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Synthesis and Nmr Studies of Heterocyclic Systems

Permanent Link: http://ufdc.ufl.edu/UFE0042344/00001

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Title: Synthesis and Nmr Studies of Heterocyclic Systems
Physical Description: 1 online resource (174 p.)
Language: english
Creator: Elgendy, Bahaa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 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: SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS The theme of this work is to develop new synthetic routes to organic compounds of biological interest and to apply different NMR techniques in their structural analysis. Chapter 1 provides a general introduction to the subsequent chapters and an introduction to the methods used throughout the thesis. Chapter 2 describes a new microwave assisted synthesis of enantiomerically pure amides containing wide variety of heterocyclic systems. In this novel methodology, N-acylbenzotriazoles, N-(protected ?-aminoacyl)benzotriazoles and N-(protected dipeptidoyl)benzotriazoles were reacted under microwave irradiation with various heterocyclic amines including weakly nucleophilic examples and enantiopure amides were synthesized in high yields. Chapter 3 describes the synthesis of 2-hydrazinoquinazoline derivatives, a group of compounds of intense current interest in the development of commercial drugs for analgesic and anti-inflammatory activity. The tautomeric equilibria of these compounds were studied by NMR techniques and the compounds were found to exist predominantly in the imino form in DMSO solution, following the tautomeric preferences of the aminoguanidines. The synthesis of N-(?-aminoalkyl)tetrazoles is described in Chapter 4 and their tautomeric behavior studied by NMR. Thermodynamic and kinetic parameters of the equilibrium are calculated and the detailed mechanism of interconversion between the two tautomers is reported. In Chapter 5, the conformational equilibria and barriers to rotation in some novel nitroso derivatives of indolizines and 3- and 5- azaindolizines were studied by NMR. 13C NMR substituent chemical shifts are used to differentiate the monomers from the dimers. Molecular modeling is used for interpretation of the conformational preferences in the monomers. In Chapter 6, full 1H, 13C, and 15N chemical shift assignments of a series of pyridazines is reported. Long range 1H-15N NMR correlation experiments were used to identify the site of N-oxidation and to determine the site of N-alkylation in some of these pyridazines. In Chapter 7, diverse applications of 15N NMR spectroscopy in structural analysis are described. 1H, 13C, and 15N NMR chemical shifts of some N- and S-acylcysteines and some pH sensitive GFP chromophore analogous are reported. Long range 1H-15N NMR correlation spectra are used to differentiate between the N- and S-acylcysteines and to study protonation.in imidazolinone derivatives. A summary of achievements together with conclusions are presented in Chapter 8.
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 Bahaa Elgendy.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042344:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042344/00001

Material Information

Title: Synthesis and Nmr Studies of Heterocyclic Systems
Physical Description: 1 online resource (174 p.)
Language: english
Creator: Elgendy, Bahaa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 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: SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS The theme of this work is to develop new synthetic routes to organic compounds of biological interest and to apply different NMR techniques in their structural analysis. Chapter 1 provides a general introduction to the subsequent chapters and an introduction to the methods used throughout the thesis. Chapter 2 describes a new microwave assisted synthesis of enantiomerically pure amides containing wide variety of heterocyclic systems. In this novel methodology, N-acylbenzotriazoles, N-(protected ?-aminoacyl)benzotriazoles and N-(protected dipeptidoyl)benzotriazoles were reacted under microwave irradiation with various heterocyclic amines including weakly nucleophilic examples and enantiopure amides were synthesized in high yields. Chapter 3 describes the synthesis of 2-hydrazinoquinazoline derivatives, a group of compounds of intense current interest in the development of commercial drugs for analgesic and anti-inflammatory activity. The tautomeric equilibria of these compounds were studied by NMR techniques and the compounds were found to exist predominantly in the imino form in DMSO solution, following the tautomeric preferences of the aminoguanidines. The synthesis of N-(?-aminoalkyl)tetrazoles is described in Chapter 4 and their tautomeric behavior studied by NMR. Thermodynamic and kinetic parameters of the equilibrium are calculated and the detailed mechanism of interconversion between the two tautomers is reported. In Chapter 5, the conformational equilibria and barriers to rotation in some novel nitroso derivatives of indolizines and 3- and 5- azaindolizines were studied by NMR. 13C NMR substituent chemical shifts are used to differentiate the monomers from the dimers. Molecular modeling is used for interpretation of the conformational preferences in the monomers. In Chapter 6, full 1H, 13C, and 15N chemical shift assignments of a series of pyridazines is reported. Long range 1H-15N NMR correlation experiments were used to identify the site of N-oxidation and to determine the site of N-alkylation in some of these pyridazines. In Chapter 7, diverse applications of 15N NMR spectroscopy in structural analysis are described. 1H, 13C, and 15N NMR chemical shifts of some N- and S-acylcysteines and some pH sensitive GFP chromophore analogous are reported. Long range 1H-15N NMR correlation spectra are used to differentiate between the N- and S-acylcysteines and to study protonation.in imidazolinone derivatives. A summary of achievements together with conclusions are presented in Chapter 8.
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 Bahaa Elgendy.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Katritzky, Alan R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042344:00001


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1 SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS By BAHAA EL DIEN M. EL GENDY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Bahaa El Dien M. El Gendy

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3 To my mother Ms. Fatema Al Atrash, my father Dr. Mostafa El Gendy, and my brothers Malek, Ahmed, Zeyad, Seif El Islam, Elias and finally, to my wonderful wife Lamees Hegazy and my beautiful kids Albaraa and Darine.

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4 ACKNOWLEDGMENTS I ultimately thank my Lord for carrying me through this journey. I am heartily thankful to my supervisor, Prof. Alan R. Katritzky, whose encouragement, guidance and support from the initial to the final level enabled me to accomplish this work. This thesis would have not been possible without the help and support of Dr. Ion Ghiviriga who taught me the NMR and held his door open to me all times. I am grateful to my committee members (Dr. Ion Ghiviriga, Dr. Margret James, Dr. Ronald Castellano, and Dr. Sukown Hong) for their continuous assistance and support. I owe my deepest gratitude to Dr. C. Dennis Hall for the incredible help and support I received from him throughout the years and during the preparation of this thesis. I am grateful to my friend Henry M artinez for carrying out the computational study in Chapter 5 of this thesis. I would like to thank former and current members of the Katritzky group, and professors of the Chemistry Department especially Dr. Ben Smith. I am indebted to my master advisor, the late Prof. Samy A. Essawy. Without his help and encouragements; it would have been very difficult to me to achieve my goals. Very special thanks go to Prof. Mohamed N. Mosaad and my friends at Benha University. I can not express my gratitude to my pare nts and my brothers. Their love, prayers and support have meant the world to me. I have been blessed with a wonderful wife, Lamees, and beautiful kids Albaraa and Darine ; we share in this accomplishment.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF SCHEMES ................................ ................................ ................................ ...................... 13 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 14 ABSTRACT ................................ ................................ ................................ ................................ ... 18 CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .............................. 20 2 ( AMINOACYL)AMINO SUBSTITUTED HETEROCYCLES AND RELATED COMPOUNDS ................................ ................................ ................................ ....................... 23 2.1 Introduction ................................ ................................ ................................ ....................... 23 2.2 Results and Discussion ................................ ................................ ................................ ..... 25 2.2.1 Preparation of Acylamino Thiazoles, Benzothiazoles, Benzimidazoles and Thiadiazoles ................................ ................................ ................................ ................. 25 2.2.2 Acylation of Aminopyrimidones ................................ ................................ ............ 28 2.2.3 Acylation of Aminopyrazoles ................................ ................................ ................. 28 2.2.4 Acylation of Aminopyridines ................................ ................................ ................. 29 2.3 Conclusions ................................ ................................ ................................ ....................... 32 2.4 Experimental ................................ ................................ ................................ ..................... 32 2.4.1 General Methods ................................ ................................ ................................ .... 32 2.4.2 General Procedure for the Preparation of 2.6a j, 2.6a' c' ................................ ....... 32 2.4.3 General Procedure for the Preparation of N Substituted Amides 2.8a d, 2.8a' c', 2.14a b, 2.14a', 2.16a b, 2.18a g, 2.18d' and Dipeptide Amides 2.12a,b ................ 32 3 TAUTOMERISM OF 2 HYDRAZONO 3 PHENYLQ UINAZOLIN 4(3 H ) ONES STUDIED BY 15 N NMR ................................ ................................ ................................ ........ 41 3.1 Introduction ................................ ................................ ................................ ....................... 41 3.2 Results and Discussion ................................ ................................ ................................ ..... 46 3.2.1 Syntheses ................................ ................................ ................................ ................ 46 3.2.2 Tautomerism and NMR ................................ ................................ .......................... 48 3.2.3 Stereochemistry of the C=N Bonds ................................ ................................ ........ 57 3.3 Conclusions ................................ ................................ ................................ ....................... 58 3.4 Experimental ................................ ................................ ................................ ..................... 59 3.4.1 General Methods ................................ ................................ ................................ .... 59 3.4.2 Preparation of 3 phenyl 2 thioxo 2,3 dihydroquinazolin 4(1 H ) one (3.21) .......... 60 3.4.3 Preparation of 2 hydrazino 3 phenylquinazolin 4(3 H ) one (3.22) ........................ 60 3.4.4 General Procedure for Preparing Compounds 3.11a i ................................ ........... 61 3.4.5 Preparation of 4 [(2 (1 H benzimidazol 2 yl )hydrazono)methyl] N,N dimethylaniline (3.12) ................................ ................................ ................................ .. 64

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6 3.4.6 Preparation of 3 [2 (4,6 dimethylpyrimidin 2 yl)hydrazono] 1 methylindolin 2 one (3.13) ................................ ................................ ................................ .................. 64 3.4.7 Preparation of 2 (methylsulfanyl) 3 phenyl 4(3 H ) quinazolinone (3.24) ............. 65 3.4.8 Preparation of 3 amino 2 anilino 4(3 H ) quinazolinone (3.25) .............................. 65 4 NMR STUDY OF THE TAUTOMERIC BEHAVIOUR OF N ( AMINOALKYL) TETRAZOLES ................................ ................................ ................................ ....................... 66 4.1 Introduction ................................ ................................ ................................ ....................... 66 4.2 Results and Discussion ................................ ................................ ................................ ..... 69 4.2. 1 Preparation of 5 Substituted Tetrazoles ................................ ................................ 69 4.2.2 Preparation of N Aminoalkyl)Tetrazoles ................................ .......................... 70 4.2.3 NMR Characterization and Solvent Effects on Tautomeric Equilibrium of 1 and 2 Substituted Tetrazoles ................................ ................................ ........................ 70 4.2.4 Thermodynamic and Kinetic Parameters ................................ ............................... 75 4.2.5 Cross Over Experiment ................................ ................................ .......................... 78 4.3 Conclusions ................................ ................................ ................................ ....................... 81 4.4 Experimental ................................ ................................ ................................ ..................... 81 4.4.1 General Methods ................................ ................................ ................................ .... 81 4.4.2 General Procedure for P reparation of Compound 6.4b (Method A) ..................... 82 4.4.3 General Procedure for P reparation of Compounds 4.9c and 4.9e (Method B) ..... 83 4.4.4 General Procedure for Preparation of Compounds 4.11a, 4.11c h ........................ 84 4.4.5 Preparation of N hydroxymethylsaccharin (4.12). ................................ ................. 88 4.4.6 Preparation of N chloromethylsaccharin (4.13). ................................ .................... 88 4.4.7 Preparation of N ((1 H tetrazol 1 yl)methyl) 1,2 benzisothiazole 3(2 H ) one 1,1 dioxide (4.11b). ................................ ................................ ................................ ...... 88 5 CONFORMATIONAL EQUILIBRIA AND BARRIERS TO ROTATION IN SOME NOVEL NITROSO DERIVATIVES OF INDOLIZI NES AND 3 AND 5 AZAINDOLIZINES AN NMR AND MOLECULAR MODELLING STUDY ................. 90 5.1 Introduction ................................ ................................ ................................ ....................... 90 5.2 Results and Disscussion ................................ ................................ ................................ .... 93 5. 2.1 NMR Spectroscopy ................................ ................................ ................................ 93 5.2.1.1 2,3 Dimethyl 1 nitrosoindolizine (5.1) ................................ ........................ 93 5.2.1.2 2,6,7 Trimethyl 5 nitrosopyrrolo[1,2 b ]pyridazine (5.5) ............................ 95 5.2.1.3 Methyl 2 methyl 1 nitrosoindolizine 3 carboxylate (5.2) ........................... 96 5.2.1.4 Ethyl 2 (methylamino) 1 nitrosoindolizine 3 carboxylate (5.3) ................. 97 5.2.1.5 4 Methoxy 3 nitrosopyrazolo[1,5 a ]pyridine (5.6) ................................ ... 100 5.2.1.6 2 Methyl 3 nitrosoindolizine (5.4) ................................ ............................. 101 5.2.2 15 N Chemical Shifts ................................ ................................ .............................. 102 5.2.3 Barriers to Rotation ................................ ................................ .............................. 103 5.2.4 Molecular Modelling ................................ ................................ ............................ 103 5.3 Conclusions ................................ ................................ ................................ ..................... 112 5.4 Experimental ................................ ................................ ................................ ................... 114 5.4.1 General Methods ................................ ................................ ................................ .. 114 5.4.2 Characterization of Compounds 5.1 5.6 ................................ ............................... 115

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7 6 1 H, 13 C, AND 15 N NMR SPECTRA OF SOME PYRIDAZINE DERIVATIVES .............. 116 6.1 Introduction ................................ ................................ ................................ ..................... 116 6.2 Results and Discussion ................................ ................................ ................................ ... 118 6.2.1 1 H NMR Spectra ................................ ................................ ................................ ... 118 6.2.2 13 C NMR Spectra ................................ ................................ ................................ .. 119 6.2.3 15 N NMR Spectra ................................ ................................ ................................ 119 6.3 Conclusions ................................ ................................ ................................ ..................... 125 6.4 Experimental ................................ ................................ ................................ ................... 126 6.4.1 General Methods ................................ ................................ ................................ .. 126 6.4.2 Characterization of Compounds 6.7 6.20 ................................ ............................. 127 7 DIVERSE APPLICATIONS OF 15 N NMR IN STRUCTURAL ANALYSIS .................... 129 7.1 Introduction ................................ ................................ ................................ ..................... 129 7.1.1 Literature Background to Structural Elucidation by 15 N NMR ............................ 130 7.1.2 Literature Background to Protonation Studies by 15 N NMR ................................ 131 7.2 Results and Discussion ................................ ................................ ................................ ... 135 7.2.1 Structural Elucidation of S and N Acylcysteines ................................ ................ 135 7.2.1.1 Structural elucidation of S (4 methoxybenzoyl) L cysteine (7.13a) .......... 135 7.2.1.2 Structural elucidation of S (2 naphthoyl) L cysteine (7.13b) .................... 138 7.2.1.3 Structural elucidation of N (4 methoxybenzoyl) L cysteine (7.14a) ......... 138 7.2.1.4 Structural elucidation of N (2 naphthoyl) L cysteine (7.14b) .................... 140 7.2.2 1 H, 13 C, and 15 N NMR Chemical Shift Assignments and Protonation Study of pH Sensitive GFP Chromophore Analogues ................................ ............................. 141 7.2.2.1 1 H, 13 C, and 15 N NMR of 2 phenyl 4 (thiophen 2 ylmethylene)oxazol 5(4 H ) one (7.16a) ................................ ................................ ............................... 1 43 7.2.2.2 1 H, 13 C, and 15 N NMR and protonation study of 1 {2 (dimethylamino)ethyl) 4 (5 methylfuran 2 yl)methylene} 2 phenyl 1 H imidazol 5(4 H ) one (7.17a) ................................ ................................ ................ 143 7.2.2.3 1 H, 13 C, and 15 N NMR and protonation study of 4 {(1 H pyrrol 2 yl) methylene} 1 (2 dimethylaminoethyl)} 2 phenyl 1 H imidazol 5(4 H ) one (7.17b) ................................ ................................ ................................ ................. 145 7.3 Conclusions ................................ ................................ ................................ ..................... 146 7.4 Experimental ................................ ................................ ................................ ................... 148 7.4.1 General Methods ................................ ................................ ................................ .. 148 7.4.2 Characterization of Compounds 7.13a,b, 7.14a,b, 7.16a. and 7.17a.b ................. 149 8 CONCLUSIONS AND SUMMARY OF ACHIEVEMENTS ................................ ............. 152 LIST OF REFERENCES ................................ ................................ ................................ ............. 155 BIOGRAPHIC AL SKETCH ................................ ................................ ................................ ....... 174

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8 LIST OF TABLES Table page 2 1 Preparation of acylamino thiazoles, benzothiazoles, benzimidazoles and thiadiazoles ................................ ................................ ................................ ........................ 27 2 2 Preparation of (acylamino)pyridines from N acyl and N (aminoacyl) benzotriazoles ...... 31 3 1 1 H chemical shifts (ppm) in compounds 3.11a f 3.12 3.21 3.22 and 3.24 ..................... 54 3 2 13 C chemical shifts (ppm) in compounds 3.11a f 3.12 3.21 3.22 and 3.24 .................... 55 3 3 15 N chemical shifts (ppm) in compounds 3.11a f 3.12 3.21 3.22 and 3.24 Protons which couple to a 15 N are given in parentheses ................................ ................................ 55 3 4 1 H chemical shifts (ppm) in compounds 3.11g i and 3.13 ................................ ................. 56 3 5 13 C chemical shifts (ppm) in compounds 3.11g i 3.13 and 3.29a c ................................ 57 3 6 15 N chemical shifts (ppm) in compounds 3.11g i and 3.13 Protons which couple to a 15 N are given in parentheses ................................ ................................ .............................. 58 4 1 Synthesis of Tetrazoles 4.9b,c,e ................................ ................................ ........................ 69 4 2 Synthesis of N Aminoalkyl)tetrazoles 4.11a h ................................ ............................. 70 4 3 Percentage of N1 Isomer Observed in Different Solvents ................................ ................. 72 4 4 1 H, 13 C, and 15 N Chemical shift assignments in 4.11b,c ................................ .................... 73 4 5 Coalescence Temperatures (T c ), Equilibrium Constants (K), Chemical Shift v ), and Natural Logarithm of Exchange Rate Constant (k r ) from the 1 H and 13 C NMR Spectra of 4.11c (Acetonitrile d 3 as Solvent) ................................ ................................ ................................ .............................. 77 4 6 Signal Assignments and Molar Percentage Ratios of Compounds 4.11a 4.11d 4.11e and 4.11i in Cross Over Experiment ................................ ................................ ....... 80 5 1 1 H chemical shifts in compounds 5. 1 5. 9 ................................ ................................ .......... 95 5 2 13 C chemical shifts in compounds 5.1 5. 9 ................................ ................................ ......... 98 5 3 1 H chemical shifts in compounds 5.1 5.7 and 5.9 ................................ ............................ 102 5 4 Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5. 1 ................................ ................................ ................................ ............. 105 5 5 Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5.5 ................................ ................................ ................................ ............. 106

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9 5 6 Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5.2 ................................ ................................ ................................ ............. 106 5 7 Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5. 3 ................................ ................................ ................................ ............. 108 5 8 Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5. 6 ................................ ................................ ................................ ............. 109 5 9 Calculated energy differences (kcal/mol) and molar fractions, at 25 C, for conformers of 5. 4 ................................ ................................ ................................ ............. 110 5 10 Calculated energy differences (kcal/mol) for conformers of model compounds 5. 10 5. 12 ................................ ................................ ................................ ................................ ... 111 5 11 (trans cis ) (kcal/mol) for the donor acceptor interaction of interest in model compounds 5. 10 5. 12 ................................ ................... 111 5 12 Distances () for some bonds at the lowest (0) and highest point of the rotation (90) in model compounds 5. 10 5. 12 ................................ ................................ .............. 111 5 13 Electron Donor Acceptor interactions and N4 occupancy at the lowest (0) and highest point of the rotation (90) in model compounds 5. 10 5. 12 ................................ 112 6 1 1 H NMR (ppm) chemical shifts (DMSO d6 ) ................................ ................................ ... 122 6 2 13 C NMR (ppm) chemical shifts (DMSO d6 ) ................................ ................................ .. 123 6 3 15 N NMR (ppm) chemical shifts (DMSO d6 ) ................................ ................................ 125 7 1 1 H, 13 C, 15 N NMR chemical shifts of 7.16a 7.17a and 7.17b ................................ ........ 144

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10 LIST OF FIGURES Figure page 2 1 Biologically active ( aminoacyl)amino substituted heterocycles ................................ ... 23 3 1 Dominant tautomeric forms of amino hydroxy mercapto and methyl pyridines ....... 41 3 2 Compounds investigated in this thesis ................................ ................................ ............... 42 3 3 Tautomers A and B of 2 hydrazino 3 phenylquinazolin 4(3 H ) ones 3. 11a i and their common cations C and D ................................ ................................ ................................ ... 43 3 4 Tautomers of hydrazinopyrimidin 4(3 H ) ones ................................ ................................ .. 44 3 5 Tautomers of disubstituted guanidines ................................ ................................ .............. 44 3 6 Tautomers of 2 pyrimidinamine and of isocytosine ................................ .......................... 45 3 7 Tautomeric forms of 2 quinolylhydrazones 3.18 ................................ ............................... 46 3 8 Expansions of the 1 H 13 C gHMBC spectrum of compound 3.11b ................................ .... 49 3 9 Expansions of the 1 H 15 N CIGAR spectrum of compound 3.11b ................................ ..... 50 3 10 X ray structure of 3.11b ................................ ................................ ................................ ..... 50 3 11 15 N chemical shifts in related heterocycles from ref.[1995MRC389] ............................... 51 3 12 Isomers/rotamers of compound 3. 13 ................................ ................................ .................. 56 4 1 Some examples of bioactive compounds containing N aminoalkyl)tetrazole scaffolds ................................ ................................ ................................ ............................. 67 4 2 X ray structure of 4.11f ................................ ................................ ................................ ...... 75 4 3 1 H spectra of 4.11c in acetonitrile d3 ........................ 76 4 4 Plot of ln k r vs 1/ T c in case of inter conversion of A B and B A. ............................. 78 4 5 Different isomers expected from crossover experiment between 4.11a and 4.11e ........... 78 4 6 NOESY spectrum of crossover experiment between 4.11a and 4.11e in toluene d8 at ................................ ................................ ................................ ................................ 80 5 1 C Nitroso derivatives of indolizines and 3 and 5 azaindolizines taken into study .......... 90 5 2 Monomer dimer equilibria in heteroaromatic nitroso compounds ................................ .... 90 5 3 Rotamer equilibrium in 5. 1 ................................ ................................ ................................ 91

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11 5 4 Possible conformations for 5. 3 relevant proton chemical shifts, and nOes ...................... 99 5 5 Expansion of the NOESY spectrum of 5. 3 ................................ ................................ ...... 100 5 6 Parent compounds for 5.5 and 5.6 ................................ ................................ ................... 101 5 7 Parent compound of 5.4 ................................ ................................ ................................ ... 102 5 8 Conformers of 5.1 ................................ ................................ ................................ ............ 105 5 9 Conformers of 5.5 ................................ ................................ ................................ ............ 105 5 10 Conformers of 5.2 ................................ ................................ ................................ ............ 106 5 11 Conformers of 5.3 ................................ ................................ ................................ ............ 108 5 12 Conformers of 5.6 ................................ ................................ ................................ ............ 109 5 13 Conformers of 5.4 ................................ ................................ ................................ ............ 109 5 14 Model compounds for the s cis vs. s trans conjugation ................................ .................. 110 6 1 Isoxazolo[3,4 d ]pyridazin 7(6 H ) ones 6. 1, tetrazolopyridazines 6.2 and triazolo pyridazines 6. 3 ................................ ................................ ................................ ................. 117 6 2 Compounds investigated in this thesis ................................ ................................ ............. 118 6 3 1 H NMR of 3 diethylamino 1 ethyl 6 iodopyridazin 1 ium iodide (6.17) ..................... 119 6 4 The 1 H 13 C gHMBC spectrum of 6.17 in DMSO d6 ................................ ....................... 120 6 5 The 1 H 15 N CIGAR HMBC spectrum of 6.10 with expansion in DMSO d6 ................. 121 6 6 The 1 H 15 N CIGAR HMBC spectrum of 6.17 with expansion in DMSO d6 ................. 121 7 1 Spiro[pyrrolidine 2,3' oxindoles] (7.5a,b,c) ................................ ................................ .... 131 7 2 Nitrogen chemical shifts of 1 pheny1 3 methyl 5 N benzylidene aminopyrazole (7.6) in CDCl 3 and in TFA d ................................ ................................ ................................ .... 132 7 3 Ethenoadenosine ( 7.7 ) in the neutral form (asterisks are used to show the rotation of the outer imidazole ring with respect to the inner one) ................................ ................... 132 7 4 Equilibrium between [5,6]pinene bpy ( 7.8 ) and its monoprotonated form ( 7.8H + ) ........ 133 7 5 [4,5]CHIRAGEN[0] (7.9 ) AND [4,5]CHIRAGEN[0] ( 7.10 ) ................................ .......... 134 7 6 1 H, 13 C, and 15 N chemical shifts of 7.13a ................................ ................................ ........ 136

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12 7 7 1 H 1 H gDQCOSY spectrum of c ompound 7 .13a ................................ ............................ 136 7 8 1 H 13 C gHMBC spectrum of 7.13a ................................ ................................ .................. 137 7 9 1 H 15 N CIGAR gHMBC spectrum of 7.13a ................................ ................................ .... 137 7 10 1 H, 13 C, and 15 N chemical shifts of 7.13b ................................ ................................ ........ 138 7 11 1 H, 13 C, and 15 N chemical shift assingments of 7.14a ................................ ..................... 139 7 12 1 H 13 C gHMBC spectrum of 7.14a ................................ ................................ .................. 139 7 13 1 H 15 N CIGAR gHMBC spectrum of 7.14a with expansion ................................ ........... 140 7 14 1 H, 13 C, and 15 N chemical shift assignments of 7.14b ................................ ..................... 140 7 15 1 H 13 C gHMBC spectrum of 7.14b ................................ ................................ .................. 141 7 16 1 H 15 N CIGAR gHMBC spectrum of 7.14b with expansion ................................ .......... 142 7 17 Compounds 7.16a 7.17a and 7.17b with numbering ................................ ..................... 143 7 18 1 H 15 N CIGAR gHMBC spectrum of 7.17a in CDCl 3 ................................ .................... 145 7 19 1 H 15 N CIGAR gHMBC spectrum of 7.17a in TFA d ................................ .................... 14 5 7 20 1 H 15 N CIGAR gHMBC spectrum of 7.17b in CDCl 3 with expansion ........................... 147 7 21 1 H 15 N CIGAR gHMBC spectrum of 7.17b in TFA d ................................ .................... 147 7 22 Expansions of 1 H 15 N CIGAR gHMBC spectrum of 7.17b in TFA d ............................ 148

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13 LIST OF SCHEMES Scheme page 2 1 Preparation of N substituted amides 2.8a d and 2.8a' c' ................................ .................. 25 2 2 Preparation of dipeptidoyl amides 2.12a,b ................................ ................................ ........ 28 2 3 Preparation of (acylamino)pyrimidones 2.14a,b and 2.14a' ................................ ............. 28 2 4 Preparation of (acylamino)pyrazoles 2.16a,b ................................ ................................ .... 29 2 5 Preparation of acylamino pyridines 2.18a g and 2.18d' ................................ .................... 30 3 1 Reagents and conditions: a) EtOH, reflux 2 h ; b) n BuOH, N 2 H 4 .H 2 O, reflux 2 h .......... 47 3 2 Reagents and conditions: a) (CH 3 O) 2 SO 2 2 % Ethanolic sodium hydroxide, rt, 1 h ; b) N 2 H 4 .H 2 O, EtOH, reflux, 20 h .; c) EtOH, 3. 23d reflux, 7 h ................................ ....... 48 4 1 N 1 to N 2 Substituent isomerization of ( N N disubstituted aminomethyl) benzotriazoles ................................ ................................ ................................ .................... 68 4 2 (i) N1 N2 and N4 Substituted isomers of N aminoalkyl) 1,2,4 triazoles. (ii) N1 and N2 Substituted isomers on N aminoalkyl) 1,2,3 triazoles .............................. 69 4 3 N 1 to N 2 Substituent isomerization of 4.11c ................................ ................................ .. 77 7 1 ................................ ................................ ................................ ................................ .......... 130 7 2 Selective synthesis of S and N acyl L cysteines 7.8a,b and 7.9a,b ................................ 135 7 3 Synthesis of compounds 7.16a 7.17a and 7.17b ................................ ........................... 142 7 4 ................................ ................................ ................................ ................................ .......... 146

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14 LIST OF ABBREVIATION S Alpha locant D Specific rotation Ala Alanine Ar Aryl Beta locant Bn Benzyl Boc t Butoxycarbonyl BOP Benzotriazole 1 yl oxy tris (dimethylamino) phosphonium hexafluorophosphate br Broad Bt Benzotriazol 1 yl C Carbon Degree Celcius Calcd Calculated Cbz Carbobenzyloxy CDCl 3 Deuterated chloroform CIGAR HMBC Constant time inverse detection gradient accordi on rescaled heteronuclear multiple bond correlation spectroscopy (NMR technique) Cys Cysteine Chemical shift in parts per million downfield from tetramethylsilane d Douplet D Dextrorotatory (right) DCC N N' Dicyclohexylcarbodiimide DCM Dichloromethane DMF Dimethylformamide

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15 DMSO Dimethylsulfoxide D 2 O Deuterium oxide EDC 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide (stands as an abbreviation for EDAC and EDCI as well) Et Ethyl et al. And others Et 3 N Triethylamine EtOAc Ethyl acetate g Gram(s) gDQCOSY Gradient d ouble q uantum c orrelation s pectroscopy (NMR technique) GFP Green fluorescent protein gHMQC Gradient heteronuclear multiple quantum coherence (NMR technique) gHMBC Gradient heteronuclear multiple bond coherence (NMR technique) Gly Glycine h Hour H Hydrogen HBTU O Benzotriazole N,N,N',N' tetramethyl uronium hexafluoro phosphate HOBt 1 Hydroxybenzotriazole HPLC High performance liquid chromatography HRMS High resolution mass spectrometry Hz Hertz IR Infrared J Coupling constant L (10 point) Levorotat ory (left) Lit Literature m Multiplet

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16 M Molar Me Methyl MeCN Acetonitrile MeOH Methanol Met Methionine min Minute(s) MgSO 4 Magnesium sulfate mol Mole(s) mp Melting point MW Microwave m/z Mass to charge ratio N Nitrogen Na 2 CO 3 Sodium carbonate NaOH Sodium hydroxide NMR Nuclear magnetic resonance NOE Nuclear Overhauser effect NOESY Nuclear Overhauser effect spectroscopy o Ortho locant O Oxygen OEt Ethoxy OH Hydroxyl group OMe methoxy p Para locant Ph Phenyl Phe Phenylalanine

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17 ppm Part per million Pro Proline Py Pyridine q Quartet R Rectus (right) ref. Reference rt Room temperature s Singlet S Siister (left) SCS Substituent chemical shift SOCl 2 Thionyl chloride t Triplet t Tertiary TFA Trifluoroacetic acid TLC Thin layer chromatography TMS Trimethylsilane TOCSY Total Correlation Spectroscopy (NMR technique) Trp Tryptophan Val Valine W Watt(s)

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18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS By Bahaa El Dien M. El Gendy December 2010 Chair: Alan R. Katritzky Major: Chemistry The theme of this work is to develop new synthetic routes to organic compounds of biological interest and to apply different NMR techniques in their structural analysis. Chapter 1 provides a general introduction to the subsequent chapters and an introduction to the methods used throughout the thesis. Chapter 2 describes a new microwave assisted synthesis o f enantiomerically pure amides containing wide variety of heterocyclic systems. In this novel methodology, N acylbenzotriazoles, N aminoacyl)benzotriazoles and N (protected dipeptidoyl)benzotriazoles were reacted under microwave irradiation wi th various heterocyclic amines including weakly nucleophilic examples and enantiopure amides were synthesized in high yields. Chapter 3 describes the synthesis of 2 hydrazinoquinazoline derivatives, a group of compounds of intense current interest in the d evelopment of commercial drugs for analgesic and anti inflammatory activity. The tautomeric equilibria of these compounds were studied by NMR techniques and the compounds were found to exist predominantly in the imino form in DMSO solution, following the t automeric preferences of the aminoguanidines.

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19 The synthesis of N aminoalkyl)tetrazoles is described in Chapter 4 and their tautomeric behavior studied by NMR. Thermodynamic and kinetic parameters of the equilibrium are calculated and the detailed mechan ism of interconversion between the two tautomers is reported. In Chapter 5, the conformational equilibria and barriers to rotation in some novel nitroso derivatives of indolizines and 3 and 5 azaindolizines were studied by NMR. 13 C NMR substituent chemic al shifts are used to differentiate the monomers from the dimers. Molecular modeling is used for interpretation of the conformational preferences in the monomers. In Chapter 6, full 1 H, 13 C, and 15 N chemical shift assignments of a series of pyridazines is reported. Long range 1 H 15 N NMR correlation experiments were used to identify the site of N oxidation and to determine the site of N alkylation in some of these pyridazines. In Chapter 7, diverse applications of 15 N NMR spectroscopy in structural analysis are described. 1 H, 13 C, and 15 N NMR chemical shifts of some N and S acylcysteines and some pH sensitive GFP chromophore analogous are reported. Long range 1 H 15 N NMR correlation spectra are used to differentiate between the N and S acylcysteines and to study protonation.in imidazolinone derivatives. A summary of achievements together with conclusions are presented in Chapter 8.

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20 CHAPTER 1 GENERAL INTRODUCTION N Acylbenzotriazoles are powerful acylating reagents and can efficiently acylate proton labile compounds such as amines, alcohols, and acids. N Acetylbenzotriazoles have been widely used for protein acetylation and have advantage over other co mmon acetylating reagents such as acetic anhydride or acetylimidazole [1976EJB25]. Major advantages of N acylbenzotriazoles include: (i) relative stability to hydrolysis. (ii) they are crystalline and easy to prepare, handle, and store [2009SL2392]. Chapte r 1 describes reactions of N acylbenzotriazoles, N aminoacyl)benzotriazoles and N (protected dipeptidoyl)benzotriazoles under microwave irradiation with various heterocyclic amines including weakly nucleophilic one s in order to synthesize enantiopure amides in high yields. Nuclear magnetic resonance (NMR) is a powerful method for structure elucidation, as it reveals correlations through bonds (based on scalar couplings) and correlations through space (based on dipolar couplings). In many cases, this information alone can reveal the structure of a compound. The use of NMR in organic chemistry is ubiquitous, from the very first steps in a synthesis to the characterization and confirmation of the structure of a final product. The hy drocarbon skeleton of a compound is revealed step by step through the long range (two or three bonds) couplings between protons and carbons, seen in experiments of the HMBC type. In heterocycles the heteroatoms often break this network of couplings, leavin g many structural possibilities. When the heteroatom is nitrogen, it is possible to use the proton nitrogen correlations to elucidate the structure. With the advent of indirect detection and gradients, such correlations can be obtained at natural abundance in a couple of hours using 10 30 mg of sample. NMR is appropriate for investigating chemical equilibria, since it does not interfere with the reaction. The advantage of NMR is that species in exchange can be identified by the

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21 correlations mentioned above. Heterocyclic compounds sometimes display tautomeric equilibria, and the identification of tautomers is, in most cases, not trivial. The proton involved in the exchange usually displays a broad line, which precludes the observation of its cross peaks. Even when the NMR spectra display the signals of one compound, one has to consider the case of more tautomers in rapid exchange. Methods to determine the position of the tautomeric equilibrium of species in fast exchange include the analysis of 13 C chemical sh ifts and deuterium induced shifts. In Chapter 3, the tautomers of some 2 hydroaz i noquinazolines were identified based on the chemical shifts of the nitrogens which may or may not be protonated in the two tautomeric forms. The method is applicable to cases where the proton responsible for the tautomerism displays a broad line, since the chemical shifts of nitrogen are revealed by correlations to other protons. We also present a case where the proton nitrogen correlations were necessary to discriminate betwe en isomers from the same reaction In Chapter 4, N aminoalkyl)tetrazoles were synthesized and their tautomeric behavior studied by NMR. The equilibrium between N 1 and N 2 tautomers in solvents of different polarity is described. Thermodynamic and kinet ic parameters of this equilibrium are calculated and the detailed mechanism of interconversion between the two tautomers is reported. In Chapter 5, the conformational equilibria and barriers to rotation in some novel nitroso derivatives of indolizines and 3 and 5 azaindolizines were studied by NMR. 13 C NMR substituent chemical shifts are used to differentiate the monomers from the dimers. Molecular modeling is used for interpretation of the conformational preferences in the monomers.

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22 In Chapter 6, a serie s of pyridazines is reported. Long range 1 H 15 N NMR correlation experiments were used to identify the site of N oxidation and to determine the site of N alkylation in some of these pyridazines. In Chapter 7, diverse applications of 15 N NMR spectroscopy in structural analysis are described. 1 H, 13 C, and 15 N NMR chemical shifts of some N and S acylcysteines and some pH sensitive GFP chromophore analogous are reported. Long range 1 H 15 N NMR correlation spectra are used to differentiate between the N and S ac ylcysteines and to study protonation in some imidazolinone derivatives. Chapter 8 presents a summary of achievements together with conclusions.

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23 CHAPTER 2 ( AMINOACYL)AMINO SUBSTITUTED HETEROCY CLES AND RELATED COMPOUNDS 1 2.1 Introduction N Substituted heterocycles show anti inflammatory [ 19 98 WOP9822475 ] antiproliferative [ 200 06 US P009457 ] antithrombotic [ 19 98 USP5780590 ] antifungal [ 19 82F A 450] and antineurological biological activities [ 20 07 USP066613 ] Such units occur in diverse pharmacologically active molecules including cell adhesion inhibitors [ 2001WOP012183 ] platelet activating factor (RAF) or angiotensin II antagonists [ 1993WOP9314069 ] mitogen activated protein (MAP) kinase [ 2003WOP035638 ] and mitotic kinesin KSP inhibitors [2003WOP103575]. ( Aminoacyl)amino substituted heterocycles are useful synthetic intermediates (Figure 2 1) for endomorphin 2 (EM 2) analogues ( 2. 1 ) [ 20 0 4 JM E 3591] bacterial RND efflux pump inhibitors (EPIs) such as MC 04,124 ( 2 .2 ) [ 20 03BM C 4241] and MC 02,595 ( 2. 3 ) [ 20 03BMC2755] secretase inhibitor LY411575 ( 2. 4 ) [ 20 04TL2323] and inhibitors of tumor necrosis factor 2. 5 ) [2001JME4252] Figure 2 1. Biologically active ( aminoacyl)amino substituted heterocycles 1 Reproduced in part with permission from The Journal of Organic Chemistry 2008 73 5442 5445 Copyright 2008 American Chemical Society

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24 N Acylbenzotriazoles [ 20 05S L 1656] have been employed for: (i) N acylation [ 20 07S3673] in the preparation of primary, secondary, tertiary [ 20 00JOC8210] and Weinreb amides [ 20 02ARK39] ; (ii) C acylation for the preparation of ketosulfones [ 20 03JOC1443] primary and secondary cyanonitril es [ 20 03JOC4932] nitroketones [ 20 05JOC9211] ketones [ 20 06JOC9861] and ketoazines [ 20 05ARK329] ; and (iii) O acylation of aldehydes [ 19 99JHC777] and of steroids [ 2006ST660 ] to give esters. N (Boc aminoacyl)benzotriazoles and chiral amines give N (Boc amino)amides with no detectable racemization [2002ARK134] Numerous N (protected aminoacyl)benzotriazoles couple with unprotected amino acids in mixed organic/aqueous solution with complete preservation of the original chirality [2007JOC5794, 2004S2645] In continuation of this considerable research in our group, I now report the synthesis of N substituted amides 2. 8 a d, 2. 8 a' c' 2. 9a b, 2. 14 a', 2. 16 a b, 2. 18 a g, 2. 1 8 d' and N protected dipeptidoyl amides 2. 12 a b by treatment of the corresponding N (pro tected aminoacyl)benzotriazoles 2. 6 a e,g,i,j, 2. 6 a' c' N acylbenzotriazoles 2. 6 f,h or N (protected peptidoyl)benzotriazoles 2. 6a b with heterocyclic amines under microwave irradiation. Microwave irradiation is kno wn to accelerate the reaction rates and shorten the reaction times substantially [2002ACR717] This acceleration is mainly due to thermal effects but it could also be due to some microwave effects. The rmal effects are more generally accepted by the scientific community to be the main reason for drastic enhancement of reaction rates by microwave I n microwave organic assisted reactions, heating process is rapid and produces heat profiles that can no t be obtained through conventional heating [2001T9225] The energy transfer to reactants is instantaneous in case of microwave while it takes place via classical conduction in Compound numbers written with primes represent racemates, e.g. 2.6a' c' 2.8a' c' and 2.18d'

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25 conventional heating Moreover, heaing p r o cess is highly controlled, energy efficient, smooth and homogeneous and that is could be the reason why it leads to formation of products of higher purity and higher yields 2.2 R esults and Discussion 2.2.1 Preparation of A cylamino T hiazoles, B enzothiazoles B enzimidazoles and T hiadiazoles T he starting N (protected aminoacyl )benzotriazoles 2. 6 a d, 2. 6 a', 2. 6 b', 2. 6 c' were prepared from N protected amino acids following a published one step procedure [2003S2795, 2006S411] Treatment of 2 aminothiazole ( 2. 7 a ), 2 amino 6 methoxybenzothiazole ( 2. 7 b ), N benzyl 2 aminobenzimidazole ( 2. 7 c ) and 5 amino 3 methoxy 1,2,4, thiadizole ( 2. 7 d ) and N (protected aminoacyl)benzotriazoles 2. 6 a d 2. 6 a' c' under microwave irradiation at 70 C for 30 min (150 min for 2. 7 d with 2. 6 d ) gave the N substituted amides 2. 8 a d and 2. 8 a' c' in 50 98% yields (Scheme 2 1 and Table 2 1). Scheme 2 1. Preparation of N substituted amides 2.8a d and 2 8a' c' The enantiopurity of compounds 2. 8 a c was confirmed by HP LC analysis. As expected, HPLC analysis of enantiopure 2. 8 a c gave a single peak fo r each compound. In contrast, two peaks were observed for the corresponding racemic N substituted heterocycles 2. 8 a' 2. 8 b' and 2. 8 c' (Table 2 1).

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26 As a further application of this synthetic approach, Cbz L Met L Trp OH ( 2. 10 a ) (prepared as reported [2004S2645] by coupling of Cbz L Met Bt ( 2. 6 e ) with unprotected L Ala ( 2. 9 a ) in aqueous acetonitrile) was treated with benzotriazole and SOCl 2 to provide the N (protected dipeptidoyl)benzotriazole Cbz L Met L Trp Bt ( 2. 11 a ). Compound 2. 11 a was reacted with 2. 7 a under microwave irradiation (100 W) in DMF at 70 C for 30 minutes to give dipeptidoyl amide 2. 12 a in 60% yield (Scheme 2 2). D ipeptidoyl amide 2. 12 b was prepared in 52 % by coupling 6 methoxybenzothiazol 2 a mine ( 2. 7 b ) with Cbz L Phe L Ala Bt ( 2. 11 b ) as described above (Scheme 2 2). Scheme 2 2. Preparation of dipeptidoyl amides 2.12a,b aminoacyl derivatives of heterocyclic amines, i.e., carboxamides of type 2.8 Kraus et al. [2004JCO695, 2005OBC612] investigated the coupling reaction between amino acids and weakly nucleophilic heteroar omatic amines including substituted 2 aminothiazole and substituted 2 aminobenzothiazole using four different coupling reagents such as (i) DCC/HOBt (ii) EDC, (iii) HBTU (iii) Benzotriazole 1 yl oxy tris (dimethylamino) phosphonium hexafluorophosphate (BOP ) and (iv) Phosphorus oxychloride

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27 POCl 3 /pyridine. Use of the uronium coupling reagent (HBTU) failed. The best literature yields (41 93%) were achieved with POCl 3 N acylation of weakly nucleophilic heterocyclic amines by protected amino acids is not a straight forward Other literature methods utilizing DCC/HOBt [2003BMC5529] or EDC [2001JMC4252] as coupling reagents reported yields of 27 36% and reaction times of 4 16 h. Table 2 1 Preparation of a cylamino thiazoles, benzothiazoles, benzimidazoles and thiadiazoles Entry Reactant Product Yield a (%) 25 D R.T. (min) 1 Cbz L Trp Bt (2. 6 a) 2. 8 a 81 39.8 3.57 2 Cbz D L Trp Bt (2.6 a ') 2. 8 a 66 racemic 3.52 and 5.36 3 Cbz L Ala Bt (2.6 b) 2. 8 b 98 49.8 3.41 4 Cbz D L Ala Bt (2. 6 b ') 2. 8 b 78 racemic 3.46 and 4.01 5 Cbz L Val Bt (2. 6 c) 2. 8 c 98 44.6 3.39 6 Cbz D L Val Bt (2. 6 c ') 2. 8 c 82 racemic 2.97 and 3.56 7 Cbz L Phe Bt (2. 6 d) 2. 8 d 50 (27) b 63.9 NM c a Isolated yield. b [2003BMC5529]. c Not measured.

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28 2.2.2 Acylation of A mino p yrimidones Procedures similar to those of Section 2.2.1 above coupled Cbz L Ala Bt (2. 6 b) Cbz DL Ala Bt (2. 6 b') and 4 ClPhCOBt (2. 6 f) with 4 amino 1 benzylpyrimidin 2 one (2. 13 a) under microwave irradiation to give novel 2. 14 a b and 2. 14 a' in 7 6 98% yields (Scheme 2 3). The structures of compounds 2. 14 a,b and 2. 14 a' were supported by spectroscopic data together with microanalyses. The 13 C NMR and 1 H NMR spectra of N substituted amides 2. 14 a b and 2. 14 a' showed characteristic signals in the regions 165.9 175.2 ppm and 10.94 11.31 ppm which were assigned to the N heteroaryl amide carbonyl carbon and the proton of the NH respectively. Scheme 2 3. Preparation of (acyl amino ) pyrimidones 2.14a,b and 2.14a' Previous preparations of N substituted aminopyrimidones reported yields of 19 to 79% and reaction times of 17 40 h using carbodiimide based reagents such as DCC [1995EJM789] 1 ethyl 3 (3 dimethyla minopropyl) carbodiimide (EDAC) [2000BMC539] or (1 ethyl 3 (3 dimethylaminopropyl)carbodiimide (EDCI) [1992JM E 3344] in the presence of HOBt. Kenner et al. [1955JCS855] acylated 3 methylcytosine with benzoyl chloride in pyridine at 100 C (1.5 h) in 65% yield. 2.2.3 Acylation of A minopyrazoles N Substituted pyrazoles were prepared in yields of 23 to 89% and reaction times of 5 10 h. from activated aromatic acids and N protected amino acids via isolated intermediates includ ing acyl chlori des [1996EJM 461, 1998EJM 375] or N protected aminoacyl chlorides [1982FA450]

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29 that are not easily storable and sensitive t o degradation and racemization [1990JA9651, 1991TL1303] Literature couplings without isolation of inter mediates include activation by HCTU/HATU, EDC/HOBt or phosponate anhydrides (T3P) in yields ranging of up to 4 2% in reaction times up to 16 h [2004JOC5168] C oupl ing of 4 ClPhCOBt 2. 6 f and Cbz Gly Bt 2. 6 g with 5 amino 3 methyl 1 phenyl pyrazole 2. 1 5 a was achieved in DMF under microwave irradiation (100 W, 70 C) during 30 min (Scheme 2 4) to obtain 2. 1 6 a b (40% and 75%, respectively). N (Aminoacyl)benzotriazoles are stable, easy to handle reagents and can be stored at 20 C for months. Scheme 2 4. Preparation of (acylamino)pyrazoles 2.16a,b 2.2.4 Acylation of A mino p yridines Microwave irradiation of 2. 6 f and 2 aminopyridine (2.1 7 a) at 70 C for 30 minutes gave N (4 chloropyridin 2 yl)benzamide (2.18 a) in 94% yield (heating 2. 6 f and 2.1 7 a in DMF at 100 C for 6 h gave 2.1 8 a in 75%). The microwave conditions were applied to the reactions of N acylbenzotriazoles 2. 6 d,e,g j and 2. 6 d' with 2 aminopyridine (2.1 7 a) 2 amino 4 methylpyridine (2.1 7 b) and 2 amino 4,6 dimethylpyridine (2.1 7 c) thus providing 55 98% of the corresponding heteroaryl carboxamides 2. 1 8a g and 2. 1 8 d' (Scheme 2 5 and Table 2 2 ). The absence of racemization was confirmed for 2.18d by HPLC analysis, which showed a single peak at 3.66 min, while two peaks of equal intensity at retention times 3.63 min and 5.74 min were observed for racemic Cbz DL Phe NHPy 2 ( 2.18d' ).

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30 Scheme 2 5. Preparation of acylamino pyridines 2.18a g and 2.18d' This methodology is advantageous compared to several recent approaches to ( amino acyl)amino substituted pyridines that was previously prepared in yields from unreported to 77% and reaction times fro m unreported to 54 h using: (i) N,N` Dicyclohexyl carbodiimide (DCC) and 1 hydroxybenzotriazole HOBt [2001TL4799], (ii) 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide (EDC) and HOBt [1998BMCL1359], (iii) 1,1` Carbonyldiimidazole (CDI) [1998EJM635], (iv) Et hyl chloroformate [2006AXEo3947], (v) Phosphorus trichloride (PCl 3 ).[2004JME3591] and (vi) Acid chloride method [2004EJO3254]. Our approach provides known compounds 2.18a b d f g in better or comparable yields to those reported in the literature (Table 2 2) and afforded previously unreported N substituted amides 2.18c 2.18d' 2.18e and 2.12b in isolated yields of 52 98%. The previously reported method [1998EJMC635] i.e. activatin g the corresponding N protected amino acids with CDI followed by treatment with 2 amino 4,6 dimethylpyridine is advantageous for N substituted amides from 2 amino 4,6 dimethylpyridine, and 2.18f was prepared (68%, cf 77%) by this procedure. However, similar treatment of 2 amino 4 methylpyridine failed to yield compound 2.18g which was prepared in high yield (98%) by our alternative methodology, demonstrating the wide scope of benzotriazole approach.

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31 Table 2 2. Preparation of (a cylamino)pyridines from N acyl and N (aminoacyl) benzotriazoles Entry Reactant Product Yield a (%) Mp ( o C) 25 D 1 2.6f 2.18a i 94 (30 d ) 130 131 Non chiral 2 2.6h 2.18b 82 (66 e ) 76 78 Non chiral 3 Boc Ala Bt 2.6i 2.18c i 82 122 123 Non chiral 4 Cbz L Phe Bt 2.6d 2.18d b 55 (60 f ) 129 131 18.3 5 Cbz D L Phe Bt 2.6d' 2.18d' c 70 51 53 Racemic 6 Cbz L Met Bt 2.6e 2.18e 68 oil 24.0 7 Cbz Gly Bt 2.6g 2.18f 76 (77 g ) 108 110 Non chiral 8 Cbz L Pro Bt 2.6j 2.18g h 98 (N/A h ) 125 126 92.5 a Isolated yield, b HPLC for 2.18d : 3.66 min; c HPLC for 2.18d : 3.63 and 5.74 min. d Ref. [1958JOC1909], e Ref. [2004EJO3254], f Ref. [2004JME3591] g Ref. [1998EJM635], h no yield stated Ref. [2006AXEo3947], i Compounds 2.18a and 2.18c were prepared by colleague.

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32 2.3 Conclusions In summary, a general and convenient route for the preparation of N substituted amides derived from diverse heterocyclic amines and carboxylic acids under simple reaction conditions have been developed. 2.4 Experimental 2.4.1 General M ethods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl 3 or DMSO d 6 with TMS for 1 H (300 MHz) and 13 C (75 MHz) as an internal reference. N Cbz Amino acids and amino acids were used without further pu rification. All the reactions were carried out under microwave irradiation with a single mode cavity microwave s ynthesizer producing a continuous irradiation at 2450 MHz (with infrared temperature control system). Optical rotation values were measured usin g the sodium D line. Column chromatography was performed on silica gel (200 425 mesh). HPLC analyses were performed using Chirobiotic T column (4.6 1 250 mm), detection at 254 nm, flow rate 1 mL/min, and methanol as solvent. 2.4.2 General P rocedure for the P reparation of 2. 6 a j, 2. 6 a' c' N Aroyl ( 2. 6 f,h ), N (Boc aminoacyl) and N (Cbz aminoacyl) benzotriazoles ( 2. 6 a e,g,i,j, 2. 6 a' c' ) and dipeptidoyl benzotriazoles ( 2. 11 a,b ) were prepared according to literature procedures [2003S2795, 2006S411] 2.4.3 General P rocedure for the P reparation of N S ubstituted A mides 2. 8 a d, 2. 8 a' c', 2. 14 a b, 2. 14 a', 2. 1 6 a b, 2. 1 8 a g, 2. 1 8 d' and D ipeptide A mides 2. 12 a,b A dried heavy walled Pyrex tube containing a small stir bar was charged with benzotriazole adduct (0.25 mmol) and aminoheterocycle 2. 6 (0.25 mmol) dissolved in DMF (1 mL). The reaction mixture was exposed to microwave irradiation (100 W) for 30 minutes at a

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33 temperature of 70 C. The mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30 C (ca. 10 min). The reaction mixture was quenched with water and extracted with EtOAc (3 25 mL). The extracts were washed with (1 0%) Na 2 CO 3 (3 50 mL), water (3 50mL), and were dried over MgSO 4 The solvent was removed under reduced pressure and the residue was subjected to silica gel column using EtOAc/Hexane (1:1) as an eluent to give the corresponding N substituted amide. Benzyl N [(1S) 1 (1H indol 3 ylmethyl) 2 oxo 2 (1,3 thiazol 2 ylamino) ethyl] carbamate ( 2. 8 a ) White microcrystals (81%) ; mp 94 .0 96 .0 C ; D 25 = 39.8 (c 1.7, CHCl 3 ). 1 H NMR (300 MHz, CDCl 3 ) 4.84 4.86 (m, 1H), 3.21 3.35 (m, 2H). 5.06 (d, J = 12.1 Hz, 1H), 5.11 (d, J = 12.5 Hz, 1H), 5.90 (d, J = 8.0 Hz, 1H), 6.76 (s, 2H), 6.95 (t, J = 7.1 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.19 7.32 (m, 7H), 7.45 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 11.73 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 29.0, 55.6, 67.2, 10 9.5, 111.2, 113.7, 118.4, 119.7, 122.3, 123.0, 127.1, 128.0, 128.2, 128.5, 135.9, 136.9, 156.1, 158.3, 170.0. Anal. Calcd for C 22 H 20 N 4 O 3 S: C, 62.84; H, 4.79; N, 13.32. Found: C, 62.65; H, 4.74; N, 13.14. Benzyl N [1 (1H indol 3 ylmethyl) 2 oxo 2 (1,3 thiazol 2 ylamino)ethyl] carbamate ( 2. 8 a' ). White solid (66%) ; mp 188 .0 190 .0 C. 1 H NMR (300 MHz, DMSO d 6 ) 3.00 3.04 (m, 1H), 3.16 3.19 (m, 1H), 4.60 (br s, 1H), 4.95 (s, 2H), 6.97 7.04 (m, 3H), 7.20 7.31 (m, 8H), 7.49 (s, 1H), 7.70 7.77 (m, 1H), 10.85 (s, 1H), 12.49 (s, 1H). 13 C NMR (75 MHz, DMSO d 6 ) 27.6, 55.4, 65.5, 109.4, 111.3, 113.7, 118.2, 118.8, 120.9 124.3, 127.2, 127.7, 127.8, 128.4, 136.1, 136.9, 137.8, 155.9, 157.9, 171.2. Anal. Calcd for C 22 H 20 N 4 O 3 S: C, 62.84; H, 4.79; N, 13.32. Found: C, 62.61; H, 4.91; N, 12.95. Benzyl N {(1S) 2 [(6 methoxy 1,3 benzothiazol 2 yl)amino] 1 methyl 2 oxoethyl} carbamate ( 2. 8 b ). White microcrystals (98%) ; mp 90 .0 92 .0 C ; D 25 = 49.8 (c 2.1, CHCl 3 ).

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34 1 H NMR (300 MHz, CDCl 3 ) 1.50 (d, J = 7.0 Hz, 3H), 3.85 (s, 3H), 4.78 (qui, J = 7.0 Hz, 1H), 5.13 (d, J = 12.2 Hz, 1H), 5.22 (d, J = 12.2 Hz, 1H), 6.19 (d, J = 7.7 Hz, 1H), 6.98 (dd, J = 8.9, 2.5 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 7.30 7.33 (m, 5H), 7.56 (d, J = 8.9 Hz, 1H), 11.06 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 18.4, 50.7, 55.7, 67.4, 104.1, 115.3, 121.6, 128.1, 128.2,128.5, 133.3, 135.9, 142.3, 155.9, 156 .3, 156.8, 171.6. Anal. Calcd for C 19 H 19 N 3 O 4 S: C, 59.21; H, 4.97; N, 10.90. Found: C, 58.86; H, 4.97; N, 10.63. Benzyl N 2 [(6 methoxy 1,3 benzothiazol 2 yl)amino] 1 methyl 2 oxoethylcarbamate ( 2. 8 b' ) Colorless microcrystals (78%) ; mp 83 .0 85 .0 C 1 H NMR (300 MHz, CDCl 3 ) 1.50 (d, J = 7.1 Hz, 3H), 3.86 (s, 3H), 4.75 4.81(m, 1H), 5.18 (dd, J = 29.1, 12.3 Hz, 2H), 5.92 (br s, 1H), 6.99 (dd, J = 8.8, 2.5 Hz, 1H), 7.26 7.34 (m, 6H), 7.65 (d, J = 8.8 Hz, 1H), 10.74 (br s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 18.5, 51.1, 67.9, 56.1, 104.5, 115.7, 121.9, 128.5, 128.7, 128.9, 133.7, 136.1, 142.8, 156.1, 156.7, 157.2, 171.7. Anal. Calcd for C 19 H 19 N 3 O 4 S: C, 59.21; H, 4.97; N, 10.90. Found: C, 59.55; H, 5.18; N, 10.55. Benzyl N ((1S) 1 [(1 benzyl 1H benzimidazol 2 y l)amino]carbonyl 2 methylprop yl) carbamate ( 2. 8 c ). Colorless prisms (98%) ; mp 70 .0 72 .0 C ; D 25 = 44.6 (c 2.2, CHCl 3 ). 1 H NMR 0.9 (d, J = 6.7 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H), 2.33 2.39 (m, 1H), 4.38 (dd, J = 8.7, 4.3 Hz, 1H), 5.12 (t, J = 12.8 Hz, 2H), 5.31 (s, 2H), 5.76 (d, J = 8.7 Hz, 1H), 7.18 7.36 (m, 14H), 12.04 (s, 1H). 13 C NMR 17.4, 19.5, 32.2, 45.7, 62.5, 66.6, 108.1, 109.9, 111.2, 123.3, 127.7, 127.9, 128.0, 128.1, 128.4, 128.8, 129.1, 135.1, 135.4, 136.8, 153.4, 156.5, 182.7. Anal. Calcd for C 27 H 28 N 4 O 3 : C, 71.03; H, 6.18; N, 12.27. Found: C, 71.06; H, 5.92; N, 11.89. Benzyl N (1 [(1 benzyl 1H benzimidazol 2 yl)amino]carbonyl 2 methylpropyl)carbamate ( 2. 8 c' ) Colorless prisms (82%) ; mp 131 .0 133 .0 C. 1 H NMR (300 MHz, CDCl 3 ) 0.90 (d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H), 2.33 2.38 (m, 1H), 4.38 (dd, J = 8.8, 4.1 Hz, 1H), 5.12

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35 5.18 (m, 2H), 5.30 (s, 2H), 5.77 (d, J = 8.5 Hz, 1H), 7.17 7.35 (m, 14H), 12.05 (br s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 17.8, 19.9, 32.5, 46.0, 62.8 66.9, 110.2, 111.6, 123.6, 128.0, 128.3, 128.4, 128.8, 129.2, 129.5, 135.8, 137.1, 153.8, 156.9, 183.0. Anal. Calcd for C 27 H 28 N 4 O 3 : C, 71.03; H, 6.18; N, 12.27. Found: C, 71.20; H, 6.44; N, 11.94. Benzyl N (1S) 1 benzyl 2 [(3 methoxy 1,2,4 thiadiazol 5 y l)amino] 2 oxoethylcarbamate ( 2. 8 d ). White microcrystalls (50%) ; mp 150 .0 152 .0 C (lit. mp not reported) [2003BMC5529] [ ] D 25 = 63.9 (c 0.18, CHCl 3 ). 1 H NMR (300 MHz, CDCl 3 ) 3.12 3.14 (m, 2H), 4.00 4.04 (m, 3H), 5.04 5.17 (m, 3H), 5.81 5.93 (dd, J = 25.2, 8.06 Hz, 1H), 7.02 7.32 (m, 10H), 12.45 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 39.6, 55.6, 56.8, 67.2, 127.4, 128.1, 128.4, 128.6, 128.7, 129.3, 135.2, 136.1, 155.6, 166.5, 172. 4, 176.3. Anal. Calcd for C 20 H 20 N 4 O 4 S: C, 58.24; H, 4.89; N, 13.58. Found: C, 58.34; H, 4.90; N, 13.33. Benzyl (S) 1 ((S) 3 (1H indol 3 yl) 1 oxo 1 (thiazol 2 ylamino)propan 2 ylamino) 4 (methylthio) 1 oxobutan 2 ylcarbamate ( 2. 12 a ). White microcrystals (60 %) ; mp 145 .0 147 .0 C ; D 25 = 20.7 (c 1.9, CHCl 3 ). 1 H NMR (300 MHz, CDCl 3 ) 0.88 0.90 (m, 1H), 1.25 1.26 (m, 1H), 1.94 (s, 3H), 2.31 2.46 (m, 2H), 3.13 (dd, J = 14.3, 6.4, 1H), 3.24 (dd, J = 14.7, 7.2, Hz, 1H), 4.54 4.58 (m, 1H), 5.09 (d, J = 12.9 Hz, 1H), 5.15 (d, J = 12.9 Hz, 1H), 5.32 5.34 (m, 1H), 6.82 6.85 (m, 1H), 6.86 6.94 (m, 1H), 7.00 (t, J = 7.4 Hz, 2H), 7.12 7.18 (m, 1H), 7.21 7.31 (m, 6H), 7.36 7.41 (m, 2H), 7.76 (d, J = 8.2, 1H ), 8.12 (s, 1H), 11.80 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 15.1, 28.5, 30.0, 31.5, 31.9, 53.6, 53.8, 67.0, 109.2, 111.1, 113.7, 118.3, 119.4, 121.9, 123.4, 127.1, 127.9, 128.1, 128.5, 135.8, 136.4, 137.3, 156.6, 158.2, 169.9, 170.0, 172.4. Anal. Calcd for C 27 H 29 N 5 O 4 S 2 : C, 58.78; H, 5.30. Found: C, 59.13; H, 5.68. m/z ( TOF.MS): 551.1661 [M + + H] 552.1739.

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36 Benzyl {(S) 1 [(S) 1 (6 Methoxy benzothiazol 2 ylcarbamoyl)ethylcarbamoyl] 2 phenyl ethyl}carbamate ( 2. 12 b ). White prisms (52%) ; mp 190 .0 192 .0 C ; [ ] D 25 = 68.8 (c 2.3, CHCl 3 ). 1 H NMR 12.05 (br s, 1H), 8.15 (d, J = 8.9 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.27 7.23 (m, 6H), 7.03 7.00 (m, 5H), 6.86 (dd, J = 8.9, 2.3 Hz, 1H), 5.42 5.35 (m, 2H), 5.12 (d, J = 12.6 Hz, 1H), 5.03 (dd, J = 16.2, 8.4 Hz, 1H), 3.85 (s, 3H), 3.13 2.98 (m, 2H), 1.49 (d, J = 6.6 Hz, 3H). 13 C NMR 172.8, 170.7, 156.8, 156.7, 156.0, 142.5, 136.6, 136.0, 133.2, 129.3, 128.3, 127.8, 127.7, 126.9, 121.9, 115.1, 103.9, 103.3, 66.9, 56.5, 55.7, 48.7, 39.9, 19.2. An al. Calcd for C 28 H 28 N 4 O 5 S: C, 63.14; H, 5.30; N, 10.52. Found: C, 62.77; H, 5.34; N, 10.34. Benzyl N (1S) 2 [(1 benzyl 2 oxo 1,2 dihydro 4 pyrimidinyl)amino] 1 methyl 2 oxo ethylcarbamate ( 2. 14 a ) Colorless needles (97%) ; mp 152 .0 153 .0 C ; D 25 = +10.7 (c 0.15, DMF). 1 H NMR (300 MHz, DMSO d 6 ) 1.26 (d, J = 7.0 Hz, 3H), 4.23 4.26 (m, 1H), 5.02 (s, 4H), 7.19 (d, J = 7.1 Hz, 1H), 7.31 7.35 (m, 10H), 7.69 (d, J = 6.7 Hz, 1H), 8.26 (d, J = 7.1 Hz, 1H), 10.94 (s, 1H). 13 C NMR (75 MHz, DMSO d 6 ) 17.3 50.9, 52.3, 65.5, 95.4, 127.6, 127.7, 127.8, 128.4, 128.6, 136.8, 150.6, 155.2, 155.8, 162.5, 174.2. Anal. Calcd for C 22 H 22 N 4 O 4 : C, 65.01; H, 5.46; N, 13.78. Found: C, 64.62; H, 5.50; N, 13.48. Benzyl N 2 [(1 benzyl 2 oxo 1,2 dihydro 4 pyrimidinyl)amino] 1 methyl 2 oxoethyl carbamate ( 2. 14 a' ). Colorles needles (76%) ; mp 194 .0 1 96 .0 C. 1 H NMR (300 MHz, DMSO d 6 ) 1.26 (d, J = 7.0 Hz, 3H), 4.27 4.23 (m, 1H), 5.03 (s, 4H), 7.18 7.15 (m, 1H), 7.36 7.32 (m, 10H), 7.70 (d, J = 6.7 Hz, 1H), 8.26 (d, J = 7.0 Hz, 1H), 10.95 (s, 1H). 13 C NMR (75 MHz, DMSO d 6 ) 17.3, 50.9, 52.3, 65.5, 95.5, 127.7, 127.8, 127.9, 128.4, 128.6, 136.8, 136.9, 150.6, 155.2, 155.9, 162.5, 174.3. Anal. Calcd for C 22 H 22 N 4 O 4 : C, 65.01; H, 5.46; N, 13.78. Found: C, 64.86; H, 5.37; N, 13.63.

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37 N (1 B enzyl 2 oxo 1,2 dihydro 4 pyrimidinyl) 4 chlorobenzamide ( 2.14 b ). White needles (98%) ; mp 253 .0 255 .0 C. 1 H NMR (300 MHz, DMSO d 6 ) 5. 05 (s, 2H), 7.31 7.40 (m, 6H), 7.59 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 8.5 Hz, 2H), 8.31 (d, J = 7.0 Hz, 1H), 11.31 (s, 1H). 13 C NMR (75 MHz, DMSO d 6 ) 45.5, 52.3, 96.3, 127.6, 127.8, 128.5, 128.6, 128.8, 130.4, 131.2, 132.1, 136.7, 137.6, 150.3, 163.0, 166.5. Anal. Calcd for C 18 H 14 ClN 3 O 2 : C, 63.63; H, 4.15; N, 12.37. Found: C, 63.46; H, 4.01; N, 12.20. 4 C hloro N (3 methyl 1 phenyl 1H pyrazol 5 yl)benzamide ( 2. 1 6 a ). White microcrystals (75%) ; mp 151 .0 153 .0 C (lit mp 156 .0 157 .0 C) [1996EJMC461] 1 H NMR (300 MHz, CDCl 3 ) 2.35 (s, 3H), 6.63 (s, 1H), 7.39 7.56 (m, 7H), 7.66 (d, J = 8.5 Hz, 2H), 7.96 (br s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 14.0, 98.8, 124.5, 128.4, 128.5, 129.2, 129.6, 129.9, 131.6, 135.6, 137.9, 138.8, 149.8, 162.7. Anal. Calcd for C 17 H 12 ClN 3 O: C, 65.49; H, 4.53; N, 13.48. Found: C, 65.24; H, 4.44; N, 13.31. Benzyl N 2 [(3 methyl 1 phenyl 1H pyrazol 5 yl)amino] 2 oxoethylcarbamate ( 2. 1 6 b ). Colorless microcrystals (40%) ; mp 152 .0 153 .0 C (lit. mp 153 .0 155 .0 C) [1982FA450] 1 H NMR (300 MHz, CDCl 3 ) 2.30 (s, 3H), 3.88 (d, J = 5.6 Hz, 2H), 5.03 (s, 2H), 5.46 (br s, 1H), 6.48 (s, 1H), 7.20 7.44 (m, 10H), 8.31 (br s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 13.9, 45.3, 67.5, 86.3, 98.6, 100.2, 124.6, 128.1, 128.5, 128.6, 129.7, 135.2, 135.6, 137.7, 149.6, 166.1, 169.9. Anal. Calcd for C 2 0 H 20 N 4 O 3 : C, 65.92; H, 5.53; N, 15.37. Found: C, 66.16; H, 5.67; N, 15.33. 4 Chloro N (2 pyridinyl)benzamide ( 2. 1 8 a ). Yellowish needles (94%) ; mp 130 .0 131 .0 C (lit. mp. 139.0 C) [1958JOC1909] 1 H NMR (300 MHz, CDCl 3 ) 7.04 7.08 (m, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.73 7.79 (m, 1H), 7.87 (d, J = 8.4 Hz, 2H), 8.19 (d, J = 4.5 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 9.02 (br s, 1H); 13 C NMR (75 MHz, CDCl 3 ) 114.3, 120.1, 128.7, 129.0, 132.7,

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38 138.5, 147.8, 151.4, 164.8. Anal. Calcd For C 12 H 9 ClN 2 O: C, 61.9 5; H, 3.90; N, 12.04. Found: C, 61.98; H, 3.83; N, 12.12. N (2 P yridinyl)benzamide ( 2. 1 8 b ). White needles (82%) ; mp 76 .0 78 .0 C (lit. mp. 82 .0 84 .0 C) [2004EJO3254] 1 H NMR (300 MHz, CDCl 3 ) 7.02 7.06 (m, 1H), 7.46 7.59 (m, 3H), 7.73 7.78 (m, 1H), 7.94 (d, J = 7.3 Hz, 2H), 8.19 (d, J = 4.3 Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 9.06 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 114.3, 119.9, 127.3, 128.7, 132.2, 134.3, 138.5, 147.8, 151.6, 165.9. Anal. Calcd For C 12 H 10 N 2 O: C, 72.71; H, 5.08; N, 14.13. Found: C, 72.48; H, 5.03; N, 13.97. 3 (tert Butoxyamino) N (2 pyridinyl)propanamide ( 2. 1 8 c ). White microcrystals (82%) ; mp 122 .0 123 .0 C. 1 H NMR (300 MHz, CDCl 3 ) 1.43 (s, 9H), 2.63 2.66 (m, 2H), 3.47 3.53 (m, 2H), 5.30 (br s, 1H), 7.03 7.07 (m, 1H), 7.69 7.74 (m, 1H), 8.19 (d, J = 8.1 Hz, 1H), 8.28 (d, J = 3.6 Hz, 1H), 8.98 (br s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 28.4, 36.2, 37.2, 79.4, 114.3, 119.8, 138.5, 147.7, 15 1.3, 156.0, 170.5. Anal. Calcd For C 13 H 19 N 3 O 3 : C, 58.85; H, 7.22; N, 15.84. Found: C, 58.51; H, 7.42; N, 15.79. Benzyl N [(1S) 1 benzyl 2 oxo 2 (2 pyridinylamino)ethyl]carbamate ( 2. 1 8 d ). White needles (55%) ; mp 129 .0 131 .0 C (lit. mp. 137 139 .0 C) [ 2004JME3591] ; [ ] D 25 = 18.3 (c 0.3, CHCl 3 ). 1 H NMR (300 MHz, CDCl 3 ) 3.08 (dd, J = 13.2 7.0 Hz, 1H), 3.17 (dd, J = 13.9 6.2 Hz, 1H), 4.73 4.76 (m, 1H), 5.05 (d, J = 12.3 Hz, 1H), 5.12 (d d J = 20.7, 12. 2 Hz, 1H), 5.81 (d, J = 7.6 Hz, 1H), 6.99 7.04 (m, 1H), 7.09 7.12 (m, 2H), 7.18 7.21 (m, 3H), 7.23 7.29 (m, 5H), 7.66 7.71 (m, 1H), 8.20 8.25 (m, 2H), 9. 15 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 38.6, 56.8, 67.2, 114.3, 120.1, 127.1, 128.0, 128.1, 128.5, 128.6, 129.2, 135.9, 136.0 138.5, 147.8, 150.9, 156.1, 170.2. Anal. Calcd For C 22 H 21 N 3 O 3 : C, 70.38; H, 5.64; N, 11.19. Found: C, 70.07; H, 5.78; N, 11.04.

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39 Benzyl N [1 benzyl 2 oxo 2 (2 pyridinylamino)ethyl]carbamate ( 2. 1 8 d' ). White needles (70%) ; mp 51 .0 53 .0 C. 1 H NMR (300 MHz, CDCl 3 ) 3.11 3.13 (m, 1H), 3.19 (dd, J = 13.9, 6.3 Hz, 1H), 4.69 4.71 (m, 1H), 5. 10 ( d d, J = 20.6, 12.2 Hz, 1H), 5.65 (br s, 1H), 7.01 7.05 (m, 1H), 7.13 7.30 (m, 10H), 7.67 7.72 (m, 1H), 8.20 8.25 (m, 2H), 8.96 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 38.5, 56 .8, 67.2, 114.2, 120.1, 127.1, 128.0, 128.2, 128.5, 128.7, 129.2, 135.8, 138.5, 147.8, 150.8, 156.0, 169.9, 170.0. Anal. Calcd For C 22 H 21 N 3 O 3 : C, 70.38; H, 5.64; N, 11.19. Found: C, 70.00; H, 5.84; N, 11.18. Benzyl N {(1S) 3 (methylsulfanyl) 1 [(2 pyridylamino)carbonyl]propyl}carbamate ( 2. 1 8 e ). Colorless oil (68%) ; [ ] D 25 = 24.0 (c 2.8, CHCl 3 ) 1 H NMR (300 MHz, CDCl 3 ) 2.00 2.05 (m, 1H). 2.07 (s, 3H), 2.17 2.24 (m, 1H), 2.54 2.60 (m, 2H), 4.59 4.69 (m, 1H), 5.12 (d, J = 12.2 Hz, 1H), 5.18 (d, J = 12.2 Hz, 1H), 5.82 (br s, 1H), 7.35 7.62 (m, 5H), 7.04 7.08 (m, 1H), 7.69 7.74 (m, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.30 8.31 (m, 1H), 9.02 (br s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 15.2, 3 0.0, 32.1, 54.8, 67.2, 114.4, 120.2, 128.0, 128.1, 128.5, 136.0, 138.5, 147.8, 150.9, 156.2, 170.6. Anal. Calcd For C 18 H 21 N 3 O 3 S: C, 60.15; H, 5.89; N, 11.69. Found: C, 59.79; H, 6.02; N, 11.69. Benzyl N 2 [(4,6 dimethyl 2 pyridinyl)amino] 2 oxoethylcarbamate ( 2. 1 8 f ). White microcrystals (76%) ; mp 108 .0 110 .0 C (lit. mp. 103 .0 C) [1998EJM635] 1 H NMR (300 MHz, CDCl 3 ) 2.26 ( s, 3H), 2.34 (s, 3H), 4.00 (d, J = 4.8 Hz, 2H), 5.08 (s, 2H), 5.67 (s, 1H), 6.69 (s, 1H), 7.28 (s, 5H), 7.80 (s, 1H), 8.89 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ) 21.4, 23.1, 45.3, 67.3, 111.9, 120.8, 128.2, 128.5, 136.0, 149.6, 151.5, 155.3, 156.6, 167.8. Anal. Calcd For C 17 H 19 N 3 O 3 : C, 65.16 ; H, 6.11; N, 13.41. Found: C, 65.00; H, 6.03; N, 13.32. Benzyl (2S) 2 [(4 methyl 2 pyridinyl)amino]carbonyltetrahydro 1H pyrrole 1 carboxylate ( 2. 1 8 g ). White microcrystals (98%) ; mp 125 .0 126 .0 C (lit. mp. not reported)

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40 [2006A XE o3947] ; [ ] D 25 = 92.5 (c 2.4, CHCl 3 ). 1 H NMR (300 MHz, CDCl 3 ) [two rotamers] 1.94 2.20 (m, 3H), 2.21 2.27 (m, 1H), 2.35 (s, 3H), 3.50 3.60 (m, 2H), 4.39 4.52 (m, 1H), 5.15 5.72 (m, 2H), 6.78 (s, 1H), 7.18 7.37 (m, 5H), 8.05 (s, 1H), 8.31 (s, 1H), 8.49 (s, 0.4H), 9.22 (s, 0.6H). 13 C NMR (75 MHz, CDCl 3 ) [two rotamers] 21.3, 23.8, 24.5, 28.6, 31.3, 47.2, 47.6, 61.5, 67.5, 114.5, 121.0, 128.0, 128.4, 136.2, 147.4, 149.7, 151.2, 156.3, 169.9, 170.3. Anal. Calcd for C 19 H 21 N 3 O 3 : C, 67.24; H, 6.24; N, 12.38. Found: C, 66.93 ; H, 6.43; N, 12.62.

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41 CHAPTER 3 TAUTOMERISM OF 2 HYDRAZONO 3 PHENYLQUINAZOLIN 4(3 H ) ONES STUDIED BY 15 N NMR 1 3.1 Introduction The tautomeric equilibria of heterocycles are extremely important for understanding the function of numerous biologically importa nt components of living systems [1997MI 1 ] Thus the genetic code could only be deciphered after the dominant structures of the nucleotide bases were correctly represented. Moreover, it is now realized that the whole basis of evolution depends on the occurrence of minor proportions of the less stable nucleotide base tautomers which caus e advantageous genetic mistakes [1953MI 2 ] Figure 3 1. Dominant tautomeric forms of amino hydroxy mercapto and methyl pyridines The tautomeric equilibria of heterocycles have been investigated extensively [1963AHC1, 1976AHS1, 2000AHC1, 2006AHC1] and the following major trends are now clear ( Fig ure 3 1 ). (i) Most aminohetero aromatic compounds exist predominantly in the amino form cf 3.1 (and not the imino form cf 3.2 ) under normal conditions (aqueous solution or the crystalline state). (ii) Under these conditions most (although not all) hydroxyheteroaromatic compounds exist predominantly in the tautomeric carbonyl form (for example 2 hy droxypyridine 3.3 and 4 hydroxypyridine 3.5 exist as 2 pyridone 3.4 and 4 pyridone 3.6 respecti vely). (iii) M ercapto 1 Org. Biomol. Chem. 2009 7 4110 4119. Reproduced in part by permission of the Royal Society of Chemistry.

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42 derivatives of 6 membered heteroaromatic tend to follow their hydroxy analogs and exist as the thione, e.g. 3.8 and not as mercapto form e.g., 3.7 In contrast mercapto derivatives of five membered heterocyclic rings exist as mercaptans (fol lowing the amino analogs). (iv) Methyl groups and most substituted methyl groups exist largely as the methyl tautomer e.g. 3.9 and not the possible methylene tautomer e.g. 3.10 which is far less stable. Knowledge of the tautomerism of po tential drugs is relevant to the modeling of their interaction with a receptor since different tautomers have different affinit ies for the receptor. It is also important in the tuning of a desired pharmacological activity. Figure 3 2. Compounds investigat ed in this thesis The present chapter discusses the tautomeric equilibrium of a set of 2 hydrazono 3 phenyl 3 H quinazolin 4 ones 3. 11a i all representatives of a group of compounds of intense current interest in the development of comme rcial drugs for analgesic and anti inflammatory activity [2007BMC235, 2007CPB76, 2007AP41, 2008AF174] T he related 2 hydrazinobenzimidazole 3. 12 and 2 hydrazinopyrimidine 3.13 derivatives (Fig ure 3 2 ) were also include d in the study. Tautomeric equilibria can depend significantly on the nature of the medium. There are two major factors Firstly, the dielectric constant of the medium is important and of course this can

PAGE 43

43 range from very low in the vapor phase and nonpolar solvents (favori ng the tautomeric form with the lowest dipole moment) to very high for certain polar solvents (favoring the form with the highest dipole moment). The second major effect of medium is its hydrogen bonding donor and acceptor a bility which can lead to differ ent interactions of the t automeric forms. In the present chapter we deal with equilibrium positions in DMSO as a solvent. Given the poor solubility of these compounds in water, this is probably the best medium to use for comparisons with biological systems. Figure 3 3. Tautomers A and B of 2 hydrazino 3 phenylquina zolin 4(3 H ) ones 3. 11a i and their common cation s C and D Compounds 3.11a i 3.12 and 3.13 could exist in two tautomeric forms, amino forms A or imino forms B as shown in Fig ure 3 3 for 3.11a f Apparently there has been no previous discussion published of the tautomerism of 2 hydrazinoquinazolin 4(3 H ) ones 3.11 T he 404 hits for substructure 3. 11 in a Beilstein search are depicted in the presumed amino form 3. 11 A and none in the imino form 3. 11 B However, no supporting evidence such as interatomic distan ces or

PAGE 44

44 angles from solid state crystallographic data has been provided for their existence in the amino form A Figure 3 4. Tautomers of hydrazinopyrimidin 4(3 H ) ones The tautomerism of 2 hydrazinopyrimidin 4(3 H ) ones 3.14 (Fig ure 3 4) has also not been studied. Derivatives of 3.14 are usually represented in the amino form 3. 14A (308 Beilstein hits). The 18 hits for the alternative imino form 3. 14B were perhaps due to the fact that they represent ed compounds synthesized from uracil. Figur e 3 5. Tautomers of disubstituted guanidines The tautomerism of compounds 3.11a i 3.12 and 3.13 each involves a guanidine moiety 3.15 In substituted guanidines 3.15a c (Fig ure 3 5), the most stable tautomer is 3.15a with the double bond attached to the nitrogen carrying the most electron attracting substituent (e.g. R = NO 2 CN, SO 2 C 6 H 4 NH 2 ) [ 1 994JCS(P2)1849] This can be rationalized by realizing that of the two exchangeable protons in the common cation ( 3. 1 1 a f C and 3.1 1 a f D in Fig ure 3 3 ) the hydrogen attached to the most electronegative nitrogen is the most acidic. The experimental evidence for such tautomerism includes 15 N NMR [1995MRC383] and crystal structures [1965AXA47] When two of the guanidine nitrogens are part of an aromatic sy stem, the most aromatic tautomer is usually favored. The tautomerism of 2 aminopyrimidines has been studied extensively; both experimental data (low temperature IR

PAGE 45

45 spectroscopy) [1987JST275] and semiempirical and ab initio calculations [1995MI 3 1996JST375] confirm the 2 amino tautomer as the most stable. Spectroscopic studies (UV, IR and 1 H NMR) of N substituted 2 pyrimidinamine 3. 16 (R =H, NO 2 ) show that neither the phenyl [1980BCJ717] nor the 2,4,6 trinitrophenyl substituents [1992JHC1461] ar e electronegative enough to shift the equilibrium from the amino form 3. 16a to the imino form 3. 16b Figure 3 6. Tautomers of 2 pyrimidinamine and of isocytosine 2 Amino 3 H pyrimidin 4 one 3. 17 (isocytosine), which was studied extensively, both experimentally [1993T7627, 1993T595, 1980BCJ3073] and computationally [2007MI4] exists in aqueous solution mainly as 2 amino 3 H pyrimidin 4 one 3. 17a with some 2 amino 1 H pyrimidin 4 one 3. 17b Calculations suggest the 2 imino form 3. 17c not detected experimentally, is 5.6 kcal/mol less stable in aqueous solution than the 2 amino form 3. 17b The tautomerism of 2 hydrazinopyrimidine has apparently not been studied. 2 Quinolylhydrazones 3 18 (Fig ure 3 7) exist in solution predominantly in the amino form 3. 18a although the imino form was also detected [1976JOC2491, 1975JOC2512, 1975JCS(P1)2036] In solid state compounds 3. 18 were found in the amino form when Ar = thienyl or 5 chlorothienyl and in the imino form when Ar = 5 bromo 2 thienyl [1997AXC973]

PAGE 46

46 Figure 3 7. Tautomeric forms of 2 quinolylhydrazones 3.18 T he present work aims to identify the tautomeric preferences of the title compounds 3. 11a i 3. 12 3. 13 The literature data presented above support both the most aromatic amino form A and the imino form B which has the double bond on the most electronegative nitrogen. Since the two tautomeric forms differ in the protonation of two different nitrogens, 15 N NMR is the method of choice since a large difference in the chemical shifts of the alternative structures is expected. Measurement of the 15 N chemical shifts at natural abundance is now facile with the advent of indirect detec tion and pulsed field gradie nts [2002 CO R 35 2000JNP543] 3.2 Results and Discussion 3.2.1 Syntheses Products 3. 11a i were prepared by the literature procedure described in Scheme 3 1 [1985JHC1535] Compounds 3. 12 and 3. 13 were prepared by condensing 2 hydrazino 1 H benzimidazole and 2 hydrazino 4,6 dimethyl pyrimidine with 4 (dimethylamino) benzaldehyde and 1 methyl 1 H indole 2,3 dione respectively. Our initial attempt to prepare compound 3. 22 following the previously reported procedure [2007BMC235, 2007AP41, 2005BML1877, 2005BPB1531, 2006AF834, 2006JPP1249, 2008JHC709] described in Scheme 3 2 gave 3 amino 2 anilino 4(3 H ) quinazolinone 3. 25 instead. There is a single literature report [1985JHC1 535], in which 3. 25 was characterized only by melting point and elemental analysis.

PAGE 47

47 Scheme 3 1. Reagents and conditions: a) EtOH, reflux 2 h ; b) n BuOH, N 2 H 4 .H 2 O, reflux 2 h Our proof for the structure of compound 3.25 is based on NMR data. The nitrogen which couples with the ortho protons of the phenyl ring was at 102.2 ppm and bears the proton at 9.36 ppm. This proton couples with carbons C1 and C5a and with three nitrogens at 187.5, 165.5, and 64.3. The nitrog en at 187.5 ppm was assigned to N1, because it couples with H8. The nitrogen at 64.3 has the chemical shift of an amino group and it carries two protons at 5.71. These latter protons couple with the nitrogen at 165.5 (N3 in 3. 25) and with two carbons, C4 a nd another one assigned as C2 (see experimental section for atom labeling) Compound 3.25 reportedly condensed with benzaldehydes to give the corresponding 3.26 (Scheme 3 2) [1985JHC1535] In our case 3.25 with p nitrobenzaldehyde

PAGE 48

48 ( 3.23d ), gave compound 3.11d instead of the corresponding 3.26 The reaction was much slower than in the case of 3.22 Scheme 3 2. Reagents and conditions: a) (CH 3 O) 2 SO 2 2 % Ethanolic sodium hydroxide, rt, 1 h ; b) N 2 H 4 .H 2 O, EtOH, reflux, 20 h ; c) EtOH, 3. 23d reflux, 7 h 3.2.2 Tautomerism and NMR 1 H and 13 C chemical shifts were assigned based on 1 H 1 H, and one bond and long range 1 H 13 C couplings, seen in the gDQCOSY, gHMQC and gHMBC spectra. They are presented in Tables 3 1, 3 2, 3 4 and 3 5. Position numbering is given at the top of the tables. A typical assignment started with identifying the sequence H5 H8 in the gDQCOSY spectrum. H 5 and C4 w ere then assigned by their cross peak in the gHMBC spectrum. The 1 H 13 C gHMBC spectrum of 3.11b is presented in Fig. 3 8. Cross peaks of C4a with H6 and H8, and of C8a with H5 and H7 reveal these quaternary carbons. The phenyl protons, H1' H3' ca n be assigned from their intensity and coupling pattern and C5a' couples with H2'. In 2 hydrazono 3 phenyl 3 H quinazolin 4 ones, H3'', the singlet on a carbon at ca 150 ppm couples with C1''' and C5a'''. Other cross peaks in the gHMBC spectrum were then used to complete the assignments on the substituted benzylidene moiety C1''' C5a'''.

PAGE 49

49 In 2 [ N (2 oxo 1,2 dihydro indol 3 ylidene) hydrazono] 3 phenyl 3 H quinazolin 4 ones 3. 11h,i having a substituent on N1''', the alpha substituent protons couple with C7a' ''. Carbon C7a''' also couples with H6''' and H4''' which can be discriminated based on their multiplicity. Other cross peaks in the gHMBC spectrum were then used to complete the assignments on the substituted indole moiety C1''' C7a'''. In compound 3. 11g lacking the substituent on N1''', H7''' is the proton on the carbon at ca 110 ppm. A sharp signal of the exchangeable proton in compound 3. 11b afforded cross peaks with C8, C8a, C4a and with another quaternary carbon, assigned as C2 (Fig ure 3 8). This i ndicates that this exchangeable proton is linked to N1, meaning that 3. 11b is present in DMSO solution mainly as the imino tautomer. The chemical shift of C8, ca 117 ppm, further supports this assignment of tautomerism. Final proof for the imino tautomer comes from the one bond cross peak in the 1 H 15 N CIGAR spectrum (Fig ure 3 9) between the exchangeable proton and the nitrogen at 99.7 ppm, which is N1, because it displays a long range coupling with H8. Figure 3 8. Expansions of the 1 H 13 C gHMBC spectrum of compound 3.11b

PAGE 50

50 Figure 3 9. Expansions of the 1 H 15 N CIGAR spectrum of compound 3.11b The X ray structure of compound 3.11b (Figure 3 10) reveals the imino tautomer in the solid state also. Figure 3 10. X ray structure of 3.11b

PAGE 51

51 There is one more long range cross peak of the exchangeable proton in the 1 H 15 N CIGAR spectrum of 3.11b with one of the nitrogens three bonds away, which was assigned as N3 (151.3 ppm), because it also couples with H1'. N1'' and N2'' both couple wi th H3'' only, therefore, they could not be dis tinguished based on their couplings with protons. Their chemical shifts at 329.1 and 247.2 ppm, are different enough however to allow assignment based on chemical shifts seen in related compounds. A p articularly interesting example is presented in Fig ure 3 11 [1995MRC389] which ha s the same sequence of nitrogen atoms as in amino guanidines, in the amino form in 3.27 and in the imino form in 3.28 Based on the 15 N chemical shifts in compound 3.28 the signal at 247.2 ppm was assigned to N1'', and the signal at 329.1 ppm to N2''. The heterocycles in Figure 3 11 demonstrate that the chemical shifts of N1 and N1'' are good reporters of the amino imino tautomerism of 2 hydrazono 3 phenyl 3 H quinazolin 4 on es, as a difference of ca 100 ppm is to be expected in the two tautomers. The value of 99.7 ppm for N1 in 3.11b is in the range found for N1 in a series of 2,3 dihydro 1 H quinazolin 4 ones, 92 100 ppm; in a series of quinazolin 4(3 H ) ones N1 was at 253 27 0 ppm. In both series, N3 was at 140 190 ppm [2000JHC831] Figure 3 11. 15 N chemical shifts in related heterocycles from ref.[1995MRC389] Of all the compounds in this study, 3.11a and 3.11b were the only ones in which the signal of the exchangeable proton was sharp enough to afford cross peaks in the 1 H 13 C gHMBC spectrum. Cross peaks with C4a, C8 and C8a identified the imino tautomer. A cross peak with

PAGE 52

52 C3''' would have been a proof for the amino tautomer. For all of the other compounds, exchange with water or between tautomeric forms broadens the signal of the exchangeable proton too much for it to display any couplings with carbons or with nitrogens. In all of these cases, the tautomerism w as assigned based solely on 15 N chemical shifts. In both 2 hydrazono 3 phenyl 3 H quinazolin 4 ones 3. 11a f and in the 2 [ N (2 oxo 1,2 dihydro indol 3 ylidene) hydrazono] 3 phenyl 3 H quinazolin 4 ones 3. 11g i N1 displayed a cross peak with H8 in the CIGAR spectrum. The range of chemical shifts for N1 was 100 114 ppm. In the first series, the chemical shift of N1 '' was detected through the coupling between N1 '' and H3 '' and t he values for N1 '' chemical shifts were 247 251 ppm. These chemical shifts identify the imino form as the pre dominant tautomer in solution for compounds 3. 11a i The N1 chemical shift is expected to depend mainly on the position of the tautomeric equilibrium, and could be used to estimate this position, if values of the two tautomers are known. When the guanidine unit is part of a more aromatic structure, the amino form prevail s This is illus trated by compounds 3. 12 and 3. 13 In 3. 12 where rapid exchange of the proton between N1 and N3 (numbering relative to the aminoguanidine moiety, as in 3. 11a i ) produced an AA 'XX' pattern for the H5 H8 signals The NH protons were in fast exchange with the residual water in DMSO d 6 and did not produce a separate signal. The chemical shift of N1, N3 was revealed by coupling with H5, H8; cross peaks with H3'' revealed N1'' and N2''. The chemical shifts in the pair N1'', N2'' (142.9, 305.1) were closer to the values in 3. 27 (133.0, 328.0) than to th ose in 3. 28 (238.0, 384.0), indicating that compound 3. 12 is present in DMSO solution predominantly in the amino form. The chemical shift of the azole nitrogen cannot be used for the assignment of the tautomerism, at least in the presence of fast exchange. The va lue in 3. 12 136.3 ppm, is closer to the value for N1 in 1 methyl 2 aminobenzimidazole (134.6 ppm), than to their

PAGE 53

53 average (N 3 is at 191.9 ppm), which suggest s that 3. 12 is in the imino form. However, in 1 methylbenzimidazole N1 and N3 are at 143.8 and 243.9 ppm, correspondingly, while in benzimidazole, the equivalent nitrogens are at 143.2 ppm [ 1997MRC35] Compound 3. 13 did not dissolve in DMSO d 6 at room temperature, bu t did so at 70 C. About 30 minutes after dissolution, the proton spectrum of 3. 13 displayed the signals of two compounds, in a ratio 2:1. 1 H 13 C correlations indicated that both compounds contain the 4,6 dimethylpyrimidine and the 1,3 dihydroindole 2 one moieties. After one day, the sample consisted entirely of what was ini tially the minor compound. Correlations to the methyl protons identified the pyrimidine nitrogens at 254.2 and 252.4 ppm in the initially major and minor compounds, respectively These values are comparable to the value in 2 amino 4,6 dimethylpyrimidine, at 242 ppm [ 1981OMR106], indicating that both isomers are in the amino form. Further evidence comes from the initial minor compound which displayed a sharp signal for the exchangeable p roton at 12.80 ppm. This proton is attached to the nitrogen at 164.3, and displays long range couplings with the nitrogens at 252.4 and 340.0 ppm. Since these are amino tautomers, the y must differ in the configuration of the C3' '' =N2'' double bond The sharp, deshielded signal of the NH proton in the initially minor compound suggests an intramolecular hydrogen bond, possible only in the Z isomer. Some of the signals in the other isomer, E are broadened, particularly H4 ''' and the pyrimidine CH pro bably due to restricted rotation. The bonds to be considered as having partial double bond character are C2 N1 '' and N1 '' N2 '' Restricted rotation about C2 N1 '' would produce broadening of the signals of the methyl groups in the 4,6 dimethylpyrimidine moi ety, but not of the pyrimidine CH or of H4 ''' Broadening of these lat t er signals is due to restricted rotation about the N1 '' N2 '' bond (Figure 3 12). The assignment of the Z and E isomers was also confirmed by 13 C chemical shifts, as described later

PAGE 54

54 Discrimination between compounds 3.22 and 3.25 (Scheme 3 2) was based on the 15 N NMR data. The 1 H 15 N correlations are presented in the experimental part for 3.25 and in Table 3 3 for 3.22 N1 was identified in both compounds by its cross peak with H8 in the CIGAR gHMBC spectrum. Chemical shift values for N1 of 183.9 ppm in 3.22 and 188.0 ppm in 3.25 demonstrate that these compounds are both present in solution predominantly as the amino tautomer. This is to be expected for 3.25 in which the exocyclic guanidine nitrogen does not carry nitrogen. The preference for the amino tautomer of 3.22 is surprising, considering that its derivatives 3.11a i prefer the imino form, but can be explained by the greater electronegativity of N2'' in the latter compounds. Table 3 1 1 H chemical shifts (ppm) in compounds 3.11a f 3.12 3.21 3.22 and 3.24 \ Position Compd. \ 1 5 6 7 8 1' 2' 3' 3'' 1''' 2''' 3''' 4''' 5''' 3. 11a 10.61 7.91 7.16 7.69 7.69 7.34 7.49 7.40 8.07 7.93 7.41 7.38 7.41 7.93 3. 11b 10.41 7.88 7.11 7.63 7.64 7.31 7.48 7.40 7.87 7.68 6.64 3.37, 1.09 a 6.64 7.68 3. 11c NM b 8.00 7.37 7.83 8.04 7.49 7.61 7.59 8.59 3.80 6.60 3.83 6.65 8.38 3. 11d NM 7.97 7.22 7.72 7.72 7.36 7.52 7.44 8.19 8.16 8.21 8.21 8.16 3. 11e 10.83 7.96 7.22 7.72 7.72 7.36 7.52 7.44 8.16 8.11 7.86 7.86 8.11 3. 11f 10.68 7.94 7.18 7.70 7.70 7.35 7.50 7.42 8.12 9.09 8.55 7.43 8.31 3. 12 NM 7.40 7.16 7.16 7.40 8.19 7.68 6.74 2.96 6.74 7.68 3. 21 13.03 7.96 7.35 7.78 7.46 7.29 7.50 7.42 3. 22 c 8.87 d 8.50 7.29 7.79 7.88 7.53 7.43 7.29 3. 24 2.50 e 8.10 7.49 7.84 7.65 7.47 7.58 7.58 a CH 2 and CH 3 correspondingly. b Not measured. c Measured in pyridine d5 at 30 C. d H''. e CH 3 S in position 2.

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55 Table 3 2. 13 C chemical shifts (ppm) in compounds 3.11a f 3.12 3.21 3.22 and 3.24 \ Position Compd. \ 2 4 4a 5 6 7 8 8a 1 2 3 5a 3. 11a 152.2 161.3 115.1 128.0 122.6 135.8 116.4 140.6 129.9 129.5 128.7 137.1 3. 11b 150.6 161.3 114.9 128.1 122.1 135.7 116.2 140.8 130.0 129.6 128.6 137.3 3.11c NM 160.4 116.0 128.0 124.9 136.4 118.0 139.4 130.0 130.4 130.4 134.2 3.11d NM 161.2 115.9 128.2 123.0 135.8 116.8 140.3 129.9 129.5 128.6 137.0 3.11e NM 161.3 115.4 128.1 122.9 135.8 116.7 140.4 129.9 129.5 128.7 136.9 3.11f NM 161.4 115.3 128.1 122.7 135.8 116.4 140.5 129.9 129.5 128.6 137.0 3.12 NM 133.5 112.6 122.7 122.7 112.6 133.5 3.21 NM 160.5 116.9 128.1 125.0 136.3 116.4 140.3 129.7 129.6 128.8 140.0 3.22 a NM 163.1 118.7 128.0 122.9 135.4 125.8 150.9 130.2 130.8 130.0 136.0 3. 24 158.7 161.5 120.3 127.3 126.5 135.6 126.8 148.1 130.1 130.7 130.0 137.0 3 '' 1 ''' 2 ''' 3 ''' 4 ''' 5 ''' 5a ''' other 3. 11a 153.9 128.5 129.0 130.3 129.0 128.5 135.9 3. 11b 154.5 130.3 111.4 149.3 111.4 130.3 122.6 44.4 (CH 2 ), 13.2 (CH 3 ) 3. 11c 149.7 160.5 98.6 164.0 107.3 129.4 114.9 56.4 (CH 3 O 1 ''' ); 56.3 (CH 3 O 3 ''' ) 3. 11d 151.5 129.2 124.2 148.4 124.2 129.2 142.3 3. 11e 152.0 128.9 132.9 112.1 132.9 128.9 140.4 119.5 (CN) 3. 11f 151.0 149.8 150.7 124.3 135.1 131.7 3. 12 147.0 129.1 112.3 152.1 112.3 129.1 122.2 40.5 (CH 3 ) 3. 24 15.7 (CH 3 ) a Measured in pyridine d5 at 30 C. Table 3 3. 15 N chemical shifts (ppm) in compounds 3. 11a f 3. 12 3. 21 3. 22 and 3. 24 Protons which couple to a 15 N are given in pare ntheses \ Position Compd. \ 1 3 1 '' 2 '' other 3. 11a 100.4 (H8) 151.9 (H1 ) 247.0 (H3 '' ) 346.0 (H3 '' ) 3. 11b 99.7 (H1, H8) 151.3 (H1 '' ) 247.2 (H3 '' ) 329.1 (H3 '' ) 78.8 (H2 ''' CH 2 ) 3. 11c a 110.5 (H8) 156.3 (H1 ) N m 318.5 (H3 '' ) 3. 11d 113.9 (H8) 157.2 (H1 ) 251.7 (H3 '' ) 369.2 (H3 '' ) 372.9 (H2 ''' ) 3. 11e a NM 157.1 (H1 ) 251.1 (H3 '' ) 361.2 (H3 '' ) 3. 11f 104.4 (H8) 156.4 (H1 ) 251.2 (H3 '' ) 354.7 (H3 '' ) 318.2 (H1 ''' H3 ''' H4 ''' ) 3. 12 136.3 (H7,H8) 136.3 (H4,H5) 142.9 (H3 '' ) 305.1 (H3 '' H1 ''' ) 55.5 (H2 ''' CH 3 ) 3. 21 150.9 (H1, H8) 191.1(H1, H1 ) 3. 22 b 183.9 (H8, H1 '' ) 161.4 (H1 ) 106.5 (H1 '' ) 62.7 (H1 '' ) 3. 24 230.6 (H8) 180.6 (H1 ) a Measured at 70 C. b Measured in pyridine d 5 at 30 C.

PAGE 56

56 The substituents on N1'' could be used to control the tautomeric equilibrium of 2 hydrazino 3 substitutedquinazolin 4(3 H ) ones and related compounds, in order to fine tune their pharmacological and optical properties. Figure 3 12. Isomers/rotamers of compound 3. 13 Table 3 4. 1 H chemical shifts (ppm) in compounds 3.11g i and 3.13 \ Position Compd. \ 1 5 6 7 8 1 2 3 1 ''' 4 ''' 5 ''' 6 ''' 7 ''' 3. 11g 11.70 7.99 7.27 7.75 7.75 7.45 7.60 7.53 10.42 6.81 1.97 a 6.93 6.63 3. 11h 11.76 8.01 7.33 7.77 7.87 7.45 7.60 7.55 3.14 a 7.11 7.39 6.90 3. 11i 11.80 8.03 7.29 7.75 7.86 7.45 7.60 7.55 b 6.94 6.59 7.19 6.85 3. 13 (E) c 10.59 d 6.86 (CH), 2.40 (CH3) 3.19 a 8.06 7.10 7.40 7.04 3. 13 (Z) c 12.80 d 6.85 (CH), 2.38 (CH3) 3.24 a 7.58 7.12 7.37 7.08 a CH 3 b 4.34 (CH 2 ), 5.09 (CH 2 cis to H ), 5.12 (CH 2 trans to H ). c At 70 C. d H1''

PAGE 57

57 3.2.3 Stereochemistry of the C=N B onds The barrier to rotation about a C=N bond is lower than that about a C=C bond, and decreases with the electronegativity of the substituents on the N atom. In hydrazones, often the E and Z forms equilibrate in a matter of hours or days [ 1984JCS(P1)2109]. NOe experiments when both forms were available identified the isomers and demonstrated that 13 C chemical shifts can be diagnostic for the stereochemistry. In particularly, carbons alpha to the C=N carbons are shifted upfield when syn to the vicinal nitroge n, relative to the situation when they are anti This is the gamma effect and it is due to steric compre s sion. Table 3 5 13 C chemical shifts (ppm) in compounds 3. 11g i 3. 13 and 3. 29a c \ Position Compd. \ 2 4 4a 5 6 7 8 8a 1 2 3 5a 3. 11g NM a 161.2 116.1 128.0 123.8 136.1 117.2 140.0 129.6 129.9 129.3 137.3 3.11h NM 161.2 116.3 128.0 124.1 136.0 117.8 139.8 129.2 129.9 129.9 136.9 3.11i NM 161.2 116.3 128.1 123.9 138.0 117.6 140.0 129.6 130.0 128.8 137.0 3.13 (E) b NM 168.6 (q), 115.4 (CH), 24.1 (CH 3 ) 3.13 (Z) b NM 168.8 (q), 115.4 (CH), 24.0 (CH 3 ) 2 ''' 3 ''' 3a ''' 4 ''' 5 ''' 6 ''' 7 ''' 7a ''' other 3. 29a (E) c 166.8 134.6 118.9 125.5 120.8 128.1 109.0 140.7 3. 29a (Z) c 158.9 132.2 124.3 118.5 120.6 127.1 109.0 139.6 3. 29b (E) c 165.1 133.8 118.0 125.2 121.3 128.0 107.5 141.9 3. 29b (Z) c 156.9 130.8 123.4 118.0 120.9 126.8 107.4 140.7 3. 29c (E) c 166.8 134.8 118.9 126.1 129.4 128.4 108.6 138.5 3. 11g 166.8 144.8 118.2 128.0 131.0 131.9 110.1 141.0 21.3 (CH 3 ) 3. 11h 164.1 142.2 114.6 129.0 118.8 133.6 110.7 143.2 25.9 (CH 3 ) 3. 11i 164.9 NM 117.6 127.7 122.3 131.1 109.5 143.5 41.9 (C ), 132.8 (C ), 117.2 (C ) 3. 13 (E) b 164.6 NM 116.2 125.5 122.7 131.7 109.4 144.5 3. 13 (Z) b 161.9 131.7 120.7 120.1 123.4 130.4 110.1 143.0 a Not measured. b At 70 C. c From ref.[1996JHC675]

PAGE 58

58 Table 3 6 15 N chemical shifts (ppm) in compounds 3. 11g i and 3. 13 Protons which couple to a 15 N are given in parentheses \ Position Compd. \ 1 3 1 '' 2'' 1 ''' 3. 11g 104.2 (H8) 156.7 (H1 ) NM a 377.6 (H1 ''' ) 133.0 (H1 ''' H7 ''' ) 3.11h 109.4 (H8) 161.8 (H1') NM NM 131.8 (CH 3 H7''') 3.11i 105.1 (H8) 157.5 (H1') NM NM 136.2 (CH 2 H7''') 3. 13 (E) b 254.2 (CH 3 ) NM NM NM 3. 13 (Z) b 252.4 (CH 3 CH,NH ) 164.3 (NH, CH) 340.0 (NH, H7 ''' ) 133.1 (CH 3 H7 ''' H6 ''' ) a Not measured. b At 70 C The E Z pairs of the related isatin guanylhydrazones [ 1996JHC675] 3. 29a and 3. 29b (Table 3 4 ) display 13 C chemical shift differences of ca 6 ppm in positions 2 ''' 3a ''' and 4 ''' The 13 C chemical shifts of these carbo ns in 3. 11g i demonstrate the E configuration for the C3 ''' =N2 '' double bond in these compounds. The chemical shifts of the same positions in 3. 13 (E) and 3. 13 (Z) confirm the assignment. The X ray structure of 3. 11b (Fig ure 3 10) shows the E configuration of th e C3 ''' =N2 '' double bond. Since this is the expected configuration of hydrazones of aldehydes [ 1984JCS(P1)2109] it is reasonable to assume the same all of the 3. 11a f The C2=N1 '' double bond is in the E configuration in 3. 11b The same configuration is expected for all of 3. 11a i because the 2 hydrazono 3 phenyl 3 H quinazolin 4 one moiety is common to all of these compounds. The Z configuration would be higher in energy due to the steric hindrance between the phenyl in positi on 3 and N2 '' T here is also a hydrogen bond between H1 and N2 '' which stabilizes the E form. 3.3 Conclusions 15 N NMR is a powerful technique for the elucidation of tautomerism involving protonation of a nitrogen atom, since a large chemical shift, ca 100 ppm, is expected for the nitrogen of the two tautomeric forms. 15 N chemical shifts can be measured by indirect detection, through

PAGE 59

59 coupling of the nitrogen reporter of tautomerism with non exchangeable protons 2 or 3 bonds away. With typical samples of 15 30 mg, at natural abundance of 15 N, the total experiment time was ca 2 hours. 2 (2 Substituted methylenehydrazinyl) 3 phenylquinazolin 4(3 H ) ones ( 3. 11a i ) were found to be predominantly in the imino form in DMSO solution following the tautomeric pref erences of the aminoguanidines. 2 Hydrazino 3 phenyl 3 H quinazolin 4 one ( 3. 22 ) itself is in the amino form, demonstrating that the terminal nitrogen in the hydrazine moiety has to be involved in a double bond for the imino form to prevail. When the 3,4 dihydro 4 oxo 3 phenylquinazolin 2 yl moiety of 3. 11a i is replaced by a more aromatic one, as in benzimidazol 2 yl in 3. 12 or 4,6 dimethylpyrimidin 2 yl in 3. 13 the amino tautomer dominates the equilibrium in DMSO solution. 3.4 Experimental 3.4.1 Gen eral M ethods Melting points were determined on a capillary point apparatus equip ped with a digital thermometer. The NMR spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1 H, 125 MHz for 13 C and 50 MHz for 15 N, equipped with a three channel, 5 mm, indirect detection probe, with z axis gradients. The solvent was DMSO d 6 and the temperature was 25 C, unless specified otherwise. The chemical shifts for 1 H and 13 C were reference to the residual solvent signal, 2.50 ppm for 1 H and 39.5 ppm for 13 C, on the tetramethylsilane scale. The chemical shifts for 15 conversion to the nea t nitromethane scale, subtract 381.7 ppm [2002CO R 35]

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60 1 H spectra were acquired in one transient, with a 90 pulse, no relaxation delay and an acquisition time of 5 s, over a spectral window from 16 to 2 ppm. The FID was zero filled to 131072 points prior to Fourier transform. Typically, 1 H 13 C gHMBC spectra were acquired in 2048 points in f2 on a spectral window from 6.5 to 11 ppm, and 1 s relaxation delay. In f1 256 increments were acquired in 1 transient over a spectral window from 110 to 170 ppm, and then the corresponding FID s were zero filled twice prior to the second Fourier transform. 1 H 15 N CIGAR gHMBC spectra were acquired with a pulse sequence optimized for 15 N, as de s cribed in ref. [2003MRC307] 2048 points were acquired in f2, over a spectral window typically from 6.5 to 11 ppm, with 1 s relaxation delay. 1024 increments were acquired in f1 on a spectral window from 0 to 400 ppm, and the corresponding FID was zero filled twice prior to Fourier transform. The accordion delay wa s optimized for a value of 1 H 15 N coupling constants between 3 and 10 Hz. The number of transients per increment was between 4 and 64, depending on the concentration of the sample. Total experiment time was in most cases, ca 2 h 3.4.2 Pre paration of 3 phenyl 2 thioxo 2, 3 dihydroquinazolin 4(1 H ) one ( 3. 21) Anthranilic acid (1.37 g, 0.01 mol) and phenylisothiocyanate (1.35 g, 0.01 mol) were heated under reflux in 50 mL ethanol for 2 h. The solid obtained was filtered off and purified by crystallization fr om DMF: white microcrystals (2.03 g, 80%) ; mp 313 [1985JHC1535]. Anal. Calcd. for C 14 H 10 N 2 OS (254.31): C, 66.12; H, 3.96; N, 11.02. Found: C, 66.11; H, 3.78; N, 10.94. 3.4.3 Preparation of 2 hydrazino 3 phenylquinazolin 4(3 H ) one (3.22) A mixture of 3 phenyl 2 thioxo 2,3 dihydroquinazolin 4( 1H ) one 3. 21 (0.25 g, 1 mmol) and hydrazine hydrate (99 %, 0.05 g, 10 mmol) was heated under reflux in n butanol for 3 h. After the reaction was cooled down, a white precipitate separated that w as recrystallized from n

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61 butanol to give the desired product in 79% yield (0.20 g, 0.8 mmol). White needles ; mp 193 195 Anal. Calcd. for C 14 H 12 N 4 O (252.28): C, 66.65; H, 4.79; N, 22.21. Found: C, 66.27; H, 4.70; N, 21 .96. 3.4.4 General P rocedure for P reparing C ompounds 3.11a i 2 Hydrazinyl 3 phenylquinazolin 4( 3 H ) one 3. 22 (0.25 g, 1 mmol) was heated under reflux in ethanol (25 mL) for 15 min. to 1 h with 1 mmol of the corresponding aldehyde or ketone 3. 23a i The precipitate formed was collected and crystallized from the appropriate solvent to give the desired products in quantitative yields. (E) 2 ((E) B enzylidenehydr azono) 3 phenyl 2,3 dihydroquin azolin 4(1H) one ( 3. 11a ). The product was crystallized from EtOH to give white needles (98%) ; mp 215 [1985JHC1535] Anal. Calcd. for C 21 H 16 N 4 O (340.39): C, 74.10; H, 4.74; N, 16.46. Found: C, 74.21; H, 4.63; N, 16.46. (E) 2 ((E) (4 ( D iethylamino)benzylidene)hydrazono) 3 phenyl 2,3 dihydro qu inazolin 4(1H) one ( 3. 11b ). The solid obtained was crystallized from DCM/hexanes to give the desired product as yellow needles (97%) ; mp 202 Calcd. for C 25 H 25 N 5 O (411.51): C, 72.97; H, 6.12; N, 17.02. Found: C, 72.81; H, 6.12; N, 17.11. X ray experimental for 3.11b D ata were collected with a APEX II CCD area detector, using graphite monochromatised [1990AXA467] and refined on F 2 using all data by full matrix least squares procedures with SHELXL 97 [ G. M. Sheldrick, SHELXL 97 University of Gttingen, 1997 ] Hydrogen atoms were included in calculated positions with isotropic displacement parameters 1.3 times the isotropic equivale nt of their carrier atoms.

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62 Crystal data and structure refinement for 3.11b Identification code pjs Empirical formula C25 H25 N5 O Formula weight 411.50 Temperature 153(2) K Wavelength 0.71073 Crystal system Orthorhombic Space group P 21 21 21 Unit cell dimensions a = 5.1252(10) = 90. b = 17.913(4) = 90. c = 23.305(5) = 90. Volume 2139.6(7) 3 Z 4 Density (calculated) 1.277 Mg/m 3 Absorption coefficient 0.081 mm 1 F(000) 872 Crystal size 0.20 x 0.08 x 0.05 mm 3 Theta range for data collection 3.47 to 25.10. Index ranges 5<=h<=5, 21<=k<=21, 27<=l<=27 Reflections collected 14854 Independent reflections 2209 [R(int) = 0.0258] Completeness to theta = 25.10 98.4 % Absorption correction None

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63 Refinement method Full matrix least squares on F 2 Data / restraints / parameters 2209 / 0 / 283 Goodness of fit on F 2 1.082 Final R indices [I>2sigma(I)] R1 = 0.0313, wR2 = 0.0814 R indices (all data) R1 = 0.0328, wR2 = 0.0827 Absolute structure parameter 10(10) Extinction coefficient 0.0099(17) Largest diff. peak and hole 0.124 and 0.170 e. 3 (E) 2 ((E) (2,4 D imethoxybenzylidene)hydrazono) 3 phenyl 2,3 dihydro quinazolin 4(1H) one ( 3. 11c ). The solid obtained was filtered off and crystallized from n butanol to give yellow microcrystals (98%) ; m p 257 Calcd. for C 23 H 20 N 4 O 3 (400.44): C, 68.99; H, 5.03; N, 13.99. Found: C, 68.73; H, 5.02; N, 13.79. (E) 2 ((E) (4 N itrobenzylidene)hydrazono) 3 phenyl 2,3 dihydroquinazolin 4(1H) one ( 3. 11d ). The precipitate was coll ected and crystallized from n butanol to give yellow microcrystals (97%) ; mp 275.0 [2007BMC235] Anal. Calcd. for C 21 H 15 N 5 O 3 1/3 H 2 O (391.39): C, 64.44; H, 4.03; N, 17.89. Found: C, 64.66; H, 3.85; N, 17.67. 4 ((E) ((E) (4 O xo 3 phenyl 3,4 dihydroquinazolin 2(1H) ylidene)hydrazono) methyl)benzonitrile ( 3. 11e ). The precipitate was collected and crystallized from n butanol to give pale yellow microcrystals (90%) ; mp 312 Calcd. for C 22 H 15 N 5 O (365.40): C, 72.32; H, 4.14; N, 19.17. Found: C, 72.07; H, 3.99; N, 19.03. (E) 3 P henyl 2 ((E) (pyridin 3 ylmethylene)hydrazono) 2,3 dihydroquinazolin 4(1H) one ( 3. 11f ). The precipitate was collected and crystallized from n butanol to give white crystals

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64 (97%) ; mp 216.0 Anal. Calcd. for C 20 H 15 N 5 O.H 2 O (359.39): C, 66.84; H, 4.77; N, 19.49. Found: C, 67.04; H, 4.56; N, 19.38. (E) 2 ((E) (5 M ethyl 2 oxoindolin 3 ylidene)hydrazono) 3 phenyl 2,3 dihydro quinazolin 4(1H) one ( 3.11g ). The product was crystallized from n butanol to give pale yellow needles (96 %) ; mp 348 Anal. Calcd. for C 23 H 17 N 5 O 2 .H 2 O (413.44): C, 66.82; H, 4.63; N, 16.94. Found: C, 66.66; H, 4.50; N, 16.88. (E) 2 ((E) (5 B romo 1 methyl 2 oxoindolin 3 ylidene) hydrazono) 3 phenyl 2,3 dihydroquinazolin 4(1H) one ( 3.11h ). The product was crystallized from EtOH to give orange needles (95 %) ; mp 363 Calcd. for C 23 H 16 BrN 5 O 2 (474.32): C, 58.24; H, 3.40; N, 14.77. Found: C, 57.98; H, 3.35; N, 14.60. (E) 2 ((E) (1 A llyl 2 oxoindolin 3 ylidene)hydrazono) 3 phenyl 2,3 dihydro quinazolin 4(1H) one ( 3.11i ). The product was crystallized from EtOH to give yellow needles (84 %) ; mp 242 Calcd. for C 25 H 19 N 5 O 2 .H 2 O (474.32): C, 68.33; H, 4.82; N, 1 5.94. Found: C, 68.79; H, 4.58; N, 15.94. 3.4.5 Preparation of 4 [(2 (1 H b enzimidazol 2 yl )hydrazono)methyl] N,N dimethylaniline ( 3. 12) 2 Hydrazinyl 1 H benzoimidazole (0.074 g, 0.5 mmol) was heated under reflux in ethanol (25 mL) with 4 (dimethylamino)benzaldehyde (0.075 g, 0.5 mmol) for 3 h. The precipitate formed was collected and crystallized from EtOH to give brown microcrystals (50%) ; mp 262 [1988BCJ1339]. HRMS (ESI) calcd. for C 16 H 17 N 5 (MH) + 280.1557, found 280.1557. 3.4.6 Preparation of 3 [2 (4,6 dimethylpyrimidin 2 yl)hydrazono] 1 methylindolin 2 one ( 3. 13) 2 Hydrazinyl 4,6 dimethylpyrimidine (0.069 g, 0.5 mmol) was heated under reflux in ethanol (25 mL) with 1 methyl 1 H indole 2,3 dione (0.08 g, 0.5 mmol) for 1 h. The percipitate

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65 formed was collected and purified by column chromatography using ethyl acetane/hexanes (1:3) as eluent. The sharp yellow needle crystals were pur e enough for elemental analysis. This compound was obtained in 71% yield and has mp 191 Calcd. for C 15 H 15 N 5 O 1/4 H 2 O (285.83): C, 63.03; H, 5.47; N, 24.50. Found: C, 63.32; H, 5.39; N, 24.10. 3.4.7 Preparation of 2 (methylsulfanyl) 3 phenyl 4(3 H ) quinazolinone ( 3. 24) This compound was prepared according to a literature method [2007BMC235] The solid obtained was crystallized from dichloromethane/diethyl ether to give compound 24 as colorless needles (75 %) ; mp 118 [2007BMC235] Anal. Calcd. for C 15 H 12 N 2 OS (268.34): C, 67.14; H, 4.51; N, 10.44. Found: C, 67.36; H, 4.50; N, 10.46. 3.4.8 Preparation of 3 amino 2 anilino 4(3 H ) quinazolinone (3.25) This compound was prepared according to a literature method [1985JHC1535] The solid obtained was crystallized from dichloromethane/ hexanes to afford compound 3. 25 as colorless prisms (80 %) ; mp 153 1 H NMR (500 MHz, DMSO d 6 ) 9.37 (s, 1H, H1''), 8.00 (dd, J = 7.9, 1.3 Hz, 1H, H5), 7.93 (dd, J = 8.7, 1.1 Hz, 2H, H1'), 7.65 (ddd, J = 8.5 Hz, 7.2, 1.7, 1H, H7), 7.39 (d, J = 8.1 Hz, 1H, H8), 7.36 (t, J = 8.0 Hz, 2H, H2'), 7.23 (ddd, J = 8.0, 7.9, 1.1 Hz, 1H, H6), 7.07 (tt, J = 7.4, 1.0 Hz, 1H, H3'), 5.72 (s, 2H, H2''). 13 C NMR (125 MHz, DMSO d 6 ) 161.7 (C4), 148.7 (C2), 148.4 (C8a), 139.3 (C5a'), 134.9 (C7), 129.3 (C2'), 126.8 (C5), 125.8 (C8), 123.6 ( C3'), 123.4 (C6), 121.2 (C1'), 118.3 (C4a). 15 N NMR (50 MHz, DMSO d 6 ) 188.0 (N1), 164.4 (N3), 101.9 (N1 '' ), 64.1 (N2 '' ). Anal. Calcd. for C 14 H 12 N 4 O (252.28): C, 66.65; H, 4.79; N, 22.21. Found: C, 66.45; H, 4.73; N, 22.34.

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66 CHAPTER 4 NMR STUDY OF THE TAU TOMERIC BEHAVIOUR OF N ( AMINOALKYL)TETRAZOLE S 1 4.1 Introduction Tetrazoles have wide application in chemistry [1996CHEC(2)621]: the ring can serve as a metabolically stable analogue for the carboxyl group [1980PMC151, 1977AHC323, 1968 MI 5 ] and confer useful biological activity. Tetrazoles bind anions tightly in polar solution when compared to the corresponding carboxylic acids. Tetrazole times stronger than the corresponding acidic hosts. This remark able difference in binding strength is rationalized by considering the tautomeric equilibria in tetrazoles and the conformational preferences in carboxylic acids. Tetrazole 1H tautomer is energetically more favored than the 2H tautomer by 3 kcal/mol and it resembles the anti conformation of carboxylic acid which is disfavored energetically but is the preferred conformation for binding [2008OL4653]. The tetrazole ring appears in some well known drugs such as Diovan, Benicar, Avapro, Atacand, and Hyzaar, that are generally used as cardiovascular or hypertension drugs. In biological systems, free NH tetrazoles usually exist mainly in the anionic form [2010 MI 6 ]. N Aminoalkyl)tetrazoles (4.1 4.3) have recently provided modified protein formation inhibitors f or the prevention and treatment of diseases associated with AGEs (advanced glycation end products) and ALEs (advanced lipoxidation end products) (Figure 4 1) [2007WOP051930]. Benzodiazepine analogs (4.4, 4.5) was found to be effective in treating cardiac a rrhythmias 1998USP5817658] in mammals by modulating the slowly activating delayed rectifier potassium 1 Reproduced in part with permission from The Journal of Organic Chemistry 20 1 0 75 6468 6476 Copyright 20 1 0 American Chemical Society

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67 current (IKs) and the rapidly activating and deactivating relayed rectifier potassium current (IKr). All of these compounds are of particular interest as therapeutic agents and all of them are repor ted without considering their tautomeric equilibria. Individual tautomers may interact differently with a particular receptor. Figure 4 1. Some examples of bioactive compounds containing N aminoalkyl)tetrazole scaffolds NMR study of the tautomeric equilibria of a series of tetrazoles in different solvents is now reported in order to achieve better understanding of the effects of substituents and solvents on this equilibrium. The equilibria are compared with tho se previously reported for analogous N substituted benzotriazoles and 1,2,3 triazoles. N ( N N Disubstituted aminomethyl)benzotriazoles exist in solution as equilibrium mixtures of the corresponding 1 ( N N disubstituted aminomethyl) and 2 ( N N disubstituted aminomethyl) benzotriazoles [1975JCS(P1)1181] with interconversion taking place via a dissociation recombination mechanism [1987JCS(P1)2673, 1989H(28)1121], involving an iminium cation and a benzotriazole anion, the formation which is facili tated by easy cleavage of the C N bond. This type of tautomerism is known as cationotropy since it is the cation which moves from one position to another in a molecule. The equilibrium position of such

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68 benzotriazoles depends on the polarity of the solvent and on the substrate structure. Increasing solvent polarity favors the 1 isomer as expected from dipole moment measurements which showed that 1 substituted benzotriazoles are more polar than their 2 substituted isomers [1961MI 7 1990JOC5683]. However the N 2 tautomer may predominate when the dialkyaminoalkyl substituent is bulky [1975JCS(P1)1181, 1989H(28)1121]. Crossover experiments showed that the isomerization process is intermolecular and occurs by a dissociation recombination mechanism (Scheme 4 1 (i)) rather than an intramolecular concerted mechanism (Scheme 4 1 (ii)) [1987JCS(P1)2673]. Scheme 4 1. N 1 to N 2 Substituent isomerization of ( N N disubstituted aminomethyl) benzotriazoles N Aminoalkyl) 1,2,4 triazoles also exist in solution as mixtures of N 1 { 4. 6(A) } and N 2 { 4.6 (B) } tautomers and a previous report [1990T633] demonstrated that a series of such compounds convert easily between the N1 and N2 tautomers, but no evidence for the N4 tautomer { 4.6 (C) } was found {Scheme 4 2 (i) } Recent work on the tautomerism of N aminoalkyl) 1,2,3 triazoles [2010H1] showed that these compounds exist in non polar CDCl 3 predominantly as the 2 isomer { 4.7 (B) } although in polar DMSO (and D 2 O, when soluble) some of the 1 is omer { 4.7 (A) } is present {Scheme 4 2 (ii) } The present study reports the analogous intermediates of 1 and 2 tetrazoles.

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69 Scheme 4 2. (i) N1 N2 and N4 Substituted isomers of N aminoalkyl) 1,2,4 triazoles. (ii ) N1 and N2 Substituted isomers on N aminoalkyl) 1,2,3 triazoles 4.2 Results and Discussion 4.2.1 Preparation of 5 S ubstituted Tetrazoles 1 H Tetrazole (4.9 a) and 5 phenyl 1 H tetrazole (4.9d ) are commercially available. The other parent tetrazoles were synthesized according to known methods A [2001JOC7945] ( 4.9 b ) and B [1958JA3908] (4.9c and 4.9e ) [see experimental section for details]. Table 4 1. Synthesis of Tetrazoles 4.9b,c,e Entry Nitriles 4.8a,b,c Tetrazoles 4.9b,c,e Mp (lit. mp Yield % 1 4.8a 4.9b 98.0 100.0 (113.0 114.0) [1950JOC1082] 32 2 4.8b 4.9c 118.0 120.0 (125.5.0 126.0) [1950JOC1082] 81 3 4.8c 4.9e 218.0 220.0 (219.0 220.0) [2008TL2824] 94

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70 4.2.2 Preparation of N Aminoalkyl)Tetrazoles The parent tetrazoles 4.9a e were used to prepare compounds 4.11a,c h (Table 4 2) by the standard procedure of Bechmann and Heisey [1946JA2496]. Compound 4.11b was prepared by the synthetic route shown in Table 4 2 [1993JOC4913] because the Bechmann and Heisey procedure [1946JA2496] failed (see experimental section for details). Table 4 2. Synthesis of N Aminoalkyl)tetrazoles 4.11a h Compound N Aminoalkyl)tetrazoles Yield % M R 1 R 2 R 3 4.11a H (CH 2 ) 2 O (CH 2 ) 2 75 81.0 82.0 (80.0 82.0) a 4.11b H CO o C 6 H 4 SO 2 60 177.0 179.0 4.11c (CH 3 ) 2 CH (CH 2 ) 2 O (CH 2 ) 2 80 Oil 4.11d PhCH 2 (CH 2 ) 2 O (CH 2 ) 2 70 59.0 61.0 4.11e PhCH 2 (CH 2 ) 5 60 Oil 4.11f Ph (CH 2 ) 2 O (CH 2 ) 2 75 57.0 59.0 (60.0 61.0) b 4.11g Ph (CH 2 ) 5 74 67.0 69.0 (65.0 66.0) c 4.11h p NO 2 C 6 H 4 (CH 2 ) 2 O (CH 2 ) 2 86 133.0 135.0 a [1990T633]. b [1992PJC1257]. c [1997RJO524] 4.2.3 NMR C haracterization and S olvent E ffects on Tautomeric E quilibrium of 1 and 2 S ubstituted T etrazoles Preliminary work on the tautomeric composition of N aminoalkyl)tetrazoles reported that 4 (1 H tetrazol 1 ylmethyl)morpholine ( 4.11a ) exists predominately as the N2 isomer { 4.11a(B) } in CDBr 3 =17.6 kcal/mole [1990T633]. The N2 isomer of { 4.11a(B) } was also predominant in toluene d 8, but substantial amounts of the N1

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71 isomer { 4.11a(A) } appeared in CD 3 NO 2 Similarly, for 4 ((5 methyl 1 H tetrazol 1 yl)methyl)morpholine, the N2 isomer was the dominant isomer in CDBr 3 =20.0 kcal/mole for the inte rconversion of tautomers [1990T633]. N1 Substituted tetrazoles have higher dipole moments than N2 substituted tetrazoles [1984ZOR2464, 1956JA4197, 1958JPC508] (e.g. 1 methyl 5 phenyltetrazole, = 5.88 D, and its 2 methyl isomer ( = 2.52 D); this higher p olarity is consistent with the N1 isomer predominating in polar solvents [1984ZOR2464]. Nonempirical quantum mechanical calculations (MP2/6 31G* and MP2/6 31G*//HF/6 31G*) concluded that N2 substituted tetrazoles are more stable than the N1 isomers in the gas phase and in nonpolar solvents and also suggested that the equilibrium would be displaced toward the N1 tautomer by increasing solvent polarity [2003RJC275]. The 1 H NMR spectra of compounds 4.11a h were analyzed in a wide range of solvents of different polarity and the ratio of both N1 and N2 isomers was calculated based on their integration. N Aminoalkyl)tetrazoles 4.11a d occurred as mixtures of two isomers in solution and the mole fractions of the two isomers 4.11a d(A) and 4.11a d(B) were determ ined easily. Compounds 4.11e h showed the N2 isomer exclusively in different solvents. 1 H NMR chemical shifts were deduced from the relative intensities of the peaks and literature data for model tetrazoles [1976T499]. 13 C NMR chemical shift assignments were made according to literature values [ 1988MRC134]. Further 2D NMR experiments including gDQCOSY, gHMQC, gHMBC, and CIGAR HMBC were carried out to support chemical shift assignments and to assign 15 N chemical shifts in 4.11b,c (See experimental section for details). The analysis of 1 H NMR spectra for 4 (1 H tetrazol 1 ylmethyl)morpholine ( 4.11a ) showed clearly that the N2 isomer 4.11a(B) predominates in nonpolar solvents, C 6 D 6 and CDCl 3 but the

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72 N1 isomer 4.11a(A) predominates i n polar solvents (Table 4 3). 1 H and 13 C NMR spectra of 4.11a in CDCl 3 gave the characteristic chemical shifts of both N1 and N2 isomer with N2 isomer 4.11a(B) being the major isomer (83%). In 1 H NMR spectra, H 5 has a chemical shift of 8.65 ppm (downfiel d) in 4.11a(A) and 8.53 ppm (upfield) in 4.11a(B) In contrast, the chemical shifts of N (CH 2 ) N are 5.29 (upfield) ppm in 4.11a(A) and 5.50 ppm (downfield) in 4.11a(B) In 13 C NMR spectra, C 5 appears at 142.8 ppm (upfield) in 4.11a(A) and at 152.8 ppm (d ownfield) in 4.11a(B) However, N (CH 2 ) N appears at 70.0 ppm (upfield) in 4.11a(A) and at 74.1 ppm (downfield) in 4.11a(B) Table 4 3. Percentage of N1 Isomer Observed in Different Solvents Comp. D 2 O (62.8) a (CD 3 ) 2 SO (45.1) a CD 3 CN (45.6) a CD 3 OD (55.4) a (CD 3 ) 2 CO (42.2) a CDCl 3 (39.1) a C 6 D 6 (34.3) a 4.11a 85 >99 67 81 59 17 17 4.11b 58 80 79 62 83 57 24 4.11c NS b 29 26 86 20 5 <1 4.11d 92 44 33 20 30 11 10 4.11e NM d <1 <1 NM d NM d <1 NM d 4.11f <1 <1 <1 <1 <1 <1 <1 4.11g NM d <1 <1 NM d NM d <1 NM d 4.11h <1 <1 <1 56/44 Reacts c <1 <1 a Dimroth Reichardt polarity index E T (30). b Insoluble. c Reacts with the solvent. d Not measured. Compounds 4.11b and 4.11c were analyzed using 2D NMR in addition to the conventional 1D NMR. 1 H, 13 C, and 15 N Chemical shifts of 4.11b,c are given in Table 4 4. N ((1 H Tetrazol 1 yl)methyl) 1,2 benzisothiazole 3(2 H ) one 1,1 dioxide (4.11b) exists in benzene d6 mainly as a 2 substi tuted tetrazole but in all other solvents the 1 isomer

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73 predominates (Table 4 3). The 1 H, 13 C and four 15 N chemical shifts were assigned in both isomers in DMSO d6 using 2D NMR techniques (Table 4 4). Table 4 4. 1 H, 13 C, and 15 N Chemical shift assignments in 4.11b,c 1 H NMR Comp. H 5 H 1 H 2 '' H 3 '' Other 4.11b 4.11b(A) 9.59 6.57 H 4'' (8.36), H 5'' (8.10), H 6'' (8.03), H 7'' (8.18) 4.11b(B) 9.02 6.72 H 4'' (8.36), H 5'' (8.10), H 6'' (8.03), H 7'' (8.18) 4.11c 4.11c(A) 5.51 2.53 3.57 H 5' (3.23), H 5'' (1.33) 13 C NMR C 5 C 1' C 2'' C 3'' Other 4.11b 4.11b(A) 145.4 49.2 158.6 C 4a'' (137.7), C 4'' (122.7), C 5'' (137.3),C 6'' (136.3),C 7'' (126.5) 4.11b(B) 154.2 53.7 158.6 C 4a'' (137.7), C 4'' (122.7), C 5'' (137.3),C 6'' (136.3),C 7'' (126.5) 4.11c 4.11c(A) 161.0 73.9 50.0 66.6 C 5' (26.0), C 5 '' (50.0) 15 N NMR N1 N2 N3 N4 Other 4.11b 4.11b(A) 239.0 370.2 NM a 396.4 N 2'' (160.9) 4.11b(B) 384.4 308.2 285.1 NM a N 2'' (160.9) a Not measured. The assignments for 4.11b(A) were based on the correlation seen in gHMBC between the methylene protons (H1') and C 5 of the tetrazole ring; such a correlation was not seen in the case of 4.11b(B) The 1 H and 13 C chemical shifts of N CH=N (H 5 and C 5) and N CH 2 N (H 1' and C 1') follow the trend found in 4.11a 15 N Chemical shifts of all four nitrogens in both isomers were recorded. In 4.11b(A) the methylene protons H 1' (6.57 ppm) showed two bond correlation to N1 (239. 0 ppm) and to saccharine nitrogen N2'' (160.9 ppm) and three bond correlation to N2

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74 (370.2 ppm). The tetrazole proton H 5 (9.59 ppm) showed two bond correlations to N1 (239.0 ppm) and to N4 (396.4 ppm) and a three bond correlation to N2 (370.2 ppm). In the case of 4.11b(B) N1 (384.4 ppm) was identified through a two bond correlation to H 5 (9.09 ppm) and three bond correlations to the methylene protons H 1' (6.72 ppm). The methylene protons correlate to N2 (308.2 ppm), N3 (285.1 ppm) and to saccharine nitr ogen N2'' (160.9 ppm) (Table 4 4). 4 ((5 Isopropyl 1 H tetrazol 1 yl)methyl)morpholine ( 4.11c ) exists exclusively as the N2 isomer {4.11c(B)} in C 6 D 6 and CDCl 3 (non polar solvents) as is clear from the 13 C chemical shift of tetrazole C 5 (171.5 ppm) (Table 4 4). On increasing the polarity of the solvent, the N 1 isomer {4.11c(A)} appears in the 1 H NMR spectra. The highest ratio of N1 isomer (1 to 2) was obtained in DMSO d6 which is the most polar in the series of solvents used (Table 4 3). The 1 H and 13 C ch emical shifts for 4.11c(A) ( cf Tables 4 4) were fully assigned but chemical shifts for 4.11c(B) could not be assigned due to broad peaks of low intensity in DMSO d6 4 ((5 Benzyl 1 H tetrazol 1 yl)methyl)morpholine (4.11d) exists in C 6 D 6 and in CDCl 3 as a mixture of N2:N1 isomers with a ratio of 9:1. The proportion of the N1 isomer increases with increasing solvent polarity so the N1 isomer is dominant in D 2 O with a ratio of 9:1 (Table 4 3). 1 ((5 Benzyl 2 H tetrazol 2 yl)methyl)piperidine (4.11e) exis ts in CDCl 3 CD 3 CN, and DMSO d6 and toluene d8 at isomer with no evidence for N1 isomer in any of these solvents (Table 4 3). For N aminoalkyl)tetrazoles 4.11f,g,h only the N2 isomers 4.11f,g,h (B) were observed in solution ( cf. Table 4 3), probably because of the steric bulk of the C 5 substituent. The 13 C chemical shifts of C 5 were 165.1, 164.7, and 163.3 for 4.11f 4.11g and 4.11h respectively. These chemical shift values are in agreement with literature values for the N2 isomer repo rted by

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75 Begtrup et al. [1988MRC134]. It was also shown by Binda et al. [1992PJC1257] that the aminomethylation of 5 substituted tetrazoles bearing aromatic substituents at C 5 occurs regioselectively at the N2 of the tetrazole ring. Finally, an X ray cryst al structure for 4.11 f (Figure 4 2) showed clearly that this compound exists exclusively in the solid state as the N2 isomer thus avoiding steric interaction between the substituents at N1 and C 5. Figure 4 2. X ray structure of 4.11f 4.2.4 Thermodynamic and K inetic P arameters Free energy of activation for the isomerization of 4.11 a was measured previously [1990T633]. A variable temperature 1 H NMR study of compounds 4.11b failed to detect d6 probably b ecause the electron withdrawing groups on nitrogen destabilized the iminium ion thus slowing down the isomerization process. Variable temperature 1 H and 13 C NMR were carried out for 4.11 c in CD 3 CN; seven different sites within the molecule showed coalesce nce at six different temperatures (Figure 4 3 for VT 1 H NMR). ), calculated using the method of Shanan Atidi and Bar Eli [1970JPC961] for the case of unequal populations (P A B ) (Table 4 5), showed that the 2 substituted isomer 4.11c(B) is less stable than the 1 substituted isomer 4.11c(A) in CD 3 CN by an average of 0.56 kcalmol 1

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76 Figure 4 3. 1 H spectra of 4.11c in acetonitrile d3 For inter conversion of A B, a plot of ln k r vs 1/T c gave a straight line (Figure 4 3) from which values of E a (16.5 kcalmol 1 # (15.9 kcalmol 1 # ( 4.5 e.u.) were derived. For inter conversion of B A, a similar plot (Figure 6 3) gave values of E a (18.5 kcalmol 1 # (17.9 kcalmol 1 # ( 6. 0 e.u.). The low entropies of activation in both directions suggest that the isomerization process occurs via a unimolecular dissociation recombination mechanism involving a tight ion pair (Scheme 4 3). This is entirely consistent with a polar solvent (e.g CD 3 CN) facilitating the isomerization. By contrast, in the less polar solvent CDCl 3 solution no coalescence was observed

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77 Scheme 4 3. N 1 to N 2 Substituent isomerization of 4.11c Table 4 5. Coalescence Temperatures (T c ), Equilibrium Constants (K), Chemical Shift v ), and Natural Logarithm of Exchange Rate Constant (k r ) from the 1 H and 13 C NMR Spectra of 4.11c (Acetonitrile d 3 as Solvent) T c (K) K c (Hz) (T c ) (kcal mol 1 ) ln k r (T c ) (kcal mol 1 ) ln k r N(CH 2 )N a 338 0.54 195.0 17.5 3.5 15.8 6.0 Ph CH 2 a 325 0.55 40.7 17.2 2.9 15.8 5.1 Ph CH 2 b 343 0.55 d 271 17.4 4.1 15.8 6.4 N(CH 2 ) 2 a 318 0.58 30.3 17.1 2.4 16 4.2 N(CH 2 ) 2 b 323 0.58 d 45 17.2 2.7 16.2 4.3 O(CH 2 ) 2 a 318 0.55 38.7 17.3 2.1 15.8 4.5 O(CH 2 ) 2 b 308 0.55 d 13.8 17.4 1.0 16 3.3 a T c calculated from 1 H NMR. b T c calculated from 13 C NMR. c Calculated at 273 K, K= P A /P B where P A and P B are the population of 1 and 2 isomers (A and B). d Assumed to be the same as in 1 H NMR. Figure 4 4. Plot of ln k r vs 1/ T c in case of inter conversion of A B and B A.

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78 4.2.5 Cross O ver E xperiment An equimolar mixture of 4.11a with 4.11e in toluene d8 1 H NMR recorded at 3.7 ppm, corresponding to the eight possible isomers resulting from the cross reaction (Figure 4 5). The four signals for the methylene protons in the benzyl moiety in 4.11d(A) 4.11d(B) 4.11e(A) and 4.11e(B) were identified by their cross peaks in the gHMBC spectrum with three sp 2 carbons, namely C ipso and C ortho on the phenyl ring, and C 5 on the tet razole moiety. Two of these methylene protons, at 3.77 {4.11d(A)} and 3.82 {4.11e(A)} ppm coupled with C 5 signal at 153.7 ppm, while the other two at 4.05 {4.11d(B)} and 4.08 {4.11e(B)} ppm coupled with C 5 signals at 165.4 and 165.1 ppm, indicating that the first pair is 1 substituted tetrazole and the second pair is the 2 substituted isomers. Figure 4 5. Different isomers expected from crossover experiment between 4.11a and 4.11e The signals for the H 5 proton in tetrazole were assigned in a similar manner as 7.50 {4.11a(A)} and 7.49 {4.11i(A)} on carbons at 142.4 assigned to 1 substituted compounds and 7.99 {4.11a(B)} and 8.04 {4.11i(B)} on the carbons at 152.3 and 152.5 corresponding to 2 substituted compounds.

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79 The triplets at 3.30, 3.29, 3.26 and 3.18 have the distinct chemical shifts of the methylene hydrogens alpha to the oxygen in morpholine. They display cross peaks in the NOESY spectrum with triplets at 2.18, 2.12, 1.78 and 1.82, which are the methylene protons alpha to nitrogen. These protons display nOe peaks with protons at 4.66 {4.11d (B)} 4.70 {4.11a(B)} 4.14 {4.11a(A)} and 3.98 {4.11d(A)} which are the methylene protons attached to the morpholine moiety. The other four signals at 4.84 {4.11i(B)} 4.80 {4.11e(B)} 4.29 {4.11i(A)} and 4.15 {4.11e(A)} are for the methylene protons att ached to the piperidine group, and show nOes with the two protons at 2.33 and 2.28. A nOe between the benzyl protons at 3.77 ppm and the morpholine protons at 3.98 ppm identified these signals as from the 4.11d(A) compound, as confirmed by their equal inte grals. The other signals of the methylene groups bearing piperidine or morpholine moieties were assigned by matching their integrals to the integrals of H5 or to the integrals of the benzyl protons. The assignment of the significant proton signals and the molar percentage of compounds in the mixture are given in Table 4 6. It is worth mentioning here that the molar percentage of the 2 isomers is higher than the 1 isomer because 2 isomer predominates in non polar solvents. The size of the exchange peaks in t he NOESY spectrum demonstrates that piperidine isomerises faster than morpholine and that exchange between positions 1 and 2 is faster than the intermolecular crossover process (Figure 4 6). The cross over experiment was initially carried out in polar solv ent (acetonitrile d3 ) and it gave almost the same cross over products with different molar ratios, but the NCH 2 N protons of the eight isomers were not well separated in this solvent.

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80 Table 4 6. Signal Assignments and Molar Percentage Ratios of Compoun ds 4.11a 4.11d 4.11e and 4.11i in Cross Over Experiment Compound 4.11a (A) 4.11a (B) 4.11d (A) 4.11d (B) 4.11e (A) 4.11e (B) 4.11i (A) 4.11i (B) a 4.14 4.70 3.98 4.66 4.15 4.80 4.29 4.84 Molar % 5.2 24.8 2.6 26.1 1.7 17.4 4.5 17.6 b 7.50 7.99 3.77 4.05 3.82 4.08 7.49 8.04 a Protons in the methylene group carrying a piperidine or a morpholine moiety. b Benzyl protons or H5. Figure 4 6. NOESY spectrum of crossover experiment between 4.11a and 4.11e in toluene d8 at

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81 4.3 Conclusions In conclusion, the equilibrium between the N 1 and N 2 tautomers of N aminoalkyl) tetrazoles was shown to favor the N 2 tautomer in non polar solvents, but the equilibrium shifts to favor the N 1 tautomer with increasing solvent polarity. Bu lky substituents in the 5 position of the tetrazole ring favor the N 2 tautomer. The detailed mechanism of interconversion between the N 1 and N 2 tautomers is shown to involve both tight ion pairs, which can relax to give isomerisation without crossover o r may become solvent separated and thus lead to crossover between different components in a mixture of two tetrazoles in which both the tetrazole ring and the amino substituents differ. 4.4 Experimental 4.4.1 General M ethods Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. 1 H Tetrazole was purchased from Fluka Chemie Gmbh as solution (0.45 M in acetonitrile). The 1 H and 13 C NMR spectra for starting materials were recorded on a Varian Gemini instrument, operating at 300 MHz for 1 H and 75 MHz for 13 C. The NMR spectra for final products were recorded on a Varian Inova instrument, operating at 500 MHz for 1 H, 125 MHz for 13 C an d 50 MHz for 15 N, equipped with a three channel, 5 mm, indirect detection probe, with z axis gradients. The 1 H NMR spectra were recorded in deuterium oxide, dimethylsulfoxide d 6 acetonitrile d 3 methanol d acetone d 6 chloroform d benzene d 6 with TMS f or 1 H (500 MHz) and 13 C (125 MHz) as an internal reference. The chemical shifts for 15 tetramethylsilane is 100.0000000 MHz. For conversion to the neat nitromethane scale, subtract 381.7 ppm.

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82 1 H spectra were acquired in one transient, with a 90 pulse, no relaxation delay and an acquisition time of 5 s, over a spectral window from 16 to 2 ppm. The FID was zero filled to 131072 points prior t o Fourier transform. Typically, 1 H 13 C gHMBC spectra were acquired in 4096 points in f2 on a spectral window from 1.5 to 11 ppm, and 1 s relaxation delay. In f1 512 increments were acquired in 1 transient over a spectral window from 170 to 10 ppm, then t he corresponding FID s were zero filled twice prior to the second Fourier transform. 1 H 15 N CIGAR gHMBC spectra were acquired with a pulse sequence optimized for 15 N, as decribed in ref. 1. 2048 points were acquired in f2, over a spectral window typically from 1.5 to 11 ppm, with 1 s relaxation delay. 1024 increments were acquired in f1 on a spectral window from 0 to 400 ppm, and the corresponding FID was zero filled twice prior to Fourier transform. The accordion delay was optimized for a value of 1 H 15 N coupling constants between 3 and 10 Hz. The number of transients per increment was between 4 and 64, depending on the concentration of the sample. 4.4.2 General P rocedure for P reparation of C ompound 6.4b (Method A) [2001JOC7945] A mixture of isopropyl nit rile 4.8a (0.9 mL, 10 mmol), sodium azide (0.72 g, 11 mmol), zinc bromide (2.25 g, 10 mmol) and water (20 mL) was heated under reflux for 24 h. The reaction mixture was allowed to cool down and acidified with HCl (6 N) to pH 1. The product was extracted wi th ethyl acetate (3 50 mL). The solvent was then evaporated and 0.25 N NaOH (100 mL) was added. The mixture was stirred for 30 min. until the solid dissolved and a suspension of zinc hydroxide was formed. The suspension was filtered and the solid was was hed with 1 N NaOH (10 mL). The filtrate was acidified again with HCl (6 N) and stirred vigorously until the tetrazole precipitated. If tetrazole did not precipitate from water, it was extracted with

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83 ethyl acetate (3 50 mL). The combined ethyl acetate ext racts were dried over MgSO 4 then distilled under reduced pressure to give the desired product. 5 Isopropyl 1H tetrazole ( 4.9b ). The product was crystallized from DCM/Hexanes to give white needles (32%) ; mp 98.0 [1950JOC1082] 1 H NMR (300 MHz, CDCl 3 ) 12.42 (br s, 1H), 3.60 3.46 (septet, J = 7.0 Hz, 1H), 1.50 (d, J = 7.0 Hz, 6H). 13 C NMR (75 MHz, CDCl 3 ) 161.9, 24.8, 21.3. Anal. Calcd. for C 4 H 8 N 4 (112.14): C, 42.84; H, 7.19; N, 49.96. Found: C, 43.07; H, 7.32; N 49.88. 4 .4.3 General P rocedure for P reparation of C ompounds 4.9c and 4.9e (Method B) [1958JA3908] A mixture of benzyl nitrile (4.8b) (0.12 g, 1mmol) or 4 nitrobenzonitrile (6.3c) (0.148 g, 1mmol), sodium azide (0.195 g, 3 mmol), ammonium chloride (0.214 g, 4 mmol), and dimethyl formamide (5 mL) was heated under reflux for 12 h. After allowing the reaction to cool to room temperature, water (20 mL) was added with continuous stirrin g. The mixture was then acidified with HCl (6 N) to pH 2. The reaction mixture was extracted with ethyl acetate (3 50 mL), dried over MgSO 4 and the solvent was removed under reduced pressure. The resultant solid was recrystallized to give compounds 4.9c and 4.9e respectively. 5 Benzyl 1H tetrazole ( 4.9c ). The product was crystallized from DCM/hexanes to give gray needles (81 %) ; mp 118.0 [1995TL1759] 1 H NMR (300 MHz, CDCl 3 ) 7.32 7 .23 (m, 5H), 4.34 (s, 2H). 13 C NMR ( 75 MHz, CDCl 3 ) 156.2, 134.4, 129.2, 128.9, 127.8, 30.0. Anal. Calcd. for C 8 H 8 N 4 (160.18): C, 59.99; H, 5.03; N, 34.98. Found: C, 60.13; H, 5.11; N, 35.10. 5 (4 Nitrophenyl) 1H tetrazole ( 4.9e ). The product was crystallized from EtOH to give brown microcrystals (94%) ; mp 218.0 [1987JMC552] 1 H NMR (300 MHz, DMSO d 6) 8.44 (d, J = 8.8 Hz, 2H), 8.30 (d, J = 8.8 Hz, 2H). 13 C NMR (75

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84 MHz, DMSO d 6) 155.5, 148.7, 130.7, 128.2, 124.6. Anal. Calcd. for C 7 H 5 N 5 O 2 (191.15): C, 43.98; H, 2.64; N, 36.64. Found: C, 44.35; H, 2.17; N, 36.25. 4 .4. 4 General P rocedure for P reparation of C ompounds 4.11a, 4.11c h [1946JA2496] Tetrazoles 4.9a e were dissolved in methanol (5 mL) unless otherwise specified and the solutions were cooled in an ice bath. Morpholine (4.10a) or piperidine (4.10c) (1.1 mmol) was added and the mixture was allowed to stir for 15 min. Formalin 37% (1.2 mmol, 0.096 mL) was added dropwise and the mixture stirred for 1 hr. The ice bath was then removed and the reaction was stirred overnight. The solvent was evaporated and the residue was recrystallized to give 4.11a and 4.11c h 4 (1H Tetrazol 1 ylmethyl)morpholine ( 4.11a ). The product was obtained as colorless prisms after recrystallization from DCM/hexanes (75%) ; mp 81.0 82.0 1 H NMR (300 MHz, CDCl 3 ) 8.65 (s, 0.3H from A), 8.53 (s, 0.7H from B), 5.50 (s, 1.6 H from B), 5.29 (s, 0.4 H from A), 3.69 (t, J = 4.7 Hz, 4H), 2.63 (t, J = 4.7 Hz, 3.3H from B), 2.58 (t, J = 4.5 Hz, 0.7 H from A). Two tautomers: 13 C NMR (75 MHz, CDCl 3 ) 152.8 (B), 142.8 (A), 7 4.1 (B), 70.0 (A), 66.8 (B), 66.6 (A), 49.9. Anal. Calcd. for C 6 H 11 N 5 O (169.19): C, 42.59; H, 6.55; N, 41.39. Found: C, 42.92; H, 6.67; N, 41.41. 4 ((5 Isopropyl 1H tetrazol 1 yl)methyl)morpholine ( 4.11c ). The reaction was carried out in water and the prod uct was obtained as a colorless oil (80%). 1 H NMR (300 MHz, CDCl 3 ) 5.53 (s, 2H), 3.64 (t, J = 4.7 Hz, 4H), 3.22 (septet, J = 7.0 Hz, 1H), 2.59 (t, J = 4.7 Hz, 4H), 1.35 (d, J = 7.0 Hz, 6H). 13 C NMR (75 MHz, CDCl 3 ) 171.5, 77.6, 73.6, 66.7, 49.9, 26.1, 2 1.5. Anal. Calcd. For C 9 H 17 N 5 O (211.27): C, 51.17; H, 8.11; N, 33.15. Found: C, 50.99; H, 8.32; N, 32.96.

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85 4 ((5 Benzyl 1H tetrazol 1 yl)methyl)morpholine ( 4.11d ). The product was obtained as colorless needles after recrystallization from DCM/hexanes (70%) ; mp 59.0 1 H NMR (300 MHz, CDCl 3 ) 7.27 7.15 (m, 5H), 5.36 (s, 1.8H from B), 4.77 (s, 0.2H from A), 4.33 (s, 0.2H from A), 4.22 (s, 1.8H from B), 3.64 (t, J = 4.7 Hz, 3.5H from B), 3.57 (t, J = 4.5 Hz, 0.5H from A), 2.59 (t, J = 4.7 Hz, 3.5H from B), 2.43 (t, J = 4.5 Hz, 0.5H from A). 13 C NMR (75 MHz, CDCl 3 ) Two tautomers: 165.6 (B), 136.8 (A), 129.3 (A), 128.9 (B), 128.8 (B), 128.7 (A), 127.9 (A), 127.0 (B), 73.9 (B), 68.8 (A), 66.8 (B), 66.6 (A), 50.5 (A), 50.0 (B), 32.0 (B), 29.9 (A). Anal. Calcd. for C 13 H 17 N 5 O (259.13): C, 60.12; H, 6 .61; N, 27.01. Found: C, 60.59; H, 6.74; N, 27.12. 1 ((5 Benzyl 2H tetrazol 2 yl)methyl)piperidine ( 4.11e ). The reaction was carried out in water and the product was obtained as a sticky yellow oil (60 %). 1 H NMR (500 MHz, CD 3 CN) 7.34 7.24 (m, 5H), 5.27 (br s, 2H), 4.26 (s, 2H), 2.51 (t, J = 5.1 Hz, 4H), 1.54 1.49 (m, 4H), 1.34 1.29 (m, 2H). 13 C NMR (125 MHz, CD 3 CN) 129.8, 129.7, 128.0, 51.8, 26.6, 24.4. Anal. Calcd. for C 14 H 19 N 5 (257.34): C, 65.34; H, 7.44; N, 27.21. Found: C, 65.13; H, 7.46; N, 27.50. 4 ((5 Phenyl 2H tetrazol 2 yl)methyl)morpholine ( 4.11f ). The reaction was carried out in water and the product was obtained as colorless plates (75%) ; mp 57.0 61.0 1 H NMR (300 MHz, CDCl 3 ) 8.18 8.15 (m, 2H), 7.51 7.48 (m, 3H), 5.50 (s, 2H), 3.71 (t, J = 4.7 Hz, 4H), 2.71 (t, J = 4.7 Hz, 4H). 13 C NMR (75 MHz, CDCl 3 ) 165.1, 130.5, 129.0, 127.5, 127.1, 74.2, 66.8, 50.0. Anal. Calcd. for C 12 H 15 N 5 O (245.29): C, 58.76; H, 6.16; N, 28.55. Found: C, 58.91; H, 6.24; N, 28.63.

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86 X ray experimental for 4.11f D ata were collected with a APEX II CCD area detector, using graphite monochromatised [1990AXA467] and refined on F 2 using all data by full matrix least squares procedures with SHELXL 97 [ G. M. Sheldrick, SHELXL 97 University of Gttingen, 1997 ] Hydrogen atoms were included in calculated positions with isotropic displacement parameters 1.3 times the isotropic equivale nt of their carrier atoms. Crystal data and structure refinement for 4.11f Identification code 3mao Empirical formula C12 H15 N5 O Formula weight 245.29 Temperature 108(2) K Wavelength 0.71073 Crystal system Monoclinic Space group P 21/c Unit cell dimensions a = 19.7973(19) = 90. b = 5.0600(4) = 100.964(4). c = 12.4821(11) = 90. Volume 1227.56(19) 3 Z 4 Density (calculated) 1.327 Mg/m 3 Absorption coefficient 0.091 mm 1 F(000) 520

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87 Crystal size 0.45 x 0.20 x 0.02 mm 3 Theta range for data collection 3.14 to 25.04. Index ranges 23<=h<=23, 4<=k<=6, 12<=l<=14 Reflections collected 10395 Independent reflections 2159 [R(int) = 0.0832] Completeness to theta = 25.04 99.9 % Absorption correction Semi empirical from equivalents Max. a nd min. transmission 1.000 and 0.842 Refinement method Full matrix least squares on F 2 Data / restraints / parameters 2159 / 0 / 163 Goodness of fit on F 2 0.810 Final R indices [I>2sigma(I)] R1 = 0.0426, wR2 = 0.0762 R indices (all data) R1 = 0.1089, wR2 = 0.0875 Largest diff. peak and hole 0.194 and 0.218 e. 3 1 ((5 Phenyl 2H tetrazol 2 yl)methyl)piperidine ( 4.11g ). The product was obtained as white prisms after recrystallization from DCM/hexanes (74 %) ; mp 67.0 1 H NMR (500 MHz, CDCl 3 ) 8.18 8.16 (m, 2H), 7.51 7.46 (m, 3H), 5.50 (s, 2H), 2.65 (t, J = 5.4 Hz, 4H), 1.62 1.57 (m, 4H), 1.36 1.33 (m, 2H). 13 C NMR (125 MHz, CDCl 3 ) 164.7, 130.3, 129.0, 127.7, 127.0 126.9, 75.2, 51.0, 25.9, 23.5. Anal. Calcd. for C 13 H 17 N 5 (243.31): C, 64.17; H, 7.04; N, 28.78. Found: C, 64.08; H, 7.15; N, 28.70. 4 ((5 (4 Nitrophenyl) 2H tetrazol 2 yl)methyl)morpholine ( 4.11h ). The product was crystallized from DCM/hexanes to give yellow microcrystals (86%) ; mp 133.0 1 H NMR (300 MHz, CDCl 3 ) 8.36 (s, 4H), 5.55 (s, 2H), 3.72 (t, J = 4.7 Hz, 4H), 2.72 (t, J = 4.7 Hz,

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88 4H). 13 C NMR (75 MHz, CDCl 3 ) 163.3, 149.1, 133.5, 12 7.9, 124.4, 74.7, 66.8, 50.0. Anal. Calcd. for C 12 H 14 N 6 O 3 (290.28): C, 49.65; H, 4.86; N, 28.95. Found: C, 49.87; H, 4.48; N, 28.91. 4 .4. 5 Preparation of N h ydroxymethyl s accharin (4.12). A mixture of 1,2 benzisothiazol 3(2 H ) one, 1,1 dioxide ( 4.10b ) (2.5 g, 13.5 mmol) and formalin (2.5 mL, 34 mmol) in water (10 mL) was heated under reflux for 10 min. The solid pricipitated on cooling was filtered, washed with cold water, and dried to give N hydroxymethyl saccharine ( 4.12 ) (2.73 g, 95%) ; mp 139.0 141 .0 [2007SC767]. 1 H NMR (300 MHz, DMSO d 6) 8.30 8.27 (m, 1H), 8.16 7.92 (m, 3H), 5.17 (s, 2H), 4.60 (s, 1H). 13 C NMR (75 MHz, DMSO d 6) 158.5, 137.2, 136.2, 135.3, 125.3, 124.9, 121.5, 62.8. 4 .4. 6 Preparation of N c hloromethyl s accharin (4.13). A mixture of N hydroxymethyl saccharin ( 6.7 ) (1.07 g, 5 mmol) and thionyl chloride (2 mL) was heated under reflux for 0.5 h. Excess of thionyl chloride was distilled off and the residue was dried under vacuum to give 6.8 ( 1.2 g, 99%) ; mp 142.0 144.0 [1959JPC150]. 1 H NMR (300 MHz, CDCl 3 ) 8.15 8.12 (m, 1H), 7.99 7.86 (m, 3H), 5.58 (s, 2H). 13 C NMR (75 MHz, CDCl 3 ) 157.7, 137.9, 135.8, 134.9, 126.7, 126.0, 121.5, 45.5. 4 .4. 7 Preparation of N ((1 H tetrazol 1 yl)methyl) 1,2 benzisothiazole 3(2 H ) one 1,1 dioxide (4.11b). Equimolar amounts of N chloromethylsaccharin ( 4.13 ) (0.46 g, 2 mmol), potassium carbonate (0.28 g, 2 mmol), sodium iodide (0.3 g, 2 mmol), and 1 H tetrazole ( 4.9a ) (4.4 mL, 2 mmol) were added to dry acetone (5 mL) and stirred overnight. The mixture was diluted with methylene chloride (50 mL) and filtered. The filtrate was extracted with of 0.5% NaOH solution (10 mL). T he organic phase was collected, dried over anhydrous MgSO 4 filtered and the solvent evaporated to give 4.11b as yellow crystals

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89 that turned white upon washing with methylene chloride (60%); mp 177.0 179.0 tautomers: 1 H NMR (500 MHz, DMSO d6 ) 9.5 9 (s, 0.5H from B), 9.09 (s, 0.1H from A), 8.39 8.37 (m, 1H), 8.22 8.18 (m, 1H), 8.12 8.09 (m, 1H), 8.07 8.03 (m, 1H), 6.71 (s, 0.5H from A), 6.56 (s, 2H from B). Two tautomers: 13 C NMR (75 MHz, DMSO d6 ) 158.0, 153.9 (C5 from B), 144.7 (C5 from A), 136.6 135.6, 125.7, 125.6, 121.9, 48.4. Anal. Calcd. for C 9 H 7 N 5 O 3 S.1/2 H 2 O (283.27): C, 39.41; H, 2.94; N, 25.54. Found: C, 39.37; H, 2.80; N, 24.60.

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90 CHAPTER 5 CONFORMATIONAL EQUIL IBRIA AND BARRIERS T O ROTATION IN SOME NOV EL NITROSO DERIVATIVES OF INDOLIZINES AND 3 AND 5 AZAINDOLIZINES AN NMR AND MOLECULAR MO DELLING STUDY 1 5.1 Introduction In connection with the synthesis of a series of heterocyclic derivatives, some C nitroso heterocycles, namely ind olizines 5.1 5. 4 pyrrolo[1,2 b ]pyridazine 5. 5 and 3 nitroso pyrazolo [1,5 a ]pyridine 5. 6 (Figure 5 1), were prepared as intermediates. Figure 5 1. C Nitroso derivatives of indolizines and 3 and 5 azaindolizines taken into study The 1 H NMR spectra of some of these compounds, 5. 1 5. 3 and 5. 5 displayed in certain conditions two sets of signals, characteristic for two compounds in exchange. In principle, these sets of signals could arise from either of two isomeric azodioxy dimers (Figur e 5 2) or include one or both of the two rotamers arising from restricted rotation about the C NO bond (Figure 5 3). Figure 5 2. Monomer dimer equilibria in heteroaromatic nitroso compounds 1 Org. Biomol. Chem. 2010 8 3518 3527 Reproduced in part by permission of the Royal Society of Chemistry

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91 Figure 5 3. Rotamer equilibrium in 5. 1 Spectroscopic methods can differentiate monomers from dimers. Most nitroso m onomers are green blue, due to a n transition around 750 nm, while most nitroso dimers are colorless [2007JCP094102]. The N=O stretching frequency in the IR spectra of nitrosobenzenes is at 1485 1515 cm 1 in the monomer, 1250 1300 cm 1 in the E dimer, w hile the Z dimer has two bands in the region 1350 1400 cm 1 [1996JCS(P2)191, 1992JCS(P2)243, 2000JCS(P2)2280]. Monomers and dimers are also characterized by the 13 C chemical shift of the carbon bearing the nitroso group, the substituent chemical shift (SCS ) on C ipso in nitrosobenzenes being ca 25 30 ppm downfield in the monomers than in the dimers [1996JCS(P2)191, 1994CJC514, 1999JCM202, 2001JCS(P2)1904]. Monomer syn and anti rotamers are distinguished by the 13 C chemical shifts of the carbons gamma to the oxygen. The carbon syn to the oxygen would be shielded by 25 35 ppm compared to the one anti gamma 1976JCS(P2)1791]. The monomer dimer ratio depends on conditions and on structural features Dimers are favored in solid state or at lower temperatures in solution. A high double bond character of the C NO bond favours the monomer. Electron donating substituents in the para position in nitrosobenzenes stabilize the monomer: 4 alkyl or 4 di alky laminonitrosobenzenes exist as monomers in all conditions; 4 methoxynitrosobenzene is thought to dimerize to a very small extent in concentrated solutions.

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92 Nitrosobenzene itself is an equilibrium mixture of the monomer and the two dimers in solution, in wh ich the monomer predominates. 2 Nitrosopyridines display the same equilibrium, but here the dimer dominates [2000JCS(P2)2280]. Methyl or chlorine substituents in both of the 2 and 6 positions in nitrosobenzenes tilt the equilibrium from monomers towards th e E dimers, presumably by hindering the coplanarity of the nitroso group and the benzene ring. Larger substituents destabilize the dimer. The interplay of steric and electronic factors similarly affects the barrier to rotation of the monomer about the C NO bond; G # values are good predictors for the tendency of nitroso compounds to dimerize [2001JCS(P2)1904]. Although aromatic nitroso compounds have been known since the early days of organic chemistry, heterocyclic nitroso compounds are relatively rare. A Beilstein search disclosed seven 1 nitrosoindolizines similar to 5. 1 5. 3 and fourty seven 3 nitrosoindoliz ines similar to 5. 4 but no reports on the conformation of these compounds was found, nor were any 13 C NMR data uncovered. A search for the 3 nitrosopyrrole substructure of 5. 1 5. 3 produced 118 hits, and 85 hits were found for the 2 nitrosopyrrole substruc ture of 5. 4. The single conformational study found, was theoretical and dealt with the conformation of the parent compounds, 2 and 3 nitrosopyrrole [1979NJC473]. We found no 5 nitrosopyrrolo[1,2 b ]pyridazine, of type 5. 5 There are 14 examples of 3 nitro sopyrazolo[1,5 a ]pyridines like 5. 6 out of which only one has 13 C NMR data [1994AJC1009]. There are three hundred and ninety three 4 nitrosopyrazoles (they have been known for more than 100 years), but only a single conformational study [1997JCS( P2)721, 1996JCS(P2)2271]. 1,3,5 Trisubstituted 4 nitrosopyrazoles are mixtures of syn and anti rotamers; with identical substituents in positions 3 and 5, the dominant rotamer had the nitroso oxygen anti to

PAGE 93

93 N1. NMR and X ray data confirmed that the shield ing anisotropy of the nitroso group in 4 nitrosopyrazoles is similar to that in nitrosobenzenes [1997JCS(P2)721]. Nitroso derivatives of electron rich heterocycles are not expected to dimerize, however the dimer of 3,5 dimethyl 4 nitrosopyrazole was recen tly prepared in solid state. Upon heating its ethanol solution, this dimer dissociates into monomer [2007TL5547]. No other dimers of nitrosopy r roles, nitrosopyrazoles, or nitrosoimidazoles were found. The lack of data on the dimerization and conformational equilibria of nitroso derivatives of indolizines, pyrrolo[1,2 b ]pyridazine and pyrazolo[1,5 a ]pyridines (and the paucity of data on related heterocycles) was the real motivation to study compounds 5.1 5. 6 5.2 Results and Disscussion 5.2.1 NMR S pectroscopy The 1 H and 13 C chemical shifts in the isomers of these compounds were assigned based on the 1 H 13 C correlations seen in the gHMBC and gHMQC spectra, and are given in Tables 5 1 and 5 2. For the purpose of consistency, the same position numberin g as in indolizine (Figure 5 1) was used for all of the compounds. 5.2.1.1 2,3 Dimethyl 1 nitrosoindolizine (5.1) Compound 5. 1 displayed in the proton spectrum in acetone d6 at 65 C the signals for two isomers, the molar fraction of the major being 0.62. Of the two methyl protons of the major isomers, at 2.74 and 2.45 ppm, the former displays cross peaks with three aromatic carbons at 154.5, 126.7 and 123.4 ppm, therefore it is the methyl in position 2. The la st two carbons couple with 2.45, and are in po sitions 2 and 3, leaving 154.5 for position 1. The carbon at 123.4 couples with the doublet at 8.37, therefore it is in position 3, and 8.37 is the proton in position 5. The proton at 8.37 is directly attached to the carbon at 125.0 and coup les with both c arbons at 119.7 and 135.4. The lat t er carries the triplet at 7.83, therefore they are in positions 8a and 7,

PAGE 94

94 respectively. The remaining signals of the major, the doublet at 8.25 and the triplet at 7.36 have been assigned to positions 8 and 6, correspondingly. The signals in the minor have been assigned with confidence in a similar way. The 13 C chemical shifts o f C1 indicate that both isomers of 5. 1 are monomers. In the related 3,5 dimethyl 4 nitrosopyrazole the chemical shift of C4 is 161.0 in the monomer [1996JCS(P2)2271] (SCS = 56.2 ppm) and 132.6 ppm in the dimer [2007TL5547] (SCS = 27.8 ppm). The chemical s hift of C1 in indolizine being 99.5 ppm [1980JST15], the calculated values for 1 nitrosoindolizine are 155.7 in the monomer and 127.3 in the dimer. The chemical shifts of C1 in the two isomers of 5. 1 are 154.3 and 154.5, clearly indicating that they are bo th monomers, evidently rotamers about the C1 NO bond in the monomer (Figure 5 3). The two rotamers will be called syn and anti through out this chapter, referring to the orientation of the nitroso oxygen relative to N4. The rotamers of 5. 1 have been identi fied on the basis of the chemical shifts of C2 and C8a. The chemical shift differences between isomers, 17.3 ppm in position 2 and 23.3 ppm in position 8a, are similar to those seen for rotamers of 1,3,5 trimethyl 4 nitrosopyrazole, for which an NMR and X ray study demonstrated that the carbon gamma and syn to the oxygen is shielded [1997JCS(P2)721]. In the major isomer of 5. 1 in acetone d6 at 65 C, C8a is at 119.7 ppm (Table 5 2), while in the minor it is at 143.0 ppm, therefore in the major the nitroso oxygen and the indolizine nitrogen are syn Chemical shifts of C2 in the two isomers are in agree ment with this assignment. Nitroso dimers do not display this type of isomerism [1994CJC514]. The proton chemical shifts (Table 5 1) in general agree with ea rlier studies [2001JCS(P2)1904, 1997JCS(P2)2201] which found large differences, up to 3 ppm, between the

PAGE 95

95 protons syn and anti to the oxygen in the nitroso group, the former being more shielded. The only exception is compound 5. 4 in which H5 is more shielded in the anti conformer. Table 5 1. 1 H chemical shifts in compounds 5. 1 5. 9 \ Position Compd. \ 1 2 3 5 6 7 8 5.1 syn a 2.74 b 2.45 b 8.37 7.36 7.83 8.25 5.1 anti a 2.14 b 2.36 b 8.45 7.37 7.78 8.56 5.2 syn c 3.16 b 3.98 b 9.70 7.47 7.95 8.33 5.2 anti c 2.41 b 3.92 b 9.68 7.47 7.95 8.87 5.2 syn d 3.21 b 3.40 b 9.29 5.94 6.55 8.24 5.2 anti d 2.55 b 3.26 b 9.29 5.94 6.55 8.49 5.3 syn a d 3.28 b 8.28 e 1.03 b 4.07 f 8.29 6.12 6.69 8.67 5.3 syn b d 3.40 b 6.86 e 1.06 b 4.12 f 9.48 6.10 6.65 8.63 5.3 syn a a 3.55 b 8.39 e 1.40 b 4.38 f 9.21 7.4 7.80 8.52 5.3 syn b a 3.48 b 7.87 e 1.34 b 4.39 f 9.71 7.46 7.84 8.53 5.4 syn g 5.84 2.72 b 10.07 6.14 6.60 6.65 5.4 anti g 5.70 2.22 b 9.21 6.10 6.62 6.65 5.5 syn d 2.85 b 2.01 b 1.87 b 5.87 7.93 5.5 anti d 2.22 b 1.89 b 1.95 b 5.87 8.36 5.5 syn h 2.77 b 2.31 b 2.46 b 7.36 7.95 5.5 anti h 2.04 b 2.22 b 2.55 b 7.42 8.75 5.6 anti a 7.76 8.69 7.47 7.65 4.25 b 5.6 anti i 7.72 8.58 7.31 7.53 4.14 b 5.7 h 6.28 2.23 b 2.39 b 2.39 b 6.41 7.67 5.8 i 6.57 7.87 8.23 6.77 6.58 3.92 b 5.9 j 6.24 2.33 b 7.10 7.80 6.37 6.58 7.25 a In acetone d6 at 65 C. b M ethyl. c I n acetone d6 at 0 C. d I n toluene d8 at 65 C. e NH. f M ethylene. g I n toluene d8 at 25 C. h I n DMSO d6 at 25 C. i I n DMSO d6 at 70 C. j I n chloroform d at 25 C. 5.2.1.2 2,6,7 Trimethyl 5 nitrosopyrrolo[1,2 b ]pyridazine (5.5) Compound 5. 5 displayed the signals for two isomers in equal amounts in the proton spectrum in DMSO d6 at 25 C. In acetone d6 at 65 C, the molar fraction of the major isomer was 0.58. The assignment of 1 H and 13 C chemical shifts in compound 5. 5 followed the same procedure as for 5. 1 up to the point where the lack of a proton in position 5 made the discrimination between C2 an d C3 impossible. The syn and anti rotamers were identified by the

PAGE 96

96 chemical shifts in position 8, and then C2 and C5 were assigned based on the chemical shift trends seen in 5. 1 The chemical shift differences between the rotamers of 5. 5 19.3 ppm in positi on 2 and 24.5 ppm in position 8a, are very similar to the ones found in 5. 1 The chemical shifts of the methyl carbons in positions 2 and 3 are practically the same in compounds 5. 1 and 5 .5 5.2.1.3 Methyl 2 methyl 1 nitrosoindolizine 3 carboxylate (5.2) Proton spectra of compound 5. 2 were recorded in 5 C increments in acetone d6 in the interval 65.0 45.0 C and in toluene d8 in the interval 65.0 75.0 C. In both solvents, at 65.0 C, there were two isomers in the mixture, the molar fraction of the major being 0.95. As the temperature increased, the signals of the minor which did not overlap the signals for the same position in the major broadened and then disappeared. The signals for the same position in the major broadened then became sharp again. 13 C chemical shifts were measured for the major isomer only, due to limited solubility and dynamic range, in acetone d6 at 0 C and in toluene d8 at 6 5 C. Of the two doublets in 5. 2 only one coupled in the gHMBC spectrum with a quaternary carbon (C8a), and was assigned as H5. H5 also coupled with an aromatic carbon bearing a proton, which identified C7 and H7. The remaining doublet and triplet were a ssigned to H8 and H6, correspondingly. The methyl protons at 3.98 ppm, coupled with only one carbon which had the chemical shift of an ester, therefore this is the methoxy methyl. The methyl protons at 3.16 ppm coupled with three carbons, at 154.7, 140.9 a nd 114.8 ppm. None of these carbons displayed any cross peak with H5 or H8, and they had to be assigned based on chemical shift values. The chemical shift of C8 in the major conformer of 5. 2 is 3.2 ppm higher than that in 5. 1 syn and 22.1 ppm lower than in 5. 1 anti therefore the major conformer of 5. 2 is syn 154.7 is

PAGE 97

97 basically identical to the chemical shift of C1 in 5. 1 syn and was assigned as such. The methoxycarbonyl group is expected to produce shielding of C3 and deshielding of C2 in 5. 2 as compar ed to 5. 1 so 140.9 was assigned to C2 and 114.8 to C3. Conjugation of the carboxyl group with the electron donor heterocycle could raise barriers for the rotation about the C3 COOMe bond enough to observe two distinct rotamers. Since the 13 C chemical shi fts in the minor could not be measured, the question arises if the two rotamers observed are due to restricted rotation about the C1 NO bond, or about the C3 COOMe bond. Proton chemical shift differences between rotamers, in positions 5 and 8, demonstrate that they are the syn and anti orientations of the nitroso group. The difference in position 8, 0.54 ppm, is comparable with that found for the syn anti pair of 5. 1 0.31 ppm. The difference in position 5 is 0.02 ppm for 5. 2 and 0.08 ppm for 5. 1 The simi larity of the chemical shifts of H5 in both isomers of 5. 2 with that in 5. 3B ( Figure 5 4 ) indicates that in both isomers of 5. 2 the carbonyl oxygen and the indolizine nitrogen are syn 5.2.1.4 Ethyl 2 (methylamino) 1 nitrosoindolizine 3 carboxylate (5.3) Compound 5. 3 displayed in the proton spectrum in toluene d8 at 65 C the signals for two isomers; the molar fraction of the major was 0.52. The methyl protons in the major, doublet at 3.28, coupled with a quaternary carbon at 151.9, assigned as C2. The NH quartet coupled with two carbons, at 150.6 and 99.7 ppm, which were assigned as C1 and C3 respectively, based on their chemical shifts. C3 coupled with the doublet at 8.29, which was assigned as H5. H5 coupled with a quaternary carbon at 123.5, assigned a s C8a, and with a carbon at 133.7, which caries the triplet at 6.69, assigned to position 7. The remaining triplet, at 6.12, and doublet, at 8.67, were assigned to H6 and H8. The assignment of the chemical shifts in the minor parallels the one in the major

PAGE 98

98 Table 5 2. 13 C chemical shifts in compounds 5.1 5. 9 \ Position Compd. \ 1 2 3 5 6 7 8 8a Other 5.1 syn a 154.5 126.7 123.4 125.0 118.4 135.4 118.2 119.7 8.7 (CH 3 2); 8.6 (CH 3 3) 5.1 anti a 154.3 109.4 125.0 126.2 117.0 129.8 115.9 143.0 10.9 (CH 3 2); 7.6 (CH 3 3) 5.2 syn b 154.7 140.9 114.8 128.5 119.4 136.7 117.8 122.9 10.9 (CH 3 2); 51.9 (CH 3 O); 162.5 (C=O) 5.2 syn c 154.9 141.1 113.8 127.4 117.6 134.8 118.0 122.5 11.7 (CH 3 2); 51.1 (CH 3 O); 162.5 (C=O) 5.3 syn a c 150.6 151.9 99.7 128.1 118.2 133.7 117.3 123.5 34.1 (CH 3 2); 14.6 (CH 3 3); 60.1 (CH 2 O); 162.0 (C=O) 5.3 syn b c 150.8 149.2 99.4 127.5 118.6 134.2 116.9 124.4 34.5 (CH 3 2); 14.9 (CH 3 3); 59.8 (CH 2 O); 161.3 (C=O) 5.3 syn a a NM d NM NM 129.6 120.3 135.5 116.8 NM 34.3 (CH 3 2); 14.3 (CH 3 3); 60.5 (CH 2 O); 161.4 (C=O) 5.3 syn b a NM NM NM 128.3 120.3 135.6 116.8 NM 34.9 (CH 3 2); 14.8 (CH 3 3); 59.8 (CH 2 O); 161.4 (C=O) 5.4 syn e 107.3 141.6 153.5 125.0 117.4 130.8 117.0 136.3 11.7 (CH 3 2) 5.4 anti e 107.5 118.1 159.2 121.8 114.3 129.1 117.8 138.0 13.8 (CH 3 2) 5.5 syn c 153.1 126.4 f 125.5 f 153.4 123.7 126.4 110.8 9.0 (CH 3 2); 8.5 (CH 3 3); 21.2 (CH 3 6) 5.5 anti c 152.8 106.7 127.4 152.8 119.5 124.2 135.3 11.0 (CH 3 2); 7.6 (CH 3 3); 21.2 (CH 3 6) 5.5 syn g 152.3 125.8 125.8 154.7 125.8 125.8 113.3 8.5 (CH 3 2); 8.3 (CH 3 3); 21.3 (CH 3 6) 5.5 anti g 151.9 106.2 128.0 154.5 121.7 124.1 135.5 10.2 (CH 3 2); 7.4 (CH 3 3): 21.3 (CH 3 6) 5.6 a 158.0 124.5 123.2 117.0 111.2 153.1 135.1 57.6 (CH 3 O 8) 5.6 h 158.3 126.3 123.8 117.4 112.5 153.2 134.7 57.7 (CH 3 O 8) 5.7 g 100.0 120.6 122.4 149.4 110.0 126.4 124.3 12.4 (CH 3 2); 9.6 (CH 3 3); 22.3 (CH 3 6) SCS syn i 52.3 5.2 3.4 5.3 15.8 0.6 11 SCS anti j 51.9 14.4 5.6 5.1 11.7 2.3 11.2 5.8 h 95.4 141.2 122.3 112.5 100.7 151.2 135.2 56.5 (CH 3 O 8) SCS k 62.9 14.9 1.5 4.9 11.8 2 0.5 5.9 l 100.1 125.1 111.4 125.1 109.7 116.8 118.5 133.1 12.8 (CH 3 2) SCS syn m 7.2 16.5 42.1 0.1 7.7 14 1.5 3.2 SCS ant i n 7.4 7 47.8 3.3 4.6 12.3 0.7 4.9 a In acetone d6 at 65 C. b I n acetone d6 at 0 C. c I n toluene d8 at 65 C. d N ot measured. e I n toluene d8 at 25 C. f I nterchangeable. g I n DMSO d6 at 25 C. h I n DMSO d6 at 70 C. i ( 5. 5 syn ) ( 5. 7 ). j ( 5. 5 anti ) ( 7.7 ). k ( 5. 6 ) (5. 8 ). l I n chloroform d at 25 C. m ( 5. 4 syn ) ( 5. 9 ). n ( 5. 4 anti ) ( 5. 9 ).

PAGE 99

99 The chemical shift of C8a indicates that in both of the isomers of 5. 3 the indolizine nitrogen and the nitroso oxygen are syn This is confirmed by the chemical shift of H8 which is basically the same for the two isomers. Large chemical shifts differences between isomers are seen for the NH protons in position 2 and for H5. The isomers of 5. 3 are two of the four rotamers arising from the restricted rotation about the C2 N and C3 COOEt bonds (Figure 5 4). Figure 5 4. Possible conformations for 5. 3 relevant proton chemical shifts, and nOes The chemical shift of the NH proton in the major, 8.28 ppm, suggests a hydrogen bond with the carbonyl group, as in 5. 3A This is confirmed by the nOe between the CH 2 O protons and H5 The chemical shifts of H5 in the major (8.29 ppm) and in the minor (9.48 ppm) indicate that they have different orientations of the ethoxycarbonyl group; of 5. 3B and 5. 3D the former agrees with the nOe between the NH and the CH 2 O protons, seen in the mi nor (Figure 5 5). To our knowledge, this is the first NMR report to observ e rotamers arising from restricted rotation about the C COOR bond in pyrrole carboxylates. Such rotamers of 2 and 3 pyrrolecarboxylates have been demonstrated by IR spectroscopy [19 80JCS(P2)737, 1980JCS(P2)1631]. The intramolecular hydrogen bond in 5. 3A is confirmed by the shift of the molar fraction of the isomer with the deshielded NH from 0.52 to 0.35 when spectra were taken in acetone d6 at 65 C. The hydrogen bond acceptor solv ent competes with the carbonyl group for the hydrogen bond donor, the NH. The chemical shift of the NH in 5. 3A in toluene d8 (8.28 ppm) is close to the one in acetone d6 (8.39 ppm), as it is determined mostly by the strength of the hydrogen

PAGE 100

100 bond. The chemical shift of the NH in 5. 3B in toluene d8 is 6.86 ppm, while in acetone d6 is 7.87 ppm, because of the hydrogen bonding with the solvent. Figure 5 5. Expansion of the NOESY spectrum of 5. 3 5.2.1.5 4 Methoxy 3 nitrosopyrazolo[1,5 a ]pyridi ne (5.6) Coupling with the methoxy protons in 5. 6 identifies C8. The triplet at 7.47 is H6, and the singlet at 7.76 is H2. The remaining doublets, at 8.69 and 7.65 ppm, have been assigned to positions 5 and 7 correspondingly, based on the chemical shifts o f the carbons that carry them. Both of these protons couple with a quaternary carbon at 135.1 ; this carbon couples with H2 and was assigned to position 8a. C8a also couples with H2. The remaining quaternary carbon which couples with H2 is C1. A single set of signals was detected in the proton spectrum of 5. 6 both in DMSO d6 at 70 C and in acetone d6 at 65 C, however, in a series of spectra in acetone d6 in which the temperature was increased from 65 C to 45 C in steps of 5 C, the signal of H2 broadened between 45 C and 15 C, then sharpened again between 15 C and 10 C. This indicates that at temperatures above 45 C a minor conformer is presen t in exchange with the major, and that the exchange becomes fast on the NMR time scale at temperatures above 10 C. Comparison of

PAGE 101

101 the 13 C chemical shifts of 5. 6 between acetone d6 at 65 C and DMSO d6 at 70 C indicates that the composition of the rotamer ic mixture is about the same. The largest chemical shift difference is in position 2, ca. 2 ppm, which a ccounts for 10 % of the minor at 70 C, considering that the chemical shift difference in position 2 is ca. 20 ppm between the syn and anti rotamers. T he major rotamer of 5. 6 was identified by the SCSs for the nitroso group, inferred from the parent compounds of 5. 5 and 5. 6 5. 7 and 5. 8 correspondingly (Figure 5 6). While 5. 7 was available pure, 5. 8 occurred as a 10 % impurity in the sample of 5. 6 Limi ted solubility precluded the measurement of the 13 C chemical shifts for 5. 8 in acetone at 65 C ; therefore they were measured in DMSO d6 at 70 C, where the major rotamer of 5. 6 was in exchange with ca. 10 % of the minor. The SCS in position 2 indicates that the major rotamer of 5. 6 is anti The SCS in position 8a however, is neutral. This is due perhaps to the influence of the conformational equilibrium of the methoxy group on the chemical shift of C8a; this equilibrium is not the same in 5. 6 and in 5. 8 This may also be the explanation why the SCS in position 1 is 11 ppm larger than expected. Figure 5 6. Parent compounds for 5.5 and 5.6 5.2.1.6 2 Methyl 3 nitrosoindolizine (5.4) The proton spectrum of 5. 4 in toluene d8 at 25 C displayed two sets of signals, the molar fraction of the major isomer being 0.92. The barrier to rotation was quite high, since selective irradiation for 10 s did not display the interconversion of isomers at room temperature but at 80 C.

PAGE 102

102 The methyl protons at 2.72 ppm couple with three carbons, at 107.3, 141.6 and 153.5. The former caries the proton at 5.84 ppm and was assigned to position 1. The la t ter couples with the proton at 10.07 ppm, therefore they were assigned as C 3 and H5. C8a and C7 were identified by their coupling with H5. Figure 5 7. Parent compound of 5.4 The rotamers of 5. 4 were identified by the SCSs for the nitroso group, inferred from 5. 9 the parent compound of 5. 4 (Figure 5 7). The 13 C SCS in position 2, 16.5 ppm, indicates that the major rotamer of 5. 4 is syn The chemical shift of N4 is also 17 ppm lower in 5. 4 as compared to 5. 9 as expected for the syn conformer. 5.2.2 15 N C hemical S hifts The 15 N chemical shifts, given in Table 5 3, were measured in 1 H 15 N CIGAR gHMBC spectra acquired with a pulse sequence optimized for 15 N [2003MRC307]. Unfortunately, the chemical shifts for the nitroso group could not be determined, because these compounds do n ot have any protons within two or three bonds from the nitroso nitrogen. Table 5 3. 1 5 N chemical shifts in compounds 5.1 5.7 and 5.9 Compd. N 4 Other 5.1 syn a 200.0 5.1 anti a 200.6 5.2 syn b 195.2 5.3 b 194.0 75.9 (NHCH 3 ) 5.4 syn c 174.4 5.5 syn a 230.1 302.7 (N5) 5.5 anti a 230.4 300.8 (N5) 5.6 anti b 242.9 289.1 (N3) 5.7 a 221.6 295.8 (N5) 5.9 d 191.3 a In DMSO d6 at 25 C. b In DMSO d6 at 70 C. c In toluene d8 at 25 C. d In chloroform d at 25 C.

PAGE 103

103 5.2.3 Barriers to R otation The barriers to rotation were measured by variable temperature NMR in toluene d8 The exchange rates were determined by line shape simulation in gNMR [2005MI 8 ] The free en ergies of activation at 25 C ( G # 298 ) for the process syn anti in compounds 5. 1 5. 2 5. 4 and 5. 5 have been calculated from the enthalpy and entropy of activation and are 16.6, 15.6, 24.0 and 15.7 kcal/mol, correspondingly. These values are significantly larger than those found in nitrosobenzene (8.2 kcal/mol), 4 methoxynitrosobenene (9.8 kcal /mol) and 4 dimethylaminonitrosobenzene (12.6 kcal/mol) [1998JCS(P2)797]. It was demonstrated that in 4 substituted nitrosobenzenes, the barrier to rotation increases with the p donor capability of the substituent [1998JCS(P2)797]. Smaller values for compo unds 5. 2 and 5. 5 compared to 5. 1 are due to a less electron donating pyrrole moiety in the former two. The barrier for the process 5. 3B 5. 3A in toluene d8 is 14.4 kcal/mol, significantly larger than the barrier for the carboxylate rotation in methyl benzoate, 5.0kcal/mol [2000JOC1552], methyl 4 methylamino 3 nitrobenzoate, 6.3 kcal/mol [1982JOC3759], methyl 8 isopropyl 1 napht h oate, 8.9 kcal/mol [1975 CC942] ethyl 7 ethyloxepine 2 ca rboxylate, 6 8 kcal/mol [1986 CC970], or methyl 2 dimethylamino 3 nitrothiophene 1 carboxylate, 7.7 kcal/mol [1982JOC3759]. 5.2.4 Molecular M odelling Calculations were performed using the MM+ and the ab initio methods, as implemen ted in the HyperChem program. Ab initio calculations for compounds 5. 1 and 5. 3 were run in Gaussian 03. First, a conformational search was run using the MM+ method, retaining all conformations within 10 kcal/mol from the minimum. The energy of these confor mers was also calculated with the a b initio method with the 6 31 g(d,p) basis set.

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104 The eight conformations for compound 5. 1 are depicted in Figure 5 8, and their energies are given in Table 5 4. The calculated molar fraction of the syn conformer is 0.55 by MM+ and 0.43 by ab initio vs 0.62 experimental. Both methods indicate that the conformations in which a hydrogen of the methyl group in population. In the significant conformations, the methyl groups are either geared ( 5. 1a and 5. 1a' ) or face to face ( 5. 1b and 5. 1b' ). In the geared conformations, one of the hydrogens of the methyl group in position 2 is facing the NO group and it is sterically more demanding than H8. Both methods found 5. 1a more stable than 5. 1a'. In the face to face arrangement of the methyl groups, the methyl in position 2 presents its back to the NO group, and it is sterically less demanding than H8. Both methods found 5. 1b' more stable th an 5. 1b Assuming no interaction between the lone pair on the nitroso nitrogen and the methyl in position 2, the face to face arrangement of the methyls is less stable than the geared one ( 5. 1b 5. 1a ) by 0.66 kcal/mol in MM+ and by 0.18 kcal/mol in ab initi o The difference between the syn and anti orientations of the nitroso group is 0.35 kcal/mol ( MM+ ) or 0.78 kcal/mol ( ab initio ) for geared methyls ( 5. 1a' 5. 1a ) and 0.39 kcal/mol ( MM+ ) or 0.29 kcal/mol ( ab initio ) for the face to face arrangement ( 5. 1b' 5. 1b ). In conclusion, the conformational preferences of 5. 1 are dominated by steric effects; the strongest is the interaction between the methyl group in position 3 and H5, which leaves the former with a hydrogen facing the methyl group in position 2. The energy difference for the interaction of the two methyl groups in the face to face or geared conformations is comparable to the energy difference of the two orientations of the nitroso group.

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105 Figure 5 8. Conformers of 5.1 Table 5 4. Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5. 1 Conformer MM+ x Ab initio x 5.1a 0.00 0.43 0.47 0.17 5.1b 0.66 0.09 0.29 0.26 5.1c 1.47 0.01 2.69 0.00 5.1d 1.35 0.02 2.79 0.00 5.1a' 0.35 0.18 1.25 0.03 5.1b' 0.27 0.22 0.00 0.53 5.1c' 0.93 0.05 2.20 0.00 5.1d' 2.62 0.00 3.35 0.00 A conformation search with MM+ found three conformers for compound 5. 5 (Figure 5 9). Their energies are given in Table 5 5. The calculated molar fraction of the syn conformer is 0.68, vs. 0.58 experimental. The conformational preferences of 5. 5 are very similar to those of 5. 1 Compound 5.5 however misses conformations in which the methyl groups are face to face, because there is no need to minimize the interaction b etween the methyl in position 3 and H5. Figure 5 9. Conformers of 5.5

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106 Table 5 5. Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5.5 Conformer MM+ x Ab initio x 5.5a 0.00 0.53 0.08 0.43 5.5b 0.22 0.31 0.97 0.05 5.5c 0.52 0.15 0.00 0.52 Compounds 5. 1 and 5.5 which have different heterocycles but the same substituents in positions 2 and 3, display similar conformational equilibria, indicating that, in these cases, steric interactions of the methyl groups are more important than the electronic interactions bet ween the nitroso group and the heterocyclic frame. A conformation search with MM+ found four conformers for compound 5. 2 (Figure 5 10). Their energies are given in Table 5 6. The calculated molar fraction of the syn conformer is 0.81 by MM+ and 0.99 by ab initio vs 0.95 experimental. Figure 5 10. Conformers of 5.2 Table 5 6. Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5.2 Conformer MM+ x Ab initio x 5.2a 0.00 0.78 0.00 0.99 5.2b 0.60 0.18 2.04 0.01 5.2c 1.41 0.03 2.82 0 5.2d 1.94 0.01 4.66 0 The conformational preference for compound 5. 2 can also be explained by steric effects. The carboxyl group in the plane of the heterocycle forces the methyl in position 2 to have a

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107 hydrogen pointing towards the nitroso group in all of the conformers. As seen in 5. 1 and 5. 5 for this conformation of the methyl group, the syn conformer of the nitroso group is more stable. The molar fraction of the syn conformers in the equilibrium of 5. 3 was 0 .91 by MM+ and 1.00 by ab initio (Figure 5 11 and Table 5 7). The anti conformers in MM+ had the NMe group out of the plane of the heterocycle and the method does not account for the electronic destabilization of these conformers. Conformers of type 5. 3A i n Figure 5 4 and 5. 3d f in Figure 5 11, represent a fraction of 0.14 by MM+ and 0.20 by ab initio compared with 0.35 observed in acetone d6 The coplanarity of the carboxyl group and the heterocycle precludes conformations 5. 3C and 5. 3D in Figure 5 4, in which there is a strong repulsion between the methyl in position 2 and the carboxyl in position 3. In the accessible conformations of the methylamino group, the NH bond is in the plane of the heterocycle, with the hydrogen pointing towar ds the carboxyl. The methyl group pointing towards the nitroso is much bulkier than the hydrogen in 5. 2 therefore the anti orientation of the nitroso is not possible anymore. The NH group in 5. 3 appears to the carboxyl group bulkier than the methyl group in 5. 2 and comparable to H5, making the two orientations of the carboxyl of comparable energies. In addition, conformations of type 5. 3A in Figure 5 4, 5. 3d f in Figure 5 11, are stabilized by the hydrogen bond between the NH and the C=O. The energies of the conformers of 5. 6 calculated with MM+ (Figure 5 12 and Table 5 8) reproduce very well the fraction of the syn conformer found by NMR, 0.00 at 65 C and 0.10 at 70 C. The methoxy group appears to the nitroso larger than H2 and the anti isomer is fav ored by ca 2 kcal/mol.

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108 Figure 5 11. Conformers of 5.3 Table 5 7. Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5. 3 Conformer MM+ x Ab initio x 5.3a 0.00 0.34 0.00 0.47 5.3b 0.20 0.21 0.43 0.16 5.3c 0.20 0.21 0.43 0.16 5.3d 0.71 0.06 0.58 0.12 5.3e 0.88 0.04 0.97 0.04 5.3f 0.88 0.04 0.97 0.04 5.3a 0.93 0.04 5.14 0.00 5.3b 1.21 0.02 5.20 0.00 5.3c 1.21 0.02 5.20 0.00 5.3d 1.56 0.01 5.34 0.00 5.3e 1.77 0.00 5.59 0.00 5.3f 1.77 0.00 5.59 0.00

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109 Figure 5 12. Conformers of 5.6 Table 5 8 Calculated energy differences (kcal/mol) and molar fractions, at 65 C, for conformers of 5. 6 Conformer MM+ x Ab initio x 5.6a 0.00 0.79 0.00 0.72 5.6b 0.84 0.10 0.68 0.14 5.6c 0.84 0.10 0.68 0.14 5.6d 2.01 0.01 3.32 0.00 5.6e 2.70 0.00 3.10 0.00 5.6f 2.70 0.00 3.10 0.00 MM + calculations (Figure 5 13 and Table 5 9) predict that 5. 4 would be 82% anti as would be expected from the pattern of steric interactions seen in 5. 1 5. 2 and 5. 5 However, 13 C chemical shifts in position 2 indicate that 5. 4 is 92% syn This resembles the preference for the syn orientation of the carbonyl group s of 2 carboxy and 2 formyl pyrroles to the nitrogen (methylated or not), which was explained by an electrostatic attr action between the positive nitrogen and the negative carbonyl oxygen [1980JCS(P2)1631]. Ab initio calculations reflect the observed equilibrium better, and predict 82% syn conformer at 25 C. This is the only case in this study in which the two methods pr oduced a different order of the energy of the conformers. Figure 5 13. Conformers of 5.4

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110 Table 5 9. Calculated energy differences (kcal/mol) and molar fractions, at 25 C, for conformers of 5. 4 Conformer MM+ x Ab initio x 5.4a 0.00 0.82 1.15 0.18 5.4b 1.21 0.11 0.00 0.82 5.4c 1.43 0.07 Converged to 5. 4b In order to evaluate the size of the electronic effects, in particular the difference between conjugation of the nitroso or carbonyl group and the double bond on the pyrrole moiety in the s cis and s trans geometry, we considered model compounds in Figure 5 14, for which the ground states and Natural Bond Orbital Analysis (NBO) have been calculated at HF/6 31g(d,p) and B3LYP/6 31g(d,p) level respectively. The s trans geometry was found to be the lowest energy one for all of the compounds 5. 10 5. 12 (Table 5 10). Comparison with calculations in MM+ indicated that this stabilization is mostly steric in the case of 5. 10 and 5. 11 and mostly electronic in the case of 5. 12 The explanation for this came from the NBO analysis which indicated that the extra stabilization in the s trans geometry coming from the donor acceptor interaction between the orbital of the pyrrole double bond and the orbital of the nitroso or carbo nyl group, (trans cis ), is significantly larger in 5. 12 than in 5. 10 or 5. 11 (Table 5 11). Figure 5 14. Model compounds for the s cis vs. s trans conjugation Barriers for rotation about the C NO bond or the C COOMe bond in model compounds 5. 10 5. 12 calculated with the HF/6 31g(d,p) method, are 15.1, 14.4 and 17.6 kcal/mol, respectively. The barrier in 5. 10 compares well with the barriers in 5. 1 and 5. 2 16.6 and 15.6

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111 kcal/mol respectively. The calculated barrier in 5. 11 is identical to that found i n 5. 3 14.4 kcal/mol. The barrier in 5. 12 is smaller than the barrier found for 5. 4 17.6 vs 24 kcal/mol, but the value re f lects the experimental trend. Table 5 10. Calculated energy differences (kcal/mol) for conformers of model compounds 5. 10 5. 12 Compound HF/6 31g(d,p) MM+ trans cis trans cis 5.10 0.00 0.69 0.00 0.92 5.11 0.00 1.13 0.00 1.40 5.12 0.00 3.14 0.00 0.86 Table 5 11. Calculated stabilization energy and E (trans cis ) (kcal/mol) for the donor acceptor interaction of interest in model compounds 5. 10 5. 12 Compound Donor ( ) Acceptor ( *) trans cis E 5.10 C1=C8a N=O 29.94 27.49 2.45 5.11 C2=C3 C=O 27.77 26.59 1.18 5.12 C2=C3 N=O 38.4 30.69 7.71 The large barriers in the indolizine system can be explained by the partial double bond character of the C X=O bond (X is N or C) and the increased aromaticity of the pyridine ring in the ground state. Results of the NBO analysis for the ground state and the highest energy p oint on the rotation pathway are presented in Tables 5 12 and 5 13. Table 5 12. Distances () for some bonds at the lowest (0) and highest point of the rotation (90) in model compounds 5. 10 5. 12 Compd. 10 11 12 Distance 0 90 0 90 0 90 C XO 1.384 1.443 1.453 1.493 1.36 1.442 N4 C8a 1.369 1.384 1.375 1.386 1.371 1.387 N4 C5 1.369 1.376 1.373 1.383 1.351 1.378 N4 C3 1.386 1.364 1.386 1.368 1.393 1.365 In the transition state, the length of the C XO bond is larger than in the ground state, due to the loss of the partial double bond character. The length of the N4 C8a and N4 C5 bonds also

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112 increases, due to some loss of the aromaticity of the pyridine ring The N4 C3 bond is shorter in the transition state, suggesting a higher donation from the lone pair into the C2=C3 bond. Table 5 13. Electron Donor Acceptor interactions and N4 occupancy at the lowest (0) and highest point of the rotation (90) in model compounds 5. 10 5. 12 Compd. 5.10 5.11 5.12 0 90 0 90 0 90 40.0 35.0 40.1 34.9 42.0 34.6 C5=C6 34.9 33.9 36.0 34.4 35.0 33.4 25.6 33.3 29.2 33.6 29.4 34.2 29.9 1.6 27.8 4.5 38.4 3.2 Nitrogen Occupancy 1.480 1.495 1.479 1.491 1.478 1.506 Data in Table 5 13 demonstrate a large decrease in the donation to the X=O bond in the transition state in compounds 5. 10 5. 12 and particularly in the lat t er, which correlates well with the increase in the C XO bond distance, which loses its double bond c haracter in the transition state. The N4 lone pair donation decre a ses in the transition state, while its occupancy increases. 5.3 Conclusions 13 C substituent chemical shifts (SCS) in the alpha position to the nitroso group identified compounds 5. 1 5. 6 as monomers, as expected for nitroso derivatives of electron rich heterocycles. The SCS in the beta position to the nitroso group identified the syn and anti rotamers of the monomers. Compounds 5. 1 and 5. 5 displayed both rotamers in comparable amounts, 5. 2 and 5. 4 were mostly syn and 5. 6 was mostly anti Compound 5. 3 had two rotamers in comparable amounts, but they were both syn and they were due to restricted rotation of the carboxyl group. Molecular modelling provided the interpretation for the conformational preferences of the monomers. Both MM+ and ab initio with the 6 31g(d,p) basis set calculations gave the same order of stability for conformers, except for the case of 5. 4 This is becau se in all of the

PAGE 113

113 compounds but 5. 4 steric interactions prevail over electronic ones. The extra stabilization coming from better delocalization in the s trans geometry of the endocyclic double bond and the N=O bond is significantly larger in 3 nitrosoindol izines than in 1 nitrosoindolizines, and this is the determining factor in the conformational equilibrium of 5. 4 In 1 nitrosoindolizines, steric interactions were found to prevail over the electronic interactions between the nitroso group and the heterocy cle. The syn and anti orientations of the nitroso group flanked by a peri hydrogen (H8) and a freely rotating methyl in position 2 are of comparable energies. This is the case for compounds 5. 1 and 5. 5 In compound 5. 2 the methyl in position 2 has a hydro gen pointing towards the nitroso, and the syn conformation prevails. The methyl is forced in this conformation by the carboxyl group in position 3, which has to be in the plane of the heterocycle. The carboxyl group has the carbonyl facing H5, which appear s larger than the methyl. In compound 5. 3 both the carboxyl and the methylamino group are in the plane of the heterocycle. The methylamino group has the methyl pointing towards the nitroso, and 5. 3 is entirely syn The methylamino group in 5. 3 appears to the carboxyl group larger than the methyl group in 5. 2 and the two orientations of the carboxyl group in 5. 3 are of comparable energies. Barriers to rotation about the C NO bond were larger than in other C nitroso compounds, and more so for 3 nitrosoindo lizines than for 1 nitrosoindolizines. The barrier to rotation about the C COOR bond in 5. 3 is also exceptionally large. Molecular modeling demonstrated that this is because the stabilization of the s trans geometry coming from the donor acceptor interacti on between the orbital of the pyrrole double bond and the orbital of the nitroso or carbonyl group is significantly larger when these substituents are in position 3, than when they are in position 1.

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114 5.4 Experimental 5.4.1 General M ethods Melting points were determined on a capillary point apparatus equipped with a digital thermometer and are uncorrected. The NMR spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1 H, 125 MHz for 13 C and 50 MHz for 15 N, equipped wi th a three channel, 5 mm, indirect detection probe, with z axis gradients. The solvent was DMSO d 6 and the temperature was 25 C, unless specified otherwise. The chemical shifts for 1 H and 13 C were referenced to the residual solvent signal, 2.50 ppm for 1 H and 39.5 ppm for 13 C, on the tetramethylsilane scale. The chemical shifts for 15 conversion to t he neat nitromethane scale, subtract 381.7 ppm. 1 H spectra were acquired in one transient, with a 90 pulse, no relaxation delay and an acquisition time of 5 s, over a spectral window from 16 to 2 ppm. The FID was zero filled to 131072 points prior to Fo urier transform. Typically, 1 H 13 C gHMBC spectra were acquired in 4096 points in f2 on a spectral window from 1.5 to 11 ppm, and 1 s relaxation delay. In f1 512 increments were acquired in 1 transient over a spectral window from 170 to 10 ppm, then the c orresponding FID s were zero filled twice prior to the second Fourier transform. 1 H 15 N CIGAR gHMBC spectra were acquired with a pulse sequence optimized for 15 N, as decribed in ref. 41. 2048 points were acquired in f2, over a spectral window typically fro m 1.5 to 11 ppm, with 1 s relaxation delay. 1024 increments were acquired in f1 on a spectral window from 0 to 400 ppm, and the corresponding FID was zero filled twice prior to Fourier transform. The accordion delay was optimized for a value of 1 H 15 N cou pling constants between 3 and 10

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115 Hz. The number of transients per increment was between 4 and 64, depending on the concentration of the sample. Total experiment time was in most cases, ca 2 hrs. 5.4.2 Characterization of Compounds 5.1 5.6 C ompounds 5.1 5.6 were obtained from ARKIVE, purified, and properly characterized before use. (See ref. [2010OBC 3518] for details) 2,3 Dimethyl 1 nitrosoindolizine ( 5.1 ). G reen microcrystals (83 %); mp 147.0 HRMS calc. for [C 10 H 10 N 2 O+H] + 175.0866; Found: 175.0862. Methyl 2 methyl 1 nitrosoindolizine 3 carboxylate ( 5.2 ). G reen microcrystals (82 %); mp 131.0 Ethyl 2 (methylamino) 1 nitrosoindolizine 3 carboxylate ( 5.3 ). G reen microcrystals (77 %) ; mp 156.0 Anal. Calc. for C 12 H 13 N 3 O 3 (247.26): C, 58.29; H, 5.30; N, 16.99. Found: C, 57.96; H, 5.194; N, 17.36. 2 Methyl 3 nitrosoindolizine ( 5.4 ). Green needles ; m p 99.0 106.0 Anal. Calc. for C 9 H 8 N 2 O ( 160.18): C, 67.49; H, 5.03; N, 17.48. Found: C, 67.17; H, 5.13; N, 17.48. 2,6,7 Trimethyl 5 nitrosopyrrolo[1,2 b]pyridazine ( 5.5 ). G reen crystals (97%) ; mp 132.0 133.0 C Anal. Calc. for C 10 H 11 N 3 O (189.22): C, 63.48; H, 5.86; N, 22.21. Found: C, 63.82; H, 5.94; N, 22.17. HRMS (ESI) Calc. For [ C 10 H 11 N 3 O+H] + : 190.0975. Found: 190.0967. 4 Methoxy 3 nitrosopyrazolo[1,5 a]pyridine ( 5.6 ). Green crystals (32%) ; mp 216.0 218.0 C. HRMS (ESI) calc. for [C 8 H 7 N 3 O 2 +H] + 178.0611; Found: 178.0615.

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116 CHAPTER 6 1 H, 13 C, AND 15 N NMR SPECTRA OF SOM E PYRIDAZINE DERIVAT IVES 1 6.1 Introduction Pyridazines are important biologically active scaffolds, common for analgesic [2007JPR166, 1996CPB980], anti inflammatory [2007JPR166, 2002JMC563], antihypertensive [2004JMC1089, 1980JMC1398], antidepressant [1974EJMC644, 2009WOP079683], antibacterial [2009H961, 2009BMC2823], antiaggregative [1999BPB1376], anticancer [2009WO035568, 2009WOP016286], cardiotonic [2009PCJ87, 2008 AF569] and nephrotopic drugs [1995EPA661273]. Pyridazines also inhibit acetylcholinesterase [1999JMC730], aldose 1 adrenoceptor [2008BML5140], cyclin dependent kinase (CDKs) [2008BML5758, 2007ARK247], cyclooxyge nase enzymes (COX 1 and COX 2) [ 2007MI 9 2003AP406] and p38 MAP kinase [2006BML5809]. Especially, highly bioactive 3,6 disubstituted pyridazines have attracted particular attention. We know of only one systematic study for the assignment of 1 H and 13 C che mical shifts of pyridazines [1991CJC972] t h is report uses 1 H and 13 C coupling constants and J modulated spin echo sequences to differentiate quaternary carbon atoms from those with attached protons. Selective heteronuclear decoupling and semiempirical mol ecular orbital calculations assisted the assignment [1991CJC972]. 15 N NMR has been used for the structural elucidation and study of electronic properties of several heterocyclic systems containing a pyridazine moiety. Complete assignment of all nitrogens in a series of isoxazolo[3,4 d ]pyridazin 7(6 H ) ones (6.1) was reported by Holzer et. al. [2005MRC240]. The chemical shift for these pyridazinone nitrogens varies from 307.1 to 324.0 ppm for N 5 and from 188.2 to 215.7 ppm for N 6 (Figure 6 1). 15 N NMR chemical shift values 1 Reproduced in part with permission from The Journal of Magnetic Resonance in Chemistry 20 1 0 48 397 402 Copyright 20 1 0 John Wiley and Sons

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117 were used to establish the structures of potassium salts of 6 (3 cyano 1 triaz i no)tetrazolo[1,5 b ]pyridazines and their amido derivatives [2002MRC507]. Since it is highly sensitive toward changes in the electronic environment of the nitrogen atom, 15 N NMR was used to study the valence tautomerization and prototropy of triazolo and tetrazolo pyridazines. It was shown by 15 N NMR that the tetrazole tautomer 6.2a is more stable than the azide tautomer 6.2b The protonation site in 6.2 was easily determined as N 1 due to the large difference in chemical shift between the protonated and non protonated nitrogens [1999MRC493]. By comparison with 6.2 significant s hielding in excess of 50 ppm was observed in 6.3 for N 2 and N 4 due to the absence of N 3. Figure 6 1. Isoxazolo[3,4 d ]pyridazin 7(6 H ) ones 6. 1, tetrazolopyridazines 6.2 and triazolo pyridazines 6. 3 The structure of 6 substituted 4 (subst amino) 5 aryldiazenyl 1 arylpyridazinium salts have been studied by 15 N NMR [2005T8130]. Metal nitrogen coordination bonding in Zn(II), Cd(II) and Hg(II) chloride complexes involving coordination with pyridazine nitr ogens results in significant NMR shielding of the 15 N nuclei; the magnitude decreased with the atomic mass of the zinc triad metal [2004JS T 143]. 15 N NMR was informative in establishing the structures of mononuclear [Rh(dppn)(NBD)]BF 4 and dinuclear [Rh 2 (dpp n 1 C 7 H 9 )(CH 3 OH) 2 (CH 3 CN)](BF 4 ) 2 complexes both containing the tetradentate ligand 3,6 bis(2 pyridyl)pyridazine (dppn). Binding to the metal causes significant shifts of the nitrogen atom compared to that of the free ligand. The

PAGE 118

118 coupling between the coordinating pyridazine nitrogen atom and the corresponding rhodium was found to be in agreement with previously reported values [2003EJI70]. I now report the 1 H, 13 C, and 15 N chemical shifts of pyridazines 6. 4 6.20 (Figure 6 2) by fast and accurate 2D NMR techniques using pulsed field gradients and indirect detection. Figure 6 2. Compounds investigat ed in this thesis 6.2 Results and Discussion 6.2.1 1 H NMR Spectra The proton chemical shifts are reported in Table 6 1 The 1 H NMR spectra of 6.5 6.18 could be completely assigned based on 1D NMR, because of the simplicity of the structures (Figure 6 3 as an example). These assignments were confirmed by correlation with different carbons that was observed in 1 H 13 C gHMBC experiment. 1 H 1 H gDQCOSY was needed to assign H5 H8 in cinnolin 4 ol (6.19) TOCSY 1D was needed to assign the phenyl protons in 4 methyl 3,6 diphenylpyridazine (6.20) Compound (6.19) was previously reported by Holzer and

PAGE 119

119 his co workers [2008H (75) 77] to exist exclusively as the NH isomer (6.19 B) and our assignment is in a total agreement with this literature. Figure 6 3. 1 H NMR of 3 diethylamino 1 ethyl 6 iodopyridazin 1 ium iodide ( 6.17 ) 6.2.2 13 C NMR Spectra The carbon chemical shifts are reported in Table 6 2 Protonated carbons were assigned using one bond gHMQC correlations. The quaternary carbons (C 3 and C 6) were assigned based on gHMBC correlations (Figure 6 4 for 6.17 ) with both protons at C 4 and C 5 and also with substituent protons on C 3 and C 6. The C 3 and C 6 chemical shifts depend significantly on the nature of the attached substituent. For example, in compound 6. 14 where C 3 and C 6 are attached to iodine, C 3 and C 6 chemical shifts were shielded by 24.8 and 30.2 ppm from the corres ponding 6. 4 and 6. 7 chemical shifts respectively because of the heavy atom effect. 6.2.3 15 N NMR Spectra Nitrogen chemical shifts are reported in Table 6 3 and were assigned based on long range correlation in 1 H 15 N CIGAR HMBC experiment s The chemical sh ift assignment of N 1 was based on three bond correlation with either H 5, or the protons attached to the substituent at C 6,

PAGE 120

120 or both. Similarly, the chemical shift assignment of N 2 was based on three bond correlation with either the proton at C 4, or the protons attached to the substituent at C 3, or both. Figure 6 4. The 1 H 13 C gHMBC spectrum of 6.17 in DMSO d6 15 N NMR was used to identify the site of N oxidation in N oxides 6. 9 and 6.10 In both compounds the N 1 chemical shift was revealed by three bond correlation with H 5 and H 6' and the N 2 chemical shift was revealed by three bond correlation of H 4 and H 3' in 6. 9 and H 3' in 6. 10 15 N NMR is very useful in determining the site of N oxidation especially in the case of unsymmetrical compounds like 6. 10 (Figure 6 5) In compounds 6. 9 and 6. 10 the site of oxidation was assigned based on 15 N chemical shift values. The site of oxidation was assigned as N 1 which was found to be more shielded than N 2 by 21.8 ppm and 17.4 ppm in both 6. 9 and 6. 10 respectively. 15 N NMR was also used to determine the site of alkylation in 6.17 (Figure 6 6). N 1 was found to be the site of alkylation as it was shielded by 41.5 ppm from N 2. In 6.17 the N 1 chemical shift was revealed by the two bond correlation to H 1' (large coupling) and the three bond correlation to H 5 and H 1'' (large coupling). This assignm ent was confirmed by three bond correlation between H 1' and C 6 that was seen in the 1 H 13 C gHMBC spectra (Figure 6 4).

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121 Figure 6 5. The 1 H 15 N CIGAR HMBC spectrum of 6.10 with expansion in DMSO d6 Figure 6 6. The 1 H 15 N CIGAR HMBC spectrum of 6.17 with expansion in DMSO d6

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122 Table 6 1. 1 H NMR (ppm) chemical shifts (DMSO d6 ) a For numbering see scheme 1. b Compounds are symmetric. c Chemical shift values were taken from ref. [2010MRC397] and used for comparison. Comp. a R 3 R 6 1 H Chemical Shifts ( ppm) H 4 H 5 Other H 6.4 b,c H H 7.65 (t, J = 3.5 Hz) 7.65 (t, J = 3.5 Hz) 9.23 (t, J = 3.5 Hz, H 3,6) 6.5 Me H 7.59 (m) 7.61 (m) 9.10 (d, J = 4.3 Hz, H 6), 2.65 (s, CH 3 3') 6.6 H H 7.55 (d, J = 5.3 Hz) 9.14 (br s, H 3), 9.10 (d, J = 5.3 Hz, H 6), 2.35 (s, CH 3 4') 6.7 b Me Me 7.45 (s) 7.45 (s) 2.58 (s, CH 3 3',6') 6.8 b CH(CH 3 ) 2 CH(CH 3 ) 2 7.51 (s) 7.51 (s) 3.18 (septet, J = 7.0 Hz, CH 3',6'), 1.26 (d, J = 7.0 Hz, (CH 3 ) 2 3'',6'') 6.9 Me Me 7.09 (d, J = 8.4 Hz) 7.81(d, J = 8.4 Hz) 2.40 (s, CH 3 3'), 2.32 (s, CH 3 6') 6.10 Me Me 8.72 (s) 2.68 (s, CH 3 3'), 2.39 (s, CH 3 6') 6.11 Me Me 6.88 (s) 2.20 (s, CH 3 3'), 2.21 (s, CH 3 6'), 6.00 (br s, NH 2 4') 6.12 b OMe OMe 7.16 (s) 7.16 (s) 3.92 (s, OCH 3 3',6') 6.13 Cl Me 7.78 (d, J = 9.0 Hz) 7.66 (d, J = 9.0 Hz) 2.60 (s, CH 3 6') 6.14 b I I 7.84 (s) 7.84 (s) 6.15 N(CH 2 CH 3 ) 2 Me 6.91 (d, J = 9.3 Hz) 7.17 (d, J = 9.3 Hz) 3.50 (q, J = 7.1 Hz, (CH 2 ) 2 3''), 1.08 (t, J = 7.1 Hz, (CH 3 ) 2 3'''), 2.38 (s, CH 3 6') 6.16 N(CH 2 CH 3 ) 2 I 6.82 (d, J = 9.5 Hz) 7.59(d, J = 9.5 Hz) 3.49 (q, J = 7.0 Hz, (CH 2 ) 2 3''), 1.09 (t, J = 7.0 Hz, (CH 3 ) 2 3''') 6.17 N(CH 2 CH 3 ) 2 I 7.53 (d, J = 9.7 Hz) 8.36 (d, J = 9.7 Hz) 3.55 (q, J = 7.1 Hz, (CH 2 ) 2 3''), 1.13 (t, J = 7.1 Hz (CH 3 ) 2 3'''), 4.68 (q, J = 7.3 Hz, CH 2 1'), 1.50 (t, J = 7.3 Hz, CH 3 1'') 6.18 Cl O 6.96 (d, J = 9.8 Hz) 7.49 (d, J = 9.8 Hz) 13.16 (s, NH 1) 6.19 b 9.76 (s, H 1, H 4), 8.22 (m, H 5, H 8), 8.08 (m, H 6, H 7) 6.20 Ph Ph 8.17 2.39 (H 4'''), 7.67 (H 2'), 7.54 (H 3'), 7.51 (H 4'), 8.20 (H 2''), 7.56 (H 3''), 7.53 (H4'')

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123 Table 6 2. 13 C NMR (ppm) chemical shifts (DMSO d6 ) a Comp b R 3 R 6 13 C Chemical Shifts ( ppm) C 3 C 4 C 5 C 6 Other C 6.4 c,d H H 152.5 (H4) 127.4 (H3, H6) 127.4 (H3, H6) 152.5 (H5) 6.5 Me H 160.6 (H3', H4, H5) 127.6 (H3', H6) 127.2 (H6) 150.3 (H4, H5) 22.5 (CH 3 3') (H4) 6.6 H H 154.0 (H4', H5 138.4 (H4', H6) 127.6 (H4', H6) 151.6 (H5) 18.4 (CH 3 4' (H3, H5)) 6.7 c Me Me 157.9 (H3', H4) 127.4 (H3') 127.4 (H6') 157.9 (H6', H5) 22.0 (CH 3 3' (H4),6' (H5)) 6.8 c CH(CH 3 ) 2 CH(CH 3 ) 2 166.4 (H3', H3'') 125.7 (H3') 125.7(H6') 166.4 (H6', H6'') 34.5 (CH 3' (H3''),6' (H6'')), 22.9 (CH 3 3'' (H3', H3''),6'' (H6', H6'')) 6.9 Me Me 158.6 (H3', H4, H5) 117.4 (H3') 135.2 (H6') 140.8 (H4, H5, H6') 21.5 (CH 3 3' (H4)), 18.0 (CH 3 6' (H5)) 6.10 Me Me 153.9 (H3', H5, H6') 137.1 (H3', H5) 130.9 (H6') 143.7 (H3', H5, H6') 21.6 (CH 3 3' (H5)), 17.5 (CH 3 6' (H5)) 6.11 Me Me 144.1(H3', H4', H5) 139.4 (H3', H5) 116.1 (H4', H6') 141.5 (H6') 18.2 (CH 3 3'), 18.2 (CH 3 6') 6.12 c OMe OMe 162.4 (H3', H4) 122.1 122.1 162.4 (H5, H6') 54.8 (OCH 3 3',6') 6.13 Cl Me 155.1(H5) 129.1 131.2 (H6') 160.7 (H4, H6') 21.7 (CH 3 6' (H5)) 6.14 c I I 127.7 (H4, H5) 139.0 139.0 127.7 (H4, H5) 6.15 N(CH 2 CH 3 ) 2 Me 157.2 (H3'', H5) 112.4 (H5) 128.5 (H5, H6') 149.2 (H4, H5, H6') 42.6 ((CH 2 ) 2 3'' (H3'', H3''')), 13.4 ((CH 3 ) 2 3''' (H3'')), 21.4 (CH 3 6' (H5)) 6.16 N(CH 2 CH 3 ) 2 I 157.9 (H3'', H4, H5) 114.8 137.3 (H4) 111.5 (H4, H5) 42.8 ((CH 2 ) 2 3'' (H3'', H3''')), 13.2 ((CH 3 ) 2 3''' (H3''))

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124 Table 6 2. Continued Comp b R 3 R 6 13 C Chemical Shifts ( ppm) C 3 C 4 C 5 C 6 Other C 6.17 N(CH 2 CH 3 ) 2 I 158.0 (H3'', H4, H5) 121.5 145.1 114.4 (H1', H4, H5) 43.8 ((CH 2 ) 2 3'' (H3'', H3''')), 12.8 ((CH 3 ) 2 3''' (H3'')); 64.8 (CH 2 1'(H1'')), 14.7 (CH 3 1''(H1')) 6.18 Cl O 160.4 (H5) 133.3 135.4 138.1(H4) 6.19 H 140.9 170.9 (H3, H5, H8) 124.5 (H7) 125.5 (H8) 123.5 (C 4a (H3, H6, H8)), 141.5 (C 8a (H5, H7)), 134.5 (C 7 (H5)), 117.1 (C 8 (H6, H7) 6.20 Ph Ph 160.9 (H2', H4''', H5) 137.3 (H4''') 126.3(H4''') 157.4 (H5) 19.9 (CH 3 4), 137.7 (C 1' (H3')), 129.8 (C 2' (H4')), 128.1 (C 3'), 129.4 (C 4' (H2')), 136.6 (C 1'' (H3'', H5)), 127.5 (C 2'' (H2'')), 129.7 (C 3'' (H3'')), 130.5 (C 4'' (H2'')) a Protons that shows 1 H 13 C gHMBC correlations are given in parentheses. b For numbering see scheme 1. c Compounds are symmetric. d Chemical shift values were taken from ref. [2010MRC397] and used for comparison.

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125 Table 6 3. 15 N NMR (ppm) chemical shifts (DMSO d6 ) a Comp b R 3 R 6 15 N Chemical Shifts ( ppm) N 1 N 2 Other N 6.4 c,d H H 402.7 (H3, H4, H5, H6) 402.7 (H3, H4, H5, H6) 6.5 Me H 401.6 (H5, H6) 397.6 (H3', H4, H6) 6.6 H H 391.0 (H3, H5) 398.3 (H6) 6.7 c Me Me 395.9 (H5, H6') 395.9 (H3', H4) 6.8 c CH(CH 3 ) 2 CH(CH 3 ) 2 393.4 (H5, H6') 393.4 (H3', H4) 6.9 Me Me 323.4 (H5, H6') 345.2 (H3', H4) 6.10 Me Me 330.0 (H5, H6') 347.4 (H3', H5, H6') 367.3 (NO 2 4' (H3', H5)) 6.11 Me Me 294.0 (H5, H6') 342.0 (H3') 66.6 (NH 2 4 (H4' (d, J = 86.5 Hz), H5)) 6.12 c OMe OMe 322.2 (H 5) 322.2 (H 4) 6.13 Cl Me 399.5 (H4, H5, H6') 385.6 (H4, H6') 6.14 c I I 420.4 (H5) 420.4 (H4) 6.15 N(CH 2 CH 3 ) 2 Me 388.3 (H4, H5, H6') 339.8 (H3'', H4, H5, H6') 86.0 (N 3' (H3'', H3''', H4, H5, H6')) 6.16 N(CH 2 CH 3 ) 2 I 403.3 (H4, H5) 353.2 (H3'', H4, H5) 91.9 (N 3' (H3'', H3''', H5)) 6.17 N(CH 2 CH 3 ) 2 I 258.0 (H1', H1'', H4, H5) 300.5 (H1', H3'', H4) 105.3 (N 3' (H3'', H3''', H4, H5)) 6.18 Cl O 325.0 (H4, H5) 208.6 (H4, H5) 6.19 H 176.3 (H3, H 8) 340.9 (H 3) 6.20 Ph Ph 386.8 (H5) 399.9 (H5) a Protons which couple to 15 N given in parentheses. b For numbering see Scheme 1. c Compounds are symmetric, i.e. N1 and N2 have the same chemical shift values. d Chemical shift values were taken from ref. [2010MRC397] and used for comparison. 6.3 Conclusions 1 H, 13 C, and 15 N NMR chemical shifts for pyridazines 6.4 6.20 were measured using 1D and 2D NMR spectroscopic methods including 1 H 1 H gDQCOSY, 1 H 13 C gHMQC, 1 H 13 C gHMBC, and 1 H 15 N CIGAR HMBC experiments. 15 N NMR spectroscopy was very useful in identifying the site of N oxidation in 6.9 and 6.10 and the site of N alkylation in 6.17

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126 6.4 Experimental 6.4.1 General M ethods Melting points were determined on a capillary point apparatus equipped with a digital the rmometer. The NMR spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1 H, 125 MHz for 13 C and 50 MHz for 15 N, equipped with a three channel, 5 mm, indirect detection probe, with z axis gradients. The solvent was DMSO d 6 and the t emperature was 25 C, unless specified otherwise. The chemical shifts for 1 H and 13 C were referenced to the residual solvent signal, 2.50 ppm for 1 H and 39.5 ppm for 13 C, on the tetramethylsilane scale. The chemical shifts for 15 .1328898, corresponding to 0 for neat conversion to the neat nitromethane scale, subtract 381.7 ppm [2002CO R 35]. 1 H spectra were acquired in one transient, with a 90 pulse, no relaxation delay and an acquisition time of 5 s, over a spectral window from 16 to 2 ppm. The FID was zero filled to 131072 points prior to Fourier transform. Typically, 1 H 13 C gHMBC spectra were acquired in 2048 points in f2 on a spectra l window from 0 to 11 ppm, and 1 s relaxation delay. In f1 256 increments were acquired in 1 transient over a spectral window from 0 to 170 ppm, and then the corresponding FID s were zero filled twice prior to the second Fourier transform. 1 H 15 N CIGAR gH MBC spectra were acquired with a pulse sequence optimized for 15 N, as described in the literature [2003MRC307] ; 2048 points were acquired in f2, over a spectral window typically from 0 to 11 ppm, with 1 s relaxation delay. 1024 Increments were acquired in f1 on a spectral window from 0 to 400 ppm, and the corresponding FID was zero filled twice prior to Fourier transform. The accordion delay was optimized for a value of 1 H 15 N coupling

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127 constants between 3 and 10 Hz. The number of transients per increment w as between 4 and 64, depending on the concentration of the sample. Total experiment time was in most cases, ca 2 hrs. 6.4.2 Characterization of Compounds 6.7 6.20 Pyridazines 6. 5 and 6. 6 were purchased from Aldrich Chemical Company, Inc. and were found to be pure by NMR Compounds 6. 7 6.20 were obtained from ARKIVE (see ref. [2010MRC397] for details) and were purified and properly characterized before use. 3,6 Dimethylpyridazine ( 6.7 ). Mp 32.0 [1956JA1961] 3,6 Diisopropylpyridazine ( 6.8 ). Mp 63.5 10 H 16 N 2 : C, 71.17; H, 9.85; N, 16.60. Found: C, 71.46; H, 9.64; N, 16.73. 3,6 Dimethylpyridazine 1 oxide ( 6.9 ). M p 114.0 [1980CPB529] 3,6 Dimethyl 4 nitropyridazine 1 oxide ( 6.10 ). M p 116.0 188.0 [1961CPB149] 3,6 Dimethyl 4 aminopyridazine ( 6.11 ). M p 162 .0 163 .0 [1962YZ253]. 3,6 Dimethoxypyridazine ( 6.12 ). M p 106.0 p 104.5 [1959JA6511] 3 Chloro 6 methylpyridazine ( 6.13 ). Mp 53.0 p 58 .0 [1947JCS239] 3,6 Diiodopyridazine ( 6.14 ). M p 157.0 [1963JOC218] Diethyl(6 methylpyridazin 3 yl)amine ( 6 .15 ). B rown oil. Anal. Calcd. for C 9 H 15 N 3 : C, 65.42; H, 9.15; N, 25.43. Found: C, 65.50; H, 9.41; N, 25.26. Diethyl (6 iodo pyridazin 3 yl) amine ( 6.16 ). M p 90 .0 92 .0 Calcd. for C 8 H 12 N 3 : C, 33.58; H, 4.58; N, 14.69. Found: C, 33.69; H, 4.09; N, 14.47.

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128 3 Diethylamino 1 ethyl 6 iodopyridazin 1 ium iodide ( 6.17 ). Y ellow crystals; mp 173 177 10 H 17 N 3 I 2 : C, 27.73; H, 3.96; N, 9.70. Found: C, 28.09; H, 3.91; N, 9.63. 6 Chloro 2H pyridazin 3 one ( 6.18 ). Mp 140.0 p 137 140. [1962CPB580] Cinnolin 4 ol ( 6.19 ). Mp 238.5 [2008H(75)77] 4 Methyl 3,6 diphenylpyridazine ( 6.20 ). M p 126.0 [1983JCS(P1)1601]

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129 CHAPTER 7 DIVERSE APPLICATION S OF 15 N NMR IN STRUCTURAL ANALYSIS 7.1 Introduction Nitrogen is one of the most important elements in organic and biochemistry. Amines, amides, imines, enamines, and nitro compounds are among the major classes of organic compounds that contain nitrogen. Moreover, nitrogen can be found in all living organism s because it forms an essential part of the structural motif of nucleic acids, proteins, alkaloids, and cytokinins. The large number and wide diversity of nitrogen containing molecules made 15 N NMR one of the major NMR probes beside 1 H and 13 C NMR in solvi ng structural problems in organic and bioorganic molecules. 15 N NMR has an inherited sensitivity problem because of the low natural abundance N = 2.710 7 rad T 1 s 1 ) of the 15 N isotope. However, the great advances in inverse detection techniques for 1 H 15 N heteronuclear correlation [2000JNP543], new probe technologies, and high field magnets have now made the detection of submilligram sample s achievable. 15 N NMR spectroscopy ha s become a very powerful technique for the structural analysis of compounds that contain nitrogen [2000JNP543, 2002COR35, 2007COR1154]. For example, 15 N NMR spectroscopy was used successfully to study: (i) S tructural elucidation [2000JHC831] ; (ii) C hanges in electron structure [2000EJO2947] ; (iii) I nteraction with H + [1988BSB23] and metals [1998EJI1555] ; (iv) A lkylation, arylation, and acylation of nitrogen heterocycles [1999MRC195] ; (v) E ffect of N oxidation [1997H (46) 645] ; (vi) T automerism [2000AHC1] ; (vi i) C onfigurational [1996CCC589, 1997T10899] and conformational changes [1998MRC587] and rotational barriers [1997MRC323] ; (viii) D ifferentiation of regioisomers [1998MRC35].

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130 7.1.1 Literature Background to Structural Elucidation by 15 N NMR 15 N NMR spectroscopy has been used in addition to 1 H and 13 C NMR to solve structural elucidation problems in organic compounds for a long time. For example, Hardil e t al. [2000JHC831] used 1 H 15 N NMR correlation spectra to elucidate the structure of the prod uct of the reaction of phenacyl anthranilate (7.1) with ammonium acetate (Scheme 7 1). This reaction was expected to give product 7.2 but 1 H and 13 C NMR did not support structure 7.2 1 H 15 N NMR correlation spectra proved the structure to be 7.3 which was obtained through the rearrangement shown in Scheme 7 1. Scheme 7 1. The most distinct observations in support of structure 7.3 came from 1 H 15 N NMR correlation spectra : (i) T he reaction product have two non equivalent NH groups instead of the expected NH and NH 2 groups ; (ii) A three bond couplings were found between each NH proton and the other nitrogen. To further confirm the structure, quinazoline derivative (7.3) was acylated with acetic anhydride to give 7.4 1 H 15 N NMR correlation spectra of 7.4 gave unambiguous evidence that the proposed structure was the correct one. First ly 1 J H,N for both nitrogens was found to be 90 Hz (characteristic of one bond coupling) showing the existence of two NH

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131 groups. Secondly, CH 2 protons showed three bond correlation with both N1 and N3. Finally, H8 showed three bond correlation with only one nitrogen which was found to be N1. ( 1 H 15 N NMR correlations are indicated in Scheme 7 1 by single headed arrows for 7.3 and 7.4 ) 15 N NMR spe ctroscopy was used by Laihia et. al. [2006JST100] for the structural characterization of three spiro[pyrrolidin e 2,3' oxindoles] ( 7.5a,b,c ). It was easy to differentiate between the amidic nitrogen (N1) and the tertiary nitrogen (N4') by means of 15 N NMR spectroscopy because of the large difference in their chemical shifts (>50 ppm). The chemical shift of N1 varied from 131.4 to 135.2 ppm and the chemical shift of N4' varied from 65.7 to 74.2 ppm. Figure 7 1. Spir o[pyrrolidin e 2,3' oxindoles] (7.5a,b,c) 7.1.2 Literature Background to Protonation Studies by 15 N NMR 15 N NMR spectroscopy is very useful for studying the electronic changes resulting from protonation or interaction with metals in nitrogen containing comp ounds because of the high sensitivity of nitrogen chemical shifts to protons and substituents. 15 N NMR was used [1996JCS(P2)2383] to study the protonation in 1 pheny1 3 methyl 5 N benzylidene aminopyrazole ( 7. 6 ). In TFA d all three nitrogens were protonated and this is consistent with the large upfield shift (Figure 7 2 ). Ab initio MO calculations at HF/6 31G* level supported the NMR data.

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132 Figure 7 2 Nitrogen chemical shifts of 1 pheny1 3 methyl 5 N benzylidene aminopyrazole ( 7. 6 ) in CDCl 3 and in TFA d In another literature example, Mki et al. have studied the restricted rotation in 5 amino 4 imidazol 2'' yl 1 ( D ribofuranosyl) 1 H imidazole ( 7. 7 ), which is a bi imidazole nucleoside generally known as ethenoadenosine [2001JCS(P1)1216]. This nucleoside is a common adduct usually formed by the action of genotoxic compounds on adenosine. The prototropic equilibrium and protonation of this adduct was studied by 1 H, 13 C, and 15 N NMR spectroscopy with the latter being the essential technique to assign the structures of different tautomers and the protonation site. Figure 7 3 Ethenoadenosine ( 7. 7 ) in the neutral form (asterisks are used to show the rotation of the outer imidazole ring with respect to the inner one) In the neutral form, there was an intramolecular hydrogen bond between N3'' and N6 protons that gave rise to tautomeric forms 7.7a and 7.7b arising from restricted rotation about C4 C2'' b ond (Figure 7 3). In the protonated form, the nitrogen chemical shift values of N1, N3, N6, and N1'' have very small upfield shift providing no proof for protonation at these sites. The N3'' was found by 15 N NMR spectroscopy to be the exclusive site of pro tonation because of the observed upfield shift of 79.2 ppm which is expected for such protonated pyridine like nitrogens.

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133 Protonation at N3'' reduced the intramolecular H bond between N3'' and N6 protons and led to unrestricted rotation. Recently, Zelewsk y and his co workers studied the protonation behavior of chiral pinene using NMR measurements including 15 N chemical shift measuremens and UV visible spectroscopy [2005CEJ185]. The UV visible spectra shows that mon oprotonation of [5,6]pinene bypyridine leads to an equilibrium between the free ligand ( 7. 8 ) and the monoprotonated species ( 7. 8 H + ) and changes the conformation from trans to cis with the proton acting as a small cation being shared between the two nitrogens. This result was supported by 15 N N MR spectroscopy; both nitrogens in the free ligand at 304.7 ppm (N1) and 309.7 ppm (N1') shifted upfield by 57.0 and 62.0 ppm respectively u pon protonation. Figure 7 4 Equilibrium between [5,6]pinene bpy ( 7. 8 ) and its monoprotonated form ( 7. 8 H + ) In ligand 7. 9 (Figure 7 5 ), the bypyridine rings adopt the cis conformation on protonation without major conformational changes in the ligand. A 1 H 15 N HMBC experiment showed that both N1 and N1' chemical shifts moved upfield by 79.0 and 40.0 ppm respectively indicating that the proton is shared between both nitrogens. Ligand 7.1 0 (Figure 7 5 ) was the most interesting case because the ligand changes its conformation to a closed form on first protonation. Upon second protonation, the bypyridine units adopt the cis conformation and the ligand keeps its closed form conformation. In this case, 1 H 15 N HMBC experiment was the most significant experiment to explain the electronic changes in nitrogen upon protonation [2005CEJ185]. This

PAGE 134

134 example shows clearly that protons can act in a similar way to metal cations and fit into the pocket of the ligand to fix a helical conformation. Figure 7 5 [4,5]CHIRAGEN[0] (7. 9 ) AND [4,5]CHIRAGEN[0] ( 7.1 0 ) Imidazoles are of great importance in biology and imidazole nitrogens play a crucial role in many enzyme systems such as carbonic anhydrase and serine protease. 15 N NMR was used extensively to study different electronic changes for imidazole nitrogens, for example, 15 N chemical shifts of imidazole nitrogen was studied as a function of pH in histidine [1975CL1305] and in the histidine of lytic protease [1978JA804 1] in aqueous solution. It was also used to study complexation between imidazole and Zn(II) [1978IC2288] or Cd(II) [1978IC2288] in aqueous solution. Alei et al. [1980JA2881] have used 15 N NMR to study protonation in imidazole and 1 methylimidazole in aqueo us solution and concluded that the pyridine like nitrogen is the most sensitive indicator of protonation in imidazole and 1 methylimidazole because a large upfield shift of about 73.0 ppm was observed on protonation. On the contrary, the pyrrole like nitro gen moves downfield about 8.0 ppm relative to the unprotonated species.

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135 7.2 Results and Discussion 7.2.1 Structural Elucidation of S and N Acylcysteines 1 S Acyl L cysteines ( 7.13a,b ) and N Acyl L cysteines ( 7.14a,b ) were selectively synthesized by colleagues from the corresponding L cysteine (7.11) and N (acyl) 1 H benzotriazoles ( 7.12a,b ) under mild reaction conditions (Scheme 7 2) [2009JOC7165]. Structural elucidation of 7.13a,b and 7.14a,b was achieved using diffe rent 1D and 2D NMR techniques. 1 H and 13 C chemical shifts were assigned based on the 1 H 1 H, one bond and long range 1 H 13 C couplings seen in the gDQCOSY, gHMQC, and gHMBC spectra. 1 H 15 N CIGAR gHMBC spectra were acquired with a pulse sequence optimized for 15 N as described in ref. [2003MRC307]. Scheme 7 2. Selective synthesis of S and N acyl L cysteines 7. 13 a,b and 7. 14 a,b 7.2.1.1 Structural elucidation of S (4 methoxybenzoyl) L cysteine (7. 13 a) The spectra for S (4 methoxybenzoyl) L cysteine 7. 13 a were recorded in TFA d at room temperature. This compound was fully assigned based on different 2D NMR experiments (Figure 7 6 ). The gDQCOSY spectrum (Figure 7 7 ) revealed all 1 H 1 H correlations and the gHMQC spectrum revealed all 1 H 13 C one bond correlat ions. The gHMBC spectrum (Figure 7 8) revealed all long range 1 H 13 C correlations and shows that both diastereotopic protons at 3.82 and 4.07 1 Reproduced in part with permission from The Journal of Organic Chemistry 2009 74 7165 7167. Copyright 2009 American Chemical Society

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136 ppm have three bond correlations with the thiocarbonyl carbon (S CO) at 196.4 ppm. Also, the two aromatic protons at ortho position (8.05 ppm) and at meta position (7.13 ppm) show three and four bond correlations respectively to the thiocarbonyl carbon ( S CO ) The nitrogen chemical shift of 39.5 ppm was recorded in CIGAR gHMBC experiment (Figure 7 9 ). This chemical shift was revealed through the three bond correlation with the two diastereotopic protons at 3.82 and 4.07 ppm. The two NH 2 protons and the adjacent proton at 4.93 ppm were too broad to show any correlation to the nitrogen atom. The nitrogen chemical shift appears in the expected region of free NH 2 reported in literature [2002COR35]. These correlations further confirm that the compound exists exclusively in the S acyl form. Figure 7 6 1 H, 13 C, and 15 N chemical shift s of 7. 13 a Figure 7 7 1 H 1 H gDQCOSY spectrum of c ompound 7 13 a

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137 Figure 7 8 1 H 13 C gHMBC spectrum of 7. 13 a Figure 7 9 1 H 15 N CIGAR gHMBC spectrum of 7. 13 a

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138 7.2.1.2 Structural elucidation of S (2 naphthoyl) L cysteine (7. 13 b) Compound 7. 13 b exists exclusively in the S acyl form (Figure 7 10 ) and that was easily confirmed using different 2D NMR techniques. In gHMBC experiment, there is a three bond correlation between the thiocarbonyl carbon (C at 199.2 ppm) and the aromatic proton at 7.99 ppm. Moreover, the two protons at 3.65 and 3.88 ppm h ave three bond correlations with the same carbon. The assignment is confirmed by the nitrogen chemical shift (39.0 ppm) observed in CIGAR gHMBC experiment, which falls within the range of free NH 2 reported in the literature [2002COR35]. The chemical shift value of this nitrogen is slightly upfield because of protonation since the spectrum was taken in TFA d This chemical shift assignment was based on two bond correlation with the proton at 4.76 ppm and three bond correlations with the two protons at 3.65 and 3.88 ppm seen in the 1 H 15 N CIGAR gHMBC spectrum (important correlations are indicated by single headed arrow in Figure 7 10 ). Figure 7 10 1 H, 13 C, and 15 N chemical shifts of 7. 13 b 7.2.1.3 Structural elucidation of N (4 methoxybenzoyl) L cysteine (7. 14 a) The NMR spectra for N (4 methoxybenzoyl) L cysteine 7. 14 a were recorded in DMSO d 6 at room temperature. The gDQCOSY spectrum showed that the NH proton (doublet at 8.49 ppm) couples with the adjacent tertiary proton at 4.51 ppm. In gHMBC (Figure 7 12 ), the amino proton (8.49 ppm) shows a two bond correlation with the carbonyl carbon at 166.7 ppm T his chemical shift value is characteristic of amidic carbonyl and not thiocarbonyl. Th e carbonyl

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139 carbon shows further corr elations with the aromatic protons at 7.89 ppm which confirms the N acyl configuration. In 1 H 15 N CIGAR gHMBC experiment, the NH proton at 8.49 ppm shows a one bond correlation to the nitrogen atom at 114.2 ppm (Figure 7 13 ). This chemical shift is in agre ement with the previously reported values for amide nitrogens [2002COR35]. Th e NH proton appeared as a doublet with a typical one bond coupling constant of 90.3 Hz. Th e nitrogen shows three other correlations, a two bond correlation to the proton at 4.51 p pm and three bond correlations to the protons at 2.89 and 2.99 ppm (Figure 7 13 ). Figure 7 11 1 H, 13 C, and 15 N chemical shift assingments of 7. 14 a Figure 7 12 1 H 13 C gHMBC spectrum of 7. 14 a

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140 Figure 7 13 1 H 15 N CIGAR gHMBC spectrum of 7. 14 a with expansion 7.2.1.4 Structural elucidation of N (2 naphthoyl) L cysteine (7. 14 b) The NMR spectra for N (2 naphthoyl) L cysteine (7. 14 b) were recorded in DMSO d 6 at room temperature. 1 H, 13 C, and 15 N chemical shifts were fully assigned following the same approach as for 7. 13 a,b and 7. 14 a (Figure7 1 4 ). The most distinct correlations that confirm the N acyl configuration were seen in 1 H 13 C gHMBC and 1 H 15 N CIGAR gHMBC spectra. Figure 7 1 4 1 H, 13 C, and 15 N chemical shift assignments of 7. 14 b In the 1 H 13 C gHMBC spectrum, both NH proton (at 8.84 ppm) and aromatic protons (at 8.53 and 7.99 ppm) show two and three bond correlations respectively to the amidic carbonyl carbon (at 167.3 ppm). Moreover, the two diastereotopic protons at 2.96 and 3.07 ppm show

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141 three bond correlations to the carbonyl carbon of the carboxylic group (at 172.6 ppm) and do not correlate to the amidic carbonyl which is four b onds away. Figure 7 1 5 1 H 13 C gHMBC spectrum of 7. 14 b In the 1 H 15 N CIGAR gHMBC spectrum (Figure 7 1 6 ), four different protons coupled with the amidic nitrogen at 116.7 ppm. The first is the proton carried by the same nitrogen (at 8.84 ppm) which appears as doublet with one bond coupling constant ( 1 J N,H ) of 92.7 Hz. The second (at 4.63 ppm) is the proton attached to the carbon alpha to this nitrogen and shows two bond correlations. The third and fourth protons (at 2.96 and 3.07 ppm) are three bonds away and couple to the same nitrogen. 7.2.2 1 H, 13 C, and 15 N NMR Chemical Shift Assignments and Protonation Study of pH Sensitive GFP Chromophore Analogues Compounds 7.16a 7.17a and 7.17b were synthesized by Megumi Yoshioka Tarver [2009MI10] as pH sensi tive GFP chromophore analogous. Herein, we report 1 H, 13 C, and 15 N NMR chemical shifts of 7.16a 7.17a and 7.17b (Table 7 1) and a study of the protonation of 7.17a and 7.17b in TFA d using 15 N NMR spectroscopy.

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142 Figure 7 1 6 1 H 15 N CIGAR gHMBC spectrum of 7. 14 b with expansion 1 H and 13 C chemical shifts were assigned based on the 1 H 1 H, one bond and long range 1 H 13 C couplings, of the gDQCOSY, gHMQC and gHMBC spectra. 15 N chemical shifts were measured for all compounds in CDCl 3 and protonation of 7.17a and 7.17b was studied by 15 N NMR in trifluoroacetic acid d (TFA d ) using 1 H 15 N CIGAR gHMBC experiment (See Figure 7 17 for numbering in 7.16a 7.17a and 7.17b ). Scheme 7 3. Synthesis of compounds 7.16a 7.17a and 7.17b

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143 Figure 7 17. Compounds 7.16a 7.17a and 7.17b with numbering 7.2.2.1 1 H, 13 C, and 15 N NMR of 2 phenyl 4 (thiophen 2 ylmethylene)oxazol 5(4 H ) one (7.16a) 1 H, 13 C, and 15 N NMR chemical shifts of 2 phenyl 4 (thiophen 2 ylmethylene)oxazol 5(4 H ) one (7.15) are reported in Table 7 1. This compound was found to exist exclusively as the Z isomer in CDCl 3 and this was confirmed by comparing 1 H and 13 C chemical shift data with the literature [2010TL625] and by the fact that this compound was synthesized under Erlenm eyer conditions that are generally known to give only the thermodynamically stable Z isomer [1976JOC722] A 1 H 15 N CIGAR gHMBC experiment revealed the nirogen chemical shift of N3 (240.0 ppm) through three bond correlation with H6. 7.2.2.2 1 H, 13 C, and 15 N NMR and protonation study of 1 {2 (dimethylamino)ethyl) 4 (5 methylfuran 2 yl)methylene} 2 phenyl 1 H imidazol 5(4 H ) one (7.17a) 1 H, 13 C, and 15 N NMR chemical shifts are reported in Table 7 1. The 15 N chemical shift of N 1 was identified by long range cor relation with the protons of the two methylene groups (H1''' and H2'''). The 15 N NMR chemical shift of N 3 was identified by a three bond correlation to H6. The dimethylamino nitrogen (N 3''') chemical shift was revealed by long range correlation with the protons of the two methyl groups (H 4''') and the two methylene groups (H 1''' and H 2''') (Figure 7 18 and Figure 7 19). In CDCl 3 compound 7.17a exists exclusively as the thermodyamically stable Z isomer. In TFA d all three nitrogens were protonated and this was clear from the change in nitrogen chemical shift; sp 3 hybridized nitrogens, N1 and N3''', moved

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144 downfield by 9.2 and 17.5 ppm respectively. The 15 N chemical shift of N3, the sp 2 hybridized nitrogen, moved upfield by 90.5 ppm consistent with protonation and formation of the Z isomer of 7.17a by intramolecular hydrogen bonding with furyl oxygen. The coupling of 3.7 Hz between H 6 and C 5 further confirms the Z isomer. Table 7 1. 1 H, 13 C, 15 N NMR chemical shi fts of 7.16a 7.17a and 7.17b a Atom 7.16a Z 7.17a Z 7.17a3H + Z 7.17b Z 7.17b E 7.17b3H + E CDCl 3 CDCl 3 TFA d CDCl 3 CDCl 3 TFA d H 6 7.47 7.03 7.52 7.35 7.35 7.61 H 11.12 13.20 b H 7.62 7.31 7.48 6.29 6.39 6.72 H 7.16 6.13 6.50 6.69 6.77 7.29 H 7.72 7.09 7.17 7.71 H 2.33 2.40 H 8.17 7.71 7.66 7.74 7.70 7.63 H 7.53 7.44 7.66 7.53 7.50 7.67 H 7.60 7.44 7.80 7.53 7.50 7.79 H 3.80 4.41 3.85 3.91 4.54 H 2.35 3.69 2.42 2.47 3.77 H 2.04 3.07 2.10 2.15 3.17 C 2 162.8 b b 159.2 156.0 151.7 C 4 135.6 135.2 134.9 b 133.1 113.6 C 5 167.2 171.3 164.9 170.4 169.5 162.4 C 6 125.1 116.2 120.0 128.2 128.2 130.5 C 137.9 150.1 148.5 129.9 129.8 130.8 C 135.6 121.5 134.8 111.4 112.5 118.1 C 128.1 110.8 114.9 119.5 121.5 132.6 C 135.2 157.3 169.6 125.9 119.0 138.0 C 14.5 13.2 C 125.9 143.8 119.8 131.1 130.7 130.5 C 128.5 128.6 128.9 128.4 128.4 128.5 C 129.2 129.0 130.4 129.0 130.7 128.5 C 133.4 131.2 136.1 129.0 130.7 135.2 C 40.2 39.2 40.0 40.4 39.4 C 57.7 57.7 57.6 57.6 58.1 C 45.7 43.9 45.7 45.7 44.2 N 1 160.2 169.4 160.5 163.0 167.9 N 3 240.0 241.7 151.2 237.6 250.4 154.9 N 158.1 163.0 166.5 N 24.6 42.1 23.8 24.0 42.0 a Spectra were recorded on Varian Inova instrument, operating at 500 MHz for 1 H, 125 MHz for 13 C and 50 MHz for 15 N. b Not determined

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145 Figure 7 18. 1 H 15 N CIGAR gHMBC spectrum of 7.17a in CDCl 3 Figure 7 19. 1 H 15 N CIGAR gHMBC spectrum of 7.17a in TFA d 7.2.2.3 1 H, 13 C, and 15 N NMR and protonation study of 4 {(1 H pyrrol 2 yl) methylene} 1 (2 dimethylaminoethyl)} 2 phenyl 1 H imidazol 5(4 H ) one (7.17b) 1 H, 13 C, and 15 N NMR chemical shifts are reported in Table 7 1. Compound 7.17b exists in CDCl 3 as a mixture of E (75 %) and Z isomer (25%), but in TFA d only one isomer is found

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146 with 15 N chemical shift of N 3 shifted upfield by 95.9 ppm from the E isomer and 82.7 ppm from the Z isomer (Figures 7 20, 7 21, and 7 22). We suggest that in this case, the E isomer ( 7.17b3H + ) predominates in the protonated system due to hydrogen bonding between the NH proton of the pyrryl group and the carbonyl oxygen (Scheme 7 4). This hypothesis was confirmed by a coupling of 8.5 Hz between H 6 and C 5. Furthermore, the 1 H 15 N CIGAR experiment failed to detect 3 J H6 N3 coupling which suggests a value < 3.0 Hz and further supports the E isomer assignment. The amide nitrogen of the imidazolinone ring (N 1) however, shifted downfield 9.2 ppm (from E isomer), and by 4.9 ppm (from Z isomer) upon protonation. For dimethylamino nitrogen (N 3'''), prominent deshieding was observed in protonated 7.17b3H + (+16.0 ppm from E isomer and 16.2 ppm from Z isomer). In contrast to sp 2 nitrogens, sp 3 nitrogens are known to be deshielded by protonation [2002COR35]. Scheme 7 4. 7.3 Conclusions In conclusion, different NMR techniques were used to fully assign 1 H, 13 C, and 15 N NMR chemical shifts in 7.13a,b 7.14a,b 7.16a and 7.17a,b 15 N NMR spectroscopy was used successfully to differentiate between S and N acyl cysteines ( 7.13a,b and 7.14a,b ) and to study protonation in 7.17a,b 1 H 15 N CIGAR gHMBC experiments were very e ffective in both cases.

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147 Figure 7 20. 1 H 15 N CIGAR gHMBC spectrum of 7.17b in CDCl 3 with expansion Figure 7 21. 1 H 15 N CIGAR gHMBC spectrum of 7.17b in TFA d

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148 Figure 7 22. Expansions of 1 H 15 N CIGAR gHMBC spectrum of 7.17b in TFA d 7.4 Experimental 7.4.1 General M ethods NMR spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1 H, 125 MHz for 13 C and 50 MHz for 15 N, equipped with a three channel, 5 mm, indirect detection probe, with z axis gradients. The solvents w ere chloroform d DMSO d6 and TFA d and the temperature was 25 C. The chemical shifts for 1 H and 13 C were reference to the residual solvent signal, 7.26 ppm for 1 H in CDCl 3 2.50 ppm in DMSO d6 and 11.50 ppm in TFA d on the tetramethylsilane scale. The chemical shifts for 15 tetramethylsilane is 100.0000000 MHz. For conversion to the neat nitromethane sc ale, subtract

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149 381.7 ppm [2002COR35]. 1 H spectra were acquired in one transient, with a 90 pulse, no relaxation delay, and an acquisition time of 5 s. Typically, 1 H 13 C gHMBC spectra were acquired in 2048 points in f2 and 1 s relaxation delay. In f1 256 i ncrements were acquired in 1 transient and the corresponding FID s were zero filled twice prior to the second Fourier transform. 1 H 15 N CIGAR gHMBC spectra were acquired with a pulse sequence optimized for 15 N as decribed in ref. [2003MRC307]; 2048 points were acquired in f2, with 1 s relaxation delay. 1024 increments were acquired in f1 on a spectral window from 0 to 400 ppm, and the corresponding FID was zero filled twice prior to Fourier transform. The accordion delay was optimized for a value of 1 H 15 N coupling constants between 3 and 10 Hz. The number of transients per increment was between 4 and 64, depending on the concentration of the sample. Total experiment time was in most cases, ca 2 h. Coupling constants 3 J H6 C5 in 7.17a3H + and 7.17b3H + were m easured from the trace seen in gHMBC experiment with a linewidth of 3.0 Hz in 1 H NMR and digital resolution of 0.1 Hz in f2 The measure ment of 3 J H6 N3 in 7.17b3H + was also attempted with a linewidth of 3.0 Hz in 1 H NMR and digital resolution of 0.1 Hz in f2 7.4.2 Characterization of Compounds 7. 13 a,b, 7. 14 a,b, 7.16a. and 7.17a.b Synthetic procedures and C,H,N analysis for compounds 7. 13 a,b and 7. 14 a,b see reference [2009JOC7165] and for compounds 7.16a 7.17a and 7.17b see reference [2009MI 10 ]. S (4 M ethoxybenzoyl) L cysteine ( 7. 13 a ). White microcrystals; mp 160.0 162.0 C. 1 H NMR (500 MHz, TFA d ) 8.05 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 4.93 (br s, 1H), 4.07 (dd, J = 16.2, 3.2 Hz, 1H), 4.03 (s, 3H), 3.82 (dd, J = 15.9, 6.6 Hz, 1H); 13 C NMR (125 MHz, d TFA d ) 196.4, 170.9, 165.8, 130.7, 128.0, 114.8, 55.3, 28.4. S (2 Naphthoyl) L c ysteine ( 7.8b ). White microcrystals; mp 167.0 168.0 C. 1 H NMR (500 MHz, TFA d ) 8.24 (d, J = 8.7 Hz, 1H), 7.99 (d, J = 7.1 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H),

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150 7.71 (d, J = 8.0 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 4.76 (dd, J = 5.6, 3.7 Hz, 1H), 3.88 (dd, J = 15.7, 3.7 Hz, 1H), 3.65 (dd, J = 15.7, 6.6 Hz, 1H); 13 C NMR (125 MHz, d 34.1, 132.1, 129.9, 129.0, 128.6, 127.0, 124.0, 123.7, 54.7, 29.2. N (4 M ethoxybenzoyl) L cysteine ( 7.9a ). White microcrystals; mp 99.0 101.0 C. 1 H NMR (500 MHz, DMSO d6 ) 12.83 (br s, 1H), 8.49 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 7.9 Hz, 2H), 7.01 (d, J = 7.8 Hz, 2H), 4.51 (br s, 1H), 3.81 (s, 3H), 2.99 (br s, 1H), 2.89 (br s, 1H), 2.55 (br s, 1H); 13 C NMR (125 MHz, DMSO d6 ) 172.7, 166.7, 162.5, 130.1, 126.7, 114.2, 56.1, 25.9. N (2 Naphthoyl) L cysteine ( 7.9b ). White microcrystals; mp 151.0 152.0 C. 1 H NMR (500 MHz, DMSO d6 ) 12.94 (br s, 1H), 8.84 (br s, 1H), 8.53 (s, 1H), 8.05 (dd, J = 7.2, 1.8 Hz, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.99 (dd, J = 8.1, 1.5 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 6.3 Hz, 1H), 4.67 4.59 (m, 1H), 7.60 (t, J = 6.6 H z, 1H), 3.11 3.03 (m, 1H), 3.00 2.92 (m, 1H), 2.64 (t, J = 8.2 Hz, 1H); 13 C NMR (125 MHz, DMSO d6 ) 172.6, 167.3, 134.9, 132.8, 129.6, 128.5, 127.5, 125.0, 56.4, 26.0. 2 Phenyl 4 (thiophen 2 ylmethylene)oxazol 5(4H) one ( 7.16a ). Yellow microcrystals; mp 175.5 C). 1 H NMR (500 MHz, CDCl 3 ) 8.17 ( d t, J = 7.2, 0.7 Hz, 2H), 7.72 (dt, J = 5.1, 0.9 Hz, 1H), 7.62 (dt, J = 3.8, 0.9 Hz, 1H), 7.60 (tt, J = 7.4, 0.7 Hz, 1H), 7.53 (tt, J = 7.5, 0.7 Hz, 2H), 7.47 (s, 1H), 7.16 (dd, J = 5.1, 3.8 Hz, 1H); 13 C NMR (125 MHz, CDCl 3 ) 167.2, 162.8, 137.9, 135.6, 135.2, 133.4, 129.2, 128.5, 128.1, 125.9, 125.1. 1 {2 (Dimethylamino)ethyl ) 4 (5 methylfuran 2 yl)methylene} 2 phenyl 1H imidazol 5(4H) one ( 7.17a ). 1 H NMR (500 MHz, CDCl 3 ) 7.71 (dt, J = 6.1, 1.8 Hz, 2H), 7.46 7.41 (m, 3H), 7.31 (d, J = 3.3 Hz, 1H), 7.03 (s, 1H), 6.13 (d, J = 3.3 Hz, 1H), 3.80 (t, J = 7.0 Hz, 2H), 2.35 (t, J = 7.0 Hz, 2H), 2.04 (s, 6H); 13 C NMR (125 MHz,

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151 CDCl 3 ) 171.3, 161.3, 157.3, 150.1, 143.8, 135.2, 131.2, 129.0, 128.6, 121.5, 116.2, 110.8, 57.7, 45.7, 40.2, 14.5. 4 {(1H Pyrrol 2 yl)methylene} 1 (2 dimethylaminoethyl)} 2 phenyl 1H imidazol 5(4H) one ( 7.17b ). Yellow gum Two isomers: for Z isomer (25%): 11.12 (br s, 0.3H), 7.75 7.71 (m, 0.6H), 7.53 7.50 (m, 0.8H), 7.17 (s, 0.3H), 7.09 7.06 (m, 0.3H), 6.70 6.67 (m, 0.3H), 6.30 6.26 (m, 0.3H), 3.85 (t, J = 7.0 Hz, 0.7H), 2.42 (t, J = 7.0 Hz, 0.7H), 2.10 (s, 2H); for E isomer (57%): 13.20 (brs, 0.9H), 7.71 7.67 (m, 2H), 7.50 7.46 (m, 3H), 7.35 (s, 1H), 7.17 7.15 (m, 1H), 6.78 6.74 (m, 1H), 6.40 6.37 (m, 1H), 3.91 (t, J = 7.0 Hz, 2H), 2.47 (t, J = 7.0 Hz, 2H), 2.15 (s, 6H). 13 C NMR (125 MHz, CDCl 3 ) for Z isomer: 170.4, 159.2, 131.1, 129.9, 129.0, 128.4, 125.9, 119.5, 111.4, 57.6, 45.7, 40.0; for E isomer: 169.5, 156.0, 133.1, 130.7, 129.8, 128.4, 128.2, 121.5, 119.0, 112.5, 57.6, 45.7, 40.4

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152 CHAPTER 8 CONCLUSIONS AND SUMM ARY OF ACHIEVEMENTS Chapter 2 o f this thesis presents a convenient route for the synthesis of ( aminoacyl) amino substituted heterocycles from N acyl benzotriazoles. These heterocyclic derivatives are useful intermediates for the synthesis of endomorphin 2 (EM 2) analogues [2004JME3591], secretase inhibitors [2004TL2323], and inhibitors of tumor necrosis factor [2001JME4252]. I describe a variety of acylamino thiazoles, benzothiazo les, benzimidazoles, thiadiazoles, pyrimidones, pyrazoles, and pyridines, all prepared in high yields under microwave irradiation. This methodology was proved to be general and overcomes the common obstacles faced in alternative syntheses of compounds of this type such as long reaction times, harsh conditions, and low yields. The tautomeric equilibria of heterocyclic systems are of great importance for understanding the function of many important biological components of living systems such as nucleoti de bases. NMR techniques are very powerful in elucidating tautomerism. Chapter 3 describes the synthesis and an NMR study of tautomeric equilibria in a series of 2 (2 substituted methylenehydrazinyl) 3 phenylquinazolin 4(3 H ) ones. 15 N NMR spectroscopy was the main tool in this study and it was found that the imino tautomer is dominant in DMSO solution and in the solid state, thus following the tautomeric preferences of aminoguanidines. When the 3,4 dihydro 4 oxo 3 phenylquinazolin 2 yl moiety is replaced by a more aromatic moiety such as benzimidazol 2 yl or 4,6 dimethylpyrimidin 2 yl, the amino tautomer prevails in DMSO solution. Chapter 4 concerns N aminoalkyl)tetrazoles, which have received considerable attention recently because they provide modified protein formation inhibitors and some benzodiazepine

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153 derivatives were found effective in treating cardiac arrhythmias mammals These compounds have previously been reported without considering their tautomeric equilibria, which ar e of great importance since different tautomers can have different interactions with diverse receptors. I synthesized some N aminoalkyl)tetrazoles and studied their tautomeric behavior by NMR. The equilibrium between the N 1 and N 2 tautomers of N am inoalkyl)tetrazoles was shown to favor the N 2 tautomer in non polar solvents, but the equilibrium shifts to favor the N 1 tautomer with increasing solvent polarity. Bulky substituents in the 5 position of the tetrazole ring favor the N 2 tautomer. Thermod ynamic and kinetic parameters of this interconversion were calculated and a detailed mechanism of interconversion between the N 1 and N 2 tautomers has shown to involve both tight ion pairs which can relax to give isomerisation without crossover or may bec ome solvent separated and thus lead to crossover between different components in a mixture of two tetrazoles in which both the tetrazole ring and the amino substituents differ. Chapter 5 studies the conformational equilibria and barriers to rotation in so me novel nitroso derivatives of indolizines and 3 and 5 azaindolizines by a variety of NMR techniques. These nitroso compounds were identified as monomers by 13 C substituent chemical shifts (SCS), which exist in two rotameric, syn and anti forms and the barriers of rotation between these rotamers were calculated using VT NMR. Molecular modeling study is in good agreement with the experimental NMR study and it showed that in most cases steric interactions govern the ratio of syn to anti The only exception was 2 methyl 3 nitrosoindolizine (5.4) where electronic factors governed the conformational equilibrium. Chapter 6 deals with pyridazines, biologically active scaffolds in many important therapeutic reagents. Pyridazines are also frequently used as ligand s in organometallic catalysts

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154 for important synthetic transformations. 1 H, 13 C, and 15 N NMR chemical shifts were recorded for some 3,6 disubstituted pyridazines using different 1D and 2D NMR techniques. 15 N NMR spectroscopy was used to determine the site of N oxidation and N alkylation in some of these pyridazine derivatives. Chapter 7 uses different NMR techniques to elucidate the structures of some S and N acylcysteines and pH sensitive GFP chromophore analogous and illustrates that 15 N NMR spectroscopy has become a very powerful technique for the structural analysis of compounds that contain nitrogen. It is now considered the third major probe beside 1 H and 13 C NMR in solving structural problems in organic and bioorganic molecules. 15 N NMR spectroscopy was the main tool in distinguishing the S acyl from N acylcysteines because of the large difference in nitrogen chemical shift between the free nitrogen and the acylated one. Moreover, it was used to study protonation in some imidazolinon e derivatives that were used as pH sensitive GFP chromophore analogs. Upon protonation, compound 7.17a adopts the Z configuration while compound 7.17b adopts the E configuratio n.

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155 LIST OF REFERENCES The reference citation employed throughout the thesis is f (Vol. 99) Academic Press, 2010 (Ed. Alan R. Katritzky). In this system references are cited in the text by giving two or three letter code to every journal. This code is preceded by the year (using all four digits) and followed by the page number. Additional notes: i Each reference code is followed by the conventional literature citation as depicted in the Advances in Heterocyclic Chemistry instruction for authors ii ess co mmonly used books and journals. iii The list of references is arranged according to the designated code in the order of (a) year, (b) journal in alphabetical order, (c) volume or part number, and ( d ) page numb er. 1946JA2496 G. B. Bechmann and L. V. Heisey, J. Am. Chem. Soc ., 68 2496 ( 1946) 1946JCS1075 E. T. Borrows, D. O. Holland, and J. Kenyon, J. Chem. Soc. 1075 ( 1946) 1947JA254 R. B. Saul, J. Am. Chem. Soc., 69 254 (1947). 1947JCS239 W. G. Overend and L. F.Wiggins, J. Chem. Soc,. 239 (1947). 1950JOC81 B .F. Crowe and F. F. Nord, J. Org. Chem ., 15 81 ( 1950) 1950JOC1082 J. S. Mihina and R. M. Herbst, J. Org. Chem., 15 1082 ( 1950). 1953MI2 J. D. Watson and F. H. C. Crick, Cold Spring Harbor Symposia on Quantitative Biology, 18 123 (1953). 1955JCS855 G. W. Kenner, C. B. Reese, and A. R. Todd, J. Chem. Soc., 855 (1955). 1956JA1961 C. G. Overberger,N. R. Byrd, and R. B. Mesrobian, J. Am. Chem. Soc., 78 1961 (1956). 1956JA4197 M. H. Kaufman, F. M. Ernsberger, and W. S. McEwan, J. Am. Chem. Soc.,

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174 BIOGRAPHICAL SKETCH Bahaa El Dien M El Gendy was born in Al Kanater Al Khairia, Egypt. He worked under Professor Samy A. Essawy at Benha University, where he received his Master of Science in July 2003. In 2005, he was awarded the Monbukagakusho Scholarsh ip from Japanese government to pursue his Ph.D studies in Japan. At the same time he had an invitation to join the University of Florida Center of Heterocyclic Compounds supervised by Professor Alan R. Katritzky where he started in February 2006. During 20 06, Bahaa received an Egyptian government fellowship and started his Ph.D in the Chemistry Department of the University of Florida in January 2007 working under Professor Alan R. Katritzky. His research involves the synthesis and NMR studies of different h eterocyclic systems. During his course of study, Bahaa was awarded the Certificate of Outstanding Achievement twice from the University of Florida, a Travel Award from The College of Liberal Arts and Sciences, and the Proctor and Gamble Award for Excellenc e in Graduate Research.