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 2 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 O 1 1 O O OR O R H H H H 2 H H O R 2 2 8 O O R O O O O O OR3 R1 R2 R1 R2 R3 1a 1d Ac OH 28a OH H H 1e H OAc 28b OH OH Me 1f H H 28c H OAc H 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. O OH O H O O O O 59 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.thethesisislessthan100,000wordsin length, exclusive of tables, maps, bibliographies and appendices. Thomas Munro 5 6 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, andanImprovedMethodforthe 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 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 1.1Botany................................ 31 1.2Ethnopharmacology......................... 33 1.3Chemistry............................... 36 1.3.1 Terpenoids.......................... 36 1.3.2 Allegedalkaloids....................... 43 1.4Pharmacology............................. 47 11 12 CONTENTS 1.4.1 AnimalTesting........................ 47 1.4.2 HumanTesting........................ 51 1.4.3 In vitro Testing........................ 53 1.4.4 Mechanism: κ Opioids.................... 54 1.5Toxicology............................... 61 1.6Socialimpact............................. 62 1.6.1 RecreationalUse....................... 62 1.6.2 LegalStatus......................... 63 1.7Summary............................... 66 2Isolation. 67 2.1IsolationProcedure.......................... 67 2.1.1 ExtractionConditions.................... 67 2.1.2 ProblemsCausedbyPigments............... 69 2.1.3 UseofActivatedCarbon................... 72 2.1.4 SeparationofTerpenoids.................. 82 2.2StructureElucidation........................ 90 2.2.1 RevisedNMRAssignmentsforSalvinorinA(1a)..... 90 2.2.2 RevisedNMRAssignmentsforSalvinorinC(1c)..... 92 2.2.3 OtherKnownDiterpenoids................. 92 2.2.4 KnownTriterpenoids..................... 96 2.2.5 SalvinorinsD-F(1d-1f)................... 98 2.2.6 DivinatorinsA-C(28a-28c).................110 2.2.7 Subsequent isolations. .................118 CONTENTS 13 3 Synthesis. 119 3.1Knownderivatives..........................119 3.2 Epimerisation at C-8 under Basic Conditions. ..........120 3.2.1 PreviousReports.......................120 3.2.2 8-epi-SalvinorinsAandB(37aand37b)..........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 ChromatographicIdentificationofEpimers........125 3.2.6 Mechanism..........................126 3.2.7 Attempted Deacetylation under Acidic Conditions. 128 3.3SimpleDerivatives..........................128 3.3.1 Esters(46and47)......................128 3.3.2 AttemptedBenzylEtherFormation(48)..........129 3.3.3 17-Deoxy Compounds (49 and 50). ............130 3.3.4 TetrahydrosalvinorinA(51).................131 3.3.5 (+)-Hardwickiic Acid (ent-29a)...............134 3.4ModificationoftheMethylEster..................134 3.4.1 RelevantResultsfromPreviousWork...........134 3.4.2 TreatmentofSalvinorinAwithKOHinMeOH......135 3.4.3 O-Demethylsalvinorin A (67a). ..............144 3.4.4 O-Demethyl-18-deoxysalvinorinA(77)...........150 3.5ModificationoftheKetone.....................153