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Innovative Strategies for the Synthesis of Heterocycles of Potential Interest in Medicinal Chemistry

Permanent Link: http://ufdc.ufl.edu/UFE0044496/00001

Material Information

Title: Innovative Strategies for the Synthesis of Heterocycles of Potential Interest in Medicinal Chemistry
Physical Description: 1 online resource (128 p.)
Language: english
Creator: Beagle, Lucas K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diketopiperazine -- nitroso -- staudinger -- triazole
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This thesis focuses on expanding the role of benzotriazole-mediated reactions leading to compounds of potential interest in medicinal chemistry. Chapter 1 reviews the common themes covered in this research which includes: the role in synthesis of 1H-benzotriazole, a brief overview of peptides and microwave-assisted synthesis. In Chapter 2, 3,5-diamino-1,2,4-triazoles were synthesized under optimized microwave irradiation conditions. Exocyclic and ring acylation of 3,5-diamino-1,2,4-triazoles were studied using N-(protected a-aminoacyl)benzotriazoles and N-(protected dipeptidoyl)benzotriazoles. Chapters 3 and 4 describe the formation of cyclic 2,5-diketopiperazines using novel cyclization methodology. In Chapter 3, the turn-inducer proline was used to facilitate head-to-tail cyclization of N-(Cbz-protected dipeptidoyl)benzotriazoles to selectively form cis- or trans-2,5-diketopiperazines through a tandem deprotection/cyclization or cyclization/epimerization strategy. Chapter 4 discusses the use of Staudinger protocols in forming 2,5-diketopiperazines from a head-to-tail cyclization of azido-protected dipeptidoyl thioesters. In Chapter 5, the synthesis of a novel class of alpha-leaving group nitroso compounds is discussed. These compounds, which utilize benzotriazole as the leaving group, were investigated for reactivity as dienophiles in hetero Diels-Alder reactions and as potential NO donors.
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.
Statement of Responsibility: by Lucas K Beagle.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044496:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044496/00001

Material Information

Title: Innovative Strategies for the Synthesis of Heterocycles of Potential Interest in Medicinal Chemistry
Physical Description: 1 online resource (128 p.)
Language: english
Creator: Beagle, Lucas K
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: diketopiperazine -- nitroso -- staudinger -- triazole
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This thesis focuses on expanding the role of benzotriazole-mediated reactions leading to compounds of potential interest in medicinal chemistry. Chapter 1 reviews the common themes covered in this research which includes: the role in synthesis of 1H-benzotriazole, a brief overview of peptides and microwave-assisted synthesis. In Chapter 2, 3,5-diamino-1,2,4-triazoles were synthesized under optimized microwave irradiation conditions. Exocyclic and ring acylation of 3,5-diamino-1,2,4-triazoles were studied using N-(protected a-aminoacyl)benzotriazoles and N-(protected dipeptidoyl)benzotriazoles. Chapters 3 and 4 describe the formation of cyclic 2,5-diketopiperazines using novel cyclization methodology. In Chapter 3, the turn-inducer proline was used to facilitate head-to-tail cyclization of N-(Cbz-protected dipeptidoyl)benzotriazoles to selectively form cis- or trans-2,5-diketopiperazines through a tandem deprotection/cyclization or cyclization/epimerization strategy. Chapter 4 discusses the use of Staudinger protocols in forming 2,5-diketopiperazines from a head-to-tail cyclization of azido-protected dipeptidoyl thioesters. In Chapter 5, the synthesis of a novel class of alpha-leaving group nitroso compounds is discussed. These compounds, which utilize benzotriazole as the leaving group, were investigated for reactivity as dienophiles in hetero Diels-Alder reactions and as potential NO donors.
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.
Statement of Responsibility: by Lucas K Beagle.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044496:00001


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1 INNOVATIVE STRATEGIES FOR THE SYNTHESIS OF HETEROCYCLES OF POTENTIAL INTEREST IN MEDICINAL CHEMISTRY By L UCAS K. BEAG L E A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT O F THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 L ucas K. Beag l e

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3 To my wife, without whom this would not have been possible

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4 ACKNOWLEDGMENTS I would like to thank my advisor, Professor Alan R. Ka tritzky for his guidance, mentorship and availability I would like to thank the members of my committee, Dr. Ronald Castellano, Dr. Eric Enholm, Dr. Jon Stewart and Dr. Margaret James, for their helpful comments and instruction. I would like to thank th e University of Florida chemistry department for your tireless labor and assistance, especially Dr. Benjamin Smith and Mrs. Lori Clark in the graduate office for your guidance and endless support. Special thanks for the entire Katritzky research group, yo u have all become an important part of my life. Dr. Jean Christophe Monbaliu, you have been an inspiration and guide in my last years of study, I am glad we were on the journey together. Dr. Finn Hansen, your welcoming spirit was priceless and comfort ing a long the way. Ms. Judit Kovacs thank you for your friendship and encouragement for the last two years. Dr. C. Dennis Hall, thank you for your guidance and constructive criticism s I would like to thank my family for their support and encouragement, especi ally my parents Timothy and Ellen Beagle. I would like to give needed thanks to my loving wife Kristen Beagle who supported me and was extremely understanding during the entire process of my studies. Your encouragement and love got me through the toughest and most difficult times. It has been a honor and privilege for me to learn and be able to express my passion for chemistry at the University of Florida.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF SCHEMES ................................ ................................ ................................ ...... 12 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 19 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .................. 21 1.1 Heterocycles in Synthesis ................................ ................................ ................. 21 1.2 Peptides in Synthes is ................................ ................................ ........................ 25 1.3 Microwaves in Synthesis ................................ ................................ ................... 27 2 EFFICIENT MICROWAVE ASSISTED SYNTHESIS OF 1,2,4 TRIAZOLE BASED PEPTIDOMIMETICS USING BENZOTRIAZOLE METHODOLOGY ......... 30 2.1 Literature Overview ................................ ................................ ........................... 30 2.1.1 Biological Properties o f Selected 3,5 Disubstituted 1,2,4 Triazoles ........ 30 2.1.2 Previous Methods for the Synthesis of 1,2,4 Triazole 3,5 diamines ........ 31 2.1.3 Peptidomimetics ................................ ................................ ...................... 31 2.2 Results and Discussion ................................ ................................ ..................... 32 2.2.1 Starting Material Syn thesis ................................ ................................ ...... 33 2.2.2 Ring Acylation of 2.15 and 2.17. ................................ .............................. 34 2.2.3 Exocyclic Acylation of 2.18 ................................ ................................ ...... 36 2.2.4 Determination of Acylation Site by 1 H NMR Experiments ........................ 36 2.3 Summary ................................ ................................ ................................ .......... 37 2.4 Experimental ................................ ................................ ................................ ..... 37 2.4.1 General Methods ................................ ................................ ..................... 37 2.4.2 Synthesis 2.12 14 and 2.15 17 ................................ ................................ 38 2.4.2.1 General procedures for the microwave assisted preparation of isothioureas 2.12 14 ................................ ................................ ............... 38 2.4.2.2 General procedures for microwave assisted synthesis of 1,2,4 triazoles 2.15 17 ................................ ................................ ..................... 39 2.4 .2.3 Synthesis of ring protected 1,2,4 trizaole 2.18 ............................... 40 2.4.3 Ring Acylation of 1,2,4 Triazoles 2.23 27 and 2.31 33 ............................ 40 2.4.3.1 General procedures for microwave assisted synthesis of compounds 2.23 27 ................................ ................................ ................ 40

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6 2.4.3.2 General procedures for microwave assisted synthesis of compounds 2.31 33. ................................ ................................ ............... 42 2.4.4 Exocyclic A cylation of 1,2,4 Triazoles 2.34 35 ................................ ........ 44 3 A NEW BENZOTRIAZOLE MEDIATED, STEREOFLEXIBLE GATEWAY TO HETERO 2,5 DIKETOPIPERAZINES ................................ ................................ ..... 46 3.1 Literature Overview ................................ ................................ ........................... 46 3.1.1 Properties of Proline Containing Cyclic Peptides ................................ .... 46 3.1.2 Turn inducers in Peptide Synthesis ................................ ......................... 48 3.2 Results and Discussion ................................ ................................ ................. 49 3.2.1 Starting Material Synthesis ................................ ................................ ...... 49 3.2.2 Synthesis of trans 2,5 Diketopiperazines. ................................ ............... 50 3.2.3 Synthesis of cis 2,5 Diketopiperzines ................................ ...................... 57 3.2.4 Reaction Kinetics for the Formation of 3.31. ................................ ............ 58 3.3 Summary ................................ ................................ ................................ .......... 59 3.4 Experimental ................................ ................................ ................................ ..... 60 3.4.1 General Methods. ................................ ................................ .................... 60 3.4.2 General Procedures for the Tandem Cyclization/Epimerization Sequence and Characterization of the Corresponding rac Diketopiperazines 3. 31 and 3.37 and trans Diketopiperazines 3.38 39 and 3.44 47 ................................ ................................ ................................ .......... 61 3.4.3 Deprotection of Compound 3.39 and Characterization of Compoun d 3.48 ................................ ................................ ................................ ............... 64 3.4.4 General Procedures for the Tandem Deprotection/Cyclization Sequence and Characterization of Compound 3.49 51 ................................ 65 3.4.5 X Ray Data for 3.38 39 ................................ ................................ ............ 66 3.4.6 Kinetic Data for the Formation of 3.31 ................................ ..................... 67 4 STAUDINGER LIGATION IN THE FORMATION OF 2,5 DIKETOPIPERAZINES 71 4.1 Literature Overview ................................ ................................ ........................... 71 4.1.1 Biological Properties of 2,5 Diketopiperazines ................................ ........ 71 4 .1.2 Synthesis of 2,5 Diketopiperazines ................................ ......................... 72 4.1.3 Solid Phase Supported Synthesis ................................ ........................... 75 4.1.4 Staudinger Ligation ................................ ................................ .................. 76 4.2 Results and Discussion ................................ ................................ ..................... 78 4.2.1 Synthesis of Starting Materials for Solution Phase Staudinger Ligation Reactions of 4.38 40 and 4.43 ................................ ................................ ...... 78 4.2.1.1 Synthesis of chloro dipeptides 4.32 34 ................................ .......... 78 4.2.1.2 Synthesis of azido dipeptides 4.35 37 ................................ ............ 78 4.2.1.3 Synthesis of azido thioester dipeptides 4.38 40 ............................. 79 4.2.2 Synthesis of Starting Materials for Solid Phase Staudinger Ligation of 4.59 61 and 4.68 69 ................................ ................................ ...................... 80 4.2.2.1 Synthesis of boc protected amino acid linkers 4.48 50 .................. 80 4.2.2.2 Synthesis of azido protected solid phase supported dipeptides 4.59 61. ................................ ................................ ................................ .. 80

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7 4.2.2.3 Synthesis of azido protected solid phase supported t ripeptides 4.68 69 ................................ ................................ ................................ ... 81 4.2.3 Staudinger Ligation of 4.38 40, 4.43, 4.59 61, and 4.68 69 for the Formation of 2,5 Diketopiperazines 4.63 4.65 ................................ .............. 82 4.3 Summary ................................ ................................ ................................ .......... 86 4.4 Experimental ................................ ................................ ................................ ..... 87 4.4.1 General Methods ................................ ................................ ..................... 87 4.4.2 General Methods for the Synthesis of 4.70 72 ................................ ........ 87 4.4 .3 General Methods for the Synthesis of Compounds 4.32 40 .................... 89 4.4.3.1 General procedures for the synthesis of compounds 4.32 34 ........ 89 4.4.3.2 General procedures for the synthesis of compounds 4.35 37 ........ 90 4.4.3.3 General methods for the synthesis of 4.38 40 ............................... 91 5 BENZOTRIAZOYL NITROSO DERIVATIVES: POTENTIAL NOVEL NO DONORS ................................ ................................ ................................ ................ 93 5.1 Literature Overview ................................ ................................ ........................... 93 5.1.1 Biological Properties of Selected Nitroso Compounds ............................ 93 5.1.2 Different Forms of Nitroso Compounds ................................ ................... 94 5.1.3 Reactions of Nitroso Compounds ................................ ............................ 97 5.1.3.1 Diels Ald er reactions of nitroso compounds ................................ ... 97 5.1.3.2 Nitroso ene reactions ................................ ................................ ..... 98 5.1.3.3 Nitroso aldol reactions ................................ ................................ .... 99 5.1.3.4 Release of NO and HNO ................................ ................................ 99 5.2 Results and Discuss ion ................................ ................................ ................... 101 Leaving Group Benzotriazole Nitroso Compounds ........ 101 5.2.2 Hetero Diels Alder Reaction of 5.46 ................................ ...................... 102 5.2.3 Computational Investigation into the Release of NO ............................. 104 Benzotriazole Nitroso Compounds 5.46 51 .................. 106 5.3 Summary ................................ ................................ ................................ ........ 109 5.4 Experimental ................................ ................................ ................................ ... 109 5.4.1 General Methods ................................ ................................ ................... 109 5.4.2 Synthesis of Nitroso Compounds 5.46 51 ................................ ............. 110 5.4.3 X R ay Data for 5.46 and 5.69 ................................ ................................ 111 6 SUMMARY OF ACHIEVEMENTS ................................ ................................ ........ 113 LIST OF REFERENCES ................................ ................................ ............................. 115 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 128

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8 LIST OF TABLES Table page 1 1 ................................ ............ 28 2 1 Synthesis of Isothioureas intermediates 2.12 14 ................................ ................ 33 2 2 Synthesis of 3,5 di amino 1,2,4 triazoles 2.15 17 from isothiourea intermediates 2.12 14 ................................ ................................ ......................... 34 2 3 Synthesis of ring acylated 3,5 diamino 1,2,4 triazoles 2.23 27 .......................... 35 2 4 Ring acylation using Cbz protected dipeptidoyl benzotriazoles on 2.15 ............. 35 3 1 Synthesis of Cbz protected dipeptides 3.17 23 through benzotriazole mediated coupling ................................ ................................ .............................. 50 3 2 Optimization of cyclization conditions ................................ ................................ 51 3 3 Cyclization of 3.25 and 3.27 to form trans 3.38 and 3.39 [a] ................................ 53 3 4 Tandem cyclization/epimerization for the formation of 3.28 29 and 3.44 47 ...... 56 3 5 Data obtained from reaction of 3.24 with 1 equivalent of triethyl amine. .............. 67 4 1 Advantages and disadvantage of SPPS methodology ................................ ....... 75 4 2 Initial conditions for optimization of preperation of 4.70 ................................ ...... 83 4 3 Reaction of 4.38 40 and 4.59 61 to form 4.70 72 ................................ ............... 84 5 1 benzotriazoyl nitroso compounds 5.46 51 ................................ 101 5 2 Reaction of 5.46 in the nitroso Diels Alder reaction ................................ .......... 103 5 3 Isod esmic heat ( H iso ) for radical exchange on different cyclohexyl substrates and corresponding radical stabilization energy (RSE) ..................... 104 5 4 Homolytic bond dissociation energies (BDE) for compounds 5.8, 5.10 and 5.46 ................................ ................................ ................................ .................. 105 5 5 Heterolytic bond dissociation energies (BDE) for compounds 5.8, 5.36, and 5.46 ................................ ................................ ................................ .................. 105

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9 LIST OF FIGURES Figure page 1 1 Selected examples of common heterocycles ................................ ..................... 21 1 2 Tautomerization of 1 H and 2 H benzotriazole ................................ ................... 21 1 3 Important characteristics of benzotriazole ................................ .......................... 22 1 4 Synthesis of acyl benzotriazolides ................................ ................................ ...... 23 1 5 Acyl ation reactions involving C O N and S nucleophiles .............................. 24 1 6 Synthesis of peptides ................................ ................................ ......................... 25 2 1 Selected biologically active 1,2,4 triazole derivatives ................................ ......... 31 2 2 Synthesis of 1,2,4 triazoles 3,5 diamine 2.7 from S dimethyl N cyanodithioimidocarbonate 2.4 ................................ ................................ ........... 31 2 3 1,2,4 triazoles as surrogates of cis amide bonds ................................ ............... 32 2 4 Peptidomimetic drugs which showed anti hypertensive properties ..................... 32 2 5 1 H NMR shifts of ring acylated compounds ................................ ........................ 37 3 1 Selected examples of proline containing 2,5 diketopiperazines ......................... 47 3 2 Proline as a turn inducer in the synthesis of larger cyclic peptides ..................... 49 3 3 Cyclization and racemization of substrates 3.24 and 3.32 ................................ .. 52 3 4 Single crystal X ray diffraction structure of (L,D) 3.38 showing the trans configuration ................................ ................................ ................................ ....... 54 3 5 Single Crystal X ray diffraction structure of 3.39 showing the absolute stereochemistry of the L,D configuration ................................ ............................ 54 3 6 Thermodynamic stabilities of enols and mother DKPs showing the effect of the protecting group ................................ ................................ ............................ 55 3 7 P ictures of the transition states associated with the unimolecular unassisted mechanism (TS uni left) and with the bimolecular assisted mechanism (TS bi right) ................................ ................................ ................................ ................... 56 3 8 Arrhenius plot for the cyc lization of 3.24 to 3.31 under conventional heating ..... 58 3 9 Eyring plot for the cyclization of 3.24 to 3.31 under conventional heating .......... 59

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10 3 10 Plot showing the percent completion over time ................................ .................. 68 3 11 Plot showing the natural log of the concentration over time ............................... 69 3 12 Arrhenius plot for the cyclization of 3.50 ................................ ............................. 69 3 13 Eyring plot for the cyclization of 3.24 ................................ ................................ .. 70 3 14 Data extrapolated fro m the Arrhenius and Eyring plots ................................ ...... 70 4 1 Selected examples of biologically active 2,5 diketopiperazines .......................... 72 4 2 Common synthetic m ethods for cyclic peptides ................................ .................. 72 4 3 Selected resin solid phase supports ................................ ................................ ... 76 4 4 Mechanism of the formation of the activated iminoph osphorane ........................ 77 4 5 Competitive cyclization through TS 6 and TS 9 for the formation of 4.72 .............. 86 5 1 N Nitroso compounds showing intere sting biological activity ............................. 93 5 2 S Nitroso compounds of biological relevance ................................ ..................... 94 5 3 Examples of various nitroso compounds ................................ ............................ 94 5 4 Global electrophilicity scale of common nitroso compounds[10JST49] .............. 95 5 5 Transient acyl nitroso compound 5.15 and their deriva tives 5.17 19 on reaction with nucleophiles ................................ ................................ .................. 96 5 6 Three step process for the formation of 1,2 oxazines in nitroso hetero Diels leaving group nitroso 5.8 ................................ ............... 97 5 7 Asymmetric hDA utilized in the synthesis of ( ) epibatidine from chiral nitroso reagent 5.25 ................................ ................................ ................................ ....... 98 5 8 The nitroso ene reaction involving an allylic system and a nitroso compound ... 98 5 9 The nitroso aldol reaction of nitrosobenzene and diethyl malonate .................... 99 5 10 Select ive addition to the O or N moiety of nitroso compounds ......................... 99 5 11 acetoxy nitroso compounds ................................ ........ 100 5 12 Releas cyano nitroso compound 5.10 ........................... 100 5 13 benzotriazoyl nitroso 5.53 ................................ ................................ ................................ ....... 101

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11 5 14 Benzotriazoyl nitroso derived from methyl ketoxime 5.54 ............................. 102 5 15 Picture of the TSs associated with the cycloaddition of the selected nitroso compounds with buta diene ................................ ................................ ............... 103 5 16 Indirect proof for the release of NO through radical intermediate 5.70 ............. 106 5 17 X benzotriazoy l nitro 5.69 ................................ ..................... 106 5 18 X ray structure of 5.46 with minor impurities from the nitro compound 5.69 ..... 107 5 19 Half lives of 5.46 as measured in various solvents ................................ ........... 108 5 20 Kinetics for dissociation at various concentrations in methanol of 5.46 ............ 108 5 21 Compariso n of the half lives of 5.46 and 5.50 51 in methanol .......................... 109

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12 LIST OF SCHEMES Scheme page 2 1 Formation of ring protected 3,5 diamino 1 ,2,4 triazole 2.18 ............................... 34 2 2 Exocyclic acylation of 2.18 under microwave irradiation ................................ ..... 36 3 1 Formation of dipeptidoyl benzotriazole compounds 3.24 30 .............................. 50 3 2 Formation of racemic 3.31 from chiral 3.32 ................................ ........................ 51 3 3 Cyclization/racemization of 3.36 ................................ ................................ ......... 52 3 4 Formation of 3.48 as the free 2,5 diketopiperazine form Cbz protected 3.39 ..... 57 3 5 Tandem deprotection/cyclization for the formation of cis DK Ps 3.49 51 ............ 58 4 1 Head to tail condensation of N ketoacyl amino acid amides ........................... 73 4 2 Synthesis of symmetrical DKPs from dimeriz ation of two identical subunits ..... 74 4 3 Intramolecular Negishi peptide coupling strategy ................................ ............... 74 4 4 Raines and co ligation methodology using phosphinothioester and azide ................................ ................................ ............. 77 4 5 Synthesis of chloro dipeptides 4.32 34 from L amino acid and chloroacetyl chloride ................................ ................................ ................................ ............... 78 4 6 Synthesis of azido dipeptides 4.35 37 from chloro dipeptides 4.32 34 ............... 79 4 7 Synthesis of azido thioester dipeptides 4.38 40 from azido dipeptides 4.35 37 79 4 8 Synthesis of azido methyl ester dipeptide 4.42 from L leucine methyl ester 4.41 ................................ ................................ ................................ .................... 80 4 9 Synthesis of Boc protected amino acid linkers 4.48 50 ................................ ...... 80 4 10 Synthesis of solid phase supported azido protected dipeptides 4.59 61 ............ 81 4 11 Recyclability of resin 4.63 to giv e intermediate 4.53 ................................ ........... 81 4 12 Synthesis of azido protected solid supported tripeptides 4.68 69 ....................... 82 4 13 Reaction of methyl ester de rivative 4.43 in the Staudinger ligation under optimized conditions ................................ ................................ ........................... 84

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13 4 14 Attempted solid phase cyclic tripeptide formation of 4.73 and 4.74 under Staudinger ligation protocols ................................ ................................ .............. 85 4 15 Formation of the unexpected cyclic peptide 4.72 from the linear tripeptides 4.68 and 4.69 ................................ ................................ ................................ ...... 85

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14 LIST OF ABBREVIATION S Angstrom AcOH Acetic acid Ac 2 O Acetic anhydri de OAc Acetoxy pK a Acid dissociation constant Anal. Analytical aq. Aqueous Bn Benzyl br s B road singlet BtH Benzotriazole Bt Benzotriazole (1 Substituted) 2 Bt Benzotriazole (2 substituted) BtMS Benzotriazole mesylate BINAP Binaphathol BDE Bond dissociation Energy Cbz Carboxybenzyl C Celsius degree Chemical shift (in ppm) Calcd. Calculated 13 C NMR 13 Carbon nuclear magnetic resonance Chemical shift CHCl 3 Chloroform Conv Conventional heating Cu C o pper

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15 CRF Corticotropin releasing factor J Coupling constant CDK Cyclin dependent Kinase Degree D Dext arotary DKP Diketopiperazine d Doublet dd Doublet of doublet CH 2 Cl 2 Methylene chloride DCM Methylene chloride DMF Dimethylformamide CDCl 3 Deuterated chloroform DMSO d 6 Deuterated dimethyl sulfoxide eV Electron volts Electrophilicity e.g. Exempli gratia E t Ethyl et al And others Et 3 N Triethylamine EtOH Ethanol OEt Ethoxy eq Equivalent Fmoc Fluorenylmethyloxycarbonyl G 1 Phase G 1 phase of cell division g Gram

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16 hDA Hetero Diels Alder h Hour HCl Hydrochloric acid N 2 H 4 Hydrazine H 2 Hydrogen (gas) NH 2 OH Hydroxyla mine H 2 SO 4 Sulfuric acid t 1/2 Half life Hz Hertz IC 50 Half maximal inhibitory concentration HIV Human Immunodeficiency Virus IRC Intrinsic reaction coordinate Fe Iron i Bu iso butyl H iso Isodesmic heat JAKs 1 3 Janus associated k inases 1 3 L Levorotary li t Literature Loss factor MgSO 4 Magnesium Sulfate m.p. M elting point m Meta Me Methyl MeOH Methanol M Mi c romol e

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17 mL Milliliter mm Millimeter mmol Millimole min Minute MW Molecular weight m Multiplet NO Nitric oxide NMOR Nitrosomorpholine HNO Nitroxyl N Normal NMR Nuclear magnetic resonance ox oxidize p Para ppm Parts per million % Percent % w/w Percent weight by weight Ph Phenyl K 2 CO 3 Potassium carbonate PG Protecting group 1 H NMR Proton nuclear magnetic resonance q Quartet RSE Radical stabilization ene rgy rt Room temperature sec S econd s Singlet

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18 Na 2 CO 3 Sodium carbonate NaOH Sodium hydroxide SPPS Solid phase peptide synthesis Boc tert butyl carboxylate THF Tetrahydrofuran TMS Tetramethylsilane TLC Thin layer chromatography SOCl 2 Thionyl chloride t Time T s Tosyl TS Transition State Et 3 N Triethylamine Tf Triflate TFA Trifluoroacetate anion TFA Trifluoroacetatic acid TMS Trimethyl silyl t Triplet TYK2 Tyrosine a ssociated k inase 2 uv Ultraviolet vs. Versus W Weight

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19 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 INNOVATIVE STRATEGIES FOR THE SYNTHESIS OF HETEROCYCLES OF POTENTIAL INTEREST IN MEDICINAL CHEMISTRY By L ucas K. Beag l e August 2012 Chair: Alan R. Katritzky Major: Chemistry This thesis focuses on expanding the role of benzotriazole mediated reactions leading to compounds of potential interest in medicinal chemistry. Chapter 1 reviews the common themes covered in this research which includes: the role in synthesis of 1 H benzotriazole, a brief overview of peptides and microwave assisted synthesis. In C hapter 2, 3,5 diamino 1,2,4 triazoles were synthesized under optimized microwave irradiation conditions Exocyclic and ring acylation of 3,5 diamino 1,2,4 triazoles were studied using N aminoacyl)benzotriazoles and N (protected dipeptidoyl)benzotriazoles. Chapter s 3 and 4 describe the formation of cyclic 2,5 diketopiperazines using novel cyclization method olog y In C hapter 3 the turn inducer proline was used to facilitate head to tail cyclization of N (Cbz protected dipeptidoyl)benzotriazoles to selectively form cis or trans 2,5 diketopi p erazines through a tandem deprotection/cyclization or cyclization/ep imerization strategy Chapter 4 discusses the use of Staudinger protocols in forming 2,5 diketopiperazines from a head to tail cyclization of azido protected dipeptidoyl thioesters

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20 In C hapter 5 the synthesis of a n leaving group nitroso compounds is discussed These compounds, which utilize benzotriazole as the leaving group, were investigated for reactivity as dienophiles in hetero Diels Alder reactions and as potential NO donors.

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21 CHAPTER 1 GENERAL INTRODUCTION The three themes that pervade this thesis are heterocycles, cyclic peptides and microwave assisted synthesis that are mediated by the use of benzotriazole as a synthetic auxiliary. 1.1 Heterocycles in S ynthesis Heterocycles are cyclic compoun ds composed of more than one type of atom in the ring (Figure 1 1). The natural antithesis of a heterocycle is the homocycle, in which cyclic compounds have a single type of atom in the ring.[86JCE8 60 ] Heterocycles include multiple heteroatoms, varying deg rees of unsaturation, single to multiple rings, and a range of ring sizes. Figure 1 1. Selected examples of common heterocycles Heterocycles are extremely important in organic chemistry and many research programs have been dedicated to their synthesis, structure and reactivity. [ 03HC1, 08CHC1, 11PHC1 ] In particular 1 H benzotriazole is of great importance to this research, and some of its properties and reactivity are described below. Figure 1 2. Tautomerization of 1 H and 2 H benzotriazole

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22 Benzotriazole is an interesting molecule in which there are two fused rings, a six membered benzenoid ring and a five membered 1,2,3 triazole ring.[54SCI989] It naturally exists in two tautomeric forms (Fi gure 1 2) [09MRC142, 75JCS(P1)1181] and has the ability to both accept (pK aH < 0) and donate a proton (pK a = 8.2).[48JCS2240, 0 0JACS5849, 75JCS(P1)1181, 91T2683] Figure 1 3. Important characteristics of benzotriazole Benz otriazole has the ability to act as a proton activator 1.10 [98CR409, 06S3231, 91CB1819] a leaving group 1.11 [94CSR363] an ambient ion director 1.12 [90HC21, 92LAC843] a radical activator 1.13 [04CC2356, 05JOC9521] and an anion precursor 1.14 (Figure 1 3) [97JOC4148, 92JCS(P1)1111 ] Often behaving like a mild halogen in most reactions, the synthetic utility of benzotriazole coupled with its stabilizing ability through electron donation offers the organic chemist a valuable tool. The use of benzotriazole as a leaving group is of greatest importance to this research. Comparable to cyano and sulfonyl groups in its pure leaving group ability, it forms a stable anion in solution.[98CR409, 95S1315] Relative to acyl leaving groups,

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23 benzotriazole offers a modulated reactivity due to its ability to stabilize the carbonyl, which offers an advantage over the more reactive a cyl halides and acyl tosylates.[ 91T268 3 98CR409, 03CEJ4586] Easy displacement by nucleophilic reagents offers a wide substrate scope for activation by benzotriazole methodologies. [94CSR363] Synthesis of acyl benzotriazolide 1.17 occurs under mild conditions starting from acyl chlorides or carboxylic acids (Figure 1 4). Formation from the carboxylic acid 1.15 involves in situ generation of thionyl ben zotriazole (from an excess of 1 H benzotriazole and thionyl chloride) or benzotriazol e mesylate and triethylamine.[00JOC8069, 03S2795, 92T7817] Figure 1 4. Synthesis of acyl benzotriazolides Benzotriazole mediated acylation has been shown to be effective for C O N and S nucleophiles (Figure 1 5).[03JOC4932, 03JOC5720, 00S2029] The unique reactivity and stabilization effect of benzotriazole allow these transformations under mild

PAGE 24

24 conditions and often in high yields compa red to literature methods. [96JOC1624, 00JOC8240, 06S411] Figure 1 5. Acylation reactions involving C O N and S nucleophiles Benzotriazole based reagents have become an important part of substitution chemistry, espec ially involving acylation of peptides with other compounds. Benzotriazole mediated reactions are an important part of the research included in this thesis. The use of benzotriazole based acylating reagents are presented in C hapters 2 4, and the ability of benzotriazole as a stabilizing agent is described in C hapter 5. In C hapter 2, N (p aminoacyl)benzotriazoles and N (protected dipeptidoyl)benzotriazoles are used to acylate 3,5 diamine 1,2,4 triazole compounds giving novel compounds as potential peptidomimetics. Proline containing N (protected dipeptidoyl)benzotriazoles are use d in C hapter 3 to selectively form cis and trans 2,5 diketopiperazines under mild conditions. Aminoacyl benzotriazoles are used in the synthesis of starting materials which form 2,5 diketopiperazines under Staudinger ligation conditions in C hapter 4.

PAGE 25

25 In C hapter 5, benzotriazole plays a different role, being used a stabilizing agent in benzotriazoyl nitroso compounds. 1.2 Peptides in S ynthesis Peptides are oligomeric sequences made from natural and non natural amino acids, connected through amide (peptide) bonds.[02PBC5] Peptides are generically differentiated from proteins by the length of their sequences; peptides are generally under 50 residues whereas proteins are longer. They are named and characterized directionally going from an N to a C terminus on the amino acid sequence. Some small peptides have shown biological activity but suffer from hydrolysis especially by native peptidases in vivo .[99QJM1] While peptides are biosynthesized from N to C terminus, chemical synthesis u sually occurs from C to N terminus (Figure 1 6).[02PBC 5 ] Synthetic preparation occurs by attack of the amine moiety on an activated carboxyl group in a sequential manner.[12CSR1826] While many peptides are available from natural sources, synthesis has be come a more reliable method, often at lower cost and in higher purity. Figure 1 6. Synthesis of peptides

PAGE 26

26 Two main methods are available for the preparation of peptides in the laboratory. The classical method relies on solu tion phase coupling by which chemists have successfully prepared many peptides. The methods are often tedious, require complicated purification and long reaction times.[12CSR1826] Solid phase peptide synthesis (SPPS) is a paradigm shift that allowed signif icantly simpler purification, shorter reaction times and the ability to solubilize larger peptides.[88ARB957] SPPS relies on attachment of peptides to polymer resins in which they are immobilized, while the reactants and byproducts are easily washed away. Peptide synthesis using SPPS methods is a four part cycle (couple wash deprotection wash) that is continued until synthesis is complete and ready for cleavage from the resin. For the most part, SPPS has replaced solution phase synthesis due to the above ad vantages, but solution phase synthesis remains more effective for industrial preparation of peptides.[07EJB279] Chapter 3 uses the classical solution phase approach in the selective synthesis of cis and trans 2,5 diketopiperazines. Whereas both methods ar e used in C hapter 4 for the synthesis of 2,5 diketopiperazines using Staudinger ligation protocols. An overview comparing both solution and solid phase peptide syntheses is included in C hapter 4. Peptide conjugates are an important class of compounds invo lved in peptide chemistry, since they merge the properties of two compounds often leading to enhanced activity.[08PBDD1] Common peptide conjugates include phosphopeptides,[90TL2497] glycopeptides,[06G113R] lipopeptides,[09CEJB258] and pharmaceutical peptid e conjugates.[01JMC1341, 08PBDD1] These compounds are valuable targets for a library of synthetic materials as one target molecule can simultaneously be reacted with many different peptides.

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27 1.3 Microwaves in S ynthesis Microwave assisted synthesis, once on e of the hottest topics in synthesis, is an area that continues to receive intense investigation. The use of microwave reactors has grown considerably since their introduction in 1986.[86TL279] Almost any reaction can be performed under microwave irradiati on, and since its inception, reactions involving microwave assisted chemistry include, but are not limited to, reductions, catalytic reactions, and substitutions.[04AA66] Many reviews have been written on the subject and the methodology has been applied to virtually every kind of substrate. Microwave assisted chemistry is characterized by its efficient heating and drastic decreases in reaction times while often providing superior yields.[03N571, 04A CI E6250] These advantages are seen in C hapters 2 4 where de creases in reaction times and improved yields using microwave assisted synthesis are integral. Microwave assisted heating has been an area of debate as to what causes the acceleration of reactions. Although pronounced effects are seen, the energy of microw ave photons in the frequency of microwave reactors (2.45 GHz) are too low to break chemical bonds and therefore cannot induce chemical reactions which are rationalized by simple thermal/kinetics concerns.[91CSR1, 98CSR213] Heating from microwaves occurs in one of two ways: i) dipolar polarization and ii) ionic conduction.[05CSR164] Dipolar polarization is the most common mode of heating in microwave chemistry, as it is dependent on the solvent. Solvents which are dipolar and therefore have high dielectric c onstants interact with the microwave fields. These molecules attempt to align with the field causing motion and therefore heat transfer to the system.[04AA66] A measure of the ability of solvents to interact with microwave irradiation is known as loss

PAGE 28

28 fact 7).[04 ACIE 6250] Solvents with extremely low loss factors are known as microwave transparent (i.e. benzene, hexanes) and show little to no affect from microwave heating. Heating of transparent solvents can still be ac complished by the addition of electrical conductive materials such as catalysts or ionic liquids which are intense microwave absorbers.[05CSR164] Table 1 CIE 6250] Solvent Solvent ethylene glycol 1.350 chl oroform 0.091 ethanol 0.941 acetonitrile 0.062 DMSO 0.825 ethyl acetate 0.059 methanol 0.659 acetone 0.054 acetic acid 0.174 THF 0.047 DMF 0.161 DCM 0.042 water 0.123 hexane 0.020 Ionic conduction is an important characteristic in microwave heating The phenomenon is due to microwave electric fields causing oscillations of ions, which in A66] Advantages of microwave heating versus conventional heating have been extensively discussed.[04A CI E6250, 02MOS253] In contrast, conventional heating relies simply on convection currents, thus is limited by the ability of the reactor to transfer heat f rom the source to the reaction medium. Conventional heating depends on the thermal conductivity of the solvent, whereas microwave irradiation relies on the dielectric properties of the solvent to increase heating.[04AA66] Superheating of a solvent is an im portant consideration; reactions carried out in sealed tubes under microwave reactions employ this technique efficiently.[92JCSCC674]

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29 Libraries of compounds may be synthesized in short time periods using microwave assisted heating and are utilized in prosp ective drug discovery. With sensitive catalytic systems, efficient heating and reduction of reaction times often enables the catalyst to last longer, often resulting in higher yields.[04AA66, 06NR51]

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30 CHAPTER 2 EFFICIENT MICROWAVE ASSISTED SYNTHESIS O F 1, 2,4 TRIAZOLE BASED PEPTIDOMIMETICS USIN G BENZOTRIAZOLE METH ODOLOGY 1 2.1 Literature Overview 1,2,4 Triazole derivatives have attracted interest among medicinal chemists because of their versatile biological properties and the bioisosteric replacement of cis amide bonds. By coupling microwave assisted reactions with traditional organic synthesis, a large range of compounds can be synthesized using shorter reaction times and with increased yields. 2.1.1 Biological Properties of Selected 3,5 Disubstituted 1,2,4 Triazoles Compounds bearing the 1,2,4 triazole moiety show interesting biological activities that including: potent inhibitors of CRF1 antagonist, regulatiors of long term stress responses in the pituitary gland [01BMCL3165], and binding with muscarinic r eceptor ligands as [92JMC2392] Several examples of 1,2,4 triazoles have undergone biological testing showing activity in vivo (Figure 2 1). Ribavirin (2.1) is effective against a number of viral types, including HIV, and has been used to treat leukemic cell proliferation. [73JMC935, 09AR1971] JNJ 07706621 (2.2) inhibits Janus associated kinases (TYK2 and JAKs 1 3), and cyclin dependent kinases (CDKs) by affecting the early G1 phase of the cell cycle. [10BMCL7454, 06BMCL3639] AH22216 (2.3) is a H 2 receptor antagonist, and in vivo testing shows it to be 20 30 times more effective than cimetidine in oral potency. [83BR871] 1 Reproduced with permission from Heterocycles 2012 84 515 Copyright 2012 The Japan Institute of Heterocyclic Chemistry

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31 Figure 2 1. Selected biologi cally active 1,2,4 triazole derivatives 2.1.2 Previous Methods for the Synthesis of 1,2,4 Triazole 3,5 diamines Efficient methods for the preparation of 1,2,4 triazole 3,5 diamine derivatives include: N cyanoguanidines,[58JACS3929, 77JHC443] S S dimethyl N cyanodithioimidocarbonate,[86JHC401] and diphenyl cyanocarbonimidate.[82JHC1205, 87JHC275, 93T165, 98TL7983] Due to the limited scope of N cyanoguanidines and the relatively expensive starting materials of diphenyl cyanocarbonimidate, S dimethyl N cyan odithioimidocarbonate 2.4 is the most widely used reagent (Figure 2 2). Figure 2 2. Synthesis of 1,2,4 triazoles 3,5 diamine 2.7 from S dimethyl N cyanodithioimidocarbonate 2.4 2.1.3 Peptidomimetics Bioactive peptides f unction as hormones, enzyme inhibitors and neurotransmitters, but their clinical application is limited due to their rapid hydrolysis by peptidase enzymes.[00CMC945, 02DD847] One approach to overcome this is the use of peptidomimetics. These are small prot ein or peptide like molecules designed to mimic natural peptides.[09EJOC5099] The bioisosteric replacement of the amide bond is

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32 important in the design of peptidomimetics (Figure 2 3).[96CR3147] In particular, 1,2,4 triazole derivatives are utilized as am ide bond mimetics with increased hydrolytic stability.[98BMCL775, 09EJOC5099] Figure 2 3. 1,2,4 triazoles as surrogates of cis amide bonds Peptidomimetic drugs, such as Remikiren 2.10, resist degradation by peptidases (Fig ure 2 4), and compound 2.10 has shown great efficacy in vivo towards hypertension as an inhibitor of the liver enzyme renin. Unfortunately, it also has a short half life on oral delivery and was recently replaced by Aliskiren 2.11.[ 08NRDD399] Figure 2 4. Peptidomimetic drugs which showed anti hypertensive properties 2.2 Results and Discussion N Acylbenzotriazoles are stable solids, easy to handle and useful for N O C and S acylation.[09S2392] A novel microwave assisted approach for the synthesis of 1,2,4 triazole based peptidomimetics using benzotriazole methodology and starting from inexpensive and versatile starting materials is described.

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33 2.2.1 Starting Material Synthesis Table 2 1. Synthesis of Isothioureas interme diates 2.12 14 Entry R 1 R 2 Product Conv. Method Microwave Mp (C) Time (min) Yield (%) Time (min) Yield (%) 1 (CH 2 ) 2 O(CH 2 ) 2 2.12 240 63 a 5 76 d 131 132 2 PhCH 2 H 2.13 300 89 b 5 86 d 158 161 3 Ph H 2.14 240 63 c 30 61 e 195 198 a CHCl 3 reflux, b Diethyl ether, RT, c EtOH, reflux, d Diethyl ether, MW, 45 C, 50 W, e EtOH, MW, 90 C, 100 W Isothiourea intermediates 2.12 14 were prepared following literature procedures by reacting commercially available S dimethyl N cya nodithioimidocarbonate 2.4 with primary (aniline and benzyl amine) and secondary amines (morpholine) (Table 2 1).[86JHC401, 87AP608, 70JOC2067] The conventional heating method gave isothioureas 2.12 14 (63 89% yield), but long reaction times were necessary (4 5 hours). Carrying out the reaction under microwave irradiation led to significantly reduced reaction times (5 30 minutes) and afforded 2.12 14 in comparable yields (61 86%). Isothiourea intermediates 2.12 14 were reacted with hydrazine hydrate in refl uxing ethanol to give 3,5 diamino 1,2,4 triazoles 2.15 17 (75 90% yield). Significant reduction in reaction times was again seen under microwave irradiation giving 2.15 17 in 85 95% yield (4 5 h to 5 10 min). The optimized microwave assisted protocol was c arried out at 80 C at 100 W under a continuous flow of nitrogen.

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34 Table 2 2. Synthesis of 3,5 diamino 1,2,4 triazoles 2.15 17 from isothiourea intermediates 2.12 14 Entry R 1 R 2 Product Conv. heating a Microwave b Mp (C) Time (min) Yield (%) Time (min) Yield (%) 1 (CH 2 ) 2 O(CH 2 ) 2 2.15 240 83 5 89 165 166 2 PhCH 2 H 2.16 300 75 10 90 147 148 3 Ph H 2.17 240 90 10 95 166 169 a EtOH, reflux, b EtOH, MW, 80 C, 100 W Isothiourea intermediate 2.13 was used in the synthesis of N 1 methyl protected 3,5 diamino 1,2,4 triazole 2.18 (Scheme 2 1). Methyl hydrazine (2 equivalents) was reacted with isothiourea 2.13 in refluxing ethanol to give 2.18 (51% yield).[86JHC401] Under microwave heating a 50/50 mixture of regioisomers was obt ained and could not be separated. Scheme 2 1. Formation of ring protected 3,5 diamino 1,2,4 triazole 2.18 2.2.2 Ring Acylation of 2.15 and 2.17. Initial experiments showed reaction of 2.15 with Cbz Ala Bt under reflux cond itions was incomplete after 12 hours. However, when heating under microwave irradiation was employed, 2.23 was obtained in 95% yield (Entry 1, Table 2 3). Compound 2.15 (Entries 2 4) was therefore reacted with N aminoacyl)benzotriazoles 2.20 2 2 to afford the corresponding N aminoacyl triazoles 2.24 26 in good to excellent yields (70

PAGE 35

35 95%). Reaction of 2.17 (Table 2 2) with Boc protected glycyl benzotriazole gave 2.27 in 65% yield (Entry 5). This methodology showed compatibility with a wide range of amino acid protecting groups. Table 2 3. Synthesis of ring acylated 3,5 diamino 1,2,4 triazoles 2.23 27 Entry Product R 1 R 2 R 3 PG Yield (%) Mp (C) 1 2.23 (CH 2 ) 2 O(CH 2 ) 2 CH 3 Cbz 95 205 207 2 2.24 (CH 2 ) 2 O(CH 2 ) 2 H F moc 70 211 214 3 2.25 (CH 2 ) 2 O(CH 2 ) 2 PhCH 2 Cbz 95 217 218 4 2.26 (CH 2 ) 2 O(CH 2 ) 2 H Boc 73 212 215 5 2.27 Ph H H Boc 65 230 233 Table 2 4. Ring acylation using Cbz protected dipeptidoyl benzotriazoles on 2.15 Entry Pr oduct R 1 R 2 Yield (%) Mp (C) 1 2.31 CH 3 PhCH 2 95 188 190 2 2.32 PhCH 2 CH 3 86 194 195 3 2.33 i Bu H 76 102 106

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36 Ring acylation was observed in good to excellent yields (65 95%) with N aminoacyl)benzotriazoles 2.19 22 and N (protected dipeptidoyl)benzotriazoles 2.28 30, showing tolerance for an array of standard amino acid and peptide protecting groups. 2.2.3 Exocyclic Acylation of 2.18 Ring acylation was seen exclusively with 2.23 27 and 2.31 33, but exocyclic acylation was also required. Reaction of 2.18 with N Cbz Alaninyl benzotriazole 2.19, gave exocyclic product 2.34 in 57% yield (Scheme 2 2), and reaction with dipeptidoyl 2.28 gave exocyclic dipeptidoyl acylation of 2.35 in 71% yield. Scheme 2 2. Exocyclic acylation of 2.18 under microwave irradi ation 2.2.4 Determination of Acylation Site by 1 H NMR Experiments Acylation of the ring nitrogen was confirmed by observation of an amino sig nal (2H) in the 1 H NMR spectrum. The downfield shift of the amino group signal (> 7 ppm) indicates that ring acylation has taken place at the N 1 position proximal to the amino group. This is consistent with the findings of Reiter et al who showed that the exocyclic

PAGE 37

37 amino group proximal to the methyl substituted ring nitrogen in 1 methyl 1 H 1,2,4 triazole 3,5 diamine induced a downfield shift whereas the distal exocyclic amino group t al. found a similar trend with an analog of compound JNJ 7706621 2.2, for which the chemical shift of the amino group upon acylation at N 2 was upfield (6.25 ppm) whereas acylation at N 1 showed a considerable downfield shift of 7.95 ppm.[10BMCL7454] Compo unds (2.23 27 and 2.31 33) share this chemical shift pattern where the downfield signal of the amino group indicates acylation of the ring nitrogen ( N 1 ) proximal to the primary amino group (Figure 2 5). Figure 2 5. 1 H NMR shifts of ring acylated compounds 2.3 Summary An efficient, fast and convenient method for microwave assisted preparation of 1,2,4 triazole substituted amino acids and dipeptides was developed. This method offers short reaction times, good to excellent yie lds (57 95%) and is compatible with a variety of standard protecting groups 2.4 Experimental 2.4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl 3 or DMS O d 6 on Gemini or

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38 Varian NMR operating at 300 MHz for 1 H and 75 MHz for 13 C with TMS as an internal standard. Elemental analyses were performed on a Carlo Erba 1106 instrument. All microwave assisted reactions were carried out with a single mode cavity Dis cover Microwave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon septum and the reaction mixture was subjected to m icrowave irradiation (Discover mode; run time: 60 sec.; PowerMax cooling mode). All N (protected aminoacyl)benzotriazoles and N (protected dipeptidoyl)benzotriazoles used have been prepared according to our previously published methods. 25 2.4.2 Synthesis 2.12 14 and 2.15 17 2.4.2.1 General p rocedure s for the m icrowave a ssisted p reparation of i sothioureas 2.12 14 A mixture of S dimethyl N cyanodithioimidocarbonate 2.4 (0.44 g, 3.0 mmol) and the respective primary or secondary amine (3.0 mmol) in diethyl ether (5 mL) or ethanol (5 mL) was subjected to microwave irradiation (Table 1). Compounds 2.12 14 were collected, washed with diethyl ether (2 x 5 mL) and dried under vacuum. Compound 2.14 was crystallized from ethanol:hexanes, filtered, washed with hexa nes (2 x 5 mL) and dried under vacuum. Methyl N cyanomorpholine 4 carbimidothioate 2.12. White microcrystals, 76% yield, mp 131 132 C (lit. mp 125 126 C) ;[81TL2285] 1 H NMR (300 MHz, CDCl 3 ) 3.82 (t, J = 4.7 Hz, 4H), 3.70 (t, J = 4.7 Hz, 4H), 2.76 (s, 3H); 13 C NMR (75 MHz, CDCl 3 ) 169.4, 114.9, 66.4, 48.7, 16.3. Anal. Calcd for C 7 H 11 N 3 OS: C 45.39; H 5.99; N 22.68. Found: C 45.24; H 5.96; N 22.64.

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39 Methyl N benzyl N cyanocarbamimidothioate 2 .13. White microcrystals, 86% yield, mp 158 161 C (lit. mp 156 157 C);[05BCSJ873] 1 H NMR (300 MHz, DMSO d 6 ) 8.91 (s, 1H), 7.38 7.25 (m, 5H), 4.50 (s, 2H), 2.63 (s, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 170.3, 137.4, 128.4, 127.3, 127.2, 115.8, 46.2, 14.1. A nal. Calcd for C 10 H 11 N 3 S: C 58.51; H 5.40; N 20.47. Found: C 58.13; H 5.29; N 20.68. Methyl N cyano N phenylcarbamimidothioate 2.14. White microcrystals, 61% yield, mp 195 198 C (lit. mp 194 196 C);[70JOC2067] 1 H NMR (300 MHz, DMSO d 6 ) 10.16 (s, 1H), 7.52 7.18 (m, 5H), 2.70 (s, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 170.2, 137.2, 128.8, 126.4, 124.2, 114.8, 14.9. Anal. Calcd for C 9 H 9 N 3 S: C 56.52; H 4.74; N 21.97. Found: C 56.25; H 4.60; N 21.95. 2.4.2.2 General p rocedure s for m icrowave a ssisted s ynthesis of 1,2,4 t riazoles 2.15 17 A reaction mixture of the appropriate isothiourea 2.12 14 (2.0 mmol) and 72% hydrazine hydrate (0.2 g, 4.0 mmol) in ethanol (5 mL) was subjected to microwave irradiation (80 C, 100 W, 5 10 min). On completion of the reaction (TLC) the solvent was removed under reduced pressure and the residue was crystallized from CHCl 3 :hexanes. 3 Morpholino 1 H 1,2,4 triazol 5 amine 2.15. White microcrystals, 89% yield, mp 165 166 C (lit. mp 167 168 C);[86JHC401] 1 H NMR (300 MHz, DMSO d 6 ) 10.90 (br s, 1H), 5.99 (br s, 2H), 3.66 (t, J = 4.4 Hz, 4H), 3.15 (t, J = 4.4 Hz, 4H); 13 C NMR (75 MHz, DMSO d 6 ) 163.1, 156.9, 66.3, 47.4. N 3 Benzyl 1 H 1,2,4 triazole 3,5 diamine 2.16. White microcrystals, 90% yield, mp 147 148 C (lit. mp 151 153 C) ; [86JHC401] 1 H NMR (300 MHz, DMSO d 6 ) 7.47 7.10 (m, 5H), 6.20 (s, 1H), 5.42 (s, 2H), 4.23 (d, J = 5.7 Hz, 2H); 13 C NMR (75 MHz, DMSO d 6 ) 160.1, 157.8, 141.1, 128.0, 127.2, 126.4, 46.2.

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40 N 3 Phenyl 1 H 1,2,4 triazole 3,5 diamine 2.17. White microcrystals, 95% yield, mp 166 169 C (lit. mp 161 162 C); [86JHC401] 1 H NMR (300 MHz, DMSO d 6 ) 11.20 (br s, 1H), 8.62 (s, 1H), 7.49 (d, J = 7.9 Hz, 2H), 7.15 (t, J = 7.8 Hz, 2H), 6.71 (t, J = 7.3 Hz, 1H), 5.87 (br s, 2H); 13 C NMR (75 MHz, DMSO d 6 ) 157.6, 155.6, 1 42.6, 128.4, 118.3, 115.5. Anal. Calcd for C 8 H 9 N 5 : C 54.85; H 5.18; N 39.98. Found: C 54.48; H 5.08; N 39.81. 2.4.2.3 Synthesis of r ing p rotected 1,2,4 t rizaole 2.18 N 5 Benzyl 1 methyl 1 H 1,2,4 triazole 3,5 diamine 2.18. Methyl N benzyl N cyanocarbamimido thioate (2.0 g, 10.0 mmol) and methylhydrazine (1.1 mL, 20.0 mmol) were heated under reflux in ethanol (50 mL) for 4 hours. The solvent was removed under reduced pressure and the crude residue was recystallized from acetonitrile/hexanes. White microcrystal s, 51% yield, mp 159 162 C (lit. mp 159 161 C) ; 86JHC401] 1 H NMR (300 MHz, DMSO d 6 ) 7.40 7.16 (m, 5H), 6.74 (t, J = 6.0 Hz, 1H), 4.90 (br s, 2H), 4.39 (d, J = 6.0 Hz, 2H), 3.34 (s, 3H) ; 13 C NMR (75 MHz, DMSO d 6 ) 160.2, 154.8, 140.4, 128.2, 127.1, 126 .7, 46.6, 32.5. 2.4.3 Ring Acylation of 1,2,4 Triazoles 2.23 27 and 2.31 33 2.4.3.1 General p rocedure s for m icrowave a ssisted s ynthesis of c ompounds 2.23 27 A mixture of the respective N (protected aminoacyl)benzotriazole (1.0 mmol) and 1,2,4 triazole 2. 15 and 2.17 (1.0 mmol) in dry THF (3 mL) was subjected to microwave irradiation (70 C, 100 W, 30 min). The products were isolated and purified according to the following procedures. The reaction mixtures of compounds 2.23 and 2.25 were quenched with water (2 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with Na 2 CO 3 solution (10% w/w, 3 x 20 mL), water (3 x 20 mL), dried over MgSO 4 and the solvent was removed under reduced

PAGE 41

41 pressure. The residues were recrystallize d from CH 2 Cl 2 :hexanes to give the desired products 2.23 and 2.25. In case of compounds 2.24 and 2.26 the reaction mixtures were evaporated under reduced pressure and the crude products were recrystallized from methanol. The reaction mixture of compound 2.2 7 was allowed to cool to room temperature and crystallized from a mixture of THF, CH 2 Cl 2 and hexanes. The precipitate was collected, washed with CH 2 Cl 2 (2 x 10 mL) and dried under vacuum. ( S ) Benzyl (1 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 1 oxoprop an 2 yl)carbamate 2.23. White microcrystals, 95% yield, mp 205 207 C; 1 H NMR (300 MHz, DMSO d 6 ) 7.85 (d, J = 7.2 Hz, 1H), 7.61 (br s, 2H), 7.39 7.31 (m, 5H), 5.04 5.01 (m, 2H), 4.91 4.81 (m, 1H), 3.67 3.62 (m, 4H), 3.31 3.24 (m, 4H), 1.35 (d, J = 7.2 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 172.5, 163.0, 157.2, 155.9, 136.9, 128.4, 127.9, 65.6, 49.4, 4 5.6, 34.4, 16.2. HRMS calcd. for C 17 H 22 N 6 O 4 [M+H] + : 375.1775. Found [M+H] + : 375.1786. (9 H Fluoren 9 yl)methyl (2 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 2 oxoethyl)carbamate 2.24. White microcrystals, 70% yield, mp 211 214 C; 1 H NMR (300 MHz, DMSO d 6 ) 7.90 (d, J = 7.2 Hz, 2H), 7.84 7.67 (m, 3H), 7.58 (br s, 2H), 7.48 7.30 (m, 4H), 4.44 4.09 (m, 5H), 3.63 (br s, 4H), 3.28 (br s, 4H); 13 C NMR (75 MHz, DMSO d 6 ) 169.3, 163.7, 157.5, 157.3, 144.4, 141.4, 128.3, 127.7, 125.9, 120.8, 66.5, 66.2, 47.3, 46.2 44.0. Anal. Calcd for C 23 H 24 N 6 O 4 : C 61.60; H 5.39; N 18.74. Found: C 61.91; H 5.53; N 18.53. ( S ) Benzyl (1 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 1 oxo 3 phenylpropan 2 yl)carbamate 2.25. White microcrystals, 95% yield, mp 217 218 C; 1 H NMR (300 M Hz, DMSO d 6 ) 7.88 (d, J = 7.2 Hz, 2H), 7.76 7.75 (m, 4H) 7.57 (br s, 2H), 7.45 7.40

PAGE 42

42 (m 4 H), 4.34 4.30 (m, 2H), 4.27 4.21 (m, 2H), 3.63 (d, J = 4.8 Hz, 4H), 3.28 (d, J = 4.8 Hz, 4H); 13 C NMR (75 MHz, DMSO d 6 ) 166.7, 163.0, 156.8, 156.6, 143.8, 140.7, 127.6, 127.0, 125.2, 120.1, 65.8, 65.5, 46.6, 45.5, 43.4. Anal. Calcd for C 23 H 26 N 6 O 4 : C 61.32; H 5.82; N 18.65. Found: C 61.44; H 5.45; N 18.28. tert Butyl (2 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 2 oxoethyl) carbamate 2.26. White microcrystals, 73% yield, mp 212 215 C; 1 H NMR (300 MHz, DMSO d 6 ) 7.55 (br s, 2H), 7.14 (t, J = 6.1 Hz, 1H), 4.14 (d, J = 6.1 Hz, 2H), 3.64 (t, J = 4.7 Hz, 4H), 3.27 (t, J = 4.7 Hz, 4H), 1.39 (s, 9H); 13 C NMR (75 MHz, DMSO d 6 ) 169.0, 163.0, 156.8, 155.9, 78.2, 65.6, 45.5, 43.0, 28.2. Anal. Calcd for C 13 H 22 N 6 O 4 : C 47. 84; H 6.79; N 25.75. Found: C 48.17; H 6.97; N 25.95. tert Butyl (2 (5 amino 3 (phenylamino) 1 H 1,2,4 triazol 1 yl) 2 oxoethyl) carbamate 2.27. White microcrystals, 65% yield, mp 230 233 C; 1 H NMR (300 MHz, DMSO d 6 ) 9.29 (s, 1H), 7.65 7.52 (m, 4H), 7. 29 7.17 (m, 3H), 6.85 (t, J = 7.2 Hz, 1H), 4.27 (d, J = 6.0 Hz, 2H), 1.41 (s, 9H); 13 C NMR (75 MHz, DMSO d 6 ) 168.9, 158.4, 155.8, 140.9, 128.6, 120.0, 116.7, 78.3, 43.2, 28.2. Anal. Calcd for C 15 H 20 N 6 O 3 : C 54.21; H 6.07; N 25.29. Found: C 54.43; H 6.23 ; N 24.57. 2.4.3.2 General p rocedure s for m icrowave a ssisted s ynthesis of c ompounds 2.31 33. A mixture of the appropriate N (protected dipeptidoyl)benzotriazole (1.0 mmol) and 3 morpholino 1 H 1,2,4 triazol 5 amine (0.169 g, 1.0 mmol) in dry THF (3 mL) was subjected to microwave irradiation (70 C, 100 W, 30 min). The reaction mixtures were allowed to cool to room temperature and evaporated to give crude products. Compound 2.31 was dissolved in ethyl acetate (30 mL), washed with Na 2 CO 3 solution (10% w/w, 3 x 20 mL), water (3 x 20 mL), dried over MgSO 4 and the solvent was removed under reduced pressure to give the desired product. Compound 2.32 was recrystallized from

PAGE 43

43 methanol. Compound 2.33 was recrystallized from diethyl ether:hexanes. The precipitates were collected, washed with hexanes (2 x 5 mL) and dried under vacuum. Benzyl (( S ) 1 ((( S ) 1 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 1 oxopropan 2 yl)amino) 1 oxo 3 phenylpropan 2 yl)carbamate 2.31. White microcrystals, 95% yield, mp 188 190 C; 1 H NMR (3 00 MHz, DMSO d 6 ) 8.36 (d, J = 6.5 Hz, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.7 Hz, 2H), 7.57 (m, 3H), 7.46 7.28 (m, 6H), 5.10 5.00 (m, 1H), 4.30 4.17 (m, 3H), 3.68 3.62 (m, 6H), 3.32 3.24 (m, 4H), 1.36 (d, J = 7.0 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 171.9, 169.0, 162.9, 157.1, 156.4, 143.8, 140.7, 127.6, 127.1, 125.2, 120.1, 65.7, 65.6, 47.6, 46.6, 45.5, 42.9, 16.3. Anal. Calcd for C 26 H 31 N 7 O 5 : C 59.87; H 5.99; N 18.80. Found: C 60.21; H 5.60; N 18.42. Benzyl (( S ) 1 ((( S ) 1 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 1 oxo 3 phenylpropan 2 yl) amino) 1 oxopropan 2 yl)carbamate 2.32. White microcrystals, 86% yield, mp 194 195 C; 1 H NMR (300 MHz, DMSO d 6 ) 8.26 (d, J = 7.7 Hz, 1H), 7.56 (br s, 2H), 7.40 7.20 (m, 10H), 7.19 7.11 (m, 1H), 5.28 5.05 ( m, 1H), 4.93 (d, J = 2.6 Hz, 2H), 4.12 3.96 (m, 1H), 3.67 3.54 (m, 4H), 3.37 3.22 (m, 4H), 3.14 (dd, J = 13.6, 3.3 Hz, 1H), 2.81 (dd, J = 13.8, 9.8 Hz, 1H), 1.14 (d, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 172.8, 170.6, 163.0, 157.1, 155.5, 137.6, 137 .0, 129.0, 128.3, 128.2, 127.8, 126.5, 65.6, 65.3, 53.8, 49.7, 45.5, 35.7, 18.2. Anal. Calcd for C 26 H 31 N 7 O 5 : C 59.87; H 5.99; N 18.80. Found: C 59.72; H 6.04; N 18.92. ( S ) Benzyl (2 ((1 (5 amino 3 morpholino 1 H 1,2,4 triazol 1 yl) 4 methyl 1 oxopentan 2 yl )amino) 2 oxoethyl)carbamate 2.33. White microcrystals, 76% yield, mp 102 106 C; 1 H NMR (300 MHz, DMSO d 6 ) 8.26 (d, J = 8.0 Hz, 1H), 7.57 (br s, 2H), 7.43 (t, J = 6.2 Hz, 1H), 7.39 7.29 (m, 5H), 5.18 5.07 (m, 1H), 5.02 (s, 2H), 3.74 3.58

PAGE 44

44 (m, 6H), 3.31 3.22 (m, 4H), 1.79 1.36 (m, 3H), 0.97 0.78 (m, 6H); 13 C NMR (75 MHz, DMSO d 6 ) 171.9, 169.4, 162.8, 157.1, 1 56.4, 137.0, 128.3, 127.7, 65.5, 65.4,50.3, 45.5, 43.1, 24.6, 23.2, 20.9. Anal. Calcd for C 22 H 31 N 7 O 3 : C 55.80; H 6.60; N 20.71. Found: C 55.86; H 6.55; N 20.33. 2.4.4 Exocyclic Acylation of 1,2,4 Triazoles 2.34 35 ( S ) Benzyl (1 ((1 methyl 5 (phenylamino) 1 H 1,2,4 triazol 3 yl)amino) 1 oxopropan 2 yl) carba mate 2.34. A mixture of Cbz L Ala Bt 2.19 (0.32 g, 1.0 mmol) and N 5 benzyl 1 methyl 1 H 1,2,4 triazole 3,5 diamine 2.18 (0.20 g, 1.0 mmol) in dry THF (3 mL) was subjected to microwave irradiation (70 C, 10 0 W, 30 min). The reaction was quenched with water (2 mL), and extracted with ethyl acetate (3 x 10 mL). The combined organics were washed with Na 2 CO 3 solution (10% w/w, 3 x 20 mL), water (3 x 20 mL) and dried over MgSO 4 The solvent was then removed und er reduced pressure and the residue was recrystallized from CH 2 Cl 2 /hexanes. White microcrystals, 57% yield, mp 184 185 C; 1 H NMR (300 MHz, DMSO d 6 ) 7.47 (d, J = 7.6 Hz, 1H), 7.38 7.29 (m, 11H), 7.28 7.18 (br s, 1H), 5.00 (s, 2H), 4.41 (d, J = 5.9 Hz, 2H), 4.14 (d, J = 1.3 Hz, 1H), 3.48 (s, 3H), 1.21 (d, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 155.6, 154.7, 140.0, 128.3, 128.2, 127.7, 127.0, 126 .7, 65.3, 46.4, 33.0, 18.0. Anal. Calcd for C 21 H 24 N 6 O 3 : C 61.75; H 5.92; N 20.57. Found: C 61.37; H 5.92; N 20.63. Benzyl (( S ) 1 ((( S ) 1 ((5 (benzylamino) 1 methyl 1 H 1,2,4 triazol 3 yl)amino) 1 oxo 3 phenyl propan 2 yl)amino) 1 oxopropan 2 yl)carbamate 2. 35. A mixture of Cbz L Ala L Phe Bt 2.28 (0.48 g, 1.0 mmol) and N 5 benzyl 1 methyl 1 H 1,2,4 triazole 3,5 diamine 2.18 (0.20 g, 1.0 mmol) in dry THF (3 mL) was subjected to microwave irradiation (70 C, 100 W, 30 min). The reaction was quenched with water ( 2 mL), and extracted with ethyl acetate (3 x 10 mL). The combined organics were washed with

PAGE 45

45 Na 2 CO 3 solution (10% w/w, 3 x 20 mL), water (3 x 20 mL) and dried over MgSO 4 The solvent was then removed under reduced pressure and the residue was recrystallized from CH 2 Cl 2 /hexanes. White microcrystals, 71% yield, mp 107 109 C; 1 H NMR (300 MHz, DMSO d 6 ) 10.27 (br s, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.45 7.14 (m, 16H), 7.07 (br s, 1H), 5.06 4.91 (m, 2H), 4.72 4.51 (m, 1H), 4.42 (d, J = 6.0 Hz, 2H), 4.10 3.94 (m, 1H), 3.50 (s, 3H), 3.09 2.93 (m, 1H), 2.89 2.68 (m, 1H), 1.12 (d, J = 7.1 Hz, 3H); 13 C NMR (75 MHz, DMSO d 6 ) 172.9, 169.5, 156.2, 155.3, 152.4, 140.6, 138.1, 137.6, 130.0, 129.0, 128.9, 128.6, 128.4, 127.7, 127.4, 126.9, 66.1, 54.7, 50.7, 47.1, 38.2, 33.7, 18.9. Anal. Calcd for C 30 H 33 N 7 O 4 : C 64.85; H 5.99; N 17.65. Found: C 64.55; H 6.04; N 17.55.

PAGE 46

46 CHAPTER 3 A NEW BENZOTRIAZOLE MEDIATED, STEREOFLEX IBLE GATEWAY TO HETE RO 2,5 DIKETOPIPERAZINES 1 3.1 Literature Overview Small cyclic peptides are an important subgroup of peptidic structures. A large library of cyclic structures can be generated from av ailable natural or synthetic amino acids. The 2,5 diketopiperazine (DKP) backbone, the smallest cyclic peptidoyl sequence, appears in many naturally occurring molecules.[11CEJ1388, 11OL2770, 11TL2262, 09P833] Proline plays a prominent role in protein foldi ng conferring on DKPs turns.[11EJOC217] The biological activity associated with DKPs includes antibiotic [08JACS6281, 03JNP1299], insecticidal [03TL6003], antimitotic [08BMC4626], chemosensitizing [02MCT417, 8 6TL6361], and antiviral [10OBC5179] properties. 3.1.1 Properties of Proline Containing Cyclic Peptides The proline containing 2,5 diketopiperazine (DKP) scaffold 3.1 has three positions available for functionalization, two of which are stereocenters (Figur e 3 1). [10BC210, 09JPS474, 08OBC3989, 08TL906, 07ACIE7488, 07JOC195] Cyclo( L Leu D Pro) (3.2) exhibits antibiotic properties against Vibrio anguillarium concentrations.[03JNP1299] Spirostyrptostatin A 3.3 [99JACS2417, 96T12651] and tryptostatin A and B 3.5 [98TL7009] show ability to inhibit the mammalian cell cycle at the G2/M phase during cell division, making them potential therapeutics fo r cancer treatment. Fumitremorgin B 3.4 [86TL6217], isolated from toxigenic food borne fungi Aspergillus fumigates exhibits strong tremorgenic actions in mice. Protubonines A and 1 Reproduced with permission from Chem. Eur. J. 2012, 18 2632 Copyright 2012 John Wiley & Sons, Inc.

PAGE 47

4 7 B 3.6 [11JNP1284], isolated from marine fungus, are effective against human cancer cells lines. Figure 3 1. Selected examples of proline containing 2,5 diketopiperazines Cyclic 2,5 diketopiperazines show improved bioavailability and increased resistance to enzymatic degradation relative to linear analogs. The lipophilicity of the lateral chains can be tuned by structural changes [07JACS11802], thus making DKPs important building blocks for the generation of new therapeutic agents. Most naturally occurring DKPs posses a cis configuration since they originate from ( L ) amino acids and considerable attention has been given to their synthesis.[11EJOC217, 07T9923, 02T3297] cis Cyclic dipeptide synthesis usually starts from protected amino acids or peptide subunits in solution or on a solid phase.[07T9923, 02T3297, 07CCH TS857] Literature cyclizations fall into three groups:

PAGE 48

48 head to tail condensation [10ACIE9262, 10TL1303, 09ACB1051, 09T5343, 08ACIE1485, 06JCC915, 06T4784 ] dimerization of two peptidic subunits [10TL4558, 08EJOC5418 ] and non peptidic coupling methods.[10J ACS2889, 09JOC4267] Several recent reports include trans DKPs bearing non proteinogenic ( D ) amino acids.[09ACB1051] trans DKPs have also been used as building blocks for foldamers.[06JOC8691] ( D ) Proline containing DKP backbones show a number of properties including retardation of metabolism pharmaceuticals [02JMC1559], and increased mimicry of substrates.[04 J MC5713, 12BJ23] trans DKPs have been synthesized from the more expensive ( D ) amino acids [10OBC5179, 02JMC1559, 10OL2162, 10BMCL7327] or by epimerization of cis DKPs.[03JNP1299, 74JACS3985] Epimerization, often reported as a side reacti on upon cyclization [10OL2418, 05S3412] or further functionalization [03JNP1299, 08T3713, 00BMC2407] leads to mixtures of cis / trans DKPs. 3.1. 2 Turn inducers in Peptide Synthesis Turn inducing moieties are common features in proteins and other molecules. Their incorporation in small peptide sequences mimics helices and turns) is used to study complex interactions in the secondary structure.[11EJOC217] Turn inducers allow favorable geometric conformations and facilitate cyclizations. Common turn inducers include proline,[88JMB221] pseud op rolines, [10OL3136] unnatural amino acids [06JMC616, 08CBDD125] and non proteinogenic residues.[09T240]

PAGE 49

49 Figure 3 2. Proline as a turn inducer in the synthesis of larger cyclic peptides 3.2 Results and Discussion Despite con siderable research, there is a need for further development of alternative, flexible and cost effective strategies for the synthesis of cyclic peptides. Our longstanding involvement in benzotriazole mediated oligopeptide chemistry prompted the design of a new, versatile and flexible strategy to provide cis or trans configured DKPs starting from identical ( L L ) dipeptidoyl benzotriazolides. trans DKPs were synthesized by a tandem triethylamine catalyzed cyclization/epimerization, whereas a tandem deprotecti on/cyclization strategy led to cis DKPs. 3.2.1 Starting Material Synthesis Dipeptides 3.17 23 were prepared from N (Cbz aminoacyl)benzotriazoles 3.9 15 and L proline 3.16. Coupling was performed without racemization using procedures optimized by Katritzky et al. [09S2392] The reaction was complete in acetonitrile/water (3:1) in the presence of triethylamine (Et 3 N) at room temperature in less than 3 hours (Table 3 1 Condition A). Significant improvements in yield and reaction times were seen under microwave heating giving dipeptides 3.17 23 in 15 min and in 85 95% yield (Table 3 1 Condition B).

PAGE 50

50 Table 3 1. Synthesis of Cbz pr otected dipeptides 3.17 23 through benzotriazole mediated coupling Compound R 1 A [a] B [b] Time (min) Yield (%) Time (min) Yield (%) 3.17 H 120 76 5 87 3.18 Me 120 92 5 92 3.19 PhSCH 2 180 89 10 91 3.20 i Pr 300 77 15 85 3.21 i Bu 180 91 10 94 3.22 Bn 180 89 10 95 3.23 INDM [c] 180 91 10 90 [a] Condition A: MeCN/water 3:1, 1 eq. Et 3 N, rt. [b] Condition B:MeCN/water 3:1, 1 eq. Et 3 N, MW, 50W, 50 C. [c] 1 H Indol 3 ylmethyl. Benzotriazole activated dipeptides 3.24 30 ( Scheme 3 1) were synthesized from dipeptides 3.17 23. The process was carried out at 10 C with thionyl benzotriazole generated in situ to yield 3.24 30 (61 87%). [09S2392] Scheme 3 1. Formation of dipeptidoyl benzotriazo le compounds 3.24 30 3.2.2 Synthesis of trans 2,5 Diketopiperazines. Conditions for cyclization were optimized using 3.24 (Table 3 2). Different solvents, co reagents, and reaction conditions including the effects of microwave irradiation, were studied. Un der reflux conditions for 18 hours in acetonitrile and in the absence of co reagent, 14% of 3.31 was isolated, while no product was detected under

PAGE 51

51 microwave irradiation ( E ntry 1). Attempts using pyridine and sodium carbonate also led to no product formatio n ( E ntries 2 and 3). Table 3 2. Optimization of cyclization conditions Entry Solvent Co reagent (eq.) t (min) [a] Isolated 3.31 (%) 1 MeCN None 60 0 2 MeCN Pyridine (1) 60 0 3 MeCN NaHCO 3 (5) 60 0 4 MeCN Et 3 N (1) 20 83 5 THF Et 3 N (1) 40 83 6 MeCN Et 3 N (0.1) 25 75 7 MeCN Et 3 N (0.01) 40 72 8 MeCN DBU (1) 20 0 [b] [a] 80 C, 70 W. [b] Complete degradation was observed. Reaction in the presence of 1 equivalent of triethylamine (Et 3 N), however gave 3.31 in 83% yield ( E nt ry 4), with THF as an effective solvent ( E ntry 5). Catalysis by sub stoichiometric amounts of Et 3 N, 0.1 equivalents (Entry 6) led to a 75% yield of 3.31 and 0.01 equivalents of Et 3 N gave 3.31 in 72% yield ( E ntry 7). Chiral HPLC on 3.31 revealed complete ra cemization. Scheme 3 2. Formation of racemic 3.31 from chiral 3.3 2 Cyclization with enantiomer 3.32 again showed racemization and which led to an investigation of the reaction mechanism.

PAGE 52

52 The unexpected racemization, was pr obably due to base catalyzed enolization of the 2,5 diketopiperazine. Lacking a stereo center at position 3, there was no chiral memory to induce stereoselectivity during reprotonation (Figure 3 3 ). Literature sources suggest that proline containing DKPs a re prone to acid or base induced prototropy, although racemization was also observed in the cyclization of Cbz N glycl ( L ) leucyl benzotriazole 3. 36 (Scheme 3 3).[74JACS3985, 01JOC6333] According to recent literature however, cyclization under harsh condi tions utilizing a methoxybenzyl protection scheme proceeded without racemization.[08T3713] Figure 3 3. Cyclization and racemization of substrates 3.24 and 3.32 Scheme 3 3. Cyclization/rac emization of 3.36 On the basis of our preliminary observations, we postulated that inclusion of a stereocenter at position 3 of the DKP would influence the stereochemistry of

PAGE 53

53 reprotonation. Authentic stereoisomeric samples of 3.25 and 3.27 were synthesized using previously described procedures. Cyclization of 3.25 and 3.27 using the optimized reaction conditions was then examined by chiral HPLC (Table 3 3). Table 3 3. Cyclization of 3.25 and 3.27 to form trans 3.38 and 3.39 [a] Entry (X,Y) Yield (%) R t (min) Stereochem. 1 ( DL,L ) 3.25 70 42.0, 43.3 ( L,D )+( D,L ) 3.38 2 ( L,L ) 3.25 69 43.3 ( L,D ) 3.38 3 ( L,D ) 3.25 72 43.3 ( L,D ) 3.38 4 ( D,L ) 3.25 71 41.9 ( D,L ) 3.38 5 ( D,D ) 3.25 63 42.0 ( D,L ) 3.38 6 ( L,L ) 3.27 75 35.2 ( L,D ) 3.39 7 ( L,D ) 3.27 77 35.1 ( L,D ) 3.39 8 ( D,L ) 3.27 79 37.9 ( D,L ) 3.39 9 ( D,D ) 3.27 69 37.9 ( D,L ) 3.39 [a] Chiral HPLC was performed using a Chirobiotic T column. Flow rate = 0.1 mL / min; eluent = methanol. The initial condition (Entry 1), starting from a mixture of DL alanine and L proline yielded a racemic mixture of thermodynamically favored trans DKPs ( L,D ) 3.38 and ( D,L ) 3.38. The enantiomers were co crystallized and the relative stereochemistry was assigned unambiguously by single crystal X ray diff raction (Figure 3 4 ). Compound ( L,D ) 3.38, obtained from entries 2 and 3, resulted in identical HPLC profiles with a retention time of 43.3 minutes. The diastereomer ( D,L ) 3.38 (Entries 4 and 5) showed similar results with a unique retention time of 42.0 m inutes. The results were supported

PAGE 54

54 by optical rotation measurements showing ( L,D ) 3.38 having [ ] D 21 = 101.0 and ( D,L ) 3.38 having [ ] D 21 = 129.7 in agreement with literature values. This data allowed the absolute stereochemical assignments of the trans DKPs regardless of the stereochemistry of the starting material. Figure 3 4 Single crystal X ray diffraction structure of ( L D ) 3.38 showing the trans configuration Results obtained for the 3.39 series ( E ntries 6 9) were also consistent with formation of trans DKPs. In entry 6, the absolute stereochemistry was assigned unambiguously by X ray diffraction (Figure 3 5 ). Figure 3 5 Single Crystal X ray diffraction structure of 3.39 showing the absolute stereochemistry of the L,D configuration

PAGE 55

55 Computati ons performed by Dr. Jean Christophe Monbaliu, utilizing the B3LYP/6 31+G(d) level of theory (Figure 3 6 ) to compare the thermodynamic stability of the enols and the parent DKPs. The presence of a Cbz protecting group (modeled as a methyl carbamate) consid erably increased the thermodynamic stability of the corresponding enol (3.40 41).[10EJOC1711] Figure 3 6 Thermodynamic stabilities of enols and mother DKPs showing the effect of the protecting group Computational conforma tional analysis, by Dr. Jean Christophe Monbaliu, showed the most stable conformer of the proline containing dipeptide is the twisted, ready to cyclize conformation. Nonproline containing dipeptides, however, were in a linear transoid configuration in thei r lowest energy conformation. Investigation revealed two possible mechanisms: a unimolecular transition state with benzotriazole as a base and a leaving group, and a bimolecular transition state with Et 3 N as a catalyst and benzotriazole solely as a leaving group. The latter was energetically favored over the

PAGE 56

56 unimolecular mechanism, a result in agreement with the experimental observations (Figure 3 7 ).[12CEJ2632] Figure 3 7 Pictures of the transition states associated with the unimolecular unassisted mech anism (TS uni left) and with the bimolecular assisted mechanism (TS bi right). Cbz and benzotriazole groups are modeled by a methyl carbamate and a triazole group, respectively [12CEJ2632] Table 3 4. Tandem cyclization/epimerization for the formation of 3. 28 29 and 3.44 47 Compound R 1 Isolated yield (%) 3.38 Me 69 3.44 BnSCH 2 72 3.45 i Pr 70 3.39 i Bu 75 3.46 Bn 71 3.47 INDM [a] 73 [a] 1 H Indol 3 ylmethyl.

PAGE 57

57 A small library of proline containing DKPs 3. 38 39, 3.44 47 we re synthesized in 69 75% yield (Table 3 4), to give exclusively the trans DKPs 3.38 39, 3.44 47 by the tandem cyclization/epimerization reaction. All reactions were carried out with 1 equivalent of Et 3 N to ensure short reaction times. Reaction products wer e purified through flash column chromatography and fully characterized. Compound 3.39 was deprotected by hydrogenation in the presence of Pd/C for 24 hours at room temperature (Scheme 3 4), affording 3.48 in 87% yield as the free trans 2,5 diketopiperazine after recrystallization. Scheme 3 4. Formation of 3.48 as the free 2,5 diketopiperazine form Cbz protected 3.39 3.2.3 Synthesis of cis 2,5 D iketopiperzines Complimentary to the trans 2,5 diketopiperazines, formation of th e cis configured DKPs was accomplished by a tandem deprotection/cyclization strategy. Compound 3.24 was stirred under hydrogen with Pd/C (10% wt) for 18 hours at room temperature. Filtration through celite removed palladium yielding compound 3.49 (Scheme 3 5) in 72% as a single enantiomer by recrystallization. The optical rotation value of 3.49 coincided with the known literature value, thus confirming the tandem deprotection/cyclization without epimerization. Compounds 3.25 and 3.27 were cyclized producin g 3.50 51 (65 69% yield) as cis 2,5 diketopiperazines. Compounds 3.51 and

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58 3.48 had [ ] D 20 values that coincided with the literature cis and trans DKP values.[09ACB1051] Scheme 3 5. Tandem deprotection/cyclization for the formation of cis DKPs 3.49 51 3.2.4 Reaction Kinetics for the Formation of 3.31. The kinetics of the cyclization were studied in CD 3 CN (0.34 M) over a temperature range of 25 C 65 C (Table 3 2). Pseudo first order kinetics were observed and an Arrheniu s plot (ln(k) vs 1/T) (Figure 3 8 ) and an Eyring plot (ln(k/T) vs 1/T) were constructed (Figure 3 9 ). The Arrhenius activation energy was 40 kJ / =37 kJ / = 146 J / K coincides with the proposed highl y ordered 6 membered cyclic transition state. Figure 3 8 Arrhenius p lot for the cyclization of 3.24 to 3.31 under conventional heating

PAGE 59

59 Fi gu re 3 9 Eyring p lot for the cyclization of 3.24 to 3.31 under conventional heating Significant effects of microw ave irradiation on cyclization were shown by pseudo first order rate constants of k obs MW = 0.59 s 1 at 65 C under microwave heating and k obs CONV = 0.31 s 1 under conventional heating. 3.3 Summary Proline containing 2,5 diketopiperazines were selectively synthesized in cis and trans configurations from their corresponding aminoacyl benzotriazolides. The D amino acids were accessed from inexpensive L amino acid precursors under mild conditions showing compatiblity with a wide range of amino acids. Mechani sm and stereoselectivity were rationalized by chiral HPLC, kinetics, and computational methods. Compared to literature precedent, the methodology illustrates stereoflexibility and atom efficiency as well as shorter reaction times.

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60 3.4 Experimental 3.4.1 Ge neral M ethods. 1 H NMR spectra were recorded at 300 MHz and 13 C NMR spectra were recorded at 75 MHz on Gemini or Varian spectrometers at room temperature. The chemical shifts are reported in ppm relative to TMS as internal standard ( 1 H NMR) or to solvent re sidual peak ( 13 C NMR). The NMR experiments at variable temperatures (35, 45, 55 and 65 C) were recorded on a Varian Inova NMR spectrometer operating at 500 MHz. Chiral HPLC experiments were performed on a Chirobiotic T column using methanol as mobile phas e. Compounds were analyzed at a flow rate of 0.1 mL/min (detection wavelength = 230 nm, solvent = methanol). HRMS spectra were recorded on a LC TOF (ES) apparatus. Elemental analysis was performed on a Carlo Erba 1106 instrument. Melting points were determ ined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. [ ] D 20 values were recorded on a Perkin Elmer polarimeter. [ ] D 20 values are given in degcmcm 1 g 1 and concentration are given in mg/100cm. Flash chromatograp hy was performed on silica gel 60 (230 400 mesh). All solvents were dried according to standard procedures. Triethylamine was distilled prior use. All commercially available substrates were used as received without further purification. All microwave assis ted reactions were carried out with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon septum and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 60 sec.; PowerMax

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61 cooling mode). N Cbz protected amino acids were purchased from Chem Impex International. 3.4.2 General P rocedure s for the T andem C yclization/ E pimerization S equence and C haracterization of the C orresponding rac D iketopiperazines 3.31 and 3.37 and trans D iketopiperazines 3.38 39 and 3.44 47 A solution of Cbz N protected dipeptidoyl benzotriazole 3.24 30 (1 mmol) and triethylamine (1 mm ol) in dry acetonitrile (4 mL) was subjected to microwave irradiation (20 min, 70W, 80 C). Upon completion, the reaction mixture was concentrated under vacuum and the crude mixture was purified by column chromatography (hexanes/ethyl acetate gradient) to give the corresponding rac diketopiperazines 3.31, 3.37 or trans diketopiperazines 3.38 39 and 3.43 46. cyclo(Z Gly D Pro) 3.31 White microcrystals, y ield: 83% (0.23 g), mp 110 111 C. [ ] D 20 = 0 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): = 7.43 7.27 (m, 5H), 5.29 (d, J = 12.3 Hz, 1H), 5.26 (d, J = 12.3 Hz, 1H), 4.72 (d, J = 16.5 Hz, 1H), 4.29 4.08 (m, 2H), 3.54 (dd, J = 8.2, 5.8 Hz, 2H), 2.50 2.20 (m, 2H), 2.08 1.82 (m, 2H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 167.4, 163.1, 151.9, 134.7, 12 8.8, 128.4, 69.5, 60.4, 50.0, 45.4, 28.2, 23.2 ppm. Anal. calcd. for C 15 H 16 N 2 O 4 : C 62.49; H 5.59; N 9.72. F ound: C 62.09; H 5.61; N 9.64. cyclo(Z L Ala D Pro) ( L D ) 3.38. White microcrystals, y ield: 69% (0.20 g), mp 149 150 C. [ ] D 20 = 101.0 (c = 0. 2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): = 7.50 7.31 (m, 5H), 5.30 (s, 2H), 4.86 (q, J = 7.2 Hz, 1H), 4.20 (dd, J = 9.3, 7.2 Hz, 1H), 3.62 3.52 (m, 2H), 2.49 2.39 (m, 1H), 2.25 1.85 (m, 3H) 1.53 (d, J = 7.2 Hz, 3H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 167.4 165.9, 151.9, 134.8, 128.8, 128.7, 128. 3, 69.3, 59.4, 57.6, 45.6,

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62 29.2, 22.8, 17.4 ppm. Anal. calcd. for C 16 H 18 N 2 O 4 : C 63.56; H 6.00; N 9.27 F ound: C 63.92; H 6.08; N 9.29. cyclo(Z D Ala L Pro) ( D L ) 3.38. White solid, y ield: 71% (0.21 g ), mp 153 155 C. [ ] D 20 = 129.7 (c = 0.2 in CH 2 Cl 2 ). 1 H and 13 C NMR were identical to its enantiomer (L,D) 4b. Anal. calcd. for C 16 H 18 N 2 O 4 : C 63.56; H 6.00; N 9.27; F ound: C 63.62; H 6.09; N 9.21. cyclo(Z L (BnS) Cys D Pro) 3.44. White solid, y ield: 72% (0.31 g), m p 115 116 C. [ ] D 20 = 79.8 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): = 7.44 7.16 ( m, 10H), 5.29 (d, J = 12.0, 1H), 5.26 (d, J = 12.0, 1H), 5.01 (t, J = 6.0 Hz, 1H), 4.41 (dd, J = 10.5, 7.5 Hz, 1H), 3.71 3.43 (m, 4H), 2.96 (t, J = 6.0 Hz, 2H), 2.46 2.29 (m, 1H), 2.14 1.74 (m., 3H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 167.4, 164.0, 152.3, 137.1, 134.7, 129.2, 128.8, 128.5, 127.5, 69.6, 61.0, 60.2, 45.7, 37.0, 33.6, 29.7, 22.5 ppm. HRMS (ESI): m/z calcd for C 23 H 24 N 2 O 4 S+Na + : 447.1354 [ M +Na + ]; found 447.1356. cyclo(Z L Val D Pro) 3.45. White microcrys tals, y ield: 70% (0.23 g), mp 117 119 C. [ ] D 20 = 108.9 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): = 7.50 7.16 (m, 5H), 5.29 (s, 2H), 4.62 (d, J = 9.6 Hz, 1H), 4.26 (t, J = 8.1 Hz, 1H), 3.68 3.45 (m, 2H), 2.51 2.36 (m, 1H), 2.22 1.81 (m, 4H), 1.08 ( d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 168.1, 164.9, 152.4, 134.7, 128.6, 128.5, 128.3, 69.2, 66.6, 59.8, 45.6, 31.4, 29.6, 22.7, 19.5, 19.4 ppm. Anal. calcd. for C 18 H 22 N 2 O 4 : C 65.44; H 6.71; N 8.48; F ound: C 65.15; H 6.77; N 8.18. cyclo(Z L Leu D Pro) ( L D ) 3.39. White microcrystals, y ield: 75% (0.26 g), mp 114 115 C. [ ] D 20 = 109.0 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): 7.44 7.28 (m, 5H), 5.28 (s, 2H), 4.85 (dd, J = 8.9, 6.5 Hz, 1H), 4.22 (dd, J = 9.0, 7.2 Hz, 1H), 3.64

PAGE 63

63 3.45 (m, 2H), 2.50 2.30 (m, 1H), 2.26 2.09 (m, 1H), 2.06 1.81 (m, 2H), 1.77 1.54 (m, 3H), 0.95 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 167.9, 165.4, 152.1, 134.7, 128.7, 128.5, 69.3, 60.0, 59.6, 45.7, 41.1, 29.4, 24.9, 23.0, 22.8, 22.0 ppm. Anal. Calcd. for C 19 H 24 N 2 O 4 : C 66.26; H 7.02; N 8.13; F ound: C 66.18; H 7.25; N 8.07. cyclo(Z D Leu L Pro) ( D L ) 3.39. Yield: 79% (0.27 g), white solid. m.p. 118 119 C. [ ] D 20 = 102.2 (c = 0.2 in CH 2 Cl 2 ). 1 H and 13 C NMR were identical to its enantiomer (L,D) 4e. Anal. Calcd. for C 19 H 24 N 2 O 4 : C 66.26; H 7.02; N 8.13; found: C 66.11; H 7.31; N 8.01. cyclo(Z L Phe D Pro) 3.46. Colorless gel, y ield: 71% (0.27 g) [ ] D 20 = 79.89 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDC l 3 ): = 7.43 7.34 (m, 5H), 7.28 7.23 (m, 3H), 7.13 7.09 (m, 2H), 5.29 (d, J = 12.3 Hz, 1H), 5.23 (d, J = 12.3 Hz, 1H) 5.09 (t, J = 5.0 Hz, 1H), 3.58 3.48 (m, 1H), 3.44 3.36 (m, 1H), 3.31 3.18 (m, 2H), 2.60 (dd, J = 9.8, 6.8 Hz, 1H), 2.18 2.04 (m, 1H), 1.9 2 1.77 (m, 2H), 1.70 1.56 (m, 1H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 167.4, 164.3, 152.1, 135.1, 134.8, 130.1, 128.8, 128.8, 128.4, 127.8, 69.3, 62.6, 58.9, 45.1, 38.5, 29.4, 22.1 ppm. Anal. Calcd. fo r C 22 H 22 N 2 O 4 1/2 H 2 O: C 68.20; H 5.98; N 7.23; found: C 68 .30; H 6.01; N 6.92. cyclo(Z L Trp D Pro) 3.47 White solid, y ield: 73% (0.27 g ), mp 77 79 C. [ ] D 20 = 135.5 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): = 8.70 (br s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.43 7.28 (m, 6H), 7.20 7.04 (m, 2H), 6.86 (d, J = 2 .4 Hz, 1H), 5.31 (d, J = 12.0, 1H), 5.20 (d, J = 12.3 Hz, 1H), 5.11 (dd, J = 5.1, 3.6 Hz, 1H), 3.58 (dd, J = 15.0, 3.6 Hz, 1H), 3.48 3.28 (m, 2H), 3.16 3.03 (m, 1H), 2.31 2.27 (m, 1H), 1.99 1.84 (m, 1H), 1.79 1.57 (m, 2H), 1.30 1.07 (m, 1H) ppm 13 C NMR (7 5 MHz, CDCl 3 ): = 168.1,

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64 165.1, 152.1, 136.3, 134.8, 128.8, 128.5, 127.2, 124.8, 122.6, 119.9, 118.9, 111.5, 109.0, 69.3, 62.2, 59.1, 45.1, 29.5, 28.6, 21.8 ppm. Anal. Calcd. for C 24 H 23 N 3 O 4 : C 69.05; H 5.55; N 10.07, F ound: C 69.04; H 5.56; N 10.07. cyclo(Z G ly Leu) 3.37. White Solid, Y ield: 42% (0.13 g), mp 96 98 C. [ ] D 20 = 0 (c = 0.2 in CH 2 Cl 2 ). 1 H NMR (300 MHz, CDCl 3 ): = 7.72 (br s, 1H), 7.44 7.25 (m, 5H), 5.31 (s, 2H), 4.42 (d, J = 17.4 Hz, 1H), 4.32 (d, J = 17.4 Hz, 1H), 4.08 3.93 (m, 1H), 1.51 1.88 ( m, 3H), 0.97 (d, J = 6.0 Hz, 3H), 0.94 ( d, J = 5.7 Hz, 3H) ppm 13 C NMR (75 MHz, CDCl 3 ): = 167.2, 168.9, 152.2, 134.7, 128.8, 128.4, 69.5, 55.2, 48.0, 41.8, 24.4, 23.1, 21.4 ppm. Anal. Calcd. for C 16 H 20 N 2 O 4 : C 63.14; H 6.65; N 9.20; Found: C 62.81; H 6.6 5; N 9.25. 3.4.3 Deprotection of C ompound 3.39 and C haracterization of C ompound 3.48 A solution of (3 S ,8a S ) benzyl 3 isobutyl 1,4 dioxohexahydropyrrolo[1,2 a]pyrazine 2(1 H ) carboxylate (3.39) (5.8 mmol) in dry ethanol (40 mL) in the presence of Pd / C (10 wt %) was stirred for 24 hours at room temperature under an atmosphere of hydrogen. Upon completion, the crude mixture was filtered on Celite and concentrated under reduced pressure. (3 S ,8a R ) 3 isobutyl hexahydropyrrolo[1,2 a]pyrazine 1,4 dione (3.48) was r ecrystallized from an ethyl acetate/hexanes mixture. cyclo(L Leu D Pro) 3.48. White Solid, y ield: 87% (1.06 g), mp 142 145 C (lit. 146 149 C). [ ] D 21 = 88.6 (c = 0.2 in EtOH) (lit. [ ] D 21 = 98.9, c = 0.9 in EtOH). [13c] 1 H NMR (300 MHz, DMSO d 6 ): = 8.36 (br d, J = 4.1 Hz, 1H), 4.17 (dd, J = 8.8, 6.8 Hz, 1H), 3.65 3.57 (m, 1H), 3.48 3.24 (m, 2H), 2.19 2.06 (m, 1H), 1.90 1.62 (m, 4H), 1.60 1.49 (m, 1H), 1.47 1.35 (m, 1H), 0.91 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.3 Hz, 3H) ppm. 13 C NMR (75 MHz, DMSO d 6 ): = 168.7, 166.0, 57.3, 55.2, 45.0, 42.1, 28.4, 23.8, 22.8,

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65 21.8, 21.5 ppm. Anal. Calcd. for C 11 H 18 N 2 O 2 : C 62.83; H 8.63; N 13.32; F ound: C 62.63, H 8.96; N 1 3.09. 3.4.4 General P rocedure s for the T andem D eprotection/ C yclization S equence and C haracterizatio n of C ompound 3.49 51 A solution of N Cbz protected ( L L ) dipeptidoyl benzotriazole 3.24 25 or 3.27 (5 mmol) in dry ethanol (50 mL) was stirred for 18 h at room temperature in the presence of Pd / C (10 wt. %) under an atmosphere of hydrogen. Upon completion the crude mixture was filtered on Celite and concentrated under reduced pressure. (S) Hexahydropyrrolo[1,2 a]pyrazine 1,4 dione (3.49), (3 S ,8a S ) 3 methyl hexahydropyrrolo[1,2 a]pyrazine 1,4 dione (3.50) and (3 S ,8a S ) 3 isobutyl hexahydropyrrolo[1,2 a]pyra zine 1,4 dione (3.51) were recrystallized from ethanol/hexanes mixtures. cyclo(Gly L Pro) 3.49. White microcrystals y ield: 72% (0.55 g), mp 218 221 C (lit. mp 220 223 C ). [ ] D 2 0 = 194.2 (c = 0.2 in EtOH) (lit. [ ] D 2 0 = 179.6, c = 0.8 in EtOH). 1 H NMR ( 300 MHz, CDCl 3 ) = 7.25 (s, 1H), 4.15 4.04 (m, 2H), 3.90 (dd, J = 16.6, 4.4 Hz, 1H), 3.69 3.51 (m, 2H), 2.43 2.33 (m, 1H), 2.15 1.83 (m, 3H) ppm ; 13 C NMR (75 MHz, CDCl 3 ) = 170.3, 163.7, 58.7, 46.8, 45.5, 28.6, 22.6. Anal. Calcd. for C 7 H 10 N 2 O 2 : C 54.54; H 6.54; N 18.17; Found: C 54.35; H 6.55; N 18.10. cyclo(L Leu L Pro) 3.51. White microcrystals, y ield: 69% (0.73 g), mp 160 163 C (lit. m p 162 168 C). [ ] D 2 0 = 135.19 (c = 0.2 in EtOH) (lit. [ ] D 2 0 = 134.3, c = 1.1 in EtOH). 1 H NMR (300 MHz, DMSO d 6 ) = 8.02 (br s, 1H), 4.19 (t, J = 7.9 Hz, 1H), 4.00 (t, J = 6.3 Hz, 1H), 3.42 3.26 (m, 2H), 2.17 2.05 (m, 1H), 1.99 1.68 (m, 5H), 1.35 (ddd, J = 13.6, 7.5, 5.7 Hz, 1H), 0.87 (d, J = 2.4 Hz, 3H), 0.85 (d, J = 2.7 Hz, 3H) ppm ; 13 C NMR (75 MHz, DMSO d 6 ) = 1 70.2, 166.4, 58.4, 52.5, 44.8, 37.7, 27.4, 24.0, 22.8,

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66 22.4, 21.8 ppm. Anal. Calcd. for C 11 H 18 N 2 O 2 : C 62.83; H 8.63; N 13.32; F ound C 62.63; H 8.96; N 13.09. 3.4. 5 X R ay D ata for 3.38 39 Crystal data for compound 3.38: colorless crystal (block), dimensions 0.38 x 0.25 x 0.23 mm, crystal system monoclinic, space group P2 1 /n, Z = 4, a = 10.0745(15), b = 11.0092(19), c = 12.972(2) 3 3 T = scans with CCD area detector, covering a whole sphere in reciprocal space, 9260 reflections measured, 1681 unique (R int = 0.0554), 2770 o and polarization effects, an empirical absorption correction was applied using SADABS22 based on the Laue symmetry of the reciprocal space, m = 0.102 mm 1 Tmin = 0.795, Tmax = 1.00, structure sol ved by directmethods and refined against F2 with a Full matrix least squares algorithmusing the SHELXL 97 software package, 199 parameters refined, hydrogen atoms were treated using appropriate riding models, goodness of fit = 0.930 for observed reflection s, final residual values R1(F) = 0.0473, wR(F2) = 0.1129 for observed reflections.CCDC 844865, 844866 Crystal data for compound 3.39: colorless crystal (block), dimensions 0.12 x 0.10 x 0.09 mm, crystal system monoclinic, space group P2(1), Z = 2, a = 9.8 547(5), b = 3 3 T = scans with CCD area detector, covering a whole sphere in reciprocal space, 9033 reflections me asured, 2926 unique (R int and polarization effects, an empirical absorption correction was applied using SADABS22 based on the Laue symmetry of the reciprocal space, m = 0.744 mm 1

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67 Tmin = 0.7086, Tmax = 0.7528, structure solved by directmethods and refined against F2 with a Full matrix least squares algorithmusing the SHELXL 97 software package, 228 parameters refined, hydrogen atoms were treated using appropriate riding models, g oodness of fit = 1.055 for observed reflections, final residual values R1(F) = 0.0233, wR(F2) = 0.0611 for observed reflections.CCDC 846343. 3.4. 6 Kinetic Data for the Formation of 3.31 Table 3 5 Data obtained from reaction of 3.24 with 1 equivalent of tri ethylamine. Peak SM Peak BtH time (min) ratio percent conc pro conc R ln conc R 1/conc R 2 0 0 0 0.00 0.00 0.34 1.08 2.94 2 0.23 2 0.103139 10.31 0.04 0.30 1.19 3.28 2 1.59 4 0.442897 44.29 0.15 0.19 1.66 5.28 2 3.04 6 0.603175 60.32 0.21 0.13 2.00 7.41 2 4.54 8 0.69419 69.42 0.24 0.10 2.26 9.62 2 5.62 10 0.737533 73.75 0.25 0.09 2.42 11.21 2 7.81 12 0.796126 79.61 0.27 0.07 2.67 14.43 2 7.82 14 0.796334 79.63 0.27 0.07 2.67 14.44 2 7.22 16 0.78308 78.31 0.27 0.07 2.61 13.56 2 8.64 18 0.8 1203 81.20 0.28 0.06 2.75 15.65 2 10.61 20 0.841396 84.14 0.29 0.05 2.92 18.54 2 8.7 22 0.813084 81.31 0.28 0.06 2.76 15.74 2 9.26 24 0.82238 82.24 0.28 0.06 2.81 16.56 2 9.86 26 0.831366 83.14 0.28 0.06 2.86 17.44 2 10.97 28 0.845798 84.58 0.29 0.05 2.95 19.07 2 9.25 30 0.822222 82.22 0.28 0.06 2.81 16.54 Reaction kinetics were studied using a 0.34 M solution of 3.24 in CD 3 CN, monitoring the disappearance of 3.24 and formation of 3.31 using the benzotriazole aromatic protons in 1 H NMR. One m illiter of the solution of 3.31 in CD 3 CN (0.34 mmol)

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68 was injected via syringe into a standard 5 mm NMR sample tube. The lock and shim was set with the initial sample in order to maximize the efficiency of the experiment. At t=0, the sample was removed from the machine and 1 equivalent of Et3N (0.05 mL, 0.34 mmol) was injected into the sample. The tube was shaken to ensure proper mixing and injected into the NMR probe. Proper adjustments to the lock and shim were done prior to each scan. Scans were taken eve ry 2 minutes for 30 minutes. Integration of the appropriate signals allowed for determination of concentration and the percentage of product formed. (Table 3 5) As the reaction is in a 1:1 ratio with respect to the starting material and product, a simple d etermination of concentration is possible. Figure 3 1 0 Plot showing t he percent completion over time The data obtained from the kinetics experiment was then plotted to show percent completion versus time which shows the useful data to be between 0 12 mi nutes (Figure 3 1 0 ). A plot of the natural log of the concentration versus time (0 12 minutes) shows the reaction to be pseudo first order (Figure 3 1 1 ). By linear regression, the rate constant ,k obs is obtained as the slope of the determined linear regre ssion line equation as 0.1465 s 1.

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69 Figure 3 1 1 Plot showing the natural log of the concentration over time Figure 3 1 2 Arrhenius plot for the cyclization of 3.50 The procedure was repeated at various temperature (25, 35, 45, 55, 65 C) as well as the analysis to determine the value of k obs After analysis and acquisition of the rate constant data, an Arrhenius plot was constructed (Figure 3 1 2 ). The equation obtained from linear regression analysis allows for the determination of the Arrhenius activat ion parameters (k=Ae Ea/RT ) as the slope of the regression is the E a parameter. From the Arrhenius activation parameters, an Erying plot was constructed using the natural log of

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70 rate constant divided by the temperature versus the inverse of the temperature (Figure 3 1 3 ). The equation obtained by linear regression analysis shows the slope to be the negative value of the enthalpy of the reaction divided by the universal gas constant ( Figure 3 1 3 Eyring plot for the cyclization of 3.24 Z Gly D Pro Bt 3.24 Conv k ln (k) C K 1/K 1/T k/T ln (k/T) 0.0748 2.59294 35 308.15 0.00325 0.00325 0.00024 8.32352 0.1379 1.98123 45 318.15 0.00314 0.00314 0.00043 7.74375 0.1811 1.70871 55 328.15 0.00305 0.00305 0.00055 7.50218 0.3146 1.15645 65 338.15 0.00296 0.00296 0.00093 6.97994 ln [A]= 12.937 4453.3 ln(k b 6.160 E a /R= 4775.9 37006.9 146.2 E a= 39687.73 ln(k b /h)= 23.7595 Figure 3 1 4 Data extrapolated from the Arrhenius and Eyring plots The same general procedure was followed for determination of the kineti cs for formation of 3.31 were used with one exception, the samples were heated under microwave irradiation then checked immediately after every minute by 1 H NMR.

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71 CHAPTER 4 STAUDINGER LIGATION IN THE FORMATION OF 2,5 DIKETOPIPERAZINES 4.1 Literature Overvi ew There is great interest in the study of cyclic peptides, the smallest of which are the 2,5 diketopiperazines (DKPs). A large number of cyclic peptides are available from natural sources, but their synthesis is often considered difficult and a considerab le challenge. Solid phase peptide synthesis (SPPS) is a convenient method for the formation of peptides, having many advantages over conventional solution phase procedures. Staudinger ligation is also a powerful method for the formation of amide bonds and has been used in the synthesis of large cyclic peptides. 4.1.1 Biological Properties of 2,5 Diketopiperazines Small peptides and peptide like structures show an interesting array of biological properties. These are limited in vivo due to enzymatic degradat ion and hydrolysis. Cyclic peptides, with their constrained structure, are more resilient to peptidases and hydrolysis, thereby making them valuable targets for medicinal and synthetic chemists. The 2,5 diketopiperazine scaffold (DKP) 4.1 appears in many n atural products (Figure 1 1). Cyclic glycine leucine 4.2 is a natural antibiotic that is effective against Bacillius subtilis interacting with the cytochrome P450 complex.[10B7282] Cyclic tyrosine tyrosine 4.3 and cyclic tyrosine phenylalanine 4.4 are effi opoid receptor [05JP305], and with L type calcium channels.[04BMCL1717] Cyclic concentrations [04NNNA1815] Cyclic glycine dimer 4.7 is an effective inhibi tor of glycogen phosphorylase (while not affecting the glucosidases) and an interesting target for type 2 diabetes.[03G93R]

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72 Figure 4 1 Selected examples of biologically active 2,5 diketopiperazines 4.1.2 Synthesis of 2,5 Diketopiperazines Figure 4 2 Common synthetic methods for cyclic peptides

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73 The synthesis of small cyclic peptides can be divided into three groups: i) head to tail condensation of linear peptides, ii) dimerization of two amino acid subunits and iii) transition metal mediated peptide coupling reactions (Figure 4 2).[97CR2243, 04T2447] These methods often afford low yields, require protection deprotection schemes, long reaction times and harsh conditions.[68JOC864, 83BCSJ568 02BJ23, 06CC2884, 06T7484, 09JACS3033] A recent example (Scheme 4 1) demonstrates the use of Brnsted acid to activate the C terminus for attack followed by dehydration.[09T3688] Inherent stereochemistry is retained under acidic conditions, but when the cyclization or the dehydration step is carried out under basic conditions racemization is observed. Scheme 4 1. Head to tail condensation of N ketoacyl amino acid amides Dimerization of two subunits (4.12), using phosph orus promoted cyclization under continuous microwave irradiationformed symmetrical and unsymmetrical DKPs (Scheme 4 2).[08EJOC5418] Unsymmetrical cases require a highly hindered substrate coupled with a minimally hindered substrate. The less hindered amino acid was in excess and was added after activation of the hindered substrate to minimize homodimer formation.

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74 Scheme 4 2. Synthesis of symmetrical DKPs from dimeriz ation of two identical subunits Organ o metallic coupling m ethods have been reported, ranging from oxidative phenolic coupling to olefin metathesis using Grubbs second generation catalyst.[01T353, 02JOC8247] Jackson et al. featured an intramolecular Negishi cross coupling reaction to form cyclic peptides (Scheme 4 3).[09JOC8280] Scheme 4 3. Intramolecular Ne gishi peptide coupling strategy Recent developments relied on the use of turn inducers requiring specific residues to be included in the amino acid sequence, but these methodol ogies limit the scope of the reaction and leave turn inducer residues in the cyclic peptide.[12CEJ2632, 10OL3136] Head to tail condensation of N to C terminus of linear dipeptides is of most relevance to this research. The method often leads to formation of the cyclic homodimer of the linear dipeptide and not the cyclic heterodimer. Formation of the homodimer was

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75 minimized by high dilution conditions.[09BMCL3928] Investigations into efficient and atom economical syntheses are important in the cyclic pepti de field.[97CR2243, 04T2447] 4.1.3 Solid Phase Supported Synthesis Table 4 1. Advantages and d isadvantage of SPPS methodology Advantages Disadvantages Easy separation of byproducts or excess starting material Incompatibility of resin with growing peptide chain. No purification between steps Lack of stability of peptide resin linkers. No mechanical loss in purification or mechanical transfer. Interaction with functional groups on supports. Solubility of growing peptide independent of its solubility. For mation of erroneous peptides due to truncation or failed coupling sequences. Automatization (e.g. automated peptide synthesizers).. Peptide conformational changes in macroscopic environments. Single cleavage step for final product Shorter reaction time s. Recyclability of spent resin. Bruce Merrifield pioneered SPPS in 1963, and optimized a C to N terminus synthesis under mild conditions.[63JACS2149] Many advantages over solution phase synthesis exist in SPPS methodology (Table 4 1) since column c hromatography or recrystallization are avoided thereby reducing mechanical loss of the product during purificaiton. Some SPPS methods enable the coupling of peptides in as little as 30 minutes, and coupling can be accomplished on the solid support independ ent of the solubility of growing residues. A more complete review of the advantages and disadvantages of SPPS is shown in Table 4 1.[88ARB957, 12CSR1826] The solid phase in SPPS is a polymer support having a functional group attached (Figure 4 3). Resins 4 .16 18 are used for Boc protected amino acid SPPS, while resins 4.19 20 are used for Fmoc protected peptides. A synthetic linker is used to bind the

PAGE 76

76 resin and substrate, acting as a spacer varying in length from four to six atoms.[00LPS17, 98JPR303] Figure 4 3. Selected resin solid phase supports DMF is used to swell the resin and effectively solvate the attached peptide. Use of Boc protecting groups allows for simple deprotection and neutralization performed in situ .[88ARB 957, 97ME14, 92IJPPR180] Elongation may involve many coupling reagents, but commonly DCC is used due to easy removal of the urea salt.[04T2447, 12CSR1826] Monitoring reactions in SPPS is accomplished by ninhydrin tests, and much care must be taken to chara cterize the product upon removal from the resin since the intermediate stages are not characterized.[88ARB957] 4.1. 4 Staudinger Ligation Staudinger ligation is a powerful tool for the creation of amide bonds,[09JOC2203,10BMC3679] and has been used for prot ein engineering,[00S2007, 02PNAS19] labeling of specific nucleic acids,[03BC697] studies in

PAGE 77

77 proteomics,[03PNAS9116, 03PNAS14846] and bioconjugation.[03JACS11790, 03ACIE5830] An azide reacts with triphenylphosphine to form an iminophosphorane 4.24, with enh anced nucleophilic activity.[05JACS2686, 06JACS8820] F igure 4 4. Mechanism of the formation of the activated i minophosphorane [05JACS2686] Raines et al. used a phosphinthioester (4.25) in a Staudinger ligation to form a l inear dipeptide 4.27 (Scheme 4 4 ).[00OL1939] While effective, this methodology cannot be used to form cyclic peptides since the azide is reduced before the desired reaction. Scheme 4 4 Raines and co igation methodology using phosph inothioester and azide

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78 4.2 Results and Discussion In pursuit of efficient methods for the preparation of small cyclic peptides a novel, mild and atom economic tandem deprotection cyclization strategy is demonstrated. Utilizi ng a solution and solid phase Stuadinger ligation allowed rapid, convenient and cost effective cyclization. 4.2.1 Synthesis of Starting Materials for Solution Phase Staudinger Ligation Reactions of 4.38 40 and 4.43 4.2.1.1 Synthesis of c hloro d ipeptides 4 .32 34 Commercially available ( L) amino acids 4.28 30 were reacted with chloroacetyl chloride 4.31 under reflux conditions in THF yielding chloro dipeptides 4.32 34 (Scheme 4 5 ). Purification by crystallization from ether gave 4.32 34 in 38 78% yields. [03 RCB2197, 12ACIE548] Scheme 4 5 Synthesis of chloro dipeptides 4.32 34 from L amino acid and chloroacetyl chloride 4.2.1.2 Synthesis of a zido d ipeptides 4.35 37 Chloro dipeptides 4.32 34 were reacted with sodium azide in D MF/water, since water increased the solubility of the azide ion. Azido dipeptides 4.35 37 were obtained in 61 78% yields after recrystallization from ethyl acetate and hexanes (Scheme 4 6 ).

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79 Scheme 4 6 Synthesis of azido d ipeptides 4.35 37 from chloro dipeptides 4.32 34 4.2.1.3 Synthesis of a zido t hioester d ipeptides 4.38 40 Azido protected dipeptide thioesters 4.38 40 were synthesized by mixed anhydride coupling of 4.35 37 with thiophenol, utilizing an in situ activation o f the dipeptide. Compounds 4.38 40 were purified by a recrystallization from ethyl acetate and hexanes (Scheme 4 7 ) in 42 75% yield. Scheme 4 7 Synthesis of azido thioester dipeptides 4.38 40 from azido dipeptides 4.35 37 Azido methyl ester dipeptide 4.43 was synthesized (Scheme 4 8 ) to examine the effect of the leaving group on cyclization. The L leucine methyl ester was acylated with chloroacetyl chloride 4.31 yielding chloro methyl ester dipeptide 4.42 in 65% yield. Com pound 4.42 was treated with sodium azide to give azido methyl ester dipeptide 4.43 in 35% yield.

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80 Scheme 4 8 Synthesis of azido methyl ester dipeptide 4.42 from L leucine methyl ester 4.41 4.2.2 Synthesis of Starting Mater ials for Solid Phase Staudinger Ligation of 4.59 61 and 4.68 69 4.2.2.1 Synthesis of b oc p rotected a mino a cid l inkers 4.48 50 Compounds 4.48 50 were prepared by reaction of commercially available 3 mercaptopropionic acid 4.47 with Boc protected aminoacyl b enzotriazolides 4.44 46. Acylation of the thiol afforded the B oc protected amino acid linkers in good yields (57 85%) (Scheme 4 9 ). [92IJPPR180] Scheme 4 9 Synthesis of Boc protect ed amino acid linkers 4.48 50 4.2.2.2 Syn thesis of a zido p rotected s olid p hase s upported d ipeptides 4.59 61. Boc protected amino acid linkers 4.48 50, were attached to the aminomethyl (AM) resin (4.51), chosen due to its high loading capacity (1.66 mmol / g), using DCC (0.5 M in DCM) coupling overn ight in a mechanical shaker. Boc deprotection gave the TFA salts 4.55 57. Azido glycyl benzotriazole 4.58 was then reacted with 4.55 57 under the basic conditions to yield the azido protected solid phase supported dipeptides 4.59 61 (Scheme 4 1 0 ).

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81 Scheme 4 1 0 Synthesis of solid phase supported azido protected dipeptides 4.59 61 The spent resin 4.63 was recycled for a second cycle (Scheme 4 1 1 ). Boc protected amino acid 4.62 was reacted with 4.63 to yield 4.64. The solid ph ase supported amino acid was then carried out through the synthesis to give azido protected solid supported dipeptide 4.60 (Scheme 4 1 1 ). Scheme 4 1 1 Recyclability of resin 4.63 to give intermediate 4.53 4.2.2.3 Synthesis of a zido p rotected s olid p hase s upported t ripeptides 4.6 8 6 9 Due to the modular design of the SPPS, intermediates from the dipeptide synthesis were used as starting materials in the tripeptide synthesis (Scheme 4 12). TFA salts 4.55 and 4.56 were reacted with Boc protected alaninyl benzotriazolide 4.46 under basic conditions to give Boc protected solid supported dipeptides 4.64 and 4.65

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82 respectively. Deprotection with TFA, gave salts 4.66 and 4.67 which were reacted with azido glycyl benzotriazole 4.58 to give the solid supported azido protected tripeptides 4.68 69. Scheme 4 1 2 Synthesis of azido protected solid supported tripeptides 4.68 69 4.2.3 Staudinger Ligation of 4.38 40, 4.43, 4.59 61, and 4.68 69 for the Formation of 2,5 Diketopiperazines 4.63 4.65 A screening of the reaction was undertaken to indentify optimal conditions for the proposed Staudinger ligation. Initial investigation with 4.38 utilizing 1.5 equivalents of tributylphosphine in dry DCM under standard re flux conditions (Entry 1) served as the starting point (Table 4 2). Solvent, addition or exclusion of water, and different phosphines were examined for optimization. Microwave irradiation gave a higher yield

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83 with shorter reaction time (Table 4 2, E ntry 2). An increase in yield was seen upon addition of 5 equivalents of water, facilitating hydrolysis of the phosphonium salt (Table 4 2, E ntry 3). The lower polarity of DCM aids in filtration of insoluble 4.70, indentifying it as the best solvent (Table 4 2, E n tries 4 6). Reaction with triphenylphosphine resulted in lower yields and difficulty was seen in separation of triphenylphosphine oxide from 4.70 (Table 4 2, E ntry 7). Thus using tributylphosphine, in DCM, under continuous microwave irradiation (50 W, 50 C, 5 min), with 5 equivalents of water (Table 4 2, E ntry 3) gave the best yield of 4.70. Table 4 2. Initial conditions for optimization of preperation of 4.70 Entry Reaction conditions Yield (%) 1 PBu 3 CH 2 Cl 2 (dry) a rt, 12 h 55 2 PBu 3 CH 2 Cl 2 (dry) a MW b 30 min 66 3 PBu 3 CH 2 Cl 2 H 2 O (5 eq.), MW b 5 min 78 4 PBu 3 THF, H 2 O (5 eq.), MW b 5 min 70 5 PBu 3 CH 3 CN, H 2 O (5 eq.), MW b 5 min 65 6 PBu 3 toluene, H 2 O (5 eq .), MW b 5 min 63 7 PPh 3 CH 2 Cl 2 H 2 O (5 eq.), MW b 5 min 58 a Reaction was quenched with H 2 O; b 50 C, 50 W Methyl ester 4.43 was treated under the optimum conditions (60% yield of 4.70) but was less reactive then the thioester 4.38 (Scheme 4 1 3 ). A computational investigation, at the B3LYP/6 31G(d ) level of theory, supported the experimental evidence by showing a lower activation barrier for thiophenol (21.6 kcal/mol) versus the methyl ester (30.0 kcal/mol). [Unpublished work by Dr. Jean Christophe Monbaliu]

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84 Scheme 4 1 3 Reaction of methyl ester derivative 4.43 in the Staudinger ligation under opt imized conditions Compounds 4.39 and 4.40 gave 4.71 and 4.72 (74% and 78% yields) using method A for solution phase synthesis (Table 4 3). Table 4 3. Reaction of 4.38 40 an d 4.59 61 to form 4.70 72 Entry R 1 Yield (%) 1 i Bu ( 4.38) 78 a 2 Bn ( 4.39 ) 74 a 3 Me ( 4.40 ) 78 a 4 i Bu ( 4.59 ) 79 b 5 Bn ( 4.60 ) 72 b 6 Me ( 4.61 ) 81 b 7 Bn ( 4.60 ) 82 c a Method A (solution phase); b Method B (solid phase); c Linker aminomethyl resin 4.63 was recovered and reused

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85 Reactions of the solid phase substrates 4.59 4.61 were carried out using the optimized solution phase conditions (Table 4 3), and gave similar yields to those found in the solution phase method. Recy cled resin 4.60 ( E ntry 7) was reacted under the optimized Staudinger protocols yielding 4.71 in 83% yield thus demonstrated recyclability. Formation of cyclic tripeptides was attempted (Scheme 4 1 4 ) by solid phase methodology. Unfortunately, no evidence wa s obtained for cyclic tripeptide formation under Staudinger ligation protocols. Scheme 4 1 4 Attempted solid phase cyclic tripeptide formation of 4.73 and 4.74 unde r Staudinger ligation protocols However, formation of cycl ic dipeptide 4.72 was observed from 4.68 and 4.69 due to an unprecedented cyclization/cleavage of the amide bond (Scheme 4 1 5 ). Scheme 4 1 5 Formation of the unexpected cyclic peptide 4.72 from the linear tripeptides 4.68 and 4.69 Computational investigation, performed by Dr. Jean Christophe Monbaliu, utilizing the B3LYP/6 31G(d) level of theory on model compounds, including PH 3 as a model

PAGE 86

86 phosphine, showed the energy difference between the competitive cyclization pathways The 9 membered cyclic transition state (TS 9 4.78) was favored by only 2.4 kcal/mol over the 6 membered cyclic transition state (TS 6 4.77). Increase in steric congestion around the thioester and the higher phosphine may lead to a reversal of the tendency to cyclize to the tripeptide. Figure 4 5. Competitive cyclization through TS 6 and TS 9 for the formation of 4.72 4.3 Summary 2,5 Diketopiperazines (4.70 72) were synthesized from the corresponding azido thioester dipeptides in excellent yields, using a st raightforward Staudinger ligation amino acids by known procedures, and syntheses were accomplished with short reaction times and simple purification procedures. The modified am inomethyl resin 4.63 was recycled and reused without loss in yield providing a cost efficient and eco friendly method compared to conventional SPPS procedures. Further work should include revisiting the tripeptide sequence design, but taking steric factors into consideration.

PAGE 87

87 4.4 Experimental 4.4.1 General Methods 1 H NMR spectra were recorded at 300 MHz and 13 C NMR spectra were recorded at 75 MHz on Gemini or Varian spectrometers at room temperature. The chemical shifts are reported in ppm relative to TMS as internal standard ( 1 H NMR) or to solvent residual peak ( 13 C NMR). HRMS spectra were recorded on a LC TOF (ES) apparatus. Elemental analysis was performed on a Carlo Erba 1106 instrument. Melting points were determined on a capillary point apparatus equi pped with a digital thermometer and are uncorrected. Flash chromatography was performed on silica gel 60 (230 400 mesh). All solvents were dried according to standard procedures. Triethylamine was distilled prior use. All commercially available substrates were used as received without further purification. All microwave assisted reactions were carried out with a single mode cavity Discover Microwave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure microwa ve tube equipped with a magnetic stir bar. The tube was closed with a silicon septum and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 120 sec.; PowerMax cooling mode). Amino acids were purchased from Chem Impex Inte rnational, and the aminomethyl (AM) resin was purchased from Chempep. Quantum chemical calculations were done using Gaussian 03W version 6.1. 4.4.2 General Method s for the Synthesis of 4.70 72 To a stirred solution of 4.38 40 (1 mmol) in CH 2 Cl 2 (4 mL), tri butylphosphine (0.37 mL, 1.5 mmol) was added and stirring was continued for 5 min at room temperature. Water (0.1 mL, 5 mmol) was added and stirring was continued for 5 min. The reaction mixture was then subjected to microwave irradiation (50 W, 50 C, 5 m in). Hexane (4

PAGE 88

88 ml) was then added to the reaction to induce crystallization and the mixture was placed in the freezer. The reaction was filtered and washed with DCM (5 mL) and hexanes (15 mL) and dried under vacuum to yield pure 4.70 72. To a stirred suspe nsion of 4.59 61 (1 mmol) in CH 2 Cl 2 (4 mL), tributylphosphine (0.37 mL, 1.5 mmol) was added and stirring was continued for 5 min at room temperature. Water (0.1 mL, 5 mmol) was added and stirring was continued for 5 min. The reaction mixture was then subje cted to microwave irradiation (50 W, 50 C, 5 min). The solids were filtered and washed with CH 2 Cl 2 the remaining solid was treated with hot methanol and the mother liquor was collected, cooled in the freezer and the precipitate collected to yield pure 4. 70 72. To a stirred suspension of 4.68 69 (1 mmol) in CH 2 Cl 2 (4 mL), tributylphosphine (0.37 mL, 1.5 mmol) was added and stirring was continued for 5 min at room temperature. Water (0.1 mL, 5 mmol) was added and stirring was continued for 5 min. The reacti on mixture was subjected to microwave irradiation (50 W, 50 C, 5 min), the solids filtered and washed with CH 2 Cl 2 The remaining solid was treated with hot methanol and the resulting mother liquor was collected, cooled in the freezer and the precipitate c ollected and dried under vacuum to yield pure 4.72. (S) 3 Isobutylpiperazine 2,5 dione 4.70. White microcrystals, y ield: 78% (0.13 g), mp 248.0 250.0 C. 1 H NMR (300 MHz, DMSO d 6 ): = 8.26 (br s, 1H), 8.26 (br s, 1H), 7.99 (br s, 1H), 3.89 3.79 (m, 1H), 3.70 3.56 (m, 2H), 1.84 1.69 (m, 1H), 1.56 1.49 (m, 2H), 0.89 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H) ppm. 13 C NMR (75 MHz, DMSO d 6 ): = 168.8, 166.3, 52.9, 44.2, 42.1 23.6, 22.9, 21.8 ppm. Anal. Calcd. for C 8 H 14 N 2 O 2 : C 56.45; H 8.29; N 16.46; F ound: C 56.63; H 8.41; N 16.28.

PAGE 89

89 (S) 3 Benzylpiperazine 2,5 dione 4.71. White microcrystals, y ield: 74% (0.15 g), mp 251.0 252.0 C. 1 H NMR (300 MHz, DMSO d 6 ): = 8.18 8.11 (m, 1H), 7.90 7.84 (m, 1H), 7.32 7.21 (m, 3H), 7.18 7.10 (m, 2H), 4.09 4.02 (m, 1H), 3.42 3.26 (m, 2H), 3.08 (dd, J = 13.5, 4.4 Hz, 1H), 2.87 (dd, J = 13.5, 4.9 Hz, 1H) ppm. 13 C NMR (75 MHz, DMSO d 6 ): = 167.4, 166.0, 136,1, 130.2, 128.3, 127.0, 55.7, 43.8 pp m. Anal. Calcd. for C 11 H 12 N 2 O 2 : C 64.49; H 5.92; N 1 3.72; F ound: C 64.43; H 6.06; N 13.65. (S) 3 Methylpiperazine 2,5 dione 4.72. White microcrystals, y ield: 78% (0.10 g), mp 236 238 C. 1 H NMR (300 MHz, DMSO d 6 ): = 8.17 (br s, 1H), 7.99 (br s, 1H), 3.88 (dq, J = 0.6, 6.9 Hz, 1H), 3.76 (s, 2H), 1.30 (d, J = 6.9 Hz, 3H) ppm. 13 C NMR (75 MHz, DMSO d 6 ): = 168.8, 166.2, 49.7, 44.5, 18.6. ppm. 4.4.3 General Methods for the Synthesis of Compounds 4.32 40 4.4.3.1 General p rocedure s for the s ynthesis of c ompound s 4.32 34 To a suspension of the appropriate amino acid 4.28 30 (20 mmol) in THF (50 mL), chloroacetyl chloride 4.31 (2.4 mL, 30 mmol) was added and the mixture was heated under reflux for 2 h. After cooling, water (20 mL) and brine (30 mL) were added and the mixture was extracted with ethyl acetate (3 x 75 mL). The combined organics were dried over MgSO 4 and the solvent was removed under reduced pressure. The residue was dissolved in diethyl ether and stirred at 0 C for 30 min. The mixture was filtered a nd the white solid was collected to yield pure 4.32 34. (S) 2 (2 Chloroacetamido) 4 methylpentanoic acid 4.32. White microcrystals, y ield: 77% (3.19 g), mp 139.0 141.0 C. 1 H NMR (300 MHz, DMSO d 6 ): = 8.46 (d, J = 7.9 Hz, 1H), 4.28 4.17 (m, 1H), 4.13 4.02 (m, 2H), 1.70 1.58 (m, 1H), 1.58 1.48 (m, 2H), 0.88 (d, J = 6.3 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H), 0.84 (d, J = 6.3 Hz, 3H) ppm. 13 C

PAGE 90

90 NMR (75 MHz, DMSO d 6 ): = 21.9, 23.4, 25.0, 40.6, 43.0, 51.2, 166.6, 174.2 ppm. Anal. Calcd. for C 8 H 14 ClNO 3 : C 46.27; H 6.80; N 6.75; found: C 46.64; H 6.61; N 6.70. (S) 2 (2 Chloroacetamido) 3 phenylpropanoic acid 4.33. White microcrystals, y ield: 61% (2.97 g), mp 125.0 127.0 C. 1 H NMR (300 MHz, DMSO d 6 ): = 8.53 (d, J = 7.9 Hz, 1H), 7.37 7.21 (m, 5H), 4.58 4.46 (m, 1H), 4.10 (s, 1H), 4.09 (s, 1H) 3.14 (dd, J = 13.9, 5.0 Hz, 1H), 2.98 (dd, J = 13.8, 8.9 Hz, 1H) ppm. 13 C NMR (75 MHz, DMSO d 6 ): = 172.5, 170.0, 137.3, 129.2, 128.3, 126.6, 53.9, 42.4, 36.7 ppm. Anal. Calcd. for C 11 H 12 ClNO 3 : C 54.67; H 5.00; N 5.80 ; found: C 54.63; H 5.04; N 5.71. (S) 2 (2 Chloroacetamido)propanoic acid 4.34. White microcrystals, y ield: 38% (1.29 g), mp 84.0 87.0 C. 1 H NMR (300 MHz, DMSO d 6 ): = 12.79 12.58 (m, 1H), 8.51 (d, J = 7.2 Hz, 1H), 4.41 4.14 (m, 1H), 4.12 (s, 1H), 1.32 ( d, J = 7.3 Hz, 3H) ppm. 13 C NMR (75 MHz, DMSO d 6 ): = 173.7, 165.7, 47.9, 42.4, 17.1 ppm. Anal. Calcd. for C 5 H 8 ClNO 3 : C 36.27; H 4.87; N 8.46; found: C 36.63; H 4.96; N 8.29. 4.4.3.2 General p rocedure s for the s ynthesis of c ompounds 4.35 37 4.32 34 (10 mmo l) was dissolved in a DMF/water 2:1 mixture (45 mL), sodium azide (2.92 g, 45 mmol) was added and the suspension was stirred for 12 h at room temperature. Water (30 mL) was added and the mixture stirred for 30 min, and extracted with ethyl acetate (3 x 50 mL). The combined organics were washed with water (3 x 50 mL) and brine (3 x 50 mL), dried over MgSO 4 and solvent was removed under reduced pressure. The residue was recrystallized from ethyl acetate/hexanes to afford pure 4.35 37. (S) 2 (2 Azidoacetamido ) 4 methylpentanoic acid 4.35. White microcrystals, y ield: 61% (1.31 g), mp 105.1 107.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 12.75 (s, 1H), 8.47 (dd, J = 28.6, 7.9 Hz, 1H), 4.35 4.20 (m, 1H), 4.13 (d, J = 2.0 Hz, 1H), 3.89 (d, J = 0.8

PAGE 91

91 Hz, 1H), 1.75 1.49 (m, 3H ), 0.91 (dd, J = 12.7, 6.3 Hz, 6H). 13 C NMR (75 MHz, CDCl 3 ): = 173.7, 167.5, 50.6, 50.4, 50.3, 42.4, 24.3, 22.9, 21.3 ppm. HRMS calcd for C 8 H 14 N 4 O 3 [M H]: 213.1005. Found 213.1002 (S) 2 (2 Azidoacetamido) 3 phenylpropanoic acid 4.36. White microcrystals, y ield: 78% (1.94 g), mp 99.4 103.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 12.86 (br s, 1H), 8.42 (d, J = 8.0 Hz, 1H), 7.31 7.15 (m, 5H), 4.47 (dt, J = 8.7, 5.0 Hz, 1H), 3.79 (d, J = 5.3 Hz, 2H), 3.08 (dd, J = 13.8, 5.0 Hz, 1H), 2.89 (dd, J = 13.8, 9.2 Hz, 1H) p pm. 13 C NMR (75 MHz, CDCl 3 ): = 172.6, 167.4, 137.4, 129.1, 128.3, 126.6, 53.6, 50.5, 36.7 ppm. Anal. Calcd for C 11 H 12 N 4 O 3 : C 53.22; H 4.87; N 22.57; found: C 53.46; H 4.92; N 22.45. (S) 2 (2 Azidoacetamido)propanoic acid 4.37. Yield: 65% (1.12 g), wh ite microcrystals. m.p. 100.1 101.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 12.68 (br s, 1H), 8.45 (d, J = 7.3 Hz, 1H), 4.33 4.22 (m, 1H), 3.88 (br s, 2H), 1.32 (d, J = 7.3 Hz, 3H) ppm. 13 C NMR (75 MHz, CDCl 3 ): =173.7, 167.1, 50.4, 47.6, 17.2. ppm. Elemental ana lysis calcd (%) for C 5 H 8 ClNO 3 : C, 36.27; H, 4.87; N, 8.46; found: C, 36.63; H, 4.96; N, 8.29. 4.4.3.3 General m ethod s for the s ynthesis of 4.38 40 Isobutyl chloroformate (0.72 mL, 5.5 mmol) and N methyl morpholine (0.60 mL, 5.5 mmol) were mixed and reacted for 5 minutes with a stirred solution of 4.35 37 (5 mmol) in THF (30 mL). Thiophenol (0.56 mL, 5.5 mmol) was added and the reaction was warmed to room temperature and allowed to stir for 18 h. The solvent was removed under reduced pressure, the residue wa s dissolved in ethyl acetate (50 mL), washed with 10% NaOH (3 x 25 mL), and brine (25 mL) then dried over MgSO 4 The solvent was removed under reduced pressure, and the residue was recrystallized from a mixture of DCM/hexanes to yield pure 4.38 40.

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92 (S) S P henyl 2 (2 azidoacetamido) 4 methylpentanethioate 4.38. Yield: 75% (1.15 g), white microcrystals. m.p. 138.0 140.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 7.50 7.35 (m, 5H), 7.34 7.27 (m, 1H), 7.09 (d, J = 8.3 Hz, 1H), 4.91 4.74 (m, 1H), 4.13 4.06 (m, 2H), 4.01 (s, 2H), 1.84 1.53 (m, 3H), 0.96 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H) ppm. 13 C NMR (75 MHz, CDCl 3 ): = 198.9, 168.6, 168.6, 16 7.8, 134.8, 129.8, 129.5, 126.9, 58.0, 52.6, 43.2, 41.5, 25.1, 23.2, 21.7 ppm. Elemental analysis calcd (%) for C 16 H 21 N 4 O 3 S: C, 52.88; H, 5.82; N, 19.27; found: C, 52.94; H, 6.01; N, 19.14. (S) S Phenyl 2 (2 azidoacetamido) 3 phenylpropanethioate 4.39. Yie ld: 57% (0.97 g), white microcrystals. m.p. 135.0 137.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 12.68 (br s, 1H), 8.45 (d, J = 7.3 Hz, 1H), 4.33 4.22 (m, 1H), 3.88 (br s, 2H), 1.32 (d, J = 7.3 Hz, 3H) ppm. 13 C NMR (75 MHz, CDCl 3 ): = 173.7, 167.1, 50.4, 47.6, 17.2 ppm. Elemental analysis calcd (%) for C 17 H 16 N 4 O 3 S: C, 59.98; H, 4.74; N, 16.46; fou nd: C, 59.74; H, 4.68; N, 16.22. (S) S Phenyl 2 (2 azidoacetamido)propanethioate 4.40. Yield: 42% (0.56 g), white microcrystals. m.p. 124.5 126.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 7.29 7.22 (m, 5H), 6.65 (d, J = 7.7 Hz, 1H), 4.71 (qd, J = 14.4, 7.2 Hz, 1H), 3.90 (d, J = 1.3 Hz, 2H), 1.43 1.39 (m, 2H), 1.36 (d, J = 7.2 Hz, 3H)ppm. 13 C NMR (75 MHz, CDCl 3 ): = 173.0, 166.6, 134.9, 130.0, 129.6, 54.9, 52.8, 19.1 ppm. Elemental analysis calcd (%) for C 11 H 12 N 4 O 3 S: C, 49.99; H, 4.58; N, 21.20; found: C, 50.12; H, 4 .43; N, 21.57.

PAGE 93

93 CHAPTER 5 BENZOTRIAZOYL NITROS O DERIVATIVES: POTEN TIAL NOVEL NO DONORS 5.1 Literature Overview Nitroso compounds are of great interest due to their reactivity and transient nature. While many of these compounds have shown both therapeutic and mutagenic activity, attention has also been focused on their use in synthetic processes. Nitrogen oxides and their derivatives form a family of compounds involved in many physical, chemical and biological phenomena, ranging from atmospheric pollution to immune response. 5.1.1 Biological Properties of Selected Nitroso Compounds N Nitroso amines are a class of nitrosation agents that act as potent mutagens to mammalian cells which may lead to heptacarcinoma (liver cancer).[81CR5039] Nitrosomorpholine (NM OR) 5.1, is a potent mutagen forming liver cancer in rats in vivo [79JOC1563] Although nitroso proline 5.2 and nitroso hydroxyproline 5.3 are non carcinogenic compounds found in nature, consumption may lead to trans nitrosation with morpholine to form NMO R.[81CR5 03 9] Cyclic N nitroso amines have been shown to be more carcinogenic than their acyclic counter parts.[ 81CR5039 ] Figure 5 1. N Nitroso compounds showing interesting biological activity However, NO plays an importan t role in neurotransmission,[91TN60] immune regulation,[88B8706, 89JEM1011, 91RI565] vascular smooth muscle relaxation,[86PNAS9164, 89PR651] and inhibition of platelet aggregation.[86JP411, 87JP687, 87BPRC1482] Naturally occurring S nitrosothiols, S nitros o L cysteine (5.4)

PAGE 94

94 and S nitroso L glutathione (5.5), are important NO releasers in vivo .[00CS507] An important class of enzymes known as NO synthase enzymes (NOS), catalyze the sequential oxidation of L arginine to release NO and L citrulline.[ 89PNAS444 ] The mechanism involves a Cu + species for the in vivo release of NO from thiols and thiol based NO releasing drugs. [ 00CS507] Figure 5 2. S N itroso compounds of biological relevance 5.1.2 Different Forms of Nitroso C ompound s Many compounds with the nitroso moiety have been studied (Figure 5 3).[11ACIE5630, 05CC3514] Our research will pay a particular interest to compounds 5.8 9 involving an leaving group. Figure 5 3. Examples of various nitroso compounds Recent literature has shown that reactivity is significantly affected by the structure of the nitroso compound.[10JSST49] The electrophilicity scale (Figu re 5 4) shows that acyl nitrosos are the most reactive, thus they must be generated in situ .[11ACIE5630]

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95 While the alkyl nitrosos are the least electrophilic, making them more stable and sluggish to react. Figure 5 4. Glob al electrophilicity sc ale of common nitroso compounds [10JST49] Nitroxyl 5.6 is the simplest form of a nitroso compound and is highly reactive. It is not usually used in synthesis due to its fleeting nature and is a decomposition product of nitroso compound s.[05CCR433] Dimerization of 5.6 is a major issue, leading to loss of H 2 O and formation of N 2 O.[96JACS3550, 06JACS9687] Chloronitroso compounds 5.8 are subject to rapid decomposition, due to their highly polarized nature.[98T1317] With chloro as a weak e lectron withdrawing leaving group, the nitroso is slow to react as an electrocyclization reagent, but has been well Chloronitroso compounds are readily synthesized from oximes and electrophilic c hlorine reagents (e.g. t BuOCl).[84TL5377, 00JCS(P1)329] chloro nitroso analogs, compound 5.9 can be synthesized from oxime precursors by oxidation with lead (IV) tetraacetate or IBX.[06JACS9687, 0 Acetoxy nitroso compounds are also important in studying the release of NO and HNO.[05CCR433, 11ARS1637] Acyl nitroso species 5.15 are extremely unstable, but can be reacted in situ as transient species.[81T4007] They are commonly synthesized by oxidation of oxime or isocyanate precursors and either reacted or trapped as cycloadducts with 9,10 dimethylanthracene.[79CJC1712, 81T4007] They have found considerable use in

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96 hetero Diels Alder reactions because of their high reactivity.[11AGE5630] Th eir existence was first proved by reaction with various nucleophiles (Figure 5 5),[77CSR1] although the first direct proof was provided by Schwarz and co workers in 1991 through neutralization reionization mass spectrometry when liberated from 9,10 dimethy l anthracene adducts.[91HCA2068] Figure 5 5. Transient acyl nitroso compound 5.15 and their derivatives 5.17 19 on reaction with nucleophiles Aryl nitroso compounds 5.13 14 are stable compounds whose aryl group acts to m odulate the reactivity of the nitroso moiety.[94CR1621] This dampening of reactivity can be regulated by introducing electron withdrawing groups into the aryl ring which enhances reactivity.[10TL328] Normally these compounds are synthesized by oxidation of the corresponding oximes, but may also be generated by oxidation in situ of the corresponding aromatic amines.[98T1317] Cyano based nitroso compounds fall into two main groups: nitrosocyanamide 5.7 (direct attachment of the nitroso to the cyano group) and cyano nitroso compounds leaving group).[98T1317, 11AGE5630] The use of nitrosocyanamide 5.7 is limited due to its high reactivity and is usually stored as a

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97 cycloadduct with 9,10 dimethyl anthracene.[80JCS(P1)1587, 81JCS(P1)1802] chloro nitroso compounds where excessive reactivity is a concern.[96T7585, 09JOC1450] 5.1.3 Reactions of Nitroso Compounds Three common reactions of nitroso compounds described in the litera ture are: i) nitroso Diels Alder reactions, ii) nitroso ene reactions and iii) nitroso aldol reactions. HNO is released. 5.1.3.1 Diels Alder r eactions of n itroso c ompound s The Diels Alder reaction is a pericyclic reaction discovered in 1928 by Otto Paul Hermann Diels and Kurt Alder.[28JLAC98] Nitroso compounds act as activated dienophiles, the exception being vinyl nitrosos that can also act as a diene.[11ACIE5630, 98T1317 ] The nitroso Diels Alder reaction results in useful building blocks such as 1,2 leaving group nitroso, the nitroso Diels Alder reaction is a three step process (Figure 5 6 ). Initially the 1,2 oxazine ring 5.21 is fo rmed by pericyclic reaction of 5.8 and 5.20. The acetoxy or chloro group leaves forming iminium salt 5.22 which is then hydrolyzed yielding the free 1,2 oxazine 5.23. If the reaction takes place in a polar protic solvent such as methanol, solvolysis occurs immediately on formation of the iminium salt 5.22.[10JST49] Figure 5 6 Three step process for the formation of 1,2 oxazines in nitroso hetero Diels Alder reactions using leaving group nitroso 5.8

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98 Much attention has been given to enantio and diastereoselective Diels Alder r eactions, and chiral nitroso compounds are used to induce stereoselectivty.[05CC3514, 98T1317, 07BCJ595] Yamamoto et al. have recently written a comp rehensive review on stereoselective nitroso hetero Diels Alder reactions.[06EJOC2031] For example, chiral synthesis of ( ) epibatidine (Figure 5 7 ).[98JOC8397] Figure 5 7 Asymmetric hDA utilized in the synthesis of ( ) epibatidine from chiral nitroso reagent 5.25 5.1.3.2 Nitroso e ne r eactions The nitroso ene reaction is another reaction associated with nitroso compounds (Figure 5 8 ).[05CC3514, 07 BCJ595] The electron deficient nitroso 5.28 acts as an enophile that reacts with the allylic system 5.27 to give the pericylic product 5.29.[01JCSPT(1)1908] Asymmetric versions have also been developed using chiral nitroso compounds.[81JACS3173, 82JOC1302, 00JACS9846] Figure 5 8 The nitroso ene reaction involving an allylic system and a nitroso compound

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99 5.1.3.3 Nitroso a ldol r eactions In the nitroso aldol reaction the nitroso acts as a surrogate carbonyl compound. Mukaiyam a and co workers report the reaction in 1963, isolating azomethine 5.32 from reaction of nitrosobenzene 5.31 with diethyl malonate 5.30.[63BCJ970] Figure 5 9 The nitroso aldol reaction of nitrosobenzene and diethyl malona te Later, however, it was found that both the nitrogen and oxygen could be involved in the nitroso aldol reaction (Figure 5 10 ).[07BCJ595] Yamamoto and co workers also reported the catalytic enantioselective addition of enolates to nitroso. By varying the equivalents of silver in the catalyst, they were able to induce selective addition to either the O or N moiety of the nitroso group.[04JACS5360 04JACS5962 ] Figure 5 1 0 Selective addition to the O or N moiety of nitro so compounds 5.1.3.4 Release of NO and HNO The release of NO or HNO by decomposition of nitroso compounds is important in medicinal and pharmaceutical chemistry,[11JMC1059, 11ARS1637] since NO and HNO have therapeutic value as vasodilators (Chapter 5.1.1). [00PR471] King and co workers investigated the release of HNO from acetoxy nitroso compound 5.36 and found

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100 addition of base gave 5.37, which decomposed to release HNO 5.6 and cyclohexanone 5.38 (Figure 5 17).[06JACS9687] Figure 5 1 1 Release o acetoxy nitroso compounds The bond dissociation energy of C Nitroso compounds is approximately 36 40 kcal/mol which is similar to the O NO BDE of organic nitrate esters (a common NO donor).[95JPC10815, 78JCS(P2)1110, 02JPCA12386, 05CTMC687] Th us NO release occurs by homolytic bond cleavage of the nitroso moiety from the parent C nitroso compound.[01JACS8868] Nitrate esters are effective NO donors, but unfortunately require co catalysts (protons, base, enzymes) and discharge NO in various oxidat ion states ( NO vs + NO).[05CCR433, 92S1898, 99BPA1411, 05CRT790, 10CC3788] However, Toone et al. used cyano nitroso compound 5.10 to selectively release NO without a co catalyst (Figure 5 18).[09JOC1450] Figure 5 1 2 cyano nitroso compound 5.10

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101 5.2 Results and Discussion L eaving G roup B enzotriazole N itroso C ompounds The starting oximes 5.40 45 were synthesized in 67 91% yields by literature methods. Lead (IV) acetate/benzotriazole reagent, generated in situ from lead (IV) tetracetate with 10 equivalents of 1 H benzotriazole in DCM or THF on addition of 5.40 45, the reaction mixture turned blue green signaling formation of 5.46 51 (Table 5 1). Table 5 benzotriazoyl nitroso compounds 5.46 51 Compound R 1 R 2 Solvent Yield(%) 5.46 Cyclohexyl DCM 59 5.46 Cyclohexyl THF 85 5.47 Cyclopentyl THF 48 5.48 Cycloheptyl THF 37 5.49 Cyclooctyl THF 39 5.50 Me Me THF 76 5.51 Me Et THF 72 aromatic oxime 5.52, derived from acetophenone, failed to produce nitroso compound 5.53. Figure 5 1 3 Aromatic substrate 5.52 which failed to react to prod benzotriazoyl nitroso 5.53 Aromatic nitroso 5.54 (Figure 5 14 ), derived from the methyl ibuprofen ketoxime, decomposed rapidly (within minutes), and 5.55 could not be isolated.

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102 Figure 5 14 B enzotriazoyl nitroso derived from methyl ketoxime 5.54 5.2.2 Hetero Diels Alder Reaction of 5.46 No reaction was observed between 5.46 and either 2,3 methyl butadiene 5.56 or cyclopentadiene 5.57 (Table 5 2), and it was concluded that 5.46 was not a suitable substrate for nitr oso Diels Alder reactions. In an effort to understand the lack of reactivity, a computational investigation, performed by Dr. Jean Christophe Monbaliu, on the cycloaddition of X NO derivatives with dienes 5.56 57. The computations were carried out using 2 (2 nitrosopropan 2 yl) 2 H benzo[ d ][1,2,3]triazole (5.61, X=Bt), 2 chloro 2 nitrosopropane (5.62, X=Cl) and 2 nitrosopropan 2 yl acetate (5.63, X=AcO) as model dienophiles. 2 Nitrosopropane (5.64, X=H) was selected as a reference for studying the nature of the leaving group on the process. The global electrophilicity was obtained by the method of Domingo. [07CPL341] [06EJOC2570] Full geometry optimization and verification of the Hessian were performed by the G03 program package (revision E.01 ). [09GE01] Tra nsition states were optimized and localized at the B3LYP/6 31+G* level with zero point energy correction and were verified by frequency and IRC calculations. All reactants were optimized in gas ph ase at the B3LYP/6 31+G* level.

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103 Table 5 2. Reaction of 5. 46 in the nitroso Diels Alder reaction BtNO Diene Solvent Conditions Time (h) Yield (%) 5.46 5.56 neat rt 48 0 5.46 5.56 toluene rt 48 0 5.46 5.56 DCM rt 48 0 5.46 5.56 diethyl ether rt 48 0 5.46 5.56 THF rt 48 0 5.46 5.56 DCM/EtOH rt 48 Degradation 5.46 5.56 MeOH rt 48 0 5.46 5.57 MeOH rt 48 0 5.46 5.57 MeOH MW 48 0 The computed global electrophilicity ( ) allowed for a classification of the selected nitroso compounds according to their reactivity. The following e lectrophilicity scale was found: 5.64 ~ 5.61 ( =2.6 eV) < 5.62 ( =2.7 eV) < 5.63 ( =2.9 eV), showing that these compounds are moderate electrophiles within the electrophilicity scale, and that the nature of leaving group X has minimal effect on global reac tivity. Figure 5 1 5 Picture of the TSs associated with the cycloaddition of the selected nitroso compounds with butadiene. For each situation, 4 isomeric TSs have been isolated from endo/exo approach of the dienophile and the syn/anti orientation of the X group vs N=O. The results shown are the most stable TSs (endo/anti) isolated

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104 Activation barriers for the cycloaddition step were calculated, and showed relative insensitivity towards the nature of X (Figure 5 1 5 ). The transition state associated with th e most electrophilic species (5.63 ) is also the lowest in energy. 5.2.3 Computational Investigation into the R elease of NO The possibility of 5.46 as a NO releasing agent was investigated, since 5.46 did not participate in a hDA. Attempts were made to show NO release in our facilities, and an outside laboratory was also utilized. In an effort to provide evidence for the formation of NO, a computational investigation was undertaken. The computational work was carried out by Dr. Jean Christophe Monbaliu. Isod esmic heat ( H iso ) for radical exchange and radical stabilization energies (RSE) [10PCCP9597] were computed for a variety of substituted cyclohexyl substrates, showing that the Bt 2 substituent stabilizes a radical better than phenyl does (Table 5 3). Table 5 3. Isodesmic heat ( H iso ) for radical exchange on different cyclohexyl substrates and corresponding radical stabilization energy (RSE) Compound R 1 = H iso (kcal/mol) RSE (kcal/mol) 5.65 Me 10.8 10.8 5.66 Cl 10.2 10.2 5.67 Ph 19.3 19.3 5.68 2 Bt 30.9 30.9 Homolytic bond dissociation energies (BDE) for the release of nitric oxide were computed for compounds 5.65 68, and revealed that the homolytic bond rupture for 5.46 is similar to that of reference compound 5.10 ( Table 5 4),[09JOC1450]

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105 emphasizing that compound 5.46 could release nitric oxide forming a stable tertiary radical. Table 5 4. Homolytic bond dissociation energies (BDE) for compounds 5.8, 5.10 and 5.46 Compound X BDE (kca l/mol) 5.46 2 Bt 22.4 5.10 CN 19.8 5.8 Cl 30.6 In contrast, the heterolytic bond dissociation leading to nitroxyl was found to be extremely disfavored for 5.46 compared to 5.8 [06JACS9687], the reference for HNO donors (Table 5 5). Table 5 5. Heterol ytic bond dissociation energies (BDE) for compounds 5.8, 5.36, and 5.46 Compound X BDE (kcal/mol) 5.46 2 Bt 189.4 5.36 O 37.1 5.8 Cl 211.1 Indirect proof for the release of NO in situ was found when 5.46 was dissolve d in methanol and allowed to stir for 4 hours open to air (Figure 5 1 6 ). The characteristic blue color of 5.46 disappeared and on removal of the solvent a white solid was obtained. The structure was determined unambiguously by X ray diffraction of a single crystal as 5.69 (Figure 5 1 7 ).

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106 Figure 5 1 6 Indirect proof for the release of NO through radical intermediate 5.70 Figure 5 1 7 X benzotriazoyl nitro 5.69 The formation of nitro compound 5.69 from the nitroso has literature precedent and involves oxidation by molecular oxygen.[08EJOC3279] Oxidized N 2 O reacts with the stabilized radical 5.70 to yield 5.69. The f ormation of NO 2 from NO is a well known and has been investigated by the oxidation of nitroso compounds by N 2 O 4 .[08EJOC3279] Benzotriazole Nitroso C ompounds 5.46 51 C Nitroso compounds have a characteristic blue green to deep blue col or associated with their monomeric form.[96JOC1047, 09JOC1450] This blue coloration is compounds have a tendency to dimerize since the dimer is a lower energy state (6 10 kca l/mol for the dimerization and 20 30 kcal/mol for dissociation).[70JACS1460,

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107 71JOC3055, 09JOC1450] The dimers are unreactive, and must dissociate for a reaction to occur.[96JOC1047] Compound 5.46 is deep blue in color in both solution and solid state indi cating the monomer. In contrast, compounds 5.50 51 dimerize in the solid state forming a white powder. Compound 5.46 is both air and moisture stable for at least six months, and a X ray structure showed a 95:5 ratio of nitroso to nitro compound, probably d ue to a small amount of NO release and oxidation to NO 2 in solution during recrystallization. Figure 5 1 8 X ray structure of 5.46 with minor impurities from the nitro compound 5.69 Kinetic information was obtained by Ms. Judit Kovacs for the release of nitric oxide from compound 5.46 and 5.50 51 using UV spectrophotometry at 20 C. Effects of different solvents were seen by following the disappearance of the nitroso signal ( max = 655.7 nm, 10 mg mL 1 ). The observed dependency of the reaction rates on th e solvent polarity supports a homolytic mechanism for release of NO since the half life of 5.46 in MeOH, CH 3 CN, and CH 2 Cl 2 was 2.47, 345.6, and 495.1 min, respectively. Inclusion of water in acetonitrile (7:3 acetonitrile/water mixture) increased the half life slightly ( t 1/2 = 385.1 min). First order rate constants k obs = 3.1, 2.0, 1.8 and 1.4 x10 3 s 1 were determined in methanol, acetonitrile/water, acetonitrile and dichloromethane (Figure 5 1 9 ).

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108 Figure 5 1 9 Half li ves of 5.46 as measured in various so lvents Additional experiments using different concentrations (5 and 20 mg mL 1 ) of compound 5.46 in acetonitrile gave half lives of t 1/2 = 266.6 and 495.1 min. This is azodioxy dimer at higher concentration (Figure 5 20 ). As per Figure 5 2 1 the steric hindrance of the backbone on carbon has an impact on the release of NO ( t 1/2 = 223.6 (5.50), 247.6 (5.51) and 495.1(5.46) min). Figure 5 20 Kinetics for dissociation at various concentrations in methanol of 5.46

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109 Figure 5 21 Comparison of the half li ves of 5.46 and 5.50 51 in m ethanol 5.3 Summary benzotriazoyl nitroso reagents 5.26 51 were synthesized in good yields utilizing a lead (IV) benzotriazole/acetate reagent. This method provided 5.46 as a stable reagent that is air and moisture insensitive for at lea st six months. The benzotriazoyl nitrosos did not to react as dienophiles in hetero Diels Alder reactions, but their potential as NO donors was investigated. A computation investigation benzotriazoyl nitro 5.69 was obtained. Further laboratory testing is needed to learn more about the nature of 5.46 as a candidate for NO release. 5.4 Experimental 5.4.1 General Methods 1 H NMR spectra were recorded at 300 MHz and 13 C NMR spectra were record ed at 75 MHz on Gemini or Varian spectrometers at room temperature. The chemical shifts are reported in ppm relative to TMS as internal standard ( 1 H NMR) or to solvent residual peak ( 13 C NMR). The NMR experiments at variable temperatures (35, 45, 55 and 65 C)

PAGE 110

110 were recorded on a Varian Inova NMR spectrometer operating at 500 MHz. Chiral HPLC experiments were performed on a Chirobiotic T column using methanol as mobile phase. Compounds were analyzed at a flow rate of 0.1 mL/min (detection wavelength = 230 nm solvent = methanol). HRMS spectra were recorded on a LC TOF (ES) apparatus. Elemental analysis was performed on a Carlo Erba 1106 instrument. Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorr ected. Flash chromatography was performed on silica gel 60 (230 400 mesh). All commercially available substrates were used as received without further purification. All microwave assisted reactions were carried out with a single mode cavity Discover Microw ave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon septum and the reaction mixture was subjected to microwave irr adiation (Discover mode; run time: 60 sec.; PowerMax cooling mode). Quantum chemical calculations were done using Gaussian 03W version 6.1. 5.4.2 Synthesis of N itroso C ompounds 5.46 51 A solution of lead (IV) tetraacetate (4.43 g, 10.0 mmol) and 1H benzotr iazole (11.9 g, 100.0 mmol) in THF (100 mL) was stirred for 15 min at 0 C. The resulting homogeneous solution was treated dropwise by a solution of oxime 5.40 45 (10.0 mmol) in THF (25 mL) over 15 min at 0C. After 2 h, the solvent was removed under redu ced pressure and the brown residue was washed several times with hexanes (5 x 50 mL). The combined organic fractions were evaporated under reduced pressure and the greenish oily residue was purified over silica gel (hexanes /ethyl acetate 10/1) to

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111 yield p ure benzotriazole nitrosos 5.46 51. Slow crystallization from a hexanes/diethyl ether mixture gave 5.46 as blue crystals. 2 (1 Nitrosocyclohexyl) 2H benzo[d][1,2,3]triazole 5.46. Yield: 48% (1.95 g), blue microcrystals. m.p. 125.0 127.0 C. 1 H NMR (300 MHz, CDCl 3 ): = 1.34 1.51 (m, 2H), 1.55 1.67 (m, 1H), 1.67 1.80 (m, 1H), 1.94 2.06 (m, 2H), 2.65 2.86 (m, 4H), 7.36 7.45 (m, 2H), 7.84 7.93 (m, 2H) ppm. 13 C NMR (75 MHz, CDCl 3 ): = 21.7, 24.6, 29.2, 118.7, 127.0, 128.1, 144.9 ppm. Elemental analysis calcd (%) for C 12 H 14 N 4 O 1 : C, 62.59; H, 6.13; N, 24.33; found: C, 62.20; H, 5.90; N, 24.41. 5.4.3 X R ay D ata for 5.46 and 5.69 Crystal data for compound 5.46: blue crystal (plates), dimensions 0.4 x 0.1 x 0.04 mm, crystal system monoclinic, space group P2(1)/c, Z = 4, a = 11.7352(5), b = 8.5659(3), c = 12.1021(4) 1138.63(7) 3 3 T = scans with CCD area detector, covering a whole sphere in reciprocal space, 14435 reflections measured, 3407 unique (R int = 0.0218), Lorentz and polarization effects, an empirical absorption correction was applied using SADABS22 based on the Laue symmetry of the reciprocal space, m = 0.091 mm 1 Tmin = 0.6840, Tmax = 0.7461, str ucture solved by directmethods and refined against F2 with a Full matrix least squares algorithmusing the SHELXL 97 software package, 164 parameters refined, hydrogen atoms were treated using appropriate riding models, goodness of fit = 1.069 for observed reflections, final residual values R1(F) = 0.0376, wR(F2) = 0.0976 for observed reflections.CCDC 883509 Crystal data for compound 5.69: white crystal (rods), dimensions 0.55 x 0.29 x 0.26 mm, crystal system orthorhombic, space group P2(1)2(1)2(1), Z = 4, a = 5.9246(9),

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112 1172.5(3) 3 3 T = scans with CCD area detector, covering a whole sphere in reciprocal space, 8626 reflections measured, 2124 unique (R int and polarization effects, an empirical absorption correction was applied using SADABS22 based on the Laue symmetry of the reciprocal space, m = 0.099 mm 1 Tmin = 0.6747, Tmax = 0.7463, structure solved by directmethods and refined against F2 with a Full matrix least squares algorithmusing the SHELXL 97 software package, 163 parameters refined, hydrogen atoms were treated using appropriate riding models goodness of fit = 1.071 for observed reflections, final residual values R1(F) = 0.0346, wR(F2) = 0.0888 for observed reflections.CCDC 883510

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113 CHAPTER 6 SUMMARY OF ACHIEVEME NTS Synthetic organic chemistry continues to play an important role in materials science, medicinal chemistry, and biochemistry. Heterocyclic compounds are found in a significant segment of biologically active compounds, and the work in this thesis extends the use of mild and efficient methods for the synthesis of heterocyclic compound s. Chapter 1 includes the common research themes: heterocyclic compounds, peptides and microwave assisted synthesis. In addition, the properties and reactivity of benzotriazole are reviewed, as developed in the Katritzky laboratories. Properties and genera l information on the synthesis of peptides, as well as general information regarding microwave heating are outlined. Chapter 2 discusses microwave assisted synthesis of 3,5 diamino 1,2,4 triazole compounds. Novel compounds were obtained by ring acylation u sing N aminoacyl)benzotriazoles and N (protected dipeptidoyl)benzotriazoles, however with the ring protected, exocyclic acylation was exclusively observed. Chapter 3 discusses the formation of proline containing 2,5 diketopiperazines; stereoflexible strategies lead selectively to cis or trans configured DKPs. Starting from Cbz protected dipeptidoyl benzotriazoles cis configured DKPs were synthesized from a tandem deprotection/cyclization step, whereas trans DKPs were formed utilizing a tandem cycl ization/epimerization strategy. Through a series of computational and experimental investigations, a full rationalization of the phenomenon was accomplished. Chapter 4 discusses the formation of cyclic peptides without the use of a turn inducer. The use o f Staudinger protocols in the formation of a phospho aza ylide

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114 allowed for the convenient synthesis of 2,5 diketopiperazines from easily prepared starting materials. The methodology was extended to a novel solid phase protocol involving an aminomethyl (AM) resin solid support. Finally, C benzotriazoyl nitroso compounds to be poor dienophiles for nitroso Diels Alder reactions. Computational investigation of NO benzotriazoyl compounds gave promising results, including indirect experimental evidence. Further experimental testing is needed to benzotriazoyl nitroso compounds as NO release candidates.

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115 LIST OF REFERENCES [ 28JLAC98] O. van Diels, and K. Alder, Justus Lebigs Ann. Chem. 1928, 460 98. [48JCS2240] A. Albert, R. Goldacre, and J. Phillips, J. Chem. Soc. 1948, 2240. [54SCI989] D. Davis, Science 1954, 120 989. [58JACS3929] A. Steck, R. Pauline, and T. Fletcher J. Am. Chem. Soc. 1958, 80 3929. [58QRCS321] W. Gowenlock, B.G., Luttke, Q. Rev. Chem. Soc. 1958, 12 321. [63BCJ970] M. Tsuge, O., Tashiro, Bull. Chem. Soc. Jpn. 1963, 36 970. [63JACS2149] R. B. Merrifield, J. Am. Chem. Soc. 1963, 85 2149. [ 68JOC 864] J. W. Nitecki, D.E., Halpern, B., Westley, J. Org. Chem. 1968, 33 864 [70JACS1460] R. Hoffmann, R. Gleiter, and F. B. Mallory, J. Am. Chem. Soc. 1970, 92 1460. [70JOC2067] D. M. McCarty, C.G., Parkinson, J.E., Wieland, J. Org. Chem. 1970, 35 20 67. [71JOC3055] C. Stowell, J. Org. Chem. 1971, 36 3055. [73JMC935] J. T. Witkowski, R. K. Robins, G. P. Khare, and R. W. Sidwell, J. Med. Chem. 1973, 16 935. [74JACS3985] C. Eguchi and A. Kakuta, J. Am. Chem. Soc. 1974, 96 3985 [75JCS(P1)1181] J. R L. Smith and J. S. Sadd, J. Chem. Soc., Perkin Trans. 1 1975, 1181. [76T467] H. B. and G. W. V. Bhujle, U. P. Wild, Tetrahedron 1976, 32 467. [77CSR1] G. W. Kirby, Chem. Soc. Rev. 1977, 6 1 [77JHC443] M. T. Wu, J. Heterocyclic Chem. 1977, 14 443. [78JCS(P1)1110] L. R. Medici, A., Rosini, G., Serantoni, E.F., di Sanseverino, J. Chem. Soc., Perkin Trans. 1 1978, 1110 [79CJC1712] K. N. Dao, L.H. Dust, J. M. Mackay, and D. Watson, Can. J. Chem. 1979, 57 1712. [79JOC1563] C. H. B. Hecht, and S. S., Chen, J. Org. Chem. 1979, 44 1563

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118 [92S1898] J. S. Stamler, D. J. Singel, and J Lascalzo, Science 1992, 258 1898. [92T7817] A. R. Katritzky, N. Shobana, J. Pernak, A. S. Afhdi, and W. qiang Fan, Tetrahedron 1992, 48 7817. [93T165] P. J. Garratt, S. N. Thorn, and R. Wrigglesworth, Tetrahedron 1993, 49 165. [94CR1621] P. Zuman and B. Shah, Chem. Rev. 19 94, 94 1621. [94CSR363] A. R. Katritzky and X. Lan., Chem. Soc. Rev. 1994, 363. [95JPC10815] A. A. Boyd, B. Nozikre, and R. Lesclaux, J. Phys. Chem. 1995, 99 10815. [94S1107] J. Streith, and A. Defoin, Synthesis 1994, 1107. [95S1315] A. R. Katritzky, H. Wu, L. Xie, S. Rachwal, R. Rachwal, J. Jiang, G. Zhang, and H. Lang, Synthesis 1994, 1315. [96CR3147] G. A. Patani and E. J. Lavoie, Chem. Rev. 1996, 96 3147. [96JACS3550] N. Bahr, R. Gu, J. louis Reymond, and R. A. Lerner, J. Am. Chem. Soc. 1996, 1 18 3550. [96JOC1047] R. Glaser, R. K. Murmann, and C. L. Barnes, J. Org. Chem. 1996, 61 1047. [96T7585] V. Gouverneur and L. Ghosez, Tetrahedron 1996, 52 7585 [96T12651] C. B. Cui, H. Kakeya, and H. Osada, Tetrahedron 1996, 52 12651. [97JOC4148] A. R. Katritzky, C. N. Fali, and J. Li, J. Org. Chem. 1997, 62 4148. [97ME14] P. Alewood, D. Alewood, L. E. S. Miranda, S. Love, and D. Wilson, Methods in Enzymology 1997, 289 14 [98BMCL775] J. V. Duncia, J. B. Santella III, C. A. Higley, M. K. Vanatt en, P. C. Weber, R. S. Alexander, C. A. Kettner, J. R. Pruitt, A. Y. Liauw, M. L. Quan, R. M. Knabb, and R. R. Wexler, Bioorg. Med. Chem. Lett. 1998, 8 775. [98CR409] A. R. Katritzky, X. Lan, J. Z. Yang, and O. V. Denisko, Chem. Rev. 1998, 98 409. [98C SR213] C. Gabriel, S. Gabriel, E. H. Grant, S. J. Halstead, D. P. Michael, E. H. Grant, and B. S. J. Halstead, Chem. Soc. Rev. 1998, 27 213. [98JOC8397] S. Aoyagi, R. Tanaka, M. Naruse, and C. Kibayashi, J. Org. Chem. 1998, 63 8397.

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119 [98JPR303] J. A. Ca marero, G. J. Cotton, A. Adeva, and T. W. Muir, J. Peptide. Res., 1998, 51 303 [98T1317] P. F. Vogt and M. J. Miller, Tetrahedron 1998, 54 1317 [98TL7009] S. Zhao, T. Gan, P. Yu, and J. M. Cook, Tetrahedron Lett. 1998, 39 7009 [98TL7983] L. E Iwers, A. R. Dunstan, H. peter Weber, G. Rihs, H. Widmer, and E. K. Dzladulewlcz, Tetrahedron Lett. 1998, 39 7983. [99BPA1411] M. N. Hughes, Biochim. Biophys. Acta 1999, 263 1411. [99JACS2147] S. Edmondson, S. J. Danishefsky, L. Sepp lorenzino, and N Rosen, J. Am. Chem. Soc. 1999, 121 2147 [99QJM1] C. M. B. Edwards, M. A. Cohen, and S. R. Bloom, Q. J. Med. 1999, 92 1. [00BMC2407] J. F. Sanz Cervera, E. M. Stocking, T. Usui, H. Osada, and R. M. Williams, Bioorg. Med. Chem. 2000, 8 2407. [0 0CMC945] V. J. Hruby and P. M. Balse, Curr. Med. Chem. 2000, 7 945. [00CS507] H. Al Clinical Science 2000, 98 507 [00JACS5849] H. Wang, C. Burda, G. Persy, and J. Wirz, J. Am. Chem. Soc. 2000, 122 5849. [00JACS9846] W. Adam an d N. Bottke, J. Am. Chem. Soc. 2000, 122 9846. [00JCS(P1)329] A. Hall, P. D. Bailey, D. C. Rees, M. Rosair, and R. H. Wightman, J. Chem. Soc., Perkin Trans. 1 2000, 329 [00JOC8 069 ] A. R. Katritzky, T. B. Huang, M. V. Voronkov, and P. J. Steel, J. Org. Chem. 2000, 65 8069. [00JOC8 402 ] Y. Hayashi, S. Orikasa, K. Tanaka, K. Kanoh, and Y. Kiso, J. Org. Chem. 2000, 65 8402. [ 00LPS17] J. A. Camarero, A. Adeva, and T. W. Muir, Letters in Peptide Science 2000, 7 17. [00OL1939] B. L. Nilsson, L. L. Kiessli ng, and R. T. Raines, Org. Lett. 2000, 2 1939. [00PR471] A. D. I. Stilo, C. Medana, B. Ferrarotti, L. Gasco, D. Ghigo, A. Bosia, P. A. Martorana, and A. Gasco, Pharm. Res. 2000, 41 469 [00S2007] E. Saxon, and C. R. Bertozzi, Science 2000, 287 2007.

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128 BIOGRAPHICAL SKETCH Lucas K Beagle, born in 1982 i n Beaver County, Pennsylvania, was the first of two children of Timothy C. and Ellen K. Beagle. He graduated with a Bachelor of Science in b iological c ciences from Wright State University in Dayton, Ohio in 2005. In 2007, he enrolled at Youngstown State Un iversity studying under Dr. Peter Norris and Dr. Allen Hunter earning a Master of Science in c hemistry in 2008. Immediately following he enrolled at the University of Florida, joining the Florida Center for Heterocyclic Compounds in 2010 under the directio n of Prof Alan R. Katritzky. Lucas received his Ph. D. from the University of Florida in the summer of 2012, and accepted an assistant professor position in the Department of Chemistry at the University of Georgia in August 2012. includes benzotriazole activation methodology, cyclic peptide synthesis, and novel stabilized nitroso compounds.