Thomas Anthony Munro- The Chemistry of Salvia divinorum

The Chemistry of Salvia divinorum

Thomas Anthony Munro

Submitted in total fulfilment of the requirements

of the degree of Doctor of Philosophy

April 2006

Department of Chemistry

The University of Melbourne

Abstract

Salvia divinorum is a hallucinogenic sage used to treat illness by the Mazatec

Indians of Mexico. Salvinorin A (1a), a neoclerodane diterpenoid isolated

from the plant, is a potent, selective agonist at the κ opioid receptor (KOR),

and is the first non-nitrogenous opioid. The plant is used recreationally as

a hallucinogen, but is unpopular due to its dysphoric effects. 1a has been

prohibited in Australia under an invalid systematic name.

An early report of psychoactive alkaloids in S. divinorum proved to be irre-

producible. Similarly, tests in mice suggesting the presence of psychoactive

compounds other than 1a were confounded and therefore unreliable.

O

O

O

H HOR1

R2

O O

R2

O

H HR1

O OR3

1a

R1

AcHH

R2

OHOAcH

1d1e1f

28a28b28c

R1

OH OH H

R2

HOHOAc

R3

HMeH

22 88

O

O

O

H HOO

O

O O

In this work, an improved isolation method for 1a was developed, using fil-

tration through activated carbon to decolourise the crude extract. Six new

diterpenoids were isolated: salvinorins D–F (1d–1f) and divinatorins A–C

(28a–28c). Five known terpenoids not previously reported from this species

were also isolated.

3

4

The structure–activity relationships of 1a were evaluated via selective mod-

ifications of each functional group. Useful synthetic methods are reviewed,

including the first thorough review of furanolactone hydrogenations. Testing

of the derivatives at the KOR suggests that the methyl ester and furan ring

of 1a are required for activity, but that the lactone and ketone functionali-

ties are not. Other compounds from S. divinorum did not bind to the KOR,

suggesting that 1a is the plant’s active principle.

The structure of the 8-epimer of 1a, reported previously without supporting

evidence, was firmly established. This epimerisation proved to be a general

phenomenon among salvinorins and related furanolactones, occurring via eno-

lisation of the lactone. The more complex mechanism proposed by Koreeda

and co-workers was inconsistent with subsequent data. Under strongly basic

conditions, autoxidation of 1a occurred to give the enedione 59 as the major

product. A previously proposed structure was shown to be incorrect.

Salvinorins and divinatorins were tested and found to be inactive against in-

sects, bacteria, fungi, HIV, tumour cell lines and protein synthesis.

59

O

O

O

HOH

O

O O

Declaration

This is to certify that

1. the thesis comprises only my original work except where indicated in the

preface,

2. due acknowledgement has been made in the text to all other material

used,

3. the thesis is less than 100,000 words in length, exclusive of tables, maps,

bibliographies and appendices.

Thomas Munro

5

Preface

Some of the bioassays described in Chapter 4 were performed by others.

• Opioid receptor binding assays were performed in the laboratories of

Bryan Roth, Case Western Reserve University (Cleveland, Ohio), by

Glenn Goetchius, Beth Ann Toth, Feng Yan, and Timothy Vortherms.

• Protein synthesis inhibition assays were performed in the laboratories of

Jerry Pelletier, McGill University (Montreal, Canada).

• HIV replication assays (NL4.3 and AD8 strains) were performed in the

laboratories of Sharon Lewin, Monash University, by Ajantha Solomon.

• Other HIV assays were performed at Southern Research Institute (Fred-

erick, Maryland), under the direction of Dr Stephen Turk, U. S. National

Institute of Allergy and Infectious Diseases (NIAID).

• Tumour cell growth inhibition assays were performed by the U. S. Na-

tional Cancer Institute (NCI).

Other assays were performed collaboratively:

• Antibacterial and antifungal assays were performed in the laboratories

of Professor Roy Robins-Browne, University of Melbourne, with Andrea

Bigham.

• Insect antifeedant assays were performed using supplies and facilities

provided by David Heckel and Charles Robin, University of Melbourne.

7

8

Some photographs were provided by others, as credited.

Parts of this work have been published previously:

• Munro, T. A.; Rizzacasa, M. A. Salvinorins D–F, New Neoclerodane

Diterpenoids from Salvia divinorum, and an Improved Method for the

Isolation of Salvinorin A. J. Nat. Prod. 2003, 66, 703–705. http://dx.

doi.org/10.1021/np0205699

• Bigham, A. K.; Munro, T. A.; Rizzacasa, M. A.; Robins-Browne, R. M.

Divinatorins A–C, New Neoclerodane Diterpenoids from the Controlled

Sage Salvia divinorum. J. Nat. Prod. 2003, 66, 1242–1244. http://dx.

doi.org/10.1021/np030313i

• Munro, T. A.; Rizzacasa, M. A.; Roth, B. L.; Toth, B. A.; Yan, F.

Studies toward the Pharmacophore of Salvinorin A, a Potent κ Opioid

Receptor Agonist. J. Med. Chem. 2005, 48, 345–348. http://dx.doi.

org/10.1021/jm049438q

• Munro, T. A.; Goetchius, G. W.; Roth, B. L.; Vortherms, T. A.; Rizza-

casa, M. A. Autoxidation of Salvinorin A under Basic Conditions. J. Org.

Chem. 2005, 70, 10,057–10,061. http://dx.doi.org/10.1021/jo051813e

http://dx.doi.org/10.1021/np0205699


http://dx.doi.org/10.1021/np030313i


http://dx.doi.org/10.1021/jm049438q


http://dx.doi.org/10.1021/jo051813e

Acknowledgments

Thanks to the Commonwealth Government for an Australian Postgraduate

Award.

Thanks Mark for betting tight resources on a risky project. I’m glad it paid

off. Thanks to the man with the coolest name in chemistry, Leander Jerome

Julian Valdés III, for generously sharing ideas and advice. Daniel Siebert for

helpful advice on lots of stuff, and for introducing me to Bryan Roth. Torsten

for introducing me to Daniel; Carl Turney for introducing me to Torsten (and

everything else); and Erik for introducing me to Carl. Six degrees of separation.

Mike and Heike, Antoine and Lara for being so generous. Frances for changing

my life, and for the world’s coolest lab coat and bestest present. TK for

teaching me NMR, from setting the trash hole to running a NOESY. Les Gamel

for all that masterful glassblowing. Carl Schiesser for the radical initiator

that dare not speak its name. Sammy for letting me use windoze. Danny

for letting me use Adobe Creative Suite. Ben for introducing me to Hoye’s

NMR papers. The man with the second-coolest name in chemistry, Carlos

Rodríguez, for translating Díaz. Vic Iwanov for letting me use the safe, and

filling out those annoying manifests. Max Hem for the photographs, glorious

as always. A man with another cool name, Slava Olcheski, for the Oaxaca

shot. Scott Crawford for getting those stunning shots out of the SEM. Richard

Westkaemper for providing the binding model data. Tom for proofreading – I

owe you one big fella. Same goes for Caroline. Cheese, Gromit! Thanks Dad

for keeping life interesting. Mojave, Ilulisaat, Peshawar, terror australis, silver

shark, helibagging. What can I say: my dad’s better than yours. Thanks mum

9

10

for the sacrifices you made and the love and hard slog you put into raising kids

and working full time. And for helping with the move so I could keep writing

this till five days before leaving for my postdoc!

Contents

Abstract 3

Declaration 5

Preface 7

Acknowledgments 9

List of Figures 17

List of Schemes 23

List of Tables 25

Acronyms 27

1 Introduction. 31

1.1 Botany. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

1.2 Ethnopharmacology. . . . . . . . . . . . . . . . . . . . . . . . . 33

1.3 Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

1.3.1 Terpenoids. . . . . . . . . . . . . . . . . . . . . . . . . . 36

1.3.2 Alleged alkaloids. . . . . . . . . . . . . . . . . . . . . . . 43

1.4 Pharmacology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

11

12 CONTENTS

1.4.1 Animal Testing. . . . . . . . . . . . . . . . . . . . . . . . 47

1.4.2 Human Testing. . . . . . . . . . . . . . . . . . . . . . . . 51

1.4.3 In vitro Testing. . . . . . . . . . . . . . . . . . . . . . . . 53

1.4.4 Mechanism: κ Opioids. . . . . . . . . . . . . . . . . . . . 54

1.5 Toxicology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

1.6 Social impact. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

1.6.1 Recreational Use. . . . . . . . . . . . . . . . . . . . . . . 62

1.6.2 Legal Status. . . . . . . . . . . . . . . . . . . . . . . . . 63

1.7 Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

2 Isolation. 67

2.1 Isolation Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . 67

2.1.1 Extraction Conditions. . . . . . . . . . . . . . . . . . . . 67

2.1.2 Problems Caused by Pigments. . . . . . . . . . . . . . . 69

2.1.3 Use of Activated Carbon. . . . . . . . . . . . . . . . . . . 72

2.1.4 Separation of Terpenoids. . . . . . . . . . . . . . . . . . 82

2.2 Structure Elucidation. . . . . . . . . . . . . . . . . . . . . . . . 90

2.2.1 Revised NMR Assignments for Salvinorin A (1a). . . . . 90

2.2.2 Revised NMR Assignments for Salvinorin C (1c). . . . . 92

2.2.3 Other Known Diterpenoids. . . . . . . . . . . . . . . . . 92

2.2.4 Known Triterpenoids. . . . . . . . . . . . . . . . . . . . . 96

2.2.5 Salvinorins D-F (1d-1f). . . . . . . . . . . . . . . . . . . 98

2.2.6 Divinatorins A-C (28a-28c). . . . . . . . . . . . . . . . . 110

2.2.7 Subsequent isolations. . . . . . . . . . . . . . . . . . . . 118

CONTENTS 13

3 Synthesis. 119

3.1 Known derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . 119

3.2 Epimerisation at C-8 under Basic Conditions. . . . . . . . . . . 120

3.2.1 Previous Reports. . . . . . . . . . . . . . . . . . . . . . . 120

3.2.2 8-epi-Salvinorins A and B (37a and 37b). . . . . . . . . . 121

3.2.3 Control of Epimerisation and Separation of Epimers. . . 122

3.2.4 8-epi-Salvinorin C (37c) and Related Compounds. . . . . 124

3.2.5 Chromatographic Identification of Epimers. . . . . . . . 125

3.2.6 Mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . 126

3.2.7 Attempted Deacetylation under Acidic Conditions. . . . 128

3.3 Simple Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . 128

3.3.1 Esters (46 and 47). . . . . . . . . . . . . . . . . . . . . . 128

3.3.2 Attempted Benzyl Ether Formation (48). . . . . . . . . . 129

3.3.3 17-Deoxy Compounds (49 and 50). . . . . . . . . . . . . 130

3.3.4 Tetrahydrosalvinorin A (51). . . . . . . . . . . . . . . . . 131

3.3.5 (+)-Hardwickiic Acid (ent-29a). . . . . . . . . . . . . . . 134

3.4 Modification of the Methyl Ester. . . . . . . . . . . . . . . . . . 134

3.4.1 Relevant Results from Previous Work. . . . . . . . . . . 134

3.4.2 Treatment of Salvinorin A with KOH in MeOH. . . . . . 135

3.4.3 O-Demethylsalvinorin A (67a). . . . . . . . . . . . . . . 144

3.4.4 O-Demethyl-18-deoxysalvinorin A (77). . . . . . . . . . . 150

3.5 Modification of the Ketone. . . . . . . . . . . . . . . . . . . . . 153

3.5.1 Attempted Methylenation. . . . . . . . . . . . . . . . . . 153

3.5.2 Attempted Direct Deoxygenation. . . . . . . . . . . . . . 154

3.5.3 Indirect Deoxygenation. . . . . . . . . . . . . . . . . . . 155

14 CONTENTS

4 Bioassays. 161

4.1 Insect Antifeedant Activity. . . . . . . . . . . . . . . . . . . . . 161

4.2 Eukaryotic Protein Synthesis Inhibition. . . . . . . . . . . . . . 163

4.3 Antimicrobial Activity. . . . . . . . . . . . . . . . . . . . . . . . 164

4.3.1 Bacteria and Fungi. . . . . . . . . . . . . . . . . . . . . . 164

4.3.2 HIV-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

4.4 NCI Anticancer Screen. . . . . . . . . . . . . . . . . . . . . . . . 168

4.5 Activity at the κ Opioid Receptor. . . . . . . . . . . . . . . . . 170

4.5.1 Other Salvinorins and Divinatorins. . . . . . . . . . . . . 171

4.5.2 Modification of the Ketone. . . . . . . . . . . . . . . . . 173

4.5.3 Modification of the Acetoxy Group. . . . . . . . . . . . . 174

4.5.4 Modification of the Methyl Ester. . . . . . . . . . . . . . 175

4.5.5 Modification of the Lactone. . . . . . . . . . . . . . . . . 176

4.5.6 Modification of the Furan Ring. . . . . . . . . . . . . . . 176

4.5.7 Incorporation into a Revised Binding Model. . . . . . . . 177

4.5.8 Subsequent Results. . . . . . . . . . . . . . . . . . . . . . 179

5 Experimental. 181

5.1 General Conditions . . . . . . . . . . . . . . . . . . . . . . . . . 181

5.1.1 Instruments and Procedures. . . . . . . . . . . . . . . . . 181

5.1.2 Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . 183

5.1.3 Plant Materials. . . . . . . . . . . . . . . . . . . . . . . 183

5.1.4 Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

5.2 Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

5.2.1 Extraction of Commercial S. divinorum. . . . . . . . . . 185

CONTENTS 15

5.2.2 Extraction of Australian S. divinorum. . . . . . . . . . . 187

5.2.3 Salvinorin A (1a). . . . . . . . . . . . . . . . . . . . . . 188

5.2.4 Salvinorin B (1b). . . . . . . . . . . . . . . . . . . . . . . 189

5.2.5 Salvinorin C (1c). . . . . . . . . . . . . . . . . . . . . . 189

5.2.6 Salvinorin D (1d). . . . . . . . . . . . . . . . . . . . . . . 190

5.2.7 Salvinorin E (1e). . . . . . . . . . . . . . . . . . . . . . . 191

5.2.8 Salvinorin F (1f). . . . . . . . . . . . . . . . . . . . . . . 192

5.2.9 Divinatorin A (28a). . . . . . . . . . . . . . . . . . . . . 194

5.2.10 Divinatorin B (28b). . . . . . . . . . . . . . . . . . . . . 195

5.2.11 Divinatorin C (28c). . . . . . . . . . . . . . . . . . . . . 196

5.2.12 (–)-Hardwickiic Acid (29a) and methyl ester 29b. . . . . 197

5.2.13 Oleanolic Acid (31). . . . . . . . . . . . . . . . . . . . . 198

5.2.14 Presqualene Alcohol (32). . . . . . . . . . . . . . . . . . 198

5.2.15 Peplusol (33). . . . . . . . . . . . . . . . . . . . . . . . . 199

5.3 Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

5.3.1 Salvinorin C (1c) via acetylation of salvinorin D (1d). . . 199

5.3.2 Salvinorins D (1d) and E (1e) via acetylation of 1h. . . . 200

5.3.3 Salvinorins C (1c) and E (1e) via acetylation of 1h. . . . 200

5.3.4 Dideacetylsalvinorin C (1h) from 1c. . . . . . . . . . . . 201

5.3.5 (+)-Hardwickiic acid (ent-29a). . . . . . . . . . . . . . . 202

5.3.6 Salvinorin A lactol (35). . . . . . . . . . . . . . . . . . . 203

5.3.7 (4R)-3,4-Dihydrosalvinorin C (36c). . . . . . . . . . . . . 205

5.3.8 (4R)-3,4-Dihydrosalvinorin E (36e). . . . . . . . . . . . . 206

5.3.9 (4R)-Dideacetyl-3,4-dihydrosalvinorin C (36h). . . . . . . 207

5.3.10 8-epi-Salvinorin A (37a). . . . . . . . . . . . . . . . . . . 208

16 CONTENTS

5.3.11 8-epi-Salvinorin B (37b). . . . . . . . . . . . . . . . . . . 210

5.3.12 8-epi-Salvinorin C (37c). . . . . . . . . . . . . . . . . . . 211

5.3.13 8-epi-Salvinorin D (37d). . . . . . . . . . . . . . . . . . . 212

5.3.14 8-epi-Salvinorin E (37e). . . . . . . . . . . . . . . . . . . 214

5.3.15 8-epi-Dideacetylsalvinorin C (37h). . . . . . . . . . . . . 215

5.3.16 Salvinorin B formate (46). . . . . . . . . . . . . . . . . . 216

5.3.17 Dideacetylsalvinorin C 2-O-(4-bromobenzoate) (47). . . . 218

5.3.18 17-Deoxysalvinorin A (49). . . . . . . . . . . . . . . . . . 219

5.3.19 8,17-Didehydro-17-deoxysalvinorin A (50). . . . . . . . . 220

5.3.20 13,14,15,16-Tetrahydrosalvinorin A (51). . . . . . . . . . 222

5.3.21 Autoxidation of 1a in KOH/MeOH. . . . . . . . . . . . . 223

5.3.22 NaBH4 reduction of 59. . . . . . . . . . . . . . . . . . . . 228

5.3.23 O-Demethyl-18-deoxysalvinorin A (77). . . . . . . . . . 229

5.3.24 1-Deoxysalvinorin A (81a). . . . . . . . . . . . . . . . . . 233

Bibliography 239

List of Figures

1.1 Flowering specimen of S. divinorum.1 . . . . . . . . . . . . . . . 31

1.2 Location of Oaxaca and the Sierra Mazateca within Mexico. . . 32

1.3 Young Salvia divinorum plant, Oaxaca.8 . . . . . . . . . . . . . 33

1.4 S. divinorum flower in bud (stereoview).1 . . . . . . . . . . . . . 33

1.5 Ortega et al’s X-ray structure of 1a (stereoview). . . . . . . . . 37

1.6 Salvinorins B (1b) and C (1c). . . . . . . . . . . . . . . . . . . 38

1.7 Known terpenoids. . . . . . . . . . . . . . . . . . . . . . . . . . 38

1.8 Clerodin (5) and standard clerodane numbering. . . . . . . . . . 39

1.9 Biosynthetic precursors of diterpenoids. . . . . . . . . . . . . . . 40

1.10 Peltate glandular trichome (SEM stereoview).39 . . . . . . . . . 42

1.11 Underside of S. divinorum leaves (SEM stereoview).39 . . . . . . 43

1.12 Activity of pure 1a versus mixtures in mice (open field assay). . 48

1.13 Morphine (11) and derivatives. . . . . . . . . . . . . . . . . . . 54

1.14 Arylacetamide κ opioids. . . . . . . . . . . . . . . . . . . . . . . 56

1.15 Structurally diverse κ opioids. . . . . . . . . . . . . . . . . . . . 59

1.16 Relevant IUPAC fused-ring numbering schemes. . . . . . . . . . 64

2.1 Siebert’s TLC analysis of crude CHCl3 extracts.40 . . . . . . . . 69

2.2 Representative major plant pigments. . . . . . . . . . . . . . . . 71

17

18 LIST OF FIGURES

2.3 Ortho- and non-ortho-substituted PCBs. . . . . . . . . . . . . . 76

2.4 Apparatus for Filtration through Activated Carbon. . . . . . . . 78

2.5 Flavonoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

2.6 SEM image of salvinorin A crystals (blade morphology).39 . . . 82

2.7 Other salvinorin A crystal morphologies (stereoview).39 . . . . . 84

2.8 Terpenoids isolated from S. divinorum. . . . . . . . . . . . . . . 85

2.9 TLC data of isolated compounds. . . . . . . . . . . . . . . . . . 86

2.10 Isolation of terpenoids from commercial S. divinorum. . . . . . . 87

2.11 Isolation of terpenoids from Australian S. divinorum. . . . . . . 88

2.12 1H NMR spectrum of 1a (800 MHz, CDCl3). . . . . . . . . . . . 91

2.13 HSQC spectrum of 1a (800 MHz, CDCl3). . . . . . . . . . . . . 91

2.14 Revised NMR assignments for 1a (stereoview). . . . . . . . . . . 92

2.15 1H NMR spectrum of 1c (400 MHz, CDCl3). . . . . . . . . . . . 93

2.16 Revised NMR assignments for 1c (stereoview). . . . . . . . . . . 93

2.17 HMQC and HMBC spectra of 1c (400 MHz, CDCl3). . . . . . . 94

2.18 Single-crystal X-ray structure of 29a (stereoview). . . . . . . . . 95

2.19 (E)-Phytol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

2.20 Oleanolic acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

2.21 Presqualene alcohol and peplusol. . . . . . . . . . . . . . . . . . 97

2.22 1H NMR spectrum of 1d (400 MHz, CDCl3). . . . . . . . . . . . 99

2.23 NMR assignments and key 2D correlations for 1d (stereoview). . 100

2.24 HMQC spectrum of 1d (400 MHz, CDCl3). . . . . . . . . . . . 100

2.25 HMBC spectrum of 1d. . . . . . . . . . . . . . . . . . . . . . . 101

2.26 1H NMR spectrum of 1e (400 MHz, CDCl3). . . . . . . . . . . . 102

2.27 NMR assignments and key 2D correlations for 1e (stereoview). . 102

LIST OF FIGURES 19

2.28 HMQC spectrum of 1e. . . . . . . . . . . . . . . . . . . . . . . 103

2.29 HMBC spectrum of 1e. . . . . . . . . . . . . . . . . . . . . . . . 103

2.30 1H NMR spectrum of 1h (400 MHz, CDCl3). . . . . . . . . . . . 104

2.31 NMR assignments and key 2D correlations for 1h (stereoview). . 105

2.32 HMQC and HMBC spectra of 1h. . . . . . . . . . . . . . . . . . 105

2.33 NOESY spectrum of 1h. . . . . . . . . . . . . . . . . . . . . . . 105

2.34 1H NMR spectrum of 1f (400 MHz, CDCl3). . . . . . . . . . . . 108

2.35 NMR assignments and key 2D correlations for 1f (stereoview). . 109

2.36 HMQC spectrum of 1f. . . . . . . . . . . . . . . . . . . . . . . . 109

2.37 HMBC spectrum of 1f. . . . . . . . . . . . . . . . . . . . . . . . 109

2.38 Divinatorins A–C (28a-28c) and hardwickiic acid (29a). . . . . 110

2.39 1H NMR spectrum of 28a (400 MHz, CDCl3). . . . . . . . . . . 110

2.40 NMR assignments and key 2D correlations for 28a (stereoview). 111

2.41 HMBC spectrum of 28a. . . . . . . . . . . . . . . . . . . . . . . 111

2.42 NOESY spectrum of 28a. . . . . . . . . . . . . . . . . . . . . . 112

2.43 1H NMR spectrum of 28b (400 MHz, CDCl3). . . . . . . . . . . 113

2.44 NMR assignments and key 2D correlations for 28b (stereoview). 113

2.45 HMBC spectrum of 28b. . . . . . . . . . . . . . . . . . . . . . . 114

2.46 NOESY spectrum of 28b. . . . . . . . . . . . . . . . . . . . . . 114

2.47 1H NMR spectrum of 28c (400 MHz, CDCl3). . . . . . . . . . . 115

2.48 NMR assignments and key 2D correlations for 28c (stereoview). 115

2.49 HMBC spectrum of 28c. . . . . . . . . . . . . . . . . . . . . . . 116

2.50 NOESY spectrum of 28c. . . . . . . . . . . . . . . . . . . . . . 116

2.51 Subsequently isolated compounds. . . . . . . . . . . . . . . . . . 118

3.1 Key NOESY correlations for 37a (stereoview). . . . . . . . . . . 123

20 LIST OF FIGURES

3.2 TLC comparison of epimers using vanillin/H2SO4. . . . . . . . . 126

3.3 Ester and ether derivatives. . . . . . . . . . . . . . . . . . . . . 129

3.4 Furanolactones 53, 54 and 55. . . . . . . . . . . . . . . . . . . . 131

3.5 (+)-Hardwickiic acid (ent-29a). . . . . . . . . . . . . . . . . . . 134

3.6 Key HMBC correlations of 59 and 60a. . . . . . . . . . . . . . 137

3.7 UV/Visible spectra of 1a, 1c and 59 in MeCN. . . . . . . . . . 138

3.8 Diol 36h and proposed autoxidation product 62. . . . . . . . . 139

3.9 O-Demethyl salvinorins A and B. . . . . . . . . . . . . . . . . . 145

3.10 Useful non-hydrogen bond donor solvents. . . . . . . . . . . . . 145

3.11 Methylenated target compound 79. . . . . . . . . . . . . . . . . 154

3.12 RP-LCMS traces of early fractions versus 81b/82b. . . . . . . . 159

3.13 1-Deoxysalvinorin A (81a). . . . . . . . . . . . . . . . . . . . . 160

4.1 Luciferase assay results for salvinorins and divinatorins (50 µM). 163

4.2 (-)-Hardwickiic acid and divinatorins A-C. . . . . . . . . . . . . 164

4.3 Disk and microdilution assays for ent-29a and crude extract.428 165

4.4 HIV-1 replication assays (NL43 and AD8 strains). . . . . . . . . 166

4.5 HIV-1 replication assays (ROJO isolate). . . . . . . . . . . . . . 167

4.6 NCI 60 cell line results for salvinorins and divinatorins . . . . . 169

4.7 CNS cell line results for divinatorin B and salvinorin B. . . . . . 170

4.8 KOR binding affinity and potency of salvinorin A. . . . . . . . . 171

4.9 KOR binding affinities of salvinorins and divinatorins. . . . . . . 172

4.10 KOR activity after ketone modifications. . . . . . . . . . . . . . 174

4.11 KOR activity after acetoxy group modifications. . . . . . . . . . 174

4.12 KOR activity after methyl ester modifications. . . . . . . . . . . 175

4.13 KOR activity after lactone modifications. . . . . . . . . . . . . . 176

LIST OF FIGURES 21

4.14 KOR activity after furan modifications. . . . . . . . . . . . . . . 176

4.15 Westkaemper’s original binding model. . . . . . . . . . . . . . . 177

4.16 Westkaemper’s revised binding model (stereoview). . . . . . . . 178

4.17 KOR binding affinities and potencies of recent derivatives. . . . 179

22 LIST OF FIGURES

List of Schemes

1.1 Cyclization of 7 to the trans-neoclerodane skeleton(stereoview). 41

1.2 Cyclization of 7 to the cis-neoclerodane skeleton. . . . . . . . . 41

2.1 Interconversion of 1c-1h. . . . . . . . . . . . . . . . . . . . . . . 104

3.1 Preparation of known derivatives. . . . . . . . . . . . . . . . . . 119

3.2 Formation of 8-epi-salvinorins A and B. . . . . . . . . . . . . . 121

3.3 Synthesis of 37c-37h. . . . . . . . . . . . . . . . . . . . . . . . 125

3.4 Koreeda et al’s proposed mechanism of epimerisation. . . . . . . 127

3.5 Epimerisation of related natural products with base. . . . . . . . 127

3.6 Brown’s deuteration of 1b. . . . . . . . . . . . . . . . . . . . . . 128

3.7 Deoxygenation of lactol 35. . . . . . . . . . . . . . . . . . . . . 130

3.8 Hydrogenation of 1a and other furanolactones. . . . . . . . . . . 131

3.9 LiAlH4 and Li/NH3 reductions of 1a. . . . . . . . . . . . . . . . 135

3.10 Autoxidation of 1a. . . . . . . . . . . . . . . . . . . . . . . . . . 136

3.11 Proposed mechanism of the autoxidation. . . . . . . . . . . . . . 141

3.12 Unexpected oxidation product 65. . . . . . . . . . . . . . . . . . 141

3.13 Attempted reductions of 59. . . . . . . . . . . . . . . . . . . . . 143

3.14 BAl2 and BAc2 ester cleavage mechanisms. . . . . . . . . . . . . 144

3.15 Formation of mixed anhydride 68. . . . . . . . . . . . . . . . . . 147

3.16 Some previously reported BH3·THF reductions. . . . . . . . . . 151

3.17 Borane reduction of 67a. . . . . . . . . . . . . . . . . . . . . . . 152

3.18 Ketone deoxygenation via a tosylhydrazone. . . . . . . . . . . . 154

3.19 Formation of cyclic thionocarbonate 80. . . . . . . . . . . . . . 156

3.20 Radical deoxygenation of 80. . . . . . . . . . . . . . . . . . . . 158

23

24 LIST OF SCHEMES

List of Tables

1.1 Relative potency of 1a at cloned κ opioid receptors. . . . . . . . 60

1.2 Alternate systematic names for 1a. . . . . . . . . . . . . . . . . 64

2.1 Effects of solvent and temperature on recovery of 1a. . . . . . . 68

2.2 Data and sources used to identify known compounds. . . . . . . 94

3.1 Coupling constants (Hz) at H-8 for 1a, 1b and 8-epimers. . . . . 122

3.2 Some previously reported furanolactone hydrogenations. . . . . 133

3.3 Summary of results - nucleophilic cleavage of 1a methyl ester. . 148

3.4 Unsuccessful treatment of 1a with excess tosylhydrazide. . . . . 155

4.1 Antifeedant test results. . . . . . . . . . . . . . . . . . . . . . . 162

4.2 KOR radioligand and functional assay results. . . . . . . . . . . 172

5.1 Yields and TLC data (hRf) of isolated compounds. . . . . . . . 187

25

26 LIST OF TABLES

Acronyms

AIBN Azobisisobutyronitrile [2,2’-Azobis(2-methylpropionitrile)]

AZT Azidovudine

Borsm Based on recovered starting material

CNS Central nervous system

COSY Correlation spectroscopy 2D NMR (2−4JHH)

DEPT Distortionless enhancement by polarization transfer NMR (13C mul-

tiplicity)

DMAP 4-Dimethylaminopyridine

DMF Dimethylformamide

DMPU 1,3-dimethyltetrahydropyrimidin-2-one

EC50 Concentration causing 50% of maximal efficacy

GC/MS Gas chromatography/mass spectrometry

HMBC Heteronuclear multiple bond correlation 2D NMR (2−3JCH )

HMQC Heteronuclear multiple quantum coherence 2D NMR (1JCH)

HMPA Hexamethylphosphoric triamide (a.k.a. HMPT)

HPLC High performance liquid chromatography

HRESIMS High resolution electrospray ionisation mass spectrometry

27

28 LIST OF TABLES

hRf = Rf × 100

HSQC Heteronuclear single quantum coherence 2D NMR (1JCH)

IC50 Concentration causing 50% inhibition

InChI IUPAC International Chemical Identifier

K i Receptor binding affinity constant

KOR κ (kappa) opioid receptor

LC/MS Liquid chromatography/mass spectrometry

LD50 Dose lethal to 50% of test animals

MIC Minimum inhibitory concentration

NDPSC National Drugs and Poisons Schedule Committee

NMR Nuclear magnetic resonance

nOe Nuclear Overhauser effect (through–space signal enhancement)

NOESY Nuclear Overhauser enhancement spectroscopy (through–space 2D

NMR)

PCB Polychlorinated biphenyl

PCR Polymerase chain reaction

Rf Retardation factor af

[distances of analyte (a) and solvent front (f) from

origin]

ROESY Rotating frame Overhauser enhancement spectroscopy (through–

space 2D NMR)

RP Reverse phase (nonpolar stationary phase, polar mobile phase)

SEM Scanning electron microscopy / standard error of the mean

THF Tetrahydrofuran

LIST OF TABLES 29

TLC Thin layer chromatography

30 LIST OF TABLES

Chapter 1

Introduction.

Figure 1.1: Flowering specimen of S. divinorum.1

1.1 Botany.

The genus Salvia, containing over 900 species internationally, is one of the

largest in the family Lamiaceae (or Labiatae). The largest subgenus is the

31

32 CHAPTER 1. INTRODUCTION.

Mexico

N

Oaxaca

Sierra Mazateca

0 300 km200100

Figure 1.2: Location of Oaxaca and the Sierra Mazateca within Mexico.

South American Calosphace (or Jungia),2 of which over 300 species are found

in Mexico.3 Among these is Salvia divinorum Epling & Játiva.4, 5 Discovered

in the highlands of northern Oaxaca (Figure 1.2), the plant is a perennial shrub

growing to about 1.5 m, preferring moist shady sites at high elevations.6 While

the flowers are distinctive (Figure 1.1), the plant’s appearance is nondescript

during vegetative growth (Figure 1.3), apart from the unusual square stem.

In the original botanical description, based on a dried specimen and witness

reports, the flower (corolla) was erroneously described as being blue.4 This

error was incorporated into several colour botanical illustrations.7 The error

originated in observations by Robert Gordon Wasson and Albert Hofmann,

who obtained the type specimen but were not botanists.6 In fact, the corolla is

white, emerging from a violet calyx (Figure 1.4). This error has been corrected

in an amended botanical description,6 which also has the advantage of being

in English rather than Latin.

Several others had investigated the species and collected specimens,9 but Was-

son and Hofmann were the first to obtain a flowering specimen, which is essen-

tial for species identification. It should be pointed out that, although Wasson

and Hofmann were credited as the collectors of the type specimen,4 it was in

fact given to them by Natividad Rosa, a Mazatec healing woman.10, 11 Wasson

and Hofmann have also been incorrectly credited with bringing live plants to

1.2. ETHNOPHARMACOLOGY. 33

Figure 1.3: Young Salvia divinorum plant, Oaxaca.8

the U.S.A., which was actually done by botanist Sterling Bunnell.12 Most of

the S. divinorum plants in cultivation internationally are clones of this mis-

named “Wasson and Hofmann” strain.12

Figure 1.4: S. divinorum flower in bud (stereoview).1

There has been no report of growing the plant from seed: propagation is ex-

clusively vegetative.6, 13 Moreover, there are no published first-hand reports of

wild populations;7 despite thorough searches in the region by several workers,

all known stands appear to have been planted.6, 14

1.2 Ethnopharmacology.

The leaves of S. divinorum are used as a traditional medicine by the Mazatec

Indians of the Oaxaca region. The disorders treated include gastrointestinal


problems, headaches, rheumatism, anaemia and swelling of the stomach.15, 11, 13

An infusion is prepared by crushing or rubbing fresh leaves in water; a frothy

infusion is considered a sign of potency. The leaf residue may also be applied

to the patient’s forehead as a poultice afterwards.11

In addition, the healers (curanderos) themselves drink the infusion to induce

visions.5 They believe these visions allow them to divine the cause of the

illness. Hence the name S. divinorum, meaning “sage of the seers.”4 For

these effects, larger doses of the infusion are used, or the leaves themselves

are slowly chewed and eaten. Eating the leaves is very difficult, due to their

sickeningly bitter taste, and often induces vomiting. First-hand accounts of

the effects vary from barely perceptible (an overlay of “dancing colours”)5 to

powerful experiences, in which awareness of reality is lost and bizarre visions

are perceived as real:

I saw a pulsating purplish light that changed to an insect-like shape,

perhaps a bee or a moth, and then into a pulsating sea anemone.

It expanded into a desert full of prickly pear cacti, and remained

so for several minutes. During the first session and throughout the

night, my visions had all appeared to be something like a cross

between a silent moving picture and a cartoon. I felt myself to

be an observer of these mute visions, rather than being an actual

part of them. Suddenly, however, I was in a broad meadow with

brightly colored flowers. I had just crossed a stream by way of a

small wooden bridge. Next to me was something that seemed to

be the skeleton of a giant model airplane made of rainbow colored

inner tubing. The sky was bright blue and I could see ... woods in

the distance. I found myself talking to a man in a shining white

robe who was either shaking my hand, or else holding on to it.

It was an amazing hallucination, as I truly believed I was in the

meadow.14

This is in marked contrast to the pseudohallucinations induced by compounds

1.2. ETHNOPHARMACOLOGY. 35

such as LSD. The visions were accompanied by impaired physical coordination

and slurred speech.16 Notwithstanding the intensity of some such experiences,

the Mazatecs consider the plant the weakest of their visionary substances.13

They refer to it as ška María pastora (the leaves of Mary shepherdess), appar-

ently from an obscure Catholic term for the Virgin Mary.17

Ott has argued7 that the lack of an indigenous name indicates that the plant

is not indigenous to the region – that, like sheep and Catholicism themselves,

it came to the Mazatecs after European settlement. He points out that the

introduced European hallucinogenic mushroom Psilocybe cubensis also has no

indigenous name, unlike its indigenous equivalents, and is regarded as an in-

ferior substitute. S. divinorum is regarded in the same way. Furthermore,

the Mazatecs consider the plant as part of the same family as two introduced

Coleus species. As further evidence, he points out that the Mazatec belief that

drying the leaves destroys their potency has been disproven. Similarly, drink-

ing an infusion has been shown to be very inefficient; much stronger effects

are produced by retaining the infusion,18 or alternatively a “quid” of chewed

leaves,7 in the mouth. Ott argues that each of the above points suggests a

lack of cultural tradition, and concludes that the plant was adopted relatively

recently from another tribe.

Ott has also endorsed Wasson’s suggestion19 that S. divinorum may represent

the divinatory plant pipiltzintzintli cultivated by the Aztecs.7 The Aztecs pre-

pared an infusion from the plant, including the leaves, and also applied it as

a poultice; no other plant was used in both these ways. The only Mexican

plant whose leaves are presently so used is S. divinorum. Accounts of pipiltz-

intzintli make no mention of seeds, unlike other Aztec visionary plants; and S.

divinorum is effectively seedless, unlike other contemporary Mexican visionary

plants. Here, then, we have a plant without an indigenous name, and an in-

digenous name referring to an unknown plant. One has no present, the other

has no past, and they have several very distinctive characteristics in common.

Others reject this hypothesis. Díaz has pointed out that male and female gen-


ders of pipiltzintzintli were reported, that the plant was dried before use, and

that the infusion was prepared from the entire plant, none of which is true

of S. divinorum.20 Ott counters that the Aztecs may have been using gender

metaphorically, as the Mazatecs do today. Drying does not affect the plant’s

potency, and the addition of other plant parts, while superfluous, would not

affect the infusion. Moreover, Díaz’s preferred candidate, Cannabis sativa, can-

not be correct, since it was introduced after European settlement.7 Valdés also

rejects Wasson’s hypothesis, preferring Beltrán’s proposal that pipiltzintzintli

was a synonym for the morning glory, ololiuhqui.13 Ott has shown, however,

that these were explicitly described as different plants, and that S. divinorum

is thus

... the only Mexican entheogenic plant which fits the criteria for

pipiltzintzintli, and ... remains our best guess for the identity of

the lost Aztec entheogen.7

Both parties in this surprisingly bitter dispute concede that the evidence is

inconclusive, and both suspect that S. divinorum has been used by tribes

other than the Mazatecs. Valdés notes early anecdotal reports of use by the

Cuicatec and Otomí tribes, whose lands adjoin the Sierra Mazateca.21, 9

1.3 Chemistry.

1.3.1 Terpenoids.

1.3.1.1 Isolation.

The first compound isolated from S. divinorum was 1a, a clerodane diter-

penoid discovered by Ortega et al in 1982 and named salvinorin.22 The ter-

minology of these terpenoids will be discussed in the next section. Extraction

of the dried leaves in refluxing CHCl3, chromatography on “Tonsil” activated

clay and crystallisation from MeOH gave 1a in unstated yield. The structure

1.3. CHEMISTRY. 37

1a

O

O

O

H HOO

O

O O

Figure 1.5: Ortega et al’s X-ray structure of 1a (stereoview).

was elucidated spectroscopically, and confirmed by X-ray crystallography.22

Absolute stereochemistry was tentatively assigned by comparing the circular

dichroism spectrum with known compounds.

Subsequently, Valdés isolated the same compound. Unaware of Ortega’s work,

he named the compound divinorin A in his thesis.15 He also isolated the

deacetyl analogue 1b, which he named divinorin B. When the work was pub-

lished, this oversight was corrected, and the compounds were named salvi-

norins A and B.23 Valdés’s isolation procedure was more complex. The Et2O

extract was dissolved in MeOH and washed with hexanes. Repeated chro-

matography on silica gel and repeated recrystallisation from EtOH gave 1a

in 1.8 g/kg yield based on dry weight. The yield of 1b was much lower (74

mg/kg). Spectroscopic structure elucidation of 1a was again confirmed by

X-ray crystallography. The fit of the model was in excellent agreement with

the diffraction data (R-factor = 8.7 %), though not quite as good as Ortega

et al’s (5.2 %).


1b

O

O

O

H HOHO

O O 1c

O

O

O

H HOO

O

O

O O

Figure 1.6: Salvinorins B (1b) and C (1c).

Ortega et al’s model also has the advantage of including the positions of hydro-

gen atoms. Unfortunately, the absolute stereochemistry of Valdés et al’s model

is incorrect, although it was correctly assigned in the paper, again on the basis

of circular dichroism. This stereochemistry has since been definitively con-

firmed by the use of exciton chirality circular dichroism,24 and more recently

X-ray crystallography,25 on suitable derivatives. This absolute stereochemistry

is common to all clerodanes isolated from the Lamiaceae.26

Valdés et al subsequently isolated salvinorin C (1c).27 Repeated chromatog-

raphy of the Et2O extract followed by HPLC gave 1c in 78 mg/kg yield.

Spectroscopic structure elucidation was supported by partial synthesis of ana-

logues.

4

HOH

H

H

3

HO OO

H

2

Figure 1.7: Known terpenoids.

In addition to these new diterpenoids, several known terpenoids were detected.

The monoterpenoid loliolide (2) was isolated and fully characterised by Valdés,

in the same manner as 1c, in 4.4 mg/kg yield.28 GC/MS analysis by Giroud

1.3. CHEMISTRY. 39

et al also detected compounds whose MS data were consistent with the nor-

triterpenoid stigmasterol (3) and the diterpenoid neophytadiene (4).29

1.3.1.2 Terminology.

5

H

OOAc

O

O

H H

H

OAc

22

3344

55

101011

66

77

8899 1717

12121111

1313

1616

1515

1414

1818

1919

2020

Figure 1.8: Clerodin (5) and standard clerodane numbering.

Clerodane diterpenoids30 are named after clerodin (5). Conclusively establish-

ing the structure of this compound was a tortuous process, which has been

lucidly reviewed by Rodriguez-Hahn et al.26 The absolute stereochemistry was

initially proposed as ent-5. Compounds with stereochemistry matching 5 were

therefore termed ent-clerodane for many years. Extensive crystallographic and

degradation studies ultimately proved the true structure of clerodin to be 5.

To avoid ambiguity, the term “neoclerodane” was therefore coined for com-

pounds matching 5.31 Compounds previously known as clerodanes would be

termed ent-neoclerodane. For further detail, consult Rodriguez-Hahn et al.26

Clerodanes are further subdivided into trans- and cis- varieties, according to

the configuration of C-5 relative to C-10 in the standard32 numbering scheme

(Figure 1.8). All clerodane diterpenoids isolated from S. divinorum to date

are trans-neoclerodane.

1.3.1.3 Biosynthesis.

Terpenoids are synthesised from C5 “isoprene” units, derived from isopen-

tenyl diphosphate (6).33 Assembly of four isoprene units gives geranylger-

anyl diphosphate (7), the final common biosynthetic intermediate of all diter-


OPP

7

P

O

O OHOH

OP

O

OH6

= OPP

Figure 1.9: Biosynthetic precursors of diterpenoids.

penoids.34 Formation of clerodanes begins with protonation at C-14 of 7

(Scheme 1.1). This initiates a cationic cascade leading to the labdane in-

termediate 8. A sequence of 1,2-hydride and methyl shifts then gives the

trans-clerodane skeleton 9.

It is unknown whether these 1,2- shifts are concerted. In the formation of cis-

clerodanes, a discrete halimane intermediate (10) is formed (Scheme 1.2).34

These pathways have been substantiated by feeding plants isotopically labelled

mevalonic acid, a precursor to 6. Notably, labelling of the terminal carbon of

7, which becomes C-18 in labdane intermediate 8, results in labelling at C-18

in trans-clerodanes,35 but C-19 in cis-clerodanes,36 as expected (Schemes 1.1

and 1.2). Tritium labelling also confirms the hydride shift from H-5 to H-10.36

Mevalonic acid was used in these studies because it was long assumed to be

the sole precursor to 6. Recently, revolutionary work has overturned this as-

sumption.37, 38 An alternate pathway, via 1-deoxyxylulose 5-phosphate, has

been established. Indeed, the mevalonic acid pathway makes a negligible con-

tribution to diterpenoid biosynthesis. Nonetheless, as reflected in the above

results, the pathways are not mutually exclusive. Some “crosstalk” occurs,

which is increased by the feeding of precursors.38 Nonetheless, incorporation

of mevalonate into diterpenoids is very low: below 0.01% in some cases.36

1.3. CHEMISTRY. 41

H+

++

7

14

1819

1819

15

14

15

7

99

H+

OPP

++

18 1918 19

88

CH2OPP

CH2OPPCH2OPP

CH2OPP

OPP

Scheme 1.1: Cyclization of 7 to the trans-neoclerodane skeleton(stereoview).

55

101088

99

1818 1919

H 19191818

H

+++

8 10

H

H+

H

10

7

OPP OPP OPP OPP

Scheme 1.2: Cyclization of 7 to the cis-neoclerodane skeleton.


0 20 µm0 20 µm

Figure 1.10: Peltate glandular trichome (SEM stereoview).39

1.3.1.4 Distribution.

In a series of simple and elegant experiments, Siebert has demonstrated that

the salvinorins are not evenly distributed through the tissues of S. divinorum,

but are localised in particular structures: peltate glandular trichomes (Fig-

ure 1.10).40 These are found particularly on the undersides of the leaves -

densely packed on newly formed leaves, more sparsely distributed on mature

ones (Figure 1.11). Other types of trichome, glandular and non-glandular, are

also visible. Terpenoid accumulation in glandular trichomes is typical of the

Lamiaceae family.40

This finding is significant for future isolation work. Siebert found that dipping

fresh leaves in CHCl3 (30 seconds × 3) gave nearly complete recovery of salvi-

norins.40 Powdering the leaves, as has been done in all isolation procedures to

date, is therefore unnecessary (see Section 2.1.1 on page 67).

1.3. CHEMISTRY. 43

0 200 µm

0 200 µm 0 200 µm

0 200 µm

Above: immature leaf (∼1 mm wide); below: mature leaf (∼10 cm wide).

Figure 1.11: Underside of S. divinorum leaves (SEM stereoview).39

1.3.2 Alleged alkaloids.

1.3.2.1 Summary of the Original Report.

The first investigation into the chemistry of S. divinorum was reported by

José Luis Díaz in 1975.41 The report is in Spanish, but key sections have been

translated by Valdés, who also added flowcharts clarifying Díaz’s procedures.42

The report was not peer reviewed, and the workers involved were not named.

Valdés, who later visited the lab, reports that the chemistry was performed


by “undergraduate biology and botany students with a minimal chemistry

background.”43 Many essential details were omitted. For instance, in some

cases TLC data were given without the solvent system. Administration of

certain extracts reportedly caused abnormal behaviour and posture in cats.

These are, however, not evident in the accompanying photographs. Moreover,

there were apparently “great variations” and “inconsistency” in the results,

which are not specified: “the described behaviour is not always present.” The

assay results are reported simply as “active”, “inactive” or “dubiously active”

without further detail, and without specifying the number of subjects or trials.

No positive or negative controls were used to validate the assay.

Given these serious deficiencies, the procedures and results will not be repro-

duced here. The interested reader will find Valdés’s translation helpful.42 One

important omission should be noted: the isolation procedure Valdés describes

as method 2 was performed twice: on the first occasion, as he notes, one of

the fractions was active. On the second occasion, no fraction was active, yet

another “inconsistency.”

The results can be briefly summarised as follows: certain fractions of the

plant extract, soluble in aqueous acid but insoluble in aqueous base, some-

times caused cats to behave abnormally. Other fractions never showed clear

activity. The active, acid-soluble fractions contained at least four compounds

which gave positive reactions to Dragendorff’s reagent,44 a standard alkaloid

visualisation reagent, in both standard and modified (Lüdy-Tenger)45, 46 forms.

On this basis, Díaz concluded that “several alkaloids exist in Salvia divinorum,

two of them apparently psychoactive.” In 1977 he reported that the structures

of the two compounds were under study.20 In 1979, however, he reported that:

It has been particularly difficult to identify the substance(s) respon-

sible for these interesting effects. There exists a great variability

or instability in the constituents of S. divinorum, which has im-

peded the consistent reproduction of the mental or behavioural al-

terations, preventing the identification of the active fraction. Some

1.3. CHEMISTRY. 45

initial observations indicated the presence of nitrogenous compounds,

possibly amino acids or amines, although they now appear to be of

no pharmacological interest.47

These remarks, while vague, are not consistent with the original report. Pre-

sumably attempts were made to replicate the original experiments, but the

results proved irreproducible.

1.3.2.2 Discussion.

The phytochemistry of the genus Salvia has been thoroughly studied,3 yielding

hundreds of terpenoids.2 Yet extensive literature searches48, 49, 50 revealed no

report of an alkaloid from an American Salvia species. The saps of several

American Salvias have given positive results to Dragendorff’s reagent,51 but

far more tested negative. Furthermore, Dragendorff’s gives false positives with

numerous non-nitrogenous compounds.44

The American species S. reflexa gave false positives to several alkaloid test

reagents.52 The compound responsible proved to be choline [Me3NEtOH]+.

Being a quaternary ammonium compound rather than an alkaloid, choline

could not be extracted from basic aqueous solution by chloroform.52 This

is also true of non-nitrogenous Dragendorff’s-positive compounds,44 and thus

none of these compounds can account for Díaz’s results. Díaz41 cites a sec-

ondary source53 reporting that histamine occurs in the genus Salvia. The

primary source54 cited there stresses that histamine is a primary metabolite,

formed by decarboxylation of the amino acid histidine. It is therefore described

as a “biogenic amine” rather than a true alkaloid, which in the strict sense of

the word are secondary metabolites.

Recent work on some Mexican Salvia species has found a very close chemotaxo-

nomic relationship with Chinese species,2 some of which have yielded alkaloids.

In particular, several seco- and nor-abietane diterpenoids previously known

only from the Chinese species S. miltiorrhiza have been isolated from Mexican


Salvias.2 S. miltiorrhiza has also yielded alkaloids.55 Thus, the presence of

alkaloids in S. divinorum cannot be dismissed out of hand.

Subsequent work has failed to replicate any of Díaz’s findings, however. Valdés

found no compound in the crude extract which gave a positive reaction to Dra-

gendorff’s or other alkaloid-specific test reagents.56, 23, 57 He has hypothesised

that Díaz’s group actually used mislabelled Erlich’s reagent, which reacts with

alkaloids but also furanolactones, and thus mistook the salvinorins for alka-

loids.57

This hypothesis, however, is inconsistent with the published claims; all defi-

nitely active fractions were soluble in aqueous acid, unlike 1a. Thus, if the ac-

tive fractions contained the salvinorins then not only the visualisation reagent,

but the fractions themselves, must have been misidentified. Moreover, the ac-

tivity reported by Díaz was dramatically different from that later observed

in cats by Valdés. Díaz reported “intense attention”, “reactions of fear and

attack” and “fury”, lasting 10 minutes after intravenous injection.41, 42 The ef-

fects observed by Valdés were almost the opposite. Subcutaneous injection of

an extract caused erratic eye movements rather than intense attention, and loss

of physical coordination (the cat could not walk, much less assume postures

of fear and attack). The effects lasted over 24 hours rather than 10 minutes.58

Chemical investigations by several groups have now yielded a total of 20 ter-

penoids, none of which is soluble in aqueous acid (see below). Furthermore,

GC/MS29 and LC/MS59 analyses of the crude extract gave no indication of

the presence of alkaloids. Compounds containing an odd number of nitrogens

give characteristic molecular ions and fragments, identifiable by the nitrogen

rule.60

In summary, the claim of biologically active alkaloids was implausible and

irreproducible, and has been abandoned by its author. The claim was evidently

false.

1.4. PHARMACOLOGY. 47

1.4 Pharmacology.

1.4.1 Animal Testing.

1.4.1.1 Cats and Rats.

As discussed above, Díaz’s tests in cats yielded no useful results. The next

worker to study the plant’s pharmacology was Valdés. Although he consumed

the traditional infusion during his initial work in Oaxaca, and later tested it for

activity after freeze-drying, further use of human subjects was “precluded”61

(i.e. forbidden by his supervisor).

For the purposes of bioassay-guided fractionation, Valdés therefore had to

develop an animal assay. This presented an enormous challenge. Previous

attempts to identify the active principles of hallucinogenic plants using ani-

mal assays, even when sustained and well funded, had failed.7 In each case

the active principle was later identified, quickly and cheaply, using human

tests. Examples include mescaline (from Lophophora williamsii), psilocybin

(Psilocybe cubensis) and lysergic acid amides (Ipomoea and Turbina spp.)62

Similarly, initial testing of LSD in mice caused no apparent effect other than

“disquiet”.10 To these must of course be added Díaz’s work with S. divinorum.

Evidently, and unsurprisingly, it is practically impossible to tell if an animal

is hallucinating. In Valdés’s preliminary trials, standard hallucinogen assays

in rats and cats were not sensitive to the effects of the extract.63 However, the

cats exhibited impaired motor coordination, which reminded him of the im-

paired physical coordination and slurred speech caused by the infusion.16 The

duration of action was much longer, however (24 hours). Given the difficulty

of detecting psychological states in animals, Valdés decided to choose an assay

sensitive to this physical effect.


1.4.1.2 Mice.

A standard assay of impaired locomotor function in mice, the inverted screen

test,64 proved sensitive to the crude extract, but insufficiently so. Next, a

modified65 version of the open field assay66 was tested. The movements of mice

on a printed grid were recorded over 15 minutes. Three measures of activity

were recorded: lines crossed, number of rearings, and time spent immobile.

This assay showed unambiguous effects, with a clear dose-response relation-

ship. Assay-guided fractionation led to the identification of 1a as the active

principle. No activity was seen with 1b, the only other pure compound iso-

lated. However, various mixed fractions gave puzzling results.

lines crossed

1a

0

50

100

150

200

250

300

dose (mg/kg)

lin

es

cro

ssed

rearings

impure 1a (~10% 1c) mixtures

0

10

20

30

40

50

60

dose (mg/kg)

reari

ng

s

immobility

0

2

4

6

8

10

12

14

16

dose (mg/kg)log scalelog scalelog scale

10 20 30 40 50 10 20 30 40 50 100 10 20 30 40 50 100100

tim

e im

mo

bile (

min

)

Figure 1.12: Activity of pure 1a versus mixtures in mice (open field assay).

Using the open field assay, impure 1a (later found to contain ∼10% 1c) was

found to be “significantly more potent”27 than the pure compound.65 It was

therefore long suspected13, 27, 67 that 1c was also psychoactive.

The open field assay results are shown in Figure 1.12. The potency of the

fraction containing ∼10% 1c was at least 10× greater than pure 1a, by all

three measures (Figure 1.12). Thus, if Valdés’s interpretation68 were correct,

1c would be at least 100× more potent than 1a, making it among the most

potent psychoactive compounds ever discovered.

Three other mixed fractions containing 1a also gave higher efficacies than the

pure compound, despite containing as little as 10% 1a.65 At 100 mg/kg, the

dilute fractions almost completely immobilised the mice, while pure 1a only

reduced activity. Despite their higher efficacy at high doses, however, these


fractions were not in fact more potent: the lines of best fit suggest that pure

1a had comparable efficacy at intermediate doses, and higher efficacy at low

doses (Figure 1.12). Thus, these results are not consistent with the presence of

a more potent compound in the mixed fractions, but appear to be confounded.

The anomaly was not specific to the open field assay: the same pattern was

evident69 in the inverted screen assay.

The confounding factor was probably differences in absorption, caused by

the unusual method of administration. The test fractions were injected in

an emulsion of vegetable oil, water and Tween 80 surfactant.70 The use of

oil/water/surfactant emulsions as drug vehicles has proven effective for some

hydrophobic drugs,71 and 1a is indeed hydrophobic. However, to be effectively

delivered in an emulsion, drugs must also be highly lipophilic:

Generally the most difficult drugs are those which have limited

solubility in both water and lipids (typically with log P values of

approximately 2). It is unlikely that lipid formulation will be of

value for such drugs.71

The solubility of 1a is negligible in hexanes, and presumably in oil (predicted

log P = 1.8).72 Thus, this vehicle would be expected to give poor absorption;

1a may have been administered not in solution, but as a suspension.

The 1a tested by Valdés was crystalline. Two of the impure fractions were

explicitly described as “oily solids.”73 The other two were not described;

however, they were mixtures, and were obtained by evaporation from 10%

MeOH/CHCl3.73 Evaporation of 1a from chlorinated solvents gives an amor-

phous solid even when pure. Hence, all four of the impure fractions shown

in Figure 1.12 were amorphous. This would result in differences in solubility;

the amorphous state of a solid typically has 2-10× higher solubility than the

crystalline state.74 This results from the energy barrier to dissolution of a

crystalline solid: disruption of the ordered crystal lattice requires additional

energy (the enthalpy of fusion, ∆Hf).74, 75 Also, in a vigorously agitated emul-

sion, the amorphous solids could form a dispersion of microscopic particles,


with a higher surface area than macroscopic crystals. This in turn would in-

crease the rate of dissolution (kinetics of solubility).75 Thus, faster dissolution

and greater absorption of the amorphous fractions would be expected.

Other compounds present in the mixtures might also influence absorption.

Many instances have been reported of enhanced absorption of active com-

pounds from a crude extract relative to the pure compound.76 For instance,

some terpenoids are known to act as permeation enhancers,77 increasing trans-

dermal absorption of co-administered drugs up to 90×. Thus, inactive com-

pounds in the crude fractions may have increased absorption of 1a.

Recent evidence conclusively establishes that the emulsion was very poorly

absorbed. When injected in solution (EtOH/surfactant/H2O), 1 mg/kg of 1a

reduced locomotor activity significantly over 30 minutes.78 Reinvestigation

by Valdés et al confirmed this, showing that 0.5 mg/kg caused maximal im-

pairment in the inverted screen test;79 quadrupling the dose did not increase

efficacy. By contrast, in the original tests using the emulsion, maximal im-

pairment only occurred above 1500 mg/kg.69 Thus, absorption of 1a from the

emulsion was clearly negligible.

The use of an emulsion also seems to prolong the effect of 1a; performance on

the inverted screen test remained impaired after 30 minutes.15 By comparison,

the effects of the solution peaked within 5 minutes, and were undetectable by

15 minutes.79 Consistent with this, a recent study found a strong analgesic

effect of 1 mg/kg injected 1a solution at 10 minutes,80 while a previous study

which began testing 20-25 minutes after injection found very little analgesic

effect even at 40 mg/kg.81 Another study reported analgesic effects at 0.6

mg/kg, but unfortunately contained no detail on administration or timing.82

In summary, the open field results may have been confounded by differences

in bioavailability. The vehicle used gave very poor absorption, and superior

absorption of amorphous solids is typical. Thus, the differences in apparent

potency between pure 1a and the impure fractions do not provide reliable

evidence of the presence of other active compounds. Evidence that 1c and


other compounds in the plant are inactive will be presented in the next section,

and in section 4.5.1 on page 171.

1.4.1.3 Rhesus Monkeys (Postscript).

Studies administering 1a to rhesus monkeys have recently been reported.83, 84

Interestingly, even the highest dose tested (32 µg/kg) produced only “slight

overt behavioural effects”,83 described as “sedation-like” in both studies, con-

firming the difficulty of establishing animal models of psychoactivity.

1.4.2 Human Testing.

As mentioned above, Valdés’s open field assay results indicated that 1a was

the active principle of S. divinorum. Further testing with other compounds

in the open field indicated that 1a was approximately equal in potency to

mescaline.13, 14 However, this conclusion remained tentative. As Valdés had

written earlier,

... there is no definite evidence that divinorin A is an hallucinogen

... the results are as yet unclear. And they will probably remain

so until the divinorins are tested in human beings.68

The question was finally resolved by Siebert in 1994.18 He reported that

1a was hallucinogenic when vaporised and inhaled. Activity was percepti-

ble with doses as low as 200 µg. The effects commenced within seconds, and

the strongest effects lasted 5 – 10 minutes. No effects were detectable after

30 minutes, suggesting a half-life of under 10 minutes. Siebert also explored

the effect of the route of administration. Swallowing encapsulated 1a in very

large doses (10 mg) produced no effect. This also held for the plant: an in-

fusion prepared from ten fresh leaves, when swallowed, produced no effect in

any subject. The same amount, when held in the mouth for 10 minutes and

spat out, produced definite effects in all subjects. Thus, absorption through


the oral mucosa is clearly far more efficient than through the gastrointestinal

system. Nonetheless, an ethanolic solution of 1a gave inconsistent effects sub-

lingually. The onset of effects is slower by the sublingual route, and the effects

last longer.

Siebert’s results were soon confirmed by others.7, 85 Ott and Gartz86 also re-

ported that sublingual application of 1a was effective in acetone or Me2SO,

with potency comparable to inhalation. Thus, far from being equipotent with

mescaline as the open field assay indicated, 1a is in fact ∼1000× more po-

tent.14, 7 Indeed, it is the most potent naturally-occurring hallucinogen yet

isolated.7 The rapid metabolism of 1a has been confirmed in vitro87 and in

vivo.88

Threshold doses of 1a produce visions of coloured patterns overlaid on reality,

reminiscent of the pseudohallucinations induced by indole and phenethylamine

hallucinogens. These patterns are faint, and only perceptible under dark and

quiet conditions.14 Higher doses, however, produce intense and unique effects

like those described earlier (Section 1.2 on page 33). Awareness that the visions

are drug-induced is lost, and true dreamlike hallucinations occur. These expe-

riences frequently involve certain themes not found with other hallucinogens.

The distinction between self and surroundings is lost; subjects often feel that

they are blending into, or have become, inanimate objects.18 Similarly, the

distinction between past and present is weakened; subjects will relive events,

often from childhood, rather than simply remembering them. Siebert’s initial

reports of these unique themes have again been confirmed by others.85, 89

Further research has revealed no evidence of active compounds other than 1a.

Siebert confirmed that 1b is inactive.40 He also found that self-administration

of 3 mg90 of 1c, sublingually in acetone, had no noticeable effect;40 this is 10×a threshold dose of 1a by that route. Evidence that 1c and other terpenoids

in the plant are inactive in vitro will be presented in Section 4.5.1 on page 171.

In conclusion, although definitive proof awaited human testing, Valdés nonethe-

less correctly identified the active principle of S. divinorum using animal as-


says. The magnitude of this achievement is not widely appreciated. As noted

above, previous attempts to identify plant hallucinogens using animal assays

had invariably failed, while human assays had succeeded, quickly and cheaply.

Valdés’s supervisor had thus forbidden the only demonstrably effective tech-

nique available. Moreover, in this case even human testing had failed. And it

had been performed, independently, by arguably the two greatest authorities

in the field, Alexander Shulgin91 and Albert Hofmann92, 10 (twice in the latter

case).93 Consider also the prevailing consensus when Valdés began work: the

active principle, whose effects were barely perceptible, was water-soluble and

well-absorbed orally, but unstable and destroyed by drying. This consensus

proved false in every detail. The active compound(s) also appeared to be al-

kaloid(s), which was also false. Sadly, despite overcoming these myths, Valdés

considers the time he spent on animal testing wasted, believing it cost him

priority on the discovery of 1a.94

1.4.3 In vitro Testing.

After establishing that 1a was psychoactive, Siebert submitted the compound

for in vitro screening against potential molecular targets.18 The NovaScreen

assay tested for radioligand binding inhibition at targets including receptors

for small-molecule and peptide neurotransmitters, as well as ion channels and

enzymes. No binding was detected at 10 µM.

Subsequently, the compound was screened by Roth et al against a much larger

battery of targets, the National Institute of Mental Health’s Psychoactive Drug

Screening Program. This revealed that 1a bound with high affinity to the κ

opioid receptor (K i = 4 nM).67 Functional testing showed that it activated the

receptor with full efficacy and high potency (EC50 =1 nM). Thus, 1a is a potent

full agonist at the κ opioid receptor. This result has since been replicated

by other groups.82, 81, 95 Further confirmation has come from in vivo testing.

The effects of 1a in mice are blocked by selective κ opioid antagonists;81, 78, 80

rhesus monkeys trained to discriminate κ opioids recognised 1a as such, and


the effects were blocked by a nonselective opioid antagonist.83

No binding was apparent in vitro to any of 48 other CNS targets at 10 µM; 1a

is thus extremely selective compared to most other psychoactive compounds.67

However, the strength of this conclusion is contingent on the number of targets

tested; many orphan receptors exist for which affinity testing cannot yet be

performed.96 However, in vivo confirmation of the selectivity of 1a is available.

Siebert found that the effects of 1a were blocked by naloxone, a nonselective

opioid antagonist.97 This makes it highly unlikely that a nonopioid mechanism

contributes independently to the compound’s effects.

1.4.4 Mechanism: κ Opioids.

1.4.4.1 Discovery.

The strongest painkiller available in the preindustrial era was opium, a milky

secretion of the opium poppy Papaver somniferum. The isolation of morphine

(11) from opium was reported by Sertürner in 1806; he also showed the com-

pound to be a potent analgesic.98

OHO OH

N

11

H

HO

N

OOHO

N

12

OH

HO

N

13

O

14

H

Figure 1.13: Morphine (11) and derivatives.

Morphine has had an immense impact on science. It was the first drug – the

first known pharmacologically active compound.99 It was the first alkaloid;


indeed, the word alkaloid was coined to describe it.98 Its structure elucidation

took over a century, which is unsurprising since morphine’s discovery predated

the concept of molecular structure by several decades. Indeed it predated the

publication of Dalton’s atomic hypothesis, which was regarded with skepticism

for decades.100

Morphine’s very strong analgesic effect is accompanied by serious side effects:

nausea, respiratory depression and constipation.101 Intense euphoria also oc-

curs in some, making the drug highly addictive. The pursuit of an analgesic

lacking these side effects gave rise to medicinal chemistry. The first morphine

derivatives were synthesised in the 1850s;98 the structure-activity relationships

of morphine have since been explored more thoroughly than those of any other

compound, with thousands of derivatives synthesised.101 Simplified structures

were found to retain activity: morphinans such as butorphanol (12), ben-

zomorphans such as ketazocine (13) and remarkably even phenylpiperidines

such as pethidine (meperidine, 14).

Some of these compounds (such as 14) closely mimic morphine’s actions.

Some, however, proved to be antagonists, which have proven immensely valu-

able in reversing opioid overdose. Other derivatives caused quite distinct be-

havioural effects, but were not antagonists and did not exhibit cross-tolerance.

The study of these differences led to the discovery of opioid receptor subtypes.

The µ and κ subtypes were named after the archetypes morphine (11) and

ketazocine (13), while δ comes from the vas deferens, in which that subtype

was discovered.101, 102 These receptors are commonly abbreviated as MOR,

KOR and DOR. Neither of these terminologies is endorsed by the International

Union of Pharmacology, but the officially sanctioned names, OP1−3,102, 78 have

not gained wide acceptance. The study of opioid receptors led to the discovery

of the endogenous ligands, the endorphins.101, 102 Remarkably, recent work has

proven that morphine is endogenous in humans.103

Opioids are compounds which act at opioid receptors, and whose effects are

reversed by the antagonist naloxone.101 This terminology lends itself readily to


selective compounds; thus, 1a will be referred to below as a “κ opioid.” This

is synonymous with the common but unwieldy tautology “κ opioid receptor

agonist.”

1.4.4.2 Development.

R O

N

O

N

Cl O

16

1718

O

N

N

Cl

Cl

15

Cl

Figure 1.14: Arylacetamide κ opioids.

U50,488 (15) was devised as a structurally simplified morphine derivative.104, 105

In animal tests, U50,488 was an effective analgesic, whose effects were reversed

by naloxone. Remarkably, however, the compound did not produce physical

dependence like morphine, and was not self-administered. Initially referred to

as a non-µ opioid, 15 was soon found to be the first selective agonist at the κ

opioid receptor.

These remarkable findings inspired extensive research; numerous derivatives

were synthesised, some of which proved to be even more potent and selective.105

Examples include U69,593 (16), spiradoline (U62,066, 17) and enadoline (CI-

977, 18). Testing of these compounds confirmed the findings with 15: at last it

appeared that the long-awaited nonaddictive opioid analgesics had been found.

1.4.4.3 CNS Effects.

Addiction can be studied using animal models. The µ opioids are positively

reinforcing. This means test animals trained to self-administer them will do


so compulsively, at the expense of social activity, food and sleep. By contrast,

κ opioids (including 1a)78 are negatively reinforcing, or aversive. Test animals

will not self-administer them; if administered when an animal behaves in a

certain way, the animal will avoid that behaviour. In other words, κ opioids

act as a punishment, where µ opioids act as a reward. This is a vast field of

research in its own right, and a thorough review is available.106 The mechanism

of these effects, believed to be mediated by dopamine, has also been thoroughly

studied.78, 107

Since the objective had been to eliminate the euphoria associated with opioids,

the aversive nature of κ opioids was initially regarded as desirable. Human

tests soon revealed a problem: rather than euphoria, κ opioids produced dys-

phoria. This lowering of mood was accompanied by “psychotomimetic” effects:

confusion, hallucinations and depersonalisation.

Enadoline (18), for instance, caused side effects such as somnolence, hallucina-

tions, anxiety, depersonalisation, confusion and abnormal thinking; the sever-

ity of these effects led to termination of clinical development.108 Subsequent

tests with higher doses reported visual, auditory and tactile hallucinations,

along with impaired coordination and recall. One subject felt waves moving

through the floor, and felt his body was blending into them109 (cf. 1a).18 Spi-

radoline (17) caused altered perceptions, impaired coordination and slurred

speech; subjects reported being more irritable, anxious and sad.110 Responses

to other κ opioids range from “personality disorders and mild confusion”111

to “depersonalisation”, “dreamlike” experiences and “episodes of unmotivated

and uncontrolled laughter”112 (again cf. 1a).18

In one study, heroin users were given various substances and asked “do you

like the drug?” and “does the drug have any good effects?” They consistently

answered yes after administration of a µ opioid, but no after enadoline (18).109

The opposite was true when asked about “bad effects.” Similar results were

reported in tests of nonselective κ opioids such as 13113 and 12.114 Subjects

with extensive histories of illicit drug use reported dysphoria, hallucinations,


paranoia and confusion after the κ opioids, but euphoria after morphine. Thus,

both drug-naïve subjects and experienced illicit drug users overwhelmingly find

κ opioids dysphoric and aversive.

1.4.4.4 Therapeutic Potential.

Clinical development of these compounds as analgesics was eventually aban-

doned due to these psychological effects.115, 105 Some κ opioids which do not

cross the blood-brain barrier, and are therefore not psychoactive, remain in de-

velopment for arthritis116 and abdominal pain.117 This is interesting in light of

Mazatec use of S. divinorum infusion against abdominal pain and swelling.11, 15

Besides analgesia, many other therapeutic uses have been proposed for κ opi-

oids.115, 118, 119, 120 None of these therapies has reached clinical use.

There has been a report of an antidepressant effect of U50,488 (15) in an animal

model.121 However, there was at the time no validated protocol for that model

(learned helplessness in mice),122 and compounds of known activity were not

used as controls. In another model, the forced swim test in rats, one study

reported that a κ opioid had no effect.123 However, other studies reported

a prodepressant effect,124 one of which used 1a.107 This conclusion, which

is consistent with the aversive and dysphoric effects discussed above, is also

more consistent with prior results and recent theoretical insights.107, 125 Thus,

it seems that κ opioids exacerbate depression in animal models. Surprisingly,

however, there have been reports of antidepressant effects from S. divinorum

use.126, 127 However, the plant was used only three times weekly. It may be

that, just as the acute euphoria caused by µ opioids is followed by prolonged

dysphoria, the converse may be the case with κ opioids.

1.4.4.5 Claimed Subtypes.

There have been claims that certain κ opioids lack dysphoric effects. TRK

820 (19, Figure 1.15 on the facing page) allegedly produces “moderate be-

havioural/psychological side effects, but not psychotomimetic activity”, but


this is based on “unpublished data”.128 It is also often claimed that 19 is not

aversive in animals,128 which is untrue.129 This is reminiscent of earlier claims

that enadoline (18) “appears to be devoid of psychotomimetic activity,”130

which was subsequently and spectacularly discredited as mentioned above.

One proposed mechanism for differences between κ opioids is that there are

subtypes of κ opioid receptor.131 Differences between κ opioids might reflect

different selectivities for these subtypes. The evidence for subtypes is purely

pharmacological131 — the gene encoding the receptor has now been cloned

from several species, with no subtypes detected. For this reason, and because

of the lack of selective ligands for each proposed subtype, the claim has not

won general acceptance.132 The apparent subtypes may in fact represent dimer

formation between different receptors.131, 132

1.4.4.6 Structure, Potency and Selectivity.

S

OHN

N

NF

20

OHO N

N

OH

O

O

19H HO

N

13

O

O

N

O

N

O 18

phenylalkylamine moiety

Figure 1.15: Structurally diverse κ opioids.

The major structural classes of κ opioid are peptides such as dynorphin A, mor-

phine derivatives such as 19, benzomorphans such as 13 and arylacetamides

such as 18 (Figure 1.15). The first major departure from these categories was

the benzodiazepine tifluadom (20).105 The discovery of such compounds, and


similarly diverse ligands at other subtypes, eventually rendered generalisations

about structure-activity relationships impossible.101

Nonetheless, as diverse as these compounds may appear, they are all alka-

loids. 1a was the first non-nitrogenous opioid reported. Indeed, it was the

first opioid lacking the phenylethylamine moiety or its propyl homologue (Fig-

ure 1.15). These moieties are near-ubiquitous in hallucinogens,133 and indeed

in psychoactive compounds generally. In a random sample of compounds from

the Merck Index, 82% of psychoactive compounds contained a phenylalky-

lamine moiety, versus 8% of non-psychoactive compounds.134 Conversely, 58%

of phenylalkylamines were psychoactive, versus 3% of other compounds. Be-

ing non-nitrogenous and lacking a benzene ring, 1a is thus not merely unique

among opioids, but extremely unusual among psychoactive compounds in gen-

eral.

EC50 (nM) Ref1a 15 16 191 1.2 677 24 13 135

4.5 4.5 9545 207 1363.1 3.9 1374.6 2.2 0.025 81

1.1 0.0048 13816 0.15 139

Table 1.1: Relative potency of 1a at cloned κ opioid receptors.

The binding affinity and potency of 1a in vitro are close to those of U50,488

(15) and U69,593 (16). Potencies are shown in Table 1.1. The most potent

κ opioid to date appears to be TRK 820 (19). However, few groups have

studied this compound, and their binding affinity data are wildly discordant

(K i = 75 pM81 vs 3.5 nM).139 The compound also has lower µ/κ selectivity

than U50,488138 and other arylacetamides,104 which are in turn less selective

than 1a.67 Indeed, there has been no report of 1a showing any affinity at the

µ opioid receptor (K i > 10 µM).

In summary, κ opioids have not lived up to initial expectations as analgesics.

1.5. TOXICOLOGY. 61

Ultimately, the enormous effort to improve upon morphine has failed. Two

hundred years after its discovery, morphine remains “the opioid of first choice”

for severe pain under World Health Organization guidelines.140 There is no

superior analgesic:

Morphine remains the most widely used opioid for the manage-

ment of pain and the standard against which other opioids are

compared.141

Opioids remain the topic of extensive research, however, for other therapeutic

purposes, as mentioned above.

1.5 Toxicology.

There has been little toxicological research on S. divinorum and 1a. Valdés

made several incidental observations on the topic. One of his rats died during

oral administration of an extract, but this was due to choking on the large

volume of liquid rather than toxicity.142

The crude aqueous extract proved highly toxic by injection in cats. Subcu-

taneous injection of 0.7 g/kg, approximately equivalent to a human dose of

the infusion, caused a sterile abscess at the injection site.58 A higher dose (1.3

g/kg) caused kidney failure in two cats, one of which died. The other recovered

with a sterile abscess. Based on the known toxicity of polyphenols in crude ex-

tracts,143 and veterinary examinations suggesting this was the cause,58 Valdés

prepared a polyphenol-free extract by partitioning between CH2Cl2/MeOH

and water. The resulting organic fraction was not toxic, even in much higher

doses, estimated to be equivalent to 635× the human dose.58

Further toxicity testing was performed in mice. The ether extract of the leaves

was dissolved in aq. MeOH and washed with hexanes. The resulting methano-

lic fraction, which was presumably free of polyphenols, was nevertheless still

toxic enough to kill the mice within a week (LD50 = 340 mg/kg i.p.)69 This is


approximately equal to the toxicity of vitamin B3.144 Pure 1a caused no fatal-

ities at 1 g/kg,13 the largest dose which could be administered as an emulsion,

and more than 105 × the psychoactive dose in humans.

As discussed above, however, the emulsion was very poorly absorbed. This

would reduce the immediate systemic dose of the extracts, giving a mislead-

ingly low impression of acute toxicity. On the other hand, the undissolved

material at the injection site may have had toxic effects through another mech-

anism, such as shock. It would also presumably slowly dissolve, causing chronic

rather than acute toxicity, especially to the liver. Surfactants such as Tween

80 can also have adverse effects.145 These data therefore cannot be used to

make reliable comparisons with other substances.

Mowry et al later published a study specifically devoted to the toxicology of

1a.146 Injection of 6.4 mg/kg/day for two weeks caused no fatalities; post-

mortem examination of organs and tissues showed no apparent changes. Un-

fortunately, since the drug was administered intraperitoneally as a “fine sus-

pension,” and behavioural effects were not described, the same issue of bioavail-

ability arises as with Valdés’s results. It is also regrettable that larger doses

were not administered to determine the LD50.

Human toxicological data are not available. There have been no published

reports of toxic effects or hospitalisation related to S. divinorum or salvinorin

A.147

1.6 Social impact.

1.6.1 Recreational Use.

S. divinorum came to the attention of recreational drug users even before

chemical investigations began. Books published in 1973 described its use148

and cultivation,149 listing a mail-order source for live cuttings. By 1975, Ott

reports seeing some young Mexicans smoking the dried leaves recreationally.7

1.6. SOCIAL IMPACT. 63

Although he claims Díaz also reported this at the time, the latter was in fact

referring to a different plant.41

The plant nonetheless remained extremely obscure until Siebert’s findings were

popularised in 1996 by Turner.85 This immediately overturned the plant’s

reputation as a weak substitute for illicit drugs:

Salvinorin A is the most potent naturally occurring psychedelic

known ... it frequently induces experiences of an intensity [...] be-

yond those experienced with any other psychedelic ...85

The plant’s notoriety then began to grow. Online companies began selling live

cuttings, dried leaves, extracts and tinctures for recreational use. Interestingly,

however, after being widely available for a decade, the drug has not had any

noticeable social impact (as for instance LSD and MDMA rapidly did). As

Turner noted:

Many who have used salvinorin A find the experience extremely

unnerving, frightening and overly intense. Most have no desire to

repeat the experience.85

This is consistent with the dysphoric and aversive qualities discussed above.

1.6.2 Legal Status.

1.6.2.1 Australia.

In 2002, Australia became the first country to prohibit S. divinorum and salvi-

norin A. The committee responsible, the National Drugs and Poisons Schedule

Committee (NDPSC), justified the decision “on the basis of high potential for

abuse and risk to public health and safety.”150

Salvinorin A was prohibited under a purportedly systematic name:


8-METHOXYCARBONYL-4A,8A-DIMETHYL-6-ACETOXY-

5-KETO-3,4,4B,7,9,10,10A-SEPTAHYDRO-3-(4-FURANYL)-

2,1-NAPHTHO[4,3-E]PYRONE.150

This name is in fact meaningless. Compare the one used by Chemical Abstracts

Service:

2H -Naphtho[2,1-c]pyran-7-carboxylic acid,

9-(acetyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-,

methyl ester, (2S ,4aR,6aR,7R,9S ,10aS ,10bR)-48

NDPSC CAS- 2S,4aR,6aR,7R,9S,10aS,10bR

8-METHOXYCARBONYL- 7-carboxylic acid, methyl ester4A,8A-DIMETHYL- 6a,10b-dimethyl-

6-ACETOXY 9-(acetyloxy)-5-KETO 4,10-dioxo-

3-(4-FURANYL) 2-(3-furanyl)3,4,4B,7,9,10,10A-SEPTAHYDRO dodecahydro-2,1-NAPHTHO[4,3-E]PYRONE 2H -Naphtho[2,1-c]pyran

Table 1.2: Alternate systematic names for 1a.

Enumerating all the errors in the NDPSC name would be beyond the scope of

this work, but a brief selection will be given here. The names are contrasted in

order of functional group in Table 1.2. Note especially the nonexistent terms

“4-furanyl” and “septahydro.” The latter was presumably intended to mean

heptahydro, which is incorrect.

11

2233

44

4a4a

4b4b

5566

7788

8a8a99

1010

10a10a

phenanthrene

444a4a10b10b

1122

O33

55

666a6a

7788

991010

10a10a

2H-naphtho[2,1-c]pyran

Figure 1.16: Relevant IUPAC fused-ring numbering schemes.

The name given by the NDPSC originated in a post to the internet newsgroup

alt.drugs by William E. White:

1.6. SOCIAL IMPACT. 65

I tried to get an IUPAC name out of this; unfortunately, I gagged

when trying to decide whether it was a naphthopyrone, or a modi-

fied phenanthrene, or whether it should start from cyclohexane in-

stead of aromatic rings, or whatever. The fact that phenanthrene

is numbered funny didn’t help. The best I could do is

8-methoxycarbonyl-4a,8a-dimethyl-6-acetoxy-5-keto-3,4,4b,7,9,10,

10a-septahydro-3-(4-furanyl)-2,1-naphtho[4,3-e]pyrone.

(whew!) Sorry, I haven’t done o-chem since the Reagan adminis-

tration.151

This explains the incorrect numbering of 1a: White named it as a naph-

thopyran derivative, but numbered it as a phenanthrene derivative (see Figure

1.16).152 The resulting name was then copied without acknowledgment by the

NDPSC, with the sole change being capitalisation.

After these criticisms153 of their decision attracted publicity,154 the NDPSC

revisited the issue in late 2002. Conceding that the name used was incorrect,

they decided to adopt the CAS name.155 However, they rearranged the sub-

stituents in nonalphabetical order, in conformance with their own unspecified

“naming conventions.” The new name is therefore also incorrect.

The NDPSC is Australia’s peak body for the regulation of chemicals, whose

chief task is to list controlled chemicals by name. It is surprising and dis-

turbing, therefore, that no member of this body understands basic chemical

nomenclature. White’s posting also raises another serious issue. The news-

group alt.drugs156 is a forum for users, dealers and manufacturers of illicit

drugs to share ideas and advice. Publications which “promote, incite or in-

struct in matters of crime” cannot be legally imported into Australia, as for

instance by downloading from the internet.157 How, then, did the committee

legally obtain this material? Committee members repeatedly refused to reveal

the source to a federal shadow minister.158, 159 Similarly, documents released

under a freedom of information request160 contained nothing on this topic.


A disturbing question thus remains unanswered: how did obvious errors in a

prohibited publication find their way into Australian law?

1.6.2.2 Other Countries.

In 2003, Denmark became the second country to prohibit S. divinorum and

salvinorin A.161 William White’s erroneous name was used again, with the

locants capitalised. This new and peculiar error suggests that the authors

copied the Australian version, changing the substituents to lower case, but

leaving the locants in capitals. Ironically, the NDPSC had by this time already

admitted the name was incorrect and adopted a new one.

S. divinorum and/or salvinorin A have since been prohibited in other countries,

including Belgium, Italy, South Korea and Spain.162 There have been similar

moves in several US states, but a 2002 bill163 to prohibit the plant nationally

met with well-organised opposition,147 and was not enacted.

1.7 Summary.

Previous work had thus established that 1a was a psychoactive κ opioid. How-

ever, virtually nothing was known of its structure-activity relationships, and

only three other compounds had been isolated from S. divinorum. The suspi-

cion that other psychoactive compounds were present persisted.

Chapter 2

Isolation.

2.1 Isolation Procedure.

2.1.1 Extraction Conditions.

The isolation methods reviewed above suffer from a number of drawbacks.

Both Valdés et al23 and Ortega et al22 employed refluxing solvents for the ini-

tial extraction of the dried leaves. Subsequently, however, Gruber164 reported

that extraction at room temperature was superior, based on HPLC analysis.

He found that steeping for several days at room temperature gave markedly

better recoveries of 1a than refluxing for two hours, in either CHCl3 or MeOH

(see Table 2.1 on the next page). In optimising the room-temperature ex-

traction, he found that highest recoveries of 1a were achieved after less than

30 minutes; levels then declined steadily (by nearly 50% over two days).164

Gruber speculated that this decline might be due to 1a precipitating out of

solution as other compounds accumulated. This explanation is implausible

since 1a is freely soluble in CHCl3 (evaporation gives an amorphous resin

rather than a crystalline precipitate), and the solutions were extremely di-

lute. Furthermore, the decline is faster at reflux, when solubility is greater.

A more plausible explanation of the decline is decomposition, with the rate

proportional to temperature. This conjecture was confirmed in the course of

67

68 CHAPTER 2. ISOLATION.

this work. When chromatographically pure 1a was recrystallised from EtOH,

and the resulting mother liquor recrystallised in turn, new higher spots be-

came apparent by TLC (70% Et2O/petrol). After another recrystallisation,

the mother liquor (18% of the original fraction) consisted almost entirely of

the higher spots, with only traces of 1a. The decomposition products were

not characterised. In contrast, however, Valdés reports that he recovered 1a

unchanged after refluxing in MeOH or MeOH/H2O for 2 weeks.165 It may be

that decomposition only occurs in the presence of oxygen.

recovery (mg/g)T time CHCl3 MeOHrt 4 d 1.75 1.56

reflux 100 min 1.41 0.44

Table 2.1: Effects of solvent and temperature on recovery of 1a.

After his optimisation experiments, Gruber adopted a standard procedure of

steeping the dried, powdered leaves in CHCl3 for 30 minutes. For scale-up,

however, this presents the problem of handling and evaporating large volumes

of this carcinogenic solvent. Therefore, for this work a less toxic substitute was

sought, of lower polarity than MeOH (given the inferior recoveries achieved in

that solvent). Acetone was selected as an affordable solvent meeting both

of these criteria. Dried, powdered leaves were steeped for 30–60 minutes in

room-temperature acetone (× 3). TLC analysis (5% MeOH/CH2Cl2) showed

that the second extract was much lower in 1a than the first; the third extract

showed no detectable 1a. A further steep in CHCl3 also showed no 1a. This

extraction procedure was therefore adopted for all of the work described below.

Further improvements are possible. Powdering the leaves, for instance, appears

to be unnecessary. Pseudonymous reports166 on the internet that 1a can be

extracted in high yield from intact leaves have been confirmed: Siebert found

that dipping fresh leaves in CHCl3 (30 seconds × 3) gave excellent recovery of

terpenoids with minimal contamination by pigments.40 Drying and powdering

the leaves, followed by a further extraction, gave a pigment-rich extract with

negligible terpenoids. Thus powdering the leaves before extraction is unneces-

2.1. ISOLATION PROCEDURE. 69

sary, and indeed counterproductive. While this analytical procedure has not

yet been tested on a preparative scale or on dried leaves, these results strongly

suggest that extraction procedures based on brief steeping of intact leaves will

prove superior to the protocol given above. Claims166 that extraction in chilled

solvents gives a cleaner extract also deserve investigation.

2.1.2 Problems Caused by Pigments.

2.1.2.1 Interference with Previous Isolations.

1a1b

youngest oldest

Developed in 50% EtOAc/petrol, visualised in vanillin/H2SO4.

Figure 2.1: Siebert’s TLC analysis of crude CHCl3 extracts.40

The extract from the above procedure was a blackish-green tar. The numer-

ous pigments present in this extract rendered TLC analysis difficult. Relative

to the colourless terpenoids of interest, the pigments were present in larger

quantities and stained more vividly, obscuring the terpenoid spots. The prob-

lem is illustrated in Figure 2.1. Daniel Siebert analyzed chloroform extracts

of leaves of varying age, which had been dried and powdered.40 In all cases,

the intensities of pigment spots were equal to or greater than 1a, and much

greater than 1b. The pigments thus present a major obstacle to the isolation

of other terpenoids, which are present in lower levels. For instance, 1c is not

even detectable in the figure; in this system it appears immediately above 1a,

which is itself nearly obscured in the youngest leaf extract.

Valdés et al had addressed this problem by partitioning the crude extract be-

tween aqueous MeOH and hexanes.23 Using his partitioning procedure proved

difficult: both phases were black, and could not be distinguished even by close


inspection in direct sunlight. The interface was ultimately located by holding a

torch against the separatory funnel and looking directly into the beam, which

was dimly visible through the MeOH phase. The hexane phase was totally

opaque. Another drawback to the procedure was the tendency of the MeOH

phase to spatter during evaporation, but the water content (10%) was far too

high to use a drying agent such as MgSO4. After partitioning, while there was

some reduction in mass, the extract remained intensely coloured.

Gruber also encountered difficulties with Valdés et al’s procedure, obtaining

greenish crystals of 1a even after partitioning, repeated chromatography and

recrystallisation.167 HPLC analysis indicated purity of approximately 85%

based on UV detection at 208 nm;168 however this cannot be translated to

percentage by mass, since the coloured impurities would be expected to have

higher molar absorptivities. Gruber found that the trace pigments could be re-

moved by rinsing with cold MeOH.167 It was presumably to remove these trace

pigments that Valdés recrystallised 1a a second time, for which no explanation

was given.23 This repeated recrystallisation is clearly a major drawback to the

procedure, adding complexity and reducing recovery.

Others have simplified Valdés et al’s procedure, omitting the chromatography

and simply washing the crude extract with less polar solvents to remove the

pigments.86, 166 These procedures, while ingeniously simple, have not yet been

replicated in the peer-reviewed literature. A recent paper took the opposite

approach, omitting partitioning and recrystallisation in favour of chromatog-

raphy. Tidgewell et al169 isolated 1a in very high yield as a green powder,

after repeated chromatography of the crude extract on silica gel. Despite the

colouration, the material exhibited a reasonably sharp melting point range of

3 ◦C; other evidence of purity was not reported.

In summary, the pigments present in the extract caused considerable prob-

lems in the isolation of 1a. They therefore posed even greater obstacles to

the isolation of the other diterpenoids present in S. divinorum, since these

are present in much smaller quantities than 1a. Indeed, as noted at the start


of this section, the pigments made the mere detection of other compounds

by TLC difficult. It was therefore critical to this work that the crude ex-

tract itself be decolourised, rather than individual compounds after isolation.

Valdés et al’s partitioning procedure was unsatisfactory, as was chromatog-

raphy on silica gel. Gruber found that reverse-phase chromatography (using

a C-18 solid-phase extraction cartridge), while removing some pigmentation,

was likewise inadequate.167 Both of these techniques rely upon differences in

polarity to achieve separation. Since the pigments exhibited a very wide range

of polarities, spreading from baseline to solvent front on TLC (Figure 2.1),

another basis for separation was clearly necessary.

2.1.2.2 Chemistry of Plant Pigments.

O

O

N N

N N

Mg

O

O

O

21

22

OOH

HO O

OH

OOH

OHOH

O

OH

23

Figure 2.2: Representative major plant pigments.

The overwhelming majority of plant pigments fall into three categories: chloro-

phylls (eg. chlorophyll-a, 21, Figure 2.2), carotenoids (eg. β-carotene, 22) and

flavonoids (usually present as glycosides eg. quercitrin, 23).170, 171 In addition


to these ubiquitous classes, there are many minor pigments confined to partic-

ular taxa, which have been reviewed elsewhere.170 The common factor among

these pigments is an extended π system, often but not always aromatic, which

results in intense absorption of visible light. As a result, pigments are gener-

ally large molecules. They vary widely in polarity, from extremely hydrophilic

(eg. 23) to extremely hydrophobic (eg. 22). The chlorophylls themselves

vary: one of the earliest procedures for separating chlorophylls a and b was

partitioning between aq. MeOH and petrol.171 Thus, it is unsurprising that

Valdés’s partitioning procedure, and polarity-based fractionations in general,

are of limited effectiveness in decolourising plant extracts.

2.1.3 Use of Activated Carbon.

The standard method for decolourisation of a solution is adsorption, and the

most common adsorbent is activated carbon, often referred to as decolouris-

ing carbon.172 Activated carbon has been in widespread use, both scientifi-

cally and industrially, for centuries. The characteristics of compounds which

affect adsorption are well-established empirically.173 Within a homologous se-

ries of compounds, adsorption increases with length. In addition, aromatic

compounds are strongly adsorbed, especially polycyclic aromatic compounds.

Generally, increased polarity reduces adsorption, but this effect is smaller, and

easily overcome by size. Polyphenols, for instance, are strongly adsorbed de-

spite being freely soluble in water. Many other variables affect adsorption to

lesser extents.173 While these general trends are well-established, the underly-

ing mechanisms remain in dispute.

2.1.3.1 Mechanism of Action.

The surface chemistry of different types of activated carbon varies, from highly

oxidised forms with numerous polar functional groups distributed across the

surface, to graphitised forms with very little functionality.173 The most im-

portant common characteristic of all forms is their enormous surface area,


estimated at up to 1500 m2/g in some cases,174 resulting from the material’s

extremely porous structure. Recent work suggests that this is the key factor

controlling adsorption. In “solvophobic” theories,175, 176 adsorption is treated

as being driven by two key factors: the (unfavourable) formation of a sol-

vent cavity around the solute, and (favourable) van der Waals attraction be-

tween the solute and the carbon surface. As a result, “the theory predicts

the adsorbability to depend linearly on the nonpolar surface area of the adsor-

bate”.176 This simple model predicts very accurately the adsorption data for a

wide range of compounds.175, 176 Another recent study, based on a much larger

data set, used a more complex model (the “linear solvation energy relationship”

model).177 Interestingly, however, despite being based on different parameters,

the study reached very similar conclusions. Planar molecules were found to be

more strongly adsorbed than nonplanar molecules. This was rationalised as

follows:

Since the dispersion force of attraction is very sensitive to the dis-

tance of separation between the surface of the activated carbon

and the center of the solute molecule, it is reasonable that a pla-

nar molecule would be adsorbed more strongly than an otherwise

similar globular molecule.177

That is, consistent with the solvophobic model, surface area available for con-

tact is the key factor in determining adsorbability. Interestingly, this effect was

found not only for aromatic compounds; planar olefins were just as strongly

adsorbed. A similar effect had been observed in earlier work,178 based on an-

other model, and used to predict that square planar organometallic complexes

would adsorb more strongly than octahedral complexes. This prediction was

confirmed experimentally.178 This also helps explain the strong adsorbability

of carotenoids such as 22, which although aliphatic can assume planar confor-

mations which present a much larger surface area to the carbon surface than

less conjugated hydrocarbons. Indeed, in this work non-conjugated terpenoids


comparable in size to 22 were easily desorbed from carbon (Section 2.2.4.2 on

page 97), while 22 and other carotenoids were not.

These works establish a strong theoretical and empirical relationship between

surface area and adsorbability. Nonetheless, other factors affect adsorption,

and the underlying mechanisms remain controversial, even for particular classes

of compound.179

2.1.3.2 Use during Recrystallisation.

The most common application of activated carbon by chemists is in remov-

ing coloured impurities from crude compounds. Typically, a small amount

of powdered activated carbon (1–2% w/w relative to the crude compound) is

added during recrystallisation; the decolourised solution is then filtered before

cooling.172 In many cases, however, this is not effective. Indeed, during the

preliminary work described above, flash column chromatography of the crude

extract gave an orange solid which was rich in 1a by 1H NMR. During re-

crystallisation from MeOH, the bright yellow colour of the solution was not

diminished by activated carbon, even at 10% w/w. Upon cooling, the result-

ing crystalline 1a remained faintly yellow, and the mother liquor vividly so.

While this might be dealt with by using larger amounts of activated carbon,

this would also result in increased adsorption of 1a itself, and therefore reduced

recovery.

Even if the procedure had been effective, moreover, it would still only be useful

for removal of trace pigments from individual compounds. As noted above, it

was essential to this work that the crude extract itself be decolourised. This

would therefore require the use of a large amount of activated carbon. The

compounds of interest would then have to be desorbed, in what would be

“effectively a chromatographic procedure.”172


2.1.3.3 Use in Chromatography.

Activated carbon is rarely employed as a stationary phase in chromatography.

It is not suited to general use; for most separations, other adsorbents offer

superior selectivity and resolution.180 Nonetheless, it has found use in certain

niche applications. Perhaps the most common use has been the isolation of

antibiotics181 and enzymes182 from microbial fermentation broths. Activated

carbon remains a standard part of such procedures, although alternatives have

been proposed.183, 184

Another application where activated carbon has proven superior to conven-

tional adsorbents is in the separation of fullerenes,185, 186 giving better reso-

lution and recovery than alumina, along with higher speed and lower cost.

The larger ellipsoidal fullerenes, above C70, are very difficult to desorb.186

Activated carbon has also proven valuable in separation of polychlorinated

biphenyls (PCBs).187, 188, 189 Commercial PCB mixtures are very complex, con-

taining hundreds of PCBs differing in the number and position of chlorine sub-

stituents.189 Many of these compounds differ very little in polarity and volatil-

ity, resulting in incomplete resolution by either HPLC or GC. This problem

can be resolved by multi-dimensional GC, but activated carbon permits sepa-

ration on a different basis: extent of ortho-substitution.189 Non-o-substituted

PCBs (such as 24, Figure 2.3) can assume a coplanar conformation, and are

therefore very strongly adsorbed (as expected). Each o-substituent presents

a steric barrier to this conformation, and thus reduces adsorption, reaching

a minimum with fully o-substituted PCBs (such as 25). PCBs can therefore

be eluted from a carbon column in decreasing degree of o-substitution.190, 188

These fractions are greatly simplified relative to the initial mixture, and can

be further purified using conventional techniques.187, 188 This method also has

the attraction that degree of o-substitution is of great biological significance.

In addition to their toxicity, non-o-substituted PCBs such as 24 are extremely

teratogenic; this effect is reduced by one o-substituent, and abolished by more

than one.187


ClCl

ClCl

Cl

Cl

Cl Cl

Cl

Cl

24 25

Figure 2.3: Ortho- and non-ortho-substituted PCBs.

A surprisingly uncommon use of chromatography on activated carbon is for

decolourisation of crude extracts during isolation191, 192 or analysis193 of natural

products. In most cases this is used, as here, to permit analysis of the crude

mixture rather than purification of individual compounds.

As with any adsorbent, eluent selection is critical to achieving effective sepa-

ration. The eluotropic series on activated carbon given by Gordon180 is:

H2O < MeOH < EtOH < acetone < iPrOH < Et2O < EtOAc < hexane �benzene.

In general, then, the larger and less polar the solvent, the greater its elut-

ing strength. That benzene is the strongest eluent in the series is unsurpris-

ing given the strong adsorption of aromatic compounds. In recent decades,

toluene has been used as a less toxic and equally effective substitute for ben-

zene.189, 185 Indeed, toluene appears on the balance of the evidence to adsorb

more strongly than benzene,177 which is unsurprising given its larger surface

area. Consistent with this, recent work has shown that o-dichlorobenzene is

a much stronger eluent again.186 Based on their measured adsorption con-

stants,177 p-xylene would probably offer comparable eluting strength, with the

advantage of a lower boiling point (138 vs 178 ◦C). Another approach to elu-

tion of very strongly adsorbed compounds is exhaustive extraction with hot

solvent, using Soxhlet apparatus or similar.187

A major obstacle to practical chromatographic use of activated carbon powder

is that it packs very tightly; it is almost impermeable to solvent even under

pressure.180 One solution to this is to “fluidise” the activated carbon, using a

stream of nitrogen to remove the finest particles.187 Similarly, industrial-scale

water treatment usually employs granulated activated carbon.174 Using larger


particles in this way, however, has the undesired effect of reducing available

surface area. A more common solution is therefore to use mixed adsorbents.180

The most common choice is diatomite filter aid (eg. Celite),187 but other

possibilities include cellulose powder193 and silica gel.189, 185

2.1.3.4 Application to S. divinorum Extract.

For this work, the mixed adsorbent approach was taken. Diatomite filter aid

was selected due to its low cost, safety and ready availability. The proce-

dure was performed as standard flash column chromatography, monitored by

TLC.194 Initial testing on a small scale indicated that MeOH did not elute 1a,

while acetone did so rapidly. Additionally, 1a is far more soluble in acetone.

Some tailing was evident, so a stepwise gradient was employed based on Gor-

don’s eluotropic series,180 through Et2O, EtOAc and EtOAc/petrol. Petrol

was not used alone due to the insolubility of 1a. Gradient elution helped to

minimise tailing, but did not eliminate it – 1a was detectable from near the

solvent front into the EtOAc/hexane fractions.

No pigments were eluted when sufficient carbon was used; a carbon:solute

mass ratio of 20:1 was found adequate. At a ratio of 3:1, the adsorbent was

overloaded, and orange pigments were eluted even by acetone. After the sub-

sequent isolation of pure 1a (detailed in Section 2.1.4 on page 82), the recovery

of this compound from carbon was tested. A microscale test column on 15 mg

of 1a at 100:1 carbon ratio gave approximately 80% recovery. The loss was

more likely due to the small scale than irreversible adsorption, since stripping

the column in 50% toluene/petrol gave no additional 1a. Nonetheless, use of

minimal carbon is clearly advisable to minimise any losses. Interestingly, the

elution with toluene/petrol gave an orange fraction, presumably carotenoids.

Chlorophylls, which are more strongly adsorbed than carotenoids,195 were not

eluted.

A number of practical points should be noted:

• Activated carbon powder can contain much finer particles than flash


chromatography grade silica; these can contaminate fractions and block

glass frits. A layer of filter aid below the carbon prevents this.

• As noted by others,185 the carbon/filter aid mixture shrinks and cracks

when dry, which may contaminate the decolourised fractions with crude

extract. To prevent this, it is critical that the solvent level never be

allowed to touch the surface of the carbon. Since it is difficult to locate

the interface between the very dark crude extract and the black carbon,

it is helpful to add a thick layer of sand on top. This layer should never

be allowed to run dry.

• In later work196 filtration under vacuum proved more convenient and

practical than flash chromatography. This preferred approach is shown

in Figure 2.4. Fractions are collected by closing the separatory funnel

tap and applying vacuum. When the desired amount has been collected,

vacuum is released and the fraction dispensed.

• A gradient from 50% to 20% EtOAc/petrol gave faster elution and less

tailing, without eluting pigments. All fractions containing terpenoids by

TLC were pooled.

Separatory funnel

Vacuum adaptor

Glass fritFilter aid

Activated carbon/filter aidSand

Figure 2.4: Apparatus for Filtration through Activated Carbon.

While some separation between compounds in the decolourised fractions was


apparent (see Section 2.1.4.2 on page 85), separation of individual compounds

proved unworkable due to tailing. Thus, since the pigments were not eluted and

the eluted compounds were not separated, in practice the preferred procedure

resembled filtration more than chromatography.

2.1.3.5 General Applicability of the Method.

This procedure has some attractive features for use in isolating biologically

active compounds from plants. Activated carbon filtration provides a much

greater reduction in mass and complexity of the extract than more common

partitioning procedures. Thus, as seen with PCBs, analysis is simplified by

utilising an extra “dimension” for separation (surface area/planarity), com-

pared to the typical reliance on polarity differences. Using the procedure de-

scribed here, as discussed below, diterpenoids and triterpenoids were rapidly

eluted, while the tetraterpene carotenoids were completely removed.

Moreover, this added dimension (size) is of biological relevance. It is well

established pharmacologically that large molecules show reduced absorption

and permeation. This is embodied in Lipinski’s famous “rule of 5”,197 which

states among other things that compounds with a relative molecular mass

over 500 will be less readily absorbed in vivo, and therefore less likely to show

pharmacological activity. Thus, compounds eluted first from activated carbon

are more likely to be responsible for biological activity.

Nonetheless, as indicated previously, size is not the sole determinant of ad-

sorption. Compounds of interest may not be recovered using the procedure

described here if they are highly planar, or strongly adsorbed for some other

reason. Of particular relevance here are flavonoids, some of which are known

to be psychoactive. Apigenin (26a), isolated from S. officinalis, binds weakly

to the benzodiazepine site of the GABAA receptor (IC50 = 30 µM).198 Other

flavones isolated from plants have shown much greater potency at this site;

27 binds with affinity comparable to diazepam (K i = 6 nM).199 Interestingly,

some flavonoids appear to share the anxiolytic properties of benzodiazepines,


without the amnesic200 and sedating201 qualities.

There has been no published report of flavonoids from S. divinorum. However,

in unpublished work, Valdés isolated the known apigenin derivative 5-hydroxy-

7,4’-dimethoxyflavone (26b)202 from S. divinorum in 20 mg/kg yield.203 This

compound has been isolated from several Salvia species; identity was con-

firmed by comparison of 1H and 13C NMR, IR, MS(CI) and mp with literature

values.202 In other recent unpublished work, Claudio Medana detected a com-

pound whose ESIMS data were consistent with quercitrin (23) or a stereoiso-

mer in a crude extract of S. divinorum.204 The aglycone of 23, quercetin,

activates several CNS receptors.205 Quercetin, but not 23 itself, has been

proposed to have antidiarrhoeal effects;206 if so, this might conceivably con-

tribute to the known effects of S. divinorum on gastric motility.207 However,

quercetin’s gastrointestinal effects remain in dispute.208

O

O

2'2'O

O

HO

HO

OHO

O

OR

RO

OH R

HMe

26a26b 27

OOH

HO O

OH

OOH

OHOH

O

OH

23

Figure 2.5: Flavonoids.

Flavonoids are not recovered using the isolation procedure described above.

However, they are unlikely to play a detectable role in the plant’s effects. The

binding affinity of 26a is more than three orders of magnitude below that of

1a.23 Moreover, Valdés isolated 1a in two orders of magnitude greater yield

than 26b.

In other plants, however, where flavonoids or other strongly adsorbed com-

pounds are responsible for activity, the above procedure would clearly be in-

adequate. Nonetheless, this obstacle is readily overcome. If bioassay-guided

fractionation showed loss of activity in the decolourised extract, further elu-

tion with stronger eluents as outlined in Section 2.1.3.3 could be used. The


active fraction would then be greatly simplified relative to the initial extract,

providing another dimension of separation, as with PCBs.

2.1.3.6 Other Decolourisation Adsorbents.

Although extremely cheap for laboratory use, use of activated carbon can be

expensive on an industrial scale. For this reason, cheaper alternative adsor-

bents have been investigated. These are generally waste products: agricultural

(leaves, straw, husks, pulp) or industrial (ash, slag).174 One adsorbent which

has been used extensively is acid-activated clay.174 Activated clays (or “ac-

tivated earths”) are widely used, for instance, to decolourise cooking oils.209

Although not as effective by weight as activated carbon,195 the lower cost

makes industrial use attractive.

The adsorbent “Tonsil” used by Ortega et al in the original isolation22 of

1a is a type of activated clay used for decolourisation.209, 210 Unfortunately,

this was not explained; the material was described (in a footnote) merely as

“bentonitic earth” composed of silica (72.5%), alumina and other minerals.22

The body of the paper and the experimental procedure make no mention of

pigments or colouration. Thus, readers may assume that Tonsil is simply

a silica substitute. It is perhaps for this reason that no subsequent worker

has tested this adsorbent. Nonetheless, Dr Ortega has confirmed that Tonsil

was selected in order to decolourise the crude extract.211 He reports that the

procedure was highly effective: the collected fractions were almost colourless.

Tonsil was used for other isolations,212 but proved reactive towards certain

functional groups, especially epoxides,213 and therefore unsuited to general

use. Indeed, this substance has since found wide synthetic use as an acid

catalyst.214 Activated carbon is milder and more generally applicable.


0 100 µm

Figure 2.6: SEM image of salvinorin A crystals (blade morphology).39

2.1.4 Separation of Terpenoids.

2.1.4.1 Crystallisation of 1a.

TLC indicated that the major component of the decolourised extract (Sec-

tion 2.1.3.4 on page 77) was 1a, which was obtained as fine colourless needles

by crystallisation from MeOH. 1H NMR showed other trace components � 1%

each. Another crystallisation of the mother liquor gave additional 1a. The re-

sulting mother liquor was no longer predominantly 1a by TLC. In other tests,

EtOH was also an effective recrystallisation solvent (Lee et al subsequently

used acetone).137

Serendipitously, trituration in Et2O also proved effective for isolation of 1a

from the decolourised extract. In early tests, a chromatographic fraction rich in

1a was dissolved in Et2O for transfer; an amorphous, flaky precipitate formed

instantly. The filtrate proved, on further chromatography, to be devoid of

1a. The precipitate consisted of 1a which, despite its inferior appearance, was

highly pure by 1H NMR. Thus, trituration in cold Et2O represents a promising

alternative to recrystallisation from boiling alcohols – easier, safer and (more

significantly) much faster. Moreover, the risk of decomposition in heated solu-

tions (Section 2.1.1 on page 67) is avoided. This procedure, which was unfortu-


nately given insufficient emphasis in our initial publication,215 clearly deserves

further exploration.

The fine needles of 1a were examined by scanning electron microscopy (SEM)

(Figure 2.6). Shorter prisms were also observed, as well as plates or flakes (Fig-

ure 2.7), and close examination of crystals showed small defective, semicrys-

talline particles adhering.


0 200 µm

0 200 µm

0 20µm

0 20µm

0 20 µm 0 20 µm

Figure 2.7: Other salvinorin A crystal morphologies (stereoview).39


2.1.4.2 Chromatography.

O

O

O

H HORO

O O

O

O

O

H HOR1

R2

O O

R2

O

H HR1

O OR3

R

AcH

1a1b

R1

AcAcHH

R2

OAcOHOAcH

1c1d1e1f

28a28b28c29a

R1

OH OH H H

R2

HOHOAcH

R3

HMeHH

H

O

OH

HOH

H 31

OH

30

OH

H

32

OH 33

Figure 2.8: Terpenoids isolated from S. divinorum.

The mother liquor from crystallisation of 1a was an unexpectedly complex

mixture. Although at the time only four compounds had been reported from

S. divinorum, TLC showed many more. The spots were poorly resolved, mak-

ing a precise count impossible. Valdés et al had reported27 that 1a and 1c

were resolved in EtOAc/hexanes, but not in MeOH/CHCl3; this information

proved invaluable. 2D TLC using these systems gave much greater resolution

than either system alone. Many compounds not resolved by one system were


10 20 30 40 50 60 70 8010

20

30

40

50

60

70

80

hRf (70% Et2O/petrol)

hRf (

10%

ace

ton

e/C

H2C

l 2)

28a

1d

28b

1b

1f

1e

1a

1c

28c

32

29a

30

31

33

Figure 2.9: TLC data of isolated compounds.

resolved by the other. These differences in selectivity proved to be general

for petrol-based versus chlorinated solvent-based systems. After experimen-

tation with various petrol-based systems, Et2O gave similar resolution of 1a

and 1c to EtOAc, but better separation from other compounds. Similarly,

in CH2Cl2-based systems, acetone resolved 1a and 1c from other compounds

better than MeOH. Also, these new systems spread the mixture over a wider

range, from near the baseline to near the solvent-front. These systems were

therefore adopted for general use.

Extensive column chromatography yielded six new diterpenoids in addition to

1a–1c: salvinorins D–F (1d–1f) and divinatorins A–C (28a–28c, Figure 2.8

on the previous page). Five known terpenoids were also isolated:

(–)-hardwickiic acid (29a), (E)-phytol (30), oleanolic acid (31), presqualene

alcohol (32) and peplusol (33).


Dried leaves (860 g)

Crude extract (30.5 g)

Powder, steep in acetone (× 3)

Decolourised extract (5.7 g)

Column on activated carbonacetone to petrol gradient

Recrystallise (MeOH, EtOH)1a (2.6 g)

Column on silica5-50% acetone/CH2Cl2

Series A656 mg

Series B150 mg

Series C359 mg

Series D77 mg

Column 50-80% Et2O/petrol

1c (219 mg) 29a (7 mg)

1a (2.9 g)

1b (13 mg)


28a (36 mg)

Columns

Triturate (Et2O)

1d (75 mg)

Combine


Fraction C155 mg

Fraction C2119 mg

Fraction C357 mg

28c (23 mg) 31 (3 mg)

Columns 28b (41 mg)


Columns

1d (114 mg)

Combine

1e (3 mg) 1f (1 mg)

HPLC60% EtOAc/petrol

Combine

Figure 2.10: Isolation of terpenoids from commercial S. divinorum.

Figure 2.9 on the facing page shows the TLC data for all compounds isolated

in this work. The most dramatic differences between the two systems are


Dried leaves (224 g)

Crude extract (7 g)

Powder, steep in acetone (× 3)

Vacuum filtration on activated carbon50-20% EtOAc/petrol

1a (126 mg)32 (23 mg) 30 (12 mg)33 (1 mg)

Series E97 mg

Series F279 mg

Columns on silica

Recrystallise (MeOH × 2)

Figure 2.11: Isolation of terpenoids from Australian S. divinorum.

emphasised by dashed lines. For instance, separation of 1c from 28b or of 1a

from 1f is not possible in Et2O/petrol, but trivial in acetone/CH2Cl2. The

converse is true for separating 1f from 28c or 1a from 30.

With these solvent systems in place, separation of the components of the

mother liquor was straightforward, albeit time-consuming. Repeated column

chromatography, alternating between Et2O/petrol and acetone/CH2Cl2, re-

solved most compounds. In deciding which fractions to pool, TLC analysis

in each of the main solvent systems proved invaluable. For particular separa-

tions, other solvent mixtures were sometimes used when TLC indicated better

resolution. The overall procedure, as applied to the extraction of commercial

dried S. divinorum leaves, is summarised in Figure 2.10 on the previous page.

For more detail, see Experimental Section 5.2.1 on page 185. Yields expressed

in mg/kg are given in Table 5.1 on page 187.

A similar procedure was used on a sample of Australian-grown S. divinorum

leaves (Figure 2.11), resulting in the isolation of several additional compounds.

Partial separation of these compounds was achieved in the initial filtration

through activated carbon, but the severe tailing exhibited by all compounds


prevented full separation. Since chromatography on silica was ultimately re-

quired, attempts to separate compounds on carbon are not recommended.

2.1.4.3 Crystallisation of new compounds.

As with 1a, trituration in Et2O proved effective for purification of 1d. At-

tempted dissolution of a fraction containing ≈ 33% 1d in boiling Et2O gave

white crystals of the pure compound. Also, as reported by Tidgewell et al,169

trituration in cold MeOH is effective for purification of 1b.

The ease of crystallising these compounds varies widely. Evaporation of a so-

lution of 1b gave crystals irrespective of the solvent. 1a and 1d gave crystals

from some solvents, but an amorphous resin from others (especially CH2Cl2and CHCl3). Although Valdés reported the crystallisation of 1c,27 attempts

to replicate this were unsuccessful. Attempts to recrystallise the other new

compounds, under a variety of conditions, were also unsuccessful. On one oc-

casion, 28b gave crystals from cold CH2Cl2 on addition of petrol, but repeated

attempts to replicate this were unsuccessful.

2.1.4.4 Losses during isolation.

The recoveries of these compounds do not accurately reflect their actual pro-

portions in the crude extract. The main objective in this work was to obtain

the maximum number of pure compounds; yield was a much lower priority

except in the case of 1a. Traces of one compound were often discarded during

the isolation of another. In the most extreme case, all of the 29a isolated was

derived from a small portion (13%) of the crude extract. The actual content

of this compound in the crude extract, therefore, was much higher than the

reported yield.

There were also losses due to other causes. In one case (separation of 1e

and 1f), despite good separation by TLC (Figure 2.9 on page 86), repeated

column chromatography gave incomplete resolution. Ultimately, HPLC gave


full resolution, but unfortunately the recovery after this lengthy process was

very poor. From a 34 mg fraction containing only 1e and 1f by TLC and 1H

NMR, the final combined recovery of these two compounds was only 4 mg.

Similarly, yield of 1b (15 mg/kg) was very low compared to earlier work (75

mg/kg).23 This was probably largely due to losses in chromatography. During

subsequent synthetic work, it became apparent that column chromatography

of 1b in petrol-based systems gave very poor recoveries. Stripping the column

in acetone/CH2Cl2 gave an improvement, but full recovery was only achieved

using MeOH/CH2Cl2. Additionally, recrystallisation of 1b-rich fractions from

MeOH gave poor recoveries, and TLC showed extensive decomposition. As

noted in Section 2.1.1 on page 67, this decomposition also occurred with 1a.

2.2 Structure Elucidation.

2.2.1 Revised NMR Assignments for Salvinorin A (1a).

The published NMR assignments23 of 1a (based on decoupling experiments)

were verified on the basis of 800 MHz 1H NMR (Figure 2.12 on the next page),

along with HSQC, HMBC, DEPT and nOe experiments. These new data

supported the original 1H and 13C assignments in all cases except H-6 and -7.

The HSQC spectrum showed cross peaks from C-6 (δ 38.1 ppm) to multiplets at

δ 1.57 & 1.78 ppm, and from C-7 (δ 18.1 ppm) to δ 1.63 & 2.15 ppm (Figure 2.13

on the facing page). The 800 MHz 1H NMR spectrum also gave well-resolved

first-order multiplets for H-6β, 7α and 11β, which overlap to form a single

multiplet (δ 1.54 – 1.68 ppm) at 400 MHz. Resolution enhancement revealed

all coupling constants in these multiplets, allowing unambiguous assignment

of all peaks (Figure 2.14 on page 92).

2.2. STRUCTURE ELUCIDATION. 91

2.742.76 ppm

1.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm

1.113

1.447

1.574

1.642

1.792

2.074

2.163

2.178

2.304

2.504

2.745

3.724

5.138

5.521

6.369

7.384

7.402

2.70

2.96

1.94

1.28

1.51

1.54

5.30

2.27

0.88

1.02

3.25

1.00

0.85

0.82

1.59

1.63

7.397.40 ppm

6.366.37

5.515.525.535.135.145.155.16 ppm 2.50 2.492.512.52

2.142.162.18

2.06

1.781.80 ppm

1.561.581.601.621.641.66 ppm

2.292.312.332.351a

O

O

O

H HO

O

O

O O

Figure 2.12: 1H NMR spectrum of 1a (800 MHz, CDCl3).

1.21.41.61.82.02.22.42.62.83.03.23.43.6 ppm

20

30

40

50

60

C-6

C-7

5.25.45.65.86.06.26.46.66.87.07.27.4

80

90

100

110

120

130

140

Figure 2.13: HSQC spectrum of 1a (800 MHz, CDCl3).


171.5

171.1 171.1

51.9

35.4

38.1

42.0

171.5

169.9

30.9

20.5

64.075.0202.0

51.9

53.5

16.4

15.2

18.1

43.3

72.0

139.4

143.7

108.3

125.2

51.3

35.4

38.1

42.0

169.9

30.9

20.5

64.075.0202.0

53.5

16.4

15.2

18.1

43.3

72.0

139.4

143.7

108.3

125.2

51.3

5.14 ddt

3.72 s

2.74 dd1.58 td

1.64 tdd

1.79 dt

2.07 dd

2.17-2.14 m

2.16 s

2.31-2.28 m

2.18 br s

1.45 s

1.11 s

2.50 dd

1.57 ddd

5.52 ddd

6.37 dd

7.38 t

7.41 dt

5.14 ddt

3.72 s

2.74 dd1.58 td

1.64 tdd

1.79 dt

2.07 dd

2.17-2.14 m

2.16 s

2.31-2.28 m

2.18 br s

1.45 s

1.11 s

2.50 dd

1.57 ddd

5.52 ddd

6.37 dd

7.38 t

7.41 dt

δC

δH (REVISED ASSIGNMENT)δH

NOESY

δC

δH (REVISED ASSIGNMENT)δH

NOESY

Figure 2.14: Revised NMR assignments for 1a (stereoview).

2.2.2 Revised NMR Assignments for Salvinorin C (1c).

The published structure and NMR assignments27 of 1c were verified on the

basis of 1H NMR (Figure 2.15 on the facing page), decoupling, HMQC, HMBC

and DEPT experiments. These data were consistent with the published struc-

ture, but the assignments of H-19, H-20, C-17 and the acetates were revised as

shown in Figure 2.16 on the next page. Contrary to the original assignments,

the HMQC spectrum showed strong cross peaks between C-19 (21.8 ppm) and

H-19 (1.71 s), as well as C-20 (15.7 ppm) and H-20 (1.21 s) - see Figure 2.17a.

This was confirmed by HMBC cross peaks from H-19 to C-4 (142.6 ppm), and

H-20 to C-11 (44.2 ppm) - Figure 2.17b.

HMBC data also allowed the assignment of the acetate signals. The C-2-

O-acetate carbonyl (169.8 ppm), originally assigned to C-17,27 showed cross

peaks to H-2 (5.54 ppm) and 2.04 ppm; its C-1-O- counterpart (170.5 ppm)

showed cross peaks to H-1 (5.75 ppm) and 2.12 ppm - Figure 2.17c,d.

2.2.3 Other Known Diterpenoids.

In addition to 1a-1c, five known compounds not previously reported from S.

divinorum were isolated. The data and sources used to identify them are given


1c

2

4

117

O11

O

O

H HO

O

19

20

O

O

O O

4.7

4.65.6

5.6 5.55.8 5.74.26.2

2.1 1.13.17.18.12.2 1.2

ppm234567

0.670.70 0.88

0.781.00

1.973.00

1.031.00

5.062.95

1.144.43

1.17 4.54

062

.7

047.

3

22

1.2

730.

2

617.

1

612

.1

Figure 2.15: 1H NMR spectrum of 1c (400 MHz, CDCl3).

37.2

38.1

165.7

51.8

132.4

64.1 69.2

51.8

170.521.1

21.8

37.0

18.4

44.2

71.8

139.4

143.9

108.4

171.4

125.4

52.6

15.7

142.6

20.7169.8

37.2

38.1

165.7

51.8

132.4

64.1 69.2

51.8

170.521.1

21.8

37.0

18.4

44.2

71.8

139.4

143.9

108.4

171.4

125.4

52.6

15.7

142.6

20.7169.8

5.75 br d

2.12 s

5.54 dd

6.46 dd

3.74 s

1.71 s

1.21 s

2.59 dt

1.23 td

1.85-1.75 m

2.17-2.10 m

2.13 dd1.49 br s

2.48 dd

1.68 dd

5.53 dd

6.41 dd

7.42 t

7.45 m

2.04 s

5.75 br d

2.12 s

5.54 dd

6.46 dd

3.74 s

1.71 s

1.21 s

2.59 dt

1.23 td

1.85-1.75 m

2.17-2.10 m

2.13 dd1.49 br s

2.48 dd

1.68 dd

5.53 dd

6.41 dd

7.42 t

7.45 m

2.04 s

δC δH

HMBC

δC δH

HMBC

δC/H (REVISED ASSIGNMENT) δC/H (REVISED ASSIGNMENT)

Figure 2.16: Revised NMR assignments for 1c (stereoview).

in Table 2.2 on the following page.


151617181920212223

δH(ppm)

1.3

1.2

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

040506070809001011021031041

52.1

02.1

51.1

03.1

53.1

04.1

54.1

05.1

55.1

06.1

56.1

07.1

57.1

5.8610.9615.9610.0715.0710.1715.1710.2715.271

55.5

05.5

06.5

56.5

07.5

57.5

08.5

δC(ppm)

a) HMQC b) HMBC

c) HMBCd) HMBC

168.5169.5170.5171.5

2.02

2.00

1.98

2.04

2.06

2.08

2.10

2.12

2.14

2.16

2.18

2.20

Figure 2.17: HMQC and HMBC spectra of 1c (400 MHz, CDCl3).

Compound 1H NMR 13C NMR FTIR [α]D HRESIMS EIMS TLC29a sa, d216, 217 cs

29b* sa, d218 d218 d218 d218, 217 d217 cs30 sp,219 d220 d219, 220 d219, 220

31 d221 d221 c32 d,222 sp223 d223 d222 d223

33 d224 d224 d224 d224

* 29a was methylated with CH2N2 for full characterisation.c consistent with calculated value.cs cospotted with authentic material in petrol- and CH2Cl2-based systems.d consistent with published data.sa superimposable with spectrum of authentic material.sp superimposable with published spectrum.

Table 2.2: Data and sources used to identify known compounds.

2.2.3.1 (–)-Hardwickiic acid (29a).

(–)-Hardwickiic acid (29a) was first isolated from Hardwickia pinnata by Misra

et al,225 and the structure (including absolute stereochemistry) elucidated by


O

H H

O OR

R

HMe

29a29b

Figure 2.18: Single-crystal X-ray structure of 29a (stereoview).

spectroscopic methods and extensive degradation studies.226, 227 Recently, X-

ray crystallography has confirmed this structure (Figure 2.18).228 29a has since

been isolated from many other plant species, including Salvia regla.229 29a has

been reported to exhibit insecticidal230 and antimicrobial217 activity. Interest-

ingly, its enantiomer (+)-hardwickiic acid (ent-29a) has been isolated from

Copaifera officinalis231 and related species.232 For definitive identification, au-

thentic ent-29a was isolated as the methyl ester ent-29b from commercial

Copaiba balsam.218 The material from S. divinorum, after methylation to give

29b, cospotted by TLC and gave a superimposable 1H NMR spectrum, but

the opposite sign of optical rotation.


OH30

Figure 2.19: (E)-Phytol.

2.2.3.2 (E)-Phytol (30).

(E)-Phytol (30) is ubiquitous in photosynthetic organisms, as the sidechain

of the chlorophylls (eg. 21 on page 71). 30 displays anticancer activity,233

and may play a role in the health benefits of green-yellow vegetables.234 30

also exhibits antiplasmodial,235 antimycobacterial236, 237 and antiteratogenic238

activities.

2.2.4 Known Triterpenoids.

2.2.4.1 Oleanolic Acid (31).

H

O

OH

HOH

H 31

Figure 2.20: Oleanolic acid.

Oleanolic acid (31) occurs commonly in plants, including many Salvia species.239

It exhibits an extremely wide range of potential therapeutic activities, which

have been reviewed elsewhere:240, 241, 239 anti-inflammatory, hepatoprotective,

gastroprotective, cardiovascular, hypolipidemic and immunoregulatory effects.

Of more significance is anti-tumour activity, which appears to occur via several

independent mechanisms (inhibition of tumourigenesis, inhibition of tumour

promotion, and induction of tumour cell differentiation).240 Similarly, 31 is

active against HIV-1 by two mechanisms: inhibition of HIV protease242 and

reverse transcriptase243 enzymes. The compound also exhibits activity against


many other pathogens: herpes simplex virus,244 a range of bacteria245, 246 (in-

cluding mycobacteria)247 and Leishmania spp,248 as well as being an insect

antifeedant.249

2.2.4.2 Presqualene Alcohol (32) and Peplusol (33).

OH

H

32

OH 33

Figure 2.21: Presqualene alcohol and peplusol.

The diphosphate of presqualene alcohol (32) is a precursor in the biosynthesis

of sterols (including cholesterol), and has therefore been the subject of intense

synthetic223 and biosynthetic250 interest. The isolation of 32 from a plant is

unusual.

The closely related compound peplusol (33) was isolated from Euphorbia pe-

plus by Giner et al.224 In a paper submitted a year later, Connolly et al

reported the same compound from E. lateriflora, naming it anhydrobisfar-

nesol.251 The earlier paper by Giner et al was mentioned, but only in a foot-

note; priority was claimed252 based on a preliminary communication.253 That

communication, however, contained no experimental or characterisation data,

and the absolute configuration was not determined even in the full paper.

Additionally, the ambiguous semisystematic name anhydrobisfarnesol leaves

regio- and stereochemistry unspecified. Thus, Giner et al clearly have priority,

and their name is used here. Connolly and co-workers confirmed the proposed

structure by synthesis, obtaining the racemic compound in 6% yield over six

steps.251 However, they failed to cite a clearly superior synthesis (50% over

two steps)254 published fifteen years earlier. Connolly has proposed 33 as a

biosynthetic precursor to 32 itself.252


It is interesting to note that 33 and 32 are approximately equal in size (22-

carbon chain) to the carotenoid 22 (p.71, 24 carbons), but were easily eluted

from activated carbon in the above chromatography procedure. Since, un-

like 22, these compounds are not conjugated, this again confirms the strong

influence of planarity on adsorption.

2.2.5 Salvinorins D-F (1d-1f).

2.2.5.1 Salvinorins D and E (1d and 1e).

Inspection of the 1H NMR spectra of 1d-1f suggested that they were deriva-

tives of 1c. Compared to 1c, the 1H NMR spectra of 1d (Figure 2.22) and 1e

(Figure 2.26 on page 102) showed only one acetyl peak, and gained one new

peak (δ 2.01 in 1d; δ 1.94 in 1e) which exchanged with D2O. The presence of

a hydroxyl group was confirmed by IR spectroscopy (3475 cm−1 in 1d; 3510

cm−1 in 1e). HRESIMS established the molecular formula of both compounds

as C23H28O8, consistent with the loss of one acetyl group. These data indicated

that the compounds were monoacetates of 1c.

Relative to 1c, the H-2 signal of 1d was shifted upfield from δ 5.55 to δ 4.44,

and coupled to the hydroxylic proton (2.01 ppm) as well as to H-1 (5.70 ppm)

and H-3 (6.54 ppm). The HMBC spectrum (Figure 2.25 on page 101) showed

correlations between H-2 and C-4 (141.2 ppm), as well as H-1 and C-5 (37.6

ppm). These quaternary carbons, not apparent in the HMQC (Figure 2.24

on page 100) and DEPT spectra, were in turn located by HMBC correlations.

This established the structure of 1d as 2-deacetylsalvinorin C.

Relative to 1c, the H-1 signal of 1e was shifted from δ 5.76 to δ 4.46. Irra-

diation sharpened the H-10 singlet. The HMBC spectrum (Figure 2.29) again

showed a correlation between H-2 (5.40 ppm) and C-4 (143.4 ppm), not ap-

parent in the HMQC spectrum (Figure 2.28). Thus 1e is 1-deacetylsalvinorin

C, as shown. The quaternary carbon at 169.8 ppm was incorrectly assigned to

C-17 in our original publication;215 this has been reassigned on the basis of its


1d

22

3344

55

11

O

O

O

H HOHO

O

O O

ppm234567

0.830.85 0.92

0.870.98

0.991.00

3.002.00

4.980.97

1.054.070.99

4.43

062

.7

837.

3

051.

2

686

.1

52

2.1

1.21.3 1.1

1.61.82.02.2 2.2

2.6 2.5

5.55.65.7

4.5 4.4

6.46.56.6

7.47.5

Figure 2.22: 1H NMR spectrum of 1d (400 MHz, CDCl3).

HMBC crosspeak with the acetate protons (see Figure 2.27 on page 102).


5.70 dt

2.15 s

4.44 ddd

2.01 br d

6.54 dd

3.74 s

1.69 s

1.22 s

2.56 dt

1.20 td

1.78 dtd

2.17-2.09 m

2.13 dd1.42 br s

2.54 dd

1.64 ddd

5.53 dd

6.40 dd

7.42 t

7.44 s

5.70 dt

2.15 s

4.44 ddd

2.01 br d

6.54 dd

3.74 s

1.69 s

1.22 s

2.56 dt

1.20 td

1.78 dtd

2.17-2.09 m

2.13 dd1.42 br s

2.54 dd

1.64 ddd

5.53 dd

6.40 dd

7.42 t

7.44 s

δH δH

δC

HMBC

J (Hz)

2.4 Hz

1.3 Hz

δH δH

δC

HMBC

J (Hz)

2.4 Hz

1.3 Hz

37.0

37.6

166.2

51.7

135.7

68.6 66.5

52.5

171.621.2

21.6

37.0

18.3

43.9

71.6

139.4

143.8

108.4

171.6

125.4

51.8

15.6

141.2

37.0

141.2

37.6

166.2

51.7

135.7

68.6 66.5

52.5

171.621.2

21.6

37.0

18.3

43.9

71.6

139.4

143.8

108.4

171.6

125.4

51.8

15.6

Figure 2.23: NMR assignments and key 2D correlations for 1d (stereoview).

708090100110120130140

5.0

4.5

5.5

6.0

6.5

7.0

7.5

1520253035404550

1.6

1.4

1.2

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

δC(ppm) δC(ppm)

δH(ppm)

δH(ppm)

Figure 2.24: HMQC spectrum of 1d (400 MHz, CDCl3).


37 363839404142434445

5.50

5.45

5.55

5.60

5.65

5.70

5.75

20406080100120140160

2.5

2.0

1.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

134135136137138139140141142143

4.36

4.40

4.44

4.48

4.52

4.56

δH

(ppm)

δC(ppm)

Doublets resulting from direct coupling (1JCH) have been removed digitally.

Figure 2.25: HMBC spectrum of 1d.


1e

O

O

O

H HOH

O

O

O O

ppm2.02.53.03.54.04.55.05.56.06.57.0

0.840.91 0.93

0.921.03

1.051.06

3.022.08

5.190.870.97

3.121.18

3.16

1.20

1.53

062

.7

92

7.3

071

.2

355.

1

62

7.1

76

4.1

1.3 1.2 1.11.61.71.81.9

2.12.2

2.42.52.6

7.4

6.4

5.45.55.6

4.5 4.4

Figure 2.26: 1H NMR spectrum of 1e (400 MHz, CDCl3).

37.5

37.8

166.0

51.8

131.5

72.3 64.3

54.0

21.9

37.0

18.4

44.4

71.7

139.3

143.9

108.4

171.8

125.8

51.7

16.2

143.4

21.0169.8

37.5

37.8

166.0

51.8

131.5

72.3 64.3

54.0

21.9

37.0

18.4

44.4

71.7

139.3

143.9

108.4

171.8

125.8

51.7

16.2

143.4

21.0169.8

4.46 ddd5.40 dd

6.43 dd

3.73 s

1.72 s

1.47 s

2.52 ddd

1.19 td

1.84 dtd

2.18-2.07 m

2.18-2.07 m1.30 br s

2.46 dd

1.62 dd

5.60 ddd

6.41 dd

7.42 t

7.44 br s

2.17 s

1.94 dd

4.46 ddd5.40 dd

6.43 dd

3.73 s

1.72 s

1.47 s

2.52 ddd

1.19 td

1.84 dtd

2.18-2.07 m

2.18-2.07 m1.30 br s

2.46 dd

1.62 dd

5.60 ddd

6.41 dd

7.42 t

7.44 br s

2.17 s

1.94 dd

2.4 Hz1.6 Hz

2.4 Hz1.6 Hz

δH δH

δC

HMBC

J (Hz)

δC (REVISED ASSIGNMENT)

δH δH

δC

HMBC

J (Hz)

δC (REVISED ASSIGNMENT)

Figure 2.27: NMR assignments and key 2D correlations for 1e (stereoview).


708090100110120130140

5.0

4.5

5.5

6.0

6.5

7.0

7.5

20 1525303540455055

1.6

1.5

1.4

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

δH

(ppm) δH

(ppm)

δC(ppm) δ

C(ppm)

Figure 2.28: HMQC spectrum of 1e.

110115125 120135 130145 140150160 155165

5.6

5.4

5.8

6.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

130 125135140145150155160165170

2.0

1.8

1.6

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

20 1525303540455055606570

1.5

1.4

1.3

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

δC(ppm)

δH

(ppm)


Figure 2.29: HMBC spectrum of 1e.


2.2.5.2 Interconversion of 1c-1e via Diol 1h.

a

O

O

O

H HOR1

R2O

O O

a

a

b

R2 R1

1c Ac Ac

1d H Ac

1e Ac H

1h H H

a) Ac2O, pyridine, DMAP b) Na2CO3, CH2Cl2/MeOH (1:1), rt.

Scheme 2.1: Interconversion of 1c-1h.

ppm234567

0.780.78 0.83

0.820.95

1.011.03

3.002.06

1.952.14

1.153.16

3.18 0.991.31

062

.7

13

7.3

20

7.1

306.

1

67

4.1

1.101.151.20

1.551.601.651.701.751.801.851.90

4.24.34.4

(+D2O)

(+D2O)

(+D2O)

2.052.102.152.202.252.302.352.402.452.50

5.7 5.6

6.46.5

7.47.5

Figure 2.30: 1H NMR spectrum of 1h (400 MHz, CDCl3).

To confirm the structures of 1c-1e, diol 1h was prepared by deacetylation of

1c. Tidgewell et al’s standard conditions for deacetylation of 1a (Na2CO3 in

minimal MeOH)169 caused extensive epimerisation (see Section 3.2.4). How-

ever, epimerisation could be suppressed by addition of CH2Cl2, affording 1h


2.40 -2.30 m

2.40 -2.30 m

37.5

37.5

166.6

51.7

135.3

69.6 65.6

54.1

22.1

37.0

18.4

44.4

71.8

139.3

143.9

108.4

172.0

125.8

51.8

16.3

142.2

37.5

37.5

166.6

51.7

135.3

69.6 65.6

54.1

22.1

37.0

18.4

44.4

71.8

139.3

143.9

108.4

172.0

125.8

51.8

16.3

142.2

4.32 br d4.28 dd

6.48 dd

3.73 s

1.70 s

1.47 s

2.50-2.45 m

1.16 tdd

1.82 dtd

2.09 dq

2.14-2.10 m1.22 d

2.49 dd

1.60 ddd

5.60 dd

6.40 dd

7.42 t

7.43 br s

2.40-2.30 m4.32 br d

4.28 dd

6.48 dd

3.73 s

1.70 s

1.47 s

2.50-2.45 m

1.16 tdd

1.82 dtd

2.09 dq

2.14-2.10 m1.22 d

2.49 dd

1.60 ddd

5.60 dd

6.40 dd

7.42 t

7.43 br s

2.40-2.30 m

δH δH

δC

HMBCNOESY

J (Hz)

2.4 Hz2.4 Hz

δH δH

δC

HMBCNOESY

J (Hz)

Figure 2.31: NMR assignments and key 2D correlations for 1h (stereoview).

30 10507090110130

2.5

2.0

1.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

140145150155160165170

2.0

1.8

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

a) HMQC b) HMBC

δC(ppm)

δH(ppm)

Figure 2.32: HMQC and HMBC spectra of 1h.

1.11.21.31.41.51.61.71.8

2.1

2.2

2.3

2.4

2.5

1.21.31.41.51.61.71.81.92.02.12.22.32.42.5 ppm

4.34.44.54.64.74.84.95.05.15.25.35.45.55.6

nOe effectdirect coupling

Figure 2.33: NOESY spectrum of 1h.

from 1c or 1d in up to 86% yield. K2CO3 was equally effective.

Analysis of the NMR data (Figures 2.30 on the facing page and 2.32) was

straightforward. Carbons C-17 and -18 were distinguished on the basis of


HMBC correlations (Figure 2.32), and H-1 and 2 on the basis of coupling

constants to H-3 (Figure 2.31 on the preceding page). These couplings were

only resolved after D2O exchange. The relative stereochemistry was confirmed

using NOESY data (Figure 2.33 on the previous page). H-1 showed cross

peaks to H-2, -10 and -11α, while H-8 correlated to H-10 and -11β, and H-12

correlated to H-20 (Figure 2.31 on the preceding page). This confirms the con-

figurations shown for 1h, and therefore of 1c-1e, through the interconversions

summarised in Scheme 2.1 on page 104.

The proposed structure of 1d was verified by acetylation, which proceeded

smoothly to give 1c, identical in all respects with the isolated material - TLC

cospot, 1H and 13C NMR, IR, and optical rotation (Scheme 2.1 on page 104).

Insufficient 1e was available for acetylation. In our initial publication,215 we

assumed that 1e would be too hindered for direct acetylation under standard

conditions. This proved incorrect; the 1-hydroxy group of 1h is readily acety-

lated. However, the crude product after three hours consisted of 1d and 1e in

approximately 1:2 ratio, with only traces of 1c; thus, the 1-position of 1h is

(as expected) less reactive than the 2-position, and each of the monoacetates

is less reactive than the diol. At 45 ◦C, the starting material was consumed

in approximately 1 hour, giving a mixture of 1c and 1e, confirming that 1e is

the less reactive of the monoacetates. It also appears to be less stable; after

acetylation under these standard conditions, it was contaminated by an insep-

arable impurity, apparently an isomer other than the 8-epimer. This did not

occur with 1d. 1e was also much more prone to decomposition in storage than

1d.

During the isolation of 1c, Valdés detected traces of what appeared to be 1d,

1e, and 1h in S. divinorum;255 while we subsequently confirmed the presence

of 1d and 1e,215 no-one has yet isolated 1h. It may be that the compound

occurs in the plant in very low levels; this would be expected if its formation

were the rate-limiting step on the path to 1c–1e. Alternatively, current iso-

lation procedures may give poor recoveries. Indeed, like 1b, 1h precipitates

when loaded on silica gel in most solvent systems. Stripping the column with


MeOH/CH2Cl2 was required to achieve satisfactory recovery.

A sample of 1h was supplied to Claudio Medana for LCMS comparison with

the crude extract. This proved difficult, since 1b and 1h were not resolved by

reverse-phase HPLC, either C-18 functionalised silica or polymeric C-18. The

compounds were ultimately resolved on a porous graphitic carbon column,

eluting with a MeOH to CH2Cl2 gradient. Under these conditions, a crude

CH2Cl2 extract showed no detectable 1h.256

2.2.5.3 Salvinorin F (1f).

The molecular formula of compound 1f, C21H26O6, was established by HRES-

IMS. In contrast to 1d and 1e, 1H NMR (Figure 2.34 on the next page) showed

no acetyl peak, only one oxymethine signal, and a diastereotopic methylene

(δ 2.35 and 2.60) coupling to H-1 and H-3 (Figure 2.35 on page 109). This

implied the 2-deoxy structure shown. Protons H-2α and H-2β were distin-

guished by their coupling constants: molecular modelling257 predicted J 1,2β

= 5.4 Hz (observed: 5.5 Hz) and J 1,2α = 1.5 Hz (observed: 1.1 Hz). In our

initial publication,215 this latter coupling was not observed, but subsequent

resolution enhancement allowed direct measurement. H-2β also showed the

expected HMBC cross peaks to H-4 and -10 (Figure 2.37 on page 109).


1f

22

3344

101011

O

O

O

H HOH

O O

ppm234567

0.820.85 0.97

0.901.01

1.063.02 1.01

2.08

1.05

2.161.023.07

1.11

3.191.04

1.31

1.28

06

2.7

027

.3

655.

1

707

.1

674

.1

1.3 1.2 1.1

1.61.8 1.72.2 2.1

2.4 2.32.6 2.5

4.5 4.44.65.55.65.7

6.46.56.66.7

7.47.5

Figure 2.34: 1H NMR spectrum of 1f (400 MHz, CDCl3).


5.5 Hz

1.1 Hz

5.5 Hz

1.1 Hz

37.7

36.6

166.9

51.5

133.4

38.0 63.9

54.8

21.6

37.3

18.6

44.4

71.7

139.3

143.8

108.4

172.1

125.9

52.2

16.4

140.6

37.7

36.6

166.9

51.5

133.4

38.0 63.9

54.8

21.6

37.3

18.6

44.4

71.7

139.3

143.8

108.4

172.1

125.9

52.2

16.4

140.6

4.51 br dd2.60 ddd

6.67 ddd

3.72 s

1.71 s

1.48 s

2.53 dt

1.18 tdd

1.82 dtd

2.17-2.08 m

2.17-2.08 m1.25 br s

2.46 dd

1.62 ddd

5.60 ddd

6.41 dd

7.42 t

7.44 br s

1.29 dd

2.35 ddt

4.51 br dd2.60 ddd

6.67 ddd

3.72 s

1.71 s

1.48 s

2.53 dt

1.18 tdd

1.82 dtd

2.17-2.08 m

2.17-2.08 m1.25 br s

2.46 dd

1.62 ddd

5.60 ddd

6.41 dd

7.42 t

7.44 br s

1.29 dd

2.35 ddt

δH δH

δC

HMBC

J (Hz)

δH δH

δC

HMBC

J (Hz)

Figure 2.35: NMR assignments and key 2D correlations for 1f (stereoview).

708090100110120130140

5.0

4.5

5.5

6.0

6.5

7.0

7.5

20 1525303540455055

1.6

1.4

1.2

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

δH

(ppm)

δC(ppm)

Figure 2.36: HMQC spectrum of 1f.

110120130140150160

2.5

2.0

1.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

20 1525303540455055606570

1.4

1.3

1.2

1.1

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

δH

(ppm)

δC(ppm)


Figure 2.37: HMBC spectrum of 1f.


2.2.6 Divinatorins A-C (28a-28c).

2.2.6.1 Structure Elucidation.

28a28b28b29a

R1

OH OH H H

R2

O

H HR1

18

R2

HOHOAcH

R3

HMeHHO OR3

Figure 2.38: Divinatorins A–C (28a-28c) and hardwickiic acid (29a).

28a

O

H H

O OH

OH

3

3.2

1.1

2.1

2.1

4.2

1.1

1.1

3.1

1

1

0.92

0.99

0.87

0.88

1234567 ppm

7.27.3

6.886.906.92

6.256.26

4.474.484.494.504.51

2.32.42.52.61.81.92.02.1

1.661.681.701.72

1.21.31.41.51.6

Figure 2.39: 1H NMR spectrum of 28a (400 MHz, CDCl3).


171.8

140.8

142.8

49.037.1

39.7

39.1

38.1

38.6

37.427.4

21.4

19.8

18.2

64.715.7

138.4

125.2

136.2

110.9

171.8

140.8

142.8

49.037.1

39.7

39.1

38.1

38.6

37.427.4

21.4

19.8

18.2

64.715.7

138.4

125.2

136.2

110.9

1.67 ddd2.05 ddd

4.49 br d

2.56 ddd

6.90 ddd

1.64 s

1.15 s

1.47-1.42 m1.23-1.17 m

1.60-1.54 m

1.60-1.54 m

2.43-2.33 dt

1.45 br s

6.25 dt

7.36 t

7.20 td

0.84 d2.43-2.33 m

2.56 ddd

2.43-2.33 m

2.34 td

1.85 ddd1.67 ddd

2.05 ddd 2.34 td

1.85 ddd4.49 br d

6.90 ddd

1.64 s

1.15 s

1.47-1.42 m1.23-1.17 m

1.60-1.54 m

1.60-1.54 m

2.43-2.33 dt

1.45 br s

6.25 dt

7.36 t

7.20 td

0.84 d

4.9 Hz

2.7 Hz4.8 Hz

δH δH

δC

HMBCNOESY

J (Hz)

4.9 Hz

2.7 Hz4.8 Hz

δH δH

δC

HMBCNOESY

J (Hz)

Figure 2.40: NMR assignments and key 2D correlations for 28a (stereoview).

2030405060708090100110120130140 ppm

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Figure 2.41: HMBC spectrum of 28a.

The structures of 28a-28c were elucidated mainly by NMR spectroscopy (1H,13C, DEPT, HMQC, HMBC, COSY, and NOESY). The 1H NMR spectra

suggested that they were derivatives of hardwickiic acid (29a). The molecular

formula of 28a (C20H28O4 by HRESIMS) differed from that of 29a by one

oxygen atom, suggesting the presence of a hydroxy group, which was confirmed


0.81.01.21.41.61.82.02.22.4 ppm

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4


1.11.21.31.41.51.61.71.81.92.02.12.22.32.42.52.6 ppm

4.47

4.48

4.49

4.50

4.51

4.52

Figure 2.42: NOESY spectrum of 28a.

by a strong IR absorption at 3392 cm−1. This was located at C-1 based on 1H

NMR: the oxymethine at δ 4.49 showed couplings to H-2, -3 & -10 (COSY),

and C-3, -9 and -10 (HMBC) - see Figure 2.41 on the preceding page. The

1α configuration was confirmed by an H-1 to H-11 crosspeak in the NOESY

spectrum (Figure 2.42). NOESY cross peaks also permitted stereospecific

assignment of the rotatable H-11 and -12 methylenes, as shown in Figure 2.40

on the preceding page. In the favoured conformation thus established, C-12

is approximately anti-periplanar to the C-20 methyl group, as found in the

crystal structure of 29a228 (Figure 2.18 on page 95). Along with the cross

peaks from H-20 to H-17 and -19, these NOESY data confirm the relative

configuration of all stereogenic centres.


4.1

0.92

4.6

2.9

3.1

1

3

0.93

0.93

3

1

0.89

0.81

0.92

0.76

0.771234567 ppm

7.27.3

6.646.666.68

6.246.26 4.45 3.853.4

2.32.42.5

1.81.92.02.1

1.551.60

28b

OH

O

H HOH

O O

Figure 2.43: 1H NMR spectrum of 28b (400 MHz, CDCl3).

167.3

141.4

142.8

51.3

44.848.7

39.1

38.8

38.0

38.0

37.121.9

21.4

20.9

18.2

64.3 63.9

138.4

124.9

133.2

110.8

167.3

141.4

142.8

51.3

44.848.7

39.1

38.8

38.0

38.0

37.121.9

21.4

20.9

18.2

64.3 63.9

138.4

124.9

133.2

110.8

3.84 dd4.46 dq4.46 dq

6.65 ddd

2.53 ddd

3.71 s

1.66 s

1.18 s

1.85 dq1.19-1.13 m

1.64-1.49 m

1.64-1.49 m

2.36 dt

1.49 br s

1.49 br s

1.45 br s

6.25 dd

7.35 t

7.20 dd

3.38 dd

2.42 dddd

1.90 ddd

2.31 ddt

1.77 ddd

3.84 dd

1.49 br s

3.38 dd

2.42 dddd

1.77 ddd2.08 dddd

6.65 ddd

2.53 ddd

3.71 s

1.66 s

1.18 s

1.85 dq1.19-1.13 m

1.64-1.49 m

1.64-1.49 m

2.36 dt

1.49 br s

1.45 br s

6.25 dd

7.35 t

7.20 dd

1.90 ddd

2.31 ddt

2.08 dddd

5.1 Hz

2.8 Hz4.9 Hz

δH δH

δC

HMBCNOESY

J (Hz)

5.1 Hz

2.8 Hz4.9 Hz

δH δH

δC

HMBCNOESY

J (Hz)

Figure 2.44: NMR assignments and key 2D correlations for 28b (stereoview).

The 1H NMR spectrum of 28b suggested a methyl ester (δ 3.71) with two hy-

droxy groups (δ 1.49, 2H, D2O-exchangeable). IR showed a strong absorption

at 3434 cm−1. The molecular formula, C21H30O5 (HRESIMS), was consistent

with this. One of the hydroxy groups was again located at C-1, showing the

same couplings as in 28a. The second was located at C-17, based on the cou-


2030405060708090100110120130 ppm

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

Figure 2.45: HMBC spectrum of 28b.

1.51.61.71.81.92.02.12.22.32.4

1.2

1.3

1.4

1.5

1.6

1.7


1.21.31.41.51.61.71.81.92.02.12.22.32.42.52.6 ppm

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

Figure 2.46: NOESY spectrum of 28b.

plings of the oxymethylene signals (δ 3.38 and 3.84) to H-8 (COSY) and to

C-7, -8 and -9 (HMBC) — see Figure 2.45. The NOESY spectrum showed H-1

to H-11 and H-17 to H-20 cross peaks, confirming the configuration at these

centres (Figure 2.46). Again, NOESY data permitted full assignment of the

diastereotopic H-11, -12 and -17 methylene protons.

The 1H NMR spectrum of 28c showed an acetyl methyl signal (δ 2.03). Com-


28c

O

O

H H

O OH

O

2.9

1.2

3.8

3.3

5.4

3

2

2.1

0.99

1

1

0.87

1

0.81

0.8

1234567 ppm

38.0

72.1

30.2

7.27.3

6.876.886.896.906.91

6.286.29

4.25 3.753.80

2.22.32.42.5

1.11.2

1.41.51.61.71.8

Figure 2.47: 1H NMR spectrum of 28c (400 MHz, CDCl3).

171.9

171.2

141.2

142.8

40.946.8

38.4

38.9

27.4

35.2

37.422.3

20.5

21.0

171.2

21.0

19.0

18.3

17.066.1

138.5

110.9

140.3

125.2

171.9

141.2

142.8

40.946.8

38.4

38.9

27.4

35.2

37.422.3

20.5

19.0

18.3

17.066.1

138.5

110.9

140.3

125.2

4.26 dd

1.64 ddd

1.82-1.67 m1.82-1.67 m

6.89 dd

2.24-2.16 m2.24-2.16 m

2.03 s

1.27 s

0.83 s

1.82-1.67 m1.15 td

1.54-1.44 m

1.82-1.67 m

1.54-1.44 m2.53 dt

6.28 dd

7.35 t

7.22 dd

3.79 dd

4.26 dd

2.03 s

3.79 dd

2.40 td1.64 ddd

2.40 td

2.35 dt

6.89 dd

1.27 s

0.83 s

1.82-1.67 m1.15 td

1.54-1.44 m

1.82-1.67 m

1.54-1.44 m2.53 dt

1.42 br d 1.42 br d

6.28 dd

7.35 t

7.22 dd

2.35 dt

δH δH

δC

HMBCNOESY

δH δH

δC

HMBCNOESY

Figure 2.48: NMR assignments and key 2D correlations for 28c (stereoview).


203040506070 ppm

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Figure 2.49: HMBC spectrum of 28c.

0.80.91.01.11.21.31.41.51.61.71.8

3.8

3.9

4.0

4.1

4.2

4.3

1.31.41.51.61.71.81.92.02.12.22.32.42.5 ppm

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8


Figure 2.50: NOESY spectrum of 28c.

pared to 28b, the C-1 oxymethine was absent, while the oxymethylene signals

were shifted downfield to δ 3.79 and 4.26, with the same COSY and HMBC

couplings (Figure 2.49), establishing the 17-acetoxy structure shown. This was

consistent with the molecular formula, C22H30O5 (HRESIMS). As with 28b,

NOESY data (Figure 2.50) confirmed the configuration of all stereogenic cen-

tres and permitted full assignment of the H-11, -12 and -17 methylene protons.

The neoclerodane absolute stereochemistry shown for the salvinorins and div-

inatorins is common to all clerodanes isolated from the Lamiaceae,26 including


1a24, 25 and 29a.226

2.2.6.2 Derivation of the Name Divinatorin.

The inspiration for the name “divinatorin” was a comment by Albert Hofmann,

who obtained the type specimen of S. divinorum for botanical identification:

It was determined at the Botanical Department at Harvard that it

was a new species of Salvia and it got the name Salvia divinorum.

It is a wrong name, bad Latin; it should be actually Salvia divinato-

rum. They do not know very good Latin, these botanists. I was not

very happy with the name because Salvia divinorum means “Salvia

of the ghosts” whereas Salvia divinatorum, the correct name, means

“Salvia of the priests” ...258

The original name was coined by Carl Epling, a botanist fluent in Latin, and

was intended to mean “of the seers.”4 According to classicist Dr Parshia Lee-

Stecum, the original name is in fact correct, being taken from the adjective

divinus:259

divinus ... can be (and was as early as classical antiquity) used as

a substantive, meaning “soothsayer/prophet/seer”. Hence divino-

rum: “of the prophets/soothsayers/seers”.

Hofmann’s alternate rendering, derived from the medieval Latin noun “div-

inator”, would have had the same meaning in the medieval period. However,

this word was not used in antiquity, and “divinatorum” would have been in-

terpreted as a form of the verb divino (I foresee):

To someone of ... Virgil’s, Ovid’s or even Tacitus’ time, Salvia div-

inatorum would thus mean “salvia of those who have been foretold”

...

Nonetheless, in honour of Hofmann’s key role in the scientific identification of

the plant, compounds 28a-28c were named divinatorins.


2.2.7 Subsequent isolations.

1g

O

O

O

HO

O

O O

O

O

O

O

H HOO

O

O O

HHO

R

RHO

OMe

OMe

34a

34b

R

28d

28e

R

CH2OAc

CHO

R

O

H HOH

O O

4

Figure 2.51: Subsequently isolated compounds.

Five new diterpenoids have been isolated from S. divinorum since the initial

publication of this work. Lee et al isolated salvinorin G (1g) and divinatorins

D and E (28d & 28e)137 after decolourisation of the crude extract as described

above. The structures of the three compounds, elucidated using 2D NMR, were

incorrectly drawn with tetrahedral geometry at C-4 in the original publication;

this has been corrected in Figure 2.51. Harding et al isolated salvinicins A and

B (34a & 34b)25 without decolourisation. The structure of 34a was elucidated

using 2D NMR, and definitively confirmed by X-ray crystallography.

Chapter 3

Synthesis.

In order to study the structure-activity relationships of salvinorin A (1a) at

opioid receptors, various synthetic modifications were made to the compound.

3.1 Known derivatives.

1a

O

O

O

H HOO

O

O O

35

O

OH

O

H HOO

O

O O

O

O

O

H HOR1

R2O

O O

R2 R1

36h H H

36e Ac H

36d H Ac

a bc

d

36c Ac Acc

Conditions: a) iBu2AlH, THF, –78◦C, 25 min, 65% (81% borsm); b) NaBH4, EtOH, 40◦C, 56%; c) Ac2O, pyridine, DMAP, rt; d) i) trimethyl orthoacetate, AcOH, 105 ◦C; ii)AcOH, cat. HCl, THF/H2O, rt, 74% over 2 steps.

Scheme 3.1: Preparation of known derivatives.

Several known derivatives of 1a were prepared by published procedures with

minor modifications. Lactol 35 was prepared using iBu2AlH following Brown’s

119

120 CHAPTER 3. SYNTHESIS.

procedure,260 albeit in lower yield. Brown reported a yield of 89% after 6

minutes at –78 ◦C. In my hands, the reaction was not complete even after 25

minutes; yield plateaued at 65% (81% based on recovered starting material).

Although warming to –40 ◦C forced the reaction to completion, the yield was

not improved due to side reactions.

Diol 36h was originally prepared by Valdés from 1a using 0.6 equivalents of

NaBH4 in iPrOH;23, 15 as found by Brown,261 EtOH proved equally satisfactory;

also, the precise stoichiometry of NaBH4 proved unimportant, although a large

excess (10 eq) reduced the yield.

Acetylation of 36h under standard conditions gave dihydrosalvinorin E (36e).23

Several trace byproducts could not be completely removed, even by HPLC; this

problem was solved by HPLC purification of the starting diol 36e itself.

Using Ac2O/pyridine/DMAP, direct formation of dihydrosalvinorin C (36c)

from 36h occurs in negligible yield, even after prolonged reflux.262 Valdés et

al’s indirect route via the orthoacetate and 36d27 was therefore used. Satis-

factory purification of 36c required HPLC (40% EtOAc/petrol). Harding et al

have since reported that 36h can be directly diacetylated using NEt3 instead

of pyridine.82

3.2 Epimerisation at C-8 under Basic Condi-

tions.

3.2.1 Previous Reports.

In the NaBH4 reduction of 1a described above, Valdés isolated equal amounts

of 36h and a byproduct which “appears to be stereoisomeric at C-8 and/or

C-9”.23 Acetylation and oxidation of this compound gave “a thus far undeter-

mined stereoisomer” of 1a. Subsequently, Brown identified this compound as

8-epi-salvinorin A (37a, Scheme 3.2 on the facing page), and also reported that

deacetylation of 1a under basic conditions gave 8-epi-salvinorin B (37b).263

3.2. EPIMERISATION AT C-8 UNDER BASIC CONDITIONS. 121

No characterisation data was reported for any of these compounds. Further

references to these epimerisations appeared over the following decade,24, 14 but

no data was published until 2001, when Valdés et al characterised diol 38h.27

No basis was given for this proposed structure, however.

1a

O

O

O

H HOO

O

O O

88

O

O

O

H HOH

HO

O O

88

O

O

O

H HOHO

O O

37a

88

O

O

O

H HOO

O

O O

37b

88

O

O

O

H HOH

O O

O

O

38h 38e

NaHCO3, DMF

Ac2O / pyr

NaBH4

Ac2O / pyr

PCC

B-

ROH

Scheme 3.2: Formation of 8-epi-salvinorins A and B.

3.2.2 8-epi-Salvinorins A and B (37a and 37b).

Deacetylation of 1a under mildly basic conditions (saturated NaHCO3 in

MeOH) gave a mixture of 1b and another compound. The new compound

proved, as Brown had claimed, to be 8-epi-salvinorin B (37b). Acetylation

with Ac2O/pyridine gave 8-epi-salvinorin A (37a).

The structures of 37a and 37b were elucidated using 2D NMR (HMQC,


1a 11.6, 3.1 37a 5.0, 2.21b 11.7, 3.0 37b 4.8, 2.2

Table 3.1: Coupling constants (Hz) at H-8 for 1a, 1b and 8-epimers.

HMBC, COSY and NOESY). The configuration of H-8 was apparent from the

loss of the trans-diaxial coupling constant, establishing an equatorial configu-

ration (Table 3.1). Also, irradiation of H-12 gave a strong nOe enhancement

of H-8 (Figure 3.1 on the next page). The corresponding experiment on 1a

caused an enhancement of H-20 rather than H-8 (Figure 2.14 on page 92). The

coupling constants and NOESY correlations shown in Figure 3.1 also establish

an approximately trans-diaxial relationship between H-11β and H-12. This

suggests the preferred conformation is approximately as shown, with the furan

equatorial and the C ring in a pseudoboat conformation. Consistent with this,

X-ray crystallography of stereoisomeric furanolactones has shown that in the

solid state, the furan is invariably equatorial264, 265, 266 even where the C ring is

forced into a pseudoboat conformation.267 NMR analysis, based on coupling

constants and NOESY spectra, suggests that this also holds in solution.266

It should be noted that both 37a and 37b lack the distinctive infrared car-

bonyl absorption reported for pseudoboat δ–lactones (≈ 1760 cm−1).268 How-

ever, those values were obtained in solution, and are subject to solvent effects;

recording the spectrum as a mull gives typical carbonyl absorptions ( ≤ 1730

cm−1).269 The spectra of 37a and 37b were recorded as neat films.

Our initial publication270 contained characterisation data for 37a but not for

37b. Harding et al82 and Lee et al271 subsequently published 1H NMR data

for 37b which is inconsistent with our own.

3.2.3 Control of Epimerisation and Separation of Epimers.

Brown reported that treating 1a with KCN in refluxing MeOH/THF gave 1b

in quantitative yield,272 but in my hands 37b was the major product, as had

been the case with NaHCO3. Valdés noted the same result.273 Additionally,

the epimers were not resolved on silica using Brown’s solvent system (3%


5.25 dd (12.0, 2.2 Hz)

5.25 dd (12.0, 2.2 Hz)

1.50 dd (15.0, 12.0 Hz)

2.24 br s2.24 br s

2.36 dd (15.0, 2.2 Hz)1.50 dd (15.0, 12.0 Hz)

2.36 dd (15.0, 2.2 Hz)

2.45 dd(5.0, 2.2 Hz)

2.45 dd(5.0, 2.2 Hz)

δH δH

NOESY

δH δH

NOESY

Figure 3.1: Key NOESY correlations for 37a (stereoview).

MeOH/CH2Cl2). Petrol-based systems resolved the epimers, but gave poor

recoveries: some precipitation occurred when loading in these systems. Thus,

effective separation required elution with petrol-based systems for separation,

followed by stripping with MeOH/CH2Cl2 to achieve full recovery of 1b. Yield

was generally below 50%, making this an unsatisfactory route to 1b.

Both of these problems were elegantly overcome by Tidgewell et al,169 using

a suspension of 1a and Na2CO3 in minimal MeOH at room temperature.

In my hands, very little 37b was evident by TLC under these conditions.

This was removed by trituration in MeOH, giving 1b in 76% yield without

chromatography. The supernatant from trituration was a complex mixture

containing very little 37b.

These results were published without discussion, but demand explanation. It

is puzzling that Na2CO3 appears to cause less epimerisation than the much

weaker base NaHCO3 (the conjugate acid of Na2CO3). When Tidgewell et

al’s procedure was repeated in oxygen-free MeOH, the supernatant contained

almost pure 37b, which was obtained as an amber resin in 23% yield by evap-

oration. The precipitate consisted again of crystalline 1b (74%). Thus, 1b

is nearly insoluble in room-temperature MeOH, while 37b is freely soluble.

Additionally, these results confirm that as expected, epimerisation does occur


under Tidgewell et al’s conditions, but is usually not apparent due to decom-

position in oxygenated solution. Nonetheless, the yield of 1b is not affected, so

the reaction can be performed without precautions against oxygen or moisture.

Apparently, then, the improved epimeric ratio results from performing the

reaction as a suspension in minimal MeOH. The reaction solution must rapidly

become saturated with 1b; as additional 1b is generated, it precipitates, unlike

37b. Thus, although an equilibrium mixture of the epimers is present in

solution, this represents only a small proportion of the total reaction mixture.

When the deacetylation is performed entirely in solution, for example by the

use of cosolvents such as THF or CH2Cl2, the epimeric mixture ultimately

comprises the entire reaction mixture. Similarly, as will be discussed below,

the procedure causes extensive epimerisation when applied to 1c, where there

are no dramatic differences in MeOH solubility among starting material and

products.

Substituting NaHCO3 for Na2CO3 under Tidgewell et al’s conditions gave no

reaction after one day. Conversely, Na2CO3 in MeOH/CH2Cl2 gave a complex

mixture; the distinctive H-12 signals of 8-epimers were apparent by 1H NMR.

The above deacetylations proceed via base-catalysed solvolysis; the use of po-

lar “aprotic” solvents (strictly, non-hydrogen bond donor solvents) permits

epimerisation without deacetylation. Thus, treatment of 1a with NaHCO3 in

dry DMF or DMPU at 150 ◦C gives approximately 50% epimerisation to 37a

(addition of water caused deacetylation). In commercial DMPU (98% purity),

epimerisation occurred in the absence of NaHCO3, presumably due to a ba-

sic impurity; heating in distilled DMPU or DMF alone gave no reaction. Of

course, this reaction can also be used to regenerate the natural compounds

from the 8-epimers.

3.2.4 8-epi-Salvinorin C (37c) and Related Compounds.

As discussed above (Section 2.2.5.2), application of Tidgewell et al’s deacety-

lation conditions to 1c (Na2CO3 in minimal MeOH)169 gave predominantly


the 8-epi-diol 37h (in a 4:1 ratio with 1h) — see Scheme 3.3. Again, KCN in

MeOH gave the same result. Both epimers were too soluble in MeOH to permit

trituration. The 8-epimer 37h was freely soluble in Me2SO, but poorly soluble

in acetone and chloroform, with a tendency to precipitate during preparation

of NMR samples.

O

O

O

H HOR1

R2O

O Oc

b

R2 R1

37c Ac Ac

37d H Ac

37e Ac H

37h H H

b

1c

O

O

O

H HOO

O

O

O O

a

Conditions: a) Na2CO3, MeOH, rt × 4 h. b) Ac2O, pyridine, (± DMAP) c) CDCl3, rtovernight

Scheme 3.3: Synthesis of 37c-37h.

Acetylation of 37h under mild conditions gave the 2-acetate 37e in high yield.

Surprisingly, standing 37e in CDCl3 overnight gave 50% conversion to the 1-

acetate 37d, presumably due to traces of DCl. Filtration of either compound

through a plug of basic Al2O3 also caused this unexpected acetate migration.

Alumina-catalyzed acyl migrations have been reported previously274 in 1,2-

dihydroxy terpenoids. No such migration was ever observed in CDCl3 solutions

of the natural epimers 1d and 1e. Acetylation of 37d or 37e under forcing

conditions (with catalytic DMAP at 50 ◦C) gave 37c.

3.2.5 Chromatographic Identification of Epimers.

The epimers gave different colours when visualised with vanillin/H2SO4 in

EtOH. After brief heating, the natural H-8β compounds slowly developed a

pink/purple colour, while the 8α compounds turned blue. These colours were

observed for salvinorins A–E (1a, 1b and 1c–1e) and the diol 1h. Addition-

ally, in each case, the 8-epimer gave a higher Rf in Et2O or EtOAc/petrol.


eluent:1a 37a 1b 1h37b 37h

Et2O Et2O 50% EtOAc/petrol

Figure 3.2: TLC comparison of epimers using vanillin/H2SO4.

These relationships also held for many derivatives to be discussed below; thus,

configuration at C-8 can be confidently inferred from TLC alone. Given the

tendency for salvinorins to epimerise at C-8 under basic conditions, this infor-

mation should prove useful for future synthetic work.

3.2.6 Mechanism.

Koreeda and co-workers have on several occasions23, 24, 263 proposed a complex

mechanism for the epimerisation of 1b, involving cleavage of the C-8/9 bond

(see Scheme 3.4 on the next page). No explanation was given for rejecting the

obvious mechanism of lactone enolate formation, followed by protonation from

the opposite face. Presumably Koreeda and co-workers rejected this simpler

mechanism because the α-protons of ketones are more acidic than those of

esters, by several orders of magnitude.275 Why, therefore, should epimerisation

occur at C-8 rather than C-2 or 10? The more complex mechanism reconciles

this apparent contradiction: abstraction of H-10 inverts the configuration of


H-8 indirectly. This mechanism has been tentatively endorsed by Lee et al.271

O

O

O

H HORO

O O

1010 8899

O

O-

O

ORO

O O

O

O

O

H HORO

O O

B-

H+

Scheme 3.4: Koreeda et al’s proposed mechanism of epimerisation.

This mechanism is not consistent with the above results. Since epimerisation of

1c occurs under identical conditions, the ketone is not essential to the process.

This is also true of many derivatives of 1a lacking the ketone, which will

be discussed below. Other furanolactone terpenoids undergo epimerisation

at C-8 under basic conditions,268, 276, 277 despite lacking the ketone (Scheme

3.5). Several of the structures have been definitively established by X-ray

crystallography: columbin (39),265 isocolumbin (40),267 palmarin (42)266 and

jateorin (43).264

O

O

OO

OH

RO

O

OO

OH

R'

88

R

R

H

H

39

40

R R'

H H

H H

H H

41

42

43

44H H

a

aa

O O

O

Conditions: a) ROH, OH−, ∆.

Scheme 3.5: Epimerisation of related natural products with base.

Conclusive evidence against the mechanism can be found in Brown’s own re-

sults, performed under Koreeda’s guidance. Refluxing 1a with KCN in CD3OD

gave 45,278 deuterated at C-2, -8 and -10 (Scheme 3.6 on the following page).

This establishes that exchange occurs at H-8 in a methanolic solution of CN−


(a weaker base than CO2−3 ).275 Thus their proposed mechanism, in which this

deprotonation does not occur, is incorrect. Both the ketone and lactone are

enolised; evidently, however, inversions at C-2 or -10 are thermodynamically

unfavourable relative to C-8.

1a

O

O

O

H HOO

O

O O

22 1010 88

O

O

O

D DOHO

O O

D

KCN, CD3OD

45

Scheme 3.6: Brown’s deuteration of 1b.

3.2.7 Attempted Deacetylation under Acidic Conditions.

Given the instability of 1a under basic conditions, deacetylation under acidic

conditions was investigated. In collaboration with us, Ken G. Holden treated

1a with 5% methanesulfonic acid in CH2Cl2/MeOH at room temperature.

Monitoring by TLC showed initial formation of 1b and byproducts; after

2 days, these compounds were consumed, giving two barely resolved spots.

NMR analysis showed that each spot consisted of two compounds; deacetyla-

tion appeared to have been accompanied by lactone methanolysis (additional

methoxy peaks and an upfield shift of H-12). Since this route is clearly inferior,

the products were not characterised further.

3.3 Simple Derivatives.

3.3.1 Esters (46 and 47).

Treatment of 1b with HCO2H/Ac2O279 in pyridine280 gave formate 46. Treat-

ment in neat HCO2H281 proved too vigorous, giving inseparable byproducts.

NMR assignments of 46 were inferred from the near-identical spectra of 1a.

3.3. SIMPLE DERIVATIVES. 129

46

O

O

O

H HOO

O

O O 47

2211

O

O

O

H HOH

O

O O

O

Br

48

O

O

O

H HOO

O O

Figure 3.3: Ester and ether derivatives.

In the hope of obtaining crystallographic confirmation of the structure and ab-

solute stereochemistry of 1h, the para-bromobenzoate 47 was prepared using

the acyl chloride, DMAP and NEt3. As found in benzoylation of 3,4-dihydro

analogues,282, 24 no dibenzoylated compound was detected. Unfortunately, 47

was obtained as a powder. Recrystallisation from MeOH, and slow evaporation

of a CH2Cl2/Et2O solution, failed to give crystals suitable for X-ray analysis.

3.3.2 Attempted Benzyl Ether Formation (48).

Inspired by a report that salvinorin B benzoate is a µ opioid agonist,82 for-

mation of the benzyl ether 48 was attempted. Benzyl trichloroacetimidate

and catalytic Me3SiOTf in CH2Cl2283, 284 gave a complex, inseparable mixture

which did not appear to contain 48 by 1H NMR. An alternative route, benzyl

bromide with freshly prepared285 Ag2O in CH2Cl2,286 gave negligible reaction

after 15 hours, despite both reagents being in excess. Addition of Bu4NI287

gave completion in 4 hours. Again a complex mixture of benzylated compounds

resulted, which appeared to contain traces of 48.

Subsequently, Béguin et al reported successful preparation of 48 using BnBr

and Ag2O in MeCN.95 This is surprising, since in previous work MeCN was

the worst solvent tested, giving hydrolysis of BnBr and decreased alkylation

rates.286


3.3.3 17-Deoxy Compounds (49 and 50).

17-Deoxysalvinorin A (49) was synthesised by deoxygenation of lactol 35 using

Et3SiH and BF3·OEt2.288 The enol ether (50) was also formed as a byproduct

(Scheme 3.7). To improve the yield of 49, other routes289 were explored. Use

of Et3SiH with Amberlyst 15 sulfonic acid resin290 instead gave 50 exclusively

in 76% yield. Lactol 35 proved extremely prone to acid-catalysed elimination;

storage overnight in CDCl3 at -20 ◦C gave 52% yield of 50. Such dehydrations

typically require much harsher conditions;291, 292 however, low-temperature de-

hydration of a hemiacetal with BF3·OEt2 has been reported.293 In rare cases,

lactols may be so prone to dehydration that they cannot be isolated.294 In

this case, as with epimerisation at C-8, the elimination may be driven by relief

of steric interactions or strain in the natural H-8β configuration. The furan

substituent may also stabilise the oxonium intermediate through electron do-

nation.

49

6677

881717

O

O

H HOO

O

O O

35

O

OH

O

H HOO

O

O O

O

O

HO

O

O

O O

50

+Et3SiH, BF3•OEt2,

CH2Cl2, 0 °C48% 23%

13C1HHMBC

Et3SiH, Amberlyst 1576%

CH2Cl2

Scheme 3.7: Deoxygenation of lactol 35.

By 1H NMR, the H-17 oxymethylene of 49 appeared as a doublet, δ 3.58,

coupling to H-8 (COSY crosspeak). As with 1a (Figure 2.14 on page 92),

irradiation of H-12 gave a strong nOe enhancement of H-20 rather than H-8,


confirming the configuration at C-8. The quaternary C-8 peak of 50 showed

HMBC correlations to H-6, -7 and -17 (Scheme 3.7). A long range coupling

(1.8 Hz) was evident between one of the H-7 protons and H-17.

3.3.4 Tetrahydrosalvinorin A (51).

52

OH

O

O

H HOO

O

O O

H

51

O

O

O

H HOO

O

O O

HH13

1aH2, Pd/C H2 (4 atm), Rh/C

59%94%

MeOH MeOH/CH2Cl2

O

O

O

R'

OHR

O

O

R'

OR

O

O

R'

OHR

O

O

+ +

A B C D

H2

cat.

O

O

O

Scheme 3.8: Hydrogenation of 1a and other furanolactones.

O

O

O

OO

OO

O

HH

H

Limonin (53)

OO

O

H

O OAcOAc

H

Montanin C (54)

OO

H

O

H

Teucrin A (55)

O

OH

O

Figure 3.4: Furanolactones 53, 54 and 55.

To explore the role of the furan ring in the effects of 1a, tetrahydrosalvinorin


A (51) was prepared. Valdés had reported15, 23 that catalytic hydrogenation

of 1a over palladium on carbon gave hexahydrosalvinorin A (52) in near-

quantitative yield after 24 hours (Scheme 3.8). Saturation of the furan ring

was accompanied by hydrogenolysis of the lactone. This is typical of the pseu-

dobenzylic bond of furanolactones (A): although high yields of tetrahydro

compounds C have been reported with Pd/C, hexahydro compounds D gen-

erally predominate (see Table 3.2 on the facing page). In some cases, use of

Pd/BaSO4 also gave B. The highest reported yields of C have been achieved

with PtO2 in acetic acid; however, results are highly variable. This is true of

other catalysts: different groups report very different results for the same sub-

strate under similar conditions (compare the divergent results for 53 and 54).

This may be due to differences in the catalyst; for instance, some batches of

Pd/C are acidic.295 Generally, acidic conditions favour hydrogenolysis, while

bases (especially nitrogenous bases)296 suppress it.297 Consistent with this,

hydrogenation of 1a at rtp over Pd/C in 0.1% H2SO4/MeOH gave complete

conversion to 52 in 10 minutes. The substrate also affects the outcome: differ-

ent compounds sometimes give dramatically different results under identical

conditions (compare 55 and 54 in Ref. 298).

Rhodium on carbon is reputed172, 319, 297 to cause less hydrogenolysis than pal-

ladium catalysts. There have been reports of selective reduction of furanolac-

tones using this catalyst, giving C without D, albeit in low yields.317, 318 When

applied to 1a, Rh/C gave negligible progress at atmospheric pressure. At 4

atm, the reaction proceeded smoothly to give 51 in unusually high yield (59%).

For characterisation, the less polar 13-epimer was separated by repeated HPLC

(baseline resolution was not achieved). By 1H NMR, H-12 showed a new cou-

pling to H-13, but its coupling constants to H-11 were scarcely affected, sug-

gesting little change of conformation in the lactone.

Determination of the configuration at the tetrahydrofuran C-13 position is

challenging. This has recently been achieved for the tetrahydro derivative C

of limonin (53) by comparison of nOe (ROESY) data for each epimer with

predictions from molecular modelling.307 Since only one epimer of 51 was


Catalyst Solvent Time B C D Ref.% % %

30% Pd/C EtOAc 22 0 0 29910% Pd/C MeOH 24 h* 65 298(55)

24 h* 53 20 298(54)14 78 300(54)

8 h 10 76 30135 min 6 89 2681 h 10 84 2681 h 7 85 268

EtOH 30 min 17 56 30220 80 303

45 min 29 67 304AcOH 5 h 50 305

24 h 10 59-69 306(53)12 41 2768 44 276

CH2Cl2 24 h* 48 307(53)dioxane 4 59 308EtOAc 36 h 74 309H2O 30 310

5% Pd/C AcOH 17 h 20 77 311(53)EtOH 6 h 2 51 312(53)

10% Pd/BaSO4 MeOH 22 24 39 31318 20 35 313

EtOH n.s. n.s. 314(55)AcOH 55 314

PtO2 AcOH 5 h 34 10 31520 h 42 <55 3167 h 53 30530 h 30 299

AcOH/dioxane 48 h 88 311(53)96 h 86 311(53)

5% Rh/C AcOH 10 h 42 317(55)EtOAc 48 h 36 318

* = 2 Atm. n.s. = not stated

Table 3.2: Some previously reported furanolactone hydrogenations.

obtained pure, this was not attempted in this case. Since this work, Béguin et

al have synthesised 51 using Pt/C catalyst, albeit in lower yield (36%).125


O

H H

O OR

R

H

Me

ent-29a

ent-29b

Figure 3.5: (+)-Hardwickiic acid (ent-29a).

3.3.5 (+)-Hardwickiic Acid (ent-29a).

(+)-Hardwickiic acid (ent-29a) occurs in commercially-available copaiba bal-

sam. The compound is difficult to separate from other acids in the crude

mixture, and was therefore isolated as the methyl ester ent-29b following

a published procedure.218 Cleavage of the methyl ester proved challenging.

Heating in KOH/MeOH172 under reflux gave only slow decomposition, but

microwave irradiation on KF/Al2O3320 provided ent-29a in low yield. The

reaction did not proceed in the absence of KF.

3.4 Modification of the Methyl Ester.

3.4.1 Relevant Results from Previous Work.

To explore the pharmacophore of 1a, a selective modification of the C-18

methyl ester was desirable. It was apparent from previous work that hydride

reduction was unsuitable. As discussed above, Valdés and Brown had achieved

selective reductions of the ketone and lactone respectively (Scheme 3.1 on

page 119). Varying the solvent in the iBu2AlH reduction was of no benefit.260

Use of LiAlH4 at –78 ◦C gave the triol 56 (Scheme 3.9). Reduction of the

methyl ester was not observed in any of these reactions, demonstrating its

very low reactivity. Béguin et al have recently reported full reduction of all

carbonyl groups with LiAlH4 at room temperature, giving pentaol 57.125

3.4. MODIFICATION OF THE METHYL ESTER. 135

56

O

OH

O

H HOH

HO

O O 57

OH

OH

O

H HOH

HO

OH 58

O

O

H HOH

OH

OH

1aLiAlH4 LiAlH4

Li/NH3, -100 °C

-78 °Crt

Scheme 3.9: LiAlH4 and Li/NH3 reductions of 1a.

Similarly, Brown reduced the methyl ester using lithium in ammonia,321 but

this was accompanied by deoxygenation at C-2, epimerisation at C-1, and

cleavage of the lactone, giving 58. Brown gives two conflicting structures for

58; the hybrid structure shown here incorporates those features consistent with

the 1H NMR data. The furan peaks downfield of 6 ppm rule out saturation of

the furan ring. The C-18 oxymethylene δ and J values are nearly identical to

those of 18-hydroxy derivative 77 (see Scheme 3.17 on page 152), ruling out

epimerisation at C-4.

Low reactivity at the methyl ester was also evident under other conditions.

As noted earlier, KCN/CD3OD gave deuteration at C-2, -8 and -10, but not

C-4, α to the methyl ester (see Scheme 3.6 on page 128). Clearly none of

these approaches offered the possibility of a selective reaction at C-18. As

an alternative approach, cleavage of the C-18 methyl ester to the acid would

permit selective borane reduction to the hydroxyl, as in 57 and 58, in the

presence of other carbonyls.

3.4.2 Treatment of Salvinorin A with KOH in MeOH.

The first route attempted for methyl ester cleavage was basic hydrolysis. Since

the methyl ester was unaffected by Na2CO3 in MeOH, 1a was treated with 1M

KOH in MeOH . The solution turned a deep orange, and the starting material

was rapidly consumed. The major product, found in the neutral fraction,


was enedione 59 (Scheme 3.10). The base-soluble fraction was difficult to

analyze, smearing on TLC and giving a poorly-resolved 1H NMR spectrum.

Surprisingly, at least eight peaks were apparent in the methoxy region. After

methylation with Me3SiCHN2,322 TLC showed only a single spot. 1H NMR

analysis, however, revealed three major compounds (60a, 60b and 60c), which

were separated with difficulty by HPLC. Baseline resolution was not achieved,

necessitating repeated repurification and poor recoveries.

3.4.2.1 Structure Elucidation of the Products.

+1. KOH / MeOH

2. Me3SiCHN2

O

O

O

R'RO

O

O

O

R R'

H H

H H

H H

60a

60b

60c

10 8

59

53%47%

1a

O

O

O

H HOO

O

O O

O

O

O

HOH

O

O O O O

Scheme 3.10: Autoxidation of 1a.

The 1H spectrum of 59 showed two new singlets at δ 6.91 and 6.99 ppm. The

peak at 6.91 showed no COSY or HMQC crosspeak, and exchanged with D2O.

Such strongly deshielded exchangeable peaks are typical of cyclic α-diones,

whose enol tautomers are stabilised by internal H-bonds.323 Consistent with

this, the compound exhibited strong IR absorptions at 3373 and 1651 cm−1

(OH and enol C=C). The structure was further elucidated by analysis of the

HMBC spectrum. The quaternary C-10 peak, located unambiguously by its

correlations to the H-19 and 20 methyls, showed a correlation to the enolic

proton, placing the enol at C-1 and the ketone at C-2. The vinylic H-3 peak

showed the expected correlations to C-1, 4, 5 and 18 (see Figure 3.6). UV

absorptions at 215, 249 and 324 nm confirmed an extended π system, as in

methyl 4-oxopentenoate (61, Figure 3.7 – 222 and 324 nm).324 Compare the

spectra of 1a and 1c. Note that the longest-wavelength peak approaches vis-

ible wavelengths. The absorptions of α-diones exhibit a bathochromic shift


towards visible wavelengths in basic solution:325, 326, 327 the orange colour ob-

served during formation of 59 in basic MeOH is consistent with this.

22

3344

55

101011

O

O

O

HO

O

1919

2020

H

H

O

O

O

HHO

O

O

O

60aH

59

13C1H

18

O O O O

Figure 3.6: Key HMBC correlations of 59 and 60a.

HRESIMS confirmed the molecular formula. The remainder of the structure,

unchanged from the salvinorins, was fully elucidated and assigned by NMR

experiments (DEPT, COSY, HMQC, HMBC, and nOe). The H-12 coupling

constants were closer to those of 37a than of 1a, suggesting that epimerisation

at C-8 might have occurred. However, the β configuration of H-8 was evidenced

by a trans-diaxial coupling constant (9.7 Hz); in addition, irradiation of H-12

gave an nOe enhancement of H-20. The structure of 59 thus established is

remarkably similar to salvinorin G (1g, Figure 2.51 on page 118).137

The major product from the base-soluble fraction was identified as 1,2-secotriester

60a (Figure 3.6) based on extensive NMR experiments. The H-10 singlet

showed an HMBC correlation to the new C-1 ester carbonyl (Figure 3.6). H-4

showed correlations to C-3, 5 and 10, and formed an isolated spin system with

the two deshielded H-3 peaks. This confirmed the location of the new methyl

esters at C-1 and 2, although the three esters were not sufficiently resolved in

the 2D spectra to allow individual assignment. The remaining NMR data was

very similar to 1a. The chemical shift and coupling constants of H-12 were

near-identical to those of 1a, confirming the configuration at C-8. HRESIMS

confirmed the molecular formula. The second major product was identified

as the 8-epimer 60b based on the shifts and coupling constants of H-8 and

12 (near-identical to those of 37a).270 Assignment of the remaining data was

straightforward. 2D NMR showed the same correlations as 60a; HRESIMS


1c

59

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

400380360340320300280260240220200

1a

O

O

O

H HO

O

O

O O

O

O

O

H HO

O

O

O

O O

O

O

O

HOH

O

O O

1a

1c59

61

ε

λ (nM)

OO

O

Figure 3.7: UV/Visible spectra of 1a, 1c and 59 in MeCN.

again confirmed the molecular formula. Interestingly, although 60a and 60b

cospotted by TLC, they gave the expected colours with vanillin/H2SO4 in

EtOH (Figure 3.2 on page 126).

The third (minor) compound decomposed in CDCl3 before characterisation

was completed, but was tentatively assigned as 60c (Scheme 3.10 on page 136).

HRESIMS established that the compound was also an isomer of 60a, and the

appearance of the same couplings in the COSY spectrum suggested another

stereoisomer. The coupling constants of H-8 and 12 established that the C-

ring configurations matched those of 60a. Indeed, all of the coupling constants

determined were close to those in 60a, whereas many chemical shifts showed

large changes. This implied a change in the electronic environment of the

coupling protons, without a change in their configuration. The most plausible

candidate structure was therefore the 10-epimer 60c, since H-10 is not coupled.

Placing a large substituent in an axial configuration would be expected to

affect the conformation of both remaining rings, and hence the chemical shifts

around those rings. By contrast, inversion at C-4 would not be expected


to dramatically alter the conformations of the rings, but would be expected

to alter the coupling constants with the H-3 protons. These couplings were

scarcely changed, while the chemical shifts of H-4, 7, 8, 10, 11, and 12 (but not

H-3) were dramatically altered. Thus 60c is the more plausible structure. The

particularly large change at H-4, shifted downfield by 0.83 ppm, might be due

to falling within the deshielding region of the C-1 carbonyl. Also, as mentioned

earlier, H-10 is much more readily exchangeable than H-4. Brown reported278

that when 1a was refluxed with KCN in CD3OD, deuterium exchange occurred

at H-2, 8 and 10 but not H-4 (Scheme 3.6 on page 128). In the absence of nOe

data, however, the proposed structure 60c must remain tentative.

3.4.2.2 Comparison with Previous Reports.

These results conflict with those published previously. Tidgewell et al169 re-

ported that heating 1a with NaOH in MeOH caused cleavage of the methyl

ester and opening of the lactone, without giving further detail. Since lactone

hydrolysis would be reversed upon neutralisation, this presumably refers to

methanolysis. Ester cleavage may have been inferred from the formation of

a base-soluble fraction, and lactone methanolysis from the methoxy peaks in

the 1H NMR spectrum. As shown above, however, the acidic fraction and its

methoxy peaks result from cleavage of the α-hydroxy ketone. When the 1,2-diol

36h23 (Figure 3.8) was refluxed in KOH/MeOH for 30 min, an epimeric mix-

ture at C-8 was recovered in near-quantitative yield, confirming that neither

methyl ester hydrolysis nor lactone methanolysis occur under these conditions.

62

O

O

O

HO

O O

O

O

O

H HOH

HO

O O 36h

Figure 3.8: Diol 36h and proposed autoxidation product 62.


More recently, Lee et al treated 1a with Ba(OH)2 in MeOH, reportedly obtain-

ing 62 in 75% yield.271 The 1H and 13C NMR data quoted for 62 are identical

to those of 59, apart from the omission of the H-6 multiplet at δ 1.77-1.67

ppm; they are evidently the same compound. Their proposed structure 62 is

not consistent with the additional data presented here. Specifically, HRMS es-

tablished the molecular formula as C21H22O7 (59) rather than C21H22O6 (62).

Further, the singlet at 6.91 cannot be attached to C-1, since it exchanges with

D2O, has no HMQC crosspeak, and lacks the expected HMBC correlations to

C-3, 5, and 9. A corresponding methine peak is also absent from the DEPT

spectra. The red colour of the reaction mixture125 again suggests the distinc-

tive red shift of α-diones. The presence of the C-1 ketone was confirmed by

reduction (Section 3.4.2.5 below). Finally, deoxygenation of a ketone would

not be expected under these conditions.

3.4.2.3 Proposed Mechanism.

Autoxidation of α-hydroxy ketones (“acyloins” or “α-ketols”) to α-diones (“diosphe-

nols”) under basic conditions is well-established.325, 328, 329 The reaction con-

sumes one equivalent of O2, generating H2O2.329 While the autoxidation of

unsubstituted ketones requires stronger bases such as tBuOK, α-hydroxy ke-

tones are more readily enolised, and the reaction proceeds with KOH.328 A

proposed mechanism, via saturated dione 63, is shown in Scheme 3.11.

The alternate pathway, to seco-diester 64, also has numerous precedents.328, 330

We based our proposed mechanism on the generally-accepted formation of hy-

droperoxide intermediates,328 although this mechanism has been disputed.330

Tautomerisation of the enolate or radical will give the regioisomeric diester,

which along with epimerisation at C-8 and 10 explains the numerous methoxy

peaks in the 1H NMR spectrum of the crude product.

Dehydrogenation of 63 to form 59 is unusual. While there have been sev-

eral reports of dehydrogenation of 1,4-diones in alcoholic KOH,331 literature

searches48 revealed no such reaction involving a 4-ketoester. However, α-diones


O

O

O

H HOHO

O

O

O

H HOHO

O2-

O

O

O

H HO

HO

HOOO

O

O

H HOO

O

O

O

H HO

HO

OO

O

O

O

HHO

O

O

HO

O2-OH

1) -OH

O

O

O

H HOO

HOO

H

MeOH/MeO-

HO

59

63

2) O2

O

O

O

H HOO

O2

MeOH

-OH

-OH

MeOH

1a

O

O

O

H HOO

O

O O O O O O

O OO OO O

O O OO

O O 64

Scheme 3.11: Proposed mechanism of the autoxidation.

65

O

O

O

O

HO

O O

H

66

O

O

O

O

HO

O O

H

HO

1b

O

O

O

H HOHO

O O

CrO3

pyridine

-H2O

Scheme 3.12: Unexpected oxidation product 65.

are much more readily enolised than unsubstituted ketones; in the case of 59,

no trace of the 1-keto tautomer was detectable by NMR. It is therefore plausi-

ble that 63 should be extremely reactive, and unsurprising that this compound


was not isolated. Consistent with this, Brown’s attempts to prepare 63 via

PCC oxidation of 1b gave no isolable product.332 More recently, Harding et

al oxidised 1b using similar conditions (CrO3 in pyridine), unexpectedly ob-

taining the lactone 65136 (Scheme 3.12) with the loss of C-2, rather than the

expected 63. No mechanism was proposed. It is interesting to note that lactols

analogous to 66 have been reported from autoxidation328, 333 or ozonolysis with

oxidative workup334 of α-diones, and that dehydration of 66 would give 65.

However, if 66 were formed in the presence of excess CrO3, oxidation rather

than elimination would be expected.

3.4.2.4 Variation of Reaction Conditions.

The yield and selectivity of autoxidations of this type can be subject to strong

solvent effects.328 The reaction was therefore repeated in EtOH, iPrOH andtBuOH. The resulting neutral and acidic fractions were noticeably more com-

plex. In each case, 59 was contaminated by inseparable impurities (presumably

including the 8-epimer); the original selection of MeOH thus proved fortuitous.

The reaction proceeded with only traces of oxygen, for instance when per-

formed under N2 in MeOH pre-saturated with N2. This is again typical.328, 330

Nonetheless, the reaction was faster and more consistent when the solution

was saturated with O2. Anther useful refinement was the use of dilute KOH

rather than NaHCO3 to extract the extremely hydrophobic acid diesters during

workup.

3.4.2.5 Attempted Reductions of 59.

The unexpected installation of the 3,4-double bond in 59 suggested the com-

pound might provide a route to diol 1h. However, reduction with NaBH4

in EtOH/CH2Cl2 was accompanied by conjugate addition and epimerisation,

giving 8-epi-diol 38h27 in low yield (Scheme 3.13). Attempted Luche reduc-

tion with NaBH4–CeCl3 in MeOH (with335 or without336 sonication) was also

unsuccessful, giving a complex mixture whose unstable major components re-


O

O

O

H HOH

HO

1h59

O

O

O

H HOH

HO

NaBH4 X38h

O O O ONaBH4

CeCl3

Scheme 3.13: Attempted reductions of 59.

tained the characteristic enedione peaks at δ 6.8 and 7.0 ppm. These products

cospotted with the starting material in petrol-based systems, making the reac-

tion difficult to follow, but were resolved by acetone/CH2Cl2 and gave a darker

purple with vanillin. There are precedents for ketones which are smoothly re-

duced by NaBH4 alone, but not in the presence of CeCl3.335 Note that use

of “forcing conditions”337 (reflux for 12 hours) was futile. NaBH4 decomposes

to unreactive tetramethoxyborate within 5 minutes under the standard condi-

tions (with CeCl3 in MeOH at room temperature).336 Prolonging or heating

the reaction will therefore achieve nothing. Continued reaction would require

frequent additions of fresh reagent.

Commonly used drying procedures for CeCl3 can cause partial decomposi-

tion.338 The quality of the CeCl3 used was verified by reduction of 2-cyclohexen-

one,336 which gave 2-cyclohexenol rapidly and quantitatively.

The enols of α-diones form complexes with a wide variety of metal salts such

as FeCl3;339 it appears likely that such an enolic complex forms with 59 in

preference to the desired ketone–solvent–Ce3+ complex,336 and the reaction

therefore does not follow the desired course. The only successful precedent

located for this reduction involved non-enolisable enediones.340 It might be

possible to prevent this problem by protection of the enol, but an attempt at

Et3Si protection was unsuccessful.


3.4.3 O-Demethylsalvinorin A (67a).

To summarise the above results, previous work showed the C-18 methyl ester

to be the least reactive carboxyl of 1a under a variety of conditions. The

few reactions vigorous enough to attack this position gave undesired side reac-

tions at other positions; no selective transformation had been reported. The

preliminary results above were consistent with this.

The selective cleavage of methyl esters has been the topic of extensive research;

thorough reviews are available.341, 342 The most effective procedures involve

nucleophilic substitution at the alkoxy (or carbinol) carbon, via an SN2 mech-

anism, with the carboxylate as leaving group. This mechanism (BAl2 ester

cleavage) contrasts with the BAc2 mechanism typical of basic hydrolysis, in-

volving attack at the carboxyl carbon (Scheme 3.14).343 The tendencies of

different nucleophiles to act via these mechanisms can be understood in terms

of hard/soft acid-base theory.342 Soft nucleophiles such as I− attack the soft

electrophilic carbinol via the BAl2 mechanism, while hard nucleophiles such

as F− favour attack at the hard electrophilic carboxyl carbon, via the BAc2

mechanism. Thus soft nucleophiles are less affected by hindrance around the

carboxyl carbon, but more affected by hindered alkyl chains; hence the se-

lectivity for methyl esters. Given the severe hindrance exhibited by the C-18

carboxyl carbon of 1a, the desirability of methyl ester selectivity, and the dis-

astrous effects of hydroxide, the use of soft nucleophiles was clearly preferable.

CH3Nu+

BAl2

OR

O H

HH

Nu-

O-R

O

BAc2

OR

O

OHR

-OO

-OH

+O-R

O

CH3OH

Scheme 3.14: BAl2 and BAc2 ester cleavage mechanisms.


3.4.3.1 Cleavage with Iodide and Cyanide Reagents.

Perhaps the most widely used source for a soft nucleophile is LiI. Early re-

ports used pyridine and 2,6-lutidine344 as solvent, but subsequent work found

more polar non-hydrogen bond donor solvents such as DMF345 and especially

HMPA346 (Figure 3.10) to be superior, as would be expected for an SN2 reac-

tion.347 Anhydrous LiI has been reported to give greater selectivity for methyl

esters, but lower yield, than the hydrate.348

1a

O

O

O

H HOO

O

O O 67a

O

O

O

H HOO

O

O OH 67b

O

O

O

H HOHO

O OH

Figure 3.9: O-Demethyl salvinorins A and B.

An initial trial of anhydrous LiI in dry DMF, after refluxing for 24 h, gave a

29% yield of the epimerised acid 67a. The dark brown colour and fishy odour

of the reaction mixture suggested decomposition. The neutral fraction was a

complex mixture containing little starting material.

Adding sodium acetate has been reported to lower the required temperature

and reaction time for this procedure.349 However, in this case the reaction

showed no progress after 6 hours at 130 ◦C, and the yield after refluxing

overnight was not improved.

N

2,6-lutidine

O

N

HMPA

N N

O

DMPUDMF

N N

O

NP

Figure 3.10: Useful non-hydrogen bond donor solvents.

The use of HMPA was avoided due to its carcinogenicity,347 but work with

other nucleophiles has found that the safe substitute DMPU350 (Figure 3.10)


gives comparable rate enhancements.351, 352 Substituting this solvent for DMF

(150 ◦C for 25 h) gave no improvement in absolute yield (27%), but a much

cleaner neutral fraction of epimerised 1a, giving a 79% yield based on recovered

starting material. The reaction mixture again showed a dark colour and a fishy

smell suggestive of decomposition.

A run at a higher temperature (190 ◦C) gave a different base-soluble product

which was not identified. The NMR spectrum showed an epimeric mixture

lacking the acetyl peaks of 67a. It did not match 67b, however, and was not

acetylated by Ac2O/DMAP in pyridine.

The use of NaI in DMPU at 150 ◦C for 23 h gave a light-coloured reaction

mixture and a greatly improved yield of 67a (73%). This was surprising, since

lithium has been found to be superior to sodium as a counterion with iodide345

and other353 nucleophiles. Unfortunately, two careful attempts to replicate the

reaction gave the same yield as LiI.

Sodium cyanide has been reported to give superior results to lithium halides,

including LiI, especially in HMPA.346, 343 Treatment of 1a with KCN in DMPU

at 90 ◦C for 27 h, however, gave a complex mixture with only traces of 67a.

With NaCN the reaction proceeded more slowly, but again gave a complex

mixture after consumption of starting material (70 h).

3.4.3.2 Cleavage with Thiolates.

Thiolates (RS−) are another group of soft nucleophiles commonly used for

methyl ester cleavage. A recent study reported excellent results using arylthiols

and catalytic base at 190 ◦C, generating thiolate in situ.351 Attempts to apply

this procedure were unsuccessful: treatment of 1a with 4-methylbenzenethiol

with K2CO3 in DMPU gave no reaction after 10 minutes. Prolonged reaction

(16 h) gave decomposition, with no indication of acid formation.

Aliphatic thiolates have found greater use. Lithium methanethiolate (LiSMe),354

as well as the ethyl355 and propyl353 homologues all cleave methyl esters at room

temperature, whereas halide and cyanide reagents are typically used above 100


◦C. Again lithium353 appears to be a superior counterion to sodium,356 and

HMPA353 and DMPU352 superior solvents to DMF.

Alkylthiolate solutions are unstable.353, 355 However, LiSMe is easily prepared

and air-stable;354 ethanethiolates are now commercially available. For this

work, LiSEt was chosen since ethanethiol (bp 35 ◦C) is much less volatile

than methanethiol (bp 6 ◦C). The sickening thiol odour is therefore greatly

reduced. The salt was prepared from ethanethiol and nBuLi using a simplified

version of published procedures for LiSMe354 and LiSPr.357 Again, DMPU was

substituted for the carcinogen HMPA.

68

O

O

O

H HOO

O

O O

O

67b

O

O

O

H HOHO

O OH

Ac2O, DMAP

DMPU

Scheme 3.15: Formation of mixed anhydride 68.

The reaction between 2a and LiSEt showed little progress after 6 hours at

room temperature. After heating to 55 ◦C for 23 hours, the reaction went to

completion, with little change in colour. This was in marked contrast to the

high-temperature procedures. Ester cleavage was accompanied by deacetyla-

tion, giving 67b; standard acetylation conditions gave the epimeric acids 67a

in good yield (typically 73% over two steps).

On one occasion the acetylation was performed in one pot, after quenching the

thiolate with acetic acid (the orange colour faded to faint yellow). Addition

of Ac2O/DMAP gave 67a in four hours, unfortunately accompanied by the

mixed anhydride 68 (crude 1H NMR of the neutral fraction showed additional

characteristic peaks at δ 2.24 and 2.25 ppm; Scheme 3.15). The anhydrides

proved remarkably stable. Some starting material remained after reflux in

THF/H2O for 1 hour; addition of NaHCO3 was necessary for complete hydrol-


ysis to 67a. This process was more laborious than a separate acetylation step,

and was therefore abandoned.

The results of the various cleavage attempts are summarised in Table 3.3.

Yield of 67aNu Base Solvent T time Isolated borsm*

◦C h % %LiI - DMF 150 24 29LiI NaOAc 130-150 21 26LiI - DMPU 150 25 27 79LiI - 190 24 0NaI - 150 14 23

NaCN - 90 70 0KCN - 90 27 0

p-MePhSH cat. K2CO3 200 16 0LiSEt - 55 23 73 (after acetylation)

* = based on recovered starting material

Table 3.3: Summary of results - nucleophilic cleavage of 1a methyl ester.

3.4.3.3 Confirmation of Structure of 67a.

The mixed acids 67a streaked on TLC, and reduction by BH3 was also ex-

pected to cause epimerisation, so chromatographic separation was not at-

tempted. The structure of 67a was definitively confirmed by methylation with

CH2N2, giving a mixture of 1a and 37a by 1H NMR. The products cospotted

with authentic material in both petrol– and CH2Cl2–based TLC systems and

gave identical colours with vanillin dip.

The 1H NMR spectrum of 67a lacked the methoxy peak, but was otherwise

near-identical to a mixture of 1a and 37a. One interesting difference was the

first-order H-4 multiplet at 2.80 (dd, J = 5.2, 3.5). The H-4 multiplet of 1a is

slightly non-first order due to the almost coinciding H-3 peaks, but at 800 MHz

can be approximated as 2.74 (dd, J = 11.3, 5.6). Apparently the formation

of a carboxylic acid dimer (as with hardwickiic acid, Figure 2.18) pushes the

C-4/18 bond away from the equatorial position, altering the conformation of

the A ring. Thus the trans-diaxial coupling constant for H-4 is lowered, and

the H-3 multiplets are no longer coincident.


3.4.3.4 Subsequent Developments and Discussion.

Since this work, Me3SnOH has been reported to cleave methyl esters selec-

tively under mild, neutral conditions in complex substrates too sensitive for

previous methods.358 While the method appears very promising, it has several

drawbacks. Firstly, the compound is extremely neurotoxic.359 Secondly, like

other oxytin reagents, Me3SnOH is a hard nucleophile, attacking the carboxyl

carbon.360 The reactivity of other oxytin reagents is greatly reduced against

hindered carboxyls, including terpenoids resembling 1a.360

Also since the publication of the above work,270 Lee et al have reported an

improved yield of 1a via the LiI method (56%125 or 72%),361 by refluxing in

pyridine for 36 hours. The LiI was presumably a hydrate since no drying is

mentioned. If the higher yield proves reproducible, this method offers clear

advantages over the thiolate route above: the acetate remains intact, and the

reagents are common, stable and odourless. This result is surprising in light

of the early results on solvent effects discussed in Section 3.4.3.1 on page 145.

By the same token, the alkylthiolate route has not been optimised. This

route offers the inherent advantage of proceeding at or near room temperature.

Given the accumulation of the basic carboxylate during BAl2 ester cleavage,

and the base-sensitivity of 1a, this should permit higher maximum yields.

The deacetylation observed with LiSEt is puzzling, since alkylthiolates have

been found previously to cause less deacetylation than LiI.353 One potential

explanation is contamination, since the thiolate was not purified before use.

On several occasions after the cleavage and acetylation, 2-hydroxyethyl ac-

etate362 was unexpectedly isolated. If one of the reagents used (presumably

the thiol) was contaminated with ethanediol, the resulting thiolate would be

contaminated with the dihydroxide. Also, any LiOH present in the nBuLi used

(from exposure to moisture) would also have been present in the thiolate. ThenBuLi solution used was labelled 2.5 M, but titration363 gave a value of 2.1 M.

Finally, as discussed in Section 3.2.3 on page 124, the commercial DMPU used

subsequently proved to contain an impurity capable of causing epimerisation.


However, this was not accompanied by deacetylation, even at 150 ◦C. One of

these possible contaminants may have been responsible for the deacetylation

observed, and may also have lowered the yield. The use of demonstrably pure

solvents and reagents may improve the outcome of this reaction.

3.4.4 O-Demethyl-18-deoxysalvinorin A (77).

3.4.4.1 Borane Reduction of 67a.

With the acid 67a in hand, the planned borane reduction could be attempted.

BH3·THF reduces carboxylic acids rapidly at low temperatures (0 ◦C or below

in many cases), even when hindered (69, Scheme 3.16).364 Esters and lactones

are generally reduced at a much lower rate,365 allowing mild, selective reduc-

tions of polyfunctional acids such as 70366 and 71.367 However, some acids

are much less reactive. For example, many aromatic acids such as 72368 re-

quire excess BH3, longer reaction times and higher temperatures.369 In some

extreme cases, refluxing for several hours with excess BH3 is required (73370

and 74).371

Conversely, some lactones (75 and 76) are rapidly reduced to lactols under

mild conditions, and even to cyclic ethers and acyclic diols with prolonged

reaction.372 Thus, the selectivity achieved depends on the substrate and precise

conditions. An excellent review is available.365

Slightly different protocols have been used, but generally borane is added drop-

wise at low temperature, then the mixture is warmed gradually to the desired

reaction temperature.364, 373 During the initial addition, deprotonation of the

acid gives intermediate acyloxyboranes374 (with visible evolution of H2), which

are reduced much faster than other functionalities, accounting for the selectiv-

ity of the reagent. The low temperature and slow addition of BH3 minimises

side reactions until these intermediates are formed.

Unfortunately, attempts to apply the mildest possible protocol to 67a were

unsuccessful. Addition of BH3·THF at -25 ◦C gave no visible evolution of H2.


X

OH

X

HO

4 eq, reflux, 4h, 94%

X

HO

OR

OR

OR

OR X

OH

4 eq, reflux, 3h, 77%

O

O

O

O

OHX

XHO

3.5 eq, rt, 48h, 71%

O O

O O

OHX

1.3 eq, 0°C, 30 min, 66%

OHX

1.3 eq, 0°C to rt, 1h, 95%

OXH

H

H

O

H

H

XH

HOH

3 eq, rt, 3 days, 55%

1 eq, rt, 30 min, 80%

XO

H2BH3•THF

Br

OO

OH

X

1 eq, 0 °C, 3h, 86%

XO

HO

70 7169

73 72

74 (R= Me/Et)

75 76

H2

Scheme 3.16: Some previously reported BH3·THF reductions.

Gradual warming to 0 ◦C and addition of excess borane had no effect. Warming

to room temperature, then 45 ◦C, over several days gave no reaction. Starting

material was recovered almost quantitatively. In later trials, borane was added

at room temperature, giving the first visible signs of H2 evolution. The actual


reduction, however, required a large excess of borane. Small amounts of the

8-epi-alcohol 78 were detected (Scheme 3.17), but not the desired alcohol 77.

Refluxing with excess borane gave 78 accompanied by unidentified byproducts.

Therefore a smaller excess was tried at intermediate temperature. The most

successful conditions are shown in Scheme 3.17, giving the epimeric products

in 48% total yield.

67a

O

O

O

H HOO

O

O OH

O

O

O

H ROO

O

OH

BH3•THF (1.3 eq)

dropwise, rt x 5 min,then 55 °C x 90 min

R

H

H

77

78

23%

25%

1H1HCOSY

Scheme 3.17: Borane reduction of 67a.

These conditions are not optimised. None of the target 77 was isolated until

the last few experiments, when it was found to cospot with the starting mate-

rial in the TLC system used previously (1% AcOH in 20% acetone/CH2Cl2).

Thus, in earlier experiments, clean formation of the desired product may have

gone undetected, while decomposition of that product may have been mis-

taken for consumption of starting material. An alternate solvent system, 1%

NEt3/EtOAc, resolved all compounds.

Earlier trials may have therefore been more successful than they appeared, and

milder conditions warrant reinvestigation. Nonetheless, it is apparent from

the lack of hydrogen evolution at 0 ◦C that 67a is an exceptionally unreactive

substrate.374 This is unsurprising given the near-inertness of the C-18 position

to all other procedures tried - hydride reagents, deuteration, strong base and

soft nucleophiles. On the other hand, given the side reactions apparent at

reflux with BH3, 67a is also a sensitive substrate. It is evident that the optimal

conditions will be intermediate between the mild, original protocols364, 373 and

the harsh conditions required for stubborn, resilient substrates such as 74.

3.5. MODIFICATION OF THE KETONE. 153

3.4.4.2 Structure Elucidation of 77.

Although the H-4 multiplet of 77 (δ 1.89 ppm) was too complex for determi-

nation of coupling constants, the COSY spectrum showed cross peaks with the

diastereotopic H-18 oxymethylene (δ 3.94 & 3.49 ppm). The chemical shifts

and coupling constants for H-8 and H-12 were near-identical to those of 1a,

and irradiation of H-12 gave a strong nOe enhancement of H-20 rather than

H-8 (compare Figure 2.14 on page 92), confirming the configuration at C-8. A

strong infrared absorption at 3468 cm−1 confirmed the presence of a hydroxyl.

HRMS confirmed the molecular formula.

3.5 Modification of the Ketone.

Selective reduction of the C-1 ketone in 1a had already been reported (36e).15, 23

Since the 1α-hydroxy of this compound was axial, however, any change in bi-

ological activity would be ambiguous; attributable either to the loss of the

ketone, or the presence of a new protruding H-bond donor. Other modifica-

tions of the ketone were therefore investigated. One obvious alternative was

deoxygenation. Methylenation was also attempted, since this would preserve

the sp2 hybridisation of C-1 and thus have less effect on the conformation of

ring A, albeit with a much larger substituent.

3.5.1 Attempted Methylenation.

Initially, Wittig olefination was attempted. Treatment of 1a with the ylide

formed from Ph3PMeBr375 and nBuLi (1.6 eq, THF, 35 ◦C, 20 hours) gave only

epimerisation and partial deacetylation. No trace of the desired methylenated

compound 79 was detected.

The Wittig reaction often fails with hindered substrates, and epimerisation is

also common due to the basicity of the ylide.376 The Tebbe reagent and related

titanium compounds377 allow the methylenation of some carbonyl compounds


O

O

O

H HO

O

O O 79

Figure 3.11: Methylenated target compound 79.

for which Wittig conditions fail. Treatment of 1a with Zn-CH2Br2-TiCl4reagent378 (room temperature, CH2Cl2, 1 hour) gave an extremely viscous,

black reaction mixture. After standard workup, however, only starting mate-

rial was detectable (72% recovery). The reagent was not tested for activity

against a known substrate, but was freshly prepared379 and had the reported

grey colour and thick consistency. On addition to water, the reagent blackened

and effervesced vigorously.

3.5.2 Attempted Direct Deoxygenation.

R R

O

R R

NNH

SO

O

R R

TsNHNH2 NaBH4

Scheme 3.18: Ketone deoxygenation via a tosylhydrazone.

The next modification of the ketone to be attempted was deoxygenation. The

Clemmensen reduction (Zn/HCl) was not suitable, since α-acetoxy groups are

eliminated,380 even under the mildest conditions.381 An alternative mild ap-

proach is via a tosylhydrazone, which can be reduced to the hydrocarbon by

hydride reagents (Scheme 3.18).382, 383 In attempts to form the tosylhydrazone,

1a was treated with tosylhydrazide384 under a variety of conditions (Table 3.4).

Since no reaction occurred in solution (even with sonication), microwave irradi-


ation of the neat reagents was attempted. Under normal conditions, this is not

effective for ketones.385 However, some ketones react readily when the flask is

supported in an alumina bath, generating much higher temperatures.386 Since

these conditions proved ineffective, a procedure for oxime formation on silica

gel387 was adapted. Basic and acidic alumina were also tried as substitutes.

Unreacted starting material was recovered in all cases. It is interesting to note

that 1a is stable at room temperature in acetic acid (compare formic acid,

Section 3.3.1 on page 128).

Solvent Catalyst Conditions Time Ref.THF - reflux 6 h 388

AcOH - rt 18 h 389AcOH - ultrasound, rt 2 h 390EtOH basic Al2O3 reflux 3 h 391

- - microwave, rt to 115 ◦C 5 min 386- silica gel microwave, rt to 115 ◦C 5 min 387- basic Al2O3 microwave, rt to 115 ◦C 5 min 387- acidic Al2O3 microwave, rt to 115 ◦C 5 min 387

Ultrasound: 40 kHz, 50 W transmitted. Microwave: 1400 W (700 W output).

Table 3.4: Unsuccessful treatment of 1a with excess tosylhydrazide.

The tosylhydrazide used was prepared by a published procedure,384 omitting

the optional recrystallisation, which caused decomposition. Identity and purity

(especially the absence of the typical384 impurity ditosylhydrazide)392 were

confirmed by TLC and 1H NMR.393

No other promising methods of tosylhydrazone formation were located. El-

Sayed’s recent review of sulfonohydrazides,394 although published in 2004, ap-

pears to have been written decades earlier. The only post-1971 references cited

are the author’s own, and these are cited only in the final sentence.

3.5.3 Indirect Deoxygenation.

3.5.3.1 Formation of Cyclic Thionocarbonate (80).

Given the failure of the direct reductions, deoxygenation of 36h was at-

tempted. The usual approaches to hydroxyl deoxygenation,395 involving either


36h

O

O

O

H HOH

HO

O O

N

S

NN N

80

O

O

O

H HO

O

O O

S

67% from 1a

DMF, 90 °C

Scheme 3.19: Formation of cyclic thionocarbonate 80.

hydride reduction of sulfonate derivatives or radical reduction of thiocarbonyl

derivatives,396 would require derivatisation of the extremely hindered and un-

reactive 1α-hydroxyl group. While direct acetylation has been achieved using

NEt3,82 this is ineffective for benzoylation (as seen with the less-hindered 1h,

Section 3.3.1 on page 128). 1b has been mesylated at C-2 under mild condi-

tions;82 however, the yield was very low (32%), and C-1 is much more hindered.

Thus, direct installation of hindered functionalities at C-1 is likely to be dif-

ficult. Given that borohydride reduction of 1a proceeds exclusively from one

face, and sluggishly, the reduction step is also likely to be challenging. Of

the two approaches, ionic versus radical reduction, radical reductions are less

susceptible to hindrance.396 Inspired by the indirect diacetylation of 36h via

the 1,2-orthoacetate (Section 3.1),27 radical deoxygenation of a cyclic thiono-

carbonate was attempted.

Treatment of 36h with 1,1’-thiocarbonyldiimidazole (Scheme 3.19) in DMF

gave 80 in high yield. Since this gave inseparable 8-epimers, the reaction was

subsequently performed in two steps from 1a without separation of interme-

diate 36h and 38h. By 1H NMR, the H-1 and -2 signals of 80 were shifted

downfield, and the characteristic 13C peak (191 ppm) of cyclic thionocarbon-

ates was present.


3.5.3.2 Unsuccessful Radical Deoxygenation Attempts.

Although radical reductions have traditionally been performed with organos-

tannanes, these compounds are highly toxic, and purification of the products

tends to be difficult.397 This has led to extensive and fruitful research into al-

ternatives, such as silanes and phosphites.396 Perhaps the most promising396 of

these alternatives is hypophosphorous acid, H3PO2. Typically, this is buffered

with NEt3, and AIBN serves as radical chain initiator.398 However, other

initiators and bases have been successfully substituted.399 The standard396

conditions of excess H3PO2 and NEt3 in refluxing 1,4-dioxane were used; how-

ever, benzoyl peroxide was substituted for AIBN because the latter was not

at hand. (BzO)2 gives superior results to AIBN with alkyl phosphites.396 De-

spite adding seven portions of (BzO)2 over five hours at reflux, most starting

material was recovered (70%), along with 15% deprotected diols 36h/38h. No

deoxygenated products were detected.

One report used intriguingly simple conditions: magnesium in methanol.400

Although the method had only been proven for cyclic thionocarbonates of

2,3-dihydroxy esters, its extreme simplicity and nontoxicity were attractive.

Treatment of 80 with excess Mg turnings in MeOH at reflux for 40 minutes

gave no reaction. Addition of activated401 Mg turnings had no effect after a

further two hours’ reflux. Starting material was recovered (94%).

3.5.3.3 Radical Deoxygenation using nBu3SnH.

The traditional nBu3SnH/AIBN route was then attempted. This is the best--

established route (especially for cyclic thionocarbonates),402, 403, 404 but often

gives side reactions,405, 406 sometimes to the exclusion of the desired deoxy

products.407 An indispensible paper by Kanemitsu et al thoroughly describes

the theory and practice of the procedure, including control of side reactions.405

The nBu3SnH used was prepared by a published procedure408 with reduced

reaction time (10 minutes),409 distilled and stored in darkness under argon at


O

O

O

H RHO

O O

36f

O

O

O

H H

O O

83

O

O

O

H HO

O

O O

OOH

80 + +

detected by LCMS

4%

1) nBu3SnH

AIBNtoluene

80 °C, 6 h

2) silica gel

H

H

81b

82b

22%

25%

R

Scheme 3.20: Radical deoxygenation of 80.

-20 ◦C. A mixture410 of this and AIBN was added, in small portions, to 80 in

deoxygenated405 toluene at 80 ◦C over 6 hours. Flash chromatography gave the

desired 81b (25%) and its 8-epimer 82b (22% — see Scheme 3.20). Further

chromatography gave a small amount of the cyclic carbonate 83, a common405

byproduct of this procedure. The expected 2-deoxy regioisomers 36f were

not isolated; however, the less polar fractions eluted first contained a complex

mixture of products, heavily contaminated with organotin compounds. Analy-

sis of this mixture by reverse-phase liquid chromatography/mass spectrometry

(RP-LC/MS) was performed by Claudio Medana at Turin University. Com-

parison of the early fractions with 81b and 82b confirmed the presence of

two less polar compounds isomeric with 81b (Figure 3.12). Surprisingly, the

presumed 36f coeluted with the tin contaminants on C-18 reverse-phase, as on

silica gel. Also, total ion count detection showed only a single broad organotin

peak. Mass-selective detection, however, gave well-resolved peaks for the four

products, illustrating the power of LCMS; none of the peaks were apparent

by UV detection. The presence of 81b/82b in the early fractions shows that

these were the major products, at least 2:1 relative to 36f.

3.5.3.4 Potential Improvements.

Given the incomplete recovery of 81b/82b, the yield could clearly be in-

creased. One simple and effective way of removing organostannane reagents


0

20

40

60

80

100

0

20

40

60

80

100total ion count

50 ≤ m/z ≤ 700 (early fractions) re

lativ

e ab

und

ance

18.60

m/z = 377 (early fractions)

m/z = 377 (pure 81b/82b)

82b

81b

19.03

15.95

19.3915.21

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Time (min)

15.95

15.24

0

20

40

60

80

100

Figure 3.12: RP-LCMS traces of early fractions versus 81b/82b.

and byproducts is by washing an acetonitrile solution with hexanes.411, 410

However, in this case, the early fractions remained complex after washing,

despite a dramatic reduction in organostannanes (mass reduced by a third).

An alternative method is C-18 reverse phase chromatography.412 In this case,

C-18 RP-TLC in 80% MeCN/H2O confirmed the effectiveness of the wash: the

petrol layer consisted of baseline material which was absent from the MeCN

layer. However, the desired products were not resolved by RP-TLC, smear-

ing badly. It would not be advisable to apply either of these methods to the

crude reaction mixture: the initial reaction products are themselves alkyltin-

substituted thiocarbonates, which are cleaved on silica to give the desired

alcohols.405 Two more complex methods of organostannane removal, involv-

ing treatment with NaBH3CN413 or I2 and KF,397 are harsher and have the

potential for side reactions.


Thus, the organostannane route used above has serious disadvantages: the

requirement for freshly prepared reagents, the difficulty of purification, the

low recovery of products and most importantly toxicity. While nBu3SnH

is less toxic than other organostannanes,414 it is prepared from the highly

toxic (nBu3Sn)2O,415 and the toxicity of the organostannane byproducts is

unknown. This route is therefore not recommended. There are now many

alternatives,398, 416, 417 some of which have been successfully applied to cyclic

thionocarbonates.418, 419

3.5.3.5 1-Deoxysalvinorin A (81a).

81a

22 101011

O

O

O

H HO

O

O O

Figure 3.13: 1-Deoxysalvinorin A (81a).

Acetylation of 81b (Ac2O/pyridine/DMAP, room temperature) gave 81a.

Both H-2 (δ 4.74, tt) and H-10 (δ 1.10, dd) showed new trans-diaxial cou-

plings to H-1α (δ 1.50, td), and smaller couplings to H-1β (δ 1.95-1.89, m).

The HMQC spectrum correlated these peaks with a new 13C peak at 26.7 ppm.

The DEPT spectrum confirmed C-1 as a methylene, and HRMS confirmed the

molecular formula. The remaining data was extremely similar to 1a.

Chapter 4

Bioassays.

4.1 Insect Antifeedant Activity.

A number of neoclerodane diterpenoids, very similar in structure to the salvi-

norins, display a broad range of activities against insects.2 Some act as in-

sect antifeedants, or appetite suppressants, rather than insecticides. Hundreds

of clerodane diterpenoids have been tested; a thorough review is available.420

Salvinorins C-F (1c-1e and 1f) fit an empirical pharmacophore for antifeedant

activity against Tenebrio molitor:421 an α,β-unsaturated carbonyl approxi-

mately 10 Å from the oxygen of a conformationally constrained furan. By

contrast, a saturated C-18 carbonyl, as found in salvinorins A and B (1a and

1b), seems to confer activity against Spodoptera littoralis.422

A selection of these compounds were therefore screened for antifeedant ac-

tivity. This work was done in collaboration with Dr Charles Robin and Dr

David Heckel in the Department of Genetics at the University of Melbourne.

The species selected was Helicoverpa armigera, which has been widely used in

antifeedant tests.420 A standard choice assay employing sweetened fibreglass

discs422, 423 was used, with one modification. In early tests, the larvae refused

to eat dry discs; the discs were therefore moistened, which gave satisfactory

results. Larvae were presented with two discs: both were sweetened with su-

crose, and one was treated with test compound. Tests were terminated when

161

162 CHAPTER 4. BIOASSAYS.

more than half of one disc had been eaten. For full detail, see the Experimental

Section.

The change in masses of the control (C) and treated (T) discs allow the cal-

culation of the antifeedant index, AI:

AI =100 × (∆C − ∆T )

∆C + ∆T

Antifeedant indices fall between +100 (maximal antifeedant effect) and -100

(maximal phagostimulant effect); a value of zero indicates no effect.

The results are shown in Table 4.1.

Compound AI SEMsalvinorin A 1a 0 30salvinorin C 1c 10 26

divinatorin A 28a 6 11divinatorin B 28b 2 23divinatorin C 28c 16 33

Table 4.1: Antifeedant test results.

None of these compounds had a statistically significant effect. While the fact

that four of the five compounds had a slightly positive AI suggests a weak

(nonsignificant) effect, the standard error of the mean (SEM) is in each case

larger than the mean itself. By contrast, potent antifeedants often have AI

values of close to 100, with SEM < 5.420 The low means and high variabil-

ity observed here suggest that these compounds are not potent antifeedants

against this species. Nonetheless, the value of this experiment was limited by

the unavailability of a proven antifeedant as a positive control compound. Also,

there are significant differences between species; compounds inactive against

one species are often active against others.422, 420 The most common larvae

observed feeding on S. divinorum in Oaxaca were of the genus Chryocerinae.41

4.2. EUKARYOTIC PROTEIN SYNTHESIS INHIBITION. 163

4.2 Eukaryotic Protein Synthesis Inhibition.

Dr Jerry Pelletier at McGill University (Montreal) tested salvinorins A-D (1a-

1d) and divinatorins A-C (28a-28c) for inhibition of eukaryotic protein syn-

thesis. The assay used has been described in detail elsewhere.424 Briefly, lu-

ciferase, the protein responsible for chemiluminescence in fireflies, is produced

in vitro in a cell culture. Addition of a protein synthesis inhibitor (such as

anisomycin) causes a reduction in light output relative to the control. The

light output thus provides a simple measure of protein synthesis. The results

are shown in Figure 4.1. Luciferase from two species (firefly and Renilla) was

used. None of the compounds tested caused significant inhibition at 50 µM

(significant inhibition is defined as a relative light output of 30% or less). The

positive control, anisomycin, caused complete inhibition at 10 µM.

Relative light output (%)

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

anisomycin (+ve control)

1a

1b

1c

1d

28a

28b

28c

RenillaFirefly

Figure 4.1: Luciferase assay results for salvinorins and divinatorins (50 µM).


4.3 Antimicrobial Activity.

4.3.1 Bacteria and Fungi.

28a28b28b29a

R1

OH OH H H

R2

O

H HR1

18

R2

HOHOAcH

R3

HMeHHO OR3

Figure 4.2: (-)-Hardwickiic acid and divinatorins A-C.

(-)-Hardwickiic acid (29a) has been reported to display potent, broad-spectrum

activity against bacteria and fungi.217 Divinatorins A-C (28a-28c) were there-

fore screened against standard antibiotic susceptible strains of Escherichia coli,

Staphylococcus aureus, Bacillus subtilis, and Candida albicans, using broth mi-

crodilution425, 426 and disk diffusion427 assays in each case.196 This work was

done in collaboration with Professor Roy Robins-Browne and Andrea Bigham,

in the Department of Microbiology and Immunology at the University of Mel-

bourne.

No activity was apparent against any of the test organisms at 100 µg/mL or 100

µg/disk. These data extend the stringent structure-activity requirements of

29a.217 In particular, a hydrogen bond donor at C-18 seems to be a necessary,

but not sufficient, condition of activity.217

To probe this further, we decided to screen (+)-hardwickiic acid (ent-29a).

Ent-29a proved active against Staph. aureus (minimum inhibitory concen-

tration (MIC) 25 µg/mL) and B. subtilis (MIC 12.5 µg/mL, 10 mm zone

of inhibition), but much less potent than its enantiomer (MIC 0.78 µg/mL

against B. subtilis).217 The assays are shown in Figure 4.3. A very small zone

of inhibition is apparent around ent-29a in the disk diffusion assay.

An undergraduate investigation published without peer review429 reported that

4.3. ANTIMICROBIAL ACTIVITY. 165

E. coli(+)-hardwickiic acid (ent-29a)

(+)-hardwickiic acid (ent-29a)

MICMIC

1005025

12.56.253.12

1.56

0.78

0.390.19

µg/mL

B. subtilis S. aureus

E. coli

C. albicans

Staph. aureus

B. subtilis

Crude extract

streptomycin sulphate(positive control)

acetone(negative control)

Figure 4.3: Disk and microdilution assays for ent-29a and crude extract.428

the acetone extract of S. divinorum was active against a wide range of bacteria.

We were unable to confirm these results. The acetone extract of the commercial

material, as well as 1a, showed no activity at 100 µg/mL or 100 µg/disk.

Apparently insufficient 29a and oleanolic acid (31) were present to elicit an

effect (31, like 29a, is active against B. subtilis and Staph. aureus).245

4.3.2 HIV-1.

Several κ opioids have been shown to inhibit HIV replication in vitro in several

human cell types.430 They appear to act as viral entry inhibitors, by causing

downregulation of the CXCR4 co-receptor, used by the virus to attach to the

cell.431 HIV entry inhibitors are currently the focus of intensive research.432

The use of synergistic drug cocktails has already produced dramatic progress,

and the addition of a further mechanism is expected to offer still greater syn-

ergy. KOR antagonists, which do not themselves inhibit viral replication,

have been shown to enhance the effects of the standard therapy azidovudine

(AZT).433

Beside their effect on viral replication, κ opioids also appear to counteract the


neurotoxic effects of HIV in infected cells.434, 435 Finally, since nausea is a com-

mon side effect of standard therapies,436 the possible antiemetic activity437, 438

of κ opioids may be advantageous.

4.3.2.1 NL4.3 and AD8 Strains.

NL4.3

HIV strain:

AD8

1a (10-6 M)

Me2SO only

control

101

102

103

104

105

106

107

0 2 4 6 8

Days post infection

HIV

co

pie

s /

106 c

ells

Figure 4.4: HIV-1 replication assays (NL43 and AD8 strains).

Based on the above rationale, we submitted salvinorin A (1a) for testing

against HIV in vitro. Testing was performed by Dr Sharon Lewin and Ajantha

Solomon at Monash University’s medical department (Alfred Hospital). Two

strains of HIV-1 were tested: NL4.3 (T cell tropic) and AD8 (macrophage

tropic). HIV was quantitated using real-time PCR.439, 440 Incubation of pe-

ripheral blood mononuclear cells (PBMCs) with 1 µM salvinorin A had no

effect on viral replication after one week (Figure 4.4). There was also no

change in expression of CXCR4, CCR5 or CD4 receptors after one day, as

determined using FACS staining.440

4.3.2.2 ROJO and TEKI Strains.

Two other HIV-1 strains were tested for the US National Institute of Allergy

and Infectious Diseases (NIAID), under the direction of Dr Stephen Turk.

4.3. ANTIMICROBIAL ACTIVITY. 167

The isolates tested were ROJO (syncytium inducing/lymphocyte tropic) and

TEKI (non-syncytium inducing/monocyte tropic). Again, salvinorin A (up

to 230 µM) had no effect on viral replication in PBMCs after one week of

incubation (eg. Figure 4.5).441 The positive control, AZT, strongly inhibited

viral replication (IC50 = 13 nM). Tests were performed in triplicate.

20

40

60

80

100

120

140

160

0.00 0.02 0.07 0.23 0.73 2.30 7.27 23.0 72.7 230

0.00 0.10 0.32 1.00 3.16 10.0 31.6 100 316.2 1000

CONCENTRATION (nM)

CONCENTRATION (µM)

20

40

60

80

100

120

140

160

% VIRUS CONTROL

% CELL CONTROL

% VIRUS CONTROL

% CELL CONTROL

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140 PE

RC

EN

T O

F C

EL

L C

ON

TR

OL

PE

RC

EN

T O

F V

IRU

S C

ON

TR

OL

PE

RC

EN

T O

F V

IRU

S C

ON

TR

OL P

ER

CE

NT

OF

CE

LL

CO

NT

RO

L

AZT

salvinorin A (1a)

Figure 4.5: HIV-1 replication assays (ROJO isolate).

4.3.2.3 Discussion.

These results were surprising given the established activity of the κ opioids

U50,488 and U69,593, which are of comparable potency to 1a. However, the

extremely potent endogenous κ opioid dynorphin A showed no activity;442, 443

this has been speculatively attributed to metabolism by peptidases in the cell

culture. Consistent with this, the potent fragment dynorphin A1−13, which has

a half life in plasma of less than one minute,444 caused negligible inhibition of

HIV-1 replication.442 This suggests that the rapid metabolism87, 88 of 1a may

account for its lack of activity.

However, this hypothesis cannot account for other evidence. The intact peptide

dynorphin A1−17, which has a half life in plasma of three hours,444 also caused


no inhibition.442 Moreover, only brief exposure to U50,488 (< 30 minutes)

is necessary to inhibit HIV-1 replication.431 Thus, there appear to be other

factors at work. Wang et al found that 1a causes much less κ opioid receptor

internalisation than U50,488, despite their similar potencies.81 Such differences

may involve the apparent subtypes445 of κ opioid receptors.

There is an anecdotal report of improved health following S. divinorum use by

an AIDS patient.446 While the above data suggest that 1a itself has no activity

against HIV, oleanolic acid (31) is known to be active by two complementary

mechanisms,243, 242 as discussed in Section 2.2.4.1 on page 96. Given a sufficient

dosage, therefore, the crude plant extract would probably exhibit some activity.

No relevant data was located regarding other compounds in the plant.

4.4 NCI Anticancer Screen.

Salvinorins B and C (1b and 1c) and divinatorins A-C (28a-28c) were tested

by the US National Cancer Institute, in the standard in vitro assay against 60

tumour cell lines.447 The standard measure of tumour cell growth inhibition

is the GI50 – the drug concentration at which growth is reduced to 50% of the

control value. In most cases, this degree of inhibition was not achieved. Where

substantial inhibition occurred, the GI50 was above 10−5 M in all cases (Figure

4.6). In the one apparent exception, 28b exhibited a GI50 of just under 10−6

M against the SF-539 brain tumour cell line. However, a significant inhibition

of growth was already apparent at 10−8 M (59% of control), which scarcely

increased up to 10−5 M (45%), a 1000-fold increase in concentration (Figure

4.7). The GI50 value was therefore clearly artefactual. In all other cases,

inhibition of growth increased sharply at higher doses: see Figure 4.6. GI50values this high are strongly predictive of poor performance in subsequent

assays,448 and the NCI therefore did not select the compounds for further

testing.

Salvinorin B (1b) completely inhibited growth, giving negative growth rates,

4.4. NCI ANTICANCER SCREEN. 169

All Cell Lines

% G

row

th

-8 -7 -6 -5 -4

Log10

Concentration (M)Log10

Concentration (M)

-8 -7 -6 -5 -4

-8 -7 -6 -5 -4

All Cell Lines

-8 -7 -6 -5 -4

100

50

-50

-100

0

100

50

-50

-100

0

100

50

-50

-100

0

100

50

-50

-100

0

100

50

-50

-100

0

% G

row

th

-8 -7 -6 -5 -4

salvinorin B (1b)

(NSC D737715)

divinatorin A (28a)

(NSC D737768)

divinatorin B (28b)

(NSC D737769)

divinatorin C (28c)

(NSC D737770)

salvinorin C (1c)

(NSC D737716)

Figure 4.6: NCI 60 cell line results for salvinorins and divinatorins

in 8 cell lines. The “Total Growth Inhibition” concentration (TGI) was above

10−5 M in each case. No other compound gave this degree of inhibition. Only

one cell line gave below -50% growth, the standard threshold for clear cyto-

toxicity: the SNB-75 CNS tumour line, with an LC50 slightly below 10−4 M

(Figure 4.7). It is interesting that CNS tumour cells responded more strongly

to 1b than the other categories. Nonetheless, the potencies even in these cases

are unremarkable.

Salvinorin A (1a) was not accepted even for in vitro testing. Given the ap-


Log10

Concentration (M)

100

50

-50

-100

0

-8 -7 -6 -5 -4

Log10

Concentration (M)

100

50

-50

-100

0

% G

row

th

-8 -7

SF-268SNB-75 SNB-19

SF-295 SF-539U251

-6 -5 -4

salvinorin B (1b)

(NSC D737715)

divinatorin B (28b)

(NSC D737769)

CNS tumour cell line:

Figure 4.7: CNS cell line results for divinatorin B and salvinorin B.

parently rapid deacetylation of 1a in cell culture, however, it seems likely that

the results would in any case have been close to those of 1b. Surprisingly,

the reason for the rejection of 1a was that it had already been tested in vivo

more than 20 years earlier.449 The results have apparently not been published

previously. Using a standard protocol,450 mice were implanted with P388 tu-

mour cells (lymphocytic leukaemia); test animals were injected with 1a daily

for 5 days. Doses of 1a up to 76 mg/kg had no effect, while 152 mg/kg was

slightly toxic (survival time 79% of control).451 The value of 0% given on the

NCI website449 is a typographical error.451

4.5 Activity at the κ Opioid Receptor.

Previous work found that 1b was inactive at the κ opioid receptor, while re-

placement of the acetoxy group in 1a with more hindered esters dramatically

reduced binding affinity.135 No information was available about other func-

tional groups.

In order to further explore salvinorin A’s structure-activity relationships, iso-

lated compounds and derivatives were tested in vitro for binding affinity at

cloned opioid receptors using radioligand assays. Those compounds with sub-

micromolar affinity were also screened for agonist potency and efficacy using

4.5. ACTIVITY AT THE κ OPIOID RECEPTOR. 171

a functional assay (see Experimental Section for details). The testing was

done under the direction of Dr Bryan Roth, under the auspices of the National

Institute of Mental Health’s Psychoactive Drug Screening Program.

No compound showed affinity for µ or δ subtypes (K i > 1 µM), and the

following discussion will therefore discuss the κ opioid receptor exclusively.

The raw data are shown in Table 4.2; structures can be found in the discussion

below. The following discussion will express affinities and potencies relative to

1a:

Krel(x) =Ki(x)

Ki(1a)

and

ECrel(x) =EC50(x)

EC50(1a)

A value of 10 thus signifies a potency or affinity 10 × lower than 1a.

O

O

O

H HOO

O

O O

1a

4 ± 1 nM

46 ± 8 nM

Ki

EC50

Figure 4.8: KOR binding affinity and potency of salvinorin A.

4.5.1 Other Salvinorins and Divinatorins.

4.5.1.1 Radioligand Binding Results.

There have been conflicting results on salvinorin B (1b). Initial work by the

Roth group found it inactive (K i > 10 µM),135 but subsequent work by Béguin

et al reported high affinities (K i = 66,137 111271 or 155125 nM). Retesting by the

Roth group indicated very weak affinity (3.1 µM versus 1a control 44 nM).452


K i s.e.m. K rel EC50 s.e.m. ECrel Emax s.e.m.nM nM %

1a 4 1 46 8 100 191b 3,153* 71*1c 1,022 262 2551d >10,000 >2,5001e >10,000 >2,500

28a >10,000 >2,50028b >10,000 >2,50028c >10,000 >2,50046 18 2 4.5 315 35 6.8 108 11

37a 163 50 41 244 102 5.3 78 1736e 1,125 28136c >10,000 >2,50051 156 18 39 126 36 2.7 108 1435 59 11 15 78 21 1.7 107 549 6 1 1.5 223 60 4.8 103 1350 6 2 1.5 624 200 14 116 1077 347 53 87 >10,000 >217

81a 18 2 4.5 141 43 3 122 2759 >10,000 >2,500

60a 2,900 400 725* = direct comparison to 1a (44 nM) against [3H]diprenorphine

Table 4.2: KOR radioligand and functional assay results.

O

O

O

H HOHO

O O

O

O

O

H HOR1

R2O

O O

28a28b28c

R1

OH OH H

R2

O

H HR1

R2

HOHOAc

R3

HMeH

O OR3

Krel

>2,500>2,500>2,500

1c1d1e

R2

Ac H Ac

R1

AcAcH

Krel

255>2,500>2,500

1b

Krel

71

Figure 4.9: KOR binding affinities of salvinorins and divinatorins.

The discrepancy is not as great as it appears, since the two groups obtained

different absolute affinities for 1a. Béguin et al reported values of K rel = 66,137

85271 or 120.125 The Roth group’s latest result (K rel = 71)452 is concordant.


The nonconcordant earlier result (K rel > 500) may have resulted from the use

of a different radioligand – [3H]bremazocine rather than [3H]diprenorphine.

Moreover, in all cases the binding affinity of 1b was negligible relative to 1a.

Salvinorin C (1c) also showed negligible binding affinity compared to 1a (K rel

= 255, Figure 4.9). Salvinorins D (1d) and E (1e) and divinatorins A-C (28a-

28c) showed no affinity (K rel > 2,500). Based on these results, we tentatively

concluded270 that 1a is the sole κ opioid present in the plant. Recently, Lee et

al have reported that salvinorins B (1b) and G (1g) and divinatorin D (28d,

Figure 2.51 on page 118) bind to the KOR, but with much lower affinities

and potencies than 1a ( K rel = 66 – 418).137 Since the concentrations of

these compounds are also orders of magnitude lower, their contribution to

the activation of the KOR by S. divinorum is negligible. Nine other isolated

compounds were inactive. Thus, these results strongly suggest that 1a is

effectively the sole active principle of S. divinorum.

The weak activity of 28d shows that the C ring can be cleaved without total

loss of affinity. Apart from this greater conformational freedom, 28d not only

lacks the 2-acetoxy function, but also possesses a 1α-hydroxy group, both of

which drastically reduce activity in analogues of 1a, as will be discussed be-

low. The structure-activity relationships of this compound are thus markedly

different from those of 1a. Given that its deacetyl analogues 28b and 28e137

are inactive, perhaps the 17-acetoxy function substitutes for the furan ring in

binding. However 28c, which also possesses this function, is inactive. Further

exploration of this productive series of compounds is clearly warranted.

4.5.2 Modification of the Ketone.

Reduction of the ketone to an α-hydroxy group (giving 36e) dramatically

reduced binding affinity (Figure 4.10). Acetylation (giving 36c) abolished

binding entirely. These results were surprising, since molecular modelling had

indicated that these modifications would not affect binding.67 Although the

very low affinities of 1e, 1c, 36e, and 36c might appear to suggest that the


O

O

O

H HOO

O

O

O O

O

O

O

H HOH

O

O

O O 36c

>2,500Krel

36e

281Krel

O

O

O

H HO

O

O O 81a

4.5

3

Krel

ECrel

Figure 4.10: KOR activity after ketone modifications.

ketone is part of the pharmacophore, the relatively high affinity and potency

of 1-deoxy compound 81a show that it is not. This suggests that 1α-hydroxy

or acetoxy groups interact unfavourably with the KOR.

Curiously, saturating the 3,4-double bond in 1e (inactive) gives 36e (active),

while the opposite is true of 1c (active) and 36c (inactive). However, none

of these compounds show appreciable (sub-micromolar) affinity. The effect of

this double bond itself therefore remains unclear.

4.5.3 Modification of the Acetoxy Group.

O

O

O

H HOO

O

O O 46

4.5

6.8

Krel

ECrel

O

O

O

HOH

O

O O

O

O

O

O

HO

O O

O

O

H

60a

725Krel

59

>2,500Krel

Figure 4.11: KOR activity after acetoxy group modifications.

It had previously been shown that substituting more hindered esters for the

2-acetoxy group reduced binding affinity.135 This suggested that the less-


hindered formate 46 (Figure 4.11 on the preceding page) might prove more

potent, but in fact both affinity and potency were reduced. The acetoxy group

therefore appears to be the optimal alkyl chain length.

The autoxidation products 59 and 60a were also screened. 59 was inactive, as

Lee et al reported for the incorrect structure 62 (Figure 3.8 on page 139).271

Surprisingly, 60a showed weak affinity at the KOR (K rel = 725). This provides

further evidence that the 2-acyloxy function in 1a is not essential for binding,

but that modifying this function usually reduces affinity dramatically.

4.5.4 Modification of the Methyl Ester.

O

O

O

H HOO

O

OH 77

87

>217

Krel

ECrel

O

O

O

H HOO

O

O OH 67a

-

Figure 4.12: KOR activity after methyl ester modifications.

The role of the methyl ester in binding was strongly confirmed by the results for

18-hydroxy derivative 77, which appeared to be an antagonist, since it bound

but did not activate the receptor. However, no functional tests of antagonist

potency were performed.

The precursor acid 67a was not tested, since the 8-epimers streaked on silica,

so separation was not attempted. Lee et al subsequently separated the epimers

using repeated chromatography. They reported, surprisingly, that the natural

H-8β epimer was inactive at the KOR, while the H-8α epimer had high affinity

(K i = 48 nM).361


4.5.5 Modification of the Lactone.

O

OH

O

H HOO

O

O O

O

O

H HOO

O

O O

O

O

HO

O

O

O O50

1.5

14

49

1.5

4.8

35

15

1.7

Krel

ECrel

Krel

ECrel

Krel

ECrel

Figure 4.13: KOR activity after lactone modifications.

The high affinities of lactol 35, ether 49, and enol ether 50 (all full agonists)

show that the lactone carbonyl is not essential for binding or activity. How-

ever, the latter two compounds, especially 50, showed reduced potency in the

functional assay.

4.5.6 Modification of the Furan Ring.

O

O

O

H HOO

O

O O

HH13

51

39

2.7

Krel

ECrel

O

O

O

H HOO

O

O O

8

37a

41

5.3

Krel

ECrel

Figure 4.14: KOR activity after furan modifications.

The substantially reduced affinity of tetrahydrofuran 51 suggests that the fu-

ran ring is involved in binding. As described earlier, one 13-epimer of 51

was isolated for characterisation. The binding affinity did not differ signif-

icantly from the 1:1 mixture (123 nM vs 156 nM). The reduced affinity of


8-epi-salvinorin A (37a) also supports the role of the furan; since the lactone

carbonyl is evidently not necessary for binding, the orientation of the furan is

the salient difference between this compound and 1a. The reduction in potency

of these compounds was far smaller than the reduction in affinity, particularly

in 51.

4.5.7 Incorporation into a Revised Binding Model.

O

O

O

H HOO

O

O O

OTyr 313

OTyr 312

Gln 115N

OH

H

H

H

O

Tyr 139

H

Figure 4.15: Westkaemper’s original binding model.

The original report of the activity of 1a at the KOR67 included a proposed

model of its interactions with the receptor (Figure 4.15). As shown, the furan

oxygen and the acetyl, methyl ester and lactone carbonyls act as hydrogen

bond acceptors. The model, created by Dr Richard Westkaemper, was derived

from a model of U69,593 binding reported previously.

While the proposed roles of the furan, acetoxy group and methyl ester were

consistent with our data above, the proposed interaction with the lactone

seemed unlikely given the high affinities of 35, 49 and 50 mentioned above.

Westkaemper subsequently proposed a revised model,453 which drew upon the

above data on the roles of each functional group in binding, although this was

regrettably not acknowledged in the finished paper. Additional data on the

role of the 2-acetoxy group was provided by others.453 Complementing these

chemical data, site-directed mutagenesis in the Roth group allowed selective


modification of the receptor itself, identifying the residues involved in binding

to 1a.453 The revised model is shown in Figure 4.16, visualised from coordi-

nates kindly provided by Dr Westkaemper. For clarity, hydrogen atoms are

not shown.

Tyr 119 Tyr 119

Tyr 320

- - - H-bonds*

*not present simultaneously *not present simultaneously

Tyr 320

Glu 297 Glu 297

Tyr 313 Tyr 313

Ile 294- - - hydrophobic interactions

- - - hydrophobic interactions

- - - H-bonds*

Ile 294

Figure 4.16: Westkaemper’s revised binding model (stereoview).

The revised model has only one residue in common with the original: tyrosine

313. The proposed interaction is a hydrophobic one with the acetyl methyl

group, however, rather than the original H bond to the carbonyl oxygen. This

was suggested by the surprising result that mutation of this tyrosine residue to

phenylalanine (lacking the hydroxyl) did not affect binding affinity.453 Three

of the five proposed interactions in the new model are hydrophobic. The

mutagenesis data suggest that the furan can form an H bond to either of

tyrosines 199 or 320.453 While these bonds are both shown in Figure 4.16 for

clarity, they cannot be present simultaneously, since only one nonbonded pair

is available on the furan oxygen.

One interesting aspect of this model is that three of the five residues involved

(isoleucine 294, glutamic acid 297 and tyrosine 313) are unique to the κ sub-

type. This provides a plausible explanation for the striking κ selectivity of


1a and analogues; very few derivatives show submicromolar affinity δ or µ

subtypes, and none of these are selective.25, 82, 136

4.5.8 Subsequent Results.

O

O

O

H HORO

O O

O OO

O

O

O

O

O

H HOO

O

O OH67a (H-8b)

>770Krel

O

O

O

H HOO

O

O O

HHO

34a

102ECrel

O

OHO

Krel

ECrel

84

6

4

85

0.31

0.13

86

12

0.13

Ki (m)

Krel

(m/k)

Figure 4.17: KOR binding affinities and potencies of recent derivatives.

Since publication of these results, numerous 1a analogues have been tested

in vitro by other groups. These results again confirm that replacement of the

2-acetoxy group (with other esters, ethers, carbonates, amides etc) almost in-

variably reduces binding affinity.95, 271, 82 There are exceptions, however. The

high affinity and potency of ethyl ether 8495 indicate that the acetyl carbonyl

is not essential for activity. This result provides strong support for the hy-

drophobic interaction at C-2 in Westkaemper’s revised model (Figure 4.17).

Another notable derivative is methoxymethyl ether 85,271 to date the only

derivative significantly more potent than 1a. Surprisingly, benzoate 8682 and

some related derivatives activate the µ opioid receptor, but are not selective.

Other results indicate that epimerisation at C-2 does not abolish activity in

all derivatives.125

There is less data on modifications at C-18,361, 95 but all esters and amides


tested showed large reductions in affinity and potency. The loss of affinity on

O-demethylation (giving 67a)361 strongly supports the carbinol hydrophobic

interaction in Westkaemper’s model over the more intuitive carbonyl H bond.

Even less data has been reported on furan modifications, but the greatly re-

duced potency of salvinicin A (34a)25 confirms that hindrance in this region

interferes with binding. Some synthetic modifications of the furan ring have

been prepared,136 but biological data have not been reported.

Chapter 5

Experimental.

5.1 General Conditions

5.1.1 Instruments and Procedures.

Flash Chromatography: Flash column chromatography was performed ac-

cording to Leonard’s194 procedure using Scharlau silica gel 60 (particle

size 0.04 - 0.06 mm) or Merck silica gel 60. Mass ratios up to 400:1 versus

crude product were used for difficult separations (∆hRf < 5). The ac-

tivated carbon used was Merck aktivkohle 2183 powder (20:1 mass ratio

versus crude product).

HPLC: Spherex 5 µm silica column (250 × 10 mm), flow rate 2 mL min−1

with refractive index detection. Solvent front reached detector at 6.8

min.

HRESIMS: Bruker 4.7T BiOAPEX FTMS.

InChIs: IUPAC International Chemical Identifiers454 were created using winChI

version 1.455

IR: Bio-Rad FTS 165 FT-IR (running WIN-IR v 4.14) and Shimadzu FTIR

8400 (running HYPER IR v 1.57), using thin films on NaCl discs.

181

182 CHAPTER 5. EXPERIMENTAL.

LCMS: Phenomenex Luna 3 µm C-18 column (150 × 2 mm). Gradient elu-

tion: 0.05 % HCO2H in 20-100% MeCN/H2O over 40 minutes. ESI in-

terface with ion trap detector. Detailed conditions have been published

elsewhere.59

NMR: Varian Inova 400, Inova 500 and Unity Plus 400 (running Vnmr 6.1;

some processing on VnmrJ 1.1D, some on iNMR). Bruker US2 800 (run-

ning TopSpin 1.3). 1H NMR are 400 MHz, and 13C NMR 100 MHz, un-

less otherwise stated. 13C multiplicities are based on DEPT experiments.

Assignments are given according to the standard32 clerodane numbering

scheme below. Where 13C assignments are given, all assignments (1H and13C) are based on 2D NMR data (COSY, HMQC, HMBC). In other cases,1H assignments were made by comparison with related compounds for

which such data was available. Stereochemical assignments were based

on coupling constants where possible, and NOESY data elsewhere (eg.

pro-R vs. pro-S in rotatable methylenes). Peaks whose stereochemistry

could not be unambiguously assigned on these bases are listed as “a”

and “b”. Complex first-order multiplets were analysed using Hoye’s al-

gorithms,456, 457 with resolution enhancement using the line-broadening

window function where necessary (not shown in the reproduced spectra).

Abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m =

multiplet, quin = quintet, sext =sextet, sept = septet, b = broad. The δ

values of solvent residual peaks (eg. CHCl3 = 7.26 ppm) and impurities

were taken from Gottlieb et al.458

22

3344

55

101011

66

77

8899 1717

12121111

1313

1616

1515

1414

18181919

2020

Polarimetry: JASCO DIP-1000. Concentration c is in g/l00 mL; the units

5.1. GENERAL CONDITIONS 183

of the specific rotation are (◦·mL·g−1·dm−1).

SEM: Samples were sputter-coated with gold. Fresh leaves were fixed in 2.5%

glutaraldehyde in 0.1 M phosphate buffer at rt for 24 h. The samples were

rinsed in the buffer for 30 min (× 3) and then dehydrated in increasing

concentrations of aq. EtOH for 30 min each (10, 30, 50, 70, 90, and

100%). After two further rinses in fresh 100% EtOH, the leaf samples

were dried in a critical point dryer.

TLC: Merck silica gel 60 F254 plates, visualised with phosphomolybdic acid

in EtOH and heated unless otherwise indicated. “hRf” = Rf × 100.

UV: Shimadzu UV-2401PC (quartz cell).

5.1.2 Reagents.

DMPU and EtSH (Aldrich, 98%) were stored over 4Å sieves.

“Petrol” refers to the fraction boiling at 40-60 ◦C.

Reactions “under Ar” were performed using freshly distilled solvents unless

otherwise indicated.

5.1.3 Plant Materials.

Commercial material Dried S. divinorum leaves, cultivated in Oaxaca Mex-

ico, were purchased from Salvia Space Ethnobotanicals (Berkeley, Cal-

ifornia). Voucher specimens were deposited at the National Herbarium

of Victoria (accession number MEL 2101361) and the University of Mel-

bourne Herbarium (MELU s.n.)

Australian material Additional S. divinorum plants were cultivated in Mel-

bourne. A voucher specimen of the dried leaves was deposited at the

National Herbarium of Victoria (accession number MEL 2145478).


Copaiba balsam was donated by Australian Botanical Products (Hallam,

Victoria).

5.1.4 Assays.

Radioligand Binding Assays performed as previously detailed459, 67 using

cloned receptors stably expressed in HEK 293 cells.

κ: rat KORs with [3H]diprenorphine (50 Ci/mmol, PerkinElmer Inc) or [3H]U69,593

(41.4 Ci/mmol, PerkinElmer Inc) as radioligand.

δ: human DORs with [3H]DADLE (51.5 Ci/mmol, PerkinElmer Inc) as radi-

oligand.

µ: human MORs with [3H]diprenorphine as radioligand. K i values were calcu-

lated using Prism 4.01 (GraphPad Software, Inc) as the mean ±SEM of

quadruplicate (n ≥ 4) determinations. Nonspecific binding was defined

using 10 µM naloxone.

Calcium Flux Functional Assay. Performed as previously detailed135, 460

using cloned rat KORs stably expressed in HEK 293 cells, cotransfected

with the universal G protein Gα16. Ca2+ mobilisation was quantified

using a 96-well FlexStationII with the calcium flux assay kit (Molecular

Devices Corp, Sunnyvale, CA). EC50 and Emax values were calculated

using Prism 4.01 (GraphPad Software, Inc), as the mean ±SEM of qua-

druplicate (n ≥ 4) determinations.

Antimicrobial Tests. The extract and compounds were tested against Es-

cherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 29213),

Bacillus subtilis (ATCC 6633), and Candida albicans (ATCC 90028) us-

ing standard broth microdilution426, 425 (100 - 0.19 µg/mL using two-fold

serial dilutions) and disc-diffusion427 assays (100 µg/disk). All measure-

ments were performed in duplicate. Streptomycin sulfate and ampho-

tericin B were used as positive controls.

5.2. ISOLATION 185

Insect Antifeedant Tests. A standard choice assay employing sweetened fi-

breglass discs423 was used, except that the discs were moistened. Final

instar H. armigera larvae were selected on the basis of head size. Squares

of Whatman GF/A filter (3.6 cm2) were treated with 100 µL of 0.05 M

sucrose solution. Test discs were then treated with 10 µg of test com-

pound in acetone, usually423 expressed as 100 µL of a 100 ppm (100 mg

L−1) solution. Control discs were treated with the same volume of ace-

tone. Discs were dried, numbered and weighed. Before performing the

assay, the discs were moistened with distilled water. After more than half

of one disc had been eaten, the discs were dried and reweighed. Tests

were performed with 5 replicates per compound.

HIV-1 Replication Assays.440 Alfred Hospital: PBMC were stimulated for

72 hours, and then infected with NL4.3 or AD8 (30 ng/ml virus) for 2

hours. Cells were then washed and seeded in 24 well plates. Salvinorin

A was dissolved in Me2SO before addition. Me2SO at the same concen-

tration was also compared to the control. Cells were lysed on day 0,

2, 5 and 7 of infection. HIV was quantitated using real-time PCR.439

NIAID/Southern Research Institute: detailed assay conditions can be

found elsewhere.441

5.2 Isolation

5.2.1 Extraction of Commercial S. divinorum.

Dried S. divinorum leaves (860 g) were powdered and steeped for 1 h in acetone

(3 × 1 L). Filtration and evaporation under reduced pressure gave a dark

green tar (30.5 g). This was purified by flash column chromatography on

an equal mixture of activated carbon and diatomite filter aid, eluting with

a stepwise gradient from acetone to petrol, to give an amber semicrystalline

syrup (5.73 g). Recrystallisations from MeOH and EtOH gave 1a (2.64 g).

The mother liquor was purified by flash column chromatography on silica gel


(5-50% acetone/CH2Cl2 gradient). This was divided, based on TLC (10%

acetone/CH2Cl2), into four series: A (656 mg), B (150 mg), C (359 mg) and

D (77 mg).

TLC: hRf of series A B C D

10% acetone/CH2Cl2 62 36-53 18 12

Series A: flash column chromatography, eluting with a gradient from 50-80%

Et2O/petrol, gave 1c (total yield 219 mg, 0.25 g/kg) and additional 1a, which

was recrystallised from EtOH (total yield 2.9 g, 3.4 g/kg). Further elution

gave 29a (7 mg).

Series B: flash column chromatography on silica gel (35 g) in 70-90% Et2O/petrol,

and recrystallisation from MeOH, gave 1b (13 mg, 0.015 g/kg).

Series C: Trituration in hot Et2O gave 1d (75 mg). Flash column chromatog-

raphy of the mother liquor (60-100% Et2O/petrol) gave four fractions based

on TLC (70% Et2O/petrol): C1 (55 mg), C2 (119 mg), C3 (57 mg) and C4

(39 mg).

TLC: hRf of fraction C1 C2 C3 C4

70% Et2O/petrol 40-43 28-21 17 13

Fraction C1: Repeated flash column chromatography (20% acetone/petrol

and 40-60% Et2O/petrol) gave 28c (23 mg) and 31 (3 mg).

Fraction C2: Repeated flash column chromatography (25% acetone/petrol

and 60-100% Et2O/petrol) gave 28b (41 mg).

Fraction C3: Extensive flash column chromatography (Et2O/petrol, ace-

tone/petrol and EtOAc/petrol) gave additional 28b (total yield 41 mg) and a

mixture of 1e and 1f. Final purification by HPLC (60% EtOAc/petrol) gave

1e (2.8 mg) and 1f (1.1 mg).

Fraction C4 gave additional 1d (total yield 114 mg, 0.13 g/kg).

Series D: Repeated flash column chromatography (60% Et2O/petrol and 4%

MeOH/CH2Cl2) gave 28a (36 mg).

5.2. ISOLATION 187

5.2.2 Extraction of Australian S. divinorum.

Dried, powdered S. divinorum leaves (224 g) were steeped in acetone for 30

min (3 × 250 mL). Filtration and evaporation under reduced pressure gave

a dark green tar (7 g). This was purified by vacuum filtration through a 1:1

mixture of activated carbon (75 g) and diatomite filter aid, eluting with a

gradient from 50-20% EtOAc/petrol, to give series E (97 mg) and F (279 mg)

based on TLC (70% Et2O/petrol).

TLC: hRf of series E F

70% Et2O/petrol 55-68 16

Series E: Repeated flash column chromatography (1% acetone/CH2Cl2 and

20% Et2O/petrol) gave 33 (1 mg). Further flash column chromatography

(0.75% MeOH/CH2Cl2 and 1% EtOH/CHCl3) gave 32 (23 mg) and 30 (12

mg).

Series F: Two recrystallisations from MeOH gave 1a (126 mg).

yield 70% Et2O/ 10% acetone/(mg/kg) petrol CH2Cl2

Salvinorin A 1a 3,400 24 57B 1b 15 14 37C 1c 254 31 60D 1d 132 18 25E 1e 3 23 47F 1f 1 24 40

Divinatorin A 28a 42 37 15B 28b 48 31 31C 28c 27 50 39

(–)-Hardwickiic acid 29a 8 64 45Oleanolic acid 31 3 66 65

Presqualene alcohol 32 3 44 34Peplusol 33 4 75 73

(E)-Phytol 30 3 59 58

Table 5.1: Yields and TLC data (hRf ) of isolated compounds.


5.2.3 Salvinorin A (1a).

InChI=1/C23H28O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h6,8,11,14-17,19H,5,7,9-10H2,1-

4H3/t14-,15-,16-,17-,19-,22-,23-/m0/s1

1a

O

O

O

H HOO

O

O O

colourless crystals, mp (from EtOH) 236-238 ◦C;

lit. (from MeOH) 238-240 ◦C;22

TLC: See Table 5.1 on the previous page.

UV (MeCN): λmax (log ε) 208 (3.76) nm;

lit.23 UV (MeOH): λmax (log ε) 211 (3.72) nm;

1H NMR (800 MHz, CDCl3): δ 7.41 (1H, dt, J = 1.7, 0.9 Hz, H-16), 7.38 (1H,

t, J = 1.7 Hz, H-15), 6.37 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.52 (1H, ddd, J

= 11.7, 5.2, 0.8 Hz, H-12), 5.14 (1H, ∼ddt, J ≈ 11.9, 9.0, 0.9 Hz, H-2), 3.72

(3H, s, CO2CH 3), 2.74 (1H, ∼dd, J ≈ 11.3, 5.6 Hz, H-4), 2.50 (1H, dd, J =

13.5, 5.2 Hz, H-11α), 2.31-2.28 (2H, m, H-3), 2.18 (1H, br s, H-10), 2.17-2.14

(1H, m, H-7β), 2.16 (3H, s, OCOCH 3), 2.07 (1H, dd, J = 12.0, 3.1 Hz, H-8),

1.79 (1H, dt, J = 13.4, 3.1 Hz, H-6α), 1.64 (1H, tdd, J = 13.5, 12.1, 3.4 Hz,

H-7α), 1.58 (1H, td, J = 13.5, 0.9 Hz, H-6β), 1.57 (1H, ddd, J = 13.5, 11.7,

0.8 Hz, H-11β), 1.45 (3H, s, H-20), 1.11 (3H, s, H-19);

13C NMR (CDCl3) data matched previously reported values.22

5.2. ISOLATION 189

5.2.4 Salvinorin B (1b).

InChI=1/C21H26O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7,10,12-15,17,22H,4,6,8-9H2,1-3H3/

t12-,13-,14-,15-,17-,20-,21-/m0/s1

1b

O

O

O

H HOHO

O O

colourless crystals, mp (from hot MeOH) 239-240 ◦C; (from cold Et2O/petrol)

244-245 ◦C;

lit. (from hot MeOH) 213-216 ◦C;23 251-254 ◦C;15, 260 (from cold MeOH) 211-

214◦C;169

1H and 13C NMR (CDCl3) data matched previously reported values.23

5.2.5 Salvinorin C (1c).

InChI=1/C25H30O9/c1-13(26)32-18-10-17(22(28)30-5)24(3)8-6-16-23(29)34-19

(15-7-9-31-12-15)11-25(16,4)21(24)20(18)33-14(2)27/h7,9-10,12,16,18-21H,6,8,

11H2,1-5H3/t16-,18-,19-,20-,21-,24-,25-/m0/s1

1c

O

O

O

H HOO

O

O

O O


clear resin;

TLC: See Table 5.1 on page 187.

[α]16D +70 (c 0.6, CHCl3);

[α]22D +49 (c 0.6, CHCl3) lit;27

UV (MeCN): λmax (log ε) 208 (4.10) nm;

FTIR (film): ν̃max 3145, 2952, 1740, 1643, 1506, 1435, 1372, 1315, 1226, 1174,

1142, 1075, 1041, 961, 950, 910, 875, 789, 754, 695, 667 cm−1;

1H and 13C NMR (CDCl3) data matched previously reported values.27

5.2.6 Salvinorin D (1d).

InChI=1/C23H28O8/c1-12(24)30-18-16(25)9-15(20(26)28-4)22(2)7-5-14-21(27)

31-17(13-6-8-29-11-13)10-23(14,3)19(18)22/h6,8-9,11,14,16-19,25H,5,7,10H2,

1-4H3/t14-,16-,17-,18-,19-,22-,23-/m0/s1

1d

O

O

O

H HOHO

O

O O

fine colourless crystals, mp (from cold Et2O) 185-187 ◦C;


[α]17D +67 (c 1.0, CH2Cl2);

FTIR (film): ν̃max 3475, 3146, 2952, 2861, 1723, 1505, 1435, 1371, 1315, 1228,

1195, 1174, 1142, 1071, 1056, 1027, 949, 875, 788, 744, 699 cm−1;

1H NMR (CDCl3): δ 7.44 (1H, br s, H-16), 7.42 (1H, t, J = 1.8 Hz, H-15),

6.54 (1H, dd, J = 2.4, 1.3 Hz, H-3), 6.40 (1H, dd, J = 1.9, 1.0 Hz, H-14), 5.70

5.2. ISOLATION 191

(1H, dt, J = 5.5, 1.3 Hz, H-1), 5.53 (1H, dd, J = 11.2, 5.8 Hz, H-12), 4.44

(1H, ddd, J = 6.7, 5.5, 2.4 Hz, H-2), 3.74 (3H, s, CO2CH 3), 2.56 (1H, dt, J

= 13.3, 3.4 Hz, H-6α), 2.54 (1H, dd, J = 13.1, 5.9 Hz, H-11α), 2.17-2.09 (1H,

m, H-7β), 2.15 (3H, s, OCOCH 3), 2.13 (1H, dd, J = 13.5, 3.5 Hz, H-8), 2.01

(1H, br d, J = 6.7 Hz, OH ), 1.78 (1H, dtd, J = 15.1, 13.3, 3.5 Hz, H-7α), 1.69

(3H, s, H-19), 1.64 (1H, ddd J = 13.1, 11.2, 1.0 Hz, H-11β), 1.42 (1H, br s,

H-10), 1.22 (3H, s, H-20), 1.20 (1H, td, J = 13.5, 3.5 Hz, H-6β);

13C NMR (CDCl3): δ 171.57 (C, C-17/OCOCH3), 171.55 (C, C-17/OCOCH3),

166.2 (C, C-18), 143.8 (CH, C-15), 141.2 (C, C-4), 139.4 (CH, C-16), 135.7

(CH, C-3), 125.4 (C, C-13), 108.4 (CH, C-14), 71.6 (CH, C-12), 68.6 (CH,

C-2), 66.5 (CH, C-1), 52.5 (CH, C-10), 51.8 (CH, C-8), 51.7 (CH3, CO2CH3),

43.9 (CH2, C-11), 37.6 (C, C-5), 37.03 (C, C-9), 36.97 (CH2, C-6), 21.6 (CH3,

C-19), 21.2 (CH3, OCOCH3), 18.3 (CH2, C-7), 15.6 (CH3, C-20);

HRESIMS: [M + Na]+ m/z 455.1672 (calcd for C23H28O8Na+, 455.1676).

5.2.7 Salvinorin E (1e).

InChI=1/C23H28O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h6,8-9,11,14,16-19,25H,5,7,10H2,

1-4H3/t14-,16-,17-,18-,19-,22-,23-/m0/s1

1e

O

O

O

H HOH

O

O

O O

clear resin;


HPLC: tR (min) 1e 1f

60% EtOAc/petrol 9.8 10.7


[α]17D +46 (c 0.14, CHCl3);

FTIR (film): ν̃max 3510, 3144, 2952, 2858, 1722, 1641, 1505, 1436, 1374, 1316,

1228, 1142, 1070, 1029, 949, 935, 897, 875, 805, 788, 763, 708, 690, 679 cm−1;


6.43 (1H, dd, J = 2.4, 1.6 Hz, H-3), 6.41 (1H, dd, J = 1.9, 1.0 Hz, H-14), 5.60

(1H, ddd, J = 11.0, 5.8, 0.9 Hz, H-12), 5.40 (1H, dd, J = 4.9, 2.4 Hz, H-2),

4.46 (1H, ddd, J = 4.7, 1.6, 1.3 Hz, H-1), 3.73 (3H, s, CO2CH 3), 2.52 (1H,

ddd, J = 12.5, 3.0, 2.6 Hz, H-6α), 2.46 (1H, dd, J = 13.1, 6.0 Hz, H-11α),

2.17 (3H, s, OCOCH 3), 2.18-2.07 (2H, m, H-7β & 8), 1.94 (1H, dd, J = 2.3,

1.5 Hz, OH ), 1.84 (1H, dtd, J = 14.2, 12.0, 3.7 Hz, H-7α), 1.72 (3H, s, H-19),

1.62 (1H, dd, J = 13.1, 11.2 Hz, H-11β), 1.47 (3H, s, H-20), 1.30 (1H, br s,

H-10), 1.19 (1H, td, J = 13.3, 3.7 Hz, H-6β);

13C NMR (CDCl3): δ 171.8 (C, OCOCH3), 169.8 (C, C-17), 166.0 (C, C-18),

143.9 (CH, C-15), 143.4 (C, C-4), 139.3 (CH, C-16), 131.5 (CH, C-3), 125.8

(C, C-13), 108.4 (CH, C-14), 72.3 (CH, C-2), 71.7 (CH, C-12), 64.3 (CH, C-

1), 54.0 (CH, C-10), 51.8 (CH3, CO2CH3), 51.7 (CH, C-8), 44.4 (CH2, C-11),

37.8 (C, C-5), 37.5 (C, C-9), 37.0 (CH2, C-6), 21.9 (CH3, C-19), 21.0 (CH3,

OCOCH3), 18.4 (CH2, C-7), 16.2 (CH3, C-20);


5.2.8 Salvinorin F (1f).

InChI=1/C21H26O6/c1-20-8-6-14-19(24)27-16(12-7-9-26-11-12)10-21(14,2)17

(20)15(22)5-4-13(20)18(23)25-3/h4,7,9,11,14-17,22H,5-6,8,10H2,1-3H3/t14-,15

-,16-,17-,20-,21-/m0/s1

5.2. ISOLATION 193

1f

O

O

O

H HOH

O O

clear resin;


HPLC: see table on page 191.

[α]16D −20 (c 0.05, CHCl3);

FTIR (film): ν̃max 3514, 3147, 2951, 2857, 1712, 1637, 1505, 1436, 1372, 1318,

1232, 1194, 1144, 1070, 1028, 978, 947, 896, 875, 797, 756, 686 cm−1;


6.67 (1H, ddd, J = 4.7, 3.0, 1.0 Hz, H-3), 6.41 (1H, dd, J = 1.9, 1.0 Hz, H-14),

5.60 (1H, ddd, J = 11.2, 5.8, 0.9 Hz, H-12), 4.51 (1H, br dd, J = 5.5, 4.7 Hz,

H-1), 3.72 (3H, s, CO2CH 3), 2.60 (1H, ddd, J = 20.1, 5.5, 3.0 Hz, H-2β), 2.53

(1H, dt, J = 13.2, 3.2 Hz, H-6α), 2.46 (1H, dd, J = 13.2, 5.8 Hz, H-11α), 2.35

(1H, ddt, J = 20.1, 4.6, 1.1 Hz, H-2α), 2.17-2.08 (2H, m, H-7β, 8), 1.82 (1H,

dtd, J = 15.0, 13.2, 3.5 Hz, H-7α), 1.71 (3H, s, H-19), 1.62 (1H, ddd, J =

13.2, 11.4, 0.9 Hz, H-11β), 1.48 (3H, s, H-20), 1.29 (1H, dd, J = 4.7, 0.7 Hz,

OH ), 1.25 (1H, br s, w 12

= 3.8 Hz, H-10), 1.18 (1H, tdd, J = 13.3, 3.6, 0.9 Hz,

H-6β);

13C NMR (CDCl3): δ 172.1 (C, C-17), 166.9 (C, C-18), 143.8 (CH, C-15), 140.6

(C, C-4), 139.3 (CH, C-16), 133.4 (CH, C-3), 125.9 (C, C-13), 108.4 (CH, C-

14), 71.7 (CH, C-12), 63.9 (CH, C-1), 54.8 (CH, C-10), 52.2 (CH, C-8), 51.5

(CH3, CO2CH3), 44.4 (CH2, C-11), 38.0 (CH2, C-2), 37.7 (C, C-9), 37.3 (CH2,

C-6), 36.6 (C, C-5), 21.6 (CH3, C-19), 18.6 (CH2, C-7), 16.4 (CH3, C-20);



5.2.9 Divinatorin A (28a).

InChI=1/C20H28O4/c1-13-6-9-20(3)15(18(22)23)4-5-16(21)17(20)19(13,2)10-

7-14-8-11-24-12-14/h4,8,11-13,16-17,21H,5-7,9-10H2,1-3H3,(H,22,23)/t13-,16+,

17-,19+,20+/m1/s1/f/h22H

28a

O

H H

O OH

OH

amber resin;


[α]19D −53 (c 1.8, CH2Cl2);

FTIR (film): ν̃max 3392, 2927, 2874, 2648, 1684, 1634, 1503, 1456, 1411, 1386,

1262, 1245, 1221, 1162, 1102, 1066, 1025, 1003, 966, 924, 894, 874, 782, 760,

703, 670 cm−1;

1H NMR (CDCl3): δ 7.36 (1H, t, J = 1.6 Hz, H-15), 7.20 (1H, td, J = 2.5,

1.0 Hz, H-16), 6.90 (1H, ddd, J = 4.8, 2.7, 1.0 Hz, H-3), 6.25 (1H, dt, J =

1.8, 1.0 Hz, H-14), 4.49 (1H, br d, J = 4.8 Hz, H-1), 2.56 (1H, ddd, J = 20.1,

5.1, 2.8 Hz, H-2β), 2.43-2.33 (2H, m, H-2α, 6α), 2.34 (1H, br td, J = 13.5, 4.2

Hz, H-12-pro-R), 2.05 (1H, br ddd, J = 14.3, 12.9, 4.7 Hz, H-12-pro-S), 1.85

(1H, ddd, J = 14.7, 12.8, 4.7 Hz, H-11-pro-S), 1.67 (1H, ddd, J = 14.8, 12.8,

4.4 Hz, H-11-pro-R), 1.64 (3H, s, H-19), 1.60-1.54 (2H, m, H-7α, 8), 1.47-1.42

(1H, m, H-7β), 1.45 (1H, br s, H-10), 1.23-1.17 (1H, m, H-6β), 1.15 (3H, s,

H-20), 0.84 (3H, d, J = 6.2 Hz, H-17);

13C NMR (CDCl3): δ 171.8 (C, C-18), 142.8 (CH, C-15), 140.8 (C, C-4), 138.4

(CH, C-16), 136.2 (CH, C-3), 125.2 (C, C-13), 110.9 (CH, C-14), 64.7 (CH,

C-1), 49.0 (CH, C-10), 39.7 (C, C-9), 39.1 (CH2, C-11), 38.6 (CH2, C-6), 38.1

5.2. ISOLATION 195

(CH2, C-2), 37.4 (C, C-5), 37.1 (CH, C-8), 27.4 (CH2, C-7), 21.4 (CH3, C-19),

19.8 (CH3, C-20), 18.2 (CH2, C-12), 15.7 (CH3, C-17);


5.2.10 Divinatorin B (28b).

InChI=1/C21H30O5/c1-20(9-6-14-8-11-26-13-14)15(12-22)7-10-21(2)16(19(2

4)25-3)4-5-17(23)18(20)21/h4,8,11,13,15,17-18,22-23H,5-7,9-10,12H2,1-3H3/

t15-,17-,18+,20-,21-/m0/s1

28b

OH

O

H HOH

O O

amber resin;


[α]20D −54 (c 2.1, CHCl3);

FTIR (film): ν̃max 3434, 2930, 2881, 1714, 1503, 1461, 1437, 1385, 1356, 1236,

1196, 1162, 1097, 1067, 1025, 979, 943, 921, 874, 781, 759, 734 cm−1;

1H NMR (500 MHz, CDCl3): δ 7.35 (1H, t, J = 1.7 Hz, H-15), 7.20 (1H, dd, J

= 1.6, 1.0 Hz, H-16), 6.65 (1H, ddd, J = 4.9, 2.8, 1.0 Hz, H-3), 6.25 (1H, dd,

J = 1.8, 0.9 Hz, H-14), 4.46 (1H, dq, J = 5.1, 1.3 Hz, H-1), 3.84 (1H, dd, J =

10.5, 3.5 Hz, H-17-pro-R), 3.71 (3H, s, CO2CH 3), 3.38 (1H, dd, J = 10.5, 8.0

Hz, H-17-pro-S), 2.53 (1H, ddd, J = 19.9, 5.1, 2.8 Hz, H-2β), 2.42 (1H, dddd,

J = 14.5, 12.7, 4.7, 1.1 Hz, H-12-pro-R), 2.36 (1H, dt, J = 13.0, 3.4 Hz, H-6α),

2.31 (1H, ddt, J = 19.9, 4.9, 1.4 Hz, H-2α), 2.08 (1H, dddd, J = 14.5, 12.5,

4.8, 1.1 Hz, H-12-pro-S), 1.90 (1H, ddd, J = 15.0, 12.4, 4.9 Hz, H-11-pro-S),

1.85 (1H, dq, J = 13.2, 3.4 Hz, H-7β), 1.77 (1H, ddd, J = 15.0, 12.6, 4.6 Hz,


H-11-pro-R), 1.66 (3H, s, H-19), 1.64-1.49 (2H, m, H-8 & 7α), 1.49 (2H, br s,

OH ), 1.45 (1H, br s, H-10), 1.19-1.13 (1H, m, H-6β), 1.18 (3H, s, H-20);

13C NMR (CDCl3): δ 167.3 (C, C-18), 142.8 (CH, C-15), 141.4 (C, C-4), 138.4

(CH, C-16), 133.2 (CH, C-3), 124.9 (C, C-13), 110.8 (CH, C-14), 64.3 (CH, C-

1), 63.9 (CH2, C-17), 51.3 (CH3, CO2CH3), 48.7 (CH, C-10), 44.8 (CH, C-8),

39.1 (C, C-9), 38.8 (CH2, C-11), 37.97 (CH2, C-2/6), 37.96 (CH2, C-2/6), 37.1

(C, C-5), 21.9 (CH2, C-7), 21.4 (CH3, C-19), 20.9 (CH3, C-20), 18.2 (CH2,

C-12);


5.2.11 Divinatorin C (28c).

InChI=1/C22H30O5/c1-15(23)27-14-17-8-11-22(3)18(20(24)25)5-4-6-19(22)21

(17,2)10-7-16-9-12-26-13-16/h5,9,12-13,17,19H,4,6-8,10-11,14H2,1-3H3,(H,24,

25)/t17-,19+,21-,22-/m0/s1/f/h24H

28c

O

O

H H

O OH

O

amber resin;


[α]25D −110 (c 1.1, CHCl3);

FTIR (film): ν̃max 2960, 2938, 2873, 2650, 1738, 1681, 1629, 1502, 1459, 1420,

1385, 1367, 1236, 1161, 1064, 1025, 1000, 975, 873, 785, 759, 730 cm−1;

1H NMR (500 MHz, CDCl3): δ 7.35 (1H, t, J = 1.6 Hz, H-15), 7.22 (1H, dd,

J = 1.6, 0.8 Hz, H-16), 6.89 (1H, dd, J = 4.8, 2.9 Hz, H-3), 6.28 (1H, dd, J

5.2. ISOLATION 197

= 1.8, 0.9 Hz, H-14), 4.26 (1H, dd, J = 11.0, 4.1 Hz, H-17-pro-R), 3.79 (1H,

dd, J = 11.0, 8.4 Hz, H-17-pro-S), 2.53 (1H, dt, J = 13.2, 3.2 Hz, H-6α), 2.40

(1H, td, J = 13.8, 4.2 Hz, H-12-pro-R), 2.35 (1H, dt, J = 20.2, 5.3 Hz, H-2α),

2.24-2.16 (2H, m, H-2β & 12-pro-S), 2.03 (3H, s, OCOCH 3), 1.82-1.67 (4H,

m, H-1β, 7β, 8, 11-pro-S), 1.64 (1H, ddd, J = 15.0, 12.6, 4.3 Hz, H-11-pro-R),

1.54-1.44 (2H, m, H-1α & 7α), 1.42 (1H, br d, J = 12.1 Hz, H-10), 1.27 (3H,

s, H-19), 1.15 (1H, td, J = 13.2, 3.6 Hz, H-6β), 0.83 (3H, s, H-20);

13C NMR (CDCl3): δ 171.9 (C, C-18), 171.2 (C, OCOCH3), 142.8 (CH, C-15),

141.2 (C, C-4), 140.3 (CH, C-3), 138.5 (CH, C-16), 125.2 (C, C-13), 110.9 (CH,

C-14), 66.1 (CH2, C-17), 46.8 (CH, C-10), 40.9 (CH, C-8), 38.9 (CH2, C-11),

38.4 (C, C-9), 37.4 (C, C-5), 35.2 (CH2, C-6), 27.4 (CH2, C-2), 22.3 (CH2,

C-7), 21.0 (CH3, OCOCH3), 20.5 (CH3, C-19), 19.0 (CH3, C-20), 18.3 (CH2,

C-12), 17.0 (CH2, C-1);


5.2.12 (–)-Hardwickiic Acid (29a) and methyl ester 29b.

InChI=1/C20H28O3/c1-14-7-10-20(3)16(18(21)22)5-4-6-17(20)19(14,2)11-8-

15-9-12-23-13-15/h5,9,12-14,17H,4,6-8,10-11H2,1-3H3,(H,21,22)/t14-,17-,19+,

20+/m1/s1

O

H H

O OR

R

HMe

29a29b

1H NMR (CDCl3) matched previously reported values;216, 217 the spectrum

was superimposable with that of authentic (+)-hardwickiic acid (ent-29a),

prepared as detailed in Section 5.3.5 on page 202. Methylation with CH2N2 in

Et2O gave the methyl ester 29b;


InChI=1/C21H30O3/c1-15-8-11-21(3)17(19(22)23-4)6-5-7-18(21)20(15,2)12-

9-16-10-13-24-14-16/h6,10,13-15,18H,5,7-9,11-12H2,1-4H3/t15-,18-,20+,21+

/m1/s1

[α]18D −86 (c 0.04, CHCl3);

[α]23D −104 (c 1.1, CHCl3) lit;217

1H and 13C NMR (CDCl3), FTIR (film) and EIMS (70 eV) matched previ-

ously reported values.218, 217 The 1H NMR spectrum was superimposable with

that of authentic (+)-methyl hardwickiate (ent-29b), prepared as detailed in

Section 5.3.5 on page 202.

5.2.13 Oleanolic Acid (31).

InChI=1/C30H48O3/c1-25(2)14-16-30(24(32)33)17-15-28(6)19(20(30)18-25)

8-9-22-27(5)12-11-23(31)26(3,4)21(27)10-13-29(22,28)7/h8,20-23,31H,9-18H2,

1-7H3,(H,32,33)/t20-,21-,22+,23-,27-,28+,29+,30-/m0/s1

H

O

OH

HOH

H 31

HRESIMS: [M + Na]+ m/z 479.3516 (calcd for C30H48O3Na+, 479.3496);

1H and 13C NMR (CDCl3) matched previously reported values.221

5.2.14 Presqualene Alcohol (32).

InChI=1/C30H50O/c1-23(2)13-9-15-25(5)17-11-18-27(7)21-28-29(22-31)30(28,

8)20-12-19-26(6)16-10-14-24(3)4/h13-14,17,19,21,28-29,31H,9-12,15-16,18,20,22

H2,1-8H3/b25-17+,26-19+,27-21+/t28-,29-,30-/m1/s1

5.3. SYNTHESIS 199

OH

H

32

[α]21D +45 (c 1.2, CHCl3);

[α]20D +49 (c 4.8, CHCl3) lit;223

1H NMR (CDCl3),222 13C NMR (C6D6)223 and FTIR (film)222 matched previ-

ously reported values. The 1H NMR spectrum in C6D6 was superimposable

with a previously published spectrum.223

5.2.15 Peplusol (33).

InChI=1/C30H50O/c1-24(2)13-9-15-26(5)17-11-18-28(7)21-22-30(23-31)29(8)

20-12-19-27(6)16-10-14-25(3)4/h13-14,17,19,21,30-31H,8-12,15-16,18,20,22-23

H2,1-7H3/b26-17+,27-19+,28-21+/t30-/m0/s1

OH 33

[α]20D −6 (c 0.07, CHCl3);

[α]25D −18 (c 0.74, iPrOH) lit;224

1H and 13C NMR (CDCl3) and FTIR (film) matched previously reported val-

ues.224

5.3 Synthesis

5.3.1 Salvinorin C (1c) via acetylation of salvinorin D

(1d).

Ac2O (250 µL, 2.6 mmol) was added to a solution of 1d (11.0 mg, 25.4

µmol) in dry pyridine (2.5 mL) under Ar. After stirring for 3.5 h, TLC


(10% acetone/CH2Cl2) indicated completion. The reaction mixture was di-

luted with ice water and extracted with Et2O (× 3). The organic phase was

washed with saturated NaHCO3, saturated CuSO4, water and brine. Drying

(MgSO4), evaporation in vacuo and flash column chromatography on silica gel

(55% Et2O/petrol) gave 1c as a clear resin (8.4 mg, 70%);

TLC: See Table 5.1 on page 187. Cospotted with isolated material.

[α]16D +69 (c 0.4, CHCl3).

Other spectra (FTIR, 1H and 13C NMR) superimposable with those of the

isolated material.

5.3.2 Salvinorins D (1d) and E (1e) via acetylation of

1h.

1h (2.8 mg, 7.2 µmol), Ac2O (7 µL, 74 µmol) and catalytic DMAP (�1 mg)

were stirred in pyridine (3 mL) for 3 h, when TLC (10% acetone/CH2Cl2,

visualised with KMnO4) showed no starting material. Evaporation in vacuo

gave a ≈1:2 mixture of 1d and 1e.

TLC: See Table 5.1 on page 187 and table on the facing page. Cospotted with

isolated materials.

1H NMR spectrum superimposable with those of the isolated materials.

5.3.3 Salvinorins C (1c) and E (1e) via acetylation of

1h.

1h (4.1 mg, 10.5 µmol), Ac2O (8 µL, 85 µmol) and catalytic DMAP (�1

mg) were stirred in pyridine (500 µL) at 45 ◦C for 75 min, when TLC (10%

acetone/CH2Cl2, visualised with KMnO4/H2SO4 dip) showed no starting ma-

terial. Evaporation in vacuo and flash column chromatography (2.5 – 10%

acetone/CH2Cl2) gave 1c and 1e; the latter was contaminated by an insepa-

rable byproduct.

5.3. SYNTHESIS 201

TLC: See Table 5.1 on page 187 and table on this page. Cospotted with

isolated materials.

1H NMR spectra superimposable with those of the isolated materials.

5.3.4 Dideacetylsalvinorin C (1h) from 1c.

To a solution of 1c (5.8 mg, 12.2 µmol) and Na2CO3 (5.1 mg, 41.1 µmol) in

CH2Cl2 (1 mL) was added MeOH (1 mL), and the solution stirred at rt for 2

h, when TLC (10% acetone/ CH2Cl2) showed considerable starting material.

After heating at 45 ◦C for a further 90 min, TLC indicated completion. The

solution was partitioned between brine (acidified with 10% HCl) and CH2Cl2(× 3). Drying (MgSO4), evaporation in vacuo, and flash column chromatogra-

phy (loaded in CH2Cl2, eluted with 33 - 50% EtOAc/petrol, then 25% MeOH/

CH2Cl2) gave 1h as a resin (4.1 mg, 86%);

InChI=1/C21H26O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7-8,10,12,14-17,22-23H,4,6,9H2,1-3H3

/t12-,14-,15-,16-,17-,20-,21-/m0/s1

1h

O

O

O

H HOH

HO

O O

TLC: hRf 1c 1h

10% acetone/CH2Cl2 60 18

[α]18D +27 (c 0.2, CH2Cl2);

FTIR (film): ν̃max 3456, 2951, 1714, 1504, 1435, 1379, 1314, 1229, 1177, 1144,

1075, 1049, 1027, 949, 875, 788, 736, 685 cm−1;


1H NMR (CDCl3): δ 7.43 (1H, m, H-16), 7.42 (1H, t, J = 1.8 Hz, H-15), 6.48

(1H, dd, J = 2.4, 1.6 Hz, H-3), 6.40 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.60 (1H,

dd, J = 11.1, 5.9 Hz, H-12), 4.32 (1H, br d,∗ J = 5.1 Hz, H-1), 4.28 (1H, dd,∗

J = 5.1, 2.4 Hz, H-2), 3.73 (3H, s, CO2CH 3), 2.49 (1H, dd, J = 13.2, 6.0 Hz,

H-11α), 2.50-2.45 (1H, m, H-6α), 2.40-2.30 (2H, m, OH ), 2.14-2.10 (1H, m,

H-8), 2.09 (1H, dq, J = 14.6, 3.6 Hz, H-7β), 1.82 (1H, dtd, J = 15.0, 13.2, 3.3

Hz, H-7α), 1.70 (3H, s, H-19), 1.60 (1H, ddd, J = 13.0, 11.1, 0.8 Hz, H-11β),

1.47 (3H, s, H-20), 1.22 (1H, d, J = 1.0 Hz, H-10), 1.16 (1H, tdd, J = 13.3,

3.6, 0.9 Hz, H-6β);

13C NMR (CDCl3): δ 172.0 (C, C-17), 166.6 (C, C-18), 143.9 (CH, C-15), 142.2

(C, C-4), 139.3 (CH, C-16), 135.3 (CH, C-3), 125.8 (C, C-13), 108.4 (CH, C-

14), 71.8 (CH, C-12), 69.6 (CH, C-2), 65.6 (CH, C-1), 54.1 (CH, C-10), 51.8

(CH, C-8), 51.7 (CH3, CO2CH3), 44.4 (CH2, C-11), 37.49 (C, C-5/9), 37.45

(C, C-5/9), 37.0 (CH2, C-6), 22.1 (CH3, C-19), 18.4 (CH2, C-7), 16.3 (CH3,

C-20);


5.3.5 (+)-Hardwickiic acid (ent-29a).

Following a modified version of Costa et al’s procedure,218 the acid fraction

of copaiba balsam was methylated with CH2N2 in Et2O, and the methyl ester

(ent-29b) was isolated. This ester (32 mg, 97 µmol) was dissolved in acetone.

KF/Al2O3 (220 mg, 40% w/w KF) was added and the acetone evaporated

under reduced pressure. This powder was irradiated in a microwave oven (650

W) at 100% power for 8 minutes, then cooled. Minimal water was added and

stirred for 5 minutes, then filtered, and the filter cake was rinsed with water

(× 2). Rinsing of the filter cake with CHCl3 (× 3) gave, after drying (MgSO4)

and evaporation, starting material ent-29b (11 mg). The aqueous filtrate was

acidified with 10% HCl and extracted with CHCl3 (× 4). The pooled organic

extracts were dried (MgSO4) and evaporated to give 14 mg crude product.∗After D2O exchange.

5.3. SYNTHESIS 203

Flash column chromatography in 80% Et2O/petrol gave ent-29a (5 mg, 16

µmol) as a semicrystalline film;

InChI=1/C20H28O3/c1-14-7-10-20(3)16(18(21)22)5-4-6-17(20)19(14,2)11-8-

15-9-12-23-13-15/h5,9,12-14,17H,4,6-8,10-11H2,1-3H3,(H,21,22)/t14-,17-,19+,

20+/m0/s1/f/h21H

O

H H

O OR

R

H

Me

ent-29a

ent-29b

TLC: hRf ent-29a ent-29b


[α]19D +81 (c 0.2, CHCl3);

[α]23D −85 (CHCl3) lit. value for 29a;217

FTIR, 1H and 13C NMR data217 matched reported values.

5.3.6 Salvinorin A lactol (35).

Following the published procedure,260 1a (15.8 mg, 36.5 µmol) was warmed in

THF (1 mL) under Ar until fully dissolved, then cooled to -78 ◦C. iBu2AlH

(1M in THF, 0.5 mL, 500 µmol) was added dropwise. The solution was stirred

for 25 minutes, then quenched (sat. aq. NH4Cl dropwise), evaporated in vacuo

until thick, diluted in water and extracted into Et2O (× 3). Washing (brine),

drying (MgSO4), evaporation in vacuo, and flash column chromatography (6-

10% acetone/CH2Cl2 gradient) gave 35260 as a clear resin (10.4 mg, 65% (81%

borsm));

InChI=1/C23H30O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h6,8,11,14-17,19,21,27H,5,7,9-10H

2,1-4H3/t14-,15-,16-,17-,19-,21u,22-,23-/m0/s1


35

O

OH

O

H HOO

O

O O

TLC: hRf 1a 35


FTIR (film): ν̃max 3446, 2953, 2256, 1730, 1503, 1438, 1379, 1272, 1237, 1203,

1161, 1123, 1053, 1010, 978, 914, 875, 785, 732, 647 cm−1;

1H NMR, major (17β) anomer (CDCl3): δ 7.36 (1H, br s, H-16), 7.34 (1H, t,

J = 1.8 Hz, H-15), 6.38 (1H, dd, J = 1.8, 0.9 Hz, H-14), 5.14-5.09 (1H, m,

H-2), 4.87 (1H, dd, J = 11.6, 2.4 Hz, H-12), 4.80 (1H, d, J = 8.7 Hz, H-17),

3.70 (3H, s, CO2CH 3), 2.76-2.72 (1H, m, H-4), 2.28-2.23 (2H, m, H-3), 2.14

(3H, s, OCOCH 3), 2.11 (1H, dd, J = 13.2, 2.4 Hz, H-11α), 2.07 (1H, d, J =

0.9 Hz, H-10), 1.80 (1H, dq, J = 13.9, 3.3 Hz, H-7β), 1.70 (1H, dt, J = 13.5,

3.2 Hz, H-6α), 1.64-1.57 (1H, m, H-6β), 1.38 (3H, s, H-20), 1.43-1.32 (1H, m,

H-7α), 1.21 (1H, ddd, J = 13.2, 11.6, 0.9 Hz, H-11β), 1.15-1.09 (1H, m, H-8),

1.08 (3H, s, H-19);

these data are broadly consistent with incomplete data published previously;260

13C NMR, major (17β) anomer (CDCl3): δ 202.5 (C, C-1), 171.9 (C, C-18),

169.9 (C, OCOCH3), 143.0 (CH, C-15), 139.1 (CH, C-16), 126.2 (C, C-13),

108.8 (CH, C-14), 94.2 (CH, C-17), 75.0 (CH, C-2), 66.2 (CH, C-12), 65.4

(CH, C-10), 53.6 (CH, C-4), 52.1 (CH, C-8), 51.8 (CH3, CO2CH3), 44.7 (CH2,

C-11), 42.4 (C, C-5), 38.8 (CH2, C-6), 35.6 (C, C-9), 30.8 (CH2, C-3), 20.6

(CH3, OCOCH3), 17.6 (CH2, C-7), 16.7 (CH3, C-19), 15.0 (CH3, C-20).

5.3. SYNTHESIS 205

5.3.7 (4R)-3,4-Dihydrosalvinorin C (36c).

Following a published procedure,27 formation of the orthoacetate of 36h (which

did not go to completion in 10 h), followed by acid-catalyzed hydrolysis, gave

36d which was used in the next reaction without purification;

InChI=1/C23H30O8/c1-12(24)30-18-16(25)9-15(20(26)28-4)22(2)7-5-14-21(27)

31-17(13-6-8-29-11-13)10-23(14,3)19(18)22/h6,8,11,14-19,25H,5,7,9-10H2,1-4H3

/t14-,15-,16-,17-,18-,19-,22-,23-/m0/s1

36d

O

O

O

H HOHO

O

O O

TLC: hRf 36h Orth.Ac. 36d

Et2O 33 60 29


6.41 (1H, br d, J = 2.0 Hz, H-14), 5.61 (1H, br s, H-1), 5.47 (1H, dd, J = 11.5,

5.3 Hz, H-12), 3.76-3.72 (1H, m, H-2), 3.69 (3H, s, CO2CH 3), 2.49 (1H, dd, J

= 13.1, 5.6 Hz, H-11α), 2.22-2.08 (3H, m), 2.16 (3H, s, OCOCH 3), 1.86-1.77

(3H, m), 1.71-1.61 (2H, m), 1.35 (3H, s, H-20), 1.35-1.24 (1H, m), 1.18 (3H, s,

H-19), 1.13 (1H, d, J = 1.9 Hz, H-10);

1H NMR [(CD3)2CO] matched values previously reported for “CDCl3”.27

Acetylation of 36d with Ac2O/pyridine27 and purification by HPLC (40%

EtOAc/ petrol) gave 36c;

InChI=1/C25H32O9/c1-13(26)32-18-10-17(22(28)30-5)24(3)8-6-16-23(29)34-

19(15-7-9-31-12-15)11-25(16,4)21(24)20(18)33-14(2)27/h7,9,12,16-21H,6,8,10-

11H2,1-5H3/t16-,17-,18-,19-,20-,21-,24-,25-/m0/s1


36c

O

O

O

H HOO

O

O

O O

TLC: hRf 36d 36c

10% acetone/CH2Cl2 33 77 (developed in KMnO4).

HPLC: tR = 18.5 min (40% EtOAc/petrol).

1H NMR (CDCl3): δ 7.45 (1H, br s, H-16), 7.41 (1H, t, J = 1.8 Hz, H-15), 6.41

(1H, dd, J = 2.0, 0.9 Hz, H-14), 5.67 (1H, dt, J = 3.5, 1.5 Hz, H-1), 5.46 (1H,

dd, J = 11.6, 5.5 Hz, H-12), 4.79-4.74 (1H, m, H-2), 3.70 (3H, s, CO2CH 3),

2.43 (1H, dd, J = 13.3, 5.4 Hz, H-11α), 2.31-2.24 (2H, m), 2.16-2.09 (2H, m),

2.14 (3H, s, OCOCH 3), 1.98 (3H, s, OCOCH 3), 1.84-1.79 (2H, m), 1.72-1.53

(m, obscured by H2O), 1.69 (1H, ddd, J = 13.4, 11.6, 0.9 Hz, H-11β), 1.40-1.31

(2H, m), 1.38 (3H, s, H-20), 1.20 (1H, d, J = 1.9 Hz, H-10), 1.17 (3H, s, H-19);

these data are broadly consistent with those published previously82 (assign-

ments differ). 1H NMR [(CD3)2CO]23 and 13C NMR (CDCl3)27 matched pre-

viously reported values.

5.3.8 (4R)-3,4-Dihydrosalvinorin E (36e).

Following the published procedure,23 acetylation in Ac2O/pyridine27 of 36h

(which had been purified by HPLC in EtOAc) gave 36e;

InChI=1/C23H30O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h6,8,11,14-19,25H,5,7,9-10H2,1-4

H3/t14-,15-,16-,17-,18-,19-,22-,23-/m0/s1

5.3. SYNTHESIS 207

36e

O

O

O

H HOH

O

O

O O

TLC: hRf 36h 36e

50% EtOAc/petrol 20 48 (developed in KMnO4).


6.41 (1H, dd, J = 1.8, 0.9 Hz, H-14), 5.55 (1H, dd, J = 11.4, 5.4 Hz, H-12),

4.70 (1H, ddd, J = 11.7, 4.6, 3.2 Hz, H-2), 4.29 (1H, br s, H-1), 3.68 (3H, s,

CO2CH 3), 2.41 (1H, dd, J = 13.2, 5.4 Hz, H-11α), 2.31 (1H, q, J = 12.6 Hz,

H-3α), 2.20(1H, dd, J = 13.2, 2.4 Hz, H-4), 2.14-2.08 (1H, m, H-8), 2.09 (3H,

s, OCOCH 3), 1.90 (1H, br s, w 12

= 11 Hz, OH ), 1.82 (1H, dddd, J = 12.5,

4.9, 2.5, 1.1 Hz, H-3β), 1.76 (1H, dq, J = 13.3, 3.2 Hz, H-7β), 1.74-1.69 (1H,

m, H-6a), 1.64 (1H, ddd, J = 13.0, 11.6, 0.9 Hz, H-11β), 1.45 (3H, s, H-20),

1.38 (3H, s, H-19), 1.38-1.26 (2H, m, H-6b,7α), 1.00 (1H, br s, H-10);

these data are consistent with incomplete data published previously.23 13C

NMR (CDCl3) matched previously reported values.27

5.3.9 (4R)-Dideacetyl-3,4-dihydrosalvinorin C (36h).

A slight modification of a published procedure was used:23, 260 to 1a (37.6 mg,

86.9 µmol) and NaBH4 (4.4 mg, 116 µmol) was added CH2Cl2 (200 µL), fol-

lowed by EtOH (3 mL), and the cloudy solution stirred under Ar at 40 ◦C.

After 4 h, TLC (Et2O) indicated completion. The solution was cooled to 0◦C, and 0.5% H2SO4/MeOH added dropwise until effervescence ceased. The

solution was concentrated to ≈ 500 µL in vacuo, then partitioned between

brine (acidified with 10% HCl) and CH2Cl2 (× 3). Drying (MgSO4), evapora-

tion in vacuo and flash column chromatography (40-66% EtOAc/petrol) gave

36h15, 23 (19.1 mg, 56%);


InChI=1/C21H28O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7,10,12-17,22-23H,4,6,8-9H2,1-3H3/t

12-,13-,14-,15-,16-,17-,20-,21-/m0/s1

36h

O

O

O

H HOH

HO

O O

TLC: hRf 1a 36h 38h

Et2O 45 14 25

HPLC: tR = 8.7 min (EtOAc).


6.41 (1H, dd, J = 1.9, 1.0 Hz, H-14), 5.56 (1H, ddd, J = 11.5, 6.1, 0.9 Hz,

H-12), 4.20 (1H, br s, H-1), 3.68 (3H, s, CO2CH 3), 3.58 (1H, ddd, J = 11.6,

5.2, 3.4 Hz, H-2), 2.48 (1H, dd, J = 13.2, 5.6 Hz, H-11α), 2.25-2.07 (4H, m),

1.91 (2H, br s, w 12

= 70 Hz, OH ), 1.77-1.69 (3H, m), 1.62 (1H, ddd, J = 13.3,

11.6, 1.0 Hz, H-11β), 1.46 (3H, s, H-20), 1.37 (3H, s, H-19), 1.29 (1H, tdd, J

= 13.6, 3.1, 1.0 Hz, H-6α), 0.92 (1H, d, J = 1.9 Hz, H-10);

1H NMR ([CD3]2CO) matched previously reported values.23

5.3.10 8-epi-Salvinorin A (37a).

Distilled DMPU (60 ◦C / 0.1 mmHg) was added to 1a (21.4 mg, 49.5 µmol)

and NaHCO3 (30.1 mg, 358 µmol), and stirred at 150 ◦C for 2 h. The amber

solution was diluted with EtOAc, neutralised dropwise with 10% HCl, and

washed (10% HCl × 4, then brine). Drying (MgSO4) and evaporation in

vacuo followed by flash column chromatography (30% - 50% EtOAc/petrol

gradient) monitored by TLC (Et2O) gave 37a as a clear resin (10.8 mg, 51%

(81% borsm));

5.3. SYNTHESIS 209

InChI=1/C23H28O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h6,8,11,14-17,19H,5,7,9-10H2,1-4

H3/t14-,15+,16+,17+,19+,22+,23+/m1/s1

37a

O

O

O

H HOO

O

O O

8

TLC: hRf 1a 37a

Et2O 39 50

vanillin/H2SO4 purple blue

[α]13D −53 (c 0.6, CHCl3);

FTIR (film): ν̃max 3018, 2951, 2883, 1732, 1504, 1450, 1437, 1375, 1323, 1238,

1202, 1161, 1124, 1084, 1047, 1024, 997, 970, 937, 876, 783, 756, 667, 601 cm−1;


6.37 (1H, d, J = 1.7 Hz, H-14), 5.25 (1H, dd, J = 12.0, 2.2 Hz, H-12), 5.09

(1H, ∼dd, J ≈ 10.9, 9.3 Hz, H-2), 3.69 (3H, s, CO2CH 3), 2.79-2.72 (1H, m,

H-4), 2.45 (1H, dd, J = 5.0, 2.2 Hz, H-8), 2.36 (1H, dd, J = 15.0, 2.2 Hz,

H-11α), 2.29-2.22 (2H, m, H-3), 2.24 (1H, br s, H-10), 2.19 (1H, dq, J = 14.3,

3.1 Hz, H-7β), 2.15 (3H, s, OCOCH 3), 2.00 (1H, td, J = 13.7, 3.9 Hz, H-6β),

1.83 (1H, tdd, J = 14.2, 5.0, 3.9 Hz, H-7α), 1.62 (3H, s, H-20), 1.54 (1H, dt,

J = 13.7, 3.4 Hz, H-6α), 1.50 (1H, dd, J = 15.0, 12.0 Hz, H-11β), 1.07 (3H,

s, H-19);

13C NMR (CDCl3): δ 202.3 (C, C-1), 173.4 (C, C-17), 171.8 (C, C-18), 169.8

(C, OCOCH3), 143.6 (CH, C-15), 139.7 (CH, C-16), 123.3 (C, C-13), 108.5

(CH, C-14), 75.2 (CH, C-2), 70.1 (CH, C-12), 64.1 (CH, C-10), 52.9 (CH, C-

4), 51.8 (CH3, CO2CH3), 48.0 (CH2, C-11), 45.2 (CH, C-8), 42.2 (C, C-5),

34.7 (C, C-9), 33.9 (CH2, C-6), 30.6 (CH2, C-3), 24.6 (CH3, C-20), 20.5 (CH3,

OCOCH3), 17.6 (CH2, C-7), 15.2 (CH3, C-19);



5.3.11 8-epi-Salvinorin B (37b).

MeOH (2 mL) was refluxed under Ar for 15 minutes, and cooled to rt. 1a (32.3

mg, 74.7 µmol) and Na2CO3 (30.4 mg, 245 µmol) were added, and the resulting

suspension stirred under Ar at rt for 4.5 h, when TLC indicated completion.

The reaction was quenched with 10% HCl, then partitioned between 10% HCl

and CH2Cl2 (× 4). Drying (MgSO4) and evaporation in vacuo gave an off-

white powder. Addition of minimal MeOH (≈ 0.3 mL) and centrifugation gave

1b as a white powder (21.5 mg, 74%). The supernatant was evaporated under

reduced pressure to give 37b as an amber resin (6.6 mg, 23%);

InChI=1/C21H26O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7,10,12-15,17,22H,4,6,8-9H2,1-3H3/t

12-,13+,14+,15+,17+,20+,21+/m1/s1

37b

O

O

O

H HOHO

O O

8

TLC: hRf 1a 1b 37b

Et2O 40 24 40

50% EtOAc/petrol 37 23 37

10% acetone/CH2Cl2 69 52 54

vanillin/H2SO4 purple purple blue

[α]13D −27 (c 0.3, CHCl3);

FTIR (film): ν̃max 3475, 2952, 1728, 1504, 1438, 1386, 1274, 1201, 1156, 1120,

1051, 1026, 997, 970, 876, 786, 755 cm−1;

5.3. SYNTHESIS 211


(1H, dd, J = 1.9, 0.8 Hz, H-14), 5.29 (1H, dd, J = 11.8, 2.2 Hz, H-12), 4.00

(1H, ddd, J = 11.9, 7.6, 1.3 Hz, H-2), 3.69 (3H, s, CO2CH 3), 2.70 (1H, dd, J

= 13.5, 3.2 Hz, H-4), 2.47 (1H, dd, J = 4.8, 2.2 Hz, H-8), 2.44 (1H, ddd, J =

13.5, 7.6, 3.2 Hz, H-3β), 2.42 (1H, dd, J = 14.8, 2.2 Hz, H-11α), 2.23 (1H, d,

J = 1.3 Hz, H-10), 2.21 (1H, dq, J = 14.4, 3.1 Hz, H-7β), 2.01 (1H, td, J =

13.5, 11.9 Hz, H-3α), 1.99 (1H, td, J = 13.8, 0.8 Hz, H-6β), 1.85 (1H, tdd, J

= 14.0, 4.8, 3.7 Hz, H-7α), 1.65 (3H, s, H-20), 1.54 (1H, dt, J = 13.5, 3.5 Hz,

H-6α), 1.46 (1H, ddd, J = 14.8, 11.7, 0.7 Hz, H-11β), 1.06 (3H, s, H-19);

these data are inconsistent with those of Harding et al82 and Lee et al.271


(CH, C-15), 139.6 (CH, C-16), 123.5 (C, C-13), 108.4 (CH, C-14), 74.5 (CH, C-

2), 70.0 (CH, C-12), 63.7 (CH, C-10), 52.4 (CH, C-4), 51.7 (CH3, CO2CH3),

48.3 (CH2, C-11), 45.3 (CH, C-8), 42.7 (C, C-5), 34.6 (C, C-9), 34.3 (CH2,

C-3), 33.9 (CH2, C-6), 24.7 (CH3, C-20), 17.6 (CH2, C-7), 15.3 (CH3, C-19);


5.3.12 8-epi-Salvinorin C (37c).

37d (1.7 mg, 3.9 µmol), Ac2O (0.25 mL) and catalytic DMAP (�1 mg)

were stirred in pyridine (0.5 mL) at 50 ◦C for 90 min, when TLC (10%

acetone/CH2Cl2, visualised with KMnO4/H2SO4 dip) showed no starting ma-

terial. In a separate flask, 37e ( 2.8 mg, 6.5 µmol) was subjected to the same

conditions for 4 h, when TLC (same conditions) showed no starting material.

The crude products, which cospotted by TLC, were pooled and evaporated in

vacuo. Flash column chromatography (2-4% acetone/CH2Cl2) gave 37c (4.7

mg, 95%) as a colourless resin;

InChI=1/C25H30O9/c1-13(26)32-18-10-17(22(28)30-5)24(3)8-6-16-23(29)34-

19(15-7-9-31-12-15)11-25(16,4)21(24)20(18)33-14(2)27/h7,9-10,12,16,18-21H,6,

8,11H2,1-5H3/t16-,18+,19+,20+,21+,24+,25+/m1/s1


37c

O

O

O

H HOO

O

O

O O

8

TLC: hRf 37c 37d 37e


vanillin/H2SO4 blue blue blue

[α]23D +9 (c 0.2, CH2Cl2);

FTIR (film): ν̃max 2926, 2854, 1741, 1716, 1645, 1556, 1504, 1435, 1371, 1332,

1229, 1198, 1172, 1159, 1093, 1032, 962, 875, 802 cm−1;

1H NMR (CDCl3): δ 7.50 (1H, dd, J = 1.6, 0.8 Hz, H-16), 7.43 (1H, t, J =

1.7 Hz, H-15), 6.42 (2H, m, H-3 & 14), 5.59 (1H, ddd, J = 4.7, 1.3, 0.9 Hz,

H-1), 5.49 (1H, dd, J = 4.8, 2.3 Hz, H-2), 5.28 (1H, br d, J = 11.0 Hz, H-12),

3.72 (3H, s, CO2CH 3), 2.49 (1H, dd, J = 5.3, 2.3 Hz, H-8), 2.33 (1H, dt, J =

13.4, 3.4 Hz, H-6α), 2.26-2.16 (1H, m, H-7β), 2.14 (1H, dd, J = 14.3, 1.6 Hz,

H-11α), 2.10 (3H, s, OCOCH 3), 2.02 (3H, s, OCOCH 3), 1.97 (1H, tdd, J =

14.8, 5.5, 3.9 Hz, H-7α), 1.70 (3H, s, H-19), 1.61-1.50 (m, H-11β, 6β, H2O),

1.35 (3H, s, H-20), 1.25 (1H, br s, H-10);

13C NMR (CDCl3): δ 173.6, 170.5, 169.7, 165.7, 143.7, 142.6, 139.7, 132.1,

123.4, 108.4, 69.7, 69.6, 65.1, 51.74, 51.72, 48.9, 45.6, 38.2, 36.0, 33.5, 25.1,

21.14, 21.13, 20.6, 18.2;


5.3.13 8-epi-Salvinorin D (37d).

Standing 37e in CDCl3 at rt overnight gave a 1:1 mixture with 37d, which

was isolated by flash column chromatography (5-10% acetone/CH2Cl2) as an

amber resin;

5.3. SYNTHESIS 213

InChI=1/C23H28O8/c1-12(24)30-18-16(25)9-15(20(26)28-4)22(2)7-5-14-21(2

7)31-17(13-6-8-29-11-13)10-23(14,3)19(18)22/h6,8-9,11,14,16-19,25H,5,7,10

H2,1-4H3/t14-,16+,17+,18+,19+,22+,23+/m1/s1

37d

O

O

O

H HOHO

O

O O

8

TLC: See table on the preceding page.

[α]23D +15 (c 0.1, CH2Cl2);

FTIR (film): ν̃max 3402, 2953, 2926, 1738, 1716, 1503, 1462, 1440, 1382, 1368,

1333, 1302, 1228, 1199, 1173, 1158, 1099, 1055, 1025, 981, 875, 842, 798, 784,

732 cm−1;

1H NMR (500 MHz, CDCl3): δ 7.49 (1H, br s, H-16), 7.43 (1H, t, J = 1.8

Hz, H-15), 6.49 (1H, dd, J = 2.3, 1.5 Hz, H-3), 6.41 (1H, dd, J = 2.0, 0.8 Hz,

H-14), 5.53 (1H, br d, J = 5.0 Hz, H-1), 5.29 (1H, br d, J = 11.5 Hz, H-12),

4.41 (1H, dd, J = 5.1, 1.4 Hz, H-2), 3.72 (3H, s, CO2CH 3), 2.48 (1H, dd, J

= 5.3, 2.3 Hz, H-8), 2.30 (1H, dt, J = 13.5, 3.8 Hz, H-6α), 2.22 (1H, dtd, J

= 14.7, 3.5, 2.1 Hz, H-7β), 2.17 (1H, dd, J = 14.1, 1.5 Hz, H-11α), 2.13 (3H,

s, OCOCH 3), 1.96 (1H, tdd, J = 14.2, 5.3, 3.9 Hz, H-7α), 1.70-1.64 (1H, m,

H-11β), 1.67 (3H, s, H-19), 1.62-1.52 (m, H-6β, H2O), 1.37 (3H, s, H-20), 1.25

(1H, br s, H-10);

13C NMR (CDCl3): δ 173.7, 171.8, 166.1, 143.7, 141.4, 139.7, 135.0, 123.6,

108.4, 69.6, 69.3, 67.6, 51.7, 51.6, 49.1, 45.7, 37.8, 36.0, 33.5, 25.2, 21.3, 21.0,

18.1;



5.3.14 8-epi-Salvinorin E (37e).

37h (6.6 mg, 16.9 µmol) was dissolved in pyridine (2 mL) and Ac2O (250

µL), and stirred at rt for 3 h, when TLC (10% acetone/CH2Cl2) showed no

starting material. H2O (10 mL) was added, and the solution extracted with

Et2O (× 2). The pooled organic layers were washed (sat. NaHCO3, sat.

CuSO4, and brine). Drying (MgSO4), evaporation in vacuo and flash column

chromatography (80% Et2O/petrol) gave 37e (5.3 mg, 72%) as an amber resin;

InChI=1/C23H28O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h6,8-9,11,14,16-19,25H,5,7,10H2,1

-4H3/t14-,16+,17+,18+,19+,22+,23+/m1/s1

37e

O

O

O

H HOH

O

O

O O

8

TLC: See tables on page 212 and on the next page.

[α]22D +14 (c 0.1, CH2Cl2);

FTIR (film): ν̃max 3514, 2951, 1737, 1722, 1503, 1461, 1435, 1373, 1322, 1257,

1227, 1199, 1155, 1126, 1092, 1047, 1031, 931, 900, 837, 843, 806, 780, 735

cm−1;

1H NMR (500 MHz, CDCl3): δ 7.49 (1H, br s, H-16), 7.42 (1H, t, J = 1.9 Hz,

H-15), 6.41 (1H, dd, J = 2.0, 1.0 Hz, H-14), 6.39 (1H, dd, J = 2.4, 1.5 Hz,

H-3), 5.35 (1H, dd, J = 4.7, 2.3 Hz, H-2), 5.30 (1H, br d, J = 11.5 Hz, H-12),

4.29 (1H, dd, J = 4.4, 1.7 Hz, H-1), 3.71 (3H, s, CO2CH 3), 2.49 (1H, dd, J =

5.2, 2.1 Hz, H-8), 2.36 (1H, dt, J = 13.3, 3.6 Hz, H-6α), 2.21 (1H, dtd, J =

14.5, 3.7, 2.1 Hz, H-7β), 2.14 (3H, s, OCOCH 3), 2.12 (1H, dd, J = 13.7, 1.5

Hz, H-11α), 1.99 (1H, tt, J = 14.3, 4.4 Hz, H-7α), 1.89 (1H, dd, J = 2.5, 1.3

5.3. SYNTHESIS 215

Hz, OH ), 1.70 (3H, s, H-19), 1.64 (1H, dd, J = 13.9, 11.5 Hz, H-11β), 1.62

(3H, s, H-20), 1.54 (1H, td, J = 13.1, 3.4 Hz, H-6β), 1.30 (1H, t, J = 0.9 Hz,

H-10);

13C NMR (CDCl3): δ 174.0, 169.7, 166.0, 143.7, 143.4, 139.6, 131.2, 123.8,

108.4, 72.5, 69.6, 65.0, 53.1, 51.6, 49.4, 45.6, 37.9, 36.2, 33.4, 25.8, 21.2, 21.0,

18.1;


5.3.15 8-epi-Dideacetylsalvinorin C (37h).

1c (11.0 mg, 23.2 µmol) and KCN∗ (7.5 mg, 115 µmol) were dissolved in MeOH

(1.5 mL) and stirred at rt for 30 min, when TLC (10% acetone/CH2Cl2) showed

no starting material. The solution was evaporated in vacuo, and the residue

partitioned between H2O and CH2Cl2 (× 3). Recrystallisation from acetone,

repeated on the mother liquor, gave 37h (6.6 mg, 73%) as colourless crystals;

InChI=1/C21H26O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7-8,10,12,14-17,22-23H,4,6,9H2,1-3H3

/t12-,14+,15+,16+,17+,20+,21+/m1/s1

37h

O

O

O

H HOH

HO

O O

8

TLC: hRf 1c 37h 1h


50% EtOAc/petrol - 28 19

vanillin/H2SO4 purple blue purple

∗Use of this poisonous reagent is not recommended. K2CO3 and other bases are equallyeffective.


mp (from acetone) 243-245 ◦C;

[α]22D +15 (c 0.04, CH2Cl2);

FTIR (film): ν̃max 3461, 2952, 2926, 1716, 1507, 1459, 1437, 1378, 1259, 1228,

1200, 1180, 1156, 1095, 1052, 875, 803 cm−1;


(1H, dd, J = 2.4, 1.6 Hz, H-3), 6.40 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.30 (1H,

br d, J = 11.5 Hz, H-12), 4.25 (1H, dd, J = 4.9, 2.4 Hz, H-2), 4.16 (1H, ddd,

J = 4.9, 1.6, 0.7 Hz, H-1), 3.71 (3H, s, CO2CH 3), 2.48 (1H, dd, J = 5.5, 2.2

Hz, H-8), 2.23 (1H, dt, J = 13.5, 2.2 Hz, H-6α), 2.23-2.17 (1H, m, H-7β), 2.15

(1H, dd, J = 13.9, 1.5 Hz, H-11α), 1.98 (1H, tdd, J = 14.5, 5.3, 3.8 Hz, H-7α),

1.81 (2H, br s, w 12≈ 50 Hz, OH ), 1.68 (3H, s, H-19), 1.64 (3H, s, H-20), 1.62

(1H, dd, J = 13.8, 12.1 Hz, H-11β), 1.51 (1H, td, J = 12.9, 3.8 Hz, H-6β),

1.21 (1H, br s, H-10);

13C NMR (CDCl3): δ 174.2, 166.4, 143.7, 142.2, 139.6, 134.9, 123.9, 108.4,

69.9, 69.6, 66.5, 53.2, 51.6, 49.4, 45.6, 37.6, 36.2, 33.4, 26.0, 21.3, 18.1;


5.3.16 Salvinorin B formate (46).

A mixture of Ac2O (0.25 mL) and HCO2H (0.7 mL) was stirred at 45 ◦C

for 40 minutes. Meanwhile 1b (18.0 mg, 46.1 µmol) was added to dry pyri-

dine (1 mL), warmed to 45 ◦C until fully dissolved, then cooled to 0 ◦C.

The cooled anhydride mixture was added dropwise by pipette, with violent

bubbling. The solution was warmed to room temperature and stirred for 30

minutes, when TLC (20% acetone/CH2Cl2, visualised in KMnO4) indicated

completion. The reaction mixture was cooled to 0 ◦C, diluted dropwise with

water, and extracted into EtOAc. The organic layer was washed (1% HCl,

water, 5% NaHCO3 and brine). Drying (MgSO4), evaporation in vacuo and

flash column chromatography (2-4% MeOH/CH2Cl2 gradient) gave 46 as a

clear resin (13.8 mg, 72%);

5.3. SYNTHESIS 217

InChI=1/C22H26O8/c1-21-6-4-13-20(26)30-16(12-5-7-28-10-12)9-22(13,2)18

(21)17(24)15(29-11-23)8-14(21)19(25)27-3/h5,7,10-11,13-16,18H,4,6,8-9H2,1-3

H3/t13-,14-,15-,16-,18-,21-,22-/m0/s1

46

O

O

O

H HOO

O

O O

TLC: hRf 1b 46


[α]26D −54 (c 0.6, CH2Cl2);

FTIR (film): ν̃max 3147, 2952, 2854, 1726, 1504, 1451, 1439, 1396, 1372, 1278,

1224, 1163, 1108, 1072, 1024, 1008, 941, 899, 875, 786, 735, 701 cm−1;

1H NMR (CDCl3): δ 8.14 (1H, s, CHO), 7.41 (1H, br s, H-16), 7.40 (1H, t, J =

1.8 Hz, H-15), 6.38 (1H, dd, J = 1.8, 0.9 Hz, H-14), 5.54 (1H, dd, J = 11.7, 5.2

Hz, H-12), 5.25 (1H, ddt, J = 11.2, 8.7, 1.1 Hz, H-2), 3.73 (3H, s, CO2CH 3),

2.79-2.74 (1H, m, H-4), 2.51 (1H, dd, J = 13.5, 5.2 Hz, H-11α), 2.37-2.32 (2H,

m, H-3), 2.18 (1H, d, J = 0.8 Hz, H-10), 2.19-2.15 (1H, m, H-7α), 2.08 (1H,

dd, J = 11.5, 3.0 Hz, H-8), 1.80 (1H, ∼dt, J ≈ 12.9, 2.9 Hz, H-6α), 1.65 (1H,

tdd, J = 13.3, 11.8, 3.2 Hz, H-7β), 1.63-1.58 (1H, m, H-6β), 1.60 (1H, ddd, J

= 13.5, 11.7, 0.8 Hz, H-11β), 1.46 (3H, s, H-20), 1.13 (3H, s, H-19);


(CH, CHO), 143.7 (CH, C-15), 139.4 (CH, C-16), 125.1 (C, C-13), 108.3 (CH,

C-14), 74.5 (CH, C-2), 72.0 (CH, C-12), 64.1 (CH, C-10), 53.5 (CH, C-4), 52.0

(CH3, CO2CH3), 51.3 (CH, C-8), 43.4 (CH2, C-11), 42.1 (C, C-5), 38.1 (CH2,

C-6), 35.5 (C, C-9), 30.6 (CH2, C-3), 18.1 (CH2, C-7), 16.4 (CH3, C-19), 15.2

(CH3, C-20);



5.3.17 Dideacetylsalvinorin C 2-O-(4-bromobenzoate) (47).

To a solution of 1h (3.8 mg, 9.7 µmol), 4-bromobenzoyl chloride (8.7 mg, 39.6

µmol) and DMAP (3.1 mg, 25.4 µmol) in dry CH2Cl2 (1.5 mL) was added

NEt3 (100 µL). The bright yellow solution was stirred under Ar for 90 min,

when TLC (Et2O, visualised in KMnO4) indicated completion. The solution

was diluted in Et2O, washed with 10% HCl (× 3), sat. NaHCO3 and brine.

Drying (MgSO4) and evaporation in vacuo gave 47 as a white powder (5.0 mg,

90%);

InChI=1/C28H29BrO8/c1-27-10-8-18-26(33)37-21(16-9-11-35-14-16)13-28(18,

2)23(27)22(30)20(12-19(27)25(32)34-3)36-24(31)15-4-6-17(29)7-5-15/h4-7,9,11

-12,14,18,20-23,30H,8,10,13H2,1-3H3/t18-,20-,21-,22-,23-,27-,28-/m0/s1

47

O

O

O

H HOH

O

O O

O

Br

TLC: hRf 1h 47


mp (from cold Et2O) 161-166 ◦C;

[α]18D +86 (c 0.4, CH2Cl2);

FTIR (film): ν̃max 3509, 2950, 1717, 1590, 1504, 1484, 1434, 1398, 1269, 1233,

1175, 1143, 1104, 1071, 1012, 946, 875, 849, 786, 758, 679 cm−1;

1H NMR (CDCl3): δ 7.93 (2H, d, J = 8.6 Hz, H-2’), 7.62 (2H, d, J = 8.6 Hz,

H-3’), 7.44 (1H, br s, H-16), 7.42 (1H, t, J = 1.9 Hz, H-15), 6.54 (1H, t, J =

2.0 Hz, H-3), 6.42 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.63 (1H, dd, J = 4.8, 2.4

Hz, H-2), 5.59 (1H, dd, J = 10.9, 5.8 Hz, H-12), 4.61 (1H, br d, J = 4.6 Hz,

H-1), 3.74 (3H, s, CO2CH 3), 2.54 (1H, dt, J = 13.4, 3.6 Hz, H-6α), 2.48 (1H,

5.3. SYNTHESIS 219

dd, J = 13.3, 6.1 Hz, H-11α), 2.17 (1H, dd, J = 12.0, 3.5 Hz, H-8), 2.12 (1H,

dq, J = 12.0, 3.5 Hz, H-7β), 1.96 (1H, br s, w 12

= 14.6 Hz, OH ), 1.92-1.80

(1H, m, H-7α), 1.76 (3H, s, H-19), 1.67 (1H, dd, J = 12.9, 11.2 Hz, H-11β),

1.47 (3H, s, H-20), 1.38 (1H, s, H-10), 1.22 (1H, td, J = 13.1, 3.7 Hz, H-6β);

13C NMR (CDCl3): δ 171.8, 166.0, 164.9, 143.9, 143.6, 139.3, 132.0, 131.4,

131.2, 129.0, 128.0, 125.7, 108.4, 73.2, 71.7, 64.4, 53.9, 51.8, 51.7, 44.4, 37.9,

37.6, 37.0, 22.0, 18.4, 16.2;

HRESIMS: [M + Na]+ m/z 595.0938, 597.0920 (calcd for C28H29O8BrNa+,

595.0938, 597.0917).

5.3.18 17-Deoxysalvinorin A (49).

Et3SiH (20 µL, 125 µmol) was added to a stirred solution of lactol 35 (18.3

mg, 42.1 µmol) in dry CH2Cl2 (1 mL) under Ar. The solution was cooled to

0 ◦C, and BF3·Et2O (10 µL, 79 µmol) was added. The light brown solution

was stirred at 0 ◦C for 2 h, when TLC (10% acetone/CH2Cl2) showed no 35.

The reaction was quenched (0.5 mL sat. NaHCO3), and partitioned between

Et2O and brine. Drying (MgSO4), evaporation in vacuo and flash column

chromatography (0-4% acetone/CH2Cl2 gradient) gave enol ether 50 (4.1 mg,

23%) along with 49 as a clear resin (8.4 mg, 48%);

InChI=1/C23H30O7/c1-13(24)30-17-9-16(21(26)27-4)22(2)7-5-15-12-29-18(14

-6-8-28-11-14)10-23(15,3)20(22)19(17)25/h6,8,11,15-18,20H,5,7,9-10,12H2,1-

4H3/t15-,16-,17-,18-,20-,22-,23-/m0/s1

49

O

O

H HOO

O

O O


TLC: hRf 35 49 50


2% acetone/CH2Cl2 - 24 42

[α]20D −81 (c 0.4, CH2Cl2);

FTIR (film): ν̃max 2950, 2927, 2857, 1730, 1506, 1236, 1201, 1161, 1107, 1042,

1018, 932, 889, 875, 784, 736 cm−1;

1H NMR (CDCl3): δ 7.33 (2H, m, H-15 & 16), 6.34 (1H, t, J = 1.4 Hz, H-14),

5.14 (1H, dd, J = 10.7, 9.4 Hz, H-2), 4.70 (1H, dd, J = 11.6, 2.5 Hz, H-12),

3.70 (3H, s, CO2CH 3), 3.58 (2H, d, J = 7.6 Hz, H-17), 2.79-2.75 (1H, m, H-4),

2.29-2.24 (2H, m, H-3), 2.15 (3H, s, OCOCH 3), 2.12 (1H, dd, J = 13.1, 2.6

Hz, H-11α), 2.09 (1H, d, J = 1.0 Hz, H-10), 1.68-1.62 (2H, m, H-6), 1.54-1.43

(1H, m, H-8), 1.38 (3H, s, H-20), 1.35-1.26 (2H, m, H-7), 1.19 (1H, ddd, J =

13.1, 11.6, 1.0 Hz, H-11β), 1.08 (3H, s, H-19);

13C NMR (CDCl3): δ 202.5 (C, C-1), 171.9 (C, C-18), 169.9 (C, OCOCH3),

142.9 (CH, C-15), 138.9 (CH, C-16), 127.0 (C, C-13), 108.7 (CH, C-14), 75.0

(CH, C-2), 67.5 (CH, C-12), 67.1 (CH2, C-17), 65.6 (CH, C-10), 53.8 (CH,

C-4), 51.8 (CH3, CO2CH3), 46.4 (CH, C-8), 45.4 (CH2, C-11), 42.7 (C, C-5),

39.0 (CH2, C-6), 34.6 (C, C-9), 30.8 (CH2, C-3), 20.6 (CH3, OCOCH3), 19.6

(CH2, C-7), 16.8 (CH3, C-19), 13.7 (CH3, C-20);


5.3.19 8,17-Didehydro-17-deoxysalvinorin A (50).

Et3SiH (25 µL, 156 µmol) and Amberlyst 15 resin (22 mg) were added to

a solution of lactol 35 (15.1 mg, 34.7µmol) in CH2Cl2 (1 mL). The sealed

flask was stirred at rt for 24 h. Filtration, evaporation and flash column

chromatography (1% acetone/CH2Cl2) gave 50 as a clear resin (11.0 mg, 76%);

InChI=1/C23H28O7/c1-13(24)30-17-9-16(21(26)27-4)22(2)7-5-15-12-29-18(14-

5.3. SYNTHESIS 221

6-8-28-11-14)10-23(15,3)20(22)19(17)25/h6,8,11-12,16-18,20H,5,7,9-10H2,1-4H

3/t16-,17-,18-,20-,22-,23-/m0/s1

50

O

O

HO

O

O

O O

TLC: See table on the facing page.

[α]23D −60 (c 0.5, CH2Cl2);

FTIR (film): ν̃max 2927, 2855, 1731, 1664, 1504, 1437, 1380, 1275, 1237, 1203,

1164, 1113, 1098, 1044, 1024, 1000, 962, 944, 875, 786, 737 cm−1;


6.36 (1H, dd, J = 1.9, 0.8 Hz, H-14), 6.26 (1H, d, J = 1.8 Hz, H-17), 5.12 (1H,

dd, J = 11.0, 9.0 Hz, H-2), 4.78 (1H, dd, J = 11.7, 2.1 Hz, H-12), 3.70 (3H,

s, CO2CH 3), 2.74-2.69 (1H, m, H-4), 2.33 (1H, dd, J = 13.6, 2.1 Hz, H-11α),

2.32-2.24 (1H, m, H-7α), 2.29-2.24 (2H, m, H-3), 2.16 (3H, s, OCOCH 3), 2.12

(1H, d, J = 0.8 Hz, H-10), 1.94 (1H, ddd, J = 14.6, 4.6, 2.6 Hz, H-7β), 1.69

(1H, ddd, J = 13.2, 4.4, 2.6 Hz, H-6α), 1.52 (3H, s, H-20), 1.50 (1H, td, J =

13.2, 4.6 Hz, H-6β), 1.40 (1H, ddd, J = 13.6, 11.6, 0.8 Hz, H-11β), 1.15 (3H,

s, H-19);


143.2 (CH, C-15), 139.3 (CH, C-16), 137.1 (CH, C-17), 125.8 (C, C-13), 117.0

(C, C-8), 108.7 (CH, C-14), 75.2 (CH, C-2), 66.6 (CH, C-12), 65.5 (CH, C-10),

53.5 (CH, C-4), 51.7 (CH3, CO2CH3), 44.4 (CH2, C-11), 42.8 (C, C-5), 40.1

(CH2, C-6), 34.1 (C, C-9), 30.6 (CH2, C-3), 22.8 (CH3, C-20), 22.6 (CH2, C-7),

20.6 (CH3, OCOCH3), 15.4 (CH3, C-19);



5.3.20 13,14,15,16-Tetrahydrosalvinorin A (51).

5% Rh/C (25.3 mg) was added to a solution of 1a (20.3 mg, 46.9 µmol) in

50% CH2Cl2/MeOH (6 mL). The suspension was agitated under H2 (4 atm)

at room temperature for 90 minutes, when TLC indicated completion (hRf =

46 (51), 74 (1a) in 20% acetone/CH2Cl2). The solution was filtered through

diatomite filter aid and evaporated in vacuo. Flash column chromatography

(10% acetone/CHCl3) gave 51 (13-epimers, 1:1) as a clear resin (12 mg, 59%).

For characterisation, a portion of the less polar epimer was separated by HPLC

in EtOAc;

InChI=1/C23H32O8/c1-12(24)30-16-9-15(20(26)28-4)22(2)7-5-14-21(27)31-17

(13-6-8-29-11-13)10-23(14,3)19(22)18(16)25/h13-17,19H,5-11H2,1-4H3/t13u,1

4-,15-,16-,17-,19-,22-,23-/m0/s1

51

O

O

O

H HOO

O

O O

HH13

TLC: hRf 51 1a


HPLC: tR (min) 51

EtOAc 11.4, 11.75

[α]19D −39 (c 0.4, CHCl3);

FTIR (film): ν̃max 2953, 2879, 1730, 1452, 1438, 1379, 1277, 1235, 1203, 1165,

1110, 1078, 1050, 1005, 951, 912, 895, 755 cm−1;

1H NMR (CDCl3): δ 5.14 (1H, dd, J = 11.4, 8.6 Hz, H-2), 4.44 (1H, ddd, J

= 11.7, 6.9, 5.0 Hz, H-12), 3.85 (1H, td, J = 8.5, 5.0 Hz, H-15a), 3.78 (1H,

dd, J = 9.0, 7.6 Hz, H-16a), 3.74 (1H, dt, J = 8.5, 7.4 Hz, H-15b), 3.72 (3H,

5.3. SYNTHESIS 223

s, CO2CH 3), 3.54 (1H, dd, J = 9.0, 6.7 Hz, H-16b), 2.73 (1H, ∼dd, J ≈ 11.4,

5.4 Hz, H-4), 2.43 (1H, sext, J = 7.4 Hz, H-13), 2.31-2.26 (2H, m, H-3), 2.19

(1H, dd, J = 13.3, 5.0 Hz, H-11α), 2.17 (3H, s, OCOCH 3), 2.17-2.12 (1H,

m, H-7α), 2.12 (1H, br s, H-10), 2.08 (1H, dddd, J = 12.6, 8.7, 7.4, 5.0 Hz,

H-14a), 1.95 (1H, dd, J = 11.5, 3.1 Hz, H-8), 1.81 (1H, ddt, J = 12.6, 8.3, 7.4

Hz, H-14b), 1.78-1.74 (1H, m, H-6α), 1.65-1.56 (1H, m, H-7β), 1.60-1.50 (1H,

m, H-6β), 1.35 (3H, s, H-20), 1.26 (1H, ddd, J = 13.3, 11.7, 0.8 Hz, H-11β),

1.09 (3H, s, H-19);


(C, OCOCH3), 78.2 (CH, C-12), 75.0 (CH, C-2), 68.8 (CH2, C-16), 68.0 (CH2,

C-15), 64.0 (CH, C-10), 53.5 (CH, C-4), 52.0 (CH3, CO2CH3), 51.4 (CH, C-8),

45.1 (CH, C-13), 42.0 (C, C-5), 41.2 (CH2, C-11), 38.1 (CH2, C-6), 35.1 (C,

C-9), 30.8 (CH2, C-3), 28.2 (CH2, C-14), 20.6 (CH3, OCOCH3), 18.1 (CH2,

C-7), 16.3 (CH3, C-19), 15.1 (CH3, C-20);


5.3.21 Autoxidation of 1a in KOH/MeOH.

Oxygen was bubbled through a solution of KOH in MeOH (1 M, 2 mL) for 5

min. This was then added to a solution of 1a (21.4 mg, 49.5 µmol) in minimal

CH2Cl2 (≈ 250 µL), and oxygen bubbled through the resulting orange solution

for 20 minutes, when TLC (Et2O) showed no 1a or 1b. 10% HCl was added

dropwise until an opaque white colour persisted. The solution was diluted in

0.05 M NaOH and extracted into CH2Cl2 (× 3). Drying (MgSO4), evapora-

tion in vacuo and flash column chromatography (20 – 50% Et2O/petrol) gave

enedione 59 as a resin (9.0 mg, 47%);

InChI=1/C21H22O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7-8,10,12,15,23H,4,6,9H2,1-3H3/t12-,

15-,20-,21-/m0/s1


59

O

O

O

HOH

O

O O

TLC: hRf 1a 1b 59

Et2O 54 42 69

[α]14D +58 (c 0.7, CH2Cl2);

UV (CH3CN): λmax (log ε) 215 (4.30), 249 (3.77), 324 (3.57) nm;

FTIR (film): ν̃max 3373, 3149, 2955, 1726, 1651, 1601, 1504, 1456, 1435, 1408,

1380, 1331, 1244, 1203, 1162, 1068, 1022, 911, 875, 793, 736, 703 cm−1;


6.99 (1H, s, H-3), 6.91 (1H, s, OH ), 6.42 (1H, dd, J = 2.0, 0.9 Hz, H-14), 5.44

(1H, dd, J = 12.3, 2.9 Hz, H-12), 3.85 (3H, s, CO2CH 3), 3.11 (1H, ddd, J =

14.8, 2.9, 1.2 Hz, H-11α), 2.98 (1H, ddd, J = 9.7, 5.4, 1.2 Hz, H-8), 2.53 (1H,

ddd, J = 14.1, 7.7, 6.3 Hz, H-6a), 2.24 (1H, dtd, J = 14.6, 7.4, 5.3 Hz, H-7a),

2.02 (1H, dd, J = 15.0, 12.2 Hz, H-11β), 1.98 (1H, dddd, J = 14.3, 9.7, 7.7,

6.4 Hz, H-7b), 1.77-1.67 (1H, m, H-6b), 1.72 (3H, s, H-19), 1.67 (3H, s, H-20);


(C, C-4), 145.0 (C, C-1), 143.6 (CH, C-15), 140.0 (C, C-10), 139.6 (CH, C-16),

128.2 (CH, C-3), 124.5 (C, C-13), 108.4 (CH, C-14), 70.9 (CH, C-12), 52.6

(CH3, CO2CH3), 44.9 (CH, C-8), 42.3 (C, C-5), 37.7 (C, C-9), 36.8 (CH2,

C-11), 30.3 (CH3, C-19), 28.3 (CH2, C-6), 24.4 (CH3, C-20), 21.9 (CH2, C-7);


The aqueous layer was acidified with 10% HCl until opaque white, then ex-

tracted into CH2Cl2 (× 3), which was dried (MgSO4) and filtered. MeOH (10

mL) and Me3SiCHN2 in Et2O (2.0 M, 200 µL) were added. The yellow solution

was stirred for 30 min, then evaporated under reduced pressure. Flash column

5.3. SYNTHESIS 225

chromatography (50% EtOAc/petrol) gave a mixture of the seco triesters 60a,

60b and 60c (12.0 mg, 53%). Repeated HPLC (36% EtOAc/petrol) gave a

compound which decomposed before full characterisation but was tentatively

identified, based on HRESIMS and NMR (1H and COSY), as 60c;

InChI=1/C23H30O9/c1-22(15(19(25)29-4)10-17(24)28-3)8-6-14-20(26)32-16

(13-7-9-31-12-13)11-23(14,2)18(22)21(27)30-5/h7,9,12,14-16,18H,6,8,10-11H2,

1-5H3/t14-,15-,16-,18+,22-,23-/m0/s1

60c(tentative)

O

O

O

O

HO

O O

O

O

10

H

TLC: hRf 59 60a 60b 60c

80% Et2O/petrol 50 41 41 41

HPLC: tR (min) 60c 60a 60b

36% EtOAc/petrol 17.0 17.4 18.3


6.42 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.19 (1H, dd, J = 11.7, 5.4 Hz, H-12),

3.72 (3H, s, CO2CH3), 3.67 (3H, s, CO2CH3), 3.65 (3H, s, CO2CH3), 3.55

(1H, dd, J = 12.3, 2.9 Hz, H-4), 2.80 (1H, dd, J = 16.6, 12.3 Hz, H-3a), 2.59

(1H, dd, J = 14.9, 5.5 Hz, H-11α), 2.56 (1H, dd, J = 16.6, 2.9 Hz, H-3b),

2.53 (1H, br s, H-10), 2.39 (1H, dd, J = 12.6, 3.7 Hz, H-8), 1.94-1.83 (1H, m,

H-7α), 1.86 (1H, dd, J = 14.9, 11.8 Hz, H-11β), 1.78 (1H, dq, J = 14.4, 3.8

Hz, H-7β), 1.60-1.41 (2H, m, H-6 [obscured by H2O]), 1.41 (3H, s, H-19), 1.20

(3H, s, H-20);


Further elution gave 60a;


InChI=1/C23H30O9/c1-22(15(19(25)29-4)10-17(24)28-3)8-6-14-20(26)32-16

(13-7-9-31-12-13)11-23(14,2)18(22)21(27)30-5/h7,9,12,14-16,18H,6,8,10-11H2,

1-5H3/t14-,15-,16-,18-,22-,23-/m0/s1

60a

O

O

O

O

HO

O O

O

O

H

TLC, HPLC: see table on the previous page.

[α]24D +6 (c 0.1, CH2Cl2);

FTIR (film): ν̃max 2953, 1732, 1506, 1436, 1393, 1373, 1261, 1226, 1202, 1163,

1137, 1079, 1025, 875, 792 cm−1;


H-15), 6.41 (1H, m, H-14), 5.46 (1H, dd, J = 11.6, 5.4 Hz, H-12), 3.69 (3H, s,

CO2CH3), 3.68 (3H, s, CO2CH3), 3.65 (3H, s, CO2CH3), 2.82 (1H, dd, J =

15.9, 11.8 Hz, H-3a), 2.72 (1H, dd, J = 11.8, 2.0 Hz, H-4), 2.43 (1H, dd, J =

15.9, 2.0 Hz, H-3b), 2.25 (1H, s, H-10), 2.17-2.08 (2H, m, H-7β, 8), 2.01 (1H,

dd, J = 13.7, 5.5 Hz, H-11α), 1.84 (1H, ddd, J = 13.7, 12.0, 0.8 Hz, H-11β),

1.74-1.63 (2H, m, H-6a, 7α), 1.52-1.48 (1H, m, H-6b), 1.38 (3H, s, H-19), 1.30

(3H, s, H-20);

13C NMR (CDCl3): δ 174.0 (C, C-2/18), 172.5 (C, C-2/18), 171.7 (C, C-1),

171.0 (C, C-17), 143.8 (CH, C-15), 139.6 (CH, C-16), 125.1 (C, C-13), 108.5

(CH, C-14), 71.6 (CH, C-12), 58.6 (CH, C-10), 52.0 (CH3, CO2CH3), 51.9

(CH, C-4), 51.7 (CH3, CO2CH3), 51.4 (CH3, CO2CH3), 50.5 (CH, C-8), 44.6

(CH2, C-11), 38.7 (C, C-5), 36.9 (C, C-9), 35.1 (CH2, C-6), 32.4 (CH2, C-3),

18.8 (CH3, C-19), 18.2 (CH2, C-7), 15.6 (CH3, C-20);


5.3. SYNTHESIS 227

Further elution gave 60b;

InChI=1/C23H30O9/c1-22(15(19(25)29-4)10-17(24)28-3)8-6-14-20(26)32-16

(13-7-9-31-12-13)11-23(14,2)18(22)21(27)30-5/h7,9,12,14-16,18H,6,8,10-11H2,

1-5H3/t14-,15+,16+,18+,22+,23+/m1/s1

60b

O

O

O

O

HO

O O

O

O

8

H

TLC, HPLC: see table on page 225.

[α]23D +4 (c 0.4, CH2Cl2);

FTIR (film): ν̃max 2953, 1733, 1504, 1436, 1392, 1361, 1247, 1200, 1166, 1091,

1062, 1025, 998, 968, 909, 875, 849, 794, 735 cm−1;


H-15), 6.42 (1H, dd, J = 1.8, 0.7 Hz, H-14), 5.26 (1H, dd, J = 12.0, 2.1 Hz,

H-12), 3.67 (3H, s, CO2CH3), 3.66 (3H, s, CO2CH3), 3.64 (3H, s, CO2CH3),

2.84 (1H, dd, J = 15.9, 11.9 Hz, H-3a), 2.76 (1H, dd, J = 11.9, 1.6 Hz, H-4),

2.53 (1H, dd, J = 4.6, 2.8, H-8), 2.39 (1H, dd, J = 16.0, 1.8 Hz, H-3b), 2.28

(1H, br s, H-10), 2.19-2.14 (1H, m, H-7β), 2.02 (1H, dd, J = 14.5, 11.9 Hz,

H-11β), 1.94-1.86 (2H, m, H-6β, 7α), 1.80 (1H, dd, J = 14.4, 2.1 Hz, H-11α),

1.48 (3H, s, H-20), 1.32-1.27 (1H, m, H-6α), 1.29 (3H, s, H-19);


(C, C-1), 143.7 (CH, C-15), 139.7 (CH, C-16), 123.6 (C, C-13), 108.4 (CH,

C-14), 69.8 (CH, C-12), 57.8 (CH, C-10), 51.9 (CH3, CO2CH3), 51.7 (CH3,

CO2CH3), 51.7 (CH, C-4), 51.5 (CH3, CO2CH3), 46.5 (CH2, C-11), 44.4 (CH,

C-8), 38.5 (C, C-5), 35.8 (C, C-9), 32.2 (CH2, C-3), 31.6 (CH2, C-6), 25.1

(CH3, C-20), 18.2 (CH3, C-19), 18.2 (CH2, C-7);



5.3.22 NaBH4 reduction of 59.

Enedione 59 (41.3 mg, 107 µmol) and NaBH4 (10 mg, 264 µmol) were dissolved

in CH2Cl2 (500 µL), followed by EtOH (2 mL), and stirred under Ar at 40◦C. The initial orange colour faded to faint yellow within 1 h. After 4 h,

TLC (Et2O) indicated completion. The solution was cooled to 0 ◦C, and 0.5%

H2SO4/MeOH added dropwise until effervescence ceased. The solution was

concentrated to ≈ 500 µL in vacuo, then partitioned between brine (acidified

with 10% HCl) and CH2Cl2 (× 3). Drying (MgSO4), evaporation in vacuo

and flash column chromatography (70-100% Et2O/petrol) gave 38h27 (15.7

mg, 37%);

InChI=1/C21H28O7/c1-20-6-4-12-19(25)28-15(11-5-7-27-10-11)9-21(12,2)17

(20)16(23)14(22)8-13(20)18(24)26-3/h5,7,10,12-17,22-23H,4,6,8-9H2,1-3H3/t

12-,13+,14+,15+,16+,17+,20+,21+/m1/s1

38h

O

O

O

H HOH

HO

O O

8

TLC: hRf 38h 59

Et2O 53 69

1H NMR (CDCl3): δ 7.48 (1H, dt, J = 1.7, 0.9 Hz, H-16), 7.42 (1H, t, J =

1.7 Hz, H-15), 6.41 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.29 (1H, dd, J = 11.7,

1.5 Hz, H-12), 4.07 (1H, br s, H-1), 3.65 (3H, s, CO2CH 3), 3.54 (1H, ddd, J

= 11.1, 4.7, 3.2 Hz, H-2), 2.45 (1H, br d, J = 4.7 Hz, H-8), 2.23-2.10 (4H, m),

1.90 (1H, tdd, J = 14.4, 5.5, 4.0 Hz, H-7α), 1.73-1.53 (m, obscured by H2O &

OH ), 1.66 (3H, s, H-20), 1.32 (3H, s, H-19), 0.90 (1H, d, J = 1.6 Hz, H-10);

1H NMR ([CD3]2CO) and 13C NMR (CDCl3) matched literature values.27

5.3. SYNTHESIS 229

5.3.23 O-Demethyl-18-deoxysalvinorin A (77).

To dry EtSH (1.5 mL, 20.2 mmol) at 0 ◦C, stirred rapidly under a stream of Ar,

was added nBuLi in hexanes (2.1 M, 8 mL, 16.9 mmol). Immediate, violent gas

evolution was accompanied by the sudden formation of a white solid. Rapid

stirring was continued while the remaining nBuLi was added, swirling the flask

when necessary to free the stir bar. After warming to room temperature, the

solution was evaporated under reduced pressure and dried under high vacuum

at 50 ◦C for 30 min, giving LiSEt as a white powder. This was stored at room

temperature in a sealed flask under Ar for up to a year without losing activity.

1a (42.2 mg, 97.6 µmol) and LiSEt (13.7 mg, 201 µmol) under Ar were dis-

solved in DMPU (1 mL). The yellow solution was stirred at 55 ◦C for 23

h, when TLC (1% H2SO4/10% MeOH/CH2Cl2) indicated consumption of 1a

and intermediate 1b. The cooled orange solution was diluted with EtOAc

and washed (10% HCl × 3, then water), then extracted into 1% NaHCO3 (×3). The pooled aqueous fractions were acidified at 0 ◦C with 10% HCl, then

extracted into CH2Cl2 (× 3). Drying (MgSO4) and evaporation in vacuo gave

an amber resin, which was treated with Ac2O (0.4 mL) in pyridine (1 mL)

and catalytic DMAP at room temperature for 17 h. After cooling to 0 ◦C and

quenching (water), the solution was diluted in EtOAc and washed (10% HCl

and sat. NH4Cl). Drying (MgSO4) and evaporation in vacuo gave the mixed

acids 67a (H-8α : β, ∼1.4:1) as an amber resin (29.7 mg, 73% over two steps);

InChI=1/C22H26O8/c1-11(23)29-15-8-14(19(25)26)21(2)6-4-13-20(27)30-16

(12-5-7-28-10-12)9-22(13,3)18(21)17(15)24/h5,7,10,13-16,18H,4,6,8-9H2,1-

3H3,(H,25,26)/t13u,14-,15-,16-,18-,21-,22-/m0/s1/f/h25H

67a

O

O

O

H HOO

O

O OH


TLC: hRf 1a 1b 67a 77 78

1% H2SO4/10% MeOH/CH2Cl2 71 63 0-20

1% NEt3/EtOAc 0-30 56 56

Et2O 20 30

vanillin/H2SO4 purple purple purple blue

1H NMR (CDCl3): δ 7.43 (1H, br s, H-16∗), 7.41 (1H, br s, H-16), 7.39-7.38

(2H, m, H-15, 15∗), 6.37 (2H, m, H-14, 14∗), 5.52 (1H, dd, J = 11.6, 5.2 Hz,

H-12), 5.26 (1H, dd, J = 11.9, 1.8 Hz, H-12∗), 5.16 (1H, dd, J = 12.4, 7.6

Hz, H-2), 5.11 (1H, dd, J = 12.4, 7.2 Hz, H-2∗), 2.80 (1H, dd, J = 5.2, 3.5

Hz, H-4), 2.76 (1H, dd, J = 5.2, 3.5 Hz, H-4∗), 2.49 (1H, dd, J = 13.4, 5.2

Hz, H-11α), 2.45 (1H, dd, J = 4.9, 2.0 Hz, H-8∗), 2.37 (1H, dd, J = 14.8, 2.0

Hz, H-11α∗), 2.37-1.94 (m), 2.28 (1H, br s, H-10∗), 2.20 (1H, br s, H-10), 2.17

(3H, s, OCOCH 3), 2.15 (3H, s, OCOCH 3∗), 1.84 (1H, tt, J = 14.2, 4.2 Hz,

H-7α∗), 1.73 (1H, dtd, J = 13.7, 3.5, 0.8 Hz, H-6α), 1.67-1.52 (m), 1.63 (3H,

s, H-20∗), 1.50 (1H, dd, J = 15.1, 12.1 Hz, H-11β∗), 1.45 (3H, s, H-20), 1.13

(3H, s, H-19), 1.09 (3H, s, H-19∗).

The mixed acids 67a (35.2 mg, 84.0 µmol) were stirred in dry THF (1 mL) at

rt, under Ar, in a flask fitted with a reflux condenser. BH3·THF (1.0 M, 110 µL,

110 µmol) was added dropwise; the solution was heated to 55 ◦C and stirred

at this temperature for 90 minutes, when TLC (1% NEt3/EtOAc) indicated

completion. The solution was cooled to room temperature, quenched with

water (dropwise), and evaporated under reduced pressure to a cloudy paste.

This was diluted with sat. NaHCO3 and extracted into CH2Cl2 (×3). Drying

(MgSO4), evaporation in vacuo and flash column chromatography (10-25%

acetone/Et2O gradient) monitored by TLC (Et2O) gave 78 as a clear resin

(8.4 mg, 25%);

InChI=1/C22H28O7/c1-12(24)28-16-8-14(10-23)21(2)6-4-15-20(26)29-17(13-

5-7-27-11-13)9-22(15,3)19(21)18(16)25/h5,7,11,14-17,19,23H,4,6,8-10H2,1-

3H3/t14-,15+,16-,17-,19-,21-,22-/m0/s1

∗Unnatural (H-8α) epimer.

5.3. SYNTHESIS 231

78

O

O

O

H HOO

O

OH

8

TLC: see table on the preceding page.

[α]24D −31 (c 0.3, CH2Cl2);

FTIR (film): ν̃max 3547, 3148, 2942, 2880, 1725, 1504, 1468, 1450, 1440, 1376,

1238, 1202, 1159, 1085, 1050, 1024, 950, 934, 875, 802, 784, 734, 703 cm−1;


6.38 (1H, dd, J = 1.9, 0.9 Hz, H-14), 5.25 (1H, dd, J = 12.0, 2.2 Hz, H-12),

5.09 (1H, ddd, J = 12.3, 7.2, 1.0 Hz, H-2), 3.93 (1H, dd, J = 10.5, 3.9 Hz,

H-18a), 3.46 (1H, ddd, J = 10.8, 8.0, 0.6 Hz, H-18b), 2.52 (1H, ddd, J = 12.3,

7.0, 2.8 Hz, H-3a), 2.45-2.43 (1H, m, H-8), 2.36 (1H, dd, J = 15.0, 2.2 Hz,

H-11α), 2.24 (1H, d, J = 0.9 Hz, H-10), 2.22-2.09 (2H, m, H-6β,7β), 2.15 (3H,

s, OCOCH 3), 1.91-1.72 (4H, m, H-3b, 4, 6α, 7α), 1.64 (3H, s, H-20), 1.52 (1H,

dd, J = 14.8, 12.0 Hz, H-11β), 0.92 (3H, s, H-19);

13C NMR (CDCl3): δ 204.0, 173.6, 169.9, 143.6, 139.7, 123.4, 108.6, 76.2, 70.1,

64.5, 61.7, 50.3, 48.1, 45.4, 42.1, 34.6, 33.9, 31.6, 24.8, 20.6, 17.7, 15.5;


Further elution gave 77 as a clear resin (7.7 mg, 23%);

InChI=1/C22H28O7/c1-12(24)28-16-8-14(10-23)21(2)6-4-15-20(26)29-17(13-

5-7-27-11-13)9-22(15,3)19(21)18(16)25/h5,7,11,14-17,19,23H,4,6,8-10H2,1-

3H3/t14-,15-,16-,17-,19-,21-,22-/m0/s1


77

O

O

O

H HOO

O

OH

TLC: see table on page 230.

[α]25D −19 (c 0.3, CH2Cl2);

FTIR (film): ν̃max 3468, 2944, 1727, 1504, 1452, 1376, 1237, 1162, 1095, 1053,

1023, 952, 875, 794, 735, 703 cm−1;


(1H, dd, J = 1.9, 0.8 Hz, H-14), 5.52 (1H, dd, J = 11.7, 5.3 Hz, H-12), 5.15

(1H, dd, J = 12.3, 7.6 Hz, H-2), 3.94 (1H, dd, J = 10.3, 3.9 Hz, H-18a), 3.49

(1H, dd, J = 10.3, 8.0 Hz, H-18b), 2.54 (1H, ddd, J = 12.3, 7.0, 2.6 Hz, H-3a),

2.49 (1H, dd, J = 13.3, 5.3 Hz, H-11α), 2.17 (1H, d, J = 0.9 Hz, H-10), 2.16

(3H, s, OCOCH 3), 2.20-2.13 (1H, m, H-7β), 2.06 (1H, dd, J = 12.1, 3.0 Hz,

H-8), 1.99 (1H, dt, J = 13.3, 3.3 Hz, H-6α), 1.94-1.87 (1H, m, H-4), 1.80 (1H,

q, J = 12.3 Hz, H-3b), 1.64 (1H, dddd, J = 14.0, 13.5, 12.1, 3.4 Hz, H-7α),

1.58 (1H, ddd, J = 13.3, 11.7, 0.9 Hz, H-11β), 1.45 (3H, s, H-20), 1.43 (1H,

td, J = 13.2, 3.7 Hz, H-6β), 0.96 (3H, s, H-19);


143.7 (CH, C-15), 139.4 (CH, C-16), 125.2 (C, C-13), 108.4 (CH, C-14), 76.0

(CH, C-2), 72.1 (CH, C-12), 64.5 (CH, C-10), 61.7 (CH2, C-18), 51.5 (CH,

C-8), 50.8 (CH, C-4), 43.4 (CH2, C-11), 41.9 (C, C-5), 38.1 (CH2, C-6), 35.3

(C, C-9), 31.8 (CH2, C-3), 20.6 (CH3, OCOCH3), 18.1 (CH2, C-7), 16.7 (CH3,

C-19), 15.3 (CH3, C-20);


5.3. SYNTHESIS 233

5.3.24 1-Deoxysalvinorin A (81a).

A solution of NaBH4 (11.6 mg, 307 µmol) and 1a (108 mg, 249 µmol) in dry

EtOH (9 mL)/CH2Cl2 (2 mL) was stirred under Ar at 40 ◦C. The cloudy

solution gradually cleared. At 4 h, TLC (Et2O) indicated completion (for hRf

values, see table on page 208). The solution was evaporated in vacuo, and

the residue partitioned between brine and CH2Cl2 (× 3). Drying (MgSO4)

and evaporation in vacuo gave an approximately equal mixture of 36h23 and

38h27 (total 103 mg), which was used without purification.

This crude mixture and 1,1’-thiocarbonyldiimidazole (118 mg, 662 µmol) were

dissolved in dry DMF and stirred under Ar at 90 ◦C for 6 h, when TLC

(40% acetone/ CH2Cl2) indicated completion. The cooled solution was diluted

in EtOAc/Et2O and washed (10% HCl × 3, then brine). Drying (MgSO4)

and evaporation in vacuo followed by flash column chromatography (1-5%

acetone/CH2Cl2 gradient) gave the cyclic thionocarbonates 80 (H-8α : β, ≈1:2), as a clear resin (72 mg, 67% over two steps);

InChI=1/C22H26O7S/c1-21-6-4-12-19(24)27-15(11-5-7-26-10-11)9-22(12,2)17

(21)16-14(28-20(30)29-16)8-13(21)18(23)25-3/h5,7,10,12-17H,4,6,8-9H2,1-3H3

/t12u,13-,14-,15-,16-,17-,21-,22-/m0/s1

80

O

O

O

H HO

O

O O

S

TLC: hRf 36h/38h 80 83 81b 82b 81a

Et2O 47 40 47 74

10% acetone/CH2Cl2 55 70 20 20


vanillin/H2SO4 purple blue purple


1H NMR (CDCl3): δ 7.49 (1H, br s, H-16∗), 7.45 (1H, br s, H-16), 7.43-7.42

(2H, m, H-15, 15∗), 6.40 (1H, br s, H-14, 14∗), 5.58 (1H, dd, J = 11.2, 5.2

Hz, H-12), 5.34 (1H, br d, J = 10.8 Hz, H-12∗), 5.03 (1H, dd, J = 6.4, 2.8

Hz, H-1), 4.93 - 4.78 (3H, m, H-2, 1∗, 2∗), 3.70 (3H, s, CO2CH 3), 3.67 (3H,

s, CO2CH 3∗), 2.56 (1H, dd, J = 5.3, 1.9 Hz, H-8∗), 2.45 (1H, dd, J = 12.8,

5.2 Hz, H-11α), 2.27 - 2.10 (m), 1.94 (1H, tdd, J = 14.2, 4.8, 3.9 Hz, H-7α∗),

1.82 - 1.69 (m), 1.71 (1H, dd, J = 13.5, 11.0 Hz, H-11β), 1.64 (3H, s, H-20∗),

1.59-1.49 (m), 1.47 (3H, s, H-20), 1.37 - 1.20 (m), 1.33 (3H, s, H-19), 1.30 (3H,

s, H-19∗);

13C NMR (CDCl3): δ 191.0, 190.9, 173.4, 171.6, 171.4, 170.8, 144.0, 143.8,

139.7, 139.4, 125.1, 123.3, 108.22, 108.19, 79.8, 79.0, 78.9, 78.8, 71.5, 69.6,

53.9, 53.1, 52.1, 52.0, 51.85, 51.79, 51.2, 48.0, 45.9, 43.5, 38.7, 37.5, 36.4, 36.2,

35.9, 34.9, 26.4, 26.3, 26.2, 18.3, 17.6, 16.5, 16.2, 14.8.

nBu3SnH was prepared by a published procedure408 with reduced reaction time

(10 minutes),409 distilled (80 ◦C/0.5 mmHg) and stored in the dark under argon

at -20 ◦C (when used, it remained clear and unclouded). The thionocarbonate

mixture was dissolved in dry toluene (2 mL). Ar was bubbled through the

resulting cloudy solution for 2 min; it was then stirred under Ar at 80 ◦C. A

solution of nBu3SnH408 (150 µL, 550 µmol) and AIBN (5.4 mg, 33 µmol) in dry

toluene (2 mL, also deoxygenated) was added in small portions over 4 h; the

solution gradually cleared. After a further 2 h, TLC (10% acetone/ CH2Cl2)

indicated completion. The solution was cooled and loaded directly onto silica

gel, rinsing the flask with CH2Cl2 (× 2). Repeated flash column chromatog-

raphy (50-70% EtOAc/petrol gradient) monitored by TLC (Et2O) gave two

fractions, A and B. Fraction A was subjected to flash column chromatography

(10% acetone/ CH2Cl2) to give carbonate 83 as a clear resin (2.7 mg, 4%);

InChI=1/C22H26O8/c1-21-6-4-12-19(24)28-15(11-5-7-27-10-11)9-22(12,2)17

(21)16-14(29-20(25)30-16)8-13(21)18(23)26-3/h5,7,10,12-17H,4,6,8-9H2,1-3H3

/t12-,13-,14-,15-,16-,17-,21-,22-/m0/s1

∗Unnatural (H-8α) epimer.

5.3. SYNTHESIS 235

83

O

O

O

H HO

O

O O

O


[α]22D +5 (c 0.1, CH2Cl2) ;

FTIR (film): ν̃max 2952, 1797, 1727, 1502, 1452, 1441, 1370, 1276, 1229, 1200,

1163, 1073, 1043, 1023, 956, 875, 790, 736 cm−1;


(1H, dd, J = 1.9, 0.9 Hz, H-14), 5.58 (1H, dd, J = 11.4, 5.4, H-12), 4.90 (1H,

dd, J = 6.3, 2.9 Hz, H-1), 4.67 (1H, tdd, J = 7.9, 6.2, 2.2 Hz, H-2), 3.70 (3H,

s, CO2CH 3), 2.46-2.41 (2H, m), 2.27-2.10 (4H, m), 1.87-1.67 (m, overlapping

with H2O), 1.42 (3H, s, H-20), 1.36-1.24 (m), 1.34 (3H, s, H-19), 1.27 (1H, d,

J = 2.9 Hz, H-10);

13C NMR (CDCl3): δ 171.6, 170.8, 154.1, 144.0, 139.4, 125.2, 108.2, 75.0, 74.6,

71.5, 54.5, 52.2, 52.0, 43.7, 38.8, 37.5, 35.9, 26.9, 24.3, 18.3, 16.4, 16.3;


Further elution gave 82b as a clear resin (15.5 mg, 25%);

InChI=1/C21H28O6/c1-20-6-4-14-19(24)27-16(12-5-7-26-11-12)10-21(14,2)17

(20)9-13(22)8-15(20)18(23)25-3/h5,7,11,13-17,22H,4,6,8-10H2,1-3H3/t13-,14+,

15-,16-,17-,20-,21-/m0/s1

82b

O

O

O

H HHO

O O

8



[α]21D −4 (c 0.1, CH2Cl2);

FTIR (film): ν̃max 3432, 2948, 2871, 1728, 1502, 1450, 1438, 1390, 1363, 1277,

1255, 1199, 1167, 1091, 1054, 1022, 1000, 875, 840, 785, 727 cm−1;


(1H, dd, J = 2.0, 0.9 Hz, H-14), 5.24 (1H, dd, J = 11.8, 1.5 Hz, H-12), 3.69 -

3.60 (1H, m, H-2), 3.64 (3H, s, CO2CH 3), 2.48 (1H, dd, J = 5.0, 1.6 Hz, H-8),

2.17 (1H, dtd, J = 14.4, 3.5, 2.1 Hz, H-7β), 2.12 (1H, dd, J = 13.2, 3.6 Hz,

H-4), 1.99 (1H, dd, J = 14.0, 1.6 Hz, H-11α), 1.91 (1H, dddd, J = 12.9, 5.0,

3.3, 1.9 Hz, H-1β), 1.87 - 1.58 (m), 1.81 (1H, dd, J = 13.0, 11.0 Hz, H-11β),

1.52 (1H, dt, J = 13.6, 3.2 Hz, H-6α), 1.43 (1H, td, J = 12.6, 10.7 Hz, H-1α),

1.28-1.24 (1H, m, H-6β), 1.24 (3H, s, H-19), 1.08 (3H, s, H-20), 1.00 (1H, dd,

J = 12.7, 2.2 Hz, H-10);

13C NMR (CDCl3): δ 174.1, 173.3, 143.7, 139.7, 123.8, 108.4, 70.1, 69.7, 53.8,

52.4, 51.3, 49.1, 44.8, 36.4, 35.8, 34.1, 34.0, 31.3, 24.1, 17.9, 14.1;


Fraction B gave 81b as a resin (13.5 mg, 22%);

InChI=1/C21H28O6/c1-20-6-4-14-19(24)27-16(12-5-7-26-11-12)10-21(14,2)17

(20)9-13(22)8-15(20)18(23)25-3/h5,7,11,13-17,22H,4,6,8-10H2,1-3H3/t13-,14-,

15-,16-,17-,20-,21-/m0/s1

81b

O

O

O

H HHO

O O


5.3. SYNTHESIS 237


6.40 (1H, dd, J = 1.9, 1.0 Hz, H-14), 5.50 (1H, ddd, J = 11.4, 5.7, 0.8 Hz,

H-12), 3.70-3.63 (1H, m, H-2), 3.67 (3H, s, CO2CH 3), 2.32 (1H, dd, J = 13.4,

5.8 Hz, H-11α), 2.13 (2H, br dd, J = 12.9, 3.2 Hz, H-4, 8), 2.09 (1H, dq, J =

14.1, 3.5 Hz, H-7β), 1.96-1.82 (3H, m, H-1β, 3), 1.74 (1H, dt, J = 13.6, 3.2

Hz, H-6α), 1.65-1.58 (m, overlapping with H2O), 1.44 (1H, td, J = 12.6, 10.9

Hz, H-1α), 1.28 (1H, td, J = 13.6, 4.0 Hz, H-6β), 1.10 (3H, s, H-19), 1.07 (3H,

s, H-20), 1.04 (1H, dd, J = 12.8, 2.5 Hz, H-10);

13C NMR (CDCl3): δ 173.0, 172.0, 143.8, 139.3, 125.7, 108.4, 71.8, 70.0, 54.4,

53.0, 51.5, 51.2, 44.0, 38.0, 36.9, 36.2, 33.7, 30.4, 18.2, 15.0, 14.6.

This was fully characterised as the acetate by dissolving in pyridine (0.3 mL)

and Ac2O (0.3 mL) with a crystal of DMAP. The solution was stirred at rt

for 2.5 h, when TLC (Et2O, visualised in KMnO4) indicated completion, then

evaporated in vacuo. Flash column chromatography (60% Et2O/n-pentane)

gave 81a as a clear resin (12.3 mg, 82%);

InChI=1/C23H30O7/c1-13(24)29-15-9-17(20(25)27-4)22(2)7-5-16-21(26)30-18

(14-6-8-28-12-14)11-23(16,3)19(22)10-15/h6,8,12,15-19H,5,7,9-11H2,1-4H3/t15

-,16-,17-,18-,19-,22-,23-/m0/s1

81a

22 101011

O

O

O

H HO

O

O O


[α]21D −8 (c 0.1, CH2Cl2);

FTIR (film): ν̃max 2952, 1729, 1503, 1450, 1434, 1365, 1319, 1245, 1202, 1153,

1099, 1024, 954, 901, 875, 783, 734 cm−1;



6.41 (1H, dd, J = 1.9, 1.0 Hz, H-14), 5.47 (1H, ddd, J = 11.2, 5.6, 0.7 Hz,

H-12), 4.74 (1H, tt, J = 11.0, 5.5 Hz, H-2), 3.66 (3H, s, CO2CH 3), 2.28 (1H,

dd, J = 13.5, 5.9 Hz, H-11α), 2.18 (1H, dd, J = 10.9, 3.5 Hz, H-4), 2.15 (1H,

dd, J = 10.0, 3.5 Hz, H-8), 2.09 (1H, dq, J = 14.2, 3.4 Hz, H-7β), 2.05 (3H, s,

OCOCH 3), 1.95-1.89 (1H, m, H-1β), 1.92-1.83 (2H, m, H-3), 1.74 (1H, dt, J

= 13.5, 3.3 Hz, H-6α), 1.68-1.57 (1H, m, H-7α), 1.64 (1H, dd, J = 13.6, 11.5

Hz, H-11β), 1.50 (1H, td, J = 12.8, 11.2 Hz, H-1α), 1.31 (1H, td, J = 13.6,

3.7 Hz, H-6β), 1.11 (3H, s, H-19), 1.10 (1H, dd, J = 12.9, 2.2 Hz, H-10), 1.06

(3H, s, H-20);


143.8 (CH, C-15), 139.4 (CH, C-16), 125.6 (C, C-13), 108.4 (CH, C-14), 71.82

(CH, C-2/12), 71.77 (CH, C-2/12), 54.2 (CH, C-4), 52.9 (CH, C-10), 51.5

(CH3, CO2CH3), 51.2 (CH, C-8), 43.9 (CH2, C-11), 38.0 (CH2, C-6), 36.9 (C,

C-9), 36.2 (C, C-5), 29.6 (CH2, C-3), 26.7 (CH2, C-1), 21.3 (CH3, OCOCH3),

18.2 (CH2, C-7), 15.0 (CH3, C-19), 14.6 (CH3, C-20);


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Thomas Anthony Munro- The Chemistry of Salvia divinorum

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