Application of Transition Metal-Mediated Conjugate Addition Reactions to the Synthesis of Novel Anti-Tumour Agents

Application of Transition Metal- Mediated Conjugate Addition Reactions to the Synthesis of Novel Anti-tumour Agents

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Engineering and Physical Sciences

2014

Stephania Christou

School of Chemistry Table of Contents Abstract ...... 6 Declaration and Copyright Statement...... 7 Acknowledgements ...... 8

1.0 Introduction ...... 9 1.1 Copper Catalysis ...... 10 1.1.1 The Background to Copper-catalysed Conjugate Additions ...... 10 1.2 Rhodium Catalysis ...... 13 1.2.1 Rhodium-catalysed Conjugate Addition of Organoboronic Acids ...... 13 1.2.2 Rhodium-catalysed Tandem Transformations with Organoboron Reagents...... 15 1.2.4 Development of Mild and Reusable Organosilanes ...... 16

2.0 Application of Conjugate Addition Reactions to the Synthesis of α-oxymethyl-α,β- cyclohex-2-enones ...... 20 2.1 Isolation of COTC ...... 21 2.2 The Glyoxalase System ...... 21 2.3 The Role of Glutathione ...... 22 2.4 Mechanism of Action of COTC and COMC...... 23 2.3 Natural Products Structurally Related to COTC ...... 26 2.3.1 The Antheminones and Carvotacetones ...... 26 2.3.2 Carvotacetone derivatives ...... 26 2.3.3 Antheminones ...... 28 2.3.4 The Phorbasins ...... 29 2.3.5 The Gabosines ...... 30 2.5 Previous Syntheses of COTC ...... 32 2.6 Previous work by the Whitehead Group ...... 34 2.6.1 Synthesis of Analogues from (-)-Quinic Acid ...... 35 2.6.2 Synthesis of Racemic Monohydroxylated and Trihydroxylated Analogues ...... 37 2.6.3 Cytotoxicity Assay Results ...... 39

3.0 Results and Discussion ...... 41 3.1 Project Aims ...... 41

2 3.2 Synthesis of Butane-1,2-Diacetal Protected Enone ...... 42 3.2.1 Butane-1,2-Diacetal Protection of (-)-Quinic Acid ...... 42 3.2.2 Completion of BDA-protected Enone Synthesis ...... 43 3.3 Synthesis of Cyclohexylidene Protected Enone ...... 44 3.4 Conjugate Addition Reactions ...... 46 3.4.1 Stereochemistry of the Conjugate Addition ...... 46 3.4.2 Chlorotrimethylsilane as an Additive in Organocuprate Reactions ...... 50 3.4.3 Organocuprate Conjugate Addition Reactions on BDA-Protected Enone ...... 52 3.4.4 Organocuprate Conjugate Addition Reactions on Cyclohexylidene-Protected Enone 35 ...... 55 3.4.5 Rhodium Catalysed Conjugated Addition Reactions of Boronic Acids ...... 57 3.4.6 Overview of Conjugate Addition Reactions ...... 62 3.5 Application of Conjugate Addition Reactions to Synthesis of Bioactive Natural Product Analogues...... 63 3.5.1 Elimination-Protection of the Cyclohexylidene Protected Adducts ...... 64 3.5.2 Perhydro-dibenzofuranone by-products ...... 68 3.5.3 Deprotection and Elimination of the BDA Protected Adducts ...... 71 3.5.4 Morita Bayllis Hillman Reaction ...... 74 3.5.5 Completion of the Synthesis of Novel Antitumour Agents ...... 77

4.0 Biological Results ...... 78 4.1 MTT Cell Viability Assay ...... 78 4.2 Assessing the Cytotoxicity of the Novel Compounds...... 79

4.3 Determination of the IC50 Concentrations ...... 80 4.4 Results of MTT Assays of the Novel Compounds ...... 80

5.0 GST/GSH-activated Prodrugs ...... 84 5.1 COMC-estradiol Conjugate as a Tissue Selective Prodrug ...... 85 5.2 Substituting the Crotonate ester with 4-Hydroxycoumarin Derivatives ...... 86 5.2.1 NAD(P)H: Quinone Oxidoreductase-1 (NQO1) ...... 86 5.2.2 NQO1 as a Target for Selective Antitumour Therapy ...... 88 5.2.3 Synthesis of NQO1 Inhibitor 253 ...... 90 5.2.4 GSH-activated prodrugs for the intracellular release of an NQO1 inhibitor ...... 91

3 6.0 Displacement of the Crotonate Side-chain by Thiols ...... 96

7.0 Conclusion and Future Work ...... 99 7.1 Transition Metal-mediated Conjugate Addition Reactions ...... 99 7.1.1 Conjugate Addition Reactions on BDA-protected Enone ...... 99 7.1.2 Conjugate Addition Reactions on the Cyclohexylidene-protected Enone ...... 100 7.2 Synthesis of Novel Anti-tumour Agents ...... 101 7.2.1 MTT viability assay ...... 102 7.2.2 Perhydro-dibenzofuranones Related to the Natural Product Incarviditone ...... 103 7.3 GSH-activated Prodrugs for Intracellular Release of NQO1 Inhibitor ...... 104

8.0 Experimental ...... 106 8.1 Experimental Procedures and Analysis of Compounds ...... 107

9.0 Biological Assays Methods and Materials ...... 175 9.1 Cell Culture ...... 175 9.2 Cell Storage ...... 175 9.3 Cell Thawing ...... 176 9.4 MTT Cell Viability Assay ...... 176 9.4.1 Counting and Seeding Cells...... 176 9.4.2 Treating the Cells ...... 177 9.4.3 Ending the viability assay ...... 178

References ...... 179 Appendix I ...... 184 Published work ...... 184 Appendix II ...... 186 MTT viability graphs for novel analogues 177-180 ...... 186 Appendix III ...... 189 X-ray Crystallography data ...... 189

Words: 48418

4 Abbreviations acac Acetylacetone Binap (2,2’-bisphenylphosphine)-1,1’-binaphthyl) δ Chemical Shift (delta) °C Degree Celsius 13C Carbon-13 COMC 2-Crotonyloxymethyl-cyclohex-2-enone COTC 2-Crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone CSA Camphorsulfonic Acid CTAB Cetrimonium bromide cod Cyclooctadiene DMSO Dimethylsulfoxide DCM Dichloromethane dppb 1,4-Bis(diphenylphosphino)butane dppe 1,2-Bis(diphenylphosphino)ethane dppp 1,4-Bis(diphenylphosphino)propane GSH Glutathione GST Glutathione transferase DABCO 1,4-Diazabicyclo[2.2.2]octane DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DMAP 4-(Dimethylamino)pyridine EtOAc Ethyl Acetate eq Equivalent(s) g Gram h Hour(s) 1H Proton-1 Hz Hertz J Coupling constant value MeOH Methanol min Minute(s) mL Millilitre NMR Nuclear Magnetic Resonance py Pyridine SDS Sodium Dodecyl Sulphate TBSOTf tert-Butyl Dimethylsilyl Trifluoromethanesulfonate TESOTf Triethylsilyl Trifluoromethanesulfonate TFA Trifluoroacetic acid TFP Tri(2-furyl)phosphine THF Tetrahydrofuran` TMSCl Trimethylsilyl Chloride

5 Abstract

The Streptomyces metabolite 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6- trihydroxycyclohex-2-enone (COTC), the antheminones and the carvotacetone derivatives are all bioactive natural products, whose structure is based on the α-oxymethyl-a,β-cyclohexenone moiety. Both COTC and antheminone A have been shown to exhibit cytotoxic and cancerostatic activity with low toxicity. The potent biological activity of these natural products has instigated numerous investigations into the synthesis of novel analogues in an attempt to determine the key structural features necessary for optimum bioactivity. The synthesis of a small library of novel anti-tumour agents which are structurally related to the natural products COTC and antheminone A is described, using the chiral pool material (-)-quinic acid as a starting material. At the outset, the aim of this project was to develop and optimise copper-mediated conjugate addition reactions and rhodium catalysed conjugate addition reactions of organoboron reagents to functionalised cyclic enones and subsequently, to apply the methodologies to the synthesis of the novel analogues. A range of novel mono-hydroxylated analogues bearing aryl side chains were prepared and their antiproliferative activity was assessed towards the A549 non- small cell cancer cell line. The biological assays revealed important structure-activity relationships and the most bioactive compound of this series had an IC50 value of 1.2 µM. In addition, the design and synthesis of a new class of GSH-activated prodrugs is described. These novel compounds are activated by GSH leading to intracellular release of an NQO1 inhibitor. The most potent compound of this new class of compounds had an IC50 value of 710 nm.

Declaration and Copyright Statement

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses.

Acknowledgements

Firstly, I am extremely grateful to Dr Roger Whitehead for giving me the opportunity to work in his group. His guidance, patience, and support throughout my time at the University of Manchester have been invaluable. Thanks also to Dr Peter Quayle for all the help and advice. I would also like to thank the members of the Whitehead and Quayle group, past and present, for their support and friendship (and cakes!) over the past few years. I would also like to thank Prof. Ian Stratford for allowing me to use his lab for the biological assays. Many thanks go to Joana and Elham for teaching me how to carry out the assays and were always happy to help. Also, thanks to Rehana and Gareth for all the help with mass spectroscopy and HPLC, and Dr Robin Pritchard for the X-ray crystallography data. A massive thanks has to go to my friends and family for their never ending support and love. A special thanks to my Mum for her unconditional love and always believing in me. Last but not least, a huge thank you to my Mikey for always being there; I couldn’t have done it without you.

1.0 Introduction

The transition metal catalysed conjugate addition reaction of organometallic reagents to electron-deficient olefins is one of the most important and reliable methods for carbon-carbon bond formation.1 C–C bond formation is fundamental for the synthesis of organic molecules; therefore the development of novel synthetic procedures for the selective formation of new carbon-carbon bonds is the focus of ongoing research. The main group organometallic reagents of boron and silicon are the most commonly employed reagents for the formation of C-C bonds.2 Organosilicon reagents have found many synthetic applications due to the fact that they are inexpensive, stable, nontoxic and environmentally benign. Also they are readily prepared and produce relatively safe and easily removed byproducts.2, 3 Organoboron reagents are also popular because they are stable and tolerant towards many functional groups, as well as being commercially available and easily handled.3 When compared to arylboronic acids, purification of arylsiloxanes is reasonably straight forward by distillation or chromatography.4 Traditionally, copper complexes have been applied to conjugate addition catalysis.5 More recently, owing to the surge in research into transition metal complexes as tools for many C-C bond forming processes, there has been a heightened interest into these alternative transition metal catalysts, such as palladium,6 rhodium,7 nickel8 and ruthenium9 complexes. Over the past two decades, numerous transition metal catalysed reactions have been developed to control the primary selectivity issues of conjugate addition processes:  Regioselectivity (1,2- vs. 1,4-addition)  Stereoselectivity (asymmetric formation of the new C–C bond). 1

1.1 Copper Catalysis

Of the many methods reported in the literature for the transition metal mediated conjugate addition of organometallic reagents, those involving catalytic amounts of Cu(I) have been developed furthest. The earliest example of a copper catalysed conjugate addition was described by Kharasch and co-workers in 1941. The traditional method for the preparation of stoichiometric organocopper reagents involved the transmetallation of an organolithium or a Grignard reagent.10 This method was revolutionised in 1993 when Alexakis and co-workers introduced dialkylzinc reagents for enantioselective conjugate addition reactions, and this procedure has since then become the literature precedent.5

1.1.1 The Background to Copper-catalysed Conjugate Additions

Organocopper(I) reagents are nucleophilic species which can effectively deliver anionic carbon nucleophiles such as alkyl and vinyl anions to electrophilic carbon centres. The first evidence of organocuprate chemistry was reported in 1941 by Kharasch and co-workers. It was observed that the reaction between a Grignard reagent and an α,β-unsaturated ketone 1 in the presence of 1 mol% of copper(I) chloride led to conjugate adduct 4 whereas, in the absence of the Cu(I) salt, the principal product was the 1,2-adduct 2 (Scheme 1).10

Scheme 1. Conjugate addition reaction of a Grignard reagent to an α,β-unsaturated ketone in the presence or in the absence of copper(I) chloride.

Organocopper reagents are formed by the addition of a main group organometallic, such as an organolithium or organomagnesium reagent, to a copper (I) salt (Scheme 2). In 1952, Gilman and colleagues reported that the monoorganocopper compound methyl copper which is formed from CuCl and one equivalent of MeLi, is insoluble in ether. Reaction of two equivalents of MeLi with the Cu(I) salt, however, resulted in a dimethylcuprate reagent which was soluble in ether.11 These results were also confirmed by House and co-workers through the reaction of CuI with MeLi.12

Scheme 2.13 Formation of organocopper reagents

The reactive organocopper species in conjugate addition reactions is the Gilman reagent, which is the soluble lithium diorganocuprate reagent with the stoichiometry of R2CuLi.12 Even though the reactive species is described as R2CuLi, this does not correspond to its actual structure or aggregation state.14 The composition and the aggregation state of the organocopper reagent can be affected by both the solvent and the salt used. There are, therefore, several structural possibilities for organocuprate reagents (Figure 1), but all of them are based on the stable linear R1-Cu-R2 arrangement.13, 15 Although organocuprate reagents are commonly synthesised from Grignard reagents, it is still uncertain if the real species in solution can be represented by the description R2CuMgX.13

Figure 1. 13 Structural possibilities of organocuprates

Synthetic applications of organocuprate reagents in conjugate addition reactions have outpaced mechanistic studies into the reactions. The reason for this is the complex nature of the organocuprate cluster structures.13, 16 In the 1970s, House and co-workers proposed a reaction mechanism which involved a single electron transfer (SET) from a dimer, leading to a CuIII intermediate.17, 18 This mechanism is no longer accepted because further studies have indicated that, although an organocopper(III) species is formed, this does not proceed via a radical intermediate.13 Spectroscopic and kinetic studies by Krauss and co-workers have revealed that the reaction proceeds via an organocuprate dimer-enone complex which is formed due to favourable lithium-carbonyl (hard-hard) and copper-olefin (soft-soft) interactions.19 This π complex unimolecularly rearranges to an organocopper(III) species. Rapid injection NMR studies carried out by Bertz and co-workers have proved that the mechanism proceeds through this organocopper(III) species.20 The rate determining step (RDS) of the reaction is the carbon-carbon bond formation by reductive elimination, which converts the CuIII to a CuI intermediate, generating RCuI and the β-alkylated enolate (Scheme 3).14, 21

Scheme 3. Mechanism of cuprate conjugate addition reaction.

1.2 Rhodium Catalysis

Over recent years, there has been a growing interest in rhodium-catalysed carbon-carbon bond forming reactions of organometallics. Rhodium catalysts represent an attractive alternative to other transition metal catalysts because they are insensitive to moisture, therefore reactions can be performed in the presence of water, either as a co-solvent or as the exclusive solvent. Rhodium catalysis shows complementary reactivity to other transition metal catalyst systems, under mild conditions, and tolerates various functional groups.7 The first reported example of rhodium-catalysed conjugate addition was published in 1997, by Miyaura and colleagues.22 Significant recent developments within the field of carbon-carbon bond formation reactions of organometallics, using rhodium catalysis, can trace their roots back to this seminal report by the Miyaura group. Today, high yielding achiral as well as enantioselective conjugate addition reactions can be successfully performed on a variety of substrate groups.7

1.2.1 Rhodium-catalysed Conjugate Addition of Organoboronic Acids

In 1997, Miyaura and Hayashi reported the first conjugate addition of aryl and alkenyl boronic acids to enones, catalysed by a rhodium(I) complex. The rhodium(I) complex was generated in situ from [Rh(acac)(CO)2] and an achiral phosphine ligand. A number of ligands were studied and it was concluded that bis(phosphine) ligands with large bite angles (dppb > dppp > TFP > dppe) were the most efficient. The reaction between methyl vinyl ketone 5 and phenylboronic acid 6, catalysed by the rhodium complex generated from Rh(acac)(CO)2 and 1,4- bis(diphenylphosphino)butane (dppb), gave a 99% yield of 4-phenylbutan-2-one 7.22

Scheme 4. Reaction between methyl vinyl ketone 5 and phenylboronic acid 6.

In 1998, Miyaura and co-workers published the first rhodium-catalysed asymmetric conjugate addition, which was a modification of the procedure used for the corresponding achiral reaction. Enantioselectivity was achieved by changing the rhodium catalyst precursor into Rh(acac)(C2H4)2, using (S)-binap as the chiral bisphosphine ligand, increasing the reaction temperature to 100 °C and using a 10:1 mixture of dioxane and water as a solvent. The reaction between 2-cyclohexenone 8 and 1.4 eq of phenylboronic acid, in the presence of 3 mol% of the rhodium catalyst was heated at 100 °C in dioxane and water (10:1) to give (S)-3-phenylcyclohexanone

9 in a 64% yield and 97% ee (Scheme 5). The relatively moderate yield is due to the formation of benzene byproduct by the proteolysis of phenylboronic acid at this elevated reaction temperature. When the reaction was attempted at 60 °C, however, the yield was less than 3%. To improve the yield a large excess of the boronic acid (2.5 eq) was used and this increased the yield to 93%.23

Scheme 5. The rhodium catalysed reaction between 2-cyclohexenone 5 and phenylboronic acid.

Detailed mechanistic studies of the rhodium-catalysed conjugate additions of aryl and alkenylboronic acids to α,β-unsaturated compounds were published in 2002 by Hayashi and his group (Scheme 6). Initially the rhodium bis(phosphine) complex is converted to the more reactive rhodium hydroxyl precursor 10 by loss of the acac ligands. It was suggested that this rhodium hydroxyl precursor 10 would react faster with the boronic acid than the corresponding acac complex, due to coordination of the hydroxyl group of the catalyst with the oxophilic boron. This interaction brings the two species closer together hence assisting transmetallation of the aryl group from boron to rhodium to give the rhodium-aryl complex 11. Coordination of complex 11 to the alkene leads to the regio- and enantioselective insertion of the alkene into the Rh-C bond: this results in the formation of a new carbon-carbon bond and the oxa-π-allylrhodium intermediate 12. Finally, hydrolysis of the rhodium

14 enolate affords the conjugate addition product 13 and regenerates the active catalyst 10.24

Scheme 6. Catalytic cycle for rhodium-catalysed conjugate additions of aryl and alkenylboronic acids to α,β-unsaturated compounds. 24

1.2.2 Rhodium-catalysed Tandem Transformations with Organoboron Reagents

Rhodium-catalysed tandem transformations triggered by the conjugate addition of organoboron reagents to activated alkenes followed by inter- or intramolecular trapping of the resulting enolate intermediates to give carbocycles and heterocycles represent efficient methods for the synthesis of complex molecules.25, 26

In 2003, Krishe and co-workers reported an intramolecular tandem conjugate addition - aldol cyclisation in which the two C-C bond forming reactions were catalysed by a rhodium complex in a single catalytic cycle. The procedure afforded five and six membered rings by the tandem cyclisation of keto-enones with

15 phenylboronic acid, in the presence of a catalytic amount of [RhCl(cod)]2 and (R)- BINAP and an excess of water.

Scheme 7. Intramolecular tandem conjugate addition - aldol cyclisation.

A more recent example is a Rh (I)-catalysed domino conjugate addition – the cyclisation reaction of α,β-unsaturated esters bearing amino and hydroxyl moieties to afford N- and O- heterocycles. Youn and co-workers reported the synthesis of 3,4- dihydroquinolin-2(1H)-ones and 3,4-hydrocoumarins, using various arylboroxines and [Rh(OH)(cod)]2 as a catalyst (Scheme 8).25

Scheme 8. Rh (I)-catalysed domino conjugate addition – cyclisation reaction.

1.2.4 Development of Mild and Reusable Organosilanes

Silicon-based reactions reported previously relied on the use of moisture sensitive organosilanediols and organotri(alkoxy)silanes reacting under relatively harsh conditions.27 Unlike these previous accounts, the methodologies described by Hiyama and co-workers using organo-[2-(hydroxymethyl)phenyl]dimethylsilane reagents involved milder conditions without the use of any activators. In 2007 Hiyama and co-workers introduced the conjugate addition reaction of organo-[2-(hydroxymethyl)phenyl]dimethylsilane reagents (20) to α,β-unsaturated ketones, in the presence of a rhodium catalyst. These tetraorganosilicon reagents

16 were initially developed by Hiyama and co-workers for fluoride-free cross-coupling reactions. The main advantage of this reaction is that the cyclic silyl ether 23 can be recovered from the reaction and reused - hence the reaction is efficient and “environmentally friendly”.3

Scheme 9. Conjugate addition of tetraorganosilicon reagent to an α,β-unsaturated ketone3

The organo-[2-(hydroxymethyl)phenyl]dimethylsilane reagents can be synthesised from the cyclic silyl ether, 1,1-dimethyl-2-oxa-1-silaindan (23) via a ring opening reaction using the corresponding Grignard reagent (Scheme 10). A diverse range of alkenyl and aryl-[2-(hydroxymethyl)phenyl]dimethylsilane reagents have been synthesised which proved to be both stable and reactive. 3

Scheme 10. Synthesis of the tetraorganosilicon reagent using the cyclic silyl ether.

The reaction mechanism suggested by the Hiyama group involves an initiation step between rhodium hydroxide and the tetraorganosilane reagent to afford organorhodium intermediate 25, followed by a conjugate addition reaction between α,β-unsaturated ketone and the R group in 26, to give rhodium enolate 27. Enolate 27 then reacts with the tetraorganosilane reagent 20 leading to the formation of conjugate addition product 28 and rhodium alkoxide 29. Finally, transmetallation of the R group from silicon to rhodium regenerates organorhodium intermediate 25 and the cyclic silyl ether 23.3

Scheme 11. Catalytic cycle of the organosilicon conjugate addition reaction 3

In 2012, a member of the Whitehead group investigated the rhodium catalysed conjugate addition reaction of organo-[2-(hydroxymethyl)phenyl] dimethylsilane reagents with the cyclohexylidene protected enone 35 (Scheme 14).28 The organosilane reagents were synthesised following the procedures reported by Hiyama and co-workers. Initially, 2-bromophenylmethanol was protected to give the THP-ether (31) in 92% yield. The protected intermediate 31 then underwent a lithium-halogen exchange followed by silylation to afford arylsilane 32 in 72% yield after purification. Reaction of silane 32 with p- toluenesulfonic acid afforded cyclic silyl ether 23.

Scheme 12. Synthesis of cyclic ether. Reagents and conditions: i) 3,4-dihydro-2H-pyran, conc. HCl, rt, 18 h, 92%; ii) BuLi, THF, -78°C, 90 mins then HSi(CH3)2Cl, rt, 18 h, 72%; iii) p-TSA.H2O, CH3OH, rt, 18 h, 66%.

Phenylsilane (34) and iso-propenylsilane (33) were synthesised via a ring opening reaction of the cyclic silyl ether 23 with the corresponding Grignard reagent.28

Scheme 13. Synthesis of phenylsilane and iso-propenylsilane

Phenylsilane (34) and iso-propenylsilane (33) were then employed in rhodium catalysed conjugate addition reactions with the cyclohexylidene protected enone 35. Both reactions afforded the corresponding conjugate adduct in reasonable yields, however, a much higher catalyst loading was required compared to that reported in Hiyama’s literature procedure.28

Scheme 14. Conjugate addition reactions of a phenyl and iso-propenyl group using tetraorganosilanes

2.0 Application of Conjugate Addition Reactions to the Synthesis of α-oxymethyl-α,β-cyclohex-2-enones

The transition metal catalysed conjugate addition reaction of organometallic reagents to electron-deficient olefins is one of the most reliable methods for the selective formation of new carbon-carbon bonds.1 The methodologies developed throughout the course of this project were applied to the synthesis of α-oxymethyl- α,β-cyclohex-2-enones, whose structure was related to the bioactive natural products COTC, antheminone A and phorbasin E. The key step in the synthesis of these compounds was the introduction of an alkyl or aryl substituent via a stereo- controlled conjugate addition reaction.

The α-oxymethyl-α,β-cyclohexenone moiety is present in several bioactive natural products, such as 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex- 2-enone (COTC, 38)29, carvotacetone derivatives (40)30 and antheminone A (41)31 (Figure 2). The significant toxicity of both COTC and antheminone A towards multiple cancer cell lines has prompted scientific interest in the synthesis of analogues of these natural compounds. COMC (39) is a simpler non-hydroxylated analogue of COTC, which was originally synthesised by Douglas and co-workers, and displayed a higher toxicity than COTC.32

Figure 2. COTC(38), COMC (39), Carvotacetone derivative (40), Antheminone A (41)

2.1 Isolation of COTC

COTC was isolated by Takeuchi and co-workers, in 1975, from cultures of the bacterium Streptomyces griseosporeus. The absolute configuration and relative stereochemistry of the compound was determined by X-ray crystallography.33 Takeuchi and co-workers reported the inhibition of glyoxalase enzymes, prepared from rat liver and yeast, by COTC. The inhibition was shown to be stronger in the presence of reduced glutathione (GSH). Further studies, had displayed that daily injections of COTC into mice intraperitoneally inoculated with cancer cells led to strong inhibition of growth of HeLa cells and EHRLICH ascites carcinoma and a weak inhibition of L-1210 leukaemia cells, with low toxicity.29 The chemical reactivity of COTC was investigated by carrying out reactions with the sulfhydryl compound, 2-mercaptoethanol. The experiments showed that the crotonate group in COTC was easily displaced by the 2-hydroxyethylthio group of 2-mercaptoethanol. This observation led to the conclusion that the biological activity of COTC was based on the simple nucleophilic displacement of the crotonyl group by sulfhydryl (-SH) compounds, such as GSH (Scheme 15).29

Scheme 15. Displacement of crotonyl group by sulfhydryl compounds.

2.2 The Glyoxalase System

The Glyoxalase system, first described in 1913, involves two enzymes, glyoxalase I and glyoxalase II, and the associated cofactor glutathione (GSH, 44).32 The glyoxalase enzymes catalyse the conversion of α-ketoaldehydes into α-hydroxy acids. This transformation is vital for the removal of intracellular methylglyoxal, which is a toxic side product of carbohydrate metabolism.34 The mechanism of action of the glyoxalase system is illustrated in Scheme 16.

Initially, the monohemithioacetal, 46, which is formed from methylglyoxal and GSH, 21 is converted by glyoxalase I (GlxI) into S-lactoylglutathione. Glyoxalase II then catalyses the hydrolysis of thioester 47 to give D-lactic acid and regenerate free glutathione.32

Scheme 16. The Glyoxalase System

The ability of the glyoxalase system to rapidly convert the cytotoxic methylglyoxal into non-toxic lactate is believed to have an important role in cell growth. Inhibition of GlxI would therefore result in an increased concentration of methylglyoxal in cells, leading to inhibition of cellular growth.34, 35 This proposal has led to substantial interest in the study of GlxI inhibitors as potential antitumour agents.29, 32

2.3 The Role of Glutathione

Glutathione is one of the most abundant tripeptides in eukaryotic cells. It is synthesised in the cytosol of cells from cysteine, glutamic acid and glycine, and it exists predominantly as reduced glutathione (GSH) but also as glutathione disulphide (GSSG).36 GSH has multiple cellular functions but one of its most important roles is as a defence “weapon” against toxic metabolytes and reactive oxygen species (ROS). It can act as an antioxidant and a reducing agent, hence it protects cells from DNA- damaging free radicals and it is involved in the metabolism of xenobiotics. Conjugation of GSH with alkylating drugs via its thiol leads to the detoxification of chemotherapeutic agents, making malignant cells drug resistant.36, 37 It has been

22 reported that cells lines which are resistant to alkylating drugs such as melphalan and cisplatin (Figure 3), display elevated levels of GSH.38

Figure 3. Chemotherapeutic agents: Melphalan and Cisplatin

In 2005, Ichikawa and collaborators reported that drug resistance in apoptosis- resistant pancreatic adenocarcinoma AsPC-1 cells could be suppressed by COTC. Firstly, they reported that the GSH levels in AsPC-1 cells dropped to 40% of the initial concentration after 1-2 h of adding COTC. Furthermore, they investigated the reaction between COTC and GSH in the presence of the alkylating chemotherapy drug melphalan; this experiment showed that COTC forms a conjugate with GSH (COTC- GSH), faster than with melphalan hence increasing the cytotoxicity of the latter. In addition, COTC-GSH inhibits Glx1 which results in increased concentration of toxic methylglyoxal in cells, leading to inhibition of cellular growth. Depletion of GSH and inhibition of Glx1 by COTC, therefore leads to higher intracellular concentration of the therapeutic agent as well as methylglyoxal, consequently increasing chemotherapy-mediated apoptosis.38

2.4 Mechanism of Action of COTC and COMC

The potent toxicity towards murine and human cancer cell lines, displayed by both COTC and COMC, was initially believed to be due to inhibition of glyoxalase via a “prodrug” mechanism.39 The bioactivity of COTC and COMC was dependant on the susceptibility of the crotonate moiety towards nucleophilic displacement by -SH compounds, such as glutathione. The proposed mechanism proceeded via an SN2 displacement of the crotonate group by glutathione (GSH) (Scheme 17), to give glutathione adducts 51 and 58 which were thought to inhibit glyoxalase I.29

Scheme 17. Displacement of crotonate group by GSH.

Further investigations, however, have suggested that the antitumor activity of COTC and COMC is not due to inhibition of GlxI by the glutathinone adducts 51 and 52.34 Ganem and co-workers carried out comparison studies for the inhibition of GlxI by enediol analogue inhibitors and by the GSH adducts of COTC and COMC. The results of the investigation showed that the glutathione adducts were poor competitive inhibitors of GlxI. These findings have led to the conclusion that the potent antitumor activity of COTC and COMC is unlikely to be due to GlxI inhibition. There is however, a possibility that the GSH adducts are toxic to tumour cells by an alternative mechanism.35, 40 In 2002, Ganem and collaborators put forward a new hypothesis for the antitumour activity of COMC. Their proposal involved the formation of an intermediate exocyclic enone via a conjugate addition reaction between glutathione and the cyclohexenone (Scheme 18). The conjugate addition of GSH to the cyclohexenone was found to be catalysed by glutathione transferase (GST), and led to elimination of the crotonate group to give the reactive enone intermediate, 53.35, 40 This exocyclic enone intermediate is an excellent Michael acceptor which may undergo alkylation reactions with reactive groups on intracellular proteins and/or nucleic acids which are critical to cell function, leading to cell death.41

Scheme 18. Formation of exocyclic enone and alkylation of proteins/nucleic acids

Preliminary kinetic studies and intermediate trapping experiments were undertaken, in order to further investigate the mechanism of the reaction. It was

24 found that the non-enzymatic reaction of COMC with GSH proceeded via a first order decay, without any evidence of an intermediate species. For the same reaction in the presence of human placental glutathione transferase (GSTP1-1) the profile of the reaction changed into a double exponential decay. The reaction profile consisted of a rapid, enzyme-dependent initial step involving COMC, followed by a slow enzyme- independent first order phase. The mechanism of the reaction which is based on these findings is shown in Scheme 19. Initially, an enzyme catalysed Michael addition of GSH to COMC gives the intermediate exocyclic enone, which after dissociation from the enzyme and reacts with GSH non-enzymatically to form GSMC (52).35, 40

Scheme 19. Reaction between COMC and GSH.

Ganem and co-workers suggested that the cytotoxicity of COMC arises from the ability of the exocyclic enone to undergo alkylation reactions with reactive groups on intracellular proteins and/or nucleic acids which are critical to cell function, leading to cell death. To further support this hypothesis, the in vitro reaction of COMC and GSH, with oligonucleotides or dinucleotides was monitored by mass spectrometry.42 The experiments provide evidence that COMC does form stable adducts in vitro with both GSH and nucleic acids. The mass spectral data indicated that the alkylation of nucleic acids by COMC could take place with or without GSH, but since GSH levels are high in cancer cells it was assumed that it was involved in the mechanism of action. Furthermore, it was observed that COMC could react with all nitrogenous bases apart from thymine and uracil: hence it was concluded that the site of

25 alkylation was the exocyclic amino groups of adenine, cytosine and guanine. COMC also appeared to exhibit sequence specificity, since in a reaction with guanosylyl(3´- 5´)-adenosine (GA), it was observed that COMC preferentially modified guanine (Figure 4).42

Figure 4. Adduct between COMC and GA

2.3 Natural Products Structurally Related to COTC

2.3.1 The Antheminones and Carvotacetones

The antheminones and the carvotacetones are two alternative types of bioactive natural products. Both are cyclohexenone derivatives which have been extracted from plants.31, 43 Similarly to COTC, the carvotacetone derivatives and the antheminones, have a cyclohexenone core and are considered a potential new class of anticancer drugs, which can target multidrug-resistant tumours.31 This has led to significant scientific interest in the synthesis of carvotacetone and antheminone analogues as potential antitumour drugs.

2.3.2 Carvotacetone derivatives

A large number of carvotacetone derivatives have been isolated from the aerial parts, or the leaves of different species of the Sphaeranthus genus.30, 43-46 The genus Sphaeranthus is mainly distributed around the tropical and subtropical areas of Australia, Africa and Southern Asia. Some species of this genus are used in folk

26 medicine for the treatment of glandular swellings, bronchitis, skin infections and nervous depression.30 Only a subsection of the species has been studied chemically and the main compounds isolated include thiophenacetylenes, inositol esters, sesquiterpene lactones and the carvotacetone derivatives. A number of carvotacetone derivatives are illustrated in Figure 5.30 The structures and stereochemistry of the carvotacetone derivatives have been confirmed by 1H NMR, 13C NMR and 2D NMR studies.

Figure 5. Carvotacetone Derivatives

In 2012, Midiwo and co-workers reported the first evidence of antimalarial and anti-tumour activity of carvotacetone derivatives 61-63. Carvotacetone derivatives 61-63 were isolated from the aerial parts of Sphaeranthus bullatus and have displayed anti-cancer activity towards melanoma, breast and ovarian cancer cell lines with IC50 values in the range of 1.1-5.3 µg/mL.

Figure 6. Carvotacetone derivatives that exhibit antimalarial and anti-cancer activity

2.3.3 Antheminones

The antheminones were isolated from the leaves of Anthemis maritium by Collu and co-workers, in 2008. The A. maritium species is an aromatic herb which can be found on the sandy beaches of eastern Corsica and specific sites in Sardinia.31, 47 Collu and co-workers were the first investigators to study the chemical constituents of this species. Collu and collaborators isolated two new cyclohexenones (antheminones A (41) and B (64)) and a new cyclohexanone (antheminone C (65)) (Figure 7) from the leaves of A.maritium. 1D and 2D NMR experiments together with mass spectrometry were used to determine the structure of these new compounds, however the absolute configuration could not be deduced, due to difficulties in the isolation of a crystalline derivative suitable for X-ray structure determination.31

Figure 7. Antheminones A, B and C

All three antheminones have been assessed for their toxicity against human cancer cells. Antheminones A (41) and C (65) demonstrated greater potency than antheminone B (64) against all cell lines and exhibit inhibition of cellular proliferation of leukaemia cells, with IC50 values ranging from 3.2 to 14 μM. Antheminone C was found to be the most active of the three. Antheminone A displayed comparable cytotoxic activity to COMC, therefore it was assumed that it undergoes reactions with GSH in the same way as COMC. However, unlike antheminone A, antheminone C is based around a cyclohexanone ring, rather than a cyclohexenone ring. As a result of this structural difference, antheminone C does not react via the mechanism proposed by Ganem and collaborators. Collu and co-workers suggested that the exocyclic enonic structure is intrinsic to the

28 cytotoxicity of antheminone C, the reason being that activation by GSH is not required and it could directly alkylate nucleic acids (Scheme 20).31

Scheme 20. Direct alkylation of nucleic acids by antheminone C

2.3.4 The Phorbasins

The phorbasins are a family of natural products, isolated from the southern Australian Phorbas species of marine sponge. The eleven phorbasins isolated to date are diterpenes and their structures resemble the carvotacetone.48-51 Phorbasins A-C are polyene diterpenes and were the first members of the family to be isolated by Capon and co-workers.48 Their absolute stereochemistry could not be assigned due to their instability and the lack of material, therefore their structure was elucidated by comparison with the spectroscopic data of carvotacetone 56. The absolute stereochemistry of phorbasin C was determined in 2008 by Macklin and Micalizio who reported its first total synthesis.52

Figure 8. Phorbasins A-I

In 2008, Zhang and Capon reported the isolation of phorbasins D-F which are unprecedented examples of diterpenyl-taurines linked by an amine. Phorbasins E and F are both dimers which comprise a unique 7-membered heterocycle.50 Capon and co-workers carried out in vitro cytotoxicity assays on phorbasins B-K, against the human cancer cell lines A549, HT29 and MM96L and normal neonatal foreskin fibroblast (NFF) cells. Their results suggested that the presence of the conjugated enone moiety was essential for cytotoxicity, since phorbasins B-I showed higher and more selective cytotoxicities than phorbasins J and K. They suggested that the cytotoxicity of the phorbasins depends on a Michael acceptor mechanism, in addition to their degree of hydroxylation and the presence of a lipophilic side chain.51

2.3.5 The Gabosines

The gabosines are a family of carbasugars which have been isolated from several strains of Streptomyces.53 KD16-U1, which was later renamed as gabosine C, was the first one to be isolated in 1974 by Umezawa and coworkers. In 1993, Thiericke and

30 collaborators isolated gabosines A-K from Streptomyces strains collected from soil samples.54 The structures were elucidated by NMR spectroscopy and the molecular formulae were confirmed by high-resolution mass spectrometry. A further three gabosines, L-O, were isolated by Thiericke and coworkers in 2000.55 The gabosines are secondary metabolites which share the common structural feature of a hydroxylated branched cyclohexenone or cyclohexanone core bearing methyl or hydroxymethyl substituents at C2 or C3. They do not demonstrate any significant biological properties, apart from weak antiprotozoal activity. Gabosines A, B, F, N and O, however, have shown weak DNA-binding properties, while gabosine E is a weak inhibitor of cholesterol biosynthesis in HEP-G2 liver cells.53-55

Figure 9. The gabosines

In 2011, Shing and collaborators reported the synthesis and biological evaluation of a series of gabosine analogues. The aim of their investigation was to prepare gabosine analogues with hydrophobic side chains, which would act as GST inhibitors and therefore suppress drug resistance. The synthetic analogue 4-O-decyl-gabosine D (Figure 10) was one of the most potent inhibitors of glutathione s-transferase M1

(GSTM1) with an IC50 of 13.4 µM, however it did not inhibit the growth of A549 cells at concentrations of 0-30 µM.56 When A549 cells were treated with cisplatin in the presence of 92, there was a significant enhancement of the cytotoxicity of cisplatin. This result suggests that inhibition of GSTM1 by analogue 92 has suppressed cisplatin resistance in human lung cancer cells.

Figure 10. 4-O-decyl-gabosine D

The synthetic analogue 4-O-decyl-gabosine D was synthesised from D-glucose in fourteen steps.56 Initially, the key enone intermediate 94 was prepared in 8 steps from D-glucose in an overall yield of 25%.57 Stereoselective reduction of enone 94 with K-selectride®, followed by alkylation of the free alcohol afforded ether 95. Ether 95 was then converted to the corresponding enone in three steps: a deacetonation, regioselective acetylation of the primary hydroxyl group and finally oxidation with pyridinium dichromate (PDC). Hydrolysis of the butane diacetal protecting group in 96, under acidic conditions, afforded the target compound 92.56

Scheme 21. Reagents and conditions: (i) K-selectride®, THF, -78 °C, 99%; (ii) NaH, THF, 0 °C, 2 h, then 1-bromodecane, tetra-n-butylammonium iodide, reflux, 1 h, 75%; (iii) 80% aq. AcOH, rt, 1.5 h, 87%; (iv) 2,4,6-collidine, acetyl chloride, CH2Cl2, -78 °C, 15 h, 89%; (v) pyridinium dichromate, 3Å molecular sieves, CH2Cl2, rt, 20 h, 99%; (vi) TFA:H2O (10:1), CH2Cl2, rt, 3h, 88%.

2.5 Previous Syntheses of COTC

The first enantioselective synthesis of COTC (38) was reported by Mirza and co- workers in 1985. The synthesis was achieved in a yield of 18% over eight steps, starting from the monosaccharide methyl α-D-mannopyranoside.58 An alternative

32 synthesis was reported in 1986, by Koizumi and collaborators. The key step in their synthetic sequence was an asymmetric Diels-Alder reaction with a chiral arylsulfinylacrylate dienophile.59 In 1990, Shing and Tang described the synthesis of COTC from the chiral staring material, (-)-quinic acid in a fourteen step sequence with an overall yield of 13%.60 The most efficient synthesis of COTC to date was reported by Ganem and co- workers starting from the quinic acid derivative, cyclohexylidene-protected methyl quinate 97 and involved an eight-step reaction sequence.61 Initially, protected quinate 97 was treated with triflic anhydride to give a bis-triflate, which upon stirring at room temperature underwent a spontaneous elimination to give mono- triflate 98. Reaction of compound 98 with caesium acetate gave diene 99, which underwent face-selective bromo-formylation to give formate ester 100. Reduction of both ester moieties of 100 with DIBAL-H, followed by reaction with lithium hexamethyldisilazide furnished epoxide 102. Epoxide 102 was then reacted with methanesulfonic acid in DMSO to give an oxysulfonium salt, which was treated with an excess of triethylamine to form dihydroxyenone 103. Selective crotonylation of 103 at the primary hydroxyl group with crotonic anhydride, followed by deprotection with TFA:H2O gave (-)-COTC (38).61

Scheme 22. Reagents and conditions: (i) Tf2O, py, DCM, 65%; (ii) CsOAc, DMF; (iii) NBS-H2O, DMF, 72% over 2 steps; (iv) DIBAL-H, benzene-toluene, 65%; (v) LiN(TMS)2, THF, -78 ˚C, 87%; (vi) CH3SO3H, DMSO, rt, 1.5 h then Et3N, rt, 5 min, 71%; (vii) crotonic anhydride, DCC, DMAP, THF, 54%; (viii) 1:1 TFA:H2O, 73%.

2.6 Previous work by the Whitehead Group

The Whitehead group is one of several research groups that have been intrigued by the potent biological activity of natural products such as COTC, and members of the group are currently investigating the synthesis of analogues of COTC and the antheminones. An array of α-oxyalkyl-α,β-enones, the general structure of which is shown in Figure 11, have been synthesised and screened against non-small-cell lung cancer cell lines A549 and H460. The general structure possesses five potential loci for variation and the aim of the research programme has been to determine the key structural features necessary for optimum bioactivity.62-65

Figure 11. General structure of analogues

Figure 12 depicts a number of analogues that have been synthesised by previous members of the Whitehead group. The trihydroxylated compound 105, 6-epi-COTC63, and the monohydroxylated compound, 10862 have been prepared in both racemic and enantiomerically pure forms, with the intention of investigating the effect of absolute stereochemistry and degree of hydroxylation on anticancer activity. The α,β-cyclopropyl ketone, 106, was synthesised with a view to determine whether the conjugated ketone moiety was essential for antitumour activity. Compound 107 is a ‘doubly activated’ analogue, which possesses two potential electrophilic sites susceptible to attack by GSH, and could therefore enhance anticancer efficacy.64 Finally, analogues 109-112 were chosen in order to examine the effect of an aryl and an alkyl side chain at R4, as well as the requirement of a reasonable leaving group at R2.28 The crotonylated analogues 109 and 111 are hybrids of COTC and the antheminones, while the diol compounds 110 and 112 are structurally related to the antheminones.

Figure 12. Analogues synthesised by previous members of the Whitehead group.

2.6.1 Synthesis of Analogues from (-)-Quinic Acid

The synthetic analogues 105-112 were all prepared from the versatile chiral pool starting material (-)-quinic acid.62, 64 The syntheses of both compounds 106 and 107, involved the butane diacetal (BDA)-protected enone 117. Initially, a selective protection of the trans-1,2-diol of (-)-quinic acid afforded the BDA-protected methyl quinate 114. Quinate 114 then underwent reduction to the corresponding triol 115, and this was followed by oxidative cleavage of the vicinal diol using silica supported sodium periodate to give the β-hydroxyketone 116. Finally, dehydration using methanesulphonyl chloride in the presence of triethylamine gave BDA-protected enone 117.62

Scheme 23. Reagents and conditions: (i) butane-2,3-dione, (CH3O)3CH, camphorsulfonic acid, MeOH, reflux, 12h, 98%; (ii) NaBH4, MeOH, 0°C to rt, 24 h; (iii) NaIO4 on silica gel, CH2Cl2, 1.5 h, 75% over two steps; (iv) Et3N, CH3SO2Cl, CH2Cl2, 3 h, 98%.

The synthesis of cyclopropane 106 began with a Morita-Baylis-Hillman (MBH) reaction between enone 117 and formaldehyde, followed by protection of the resulting primary alcohol with tert-butyldimethylsilyl chloride (TBSCl) to give intermediate 118. Reaction of compound 118 with an excess of Corey’s ylid 35

(dimethylsulfoxonium methylide) afforded the cyclopropanated intermediate 119. Finally, deprotection of the TBS-ether, followed by crotonylation and removal of the BDA protecting group yielded the target compound 106.64

Scheme 24. Reagents and conditions: (i) DMAP (cat.), H2CO, THF/H2O (1:1), 40 °C, 24 h, 80%; (ii) TBSCl, DMAP (cat.), Et3N, CH2Cl2, 72 h, 98%; (iii) trimethylsulfoxonium iodide, NaH, DMSO, 48 h, 49%; (iv) TBAF, THF, 0 °C, 98%; (v) crotonic anhydride, pyridine, DMAP (cat.), CH2Cl2, rt, 6 h, 46%; (vi) TFA/H2O (7:1), 0 °C, 30 min, then HPLC, 99%.

The α,β-cyclopropyl-α´,β´-enone 107 was also synthesised via the BDA-protected enone 117. The first step involved reaction of enone 117 with Corey’s ylid to give cyclopropane 120, which was then treated with aqueous TFA to yield alcohol 121. Protection of the free hydroxyl group in alcohol 121 as a silyl ether, followed by a M-B-H reaction gave the hydroxymethylated intermediate 122. The primary hydroxyl of intermediate 122 was then crotonylated and the TBS protecting group was removed to give the target compound 107.64

Scheme 25. Reagents and conditions: (i) trimethylsulfoxonium iodide, NaH, DMSO, rt, 1.5 h, 61%; (ii) TFA/H2O (7:1), rt, 12 h, then K2CO3, H2O, MeOH, rt, 30 min, 99%; (iii) TBSCl, DMAP (cat.), Et3N, CH2Cl2, rt, 6 d, 78%; (iv) imidazole (cat.), H2CO, THF, Na2CO3 (aq), 40 °C, 7 d, 23%; (v) crotonic anhydride, pyridine, DMAP (cat.), CH2Cl2, rt, 24 h, 48%; (vi) TFA/H2O (7:1), 0 °C, 1 h, 94%.

The enantiopure monohydroxylated analogue 108, was also synthesised from (-)-quinic acid via the cyclohexylidene protected enone 35. The synthesis of the cyclohexylidene protected enone, involved the selective protection of the cis-diol moiety in (-)-quinic acid using cyclohexanone, to afford cyclohexylidene quinide 123 in a 60% yield. Reductive ring opening of the γ-lactone in 123, with NaBH4, followed by oxidative cleavage of the vicinal diol using silica supported sodium periodate

36 resulted in the β-hydroxyketone, 125. Finally, the β-hydroxyketone was reacted with methanesulfonyl chloride and triethylamine to give α,β-enone, 35, in an 82% yield.64

® Scheme 26. Reagents and conditions: (i) Cyclohexanone, C6H6/DMF (1:1), Amberlite IR 120 (H) , reflux (Dean and Stark), 5 h, 60%; (ii) NaBH4, MeOH, 0 °C to rt, 24 h; (iii) NaIO4 on silica gel, CH2Cl2,1.5 h, 58% over two steps; (iv) Et3N, CH3SO2Cl, CH2Cl2,4 h, 82%.

The cyclohexylidene protected enone then underwent a catalytic hydrogenation to give cyclohexanone 126. Elimination of the cyclohexylidene protecting group with simultaneous silyl protection of the free hydroxyl resulted in intermediate 127, which was hydroxymethylated using a DMAP catalysed M-B-H reaction to give compound 128. The target compound was obtained by crotonylation of intermediate 128 followed by acid-mediated deprotection.64

Scheme 27. Reagents and conditions: (i) H2, 10% Pd on C, EtOAc, rt, 17 h, 92%; (ii) DBU, TBSCl, C6H6, reflux, 6 h, 80%; (iii) DMAP (cat.), H2CO, THF/H2O (1:1), 40 °C, 24 h, 52%; (iv) crotonic anhydride, pyridine, DMAP (cat.), CH2Cl2, rt, 1.5 h, 68%; (v) TFA/H2O (7:1), 0 °C, 1 h, 98%.

2.6.2 Synthesis of Racemic Monohydroxylated and Trihydroxylated Analogues

The racemic monohydroxylated analogue (±)108 was synthesised from 1,3-cyclohexadiene.65 Initially, a palladium catalysed cis-1,4-diacetoxylation of 1,3-cyclohexadiene was carried out to give the meso-diacetate intermediate 130. Compound 130 was then enzymatically de-symmetrized utilising electric eel cholinesterase to yield racemic mono-acetate 131. Silyl-protection of the free 37 hydroxyl in 131, followed by methanolysis of the acetate and oxidation using TPAP, provided enone (±)127. Finally, enone (±)127 was converted to the monohydroxylated analogue (±)108, using the procedures described previously for the preparation of the corresponding enantiopure analogue 108.

Scheme 28. Reagents and conditions: (i) MnO2, LiCl, p-benzoquinone, Pd(OAc)2, LiOAc·2H2O, AcOH, rt, 3 days, 69%; (ii) electric eel cholinesterase, NaN3, phosphate buffer (pH 6.85), 20 °C, 30 h, 63%; (iii) TBDMSOTf, Et3N, DMAP, CH2Cl2, 0 °C, 1 h, 82%; (iv) K2CO3, MeOH, rt, 3h, 88%; (v) TPAP, NMO, 4 Å mol. sieves, CH2Cl2, rt, 7 h, 65%; (vi) DMAP (cat.), H2CO, THF/H2O (1:1), 40 °C, 24 h, 20%; (vi) crotonic anhydride, pyridine, DMAP (cat.), CH2Cl2, rt, 1.5 h, 63%; (vii) TFA/H2O (7:1), 0 °C, 30 min, 98%.

The racemic trihydroxylated analogue (±)105 was prepared from the meso arene-dihydrodiol 132.63 The synthesis began with protection of the diol as its acetonide, followed by cis-dihydroxylation using catalytic osmium tetroxide, to give diol 133. The allylic hydroxyl in 133 was then selectively protected as its 2- naphthylmethyl ether via an intermediate stannylene acetal, to yield compound 134 in a 76% yield after purification by flash silica chromatography.

Scheme 29. Reagents and conditions: (i) (CH3)2C(OCH3)2, p-TSA, acetone, 0 °C to rt, 3 h; (ii) OsO4 t (cat.), NMO, BuOH, H2O, rt, 24 h, 57% over 2 steps; (iii) Bu2SnO, C6H5CH3, MeOH, Δ, then 2- bromomethylnaphthalene, Bu4NI, C6H5CH3, 130 °C, 9 h, 76 %.

Compound 134 was converted to the BDA-protected intermediate 135 by removal of the acetonide protecting group followed by protection of the vicinal trans- diol as a butane diacetal. The allylic hydroxyl of 135 was then oxidised and a M-B-H reaction was carried out in order to introduce a hydroxymethyl side chain. The final steps of the synthetic sequence involved, crotonylation of the primary hydroxyl in compound 137 and removal of the protecting groups to give the target compound 105.63

Scheme 30. Reagents and conditions: (i) H2O:TFA:THF (5:2:1), rt, 2 h, then butane-2,3-dione, (CH3O)3CH, camphorsulfonic acid, MeOH, reflux, 16 h, 46%; (ii) PCC, CH2Cl2, rt, 2 h, 76%; (iii) H2CO, imidazole, 1M NaHCO3 (aq), THF, 40 °C, 32 h, 68%; (iv) crotonic anhydride, pyridine, DMAP (cat.), CH2Cl2, rt, 3 h, 50%; (v) DDQ, CH2Cl2:MeOH (4:1), rt, 6 h, 66%; (vii) TFA/H2O (7:1), rt, 3 h, 98%.

2.6.3 Cytotoxicity Assay Results

Table 1 summarises the antiproliferative activity of the synthetic analogues depicted in Figure 12 against the non-small cell lung cancer cell lines A549 and H460. Based on the bioactivity data, a number of conclusions could be reached:  Almost all of the analogues were more potent towards the H460 cell line  The conjugated ketone moiety was crucial for anticancer activity  Crotonylated analogues were more cytotoxic than the corresponding enone diols  Monohydroxylated compounds with aryl substituents were the most potent hybrid analogues.62-64 6-epi-COTC (105) exhibited low toxicity against both cancer cell lines, while the mono-hydroxylated analogue 108 was shown to be ten times more cytotoxic than the trihydroxylated compound and up to four times more potent than COMC (39). The ‘doubly activated’ analogue 107 was more potent than COMC, but not to the same extent as 108.64 Finally, both the phenyl and iso-propyl substituted crotonylated analogues were more potent than the mono-hydroxylated analogue 108 as well as

39 their non-esterified counterparts. This latter finding, suggests that a good leaving group on the oxyalkyl side-chain is important for the mode of action of the drugs.28

IC50(μM) Compound A549 H460 COMC (39) 55 40 (±) – 105 184 160 (-) – 105 170 158 106 >200 >200 107 18 20 (±) - 108 24 10 (-) – 108 17 11 109 2.2 ± 0.8 - 110 90 ± 48 - 111 14 ± 4 - 112 159 ± 28 -

Table 1. Bioactivity of COTC analogues.

The phenyl substituted crotonated ester 109 was the most potent antiproliferative agent synthesised by the Whitehead group, with an IC50 of 2.2 ± 0.8 µM. Prior to these studies COMC was accepted to be the most potent analogue of COTC, as demonstrated by Douglas and co-workers.32 These new findings have stimulated further scientific research into substituted analogues of COMC.

3.0 Results and Discussion

3.1 Project Aims

At the outset, the aim of this project was to develop and optimise transition metal mediated conjugate addition reactions to functionalised cyclic enones and subsequently, to apply the methodologies to the synthesis of potential anti-tumour agents which are structurally related to the natural products COTC and antheminone A. Initially the research would focus on copper-mediated conjugate addition reactions and rhodium catalysed reactions of organoboron reagents. The conjugate addition reactions would be carried out on the quinate derived enones 117 and 35, which would be prepared from (-)-quinic acid as described in the introduction. The main challenge of the conjugate addition reactions was the stereoselectivity of the addition; it was predicted that conjugate addition reactions on the cyclohexylidene protected enone 35 would only afford the anti diastereoisomer, while the corresponding reactions on the BDA protected enone 117 could afford either the syn or the anti diastereoisomer, depending on the nucleophile.

Figure 13. Conjugate addition reactions on the BDA- and cyclohexylidene-protected enones

The methodologies developed throughout the project would then be applied to the syntheses of hybrid analogues of the bioactive natural products: COTC and antheminone A (Figure 14). Based on the results of previous investigations by members of the Whitehead group it was decided to synthesise a small library of 41 mono-hydroxylated analogues bearing aryl side chains. The crotonylated compounds would be analogues of COTC, while the dihydroxylated compounds would be structurally related to the antheminones and carvotacetones.

Figure 14. General templates of target molecules.

The cytotoxicity of all novel analogues towards non-small cell lung cancer cell line A549 would be assessed using an MTT viability assay. The ultimate goal of the investigation was to determine the structural features required for optimum antitumour activity.

3.2 Synthesis of Butane-1,2-Diacetal Protected Enone

3.2.1 Butane-1,2-Diacetal Protection of (-)-Quinic Acid

The selective protection of trans-1,2-equatorial diols as cyclic diacetals was first developed by Ley and co-workers who introduced the dispiroketal (Dispoke) and the cyclohexane-1,2-diacetal (CDA) protecting groups for the selective protection of vicinal diequatorial diols in carbohydrates.66 In 1996, two research groups reported the selective protection of the 1,2- diequatorial diol in (-)-quinic acid, as diacetals.67, 68 Gebauer and Brückner used a similar approach to that described by Ley and coworkers to protect the vicinal trans-diol of (-)-quinic acid as a cyclohexane-diacetal, using 1,1,2,2-tetra- methoxycyclohexane.67 Frost and collaborators reported the reaction between butane-2,3-dione and methyl quinate, which afforded butane-1,2-diacetal (BDA) protected quinic acid in 87% yield.68 Employing a similar methodology to Frost and co-workers, the butane-diacetal (BDA) protected methyl quinate, 114, was synthesised in an 88% yield. The reaction

42 was carried out using butan-2,3-dione, trimethyl orthoformate and camphorsulfonic acid in methanol, heating at reflux.

Scheme 31. Butane-1,2-diacetal protection of (-)-quinic acid.

The protection of the vicinal equatorial diol of (-)-quinic acid as a butane diacetal is both highly selective and leads to strong conformational rigidity. There are two main factors that dictate the selectivity of this reaction: the formation of a sterically undemanding trans-decalin ring and favourable anomeric effects. Anomeric stabilisation is maximised by placing the two methoxy groups of the 1,4-dioxane derivative in axial positions.66, 68, 69 The trans-1,2-diol of (-)-quinic acid is selectively protected, as protection of the cis-1,2-diol would lead to flattening of the 1,4-dioxane ring due to steric effects and loss of the anomeric stabilisation (Figure 15).69

Figure 15. Selective protection of the trans-1,2-diol.

3.2.2 Completion of BDA-protected Enone Synthesis

The synthesis of the key intermediate, butane-diacetal (BDA)-protected dihydroxycyclohexenone 117, had previously been optimised by members of the Whitehead group. Accordingly, it was prepared from methyl quinate 114 in three straight-forward steps. Firstly, 114 was reduced to the triol intermediate 115 using

NaBH4. The crude product was then progressed to the next step where oxidative cleavage of the vicinal diol using silica supported sodium periodate furnished the β- hydroxyketone 116 in a 79% overall yield from ester 114.

Scheme 32. Reagents and Conditions: (i) NaBH4, MeOH, 18 h; (ii) NaIO4 on silica gel, CH2Cl2, 1.5 h,

79% over two steps; (iii) Et3N, CH3SO2Cl, CH2Cl2, 3 h, 90%.

Sodium periodate (NaIO4) is a selective, stable and inexpensive reagent which reacts under mild conditions, and is widely used for the oxidative cleavage of vicinal diols to give carbonyl compounds.70, 71 The main disadvantage, however, concerning the use of NaIO4 is its low solubility in organic solvents. To overcome this problem,

Zhong and coworkers introduced the use of silica-gel supported NaIO4 in powder form.71 In order to facilitate the synthesis of β-hydroxyketone 116, the silica-gel supported NaIO4 was prepared by the addition of silica gel to an aqueous solution of

NaIO4, until a free flowing powder was obtained. The solid supported reagent was easy to handle and could be isolated from the reaction mixture by simple filtration. Mesylation of the resulting β-hydroxyketone, 116, using methanesulfonyl chloride (MsCl) converted the hydroxyl into a better leaving group, which was eliminated in the presence of triethylamine (Et3N) to give the α,β-enone 117 in a 90% yield.

3.3 Synthesis of Cyclohexylidene Protected Enone

The synthesis of the cyclohexylidene protected enone 35 was first described in 1980, by Géro and co-workers, using a four step synthesis, starting from (-)-quinic

44 acid. The procedure involved the protection of the vicinal cis-diol of (-)-quinic acid as a ketal, using cyclohexanone, to give cyclohexylidene quinide 123.72 In 1989, Danishefsky and co-workers reported a large scale synthesis of the isopropylidene protected enone, from (-)-quinic acid involving a four step procedure.73 Initially, the Whitehead group employed the methodology described by Danishefsky and collaborators, for the synthesis of a cis-diol protected enone. Unfortunately the isolation of some intermediates was problematic and the yields were not reproducible.65 Instead, the cyclohexylidene protection of (-)-quinic acid described by Géro and co-workers in 1971 was utilised, as illustrated in Scheme 33.74

Scheme 33. Reagents and conditions: (i) Cyclohexanone, toluene, Amberlite IR 120 (H)®, 5 h, 63%;

(ii) NaBH4, MeOH, 0 °C to rt, 20 h; (iii) NaIO4 on silica gel, CH2Cl2,1.5 h, 92% over two steps; (iv) Et3N,

CH3SO2Cl, CH2Cl2,4 h, 82%.

Selective protection of the cis-diol moiety in (-)-quinic acid was achieved using cyclohexanone, to afford cyclohexylidene quinide 123 in a 63% yield. The original procedure involved the use of benzene as a solvent, but for health and safety reasons this was replaced with toluene. Reductive ring opening of the γ-lactone in 123, was carried out with NaBH4 to give triol, 124. Oxidative cleavage of the resulting vicinal diol was achieved using silica supported sodium periodate to furnish the β- hydroxyketone, 125, in a 92% yield over two steps. Finally, elimination of the hydroxyl group was carried out following the method described by the Danishefsky group.73 The β-hydroxyketone was reacted with methanesulfonyl chloride (MsCl) and triethylamine to give α,β-enone, 35, in an 82% yield.

3.4 Conjugate Addition Reactions

The introduction of alkyl and aryl substituents at C3 of the two quinate enones was achieved using organocuprate chemistry and rhodium catalysed conjugate additions of organoboron reagents. It was predicted that conjugate addition reactions on the cyclohexylidene protected enone would only afford the diastereoisomer 141: wherein the newly added group is anti to the C4-oxygen substituent. The corresponding reactions on the BDA protected enone could afford either the syn or the anti diastereoisomer (140), depending on the nucleophile.

Scheme 34. General schemes for conjugate addition reactions of the BDA- and cyclohexylidene- protected enones.

3.4.1 Stereochemistry of the Conjugate Addition

Generally, unsaturated six-membered rings undergo axial attack where the reaction proceeds via a “chair-like” transition state. If the nucleophile attacks from an equatorial direction, the ring has to adopt a twist boat conformation: the ring then flips back into a chair conformation to give the final product, leaving the substituent in an equatorial position. If the nucleophile approaches from an axial direction, it forms a chair directly, with the substituent in an axial position: conformational relaxation may then occur in order to locate the substituent in an equatorial position.

Figure 16. A simplified account of the nucleophile attack.

Due to the rigid trans-decalin nature of the BDA-protected enone, it was predicted that an equatorial attack would be more energy demanding hence an axial attack by the organometallic reagent would be more favourable.75 Steric effects, however, between the approaching nucleophile and C(3)H may promote equatorial attack by bulkier groups. For the cyclohexylidene protected enone it was expected that the convex surface of the molecule would be more accessible due to steric hindrance from the protecting group, hence anti-attack would be more favourable.

3.4.1.1 Investigations into Stereoselectivity

In 1985, Corey and Boaz investigated the stereoselectivity of conjugate addition reactions between lithium dimethylcuprate and spirocyclic enone 145. They were testing the hypothesis that the nucleophile would add anti to the substituent at C(γ) due to stereoelectronic effects. They argued that anti addition would be more favourable because hyperconjugation between the γ-heteroatom and the Cu(III) adduct would stabilise the complex.76 When they carried out a reaction between the spiro enone 145 and lithium dimethylcuprate, however, they observed a preference for syn addition (92:8, syn:anti). Repeating the reaction in the presence of 5 eq. of chlorotrimethylsilane, only the anti product was observed.

The rationale that was put forward to explain the results was based on the reversible formation of the π-complex and the Cu(III) adduct. The reaction between the enone with the organocuprate affords syn and anti π-complexes: in the presence of TMSCl the anti π-complex is trapped which leads to formation of the anti product, while in the absence of TMSCl, the syn product is formed faster from the syn π- complex.

Scheme 35. The reaction between the spiro enone 145 and lithium dimethylcuprate in the presence and absence of TMSCl.76

Danishefsky and co-workers have also investigated the stereoselectivity of conjugate addition reactions of cyclic enones. In 1988, while working on the synthesis of mevinoid ML236A, these scientists observed the syn addition of silyl ketene acetal 148 to cyclohexenone 147.77 The reaction was Lewis acid catalysed and afforded the syn product 149 in a 90% yield and the anti product in 4% yield. A year later, however, during investigations into the synthesis of the immunosuppressive agent, FK-506 78, they observed anti addition of the methyl group from lithium dimethylcuprate to the cyclohexenone 150.

Scheme 36. The contrasting results observed by Danishefsky and co-workers.77, 78

These contradictory results prompted an investigation into the stereoselectivity of Lewis acid catalysed carbon-carbon bond formation reactions on cyclic enones.79 Initially, a Sakurai reaction and a Lewis acid catalysed Diels-Alder reaction were carried out on 5- and 6-membered cyclohexenones (Scheme 37). Both reactions displayed high syn selectivity with respect to the γ-OTBS group. The Sakurai reaction was carried out in the presence of titanium tetrachloride and afforded the syn isomer as the sole product. The Diels-Alder reaction catalysed by aluminium chloride, afforded the syn adduct 154a as the major product together with trace amounts of the anti product 154b.

Scheme 37. Lewis acid catalysed Sakurai and Diels-Alder reaction79

Danishefsky and co-workers proposed that both Lewis acid catalysis and an electron withdrawing group in the γ-position were necessary in order to achieve syn selectivity with systems similar to the γ-OTBS cyclohexenones. They proposed that a syn addition is more favourable because a syn attack by the nucleophile aligns the 49 forming C-C σ* orbital with the C-H σ orbital at the γ-C, hence resulting in stabilisation.79 Further exploratory work was carried out using the bicyclic enone 155: conjugate addition reaction between enone 155 and lithium dimethylcuprate resulted in anti addition of a methyl group. Similarly, the reaction with silyl ketene acetal 148 afforded the anti substituted adduct 156 in 89% yield. The Danishefsky group suggested that the anti selectivity with this cyclohexenone was a consequence of steric effects: anti attack from the less hindered convex face of the bicyclic ring is more favourable than the syn.

Scheme 38. Conjugate addition reactions of the bicyclic enone 113.79

3.4.2 Chlorotrimethylsilane as an Additive in Organocuprate Reactions

The rate of reaction and the yield of conjugate additions can be improved by additives such as chlorotrimethylsilane (TMSCl). One of the first reports of these effects was by Corey and Boaz in 198576 when it was demonstrated that in the presence of TMSCl, organocuprate reactions underwent faster conjugate addition reactions and afforded better yields. A year later, Alexakis and co-workers observed that esters and amides, which are usually unreactive to conjugate additions, would react with organocuprate reagents in the presence of TMSCl to give the corresponding 1,4-adducts in excellent yields. Over the years, various research groups have tried to rationalise these effects resulting in a number of conflicting theories.80 Initially, Corey and Boaz proposed that TMSCl traps the d,π*-complex to form a silyl enol ether of the CuIII intermediate.76 In 1989, Kuwajima and coworkers proposed that the TMSCl acts as a

Lewis acid and coordinates to the carbonyl group of the enone thereby activating the system resulting in modification of the stereochemistry of the conjugate addition.81

Scheme 39. Proposed interactions of TMSCl mediated cuprate reactions

Lipshutz and coworkers carried out detailed 7Li and 35Cl NMR studies and concluded that cuprate reactions are accelerated in the presence of TMSCl due to interactions between the TMSCl and R2CuLi via the Cl atom. This interaction increases the “Lewis acidity” of the silicon in TMSCl towards the carbonyl of the enone and also increases the “net size” of the organocuprate reagent (Scheme 40).82

Scheme 40. Interactions between TMSCl and organocuprates.

In 1995, Bertz and co-workers challenged the “Lipshutz theory” and proposed an alternative mechanism. They suggested that the Cl atom of TMSCl interacts with the Cu atom of the cuprate reagent, instead of the lithium, resulting in a reactive tetravalent copper intermediate which can readily undergo a reductive elimination.83

Scheme 41. Mechanism proposed by Bertz and coworkers

In a more recent publication, Frantz and Singleton have determined the kinetic isotope effects (KIE) of organocuprate conjugate additions carried out in the presence of TMSCl.80 Their results indicate that silylation of the oxygen in the carbonyl group is the rate determining step, in accord with Corey’s theory that the d,π*-complex is trapped by TMSCl to form a silyl enol ether.76 They suggested that the main reason for the effects of TMSCl is a change in the rate determining step, but no further evidence was provided to contradict Lipshutz’s proposal.

3.4.3 Organocuprate Conjugate Addition Reactions on BDA-Protected Enone

Conjugate addition reactions on the BDA protected enone were carried out using an adaptation of the procedure described by Kornienko and co-workers for aryl cuprate reactions.16 The organocuprate reagent was prepared in situ from 1.1 eq. of copper(I)iodide and 2.2 eq. of the corresponding Grignard reagent. As predicted, the conjugate addition to enone 117 occurred both syn and anti to the C4 substituent to give a mixture of the two diastereoisomers. The stereochemical outcome of the reactions with a range of organocuprate reagents is summarised in Table 2.

Grignard Ratio/ Isolated Entry Additive T/°C Time/h Product reagent syn:anti Yield/% anti- 1 TMSCl rt 2.5 1:3 51 140a syn- 2 TMSCl 0 3.5 3:1 37 140a

3 - rt 2.5 - Biphenyl -

4 - 0 2.5 - Biphenyl -

5 TMSCl rt 3 1.2:1 - -

syn- 6 TMSCl 0 2 3.5:1 40 140b

7 TMSCl 0 3 - Biphenyl -

anti- 8 TMSCl -78 1.5 1:5 33 140c 9 - -78 2 1:3 - - 10 TMSCl 0 2 - Enone -

11 CH3MgBr TMSCl -78 40 mins 3:1 - -

12 CH3MgBr - -78 2.5 3:1 - -

13 CH3MgBr TMSCl 0 1 8.5:1 - -

14 CH3MgBr - 0 15 mins 2:1 - -

Table 2. Summary of results for the organocuprate conjugate addition reactions on the BDA-protected enone

Previous exploratory work carried out by members of the Whitehead group had shown that for the preparation of the diphenylcuprate reagent an excess of CuI and PhMgBr was required, compared to standard procedures. The diphenylcuprate reagent was therefore prepared from 1.5 eq. of CuI and 3 eq. of PhMgBr at 0 °C and the conjugate addition reaction between the organocuprate reagent and the BDA- protected enone was carried out at room temperature (entry 1) and 0 °C (entry 2). In the presence of TMSCl, at room temperature the anti diastereoisomer anti-140a was furnished in excess, however at 0 °C the syn-140a adduct was the major product. These opposing results suggest that the syn diastereoisomer wherein the phenyl substituent lies in an axial position may be the kinetic product, while the corresponding equatorially substituted compound, may be the thermodynamic product. Repeating both reactions without TMSCl did not yield any product and only the biphenyl by-product and enone 117 were isolated.

The outcome of the corresponding reaction with di(4-fluorophenyl)-cuprate at 0 °C, was similar to the diphenylcuprate reaction: the syn-isomer was the major product and was isolated in a 40% yield. Initially, the preparation of the organocuprate reagent and the conjugate addition reaction were carried out at -78 °C, but a mixture of 1,2- and 1,4-addition products was obtained. This result implies that the transmetallation during the in situ synthesis of the organocuprate reagent at -78 °C had only progressed partially. The reaction was also attempted at room temperature (entry 3), however, there was no apparent facial discrimination, which correlates well with the diphenylcuprate reaction results. The stereochemistry of the adducts was assigned based on the vicinal coupling constants between C(3)H and C(4)H: for example, for the phenyl syn-adduct J3,4 = 5.1

Hz whereas for the anti-isomer J3,4 = 11.0 Hz. The assignment was also confirmed by X-ray crystallographic analysis of the syn-4-fluorophenyl compound syn-140b (Figure 17).

Figure 17. Oak Ridge Thermal Ellipsoid Plot (ORTEP) of compound syn-140b, obtained by X-ray crystallographic analysis

A few non aryl Gilman reagents, such as divinylcuprate and dimethyl cuprate, were also investigated. These cuprates are generally more reactive and less stable than their aromatic counterparts, so their preparation and conjugate addition reactions were carried out at lower temperatures (-78 °C and 0 °C). The addition of divinylcuprate to enone 117 proceeded to completion at -78 °C to give a 5:1 mixture of anti and syn isomers. The major anti-isomer was isolated by flash silica chromatography followed by crystallisation in 33% yield. In the absence of TMSCl, a

54 longer reaction time of 2 hours was needed, however the facial selectivity remained the same. The corresponding dimethylcuprate reaction was initially carried out at -78 °C, with and without TMSCl: in both cases the major product was the syn adduct but the reaction time was considerably shorter for the TMSCl mediated reaction (entries 11 and 12, Table 2). Increasing the reaction temperature to 0 °C, did not affect the facial selectivity of the addition – axial attack was still favourable - and in the absence of TMSCl the reaction time had greatly improved compared to the corresponding reaction with TMSCl (entries 13 and 14, Table 2). Unfortunately, a definitive conclusion concerning the likely stereoselectivity of the organocuprate reactions could not be drawn from the above results: their variability indicates that the organocuprate reaction is quite unpredictable and the outcome can be affected by the nature of the reagent, the additive and the reaction temperature.

3.4.4 Organocuprate Conjugate Addition Reactions on Cyclohexylidene- Protected Enone 35

The only previous example of an organocuprate 1,4-addition to the cyclohexylidene protected enone was reported by Falck and co-workers.84 In 1996, they reported the conjugate addition of an organocuprate reagent to various α,β- unsaturated ketones. The cyanocuprate was prepared in situ from α- thionocarbamoyl stannane 169 and CuCN: upon reaction with enone 35 the conjugate addition took place from the less hindered convex face to give 170 in a 50 % yield.84

Scheme 42

There have been numerous reports of organocuprate conjugate addition reactions on the iso-propylidene protected enone 155. In 1997, Maycock and co- workers utilised organocopper reagents for the introduction of a vinyl group at C3 of enone 155.85 They first attempted the reaction with vinylmagnesium bromide and CuI or CuCN, but this resulted in a mixture of the desired product and 1,2-addition adduct. To minimise the 1,2-addition they utilised a higher-order cyanocuprate, which was prepared in situ from vinyl lithium and CuCN. The reaction between

(CH=CH2)2Cu(CN)Li2 and enone 155, afforded the required product 171 and alcohol 172.

Scheme 43

Another example of a cuprate conjugate addition to the iso-propylidene protected enone, was described by Matsuo and co-workers during the synthesis of (-)-Sugiresinol dimethyl ether.86 They described the conjugate addition of a 4-methoxyphenyl group using different organometallic reagents and copper salts, under various conditions and concluded that the best procedure utilised 4- methoxyphenylmagnesium bromide and CuBr.Me2S at -15 °C and gave 50 % yield of the required product.

Scheme 44

A more extensive investigation was carried out in 2007 by Bräse and co- workers.87 They initially studied the effect of temperature on the conjugate addition of Me2CuMgBr·SMe2 to enone 155: their results suggested that the optimum temperature for the reaction was -50 °C. To investigate the scope of these reaction

56 conditions, various other cuprate nucleophiles were tested, including ethyl, vinyl and phenyl. Overall, the best yield was achieved with the dimethyl cuprate (73 %) and all additions took place from the less hindered “convex” face of the enone.87 In our investigation conjugate addition reactions were attempted on the cyclohexylidene-protected enone 35 using 4-tbutylphenylmagnesium bromide, phenylmagnesium bromide and 4-methoxyphenylmagnesium bromide. The reactions were carried out at 0 °C and the organocopper reagents were prepared in situ from 2.2 eq. of the Grignard reagent and 1.1 eq. of copper (I) iodide. Both the 4- tbutylphenyl and phenyl conjugate additions reached completion in 2 h, to give the required product in 73% yield. The 4-methoxyphenyl addition, however, did not proceed as smoothly due to large amounts of biphenyl by-product that were inseparable from the product.

Scheme 45. Organocuprate conjugate additions on the cyclohexylidene protected enone

3.4.5 Rhodium Catalysed Conjugated Addition Reactions of Boronic Acids

Alternatives to the commonly used organocuprate reagents are rhodium- catalysed reactions of organoboron reagents. It has been demonstrated by several research groups that rhodium(I) complexes can be employed as efficient catalysts for the conjugate addition reactions of boronic acid reagents to α,β-unsaturated ketones. For the investigation described herein, the catalysts utilised were chloro(1,5- cyclooctadiene)rhodium(I) dimer, [RhCl(cod)]2 (175) and hydroxy(1,5- cyclooctadiene)rhodium(I) dimer, [RhOH(cod)]2 (176).

Figure 18. Chloro and hydroxyl (1,5-cyclooctadiene)rhodium(I) dimer

Both catalysts are commercially available but the chloro substituted catalyst was also prepared following the procedure described by Giordano and Crabtree.88 The synthesis involved the reduction of rhodium trichloride in the presence of 1,5-cyclooctadiene and sodium bicarbonate in aqueous ethanol.

The choice of rhodium catalysts was based on investigations by Miyaura and co-workers into the effect of ligands and bases in the rhodium catalysed conjugate addition reactions of aryboronic acids to enones. They reported that rhodium-cod complexes were better catalysts than rhodium-alkene complexes and the reaction between p-tolylboronic acid and 2-cyclohexen-1-one was faster with [RhOH(cod)]2 than with [RhCl(cod)]2. In the presence, however, of an inorganic base such as KOH both reactions were significantly accelerated and exhibited almost identical reaction rates and yields. The reaction was also accelerated in the presence of triethylamine but to a lesser extent. Miyaura and co-workers suggested that the bases play two important roles in the catalytic cycle of the reaction. Firstly, the presence of base in the [RhCl(cod)]2 catalysed reaction is crucial in the generation of the active species, Rh-OH, which is required for transmetalation. In the case of the [RhOH(cod)]2 catalysed reaction, however, it must have another role which was proposed to be activation of the boronic acid towards transmetalation. The conjugate addition reactions in this research project were carried out at room temperature using 2 or 2.5 eq. of boronic acid and 5mol% of [RhCl(cod)]2 or

[RhOH(cod)]2, in the presence of triethylamine, in dioxane:water (10:1). Table 3

58 summarises the results of the rhodium catalysed conjugate addition reactions of various boronic acids with the BDA-protected enone.

Ratio/ Isolated Entry Boronic acid Time/h Product syn:anti Yield/% 1 4 2.2:1 syn-140a 39

2 50 (5)a 3:1 syn-140b 40 (50)a

syn-140d 27 3 4 1:1 anti-140d 20

syn-140e 16 4 6 3:1 anti-140e 8

5 18 - - -

6 24 - - -

7 20 - - -

8 18 - - -

a 2.5 eq. of boronic acid Table 3. Summary of results for the rhodium catalysed conjugate addition reactions of boronic acids with the BDA-protected enone.

The reactions with phenyl (entry 1) and 4-fluorophenyl (entry 2) boronic acids, afforded the syn-substituted diastereoisomer as the main product, which was in

59 agreement with the outcome of the corresponding organocuprate reactions carried out at 0°C. The reaction with 4-fluorophenyl boronic acid was initially carried out with 2 eq. of boronic acid but the reaction time was very long (50 h). It was, therefore, repeated using 2.5 eq. of boronic acid and this led to a significantly reduced reaction time (5h) and an improved yield. To investigate the effect of steric hindrance, experiments with 1-naphthalene and 2-naphthalene boronic acids were carried out. The reacting centre in the 1-naphthalene boronic acid is more sterically congested than that of the 2-naphthalene boronic acid, hence it was predicted that an equatorial attack would be more likely for the 1-naphthyl nucleophile. The results, to some extent, concur with this proposal: the crude ratio of diastereoisomers for the 1-naphthalene adduct was 1:1, while for the less sterically hindered 2-naphthalene nucleophile, axial attack was preferred resulting in a product ratio of 3:1 (syn-140e:anti-140e). Furthermore, it was decided to attempt a reaction using a heterocyclic boronic acid. The reagent chosen was 4-pyridyl boronic acid, however this was insoluble in the dioxane:water (10:1) solvent system. Changing the solvent system to 3:1 dioxane:water and warming to 50 °C, unfortunately did not improve the boronic acid’s solubility: as a result no reaction took place and the starting material was recovered. In order to compare results with the vinyl cuprate reaction, a rhodium catalysed reaction with 2,4,6-trivinylcyclotriboroxane-pyridine complex (entry 6) was carried out. This reagent has been previously utilised by Kerins and O’Shea for a Suzuki reaction89 and it was anticipated that under the aqueous conditions of the aforementioned solvent system the boronic anhydride would be converted to the corresponding acid. Unfortunately, this did not transpire and even when warming the reaction to 50 °C, the reaction did not proceed. The two final attempts involved 4-methoxyphenyl and 2-methoxyphenyl boronic acids. In both cases, the reaction led to a mixture of a biphenyl adduct and the enone starting material. Following the same experimental procedure, the rhodium catalysed conjugate addition reactions of boronic acids with the cyclohexylidene protected enone were

60 carried out. It was predicted that anti attack by the nucleophile onto the enone would be more favourable due to steric effects. Table 4 provides a summary of the reactions attempted.

Isolated Entry Boronic acid Time/h Product Yield/%

1 5 141c 82

2 20 141d 89

3 1.5 141e 69

4 1 141f 83

5 30 141g 27

Table 4. Summary of results for the rhodium catalysed conjugate addition reactions of boronic acids with the cyclohexylidene-protected enone.

As predicted, all reactions furnished a single diastereoisomer with the substitutent anti to the protecting group. The reaction proceeded in shorter reaction times and gave higher yields compared to the corresponding reactions on the BDA- protected enone. The reactions using 4-methoxyphenyl, 4-fluorophenyl and 4- iodophenyl boronic acid were all carried out with 2.5 eq. of the corresponding boronic acid, whereas both 1-naphthalene (entry 3) and 2-naphthalene (entry 4) boronic acid experiments were carried out using 2 eq. and had relatively short reaction times.

The initial assignment of the relative stereochemistry was accomplished by inspection of the vicinal proton coupling constants, but was later confirmed by NOE experiments on the 2-naphthalene substituted adduct and X-ray crystallographic analysis of the biphenyl substituted adduct. When the C(4)H of adduct 99d was irradiated, an NOE was observed with protons C(1’)H and C(3’)H of the naphthalene substituent, while no transannular NOE was observed between C(3)H and C(5)H. The structure of the substituted adducts was confirmed by X-ray crystal analysis of the biphenyl adduct 141h, synthesized by another member of the Whitehead group.90

Figure 19. Oak Ridge Thermal Ellipsoid Plot (ORTEP) of compound 141h, obtained by X-ray crystallographic analysis

3.4.6 Overview of Conjugate Addition Reactions

Despite the numerous reactions carried out during this project, the stereoselectivity of the conjugate addition reactions on the BDA-protected enone proved to be unpredictable and in all cases a mixture of syn and anti products was afforded. It was concluded that the reaction temperature, additive and type of nucleophile affect the stereochemical outcome of the reaction. For example, reaction of the BDA-protected enone with diphenyl cuprate at 0°C afforded the syn adduct (syn-140) as the major product; however when the temperature was increased to room temperature the opposite selectivity was observed. The corresponding reaction with di-(4-fluorophenyl)cuprate displayed similar selectivity at 0°C, with the syn diastereoisomer being the major product, but no significant facial preference was observed at room temperature. These results suggest that the syn adduct is the kinetic product whereas the anti diastereisomers is the thermodynamic product; 62 hence the formation of the π-complex could be reversible, as Corey and Boaz have suggested.76 The rhodium catalysed reactions with phenyl and 4-fluorophenyl boronic acids, both favoured syn selectivity. In contrast to the reactions involving the BDA-protected enone, the organocopper and rhodium catalysed conjugate additions on the cyclohexylidene- protected enone were high yielding and diastereoselective. Only the anti substituted product was obtained from these reactions due to steric effects from the protecting group that would only allow addition of the nucleophile from the “convex” face of the molecule. Both the organocuprate reactions and the rhodium catalysed reactions of boronic acids gave relatively high yields and the best yield was achieved with 4-fluorophenyl boronic acid (89%).

3.5 Application of Conjugate Addition Reactions to Synthesis of Bioactive Natural Product Analogues

The procedures developed for the introduction of an aryl substituent at C3 of the quinate enones, were then employed for the synthesis of the COTC and antheminone natural product hybrid analogues, 177-180.

Figure 20. Target molecules

In order to complete the synthesis, it would be necessary to remove the diol protecting group and then protect the liberated free hydroxyl group as a silyl ether. A Morita-Baylis-Hillman (M-B-H) reaction would then be carried out on the γ-silyloxy

63 cyclohexenone in order to introduce a hydroxymethyl group at C2. Deprotection of the silyl ether group would afford compound 183, which is structurally related to the antheminones and carvotacetones, while crotonylation and then deprotection would give compound 185 which is a hybrid analogue of COTC and the antheminones.

Scheme 46. Proposed routes to the target molecules-Reagents and conditions: (i) TBSOTf, DBU, CH2Cl2; (ii) formaldehyde 37%, DMAP or imidazole, THF; (iii) TFA:H2O; (iv) crotonic anhydride, DMAP, py, CH2Cl2; (v) TFA: H2O.

3.5.1 Elimination-Protection of the Cyclohexylidene Protected Adducts

In 1989, Danishefsky and co-workers reported the one step conversion of cyclohexanone 186 to cyclohexenone 187 in 87 % yield, using tert-butyldimethylsilyl chloride (TBSCl) and DBU.73 Following the same procedure, in 2007, Bräse and co- workers, performed experiments on various isopropylidene protected cyclohexanones.87 In this report, it was concluded that the overall transformation was affected by steric hindrance, consequently, the bigger the substituent at C5, the lower the yield. Exploratory work by previous members of the Whitehead group has shown that conversions using TBSOTf with DBU are more efficient than those carried out under Danishefsky conditions, for the elimination-protection of cyclohexylidene protected cyclohexanones.

Scheme 47. Deprotection and subsequent protection of isopropylidene protected cyclohexanone.73

For the synthesis of compound 188, the naphthyl substituted intermediate 141f was treated with 1.2 eq. of TBSOTf in the presence of 1.4 eq. of DBU to give a mixture of the required compound and alcohol 189. The isolated yield of 188 was 43%. Unfortunately, the reaction was not reproducible, since in further attempts under the same conditions no product or alcohol was observed: instead various unidentified unsaturated and aromatic impurities were detected by 1H NMR spectroscopy.

Scheme 48. Reagents and conditions: TBSOTf, DBU, CH2Cl2, rt, 4.5 h, 43%.

It was decided, therefore, to explore an alternative method. The first option was to use a different base; hence DBU was substituted with Et3N. This however, led to enolisation of the compound to give silyl enol ether 190. Treatment of 190 with TBAF afforded alcohol 189 in 71% yield. Attempts to protect the alcohol with TBSOTf and DBU, led to the formation of the same unknown impurities as those observed during the reaction of 141f.

Scheme 49. Reagents and conditions: (i) TBSOTf, Et3N, CH2Cl2, rt, 24 h, 76%; (ii) TBAF, THF, 0 °C, 1 h, 71%.

The second option was to explore different protecting groups, so triethylsilyl triflate (TESOTf) was employed instead of TBSOTf. Treatment of cyclohexenone 141f with TESOTf in the presence of 2,6-lutidine resulted in formation of silyl enol ether 191. The reaction did not progress to completion and consequently, after purification, silyl enol ether 191 was isolated in 39 % yield and starting material was also recovered.

Scheme 50. Reagents and conditions: TESOTf, 2,6-lutidine, CH2Cl2, 0 °C, 1 h, 39%.

The same reaction with the alcohol 189, however, afforded intermediate 192. Purification of 192 by flash silica chromatography led to the hydrolysis of the silyl enol ether to give the required product in a 54 % yield.

Scheme 51. Hydroxyl protection with TESOTf- Reagents and conditions: TESOTf, 2,6-lutidine, CH2Cl2, 0 °C, 1 h, 54 %.

Based on these findings, it was decided to carry out the deprotection and protection as two separate steps. The deprotection was achieved with DBU, to give a 75% yield of the alcohol 189, after purification by flash silica chromatography. Protection of the resulting alcohol was attempted with TESOTf in the presence of 2,6- lutidine at -40 °C, but these conditions afforded a mixture of the required product 193 and diene 192. It was assumed that the formation of the diene was due to enolisation caused by traces of triflic acid present in TESOTf. To overcome this problem, TESOTf and 2,6-lutidine were premixed before addition of alcohol 189 in

66 order to neutralise any triflic acid present and the reaction was carried out at -78 °C. The reaction reached completion in just 15 mins and after purification by flash silica chromatography the required product 193 was isolated in a 71% yield.

Scheme 52

The same reaction conditions were applied successfully to the synthesis of the 4-tert-butylphenyl and 4-methoxyphenyl substituted analogues.

Scheme 53

An alternative procedure described by Maycock and co-workers during the total synthesis of (+)Eutypoxide B, was employed for the eliminative deprotection of the 4-fluorophenyl substituted adduct 141d. The 4-fluorophenyl adduct was treated with a few drops of aqueous NaOH (0.5M) in THF at 0 °C, to give the corresponding alcohol in 71% yield. The free hydroxyl of alcohol 198 was protected as its silyl ether using the methodology described previously (Scheme 54).

Scheme 54

3.5.2 Perhydro-dibenzofuranone by-products

During an investigation by another member of the Whitehead group into the eliminative deprotection of the biphenyl conjugate adduct 141h, using a few drops of aqueous NaOH (0.5M) in THF, perhydro-dibenzofuranone 201 was isolated as a side-product.90 It was assumed that under the basic reaction conditions the resulting alcohol could act as both a nucleophile and an electrophile leading to an unexpected dimerisation.

Scheme 55

In order to further investigate the scope of this dimerisation process, conjugate adducts 141b-141d and 141g were treated with a catalytic amount of aqueous NaOH (0.5M) in THF, at room temperature, for a prolonged period of time. The corresponding diastereoisomerically pure perhydro-dibenzofuranones were isolated by flash silica chromatography in 55-86% yields.

Scheme 56

The structures of these cyclodimers were determined by extensive 1D and 2D NMR experiments and confirmed by X-Ray crystallographic analysis.

Figure 21. Oak Ridge Thermal Ellipsoid Plot (ORTEP) of compound 201b, obtained by X-ray crystallographic analysis

The mechanism of the dimerisation is a stereoselective Michael type domino reaction. An initial conjugate addition reaction of the alkoxide intermediate to the less hindered face of enone 200 takes place, followed by a second intramolecular conjugate addition to give the thermodynamically favourable, cis-fused ring system. The syn (C(5)H-C(6)H) -anti (C(6)H-C(9)H) -syn (C(9)H-C(10)H) arrangement agrees with two consecutive suprafacial conjugate addition reactions, where the alkoxide anion adds to the C3 of the second molecule, anti to the C5 substituent.

Scheme 57. Mechanism of the dimerisation process

Dimerisation of quinols is assumed to occur during the biosynthesis of several natural products.91 The complex molecular structure of these dimeric natural

69 products and their biological properties, have stimulated investigations into their biomimetic total synthesis. Carreño and co-workers have studied the base-induced dimerisation of sulfinyl-p-quinols and sulfinyl-p-cyclohexenones and have shown that in both cases diastereoselective successive conjugate additions take place. They have reported that treatment of 4-hydroxy-4-[(p-tolylsulfinyl)methyl]-5-methyl cyclohexenone with sodium hydride in dichloromethane gave the dimeric compound 204 as a single diastereoisomer in a 57% yield.91

Scheme 58. Dimerisation of 4-hydroxy-4-[(p-tolylsulfinyl)methyl]-5-methylcyclohexenone

In 2012, Wu and collaborators reported the biomimetic one-pot synthesis of incarvilleatone and incarviditone via the stereoselective homo- and heterodimerisation of (±)-rengyolone.92 The reaction was carried out by exposing racemic rengyolone to sodium hydride in dichloromethane for 12 h. Homodimerisation of (+)-rengyolone or (-)-rengyolone, furnished incarviditone 206 via a tandem oxa-Michael/carba-Michael addition. Heterodimerisation of (+)- rengyolone and (-)-rengyolone afforded incarvilleatone 208, probably through an initial domino conjugate addition reaction to give the perhydro-dibenzofuranone 207, followed by an intramolecular aldol cyclisation.

Scheme 59. One-pot synthesis of incarvilleatone and incarviditone

It has been reported that both incarviditone (206) and rengylolone (205) display toxicity towards cancer cell lines71, therefore the toxicity of the novel dimers towards the A549 non-small-cell lung cancer cell line was assessed (Section 4.4).

3.5.3 Deprotection and Elimination of the BDA Protected Adducts

In 1996, Gebauer and Brückner reported the removal of a cyclohexane- diacetal protecting group with concominant dehydration to give alcohol 210. The experiment was carried out by heating ketone 209 at reflux in the presence of acetic acid, to give alcohol 210 in a 48% yield (Scheme 60).67

Scheme 60

The corresponding transformation of a BDA protected ketone was reported by Maycock and co-workers, in 2001. They found that treatment of ketone 211 with a mixture of trifluoroacetic acid and water at reflux, afforded alcohol 210 in 85% yield.93

Scheme 61

An adaptation of Maycock’s procedure was attempted by a previous member of the Whitehead group on adduct anti-140a. Treatment of anti-140a with TFA:H2O (7:1), however, resulted in a mixture of alcohol 212 and its trifluoroacetate ester 213 (Scheme 62).94 It was decided, therefore, that an additional goal of this investigation, would be to develop an alternative, more efficient method for the synthesis of alcohol 212.

Scheme 62. Deprotection utilising TFA:H2O (5:1)

In 1999, Kobayashi and co-workers developed a Brønsted acid surfactant combined catalyst (BASC) for carrying out a range of reactions in water.95 BASCs are composed of an extended hydrophobic moiety and a Brønsted acid group which is necessary for acid catalysis. An example of a BASC is dodecylbenzenesulfonic acid (DBSA) which forms hydrophobic colloids in water due to its long alkyl chain.96

Scheme 63. Dodecylbenzenesulfonic acid

Both the Kobayashi97 and the Whitehead98 groups have previously used DBSA for dehydration reactions. It was suggested that even though it is unusual to carry out dehydration reactions in water, the reaction with DBSA is driven by the expulsion of water molecules from the hydrophobic emulsion droplets.97 72

The elimination of the BDA group from the anti-140a was initially attempted using 0.2 equivalents of DBSA in water. After stirring at room temperature for 24 hours, no product was observed and the starting material was recovered. The reaction was then repeated at 100 °C for 1.5 hours and this gave the required product in a 60% yield. The same reaction conditions were applied successfully to the deprotection of the anti-140a analogue, to give alcohol 215 in 45% yield.

Scheme 64. Removal of the BDA group with DBSA in water at 100 °C

The resulting alcohols 212 and 215 were protected as their silyl ethers, by reaction with pre-mixed TESOTf and 2,6-lutidine at -50 °C (Scheme 65).

Scheme 65. Silylation using TESOTf and 2,6-lutidine at -50 °C

3.5.4 Morita Bayllis Hillman Reaction

The Morita-Baylis-Hillman (M-B-H) reaction is a coupling reaction between an activated alkene and a carbon electrophile, catalysed by a tertiary amine. It is an important carbon-carbon bond forming reaction, which introduces new functional groups in a selective manner under relatively mild conditions. The main limitation of the reaction is its slow reaction rate since it may require several days or even weeks for the reaction to progress to completion. As a result, many investigations have been carried out with the aim of improving the rate of the reaction.99 The most commonly used catalyst for the reaction of acyclic alkenes with aldehydes is DABCO. In 1998, however, Rezgui and Gaied reported that M-B-H reactions involving cyclic enones did not proceed when DABCO was used as a catalyst.100 Rezgui and co-workers were the first to report the use of DMAP as a catalyst in an aqueous medium for the coupling of cyclic enones with formaldehyde. In their procedure, 10 mol% DMAP was used and yields of 75-82 % (Table 5, entry 1) were achieved. In 2002, Gatri and co-workers reported that imidazole is another efficient catalyst for the M-B-H reaction of cyclic enones.101 The imidazole catalysed reaction of cyclohexenone with formaldehyde proceeded with an increased 93 % yield, but the reaction required stirring at room temperature for 17 days (Table 5, entry 2).101 Luo and co-workers improved the reaction time of this step by adjusting the pH of the solution.102 The use of a mixture of aqueous 1M NaHCO3 and THF as a solvent accelerated the reaction significantly; however the yield was reduced (Table 5, entry 3).

Entry Catalyst Catalyst Solvent Reaction Yield/% loading/mol% time 1 DMAP 10 THF 15 h 82

2 Imidazole 20 THF-H2O 17 days 93

3 Imidazole 100 THF-1M NaHCO3 15 h 65 Table 5. The Morita Bayllis Hillman reaction carried out in various methods by different groups.

Previous exploratory studies carried out by members of the Whitehead group indicated that imidazole and DMAP were the most reliable nucleophilic catalysts for the hydroxymethylation of the cyclohexenones used throughout this investigation. For the hydroxymethylation of enone 188 the M-B-H reaction was attempted with DMAP and imidazole (Table 6). In both cases, however, the reaction time required was very long and the yields were very poor. It was therefore necessary to explore alternative methods.

Entry Catalyst Catalyst Solvent Reaction Yield/% loading/mol% time 1 DMAP 10 THF: H2O (1:1) 16 days 22

THF:1M NaHCO3 2 Imidazole 10 11 days 33 (1:2)

Table 6. Summary of results for the M-B-H reaction.

Williams and co-workers reported that the use of surfactants, such as sodium dodecyl sulphate (SDS) and cetrimonium bromide (CTAB), in water increased the rate of reaction of M-B-H reactions of cyclic enones with aldehydes.103, 104 It was suggested that using a surfactant in water instead of organic solvents is ideal, due to the zwitterionic nature of the reaction and the lipophilicity of substituted cyclohexenones. The proposed mechanism involved embedding of the lipophilic side group of the enone into the hydrophobic region of the surfactant, while the enone moiety of the cyclohexenone is located at the interface with the water phase.

Figure 22. Proposed mechanism for the M-B-H reaction under surfactant conditions.103

It was assumed, therefore, that due to the lipophilic nature of the substituted cyclohexenones under investigation, the reaction conditions reported by Williams and co-workers would be ideal for the synthesis of the COTC analogues. The M-B-H reaction on compounds 142, 152 and 153 was carried out in a 10 mol% solution of SDS in water and in the presence of 1 eq. of DMAP for 18 hours, to give the products in yields of 64-74%.

Scheme 66. Morita-Baylis Hillman reaction

3.5.5 Completion of the Synthesis of Novel Antitumour Agents

Deprotection of the silyl-protected hydroxyl group in compounds 220-223 would afford compounds structurally related to the antheminones and carvotacetones, whereas crotonylation followed by deprotection would provide hybrid analogues of COTC. Both the crotonylation reaction and the deprotection of the hydroxyl group have been investigated by previous members of the Whitehead group.

Scheme 67. Deprotection of the silyl ether

Removal of the triethylsilyl protecting group was achieved with TFA:H2O (7:1) at room temperature to give the corresponding diol which is structurally related to the antheminones and carvotacetones. To synthesise the analogues which are structurally related to COTC, crotonylation of the primary hydroxyl group was carried out using crotonic anhydride in the presence of DMAP and pyridine, followed by removal of the silyl protecting group with TFA:H2O (7:1).

Scheme 68. Reagents and Conditions: (i) Crotonic anhydride, DMAP, py, CH2Cl2, rt; (ii) TFA:H2O (7:1), rt.

4.0 Biological Results

4.1 MTT Cell Viability Assay

In 1983, Mosmann introduced the use of (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) in cytotoxicity assays.105 MTT forms a yellow solution and is cleaved in living cells by mitochondrial succinate dehydrogenase, to give blue crystals of formazan. The formazan crystals are dissolved in a suitable solvent and readings are taken on a scanning multiwell spectrophotometer. The amount of formazan produced is proportional to the number of living cells.105-107

Scheme 69. Reduction of MTT to formazan by mitochondrial succinate dehydrogenase

The use of the MTT assay as a chemosensitivity test was assessed in 1987 by Carmichael and co-workers.107 They compared the MTT assay with the more conventional methods at the time, such as the dry exclusion assay and the clonogenic assay, by using lung cancer cell lines. Initially, they had to modify the original procedure by Mosmann due to solubility problems with the formazan crystals: instead of using acidic iso-propanol to dissolve formazan, they used DMSO or mineral oil and read the absorption at 540 nm. They reported that in order to get accurate results, the cells have to be in an exponential growth rate throughout the assay; hence an optimal seeding density has to be determined for different cell lines. They suggested that the optimum incubation time is four days, since this is long enough for the drug to act but also avoids the need to re-feed the cells. The conclusions of the investigation were that the MTT assay can rapidly provide valid and reproducible results but its main limitation is the inability to distinguish between cytocidal and cytostatic affects.

4.2 Assessing the Cytotoxicity of the Novel Compounds

The cytotoxicity of the novel compounds towards A549 cells was assessed using an MTT viability assay. The A549 cell line is a human non-small cell lung cancer cell line which was obtained from the American Type Culture Collection (ATCC). This cell line has been previously used by members of the Whitehead group for MTT assays of COTC analogues, due to its high concentration of glutathione compared to other human tumour cell lines.63 The assays were carried out using the procedure described by Stratford and co-workers.108 Initially an optimal seeding density had to be determined to ensure that the cells were in an exponential growth rate throughout the assay; this was determined by another member of the Whitehead group.28 It was suggested that seeding 1000 cells/mL in each well would maintain an exponential growth rate throughout the assay and provide enough cellular sensitivity towards a tested compound. Consequently, A549 cells were seeded in 96-well plates and allowed to adhere for 24 hours at 37 °C in a humidified incubator. The novel compounds were dissolved in DMSO and serial dilutions from 1 nM to 4 mM were prepared in growth media for each compound. 20 µL of each dilution were added to the wells in quadruplicate across the plate to give a final drug concentration of 0.1 nM to 400 µM on the plate and incubated for 96 hours. The assay was stopped by addition of the MTT solution in each well and incubated for a further 4 hours. The formazan crystals formed were dissolved with DMSO and the absorbance for each well was measured at a wavelength of 540nm using a multi-well scanning spectrophotometer.

4.3 Determination of the IC50 Concentrations

The IC50 value is the concentration of the compound required to reduce the number of viable cells by 50%. The spectrophotometer readings were used to calculate the optical density (OD) values which are proportional to the number of living cells. A dose response curve was then generated by plotting the OD values as a percentage of the control OD values against the concentration of the compound. The

IC50 was then read from the dose response curve.

Figure 23. Reading the IC50 value from a dose response curve

4.4 Results of MTT Assays of the Novel Compounds

The MTT viability assay was repeated three times for each compound and a dose response graph was generated using the average percentage of viability. The IC50 concentrations were determined from the graph (Appendix). The results of the assays on A549 cells are presented in Table 7.

Compound IC50 (µM) COMC 54.5 177a 17 ± 5 177b 19 ± 5 177c 75 ± 11 177d 42 ± 27 178a 8.6 ± 2.1 178b 3.6 ± 3.2 178c 2.6 ± 1.2 178d 1.2 ± 0.5 179 1.3 ± 0.2

180 7.9 ± 5.4

Table 7. IC50 values for novel compounds towards A549 cells: the IC50 values given were calculated from the dose response curve plotted from 3 experiments. Standard deviations were calculated from IC50 values derived from independent dose response curves of each individual experiment.

All novel analogues, apart from 177c, were more potent than COMC: this suggests that the presence of a hydroxyl group at C4 and an aromatic substituent at C5 enhance the activity of the compounds. Furthermore, the crotonylated analogues 178a-d were markedly more toxic than the corresponding dihydroxylated compounds 177a-d. This highlights the importance of a reasonable leaving group, such as crotonate, attached to the oxymethyl side chain, in order to generate the reactive enone intermediate in accord with the mechanism proposed by Ganem and co-workers.35, 40 The moderate toxicity, however, of 177a and 177b suggests that the requirement of a good leaving group is not absolute and this is further supported by the fact that Antheminone A which lacks a leaving group displays comparable cytotoxicity to COMC. The data therefore suggests that a lipophilic side chain may influence the cytotoxicity of the drug. Finally, the results indicate that relative stereochemistry at C4 and C5 influences the potency, since the anti analogue 179 was more active than its syn counterpart 180 by a factor of 6. It has been reported that several GST isoenzymes as well as GSH are overexpressed in tumour cells; hence compounds structurally related to COTC may

81 display selective toxicity towards tumour cells. In 2012, Shing and co-workers reported that some aliphatic ether analogues of COTC demonstrated selective antiproliferative activity when assayed against WRL-68 normal cell line and HepG2 and HL-60 cancer cell lines. In order to investigate the selectivity of the novel analogues, a selection of the compounds (177b, 177d, 178a, 178d, 179) was assayed against MCF10A, a non-tumourigenic breast cancer cell line. The results of the MTT assays against both A549 and MCF10A for the selected compounds are presented in Table 8. Unfortunately, taking into consideration the error values, there was not a significant decrease in the potency towards the MCF10A cells.

IC50 (µM) Compound MCF10A A549 177b 12.2 ± 0.1 19 ± 5 177d 90 ± 33 42 ± 27 178a 9.7 ± 1.0 8.6 ± 2.1 178d 4.4 ± 2.7 1.2 ± 0.5 179 1.8 ± 0.9 1.3 ± 0.2

Table 8. IC50 values for novel compounds towards A549 and MCF10A cells: the IC50 values given were calculated from the dose response curve plotted from 3 experiments. Standard deviations were calculated from IC50 values derived from independent dose response curves of each individual experiment.

Prompted by the reported anticancer activity of both rengylolone and incarviditone, the antiproliferative activity of the perhydro-dibenzofuranones (201b- e) and one of the precursors 194 was assessed against A549 cells. The IC50 of the novel compounds are shown in Table 9. Compound 194 was found to be the most potent, presumably due to the fact that it is a good Michael acceptor that could alkylate intracellular proteins. Dimers 201c-d did not show any activity, while dimer

201e displayed a moderate potency with an IC50 of 103 µM. The most potent compound was the t-butylphenyl substituted dimer 201b which demonstrated a significant antiproliferative activity with an IC50 of 12 µM. The biological mechanism

82 of action of incarviditone and dimer 201b has not been determined at the present time.

Compound IC50 (µM) 201b 12.0 ± 1.6 201c Not toxic at 100 µM 201d Not toxic at 100 µM 201e 102.6 ± 41.6 194 1.96 ± 0.38

Table 9. IC50 values for novel compounds towards A549 cells: the IC50 values given were calculated from the dose response curve plotted from 3 experiments. Standard deviations were calculated from IC50 values derived from independent dose response curves of each individual experiment.

5.0 GST/GSH-activated Prodrugs

The fact that GST enzymes are overexpressed in cancer cells and catalyse the conjugation of glutathione to various alkylating drugs, has led to the design and synthesis of GST-activated cytotoxic prodrugs. 109 In 1999, Gunnarsdottir and Elfarra reported the GSH-dependent prodrug cis-3- (9H-purin-6-ylthio)acrylic acid (PTA) (231), which upon reaction with GSH, both in vitro and in vivo, released the antitumour agent 6-mercaptopurine.110 The prodrug could be activated via two distinct mechanisms: a non-enzymatic indirect process or a GST-catalysed one (Figure 24). The non-enzymatic process involved nucleophilic attack by GSH on the C6 of the purine ring to give GSH adduct 233, which was then enzymatically converted to 6-mercaptopurine. The second pathway proceeded via a GST-catalysed conjugate addition reaction of GSH onto the β-carbon of the acrylic acid moiety, followed by an elimination reaction to give 6-mercaptopurine (232).109, 110

Figure 24. Mechanisms of activation of cis-3-(9H-purin-6-ylthio)acrylic acid

Another example of a GSH-activated prodrug was reported by Shami and co- workers.111 They screened a range of arylated diazeniumdiolates which upon reaction with with thiols, such as GSH, would release nitric oxide (NO). Nitric oxide inhibits cell growth in leukemia cells: hence the aim of the investigation was to

84 identify a prodrug that would deliver NO directly to leukemia cells, without inducing NO-mediated systemic hypertension. The most potent prodrug screened was JS-K

(235) which inhibited cell growth of HL-60 human leukemia cancer cells with an IC50 of 200-500 nM. The mechanism of action of JS-K involved reaction with GSH to give the Meisenheimer complex 236, which would then dissociate into the GSH conjugate (DNP-SG, 237) and diazeniumdiolate 238. At physiological pH, the diazeniumdiolate 238 would undergo a spontaneous decomposition to generate NO (Figure 25).111

Figure 25. Mechanism of action of JS-K

5.1 COMC-estradiol Conjugate as a Tissue Selective Prodrug

In 2009, Ganem and Oaksmith reported the use of an estradiol conjugate of COMC as a tissue selective chemotherapy prodrug. The aim of the investigation was to replace the crotonate side chain with a different carboxylate ester which was attached to a hormone: this would enable the delivery of the drug to specific tissues expressing the relevant hormone receptor prior to GSH activation.112 Estradiol is a hormone with a high affinity for oestrogen receptors (ER) which are overexpressed in breast, gonad and ovary tissues. The COMC-estradiol conjugate 240 was, therefore, synthesised to enable delivery of the drug to tissues expressing the estrogen receptor prior to activation by GSH. The bioactivity of the COMC-estradiol conjugate has not yet been reported.112

Figure 26. COMC-estradiol conjugate

5.2 Substituting the Crotonate ester with 4-Hydroxycoumarin Derivatives

The main objective of this study was to determine the key structural features necessary for optimum bioactivity: after synthesising a small library of novel hybrid analogues of COTC with variable aryl side chains (Section 3.0), it was decided to investigate the replacement of the crotonyl ester group by 4-hydroxycoumarin derivatives. Previous literature accounts have reported that 4-hydroxycoumarin analogues can act as inhibitors of the oxidoreductase enzyme NQO1113 and inhibition of the enzyme could lead to increased oxidative stress and ultimately to cell death.114 The concept behind the design of these prodrugs was that GSH activation would generate the exocyclic enone but also release the NQO1 inhibitor, leading to an enhanced bioactivity.

Scheme 70

5.2.1 NAD(P)H: Quinone Oxidoreductase-1 (NQO1) NAD(P)H quinone oxidoreductase-1 (NQO1), previously known as DT- diaphorase, is a homodimeric flavoenzyme located mainly in the cytosol of almost all mammalian tissues. It is a detoxifying enzyme, which catalyses the NAD(P)H dependant two-electron reduction of quinones to hydroquinones.115, 116

Q + NAD(P)H + H+ → QH2 + NAD(P)+ (Q = quinone)

Quinones are ubiquitous, toxicological intermediates which are commonly found in natural products or can be formed through the metabolism of hydroquinones and catechols. Both their potent redox activity and electrophilicity contribute to the quinone toxicity. Quinones are excellent Michael acceptors which can alkylate intracellular proteins and/or DNA leading to cell damage. In addition, they are highly redox active molecules which can generate reactive oxygen species (ROS) leading to oxidative stress within the cells.117 In mammalian cells there are two competing reactions by which quinones can be reduced; a one-electron process catalysed by enzymes such as NAD(P)H-cytochrome-

P450 and NADPH-cytochrome b5 reductase, and a two-electron reduction catalysed by NQO1.115, 116 The one-electron reduction of quinones leads to the formation of semiquinone radicals which in the presence of oxygen can generate superoxide anion radicals. These ROS are powerful oxidising agents and can therefore increase oxidative stress in cells by reacting with essential cellular macromolecules (lipids, proteins, DNA). NQO1 competes with the formation of ROS through the one-electron process, by reducing quinones into the more stable hydroquinone via a two-electron process. The action of NQO1 can therefore protect the cell from quinone-induced cytotoxicity, oxidative-stress and mutagenicity.115, 117

Scheme 71. Alkylation and redox cycling of quinones117

5.2.2 NQO1 as a Target for Selective Antitumour Therapy

NQO1 is upregulated in most human tumour cells. It was reported that NQO1 expression is much higher in lung, liver, colon, breast and pancreas tumour tissues, compared to the corresponding normal tissues. The largest difference in NQO1 activity was observed between lung adenocarcinoma cells and normal lung cells, with a 123-fold increase in activity. It was hypothesised that the enzyme is upregulated in cancer cells in order to satisfy the needs of the rapid tumour cell metabolism. The high expression of NQO1 in tumour cells makes it an attractive therapeutic target for selective antitumour therapy. Inhibition of NQO1 would lead to an increased production of reactive oxygen species and hence induce oxidative stress in cells that would eventually lead to inhibition of cell growth. The most commonly used inhibitor of NQO1 is dicoumarol (247), a naturally occurring coumarin-derived anticoagulant. It acts by competing with NAD(P)H for the binding site of NQO1, hence preventing the two-electron transfer. In 2004, Cullen and co-workers reported that inhibition of NQO1 with dicoumarol induced oxidative stress and decreased cell viability in MIA PaCa-2 pancreatic cancer cells. It was suggested that the oxidative stress was induced due to an increased production of O2·- radicals, which was evidenced by elevated levels of glutathione and oxidised glutathione.114

Figure 27. Dicoumarol

Unfortunately, however, dicoumarol is not a selective inhibitor and has numerous “off target” effects caused by extensive protein binding. This has led to numerous investigations into the development of novel NQO1 inhibitors which would be as potent as dicoumarol but lack the “off target” effects. An investigation into the synthesis and biological evaluation of novel NQO1 inhibitors was published by Moody and co-workers in 2008.118 The researchers reported the syntheses of a range of indolequinones which were found to be

88 mechanism-based inhibitors of NQO1. The mechanism of action of these heterocyclic quinones involved an irreversible alkylation of the enzyme which led to inhibition of the NQO1 function both in enzyme and cell assays. Furthermore, an MTT viability assay was carried out, which indicated that indolequinones 248 and 249 were growth inhibitors of human pancreatic MIA PaCa-2 cancer cells with an IC50 of 629 nM and 638 nM respectively.

Figure 28. Indoquinolines

The Whitehead and Stratford groups at the University of Manchester have been investigating the design, synthesis and biological evaluation of novel NQO1 inhibitors for a number of years. Initially, “dicoumarol-like” inhibitors were identified by screening the National Cancer Institute (NCI) database and in 2009 a small library of unsymmetrical and symmetrical dicoumarol analogues was synthesised. The novel dicoumarol analogues shown in Figure 29 were identified as potent NQO1 inhibitors with IC50 values in the nanomolar range.113

Figure 29. Novel NQO1 inhibitors

In 2013, a previous member of the Whitehead group reported the synthesis of a series of 4-hydroxycoumarin analogues which were potent inhibitors of NQO1.119

Analogue 253, which was reported to inhibit NQO1 with an IC50 of 816 nm, was selected as a potential replacement of the crotonyl ester group during the preparation of the GSH-activated prodrugs.

Figure 30. Novel NQO1 inhibitor

5.2.3 Synthesis of NQO1 Inhibitor 253

The 4-hydroxycoumarin analogue 253 was prepared following the procedure reported in 2013 by Chee.119 The first step of the synthesis involved a base-catalysed cyclisation of 2-hydroxy-6-methoxy-acetophenone with diethyl carbonate, in the presence of sodium hydride to give 4-hydroxy-5-methoxycoumarin (255).

Scheme 72

4-Hydroxy-5-methoxycoumarin (255) was then alkylated at the C3 position with benzyl alcohol, utilising the borrowing hydrogen methodology. Alcohols generally display limited reactivity towards alkylation reactions and hence have to be activated before they can react.120 The borrowing hydrogen methodology involves activation of alcohols by temporarily removing hydrogen, in order to generate more reactive aldehydes which subsequently can be transformed into alkenes, imines or used to form new C-C bonds.121 The most commonly used catalysts are iridium and ruthenium complexes and the mechanism of the reaction is based on three steps: oxidation, alkene formation and reduction. Oxidation of the alchohol via dehydrogenation gives the corresponding aldehyde or ketone which undergoes an aldol condensation to furnish an alkene intermediate. Finally, the alkene is reduced by hydrogen transfer from the catalyst to the double bond.122

Scheme 73. Iridium catalysed ‘borrowing hydrogen’ reaction

The most effective system reported for the alkylation of 4-hydroxy-5- methoxycoumarin with benzyl alcohol, was 5 mol% [Cp*IrCl2]2 in the presence of caesium carbonate and isopropanol.119, 123 It is believed that the carbonate activates the catalyst by binding to the metal to give an iridium-carbonate complex. The benzyl alcohol then coordinates to the complex and two hydrogen transfers occur: the first from the benzyl alcohol to the carbonate and then from the alcohol to iridium to form benzaldehyde. Dissociation of the benzaldehyde followed by an in situ aldol condensation with the coumarin leads to an intermediate alkene. Finally, the alkene is reduced by a step-by-step reverse oxidation, to give the required product.124

The reaction was carried out using 5 mol% [Cp*IrCl2]2 in the presence of caesium carbonate and isopropanol, heating at 110 °C for 20 h. The required product was isolated by flash silica chromatography in a 72% yield.

Scheme 74

5.2.4 GSH-activated prodrugs for the intracellular release of an NQO1 inhibitor

The novel compounds 257-259 shown in Figure 31 were synthesised and their antiproliferative activity towards A549 and MDA-MB-468 cell lines was assessed using an MTT assay. Compounds 257 a, b are analogues of COMC, while 259 a, b

91 were prepared in order to compare the bioactivity with the corresponding crotonylated hybrid analogues. Both the analogues which lack the enone moiety (258) and the individual coumarins were asssayed as control experiments.

Figure 31. Potential GSH-activated NQO1 prodrugs

The intermediate alcohol 218 was synthesised by a DMAP catalysed Morita- Baylis-Hillman reaction on 1-cyclohex-2-enone and the 1-hydroxymethylene cyclohexene (261) was prepared by a DIBAL reduction of the corresponding methyl ester (260). The synthesis of the t-butyl substituted analogue 221 is described in Section 3.5. The key step in the preparation of the NQO1 prodrugs was the introduction of the coumarin via a Mitsunobu reaction.

Scheme 75. Reagents and conditions: (i) DMAP (cat.), H2CO, THF, rt, 20 h, 13%; (ii) DIAD, PPh3, rt, a – 22 h, 19%, b – 24 h, 55%; (iii) 1M DIBAL in toluene, toluene, -78 °C, 30 mins, 68%; (iv) DIAD, PPh3, rt, a – 17 h, 32%, b – 20 h, 34%; (v) DIAD, PPh3, rt, a – 17 h, b – 24 h, 59%; (vi) TFA:H2O (7:1), rt, a – 40 mins, 27% over two steps, b – 30 mins, 76%.

The Mitsunobu reaction is a versatile coupling reaction which is widely used to prepare esters, aryl or cyclic ethers and C-N bonds. The reaction involves the coupling of a primary or secondary alcohol with an acidic nucleophile, in the presence of a reducing phosphine reagent such as triphenylphosphine (PPh3) and an oxidising azo reagent, such as diethyl azodicarboxylate (DEAD) or diisopropyl azocarboxylate (DIAD). The main byproducts of the reaction, which commonly cause problems during purification, are the corresponding hydrazide and phosphine oxide.125

The mechanism of the reaction involves an initial nucleophilic addition of PPh3 to DIAD, to generate the reactive betaine intermediate 264, which then deprotonates the acidic nucleophile to form an ion pair. Deprotonation of the alcohol gives DIAD-

H2, the carboxylate ion 266 and the oxyphosphonium ion 267. Finally, nucleophilic displacement of triphenylphosphine oxide by the carboxylate ion generates the coupled product.

Scheme 76. Mitsunobu reaction mechanism

The Mitsunobu reaction with alcohols 218, 261 and 221 required the use of two equivalents of the coumarin in the presence of DIAD and PPh3. Unfortunately, the yields after purification by flash silica chromatography were generally poor (19- 55%). The antiproliferative activity of all novel compounds was assessed via an MTT assay on the A549 cell line and a selection of the compounds was also assayed against MDA-MB-468 cell lines. The A549 cell line is a human non-small cell lung

93 cancer cell line with upregulated levels of NQO1, whereas MDA-MB-468 is a human breast adenocarcinoma cell line with low NQO1 activity. The IC50 values of the novel compounds, together with COMC and the COTC hybrid analogues 177b and 178b are summarised in Table 10.

IC50 (µM) Compound A549 MDA-MB-468 COMC 54.5 - 257a 10 ± 1 - 257b 0.71 ± 0.33 7.6 ± 2.2 258a Not toxic at 100 µM - 258b Not toxic at 100 µM - 259a 4.4 ± 2.5 2.8 ± 2.5 177b 19 ± 5 - 178b 3.6 ± 3.2 - 4-hydroxycoumarin Not toxic at 100 µM - 253 Not toxic at 100 µM -

Table 10. IC50 values for novel compounds towards A549 and MDA-MB-468 cells; the IC50 values given were calculated from the dose response curve plotted from 3 experiments. Standard deviations were calculated from IC50 values derived from independent dose response curves of each individual experiment.

The results of the MTT viability assay indicate that both COMC analogues 257a and 257b are more potent than COMC, suggesting that substituting the crotonyl side chain with a coumarin does enhance the potency of the drug. Compound 257a is five times more potent than COMC, while analogue 257b with an IC50 of 710 nM is about 80 times more potent and it is the most active compound synthesised by the Whitehead group to date. In order to investigate whether the increase in activity was due to inhibition of NQO1, 257b was also assayed against MDA-MB-468 cells which have low levels of the NQO1 enzyme. The IC50 against MDA-MB-468 cells increased to 7.6 µM, suggesting that NQO1 concentrations may have an impact on the activity. To confirm the hypothesis that 257b has an enhanced bioactivity due to inhibition of NQO1, the MTT assay has to be repeated on genetically modified MDA-MB-468 cells which overexpress NQO1. The data has also confirmed that compounds 258a and 258b which lack the enone moiety and hence cannot readily be activated by GSH, do not inhibit cell growth. Furthermore, the individual coumarins do not show any activity either, probably due to the fact that they are ionised at physiological pH and therefore the net diffusion of the drug across the lipid bilayer membrane of the cell is affected. Finally, the activity of 259a did not change significantly compared to 178b. Unfortunately, the enhancement in activity of 257a compared to COMC was not mirrored between 259a and 178b. The inhibitory effect of 259a was greater than 177b, demonstrating that a side chain has an impact on the bioactivity. The antiproliferative effect of compound 257b has not been assessed yet. The results for the COMC analogues 257a and 257b are promising, but in order to confirm that the enhancement in bioactivity is a combination of NQO1 inhibition and the action of the GSH activated exocyclic enone, further investigations need to be undertaken.

6.0 Displacement of the Crotonate Side-chain by Thiols

In 2002, Ganem and collaborators investigated the enzymatic and non-enzymatic reaction of COMC with GSH and cysteine. They reported that in the non-enzymatic reactions no intermediate species could be detected, whereas for the human placental glutathione transferase (GSTP1-1) catalysed reactions the profile of the reaction changed into a double exponential decay. The reaction profile consisted of a rapid, enzyme-dependent initial step generating the intermediate exocyclic enone, which after dissociation from the enzyme reacted non-enzymatically with GSH or cysteine to form the corresponding adduct.

Scheme 77

In order to confirm that the crotonate ester side could be displaced by a thiol, the BDA-protected adduct 272 was treated with cysteamine hydrochloride and N- protected cysteamine. The BDA-protected adduct 272 was prepared by an initial DMAP-catalysed Morita Baylis Hillman reaction on the BDA-protected enone 117, to give the hydroxymethylated intermediate 271 in 95% yield. The free hydroxyl group was then esterified with crotonic anhydride in the presence of DMAP and pyridine. Both steps had been optimised by previous members of the Whitehead group.

Scheme 78. Synthesis of the crotonate ester 272- Reagents and conditions: (i) formaldehyde 37%, DMAP, THF, H2O, 40 °C, 95%; (ii) crotonic anhydride, DMAP, py, CH2Cl2, rt, 87%.

The next step in the sequence involved the displacement of the crotonate ester group. Intermediate 272 was therefore treated with cysteamine hydrochloride in the presence of triethylamine: the 1H NMR of the crude reaction mixture after work up showed no signals for the crotonate ester group, but the product could not be isolated. In order to make the purification of the resulting amine easier, it was decided to synthesise its corresponding amide in a one pot reaction. The same reaction was carried out, but after reacting with cysteamine for 2 h, more triethylamine was added followed by acetic anhydride and DMAP. Purification by flash silica chromatography afforded the required amide in 17% yield.

Scheme 79. Reagents and conditions: (i) cysteamine.HCl, Et3N, CH2Cl2, 2 h, rt; (ii) acetic anhydride, Et3N, rt, 17% over 2 steps.

To investigate the reactivity of the crotonylated enone further, it was decided to N-protect the cysteamine and repeat the reaction. The cysteamine was N- protected using di-tert-butyl dicarbonate in the presence of triethylamine to give the Boc-protected product which was subsequently reacted with 272 in the presence of triethylamine to give the required product 277 in 58% yield.

Scheme 80

The displacement of the crotonate ester was successful with both cysteamine.HCl and N-protected cysteamine, which agrees with the findings reported by Ganem. In order, however, to confirm that the analogues reported in this thesis are activated by GSH, further investigations need to be undertaken.

7.0 Conclusion and Future Work

A small library of novel anti-tumour agents has been prepared from the chiral pool starting material (-)-quinic acid. The synthetic routes developed and optimised proceeded via the key enone intermediates 117 and 35. The synthesis of the BDA- protected enone 117 from (-)-quinic acid was achieved in 63% yield over four steps, whereas the cyclohexylidene protected enone 35 was prepared in 48% over four steps. The introduction of alkyl and aryl substituents at C3 of the two quinate enones was achieved using organocuprate chemistry and rhodium catalysed conjugate additions of organoboron reagents. The conjugate addition reactions on the cyclohexylidene protected enone afforded only the anti product 141, while the corresponding reactions on the BDA protected enone resulted in a mixture of the syn and anti products (anti-140 and syn-140).

Scheme 81

7.1 Transition Metal-mediated Conjugate Addition Reactions

7.1.1 Conjugate Addition Reactions on BDA-protected Enone

The conjugate addition reactions on the BDA-protected enone were investigated with a range of aryl and alkyl nucleophiles, using organocuprate

99 chemistry and rhodium catalysed reactions of boronic acids. In both cases, a mixture of syn and anti products was afforded. The rhodium catalysed reactions with various boronic acids were carried out with 5mol% of [RhCl(cod)]2 or [RhOH(cod)]2 in the presence of triethylamine, using dioxane:water (10:1) as a solvent, at room temperature. The final products were isolated by HPLC or crystallisation in relatively poor yields. The organocuprate conjugate addition reactions on the BDA-enone were investigated extensively: a range of nucleophiles were reacted under a variety of reaction conditions. The stereoselectivity of the reaction was explored by altering the reaction temperature and by the addition of TMSCl. Unfortunately, due to the variability of the results a definite conclusion regarding the stereoselectivity of the organocuprate reaction could not be drawn. It was concluded therefore that the stereoselectivity of the organocuprate conjugate addition of nucleophiles to the BDA substrate was unpredictable and the outcome could be affected by the substrate, the additive and temperature.

7.1.2 Conjugate Addition Reactions on the Cyclohexylidene-protected Enone The conjugate addition reactions of the cyclohexylidene-protected enone were diastereoselective: only the anti product was afforded due to steric effects from the protecting group that would only allow addition of the nucleophile from the “convex” face of the molecule. This led to improved reaction times and yields compared to the corresponding reactions on the BDA-protected enone. Rhodium catalysed reactions of boronic acids gave relatively high yields with the best yield being achieved with 4-fluorophenyl boronic acid (89%). Prior to this investigation there was only one literature report describing an organocuprate 1,4-addition on the cyclohexylidene protected enone which was published by Falck and co-workers in 1996.84 In our investigation conjugate addition reactions were attempted on the cyclohexylidene-protected enone 35 using cuprate reagents derived from 4-tbutylphenylmagnesium bromide, phenylmagnesium bromide and 4-methoxyphenylmagnesium bromide. Both the 4-tbutylphenyl and

100 phenyl conjugate adducts were isolated in a 73% yield, however, the reaction with 4- methoxyphenyl cuprate afforded a mixture of the required product and the biphenyl by-product.

7.2 Synthesis of Novel Anti-tumour Agents

The transition metal-mediated conjugate addition reaction procedures developed for the introduction of an aryl substituent to C3 of the quinate enones, were then applied to the synthesis of hybrid analogues of COTC and antheminone A, 177-180.

Figure 32. Novel hybrid analogues of COTC and antheminone A

To complete the synthesis, the BDA protecting group was removed using DBSA in water and the cyclohexylidene protecting group was eliminated using either DBU or a catalytic amount of aqueous NaOH. The resulting allylic alcohols were protected as their silyl ethers: the silylation was achieved with TESOTf and 2,6- lutidine at -50 °C. A Morita-Baylis-Hillman (M-B-H) reaction under surfactant conditions was then carried out on the γ-silyloxy cyclohex-2-enones in order to introduce a hydroxymethyl group at C2. Deprotection of the silyl-protected hydroxyl group afforded the diol enone compounds, which are structurally related to the antheminones and carvotacetones, while crotonylation and then deprotection generated the crotonylated compounds which are hybrid analogues of COTC.

101

Scheme 82. Optimised routes to the target molecules-Reagents and conditions: (i) DBSA, H2O, 100 °C; (ii) DBU, CH2Cl2, rt; (iii) TESOTf, 2,6-lutidene, CH2Cl2, -50 °C; (iv) SDS, formaldehyde 37%, DMAP, H2O, rt; (v) crotonic anhydride, DMAP, py, CH2Cl2; (vi) TFA:H2O (7:1).

7.2.1 MTT viability assay

The antiproliferative activity of the novel compounds towards A549 non- small cell lung cancer cells was assessed using the MTT assay and this showed that almost all of the analogues were more potent than COMC. This suggests that the combination of a hydroxyl group at C4 and a lipophilic side chain at C5 enhances the activity of the compounds. The assays also demonstrated that the crotonylated analogues 178a-d were significantly more active than the corresponding diols 177a- d. The most potent compounds of this series were 178d and 179, with IC50 values of 1.2 ± 0.5 µM and 1.3 ± 0.2 µM respectively. Unfortunately both compounds were also toxic towards the non-tumourigenic breast cancer cell line MCF10A.

102

Figure 33. Most potent analogues

In order to further investigate the structural features necessary for optimum bioactivity, compounds bearing different side chains could also be prepared. Previous investigations by members of the Whitehead group have indicated that cytotoxicity towards A549 cells increases with an increase in leaving group ability. Compound 278 is a COMC analogue which has a pentafluorobenzoate side chain instead of the crotonate ester and it was found to be more potent than COMC with an

IC50 of 1.3 µM against A549 cells. Analogues 178d and 179 could therefore be altered in order to incorporate the pentafluorobenzoate side chain instead of the crotonate ester.

Figure 34. COMC analogue with a pentafluorophenyl side chain

7.2.2 Perhydro-dibenzofuranones Related to the Natural Product Incarviditone

During the preparation of the COTC hybrid analogues a fortuitous discovery led to the synthesis of a range of diastereoisomerically pure perhydro-dibenzofuranones. These dimers are structurally related to the natural product incarviditone which has been found to display anticancer activity. The antiproliferative activity of the perhydro-dibenzofuranones (201b-e) was assessed towards A549 cells and the t-butylphenyl substituted dimer 201b demonstrated the most significant activity with an IC50 of 12 µM. Further studies are necessary in order to establish the precise mode of action of this class of compounds. 103

Scheme 83. Perhydro-dibenzofuranones

7.3 GSH-activated Prodrugs for Intracellular Release of NQO1 Inhibitor

Investigations into the structural features necessary for optimum bioactivity led to the replacement of the crotonyl ester side chain by an NQO1 inhibitor. The concept behind the design of these compounds was that GSH activation would generate the exocyclic enone but also release the NQO1 inhibitor, leading to an enhanced antiproliferative activity. The novel compounds 257-259 (Figure 33) were designed and synthesised and their antiproliferative activity was assessed via an MTT assay on the A549 and MDA-MB-468 cell lines.

Compound 257b with an IC50 of 710 nM was found to be about 80 times more active than COMC and it is the most potent compound synthesised by the Whitehead group to date. The IC50 against MDA-MB-468 cells - which have low NQO1 levels- was substantially higher at 7.6 µM, suggesting that the cytotoxicity of the drug may be due to inhibition of NQO1. To confirm the hypothesis that the enhanced bioactivity of 257b is due to inhibition of NQO1, the MTT assay should be repeated on genetically modified MDA-MB-468 cells which overexpress NQO1.

Figure 35

The enhanced bioactivity of the COMC analogue 257b is intriguing, but in order to confirm that the increase in potency is a combination of NQO1 inhibition and the 104 action of the GSH activated exocyclic enone, further studies need to be carried out. For example, the crotonate ester side chain of the most potent COTC hybrid analogue 178d should be replaced by a coumarin based NQO1 inhibitor, in order to compare bioactivities. Additionally, more potent NQO1 inhibitors such as coumarins 280 and 281, which have been reported by Chee, should also be investigated as potential side chains.

Figure 36

105

8.0 Experimental

Chromatography Flash silica chromatography was performed using silica gel (Sigma-Aldrich) 40 – 63 m 60 Å. The solvent mixtures used are reported in individual procedures. Infra-red Spectroscopy Recorded as KBr discs using an ATI Mattson genesis series or Perkin Elmer FT-IR instrument. The recorded absorption maxima (νmax) are expressed in wavenumbers (cm1). The intensity and appearance of peaks are described using the following abbreviations: w, weak; m, medium; s, strong; br, broad. Mass spectroscopy Mass spectroscopy was performed by the staff in the Mass Spectrometry Laboratory, School of Chemistry, at the University of Manchester. Major peaks are reported as mass/charge (m/z) ratios. The mass values reported are within  5 ppm mass units for electrospray. Melting Points Recorded on a Sanyo Gallenkamp MPD350 heater. Nuclear Magnetic Resonance 1H NMR experiments were carried out using a Bruker Avance 400 and 500. 1H assignments were supported by 2D 1H-1H COSY. Chemical shifts (H) are given in parts per million (ppm) to the nearest 0.01 ppm and referenced to the residual non- deuterated solvent peak. The coupling constants (J) are given to the nearest 0.1 Hz. Spectral data is reported as follows: chemical shift, integration, multiplicity, coupling constant(s) and assignment. Multiplicity is described using the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; or as a combination of these dd, dt etc. 13C NMRs were recorded using a Bruker Avance 400. 13C assignments were supported by 2D one-bond 13C-1H HMQC. Chemical shifts (C) are recorded in ppm to the nearest 0.01 ppm and referenced to the residual non-deuterated solvent peak.

106

Solvents THF was distilled from sodium and benzophenone under nitrogen atmosphere and Petroleum ether (b.p. 40-60) was distilled before use. All reactions were carried out with under dry conditions in an inert nitrogen atmosphere using a nitrogen balloon, unless otherwise stated. Reaction temperatures of 0 °C were achieved using an ice water bath and -78 °C by using a dry ice acetone bath. Room temperature refers to 20-25 °C.

8.1 Experimental Procedures and Analysis of Compounds

8.1.1 (2'S,3'S,3R,4R,5R)-Methyl-4-O,5-O-(2',3'-dimethoxybutane-2',3'-diyl)- quinate (114)

C14H24O8; Mw 320.34 To a solution of (-)-quinic acid (2 g, 10.4 mmol) and CSA (0.27 g, 1.2 mmol) in methanol (50 mL) was added trimethylorthoformate (8.8 mL, 80.4 mmol) and butan- 2,3-dione (2.1 mL, 23.9 mmol). The yellow solution was heated at reflux under an atmosphere of N2 for 22 h. The reaction was allowed to cool to room temperature and it was then quenched by the addition of triethylamine (10 mL). The solution was concentrated in vacuo to give a dark red oil. The oil was dissolved in ethyl acetate (15 mL) and decolourised by activated charcoal. It was filtered through celite and concentrated in vacuo to give a yellow oil. Recrystallisation from hot ethyl acetate and cold petroleum ether, afforded the title compound (114) as yellow crystals (2.48 g, 75%); mp 136.1-137.8 °C [Lit.126 mp 138-140 °C]; [α]D24 +119.8 (c 1.00 in CH2Cl2)

[Lit.126 [α]D20 +116.3 (c 1.06 in CH2Cl2)]; νmax(film)/cm-1 3445br (O-H), 2991s (C-H),

2952s (C-H), 2835s (C-H), 1738s (C=O ester); δH(400MHz; CDCl3) 1.31 (3H, s, CH3),

1.35 (3H, s, CH3), 1.93 (1H, ~t, J 12.6, C(6)Hax), 2.05 (1H, dd, J 14.8, 3.0, C(2)Hax), 2.11

(1H, ddd, J 12.6, 4.6, 2.9, C(6)Heq), 2.19 (1H, d~t, J 14.8, 2.9, C(2)Heq), 3.27 (3H, s,

OCH3), 3.27 (3H, s, OCH3), 3.61 (1H, dd, J 10.2, 2.9, C(4)H), 3.80 (3H, s, CO2CH3), 4.19-

107

4.21 (1H, m, C(3)H), 4.32 1H, ddd, J 12.6, 10.2, 4.6, C(5)H); δC(100MHz; CDCl3) 17.7

(CH3), 17.8 (CH3), 37.4 (C(2)H2), 38.6 (C(6)H2), 47.9 (2 x acetal OCH3), 52.9 (CO2CH3), 62.4 (C(5)H), 69.2 (C(3)H), 72.7 (C(4)H), 75.8 (C(1)), 99.8 (acetal C), 100.3 (acetal C),

174.3 (CO2CH3); m/z (+ES) 343 ([M+Na]+, 100%).

8.1.2 (2'S,3'S,3R,4R,5R)-4-O,5-O-(2',3'-Dimethoxybutane-2',3'-diyl)-cyclohexan- 1-one-3,4,5-triol (116)

C12H20O6; Mw 260.28 To a solution of 114 (2.48 g, 7.74 mmol) in MeOH (60 mL) at 0 °C, sodium borohydride (2.92 g, 0.08 mmol) was added portionwise. The reaction mixture was stirred at room temperature under an atmosphere of N2 for 17 h. The reaction was quenched by pouring onto a cold saturated aqueous solution of NH4Cl (15mL). The resulting mixture was concentrated in vacuo to give a white solid. Ethyl acetate (50 mL) was added and the resulting suspension was filtered. The residue was washed with ethyl acetate (3 x 10 mL) and the combined organic filtrates were dried over

MgSO4 and concentrated in vacuo to give the crude triol (115) as a white solid (4.49 g). Sodium periodate (8.20 g, 38.3 mmol) was dissolved in hot water (30 mL). To the resulting suspension silica gel (~30 g) was added portionwise to give a silica- supported sodium periodate as a free flowing powder. To this, DCM (120 mL) was added, followed by a solution of the crude triol (4.48 g, 15.3 mmol) in DCM (60 mL).

The reaction mixture was stirred at room temperature under an atmosphere of N2 for 1.5 h, then it was filtered and the residue was washed with DCM (3 x 50 mL). The filtrate was dried over MgSO4 and concentrated in vacuo to give the title compound

(116) as a white solid (4.06 g, 79%); mp 166.8-170.1 °C [Lit.93 mp 163-165 °C]; [α]D24

+145 (c 1.00 in CH2Cl2) [Lit.93 [α]D20 +159.8 (c 0.59 in CH2Cl2)]; νmax(film)/cm-1 3443br (O-H), 2992w (C-H), 2948w (C-H), 2899w (C-H), 2834w (C-H), 1718s (C=O ketone); δH(400MHz; CDCl3) 1.32 (3H, s, CH3), 1.35 (3H, s, CH3), 2.47-2.54 (2H, m, 1 of

108

C(2)H2 and 1 of C(6)H2 ), 2.64-2.69 (2H, m, 1 of C(2)H2 and 1 of C(6)H2), 3.24 (3H, s,

OCH3), 3.32 (3H, s, OCH3), 3.90 (1H, dd, J 9.9, 2.5, C(4)H), 4.25-4.32 (2H, m, C(3)H and

C(5)H); δC(100MHz; CDCl3) 17.6 (CH3), 17.7 (CH3), 44.7 (C(2)H2 or C(6)H2), 46.1

(C(2)H2 or C(6)H2), 47.9 (acetal OCH3), 48.1 (acetal OCH3), 63.2 (C(3)H or C(5)H), 67.7 (C(3)H or C(5)H), 72.2 (C(4)H), 99.2 (acetal C), 100.3 (acetal C), 205.5 (C=O); m/z (+ES) 283 ([M+Na]+, 100%).

8.1.3 (2'S,3'S,3R,4R,5R)-4-O,5-O-(2',3'-Dimethoxybutane-2',3'-diyl)-4,5- dihydroxy-cyclohex-2-en-1-one (117)

C12H18O5; Mw 242.27 To a stirred solution of hydroxyketone 116 (3 g, 11.5 mmol) in DCM (50 mL) at 0 °C under an atmosphere of N2, methanesulfonyl chloride (1.08 mL, 13.8 mmol) and triethylamine (4.62 mL, 33.4 mmol) were added and the reaction mixture was stirred at room temperature for 3 h. The reaction was quenched by the addition of water (50 mL). The two layers were separated and the aqueous layer was extracted with DCM (2 x 50mL). The combined organic extracts were washed with a saturated aqueous solution of sodium carbonate (2 x 50 mL), dried over MgSO4 and concentrated in vacuo to give the crude product as a pale yellow solid. The residue was dissolved in DCM and preabsorbed onto silica, then it was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:4) to give the title compound (117) as a white solid (2.51 g, 90%); mp 178.6-181.3 °C [Lit.93 mp 182-184°C]; [α]D24 +65.2 (c 1.00 in CH2Cl2) [Lit.93 [α]D20 +64.4 (c 0.39 in CH2Cl2)];

νmax(film)/cm-1 3002w (C-H), 2928w (C-H), 2854w (C-H), 2836w (C-H), 1679s (C=O enone); δH(400MHz; CDCl3) 1.34 (3H, s, CH3), 1.38 (3H, s, CH3), 2.50 (1H, dd, J 16.4,

13.5, C(6)Hax), 2.75 (1H, ddd, J 16.4, 5.0, 1.3, C(6)Heq), 3.27 (3H, s, OCH3), 3.33 (3H, s,

OCH3), 4.06 (1H, ddd, J 13.5, 9.1, 5.0, C(5)H), 4.51 (1H, ddd, J 9.1, 2.7, 1.8, C(4)H), 6.01

(1H, ddd, J 10.1, 2.7, 1.3 C(2)H), 6.88 (1H, dd, J 10.1, 1.8, C(3)H); δC(100MHz; CDCl3)

17.7 (CH3), 17.7 (CH3), 42.0 (C(6)H2), 48.1 (OCH3), 48.2 (OCH3), 68.1 (C(5)H), 69.2

109

(C(4)H), 99.7 (acetal C), 100.8 (acetal C), 130.1 (C(2)H), 148.5 (C(3)H), 196.8 (C=O); m/z (+ES) 265 ([M+Na]+, 100%).

8.1.4 (1S,3R,4R,5R)-3-O,4-O-Cyclohexylidene-7-oxo-6-oxabicyclo[3.2.1]octan- 1,3,4-triol (123)

C13H18O5; Mw 254.27 A mixture of (-)-quinic acid (5 g, 26.0 mmol) and cyclohexanone (16 mL, 158.7 mmol) in DMF (20 mL) and toluene (30 mL) were refluxed under an atmosphere of N2 in a flask fitted with a Dean and Stark trap and a condenser for 30 mins. After allowing to cool to room temperature, Amberlite® resin IR (5 g) (previously washed with MeOH, filtered and washed again with diethyl ether and dried under vacuum) was added. The resulting suspension was heated at reflux for 5 h and then allowed to cool to room temperature. The resin was filtered off and the filtrate was washed with a saturated solution of sodium bicarbonate (2 x 60 mL), water (2 x 60 mL) and brine

(60 mL), then dried over MgSO4 and concentrated in vacuo to give a white solid (123)

(4.18 g, 63%). mp 141.6-143.8 °C [Lit.74 mp 139-141 °C]; [α]D30 -30.6 (c 1.00 in

CH2Cl2) [Lit.74 [α]D22 -33.0 (c 1.05 in CHCl3)];] δH(300MHz; CDCl3)127 1.55-1.75 (10H, m, 5 x CH2 of cyclohexane), 2.20 (1H, dd, J 14.7, 3.0, C(2)Hax), 2.27-2.40 (2H, m,

C(2)Heq and C(6)Heq), 2.68 (1H, d, J 11.7, C(6)Hax), 4.31 (1H, ddd, J 6.4, 2.6, 1.5,

C(4)H), 4.49 (1H, ~td, J 6.4, 3.0, C(3)H), 4.75 (1H, dd, J 6.4, 2.6, C(5)H); δC(100MHz;

CDCl3) 23.5, 23.9, 25.0, 33.6 (4 x CH2 of cyclohexane), 34.3 (C(6)H2), 36.9 (CH2 of cyclohexane), 38.5 (C(2)H2), 71.1 (C(3)H), 71.5 (C(1)), 71.7 (C(4)H), 76.0 (C(5)H); m/z (+ES) 277 ([M+Na]+, 100%).

110

8.1.5 3-O,4-O-Cyclohexylidene-(3R,4S,5R)-trihydroxycyclohexanone (125)

C12H18O4; Mw 226.27 To a solution of the lactone (123) (4.1 g, 16.1 mmol) in methanol (200 mL) at 0°C, sodium borohydride (6.1 g, 161.2 mmol) was added portionwise. Once effervescence had ceased, the reaction mixture was stirred at room temperature under an atmosphere of N2 for 20 h. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (30 mL). The resulting mixture was concentrated in vacuo to give a white solid. Ethyl acetate (100 mL) was added and the resulting suspension was filtered. The residue was washed with ethyl acetate (3 x 30 mL) and the combined organic filtrates were dried over MgSO4 and concentrated in vacuo to give the crude triol (124) as an off white solid (4.20 g). Sodium periodate (8.69 g, 40.6 mmol) was dissolved in hot water (10 mL). To the resulting suspension silica gel (~20 g) was added portionwise to give silica- supported sodium periodate as a free flowing powder. To this, DCM (50 mL) was added, followed by a solution of 124 (4.20 g, 16.3 mmol) in DCM (40 mL). The reaction mixture was stirred at room temperature under an atmosphere of N2 for 1.5 h, then it was filtered and the residue was washed with DCM (3 x 50 mL). The filtrate was dried over MgSO4 and concentrated in vacuo to give the title compound (125) as a white solid (3.34 g, 92% over two steps). mp 92.6-94.8 °C [Lit.128 mp 97-98 °C];

[α]D30 +98.6 (c 1.00 in CH2Cl2) [Lit.128 [α]D +100.3 (c 0.44 in CH3OH)]; δH(400MHz;

CDCl3)127 1.55-1.66 (10H, m, 5 x CH2 of cyclohexane), 1.75 (1H, d, J 3.3, OH), 2.46 (1H, ddd, J 17.9, 3.0, 2.2, C(6)Heq), 2.69-2.74 (2H, m, C(2)Heq and C(6)Hax), 2.81 (1H, dd, J

17.6, 3.4, C(2)Hax), 4.27 (1H, ~q, J 3.0, C(5)H), 4.30 (1H, d~t, J 7.1, 3.0, C(4)H), 4.71

(1H, d~t, J 7.1, 3.4, C(3)H); δC(100MHz; CDCl3) 23.5, 23.9, 25.1, 35.6, 36.3 (5 x CH2 of cyclohexane), 40.4 (C(2)H2), 41.9 (C(6)H2), 68.3 (C(5)HOH), 71.5 (C(3)H), 74.2 (C(4)H), 109.3 (cyclohexane C), 206.9 (C=O); m/z (+ES) 249 ([M+Na]+, 100%).

111

8.1.6 (3aR,7aS)-3a,4-dihydrospiro[benzo[d][1,3]dioxole-2,1'-cyclohexan]- 5(7aH)-one (35)

C12H16O3; Mw 208.25 To a solution of the hydroxyketone (125) (3.30 g, 14.6 mmol) in dichloromethane

(60 mL) at 0°C under N2, was added methanesulfonyl chloride (1.35 mL, 17.5 mmol) and triethylamine (5.86 mL, 13.6 mmol). The reaction mixture was stirred at room temperature for 4 h and then quenched with water (20 mL) and extracted with dichloromethane (2 x 20 mL). The combined organic extracts were washed with water (2 x 20 mL), brine (20 mL), dried over MgSO4 and concentrated in vacuo to give an orange oil. The residue was purified by flash silica chromatography eluting with ethyl acetate:40-60 petroleum ether (1:3) to yield the title compound (35) as an off-white solid (2.50 g, 82 %). mp 49.8-51.0 °C [Lit.72 mp 55-58 °C]; [α]D30 +98.6 (c

1.00 in CH2Cl2) [Lit.128 [α]D +100.3 (c 0.44 in CH3OH)]; δH(400MHz; CDCl3) 1.57-1.63

(10H, m, 5 x CH2 of cyclohexane), 2.69 (1H, dd, J 17.6, 3.8, one of C(6)H2), 2.96 (1H, ddd, J 17.6, 2.8, 1.0, one of C(6)H2), 4.68-4.74 (2H, m, C(4)H and C(5)H), 6.02 (1H, d~t,

J 10.2, 1.3, C(2)H), 6.66 (1H, ddd, J 10.2, 2.7, 2.0, C(3)H); δC(100MHz; CDCl3) 23.6,

23.8, 24.9, 35.8, 37.1 (5 x CH2 of cyclohexane), 38.9 (C(6)H2), 70.7 (C(4)H or C(5)H), 72.9 (C(4)H or C(5)H), 110.7 (cyclohexane C), 128.7 (C(2)H), 146.3 (C(3)H), 195.8 (C=O); m/z (+ES) 231 ([M+Na]+, 75%), 263 ([M+Na+MeOH]+, 70), 460 (100).

8.1.7 (2S,3S,4aR,8S,8aR)-2,3-dimethoxy-2,3-dimethyl-8-phenylhexa- hydrobenzo-[b][1,4] dioxin-6(7H)-one (syn-140a)

C18H23O5; Mw 320.38 Method 1 To a stirred suspension of copper iodide (236 mg, 1.24 mmol) in THF

(2 mL) at 0 °C, was added phenylmagnesium bromide (3.0M in Et2O) (0.83 mL, 2.48

112 mmol) and the reaction mixture was stirred for 1 h at 0 °C, under an atmosphere of

N2. Chlorotrimethylsilane (0.52 mL, 4.13 mmol) and a solution of enone (117) (200 mg, 0.83 mmol) in THF (8 mL) were added dropwise and the reaction mixture was stirred at 0 °C for 3.5 h. The reaction was quenched by the addition of a mixture of a saturated aqueous solution of NH4Cl and aqueous NH3 solution (9:1, 10 mL). The two layers were separated and the aqueous layer was extracted with diethyl ether (3 x 10 mL). The combined organic extracts were washed with brine (15 mL), dried over MgSO4 and concentrated in vacuo to give a white oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:6), and recrystallised from diethyl ether and petroleum ether, to give the title compound

(syn-140a) as an off-white solid (97 mg, 37%); mp 141.8-143.6 °C; [α]D26 +91.6 (c

1.00 in CH2Cl2); νmax(film)/cm-1 2990w (C-H), 2954w (C-H), 2912w (C-H), 2830w (C-

H), 1715s (C=O ketone); δH(400MHz; CDCl3) 1.24 (3H, s, CH3), 1.25 (3H, s, CH3), 2.57

(1H, dd, J 15.4, 12.1 C(6)Hax), 2.79 (1H, ddd, J 15.4, 5.3, 2.4, C(6)Heq), 2.79 (1H, dd, J

16.0, 6.2, C(2)Hax), 2.91 (1H, d~t, J 16.0, 2.4, C(2)Heq), 2.97 (3H, s, OCH3), 3.38 (3H, s,

OCH3), 3.54 (1H, ~t, J 6.2, C(3)H), 3.75 (1H, ddd, J 12.1, 10.3, 5.3, C(5)H), 4.15 (1H, dd,

J 10.3, 6.2, C(4)H), 7.21-7.30 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 17.5 (CH3), 17.6

(CH3), 40.5 (C(3)H), 44.9 (C(2)H2), 45.2 (C(6)H2), 47.6 (acetal OCH3), 48.1 (acetal

OCH3), 63.5 (C(5)H), 71.3 (C(4)H), 98.7 (acetal C), 99.7 (acetal C), 126.7 (Ar-CH), 127.8 (Ar-CH), 129.1 (Ar-CH), 138.9 (Ar-C), 208.3 (C=O); m/z (-ES) 319 ([M-H]-,

100%); (Found 343.1505, C18H24O5Na ([M+Na]+), requires 343.1516).

Method 2: To a mixture of phenyl boronic acid (91 mg, 0.74 mmol) and [RhCl(cod)]2 (9.5 mg, 5mol%), a solution of enone 117 (90 mg, 0.37 mmol) in dioxane:water (10:1,

3 mL) was added followed by Et3N (0.05 mL, 0.37 mmol). The reaction mixture was stirred at room temperature for 4 h. The solvents were evaporated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:5) to give a white solid. Recrystallisation from diethyl ether and petroleum ether, afforded the title compound (syn-140a) as colourless crystals (40 mg, 34%).

113

8.1.8 (2S,3S,4aR,8R,8aR)-2,3-dimethoxy-2,3-dimethyl-8-phenylhexahydro benzo-[b][1,4] dioxin-6(7H)-one (anti-140a)

C18H23O5; Mw 320.38 Followed the same procedure as for compound syn-140a (Method 1), however on addition of the chlorotrimethylsilane and the enone, the reaction mixture was allowed to warm to room temperature and it was stirred at room temperature for 2.5 h. Recrystallisation from diethyl ether and petroleum ether gave compound anti-

140a as colourless crystals (135 mg, 51%). mp 92.6-95.3 °C; [α]D28 +173.5 (c 1.30 in

CH2Cl2); νmax(film)/cm-1 2990w (C-H), 2954w (C-H), 2912w (C-H), 2830w (C-H),

1715s (C=O); δH(400MHz; CDCl3) 1.17 (3H, s, CH3), 1.31 (3H, s, CH3), 2.57-2.68 (2H, m, C(6)H2), 2.68-2.78 (2H, m, C(2)H2), 2.96 (3H, s, OCH3), 2.97-2.03 (1H, m, C(3)H),

3.27 (3H, s, OCH3), 3.95 (1H, ddd, J 11.1, 9.3, 7.1, C(5)H), 4.12 (1H, dd, J 10.8, 9.3,

C(4)H), 7.24-7.36 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 17.6 (CH3), 17.6 (CH3), 43.2

(C(3)H), 44.9 (C(6)H2), 47.5 (C(2)H2), 47.9 (acetal OCH3), 48.1 (acetal OCH3), 68.5 (C(5)H), 73.5 (C(4)H), 99.5 (acetal C), 99.8 (acetal C), 127.1 (Ar-CH), 127.5 (Ar-CH), 128.4 (Ar-CH), 205.7 (C=O); m/z (+ES) 321 ([M+H]+, 100%); (Found 343.1519,

C18H24O5Na ([M+Na]+), requires 343.1516).

8.1.9 (2S,3S,4aR,8S,8aR)-8-(4-fluorophenyl)-2,3-dimethoxy-2,3-dimethylhexa hydrobenzo[b][1,4]dioxin-6(7H)-one (syn-140b)

C18H23FO5; Mw 338.37 Method 1: To a stirred suspension of copper iodide (173 mg, 0.91 mmol) in THF (2 mL) at 0 °C, was added 4-fluorophenylmagnesium bromide (1.0M in THF) (1.82 mL, 1.82 mmol) and the reaction mixture was stirred for 1 h at 0 °C, under N2.

114

Chlorotrimethylsilane (0.37 mL, 2.89 mmol) and a solution of enone (117) (200 mg, 0.83 mmol) in THF (6 mL) were added dropwise and the reaction mixture was stirred at 0 °C, under an atmosphere of N2 for 2 h. The reaction was quenched by the addition of a mixture of a saturated aqueous solution of NH4Cl and aqueous NH3 solution (9:1, 10 mL). The two layers were separated and the aqueous layer was extracted with diethyl ether (3 x 10 mL). The combined organic extracts were washed with brine (15 mL), dried over MgSO4 and concentrated in vacuo to give a white oil. The crude residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:6), and recrystallised from hot methanol and water, to give the title compound (syn-140b) as colourless crystals (111 mg, 40%); mp 133.8-136.2 °C; [α]D26 +91.6 (c 1.00 in CH2Cl2); νmax(film)/cm-1 2991w (C-H),

2949w (C-H), 2903w (C-H), 2834w (C-H), 1715s (C=O ketone); δH(400MHz; CDCl3)

1.24 (3H, s, CH3), 1.26 (3H, s, CH3), 2.57 (1H, dd, J 15.3, 12.3 C(6)Hax), 2.70 (1H, ddd, J

15.3, 5.5, 2.3, C(6)Heq), 2.80 (1H, dd, J 16.1, 5.7, C(2)Hax), 2.88 (1H, d~t, J 16.1, 2.3,

C(2)Heq), 2.98 (3H, s, OCH3), 3.38 (3H, s, OCH3), 3.51 (1H, ~t, J 5.7, C(3)H), 3.69 (1H, ddd, J 12.3, 10.3, 5.5, C(5)H), 4.15 (1H, dd, J 10.3, 5.7, C(4)H), 6.95 (1H, d, J 8.8, C(3´)H or C(5´)H), 6.97 (1H, d, J 8.8, C(3´)H or C(5´)H), 7.20 (2H, m, C(2´)H and C(6´)H);

δC(100MHz; CDCl3) 17.5 (CH3), 17.6 (CH3), 39.8 (C(3)H), 44.9 (C(2)H2), 45.2 (C(6)H2),

47.7 (acetal OCH3), 48.1 (acetal OCH3), 63.5 (C(5)H), 71.2 (C(4)H), 98.7 (acetal C), 99.7 (acetal C), 114.5 (Ar-CH), 130.6 (Ar-CH), 208.1 (C=O); m/z (-ES) 337 ([M-H]-),

100%), (Found 361.1424, C18H23O5FNa ([M+Na]-), requires 361.1422).

Method 2: To a mixture of 4-fluorophenyl boronic acid (144 mg, 1.03 mmol) and

[RhCl(cod)]2 (10 mg, 5 mol%), a solution of enone 117 (100 mg, 0.41 mmol) in dioxane:water (10:1, 3 mL) was added followed by Et3N (0.05 mL, 0.41 mmol). The reaction mixture was stirred at room temperature for 6 h. The solvents were evaporated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:5) to give a white solid. Recrystallisation from hot methanol and cold water, afforded the title compound (syn-140b) as colourless crystals (70 mg, 50%).

115

8.1.10 (2S,3S,4aR,8R,8aR)-2,3-dimethoxy-2,3-dimethyl-8-vinylhexahydro benzo[b][1,4] dioxin-6(7H)-one (anti-140c)

C14H22O5; Mw 270.32 To a stirred suspension of copper iodide (173 mg, 0.91 mmol) in THF (2 mL) at -78 °C, was added vinylmagnesium bromide (1.0M in THF) (1.82 mL, 1.82 mmol) and the reaction mixture was stirred for 1 h at -78 °C, under an atmosphere of N2. Chlorotrimethylsilane (0.37 mL, 2.89 mmol) and a solution of enone 117 (200 mg, 0.83 mmol) in THF (6 mL) were added dropwise and the reaction mixture was stirred at -78 °C for 2 h. The reaction was quenched by the addition of a mixture of asaturated aqueous solution of NH4Cl and aqueous NH3 solution (9:1, 10 mL). The two layers were separated and the aqueous layer was extracted with diethyl ether (3 x 10 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO4 and concentrated in vacuo to give a yellow oil. The crude residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:10), and recrystallised from hot petroleum ether, to give the title compound

(anti-140c) as white crystals (75 mg, 33%); mp 99.7-101.8°C; [α]D24 +203 (c 1.00 in

CH2Cl2); νmax(film)/cm-1 2995w (C-H), 2958w (C-H), 2884w (C-H), 2833w (C-H),

1721s (C=O ester); δH(400MHz; CDCl3) 1.32 (3H, s, CH3), 1.34 (3H, s, CH3), 2.31 (1H,

~t, J 14.7, C(2)Hax), 2.46-2.59 (3H, m, C(2)Heq and C(3)H and C(6)Hax), 2.63 (1H, ddd, J

14.1, 5.6, 2.2, C(6)Heq), 3.26 (3H, s, OCH3), 3.28 (3H, s, OCH3), 3.67 (1H, ~t, J 9.7,

C(4)H), 3.84 (1H, ddd, J 12.4, 9.7, 6.0, C(5)H), 5.10-5.17 (2H, m, C=CH2), 5.91 (1H, ddd,

J 17.1, 10.6, 6.0, C=CH); δC(100MHz; CDCl3) 17.6 (CH3), 17.7 (CH3), 40.1 (C(3)H), 44.7

(C(2)H2 or C(6)H2), 44.8 (C(2)H2 or C(6)H2), 48.0 (acetal OCH3), 48.1 (acetal OCH3),

68.2 (C(5)H), 73.3 (C(4)H), 99.5 (acetal C), 99.7 (acetal C), 116.3 (C=CH2), 136.5 (C=CH), 205.9 (C=O).

116

8.1.11 2,3-dimethoxy-2,3-dimethyl-8-(naphthalen-1-yl)hexahydrobenzo [b][1,4]dioxin-6(7H)-one (syn-140d, anti-140d)

C22H26O5; Mw 370.44

To a mixture of 1-naphthyl boronic acid (142 mg, 0.83 mmol) and [RhCl(cod)]2 (9 mg, 5 mol%), a solution of enone 117 (100 mg, 0.41 mmol) in dioxane:water (10:1, 3 mL) was added followed by Et3N (0.05 mL). The reaction mixture was stirred at room temperature for 4 h. The solvents were evaporated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:5) to give the title compound in a 1:1 ratio of axial:equatorial diastereoisomers, as a white solid. The diastereoisomers were separated by preparative HPLC, eluting with n-hexane:ethyl acetate (4:1) to give syn-140d (42 mg, 27%) and anti-140d (31 mg, 20%) as white solids.

Syn-140d mp 159.7-161.6 °C; [α]D38 +220 (c 0.66 in CH2Cl2); νmax(film)/cm-1 2948w

(C-H), 2831w (C-H), 1713s (C=O ketone); δH(400MHz; CDCl3) 0.94 (3H, s, CH3), 1.20

(3H, s, CH3), 2.69 (1H, dd, J 15.6, 12.2, C(6)Hax), 2.82 (1H, d~t, J 16.4, 2.0, C(2)Heq),

2.86 (1H, ddd, J 15.6, 5.5, 2.4, C(6)Heq), 2.96 (3H, s, OCH3), 2.97 (1H, dd, J 16.4, 6.4,

C(2)Hax), 3.44 (3H, s, OCH3), 4.05 (1H, ddd, J 12.2, 10.3, 5.5, C(5)H), 4.40 (1H, dd, J 10.3, 6.4, C(4)H), 4.51 (1H, ~t, J 6.4, C(3)H), 7.04 (1H, d, J 7.5, C(2´)H or C(4´)H), 7.39 (1H, ~t, J 7.5, C(3´)H), 7.44-7.49 (2H, m, C(6´)H and C(7´)H), 7.76 (1H, d, J 7.8, C(2´)H or C(4´)H), 7.81-7.83 (1H, m, C(5´)H or C(8´)H), 8.28-8.30 (1H, m, C(5´)H or C(8´)H);

δC(100MHz; CDCl3) 17.2 (CH3), 17.5 (CH3), 36.2 (C(3)H), 45.2 (C(6)H2), 45.8 (C(2)H2),

47.6 (acetal OCH3), 48.1 (acetal OCH3), 68.4 (C(5)H), 71.5 (C(4)H), 98.7 (acetal C), 99.6 (acetal C), 124.8 (Ar-CH), 124.9 (Ar-CH), 125.2 (Ar-CH), 125.3 (Ar-CH), 127.8 (Ar-CH), 128.3 (Ar-CH), 133.6 (Ar-C), 208.6 (C=O); m/z (-ES) 369 ([M-H]-, 100%);

(Found 371.1856, C22H27O5 ([M+H]+), requires 371.1853).

Anti-140d mp 152.6-155.0 °C; [α]D38 +203.1 (c 0.60 in CH2Cl2); νmax(film)/cm-1

2924w (C-H), 1718s (C=O ketone); δH(400MHz; CDCl3) 1.07 (3H, s, CH3), 1.32 (3H, s,

CH3), 2.59 (1H, t, J 14.2, C(6)Hax), 2.69-2.80 (3H, m, C(6)Heq, C(2)Hax and C(2)Heq),

117

3.09 (3H, s, OCH3), 3.30 (3H, s, OCH3), 3.92-3.99 (1H, m, C(5)H), 4.08-4.16 (1H, m, C(3)H), 4.47 (1H, ~t, J 10.0, C(4)H), 7.47-7.56 (4H, m, C(2´)H, C(3´)H, C(6´)H and C(7´)H), 7.78 (1H, d, J 7.8, C(4´)H), 7.86-7.88 (1H, m, C(5´)H), 8.11 (1H, d, J 8.1,

C(8´)H); δC(100MHz; CDCl3) 17.6 (CH3), 17.7 (CH3), 36.8 (C(3)H), 45.1 (C(2)H2), 48.1

(acetal OCH3), 48.2 (C(6)H2), 48.4 (acetal OCH3), 68.7 (C(5)H), 73.2 (C(4)H), 99.6 (acetal C), 99.8 (acetal C), 122.5 (Ar-CH), 123.1 (Ar-CH), 125.1 (Ar-CH), 125.7 (Ar- CH), 126.1 (Ar-CH), 126.7 (Ar-CH), 127.4 (Ar-CH), 128.7 (Ar-CH), 205.9 (C=O); m/z (-

ES) 369 ([M-H]-, 100%); (Found 371.1856, C22H27O5 ([M+H]+), requires 371.1853).

8.1.12 2,3-dimethoxy-2,3-dimethyl-8-(naphthalen-2-yl)hexahydrobenzo [b][1,4]dioxin-6(7H)-one (syn-140e, anti-140e)

C22H26O5; Mw 370.44

To a mixture of 2-naphthyl boronic acid (142 mg, 0.83 mmol) and [RhCl(cod)]2 (9 mg, 5 mol%), a solution of enone 117 (100 mg, 0.41 mmol) in dioxane:water (10:1, 3 mL) was added followed by Et3N (0.05 mL, 0.41 mmol). The reaction mixture was stirred at room temperature for 6 h. The solvents were evaporated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:5) to give a 3:1 mixture of syn:anti diastereoisomers, as a white solid. The diastereoisomers were separated by preparative HPLC, eluting with 4:1 n-hexane: ethyl acetate to give syn-140e (25 mg, 16%) and anti-140e (12 mg, 8%) as white solids.

Syn-140e mp 103.0-105.4 °C; [α]D38 +28.8 (c 0.56 in CH2Cl2); νmax(film)/cm-1 2911w

(C-H), 2330w (C-H), 2361w (C-H), 1718s (C=O ketone); δH(400MHz; CDCl3) 1.24 (3H, s, CH3), 1.28 (3H, s, CH3), 2.61 (1H, dd, J 15.3, 12.1, C(6)Hax), 2.75 (1H, ddd, J 15.3, 5.6,

2.4, C(6)Heq), 2.88 (1H, dd, J 16.0, 6.7, C(2)Hax), 2.92 (3H, s, OCH3), 3.04 (1H, d~t, J

16.0, 4.0, C(2)Heq), 3.42 (3H, s, OCH3), 3.71 (1H, ~t, J 4.0, C(3)H), 3.82 (1H, ddd, J 12.1, 10.3, 5.6, C(5)H), 4.27 (1H, dd, J 10.3, 4.0, C(4)H), 7.45-7.48 (2H, m, C(6´)H and C(7´)H), 7.49 (1H, dd, J 8.6, 1.8, C(3´)H), 7.59 (1H, s, C(1´)H), 7.75 (1H, d, J 8.8,

118

C(4´)H), 7.79-7.83 (2H, m, C(5´)H and C(8´)H); δC(100MHz; CDCl3) 17.5 (CH3), 17.6

(CH3), 40.6 (C(3)H), 45.0 (C(6)H2), 45.3 (C(2)H2), 47.6 (acetal OCH3), 48.1 (acetal

OCH3), 63.7 (C(5)H), 71.6 (C(4)H), 98.7 (acetal C), 99.8 (acetal C), 125.7 (Ar-CH), 125.7 (Ar-CH), 127.1 (Ar-CH), 127.4 (Ar-CH), 128.0 (Ar-CH), 128.3 (Ar-CH), 132.3 (Ar-C), 133.0 (Ar-C), 136.8 (Ar-C), 208.1 (C=O); m/z (-ES) 369 ([M-H]-, 100%).

Anti-140e mp 213.6-214.5 °C; [α]D38 +239.1 (c 0.90 in CH2Cl2); νmax(film)/cm-1

2999w (C-H), 2924w (C-H), 2903w (C-H), 1718s (C=O ketone); δH(400MHz; CDCl3)

1.19 (3H, s, CH3), 2.06 (3H, s, CH3), 2.65 (1H, ddd, J 17.0, 5.3, 1.9, C(2)Heq), 2.71-2.81

(2H, m, C(6)Hax and C(6)Heq) , 2.72 (1H, dd, J 17.0, 12.9, C(2)Hax), 2.95 (3H, s, OCH3),

3.18 (1H, ddd, J 12.9, 11.1, 5.3, C(3)H), 3.29 (3H, s, OCH3), 4.01 (1H, ddd, J 11.6, 9.5, 6.5, C(5)H), 4.28 (1H, dd, J 11.1, 9.5, C(4)H), 7.44 (1H, dd, J 8.6, 1.8, C(3´)H), 7.46-7.53 (2H, m, C(6´)H and C(7´)H), 7.73 (1H, s, C(1´)H), 7.79-7.83 (2H, m, C(5´)H and C(8´)H),

7.84 (1H, d, J 8.6, C(4´)H); δC(100MHz; CDCl3) 17.6 (CH3), 17.6 (CH3), 43.4 (C(3)H),

45.0 (C(6)H2), 47.7 (C(2)H2), 48.0 (acetal OCH3), 48.1 (acetal OCH3), 68.5 (C(5)H), 73.4 (C(4)H), 99.6 (acetal C), 99.9 (acetal C), 125.1 (Ar-CH), 125.8 (Ar-CH), 126.2 (Ar- CH), 126.9 (Ar-CH), 127.6 (Ar-CH), 127.7 (Ar-CH), 132.6 (Ar-C), 136.1 (Ar-C), 137.2 (Ar-C), 205.6 (C=O); m/z (-ES) 369 ([M-H]-, 100%).

8.1.13 (3aS,4S,7aR)-4-(4-methoxyphenyl)tetrahydrospiro[benzo [d][1,3] dioxole-2,1'-cyclohexan]-6(3aH)-one (141c)

C18H24O4; Mw 316.39 To a solution of enone 35 (260 mg, 1.25 mmol) in dioxane:water (10:1, 2.2 mL) was added 4-methoxyphenylboronic acid (474 mg, 3.12 mmol) and [RhOH(cod)]2 (28 mg,

5 mol%), followed by Et3N (0.16 mL, 1.25 mmol). The reaction mixture was stirred at room temperature for 5 h. The solvents were evaporated in vacuo to give an orange residue. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound (141c) as a pale yellow solid (325 mg, 82%). mp 109.8-111.1 °C; [α]D27 -105.6 (c 0.50 in CH2Cl2);

119

νmax(film)/cm-1 2935s (C-H), 2856s (C-H), 1718s (C=O ketone); δH(400MHz; CDCl3)

1.35-1.75 (10H, m, 5 x CH2 of cyclohexane), 2.59 (1H, dd, J 17.5, 8.4, C(2)Hax), 2.62

(1H, dd, J 17.2, 4.6, C(6)Heq or C(6)Hax), 2.73 (1H, dd, J 17.2, 4.6, C(6)Hax or C(6)Heq),

2.75 (1H, dd, J 17.5, 4.6, C(2)Heq), 3.38 (1H, dt, J 8.4, 4.6 C(5)H), 4.54-4.60 (1H, m, C(3)H and C(4)H), 6.86-6.90 (2H, m, 2x Ar-H), 7.14-7.18 (2H, m, 2x Ar-H);

δC(100MHz; CDCl3) 23.6 (cyclohexane CH2), 23.9 (cyclohexane CH2), 25.1

(cyclohexane CH2), 33.7 (cyclohexane CH2), 36.9 (cyclohexane CH2), 40.9 (C(2)H2),

41.8 (C(5)H), 42.3 (C(6)H2), 55.2 (OCH3), 72.1 (C(3)H or C(4)H), 76.7 (C(3)H or C(4)H), 109.3 (cyclohexane C), 114.1 (Ar-CH), 128.4 (Ar-CH), 132.0 (Ar-C), 158.4 (Ar- C), 209.0 (C=O); m/z (+ES) 339 ([M+Na]+, 100%).

8.1.14 (3aS,4S,7aR)-4-(4-fluorophenyl)tetrahydrospiro[benzo[d][1,3] dioxole- 2,1'-cyclohexan]-6(3aH)-one (141d)

C18H21FO3; Mw 304.36 To a solution of enone 35 (270 mg, 1.30 mmol) in dioxane:water (10:1, 2.2 mL) was added 4-fluorophenyl boronic acid (453 mg, 3.24 mmol) and [RhOH(cod)]2 (29 mg, 5 mol%), followed by Et3N (0.17 mL, 1.30 mmol). The reaction mixture was stirred at room temperature for 20 h. The solvents were evaporated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound (141d) as a white thick oil

(350 mg, 89%). [α]D34 -54.8 (c 1.00 in CH2Cl2); νmax(film)/cm-1 2932s (C-H), 2856s (C-

H), 1718s (C=O); δH(400MHz; CDCl3) 1.35-1.75 (10H, m, 5 x CH2 of cyclohexane), 2.57

(1H, dd, J 17.2, 9.6, C(6)Hax), 2.65 (1H, dd, J 17.2, 5.6, C(6)Heq), 2.72 (1H, dd, J 14.0,

4.9, C(2)Hax or C(2)Heq), 2.76 (1H, dd, J 14.0, 4.9, C(2)Hax or C(2)Heq), 3.38 (1H, ddd, J 9.6, 6.0 , 5.6, C(5)H), 4.51 (1H, ~t, J 6.0, C(4)H), 4.59 (1H, d~t, J 6.0, 4.9, C(3)H), 7.27 (2H, ~t, J 8.6, C(3')H and C(5')H), 7.44 (2H, dd, J 8.6, 5.1, C(2')H and C(6')H);

δC(100MHz; CDCl3) 23.6 (cyclohexane CH2), 23.9 (cyclohexane CH2), 25.1

(cyclohexane CH2), 33.8 (cyclohexane CH2), 37.1 (cyclohexane CH2), 41.2 (C(2)H2),

120

42.2 (C(5)H), 42.4 (C(6)H2), 72.1 (C(3)H), 77.5 (C(4)H), 109.5 (cyclohexane C), 115.7 (d, J 21, C(3')H and C(5')H), 128.9 (d, J 9, C(2')H and C(6')H), 135.9 (d, J 3, C(1')H), 161.8 (d, J 244, C(F)), 208.4 (C=O); m/z (+ES) 327 ([M+Na]+, 100%); (Found

327.1362, C18H21O3FNa ([M+Na]+), requires 327.1367).

8.1.15 (3aS,4S,7aR)-4-(naphthalen-1-yl)tetrahydrospiro[benzo[d][1,3] dioxole- 2,1'-cyclohexan]-6(3aH)-one (141e)

C22H24O3; Mw 336.42

To a mixture of the 1-naphthylboronic acid (83 mg, 0.48 mmol) and [RhCl(cod)]2 (6 mg, 5 mol%), a solution of enone 35 (50 mg, 0.24 mmol) in dioxane:water (10:1,

2 mL) was added followed by Et3N (0.03 mL, 0.24 mmol). The reaction mixture was stirred at room temperature for 1.5 h. The solvents were evaporated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound (141e) as an off-white solid (56 mg, 69%). mp 123.2-125.7 °C; δH(400MHz; CDCl3) 1.32-1.79 (10H, m, 5 x CH2 of cyclohexane), 2.70-2.80 (3H, m, C(2)H2 and C(6)Hax), 3.02 (1H, dd, J 17.9,

5.4, C(6)Heq), 4.22-4.68 (1H, m, C(5)H), 4.65 (1H, d~t, J 6.9, 4.3, C(3)H), 4.75 (1H, dd, J 6.9, 3.6, C(4)H), 7.19 (1H, d, J 7.8, C(2´)H or C(4´)H), 7.42 (1H, ~t, J 7.8, C(3´)H), 7.48-7.63 (2H, m, C(6´)H and C(7´)H), 7.79 (1H, d, J 7.8, C(2´)H or C(4´)H), 7.89 (1H, d,

J 8.3, C(5´)H or C(8´)H), 8.15 (1H, d, J 8.3, C(5´)H or C(8´)H); δC(100MHz; CDCl3) 23.6

(cyclohexane CH2), 23.9 (cyclohexane CH2), 25.2 (cyclohexane CH2), 33.5

(cyclohexane CH2), 36.7 (cyclohexane CH2), 39.5 (C(2)H2), 40.5 (C(6)H2), 41.2 (C(5)H), 72.2 (C(3)H), 76.1 (C(4)H), 109.1 (cyclohexane C), 123.3 (Ar-CH), 123.8 (Ar- CH), 125.2 (Ar-CH), 125.9 (Ar-CH), 126.5 (Ar-CH), 128.0 (Ar-CH), 129.2 (Ar-CH), 136.3 (Ar-C), 209.4 (C=O); m/z (+ES) 359 ([M+Na]+, 100%); (Found 359.1624,

C22H24O3Na ([M+Na]+), requires 359.1618).

121

8.1.16 (3aS,4S,7aR)-4-(naphthalen-2-yl)tetrahydrospiro [benzo [d][1,3] dioxole-2,1'-cyclohexan]-6(3aH)-one (141f)

C22H24O3; Mw 336.42

To a mixture of 2-naphthylboronic acid (165 mg, 0.96 mmol) and [RhCl(cod)]2 (12 mg, 5 mol%), a solution of enone 35 (100 mg, 0.48 mmol) in dioxane:water (10:1,

2 mL) was added followed by Et3N (0.07 mL, 0.48 mmol). The reaction mixture was stirred at room temperature for 1 h. The solvents were concentrated in vacuo to give an orange residue which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound (141f) as a colourless crystalline solid (134 mg, 83%). mp 83.3-85.3 °C; [α]D24 -74.4 (c 1.00 in

CH2Cl2); νmax(film)/cm-1 3047w (C-H), 3017w (C-H), 2933s (C-H), 2861s (C-H), 1718s

(C=O); δH(400MHz; CDCl3) 1.37-1.71 (10H, m, 5 x CH2 of cyclohexane), 2.68 (1H, dd, J

17.4, 4.9, C(2)Hax or C(2)Heq), 2.76 (1H, dd, J 17.7, 8.1, C(6)Hax), 2.77 (1H, dd, J 17.4,

4.9, C(2)Hax or C(2)Heq), 2.87 (1H, dd, J 17.7, 5.9, C(6)Heq), 3.60 (1H, d~t, J 8.1, 5.9, C(5)H), 4.64 (1H, d~t, J 5.9, 4.9, C(3)H), 4.75 (1H, ~t, J 5.9, C(4)H), 7.43 (1H, dd, J 8.7, 1.9, C(3´)H), 7.46-7.53 (2H, m, C(6´)H and C(7´)H), 7.62 (1H, s, C(1´)H), 7.78-7.86 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3) 23.6 (cyclohexane CH2), 23.9

(cyclohexane CH2), 25.1 (cyclohexane CH2), 33.7 (cyclohexane CH2), 36.9

(cyclohexane CH2), 40.6 (C(2)H2), 42.3 (C(6)H2), 42.8 (C(3)H), 72.2 (C(4)H), 77.1 (C(5)H), 109.3 (cyclohexane C), 125.7 (Ar-CH), 125.9 (Ar-CH), 126.4 (Ar-CH), 127.7 (Ar-CH), 128.5 (Ar-CH), 132.3 (Ar-C), 205.9 (C=O); m/z (+ES) 359 ([M+Na]+, 100%);

(Found 359.1616, C22H24O3Na ([M+Na]+), requires 359.1618).

8.1.17 Di-μ-chlorido-bis[η2,η2-(cycloocta-1,5-diene)rhodium] (175)

122

C20H32Cl2Rh2; Mw 549.18

To a mixture of rhodium trichloride (2 g, 9.56 mmol) and Na2CO3 (1.01 g, 9.56 mmol) under N2 was added deoxygenated ethanol:water (5:1, 25 mL) and 1,5- cyclooctadiene (4 mL, 28.7 mmol). The reaction mixture was heated at 40 °C for 19 h and it was then allowed to cool to room temperature. The yellow-orange precipitate was filtered off and washed with pentane (50 mL), followed by methanol:water (1:5, 60 mL) until the washings no longer contained Cl-. Recrystallisation from hot dichloromethane afforded the title compound (175) as orange crystals (330 mg, 7%). mp 220 – 224 °C (It darkens from about 190 °C) [Lit.129 mp 256 °C, darkens from 220

°C]; δH(400MHz; CDCl3) 1.76 (8H, m, 4 x CH2), 2.51 (8H, m, 4 x CH2), 4.24 (8H, s, 8 x CH).

8.1.18 (4R,5S)-4-((tert-butyldimethylsilyl)oxy)-5-(naphthalen-2-yl)cyclohex-2- enone (188)

C23H28O2Si; Mw 352.19 To a solution of 141f (110 mg, 0.33 mmol) in dichloromethane (5 mL), was added TBSOTf (0.10 mL, 0.39 mmol) followed by DBU (0.05 mL, 0.36 mmol). The reaction mixture was stirred at room temperature under an atmosphere of nitrogen for 2 h. More DBU (0.01 mL, 0.10 mmol) was added and the reaction was stirred for a further 2.5 h. The reaction was quenched by the addition of water (3 mL) and diluted with dichloromethane (5 mL). The two layers were separated and the organic layer was washed with 1M HCl (5 mL), 1M NaHCO3 (5 mL) and brine (5 mL). The organic extract was dried over MgSO4 and concentrated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound (188) as a pale yellow solid (50 mg,

43%). mp 56.2-58.3 °C; [α]D34 -86.8 (c 0.28 in CH2Cl2); νmax(film)/cm-1 2955s (C-H),

2931s (C-H), 2856s (C-H), 1685s (C=O ketone); δH(400MHz; CDCl3) -0.63 (3H, s,

Si(CH3)), -0.17 (3H, s, Si(CH3)), 0.72 (9H, s, SiC(CH3)3), 2.75 (1H, ddd, J 16.5, 4.1, 1.2,

123

C(6)Heq), 2.91 (1H, dd, J 16.5, 13.8 C(6)Hax), 3.45 (1H, ddd, J 13.8, 9.5, 4.1, C(5)H), 4.64 (1H, d~t, J 9.5, 1.8, C(4)H), 6.07 (1H, d~t, J 10.3, 1.8, C(2)H), 6.89 (1H, dd, J 10.3, 1.8, C(3)H), 7.40 (1H, dd, J 8.4, 1.8, C(3´)H), 7.45-7.52 (2H, m, C(6´)H and C(7´)H), 7.70

(1H, s, C(1´)H), 7.80-7.85 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3) -5.8

(Si(CH3)), -5.2 (Si(CH3)), 17.8 (SiC(CH3)3), 25.5 (SiC(CH3)3), 42.6 (C(6)H2), 50.8 (C(5)H), 72.9 (C(4)H), 125.7 (Ar-CH), 125.9 (Ar-CH), 126.2 (Ar-CH), 127.3 (Ar-CH), 127.6 (Ar-CH), 128.1 (Ar-CH), 128.5 (C(2)H), 132.7 (Ar-C), 133.3 (Ar-C), 138.0 (Ar-C), 153.9 (C(3)H), 198.5 (C=O); m/z (-ES) 387 ([M+Na]-, 100%); (Found 375.1765,

C22H28O2NaSi ([M+Na]+), requires 375.1751).

8.1.19 tert-Butyldimethyl(((3aS,4S,7aR)-4-(naphthalen-2-yl)- 3a,4,5,7a - tetrahydro spiro [benzo[d][1,3]dioxole-2,1'-cyclohexan]-6- yl)oxy)silane (190)

C28H38O3Si; Mw 450.69 To a solution of 141f (146 mg, 0.43 mmol) in dichloromethane (2 mL), under an atmosphere of N2, was added Et3N (0.06 mL, 0.43 mmol). The reaction mixture was stirred at room temperature for 20 h. More Et3N (0.06 mL, 0.43 mmol) and TBSOTf (0.12 mL, 0.52 mmol) was added and the reaction mixture was stirred for 4 h. The reaction was quenched by the addition of H2O (10 mL) and extracted with dichloromethane (2 x 10 mL). The combined organic extracts were washed with 1M

HCl (10 mL), 1M NaHCO3 (10 mL) and brine (10 mL). The organic extract was dried over MgSO4 and evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:90) to give the title compound (190) as a white oil (150 mg, 76%). δH(400MHz;

CDCl3) 0.20 (3H, s, Si(CH3)), 0.22 (3H, s, Si(CH3)), 0.93 (9H, s, SiC(CH3)3), 1.51-1.85

(10H, m, 5 x CH2 of cyclohexane), 2.34 (1H, dd, J 17.2, 5.0, C(6)Heq), 2.57 (1H, dm, J

17.2, C(6)Hax), 3.37 (1H, ddd, J 15.4, 10.1, 5.0, C(5)H), 4.32 (1H, dd, J 10.1, 5.1, C(4)H), 4.70 (1H, t, J 5.1, C(3)H), 5.10 (1H, dd, J 5.1, 1.9, C(2)H), 7.41-7.48 (3H, m, C(3´)H,

124

C(6´)H and C(7´)H), 7.70 (1H, s, C(1´)H), 7.79-7.83 (3H, m, C(4´)H, C(5´)H and

C(8´)H); δC(100MHz; CDCl3) -4.6 (Si(CH3)), -4.2 (Si(CH3)), 18.0 (SiC(CH3)3), 23.7

(cyclohexane CH2), 24.1 (cyclohexane CH2), 25.1 (cyclohexane CH2), 25.6 (SiC(CH3)3),

35.5 (cyclohexane CH2), 35.7 (C(6)H2), 38.7 (cyclohexane CH2), 44.0 (C(5)H), 72.6 (C(3)H), 77.6 (C(4)H), 100.0 (C(2)H), 109.6 (cyclohexane C), 125.4 (Ar-CH), 125.9 (Ar-CH), 126.4 (Ar-CH), 126.5 (Ar-CH), 127.6 (Ar-CH), 127.7 (Ar-CH), 128.0 (Ar-CH), 132.5 (Ar-C), 133.6 (Ar-C), 139.4 (Ar-C), 155.5 (C(1)); m/z (+ES) 473 ([M+Na]+, 100%).

8.1.20 (4R,5S)-4-hydroxy-5-(naphthalen-2-yl)cyclohex-2-enone (189)

C16H14O2; Mw 238.10 Method 1: To a solution of 141f (70 mg, 0.21 mmol) in dichloromethane (4 mL) was added DBU (30 μL, 0.23 mmol) and the reaction mixture was stirred at room temperature for 2 h 45 min. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL) and organic material was extracted into dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine

(10 mL), dried over MgSO4 and evaporated in vacuo to give a brown gum. This residue was purified by flash silica chromatography eluting with ethyl acetate:40-60 petroleum ether (1:2) to give the title compound (189) as a cloudy oil (37 mg, 75%).

[α]D33 -88.5 (c 0.40 in CH2Cl2); νmax(film)/cm-1 3399br (O-H), 2355s, (C-H), 2310s

(C-H), 1692s (C=O); δH(400MHz; CDCl3) 1.95 (1H, d, J 3.8, OH), 2.77 (1H, ddd, J 16.8,

4.8, 1.2, C(6)Heq), 2.85 (1H, dd, J 16.8, 13.4, C(6)Hax), 3.44 (1H, ddd, J 13.4, 9.7, 4.8, C(5)H), 4.83 (1H, ddt, J 9.7, 3.8, 2.2, C(4)H), 6.12 (1H, ddd, J 10.3, 2.2, 1.2, C(2)H), 7.05 (1H, dd, J 10.3, 2.0, C(3)H), 7.45 (1H, dd, J 8.4, 1.9, C(3´)H), 7.49-7.56 (2H, m, C(6´)H and C(7´)H), 7.77 (1H, s, C(1´)H), 7.84-7.88 (2H, m, C(5´)H and C(8´)H), 7.91 (1H, d, J

8.4 C(4´)H); δC (100MHz; CDCl3) 43.1 (C(6)H2), 55.1 (C(5)H), 71.8 (C(4)H), 126.3, 124.8, 127.5, 127.6, 128.6, (Ar-CH), 129.4 (C(2)H), 134.7 (Ar-C), 154.2 (C(3)H), 199.5

125

(C=O); m/z (-ES) 237 ([M-H]-, 100%); (Found 237.0916, C16H13O2 ([M-H]-), requires 237.0921).

Method 2: To a solution of 190 (112 mg, 0.25 mmol) in THF (1 mL) at 0°C under N2, was added 1M TBAF (0.30 mL, 0.30 mmol). The reaction mixture was stirred at 0°C for 1 h. The reaction was quenched by the addition of water (5 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO4 and evaporated in vacuo. The crude product was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:3) to give the title compound (189) as a cloudy oil (42 mg, 71%).

8.1.21 Triethyl(((4S)-4-(naphthalen-2-yl)-3a,4,7,7a-tetrahydrospiro [benzo [d][1,3] dioxole-2,1'-cyclohexan]-6-yl)oxy)silane (191)

C28H38O3Si; Mw 450.69

To a solution of 141f (75 mg, 0.22 mmol) in dichloromethane (5 mL) at 0°C under N2 was added 2,6-lutidine (0.04 mL, 0.34 mmol) and TESOTf (0.05 mL, 0.25 mmol). The reaction mixture was stirred at 0°C for 1 h. The reaction was quenched by the addition of saturated aqueous solution of NH4Cl (10 mL) and extracted with dichloromethane (3 x 10 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO4 and evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography eluting with ethyl acetate : 40-60 petroleum ether (1:90) to give the title compound (191) as a cloudy oil (39 mg,

39%). [α]D28 -54.3 (c 0.70 in CH2Cl2); δH(400MHz; CDCl3) 0.73 (6H, q, J 8.0,

Si(CH2CH3)3), 1.00 (9H, t, J 8.0, Si(CH2CH3)3), 1.54-1.83 (10H, m, 5 x CH2 of cyclohexane), 2.38 (1H, dd, J 17.4, 5.2, C(6)Heq), 2.46 (1H, dm, J 17.4, C(6)Hax), 3.28 (1H, ddd, J 15.4, 10.0, 5.2, C(5)H), 4.31 (1H, dd, J 10.0, 5.3, C(4)H), 4.69 (1H, t, J 5.3, C(3)H), 5.09 (1H, dd, J 5.3, 1.8, C(2)H), 7.41-7.48 (3H, m, C(3´)H, C(6´)H and C(7´)H),

126

7.70 (1H, s, C(1´)H), 7.79-7.83 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3)

5.0 (Si(CH2CH3)3), 6.7 (Si(CH2CH3)3), 23.7 (cyclohexane CH2), 24.0 (cyclohexane CH2),

25.1 (cyclohexane CH2), 35.4 (cyclohexane CH2), 35.5 (C(6)H2), 38.6 (cyclohexane

CH2), 43.9 (C(5)H), 72.6 (C(3)H), 77.6 (C(4)H), 99.8 (C(2)H), 109.6 (cyclohexane C), 125.4 (Ar-CH), 125.9 (Ar-CH), 126.4 (Ar-CH), 126.5 (Ar-CH), 127.6 (Ar-CH), 127.7 (Ar-CH), 128.0 (Ar-CH), 132.5 (Ar-C), 133.5 (Ar-C), 139.4 (Ar-C), 155.3 (C(1)); m/z (+ES) 473 ([M+Na]+, 75%), 101 (100).

8.1.22 (4R,5S)-5-(naphthalen-2-yl)-4-((triethylsilyl)oxy)cyclohex-2-enone (193)

C22H28O2Si; Mw 352.54 To a solution of 2,6-lutidine (50 µL, 0.50 mmol) and TESOTf (0.11 mL, 0.37 mmol) in dichloromethane (2 mL) at -78 °C, a solution of alcohol 189 (40 mg, 0.17 mmol) in dichloromethane (2 mL) was added dropwise. The reaction mixture was stirred at -

78 °C, under an atmosphere of N2 for 15 mins. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL) and extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine

(10 mL), dried over MgSO4 and concentrated in vacuo to give a yellow oil. This residue was purified by flash silica chromatography eluting with ethyl acetate:40-60 petroleum ether (1:19) to give the title compound (193) as a viscous oil (42 mg,

71%). [α]D29 -11.8 (c 1.65 in CH2Cl2); νmax(film)/cm-1 3408br (O-H), 2955w (C-H),

2877s (C-H), 1683s (C=O); δH(400MHz; CDCl3) 0.16-0.38 (6H, m, Si(CH2CH3)3), 0.69

(9H, t, J 8.1, Si(CH2CH3)3), 2.77 (1H, ddd, J 16.6, 4.1, 1.2, C(6)Heq), 2.88 (1H, dd, J 16.6,

13.6, C(6)Hax), 3.45 (1H, ddd, J 13.6, 9.3, 4.1, C(5)H), 4.64 (1H, d~t, J 9.3, 2.0, C(4)H), 6.07 (1H, d~t, J 10.3, 2.0, C(2)H), 6.89 (1H, dd, J 10.3, 2.0, C(3)H), 7.41 (1H, dd, J 8.6, 1.8, C(3´)H), 7.45-7.52 (2H, m, C(6´)H and C(7´)H), 7.70 (1H, s, C(1´)H), 7.80-7.85 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3) 4.4 (Si(CH2CH3)3), 6.5

(Si(CH2CH3)3), 42.8 (C(6)H2), 50.8 (C(5)H), 72.6 (C(4)H), 125.7 (C(6´)H and C(7´)H),

127

126.2 (C(3´)H), 127.1 (C(1´)H), 127.6, 127.7, 128.2 (C(4´)H, C(5´)H and C(8´)H), 128.5 (C(2)H), 132.7 (Ar-C), 133.4 (Ar-C), 138.0 (Ar-C), 153.8 (C(3)H), 198.4 (C=O); m/z

(+ES) 375 ([M+Na]+, 100%); (Found 353.1947, C22H29O2Si ([M+H]+), requires 353.1932).

8.1.23 (4R,5S)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5- (naphthalen-2-yl)cyclohex-2-enone (219)

C23H30O3Si; Mw 382.20 Method 1: To a solution of compound 188 (50 mg, 0.14 mmol) in tetrahydrofuran

(0.5 mL) was added 1M NaHCO3 (aq) (1 mL), followed by formaldehyde 37% (0.02 mL, 0.21 mmol) and imidazole (1 mg, 0.014 mmol). The reaction mixture was stirred at room temperature under an atmosphere of N2 for 11 days. The reaction was acidified with 1M HCl (1mL), diluted with water (10 mL) and extracted with dichloromethane (3 x 15 mL). The combined organic extracts were washed with 1M

NaHCO3 (aq) (20 mL) and brine (20 mL), dried over MgSO4 and evaporated in vacuo. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:5) to give the title compound (219) as a white solid

(18 mg, 33 %). mp 139.3-141.4 °C; νmax(film)/cm-1 3733b (-OH), 2951w (C-H), 2359s

(C-H), 1676s (C=O ketone); δH(400MHz; CDCl3) -0.65 (3H, s, CH3), -0.15 (3H, s, CH3),

0.71 (9H, s, 3 x CH3), 2.77 (1H, dd, J 16.7, 4.1, C(6)Heq), 2.94 (1H, dd, J 16.7, 13.7,

C(6)Hax), 3.44 (1H, ddd, J 13.7, 9.6, 4.1, C(5)H), 4.31 (1H, d, J 13.5, C(7)HaHbOH), 4.41

(1H, d, J 13.5, C(7)HaHbOH), 4.67 (1H, dm, J 9.6, C(4)H), 6.80 (1H, q, J 1.7, C(3)H), 7.39 (1H, dd, J 8.6, 2.0, C(3´)H), 7.46-7.52 (2H, m, C(6´)H and C(7´)H), 7.69 (1H, s, C(1´)H),

7.80-7.85 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3) -5.8 (Si(CH3)), -5.1

(Si(CH3)), 17.8 (SiC(CH3)3), 25.5 (SiC(CH3)3), 42.9 (C(6)H2), 50.9 (C(5)H), 61.4

(C(7)H2OH), 72.8 (C(4)H), 125.7 (Ar-CH), 125.8 (Ar-CH), 126.2 (Ar-CH), 127.3 (Ar- CH), 127.6 (Ar-CH), 128.1 (Ar-CH), 132.7 (Ar-C), 133.3 (Ar-C), 136.9 (Ar-C), 137.7

128

(C(2)), 149.7 (C(3)H), 199.2 (C=O); m/z (+ES) 405 ([M+Na]+, 100%); (Found

383.2035, C23H31O3Si ([M]+), requires 383.2037).

Method 2: To a suspension of enone 188 (25 mg, 0.071 mmol) in water (0.2 mL) was added SDS (6 mg, 0.02 mmol) and DMAP (8.6 mg, 0.071 mmol). After stirring for 5 mins formaldehyde 37% (0.09 mL, 4.73 mmol) was added and the resulting suspension was stirred at room temperature for 22 h. The reaction was quenched with brine (1 mL) and diluted with ethyl acetate (5 mL). The two layers were separated and the aqueous layer was extracted with ethyl acetate (5 mL). The combined organics were washed with brine (5 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:6) to give the title compound (219) as a white solid (19 mg, 70 %).

8.1.24 (4R,5S)-2-(hydroxymethyl)-5-(naphthalen-2-yl)-4-((triethyl silyl)oxy) cyclohex-2-enone(220)

C23H30O3Si; Mw 382.57 To a suspension of compound 193 (50 mg, 0.14 mmol) in water (0.4 mL) was added SDS (12 mg, 0.04 mmol) and DMAP (17 mg, 0.14 mmol). After stirring for 5 mins, formaldehyde 37% (0.19 mL, 1.96 mmol) was added and the resulting suspension was stirred at room temperature for 22 h. The reaction was quenched by the addition of brine (1 mL) and diluted with ethyl acetate (5 mL). The two layers were separated and the aqueous layer was washed with ethyl acetate (5 mL). The combined organic extracts were washed with brine (5 mL), dried over MgSO4 and concentrated in vacuo to give a waxy solid. Purification by flash silica chromatography, eluting with ethyl acetate:40-60 petroleum ether (1:6) gave the title compound (220) as a cloudy oil

(35 mg, 64 %). [α]D29 -103.3 (c 1.2 in CH2Cl2); νmax(film)/cm-1 3420br (O-H) 2955s (C-

129

H), 2910s (C-H), 2874s (C-H), 1672s (C=O); δH(400MHz; CDCl3) 0.16-0.37 (6H, m, Si(CH2CH3)3), 0.69 (9H, t, J 7.6, Si(CH2CH3)3), 2.78 (1H, dd, J 16.4, 4.2, C(6)Heq),

2.91 (1H, dd, J 16.4, 13.6, C(6)Hax), 3.44 (1H, ddd, J 13.6, 9.5, 4.2, C(5)H), 4.30 (1H, d, J

13.6, C(7)HaHb), 4.42 (1H, d, J 13.6, C(7)HaHb), 4.73 (1H, dd, J 9.5, 0.8, C(4)H), 6.82 (1H, s, C(3)H), 7.40 (1H, dd, J 8.8, 1.5, C(3´)H), 7.45-7.51 (2H, m, C(6´)H and C(7´)H),

7.70 (1H, s, C(1´)H), 7.80-7.85 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3)

4.4 (Si(CH2CH3)3), 6.5 (Si(CH2CH3)3), 43.1 (C(6)H2), 50.9 (C(5)H), 61.3 (C(7)H2), 72.6 (C(4)H), 125.7 (Ar-CH), 125.8 (Ar-CH), 126.2 (Ar-CH), 127.1 (Ar-CH), 127.6 (Ar- CH), 127.6 (Ar-CH), 128.2 (Ar-CH), 132.7 (Ar-C), 133.3 (Ar-C), 137.0 (Ar-C), 137.8 (C(2)), 149.5 (C(3)H), 199.1 (C=O); m/z (+ES) 383 ([M+H]+, 100%); (Found

383.2048, C23H31O3Si ([M]+), requires 383.2037).

8.1.25 (E)-((3R,4S)-3-((tert-butyldimethylsilyl)oxy)-4-(naphthalen-2-yl)-6- oxocyclo hex-1-en-1-yl)methyl but-2-enoate (219a)

C27H34O4Si; Mw 450.64 To a solution of 219 (28 mg, 0.073 mmol) in dichloromethane (0.5 mL) at room temperature under an atmosphere of nitrogen, was added crotonic anhydride (0.02 mL, 0.16 mmol) followed by DMAP (0.89 mg, 0.007 mmol) and pyridine (0.05 mL, 0.65 mmol). The reaction mixture was stirred at room temperature for 3 h and then quenched by the addition of a saturated aqueous solution of NaHCO3 (0.5 mL). The reaction mixture was diluted with water (5 mL) and extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (10 mL), dried over MgSO4 and evaporated in vacuo. The residue was purified by flash silica chromatography eluting with ethyl acetate:40-60 petroleum ether (1:19) to give the title compound as a waxy white solid (15 mg, 45

%). [α]D34 -59.6 (c 0.26 in CH2Cl2); νmax(film)/cm-1 3051w (C-H), 2930s (C-H), 2861s

(C-H), 2362s (C-H), 1726s (C(8)=O), 1681s (C(1)=O); δH(400MHz; CDCl3) -0.59 (3H, s,

Si(CH3)), -0.18 (3H, s, Si(CH3)), 0.72 (9H, s, SiC(CH3)3), 1.90 (3H, dd, J 7.0, 1.8,

130

C(11)H3), 2.79 (1H, dd, J 16.4, 4.2, C(6)Heq), 2.93 (1H, dd, J 16.4, 13.7, C(6)Hax), 3.45 (1H, ddd, J 13.7, 9.5, 4.2, C(5)H), 4.69 (1H, dq, J 9.5, 1.8, C(4)H), 4.96 (1H, dt, J 13.9,

1.8, C(7)HaHbOH), 4.83 (1H, dt, J 13.9, 1.8, C(7)HaHbOH), 5.91 (1H, dq, J 15.5, 1.8, C(9)H), 6.83 (1H, q, J 1.8, C(3)H), 7.06 (1H, dq, J 15.5, 7.0, C(10)H), 7.39 (1H, dd, J 8.4, 1.9, C(3´)H), 7.45-7.52 (2H, m, C(6´)H and C(7´)H), 7.68 (1H, s, C(1´)H), 7.79-7.85 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3) -5.6 (Si(CH3)), -5.1 (Si(CH3)), 17.8

(SiC(CH3)3), 25.5 (SiC(CH3)3), 42.8 (C(6)H2), 50.8 (C(5)H), 60.3 (C(7)H2), 72.9 (C(4)H), 122.2 (C(9)H), 125.8 (Ar-CH), 127.2 (Ar-CH), 127.6 (Ar-CH), 128.2 (Ar- CH), 136.9 (Ar-C), 149.7 (C(3)H), 164.8 (C(8)=O), 196.7 (C(1)=O); m/z (+ES) 473

([M+Na]+, 100%); (Found 473.2110, C27H34O4NaSi ([M+Na]+), requires 473.2119).

8.1.26 (E)-((3R,4S)-4-(naphthalen-2-yl)-6-oxo-3-((triethylsilyl)oxy) cyclohex-1- en-1-yl)methyl but-2-enoate (226a)

C27H34O4Si; Mw 450.64 To a solution of 220 (11 mg, 0.03 mmol) in dichloromethane (0.2 mL) at room temperature under an atmosphere of nitrogen, was added crotonic anhydride (0.01 mL, 0.06 mmol) followed by DMAP (0.4 mg, 0.003 mmol) and pyridine (0.02 mL, 0.25 mmol). The reaction mixture was stirred at room temperature for 2.5 h and it was then quenched by the addition of a saturated aqueous solution of NaHCO3 (0.2 mL). The reaction mixture was diluted with water (5 mL) and organic material was extracted into dichloromethane (3 x 5 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (10 mL), dried over MgSO4 and concentrated in vacuo to give a yellow oil. This residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give the title compound (226a) as a colourless oil (9 mg, 69 %). [α]D29 -36.7 (c 0.45 in CH2Cl2);

νmax(film)/cm-1 2958w (C-H), 2934w (C-H), 2918w (C-H), 2876w (C-H), 1726s

(C(8)=O), 1683s (C(1)=O), 1103s (Si-O-C); δH(400MHz; CDCl3) 0.17-0.38 (6H, m,

131

Si(CH2CH3)3), 0.69 (9H, t, J 7.8, Si(CH2CH3)3), 1.91 (3H, dd, J 6.9, 1.7, C(11)H3), 2.79

(1H, dd, J 16.5, 4.2, C(6)Heq), 2.91 (1H, dd, J 16.5, 13.6, C(6)Hax), 3.45 (1H, ddd, J 13.6,

9.3, 4.2, C(5)H), 4.73 (1H, dq, J 9.3, 1.5, C(4)H), 4.86 (1H, dt, J 14.0, 1.5, C(7)HaHb),

4.94 (1H, dt, J 14.0, 1.8, C(7)HaHb), 5.91 (1H, d~q, J 15.6, 1.7, C(9)H), 6.82 (1H, ~q, J 1.5, C(3)H), 7.05 (1H, dq, J 15.6, 6.9, C(10)H), 7.40 (1H, dd, J 8.6, 2.0, C(3´)H), 7.45- 7.51 (2H, m, C(6´)H and C(7´)H), 7.69 (1H, s, C(1´)H), 7.79-7.85 (3H, m, C(4´)H, C(5´)H and C(8´)H); δC(100MHz; CDCl3) 4.4 (Si(CH2CH3)3), 6.5 (Si(CH2CH3)3), 18.1 (C(11)H3),

42.9 (C(6)H2), 50.7 (C(5)H), 60.3 (C(7)H2), 72.6 (C(4)H), 122.2 (C(9)H), 125.7 (C(3´)H), 125.8 (C(6´)H or C(7´)H), 126.2 (C(6´)H or C(7´)H), 127.1 (C(1´)H), 127.6 (C(4´)H or C(5´)H or C(8´)H), 127.7 (C(4´)H or C(5´)H or C(8´)H), 128.2 (C(4´)H or C(5´)H or C(8´)H), 132.7, 133.3, 133.5, 137.8 (Ar-C), 145.5 (C(10)H), 150.0 (C(3)H), 165.9 (C(8)=O), 196.7 (C(1)=O); m/z (+ES) 473 ([M+Na]+, 100%); (Found 473.2110,

C27H34O4NaSi ([M+Na]+), requires 473.2119).

8.1.27 (E)-((3R,4S)-3-hydroxy-4-(naphthalen-2-yl)-6-oxocyclohex-1-en-1- yl)methyl but-2-enoate (178a)

C21H20O4; Mw 336.38

A solution of 226a (13 mg, 0.03 mmol) in TFA:H2O (7:1, 0.32 mL) was stirred at room temperature, under an atmosphere of nitrogen, for 1.5 h. The solvents were removed in vacuo to give a brown oil which was purified by flash silica chromatography, eluting with ethyl acetate:40-60 petroleum ether (1:3), to give the title compound

(178a) as a yellow oil (8 mg, 83 %). [α]D29 -57.0 (c 0.4 in CH2Cl2); νmax(film)/cm-1 3426br (O-H), 2962m (C-H), 2915w (C-H), 1722s (C(8)=O), 1682s (C(1)=O);

δH(400MHz; CDCl3) 1.92 (3H, dd, J 7.0, 1.8, C(11)H3), 2.81 (1H, dd, J 16.6, 5.0,

C(6)Heq), 2.88 (1H, dd, J 16.6, 12.9, C(6)Hax), 3.44 (1H, ddd, J 12.9, 9.8, 5.0, C(5)H),

4.83-4.86 (1H, m, C(4)H), 4.87 (1H, dt, J 14.4, 1.8, C(7)HaHb), 4.97 (1H, dt, J 14.4, 1.8,

C(7)HaHb), 5.92 (1H, dq, J 15.5, 1.8, C(9)H), 7.00 (1H, q, J 1.8, C(3)H), 7.07 (1H, dq, J 15.5, 7.0, C(10)H), 7.43 (1H, dd, J 8.4, 2.0, C(3´)H), 7.50-7.56 (2H, m, C(6´)H and

132

C(7´)H), 7.76 (1H, s, C(1´)H), 7.84-7.88 (2H, m, C(5´)H and C(8´)H), 7.91 (1H, d, J 8.4,

C(4´)H); δC(100MHz; CDCl3) 18.1 (C(11)H3), 43.2 (C(6)H2), 50.8 (C(5)H), 60.2

(C(7)H2), 71.8 (C(4)H), 122.2 (C(9)H), 125.0 (C(3´)H), 126.3 (C(6´)H or C(7´)H), 126.7 (C(6´)H or C(7´)H), 127.0 (C(1´)H), 127.7 (C(5´)H and C(8´)H), 129.2 (C(4´)H), 133.0, 133.5, 134.3, 136.5 (Ar-C), 145.7 (C(10)H), 148.0 (C(3)H), 165.9 (C(8)=O), 196.7 (C(1)=O).

8.1.28 (4R,5S)-4-hydroxy-2-(hydroxymethyl)-5-(naphthalen-2-yl) cyclohex-2- enone (177a)

C17H16O3; Mw 268.31

A solution of allylic alcohol 220 (25 mg, 0.07 mmol) in TFA/H2O (7:1, 0.56 mL) was stirred at room temperature for 30 min. The solvents were removed in vacuo to give a yellow gum which was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2), to give the title compound (177a) as a colourless oil (18 mg, 93 %). [α]D28 -96.4 (c 0.50, CH2Cl2); δH(400MHz; CDCl3) 2.78

(1H, dd, J 16.7, 4.8, C(6)Heq), 2.87 (1H, dd, J 16.7, 13.2, C(6)Hax), 3.42 (1H, ddd, J 13.2,

9.8, 4.8, C(5)H), 4.34 (1H, d, J 13.6, C(7)HaHb), 4.40 (1H, d, J 13.6, C(7)HaHb), 4.83 (1H, dd, J 9.8, 0.9, C(4)H), 6.99 (1H, s, C(3)H), 7.43 (1H, d, J 8.6, C(3´)H), 7.49-7.56 (2H, m, C(6´)H and C(7´)H), 7.76 (1H, s, C(1´)H), 7.84-7.88 (2H, m, C(5´)H and C(8´)H), 7.90

(1H, d, J 8.6, C(4´)H); δC(100MHz; CDCl3) 43.3 (C(6)H2), 50.9 (C(5)H), 61.1 (C(7)H2), 71.7 (C(4)H), 124.9 (C(3´)H), 126.3 (C(6´)H or C(7´)H), 126.6 (C(6´)H or C(7´)H), 127.0 (C(1´)H), 127.7 (C(5´)H and C(8´)H), 129.1 (C(4´)H), 132.9 (Ar-C), 133.4 (Ar-C), 136.5 (Ar-C), 137.7 (Ar-C), 147.8 (C(3)H), 198.5 (C=O); m/z (+ES) 291 ([M+Na]+,

100%); (Found 291.0998, C17H16O3Na ([M+Na]+), requires 291.0992).

133

8.1.29 (1R,6S)-6-hydroxy-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (212)

C12H12O2; Mw 188.22 A mixture of anti-140a (130 mg, 0.41 mmol) and DBSA (26 mg, 0.08 mmol) in water (2.5 mL) was stirred at 100 °C for 1.5 h. The reaction was quenched by the addition of brine (2 mL) and a saturated aqueous solution of NaHCO3 (2 mL). Organic material was extracted into ethyl acetate (3 x 5 mL) and the combined extracts were washed with brine (10 mL), dried over MgSO4 and concentrated in vacuo to give the ctude product as a yellow oil. Purification by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:2), gave the title compound (212) as a yellow oil

(50 mg, 65 %). νmax(film)/cm-1 3389br (O-H), 2357w (C-H), 1671s (C=O);

δH(400MHz; CDCl3) 2.07 (1H, s, C(4)OH), 2.68-2.71 (2H, m, C(6)H2), 3.25 (1H, dd, J 11.6, 9.8, 6.4, C(5)H), 4.68 (1H, dt, J 9.8, 1.9, C(4)H), 6.07 (1H, ddd, J 10.2, 2.5, 1.8,

C(2)H), 7.01 (1H, dd, J 10.2, 1.9, C(3)H), 7.30-7.43 (5H, m, 5 x Ar-H); δC(100MHz;

CDCl3) 43.0 (C(6)H2), 50.7 (C(5)H), 71.8 (C(4)H), 127.7 (Ar-CH), 127.9 (Ar-CH), 128.9 (C(2)H), 129.2 (Ar-CH), 152.1 (C(3)H), 198.0 (C=O); m/z (+ES) 211 ([M+Na]+, 100%);

(Found 187.0759, C12H11O2 ([M]-), requires 187.0764).

8.1.30 (1S,6S)-6-hydroxy-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (215)

C12H12O2; Mw 188.22 A mixture of syn-140a (105 mg, 0.33 mmol) and DBSA (21 mg, 0.07 mmol) in water (2.5 mL) was stirred at 100 °C for 2 h. The reaction was quenched by the addition of brine (2 mL) and a saturated aqueous solution of NaHCO3 (2 mL). Organic material was extracted into ethyl acetate (3 x 5 mL) and the combined extracts were washed with brine (10 mL), dried over MgSO4 and concentrated in vacuo to give a yellow oil.

134

The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:2) to give the title compound (215) as a yellow oil (30 mg,

49 %). [α]D30 +157.4 (c 0.54 in CH2Cl2); νmax(film)/cm-1 3393br (O-H), 2341w (C-H),

2357w (C-H), 1675s (C=O); δH(400MHz; CDCl3) 1.70 (1H, d, J 4.5, C(4)OH), 2.60 (1H, dd, J 16.3, 3.8, C(6)Heq), 3.16 (1H, dd, J 16.3, 12.5, C(6)Hax), 3.48 (1H, dt, J 12.5, 4.5, C(5)H), 4.48 (1H, ~q, J 4.5, C(4)H), 6.16 (1H, d, J 10.1, C(2)H), 7.03 (1H, dd, J 10.1, 4.5,

C(3)H), 7.28-7.43 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 37.6 (C(6)H2), 45.0 (C(5)H), 66.6 (C(4)H), 127.7 (Ar-CH), 128.1 (Ar-CH), 128.9 (Ar-CH), 130.7 (Ar-CH), 139.3 (C(2)H), 147.0 (C(3)H), 199.4 (C=O); m/z (+ES) 211 ([M+Na]+, 100%); (Found

187.0759, C12H11O2 ([M]-), requires 187.0764).

8.1.31 (1R,6S)-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (216)

C18H26O2Si; Mw 302.48 To a solution of 2,6-lutidine (0.05 mL, 0.58 mmol) and TESOTf (0.09 mL, 0.32 mmol) in dichloromethane (2 mL), at -78 °C under an atmosphere of N2, was added a solution of 212 (55 mg, 0.29 mmol) in dichloromethane (3 mL), dropwise. The reaction mixture was stirred at -78 °C for 10 min. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over MgSO4 and evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give the title compound (216) as a yellow oil (65 mg, 74 %). νmax(film)/cm-1 2952m (C-H),

2909w (C-H), 2876m (C-H), 2357w (C-H), 1682s (C=O); δH(400MHz; CDCl3) 0.24-

0.40 (6H, m, Si(CH2CH3)3), 0.77 (9H, t, J 7.8, Si(CH2CH3)3), 2.69 (1H, ddd, J 16.5, 4.4,

1.4, C(6)Heq), 2.74 (1H, dd, J 16.5, 13.5, C(6)Hax), 3.27 (1H, ddd, J 13.5, 9.3, 4.4, C(5)H), 4.58 (1H, dt, J 9.3, 2.0, C(4)H), 6.04 (1H, ddd, J 10.3, 2.3, 1.4, C(2)H), 6.85 (1H, dd, J

135

10.3, 2.0, C(3)H), 7.25-7.36 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 4.4 (Si(CH2CH3)3),

6.6 (Si(CH2CH3)3), 42.8 (C(6)H2), 50.6 (C(5)H), 72.7 (C(4)H), 127.3 (Ar-CH), 128.0 (Ar-CH), 128.4 (C(2)H), 128.5 (Ar-CH), 140.7 (Ar-C), 153.8 (C(3)H), 198.5 (C=O); m/z

(+ES) 325 ([M+Na]+, 30%), 420 (100); (Found 303.1785, C18H27O2Si ([M]+), requires 303.1775).

8.1.32 (1S,6S)-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (217)

C18H26O2Si; Mw 302.48 To a solution of 2,6-lutidine (0.07 mL, 0.74 mmol) and TESOTf (0.12 mL, 0.41 mmol) in dichloromethane (3 mL), at -78 °C under an atmosphere of N2, was added a solution of 215 (70 mg, 0.37 mmol) in dichloromethane (3 mL), dropwise. The reaction mixture was stirred at -78 °C for 1.5 h. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 5 mL). The combined organic extract were washed with brine (10 mL), dried over MgSO4 and evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give the title compound (217) as a yellow oil (86 mg, 77 %). [α]D28 +200.3 (c 1.00 in CH2Cl2);

νmax(film)/cm-1 2954m (C-H), 2904w (C-H), 2876m (C-H), 2359w (C-H), 1685s (C=O);

δH(400MHz; CDCl3) 0.30-0.40 (6H, m, Si(CH2CH3)3), 0.77 (9H, t, J 7.8, Si(CH2CH3)3),

2.52 (1H, dd, J 15.9, 3.2, C(6)Heq), 3.26 (1H, dd, J 15.9, 12.9, C(6)Hax), 3.36 (1H, dt, J 12.9, 3.2, C(5)H), 4.35 (1H, dt, J 5.4, 3.2, C(4)H), 6.07 (1H, dd, J 9.9, 1.0, C(2)H), 6.96

(1H, dd, J 9.9, 5.4, C(3)H), 7.23-7.35 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 4.6

(Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 38.0 (C(6)H2), 45.7 (C(5)H), 67.1 (C(4)H), 127.1 (Ar-CH), 128.2 (Ar-CH), 128.6 (C(2)H), 129.4 (Ar-CH), 140.5 (Ar-C), 148.4 (C(3)H),

200.4 (C=O); m/z (+ES) 325 ([M+Na]+, 100%); (Found 303.1785, C18H27O2Si ([M]+), requires 303.1775).

136

8.1.33 (1R,6S)-4-(hydroxymethyl)-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'- biphenyl]-3(2H)-one (224)

C19H28O3Si; Mw 332.52 To a suspension of compound 216 (55 mg, 0.18 mmol) in water (0.5 mL) was added SDS (14 mg, 0.05 mmol) and DMAP (22 mg, 0.18 mmol). After stirring for 5 mins formaldehyde 37% (0.19 mL, 2.55 mmol) was added and the resulting suspension was stirred at room temperature for 18 h. The reaction was quenched by the addition of brine (1 mL) and diluted with ethyl acetate (5 mL). The two layers were separated and organic material was extracted into ethyl acetate (2 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over MgSO4 and concentrated in vacuo to give the crude product as an orange oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:3) to give the title compound (224) as a colourless oil (35 mg, 58 %).

νmax(film)/cm-1 3395br (O-H), 2956m (C-H), 2910m (C-H), 2878m (C-H), 1674s

(C=O); δH(400MHz; CDCl3) 0.22-0.41 (6H, m, Si(CH2CH3)3), 0.77 (9H, t, J 7.8,

Si(CH2CH3)3), 2.38-2.41 (1H, m, C(7)OH), 2.70 (1H, dd, J 16.6, 4.5, C(6)Heq), 2.79 (1H, dd, J 16.6, 13.4, C(6)Hax), 3.25 (1H, ddd, J 13.4, 9.2, 4.5, C(5)H), 4.39 (1H, d, J 13.6, 4.3,

C(7)HaHbOH), 4.26 (1H, dd, J 13.6, 3.8, C(7)HaHbOH), 4.60 (1H, ~dq, J 9.2, 1.7, C(4)H),

6.78 (1H, ~q, J 1.7, C(3)H), 7.24-7.36 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 4.5

(Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 42.8 (C(6)H2), 50.6 (C(5)H), 61.4 (C(7)H2), 72.9 (C(4)H), 127.3 (Ar-CH), 128.2 (Ar-CH), 128.5 (Ar-CH), 136.9 (C(2)H), 140.5 (Ar-C), 149.4 (C(3)H), 198.9 (C=O); m/z (+ES) 355 ([M+Na]+, 100%); (Found 333.1884,

C19H29O3Si ([M]+), requires 333.1881).

137

8.1.34 (1S,6S)-4-(hydroxymethyl)-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'- biphenyl]-3(2H)-one (225)

C19H28O3Si; Mw 332.52 To a suspension of enone 217 (35 mg, 0.116 mmol) in water (0.3 mL) was added SDS (8 mg, 0.03 mmol) and DMAP (14 mg, 0.116 mmol). After stirring for 5 mins formaldehyde 37% (0.19 mL, 1.62 mmol) was added and the resulting suspension was stirred at room temperature for 18 h. The reaction was quenched by the addition of brine (1 mL) and diluted with ethyl acetate (5 mL). The two layers were separated and organic material was extracted into ethyl acetate (2 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:3) to give the title compound

(225) as a colourless oil (17 mg, 44 %). [α]D32 +130.7 (c 0.75 in CH2Cl2);

νmax(film)/cm-1 3439br (O-H), 2956m (C-H), 2914m (C-H), 2874m (C-H), 2361w (C-

H), 1677s (C=O); δH(400MHz; CDCl3) 0.26-0.42 (6H, m, Si(CH2CH3)3), 0.76 (9H, t, J

7.8, Si(CH2CH3)3), 2.38 (1H, t, J 5.5, C(7)OH), 2.54 (1H, dd, J 15.9, 3.2, C(6)Heq), 3.30

(1H, dd, J 15.9, 12.9, C(6)Hax), 3.40 (1H, dt, J 12.9, 3.2, C(5)H), 4.41 (1H, dd, J 13.6, 5.5,

C(7)HaHbOH), 4.29 (1H, dm, J 13.6, C(7)HaHbOH), 4.41 (1H, dd, J 5.3, 3.2, C(4)H), 6.89

(1H, ~dt, J 5.3, 1.2, C(3)H), 7.22-7.35 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 4.6

(Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 38.1 (C(6)H2), 45.8 (C(5)H), 61.5 (C(7)H2), 67.3 (C(4)H), 127.1 (Ar-CH), 128.2 (Ar-CH), 128.5 (Ar-CH), 137.7 (C(2)H), 140.3 (Ar-C), 144.0 (C(3)H), 201.07 (C=O); m/z (+ES) 355 ([M+Na]+, 100%); (Found 355.1702,

C19H28O3NaSi ([M+Na]+), requires 355.1700).

138

8.1.35 (E)-((1R,6S)-3-oxo-6-((triethylsilyl)oxy)-1,2,3,6-tetrahydro-[1,1'- biphenyl]-4-yl)methyl but-2-enoate (227)

C23H32O4Si; Mw 400.58 To a solution of 224 (28 mg, 0.084 mmol) in dichloromethane (0.5 mL) at room temperature under an atmosphere of nitrogen, was added crotonic anhydride (0.03 mL, 0.19 mmol) followed by DMAP (1 mg, 0.008 mmol) and pyridine (0.06 mL, 0.74 mmol). The reaction mixture was stirred at room temperature for 2 h and then quenched by the addition of a saturated aqueous solution of NaHCO3 (0.5 mL). The reaction mixture was diluted with water (5 mL) and extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (10 mL) and brine (10 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give the title compound (227) as a colourless oil (29 mg, 86 %). νmax(film)/cm-1 2955m (C-H), 2914m (C-H), 2876m (C-H), 2358w (C-H), 1725s (C(8)=O), 1679s

(C(1)=O); δH(400MHz; CDCl3) 0.22-0.42 (6H, m, Si(CH2CH3)3), 0.76 (9H, t, J 8.1,

Si(CH2CH3)3), 1.91 (3H, dd, J 7.0, 1.7, C(11)H3), 2.71 (1H, dd, J 16.4, 4.5, C(6)Heq), 2.80

(1H, dd, J 16.4, 13.2, C(6)Hax), 3.26 (1H, ddd, J 13.2, 9.4, 4.5, C(5)H), 4.60 (1H, ddd, J

9.4, 3.3, 1.6, C(4)H), 4.83 (1H, dt, J 14.0, 1.4, C(7)HaHbO), 4.91 (1H, dt, J 14.0, 1.4,

C(7)HaHbO), 5.90 (1H, dq, J 15.5, 1.7, C(9)H)), 6.78 (1H, ~dt, J 3.3, 1.4, C(3)H), 7.04

(1H, dq, J 15.5, 7.0, C(10)H), 7.24-7.36 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 4.4

(Si(CH2CH3)3), 6.5 (Si(CH2CH3)3), 18.0 (C(11)H3), 42.9 (C(6)H2), 50.7 (C(5)H), 60.3

(C(7)H2), 72.6 (C(4)H), 122.2 (C(9)H), 127.4 (Ar-CH), 128.0 (Ar-CH), 128.5 (Ar-CH), 133.4 (C(2)H), 140.4 (Ar-C), 145.5 (C(10)H), 150.0 (C(3)H), 165.8 (C(8)=O), 196.7

(C(1)=O); m/z (+ES) 423 ([M+Na]+, 100%); (Found 401.2139, C23H33O4Si ([M]+), requires 401.2143).

139

8.1.36 (E)-((1S,6S)-3-oxo-6-((triethylsilyl)oxy)-1,2,3,6-tetrahydro-[1,1'- biphenyl]-4-yl)methyl but-2-enoate (228)

8.1.37 (E)-((1S,6S)-6-hydroxy-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-4- yl)methyl but-2-enoate (180)

C17H18O4; Mw 286.32 To a solution of 225 (35 mg, 0.105 mmol) in dichloromethane (0.5 mL) at room temperature under an atmosphere of nitrogen, was added crotonic anhydride (0.03 mL, 0.23 mmol) followed by DMAP (1 mg, 0.011 mmol) and pyridine (0.07 mL, 0.93 mmol). The reaction mixture was stirred at room temperature for 1.5 h and then quenched by the addition of a saturated aqueous solution of NaHCO3 (0.5 mL). The reaction mixture was diluted with water (5 mL) and extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (10 mL) and brine (10 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography eluting with ethyl acetate:40-60 petroleum ether (1:19) to give a mixture of compound (228) and crotonic acid.

A solution of the crude product (228) (20 mg, 0.05 mmol) in TFA:H2O (7:1, 0.32 mL) was stirred at room temperature, under an atmosphere of nitrogen, for 1 h. The solvents were evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:3) to give the title compound (180) as a colourless oil (10 mg,

33% over 2 steps). [α]D29 +47.7 (c 0.30 in CH2Cl2); νmax(film)/cm-1 3437br (O-H), 2961w (C-H), 2917m (C-H), 2847w (C-H), 2362w (C-H), 1723s (C(8)=O), 1680s

(C(1)=O); δH(400MHz; CDCl3) 1.62 (1H, d, J 4.8, C(7)OH), 1.91 (3H, dd, J 6.9, 1.7,

C(11)H3), 2.65 (1H, dd, J 16.4, 4.2, C(6)Heq), 3.22 (1H, dd, J 16.4, 12.4, C(6)Hax), 3.52 (1H, dt, J 12.4, 4.2, C(5)H), 4.55 (1H, ~q, J 4.2, C(4)H), 4.88 (1H, ~d, J 14.4,

C(7)HaHbO), 4.98 (1H, dt, J 14.4, 1.5, C(7)HaHbO), 5.90 (1H, dq, J 15.6, 1.7, C(9)H)),

140

6.98 (1H, ~dt, J 4.2, 1.5, C(3)H), 7.06 (1H, dq, J 15.6, 6.9, C(10)H), 7.29-7.43 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 18.1 (C(11)H3), 37.6 (C(6)H2), 44.9 (C(5)H), 60.4

(C(7)H2), 66.8 (C(4)H), 122.2 (C(9)H), 127.7 (Ar-CH), 128.1 (Ar-CH), 129.0 (Ar-CH), 135.8 (C(2)H), 139.2 (Ar-C), 143.0 (C(10)H), 145.7 (C(3)H), 165.9 (C(8)=O), 197.6

(C(1)=O); m/z (+ES) 309 ([M+Na]+, 100%); (Found 309.1098, C17H18O4Na ([M]+), requires 309.1098).

8.1.38 (E)-((1R,6S)-6-hydroxy-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-4- yl)methylbut-2-enoate (179)

C17H18O4; Mw 286.32

A solution of 227 (20 mg, 0.05 mmol) in TFA:H2O (7:1, 0.48 mL) was stirred at room temperature, under an atmosphere of nitrogen, for 1 h. The solvents were evaporated in vacuo to give a colourless oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:3) to give the title compound (179) as a colourless oil (10 mg, 70 %). [α]D29 +82.0 (c 0.6 in CH2Cl2);

νmax(film)/cm-1 3379br (O-H), 2972w (C-H), 2961w (C-H), 2925w (C-H), 1716s

(C(8)=O), 1675s (C(1)=O); δH(400MHz; CDCl3) 1.92 (3H, dd, J 6.9, 1.9, C(11)H3), 1.96

(1H, d, J 4.3, C(7)OH), 2.74-2.77 (2H, m, C(6)H2), 3.27 (1H, dt, J 9.9, 7.8, C(5)H), 4.71

(1H, dq, J 9.9, 1.9, C(4)H), 4.87 (1H, dt, J 14.4, 1.9, C(7)HaHbO), 4.92 (1H, dt, J 14.4, 1.9,

C(7)HaHbO), 5.91 (1H, dq, J 15.6, 1.7, C(9)H)), 6.96 (1H, ~q, J 1.9, C(3)H), 7.05 (1H, dq, J 15.6, 6.9, C(10)H), 7.30-7.44 (5H, m, 5 x Ar-H); δC(100MHz; CDCl3) 18.1

(C(11)H3), 43.2 (C(6)H2), 50.6 (C(5)H), 60.1 (C(7)H2), 71.9 (C(4)H), 122.1 (C(9)H), 127.7 (Ar-CH), 128.0 (Ar-CH), 129.2 (Ar-CH), 134.2 (C(2)H), 139.1 (Ar-C), 145.7 (C(3)H), 148.1 (C(10)H), 165.9 (C(8)=O), 196.3 (C(1)=O); m/z (+ES) 309 ([M+Na]+,

100%); (Found 309.1107, C17H18O4Na ([M]+), requires 309.1098).

141

8.1.39 (3aS,4S,7aR)-4-(4-(tert-butyl)phenyl)tetrahydrospiro [benzo[d][1,3] dioxole-2,1'-cyclohexan]-6(3aH)-one (141a)

C22H30O3; Mw 342.47 To a stirred suspension of copper iodide (503 mg, 2.64 mmol) in THF (5 mL) at 0 °C, was added 4-tert-butylphenylmagnesium bromide (0.5M in THF) (10.6 mL, 5.28 mmol) and the reaction mixture was stirred for 1 h at 0 °C, under an atmosphere of

N2. A solution of enone 35 (500 mg, 2.40 mmol) in THF (15 mL) was added dropwise and the reaction mixture was stirred at 0 °C, under an atmosphere of N2 for 2 h. The reaction was quenched by the addition of a mixture of saturated aqueous solution of

NH4Cl and aqueous NH3 solution (9:1, 10 mL). The two layers were separated and the aqueous layer was extracted with diethyl ether (3 x 15 mL). The combined organic extracts were washed with brine (25 mL), dried over MgSO4 and concentrated in vacuo. The crude residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound (141a) as a white solid (599 mg, 73 %). mp. 125.6-127.3 C; [α]D23 -64.50 (c 1.15, CH2Cl2); νmax

(film)/cm-1 2941w (C-H), 2902w (C-H), 2863w (C-H), 1710s (C=O); H (400MHz;

CDCl3) 1.31 (9H, s, C(CH3)3, 1.58-1.76 (10H, m, 5 x CH2 of cyclohexane), 2.59-2.67 (2H, m, C(2)Hax and C(6)Hax), 2.72 (1H, dd, J 17.1, 4.0, C(2)Heq), 2.75 (1H, dd, J 17.9, 4.8,

C(6)Heq), 3.43 (1H, d~t, J 7.8, 4.8, C(5)H), 4.57-4.68 (2H, m, C(3)H and C(4)H), 7.17

(2H, d, J 8.2, 2 x Ar-H), 7.36 (2H, d, J 8.2, 2 x Ar-H); δC(100MHz; CDCl3) 23.6

(cyclohexane CH2), 24.0 (cyclohexane CH2), 25.1 (cyclohexane CH2), 31.3 (C(CH3)3)

33.7 (cyclohexane CH2), 34.4 (cyclohexane CH2), 36.8 (C(CH3)3), 40.5 (C(6)H2), 42.0

(C(5)H), 42.2 (C(2)H2), 72.2 (C(3)H or C(4)H), 76.7 (C(3)H or C(4)H), 109.2 (cyclohexane C), 125.7 (Ar-CH), 127.1 (Ar-CH), 137.0 (Ar-C), 209.2 (C=O); m/z (+ES)

363 ([M+Na]+, 100%); (Found 341.1598, C17H25O7 ([M]+), requires 341.1595).

142

8.1.40 (1S,6R)-4'-(tert-butyl)-6-hydroxy-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (194)

C16H20O2; Mw 244.33 To a stirring solution of 141a (210 mg, 0.61 mmol) in dichloromethane (10 mL), under an atmosphere of nitrogen, was added DBU (0.10 mL, 0.68 mmol). The reaction mixture was stirred at room temperature for 2.5 h. The reaction was quenched by the addition of 0.1M HCl (aq) (5 mL). The two layers were separated and the aqueous was extracted with dichloromethane (2 x 10 mL). The combined organic extracts were washed with brine (15 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as a pale yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2) to give the title compound (194) as a white solid (120 mg, 80 %). mp. 119.3-121.7 °C;

[α]D26 -134 (c 0.9, CH2Cl2); νmax (film)/cm-1 3494br (O-H), 2961w (C-H), 2895w (C-H),

1665s (C=O), 1650s (C=C); H (400MHz; CDCl3) 1.34 (9H, s, C(CH3)3, 1.95 (1H, d, J 3.9,

C(4)OH), 2.66-2.76 (2H, m, C(6)H2), 3.23 (1H, ddd, J 11.3, 10.0, 6.8, C(5)H), 4.67 (1H, ddt, J 10.0, 3.9, 2.0, C(4)H), 6.08 (1H, dd, J 10.3, 2.0, C(2)H), 7.01 (1H, dd, J 10.3, 2.0,

C(3)H), 7.24 (2H, d, J 8.3, 2 x Ar-H), 7.42 (2H, d, J 8.3, 2 x Ar-H); δC(100MHz; CDCl3)

31.3 (C(CH3)3, 34.6 (C(CH3)3, 43.1 (C(6)H2), 50.3 (C(5)H), 72.0 (C(4)H), 126.1 (Ar- CH), 127.4 (Ar-CH), 129.0 (C(2)H), 150.9 (Ar-C), 152.0 (C(3)H), 198.2 (C=O); m/z (-

ES) 279 ([M+35Cl]-, 100%), 281 ([M+37Cl]-, (30); (Found 245.1547, C16H21O2 ([M]+), requires 245.1537).

8.1.41 (1S,6R)-4'-(tert-butyl)-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'-biphenyl] -3(2H)-one (196)

C22H34O2Si; Mw 358.59

143

To a solution of 2,6-lutidine (0.21 mL, 2.27 mmol) in dichloromethane (10 mL), at -78 °C under an atmosphere of nitrogen, was added TESOTf (0.48 mL, 1.67 mmol) followed by a solution of alcohol 194 (185 mg, 0.76 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at -78 °C for 15 min. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (10 mL) and allowed to warm to room temperature. The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO4 and evaporated in vacuo. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give the title compound (196) as a yellow oil (240 mg, 88 %). [α]D28 -132.5 (c 1.00, CH2Cl2); νmax (film)/cm-1 2958s (C-H),

2909m (C-H), 2876m (C-H), 1685s (C=O); H (400MHz; CDCl3) 0.22-0.37 (6H, m,

Si(CH2CH3)3), 0.75 (9H, t, J 8.1, Si(CH2CH3)3), 1.32 (9H, s, C(CH3)3, 2.69 (1H, ddd, J

16.5, 4.5, 1.1, C(6)Heq), 2.74 (1H, dd, J 16.5, 13.2, C(6)Hax), 3.24 (1H, ddd, J 13.2, 9.2, 4.5, C(5)H), 4.52 (1H, dt, J 9.2, 2.1, C(4)H), 6.02 (1H, ddd, J 10.3, 2.1, 1.1, C(2)H), 6.83 (1H, dd, J 10.3, 2.1, C(3)H), 7.18 (2H, d, J 8.3, 2 x Ar-H), 7.42 (2H, d, J 8.3, 2 x Ar-H);

δC(100MHz; CDCl3) 4.3 (Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 31.3 (C(CH3)3, 34.4 (C(CH3)3,

42.6 (C(6)H2), 50.2 (C(5)H), 72.9 (C(4)H), 125.3 (Ar-CH), 127.7 (Ar-CH), 128.4 (C(2)H), 137.6 (Ar-C), 150.4 (Ar-C), 154.0 (C(3)H), 198.7 (C=O); m/z (-ES) 393

([M+35Cl]-, 100%), 395 ([M+37Cl]-, (45); (Found 359.2392, C22H35O2Si ([M]+), requires 359.2401).

8.1.42 (1S,6R)-4'-(tert-butyl)-4-(hydroxymethyl)-6-((triethylsilyl)oxy)-1,6- dihydro-[1,1'-biphenyl]-3(2H)-one (221)

C23H36O3Si; Mw 388.62 To a suspension of compound 196 (160 mg, 0.45 mmol) in water (1.5 mL), was added SDS (43 mg, 0.15 mmol) and DMAP (55 mg, 0.45 mmol). The reaction mixture

144 was stirred for 5 min at room temperature and then formaldehyde 37% (0.47 mL, 6.35 mmol) was added. The resulting mixture was stirred at room temperature for 22 h. The reaction was quenched by the addition of brine (2 mL) and extracted with ethyl acetate (2 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as an off white wet solid. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:4) to give the title compound (221) as a white solid (130 mg, 74 %). mp 67.5- 69.2 °C; [α]D29 -111.3 (c 0.80, CH2Cl2); νmax

(film)/cm-1 3431br (O-H), 2955s (C-H), 2905m (C-H), 2877m (C-H), 1673s (C=O); H

(400MHz; CDCl3) 0.21-0.37 (6H, m, Si(CH2CH3)3), 0.75 (9H, t, J 8.1, Si(CH2CH3)3), 1.32

(9H, s, C(CH3)3, 2.37 (1H, t, J 6.5, C(7)OH), 2.73 (1H, dd, J 16.6, 4.5, C(6)Heq), 2.77 (1H, dd, J 16.6, 13.5, C(6)Hax), 3.24 (1H, ddd, J 13.5, 9.5, 4.5, C(5)H), 4.25 (1H, ddt, J 13.4,

6.5, 1.6, C(7)HaHb), 4.38 (1H, ddt, J 13.4, 6.5, 1.6, C(7)HaHb), 4.54 (1H, dq, J 9.5, 1.6, C(4)H), 7.76 (1H, d, J 1.6, C(3)H), 7.18 (2H, d, J 8.3, 2 x Ar-H), 7.42 (2H, d, J 8.3, 2 x Ar-

H); δC(100MHz; CDCl3) 4.3 (Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 31.3 (C(CH3)3), 34.5

(C(CH3)3), 42.9 (C(6)H2), 50.3 (C(5)H), 61.4 (C(7)H2), 72.9 (C(4)H), 125.3 (Ar-CH), 127.7 (Ar-CH), 136.9 (Ar-C), 137.3 (Ar-C), 149.7 (C(3)H), 165.3 (C(2)), 199.4 (C=O); m/z (-ES) 423 ([M+35Cl]-, 100%), 425 ([M+35Cl]-, (45); (Found 389.2494, C23H37O3Si ([M]+), requires 389.2507).

8.1.43 (1S,6R)-4'-(tert-butyl)-6-hydroxy-4-(hydroxymethyl)-1,6-dihydro-[1,1'- biphenyl]-3(2H)-one (177b)

C17H22O3; Mw 274.35

A solution of compound 221 (65 mg, 0.167 mmol) in TFA:H2O (7:1) (1.6 mL) was stirred at room temperature for 40 mins. The solvents were evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:1) to give the title compound (177b) as a white solid (17 mg, 37 %). mp 159.3-161.1 °C; [α]D28 -99.3 (c 0.40, CH2Cl2); νmax

145

(film)/cm-1 3448br (O-H), 2952s (C-H), 2893m (C-H), 2861m (C-H), 1667s (C=O);

H(400 MHz; CDCl3) 1.34 (9H, s, C(CH3)3), 1.95 (1H, d, J 4.1, C(4)OH), 2.31-2.35 (1H, m, C(7)OH), 2.70-2.78 (2H, m, C(6)H2), 3.23 (1H, ddd, J 11.1, 9.9, 6.8, C(5)H), 4.31 (1H, dt, J 13.8, 1.4, C(7)HaHb), 4.39 (1H, dt, J 13.8, 1.4, C(7)HaHb), 4.70 (1H, dq, J 9.9, 1.8, C(4)H), 6.96 (1H, ~d, J 1.8, C(3)H), 7.24 (2H, d, J 8.5, 2 x Ar-H), 7.42 (2H, d, J 8.5, 2 x

Ar-H); δC(100MHz; CDCl3) 31.3 (C(CH3)3), 34.6 (C(CH3)3), 43.3 (C(6)H2), 50.3 (C(5)H),

61.2 (C(7)H2), 71.9 (C(4)H), 126.1 (Ar-CH), 127.3 (Ar-CH), 136.0 (C(2)H), 137.6 (Ar- C), 147.8 (C(3)H), 184.4 (C(1)=O); m/z (-ES) 297 ([M+Na]+, 100%); (Found 297.1457,

C17H22O3Na ([M+Na]+), requires 297.1462).

8.1.44 (E)-((1S,6R)-4'-(tert-butyl)-6-hydroxy-3-oxo-1,2,3,6-tetrahydro-[1,1'- biphenyl]-4-yl)methyl but-2-enoate (178b)

C21H26O4; Mw 342.43 To a solution of 220 (90 mg, 0.23 mmol) in dichloromethane (1 mL), was added crotonic anhydride (0.08 mL, 0.51 mmol), DMAP (3 mg, 0.02 mmol) and pyridine (0.16 mL, 2.04 mmol). The reaction mixture was stirred at room temperature for 2.5 h. The reaction was quenched by the addition of a saturated aqueous solution of

NaHCO3 (2mL) and diluted with H2O (3mL) and dichloromethane (5mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over

MgSO4 and evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give a mixture of the crotonylated compound (226b) and crotonic acid, as a colourless oil (94 mg).

A solution of crude ester 226b (94 mg, 0.206 mmol) in TFA:H2O (7:1, 1.6 mL) was stirred at room temperature for 30 mins. The solvents were evaporated in vacuo to give a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (5:1) to give the title compound (178b) as 146 a pale yellow sticky solid (28 mg, 35 % over 2 steps). [α]D28 -41.3 (c 0.40, CH2Cl2);

δH(400MHz; CDCl3) 1.34 (9H, s, C(CH3)3), 1.91 (3H, dd, J 6.9, 1.6, C(11)H3), 2.71-2.80

(2H, m, C(6)H2), 3.24 (1H, td, J 10.0, 7.3, C(5)H), 4.70 (1H, dq, J 10.0, 1.7, C(4)H), 4.87

(1H, d~t, J 14.4, 1.7, C(7)HaHbO), 4.91 (1H, d~t, J 14.4, 1.7, C(7)HaHbO), 5.90 (1H, dq, J 15.5, 1.6, C(9)H), 6.96 (1H, ~q, J 1.7, C(3)H), 7.05 (1H, dq, J 15.5, 6.9, C(10)H), 7.23

(2H, d, J 8.2, 2 x Ar-H), 7.42 (2H, d, J 8.2, 2 x Ar-H); δC(100MHz; CDCl3) 18.1 (C(11)H3),

31.3 (C(CH3)3), 34.5 (C(CH3)3), 43.2 (C(6)H2), 50.2 (C(5)H), 60.2 (C(7)H2), 72.0 (C(4)H), 122.2 (C(9)H), 126.1 (Ar-CH), 127.3 (Ar-CH), 134.1 (C(2)H), 135.9 (Ar-C), 145.6 (C(3)H), 148.0 (C(10)H), 165.9 (C(8)=O), 196.5 (C(1)=O); m/z (+ES) 365

([M+Na]+, 100%); (Found 343.1907, C21H27O4 ([M]+), requires 343.1904).

8.1.45 (1S,6R)-6-hydroxy-4'-methoxy-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (195)

C13H14O3; Mw 218.25 To a stirring solution of 141c (460 mg, 1.34 mmol) in dichloromethane (15 mL), under an atmosphere of nitrogen, was added DBU (0.22 mL, 1.478 mmol). The reaction mixture was stirred at room temperature for 2.5 h. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (2 x 10 mL). The combined organic extracts were washed with brine (20 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2) to give the title compound (195) as a white solid (187 mg, 57

%). mp. 119.3-121.7 ºC; [α]D26 -134 (c 0.9, CH2Cl2); νmax (film)/cm-1 3494br (O-H),

2961w (C-H), 2895w (C-H), 1665s (C=O), 1650s (C=C); H(400MHz; CDCl3) 2.62-2.73

(2H, m, C(6)H2), 3.20 (1H, dt, J 10.0, 7.8, C(5)H), 3.83 (3H, s, OCH3), 4.62 (1H, dt, J 10.0, 2.1, C(4)H), 6.06 (1H, dd, J 10.3, 2.1, C(2)H), 6.94 (2H, d, J 8.6, 2 x Ar-H), 7.01

(1H, dd, J 10.3, 2.1, C(3)H), 7.23 (2H, d, J 8.6, 2 x Ar-H); δC(100MHz; CDCl3) 43.3

147

(C(6)H2), 50.0 (C(5)H), 55.4 (OCH3), 72.1 (C(4)H), 114.6 (Ar-CH), 128.7 (Ar-CH), 129.0 (C(2)H), 131.3 (Ar-C), 152.1 (C(3)H), 159.2 (Ar-C), 198.2 (C=O); m/z (+ES) 241

([M+Na]+, 100%), 403 (50); (Found 241.0837, C13H14O3Na ([M]+), requires 241.0836).

8.1.46 (1S,6R)-4'-methoxy-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'-biphenyl]- 3(2H)-one (197)

C19H28O3Si; Mw 332.18 To a solution of 2,6-lutidine (0.10 mL, 1.10 mmol) in dichloromethane (4 mL), at - 78 °C under an atmosphere of nitrogen, was added TESOTf (0.23 mL, 0.81 mmol) followed by a solution of alcohol 195 (80 mg, 0.37 mmol) in dichloromethane (4 mL). The reaction mixture was stirred at -78 °C for 20 mins. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL) and allowed to warm to room temperature. The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine (5 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give the title compound (197) as a yellow oil (105 mg, 86 %). [α]D26 -149.6 (c 0.50, CH2Cl2);

νmax (film)/cm-1 2957m (C-H), 2908m (C-H), 2874m (C-H), 1684s (C=O); H(400MHz;

CDCl3) 0.25-0.44 (6H, m, Si(CH2CH3)3), 0.79 (9H, t, J 8.1, Si(CH2CH3)3), 2.66 (1H, ddd, J

16.4, 4.8, 1.0, C(6)Heq), 2.73 (1H, dd, J 16.4, 13.4, C(6)Hax), 3.21 (1H, ddd, J 13.4, 9.7,

4.8, C(5)H), 3.82 (3H, s, OCH3), 4.51 (1H, dt, J 9.7, 1.9, C(4)H), 6.02 (1H, ddd, J 10.2, 1.9, 1.0, C(2)H), 6.84 (1H, dd, J 10.2, 1.9, C(3)H), 6.88 (2H, d, J 8.6, 2 x Ar-H), 7.17 (2H, d, J 8.6, 2 x Ar-H); δC(100MHz; CDCl3) 4.4 (Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 43.0

(C(6)H2), 49.8 (C(5)H), 55.4 (OCH3), 73.2 (C(4)H), 113.8 (Ar-CH), 128.4 (C(2)H), 128.9 (Ar-CH), 131.3 (Ar-C), 153.9 (C(3)H), 159.2 (Ar-C), 198.2 (C=O); m/z (+ES) 355

([M+Na]+, 100%); (Found 355.1692, C19H28O3NaSi ([M+Na]+), requires 355.1700).

148

8.1.47 (1S,6R)-4-(hydroxymethyl)-4'-methoxy-6-((triethylsilyl)oxy)-1,6- dihydro-[1,1'-biphenyl]-3(2H)-one (222)

C20H30O4Si; Mw 362.19 To a suspension of 197 (70 mg, 0.21 mmol) in water (0.6 mL), was added SDS (17 mg, 0.06 mmol) and DMAP (26 mg, 0.21 mmol). The reaction mixture was stirred for 5 min at room temperature and then formaldehyde 37% (0.22 mL, 2.95 mmol) was added. The resulting mixture was stirred at room temperature for 18 h. The reaction was quenched by the addition of brine (2 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over

MgSO4 and evaporated in vacuo to give the crude product as a yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:3) to give the title compound (222) as a colourless oil (50 mg, 65

%). [α]D26 -91.2 (c 0.50, CH2Cl2); νmax (film)/cm-1 3423br (O-H), 2955s (C-H), 2911m

(C-H), 2876m (C-H), 1674s (C=O); H(400MHz;CDCl3) 0.25-0.44 (6H, m, Si(CH2CH3)3),

0.79 (9H, t, J 7.8, Si(CH2CH3)3), 2.68 (1H, dd, J 16.7, 4.8, C(6)Heq), 2.75 (1H, dd, J 16.7,

13.4, C(6)Hax), 3.20 (1H, ddd, J 13.4, 9.3, 4.8, C(5)H), 3.81 (3H, s, OCH3), 4.25 (1H, dt, J

13.5, 1.4, C(7)HaHb), 4.38 (1H, ddt, J 13.5, 1.4, C(7)HaHb), 4.54 (1H, dq, J 9.3, 1.4, C(4)H), 6.76 (1H, d, J 1.4, C(3)H), 6.88 (2H, d, J 8.6, 2 x Ar-H), 7.17 (2H, d, J 8.6, 2 x Ar-

H); δC(100MHz; CDCl3) 6.4 (Si(CH2CH3)3), 6.8 (Si(CH2CH3)3), 43.5 (C(6)H2), 50.0

(C(5)H), 55.3 (OCH3), 61.2 (C(7)H2), 72.0 (C(4)H), 114.5 (Ar-CH), 128.7 (Ar-CH), 131.0 (Ar-C), 137.6 (Ar-C), 147.8 (C(3)H), 159.2 (C(2)), 198.8 (C=O); m/z (+ES) 385 ([M+Na]+, 100%).

149

8.1.48 (E)-((1S,6R)-6-hydroxy-4'-methoxy-3-oxo-1,2,3,6-tetrahydro-[1,1'- biphenyl]-4-yl)methyl but-2-enoate (178c)

C18H20O5; Mw 316.35 To a solution of compound 222 (55 mg, 0.152 mmol) in dichloromethane (1 mL), was added crotonic anhydride (0.05 mL, 0.334 mmol), DMAP (2 mg, 0.015 mmol) and pyridine (0.11 mL, 1.34 mmol). The reaction mixture was stirred at room temperature for 2.5 h. The reaction was quenched by the addition of a saturated aqueous solution of NaHCO3 (2mL) and diluted with H2O (3mL) and dichloromethane (5mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine

(10 mL), dried over MgSO4 and evaporated in vacuo. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:19) to give a mixture of the crotonylated compound 226c and crotonic acid, as a colourless oil (15 mg).

A solution of crude ester 226c (15 mg, 0.035 mmol) in TFA:H2O (7:1, 0.4 mL) was stirred at room temperature for 30 mins. The solvents were evaporated in vacuo to give a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (2:1) to give the title compound (178c) as a pale yellow foam (5 mg, 10 % over 2 steps). [α]D27 -72.5 (c 0.50, CH2Cl2); νmax (film)/cm-1 3415br (O-H), 2922w (C-H), 2901w (C-H), 1717 (C(8)=O), 1699s

(C(1)=O); δH(400MHz;CDCl3) 1.91 (3H, dd, J 6.8, 1.6, C(11)H3), 2.71 (2H, d, J 9.3,

C(6)H2), 3.20 (1H, q, J 9.3, C(5)H), 3.83 (3H, s, OCH3) 4.66 (1H, dd, J 9.3, 1.8, C(4)H),

4.87 (1H, d~t, J 14.4, 1.6, C(7)HaHbO), 4.91 (1H, d~t, J 14.4, 1.6, C(7)HaHbO), 5.90 (1H, dd, J 15.5, 1.6, C(9)H)), 6.94 (3H, ~d, J 8.7, 2 x Ar-H and C(3)H), 7.05 (1H, dq, J 15.5,

6.8, C(10)H), 7.23 (2H, d, J 8.7, 2 x Ar-H); δC(100MHz; CDCl3) 18.1 (C(11)H3), 43.4

(C(6)H2), 49.9 (C(5)H), 55.3 (60.2 (C(7)H2), 72.1 (C(4)H), 114.6 (Ar-CH), 122.1 (C(9)H), 128.7 (Ar-CH), 131.0 (Ar-C), 134.2 (C(2)H), 145.7 (C(3)H), 148.1 (C(10)H),

150

159.2 (Ar-C), 165.9 (C(8)=O), 196.5 (C(1)=O); m/z (+ES) 339 ([M+Na]+, 100%);

(Found 317.1389, C18H21O5 ([M+H]+), requires 317.1384).

8.1.49 (1S,6R)-6-hydroxy-4-(hydroxymethyl)-4'-methoxy-1,6-dihydro-[1,1'- biphenyl]-3(2H)-one (177c)

C14H16O4; Mw 248.28

A solution of compound 222 (40 mg, 0.110 mmol) in TFA:H2O (7:1) (0.75 mL) was stirred at room temperature for 15 mins. The solvents were evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (2:1) to give the title compound (177c) as a white solid (18 mg, 66 %). mp. 108.7-110.9 C; [α]D28 -97.5 (c 0.45, CH2Cl2); νmax

(film)/cm-1 3367br (O-H), 2926w (C-H), 2901w (C-H), 1668s (C=O); H(400 MHz;

CDCl3) 1.97 (1H, s, OH), 2.32 (1H, br s, OH), 2.67-2.77 (2H, m, C(6)H2), 3.21 (1H, td, J

9.8, 8.1, C(5)H), 3.83-3.84 (3H, m, (OCH3), 4.31 (1H, d, J 13.5, C(7)HaHb), 4.39 (1H, d,

J 13.5, C(7)HaHb), 4.66 (1H, d, J 9.8, C(4)H), 6.93-6.95 (3H, m, C(3)H and 2 x Ar-H),

7.22-7.24 (2H, m, 2 x Ar-H); δC(100MHz; CDCl3) 43.5 (C(6)H2), 50.0 (C(5)H), 55.3

(OCH3), 61.2 (C(7)H2), 72.0 (C(4)H), 114.6 (Ar-CH), 128.7 (Ar-CH), 131.0 (C(2)H), 137.6 (Ar-C), 147.8 (C(3)H), 192.0 (C(1)=O); m/z (+ES) 297 ([M+Na]+, 60%), 129

(100%); (Found 271.0939, C14H16O4Na ([M+Na]+), requires 271.0941).

8.1.50 (1S,6R)-4'-fluoro-6-hydroxy-1,6-dihydro-[1,1'-biphenyl]-3(2H)-one (198)

C12H11FO2; Mw 206.07 To a stirring solution of 141d (100 mg, 0.33 mmol) in tetrahydrofuran (2.5 mL) at 0°C, was added 2 drops of an aqueous solution of NaOH (0.5M). The reaction mixture

151 was stirred at 0 °C for 5 h and 2 more drops of an aqueous solution of NaOH (0.5M) were added every hour. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (5 mL). Organic material was extracted into diethyl ether (2 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over MgSO4 and evaporated in vacuo to give a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2) to give the title compound (198) as a waxy white solid (48 mg, 71%). [α]D28 -122 (c

0.5, CH2Cl2); νmax (film)/cm-1 3365br (O-H), 2889w (C-H), 1685s (C=O), 1672s (C=C);

H (400MHz; CDCl3) 2.68 (2H, d, J 9.3, C(6)H2), 3.25 (1H, q, J 9.3, C(5)H), 4.65 (1H, ~d, J 9.3, C(4)H), 6.09 (1H, dd, J 10.3, 2.2, C(2)H), 7.01 (1H, dd, J 10.3, 1.6, C(3)H), 7.10

(2H, t, J 8.6, 2 x Ar-H), 7.28-7.31 (2H, m, 2 x Ar-H); δC(100MHz; CDCl3) 43.1 (C(6)H2), 49.9 (C(5)H), 71.9 (C(4)H), 116.1 (d, J 22, C(3’)H and C(5’)H),), 129.2 (d, J 11, C(2’)H and C(6’)H), 129.3 (C(2)H), 135.2 (Ar-C), 151.9 (C(3)H), 162.2 (d, J 256, C(F)), 197.7 (C=O); m/z (+ES) 413 ([2M+H]+, 100%).

8.1.51 (1S,6R)-4'-fluoro-6-((triethylsilyl)oxy)-1,6-dihydro-[1,1'-biphenyl]- 3(2H)-one (199)

C18H25FO2Si; Mw 320.47 To a solution of 2,6-lutidine (0.13 mL, 1.46 mmol) in dichloromethane (5 mL), at -78 °C under an atmosphere of nitrogen, was added TESOTf (0.31 mL, 1.07 mmol) followed by a solution of alcohol 198 (100 mg, 0.49 mmol) in dichloromethane (5 mL). The reaction mixture was stirred at -78 °C for 30 mins. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl (8 mL) and allowed to warm to room temperature. The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic extracts were washed with brine (15 mL), dried over MgSO4 and evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:19) to

152 give the title compound (199) as a pale yellow oil (140 mg, 90 %). [α]D28 -129.4 (c

0.45, CH2Cl2); νmax (film)/cm-1 2953m (C-H), 2874m (C-H), 2368w (C-H), 1684s

(C=O); H (400MHz; CDCl3) 0.25-0.44 (6H, m, Si(CH2CH3)3), 0.78 (9H, t, J 7.8,

Si(CH2CH3)3), 2.66 (1H, ddd, J 16.5, 4.8, 1.2, C(6)Heq), 2.71 (1H, dd, J 16.5, 13.4,

C(6)Hax), 3.25 (1H, ddd, J 13.4, 9.3, 4.8, C(5)H), 4.51 (1H, dt, J 9.3, 1.9, C(4)H), 6.03 (1H, ddd, J 10.3, 1.9, 1.2, C(2)H), 6.84 (1H, dd, J 10.3, 1.9, C(3)H), 7.04 (2H, d, J 8.5, 2 x

Ar-H), 7.23 (2H, d, J 8.5, 2 x Ar-H); δC(100MHz; CDCl3) 4.4 (Si(CH2CH3)3), 6.6

(Si(CH2CH3)3), 42.8 (C(6)H2), 49.9 (C(5)H), 72.8 (C(4)H), 115.3 (d, J 21, C(3’)H and C(5’)H), 128.4 (C(2)H), 129.5 (d, J 8, C(2’)H and C(6’)H), 136.5 (d, J 2.8, (C(1’)), 153.7

(C(3)H), 162.1 (d, J 245, C(F)), 198.2 (C=O); δF(376MHz; CDCl3) -115.47 (1F, s); m/z (+ES) 343 ([M+Na]+, 100%).

8.1.52 (1S,6R)-4'-fluoro-4-(hydroxymethyl)-6-((triethylsilyl)oxy)-1,6-dihydro- [1,1'-biphenyl]-3(2H)-one (223)

C19H27FO3Si; Mw 350.50 To a suspension of 199 (135 mg, 0.42 mmol) in water (1.6 mL), was added SDS (46 mg, 0.16 mmol) and DMAP (51 mg, 0.42 mmol). The reaction mixture was stirred for 5 min at room temperature and then formaldehyde 37% (0.44 mL, 5.89 mmol) was added. The resulting mixture was stirred at room temperature for 22 h. The reaction was quenched by the addition of brine (2 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over

MgSO4 and evaporated in vacuo to give the crude product as a yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2) to give the title compound (223) as a white solid (100 mg, 68

%). mp. 68.9-70.1°C; [α]D29 -38.7 (c 0.20, CH2Cl2); νmax (film)/cm-1 3434br (O-H),

2927w (C-H), 2866w (C-H), 1668s (C=O); H (400MHz; CDCl3) 0.24-0.44 (6H, m,

Si(CH2CH3)3), 0.79 (9H, t, J 8.1, Si(CH2CH3)3), 2.67 (1H, dd, J 16.4, 4.9, C(6)Heq), 2.76

153

(1H, dd, J 16.4, 13.6, C(6)Hax), 3.24 (1H, ddd, J 13.6, 9.5, 4.9, C(5)H), 4.26 (1H, dt, J

13.6, 1.5, C(7)HaHb), 4.38 (1H, dt, J 13.6, 1.5, C(7)HaHb), 4.54 (1H, dq, J 9.5, 1.5, C(4)H), 6.77 (1H, d, J 1.5, C(3)H), 7.04 (2H, t, J 8.8, 2 x Ar-H), 7.22 (2H, dd, J 8.8, 5.3, 2 x Ar-H);

δC(100MHz; CDCl3) 6.4 (Si(CH2CH3)3), 6.8 (Si(CH2CH3)3), 43.4 (C(6)H2), 50.0 (C(5)H),

61.2 (C(7)H2), 70.1 (C(4)H), 116.1 (d, J 21, C(3’)H and C(5’)H), 129.2 (d, J 7.4, C(2’)H and C(6’)H), 147.8 (C(3)H), 157.8 (C(2)), 161.4 (Ar-C), 204.9 (C=O); m/z (+ES) 102 (100%), 259 ([(M-TES)+Na]+, 80%).

8.1.53 (E)-((1S,6R)-4'-fluoro-3-oxo-6-((triethylsilyl)oxy)-1,2,3,6-tetrahydro- [1,1'-biphenyl]-4-yl)methyl but-2-enoate (226d)

8.1.54 (E)-((1S,6R)-4'-fluoro-6-hydroxy-3-oxo-1,2,3,6-tetrahydro-[1,1'- biphenyl]-4-yl)methyl but-2-enoate(178d)

C17H17FO4; Mw 304.11 To a solution of alcohol 223 (100 mg, 0.29 mmol) in dichloromethane (1 mL), was added crotonic anhydride (93 µL, 0.63 mmol), DMAP (4 mg, 0.029 mmol) and pyridine (0.20 mL, 2.51 mmol). The reaction mixture was stirred at room temperature for 2.5 h. The reaction was quenched by the addition of a saturated aqueous solution of NaHCO3 (2 mL) and diluted with H2O (3 mL) and dichloromethane (5 mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (3 x 5 mL). The combined organic extracts were washed with brine (10 mL), dried over MgSO4 and evaporated in vacuo to give a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give a mixture of the crotonylated compound (226d) and crotonic acid, as a colourless oil (80 mg).

A solution of crude ester 226d (80 mg, 0.19 mmol) in TFA:H2O (7:1, 1.6 mL) was stirred at room temperature for 40 mins. The solvents were evaporated in vacuo to give the crude product as a brown oil. The residue was purified by flash silica

154 chromatography, eluting with ethyl acetate: 40-60 petroleum ether (2:1) to give the title compound (178d) as a white thick oil (33 mg, 38 % over 2 steps). [α]D29 -112.7

(c 0.40, CH2Cl2); νmax (film)/cm-1 3446br (O-H), 2948w (C-H), 2924w (C-H), 1718

(C(8)=O), 1675s (C(1)=O); δH(400MHz; CDCl3) 1.91 (3H, dd, J 6.8, 1.6, C(11)H3), 2.66-

2.75 (2H, m, C(6)H2), 3.25 (1H, td, J 9.8, 7.8, C(5)H), 4.67 (1H, dd, J 9.8, 1.6, C(4)H),

4.86 (1H, d~t, J 14.4, 1.6, C(7)HaHbO), 4.91 (1H, d~t, J 14.4, 1.6, C(7)HaHbO), 5.90 (1H, dd, J 15.5, 1.6, C(9)H)), 6.94-6.95 (1H, m, C(3)H), 7.00-7.07 (1H, m, C(10)H), 7.08-7.10

(2H, m, 2 x Ar-H), 7.26-7.29 (2H, m, 2 x Ar-H); δC(100MHz; CDCl3) 18.1 (C(11)H3),

43.3 (C(6)H2), 49.8 (C(5)H), 60.1 (C(7)H2), 71.5 (C(4)H), 116.1 (d, J 21, C(3’)H and C(5’)H), 122.1 (C(9)H), 129.2 (d, J 8.3, C(2’)H and C(6’)H), 134.3 (d, J 3.7, C(1’), 145.8

(C(3)H), 148.0 (C(10)H), 165.9 (C(8)=O), 195.4 (C(1)=O); δF(376MHz; CDCl3) -114.12 (1F, s); m/z (+ES) 305 ([M+H]+), 100%.

8.1.55 (1S,6R)-4'-fluoro-6-hydroxy-4-(hydroxymethyl)-1,6-dihydro-[1,1'- biphenyl]-3(2H)-one (177d)

C13H13FO3; Mw 236.24

A solution of compound 223 (35 mg, 0.10 mmol) in TFA:H2O (7:1, 0.72 mL) was stirred at room temperature for 30 mins. The solvents were evaporated in vacuo to give a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (2:1) to give the title compound (177d) as a colourless film (10 mg, 42 %). [α]D29 -63.7 (c 0.40, CH2Cl2); νmax (film)/cm-1 3424br

(O-H), 2945w (C-H), 2919w (C-H), 1683s (C=O); H(400 MHz; CDCl3) 2.68-2.76 (2H, m, C(6)H2), 3.25 (1H, ~q, J 9.5, C(5)H), 4.31 (1H, dt, J 13.9, 1.5, C(7)HaHb), 4.38 (1H, dt, J 13.9, 1.3, C(7)HaHb), 4.67 (1H, dd, J 9.5, 1.4, C(4)H), 6.96 (1H, ~d, J 1.4, C(3)H),

7.10 (2H, t, J 8.7, 2 x Ar-H), 7.28 (2H, dd, J 8.7, 5.0, 2 x Ar-H); δC(100MHz; CDCl3) 43.4

(C(6)H2), 49.9 (C(5)H), 61.0 (C(7)H2), 71.9 (C(4)H), 116.1 (d, J 21, C(3’)H and C(5’)H), 129.2 (d, J 8.3, C(2’)H and C(6’)H), 135.0 (d, J 3.7, C(1’), 137.7 (C(2)H), 147.7 (C(3)H),

198.4 (C(1)=O); δF(376MHz; CDCl3) -114.15 (1F, s); m/z (+ES) 259 ([M+Na]+, 100%);

(Found 259.0759, C13H13NaO3F ([M+Na]+), requires 259.0746).

155

8.1.56 (4S)-4-Phenyltetrahydrospiro[benzo[d][1,3]dioxole-2,1'-cyclohexan]- 6(3aH)-one (141b)

C18H22O3; Mw 286.16 To a stirred suspension of copper iodide (100 mg, 0.528 mmol) in THF (1 mL) at 0 °C, was added phenylmagnesium bromide (3M in Et2O) (0.35 mL, 1.06 mmol) and the reaction mixture was stirred for 1 h at 0 °C, under an atmosphere of N2. A solution of enone (35) (100 mg, 0.480 mmol) in THF (5 mL) was added dropwise and the reaction mixture was stirred at 0 °C, under N2 for 2 h. The reaction was quenched by the addition of a mixture of a saturated aqueous solution of NH4Cl and aqueous NH3 solution (9:1, 10 mL). The two layers were separated and the aqueous layer was extracted with diethyl ether (3 x 15 mL). The combined organic extracts were washed with brine (30 mL), dried over MgSO4 and concentrated in vacuo to give a yellow oil. The crude residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound as a pale yellow oil (98 mg, 73%). [α]D28 -42.8 (c 1.0, CH2Cl2); νmax (film)/cm-1 2934w (C-H),

2883w (C-H), 1712s (C=O); H (400MHz; CDCl3) 1.37-1.76 (10H, m, 5 x CH2 of cyclohexane), 2.60-2.66 (2H, m, one of C(2)H2 and one of C(6)H2), 2.73 (1H, dd, J 16.7,

4.4, one of C(2)H2), 2.79 (1H, dd, J 17.6, 4.8, one of C(6)H2), 3.44 (1H, d~t, J 8.5, 4.8, C(5)H), 4.57-4.68 (2H, m, C(3)H and C(4)H), 7.23-7.28 (3H, m, 3 x Ar-H), 7.33-7.37

(2H, m, 2 x Ar-H); δC(100MHz; CDCl3) 23.6 (cyclohexane CH2), 24.0 (cyclohexane

CH2), 25.1 (cyclohexane CH2), 33.7 (cyclohexane CH2), 36.9 (cyclohexane CH2), 40.7

(C(6)H2), 42.3 (C(5)H), 42.7 (C(2)H2), 72.2 (C(3)H or C(4)H), 76.7 (C(3)H or C(4)H), 109.3 (cyclohexane C), 127.0 (Ar-CH), 127.5 (Ar-CH), 128.8 (Ar-CH), 140.2 (Ar-C), 208.9 (C=O); m/z (+ES) 309 ([M+Na]+, 100%), 341 (65); (Found 309.1461,

C18H22NaO3 ([M+Na]+), requires 309.1462).

156

8.1.57 (3aS,4S,7aR)-4-(4-iodophenyl)tetrahydrospiro[benzo[d][1,3]dioxole- 2,1'-cyclohexan]-6(3aH)-one (141g)

C18H21IO3; Mw 412.27 To a solution of enone 35 (200 mg, 0.96 mmol) in dioxane:water (10:1, 2.2 mL) was added 4-iodophenyl boronic acid (595 mg, 2.40 mmol) and [RhOH(cod)]2 (22 mg, 5 mol%), followed by Et3N (0.12 mL, 0.96 mmol). The reaction mixture was stirred at room temperature for 30 h. The solvents were evaporated in vacuo to give a brown oil which was purified by flash silica chromatography, eluting with ethyl acetate: 40- 60 petroleum ether (1:5) to give the title compound (141g) as a yellow thick oil

(106 mg, 27%). [α]D28 -80.7 (c 1.6, CH2Cl2); νmax(film)/cm-1 2932m (C-H), 2858w (C-

H), 1716s (C=O); δH(400MHz; CDCl3) 1.56-1.74 (10H, m, 5 x CH2 of cyclohexane), 2.55

(1H, dd, J 17.5, 9.3, C(6)Hax), 2.64 (1H, dd, J 17.0, 5.2, C(2)Hax or C(2)Heq), 2.70 (1H, dd, J 17.5, 4.4, C(6)Heq), 2.73 (1H, dd, J 17.0, 5.2, C(2)Hax or C(2)Heq), 3.34 (1H, ddd, J 9.3, 6.6, 4.4, C(5)H), 4.50 (1H, ~t, J 6.6, C(4)H), 4.58 (1H, d~t, J 6.6, 4.4, C(3)H), 7.00

(2H, d, J 8.4, 2 x Ar-H), 7.67 (2H, d, J 8.4, 2 x Ar-H); δC(100MHz; CDCl3) 23.6

(cyclohexane CH2), 23.9 (cyclohexane CH2), 25.1 (cyclohexane CH2), 33.8

(cyclohexane CH2), 37.0 (cyclohexane CH2), 40.9 (C(6)H2), 42.4 (C(2)H2), 42.5 (C(5)H), 72.1 (C(3)H), 77.2 (C(4)H), 92.5 (C(I), 109.5 (cyclohexane C), 129.4 (Ar-CH),

137.8 (Ar-CH), 139.9 (Ar-C), 208.2 (C=O); (Found 435.0440, C18H21NaO3I ([M+Na]+), requires 435.0433).

8.1.58 (2S,3S,4aR,8aR)-7-(hydroxymethyl)-2,3-dimethoxy-2,3-dimethyl- 2,3,4a,5-tetrahydrobenzo[b][1,4]dioxin-6(8aH)-one (271)

C13H20O6; Mw 272.29

157

To a solution of enone 117 (500 mg, 2.06 mmol) in THF:H2O (1:1, 20 mL) was added 37% formaldehyde (0.31 mL, 4.13 mmol) followed by DMAP (25 mg, 0.21 mmol). The reaction mixture was stirred at 40 °C for 20 h. The reaction was quenched by the addition of 1M HCl (1 mL) and extracted with dichloromethane (3 x 15 mL). The combined organic extracts were washed with a saturated aqueous solution of

NaHCO3 (20 mL), brine (20 mL) and dried over MgSO4. The solvents were evaporated in vacuo to give the title compound as a white solid (535 mg, 95%). mp. 179.8-181.2

C; []D23 +66.9 (c 1.0, CH2Cl2); max(film)/cm-1 3532br (O-H), 2957w (C-H), 2946w

(C-H), 1669s (C=O, enone); δH(400MHz; CDCl3) 1.34 (3H, s, CH3), 1.37 (3H, s, CH3),

2.18 (1H, t, J 6.3, C(7)OH), 2.53 (1H, dd, J 16.5, 13.5, C(6)Hax), 2.78 (1H, dd, J 16.5, 4.9,

C(6)Heq), 3.27 (3H, s, OCH3), 3.33 (3H, s, OCH3), 4.04 (1H, ddd, J 13.5, 9.1, 4.9, C(5)H),

4.17 (1H, ddt, J 13.9, 6.3, 1.6, C(7)HaHb), 4.39 (1H, ddt, J 13.9, 6.3, 1.6, C(7)HaHb), 4.53

(1H, dq, J 9.1, 1.6, C(4)H), 6.85 (1H, ~q, J 1.6, C(3)H); C(100MHz; CDCl3) 17.6 (CH3),

17.7 (CH3), 40.1 (C(6)H2), 46.0 (acetal OCH3), 48.1 (acetal OCH3), 60.9 (C(7)H2), 68.2 (C(5)H), 69.3 (C(4)H), 100.0, 101.0 (2 x acetal C), 139.1 (C(2)), 144.4 (C(3)H), 197.5

(C(1)=O); m/z (-ES) 271 ([M-H]-, 100%); (Found 295.1152, C13H20O6Na ([M]-), requires 295.1153).

8.1.59 (E)-((2S,3S,4aR,8aR)-2,3-dimethoxy-2,3-dimethyl-7-oxo-2,3,4a,7,8,8a- hexahydro benzo [b][1,4]dioxin-6-yl)methyl but-2-enoate (272)

C17H24O7; Mw 340.37 To a solution of 271 (1 g, 3.67 mmol) in dichloromethane (20 mL) at room temperature under an atmosphere of nitrogen, was added crotonic anhydride (1.20 mL, 8.08 mmol), DMAP (45 mg, 0.37 mmol) and pyridine (2.61 mL, 32.3 mmol). The reaction mixture was stirred at room temperature for 2 h and then quenched by the addition of a saturated aqueous solution of NaHCO3 (10 mL). The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (3 x 20 mL).

158

The combined organic extracts were washed with a saturated aqueous solution of

NaHCO3 (30 mL) and brine (30 mL), dried over MgSO4 and evaporated in vacuo. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:9) to give the title compound as a white solid (1 g, 87 %). mp.

79.0-81.6 °C; [α]D27 +58.0 (c 1.00, CH2Cl2); νmax (film)/cm-1 2958w (C-H), 2948w (C-

H), 1715s (C(8)=O), 1671s (C(1)=O); H(400MHz; CDCl3) 1.34 (3H, s, CH3), 1.37 (3H, s, CH3), 1.90 (3H, dd, J 7.1, 1.8, C(11)H3), 2.53 (1H, dd, J 16.4, 13.5, C(6)Hax), 2.80 (1H, dd, J 16.4, 4.8, C(6)Heq), 3.27 (3H, s, OCH3), 3.33 (3H, s, OCH3), 4.04 (1H, ddd, J 13.5,

9.0, 4.8, C(5)H), 4.54 (1H, dq, J 9.0, 2.6, C(4)H), 4.77 (1H, d~t, J 14.4, 1.6, C(7)HaHb),

4.86 (1H, ddd, J 14.4, 2.6, 1.6, C(7)HaHb), 5.87 (1H, dq, J 15.6, 1.8, C(9)H), 6.83 (1H, ~q,

J 1.6, C(3)H), 7.03 (1H, dq, J 15.6, 7.1, C(10)H); C (100 MHz; CDCl3) 17.6 (CH3), 17.7

(CH3), 18.0 (C(11)H3), 42.0 (C(6)H2), 48.1 (acetal OCH3), 48.2 (acetal OCH3), 60.0

(C(7)H2), 67.9 (C(5)H), 69.2 (C(4)H), 99.7 (acetal C), 100.8 (acetal C), 122.1 (C(9)H), 135.5 (C(2)), 144.8 (C(3)H), 145.7 (C(10)H), 165.8 (C(8)=O), 195.1 (C(1)=O); m/z

(+ES) 363 ([M+Na]+, 100%); (Found 341.1598, C17H25O7 ([M]+), requires 341.1595).

8.1.60 tert-butyl (2-mercaptoethyl)carbamate (276)

8.1.61 tert-butyl(2-((((2S,3S,4aR,8aR)-2,3-dimethoxy-2,3-dimethyl-7-oxo- 2,3,4a,7,8,8a-hexahydrobenzo[b][1,4]dioxin-6yl)methyl)thio) ethyl)carbamate (277)

C20H33NO7S; Mw 431.54 To a suspension of cysteamine hydrochloride (200 mg, 1.76 mmol) in dichloromethane (4 mL), triethylamine (0.27 mL, 1.94 mmol) was added dropwise followed by di-tert-butyl dicarbonate (0.44 mL, 1.90 mmol). The reaction mixture was stirred at room temperature for 1 h 40 mins when the solvents were evaporated in vacuo to give a white solid. The residue was dissolved in ethyl acetate (5 mL) and water (5mL). The two layers were separated and the organic layer was washed with water (5 mL), a saturated aqueous solution of NaHCO3 (5 mL) and brine (5 mL). The

159 organic extract was then dried over MgSO4 and evaporated in vacuo to give a mixture of compound 276 and t-butanol, as a colourless oil (300 mg, 96%). H(500 MHz;

CDCl3) 1.36 (1H, t, J 8.6, SH), 1.45 (9H, s, C(CH3)3), 2.65 (2H, dt, J 8.6, 6.4, C(2)H2), 3.31

(2H, ~q, J 6.4, C(3)H2), 4.93 (1H, s, NH); m/z (+ES) 200 ([M+Na]+, 100%); (Found

200.0715, C7H15O2NNaS ([M+Na]+), requires 200.0716). To a solution of crude 276 (170 mg, 0.961 mmol) in dichloromethane (2 mL) was added triethylamine (0.11 mL, 0.768 mmol), followed by a solution of ester 272 (200 mg, 0.640 mmol) in dichloromethane (2 mL). The reaction mixture was stirred at room temperature for 18 h. The reaction was quenched by the addition of water (4 mL). The two layers were separated and the aqueous layer was extracted with dichloromethane (2 x 5 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (10 mL), brine (10 mL), dried over MgSO4 and evaporated in vacuo to give a yellow oil. The residue was purified by flash silica chromatography eluting with ethyl acetate: 40-60 petroleum ether (1:5) to give the required compound 173 as a colourless oil (160 mg, 58 %). H(400 MHz; CDCl3) 1.33

(3H, s, CH3), 1.37 (3H, s, CH3), 1.45 (9H, s, 1.45 (9H, s, C(CH3)3), 2.51 (1H, dd, J 16.4,

13.6, C(6)Hax), 2.61 (2H, t, J 6.6, C(8)H2), 2.80 (1H, dd, J 16.4, 4.9, C(6)Heq), 3.23 (3H, s,

OCH3), 3.26-3.32 (4H, m, C(7)H2 and C(9)H2), 3.32 (3H, s, OCH3), 4.01 (1H, ddd, J 13.6, 9.3, 4.9, C(5)H), 4.52 (1H, dq, J 9.3, 1.8, C(4)H), 4.92 (1H, s, NH), 6.82 (1H, s, C(3)H);

C(100MHz; CDCl3) 17.6 (CH3), 17.7 (CH3), 28.4 (C(CH3)3), 28.9 (C(7)H2 or C(9)H2),

32.5 (C(8)H2), 39.4 (C(7)H2 or C(9)H2), 42.0 (C(6)H2), 48.1 (acetal OCH3), 48.2 (acetal

OCH3), 67.9 (C(5)H), 69.2 (C(4)H), 99.7(acetal C), 100.7 (acetal C), 137.0 (C(2)), 144.9 (C(3)H), 155.7 (C(10)=O), 195.3 (C(1)=O); m/z (-ES) 466 ([M+Cl]-, 100%).

8.1.62 N-(2-((((2S,3S,4aR,8aR)-2,3-dimethoxy-2,3-dimethyl-7-oxo- 2,3,4a,7,8,8a-hexahydro benzo[b][1,4]dioxin-6-yl)methyl)thio) ethyl)acetamide (274)

C17H27NO6S; Mw 373.46

160

To a suspension of cysteamine hydrochloride (40 mg, 0.35 mmol) in dichloromethane (1 mL), was added triethylamine (88 μL, 0.64 mmol) followed by a solution of ester 272 (100 mg, 0.32 mmol) in dichloromethane (1 mL). The reaction mixture was stirred at room temperature for 2 h. More triethylamine (48 μL, 0.35 mmol) was added, followed by acetic anhydride (36 μL, 0.38 mmol) and DMAP (4 mg, 0.032 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction was quenched by the addition of water (2 mL) and extracted with dichloromethane (2 x 5 mL). The combined organic extracts were washed with a saturated aqueous solution of NaHCO3 (5 mL), 0.1M HCl (5 mL), brine (5 mL), dried over MgSO4 and evaporated in vacuo to give an off white foam. The residue was purified by flash silica chromatography eluting with 1-3% methanol in dichloromethane to give the title compound 274 as a yellow gum (20 mg, 17 %).

H(400MHz; CDCl3) 1.33 (3H, s, CH3), 1.37 (3H, s, CH3), 2.02 (3H, s, C(11)H3), 2.51

(1H, dd, J 16.6, 13.5, C(6)Hax), 2.64 (2H, t, J 6.0, C(8)H2), 2.80 (1H, dd, J 16.6, 4.8,

C(6)Heq), 3.26 (3H, s, OCH3), 3.30 (2H, ~s, C(7)H2), 3.32 (3H, s, OCH3), 3.45 (2H, q, J

6.0, C(9)H2), 4.01 (1H, ddd, J 13.5, 9.3, 4.8, C(5)H), 4.51 (1H, d~t, J 9.3, 1.6, C(4)H),

6.18 (1H, s, NH), 6.81 (1H, s, C(3)H); C(100MHz; CDCl3) 17.6 (CH3), 17.7 (CH3), 23.2

(C(11)H3), 29.1 (C(7)H2), 32.3 (C(8)H2), 38.3 (C(9)H2), 42.0 (C(6)H2), 48.1 (acetal

OCH3), 48.2 (acetal OCH3), 67.9 (C(5)H), 69.2 (C(4)H), 99.7(acetal C), 100.8 (acetal C), 136.9 (C(2)), 145.4 (C(3)H), 170.2 (C(10)=O), 195.5 (C(1)=O); m/z (+ES) 396

([M+Na]+, 100%); (Found 374.1632, C17H28NO6S ([M]+), requires 374.1632).

8.1.63 2-(Hydroxymethyl)cyclohex-2-enone (218)

C7H10O2; Mw 126.15 To a solution of 1-cyclohex-2-enone (2 mL, 20.6 mmol) in tetrahydrofuran (10 mL) was added 37% aqueous formaldehyde (3.8 mL, 51.6 mmol) followed by DMAP (240 mg, 2.66 mmol). The resulting reaction mixture was stirred at room temperature, under an atmosphere of N2, for 20 h and was then acidified by the addition of 1M HCl (2 mL). Organic material was extracted into dichloromethane (2 x 20 mL). The 161 combined organic extracts were washed with brine (10 mL), dried over MgSO4 and concentrated in vacuo to give the crude product as a dark orange oil. Purification of the residue by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2), gave the title compound as a yellow oil (350 mg, 13 %). νmax

(film)/cm-1 3980br (O-H), 2930w (C-H), 1660s (C=O); H(400MHz; CDCl3) 2.02 (2H, quintet, J 6.0, C(5)H2), 2.08 (1H, s, C(7)OH), 2.39-2.49 (4H, m, C(4)H2 and C(6)H2),

4.25 (2H, q, J 1.3, C(7)H2), 6.95 (1H, t, J 4.2, C(3)H); C (100MHz; CDCl3) 23.0 (C(5)H2),

25.8 (C(4)H2), 38.6 (C(6)H2), 62.3 (C(7)H2), 138.5 (C(2)), 147.2 (C(3)H), 200.8 (C=O); m/z (+ES) 127 ([M+H]+, 100%); (Found 126.0673, C7H10O2 ([M]+) requires 126.0675).

8.1.64 4-((6-oxocyclohex-1-en-1-yl)methoxy)-2H-chromen-2-one (257a)

C16H14O4; Mw 270.09

To a solution of alcohol 218 (100 mg, 0.79 mmol) in tetrahydrofuran (3 mL), under an atmosphere of nitrogen, was added triphenylphosphine (208 mg, 0.79 mmol) and 4-hydroxycoumarin (135 mg, 0.83 mmol). The reaction mixture was cooled to 0 °C, under an atmosphere of N2, and DIAD (0.16 mL, 0.83 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 30 mins and then at room temperature for 22 h. The solvents were evaporated in vacuo to give the crude product as a thick yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2), to give the title compound as a white solid (40 mg, 19 %). mp. 161.5-163.1 C; νmax (film)/cm-1 2927w (C-H), 2928w (C-H), 1710s

(C(10)=O), 1669s (C(1)=O); H(400MHz; CDCl3) 2.10 (2H, quin, J 6.4, C(5)H2), 2.50-

2.53 (2H, m, C(4)H2), 2.55 (2H, t, J 6.4, C(6)H2), 4.15 (2H, s, C(7)H2), 5.65 (1H, s, C(9)H), 7.17 (1H, ~t, J 4.0, C(3)H), 7.26-7.30 (1H, m, Ar-H), 7.33 (1H, dd, J 8.4, 0.5, Ar-

H), 7.56 (1H, ddd, J 8.4, 7.6, 1.6, Ar-H), 7.83 (1H, dd, J 7.6, 1.6, Ar-H); C (100MHz;

162

CDCl3) 22.7 (C(5)H2), 25.6 (C(4)H2), 37.9 (C(6)H2), 65.9 (C(7)H2), 89.9 (Ar-CH); m/z (+ES) 293 ([M+Na]+, 100%).

8.1.65 Cyclohex-1-en-1-ylmethanol (261)

C7H12O; Mw 112.17 To a solution of methyl cyclohex-1-enecarboxylate (0.5 mL, 3.67 mmol) in toluene (5 mL), at -78 °C under an atmosphere of nitrogen, was added a solution of 1M DIBAL (in toluene) (9.5 mL, 9.54 mmol). The reaction mixture was stirred at -78 °C for 30 mins and then allowed to warm to room temperature over 40 mins. It was then poured onto a cold saturated aqueous solution of NH4Cl (10 mL). The two layers were separated and the emulsion at the interface of the two layers was passed through a pad of NaCl. The aqueous layer was extracted with diethyl ether (2 x 10 mL). The combined organic extracts were dried over MgSO4 and evaporated in vacuo to give the crude product as a colourless oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:5), to give the title compound as a colourless oil (279 mg, 68 %). νmax (film)/cm-1 3343br (O-H),

2926m (C-H), 2857w (C-H), 2835w (C-H), 1667w (C=C); δH(400MHz; CDCl3) 1.56-

1.69 (4H, m, C(4)H2 and C(5)H2), 2.00-2.06 (4H, m, C(3)H2 and C(6)H2), 3.99 (2H, s,

C(7)H2), 5.69 (1H, spt, J 1.8, C(2)H); δC(100MHz; CDCl3) 22.4 (C(4)H2 or C(5)H2), 22.5

(C(4)H2 or C(5)H2), 24.9 (C(3)H2 or C(6)H2), 25.6 (C(3)H2 or C(6)H2), 67.7 (C(7)H2), 123.1 (C(2)H), 137.5 (C(1)).

8.1.66 4-(Cyclohex-1-en-1-ylmethoxy)-2H-chromen-2-one (258a)

C16H16O3; Mw 256.30

163

To a solution of alcohol 261 (150 mg, 1.34 mmol) in tetrahydrofuran (5 mL), under an atmosphere of nitrogen, was added triphenylphosphine (702 mg, 2.67 mmol) and 4-hydroxycoumarin (435 mg, 2.67 mmol). The reaction mixture was cooled to 0 °C and DIAD (0.53 mL, 2.67 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 30 mins and then at room temperature for 17 h. The solvents were evaporated in vacuo to give the crude product as a thick yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:3), to give the title compound as a white gum (110 mg, 32 %). νmax (film)/cm-

1 2929w (C-H), 2859w (C-H), 2838w (C-H), 1716s (C=O), 1622w (C=C); H(400MHz;

CDCl3) 1.62-1.75 (4H, m, C(4)H2 and C(5)H2), 2.10-2.13 (4H, m, C(3)H2 and C(6)H2),

4.54 (2H, s, C(7)H2), 5.71 (1H, s, C(9)H), 5.90 (1H, br s, C(2)H), 7.26-7.30 (1H, m, C(12)H), 7.33 (1H, dd, J 8.5, 0.8, C(11)H), 7.55 (1H, ddd, J 8.5, 7.6, 1.6, C(13)H), 7.85

(1H, dd, J 7.6, 1.6, C(14)H); δC(100MHz; CDCl3) 22.0 (C(4)H2 or C(5)H2), 22.2 (C(4)H2 or C(5)H2), 25.0 (C(3)H2 or C(6)H2), 25.7 (C(3)H2 or C(6)H2), 73.9 (C(7)H2), 90.8 (C(9)H), 115.9 (Ar-C), 116.7 (C(11)H), 123.1 (C(14)H), 123.8 (C(12)H), 127.8 (C(2)H), 131.6 (C(1)), 132.3 (C(13)H), 153.3 (Ar-C), 163.1 (C), 165.5 (C=O); m/z

(+ES) 279 ([M+Na]+, 100%); (Found 279.0987, C16H16O3Na ([M+Na]+) requires 126.0675).

8.1.67 4-Hydroxy-5-methoxy-2H-chromen-2-one (255)

C10H8O4; Mw 192.17 To a suspension of NaH (722 mg, 30.1 mmol) in diethyl carbonate (7.5 mL), under an atmosphere of N2, at 0 °C, was added a solution of 2-hydroxy-6-methoxy- acetophenone in diethyl carbonate (7.5 mL), dropwise. Once effervescence had ceased, the reaction mixture was heated at reflux for 3h. The reaction was then allowed to cool to room temperature and it was quenched by the addition of H2O (10 mL). The two layers were separated and the aqueous layer was washed with diethyl ether (10 mL). The aqueous extracts were cooled to 0 °C and then acidified by

164 the addition of concentrated HCl to give a pale yellow solid. The precipitate was filtered off, washed with H2O (10 mL) and dried under vacuum to give the title compound as an off-white solid (560 mg, 48%). mp. 157.3-158.9 °C (Lit 155 °C)130;

νmax (film)/cm-1 3266br (O-H), 2949w (C-H), 1706s (C=O), 1644s (C=C); H(400MHz;

CDCl3) 4.09 (3H, s, OCH3), 5.71 (1H, s, C(3)H), 6.81 (1H, d, J 8.4, C(6)H or C(8)H), 7.04 (1H, dd, J 8.4, 0.9, C(6)H or C(8)H), 7.50 (1H, t, J 8.4, C(7)H), 9.55 (1H, s, C(4)OH);

δC(100MHz; CDCl3) 57.0 (OCH3), 92.9 (C(3)H), 104.9 (C), 105.5 (C(6)H or C(8)H), 111.4 (C(6)H or C(8)H), 132.5 (C(7)H), 155.0 (Ar-C), 156.2 (Ar-C), 162.8 (C), 165.9 (C=O); m/z (+ES) 193 ([M+H]+, 100%).

8.1.68 3-Benzyl-4-hydroxy-5-methoxy-2H-chromen-2-one (253)

C17H14O4; Mw 282.29 To a mixture of 4-hydroxy-5-methoxy-2H-chromen-2-one (255) (300, 1.56 mmol), (pentamethylcyclopentadienyl)iridium(III) chloride dimer (62 mg, 5mol%) and caesium carbonate (51 mg, 0.16 mmol) in toluene (1.1 mL), was added benzyl alcohol (0.81 mL, 7.81 mmol) and isopropanol (0.02 mL). The reaction mixture was heated under reflux at 110 °C for 20 h. The resulting mixture was allowed to cool to room temperature and then concentrated in vacuo to give a brown thick oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2), to give the title compound as an off white solid (315 mg, 72

%). mp. 175.6-178.1 °C; νmax (film)/cm-1 3320br (O-H), 2980w (C-H), 1686s (C=O),

1644s (C=C); H(400MHz; CDCl3) 3.91 (2H, s, C(9)H2), 4.06 (3H, s, OCH3), 6.78 (1H, d, J 8.4, C(6)H or C(8)H), 7.02 (1H, d, J 8.4, C(6)H or C(8)H), 7.18 (1H, d, J 7.1, Ar-H),

7.29-7.25 (2H, m, Ar-H), 7.45-7.51 (3H, m, Ar-H), 9.73 (1H, s, C(4)OH); δC(100MHz;

CDCl3) 29.2 (C(9)H2), 57.0 (OCH3), 105.2 (C), 105.3 (C), 105.4 (C(6)H or C(8)H), 111.2 (C(6)H or C(8)H), 126.0 (Ar-CH), 128.2 (Ar-CH), 128.8 (Ar-CH), 131.5 (Ar-C), 140.1 (Ar-C), 153.6 (Ar-C), 155.7 (Ar-C), 161.0 (C), 163.4 (C=O); m/z (+ES) 283 ([M+H]+, 100%).

165

8.1.69 3-Benzyl-4-(cyclohex-1-en-1-ylmethoxy)-5-methoxy-2H-chromen-2-one

C24H24O4; Mw 376.44

To a solution of alcohol 261 (26 mg, 0.24 mmol) in tetrahydrofuran (1.5 mL), under an atmosphere of nitrogen, was added triphenylphosphine (121 mg, 0.46 mmol) and 3-benzyl-4-hydroxy-5-methoxy-2H-chromen-2-one 253 (100 mg, 0.35 mmol). The reaction mixture was cooled to 0 °C and DIAD (0.09 mL, 0.46 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 30 mins and then at room temperature for 20 h. The solvents were evaporated in vacuo to give the crude product as a thick yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:5), to give the title compound as a colourless oil (30 mg, 34 %). νmax (film)/cm-1 2932w (C-H),

2856w (C-H), 1713s (C=O), 1620w (C=C); H(400 MHz; CDCl3) 1.62-1.75 (4H, m,

C(4)H2 and C(5)H2), 2.09-2.22 (4H, m, C(3)H2 and C(6)H2), 3.94 (5H, s, C(7)H2 and

OCH3), 4.26 (2H, s, C(15)H2), 5.81 (1H, brs, C(2)H), 6.76 (1H, dd, J 8.3, 0.8, C(11)H or C(13)H), 6.97 (1H, dd, J 8.3, 0.8, C(11)H or C(13)H), 7.17 (1H, tt, J 7.6, 1.5, Ar-H), 7.24

(2H, ~d, J 7.6, Ar-H), 7.37 (1H, ~d, J 7.6, Ar-H), 7.42 (1H, t, J 8.3, C(12)H); δC(100MHz;

CDCl3) 19.7 (C(4)H2 or C(5)H2), 21.4 (C(4)H2 or C(5)H2), 26.3 (C(3)H2 or C(6)H2),

27.8 (C(3)H2 or C(6)H2), 55.9 (OCH3), 73.0 (C(7)H2), 109.1 (C(11)H or C(13)H), 109.4 (C(11)H or C(13)H), 118.1 (C), 128.0 (Ar-CH), 128.2 (Ar-CH), 129.9 (Ar-CH), 139.2 (C), 161.4 (C), 165.7 (C(10)=O); m/z (+ES) 399 ([M+Na]+, 100%).

166

8.1.70 3-Benzyl-5-methoxy-4-((6-oxocyclohex-1-en-1-yl)methoxy)-2H- chromen-2-one (257b)

C24H22O5; Mw 390.44 To a solution of 2-(hydroxymethyl)cyclohex-2-enone 218 (50 mg, 0.39 mmol) in tetrahydrofuran (1.5 mL), under an atmosphere of nitrogen, was added triphenylphosphine (208 mg, 0.79 mmol) and 3-benzyl-4-hydroxy-5-methoxy-2H- chromen-2-one 253 (224 mg, 0.79 mmol). The reaction mixture was cooled to 0 °C and DIAD (0.16 mL, 0.79 mmol) was added dropwise. The reaction was stirred at room temperature for 24 h. The solvents were evaporated in vacuo to give the crude product as a thick yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:2), to give the title compound as a yellow oil (85 mg, 55 %). νmax (film)/cm-1 2937w (C-H), 1712s

(C(10)=O), 1671s (C(1)=O); H(400 MHz; CDCl3) 2.07 (2H, quin, J 6.3, C(5)H2), 2.49-

2.53 (4H, m, C(4)H2 and C(6)H2), 3.88 (3H, s, OCH3), 3.93 (2H, s, C(15)H2), 4.61 (2H, d,

J 1.3, C(7)H2), 6.75 (1H, d, J 8.3, C(11)H or C(13)H), 6.98 (1H, d, J 8.3, C(11)H or C(13)H), 7.14-7.20 (2H, m, C(3)H and Ar-H), 7.24 (2H, t, J 7.4, 2 x Ar-H), 7.33 (1H, d, J

7.4, 2 x Ar-H), 7.42 (1H, t, J 8.3, C(12)H); δC(100MHz; CDCl3) 22.8 (C(5)H2), 25.9

(C(4)H2 or C(6)H2), 30.2 (C(15)H2), 38.2 (C(4)H2 or (C(6)H2), 56.1 (OCH3), 70.4

(C(7)H2), 106.3 (C(11)H or C(13)H), 107.5 (C), 109.9 (C(11)H or C(13)H), 117.6 (C), 126.2 (Ar-CH), 128.3 (Ar-CH), 128.7 (Ar-CH), 131.6 (Ar-CH), 135.5 (C), 139.5 (C), 147.6 (C(3)H), 154.6 (Ar-C), 156.0 (Ar-C), 163.1 (C), 163.4 (C(10)=O), 198.0

(C(1)=O); m/z (+ES) 391 ([M+H]+, 100%); (Found 413.1374, C24H22NaO5 ([M+Na]+), requires 413.1365).

167

8.1.71 4-(((1S,6R)-4'-(tert-butyl)-6-hydroxy-3-oxo-1,2,3,6-tetrahydro-[1,1'- biphenyl]-4-yl)methoxy)-2H-chromen-2-one (259a)

C26H26O5; Mw 418.49 To a solution of compound 221 (125 mg, 0.32 mmol) in tetrahydrofuran (2 mL), under an atmosphere of nitrogen, was added triphenylphosphine (169 mg, 0.64 mmol) and 4-hydroxycoumarin (104 mg, 0.64 mmol). The reaction mixture was cooled to 0 °C and DIAD (0.13 mL, 0.64 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 30 mins and then at room temperature for 17 h. The solvents were evaporated in vacuo to give the crude product as a thick yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:7), to give a mixture of the required product (262a) and unknown impurities, as a white solid (55 mg, 32 %).

A solution of the crude adduct (262a) (48 mg, 0.09 mmol) in TFA:H2O (7:1, 0.72 mL) was stirred at room temperature for 40 mins. The solvents were evaporated under reduced pressure to give an off white solid. The crude residue was purified by flash silica chromatography, eluting with ethyl acetate:40-60 petroleum ether (4:6), to give the title compound as a white solid (36 mg, 97%). mp. 147.3-148.5 °C; [α]D28 – 45.7 (c

0.6, CH2Cl2); νmax (film)/cm-1 3433br (O-H), 2962w (C-H), 1676s (C=O), 1612s (C=C);

H(400MHz; CDCl3) 1.35 (9H, s, C(CH3)3), 2.78-2.88 (2H, m, C(6)H2), 3.31 (1H, td, J

10.1, 7.6, C(5)H), 4.78 (1H, dd, J 10.1, 1.8, C(4)H), 4.93 (2H, ~q, J 1.8, C(7)H2), 5.76 (1H, s, C(9)H), 7.16 (1H, ~d, J 1.8, C(3)H), 7.26-7.36 (4H, m, 4 x Ar-H), 7.45 (2H, d, J 8.3, 2 x Ar-H), 7.58 (1H, ddd, J 8.3, 7.2, 1.6, Ar-H), 7.88 (1H, dd, J 8.3, 1.6, Ar-H);

δC(100MHz; CDCl3) 31.3 (C(CH3)3), 34.6 (C(CH3)3), 43.1 (C(6)H2), 50.1 (C(5)H), 65.1

(C(7)H2), 71.9 (C(4)H), 91.3 (C(9)H), 115.5 (C), 116.9 (Ar-CH), 123.0 (Ar-CH), 124.0 (Ar-CH), 126.3 (Ar-CH), 127.3 (Ar-CH), 132.6 (C), 132.7 (Ar-CH), 135.7 (C), 149.0 (C(3)H), 151.2 (C), 153.3 (C), 162.7 (C), 164.9 (C(10)=O), 196.2 (C(1)=O); m/z (+ES)

419 ([M+H]+, 100%); (Found 419.1847, C26H27O5 ([M+H]+), requires 419.1858). 168

8.1.72 3-Benzyl-4-(((1S,6R)-4'-(tert-butyl)-3-oxo-6-((triethylsilyl)oxy)-1,2,3,6- tetrahydro-[1,1'-biphenyl]-4-yl)methoxy)-5-methoxy-2H-chromen-2-one (262b)

C40H48O6Si; Mw 652.90 To a solution of alcohol 221 (40 mg, 0.10 mmol) in tetrahydrofuran (1 mL), under an atmosphere of nitrogen, was added triphenylphosphine (54 mg, 0.21 mmol) and 3- benzyl-4-hydroxy-5-methoxy-2H-chromen-2-one 253 (58 mg, 0.21 mmol). The reaction mixture was cooled to 0 °C and DIAD (40 µL, 0.21 mmol) was added dropwise. The reaction mixture was stirred at 0 °C for 30 mins and then at room temperature for 24 h. The solvents were evaporated in vacuo to give the crude product as a thick yellow oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate: 40-60 petroleum ether (1:6), to give the title compound as a white thick oil (40 mg, 59 %). [α]D28 –142.8 (c 1.0, CH2Cl2); νmax (film)/cm-1 2953m (C-H), 2938w (C-H), 2909w (C-H), 2976w (C-H), 1718s (C=O);

H(400 MHz; CDCl3) 0.23-0.42 (6H, m, Si(CH2CH3)3), 0.76 (9H, t, J 7.9, Si(CH2CH3)3),

1.33 (9H, s, C(CH3)3), 2.77 (1H, dd, J 16.5, 5.0, C(6)Heq), 2.82 (1H, dd, J 16.5, 12.3,

C(6)Hax), 3.26 (1H, ddd, J 12.3, 8.8, 5.0, C(5)H), 3.88 (3H, s, OCH3), 3.93 (2H, s,

C(15)H2), 4.58-4.66 (3H, m, C(7)H2 and C(4)H), 6.76 (1H, d, J 8.1, C(11)H or C(13)H), 6.98 (1H, d, J 8.1, C(11)H or C(13)H), 7.07 (1H, d, J 1.5, C(3)H), 7.15-7.29 (5H, m, 5 x

Ar-H), 7.35-7.38 (4H, m, 4 x Ar-H), 7.43 (1H, t, J 8.1, C(12)H); δC(100MHz; CDCl3) 4.4

(Si(CH2CH3)3), 6.6 (Si(CH2CH3)3), 30.1 (C(15)H2), 31.3 (C(CH3)3), 34.5 (C(CH3)3), 42.7

(C(6)H2), 50.3 (C(5)H), 56.3 (OCH3), 69.6 (C(4)H or C(7)H2), 73.0 (C(4)H or C(7)H2), 106.3 (C(11)H or C(13)H), 107.4 (C), 109.9 (C(11)H or C(13)H), 125.4 (Ar-CH), 126.2 (C), 128.3 (Ar-CH), 128.7 (Ar-CH), 131.6 (C(12)H), 133.9 (C), 139.4 (C), 149.8 (C(3)H), 169.5 (C(10)=O), 199.7 (C(1)=O); m/z (+ES) 60 (100%), 653([M+H]+, 60%);

(Found 653.3301, C40H49O6Si ([M+H]+), requires 653.3298).

169

8.1.73 3-Benzyl-4-(((1S,6R)-4'-(tert-butyl)-6-hydroxy-3-oxo-1,2,3,6-tetrahydro- [1,1'-biphenyl]-4-yl)methoxy)-5-methoxy-2H-chromen-2-one (259b)

C34H34O6; Mw 538.24

A solution of compound 262b (35 mg, 0.05 mmol) in TFA:H2O (7:1, 0.72 mL) was stirred at room temperature for 30 mins. The solvents were removed under reduced pressure to give a brown oil. The residue was purified by flash silica chromatography, eluting with ethyl acetate:40-60 petroleum ether (1:2), to give the title compound as white foam (22 mg, 76%). [α]D30 –77.7 (c 0.4, CH2Cl2); νmax (film)/cm-1 3445 (O-H), 2961w (C-H), 2903w (C-H), 1715s (C(1)=O), 1680s

(C(10)=O); H(400MHz; CDCl3) 1.35 (9H, s, C(CH3)3), 2.04 (1H, br s, C(4)OH), 2.71-

2.80 (2H, m, C(6)H2), 3.24 (1H, q, J 9.7, C(5)H), 3.91 (3H, s, OCH3), 3.95 (1H, d, J 14.1,

C(15)HaHb), 3.98 (1H, d, J 14.1, C(15)HaHb), 4.59 (1H, dt, J 13.0, 1.8, C(7)HaHb), 4.66

(1H, dt, J 13.0, 1.8, C(7)HaHb), 4.73 (1H, br d, J 9.7, C(4)H), 6.77 (1H, d, J 8.4, C(11)H or C(13)H), 6.99 (1H, d, J 8.4, C(11)H or C(13)H), 7.18 (1H, ~t, J 7.1, Ar-H), 7.22 (1H, d, J 1.5, C(3)H), 7.24-7.28 (5H, m, 5 x Ar-H), 7.35 (2H, ~d, J 7.1, 2 x Ar-H), 7.43 (1H, t, J 8.5,

C(12)H), 7.44 (2H, ~d, J 8.5, 2 x Ar-H); δC(100MHz; CDCl3) 30.1 (C(15)H2), 31.3

(C(CH3)3), 34.6 (C(CH3)3), 43.2 (C(6)H2), 50.3 (C(5)H), 56.4 (OCH3), 69.7 (C(7)H2), 72.1 (C(4)H), 106.4 (C(11)H or C(13)H), 110.0 (C(11)H or C(13)H), 117.7 (C), 126.2 (Ar-CH), 126.3 (C), 127.4 (Ar-CH), 128.4 (Ar-CH), 128.7 (Ar-CH), 129.6 (C), 131.7 (C), 134.6 (C), 136.0 (C), 138.7 (C), 139.5 (C), 148.7 (C(3)H), 156.1 (C), 163.2 (C(10)=O), 200.4 (C(1)=O); m/z (+ES) 238 (100%), 539 ([M+H]+, 65%); (Found 561.2265,

C34H34NaO6 ([M+Na]+), requires 561.2253).

170

8.1.74 Perhydro-dibenzofuranones – General Method

To a solution of the appropriate conjugate adduct (141a,c,d,g) in THF (approx. concentration 0.1M) was added a catalytic amount of 0.5 M aqueous solution of NaOH (a few drops) and the reaction mixture was stirred at room temperature for 20-48 h. The reaction was quenched by the addition of a saturated aqueous solution of NH4Cl and organic material was extracted into dichloromethane. The organic extracts were dried over

MgSO4 and concentrated in vacuo. The crude product was purified by flash silica chromatography eluting with EtOAc:petroleum ether (40/60) in a 1:1 ratio for 201b, c, g and 2:1 ratio for 201d.

8.1.74.1 (3S,4S,4aR,5aR,6S,9aS,9bS)-3,6-bis(4-(tert-butyl)phenyl)-4-hydroxy octahydrodibenzo[b,d]furan-1,8(2H,5aH)-dione (201b)

C32H40O4; Mw 488.67 Using the general procedure above, the title compound 201b was obtained as a white solid (20 mg, 62%). mp. 238.6-240.1 °C; [α]D28 -184.8 (c 0.35, CH2Cl2); νmax (film)/cm-1 3463br (O-H), 2964m (C-H), 2955m (C-H), 2950w (C-H), 2899w (C-H),

2868w (C-H), 1715s (C=O); H (500MHz; CDCl3) 1.31 (9H, s, C(CH3)3), 1.32 (9H, s,

C(CH3)3), 2.15 (1H, d, J 7.2, C(4)OH), 2.52-2.64 (4H, m, C(8)H2 and C(12)H2), 2.64-

2.72 (2H, m, C(2)H2), 2.80 (1H, dd, J 5.1, 2.3, C(6)H), 3.20 (1H, ddd, J 11.7, 9.1, 5.4, C(11)H), 3.31 (1H, dt, J 9.3, 7.6, C(3)H), 3.52 (1H, dtd, J 11.0, 7.4, 2.3 C(9)H), 4.29 (1H, ddd, J 9.3, 7.2, 3.2, C(4)H), 4.45 (1H, dd, J 9.1, 7.4, C(10)H), 4.72 (1H, dd, J 5.1, 3.2, C(5)H), 7.16 (2H, d, J 8.3, 2 x Ar-CH), 7.17 (2H, d, J 8.3, 2 x Ar-CH), 7.37 (2H, d, J 8.3, 2

171 x Ar-CH), 7.38 (4H, dd, J 8.3, 2 x Ar-CH); δC(100MHz; CDCl3) 31.3 (C(CH3)3), 34.5

(C(CH3)3), 37.1 (C(9)H), 40.2 (C(2)H2 or C(8)H2 or C(12)H2), 42.6 (C(11)H), 42.8

(C(2)H2 or C(8)H2 or C(12)H2), 43.9 (C(3)H), 45.0 (C(2)H2 or C(8)H2 or C(12)H2), 56.7 (C(6)H), 72.8 (C(4)H), 78.4 (C(5)H), 81.6 (C(10)H), 125.7, 125.9, 127.1 (Ar-CH), 136.8 (Ar-C), 138.1 (Ar-C), 150.0 (Ar-C), 150.3 (Ar-C), 206.7 (C=O), 209.8 (C=O); m/z (+ES)

506 ([M+NH4]+, 100%); (Found 511.2807, C32H40O4Na ([M+Na]+) requires 511.2819).

8.1.74.2 (3S,4S,4aR,5aR,6S,9aS,9bS)-4-hydroxy-3,6-bis(4-methoxyphenyl) octahydrodibenzo[b,d]furan-1,8(2H,5aH)-dione (201c)

C26H28O6; Mw 436.50 Using the general procedure above, the title compound 201c was obtained as a white solid (36 mg, 86%). mp. 179.1-181.9 °C; [α]D28 -192.7 (c 0.55, CH2Cl2);

νmax(film)/cm-1 3437br (O-H), 2958w (C-H), 2911w (C-H), 2836w (C-H), 1711s

(C=O); H (400MHz; CDCl3) 2.53-2.60 (4H, m, C(8)H2 and C(12)H2), 2.65 (2H, d, J 8.1,

C(2)H2), 2.79 (1H, dd, J 4.9, 2.0, C(6)H), 3.15 (1H, ddd, J 11.4, 9.2, 5.8, C(11)H), 3.27

(1H, dt, J 9.8, 8.1, C(3)H), 3.52 (1H, ddd, J 11.1, 7.4, 2.0, C(9)H), 3.80 (3H, s, OCH3),

3.81 (3H, s, OCH3), 4.24 (1H, dd, J 9.8, 3.2, C(4)H), 4.40 (1H, dd, J 9.2, 7.4, C(10)H), 4.71 (1H, dd, J 4.9, 3.2, C(5)H), 6.89 (4H, d J 8.4, 4 x Ar-CH), 7.15 (4H, d, J 8.4, 4 x Ar-

CH); δC (100MHz; CDCl3) 36.8 (C(9)H), 40.3 (C(8)H2 or C(12)H2), 42.3 (C(11)H), 43.0

(C(8)H2 or C(12)H2), 43.6 (C(3)H), 45.3 (C(2)H2), 55.3 (OCH3), 56.8 (C(6)H), 73.0 (C(4)H), 78.4 (C(5)H), 81.9 (C(10)H), 114.2 (Ar-CH), 114.3 (Ar-CH), 128.4 (Ar-CH), 131.9 (Ar-C), 133.3 (Ar-C), 147.5 (Ar-C), 158.7 (Ar-C), 158.8 (Ar-C), 206.5 (C=O),

207.3 (C=O); m/z (+ES) 454 ([M+NH4]+, 100%); (Found 459.1771, C26H28O6Na ([M+Na]+) requires 459.1778).

172

8.1.74.3 (3S,4S,4aR,5aR,6S,9aS,9bS)-3,6-bis(4-fluorophenyl)-4-hydroxyocta hydrodibenzo[b,d]furan-1,8(2H,5aH)-dione (201d)

C24H22F2O4; Mw 412.43

Using the general procedure above, the title compound 201d was obtained as a white solid (30 mg, 59%). [α]D28 -188.5 (c 0.5, CH2Cl2); νmax (film)/cm-1 3431br (O-H),

2963w (C-H), 2927w (C-H), 2890w (C-H), 1715s (C=O); H (500MHz; CDCl3) 2.02 (1H, d, J 9.0, C(4)OH), 2.49-2.69 (6H, m, C(2)H2, C(8)H2 and C(12)H2), 2.81 (1H, dd, J 4.7, 2.0, C(6)H), 3.18 (1H, ddd, J 12.6, 9.3, 4.7, C(11)H), 3.28 (1H, td, J 9.9, 6.6, C(3)H), 3.56 (1H, dtd, J 12.0, 7.4, 2.0, C(9)H), 4.26 (1H, ddd, J 9.9, 9.0, 3.3, C(4)H), 4.39 (1H, dd, J 9.3, 7.4, C(10)H), 4.72 (1H, dd, J 4.7, 3.3, C(5)H), 7.05 (4H, t, J 8.5, 4 x Ar-CH), 7.19 (2H, dd, J 8.5, 5.1, 2 x Ar-CH), 7.21 (2H, dd, J 8.5, 5.1, 2 x Ar-CH); δC (100MHz; CDCl3) 36.6

(C(9)H), 40.2 (C(8)H2 or C(12)H2), 42.4 (C(11)H), 42.8 (C(8)H2 or C(12)H2), 43.9

(C(3)H), 45.4 (C(2)H2), 56.8 (C(6)H), 72.9 (C(4)H), 78.5 (C(5)H), 81.8 (C(10)H), 115.7 (d, J 21, C(3’)H and C(5’)H), 115.9 (d, J 21, C(3’)H and C(5’)H), 128.9 (d, J 7.4, C(2’)H and C(6’)H), 129.0 (d, J 7.4, C(2’)H and C(6’)H), 135.6 (d, J 2.8, C(1’)H), 136.9 (d, J 2.8,

C(1’)H), 147.5 (Ar-C), 158.7 (Ar-C), 158.8 (Ar-C), 206.5 (C=O), 207.3 (C=O); δF

(376MHz; CDCl3) -14.95 (1F, s), -115.17 (1F, s); m/z (-ES) 457 ([M+HCO2]-, 100%);

(Found 435.1378, C24H22O4F2Na ([M+Na]+) requires 435.1378).

8.1.74.4 (3S,4S,4aR,5aR,6S,9aS,9bS)-4-hydroxy-3,6-bis(4-iodophenyl) octahydrodibenzo[b,d]furan-1,8(5aH,9bH)-dione (201e)

C24H22I2O4; Mw 628.24

173

Using the general procedure above, the title compound 201e was obtained as a white solid (27 mg, 60%). mp. 257.9-259.6 °C; [α]D28 -169.7 (c 0.6, CH2Cl2); νmax (film)/cm-1

3419br (O-H), 2941w (C-H), 2905w (C-H), 1712s (C=O); H (500MHz; Acetone-d6)

2.37 (1H, dd, J 17.8, 3.4, C(12)Heq), 2.38 (1H, dd, J 13.7, 3.9, C(2)Heq), 2.43 (1H, dd, J

16.2, 5.8, C(8)Heq), 2.64 (1H, dd, J 17.8, 13.9, C(12)Hax), 2.78 (1H, dd, J 16.2, 13.6,

C(8)Hax), 2.88 (1H, t, J 13.7, C(2)Hax), 3.05 (1H, d, J 4.0, C(6)H), 3.19 (1H, ddd, J 13.7, 10.4, 3.9, C(3)H), 3.32 (1H, ddd, J 13.9, 9.8, 3.4, C(11)H), 3.41 (1H, ddd, J 13.6, 7.7, 5.8, C(9)H), 4.32 (1H, dd, J 9.8, 7.7, C(10)H), 4.39 (1H, dd, J 10.4, 2.9, C(4)H), 4.74 (1H, dd, J 4.0, 2.9, C(5)H), 7.13 (2H, d, J 8.6, 2 x Ar-CH), 7.16 (2H, d, J 8.6, 2 x Ar-CH), 7.62 (2H, d, J 8.6, 2 x Ar-CH), 7.64 (2H, d, J 8.6, 2 x Ar-CH); δC (100MHz; CDCl3) 36.6 (C(9)H),

40.1 (C(8)H2 or C(12)H2), 42.5 (C(11)H), 42.7 (C(8)H2 or C(12)H2), 44.2 (C(3)H), 45.1

(C(2)H2), 56.8 (C(6)H), 72.7 (C(4)H), 78.5 (C(5)H), 81.6 (C(10)H), 92.7 (Ar-CI), 92.9 (Ar-CI), 129.4 (Ar-CH), 137.9 (Ar-CH), 138.0 (Ar-CH), 139.6 (Ar-C), 140.8 (Ar-C), 205.6 (C=O), 208.7 (C=O); m/z (+ES) 651 ([M+Na]+, 100%)%); (Found 650.9518,

C24H22O4NaI2 ([M+Na]+) requires 650.9505).

174

9.0 Biological Assays Methods and Materials

9.1 Cell Culture

The three cell lines utilised for the biological evaluation of the novel compounds were the A549 and MDA-MB-468 human adenocarcinoma cell lines and MCF10A, a non-tumorigenic epithelial cell line. All cell lines were cultured in T-75 tissue culture flasks (Falcon, Becton Dickinson, USA) and were maintained in a humidified incubator at 37 °C within a 5% CO2 atmosphere. All the cell culture work was carried out under sterile conditions in a Class II laminar flow microbiological safety cabinet. The A549 and MDA-MB-468 cell lines were grown in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS) and 2mM L-glutamine. The MCF10A cell line was grown in DMEM/F12 medium supplemented with 5% horse serum, human insulin (10 µg/mL), epidermal growth factor (10 ng/mL) and hydrocortisone (0.5 µg/mL) . The cell lines had to be sub-cultured when the cell monolayer in the culture flask reached 70% confluence. The growth media was aspirated and the cells were washed with sterile phosphate buffered saline (PBS). The cells were detached from the flask by treating them with 2 mL of 0.25% trypsin/0.2% EDTA in PBS and incubating at 37 °C for 2-10 minutes. The trypsin/EDTA was then neutralised by adding 8 mL of fresh growth media. The cells were re-seeded in a new T-75 flask containing fresh growth media. The overall dilution ratio varied from 1:2 to 1:4.

9.2 Cell Storage

The cells were frozen to -80 °C in order to preserve them for a short time. The freezing was carried out when the cells were 70% confluent. The cells were treated with trypsin/EDTA, as described in section 9.1, and then resuspended in growth media. The resulting suspension was centrifuged for 5 minutes at 1200 rpm (Megafuge 1.0, Heraeus, Hanau, Germany). The media was aspirated and the cell pellet was resuspended in freezing media (90% FCS, 10% DMSO). Approximately 1 mL of the resulting cell suspension was transferred into a 1.5 mL cryovial (Greiner 175

Bio-one, Frickenhausen, Germany). The cells were frozen down gradually, in order to prevent cell death, by first placing them in a -20 °C freezer and then stored at -80 °C.

9.3 Cell Thawing

The cells were thawed rapidly by warming the cryovial to 37 °C and transferring the cell suspension in 3 mL of pre-warmed growth media. The suspension was centrifuged at 1200rpm for 5 minutes and the media was aspirated. The cell pellet was re-suspended in 3 mL of growth media and split into two T-25 tissue culture flasks (Falcon, Becton Dickinson, USA). 2 mL of the cell suspension with 3 mL of media was added to one T-25 culture flask and 1 mL of the cell suspension with 4 mL of fresh media was added to the other one. The cells were allowed to adhere by incubating them at 37 °C in standard culture conditions and they were only used for studies after two sub-cultures.

9.4 MTT Cell Viability Assay

9.4.1 Counting and Seeding Cells

The cells were seeded into 96-well plates. 180 μL of the cell suspension at the appropriate seeding density were added to each well and allowed to adhere for 24 hours in the incubator before the drug treatment. In order to calculate the seeding density, the concentration of cells in the cell suspension had to be determined using a haemocytometer (Neubauer, Germany). The cover-slip was placed over the counting chamber of the haemocytometer and 10 μL of the cell suspension was transferred at the edge of the chamber. The suspension was drawn under the cover-slip onto the grid by capillary action. The grid was placed under a light microscope and the cells in the four corner squares were counted.

176

Figure 37. The haemocytometer grid

The cell concentration per mL was calculated using the following equation:

Cells/mL=Mean cell number of corner squares x 104

9.4.2 Treating the Cells

The novel compounds were dissolved in DMSO and serial dilutions from 1 nM to 4 mM were prepared in growth media for each compound. 20 µL of each dilution were added to the wells in quadruplicate across the plate to give a final drug concentration of 0.1 nM to 400 µM on the plate. The 0.1nM to 100 µM solutions contained 0.4% DMSO and the 400 µM solution contained 1.6% DMSO; hence the ends of the plates were control wells which contained 0.4% and 1.6% DMSO (v/v). The plates were incubated for 96 hours.

177

Figure 38. 96-well plate layout: two compounds were tested in quadruplicate on each plate; white wells represent the control wells, black wells are the solvent control wells (column 2 is 0.4%DMSO and column 11 is 1.6% DMSO), grey wells (left to right) are increasing concentrations of the tested compound.

9.4.3 Ending the viability assay

The experiment was terminated by the addition of 50 μL of MTT solution (2.5 mg/mL) at 96 h. The plate was incubated for 4 hours, after which the solution was aspirated from the wells and 200 μL of DMSO was added to dissolve the formazan crystals formed. The plates were shaken for 5 minutes and then the absorbance for each well was measured at a wavelength of 540 nm, on a multi-well scanning spectrophotometer (μQuant Microplate Spectrophotometer, BioTek, Potton, UK) using the Gen5 software package (BioTek, UK). The average absorption values were used to calculate the optical density (OD) values, which are proportional to the number of living cells. The OD values were then used to calculate the IC50 concentrations, as described in Section 4.3.

178

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Appendix I

Published work

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Appendix II

MTT viability graphs for novel analogues 177-180

100