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FAIR-CT96-1781
Terpenes as Natural Chiral Starting Materials For the Synthesis of Flavours, Fragrances, Pharmaceuticals and Biocontrol Agents |
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Proposal No: | FAIR-CT96-1781 |
| Date Prepared: | June 2001, May 1999 | |
| Source: | Final Report Executive Summary
Second Annual Progress Report First Annual Progress Report |
Introduction
These are the objectives of this project.
Agricultural producers, industrial producers of (collected) natural products, academic researchers from several EU countries (Sweden, The Netherlands, Spain, Italy, and Greece) and potential industrial users (from Great Britain, Spain and The Netherlands) of the isolated or produced natural products will co-operate closely together in a joint project. The starting plant material can be produced by agriculture, it may be collected from nature or it can be a waste product from industrial processes based on natural starting materials. The "green" agricultural product is a renewable source of starting material for the production of useful products for the industry (flavours, fragrances, pharmaceuticals and biocontrol agents such as pheromones). The academic research laboratories involved, all posses extensive specialistic knowhow on isolation,'synthetic conversion and physiological activities of terpenoid natural products. The costs of the explorative, pre-competitive, basic research are to high for SME or national agricultural organisations and can be performed only in projects which will be stimulated by the European Union. This project will contribute and extend these possibilities of industrial use of sesqui- and diterpenes significantly because it is focusses on:
The terpenes that will be investigated as chiral starting material are carvone, aromadendrane, guaiol, santonine, lactaranes, uvidins, labdanolic acid and related labdanes, ingenol and taxanes, and eventually other terpenes when they will be disvovered during the project. The sesquiterpenes that will be investigated belong to the classes of the germacranes, the eudesmanes, the guaianes, the lactaranes, the drimanes, and the aromadendranes; the diterpenes that will be investigated are members of the classes of the labdanes, clerodanes, ingenanes and taxanes. Phytochcmical investigations of mostly endemic plants of the homecountries of the participants will be carried out to search for new bioactive terpenes or new starting materials that can be collected from nature.
The research tasks on sesqui- and diterpenes, that will be carried out in this project, will be performed by one or more of the participating research groups mentioned in table 1. Most of the tasks mentioned in this table are subdivided in subtasks and will be performed by junior researchers, technicians or post doctoral researchers. The methodology that will be applied is that of normal practice in Organic Chemistry. All tasks show a perfect fit in the ongoing research programs of the participants. It is also clear from this table that a close interaction and cooperation between the participan 'ts will be ensured, based on these longstanding common interests in terpene chemistry.
Objectives
Technical Approach
Agricultural producers, industrial producers of (collected) natural products, academic researchers from several EU countries (Sweden, The Netherlands, Spain, Italy, and Greece) and potential industrial users (from Great Britain, Spain and The Netherlands) of the isolated or produced natural products will co-operate closely together in a joint project. The starting plant material may be produced by agriculture, it may be collected from nature or it can be a waste product from industrial processes based on natural starting materials. The "green" agricultural product is a renewable source of starting material for the production of useful products for the industry (flavours, fragrances, pharmaceuticals and biocontrol agents such as pheromones).The academic research laboratories involved, all posses extensive specialistic knowhow on isolation, synthetic conversion and physiological activities of terpenoid natural products. The costs of the explorative, pre-competitive, basic research are to high for SME or national agricultural organisations and can be performed only in projects which will be stimulated by the European Union. This project will contribute and extend these possibilities of industrial use of sesqui- and diterpenes significantly because it is focusses on:
The terpenes that will be investigated as chiral starting material are carvone, aromadendrane, guaiol, santonine, lactaranes, uvidins, labdanolic acid and related labdanes, ingenol and taxanes, and eventually other terpenes when they will be disvovered during the project. The sesquiterpenes that will be investigated belong to the classes of the germacranes, the eudesmanes, the guaianes, the lactaranes, the drimanes, and the aromadendranes; the diterpenes that will be investigated are members of the classes of the labdanes, clerodanes, ingenanes and taxanes. Phytochemical investigations of mostly endemic plants of the homecountries of the participants will be carried out to search for new bioactive terpenes or new starting materials that can be collected from nature.
Summary
The project has two main goals:Research has been carried out to find an economically feasible routes for the synthesis of usefull products and of the colouring substance guaiazulene from aromadendrene, derived from the distillation tail of eucalyptus oil. Existing syntheses for guaiazulene are expensive and a good industrially applicable procedure for this compound is not available. However, in this period little progress has been made and the prospectives are not favourable. A second goal is the synthesis of the chiral pheromones from aromadendrene. Here progress has been reasonable and the crucial stepe have been achieved.
alpha-Santonin has been investigated as the second natural starting material, especially for the synthesis of eudesmanes and guaianes. Irradiation of santonin solutions with UV light gives iso photosantonic lactone and this compound was subjected to functional modification in order to prepare a number of natural guaianolides. Preliminary work on the synthesis of sesquiterpene lactones bearing a dihydrooxepine moiety and other oxygenated moieties on the A ring, has led to the preparation of seco-isoerivanin pseudo acid, a bis-lactone isolated from Artemisia judaica which is structurally related to the antitumour compound (+)-8 deoxyvernolepin.
Catalytic hydrogenation of alpha-santonin and subsequent epimerization gave an intermediate which was converted into a 2H-pyranone derivative. The alpha, beta-unsaturated carbonyl backbone of this molecule was used as Michael acceptor for the synthesis of several interesting compounds that are available now for testing.
The considerable biological activities of various chamazulenes, guaianolides and eudesmanolides are well recognized and analogues of these compounds with diverse lipophilicity have been prepared to determine their biological activity. Furthermore the discovery and development of a biotechnological, cost-efficient method for the preparation of useful compounds has led to the development of a protocol for induction of somatic embryogenesis and in vitro flowering from chamomile flower tissues collected in central and southern Greece. Approximately 10,000 callus pieces induced from both disk and ray flowers are being repeatedly cultivated in order to select somaclonal variants with increased chamazulene productivity or altered product production via biotransformation.
The gum of the easily to collect species Cistus ladaniferus consists of a mixture of labdane diol, labdanolic acid, and a number of more simple carboxylic acids. It is of industrial importance to investigate possible uses for this gum as starting material in the synthesis of suitable target molecules. Labdanolic acid has been isolated from tha acidic fraction of the gum in 25% yield and its conversion into Ambrox® is presently investigated. Ambrox® has been synthesized from natural occurring labdanes like labdanolic acid, sclareol, manool, manoyl oxide and now also larixol is investigated as a possible starting material for this compound. For this purpose the oxidative degradation of the sidechain in larixol and other labdanes has been studied systematically and a new protocol has emerged from these investigations.
Paclitaxel (Taxol®) is a new antitumor compound, initially isolated from the bark of Taxus brevifolia. This compound has gained considerable attention due to its novel mode of action and its efficacy in the treatment of various cancers including ovarian, breast and lung carcinoma. Development of new analogues goes tohether with the necessity of the synthesis of more water soluble derivatives. Up to now synthesis of paclitaxel has been achieved by total synthesis and semisynthesis starting from 10-deacetylbaccatin III. Synthesis of analogues has been mostly accomplished by structural modifications of paclitaxel and in some cases from precursors isolated from the needles of several Taxus species (e.g. baccatin III, brevifoliol and 13-acetyl-9-dihydrobaccatin III). Extraction and isolation of crude Taxine B from the needles of the T. baccata has been scaled up and about 500 gr crude Taxine B has been isolated from T. baccata needles last year. A new analytical HPLC method for the detection of taxine alkaloids in yew needles was developed also and figure 2 gives a typical example of a HPLC chromatogram of a T. baccata sample. With the help of this analytical method, several yew species were screened for their taxine alkaloid content. T. baccata has the highest taxine content, while T. brevifolia does not contain any taxine alkaloids at all. Very interesting for the synthesis of 1,7-dideoxy paclitaxel derivatives is the T. cuspidata because this species does not contain any 1-hydroxy taxines. Furthermore, T. cuspidata contains a relatively large amount of taxinine, which is also an interesting precursor for the semi-synthesis of 1,7-dideoxy paclitaxel derivatives.
For the synthesis of new derivatives, a Pd catalysed allylic substitution using hydride as the nucleophile has been developed to remove the C5 substituent in a short procedure which ultimately led to an enol ether. The 4(5)-double bond in this enoether enables the introduction of new D-rings via cyclo-addition reactions.
Large amounts (approxirnately 50 kg) of Lactarius vellereus, were collected and extracted from which the sesquiterpene isovelleral was isolated and used for chemical investigations. The synthesis of bioactive lactaranes and marasmanes from isovelleral has concentrated on the preparation of novel tropinones, it has been demonstrated that this reaction only works with isovelleral and isovelleral-like compounds, not with for example dihydroisovelleral or polygodial (isolated from the plant Polygonum hydropiper). The product obtained with isovelleral has been further transformed by reduction of the keto function. A lot of experiments to produce completely reduced (and volatile) marasmane sesquiterpenes have been carried out, but so far with little success.The intermediates produced during these attempts, of which some have odours, will be sent to Quest for analysis.
The transformation of marasmanes to lactaranes by opening of the cyclopropane ring has been studied. In the presence of an primary amine and chloride or iodide this reaction is feasible in moderate yields. The synthesis of tridemethylisovelleral has been finished in asatisfactory way, and the biological testing of this presumably highly active unsaturated dialdehyde is due to take place in a near future.
The enantioselective synthesis of saponaceolide structure has been investigated and a viable strategy for assembling the saponaceolide structure was found, as was demonstrated by the highly stereoselective synthesis of 2-epi-saponaceolide B. The two important enantiomerically enriched (> 90% ee) building blocks were available from previous synthetic work. Their conversion into the two building blocks and coupling of these two parts by a Wittig olefination gave finally the O-methyl ether of 2-epi-saponaceolide B in six additional steps. This excellent piece of work will certainly lead to the synthesis of saponaceolide itself in the near future.
An original and rather efficient enantioselective synthesis of (1S, 5R)-Karahana lactone and (1S,5R)-Karahara ether has also been completed. Comparing the fragrancy properties of the two antipodal series, we found that (1S, 5R)-Karahana lactone and (1S,5R)-Karahara ether have a much more intense perfume; particularly (1S,5R)-Karahara ether has a pleasant camphoric smell. Uvidin B has been converted into a diene, which will be employed for preparing an important intermediate for the formal enantioselective synthesis of forskolin.
The second main goal of the project is devoted to phytochemical research with the aim to discover new useful substances from plants. In this area, results are as follows.
The extracts of two plants used in Ecuadorian herbal medicine have been investigated. The CH2Cl2 extract of Schistoscarpa aff eupatorioides afforded safranol and a mixture of sesquiterpene hydrocarbons that has been analyzed by GCMS. The following compounds have been identified: beta-pinene, p-cymene, limonene, gamma-terpinene, caryophyllene, aromadendrene, selinene, beta-patchoulene, germacrene D, alpha-himachalene, beta-gurjunene, alpha-cedrene, delta-guaiene, gamma-muurolene, alpha-humulene.
Cyperus odoratus L. is used in Ecuador against dysentery; the plant has a pleasant scent. Eleven compunds was indentified from this material, one of whioch was new.
Fruiting bodies (250 g) of Lactarius subumbonatus Lindgr. [syn L. serifluus (D. C. ex Fr.) Fr.] were minced at - 20° C and extracted with chloroform. Chromatographic separation of the extract (150 mg) gave glycerides and the new caryophyllane ester., while chromatographic separation of the chloroform extract of Russula lepida yielded two new nardosinane sesquiterpenes, (+)-aristolone, and the new naturally occurring aristoladiene. These compounds are the first examples of sesquiterpenes of these types isolated from Basidiomycetes. They belong to the antipodal series of the corresponding sesquiterpenes typical of liverworts and Octocorallia.
Tricholoma vaccinum (pers ex Fr. ) Kummer was extracted with EtOAc at -20 °C. The extract contained five very polar compounds named tricholomenyn C-G.
Parsley (Petroselinum sativum Hoffm.) is a popular culinary herb. From the seeds of parsely cultivated in Tuscany, crispanone was isolated, and found to be identical to siol angelate, a daucane ester whose structure was solved by X-ray. Barton deoxygenation of crispanone afforded crispane, whose structure should also revised to the one of lasidiol angelate. A novel phenylpropane aldehyde was also obtained.
The seeds of pumpkin are a popular snack and the source of an edible and medicinal oil, which is a constituent of several non-proprietary drugs for the treatment of benign prostate hyperplasia (BHP). A series of triterpenoids of the multiflorane type esterified with p-aminobenzoic acid (PABA) was found in the seeds. These compounds were also isolated from the seeds of squash (C. pepo L.), cucumber (Cucumis sativus L.) and buffalo gourd (C. foetidissima H.B.K.), a plant of great potential as a non-food crop.
A three step isolation procedure to obtain ingenol from the seeds of Euphorbia lathyris L., an agricultural commodity, was developed. As a byproduct of ingenol production, large amounts of lathyrane triesters were obtained. A sub-project concerning the transannular cyclisation of macrocyclic lathyrane diterpenoids was initiated, in order to investigate the posdsibility of producing novel compounds with interesting biological activities.
Ingenol 3-monoesters exert their irritant and tumour-promoting activity by binding to and activating the enzyme protein kinase C (PKC). The presence of a free 20-hydroxyl is essential for PKC binding, but its role is unknown. Replacement of the 20-hydroxyl with a fluorine atom was investigated and achieved, to assess if this modification could decrease PKC binding. The fluorine derivative showed only very low PKC binding, indicating that 20-deoxy-20-fluoroingenol esters can be used in establishing the mode of action and activities of ingenol esters.
Ingenol 3,20-dibenzoate is the prototype of anticancer ingenol esters. To increase the in vivo stability of the 20-ester group, the latter was replaced with hydrolytically more stable isosteric groups, such as an amide, urea or carbamate. The cloning of the vanilloid receptor requires the development of high-affinity radioactive probes, such as iodinated compounds. Using a suitable iodination protocol, iodo-CPS was synthesised. The affinity of the compounds for the VR-2 receptor was measured by the displacement of 3H-RTX from dorsal root ganglia. Iodination led to substantial retention of affinity for CPS and RTX, but to a marked increased for RTX. Indeed, iodo-RTX represents the most powerful vanilloid known.
Phorbol is a versatile template, which can be directed to different end-points by suitable molecular decoration. To test this strategy, elements of both RTX (the homovanillyl moiety) and the CPS pharmacophores (alkyl chain) were implanted on phorbol. Since the methylation of the 4-hydroxyl decreases affinity for PKC but not for the VR, this modification was also explored. The results of the biological evaluation of these compounds showed high affinity for the VR-1 receptor, as measured by calcium uptake, but a negligible affinity for the VR-2 receptor, as judged by displacement of 3H-RTX. These phorboid bisnonanoate homovanillates are the first vanilloid selective for the VR-1 receptor.
From the summary above it is clear that a number of new compounds have been synthesised or isolated from different sources and are available for testing now by the industrial partners in the project.
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