) in ionic liquid was studied with the stabilization and modification of cinchona alkaloids. TEM characterization showed that well‐dispersed Rh NPs of about 1.96 nm were obtained in ionic liquid. The results showed that cinchona alkaloids not only had good enantiodifferentiating ability but also accelerated the catalytic reaction. Under the optimum reaction conditions, the enantiomeric excess in ethyl benzoylformate hydrogenation could reach as high as 60.9%.     

Efficient nitrogen alkylation with carbohydrates

Efficient nitrogen alkylation of various primary and secondary amines, including cyclic, heterocyclic and alkaloid type amines, with a sugar oxetane 3,5-anhydro-1,2-O-cyclohexylidene-alpha-D-xylofuranose is described. As a result, 5-amino-5-deoxy derivatives of xylofuranose were obtained in good yields.     

Furoquinoline alkaloids from Teclea nobilis

Five new furoquinoline alkaloids, namely tecleabine (1), tecleoxine (2), isotecleoxine (3), methylnkolbisine (4) and chlorodesnkolbisine (5) were isolated from the aerial parts of Teclea nobilis, together with seven known furoquinoline derivatives; one acridone alkaloid, and one known flavanone. The structures of the alkaloids 1-5 were established by 1D and 2D NMR spectral data, including COSY, HMQC and HMBC experiments, as well as HRMS.     

Theoretical bases of optimization of the modes of postharvest treatment of alkaloid-synthesizing medicinal plants

The method of disks in combination with thin-layer chromatography and spectrophotometry was used for a comparative evaluation of the efficiency of different methods of fixing and fermentation of medicinal plants (leaves). Medicinal plant species capable of synthesizing alkaloids of various structural types (isoquinoline derivatives including benzophenanthridines, bisbenzylisoquinoline, quaternary protoberberines, and aporphines, as well as steroid and diterpene alkaloids) were used. The methods of fixing and fermentation were shown to exert a substantial effect on both the qualitative and quantitative composition of alkaloids. This difference in the alkaloid composition was found to represent the superposition of three independent and, to a large extent, opposite processes: catabolism of alkaloids, interaction of alkaloids with each other, and resynthesis from primary precursors. The use of different methods of fixing and fermentation provides activation or inhibition of these processes, thereby making it possible to carry out target-oriented correction of the alkaloid composition of the medicinal plants studied.     

Theoretical Bases of Optimization of the Modes of Postharvest Treatment of Alkaloid-Synthesizing Medicinal Plants

The method of disks in combination with thin-layer chromatography and spectrophotometry was used for a comparative evaluation of the efficiency of different methods of fixing and fermentation of medicinal plants (leaves). Medicinal plant species capable of synthesizing alkaloids of various structural types (isoquinoline derivatives including benzophenanthridines, bisbenzylisoquinoline, quaternary protoberberines, and aporphines, as well as steroid and diterpene alkaloids) were used. The methods of fixing and fermentation were shown to exert a substantial effect on both the qualitative and quantitative composition of alkaloids. This difference in the alkaloid composition was found to represent the superposition of three independent and, to a large extent, opposite processes: catabolism of alkaloids, interaction of alkaloids with each other, and resynthesis from primary precursors. The use of different methods of fixing and fermentation provides activation or inhibition of these processes, thereby making it possible to carry out target-oriented correction of the alkaloid composition of the medicinal plants studied.     

Induction of apoptosis by carbazole alkaloids isolated from Murraya koenigii.

In the current study, we isolated 10 carbazole alkaloids from the plant species Murraya koenigii (Rutaceae), and examined their effects on the growth of the human leukemia cell line HL-60. Three carbazole alkaloids, mahanine (6), pyrayafoline-D (7) and murrafoline-I (9), showed significant cytotoxicity against HL-60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin was increased in a time-dependent manner after treatment with each alkaloid. Interestingly, each carbazole alkaloid induced the loss of mitochondrial membrane potential. In addition, both caspase-9 and caspase-3 were also time-dependently activated upon treatment with the alkaloids. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by these alkaloids. The results suggest that these three alkaloids induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, through mitochondrial dysfunction.     

小果博落回的生物碱分离与鉴定

以D101大孔吸附树脂为吸附剂,将对小果博落回全草的酸水提取物分为50%乙醇洗脱部分和丙酮洗脱部分.对两部分洗脱物分别进行硅胶柱色谱分离,由50%乙醇洗脱部分得到5个生物碱:二氢血根碱(1),二氢白屈菜红碱(2),6-乙氧基血根碱(3),6-丙酮基血根碱(4)和Bocconarborine A (5).由丙酮洗脱部分得到原阿片碱(6),α-别隐品碱(7),β-别隐品碱(8)和小檗红碱(9).采用波谱分析对化合物进行了结构表征,结果表明,化合物3、4、5、9是首次从该植物中分离得到.