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SEMI-SYNTHETIC APPROACHES TOWARDS ANALOGUES OF
PACLITAXEL, FROM BREVIFOLIOL AND TAXCHININ A
Thesis submitted for the degree of
Doctor of Philosophy
Lucy Anne Swallow
Department of Chemistry
University of Leicester
October 1997
UMI Number: U105953
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STATEMENT
This thesis, submitted for the degree of Doctor of Philosophy, entitled ‘Semi-Synthetic
Approaches Towards Analogues of Paclitaxel, from Brevifoliol and Taxchinin A’, is based on
work carried out by the author, Lucy Anne Swallow, in The Department of Chemistry, at The
University of Leicester, between October 1994 and October 1997. All the work presented,
herein, is original unless otherwise stated and referenced, none of this thesis has been
submitted for any other degree at this, or any other, university or establishment.
Signpri- U m j . ^ -'.W oX U lJ1
SEMI-SYNTHETIC APPROACHES TOWARDS ANALOGUES OF PACLITAXEL, FROM BREVIFOLIOL AND TAXCHININ A
ABSTRACT
This thesis describes our approaches towards the construction of a new taxol analogue, beginning with brevifoliol 1 or taxchinin A 2 both isolated, here in Leicester, from an Indian Yew resin.
HOBzO BzO PH OHjPAc OAc OAc
HO....<HO..... HO....< 'OH'OH
OAc HOHO HO31 2
Attempts to selectively protect either OH(5) or OH(13), or construct the oxetane ring across positions C-4/C-5, directly on brevifoliol 1, were unsuccessful. Hence, we hydrolysed all three ester groups, affording compound 3 for further investigations. On treating this derivative with di-terf-butylsilyl ditriflate, then triethylsilyl chloride, we achieved the successful formation of compounds 4 and 5. Conformational analysis of derivatives 3, 4, and 5, using 2-D, NOESY and 29Si-!H NMR spectroscopy, allowed us to determine the twist- boat/chair conformation for both these compounds. Assignment of these conformations, containing the unusual hydroxysilyl ether groups, strengthened our knowledge of 11(15- l)a&eo-taxanes, allowing us to continue work towards oxetane ring construction.
4 5
In addition, hydrolysis of taxchinin A 2, furnished compound 6 . Variable temperature NMR analysis showed this derivative exists, in solution, in conformational equilibrium. Treatment with di-terr-butylsilyl ditriflate then triethylsilyl chloride, gave compounds 7 and 8 in the twist-boat/chair and twist-chair/boat conformations respectively. This was again confirmed using 2-D and NOESY NMR spectroscopy.
™,OTES
OTES
H,CCH,
OH
76
We have shown, that in carrying out work on these rearranged taxanes, conformational behaviour is crucial to the outcome of the reaction and, as such, must be taken into account when constructing a new taxol analogue.
ACKNOWLEDGEMENTS
I would initially like to thank my superviser Dr. Paul Jenkins for all his advice and
encouragement, particularly over the last three months. My thanks and appreciation also go
to Pharmachemie BV, The Netherlands, for their financial support. I would also like to give
huge thanks to Dr. Gerry Griffiths, for all his help running all the 2-Dimensional, low
temperature and 29Si NMR spectra, my thanks also go, to Dr. Mike Sutcliffe for all his help
with the molecular modelling, to Roy for building the extraction apparatus and to Mick Lee
for all his technical support. I am also grateful to the rest of the group, past and present,
Claire, Sam, Andy, Gary, Michelle, David and Frank, for all their support when things were
not quite going to plan.
I would especially like to thank all my friends, Abby, Helen, Zoe and Julie, for lots of good
times in between all the work, thanks to Will, for putting up with me, to Russ and Vicky, for
being brilliant house mates over the last three years and also to Kev and Ange, the newest
residents of Stanfell Road.
Finally, I would like to dedicate this thesis to my family, Mum, Dad and Catherine, who have
always been there to give me loads of support, advice and encouragement in everything I have
chosen to do.
ABBREVIATIONS
a proton down LDA lithium
(spectral) diisopropylamide
[a] specific rotation LTMP lithium tetramethyl
piperidine
Ac acetyl m multiplet (spectral)
p proton up (spectral) mmol millimole(s)
Boc terf-butoxycarbonyl m/e mass to charge ratio
br broad (spectral) Me methyl
BOM benzyloxymethyl MHz megahertz
Bu butyl min minute(s)
s-Bu sec-butyl mp melting point
t-Bu tert- butyl Ms methanesulfonyl
Bz benzyl (mesyl)
°C degrees Celcius NMO 4-methylmorpholine
COSY correlation spectroscopy N-oxide
6 chemical shift in parts per NMR nuclear magnetic
million, downfield from resonance
tetramethylsilane nOe nuclear Overhauser
DABCO 1,4-diazabicyclo[2.2.21octane effect
DCC dicyclohexylcarbodiimide NOESY nuclear Overhauser
d doublet (spectral) effect spectroscopy
DMAP 4-(dimethylamino)pyridine np Neumann projection
DMF dimethylformamide Ph phenyl
El electron ionisation ppm parts per million
eq./equiv. molar equivalent(s) Pr propyl
Et ethyl i-Pr isopropyl
FAB fast atom bombardment Py pyridine
g gramme(s) q quartet (spectral)
hs hours s singlet (spectral)
HPLC high performance liguid t triplet (spectral)
Hz hertz TLC thin layer
chromatography
JR infra-red TBAF tetra-n-butyl
ammonium
J coupling constant fluoride
TES triethylsilyl TMS trimethylsilyl
Tf trifluoromethane Ts p-toluenesulfonyl
sulfonyl TsOH p-toluenesulfonic
THF tetrahydrofuran acid
TIPS tri-iso-propylsilyl Xs excess
TBDMS terf-butyldimethyl
silyl
CONTENTS
CHAPTER ONE
INTRODUCTION
1. CANCER AND THE CELL
1.1. Healthy Cells and Cell Division 1
1.2. The Role of Microtubules 2
1.3. Cancerous Cells 3
2. NATURAL PRODUCTS AND THEIR PART IN DRUG DEVELOPMENT
2.1. Background 4
2.2. Taxol & its Role In Cancer Chemotherapy 5
2.2.1. Discovery Development and the Yew Tree 5
2.2.2. Taxol’s Unique Mode of Action 6
2.2.2.1. Spindle Destroyers 6
2.2.22. Spindle Promoters 7
2.3. The Problems with Taxol'.Ecological and Biological 7
3. SOLUTIONS TO THE TAXOL PROBLEM
3.1. Biological Methodology 8
3.1.1. Plant Cell and Tissue Culture 8
3.1.2. Biosynthesis and Genetic Engineering 10
3.1.2.1. From Geranylgeranyl Pyrophosphate 10
3.1.2.2. Taxa-4(5),ll,12-diene 1 1
3.2. Total Synthesis of Taxol 12
3.2.1. Holton 12
3.2.2. Nicolaou 14
3.2.3. Danishefsky 15
3.2.4. Wender 16
3.3. Semi-Synthesis of Taxol & Taxol Analogues 17
3.3.1. Increasing Water Solubility Using Semi-Synthesis 18
3.3.2. Semi-Synthesis of Taxol 19
3.3.3. Taxotere 20
4. REARRANGED TAXANES
4.1. Discovery and Nomenclature 21
4.2. Kingston’s Discovery of Ring-A Contracted Taxanes 22
4.3. Studies by Francoise Gueritte-Voeglein 25
4.4. Studies by GiovanniAppendino 25
4.4.1. Suggested Mechanism For Acid Catalysed Ring-A Contraction of
10-Deacetyl Baccatin HI 26
4.5. Biological Evaluation and Potential for 1 l(15-l)<2&eo-taxanes 27
5. NATURAL PRODUCTS POSSESSING THIS REARRANGED TAXANE
SKELETON
5.1. Discovery, Nomenclature and Problems in Identification 28
5.2. Determination of Brevifoliol’s True Structure 29
5.2.1. Evidence for the Revised Structure 29
6 . 11(15-1 )ARE(9-TAXANES
6 .1. History and Development of New 11(15-1 )a&eo-taxanes 31
6.2. Correctly Assigned New 11(15-1 )<zfceo-taxanes and Conformational
Isomerism 32
6.2.1. Cycloheptane 33
6.2.2. Studies by Fuji into Conformational Isomerism in 11(15-1 )abeo-
taxanes 34
6.2.3. Appendino, Investigations into Conformational Isomerism 40
6.3. Growing Interest in 11 (15-1 )a&£<?-taxanes 42
7. THE PURPOSE, AIMS AND INTENTIONS BEHIND THIS THESIS 43
CHAPTER TWO
INITIAL STUDIES
1. THE TAXOL SIDE-CHAIN
1.1. Denis and Greene’s Original Synthesis 45
1.2. Improved and Adapted Synthesis 46
1.3. Alternative Syntheses, e.g. p-Lactam Approach 47
2. BREVIFOLIOL, STRUCTURE DETERMINATION
2.1. Introduction 49
2.1.1. The Twist-boat/Chair Conformation 50
2 .1 .2 . The Twist-Chair/Boat Conformation 51
2.2. Brevifoliol, Conformational Analysis 53
2.2.1. Conclusion from These Studies on Brevifoliol 54
3. C-5 Vs C-13 REACTIVITY IN BREVIFOLIOL 55
4. CINNAMIC ACID COUPLING 57
4.1. G. I. Georg, Side-Chain Attachment to Brevifoliol 58
5. TOWARDS THE OXETANE RING
5.1. Approaches by Ettouati 59
5.2. Proposed Scheme, Towards Oxetane Ring Construction 61
5.3. Revised Scheme 65
6 . RING CLOSURE 6 6
6.1. Conclusion 67
7. NATURAL PRODUCT ISOLATION
7.1. Yew Tree Pharmaceuticals, Extraction of Brevifoliol 6 8
7.2. Laboratory Scale Extraction Procedure 70
CHAPTER THREE
NEW APPROACHES
1. BREVIFOLIOL HYDROLYSIS 72
1.1. NMR and Conformational Analysis 73
2. INVESTIGATING HYDROXYL PROTECTION 75
2.1. Results and Discussion 76
2.1.1. Initial Investigations into Acetonide Formation 76
2.1.2. Alternative Protecting Groups 78
3. REACTION WITH DI-terf-BUTYLSILYL DITRIFLATE 79
3.1. Initial Thoughts & Hypotheses 79
4. REACTION WITH TRIETHYLSILYL CHLORIDE 81
4.1. Reaction between Unknown A and Et3SiCl 81
4.1.1. NMR Analysis of the Compound Possessing Two Triethylsilyl
Groups 83
4.1.1.1. Beginning With The NOESY Spectrum, NMR 1 84
4.1.1.2. 29SiNM R,NM R2 85
4.1.1.3. Returning To The NOESY Spectrum 85
4.1.2. NMR Analysis of the Compound Possessing One Triethylsilyl
Group 87
4.1.2.1. Beginning With The NOESY Spectrum, NMR 3 89
4.1.2.2. The !H-29Si NMR Spectrum, NMR 4 89
4.1.2.3. Returning To The NOESY Spectrum 90
4.2. Reaction Between Crude Unknown B/Unknown C and
Triethylsilylchloride 91
5. DI-fert-BUTYLSILANE DIOL 92
6 . HYDROXYSILYLMONOETHERS 94
7. SUMMARY AND CONCLUSIONS 95
7.1. Revised Structural Identification of Unknown A 95
7.2. Summary & Conclusion of Reaction with Triethylsilyl Chloride 97
8 . FURTHER SYNTHESES 98
8.1. Determining Position of Triethylsilyl Protection 98
8.2. Selective Deprotection of One Triethylsilylether 102
9. FURTHER APPROACHES TOWARDS OXETANE RING CONSTRUCTION
9.1. Dihydroxylation 103
9.2. Approaches Towards Ring Closure 104
9.2.1. Nicolaou, Mesylate Approach 104
9.2.2. Nicolaou Approach, Triflate Method 105
9.3. Conclusion 106
CHAPTER FOUR
TAXCHININ A
1. TAXCHININ A, STRUCTURE DETERMINATION 107
2. HYDROLYSIS OF TAXCHININ A 109
2.1. NMR Analysis of Hydrolysed Taxchinin A 111
2.2. Conformational Analysis 115
3. REACTION WITH DI-rm-BUTYLSILYL DITRIFLATE 118
3.1. Initial Identification of Unknown B 119
4. REACTION WITH TRIETHYLSILYLCHLORIDE 120
4.1. NMR Analysis of Compound 198 121
4.1.1. Analysis of the NOESY Spectrum, NMR 6 123
5. FURTHER REACTIONS AND STRUCTURE IDENTIFICATIONS
5.1. Reaction with AD-mix-a 125
5.1.1. NMR Analysis of Compound 200 126
5.1.1.2. Analysis of the NOESY Spectrum, NMR 7 128
5.2. Revised Structural Identification of Unknown B 129
6 . CONCLUSION AFTER STRUCTURAL IDENTIFICATIONS 130
7. FURTHER APPROACHES TOWARDS THE OXETANE RING 132
8 . FINAL CONCLUSION 134
CHAPTER 5
EXPERIMENTAL
1.1. General Experimental 135
1.2. Nomenclature 136
2. SIDE CHAIN 137
3. NATURAL PRODUCT EXTRACTION 141
4. INITIAL STUDIES 144
5. NEW APPROACHES 154
6 . TAXCHININ A 177
REFERENCES 185
Appendix 1 190
Appendix 2 191
Appendix 3 192
Appendix 4 193
CHAPTER ONE
INTRODUCTION
DISEASE TODAY
Over the last one hundred years a spectacular improvement in life expectancy has taken
place, for people living in developed countries, indeed by the 1980s the average overall
life span had increased by 50%. At the beginning of the century, death was common after
only 45 years, however, by 1985 this life span had approximately doubled. 1 This
phenomenon (see attached supplement) can be attributed to a cleaner environment, better
sanitation, and to the control of infectious diseases, such as cholera and tuberculosis. We
cannot, however, rest on our laurels, with the increase in life expectancy has come a
marked change in the most common causes of death. Since the advent of antibiotics, these
are no longer infectious illnesses but are what we might call degenererative diseases, for
example, heart disease, cancer and AIDS. The new challenge, as we approach the
millennium, is to further increase life expectancy and improve quality of life, by
controlling such diseases to the extent that some of these life threatening illnesses will,
effectively, be cured. Great progress has been made in the understanding and treatment of
heart disease, cancer treatment, however, is at an earlier stage of development and is
currently an important area of research.
1. CANCER AND THE CELL
1.1. Healthy Cells and Cell Division
Most of the 1013 cells in the human body spend their time in an inactive state outside the
cell division cycle, nevertheless, an adult human still produces millions of new cells each
second to compensate for general wear within the body.2 Cells replicate by duplicating
their contents and dividing into two, when more cells are needed, each cell is induced into
1
entering a highly controlled, efficient cell division sequence, mitosis, fig. 1 , at Gi in
interphase. During S-phase, new DNA, within the parent cell, is synthesised, allowing
each chromosome to divide in two, each with a full complement of DNA. After G2 phase,
each replicated chromosome begins to condense and M-phase commences during which
time the mitotic spindle forms accompanied by nuclear breakdown, allowing the formation
of two nuclei. Following this, chromosomes align on the equator of the mitotic spindle,
then division occurs as they pull apart into separate chromatids. As the cytoplasm divides,
two new daughter cells are produced, each with a single nucleus, with all characteristics
identical to that of the parent cell.
V-;
w m m:&2$«d»KK
fig. I3
1.2. The Role of Microtubles
Microtubles play a crucial part in the body’s cell activity, for example, they have vital roles
in the formation of the cytoskeleton and in the communication of cellular signals. 3 Most
pointedly, however, is their essential role in the formation of the mitotic spindle, during
cell division. A microtuble is made up, mainly from 2 protein sub-units, a and p tubulin,
polymerisation begins, shown in fig. 2 , with the joining of one molecule of a and one
molecule of p-tubulin. In the presence of MAPS (microtubule associated proteins), Mg2+
2
and GTP, the heterodimers join head to tail forming a nucleation centre for protofilament
formation. Continuing growth leads to microtubule development, which forms the basis of
the mitotic spindle during cell division, thereafter, an equilibrium is usually set up at each
end of the microtubule, with constant loss and gain of tubulin sub-units until the
microtubule and free tubulin reach a critical concentration. Crucially, if tubulin
concentration is below the level required to achieve equilibrium, microtubule assembly
cannot take place, seriously affecting the cell division cycle.
fig.23
1.3. Cancerous Cells
The cell cycle is a linear pathway whereby completion of the previous phase is required
before beginning of the next. Healthy cells possess ‘checkpoints’, or mechanisms, forcing
this to occur, cancerous cells, however, lack these constraints and hence division is
uncontrolled.2, 3 Tumours form as abnormal cells proliferate continuously and are
malignant when cells begin to invade surrounding tissue, forming secondary tumours or
metastases, cancers from epithelial cells are carcinomas, those from muscle cells are
sarcomas and other cancers usually fit into the broad category of leukaemias . 2
3
2. NATURAL PRODUCTS & THEIR PART IN DRUGDEVELOPMENT
2.1. Background
Chemical compounds in all living organisms are synthesised and broken down by a series
of reactions controlled by enzymes, that is, each cell can undergo a sequence of pathways
collectively known as metabolism .4 Primary metabolic pathways produce compounds
essential for life, particular examples being, proteins and DNA, secondary metabolic
pathways, however, generate compounds with no obvious apparent use, so called ‘natural
products’. It is these compounds that have formed the basis for many dmgs and
pharmaceuticals developed since mediaeval times and constitute a large part of the
pharmaceutical industry today.
Since the beginning of the nineteenth century, plants have been screened for secondary
metabolites, possessing crucial biological modes of action, for the treatment of all kinds of
illnesses, ranging from heart disease to cancer. There are between 300 and 500 thousand
plant species in the vegetable Kingdom,5 a plethora of potential sources for anti-tumour
drugs. However, cancer is an extremely complex disease in which uncontrolled cell
division may be induced by environmental factors such as exposure to radiation, or after
prolonged contact with a carcinogenic compound, or, it may be determined by some form
of genetic mutation taking place within the body. Once the body is producing tumours,
current treatments are surgery, radiotherapy or chemotherapy, with the effectiveness of
these procedures being evaluated by the improvement in life expectancy they produce.
Natural products form the basis of chemotherapy,6 they can be used as they stand or
alternatively they are often modified generating natural product analogues, or new
synthetic equivalents are developed with their structure based on a naturally occurring
similar compound. Each of these compounds are classified depending on their mode of
action within the cell.
4
2.2. Taxol & Its Role In Cancer Chemotherapy
2.2.1. Discovery, Development and The Yew Tree
In the quest for new therapeutics, the NCI (National Cancer Institute) set up a programme
in 1960, in the USA, in collaboration with the USDA (US Department of Agriculture),
where over 35 000 plants were screened for compounds possessing anti-cancer activity.3
As part of this ongoing programme, the Pacific Yew, Taxus brevifolia Nutt., a slow
growing yew from the Pacific Northwest, was collected by botanist A. Barclay.7 The Yew
tree is classified, taxonomally, in the genus, Taxus, which contains around 10 species in
the Northern Hemisphere, exemplified not only by the Pacific (or Western) Yew, Taxus
brevifolia Nutt., but also by the European Yew, Taxus baccata Linn., the Japanese Yews,
Taxus cuspidata Zieb. and Zucc. and the Himalayan Yew, Taxus wallichiana Zucc.
In 1964 a compound was isolated from the bark of Taxus brevifolia and was identified, by
Wahl and Wani, in North Carolina, USA, as being cytotoxic towards KB cells,7 by 1966
the pure active substance had been isolated showing in vivo anti-tumour activity towards
murine P I534, LI210 and P388 leukemia. In 1971, the structure of this active substance
was determined by X-ray crystallography, and was shown to possess a highly
functionalised diterpenoid skeleton, attached to a p-amino acid side chain, it belonged in
the largest group of secondary metabolites, the isoprenoids, or terpenoids, and was named
Taxol®’ l .8
AcO OH
OHHO OAc
OBz
1
The initial excitement, generated by these discoveries, died down during the early
seventies when other new drugs were considered more promising, however, between 1974
* Taxol® 1 is a registered trademark of the Bristol-Myers Squibb Company, the generic term is actually Paclitaxel, however, the name ‘Taxol’ will be used throughout this thesis, when referring to compound 1, as named by Wahl and Wani in their original paper. 8
5
and 1975 taxol was found to be highly active against the murine B16 melanoma model 7
and interest was renewed to the extent that in 1977 it was selected for preclinical trials.7 , 9
In addition to leukaemias, taxol also exhibited higher activity against solid human tumour
xenografts, than murine tumours, in contrast to the rates of activity usually shown by anti
tumour compounds. It was also active against forms of breast, bronchial and ovarian
cancer.
These extremely encouraging results led clinical trials to begin in earnest in 1983, although
initially there were problems surrounding the development of a suitable formulation for
administration. By 1989 taxol had been reported as ‘the most promising anti-cancer drug
of the past 15 years’ a 30% response rate had been found for patients with incurable
ovarian cancer where good response rates are usually considered to be 15-20%!5 The task
of developing this drug further was enormous and in 1991 Bristol-Myers Squibb won the
franchise to develop taxol, in collaboration with the NCI. After phase II trials taxol
showed excellent activity against metastatic breast cancer, some forms of lung cancer and,
head and neck cancer, in 1992 it was finally approved by the FDA (Food and Drug
Administration) as a chemotherapeutic drug for the treatment of refractory ovarian cancer.
More recently, in 1994, preliminary approval was achieved for the treatment of metastaticn obreast cancer and research is still continuing. ’
2.2.2. Taxol’s Unique Mode of Action
Taxol belongs to the ‘spindle poison’ class of cancer therapeutics which either prevent, or
stabilise, the formation of the mitotic spindle during cell division,6 with tubulin being the
major target.
2.2.2.I. spindle destroyers
The majority of cancer chemotherapeutic dmgs in the spindle poison class inhibit
uncontrolled cell division by preventing the formation of the essential mitotic spindle.
Colchicine 2 from Colchicum autumnale L., one of the first to be discovered, prevents
polymerisation of tubulin into microtubules, other examples include vincristine 3 and
6
vinblastine 4,6 two of the most important chemotherapeutic drags developed to date, these
drags have lead to a substantial improvement in life expectancy for cancer patients.
OH
MeO.
► NHCOMe
MeOOMe
MeO 'OAc
Me
2 3 R=Me4 R=CHO
2 .2 .2 .2 . spindle promoters 3
In 1979, Susan Horwitz and co-workers discovered taxol has a unique mode of action, 10 in
contrast to typical spindle poisons, taxol promotes microtubule polymerisation and
stabilises the formation of the mitotic spindle, inhibiting depolymerisation back into
microtubules, 9 in fact, depolymerisation is inhibited even when subjected to ionic calcium
and cooling to 0°C.3, 7 Taxol is thought to bind to the p-subunit of tubulin causing the
equilibrium to shift in favour of microtubule assembly, so decreasing the critical
concentration of tubulin required for microtubule polymerisation,3 it actually affects the
microtubules in all phases of the cell division cycle by causing the formation of unnatural
bundles of microtubules, anti-cancer activity results since in stimulating unnatural
microtubule formation, then preventing depolymerisation, uncontrolled cell proliferation,
that is cancer, can be prevented.
2.3. The Problems with Taxol: Ecological and Biological
The potential for taxol to be a major force in the treatment of cancer is obviously
enormous, why then is it not now widely available for use in chemotherapy?
Unfortunately, the general use of the drug is being thwarted by a number of problems, the
first one being taxol’s high insolubility in water, causing problems with the development
of a suitable formulation for administration, unsuitable formulations ran the risk of
causing hypersensitivity and other allergic reactions.5 , 7 The biggest problem, however, is
7
the issue of supply.5 Taxol is isolated from the bark of the Yew tree, however, a century
old tree yields, on average, only 300 mg of taxol. Six, century old, trees are needed to
provide the 20 Kg of bark necessary for the isolation of only 2 g of taxol, which is the
amount required for a course of treatment for only one person. Furthermore, a new tree
takes approximately 1 0 0 years to reach the maturity necessary for taxol extraction, as a
result, alternative methods for taxol production are urgently needed, to avoid eradicating
the entire Taxus brevifolia species.
3. SOLUTIONS TO THE TAXOL PROBLEM
Although the problems facing the large scale production of taxol are fairly substantial, they
are not insurmountable, there are a number of solutions, some more realistic than others in
terms of practical, long term commercial supply solutions. Total synthesis, of this hugely
complex compound, is a major challenge and has proved so for many groups around the
world. Chemically, semi-synthesis, of taxol, or a potent taxol analogue, from suitable
renewable taxane precursors, is realistically a much more viable route forward.
Furthermore, in addition to synthetic chemistry methodology, biological routes towards
taxol production are also being investigated and in fact look extremely promising.
3.1. Biological Methodology
Biological approaches will probably provide the answers to the long term taxol supply
problem, however, presently, work is still only in preliminary stages and efforts need to be
concentrated in this area, biological routes to taxol are based around plant cell tissue
cultures and even more hopefully, genetic engineering.
3.1.1. Plant Cell and Tissue Culture
Plant cell cultures begin by obtaining an explant from a taxus species, that is, a piece of
living tissue, probably containing a source of secondary metabolite, cell proliferation must
then be induced. Cell cultures can be differentiated or undifferentiated, however the
majority of taxol production, so far, has come from undifferentiated, or callus, cultures,
fig. 3, which is an unorganised, proliferating mass of undifferentiated cells and is induced
8
by subjecting the explant to specific, highly controlled conditions. 11 Once cell division is
underway sub-culturing can take place, for example stable callus cultures, sub-cultured 4-5
times per week, have been maintained via continuous sub-culturing over 4 year periods. 11
fig. 312
Alternatively, differentiated cell cultures can be established from organised tissues or
embryos, for example, germination of Taxus baccata has been successfully achieved from10its embryo state, fig. 4.
fig. 412
9
Although callus cultures are highly susceptible to bacterial attack and fungal infection,
once cell growth media and conditions have been optimised, large scale taxol production
should be possible. HPLC is the technique used to monitor taxane production, together
with extraction from the culture medium being relatively straight forward. The potential
for taxol production from cell cultures is enormous, advantages include preserving the
endangered Taxus species, in addition, the technology is not subject to climate and season
variation, as are the trees, so allowing year round production, it has in fact been reported
that the yield of taxol from cell culturing is 1 0 times higher than that obtained from the
bark of a mature Yew tree and 40 times higher than that found in the needles. 12 Presently,
work is continuing to optimise conditions for growth media to eventually scale up to
industrial scale, plant cell bioreactors, Phyton Catalytic Inc. (Ithaca, N. Y) is one company
currently involved with commercialising cell culturing for taxol production.
3.1.2. Biosynthesis and Genetic Engineering
Ultimately, a firm understanding of the biosynthetic intermediates and pathways towards
taxol, including, particularly, the enzymes catalysing the rate limiting steps, should allow
genetic manipulation of the genes coding for these important proteins, permitting efficient
taxol generation under highly controlled conditions. 13
3.1.2.1 From Geranylgeranyl Pyrophosphate
Taxol belongs to the group of secondary metabolites known as the isoprenoids, or
terpenoids, which possess a 20 carbon diterpenoid skeleton. Biosynthesis of these C20
compounds begins with the acyclic precursor GGPP 5 geranylgeranylpyrophosphate, from
the C5 isoprenoid precursor, the isoprene unit 6 . 4
6
10
3.I.2.2. Taxa-4,ll-diene
In 1856, Lucas and co-workers isolated an alkaloidal substance from the leaves of the
European Yew Taxus baccata L . , 14 now given the name taxine. During the 1960s work
was carried out by several groups, into the nature of this alkaloid, producing some
interesting results. Using substances isolated from the leaves of Taxus baccata, Lythgoe
and co-workers showed taxine to be made up from nitrogen free polyhydroxylic
compounds, esterified with a (3-amino acid side-chain and acetic acid, 15 by 1966 the
structures of the two major polyols had been determined as taxicin 17 and taxicin I I 8 . 16’ 17
OHHO
OHHO
OH
7
HO. OH
OH
OH
8
From the identification of these compounds, Lythgoe et al proposed a logical biosynthetic
pathway starting from GGPP 5, Scheme 1, to the taxane precursor taxa-4,20,11-diene 12.17
Since a large number of taxane compounds bearing a C-4, C-20 exocyclic double bond1 fihave been isolated, this biogenetic intermediate was assumed for many years. However,
very recently, using labelled [1-3H]GGPP Croteau and co-workers, in the USA, were able
to show cyclisation actually occurs to taxa-4,11-diene 13, via verticillene 10, and not to 12
as originally postulated. 19 Further oxidations, hydroxylations and acylations, including
side chain attachment, must then proceed before taxol 1 production is complete. In fact,
Croteau and his co-workers have just identified taxadiene synthase as being the enzyme
catalysing cyclisation,20 furthermore, they have also identified a cytochrome P450
hydroxylase that catalyses the first hydroxylation, at position 5, on the taxane skeleton,
affording compound 14.13,18
11
OPP10
TAXOL
Scheme 1
3.2. Total Synthesis of Taxol
Due to the complexity, of the taxol structure, it is unlikely that any total synthesis
developed, will be commercially viable, nevertheless, four groups, all from the USA, have
successfully completed the challenge and in doing so have contributed enormously to the
development and understanding of new synthetic chemistry methodology. In the following
section I have attempted to summarise each of the successful routes, however, the schemes
are by no means fully detailed and are just intended to give a general overview of the main
reactions involved in constructing the complex tricyclic system.
3.2.1. Holton21
Holton and co-workers, in 1993, completed their route to taxol, Scheme 2, from
intermediate 15, easily obtained from camphor. Following the novel epoxy alcohol
fragmentation to compound 16, aldol reaction then hydroxylation at C-2, compound 17
was obtained.
12
H olton’s Synthesis
TESO
OTES TBSO"-
TESO
2 steps0 C 0 2Et
TBSO.....
OH
15 16 17
2 steps
TESOTESO
LTMPTBSO -TBSOi"
OH
O1920
TESO
SwemTBSO»-<
OH18
4 steps
TESO TESO
TBSO"-*2 steps
TESO OH
OH
21 22 23
5 steps
TAXOL
TESO OBOM
TBSO .
24
Scheme 2
After reduction to a triol and conversion to the cyclic carbonate 18, Swem oxidation gave
the ketone 19, which rearranged to lactone 20 after treatment with LTMP, setting the stage
for functionalisation at positions 1, 2 and 3. C-3 hydroxyl reduction and enolate formation
13
followed by C-2 reduction then treatment with phosgene, gave compound 21. Oxidative
cleavage of the terminal olefin and esterification afforded 22, after which, Dieckmann
cyclisation generated the desired tricyclic system 23. Following straightforward functional
group manipulation, oxetane ring and side chain construction, taxol 1 was successfully
synthesised via 24 in a 4-5% yield.
3.2.2. Nicolaou22
Almost simultaneously, Nicolaou and his group completed and published their route to
taxol, a convergent synthesis based on the separately constructed compounds 26 and 27,
Scheme 3. Conversion of 25 into aldehyde 26 was achieved after protecting group
manipulation, reductive opening of the lactone, protection and oxidation. Compound 26
was then used in the first key reaction of the synthesis, Shapiro coupling with 27, joining
the A and C rings affording compound 28. Following conversion into 29, via epoxidation
at C-14, C-l, allowing preparation for C-l, C-2 functionalisation and oxidation, the second
key reaction, McMurray coupling, was possible, generating the desired tricyclic
intermediate 30 for conversion into taxol via intermediate 31.
Nicolaou’s Synthesis
Et02C
OTBS
OTPSOBn TBSO OBn
7 steps TPSO'
14
OH
2826
AcO. OTES
OAc
31
HO OH OBn
1 2 steps'" / /
O30
Scheme 3
4 steps
OBn
o29
14
3.2.3. Danishefsky23
1995 Saw completion of a third successful total synthesis by a group led by Samuel
Danishefsky, they set a new precedent in introducing the oxetane ring early in the
sequence. The route begins, Scheme 4, with the catalytically induced asymmetric aldol
condensation of compound 32, affording 33, already showing the correct chirality at C-8 ,
in the eventual taxane skeleton. The oxetane ring was constructed via compounds 34 and
35 allowing formation of aldehyde 36 after ring opening, then, coupling with the
previously prepared compound 37 gave the advanced key intermediate 38. Danishefsky’s
key reaction, in achieving the tricyclic skeleton, is the Heck reaction, which was carried
out on compound 39 successfully affording 40. Through functional group manipulation
the diene was converted to the desired C-9, C-10 functionalities via an epoxide at C-l 1, C-
12, finally affording Baccatin El 41 for simple transformation into taxol.
Danishefsky’s Synthesis
OTBS
0 ««i
33, 0 H
4 stepso
34
,OMeMeO,
OTBS
OBnOH
OTMS
37
OMeMeO.OTBS
OBnO
OTBS
5 steps
38 36steps |
OTBSOTf
OBn
HeckOTBS
OBn
O
1 1 stepsAcO.
HO......
HO OAcOBz
39 40 41
Scheme 4
3.2.4. Wender24
Most recently, in the last year, Wender and his co-workers added their contribution to the
taxol story, with their total synthesis. Wender’s synthesis, Scheme 5, based on the natural
product pinene, isolated from pine trees, is the shortest yet.
Wender’s Synthesis
42 43 44
10 OTMSj 2 steps
COjEt
OH
45
c h 2o h
484 steps
3 stepsTIPSO .
OTBS
47
2 steps 13
OH
OTBS
46
AcO AcOOTES OH
7 steps ► TIPSO .TIPSO* .
OBOfOBOMHO HO
OBz OBz
49
Scheme 5
Beginning with C-10, C -ll construction from verbenone 42, the air oxidation product of
pinene, via prenyl bromide addition, then selective ozonolysis of the terminal alkene,
compound 43 was afforded. After photorearrangement, carbon connectivity was extended
to allow construction to move towards completion of ring B. That is, the Li salt of ethyl
propiolate was added to aldehyde 43, giving 44 after trapping of the alkoxide with TMSC1.
16
Introduction of the C- 8 methyl group using Me2CuLi led to carbanion generation at C-3
allowing cyclisation to proceed. Immediately afterwards the hydroxyl at C-9 was oxidised
allowing C-10 oxygen introduction using Davis’ oxaziridine, following carbonyl
reduction, compound 45 was afforded. Acetonide formation across C-9, C-10 introduced
conformational rigidity into the system, generating compound 46, the advanced taxane
intermediate 47 was then constructed following epoxidation at C -l2, C -l3 then a DABCO
fragmentation. Subsequent triol formation across C-l, C-2 and C-3 was achieved in one
sequence via reaction with KOt-Bu and P(OEt>3 under O2 then C-2 reduction with NaBFL*.
Hydrogenation across C-3, C-8 , phosgene protection across C-l, C-2 and oxidation at C-4
generated the AB ring system intermediate 48 ready for C-ring construction. Carbon
extension at C-4 was achieved with PhsPHCOMe, together with C-20 introduction using
[Me2NCH2]I, finally affording 49, which was elaborated to 50 allowing the desired aldol
condensation to the tricyclic taxane skeleton 51 for subsequent conversion to taxol.
3.3. Semi-Synthesis of Taxol & Taxol Analogues
Chemically, the most realistic solution towards solving the supply problem is semi-
synthesis of taxol from, a suitable precursor, or semi-synthesis of a new taxol analogue
with biological activity equal to, or better than, that of taxol its self. Semi-synthesis
involves isolating easily obtainable, renewable taxane natural products, from the Yew tree,
then adapting them into biologically active compounds using synthetic chemistry
methodology. Development of a potent taxol analogue requires an understanding of the
structure-activity relationships between taxol and the microtubules in the cell, from a
synthetic chemist’s point of view, knowledge of the functional groups and skeleton
conformation, essential for biological activity, is required and over the last 15-20 years
thorough research has been carried out into this area of taxane chemistry. During the early
1980s, two principal groups from the USA and France, led by Kingston and Potier
respectively, carried out much of the groundwork and initial research, taxane analogues
with differing functional groups were isolated, and sometimes modified, then tested for
cytotoxicity. For example, Kingston and co-workers, in 1982, found taxanes, without the
(3-amino acid side-chain, to be inactive in all biological tests,25 and this was later
confirmed by the Potier group. Work on these structure-activity relationships continued
throughout the 80s and early 90s and the results are summarised in fig. 5,27 in general
17
cytotoxicity is determined in vivo using cell culture systems and in vitro by monitoring
ability to induce microtubule assembly in the absence of GTP.25
N-acyl group Functionalities at C-9, C-10required and C-7 may be modified
J LPhenyl group or ™ o Oxetane ringclose analogue essentialrequired V y oh 0
AcO OH
HO OAcOBzFree OH orhydroiysable ester Benzoyloxy groupnecessary needed; som e substituted
groups have improved activity
fig. 5
3.3.1. Using Semi-Synthesis To Increase Water Solubility
In determining which functionalities are essential for activity and which groups have little
effect, the opportunity to modify non-essential groups, to crucially increase solubility in
water is presented.
oAcO
Ph' NH
Ph‘
HO OAcOBz
52 R1 = R2 = OCOCH3
53 R1 = OCOCH3 , R2 = H
54 R1 = H, R2 = OCOCH3
OAcO OR'
Ph' NH
ORHO OAc
55 R1 = C0(CH2 )2 C02 X;
X = (HOCH2 CH2 )3 NH, R 2= H
56 R1 = CO(CH2 )2 COX;
X = N-methylglucammonium, F? = H
57 R1 = C0(CH2 )3 C02 X; X = Na, R2 = H
In 1984, during their investigations into the biological activity of different taxol acetates,
Kingston and co-workers prepared compounds 52, 53 and 54,28 compound 52 showed no
18
activity, 53 and 54, 2’-acetyltaxol and 7-acetyltaxol respectively, however, were more
promising, with both showing in vivo activity. Although compounds 53 and 54 are not
themselves more water soluble than taxol, the opportunity to introduce new groups in
these positions, to increase solubility, had been presented and was indeed followed up in
1987 by M. Suffness and his colleagues, again in the USA. Their work involved the
reaction of taxol with either succinic or glutaric anhydride in pyridine solution.29
Crucially, in their investigations, preparation of sodium, triethanolamine and N-
methylglucamine taxane salt derivatives resulted in the development of compounds 55, 56
and 57, all three salts had vastly improved water solubility and showed good in vivo
activity, with the 2'-monoglutarate derivative 57 showing better overall properties to that
of its corresponding succinates.
3.3.2. Semi-Synthesis of Taxol
During an investigation of the reactivity of each functional group around the skeleton a
semi-synthetic route to taxol was developed by the French group led by Potier from 10-
deacetylbaccatin IE (10-DAB ID), 58 Scheme 6 . 30 Critically the route has provided an
alternative, renewable and efficient supply of this chemotherapeutic drug. 10-DAB (III),
compound 58 is isolated from the leaves of the European Yew, Taxus baccata L. Since the
leaves are quickly regenerated, careful harvesting should provide a good yield without
endangering the Yew species. In fact, 10-DAB (IE), 58, differs from taxol in only two
positions, that is, the acetate functionality is missing at C-10 together with the side chain
moiety at C -l3. Potier’s route to taxol is short and efficient, following selective protection
of the free hydroxyl group at C-7 affording compound 59, acetylation at C-10 can be
successfully achieved producing compound 60, allowing the desired attachment of the
suitably protected side chain 61 at C-l3. Protecting group removal from compound 62
gave taxol in an impressive 89% yield.
19
HO. .0
HO'"
1. Et3SiCl, Py2. CH3COCI, Py
RQ. .0 OSKCHzCHjV
HO"
OBz
58 59 R = H60 R = COCH3
61 R = CHCH3OC2H5
TAXOLDeprotection
AcONH O
ORHO OAc
OBz
62
Scheme 6
3.3.3. Taxotere
The hard work involved in investigating these complex compounds finally paid off with
the discovery and development of Taxotere®* 63,31 a taxol analogue possessing in vivo and
in vitro activity slightly better than taxol its self. In investigating modified side-chains for
use in their taxol semi-synthesis, Potier and co-workers introduced the t-BOC protecting
group to the N'-acyl group of the side chain. After coupling of the modified protected
side-chain 64 to intermediate 65, compound 6 6 was generated which was converted to
taxotere 63 after protecting group removal, Scheme 7.
* Again, Taxotere® 63 is a registered tradename, the generic term is Docetaxel, however, the name ‘Taxotere’ will be used, when referring to compound 63, throughout this thesis. 3 1
20
HO
HO OAcOBz
Protection at C - l and C-10
CI3 CCH2 O2 CO o
HOi»-
oco2CH2ca3
HO 1 H OAc OBz
58
Esterification with taxotere side chain
65
0t-BuO NH O
P h ' ' 0H
OR
64
HO. OHBuO NH
....." " / /
OHHO OAc
Deprotection
OBz
t-BuO' NH
Ph' On-'OR
HO OAcOBz
63 66
Scheme 7
4. REARRANGED TAXANES
4.1. Discovery & Nomenclature
My research begins with another group of taxanes, that is, rearranged taxanes possessing a
contracted A-ring, with the basic skeleton shown in structure 6 8 , fig. 6 .
18 10 9 1 9
67
NORMAL TAXANE SKELETON
68
RING-A CONTRACTED 11(15-1) ABEO-TAXANE SKELETON
fig. 6
21
In the following section the numbering system shown in fig. 6 , for both the normal 67 and
rearranged 6 8 taxane skeletons, will be used, as has been precedented in the literature.31,32,
3 3 ,34 During the years 1990-1993, three separate groups, from America, France and Italy,
independently published work introducing the novel rearranged taxane compounds. In
general, they were discovered by accident during the course of their reactions investigating
the nature and reactivity of taxol and other taxane compounds. Appendino and his co
workers introduced the name ll(15-l)a&e0-taxane, to describe the general class of these
rearranged taxane compounds,34 the numbering system, 11(15-1), refers to the position of
contraction in the taxane skeleton, see 6 8 , fig. 6 , accordingly this general term is used
throughout the chapter.
4.2. Kingston’s Discovery of Ring-A Contracted Taxanes
Ring-A contracted taxanes were first discovered by Kingston and co-workers, in 1991,32
during their studies into the structure-activity relationships between taxol and the
microtubules in the cell, see fig. 7.
i.o o
AcO AcOOH OHNHPh- NH
0 «»-Ph- Ph- ......OH OHOH
'OHHO OAc HOOBz OBz OAc
1
(CH3)2C(OMe) 2
p-TsOH
oAcO OHO
Ph- ......OH
OBz OAc
70
22
11.o
A AcO OPh NH O
1A J U mu-/
OA AcO. O
Refluxing P h ' NH O OAc
...
HO L H OAc OBz
Ph" 'r" 'o"0 CH3COC1 =H = T* V,//OAc
H * OAc
OAc
71
111.
? S,Et3 MsClPh' NH O Ph' NH O
OSiEt-i Et,NPh^^V ^O (
OHH° =„ OAc OBz
72 73
fig. 7
In investigating the necessity of the oxetane ring for biological activity they reacted taxol 1
with various electrophilic reagents since this functionality was known to be reactive
towards these compounds. Reactions were carried out, under acidic conditions, with
electrophiles such as Meerwein’s reagent, reaction i, with acetyl chloride, reaction ii, then
later, mesyl chloride, in the presence of triethylamine, reaction iii, was also investigated.
They found, Meerwein’s reagent induced oxetane ring opening, possibly via the
mechanism shown in fig. 8. However, in addition, when compound 69 was subsequently
treated with 2 ,2 -dimethoxypropane, under acidic conditions, unexpectedly, acetonide
formation was accompanied by A-ring contraction, affording compound 70. Accordingly,
Kingston et al postulated an acid induced dehydration mechanism (this mechanism is
discussed in detail later).
23
OH OH
OO. .0
CH3 c h 3
'""'OH
OH
‘" " O
CH.OEt
OH
Me
Nu:-
OH1 -OH
EtNu
Me
fig. 8
In addition, oxetane ring opening was also induced using the electrophile, acetyl chloride,
see ii, fig. 7, however, again, unusually, ring opening was accompanied by ring-A
contraction, generating compound 71. In this case, Kingston suggested an electrophile
induced ring-A contraction could be taking place, with rearrangement occurring via acetate
formation at C-l, as shown in fig. 9. In a similar vein, compound 72 probably rearranged
to eventually form the ring-A contracted derivative 73 via a mesylate at C-l, in place of
the acetate functionality, although here, the oxetane ring remained intact indicating acidic
conditions are needed to encourage ring cleavage.
..........
OCOR
fig. 9
24
4.3. Studies by Francoise Gueritte-Voegelein
Following the work of the American group, in 1992 a French group, led by Francoise
Gueritte-Voegelein in Gif-sur-Yvette, reported their findings regarding similar
rearrangements.33
TROCO OTROC
HO......
HO OAcOBz
TROCO OTROC
OAcOBz
74
ITROCO, OTROC
HO......
OAcOBzHO
75 76
TROCO, OTROC
HO'.....
'OHOH
OBz
78OAc
TROCO. OTROC
HO.....""OH
'OH
OBz OH
77
Scheme 8
They carried out reactions, under acidic conditions, on a 10-deacetylbaccatin HI derivative
74, again producing some interesting results, Scheme 8.31 Under a variety of acidic
conditions, ring-A contracted compounds 75 and 76 were afforded followed by oxetane
ring opening generating 77 and 78.
4.4. Studies by Giovanni Appendino
In 1993, hot on the heels of the Americans and the French, the Italian group, under
Appendino, also reported their investigations into ring-A contractions, leading to 11(15-
l)a£e<?-taxanes.34 During their work investigating renewable taxane sources, they isolated
25
a rearranged taxane from the needles of the Himalayan Yew Taxus wallichiana Zucc.,
compound 79. The structure of this strange compound was identified using NMR data and
by studying the acid catalysed rearrangement of 10-DAB HI.
OH
HO.......
OAcOBz
79
4.4.1. Suggested Mechanism For The Acid Catalysed Ring-A Contraction Of
10-Deacetyl Baccatin HI
HO. OH
OAcOBz
58
HO OH
HO"'-'
OAcOBz
80
OH
OAcOBz
HO"-
OH
HO......
OAcOBz
84 82 R=H, OH
83 R =0
Scheme 9
Kingston had already postulated that acid induced rearrangement could proceed via a
cation at C -l ,32 Appendino and his co-workers agreed with this idea after investigating the
acid catalysed ring-A contraction of 10-DAB HI, as shown in Scheme 9. 34 Protonation at
the tertiary C-l hydroxyl position, of compound 58, allows dehydration leaving cation 80,
migration of the bond at C-l 1 is induced, producing a ring-A contracted product via the C-
15 tertiary carbenium ion 81. Compounds 82-84 were isolated from the reaction, it seems
26
reasonable that they arise from proton loss at C -l6 route a or from trapping of the C-10
hydroxyl group route b. Production of derivative 84 helped confirm the assigned structure
of compound 79. Indeed this mechanism was among the two suggested by Appendino and
co-workers, later, in 1996,35 for the biogenesis of ll(15-l)afceo-taxanes, as illustrated in
fig. 10. Route a proceeds from a normal 1-hydroxy taxane via the dehydration mechanism
already described, route b, however, proceeds via the transannular cyclisation of an
epoxybriarene.
HO
b
HO
HO
fig . 10
4.5. Biological evaluation & potential for ll(15-l)aZ>eo-taxanes
As interesting as these structures were, the question was raised as to whether they had any
potential as possible precursors to new biologically active taxol analogues. Kingston’s
compounds 69, 70 and 73, fig. 7, were all tested for biological activity, compounds 70 and
71 were inactive, indicating the need for an intact oxetane ring, compound 73, however,
was almost as active as taxol in the tubulin depolymerisation assay. 32 Although it was
inactive in the cytotoxicity test there is obviously great potential for these compounds to
27
become the basis of new biologically active taxol analogues and at the very least any new
and interesting results, available on these novel compounds, can only enhance our
understanding of taxane compounds in general, increasing our knowledge, allowing the
development of the most efficient drugs possible.
5. NATURAL PRODUCTS POSSESSING THIS REARRANGEDTAXANE SKELETON
5.1. Discovery, Nomenclature & Problems in Identification
BzO,
10 9
HO <13OH
HO
85BREVIFOLIOL TRUESTRUCTURE 5,7, 6 RING SYSTEM
AcO PAc OBz
HO»-<13 15X'OH
HO
86
BREVIFOLIOL ORIGINAL ASSIGNMENT 6, 8, 6, RING SYSTEM
BzO PAc OAc
HO.... .'OH
OAcHO
87
TAXCHININ A
BzO PAc OAc
OAcOAcHO
88
TAXCHININ BR=Cinnamoyl
There has been a fair amount of confusion surrounding the discovery of natural product
taxanes with an ll(15-l)a&ec>-taxane skeleton, compound 85 was actually the first
rearranged natural product to be isolated, from Taxus brevifolia, by Balza and co-workers
in 1990, given the trivial name, brevifoliol,36 however, they assigned a normal taxane
skeleton 8 6 , a 6 , 8 , 6 ring system, as in taxol, rather than a rearranged 5, 7, 6 rearranged
ring system 85. The first natural product to be discovered and correctly identified as
28
having a rearranged taxane skeleton was compound 87, isolated from the needles and
stems of Taxus Chinensis, in 1992, by the Japanese group led by K. Fuji, its structure was
unambiguously confirmed by X-ray analysis and given the trivial name, Taxchinin A .37
The Japanese were also the first to isolate a rearranged taxane compound containing an
intact oxetane ring, compound 8 8 , it was given the trivial name, Taxchinin B .38
As such, three general, trivial names were precedented as a result of the presentation of
these three initial compounds. When describing an 11(15-1 )abeo-taxzne derivative
possessing a C-4/C-20 exocyclic double bond the general term ‘Brevifoliol derivative’ or
‘Taxchinin A type derivative’ is used, in the literature, and will also be used throughout
this thesis, however, when citing contracted A-ring compounds with an intact oxetane ring,
the expression ‘Taxchinin B type derivative’ is employed, here and also in the literature.
5.2. Determination of Brevifoliol’s True Structure
It was not until 1993, when two groups independently noticed discrepancies in the original
NMR assignment, that the spectra were reinterpreted and brevifoliol 8 6 was assigned its
true structure 85. During their investigation into the taxane content of the Indian Yew,
Taxus wallichiana Z., the American group, led by G. Georg, isolated a large amount of
brevifoliol, on interpreting the NMR spectrum they assigned the new structure, the
rearranged 11(15-1 )abeo-taxane 85.39 Georg’s findings were confirmed later in 1993 by
the Italian group, under G. Appendino, who also reported a revised structure for
brevifoliol, along with some structural revisions for some previously reported baccatin VI
derivatives.40
5.2.1. Evidence for the revised structure
i. Initially the ester groups at C-10 and C-l were interchanged, primarily after examination
of the HMBC (Heteronuclear Multiple Bond Correlation) NMR spectrum.39,40,41 A clear 3
bond correlation was found between H-10 and the benzoyl carbonyl group, together with
H-7 showing correlation to an acetate functionality.
29
ii. HMBC NMR spectroscopy also provided evidence to support a ring-A contracted
taxane, normal 6 , 8 , 6 taxanes, see fig. 10, usually show a 3-bond correlation from H-16
and H-17 to C -ll, Georg and co-workers discovered this signal was absent in the
brevifoliol HMBC spectrum, 39 indicating the atoms were actually separated by more than
3 bonds.
iii. Georg’s final evidence, to confirm further the revised structure, 39 came to light when
he noticed the unusual chemical shift of 575.9 attributable to C -l5, due to HMBC
correlations to H-14, H-16 and H-17, the signal was unusually low for a carbon atom
bearing no heteroatoms, furthermore, the signal attributable to C -l5 in taxol and other
taxanes normally appears at about 543.0, casting doubt on Balza’s original assignment but
supporting the rearranged structure with a free hydroxyl group at C-15. In addition the
chemical shift attributable to C-l normally appears around 579.0, however, in brevifoliol’s
case a signal appears at 562.5 with HMBC correlations to H-16, H-17 and, most
importantly, H-10, this signal can be attributed to C-l in the rearranged structure. 562.5 is
also unusually low for a quartemary carbon bearing no heteroatoms, however, this signal,
usually appearing between 560 and 570, has now become indicative of a taxane compound
possessing a novel 11(15-1 )abeo-taxane, skeleton, the low resonance possibly being
attributable to linear strain caused by long bond lengths C-l/C-2 and C-l/C-15.40 In
agreement with Georg, Appendino and co-workers strengthened the story surrounding the
revised structure with their results and ideas, published later in 1993,36 agreeing with those
of Georg, in addition, their detection of an ROE effect between the tertiary hydroxyl
proton and H-9 finally confirmed the brevifoliol structure as that shown in compound 85.
30
6. 11(15-1)A£E0-TAXANES
Once the problems surrounding structure identification of these compounds, had been
solved, the way was clear for the development of a whole new area of taxane chemistry,
possibly leading to the development of a new potent taxol analogue.
6.1 History and Development of New ll(15-l)a&eo-taxanes
Since 1993, when 11(15-1 )abeo-taxanes were first described, there has been a deluge of
new rearranged taxanes reported in the literature, including the republishing of some
compounds originally given the wrong structural assignment. Appendino reported 40 that
some compounds isolated from Taxus brevifolia, by R. Croteau and co-workers, originally
reported as baccatin VI 89 derivatives, should have their structural assignment revised.
For example, on the basis of NMR data, Appendino revised the structure of compound 90a
to that of 90b, in addition, on the foundation of NMR data and X-ray analysis, compound
91a was revised to that of 91b.
AcOBzO. OAcAcO. OAc
HO"-<AcO ' " - 1
'OrOHOHOHO OAcOAcOAc OBzOBzOBz
89 90a 91a
BzOBzO. £Ac qac
HO ' " - 1 HO......
.0
OAc OAcOBz OAcHO HO
90b 91b
Similarly, some compounds originally reported as taxchinin A type derivatives should also
be reassigned the rearranged skeleton. For example, compounds 92a and 93a illustrate
two derivatives assigned the incorrect structure in Appendino and co-workers early
investigations into new taxanes from the European Yew Taxus baccata, 43,44 these
31
structures were also accordingly reassigned, in their paper describing the brevioliol
structure revision, as derivatives 92b and 93b respectively.40
AcO ;P Ac OBz
HO'"-<'OH
HOOAc
HQ.
HO..... .
'OH
HOOAc
92a 93aAcO
HO"-<
'OH
OAcHO
HQ.
HO.....<
'OH
OAcHO
92b 93b
6.2. Correctly Assigned New ll(15-l)a&eo-taxanes &
Conformational Isomerism
From mid 1993 to date, between 30 and 40 new rearranged taxanes have been discovered
and reported in the literature. The two main groups working on this area of chemistry are
from Japan and Italy led by K. Fuji and G. Appendino respectively, however interest in
this subject is growing rapidly and groups in America, China, Canada and India have all
published work describing their discoveries and investigations into ll(15-l)<z&eo-taxanes,
isolated from various types of Taxus species world-wide. Furthermore, with the
presentation of these novel compounds came the discovery and reporting of a new
development in 1 l(15-l)a&eo-taxane chemistry, that is, conformational isomerism.
Normal 6 , 8 , 6 taxane skeletons are fairly rigid and only one conformation is usually
observed in solution, the unusual 5, 7, 6 ring system in 11(15-1 )a&eo-taxanes, however,
induces a flexibility into the skeleton and as a result conformational isomerism is often
observed.
32
6.2.1. Cycloheptane
Conformational analyses are primarily based around the B-ring in the 11(15-1 )abeo-taxm&
skeleton, that is, different conformations adopted by seven membered rings must be
considered, see fig. 1 1 .
CHAIR, 9.04 KJmol'1 TWIST CHAIR, 0 KJmol1
TWIST BOAT, 10.4 KJmol'1BOAT, 12.6 KJmol'1H IV
4 CONSECUTIVE RING CARBONS CO-PLANAR
V
fig. 1 1
33
J. Hendrickson, in 1961, was the first to provide realistic data on this subject with his
studies into the possible conformations of medium rings, including cycloheptane.45 In
determining the most stable arrangement of a given molecule it was deemed necessary to
calculate the total strain energy of each possible conformation of that molecule in relation
to geometrical parameters and constraints. For example, values relating to bond angle
strain, bond length, torsional strain and non-bonded interactions were used, with values
derived from thermodynamic and spectroscopic data. When considering cycloheptane,
two definable groups were found to be preferred, namely, the boat and chair families. The
two plane symmetric forms, that is with just Cs symmetry, are the chair I and boat II
conformations. However, both arrangements are flexible and can pseudorotate to relieve
severe 1,3 diaxial interactions, that is, the chair I form can pseudorotate to the twist-chair
conformation III, now with C2 symmetry, similarly, the boat II conformation can
pseudorotate to the twist-boat arrangement IV, again now with C2 symmetry. Even in the
‘twist’ forms, however, steric repulsions are not completely minimised and pseudorotation
is accompanied by ‘bond-breathing’. That is, a slight bond angle enlargement takes place,
from the usual 109.5° to between 112° and 116°. Hendrickson calculated the most stable
conformation to be that of the twist-chair III, pseudorotation over an energy barrier of 9.04
KJmof1 converts III to the chair isomer I, similarly, in pseudorotating from the boat II to
the twist-boat IV conformation, 2.2 KJmol' 1 of energy is released. It must be noted,
however, that in flipping from the chair to the boat family actual angle bending is required
rather than the simple ‘bond breathing’, in this vein the energy barrier between the twist-
chair III and the boat forms is much larger at 35.6 KJmol'1. That is, the twist-chair III is
converted to the twist-boat IV via conformation V, with four consecutive ring carbons co-
planar.
6.2.2. Studies by Fuji into Conformational Isomerism in ll(15-l)a&eo-taxanes
Conformational isomerism, in 11(15-1 )<2&eo-taxanes, was first reported by a Japanese
group led by K. Fuji, in Kyoto in 1993, it was discovered during their investigations into
the stem and needle taxane content of Taxus chinensis. To date this group have isolated 13
new 11(15-1 )abeo-ta.xane compounds, taxchinins A-M, some of which do not contain the
oxetane ring (taxchinin A type derivatives), for example compounds 94 and 95, some of
which do (taxchinin B type derivatives), exemplified by compounds 96 and 9 7 38>46’47’48’49
34
In investigating the structures of these compounds they used NMR spectroscopy, X-ray
analysis and molecular modelling, as a result they have been able to report some
interesting conformational analyses.
TAXCHININ A TYPE DERIVATIVES
BzO.
AcO"*-'
'OH
OAcHO
94
HO. •pAc OAc
A cO '"-'
'OH
OAcHO
95
TAXCHININ B TYPE DERIVATIVES
BzO. PBz OAc
OAcOAcHO
96
HO. P Bz OAc
AcO>", ,<
OAcOBzHO
97
It was shown by Hendrickson,45 that the preferred conformation, for unsubstituted
cycloheptane, is that of the twist-chair, see structure III, fig. 11, since in relaxing to this
arrangement severe 1,3-diaxial interactions are reduced. However, from their NMR
analyses, Fuji and co-workers were able to show that for some ll(15-l)a&e0-taxanes, two
conformations exist in solution, in slow equilibrium. For example, taxchinin D 94 was
unambiguously assigned the ll(15-l)afceo-taxane skeleton in the solid state, using X-ray
analysis, however, in addition, NMR spectra indicated that, in solution, two
conformational isomers were present. In CDCI3 , at room temperature, a broad set of
signals was observed, however, on cooling to -10 °C two sets of sharp signals could be
seen corresponding to the presence of two isomers, see fig.12,47 where the prime numbers
belong to the minor conformer.
35
fig. 1 2 47
Their analysis of key signals attributable to H-10, H-9, H-7, H-6 a and H-6 p in both the
major and minor conformers, in the full *H spectrum , 4 6 clearly indicated the minor isomer
as being in possession of the twist-boat/chair conformation 94a and the major
conformation to be in the twist-chair/boat arrangement 94b.
M ? 19 OAcOAc
15=
l14b OH
Sac Me‘«
94aTwist-boat/Chair
Minor
94bTwist-chair/Boat
Major
In the minor conformer, 94a, a large J value of 11 Hz placed H-9' and H-10' in an anti
arrangement, with respect to each other, corresponding to a dihedral angle of 180°, see (np
1), accordingly ring-B is forced into the twist-boat conformation 94c, as shown in fig. 13.
36
94cRing-b, Twist-boat n p 1
fig. 13
Analysis of the coupling constants attributable to H-7', allowed determination of the
conformation belonging to rinc-C, in the minor isomer. J7-,6p9 . 0 was analogous to a
dihedral angle of 180° between these two protons, see np 2, then J7 '6a'5.0 indicated a
dihedral angle of about 60° between H-7' and H-6 oc’ , also shown in np 2, assigning the
chair 94d arrangement to ring-C, as illustrated in fig. 14.
Turning to the major isomer 94b, a small J value of 3.4 Hz placed H-9 and H-10 in an
equatorial arrangement with respect to each other, indicating a dihedral angle of 60°
between the two protons, see np 3, forcing ring-B into a twist-chair arrangement, 94e, fig.
94d Ring-C, Chair
np 2
fig. 14
15.
37
Mej9 OBz
Hioh 3c
94eRing-b, Twist-chair
OAc
np 3
fig. 15
Accordingly, when ring-B adopts the twist-chair 94e conformation, ring-C flips to the boat
94f arrangement. The change in J value, corresponding to H-7, indicated this conversion.
Fuji et al observed a triplet, at 54.90, attributable to H-7, with J equal to 9 Hz, indicating a
smaller dihedral angle between H-7 and H-6 a, see np 4, confirming the boat arrangement
for ring-C 94f, as shown in fig. 16.
Interestingly, although the twist-chair/boat isomer 94b is the major conformation in
solution, X-ray analysis showed the twist-boat/chair 94a conformation in the solid
state.46,47 Furthermore, the ratios of each conformational isomer, seen in solution, varies
from compound to compound, from their thorough investigations into the nature of these
suggest that for those compounds possessing a taxchinin A type skeleton, that is, those
11(15-1 )aZ?eo-taxanes with a C-4/C-20 exocyclic double bond, conformational ratios seem
to depend on the acylation pattern around the diterpenoid skeleton. For example, in
considering taxchinin D 94 they proposed the twist-boat/chair conformation 94a is
94fRing-C, boat
np 4
fig. 16
rearranged taxanes, Fuji et al were able to postulate a number of ideas why 47,48 They
38
sterically more stable since in adopting this conformation the large steric interaction
between C-lO(OBz) and the C-1 (r-butyl like) group is drastically reduced, see fig. 17,
(from here on in when referring to the functional group at C-l the phrase C-l(f-Bu) group
will be used). This is in contrast to the simple unsubstituted cycloheptane, where the
twist-chair III conformation is the most stable, see fig. 11. However, in CDCI3 , the twist-
chair/boat type conformation 94b predominates, although this arrangement is sterically
the least favourable, this structure can now be stabilised by intramolecular H-bonding
between the C-15(OH) and C-lO(OBz) substituents, also illustrated in fig. 17.
Unfortunately, in converting to 94b steric crowding within the molecule is increased,
forcing conversion back to 94a, hence, in solution, a conformational equilibrium is in
place.
Reduced steric crowding between C-1(*Bu) & OBz(10)
H-Bonding stabilisation, but increased steric crowding between C-1(fBu) & OBz(10)
Me17 Me19 QAcw Q A c U h '
Mei6 ,„ti f / H9 ‘‘5 | h 2 / " 2 0
H 14b
\1 OAc H3
Hl H10
MejgOAc
94aTwist-boat/Chair
Minor94b
Twist-chair/BoatMajor
fig. 17
A similar analysis was carried out on taxchinin G 95, in contrast to the findings for
taxchinin D 94, the twist-chair/boat 95a type conformation, for rings B and C
respectively, was adopted in the solid state and also in solution, with H-9 and H-10 in
pseudo-equatorial positions 95a .46,47
39
H-bonding stabilisation
Ac° AcO
H13 OAc
95aTwist-chair/Boat
Fuji and his co-workers suggest the twist-chair/boat type conformation 95a can now exist
in the solid state since Taxchinin G 95 does not possess a bulky functional group at C-10,
which would sterically disfavour this conformer, as in Taxchinin D 94. In addition, this
twist-chair/boat conformation 95a is also stabilised, in solution, by intramolecular H-
bonding between substituents C-15(OH) and C-IO(OH). In conclusion, when considering
conformational isomerism in 11(15-1 )a£eo-taxanes, the ratios observed, in solution, of
each conformer, depends on the balance between H-bonding stabilisation and steric
interactions, as the molecule attempts to reach its most stable arrangement. That is, the
restrictions imposed, by the presence of the two fused rings, A and C, together with the
substituents around the skeleton, determines the ratios of each isomer observed. In a
similar vein, conformational ratios observed for taxchinin B type derivatives (with
possession of an oxetane ring) seem also to be dependent on the acylation pattern around
the ring,47 although more flexibility restrictions are now also imposed by the presence of
the oxetane ring.
6.2.3. Appendino, Investigations into Conformational Isomerism
In investigations into the taxane content of various Yew trees, namely Taxus baccata, 44,50
Taxus wallichiana 35,51,52 and the ornamental Yew, Taxus x media Rehd cv Hicksii, 53
Appendino and co-workers have also identified new 11(15-1 )a6 eo-taxanes and observed
their conformational isomerism. July 1994 saw the reporting of their discoveries and ideas
surrounding some 1 l(15-l)a&eo-taxanes, isolated from the roots of Taxus x media Rehd.
cv hicksii,53 a Yew normally found in nurseries. Since the chemistry surrounding these
novel taxanes is quite complex and only in the early stages of understanding, Appendino
40
and his co-workers only postulate and suggest ideas surrounding the observed
conformational isomerism. Having said this, however, their discoveries and suggestions
do largely agree with and corroborate those made by the Japanese group, under Fuji.
AcO.
AcO .
OAcOBzHO
98
AcO
OAcOBz
99
For example, the broad NMR spectrum of compound 98 was sharpened to two sets of clear
signals at -20°C, in a 4:1 ratio, with the major conformer possessing the twist-boat/half-
boat conformations for rings B and C respectively, see 98a, fig. 18, in this case, rinc-C is
forced to adopt a boat-like conformation to accommodate the oxetane ring.
Reduced steric crowdingbetween C-1(fBu) & OAc(10)
Mej9OBz AcO,
OAc
AcO,14a'
OAc M ' 18
98aTwist-boat/Half-boat
Major
H-Bonding stabilisation, but increased steric crowding between C-1(*Bu) & OAc(10)
(H -* ^ \^ O A c | t
I / Me19 o ^ H 5 ~ H2 H<J ^
98bTwist-chair/Boat
Minor
fig. 18
From their findings Appendino et al, in agreement with Fuji et al, suggest conformational
equilibrium is governed by a mixture of steric factors and intramolecular H-bonding due to
the acylation pattern around the diterpenoid skeleton, in an attempt to reach stability.52,53
In the case of compound 98, sterically, the twist-boat/half-boat 98a conformation is more
stable, since in adopting this arrangement steric crowding between the *Bu like group at C-
41
1 and OAc(lO) is reduced. However, in solution, intra-molecular H-bonding, between C-
15(OH) and OAc(lO), can now stabilise the unexpected twist-chair/boat conformation
98b causing the observed conformational equilibrium. In conclusion, when investigating
the conformational isomerism of 1 l(15-l)aZ?eo-taxanes, it is necessary to consider not only
the restrictions imposed by the presence of the tricycle ring system, but also the nature of
the substituents, around the skeleton, must be taken into account. On a final note, it is
possible to fix the conformation of an 1 l(15-l)afceo-taxane, that is, the skeleton becomes
anancomeric, this is induced either by steric factors, as in compound 99, or chemically, for
example, by rendering OH(15) inert to H-bonding via formation of a carbamate or by
acetylation.
6.3. Growing Interest in ll(15-l)#£e0-taxanes
Although much of the work, so far, on these new compounds, has come from Italy and
Japan there is mounting interest from other groups world-wide, as the search to find a
renewable source of taxol or taxol analogue continues. For example, in 1994,
collaborating groups from America and China, led by Tung-Ling Lee and Kuo-Hsiung, 54
reported their findings after investigation into the bark of the Chinese Yew, Taxus
chinensis.
AcO
AcO .
OAc
OAcHO
OH
OAcOBz
100 101
HO. HOPBz OAc jP Bz OAc BzO >0A c OAc
:o
OAc OAcOAc OAcHO HO HO
102 103 104
They isolated the novel rearranged compound 100 possessing a novel epoxide
functionality at the C-5 position rather than the usual exocyclic double bond or oxetane
42
ring. Continuing through 1994, the Canadians kept their interest in this field alight with
their investigations into the needles of the Himalayan Yew, Taxus wallichiana Z.,
wallifoliol 101, with the unusual structure shown, was discovered.55 In addition, D. G. I
Kingston, from Virginia State University, USA, reported more new rearranged taxanes,
isolated from the Pacific Yew, Taxus brevifolia,56 Compound 102, for example, was
discovered, possessing all characteristics typical of an 11(15-1 )abeo-taxanQ, from the
unusual downfield shift of the C-15 signal in the 13C NMR spectrum (876.4 ppm), to the
broad NMR spectrum at room temperature, which sharpened on heating to 57 °C revealing
the slow equilibrium between conformational isomers. The end of 1994 saw the
investigation, by a Chinese group, under Qi-Cheng Fang, into another Chinese Taxus
species, Taxus yunnanensis,57 taxayuntin F 103 was among two 11(15-1 )abeo-taxants
isolated from this new species. Again, thorough NMR investigation was consistent with
that expected for a rearranged taxane of this kind. In line with the results previously
discussed, regarding compound 98, X-ray analysis placed ring-B in the sterically more
stable twist-boat conformation, however, cooling to 0°C sharpened the broad room
temperature NMR spectrum to two clear sets of signals corresponding to two
conformations in slow equilibrium, with the twist-chair/boat type being the minor
conformer, stabilised by H-bonding. Finally, interest in this field has also been shown in
India since the Himalayas are also, potentially, a huge source of taxane compounds. For
example, a group led by B. Das, in Hyderabad, have reported their findings from
investigations into the needles of the Himalayan Yew,58,59 compound 104 was among
many taxanes isolated from this species, showing characteristics consistent with those of
11(15-1 )abeo-taxanes.
7. THE PURPOSES, AIMS & INTENTIONS BEHIND THIS THESIS
The ambitious aim, at the beginning of October 1994, was to develop a route towards the
synthesis of a biologically active taxol analogue, from an ll(15-l)a&eo-taxane, using
semi-synthetic methodology. Chapter One covers the history and development of 11(15-
l)abeo-taxanes, introducing the concept of conformational isomerism in these systems.
Chapter Two then begins with the initial work I carried out, constructing the taxol side
chain and determining the reactivity of the hydroxyl functionalities at positions C-5 and C-
13, on the rearranged taxane, brevifoliol 85. The chapter also contains discussion about
43
the isolation of the natural products from the Yew extract and my initial studies towards
formation of the oxetane ring. Chapter Three moves on to my endeavours to develop an
alternative route towards the oxetane ring, again working on the 11(15-1 )abeo-tzxane,
brevifoliol 85, with some interesting results. Chapter Four concentrates solely on a
different 11(15-1 )abeo-taxane, taxchinin A 87, it contains further work on conformational
analyses and, in addition, attempts towards oxetane ring construction, finally, chapter five
describes experimental work including detailed NMR analysis. I am confident that,
although the original aim of the PhD was not met, the interesting new compounds and
results I have developed and discovered, over the last three years, are of valuable use to the
world of taxane chemistry. Interest in this area of chemistry is world-wide and I am sure
that my results will contribute to the understanding, and the knowledge, we have on these
complex compounds, after all it is the build up of information and experience over many
years that leads to the successful development of a new drug, who knows what will be on
the market in 20 or 30 years time as a direct result of work carried out today.
44
CHAPTER TWO
INITIAL STUDIES
“You’re going to develop a novel, biologically active, taxol analogue”, were amongst the
first words I heard at the beginning of my PhD, “it shouldn’t be too difficult, after isolating
a suitable taxane compound from the Yew extract, all you have to do is construct the
oxetane ring and attach the side chain, no problem”. As has been the story surrounding the
subject of taxane chemistry, over the last 30 years, my initial problem was supply of a
suitable taxane compound, my studies were to involve the rearranged 11(15-1 )abeo-
taxane, brevifoliol 85, found in the Indian Yew, Taxus wallichiana Z., since a large
consignment of Yew resin from India had been acquired by a local company, Yew Tree
Pharmaceuticals bv. Whilst awaiting arrival of the resin I began work constructing the
taxol side-chain since any potential taxane anti-cancer compound requires this
functionality for biological activity.
BzO
OH
HO
85
1. THE TAXOL SIDE-CHAIN
1.1. Denis & Greene’s Original Synthesis
It is crucial that the side-chain moiety has the correct chirality at positions 2 and 3, since
any deviation from the natural product arrangement results in serious loss of activity,
Greene and co-workers, in 1986, at their research laboratories, in France, were the first to
report an efficient, enantioselective synthesis of the taxol side-chain 105, Scheme 10.60
45
Ph CH2OH Ti(0-*-Pr) 4
L-(+)-DET
106
O Ph O
H P. Hv v/ \ 2 . CH2 N2
Ph CH2OH
107
Ph O
1. RuC13, NaI04 H O H-► y V
Ph C02Me
108Me3 SiN3
„ ZnChPh O
Jl 1 il 1 11 1 . PhCOCl I J[
H : : 2. r i2, rU -L zOH OCOPh OH
105
2 : 2. H2, Pd-COCOPh OH
110 109
Scheme 10
A protected form of side-chain 105 was successfully coupled to the 10-deacetylbaccatin
IE, (10-DAB HI), derivative 58, in Greene and Potier’s semi-synthesis of taxol, see
Scheme 6 in the previous chapter. This first synthesis was based on the asymmetric
epoxidation of cis-cinnamoyl alcohol 106 using f-BuOOH and Ti(0 -i-Pr)4/L-(+)-DET as
the catalyst, in an effort to introduce chirality early in the sequence, with formation of the
2R,35-epoxyalcohol 107, which was subsequently oxidised and esterified to compound
108. Cleavage of the epoxide was carried out, with full regioselectivity, using
azidotrimethylsilane and zinc chloride as catalyst, then in situ hydrolysis afforded 109 in
90% yield. After esterification, with benzoyl chloride, and azide hydrogenation, migration
of the benzoyl functionality to the amine group was induced, yielding the taxol side chain
105 in an overall 23% yield.
1.2. Improved & Adapted Synthesis
Since this first asymmetric synthesis, there has been an abundance of new, more efficient
syntheses reported in the literature. The first successful taxol semi-synthesis, Scheme 6,
Chapter 1, utilised six equivalents of Greene’s protected side-chain 61, together with
extremely severe conditions to achieve coupling to the 10-DAB IE derivative 60.11
Although this semi-synthesis was a significant achievement, improvements were vital to
develop a more efficient route which would incorporate minimum loss of the precious 1 0 -
DAB HI. Denis and Greene published their improved side-chain synthesis in 1990,61 this
synthesis was more efficient and chemically much less complex, in addition, the side chain
46
generated from this scheme was also modified and incorporated into the semi-synthesis of
taxotere 63, Scheme 7, in the previous chapter. It was this synthesis I carried out, here in
Leicester, slightly modified by P. Wiegerinck,62 a Dutch college also working in
conjunction with the Dutch pharmaceutical company, Pharmachemie bv, to construct N-
benzoyl-(2/?,3S)-3-phenylisoserine 105, the taxol side-chain, Scheme 11.
O K 20 s 0 2(0 H )4 H P ______^ 0 H p-TsCl H(\ / ° TS
P h '^ ^ ^ ^ O C H 3 (DHQD Phthal Ph //C 02CH3 Et3N Ph /C 02CH3
111 112 113k2c o 3, d m f h20
ph. PH 1. PhCOCl, Et3N Ph. P H NaN3 y ° \
HN C 02CH3 2’ H2> Pd*C N3 C 02CH3 Ph C 02CH3
HPh
105 115 114
Scheme 11
The scheme begins with a Sharpless asymmetric dihydroxylation of methyl cinnamate
111,63 this gave the optically active diol 112 in a 6 8 % yield. Formation of the tosylate 113
was followed by cyclisation affording the epoxide 114, in 75% yield. Nucleophilic
opening of the epoxide with sodium azide furnished compound 115 which, after
subsequent reaction with benzoyl chloride, produced an ester intermediate. After
hydrogenation, in situ migration of the benzoyl functionality successfully afforded
compound 105, the taxol side chain, in 29% yield, with all characteristics identical to those
of the literature compound.60
1.3. Alternative Syntheses, eg. (3-lactam Approach
In attempts to improve the synthesis further, numerous approaches towards the side-chain,
have been undertaken by many research groups world-wide. Since direct coupling of the
protected side-chain had generated problems, from low yields, harsh conditions and loss of
10-DAB IE, to low stability of the free acid, much of the work, carried out, has
47
concentrated on developing alternative ‘surrogate’ acylating agents. 11 The AT-acyl-p-
lactam method is a prime example since these derivatives are sterically less bulky allowing
an easier approach to the hindered C-13 hydroxyl group. Georg and Ojima, two scientists
working at The State University of New York, USA, have particularly studied this area,
with their asymmetric syntheses of p-lactams via the ester enolate-imine condensation, n ’
64 with the key reactions illustrated in fig. 19.11
ROLDA LDA
'IL>O
y °___________ HN— V ^ ____________
PhCH=NTMS ) ---- \ PhCH=NTMSPh' 'OR
"ORO
S0 2 N(C6 H„ ) 2
116R=TIPS
117a R=TIPS 117b R=TMDMS
118R=TMDMS
fig. 19
p-Lactams have now been used in the semi-synthesis of taxol from 7-TES-baccatin HI 60,
as illustrated in Scheme 12. li
AcO OTES
HO....
HO OAcOBz
n-BuLiLiO'"
H 0 6 Bz 0A c
60 119
oX| I 120
P h '° *t)R
OA , AcQ .0
Ph NH OH F.Py
TAXOL ---------- Ph'
OTES
OR
H 0 6 bz ° Ac
121 R=Protecting Group
Scheme 12
48
In this sequence, the lithium alkoxide derivative 119 reacts with just 1.1 equivalents of the
p-lactam 1 2 0 affording protected taxane 1 2 1 in almost quantitative yield, a highly efficient
reaction proceeding after attack of the alkoxide on the strained carbonyl group, forcing
ring opening of the newly formed tetrahedral intermediate. In addition, in some cases
racemic p-lactams can be used since attack by the alkoxide ion is sometimes
diastereoselective.
2. BREVIFOLIOL, STRUCTURE DETERMINATION
Having prepared the taxol side-chain we were now ready to begin work on the 11(15-
l)abeo-taxane, brevifoliol 85, beginning with structure determination.
BzO OAc
HO......
HO
85
Although, at this stage, we had not received the actual Yew resin, we were lucky enough to
be given a sample of the compound, from another Dutch colleague, Erik Van Rozendaal, a
Doctor working at The Agricultural University in Wageningen, Holland, also in
collaboration with Pharmachemie.
2.1. Introduction
Conformational analysis, of 11(15-1 )abeo-taxanes, has been carried out, primarily by the
Japanese and Italien groups, led by K. Fuji and G. Appendino respectively, as discussed in
chapter one. Although one might expect, at first sight, ll(15-l)<z6eo-taxanes to be in
possession of fixed conformations, Fuji and Appendino found, in fact, a flexibility exists
in the diterpenoid skeleton inducing, in some cases, a conformational isomerism.46' 49 & 53
49
2.1.1. The Twist-boat/Chair Conformation
Both groups suggest the twist-boat/chair type conformation, type A, for rings B and C
respectively, is sterically the most stable arrangement for an 11(15-1 )abeo-taxane, in
possession of a C-4/C-20 exocyclic double bond, see fig. 19. In adopting this
conformation, it has been postulated, steric crowding between the 'Bu group at C-l and the
functional group at C-l0 is reduced, so stabilising the arrangement.47,48,53
Steric crowding between C-1(fBu) and C-10(OBz) reduced
Mf ”> RO
15=
‘14bOR
OR M e ' 8
Type A Twist-boat/Chair
fig. 19
In forming this type A conformation, protons H-9 and H-10, in ring-b, adopt an anti
arrangement, with respect to each other, separated by a dihedral angle of 180°, see np a
fig. 20. Typically in the twist-boat TB conformation, J9, i0 is in the range 9-11 Hz.46' 49 & 53
OR,
OR
H10
OR
RO,
H10
TBRing-b, Twist-boat np a
fig. 20
50
Furthermore, it has generally been found, when ring-b adopts the twist-boat arrangement
TB, ring-c preferentially converts to the chair C conformation, see fig. 21, affording, in
total, the twist-boat/chair type A conformer.47 Typically coupling constants attributable to
H-7 and H-6 a/H-6 p are indicative of this arrangement. J7>6P, in the range 9-11 Hz, is
finally, in addition, when J7>60 is equal to a value of about 5.0 Hz, this points to a dihedral
angle of about 60°, between H-7 and H-6 a , also illustrated in np b, confirming the chair C
arrangement.
Ring-C, Chair
fig. 2 1
2.1.2. The Twist-Chair/Boat Conformation
In some cases, in addition to, or sometimes in preference to, the type A conformation, fig.
19, the twist-chair/boat type conformation, type B, is seen in solution, shown in fig. 22.47,53
analogous to a dihedral angle of 180° between these two protons, see np b,‘,46'49 & 53 then
C np b
H -b o n d in g s ta b ilis a tio n
H13 or
Type B Twist-chair/Boat
fig. 22
51
In non-polar, aprotic solvents, this arrangement can be stabilised by intramolecular H-
bonding between C-15(OH) and the functionality at C-10. In forming this type B
conformation, protons H-9 and H-10, in the b-ring, contrary to the type A conformer, now
adopt an equatorial arrangement, with respect to each other, separated by a dihedral angle
of about 60°, corresponding to the twist-chair TC conformation, see np c, fig. 23.
Typically, in this arrangement, Jg, i0 is found to be in the range 3-4 Hz.46"49 & 53
On adopting the twist-chair TC arrangement, in ring-b, it has been commonly noted, that
the C-ring preferentially converts to a boat B type conformation 46,47,53 Again, those J
values attributable to H-7 and H-6 cx/H-6 p, are often the key signals to analyse when
deciphering a possible conformational change. On flipping from a chair C to a boat B
conformation the dihedral angle between H-7 and H-6 a decreases from 60° to about 30°,
increasing the coupling constant between the two protons, see np d, fig. 24. Typically the
doublet of doublets, attributable to H-7 in the chair C conformation, changes to a triplet,
on conversion to the boat B arrangement.4 6 ,47 This alteration is analogous to a J value in
the range 7-9 Hz attributable to J7,6a, 6P-
ORg
TCRing-b, Twist-chair
np c
fig. 23
H 7
np dBRing-C, boat
fig. 24
52
2.2. Brevifoliol, Conformational Analysis
If we now consider brevifoliol 85, in light of the work carried out by Fuji and Appendino,
we determined compound 85 to exist, in solution, entirely in the twist-boat/chair, 85a,
conformation.
M ?19 OAcOAc
15=
H 14bOH
Hio
OH MCl8
85a
The presence of only one one set of sharp signals, in both the *H and 13C NMR spectra,
indicated the presence of only one conformer. The coupling constant attributable to H-
9/H-10 was calculated to be 10.4 Hz, indicating separation of these two protons by a large
dihedral angle of 180°, see np e. Accordingly, on adopting this anti arrangement, ring-b
was forced into the twist-boat conformation, 85b, as shown in fig. 25.
OBz
OAc
OAc
BzO.
Hio
85b np e
fig. 25
The chair 85c conformation was assigned after analysis of the coupling constants
attributable to H-7 and H-6a/H-6(3, see fig. 26. The doublet of doublets, at 55.56,
exhibited J7,6p10.9, indicating a dihedral angle of 180° between these two protons, see np
53
f, J7,6a5.7 pointed to a dihedral angle of about 60°, between H-7 and H-6 a, also shown in
np f, confirming the chair 85c conformation.
fig. 26
2.2.1. Conclusion, from these Studies on Brevifoliol
Even in the polar, aprotic solvent, CDCI3 , brevifoliol 85 existed entirely in the, sterically
more stable, twist-boat/chair conformation, 85a, reducing crowding between the bulky rBu
group at C-l and the large OBz functionality at C-10. The extra stabilisation, that would
be provided by the intramolecular H-bonding between OH(15) and C-lO(OBz), is not
enough to allow any conversion to the sterically less stable twist-chair/boat 85d conformer,
see fig. 27.
85c np f
Reduced steric crowding between C-1(*Bu) & C-10(OBz)
H-bonding stabilisation, not enough to over-ride extreme steric hindrance between C-1(tBu) & C-10(OBz)
OH H13 o h
85a 85d
fig. 27
54
Throughout the rest of this chapter, functional group manipulations at positions C-4, C-5
and C -l3 are discussed, which did not affect the conformation of the main diterpenoid
skeleton. That is, all the compounds subsequently produced, from brevifoliol 85,
remained entirely in the twist-boat/chair, type A, see fig. 19, arrangement. Thus,
conformational isomerism is not debated further, in this chapter, however, in chapters
three and four, our investigations into the chemistry and characteristics of the diterpenoid
skeleton are discussed bringing in the conformational behaviour.
3. C-5 Vs C-13 REACTIVITY IN BREVIFOLIOL
Using the sample donated from The Netherlands, our initial aim was to determine the
relative reactivity of the hydroxyl functionalities at positions C-5 and C-13 .
C-13 C-5BzO «PAC OAc
HO
85
Selective protection at C-5 would allow side-chain attachment at C-13 or, alternatively,
selective protection at C-13 would allow smooth oxetane ring construction across C-4, C-
5. Unfortunately, all attempts to achieve selective protection, at either position, were
unsuccessful, despite the strongest endeavours with numerous protecting groups, see table
1 , it would appear that both positions are equally unreactive, the hydroxyl groups are
probably too highly hindered, by other functional groups in close proximity, and by the
taxane skeleton its self.
55
REAGENT NO.
EQUIVALENTS
FURTHER
COND331QNS
EEAGTION
f-BuPh2SiCl 1.5 Imidazole, CH2C12, rt, 2 0 hrs
No reaction
2.5 Imidazole, CH2C12, rt, 48 hrs
No reaction
f-BuMe2SiCl 1.5 Et3N, DMAP, DMF, rt, 7 hrs
No reaction
2 Et3N, DMAP, DMF rt, 2 0 hrs
No reaction
Et3SiCl 1 .2 Imidazole, CH2C12
rt, 18 hrsNo reaction
2 Imidazole, CH2C12, rt, 2 0 hrs
No reaction
2.5 Imidazole, CH2C12, rt, 24 hrs
No reaction
Et3SiOS02CF3 1 Pyridine, CH2C12, rt, 17.5 hrs
No recation
2 Pyridine, CH2C12, rt, 2 0 hrs
No reaction
1.5 DMAP, CH2C12, rt, 18hrs
No reaction
2.5 DMAP, CH2C12, rt, 24 hrs
No reaction
Me3SiCl 1 Imidazole, CH2C12, rt, 2 0 hrs
No reaction
CCl3CH2OCOCl 1 Pyridine, 80°C, 5hrs No reaction2 Pyridine, 80° 15 hrs No reaction3 Pyridine, 80°C, 6 hrs No reaction
CC13C0C1 1 Pyridine, DMF, rt, 24 hrs
No reaction
2 Pyridine, DMF, rt, 24 hrs
No reaction
C1CH2C0C1 1 Pyridine, rt, 18 hrs No reactionDihydropyran 1 .1 p-TsOH, CH2C12,
6 hrsNo reaction
Table 1
56
4. CINNAMIC ACID COUPLING
In our studies towards determining the relative reactivity of the hydroxyl groups at C-5 and
C-13, we also investigated the reaction shown in fig 28. Treatment of brevifoliol 85 with
one equivalent of cinnamic acid, in the presence of DCC and DMAP, afforded a mixture
of products. The C-5 and C-13 cinnamates, compounds 123 and 124 respectively, were
produced in a 3:1 ratio, determined by NMR spectroscopy.
PAcOAcpAc o a c
DCC, DMAP
R = Cinnamoyl R = Cinnamoyl
fig. 28
All major signals were attributed to the C-5 cinnamate, compound 123, after analysis of
the COSY spectrum. The signal attributable to H-5, in the major compound, had
shifted from 64.37, in brevifoliol 85, downfield to 65.49 after cinnamic acid 122 coupling.
This signal showed correlation peaks to both H-6 a (major) and H-6 p (major), see structure
123a. The broad singlet, assigned to H-13 in the major compound, remained at about
64.0, this signal showed cross peaks with both H-14a (major) and H-14p (major).
BzO PAc OAc
HO •••/',o
HO123a
All minor signals were assigned to the C-13 cinnamate, compound 124, again using the 2-
D spectrum. Although some signals were partially obscured, key peaks, allowing
structure determination, were evident. In the minor product, the broad singlet, attributable
57
to H-13 (minor), had shifted downfield, from 54.37, in brevifoliol, to 55.58, after
cinnamate formation. This signal exhibited a clear cross peak corresponding to H-14p, in
the minor compound. In addition, the signal attributable to H-5 (minor), remained at about
54.0, this broad singlet showed correlation peaks with both H-6 a (minor) together with H-
6 p (minor), see structure 124a.
4.1. G. I. George, Side-Chain Attachment To Brevifoliol
At this point in the project a paper, by G. I Georg and co-workers, describing side-chain
attachment to brevifoliol, was published.65 They directly coupled the p-lactam side-chain
derivative 125 to brevifoliol 85, giving brevifoliol 13-[A^-benzoyl-(2'/?,3'5)-3-
phenylisoserinate], 126, in 85% yield, fig. 29. The less hindered, almost planar, p-lactam
derivative 125, was obviously necessary to achieve selective attachment at C-13. The new
brevifoliol derivative 126 was tested for biological activity, no activity was seen in the
tubulin assay and the cytotoxicity assay was very poor indicating the need for the oxetane
ring and also, possibly, the benzoate functionality at C-2.
HO
124a
o
BzO p A c QAc
Py, DMAP, CH?C1? >
2. 0.5% HCl/EtOH""'OH
HO HO
85 126
fig. 29
58
5. TOWARDS THE OXETANE RING
It was now obvious that an alternative course of action was needed, accordingly, it was
decided to attempt construction of the oxetane ring across the C-4, C-5 positions,
contained in brevifoliol 85, without any prior protection at C-13.
5.1. Approaches by Ettouati
In their attempts to prepare a novel potent taxol analogue from taxane derivatives isolated
from the European Yew, Taxus baccata, using semi-synthesis, Ettouati and his co
workers, at The Natural Products Institute, in Gif-Sur-Yvette, France, were the first to
develop a synthesis towards construction of the oxetane ring moiety.66 They isolated a
large quantity of taxine B 127, with a C-4/C-20 double bond, after converting it to
derivative 128 they were ready to begin oxetane ring formation, Scheme 13.
X
oo""O H NMO
'OH
'OH
128
AcO OH
O'"O R
HOOH
R=COCH2CHNMe2PhTBDMSC1,Imidazole,DMF
XMsCl
oPy
'OH
'OR
XTBAF
Oi H r '"O H
- O ''''OTBDMS
132 131 130R=TBDMS
Scheme 13
59
Dihydroxylation across the C-4/C-20 double bond, using osmium tetroxide and NMO,
afforded diol 129. Protection of the primary hydroxyl group was achieved using t-
BuMe2SiCl, giving compound 130, which was easily converted to mesylate 131 using
mesyl chloride, protecting group removal was then accomplished, using TBAF, to afford
compound 132. Problems occurred in attempting to induce ring closure, using sodium
hydride or potassium terr-butoxide compounds 133-135 were obtained, as shown in fig.
30, probably as a result of the highly basic nature of these two reagents.
o
'o h
132
o
OH
135
o'CH2OH
134
o
133
fig. 30
Ettouati and co-workers suggested derivative 133 was probably formed via an enol
following concerted elimination of formaldehyde and the mesyl group, in addition,
compounds 134 and 135 probably occured as a result of hydroxymethylene transfer, again
with concerted mesyl departure. Eventually, however, they obtained compound 136, using
the weaker base, tefra-butylammonium acetate, H-BU4NOAC, to induce ring closure, with
only a small amount of derivative 137 being seen.
60
OH
136
X
OMsOHOAc
137
This methodology was also used by our Dutch colleage, E. Van Rozendaal, in his
investigations into the semi-synthesis of a new taxol analogue from taxine B ,67
furthermore, this synthesis had also been adapted by both Holton 21 and Nicolaou 68 in their
taxol total syntheses.
5.2. Proposed Scheme Towards Oxetane Ring Construction
As such, we devised a scheme to incorporate the oxetane ring into the brevifoliol structure,
Scheme 14, basing our ideas on Ettouati methodology.66 The scheme begins with diol
formation 138 across the C-4/C-20 double bond,60 following selective protection of the
primary hydroxyl group at C-20, using f-BuMe2SiCl (TBDMSC1), affording compound
139, we hoped to proceed via the acetonide 140,32 which would afford protection of the
1,2-diol across C-4/C-5. Having constructed derivative 140, this would then allow the
desired selective protection of the hydroxyl group at C-13, affording compound 141. It
was envisaged that a protecting group such as 'Pr3SiCl (TIPSC1) or 'P^SiOTf (TIPSOTf)
would be used for this reaction since this silyl ether is more stable than the TBDMS
analogue. Continuing through the route, removal of the isopropylidene group should be
possible, by treatment with ethanedithiol and p-TSA. D. R. Williams, in his synthesis of
phyllanthocin, successfully removed the acetonide protecting group in the presence of a
TBDMS ether, using these conditions.69 Treatment of diol 142 with mesyl chloride should
give mesylate 143 which would be converted to compound 144 on treatment with TBAF.
The TBDMS group is more labile than the TIPS ether, which will require harsher
conditions for removal, hence, allowing selective deprotection, this methodology was used
by Danishefsky in his synthesis of the immunosuppressant, rapamycin.70 Oxetane ring
closure should then be possible using W-BU4NOAC, as precedented by Ettouati and co
workers,66 affording compound 145. Acetylation at C-4, using acetic anhydride and
61
DMAP,66, 68 should give us derivative 146, leaving us poised for the production of our
initial target compound, 147, after TIPS removal and side-chain attachment.
BzQ. X)Ac^ OAc
HO"1'
BzO. OAc OAc
OSO4HO""
""OH NMO
BzQ. JOAcOAc
TBDMSC1 ................ ■►HO""
C > O H Et3N 'o h
85 139 R =TBDMS(CH3)2C(OMe) 2
p-TsOH
BzQ. OAcf OAc
R O""
BzQ. DAcOAc
EtSHR2 0 " -
TIPSC1
""OH P-TsOH
BzQ.
HO'.....
OR1HO
141R =TIPS 140
BzO,
OMs'OH
ORHO
TBAF
BzO.BzO,
OMs'OH OHOH HOHO
143 144 145Ac^O,
DMAP
BzO,NH
O'....OH
OAcHO
R Removal
Side-Chain
Attachment
BzO. OAcOAc
OAcHO
147 146
Scheme 14
On undertaking the proposed scheme, diol formation across the C-4/C-20 double bond was
successfully achieved using osmium tetroxide and NMO, affording compound 138 in a
62% yield. It was assumed that, in line with the literature precedent,66’ 67 steric hindrance
from the methyl group at C-8 forced cis-dihydroxylation below the double bond, as shown
in fig. 31,67 to give the stereochemistry of diol 138.
62
STERIC HINDERANCE FROM Me-1S BLOCKS DIOL FORMATION HERE
CIS-DIHYDROXYLATION FROM THIS FACE
fig. 31
Problems were initially encountered in the next step, on treatment of the diol 138 with 4, 6
and 8 equivalents of TBDMSC1, in the presence of Et3N and DMAP, no reaction was seen,
even after 24hrs stirring, only starting material was seen by TLC. Protection was also
attempted using TBDMSOTf, however, again, no significant reaction progression could be
seen by TLC. Although, at this stage, things were not looking promising, one final
reaction using TBDMSC1, was investigated, using 10 equivalents of reagent, again in the
presence of Et3N and DMAP, after 16 hrs stirring, a single new product was observed by
TLC. On isolating the new compound, it was determined, after NMR analysis, two silyl
ether groups had been introduced to diol 138, affording the disilyl compound 148, fig. 32,
in 78% yield, this observation was in agreement with mass spectral data.
TBDMSCl
"''O H E t3N > )H DMAP
138 148 R=TBDMS 149
fig. 32
Initially, it was difficult to determine, by NMR analysis, exactly where protection had
taken place since there were potentially five possible sites available for TBDMS reaction,
OH(4), OH(5), OH(13), OH(15) or OH(20). Hence, we elected to react the newly formed
63
disilyl compound with 2,2-dimethoxypropane, in the presence of p-TsOH, as precedented
by Kingston in his initial paper describing A-ring contraction.32 In the event, NMR
analysis indicated the formation of the acetonide 149 pointing to TBDMS protection at the
more reactive C-20 primary hydroxyl position and also at C-13. Acetonide formation was
deemed to have taken place across positions C-4/C-5 rather than C-4/C-20, after NMR
analysis. The signal attributable to H-5 moved from 83.79 to 84.49, after isopropylidene
formation, this shift, downfield, was in agreement with the analogous reaction, carried out
by Kingston.32 This acetonide formation also confirmed the original dihydroxylation,
affording diol 138, had indeed taken place below the C-4/C-20 double bond, see fig. 33,
path a. Acetonide construction across C-4/C-5 would not be possible had the
stereochemistry been reversed, as shown, see path b.
Me,M®19 O Ac-OHOAc
a
OHOH OH
H3
X b
M®19 O A c,O H
tM?l9 r OH
OH
HO
R=OAc
fig. 33
64
5.3. Revised Scheme
At this stage a revised scheme was drawn up towards compound 150, incorporating the
newly formed disilyl compound 148. Construction of the target compound 147, after
TBDMS removal and side-chain attachment, would then be possible. The new route
would, hopefully, in addition, be more efficient since it removed the need for a cyclic
protecting group, Scheme 15.
BzO.
HO
BzO £>AcOAc
HOi<"
BzO. *OAcOAc
R1©!'I-.,. 'OH
85 138 148 R'=TBDMS
MsCl,Py
BzO
'OMs'OH
OHHO
HO BzO OAc OAc
TBAF
'OH
O R 1
'OH
O R 1HO HO
154 153 151 R, =Ms
>!(t
BzO.
OH
HO
O
BzO
OHHO
BzO. OAc OAc
H O n .
'OH
OHHO
147R3=Side-chain
150 152
Scheme 15
On undertaking the revised scheme, treatment of the disilyl compound 148 with mesyl
chloride, in pyridine, successfully afforded mesylate 151 in a 59% yield. Presence of a
signal, attributable to (0 S 0 2CH3), in the NMR spectrum, together with a clear
65
downfield shift, from 83.79 to 85.05, of the broad singlet, assigned to H-5, allowed this
structure determination. On treatment with TBAF, in THF, however, the double
deprotected compound 152 was not obtained, instead a mixture of products was isolated.
NMR analysis indicated the formation of the 10-debenzoyl derivative 153, in 34% yield,
with the mono-deprotected compound 154 in a 35% yield.
6. RING CLOSURE
Initially, ring closure was attempted, using compound 154, adopting the fairly harsh
conditions employed by Nicolaou in the final stages of his taxol total synthesis,68 these
conditions had been precedented by Ettouati during his taxine B investigations.66 It was
now envisaged, protecting group removal, from C-13, would be made after oxetane ring
formation.
BzO.
TBDMSO'.....'OMs
OH
OHHO
11.8 eq. «-Bu4NOAc
^ -----Butanone, reflux,
4.5 hr
BzO.
TBDMSO""-'
OHHO
154 155
fig. 34
Diol 154 was treated with 11.8 equivalents of n-Bu4NOAc, in butanone, see fig. 34. After
refluxing, under nitrogen, for 4.5 hrs, TLC analysis indicated disappearance of starting
material, however, no pure compound could be isolated using column chromatography, it
was impossible to determine, by crude NMR, if any oxetane 155 had been formed due to
the complexity of the spectrum.
66
As such, the reaction was repeated using the conditions employed by one of our Dutch
colleagues, P. Wiegerinck, in his oxetane ring syntheses,62 fig. 35.
TBDMSO...4 eq. /1-BU4NOAC
Butanone, reflux,
1 hr
TBDMSO'""O
HO OH HO
154 155
fig. 35
In this case diol 154 was treated with only 4 equivalents of n-Bu4NOAc, in butanone.
After refluxing for just 1 hr, multiple spots were evident by TLC, indicating compound
decomposition. Column chromatography was, however, attempted in an endeavour to
isolate some oxetane 155, one compound was separated on the column, although in
negligible yield. NMR analysis, however, showed no evidence of oxetane ring formation,
instead, spectral analysis indicated a possible partied hydrolysis of the benzoate group.
6.1. Conclusion
Oxetane ring formation, under the conditions used, was unsuccessful, the tendency for the
compound to decompose could be attributed to the highly sensitive nature of the ester
functional groups at C-7, C-9 and C-10. Relatively harsh conditions seem to be required
to induce ring closure, for example, refluxing butanone is used in Ettouati’s original
synthesis.66 In the case of our brevifoliol derivative, it seems reasonable to suggest, that in
order to avoid compound decomposition, more robust protecting groups are needed to
stand up to the severe reaction conditions required. In this vein, we concluded, removal of
all ester functionalities was necessary before beginning a synthesis towards the
construction of a new taxol analogue. Our endeavours, following this reasoning, are
discussed in chapters three and four.
67
7. NATURAL PRODUCT ISOLATION
Extraction of the natural product, brevifoliol 85, was not a trivial matter, either in terms of
the actual isolation procedure, or in terms of actually acquiring the Yew tree resin, in order
to carry out the extraction. Our initial studies, into the chemistry and behaviour of
brevifoliol, were carried out on a sample isolated from Yew extract, by E. Van Rozendaal
in his laboratories in The Netherlands. He developed his extraction procedure using the
resin obtained from India by Pharamchemie’s daughter company, Yew Tree
Phamaceuticals bv, (YTP). Since YTP still had this consignment, I spent a month working
there with our colleagues, investigating the possibility of developing a large scale
extraction procedure, using a mixture of reverse phase HPLC and normal phase column
chromatography.
7.1. Yew Tree Pharmaceuticals, Extraction of Brevifoliol
An outline of the method developed has been presented below, however, since only one
month was available, the procedure was by no means optimised, there is, without a doubt,
room for improvement. Having said this, however, the potential to develop a highly
efficient, large scale brevifoliol extraction is in place. Through carrying out this extraction
procedure, a further 4g of brevifoliol was isolated.
1. Indian resin (black ‘tar-like’ substance) dissolved in methanol.
2. Back extraction into dichloromethane, leaving any polar waste material in the methanol
layer.
3. Crude cromatography, see HPLC 1 for spectrum illustrating a sample of the crude
resin.
a. Normal Phase
i.Dichloromethane extract passed through alumina. Initial eluting solvent,
dichloromethane, encouraging removal of non-polar ‘waste’ material, later
Rt-ii. Solvent changed to 10% MeOH in CH2CI2, encouraging taxane elution,
particularly brevifoliol, Rt 13.00.
b. Reverse Phase
68
Fraction containing the brevifoliol majority, passed through a C-18 column,
eluting solvent, 100% methanol. Polar components, i.e. predominantly
brevifoliol and minor taxanes, immediately eluted, waxes retained on the
column, overall, material becomes more manageable.
4. Solvents removed under reduced pressure.
5. Solid taken up in dichloromethane and dry loaded onto a normal phase silica column.
a. Initial eluting solvent system, ethyl acetate:hexane, 1:1, v/v, causing removal of
chlorophyll, Rt 11.983.
b. Solvent system changed to 10% MeOH in CH2CI2 , causing brevifoliol, and
other
taxane, elution.
6 . Solvents removed under reduced pressure.
7. Solid dissolved in MeOHiKbO, 50:50, v/v and applied to a second reverse phase C-18
column. Eluting solvent system, MeOH:H2 0 , 50:50, v/v, (isocratic), separating the
brevifoliol peak, allowing collection of pure brevifoliol, see HPLC 2, Rt 21.775.
1 0%i1e00<*
:1 .
1*4
:0
1 i m
' ' 1 Oi> 5 5 £ • S • 1 I
; 1W :,— ,,1C aj
li
HPLC 1
U i -
HPLC 2
69
7.2. Laboratory Scale Extraction Procedure
In addition to the time I spent at Yew Tree pharmaceuticals, I also spent three weeks
working with Dr. E. Van Rozendaal at The Agricultural University, in The Netherlands, he
had developed an extraction procedure for use on a laboratory scale,71 the scheme below
gives an overview of the process, full details are given in the experimental section.
INDIAN YEW EXTRACT
(Dissolved MeOH/H 20
Continuous extraction, 2 days, petroleum ether (40-60°C)
(Pet. ether discarded
Continuous extraction, 2 days, ethyl acetate
J Solvent Removal
Tar/solid dissolved CHC13, stirred overnight with decolourising charcoal
(Charcoal filtration
CHC13 extract washed 3 times with aq. NH3 solution
(Solvent removal
CHC1, extract adsorbed onto silica
x \Non-polar waste material Taxane fractioneluted, 1 :1 , eluted, 1 0 %pet. ether: ethyl acetate MeOH/CHCl3
(Solvent Removal
CHC13 extract adsorbed onto silica for chromatography ethyl acetate:pet ether, 92:8
x \TAXCHININ A 87 BREVIFOLIOL 85
70
This method proved to be highly reliable, if somewhat time consuming, and I was able to
repeat it many times here in Leicester, isolating, not only brevifoliol 85 but also a second
ll(15-l)afoo-taxane, taxchinin A 87, all characteristics, attributable to each taxane,
were in agreement with those of the literature compounds.37,39,40,41
BzQ OAc OAc
HO......'OH
HO
BzQ OAc OAc
HO".... .
OAcHO
85 87
71
CHAPTER THREE
NEW APPROACHES
I returned to Leicester, from Holland, with about 3 Kg of the Indian resin and some new
ideas and approaches towards tackling the challenges involved with taxane semi-syntheses,
after discussions with our Dutch colleagues. Now we had a reliable extraction procedure
and, therefore, a regular supply of material, we were able to go ahead with some new
syntheses.
1. BREVIFOLIOL HYDROLYSIS
Following my discussions in The Netherlands, we decided to begin work investigating the
reactivity and chemistry of the brevifoliol diterpenoid skeleton 85, starting with hydrolysis
of all three ester functionalities, fig. 36.
BzO. HO.PAc OAc OH
NaHO......HO.... .
'""OH MeOH 'OH
HOHO
85
fig. 36
Using methodology developed by S. Py, a French colleague, in her investigations into the
semi-synthesis of a new taxol analogue,71 the ester groups were easily hydrolysed with
0.125M sodium methoxide, affording polyol 156 in an 83% yield.
72
1.1. NMR & Conformational Analysis of Hydrolysed Brevifoliol
We showed, in the previous chapter, brevifoliol 85, exists entirely in the twist-boat/chair
conformation 85a, reducing steric crowding between the 'Bu group at C-l and the OBz
functionality at C-10, see fig. 19. On forming compound 156, however, the bulky benzoate
group, at C-10, is replaced by a smaller hydroxyl group, it seemed reasonable, therefore, to
predict conformational isomerism may now be possible in this compound, between the
twist-boat/chair 156a and the twist-chair/boat 156b conformations, as illustrated in fig. 37.
Even with a smaller functional group at C-10, however, the twist-chair/boat conformer,
156b, would still be sterically disfavoured, nevertheless, in comparison to a benzoate
group in this position, crowding, within the molecule, would be considerably less. As a
result intramolecular H-bonding between C-15(OH) and C-IO(OH) should, to an extent,
stabilise this twist-chair/boat 156b isomer.
For all that, compound 156 was insoluble in most organic solvents, as a result, NMR
analysis was carried out using MeOD. At room temperature a single set of sharp signals
was observed, corresponding to only one isomer, the sterically more stable twist-boat/chair
conformer, 156a. The J value corresponding to H-9/H-10 was, in agreement with values
typical of the twist-boat 156c arrangement,46*49 & 53 9.4 Hz, placing these protons in an anti
arrangement, with respect to each other, with a dihedral angle of 180°, see np A, fig. 38.
H-bonding should help stabilise this conformation
Me16 '//,
156a 156b
fig. 37
73
OH
OH
156cRing-b, Twist-boat np A
fig. 38
The doublet of doublet of doublets, at 51.71, attributable to H-6p allowed assignment of
the chair 156d conformation to ring-c, see fig. 39. J6a, 6p14 .8 was analogous to geminal
coupling, J6P, 7 1 1.7 indicated a dihedral angle of about 180° between these two protons, see
np B, then J6p, s4.3 pointed to a dihedral angle of about 60° between H-6p and H-5, shown
in np C, confirming the chair 156d arrangement.
Since in methanol, intramolecular H-bonding, within a molecule, would be expected to be
methanol, the intramolecular H-bonding, between C-15(OH) and C-IO(OH), will be
negligible, hence, in this solvent, none of the twist-chair/boat 156b conformer was seen in
our NMR analysis.
156d np B np C
fig. 39
negligible,90 this phenomenon can be explained. In protic, polar solvents, such as
74
2. INVESTIGATING HYDROXYL PROTECTION
Although it was exciting to achieve a clean successful reaction, we were now faced, once
again, with the need to carry out protecting group chemistry. Compound 156 was
particularly challenging since it possessed six free hydroxyl functional groups. Our initial
ideas, therefore, involved using cyclic protecting groups with the intention of ‘tying up’
two or four hydroxyl groups, allowing smooth construction of the oxetane ring. Since 156
was insoluble in most traditional organic solvents, such as dichloromethane, ether and
THF, any successful reaction would need to involve a polar aprotic solvent. Furthermore,
since the diterpenoid skeleton would presumably adopt the twist-boat/chair type
conformation 156a in this type of solvent, it was possible to predict, using a Dreiding
Molecular Model, the most likely positions for cyclic protection to take place, as
illustrated in fig. 40.
Cyclic protection should occur acrossOH(10)/OH(15), OH(9)/OH(10) or OH(9)/OH(7)
OH
15=
OH
l14a
Me,OH
156a
fig . 40
The most obvious positions, to accommodate cyclic protection, should be the 1,3-diol
across C-7, C-9, or the 1,2-diol across C-9, C-10, since in the twist-boat/chair type
conformation shown, these three hydroxyl groups have the correct geometry for such
protections. Furthermore, it is possible that protection could, theoretically, take place
across the diterpenoid skeleton, at positions OH(15) and OH(IO), although this would
introduce steric crowding between the t-Bu group at C-l and H-9 and, as a consequence, is
unlikely.
75
2.1. Results & Discussion
In the event, results far from that expected were obtained, providing us with some
interesting and challenging problems, regarding interpretation of the complex spectra
generated.
2.1.1. Initial Investigations into Acetonide Formation
Initially, the most obvious diol protecting group to utilise was the cyclic isopropylidene
acetal, this functional group has been used many times in numerous syntheses requiring
protection of 1, 2 and 1, 3 diols. Indeed, use of the acetonide has been successfully
employed by Ettouati66 and Van Rozendaal67 in their semi-synthetic investigations on
taxine B, furthermore, Wender used this functionality in his taxol total synthesis.24
Despite this, however, although varied conditions were investigated, see Table 2, no pure,
mono-157,158 or d i-159, protected compounds could be isolated, as shown in fig. 41.
HO
158
fig. 41
76
REAGENTS & REACTION
CONDITIONS
CUSO4 , Acetone, No Reaction
24 hrs, rt
CUSO4 , Acetone, Compound Decomposition
cat. c. H2SO4 , rt
CUSO4 , Xs Acetone, No Reaction
3 Days, rt
CuS04, Acetone, Severe streaking by TLC
p-TsOH, rt
2,2-Dimethoxypropane, No pure compound
p-TsOH, 5 hr, rt isolated
Table 2
Initially, brevifoliol 85 was treated with dry acetone and anhydrous CUSO4 as described by
Van Rozendaal.67 After stirring at room temperature, for 24 hrs, only starting material
could be seen by TLC, however adding just one drop of conc. H2SO4 afforded compound
decomposition. In the third attempt, molar equivalents of CUSO4 and acetone were
increased, following methodology utilised by Lythgoe and his co-workers in their early
investigations into o-cinnamoyltaxicin-I,73 however, again, after 3 days stirring, only
starting material was observed by TLC. In a final attempt, using acetone, a catalytic
amount of p-TsOH was added to the reaction, as used by Ettouati,66 however, severe
streaking and several spots, by TLC, indicated an unsuccessful reaction. One alternative
acetonide protection was investigated, using 2 ,2 -dimethoxypropane, in place of acetone,
the reaction, as usual, was monitored by TLC. After 5 hrs stirring with p-TsOH, multiple
spots were evident, however, since starting material had disappeared, work-up was carried
out, unfortunately, despite repeated attempts at chromatography, to achieve separation, no
pure compound could be isolated from the reaction. Although continued chromatography
could possibly have furnished a degree of purification, it was clear that this protecting
group would not be satisfactory in any reasonably efficient semi-synthesis and it was
decided to investigate alternative means of protection.
77
2.1.2. Alternative Protecting Groups
Table 3, below, illustrates the range of alternative protecting groups investigated.
REAGENTS &
CONDITIONS
REACTION
f-Bu2Si(OTf)2, 2, 6 lutidine Unexpected Reactions
DMF, rt
Ph2SiCl2, Et3N, Product unstable to column
DMF, rt chromatography
Me2SiCl2, Et3N, Product unstable to column
DMF, rt chromatography
f-Bu2SiCl2, Et3N, No reaction
DMF, rt
i-Pr2Si(OTf)2, 2 , 6 lutidine Product unstable to column
DMF, 0°C chromatography
Ph2SnCl2, NaNH2, Compound Decomposition
THF, reflux
Table 3
On treating brevifoliol with 2 equivalents of r-Bu2Si(OTf)2, in DMF, in the presence of
2,6-lutidine, as used by Evans and co-workers in their synthesis of the macrolide
antibiotic, cytovaricin,74 reaction was obvious, after 3 hrs, TLC analysis indicated
complete disappearance of starting material. However, several spots were evident on the
TLC plate, indicating the formation of multiple compounds, in addition, a white crystalline
solid was isolated from the reaction. It was obvious, this reaction needed careful
investigation to elucidate the chemistry taking place, this will be discussed, in detail, later
in the chapter. However, in addition, various other silicon protecting groups were
investigated, including both the diphenyl and the dimethyldichlorosilane since it was
thought that these groups would prove relatively easy to identify on any subsequently
produced NMR spectra. Unfortunately, although new products were identified, by TLC,
both products proved to be unstable to column chromatography. Reaction with f-B^SiCL,
78
in DMF, in the presence of Et3N, was investigated,75 however after continued stirring only
starting material was observed by TLC. Two other cyclic protecting groups were also
investigated, brevifoliol was treated with i-Pr2Si(OTf)2, in DMF, in the presence of 2,6-
lutidine, again, although disappearance of starting material was observed, by TLC,
decomposition occurred during column chromatography. One fined reaction was
investigated, using Ph2SnCl2 ,77 unfortunately, again with no success. Since different
protecting groups were not producing any improvement on the initial reaction with t-
Bu2Si(OTf)2 , it was decided to concentrate all our efforts in this area. Fortunately, after
months carefully analysing complicated spectra, we were able to decipher the chemistry
taking place, producing some interesting results.
3. REACTION WITH DI-to*-BUTYLSILYL DITRIFLATE
Initial TLC analysis of the reaction mixture indicated the formation of two products, as
illustrated in fig. 42. At first it seemed two major compounds had been formed, since two
clear new spots were evident at R f 0.25 unknown A and R f 0.66 unknown B, in the
solvent system used. Minor stains were also evident and repeated column chromatography
was carried out to achieve purification.
2 eq. r-Bu2Si(OTf)2 UNKNOWN A, R f 0.25
2, 6 lutidine, DMF UNKNOWN B, R f 0.66
fig. 42
3.1. Initial Thoughts & Hypotheses
Initial NMR analysis, of unknown A, Rf 0.25, in CDCI3 , was extremely encouraging, the
spectrum indicated the formation of a new compound possessing two f-Bu2Si groups, in
possession of a single conformation. Furthermore, the J value assigned to H-9/H-10 was
relatively large at 8 . 8 Hz, indicating these two protons had adopted an anti arrangement,
HO. :PH OH
OH
HO
156
79
with respect to each other, corresponding to a separation of 180°, see np D, indicating the
twist-boat arrangement, Unknown A i, in ring-b, see fig. 43.
Unknown A i np D
fig. 43
Accordingly, as expected, after analysis of coupling constants, attributable to H-7, H-6 a
and H-6 p, ring-c was found to be in the chair, Unknown A ii, conformation, see fig. 44.
J7, 6p10.8 indicated a dihedral angle of 180° between these two protons, see np E, in
addition, J7, 6a5.0 pointed to a dihedral angle of about 60° between H-7 and H-6 a, also
shown in np E, confirming the chair Unknown A ii arrangement.
Logically, the structure of this newly formed compound should be the twist-boat/chair
conformer, 160, since achieving two cyclic protections at any other set of positions would
be impossible due to the geometrical constraints imposed by the taxane skeleton.
Unknown A ii np £
fig. 44
80
Expected Structure After Reaction With *Bu2Si(OTf)2
(H3C)3< \ /C (C H 3)3.Si9 \
Me16/,,J,*Osr
Analysis of the second component, unknown B, Rf 0.66, was more bemusing, this white
crystalline compound was insoluble in CDCI3, furthermore, in MeOD, just one singlet, at
60.95, was present, in the *H NMR spectrum. On expanding this spectrum, however, it
appeared that, in addition, another compound was also present, suggesting, unknown B,
Rf 0.66, was actually a mixture of two compounds. Unfortunately, the minor signals were
too weak to allow structure identification of this third unknown compound unknown C,
could it be that unknown C was actually a second protected brevifoliol derivative and the
white crystalline compound, unknown B, was an impurity which, by pure coincidence, ran
at the same Rf value as unknown C? Thus, it was decided to carry out further reactions to
help confirm the exact identity of unknown A and, in addition, determine the identities of
the possible inpurity, unknown B, together with the newly discovered unknown C.
Unfortunately, unknown A was not stable to lengthy analyses and handling, it was hoped,
therefore, to construct a subsequent compound that would withstand a detailed analysis. It
was envisaged that in building up as much NMR data as possible on all compounds, both
already formed and on those to be subsequently formed, we would be able to build a good
picture of the interesting new chemistry that was taking place.
4. REACTION WITH TRIETHYLSILYL CHLORIDE
4.1. Reaction Between Unknown A & Et3SiCl
We had already postulated that, logically, the structure of unknown A should be that of
compound 160, if this was the case, the free hydroxyl at C-13 was again in need of
81
selective protection to allow oxetane ring construction across C-4, C-5. It was decided,
therefore, to attempt protection using a large excess of reagent since we already knew that
both positions, 5 and 13, were highly unreactive, we would hopefully, then, achieve some
reaction at one or both positions. On carrying out the reaction, in the presence of pyridine,
as described by S. Py,72 two new spots were observed by TLC, after overnight stirring.
Initial NMR analysis, of these two isolated compounds, indicated the formation of a
mono-triethylsilyl compound and its di-triethylsilyl analogue in 18% and 48% yields
respectively, suggesting compounds (161a) and (161b) had been formed as in fig. 45.
Expected Structures After Reaction With E t3SiCl
Me j g it,, * S i'%IS \Hl4b \ ___ 1
Expected Structure 161a
Unknown A Possible Structure
160
OR M e i 8
Expected Structure 161b R=H or TES
fig. 45
Both new compounds were highly stable and could, therefore, be subjected to detailed
NMR analysis allowing exact structure determination.
82
4.1.1. NMR Analysis of the Compound Possessing Two Triethylsilyl Groups
Unfortunately, despite our strongest endeavours, a suitable crystal could not be grown,
which would allow absolute structure determination, however, we were able to carry out a
full NMR analysis, on the new compound, employing 1H, l3C, COSY, 1H-13C
COSY, NOESY and 29Si NMR techniques. Fortunately, this new compound was soluble
in CDCI3 allowing observation of some signals attributable to OH groups. After thorough
NMR investigation we were able to show that the structure of the new triethylsilyl
diprotected compound was actually the unusual structure 162, possessing the twist-
boat/chair type conformation 162a. Our original structure postulated for the di-
triethylsilyl compound, 161b was incorrect.
Revised Structure of the Compound Possessing Two Triethylsilyl Groups
M e,« OTES O H . H
/ / II*13 / H14a
'••-I / /T y f o u H10 sir\^fBu 'R„ O = OHB U ^ / Me18
SirB if \OH
\ I S i-O H
OH OTES
OTES
'Bu OH
OH
162a 162
Again, the !H NMR spectrum exhibited one set of sharp signals corresponding to the
presence of one conformational isomer. U, 10 was equal to 9.2 Hz indicating, as usual, the
twist-boat arrangement in ring-b, in addition, although the signal attributable to H-7 was
partially obscured, J7 , 6a could be determined as 5.6 Hz, indicating, as usual, the chair
arrangement in ring-c. After each proton signal had been identified, using ^ ^ H
correlation spectroscopy appendix 1 and also the !H-13C COSY spectrum appendix 2, the
key spectra in determining structure 162 were the NOESY spectrum NMR 1 and the 29Si
spectrum NMR 2.
83
4.1.1.1. Beginning With The NOESY Spectrum, NMR 1
Considering NMR 1, the most obvious nOes to initially look for were those
corresponding to the Si(CZ/2CH3) 3 protons, attributable to each of the triethylsilyl (TES)
groups. A clear nOe was seen between Si(C/f2CH3) 3 1 (TES 1) and H-5, nOe a,
indicating triethylsilyl attachment to OH(5). In addition, an nOe was obviously evident
between Si(CZ/2CH3) 3 2 (TES 2) and H-7, nOe b, clearly showing attachment to OH(7),
see structure 162b, fig. 46.
Me,OTES
OTES
162 b
fig. 46
As the C-ring had now been identified as having structure 162b, shown in fig. 46, the
question arose as to how two cyclic f-Bu2Si protecting groups could now be arranged
around the rest of the skeleton? It was possible that one cyclic protection could take place,
either across OH(9)/OH(10) or, alternatively, across the diterpenoid skeleton at
OH(15)/OH(10), see fig. 47.
Cyclic protection is possible across OH(9)/OH(10) OR across OH(15)/OH(10), two cyclic protections, in this compound, arenot possible.
Me,OH
OTES
OHMe,
fig. 47
84
Me-18
OH-9
Me-16
H -10H-20 H-20
I I H*jlL I. A u
A— v
H-2
H-3 H-14b
Me-17
Me-19
/ t-Bu
TES 2\ TES 1
U ltH-13 H-7
b 0
< >
(>
H-14a
| vm
D I
-Srh-Tt b
*!------j r Opo o
q c
pp*
NMR 2
However, given the positions of the remaining four free hydroxyl groups it was impossible
for two cyclic protecting groups to be in place around the skeleton. From this conclusion
it was necessary to consider the possibility that some other form of ‘protection’ might be
taking place, indeed, this in fact turned out to be the case.
4.1.1.2. 29Si NMR, NMR 2
If we now turn to the 29Si NMR spectrum, NMR 2, four clear signals can be seen
corresponding to four different silicon atoms, each in a different environment. 29Si NMR
studies on silanols and silylamines were carried out in 1975 by Williams and co-workers,
in the USA.78 Particularly noteworthy is the chemical shift assigned to (Ph)3SiOH, 6-12.6
in CDCI3, in addition, the chemical shift assigned to (Ph)2S£(OH) 2 was 6-30.7, again in
CDCI3 (both values are relative to Me^Si, 60.00). Furthermore, Roesky and co-workers, in
1996,79 during their studies into metallasiloxanes from silanetriols, were able to show the
chemical shift attributable to r-Bu3S£OH as being 5-36.8. From this data we felt it was
sensible to suggest that the two signals below 50.00 in the 29Si NMR spectmm, NMR 2, at
5-5.27 and 5-10.25 should be attributed to the form r-Bu2Si(OH)OR where R is the taxane
skeleton as in compound 162, fig. 31. The two signals further downfield at about 518.92
and 522.15 can then be assigned to the two triethylsilyl groups, in the form
(CH3CH2)3SiOR, again, where R is the taxane skeleton, with the absence of free hydroxyl
groups, in these two cases, causing the difference in chemical shift.
4.1.1.3. Returning To The NOESY Spectrum, NMR 1
Having established the r-Bu2Si protecting groups as being in the form f-Bu2Si(OH)OR,
protection was theoretically possible at OH(10), OH(9), OH(13) or OH(15). On further
analysis of the NOESY spectrum, NMR 1, we were able to determine exactly which
hydroxyl groups had been protected, see table 4 for a summary of all the main nOe
signals. Take a look at the signal assigned to H-9, this doublet of doublets, at 54.11, is
obviously coupling to two different protons, the first being the expected H-10 (J=9.2 Hz),
the second, however, must come from an attached hydroxyl group since there are no other
protons in close proximity, indicating the presence of a free hydroxyl group at this position
85
OH(9), J=6 . 6 Hz. The assignment of a free hydroxyl functionality, at this position, was
confirmed by the clear nOe nOe c between H-9 and the doublet at 86.27, J=6 . 6 Hz,
attributable, therefore, to OH(9). It was then possible to assume protection, by the two t-
Bu2Si(OH) groups, had taken place at (OH)10 and (OH)13 since attachment at (OH)15
would obviously lead to extreme steric crowding within the molecule. In addition, the
clear nOe between Me-18 and the f-Bu signals, nOe n, together with nOe k, between t-Bu
and H-10, provide further evidence to support the proposed structure 162a.
PROTON nOes BETWEEN nOe
H-2 H-9 d
H-3 H-7 e
H-5 Si(Ctf2CH3) 3 1 (TES 1),
H-20
a, o
H-6 (X Si(Ctf2CH3) 3 2 (TES 2) f
H-7 Si(CH2CH3) 3 2, H-3, H-10,
OH-9
b, e, g, q
H-9 OH-9, H-2, Me-19, OH-15 c, d , h, w
H-10 OH-9, Me-18, r-Bu, Si-OH i, j, k, s
H-13 Me-16 1
Me-16 H-13, OH-15 1, m
Me-18 t-Bu n
Me-19 H-9 h
H-20 H-5 0
Si(C/f2CH3) 3 1 (TES 1) H-5 a
Si(Ctf2CH3) 3 2 (TES 2) H-7, OH-9 b, p
OH-9 Si(CfiT2CH3) 3 2 (TES 2),
H-7, H-9, Si-OH, 'Bu
p, q, c, v, r
Si-OH H-10, 'Bu, OH-9 s, U, V
OH-15 H-9, Me-16 w, m
Table 4
There is no doubt that the structure postulated for compound 162 is extremely unusual and
indeed we were initially quite sceptical, however, there all the NMR evidence points to
86
this structure. Furthermore, the mass spectrum was completely consistent with the mass
expected for derivative 162 containing two hydoxysilyloxyethers rather than two bridged
silyleneoxyethers, in fact, the more studies and investigations we carried out, on these
compounds, the more certain we were about our assignments.
4.1.2. NMR Analysis Of The Compound Possessing One Triethylsilyl Group
Again, despite our strongest endeavours, we were unable to prepare a crystal suitable for
X-ray analysis, to determine, beyond doubt, the absolute structure of this derivative. After
thorough NMR investigation, again employing !H, 13C, 1H-1H, ^ - ^ C , NOESY and 1H-
29Si spectroscopy, we were able to show that, in fact, the structure of this mono-
triethylsilyl compound was that of compound 163 and not compound 161b as originally
postulated.
Revised Structure of the Compound Possessing One Triethylsilyl Group
162a
OH
163
87
and !H-13C correlation spectroscopy, appendix 3 and appendix 4 respectively,
allowed assignment of each proton in this mono-triethylsilyl compound. Analysis of the
key coupling constant, attributable to H-9/H-10, assigned the twist-boat conformation,
163a, to ring-b. J9 , 10 was equal to 10.7 Hz indicating an anti arrangement between these
two protons, see np F, fig. 48.
OR9
OR
OR
RO.
163a np F
fig. 48
Furthermore, the doublet of doublets, at 64.35, attributable to H-7, assigned the chair
conformation, 163b, as expected, to ring-c, see fig. 49. J7 ,6pl 1.0 indicated a dihedral angle
of 180° between these two protons, together with J7 , 6o5.0 pointing to a separation of 60°
between H-7 and H-6 a, see np G.
ORMe
OR
OR
163b np G
fig. 49
However, again it was the combination of the NOESY spectrum, NMR 3, the 29Si
spectrum and also, in addition, the 'H-29Si correlation spectrum, NMR 4, that allowed us
to postulate structure 163.
4.I.2.I. Beginning With The NOESY Spectrum, NMR 3
Again, as in the case of the di-triethylsilyl protected compound, 162, the most obvious
signals to look for were those corresponding to the Si(CH2Cfir3 )3 protons. A clear nOe
was evident between Si(CH2C/ / 3)3 l a (TES la), and H-5, nOe a, furthermore, nOe b,
between Si(C//2CH3)3 lb (TES lb), and H-5, placed the triethylsilyl group on OH(5). In
addition, the signed at 54.87 could be assigned to OH(7) since a definite nOe was present
between H-7 and this singlet, nOe c, as a result, determination of ring C’s structure was,
again, relatively straight forward, see partial structure 163c, fig. 50.
Me, OH
OTES
h3
163c
fig. 50
The question then arose as to how the two f-Bu2Si protecting groups were arranged around
the skeleton. As in the case of the di-triethylsilyl protected compound, 162, two cyclic
protections were not possible due to the geometrical constraints imposed by the taxane
skeleton.
4.I.2.2. The ‘H-2,Si NMR Spectrum, NMR 4
The 'H- Si, 2-D correlation NMR spectrum, NMR 4, helped confirm the structure of the
mono-triethylsilyl protected derivative, as that of compound 163. In this spectmm three
signals corresponding to 3 different silicon atoms, in different environments, were evident.
The first signal, Si 1, 5-10.37, showed a clear correlation peak to the t-Bu signals, cross
peak a, indicating this Si group has again adopted the form f-Bu2Si(OH)OR. The second
signal Si 2 , 512.64, also showed definite correlation peaks to the f-Bu signals, cross peaks
b and c. However, the large difference in chemical shift between Si 1 and Si 2 clearly
89
TES-1b
NMR 3TES-1a
H -6aH*10, H-13 oh-7 H-20 I H-6bH-20 H-7 H-5
H-2H-14a
-0 .9
• 0
*• •
•a.o
-3.5
1■ > I 111
—r~4.5
—r— 3.5
—r~1.0
T4.0
indicated the second silicon protecting group had adopted a different form. It seems
reasonable to suggest, that in this case, a cyclic protection had taken place, in the form t-
Bu2Si(OR)2, where R is again the taxane skeleton. Furthermore, as expected, the third
signal Si 3, 818.06, showed a clear correlation with the S\(CH2CHi)^ cross peaks d and
4.I.2.3. Returning To The NOESY Spectrum, NMR 3
Having established the two t-Bu2Si protecting groups to be in the forms r-Bu2Si(OH)OR
and f-Bu2Si(OR)2 respectively, we now had to identify the positions of each protection.
The ]H spectrum provided evidence to support a cyclic protection across C-9/C-10, since
H-9, 84.48, was now a doublet, rather than a doublet of doublets as in the diprotected
compound, NMR 1, furthermore, the signal attributable to OH(9) was also absent in NMR
2. In addition, the clear nOe, nOe i, between H-9 and the *Bu signals and, nOe k, between
H-10 and *Bu, also provided further evidence for a cyclic protection. It is now reasonable
to suggest that rBuSi(OH) attachment will again be situated at OH-13 since protection at
OH-15 would introduce severe steric crowding into the molecule. In the absence of an X-
ray structure, we believe, that in taking all the NMR data into account, the structure 163
illustrated, is the most reasonable determination. Furthermore, the mass spectrum, of this
mono-triethylsilyl compound 163, is in agreement with the mass expected for the proposed
structure, containing one bridging protecting group and one free hydroxysilyl group, table
5 gives a summary of the identifiable nOe signals.
e.
OH
163
90
PROTON* SH OW NGlnpes J J „>j * ✓* * *» -' < y
y y?'!* V 1 v< <
H-3 H-7, H-10 e, f
H-5 Si(CH2CH3)3 (TES), H-20 a, b, g
H-6 a Si(CH2CH3)3 (TES) d
H-7 H-3, H-10 e, h
H-9 'Bu, Me-19 i jH-10 'Bu, Me-18 k, 1, h
Me-16 H-13 m
Me-18 H-10 1
Me-19 H-9, H-20, OH-7 j,n , o
H-20 Me-19 n
si(cjy 2cflr3)3 (TES) H-5, H-6 a a, d
Si(CH2CH3h (TES) H-5 b
OH-7 H-7, Me-19, 'Bu c, o ,p
Table 5
4.2 Reaction Between Crude Unknown B/Unknown C & Et3SiCl
On reacting this crude mixture with Et3SiCl, in pyridine, using the same conditions
employed as in the equivalent, unknown A, reaction, some surprising and interesting
results were obtained. On treatment with triethylsilyl chloride, in pyridine, protected
brevifoliol derivative unknown C, Rf 0.66 reacted to give one new product, see fig. 51.
CRUDE B/C
OH
'Bu O" .'OTES
'Bu OH
OH
Inpurity, Unknown B 163
fig. 51
91
On purification, initial ]H NMR analysis indicated, this compound to be in possession of
two 'Bu2Si groups and one triethylsilyl functionality, it was identical in all respects to the
mono-triethylsilyl compound 163 produced in the previous reaction with unknown A!
Furthermore, during purification, by column chromatography, the inpurity, unknown B,
was removed, the identity of this compound will be subsequently discussed.
5. DI-te/tf-BUTYLSILANE DIOL
On recrystallising a sample of the crude unknown B/unknown C solid we were able to
prepare a crystal suitable for X-ray analysis, and thus we determined the exact nature of
the impurity, unknown B, as being di-ferf-butylsilane diol 164, fig. 52, with all
characteristics identical to those of the literature compound. 8 0 ,81 ’ 82
fig. 52
In addition, a sample of the silanediol 164 was also prepared directly from ?-Bu2Si(OTf) 2
165, as shown in fig. 53, the compound 164a, thus produced, also had characteristics
identical to those of the literature compound, confirming further the identity of unknown
B, as being di-terf-butylsilane diol.
(H3C)3Cv /)T f H20 (H3C)3CNs/)H
(H3 C)3 C/ \ ) T f EtOH (H3 C)3 C/ \ )H
165 164a
fig. 53
92
Studies into silane diols date back as far as 1904 with Dilthey and Eduardoff s discovery
of diphenylsilane diol.83 Since then, numerous silanediols have been reported on and
investigated. In the literature, for example, F. Kipping, at The University of Nottingham,
in 1912, continued work studying diphenylsilane diol and dibenzylsilane diol, 84 however,
subsequent work has been carried out by Sommer and his co-workers, in the USA. 85 In
1953 they hydrolysed the diethyl-166, di-w-propyl-167 and di-n-butyl 168 dichlorosilanes
to give the corresponding silanediols 169,170 and 171 respectively, fig. 54.
1.5 M NaOH, 0 °CEt2SiCl2 --------------------- ► Et2Si(OH)2
166 169
1.5 M NaOH, 0°CzBu2SiCl2 ► lBu2Si(OH)2
167 1701.5 M NaOH, 0 °C
"Bu2SiCl2 ► "Bu2Si(OH)2
168 171
fig. 54
Most silane diols, along with their analogous RsSiOH, RSi(OH)3 and Si(OH) 4 derivatives,
easily undergo self condensation reactions, with elimination of water, in the presence of
acid, base or heat, to give polysiloxanes (or silicones) as illustrated in fig. 55.86
R
0 \0 4 -S i- j -0 „ R R R
. ^ t t , -h2o \ / l^ \ / J. ( JA j.R2 Si(OH ) 2 ------- ► Si R gj + HO Si O— Si-^—O Si OH
/ \ /T \ / \R 04—Si—1-0 ER \ L . S i - j - 0 R R R R
1 n
cyclics Linear Oligomers
fig. 55
In general, preparation and isolation of monomeric silane diols is quite difficult since they
are so sensitive to dehydration and polymer formation. However, in contrast to the
93
behaviour normally expected, on preparation of di-ferf-butyl silane diol, 164, Sommer
and Tyler found it to be completely inert to all chemical investigations.80 This silane diol
164 was unaffected after treatment with conc. HC1 and ZnCl2, heating in conc. H2S0 4 then
pouring into ice had no impact, refluxing in benzene did not cause condensation, the diol
was recovered unchanged from all these reactions. The enormous stability, of the
monomeric compound, is imposed by the bulky rBu groups which would severely impede
free bond rotation in a siloxane polymer. In addition, to support this phenomenon, other
bulky silane diols, such as di-r-butylphenyl and di-f-butyl-n-hexadecyl derivatives, were
also found to oppose condensation to their polymeric siloxane analogues. As a result,
sterically crowded silane diols can be obtained as reaction by-products, seen not only by
us, in our investigations, but also by Weidenbruch and co-workers, during their studies
into tri-butylsilyl radicals.87
6. HYDROXYSILYLMONOETHERS
Further evidence to support the existence of hydroxysilylmonoethers was found in a
paper by Corey, describing his work using di-terf-butylsilyl ditriflate as a reagent for the
protection of diols.88 In general, he noted, on treating 1,2, 1,3 and 1,4-diols with t-
Bu2Si(OTf)2, in the presence of 2 ,6 -lutidine, bridged silylenes are formed as shown in
compounds 172-175.
(H3 C)3 C. .^C(CH3 ) 3
o(H3C)3Cksi^C(CH3)3
\" H C6 H5 C6 Hs
172
H Hi'o^ 0
173
(H3Q 3C. / C(CH3)3 (H3Q 3C C(CH3)3.Si
O o o
(H3 C)3 Cn /C(CH3 ) 3
Si-
h 3c c h 3
174
HO O OH
RR R
175 176
However, in addition, he also found that although 1,3 and 1,4 protected silylenes, for
example, 174 and 175 were relatively stable, 1,2-diols were extremely sensitive to
94
hydrolysis. Under extremely mild conditions, for example, (5:1, THF:H2 0 , pH 7, 1 hr,
25 °C), 1,2-diols often hydrolysed to hydroxysilylmonoethers in the form shown in
compound 176. We were able to support these results with our own investigation into the
reaction between ethylene glycol 177 and r-Bu2Si(OTf)2, the product 178, shown in fig. 56
was identified by NMR analysis.
(H3 C)3 Cs ,C(CH 3 ) 3
HQ 0H 'Bu.SKOTf), HQ ° ' Si' OH
V H 2, 6 lutidine, DMF H 1 X HH H u ,UUU1UC’ H H
177 178
fig. 56
Two different CH2 environments were seen in both the *H and the 13C NMR spectra, if
cyclic protection had taken place, both CH2 groups would be equivalent. In addition, 29Si
NMR showed a single signal at 5-9.95, well upfield form 60.00, indicating silicon
attachment to a hydroxyl group, confirming the presence of a hydroxysilyl ether.
7. SUMMARY AND CONCLUSIONS
7.1. Revised Structural Identification Of Unknown A
(h3c)3cC (C H 3>3
" " S i - O H Q. O H OTES
(H 3c ) 3c v p " ’S i
(H 3C )3C o h - ^ a H O x
O T E S
162
(H 3C )3C x /C ( C H 3)3
xSi Q p
(H3O3C p ,.....
Si/ \ _
(H3 O 3 C o h h o 'O T E S
163
(H3 C)3C OH\ /
Si(H3 C)3 C ^ o h
164
95
In light of the evidence provided, from NMR analyses of both the di- and mono-
triethylsilyl protected compounds, 162 and 163 respectively, indicating the formation of
hydroxysilyl ethers, together with identification of di-te/t-butyl silane diol 164, shown on
the previous page, we returned to the original reaction, illustrated in fig. 57a.
H O .....O H
HO
156
'Bu2Si(OTf)2 HO'" .'OH
160Original Hypothesis
for Unknown A, Rf 0.25
fig. 57a
Unknown B, Rf 0.66Possible Inpurity/
+ Unknown C, Rf 0.66Possible 2nd Taxane Derivative
We now felt it reasonable to suggest, the actual reaction taking place was that shown in
fig. 57b.
(H 3C )3C / C (C H 3)3
,Si° - P O H
(H 3C ) 3Cn 0 '» -
S i/ \ _
(H 3 O 3 C O H '
(H3C)3CS
(H303C'♦ V
OH
"OH
H O O H
H O "'*Bu2Si(OTf)2
O H
156
180Unknown C, Rf 0.66
Actual Structure
164Unknown B, Rf 0.66
C (C H 3)3
(H 3C )3C S i- o h
o PH
(H 3 O 3 C oS i
(H 3C )3C / O H
179Unknown A, R f 0.25
Actual Structure
fig. 57b
96
Unknown A, Rf 0.25, was revised, from structure 160 originally hypothesised, to structure
179, after identification of compound 162. Furthermore, we also felt it reasonable to
postulate, after identifying compound 163, compound 180 as being Unknown C, R{ 0.66,
contaminated with di-terf-butylsilane diol 164. In conclusion, on reacting hydrolysed
brevifoliol 156 with fBu2Si(OTf)2, we suggest protected compounds 179 and 180 were
afforded, derivative 180 was contaminated by di-rm-butylsilane diol 164, however, this
inpurity was removed during purification, of the subsequent reaction. Hydrolysis of the
reagent ?Bu2Si(OTf)2’ during the reaction, must allow formation of these hydroxysilyl
ethers, together with the production of compound 164, which is highly stable and could,
therefore, be the driving force of the reaction. In addition, 1,2 bridged silylenes are
extremely susceptible to ring opening88 this must have allowed isolation of both the
unbridged and the bridged compounds, 180 and 179 respectively.
7.2. Summary & Conclusion of Reactions with Triethylsilyl Chloride
On treatment with triethylsilyl chloride, compound 179 was converted to the di-
triethylsilyl compound, 162, and in addition, its mono-triethylsilyl analogue, compound
163 was also afforded, see fig. 58, carrying out this reaction in pyridine probably induced
some ring closure of the hydroxysilyl ether to the silylene derivative 163. In addition, we
feel it is reasonable to suggest, the presence of the free hydroxy silyl group at C-10, in
compound 179, allowed the formation of both the mono- and di- protected compounds,
since the free rotation of this functionality would allow approach of the second triethylsilyl
group to the hydroxyl at C-7. In contrast, however, if we now consider compound 180, the
bridged silylene group, across C-9/C-10, now has a fixed conformation and cannot rotate
to accommodate the second triethylsilyl group at C-7, thus affording only compound 163,
also shown in fig. 58.
97
C(CH3)3
(H3Q3C— - S i - OH0 . OH
(H 3 O 3 C ... ....S i
(H 3 C )3C o h
Et3SiCI
OH Py
179Rf 0.25=Unknown A
C (C H 3 ) 3
(H3C)3C^ I3 S i - O Ho' o h O T E S
(H 3 C ) 3 C n O '- S i
(H3Q3C' OH* O T E S
(H3O3C / C (C H 3 ) 3
o: Q
(H3O3C p ....S i/ \ _
(H3O3C o h h o >
O T E S
163
(H 3 C ) 3 C s ,C (C H 3 > 3
o; vo O H
(H 3 C ) 3 C N P ....S i
(H 3 C )3C o h
(h3c)3cx
+ / Si\OH (H3C)3C o h
180 164fff 0.66=Unknown C R\ 0.66=Unknown B
(H3O3C / C (C H 3 ) 3
.Si\ P OH
(H3Q3C p ....S i/ \ _
(H3O3C o h Hq>O T E S
163
fig. 58
8. FURTHER SYNTHESES
8.1. Determining The Position of Triethylsilyl Protection
Accumulation and analysis of all the aforementioned spectra was not a trivial task and was
carried out over the best part of a year. Thus, in the mean time, over the same period of
time, whilst acquiring this spectral data, we also carried out further reactions, again with
the intention of building up as much information as possible on these complex
compounds.
98
Beginning with the mono-triethylsilyl compound, 163, before acquiring the NOESY
spectrum, it was difficult to determine, by simple observation of 1-D spectra, the position
at which the triethylsilyl group had attached, C-5 or C-7, with the possible formation of
163a or 163b.
(H3 C)3 CV ,C(CH3 ) 3
/Si.o
(H3C)3 Cn O'"- Si
(H3C)3C q jj
C-5
OSi(CH2 CH3 ) 3
163a
(H3 C)3CX ,C(CH3 ) 3
° - & OSi(CH2CHj)3
(H3 C)3Cn P ....Si
(H3 C)3C qpj C-7
163b
We hoped, of course, that protection had taken place at OH-7, since this would then allow
us to go ahead with oxetane ring construction across C-4/C-5. It was decided, therefore, to
embark on oxetane synthesis, in the hope that we would be able to determine the site of
triethylsilyl protection later in the route. That is, if oxetane ring formation was successful
this would place the Si(CH2CHs)3 group at OH-7 and vice versa. The proposed synthesis
was that shown below, Scheme 16.
'Bu 'Bu
OTES
'Bu p«*-<'OH
'Bu OHHO
0 s04
'o h NM0
163b
OTES
'Bu .'OH
'OH
‘OHHO
181
OTES
Bu OH OHHO
Me,SiCl
Py
OTES
'Bu O'""', "OH "OH
“OTMSHO
182MsClPy
CSAOTES
'OMs'OH
OTMS
184 183
Scheme 16
Following a route described by Nicolaou,68 it was hoped to convert diol 181 via silyl ether
182 and mesylate 183 to oxetane 184. In the event, Scheme 17 illustrates the synthesis
that actually took place.
'Bu 'Bu
OH
'OTES
Bu OHHO
0 s04NMO
163a
OH
'OTES'OH
OHHO
185
Bu 'Bu
'Bu 0»*•""OTES
Me3SiClPy
CSA
OTMS
'Bu O"OTE5
'OHOH
OHHO
186MsClPy
'Bu
OTMS
[ C 'OTES 1 'OH
OMsHO
188 187
Scheme 17
On treatment with osmium tetroxide and NMO, in THF, NMR analysis showed the
successful dihydroxylation of compound 163a, afforded diol 185. Following this,
compound 185 was treated with 10 eqs. trimethylchlorosilane in the presence of pyridine,
protecting, we believe, OH-7 instead of the expected primary hydroxyl at C-20. However,
since compound 186 was taken through to the next step without purification, following the
Nicolaou methodology,68 this structural determination was not made until the end of the
route and the final compound was analysed. Hence, in the same vein, we also believe,
treatment of the crude trimethylsilyl ether 186 with mesyl chloride, in pyridine, afforded
mesylate 187 resulting in formation of compound 188, on treatment with camphor
sulphonic acid. In spite of oxetane ring closure being unsuccessful, in carrying out this
synthesis we now had full NMR spectra on compounds 163a, 185 and 188, in addition to
the spectra obtained for the di-triethylsilyl protected compound, 162. On analysis of these
spectra, events began to fall into place, the fog, so far engulfing this work, was slowly
clearing. Considering NMR spectra generated by the final compound isolated, 188,
oxetane ring formation was immediately ruled out since a singlet attributable to the m esyl,
100
(0 S 0 2Ctf3), functionality was immediately evident at 83.06. In addition, no nOes were
observed between this signal and any other proton, indicating the reaction had taken place
at the free primary hydroxyl group, situated at C-20 as in compound 187. Furthermore, the
clear nOe between H-5 and the triethylsilyl group confirmed triethylsilyl attachment at
OH(5), the signal characteristic of OH(7) was also identified, showing a clear nOe to H-7,
identifying the final compound as derivative 188. The two alternative routes, concerning
ring-C, are illustrated in fig. 59.
OTES
OTES
bl
OH! OH OMs1b o Y l - Y T O — 1^ VN o ' /OTESP'OH
OH"OTEST OH 20 V,OTMS
/ sV ^ ',,<OTESP'OHOTMS
a2 a3 a4OH
Y S iOTMS OTMS
I y S 1: 1 J -► ; [ J ►: 1 J —* - «
> O HOH
i "oh 20V OH: / \^ x ’’,*/o'ees i PbH
OMsb2 b3 b4
OMs
'"OTES
'"OTESOTMS
b5
fig. 59
Route a, via trimethylsilyl (TMS) protection at C-20 a3 and mesylate attachment at C-7
a4, was eliminated in favour of route b, via TMS addition at C-7 b3 and mesylate reaction
at C-20 b4, after identification of compound 188, using NMR spectroscopy, previously
discussed, thus, also clarifying the structures of compounds 186 and 187. We were now in
an excellent position to offer a reasonable explanation regarding the chemistry taking
place. Formation of diol 185 required unusually harsh conditions, refluxing THF was
needed to induce reaction when dihydroxylation, across double bond C-4/C-20, usually
takes place at room temperature. The need for such severe conditions is, of course, now
explained by the presence of the bulky triethylsilyl group, at C-5, hindering any reaction at
the double bond. Furthermore, this large group probably impeded the approach qf the
trimethylsilyl (TMS) group to the primary hydroxyl, OH(20), causing preferred attachment
at C-7 as in compound 186. The final reaction, with camphor sulphonic acid, clearly,
101
simply induced trimethylsilyl removal, affording compound 188 instead of the desired
oxetane ring analogue 184.
8.2. Selective Deprotection of One Triethylsilylether
During the investigation of conditions for dihydroxylation of the C-4/C-20 double bond on
the mono-triethylsilyl protected compound, 163, test reactions were also carried out on the
diprotected di-triethylsilyl derivative, 162, to make full use of all the available material. In
the event, a method to selectively deprotect one of the triethylsilyl groups, was
inadvertently discovered, as shown in fig. 60.
OTES
'OTES
HO
OH
AD-mix
'OTES
HO
163
fig. 60
The di-triethylsilyl compound, 162 was treated with AD-mix-a in 'BuOH, 89 however,
instead of initiating dihydroxylation, selective removal of the protecting group at C-7 was
achieved, affording the mono-triethylsilyl derivative 163, identical in all respects to the
previously formed mono- protected compound.
102
9. FURTHER APPROACHES TOWARDS OXETANE RINGCONSTRUCTION
9.1. Dihydroxylation
Having determined beyond reasonable doubt, in the absence of an X-ray, the structure of
the mono-triethylsilyl protected compound as structure 163 and, in addition, the structures
of the original di-terf-butylsilyl derivatives as 179 and 180, we decided to return to these
two compounds and once again attempt oxetane ring formation.
(H3C)3Cs..'C<CH3)3jP OH
(H3 C)3 Cv ,< Si
(HjCbc' q H
C(CH3 ) 3
(H3C)3C-si_OHO' PH 0H
(H3C)3 Cv ,C(CH3 ) 3
„Si* P OH
‘"'OSi(CH2 CH3 ) 3 „ ■
(H^c'^h 'AH O '
....
'"OH (H303C'^h
163 179 180
Initially, we took a crude sample of compound 180, (contaminated with di-terf-butyl silane
diol, 164) and attempted dihyroxylation across the C-4/C-20 double bond, see fig. 61.
(H3 C)3C C(CH3 ) 3
OH
'OH
HO
OsOa
NMO
(H3C)3Cn C(CH3 ) 3 .
° - > OH
(H3C)3 CS O"- Si
(H3C)3c 'HO
180 189
fig. 61
It was extremely encouraging to find NMR analysis indicated the successful
dihydroxylation, affording diol 189 in a 14% overall yield from hydrolysed brevifoliol
156, again compound 164 (di-f-butyl silane diol) was removed during purification. We
also treated the pure compound 179 with osmium tetroxide, under the same conditions as
previously employed. We were pleased to find, NMR analysis showed a successful
103
dihydroxylation and protecting group cyclisation, affording compound 189, identical in all
respects to the diol 189 produced in the previous reaction from derivative 180, see fig. 62.
(H3C)3Cs />"••
(H3C)3C qjjNMO
0s04(H3C)3CN p » -
(HsChc' q HSi
(H3C)3Cn C(CH3)3X
OH
179 189
fig 62
9.2. Approaches Towards Ring Closure
9.2.1. Nicolaou, Mesylate Approach
Following the same methodology as previously described, we again attempted oxetane
ring closure, however, in this case, we believe Scheme 18 illustrates the sequence of
events that occurred, in preference to oxetane ring formation.
'Bu O'""
OTMS
Me3SiCl
h o v OTMS
189 190MsClPy
OTMS
OTMSOTMS
192 191
Scheme 18
104
On treatment with trimethylsilyl chloride, in the presence of pyridine, at 0°C, after 15 min.,
two new spots were observed by TLC, accompanied by the disappearance of starting
material. It seemed likely that the majority of compound 189 had been converted to
compound 190 protected at both C-7 and C-20. After work-up, 190 was taken to the next
stage without further purification, treatment with mesyl chloride, in pyridine, presumably
afforded mesylate 191. Although it was difficult to determine by TLC if the reaction had
really been successful, a work-up was carried out and the crude product taken to the next
step. Mesylate 191 was treated with camphor sulphonic acid in an attempt to induce ring
closure. Unfortunately, only a small amount of any ‘new product’ could be isolated from
the reaction. It was immediately obvious ring closure had been unsuccessful, since initial
*H NMR analysis of the product 192 clearly showed a singlet at 83.07 attributable to the
mesyl functionality. In all probability compound 192 had actually been formed although
full characterisation was not carried out due to lack of material.
9.2.2. Nicolaou Approach, Triflate Method
In a final attempt to achieve ring closure, compound 190 was treated with triflic anhydride,
in the presence of diisopropylethylamine.68 Again, although it was difficult to determine
by TLC if the the new product 193 was forming, fig. 63, the crude product was treated
with camphor sulphonic acid in the hope of eventually isolating some oxetane 194,
unfortunately, after column chromatography, no pure compound was isolated.
OTMS OTMS
OTMS OTMS
190 193 194
fig. 63
105
9.3. Conclusion
It would seem the di-terf-butylsilyl group is an unsuitable protecting agent for use in a
semi-synthesis of a new taxol analogue, from brevifoliol. Stability is only achieved on
reaction with triethylsilylchloride, however, in terms of oxetane ring construction, this is
unsuitable, since reaction occurs at the C-5 position, blocking ring formation. Other cyclic
protecting groups were investigated, in the hope of producing a stable, protected
brevifoliol intermediate, however, these were all unsuccessful. It must be concluded
therefore, that the conformational flexibility, imposed into the diterpenoid skeleton, in the
unusual, rearranged 11(15-1 )aZ?eo-taxanes, causes the difficulty in achieving stable, cyclic
protected brevifoliol derivatives. Having said this, since the chemistry of 11(15-1 )abeo-
taxanes is largely unexplored, the results and discoveries we have made, during our
investigations, will go a long way in furthering our knowledge and understanding of these
new compounds.
106
CHAPTER FOUR
TAXCHININ A
In addition to our studies and investigations surrounding brevifoliol, we also carried out
work on a second 11(15-1 )abeo-taxanQ, named taxchinin A 87, by Fuji and co-workers,
who first isolated it, in 1991, during their investigations into Taxus chinensis?1
BzO p Ac OAc
OH
OAcHOC-2
87
BzO. P Ac OAc
HO....OH
HO
Taxchinin A 87 differs from brevifoliol 85 at only one position, that is, it possesses an
additional acetate functionality at C-2. In terms of developing a potent taxol analogue, this
taxane looked extremely promising since fimctionalisation at this position is thought to be
necessary for biological activity. While developing the brevifoliol extraction procedure,
outlined in chapter 2, Erik Van Rozendaal discovered taxchinin A 87 was also present in
our Indian resin, as such, after isolation, here in Leicester, we began work on this taxoid
with the intention of developing a new taxol analogue.
1. TAXCHININ A, STRUCTURE DETERMINATION
The *H and 13C spectra (carried out in CDCI3), of compound 87, were quite complex due
to the appearance of some minor impurities, after extraction, together with the possible
presence of a minor isomer. Identification of the major compound was possible, however,
and was consistent with the literature data on taxchinin A 87.37 In addition, we established
87 as being primarily in the twist-boat/chair conformation 87a, as is the case for this
compound in the solid state.37
107
H 13"
Mejg
H6a^h5
OH
87aTwist-boat/Chair
The large J value of 10.7 Hz between H-9/H-10 indicated they were in an anti
arrangement, with respect to each other, with a dihedral angle of 180°, see np i, consistent
with the twist-boat, 87b, arrangement in ring-B, fig. 64.
Although signals attributable to H-5, H-7, H-6 a and H-6(3 were overlapping with other
protons in close proximity, we felt it reasonable to assume ring-C had adopted the chair
conformation in line with the trend seen so far when ring-B adopts the twist-boat
conformation. In adopting the twist-boat/chair 87a type arrangement in preference to the
twist-chair/boat 87c conformation, steric hindrance between the benzoate group at C-10
and the 'Bu group at C-l is reduced, together with a reduction in crowding between H-5
and Me-19, in the C-ring.
87bRing-b, Twist-boat np i
fig. 64
108
Steric H indrance
In ring-b
M e 17
M e 16
H14a'
In ring-c
q u B zO h
*13 OH
87cTw ist-chair/Boat
Although extra signals were also evident in both the !H and 13C spectra, possibly being
attributable to the presence of a minor conformer, identification was not possible at this
stage due to the complexity of the spectrum. Having said this, on carrying out the first
reaction, on compound 87, we able to carry out full analyses on the product thus produced.
2. HYDROLYSIS OF TAXCHININ A
Following methodology already used in our brevifoliol studies,72 we began with the
intention of hydrolysing all four ester functionalities, to produce compound 195, as in fig.
65.
BzO PAc OAc
HO...OH
OAcHO
Na
'"'O H MeOH
HO.
OH
OHHO
87 195
fig. 65
After overnight stirring TLC analysis indicated disappearance of starting material
accompanied by the formation of a new compound. After work-up and purification we
fully expected the NMR spectrum of the new compound to closely resemble that of our
hydrolysed brevifoliol equivalent 156.
109
HO. >0H OH
HO.....
'OH
HO
156
This new compound, like 156, was also insoluble in CDCI3 and accordingly our NMR
analyses were carried out in CD3OD. However, instead of the sharp, clean spectrum
observed at room temperature for compound 156, see fig. 6 6 , we were now faced with an
extremely broad set of signals, again at room temperature, corresponding to hydrolysed
taxchinin A, suggesting some form of conformational isomerism was taking place, see fig.
67.
i < * ■ t > ‘
fig. 67
1 1 0
2.1. NMR Analysis of Hydrolysed Taxchinin A
Clearly, limited structural determination and assignment could be obtained from this broad
spectrum, fig. 67. Fig. 6 8 , however, illustrates the same spectrum, carried out at -20°C,
400 MHz, in CD3 OD.
fig. 6 8
In stark contrast to our original room temperature spectrum, fig. 67, we now observed two
sets of sharp signals corresponding to two conformational isomers in equilibrium, in a 2 : 1
ratio, structure determination and assignment was now possible. We carried out the 2-D
COSY spectmm NMR 5, at -20°C, allowing us to assign the majority of the signals
to both the major and the minor conformer. The major isomer was determined to be in
possession of the twist-boat/chair conformation 196a and the minor isomer to have
mastery of the twist-chair/boat conformation 196b.
HO Me17 H° H \ f H10/ „ . . \ J
OH
O HO
14a Me,
OH M e i8
196a 196b
i l l
NMR 5 H-20, H-13
" ? H-10 |H-5
H-3*
H-6a'
H-14b
H -6b, H -6b '
---------H-r n -----------H-13’
- I ---------H S
-----------4H>14t
<••<
— — • H-6a
t•
ft*i
' S
J
*
f * *
8 > ; • • ft*ft•
rft*•
i *• »• ftft •
* *
11
•
#
• •*•* *• •
• «•*•
1i i
*8 ^ * *
•»»•ft ftft
\ 1
\1 • 1
•
••»•¥
ST
I
ftft h « : t:>
¥
I
V *
1f. » * »
s r f t '•
J i r
* J
B » I0
■ «t •
»
:=*,• . • •
#•I- ± _____ '
1 t a * ,
Interestingly, on initial analysis of NMR 5 we noted the signal attributable to H-2, in the
major compound, was way downfield, at 85.72, even after hydrolysis, see cross peaks a in
correlation with H-3. In addition, the signal attributable to H-2’, in the minor conformer,
was also still downfield, at 85.78, see cross peaks a ’ in correlation with H-3’.
Furthermore, the additional singlets at 81.93 and 81.90, corresponding to both the major
and minor conformers respectively, indicated the presence of an acetate functionality.
Since the signals attributable to H-7/H-7’, H-9/H-9’ and H-10/H-10’ had all moved, as
expected, upfield, we could now assign the basic skeleton 196, as being hydrolysed
taxchinin A, with the acetate, at the C-2 position, still intact, fig. 69.
BzO PAc OAc
'OH
OAcHO
Na
'"'O H MeOH
HOs?H OH
H O .....
'OH
OAcHO
87 196
fig. 69
Now look at the signals attributable to H-9 and H-10, in the major conformer, cross peaks
b. The large J value of 9.6 Hz, for these two protons, indicated they were in the anti
arrangement, at 180°, with respect to each other, see np ii, showing in the major isomer
ring-B had adopted the twist-boat 196c conformation, fig. 70.
OH
OH,
OH
HO.
196cRing-b, Twist-boat np ii
fig. 70
112
Furthermore, analysis of the coupling constants attributable to H-7 and H-6|3, cross peaks
c & d, assigned the chair conformation to ring-C, see* 196d. That is, the doublet of
doublets at 54.14, attributable to H-7, with J7, 6pll-2, indicated a dihedral angle of 180°
between the two protons, np iii. In addition, J7, 605 . 2 , indicated these two protons were
separated by a dihedral angle of 60°, also seen in np iii, illustrated in fig. 71. This
assignment was further confirmed by the overlapping doublet of doublet of doublets at
51.63, attributable to H-6|3, J6a, 6p 1 4 . 8 indicated geminal coupling, np iv, then, J6p, s5.0,
indicated a dihedral angle of 60° between H-6(3 and H-5, also np iv.
OH-M ? 19 O H
O H
196d np iii np ivRing-C, Chair
fig. 71
Having carried out this analysis we concluded the major isomer had adopted the twist-
boat/chair type conformation, 196a.
In contrast, if we now turn again to the minor isomer, on observation of cross peaks e, we
assigned the key signals H-9' and H-10'. From the 1-D lH NMR spectrum, the coupling
constant for these two doublets was clearly much smaller and was in fact calculated as 4.0
Hz. This analysis indicated H-9' and H-10' had adopted an equatorial arrangement, in
relation to each other, see np v, showing in the minor isomer, ring-B had adopted the
twist-chair conformation 196e, fig. 72.
113
196eRing-b, Twist-chair
np v
fig. 72
Although, the signal attributable to H-7' was obscured by H-10 at 54.56, cross peaks f &
g, in correlation with H-6 a ' and H-6 p' (also obscured), the signal assigned to H-5’, 53.84,
cross peaks h & i, was clearly visible. Contrary to the signal normally seen for H-5, (a
broad singlet, see H-5, 54.20, in the major conformer), we now observed a broad triplet
corresponding to H-5', in the minor isomer. Despite the fact the signal was partially
obscured, a coupling constant of 9.7 Hz could still be calculated indicating ring-C had
now adopted the boat arrangement 196f.
That is the dihedral angle between H-6 P' and H-5' had now decreased, thus increasing the
value of J6P', 5’ to 9.7 Hz, see np vi, fig. 73, confirming the twist-chair/boat type
conformation for the minor isomer, 196b.
196f Ring-C, boat
np vi
fig. 73
114
2.2. Conformational Analysis
As we had now identified each of the conformers, the question then arose as to why we
were observing this conformational equilibrium? Hydrolysed taxchinin A 196 differs from
hydrolysed brevifoliol 156 at only one position, namely, it contains an extra acetate
functionality at C-2.
HOOH
ho...'OH
OAcHO
HO. :PH OH
'OH
HO
196 156
As expected, in the polar protic solvent, methanol, 156 exists in only one conformation,
the sterically stable twist-boat/chair arrangement, 156a, since the intra-molecular H-
bonding that could stabilise the alternative twist-chair/boat arrangement 156b, is
negligible in this solvent.90
H-bonding absent in MeOH
OHOH
15=
h 2 h oOH
OHOH MC18
156a 156b
Since the NMR analyses, of hydrolysed taxchinin A, 196, were also carried out in
methanol, the observed conformational isomerism could not be accounted for by intra
molecular H-bonding, as is normally the case. As a consequence of this phenomenon, we
rationalised, there must be some other factor involved to cause the appearence of the twist-
chair/boat minor isomer 196b.
115
OH
OHHO/,14a
OH Mei8
O HO
CH-
196a 196b
Using the molecular modelling programme, Insight n, we were able to show that in the
major isomer, the twist-boat/chair conformation 196a, there was a clear overlap between
the acetate functionality at C-2 and the exocyclic double bond at C-4/C-20, causing steric
crowding within the molecule, as illustrated in fig. 74.
fig. 74
However, on flipping to the minor isomer, the twist-chair/boat conformation 196b, this
overlap was reduced, shown in fig. 75.
116
fig. 75
For all that, when the twist-chair/boat 196b type arrangement is adopted, steric crowding,
between the fBu group at C-l and the hydroxyl group at C-10, is now increased, hence, in
solution, the molecule exists in conformational equilibrium in an attempt to reach stability,
as illustrated in fig. 76.
Reduced steric crowding between C-l(Bu) & OH(10)
Increased steric crowding between C-I^Bu) & OH(10)
Slight overlap between C-2 (OAc) & C-4/C-20 double bond
No overlap between C-2(OAc) & C-4/C-20 double bond
196aTwist-boat/chair
196bTwist-chair/boat
fig. 76
117
Since hydrolysed brevifoliol 156 does not possess the additional acetate at C-2, this
molecule can remain entirely in the sterically more stable twist-boat/chair type
arrangement, 156a, and thus, conformational isomerism is not seen in solution.
OH
15=
OH
HO/,l!4a
OH C-2(OAc) absent, molecule canremain entirely in twist-boat/chair conformation
156a
3. REACTION WITH DI-terf-BUTYLSILYL DITRIFLATE
Having established the structure of hydrolysed taxchinin A as 196 we decided to go ahead
with syntheses towards construction of the oxetane ring. Since work on this taxane was
running concurrently with our work on brevifoliol, we elected to begin using the same
protecting group, /-Bu2Si(OTf) 2 ,74 again we achieved some interesting results, far from
that expected. Our initial aim was to protect either 2 or 4 hydroxyl groups using the diol
protecting group 'Bu2Si(OTf) 2 .74 On treating compound 196 with two equivalents of this
reagent, in the presence of 2 ,6 -lutidine, two new compounds were isolated, as illustrated in
fig. 77. Initially, as in the analogous brevifoliol reaction, identification of these ‘products’
was perplexing.
Bu2Si(OTf)2 Unknown A, Rf 0.58
'oh 2,6-lutidine
196
fig. 77
118
Unknown A, Rf 0.58, in the solvent system used, was again a white crystalline solid,
insoluble in CDCI3 . NMR analysis in MeOD showed a single singlet at 60.95 and was
identical in all respects to di-ferf-butylsilane diol 164 previously produced.80, 81 ’ 82 In
contrast to the equivalent brevifoliol reaction, however, no other signals were evident in
the spectrum, in this case, only one ‘real’ product was isolated form the reaction,
unknown B, Rf 0.21.
3.1. Initial Indentification of Unknown B, Rf 0.21
Initial !H NMR analysis of this compound was encouraging, it appeared that two rBu2Si
groups had become attached to compound 196, the questions were, however, at which
positions and in what form? Unfortunately, signals attributable to H-7, H-9 and H-10 were
all overlapping, or were obscured by other protons in close proximity, however, on close
analysis we were able to determine the coupling constants for H-6(3. A coupling of J6a,
6p 1 3 . 9 indicated geminal coupling, see np vii, J6p, 7 10.2 suggested a dihedral angle of 180°
between these two protons, also shown in np vii, then finally, J6P, s3.5 indicated the
dihedral angle between H-5 and H-6(3 was 60°, np viii, assigning the chair 197a
conformation to ring-C, fig. 78.
MeOR
OR
OR
OR
H<
H6a
197a np vii np vin
fig. 78
Initially, we assumed cyclic protection had taken place to form two silylene ring systems
around the taxane skeleton, see 197. This diprotection should take place across the 1,3-
diol at C-7/C-9 and also across the skeleton at C-10/C-15, since achieving two cyclic
protections across any other positions would be impossible due to the geometric
119
constraints of the molecule. In addition, although the signals attributable to H-9 and H-10
were obscured, we felt it reasonable to suggest that, again, in line with the trend seen so
far, with ring-C in the chair 197a arrangement, ring-B would preferentially adopt the twist-
boat conformation so suggesting, at this stage, the twist-boat/chair derivative, 197, as
being unknown B.
OH
W
OH Me»8
197
WBu
OH
OAc
4. REACTION WITH TRIETHYLSILYL CHLORIDE
Since this diprotected compound, with possible structure 197, was unstable to lengthy
analyses we elected to treat it with triethylsilylchloride, in the presence of pyridine,72 in an
endeavour to construct a more stable derivative and so constitute an exact structure
determination. Using an excess of reagent we achieved the formation of, and identified,
compound 198, shown in fig. 79, as being the product from this reaction.
(H3 C)3 CN ,C(CH3 ) 3
OH
Unknown BOTES
OAcHO
198
fig. 79
120
4.1. NMR Analysis of Compound 198
Proton assignment was achieved using the1!!-1!! 2-D COSY spectrum, the 1-D version is
shown below, fig. 80.
fig. 80
Three key details were immediately evident, the first being the appearance of only one set
of sharp signals, indicating the presence of only one conformational isomer. The second
was the obvious attachment of two triethylsilyl groups, shown by the appearance of two
sets of signals attributable to SiCCfi^CHa^, see 80.61 and 80.75. Finally, the third was the
unusual coupling constant attributable to H-9 and H-10, calculated as 2.9 Hz. This small
value indicated these two protons were situtated in equatorial positions, in relation to each
other, with the dihedral angle between them about 60°, see np ix, suggesting ring-B had
adopted the twist-chair arrangement, 198a, fig. 81.
198aRing-b, Twist-chair
h 9
Hio OR
np ix
fig. 81
12 1
Furthermore, the coupling constants, attributable to H-7 and H-6 a, indicated a boat, 198b,
conformation for ring-C. J7, 6a6.9 suggested a small dihedral angle between these two
protons, see np x, and hence a larger coupling constant. In addition, the signal assigned to
H-5, at 54.62, was a broad triplet, again indicating a boat arrangement, 198b, for ring-C,
fig. 82
ORMe19H5
OR
198b Ring-C, boat
fig. 82
Having identified the twist-chair/boat type conformation, in possession of two rBu2Si and
two (CHsCH2)3Si groups, logically the structure of this new compound should be 199.
'Bu 'BuM e19 H5 v J > O S i E t 3
H i 3”
199
OTES
OAc
However, after analysis of the NOESY spectrum, NMR 6 , we realised that, as in the case
of the analogous brevifoliol reaction, a different form of ‘protection’ was taking place, the
structure of this double diprotected compound was actually that of structure 198.
122
NMR 6 OAc Ma-18
Ma-19
OH-10 H-2 H-20 I H-10 H_20\ I
H-6a H-14b i
li lh U M
Ma-17
TES-2
! H-13 H-14* m n ube
M dd a
•«= |' W f E ?i i UBUTm
[ ' ' !0 m »h 6 I ( 9
\ '
• i
|R
♦dp <$>
h 5 ° ‘sO
«• £ g a | F ®v n
•q • , 6 » 0
) f e t e ’ ®
' J i ° *3
• §
I •
i e|
i •! §
g j n f ,
. © • i l •S | * s f
L___
___
o - in
8 g o n i* * / ! • T ny f \ « 0 A[ fCT ! I *7$
i! » i i . j
*
A p| © ci
i y yI I Z r
ia
** n” ! ‘ ~ ’*|R! :! i
\ * i j f i Jy*
| ® | | i d a ' oOj 0 : 5
4 P 1 *, It?
f f l j $h r'
r ! 3 ■* i ; J q |JJ® 0 d » • C. 0 „» 0 n‘6 O e
h M
\ j ? . »1 9 ; ; . , | Om j i b
. : ....... - - . . ............- l, ..
Ho.s
[!■r -1 .0
r-2-ot
(H3C)3C &CH& HO. OTES
(H3C)3C ^ s ./
(H3C)3C/' no h
H 3C
*'/yOTES
'Bu
198
4.1.1. Analysis of the NOESY Spectrum, NMR 6
Initially the key signals to look for were those corresponding to the two triethylsilyl,
(CH3CH2)3Si, groups. NOe a, between Si(C/?2CH3)3 1 (TES 1) and H-5, together with
nOe b, between an H-20 and SiiCHzCH^ 1 (TES 1), indicated one triethylsilyl group had
attached at OH(5). Identifying the position of the second triethylsily protection was more
challenging. Si(CZ/2CH3) 3 2 (TES 2) was clearly exhibiting nOes to three different
protons, nOe c, H-9, nOe d, H-10 and nOe e, an OH signal, was triethylsilyl 2 attached to
OH(9) or OH(IO)? Identification of the OH signal, at 55.19, as being attributable to
OH(IO), allowed us to be confident in assigning triethylsilyl 2 to OH(9). OH(IO) was
assigned after observing nOes f, g and h, that is, there are clear nOes between OH(IO) and
H-2, nOe f, between OH(IO) and Me-19, nOe g and between OH(IO) and Me-17, nOe h.
In addition, these three nOes, together with nOe i, between H-5 and Me-19, also
confirmed the twist-chair/boat conformation 198c, as opposed to the twist-boat/chair
conformation 198d.
H13 OR
198c 198d
123
Having established the partial structure of this compound as being 198c, we again realised
that attachment of two cyclic 'Bu2Si groups was impossible, since the second triethylsilyl
group was blocking any reaction at C-9. However, since we had now indentified the
analogous brevifoliol compounds, we now felt in a position to determine 'Bu2Si protection
as being in the form 'Bu2Si(OH)OR, where R is the taxane skeleton. After observing nOes
j & k, between H-13/'Bu and between H-7/'Bu respectively, we determined JBu2Si(OH)
protection to have occured at these two positions, a summary of all the identifiable main
nOes is shown in table 6 .
PROTON nOes BETWEEN nOe
H-2 OH-10, Me-19, Me-17 f,l,m
H-3 H-7, H-20b n, o
H-5 Si(Cir2CH3) 3 1 (TES 1)
Me-19,
H-20a
a, i, t
H-7 'Bu, H-3 k, n
H-9 Si(C/72CH3) 3 2 (TES 2),
Me-19
c,p
H-10 Si(C/f2CH3) 3 2 (TES 2),
Me-18
d, q
H-13 'Bu, Me-18 j>r
H-14(3 Me-17 s
Me-17 H-14p, OH-10, H-2 s, h, m
Me-18 H-10 qMe-19 H-9, OH-10, H-2, H-5 p, g, 1, iH-20a Si(CH2CH3) 3 1 (TES 1),
H-5
b, t
H-20b OAc, H-3 u, 0
OAc H-20b u
Si(Ctf2CH3)31 (TES 1) H-5, H-20a a, b
Si(Ctf2CH3) 3 2 (TES 2) H-9, H-10, OH-10 c, d, e
OH-10 Si(CET2CH3) 3 2 (TES 2),
Me-19, Me-17,
f, g,h
Table 6
124
Again, although we realise compound 198, now postulated, is in possession of an unusual
structure, in the light of all the NMR data and in the absence of an X-ray, we feel this
structural determination is the most accurate.
*6b OTESMe,
HO Men HO,
\ / H10/Me, HO.O TESO
Bui i ..\Me,
I*Bu
'Bu
198
5. FURTHER REACTIONS & STRUCTURE IDENTIFICATIONS
As in the case of the brevifoliol reactions, acquirement and analysis of this NMR data was
carried out over a period of months. As a result, in addition, we also carried out further
reactions with the intention of building up as much information as possible to elucidate the
interesting chemistry taking place.
5.1. Reaction with AD-mix-oc
Having already established that AD-mix-a selectively removes triethylsilyl groups, (see
chapter 3, fig. 47, we decided to investigate the same reaction using the taxchinin A
equivalent, compound 198.89 In the event, we determined the reaction shown in fig. 83, to
have taken place, affording compound 200 in 32% yield. Conformational analysis and
structural determination of compound 2 0 0 , although challenging, proved to be extremely
interesting.
125
HO.
AD-mix-a
OAcHO
HO
'OTES
Bu OHOAc
198
fig.83
5.1.1. NMR Analysis of Compound 200
Proton assignment was carried out using 2-D, ^ ^ H and !H-13C, COSY spectra, the 1-D
*H NMR version is shown below, fig. 84.
fig. 84
Immediately, it was evident one triethylsilyl group had been removed, in addition,
dihydroxylation across C-4/C-20 had not taken place, the two protons attached to C-20
remained at 64.45 and 64.96. Furthermore, a conformational change was evident.
Although the signals, attributable to H-9 and H-10, were overlapping, with other nearby
protons, a large coupling constant of 9.8 Hz between these two protons could still be
calculated. This J value placed them anti, with respect to each other, indicating ring-B
had again adopted the twist-boat conformation, 200a, fig. 85.
126
2 0 0 aRing-b, Twist-boat
fig. 85
Now look at the overlapping doublet of doublet of doublets, at 81.61, attributable to H-6p.
Analysis of the coupling constants between H-6a, H-6p, H-7 and H-5 respectively,
allowed us to determine ring-C had adopted the chair, 200b, conformation, fig. 8 6 .
A coupling of J6a, 6p13.8 (geminal coupling), np xi, J6p, 7 1 1.2 indicated an anti-arrangement
with a dihedral angle of 180°, also np xi, then finally, J6p, 5 was calculated as 3.3 Hz
corresponding to a dihedral angle of 60° between H-6 P and H-5, np xii, confirming the
chair 200b arrangement for ring-C. Having now established the skeleton as being in
possession of the twist-boat/chair skeleton, 2 0 0 c, we now had to identify the position
from which the triethylsilyl group had been selectively removed.
200b Ring-C, Chair
np xi np xii
fig.8 6
127
Me-18OAcNMR 7H-20
TESH-20
H-3H-2 H-S
t-Bu
OR
Hfia .H5
200c
5.1.1.1. Analysis o f the NOESY spectrum, NMR 7
Observation of nOe a, between H-5 and the Si(CHr2CH3)3 (TES) protons, allowed us to
determine whether the remaining triethylsilyl group was still situated at OH(5). Absolute
determination of the forms the *Bu2Si groups had now adopted was more difficult, even
after analysis of the NOESY spectrum, since nOes attributable to the rBu protons were not
absolutely definable. However, at this stage, it seemed reasonable to suggest the
compound had either structure 2 0 0 or structure 2 0 1 .
Analysis of the mass spectrum, however, indicated a peak attributable to the mass
expected for structure 200. Unfortunately, due to lack of material, we were unable to
obtain 29Si NMR spectra on these compounds, in order to absolutely distinguish between
the bridged structure, 2 0 0 and the unbridged alternative, 2 0 1 .
200 201
128
5.2. Revised Structural Identification of Unknown B
If we now return to the original reaction, shown in fig. 87.
HOOH
HO......
OH
OAcHO
196
rBu2Si(OTf)2 Unknown A, 0.58
fig. 87
In light of the data identifying the structures of compounds 198 and 200, doubt was cast on
our original structure determination of unknown B.
Mejfi///, f,\\O 'Si' \ 15 \Hl4b \ ____1 OH
OH M*18
197
OTESO
'Bu H20
198
Me16*/,
129
We had postulated the most likely identity of this compound as being structure 197.
However, on re-examining the mass spectrum we noted a peak corresponding to the
attachment of two di-terf-butylsilyl protecting groups in the form, 'Bu2Si(OH)OR. We
then felt it reasonable, taking all evidence into account, to postulate unknown B as being
compound 202, containing two 'Bu2Si(OH) groups at positions C-7 and C-13.
6. CONCLUSION AFTER STRUCTURAL IDENTIFICATIONS
In conclusion, we that postulated the reaction scheme, shown on the next page, Scheme
19, had taken place. On hydrolysing taxchinin A 87, compound 196 was afforded, in
conformational equilibrium. The major conformer adopted the twist-boat/chair, 196a,
arrangement, reducing steric crowding between the 'Bu group at C-l and OH(IO), but
forcing the acetate functionality, at C-2, and the C-4/C-20 double bond closer together. In
order to reduce this overlap the twist-boat/chair, 196a, conformation flipped to the minor
isomer, the twist-chair/boat, 196b, conformer. Unfortunately, in converting to 196b,
steric crowding between the 'Bu group, at C-l, and OH(IO), is increased, inducing
conversion back to 196a, hence a conformational equilibrium was in place. On treatment
with 'Bu2Si(OTf)2 compound 202 was afforded, with only the twist-boat/chair conformer
observed in CDCI3, on introduction of two bulky 'Bu2Si groups this arrangement was
entirely favoured. However, on treatment with (CH3CH2)3SiCl, compound 198 was
produced, entirely in the twist-chair/boat conformation. Protection by a bulky
(CH3CH2)3Si group at C-9 forced ring-B to flip to the twist-chair arrangement to reduce
the severe steric crowding between this triethylsilyl group and the 'Bu2Si group at C-7, that
would be present in the twist-boat/chair conformation, 198d.
'bu )■OH
OH
198d
130
Ofn
JS
Xo z 2
On.; J L
to
o K CQ
Oi„
rlllllllMM*
8c*
ooC*
Sche
me
19
45
On selective removal of the triethylsilyl group from C-9, this steric crowding is removed
and accordingly, on treatment with AD-mix-a, compound 198 was converted to derivative
200, now back in the sterically more stable twist-boat/chair conformation.
7. FURTHER APPROACHES TOWARDS THE OXETANE RING
Having determined the identity of unknown b, as being compound 202, we returned to
this compound in a final endeavor to construct the oxetane ring. On treatment with
osmium tetroxide and NMO, in THF, TLC analysis indicated disappearence of starting
material. After column chromatography, compound 203 was isolated, however,
unfortunately, in only a 7% yield, as illustrated in fig. 88.
Si— 'Bu PH o \
.$• V 'BuHO. HO
OsO,
'Bu OHBu OHOAc OAc OHHO HO
202 203
fig. 88
The clear upfield shift of the signals attributable to protons H-20a and H-20b, from two
singlets, at 64.55 and 65.12, in compound 202, to a multiplet, at 63.54, in diol 203,
indicated the successful dihydroxylation. In addition, the upfield shift of the broad singlet,
attributable to H-5, from 64.23 to 63.78, confirmed this assignment. Furthermore, the
mass spectrum supported structure 203, including the presence of two rBu2Si(OH)
protecting groups, providing further evidence to support our postulation regarding
structure 2 0 2 .
Although the yield of diol 203, from the reaction illustrated in fig. 88, was low, we
decided to go ahead with the oxetane ring synthesis, again using Nicolaou’s
methodology.68 Accordingly, compound 203 was treated with trimethylsilyl chloride, in
the presence of pyridine, after 35 mins stirring a new compound was identified by TLC.
132
Hopefully compound 204, illustrated in fig. 89, had been afforded, with trimethylsilyl
protection at the primary hydroxyl position OH(20).
HO HOrBu 'Bu
HO. HO.OH OH'Bu 'BuTMSC1
OH OHOH 'OHBu OH Bu OH
OAc OAcOH OTMSHO HO
203 204
fig. 89
After work-up the crude product, 204, was taken through to the next reaction, treatment
with mesyl chloride, in the presence of pyridine, see fig. 90.
HO HO
HO.
'OH'OH
,Bu OHOAc OTMSHO
204
HO.
'OMs'OH
OHOAc OTMSHO
205
fig. 90
Unfortunately, after overnight stirring, compound decomposition had occurred, mesylate
205 was not obtained. Due to lack of time and material the reaction was not repeated.
133
8. FINAL CONCLUSION
We again concluded, these compounds, in possession of ?Bu2Si(OH) groups, are only
stabilised on addition of triethylsilyl groups. However, on treatment with
triethylsilychloride reaction takes place at the C-5 position, blocking oxetane ring
construction, as in compound 198.
To successfully achieve oxetane ring formation, and so begin the synthesis of a potent
taxol analogue, alternative protecting groups, other than those already studied, must be
sought after and investigated. Having said this, however, analysis and investigation of
11(15-1 )<2 6 eo-taxanes is extremely challenging due to the complexity imposed by the
tricyclic ring system, together with the flexibility of this skeleton, inducing conformational
equilibrium. In carrying out our studies and investigations, on both brevifoliol and
taxchinin A, we feel a significant contribution has been made to this relatively new area of
chemistry.
formation
Protection at C-5 blocks oxetane ring formation
198
134
CHAPTER FIVE
EXPERIMENTAL
1.1. General Experimental
All reactions were performed under an atmosphere of nitrogen and all solvent extractions
were dried using MgSC>4 , unless otherwise stated in the text. Tetrahydrofuran (THF) was
distilled for sodium benzophenone, diethyl ether from lithium aluminium hydride,
dichloromethane and methanol were distilled from calcium hydride. Dimethyl formamide
(DMF) was purchased directly from Aldrich distilled and stored over molecular sieves.
Petroleum ether (40-60°C) was also distilled, before use during column chromatography.
Flash chromatography was carried out using sorbsil C-60 silica gel, 40-60 M, TLC
analysis was performed using silica gel 60 F254 aluminium TLC plates, Merck 5554,
vanillin stain was employed throughout all the work involving taxanes, PMA was utilised
during the side-chain synthesis. Melting points were measured using a Kofler Hotstage
and are uncorrected, elemental analyses were carried out by Butterworth Laboratories,
Middlesex. IR spectra were recorded using a Perkin Elmer 298 spectrophotometer, optical
rotations were measured using a Perkin Elmer polarimeter and mass spectra were recorded
using a Kratos Concept. NMR spectra were recorded on a Bruker ARX 250 (250 MHz 1H,
62.9 MHz 13C) or a Bruker DRX 400 (400 MHz ]H, 100.6 MHz 13C), both at Leicester
University. NMR spectra recorded in CDCI3 were calibrated to CHC13 (!H, 57.27), (13C,
577.4), all chemical shifts were then taken directly from the spectra and J values given in
Hz. Some NMR spectra were run in CD3OD, in these cases, chemical shifts were taken
directly from the spectra without any prior correction.
135
1.2. Nomenclature
The names subsequently used, in the following experimental section, are based on the
trivial names brevifoliol and 2a-acetoxybrevifoliol (taxchinin A). The systematic name
for brevifoliol 85 using the IUPAC naming system is (1/?,35,55,&R,9/?, 105,11/?,13/?)-8-
Benzoxy-9,11 -diacetoxy-5,13-dihydroxy-3-( 1 -hydroxy-1 -methylethyl)- 14-methylidenyl-
tricyclo[8.4.0.03 ,7]tetradec-6-ene.
BzO OAc
OH
OAcHO
87
BzO
OH
HO
85
We, however, felt it sensible to name our compounds as derivatives of brevifoliol 85, or
derivatives of taxchinin A 87, depending on which natural product was used at the
beginning of the synthesis. This form of nomenclature is in line with the naming systemi j J O f f A
used by Appendino in his papers describing 11(15-1 )abeo-taxanQs. ’ ’ ’
136
2. THE TAXOL SIDE CHAIN
(25, 3/?)-(-)-2,3-Methyl 2,3-dihydroxy-3-phenylpropionate:
O
Ph ^ OCH3
111
HO OH
Ph’' "'C02CH3
112
Methyl cinnamate 111 (13.3 g, 82 mmol), was added to a mixture containing
K20 s 0 2(0H ) 4 (59.4 mg, 0.16 mmol), (DHQD)2PHAL (640 mg, 0.817 mmol), K3Fe(CN)6
(80.5 g, 244 mmol), potassium carbonate (33.8 g, 2441 mmol) and methane sulfonamide
(7.8 g, 82 mmol) in tert-butyl alcohol/water (900 cm3, 1:1, v/v). The solution was stirred
at room temperature for 15 h, after which time sodium sulphite (82 g) was added and
stirring continued a further 15 min. Ethyl acetate (270 cm3) was added, layers separated
and the aqueous layer extracted three times, again with ethyl acetate. Combined organic
layers were washed with brine, dried (Na2S04), filtered and solvents removed under
reduced pressure. Recrystallisation of the crude solid yielded compound 112, (10.93 g,
6 8 % , from toluene), mp 84-85°C, Lit. mp 85-85.50;62 [ cc] d 20 -7 (c 1.00 in MeOH), lit.
[oc] D24 -10.7 (c 1.00 in CHCI3 ) ;62 ymax(film)/cm' 1 3500m, 3380m, 1710s, 1320m, 1270m,
1220m and 1110s; 6r(250 MHz; CDC13) 3.21 (1 H, d, 7 0 h . h 6.9, OH), 3.48 (1 H, d, 7 0 h . h
6.5, OH), 3.90 (3 H, s, CO2Ctf3), 4.46 ( 1 H, dd, / H, o h 3.3 and / H, h 6 . 6 #C C 0 2CH3), 5.11
(1 H, dd, JH, H 3.3 and 7H, o h 6.9, ZfCPh), 7.40-7.50 (5 H, complex m, Hm, H0 and Hp in
Ph); 8c(62.9 MHz; CDC13) 53.1 (CH3, C 0 2CH3), 75.0 (CH, CHOH), 75.5 (CH, CHOH),
126.7, 128.4, 128.8 (CH, C0, Cm and Cp in Ph), 140.4 (C, Ph) and 173.6 (C, C02CH3); m/z
(El) 196.0736 (M+ C10Hi2O4 requires 196.0732), 196 (1%), 121 (4), 119 (7), 108 (8 ), 107
(100), 91 (24), 90 (95), 79 (59), 77 (35) and 51 (10), this is a literature compound.61 ,62
137
(2S, 3U)-(-)-Methyl 3-hydroxy-3-phenyl-2-((p-tolylsulfonyl)oxy)propionate:
HO OTs
Ph C 0 2CH3
113
Triethylamine (15.5 cm3, 110.9 mmol) and p-toluene sulfonic acid (14.5 g, 76.0 mmol)
were added to a stirred solution of diol 112 (14.5 g, 73.95mmol) in dry dichloromethane
(250 cm3), the resulting solution was stirred at 0°C, under an atmosphere of nitrogen, for 3
days. Citric acid (90 cm3, 10% w/v aqueous solution) was added and the aqueous layer
extracted three times into dichloromethane, the combined organic layers were washed with
brine, dried (Na2SC>4), filtered and solvents removed under reduced pressure to give a
crude solid. Column chromatography of the residue on silica with ethyl acetate-petroleum
ether (bp 40-60°C; 2:5, v/v) as eluent, afforded the title compound, tosylate 113 (15.6 g,
60%) as a white solid, mp 111-112°C, Lit. mp 111-112°C;61 Rf [ethyl acetate-petroleum
ether (bp 40-60°C), 2:5, v/v] 0.13; [a ] D20 -56 (c 1.00 in MeOH); Ymax(film)/cm' 1 3540br
w, 1750m, 1370m, 1180s, 1020br w and 870m; 6h (250 MHz; CDC13) 2.41 (3 H, br s,
PhCiJ3), 2.75 (1 H, br s, OH), 3.61 (3 H, s, C 02CH3), 4.92 (1 H, d, 74.4, i/COTs), 5.10 (1
H, br s, HCOH), 7.18-7.27 ( 8 H, complex m, 2 x Ph) and 7.55-7.58 (2 H, m, 2 x Ph);
6C(62.9 MHz; CDC13) 22.0 (CH3, PhCH3), 53.2 (CH3, C02CH3), 73.9 (CH, CHOH), 81.8
(CH, CHOTs), 126.6, 128.2, 128.6 and 130.1 (CH, 2 x Ph), 132.6 (C, PhCH3), 138.0 (C,
Ph), 145.4 (C, Ph) and 168.0 (C, C02CH3); m/z (Cl) 368 ([M+NHU]+ Ci7H180 6SNH4
requires 368.1162), 368 (100%), 357 (15) and 300 (21), this is a literature compound. 61,62
(2R, 3S)-(+)-Methyl 3-phenyl-2,3-epoxypropionate:
HO OTs O
> - < ►Ph ,/C 0 2CH3 P IT C 0 2CH3
113 114
Potassium carbonate (17.5 g) and water (3.8 cm3) were added to a solution containing
tosylate 113 (15.19 g, 43.4 mmol) in DMF (150 cm3). The reaction was stirred at room
HO OH
XPh C 0 2CH3
112
138
temperature for 20 h, after which time water (30 cm ) was added and the solution extracted
into diethyl ether. The combined organic layers were washed with brine, dried (Na2S0 4 ),
filtered and solvents removed under reduced pressure to give a crude oil. Column
chromatography on silica with ethyl acetate-petroleum ether (bp 40-60°C; 2:8, v/v) as
eluent, furnished epoxide 114 (5.8 g, 75%) as a colourless oil; R f [ethyl acetate-petroleum
ether (bp 40-60°C), 2:8, v/v]0.5; [a ] D20 18 (c 1.00 in CHC13), lit. [ale25 14.0 (c 1.6 in
CHCI3 ) ;61 8h(250 MHz; CDCI3) 3.37 (3 H, s, C0 2Ctf3), 3.68 (1 H, d, J 4.7, HCCO2CH3),
4.73 (1 H, d, J 4.7, ffCPh) and 7.14-7.29 (CH, complex m, Hm, Hp and HOJ Ph); 8c(62.9
MHz; CDCI3) 52.3 (CH3, CO2CH3), 56.2 (CH, HCCO2CH3), 57.8 (CH, HCPh), 126.5-
128.9 (CH, Ph), 133.3 (C, Ph) and 167.4 (C, C02CH3); m!z (El) 178.0630 (M+ C10Hio03
requires 178.0627), 178 (2%), 122 (9), 118 (11), 91 (30), 89 (20), 77 (12) and 51 (6 ), this
is a literature compound. 2
(2R9 3S)-(+)-Methyl 3-azido-2-hydroxy-3-phenylpropionate:
O Ph OH
P h ^ ^ C 0 2CH3 Nf \ o 2CH3
114 115
Methyl formate (25.2 cm3) and sodium azide (9.82 g, 151.0 mmol) were added to a stirred
solution containing epoxide 114 (5.39 g, 30.3 mmol) in methanol/water (160 cm3, 8.1,
v/v). The solution was stirred at 50°C for 2 days, after which time it was concentrated
under reduced pressure to give a crude solid. Water was added to the residue and the
aqueous layer extracted into diethyl ether, combined organic extracts were washed with
brine, dried (Na2S0 4 ), filtered and solvents were removed under reduced pressure.
Column chromatography of the residue on silica with ethyl acetate-petroleum ether (bp 40-
60°C; 9:1, v/v) as eluent yielded azide 115 (6.25 g, 93%) as a white solid, mp 50-52°C,
Lit. mp 52.5-53.5°C60; Rf [ethyl acetate-petroleum ether (bp 40-60°C), 9:1, v/v] 0.12;
[oc] D20 119 (c 1.00 in CHC13), lit. [a ] D25 105 (c 2.3 in CHC13) ; 60 6h (250 MHz;CDC13) 3.30
(1 H, br s, OH), 3.64 (3 H, s CO2Ctf3), 4.25 (1 H, d, J 3.2, HCOH), 4.74 (1 H, d, J 3.2,
#CN3) and 7.21-7.33 (5 H, complex m, Hm, H0 and Hp, Ph); 6C(62.9 MHz; CDC13) 53.2
(CH3, C 0 2CH3), 67.5 (CH, HCOH), 74.4 (CH, HCN3), 128.3 (CH, Cm in Ph), 129.0 (CH,
139
Co in Ph), 129.2 (CH, Cp in Ph) 135.9 (c, Ph) and 172.8 (C, C 0 2CH3); m/z (Cl) 239
([M+NH4]+ C 1 0 HHQ3N 3NH 4 requires 239.1141), 239 (13%), 132 (26), 91 (26), 104 (1 0 0 )
and 77 (53), this is a literature compound.60,62
(2 R, 3S)-(-)-Methyl-iV-benzoyl-3-amino-2-hydroxy-3-phenyl-propionate:
Ph OH Ph OH
) — ( ► ) “ (n 3 c o 2c h 3 h n c o 2c h 3
r °Ph
115 105
Benzoyl chloride (6.3 cm3, 67.6 mmol), triethylamine (11.2 cm3, 100.6 mmol) and DMAP
(0.12 g, 1.26 mmol) were added to a solution of azide 115 (6.0 g, 33.7 mmol) in ethyl
acetate (115 cm3). The reaction was stirred at room temperature, under an atmosphere of
nitrogen, for 4 h, after which time, methanol (5.6 cm3) was added and stirring continued a
further 2 h. The solution was filtered over celite and the filtrate washed with 10% citric
acid solution then brine, dried (Na2S0 4 ), filtered and solvents removed under reduced
pressure. Column chromatography of the residue on silica with ethyl acetate-petroleum
ether (bp 40-60°C; 1:9, v/v) as eluent, gave a white powder (8.84 g), which was dissolved
in ethyl acetate and treated with palladium on charcoal (1.14 g, 5% Pd-C) and placed under
an atmosphere of hydrogen for 20h. After filtration over celite, solvents were removed
under reduced pressure. Column chromatography of the residue on silica with petroleum
ether (bp 40-60°C)-ethyl acetate, (1:1, v/v) as eluent afforded the taxol side chain 105
(2.39 g, 29%) as a white solid, mp 183-184°C, Lit. mp 184-185°C;60,61,62 Rf [ethyl acetate-
petroleum ether (bp 40-60°C), 1:1, v/v] 0.4; [o c ]d 20 -44 (c 1.00 in MeOH), lit. [o c ]d 26 -48
(0.92 in MeOH) ; 61 6h (250 MHz; CDC13) 3.32 (1 H, br s, OH), 3.76 (3 H, s, CO2C H 3),
4.56 (1 H, br s, H C OH), 5.66 (1 H, dd, J 1.9 and 9.1, tfCNHCOPh), 7.22-7.47 ( 8 H,
complex m, 2 x Ph) and 7.69 (2 H, H0 in COPh); 5c(62.9 MHz; CDC13) 52.9 (CH3,
C 0 2CH3), 55.9 (CH, HCOH), 73.7 (CH, HCNH), 127.1-128.9 (CH, 2 x Ph), 132.1 *(CH,
C p in CO Ph), 134.2 (C, Ph), 139.0 (C, CO Ph), 168.2 (C, NHCOPh), 173.4 (C, C02CH3);
m/z (Cl) 300 (MH+ Q 7H18O4N requires 300.1231), 300 (28%), 210 (40), 122 (49), 105
(100), 77 (31) and 58 (31), this is a literature compound.60,61,62
140
3. NATURAL PRODUCT EXTRACTION
Brevifoliol 85 and 2a-Taxchinin A 87:
BzO P Ac OAc
HO.......
Ph
OAcHO
BzO P A c OAc
'OH
HO
85 87
Typical extraction procedure:
Crude extract, as a thick brown tar (250 g) was dissolved in methanol (600 cm3) and water
(2.5 It), then extracted into petroleum ether (bp 40-60°C), (5-6 It), using continuous
extraction, for 2 days. After removal of the petroleum ether (bp 40-60°C), the continuous
extraction was followed by manual extraction until the petroleum ether (bp 40-60°C) was
almost colourless, ensuring complete removal of all debris. The crude mixture was then
extracted into ethyl acetate (5-6 It) for a further 2 days, again via continuous extraction,
after which time a manual extraction was carried out until the ethyl acetate extract was
almost colourless, ensuring complete removal of all taxane derivatives. Ethyl acetate was
removed under reduced pressure to again leave a crude, thick brown tar like substance (60
g) for further purification. This crude extract (60 g) was dissolved in chloroform (600
cm3) and stirred with activated charcoal (17 g) for 24 h, after which time the charcoal was
filtered over Celite and solvent removed under reduced pressure to leave a brown solid (50
g). Crude extract (50 g) was then dissolved in chloroform-methanol, (90:10, v/v) and
washed 4 times with aqueous ammonia solution (1:10, v/v, of a 35% ammonia solution),
solvent evaporation under reduced pressure yielded a brown solid (34 g), which was again
dissolved in chloroform (600cm3) and stirred with activated charcoal ( 1 0 g) for 2 h.
Charcoal filtration, over celite, and solvent removal, under reduced pressure, afforded a
brown solid (30 g), which was dissolved in chloroform and adsorbed onto silica. Column
chromatography on silica gel with ethyl acetate-petroleum ether (bp 40-60°C; 1:1, v/v) as
eluent, was carried out until the eluent ran almost colourless , removing the first dark band
141
from the column (this eluent was discarded). At this stage eluting solvent was changed to
chloroform-methanol, (95:5, v/v) and the column continued, again until the eluent ran
almost colourless, removing the second dark band from the column (the taxane fraction).
Solvent evaporation, under reduced pressure furnished a brown foam (9 g), which was
again dissolved in chloroform and adsorbed onto silica for final purification on silica gel,
with ethyl acetate-petroleum ether (bp 40-60°C; 92:8, v/v) as eluent, yielding pure
brevifoliol 85 (2-3 g) and taxchinin A 87 (1-2 g) as pale brown foams. Typically, three
columns were carried out with the ethyl acetate-petroleum ether (bp 40-60°; 92:8, v/v)
eluting system, to achieve separation and purification, since taxchinin A and brevifoliol
run extremely close together on the column. The duration of each extraction procedure,
from initial continuous extraction to isolation of pure compounds, was 2-3 weeks and was
carried out 15 times over 18 months.
Brevifoliol 85, mp 200-201 °C, (from ethanol/water), (lit., 200-203°C);39,40,41 (Found: C,
64.9; H, 7.3; C31H40O9 .H2O requires C, 64.8; H, 7.4%); R f [ethyl acetate-petroleum ether
(bp 40-60°C), 98:8, v/v] 0.22, stains purple first, in relation to taxchinin A staining, using
vanillin; [afo23 -17.6 (c 1.88 in CHC3); Yma^filmycm' 1 3600w, 3590w, 3400br w, 3060-
2900m, 1740s, 1600w, 1370m, 1240s and 1030m; 8h(250MHz;CDC13) 0.86 (3 H, s, 19-
Me), 1.02 (3 H, s, 16-Me), 1.32 (3 H, s, 17-Me) overlapping with 1.46 (1 H, m, 14a-H),
1.71 (3 H, s, 18-Me) overlapping with 1.82 (1 H, m, 2-H) overlapping with 1.82 (1 H, m,
6 a-H) overlapping with 1.98 (3 H, s, C O C H 3 ) , 2.04 (1 H, m, 6 P-H) overlapping with 2.04
(3 H, s, C O C H 3 ) , 2.31 (1 H, m, 2-H) overlapping with 2.42 (1 H, dd, / i 4p> 13 7.2 and / i 4fJ> i4(X
13.9, 14p-H), 2.75 (1 H, br d, / 3>2 8.5, 3-H), 4.37 (1 H, br s, 5-H) overlapping with 4.37 (1
H, br s, 13-H), 4.79 (1 H, s, 20-H), 5.14 (1 H, s, 20-H), 5.56 (1 H, dd, / 7, 6P 5.7 and / 7, 6„
10.9, 7-H), 6.00 (1 H, d , J 9, 10 10.4, 9-H), 6.50 (1 H, 710, 9 10.4, 10-H), 7.37-7.56 (3 H,
complex m, Hm a n d Hp i n COPh) a n d 7.83 (2 H, d , JQ, m 7.2, H0 in COPh); 5c(62.9 MHz;
CDCI3) 12.4 (CH3, COCH3), 13.3 (CH3, C-19), 2 1 . 1 (CH3, C-18), 21.8 (CH3, COCH3),
27.4 (CH3, C-17), 29.5 (CH3, C-16), 29.5 (CH2, C-2), 36.5 (CH2, C-6 ), 38.4 (CH, C-3),
45.5 (C, C-8 ), 47.7 (CH2, C-14), 62.9 (C, C-l), 70.6 (CH, C-7), 70.8 (CH, C-10), 72.9
(CH, C-5), 76.3 (C, C-15), 77.5 (CH, C-13), 77.6 (CH, C-9), 112.4 (CH2, C-20), 129.1 (Cm
in COPh), 129.8 (C, COPh), 129.9 (C0 in COPh), 134.4 (C, C-12), 133.6 (Cp in COPh),
149.6 (C, C -ll), 151.9 (C, C-4), 164.8 (C, COPh), 170.4 (C, COCH3) and 170.9 (C,
142
COCH3); rrilz 579.2570 (MNa+ C3iH4o0 9Na requires 579.2559), 579 (9%), 417 (18), 359
(39), 316 (27), 239 (23), 154 (100) and 136 (87), this is a literature compound.39,40,41
Taxchinin A 87, mp 106-108°C, (lit., 166-167°),37 taxchinin A was impure, however, it
was pure enough for identification purposes, purification takes place in the initial reaction;
Rf [ethyl acetate-petroleum ether (bp 40-60°C), 92:8, v/v] 0.38, stains green second, in
relation to brevifoliol staining, using vanillin; Ymax(film)/cm' 1 3600w, 2920w, 1740s,
1600w, 1370m, 1220m and 1020m; 8h(250MHz; CDC13) 1.03 (3 H, s, 19-Me), 1.10 (3 H,
s, 16-Me), 1.17 (3 H, s, 17-Me), 1.73 (3 H, s, 18-Me), 1.98 (3 H, s, COCH3), 2.04 (3 H, s,
COCH3), 2.08 (3 H, s, COCHj), 3.36 (1 H, br d, h , 2 9.3, 3-H), 4.31 (1 H, br s, 13-H), 4.44
(1 H, br s, 5-H), 4.57 (1 H, s, 20-H), 5.17 (1 H, s, 20-H), 5.49 (1 H, br dd, 7-H), 5.99 (1 H,
d, J% 10 10.7, 9-H), 6.06 (1 H, d, J2, 3 9 .3 ,2-H), 6.40 (1 H, d, J , 0, 9 10.7,10-H), 7.38-7.57 (3
H, complex m, Hm and Hp in COPh) and 7.85 (2 H, d, J„, m 7.6, H„ in COPh), (H-6 a , H-6|3
and H-14a, H-14P) undefinable; 8c(62.9MHz; CDC13) 12.6 (CH3, COCHj), 13.8 (CH3,
C-19), 21.1 (CH3, C-18), 21.2 (CH3, COCH3), 21.8 (CH3, COCH3), 26.1 (CH3, C-17), 28.1
(CH3, C-16), 37.6 (CH2, C-6 ), 40.9 (CH2, C-14), 42.5 (CH, C-3), 45.7 (C, C-8 ), 67.9 (C,
C-l), 69.7 (CH, C-7), 69.7 (CH, C-10), 70.1 (CH, C-2), 74.9 (CH, C-5), 75.9 (C, C-15),
77.0 (CH, C -l3), 78.0 (CH, C-9), 113.2 (C, C-20), 129.2 (Cm in COPh), 129.5 (C, COPh),
129.9 (C„ in COPh), 133.6 (Cp in COPh), 133.8 (C, C-12), 144.2 (C, C -ll), 153.3 (C, C-
4), 164.8 (C, COPh), 170.1 (C, COCH3), 170.6 (C, COCH3) and 171.8 (C, COCH3); m/z
(FAB) 637.26245 (MNa+ C33H420 nNa requires 637.2613), 637 (100%), 475 (59), 433
(70) and 417 (95), this is a literature compound.37
143
4. INITIAL STUDIES
5a-CinnamoyI brevifoliol 123 and 13a-Cinnamoyl brevifoliol 124:
BzO pA c OAc
HO.....-
HO
123
BzO PAc OAc
HO......
'OH
HO
85
BzO pAc OAc
Ph'
'OH
HO
124
A solution containing cinnamic acid (53 mg, 0.36 mmol), DCC (74 mg, 4.85 mmol) and
DMAP (44 mg, 0.36 mmol) in dry dichloromethane (6.0 cm3), was stirred at room
temperature for 30 min. Brevifoliol 85 (0.2 g, 0.36 mmol) in dry dichloromethane (16 cm3)
was added at once and the solution stirred at room temperature, under an atmosphere of
nitrogen, for 2 days, after which time it was diluted with dichloromethane ( 1 2 0 cm3),
washed with water ( 2 x 2 0 cm3), dried and solvents were removed under reduced pressure.
Column chromatography of the residue on silica using diethyl ether-petroleum ether (bp
40-60°C; 7:3, v/v) as eluent gave a mixture of the C-5 and C -l3 monocinnamate 123 and
124 (0.11 g, 43%) as a white solid, mp 113-116°C, in a 3:1 ratio, by NMR, Rf [diethyl
ether-petroleum ether (bp 40-60°C), 7:3, v/v] 0.39; ymax(film)/cm' 1 3580-3340br s, 2980-
2840s, 1740-1710s, 1640m, 1450m, 1280-1260br s, 1030m and 710m; C-5 cinnamate
(major), 8H(250 MHz; CDC13) 0.87 (3 H, s, 19-Me), 0.97 (3H, s, 17-Me), 1.26 (3H, s, 16-
Me) overlapping with 1.30 (1 H, m, 14a-H), 1.46 (1 H, m, 2-H), 1.70 (3 H, s, COCH3),
1.78-1.94 (2 H, complex m, 6 a-H and 60-H), 2.00 (3H, s, Me-18), 2.07 (3H, s, COCH3),
2.31 (1 H, m, 2-H) overlapping with 2.31 (1 H, dd, / i4p, 13 7.0 and / i 4pj Ua 13.5, 14P-H),
2.84 (1 H, d, J3, 2 8.5, 3-H), 4.42 (1 H, br t, 7,3)i4p 7.0, 13-H), 4.87 (1 H, s, 20-H), 5.49 (1
144
H, br s, 5-H), 5.24 (1 H, s, 20-H), 5.58 (1 H, dd, J7.6P 6.7 and Jly 6o 11.7,7-H), 5.96 (1 H, br
d, 9-H), 6.50 (1 H, d, JTy 3’ 16.0, 2’-H), 6.58 (1 H, d, J10y 9 10.4, 10-H), 7.28-7.39 (5 H,
complex m, H0, Hp and Hm in COPh), 7.49 (3 H, m, Hm and Hp in Ph), 7.60 (1 H, d, J3y 2*
16.0, 3’-H) and 7.80 (2 H, br d, H0 in Ph); C-5 cinnamate (major), 6 c (62.9 MHz; CDCI3)
1 2 .1 (CH3, COCH3), 13.2 (CH3, C-l9), 21.0 (CH3, COCH3), 21.6 (CH3, C-18), 25.2 (CH3>
C -l6 ), 27.3 (CH3, C-17), 29.6 (CH2, C-2), 34.1 (CH2, C-6 ), 43.3 (CH, C-3), 45.1 (C, C-8 ),
47.3 (CH2, C -l4), 63.1 (C, C-l), 70.3 (CH, C-7), 74.6 (CH, C-5), 74.6 (C, C-15), 76.1
(CH, C -l3), 77.5 (CH, C-9), 114.4 (CH2, C-20), 118.6 (CH, C-2’), 128.6-130.8 (CH, Ca
and Cm in COPh), 129.7 (C, COPh), 133.7 (C, Ph), 134.6 (Cp in COPh), 134.9 (C, C12),
145.6 (CH, C-3’), 152.4 (C, C-ll), 164.7 (C, C-4), 166.6 (C, COPh), 170.6 (C, COCH3)
and 170.7 (C, COCH3); m/z (FAB) 709.2989 (Mna+ C4oH460 1oNa requires 709.2976), 709
(81%), 565 (100), 489 (100), 399 (77).
C-13 cinnamate (minor), 6H(250 MHz; CDC13) 1.06 (3 H, s, 17-Me), 1.35 (3 H, s, 16-Me),
I.78-1.94 (2 H, complex m, 6 a-H and 6 p-H), 2.00 (3 H, s, COCtf3), 2.52 (1 H, dd, / i 4p, 13
7.2 and / i 4p, u a 14.2, 14p-H), 2.91 (1 H, d, J3, 2 9.1, 3-H), 4.26 (1 H, br s, 5-H), 4.72 (1 H,
s, 20-H), 5.05 (1 H, s, 20-H), 5.58 (1 H, br s, 13-H), 6.36 (1 H, d, JT y 16.0, 2’-H), 7.28-
7.39 (5 H, complex m, Hc, Hm and Hp in COPh), 7.49 (3 H, m, Hm and Hp in Ph), 7.62 (1
H, d, J3y 2’ 16.0, 3’-H) and 7.80 (2 H, br d, H0 in Ph); C-13 cinnamate (minor conformer),
6 c (62.9 MHz; CDC13) 12.2 (CH3, COCH3), 25.2 (CH2, C-2), 25.9 (CH2, C-6 ), 80.1 (CH,
C-13), 118.0 (CH, C-2’) and 145.9 (CH, C-3’), for minor cinnamate, only identifiable
peaks reported; m/z (FAB) 709.2989 (MNa+ GwH^OioNa requires 709.2976), 709 (81%),
565 (100), 489 (100), 399 (77).
145
4a,20-Dihydro-4a,20-dihydroxy brevifoliol:
BzO
OHOHOHHO
138
BzO
HO"-Oh
HO
85
Osmium tetroxide (2.20 cm3, 0.54 mmol, 2.5% w/v in terf-butyl alcohol) was added to a
stirred solution of brevifoliol 85 (3 g, 5.39 mmol) and NMO (0.70 cm3, 60% w/v in H2O)
in THF (30 cm3) and water (15 cm3). The resulting solution was stirred at room
temperature under an atmosphere of nitrogen for 3 days, after which time water (20 cm3)
and sodium sulphite (0.32 g, 1.83 mmol) were successively added and stirring continued a
further 10 min. The solution was then neutralised with saturated aqueous ammonium
chloride and then extracted with ethyl acetate (3 x 20 cm3). The combined extracts were
washed with brine (20 cm3), dried and solvents removed under reduced pressure. Column
chromatography of the residue on silica with dichloromethane-methanol (90:10, v/v) as
eluent gave the diol 138 (2.27 g, 62%) as a white solid, mp 131-133°C, Rf
(dichloromethane-methanol, 90:10, v/v) 0.47; [a]o23 -23.9° (c 9.5 in CHCI3);
ymax(film)/cm‘ 1 3580w, 3420br s, 2980-2880m, 1740s, 1720s, 1450w, 1370m, 1230s and
1070-1030s; 8h(250MHz; CDC13) 1.05 (3 H, s, 19-Me), 1.10 (3 H, s, 16-Me), 1.34 (3 H,
s, 17-Me), 1.70 (3 H, s, 18-Me), 1.81 (3 H, m, 2-H, 6 a-H and 14a-H) overlapping with
2.01 ( 6 H, s, 2 x C O C H 3 ) , 2.28 (2 H, m, 2-H and 6 p-H), 2.38 (1 H, dd, JUf, 13 7.1 and
/ i 4P,i4a 14.2, 14p-H), 3.09 (1 H, br s, 3-H), 3.55 (2H, br s, 20-H), 4.08 (1 H, br s, 5-H),
4.59 (1 H, br s, 13-H), 5.39 (1 H, dd, J7,6p 4.1 and J1>6a 10.7, 7-H), 6.02 (1 H, br s, 9-H),
6.33 (1 H, d, J\o,9 10.1, 10-H), 7.39-7.58 (3 H, complex m, Hm and Hp in COPh), and 7.87
( 2 H, d, J0,m 7.9, H0 in COPh); 8C(62.9 MHz; CDC13) 12.4 (CH3, COCH3), 14.8 (CH3, C-
19), 2 1 . 1 (CH3, C -l8 ), 21.9 (CH3, COCH3), 27.5 (CH3, C-17), 27.5 (CH2, C-2 ), 27.7 (CH3,
C -l6 ), 32.7 (CH2, C-6 ), 41.3 (CH, C-3), 44.7 (C, C-8 ), 44.9 (CH2, C-14), 62.6 (C, C-l),
63.4 (CH2, C-20), 68.0 (CH, C-5), 69.9 (CH, C-l), 70.9 (CH, C-10), 75.2 (C, C-15), 76.8
(CH, C-9), 78.0 (CH, C-13), 129.1 (Cm in COPh), 129.8 (C, in COPh), 129.9 (Q, in
COPh), 133.7 (Cp in COPh), 135.5 (C, C-12), 149.5 (C, C -ll), 165.0 (C, COPh), 170.5 (C,
146
COCH3) and 171.8 (C, COCH3); m/z (FAB) 613.2625 (MNa+ C3 iH42OnNa requires
613.2613), 613 (41%), 393 (42), 333 (81) and 154 (100).
13a,20-Bis(ter/-butyldimethylsilyl)-4a,20-dihydro-4a,20-dihydroxy brevifoliol:
BzO. PAc OAc
t-BuM^SiOii'"
HO
148
BzO PA c OAc
OHHO
A solution of tert-butyldimethylsilyl chloride (2.55 g, 16.92 mmol) and triethylamine (1.40
cm3, 10.18 mmol) in DMF (7.0 cm3), was added to a stirred solution of 138 (1.00 g, 1.69
mmol), triethylamine (5.0 cm3, 35.58 mmol) and DMAP (0.02 g, 0.17 mmol). The
solution was stirred at room temperature, under an atmosphere of nitrogen, for 16 h after
which time the DMF was removed under reduced pressure. The residue was taken up in
ethyl acetate (80 cm3), filtered over celite and solvent was removed, again under reduced
pressure. Column chromatography of the residue on silica with diethyl ether-petroleum
ether (bp 40-60°C; 6:4 v/v) as eluent yielded the disilyl compound 148 (1.08 g, 78%) as a
white solid, mp 106-108°C (Found: C, 62.7; H, 8.5; C43H7oOnSi2 requires C, 63.05; H,
8 .6 %); Rf [diethyl ether-petroleum ether (bp 40-60°C), 6:4, v/v] 0.55; [oc]d 2 5 15.7 (c 2.0 in
CHCI3); ymax(filni)/cm‘1 3560br m, 2960-2860m, 1730s, 1470-1360w, 1240m, 1080s,
840m and 780-680m; 6h (250 MHz; CDC13) 0.05 ( 6 H, d, Si(CH3)2), 0.10 ( 6 H, d,
Si(Ctf3)2), 0 . 8 8 (9 H, s, SiC(Ctf3)3), 0.91 (9 H, s, SiC(Ctf3)3), 1 .0 1 ( 6 H, br s, 16-Me and
19 Me), 1.29 (3H, s, 17-Me), 1.89 (9 H, complex m, C O C H 3 , 18-Me, 2-H, 6 a-H and 14a-
H), 1.97 (3 H, s, C O C H s ) , 2.04 (1 H, ddd, / 6p, 5&7, 4.4, 6 p-H), 2.14 (2 H, complex m, 2-H
and 14|3-H), 2.38 (1 H, br d, 7 3,2 8.5, 3-H), 3.36 (1 H, br s, OH), 3.66 (2 H, m, 20-H), 3.79
(1 H, br s, 5-H), 4.50 (1 H, br t, 13-H), 5.58 (1 H, dd, h , 6|J 4.4 and / 7,6„ 11.3, 7-H), 6.41 (1
H, br d, 10-H), 7.39-7.57 (3 H, complex m, Hm and Hp in COPh) and 7.91 (2 H, br d, H0 in
COPh); 5C(62.9 MHz; CDC13) -5.0 (CH3, Si(CH3)2), -4.4 (CH3, (Si(CH3)2), -3.9 (CH3,
Si(CH3)2), 1 2 . 8 (CH3, COCH3), 14.8 (CH3, C-19), 15.6 (CH3, C-16), 18.3 (C, SiC(CH3)3),
18.7 (C, SiC(CH3)3), 2 1 . 2 (CH3,COCH3), 21.7 (CH3, C-18), 26.1 (CH3, SiC(CH3)3), 26.2
147
(CH2, c-2), 26.3 (CH3, SiC(CH3)3), 27.5 (CH2, C-6 ), 27.8 (CH3, C-17), 38.0 (CH, C-3),
44.9 (C, C-8 ), 45.7 (CH2, C-14), 62.8 (C, C-l), 64.0 (CH* C-20), 66.2 (C, C-4), 69.1 (CH,
C-5), 69.5 (CH, C-7), 71.2 (CH, C-10), 74.9 (CH, C-9), 74.9 (C, C-15), 78.4 (CH, C-13),
129.1 (Cm in COPh), 129.9 (C„ in COPh), 129.9 (C, in COPh), 133.1 (Cp in COPh), 133.6
(C, C-12), 151.3 (C, C -ll), 167.7 (C, COPh) 169.9 (C, COCH3) and 170.5 (C, COCH3);
m/z (FAB) 841.43545 (MNa* C43H7oOn Si2Na requires 841.4335), 841 (63%), 679 (69),
619 (100), 578 (51), 447 (33) and 136 (57).
13a,20-Bis(/<?rt-butyIdimethylsilyl)-4a,20-dihydro-4a3-O-isopropylidene brevifoliol:
BzOP Ac OAc
t-BuMejSiO"1
Na
HO
149
BzO PA cOAc
P HPH
HO
148
2, 2 Dimethoxypropane (5.1 cm3, 41.77 mmol) was added to a solution of 148 (0.15 g,
0.18 mmol) and p-toluene sulphonic acid (3.5 mg, 0.02 mmol), in dry dichloromethane
(12.5 cm3). The solution was stirred at room temperature, under an atmosphere of
nitrogen, for 1 h after which time it was washed successively with sodium hydrogen
carbonate solution and brine, dried and solvent removed under reduced pressure. Column
chromatography of the residue on silica using petroleum ether (bp 40-60°C)-diethyl ether,
(6:4, v/v) as eluent afforded the acetonide 149 (0.13 g, 81%) as a colourless film, Rf
[petroleum ether (bp 40-60°C)-diethyl ether, 6:4, v/v] 0.55; [a]o21 -2.0 (c 2.55 in CHCI3);
ymax(film)/cm ' 1 3580m, 2960-2860s, 1740s, 1470m, 1370s, 1240s, 1090s, 840s and 780-
690m; 6h(250MHz; CDC13) -0 . 0 1 ( 6 H, s, Si(Ctf3)2), 0.06 (6 H, s, Si(Ctf3)2), 0.84 (9 H, s,
SiC(Ctf3)3), 0.89 (9 H, s, SiC( C H 3) 3) , 1.06 ( 6 H, br s, 16-Me and 19-Me), 1.32 ( 6 H, br s,
C(CHs)2 acetonide), 1.48 (3 H, s, 17-Me) overlapping with 1.48 (1 H, m, 2-H), 1.92 ( 6 H,
br s, 18-Me and C O C H 3 ) overlapping with 1.92 (2 H, m, 6 a-H and 14a-H), 2.02 (3 H, s,
C O C H 3 ) , 2.13 (4H, complex m, 2-H, 3-H, 6 p-H and 140-H), 3.78 (2 H, m, 20-H), 4.49 (2
H, br s, 5-H and 13-H), 5.53 (1 H, br m, 7-H), 6.09 (1 H, br d, 9-H), 6.57 (1 H, br d, 10-H),
7.39-7.51 (3 H, complex m, Hm and Hp in COPh) and 7.83 (2 H, br d, H0 in COPh);
148
8C(62.9 MHz; CDC13) -5.0, -4.6, -4.0 (CH3, Si(CH3)2), 12.4 (CH3, COCH3), 14.3 (CH3, C-
19), 15.6 (CH3, C -l6 ), 18.7 (C, SiC(CH3)3), 18.7 (C, SiC(CH3)3), 21.1 (CH3, COCH3),
21.7 (CH3, C-18), 25.5 (CH3, C(CH3)2), 26.3 (CH3, SiC(CH3)3), 26.3 (CH3, SiC(CH3)3),
27.9 (CH3, C -l7), 28.1 (CH2, C-2), 29.7 (CH3, C(CH3)2), 30.8 (CH2, C-6 ), 44.2 (CH, C-3),
44.6 (C, C-8 ), 46.1 (CH2, C-14), 61.8 (C, C-l), 64.3 (CH2, C-20), 66.2 (C, C-4), 70.2 (CH,
C-7), 70.6 (CH, C-10), 73.0 (CH, C-5), 76.7 (C, C-15), 77.0 (CH, C-9), 78.0 (CH, C-13),
84.1 (C, C(CH3)2), 129.0 (Cm in COPh), 129.8 (C in COPh), 129.9 (Cc in COPh), 133.5
(Cp in COPh), 133.5 (C, C-12), 152.0 (C, C -ll), 164.4 (C, COPh), 170.0 (C, COCH3) and
170.4 (C, COCH3); miz (FAB) 881.4667 (MNa+ C46H740iiSi2Na requires 881.4647), 881
(67%), 759 (22), 719 (100), 619 (62), 559 (41), 369 (28) and 287 (41).
13a,20-Bis(terf-butyldimethyIsilyl)-4(X,20-dihydro-4oc-hydroxy-5a-methanesulfonyl
brevifoliol:
BzO. PAcOAc
HO
149
BzO PA c OAc
HO
151
Methanesulfonyl chloride (0.30 cm3, 3.67 mmol) was added to a solution of 149 (0.67 g,
0.82mmol) in dry pyridine (13.5 cm3) and the resulting solution stirred at room
temperature, under an atmosphere of nitrogen, for 3 h. Dichloromethane (45 cm3) was
added and the solution washed with citric acid (3 x 40 cm3, 10% aqueous solution) and
brine (40 cm3) before being dried and solvent removed under reduced pressure. Column
chromatography of the residue on silica with diethyl ether-petroleum ether (bp 40-60°C;
6:4, v/v) as eluent furnished the mesylate compound 151 (0.44 g, 59%) as a white foam,
mp 126-128°C, Found: C, 59.0; H, 8.05; C44H72Oi3Si2S requires C, 58.9; H, 8.1%); Rf
(diethyl ether-petroleum ether (bp 40-60°C), 6:4, v/v) 0.32; [cc]d21 11.1 (c 2.07 in CHC13);
Ymax(film)/cm' 1 3580br m, 2960-2860s, 1740s, 1470m, 1380-1340m, 1240s, 1180s, 990-
970s, 940m and 840s; 8H(250 MHz; CDC13) 0.04 ( 6 H, d, J 1.6, Si(Ctf3)2), 0.11 ( 6 H, d, J
2.2, Si(Ctf3)2), 0.87 (9 H, s, SiC(Ctf3)3), 0.92 (9 H, s, SiC(Ctf3)3), 1.05 (3 H, s, 19-Me)
149
overlapping with 1.12 (3 H, br s, 16-Me), 1.30 (3 H, s, 17-Me), 1.86 (6 H, s, Me-18 and
COCH3) overlapping with 1.86 (3 H, complex m, 2-H, 6 a-H and 14a-H), 1.97 (3 H, s,
COCH3), 2.12-2.34 (4 H, complex m, 2-H, 3-H, 6 p-H and 140-H), 3.12 (3 H, s,
OSO2CH3), 3.58 ( 1 H, br s, 20-H), 3.71 ( 1 H, br d, 2 0 -H), 4.55 (1H, br t, J I3, 14p 6.3, 13-H),
5.05 (1 H, br s, 5-H), 5.50 (1 H, br dd, 7-H), 6.39 (1 H, br s, 10-H), 7.40-7.57 (3 H,
complex m, Hm and Hp in COPh) and 7.92 (2 H, br d, H„ in COPh); 6 c (62.9 MHz CDCI3)
-4.3 (CH3, Si(CH3)3), -4.0 (CH3, Si(CH3)3), 12.9 (CH3, COCH3), 15.1 (CH3, C-19), 18.6
(C, SiC(CH3)3), 18.6 (C, SiC(CH3)3), 21.1 (CH3, COCH3), 21.5 (CH3, C-18), 26.2 (CH3,
SiC(CH3)3), 26.2 (CH3, C-17), 26.3 (CH3, SiC(CH3)3), 26.3 (CH2, C-2), 27.1 (CH2, C-6 ),
28.0 (CH3, C -l6 ), 39.6 (CH, C-3), 39.6 (CH3,0 S 0 2CH3), 44.8 (C, C-8 ), 45.8 (CH2, C-14),
62.0 (C, C-l), 64.0 (CH2, C-20), 66.2 (C, C-4), 68.9 (CH, C-7), 70.5 (CH, C-10), 74.5 (C,
C -l5), 76.6 (CH, C-9), 78.6 (CH, C-13), 79.6 (CH, C-5), 129.1 (C„ in COPh), 129.8 (C„
on COPh), 129.9 (C in COPh), 133.7 (Cp in COPh), 133.7 (C, C-12), 150.8 (C, C -ll),
164.8 (C, COPh), 169.8 (C, COCH3) and 170.3 (C, COCH3); m/z (FAB) 919.4132 (MNa+
C44H72Oi3Si2SNa requites 919.411), 919 (38%), 775 (42), 757 (50) and 697 (100).
13a,20-Bis(terf-butyldimethylsilyI)-10-debenzoyl-4a,20-dihydro-4a-hydroxy-5a-
methanesulfonyl brevifoliol 153 and 20-fcrt-Butyldimethylsilyl-4a,20-dihydro-4,20-
dihydroxy-5a-methanesulfonyl brevifoliol 154:
HO. P A c OAc
HO
153
BzO PAc OAc
HO
151
BzO. P A c OAc
'OMs'OH
OHHO
154
tetra-Butyl ammonium fluoride (0.04 cm3, 0.12 mmol) was added to a solution containing
151 (0.1 g, 0.11 mmol) in dry THF (5.9 cm3). The solution was stirred at room
150
temperature, under an atmosphere of nitrogen, for 15 min, after which time sodium acetate
(9.6 mg, 0.12 mmol) was added and stirring continued under the same conditions for 15 h.
The resulting solution was then taken up in ethyl acetate (20 cm3), washed with saturated
ammonium chloride solution ( 2 x 1 2 cm3), dried and solvents removed under reduced
pressure. Column chromatography of the residue on silica gel using diethyl ether-
petroleum ether (bp 40-60°C; 9:1, v/v) as eluent gave 2 isolable compounds, 154 (0.03 g,
35%) as a white solid, mp 120-122°C; R f [diethyl ether-petroleum ether (bp 40-60°C),
9:1,v/v] 0.38; [a ] D 21 26.4 (c 3.07 in CHCI3); W filnO /cm ' 1 3580w, 3320br w, 2960-
2860m, 1730s, 1370m, 1250s, 1180m, 1090m, 910m, 840m, 780-690m and 153 (0.03 g,
34%) as a colourless film; Rf [diethyl ether-petroleum ether (bp 40-60°C), 9:1, v/v] 0.60;
[a ] D 18 (1.96 in CHC13); Ymax(film)/crn1 3600w, 3320br w, 2980-2860m, 1730s, 1370m,
1240m, 1180m, 1080m, 840m, 780-690m; Compound 154: §h (250 MHz, CDC13) 0.07 ( 6
H, s, Si(Cff3)3), 0.90 (9 H, s, SiC(Ctf3)3), 1.13 (3 H, s, 19-Me), 1.39 (3 H, s, 16-Me), 1.42
(3 H, s, 17-Me), 1.63 (3 H, s, COCH3) overlapping with 1.73 (1 H, m, 14cx-H), 1.95 (2 H,
m, 2-H and 6 cx-H) overlapping with 2.01 (3 H, s, 18-Me), 2.03 (3 H, s, COCH3), 2.11-2.39
(4 H, complex m, 2-H, 3-H, 6 p-H and 140-H), 3.18 (3H, s, OS02CH3), 4.50 (2H, m, 20-H)
overlapping with 4.56 (1 H, br t, 13-H), 4.79 (1 H, br d, 9-H), 5.01 (1 H, br s, 10-H), 5.24
(1 H, br s, 5-H), 5.39 (1 H, dd, / 7, 6„ 4.6 and J7,6a 12.6, 7-H), 7.43-7.62 (3 H, complex m,
Hm and H, in COPh) and 8.02 (2 H, d, J0,m 6.9, H0 in COPh); §c(62.9 MHz; CDC13) -4.3
(CH3, Si(CH3)3), -3.8 (CH3, Si(CH3)3), 13.1 (CH3, COCH3), 15.5 (CH3, C-16), 18.4 (C,
SiC(CH3)3), 21.3 (CH3, COCH3), 21.5 (CH3, C-18), 26.2 (CH3, SiC(CH3)3), 26.8 (CH2, C-
2), 26.8 (CH3, C -l7), 27.6 (CH2, C-6 ), 27.9 (CH3, C-19), 39.6 (CH3, 0 S 0 2 CH3), 40.1
(CH, C-3), 45.0 (C, C-8 ). 45.1 (CH2, C-14), 60.7 (C, C-l), 65.7 (CH2, C-20), 68.3 (CH. C-
10), 69.2 (CH, C-7), 74.5 (C, C-15), 76.9 (CH, C-9), 78.9 (CH, C-13), 79.6 (CH, C-5),
129.1 (Cm in COPh), 129.5 (C, COPh), 130.1 (C0 in COPh), 134.0 (Cp in COPh), 134.0 (C,
C-12), 146.1 (C, C -ll) , 166.8 (C, COPh), 170.6 (C, COCH3) and 171.0 (C, COCH3); m/z
(FAB) 805.3266 (MNa+ C38H58Oi3SiSNa requires 802.3249), 805 (13%), 765 (32), 687
(50), 237 (50) and 136 (100); Compound 153: 8h (250 MHz; CDC13) 0.06 ( 6 H, d, J 1.4,
Si(C//3)3), 0.10 ( 6 H, d, J 1.4, Si(Ctf3)3), 0.89 (9 H, s, SiC(Ctf3)3), 0.92 (9 H, s,
SiC(Ctf3)3), 1.12 (3 H, s, 19-Me), 1.23 (3 H, s, 16-Me), 1.36 (3H, s, 17-Me), 1.61 (3H, s,
C O C H 3 ) , 1.69-1.89 (3 H, complex m, 2-H, 6 a-H and 14a-H), 1.99 (3 H, s, 18-Me), 2.03
(3 H, s, COCtf3), 2.11-2.27 (4 H, complex m, 2-H, 3-H, 60-H and 14(3-H), 3.12 (3 H, s,
151
OSO2CH3), 3.64 (2H, m, 20-H), 4.56 (1 H, br t, Ji3>Mp7.9, 13-H), 4.77 (1 H, d, J9t i0 6.0, 9-
H), 4.93 (1 H, br s, 10-H), 5.06 (1 H, br s, 5-H) and 5.39 (1 H, dd, Jlt6fJ 4.6 and J7,6„ 12.5,
7-H); 6C(62.9 MHz; CDC13) -4.3 (CH3, Si(CH3)3), -3.8 (CH3, Si(CH3)3), 13.1 (CH3,
COCH3), 15.5 (CH3, C -l9), 18.5 (C, SiC(CH3)3), 18.6 (C, SiC(CH3)3), 21.4 (CH3, C-18),
21.4 (CH3, CH3CO), 26.2 (CH3, SiC(CH3)3), 26.2 (CH3, SiC(CH3)3), 26.8 (CH3, C-17),
27.3 (CH2, C-6 ), 28.1 (CH3, C-16), 29.9 (CH2, C-2), 37.9 (CH, C-3), 39.4 (CH3,
0 S 0 2CH3), 45.0 (CH2, C-14), 60.6 (C, C-l), 64.0 (CH2, C-20), 68.3 (CH, C-9), 69.4 (CH,
C-7), 75.4 (C, C -l5), 76.9 (CH, C-10), 76.9 (C, C-4), 79.0 (CH, C-13), 80.5 (CH, C-5),
146.1 (C, C ll) , 170.5 (C, COCH3) and 171.0 (C, COCH3); m/z (FAB) 815.3868 (MNa+
C37H680 12Si2SNa requires 815.3849), 815 (16%), 775 (30), 697 (43), 237 (62) and 136
( 100).
Attempts towards oxetane ring formation:
BzO PA c OAc
OHHO
154
BzO. jPAc OAc
OHHO
155
A solution containing 154 (9.3 mg, 0.012 mmol) in butanone (0.69 cm3), was treated with
tetra-butyl ammonium acetate (42 mg, 0.14 mmol). The solution was stirred at reflux,
under an atmosphere of nitrogen, for 4.5 h, after which time it was allowed to cool to room
temperature and partitioned between diethyl ether and water. The organic layers were
combined, washed with brine, dried and solvents removed under reduced pressure. TLC
analysis indicated the possible formation of 2 new components, Rf [diethyl ether-petroleum
ether (40-60°C), 9:1, v/v] 0.33 and 0.58. Accordingly, column chromatography on silica
with diethyl ether-petroleum ether (bp 40-60°C; 9:1, v/v) as eluent, was attempted in an
effort to isolate the 2 components. Unfortunately, no pure compound could be isolated,
TLC streaking indicated compound decomposition.
152
BzO PA c OAc
'OMsOH
OHHO
154
BzO, sPAc OAc
OHHO
155
A solution containing 154 (59 mg, 0.075 mmol) and tetra-butyl ammonium acetate (8.5
mg, 0.28 mmol) in butanone (2.35 cm3), was stirred at reflux, under an atmosphere of
nitrogen, for 1 h, after which time, it was allowed to cooled to room temperature, diluted
with ethyl acetate (4.5 cm3), washed with saturated ammonium chloride solution and brine,
then dried (Na2SC>4), filtered and solvents removed under reduced pressure. Column
chromatography of the residue on silica with ethyl acetate-dichloromethane (1 :1 , v/v) as
eluent, was attempted in an endeavor to isolate the one identifiable new component at R f
(ethyl acetate-dichloromethane, 1:1, v/v) 0.15. Although some product (3.6 mg) was
isolated, initial NMR analysis showed no evidence of oxetane ring formation, instead
NMR analysis indicated possible partial hydrolysis of the benzoate group.
153
5. NEW APPROACHES
10p-Debenzoyl-7p,9a-dideacetyl brevifoliol:
BzO. P c OAc
HO.....OH
HO
85
HO.OH
HO.....
OH
HO
156
A solution of brevifoliol 85 (4.15 g, 7.46 mmol) in dry methanol (7.0 cm3)was added to a
stirred solution of sodium (0.19 g, 8.26 mmol) in dry methanol (66.0 cm3) at 10°C. The
reaction was stirred at 10°C, under an atmosphere of nitrogen, for 24 h after which time
the methanol was removed under reduced pressure to leave an orange/brown solid.
Column chromatography of the residue on silica with dichloromethane-methanol (90:10,
v/v) as eluent yielded hydrolysed compound 156 (2.29 g, 83%) as a white solid, mp 228-
230°C (from ethanol-water) (Found: C, 65.2; H, 8 .8 ; C20H32O6 requires C, 65.2; H,
8.75%); Rf (dichloromethane-methanol, 90:10, v/v) 0.24; [cxJd2 0 -41 ( c 2.00 in MeOH);
Y m a x ( K B r Discycm*1 3340br s, 2980-2880s, 1660s, 1460-1150br s, 1040br s, 900m;
6h(250MHz; CD3OD 0.96 (3 H, s, 19-Me), 1.10 (3 H, s, 16-Me) overlapping with 1.14 (1
H, m, 14a-H), 1.30 (3 H, s, 17-Me) overlapping with 1.38 (1 H, m, 2-H), 1.71 (1 H, ddd,
7 6 a,5 4.3, Jeaj 11.7 and / 6a,6p 14.8, 6 a-H), 1.90 (3 H, s, 18-Me) overlapping with 1.90 (2 H,
m, 2-H and 60-H), 2.34 (1 H, dd, J 14)W l3 7.1 and J lAfk Ua 13.9, 14P-H), 2.62 (1 H, d, Jx 2
7.9, 3-H), 4.08 (1 H, d, J9, 1 0 9.4, 9-H), 4.25 (2 H, m, 5-H and 7-H), 4.43 (1 H, br t, J13r i4p
7.1, 13-H), 4.56 (1 H, d, 7io, 9 9.4, 10-H), 4.74 (1 H, br s, 20-H) and 5.09 (1 H, br s, 20-H);
8C(62.9 MHz; CD3OD) 11.9 (CH3, C-19), 13.9 (CH3, C -l8 ), 25.3 (CH3, C-17), 27.9 (CH3,
C -l6 ), 30.4 (CH2, C-2), 39.1 (CH3, C-3), 40.0 (CH2, C-6 ), 45.8 (C, C-8 ), 47.9 (CH2, C-14),
63.3 (C, C-l), 71.3 (CH, C-10), 72.8 (CH, C-l), 74.2 (CH, C-5), 77.6 (C, C-15), 79.0 (CH,
C-13), 83.2 (CH, C-9), 111.5 (CH2, C-20), 140.3 (C, C-12), 146.7 (C, C -ll) and 152.8 (C,
C-4); m/z (FAB) 391.2097 (MNa+ C2oH32 0 6 Na require 391.2088), 391 (62%), 351 (36),
333 (74), and 307 (100).
154
Attempts towards C-9, C-10 cyclic protection:
1. Towards acetonide formation
HO
HO'....OH
HO
157HO. OH
HO.....'OH
HO
156
H O "-''OH
XOH
'OH
HO
158
Hydrolysed brevifoliol 156 (0.05 g, 0.14 mmol) was added to a stirred suspension
containing anhydrous copper sulphate (0.2 g) in dry acetone (3 cm3), the mixture was
stirred at room temperature, under an atmosphere of nitrogen and monitored by TLC.
After stirring for 24 h under these conditions, no reaction progression had taken place.
Attempt 2
Concentrated sulphuric acid (1 drop) was added to a stirred solution of hydrolysed
brevifoliol 156 (0.05 g, 0.14 mmol) in dry acetone (0.15 cm3), after stirring at room
temperature, under an atmosphere of nitrogen, for 2.5 h, TLC analysis showed compound
decomposition.
155
Attempt 3
Hydrolysed brevifoliol 156 (0.05 g, 0.14 mmol) was stirred in dry acetone (6.0 cm3) with
anhydrous copper sulphate (0.6 g). After stirring at room temperature, under an
atmosphere of nitrogen, for 3 days, TLC showed no reaction progression.
Attempt 4
Hydrolysed brevifoliol 156 (0.05 g, 0.14 mmol) was stirred with anhydrous copper
sulphate (0.6 g) and /7-toluene sulfonic acid (2 mg) in dry acetone ( 6 cm3). The solution
was stirred at room temperature, under an atmosphere of nitrogen, however severe
streaking, using TLC, indicated an unsuccessful reaction.
Attempt 5
Hydrolysed brevifoliol 156 (0.05 g, 0.14 mmol) was stirred in 2, 2 dimethoxypropane (1.6
cm3) with p-toluene sulfonic acid (2 mg). The resulting solution was stirred at room
temperature, under an atmosphere of nitrogen, for 5 h, after which time TLC analysis
indicated disappearance of starting material. The solution was then neutralised with
saturated sodium hydrogen carbonate solution, extracted into ethyl acetate, dried and
solvents removed under reduced pressure. Although TLC analysis showed multiple spots,
column chromatography was attempted on silica using petroleum ether (bp 40-60°C)-ethyl
acetate, 1:9, v/v as eluent, in an attempt to isolate compound x. A small amount was
possibly isolated, however NMR analysis indicated the isolated compound to be impure
even after column chromatography, at this stage alternative protecting groups were sought
after.
156
Reaction with Ph2SiCl2
Product unstable to column chromatograpy
Triethylamine (0.08 cm3, 0.54 mmol) then dichlorodiphenylsilane (0.057 cm3, 0.27 mmol)
were added to a solution of hydrolysed brevifoliol 156 (0.1 g, 0.27 mmol) in dry DMF<5
(0.52 cm ), and the reaction stirred at 0°C, under an atmosphere of nitrogen, for 15 min.,
after which time TLC analysis indicated disappearance of starting material. The solution
was extracted into ethyl acetate, washed with saturated sodium hydrogen carbonate
solution then brine, dried and solvents removed under reduced pressure. Column
chromatography on silica with ethyl acetate-petroleum ether (bp 40-60°), 1:1, v/v as
eluent, was attempted in an effort to isolate the new component at R{ 0.32, in this solvent
system, unfortunately this compound was unstable to column chromatography.
Reaction with Me2SiCl2
HO.
OH
HO
156
*Product unstable to column chromatograpy
A similar reaction procedure, to that shown above, was followed using
dichlorodimethylsilane, however again, this compound was not stable to column
chromatography.
157
Reaction with 'Bu2SiCl2
HO.
H O '"-OH
HO
No Reaction
156
Triethylamine (0.081 cm3, 0.58 mmol) then di-r-butyldichlorosilane (0.057 cm3, 0.27
mmol) were added to a solution containing hydrolysed brevifoliol 156 (0.05 g, 0.14 mmol)
in dry DMF (0/19 cm3) and the reaction stirred at room temperature, under an atmosphere
of nitrogen. After overnight stirring only starting material was seen using TLC analysis.
Reaction with *Pr2Si(OTf)2
HO PK OH
HO.....OH
HO
156
* Compound Decomposition
2,6-Lutidine (0.95 cm3, 0.81 mmol), then diisopropylsilylbistrifluoremethanesulfonate
(0.16 cm3, 0.53 mmol), were added to a solution of hydrolysed brevifoliol 156 (0.1 g, 0.27
mmol) in dry DMF (0.5 cm3) at 0°C, under an atmosphere of nitrogen, the solution was
allowed to warm to room temperature then stirred, under an atmosphere of nitrogen, for
0.5 h. Column chromatography of the residue on silica with petroleum ether (bp 40-
60°C)-ethyl acetate, 7:3, v/v as eluent, was attempted in an endeavour to isolate the one
identifiable new component at Rf 0.38, in this solvent system. Unfortunately again,
compound decomposition occurred on the column.
158
Reaction with PhjSnClj
HO
HO.....OH
HO
156
y£—► Compound Decomposition
A solution of hydrolysed brevifoliol 156 (0.1 g, 0.27 mmol) in dry THF (0.5 cm3) was
added to a stirring slurry of dichlorodiphenylstannane (0.19 g, 0.55 mmol) and sodium
amide (0.02 g, 0.51mmol) in dry THF (0.5 cm3). The solution was stirred at reflux, under
an atmosphere of nitrogen, for 16 h after which time a black tar had resulted and the
reaction was abandoned.
159
10p,13a-Bis(di-terf-butylhydroxysilyl)-10p-debenzoyl-7p,9a-dideacetyl brevifoliol
179 and 13a-Di-te/t-butylhydroxysilyl-9a,10p-di-terf-butylsilylene-10P-debenzoyl-
7p,9a-dideacetyl brevifoliol 180:
(H3C)3Cn /C(CH3)3S i-O H
/
.......
HO
179HO.
OH
HO
156
(H3C)3C ^ ^c (CH3)3
OH
HO
180
2,6-Lutidine (0.8 cm3, 6.81 mmol) then di-terf-butylsilylditriflate (1.94 cm3, 5.99 mmol)
were added to a stirred solution of 156 (0.85 g, 2.30 mmol) in dry DMF (4.40 cm3), and
the solution stirred at room temperature, under an atmosphere of nitrogen, for 3 h. Water
(20 cm3) was added and the solution extracted into ethyl acetate (3 x 30 cm3). Organic
extracts were successively washed with saturated aqueous sodium hydrogen carbonate
solution then brine, dried and solvents removed under reduced pressure to leave a
brown/orange jelly. Column chromatography of the residue on silica with petroleum ether
(bp 40-60°C)-ethyl acetate (7:3, v/v) and petroleum ether (bp 40-60°C)-ethyl acetate (6:4,
v/v) as eluents, afforded crude 180 (0.67 g, contaminated with terf-butylsilanediol 164) as
a white solid and 179 (0.43 g, 28%) as a white solid. For analytical purposes,
recrystallisation of crude 164/180 gave pure te/t-butylsilanediol as a white crystalline
solid, mp 150-152°C (from dichloromethane) (Found: C, 53.7; H, 11.6; Ci6H2o0 2Si
requires C, 54.5; H, 11.4%); Rf (petroleum ether (bp 40-60°C)-ethyl acetate, 7:3, v/v) 0.66;
ymax(KBr D iscern ' 1 3380br s, 2940-2860s, 1470m, 1370m and 820br s; 6 h (2 5 0 M H z;
160
CD3OD) 0.95 (18 H, s, Si(C(CiJ3)3)2), identical with an authentic sample of the silane diol.
Compound 179, mp 131-134°C (Found: C, 62.8; H, 10.2; C36H680 8Si2 requires C, 63.1; H,
10.0%); Rf (petroleum ether (bp 40-60°C)-ethyl acetate, 7:3, v/v) 0.25; [a]o25 16.7 (c 1.38
inMeOH); ymax(KBr D iscern ' 1 3440 br s, 2970-2860s, 1660m, 1470s, 1370m, 1040s,
900m, 870s amd 820s; §h(250MHz; CDC13) 0 . 8 8 (9 H, s, Si(Ctf3)3), 0.92 (18 H, s
Si(C(Ctf3)3)2), 0.97 (3 H, s, 19-Me), 0.99 (9 H, s, Si(CH3)3), 1.04 (3 H, s, 16-Me), 1.16 (3
H, s, 17-Me) overlapping with 1.26 (2 H, m, 2-H and 14a-H), 1.60 (1 H, ddd, Jea, 5 4.2, Jea,
7 10.8 and J6a. 6P 14.8, 6 a-H), 1.87 (3 H, s, 18-Me) overlapping with 1.87 (2 H, m, 2-H and
6 p-H), 2.25 (1 H, dd, / 14p, 13 6 . 8 and JUfit 14<x 14.6,140-H), 2.66 (1 H, d, J3t 2 8.2, 3-H), 3.93
(1 H, d, J9 , 10 8.7, 9-H), 4.07 (1 H, dd, Jly6P 5.0 and Jly6a 10.8, 7-H), 4.16 (1 H, br s, 5-H),
4.61 (1 H, br s, 20-H), 4.74 (1 H, br t, 13-H), 4.96 (1 H, s, 20-H) and 5.02 (1 H, d, 7i0, 9
8.7, 10-H); 6C(62.9 MHz; CDC13) 13.4 (CH3, C-18), 2 0 .6 , 20.8, 20.9 (C, Si(C(CH3)3)2),
24.5 (CH3, C-17), 27.4, 27.9 (CH3, Si(C(CH3)3)2), 28.1 (CH3, C-16), 28.5 (CH3,
Si(C(CH3)3)2), 29.2 (CH2, C-2 ), 37.1 (CH, C-3), 38.7 (CH2, C-6 ), 44.7 (C, C-8 ), 46.9
(CH2, C-14), 63.5 (C, C-l), 71.6 (CH, C-5), 72.9 (CH, C-10), 75.3 (C, C-15), 75.5 (CH, C-
7), 78.7 (CH, C-13), 83.1 (CH, C-9), 110.8 (CH2, C-20), 135.8 (C, C-12), 147.2 (C, C -ll)
and 151.5 (C, C-4); 6Si(79.5 MHz;CDC13) -13.02 and -6.28 (Si, 2 x Si(C(CH3)3)2OH);
m/z (FAB) 707.4351 (MNa+ C36H680 8Si2Na require 707.4332) 707 (26%), 667 (6 8 ), 649
(100), 631 (6 6 ), 607 (70) and 591 (79).
161
10p,13a-Bis(di-ter*-butylhydroxysilyl)-5a,7p-bis(triethylsilyl)-10p-debenzoyl-7p,9(X-
dideacetyl brevifoliol 162 and 10P-Debenzoyl-13oc-di-ter/-butylhydroxysilyl-9oc,10p-
di-ter/-butylsilylene-7p,9a-dideacetyl-5a-triethylsilyl brevifoliol 163:
(H3C)3Cv C(CH3)3
S i-O H
«PH OH
O '1"(H3C)3C ^ s ./
/ \(H3C)3C o h
HO
179
(H3C)3Cn C(CH3)3
S i-O H
HO
162
(H3C)3C OHHO
163
A solution containing 179 (0.02 g, 0.03 mmol) and triethylsilylchloride (0.10 cm3, 0.62
mmol) in dry pyridine (1.54 cm3), was stirred at room temperature, under an atmosphere of
nitrogen, for 16 h. The solution was neutralised with saturated sodium hydrogen carbonate
solution, extracted into dichloromethane (3 x 25 cm3), dried and solvents removed under
reduced pressure. Column chromatography, of the residue, on silica using petroleum ether
(bp 40-60°C)-diethyl ether (7:3, v/v) as eluent afforded two products as white solids, the
diprotected compound 162 (13 mg, 48%), mp 199-201°C, (Found: C, 63.2; H, 10.8;
C48H9 6 0 8Si4 requires C, 63.1; H, 10.6); R{ [petroleum ether (bp 40-60°C)-diethyl ether,
7:3, v/v] 0.71; [a ] D20 9.4 (c 2.05 in CH2C12); Yimx(film)/cm'‘ 3680w, 3340br w, 2960-
2860s, 1470m, 1080- 1000m, 880m, 820m, and the monoprotected compound (163) (4.2
mg, 18%) as a white solid, mp 231-233°C, (Found: C, 64.9; H, 10.3; C42Hgo0 7 Si3 requires
C, 64.6; H, 10.3%); Rf [petroleum ether (bp 40-60°C)-diethyl ether, 7:3, v/v] 0.42; [a ]© 23
4.7 (c 1.48 in CHC13); Ym»(film)/cin1 3660w, 3480br w, 2960-2860s, 1470-1370m, 1080s
and 820; Diprotected derivative 162: 5h (250 MHz; CDCI3) 0.50-0.63 ( 6 H, m
Si(Ctf2CH3)3), 0.62-0.72 ( 6 H, m, Si(Ctf2CH3)3), 0.91 (18 H, t, 2 x Si(CH2Ctf3)3), 0.94 (3
162
H, s, 19-Me), 1.00 (18 H, d, Si(C(Cff3)3)2), 1.06 (18 H, d, Si(C(Cif3)3)2), 1-16 (3 H, s, 16-
Me), 1.26 (3 H, s, 17-Me) overlapping with 1.29 (1 H, m, 2-H) overlapping with 1.33 (1
H, m, 14a-H), 1.77 (2 H, complex m, 6 a-H and 6 p-H), 1.93 (3 H, s, 18-Me), 2.08 (1 H, m,
2-H) overlapping with 2.18 (1 H, dd, J\4p, i3 6 . 6 and 7i4p, i4o 12.9, 14p-H), 2.68 (1 H, d, 73, 2
8 .8 , 3-H), 4.11 (1 H, dd, 79,0h 6.7 and 79, 10 9.2, 9-H), 4.17 (1 H, br s, 5-H), 4.39 (1 H, br s,
OH-15), 4.40 (1 H, dd, 77, 6P 5.7, 7-H), 4.63 (1 H, s 20-H), 4.87 (1 H, t, Jl3,4p 7.6, 13-H),
4.92 (1 H, s, 20-H), 5.02 (1 H, d, 7io,9 9.2, 10-H), 5.31 (1 H, s, OH) and 6.27 (1 H, d, 7OH, 9
6.7, OH-9); 5C(62.9 MHz; CDC13) 4.7, 5.1, 5.6, 6.1 and 6.7 (CH2, 2 x Si(CH2CH3)3), 7.1
and 7.3 (CH3, Si(CH2CH3)3), 14.0 (CH3, C-18), 14.5 (CH3, C-19), 20.7, 20.9, 21.0 and
21.1 (C, Si(C(CH3)3)2), 25.2 (CH3, C-17), 27.6-28.2 (CH3, 2 x Si(C(CH3)3)2), 28.2 (CH3,
C-16), 30.0 (CH2, C-2), 37.4 (CH, C-3), 41.8 (CH2, C-6 ), 44.9 (C, C-8 ), 47.4 (CH2, C-14),
61.7 (C, C-l), 73.1 (CH, C-5), 74.8 (CH, C-7), 75.2 (C, C-15), 75.4 (CH, C-10), 77.9 (CH,
C-13), 82.8 (CH, C-9), 110.0 (C, C-20), 136.3 (C, C-12), 146.3 (C, C -ll) and 151.5 (C, C-
4); 6Si(79.5 MHz; CDC13) -10.25 (Si, 05i(C(CH3)3)20H at C-13), -5.97 (Si,
05/(C(CH3)3)20H at C-10), 18.92 (Si, Si(CH2CH3) 3 at C-5), 22.15 (Si, Si(CH2CH3) 3 at C-
7); m/z (FAB) 935.60825 (M+ C48H96 0 8Si4 requires 935.6054), 936 (17%), 719 (100), 705
(39), 689 (28), 661 (5) and 605 (30). Monoprotected derivative 163: 6h (250 MHz; CDC13)
0.57 ( 6 H, q, 7 7.8, Si(CH2CH3)3), 0.90 (9 H, t, 77.8, Si(CH2CH3)3), 0.98 (3 H, s, 19-Me),
I.01 (9 H, s, SiC(Ctf3)3), 1.05 (18 H, s, Si(C(Ctf3)3)2), 1.15 (9 H, s, Si(Ctf3)3), 1.17 (3 H,
s, 16-Me), 1.29 (3H, s, 17-Me) overlapping with 1.29 (1 H, m, 2-H) overlapping with 1.37
(1 H, m, 14ot-H), 1.64 (1 H, ddd, 76(X, 5 3.1, 76<x, 7 11-0 and 76<x, 6p 13.8, 6 oc-H), 1.94 (3 H, s,
18-Me) overlapping with 1.94 (2 H, m, 2-H and 6 p-H), 2.24 (1 H, dd, 7i4p, i3 6.3 and 7i4p
14* 12.6, 14p-H), 2.89 (1 H, d, 73, 2 8.5, 3-H), 4.24 (1 H, br s, 5-H), 4.35 (1 H, dd, 77,6p 5.0
and 77, 6„ 11-0, 7-H), 4.48 (1 H, d, 79, i0 10.7, 9-H), 4.59 (1 H, br s, 20-H), 4.87 (1 H, s,
OH-7) and 4.94 (3 H, m, H-10, H-13 and H-20); 6C(62.9 MHz; CDC13) 4.8 (CH2,
Si(CH2CH3)3), 6.9 (CH3, Si(CH2CH3)3), 12.3 (CH3, C-18), 12.4 (CH3, C-19), 20.3, 20.5,
20.8 and 21.5 (C, Si(C(CH3)3)2), 24.8 (CH3, C-17), 27.3-27.7 (CH3, 2 x Si(C(CH3)3)2),
28.2 (CH3, C-16), 29.6 (CH2, C-2), 36.2 (CH, C-3), 39.4 (CH2, C-6 ), 43.6 (C, C-8 ), 48.9
(CH2, C-14), 61.3 (C, C-l), 69.8 (CH, C-5), 72.8 (CH, C-10), 73.2 (CH, C-7), 76.8 (CH,
C-13), 77.3 (C, C-15), 85.9 (CH, C-9), 108.6 (CH2, C-20), 135.4 (C, C-12), 151.0 (C, C-
11) and 151.3 (C, C-4); 8si(79.5 MHz; CDC13) -10.4 (Si, OSz(C(CH3)3)2OH at C13), 12.64
(Si, Si(C(CH3)3)2)0 2 at C-9/C-10), 18.1 (Si, 5/(CH2CH3) 3 at C-5); m/z (FAB) 780.5212
163
(M+ C4 2H8o0 7Si3 requires 780.519), 781 (21%), 745 (36), 721 (43), 587 (48), 237 (70) and
136(100)
10p-Debenzoyl-13a-di-te/t-butylhydroxysilyl-9a,10p-di-te/t-butylsilylene-7p,9a-
dideacetyl-5a-triethylsilyl brevifoliol:
(H3C)3C ^ ✓C(CH3>3
OH
...... .'OH
HO
(H3C)3C ^ y C(CH3)3
OH
O '....(H3C)3C . /
HO
180 163
A solution containing crude 180 (0.12 g) and triethylsilyl chloride (0.62 cm3, 3.60 mmol)
in dry pyridine (9.25 cm3), was stirred at room temperature under an atmosphere of
nitrogen for 16 h. Work-up was carried out, as in the previous reaction, then column
chromatography of the residue on silica with (petroleum ether (bp 40-60°)-diethyl ether
(6:4, v/v) as eluent again furnished compound 163 as a white solid (48 mg, 15% overall
yield from 156), identical, in all respects, to the monoprotected compound made in the
previous reaction, Rf [petroleum ether (bp 40-60°C)-diethyl ether, 6:4, v/v] 0.5.
Di-terf-butylsilanediol:
OTf
OTf
165
(H3 C)3C p H
Si'
(H3O 3C OH
164
A solution containing di-terf-butylsilylditriflate 165 (0.50 g, 1.14 mmol) and water (0.21 g,
11.65 mmol) in ethanol (1.15 cm3), was stirred at room temperature, under an atmosphere
of nitrogen, for 1 h, after which time it was extracted into ethyl acetate, washed with
saturated aqueous sodium hydrogen carbonate solution and brine, dried and solvents
164
removed under reduced pressure. Column chromatography of the residue on silica with
petroleum ether (bp 40-60°C)-ethyl acetate, (8:2, v/v) as eluent gave di-terf-butylsilanediol
164 ( 6 6 mg, 33%) as a white crystalline solid, mp 150-152° (from dichloromethane), (lit.,
150-152°C); (Found: C, 54.3; H, 11.8; C8H2o02Si requires C, 54.5; H, 11.4%); R{
[petroleum ether (bp 40-60°C)-ethyl acetate, 8:2, v/v] 0.39; ymax(KBr DiscVcm' 1 3500-
3260br s, 2980-2860s, 1480-1470s, 1360m and 880-800br s; 8h(250 MHz; DMSO) 0.95
(18 H, s, Si(C(Ctf3)3)2), 5.50 ( 2 H, s, 2 x OH); 6c(62.9 MHz; DMSO) 2 0 . 1 (C,
Si(C(CH3)3)2), 28.0 (CH3, Si(C(CH3)3)2); 6Si(75.5 MHz; DMSO) -13.8 (Si,
5/(C(CH3)3)2(OH)2); m/z (El) 176.1232 (M+ C8H20O2Si requires 176.1227), 176 (6.2%),
119 (16.9) and 77(100), this is a literature compound, all characteristics identical to
literature data on this compound and to the characteristics of the silane diol derivatives
produced during the formation of 179 and 180.
l-Di-terf-butylhydroxysilyloxy-ethan-2-ol:
(H3C)3Cn^ 7C(c h 3)3
177 178
2,6-Lutidine (1.15 cm3, 9.61 mmol) then di-terf-butylsilylditriflate (3.15 cm3, 9.67 mmol)
were added to a solution of compound 177 (0.3 g, 4.83mmol) in dry DMF (4.80 cm3), the
solution was stirred at room temperature, under an atmosphere of nitrogen, for 30 min.
Water (20 cm3) was then added and the solution extracted into ethyl acetate (3 x 30 cm3),
washed with brine, dried and solvents removed under reduced pressure. Column
chromatography of the residue on silica gel with diethyl ether-petroleum ether (bp 40-
60°C; 6:4; v/v) as eluent, furnished compound 178 (0.47g, 45% yield) as a white
crystalline solid, mp 50-52 °C, (Found; C, 54.3; H, 11.3; CioH240 3Si requires C, 54.5; H,
11.0%); Rf [diethyl ether-petroleum ether (bp 40-60°C), 6:4, v/v] 0.35; Ymax(film)/cm' 1
3660w, 3440br w, 2940-2860s, 1470m, 1120m and 820s; 8h (250 MHz; CDC13) 0.99 (18
H, s, Si(C(C//3)3)2), 3.66 (3 H, br m, CH2OH), 3.90 (2 H, br m, C#2OSi) and 4.48 (1 H, br
165
s, SiOH); 6c(62.9 MHz; CDC13) 19.8 (C, Si(C(CH3)3)2), 26.0, 26.3, 26.5, 26.7 and 26.9
(CH3, Si(C(CH3)3)2), 63.2 (CH2, CH2OH) and 63.0 (CH2, CH2OSi); 6Si(75.5 MHz;
CDC13) -9.95 (Si, HOSi(C(CH3)3)2OCH2); m/z (FAB) (MH+ 221 requires 221.8660), 221
(4%), 203 (90), 163 (26), 103 (34), 76 (100).
10p-Debenzoyl-13a-di-te/*-butylhydroxysilyl-9a,10p-di-terf-butyIsilylene-7p,9a-
dideacetyl-4a,20-dihydro-4oc,20-dihydroxy-5a-triethylsilyl brevifoliol:
(H3C)3C ^ _ C(CH3>3
OH
HO
163
OH
O '....(H3C)3C . /
OTESOH
HO OH
185
Osmium tetroxide (0.12 cm3, 2.5% solution in terf-butyl alcohol) was added to a stirred
solution containing 163 (0.11 g, 0.14 mmol) and NMO (0.24 g, 2.05 mmol), in dry THF
(0.80 cm3) and water (0.40 cm3). The resulting solution was stirred at reflux, under an
atmosphere of nitrogen, for 6.5 h, after which time it was cooled to room temperature,
water (0 . 8 cm3) and sodium sulphite ( 8 mg) were successively added and the solution
stirred a further 10 min. The aqueous layer was separated and extracted 3 times with ethyl
acetate, organic layers were combined, washed with brine, dried and solvents removed
under reduced pressure. Column chromatography of the residue on silica with diethyl
ether-petroleum ether (bp 40-60°C; 7:3, v/v) as eluent yielded diol 185 (36 mg, 33%) as a
white solid and starting material 163 (14 mg, 13%), compound 185: Rf [diethyl ether-
petroleum ether (bp 40-60°C), 7:3, v/v] 0.33; [oc]d23 31.6 (c 0.76 in CHC13); Ymax(film)/cm'
1 3680w, 3500br w, 2960-2860s, 1470-1370m, 1080m, 820m and 780-690br m;
5h(400MHz; CDC13) 0.58 (6 H, q, 7 7.8, Si(C/72CH3)3), 0.89 (9 H, t, 7 7.8, Si(CH2C/73)3),
0.94, 0.96 and 0.98 (27 H, 3 x s, 2 x Si(C(Ctf3)3)2), 1.04 (3 H, s, 19-Me), 1.06 (9 H, s,
Si(C(Ctf3)3)2), 1.09 (3 H, s, 16-Me), 1.09 (3 H, s, 17-Me), 1.38 (1 H, d, J2, 2 13.4, 2-H),
1.66 (1 H, dd,7 i4a, 13 6.4 and 7i4a, i4p 13.0, 14a-H), 1.72 (2 H, m, 2-H and 3-H), 1.78 (1 H,
m, 6 a-H) overlapping with 1.84 (3 H, s, Me-18), 1.92 (1 H, ddd, 3.9 and 76p,6a 14.6,
166
6 P-H), 2.17 ( 1 H, dd, 714p. 13 6.4 and 7[4ft u « 13.0, 14p-H), 2.35 (1 H, br d, 20-OH), 2.68 (1
H, s, 4-OH), 3.43 (2H, m, 20-H), 4.08 (1 H, br s, 5-H), 4.11 (1 H, dd, 7?, 3.9 and h , 6a
11.5, 7-H), 4.40 (1 H, d, J% 10 10.5, 9-H), 4.52 (1 H, s, 7-OH), 4.74 (1 H, d, 710, 9 10.5, 10-
H) and 4.89 (1 H, t, 7,3, i4|> 6.4, 13-H); 5c(100.6 MHz; CDCI3) 4.9 (CH2, Si(CH2CH3)3),
6 . 8 (CH3, Si(CH2CH3)3), 12.3 (CH3, C-18), 14.2 (CH3, C-19), 20.5, 2 1 . 1 and 21.9 (C,
Si(C(CH3)3)2), 24.8 (CH3, C-17), 27.7 and 27.8 (CH3, Si(C(CH3)3)2), 28.1 (CH2, C-2), 28.1
Si(C(CH3)3)2), 28.1 (CH3, C-16), 34.3 (CH2, C-6 ), 40.9 (CH, C-3), 42.7 (C, C-8 ), 47.8
(CH2, C-14), 62.4 (CH2, C-20), 67.8 (C, C-l), 68.3 (CH, C-7), 69.8 (CH, C-5), 72.7 (CH,
C-10), 74.7 (C, C-4), 76.8 (C, C-15), 77.9 (CH, C-13), 85.2 (CH, C-9), 136.4 (C, C-12)
and 150.4 (C .C -ll); mJz (FAB) 815.5345 (MH+ C42H8309Si3 requires 815.5322), 837
(46%), 815 (64), 797 (76) and 755 (100).
10P-Debenzoyl-13a-di-ferf-butylhydroxysiIyl-9(x,10P-di-4erf-butylsilylene-7P,9a-
dideacetvl-4 a,20-dihydro-4a,20-dihydroxy-5a-triethylsilyl brevifoliol:
O H
HO OH
185
O T M S
....( H 3 Q 3 C . /
'OTESOH
HO OH
186
Dry pyridine (0.065 cm3, 0.81 mmol) then trimethylsilyl chloride (0.034 cm3, 0.27 mmol)
were added to a stirred solution of 185 (0.022 g, 0.027 mmol) in dry dichloromethane (0.5
cm3) at 0°C, under an atmosphere of nitrogen. The solution was allowed to warm to room
temperature and stirred for 1.5 h, after which time it was quenched with saturated aqueous
sodium hydrogen carbonate solution and extracted 3 times with diethyl ether, organic
layers were combined and washed with brine, dried and solvents removed under reduced
pressure to leave a crude product 186 (16 mg), Rf [ethyl acetate-petroleum ether (40-
60°C), 7:3, v/v] 0.71, which was taken to the next step without further purification.
167
10p-Debenzoyl-13a-di-ter/-butylhydroxysilyl-9a,10P-di-terf-butylsilylene-7p,9a-
dideacetyl-4a,20-dihydro-4a-hydroxy-20-methanesiilfonyl-5a-triethylsilyl-7p-
trimethylsilyl brevifoliol:
(H3C)3C ^ xC(CH3>3
OTMS
HO OH
186
OTMS
OTESOH
OMsHO
187
A solution containing crude 186 (16 mg) in dry pyridine (0.30 cm3) with methane sulfonyl
chloride (0.0065 cm3, 0.085 mmol) was stirred at room temperature, under an atmosphere
of nitrogen, for 2 h. The solution was diluted with dichloromethane and neutralised with
sodium hydrogen carbonate solution after which time the aqueous layer was extracted 3
times with dichloromethane, organic layers combined, washed with brine, dried and
solvents removed under reduced pressure to leave a crude product 187 (14.5 mg), Rf
[diethyl ether-petroleum ether (bp 40-60°C), 7:3, v/v] 0.74, which was taken to the next
step without further purification.
10p-Debenzoyl-13a-di-te/t-butylhydroxysilyl-9a,10P-di-te/t-butylsaylene-7p,9a-
dideacetyl-4(x,20-dihydro-4a-hydroxy-20-methanesiilfonyl-5a-triethylsilyl
brevifoliol:
(H3C)3C v ✓C(CH3)3
O I"-'
OMsHO
188
(H3C)3C ^ ✓C(CH3>3
OTMS
.......
OMsHO
187
A solution containing crude 187 (14.5 mg) in dry methanol (1.50 cm3) with camphor
sulfonic acid (1.0 mg, 0.0043 mmol) was stirred at room temperature, under an atmosphere
168
of nitrogen, for 15 min., after which time it was quenched with saturated sodium hydrogen
carbonate solution, extracted 3 times with dichloromethane, washed with brine, dried and
solvents removed under reduced pressure. The residue was dissolved in dichloromethane
and stirred with silica gel (0.1 g) for 1 h, after which time the silica was filtered and the
solvents removed, again under reduced pressure. Column chromatography of the residue
on silica with ethyl acetate-dichloromethane (3:7, v/v) as eluent, furnished compound 188
(6.6 mg, 28% from diol 185) as a colourless film, R f (ethyl acetate-dichloromethane, 3:7,
v/v) 0.70; [a]D23 (c 0.3 in CHC13); Ymax 3680br w, 3500br w, 2960-2860m, 1470-1360m,
1180m, 1080m, 820m and 760-680br w; §h (250 MHz; CDC13) 0.66 (6 H, q, J 8.0,
Si(Ctf2CH3)3), 0.97 (9 H, t, J 8.0, Si(CH2Ctf3)3), 1.01, 1.04 and 1.16 (30 H, 3 x s, 2 x
Si(C(Ctf3)3)2), 1.14 (6 H, s, Si(C(Ctf3)3)2), 1.16 (CH3, s, 16-Me), 1.18 (3H, s, 19-Me),
1.27 (3 H, s, 17-Me), 1.46 (1 H, m, 2-H), 1.67-1.88 (4 H, complex m, 2-H, 3-H, 6a-H and
14oe-H), 1.92 (3 H, s, 18-Me), 2.02 (1 H, ddd, / 6p, 5&7 4.4 and J6 6a 15.0, 6p-H), 2.25 (1 H,
dd, /up i3 6.3 and J i4p, u a 13.2, 140-H), 2.63 (1 H, br s, 4-OH), 3.06 (3 H, s, 0 S 0 2Ctf3),
4.07 (1 H, br s, 5-H), 4.19 (2 H, m, 7-H and 20-H), 4.39 (1 H, d, 20-H), 4.50 (1 H, d J9> i0
10.6, 9-H), 4.54 (1 H, s, 7-OH), 4.81 (1 H, d, / i 0,9 10.6, 10-H) and 4.93 (1 H, br t, 13-H);
5C(62.9 MHz; CDC13) 5.2 (CH2, Si(CH2CH3)3), 7.1 (CH3, Si(CH2CH3)3), 12.7 (CH3, C-
18), 14.9 (CH3, C-19), 20.5, 21.1, 21.2 and 21.9 (C, Si(C(CH3)3)2), 25.2 (CH3, C-17), 27.6,
27.8, 27.9 and 28.1 (CH3, Si(C(CH3)3)2), 27.9 (CH2, C-2), 28.6 (CH3, C-16), 34.6 (CH2, C-
6), 38.2 (CH, C-3), 43.2 (CH3, 0 S 0 2CH3), 43.2 (C, C-8), 48.2 (CH3, C-14), 62.9 (C, C-
15), 68.3 (CH, C-7), 70.1 (CH, C-5), 72.9 (CH, C-10), 73.9 (C, C-15), 78.3 (CH, C-13),
85.4 (CH, C-9), 136.6 (C, C-12) and 150.9 (C, C -ll); m/z (FAB) 893.5121 (MH+
C43H85OnSi3S requires 893.5097), 915 (26%), 893 (42), 875 (84), 857 (70), 833 (100),
817(23) and 717 (26).
169
10P-Debenzoyl-13a-di-te/t-butylhydroxysilyl-9(x,10P-di-terf-butylsilylene-7p,9a-
dideacetyl-5a-triethylsilyl brevifoliol:
(H3C)3Cn C(CH3)3S i- O H
/jPH OSi(CH2CH3)3
<
HO
OH
HO
162 163
Compound 162 (0.05 g, 0.055 mmol) was added to a mixture containing AD-mix-a
(0.20g) in terf-butyl alcohol (0.30 cm3) and water (0.30 cm3). The resulting solution was
stirred at reflux, under an atmosphere of nitrogen, for 6 h, after which time it was cooled to
room temperature, water (0.40 cm3) then sodium sulphite (0.1 g) were successively added
to the solution and stirring continued a further 30 min. The aqueous layer was extracted 3
times with dichloromethane, organic extracts were combined and washed with brine, dried
and solvents removed under reduced pressure. Column chromatography of the residue on
silica using petroleum ether (40-60°C)-diethyl ether (6:4, v/v) as eluent, gave 163 (26 mg,
62%, overall yield of monotessilyl ether from hb, 30%) as a white solid, with all
characteristics identical to the previously made mono-triethylsilyl protected compound.
10p-Debenzoyl-13a-di-terf-butylhydroxysilyl-9a,10p-di-terf-butylsilylene-7p,9a-
dideacetyl-4a,20-dihydro-4a,20-dihydroxy brevifoliol:
.(H3C)3C. /
OHHO189
O i" -1
OH
HO180
Osmium tetroxide (2.60 cm3, 2.5% solution in tert-butyl alcohol) was added to a stirred
solution containing crude 180 (0.20 g) and NMO (0.54 g, 4.61 mmol), in dry THF (1.80
170
cm ) and water (0.90 cm3), the solution was stirred at reflux, under an atmosphere of
nitrogen, for 4-5 h. Work-up as for compound 185 then column chromatography of the
residue on silica with ethyl acetate-petroleum ether (bp 40-60°C; 6:4, v/v) as eluent
afforded diol 189 (65.5 mg, 14% overall yield from 156) as a white solid, mp 189-191°C;
R f [ethyl acetate-petroleum ether (bp 40-60°C), 6:4, v/v] 0.45; [alo26 9.6 (c 1.06 in
CHC13); Ymax(KBr Disc)/cm'' 3450br s, 2980-2860s, 1740br w, 1480s, 1390-1360m,
1090m and 820s; 8h (250 MHz; CDCI3) 1.00 (9 H, s, SiC(0?3)3), 1.05 (18 H, s,
Si(C(C//3)3)2), 1.10 (3 H, s, 19-Me), 1.14 (9 H, s, SiC(Cff3)3), 1.24 (3 H, s, 16-Me), 1.39
(3H, s, 17-Me), 1.44 (1 H, br d, 2-H), 1.88 (4 H, complex m, 2-H, 3-H, 6 a-H and 14-a-H),
2.10 (1 H, ddd, 5&7 4.4 and \ 6„ 15.1, 6 P-H), 2.18 (3 H, s, 18-Me), 2.24 (1 H, dd,
13 6.3 and 14o 12.6, 14p-H), 3.36 (1 H, br s, OH), 3.49 (1 H, J 11.2, 20-H), 3.69 (1 H,
d, J 11.2, 20-H), 3.95 (1 H, br s, 5-H), 4.24 (1 H, dd, h , 6„ 4.4 and77,6a 11.1, 7-H), 4.39 (1
H, d, h , 10 10.1, 9-H), 4.56 (1 H, s, 7-OH) and 4.85 (1 H, d, 710, 9 10-H) overlapping with
4.89 (1 H, br t, 13-H); 8c(62.9 MHz; CDClj) 12.8 (CH3, C-18), 14.5 (CH3, C-19), 20.5,
21.2 and 21.8 (C, Si(C(CH3)3)2), 24.6 (CH3, C-17), 27.7 (CH2, C-2), 27.7, 27.8 and 28.2
(CH3, Si(C(CH3)3)2), 28.2 (CH3, C-16), 34.0 (CH2, C-6 ), 39.7 (CH, C-3), 43.0 (C, C-8 ),
47.2 (CH2, C-14), 62.8 (CH2, C-20), 63.8 (C, C-l), 68.9 (CH, C-5), 73.1 (CH, C-7), 73.1
(CH, C-10), 75.0 (C, C-15), 79.1 (CH, C-13), 85.6 (CH, C-9), 136.2 (C, C-12) and 151.4
(C, C -ll); m/z (FAB) 701.4482 (MH+ C36H690 9Si2 requires 701.4461), 723 (18%), 701
(23), 641 (25), 507 (35), 467 (50), 291 (35) and 136(100).
171
10p-Debenzoyl-13a-di-tert-butylhydroxysilyl-9a,10p-di-ter*-butylsilylene-7p,9a-
dideacetyl-4a,20-dihydro-4cc,20-dihydroxy brevifoliol:
(H3C)3Cn C(CH3)3Si-OH
/OH OH
......
HO
OH
....(H3C)3C. /
OHHO179 189
Osmium tetroxide (0.24 cm3, 2.5% solution in terT-butyl alcohol) was added to a solution
containing 179 (0.18 g, 0.27 mmol) and NMO (0.49 g, 4.18 mmol), in dry THF (1.60 cm3)
and water (0.80 cm3). The solution was stirred at reflux, under an atmosphere of nitrogen,
for 5 h after which time work-up was carried out, as for compound 180. Column
chromatography of the residue on silica with ethyl acetate-petroleum ether (bp 40-60°C;
1:1, v/v) as eluent gave diol 189 (77.8 mg, 40%) as a white solid, mp 189-191°C; Rf [ethyl
acetate-petroleum ether (40-60°C), 1:1, v/v] 0.48, with all characteristics identical to diol
189, synthesised in the previous reaction.
7P,20JJis(trimethylsilyl)-10p-debenzoyl-13a-di-terf-butylhydroxysilyl-9oc,10p-di-terf-
butylsilylene-7p,9a-dideacetyl-4oc,20-dihydro-4a-hydroxy brevifoliol:
O'1"(H3C)3C^s.// \(H3C)3C oh
(H3C)3Cx^ C(CH3)3
oh
(H3C)3Cn C(CH3)3
OTMS
OTMSHO
189 190
Dry pyridine (0.15 cm3, 1.77 mmol) then trimethylsilylchloride (0.076 cm3, 0.60 mmol)
were added to a stirred solution of 189 (42 mg, 0.06 mmol) in dry dichloromethane (0.5
cm3) at 0°C, under an atmosphere of nitrogen, and the solution stirred, under these
172
conditions, for 15 min. Work-up was carried out, as for compound 186, to afford crude
compound 190 (69 mg) which was taken to the next step without further purification, Rf
(petroleum ether (bp 40-60°C)-diethyl ether, 1:1, v/v) 0.57 and 0.70.
7p,20-Bis(trimethylsilyI)-10p-debenzoyI-13a-di-terf-butylhydroxysilyl-9a,10p-di-terf-
butylsilylene-7p,9a-dideacetyl-4a,20-dihydro-4a-hydroxy-5a-methanesulfonyl
brevifoliol:
(H3C)3Cs ^C(CH3)3
OTMS
......
OTMSHO
(H3C)3Cs ^C(CH3)3
OTMS
O '....(H3C)3Cv_ /
OMsOH
OTMSHO
190 191
A solution containing crude 190 (70 mg) in dry pyridine (0.70 cm3) with
methanesulfonylchloride (0.031 cm3, 0.27 mmol) was stirred at room temperature, under
an atmosphere of nitrogen, for 2 h. Although difficult to determine, by TLC, if the reaction
had been successful, work-up was carried out, as for compound 187, to furnish crude
compound 191, (70 mg), which was taken to the next step without further purification.
173
10p-Debenzoyl-13a-di-ter/-butylhydroxysilyl-9a,10p-di-terf-butyl-siIylene-7p,9a-
dideacetyl-4oc,20-dihydro-4a-hydroxy-5a-methanesulfonyl-20-trimethylsilyl
brevifoliol:
(H3C)3Cn C(Ch 3)3
' ^ OTMS
O "" (HsOjC // \
(H3O3C OHOTMS
OMs
191
(H3Q3C C(CH3)3
^SiC0 - > OH
O'"*(H3C)3C ^ s /
(H3O3C OHOTMS
OMs
192
A solution containing crude 191 (70 mg) in dry methanol (7.0 cm3) with camphor
sulphonic acid (1.6 mg, 0.0072 mmol), was stirred at room temperature, under an
atmosphere of nitrogen, for 15 mins, after which time TLC indicated reaction completion
and a work-up was carried out, as for compound 188. Column chromatography of the
residue on silica with diethyl ether-petroleum ether (bp 40-60°C; 8:2, v/v) as eluent
yielded only one isolable compound (8.7 mg, 17%) as a colourless film, Rf [diethyl ether-
petroleum ether (bp 40-60°C), 8:2, v/v] 0.51, initial !H NMR analysis indicated the
isolated compound to be derivative 192. Accordingly, this compound was again dissolved
in dry methanol (0.43 cm3) and stirred with camphor sulphonic acid (1.2 mg, 0.052 mmol),
the mixture was heated gently in an attempt to encourage ring closure, however, this led to
compound decomposition. Unfortunately, full characterisation was not carried out on
compound 192, however *H NMR was recorded, indicating the structure to be that
described above; 8h (2 5 0 MHz; CDCI3) 0.00 (9 H, s, 1.00 (9 H, s, SiCXCiT^),
1.05 (18 H, s, Si(C(Ctf3)3)2), 1-15 (9 H, s, Si(C(CH3)3)2), 1.16 (3 H, s, 19-Me), 1.27 (3 H,
s, 16-Me), 1.60 (3 H, s, 17-Me), 1.67-1.87 (5 H, complex m, 2-H, 2-H, 3-H, 6 a-H and
14oc-H), 1.94 (3 H, s, 18-Me), 2.01 (1 H, ddd, J6fi, 5&7 4.1 and / 6p, 6a 15.1, 6 £-H), 2.25 (1 H,
dd, /up, 13 6.3 and Ji4p, i4(t 13.2, 140-H), 3.07 (3 H, s, OSO2CH3), 4.02 (1 H, br s, 5-H),
4.18 (1 H, dd, 7 7 ,6 3 4.1 6« H*7’ 7_H) overlapping with 4.20 (1 H, d, 9-H), 4.41 (2 H,
m, 20-H), 4.51 (1 H, br s, 7-OH), 4.81 (1 H, d, 7i0, 9 10.4, 10-H) and 4.93 (1 H, br t, 13-H).
174
10p-Debenzoyl-13a-di-tert-butylhydroxysilyl-9a,10p-di-tert-butylsilylene-7p,9a-
dideacetyl-4(x,20-dihydro-4a-hydroxy-5a-trifluoromethanesulfonyl-20-trimethylsilyl
brevifoliol:
(H3C)3Cn XC(CH3)3
OTMS
......
HO OTMS
190
0 " '- < ^ (H3C)3C ^ /
OTfOH
OTMSHO
193
Diisopropylethylamine (0.38 cm3, 2.17 mmol) then triflic anhydride (0.16 cm3, 1.10
mmol) were added to a solution containing crude 190 (45 mg) in dry dichloromethane
(8.60 cm3) at 0°C, under an atmosphere of nitrogen, and the resulting solution stirred under
these conditions for 0.5 h. Although it was difficult to determine, by TLC, if the reaction
had been successful, starting material had disappeared and accordingly a work-up was
carried out. The solution was allowed to warm to room temperature and quenched with
saturated sodium hydrogen carbonate solution, extracted 3 times with diethyl ether,
washed with brine, dried and solvents removed under reduced pressure to leave a crude
compound (0.12 g) which was taken to the next step without further purification.
(H3C)3Cn ^C(CH3)3
Oi"-<O H '-'
OHHOOTMSHO
193
Crude compound 193 (0.12g) in dry methanol (8.60 cm3) with camphor sulphonic acid (2
mg, 0.009 mmol), was stirred at room temperature, under an atmosphere of nitrogen, for
15 min, after which time the reaction was quenched with saturated sodium hydrogen
carbonate solution, extracted 3 times into dichloromethane, washed with brine, dried and
solvents removed under reduced pressure to a leave a crude compound (91 mg). Column
175
chromatography of the residue on silica gel with ethyl acetate-petroleum ether (bp 40-60°;
8 :2 , v/v) as eluent, was attempted in an effort to isolate the one identifiable new
component at Rf [ethyl acetate-petroleum ether (bp 40-60°C), 8:2, v/v] 0.47, but
unfortunately this was to no avail, no pure compound 194 could be isolated from the
column, decomposition appeared to take place.
176
6. TAXCHININ A
10p-Debenzoyl-7p,9a-dideacetyl taxchinin A:
HOBzO.
HO.....HO.....'OH
OAcOAc HOHO
87 196
A solution of taxchinin A 87 (0.67 g, 1.10 mmol) in dry methanol (1.20 cm3), was added
to a stirred solution of sodium (28 mg, 1.22 mmol) in dry methanol (9.70 cm3) at 10°C.
The resulting solution was stirred at this temperature, under an atmosphere of nitrogen,
overnight, after which time the methanol was removed under reduced pressure. Column
chromatography of the residue on silica with dichloromethane-methanol (90:10, v/v) as
eluent yielded hydrolysed compound 196 (0.25 g, 54%), as a white solid, mp 139-141 °C
(from ethanol/water) (Found: C, 59.1; H, 8.45; C22H35O8.H2O requires C, 59.4; H, 8.2%);
Rf (dichlomethane-methanol, 90:10, v/v) 0.21; [<x]d20 -26.0 (c 2.00 in CHCI3); ymax(KBr
DiscVcm' 1 3360br s, 2980-2880m, 1710s, 1700m, 1470-1370s, 1260s and 1040s;
5h (4 0 0 M H z; CD3OD) Major conformer 253K, 1.08 (3 H, s, 16-Me), 1.11 (3H, s, 19-Me),
1.19 (3H, s, 18-Me), 1.63 (1H, overlapping ddd, Jea,5 5.0 and / 6a, 6p 14.8, 6 a-H), 1.93 (3 H,
s, COCH3) overlapping with 1.93 (1 H, m, 14a-H), 2.02 (3 H, s, 18-Me) overlapping with
2.08 (1 H, m, 6 p-H), 2.10 (1 H, dd, Ji4p, 13 7.1 and Ju , u a 14.6, 14P-H), 3.29 (1 H, d, J3y 2
8.9, 3-H), 4.05 (1 H, J9j 10 9.6, 9-H), 4.14 (1 H, dd, h , 6p 5.2 and / 7, 6a 11.2, 7-H), 4.20 (1 H,
br s, 5-H), 4.46 (1 H, s, 20-H), 4.48 (1 H, br s, 13-H), 4.56 (1 H, d, Jw , 9 9.6, 10-H), 5.13 (1
H, s, 20-H) and 5.72 (1 H, d, J2,3 8.9, 2-H); 5c(100.6MHz; CD3OD) Major conformer
253K, 9 . 6 (CH3, COCH3), 11.6 (CH3, C-19), 19.9 (CH3, C-18), 23.1 (CH3, C-17), 26.0
(CH3, C-16), 37.9 (CH2, C-6 ), 38.8 (CH2, C-14), 40.3 (CH, C-3), 43.5 (C, C-8 ), 65.3 (CH,
C-10), 66.7 (C, C-l), 67.6 (CH, C-2), 69.7 (CH, C-7), 73.7 (CH, C-5), 74.9 (C, C-15), 76.0
(CH, C-13), 78.7 (CH, C-9), 109.9 (C, C-20), 136.0 (C, C-12), 145.4 (C, C -ll), 147.8 (C,
C-4) and 171.3 (C, COCH3); 6h (400M H z; CD3OD) Minor conformer 253K, 1.06 (3 H, s,
177
16-Me), 1.19 (3 H, s, 17-Me), 1.38 (3H, s, 19-Me), 1.90 (3 H, s, COCff3), 1.99 (3 H, s, 18-
Me), 2.33 (1 H, br dd, 140-H), 2.91 (1 H, J3, 2 9.3, 3-H), 3.80 (1 H, d, J% 10 4.0, 9-H), 3.84
(1 H, br t, J5, 6a, 6p 9.7, 5-H), 4.48 (1 H, m, 13-H), 4.56 (1 H, m, 7-H), 4.88 (1 H, d, J , 0 . 9
4.0, 10-H) and 5.78 (1 H, d, J3. 3 9.3, 2-H); 8c(100.6MHz; CD3OD) Minor conformer
253K, 1 1 . 2 (CH3, COCHj), 19.8 (CH3, C-18), 24.7 (CH3, C-17), 26.0 (CH3, C-16), 37.4
(CH2, C-6 ), 40.3 (CH2, C-14), 43.8 (C, C-8 ), 43.8 (CH, C-3), 67.9 (CH, C-7), 68.1 (CH,
C-5), 70.0 (CH, C-2), 70.7 (CH, C-10), 73.8 (CH, C-9), 76.3 (CH, C-13), 110.9 (CH2, C-
20), 134.3 (C, C-12), 145.5 (C, C -ll), 146.4 (C, C-4) and 170.1 (C, COCH3), for minor
conformer, only identifiable peaks reported; m/z (FAB) 427.2332 (MH+ C22H35Og requires
427.2322), 449 (26%), 427 (7), 409 (13), 351 (15), 255 (25)and 176 (100).
7P,13a-Bis(di-fcrt-butylhydroxysilyl)-10P-debenzoyl-7P,9a-dideacetyl taxchinin A:
HO yC(CH3 ) 3
HO.
► (H3 C)3C .. .....OH
OAcHO
202
HO. PH OH
HO.....OH
OAcHO
2 ,6 -lutidine (0.5 cm3, 4.12 mmol) then di-terf-butylsilylditriflate (1.2 cm3, 3.59 mmol)
were added to a stirred solution of 1 9 6 (0.59 g, 1.38 mmol) in dry DMF (3.1 cm3). The
resulting solution was stirred at room temperature, under an atmosphere of nitrogen, for 3
h after which time water ( 2 0 cm3) was added and the solution extracted into ethyl acetate
(3 x 40 cm3) then washed with sodium hydrogen carbonate solution (40 cm3) and brine (40
cm3). The combined organic extracts were dried and solvents removed under reduced
pressure to leave a brown/orange jelly, which was purified by column chromatography, on
silica gel, with petroleum ether (bp 40-60°C)-ethyl acetate (7:3, v/v) as eluent, furnishing
compound 2 0 2 (0.23 g, 22%) as a white solid, mp 137-140°C; Rf [petroleum ether (40-
60°C)-ethyl acetate, 7:3, v/v] 0.21; [a ] D24 -13.3 (c 2.56 in CHC13); ymax(KBr Disc)/cm_1
3380 br s, 2980-2860s, 1710s, 1700s, 1480s, 1370s, 1260m, 1120-970br s, 820s and di-
terf-butylsilanediol 1 6 4 (0.15 g, 24%) as a white crystalline solid, mp 150-152°C; R f
178
[petroleum ether (40-60°C)-ethyl acetate, 7:3, v/v] 0.58; §h (250 MHz, CD3OD) 0.95 (18
H, s, Si(C(Ctf3)3)2) identical to silanediol derivative produced in equivalent brevifoliol
reaction. Compound 202: 5h (250 MHz; CDC13) 1.04, 1.04, 1.07 and 1.09 (36 H, 4 x s, 2
x Si(C(C/73)3)2), 1.13 (3 H, s, 16-Me), 1.14 (3 H, s, 17-Me), 1.19 (3 H, s, 19-Me), 1.66 (1
H, ddd, /6a, 5 3.5, Jea, 7 10.2 and Jea, 6P 13.9, 6a-H), 2.00 (1 H, m, 6p-H) overlapping with
2.00 (3 H, s, 18-Me) and 2.02 (3 H, s, COCtf3), 2.20 (2 H, complex m, 14a-H and 14P-H),
3.39 (1 H, d, / 3 , 2 9.0, 3-H), 3.50 (1 H, br s, OH), 4.16 (1 H, m, 7-H) overlapping with 4.16
(1 H, m, 9-H), 4.23 (1 H, br s, 5-H), 4.55 (1 H, br d, 20-H), 4.67 (1 H, br s, OH), 4.88 (1
H, t, / i 3,i4« 7.6, 13-H), 5.12 (1 H, s, 20-H), 5.14 (1 H, d, Jm 8.8, 10-H), 5.59 (1 H, br s,
OH) and 5.83 (1 H, d, / 2,3 9.0, 2-H); 5c(62.9 MHz; CDC13) 13.9 (CH3, C-19), 14.1 (CH3,
COCH3), 20.9 (C, Si(C(CH3)3)2), 21.0 (C, Si(C(CH3)3)2), 21.2 (C, Si(C(CH3)3)2), 21.5 (C,
Si(C(CH3)3)2), 22.3 (CH3, C-18), 25.5 (CH3, C-16), 27.4 (CH3, Si(C(CH3)3)2), 27.7 (CH3,
Si(C(CH3)3)2), 28.2 (CH3, Si(C(CH3)3)2), 28.4 (CH3, C-17), 28.8 (CH3, Si(C(CH3)3)2),
40.3 (CH2, C-6), 41.1 (CH2, C-14), 41.5 (CH, C-3), 45.1 (C, C-8), 68.9 (C, C-l), 68.9
(CH, C-5), 69.4 (CH, C-2), 71.1 (CH, C-7), 74.9 (CH, C-10), 76.9 (C, C-15), 78.7 (CH, C-
13), 80.9 (CH, C-9), 134.9 (C, C-12), 145.5 (C, C -ll), 148.7 (C, C-4) and 172.2 (C,
COCH3); m/z (FAB) 765.4406 (MNa+ C38H35Oi0Si2Na requires 765.4488), 765 (4%), 549
(100), 489 (25), 449 (13) and 133 (6).
7p,13a-Bis(di-tert-butylhydroxysilyl)-5a,9a-bis(triethylsilyl)-10p-debenzoyl-7p,9a-
dideacetyl taxchinin A:
HO y C(CH3 ) 3 H<D /C (CH3 ) 3
HO.
OH
OAcHO
202
HO.
(H3 C)3C .. .....OTES
OAcHO
198
Triethylsilylchloride (1.90 cm3, 5.64 mmol) was added to a solution containing 202 (0.20
g, 0.28 mmol) in dry pyridine (14.0 cm3), and the solution stirred at room temperature,
under an atmosphere of nitrogen, for 16 h, after which time it was neutralised using
179
saturated aqueous sodium hydrogen carbonate solution, then extracted with
dichloromethane (3 x 20 cm3). The combined organic extracts were washed with brine,
dried and solvents removed under reduced pressure. Column chromatography of the
residue on silica with petroleum ether (bp 40-60°C)-diethyl ether (6:4, v/v) as eluent
afforded 198 (0.15 g, 58%) as a white solid, mp 232-235°C; R f [petroleum ether (bp 40-
60°C)-diethyl ether, 6:4, v/v] 0.49; [a]D24 -30.9 (c 0.57 in CHC13); y^/film l/cm '1 3680s,
3360s, 2960-2860s, 1730s, 1470-1360, 1020-1000s and 820s; 6h (250 MHz; CDC13) 0.61
(6 H, q, J 7.3, CH3Cff2Si), 0.75 (6 H, q, J 7.3, CH3Ctf2Si), 0.96 (18 H, t, J 7.3,
Ctf3CH2Si), 1.03 (9 H, s, SiC(Ctf3)3) overlapping with 1.03 (3 H, s, 16-Me), 1.05 (18-H, s,
Si(C(C//3)3)2), 1.13 (9 H, s, SiC(Cff3)3), 1.25 (3 H, s, 17-Me), 1.36 (3 H, s, 19-Me), 1.68
(1 H, br ddd, J6a, 5 6.5 and J6a, 6, 12.3, 6a-H), 1.87 (3 H, s, 18-Me), 1.89 (3 H, s, COCH 3),
2.10 (1H, dd, 7i40 J3 7.0 and Ju a, i4p 14.5,14a-H), 2.17 (1 H, m, 6(3-H overlapping 14a-H),
2.40 (1 H, dd, Ji^ ,3 7.0 and 7i4s, h„ 14.5, 140-H), 3.03 (1 H, d, J3, 2 8.2, 3-H), 3.92 (1 H,
d, J% io 2 .9 ,9-H), 3.96 (1 H, dd, h , 6S 6.9 and h , 6„ 11.6, 7-H), 4.62 (1 H, br t, 5-H), 4.74 (1
H, s, 20-H), 4.96 (1 H, t, J n , 14„ 6.7, 13-H), 5.09 (1 H, d, 7,0,9 2.9, 10-H), 5.19 (1 H, s, 10-
OH), 5.46 (1 H, s, 20-H) and 5.58 (1 H, d, J%3 8.2,2-H); 8c(62.9 MHz; CDC13) 5.0 (CH2,
CH3CH2Si), 5.2 (CH2, CH3CH2Si), 7.1 (CH3, CH3CH2Si), 7.3 (CH3, CH3CH2Si), 13.6
(CH3, C-18), 17.4 (CH3, C-19), 20.6 (C, Si(C(CH3)3)2), 21.2 (C, Si(C(CH3)3)2), 21.3 (C,
Si(C(CH3)3)2), 21.5 (C, Si(C(CH3)3)2), 22.4 (CH3, COCHj), 28.0 (CH3, Si(C(CH3)3)2),
28.2 (CH3, C-18), 28.4 (CH3, Si(C(CH3)3)2), 28.9 (CH3, Si(C(CH3)3)2), 29.1 (CH3,
Si(C(CH3)3)2), 39.3 (CH2, C-6), 42.0 (CH2, C-14), 46.1 (CH, C-3), 46.7 (C, C-8), 66.8 (C,
C-l), 68.1 (CH, C-5), 70.1 (CH, C-2), 73.9 (CH, C-7), 74.1 (C, C-15), 75.0 (CH, C-10),
79.5 (CH, C-13), 82.6 (CH, C-9), 114.0 (CH2, C-20), 135.0 (C, C-12), 146.1 (C, C -ll),
147.3 (C, C-4) and 170.2 (C, COCH3); m/z (FAB) 803.5134 ((M-[OSi(CH2CH3)3 +
2H20]))+.C44H790 7Si3 requires 803.5112), 803 (56%), 744 (40), 703 (32), 570 (43), 395
(47), 227 (100) and 133 (96).
180
10p-Debenzoyl-13a-di-tert-butylhydroxysilyl-7p,9a-di-tert-butylsilylene-7P,9a-
dideacetyl-5a-triethylsilyl taxchinin A:
(H3 C)3 CN C(CH3 ) 3
HO. jP t e S q -^ ^ o h
(H3 C)3C P ....OTES
OAcHO
198
(H3 C)3 Cv .C(CH3 ) 3
HO.
OTES
OAcHO
200
A solution containing 198 (0.14 g, 0.15 mmol) and AD-mix-a (0.15 g) in tert-butyl
alcohol (0.90 cm3) and water (0.90 cm3), was stirred at reflux, under an atmosphere of
nitrogen, for 6.5 h. The solution was then cooled to room temperature, sodium sulphite
(0 . 2 2 g) added and the stirring continued for a further 2 0 min., before being diluted with
dichloromethane (20 cm3). The aqueous layer was extracted with dichloromethane (3 x 20
cm3) and combined organic extracts washed with brine, dried and solvents removed under
reduced pressure. Column chromatography of the residue on silica with diethyl ether-
petroleum ether (bp 40-60°C; 6:4, v/v) as eluent furnished 200 (38 mg, 32%) as a white
solid, mp 101-104°C, R f [diethyl ether-petroleum ether (bp 40-60°C), 6:4, v/v] 0.35;
[a ] D25 -17.4 (c 1.15 in CHCI3); ymax(film)/cm' 1 3680m, 3540w, 3440m, 2940-2860s,
1730s, 1480-1370s, 1240s, llOO-lOOOs and 820s; 8H(250 MHz; CDC13) 0.57 ( 6 H, q, J
8.1, CH3Ctf2Si), 0.92 (9 H, t, J 8.1, Ctf3CH2Si), 1.04, 1.06, 1.07, 1.13 (36 H, 4 x s, 2 x
Si(C(Ctf3)3)2), 1.11 (3 H, s, 16-Me), 1.16 (3 H, s, 17-Me), 1.30 (3 H s, 19-Me), 1.61 (1 H,
ddd, J6a, 5 3.3, J6a,i 11.3 and /6a,6p 13.8, 6 a-H), 1.93 (3 H, s, 18-Me) overlapping with 1.93
(1 H, m, 6 p-H) overlapping with 1.93 (1 H, m, 14a-H), 1.98 (3 H, s, COC/f3), 2.06 (1 H,
dd, / 14p, 13 6 . 6 and 14p, 14a 13.5, 14P-H), 3.31 (1 H, d, J3, 2 8.5, 3-H), 4.14 (1 H, br s, 5-H),
4.40 (1 H, dd, / 7 , 6P 5.8 and Jlt 6a 11.3 ,7-H), 4.41 (1 H, d, J9, 10 9.8, 9-H), 4.45 (1 H, s, 20-
H), 4.90 (1 H, d, 7i0) 9 9.8, 10-H) overlapping with 4.92 (1H, br t, 13-H), 4.96 (1 H, s, 20-
H) and 5.67 (1 H, d, J2, 3 8.5, 2-H); 8C(62.9 MHz; CDC13) 4.8 (CH2, CH3CH2Si), 5.1
(CH2, CH3CH2Si), 5.4 (CH3Ctf2Si), 7.3 (CH3, (CH3CH2)3Si), 12.3 (CH3, C-18), 15.5
(CH3, C-19), 20.7 (C, Si(C(CH3)3)2), 20.9 (C, Si(C(CH3)3)2), 21.7 (C, Si(C(CH3)3)2), 22.2
(CH3, COCH3), 22.9 (C, Si(C(CH3)3)2), 25.1 (CH3, C-16), 27.7 (CH3, Si(C(CH3)3)2), 28.1
181
(CH3, Si(C(CH3)3)2), 28.2 (CH3, C-17), 28.7 (CH3, Si(C(CH3)3)2), 28.8 (CH3,
Si(C(CH3)3)2), 42.0 (CH2, C-6), 42.2 (CH2, C-14), 42.3 (CH, C-3), 45.0 (C, C-8), 67.1 (C,
C-l), 70.3 (CH, C-2), 70.7 (CH, C-7), 70.8 (CH, C-10), 75.8 (CH, C-5), 76.4 (C, C-15),
77.6 (CH, C -l3), 83.9 (CH, C-9), 110.7 (C, C-20), 134.6 (C, C-12), 146.5 (C, C -ll), 148.9
(C, C-4) and 171.7 (C, COCH3); m/z (FAB) 820.516 ([M-H20]+.C44H8o08Si3 requires
820.5139), 861 (11%), 821 (15), 803 (90), 743 (53), 703 (43), 588 (47), 395 (43) and 227
(100).
13a,7p-Bis(di-tert-butylhydroxysilyI)-10p-debenzoyl-7p,9a-dideacetyl-4a,20-
dihydro-4a-hydroxy taxchinin A:
HO.HO. PHPH
(h 3 c )3c P '" -OHrOH
PHOAcOAc OHHOHO
203202
Osmium tetroxide (0.19 cm3, 2.5% solution in terf-butyl alcohol) was added to a stirred
solution containing 202 (0.16 g, 0.23 mmol) and NMO (0.40 g, 3.41 mmol), in dry THF
(1.30 cm3) and water (0.60 cm3), the resulting solution was stirred at reflux, under an
atmosphere of nitrogen, for 1 h. After cooling to room temperature, water (0.1 cm3) was
added, then sodium sulphite (10 mg) and the solution stirred a further 10 min. The organic
layer was separated and the aqueous layer extracted with ethyl acetate, washed with brine,
dried and solvents removed under reduced pressure. Column chromatography, of the
residue, on silica using ethyl acetate-petroleum ether (bp 40-60°C; 6:4, v/v) afforded diol
203 (12 mg, 7%) as the only isolabe compound; Rf [ethyl acetate-petroleum ether (bp 40-
°C), 6:4, v/v] 0.48; [cx]d2 4 -17.8 (c 1.46 in CHC13); y ^ film y c m '1 3680w, 3400br m,
2980-2860s, 1750m, 1470m, 1370m, 1220m, 1100-990br s and 830s; 5h (400 MHz;
CDC13) 1.02, 1.06, 1.08 and 1.10 (36 H, 4 x s, Si(C(Ctf3)3)2), 1.11 (3 H, s, 16-Me), 1.13
(3H, s, 17-Me), 1.26 (3 H, s, 19-Me), 1.73 (1 H, m, 6a-H) overlapping with 1.99 (3 H, s,
18-Me) overlapping with 2.03 (1 H, m, 6P-H), 2.10 (3 H, s, COCH3) overlapping with
182
2.14 (1 H, m, 14a-H), 2.31 (1 H, dd, JlA> 13 7.2 and i4o 14.4, 14p-H), 2.64 (1 H, br s,
20-OH), 2.99 (1 H, d, 73, 2 7.4, 3-H), 3.54 (2 H, m, H-20), 3.78 (1 H, br t, 5-H), 4.13 (1 H,
dd, 7?,6p 4.9 and J~, 6„11.9, 7-H) overlapping with 4.17 (1 H, dd, 7 9 o h -9 3.4 and J9 i0 9.0,9-
H), 4.22 (1 H, br s, OH), 4.28 (1 H, b s, OH), 4.88 (1 H, br s, OH), 4.93 (1 H, t, Jl3, 14, 3.5,
13-H), 5.10 (1 H, d, 7 ,0 ,9 9.0, 10-H), 5.89 (1 H, d, 72, 3 7.4, 2-H) and 6.01 (1 H, d, 7OH-9,9
3.4, OH-9; 8c(100.6 MHz; CDC13) 13.9 (CH3, C-18), 14.7 (CH3, C-19), 20.0, 20.7, 21.0,
21.0, 21.1 and 21.3 (C, Si(C(CH3)3)2), 22.4 (CH3, COCH3), 25.3 (CH3, C-16), 27.8, 28.0
and 28.2 (CH3, Si(C(CH3)3)2), 28.7 (CH3, C-17), 28.9 (CH3, Si(C(CH3)3)2), 33.9 (CH2, C-
6), 40.1 (CH2, C-14), 43.3 (CH, C-3), 44.1 (C, C-8), 63.2 (CH2, C-20), 69.1 (C, C-l), 69.9
(CH, C-7), 70.6 (CH, C-5), 70.9 (CH, C-2), 74.8 (CH, C-10), 75.2 (C, C-4), 76.0 (C, C-
15), 78.9 (CH, C-13), 81.3 (CH, C-9), 136.1 (C, C-12), 147.9 (C, C -ll) and 171.1 (C,
COCH3); mJz (FAB) 799.4460 (MNa+ C38H720 , 2Si2Na requires 799.4542), 799 (7%), 583
(23), 307 (26), 207 (25) and 147 (64).
13ot,7p-Bis(di-tert-butyIhydroxysilyl)-10P-debenzoyl-7P,9a-dideacetyl-4cq20-dihydro-
4a-hydroxy-20-trimethylsilyl taxchinin A:
HO.HQ. PHPH
(H3C)3C .. .....OH'OH
'OHOHOAcOAc OTMSOH HOHO
204203
Dry pyridine (0.012 cm3, 0.15 mmol) then trimethylsilyl chloride (0.0064 cm3, 0.050
mmol) were added to a solution of 203 (7.6 mg, 0.10 mmol) in dry dichloromethane (1.20
cm3), and the resulting solution stirred for 35 min., under these conditions, after which
time, the reaction was quenched with saturated ammonium chloride solution and allowed
to warm to room temperature. The aqueous layer was extracted 3 times with diethyl ether,
washed with brine, dried and solvents removed under reduced pressure to leave a crude
product (5.2 mg), Rf [diethyl ether-petroleum ether (bp 40-60°C), 7:3, v/v] 0.87, which
was taken to the next step without further purification.
183
HO. PH HO. PH
(H3O 3C O"*- (H3O 3C O'....*V/OH
PH OMsPH
OAc OTMSHO OAc OTMSHO
204 205
Methanesulfonyl chloride (0.0035 cm3, 0.045 mmol) was added to a stirred solution of
crude silyl ether 204 (5.2 mg) in dry pyridine (0.16 cm3) and the reaction was monitered by
TLC for 3 h after which time no progression had taken place. After overnight stirring,
TLC analysis indicated an unsuccessful reaction and compound decomposition.
184
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Appendix 2
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Appendix 3
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