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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