Frontiers in Natural Product Chemistry

This Proceedings volume entitled “Frontiers in Natural Product Chemistry” contains a feast of original articles contributed by experts in the
interdisciplinary field of natural product science. They include articles on the synthesis of several classes of natural products, isolation and structure elucidation of natural compounds, pharmacology and bioassay screening of natural substances and other related topics. These papers were presented as Plenary and Invited Lectures during the 9th International Symposium on Natural Product Chemistry (9th ISNPC) held in Karachi during January 10-13, 2004. This series of symposia is well reputed for its excellence and focused theme.

Asymmetric 1,4-arylation and -alkenylation was achieved by use of organoboronic acids or their derivatives in the presence of a rhodium catalyst coordinated with binap or its related ligands. The scope of this asymmetric addition is very broad, α,β-unsaturated ketones, esters, amides, 1- alkenylphosphonates, and 1-nitroalkenes being efficiently converted into the corresponding 1,4-addition products with over 95% enantioselectivity. The catalytic cycle of the reaction in water is proposed to involve three intermediates (aryl- or alkenyl-rhodium, (oxa-π-allyl)rhodium, and hydroxorhodium) by NMR studies on the rhodium intermediates. The asymmetric addition of B-aryl-9BBN and ArTi(OPr-i)3 in aprotic solvents proceeded with high enantioselectivity under mild conditions to give the corresponding metal enolates as the 1,4-addition products.

Protected trihydroxystilbenes have been synthesized by Heck coupling methodology in three steps. Treatment of 3,4-dimethoxy-12-benzyloxymethyl stilbene with ferric chloride in dichloromethane (room temperature), gave catechol analogues of ampelopsin F and of restrytisol C, while 3,4-dimethoxy-12- acetoxymethyl stilbene treated in the same conditions yielded two other analogues of restrytisol C (but no trace of ampelopsin F analogues). All the structures were unambiguously confirmed by 1D- and 2D- homo- and heteronuclear nmr experiments. All transformations were stereospecific. The ampelopsin F-type compounds are the result of radical cation pathways. By contrast, the restrytisol Ctype compounds are the products of pericyclic pathways.

The cephalostatins ( Cephalodicus gilchristi ) and the agelorins (Agelas oroides ) represent two groups of marine natural products showing high biological activity. The total synthesis of enantiopure analogues reveals for the cephalostatins and the decisive role of molecular dissymmetry and of a chiral curvature, while the agelorins were shown to be prodrugs, which under stress conditions are undergoing an enzymatically induced fragmentation, generating the highly active cyclohexadiene aeroplysinin. Finally a simple diversity oriented enantioselective synthesis of wistarin precursor is reported.

The nitroaldol (Henry) reaction although has been discovered more than one century ago is still largely employed as the key step for the synthesis of many targets. The reaction is performed under basic condition by mixing primary or secondary nitroalkanes (and nitromethane) with a carbonyl, allowing the formation of a β-nitroalkanol. The latter shows an high versatility since it can be converted into a lot of different functionalities and many natural products have been prepared using the Henry reaction as the key step. Some of these synthesis will be reported.

The bisindole alkaloids of the Madagascan periwinkle Catharanthus Roseus vincristine 1 and vinblastine 2 are widely used clinically in the chemotherapy of cancer. Many efforts were made to synthesize more efficient new derivatives having less side effects than vinblastine 2. New anhydrovinblastine derivatives were obtained by the ferric chloride mediated coupling reaction of catharanthine and vindoline and their derivatives. The nitro- dinitro-, amino- and hydroxymethyl analogues of the alkaloids 1, 2 and 3 showed promising antitumor activity.

The bifunctional taxoid-colchicinoid hybrids were synthesised and evaluated in assays of cytotoxicity and tubulin assembly/disassembly. All compounds showed a high degree of cytotoxicity.

Electronic control of diastereoselectivity is discussed; only electrophilic attack adjacent to a stereogenic centre carrying an electropositive element 3 makes a coherent story.

Natural photoreceptors evoke a biological response on absorption of light signal. A reversible change in the structure of photoreceptors occurs by energy or sensory transduction. Chemically they are conjugated trans-membrane proteins with retinal or tetrapyrrole based chromophores in most of them. Photosynthesis, photomorphogenesis, photoperiodism, vision and phototaxis are various functions of photoreceptors.

Bacteriorhodopsin (bR), from H. salinarium acts as a light driven proton pump for the synthesis of ATP. bR undergoes an interesting reversible photocycle. During the photocycle, retinylidene chromophore undergoes a reversible all-trans-to 13- cis- isomerisation, and proton translocation takes place.Chemical modifications of bR and 3D - structure of bR at 1.5-Angstrom resolution help to know the binding site of chromophore and proton translocation at molecular level. Many colour control mechanisms have been suggested for bR. A large number of bR analogues can be made as follows:

bR + NH2OH.HCl -→ Bacterial opsin (bOP) + Retinal, and bOP + retinal analogue → bR analogue, which can be used to study structure, function and likely applications. bR can act as a bistable photobiological switch with potential applications in optical memory, optical data processing.

Photoactive Yellow Protein (PYP), a cytosolic photoreceptor from E. halophila also undergoes a photocycle and can be put to similar uses. PYP uses p-hydroxy cinnamoyl thioester, a novel chromophore that isomerises with change in conformation of protein during the photocycle, resulting in a signalling state in yet unknown sensory path.

Natural photoreceptors constitute a novel and unique field of natural product chemistry.

The binding of betamethasone sodium phosphate and prednisolone, two steroidal antiinflammatory drugs and theophylline sodium glycinate, a bronchodilator, to bovine serum albumin (BSA), has been studied by equilibrium dialysis (ED) method at different temperature and pH values for characterizing the binding of these drugs to BSA. Binding was exothermic, entropically driven and spontaneous, as indicated by the thermodynamic analysis. The major part of the binding energy at site II results from electrostatic and hydrophobic interactions. The free fraction of either betamethasone or prednisolone in the presence of theophylline sodium glycinate and vice versa was monitored in the presence and absence of site specific probes. The free fraction of betamethasone sodium phosphate by theophylline sodium glycinate and vice versa was increased during concurrent administration causing reduced binding of these drugs to BSA. This increment of free fraction was more prominent in the presence of site I specific probe, which suggested that in the absence of site I specific probe, betamethasone after being displaced by theophylline from its high affinity site rebound to its low affinity site. Similar type of result was observed in case of prednisolonetheophylline interaction.

Cellulases are attractive source for utilization of the agro-industrial waste materials. Exoglucanase (EC; 3.2.1.91), Endoglucanase (EC. 3.2.1.4) and b- glucosidase (EC. 3.2.1.21) were isolated from Trichoderma harzianum (E-58 strain). The fungus was grown on Vogel's medium with different carbon sources. Maximal production of the enzymes was achieved at 28 0C, pH 5.5 under continuous shaking at 120 rpm for 5 days. Glucose repressed the synthesis of the enzymes whereas carboxymethylcellulose (CMC) produced the enzymes in substantial amounts. Maximum activity of cellulases (exoglucanase, endoglucanase and b-glucosidase) were found to be 2.764, 14.4 and 0.629IU mL-1, respectively. Corresponding genes for cellulases were isolated with the help of RTPCR. RNA was isolated from mycelia of T. harzianum grown on CMC and xylan. First strand of cDNA was synthesized using oligo dT (18) primer and subjected to PCR with specific primers. The amplified products were purified through agarose gel electrophoresis and ligated into SmaI site of pUC18. The plasmids containing exg, egl and bgl genes were transformed into E. coli for further characterization.

Most bacteria have developed specific systems to procure sufficient amounts of iron which is not generally available in water soluble form. One possibility is the production of Fe3+ chelating substances, so-called siderophores. Structural varieties, the transport mechanism through the cell wall, and the importance of siderophores in health, agriculture and environment will be discussed.

In this study, the method of determining the absolute configurations of 14b-hydroxy-3β-O-(L-thevetosyl)-5β-card-20(22)-enolide (I) which was isolated from the air-dried fruits of Cerbera odollam and 3α-feruloyl-taraxerol (II) which was isolated from the air-dried fruits of Bruguiera cylindrica will be presented. The novel method [1] of incorporating heavy-atom-containing solvent molecules into the single crystal structure of the compounds and refinement of the Flack parameter [2] was used to find the absolute configurations of these natural products. Compounds (I) has a cardenolide skeleton whereas compound (II) has a taraxerol skeleton. The present X-ray study shows the absolute L-form of (I) and the absolute α-form of (II). We have clearly demonstrated for the first time a novel approach which is an extremely useful and easy method to determine the absolute stereochemistry of natural products by X-ray diffraction. The importance of determining the absolute stereochemistry of natural products is emphasised in this work.

Anacardium occidentale (cashew) containing the principal component phenols, anacardic acid, cardol, 2-methylcardol and cardanol is a unique source of materials useful both for industrial technology, in semi-synthesis and for biological/pharmaceutical applications. Thus, by industrial decarboxylation, anacardic acid in the natural cashew gives predominantly cardanol in the product; technical cashew nutshell liquid (CNSL) together with cardol/2-methylcardol and the cashew kernel is the valuable desired main edible commercial product. Anacardic acid itself together with cardol is best recovered by cold cutting of raw cashew shells followed by solvent extraction to afford natural CNSL. Either from technical or natural CNSL, the individual component phenols, cardanol, anacardic acid, cardol, 2-methylcardol, respectively can be separated by several different techniques notably by phase separation. Each separated component phenol contains saturated, 8(Z)-monoene, 8(Z),11(Z)-diene and 8(Z).11(Z),14-triene constituents. Subsidiary chromatographic separation can give the individual constituents. Technical CNSL without separation is widely used in the production of friction dusts for the automobile industry and in certain polymeric/surface coating applications. Cardanol separated from technical CNSL has uses in semisynthesis, e.g. in the formation of polyethoxylate surfactants, chelatants for metals and for boron. Natural CNSL and anacardic acid separated from it, has potential industrial applications in semi-synthesis and in biological studies. Cardol separated, either from technical or natural CNSL also has potential interest in semi-synthesis, and its homologues, more recently as a potential biological marker.

The objective of this paper is to give an overview on natural resources in terms of renewable resources as well as non-renewable resources for the development of new pharmaceuticals, chemical and food products, the roles of microbial cells in solid substrate fermentation and submerged fermentation. The benefit of this paper is to present current information on the role of traditional and modern biotechnology.

The methods used, namely traditional and modern biotechnology and chemical synthesis are developed and implemented to produce pharmaceutical and chemical products. Results can be shown that microorganisms have been engineered to commercially produce such substances as the hormone insulin, the virus fighting compound interferon, and a new vaccine against foot-and-mouth disease. Traditional fermentation technology has been done to produce low cost protein food using natural product as a substrate and the development of palm oil derived products for food, oleo chemicals, bio-plastic, bio-diesel and lubricants, respectively.

Microbial transformation is an effective tool for the structural modification of bioactive natural and synthetic compounds. Its application in asymmetric synthesis is increasing due to its versatility and ease. The present article reviews our contributions in the field of microbial transformation of different classes of bioactive natural products. The microbial transformation of monoterpenoids [(1R, 2S, 5R)-(-)-menthol and (-)-a-pinene], sesquiterpenoids [(+)-sclareolide, 7a-hydroxyfrullanolide, nootkatone, a-santonin, (-)-ambrox and isolongifolen-4-one], sapogenins (sarsasapogenin), alkaloids (vindoline) and steroids (E-guggulsterone, withaferin-A, (+)-adrenosterone, androst-1,4-dien-3,17- dione, dehydroepiandrosterone, norethisterone, 17a-ethynylestradiol, prednisone, testosterone and danazol) are described here along with biological activities of transformed products.

Based on isolation, sequence determination and X-ray studies, the primary and three-dimensional structure of the glycoprotein mistletoe lectin I (MLI) are determined. ML-I is constituted of two chains (A chain: 254 amino acid residues; B chain: 264 amino acid residues) linked by a disulfide bridge. Three different structurally identified oligosaccharides (I, II, III) are attached to four Ntype glycosylation sites (NA112, NB61, NB96 and NB136). According to these structural characterizations, ML-I is a member of ribosome inactivating proteins (RIP) of type II. The three-dimensional X-ray structure allows a clear-cut picture of the highly toxic effects of ML-I caused by its RNA-N-glycosidase activity, which is in contrast to its immunomodulating activity, applied for the treatment of cancer patients.

The aerial parts of Chrysanthemum coronarium were extracted in MeOH, and the extract was partitioned using EtOAc, n-BuOH and H2O. The repeated column chromatography of EtOAc fraction gave four sterols, whose chemical structures were identified as stigmast-4-en-6b-o1-3-one (1), stigmast-4- en-6a-ol-3-one (2), which have been so far reported only in the aquatic plants and were isolated for the first time from the land plants, b-sitosterol (3) and daucosterol (4) based on several spectral data including gCOSY, gHSQC, gHMBC and comparison of the data with those of literature. And the repeated column chromatography of EtOAc and n-BuOH fractions gave nine polyacetylens, which were identified as 2-[(lZ,4Z)-5-methylsulfinyl-2-pentyn-4-enyliden]-1,6- dioxaspiro[4,4]non-3-ene (5), 2-[(IE,4Z)-5-methylsulfinyl-2-pentyn-4-enyliden]- 1,6-dioxaspiro[4,4]non-3-ene (6), 2-[lZ-2,4-dipentyn-6-methyliden]-1,6-dioxaspiro [4,4]non-3-ene (7), 2-[lE-2,4-dipentyn-6-methyliden]-1,6-ioxaspiro[4,4]non-3- ene (8), 2-[lE-2,4-dipentyn-6-methyliden]-1,6-dioxaspiro[4,5]non-3-ene (9), 2-[lZ- 2,4-dipentyn-6-methylidene]-1,6-dioxaspiro[4,4]non-3,7-dien-9a-ol (10), 2-[lE-2, 4-dipentyn-6-methyliden]-1,6-dioxaspiro[4,4]non-3,7-dien-9b-ol (11), 2-[lE-2,4- dipentyn-6-methyliden]-1,6-dioxaspiro[4,4] non-3,7-dien-9a-ol (12) and 2-[1Z- 2,4-dipentyn-6-methyliden ]-1,6-dioxaspiro[4,4]non-3,7-dien-9b-o1 (13). Some of them showed the inhibitory effect on the activity of ACAT (Acyl-CoA: cholesterol acyltransferase), the catalyzing enzymes of the intracellular esterification of cholesterol, and FPTase (Farnesyl-protein transferase), the farnesylation enzymes for Ras protein in charge of cancer promotion, PLT aggregation, and the growth of HUVEC (Human umbilical vascular endothelial cell) or A549 cells.

All of the possible conduritol isomers have already been synthesized starting from completely different materials and their biological importance studied. Conduritols are useful precursors in the preparation of cyclitols such as myo-inositols phosphates, and pseudo-sugars, some of which are important mediators in many cellular processes. The synthesis of conduritpls, quercitols and the development of new synthetic methods are discussed in this paper.

We undertook the (re-)investigations of the chemical composition of the phenolic contents of Malaysian timber species. In order to efficiently isolate these constituents, we developed semi-automated separation procedures. It includes the extensive use of HPLC systems, operated at low or high pressure, in conjunction with automatic injection and automatic fraction collection. The efficiency of this procedure is demonstrated by the isolation of an array of oligostilbenoids from the heartwood of chengal (Neobalanocarpus heimii, Dipterocarpaceae), when only a single compound was isolated by the previous investigators.

Essential oil (0.51%) has been isolated from the rhizomes of Zingiber zerumbet from which a white crystalline fraction-A (m.p.620C) has been separated and purified. Another fraction-B (m.p. 660C) has been isolated from petroleum ether (40-600C) extract. The fraction-A & B have been analyzed by, IR, GC/MS. From the above observation it appears that the major constituent of fraction-A & B is same and the structure is possibly 2,6,10- cycloundecatriene-1-one 2,6,9,9- tetramethyl.

Bioflavonoids were once known as vitamin P and are also considered semi-essential nutrients that are responsible for the color of fruits and flowers. They are used in the treatment or prevention of various diseases. There are four categories and these are the proanthocyanidins, quercetin, the citrus flavonoids and green tea polyphenols. The major sources of these bioflavonoids are from plants. The bioflavonoids are known for their antioxidant activity but a great number of other bioactivities are also attributed to them. Some of these bioactivities will be presented as well as the bioactivities of the rare flavonoids isolated from Syzygium samarangense (Blume) Merr. & L.M. Perry. Plant natural products, arguably, will still play a major role in providing the world with an alternative and a more effective health care.

In a continuation of our studies towards the discovery of biologically active compounds from Sri Lankan plants, recently we have chemically investigated the various parts of Diploclisia glaucescens (Menispermaceae), Filicium decipiens, Pometia eximia (Sapindaceae), Artocarpus nobilis (Moraceae) and Bridelia retusa (Euphorbiaceae). These work led to the isolation of a number of ecdysones, triterpenes, saponins, chalcones, stilbenes, flavonoids including over twenty-five new natural products. Some of these compounds showed high molluscicidal, insecticidal, antifungal activities and radical scavenging properties towards DPPH.

There are approximately 100 species of Zanthoxylum in the tropical and subtropical regions, and 10 of them are indigenous to Taiwan. For the last 10 years, our laboratory has examined six species of Zanthoxylum in Taiwan (Z. ailanthoides, Z. integrifoliolum, Z. nitidum, Z. pistaciiflorum, Z. schinifolium and Z. simulans) for their chemical and bioactive compounds. This research has led to the isolation of 59 new compounds and around 200 known compounds. The structures of the new compounds were established by spectroscopic data and chemical evidence. These new compounds showed diversity of structures including benzo[c]phenanthridine, 2-phenyl-1-N-methylphthaliamide, pyranoquinoline, 2-quinolone, isobutylamide, indolopyridoquinazoline, aporphine, phenylacetonitrile, coumarin, phenylpropanoid, lignan, and flavonoid. Several isolates showed antiplatelet aggregation activity in vitro.

Leaf, stem and root of Murraya paniculata (L.) Jack was extracted in four different organic solvents (petroleum ether, ethyl acetate, acetone and methanol). The extracts were tested on the mortality of adult male and female of Callosobruchus maculatus F. All the evaluated extract was toxic to both the sexes. Males generally showed higher susceptibility compared to females. Petroleum ether extract was more toxic than others. It was in the order: pet. ether > EtOAc > acetone > MeOH extract. Leaf extract was most toxic in comparison to stem and root extracts. It was in order: Leaf > root > stem. Generally, 72 hours exposure exhibited lower LD50 values compared to 24 and 48 hours.