some synthetic carbohydrate chemistry...bsa bovine serum albumin conc. concentrated csa...

Some Synthetic Carbohydrate Chemistry

Natural Product Synthesis, Rational Inhibitor Design and the Development of a New Reagent

Ethan D. Goddard-Borger B.Sc. (Hons)

Chemistry

School of Biomedical, Biomolecular and Chemical Sciences

This thesis is presented for the degree of

Doctor of Philosophy of The University of Western Australia

2008

Candidate Declaration

The work described in this thesis was carried out by the author in the School of

Biomedical, Biomolecular and Chemical Sciences at The University of Western

Australia under the supervision of Professor Robert V. Stick. Unless duly referenced,

the work described is original.

__________________________

Ethan D. Goddard-Borger

September 2008

Contents

Summary III

Acknowledgements V

Glossary VII

Chapter One

“The Synthesis and Assay of Some Germination Stimulants Associated with Smoke”

Introduction 3

Results and Discussion 7

Experimental 27

Chapter Two

“Isofagomine-quercetin Conjugates as Putative Inhibitors of a Glycosyltransferase”

Introduction 51


Experimental 83

Chapter Three

“α-L-Arabinofuranosylated Pyrrolidines as Putative Arabinanase Inhibitors”

Introduction 105


Experimental 127

Chapter Four

“Imidazole-1-sulfonyl Azide Hydrochloride: A Novel Diazotransfer Reagent”

Introduction 143


Experimental 165

References 175

Appendix

Summary

Earnest carbohydrate research was initiated in the nineteenth century by several talented

organic chemists. Carbohydrates, now known to play essential roles in a range of

fundamental biological processes, are presently studied by a throng of scientists from

many fields, including: biochemistry, molecular biology, immunology, structural

biology, medicine, agriculture, pharmacology and, of course, chemistry. Organic

chemistry remains as relevant to carbohydrate research as it has ever been; its

practitioners, with their skills in synthesis and fundamental understanding of molecules,

are truly indispensable.

This thesis details various synthetic endeavours within the field of carbohydrate

chemistry. It describes four projects with goals as diverse as natural product synthesis,

rational inhibitor design and the development of new reagents in organic synthesis.

The first chapter provides an account of the synthesis of compound 1, a potent

germination stimulant present in smoke, from D-xylose. Many analogues of 1 were

prepared from carbohydrates and evaluated as germination stimulants, which permitted

the dissemination of several structure-activity relationships.

O

O

O

D-Xylose

1

Subsequent chapters describe the design and preparation of inhibitors for various

carbohydrate-processing enzymes.

III

Compounds 55 and 56 were sought after as putative synergistic inhibitors of a Vitis

vinifera (grape) uridine diphospho-glucose:flavonoid 3-O-glucosyltransferase (VvGT1).

It was hoped that crystallographic investigations of VvGT1-UDP-2/3 complexes by a

collaborator, structural biologist Professor Gideon Davies, would aid in clarifying

mechanistic aspects of this enzyme.

O

O OH

OH

HO

HON

HOHO

OH

n

n = 1n = 2

5556

n = 0n = 1n = 2

118114115

n

HN

OH

HO

OO

OH

HO

HO

Compounds 114, 115 and 118 were prepared as putative arabinanase inhibitors. Once

again, this work was undertaken to assist in crystallographic studies that might provide a

better understanding of how these enzymes operate.

The thesis concludes by describing the development of compound 152.HCl, a novel

reagent for the diazotransfer reaction. Previously, this reaction utilised

trifluoromethanesulfonyl azide (TfN3), an expensive and explosive liquid with a poor

shelf-life, to convert a primary amine directly into an azide. Reagent 152.HCl was

developed to replace TfN3 in this useful synthetic transformation. A one-pot procedure

enabled the simple and inexpensive preparation of 152.HCl, which was demonstrated to

be shelf-stable, crystalline and, crucially, effective in the diazotransfer reaction.

HCl.NN S N3

O

O

152.HCl

IV

Acknowledgements

It has been my privilege to pursue graduate studies under the tutorship of Prof. Robert

V. Stick. I am most thankful for his encouragement, wisdom and friendship, which were

instrumental in my progression to this point.

I am indebted to my laboratory colleagues, both past and present. Particular mention is

owed to some good friends: Dr. Adrian Scaffidi, Dr. Keith A. Stubbs and Dr. Gavin R.

Flematti.

Acknowledgements are owed also to Assoc. Prof. Emilio L. Ghisalberti and Dr.

Matthew J. Piggott, who were my ‘care-taker’ supervisors whilst R. V. Stick was on

sabbatical leave.

I wish to thank several collaborators: Kings Park and Botanical Gardens (Western

Australia) for their generous gift of some Solanum orbiculatum seeds, Prof. Gideon

Davies (The University of York, U.K.) with whom several projects were conducted and

Assoc. Prof. David J. Vocadlo (Simon Fraser University, Canada) for accommodating

me in his laboratory for several weeks.

An expression of gratitude is extended to: Dr. Brian W. Skelton, for performing the

single crystal X-ray structure determinations in this thesis; Dr. Lindsay T. Byrne, for his

technical assistance with the acquisition and simulation of some NMR spectra; and Dr.

Anthony Reeder, for conducting high-resolution mass spectrometry on my samples.

V

The patience and support of those who are dear to me have made these years of work all

the more pleasant. I extend a heartfelt thank-you to these people. My mother and father,

both graduates of ‘The University of Life’, are particularly deserving of my praise.

The financial assistance of a Hackett Postgraduate Scholarship from the Hackett

Foundation is gratefully acknowledged.

VI

Glossary

Abbreviations 18[crown]-6 1,4,7,10,13,16-hexaoxacyclooctadecane

AIBN 2,2′-azobis(2-methylpropionitrile)

aq. aqueous

B base

BSA bovine serum albumin

conc. concentrated

CSA camphor-10-sulfonic acid

d day(s)

DAST diethylaminosulfur trifluoride

DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

DEAD diethyl azodicarboxylate

DHA dihydroxyacetone

DHAP dihydroxyacetone phosphate

DIAD diisopropyl azodicarboxylate

DIBAL diisobutylaluminium hydride

DMAP 4-N,N-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

DSC differential scanning calorimetry

E electrophile

EI electron ionisation

ES electrospray ionisation

FAB fast atom bombardment

FAD flavin adenine dinucleotide

GDP guanosine diphosphate

GH glycoside hydrolase (glycosidase)

GMP guanosine monophosphate

GT glycosyltransferase

h hour(s)

HRMS high resolution mass spectrometry

HRP horse-radish peroxidase

HWE Horner-Wadsworth-Emmons (reaction)

IR infrared (absorption spectrometry)

LPH lactase-phlorizin hydrolase

mCPBA 4-chloroperbenzoic acid

min minute

m.p. melting point

NBS N-bromosuccinamide

NMR nuclear magnetic resonance

VII

Nu nucleophile

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

PDC pyridinium dichromate

Pd/C palladium on carbon

Pd(OH2)/C palladium hydroxide on carbon (Pearlman’s catalyst)

pTSA 4-toluenesulfonic acid

r.t. room temperature

sat. saturated

SDS sodium dodecyl sulfate

TBAF tetrabutylammonium fluoride

TCEP tris(2-carboxylethyl)phosphine

TFA trifluoroacetic acid

THF tetrahydrofuran

t.l.c. thin layer chromatography

Tris tris(hydroxymethyl)aminomethane

UDP uridine diphosphate

UMP uridine monophosphate

WT wild-type

Functional Groups Ac acetyl COCH3Ar aryl

Bn benzyl CH2C6H5Boc tert-butoxycarbonyl CO2C(CH3)3BMS tert-butyldimethylsilyl Si(CH3)2C(CH3)3But tert-butyl C(CH3)3Bu butyl (CH2)3CH3Bz benzoyl COC6H5Et ethyl CH2CH3 Im imidazolyl 1-C3H3N2Me methyl CH3Ms methanesulfonyl SO2CH3Ph phenyl C6H5Piv pivalyl COC(CH3)3Pr propyl (CH2)2CH3Pri isopropyl CH(CH3)2Tf trifluoromethanesulfonyl SO2CF3TMS trimethylsilyl Si(CH3)3 Tr triphenylmethyl C(C6H5)3

VIII

Chapter One

The Synthesis and Assay of Some Germination

Stimulants Associated with Smoke

Chapter One

2

The Synthesis and Assay of Some Germination Stimulants Associated with Smoke

Introduction

Many plant species native to areas frequented by wild-fire have evolved traits allowing

them to synchronise some of their key biological events, such as regrowth, reproduction

and germination, with an incidence of fire.[1] Such adaptations allow these species to

capitalise upon the opportunities offered by their post-fire environment. Perhaps the

most remarkable of these adaptations is the tendency for the seeds of some species to

germinate more readily, or only, after an outbreak of fire.[2, 3]

Smoke generated by the combustion of plant matter provides the cue for this post-fire

emergence; species receptive to this stimulus are referred to as ‘smoke-responsive’.[4]

The charred remnants and leachates of burnt plant material are also capable of initiating

the germination of smoke-responsive species.[5-7] Evidently, the stimulus eliciting this

germination response was chemical in origin.[8] Over the years, many attempts at

elucidating the nature of the compound(s) in smoke responsible for promoting

germination went unrewarded, in no small part due to the complexity of the concoction

of chemicals that is smoke.[9]

In 2004, Flematti et al. isolated and characterised 3-methyl-2H-furo[2,3-c]pyran-2-one

1 from ‘cellulose-derived’A smoke and demonstrated its ability to promote the

germination of both smoke-responsive and non-smoke-responsive plant species from

Africa, Australia and North America.[10] This remarkable germination stimulant

exhibited incredible potency, promoting germination at concentrations of less than one

part-per-billion (< 10-9 M). A The smoke obtained by the slow combustion of pure cellulose has been shown to illicit a very similar germination response to that obtained by burning plant material.[10] Cellulose-derived smoke is a less complex mixture than plant-derived smoke, which is why it was used in the isolation of 1.

3

Chapter One

The possible benefits that the potent germination stimulant 1 could offer the

agricultural, horticultural and even mining (environmental rehabilitation) industries is

obvious.[10]

O

O

O

1

Only one, rather inefficient, synthesis of 1 had been reported, which was somewhat

surprising given its potency, efficacy and potential applications.[11] This synthesis began

with kojic acid 2 which, over four steps, was converted into pyromeconic acid 3

(Scheme 1.1).[12] The thione 4, easily obtained from 3, was esterified to give 5. Heating

5 in acetic anhydride with triphenylphosphine and sodium acetate provided 1. The

global yield for this seven step synthesis was an unenviable 1.4% and, regrettably, the

final step offered the poorest yield; just 22%. Further, lower yields were obtained on

larger scale reactions, with the isolation of 1 from the complex mixture of products

obtained in the final step proving to be laborious, impractical and inefficient.[11]

O

O

O

OHO

O

SHO

SO

OCl

O

OHO

OH

O O

1

2 3

4 5

4 steps

12%

a

67%

79% 22%

b c

Scheme 1.1 a) P2S5, NaHCO3, THF; b) CH3CHClCOCl, Et3N, CH2Cl2;

c) NaOAc, Ph3P, Ac2O.

4


The intriguing, albeit deceptively simple, nature of the novel ring system of 1, its potent

biological activity and potential applications encouraged an investigation of alternative

syntheses for this compound. This task was embarked upon with the intention to

concurrently prepare a number of analogues of 1, and to evaluate their ability to

promote the germination of a smoke-responsive plant species. It was hoped that such an

investigation would lead to the delineation of some structure-activity relationships.

Retrosynthetic Analysis

Retrosynthetic analysis was performed on the parent ring system 2H-furo[2,3-c]pyran-

2-one 6, the functionalisation of which, at a later stage, would offer a convergent

synthesis of 1 and a number of its analogues differently substituted at C3 (Scheme 1.2).

Presumably 6 could be obtained from a molecule of general structure 7 by way of

lactonisation and eliminations across both C4−C5 and C7−C7a. A molecule of structure

7 could, in turn, be obtained by olefination of a ketone resembling generic structure 8.

Such a ketone is quite clearly the product of a carbohydrate; a pentose in this instance.

Notably, the application of this synthetic strategy to a hexose would yield compounds

analogous to 6, but with an extra carbon at C5. Thus, this approach promised a route to

both 1 and a range of its analogues different at both C3 and C5.

O

O

O R

O

O

O

O

RORO

RO

OR

O

ORO

RO

OR

O

OHHO

HO

OH

1

3

3a4

5

6

7a

7

2

1

3 4

52

6 7

8

O

Scheme 1.2 Retrosynthetic analysis of 1.

5

Chapter One

Given that the germination stimulant’s origin within Nature appeared to lie in

carbohydrates (cellulose)[10] and in keeping with the human inclination to mimic Nature,

it seemed fitting that this retrosynthetic analysis had ultimately led to a carbohydrate

starting material (albeit one unrelated to cellulose).

Investigations into the use of carbohydrates to prepare the potent germination stimulant

1 and a number of its analogues are described forthwith. The ability of these novel

analogues to promote the germination of Solanum orbiculatumB and some emerging

structure-activity relationships are detailed in the latter part of this chapter.

B A highly smoke-responsive perennial native to Western Australia.

6


Results and Discussion

The Synthesis of 6 from D-Xylose

Retrosynthetic analysis of 6 had revealed that a pentose was required for the proposed

synthesis; the obvious choice was abundant and inexpensive D-xylose. Given that

oxidation (and subsequent olefination) was to occur at C3, D-xylose required protection

such that only the hydroxyl group at this position remained unmasked. This requirement

was met by the regioselective tritylation of diol 9 (an inexpensive, commercially

available substrate easily obtained from D-xylose[13]), which provided the alcohol 10

(Scheme 1.3).[14] Oxidation of 10, in accordance with literature procedure, returned the

ketone 11.[14] Several attempts at Wittig olefination of 11 went unrewarded, indeed, no

reaction was observed. In contrast, the Horner-Wadsworth-Emmons (HWE) method

provided the desired (Z)-olefin 12 in high yield and in nineteen-fold excess of the (E)-

isomer 13.

O

O

O

HO

HOO

O

O

HO

TrO

O

O

O

TrOO

O

O

TrO

CO2EtEtO2C

a b

109

O

O

O

O

TrO

11

c

d

D-Xylose

12 13

Scheme 1.3 a) i) Me2CO, H2SO4 ii) AcOH, H2O; b) TrCl, Et3N, CH2Cl2; c) Ac2O,

Me2SO; d) NaH, (EtO)2POCH2CO2Et, THF (12:13, 19:1).

7

Chapter One

It is tempting to suggest that steric effects in 11, particularly those associated with the

trityl group, were directly responsible for the favourable stereochemical outcome of this

olefination. In truth, such a rationalisation is probably an oversimplification; the

stereoselectivity of a HWE reaction is the product of a complex interplay of electronic

effects (mainly with respect to the phosphonate), steric effects (from both the carbonyl

compound and phosphonate), solvent effects and even the nature and concentration of

the cations present.[15]

Treatment of 12 with aqueous trifluoroacetic acid, in a rapid reaction, resulted in

hydrolysis of the trityl ether and acetonide, lactonisation and rearrangement of the

resulting hemiacetal (though not necessarily in that order) to give the butenolide 14 in

excellent yield (83%) (Scheme 1.4). Following the publication of the work presented in

this chapter,[16] a similar reaction, producing 14 from silyl ether 15, was reported by

Xavier and Rauter.[17]

O

O

O

TrO

CO2Et O

O

O

OH

HO

12 14

O

O

O

BMSO

CO2Et

15

a

Scheme 1.4 a) CF3CO2H, H2O.

Hypothetically, compound 6 could be obtained from 14 by way of two eliminations,

formally dehydrations, one across C4−C5 and the other across C7−C7a (formerly

C1−C2 of D-xylose). All attempts at the dehydration of 14 directly to 6 were

unsuccessful, typically resulting in dark intractable mixtures of unidentified products. It

was evident that a less direct approach was required.

8


Treatment of 14 with acetic anhydride in pyridine gave the diacetate 16, which, in the

presence of triethylamine, underwent elimination across C7-C7a to give 17 (Scheme

1.5).

O

O

O

OH

HO O

O

O

OAc

AcO O

O

O

OAca b

14 16 17

Scheme 1.5 a) Ac2O, C5H5N; b) Et3N, CH2Cl2.

It was apparent that 17 could perhaps be obtained directly from 14 by performing an

acetylation in the presence of a base stronger than pyridine. Curiously, doing so with

triethylamine resulted in the exclusive formation of the furan 18 (Scheme 1.6).

O

O

AcO

OAc

AcOO

O

O

OH

HO

a

14 18

Scheme 1.6 a) Ac2O, Et3N, C5H5N.

If nothing else, this result suggested that perhaps the elimination across C7−C7a of 16

occurred by way of a stabilised furyloxide intermediate (Figure 1.1).

O

O

O

OAc

AcO O

O

-O

OAc

AcO O

O

O

OAcHB:

AcO_

16 17

Figure 1.1 Elimination possibly occurs by way of the furyloxide intermediate.

9

Chapter One

Efforts at conducting the remaining elimination across C4−C5 of 17, through the use of

various acids or bases, went unrewarded. In contrast to the previous case, electronic and

stereochemical factors disfavour this second elimination.C With both product and

substrate possessing acid sensitive (enol-ether) and base sensitive (lactone) moieties, an

alternative approach to this elimination was required.

A solution to this problem was drawn from the work of Tsuji and Trost,[18, 19] who

independently demonstrated that the treatment of allylic acetates, carbonates or halides

with palladium(0) catalysts, in the absence of suitable nucleophiles and where possible,

resulted in the formation of 1,3-dienes.[18-22] This mild method of elimination may be

conducted under neutral conditions; a feature that appeared ideal for substrate 17,

which, fortuitously, also happened to be an allylic acetate. Treatment of 17 with

tetrakis(triphenylphosphine)palladium(0) in refluxing THF returned the desired

compound 6 (Scheme 1.7). However, somewhat unsatisfactory was the reaction’s

necessarily high catalyst loading (20 mol %), long reaction time (2 days) and moderate

yield (61%).

O

O

O

OAc

O

O

O

a

17 6

Scheme 1.7 a) (Ph3P)4Pd, THF.

C With respect to 17: H4 is more acidic than both protons at C5, the departing acetate at C4 has a gauche relationship to both protons at C5 and the development of positive charge at C4 and negative charge at C5 is discouraged by their respective adjacent groups.

10


In general, allylic carbonates have proven to be better substrates than allylic acetates in

these ‘Tsuji-Trost eliminations’.[23] With this in mind, the biscarbonate 19 was prepared,

which, when treated with triethylamine, underwent elimination to give 20 (Scheme 1.8).

When the carbonate 20 was heated at reflux in THF with the same palladium(0) catalyst

as before, a smooth conversion into 6 was observed in high yield (84%) and reasonable

time (8 hours) at standard catalyst loadings (4 mol %).

O

O

O

OH

HO O

O

O

OCO2Et

EtO2CO

O

O

O

OCO2Et

a

b

O

O

O

c

14 19

20 6

Scheme 1.8 a) EtOCOCl, C5H5N; b) Et3N, CH2Cl2; c) (Ph3P)4Pd, THF.

It is noteworthy that this method has proven to be reliable on reasonably large scales; it

has been used to prepare multigram quantities of 6.

Extension of the Synthetic Strategy to a Hexose

Having established a good synthetic route to 6, the application of a similar synthetic

strategy to a hexose was investigated in order to prepare analogues of 6 substituted at

C5. By utilising a hexuronic acid ester substrate it was hoped that, owing to the

enhanced acidity of H5, the elimination across C4-C5 would occur with greater ease

than in the synthesis of 6, obviating the necessity of a palladium catalyst for this final

elimination. A convenient substrate for this purpose was inexpensive and commercially

11

Chapter One

available D-glucuronic acid γ-lactone 21 (Scheme 1.9). The acetonide 22 was prepared

from 21 in accordance with the procedure of Fleet and co-workers.[24] A modified

method for the benzoylation and methanolysis of the lactone 22 gave the alcohol 23.[25]

Oxidation of 23 presumably gave the ketone, which underwent Wittig olefination to

give the desired (Z)-isomer 24 in vast excess of the (E)-isomer 25.

O

OO

HO

OH

OH O

OO

HO

O

O

O

O

O

HO

MeO2C

BzO

O

O

OMeO2C

BzO

CO2Et

O

O

OMeO2C

BzO

EtO2C

a

b, c d, e

21 22

23

24 25

Scheme 1.9 a) Me2CO, H2SO4; b) BzCl, C5H5N; c) Et3N, MeOH;

d) PDC, Ac2O, CH2Cl2; e) Ph3PCHCO2Et, CH2Cl2 (24:25, 99:1).

Transesterification of the benzoate 24 by the catalytic action of alkali in methanol

resulted in a complex mixture of products. A catalytic, nucleophilic transesterification

using sodium cyanide in methanol was more successful in unmasking O5, however, a

mixture of two products was obtained owing to the slow concomitant transesterification

of the ethyl ester. This mixture was treated with aqueous trifluoroacetic acid, incurring a

transformation analogous to that observed before, to return the butenolides 26 in good

yield (Scheme 1.10).

12


O

O

OMeO2C

BzO

EtO2C O

O

O

OH

HO CO2Me O

O

O

CO2Me

a, b c, d

24 26 27

Scheme 1.10 a) NaCN, MeOH; b) CF3CO2H, H2O; c) Ac2O, C5H5N; d) DBU, CH2Cl2.

Acetylation of 26, using the usual protocol, gave a mixture of three compounds that, by

NMR spectroscopy, appeared to be the α- and β-anomers of the diacetate in addition to

the product of C7-C7a elimination. This mixture of compounds was treated with DBU

in dichloromethane to furnish 27 in excellent yield over the two steps. Several attempts

at the direct conversion of 26 into 27 by performing the acetylation in the presence of

strong base were unsuccessful, most likely owing to the formation of acetoxyfurans as

was observed in the synthesis of 6 (Scheme 1.6, Figure 1.1).

Electrophilic Substitution at C3 of 6 and 27

With the parent heterocycle 6 and an anlogue 27 now available, the challenge remained

to elaborate these molecules at C3 to prepare the natural product 1 (from 6) and a

number of its analogues (from both 6 and 27). By inspection, C3 appears to be the most

nucleophilic carbon in compounds 6 and 27 and for this reason electrophilic substitution

was investigated as a means of elaborating these molecules at this position.

The germination stimulant 1 required the extension of compound 6 by a single carbon at

C3 and so an electrophilic formylation of 6 was the first transformation attempted.

Treatment of 6 with the Vilsmeier reagent,[26] followed by base hydrolysis, furnished the

aldehyde 28 exclusively and in excellent yield (Table 1.1). A similar treatment of 27

furnished the aldehyde 29, though this reaction required more time at a greater

13

Chapter One

temperature to reach completion. This is most probably a result of the diminished

nucleophilicity of C3 in 27, a result of the conjugation of the relevant double bond (C3-

C3a) with the electron-withdrawing methyl ester at C5.

Table 1.1 Electrophilic substitution of compounds 3 and 27 at C3.

R = HR = HR = CHOR = CHOR = HR = COMeR = COEt3

R' = HR' = CO2MeR' = HR' = CO2MeR' = CONMe2R' = HR' = H

O

O

O R

R'

627

128129130131132

R = COCHCH2CH2R = NO2

33034

R' = H1R' = H

Substrate Product Conditions† Yield %

6 28 a 92

27 29 a 90

27 30 b 73

6 31 c 91

6 32 c 89

6 33 c 85

6 34 d 48

† a) i) POCl3, DMF ii) sat. aq. NaHCO3; b) POCl3, Me2NCOMe; c) AlCl3, CH2Cl2, RCl; d) NaNO3, CF3CO2H.

Attempts at performing a Vilsmeier-Haack acetylation (employing phosphoryl chloride

and N,N-dimethylacetamide) were unsuccessful, with 6 offering no reaction and 27

yielding the amide 30, presumably through reaction with liberated dimethylamine.

Friedel-Crafts acylation proved to be more fruitful for the preparation of ketones.

Indeed, the action of a number of acyl chlorides and aluminium(III) chloride on 6

produced the ketones 31−33, all in excellent yield. These acylations, as for the

aforementioned formylations, occurred only at C3.

14


Whilst several nitration protocols were tried on substrate 6, only the combination of

sodium nitrate and trifluoroacetic acid returned isolable quantities of the nitro

compound 34, albeit in modest yield.[27] Once again, substitution occurred exclusively at

the C3 position.

The regiospecificity and facile nature of these electrophilic substitutions is not

unexpected. By inspection, C3 appears to be the most electron-rich and therefore most

nucleophilic carbon in compounds 6 and 27; one would anticipate favourable kinetics

for the formation of the C3-electrophile adduct. In addition, attack of an electrophile at

C3 may give a stable (aromatic) pyrilium ion intermediate, further enhancing the

process (Figure 1.2).

O

O

O

O+

O

O

O

O

OE E+ E+

– E+

– H+

Figure 1.2 Rationalisation for the exclusive electrophilic substitution at C3.

Reductions in the Preparation of 1 and Analogues Thereof

A sufficiently mild method for the deoxygenation of the aldehyde 28 was sought in

order to complete the synthesis of 1. Whilst several methods for deoxygenation of

aldehydes were investigated, it was the combination of tert-butylamine-borane and

aluminium(III) chloride that worked best on the aldehyde 28;[28] compound 1 was

obtained in high yield (Table 1.2). This completed the synthesis of the natural

germination stimulant 1, obtained from D-xylose in ten steps and an overall yield of

19%, or 30% in nine steps from commercially available 1,2-O-isopropylidene-D-

15

Chapter One

xylofuranose 9. The complete ten step synthesis has been conducted in eight days to

provide gram quantities of 1.

The same borane reagent system was used in the preparation of analogues of 1; it

proved effective in the deoxygenation of the ketones 31 and 32 to compounds 35 and

36, respectively. An analogous reduction of the aldehyde 29 gave compound 37, albeit

in modest yield, where the formyl group had been completely reduced but reduction of

the ester moiety ceased at the primary alcohol. Given this result, identical reaction

conditions were employed to produce the alcohol 38 from the ester 27; prolonged

reaction times produced the chloride 39. The alcohol 38 was converted into a further

halomethyl analogue, the fluoride 40, by treatment with DAST.

Table 1.2 Reductions utilising ButNH2.BH3 and AlCl3.†

O

O

O R

R'

R = EtR = PrnR = MeR = HR = HR = H

R' = HR' = HR' = CH2OHR' = CH2OHR' = CH2ClR' = CH2F

3536

137138139140

R = MeR = HR = CHOR = CHOR = COMeR = COEt3

R' = HR' = CO2MeR' = HR' = CO2MeR' = HR' = H

127

128129

1131132

Substrate Product Yield %

28 1 83

31 35 79

32 36 77

29 37 42

27 38 74

27 39‡ 46

† Bu tNH2.BH3 (6 mol equivalents), AlCl3 (3 mol equivalents), CH2Cl2 (reflux, 30 min). ‡ An extended reaction time of 48 hours was used.

16


The Versatility of Aldehyde 28 in Preparing Analogues of 1

Further analogues of 1 were obtained by capitalising on the versatility of the formyl

moiety in organic synthesis (Scheme 1.11).

O

O

O CHO

O

O

O CH2OR

O

O

O CH2OH

O

O

O CH2NMe2

O

O

O CHF2

O

O

O

O

O

O

O

O

O CN

Br

Br

O

O

O

28

1 4

45

48

42

14344

R = MeR = Et

46

47

a

b

cd

e

f

g

N

1

OH

Scheme 1.11 a) Me2NH2Cl, NaBH3CN, MeOH; b) ButNH2.BH3, CH2Cl2;

c) MeI or EtBr, Ag2O, CH2Cl2; d) DAST, CH2Cl2; e) HONH3Cl, MeOH;

f) SOCl2, Et3N, CH2Cl2; g) CBr4, Ph3P, Zn, CH2Cl2.

Reductive amination of 28 with dimethylammonium chloride returned the amine 41.

Treatment of aldehyde 28 with tert-butylamine-borane returned the alcohol 42. This

alcohol was elaborated further, providing both the methyl 43 and ethyl 44 ethers.

Prolonged treatment of 28 with DAST produced the difluoromethyl analogue 45.

17

Chapter One

Condensation of 28 with hydroxylamine gave but one product, the oxime 46. The

stereochemistry of 46 was determined by a single-crystal X-ray diffraction experiment

(Figure 1.3). Thionyl chloride was utilised in the dehydration of 46 to provide the

nitrile 47. Olefination of the aldehyde 28 using Corey’s procedure yielded the

dibromoalkene 48; all attempts at converting this olefin into an alkyne through

treatment with strong lithium bases went unrewarded.[29]

Figure 1.3 Molecular projection of the oxime 46.

Germination Promoting Activity of the Various Analogues

The synthetic endeavours detailed in this chapter produced twenty-nine novel analogues

of 1. Of these molecules, twenty-three (6, 27−48) differed from 1 in substitution at C3

and/or C5, whilst the remaining six were dihydro- (17, 20) or tetrahydro- (14, 16, 19,

18


26) analogues. The majority of these compoundsD were evaluated as germination

stimulants on the seeds of Solanum orbiculatum.[30] This plant, commonly known as the

‘bush tomato’ owing to the striking resemblance that its seeds bear to those of the

domestic tomato Solanum lycopersicum, is a smoke-responsive species native to

Western Australia. Solanum orbiculatum is an ideal species on which to test these

analogues as it is particularly sensitive to the stimulatory effects of 1 and provides very

low control germination yields; the percentage of seeds that germinate in the absence of

1.[30]

The germination-promoting activity of compounds 6, 14, 17, 27, 28 and 30−47 was

determined using the procedure of Flematti et al. (Figure 1.4).[30] Full details are,

naturally, provided in the Experimental section of this chapter, though, in the interests

of clarifying the data presented in Figure 1.4, a summary of the procedure is warranted.

Briefly, Solanum orbiculatum seeds were treated with solutions of each compound at

five concentrations (1 ppb, 10 ppb, 100 ppb, 1 ppm and 10 ppm) and stored for six days.

The percentage of seeds that had germinated after this time was determined for each

compound at each concentration (performed in triplicate). A seed was considered to

have germinated if a root radicle had broken through the seed coat. Both positive (1 at

100 ppb) and negative (water) control experiments were used in each assay.

Note that the negative control germination yield for some compounds in Figure 1.4

appears a great deal higher than for others. This is due to the phenomenon of ‘after

ripening’ − the tendency for germination yields to become progressively better over a

period of months to years after harvesting.[31] The data presented here were not acquired

D A number of the analogues proved to be too insoluble (16, 19, 20, 29 and 48) or unstable (26 and 48) in water to provide meaningful germination data.

19

Chapter One

Figure 1.4 Germination data for the analogues.

20


Figure 1.4 … continued…

21

Chapter One

Figure 1.4 … continued.

simultaneously but collected in a continuous manner throughout the progression of this

work and so later tests had higher control levels of germination.

Inspection of the data presented in Figure 1.4 reveals several trends.

Compounds with electron-withdrawing groups at C3 appear to have greatly diminished

activity. The nitrile 47 is, arguably, inactive whilst the nitro compound 34 and ketones

31−33 illicit only a marginal response at relatively high concentration (10 ppm). The

aldehyde 28, oxime 46 and difluoromethyl 45 analogues were also much less active than

the natural stimulant 1, promoting germination effectively only at the highest

concentration tested (10 ppm). Given the similar Van der Waals radii of fluorine and

hydrogen, the relatively poor activity of the difluoromethyl analogue 45 with respect to

the natural stimulant 1 is particularly telling of this electronic effect.

22


The natural stimulant 1 is at least two orders of magnitude more active than any other

compound tested. Any increase in the size of the alkyl substituent at C3 appears to

decrease the potency of the molecule, as observed for the ethyl 35 and propyl 36

derivatives. In the absence of the methyl group the germination response is also

diminished, as observed for compound 6. Thus, it would appear that a methyl group at

C3 is optimal for germination-promoting activity.

Four of the six analogues with substitution at C5 demonstrated very poor germination-

promoting activity. It may well be that substitution at this position is not well tolerated,

although it is perhaps premature to make such a statement given the limited number of

C5 analogues tested here. Indeed, other work has suggested that substitution at C5 does

not impede activity greatly,[30] suggesting that it may be the nature of the groups at C5

that diminish the activity of these compounds.

The dihydro analogue 17 and the tetrahydro analogue 14 failed to promote germination

at any concentration. This suggests that the complete unsaturated system of 1 may be

required for activity.

The germination data in Figure 1.4 demonstrate the strong dependence germination

activity has on the likeness of the analogue to 1. It could well be that Solanum

orbiculatum is unusually selective for 1, more so than other smoke-responsive plants.

However, given that these plants have, through evolution, adopted this molecule as a

cue for an event as important as germination, it is not surprising that they should exhibit

a high degree of fidelity towards it.

23

Chapter One

Epilogue

Since the publication of the work presented in this chapter,[16] two other syntheses of 1

have been reported.[32, 33] Whilst these more recent syntheses utilised fewer steps in

preparing 1 than the method described here, they did so with markedly lower global

yields and diminished opportunity for analogue preparation.

For a period of time during the research described in this chapter, an effort was made by

DuPont (in collaboration with the author and others) to develop 1, or an analogue

thereof, into a marketable product. Alas, this goal was never realised owing to the fact

that 1, and its analogues, failed to increase the germination yields for many of the major

crop species.E Without widespread agricultural use, the ‘projected market size’ of 1

proved to be too small to sustain the interest of agrochemical companies. The

advantages 1 may yet offer to the cultivation of ‘economically unimportant’ plant

species, weed control and land restoration remains largely unexplored.

The mode of action of 1 has yet to be investigated. Given the common role and features

of 1 and (+)-strigol 49, a potent germination stimulant of parasitic weeds belonging to

the genera Striga and Orobanche,[10] it is possible that they act in similar ways.

O O

O OOOH

O

O

O

1 49

E Compound 1 did not increase the germination yield of the common wheat, maize, rice, canola or cotton varieties.

24


Both germination stimulants 1 and 49 contain a 3-methylbutenolide moiety and an α,β-

unsaturated lactone in conjugation with an enol ether. These features have, through

structure-activity relationship studies, been found to be essential to the activity of 49

and a mechanism by which 49 might act has been proposed (Figure 1.5).[34, 35] The

mechanism involves Michael addition of a nucleophile to the enol ether, followed by

expulsion of an alkoxide that tautomerises upon departure.

O O

O OO

NuO O

O OO

Nu

O O

Nu

CO2HOHC

H+

Figure 1.5 A rationalisation proposed for the action of (+)-strigol.[35]

A similar, albeit entirely conjectural, rationalisation could be proposed for the action of

1 (Figure 1.6). For 1, nucleophilic attack would be expected to occur at either C5 or C7,

though one might expect attack to proceed more readily at C7 given that the

intermediate may have some aromatic (furan) character. Expulsion of enolate ion,

protonation and tautomerisation would complete the analogy.

O

O

O

NuO

O

O

O

O

ONuNu

H+

Figure 1.6 One of many possible rationalisations for the action of 1.

Of course many modes-of-action are possible for 1 and the challenge remains to

construct a proposal based solely on empirical evidence rather than analogy; a task that

may be made simpler if 1 does indeed act on its target irreversibly.

25

Chapter One

26


Experimental

General

1H and 13C nuclear magnetic resonance (NMR) spectra were obtained with a Bruker

AM 300 (300.13 MHz for 1H and 75.5 MHz for 13C), a Bruker ARX500 (500.13 MHz

for 1H and 125.8 MHz for 13C) or a Bruker AV600 (600.13 MHz for 1H and 150.9 MHz

for 13C) spectrometer. Unless stated otherwise, 1H NMR spectra were calibrated using

the residual solvent peak and 13C NMR spectra were calibrated using the most

prominent solvent 13C resonance.[36] NMR spectra run in D2O used internal MeOH [1H,

δ = 3.34 (CH3) and 13C, δ = 49.0] as the standard.

Melting points were determined on a Reichert hot stage melting point apparatus. Optical

rotations were performed with a Perkin-Elmer 141 Polarimeter in a microcell (1 ml, 10

cm path length) at a concentration of 10 mg/ml in CHCl3, unless otherwise stated, at

room temperature. Mass spectra were recorded with a VG-Autospec spectrometer using

the fast atom bombardment (FAB) technique, with 3-nitrobenzyl alcohol as a matrix,

unless otherwise stated. Single-crystal X-ray investigations were conducted on a Bruker

AXS instrument at temperatures of 153 K or 298 K.

Flash chromatography was performed on BDH silica gel or Geduran silica gel 60 with

the specified solvents. Thin layer chromatography (t.l.c.) was effected on Merck silica

gel 60 F254 aluminium-backed plates that were stained by heating (>200˚C) with 5%

sulfuric acid in EtOH. Occasionally other stains were used, including: 2% w/v ninhydrin

in EtOH, 0.5% w/v dinitrophenylhydrazine in HCl (2 M), 10% w/v cerium(IV) sulfate in

H2SO4 (2 M) and iodine (as solid iodine crystals in a sealed tank).

27

Chapter One

Percentage yields for chemical reactions as described are quoted only for those

compounds that were purified by recrystallisation or by column chromatography and the

purity assessed by 1H NMR spectroscopy

All solvents except DMF and MeCN were distilled prior to use and dried according to

the methods of Burfield.[37]

‘Standard workup’ refers to dilution with water, repeated extraction into an organic

solvent, sequential washing of the combined organic extracts with HCl (1 M, where

appropriate), sat. aq. NaHCO3 and NaCl solutions, followed by drying over anhydrous

magnesium sulfate, filtration and evaporation of the solvent by means of a rotary

evaporator at reduced pressure.

Other General Procedures

Procedure A – Formylation: Phosphoryl chloride (0.70 ml, 7.5 mmol) was added

dropwise to the substrate (0.50 mmol) in DMF (3 ml) and the solution stirred at the

given temperature for the given period of time. The cooled solution was diluted with

CH2Cl2 (5 ml), poured onto sat. aq. NaHCO3 (30 ml) and stirred (15 min). The mixture

was extracted with CH2Cl2 (3 × 10 ml), the combined organic layers dried (MgSO4),

filtered and concentrated. Flash chromatography gave the aldehyde.

Procedure B – Acylation: Aluminium(III) chloride (0.67 g, 5.0 mmol) was added to the

acid chloride (2.5 mmol) and the substrate (0.50 mmol) in CH2Cl2 (4 ml) and the

mixture stirred at room temperature for the given period of time. The mixture was

cooled to 0˚C and HCl (10 ml, 1 M) was added dropwise with stirring. The mixture was

28


extracted with CH2Cl2 (3 × 10 ml), the combined organic layers dried (MgSO4), filtered

and concentrated. Flash chromatography gave the ketone.

Procedure C – Reduction: Aluminium(III) chloride (0.12 g, 0.90 mmol) was added to

ButNH2.BH3 (0.16 g, 1.8 mmol) and the substrate (0.30 mmol) in CH2Cl2 (6 ml) and the

mixture heated at reflux (20 min). Additional AlCl3 (40 mg, 0.30 mmol) was added

periodically (every 10 min) until the reaction was complete (t.l.c.). The mixture was

cooled to 0˚C and HCl (10 ml, 1 M) was added dropwise with stirring. The mixture was

extracted with CH2Cl2 (3 × 10 ml), the combined organic layers dried (MgSO4), filtered

and concentrated. Flash chromatography gave the product.

O

O

O

HO

TrO

10

O

O

O

TrOO

O

O

TrO

CO2EtEtO2C

O

O

O

O

TrO

11 12 13

(E)- and (Z)-3-Deoxy-3-C-[(ethoxycarbonyl)methylene]-1,2-O-isopropylidene-5-O-

triphenylmethyl-α-D-erythro-pentose, 13 and 12

Triethyl phosphonoacetate (4.0 ml, 20 mmol) was added dropwise to NaH (0.80 g, 20

mmol, 60% dispersion in mineral oil) in THF (20 ml) at –10˚C, and the mixture stirred

(15 min). The crude ketone 11 [obtained from the alcohol 10[14] (4.3 g, 10 mmol)] in

THF (20 ml) was added dropwise and the red solution stirred (15 min). Concentration of

the mixture and a standard workup (EtOAc) was followed by flash chromatography

(EtOAc/petrol, 1:19). The (E)-isomer 13 was first to elute; it was obtained as a

colourless oil (0.25 g, 5%), [α]D +186˚. 1H NMR (500 MHz, CDCl3) δ = 1.22 (ap. t, 3

H, J = 7.1 Hz, CH2CH3), 1.44, 1.50 (2 s, 6 H, C(CH3)2), 3.43 (dd, 1 H, J = 2.0, 9.9 Hz,

H-5), 3.53 (dd, 1 H, J = 2.4, 9.9 Hz, H-5), 4.00−4.09 (m, 2 H, CH2CH3), 5.31 (ddd, 1 H,

29

Chapter One

J = 1.9, 1.9, 4.6 Hz, H-2), 5.61 (dddd, 1 H, J = 1.9, 1.9, 2.0, 2.4 Hz, H-4), 6.09 (dd, 1 H,

J = 1.9, 1.9 Hz, =CH), 6.26 (d, 1 H, J = 4.6 Hz, H-1), 7.22−7.39 (m, 15 H, Ph); 13C

NMR (125.8 MHz, CDCl3) δ = 14.2 (CH2CH3), 27.9, 28.0 (C(CH3)2), 60.5 (CH2CH3),

66.0 (C-5), 81.2, 82.6 (C-2,4), 87.3 (CPh3), 104.7 (C-1), 113.5 (C(CH3)2), 116.8 (=CH),

127.2-143.8 (Ph), 160.1 (C-3), 165.2 (C=O). HRMS (EI): m/z = 500.2196; [M]+•

requires 500.2199.

Next to elute was the (Z)-isomer 12; it was obtained as a colourless oil (3.7 g, 74%),

[α]D +96.3˚. 1H NMR (500 MHz, CDCl3) δ = 1.32 (ap. t, 3 H, J = 7.1 Hz, CH2CH3),

1.48, 1.54 (2 s, 6 H, C(CH3)2), 3.27 (dd, 1 H, J = 4.0, 10.0 Hz, H-5), 3.42 (dd, 1 H, J =

4.1, 10.0 Hz, H-5), 4.25 (ap. q, 2 H, J = 7.1 Hz, CH2CH3), 4.98 (dddd, 1 H, J = 4.0, 4.1,

1.7, 1.7 Hz, H-4), 5.75−5.78 (m, 2 H, H-2,=CH), 6.08 (d, 1 H, J = 4.0 Hz, H-1),

7.24−7.48 (m, 15 H, Ph); 13C NMR (125.8 MHz, CDCl3) δ = 14.2 (CH2CH3), 27.3, 27.6

(C(CH3)2), 60.7 (CH2CH3), 65.6 (C-5), 78.8, 79.8 (C-2,4), 87.1 (CPh3), 105.5 (C-1),

113.0 (C(CH3)2), 116.7 (=CH), 127.2−143.6 (Ph), 156.4 (C-3), 165.0 (C=O). HRMS

(EI): m/z = 500.2178; [M]+• requires 500.2199.

(4S,7S,7aR)-4,7-Dihydroxy-4,5,7,7a-tetrahydro-2H-furo[2,3-c]pyran-2-one 14

Trifluoroacetic acid / H2O (5 ml, 4:1) was added to the alkene 12 (0.50 g) in CH2Cl2 (3

ml) and the yellow solution held at room temperature (5 min). The solvent was

removed, H2O added and the aqueous solution washed with EtOAc. Concentration of

the aqueous layer and recrystallisation of the residue gave the butenolide 14 as

colourless needles (0.14 g, 83%), m.p. 184−185.5˚C (MeOH), [α]D +168˚ (H2O). 1H

NMR (500 MHz, (CD3)2SO) δ = 3.42 (dd, 1 H, J = 10.1, 10.1 Hz, H-5), 3.75 (dd, 1 H, J

= 7.5, 10.1 Hz, H-5), 4.50 (dddd, 1 H, J = 1.4, 5.9, 7.5, 10.1 Hz, H-4), 4.94 (ddd, 1 H, J

= 0.8, 1.4, 4.6 Hz, H-7a), 5.43 (dd, 1 H, J = 4.6, 4.8 Hz, H-7), 5.87 (dd, 1 H, J = 1.4, 1.4

30


Hz, H-3), 5.89 (d, 1 H, J = 5.9 Hz, 4-OH), 6.97 (dd, 1 H, J = 0.8, 4.8 Hz, 7-OH); 13C

NMR (125.8 MHz, (CD3)2SO) δ = 62.6 (C-5), 65.5, 78.4 (C-4,7a), 90.3 (C-7), 111.1 (C-

3), 170.3 (C-3a), 172.8 (C-2). HRMS (FAB): m/z = 173.0449; [M + H]+ requires

173.0450.

O

O

O

OH

HO

14

O

O

O

OAc

AcO O

O

O

OAc

16 17

(4S,7R,7aR)-4,7-Diacetoxy-4,5,7,7a-tetrahydro-2H-furo[2,3-c]pyran-2-one 16

Acetic anhydride (0.76 ml, 8.0 mmol) was added to the butenolide 14 (0.34 g, 2.0

mmol) in C5H5N (8 ml) and the mixture stirred (2 h). Methanol (1 ml) was added and

the solution kept at room temperature (10 min). Concentration of the mixture and a

standard workup (EtOAc) followed by flash chromatography (EtOAc/petrol, 1:3) gave

the diacetate 16 as a colourless oil (0.49 g, 95%), [α]D +188˚. 1H NMR (500 MHz,

CDCl3) δ = 2.06, 2.18 (2 s, 6 H, OCOCH3), 3.54 (dd, 1 H, J = 10.1, 10.4 Hz, H-5), 4.16

(dd, 1 H, J = 7.2, 10.4 Hz, H-5), 5.02 (dd, 1 H, J = 1.4, 4.6 Hz, H-7a), 5.69 (ddd, 1 H, J

= 1.4, 7.2, 10.1 Hz, H-4), 6.01 (dd, 1 H, J = 1.4, 1.4 Hz, H-3), 6.52 (d, 1 H, J = 4.6 Hz,

H-7); 13C NMR (125.8 MHz, CDCl3) δ = 20.6, 20.7 (2 C, OCOCH3), 62.6 (C-5), 66.2,

76.5 (C-4,7a), 88.8 (C-7), 114.0 (C-3), 161.5 (C-3a), 168.5, 169.3, 171.2 (3 C, C-

2,C=O). HRMS (FAB): m/z = 257.0664; [M + H]+ requires 257.0661.

(4S)-4-Acetoxy-4,5-dihydro-2H-furo[2,3-c]pyran-2-one 17

Triethylamine (1 ml) was added to the diacetate 16 (256 mg) in CH2Cl2 (5 ml) and the

solution kept at room temperature (5 min). Concentration of the mixture and flash

chromatography (EtOAc/petrol, 1:3) gave the butenolide 17 as a pale yellow oil (184

mg, 94%), [α]D +84.7˚. 1H NMR (500 MHz, CDCl3) δ = 2.13 (s, 3 H, OCOCH3), 4.19

31

Chapter One

(dd, 1 H, J = 3.5, 12.6 Hz, H-5), 4.33 (dd, 1 H, J = 4.1, 12.6 Hz, H-5), 5.84 (ddd, 1 H, J

= 0.7, 3.5, 4.1 Hz, H-4), 5.92 (dd, 1 H, J = 0.7, 1.8 Hz, H-3), 7.07 (d, 1 H, J = 1.8 Hz,

H-7); 13C NMR (125.8 MHz, CDCl3) δ = 20.7 (OCOCH3), 62.7 (C-4), 69.3 (C-5), 109.5

(C-7), 133.1 (C-3), 138.3 (C-7a), 145.5 (C-3a), 168.8, 169.8 (C-2,C=O). HRMS (FAB):

m/z = 197.0448; [M + H]+ requires 197.0450.

O

O

AcO

OAc

AcO O

O

O

OCO2Et

EtO2CO O

O

O

OCO2Et

18 19 20

O

O

O

6

(4S,7R)-2,4,7-Triacetoxy-4,7-dihydro-5H-furo[2,3-c]pyran-2-one 18

Triethylamine (0.5 ml) was added to the butenolide 14 (86 mg, 0.50 mmol) and Ac2O

(0.19 ml, 2.0 mmol) in CH2Cl2 (2 ml) and the mixture stirred (30 min). Concentration of

the mixture and flash chromatography (EtOAc/petrol, 1:3) gave the furan 18 as a pale

yellow oil (0.14 g, 91%), [α]D +2.7˚. 1H NMR (600 MHz, CDCl3) δ = 2.05, 2.13, 2.27 (3

s, 9 H, OCOCH3), 4.18 (dd, 1 H, J = 6.4, 12.0 Hz, H-5), 4.41 (dd, 1 H, J = 3.9, 12.0 Hz,

H-5), 5.92 (ddd, 1 H, J = 1.1, 3.9, 6.4 Hz, H-4), 6.14 (dd, 1 H, J = 0.6, 1.1 Hz, H-3),

7.52 (d, 1 H, J = 0.6 Hz, H-7); 13C NMR (150.9 MHz, CDCl3) δ = 20.5, 20.6, 20.8 (3 C,

OCOCH3), 64.5 (C-5), 66.3 (C-4), 116.4 (C-3), 120.2 (C-7), 136.2 (C-7a), 153.6 (C-2),

166.4, 169.4, 170.5 (3 C, C=O), 166.7 (C-3a). HRMS (FAB): m/z = 299.0762; [M + H]+

requires 299.0767.

(4S,7R,7aR)-4,7-Bis(ethoxycarbonyloxy)-4,5,7,7a-tetrahydro-2H-furo[2,3-c]pyran-2-

one 19

Ethyl chloroformate (3.82 ml, 40.0 mmol) was added dropwise to the butenolide 14

(1.72 g, 10.0 mmol) in C5H5N (20 ml) at 0˚C and the mixture stirred (r.t., 1 h).

32


Concentration of the mixture and a standard workup (EtOAc) followed by flash

chromatography (EtOAc/petrol, 1:3) gave the carbonate 19 as colourless needles (2.94

g, 93%), m.p. 121−124˚C (EtOAc/petrol), [α]D +120˚. 1H NMR (300 MHz, CDCl3) δ =

1.24−1.35 (m, 6 H, CH2CH3), 3.66 (dd, 1 H, J = 10.1, 10.6 Hz, H-5), 4.15−4.28 (m, 5 H,

CH2CH3,H-5), 5.01 (ddd, 1 H, J = 0.8, 0.8, 4.6 Hz, H-7a), 5.56 (dddd, 1 H, J = 0.8, 1.9,

7.1, 10.1 Hz, H-4), 6.01 (dd, 1 H, J = 0.8, 1.9 Hz, H-3), 6.37 (d, 1 H, J = 4.6 Hz, H-7);

13C NMR (75.5 MHz, CDCl3) δ = 14.1, 14.2 (2 C, CH2CH3), 62.4 (C-5), 65.2, 65.4 (2

C, CH2CH3), 69.0, 76.3 (C-4,7a), 92.0 (C-7), 114.5 (C-3), 152.9, 153.7 (2 C, OCO2),

160.3 (C-3a), 171.0 (C-2). HRMS (FAB): m/z = 317.0870; [M + H]+ requires 317.0873.

(4S)-4-Ethoxycarbonyloxy-4,5-dihydro-2H-furo[2,3-c]pyran-2-one 20

Triethylamine (5 ml) was added to the carbonate 19 (2.85 g) in CH2Cl2 (30 ml) and the

solution kept at room temperature (5 min). Concentration of the mixture and flash

chromatography (EtOAc/petrol, 1:3) gave the butenolide 20 as a pale yellow oil (1.93 g,

95%), [α]D +84.7˚. 1H NMR (500 MHz, CDCl3) δ = 1.31 (ap. t, 3 H, J = 7.1 Hz,

CH2CH3), 4.20 (dd, 1 H, J = 3.4, 12.7 Hz, H-5), 4.24 (ap. q, 2 H, J = 7.1, CH2CH3), 4.40

(dd, 1 H, J = 4.0, 12.7 Hz, H-5), 5.70 (ddd, 1 H, J = 0.8, 3.4, 4.0 Hz, H-4), 5.98 (dd, 1

H, J = 0.8, 1.8 Hz, H-3), 7.07 (d, 1 H, J = 1.8 Hz, H-7); 13C NMR (125.8 MHz, CDCl3)

δ = 14.2 (CH2CH3), 65.3 (CH2CH3), 65.9 (C-4), 69.2 (C-5), 109.9 (C-3), 133.3 (C-7),

138.2 (C-7a), 144.8 (C-3a), 154.1 (OCO2), 168.9 (C-2). HRMS (FAB): m/z = 227.0551;

[M + H]+ requires 227.0556.

2H-Furo[2,3-c]pyran-2-one 6

(a) Tetrakis(triphenylphosphine)palladium(0) (0.12 g, 0.10 mmol) was added to the

acetate 17 (98 mg, 0.50 mmol) in THF (4 ml) and the solution heated at reflux (48 h).

Concentration of the mixture and flash chromatography (EtOAc/petrol, 1:3) gave the

33

Chapter One

butenolide 6 as tan needles (42 mg, 61%), m.p. 109−110˚C (Pri2O). 1H NMR (600 MHz,

(CD3)2CO) δ = 5.40 (dd, 1 H, J = 0.5, 1.5 Hz, H-3), 6.91 (dd, 1 H, J = 0.5, 5.5 Hz, H-4),

7.72 (d, 1 H, J = 5.5 Hz, H-5), 7.93 (d, 1 H, J = 1.5 Hz, H-7); 13C NMR (150.9 MHz,

(CD3)2CO) δ = 90.8 (C-3), 105.6 (C-4), 129.5 (C-7), 144.2 (C-7a), 146.3 (C-3a), 151.2

(C-5), 170.6 (C-2). HRMS (EI): m/z = 136.0161; [M]+• requires 136.0160.

(b) Tetrakis(triphenylphosphine)palladium(0) (0.37 g, 0.32 mmol) was added to the

carbonate 20 (1.8 g, 8.0 mmol) in THF (20 ml) and the solution heated at reflux (8 h).

Concentration of the mixture and flash chromatography (EtOAc/petrol, 1:3) gave the

butenolide 6 as tan needles (915 mg, 84%). The m.p., HRMS data, 1H and 13C NMR

spectra agreed with those reported in (a).

O

OO

HO

O

OO

O

O

HO

MeO2C

BzOO

O

OMeO2C

BzO

CO2Et

O

O

OMeO2C

BzO

EtO2C

22 23 24 25

Methyl 5-O-Benzoyl-1,2-O-isopropylidene-α-D-glucuronate 23

Benzoyl chloride (4.2 ml, 36 mmol) was added dropwise to the lactone 22[24] (6.5 g, 30

mmol) in C5H5N (24 ml) at 0˚C and the mixture stirred (r.t., 30 min). Water (1.1 ml)

was added and the mixture stirred (30 min). Concentration of the mixture and a standard

workup (EtOAc) gave the crude benzoate as a colourless glass. Triethylamine (1.0 ml)

was added to the residue in MeOH (24 ml) at 0˚C and the mixture stirred (1 h).

Filtration of the mixture gave the methyl ester 23 as colourless needles (8.4 g, 79%),

m.p. 146−148.5˚C (MeOH), [α]D +18.9˚. 1H NMR (600 MHz, CDCl3) δ = 1.32, 1.51 (2

s, 6 H, C(CH3)2), 3.23 (bd, 1 H, J = 5.0 Hz, OH), 3.84 (s, 3 H, CO2CH3), 4.30−4.34 (m,

1 H, H-3), 4.54 (dd, 1 H, J = 2.8, 7.2 Hz, H-4), 4.57 (d, 1 H, J = 3.6 Hz, H-2), 5.55 (d, 1

H, J = 7.2 Hz, H-5), 5.98 (d, 1 H, J = 3.6 Hz, H-1), 7.44−8.08 (m, 5 H, Ph); 13C NMR

34


(150.9 MHz, CDCl3) δ = 26.4, 27.0 (C(CH3)2), 53.2 (CO2CH3), 70.6, 74.9, 79.7, 84.9

(C-2,3,4,5), 105.3 (C-1), 112.4 (C(CH3)2), 128.7-134.1 (Ph), 165.9, 169.3 (C-6,C=O).

HRMS (FAB): m/z = 353.1231; [M + H]+ requires 353.1236.

Methyl (E)- and (Z)-5-O-Benzoyl-3-deoxy-3-C-[(ethoxycarbonyl)methylene]-1,2-O-

isopropylidene-α-D-erythro-penturonate, 25 and 24

Acetic anhydride (4.7 ml, 50 mmol) was added to PDC (3.8 g, 10 mmol) and the methyl

ester 23 (3.5 g, 10 mmol) in CH2Cl2 and the mixture heated at reflux (1 h). The mixture

was concentrated and rapid silica gel filtration (EtOAc/petrol, 4:1) gave the crude

ketone as a pale green oil. Ethyl (triphenylphosphoranylidene)acetate (8.7 g, 25 mmol)

was added to the crude ketone in CH2Cl2 (40 ml) and the mixture stirred (2.5 h). The

solution was poured onto HCl (80 ml, 1 M) before a standard workup (EtOAc) and flash

chromatography (EtOAc/petrol, 1:4). The (E)-isomer 25 was first to elute; it was

obtained as a colourless oil (34 mg, 0.8%), [α]D +154˚. 1H NMR (600 MHz, CDCl3) δ =

1.25 (ap. t, 3 H, J = 7.1 Hz, CH2CH3), 1.38, 1.42 (2 s, 6 H, C(CH3)2), 3.71 (s, 3 H,

CO2CH3), 4.11−4.23 (m, 2 H, CH2CH3), 5.30 (ddd, 1 H, J = 1.7, 2.3, 2.3 Hz, H-4), 5.80

(d, 1 H, J = 1.7 Hz, H-5), 5.83 (d, 1 H, J = 4.7 Hz, H-1), 5.94 (ddd, 1 H, J = 2.3, 2.3, 4.7

Hz, H-2), 6.27 (dd, 1 H, J = 2.3, 2.3 Hz, =CH), 7.42−8.12 (m, 5 H, Ph); 13C NMR

(150.9 MHz, CDCl3) δ = 14.2 (CH2CH3), 27.7, 27.8 (C(CH3)2), 52.8 (CO2CH3), 61.0

(CH2CH3), 75.4, 81.3, 82.0 (C-2,4,5), 104.8 (C-1), 113.4 (C(CH3)2), 118.6 (=CH),

128.5−133.6 (Ph), 157.4 (C-3), 165.38, 165.44, 168.3 (3 C, C-6,C=O). HRMS (FAB):

m/z = 421.1518; [M + H]+ requires 421.1499.

Next to elute was the (Z)-isomer 24; it was obtained as colourless needles (3.7 g, 88%),

m.p. 95−96˚C (Pri2O), [α]D +158˚. 1H NMR (600 MHz, CDCl3) δ = 1.30 (ap. t, 3 H, J =

7.1 Hz, CH2CH3), 1.42, 1.46 (2 s, 6 H, C(CH3)2), 3.78 (s, 3 H, CO2CH3), 4.24 (ap. q, 2

35

Chapter One

H, J = 7.1 Hz, CH2CH3), 5.37−5.39 (m, 1 H, H-4), 5.60 (d, 1 H, J = 2.8 Hz, H-5), 5.80

(ddd, 1 H, J = 1.9, 1.9, 4.2 Hz, H-2), 5.89 (d, 1 H, J = 4.2 Hz, H-1), 5.98 (dd, 1 H, J =

1.9, 1.9 Hz, =CH), 7.43−8.04 (m, 5 H, Ph); 13C NMR (150.9 MHz, CDCl3) δ = 14.2

(CH2CH3), 27.4, 27.6 (C(CH3)2), 52.9 (CO2CH3), 60.9 (CH2CH3), 74.9, 78.9, 80.4 (C-

2,4,5), 106.1 (C-1), 113.4 (C(CH3)2), 118.2 (=CH), 128.7−133.8 (Ph), 154.1 (C-3),

164.5, 165.4, 167.2 (3 C, C-6,C=O). HRMS (FAB): m/z = 421.1510; [M + H]+ requires

421.1499.

O

O

O

OH

HO CO2Me

26

O

O

O

CO2Me

27

(4S,5S,7R/S,7aR)-4,7-Dihydroxy-5-methoxycarbonyl-4,5,7,7a-tetrahydro-2H-furo[2,3-

c]pyran-2-one 26

Sodium cyanide (0.12 g, 2.4 mmol) was added to the alkene 24 (3.4 g, 8.0 mmol) in

MeOH (30 ml) and the mixture stirred (5 h). Concentration of the mixture and a

standard workup (EtOAc) returned a brown gum. Trifluoroacetic acid / H2O (10 ml,

4:1) was added to this gum and the solution kept at room temperature (5 min). The

solvent was removed, H2O added and the aqueous solution washed with Et2O.

Evaporation of the aqueous layer (r.t.) and flash chromatography (EtOAc/petrol, 7:3)

gave the esters 26 as a colourless oil (1.5 g, 81%). 1H NMR (500 MHz, (CD3)2SO) δ =

3.73 (s, 3 H, β-CO2CH3), 3.74 (s, 3 H, α-CO2CH3), 3.81 (d, 1 H, J = 9.2 Hz, α-H-5),

4.04 (d, 1 H, J = 9.2 Hz, β-H-5), 4.53 (dd, 1 H, J = 6.7, 7.0 Hz, α-H-7), 4.57−4.64 (m, 2

H, α,β-H-4), 4.74 (dd, 1 H, J = 1.7, 7.0 Hz, α-H-7a), 5.08 (dd, 1 H, J = 1.8, 4.5 Hz, β-H-

7a), 5.54 (dd, 1 H, J = 4.5, 4.8 Hz, β-H-7), 5.97 (dd, 1 H, J = 1.8, 1.8 Hz, β-H-3), 6.01

36


(dd, 1 H, J = 1.7, 1.7 Hz, α-H-3), 6.31 (d, 1 H, J = 6.7 Hz, β-4-OH), 6.37 (d, 1 H, J =

6.1 Hz, α-4-OH), 7.43 (d, 1 H, J = 4.8 Hz, β-7-OH), 7.43 (d, 1 H, J = 6.7 Hz, α-7-OH);

13C NMR (125.8 MHz, (CD3)2SO) δ = 52.5 (2 C, α,β-CO2CH3), 67.5 (β-C-5), 68.0 (α-

C-5), 72.4, 78.1 (β-C-4,7a), 77.5, 81.5 (α-C-4,7a), 91.0 (β-C-7), 98.7 (α-C-7), 112.7 (2

C, α,β-C-3), 168.4−172.5 (6 C, α,β-C-2,3a,C=O). HRMS (FAB): m/z = 231.0500; [M +

H]+ requires 231.0505.

5-Methoxycarbonyl-2H-furo[2,3-c]pyran-2-one 27

Acetic anhydride (5.7 ml, 60 mmol) was added to the ester 26 (4.6 g, 20 mmol) in

C5H5N (30 ml) and the solution kept at room temperature (2 h). Methanol (2 ml) was

added and the solution kept at room temperature (15 min). The mixture was

concentrated, diluted with EtOAc (60 ml) and poured onto HCl (50 ml, 1 M). A standard

workup (EtOAc, without the sat. aq. NaHCO3 wash) gave a dark syrup that, after rapid

silica gel filtration (EtOAc/petrol, 1:1), returned a pale yellow gum. 1,8-

Diazabicyclo[5.4.0]undec-7-ene (7.5 ml, 50 mmol) was added dropwise to this gum in

CH2Cl2 (20 ml) and the dark mixture stirred (10 min). The mixture was diluted with

CH2Cl2 (40 ml) and poured onto HCl (50 ml, 1 M). The organic layer was washed with

H2O (3 × 50 ml), dried (MgSO4), filtered and concentrated before flash chromatography

(EtOAc/CH2Cl2, 3:97) gave the ester 27 as tan needles (2.7 g, 68%), sublimed

151−154˚C (CH2Cl2/petrol). 1H NMR (600 MHz, (CD3)2CO) δ = 3.95 (s, 3 H,

CO2CH3), 5.70 (d, 1 H, J = 1.5 Hz, H-3), 7.69 (d, 1 H, J = 0.5 Hz, H-4), 8.00 (dd, 1 H, J

= 0.5, 1.5 Hz, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 53.6 (CO2CH3), 94.8 (C-3),

109.3 (C-4), 128.7 (C-7), 144.2 (C-7a), 145.7, 147.8 (C-3a,CO2CH3), 160.6 (C-5), 170.2

(C-2). HRMS (EI): m/z = 194.0216; [M]+• requires 194.0215.

37

Chapter One

3-Formyl-2H-furo[2,3-c]pyran-2-one 28

The butenolide 6 (136 mg, 1.00 mmol) was treated according to Procedure A [50˚C, 15

min, flash chromatography (EtOAc/PhMe, 1:2)] to give the aldehyde 28 as tan needles

(151 mg, 92%), m.p. 216−217.5˚C (Pri2O). 1H NMR (600 MHz, (CD3)2CO) δ = 7.73 (d,

1 H, J = 5.1 Hz, H-5), 8.41 (d, 1 H, J = 5.1 Hz, H-4), 8.60 (s, 1 H, H-7), 9.82 (s, 1 H,

CHO); 13C NMR (150.9 MHz, (CD3)2CO) δ = 100.4 (C-3), 108.1 (C-4), 136.0 (C-7),

143.7 (C-7a), 147.8 (C-3a), 156.6 (C-5), 168.6 (C-2), 185.1 (CHO). HRMS (FAB): m/z

= 165.0194; [M + H]+ requires 165.0188.

O

O

O

28

O

O

O

6

O

O

O

CO2Me

29

CHO

O

O

O

CONMe2

30

CHO

3-Formyl-5-methoxycarbonyl-2H-furo[2,3-c]pyran-2-one 29

The butenolide 27 (97 mg, 0.50 mmol) was treated according to Procedure A [80˚C, 1 h,

flash chromatography (EtOAc/petrol, 1:2)] to give the aldehyde 29 as yellow needles

(100 mg, 90%), m.p. 158.5−159˚C (Et2O). 1H NMR (600 MHz, (CD3)2CO) δ = 4.03 (s,

3 H, CO2CH3), 8.27 (s, 1 H, H-4), 8.65 (s, 1 H, H-7), 9.87 (s, 1 H, CHO); 13C NMR

(150.9 MHz, (CD3)2CO) δ = 54.1 (CO2CH3), 102.7 (C-3), 110.2 (C-4), 135.3 (C-7),

143.9 (C-7a), 147.6, 151.9 (C-3a,CO2CH3), 159.8 (C-5), 168.2 (C-2), 185.4 (CHO).

HRMS (FAB): m/z = 223.0250; [M + H]+ requires 223.0243.

5-(Dimethylamino)carbonyl-2H-furo[2,3-c]pyran-2-one 30

Phosphoryl chloride (0.70 ml, 7.5 mmol) was added dropwise to the ester 27 (97 mg,

0.50 mmol) in Me2NCOMe (5 ml) and the solution stirred (120˚C, 36 h). The cooled

solution was diluted with CH2Cl2 (5 ml), poured onto sat. aq. NaHCO3 (30 ml) and

38


stirred (15 min). Standard workup (CH2Cl2) and flash chromatography (EtOAc/petrol,

7:3) gave the amide 30 as colourless needles (76 mg, 73%), m.p. 147−149˚C

(EtOAc/petrol). 1H NMR (600 MHz, (CD3)2CO) δ = 3.03, 3.12 (2 bs, 6 H, CON(CH3)2),

5.54 (d, 1 H, J = 1.5 Hz, H-3), 7.14 (d, 1 H, J = 0.5 Hz, H-4), 7.95 (dd, 1 H, J = 0.5, 1.5

Hz, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 35.6, 38.3 (CON(CH3)2), 92.8 (C-3),

105.5 (C-4), 128.3 (C-7), 143.9 (C-7a), 146.1 (C-3a), 154.2 (CON(CH3)2), 162.3 (C-5),

170.4 (C-2). HRMS (FAB): m/z = 208.0617; [M + H]+ requires 208.0610.

3-Acetyl-2H-furo[2,3-c]pyran-2-one 31

The butenolide 6 (69 mg, 0.50 mmol) was treated according to Procedure B [AcCl, 20

min, flash chromatography (EtOAc/petrol, 2:3)] to give the ketone 31 as colourless

needles (81 mg, 91%), m.p. 185−185.5˚C (petrol). 1H NMR (600 MHz, (CD3)2CO) δ =

2.42 (s, 3 H, CH3), 7.78 (d, 1 H, J = 5.1 Hz, H-5), 8.29 (d, 1 H, J = 5.1 Hz, H-4), 8.49

(s, 1 H, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 28.9 (CH3), 101.1 (C-3), 108.5 (C-

4), 135.0 (C-7), 143.4 (C-7a), 149.0 (C-3a), 155.8 (C-5), 168.3 (C-2), 193.4 (C=O).

HRMS (EI): m/z = 178.0265; [M]+• requires 178.0266.

O

O

O

32

O

O

O

31

O O

3-Propanoyl-2H-furo[2,3-c]pyran-2-one 32

The butenolide 6 (69 mg, 0.50 mmol) was treated according to Procedure B

[CH3CH2COCl, 20 min, flash chromatography (EtOAc/petrol, 1:2)] to give the ketone

32 as colourless plates (86 mg, 89%), m.p. 185−186˚C (petrol). 1H NMR (600 MHz,

(CD3)2CO) δ = 1.08 (t, 3 H, J = 7.3 Hz, CH2CH3), 2.87 (q, 2 H, J = 7.3 Hz, CH2CH3),

39

Chapter One

7.80 (d, 1 H, J = 5.1 Hz, H-5), 8.29 (d, 1 H, J = 5.1 Hz, H-4), 8.47 (s, 1 H, H-7); 13C

NMR (150.9 MHz, (CD3)2CO) δ = 8.1 (CH2CH3), 34.8 (CH2CH3), 100.7 (C-3), 108.5

(C-4), 134.8 (C-7), 143.5 (C-7a), 149.1 (C-3a), 155.6 (C-5), 168.1 (C-2), 196.7 (C=O).


3-Cyclopropylcarbonyl-2H-furo[2,3-c]pyran-2-one 33

The butenolide 6 (69 mg, 0.50 mmol) was treated according to Procedure B

[cyclopropylcarbonyl chloride, 40 min, flash chromatography (EtOAc/petrol, 1:3)] to

give the ketone 33 as colourless plates (87 mg, 85%), m.p. 239−241˚C (petrol). 1H

NMR (600 MHz, (CD3)2CO) δ = 0.92-1.05 (m, 4 H, CH2), 3.13-3.18 (m, 1 H, CH), 7.80

(d, 1 H, J = 5.1 Hz, H-5), 8.29 (d, 1 H, J = 5.1 Hz, H-4), 8.50 (s, 1 H, H-7); 13C NMR

(150.9 MHz, (CD3)2CO) δ = 11.3 (CH2), 18.2 (CH), 101.0 (C-3), 108.7 (C-4), 135.0 (C-

7), 143.3 (C-7a), 148.8 (C-3a), 155.8 (C-5), 168.6 (C-2), 196.0 (C=O). HRMS (EI): m/z

= 204.0414; [M]+• requires 204.0423.

3-Nitro-2H-furo[2,3-c]pyran-2-one 34

Sodium nitrate (0.21 g, 2.5 mmol) was added to the butenolide 6 (69 mg, 0.50 mmol) in

TFA (3 ml) and the mixture stirred (15 min). The dark mixture was diluted with CH2Cl2

(5 ml) and poured onto sat. aq. NaHCO3 (30 ml). Standard workup (CH2Cl2) and flash

chromatography (EtOAc/petrol, 1:1) gave the butenolide 34 as yellow plates (44 mg,

48%), m.p. 207−208˚C (Et2O). 1H NMR (600 MHz, (CD3)2CO) δ = 7.91 (dd, 1 H, J =

0.6, 5.0 Hz, H-5), 8.67 (d, 1 H, J = 5.0 Hz, H-4), 8.82 (d, 1 H, J = 0.6 Hz, H-7); 13C

NMR (150.9 MHz, (CD3)2CO) δ = 107.9 (C-3,4), 137.8 (C-7), 140.5 (C-7a), 144.2 (C-

3a), 158.2 (C-5), 159.3 (C-2). HRMS (EI): m/z = 181.0018; [M]+• requires 181.0011.

40


O

O

O

33

O

O

O

34

NO2O

O

O

O

28

O

O

O

35

O

O

O

1

O

O

O

36

CHO

3-Methyl-2H-furo[2,3-c]pyran-2-one 1

The aldehyde 28 (49 mg, 0.30 mmol) was treated according to Procedure C [flash

chromatography (EtOAc/petrol, 1:3)] to give the butenolide 1 as colourless needles (37

mg, 83%), m.p. 118−120˚C (petrol, lit.[11] 118−119˚C). The 1H and 13C NMR spectra

were consistent with those previously reported.[11]

3-Ethyl-2H-furo[2,3-c]pyran-2-one 35

The ketone 31 (53 mg, 0.30 mmol) was treated according to Procedure C [flash

chromatography (EtOAc/petrol, 1:3)] to give the butenolide 35 as a colourless oil (39

mg, 79%). 1H NMR (600 MHz, (CD3)2CO) δ = 1.13 (t, 3 H, J = 7.6 Hz, CH2CH3), 2.37

(q, 2 H, J = 7.6 Hz, CH2CH3), 6.85 (d, 1 H, J = 5.5 Hz, H-5), 7.63 (d, 1 H, J = 5.5 Hz,

H-4), 7.79 (s, 1 H, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 13.2 (CH2CH3), 17.1

(CH2CH3), 104.2 (C-4), 106.0 (C-3), 128.4 (C-7), 140.2 (C-7a), 143.1 (C-3a), 150.0 (C-

5), 170.8 (C-2). HRMS (EI): m/z = 164.0472; [M]+• requires 164.0473.

3-Propyl-2H-furo[2,3-c]pyran-2-one 36

The ketone 32 (58 mg, 0.30 mmol) was treated according to Procedure C [flash

chromatography (EtOAc/petrol, 1:4)] to give the butenolide 36 as a colourless oil (41

mg, 77%). 1H NMR (600 MHz, (CD3)2CO) δ = 0.91 (t, 3 H, J = 7.4 Hz, CH2CH2CH3),

1.57 (tq, 2 H, J = 7.4, 7.4 Hz, CH2CH2CH3), 2.33 (t, 2 H, J = 7.4 Hz, CH2CH2CH3),

6.84 (d, 1 H, J = 5.5 Hz, H-5), 7.63 (d, 1 H, J = 5.5 Hz, H-4), 7.78 (s, 1 H, H-7); 13C

41

Chapter One

NMR (150.9 MHz, (CD3)2CO) δ = 14.1 (CH2CH2CH3), 22.4 (CH2CH2CH3), 25.6

(CH2CH2CH3), 104.3 (C-4), 104.5 (C-3), 128.3 (C-7), 140.9 (C-7a), 143.1 (C-3a), 150.1

(C-5), 171.0 (C-2). HRMS (EI): m/z = 170.0628; [M]+• requires 170.0630.

5-Hydroxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one 37

The aldehyde 29 (0.11 g, 0.50 mmol) was treated according to Procedure C [flash

chromatography (EtOAc/petrol, 4:1)] to give the alcohol 37 as colourless needles (38

mg, 42%), m.p. 133.5−134.5˚C (Pri2O/petrol). 1H NMR (600 MHz, (CD3)2CO) δ = 1.87

(s, 3 H, CH3), 4.44 (dd, 2 H, J = 0.8, 6.2 Hz, CH2), 4.76 (t, 1 H, J = 6.2 Hz, OH), 6.78

(d, 1 H, J = 0.8 Hz, H-4), 7.74 (s, 1 H, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 7.6

(CH3), 61.3 (CH2), 99.4 (C-4), 99.7 (C-3), 127.4 (C-7), 141.8 (C-7a), 142.6 (C-3a),

162.5 (C-5), 171.4 (C-2). HRMS (FAB): m/z = 181.0499; [M + H]+ requires 181.0501.

O

O

O

29

O

O

O

37

O

O

O

27

O

O

O

38

CHO

CO2Me CO2Me CH2OH CH2OH

5-Hydroxymethyl-2H-furo[2,3-c]pyran-2-one 38

Aluminium(III) chloride (0.12 g, 0.90 mmol) was added to ButNH2.BH3 (0.16 g, 1.8

mmol) and the ester 27 (58 mg, 0.30 mmol) in CH2Cl2 (6 ml) and the mixture heated at

reflux (10 min). The mixture was cooled to 0˚C and HCl (10 ml, 1 M) was added

dropwise with stirring. Standard workup (CH2Cl2) and flash chromatography

(EtOAc/petrol, 4:1) gave the alcohol 38 as colourless needles (37 mg, 74%), m.p.

163−164.5˚C (Et2O). 1H NMR (600 MHz, (CD3)2CO) δ = 4.47 (dd, 2 H, J = 0.9, 6.2 Hz,

CH2), 4.82 (t, 1 H, J = 6.2 Hz, OH), 5.37 (d, 1 H, J = 1.5 Hz, H-3), 6.89 (dt, 1 H, J =

0.5, 0.9 Hz, H-4), 7.87 (dd, 1 H, J = 0.5, 1.5 Hz, H-7); 13C NMR (150.9 MHz,

42


(CD3)2CO) δ = 61.2 (CH2), 90.5 (C-3), 100.8 (C-4), 128.6 (C-7), 143.7 (C-7a), 147.4

(C-3a), 163.9 (C-5), 170.8 (C-2). HRMS (FAB): m/z = 167.0344; [M + H]+ requires

167.0344.

5-Chloromethyl-2H-furo[2,3-c]pyran-2-one 39

The ester 27 (58 mg, 0.30 mmol) was treated as above for the synthesis of the alcohol

38 except that the mixture was heated at reflux for 48 h. Flash chromatography

(EtOAc/petrol, 1:4) gave the chloride 39 as pale yellow needles (26 mg, 46%), m.p.

125−125.5˚C (petrol). 1H NMR (600 MHz, (CD3)2CO) δ = 4.64 (s, 2 H, CH2), 5.49 (d, 1

H, J = 1.5 Hz, H-3), 7.07 (d, 1 H, J = 0.4 Hz, H-4), 7.95 (dd, 1 H, J = 0.4, 1.5 Hz, H-7);

13C NMR (150.9 MHz, (CD3)2CO) δ = 42.4 (CH2), 92.5 (C-3), 104.7 (C-4), 129.2 (C-7),

143.7 (C-7a), 146.8 (C-3a), 158.0 (C-5), 170.6 (C-2). HRMS (EI): m/z = 183.9930;

[M]+• requires 183.9927.

O

O

O

40

O

O

O

39

CH2Cl CH2F

5-Fluoromethyl-2H-furo[2,3-c]pyran-2-one 40

Diethylaminosulfur trifluoride (80 µl, 0.60 mmol) was added to the alcohol 38 (33 mg,

0.20 mmol) in CH2Cl2 (4 ml) and the solution stirred (0˚C, 1 h). The solution was

diluted with CH2Cl2 (5 ml), poured onto sat. aq. NaHCO3 (10 ml) and stirred (15 min).

Standard workup (CH2Cl2) and flash chromatography (EtOAc/petrol, 1:4) gave the

fluoride 40 as colourless needles (29 mg, 87%), m.p. 99−99.5˚C (petrol). 1H NMR (600

MHz, (CD3)2CO) δ = 5.30 (d, 2 H, J = 46.8 Hz, CH2F), 5.50 (dd, 1 H, J = 0.8, 1.5 Hz,

H-3), 7.06 (d, 1 H, J = 2.2 Hz, H-4), 7.96 (d, 1 H, J = 1.5 Hz, H-7); 13C NMR (150.9

MHz, (CD3)2CO) δ = 80.8 (d, J = 169 Hz, CH2F), 92.5 (d, J = 1.8 Hz, C-3), 104.5 (d, J

43

Chapter One

= 6.5 Hz, C-4), 129.0 (C-7), 143.9 (C-7a), 146.6 (C-3a), 157.1 (d, J = 18.2 Hz, C-5),

170.6 (C-2). HRMS (FAB): m/z = 169.0300; [M + H]+ requires 169.0301.

3-(Dimethylamino)methyl-2H-furo[2,3-c]pyran-2-one 41

Dimethylammonium chloride (0.24 g, 3.0 mmol) was added to the aldehyde 28 (49 mg,

0.30 mmol) in MeOH (5 ml) and the mixture stirred (4 h). Sodium cyanoborohydride

(28 mg, 0.45 mmol) was added to the yellow solution and the mixture stirred (16 h).

Water (1 ml) was added and the mixture stirred (10 min). Concentration of the mixture

and flash chromatography (EtOAc:petrol:Et3N, 90:9:1) gave the amine 41 as a pale

yellow oil (36 mg, 62%). 1H NMR (600 MHz, (CD3)2CO) δ = 2.39 (s, 6 H, CH3), 3.46

(s, 2 H, CH2), 6.98 (d, 1 H, J = 5.5 Hz, H-5), 7.71 (d, 1 H, J = 5.5 Hz, H-4), 7.89 (s, 1 H,

H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 42.7 (CH3), 53.5 (CH2), 100.0 (C-3), 105.2

(C-4), 130.2 (C-7), 142.0 (C-7a), 143.9 (C-3a), 150.7 (C-5), 171.2 (C-2). HRMS (EI):

m/z = 193.0741; [M]+• requires 193.0739.

3-Hydroxymethyl-2H-furo[2,3-c]pyran-2-one 42

tert-Butylamine-borane (78 mg, 0.90 mmol) was added to the aldehyde 28 (49 mg, 0.30

mmol) in CH2Cl2 (4 ml) and the solution stirred (30 min). Concentration of the solution

and flash chromatography (EtOAc/petrol, 4:1) gave the alcohol 42 as colourless needles

(43 mg, 86%), m.p. 144˚C (decomp.) (Et2O). 1H NMR (600 MHz, (CD3)2CO) δ = 4.04

(t, 1 H, J = 5.6 Hz, OH), 4.43 (d, 2 H, J = 5.6 Hz, CH2), 7.01 (d, 1 H, J = 5.5 Hz, H-5),

7.70 (d, 1 H, J = 5.5 Hz, H-4), 7.89 (s, 1 H, H-7); 13C NMR (150.9 MHz, (CD3)2CO): δ

= 55.2 (CH2), 104.3 (C-3), 105.1 (C-4), 129.4 (C-7), 142.2, 143.2 (C-3a,7a), 150.4 (C-

5), 169.9 (C-2). HRMS (EI): m/z = 166.0268; [M]+• requires 166.0266.

44


3-Methoxymethyl-2H-furo[2,3-c]pyran-2-one 43

Methyl iodide (0.13 ml, 2.0 mmol) was added to the alcohol 42 (33 mg, 0.20 mmol) and

silver(I) oxide (0. 12 g, 0.50 mmol) in CH2Cl2 (5 ml) and the mixture heated at reflux

(16 h). The mixture was filtered through Celite and concentrated before flash

chromatography (EtOAc/petrol, 1:4) gave the methyl ether 43 as colourless needles (31

mg, 87%), m.p. 63−63.5˚C (petrol). 1H NMR (600 MHz, (CD3)2CO) δ = 3.30 (s, 3 H,

CH3), 4.25 (s, 2 H, CH2), 6.98 (d, 1 H, J = 5.5 Hz, H-5), 7.77 (d, 1 H, J = 5.5 Hz, H-4),

7.95 (s, 1 H, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 58.2 (CH3), 64.5 (CH2), 100.7

(C-3), 104.9 (C-4), 129.9 (C-7), 143.1, 143.5 (C-3a,7a), 151.1 (C-5), 170.0 (C-2).


O

O

O

41

O

O

O

42

CH2NMe2 CH2OH

O

O

O

43

CH2OMe

O

O

O

44

CH2OEt

O

O

O

28

CHO

3-Ethoxymethyl-2H-furo[2,3-c]pyran-2-one 44

Ethyl bromide (0.15 ml, 2.0 mmol) was added to the alcohol 42 (33 mg, 0.20 mmol) and

silver(I) oxide (0. 12 g, 0.50 mmol) in CH2Cl2 (5 ml) and the mixture heated at reflux

(20 h). The mixture was filtered through Celite and concentrated before flash

chromatography (EtOAc/petrol, 1:5) gave the ethyl ether 44 as colourless needles (28

mg, 72%), m.p. 78.5−79˚C (petrol). 1H NMR (600 MHz, (CD3)2CO) δ = 1.16 (t, 3 H, J

= 7.0 Hz, CH2CH3), 3.50 (q, 2 H, J = 7.0 Hz, CH2CH3), 4.29 (s, 2 H, CH2), 6.98 (d, 1 H,

J = 5.5 Hz, H-5), 7.75 (d, 1 H, J = 5.5 Hz, H-4), 7.94 (s, 1 H, H-7); 13C NMR (150.9

MHz, (CD3)2CO) δ = 15.5 (CH2CH3), 62.7 (CH2CH3), 66.3 (CH2), 101.1 (C-3), 104.9

(C-4), 129.7 (C-7), 143.0, 143.1 (C-3a,7a), 150.9 (C-5), 169.9 (C-2). HRMS (EI): m/z =

194.0576; [M]+• requires 194.0579.

45

Chapter One

3-Difluoromethyl-2H-furo[2,3-c]pyran-2-one 45

Diethylaminosulfur trifluoride (0.20 ml, 1.5 mmol) was added to the aldehyde 28 (49

mg, 0.30 mmol) in CH2Cl2 (4 ml) and the solution stirred (35˚C, 24 h). The solution was

diluted with CH2Cl2 (5 ml), poured onto sat. aq. NaHCO3 (30 ml) and stirred (15 min).

Standard workup (CH2Cl2) and flash chromatography (EtOAc/petrol, 1:4) gave the

difluoride 45 as tan needles (44 mg, 79%), m.p. 136−138˚C (decomp.) (petrol). 1H

NMR (600 MHz, (CD3)2CO) δ = 6.77 (t, 1 H, J = 54.8 Hz, CHF2), 7.18 (d, 1 H, J = 5.4

Hz, H-5), 8.07 (d, 1 H, J = 5.4 Hz, H-4), 8.28 (s, 1 H, H-7); 13C NMR (150.9 MHz,

(CD3)2CO) δ = 96.2 (t, J = 26.7 Hz, C-3), 105.3 (C-4), 112.7 (t, J = 231.8 Hz, CHF2),

133.0 (C-7), 142.8 (C-7a), 144.7 (t, J = 2.3 Hz, C-3a), 153.6 (C-5), 167.2 (t, J = 6.9 Hz,

C-2). HRMS (FAB): m/z = 187.0213; [M + H]+ requires 187.0207.

O

O

O

45

CHF2

O

O

O

46

O

O

O

47

CN

O

O

O

48

Br

Br

NOH

(E)-3-Hydroxyimino-2H-furo[2,3-c]pyran-2-one 46

Sodium acetate (49 mg, 0.60 mmol) was added to HONH3Cl (42 mg, 0.60 mmol) and

the aldehyde 28 (49 mg, 0.30 mmol) in MeOH and the mixture heated at reflux (2 h).

The mixture was concentrated and a standard workup (CH2Cl2) before flash

chromatography (EtOAc/petrol, 3:1) gave the oxime 46 as yellow prisms (43 mg, 80%),

m.p. 197˚C (decomp.) (Et2O). 1H NMR (600 MHz, (CD3)2CO) δ = 7.24 (d, 1 H, J = 5.3

Hz, H-5), 7.93 (s, 1 H, CHNOH), 7.97 (d, 1 H, J = 5.3 Hz, H-4), 8.16 (s, 1 H, H-7),

10.42 (s, 1 H, CHNOH); 13C NMR (150.9 MHz, (CD3)2CO) δ = 96.7 (C-3), 107.2 (C-4),

131.7 (C-7), 140.7 (C-7a), 142.1 (CHNOH), 143.7 (C-3a), 153.1 (C-5), 168.8 (C-2).


46


3-Cyano-2H-furo[2,3-c]pyran-2-one 47

Thionyl chloride (73 µl, 1.0 mmol) was added to Et3N (0.14 ml, 1.0 mmol) and the

oxime 46 (36 mg, 0.20 mmol) in CH2Cl2 (3 ml) at 0˚C and the mixture stirred (30 min).

The solution was diluted with CH2Cl2 (5 ml), poured onto sat. aq. NaHCO3 (30 ml) and

stirred (15 min). Standard workup (CH2Cl2) and flash chromatography (EtOAc/petrol,

1:4) gave the nitrile 47 as colourless needles (26 mg, 82%), m.p. 192−193˚C (Pri2O). 1H

NMR (600 MHz, (CD3)2CO) δ = 7.41 (d, 1 H, J = 5.2 Hz, H-5), 8.38 (d, 1 H, J = 5.2

Hz, H-4), 8.52 (s, 1 H, H-7); 13C NMR (150.9 MHz, (CD3)2CO) δ = 75.6 (C-3), 106.7

(C-4), 113.1 (CN), 134.9 (C-7), 143.3 (C-7a), 150.8 (C-3a), 155.8 (C-5), 166.7 (C-2).


3-(2,2-Dibromovinyl)-2H-furo[2,3-c]pyran-2-one 48

Tetrabromomethane (0.73 g, 2.2 mmol) was added to Ph3P (0.58 g, 2.2 mmol) and Zn

(0.14 g, 2.2 mmol) in CH2Cl2 (3 ml) and the mixture stirred (0˚C, 15 min). The

aldehyde 28 (0.12 g, 0.75 mmol) was added and the mixture stirred (3 h). The mixture

was filtered through Celite and concentrated before flash chromatography

(EtOAc/petrol, 1:9) gave the alkene 48 as yellow needles (0.10 g, 42%), m.p.

130−132˚C (Et2O/hexane). 1H NMR (600 MHz, (CD3)2CO) δ = 7.10 (d, 1 H, J = 5.5

Hz, H-5), 7.32 (s, 1 H, CHCBr2), 7.98 (d, 1 H, J = 5.5 Hz, H-4), 8.17 (s, 1 H, H-7); 13C

NMR (150.9 MHz, (CD3)2CO) δ = 91.5 (CHCBr2), 100.1 (C-3), 107.3 (C-4), 129.5

(CHCBr2), 131.5 (C-7), 140.6 (C-7a), 143.7 (C-3a), 151.8 (C-5), 167.9 (C-2). HRMS

(FAB): m/z = 318.8600; [M + H]+ requires 318.8605.

47

Chapter One

Evaluation of Germination Activity

The Solanum orbiculatum seeds used in the germination assay were collected in the

Shark Bay region (Western Australia) in November 2004 and were stored in the dark at

ambient conditions (ca. 23˚C and 50% relative humidity) until use. Millipore (MP)

water was obtained by filtration through a Milli-Q ultra-pure water system (Millipore,

Australia). MP water was used as a negative control for the experiment and 3-methyl-

2H-furo[2,3-c]pyran-2-one 1 (100 ppb) was used as a positive control.

Stock solutions of the compounds to be tested were prepared by dissolving between 1.5

and 2.0 mg of the compound in MP water to give a concentration of 10 mg.l-1.

Sonication was used to assist in the dissolution of some compounds. Five sequential

ten-fold dilutions of the stock gave solutions with concentrations of 10 ppm, 1 ppm, 100

ppb, 10 ppb and 1 ppb. For each of these, three 90 mm Petri dishes containing two

pieces of 70 mm Whatman No. 1 filter paper and 2.5 ml of the solution were prepared.

Approximately 20−25 seeds were placed on the wet filter papers in each Petri dish. The

Petri dishes were sealed with ParafilmTM and stored in a light proof container at 22˚C

for six days. The number of seeds that had germinated and the total number of seeds in

each dish were recorded, allowing the calculation of a mean germination percentage and

standard error for each compound at each concentration.

48

Chapter Two

Isofagomine-quercetin Conjugates as Putative

Inhibitors of a Glycosyltransferase

Chapter Two

50

Isofagomine-quercetin Conjugates as Putative Inhibitors of a Glycosyltransferase

Introduction

Many processes that are fundamental to life are dependant upon glycosylation and/or

deglycosylation events;[38-40] the many disease states that arise from deficiencies in the

enzymes that mediate these reactions stand as a testament to this fact.[41, 42] Glycoside

hydrolases (glycosidases, GHs), the enzymes that catalyse deglycosylation, have been

studied in some detail and the various mechanisms that they utilise are, for the most

part, well understood.[43-45] In contrast, the mechanisms of glycosyltransferases (GTs),

enzymes that catalyse the formation of glycosidic bonds, are not nearly as well defined.

The GTs are generally considered to be, with few exceptions, highly specific enzymes

that are dedicated to a single, well-defined task; the ‘one enzyme − one linkage’

dogma.[46] However, this notion has been challenged by the continual emergence of GTs

that flaunt an appreciable promisquity, be it in vivo or in vitro.[43, 47, 48]

Plants, Nature’s most accomplished chemists, use glycosylation extensively as a means

to modulate the bioactivity, stability and solubility of hormones, secondary metabolites

and environmental toxins.[49-51] It follows that plants should have, relative to other

organisms, a very large number of GTs. Indeed, as of June 2008, 445 putative

Arabidopsis thaliana (the model flowering plant) GTs were listed on the CAZy database

compared to just 230 for Homo sapiens.A[52] Plants, with a wealth of GTs that act on a

vast array of substrates, provide an excellent opportunity to investigate the structural

and evolutionary origins of GT substrate selectivity (or lack there-of).

A The CAZy database (Carbohydrate Active Enzymes database, http://www.cazy.org/) is an excellent, well maintained, open-access resource for glycobiologists that categorises the various carbohydrate-processing enzymes into families based upon their amino acid sequence.

51

Chapter Two

The first X-ray crystal structure of a plant GT was reported in 2005 for an enzyme of

family GT1 from Medicago truncatula (the model legume).[53] Shortly after, Davies and

co-workers disclosed the structure of a GT from the same family, a uridine diphospho-

glucose (UDP-Glc):flavonoid 3-O-glucosyltransferase from Vitis vinifera (grape).[48]

This enzyme (VvGT1) is responsible for the glucosylation of anthocyanidins, such as

cyanadin 50, to yield anthocyanins, such as glucoside 51 (Figure 2.1).B[54] Davies’

group showed that, at least in vitro, VvGT1 was quite promiscuous with respect to its

choice of acceptor; it was capable of glucosylating many different phenolic compounds.

O

OH

OH

HO

OHOHO O

OH

OHHO

O

OH

OH

HO

HO

HO

UDP-Glc

UDP

VvGT1

50 51

O

OH

OH

HO

HO

R

O

R = HR =

some synthetic carbohydrate chemistry...bsa bovine serum albumin conc. concentrated csa...

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