Tryptamines as Ligands and Modulators of the Serotonin 5-HT2A Receptor and the Isolation of Aeruginascin from the Hallucinogenic Mushroom Inocybe aeruginascens Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Niels Jensen aus Hamburg Göttingen 2004 D 7 Referent: Prof. Dr. H. Laatsch Korreferent: Prof. D. E. Nichols Tag der mündlichen Prüfung: 4. November 2004 Table of Contents III _________________________________________________________________________ Table of Contents Table of Contents ................................................................................................................III List of Figures...................................................................................................................... V List of Tables ...................................................................................................................... IX List of Abbreviations ...........................................................................................................XI Theoretical Part ....................................................................................................................1 Introduction.......................................................................................................................1 Psychoactive mushrooms.............................................................................................1 Inocybe aeruginascens.................................................................................................3 Aeruginascin.................................................................................................................3 Aims of the aeruginascin project ..................................................................................4 Tryptamines as 5-HT receptor ligands .........................................................................4 G-protein coupled receptors .........................................................................................7 Mechanism of hallucinogenic action...........................................................................16 Pharmacology of 5-HT2A ligands ................................................................................23 Aims of the tryptamine project ....................................................................................26 Results and Discussion ..................................................................................................29 Isolation of aeruginascin.............................................................................................29 New route to 4-hydroxytryptamines............................................................................29 Isolation of aeruginascin.............................................................................................37 Synthesis of aeruginascin...........................................................................................42 Serotonin receptor ligand synthesis ...........................................................................49 Pharmacological testing .............................................................................................63 IP accumulation assay results ....................................................................................73 Non-competitive modulation .......................................................................................99 Alleged "high affinity" 5-HT2A receptor ligand ...........................................................104 Summary of the aeruginascin project .......................................................................107 Summary of the tryptamine project...........................................................................110 Experimental Part.............................................................................................................117 Materials and Methods .................................................................................................117 Pharmacological methods ........................................................................................117 Receptor binding experiments..................................................................................118 Chemical syntheses .................................................................................................119 Chemical Experiments..................................................................................................121 4-Hydroxytryptamines: Ene synthesis route .............................................................121 Synthesis of baeocystin and norbaeocystin .............................................................125 IV Table of Contents _________________________________________________________________________ Aeruginascin: Isolation, synthesis, and spectroscopic data .....................................131 Synthesis of alcohols................................................................................................135 Synthesis of alkyl halides .........................................................................................148 Synthesis of N-alkyl-N-methyltryptamines................................................................164 Synthesis of N-(4-bromobenzyl)-5-methoxytryptamine ............................................243 Appendix ..........................................................................................................................247 Aeruginascin spectra ................................................................................................247 Tested compounds (Table 6)....................................................................................251 Receptor binding data (Table 7) ...............................................................................255 Comparison of binding affinities (Table 8) ................................................................263 Functional 5-HT2A data (Table 9)..............................................................................271 Functional data compared to binding data (Table 10) ..............................................279 References .......................................................................................................................287 List of Figures V _________________________________________________________________________ List of Figures Figure 1: Chemical structures of the Psilocybe alkaloids and of aeruginascin........................2 Figure 2: Chemical structures of tryptamine, serotonin (5-HT), and psilocin. .........................7 Figure 3: Typical binding curve from a competitive binding experiment................................14 Figure 4: Typical dose-response curve from an IP accumulation assay. ..............................15 Figure 5: Dose-response curves of agonists, partial agonists, and antagonists. ..................16 Figure 6: 5-HT2A receptor ligands (I): Phenethylamines........................................................23 Figure 7: 5-HT2A receptor ligands (II): Lysergamides. ...........................................................24 Figure 8: 5-HT2A receptor ligands (III): Tryptamines..............................................................25 Figure 9: Alleged 5-HT2A receptor ligand...............................................................................26 Figure 10: New 4-hydroxytryptamine synthesis. ...................................................................30 Figure 11: Isomerization of diethyl-prop-2-ynyl-sulfonium bromide.......................................30 Figure 12: Mechanism of the annulation reaction. ................................................................31 Figure 13: Mechanism of the ene reaction. ...........................................................................31 Figure 14: Reaction of the exo-methylene compound with Eschenmoser's salts. ................32 Figure 15: Isomerization of exo-methylene-tetrahydrofuranone............................................33 Figure 16: Reaction mechanism of the ammonolysis............................................................33 Figure 17: Putative aromatization product.............................................................................35 Figure 18: Synthesis of baeocystin and norbaeocystin. ........................................................36 Figure 19: UV spectra of isolated and synthetic aeruginascin. .............................................40 Figure 20: 1H NMR spectra of isolated aeruginascin.............................................................41 Figure 21: Decomposition products of aeruginascin detected by ESI MS. ...........................43 Figure 22: Biosynthesis of Psilocybe alkaloids......................................................................45 Figure 23: Aeruginascin, muscarine, and phosphorylcholine................................................47 Figure 24: Superposition of aeruginascin and muscarine. ....................................................47 Figure 25: Aeruginascin and 5-HTQ......................................................................................48 Figure 26: 1H-NMR spectra of the precursors and the final tryptamine.................................52 Figure 27: Transition from a first order into a second order signal........................................54 Figure 28: Synthesis of N-methyl-N-alkyltryptamine. ............................................................54 Figure 29: Synthesis of N-monomethyltryptamine.