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Synthesis of Nicotinic Receptor Ligands and Strigolactones

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Title:
Synthesis of Nicotinic Receptor Ligands and Strigolactones
Creator:
Chojnacka, Kinga
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (362 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Chemistry
Committee Chair:
HORENSTEIN,NICOLE ALANA
Committee Co-Chair:
STEWART,JON DALE
Committee Members:
BRUNER,STEVEN DOUGLAS
APONICK,AARON
PAPKE,ROGER LEE
Graduation Date:
12/13/2013

Subjects

Subjects / Keywords:
Acetates ( jstor )
Agonists ( jstor )
Aldehydes ( jstor )
Chromatography ( jstor )
Esters ( jstor )
Ligands ( jstor )
Nicotinic receptors ( jstor )
Quaternary ammonium compounds ( jstor )
Receptors ( jstor )
Sodium ( jstor )
7 -- acetylcholine -- agonist -- alpha -- desensitization -- nicotinic -- receptor -- silent -- strigolactones
Chemistry -- Dissertations, Academic -- UF
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Chemistry thesis, Ph.D.

Notes

Abstract:
The first part of this dissertation is focused on the design and synthesis of molecules that differentially regulate activation and desensitization of the human alpha7 nicotinic acetylcholine receptor (nAChR). Alpha7 nAChR is a pentameric ligand gated ion channel that is currently a drug target for Alzheimer’s disease, schizophrenia and inflammatory disorders. Emerging evidence suggests that alpha7 in non-neuronal cells may inhibit pro-inflammatory cytokine production by a signaling mechanism that does not involve ion channel opening.  The term “silent agonists” is introduced to describe receptor ligands that bind to the conventional acetylcholine binding site, do not initiate ion channel activity, and place the receptor in a desensitized state that can be revealed in the presence of a type II positive allosteric modulator. Because these molecules convert the alpha7 nAChR from a resting state selectively into a desensitized state that can be probed by a type II PAM, they are of interest as a tool to study the functions of the alpha7 nAChR that do not involve ion conducting states, and may constitute a new modality for the development of alpha7 nAChR therapeutics. Three groups of silent agonists are characterized: the first group is represented by newly designed and synthesized KC-1 and KC-5 compounds, the second group features bulky quaternary ammonium compounds of appropriate molecular volume, and the third is exemplified by pyridinylmethylene anabaseines (PABs). The second part of this dissertation is focused on the strigolactone synthesis and biosynthesis. Strigolactones are a new class of plant hormones that inhibit shoot lateral branching. Synthesis of the strigolactone ABC-core in one step from linear precursors by an acid-catalyzed double cyclization was proposed and tested. Linear model precursors were prepared and conditions under which these molecules undergo the proposed double cyclization in good yields and with a high degree of stereocontrol were identified. These cyclization results are relevant because they suggest a plausible mechanism along the strigolactone biosynthetic pathway. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: HORENSTEIN,NICOLE ALANA.
Local:
Co-adviser: STEWART,JON DALE.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-12-31
Statement of Responsibility:
by Kinga Chojnacka.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
12/31/2015
Classification:
LD1780 2013 ( lcc )

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1 SYNTHESIS OF NICOTINIC RECEPTOR LIGANDS AND STRIGOLACTONES By KINGA CHOJNACKA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Kinga Chojnacka

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3 ACKNOWLEDGEMENTS There are many people without whom I w ould not have finish ed my Ph.D I would like to e specially thank my mentor Dr. Nicole A. Horenstein She to ok a chance on me during my complicated graduate career which I believe work out well for us both. Her continued support and guidance was critical to my success. I would also like to thank my co advisor Dr. Roger P apke for his teaching of pharmacology and neuroscience, as well as making great suggestions for my research. I would like to thank Dr. Nigel Richards for advising me in the first three years of my graduate studies and teaching me how important it is to give a good talk. I am also very grateful t o Dr. Aaron Aponick for his time in mentoring me on the strigolactone project, encouragement when reactions did not work for prolong ed times and making me a better organic chemist. I would also like to acknowledge my doctoral committee members Dr. Jon Ste wart, Dr. Steven Bruner, Dr. Harry Klee, and Dr. Radi Awartani for their advice. I am very grateful to all people from Dr. Papke Laboratory who performed the electrophysiological experime nts on OpusXpress, including Clare Stokes Matthew Kimbrell, Lu W. Co rrie, Shehd A. Al Rubaiy, Matthew D. Isaacson Thomas F. Pack Sara B. Copeland, Sarah Pinhei ro Akshatha Rao Khan A. Manther and Chrisopher W. Kinter. I thank Dr. Stefano Santoro and Dr. Fahmi Himo from Stockholm University for performing computational c alculations on the cyclization of the strigolactone ABC core aldehyde precursors. My special acknowledgements go to people from the Aponick group for many scientific conversations, sharing equipment and making me feel at home in their lab s

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4 and group meetin gs, especially in the first three years of my Ph.D studies. In particular, I would like to thank Dr. Berenger Biannic, Dr. Nick Borrero, Dr. John Ketcham, Dr. Carl Ballesteros Dr. Jean Palmes, Flavio Cardoso Thomas Ghebreghiorgis, Justin Goodwin and Pau lo Paioti. I also thank all the people from the Horenstein and Richards groups for scientific discussions and all the fun we had working together, especially Dr. Jingyi Wang Dr. Lorraine Clark Jeff Arciola and also Claribel Yanbin, Tim, Michelle, Alica n Alison, Megan, Yongmo, and Wen. I would also like to thank the best and most helpful secretary I have ever met Mrs. Lori Ball. I thank Lucas, Brad and Filip for their support, friendship, and beer when I needed it. Lastly, I thank m y family especial ly my grandparents, for supporting me all my life.

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5 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................ ................................ ............................... 3 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 15 ABSTRACT ................................ ................................ ................................ ................... 18 C HAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 20 1.1 Chemical Neurotransmission, Receptors, Ion Channels, Functions of nAChRs ................................ ................................ ................................ ........... 20 1.2 Subtypes of nAChRs ................................ ................................ ........................ 26 1.3 S tructure of the Receptor ................................ ................................ ................. 30 1.3.1 Overview ................................ ................................ ................................ 30 1.3.2 Extracellular Domain (Ligand Binding Domain) ................................ ..... 32 1.3.3 Transmembrane Domain ................................ ................................ ....... 34 1.3.4 Intracellular Domain ................................ ................................ ............... 36 1.3.5 Three Dimensional Structur es of Pentameric LGICs ............................. 36 1.3.5.1 X ray structures of acetylcholine binding protein (AChBP) ....... 36 1.3.5.2 Cryo electron microscopic structure of the nAChR from the Torpedo electric organ at 4 ................................ .................... 38 1.3.5.3 X ray structure of the ECD of a mouse 1 monomer with bungarotoxin bound at 1.94 resolution. ................................ 39 1.3.5.4 X ray structures of prokaryotic ion channels (GLIC and ELIC) 40 1.3.5.5 X ray structure of a the ECD of h 7 nAChR and AChBP chimeras ................................ ................................ ................... 41 1.3.5.6 Structure of a pentameric glutamate LGIC from C. elegans ..... 42 1.4 Studying the nAChR Ionotropic Function ................................ ......................... 43 1.5 Ligands for the nAChRs ................................ ................................ ................... 45 1.5.1 Nicotinic Agonists ................................ ................................ .................. 48 1.5.2 Nicotinic Anta gonists ................................ ................................ ............. 51 1.5.3 Positive Allosteric Modulators ................................ ................................ 53 1.5.4 7 nAChR Silent Agonists ................................ ................................ ..... 54 1.5.5 Therapeutic Opportunities of the nAChR Ligands ................................ 55 1.6 Allosterism and Desensitization ................................ ................................ ....... 55 1.7 The 7 nAChR and Inflammatory Response ................................ ................... 56 1.7.1 Cholinergic Anti inflammatory Pathway ................................ ................. 56

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6 1.7.2 Findings Indicating that the 7 nAChR M ay Have Signaling Function in Inflammation Without Ion Channel Activity ................................ ......... 58 1.7.3 Other Possible Signaling Pathways Involving 7 nAChR ...................... 59 2 KC COMPOUNDS AS NEW 7 SILENT AGONISTS ................................ ............. 60 2.1 Background ................................ ................................ ................................ ...... 60 2.2 Results and Discussion ................................ ................................ .................... 61 2.2.1 Synthesis of KC 1 ................................ ................................ .................. 61 2.2.2 Activity Profile of KC Silent Agonism ... 66 2.2.3 Synthesis of KC 1 Analogs ................................ ................................ .... 67 2.2.4 Activity Profile of KC .................... 70 2.2.5 Activity Profile of KC ................ 77 2.3 Summary ................................ ................................ ................................ ......... 79 3 SILENT AGONIST BULKY QUATERNARY AMMONIUM ................................ ...... 82 3.1 Background ................................ ................................ ................................ ...... 82 3.2 Results and Discussion ................................ ................................ .................... 84 3.2.1 Synthesis ................................ ................................ ............................... 84 3.2.2 Activity profile of quaternary ammonium compounds on the human 7 nAChR ................................ ................................ .............................. 84 3.2.3 Activity Profile of Quaternary Ammonium Compounds on the Human ................................ ................................ .......................... 95 3.3 Summary ................................ ................................ ................................ ......... 97 4 SYNTHESIS OF NEW FLUORINATED PYRIDINYLMETHYLENE ANABASEINES TO STUDY INTERACTIONS LEADING TO DESENSITIZED STATES OF THE HUMAN 7 NICOTINIC ACETYLCHOLINE RECEPTOR ......... 99 4.1 Background ................................ ................................ ................................ ...... 99 4.2 Results and Discussion ................................ ................................ .................. 101 4.2.1 Synthesis of New Fluorinated Arylidene Anabaseine s ......................... 101 4.2.2 pKa Values of New Fluorinated Arylidene Anabaseines ...................... 105 4.2.3 Activity Profile of Fluorinated Arylidene Anabaseines on the Human 7 nAChR ................................ ................................ ............................ 106 4.3 Summary ................................ ................................ ................................ ....... 109 5 SYNTHESIS OF NOVEL ARYLIDENE QUINUCLIDINES TO STUDY THE EFFECT OF H BONDING IN THE 7 nAChR SELECTIVITY POCKET ON ACTIVATION AND DESENSITIZATION ................................ ............................... 111 5.1 Background ................................ ................................ ................................ .... 111 5.2 Results and Discussion ................................ ................................ .................. 113 5.2.1 Synthesis of Arylidene Quinuclidines ................................ ................... 113 5.2.1.1 Synthesis of 2 TQN and 2 FQN Using Phosphonate Ethyl Esters ................................ ................................ ...................... 114

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7 5.2.1.2 Synthesis of 2 TQN and 2 FQN Using Phosphonate Methyl Esters ................................ ................................ ...................... 116 5.2.1.3 Synthesis of 3 TQN and 3 FQN ................................ .............. 116 5.2.1.4 Attempts to synthesize N Boc 2 (chloromethyl)pyrrole (90). ... 117 5.2.1.5 Attempt to synthesize 2 PyroQN (66ab) ................................ 118 5.2.2 Activity of QN Compounds ................................ ................................ ... 118 5.3 Summary ................................ ................................ ................................ ....... 119 6 STRIGOLACTONES ................................ ................................ ............................. 120 6.1 Background ................................ ................................ ................................ .... 120 6.1.1 Roles of Strigolactones ................................ ................................ ........ 120 6.1.2 Strigolactone Biosynthetic Pathway ................................ ..................... 122 6.1.3 Strigolactone Synthesis ................................ ................................ ....... 125 6.1.4 Importance of Work on Strigolactones ................................ ................. 128 6.2 Research Design and Specific Aims ................................ .............................. 128 6.3 Results and Discussion ................................ ................................ .................. 131 6.3.1 First Cyclization Attempts (Aliphatic Substrate) ................................ ... 131 6.3.2 Attempts to Cyclize Aromatic Substrates at the Alcohol Oxidation Level ................................ ................................ ................................ .... 137 6.3.2.1 Rationale ................................ ................................ ................. 137 6.3.2.2 GR24 Synthesis ................................ ................................ ...... 138 6.3.2.3 Synthesis of Aromatic Substrates at Alcohol Oxidation Level and Attempts to Cyclize Them ................................ ................ 140 6.3.3 Cyclization of Aromatic Substrates at the Aldehyde Oxidation Level ... 145 6.3.4 Synthesis of Proposed Structure for Solanac ol (105) Using Acid catalyzed Double Cyclization as a Key Step to Prepare the Core. ................................ ................................ ...... 158 6.4 Additional Notes ................................ ................................ ............................. 171 6.5 Summary ................................ ................................ ................................ ....... 171 7 CONCLUSIONS AND FUTURE WORK ................................ ............................... 173 APPENDIX A EXPERIMENTAL PROCEDURES ELECTROPHYSIOLOGY ............................... 177 B COMPUTATIONAL METHODS ................................ ................................ ............ 180 C ALTERNATIVE PHARMACOPHORE ................................ ................................ ... 181 D SYNTHETIC EXPERIMENTA L PROCEDURES ................................ ................... 184 E NMR SPECTRA OF NEW SYNTHESIZED COMPOUNDS ................................ .. 260 LIST OF REFERENCES ................................ ................................ ............................. 331 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 362

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8 LIST OF TABLES Table Page 2 1 IC 50 values for KC 1 analogs ................................ ................................ .............. 71 2 2 Net charge for KC co applications with PNU 120596. ................................ ........ 76 3 1 The EC 50 and I max for bulky ammonium compounds that have ionotropic agonism activity. ................................ ................................ ................................ 86 3 2 The IC 50 values for bulky quaternary ammonium compounds. ........................... 87 3 3 Net charge for quaternary ammonium compounds co applications with PNU 12059 6. ................................ ................................ ................................ ...... 90 4 1 The predicted iminium cation percentages of the fluorinated arylidene anabaseines. ................................ ................................ ................................ .... 106 6 1 Cyclization of the E aldehy de precursor. ................................ ......................... 147 6 2 Cyclization of the Z aldehyde precursor.. ................................ ......................... 151

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9 LIST OF FIGURES Figure Page 1 1 Chemical si gnaling in the nervous system. ................................ ......................... 21 1 2 Structures of some neurotransmitters. ................................ ................................ 23 1 3 Structures of ace tylcholine, muscarine, atropine, and nicotine. .......................... 24 1 4 Schematic representation of a nicotine acetylcholine receptor (nAChR) from muscle. ................................ ................................ ................................ .............. 25 1 5 Schematic representation of a few nAChR subtypes with acetylcholine binding sites shown. ................................ ................................ ........................... 27 1 6 Schematic representation of one subunit. ................................ ....................... 31 1 7 Ligand binding domain. ................................ ................................ ...................... 33 1 8 Conformational changes in the loop C in AChBPs on ligand binding. ................ 34 1 9 Cross section of a nAChR pore in the middle of the transmembrane domain. ... 35 1 10 The pentameric structure of AChBP. ................................ ................................ .. 37 1 11 Ribbon diagrams of the nAChR from Torpedo electric organ. ............................ 38 1 12 Overall structure of the mouse nAChR 1 subunit bound to Bgtx .................. 39 1 13 Schematic representation of one monomer of pentameric LGIC in prokaryotes and eukaryotes. ................................ ................................ .............. 40 1 14 GLIC and ELIC proteins. ................................ ................................ .................... 41 1 15 Structures of 7 AChBP chimera. ................................ ................................ ...... 42 1 16 Struc ture of the GluCl Fab complex. ................................ ................................ .. 43 1 17 Studying the nAChR ionotropic function.. ................................ ........................... 46 1 18 Structures of some non selective nAChR agonists. ................................ ............ 49 1 19 Structures of some 7 nAChR selective agonists. ................................ .............. 50 1 20 Structures of some 7 nAChR PAMs. ................................ ................................ 53 1 21 Representative traces showing the effects of PAMs on ACh evoked 7 nAChR responses. ................................ ................................ ...................... 54

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10 1 22 Cholinergic anti inflammatory pathway balances cytokine production. .............. 57 2 1 Struct ures of NS6740, KC 1 and one pharmacophore for silent agonists. ......... 61 2 2 KC 1 retrosynthetic analysis ................................ ................................ ............... 62 2 3 bromo anabaseine 1 ................................ ................................ .. 63 2 4 Synthesis of 5 phenylnicotinic ethyl ester 8 ................................ ....................... 63 2 5 Synthesis of KC 1. ................................ ................................ .............................. 65 2 6 100 M KC 1. ................................ ................................ ................................ ..... 67 2 7 Structures of KC compounds. ................................ ................................ ............. 68 2 8 Synthesis of KC 4 and KC 7. ................................ ................................ .............. 69 2 9 Synthesis of KC 2, KC 3, KC 5, KC 6, KC 8, KC 9. ................................ ........... 69 2 10 Ionot ropic agonism alpha 7 KCs series. ................................ ............................. 70 2 11 A KC 1/ACh competition curve. ................................ ................................ .......... 72 2 12 A KC 5/ACh competition curve. ................................ ................................ .......... 73 2 13 Desensitized state revealed by co application with type II PAM. ........................ 74 2 14 A sharp change of preference of KC 1 for D i desensitized state bet ween 300 M and 1 mM ................................ ................................ ............................... 75 2 15 Ratio of PNU 120596 (10 M) co application with KC compounds (100 M) responses to KC (100 M) ionotropic agonism responses.. ............................... 77 2 16 KC 1 to KC ................................ ............................ 78 2 17 KC 1 to KC 9 co ................................ 78 2 18 KC 1 to KC ................................ .. 79 2 19 KC 1 to KC 9 compounds summary.. ................................ ................................ 80 3 1 Structures of ASM 024, T EA and TMA. ................................ .............................. 82 3 2 Structures of bulky quaternary ammonium compounds. ................................ ..... 83 3 3 Alpha7 agonism bulky ammonium. ................................ ................................ ..... 85

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11 3 4 Desensitized states of quaternary ammonium revealed by co application with type II PAM. ................................ ................................ ................................ ........ 89 3 5 Ratio of 10 M PNU 120596 co application response at 100 M to an ionotropic response at 100 M (1 mM for choline analogs). ............................... 92 3 6 Bulky quaternary ammonium h 7 nAChR profile summary. ............................... 93 3 7 Structures of ASM 024, DMPP, diethylphenylpiperazinium and diethylphenylpiperidinium. ................................ ................................ .................. 95 3 8 Quaternary ammonium ionotropic agonism on h nAChR. ......................... 96 3 9 Quaternary ammonium antagonism on h ................................ ..... 97 4 1 Structures of 2 PAB, 3 PAB, and 4 PAB. ................................ ........................... 99 4 2 Pharmacological properties of 2 PAB, 3 PAB, and 4 PAB.. ............................... 99 4 3 Structures of fluorinated 3 PABs and pentafluorinated benzylidene anabaseine. ................................ ................................ ................................ ...... 101 4 4 Synthesis of anabaseine dihydrochloride 52 ................................ ................... 102 4 5 Synthesis of new fluorinated arylidene anabaseines. ................................ ....... 104 4 6 2F 3PAB ( 44 ): NOE enhancement used to assign the double bond geometries in fluorinated pyridinylmethylene anabaseines. ............................. 105 4 7 Synthesi s of 2,4,6 trifluoronicotinaldehyde 56 ................................ ................. 105 4 8 Ionotropic agonism of fluorinated arylidene anabaseines on h 7 nAChR. ....... 107 4 9 Antagonism of fluorinated arylidene anabaseines. ................................ ........... 107 4 10 Desensitized state of fluorinated arylidene anabasei nes revealed with PNU 120596. ................................ ................................ ................................ .... 108 4 11 Comparison of PNU 120596 co application responses for 3PAB and (2 F) 3PAB. ................................ ................................ ................................ ...... 109 5 1 Stru ctures of non selective nAChR agonists (anabaseine, quinuclidine), and h 7 nAChR selective agonists (GTS 21, (E) benzylidene quinuclidine). .......... 111 5 2 Structures of h 7 nAChR partial agonists 2PyroAB, 2FAB, and 2TAB. ............ 112 5 3 Structures of H bonding probes. ................................ ................................ ....... 113 5 4 Retrosynthetic analysis of arylidene quinuclidines. ................................ ........... 113

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12 5 5 Synthe sis of 2 TQN ( 70ab ) and 2 FQN ( 68ab ) using phosphonate ethyl esters. ................................ ................................ ................................ ............... 114 5 6 Comparison of 1 HNMR spectra for (Z) 2 TQN ( 70a ) and (E) 2 TQN ( 70b ). ..... 115 5 7 Synthesis of 2 TQN ( 70ab ) and 2 FQN ( 68ab ) using phosphonate methyl esters. ................................ ................................ ................................ ............... 116 5 8 Synthesis of 3 TQN ( 71ab ) and 3 FQN ( 69ab ). ................................ ................ 117 5 9 Attempts to prepare N Boc 2 (chloromethyl)pyrrole 90 ................................ ... 117 5 10 Attempts to synthesize 2 PyroQN. ................................ ................................ .... 118 6 1 Strigolactones secreted by host plants induce germination of parasitic weeds. 120 6 2 Structures of natural strigolactones and synthetic analog GR24. ..................... 121 6 3 Phenotype of wild type rice plant producing strigolactones and a mutant impaired in stri golactone biosynthesis. ................................ ............................ 122 6 4 Structures of plant hormones.. ................................ ................................ .......... 123 6 5 Partially predicted strigolactones biosynthetic pathway deduced from analysis of plant mutants displaying an increased branching phenotype and studies in carotenoids accumulating E. Co li and in vitro ................................ ... 124 6 6 First total syntheses of strigol. ................................ ................................ .......... 127 6 7 Enantiopure precursors of D ring. ................................ ................................ ..... 128 6 8 Proposed literature strigolactone biosynthetic pathway. ................................ ... 129 6 9 Proposed formation of the strigolactone ABC core in one step by an acid catalyzed do uble cyclization. ................................ ................................ ............ 130 6 10 Proposed and correct structures of solanacol. ................................ ................. 131 6 11 First cyclization plans. ................................ ................................ ...................... 131 6 12 Retrosynthetic analysis of the substrate 107 needed for cyclization studies. ... 131 6 13 Synthesis of the aldehyde 109 ................................ ................................ ......... 132 6 14 Reported reactivity of the diol 112 under acidic condtions. ............................... 133 6 15 Synthesis of the phosphonium salt 110 ................................ ........................... 134 6 16 of the carboxylate group of 110 ............... 135

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13 6 17 Wittig reaction and attempts to cyclize the olefin product 107 ......................... 136 6 18 Aromatic substrate at the alcohol oxidation level cyclization plan. ................. 137 6 19 Retrosynthetic analysis of 119 ................................ ................................ ......... 138 6 20 GR24 Synthesis. ................................ ................................ ............................... 139 6 21 Synthesis of the aldehyde 121 ................................ ................................ ......... 141 6 22 Synthesis of the phosphonium salt 122 ................................ ........................... 141 6 23 Synthesis of linear precursors and attempts to cyclize them. ........................... 142 6 24 Byproduct 142 formed during prolonged a reaction with sodium hydroxide in water. ................................ ................................ ................................ ................ 142 6 25 Formation of a cyclic product from the triflic ester 48 in a GC MS instrument. 143 6 26 Compounds formed in the reaction of the orthoester 138 with an HCl solution of allyl alcohol. ................................ ................................ ................................ .. 144 6 27 Cyclization of an aromatic substrate at the aldehyde oxidat ion level plan. ....... 145 6 28 Precedent for the first cyclization. ................................ ................................ ..... 145 6 29 Preparation of the linear aldehydes for cyclization. ................................ .......... 146 6 30 Graph of the Karplus relationship for ethane derivatives. ................................ 148 6 31 1 HNMR with proton assignments for 155 (trans) top spec trum and 156 (cis) bottom spectrum. ................................ ................................ ........................... 149 6 32 Mechanistic hypothesis for the cyclization reaction. ................................ ......... 152 6 33 Calculated transiti on states and relative energies for cyclization of the B ring from trans olefin 154 and cis olefin 153 initiated by protonation. ...................... 153 6 34 Calculated rotational barriers for protonated benzald ehyde. ............................ 154 6 35 Proposed biosynthetic pathway. ................................ ................................ ....... 155 6 36 Synthesis of the aldehyde free carboxylic acid 164 ................................ ......... 156 6 37 Synthesis of the aldehyde benzyl ester 166 ................................ .................... 156 6 38 Acid catalyzed double cyclization of the trans olefin benzyl ester aldehyde 166 ................................ ................................ ................................ .................. 157

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14 6 39 Acid catalyzed double cyclization of the allyl ester aldehyde 170 .................... 157 6 40 Cleavage of the allyl group. ................................ ................................ .............. 158 6 41 Retrosynthesis plan for the synthesis of proposed structure for solanacol ( 105 ), using acid catalyzed double cyclization as a key step. .......................... 159 6 42 Attempt to synthesize the alkyne aldehyde carboxylic acid 184 ...................... 160 6 43 Synthesis of the linear precursor needed for acid catalyzed double cyclization to form the ABC core of solanacol. ................................ .................. 162 6 44 Synthesis of Dess Martin Periodinane (DMP). ................................ ................. 163 6 45 Cyclization of the acetal carboxylic acid 190 ................................ ................... 164 6 46 The 1 HNMR spectrum of the crude from the reaction of 190 with 0.3 equivalent of TfOH: at least 4 cyclized products are formed. ...................... 164 6 47 The 1 194 ............................. 165 6 48 Cyclization of the aldehyde carboxylic acid 176 ................................ .............. 165 6 49 1 HNMR of the fraction A from cyclization of aldehyde carboxylic acid 176 (most probably 3 diasteromers of 195 ). ................................ ............................ 166 6 50 1 HNMR of the fraction B from cyclization of aldehyde carboxylic acid 176 (on 195 ). ................................ ................................ .... 166 6 51 Preparation of the E olefin aldehyde methyl ester 197 ................................ .... 167 6 52 Cyclization of the E ol efin aldehyde methyl ester 197 ................................ ..... 168 6 53 1 HNMR of the products obtained in cyclization of the E aldehyde methyl ester 197 ................................ ................................ ................................ .................. 168 6 54 Preparation of the Z olefin aldehyde methyl ester 203 ................................ .... 169 6 55 Cyclization of the Z olefin aldehyde methyl ester 203 ................................ ..... 169 6 56 1 HNMR of the crude in cyclization of the Z aldehyde methyl ester 203 ........... 170 6 57 1 HNMR consistent with a structure of 199 formed in cyclization of the Z aldehyde methyl ester 203 ................................ ................................ ........... 170 6 58 Isomerization of the cis lactone product. ................................ .......................... 171 6 59 Strigolactone biosynthetic pathway proposed in 2012 by Alder et al ............... 172

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15 L IST O F ABBREVIATIONS T ERM : Definition Aa Amino acid Ac Acetyl ACh Acetylcholine AChBP Acetylcholine binding protein AIBN Azobisisobutyronitrile BHP (E) 3 (6 benzylidenecyclohex 1 en 1 yl)pyridine Boc t Butoxycarbonyl b.p. Boilin g point Cat. Catalyst cDNA Complementary deoxyribonucleic acid cRNA Complementary ribonucleic acid (CHO) n Paraformaldehyde CRC Concentration response curve D i PNU 120596 insensitive desensitized state D s PNU 120596 sensitive desensitized state DART Dir ect analysis in real time DCM Dichloromethane DME 1,2 dimethoxyethane DMF Dimethylformamide DMP Dess Martin Periodinane DMSO Dimethyl sulfoxide ECD Extracellular domain ESI Electro spray ionization

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16 Et 2 O D iethyl ether EtOAc Ethyl acetate EtOH Ethanol FAB Fur anylmethylene anabaseine GTS 21 2,4 dimethoxybenzylidene anabaseine HRMS High resolution mass spectroscopy HCl Hydrochloric acid ICD Intracellular domain IR Infrared spectrometry LBD Ligand binding domain LDA Lithium diisopropylamide Li HMDS Lithium hexame thyldisilazide Li TMP Lithium 2,2,6,6 tetramethylpiperidide LGIC Ligand gated ion channel MeOH Methano l Mol% Percent molar equivalents m.p. Melting point mRNA Messenger ribonucleic acid nAChR Nicotinic acetylcholine receptor Et 3 N Triethylamine NBS N bromos uccinimide NMR Nuclear magnetic resonance NOE Nuclear Overhauser effect NOESY Nuclear Overhauser effect spectroscopy o/n Overnight

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17 PAB Pyridinylmethylene anabaseine PAM Positive allosteric modulator PCC Py ridinium chlorochromate PDC Py ridinium dichlorochro mate PNU 120596 N (5 chloro 2,4 dimethylphenyl) (5 methyl 3 isoxazolyl) urea QN Quninuclidine Red Al Sodium bis(2 methoxyethoxy)aluminium hydride R f Retention factor r.t. Room temperature S.E.M. Standard error TBAF Tetrabutylammonium fluoride TBS Tert B utyldimethylsilyl TFA Trifluoroacetic acid TfOH triflic acid THF Tetrahydrofuran TLC Thin layer chromatography TMA Tetramethylammonium TMD Transmembrane domain TMSOTf Trimethylsilyltriflate WT Wild type

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18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYNTHESIS OF NICOTINIC RECEPTOR LIGANDS AND STRIGOLACTONES By Kinga Chojnacka December 2013 Chair: Nicole A. Horenstein Major: Chemis try The first part of this dissertation is focused on the design and synthesis of molecules that differentially regulate activation and desensitization of the human alpha7 nicotinic acetyl choline receptor (nAChR). Alpha 7 nAChR is a pentameric ligand gated ion inflammatory disorders. Emerging evidence suggest s that alpha7 in non neuronal cells may inhibit pro inflammatory cytokine production by a signaling mechanism that does receptor ligands that bind to the conventional acetylcholine binding site, do not initiate ion channel activity, and place the receptor in a desensitized state that can be revealed in the presence of a type II positive allosteric modulator. Because these molecules convert the alpha7 nAChR from a resting state selectively into a desensitized state that can be probed by a type II PAM, they are of interest as a tool to stud y the functions of the alpha7 nAChR that do not involve ion conducting states, and may constitute a new modality for the development of alpha7 nAChR therapeutics.

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19 Three groups of silent agonists are characterized: the first group is represented by newly de signed and synthesized KC 1 and KC 5 compounds, the second group features bulky quaternary ammonium compounds of appropriate molecular volume, and the third is exemplified by pyri dinylmethylene anabaseine s (PABs). The second part of this dissertation is f ocused on the strigolactone synthesis and biosynthesis. Strigolactones are a new class of plant hormones that inhibit shoot lateral branching. Synthesis of the strigolactone ABC core in one step from linear precursors by an acid catalyzed double cyclizatio n was proposed and tested. Linear model precursors were prepared and conditions under which these molecules undergo the proposed double cyclization in good yields and with a high degree of stereocontrol were identified. These cyclization results are releva nt because they suggest a plausible mechanism along the strigolactone biosynthetic pathway.

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20 CHAPTER 1 INTRODUCTION This portion of my dissertation is focused on the design, synthesis and characterization of molecul es that modulate the function of the human 7 ni c otinic acetylcholine receptor ( h 7 nAChR ) particularly silent agonists Traditionally, the role of the 7 receptor was linked to its ion channel activity H owever growing evidence supports a metabotropic function independent of ion channel opening that may modulate signal transduction pathways that can regulate inflammatory response and cell death ( apoptosis ) or survival especially in non neuronal cells. Hence, the following section provides background information on ion channels ligands, and desensiti zation that might be the active state for signaling beyond ion channel activity. 1.1 Chemical N euro transmi ssion R eceptors Ion C hannels F unctions of nAChRs Communication between cells in humans comes from the central nervous system (CNS brain and spinal colu mn ) and the peripheral nervous system ( PNS nerves and ganglia) which connect CNS to the rest of the body. A nerve cell (neuron) possesses a cell body, dendrites and an axon (Figure 1 1. A) 1 Neurons transmit signals by electrical pulses that result from t he movement of ions across cell membranes due to different concentrations of ions inside and outside cells established by ion pumps (the concentration of potassium ions inside the cell is larger than the surrounding medium whereas the concentration of sod ium, calcium and chloride ions is smaller) 2 Neurons communicate with each other and with other cells through synapses, by passing an electrical s ignal or a chemical signal 1,3 In the electrical synapses (that are minor in the mammalian nervous system), n eurons are extremely close to each other and the signal is pass ed on directly through tight junctions that involve specialized ion

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21 channels called connexons 4 In the majority of synapses, the gap (s ynaptic cleft) between cells is too large for a direct tr ansmission and the communication between cells is mediated by endogenous chemicals called neurotransmitters 1 A. B. Figure 1 1. Chemical signaling in the nervous system. A) Structure of a typical nerve cell B) S cheme of a chemical synapse. Neurotransmitters are stored in vesicles at the axon terminal (Figure 1 1. B) After being released to a synaptic cleft they diffuse to pro teins (called receptors) localized in the membrane of target cells to which they bind causing a conformational change that leads t o a series of secondary effects. F or example a flow of ions across

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22 the cell membrane or the switching on (or off) of enzymes inside the target cells 2 There are several mechanisms t o avoid con t inuous activation of receptors. The first mechanism is by enzyme s in the synaptic cleft that qu ickly convert neurotransmitters to their inactive metabolites (for example, acetylcholinesterase hydrolyzes acetylcholine to choline ). The second mechanism is by reuptake of neuro tr ansmitters by presynapti c cells, the third is by internalization of the receptors and finally the fourth by desensitization ( i.e., induction of a closed state of the receptor that is unresponsive to agonists ) The concept of a receptor can be traced to the work of the German physician Paul E h rlich and especially to the English physiologist John Newport Langley during the period of 1905 1907 5,6 In 1926, drawing from studies of Henry Hallett Dale Otto Loewi identified acetylcholine (ACh) as the first ne urotransmitter by experiments on the heart muscle 7 Today t here are a large number of neurotransmitters known for example: biogenic amines (dopamine, norepinephrine, epinephrine, serotonin and histamine), amino acids aminobutyric acid GABA, a spartate and glutamate), purines (ATP, adenosine, and guanosine), and neuropeptides that can be subdivided into opioids (endorphins, enkephalins and dynorphins) and non opioids (substance P and neuropeptide Y) 1,8,9 Although they are not stored in vesicles and do not bind to the receptors on postsynaptic membranes but rather diffuse right through the postsynaptic membrane, s ome gaseous molecules (nitric oxide and carbon monoxide) are also classified as neurotransmitters because they are released by neuron s and influence the electrochemical state of adjacent cells 10 12 (Figure 1 2).

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23 Figure 1 2. Structures of some neurotransmitters. A molecule that interacts with receptors is called a ligand. There are two class es of rece ptors respond ing to acetylcholine that are named for the exogenous ligands that activate them. The first class called muscarinic acetylcholine receptors (mAChRs), is a ctivated by a mushroom alkaloid muscarine and inhibited by a plant tropane alkaloid atro pine (Figure 1 3) These receptors belong to the superfamily of G protein coupled receptors (GPCRs) 1,2 and they mediate the slow metabolic responses to ACh via coupling to second messenger cascades (such as cyclic adenosine monophosphate: c AMP, diacylgl ycerol: DAG, and inositol triphosphate: IP 3 ), and are not a subject of t his

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24 dissertation. The second class is activated by tobacco alkaloid nicotine (and is thus called nicotinic acetylc holine receptors nAChRs) and belongs to a superfamily of ligand gated ion channels (LGIC) 2,4,14 Figure 1 3. Structures of acetylcholine, muscarine, atropine, and nicotine. LGIC are membrane receptors that contain both the binding site for the natural ligand and the ion conducting pore, w hich can be opened or closed upon ligand binding, allowing fast ion flux (10 7 ions per second) across the cell membrane down electrochemical gradient s 13 There are three subfamilies of LGIC in mam mals 4,14 One family is P 2X receptors (activated by ATP), a nother family comprises glutamate receptors (N methyl D aspartate: NMDA, alpha amino 3 hydroxy 5 methylisoxazole propionate: AMPA, and kainate receptors), and finally the largest family called Cys loop family 15 17 includes nicotinic, glycine, serotonin ( 5HT 3 ), zinc (ZAC), and aminobutyric acid ( GABA A and GABA C ) receptors. Nicotinic acetylcholine receptors are pentameric transmembrane glycoproteins that are activated by ACh and nicotinic agonists 18 They are allosteric i.e. have multiple conformation al states such as open, closed, and desensitized and equilibria between those states are regulated by ligand binding 19 (see 1.6 Allost ery and desensitization). B inding of the agonists in the extracel lular domain at the interface between two subunits may pr omote fast opening (within s) of an ion channel permeable to Na + K + and in some cases Ca 2+ ions, 50 away from the ACh binding

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25 site (Figure 1 4). The open state is intrinsically unstable and o n a longer (ms to min) time scale the receptor is desensitized (i.e., non conductive an d non activatible by an agonist), in case of heteromeric receptors toward a higher affinity closed state. 20 Figure 1 4 Schematic representation of a nicotine acetylcholine receptor (nAChR) from muscle (Reproduced from Nature Reviews Neuroscience, 3/2 Karlin A., Emerging structure of the nicotinic acetylcholine receptors, 102 114, Copyright (2002 ), with permission from Macmillan Publishers Ltd ). 61 The first nAChR characterized in the 1970s, was a degenerate form of a skeletal muscle type receptor isol ated from the electric organ of fish ( Torpedo ) by Changeux, Kasai and Lee 21,22 Muscle type nAChR remains the best characteriz ed LGIC and it serves as a prototype for other ligand gated ion channels 23 Many other types of nAChRs have been discovered (see 1.2 Subty pes of nAChRs) that are not expressed in muscles, but in neurons and non neuronal cells, for example in lymphoid tissue ( e. g., B a nd T lymphocytes) macrophages ( e. g., microglia) skin keratinocytes, lung cells, vascular tissue ( e. g., blood v essels ), astrocytes (type of glial cells in CNS that provide support and protection for neurons) and certain carcinoma s (reviewed in 24 28 ) The n AChRs have well established ionotropic function in synaptic transmission of electric signals in the peripheral nervous system 1 However, the function of nAChRs in the brain is more commonly associated with modulatory events than mediation of

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26 synaptic transmission 20,29 S ince nAChRs can regulate influx of calcium (both directly or indirectly by controlling membra ne potential) they may modulate the release of other neurotransmitters (such as dopamine, norepinephrine, serotonin, glutamate, and GABA) 19,30,31 and also initiate calcium signaling that is involved in many intracellular enzymatic processes linked to cell motility, adhesion, migration, proliferation, differentiation gene expression and survival 29,32 The nAChRs also play a role during development and in neuronal/synaptic plasticity. 29 30 Moreover, there is growing apprecia tion that 7 nAChRs may also have metabotropic function without ion channel opening and this type of signaling is further described in Section 1. 7 ( The 7 nAChR and inflammatory response ) 1.2 Subtypes of nAChRs There are 17 different nAChR subunits in vertebrates iden tified so far ( 1 10, 1 4, , and ) that can fo rm many different subtypes of the pentameric nAChR associated not only with subunit composition, but also subunit stoichiometry and arrangement (reviewed in 19,33 38 Figure 1 5 ). A ll of these subun its are present in humans except 8 that has only been found in chicken. Even though many potential combinations of nAChRs are possible, a single or a few receptor combinations seem to be preferred 19,35 The nAChRs subunits come from a common ancestor an d have been highly conserved during evolution ( the same subunit has more than 80 % of amino acid identity across vertebrate species 39 however, it shares less sequence similarity with other subunits) The n AChR subtypes are named according to their known su bunit composition (sometimes using an asterisk indicate possible additional subunits). 36

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27 Figure 1 5. Sch ematic representation of a few nAChR subtypes with acetylcholine binding sites shown. The n AChR subtypes have similar basic structure but diff erent pharmacological properties (different affinities for ligands, different response to ACh and other ligands, different permeabilities for ions, different kinetics of activation and desensitization and recovery from desensitization ). T hey regulate diffe rent physiological processes and are implicated selectively in some diseases 34,40 Therefore, it is possible to some extent to design s ubtype selective nAChRs ligands and avoid cardiovascular and gastrointestinal side effects of nicotine and other non se lective nAChRs agonists and potential addiction liability 19,41 43 First four subunits of nAChR from the electric organ of Torpedo were assigned the Greek letters , on the basis of their increasing apparent molecular weights when resolved on polya crylamide gels 44 Homologous subunits at the neuromuscular junction were renamed later as 1, 1, and (adult form) or (embryonic form) 45

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28 The nAChR subunits are classified as if they contain vicinal cysteine residues in the C loop at positions analogous to Cys192 and Cys193 in Torpedo subunit 36 Subunits and other non alpha subunits lack those vicinal cysteine residues. Based on initial affinity labeling experiments with bungarotoxin it was assumed that subunits are agonist binding sub units, whereas non subunits are structural subunits. Later studies showed that agonists bind at subunit interfaces, both and some non subunits contribute to the agonist binding site, and thus the part of an sub unit that has two vicinal cysteines in the C loop and forms the main part of the bin ding site is called primary (or principal ) face, and the remaining part of the ligand binding domain formed by either an or non subunit is called complementary site face) 37 The 5 and 10 s ubunits appear to not be able to function as primary faces even though they possess vicinal cysteines in the C loop 37 There are two ligand binding sites in muscular nicotinic receptors, one at the 1 interface, and the other at the 1 (or ) interfac e. The 1 subunit is structural (accessory) and is not directly involved in a primary ligand binding. The nAChRs are traditionally classified as m uscle ( 1, 1, or ) and neuronal ( 2 10, 2 4) subunits, however und in muscles and in other non neuronal tissues The n AChR subunits are often classified into those that can form homomeric and those that form heteromeric pentamers. Homomeric receptors ( 7, 8, 9) contain five identical subunits and five identical puta tive binding sites for agonists at the interface between two subunits 46 Even though five binding sites are present, 7 homomeric

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29 receptors can be activated under conditions of submaximal agonist occupancy (one and two ligands present), and higher occupan cies of the receptor result in desensitization rather than activation 47 Some data suggest that 7 subunits can form anomalous heteromeric receptors, (for example, the 7 2 subtype was reported in basal forebrain in rodents) 48 but these combinations are rare, and whenever 7 is mentioned later on in this dissertation, the ( 7) 5 subtype is implied. Most nAChRs are heteromeric and have very diverse combinations. S toichiometry of many heteromeric receptors is believed to be ( ) 2 ( ) 3 arranged clockwise as with two binding sites for agonists at the interfaces for example ( 4 ) 2 ( 2 ) 3 (Figure 1 5 ). However receptors with a lternative stoichiometries have been also characterized in vitro and implicated in vivo such as ( 4 ) 3 ( 2 ) 2 The ( 4 ) 2 ( 2 ) 3 recepto rs have high sensitivity to nicotine and low Ca 2+ permeability, whereas ( 4 ) 3 ( 2 ) 2 has low sensitivity to nicotine and high Ca 2+ permeability 49,50 There are also nAChR subtypes in which the and subunits are not identical (for example ( 3) 2 3( 4) 2 and 3 5 2 4 ) 37,38 Early indication that there are significant differences between receptor subt ypes was revealed by difference in sensitivity to the snake toxin bungarotoxin ( B tx) 18 The 7 10 subunits bind B tx tightly, while other subunits (except those at the neuromuscular junction) do not 38 In 1985, Clarke et al. performed an autoradiographic comparison of [ 3 H] acetylcholine, [ 3 H] nicotine, and [ 125 I] bungarotoxin labeled receptors in a rat brain that revealed that nAChRs with different affini ties for the studied ligands were located in different parts of the brain 51 Much of our current knowledge about nAChR subtypes comes from studies of heterologously expressed receptors 37

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30 knock out ( targeted deletion of specif ic subunits) and knock in ( mu tations in critical receptor domains) mice 19,40 studies with subunit specific antibodies 41,25 and subtype specific ligands, for example conotoxins 52 The 7 subtype was discovered and cloned in 1990 53 The 7 nAChR is a close existing homolog to an ancestral receptor that was present before the development of nervous system. 54 Special features of 7 receptor include: high permeability to calcium ions (permeability ratio of Ca 2+ to Na + is ~ 10 ) 55 low probability of channel opening 56 quick and reversible desensitization 53 at least two characterized distinct desensitized states: D s and D i 57 activation by choline 58,59 and inhibition by bungarotoxin and methyllycaconitine (MLA). Many of these characteristics will be discussed further. 1.3 Structure of the R eceptor 1.3.1 Overview As mentioned earlier, nAChRs are membrane proteins composed of five pseudo symmetrically arranged subunits that surround a central ion conducting pore. Each nAChR subunit has a large N terminal extracellular domain ( ECD, ~200 amino acids ), a transmembrane domain ( TMD) that is composed of three helices labeled M1 M3 connected to a fourth helix (M4) by a larg e intracellular domain (ICD, ~100 270 amino acids depending on a subunit), and a short extracellular C terminus (4 30 amino acids) (Figure 1 6 ) The structure of the nAChRs has been review ed in 19,60 63

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31 Figure 1 6 Schematic representation of one subunit. As mentioned before, t he n ACh Rs belong to a Cys loop family, together with serotonin (5 HT 3 ), GABA A, GABA C zinc (ZAC), and glycine receptors The nam e of this superfamily comes from a signature sequence of 13 residues flanked by two disulfide linked cysteines, called Cys loop a closed loop situated between the extracellular ligand binding domai n and the transmembrane domain 15 17,60,64 Although Cys loop receptors are activated by different ligands and they can be permeable to either cations or anions, they share considerable sequence homology and have similar basic functionality. This similarity of Cys loop receptors is illustrated by the fact that t he ECD and TMD from different Cys loop family receptors can be coupled to form functional receptors, for example, a chimera made of the ECD of the 7 nAChR and the TMD of the 5HT 3 receptor has binding site properties of the 7 and channel domain properties of the 5HT 3 65 Our k nowledge of the nAChR structure comes from radiolabeling, photolabeling, sequence analysis s ite directed mutagenesis substit uted cysteine accessibility method (SCAM) use of snake venoms, immunological and electrophysiologic al experiments, computational methods, and three dimensional structures obtained by X ray or electron

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32 microscopy 19,60 63 The available structures of nAChR s or their homologu es are: 1) X ray structures of molluscan acetylcholine binding proteins (AChBP), which are soluble protein homologues of the extracellular domains of nAChR ; 66 71 2) the electron microscopy structure of a fish muscle type nAChR analog a t 4 resolution ; 72 3) the X ray structure of a mouse ECD portion of the 1 subunit bound to bungarotoxin ; 73 4) the X ray structures of two prokaryotic LGICs, one from the bacterium Gleobacter violaceus (GLIC protein) 74,75 and the other from the bacte rium Erwinia chrysanthemi (ELIC protein) ; 76,77 5) the X ray structure of the ECD h 7 nAChR /AChBP chimera ; 78,79 and 6) the X ray structure of a pentameric glutamate channel from C. elegans GluCl 80 As can be seen from the above list, there is no single high resolution structure of any whole nAChR protein. The available 3 D structures do provide valuable information about nAChRs, and have been used to make verifiable experimental hypotheses. H owever the use of these structures for design of subtype selec tive ligands and elucidation of pharmacological properties for distinct subtypes of the receptor is quite limite d and involves a lot of speculation because of the r eliance on the homology models ( i.e., models created using amino acid sequence of nAChRs and 3 D structures of AChBP that has less than 25 % sequence identity to nAChRs) 1.3.2 Extracellular D omain (Ligand Binding D omain) The extracellular domain (ECD) of nAChRs contains the ligand binding sites for acetylcholine and is thus also called the ligand bind ing domain (LBD) 81 Each subunit of the ECD also contains the main immunogenic region (MIR, major target for the nAChR antibodies) at least one glycosylation si te, and the Cys loop (Figure 1 6 ). The

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33 extracellular domain starts at the N terminus with a th ree turn helix, followed by a bundle of ten stands ( 1 10) and several connecting loops. The acetylcholine binding site (also called orthosteric site) is formed by loops A, B, and C on the primary face of an e compl ementary face (D and E lo ops are actually parts of strands) (Figure 1 7 A). The ligands bind under the C loop ( containing two adjacent cysteines residues linked by a disulfide bond ), in the cage created by five aromatic residues that interact by cation interactions with a positive charge on the nitrogen of the agonist 82,83 (Figure 1 7 B). A. B. Figure 1 7 Ligand binding domain. A) Two subunits of the AChBP showing the loops creating the binding site at the interface between the subunits (Repro duced from Quaterly Reviews of Biophysics, 43/4, AJ Thompson, HA Lester, SCR Lummis, The structural basis of function in Cys loop receptors, 449 499, Copyright (2010), with permission from Cambridge University Press). 60 B) (S) Nicotine binding to the ACh BP showing the residues participating in the binding of the ligand (Reprinted with permission from Journal of Medicinal Chemistry, 48, JA Jensen, B Frolund, T Liljefors, P. Krogsgaard Larsen, Neuronal nicotinic acetylcholine receptors: structural revelatio ns, target identifications, and therapeutic inspirations, 4705 4745, Copyright (2005), American Chemical Society). 19

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34 It is believed that the binding of an agonist to the nAChR causes the C loop to close, similar to what is seen in the acetylcho line bindin g protein (Figure 1 8 ). Figure 1 8 Conformational changes in the loop C in AChBPs on ligand binding. Comparison of the orientation of loop C between two extreme positions with Ctx ImI ( nAChR antagonist) or epibatidine ( nAChR agonist). T op view, sho Ctx ImI (left, red loop C) or epibatidine (right, blue loop C) (Reprinted from EMBO Journal, 24, Hansen SB, Sulzenbacher G, Huxford T, Marchot P, Taylor P, Bourne Y., Structures of a plysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations, 3635 3646, Copyright (2005), with permission fr om Macmillan Publishers Ltd ). 68 Note: terms agonist and antagonist have no meaning for t he AChBP. Displacement of the C loop may be a reflection of a larger size of the antagonist. 1.3.3 Transmembrane D omain The transmembrane domain (TMD) of each nAChR subunit is made of f our helices (M1 M4) The channel pore is lined with the 40 long M2 segments from each of the five subunits the M1 and M3 helices form a circle behind them and the M4 s are positioned at the periphery sequestering the ion channel from the membrane lipids 6 0 62 (Figure 1 9 ) The interface between the TMD helices and the ECD sheets is located ~10 on the extracellular side of the cell membrane 19 The M2 helices from all subunits have sets of homologous residues at each level, forming rings of chemically distinct environments facing the lumen of the pore 62 The narrowest part

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35 of the ion channel is called a gate, which acts as a molecular barrier (both steric and energetic), prohibiting the ions from passing through the channel The gate is created by inte in resting and desensitized states 19,61 In the resting state the channel ope ning has been estimated to be 6 in diameter, thus permeation of hydrated monovalent and divalent cations is impossible (ions cannot readily lose their hydration shells in the absence of polar su rfaces that would replace water molecules ) 72 T he diameter of an open channel has been estimated to be 9 84 Figure 1 9 Cross section of a nAChR pore in the middle of the transmembrane domain The transmembrane domain also contains two negatively charged rings at the ends of the M2 helices (mainly made of gl utamate residues), that contribute to ion conductivity through the ion pore by providing an electrostatic potential of opposite sign to that of the permeant ion : one ring on the extracellular domain side, and one ring on the intracellular domain side. The charge selectivity filter is also located in the transmembrane domain 19

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36 1.3.4 Intracellular D omain The intracellular domain (ICD) of nAChRs consists mainly of a large loop between the M3 and M4 helices that displays high variability in amino acid sequence and l ength in different subunits (110 270 residues) 62 It is the least characterized portion of the receptor because there is essentially no structural information about it, except that in case of a Torpedo receptor, part of the intracellular domain forms an helix, and most of it is intrinsically disordered 72 85 However, it is increasingly appreciated that structural disorder is important for the function of some proteins 86 The nAChR subunit intracellular domain s contain varying numbers of putative phosph orylation sites which have been suggested to play an important role in the nAChRs expression, trafficking, assembly of the subunits, and interactions with the cytoskeleton 62 87 89 It also appears that the ICD interacts with many proteins inside the cells 90 and it is believed that they play many other important functional roles (such as the regulation of transcription and translation and cellular signal transduction), most of which remain to be clarified. 1.3.5 Three D imensional S tructures of P entameric LGICs 1.3.5.1 X ray structures of a cetylcholine binding protein (AChBP) Our knowledge about nAChR structure comes in a large part from X ray structures of the water soluble pentameric acetylcholine binding protein (AChBP) f rom snail aplysia californica (ac) and lymnaea stagnalis (ls) 91 The AChBP in the mollusks is released by glial cells into a synaptic cleft wher e it binds acetylcholine to modulate neurotransmission 92 The first structure was solved in 2001 by Brejc et al. at 2.7 with five HEPES (4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid) molecules from the crystallization buffer, 10 Ca 2+ ions, and 15 water molecules bound (Figure 1 10 ) 66 The

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37 AChBP shares 24 % sequence identity with the extracellular domain of the h 7 nAChR has 80 in diameter and a height of 62 , contains 210 amino acids in each monomer and has a glycosylation site at position Asn 66 91 Each monomer of the protein has a modified immunoglobulin fold 93 in which two sheets are organized in a cu rled sandwich: an inner sheet is made of 1, 2, 3, 5, 6, and 8, and is linked by the Cys loop disulfide bond to an outer sheet that is formed by 4, 7, 9, and 10. There are also two short 3 10 helices (3 amino acids per turn, 2 helix translati on per residue) 66 Each monomer of the protein contains a pair of adjacent cysteines linked by a disulfide bond in loop C. A. B. Figure 1 10 The pentameric s tructure of AChBP. A) top view. B) view perpendicular to the five fold axis with ligand binding site shown in ball and sticks (Reprinted from Nature, 411/6835, Brejc K, van Dijk WJ, Klaassen RV, Schuurmans M, van der Oost J, Smit AB, Sixma TK. Crystal structure of an ACh binding protein reveals the ligand binding domain of nicotinic recepto rs 269 276 Copyright (2001), with permiss ion from Macmillan Publishers Ltd ). 66 There are currently more than fifty structures of AChBP crystallized with a variety of ligands (agonists, antagonists and allosteric modulators), bound at the interface betwe en two monomers, and resolved up to 2.05 67 71 The AChBP b inds [ 125 I] bungarotoxin and many cholinergic ligands with similar affinities to those of the 7 nAChR and is extensively used as a homology model to predict ligand receptor interactions 19 However, the use of the AChBP template is limited because it is not an

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38 ion channel, it l ack s an equivalent of the nAChR TMD and ICD and does no t provide infor mation about receptor function. 1.3.5.2 Cryo electron microscopic structure of the nAChR from the Torpedo electric organ at 4 First images of a muscle type nAChR in its native li pid surroundings were obtained by Unwin and co workers by electron microscopy in the late 1980s Using tubular crystals of postsynaptic membranes from Torpedo electric organ, the researchers revealed the structure of the receptor at 18 resolution. Since then, Unwin has published several new structures at increasing resolution ( up to 4 ), using the AChBP template for refining of the ECD 72 94 (Figure 1 11 ). A. B. C. Figure 1 11 Ribbon diagrams of the nAChR from Torpedo electric organ A) View fr om the synaptic cleft B) View parallel with the membrane plane C) Diagram of a single subunit, view parallel with the membrane plane, the central axis of the pentamer (vertical line) is at the back (the Trp149 from LBD is shown in gold) (Reproduced from Journal of Molecular Biology, 346/4, Unwin N, Refined structure of the nicotinic acetylcholine receptor at 4 angstrom resolution, 967 989, Copyright (2005), with permission from Elsevier ) 72

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39 In most of t hese electron microscopy images, the ion channel is in the closed state, but there are two structures in which the ion channel was freeze trapped in the open state 95 97 The structure is often considered to be the only one of a whole nAChR protein, howeve r a large part of the intracellular domain (M3 M4) is in fact missing Although the structure is helpful in elucidation of the ion channel functioning 98 the side chain orientation is quite ambiguous in this low resolution structure. 1.3.5.3 X ray structure of the ECD of a mouse 1 monomer with bungarotoxin bound at 1.9 4 resolution In 2007, Dellisanti et al (Chen group) have published an X ray structure of the ECD of a mouse 1 monomer with bungarotoxin bound at 1.94 resolution 73 The protein contains t hree mutations that enabled its crystallization. In the structure, there is an o ligosaccharide bound from Asn141 to Ser 143 which was suggested to facilitate folding and trafficking of the receptor. The structure is the first atomic resolution view of a nA ChR subunit extracellular domain, but it is not a good template for ligand binding because it is a monomer and its structure is altered by binding bungarotoxin and mutations. Figure 1 12 Overall structure of the mouse nAChR 1 subunit (cyan) bound to Bgtx (orange). The carbohydrate chain is shown as a stick model and colored in magenta. A) Front view between the inner and outer sheets. B) Top view ( Reprinted from Nature Neuroscience, 10/8, Dellisanti CD, Yao Y, Stroud JC, Wang Z, Chen L Crystal structure of the extracellular domain of nAChR alpha 1 bound to alpha bungarotoxin at 1.94 A resolution 953 962 Copyright (2007), with permiss ion from Macmillan Publishers Ltd ). 73

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40 1.3.5.4 X ray structures of prokaryotic ion channels (G LIC and ELIC) Analysis of bacterial genome sequences has revealed that 3 % of all bacteria sequenced so far contain a putative pentameric LGIC gene 63 The available X ray structures of prokaryotic pentameric ion channels homologous to nAChR are : GLIC ( Glo eobacter violaceus ) 74, 99 solved at 2.9 in an apparently open conformation, and ELIC ( Erwinia chrysanthemi ) 76,77 solved at 3.3 in a closed conformation. The prokaryotic pentameric LGIC s lack the N terminal helix, they contain a sequence homologous to the Cys loop but the disulfide linked cysteines are actually missing, and the intracellular domain is not present 99 (Figure 1 13 ) Figure 1 13 Schematic representation of one monomer of pentameric LGIC in prokaryotes and eukaryotes The GLIC protein is permeable to protons and shares 20 % sequence identity with the human 7 nAChR The ELIC protein is activated by many primary amines such as amino butanol, cysteamine, putrescine, and by high (mM) concentrations of GABA 100 and has 16 % amino acid identity with the nAChR 1 subunit. The structure s of bacterial proteins have a s imilar fold ing pattern to nAChRs ( sheets in the ECD and four transm embrane helices) and are used to study the transition from a clos ed channel to open (F igure 1 14 ) 63 The structure of a GLIC protein with anaestetics bound is used to elucidate allost eric potentiation in the transmembrane domain 75,101

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41 A. B. GLIC ELIC Figure 1 14 GLIC and ELIC proteins. A) Ribbon representation of GLIC viewed from the plane of the membrane. B) Top view of GLIC (a) and ELIC (b ) M2 helices (Reprinted from Nature, 457/7225, Bocquet N, Nury H, Baaden M, Le Poupon C, Changeux J, Delarue M, Corringer P X ray structure of a pentameric ligand gated ion channel in an apparently open conformation 111 114, Copyright (2009), with permission from Macmillan Publishers Ltd ). 74 1.3.5.5 X ray structure of a the ECD of h 7 nAChR and AChBP chimera s Several X ray structures of the ECD of h 7 nAChR and AChBP chimera s have been solved in 2011 78,79 For example, the Chen lab obtained a structu re of a protein that has 64 % identity to the human 7 nAChR with and without an agonist at 2.8 and 3.1 resolution (Figure 1 1 5 ) While the structures have more similarity to the extracellular domain of the 7 nAChR than the AChBP, they still do not cont ain transmembrane domain and can only be use d for predictions of ligand binding.

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42 Figure 1 15 Structures of 7 AChBP chimera A) Top view of the 7 AChBP chimera pentamer along the five fold axis of symmetry; each subunit is shown in a different color. B) Structure superposition between the 7 AChBP chimera (blue) and AChBP (orange) pentamers viewed from the side that is normal to the five fold axis. C) Structure superposition of subunits from the 7 AChBP chimera (blue) 1 extracellular domain (mage nta) and AChBP (orange); loops showing substantial differences are labeled (Reprinted from Nat Neurosci 14/10, Li S, Huang S, Bren N, Noridomi K, Dellisanti CD, Sine SM, Chen L Ligand binding domain of an alpha(7) nicotinic receptor chimera and its comp lex with agonist 1253 1260, Copyright (2011), with permission from Macmillan Publishers Ltd). 78 1.3.5.6 Structure of a pentameric glutamate LGIC from C. elegans The last structure that provides some information for stu dying the nAChR is the eukaryotic pentameri c glutamate ligand gated ion channel from C. elegans (GluCl) in a complex with an antibody at 3.3 resolution (human glutamate receptors are tetrameric) 80 GluCl is an anionic Cys loop glutamate receptor. In 2012, Changeux and co workers started to classi fy nAChR as pentameric LGIC rather than Cys loop receptors to include the recently discovered ELIC, GLIC, and GluCl into the same family 63

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43 A. B. C. Figure 1 16 Structure of the GluCl Fab complex. A) view from the top. B) view parallel to lip id membrane (only two Fab m olecules are shown for clarity). C) A single GluCl subunit (Reprinted from Nature, 474, Hibbs RE and Gouaux E Principles of activation and permeation in an anion selective cys loop receptor 54 80, Copyright (2011), with permi ssion from Macmillan Publishers Ltd). 80 1.4 Studying the nAChR Ionotropic F unction The f unction of ion channels can be monitored electrophysiologically by measuring ion flow through the cell membrane resulting from activation of the channels. In this research the synthesized compounds were tested in human nAChRs expressed heterologously in Xenopus oocytes, and their function was monitored by two electrode voltage clamp. All electrophysiological experiments described in this dissertation were performed in Dr. Papke Lab oratory (University of Florida, Department of Pharmacology and Therapeutics, College of Medicine), using OpusExpress6000A (Molecular Devices, Sunnyvale, CA) an automated multichannel high throughput system for oocyte recording 102,103 The Xeno pus laevis (Afric an frog) oocytes (egg cells) are widely used as an expression system for ion channels 104 Their large uniform size (approximately 1 mm in diameter), allows direct injection with genetic material (cDNA, cRNA

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44 transcribed in vitro from clon ed or mutated channel cDNA, or mRNA isolated from tissue of interest). Oocytes express faithfully channel proteins in their cell membrane, and have relatively few endogenous ion channels (expressed at low levels). Voltage clamping is considered as a gold s tandard technique for measuring the ion currents across the membrane. There is a direct linear relationship, derived from between the response measured and the fraction of the total population of ion channels that opened to create the current measured 102 In two electrode voltage clamp, glass microelectrodes (current electrode and voltage electrode) and an amplifier ar e used to inject current in such a way that the cell membrane circuit can be thus written as: I = (E m E rev ) N P open Where I is the current, E m represents the membrane potential (hol ding potential), E rev stands for reversal potential (zero current potential for the ion channel of interest ), N is the total number of channels, P open represents probability of a single channel being open, and is the conductance of a single open channel (a simplified scheme for two electrodes voltage clamp in Xenopus oocytes is shown in Figure 1 1 7 A ). OpusExpress is a high throughput electrophysiology system in which voltage control, data acquisition, fluid delivery, and real time analysis are all autom ated ( Figure 1 1 8 B ) 103 and it allows for run ning an experiment on up to 8 oocytes in parallel ( Fig ure 1 1 8 C ).

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45 A response to drug application is illustrated by the current from current electrode that is needed to keep the membrane voltage constant. The he ight of the peak is called a peak response or peak current, and the area under the peak is called a net charge (Fig ure 1 1 8 D) In this research, each oocyte received two pre controls of acetylcholine, then experimental drug applications (a pplication of an experimental compound to test for agonism, co application of ace tylcholine and experimental compound to test for antagonism, or co application of P NU 120596 and experimental compound ). This was followed by one or two post con trols of acetylcholine (Figure 1 17 E) Responses of the nAChR s were calculate d relative to preceding ACh controls to normalize the data (responses to ACh were normalized to 1) compensating for varying 1.5 Ligands for the nAChRs Ligands for the nAChRs can be classified as agonists, antagonists, and modulators. Molecules that bind in the same binding site as acetylcholine are often that bind elsewhere Agonists are molecules, whose binding to the nACh Rs activates ion channel opening in a similar manner as acetylcholine and nicotine. Agonists are usually characterized by their efficacy and potency. Efficacy (I max ) is the maximum response that the ligand can ellicit relative to a reference compound, usually acetylcholine in the case of nAChRs.

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46 A. B. C. D. E. Figure 1 1 7 Studying the nAChR ionotropic function. A) Schematic experimental set up for two electrode voltage clamp in Xenopus oocytes B) OpusExpress6000A in the Papke Laboratory 103 C) OpusExpress electrode arrays in baths containing one oocyte each, dr ug delivery (Reproduced from Methods 51/1, Papke RL and Stokes C Working with OpusXpress: Methods for high volume oocyte experiments 121 133, Copyright (2010), with permission from Elsevier). 103 D) figure representing a response to drug application E ) mock figure illustrating a typical experimental protocol and raw data

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47 Based on their I max the agonists can be subdivided into partial (I max lower than for ACh), full (I max similar to that of ACh), and super agonists (I max higher than for ACh). Potency is often represented by an EC 50 i.e., a concentration of an agonist that produces a half of a maximum response. The I max and EC 50 values can be obtained from a concentration response curve (CRC) that is fitted into a Hill equation: where n is a Hill coe fficient. Hill coefficient might be a measure of cooperativity (for example in case of a muscle nAChR ), or have no directly interpretable functional meaning (for example in case of 7). Antagonists are molecules that inhibit an agonist evoked activation of the receptor. Antagonists are characterized by an IC 50 i.e., a concentration of a ligand that reduces the test response by 50 %. Antagonists can be divided into competitive and non competitive. A competitive antagonist binds in the same site as the agoni st, thus its action can be overcome by increasing the concentration of the agonist: a competitive 50 but does not lower I max Binding of a non competitive antagonist in an allosteric site cannot be overcome by high conc entration of 50 and I max are affected. Allosteric modulators can be classified as negative when their binding diminishes a response to agonists (non competitive antagonists could be called negative allosteric modulators ) 105 and positive allosteric modula tors when their binding enhances the response to agonists. Positive allosteric modulators (PAMs) for nAChRs appear promising for develop ment of new nAChR therapeutics. 106,107

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48 There is a great interest in developing nAChR ligands that are selective for one of the subtypes of the receptor. 108 111 The selectivity can arise from a big difference in p otency for different subtypes, efficacy or both. There are some nAChR ligands that are considered selective for one subtype of the receptor, though it is important to keep in mind that these li gands are selective only in regards to what they were teste d for, and many of the tested compounds were subjected to limited pharmacological characteriz ation (usually nAChR ligands have bee n tested for 2 3 4, and 7 ) In addition, a ligand that selectively activates one nAChR subtype may bind to other subtypes and act as an antagonist. 112 114 The nAChR ligands have been reviewed in 19,43,106,115 118 1.5.1 Nicotinic A gonists As mentioned ear lier, acetylcholine (ACh) is the endogenous agonist for all nicotinic AChR subtypes Acetylcholine is susceptible to hydrolysis, especially in biological preparations that contain acetylcholin esterase. Nicotine (Figure 1 18 ), an alkaloid from tobacco, was used historically to classify the receptors and distinguishes this subset of AChR from the muscarinic family Nicotine activates all nAChR subtypes, except 9 and 9 10 for which it acts as an antagonist. 113,114 ( ) Cytisine 119 is a toxic alkaloid that occurs naturally in seeds of several plants such as Laburnum and Cytisus ( ) Cytisine usually acts as a full agonist for heteromeric receptors containing 4 subunit and a partial agonist for subtypes containing 2 120 Anatoxin A is a potent nicotinic agonist first isolated from blue green algae cyanobacteria, Anabaena flos aqua 121 Epibatidine was isolated in 1992 from skin of an Ecuadorian frog, 122 and its b oth enantiomers are very potent nAChR agonists, especially on heteromeric receptors.

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49 Epibatidine was recognized for analge s ic activity. 123 Anabaseine isolated from a marine worm 124 and certain species of ants, 125 an d its reduced analog anabasine found in the tree t obacco ( Nicotiana glauca ) plant are nicotinic agonists that are more potent on muscle and 7 nAChR than neuronal heteromeric receptors. 126 The natural products described above have been extensively used as leads for development of new nAChR ligands. 19,115 117 127 Figure 1 18 Structures of some non selective nAChR agonists. A p harmacophore is defined as a minimal set of structural elements in appropria te arrangement that is required for a molecular recognition of a ligand by the receptor. Based on the known agonists for nAChR (acetylcholine, nicotine, cytisine anatoxin A, epibatidine, and anabaseine), it was believed for a long time that a positive charge and an H bond acceptor were necessary for the molecule to act as an agonist. 128 However, in the 1990s it was found that tetramethylammonium (TMA) was a full agonist for 7, 3 4, and 2 4 58 thus reducing the nAChR agonist pharmacophore

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50 to a quaternary ammonium ion Quinuclidine is also a non selective nAChR agonist that has been found to activate 7 and 3 4 subtypes. 111 The efforts to make ligands selectiv e for a particular subtype of nAChRs have been reviewed in 108 111 117 T hr ee structural motifs that can be associated with selectivity for 7 nAChR subtype have been defined as the choline, tropane, an d benzylidene motif s (Figure 1 19 ) 111 The c holine mo tif can be defined as an ammonium group separated by two carbons from a hydrogen bon d acceptor, such as in choline 58 3 quinuclidinone 111 and AR R17779 129 The selectivity of the tropane motif results from small hydrophobi c groups affixed on the quaterna ry nitrogen, such as in tropise tron Addition of a methyl group to a non s elective agonist quinuclidines gives 7 selective N methylquinuclidine. 111 The b enzylidene motif could be described as an extended aromatic ring properly positioned to the ammonium positive charge, such as in 3 [2,4 dimethoxybenzylidene]anabaseine (GTS 21, DMXB) 130,131 (E) benzylidene quinu clidine 111 132,133 and SSR180711. 134 Figure 1 19 Structures of some 7 nAChR selective agonists.

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51 1.5.2 Nicotinic A ntagonists Competitive nAChR ant agonists, such as tubocurarine bungarotoxin, conotoxins, dihydro eryth roidine and methyllycaconitine (MLA) (Figure 1 20 ) bind at the agonist binding site, stabilizing the receptor in a closed channel conformation and preventing acc ess for agonists. Tubocurarine is the active agent of a South American arrow poison known as cu rare, it is a non selective nAChR antagonist that has been used as a skeletal muscle relaxant and it is believed that it might also have non competitive interactions with nAChRs 135 Alpha bungarotoxin ( Bgt), a 57 amino acid peptide isolated from the venom of the snake Bungarus multicinctus is an antagonist that binds selectively to 7, muscle and Torpedo nAChR it does not appear to bind to heteromeric neuronal receptors with very high affinity Alph a bungarotoxin has been used for purification of the Torpedo nACh receptor 21 and [ 125 I] Bgt can be used for selective labeling of 7 receptors in the brain 51 The association and dissociation kinetics of bungarotoxin are very slow. A lpha conotoxins, 12 18 amino acid peptides, have been isolated from venoms of various Conus snails. They often selectively antagonize one subtype of nAChRs 136 (for example, conotox in ImI is considered selective for 7 and 9 nAChR). 137 Dihydro erythroidine (DH E), an alkaloid from Erythrina seeds, is generally non selective for different nAChR subtypes. 138 Methyllycaconitine (MLA) an alkaloid produced by Delphinium species plants, 139 is a potent competitive antagonist, selective for 7 nAChR Inhibition of 7 nAChR by MLA is rapid and reversible. 140 Non competive ant agonists, such as mecamylamine, hexamethonium, TMPH, and tK3BzPB interact with different sites than the orthosteric ligand binding site, often

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52 the lumen of t he nAChR channel. Mecamylamine is a nonselecti ve, non competitive antagonist, able to cross freely the blood brain barrier. It has been introduced in 1950s as a drug for the treatment of hypertension. 141 Hexamethonium is also a nonselective, non competive antagonist, but unable to cross the blood bra in barrier. 142 2,2,2,2 Tetramethylpi peridin 4 yl heptanoate (TMPH) is a potent antagonist for heteromeric neuronal nAChRs. 143 A tetrakis azaaromatic quaternary ammonium antagonis t tkP3BzBP is selective for 7 nAChR 144 Figure 1 20 Structures of some nAChR antagonists.

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53 1.5.3 Posit i ve Allosteric M odulators A p ositive allosteric modulator (PAM) binds at the site other than the agonist binding site and It typically does not activate th e receptor by itself. Positive allosteric modulators of membrane receptors are important tools to control the receptor function, 106 and have been used as therapeutics (eg., valium, a benzodiazepine that acts as a PAM for GABA A receptor) 145 Several endoge nous PAMs of nAChRs have been identified (such as steroids, calcium and zinc ions, lynx 1 146 (and 2) and SLURP 1 (and 2) proteins). There is great interest in developing synthetic PAMs selective for 7 106 ,107 and 4 2. 147,148 Gr nlien and co workers ha ve proposed that 7 PAMs can be divided into two classes: type I and type II (Figure 1 20 ). 149 Figure 1 20 Structures of some 7 nAChR PAMs. The type I PAMs, such as 5 hydroxyindole 150 act mainly on energy barriers bet ween different conformations of the receptor and predominantly affe ct the peak current (Figure 1 21 A) Typ e II PAMs, such as PNU 120596, 151 act also on energy levels and largely increase the net charge: they are able to sl ow down desensitization and even reverse some desensitized states (Figure 1 21 B) 57 PAMs may also act by enhancing binding of agonists and increasing the number of activa ti ble receptors.

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54 A. B. Figure 1 21 Representative traces showing the effects of PAMs on ACh evoked 7 nAChR responses. A) 5 hydroxyindole, type I PAM, was used B) PNU 120596, type II PAM was used (Reproduced from Molecular Pharmacology 72, Gronlien JH, Hakerud M, Ween H, Thorin Hagene K, Briggs CA, Gopalakrishnan M, Malysz J Distinct profiles of al pha 7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes 715 724, Copyright (2007), with permission from American Society for Pharmacology and Experimental Therapeutics) 149 Closely related structural analogs TQS 152 and 4 BP TQS are also classified as type II PAMs, though 4 BP TQS is unique because it has also been shown to act as an agonist on its own. 153,154 It is believed that the type II PAMs, PNU 120596, TQS and 4 BP TQS bind in the transmembrane domain. 154 156 1.5.4 7 nAChR S ilent A gonists A new class of 7 nAChR ligands introduced in this dissertation is silent agonists. Silent agonists have little or no ionotropic activity on their own, a ppear as competitive antagonists of more efficacious ionotropic agonists and cause conformational changes that can be detected with the refe rence type II PAM PNU 120596 157 In these cases, co application of the PAM with the silent agonist to the receptor results in a conductive state, thus reporting on what would otherwise be a no n conductive state.

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55 1.5.5 Therapeutic O pportunities of the nAChR L igands The nAChRs are implicated in a range of physiological functions related to muscle contraction, cognitive functions, learning and memory, reward, motor control, analgesia, and inflammation a nd therefore are important for drug research. 117 158 160 It is believed that nAChR ligands could be used in treatment of neurodegenerative diseases 161 162 depression, some epilepsie s, 163 attention deficit hyperactivity disorder (ADHD), and inflammatory diseases 164 (Section 1.7). One important application of nACh R ligands is in nicotine addiction and the nicotinic drug for smok ing cessation on the market is v aren icline 165 (Chantix US Champix EU ) a cytisine analog There are several nAChR ligands that are currentl y in clinical trials for depression, and pain 158 Silent agonist s may fou nd use in the treatment of inflammatory disorders (Section 1.7). 1.6 Allosterism and D esensitization that bacterial enzymes were inhibited by the end product of the bio synthetic pathways, even though the end product was not structurally similar to the active site substrate 166,167 The inhibition was non competitive with substrate leading to a hypothesis that the non competitive inhibitor produced conformational alterations in the protein 1 68 The most known model of protein allostery, the Monod, Wyman, Changeux (MWC) model 169,170 proposed that proteins are dynamic structures existing in multiple discrete fu nctional states all of which are accessible to the protein under resting conditions The b inding of

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56 a ligand was predicted to alter the resting equilibrium by reversibly stabilizing the protein in the conformation to which the ligand has greatest affinity. 171 Nicotinic acetylcholine receptors are allosteric proteins that can exist in di fferent conformations classified as closed, open, and desensitized. 172,173 Desensitization can be defined as a decrease or loss of biological response following prolonged or repetitive stimulation. 174,175,176 Dr. Papke Laboratory has identified that ther e are at least two allosteric modulators (PAM)s such as PNU 120596, and Di that is insensitive to type II PAMs. 57 1.7 The 7 nAChR and Inflammatory R esponse 1.7.1 Cholinergic A nti i n flammatory P athway Cholinergic anti inflammatory pathway is a neura l regulatory mechanism comprised of the vagus nerve and the 7 nAChR that attenuates release of pro inflammatory cytokines such as tumor necrosis factor (TNF), interleukin 1 and 6 (IL 1, IL 6) and high mobility group box 1 (HMGB1) ( Figure 1 22 reviewed in 177,178 ). Hence, t he 7 nAChR is a pharmacologic al target for diseases linked to abnormal in flammatory response, for example : arthritis, asthma sepsis, atherosclerosis ulcerative colitis, and psoriasis (reviewed in 178 181 ). The anti inflammatory effect of 7 ligands may be also type 2 diabetes 180 182

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57 Figure 1 22 Cholinergic anti inflammatory pathway bala nces cytokine production. Pathogens, ischemia, and injury activate cytokine production. Efferent signals from the vagus nerve inhibit production of pro inflammarory cytokines via 7 nAChR on macrophages and other cells that are the major source of pro inf lammatory cytokines The cholinergic anti inflammatory pathway was discovered by Kevin J. Tracey and co workers. In 2000, the group showed that direct stimulation of the vagus nerve during lethal endotox emia in ra ts inhibited synthesis of TNF in liver and prevented development of circulatory shock (bodily collapse caused by inadequate oxygen delivery to the cells). 183 In 2003, the group further showed that electrical stimulation of the vagus nerve in 7 nAChR deficient mice does not inhibit TNF synthesis, indicating that 7 nAChR is essential for inhibiting cytokine synthesis by the cholinergic anti inflammatory pathway. 184 The exact molecular mechanism by which the 7 nAChR inhibits pro inflammatory c ytokine production has not been fully established yet 177,178,180,185 I t has

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58 been shown that stimulation of 7 nAChR with agonists acts on the JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription) pathway 185 187 and the transcript ion factor NF B (nuclear factor kappa B) 188,189 that are kno wn regulators of cytokine production. 190 1.7.2 Findings I ndicating that the 7 nAChR M ay H ave S ignaling F uncti on in I nflammation W ithout I on C hannel A ctivity A nti inflammato ry effects of nicotine have been observed in epidemiological studies in tobacco smo kers and it is believed that 7 nAChR agonists could be used as anti inflammatory agents in vivo 179 181 However, it is known that the net effect of nAChR agonists could depend on many factors, such a s drug dose or concentration and the length o f the time of exposure, and it has been hypothesized that some effects of n AChR agonists may be due to desensitization of the receptor rather than ion channel activation. 191 It is thus reasonable to suggest tha t ligand binding can communi cate to the intracellular domain through the non conductive states, classified as des ensi tized, or through states that are unique to receptors in non neuronal cells, and thereby effect metabotropic signaling. There are several f indings indicating that the 7 nAChR may have signaling function in inflammation without ion channel activity Mainly: 1) The 7 nAChR s are exp ressed in blood macrophages, microglia, and immune cells 26,27,184,192,193 and stimulation of these cells with the 7 nAChR agonists does not produce detectable ion currents. 179 194,195 2) The 7 nAChR partial agonists (such as GTS 21) with relatively low efficacy have been shown to attenuate cytokine production in human whole blood ex vivo and this e ffect was more potent than for the full agonist nicotine. 186 193 196 3) The p artial agonist GTS 21 and NS6740 which has practically no efficacy (classified

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59 in this dissertation as a silent agonist) sup p res s a pro inflammatory response by microglia, while more efficacio us agonist s SSR180711 and A 582941 are ineffective. 197 4) Silent agonist ASM 024 (Asmacure) which is currently in clinical trials for asthma has been shown to attenuate pro inflammatory cytokine production 5) Proteomics based analysis of the 7 nAChR interactome has revealed that the receptor interacts with many intracellular signa l transducing proteins (eg. protein kinase A, protein kinase C, and several phosphatases). 90 These observations make compounds that can put the receptor selectively into a desensitized state with little or no ion channel activation (silent agonists) of potential interest from both mechanistic and therapeutic perspectives. 1.7.3 Other Possible Signaling Pathways I nvolving 7 nAChR It is believed that stimulation of 7 nAChR s in cancer cells with agonists results in acceleration of cancer progression and that it might be possible to design appropriate 7 ligands for anti cancer therapies. 28 198,199,200 The exact mechanism by which 7 nAChR mediates survival of cancer cells h as not been established, but some evidence indicates that it involves Ras/Raf1/MEK1/ERK (extracellular signal regulated kinase) and JAK 2/STAT 3 pathways. 199

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60 CHAPTER 2 KC COMPOUNDS AS NEW 7 SILENT AGONISTS 1 2.1 Background There is growing evidence, especially in no n mediated signal transduction under conditions when no ion chann el activation can be detected (S ection 1.7). T have been reported One is sensitive to type II PAMs such as PNU 120596, (terme d D s ), and another is induced by strong episodes of activation and high occupancy, and is insensitive to PNU 120596, (termed D i ). 57 The pharmacological relevance of desensitized states is likely to extend beyond their lack of ability to conduct an ion cur rent. These findings make compounds that can put the nAChR in to the D s state, with little or no apparent agonism, of considerable potential interest from both mechanistic and therapeutic perspectives. NS6740 is such agent which, although inactive in an 7 sensitive model for cognitive improvement has been shown to be effective at modulating the release of pro inflammatory cytokines 201 Several observations lead to a working model for the design of a silent agonist. Compound NS6740 (Figure 2 1 ) has been characterized by Abbott and Neurosearch as a very weak a gonist (<2 % of the response to ACh), whose agonist like properties were revealed by adding a positive allosteric modulator PNU 120596. We found in our laboratories that 3PAB 202 also behaves as a sil ent agonist, leading to the idea that silent agonists may be groupable into structurally distinct families. NS6740 features a basic diaza [3.2.2] bicyclononane group, a central 2,5 disubstituted heterocyclic ring and a phenyl substituent at the 5 position of the central 1 Reprinted from Bioorganic Medicinal Chemistry Letters, 23/14 Kinga Chojnacka, Roger L. Papke, Nicole A. Horenstein, Synthesis and evaluation of a conditionally silent 4149, Copyright (2013), with permission from Elsevier.

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61 ring. Further, the compound offer s a hydrogen bond acceptor adjac ent to the bicyclic ring system ( an amide carbonyl ) We therefore initiated a synthesis of a molecule whose structural features and biological activity might shed further lig ht on what constitutes a set of features that would confer silent agonism. We considered a minimal pharmacophore to feature a positively charged center, a central ring with hydrogen bonding capability, and a flanking aryl substituent with an angular relat ionship between these elements as embodied in the molecule KC 1 and the cartoon shown in Figure 2 1 This minimal pharmacophore model does not take into account possible importance of the trifluoromethyl group, amide group and bicyclic ring of NS6740 for conferring silent agonism character. One will note the core anabaseine portion of KC 1 is a non selective nAChR agonist, and as will be presented we show phenyl substitution on the pyridine ring dramatically changes this profile. Figure 2 1. Structures of NS6740, KC 1 and one pharmacophore for silent agonists. 2.2 Results and Discussion 2.2.1 S ynthesis of KC 1 KC phenylanabaseine) can be considered as an analog of anabaseine, and we decided to make KC 1 by adapting well kn own ana baseine synthesis protocols. It was first planned to prepare KC 1 bromoanabaseine by organometalli c coupling (Figure 2 2). Tha t approach would have allowed flexibility in preparation of a series of KC 1 analogs. We envisaged preparing sever bromoanabaseine as its

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62 dihydrochloride salt ( 1 ) from bromonicotinic ethyl ester ( 2 ), by applying the Zoltewicz anabaseine synthetic protocol 203 that we use in our lab. That approach is based on a mixed Claisen condensation of nicotinic ester and the amide enolate ion of N aminomethyl protected 2 piperidone 3 followed by hydrolysis of the resulting sodium salt of the 3 nicotinoyl 2 piperidone intermediate in hot concentrated hydrochloric acid with concomitant decarboxylation and cyclic imine formation. Anabaseine is isolated by crystallization as its stable dihydrochloride salt. Figure 2 2. KC 1 r etrosynthetic analysis The reaction of 3 bromonicotinic ethyl ester ( 2 ) with N protected 2 piperidone 3 202,203 and sodium hydride in toluene (Figure 2 3) yielded the sodium salt 4 as a complex mixture of Z and E enolates and keto forms as supported by 1 H NMR but it could not be isolated from the crude reaction mixture so the crude product was subjected to the ne xt step, hydrolysis with concentrated hydrochloric acid. The desired bromoanabaseine dihydrochloride ( 1 ) was formed in that reaction as shown by 1 H NMR, however extensive degradation was observed, many impurities were presen t, and the product could no t be obtained in a pure form. To circumvent the problems with degradation of brominated pyridine compounds in concentrated HCl, we decided to switch the order of the bond formation and synthesize KC 1 from 5 phenylnicotinic ethyl ester ( 8 ) again following the Zoltewicz protocol

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63 Figure 2 3. Synthesis of bromoanabaseine 1 5 Phenylnicotic ethyl ester ( 8 ) is not commer cially available and it was prepared from 3 bromonic otinic acid ( 5 ) by Suzuki coupling with phenylboroni c acid ( 6 ), using palladium acetate as catalyst, potassium phosphate as a base, in a 1:1 mixture of water and 2 propanol 204 The 5 phenylnicotinic acid was then reacted with thionyl chloride, followed by reaction with ethanol to give the product 8 in 63 % yield from 5 (Figure 2 4). Figure 2 4. Synthesis of 5 phenylnicotinic ethyl ester 8 The 5 phenylnicotinic ethyl ester ( 8 ) was subjected to the mixed Claisen condensation with N protected 2 piperidone 3 using the same meth odology as presented in Figure 2 3 Unfortunately, the intermediate sodium salt did not crystallize from the reaction mixture after removal of excess NaH by filtration, and again the crude reaction mixture was subjected to acidic hydrolysis. After the reaction, 5 p henylcarboxylic acid 7 resulting from hydrolysis of unreacted ethyl ester 8

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64 crystallized first, then the KC 1 salt, which was impure. The KC 1 salt was thus transformed with 1M sodium hydroxide into its imine free base form, purified by co lumn chromatography, and then converted back into its dihydrochloride salt to yield the pure c ompound only in 2 % yield over two steps from 5 phenylnicotine ethyl ester ( 8 ). The results of these two approaches suggest ed that the synthetic route to anabase ine is not highly tolerant of substitution though the poor yields might have resulted from the fact that the sodium salts of the Claisen condensation products were not puri fied. An alternate approach for obtaining synthetically useful yields of KC 1 was n eeded because the relatively small scale of the reactions and potential instability of the salts would make it difficult for their r ecrystallization In addition, the subsequent treatment with concentrated acid would be troublesome to optimize because the reaction is not easily amenabl e to monitoring by TLC. The most successful alternate route for synthesis of KC 1 employed addition of 5 pheny lpyridi nyl lithium generated with n BuLi from the bromide 10 to N Boc protected 2 piperidone 11 in diethyl ether, f ollowed by deprotection, ring cl osure, and dehydration (Figure 2 5 ). Organometallic ring opening reaction of N alkoxycarbonyl lactams has been used by Giovanni et al. 205 to make cyclic imines. 3 Bromo 5 phenylpyridine 10 was prepared from 3,5 dibromopyridi ne 9 by Suzuki coupling with phenylboronic acid 6 using 2 mol % (triphenylphosphine)palladium tetrakis and potassium carbonate in a mixture of dimethoxyethane and water in 68 % yield following a patent procedure 206 The N Boc 2 piperidone 11 was prepared from 2 piperidone and Boc anhydride following the reported procedure 205

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65 Figure 2 5. Synthesis of KC 1. The bromide 10a was added into a solution of n and then the lactam 11 was added to yield the desired N Boc aminoketone 12a in 61 % yield. We also generated the anion of 10a using 2 eq. of t BuLi in diethyl ether, and the desired product 12a was obtained in 31 % yield When nBuLi was used in whereas obtained in 12 % yield. When n BuLi was used in THF instead of diethyl ether at a range of temperatures no product was formed. The problems with lit hium halogen exchange of bromopyridines in THF have been known in the literature. 207 It has been reported that isopropyl magnesium chloride may be used for metal halogen exchange instead of alkyl lithiums to avoid known side reactions of alkyl lithium wit h pyridines such as deprotonation, addition of n BuLi to pyridine ring, elimination of lithium bromide (to give pyridynes), reaction of 3 lithiopyridine with n BuBr, and even ring opening. 208, 209 However in our case the use of isopropyl magnesium chloride r esulted in a significantly lower yield (4%) of compound 12a N Boc aminoketone 12 a was purified and treated with TFA followed by NaOH to give KC 1 in 80 % yield after silica chromatography. The imine free base was then

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66 quantitatively transformed into it s more stable dihydrochloride salt by rotary evaporation from ethanolic HCl 2.2.2 Activity Profile of KC 1 on the H uman 7 Receptor Silent A gonism KC 1 was tested on the human 7 nAChR as described in Section 1 .4 and A ppendix A The and 300 applicat ion s of KC 1 resulted in no ion channel opening (Figure 2 6 A) showing that KC ionotropic 1 was co res ponse to ACh was observed (Figure 2 6 B) (peak cu rrent: 0.17 0.08, net charge 0.29 0.06), suggesting that KC 1 may bind in the same binding site as ACh. Finally, 1 was co 120596 a very la rge response was observed (Figure 2 6 C) (peak current: 4.1 0.7, net charg e 18 2 ), revealing that KC 1 favors the D s state without ion channel opening, and thus acts as a silent agonist. Note that PNU 120596 has n o detectable activity on its own with the 7 nAChR 151 and that it is just a probe to reveal KC 1 activity. The dat a indicate that KC 1 binding results in stabilization of an alternate receptor state The co application of 100 M KC 1 with another type II posit ive allosteric modulator TQS (30 M) also resulted in large responses of the 7 nAChR (peak response 2.1 0 .2, net charge 4.3 0.3), suggesting that type II PAM in general may be used to detect silent agonism character. The IC 50 of KC 1 was determined to be 41 5

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67 Figure 2 6. M KC 1. Blue bars represent duration of ACh applications, green bars the KC 1 application, and the red bar represents PNU 120596 application. The first two traces represent ACh pre controls (300 M ACh in A, 60 M ACh in B and C). 2.2.3 Synthesis of KC 1 A nalogs Aft er characterizing KC 1 as a new silent agonist, we set out to prepare a series of KC 1 a nalogs (KC 2 to KC 9, Figure 2 7 ) to further tease out pharmacophore. All the molecules in this series have an aryl group connected to or fused to a pyr idine ring, an H bond acceptor (nitrogen in the pyridine ring) and at physiological pH, a positive charge on the nitrogen in the third ring. KC 2 features a secondary amine that results from the reduction of imine function in KC 1, it thus contains an anab asine core which similarly to anabaseine is an agonist of the nAChR.

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68 KC 3 is a tertiary amine synthesized by N methylation of KC 2. KC 4 and KC 7 contain the anabaseine core, similar to KC 1, but the pyridine ring is fused to a benzene ring in different o rientations. KC 5 and KC 8 are secondary amines analogous to K C 2. KC 6 and KC 9 are N methylated amines analogous to KC 3. Figure 2 7 Structures of KC compounds We predicted that the compounds would act as 7 nAChR silent agonists, similarly to KC 1 but with differences that would be attributable to their structural variations KC 4 210 and KC 7 were prepared from 4 bromoisoquinoline ( 10b ) and 3 bromoquinoline ( 10c ) respectively using the same protocol as for the synthesis of KC 1. Therefore, the opening of the lactam 11 by the anions generated from 10b and 10c with nBuLi in diethyl ether, gave N Boc aminoketones 12b in 43 % yield and 12c in 42 % yield. The subsequent treatment with trifluoroacetic acid and sodium hydroxide gave KC 4 in 57 % yield an d KC 7 in 81 % yield (Figure 2 8 ).

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69 10b 12 b : 43 % 1 3b : 57 % (KC 4) 10c 12c : 42 % 1 3c : 81 % (KC 7) Figure 2 8 Synthesis of KC 4 and KC 7. KC 2, KC 5 and KC 8 were prepared from the N Boc aminoketones 12a c by treatment with TFA, NaOH, and sodium borohydride in 60 85 % yield. KC 3, KC 6, 210 and KC 9 were prepared by Eschweiler Clarke methylation 211,212 of KC 2, KC 5, and KC 8, respectively (Figure 2 9 ) In this reductive alkylatio n, the Schiff base formed from an amine and formaldehyde is reduced by a hydride transfer from formic acid. 213 12a 1 4a (KC 2) : 60 % 1 5a (KC 3) : 65 % 12b 1 4b (KC 5) : 85 % 1 5b (KC 6) : 65 % 12c 1 4c (KC 8) : 79 % 1 5c (KC 9) : 71 % Figure 2 9 Synthesis of KC 2, KC 3, KC 5, KC 6, KC 8, KC 9.

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70 2.2.4 Activity Profile of KC 1 Analogs on the H uman 7 nAChR The KC 1 analogs were screened at 100 M and 300 M on the human 7 nAChR as described before ( Section 1.4 and Appendix A ) All KC 1 analogs had no ionotropic activity above the detection limit, as was observed for KC 1, with the except ion of KC 7 (Figure 2 10 ) which was fully characterized and shown to be a weak partial agonist of the 7 nAChR with an I max = 0.23 0.02 and EC 50 = 112 19 Therefore the positioning of the aryl group on the anabaseine core appears to affect the ability of the compound to activate the 7 ion channel as seen in the series of KC 1, KC 4 and KC 7 The relative positioning of the middle ring and positive charge also seem to affect the ability of KC 7 to cause ion channel opening: the positive charge in KC 7 lies pra ctically in the same plane as the quinoline ring, while in KC 8 the conformation in which the positive charge and quinoline ring are in the same plane is disfavored and KC 8 has no ionotropic activity. Figure 2 10 Ionotropic agonism alpha 7 KCs series.

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71 All KC 1 analogs diminished responses to acetylcholine with the IC 50 values in the range from 41 to While in the KC 1 to KC 3 and KC 4 to KC 6 series methylation of the nitrogen resulted in higher IC 50 values, the same trend was not seen in the KC 7 to KC 9 series, suggesting that methylation may not be a sole determinant in ability of the compou nd to inhibit ACh responses Table 2 1. IC 50 values for KC 1 analogs Compound IC 50 KC 1 41 5 KC 2 57 4 KC 3 77 5 KC 4 94 8 KC 5 80 2 KC 6 117 10 KC 7 46 9 KC 8 71 9 KC 9 60 3 To determine how KC compounds antag onize the 7nACh receptor, an ACh concentration response curve (CRC) was compared to the curve obtained by co applying ACh at increasing concentrations with either KC 1 or KC 5 at their IC 50 These experiments are consistent with the hypothesis that KC 1 displaces acetylcholine in a competitive way, shifting the ACh EC 50 from 30 3 M to 165 11 M, suggesting that KC 1 binds at the same si te as acetylcholine (Figure 2 11 ).

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72 Figure 2 11 A KC 1 /ACh competit ion curve. The competition curve s with KC 5 appear to have a significant competi ti ve component (the ACh EC 50 increased from 30 3 M to 74 3 M) and also a small non competitive component (I max dimi ni shed from 1.32 0.04 to 1.11 0.01) that might be d ue to a channel block (Figure 2 12 ).

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73 Figure 2 12 A KC 5 /ACh c ompetition curve. Finally the compounds were co applied at 100 M with type II positive allosteric modulator 10 M PNU 120596 revealing that KC 1, KC 5, and KC 7 were putting the 7 nAChR preferentially in to a desensitized state that could be made conductive by PNU 120596 (Figure 2 13 ). KC 4 and KC 8 gave little but significant ionotropic responses when co applied with PNU 120596. All methylated KC 1 analogs (KC 3, KC 6 and KC 9) that have similar IC 50 values to other KC compounds, gave no response at 100 M when co applied with type II PAM, indicating that nitrogen methylation in these type of compounds disfavors the receptor from going into a Ds conformation. It is intriguing that redu ction of the imine function in the silent agonist KC 1 resulted in a lo ss of silent agonism activity of KC 2, while reduction of the imine function in a weak silent

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74 agonist KC 4, resulted in an increase in silent agonism of KC 5. It would be interesting to prepare KC 2, KC 5 and KC 8 in their enantiopure forms to check whethe r there is a difference in activity between the two enantiomers. Figure 2 13 Desensitized state revealed by co application with type II PAM. Most active KC compounds were fu r ther characterized in the range of 1 M to 1 mM to determine their potency when co applied with PNU 120596 The determinatio n of EC 50 value s for PAM co application responses appear ed infeasible because the data did not fit into the Hill equation. It was observed that a sudden drop in activit y for KC 1 occurred between 300 M and 1000 M (Figure 2 1 4 ), indicating that at higher concentrations KC 1 was preferentially putting the 7 receptor in a desensitized state that could not be reversed by co application with PNU 120596. Similar behavior was observed for other KC compounds that had activity when co applied with PNU 120596 (Table 2 2). KC 2, KC 3, KC 6 and KC 9 were inactive at 100 M when co applied with PNU 120596, but had some detectable activity when tested at 300 M, revealing that the y have some weak silent agonism ac tivity or strong induction of D i

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75 Figure 2 14 A sharp change of preference of KC 1 for D i desensitized state between 300 M and 1 mM (Figure courtesy of Dr. Roger L. Papke). To help discern functional differences of th e KC 1 analogs, the ratio of the PNU 120596 co application response at 100 M to the agonism response at 100 M was calculated for each compound (Figure 2 15). This ratio provides a measure of a D s state o ver channel opening. The graph shows that silent agonists KC 1, KC 4, KC 5, KC 7, and KC 8 overwhelmingly favor D s desensitization over ion channel activation. KC 1 and KC 5 favor PAM sensitive desensitization over ion channel activation by 1800 and 2300 f old, respectively. KC 7 does so by 200 fold which is due to partial agonism character of this molecule. KC 4 and KC 8 that do not activate opening of the h 7 nAChR ion channel do so by 110 and 70 fold, respectively which results from their small responses when co applied with PNU 120596.

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76 Table 2 2. Net charge for KC co applications with PNU 120596. Drug Drug concentration Net charge when co applied with 120596 Threshold: Concentration at which 10 % of maximal response was observed KC 1 30 100 300 1 000 0.4 0.2 13 3 22 4 1.7 0.4 5 0 KC 2 100 300 0 1.3 0.1 KC 3 100 300 0 0.010 0.005 KC 4 100 300 1.3 0.7 4 2 KC 5 30 100 300 1000 0.6 0.3 12 5 12 3 2.0 0.4 35 KC 6 100 300 0 0.2 0.1 KC 7 30 100 300 1000 2 1 22 5 14 3 0.9 0.3 30 KC 8 100 300 0.3 0.1 6 3 KC 9 100 300 0 1.6 0.3

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77 Figure 2 15 Ratio of PNU 120596 (10 M) co application with KC compounds (100 M) responses to KC (100 M) ionotropic agonism response s To avoid division by 0, when no ionotropic agonism response was observed, the PNU 120596 co application response was divided by 0.005, which was assumed to be a d etection limit for these experiments. The error bars were not calculated because of the inaccuracy resulting from limited detection of small responses. 2.2.5 Activity Profile of KC 1 Analogs on the H uman 4 2 nAChR The nine KC compounds were screened at 100 M on the nAChR (Figure 2 16 ). KC 3, KC 6, KC 9 had no detectable ionotropic activity, KC 1, KC 2, KC 4, KC 5, and KC 8 activated 2 nAChR very weakly. KC 7 was the most active compound in the series, similarly to the profile seen on the 7 nAChR and it was further characterized and determined to be a weak partial agonist for 2 nAChR with an I max = 0.19 0.02 and EC 50 = 51 3 M for the ( ) 2 ( ) 3 receptor and I max = 0.05 0.01 and EC 50 = 65 5 M for the ( ) 3 ( ) 2 receptor.

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78 Figure 2 16 KC 1 to KC 9 agonism on 2 nAChR When the KC 1 to KC 9 compounds were co applied with acetylcholine, a diminished response to acetylc holine was observed (Figure 2 17 ). Figure 2 17 KC 1 to KC 9 co application antagonism on 2 nAChR This antagonism by KC compounds on h 2 nAChR was even more visible when the receptor was incubated with 100 M KC compounds for 5 min, and then the response to 100 M KC co applied with 30 M ACh was measured (Figur e 2 18 ).

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79 Figure 2 18 KC 1 to KC 9 antagonism with soaking on 2 nAChR Taken together, these data suggest that KC compounds are not selective for the 7 nAChR and may also act on 2 nAChR 2.3 Summary The term silent agonism was introduced in reference to the 7 nAChR subtype to describe compounds that do not activate ion channel opening on their own but favor induction of D s desensitization that might be involved in 7 nAChR non ionotropic signaling Silent agonists could potentially be used in the treatment of inflammatory disorders without side effec ts linked to ion channel activity. Nine structurally similar compounds KC 1 to KC 9, were synthesized to test for the importance of hybridization of the positively charged nitrogen, N methylation, and relative geometry between the aryl group, the hydrog en bond acceptor and the positive charge for pharmacological properties of the ligands The molecules were characterized electrophysiologically on the human 7 and 2 nAChR s expressed in Xenopus oocytes. KC 1 and KC 5 have fully met the

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80 criteria to be characterized as silent agonists for the 7 nAChR KC 7 was shown to be a partial agonist for the 7 nAChR while KC 2, KC 3, KC 4 KC 6, KC 8 and KC 9 appear ed to be weak 7 nAChR silent agonists (Figure 2 19 ). The systematic structural changes in KC compound series did not result in systematic changes in their activity, suggesting that there are no clear trends between structure and activity in this series. For example, reduction of the imine function in the silent agonist KC 1 gave the weak silent agonist KC 2, while reduction of the imine function in the weak silent agonist KC 4 gave the silent agonist KC 5. It would be interesting to further test the chir al KC compounds in their enantiopure forms to establish whether there is a difference in pharmacological properties between the two antipodes. Figure 2 19 KC 1 to KC 9 compounds summar y. Red: silent agonists; purple : w eak silent agonists ; orange: silent agonist with partial ionotropic agonist character Although no clear trends were seen between the structure and properties of the KC molecules, and the profile of KC compounds on the 2 nAChR suggested that it

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81 may be challenging to obtain compounds selective for the 7 nAChR in this series, new silent agonists with desired pharmacological properties may be found by preparing analogs of these compounds.

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82 CHAPTER 3 SILENT AGONIST BULKY QUATERNARY AMMONIUM 3.1 Background The Papke Laboratory has identified ASM 024 ( 16 ) (from Asmacure) (Figure 3 1) as a n 7 nAChR silent agonist That discovery le d to a hypothesis that one of the silent agonist pharmacophores may consist of a bulky quaternary ammonium group. Thus, tetraethylammonium ( TEA) was tested and it was also determined to act as a silent agonist. Tetramethylammonium (TMA) is a known non selective agonist of the 7 nAChR 111 leading to a hypothesis that the size of the quaternary ammonium group may determine whether the molecule acts as an agonist or silent agonist. Figure 3 1. Structures of ASM 024, TEA and TMA. W e set out to test six series of quaternary ammonium compounds, to determine how their size influences their pharmacological properti es (Figure 3 2). To vary the molecular volume of each compound in the series, we replaced N methyl groups with N ethyl groups. Following up on earlier published work, 111 t he first series features tetramethylammonium, and then each compound has sequential replacement of a methyl with an ethyl group, leading to the tetraethylammonium cation. The second series was based on choline and its analogs in which subsequent methyl groups were

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83 replaced by ethyl groups. Choline is a selective agonist for the 7 nAChR and we were hoping to maintain 7 selec tivity in this series. The third series features Figure 3 2. Structures of bulky quaternary ammonium compounds

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84 benzylammoniu m compounds. The next three series (4 6) are represente d by pyrrolidinium, piperidinium, and hexahydroazepinium compounds In these three series, we varied the ring size to probe for the importance of the 7 membered ring size in ASM 024. While the TMA, pyrrolidinium, piperidinium and hexahydroazepinium series fe ature similar structural elements (positively charge d nitrogen and alkyl groups), the choline series differs by a hydroxyl group that can act as a hydrogen donor or acceptor, and the benzyl series differs by an aryl group that can be in different positio ns in relation to the ammonium group. 3.2 Results and D iscussion 3.2.1 Synthesis The compounds were either purchased (TMA, EtMA, diEdiMA, triEA, TEA, choline and BtEA), or prepared by reacting commercially available methyl or ethyl amines with methyl iodide or ethyl iodide in tetrahydrofuran or ethanol and purified by recrystallization Methylhexahydroazepine ( 39 ) was prepared from hexahydroazepine by the Eschweiler Clarke reaction 211,212 (formalin and formic acid), and ethylhexahydroazapine ( 40 ) was prepared from he xahydroazepine using potassium carbonate and ethyl iodide. 3.2.2 Activity profile of quaternary ammonium compounds on the human 7 nAChR The quaternary ammonium compounds were screened at 100 M on the human 7 nAChR except choline analogs which were tested at 1 mM because of the low potency of choline The results revealed that small quaternary ammonium compounds act as full agonists of the 7 nAChR and as their size increases their agonism decreases (Figure 3 3). The only small exception is seen in the cholin e series where

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85 (2 hydroxyethyl)ethyldimethyl ammonium ( 23 ) is a super agonist and is a stronger agonist than smaller choline ( 22 ). The biggest decrease in agonism occurs by changing one methyl to an ethyl in diEdiMA ( 20 ), (2HE) EdiMA ( 23 ), EtMePyrr ( 31 ), and dMe Pip ( 35 ). Figure 3 3 Alpha7 agonism bulky ammonium. The compounds that had an agonist activity in the screening were further tested to determine their I max and EC 50 (Table 3 1). The results revealed that small quaternary ammonium that acted as full agonists had similar EC 50 values (20 50 M), except

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86 choline agonists which were much less potent (EC 50 = 272 28 M for choline ( 22 ) and 1500 400 M for (2HE) EdiMA ( 23 )). Compounds that had partial agonism character were generally less potent than their full agonist analogs in the series. Table 3 1. The EC 50 and I max for bulky ammonium compounds that have ionotropic agonism activity. Compound EC 50 [ M] I max TMA 30 3 1.07 0.03 EtMA 26 5 1.20 0.15 diEdiMA 31 1 0.92 0.01 tEMA 107 43 0.15 0.01 choline 272 28 0.93 0.03 (2HE) EdiMA 1500 400 1.5 0.3 BtMA 39 5 0.47 0.02 dMePyrr 18 2 1.00 0.04 EtMePyrr 50.0 0.1 0.91 0.01 dMePip 24 2 0.81 0.02 diMHHA 183 7 0.29 0.01 The compounds that had little or no ionotropic agonism were further tested to determine their IC 50 values. Benzyl ammonium compounds had IC 50 values in a similar range (26 67 M), other compounds had generally higher IC 50 values (100 400 M), except diEHHA ( 38 ) which had an IC 50 value of 40 8 M and TEA (80 5 M) (Table 3 2)

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87 Table 3 2. The IC 50 values for bulky quaternary ammonium compounds. Finally, the quaternary ammonium compou nds were co applied at 100 M (1 mM for choline analogs series) with type II positive allosteric modulator PNU 120596 (10 M) Generally, among the compounds tested, those which acted as ionotropic agonists had the largest responses when co applied with PNU 120596 ( because of their ionotropic agonism responses being potentiated by the type II PAM and D s desensitized state being reversed), thou gh some exceptions are seen in the tetramethylammonium series and pyrrolidinium series (Figure 3 4). Interestingly, TEA ( 17 ), 2HE diEMA ( 23 ), a nd dEtPip ( 35 ) that had no ionotropic response on their own, gave big responses when Compound IC 50 [ M] tEMA 4 0 0 100 TEA 80 5 (2HE) diEMA 14000 5000 (2HE) triEA 300 100 BEdMA 67 2 BdEMA 42 4 BtEA 26 1 dEtPyrr 350 100 EtMePip 350 100 dEtPip 200 70 diMHHA 114 34 EMHHA 159 36 diEHHA 40 8

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88 co applied with PNU 120596 (normalized net charge of 6 2, 24 9, and 11 2, respectively), revealing their silent agonist character. Three other compounds that had no ionotropic agonism when applied alone BtEA ( 29 ), EMHHA ( 37 ), and diEHHA ( 38 ), gave small but significant responses with PNU 120596 (normalized net charge of 0.18 0.07, 0.14 0.06, and 0.2 0.1, respectively) revealing their weak silent agonism charac ter. BtEA ( 29 ), EMHHA ( 37 ), and diEHHA ( 38 ) are the largest compounds in the quaternary ammonium group, and their properties reveal the limits to the size of quaternary ammonium where a bulky quaternary ammonium silent agonist becomes an antagonist. Th e P NU 120596 co applications with quaternary ammonium compounds were further characterized at a range of concentrations (Table 3 3). Similarly to KC 1 to KC 9 co mpoun ds, the determination of EC 50 values for PNU 12059 6 potentiated responses appear ed not possib le because the values d id not fit into the Hill equation This was due to a big change in the net charge responses at a narrow concentration range, and to a decrease in PNU potentiated responses at higher concentratio ns of experimental compounds possibly caused by preferential induction of a D i desensitized state. 57 To illustrate the activity of quaternary ammonium compounds, a ratio of PNU 120596 co application responses at 100 M (1mM for choline analogs) to ionotropic agonism responses at 100 M (1mM fo r choline analogs) was calculated (Figure 3 5). The graphs show that silent agonist s TEA ( 17 ), 2HE diEMA ( 23 ), and dEtPip ( 35 ) favor induction of D s desensitization over ion channel activation in the absence of a PAM by 1100, 4800, 1000 fold respectively. The graphs also clearly show that as the size of the quaternary ammonium molecule increases by replacement of methyl by ethyl groups,

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89 their PNU 120596 co application to agonism ratios increase as well un til the molecule becomes too large Figure 3 4. Desensitized states of quaternary ammonium revealed by co application with type II PAM.

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90 Table 3 3. Net charge for quaternary ammonium compounds co applications with PNU 120596. Drug Drug concentration Net charge when co applied with 1 20596 Threshold: Concentration at which 10 % of maximal response was observed tEMA 100 300 68 11 78 19 35 TEA 100 30 0 1000 6 2 36 16 37 6 90 (2HE) diEMA 100 1000 3 2 24 9 (2HE triE A) 100 300 1000 3000 0.13 0.06 0.4 0 .1 5 2 1.6 0.2 330 BEdMA 10 30 100 300 3 1 23 7 103 19 44 16 27 BdEMA 10 30 100 300 0.08 0.02 0.9 0.1 33 14 34 8 37 BtEA 100 300 0.18 0.07 0.57 0.35 dMePyrr 10 30 100 300 85 15 99 20 84 10 107 46 2 EtMePyrr 1 0 30 100 300 2.4 0.5 25 4 127 30 137 46 23

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91 Table 3 3. Continued Drug Drug concentration Net charge when co applied with 120596 Threshold: Concentration at which 10 % of maximal response was observed dMePip 10 30 100 300 34 6 54 9 163 22 105 44 25 EtMePip 10 30 100 300 0.01 0.01 4 2 28 6 28 3 30 dEtPip 10 30 100 300 0.003 0.002 0.9 0.5 11 2 11 1 30 diMHHA 100 15 3 EMHHA 100 300 0.14 0.06 6 4 diEHHA 100 300 0.2 0.1 0.14 0.03 The Connolly solvent excluded volumes for bulky quaternary ammonium compounds were calculated u sing ChemDraw3D (Figure 3 6). There is a cl ear correlation between the volume of the quaternary ammonium compounds and their pharmacological properties. The molecules that have a molecular volume of 94 133 3 act as agonists, 142 150 3 act as silent agoni sts with partial ionotropic agonism character, 150 163 3 are silent agonists, and 167 186 3 are weak silent agonists

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92 Figure 3 5. Ratio of 10 M PNU 120596 co application response at 100 M to an ionotropic response at 100 M (1 mM for choline anal ogs). To avoid division by 0, when no ionotropic agonism response was observed, the PNU 120596 co application response was divided by 0.005 (detection limit). The error bars were not calculated because of the inaccuracy resulting from limited detection of small responses.

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93 Figure 3 6. Bulky quaternary ammonium h 7 nAChR profile summary. The molecular volumes given are Connolly solvent excluded volumes. Green: agonists, orange: silent agonists with partial agonism characte r, Red: silent agonists, Purple : weak silent agonists.

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94 Benzyl ammonium compounds do not follo w the exactly same criteria, most probably because the quaternary ammonium group and the aryl group can be in different orientations to each other, so they do not need to fit into the same space as other quaternary ammonium compounds. However, similarly t o other compounds, as their size increases their properties change from ionotropic agonism, through silent agonism to antagonism. The molecular volume of the 7 silent agonist ASM 024 is 24 2 3 and it might seem that the compound is not following the trends observed in the quaternary ammonium series described in this chapter. Interestingly, the molecular volume of an nAChR agonist dimethylphenylpiperazinium ( DM PP) (Figure 3 7) is 207 3 and it could be considered as an analog of dimethylpiperidinium (dMePip), characterized here as an agonist. Accordingly to the data presented above, DMPP would be expected to act as an 7 antagonist. Therefore, it appears that t he bulky quaternary ammonium constitutes one type of a core pharmacophore for silent agonism, while the addition of an aromatic ring on the side of the molecule opposite to the positively charged nitrogen does not change the character of the molecule (agon ist, silent agonist, antagonist) and may allow for optimization of the compound to gain selectivity over other ion channels and for 7 nAChR subtype For example, TEA is known to block voltage dependent potassium channels, 214 while ASM 024 is not. ASM 024 and DMPP possess an additional nitrogen in the ring bearing the quaternary ammonium group and its importance could be investigated by testing diethylphenylpiperazinium (A) and diethyphenylpiperidinium (B). Accordingly to the

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95 quaternary ammonium molecular volume hypothesis and observations made above, both compounds would act as 7 silent agonists. Fi gure 3 7. Structures of ASM 024, DMPP, diethylphenylpiperazinium and diethylphenylpiperidinium. 3.2.3 Activity P rofile of Q uaternary A mmonium C ompounds on the H uman 2 nAChR The quaternary ammonium compounds were screened at 100 nAChR. The com pounds had no detectable ionotropic activity, except TMA, EtMA, 111 BtMA, and dMePyrr (small quaternary ammonium with no OH group), that activated the receptor only weakly (Figure 3 8 ).

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96 Figure 3 8 Quaternary ammonium ionotropic agonism on h 4 2 nAChR When the quaternary ammonium compounds were co applied with ACh, diminished responses to ACh were observed ( except for choline) (Figure 3 9 ), many of the compounds appea r to do so very weakly.

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97 Figure 3 9 Quaternary a mmonium a nta gonism on h 2 nAChR 3.3 Summary Six series of the quaternary ammonium compounds have been tested on the human 7 nAChR and new 7 silent agonist s were identified. A clear correlation between the molecular volume of a quaternary ammonium compound and its 7 pharmacological properties has been found: small quaternary ammoniums act as agonists, and increasing their size results in changing their character from an agonist to silent agonist and fin ally to an antagonist.

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98 quaternary ammonium compounds selective for 7 nAChR.

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99 CHAPTER 4 SYNTHESIS OF NEW FLUORI NATED PYRIDINYLMETHYLENE ANABASEINES TO STUDY INTERACTIONS LEADING TO DE SENSITIZED STATES OF THE HUMAN 7 NICOTINIC ACETYLCHOLINE RECEPTOR 4.1 Background synthesized pyridinylmethylene anabaseines 2 PAB ( 41 ), 3 PA B ( 42 ), and 4 PAB ( 43 ) (Figure 4 1). 202,222 Fig ure 4 1. Structures of 2 PAB, 3 PAB and 4 PAB The compounds were weak agonists for the h 7 nAChR and when they were co applied with 30 M PNU 120596, their D s stabilizing ability and silent agonism character were revealed (Figure 4 2, Dr. Roger Papke unpublished data). Figure 4 2. Pharmac ological properties of 2 PAB, 3 PAB, and 4 PAB A. Agonism; B. Co application with PNU 120596.

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100 Dr. Jingyi Wan g also synthesized aryl idene anabaseines analogous to PABs ( 41 42 43 ) but with pyrrole (H bond donors) furan (H bond ac ceptors), and thiophene (non H bonding, hydrophobic ) rings instead of a pyridine (H bond acceptor) ring, and showed that H bonding in the 7 nAChR selectivity pocket influences activation and desensitization of the receptor. 222,223 However, the PABs ( 41 42 43 ) compounds were generally weaker agonists and much stronger D s desensitizers than the arylidene anabaseines with pyrrole, furan, an d thiophene rings, suggesting that other effects than H bonding in the 7 nAChR selectivity pocket may also control the state of the receptor. One noticeable difference between pyrrole, furan, thiophene and pyridine is that the first three heterocycles are electron rich, while pyridine is electron poor. These observations interactions between electron rich amino acids such as tyrosine or tryptophan in the 7 nAChR benzylid ene selectivity pocket and an electron deficient benzyliden e motif may be involved in recognition that promotes D s desensitized state. interactions are non systems. 215,216 Dr. Dennis Dougherty and co workers studied cation in the nAChR binding site, and by using fluorinated amino acid mutagenesis, they showed that cation aromatic amino acids side chains in the nAChR binding site and the positive charge of acetylcholine are very important for acetylcholine binding and a ctivation of the receptor. 82,83,217,218 Instead of introducing fluorinated amino acids in the binding site of the 7 nAChR we designed a series of fluorinated 3 PABs and penta fluorinated benzylidene anabaseine [ (2 fluoro) 3PAB ((2F) 3PAB, 44 ) (5 fluoro) 3PAB ((5F) 3PAB, 45 ), ( 2,6

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101 difluoro) 3PAB ((2,6 DF) 3 PAB, 46 ) (2,4,6 trifluoro) 3PAB ((2,4,6 TF) 3PAB, 47 ) and (2,3,4,5,6 pentafluoro) benzy lidene anabaseine ((2,3,4,5,6 PF) AB, 48 ) (Figure 4 3) ], to interactions in the 7 nAChR benzylid ene se lectivity pocket may be responsible for silent agonism character of pyridinylmethylene anabaseines ( 41 42 43 ). Figure 4 3. Structures of fluor inated 3 PABs and pentafluorinated benzylidene anabaseine. Because of the electron withdrawing effect of fluorine, t he fluorinated compounds ( 44 45 46 47 and 48 ) are even more electron poor than the PABs ( 41 42 43 ). W e acceptor/donor interactions system of the ligand and an electron rich donor in the 7 nAChR benzylidene selectivity pocket do control the Ds desensitized state of the receptor, the fluorinated 3 PABs would give high er responses than 3 PAB ( 42 ) w hen co applied with PNU 120596 because the interactions of the ligand with the receptor would be strengthened. 4.2 Results and Discussion 4.2.1 Synthesis of New Fluorinated Arylidene A nabaseines The general method for the synthesis o f arylidene anabaseines consists of a mixed aldol type condensation between anabaseine dihydrochloride ( 52 ) and aryl aldehydes 219,222 Therefore, we decided to make t he new flu orinated arylidene

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102 anabaseines 44 48 also by an aldol type condensation between anabaseine dihydrochloride ( 52 ) and an appropr iate fluorinated aryl aldehyde. Anabaseine dihydrochloride ( 52 ) was prepared on a four gram scale, following a protocol of Zoltewicz and Cruskie 203 with some modifications of the fir st step 202,222 ( Figure 4 4 ). The NH function of valerolactam 49 was protected by a base stable and acid labile aminomethyl group, which was valerolactam ( 49 ) paraformaldehyde and diethylamine in refluxing toluene in a Dean Stark appar atus to remove water formed. Then, the mixed Claisen condensation of ethyl nicotinate and the 1 (diethylaminomethyl) 2 piperidinone 2 afforded the sodium salt of the 3 nicotinoyl 2 piperidinone 51 in 65 % yield as a mixture of three compounds, most probabl y the E and Z enolate isomers 51a, 51 b and the keto isomer 51 c as indicated by 1 HNMR. The sodium salt 51 was treated with a hot 5:1 mixture of concentrated hydrochloric acid and acetone to yield anabaseine dihydrochloride 52 in 59 % yield by removal of the N protecting group, hydrolysis of the amide bond, decarboxylation of the keto carboxylic acid, and cyclization. Acetone in this reaction facilitates the precipitation of sodium chloride. Figure 4 4 Synthesis of anaba seine dihydrochloride 52

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103 The mixed aldol type condensation between aryl aldehydes and anabaseine dihydrochloride ( 52 ) gives arylidene anabaseines most probably via enamine form of anabaseine 219,222 Zoltewicz et al. reported that in the case of aryl alde hydes having electron donating substituents, the acid catalyzed condensation proceeds well; while in the case of aryl aldehydes having strong electron withdrawing substituents, a base is needed, for example acetate ion in acetic acid, most probably to faci litate dehyd ration of an aldol intermediate 219 Hence (2 fluoro) 3PAB ( 44 ) (5 fluoro) 3PAB ( 45 ) (2,6 difluoro) 3PAB ( 46 ) (2,4,6 trifluoro) 3PAB ( 47 ) and (2,3,4,5,6 pentafluoro) benzylidene anabaseine ( 48 ) were prepared by reacting suitable fluorinate d aryl aldehydes 5 3 57 with anabaseine dihydrochloride (52 ) in methanol in the presence of sodium acetate and acetic acid in a 1: 2.5 ratio at room temperature to give the desired products in 80%, 74%, 78%, 70%, and 57 % yield, respectively ( Figure 4 5 ). T he reaction between 5 fluoro 3 formylpyridine ( 54 ) and anabaseine dihydrochloride ( 52 ) required 26 h to go to completion, while more fluorine substituted aryl aldehydes required longer reaction times (48 h in the case of 2,4,6 trifluoronicotinealdehyde ( 56 ) and perfluorobenzaldehyde ( 57 ) ).

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104 Figure 4 5 Synthesis of new fluorinated arylidene anabaseines Only one double bond isomer was formed in the reaction and the aldol intermediate was not detected. According to the mol ecular models, both E and Z isomer configurations are possible, but the Z isomer of arylidene anabaseine, in which the two aromatic rings are stacked face to face, is higher in energy than the E isomer. 219,222 A ll previously reported arylidene anabaseines prepared by aldol type condensation had an E stereochemistry about the do uble bond 219,222 The NOESY experiments confirmed that the fluorinated arylidene anabaseines retained that previously observed preference for for mation of the E isomer (Figure 4 6, A ppendix ).

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105 Figure 4 6 2F 3PAB ( 44 ): NOE enhancement used to assign the double bond geometries in fluorinated pyridinylmethylene anabaseines. The fluorinated aryl aldehydes were purchased from Frontier Scientific, Matrix Scientific, and Sigma Aldrich except from 2,4,6 trifluoronicotinaldehyde 8 whose synthesis has not been reported in literature The 2,4,6 nicotinaldehyde 8 was prepared from 2,4,6 trifluoro pyridin e with n butyllithium and N methylf ormanilide in 58 % yield (Figure 4 7) Figure 4 7 Synthesis of 2,4,6 trifluoronicotinaldehyde 56 4.2.2 pKa Values of New Fluorinated Arylidene A nabaseines The pK a s of the imine nitrogen determines the concentration of the active protonated forms at p hysiological pH and thus affects arylidene potencie s 220,222 T he pK a s for fluorinated compounds were estimated using the previously reported formula pKa = (4.29 H) / 0.055, where H is the chemical shift of the protons on the carbon adjacent to the imine nitrogen in CDCl 3 with tetramethylsilane as a stand ard (Table 4 1) 219 The formula to predict the pKa of benzylidene anabaseines from the 1 HNMR wa s derived base d on the

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106 series of seventeen compounds in a study, where the range of chemical shifts for the methylene protons adjacent to the imine group of benzylidene anabaseines was only 0.11 ppm but a very good linear co rrelation (r = 0.999) was found using the Ham mett m p 219 The percentages of the iminium cation (% HA) at physiologic al pH (pH = 7.2) were then calculated via Henderson Haselbach equation: pH = pKa + log([A ]/[HA]) Table 4 1. The predicted iminium cation percentages o f the fluorinated arylidene anabaseines. Compound 3PAB (2 F) 3PAB (5 F) 3PAB (2,6 DF) 3PAB (2,4,6 TF) 3PAB (2,3,4,5,6 PF) BA H [ppm] 3.91 3.94 3.93 3.94 3.97 3.98 pKa 6.91 6.36 6.55 6.36 5.82 5.64 % HA at pH = 7.2 34 % 13 % 18 % 13 % 4 % 3 % The 3PA B ( 42 ) is predicted to be 34 % protonated at physiological pH while substitutions with fluorine lower the pK a of the compounds and the percentage of protonated form (which would be expected based on the structure of these compounds: fluorine stabilizes th e positive charge of the iminium ion by electron withdrawing inductive effect). 4.2.3 Activity P rofile of F luorinated A rylidene A nabaseines on the H uman 7 nAChR The fluorinated arylidene anabaseines ( 44 45 46 47 and 48 ) were screened at 30 M and 300 M on the human 7 nAChR and compared to 3PAB ( 42 ). The fluorinated compounds had no ionotropic activity above detection limit (Figure 4 8), indicating t hat fluorination of 3PAB ( 42 ) negatively impacts activation of the receptor.

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107 Figure 4 8. Ionotropic agonism of fluorinated arylidene anabaseines on h 7 nAChR When co applied with 60 M acetylcholine, the fluorinated compounds at 300 M diminishe d the r esponse of acetylcholine suggesting that they bind to the receptor (Figure 4 9). The level of fluorination did not correlate with the ability of the compound to inhibit the ACh response. Figure 4 9. Antagonism of fluorinated arylidene anabaseines.

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108 Fina lly, co application of fluorinated arylidene anabaseines at 300 M with PNU 120596 at 30 M, revealed that fluorination of 3PAB ( 42 ) also negatively impact D s desensitization (Figure 4 10), which was contrary to the expected results. Interestingly, there was a big difference in the responses of monofluorinated (2 F) 3P AB ( 44 ) and (5 F) 3PAB ( 45 ), suggesting that fluorine exerts a local effect with the amino acids in the binding site, possibly such as the interaction described by Parlow at al where fluorine act ed as a hydrogen bond acceptor in a crystal structure of fl uorobenzene inhibitor with a tissue factor VII a 221 Figure 4 10. Desensitized state of fluorinated arylidene anabaseines revealed with PNU 120596. 3 PAB ( 42 ) and (2 F) 3PAB ( 44 ) were further tested at 30 M, 100 M, and 300 M with PNU 120596 ( 10 M ) (Figure 4 11). The highest responses were observed at 100 M for both 3PAB and (2 F) 3PAB (net charge: 72 23 and 9 1 for 3PAB and (2 F) 3PAB, respectively). At 300 M the PNU 120596 potentiated responses were lower, suggesting that higher occupancy of the receptor by 3PAB and (2F) 3PAB was favoring

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109 the D i desensitized state. This result also indicated that lower activity of (2 F) 3PAB ( 44 ) versus 3PAB ( 42 ) with PNU 120596 did not result from lower concent ration of the fluorinated compound protonated form otherwise by increasing the concentration of (2F) 3PAB ( 44 ), a maximum response similar to that of 3PAB ( 42 ) should have been reached. Figure 4 11. Comparison of PNU 120596 co application responses for 3PAB and (2 F) 3PAB. 4.3 Summary Five new fluorinated arylidene anabaseines were synthesized : (2 F) 3PAB ( 44 ), (5 F) 3PAB ( 45 ), (2,6 DF) 3PAB ( 46 ), (2,4,6 TF) 3PAB ( 47 ), and (2,3,4,5,6 PF) BA ( 48 ) interactions between electron poo r benzylidene motif of the ligand and electron rich amino acids in the 7 selectivity pocket favor D s desensitization It was demonstrated that f luorination of 3PAB ( 42 ) negatively impacts activation of the receptor and D s desensitization. Perhaps fluor ination enhances entry into D i state but this remains to be considered after further investigation. The fact that fluorinated 3PABs are less active as 7 activators and D s desensitizers than 3PAB might be due to weaker H bonding ability of the nitrogen in the fluorinated pyridine ring. However, other

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110 factors also seem to play a role in controlling the state of the receptor, such as local interactions between fluorine atoms on the pyridine ring and amino acids in the binding site. The results do not support the hypothesis stating that interactions in the 7 nAChR benzylidene selectivity pocket influence 7 nAChR desensitization. The changes in the pK a s of fluorinated compounds did not correlate with their activity, suggestin g that lower ionotropic and silent agonist activities of flu orinated 3PABs are not due to lower concentrations of protonated forms of these compounds at physiological pH Higher occupancy of the receptor by PAB compounds produce s less of the D s state and more of the D i as observed previously with KC compounds and quaternary ammonium compounds.

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111 CHAPTER 5 SYNTHESIS OF NOVEL ARYLIDENE QUINUCLIDINES TO STUDY THE EFFECT OF H BONDING IN THE 7 nAChR SELECTIVITY POCKET ON ACTIVATION AND DESENSITIZATION 5.1 Background As introduced in Chapter 1, anabaseine ( 52 ) and quinuclidine ( 60 ) are non selective agonists for nAChRs, and the addition of benzylidene motif such as in GTS 21 ( 61 ) and (E) benzylid ene quinuclidine ( 62 ) makes these molecules selective for 7 nAChR (Figure 5 1). 111 Figure 5 1. Structures of non selective nAChR agonists (anabaseine, quinuclidine), and h 7 nAChR selective agonists (GTS 21, (E) benzyli dene quinuclidine ). Dr. Jingyi Wang (Horenstein Laboratory) synthesized and characterized arylidene anabaseines 2PyroAB ( 63 ) (H bond donor), 2FAB ( 64 ) (H bond acceptor) and 2 TAB ( 65 ) (hydrophobic, non H bonding ) (Figure 5 2) as partial agonist s of the h 7 nAChR and showed that H bonding interactions in the 7 selectivity pocket could control the state of the rece ptor (activation, rate of the receptor to enter into different desensitized states, and energy levels of open and desensitized states). 222,223 T hese results suggested that it may be possible to develop new nAChR agonists that have tailored responses to allosteric modulators

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112 To further investigate how H bonding interactions in the 7 selectivity pocket influence the state of the receptor, we desig ned a serie s of twelve H bonding probes: (Z ) 2 PyroQN ( 66a ) (E ) 2 PyroQN ( 66b ) (Z ) 3 PyroQN ( 67a ) ( E ) 3 PyroQN ( 67b ) (H bond donors), (Z ) 2 FQN ( 68a ) (E ) 2 FQN ( 68b ) (Z ) 3 FQN ( 69a ) (E ) 3 FQN ( 69b ) (H bond acceptors), and (Z ) 2 TQN ( 70a ) (E ) 2 TQN ( 70b ) (Z ) 3 TQN ( 71a ) (E ) 3 TQN ( 71b ) (non H bonding, hydrophobic) (Figure 5 3). Figure 5 2. Structures of h 7 nAChR partial agonists 2PyroAB, 2FAB, and 2TAB. The compounds have the same selectivity motifs as 2 PyroAB, 2 FAB, and 2 TAB, but different pharmacophore cores (quinuclidines vs anabaseines), which would allow us to exploit whether the interactions betw een the ligand and the receptor in the 7 selectivity pocket could be transferred between different pharmacophore cores. If H bonding of arylidene quinuclid in es in the 7 selectivity pocket would have same effects on activation and desensitization of the receptor as H bonding of arylidene anabaseines, it would suggest that their binding modes are the same. If arylidene quinuclidines would show different behavior than their analogous arylidene anabaseines, it would suggest that the molecules have different binding modes. We were also hoping to obtain ligands with better eff icacy, potency, and selectivity.

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113 Figure 5 3. Structures of H bonding probes. 5.2 Results and Discussion 5.2.1 Synthesis of Arylidene Q uinuclidines We decided to s ynthesize arylidene quinuclidines by Horner Wadsworth Emmons olefination 224,225 using 3 quinuclidinone ( 7 2 ) and appropriate phosphonate esters, similarly to the protocol used for the synthesis of benzylidene quinuclidines. 132 133,226 Figure 5 4. Retrosynthetic analysis of arylidene quinuclidines.

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114 5.2.1.1 Synthesis of 2 TQN and 2 FQN U sing P hosphonate E thyl E sters The necessary diethyl(thiophene 2 ylmethyl)phosphonate ( 77 ) and diethyl(furan 2 ylmethyl)phosphonate ( 78 ) were prepare d in 65 % yield by Michaelis Ar b u zov reaction, 227 using chlorides 75 76 and triethylphosphite (Figure 5 5) The chlorides were obtained from corresponding alcohols 73 74 by reaction with thionyl chloride and py ridine in dichloromethane, following publish ed procedures. 228,229 The phosphonate ethyl esters were treated first with sodium hydride 60 % dispersion in mineral oil to generate the phosphonate anions, and then with 3 quinuclidinone ( 7 2 ) in THF at reflux (no reaction was observed at room temperature ). The Horner Wadsworth Emmons reactions gave 2 TQN ( 70ab ) and 2 FQN ( 68ab ) in 85 % yield as a mixture of E and Z isomers in 1:1 ratio that required tedious chromatographic separation on silica gel using hexanes, ethyl acetate and freshly distilled trieth ylamine (use of DCM, methanol, and triethylamine as an eluent did not afford any separation of isomers). Figure 5 5. Synthesis of 2 TQN ( 70ab ) and 2 FQN ( 68ab ) using phosphonate ethyl esters. Horner Wadsworth Emmons reac tions normally favor the formation of E isomers, 225 but since 3 quinuclidinone ( 7 2 ) has similar steric bulk on both sides of the

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115 carbonyl group, no stere oselectivity was seen. The geometry of the double bond was unambiguously established using Nuclear Ove rhauser Enhancement Spectroscopy (Appendix) and also by analysis of the 1 HNMR spectra. The most diagnostic NMR feature was the peak for the proton marked by a red arrow in Figure 5 6: it appeared at 2.47 ppm in a Z isomer and was shifted 0.8 ppm downfield in an E isomer due to the deshielding from the aryl ring. This shift downfield in the E isomer was observed for all synthesized arylidene quinuclidines and has been also reported previously for benzylidene quinuclidines. 133 Figure 5 6. Comparison of 1 HNMR spectra for (Z) 2 TQN ( 70a ) and (E) 2 TQN ( 70b )

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116 5.2.1.2 Synthesis of 2 TQN and 2 FQN U sing P hosphonate M ethyl E sters 2 TQN ( 70ab ) and 2 FQN ( 68ab ) were also prepared using phosphonate methyl esters (Figure 5 7). However, the yields were much lower than those obtained using phosphonate ethyl esters and some impurities were difficult to separate, so this approach was not further pursued. Figure 5 7. Synthesis of 2 TQN ( 70ab ) and 2 FQN ( 68ab ) using phosphonate methyl esters. 5.2.1.3 S ynthesis of 3 TQN and 3 FQN Diethyl(thiophene 3 ylmethyl)phosphonate ( 85 ) and diethyl(furan 3 ylmethyl)phosphonate ( 86 ) were synthesized by Michaelis Arbuzov reaction, analogously to the analogues substituted at the posit i on 2 of the heteroaryl ring (Figur e 5 8). The Horner Wadsworth Emmons reaction of the heteroaryls with phosphonate group at position 3 did not work too well, though. The yields were much lower than those for 2 TQN ( 70ab ) and 2 FQN ( 68ab ) and an excess of phosphonates 85 86 was necessary to obtain reasonable yields.

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117 Figure 5 8. Synthesis of 3 TQN ( 71ab ) and 3 FQN ( 69ab ) Moreover, separation of the Z and E isomers of 3 TQN ( 71ab ) and 3 FQN ( 69ab ) was more difficult than for 2 TQN ( 70ab ) and 2 FQN ( 68a b ) with some sma ll impurities remaining after column chromatography, especially in the E isomers, and the compounds were not satisfactor il y pure for biological testing. 5.2.1.4 Attempts to synthesize N Boc 2 (chloromethyl)pyrrole (90) The synthesi s of pyrrole ph osphonates proved to be very problematic most probably due to instability of the pyrrole ring system in 90 (the chloride can eliminate from the product) The protected 2 hydroxymethyl pyrrole 89 was prepared from pyrrole 2 carboxaldehyde ( 87 ) following a l iterature procedure 230 (Figure 5 9). Several protocols for transformation of alcohols into chlorides were tried, but all of them met with failure: the substrate was consumed but no product formation was observed. Figure 5 9. Attempts to prepare N Boc 2 (chloromethyl)pyrrole 90

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118 5.2.1.5 Attempt to synthesize 2 PyroQN (66ab) To circumvent the instability of the pyrrole chloride, a known 2 (chloromethyl) 1 (methylsulfonyl)pyrrole ( 93 ) was prepared following a published procedure wit h small modifications 231 (Figure 5 10). The Arbuzov reaction of the chloride 93 with triethyl phosphite gave the phosphonate 94 in a low 12 % yield. Unfortunately, t he Horner Wadsworth Emmons between the phosphonate 94 and 3 quinuclidinone ( 7 2 ) did not wo rk, probably due to poor nucleophilicity of the anion of 94 (most of the unreacted substrate 94 was found in the crude). Therefore, the preparation of 2 PyroQN ( 66ab ) appeared not possible: the necessary chloride ( 90 ) was too unstable, and while the stabil ization of the pyrrole chloride by protection with an electron withdrawing mesyl group enabled the synthesis of a phosphonate 94 the mesyl phosphonate 94 was unreactive in the subsequent Horner Wadsworth Emmons reaction. Figure 5 10. Attempts to synthesize 2 PyroQN. 5.2.2 Activity of QN C ompounds The hydrochloride salts of (Z) 2 TQN ( 70a ) (E) 2 TQN ( 70b ) (Z) 2 FQN ( 68a ) and (Z) 3 TQN ( 68b ) were tested on the h 7 nAChR in Xenopus oocytes as described in Section 1.4 and Appendix A, and appeared to act as partial agonists. (E) 2 TQN ( 70b ) was shown to be a more potent and efficacious agonist than (Z) 2 TQN ( 70a )

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119 (I max = 0.71 0.04, EC 50 = 0.95 0.24 M for (E) 2 T QN; I max = 0.20 0.01, EC 50 = 7 2 M for (Z) 2 TQN). Higher activity of an E isomer has been reported before for benzylidene quinuclidines. 111 It was also noticed that (E) 2 TQN ( 70b ) produced large amounts of RID (residual inhibition and desensitization ) (10 3 % recovery of the receptor from the desensitized state as indicated by an ACh post control), while (Z) 2 TQN ( 70a ) did not (86 7 % recovery). It would be interesting to further pursue these observations and study the behavior of (E) 2 TQN ( 70b ) and (Z) 2 TQN ( 70a ) with type II PAMs. 5.3 Summary Eight new arylidene quinuclidines containing furan and thiophene rings were synthesized: (Z) 2 TQN ( 70a ), (E) 2 TQN ( 70b ), (Z) 2 FQN ( 68a ), (E) 2 FQN ( 68b ), (Z) 3 TQN ( 71a ), (E) 3 TQN ( 71b ), (Z) 3 FQN ( 69a ), (E) 3 FQN ( 69b ), but (E) 3 TQN ( 71b ), (Z) 3 FQN ( 69a ), and (E) 3 FQN ( 69b ) could not be obtained in a pure form. (Z) 2 TQN ( 70a ), (E) 2 TQN ( 70b ), (Z) 2 FQN ( 68a ), (Z) 3 TQN ( 71a ) were tested on h 7 nAChR and showed to act as partial agonists. Quinuclid ines contain in g a pyrrole ring were not synthesized because of t he instability of Boc protected 2 chloromethylpyrrole ( 90 ) and lack of reactivity of mesylphosphonate ( 94 ) in the Horner Wadsworth Emmons reaction Due to difficulties with purification of the quinuclidine compounds, problems with synthesis of pyrrole quinuclidines, and higher importance of other proje cts, this project was abandoned, though some interesting observations about different residual inhibition and desensitization properties of Z an d E arylidene quinuclidines were made that may be further investigated in the future.

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120 CHAPTER 6 STRIGOLACTONES 1 6.1 Background 6.1.1 Roles of S trigolactones Strigolactones have long been of interest because they induce germination of seeds of parasitic weeds of the genera S triga Orobanche and Alectra which cause massive crop losses esp ecially in the developing world 232 The seeds of these weeds can remain dormant in the soil for up to 20 years; they do not germinate unless they sense strigolactones secreted by roots of the host plants (Figure 6 1) Figure 6 1. Strigolactones secreted by host plants induce germination of parasitic weeds ( Reproduced from Annual Review of Phytopathology, 48, X. Xie, K. Yoneyama, K. Yoneyama, The strigolactone story, 93 117, Copyright (2010 ), with permission from Annual Reviews, Inc). 1 This work was performed in Dr. Ni gel G. J. Richards Lab in collabor ation with Dr. Aaron Aponick

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121 Strigol was first isolated in 1966 from cotton root exudates 233 and later, it gave the name to the strigolactones family (Figure 6 2 ) Strigolactones are composed of a tricyclic lactone (ABC core) connected vi a an enol ether linkage to a butyrolactone moiety (D ring). All natural strigolactones contain the same C, D moiety and possess (R) epioorobanchol, and same configuration of BC rings except for fabacyl acetate and solanac ol They display major differences in A and B rings Figure 6 2 Structures of natural strigolactones and synthetic analog GR24. It was not until 40 years after the isolation of the first strigolactone that the true role of these natural products started to emerge In 2005, it was shown that strigolactones are involved in symbiotic interactions between plant roots and arbuscular mycorrhizal fungi which are important for the growth of 80 % of plant species 234 Finally,

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122 st rigolactones have been identified in 2008 by studies of plant mutants displaying a bushy phenotype as a new class of plant hormones that inhibit shoot branching 235,236 (Figure 6 3), which led to renewed interest in strigolactones biosynthesis and their mod e of action and resulted in a number of reviews on strigolactones 237 243 Figure 6 3. Phenotype of wild type rice plant producing strigolactones (left), and a mutant impaired in strigolacto ne biosynthesis (right) (Reproduced from Plant Cell, 21, H. Lin R. Wang, Q. Qian, M. Yan, X. Meng, Z. Fu, C. Yan, B. Jiang, Z. Su, J. Li, Y. Wang, Dwarf27, an iron containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth, 1512 1525, Copyright (2009), with permission from American Society of Plant Physiologists ) Plant hormones are active at very low concentrations and they can function locally, at or near the site of synthesis, or in distant tissues 244 They regulate plant growth and mediate responses to various stresses Plant hor mones include: auxins, cytokinins abscisic acid gibberellins, jasmonates, ethylene, brassinosteroids and salicylic acid (Figure 6 4 ) 6.1.2 Strigolact one Biosynthetic P athway At the time when I started to work on strigolactones, little was known a bout their biosynthetic pathway. In 2005, Bouwmeester and coworkers have demonstrated that the ABC core of strigolactones is derived from carotenoids by the studies with inhibitors of carotenoid biosynthesis and isoprenoids pathways 245

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123 Figure 6 4 Stru ctu res of plant hormones. (Reproduced from Nature Chem ical Biology 5, A. Santner, L.I.A. Calderon Villalobos, M. Estelle, Plant hormones are versatile chemical regulators of plant growth, 301 307, Copyright (2009), with permiss ion from Macmillan Publisher s Ltd ). The strigolactone biosynthetic pathway has been partially predicted by extensive screening and genetic a nalysis performed in the last 20 years on various plant mutants ( Figure 6 5 ). Four mutants, max1 max4 (more axillary growth), were described in Arabidopsis thaliana 246 249 Several mutants have been described in rice (htd and d, high tillering dwarf and dwarf), pea (rms, ramosus) and petunia (dad, decreased apical dominance).

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124 Max3 have been shown to encode a carotenoid cleavage dioxygenase 7 (CCD 7) and max4 have been shown to encode a CCD8. It has been reported that in carotenoids accumulating E. Coli and in vitro carotene at the position 248 250,251 and the re apo carotenal, is fu rther cleaved by apo 13 carotenone (C18 ketone) 250 251 However, the natural substrat e and enzyme cleavage activities i n plants were not reported in the literature at the time when I was actively working on the project Figure 6 5 Partially predicted strigolactones biosynthetic pathway deduced from analysis of plant mutants displaying an increased branching phenotype and studies in carotenoids accumulating E. Coli and in vitro (t he int ermediates shown and enzymes activities appeared later to be different ). 252

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125 Research on plant carotenoid dioxygenases started with identification of maize 9 cis epoxy carotenoid dioxygenase VP14 (viviparous 14), which cat alyzes formation of xanthoxin, a p recursor of abscisic acid 253,254 Carotenoid cleavage dioxygenases are non heme iron proteins (reviews 255 260 ) These enzymes use dioxygen, but whether one or both oxygen atoms are incorporated into apocarotenoids products and whether the name dioxygenase s is correct or not, has not been clarified. D27 encodes a novel iron containing protein that is localized in chloroplasts. D27 shares no homology with any functionally identified protein 261 Max1 encodes a cytochrome P450 enzyme (CYP711) and it has sequ ence similarity to Thromboxane A2 synthase 249 which does not require molecular oxygen or an electron donor for catalysis (it catalyzes an isomerization and fragmentation of prostaglandin H2). Max2 is not involved in strigolactones biosynthesis and it e nc odes an F box protein needed for signal transduction. The elucidation of strigolactones biosynthetic pathway is not trivial because strigolactones are produced in very low, nano and picomolar concentrations and they are quite unstable. In addition, the ch aracterization of plastidic CCD7, CCD8 and D27 poses particular problems due to carotenoid hydrophobicity and the difficulty to reproduce the plastid organization outside the plants 262 6.1.3 Strigolactone S ynthesis A series of strigolactones have been synthesiz ed because of the need to confirm the proposed structures. For example, total syntheses showed that the initially reported structures for orobanchol, alectrol and solanacol were incorrect 263 266 Many synthetic strigolactones analogs have been m ade in an e ffort to find an inexpensive compound

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126 for use in agriculture to induce parasites seed germination in the absence of crops 267,268 This goal has not been realized in practice, though some promising results have been obtained in field trials with Nijmegen 1, an analog in which the AB ring system was replaced with phthalimide group 238 GR24 (for germina tion release) (Figure 6 2 ), is one of the most potent strigolactones analogs and it is now used in bioassays for shoot branching inhibition. Strigolactones synt hesis has been reviewed up to 2005 by Humphrey at al 232 Strigol was first synthesized in the 1970s by the groups of Charles Sih and Ralph Ra phael (Figure 6 6 ) 269,270 Both groups made the B and C rings in a stepwise fashion and then they attached the D ring using bromobutenolide. The Sih group used cyclocitral as a starting material and the Raphael group started their synthesis from 2,2, dimethyl cyclohexanone. Strigol has been synthesized later by modifications of those two routes 271 274 5 Deoxystrigol was first made by Frischmuth et al. 275 and later by Shoji et al. 276 Sorgolactone has been synthesized in the laboratories of Binne Zwanenburg 277 and Kenji Mori 278 Orobanchol synthesis has been published by Matsui et al 279 (Mori Lab) Most recent s ynthetic efforts to build the B ring of strigol actones relied on Diels Alder, followed by Perkin reaction, alkaline rearrangement and decarboxylation 265 reductive carbon carbon bond formation of an aldehyde in the presence of samarium (II) iodide 276 ring closing metathesis 266 and intramolecular Frie del Crafts 280 All the known ABC core syntheses consist of a stepwise formation of the rings, except samarium iodide mediated cyclization, in which tricyclic lactone has been obtained as a minor product 276 None of the literature strigolact one ABC ring s ystem syntheses had been biomimetic before this work was published 281

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127 Figure 6 6 First total synthe ses of strigol Several strigolactones and strigolactones synthetic analogs have been prepared in enantiopure forms 232 282,283 (+) Strigol was first obtained by resolution with a chiral agent 284 The first formal asymmetric synthesis has been done by Welzel research group using (S) malic acid as a starting material (chiral pool approach) 285 Strigolactones have also been obtained as pure enantiomers by chromatography on a

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128 chiral column (using cellulose triacetate) (for example 286,287 ) or by kinetic resolution using Candida Antarctica lipase B and vinyl acetate or acetic anhydride 288 266 Several methodologies ha ve been d eveloped to control the stereochemistry at C use of enantiopure D ring precursors 96 and 97 and liberation of D ring by retro Diels Alder reaction 289,290 (Figure 6 7 ). Figure 6 7 Enantiopure precursors o f D ring 6.1.4 Importance of W ork on S trigolactone s Synthetic analogs and biosynthetic inhibitors of enzymes making strigolactones could be used to manage germination of parasitic weeds responsible for massive crop losses in the developing world. They could als o be potential tools to control lateral branching and aerial part plant architecture which would be useful in agriculture and food industry (for example by enhancing tillering in rice and thus improv ing grain yields) 291 forestry (production of high qualit y woods with fewer knots ), and horticulture (p roduction of ornamental plants ) 262 6.2 Research D esign and Specific A ims This research has been focus ed on investigating whether the tricyclic core of strigolactones can be formed in one step by double cyclizatio n of simple linear precursors in model systems (aim 1) and applying that cascade reaction in synthesis of strigolactones (aim 2).

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129 Strigolactones were shown to be derived from carotenoids but only four enzymes involved in strigolactones biosynthe sis have b een discovered despite exte nsive studies on plant mutants In 2005, Bouwmeester and coworkers proposed how strigolactones could be made in plants 245 292 (Figure 6 8 ) and their biosynthetic schemes have been repeatedly reproduced in later publications on s trigolactones. However, the proposed schemes did not take into account activities of enzymes known to be involved in the bi osynthetic pathway and they relied on long sequences of unusual chemical transformations. Figure 6 8 Proposed literature s trigolactone biosynthetic pathway 292 As an alternative to those chemically complex sets of reactions, we aimed to propose a more rational way in which those natural products could be made in plants and show by chemical synthesis that B and C rings of the ABC ring system might be constructed in one step from a linear precursor type 98 (alcohol oxidation level) by an acid catalyzed double cyclization During this research, we also decided to study the feasibility of our proposed cyc lization with a linear precursor type 100 (aldeh yde

PAGE 130

130 oxidation level) (Figure 6 9 ). The demonstration that the ABC ring system of strigolactones can be made in a single step from suitable precursors would provide new insights in to strigolactones biosynthesi s, obviate the need to involve a large number of unidentified enzymes producing strigolactones, and suggest substrates that could be tested with MAX1. Figure 6 9 Proposed formation of the strigolactone ABC core in one s tep by an ac id catalyzed double cyclization During this research, we also decided to use our cascade cyclization methodology to synthesize natural strigolactones and make solanacol. Solanacol was isolated in 2007 293 and it is the fi rst natural strigolacto ne containing an aromatic ring. Solanacol has been initially proposed to have methyl substituents in the para relation ship ( 104 ) (Figure 6 10 ), but that structure has been disproved in 2009 by Takikawa at al. who suggested that the cor rect structure of sol anacol was 105 265

PAGE 131

131 Figure 6 10 Proposed and correct structures of solanacol. When work on solanacol synthesis was in progress in our lab, Chen et al. demonstrated that 106 is the correct structure for that compound 266 6.3 Results and D iscussion 6.3.1 First C yclization A ttempts (Aliphatic S ubstrate) We set out to study our proposed acid catalyzed double cyclization with a simplified aliphatic type substrate 1 07 (Figure 6 11 ). Figure 6 11 Fir st cyclization plans. We imagined that the necessary substrate 107 could be prepared by a Wittig reaction between the aldehyde 1 09 and an ylide generated from phosphonium salt 110 followed by deprotection of the TBS group from the Wittig product (Figure 6 12 ). Figure 6 12 Retrosynthetic analysis of the substrate 1 07 needed for cyclization studies.

PAGE 132

132 It is known that preparation of unsaturated acids by this type of Wittig reaction may be problematic, because o f possib le deprotonation of an hydrogen from the phosphonium salt, resulting in elimination of triphenylphosphine instead of desired hydrogen and formation of the ylide 294 296 However, Brandsma and co workers have reported that by using li thium 2,2,6,6 tetramethylpiperidide (LiTMP), they were able to prevent elimination of triphenylphosphine and they obtained a Wittig product in 70 % yield from an enantiomer of phosphonium salt 1 10 and benzaldehyde reacted in a 1:1 ratio (only E isomer was formed) 297 It was thus thought that we should be able to make the substrate needed for cyclization studies ( 107 ) by our devised route. Aldehyde 1 09 was prepared from 3,4,5,6 tetrahydrophthalic anhydride ( 1 11 ) in a low 8 % overall yield ( Figure 6 13 ). Figure 6 13 Synthesis of the aldehyde 109 3,4,5,6 tetrahydrophthalic anhydride ( 111 ) was reduced with lithium aluminium hydride (LAH) to give the diol 1 12 in 32 % yield after column chromatography. The crude product was quite impure, but based on its mass and the NMR data, the estimated yield would be above 75 %. It is thus thought that the product degrades on a silica gel chromatography column. Baldwin et al. reported that the diol 112 can be transformed into an aldehyd e 114 under acidic conditions 298 most probably by a mechanism shown in figure 6 14 It is possible that this reactivity of the diol may be the

PAGE 133

133 cause of its instability on an acidic silica gel column though no aldehyde 1 14 was observed Figure 6 14 Reported reactivity of the diol 1 12 under acidic condtions 298 Butina and Sondheimer also reported that the diol 112 was difficult to purify and the y ob tained it by reduction of dimethyl 1 cyclohexene 1,2 dicarboxylate in 18 % yield after transforming it into a diformate for easier purification and hydrolyzing it back to the diol with methanol and ammonia 299 The diol 1 12 was monoprotected in 32 % yield with one equivalent of tert butyl dimethylsilyl chloride and 1.5 molar ex cess of imidazole in dichloromethane. We tried to increase the yield by using a procedure of McDougal et al. for monosilylation of symmetric 1,n diols which employs one equivalent of sodium hydride 300 but the obtained yield was even lower (19 %). Finall y, the alcohol 1 13 was oxidized to the aldehyde 1 09 by Swern oxidation and also with activated manganese dioxide Both oxidation procedures gave the product of satisfactor y purity after the work up (see NMR spectra, appendix D) in 74 % yield Further p urif ication by chromatography column resulted in a significant yield decrease (to 43 %), and the 1 H NMR spectrum of the compound indicated that it was not significantly purer than the crude. The aldehyde 1 09 was rather unstable and gradually decomposed (after 3 tar, and the 1 H NMR indicated that it large ly degraded). Thus, the aldehyde 1 09 was prepared fresh, a day before running each Wittig reaction.

PAGE 134

134 Phosphonium salt 1 10 was prepared following a literature procedure for the synthesis of its enantiomer 297 (Figure 6 15 ). Figure 6 15 Synthesis of the phosphonium salt 110 R (+) methyl 2 methyl 3 hydroxy propanoate 115 (the S enantiomer was a bit more expensive tha n the R enantiomer, and the stereogenic center did not matter to us at that point), was transformed into its tosylate 116 with p toluenesulfonyl chloride (TsCl) in pyridine in 98 % yield. It was subsequently converted in 86 % yield into the iodide 117 by treatment with lithium iodide in tetrahydrofuran. The cleavage of the m ethyl group was a bit problematic and the carboxylic acid 118 was obtained in 50 % yield, using chlorotrimethylsilane and sodium iodide in acetonitrile (during this reaction iodotrimeth ylsilane is generated in situ ). 301,302 Finally, the phosphonium salt 1 10 was made in 80 % yield, by treating the iodide carboxylic acid 118 with an 8 fold excess of triphenylphosphine in acetonitrile The Wittig reaction employing phosphonium salt 110 tur ned out to be difficult due to preferential triphenylphosphine elimination instead of planned ylide formation despite the use of lithium tetramethylpiperidide (Figure 6 16 )

PAGE 135

135 Figure 6 16 Deprotonation at pos d of the carboxylate group of 110 Li TMP ( pKa is the most basic and least nucleophilic of the amide bases. It is reported to be kinetically faster than LDA, allowing some deprotonations that are not possible with LDA 303,304,305 We attempted to r epeat Brandsma reaction between benzaldehyde and the ylide generated from phosphonium salt 1 10 with Li TMP 297 but after several tries the alkene product was obtained only in an estimated 30 % yield, and it was difficult to purify. To circumvent tripheny lphosphine elimination problem, an excess of phosphonium salt can be used (for example 10 fold molar excess) 295 A test reaction between 1 cyclohexene 1 carbaldehyde and two equivalents of phosphonium salt 1 10 gave the Wittig product in an estimated 25 % yield that was still quite impure after purification by extractions and column chromatography. The reaction between the aldehyde 1 09 and an ylide made from phosphonium salt 1 10 (4 equivalents) gave the desired olefin 1 07 in an estimated 10 % yield (the TBS group was removed during the acidic work up) (Figure 6 17) Again, the product was not obtained pure because of a difficulty to separate the des ired carboxylic acid from other multiple impurities (especially meth acrylic acid by product).

PAGE 136

136 Figure 6 17 Wittig reaction and attempts to cyclize the olefin product 107 In all the Wittig reactions with phosphonium salt 1 10 only the E isomer was formed, the Z isomer has never been detected. It is well known that non stabilized tr iphenylphosphine ylides generally react with aldehydes to afford mainly Z alkenes, while stabilized ylides give E alkenes 306 The unusual E stereoselective behavior of oxido and carboxy ylides have been reported previously. 307 308,297 During the Wittig reaction studies we also tried to use lithium hexamethyldisilazide (Li HMDS) to generate the ylide, because of its commercial availability as 1M solution in THF, but it resulted in even faster triphenylphosphine elimination, and no Wittig product was ever obtained with that base. We also used methyl ester phosphonium salt instead of carboxylic acid phosphonium salt 1 10 because it could be directly prepared with triphe nylphospine from methyl ester iodide 117 omitting the troublesome methyl cleavage. However, the methyl ester salt was even more susceptible to triphenylphosphine elimination than the carboxylic acid salt 1 10 The cyclization of carboxylic acid alcohol 1 07 has been attempted using hydrochloric acid, sulfuric acid, PPTS (py ridinium p toluenesulfonate) and p toluenesulfonic acid at room temperature but no desired product has been observed and the unreacted substrate was mostly recovered (Figure 6 17 )

PAGE 137

137 As th e problems with preparing the cyclization precursor 1 07 piled up ( cost of the starting materials low yields in synthesis of aldehyde 1 09 and its instability problems with methyl cleavage from 117 preferential triphenylphosphine elimination instead of fo rmation of an ylide and the need to use the phosphonium salt in excess, low yields in Wittig reaction and difficulty to purify the desired product), a need of a different model substrate to test our proposed acid catalyzed cyclization became evident. 6.3.2 Attem pts to C yclize A romatic S ubstrates at the A lcohol O xidation L evel 6.3.2.1 Rationale To avoid the problems encountered in the preparation of the aliphatic precursor 1 07 for cyclization studies, a synthesis of an aromatic substrate that could be prepared relatively easily in larger amounts and at reasonable cost was next envisaged. Compound 119 contains all the necessary elements to study the double cyclization without any bulk that would complicate its synthesis or increase the cost of the starting reagents In addi tion, the double cyclization of 119 would result in a formation of 120 (Figure 6 18 ), which is the ABC core of GR24 (Figure 6 2 ) whose synthesis had been reported in the literature, and thus the cyclization reaction could be monitored using an authentic s tandard of the product. Since one of the natural strigolactones (solanacol) is aromatic, this reaction could be still considered as valid for biosynthesis of strigolactones. Figure 6 18 Aromatic substrate at the alcohol oxidation level c yclization plan.

PAGE 138

138 The substrate 119 could be prepared by a Wittig reaction between a known aldehyde 121 and a known orthoester phosphonium salt 122 that were used for other reactions in literature, and were stable (Figure 6 19 ) The orth oester OBO (2,6,7 trioxabicyclo[2.2.2]octane) protecting group was developed by Corey and Raju 309 it is generally resistant to attack by bases and strong nucleophiles and can be removed by mild acid hydrolysis to give an ester, which can be treated with b ase to release a carboxylic acid. The protection of the carboxylic acid group as an orthoester would ensure no problems with triphenylphosphine elimination. Figure 6 19 Retrosynthetic analysis of 119 6.3.2.2 GR24 Synthesis GR2 4 ( 127 128 ) a potent strigolactone analog used for bioassays, was made following a Mangnus et al literature procedure 268 (Figure 6 20 ) to supply some material for studies prepare lactone 120 needed as a standard for TLC and NMR for cyclization reactions (synthesis of 120 has been published 310 but not its NMR data).

PAGE 139

139 Figure 6 20 GR24 Synthesis. The synthesis of tricyclic lactone 120 started from 1 indanone ( 123 ) Ethoxycarbonyl group was introduced with 4 fold excess of diethyl carbonate and sodium hydride (60 % dispersion in mineral oil) to activate the position for alkylation and protect it against dialkylation. The subsequent alkylation with et hyl bromoacetate gave compound 124 in 93 % yield. Acid catalyzed hydrolysis and concomitant decarboxylation yielded the carboxylic acid 125 in 70 % yield. Reduction of the keto acid 125 with sodium borohydride, followed by treatment with catalytic p toluen esulfonic acid to complete lactonization, afforded the tricyclic lactone in 66 % yield. Formylation was performed with potassium tert butoxide and ethyl formate. The brominated furanone

PAGE 140

140 130 was prepared from 3 methyl 2(5H) furanone ( 129 ) with N bromosuccin imide (NBS), and azobisisobutyronitrile (AIBN) in carbon tetrachloride. Benzoyl peroxide was used in the literature to perform this bromination 268,270 but in my experience benzoyl peroxide (75 %, remainder water, Acros Organics, 21178) did not initiate th e reaction, and use of AIBN was necessary. The treatment of potassium enolate 126 with bromofuranone 130 gave GR24 in 75 % yield as a 1:1 mixture of diastereoisomers 127 and 128 that were separated by column chromatography on silica gel. The preference for E geometry at the enol ether double bond has been reported in the literature (no Z isomer is formed) MacAlpine et al showed that the resonance for the exocyclic vin yl proton of E isomer of strigol appears at 7.42 ppm (7.48 ppm for GR24) as a doublet wit h a coupling constant 4 J = 2.6 Hz while the Z isomer isolated from a mixture obtained by treatment of E isomer with u.v. light has that proton resonating 0.6 ppm upfield because it is no longer in the deshielding zone of lactone carbonyl group 270 6.3.2.3 Synthes is of A romatic S ubstrates at A lcohol O xidation L evel and Attempts to Cyclize T hem To make the precursor to test our cyclization aldehyde 121 and phosphonium salt 122 were needed. The known aromatic aldehyde 26 311 was synthesized analogously to the aliphat ic aldehyde 1 09 described in the previous section. In contrast to its aliphatic analogs, aromatic intermediates were stable, and all reactions had better yields (Figure 6 21 ) Phthalic anhydride ( 131 ) was reduced with lithium aluminium hydride to give the diol 132 The monoprotection could be achieved using McDougal et al. procedure 300 with sodium hydride and tert butyldimethylsilyl chloride 312 The diol and

PAGE 141

141 TBDMSCl were used in a 1:1 ratio to give the alcohol 133 in 90 % yield. O x idation with activated ma nganese dioxide afforded the aldehyde 121 in 94 % yield. Figure 6 21 Synthesis of the aldehyde 121 The phosphonium salt 122 was prepared from 3 methyl 3 oxetenemethanol ( 134 ) and 3 bromopropionyl chloride ( 135 ) follo wi ng a literature procedure 313 (Figure 6 22 ). Figure 6 22 Synthesis of the phosphonium salt 122 The coupling of the aldehyde 121 and an ylide generated from phosphonium salt 122 with Li HMDS by Wittig reaction gave the olefin product 138 in 93 % yield as mixture of E and Z iso mers in a 1:1 ratio (Figure 6 23 ).

PAGE 142

14 2 Figure 6 23 Synthesis of linear precursors and attempts to cyclize them. The alcohol acid 119 could then be produced from 138 by treatment with methanolic sulfuric acid 314 follo wed by aqueous sodium hydroxide to yield the desired product in 82 % yield and a byproduct acid 142 in 12 % yield. The optimum reaction time for the hydrolysis with sod ium hydroxide was found to be 20 mi n at 0 o C, followed by 15 min at room temperature as at that time most of the substrate was consumed and little byproduct resulting from isomerization to 141 and Michael addition was formed. When the re action time was increased mainly the ether carboxyli c acid 142 was obtained (Figure 6 24 ), and when it was run for shorter periods of time, the yield was much lower (below 50 %) and the byproduct 142 was already present. Figure 6 24 Byproduct 142 formed during prolonged a reaction with sodium hydroxide in water.

PAGE 143

143 The benzylic alcohol 119 was treated with a variety of acids, such as trifluoroacetic acid, polyphosphoric acid, triflic acid, and methanesulfonic acid with phosphorus pentoxide over a range of temperatures, and also with triphenylphosphine and diisopropyl azodicarboxylate ( DIAD ) (Mitsunobu conditions), but no cyclization product ( 120 ) was ever form ed. In these experiments, the starting material was recovered decomposition was observed, or the ester was formed be tween the benzylic alcohol and the acid used to induce cyclization ( Figure 6 25 ) Interestingly, the GC MS EI anal ysis of trifluoroacetate ester 143 gave a spectrum that was identical with the spectrum of th e standard cyclization product 120 suggesting th at the compound can undergo the double cyclization in the GC MS instrument. Figure 6 25 Formation of a cyclic product from the triflic ester 48 in a GC MS instrument. Since none of the chemical reaction s to cyclize ben zylic alcohol carboxylic acid 119 was successful, it was thought that the acidic conditions to form a carbocation were too harsh, or not compatible with other groups in the molecule, and a cyclization of the chloride 140 with silver triflate to induce elim ination of chloride was next explored. Compound 140 was made from 138 by using hydrochloric acid solution in allyl alcohol, followed by conversion of the alcohol function in 139 to a chloride 140 (Figure 6 23 ). The formation of alcohol 139 needed to be cl osely monitored because initial attempts to prepare it using H 2 SO 4 of HCl in allyl alcohol at reflux, yielded only diallylated compound 144 instead of the desired product Finally, the alcohol 139 was

PAGE 144

144 prepared in 79 % yield, using 0.25 HCl in allyl alcohol heated at 50 C for 2.5 h. Chloride 140 and diallylated compound 144 were also formed, but in small amounts and they could be removed by column chromatography ( Figure 6 26 ). Formation of the chloride 140 in this reaction indicates that a benzylic carbocat ion is formed but the cyclization does not proceed. Figure 6 26 Compounds formed in the reaction of the orthoester 138 with an HCl solution of allyl alcohol. Treatment of alcohol 139 with thionyl chloride in pyridine yi elded the chloride 140 in 51 % yield. That result could be improved by use of triphenylphosphine and carbon tetrachloride in dichloromethane (68 % yield), and eventually the best result was obtained with methanesulfonyl chloride, triethylamine and lithium chloride in THF (92 % yield) (Figure 6 23 ) The chloride 140 was treated with silver triflate and 2,6 lutidine at different temperatures, but formation of the cycli zed product was never observed Since Pd(0) salts are known to ionize allylic carboxylates 3 15,316 it was thought that the use of Pd(0) salt would help to effect the cyclization by facilitating the loss of the allyl group, so the chloride was treated with 5 mol % Pd(PPh 3 ) 4 1.1 equivalents of phthalimide, 2.2 equivalents of Na 2 CO 3 and 1.05 equi valents of silver triflate. Unfortunately these cyclization attempts also were unsuccessful.

PAGE 145

145 6.3.3 C ycliz ation of A romatic S ubstrates at the A l dehyde O xidation L evel 2 Since many strigolactones are oxygenated at C4, acid catalyzed cyclization of the aromatic ald ehyde 145 was next studied (Figure 6 27). The cyclization of an aldehyde 145 to form the B ring of strigolactones would be a 5 exo trig cyclization, which is allowed by Baldwin rules, while cyclization of precursors at the alcohol oxidation level was a 5 e ndo trig, disfavored by Baldwin rules. 317,318 Hence, we were hoping that cyclization of the aldehyde precursors would be more feasible under chemical conditions than that of the alcohol 119 or chloride 140 Figure 6 27. Cyclization of an aromatic substrate at the aldehyde oxidation level plan. The cyclization of the first cycle has a precedent in the literature 319 Magnus and Mansley studied the cycli zation of an aromatic aldehyde 147 using different acids, and were ab le to obtain cyclized products with SnCl 4 Sc(OTf) 3 TfOH and TMSOTf (yields up to 64 %). T hey recovered unreacted substrate when Et 2 AlCl, Yb(OTf) 3 or ZnCl 2 were used (Figure 6 28). Figure 6 2 8 P recedent for the first c yclization 2 Adapted from Synthetic Studies on the Solanacol ABC Ring System by Cation Initiated Cascade Cyclization: Implications for Strigolactone Biosynthesis K. Chojnacka, S. Santoro, R. Awartani, N.G.J. Richards, F. Himo, A. Aponick, Org. Biomol. Chem. 2011, 9, 5350 5353, with permission from The Royal Society of Chemistry

PAGE 146

146 The aldehyde methyl ester cyclization precursors were prepared from 138 by treatment with methanolic sulfuric acid, separation of E and Z isomers by flash column chromatography, and oxidation of 153 and 154 independently with pyridin ium chloroc hromate to give 153 and 154 res pectively (Figure 6 29 ). Figure 6 29 Preparation of the linear aldehyde s for cyclization. The double cyclization of 153 and 154 was tested under chemical conditions using both Lewis (trim ethylsilyl triflate, TMSOTf) and Brnsted (triflic acid, TfOH) acids in dichloromethane (DCM) The reaction was monitored by TLC and stopped promptly when the substrate was consumed or when TLC analysis indicated no further substrate consumption for prolon ged times. When E olefin 154 was subjected to cat alytic TMSOTf (0.2 equivalent) the cyclized products 155 and 156 were obtained in an 80:20 diastereomeric ratio in 60 % combined yield, along with 7 % of 157 resulting from a sing le cyclization (Table 6 1 entry 3 ). Interestingly, 155 and 156 were isolated as C4 methyl ethers due to the me thyl transfer from methyl ester. The best result in regard to stereo selectivity and isolated yield was obtained with catalytic amounts of a protic acid. Thus, when 0.1 e quivalent of triflic acid (TfOH) in dichl oromethane was used, 155 and 156 were formed in a 68 %

PAGE 147

147 combined yield in a 99:1 diastereomeric ratio along with 2% of 157 (entry 6 ). When TfOH was used in smaller catalytic amounts (0.05 equivalent) the product 155 and 156 was isolated only in 24 % yield (99:1 diastereomeric ratio), and the unreacted E olefin was recovered in 42 % yield despite prolonged reaction times. When stoichiometric amounts of TMSOTf and TfOH were employed to catalyze the cyclization of 154 t he yields were lower ( 15 % 33 % depending on temperature, and 12 % ) most probably due to decomposition o f the substrate and formation of other multiple byprod ucts using those conditions. S urprisingly the stereo selectivity was reversed (the mixture of 1 55 : 156 in two of those reactions was obtained in a 40:60 ratio) Table 6 1. Cyclization of the E aldehyde precursor. The yields given are isolated yields. The ratio of diastereoisomers was determined by 1 HNMR Entry Condi tions Yield 155 + 156 (Ratio 155 : 156 ) Yield 157 Yield u nreacted substrate 1 TMSOTf (1 .0 eq.) 0 C 10 min; r.t., 3 h 15 % ( 40:60 ) 0 % 0 % 2 TMSOTf (1.0 eq.), 0 C, 2 h 33 % (60:40) 5 % 0 % 3 TMSOTf (0.2 eq.) 0 C, 2 h 60 % (80:20) 7 % 0 % 4 TfOH (1. 5 eq.) 1h; 0 C, 3 h 12 % (40:60) 0 % 0 % 5 TfOH (0.2 eq.) 1h; 0 C, 3 h 60 % 99:1 1 % 1 % 6 TfOH (0.1 eq.) 78 C, 1h; 1h; 0 C, 3 h 68 % (99:1) 2 % 1 % 7 TfOH (0.05 eq.) 78 C, 1h; 78 C to 0 C, 1h; 0 C, 6 h ; 8 C, 14 h 24 % (99:1) 0 % 42 %

PAGE 148

148 The relative stereochemistry of the B ring methoxy group and the C ring was assigned based on the 1 HNMR data for a signal of the proton on C4 carbon and Karplus relationship, as wel l as by a comparison to proton assignments in the literature for a similarly oxygenated strigolactone ABC core whose relative stereochemistry of the B ring OH and the C ring was confirmed unambiguously by X ray 320 In the 1960s, Karplus deduced that the 3 J coupling constant is largest when the dihedral angle between H C C H is 180 as the orbitals of the two C H bonds are in the same plane and perfectly parallel. Coupling is nearly as large at 0 when the orbitals are in the same plane but not parallel. Th e coupling is close to 0 Hz when the dihedral angle is 90 as the orthogonal orbit als do not interact (Figure 6 30 ). 321 Figure 6 30 Graph of the Karplus relationship for ethane derivatives : 3 J HH versus dihedral angle ( Reproduced from http://www2.che mistry.msu.edu/ faculty/reusch/virttxtjml/spectrpy/nmr/nmr2.htm, with permission from Dr. William Reusch MSU ). The structures of lactone methyl ethers 155 and 156 were optimized in Chem 3D Pro 2012 using MM2 (minimize energy, minimum RMS gradient 0.001), and the dihedral angles for the H4 and H3a were measured to be 96.0 for the lactone with trans relationship as shown in 155 cis relationship as shown

PAGE 149

149 in 156 The signal for H4 proton in 155 was a singlet at 4.72 ppm (no coupling with H3a), while the signal for H4 proton in 156 was a doublet at 4.82 ppm, with 3 J H4 H3a = 6.9 Hz) (Figure 6 31 ) Thi s assignment is also in agreement with the 1 H NMR data for the AB C core of orobanchol (H4 at 4.45 ppm as a singlet for a compound with B ring OH and C ring in a trans relationship and H4 at 4.54 ppm as a doublet of doublets and 3 J H4 OH = 7.0 Hz and 3 J H4 H3a = 6.9 Hz for a compound with the B ring OH and the C ring in a cis relationship) 320 Figure 6 31 1 HNMR with proton assignments for 155 (trans ) top spectrum and 156 (cis ) bottom spectrum.

PAGE 150

150 I t was thought initially that the high ratio favoring the B ring methoxy group trans to the C ring might result from equilibration of the product to the most stable diastereomer under reaction conditions. As a control experiment, diastereomers 155 and 156 i n 40:60 ratio were treated with either TMSOTf or TfOH to determine if epimerization of the C4 stereocenter was possible In those reactions, compounds 155 and 156 were recovered in high yields with no detectable interconversion, indicating that there is n o equilibration of the product. The cyclization of the Z olefin 153 appeared to be much slower than that of the E olefin 154 requiring 1 equivalent of catalyst to attain a reasonable rate. When 1 equivalent of trimethylsilyl triflate was used, 153 gave exc lusively the cis diastereomer 156 in 46 % yield, along with 2 % of 157 (Table 6 2, entry 1). When catalytic TMSOTf was used, the reaction was slow, even at room temperature, but a mixture of 155 and 156 in a 30:70 ratio was ob tained in a highe r yield (68 % ) (Table 6 2, entry 4 ). With 1 equivalent of triflic acid, a mixture of 155 and 156 was obtained in a 48 % yield, again favoring 156 in a 13:87 diastereomeric ratio. The use of catalytic triflic acid (0.2 eq.) was not efficient, and it resulted in a furthe r loss of stereoselectivity (43:57), and lower yield of 155 and 156 (20 %) due to incomplete conversion even after several days (24 % of unreacted Z olefin was recovered).

PAGE 151

151 Table 6 2. Cyclization of the Z aldehyde precursor. The yields given are isola ted yields. The ratio of diastereoisomers was determined by 1 HNMR Entry Conditions Yield 155 + 156 (Ratio 155 : 156 ) Yield 157 Yield unreacted substrate 1 TMSOTf (1 eq.) 0 C, 10 min; rt., 6.5 h, 46 % (0:100) 2 % 9 % 2 T MSOTf (1.1 eq.), 0 C, 7 h; 8 C 15 h 53 % (10:90) 3 % 0 % 3 TMSOTf (0.5 eq.), 0 C, 16 h; rt., 3 h 55 % (29:71) 1 % 0 % 4 TMSOTf (0.2 eq.) 0 C, 3 h; rt. 22 h 68 % (30:70) 3 % 1 % 5 78 C, 1h; 0 C, 2.5 h 48 % (13:87) 0 % 0 % 6 h ; 8 C, 96 h 20 % (43:57) 0 % 24 % A probable reaction m echanism is shown in Figure 6 32 In this model, o xocarbenium ion 158 is formed by proto natio n of the aldehyde oxygen. At this stage, the substrate may form both the B and C rings in a single concerted step ( 158 160 ), but a stepwise mechanism is necessary to explain the formation of 157 Cyclization of the olefin in 154 by addition to the C4 oxoc arbenium ion produces the benzylic cation 159 which could further cyclize forming the C ring (path a) or eliminate to form 161 (path b). Demethylation of 160 may occur by alkylation of a variety of nucleophilic species present in solution such as alcohols 161 or 162 Under these catalytic conditions, the methylated tricyclic strigolactone core 155 is thus obtained and the acid catalyst regenerated.

PAGE 152

152 Figure 6 32 Mechanistic hypothesis for the cyclization reaction. DFT cal culations employing the B3LYP functional 322,323 (as implemented in the Gaussian03 software package) 324 were employed to investigate the origin of the stereoselectivity observed in the acid catalyzed cascade cyclization. These studies assumed that formation of the B and C rings proceeded in a stepwise fashion although we have found that cyclization can be concerted based on the calculated barrier to lactone formation. Standard methods 325,326 were used to locate transition states (TS) associated with attack o f the double bond on the protonated aldehyde moiety, which define the relative stereochemistry of the product if the reaction is under kinetic control. Four transition states we re located (Figure 6 31 ) corresponding to those for cyclization of either the t rans olefin 154 or the cis olefin 153 to each of the two possible diastereoisomeric products 155 and 156 The relative energies of these transition states provide a qualitative explanation of the observed cyclization stereoselectivity. Thus, the origin of the reaction selectivity appears to arise from the degree of deviation of the

PAGE 153

153 protonated aldehyde moiety from co planarity with the aromatic ring. In the energetically favored transition states ( TS trans olefin to trans lactone and TS cis olefin to cis lacto ne ) small deviations are observed ( ~ 35). On the other hand, in order to reduce steric hindrance between the protonated aldehyde oxygen and the attacking olefin, the formation of the cis lactone from the trans olefin and of the trans lactone from the cis o lefin requires a higher deviation from co planarity in the TS ( ~ 80) with the aldehyde being almost perpendicular to the aromatic ring. Figure 6 33 Calculated transition states and relative energies for cyclization of the B ring from trans olefin 1 54 and cis olefin 153 initiated by protonation. In order to estimate the energetic contribution of this deviation to the increased activation energy barrier, we calculated the rotational barrier of protonated

PAGE 154

154 benzaldehyde using identical methods to those u sed in locating the cyclization transition st ates (Figure 6 34 ). In this case, the barrier to rotation was found to be quite high (24.2 and 20.8 kcal/mol in gas phase and in dichloromethane, respectively), which is consistent with the differences in the en ergies obtained for the cyclization transition state. Figure 6 34 Calculated rotational barriers for protonated benzaldehyde. The fact that the alcohol 119 and the chloride 140 did not form cyclized products, while aldehydes 153 and 154 easily underwe nt the double cyclization, might be explained by lack of s tabilization of the benzylic carbocation generated from 119 and 140 (the p orbital system of the aromatic ring and the p orbital of the carbocation in the transition state are not in the same plane) while the oxygen long electron pair in 158 can stabilize the transi tion state. Based on this result we suggested that a linear aldehyde, such as 98 (Figure 6 35 ) might be the biosynthetic precursor of the ABC ring system in strigolactones. The C14 alde hyde 163 could be derived from the C18 ketone which has previously been proposed as a biosynthetic intermediate based on experimental evidence 250,251 After

PAGE 155

155 double cyclization, the product 99 would already contain an oxygenation at C4, a nd subsequent oxi dation of the methyl group, followed by attachment of D ring, coul d lead to orobanchol The cyclized product 99 or orobanchol could thus be precursors for other strigolactones after further biosynthetic transformation. Figure 6 35 Proposed biosynthetic pathway. To synthesize a tricyclic lactone with a free hydroxyl group, we subjected aldehyde methyl esters 153 and 154 to trimethylsily l iodide, but that resulted in a very complex mixture in which there was no double cyclization product. W e also tried to obtain the aldehyde free carboxylic acid 164 by methyl cleavage from 153 and 154 but these attempts were unsuccessful as well Treatment of methyl ester 153 with sodium hydroxide resulted in aldol type condensation a nd aromatization to form a naphthalene derivative, while treatment with TMSCl and NaI in acetonitrile at reflux resulted in a complex mixture in which there was no desired product. Also disappointingly the synthesis of aldehyde free carboxylic acid by oxi dation of alcohol carboxylic acid 119 using PCC yielded the desired pr oduct in low yields partially due to poor solubility of carboxylic acid 119 in many organic solvents and the product could not be isolated in a pure form (Fi gure 6 36 ).

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156 Figure 6 3 6 Synthesis of the aldehyde free carboxylic acid 164 Hence, we d ecided to prepare aldehyde benzyl ester 166 and aldehyde allyl ester 170 and cleave t he benzyl or allyl group after the double cyclization as many of removal methods for benzyl and allyl group exist 327,328 The alcohol benzyl ester 165 was obtained by transesterification of the methyl ester 152 in benzyl alcohol in 93 % yield. Oxidation with PCC gave the aldehyde 166 in 87 % yield (Figure 6 37 ). Figure 6 37 Synthesis of the aldehyde benzyl ester 166 The trans olefin aldehyde benzyl ester 166 (containing 4 % of Z isomer impurity), was subjected to the best double cyclization conditions determined for the trans olefin aldehy de methyl ester 153 i.e., 0.1 equivalent of triflic acid. Similarly to the trans olefin aldehyde methyl ester 153 cyclization of the trans olefin aldehyde benzyl ester 166 gave the lactones 167 and 168 in 66 % combined yield, favoring the benzyloxy group and the C ring trans to each other 95:5. The product resulting from single a cyclization ( 169 ) was also is olated in 3 % yield (Figure 6 38 ).

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157 Figure 6 38 Acid catalyzed double cyclization of the trans olefin benzyl este r aldehyde 166 The benzyl oxy lactone 167 was next treated with hydrogen gas over 10 % palladium on carbon to remove the benzyl group. The 1 HNMR spectrum of the crude product was very messy and it indicated that there was no unreacted substrate and no desi red free alcohol lactone, most probably because there are 3 benzylic positions in the lactone 167 that could be cleaved. The allyl ester aldehyde 170 was made by oxidation of the alcohol 139 with PCC in 86 % yield. The acid catalyzed double cyclization gav e the allyloxy lactone as a mixture of 171 and 172 in various yields and ratios depending on the E:Z ratio of the starting olefin 170 used (Figure 6 39 ) The stereoselectivity in this reaction seemed to follow the same trend as in cyclization of methyl and benzyl esters, but it was not investigated because the olefin 170 was a mixture of E and Z isomers. Figure 6 39 Acid catalyzed double cyclization of the allyl ester aldehyde 170 The cleavage of the allyl group from 171 and 172 was first tried with three equivalents of sodium borohydride, 4 mol% dichloro[propane 1,3 diylbis(diphenyl

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158 phosphane) nickel(II) ([NiCl 2 (dppp)] in THF/EtOH 4:1 329 The cleaved products 173 and 174 were obtained in an estimated 33 % combined yield and they were quite impure A much better result was obtained by palladium catalyzed tributyltin hydride reduction 330,331 : the desired product was obtained in 76 % combined yield (Figure 6 40 ). Figure 6 40 Cleavage of the allyl group. 6.3.4 Synthesis of Proposed S tructure for S olanacol (10 5 ) Using Acid catalyzed Double Cyclization as a Key Step to Prepare the C ABC C ore After demonstrating that the acid catalyzed double cyclization of the linear aldehyde in a model system proceeds with good yiel ds and stereocontrol, we set out to show that our cyclization can be used as a key step to synthesize natural st rigolactones, and prepare a proposed structure for solanacol ( 10 5 ) (Figure 6 41 ) That proposed structure had the OH B ring and C ring trans to each other as the 1 HNMR data for natural solanacol indicated and unknown stereoche mistry at C compounds would need to be made to completely prove the structure by comparison to the data for solanacol isolated fro m plants 293 The last steps in the synthesis (formylation and attachment of D ring) could be done by well precedented methods, analogously to those in the synthesis of GR24 (Figure 6 20). The ABC core of 10 5 ( 175 ) could be prepared by our acid catalyzed cy clization from an acid aldehyde 176 In case of problems with that cyclization or the preparation of the free carboxylic acid, an allyl ester 177 could be

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159 used, and the allyl group cleaved as shown in our work earlier. The lactone 175 could be separated in to its enantiomers by chiral column or kinetic resolution, using one of the literature methods. 232,284,286,287,288 The aldehyde acid 176 could be made from its corresponding alkyne 178 by hydrosilylation protodesilylation developed by B. M. Trost and Z. T Ball 332,333 The catalytic hydrosilylation of internal alkynes using a cationic ruthenium complex [Cp*Ru(MeCN) 3 ]PF 6 and a silane (for example (EtO) 3 SiH), followed by protodesilylation with catalytic cuprous iodide and TBAF is a protocol for chemoselecti ve reduction of alkynes to (E) alkenes that is compatible with many sensitive functional groups, for example: ketones, acetals, primary alkyl chlorides, secondary hydroxyl groups that are both benzylic and allylic. Figure 6 41 Retrosynthesis plan for the synthesis of proposed structure for solanacol ( 105 ), using acid catalyzed double cyclization as a key step The alkyne 178 could be prepared by Sonogashira coupling from a triflate 180 and butynoic acid 179 analogously to a nother literature Sonogashira coupling with butynoic acid 334 and the triflate 180 could be prepared from readily available and cheap 2,3 dimethylphenol ( 181 ) ($26 for 100 g).

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160 Therefore 2,3 d imethylphenol (181 ) was transformed into a known aldehyde 1 82 (Figure 6 42 ) The published procedure to make 182 335 consists of four steps: conversion of phenol 181 to an allyl ether, Claisen rearrangement, isomerization of the thus formed terminal double bond in aryl allyl phenol, and cleavage with osmium tetrox ide and sodium metaperiodate. The authors reported that attempts to prepare the desired aldehyde 182 by Reimer Tiemann (CHCl 3 and NaOH) and Vilsmeier Haack (POCl 3 DMF) reactions, as well as formylation with SnCl 4 and paraformaldehyde 336 were not successf ul. We found that the aldehyde 182 could be easily prepared in one step using the Hofslkken and Skattebl monoformylation method 337 That formylation is promoted by electron donating substituents and proceeds selectively ortho to the hydroxyl group. Thus 182 was obtained in 74 % yield by refluxing 2,3 dimethylphenol, paraformaldehyde, magnesium chloride, and triethylamine in acetonitrile for 45 min. The aldehyde 182 was converted into a triflate 180 with triflic anhydride and triethylamine. Figure 6 42 Attempt to synthesize the alkyne aldehyde carboxylic acid 184 Unfortunately, the coupling between the triflate 180 and 3 butynoic acid ( 179 ) 338,339 did not work. Gagnon et al. reported that a Sonogashira coupling with 3 b ut ynoic acid ( 179 ) proceeded smoothly and in good yield 334 but in our system we did

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161 not observe any coupling product neither under Gagnon coupling conditions (Pd(PPh 3 ) 4 CuI, Et 2 NH in THF) nor with a different catalyst (Pd(PPh) 3 Cl 2 ) or different base ( Et 3 N), and the starting 3 b utynoic acid ( 179 ) d egraded completely under reaction conditions. The same result was obtained with 3 b utynoic acid methyl ester ( 183 ). This failure could be explained by the fact that homopropargylic acids and esters rearrange e asily to allenes 340 and can then undergo further undesired reactions or degrade by action of heat. Since the synthesis of 178 by Sonogashira coupling between the triflate 180 and 3 bytynoic acid ( 179 ) were unsuccessful, we decid ed to couple the triflate with 3 butynoic 1 ol ( 185 ) to form compound 186 and oxidize the alcohol to the carboxylic acid in the next steps of the synthesis. Thus, alkyne 186 was obtai ned by Sonogashira reaction in 78 % yield using Pd(PPh 3 ) 4 (3 mol %) CuI (7 mol%) and Et 2 NH (6 eq .) in THF (Figure 6 43 ) When Pd(PPh) 3 Cl 2 CuI in Et 3 N were used, the yields for the coupling reaction were much lower (13 31 %). The aldehyde function in 186 was subsequently protected by an acetal group, and the triple bond in 187 was sele ctively reduced to an E olefin 188 in 95 % yield by use of lithium aluminium hydride (LAH). We also performed that reduction with sodium bis(2 methoxyethoxy)aluminium hydride (Red Al ) in refluxing tetrahydrofuran 341 but the yield was lower than that obtained with LAH ( 65 %). The reduction by Trost Ball silylation desilylation 332,333 did not work; the starting material was recovered. A direct oxidation of the alcohol 188 to the carboxylic acid 190 with sodium chlorite catalyzed by 2,2,6,6 tetramethylpiperidinyl 1 oxy (TE MPO) and bleach 342 resulted in degradation of the substrate and formation of byproducts Oxidation of

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162 alcohol 188 to aldehyde 189 was troublesome at first: Swern oxidation, pyridin ium chlorochromate pyridin ium chlorochromate and ammonium acetate protocol for acid sensitive substrates 343 all met with failure. Finally, we obtained the desired product 189 in 92 % yield after the work up, using Dess Martin periodinane 191 (DMP) 344 The Dess Martin oxidation is a reaction of choice for oxidation of primary alco hols to aldehydes and secondary alcohols to ketones often used in the synthesis of complex molecules because of its high chemosel ec tivity and tolerance of sensitive functional groups thanks to its mild conditions (room temperature slightly acidic or neut ral pH ). 345,346 In addition, organohypervalent iodine reagents are considered to be environmentally benign 346 Figure 6 43 Synthesis of the linear precursor needed for acid catalyzed double cyclization to form the ABC core of solanacol. The Dess Martin periodinane (1,1,1 tris(acetyloxy) 1,1 dihydro 1,2 benziodoxol 3 (1 H ) one) ( 191 ) is commercially available, but it is rather expensive, and it is known that different batches of DMP show different reactivity due to expos ure to moisture 347

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163 Hence DMP was prepared by conversion of 2 iodobenzoic acid ( 192 ) to IBX ( 193 ), using 3 equivalents of oxone (2KHSO 5 KHSO 4 K 2 SO 4 ) 348 followed by acylation with acetic anhydride and 0.5 % p toluenesulfonic acid 345 using literature proce dures (Figure 6 44 ). Figure 6 4 4 Synthesis of Dess Martin Periodinane (DMP) The aldehyde 189 was oxidized to carboxylic acid 190 by Pinnick oxidation 349 which is highly suited for substrates with many functional group s (it does not affect any C=C double bonds present nor alcohols, epoxides, benzyl ethers or halides, and proceeds at constant pH), and is often used in total syntheses of natural products. Therefore the aldehyde 189 was dissolved in t BuOH with a large e xcess (40 equivalents) of 2 methyl 2 butene (hypochlorous acid scavenger) and treated with sodium chlorite in potassium dihydrogen phosphate buffer at room temperature giving the acetal carboxylic acid 190 in 70 % yield. The acetal 190 was treated with T fOH and TMSOTf with intention to form cyclized lactone 175 with the B ring OH and C ring trans to each other (Figure 6 45 ) The 1 HNMR spectra of the crude from each reaction were similar to each other and quite complex (example: Figure 6 46 ) indicating th at at least four cy clization products were formed in different ratios depending on cyclization conditions. In each of those Two spots for the products were visible on TLC, and they could be separate d on chromatography column, however

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164 even after that purification the 1 HNMR spectra were often too complex to interpret. One consistent with the structure 194 (Figure 6 47 ). Figure 6 45 Cyclization of the acetal carboxylic acid 190 The IR spectrum showed that there was no OH group and HRMS ESI was also consistent with structure 194 (found 499.2108, calculated for [C 29 H 32 O 6 Na] + : 499.2093) there was no peak indicating formation of 175 The products degraded during GC MS analysis. Figure 6 46 The 1 HNMR spectrum of the crude from the reaction of 190 with 0.3 equivalent of TfOH: at least 4 cyclized products are formed.

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165 Figure 6 4 7 The 1 HNMR spectrum of two diastereomers of 194 Since the attempt s to obtain lactone 175 by treatment of acetal 190 with triflic acid and trimethylsilyl triflate met with failure, the acetal was removed with catalytic HCl in acetone and water to afford the aldehyde carboxylic acid 176 in 85 % yield (Figure 6 43 ) (no cyclization was observed under these conditions), and then the alde hyde was subjected to TMSOTf or TfOH with an intent to make 175 (Figure 6 48 ) Figure 6 48 Cyclization of the aldehyde carboxylic acid 176 Again, the crude 1 HNMR indicated that there were at least 4 6 products formed separation by column chromatography gave one fraction (A) in w hich 3 compounds were present (one nd two 6 49 ), and one fraction

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166 (B) whose 1 HNMR might be the spectrum of 175 (Figure 6 50 ). However, the IR spectrum of fraction B showed no OH group of alcohol and there was no peak in the HRMS ESI that would confirm the presence of 175 instead HRMS ESI indicated that compound 1 95 was formed (found 441.1691, calculated for [C 26 H 26 O 5 Na] + : 441.1674) The products degraded during GC MS analysis. Figure 6 49 1 HNMR of the fraction A from cyclization of aldehyde carboxylic acid 176 (most probably 3 diasteromers of 195 ). Figure 6 50 1 HNMR of the fraction B from cyclization of aldehyde carboxylic acid 176 (one diastereomer of 195 ).

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167 All reactions to cyclize the acid acetal 190 and acid aldehyde 176 resulted in a formation of complex mixtures of products in 40 70 % yield, depending on exact conditions. The analysis of those products was difficult beca use they could not be separated and some other small impurities were formed too. All results indicated that the desired free alcohol lactone 175 was not formed and that cyclized produ cts were reacting with the protonated 194 or 1 95 Even when dilute concentration of substrates 190 and 176 was used (0.0096 M), only the dimers were detected. We planned to prepare an allyl ester of 176 but initial tries to make it were not successful. However, a n aldehyde methyl ester 1 97 with E olef in geometry could be easily prepared by oxidation of 189 with pyridin ium dichromate (PDC) in the presence of methanol in dimethylformamide in 48 % yield 350 followed by removal of acetal protecting group with hydrochloric acid in acetone and water in 75 % yield (Figure 6 51 ). Figure 6 51 Preparation of the E olefin aldehyde methyl ester 1 97 The E olefin aldehyde methyl ester 1 97 was then subjected to treatment with triflic acid and trim ethylsilyl triflate to form a lactone 1 98 with the B ring methoxy group and the C ring trans to each other (Figure 6 52 ). The 1 H NMR spectrum of the isolated

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168 lactone in small er quantiti es (Figure 6 53 ). This result was very confusing because formation of only two diastereomers was expected: one trans ( 1 98 ) and one cis ( 1 99 ). Figure 6 52 Cyclization of the E olefin aldehyde methyl ester 1 97 Figure 6 53 1 HNMR of the products obtained in cyclization of the E aldehyde methyl ester 1 97 To tease out that cyclization result an aldehyde methyl ester 203 with Z olefin geometry was prepared (Figure 6 54 ). The alkyn e 187 was reduced to Z olefin 200 in 85 % yield with hydrogen gas over Lindlar catalyst in methanol in the presence of quinoline. The Z olefin methyl ester 203 was then prepared analogously to the E isomer by Dess Martin oxidation, treatment with PDC and methanol, and deprotection of acetal.

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169 Figure 6 54 Preparation of the Z olefin aldehyde methyl ester 203 The Z olefin aldehyde methyl ester 203 was then treated with 1 equivalent of triflic acid in dichloromethane to fo rm preferentially the cis lactone 199 (Figure 6 55 ) The crude 1 HNMR from this reactio n was quite complex (Figure 6 56 ), indicating that four cycl ized lactones were formed: 2 cis and 2 trans. Column chromatography allowed separation of one cis lactone ( 1 HN MR Figure 6 57 ), the other three lactones eluted together. GC MS (EI) analysis indicated that all four compounds were giving the same molecular ion [M] + =232 and almost identical fragmentation patterns. Figure 6 55 Cycl ization of the Z olefin aldehyde methyl ester 203

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170 Figure 6 56 1 HNMR of the crude in cyclization of the Z aldehyde methyl ester 203 Figure 6 57 1 HNMR consistent with a structure of 1 99 for med in cyclization of the Z aldehyde methyl ester 203 The nature of the products formed in cyclization of aldehyde methyl ester has not yet been elucidated. It was thought that the lactone in product 1 99 could reopen and

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171 close after a methyl transfer to f orm another lactone 204 (Figure 6 58 ), explaining the formation of two ci s lactone methyl ether products but the same transformation could not occur in the case of the trans lactone 1 98 Figure 6 58 Isomerization of th e cis lactone product. 6.4 Additional N otes In 2012, when I was no longer working on the strigolactone project, a paper in Science appeared showing that D27 catalyzes isomerization of all trans carotene into 9 cis carotene, which is then cleaved with CCD7 to 9 cis apo carotenal CCD8 converts 9 cis apo carotenal into a strigolactone like compound called carlactone. The authors suggested that carlactone could be oxidized and then c onverted to 5 deoxystrigol by double cyclization citing our work 252 (Figure 6 59 ) 6.5 Summary A new method for constructing the ABC ring system of strigolactones in a single step from simple linear precursors by an acid catalyzed double cyclization has been reported. The appropriate linear model precursors at alcohol oxidation level and aldehyde oxidation level were prepared and subjected to the proposed reaction. The alcohol and chloride substrates did not undergo the cyclization. However, aldehyde precurso rs easily underwent the cyclization catalyzed by triflic acid and trimethylsilyl triflate in good yields and with a high degree of stereocontrol. The aldehydic precursors with the E geometry around the double bond gave tricyclic lactones in which the B rin g

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172 alkoxy group and the C ring were trans to each other, whereas the precursors with the Z geometry around the double bond gave tricyclic lactones in which the B ring alkoxy group and the C ring were cis to each other. The stereochemical control of the reac tion was qualitatively rationalized using DFT calculations. The results suggested a new mechanism that might be operative in the strigolactone biosynthetic pathway. The cyclization substrate necessary for the synthesis of solanacol was prepared, but its c yclization gave more products than expected. It would be interesting to elucidate the identity of these additional products. Figure 6 59 Strigolactone biosynthetic pathway proposed in 2012 by Alder et al 252

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173 CHAPTER 7 CONCLU SIONS AND FUTURE WORK targeted to the 7 nAChR, which bind in the traditional agonist binding site and induce D s desensitization without antecedent ion channel opening. The 7 nAChR silent agonists can be used to study the 7 nAChR signaling that does not inv olve ion channel opening and have been suggested to be implicated in cholinergic anti inflammatory pathway responsible for inhibiti on of pro inflammatory cytokine synthesi s. If 7 nAChR silent agonists are established to have anti inflammatory properties, they would be preferred to regular 7 agonists to treat inflammatory disorders without possible side effects linked to the 7 ionotropic activity (the existing data sugge sts a hypothesis that the 7 ion channel activity is required for effects on cognition). 201 Three different groups of silent agonists were characterized that represented distinct groups of chemical structures. The first group features compounds KC 1 and K C 5. Closely related analogs of these molecules, KC 2, KC 3 KC 4, KC 6, KC 8, and KC 9 were characterized as weak silent agonists, while KC 7 appeared to have partial agonist character. The putative pharmacophore for this group was described as a positive ly charged ring, a central ring with hydrogen bonding capability, and a flanking aryl group. However, since small structural changes resulted in significant functional changes that could not be attributed to the structural differences between the molecules further investigation of what constitutes the silent agonist pharmacophore in this group is needed. It would be interesting to test pure enantiomers of chiral KC compounds to see whether both enantiomers have the same activity, different levels, or acti vity associated with only one

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174 antipode. This group of silent agonists presents some challenges for rational drug design, because it appears difficult to make predictions on what molecules would act as 7 agonists, silent agonists, and antagonists, as illustrated by properties of KC compounds and closely related NS6740 (silent agonist) versus NS6784 (agonist). 201 Moreover, the KC compounds did not show good selectivity over other nAChR subtypes: they app eared to bind and in some cases weakly activate 4 2 receptors and t he weak silent agonist KC 8 acted as an agonist for the 3 4 subtype. The second group of silent agonists features bulky quaternary ammonium compounds, such as TEA, (2 HE) diEMA, and dEtP ip. A very good correlation between the molecular volume of the quaternary ammonium compounds and their pharmacological activity was found. The molecules that have a molecular volume of 94 133 3 act as agonists, 142 150 3 act as silent agonists with part ial agonism character, 150 163 3 are silent agonists, and 167 186 3 are weak silent agonists. The s e results suggest that smaller quaternary ammonium compounds may allow conformational changes, such as full closure of C loop onto the agonist in the bindin g site that is then transmitted to the transmembrane domain and cause s ion channel opening, while bigger molecules such as silent agonists do not allow for this full movement of the C loop and instead cause a conformat ional change, described as D s desensi tized and possibly involving the nearby F loop, which might be transmitted to the intracellular domain causing metabotropic signaling. Antagonists appear too large to allow the C loop to close. It would be interesting to further pursue testing of the sel ectivity of quaternary ammonium compounds for different nAChR subtypes, and design new molecules containing the bulky quaternary ammonium that could be used as

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175 therapeutics. Since in this group a very good correlation between the structure of the molecule and it s pharmacological properties was seen, it appears the easiest to optimize bulky ammonium compounds in order to find a therapeutic 7 silent agonist. In addition, the bulky quaternary ammonium compounds presented in this dissertation have a hard positive charge and therefore would be unable to cross the blood brain barrier, which would be desirable to avoid side effects on the CNS (s ilent agonists would be expected to act in non neuronal cells in the PNS, the only exception would be that particular case, compounds of appropriate molecular volume w ith an alkyl group replaced with a hydrogen might be effective). The third group of silent agonists, drawn from the work of Dr. Jingyi Wang, is exemplified by benzylidene anabaseine type molecules such as 3 pyri dinylmethylene anabaseine (3PAB). Fluorinatio n of 3PAB on the pyridine ring that is putatively binding decreased D s desensitization of the receptor. This group poses some challenges for rational drug design because it appe ars difficult to predict the properties of these molecules based on their structure as has been observed with 2PyroAB, 2TAB, 2FAB and 2PAB, though it is not excluded that active 7 silent agonists with good druggable properties are going to be found in thi s group. This work could be continued by a thorough analysis of the 7 homology models with silent agonists docked in, obtaining crystals structures of silent agonists bound to the AChBP, and mutations of the amino acids putatively involved in the interact ions with the silent agonists or the conformational change of the 7 receptor upon silent agonist

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176 binding. Future studies could also include testing of the silent agonists in biological assays to establish their behavior related to the control of pro infla mmatory cytokine production. Furthermore, it would be interesting to clarify whether D s D i or yet another ionotropic signaling. Additionally, several new arylidene quinuclidines were prepared that act ed as (E) 3 (thiophen 2 ylmethylene)quinuclidine [(E) 2 TQN] produced large amount of residual inhibition and desensitization that could be associated with D i desensitization, while the Z isomer of 2 TQN did not. The c ompounds and their analogs should be further tested to establish the interactions on the receptor that lead to this observed difference in behavior for these two geometric isomers. In the second part of this dissertation, it was presented that the strigola ctone ABC core could be formed in one step from suitable aldehydic precursors by an acid catalyzed double cyclization. Model linear precursors were prepared and reaction conditions under which the aldehydes undergo the proposed double cyclization in good y ields and with a high degree of stereocontro l were successfully identified. The molecules at the alcohol oxidation level did not undergo the cyclization reaction. The synthetic studies on the strigolactone ABC ring system bear significance for elucidation of the strigolactone biosynthetic pathway. It would be interesting to test the double cyclization precursors and carlactone with a MAX1 enzyme to find the substrates for that protein

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177 APPENDIX A EXPERIMENTAL PROCEDURES ELECTROPHYSIOLOGY 102,103 All electrophysiolo gy experiments were performed in Dr. Roger Papke Lab (Department of Pharmacology and Therapeutics, College of Medicine, University of Florida) by Clare Stokes, Matthew Kimbrell, Lu Wenchi Corrie, Shehd Abdullah Al Rubaiy, Sara B. Copeland, Matthew D. Isaac son, Thomas F. Pack, Sarah Pinheiro Akshatha Rao Khan A. Manther, and Chrisopher W. Kinter 7 nAChR clones and site directed mutants The human 7 clone was obtained from Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia). The human RIC 3 clone, obtained from Dr. Millet Treinin (Hebrew University, Jerusalem, Israel), was co injected w i th the 7 constructs to improve the levels and speed of receptor expression. Amino acids were numbered as for the human 7 nAChR (vicinal C loop cysteines at positions 190 and 191). Mutations were introduced using the QuickChange Site Directed Mutagenesis instructions. All mutations were confirmed with automated fluorescent sequencing at the University of Florida core facility. After linearization and purification of cloned cDNA, cRNA transcripts were prepared in vitro using the appropriate mMessage mMachine kit from Ambion Inc. (Austin, TX). Expression in Xenopus laevis oocytes . Mature (>9 cm) female X. laevis African frogs (Nasco, Ft. Atkinson, WI) were used as the source of oocytes. Frogs were maintained in the Animal Care Services facility of University of Florida, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Prior to

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178 surgery, frogs were anaesthetized by placing the animal in a 1.5 g/L solution of M S222 (3 aminobenzoic acid ethyl ester; Sigma, St. Louis, MO) for 30 min. Oocytes were removed from an abdominal incision. In order to remove the follicular cell layer, harvested oocytes were treated with 1.25 mg/mL collagenase (Worthington Biochemical Coop calcium (88 mM NaCl, 1 mM KCl, 2.38 mM NaHCO 3 0.82 mM MgSO 4 15 mM HEPES (pH 7.6), 12 mg/L tetracycline). Stage 5 oocytes were isolated and injected with 50 nL (5 20 ng) ea ch of the appropria te cRNAs. Wild type and mutant 7 receptors were routinely co injected with cDNA for human RIC3, an accessory protein that improves and accelerates 7 expression without affecting the pharmacological properties of the receptor. Recordings were made 1 to 10 days after cRNA injection. E lectrophysiolog y Experiments were conducted by two electrode voltage clamp, using OpusXpress 6000A (Molecular Devices, Sunnyvale, CA). OpusExpress is an integrated system that provides automated impalement and voltage clamp of up to eight oocytes in paral lel. Cells were automatically bath mM NaCl, 10 mM HEPES, 2.5 mM KCl, and 1.8 mM CaCl 2 pH 7 to block endogenous muscarinic receptors). Both the voltage and current electrodes were filled with 3 M KCl. Cells were voltage Data were collected at 50 Hz and filtered at 20 Hz. Perfusion fl ow rates were set at 2 mL/min for experiments with 7 receptor and 4 mL/min for other subtypes. Drugs solutions were delivered from a 96 well plate via disposable tips. Drug applications alternated between ACh controls and experimental ligands at varying c oncentrations.

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179 Unless otherwise indicated, drug applications were 12 s in duration, followed by 181 s washout periods with 7 receptors and 6 s with 241 s washout periods for other subtypes. Experimental protocols and data analysis Data were analyzed by C lampfit 9.2 (Molecular Devices) and Excel (Microsoft, Redmond WA), and no rmalized to the averaged responses of t he acetylcholine pre controls 351 Data were expressed as means SEM from at least four oocytes for each experiment. For the concentration res ponse curve s, responses were normalized to the net charge ( 7) or peak current ( 4 2) of the most adjacent prior control. Data were plotted by Kaleidagraph 3.0.2 (Abelbeck Software, Reading, PA), and curves were generated as the best fit of the average values from the Hill equation.

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180 APPENDIX B COMPUTATIONAL METHODS Strig olactones These calculations have been done by Dr. Stefano Santoro in Dr. Fahmi Himo Lab (Department of Organic Chemistry, Stockholm University, Stockholm, Sweden). Geometry optimizations of protonated aldehydes, transition states and cyclized structures employed the 6 31G( d,p ) basis set for all atoms. Single point energy calculations were then performed for each of these optimized structures with the 6 311+G( 2d,2p ) basis set Solvation effects were taken into account by performing single point calculatio ns on the optimized structures using the conductorlike polarizable contiunuum model (CPCM) method with the UAKS radii The parameters for CH 2 Cl 2 were used for all solvation calculations. All stationary point structure s were confirmed either as minima (no i maginary frequencies) or transition states (only one imaginary frequency) by analytical frequency calculations at the same theory level as the geometry optimizations All energies reported for the transition states have been corrected for solvation and zer o point vibrational effec ts.

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181 APPENDIX C ALTERNATIVE PHARMACOPHORE For rationale of the project see Jingyi Wang dissertation. 222 Structures of 4 OH GTS 21, BHP, and PPP Synthesis of PPP Retrosynthetic analysis Attempts to synthesize PPP 211a

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182 PyPyr Sythesis The free base of PyPyr ( 217 ) was synthesized following a literature scheme 352 by Suzuki coupling as a key step N (Boc) pyrrole 2 boronic acid ( 213 ) was prepare d in 74 % yield using a patent procedure 353 by treating N (Boc) pyrrole ( 212 ) with LDA, then trimethyl borate, and followed by an acidic work up. The coupling product 215 was obtained in 66 % yield by heating at reflux a mixture of N (Boc) pyrrole 2 boron ic acid ( 213 ), 3 bromopyridine ( 214 ), tetrakis (triphenylphosphine)palladium (4 mol%) in 1,2 dimethoxyethane, with an excess of aqueous sodium carbonate as a base. In that reaction, three other byproducts were observed on a TLC plate, one of them was iden tified before as a homodimer of 213 resulting from deboronation and self coupling of N (Boc) pyrrole 2 boronic acid ( 213 ). 352 The Boc group was easily removed from 215 by treatment with an excess of sodium methoxide in me thanol at room temperature. The free base of PyPyr ( 216 ) was then

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183 quantitatively converted into its hydrochloride salt by treatment with hydrochloric acid in ethanol. PyPyr activity Test Receptor Control (C) Peak average Area average h 7 0.0021 0.0005 0.014 0.009 h 0.0036 0.0009 0.0010 0.0006 h 7W55G 0.047 0.007 0.055 0 .007 h 7Y188F GTS 21 0.06 0.03 0.05 0.05 yr h 0.007 0.001 0.0002 0.0006 h 0.10 0.01 0.05 0.02 h 7 1. 00 0.08 0.99 0.02 h 0.76 0.04 0.74 0.04 h 7W55G 3 1.31 0.09 1.3 0.1 h 7Y188F GTS 21 0.91 0.08 0.89 0.03 h 0.82 0.01 0.81 0.04 h 0.79 0.03 0.89 0.12 12 0596 h 7 0.011 0.003 0.011 0.004

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184 APPENDIX D SYNTHETIC EXPERIMENT AL PROCEDURES A.1 General: All reactions requiring anhydrous or oxygen free conditions were carried out under an atmosphere of argon in oven dried glassware. Anhydrous solvents were purchas ed from Sigma Aldrich in sure seal bottles and used as received. Thin layer chromatography was performed using 250 m Silica Gel 60 F 254 pre coated plates (Whatman) and the plates were visualized with UV or permanganate stain. Flash column chromatography w as performed using 230 400 Mesh 60A Silica Gel (Whatman). Proton nuclear magnetic resonance ( 1 H NMR) and carbon 13 nuclear magnetic resonance ( 13 C NMR) spectra were recorded in deuterated chloroform, CDCl 3 at the frequency indicated Chemical shifts ( ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS, 0.0 ppm) or CDCl 3 (7.27 ppm in 1 H NMR and 77.0 ppm in 13 C NMR). Multiplicities are reported using the following abbreviations: s, singlet; d d oublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; m, multiplet; br, broad. Infrared spectra were obtained on a Bruker Vector 22 IR spectrometer and are reported in wavenumbers. High resolution mass spectra (HRMS) were obtained by Mass Spectrometry Core Labora tory of University of Florida. A.2 Silent agonists KC 1 story 3 bromo 5 phenylpyridine (10): 206 (Triphenylphosphine)palladium tetrakis (125 mg, 0.08 mmol, 2 mol %) was added to a solution of 3,5 dibromopyridine (1.00 g, 4.22 mmol) in dimethoxyethane (13 mL) and

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185 the mixture was stirred for 10 min. A solution of potassium carbonate (1.75 g, 12.7 mmol) in water (6.5 mL) was added, followed by phenyl boronic acid (463 mg, 3.79 mmol), and the mixture was heated at reflux for 4 h A solution of 1M NaOH (6 mL) was added to the cooled mixture, the mixture was extracted with diethyl ether, the organic extracts were combined, dried over MgSO 4 filtered, and the solvents were evaporated. The crude was purified by column chromatography using dichloromethane as an eluent to give 608 mg of the product (68 % yield) as a white solid. 1 H NMR (CDCl 3 300 MHz): 8.76 (d, 1H, 2.0 Hz); 8.66 (d, 1H, 2.2 Hz); 8.03 (t, 1H, 2.2 Hz, 2.0 Hz); 7.43 7.59 (m, 5H). 13 CNMR (CDCl 3 75 MHz): 149.3, 146.3 138.2, 136.8, 136 .2, 129.1, 128.6, 127.1, 120.8. tert butyl (5 oxo 5 (5 phenylpyridin 3 yl)pentyl)carbamate (12 a ) : A solution of 3 bromo 5 phenylpyridine (10) (975 mg, 4.16 mmol) in diethyl ether (10 mL) was added dr opwise over 10 min to a solution of n BuLi (1.4 M in hexanes, 2.97 for 20 min. A solution of Boc protected valerolactam 11) (829 mg, 4.16 mmol) in diethyl ether (8 mL) was added and the mixture was furthe at 0 C for 5 min. The reaction was quenched with 2M HCl to pH 1 2, and the mixture was extracted with diethyl ether. The combined organic layers were washed twice with 10 % aqueous solution of sodium bicarbonate, dried over Na 2 SO 4 filtered, and the solvents were evaporated. The crude was purified by column chromatography using a gradient of hexanes/ethyl acetate 10:1 to 3:1as eluent to yield the product as

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186 yellowish solid (900 mg, 61 %). 1 H NMR (CDCl 3 300 MHz): 9.13 (d 1H, 2.2 Hz); 9.01 (d, 1H, 2.2 Hz); 8.42 (t, 1H, 2.2 Hz); 7.60 7.64 (m, 2H); 7.42 7.54 (m, 3H); 4.68 (br s, 1H); 3.16 3.22 (m, 2H); 3.09 (t, 2H, 7.0 Hz); 1.78 1.88 (m, 2H); 1.56 1.66 (m, 2H); 1.44 (s, 9H) 13 CNMR (CDCl 3 75 MHz): 198.6, 155.9, 151.6, 147.8, 136.7, 136.5, 133.4, 131.9, 129.1, 128.5, 127.1, 79.0, 40.1, 38.4, 29.5, 28.3, 20.8. IR (neat): 3369, 1685, 1516, 1248, 1162 cm 1 HRMS (ESI): [M+H] + calculated:355.2016, found: 355.2007; [M+Na] + calculated: 377.1836, found: 377.1835 3,4,5,6 tetrahydro bipyridine (KC 1): The ketone carbamate (12) (105 mg, 0.30 mmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (1 mL) was added at 0 C, the mixture was stirred at 0 10 C for 2 h and then quenched with 5 M NaOH (toward the end of addition 1 M NaOH was added to pH 12 13). The mixture was extracted with diethyl ether, dried over Na 2 SO 4 filtered, and the solvents were evaporated. The crude was purified by column chromatography usi ng a gradient of dichloromethane and methanol 100:1 to 60:1 to give the product as yellowish oil (56 mg, 80 % yield). 1 H NMR (CDCl 3 300 MHz): 8.92 (s, 1H); 8.86 (s, 1H); 8.31 (t, 1H, 2.1 Hz); 7.63 (dt, 2H, 7.0 Hz, 1.5 Hz); 7.48 (tt, 2H, 7.0 Hz, 1.5 Hz) ; 7.41 (tt, 1H, 7.0 Hz, 1.5 Hz); 3.86 3.91 (m, 2H); 2.69 (tt, 2H, 6.5 Hz, 2.2 Hz); 1.85 1.93 (m, 2H), 1.68 1.76 (m, 2H) 13 CNMR (CDCl 3 125 MHz): 163.5, 148.8, 146.1, 137.5, 136.1, 135.2, 131.7, 128.9, 128.1, 127.2, 50.1, 27.1, 21.7, 19.5. IR

PAGE 187

187 (neat) : 2930, 2855, 1636 cm 1 HRMS (ESI): [M+H] + calculated: 237.1386, found: 237.1395; [M+Na] + calculated: 259.1206, found: 259.1207 3 phenyl 5 (piperidin 2 yl)pyridine (KC 2): The ketone carbamate ( 12 a ) (550 mg, 1.55 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (6 mL) was added at 0 C, the mixtur e was stirred at 0 10 C for 2.5 h and then quenched with 5 M NaOH (toward the end of addition 1 M NaOH was added to pH 12 13). The mixture was extr acted wi th dichloromethane dried over Na 2 SO 4 filtered, and the solvents were evaporated. The crude cyclic imine was dissolved in methanol (10 mL) and water (1 mL), sodium borohydride (70 mg, 1.85 mmol) was added at 0C, and the reaction mixture was stirred at 0 15 C for 3 h. The reaction was quenched by addition of 1M HCl to pH 1 at 0 C, the mixture was stirred for 30 min, and then 1M aqueous solution of NaOH was added at 0 C to pH 13. The mixture was extr acted with dichloromethane dried over Na 2 SO 4 filtere d, and the solvents were evaporated. The crude product was purified by column chromatography using a gradient of dichloromethane and methanol 40:1 to 20 :1 to give the product as yellowish oil (221 mg, 60 % yield). 1 H NMR (CDCl 3 3 00 MHz): 8.74 (dd, 1H, 2.2 Hz, 0.7 Hz); 8.57 (d, 1H, 2.1 Hz); 7.98 (td, 1H, 2.1 Hz, 0.7 Hz); 7.63 7.59 (m, 2H); 7.50 7.44 (m, 2H); 7.43 7.37 (m, 1H); 3.75 (dd, 1H, 10.9 Hz, 2.3 Hz); 3.22 (d, 1H, 11.3 Hz); 2.92 (br s, 1H); 2.83 (td, 1H, 11.5 Hz, 3.0 Hz); 1.95 1.85 (m, 2H); 1.70 1.49 (m, 4H). 13 CNMR (CDCl 3 7 5 MHz): 147.1, 146.9, 139.6, 137.5, 136.2, 132.5, 128.8, 127.8,

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188 126.9, 59.4, 47.1, 34.2, 25.0, 24.8. IR (neat) : 3272, 3036, 2930, 2851, 2787, 1439, 1326, 1302, 1107, 1025, 888 cm 1 HRMS (ESI ): [M+H] + cal culated: 239.1543 found: 239.1552; [M+Na ] + calculated: 261.1362 found: 261.1363. 3 (1 methylpiperidin 2 yl) 5 phenylpyridine (KC 3): KC 2 (139 mg, 0.58 mmol) in formic acid (0.5 mL) and formalin (0.32 mL) were heated a t 90 C for 3 h. The reaction mixture was cooled to 0 C and 2M K 2 CO 3 was added to pH 12. The mixture was extracted with dichloromethane and dried over MgSO 4 The crude product was purified by column chromatography using a gradient of dichloromethane and methanol 50:1 to 30 :1 to give the product as colorless oil (92 mg mg, 62 % yield). 1 H NMR (CDCl 3 3 00 MHz): 8.73 (d, 1H, 2.1 Hz); 8.49 (d, 1H, 1.9 Hz); 7.89 (t, 1H, 2.1 Hz, 1.9 Hz); 7.60 (dt, 2H, 6.9 Hz, 2.1 Hz); 7.45 (t, 2H, 7.1 Hz); 7.40 7.34 (m, 1H) ; 3.04 (d, 1H, 11.5 Hz); 2.88 (dd, 1H, 11.1 Hz); 2.18 2.09 (m, 1H); 2.03 (s, 3H); 1.84 1.54 (m, 5H); 1.45 1.33 (m, 1H). 13 CNMR (CDCl 3 7 5 MHz): 148.0, 147.0, 140.0, 137.7, 136.5, 133.0, 128.9, 127.9, 127.1, 68.2, 57.3, 44.5, 36.0, 25.9, 24.7. IR (neat) : 2933, 2853, 2779, 1441, 1272, 1116, 1025, 894 cm 1 HRMS (ESI ): [M+H] + calculated: 253.1699 found: 253.1706; [M+Na ] + calculated: 275.1519 found: 275.1519.

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189 Tert butyl (5 (isoquinoli n 4 yl) 5 oxopentyl)carbamate ( 12b): The compound was prepared from 4 bromoisoquinoline ( 10b ) analogously to ( 12a ). Yield: 43 %, lightly yellow solid. 1 H NMR (CDCl 3 3 00 MHz): 9.32 (s, 1H); 8.95 (s, 1H); 8.68 (d, 1H, 8.5 Hz); 7.99 (d, 1H, 8.0 Hz); 7.80 (t, 1H, 8.0 Hz); 7.65 (t, 1H, 8 .0 Hz); 4.77 (br s, 1H); 3.18 3.13 (m, 2H); 3.10 (t, 2H, 7.3Hz); 1.87 1.77 (m, 2H); 1.64 1.55 (m, 2H); 1.41 (s, 9H). 13 CNMR (CDCl 3 7 5 MHz): 202.3, 156.2, 144.2, 132.7, 132.5, 128.6, 128.3, 128.1, 127.8, 125.0, 79.0, 41.2, 40.1, 29.5, 28.3, 21.4. IR (n eat): 3383, 1681, 1499, 1248, 1169 cm 1 HRMS (ESI ): [M+H] + calculated: 329.1860 found: 329.1863; [M+Na ] + calculated: 351.1679 found: 351.1684. 4 (3,4,5,6 tetrahydropyridin 2 yl)isoquinoline (KC 4): 210 The compound was prepared from 12b analogously to KC 1. Yield: 57 %, yellow oil. 1 H NMR (CDCl 3 3 00 MHz): 9.19 (s, 1H); 8.50 (s, 1H); 8.21 (d, 1H, 8.4 Hz); 7.95 (d, 1H, 8.0 Hz); 7.69 (t, 1H, 7.8 Hz, 7.4 Hz); 7.58 (t, 1H, 8.0 Hz, 7.1 Hz); 3.95 3.91 (m, 2H); 2.62 2. 58 (m, 2H); 1.94 1.86 (m, 2H); 1.81 1.74 (m, 2H). 1 H NMR was similar to that reported in the literature. 210 13 CNMR (CDCl 3 7 5 MHz): 166.7, 152.8, 141.0, 133.0, 132.8, 130.8, 128.5, 127.8, 127.1, 124.5, 50.0, 31.2, 21.7, 19.7. IR (neat): 2931, 2854, 1 639, 1499, 1348 cm 1 HRMS (DART ): [M+H] + calculated: 211.1230 found: 211.1222.

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190 4 (piperidin 2 yl)isoquinoline (KC 5): The compound was prepared from 12b analogously to KC 2. Yield: 85 %, lightly yellow oil. 1 H NMR (C DCl 3 3 00 MHz): 9.15 (s, 1H); 8.69 (s, 1H); 8.24 (d, 1H, 8.6 Hz); 7.97 (d, 1H, 8.2 Hz); 7.75 7.70 (m, 1H); 7.59 (t, 1H, 7.6 Hz); 4.31 (d, 1H, 11.3 Hz); 3.31 (d, 1H, 11.3 Hz); 2.96 2.88 (m, 1H); 2.37 (br s, 1H); 2.00 1.97 (m, 2H); 1.82 1.58 (4H). 13 CNMR (CDCl 3 7 5 MHz): 151.8, 140.3, 133.3, 129.9, 128.1, 126.4, 122.4, 56.8, 47.8, 33.5, 25.7, 25.3. IR (neat): 3270, 1105, 895, 857, 848, 786, 745 cm 1 HRMS (ESI ): [M+H] + calculated: 213.1386 found: 213.1394; [M+Na ] + calculated: 235.1206 found: 235.12 08. 4 (1 methylpiperidin 2 yl)isoquinoline (KC 6): 210 The compound was prepared from KC 5 analogously to KC 3. Yield: 65 %, colorless oil. 1 H NMR (DMSO d 6 5 00 MHz 85 C ): 9.16 (s, 1H); 8.69 (d, 1H, 8.7 Hz); 8.49 (s, 1H) ; 8.08 (d, 1H, 8.2 Hz); 7.75 (td, 1H, 8.5 Hz, 7.5 Hz, 1.4 Hz); 7.65 (t, 1H, 7.6 Hz); 3.47 (d, 1H, 11.4 Hz); 3.07 (dt, 1H, 12.0 Hz, 3.3 Hz); 2.20 2.14 (m, 1H); 1.95 (s, 3H); 1.88 1.68 (m, 5H); 1.50 1.42 (m, 1H). 13 CNMR ( DMSO d 6 125 MHz 85 C ): 152 .1, 142.6, 133.9, 133.4, 130.3, 128.7, 128.6, 127.2, 123.9, 66.9, 57.6, 44.4, 34.6, 26.2, 25.1. IR (neat): 2934, 2851, 2773, 1620, 1580, 1443, 1371, 1115, 1026, 898, 787 cm 1

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191 HRMS (ESI ): [M+H] + calculated: 227.1543 found: 227.1543; [M+Na ] + calculated : 249.1362 found: 249.1354. Tert butyl (5 oxo 5 (q uinolin 3 yl)pentyl)carbamate (12c ): The compound was prepared from 3 bromoquinoline analogously to ( 12a ). Yield: 42 %, lightly yellow solid. 1 H NMR (CDCl 3 3 00 MHz): 9.43 (d, 1H, 2.3 Hz); 8.73 (d, 1H, 2.1 Hz); 8.17 (d, 1H, 8.2 Hz); 7.96 (d, 1H, 8.2 Hz); 7.85 (ddd, 1H, 8.6 Hz, 7.8 Hz, 1.3 Hz); 7.64 (ddd, 1H, 8.6 Hz, 7.6 Hz, 1.1 Hz); 4.64 (br s, 1H); 3.24 3.18 (m, 2H); 3.15 (t, 2H, 7.1 Hz); 1.91 1.81 (m, 2H); 1.69 1.59 (m, 2H); 1.45 (s, 9H) 13 CNMR (CDCl 3 7 5 MHz): 198.7, 156.0, 149.7, 148.9, 136.9, 131.9, 129.3, 129.3, 129.0, 127.5, 126.8, 79.1, 40.1, 38.2, 29.5, 28.3, 20.9 IR (neat): 3383, 1685, 1525, 1365, 1246, 1164, 1001, 793, 759 c m 1 HRMS (ESI ): [M+ Na ] + calculated: 351.1679 found: 351.1669. 3 (3,4,5,6 tetrahydropyridin 2 yl)quinoline (KC 7): The compound was prepared from 12c analogously to KC 1. Yield: 81 %, lightly yellow solid. 1 H NMR (CDCl 3 3 00 MHz): 9.38 (d, 1H, 2.1 Hz); 8.35 (d, 1H, 2.1 Hz); 8.09 (d, 1H, 8. 6 Hz); 7.87 7.84 (m, 1H); 7.69 (ddd, 1H, 8.6 Hz, 6.9 Hz, 1.5 Hz); 7.51 (ddd, 1H, 8.4 Hz, 6.8 Hz, 1.0 Hz); 3.90 3.86 (m, 2H); 2.71 2.65 (m, 2H); 1.90 1.82 (m, 2H); 1.72 1.65 (m, 2H). 13 CNMR (CDCl 3 7 5 MHz): 163.5, 148.8, 148.3, 132.8, 132.2, 129.8,

PAGE 192

192 129. 1, 128.4, 127.3, 126.7, 50.0, 26.8, 21.7, 19.5. IR (neat): 3052, 2950, 2916, 2848, 1630, 1615, 1570, 1489, 1329, 1122 cm 1 HRMS (DART ): [M+H] + calculated: 211.1230 found: 211.1220. 3 (piperidin 2 yl)quinoline (KC 8 ): The compound was prepared from 12c analogously to KC 2. Yield: 79 %, lightly yellow solid. 1 H NMR (CDCl 3 3 00 MHz): 8.79 (d, 1H, 1.9 Hz); 8.01 7.99 (m, 2H); 7.67 (d, 1H, 8.0 Hz); 7.56 (t, 1H, 7.4 Hz); 7.41 (t, 1H, 7.4 Hz); 3.66 (d, 1H, 8.4 Hz); 3.13 (d, 1H, 11.5 Hz); 2.76 2.68 (m, 1H); 1.95 (br s, 1H); 1.83 1.75 (m, 2H); 1.60 1.42 (m, 4H). 13 CNMR (CDCl 3 7 5 MHz): 150.4, 147.3, 137.8, 132.4, 128.8, 128.6, 127.8, 127.5, 126.3, 59.5, 47.4, 34.8, 25.4, 25.0. IR (neat): 3303, 2927, 2847, 2789, 1494, 1441, 1317, 1123, 788 cm 1 HRMS (ESI ): [M+H] + calculated: 213.1386 found: 213.1395; [M+Na ] + calculated: 235.1206 found: 235.1206 3 (1 methylpiperidin 2 yl)quinoline (KC 9): The compound was prepared from KC 8 analo gously to KC 3. Yield: 71 %, lightly yellow oil. 1 H NMR (CDCl 3 3 00 MHz): 8.87 (s, 1H); 8.09 (s, 1H); 8.05 (d, 1H, 4.2 Hz); 7.75 (d, 1H, 8.0 Hz); 7.63 (t, 1H, 8.0 Hz, 7.0 Hz); 7.48 (t, 1H, 7.8 Hz, 7.0 Hz); 3.04 (d, 1H, 11.2 Hz); 2.96 (dd, 1H, 11.0 Hz, 2 .4 Hz); 2.17 2.08 (m, 1H); 2.00 (s, 3H); 1.83 1.57 (m, 5H); 1.43 1.32 (m, 1H) 13 CNMR (CDCl 3 7 5 MHz): 150.9, 147.6, 137.3, 133.7, 129.0,

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193 128.8, 128.0, 127.5, 126.4, 68.4, 57.3, 44.5, 36.0, 25.9, 24.7. IR (neat): 2934, 2852, 2779, 1495, 1321, 1116, 1 028, 908, 861, 787 cm 1 HRMS (DART ): [M+H] + calculated: 227.1543 found: 227.1538. A.3 Silent agonist bulky quaternary ammonium The quaternary ammonium salts were prepared by reacting commercially available methyl or ethyl amines with methyl iodide or ethyl iodide in tetrahydrofuran or ethanol and purified by recrystallization. Methylhexahydroazepine ( 39 ) was made from hexahydroazepine by the Eschweiler Clarke reaction 211,212 (formalin and formic acid), and ethylhexahydroazapine ( 40 ) was prepared from hexahyd roazepine using potassium carbonate and ethyl iodide. (2 H ydroxyethyl) ethyldimethylammonium iodide [(2 HE) EdiMA] ( 23 ) : From N,N dimethylethanolamine and ethyl iodide. White hygroscopic solid. 1 H NMR (D 2 O 3 00 MHz): 4.05 4.00 (m, 2H); 3.49 3.42 (m, 4H); 3.10 (s, 6H); 1.35 (tt, 3H, 7.3 Hz, 2.0 Hz). 13 CNMR (D 2 O, 12 5 MHz): 64.5, 61.1, 55.4, 50.9, 7.8. HRMS (ESI): ] calculated: 118.1226 found: 118.1232. (2 Hydroxyethyl) diet hylmethyl ammonium iodide [(2 HE) diEMA] (24 ) : From N,N diethylethanolamine and methyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 4.03 3.98 (m, 2H); 3.45 3.37 (m, 6H); 3.02 (s, 3H); 1.31 (tt, 6H, 7.3 Hz, 1.8 Hz).

PAGE 194

194 13 CNMR (D 2 O, 7 5 MHz): 61.6, 57.5, 55 .3, 48.1, 7.8. ] calculated: 132.1390 found: 132.1390. (2 Hydroxyethyl) triethyl ammonium iodide [(2 HE) triE A] (25 ) : From N,N diethylethanolamine and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 4.00 3.94 (m, 2H); 3.39 3.32 (m, 8H); 1.27 (tt, 9H, 7.2 Hz, 1.5 Hz). 13 CNMR (D 2 O, 7 5 MHz): 57.6, 54.9, 53.6, 7.1. ] calculated: 146.1539 found: 146.1544. Benzyltr imethylammonium iodide [BtMA] (26 ) : From N,N dimethylbenzylamine and methyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 7.54 (m, 5H); 4.47 (s, 2H); 3.07 (s, 9H). 13 CNMR (D 2 O, 12 5 MHz): 135.4, 133.3, 131.7, 129.9, 72.1, 55.0. ] calculated: 128.1434, found: 128.1438. Benzyl ethyl dimethylammonium iodide [B E d MA] (27 ): From N,N dimethylbenzylamine and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 7.52 (m, 5H); 4.44 (s, 2H); 3.36 (q, 2H, 7.2 Hz); 2.96 (s, 6H); 1.40 (t, 3H, 7.2 Hz)

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195 13 CNMR (D 2 O, 12 5 MHz): 132.9, 130.7, 129.2, 127.1, 67.3, 60.0, 49.1, 7.8. HRMS ] calculated: 150.1277 found: 150.1275. Benzyldi ethyl methylammonium iodide [Bd EMA] (28 ): From N ethyl N methylbenzaylamine and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 7.53 (m, 5H); 4.43 (s, 2H); 3.41 3.21 (m, 4H); 2.88 (s, 3H); 1.38 (t, 6H). 13 CNMR (D 2 O, 12 5 MHz): 132.9, 130.6, 129.1, 127.1, 64.5, 55.7, 46.4, 7.4. HRMS ] calculated: 178.1590 foun d: 178.1591. Dimethylp yrrolidinium iodide [dMePyrr] (30 ): From N methylpyrrolidine and methyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 3.47 (t, 4H, 6.9 Hz); 3.09 (s, 6H); 2.19 (br s, 4H). 13 CNMR (D 2 O, 12 5 MHz): 66.0 (t, 2.6 Hz); 51.9 (t, 4.3 Hz); 21.8. ] calculated: 100.1121 found: 100.1126. Ethylmethylpyrrolidinium iodide [Et MePyrr] (31 ): From N methylpyrrolidine and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 3.45 3.41 (m, 4H); 3.35 (q, 2H, 7.3 Hz); 2.97 (s, 3H); 2.17 2.14 (m, 4H); 1.32 (tt, 3H, 7.3

PAGE 196

196 Hz, 2.1 Hz). 13 CNMR (D 2 O, 12 5 MHz): 66.5, 62.2, 50.3 (t, 3.5 Hz); 24.0, 11.3. HRMS ] calculated: 114.1277 found: 114.1282. Diethylp yrrolidinium iodide [dEtPyrr] (32 ) : From N ethylpyrrolidine and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 3.43 (t, 4H, 6.8 Hz); 3.28 (q, 4H, 7.3 Hz); 2.13 2.09 (m, 4H); 1.26 (tt, 6H, 7.2 Hz, 1.9 Hz). 13 CNMR (D 2 O, 12 5 MHz): 64.4 (t, 3.5 Hz); 56.9, 24.1, 10.7. ] calculated: 128.1434 found: 128.1438. Dimethylpiperidinium iodide [dMePip ] (33 ): From N methylpiperidine and methyl iodide. White solid. 1 H NMR ( D 2 O 3 00 MHz): 3.29 (t, 4H, 5.9 Hz); 3.05 (s, 6H); 1.83 (br s, 4H); 1.60 (quintet, 2H, 5.9 Hz). 13 CNMR (D 2 O, 12 5 MHz): 65.5, 54.3, 23.0, 22.5. ] calculated: 114.1277 found: 114.1283. Ethylmethylpi peridinium iodide [EtMePip ] (34 ): From N methylpiperidine and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 3.39 (qd, 2H, 7.2 Hz, 1.0 Hz); 3.29 (t, 4H, 5.4 Hz); 2.98 (s, 3H); 1.86 (br s, 4H); 1.71

PAGE 197

197 1.57 (m, 2H); 1.31 (td, 3H, 7.2 Hz, 1.8 Hz). 13 CNM R (D 2 O, 12 5 MHz): 63.2, 61.5, 49.8, 23.2, 22.2, 9.5. ] calculated: 128.1434 found: 128.1437. Diethylpiperidinium iodide [dEtPip ] ( 35 ): From N ethylpiperidine and ethyl iodide. White solid. 1 H NMR ( D 2 O 3 00 MHz): 3.34 (q, 4H, 7.2 Hz); 3.29 (t, 4H, 5.7 Hz); 1.84 (br s, 4H); 1.65 (quintet, 2H, 5.9 Hz); 1.25 (td, 6H, 7.2 Hz, 1.6 Hz). 13 CNMR (D 2 O, 12 5 MHz): 58.3 (t, 1.9 Hz); 53.1, 20.8, 19.2, 6.5. ] calculated: 142.1590 found: 142. 1592. N Methylhexahydroazepine (39): From homopiperidine, formaline, and formic acid. Colorless oil. 1 H NMR (CDCl 3 5 00 MHz): 2.50 2.47 (m, 4H); 2.28 (s, 3H); 1.62 1.60 (m, 4H); 1.56 1.54 (m, 4H). 13 CNMR (D 2 O, 12 5 MH z): 58.4, 47.2, 27.8, 26.6. Dimeth ylhexahydroazepinium [diMHHA] (36 ): From N m ethylhexahydroazepine ( 39 ) and methyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 3.43 3.41 (m, 4H); 3.07 (s, 6H); 1.86 (m, 4H); 1.69 1. 66 (m, 4H). 13 CNMR

PAGE 198

198 (D 2 O, 12 5 MHz): 68.9, 56.4, 29.6, 24.2. ] calculated: 128.1434 found: 128.1440. Ethylmethylhexahydroazepinium [E MHHA] (37 ): From N m ethylhexahydroazepine ( 39 ) and ethyl iodide. W hite solid. 1 H NMR (D 2 O 3 00 MHz): 3.44 3.31 (m, 6H); 2.95 (s, 3H); 1.85 (br s, 4H); 1.67 1.65 (m, 4H); 1.32 (tt, 3H, 7.3 Hz, 1.9 Hz). 13 CNMR (D 2 O, 12 5 MHz): 66.4, 62.9, 52.0, 29.8, 23.8, 10.1. ] calculated: 142.1590 found: 142.1595 N Ethylhexahydroazepine (40 ): From homopiperidine, ethyl iodide and potassium carbonate. Colorless oil. 1 H NMR (CDCl 3 3 00 MHz): 2.50 (t, 4H, 4.6 Hz, 5.7 Hz); 2.42 (q, 2H, 7.0 Hz); 1.53 1.50 (m, 8H); 0.94 (t, 3H, 7 .0 Hz). Diethylhexahydroazepinium [diE HHA] (38 ): From N e thylhexahydroazepine ( 40 ) and ethyl iodide. White solid. 1 H NMR (D 2 O 3 00 MHz): 3.37 3.27 (m, 8H); 1.86 (m, 4H); 1.65 (m, 4H); 1.27 (t, 6H, 7.1 Hz). 13 CNMR

PAGE 199

199 (D 2 O, 12 5 MHz): 61.7, 55.4, 27.3, 21.1, 7.2. ] calculated: 156.1747 found: 156.1749. A.4 Synthesis of new fluorinated pyridinylmethylene anabaseines to study interactions leading to de sensitized stated of the human 7 nicotinic acetylcholin e receptor 1 (diethylam ino)methyl)piperidin 2 one (3 ): 202,203,222 Delta valerolactam (11.223 g, 113 mmol), diethylamine (23.4 mL, 226 mmol), and paraformaldehyde (6.802 g, 226 mmol) in toluene (130 mL) were heated at ref lux in a Dean Stark apparatus for 5 h. The reaction mixture was cooled to room temperature and concentrated on rotary evaporator to a volume of 45 mL. Then, brine was added (45 mL) and the pH of the mixture was adjusted to 11 using 4 M NaOH. The layers wer e separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic extracts were dried over MgSO 4 and concentrated. The crude product was purified by vacuum distillation (b.p. 120 121 C, 0.4 mmHg) to yield 3 as a colorless oil (18.019 g, 86 % yield). 1 H NMR (CDCl 3 3 00 MHz): 4.15 (s, 2H); 3.33 3.37 (m, 2H), 2.57 (q, 4H, 7.2 Hz); 2.36 2.41 (m, 2H); 1.78 (m 4H ), 1.01 (t, 6H, 7.2 Hz). 13 CNMR (CDCl 3 7 5 MHz): 170.3, 63.5, 46.0, 45.1, 32.4, 23.1, 21.2, 12.0

PAGE 200

200 Sodium salt of the aminal of 3 n icotinoyl 2 piperidinone (51 ): 202,203,222 Sodium hydride 60 % dispersion in mineral oil (4.784 g, 119.6 mmol) was added in 3 portions to a solution of 3 (11.030 g, 59.8 mmol) and ethyl nicotinate 50 (8.20 mL, 60.0 mmol) in toluene (52 mL) at room temper ature, and the reaction mixture was heated at reflux. After 4 h, sodium hydride 60 % dispersion in mineral oil (2.35 g, 58.8 mmol) was added and the reaction mixture was stirred at reflux for additional 4 h. Then, the unreacted sodium hydride was removed b y filtration of the hot reaction mixture, and washed with hot toluene (NaH was destroyed in ethanol). The filtrate was concentrated to 50 mL and placed in a 8 C refrigerator overnight to allow precipitation of the product 51 The product was collected and dried to give a slightly yellow solid (12.174 g, 65 % yield). Anabaseine dihydrochloride (52 ): 202,203,222 Sodium salt of 51 (9.69 g, 31.1 mmol) was heated at reflux for 12 h in a 5:1 mixture of 12 M hydrochloric acid (100 mL) and acetone (20 mL). The reaction was cooled, concentrated to 70 mL, acetone was added (30 mL), and sodium chloride was removed by filtration. The filtrate was concentrated to 20 mL, a 1:1 mixture of ethanol and 2 propanol (100 mL total) was adde d, and allowed to stand in a 8 C refrigerator overnight. The solids were filtered and the product was recrystallized from ethanol: 2 propanol (1:1) to yield the product as a white solid ( 4.26 g, 59 % yield ). 1 H NMR

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201 ( DMSO d 6 5 00 MHz): 9.29 (dd, 1H, 2.1 Hz, 0.6 Hz); 8.97 (dd, 1H, 5.4 Hz, 1.5 Hz); 8.67 (dt, 1H, 8.1 Hz, 1.5 Hz); 7.91 (ddd, 1H, 8.1 Hz, 5.4 Hz, 0.6 Hz); 3.20 (t, 2H, 6.6 Hz); 2.79 2.85 (m, 2H); 1.64 1.71 (m, 4H) General procedure for preparing arylidene anabaseines : 219,22 2 Anabaseine dihydrochloride ( 52 ) (50 mg, 0.21 mmol) was dissolved in a methanolic mixture (2.2 mL) of sodium acetate trihydrate (28 mg, 0.21 mmol) and acetic acid (33 L, 0.53 mmol). The appropriate aldehyde was added (3 equivalents), and the mixture was stirred at room temperature under argon until the TLC analysis (DCM:methanol 20:1) showed complete consumption of the substrate. Then, water was added (10 mL), the pH was adjusted to 10 with 2 M K 2 CO 3 the product was extracted with ethyl acetate, the org anic layers were dried over MgSO 4 and the solvents were evaporated. The crude product was purified by a column chromatography (SiO 2 gradient of DCM:methanol). (E) 3 (2 fluoropyridinylmethy lene) anabaseine [(2 F) 3PAB] ( 44 ): Reaction time: 30 h. After chromatography (DCM:methanol 100:1 to 50:1 gradient), the product was obtained in 80 % yield. 1 H NMR (CDCl 3 3 00 MHz): 8.76 (d, 1H, 1.5 Hz); 8.65 (dd, 1H, 5.0 Hz, 1.5 Hz); 8.16 (d, 1H, 4.8 Hz); 7.84 (dt, 1 H, 7.8 Hz, 1.8 Hz); 7.71 7.78 (m, 1H); 7.35 (ddd, 1H, 7.8 Hz, 5.0 Hz, 0.9 Hz); 7.20 7.25 (m, 1H); 6.61 (s, 1H); 3.94 (t, 2H, 5.7 Hz); 2.69 (td, 2H, 5.7 Hz, 1.2 Hz); 1. 85 (qt, 2H, 5.7 Hz). 13 CNMR

PAGE 202

202 (CDCl 3 12 5 MHz): 165.7, 160.5 (d, 240 Hz), 149.8 (d, 31 Hz), 147.0 (d, 15 Hz), 140.4 (d, 4 Hz), 136.2, 135.4 (d, 26 Hz), 126.1, 123.1, 121.1 (d, 4 Hz), 118.5 (d, 29 Hz), 50.5, 25.4, 22.2. HRMS (ESI ): [M+H] + calculated: 268. 1244 found: 268.1248 (E) 3 (5 fluoropyridinylmethy lene) anabaseine [(5 F) 3PAB] (45 ): Reaction time: 26 h. After chromatography (DCM:methanol 100:1 to 40:1 gradient), the product was obtained in 74 % yield. 1 H NMR (CD Cl 3 5 00 MHz): 8.75 (d, 1H, 1.5 Hz); 8.66 (dd, 1H, 4.8 Hz, 1.5 Hz); 8.39 (d, 1H, 3.0 Hz); 8.36 (s, 1H); 7.83 (dt, 1H, 8.0 Hz, 2.0 Hz); 7.35 7.38 (m, 2H); 6.60 (s, 1H); 3.93 (t, 2H, 6.0 Hz); 2.81 (td, 2H, 6.5 Hz, 2.0 Hz); 1.86 (qt, 2H, 6.5 Hz, 6.0 Hz). 13 CNMR (CDCl 3 1 2 5 MHz): 166.0, 159.1 (d, 256 Hz), 149.8 (d, 37 Hz), 146. 3 (d, 4 Hz), 137.2, 137.0, 136.2, 135.6, 134.9, 133.1 (d, 4 Hz), 130.0 (d, 2 Hz), 123.2, 122.7 (d, 18 Hz), 50.4, 25.7, 22.2. 19 FNMR (470 MHz, CDCl 3 with CFCl 3 ): [M+H] + calculated: 268.1245 found: 268.1247 (E) 3 (2,6 difluoropyridinylmethylene ) anabaseine [(2,6 DF) 3PAB)] (46 ): Reaction time: 30 h. After column chromatography (DCM:methanol 100:1 to 60:1 gradient) the product was obtained in 78 % yield. 1 H NMR (CDCl 3 5 00 MHz): 8.75

PAGE 203

203 (dd, 1H, 2.0 Hz, 0.5 Hz); 8.65 (dd, 1H, 5.0 Hz, 1.5 Hz); 7.82 7.88 (m, 2H); 7.36 (ddd, 1H, 8.0 Hz, 5.0 Hz, 1.0 Hz); 6.88 (dd, 1H, 8.0 Hz, 2.5 Hz); 6.55 (s, 1H); 3.94 (t, 2H, 5.5 Hz); 2.66 (td, 2H, 6.0 Hz, 2.0 Hz); 1.86 (qt, 2H, 6.0 Hz). 13 CNMR (CDC l 3 12 5 MHz): 165.5, 160.7 (dd, 247 Hz, 14 Hz), 158.3 (dd, 248 Hz, 14 Hz), 149.9, 149.6, 144.5 (dd, 7 Hz, 4 Hz), 136.1, 135.4, 135.2, 124.9 (d, 2Hz), 123.1, 115.1 (dd, 27 Hz, 6 Hz), 106.0 (dd, 35 Hz, 6 Hz), 50.5, 25.4, 22.1. HRMS (ESI ): [M+H] + calculate d: 286.1150 found: 286.1156 (E) 3 (2,4,6 trifluoropyridinylmethylene) anabaseine [(2,4,6 TF) 3PAB] (47 ): Reaction time: 48 h. After column chromatography (DCM: methanol 100:1 to 50:1 gradient), the product was obtained in 70 % yield. 1 H NMR (CDCl 3 5 00 MHz): 8.77 (s, 1H); 8.65 (dd, 1H, 4.9 Hz, 1.5 Hz); 7.84 (dt, 1H, 8.0 Hz, 1.9 Hz); 7.36 (ddd, 1H, 8.0 Hz, 4.9 Hz, 0.7 Hz); 6.66 (d, 1H, 8.0 Hz); 6.28 (s, 1H), 3.97 (t, 2H, 5.5 Hz); 2.47 (t, 2H, 5.5 Hz); 1.86 (qt, 2H, 5.5 Hz). 13 CNMR (CDCl 3 12 5 MHz): 169.7 ( ddd, 263 Hz, 12 Hz, 9 Hz); 164.7, 161.4 (dt, 247 Hz, 18 Hz), 158.9 (ddd, 247 Hz, 18 Hz, 12 Hz), 150.0, 149.7, 138.3, 136.2, 135.2, 123.1, 118.8 (d, 2 Hz), 105.0 (ddd, 33 Hz, 19 Hz, 7 Hz), 95. 4 (ddd, 39 Hz, 25 Hz, 7 Hz), 50.7, 25.7, 22.1. HRMS (ESI ): [M+H ] + calculated: 304.1056 found: 304.1062

PAGE 204

204 (E) 3 (2,3,4,5,6 perfluorophenylmethylene) a nabaseine [(2,3,4,5,6 PF) AB] (48 ): Reaction time: 48 h. After chromatography (DCM: methanol 100:1 to 75:1 gradient), the product was o btained in 57 % yield. 1 H NMR (CDCl 3 3 00 MHz): 8.77 (d, 1H, 1.8 Hz); 8.66 (dd, 1H, 5.0 Hz, 1.8 Hz); 7.85 (dt, 1H, 7.5 Hz, 2.1 Hz); 7.35 (ddd, 1H, 7.5 Hz, 5.0 Hz, 0.9 Hz); 6.31 (s, 1H); 3.98 (t, 2H, 6.0 Hz); 2.45 2.50 (m, 2H); 1.86 (qt, 2H, 6.0 Hz) 13 C NMR (CDCl 3 125 MHz): 164.5; 150.1; 149.7; 143.7 (dm, 247 Hz); 140.9 (dm, 254 Hz); 138.9; 137.6 (dm, 252 Hz); 136.2; 135.1; 123.1, 118.5 118.8 (m); 110.4 (td, 18 Hz, 4 Hz); 50.8; 25.8; 22.3. HRMS (ESI ): [M+H] + calculated: 339.0916 found: 339.0925 2,4,6 trifluoronicotinaldehyde (56 ) : nBuLi (2.5 M in hexanes, 1.40 mL, 3.50 mmol) was added to a solution of 2,4,6 trifluoro pyridin e ( 58 ) (0.33 mL, 3.72 mmol) in THF ( 18 mL) the mixture was stirred f or 30 min. A solution of N methylformanilide ( 59 ) (0.43 mL, 3.48 mmol) in THF (2 mL) C for 2 h, and then let warm to room temperature over 2 h. 1M HCl was added, the mixture was extracted w ith diethyl ether, dried (MgSO 4 ) and evaporated. Purification by column chromatography (hexanes: ethyl acetate, 10:1) gave the product as a white solid (325 mg, 58 % yield). 1 H NMR (CDCl 3 5 00 MHz): 10.23 (t, 1H, 1.0 Hz); 6.73 (dd, 1H, 8.5 Hz, 1.8 Hz) 13 CNMR (CDCl 3 125 MHz): 181.9 181.2 (m), 173.0 (ddd,

PAGE 205

205 278.5 Hz, 13.2 Hz, 6.6 Hz); 164.4 (dt, 253.7 Hz, 19.7 Hz); 162.9 (258.4 Hz, 19.8 Hz, 9.4 Hz); 107.0 106.7 (m), 97.3 96.5 (m). HRMS (GC/CI ): [M+H] + calculated: 162.0167 found: 162.0165 A.5 Quinuclidi nes 2 (Chloromethyl)thiophene (75 ) : 229 A solution of thionyl chloride (5.2 mL, 66 mmol) in dichloromethane (20 mL) was added at 0 C to a solution of 2 hydroxymethylthiophene ( 7 3 3.01 g, 26 mmol) and pyridine (3.8 mL, 4 7 mmol) in dichloromethane (50 mL). The mixture was stirred at room temperature for 2 h, poured into water (60 mL), the layers were separated, the aqueous layer was extracted with dichloromethane, the combined organic layers were washed twice with 10 % aq ueous sodium bicarbonate and brine, dried over MgSO 4 and evaporated to give the product as a lightly yellow oil (3.33 g, 95 % yield). 1 H NMR (CDCl 3 3 00 MHz): 7.31 (dd, 1H, 5.0 Hz, 1.2 Hz); 7.08 7.10 (m, 1H); 6.95 (dd, 1H, 5.0 Hz, 3.5 Hz); 4.82 (s, 2H) The 1 HNMR data matched the reported data. 229 13 CNMR (CDCl 3 7 5 MHz): 140.1, 127.6, 126.9, 126.9, 40.3. 2 (Chloromethyl)furan (76 ): The compound was prepared from 2 hydroxymethylfuran ( 74 ) analogously to 2 (Chloro methyl)thiophene ( 7 5 ) but the reaction

PAGE 206

206 Brown oil. Yield: 64 %. 1 H NMR (CDCl 3 300 MHz): 7.42 (dd, 1H 1.7 Hz, 0.8 Hz); 6.34 6.38 (m, 2H); 4.60 (s, 2H). 13 CNMR (CDCl 3 7 5 MHz): 150.0, 143.4, 110.7, 109.7, 37.4 The 13 CNMR for CH 2 Cl carbon differed from the reported data (37.4 vs 63.4) 228 3 (Chloromethyl)thiophene (83 ): The compound was prepared from 3 hydroxymethylthiophene ( 81 ) analogously to 2 (Chloromethyl)thiophene ( 7 5 ). Reaction time: 3 h. Yellow oil. 91 % yield. 1 H NMR (CDCl 3 3 00 MHz): 7.34 (dd, 1H, J= 4.8 Hz, 2.7 Hz); 7.29 7.31 (m, 1H); 7.14 (dd, 1H, J= 4.8 Hz, 1.2 Hz); 4.64 (s, 2H). The 1 HNMR data matched the literature data. 354 13 CNMR (CDCl 3 7 5 MHz): 138.0, 127.5, 126.6, 124.0, 40.6. 3 (Chloro me thyl)furan (84 ): The compound was prepared from 3 hydroxymethylfuran ( 82 ) analogously to 2 (Chloromethyl)thiophene ( 75 ). Reaction time: 2 h. Yield: 84 %. 1 H NMR (CDCl 3 3 00 MHz): 7.46 7.47 (m, 1H); 7.41 (t, 1H, 1.5 Hz); 6.45 6.46 (m, 1H); 4.49 (m, 2H). 13 CNMR (CDCl 3 7 5 MHz): 143.8, 140.7, 122.4, 110.3, 37.1. The NMR data matched the reported data. 355

PAGE 207

207 Diethyl (thi ophene 2 ylmethyl)phosphonate (77 ): 2 (Chloromethyl)thiophene ( 75 ) (1.035 g, 7.80 mmol) and trieth ylphosphite (1.47 mL, 8.57 mmol) were heated at 140 C for 4 h. The crude was purified by vacuum distillation to give the product as a colorless oil (1.180 g, 65 % yield, bp. 114 120 C at 0.5 Torr). 1 H NMR (CDCl 3 3 00 MHz): 7.17 7.26 (m, 1H); 6.94 7.0 0 (m, 2H); 4.02 4.12 (m, 4H); 3.37 (d, 2H, J= 20.7 Hz), 1.28 (t, 6H, J= 6.9 Hz). 13 CNMR (CDCl 3 7 5 MHz): 132.4 (d, J= 10.3 Hz); 127.2 (d, J= 8.3 Hz); 127.0 (d, J= 3.5 Hz); 124.7 (d, J= 3.8 Hz); 62.3 (d, J= 6.5 Hz); 27.9 (d, J= 143.2 Hz); 16.3 (d, J= 6.0 Hz). The NMR data matched the reported data 356 Dimethyl (thi ophene 2 ylmethyl)phosphonate (79 ): 2 (Chloromethyl)thiophene ( 75 ) (3.35 g, 25.3 mmol) and trimethylphosphite (3.28 mL, 27.8 mmol) were heated at reflux for 3 h. The crude was purified by vacuum distillation to give the pure product as a colorless oil (2.92 g, 56 %, bp. 110 112 C at 0.5 Torr). 1 H NMR (CDCl 3 5 00 MHz): 7.18 7.21 (m, 1H); 6.94 7.00 (m, 2H); 3.72 (d, 6H, J= 10.8 Hz); 3.38 (dd, 2H, J= 20.7 Hz, 0.7 Hz). 13 CNMR (CDCl 3 12 5 MHz): 131.7 (d, J= 10.9 Hz); 127.1 (d, J= 9.1 Hz); 126.9 (d, 3.2 Hz); 124.6 (d, 3.2 Hz), 52.8 (d, 6.9 Hz), 26.7 (d, 143.4 Hz). The NM R data matched the literature data. 357

PAGE 208

208 Diethyl (furan 2 ylmethyl)phosphonate (78 ): The compound was prepared from 2 (c hloromethyl)furan ( 76 ) analogously to d iethyl (thiophene 2 ylmethyl)phosphonate ( 77 ) Reflux 1 h. Co lorless oil. 65 % yield. Bp. 102 110 C at 0.5 Torr. 1 H NMR (CDCl 3 3 00 MHz): 7.34 7.36 (m, 1H); 6.33 6.34 (m, 1H), 6.23 6.26 (m, 1H); 4.03 4.13 (m, 4H); 3.24 (d, 2H, J= 20.7 Hz ); 1.29 (t, 6H, J= 7.2 Hz). The 1 HNMR data matched the reported data. 358 13 CNMR (CDCl 3 7 5 MHz): 145.6 (d, J= 9.5 Hz); 141.9 (d, J= 3.4 Hz); 110.8 (d, J= 2.9 Hz); 108.1 (d, J= 7.4 Hz); 62.2 (d, J= 6.6 Hz); 26.7 (d, J= 143.7 Hz); 16.3 (d, J= 6.0 Hz) Dimethyl (furan 2 ylmethyl)phosphonate (80 ): The compound was prepared from 2 (c hloromethyl)furan ( 76 ) analogously to dim ethyl (thiophene 2 ylmethyl)phosphonate ( 79 ) Reflux 1 h. Orange oil. 30 % yield. Bp. 85 95 C at 0.4 Torr. 1 H NMR (CDCl 3 3 00 MHz): 7.36 7.37 (m, 1H); 6.33 6.35 (m, 1H); 6.24 6.27 (m, 1H); 3.74 (d, 6H, J= 10.8 Hz); 3.27 (d, 2H, J= 21.0 Hz). The 1 HNMR data matched the literature data. 358 13 CNMR (CDCl 3 12 5 MHz): 144.9 (d, 10.1 Hz); 141.8 (d, 3.6 Hz); 110.6 (d, 3.1 Hz); 108.2 (d, 7. 8 Hz); 52.6 (d, 6.9 Hz); 25.4 (d, 143.2 Hz).

PAGE 209

209 Diethyl (thi ophene 3 ylmethyl)phosphonate (85 ): The compound was prepared from 3 (Chloromethyl)thiophene ( 83 ) analogously to d iethyl (thiophene 2 ylmethyl)phosphonate ( 77 ) Colorless oil. Yield 57 %. Bp. 105 115 C at 0.5 Torr. 1 H NMR (CDCl 3 3 00 MHz): 7.25 (dd, 1H, J= 5.0 Hz, 3.0 Hz); 7.13 (t, 1H, 3.0 Hz); 7.04 (dt, 1H, 5.0 Hz, 1.4 Hz); 3.95 4.05 (m, 4H); 3.17 (d, 2H, 21.1 Hz); 1.23 (t, 6H, 7.0 Hz). 13 CNMR (CDCl 3 7 5 MHz): 130.8 (d, J= 8.8 Hz); 128.9 (d, J= 4.6 Hz); 125.6 (d, J= 1.7 Hz), 123.0 (d, J= 9.7 Hz), 62.0 (d, J= 6.6 Hz); 28.2 (d, J= 140.5 Hz); 16.3 (d, J= 5.7 Hz). The NMR data matched the literature data. 359 Diethyl (furan 3 ylmethyl)phosphonate (86 ): The compound was prepared from 3 (Chloromethyl)furan ( 84 ) analogously to d iethyl (thiophene 2 ylmethyl)phosphonate ( 77 ) Colorless oil. Yield 54 %. Bp. 100 110 C at 0.5 Torr. 1 H NMR (CDCl 3 3 00 MHz): 7.37 7.39 (m, 2H); 6.42 (d, 1H, 1.2 Hz); 4.03 4.13 (m, 4H); 2.96 (d, 2H, 20.4 Hz); 1.29 (td, 6H, 6.9 Hz, 0.6 Hz). 13 CNMR (CDCl 3 7 5 MHz): 142.8; 140.5 (d, J= 11.1 Hz); 114.6 (d, J= 8.8 Hz); 111.7 (d, 4.9 Hz); 61.9 (d, J= 6.5 Hz); 22.9 (d, J= 143.5 Hz) ; 16.3 (d, J= 6.0 Hz).

PAGE 210

210 2TQN: ( Z) 3 (thiophen 2 ylmethylene)quinuclidine (70a ) and ( E) 3 (thiophen 2 ylmethylene)quinuclidine (70b ): A solution of d iethyl (thiophene 2 ylmethyl)phosphona te ( 77 ) (600 mg, 2.56 mmol) in THF (4 mL) was added to a suspension of sodium hydride 60 % dispersion in mineral oil (106 mg, 2.65 mmol) in THF (12 mL) at room temperature and a mixture was stirred at 50 C for 1 h. The mixture was cooled to room temperatu re, a solution of 3 quinuclidinone ( 72 247 mg, 1.97 mmol) in THF (4 mL) was added, and the mixture was heated at reflux for 1.5 h. The mixture was cooled to room temperature, water was added, and the mixture was extracted with ethyl acetate, washed with brine, dried (MgSO 4 ), and evaporated. The crude product was purified by column chromatography on silica gel pretreated with Et 3 N in hexanes (gradient of hexanes:ethyl acetate 4:1 + 0.3 % Et 3 N to hexanes:ethyl acetate 1:2 + 0.3 Et 3 N) to give 170 mg of the Z isomer (42 % yield) and 174 mg of the E isomer (43 % yield) as white solids. Z isomer : R f = 0.13 (hexanes: ethyl acetate: Et 3 N 1: 1: 0.1). 1 H NMR (CDCl 3 300 MHz): 7.23 (d, 1H, 5.1 Hz); 7.01 (dd, 1H, 3.6 Hz, 5.1 Hz); 6.88 (d, 1H, 3.6 Hz); 6.44 (t, 1H, 2.5 Hz); 3.75 (s, 2H), 2.82 3.01 (m, 4H); 2.47 (quintet, 1H, 3.3 Hz); 1.71 1.84 (m, 4H). 13 CNMR (CDCl 3 12 5 MHz): 145.0, 141.1, 127.0, 125.4, 124.4 113.5, 56.0, 47.6, 33.3, 28.1. HRMS (DART): [M+H] + calculated: 206.0998 found: 206.1000. E isomer : R f = 0.10 (hexanes: ethyl acetate: Et 3 N 1: 1: 0.1). 1 H NMR (CDCl 3 3 00 MHz): 7.16 (dd, 1H, 5.2 Hz, 0.9 Hz); 6.96 (dd, 1H, 5.2 Hz, 3.6 Hz); 6.85 (d, 1H, 3.6 Hz); 6.31 (t, 1H, 1.8 Hz); 3.55 (s, 2H); 3.30 (quintet, 1H, 3.0 Hz); 2.83 3.00 (m, 4H); 1.69 1.81 (m,

PAGE 211

211 4H). 13 CNMR (CDCl 3 12 5 MHz): 144.7, 140.1, 126.7, 126.0, 123.7, 112.8, 56.6, 47.6, 26.8, 26.7. HRMS (DART): [M+H] + calculated: 206.0998 found: 206.1002. The free base 3 quinuclidinone ( 72 ) used in the procedure above was obtained by treating quinucl idine hydrochloride with a 2 M aqueous solution of K 2 CO 3 extraction with diethyl ether, drying (MgSO 4 ), and removal of the solvent on the rotovap. ( Z) 3 (thiophen 2 ylmethylen e)quinuclidin 1 ium chloride : ( Z) 3 (thiophen 2 ylmethylene)quinuclidine ( 70a ) was dissolved in ethanol, and a solution of HCl in ethanol was added. The solvents were removed on the rotovap to give the product quantitatively. 1 H NMR (CDCl 3 5 00 MHz): 12.98 (s, 1H); 7.38 (d, 1H, 5.2 Hz); 7.08 (dd, 1H, 5.2 Hz, 3.6 Hz); 6.97 (d, 1H, 3.6 Hz); 6.69 (t, 1H, 2.5 Hz); 4.18 (s, 2H); 3.39 3.45 (m, 2H); 3.29 3.35 (m, 2H); 2.84 (quintet, 1H, 3.0 Hz); 2.11 2.15 (m, 4H). 13 CNMR (CDCl 3 125 MHz): 137.9, 129.1, 1 27.7, 127.7, 126.7, 118.7, 53.6, 46.7, 31.2, 24.3. ( E) 3 (thiophen 2 ylmethylen e)quinuclidin 1 ium chloride : 1 H NMR (CDCl 3 5 00 MHz): 12.59 (s, 1H); 7.29 (d, 1H, 5.0 Hz); 7.03 (dd, 1H, 5.0 Hz, 3.5 Hz); 6.98 (d, 1H, 3.5 Hz); 6.52 (s, 1H); 4.03 (s, 2H); 3.63 (t, 1H, 3.0 Hz); 3.42 3.46

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212 (m, 2H); 3.32 3.38 (m, 2H); 2.05 2.17 (m, 4H). 13 CNMR (CDCl 3 12 5 MHz): 137.3, 129.9, 128.5, 127.2, 125.7, 118.8, 54.3, 46.5, 25.4, 23.2. 2 FQN: ( Z) 3 (furan 2 ylmethylene)quinuclidine (68a ) and ( E) 3 (furan 2 ylmethylene)quinuclidine (68b ): The compounds were prepared from d iethyl (furan 2 ylmethyl)phosphonate ( 78 ) and 3 quinuclidinone ( 72 ) analog ously to ( Z) 3 (thiophen 2 ylmethylene)quinuclidine ( 70a ) and ( E) 3 (thiophen 2 ylmethylene)quinuclidine ( 70b ): Z isomer : white solid, yield: 43 %. R f = 0.13 (hexanes: ethyl acetate: Et 3 N 1: 1: 0.1). 1 H NMR (CDCl 3 5 00 MHz): 7.36 (d, 1H, 1.8 Hz); 6.39 (dd, 1H, 3.6 Hz, 1.8 Hz); 6.08 6.10 (m, 2H); 3.78 (s, 2H); 2.81 3.00 (m, 4H); 2.45 (quintet, 1H, 3.0 Hz); 1.72 1.80 (m, 4H). 13 CNMR (CDCl 3 12 5 MHz): 153.2, 145.5, 140.9, 111.2, 109.0, 107.2, 55.9, 47.6, 33.0, 28.0. E isomer : brown solid, yield: 42 %. R f = 0.10 (hexanes: ethyl acetate: Et 3 N 1: 1: 0.1). 1 H NMR (CDCl 3 5 00 MHz): 7.32 (d, 1H, 1.8 Hz); 6.35 (dd, 1H, 3.3 Hz, 1.8 Hz); 6.12 (d, 1H, 3.3 Hz); 5.96 (t, 1H, 2.1 Hz); 3.54 (s, 2H); 3.44 (quintet, 1H, 3.0 Hz); 2.83 3.00 (m, 4H); 1.69 1.81 (m, 4H). 13 CNMR (CDCl 3 12 5 MHz): 152.9, 144.9, 140.7, 110.9, 108.6, 107.3, 56.4, 47.6, 26.7, 26.5.

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213 2 FQN: ( Z) 3 (furan 2 ylmethylene )quinuclidin 1 ium chloride : 1 H NMR (CDCl 3 5 00 MHz): 12.77 ( s, 1H); 7.45 (d, 1H, 1.6 Hz); 6.43 (dd, 1H, 2.8 Hz, 1.6 Hz); 6.28 (d, 2H, 2.8 Hz); 4.35 (s, 2H); 3.39 3.45 (m, 2H); 3.29 3.35 (m, 2H); 2.79 (quintet, 1H, 3.0 Hz); 2.09 2.13 (m, 4H) 13 CNMR (CDCl 3 12 5 MHz): 151.0, 143.3, 129.0, 113.4, 111.5, 110.4, 54. 1, 46.6, 30.9, 24.3 3 TQN: ( Z) 3 (thiophen 3 ylmethylene)quinuclidine (71a ) and ( E) 3 (thioph en 3 ylmethylene)quinuclidine (71b ): The compounds were prepared from diethyl (thiophene 3 y lmethyl)phosphonate ( 85 ) and 3 quinuclidinone ( 72 ) analogously to ( Z) 3 (thiophen 2 ylmethylene)quinuclidine ( 70a ) and ( E) 3 (thiophen 2 ylmethylene)quinuclidine ( 70b ). The ratio of Z:E isomers based on the crude 1 H NMR was 3:2 The product s were obtained in an estimated 50 % yield (the E isomer was not obtained in a pure form). Z isomer : white solid. 1 H NMR (CDCl 3 5 00 MHz): 7.29 (dd, 1H, 5.1 Hz, 3.0 Hz); 7.02 7.07 (m, 2H); 6.26 (t, 1H, 2.7 Hz); 3.78 (s, 2H); 2.83 3.01 (m, 4H); 2.46 (quintet, 1H, 3.0 Hz) ; 1.76 1.81 (m, 4H). 13 CNMR (CDCl 3 12 5 MHz): 145.3, 139.0, 128.4, 125.1, 121.4, 114.5, 56.2, 47.7, 33.4, 28.2 E isomer : 1 H NMR (CDCl 3 5 00 MHz): 12.82 (s, 1H); 7.36 (dd, 1H, 5.0 Hz, 3.0 Hz); 7.10 (dd, 1H, 3.0 Hz, 1.0 Hz); 7.00 (dd, 1H, 5.0 Hz, 1.0 H z); 6.51 (t, 1H, 2.5 Hz); 4.18 (s,

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214 2H); 3.29 3.43 (m, 4H); 2.81 (quintet, 1H, 3.0 Hz); 2.10 2.13 (m, 4H) 13 CNMR (CDCl 3 12 5 MHz): 136.4, 130.2, 127.8, 126.8, 124.4, 120.3, 54.1, 47.0, 31.5, 24.7. ( Z) 3 (thiophen 3 yl methylen e)quinuclidin 1 ium chloride : 1 H NMR (CDCl 3 5 00 MHz): 12.82 (br s, 1H); 7.36 (dd, 1H, 5.0 Hz, 3.0 Hz); 7.10 (dd, 1H, 3.0 Hz, 1.0 Hz); 7.00 (dd, 1H, 5.0 Hz, 1.0 Hz); 6.51 (t, 1H, 2.5 Hz); 4.18 (s, 2H); 3.29 3.43 (m, 4H); 2.81 (quintet, 1H, 3.0 Hz ); 2.10 2.13 (m, 4H). 13 CNMR (CDCl 3 12 5 MHz): 136.4, 130.2, 127.8, 126.8, 124.4, 120.3, 54.1, 47.0, 31.5, 24.7 3 FQN: ( Z) 3 (furan 3 ylmethylene)quinuclidine (69a ) and ( E) 3 (furan 3 ylmethylene)quinuclidine (69b ): The compounds were prepared from diethyl (furan 3 ylmethyl)phosphonate ( 86 ) (2.7 equivalent) and 3 quinuclidinone ( 72 ) analogously to ( Z) 3 (thiophene 2 ylmethylene)quinuclidine ( 70a ) and ( E) 3 (thiophen 2 ylmethylene)qui nuclidine ( 70b ). The ratio of Z:E isomers based on the crude 1 H NMR was 3:2 The product s were obtained in an estimated 50 % yi eld (the products were not obtained in a pure form ). Z isomer : 1 H NMR (CDCl 3 3 00 MHz): 7.40 (t, 1H, 1.8 Hz); 7.36 (br s, 1H); 6.38 (d, 1H, 1.8 Hz); 6.09 (t, 1H, 2.4 Hz); 3.75 (s, 2H); 3.11 2.92 (m, 4H); 2.55 (quintet, 1H, 3.1 Hz); 1.88 1.82 (m, 4H).

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215 E isomer : 1 H NMR (CDCl 3 3 00 MHz): 7.39 (t, 1H, 1.7 Hz); 7.36 (br s, 1H); 6.36 (d, 1H, 1. 7 Hz); 6.03 (br s, 1H); 3.76 (s, 2H); 3.20 3.06 (m, 5H); 1.95 1.85 (m, 4H). Tert Butyl 2 formyl 1 H pyrrole 1 carboxylate (88 ) : 230 4 Dimethylaminopyridine (162 mg, 1.33 mmol), and di t butyldicarbonate (3.557 g, 16.3 mmo l) were added to a solutio n of pyrrole 2 carboxaldehyde ( 87 ) (1.50 g, 15.8 mmol) in acetonitrile (25 mL), and stirred at room temperature for 24 h. Solvent was evaporated on the rotovap and the residue was purified by column chromatography using hexanes:e thyl acetate (20:1) as an eluent to give the product (3.05 g, 99 % yield). R f = 0.37 (hexanes:ethyl acetate, 10:1). 1 H NMR (CDCl 3 3 00 MHz): 10.32 (s, 1H); 7.43 (dd, 1H, 3.1 Hz, 1.8 Hz); 7.19 (dd, 1H, 3.7 Hz, 1.8 Hz); 6.29 (td, 1H, 3.7 Hz, 3.1 Hz, 0.6 Hz); 1.65 (s, 9H). Tert Butyl 2 (hydroxymethyl) 1 H pyrrole 1 carboxylate (89 ) : 230 To a solution of aldehyde from above (2.726 g, 14.0 mmol) in methanol (30 mL) at 0 C was added sodium borohydride (528 mg, 14.0 mg) in p ortions. The mixture was stirred for 50 min, cold water was added, the mixture was extracted with diethyl ether, and washed wi th saturated aqueous NaHCO 3 solution and brine. The organic layer was dried over MgSO 4 and evaporated to give the product as color less oil which was satisfactory pure by 1 HNMR ( 2.47 g, 90 % yield). 1 H NMR (CDCl 3 3 00 MHz): 7.14

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216 7.16 (m, 1H); 6.15 6.17 (m, 1H); 6.08 (td, 1H, 3.3 Hz, 1.5 Hz); 4.63 (d, 2H, J= 7.2 Hz); 3.61 (t, 1H, J= 7.2 Hz, OH); 1.59 (s, 9H) 13 CNMR (CDCl 3 7 5 MHz) : 150.0, 134.7, 121.8, 113.4, 110.3, 84.4, 57.6, 27.9 The NMR data matched the reported data. 230 1 (Methylsulfonyl) 1 H pyrrole 2 carbaldehyde (91 ) : 231 A solution of 2 pyrrolecarboxaldehyde (0.997 g, 10.5 mmol) in TH F (10 mL) was added to a stirred suspension of sodium hydride (505 mg, 12.6 mmol of a 60 % dispersion in mineral oil washed twice with hexanes) in THF (30 mL) The resulting mixture was stirred at room temperature for 15 min. A solution of mesyl chloride ( 1.14 mL, 14.7 mmol) in THF (10 mL) was added dropwise, and the mixture was stirred for 1.5 h at room temperature. Water was added (50 mL), THF was removed on the rotovap, and the resulting mixture was extracted with ethyl acetate, washed with brine, dried over MgSO 4 and evaporated. The crude product was purified by column chromatography, using hexanes:ethyl acetate 10:1 as an eluent to give the product as a silver solid (931 mg, 51 %). R f = 0.17 (hexanes:ethyl acetate, 4:1). 1 H NMR (CDCl 3 300 MHz): 9.6 7 (t, 1H, 0.85 Hz); 7.58 7.60 (m, 1H); 7.20 (ddd, 1H, 3.7 Hz, 1.8 Hz, 0.6 Hz); 6.40 (ddd, 1H, 3.7 Hz, 3.1 Hz, 0.6 Hz); 3.61 (d, 3H, 0.85 Hz)

PAGE 217

217 [1 (Methylsulfonyl) 1 H pyrrol 2 yl]methanol (92 ): 231 Sodium borohydride (2 00 mg, 5.29 mmol) was added at 0 C to a solution of the aldehyde above ( 91 ) (642 mg, 3.71 mmol) in THF (4 0 mL). After stirring at 0 C for 1.5 h, water (15 mL) and then acetic acid (7 mL, 10 % solution in water) were carefully added, the mixture was extra cted with ethyl acetate, washed with water and brine, dried over MgSO 4 and evaporated. Purification by column chromatography (hexanes:ethyl acetate, 10:1 to 4:1 gradient) afforded the product as a n off white solid (586 mg, 90 %). R f = 0.16 (hexanes:ethyl acetate, 2:1). 1 H NMR (CDCl 3 3 00 MHz): 7.16 (dd, 1H, J= 3.3 Hz, J= 1.8 Hz); 6.30 (dd, 1H, J= 3.3 Hz, 1.8 Hz); 6.24 (t, 1H, J= 3.3 Hz); 4.77 (d, 2H, J= 6.0 Hz); 3.30 (s, 3H); 2.41 (t, 1H, 6.0 Hz, OH) 13 CNMR (CDCl 3 75 MHz): 133.7, 123.3, 115.4, 111.4, 56.7, 43.0 The NMR data matched the r eported data. 2 (Chloromethyl) 1 (methylsulfonyl) 1 H pyrrole (93 ): 231 Mesyl chloride (0.40 mL, 5.22 mmol) was added to a solution of alcohol ( 92 ) (571 mg, 3.26 mmol) and triethylamine (0.74 mL, 5.24 mmol) at 0 C. The m ixture was stirred at 0 C for 1.5 h, diluted with dichloromethane, and washed with ice cold water, cold 1 M HCl, saturated NaHCO 3 and brine. The organic phase was dried (MgSO 4 ) and evaporated to give the product as an orange oil that was satisfactory pu re by 1 HNMR and used without further purification (618 mg, 98 %). 1 H NMR (CDCl 3 300 MHz): 7.21

PAGE 218

218 (dd, 1H, J= 3.3 Hz, J= 1.8 Hz); 6.42 (dd, 1H, 3.3 Hz, 1.8 Hz); 6.27 (t, 1H, 3.3 Hz); 4.94 (s, 2H); 3.39 (s, 3H) The NMR data matched the literature data Diethyl {[1 (methylsulfonyl) 1 H p yrrol 2 yl]methyl}phosphonate (94 ): 2 (Chloromethyl) 1 (methylsulfonyl) 1 H pyrrole ( 93 ) (4.140 g, 21.4 mmol) and triethyl phosphite (3.68 mL, 21.4 mmol) were stirred at 130 C for 50 min. The mixture was cooled and purified by column chromatography (hexanes:ethyl acetate, 10:1 to 3:1 gradient) to give the product as a lightly yellow solid of acceptable purity (see NMR, estimated 12 % yield). 1 H NMR (CDCl 3 3 00 MHz): 7.13 7.15 (m, 1H); 6.25 6.30 (m, 2H); 4.06 4.15 (m, 4H); 3 .58 ( d 2 H J P CH 2 = 21.6 Hz) ; 3.46 (s, 3H); 1.32 (t, 6H, J= 7.1 Hz) 13 CNMR (CDCl 3 7 5 MHz): 124.0 (d, J= 8.8 Hz); 122.2 (d, J= 3.7 Hz); 114.8 (d, J= 7.4 Hz); 111.3 (d, J= 3.7 Hz); 62.1 (d, J= 6.8 Hz); 42.9; 24.2 (d, J= 143.6 Hz); 16.1 (d, J= 8.0 Hz) A.6 Strigolactones Cycl o hex 1 ene 1,2 diyldimethanol (112 ): Solution of 3,4,5,6 tetrahydrophtalic anhydride (985 mg, 6.5 mmol) in THF (15 mL) was added slowly to a s uspension of lithium aluminium hydride (401 mg, 10.6 mmol) in THF (35 mL) at 0 C and then stirred for 30 min at room temperature and for 2 h at reflux. The reaction mixture was cooled down to 0 C and water (0.4 mL), 15 % sodium

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219 hydroxide (0.4 mL), and wa ter (1.2 mL) were carefully added. The resulting mixture was diluted with ethyl acetate (50 mL), filtered through celite, eluted with ethyl acetate (100 mL), dried (MgSO 4 ), and evaporated under reduced pressure. The crude product was purified by column chr omatography (hexane ethyl acetate 5:1 to 1:2 gradient) to yield 289 mg (31 % yield) of the pure product. R f = 0.08 (petroleum ether ethyl acetate 1:1, KMnO 4 ) 1 HNMR (CDCl 3 300 MHz) : = 4.05 (4H, s); 3.75 (2H, br s); 2.11 (4H, s); 1.59 (4H, s). 13 CNMR (CDCl 3 7 5 MHz) : = 135.0, 63.0, 28.6, 22.7. (2 ((( tert butyldimethylsilyl)oxy)methyl)cyclohex 1 en 1 yl)methanol (113 ) : Solution of tert butyldimethyl silyl chloride (687 mg, 4.6 mmol) in dichloromethane (3 mL) was added at 0 C to the solution of diol (650 mg, 4.6 mmol) and imidazole (454 mg, 6.7 mmol) in dichloromethane (23 mL) and stirred at room temperature for 3 h. 1M hydrochloric acid was added a nd the mixture was extracted with dichloromethane, washed with brine, dried (MgSO 4 ) and evaporated. The crude was purified by column chromatography (hexane ethyl acetate 50:1 to 10:1 gradient) to yield 373 mg (32 % yield) of the pure product. R f = 0.20 (h exanes ethyl acetate 10:1, KMnO 4 ). 1 HNMR (CDCl 3 300 MHz) : = 4.15 (2H, s); 4.06 (2H, s); 2.13 (2H, s); 2.05 (2H, s); 1.60 (4H, m); 0.90 (9H, s); 0.09 (s, 6H). 13 CNMR (CDCl 3 7 5 MHz) : = 134.4, 134.0, 63.4, 63.0, 28.5,

PAGE 220

220 2 (((tert butyldimethylsilyl)oxy)methyl)cyclohex 1 ene 1 carbaldehyd e (109 ): DMSO (51 mg, 0.65 mmol) in dichloromethane (2 mL) was added over 15 min to a solution of oxalyl chloride (113 mg, 0.89 mmol) in dichloromethane (1 mL) at 78 o C and the reaction mixture was stirred for 20 min (making sure that the temperature of the reaction mixture did not exceed 60 C as the chlorosulfonium intermediate decomposes above that temperature) The monoprotected diol (155 mg, 0.60 mmol) in dichlo romethane (1mL) was added at 78 o C and the reaction mixture was stirred for 40 min. Triethylamine (220 L, 1.6 mmol) was add ed in one portion and the rea ction mixture was stirred at 78 o C. After 15 min the mixture was allowed to warm to room temperature and diluted with dichloromethane. The organic layer was washed with saturated ammonium chloride solution and brine, dried (Mg SO 4 ), and evaporated to give 115 mg (74 % yield) of almost pure product as colorless oil. R f = 0.61 (hexanes:ethyl acetate 10:1). 1 HNMR (CDCl 3 300 MHz) : = 10.19 (1H, s); 4.57 (2H, s); 2.32 (2H, m); 2.21 (2H, m); 1.61 (4H, m); 0.90 (9H, s); 0.09 (6H, s). 13 CNMR (CDCl 3 7 5 MHz) : Methyl (R) 2 methyl 3 (tos y loxy)propanoate (116 ): 297 To the solution of methyl (R) 3 hydroxy2 methylpropionate (10.1 g, 85.5 mmol) in pyridine (110 mL) at 5 o C tosyl chloride (17.3 g, 90.6 mmol) was added and the reaction mixture was stirred at 0 o C for 4 h and then kept in the ref rigerator (8 o C) for 2

PAGE 221

221 days. Ice and water were added and the resulting milky suspension was extracted with ether. The combined organic layers were washed with 1 M HCl, water, and brine, and dried over MgSO 4 The solvents were removed under reduced pres sur e to give the tosylate in 98 % yield (22 8 g) as colorless oil. R f = 0.54 (petroleum ether ethyl acetate 2:1). 1 HNMR (300 MHz, CDCl 3 ) : = 7.78 (2H, d, 7.8 Hz); 7.36 (2H, d, 7.8 Hz); 4.19 (1H, dd, 9.8 Hz, 6.7 Hz); 4.06 (1H, dd, 9.8 Hz, 6.0 Hz); 3.64 (3H, s ); 2.83 (1H, quintet, 7.2 Hz, 6.3 Hz); 2.46 (3H, s); 1.17 (3H, d, 7.2 Hz). Methyl ( S) 3 iodo 2 methylpropanoate (117 ) : 297 A solution of tosylate 116 (23.3 g, 85.5 mmol) in THF (50 mL) was added to a solution of lithium i odide (13.5 g, 101 mmol) in THF (110 mL) and the reaction mixture was heated at reflux for 20 min. A yellow suspension formed. Ether was added to a cooled mixture to precipitate more solids and the solids were filtered off and the residue was washed with e ther. The organic solution was washed with saturated ammonium chloride solution, 5 % sodium thiosulfate solution, brine and dried (MgSO 4 ). The solvents were removed under reduced pressure to yield iodide ester 117 in 86 % yield (16.7 g). 1 HNMR (300 MHz, C DCl 3 ) : = 3.71 (3H, s); 3.36 (1H, dd, 9.9 Hz, 6.6 Hz); 3.24 (1H, dd, 9.9 Hz, 6.2 Hz); 2.79 (1H, sextet, 6.7 Hz); 1.26 (3H, d 7.0 Hz)

PAGE 222

222 (S) 3 iodo 2 methylpropanoic acid (118 ) : 297 Iodide ester 117 (5.97 g, 15.7 mmol) was adde d to a solution of sodium iodide (15.6 g, 104 mmol) in acetonitrile (150 mL), after which trimethylsilyl chloride (13.2 mL, 104 mmol freshly distilled from CaH 2 ) was added. The mixture became very cloudy immediately due to precipitation of sodium chloride The reaction mixture was heated under reflux for 86 h. Water (40 mL) was added at room temperature and the resulting clear solution was stirred for 2h. The mixture was extracted with ether and washed with 15 % sodium thiosulfate. The acid iodide was extr acted with 10 % sodium carbonate solution, acidified to pH 2 with 2 M HCl, extracted with ether, washed with brine, dried (MgSO 4 ) and evaporated to yield 2.80 g (50 % yield) of the product. 1 HNMR (300 MHz, CDCl 3 ) : = 7.34 (s, broad, 1H); 3.39 (1H, dd, 10. 0 Hz, 6.2 Hz); 3.27 (1H, dd, 10.0 Hz, 6.2 Hz); 2.82 (1H, sextet, 7.0 Hz, 6.8 Hz, 6.2 Hz); 1.31 (3H, d 7.1 Hz ) (S) (2 carboxypropy l )triphenylphosphonium iodide (110 ) : 297 A solution of acid iodide ( 118 1 .42 g, 6.63 mmo l) in acetonitrile (25 mL) was added to a suspension of triphenylphosphine (13.76 g, 52.4 mmol) in acetonitrile (20 mL) and the reaction mixture was heated under reflux for 40 h. The solvent was distilled off under reduced pressure, 20 mL of ether was adde d and it was stirred for 2 h. The solvent was syringed out to give a gummy solid which was again stirred with ether (20 mL) for 2 h. This procedure was repeated until fine white powder remained (10 times) to yield 2.60 g

PAGE 223

223 (82 % yield). 1 HNMR (300 MHz, CDCl 3 ) : = 7.69 7.85 (15H, m); 6.14 (br s, 1H); 3.88 3.68 (2H, m); 3.30 3.23 (1H, m); 1.49 (3H, d d, 7.1 Hz, 1.7 Hz). (S) (3 methoxy 2 methyl 3 oxopropyl )triphenylphosphonium iodide : Iod ide ester 117 (1.374 g, 6.02 mmol) in aceto nitrile (15 mL) was added to a suspension of triphenylphosphine (12.334 g, 47.0 mmol) in acetonitrile (45 mL) and the reaction mixture was heated at reflux for 40 h. The solvent was removed under reduced pressure, ether (12 mL) was added and it was stirred for 2 h. Ether was syringed out to give a sticky solid. Ether was added (12 mL) and this procedure was repeated until fine white powder remained. Yield: 1.394 g, 47 %. 1 HNMR (300 MHz, CDCl 3 ) : = 7.66 7.88 (15 H, m); 4.50 (1H, m); 3.56 (1H, m); 3.21 (3H, s); 2.96 (1H, m); 1.60 (3H, d d, 7.3 Hz, 1.9 Hz ) (R,E) 4 (cyclohex 1 en 1 yl) 2 methylbut 3 enoic acid : Li TMP was prepared by adding n BuLi (1.6 M, 8.41 mL, 13.46 mmol) to 2,2,6,6, tetramethylpiperidine (2.40 mL, 14.14 mmol, freshly distilled from CaH 2 by vacuum 0 C for 35 min. The ylide was prepared by adding Li TMP (5.3 6 mL, 3.64mmol) over 1 h to a suspension of phosphonium salt 1 10 (880 mg, 1.85 mmol) in THF (10 mL) at h and at room temperature for 2 h. The

PAGE 224

224 aldehyde (97 mg, 0.88 mmol) in THF (2 mL) was added to the ylide was added (5 mL) and the mixture was acidified to pH 3 with 1M HCl, and extracted with ether. The ether layer was then extracted with 10 % sodium carb onate solution. The aqueous layer was acidified to pH 2 with 1M HCl, extracted with ether, dried (MgSO 4 ) and evaporated to give orange oil (78 mg) that was further purified by column chromatography (hexane ethyl acetate 10:1 to 3:1 gradient) to give the pr oduct (47 mg, 25 % yield) containing small impurities (2 methylacrylic acid). 1 HNMR (300 MHz, CDCl 3 ): = 11.30 (br s, 1H); 6.15 (d, 1H, 15.6 Hz); 5.58 (dd, 1H, 15.6 Hz, 8.0 Hz); 3.20 (quintet, 1H, 7.1 Hz); 2.15 2.12 (m, 4H); 1.71 1.56 (m, 4H); 1.31 (d, 3H, 7.1 Hz). 2 methylacrylic acid : 1 HNMR (300 MHz, CDCl 3 ): = 6.24 6.23 (m, 1H); 5.68 5.66 (m, 1H); 1.95 (dd, 3H, 1.6 Hz, 1.1 Hz). (R,E) 4 (2 (hydroxymethyl)cyclohex 1 en 1 yl) 2 methylbut 3 enoic acid (107 ) : Compound was prepared from 2 (((tert butyldimethylsilyl)oxy)methyl) cyclohex 1 ene 1 carbaldehyde ( 1 09 ) analogously to (R,E) 4 (cyclohex 1 en 1 yl) 2 methylbut 3 enoic acid above. Four equivalents of phosphonium salt 110 we re used. Estimated yield: 10 % (2 methylacrylic acid, and other small impurities still present after purification). 1 HNMR (300 MHz, CDCl 3 ) : = 6.67 (d, 1H, 15.6 Hz); 5.73 (dd, 1H, 15.6 Hz, 8.2 Hz); 4.24 (s, 2H); 3.24 (quintet, 1H, 7.1 Hz); 2.24 (m, 2H); 2.19 (m, 2H); 1.65 1.61 (m, 4H); 1.32 (d, 3H, 7.1 Hz).

PAGE 225

225 GR24 Synthesis (127, 128 ): The compound was prepared following a literature procedure, 2 68 with modifications described below. Ethyl 2 (2 ethoxy 2 oxoethyl) 1 oxo 2,3 di h ydro 1H indene 2 carboxylate (124 ): Addition of indanone (8.7 g) to a solution of NaH 60 % dispersion and diethyl carbonate in DMF, and the subsequent addition of ethyl bromo acetate are very exothermic, especially the first addition The reaction mixture can heat up to 65 C spontaneously, without external heating. Vacuum distillation of the crude resulted in a product that was much less pure than the crude. The product was ob tained pure by column chromatography instead (hexanes:ethyl acetate 8:1). R f = 0.55 (hexanes:ethyl acetate, 2:1). 1 HNMR (300 MHz, CDCl 3 ): = 7.77 (d, 1H, 7.6 Hz); 7.63 (td, 1H, 7.5 Hz, 1.1 Hz); 7.50 (d, 1H, 7.9 Hz); 7.39 (t, 1H, 7.6 Hz); 4.17 4.06 (m, 4H ); 3.89 (d, 1H, 17.6 Hz); 3.33 (d, 1H, 17.3 Hz); 3.20 (d, 1H, 17.6 Hz); 2.79 (d, 1H, 17.3 Hz); 1.17 (td, 6H, 7.1 Hz, 0.7 Hz). 13 C NMR (CDCl 3 75 MHz) : = 200.9, 170.8, 169.7, 153.4, 135.4, 134.7, 127.7, 126.3, 124.8, 61.9, 60.8, 58.0, 38.8, 37.8, 14.0, 13 .9. 2 (1 oxo 2,3 dihy d ro 1H inden 2 yl)acetic acid (125 ): The reaction was run for 10 h, because after 3 h there was still a lot of unreacted substrate. 1 HNMR (300 MHz, acetone d 6 ): = 10.77 (br s, 1H); 7.69 7.64 (m, 2H); 7.56 (d, 1H, 7.6 Hz); 7.43 (t, 1 H, 7.3 Hz); 3.46 (dd, 1H, 18.4 Hz, 8.8 Hz); 3.01 2.88 (m, 3H); 2.69 (dd, 1H, 17.3 Hz, 8.0 Hz). 13 C NMR ( methanol d 4 75 MHz) : = 209.7, 175.5, 155.5, 137.7, 136.3, 128.6, 127.9, 124.7, 45.1, 35.6, 34.0.

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226 Racemic (3aR,8 bS) 3,3a,4,8b tetrahydr o 2H indeno[1,2 b]furan 2 one (120 ): To a solution of sodium borohydride (214 mg, 5.64 mmol) in 0.2 M NaOH (3 mL) was added dropwise a solution of 2 (1 oxo 2,3 dihy dro 1H inden 2 yl)acetic acid ( 125 ) (507 mg, 2.66 mmol) in 1M NaOH (5 mL), and the mixture was stirred at room temperature for 20 h. The mixture was cooled to 0 o C, acidified to pH 1 with 6M HCl, stirred for 30 min, extracted with diethyl ether, washed with brine, and dried over MgSO 4 The 1 HNMR analysis of the crude has in dicated that there was substantial amount of hydroxy acid, and the crude product was heated at reflux with a crystal of p TsOH in tetrahydrofuran (25 mL) for 20 h to complete lactonization The solvent was removed on the rotovap, and the crude was purified by column chromatography (hexanes: ethyl acetate 10:1 to 4:1) to yield the product as an oil that turned into white plates on standing (308 mg, 66 % yield). R f = 0.33 (hexanes: ethyl acetate, 2:1). 1 HNMR (300 MHz, CDCl 3 ): = 7.47 (d, 1H, 7.4 Hz); 7.38 7.32 (m, 1H); 7.28 (t, 2H, 7.4 Hz); 5.88 (d, 1H, 7.4 Hz); 3.40 3.27 (m, 2H); 2.94 2.85 (m, 2H); 2.38 (dd, 1H, 18.0 Hz, 5.4 Hz). 13 C NMR (CDCl 3 75 MHz) : = 176.8, 142.5, 138.7, 129.9, 127.5, 126.3, 125.3, 87.6, 37.8, 37.3, 3 5.6. IR (neat): 1771 cm 1 1167 cm 1 Potassium salt of racemic (E) ((3aR,8bS) 2 oxo 4,8b dihydro 2 H indeno [1,2 b]fur a n 3(3aH) ylidene)methanolate (126 ): Ethyl formate was used instead of methyl formate (Either can be used).

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227 5 bromo 3 methylfuran 2(5H) o ne (130 ): 3 methyl 2(5H) furanone ( 129 225 bromosuccinimide (520 mg, 2.9 mmol), and azobisisobutyronitrile (15 mg, 0.09 mmol) in carbon tetrachloride (5 mL) were heated at reflux for 4 h. The mixture was cooled to room temperature and then olids were filtered off and the crude was purified by Kugelrohr distillation (105 C, 2 mmHg) to give the product as lightly yellow oil (371 mg, 82 % yield). 1 H NMR (CDCl 3 300 MHz) : 7.21 (quintet, 1H, 1.6 Hz); 6.84 (quintet, 1H, 1.6 Hz), 2.03 (t, 3H, 1 .6 Hz). GR24 (127, 128 ) Hexanes:ethyl acetate:diethyl ether in a 10:1:1 ratio was used as an eluent instead of diisopropyl ether/ethyl acetate. First diastereomer: white solid, 89 mg, 37 % yield. R f = 0.20 (hexanes:ethyl acetate 2:1). 1 H NMR (CDCl 3 300 MHz) : = 7.50 (d, 1H, 7.2 Hz); 7.48 (d, 1H, 2.6 Hz); 7.37 7.22 (m, 3H); 6.97 (quintet, 1H, 1.5 Hz); 6.18 (quintet, 1H, 1.5 Hz); 5.95 (d, 1H, 7.9 Hz); 3.99 3.91 (m, 1H); 3.44 (dd, 1H, 16.7 Hz, 9.4 Hz); 3.11 (dd, 1H, 16. 9 Hz, 3.4 Hz); 2.04 (t, 3H, 1.5 Hz). 13 C NMR (CDCl 3 75 MHz) : 171.2, 170.1, 150.9, 142.5, 140.9, 138.8, 136.0, 130.0, 127.5, 126.4, 125.1, 113.2, 100.5, 85.9, 38.8, 37.3, 10.7. IR (neat): 1789, 1748, 1684 cm 1 HRMS (DART): calculated for [M+H] + : 299.0912, found: 299.0919. Second diastereomer: wh ite solid, 91 mg, 38 % yield. R f = 0.18 (hexanes:ethyl acetate 2:1). 1 H NMR and 13 C NMR were the same as for the first diastereomer.

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228 1,2 phenylenedimethanol (132 ): A solution of phthalic anhydride ( 131 ) (5.04 g, 34.0 mmol) i n tetrahydrofuran (50 mL) was added at 0 o C to the suspension of lithium aluminium hydride (2.37 g, 62.4 mmol) in tetrahydrofuran (80 mL), and the mixture was stirred at room temperature for 30 min and then at reflux for 3 h. The mixture was cooled to 0 o C and water (2.4 mL) was added in a dropwise fashion at 0 o C, followed by 15 % aqueous sodium hydroxide solution (2.4 mL) and water (6 mL). The mixture was diluted with ethyl acetate (100 mL), filtered through a plug of celite, and eluted with ethyl acetate The solvents were evaporated and the crude was purified by column chromatography with hexanes ethyl acetate (5:1 to 1:1 gradient) to yield 3.36 g (71 %) of the product as a white solid. R f = 0.20 (hexanes: ethyl acetate 1:1, KMnO 4 ). 1 H NMR (CDCl 3 300 MHz ) : 7.33 (m, 4H); 4.68 (s, 4H), 3.43 (s, 2H) ; 13 C NMR (CDCl 3 75 MHz) : 139.3, 129.7, 128.5, 64.1. (2 ((( tert butyldimethylsilyl)oxy)methyl)phenyl)methanol (133 ): 312 Using a literature procedure, A solution of 1,2 phen ylenedimethanol ( 132 ) (539 mg, 3.90 mmol) in tetrahydrofuran (10 mL) was added to a suspension of sodium hydride (154 mg of a 60 % dispersion in mineral oil, 3.85 mmol) in tetrahydrofuran (25 mL) at room temperature and the mixture was stirred overnight. T he resulting solution was then cooled to 0 o C and t butyldimethylsilyl chloride (580 mg, 3.85 mmol) was added in one portion. After 10 min at 0 o C, the reaction mixture was stirred at room temperature for 1.5 h. Saturated aqueous ammonium chloride (40 mL) was added and the mixture was

PAGE 229

229 extracted with ether. The combined extracts were washed with brine, dried (MgSO 4 ) and evaporated. The crude product was purified by column chromatography with hexanes:ethyl acetate 10:1 as eluent to yield 877 mg (90 %) of the title compound. R f = 0.38 (hexanes: ethyl acetate 10:1 KMnO 4 ). 1 H NMR (CDCl 3 300 MHz) : 7.25 7.37 (m, 4H); 4.80 (s, 2H); 4.67 (d, 2H); 3.25 (t, 1H); 0.92 (s, 9H); 0.12 (s, 6H); 13 CNMR (CDCl 3 75 MHz) : 2 ((( tert butyldimethylsilyl)oxy)meth yl )benzaldehyde (121 ): 311 Activated manganese dioxide (2.0 g, 90 % technical oxidation grade 63548 Fluka ) was added to the solution of the alcohol ( 133 ) obtained above (237 mg, 0.94 mmol) in petroleum ether (10 mL) at 0 o C, and the mixture was stirred at 0 10 o C for 3 h. The solids were then removed by filtration through a celite plug and the solvent evaporated to yield 220 mg (94 %) of the ti tle compound as colorless oil. R f = 0.54 (hexanes: ethyl acetate 10:1). 1 H NMR (CDCl 3 300 MHz) : 10.18 (s, 1H); 7.82 (m, 2H); 7.63 (m, 1H); 7.45 (m, 1H); 5.16 (s, 2H); 0.97 (s, 9H); 0.14 (s, 6H); 13 CNMR (CDCl 3 75 MHz) : 193.3, (2 (4 methyl 2,6,7 t rioxabicyclo[2.2.2]octan 1 yl)ethyl ) triphenylphosphonium bromide (122 ): The compound was prepared following a literature procedure 313

PAGE 230

230 Tert butyldimethyl((2 (3 (4 methyl 2,6,7 trioxabicyclo[2.2.2]octan 1 yl)pro p 1 en 1 yl )benzyl)oxy)silane (138 ). Lithium hexamethyldisilazide 1 M in THF (53.0 mL, 53.0 mmol) was added at room temperature to a suspension of phosphonium bromide salt 122 (27.35 g, 54.8 mmol) in tetrahydrofuran (200 mL) and the mixture was heated to reflux for 1 o C and a solution of aldehyde 121 (9.14 g, 36.5 mmol) in tetrahydrofuran (100 mL) was added. The reaction mixture o C for 1 h, and room temperature for 2 h. Water (70 mL) was added and the mixture was extracted with diethyl ether. The combined organic layers were washed with brine, dried (MgSO 4 ) and evaporated. The crude product was purified by column chromatography pretreated with 3 % triethylamine in hexane and eluted with 0.5 % triethylam ine in hexane to yield 13.83 g of the product as 1:1 mixt ure (97 % yield) of E/Z isomers. Z isomer: R f = 0.26 (hexanes: ethyl acetate 10:1); E isomer : R f = 0.22 (hexanes: ethyl acetate 10:1) 1 H NMR (CDCl 3 300 MHz) : 7.22 7.55 (m, 4H); 6.60 (d, J= 11.3 Hz) and 6.65 (d, 1H, J= 15.9 Hz); 5.89 (dt, J= 7.1, 11.3 Hz) and 6.17 (dt, 1H, J= 7.1, 15.9 Hz); 4.72 and 4.82 (s, 2H); 3.93 and 3.94 (s, 6H); 2.58 and 2.68 (dd, 2H, J= 7.1, 1.7 Hz); 0.98 (s, 9H); 0.81 a nd 0.82 (s, 3 H ); 0.13 (s, 6H). 13 C NMR (CDCl 3 75 MHz) : 139.1, 137.7, 135.2, 134.2, 133.8, 133.5, 129.4, 129.0, 128.8, 128.6, 128.5, 128.4, 126.9, 126.6, 126.3, 125.8, 125.7, 125.6, 125.3, 108.6, 108.4, 72.7, 63.0, 62.8, 40.7, 35.9, 30.4, 30.3, 25.9, 18 IR (neat): 3018 cm 1 2931 cm 1 2881 cm 1 1472 cm 1 1397 cm 1 1356 cm 1 1259 cm 1 1119 cm 1 994 cm 1 839 cm 1 ; HRMS (DART): calculated for C 22 H 35 O 4 Si [M+H] + : 391.2299, found: 391.2300.

PAGE 231

231 4 (2 (hydroxym e thyl )phenyl)but 3 enoic acid (119 ): Tert butyldimethyl((2 (3 (4 methyl 2,6,7 trioxabicyclo[2.2.2]octan 1 yl)pro p 1 en 1 yl)benzyl)oxy)silane ( 138 ) (500 mg, 1.28 mmol) was dissolved in methanol (20 mL), concentrated H 2 SO 4 was added (0.25 mL), and the mixture wa s stirred at room temperature for 1h. The reaction mixture was cooled to 0 o C, 1 M NaOH (15 mL) was added, the mixture was stirred at 0 o C for 20 min, and at room temperature for 15 min. Water was added (40 mL), the aqueous layer was washed 3 times with di ethyl ether, then acidified to pH 1, extracted with DCM, dried (MgSO 4 ), and evaporated to yield the product as a white solid (231 mg, 94 % yield). Based on 1 HNMR the desired product was obtained in 82 % yield, and the 2 (isochroman 3 yl)acetic acid ( 142 ) i mpurity in 12 % yield. 1 H NMR (acetone d 6 300 MHz): = 7.53 7.49 (m, 2H); 7.43 7.40 (m, 1H); 7.32 7.19 (m, 5H); 6.86 (dt, 1H, 15.7 Hz, 1.4 Hz); 6.74 (dt, 1H, 11.4 Hz, 1.4 Hz); 6.26 (dt, 15.7 Hz, 7.2 Hz); 5.95 (dt, 11.4 Hz, 7.4 Hz); 4.69 (s, 2H); 4.60 (s, 2H); 3.27 (dd, 2H, 7.2 Hz, 1.7 Hz); 3.14 (dd, 2H, 7.4 Hz, 1.7 Hz). 13 CNMR (acetone d 6 75 MHz) : = 173.0, 172.9, 141.0, 139.7, 136.5, 135.5, 130.7, 130.4, 129.6, 128.6, 128.2, 128.2, 128.1, 128.0, 127.5, 126.4, 125.9, 125.4, 62.7, 62.5, 38.7, 34.3. IR (neat): 3300 2750 cm 1 1716 cm 1 2 (isochroman 3 yl)acetic acid (142 ): 1 H NMR ( CDCl 3 300 MHz): = 7.82 (br s, 1H); 7.17 7.13 (m, 2H); 7.08 (dd, 1H, 5.1 Hz, 3.8 Hz); 6.98 (dd, 1H, 5.1 Hz, 3.8 Hz), 4.83 (s, 2H); 4.19 4.10 (m, 1H); 2.79 (d, 2H, 6.7

PAGE 232

232 Hz); 2.74 2.56 (m, 2H). 13 C NMR ( CDCl 3 75 MHz) : = 176.0, 134.0, 132.3, 128.7, 126.5, 126.1, 124.1, 71.2, 68.1, 40.6, 33.4. 4 (2 ((2,2,2 trifluoroacetoxy)m e thyl)phenyl)but 3 enoic acid (143 ): 1 H NMR (CDCl 3 300 MHz): = 10.58 (br s, 1H); 7.53 7.23 (m, 4H); 6.76 (d, 0.6 H, 11.1 Hz); 6.73 (d, 0.4 H, 15.8 Hz); 6.22 (dt, 0.4 H, 15.8 Hz, 7.3 Hz); 6.02 (dt, 0.6H, 11.1 Hz, 7.4 Hz); 5.42 (s, 0.8H); 5.33 (s, 1.2H); 3.34 (dd, 0.8H, 7.3 Hz, 1.5 Hz); 3.17 (dd, 1.2H, 7.4 Hz, 1.5 Hz). IR (neat): 3500 2500 cm 1 1784 cm 1 1713 cm 1 1173 cm 1 1145 cm 1 Allyl 4 (2 (hydr o xymethyl)phenyl)but 3 enoate (139 ): Tert butyldimethyl((2 (3 (4 methyl 2,6,7 trioxabicyclo[2.2.2]octan 1 yl)pro p 1 en 1 yl)benzyl)oxy)silane ( 138 ) (1.435 g, 3.67 mmol) was dissolved in acidic allyl alcohol (20 mL + 0.45 mL concentrated HCl) and stirred at 50 o C for 2.5 h. The mixture was cooled to room temperature, DCM (80 mL) was added, the organic layer was washed with water and brine, dried (MgSO 4 ), and eva porated. The crude was purified by column chromatography (hexanes: ethyl acetate 20:1 to 5:1) to give 677 g of the product (79 % yield). R f = 0.17 (hexanes: ethyl acetate 4:1). 1 H NMR (CDCl 3 300 MHz): = 7.51 7.48 (m, 0.4 H); 7.45 7.42 (m, 1H); 7.36 7.23 (m, 3.2 H); 7.19 7.15 (m, 1H) ; 6.85 (d, 0.4H,

PAGE 233

233 15.9 Hz); 6. 81 (d, 1H, 11.3 Hz); 6.25 (dt, 0.4 H, 15.9 Hz, 7.4 Hz); 6.02 5.82 (m, 2.4); 5.37 5.21 (m, 2.8H); 4.74 (d, 0.8H, 5.4 Hz); 4.65 4.61 (m, 2.8H); 4.57 (dt, 2H, 5.9 Hz, 1.3 Hz); 3.32 (dd, 0.8H, 7.2 Hz, 1.6 Hz); 3.18 (dd, 2H, 7.4 Hz, 1.7 Hz); 2.00 (t, 1H, 5.4 Hz, OH); 1.71 (t, 0.4H, 5.4 Hz, OH). 13 CNMR (CDCl 3 75 MHz) : = 171.4, 171.2, 138.8, 137.4, 135.5, 134.6, 131.8, 131.7, 130.5, 130.4, 128.8, 128.1, 127.8, 127.7, 127.5, 127.5, 127.2, 126.0, 124.3, 123.8, 118.3, 65.3, 62.9, 62.7, 38.3, 33.7. IR (neat): 3448 cm 1 1733 cm 1 1165 cm 1 HRMS (ESI): calculated for [M+Na] + : 255.0992, found: 255.0988. Allyl 4 (2 chloro methyl)phenyl)but 3 enoate (140 ): Allyl 4 (2 (hydr oxymethyl)phenyl)but 3 enoate ( 139 ) (476 mg, 2.05 mmol) was dissolved in THF (25 mL). At 0 o C triethylamine (0.46 mL, 3.30 mmol) was added, followed by methane sulfonate chloride (0.25 mL, 3.23 mmol), and th e mixture was stirred at 0 o C for 15 min. Lithium chloride (287 mg, 6.76 mmol) was added at 0 o C, and the mixture was stirred at room temperature for 6 h, washed with water, dried (MgSO 4 ), and evaporated. Purification by column chromatography (hexanes: eth yl acetate, 20:1) yielded the pure product (473 mg, 92 % yield). R f = 0.55 (hexanes: ethyl acetate 4:1). 1 H NMR (CDCl 3 300 MHz): = 7.53 7.50 (m, 0.6H); 7.43 7.40 (m, 1H); 7.36 7.28 (m, 3.6H); 7.26 7.21 (m, 1.2H); 6.90 6.83 (m, 1.6H); 6.30 (dt, 0.6 H, 15.5 Hz, 7.3 Hz); 6.06 (dt, 1H, 11.4 Hz, 7.3 Hz); 6.00 5.85 (m, 1H); 5.39 5.23 (m, 2.8H); 4.66 4.60 (m, 4.2H); 4.65 (s, 1.2H); 4.57 ( s, 2H); 3.36 (dd,1.2H, 7.0 Hz); 3.19 (dd, 2H, 7.3 Hz,1.8 Hz). 13 CNMR (CDCl 3 75

PAGE 234

234 MHz) : = 171.1, 171.0, 136.5, 136.0, 135.4, 134.2, 131.9, 130.1, 129.9, 129.9, 129.8, 129.5, 129.1, 128.6, 127.9, 127.9, 126.7, 125.4, 125.1, 118.5, 118.4, 65.5, 65.4, 44.4, 3 8.5, 34.0. IR (neat): 1734 cm 1 1160 cm 1 HRMS (ESI): calculated for [M+H] + = 251.0833 found 251.0837. (E) methyl 4 (2 hydro xymethyl)phenyl)but 3 enoate (152 ) and (Z) methyl 4 (2 hydro xymethyl)phenyl)but 3 enoate (151 ) : The orthoester 138 (9.00 g, 23.0 mmol) was stirred in 0.2 M sulfuric acid methanolic solution (200 mL) at room temperature for 1 h. Water was then added and the resulting mixture was extracted with methylene chloride. The combined organic layers were was hed with brine, dried (MgSO 4 ), and evaporated. The crude product was purified by column chromatography (hexane/ethyl acetate 4:1) to give 1.646 g of 152 and 1.474 g of 151 (66 %). Data for 152 : R f = 0.21 (hexane:ethyl acetate :4:1); 1 H NMR (CDCl 3 300 MHz) : 7.47 (m, 1H); 7.19 7.32 (m, 3H); 6.78 (d, 1H, J= 15.9 Hz); 6.19 (m, 1H); 4.66 (s, 2H); 3.68 (s, 3H); 3.25 (dd, 2H, J= 7.2, 1.5 Hz); 2.01 (br s, 1H); 13 C NMR (CDCl 3 75 MHz) : 172.0, 137.4, 135.6, 130.3, 128.2, 127.9, 127.5, 126.1, 123.9, 63.0, 51.8, 38. 2; IR (neat): 3461 cm 1 1733 cm 1 ; HRMS (DART): calculated for C 12 H 15 O 3 [M+H] + : 207.1016, found: 207.1018. Data for 151 : R f = 0.27 (hexane:ethyl acetate :4:1); 1 H NMR (CDCl 3 300 MHz) : 7.39 7.42 (m, 1H); 7.23 7.31 (m, 2H); 7.12 7.15 (m, 1H); 6.76 (d, 1H, J= 11.1 Hz); 5.92 (dt, 1H, J= 7.3, 11.1 Hz); 4.59 (d, 2H, J= 5.4 Hz); 3.64 (s, 3H); 3.14 (dd, 2H, J= 7.3, 1.5 Hz); 2.11 (br s, 1H); 13 C NMR (CDCl 3 75 MHz) : 172.2, 138.9, 134.7, 130.6, 128.9, 127.9,

PAGE 235

235 127.6, 127.4, 124.5, 62.9, 51.9, 33.6; IR (neat): 3466 cm 1 1726 cm 1 ; HRMS (DART): calculated for [M+H] + : 207.1016, found: 207.1010. (E) methyl 4 (2 formylphenyl)but 3 enoate (154 ) : A solution of alcohol 152 (470 mg, 2.28 mmol) in DCM (4 mL) was added to a mixture of PCC ( 688 mg, 3.19 mmol) and celite (688 mg) in DCM (13 mL) at room temperature. After 1 h, the mixture was diluted with diethyl ether, filtered through celite, and the solvents were evaporated. Flash column chromatography (hexane ethyl acetate 10:1) provided 432 mg of 154 as a colorless oil (93 %); 1 H NMR (CDCl 3 300 MHz) : 10.26 (s, 1H); 7.81 (m, 1H); 7.34 7.59 (m, 4H); 6.28 (dt, 1H, J= 7.2, 15.9 Hz); 3.74 (s, 3H); 3.35 (dd, 2H, J= 7.2, 1.6 Hz); 13 C NMR (CDCl 3 75 MHz) : 192.4, 171.6, 139.3, 133.7, 132.6, 1 31.7, 129.6, 127.7, 127.6, 127.2, 51.9, 38.2; IR (neat): 2849 cm 1 2745 cm 1 1735 cm 1 1698 cm 1 ; HRMS (DART): calculated for C 12 H 13 O 3 [M+H] + : 205.0859, found: 205.0868. (Z) methyl 4 (2 formylphenyl)but 3 enoate (153 ) : A solution of alcohol 151 (1.074 g, 5.21 mmol) in DCM (15 mL) was added to a mixture of PCC (1.590 g, 7.38 mmol) and celite (1.590 mg) in DCM (35 mL) at room temperature. After 1 h, the mixture was diluted with diethyl ether, filtered through celite, an d the solvents evaporated. Flash column chromatography (hexane ethyl acetate

PAGE 236

236 10:1) yielded 994 mg of 153 as a colorless oil (93 %); 1 H NMR (CDCl 3 300 MHz) : 10.21 (s, 1H); 7.90 (d, 1H, J= 7.8 Hz); 7.58 (m, 1H); 7.44 (m, 1H); 7.29 (d, 1H, J= 7.5 Hz); 7.0 6 (d, 1H, J= 11.7 Hz); 6.07 6.16 (m, 1H); 3.68 (s, 3H); 3.11 (dd, 2H, J= 7.6, 1.1 Hz); 13 C NMR (CDCl 3 75 MHz) : 191.5, 171.1, 138.8, 133.4, 133.4, 129.9, 129.2, 128.9, 127.6, 126.1, 51.6, 33.5; IR (neat): 2854 cm 1 2740 cm 1 1731 cm 1 1695 cm 1 ; HRMS (DART): calculated for C 12 H 13 O 3 [M+H] + : 205.0859, found: 205.0867. Representative procedure for trimethylsilyl triflate catalyzed cyclization: Trimethylsilyl triflate (0.85 mL of a 0.11 M solution in CH 2 Cl 2 0.094 mmol) was added to solution of 154 (0.098 g, 0.48 mmol) in CH 2 Cl 2 (1.0 mL) at 0 C. After 2h, when thin layer chromatographic analysis indicated complete consumption of 154 water was added and the reaction mixture was extracted with CH 2 Cl 2 The combined organ ic extract was washed with brine, dried over magnesium sulf ate, and concentrated to 1 mL. Flash chromatography (gradient, hexanes:ethyl acetate 10:1 to 4:1) afforded 0.060 g of a mixture of 155 and 156 in an 80:20 ratio (60 % yield) and 0.007 g of 157 (7% ). D ata for 155 : 1 H NMR (CDCl 3 300 MHz): 7.43 7.54 (m, 4H), 6.04 (d, 1H, J = 7.2 Hz), 4.72 (s, 1H), 3.44 (s, 3H), 3.32 (m, 1H), 2.94 (dd, 1H, J = 18.2, 10.6 Hz), 2.35 (dd, 1H, J = 18.2, 6.9 Hz); 13 C NMR (CDCl 3 75 MHz): 176.1, 141.1, 139.6, 130.1, 130.1, 126.5, 126.3, 88.3, 85.9, 56.6, 44.4, 32.9; IR (neat) 1773 cm 1 1172 cm 1 1092 cm 1 1024 cm 1 ; HRMS (DART) calculated for C 12 H 13 O 3 [M+H] + : 205.0859, found: 205.0857.

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237 D ata for 156 : 1 H NMR (CDCl 3 300 MHz): 7.35 7.49 (m, 4H), 5.72 (d, 1H, J = 7.2 Hz), 4.82 (d, 1H, J = 6.9 Hz), 3.55 3.66 ( m, 1H), 3.48 (s, 3H), 2.73 (dd, 1H, J = 18.2, 7.2 Hz), 2.58 (dd, 1H, J = 18.2, 10.2 Hz). 13 C NMR (CDCl 3 75 MHz): 176.9, 141.9, 137.9, 130.0, 129.4, 126.0, 125. 5, 83.6, 82.0, 57.7, 41.9, 28.0. IR (neat) 1773 cm 1 1176 cm 1 1112 cm 1 1092 cm 1 1025 cm 1 HR MS (DART) calculated for C 12 H 13 O 3 [M+H] + : 205.0859, found: 205.0859. Data for 157 : 1 H NMR (CDCl 3 300 MHz): 7.45 (d, 1H, J = 7.5 Hz), 7.16 7.28 (m, 3H), 6.69 (s, 1H), 5.09 (s, 1H), 3.74 (s, 3H), 3.46 (d, 1H, J = 17.0 Hz), 3.39 (d, 1H, J = 17. 0 H z), 3.06 (s, 3H). 13 C NMR (CDCl 3 125 MHz): 171.2, 142.9, 141.8, 141.2, 131.3, 128.5, 125.4, 123.9 121.1, 83.8, 52.4, 52.0, 33.6. IR (neat) 1735 cm 1 HR MS (DART) calculated for C 13 H 15 O 3 [M+H] + : 219.1016, found: 219.1011. Representative procedure fo r triflic acid catalyzed cyclization: Triflic acid (0.21 mL of a 0.23 M solution in CH 2 Cl 2 0.048 mmol) was added to solution of 154 (0.098 g, 0.48 mmol) in CH 2 Cl 2 (4.0 mL) at 78 C. After 1h, the mixture was warmed to 0 C and stirred for 3 h, when thin layer chromatographic analysis indicated complete consumption of 154 The reaction mixture was then poured onto ice and extracted with CH 2 Cl 2 The combined organic extract was washed with brine, dried over magnesium sulf ate, and concentrated to 1 mL. Flash chromatography (gradient, hexanes:ethyl acetate 10:1 to 4:1) afforded 0.067 g of a mixture of 155 and 156 in a 99:1 ratio (68% yield) and 0.002 g of 157 (2%). The compounds exhibited spectral data identical to those obtained above.

PAGE 238

238 Benzyl (E) 4 (2 hydroxymethyl)phenyl)but 3 enoate (165 ): The E alcohol methyl ester 152 (containing 4 % of Z isomer) was added to a solution of concentrated hydrochloric acid (0.36 mL) in benzyl alcohol (8 mL), and the mixture was heated at 50 o C for 2h. Hydrochloric acid and benzyl alcohol were distilled off by vacuum distillation, and the crude product (ca. 900 mg) was purified by column chromatography (hexanes:ethyl acetate 10:1) to give the product in 95 % yield (290 mg). R f = 0.17 (hexanes :ethyl acetate, 4:1). 1 H NMR (CDCl 3 300 MHz): = 7.50 7.47 (m, 1H); 7.38 7.24 (m, 8H); 6.82 (d, 1H, 15.7 Hz); 6.24 (dt, 1H, 15.7 Hz, 7.3 Hz); 5.16 (s, 2H); 4.70 (s, 2H); 3.33 (dd, 2H, 7.3 Hz, 1.5 Hz); 1.73 (br s, 1H, OH). Benzyl (E) 4 (2 formylphenyl)but 3 enoate ( 1 66 ): A solution of alcohol 165 (290 mg, 1.03 mmol) in DCM (2 mL) was added to a mixture of PCC (321 mg, 1.49 mmol) and celite (320 mg) in DCM (10 mL). After stirring for 1h at room temperature, the mixture was diluted with diethyl ether, filtered through c elite, and evaporated. Column chromatography (hexanes: ethyl acetate, 20:1) gave the product as a colorless oil (248 mg, 86 %). R f = 0.50 (hexanes:ethyl acetate, 4:1). 1 H NMR (CDCl 3 300 MHz): = 10.22 (s, 1H); 7.79 (d, 1H, 7.4 Hz); 7.56 7.30 (m, 9H); 6.27 (dt, 1H, 15.9 Hz, 6.9 Hz); 5.17 (s, 2H); 3.38 (dd, 2H, 6.9 Hz, 1.5 Hz). 13 CNMR (CDCl 3 75

PAGE 239

239 MHz) : = 192.3, 170.9, 139.3, 135.6, 133.6, 132.6, 131.6, 129.7, 128.5, 128.3, 128.2, 127.7, 127.6, 1 27.2, 66.6, 38.3. Racemic (3aS,4S,8bS) 4 (benzyloxy) 3,3a,4,8b tetrahydro 2 H indeno[1,2 b]furan 2 one (167 ) : The product was made from b enzyl (E) 4 (2 formylphenyl)but 3 enoate ( 166 ) (117 mg, 0.42 mmol) (containing 4 % o f Z isomer), using a r epresentative procedure for triflic acid catalyzed cyclization (0.1 equivalent of TfOH). The product was obtained in 66 % yield as mixture of 167 and 168 in a 95:5 ratio. The compound 169 resulting from single cyclization was also is olated in 3 % yield. R f = 0.41 (hexanes:ethyl acetate, 2:1). 1 H NMR (CDCl 3 5 00 MHz): = 7.53 7.31 (m, 9H); 6.05 (d, 1H, 7.0 Hz); 4.90 (d, 1H, 1.5 Hz); 4.66 (s, 2H); 3.56 3.19 (m, 1H); 2.88 (dd, 1H, 18.2 Hz, 10.7 Hz); 2.29 (dd, 1H, 18.2 Hz, 6.7 Hz). 13 CNM R (CDCl 3 12 5 MHz) : = 176.1, 141.5, 139.5, 137.5, 130.1, 130.0, 128.5, 127.9, 127.7, 126.4, 126.2, 86.4, 85.9, 71.3, 44.9, 32.8. Benzyl 2 (1 (benzyloxy) 1 H inden 2 yl)acetate (169 ): 1 H NMR (CDCl 3 3 00 MHz): = 7.47 (d, 1 H, 7.1 Hz); 7.37 7.21 (m, 13H); 6.70 (s, 1H); 5.25 (s, 1H); 5.15 (s, 2H); 4.24 (s, 2H); 3.51 3.49 (m, 2H).

PAGE 240

240 Allyl 4 (2 formylphenyl)but 3 enoate (170 ): A solution of alcohol 139 (280 mg, 1.21 mmol) in dichloromethane (3 mL) was added to a mixture of PCC (364 mg, 1.69 mmol) and Celite (364 mg) in dichloromethane (9 mL). After 1h of stirring at room temperature, the mixture was diluted with diethyl ether, filtered through celite, and the solvents were evaporated. Purificati on by column chromatography (hexanes: ethyl acetate 10:1) gave the product as a c olorless oil (277 mg, 86 % yield) R f = 0.74 (hexanes:ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 10.27 (s, 1H); 10 .23 (s, 0.6 H); 7.92 (dd, 0.6 H 7.6 Hz, 1.4 Hz); 7.82 (s, 1H, 7.4 Hz); 7.64 7.53 (m, 2.6H); 7.48 7.41 (m, 2H); 7.36 7.30 (m, 1.2H); 7.09 (d, 1H, 11.3 Hz); 6.29 (dt, 1H, 15.8 Hz 7.1 Hz ); 6.15 (dt, 1H, 11.3 Hz, 7.6 Hz); 6.02 5.85 (m, 1.6H); 5.39 5.22 (m, 3.2H); 4.65 (dt, 2H, 6.0 Hz, 1.1 Hz); 4.60 (dt, 1.2 H, 6.0 Hz, 1.1 Hz); 3.38 (dd, 2H, 7.1 Hz, 1.4 Hz); 3.16 (dd, 1.2H, 7.6 Hz, 1.6 Hz). 13 CNMR (CDCl 3 75 MHz) : = 192.4, 191.9, 170.8, 170.7, 139.4, 139.1, 133.7, 132.6, 131.9, 131.8, 131.7, 130.2, 129.7, 129.6, 129.3, 127.8, 127.7, 127.6, 127.2, 126.2, 118.5, 65.5, 38.3, 34.0.

PAGE 241

241 Racemic (3aS,4S,8bS) 4 (allyloxy) 3,3a,4,8b tet rahydro 2 H indeno[1,2 b] furan 2 one (171 ) and racemic (3aS,4R ,8bS) 4 (allyloxy) 3,3a,4,8b tetrahydro 2 H indeno[1,2 b] furan 2 one (1 7 2 ): The product was made from allyl 4 (2 formylphenyl)but 3 enoate (170 ) using a r epresentative procedure for triflic aci d catalyzed cyclization (3x 0.15 equivalent of TfOH). R f = 0.51 (hexanes:ethyl acetate, 2:1). 1 H NMR (CDCl 3 3 00 MHz): 7.55 7.37 (m, 171 4H, 172 3.2 H); 6.05 (d, 171 1H, 6.9 Hz); 6.03 5.89 (m, 171 1H, 172 0.8H); 5.73 (d, 172 0.8H, 7.4 Hz); 5.41 5.22 (m, 171 2H, 172 1.6H); 5.00 (d, 172 0.8H, 6.8 Hz); 4.86 (d, 171 1H, 1.7 Hz); 4.18 4.11 (m, 171 2H, 172 1.6H); 3.67 3.56 (m, 172 0.8 Hz); 3.33 (dtd, 171 1H, 10.6 Hz, 6.9 Hz, 1.7 Hz); 2.93 (dd, 171 1H, 18.3 Hz, 10.6 Hz); 2.78 (dd, 172 0.8H, 18.4 Hz, 7.4 Hz); 2.58 (dd, 172 0.8H, 18.4 Hz, 10.3 Hz); 2.36 (dd, 171 1H, 18.3 Hz, 6.9 Hz). 13 C NMR (CDCl 3 7 176.9, 176.1, 142.0 141.2, 139.6, 137.9, 134.2, 134.1, 130.1, 130.0, 130.0, 129.4, 126.5, 126.2, 126.0, 125.5, 117.7, 117.5, 86.3, 85.9, 83.6, 79.7, 71.0, 70.2, 45.0, 42.3, 32.9, 28.5. HRMS (DART): calculated for C 1 4 H 1 4 O 3 [M+ N H 4 ] + : 248.1281, found: 248.1283.

PAGE 242

242 Racemic (3aS,4S,8bS) 4 hydroxy 3,3a,4,8b tetrahydro 2H indeno[1,2 b]furan 2 one (173 ) and racemic (3aS,4R ,8bS) 4 hydroxy 3,3a,4,8b tetrahydro 2H indeno[1,2 b]furan 2 one ( 174 ): A mixture of r acemi c (3aS,4S,8bS) 4 (allyloxy) 3,3a,4,8b tetrahydro 2 H indeno[1,2 b] furan 2 one ( 171 ) and racemic (3aS,4R,8bS) 4 (allyloxy) 3,3a,4,8b tetrahydro 2 H indeno[1,2 b] furan 2 one ( 172 ) in a 55:45 ratio (30 mg, 0.13 mmol) was dissolved in THF (2 mL). Anhydrous zin c chloride (23 mg, 0.17 mmol) was added, the mixture was stirred for 15 min, then triphenylphosphine palladium tetrakis was added (7 mg, 6.5 mmol) was added. After stirring for 1h at room temperature, the mixture was diluted with ethyl acetate (4 mL) and water (1 mL), acidified to pH4 with 1M HCl, extract ed with ethyl acetate, washed with brine, dried over MgSO 4 and evaporated. Purification by column chromatography (gradient of hexanes and ethyl acetate 6:1 to 2:1) afforded 173 in 41 % yield (10.2 mg) and 174 in 35 % yield (8.8 mg). Data for 173 : R f = 0.17 (hexanes:ethyl acetate, 1:1). 1 H NMR (CDCl 3 300 MHz): = 7.53 7.41 (m, 4H); 6.02 (d, 1H, 6.9 Hz); 5.13 (d, 1H, 1.8 Hz); 3.27 3.19 (m, 1H); 2.93 (dd, 1H, 18.3 Hz, 10.4 Hz); 2.44 (dd, 1H, 18.3 Hz, 5.9 Hz). 13 CNMR (CDCl 3 75 MHz) : = 176.3, 143.9, 138.8, 130.7, 130.1, 126.6, 125.4, 85.8, 80.4, 47.9, 33.0. Data for 174 : R f = 0.22 (hexanes:ethyl acetate, 1:1). 1 H NMR (CDCl 3 300 MHz): = 7.54 7.39 (m, 4H); 5.72 (d, 1H, 7.0 Hz); 5.29 (d, 1H, 7.0 Hz); 3.54 3.44 (m, 1H); 2.86

PAGE 243

243 (dd, 1H, 18.5 Hz, 5.9 Hz); 2.66 (dd, 1H, 18.5 Hz, 10.3 Hz). 13 CNMR (CDCl 3 75 MHz) : = 177. 2, 144.1, 138.2, 130.5, 129.6, 126.3, 125.2, 84.3, 73.4, 43.4, 28.8. 2 hyd r oxy 3,4 dimethylbenzaldehyde (182 ): Anhydrous magnesium chloride (23.4 g, 246 mmol) and dry triethylamine (84.0 mL, 603 mmo l) were added to a sol ution of 2,3 dimethylphenol (19.5 g, 160 mmol) in acetonitrile (650 mL), and the mixture was stirred for 15 min. Dry (P 2 O 5 ) paraformaldehyde (28.4 g, 946 mmol) was then added and the reaction mixture was heated under reflux for 45 min. The mixture was coo led to room temperature, 1M HCl (200 mL) was added, and the aqueous layer was extracted with ethyl acetate. The combined organic extracts were dried over magnesium sulfate, filtered through celite, evaporated, and the residue was purified by flash chromato graphy (hexane:ethyl acetate 25:1), yielding 17.9 g of 182 as a white solid (74 % yield). R f = 0.54 (hexanes: ethyl acetate, 10:1). 1 H NMR (CDCl 3 300 MHz): 11.36 (s, 1H), 9.80 (s, 1H), 7.28 (d, 1H, J= 7.8 Hz), 6.82 (d, 1H, J= 7.8 Hz), 2.33 (s, 3H), 2.18 (s, 3H). 13 CNMR (CDCl 3 75 MHz): 196.1, 159.8, 147.1, 130.7, 124.8, 121.5, 118.4, 20.9, 10.7. IR (neat): 3056 (broad), 2841, 2745, 1644, 1622, 1499, 131 1, 1277, 770, 752, 735 cm 1

PAGE 244

244 6 formyl 2,3 dimethylphe n yl trifluoromethanesulfonate (180 ): 78 C to a solution of phenol 182 (9.5 g, 63 mm ol) and triethylamine (30.0 mL, 215 mmol) in Water (20 mL) was added, the layers were separated, and the organic layer was washed with water (20 mL), dried over magnesium sulfate and evaporated. The residue was purified by column chromatography (hexane:ethyl acetate 25:1) to give 16.8 g (95 % yield) of the pure product as colorless oil R f = 0.50 (hexanes: ethyl acetate, 10:1). 1 H NMR (CDCl 3 300 MHz): 10.19 (s, 1H), 7.76 (d, 1H, J= 7.8 Hz), 7.35 (d, 1H, J= 7.8 Hz), 2.43 (s, 3H), 2.35 (s, 3H). 13 CNMR (CDCl 3 75 MHz): 186.8, 147.4, 147.2, 131.2, 129.9, 127.5, 127.4, 118.5 (q, J= 1277 Hz), 20.7, 13.0. IR: 2959, 2889, 2754, 1696, 1612, 1567, 1427, 1407, 1254, 1215, 1137, 1055, 953, 895, 809, 770 cm 1 2 (4 hydroxybut 1 yn 1 yl) 3,4 dimethylbenzaldehyde (186 ): Diethylamine (2.43 mL, 23 mmol) was added to a mixture of palladium tetrakis (136 mg, 0.12 mmol) and copper iodide (52 mg, 0.27 mm ol) in tetrahydrofuran (10 mL). A solution of triflate 180 (1.040 g, 3.91 mmol) in tetrahydrofuran (10 mL) was then ad ded, followed by a solution of 3 bu tynol (356 mg, 5.08 mmol) in tetrahydrofuran (5 mL). The mixture was heated to reflux for 3 h, cooled d own to room temperature, diluted with diethyl ether

PAGE 245

245 (80 mL), washed with 1M HCl, washed with brine, dried over magnesium sulfate, and evaporated. The residue was purified by chromatography column (hexane:ethyl acetate gradient 10:1 to 3:1) to yield 186 as a white solid (790 mg, 78 % yield). R f = 0.23 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): 10.38 (s, 1H), 7.59 (d, 1H, J= 7.8 Hz), 7.15 (d, 1H, J= 7.8 Hz), 3.88 (t, 2H, J= 6.3 Hz), 2.90 (br s, 1H), 2.79 (t, 2H, J= 6.3 Hz), 2.39 (s, 3H), 2.31 (s, 3H). 13 CNMR (CDCl 3 75 MHz): 192.6, 143.5, 139.7, 134.3, 129.3, 126.1, 125.8, 98.2, 77.4, 61.0, 2 4.1, 20.9, 17.0. IR: 3429, 2945, 2882, 2745, 2227, 1687, 1584, 1250, 1048, 821, 779 cm 1 4 (6 (1,3 dioxan 2 yl) 2,3 dimethylphenyl)but 3 yn 1 ol (187 ) : A mixture of aldehyde ( 186 ) (496 mg, 2.45 mmol), 1,3 propanediol (0.36 mL, 4.94 mmol), p TsOH (40 mg), and benzene (35 mL) was heated under reflux for 1 h. Water formed during the reaction was removed by a Dean Stark trap. The cooled reaction mixture was diluted with diethyl ether (80 mL), washed with saturated aqueous solution of sodium bicarbonate, washed with brine, dried over magnesium sulfate, and evaporated. The residue was purified by column chromatography (hexanes:ethyl acetate gradient 10:1 to 2:1) to yield 187 as a white solid (523 mg, 82 % yield). R f = 0.20 (he xanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): 7.38 (d, 1H, J= 7.8 Hz), 7.10 (d, 1H, J= 7.8 Hz), 5.83 (s, 1H), 4.23 4.28 (m, 2H), 3.96 4.04 (m, 2H), 3.81 (t, 2H, J= 6.4 Hz), 2.75 (t, 2H, J= 6.4Hz), 2.36 (s, 3H+1H), 2.24 (m, 3H+1H), 1.42 (d, 1H, J= 13.3 Hz). 13 CNMR (CDCl 3 75 MHz): 138.4, 137.6, 137.0, 129.4, 122.8, 121.3, 100.7, 94.7, 79.1,

PAGE 246

246 67.5, 61.2, 25.7, 24.1, 20.3, 17.4. IR: 3424, 2962, 2926, 2857, 2228, 1595, 1458, 1388, 1152, 1108, 1040, 992, 823 cm 1 ( E) 4 (6 (1,3 dioxan 2 yl) 2,3 dimethylphenyl)but 3 en 1 ol (188 ) : The solution of alkyne 187 (170 mg, 0.65 mmol) in tetrahydrofuran (4 mL) was added to a suspension of lithium aluminium hydride (37 mg, 0.98 mmol) in tetrahydrofuran (3 mL) at 0 C. The mix ture was then heated under reflux for 3 h, cooled down to 0 C, water was added (30 L), followed by 15 % sodium hydroxide (30 L), and water (90 L). The mixture was diluted with ethyl acetate, filter through a celite plug, and evaporated. The residue was purified by chromatography column (hexane:ethyl acetate gradient 3:1 to 2:1) to yield 188 as a white solid (163 mg, 95 % yield). R f = 0.23 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 7.40 (d, 1H, J= 8.1 Hz), 7.09 (d, 1H, J= 8.1 Hz), 6.59 (d, 1H, J= 16.2 Hz), 5.64 (dt, 1H, J= 16.2 Hz, 7.2 Hz), 5.56 (s, 1H), 4.18 4.24 (m, 2H), 3.86 3.95 (m, 2H), 3.77 (t, 2H, J= 6.3 Hz), 2.53 (dtd, 2H, J= 7.2 Hz, 6.3 Hz, 1.5 Hz), 2.19 2.26 (m, 7H), 1.72 (br s, 1H), 1.36 1.42 (m, 1H). 13 CNMR (CDCl 3 75 MHz): 13 7.2, 136.2, 134.1, 133.9, 132.1, 130.0, 128.5, 123.5, 100.9, 67.3, 61.9, 36.7, 25.7, 20.6, 16.6. IR: 3423, 2957, 2858, 1380, 1106, 983 cm 1

PAGE 247

247 Dess Martin Periodinane (191 ) The r eagent (11.7 g) was prepared by oxidation of 2 iodobenzoic acid to IBX ( 193 ) with oxone 348 followed by acylation, using 0.5 % p toluenesulfonic acid and acetic anhydride 345 (E) 4 (6 (1,3 dioxan 2 yl) 2 ,3 dimethylphenyl)but 3 enal (189 ): A solution of (E) 4 ( 6 (1,3 dioxan 2 yl) 2,3 dimethylphenyl)but 3 en 1 ol ( 188 ) (1.224 g, 4.66 mmol) in dichloromethane (15 mL) was added to a solution of DMP ( 191 ) (2.777g, 6 .55 mmol) in dichloromethane (35 mL), and the reaction mixture was stirred at room temperature for 1h. The reaction mixture was then poured into an Erlenmeyer flask containing diethyl ether (180 mL) and sodium thiosulphate (12 g) dissolved in saturated aqueous sodium bicarbonate solution (120 mL), and the mixture was stirred for 10 min. The layers were sep arated, the aqueous layer was extracted with diethyl ether, the combined organic layers were washed with saturated aqueous sodium bicarbonate and brine, dried over MgSO 4 and evaporated to yield the product as y ellow oil that was pure by 1 HNMR (1.116 g, 92 % yield) R f = 0.50 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 9.83 (t, 1H, 1.6 Hz); 7.46 (d, 1H, 7.8 Hz); 7.11 (d, 1H, 7.8 Hz); 6.59 (d, 1H, 16.2 Hz); 5.76 (dt, 1H, 16.2 Hz, 7.2 Hz); 5.62 (s, 1H); 4.16 4.23 (m, 2H); 3.91 4.00

PAGE 248

248 (m, 2H); 3.40 3.44 (m, 2H); 2.26 (s, 3H); 2.16 2.27 (m, 1H); 2.17 (s, 3H); 1.37 1.44 (m, 1H). 13 CNMR (CDCl 3 75 MHz) : = 199.4, 137.0, 135.5, 134.1, 134.0, 132.4, 128.9, 125.1, 123.4, 100.2, 67.2, 47.8, 25.7, 20.5, 16.5. IR: 2963, 2854, 2726, 1721, 1106, 986, 822 cm 1 (E) 4 (6 (1,3 dioxan 2 yl) 2,3 di m ethylphenyl)but 3 enoic acid (190 ): A solution of sodium chlorite (80 % w/w, 437 mg, 3.86 mmol) and potassium dihydrogenphosphate (527 mg, 3.86 mmol) in water (9 mL) was added via a glass pipet over 20 min into a solution of aldehyde ( 189 ) (335 mg, 1.29 mmol) and 2 methyl 2 butene (5.5 mL, 52 mmol) in tert butanol (27 mL ). The mixture was stirred at room temperatu re for 3.5 h extracted with ethyl acetate, washed with water and brine, dried (MgSO 4 ), and evaporated. The crude product was purified by column chromatography (hexanes: ethyl acetate, 10:1 to 1:1 gra dient) to give a yellow solid (305 mg) that was further purified by recrystallization. The yellow solid was dissolved in ethyl a cetate at room temperature, hexanes were added until the solution became milky, the mixture w hite solid (249 mg, 70 %) R f = 0.10 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 7.44 (d, 1H, 7.8 Hz); 7.11 (d, 1H, 7.8 Hz); 6.60 (d, 1H, 15.9 Hz); 5.78 (dt, 1H, 15.9 Hz, 7.2 Hz); 5.63 (s, 1H); 4.22 4.17 (m, 2H); 3.97 3.88 (m, 2H); 3.36 (dd, 2H, 7.2 Hz, 1.5 Hz); 2.26 (s, 3H); 2.28 2.16 (m, 1H); 2.17 (s, 3H); 1.41 1.36 (m, 1H). 13 CNMR

PAGE 249

249 (CDCl 3 75 MHz) : = 177.8, 137.0, 135.3, 134.2, 134.1, 131.5, 128.9, 126.5, 123.4, 100.2, 67.2, 38.3, 25.8, 20.5, 16.4. IR: 3500 2850, 1710, 1106, 982, 822 cm 1 (E) 4 (6 formyl 2,3 di m ethylphenyl)but 3 e noic acid (176 ): The acetal ( 190 ) (84 mg, 0.31 mmol) was dissolved in a solution of acetone (3 mL) and 1M HCl (0.3 mL), and the mixture was stirred at room temperature for 3 h. Water was added, the mixture was extracted with dichloromethane, washed with b rine, dried (MgSO 4 ), and evaporated to give the product as a white solid (56 mg, 85 %). 1 H NMR (CDCl 3 300 MHz): = 10.58 (br s, 1H); 10.19 (s, 1H); 7.71 (d, 1H, 7.8 Hz); 7.21 (d, 1H, 7.8 Hz); 6.79 (d, 1H, 15.9 Hz); 5.72 (dt, 1H, 15.9 Hz, 7.2 Hz); 3.43 (dd, 2H, 7.2 Hz, 0.9 Hz); 2.35 (s, 3H); 2.25 (s, 3H). 13 CNMR (CDCl 3 75 MHz) : = 193.2, 176.7, 143.3, 140.8, 135.5, 132.8, 130.4, 129.4, 129.2, 125.7, 38.1, 21.0, 15.8. IR: 3500 2500, 1710, 1679, 1586, 1244, 977, 822, 779 cm 1 Methyl (E) 4 (6 (1,3 dioxan 2 yl) 2, 3 dimethylphenyl)but 3 enoate (196 ): Pyridin ium dichromate (5.100 g, 13.6 mmol) was added at 0 C to a solution of aldehyde ( 189 ) (600 mg, 2.31 mmol) in methanol (0.56 mL, 13.8 mmol) and DMF (14 mL). The mixture was stirred at 0 C in the dark for 1 h and at room temperature for 1.5 h w hen the TLC analysis indicated full c onsumption of the substrate. The mixture was diluted

PAGE 250

250 with hexanes:diethyl ether (3:2, 100 mL), filtered through celite, washed with water (3 x 8 mL), and brine, dried (MgSO 4 ), and evaporated. The crude was purified by column chromatography using hexanes:et hyl acetate 10:1 as an eluent to give the product as a w hite solid (321 mg 48 % yield ) R f = 0.38 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 7.46 (d, 1H, 7.9 Hz); 7.11 (d, 1H, 7.9 Hz); 6.56 (d, 1H, 15.9 Hz); 5.79 (dt, 1H, 15.9 Hz, 7.1 Hz); 5.64 (s, 1H); 4.21 (dd, 2H, 11.6 Hz, 5.1 Hz); 4.00 3.91 (m, 2H); 3.76 (s, 3H); 3.32 (dd, 2H, 7.2 Hz, 1.3 Hz); 2.31 2.21 (m, 1H); 2.27 (s, 3H); 2.18 (s, 3H); 1.43 1.39 (m, 1H). 13 CNMR (CDCl 3 75 MHz) : = 172.1, 136.9, 135.5, 134.2, 134.1, 130.9, 128.8, 127 .2, 123.3, 100.1, 67. 2, 51.8, 38.4, 25.8, 20.5, 16.4. IR: 2948, 2843, 1737, 1153, 1107, 986, 822 cm 1 Methyl (E) 4 (6 formyl 2, 3 dimethylphenyl)but 3 enoate (197 ): The acetal ( 1 96 ) (321 mg, 1.11 mmol) was dissolved i n a solution of acetone (8 mL) and water (1.3 mL), and the mixture was stirred at room temperature for 2 h. The mixture was extracted with dichloromethane, washed with brine, dried (MgSO 4 ), and evaporated. The crude was purified by column chromatography (hexanes:ethyl acetate, 10:1) to give the product as a white solid (198 mg, 75 % yield). 1 H NMR (CDCl 3 300 MHz): = 10.18 (s, 1H,); 7.69 (d, 1H, 7.9 Hz); 7.20 (d, 1H, 7.9 Hz); 6.76 (d, 1H, 15.9 Hz); 5.71 (dt, 1H, 15.9 Hz, 7.2 Hz); 3.74 (s, 3H); 3.37 (dd, 2H, 7.2 Hz, 1.2 Hz); 2.35 (s, 3H); 2.24 (s, 3H). 13 CNMR (CDCl 3 75 MHz) : = 192.8, 171.4, 143.1,

PAGE 251

251 140.9, 135.5, 133.0, 131.0, 129.1, 128.9, 125.5, 52.0, 38.2, 21.0, 15.8. IR: 2953, 1736, 1677, 1586, 1240, 1201, 1165, 973, 840, 778 cm 1 (Z) 4 (6 (1,3 dioxan 2 yl) 2,3 d imethylphenyl)but 3 en 1 ol (200 ): Alkyne ( 187 ) (346 mg, 1.33 mmol) was 0.25 mmol), and 10 % wt. Lindlar catalyst (30 mg) were added, and the mixture was purged with hydrogen from a balloon for 1.5 h. The mixture was filtered through celite, and evaporated. Purification by column chromatography (hexanes:ethyl acetate 10:1 to 2:1 gradient) gave the product as l ightly yellow oil (301 mg, 85 %) R f = 0.3 3 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 7.39 (d, 1H, 7.8 Hz); 7.11 (d, 1H, 7.8 Hz); 6.59 (d, 1H, 11.4 Hz); 5.87 (dt, 1H, 11.4 Hz, 7.2 Hz); 5.54 (s, 1H); 4.19 (m, 2H); 3.86 3.94 (m, 2H); 3.55 3.61 (m, 2H); 2.27 (s, 3H); 2.14 2.21 (m, 1H); 2.13 (s, 3H); 2.04 2.11 (m, 2H); 1.81 (t, 1H, 5.0 Hz, O H); 1.36 1.43 (m, 1H). 13 CNMR (CDCl 3 75 MHz) : = 137.3, 134.9, 134.1, 133.9, 130.5, 129.2, 128.6, 123.1, 100.5, 67.4, 61.7, 32.2, 25.7, 20.6, 16.3. IR: 3422, 2961, 2857, 1376, 1152, 1108, 1038, 989, 822 cm 1

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252 (Z) 4 (6 (1,3 dioxan 2 yl) 2, 3 dimethylphenyl)but 3 enal (201 ): A solution of alcohol ( 200 ) (250 mg, 0.95 mmol) in dichloromethane (6 mL) was added to a solution of DMP (603 mg, 1.42 mmol) in dichloromethane (8 mL), and the mixture was stirred at room tempera ture for 50 min. The mixture was poured into an Erlenmeyer containing diethyl ether (40 mL) and a solution of sodium thiosulphate Na 2 S 2 O 3 (2.5 g) in saturated aqueous sodium bicarbonate (25 mL), and stirred for 10 min. The layers were separated, the aqueou s layer was extracted with diethyl ether, the combined organic layers were washed with sodium bicarbonate and brine, dried (MgSO 4 ), and evaporated to give the product as l ightly yellow oil (200 mg, 81 %) R f = 0.73 (hexanes: ethyl acetate, 2:1). 1 H NMR (CD Cl 3 300 MHz): = 9.59 (s, 1H); 7.42 (d, 1H, 8.1 Hz); 7.13 (d, 1H, 8.1 Hz); 6.67 (d, 1H, 11.4 Hz); 6.08 (dt, 1H, 11.4 Hz, 7.2 Hz); 5.45 (s, 1H), 4.10 4.21 (m, 2H); 3.78 3.93 (m, 2H); 2.89 3.11 (m, 2H); 2.26 (s, 3H); 2.12 2.25 (m, 1H); 2.10 (s, 3H); 1.34 1.43 (m, 1H). 13 CNMR (CDCl 3 75 MHz) : = 199.8, 137.4, 134.2, 134.1, 134.0, 131.4, 129.1, 123.7, 123.1, 100.1, 67.4, 67.3, 43.6, 25.7, 20.5, 16.3. IR: 2964, 2926, 2855, 2725, 1726, 1375, 1237, 1152, 1107, 991, 822 cm 1

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253 Methyl (Z) 4 (6 (1,3 dioxan 2 yl) 2, 3 dimethylphenyl)but 3 enoate (202 ): Pyridin ium dichromate (1.730 g, 4.60 mmol) was added at 0 C to a solution of aldehyde ( 201 ) (173 mg, 0.66 mmol) in methanol (0.19 mL, 4.69 mmol) and DMF (14 mL). The mixture was stirred at 0 C in the dark for 2 h and at room temperature for 2 h W hen the TLC analysis indicated fu ll consumption of the substrate, t he mixture was diluted with diethyl ether, filtered through celi te, washed with water (3x) and brine, dried (MgSO 4 ), and evaporated. The crude was purified by column chromatography using hexanes:ethyl aceta te 10:1 as an eluent to give the product as colorless oil (76 mg 39 % ). R f = 0.78 (hexanes: ethyl acetate, 2:1). 1 H NMR (CDCl 3 300 MHz): = 7.41 (d, 1H, 7.8 Hz); 7.11 (d, 1H, 7.8 Hz ); 6.58 (d, 1H, 11.1 Hz); 6.04 (dt, 1H, 11.1 Hz, 7.2 Hz); 5.48 (s, 1H); 4.10 4.20 (m, 2H); 3.82 3.94 (m, 2H); 3.64 (s, 3H); 2.82 2.89 (m, 2H); 2.26 (s, 3H); 2.13 2.24 (m, 1H); 2.10 (s, 3H); 1.34 1.41 (m, 1H). 13 CNMR (CDCl 3 75 MHz) : = 172.2, 137.1, 134.1 134.0, 133.9, 130.0, 128.9, 125.4, 122.9, 100.1, 67.3, 67.2, 51.5, 33.5, 25.7, 20.4, 16.2. IR: 2954, 2851, 1736, 1153, 1107, 990, 951, 925, 822 cm 1 Methyl (Z) 4 (6 formyl 2, 3 dimethylphenyl)but 3 enoate (203 ): The aceta l ( 202 acetone (2 mL), and stirred at room temperature for 2.5 h. The mixture was diluted with

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254 DCM, washed with water and brine, dried (MgSO 4 ), and evaporated. Purification by c olumn chromatography (hexanes:ethyl acetate, 10:1) gave 48 mg of the product (80 % yield). 1 H NMR (CDCl 3 300 MHz): = 10.13 (s, 1H); 7.70 (d, 1H, 7.8 Hz); 7.23 (d, 1H, 7.8 Hz); 6.77 (d, 1H, 11.4 Hz); 6.22 (dt, 1H, 11.4 Hz, 7.1 Hz); 3.63 (s, 3H); 2.84 (dd, 2H, 7.1 Hz, 1.5 Hz); 2.36 (s, 3H); 2.18 (s, 3H). 13 CNMR (CDCl 3 75 MHz) : = 192.5, 171.2, 143.9, 139.3, 135.5, 132.1, 129.3, 128.3, 127.4, 125.1, 51.8, 33.8, 21.0, 15.8. A.7 Alternative pharmacophore 3 phenyl 1 (triisopropylsi lyl) 1H pyrrole (207a): 3 Bromo 1 triisopropylsilylpyrrole ( 205 ) (203 mg, 0.67 mmol, TCI America), phenylboronic acid (174 mg, 1.43 mmol), and palladium tetrakis (50 mg, 0.032 mmol, 5 mol %) were stirred in a mixture of 2 M aqueous solution of sodium carbo nate (0.67 mL, 1.34 mmol), methanol (3 mL) and toluene (15 mL) at 70 C for 4 h. Solvents were removed on the rotovap, the remaining oil was dissolved in ethyl acetate to precipitate triphenylphosphine, and the mixture was filtered through anhydrous sodium sulphate plug. The crude was purified by column chromatography using petroleum ether as an eluent to give the product as colorless oil (121 mg, 60 % yield). R f =0.24 (petroleum ether) 1 H NMR (CDCl 3 300 MHz): 7.58 7.54 (m, 2H); 7.34 (tt, 2H, 7.6 Hz, 1.5 Hz); 7.16 (tt, 1H, 7.6 Hz, 1.2 Hz); 7.08 (t, 1H, 1.8 Hz); 6.82 (t, 1H, 2.3 Hz); 6.63 (dd, 1H, 2.8 Hz, 1.6 Hz); 1.50 (septet, 3H, 7.3 Hz); 1.15 (d, 18H, 7.3 Hz).

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255 3 (2 methoxyphenyl) 1 (triisopropylsilyl) 1H pyrrole (207b ): 3 Bromo 1 triisopropylsilylpyrrole ( 205 ) (409 mg, 1.35 mmol, TCI America), methoxyphenylboronic acid ( 206 149 mg, 0.98 mmol), and palladium tetrakis (61 mg, 0.039 mmol, 4 mol %) were stirred in a mixture of 2 M aqueous solution of sodium carbonate (1.0 mL, 2.0 mmol), methanol (2 mL) and toluene (5 mL) at 70 C for 4 h. Solvents were removed on the rotovap, the remaining oil was dissolved in ethyl acetate to precipitate triphenylphosphine, washed with brine, dried (MgSO 4 ), and evaporated. The crude was p urified by column chromatography using petroleum ether:ethyl acetate (10:1) as an eluent to give the product as colorless oil (97 mg, 60 % yield). R f =0.76 (petroleum ether:ethyl acetate, 10:1). 1 H NMR (CDCl 3 300 MHz): 7.56 (dd, 1H, 7.5 Hz, 1.8 Hz); 7.35 (t, 1H, 1.8 Hz); 7.13 (td, 1H, 7.5 Hz, 1.8 Hz); 6.98 6.91 (m, 2H); 6.79 (t, 1H, 2.5 Hz); 6.71 (dd, 1H, 2.8 Hz, 1.5 Hz); 3.88 (s, 3H); 1.48 (septet, 3H, 7.6 Hz); 1.13 (d, 18 H, 7.6 Hz). 13 CNMR (CDCl 3 7 5 MHz): 156.1, 127.8, 126.0, 124.9, 124.3, 123.8, 122.1, 120.8, 111.4, 110.0, 55.4, 17.9, 11.7. 3 phenyl 1H pyrrole (208a): 360 Tetra n bytylammonium fluoride (1M in THF, 0.40 mL, 0.40 mmol) was added to a solution of 3 phenyl 1 (tr ii sopropylsilyl) 1H pyrrole (#) (113 mg, 0.38 mmol) in THF at 0 C and the mixture was stirred at room temperature for 50 min. Water was added, the

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256 mixture was extracted with ethyl acetate, washed with brine, dried (MgSO 4 ), and evaporated. The crude was puri fied on silica gel column using petroleum ether: ethyl acetate 100:1 to 4:1 gradient as an eluent to give the product as a white solid (46 mg, 86 %). R f =0.07 (petroleum ether). 1 H NMR (CDCl 3 300 MHz): 8.31 (br s, 1H); 7.59 7.55 (m, 2H); 7.37 (t, 2H, 7 .6 Hz); 7.20 (tt, 1H, 7.3 Hz, 1.5 Hz); 7.10 (dd, 1H, 4.8 Hz, 2.1 Hz); 6.84 (dd, 1H, 5.3 Hz, 2.6 Hz, 2.1 Hz); 6.58 (dd, 1H, 5.4 Hz, 2.9 Hz, 2.6 Hz). 13 CNMR (CDCl 3 7 5 MHz): 135.8, 128.6, 125.5, 125.2, 124.9, 118.8, 114.5, 106.5. The NMR data matched the literature data. 360 3 (2 methoxyphenyl) 1H pyrrole (208b): 361 To a solution of 3 (2 methoxyphenyl) 1 (triisopropylsilyl) 1H py rrole ( 207b ) (37 mg, 0.11 mmol) in THF (3 mL) was added at 0 C a solution of TBAF in THF (1 M, 0.12 mL, 0.12 mmol), and the mixture was stirred at room temperature for 40 min. Water was added, the mixture was extracted with ethyl acetate, washed with brine, dried (MgSO 4 ), and evaporated. The crude was purified by column chromatography using petr oleum ether: ethyl acetate (10:1) to give 16 mg of the product (84 %). R f =0.16 (petroleum ether:ethyl acetate, 10:1). 1 H NMR (CDCl 3 300 MHz): 8.24 (br s, 1H); 7.55 (dd, 1H, 7.6 Hz, 1.8 Hz); 7.35 7.33 (m, 1H); 7.16 (ddd, 1H, 8.2 Hz, 6.8 Hz, 1.8 Hz); 7.3 5 7.33 (m, 1H); 7.16 (ddd, 1H, 8.2 Hz, 6.8 Hz, 1.8 Hz); 7.00 6.93 (m, 2H); 6.81 (dd, 1H, 5.6 Hz, 2.7 Hz); 6.65 6.62 (td, 2.7 Hz, 1.6 Hz); 3.89 (s, 3H). 13 CNMR (CDCl 3 7 5 MHz): 156.1, 128.0,

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257 126.3, 124.6, 120.7, 120.3, 117.9, 117.6, 111.1, 108.0, 55.3. T he NMR data matched the literature data. 361 (1 (tert Butoxycarbonyl) 1 H p yrrol 2 yl)boronic acid (213 ): 353 LDA (0.8 M in THF, 16.5 mL, 13.2 mmol, made from n BuLi and diisopropylamine) was added to a solution of N Boc py rrole (Aldrich, 2.00 g, 12.0 mmol) in THF (20 mL) at 2 h. 0.25 M aqueous hydroch loric acid was added (20 mL), the layers were separated, the aqueous layer was extracted with diethyl ether, the combined aqueous layers were washed with water (twice) and brine, dried over MgSO 4 and evaporated. The crude product, dissolved in DCM, was pu rified by column chromatography using a mixture of hexanes and ethyl acetate (10:1 to 4:1 gradient) to give ( 213 ) as an off white solid (1.857 g, 74 % yield). R f = 0.16 (hexanes : ethyl acetate 10:1). 1 H NMR (CDCl 3 3 00 MHz): 7.45 (dd, 1H, 3.1 Hz, 1.6 Hz); 7.19 (br. s, 2H, OH); 7.10 (dd, 1H, 3.1 Hz, 1.6 Hz); 6.26 (t, 1H, 3.1 Hz); 1.62 (s, 9H). 13 CNMR (CDCl 3 7 5 MHz): 152.2, 128.7, 127.1, 112.0, 85.5, 27.9 (The NMR data matched the reported dat a ). 362

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258 tert Butyl 2 (pyridin 3 yl) 1 H pyrrole 1 carboxylate (215 ): 352 Tetrakis (Triphenylphosphine)palladium (63 mg, 0.040 mmol, 4 mol %) was added to a mixture of N (Boc)pyrrole 2 boronic acid ( 213 297 mg, 1.41 mmol), 3 bromopyridine (0.10 mL, 1 .04 mmol), and 2 M Na 2 CO 3 (1.6 mL, 3.2 mmol) in 1,2 dimethoxyethane (7 mL) at room temperature. The mixture was stirred at reflux for 2.5 h, cooled, then water was added, and the mixture was extracted with ethyl acetate, washed with brine, dried over MgSO 4 and evaporated. The crude product was purified by column chromatography using a gradient of hexanes and ethyl acetate 20:1 to 10:1. R f = 0.12 (hexanes : ethyl acetate 10:1). 1 H NMR (CDCl 3 300 MHz): 8.60 (dd, 1H, 2.3 Hz, 0.8 Hz); 8.53 (dd, 1H, 4.8 Hz, 1.7 Hz); 7.67 (ddd, 1H, 7.9 Hz, 2.3 Hz, 1.7 Hz); 7.41 (dd, 1H, 3.1 Hz, 2.0 Hz); 7.28 7.31 (ddd, 1H, 7.9 Hz, 4.8 Hz, 0.8 Hz); 6.25 6.28 (m, 2H); 1.38 (s, 9H) (The NMR data matched the reported data ). 363 3 (1 H pyrrol 2 yl )pyridine (216 ): 352 N Boc protected 3 pyridylpyrrole ( 215 ) (144 mg, 0.59 mmol) was dissolved in THF (3 mL) and 25 % wt sodium methoxide in methanol (0.64 mL, 2.96 mmol) was added. The mixture was stirred at room temperature for 20 min, water was added, and the mixture was extracted with dichloromethane, washed with brine, dried (Na 2 SO 4 ), and evaporated. The crude was purified by column chromatography pretreated with triethylamine, using a gradient of hexanes: ethyl acetate 10:1 to 2:1 to yield the product

PAGE 259

259 a s a white solid (79 mg, 93 % yield). R f = 0.06 (hexanes: ethyl acetate 4:1). 1 H NMR (CDCl 3 300 MHz): 9.87 (br. s, 1H), 8.82 (dd, 1H, 2.3 Hz, 0.6 Hz); 8.40 (dd, 1H, 4.8 Hz, 1.7 Hz); 7.79 (ddd, 1H, 7.9 Hz, 2.3 Hz, 1.7 Hz); 7.25 (ddd, 1H, 7.9 Hz, 4.8 Hz, 0.6 Hz); 6.90 6.93 (m, 1H); 6.59 6.62 (m, 1H); 6.31 6.34 (m, 1H). 13 CNMR (CDCl 3 7 5 MHz): 146.5, 144 .9, 131.2, 129.1, 128.6, 123.8, 120.2, 110.2, 107.2 (The NMR data matched the reported data ). 364 3 (1 H pyrro l 2 yl)pyridin 1 ium chloride (217 ): 3 pyridylpyrrole ( 216 ) was dissolved in ethanol, a solution of concentrated HCl in ethanol was added, and the solvent was evaporated to yield the product in a quantitative yield. 1 H NMR ( D 2 O 5 00 MHz): 8.47 (d, 1H, 2.1 Hz); 8.30 (d, 1H, 5.7 Hz); 8.25 (dt, 1H, 8.4 Hz, 2.1 Hz); 7.79 (dd, 1H, 8.4 Hz, 5.7 Hz); 6.93 (dd, 1H, 2.7 Hz, 1.2 Hz); 6.58 (dd, 1H, 3.6 Hz, 1.2 Hz); 6.21 (dd, 3.6 Hz, 2.7 Hz) 13 CNMR ( D 2 O, 12 5 MHz acetone d 6 standard ): 139.4, 137.1 135.7, 133.0, 127.9, 124.8, 123.9, 111.2, 110.7. HRMS (ESI ): [M+H] + calculated: 145.0760 found: 145.0754.

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260 APPENDIX E NMR SPECTRA OF NEW SYNTHESIZED COMPOUNDS

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331 LIST OF REFERENCES 1. Lodish HF. (2008) Molecular cell biology, W.H. Freeman, New York, NY. 2. Patrick GL. (2013) An introdu ction to medicinal chemistry, Oxford University Press, Oxford, UK. 3. Valenstein ES. (2005) The war of the soups and the sparks : The discovery of neurotransmitters and the dispute over how nerves communicate, Columbia University Press, New York. 4. Hormuzdi S G, Filippov MA, Mitropoulou G, Monyer H, Bruzzone R. (2004) Electrical synapses: A dynamic signaling system that shapes the activity of neuronal networks. Biochim Biophys Acta Biomembranes 1662 :113 137. 5. Langley JN. (1905) On the reaction of cells and of nerve endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and to curari. J Physiol 33 :374 413. 6. Langley JN. (1907) On the contraction of muscle, chiefly in relation to the presence of "receptive" substances part I. J Physiol 36 :347 384. 7. Lopez Munoz F and Alamo C. (2009) Historical evolution of the neurotransmission concept. J Neural Transm 116 :515 533. 8. Hoyer D and Bartfai T. (2012) Neuropeptides and neuropeptide receptors: Drug targets, and peptide and non peptide l igands: A tribute to Prof. Dieter Seebach. Chemistry & Biodiversity 9 :2367 2387. 9. van den Pol AN. (2012) Neuropeptide transmission in brain circuits. Neuron 76 :98 115. 10. Snyder SH and Ferris CD. (2000) Novel neurotransmitters and their neuropsychiatric rele vance. Am J Psychiatry 157 :1738 1751. 11. Vincent SR. (1994) Nitric oxide a radical neurotransmitter in the central nervous system. Prog Neurobiol 42 :129 160. 12. Xue L, Farrugia G, Miller SM, Ferris CD, Snyder SH, Szurszewski JH. (2000) Carbon monoxide and ni tric oxide as coneurotransmitters in the enteric nervous system: Evidence from genomic deletion of biosynthetic enzymes. Proc Natl Acad Sci US A 97 :1851 1855. 13. Nelson DL(L, Cox MM, Lehninger AL. (2008) Lehninger principles of biochemistry, W.H. Freeman, New York.

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332 14. Ligand Gated Ion Channel Database, European Bioinformatics Institute, http://www.ebi.ac.uk/compneur srv/LGICdb/LGICdb.php 15. Sine SM and Engel AG. (2006) Recent advances in cys loop receptor structure and function. Nature 440 :448 455. 16. Dougherty DA. (2008) Cys loop neuroreceptors: Structure to the rescue? Chem Rev 108 :1642 1653. 17. Lester HA, Dibas MI, Dahan DS, Leite JF, Dougherty DA. (2004) Cys loop receptors: New twists and turns. Tr ends Neurosci 27 :329 336. 18. Itier V and Bertrand D. (2001) Neuronal nicotinic receptors: From protein structure to function. FEBS Lett 504 :118 125. 19. Jensen AA, Frolund B, Lijefors T, Krogsgaard Larsen P. (2005) Neuronal nicotinic acetylcholine receptors: St ructural revelations, target identifications, and therapeutic inspirations. J Med Chem 48 :4705 4745. 20. Dani JA and Bertrand D. (2007) Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol To xicol 47 :699 729 21. Changeux J, Kasai M, Lee C. (1970) Use of a snake venom toxin to characterize the cholinergic receptor protein. Proc Natl Acad Sci USA 67 :1241 1247. 22. Changeux JP and Edelstein SJ. (2005) Nicotinic acetylcholine receptors : From molecular biology to cognition, Odile Jacob, New York. 23. Devillersthiery A, Galzi JL, Eisele JL, Bertrand S, Bertrand D, Changeux JP. (1993) Functional architecture of the nicotinic acetylcholine receptor a prototype of ligand gated ion channels. J Membr Biol 136 :9 7 112. 24. Sharma G and Vijayaraghavan S. (2002) Nicotinic receptor signaling in nonexcitable cells. J Neurobiol 53 :524 534. 25. Gotti C and Clementi F. (2004) Neuronal nicotinic receptors: From structure to pathology. Prog Neurobiol 74 :363 396. 26. Wessler I and K irkpatrick CJ. (2008) Acetylcholine beyond neurons: The non neuronal cholinergic system in humans. Br J Pharmacol 154 :1558 1571. 27. Kawashima K and Fujii T. (2008) Basic and clinical aspects of non neuronal acetylcholine: Overview of non neuronal cholinergic systems and their biological significance. J Pha rmacol Sci 106 :167 173.

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362 BIOGRAPHICAL SKETCH Kinga Chojnacka was born in 1983 in Poznan (Poland) where she grew up. In 2003, Kinga started her studies at the Adam Mickiewicz University (Poznan), where she egree in chemistry in 2008 under supervision of Dr. Lech fluoro phosphate with potential cytostatic activity. During her studies at the Adam Mickiewicz University, King a spent the 2006/2007 academic year at the University of Strasbourg (Louis Pasteur Universit at the time, Strasbourg, France) within the Erasmus program (European Union student exchange program), taking classes in organic chemistry, biochemistry, medicina l chemistry, and pharmacology. At the end of that year, Kinga w orked in the summer on synthesis of new fluorescent thiophene substituted bodipys under directi on of Dr. Antoinette De Nicola in Dr. Raymond Z iessel Laboratory In the fall of 2008, Kinga start ed her doctoral research at the University of Florida under guidance of Dr. Nigel Richards in collaboration with Dr. Aaron Aponick, working on biosynthesis of plant hormones strigolactones. Finally, in the fall of 2011, Kinga began working with Dr. Nicole Horenstein and Dr. Roger Papke on the design and synthesis of molecules that modulate the functions of the alpha 7 nicotinic acetylcholine receptor, and received her Ph.D. at the end of 2013.