Dissertations / Theses on the topic 'Taxane synthesis][Synthesis of taxanes'

This thesis is a continuation of previous work done in the Jenkins group to produce taxanes from glucose. The reactions investigated by R. Bonnert and J. Howarth are improved upon to enable the large scale synthesis of the diol (1). After the successful protection of (1) with two tert-butyldiphenylsilyl protecting groups the benzylidene ring was opened by bromination with N-bromosuccinimide. This compound then underwent fragmentation with zinc metal to open the acetal ring and yield the highly functionalised key intermediate chiral cyclohexane (2). This aldehyde was then reduced with sodium borohydride and protected with a triethylsilyl group. The alkene side chain underwent ozonolysis cleanly followed by the addition of lithium-2,2,3,3-tetramethyl-bromocyclopropane. The cyclopropane addition was improved upon by the addition of cerium trichloride. Compounds (3) were isolated from this reaction and a series of studies were done to test the viability of these compounds to undergo further reactions, in particular to rearrange to give dienes. Compounds with bromine in were found to rearrange and those with chlorine in did not. It is believed that most of this work was done with compounds containing chlorine, although at the time this was not realised. Detailed spectroscopic studies verified the stereostructures as those desired for taxanes at the key chiral centres. Alternative sugar routes were also considered and this work gave rise to a new Robinson reaction on a carbohydrate, to give compound (4). Unfortunately this compound was found to have the wrong stereostructure for the synthesis of taxanes from it.

This thesis describes synthetic studies aimed towards developing a concise synthesis of taxinine, a member of the taxane class of natural products. In Chapter One, an overview of the strategies used for the synthesis of this class of compounds is provided. A bicyclic enone was identified as a target for the studies described in this thesis. The strategy described also called for the addition of the C-ring by ring-closing metathesis. A six step sequence to a C12-desmethyl A-ring is described in Chapter Two. The key step is the isomerization of an epoxide to an allylic alcohol. It was not possible to extend this chemistry to include C12-methylated substrates. An alternative Diels-Alder approach is also described. This approach allowed the synthesis of a number of C12-methylated A-ring structures. A concise synthesis of diene precursors suitable for exploring the possibility of RCM as a method for the ring closure to form a taxane AB-ring system is described in Chapter Three. The planned ring-closing metathesis reaction was unsuccessful under a number of conditions examined. An investigation described in Chapter Four delineates the use of ring-closing metathesis as a possible method for the introduction of the C-ring onto suitable AB-ring systems. An alternative to the unsuccessful ring-closing metathesis approach, an intramolecular Diels-Alder synthesis of a bicyclo[4.3.1]decene system, is described in Chapter Five. Preliminary investigations into the ring-expansion of this compound are also described. A brief summary and discussion of the future potential of the research conducted in this thesis is provided in Chapter Six.

Ce travail se divise en deux parties: la premiere partie s'attache a l'etude de la formation des cycles b et c du taxane sur un modele ainsi que de la stereochimie de la jonction de ces cycles b/c par voie photochimique. Le produit final obtenu: la 14,14-dimethyl-tricyclo 9. 2. 1. 0#3#,#8 tetradecanedione-2,10 possede la jonction de cycle trans 3,8 inverse de celle du taxane. La deuxieme partie decrit la synthese de la dihydroxy-2,12 trimethyl-4,8,11 tricyclo 5. 3. 1. 1#4#,#1#1 dodecene-8 one-5 qui par retroaldolisation conduirait a la formation du systeme bicyclo 5. 3. 1 undecene a/b du taxol, possedant des groupements oxygenes aptes a permettre l'annelation du cycle c. La synthese de ce systeme tricyclique decrite dans cette partie repose sur une condensation intramoleculaire de claisen de l'(oxo-10 hydroxy-7 trimethyl-1,3,9 cis bicyclo 4. 4. 0 decene-3 yl-2) acetate de methyle qui provient de l'adduit de diels-alder, le (dioxo-7,10 trimethyl-1,3,9 cis bicyclo 4. 4. 0 decadiene-3,8 yl-2) acetate de methyle, entre le methyl-4 hexadiene-3,5 oate de methyle et la dimethyl-2,6 para-benzoquinone. L'hydroxy-5 trimethyl-4,8,11 epoxy-2,5 tricyclo 5. 3. 1. 1#4#,#1#1 dodecene-8 one-12 ainsi produit conduit a la dihydroxy-2,12 trimethyl-4,8,11 tricyclo 5. 3. 1. 1#4#,#1#1 dodecene-8 one-5 dont la retroaldolisation s'est revelee infructueuse. La fragmentation de la liaison c-12-c-4 par l'amidure de sodium sur la cetone en c-12 non enolisable a fourni le trimethyl 4,8,11 epoxy-2,5 bicyclo 5. 3. 1 undecadiene-3,8 carboxamide-11 desire

This thesis details work towards the efficient production of low oxidation state taxanes either by synthetic biology or synthetic chemistry. With over 400 natural taxanes isolated to date, many low oxidation state taxanes have been eclipsed by the intense interest in Taxol®. Some of these low oxidation state taxanes have important medicinal properties, many are currently unexplored. Chapter I introduces the taxane family of natural products, their biosynthesis and biological activity. Chapter II documents our efforts to utilise synthetic biology to rapidly access low oxidation state taxanes. In this chapter we describe the semi-synthesis of a novel oxa-cyclotaxane (OCT2) and 5α-hydroxytaxadiene, both low oxidation state taxanes. Here we also report that taxadiene, extracted from genetically modified tomato fruit, can undergo epoxidation and rearrangement with a reduced iron porphyrin to form the same products encountered when taxadiene 5α-hydroxylase is expressed in foreign organisms. We conclude that the established free radical mechanism, based heavily on speculation, is most probably incorrect in favour of an epoxidation/ rearrangement mechanism. Chapter III describes our chemical synthesis of low oxidation state taxanes utilising carbon building blocks from renewable sources. We make use of the well-established Diels-Alder approach to construct the A and B taxane rings simultaneously, culminating in the synthesis of the natural product 5α-hydroxytaxadiene. This chapter then goes on to present both our work towards an asymmetric synthesis of taxanes utilising Yamamoto’s chiral BrØnsted acid catalyst and our efforts in the manipulation of taxane oxidation both by reduction and C-H oxidation.

Prostate cancer is the most common non-skin cancer for men in America. The androgen receptor exerts transcriptional activity and plays an important role for the proliferation of prostate cancer cells. Androgen receptor ligands bind the androgen receptor and inhibit its transcriptional activity effectively. However, prostate cancer can progress to hormone refractory prostate cancer (HRPC) to avoid this effect. Chemotherapies are currently the primary treatments for HRPC. Unfortunately, none of the available chemotherapies are curative. Among them, paclitaxel and docetaxel are two of the most effective drugs for HRPC. More importantly, docetaxel is the only form of chemotherapy known to prolong survival in the HRPC patients. We hypothesized that the conjugation of paclitaxel or docetaxel with an androgen receptor ligand will overcome the resistance mechanism of HRPC. Eleven conjugates were designed, synthesized and biologically evaluated. Some of them were active against androgen-independent prostate cancer, but they were all less active than paclitaxel and docetaxel. Discodermolide is a microtubule interactive agent, and has a similar mechanism of action to paclitaxel. Interestingly, discodermolide is active against paclitaxel-resistant cancer cells and can synergize with paclitaxel, which make it an attractive anticancer drug candidate. Understanding the bioactive conformation of discodermolide is important for drug development, but this task is difficult due to the linear and flexible structure of discodermolide. Indirect evidence for the orientation of discodermolide in the tubulin binding pocket can be obtained from fluorescence spectroscopy of the discodermolide tubulin complex. For this purpose, we designed and synthesized a simplified fluorescently labeled discodermolide analog, and it was active in the tubulin assembly bioassay. In addition, a conformationally constrained discodermolide was designed to mimic the bioactive conformation according to computational modeling. The synthetic effort was made, but failed during one of the final steps.
Ph. D.

博士
國立清華大學
化學系
86
The objective of this thesis is to find a rapid and efficient synthetic methodfor taxane and its biogenetic precursor -- seco- taxane. In the first section, we described the initial approach for the taxane synthesis. The ring expansion of bicyclo[3.3.0]octane to cyclooctane-ring and subsequently to taxane B,C ring and taxane A,B ring. However this approach was unsucessful. Later, we efficiently and successfully synthesized taxane A-ring precursor, compound 219, with 53% yield. Then compound 219 was subjected to Claisen rearrangement, reduction and Swern oxidation to form compound 243, a taxane A-ring analogue, with 42% overall yield. In the second section, we described the utilization of compound 243 as starting material in synthesis of taxane. Compound 243 on further alkylation, phenylselenyl cyclization, deprotection and Swern oxidation (4 steps) to yield a rigid compound 274 -- a taxane precursor. The alkylation of compound 243 resulted in two diastereomers 268 and 269. Compound 269 hence formed can be converted to 268 by Mitsunobu esterification and hydrolysis. Attempted intramolecular McMurry reductive cyclization on compound 274 failed. Although the molecular model suggests that the two carbonyls can be close enough to undergo cyclization, NOE experiments on compound 274 at various temperature revealed that the two carbonyl groups of compound 274 could be in close proximity only at temperature above 60℃ which is much higher than the McMurry coupling temperature. In the third section, we described the use of compound 219 as starting material in the synthesis of taxane and seco-taxane. The compound 219 was converted to 302 and 303 by a series of transformations including three key reactions: (1) macrolactonization (2) Tebbe reaction and (3) Claisen rearrangement. We tried to lactonize 278, 287 and 283 respectively to form 10-, 12-, and 14-member ring compounds, However only 283 underwent lactonization and afforded the 14-member ring compound 282 in 75% yield. Compound 282 was finally converted to compounds 302 and 303 in three steps, both having bicyclo[9.3.0]pentadecane backbone.

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.