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FAIR-CT96-1781 Terpenes as Natural Chiral Starting Materials For the Synthesis of Flavours, Fragrances, Pharmaceuticals and Biocontrol Agents |
Source: Final Report June 2001
Introduction
It is a generally accepted idea that mankind can explore and use the natural resources that surround us. This has to be done in a sensible way so that over exploitation should be avoided and a renewable source of plant material will be maintained. Surprisingly little is known about the available resources and their exploitation. This is even true for agriculturally produced plants, which are often grown to harvest one single product and the rest is considered as waste material, which in the worst case even may cause environmental pollution.
On the other hand, it is not so easy to mention or to find natural resources, e.g. plant material that can be collected from Nature or that can be produced by agriculture, which has been overlooked as a good source for a useful application. Many situations are known in which it is mentioned a pity to throw away plant material that still may contain valuable compounds but the nature and potential of these compounds are not known. Also the collection of herbs from Nature to cure or relieve al kinds of known, unknown or supposed diseases is widespread, but in most cases the active principles are not known. It therefore seems obvious to investigate these things and to gather more knowledge about the potentials of Nature and agriculture.
The present state of the art in chemical and medicinal sciences is such that investigations of available natural resources can be undertaken successfully. Chemical analysis is presently so advanced that structures of secondary metabolites can be elucidated in virtually all cases when the molecular weight remains below 500 D. Organic synthesis also has reached a state of perfection which allows the chemical synthesis or modification of such compounds. Testing of all these natural products is nowadays no problem and pharmaceutical companies even have an everlasting need for the new structures and new scaffolds that Nature can offer them. AB this is possible but not simple. Skilled teams of specialists are necessary and the research has to be financed.
In this project a team of university researchers and pharmaceutical and flavour and fragrance companies has been assembled, which is particularly suited to isolate, identify, synthesise, modify and test compounds from Nature. For better interaction between the participants, many of the tasks are concentrated on terpenoids, but it is clear that often also compounds from other classes of secondary metabolites have been isolated from plants.
Objectives
The objectives of this project can be divided roughly in two main goals.
Results
Part A. The main contributions to the first objective are concentrated on isolation and conversion of aromadendrene, alpha-santonin, isovelleral, drimanes, labdanes, saponaceolides, ionones, phorbol esters and taxanes.
The distillation of the oil obtained from the leaves of Eucalyptus globulus consists of about 70% aromadendrene. Participants 1, 8 and 9 have co-operated closely in this task to find economically feasible routes for the synthesis of attractive fragrance compounds and for the colouring substance guaiazulene from aromadendrene. Existing syntheses for these compounds are to long and expensive. Several methods were tried to obtain an alpha-epoxide at C7-C8, of ledene, the latter can be synthesised in one step from aromadendrene, but all attempts resulted in the beta-epoxide. Reactions with this beta-epoxide gave rearranged compounds with undesired structures. Also other methods, undertaken later in the project, to derivatise aromadendrene in the proper way, were either unsuccessful, resulted in orientated functionalities, or gave the wrong type of rearrangement.
Direct conversion of aromadendrene does result in guaiazulene, but a good industrially applicable procedure for this transformation is not available. The results from the dehydrogenation experiments under different conditions (batch vs continuous) show a very poor conversion of aromadendrene or congeners into guaiazulene. Commercial available palladium catalysts have been tested in the dehydrogenation reaction with a mixture of guaiol/bulnesol under batch conditions. Only in tetraglyme guaiazulene was formed at 200 °C in ca. 3% together with dehydrated product.
A much better and surprising result was obtained in the epoxidation of isoledene and the ring-opening of this epoxide to an ether with a guaiane skeleton.. This is the first time that the ring-opening of the cyclopropane ring in isoledene to the desired guaiane skeleton could be realised, besides the selectivity and yield of the reaction is very high. Several derivatives of the ether were prepared and their fragrance properties were tested. A new ring-opening reaction of the epoxide led to an oxy-patchoulene skeleton. Derivatives from this compound for testing as flavour substances still has to be carried out.
The cyclopropyl ring in isoledene could be opened to a mixture of guaiadienes, from which the endocyclic diene could be oxygenated to an endoperoxide, which is natural anti-fouling compound.
A successful route to pheromones starting from aromadendrene has been developed. The polycyclic aromadendrene was first converted into a chiral linear hydroxy ester by opening of the seven membered ring with a Baeyer-Villigcr reaction to a lactone followed by opening of the five membered ring with a Grob fragmentation. This fragmentation proceeds in good yield but it proved to be necessary to reduce the aldehyde immediately after its formation to a hydroxyl group. The obtained chiral hydroxy ester has been transformed into three linear pheromones with chiral centres approximately in the middle of the molecule. The field tests and commercialisation of one of these pheromones will be investigated. The synthesis of pheromones with two chiral centres is in an advanced state. A crucial intermediate has been synthesised in which both necessary chiral centres have been introduced. The project will be finished with the completion of the synthesis of this pheromone.
Alpha-santonin has been investigated by participants 6 and 7 as a second natural starting material, especially for the synthesis of eudesmanes and guaianes. Many of these sesquiterpnes show physiological activities like ichtiotoxicity and ixodicidity, and properties as cell-division inhibitor, fungistatic, antibacterial agent, antifeedant or deterrent against predators. The synthesis of two furanoeudesmanes starting from alpha-santonin has been achieved. Both these compounds show ichtiotoxicity towards a killi-fish Orizias latipes and the 15-acetoxy-derivative shows cytotoxicity against B-16 melanoma cells in vitro.
Irradiation of alpha-santonin solutions with UV light gives iso-photosantonic lactone and this compound was subjected to functional modification for the preparation of the natural guaianolides Podoandin and Zedolactone A. Podoandin, was isolated from Podocarpus andina, which has shown molluscicidal activity against the aquatic snail Biomphaiaria glabatrus, and it inhibits the germination of lettuce seedlings (Lactuca sativa). Zedolactone A was isolated, together with related compounds, from the dry rhizomes of Curcuma aeruginosa, 'Gajutsu' in Japan, which are used in oriental traditional medicine as gastrointestinal remedy.
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. A formal synthesis of this compound has been completed successfully.
The syntheses of two oxaguaianolides involved the photochemical rearrangement of the eudesmane skeleton of alpha-santonin into a guaiane skeleton followed by the degradation of the cyclopentane ring to a dicarbonyl compound, which was successfully transformed into the desired furanes.
Several amino sesquiterpenes have been prepared starting from santonin. The eudesmane compounds showed activity. In all cases treatment prevented affected insects to reach adult stage by stopping the development at the fifth nymphal stage. No feeding deterrence was observed. The compounds have been submitted also to partner 10 for testing their hormone activity.
Studies towards the synthesis of compounds with a dihydrooxepine unit have not yet been finished. One approach involved a regioselective Baeyer-Villiger reaction on C2-oxygenated tetrahydrosantonin derivatives in which oxygen insertion takes place exclusively between C2 and C3 which is the desired selectivity. Studies on the elimination of the group at C2 have been unsuccessful. A new approach, which involves a leaving group situated on C1 has been studied. An intermediate having a phenylselenenyl group at this position has been prepared and lactonization of this compound has allowed the synthesis of a suitable intermediate, although in low yield. Further transformations should allow the synthesis of spirafolide.
A second task around alpha-santonin has the objective to prepare novel derivatives and analogues from alpha-santonin and chamazulene with diverse lipophilicity and to determine their biological activity.
Catalytic hydrogenation of alpha-santonin and subsequent epimerization gave an intermediate that 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. Spiro rings containing hydroxy-enones, enones, ketones, hydroxyl groups, amino groups, acetals, lactones and lactams were obtained. The stereochemistry of the reaction products depends on thermodynamic, stereochemical and stereoelectronic effects. The anti-bacterial, anti-fungal and anti-coccidial activities of these 2H-pyran-3-one derivatives of alpha-santonin and their Michael type addition products were determined. Even though all the tested compounds showed some anti-microbial in vitro activity, the activity of the Michael addition amino intermediate adducts was of the same order of magnitude as the starting materials indicating their activity is due to a retro-Michael reaction. The reduced, more stable hydroxy compounds were found less active as anti-microbials. These compounds, however, were by far more active than their starting materials as coccidiostats. Various guaiazulene analogues and derivatives were synthesised and the antioxidant and free radical scavenging capacity of these analogues was estimated by the DPPH method. Also their effect on the proliferation of human leukaemia cells has been evaluated.
Furthermore the development of a biotechnological, cost-efficient method for the preparation of useful compounds from abundantly available native plants from Greece has been investigated. This 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 ensure the possibility of detecting somaclonal variants with increased chamazulene productivity or altered product production via biotransformation. The best results regarding the production of secondary metabolites were obtained when a solution of 26.8 icro-olar (alpha-naphthylacetic acid) and 11.5 micro-molar Kinetin was used as plant growth regulator. Furthermore, it was found that continuous sunlight is essential for the production of a measurable amount of the desired secondary metabolites. Seven secondary metabolites were identified as the major constituents of the essential oil from callus cultures of Chamomilla recutita. Chamomillol and Cubenol are the predominant ones, and the detected amounts of pharmacologically active metabolites such as (-)-alpha-bisabolol and (-)-alpha-bisabolol acetate, are better than those reported in the literature. The essential oil of native (widely found in Greece) Achillea taygeta was analyzed and found to contain several terpenes but no alpha-bisabolol or matricine (precursor of chamazulene) were detected.
Large amounts (approximately 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 has concentrated on the preparation of novel tropinones from the marasmane isovelleral. A variant of the classical Robinson-Schopf reaction has been developed and proceeds well. 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. The isolated new compounds and a series of isomeric tropinones are available for biological assaying by the industrial partners in the project.
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.
A series of other dialdehydes, natural compounds isolated from various mushrooms and plants or derivatives of natural compounds, has been prepared in order to compare their cytostatic activity towards cancer cell with that of isovelleral and the isovelleral analogues. Further insight about structure-cytostatic activity relationships for these compounds will assist in designing compounds with even higher potency.
The synthesis of tridemethylisovelleral has been finished in a nice way. The activity towards various cancer cell lines is very promising, and higher than that of several known and cancer drugs and of isovelleral itself (which has potent activity to begin with). A prodrug of it will be made, mimicking the way that the mushroom disguises isovelleral. Acidic hydrolysis (tumour cells have lower pH compared to normal cells) should generate tridemethylisovelleral in situ and kill the tumour cells.
Three intermediates having very pleasant odour were obtained during the synthesis of pterulone, an antibiotic fungal metabolite and these are currently being evaluated by Quest International.
The fruiting bodies of L. uvidus and L. luridus contain a large amount of drimane sesquiterpenes: drimenol, uvidins A and B and the corresponding fatty acid esters, which can be easily extracted and isolated in good yield. These compounds are attractive starting materials for the enantioselective semisynthesis of other important chiral terpenoids, in particular sesqui-, di-, and triterpenes possessing a trans decalin AB ring system. Both drimenol and uvidin A have already been used for the semisynthesis of biologically active compounds like cinnamodial, cinnamosmolide, and pereniporin A. Uvidin B has been converted into a diene, which will be employed for preparing an important intermediate for the formal enantioselective synthesis of forskolin. Chemistry starting from Uvidins has been developed by participant 5, but drimanes and forskolin derivatives can be obtained also starting from labdanes, as has been demonstrated by participant 1.
The gum of Cistus ladaniferus consists of a mixture of labdanediol, labdanolic acid, and a number of simple carboxylic acids. It is of industrial importance to investigate the possible use of this gum as starting material in the synthesis of Ambrox®. Ambrox® has been synthesised from natural occurring labdanes like labdanolic acid, sclareol, manool, manoyl oxide and in this task, next to labdanolic acid, also larixol has been investigated as a possible starting material for this compound. For this purpose the oxidative degradation of the side-chain in larixol and other labdanes has been studied systematically and a new protocol has emerged for the preparation of methyl ketones as side chain at C9.
With Cistus ladaniferus from season 1999, extractions have been carried out 'with a single solvent system. The preparation of Ambrox® starting from labdanolic acid has been achieved in three different ways, all using a iododecarboxylation as the key step. The third approach is the shortest one and gives the highest overall yield of 47%. The method is currently evaluated for industrial application. There proved to be a need for an analytical method for a quantitative analysis of a Cistus ladaniferus extracts and such a method has been developed.
A labdane diterpene that can be isolated from the terpentine from Larix Europaea is larixol. A number of simple derivatives of Ambrox, have been prepared from larixol in five different ways, the best routes show an average yield per step of over 80% and an overall yield of 18-20%. Acid treatment of 6-hydroxy-Ambrox in a Dean- Stark apparatus gives a mixture of delta5- and delta6-Ambroxene, which both had an interesting smell. The selective formation of delta5- and delta6 Ambroxene from larixol was studied also; the delta5 Ambroxene could be obtained straightforwardly in good yield, but a selective approach to delta6 Ambroxene proved to be complicated. A new and selective procedure has been developed for the synthesis of delta6 Ambroxene in which the ringclosure of an allylic alcohol to a cyclic ether is the key transformation. A similar approach has been applied for the selective synthesis of several C13 substituted delta6 -tricyclic tetrahydropyranyl ethers (delta6 -Ambra-oxides). A new synthesis of the flavour compound derivatives 6-oxo-sclareol and 6-oxo Ambrox® has been developed also. All the synthesised products have been send to Quest and Organon for testing.
For the synthesis of Ambrox-like structures from monoterpenes another route was investigated via reaction of the terpene epoxides with allyl magnesium bromide, ozonolysis, reduction of the aldehyde and finally the ring-closure of the diol to the tetrahydrofuran ring. With nopol or nopyl acetate as starting material the route is epoxidation, reduction and ring-closure.
Especially the higher functionalized drimane sesquiterpenoids, with their interesting biological properties, are attractive target molecules for syntheses starting from larixol. Since larixol has an hydroxyl group at C-6 as a characteristic structural element, it is particularly suited as chiral starting material for the synthesis of drimanes that have a functional group at the same position, like albrassitriol or mukaadial. The first semisynthesis of (-)-albrassitriol isolated from the fungus Alternaria Brassicae, and the enantioselective synthesis of (-)-drimenol has been accomplished. Drimenol is an inter-mediate in the synthesis of (-)-polygodial and (-)-warburganal, which have generated considerable interest because of their potent anti-feedant and molluscicidial activities. Later on the syntheses of (-)-uvidin C and (-)-epi-albrassitriol, which exhibits weak antiviral activity in in vitro tests against influenza A- and myxovirus, were completed as well.
The oxidation of the side chain of larixol and larixol derivatives to a methyl ketone has been a subject of special investigation in this project. The methyl ketones can undergo a Norrish type II photochemical degradation, which in the case of larixol and derivatives leads to an exocyclic dienes. These dienes have been shown to be excellent intermediates for the synthesis of drimanes, and the extra functionality at C6 that originates from larixol, enhances their synthetic possibilities. This has been demonstrated by the conversion of larixol into dienes and furanes that are key intermediates for drimane synthesis. The low conversion in the -photochemical reaction is however still a problem. This chemistry will be continued in co-operation with prof. Vlad in Moldova.
The conversion of larixol into analogues of forskolin has been successful with respect to the construction of ring B analogues with hydroxyl groups in the natural configurations. This research has been carried out in close co-operation with the Laboratory of Organic chemistry in Grenoble. Such compounds are useful for syntheses of 1-deoxy- or 1,9- dideoxyforskolin derivatives, which can provide further insight in structure/affinity relationships of the protein, which mediates glucose transport into the cell.
Extensive efforts have been invested in the further cyclisation of the side chain of larixol and its derivatives. The acid catalysed cyclisation, cyclisation with SmI2 and Diels-Alder cyclisation all did proceed, but with moderate results and the cyclisation did not always lead to the desired products. A key transformation in the preparation of many substrates for cyclisation was the conjugate addition of vinyl magnesium bromide to the enone in ring B. This was the only 1,4-addition that could be accomplished with this enone. A new and seldom seen aldol type of condensation has led to the synthesis of a tricyclic trienone.
The enantioselective synthesis of the saponaceolide structure has been investigated and a viable strategy for assembling of this structure has been 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 0-methyl ether of 2-epi-saponaceolide B in six additional steps. This excellent piece of work has led later on to an enantioselective synthesis of the anti-tumoral triterpenoid saponaceolide B, by duplicating the pathway already followed in the synthesis of 2-epi-saponaceolide B, but using an epimeric intermediate.
An original and efficient enantioselective synthesis of (lS,5R)-Karahana lactone and (1S,5R)-Karahara ether has been completed also. Comparing the fragrance properties of the two antipodal series, it was found that (1S,5R)-Karahana lactone and (1S,5R)- Karahara ether have a much more intense odour; particularly (1S,5R)-Karahara ether has a pleasant camphoric smell. It was realised that synthetic approaches to saponaceolides may be also applied to several cyclogeraniate and cyclofamesane terpenoids, such as karahana ether, cis-gamma-irone, alpha- and gama-ionone, and alpha- and gamma-damascone, which am important odour compounds. An intermediate of the synthesis of karahana ether was deoxygenated and has been used as precursors of gamma-ionone and gamma-damascone. The compounds were obtained in high yields and with high enantiomeric excess (ca. 90%).
The stereoselective synthesis of each stereoisomer of the tetrahydropyran linalool oxides, was achieved. These compounds have been found as constituents of many flower and fruit aromas, such as grapes and Carica papaya fruits. Two synthetic routes were developed and the four linalool oxides were produced in > 95% ee and >90% de.
A biomimetic approach was explored for the synthesis of 1,3-cis-disubstituted methylene cyclohexane substructures, which involved a new Lewis acid catalysed reaction of 1,5- dienylallyl silanes with carbonyl compounds. This method can be of general interest for the synthesis of other important natural compounds, such as taxol, gama-irone, or mispyric acid. Under optimised reaction conditions a mixture of alkylated-cyclized products was produced in 70-80% isolated yield (calculated on starting aldehyde). To our knowledge these are the best yields ever obtained in the biomimetic-like cyclization of -1,5- dienes promoted by alkyl electrophiles. A biomimetic-like cyclization of epoxygeraniol was investigated and it was discovered that hydrated ferric chloride nicely promoted cyclization, affording the desired diol and an internal ether in satisfactory yields.
Following the discovery of the powerful and selective vanilloid ligand phorbol 12,13- dinonanoate-20-homovanillate (PDNHV, 1), a phorbol-derived simplified analogue was prepared by reductive dimerisation of 0-benzyl capsaicin with zinc and titanocene dichloride. This compound was shown to be devoid of affinity for the vanilloid receptor. Nevertheless, it could induce apoptosis almost to the same extent as capsaicin.
All three natural capsiates were synthesised, and found active in assays of induction of apoptosis. A simplified analogue had the same activity as the natural products, and was .Y evaluated in vivo is assays of chemo-prevention, where it displayed powerful inhibitory activity against TPA-mediated cancer induction. This compound was chosen as a lead, and a series of derivatives were prepared.
The structure-activity study on the modification of the phorbol-RTX hybrid PPAHV was continued, focusing on the modification of the oxygen functions on the cyclopentenone moiety, a key structural element for vanilloid activity. A series of derivatives was obtained, which will be investigated in vanilloid activity assays.
In order to discover new acyl templates for vanilloid activity, a general synthesis of vanillamides was developed, based on the in situ formation of mixed phosphoric anhydrides. The procedure uses as condensation reagents DEPC (DiEthylPhosphoro-Cyanidate) or PPAA (n-Propane Phosphonic Acid Anhydride). Vanillamine could be employed as the stable hydrochloride, and the protocol could be successfully applied to unstable polyunsaturated acids (arachidonic, ximeninic, retinoic), whose transformation into acyl chlorides is not straightforward. The compounds synthesised are currently under investigation for vanilloid activity.
A large number of novel products have been prepared from the natural lathyrane diterpenoids Euphorbia factor LI and Euphorbia factor L3 as well as from two reduced derivatives of Euphorbia factor LI and Euphorbia factor L3. A number of novel and so far unknown diterpene skeletons have been identified and characterised, and the biological evaluation of these compounds is under way. Several novel derivatives of the lathyrane diterpenoids isolated from Euphorbia lathyris have been prepared in close collaboration between partners 3 and 4. A more complete understanding of the chemistry of this class of compounds has been generated.
Paclitaxel (Taxol®) is a new anti-tumour 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. Its therapeutic profile is still expanding and this has stimulated extensive research toward the synthesis of paclitaxel as well as the synthesis of new analogues with an even better therapeutic index. Development of new analogues is also connected with the synthesis of more water soluble derivatives.
It was found that next to the normal taxanes, crude taxine B contains two compounds that miss the 1-hydroxy functionality. Crude taxine B was isolated from dried needles of the Taxus baccata. From this taxine mixture the two compounds were obtained in 24% and 2% overall yield, and used as starting compound in the synthesis of new analogues. 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 this source.
Crude Taxine B is purified by methylation. The methylated 'Taxine B' precipitates from the ether solution as a salt, while the impurities remain in solution. The acetate functionalities are removed simultaneously with the ammonium group. Protection of the free hydroxyl groups affords the two products that are use as starting material. During the scaling up of the first steps of this synthesis a new spirotaxane was formed as a side product. This spirotaxane had a moderate anti-tumour activity. After these steps, partially protected 5-0-cinnamoyltaxicin II and fully protected 5-0-cinnamoyltaxicin I were obtained. The I- and 2-hydroxyl groups were protected as either a benzylidene acetal or as a carbonate group.
Due to the low availability of the starting compound that is present in only 2%, quick new analytical methods have been developed to screen Taxus cultivars for their taxine content, in order to find cultivars that can provide better and more starting material. 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 pachtaxel derivatives is 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.
Several D-ring modified 7-deoxypaclitaxel derivatives were prepared starting from taxane. A 'flexible' derivative with a 4beta-acetyloxymethylene group and a 4alpha-methoxy- carbonyloxy group was prepared in 10 steps with an overall yield of 1.8%. A 'Rigid' derivative with an alpha-epoxide ring and a 40-methoxymethylene group was synthesised in 10 steps with 0.7% overall yield. An approach towards another 'rigid' derivative failed in the last step, as epoxide ring opening occurred to afford a 'flexible' derivative. This compound was prepared in 9 steps with an overall yield of 1.2%.
The preparation of 1,7-dideoxypaclitaxel analogues from taxine, suffered from insufficient supply of starting material. Because of this another route was followed to a 1,7- dideoxypaclitaxel analogue which needed 3 more steps.
Within the framework of subtasks 12.3 & 12.4, two 'flexible' D-ring modified derivatives have been prepared. One derivative with a 40-methoxy group was prepared in 11 steps with 4.4% overall yield. Another 'Flexible' analogue with a 4beta-hydroxyl group and a 4alpha-methyl group was synthesised in 10 steps with 14% overall yield.
Four 'rigid' derivatives were also prepared. Two derivatives with a cyclohexene C- ring were synthesised in 11 and 12 steps, respectively. An analogue with a methyl- substituted double bond was prepared in 11 steps with 1.9% overall yield. Finally, the synthesis of a derivative with an alpha-cyclopropane ring and a 40-hydroxyl group was accomplished in 11 steps with an overall yield of 1.4%.
All derivatives have been handed over to participant 11 for evaluation of their cytotoxic activity in a range of seven well-established human cancer cell lines. At this moment, only the results for four derivatives have been handed back. The flexible derivatives were both 1,000 - 10,000 times less active than paclitaxel. Two other derivatives were about 500 - 2,000 times less active than paclitaxel.
Part B The second main goal of the project is devoted to phytochemical research with the aim to discover new useful substances from plants.
Compositae of agricultural relevance such as Artemisia nitida Bertol have been investigated with respect to its content of sesquiterpene lactones. This species is endemic to the Northern Appennins, and is used for the production of liqueurs owing to its bitter taste and pleasant aroma. The aerial parts gave fourteen sesquiterpene lactones and one seco caryophyllene derivative.
Two related species, Achillea erba-rotta All. and A. moschata Wulf, grow in the Western Alps and are employed in the production of bitter liqueurs. Very little was known about their constituents. Besides highly methoxylated flavonoids, a series of related sesquiterpene lactones was isolated and identified. The two plants contain closely related compounds, but have distinctive different aromas. The sesquiterpene lactones from these plants are similar to those from Artemisia genipiWeber, the most valued species for liqueur production. However, highly methoxylated flavonoids are typical of these two Achilleae, and should allow a straightforward distinction with A. genipi .
Two samples of the essential oil of Franseria artemisioides were found to be particularly rich in mono- and sesquiterpene hydrocarbons.
Extracts of several plants used in Equadorian herbal medicine have been investigated. An ETOH extract of Baccharis teindalensis afforded no new terpenoid compounds; instead, it is very rich in flavonoids. The CH2Cl2 extract of Schistoscarpa aff eupatorioides gave safranol and a mixture of the terpenoid hydrocarbons beta-pinene, p-cymene, limonene, gamma-terpinene, caryophyllene, aromadendrene, selinene, beta-patchoulene, germacrene D, alpha-himachalene, beta-gurjunene, alpha-cedrene, delta-guaiene, gamma-muurolene and alpha-humulene. Cyperus odoratus L. is used in Ecuador against dysentery; the plant has a pleasant scent. Eleven compounds were indentified from this plant, but only one of these was a new compound.
Two Ecuadorian plants, named Coussarea macrophylla and Conyza floribunda have been investigated. The former is reported to have anti-viral activity while the latter was found to have analgesic and anti-inflammatory properties. Multiple chromatographic separations of an ETOH extract of C macrophylla generated three new anthracene derivatives.
An ETOH extract of C. macrophylla, which is reported to have anti-viral activity in vitro, gave five new secodammarane triterpenes and three new cycloartane triterpenes, in addition to new secodammarane and secocycloartane triterpenes, three new anthracene derivatives and an antraquinone have been isolated. The oxygenation pattern of the quinone was confirmed by total synthesis. Compounds were inactive in the brine shrimp lethality assay.
Isolation and characterisation of new compounds from the fruiting bodies of the inedible European mushrooms Lactarius violascens (Otto) Fr. and L. rubrocinctus Fr. yielded two new protoilludane, and four new marasmane sesquiterpenes.
The fruit bodies of Russula delica have been investigated and this has yielded many new sesquiterpenes. The major component found in intact fruit bodies is stearoyldelicone, which is very sensitive to traces of acid.
Fruiting bodies (250 g) of Lactarius subumbonatus Lindgr. (syn L. serifluus (D. C. ex Fr.) Fr.] were minced at - 20° C and extracted with CH2Cl2 . Chromatographic separation of the extract (150 mg) gave glycerides and a new caryophyllane ester.
Chromatographic separation of the CH2Cl2 extract of Russula lepida produced, instead, 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.
The contents of fruiting bodies of four inedible mushrooms growing on Italian Apenninines have been investigated. Mycena inclinata gave the interesting sphaeroane diterpene tintinnadiol, which has been isolated previously from the related species Mycena tintinnabulum. This compound exhibited cytotoxic effects in vitro. Intact fruiting bodies of Lactarius atlanticus contain three protoilludane sesquiterpenes, one of them has been assigned a new structure. The latter is the first example of a naturally occurring protoilludane carrying an ester function at C-12.
The enzymatic conversions occur-ring in damaged fruiting bodies of Lactarius subdulcis have been studied in detail. 6-Chetostearoyl velutinal is contained in intact fruiting bodies and it is completely converted into known lactarane sesquiterpenes in injured samples, This conversion seems to follow a new biosynthetic pathway, not involving the intermediate formation of dialdehyde or hydroxy-aldehyde lactaranes.
Investigation of the terpenoid contents of fruiting bodies of Tricholoma ustaloides Romagn., a mushroom (Basidiomycetes) growing on Italian Apennines led to the isolation, in addition to five new trichoaurantianolides, of tricholidic acid and two of its derivatives, and of two new saponaceolides E and F. Four new cucurbitane triterpenes were isolated from the extremely bitter mushroom Leucopaxillus amarus. These compounds belong to rare classes of naturally occurring terpenoids. A few of them will be submitted to biological tests, since analogous saponaceolide and cucurbitane derivatives are known to display different kinds of bioactivity.
Phytochemical analysis of Tricholoma saponaceum and Tricholoma sejunctum, two typical mushrooms of Italian Apennines, allowed the isolation of six new lanostane triterpenes. They are characterised by the presence of depsipeptide side chains containing the aminoacids phenyl- alanine and phenylserine. From Tricholoma columbena, a mushroom with a similar habitat of the other two Tricholoma, a new non-isoprenoid compound was isolated and the structure again was confirmed by total synthesis.
Tricholoma vaccinum Kummer was extracted with EtOAc at -20 °C. The extract contained five very polar compounds named tricholomenyn C- G.
The collaboration that has been established between partners 3 and 4 has continued in investigations of the sesquiterpenoid contents of fruiting bodies of Lactarius atlanticus Bon, a mushroom (Basidiomycetes) growing in mixed forests of Central Italy. Three new oxygenated protoilludane sesquiterpenes have been discovered.
A three step isolation procedure to obtain ingenol from the seeds of Euphorbia lathylis L., an agricultural commodity, was developed. As a byproduct of ingenol production, large amounts of lathyrane triesters were obtained. The structure of the Euphorbia- factors L3 and L8 was established by 2D NMR experiments, which showed that the aromatic ester group (benzoate in L3, nicotinoate in L8) was bound to the 3-hydroxy group. The pharmacophore of ingenol is poorly known. Key probes to solve these points have been designed, and key intermediates bearing orthogonal protection at C-3 and C-20 have been prepared, e.g. ingenol 20 trityl ether-3,4-acetonide and ingenol 20 trityl ether-3- benzoate. An investigation for new scources of ingenol in spurges endemic to Europe was started, looking for species lacking cocarcenogenic compounds. The first species investigated was E. semiperfoliata Viv., a plant endemic to Sardinia, to which none of the traditional uses of spurges (cathartic and fish-poisoning etc.) had been attributed. The aerial parts of this plant turned out to be devoid of inflammatory and cocarcinogenic compounds. Two enones of a new structural type, one abietanolide, thirteen jatrophadienes, and two 4- deoxyphorbol derivatives were isolated as new compounds.
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, showing that 20-deoxy-20-fluoroingenol esters are promising candidates to dissect the various activities of ingenol esters.
Ingenol 3,20-dibenzoate is the prototype of anti-cancer ingenol esters. To increase the in vivo stability of the 20-ester group, the latter was replaced with hydrolytically more stable isosteric groups, like an amide, urea or carbamate.
As a by-product of ingenol production, large amounts of lathyrane triesters were obtained. A new collaboration between participants 3 and 4 concerning the transannular cyclisation of macrocyclic lathyrane diterpenoids is initiated, with aim of producing novel compounds with interesting biological activities.
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 could be 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, also this modification was 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.
A series of vanilloids selectivc for capsaicin-type pharmacology was prepared, as exemplified by phorbol 12,13-bisdecanoate-20-homovanillate. These compounds show negligible activity in vanilloid receptor assays (displacement by 3H RTX from DRG neurones), but are active in functional assays (calcium uptake) both in DRG- neurones and in cells transfected with the vanilloid receptor. These compounds are important new tools to investigated the pharmacology of the vanilloid receptor and to design drugs for diseases where malfunctioning of vanilloid-sensitive neurones is involved (chronic pain, post-herpatic neuropathy, urinary incontinence).
From Sardinian euphorbias. E. characias L. and E. pithyusa L: subsp. cupanii over 20 diterpenoids were isolated and identified. Of these 12 were new, belonging to various classes (atisanes, kauranes, pimaranes, lathyranes, premyrsinanes, tiglianes). Some of the isolated compouds are present in the plant material in fairly large concentration, and are promising candidates for the construction of semi-synthetic libraries.
The isolation of terpenoids from spices and edible plants of agricultural relevance has focused on the large-scale isolation of the zucchini-factor A from the seeds of pumpkin. The constituents of the flowers of a variety of Roman camomile (Anthemis nobilis L.) cultivated in Piedmont were investigated, isolating a series of known sesquiterpene lactones as well as very large amounts (ca 30 g/Kg) of the flavone apigenin.
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 crispanc, 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 (BBP). 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.
The roots of clarly sage (Salvia sclarea L.) represent an untapped source of secondary metabolites of potential use in several contexts (pharmaceutical, nutraceutical, cosmetic, perfumery). The roots of clarly sage were provided by a local grower, and turned out to contain ca 0.1% (w/w) of the deep-orange diterpenoid quinone aethiopinone. Though no biological activity was reported for this compound, its endocyclic isomer shows anticancer activity, and another derivative is a patented clinical candidate for several types of cancers.
To get a reliable source of the non-poisonous chemotype of the giant fernel, over 150 samples of latex collected in Sardinia were analysed, discovering that the distribution of this chemotype is limited to three enclaves. This guided the collection of the plant material, allowing thorough chemical characterisation. This plant gave large amounts of the phytoestrogen fenitinin, its corresponding benzoate, and the sesquiterpene alcohol, allohedycariol. Among the minor constituents, eight additional daucanes, three sesquiterpene-coumarin ethers, three phenylpropanoids as well as a new cadinanetriol were obtained.
Another activity has focused on St. John's wort (Hypericum perforatum L.). This plant is extensively used to make liqueurs, but has recently been at the forefront of phytochemical research because of the antidepressant activity of its extracts. On account of these potential beneficial activities, the chemical modification of the natural compound was investigated, with the aim of obtaining compounds more suitable for pharmaceutical development. The modifications investigated were akylative, acylative and oxidative. All resulting compounds were basically inactive in experiments of re-uptake of various biogenic amines, showing that the integrity of the enolized P-dicarbonyl system is essential for activity.
The preparation of more derivatives of hyperforin has been focused on the synthesis of new esters of hyperforin. Some of them can act as a pro-drug of the natural product. Among the esters prepared, the trimethoxybenzoate turned out to be highly crystalline, which allows its preparation directly from the crude hexane extract from the plant. The reduction of hyperforin with LiAlH4 furnished a bis-reduced product. This turned out to be totally refractory toward acylation reactions. Reaction of hyperforin with halogenating reagents resulted in halogenation of the enolized "carbonyl" system. Hyperforin has been amenable to acylative, oxidative, alkylative, and reductive modifications. A series of derivatives was prepared, whose biomedical potential is currently under investigation.
Future actions
Many new compounds have been isolated from plants or have been synthesized and testing of these compounds is still going on. Testing activity of the industrial partners in the project on substances obtained within the project has led to promising results. New cooperations between participants have started up, which have contributed to the coherence of the project, and which will continue in the future. Several total syntheses will be finished by students or otherwise. In general the cooperation and experience with this project has been very successful and stimulating. A new project, which can be considered to be the follow up of this one will be created and submitted for support to the EU.
© Copyright 2006 Policy Statements
Updated
by CPL Press
3 July, 2007
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