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NMR AND SYNTHETIC STUDIES OF HETEROCYCLES

By

BOGDAN DRAGHICI

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

2011

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© 2011 Bogdan Draghici

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Dedicated to my family and my friends, for their constant support

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ACKNOWLEDGMENTS

I would like to express my gratitude to my advisor Professor Alan R. Katritzky for

his consistent support and guidance. His knowledge for science and dedication for

education, and chemistry are impressive.

I would like to express acknowledgement to our Senior Professor Dennis C. Hall

for his constant help and very useful chemistry discussions, Dr. Ion Ghiviriga for the

NMR training and useful discourse about NMR experiments and features of structural

elucidation, Mr. Robert Harker for his support with the NMR maintenance.

Also, I would like to express my gratitude to the committee members: Professor

Ronald Castellano, Professor Eric Enholm, Dr. David Powell and Professor Fazil Najafi

for their support, useful suggestions.

This work would not have been possible without the help and support of all

Katritzky group members; I would like to thank all of them for their support, especially

Dr. Bahaa El-Dien El-Gendy, Mr. Ebrahim Ghazvini Zadeh and Mr. Zuoquan Wang for

useful discussions, and graduate students Judit Kovacs, Claudia El-Nachef, Khanh Ha,

Lucas Beagle, Mirna El-Khatib and Davit Jiskhariani for their friendship and useful

remarks.

Finally, I would like to thank my family, my father and my sister for their constant

encouragement and support.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF FIGURES .......................................................................................................... 8

ABSTRACT ................................................................................................................... 16

1 GENERAL INTRODUCTION .................................................................................. 18

2 RELATIVE STABILITIES OF N-(α-AMINOALKYL)TETRAZOLES .......................... 27

2.1 Background ................................................................................................... 27 2.2 The Proposed N-(α-Aminoalkyl)Tetrazole Substrates ................................... 31

2.4 Results and Discussion ................................................................................. 33

2.5 Cross-over Experiments ................................................................................ 39 2.6 Conclusions ................................................................................................... 41 2.7 Experimental Section .................................................................................... 42

3 EFFICIENT SYNTHESIS OF PROTECTED α-AMINOXYACYL CONJUGATES .... 49

3.1 Background ................................................................................................... 49 3.1.1 General preparative methods of N-Protected-α-Aminoxy acids .......... 51

3.1.2 Literature Methods of Acylation ........................................................... 52 3.2 Results and Discussion ................................................................................. 53

3.2.1 Synthesis of N-Protected-α-Aminoxy acids Conjugates ...................... 53 3.2.2 Synthesis of N-Cbz-Protected α-Aminoxy acids (3.18 a-d, 3.18c+c’) .. 54

3.2.3 Synthesis of N-Cbz-Protected(α-Aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’) ........................................................................................ 54

3.2.4 Synthesis of O-(Protected-α-Aminoxyacyl)steroids 3.20a-e and O-(protected-α-aminoxyacyl)terpenes 3.20e-h .................................................. 55

3.2.5 Synthesis of O-(Protected-α-Aminoxyacyl)sugar (3.24) ...................... 57 3.2.6 Synthesis of N-(Protected-α-Aminoxyacyl)nucleosides 3.26a,b .......... 60

3.3 Conclusions ................................................................................................... 61 3.4 Experimental Section .................................................................................... 61

3.4.1 General Procedure for (L)-2-Bromo-carboxylic acids synthesis (3.8a-d).......................................................................................................... 62

3.4.2 General Synthesis of α-2-(Benzyloxycarbonylaminooxy)carboxylic acid (3.18a-d, 3.18c+c’) ................................................................................. 63

3.4.3 General Synthesis of N-Cbz-Protected(α-Aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’) ........................................... 65

3.4.4 General Synthesis of O-(Protected-α-Aminoxyacyl)steroids (3.20a-d), and O-(Protected-α-Aminoxyacyl)terpenes (3.20e-h) .............................. 68

3.4.5. Preparation of D-(D-2-Hydroxy-2-((3aR,5R,6S,6aR)-6-hydroxy-2,2-dimethyltetrahydrofuro[3,2-d][1,3]dioxol-5-yl)ethyl)2 benzyloxycarbonyl-aminooxy)propanoate (3.24) ......................................................................... 73

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3.4.6 General Synthesis of O-(Protected-α-aminoxyacyl)nucleosides (3.26a,b). ....................................................................................................... 73

4 MULTINUCLEAR NMR ANALYSIS OF A VARIETY OF MOLECULAR STRUCTURES ....................................................................................................... 75

4.1 Background ................................................................................................... 75 4.1.1 Applications of Amino Sugars in drug design .......................................... 75 4.1.2 Applications of 15N in Structural Elucidation ............................................ 77

4.2 Results and Discussion ................................................................................. 80 4.2.1 Proton Correlations of Cbz-L-Phe-N-Galactopyranose ............................ 80

4.2.2 1H 13C 15N Chemical shifts of some Pyrazine derivatives .................... 86

4.2.3 Total correlation of 2-Ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6) .......... 89 4.3 Conclusions ................................................................................................... 90

4.4 Experimental Section .................................................................................... 91

5 SYNTHESIS OF 2,4-DISUBSTITUTED QUINAZOLINES, 4H-BENZO[E][1,3]OXAZINE AND 4H-BENZO[E][1,3]THIAZINE BY ANRORC REARRANGEMENTS OF 1,2,4-OXADIAZOLES ................................................... 93

5.1 Background ................................................................................................... 93 5.1.1 The importance of 1,2,4-Oxadiazoles ................................................. 94 5.1.2 Preparation of 1,2,4–Oxadiazoles ....................................................... 94

5.1.3 ANRORC rearrangements of 1,2,4-Oxadiazoles ................................ 96 5.1.4 The Boulton-Katritzky rearrangements of 1,2,4-Oxadiazoles .............. 99

5.1.5 Photochemical rearrangement of 1,2,4-Oxadiazoles ........................ 100

5.1.6 1,2,4-Oxadiazoles rearrangements using Strong Nucleophiles ........ 102

5.2 Results and Discussion ............................................................................... 103 5.2.1 Preparation of 1,2,4-Oxadiazoles (5.63a-j) ....................................... 103 5.2.2 Substrate Design............................................................................... 104

5.2.3 Rearrangement Results .................................................................... 108 5.3 Conclusions ................................................................................................. 110

5.4 Experimental Section .................................................................................. 111 5.4.1 General procedure for the preparation of N-Acylbenzotriazoles (5.64a-

e) ................................................................................................................. 111 5.4.2 Synthesis of N-Hydroxybenzimidamide (5.65a-c) ............................. 113

5.4.3 Preparation of 1,2,4-Oxadiazoles (5.63a-f) ....................................... 114 5.4.4 Preparation of 1,2,4-Oxadiazoles (5.63h,i) ........................................ 117 5.4.5 Synthesis and characterization data of the addition products 5.76

and rearranged products 5.77 ..................................................................... 118

6 FINAL CONCLUSIONS AND ACHIEVEMENTS ................................................... 121

LIST OF REFERENCES ............................................................................................. 124

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LIST OF TABLES Tables page 2-1 Synthesis of tetrazoles (2.14a-f). ........................................................................ 32

2-2 Preparation of N-(α-aminoalkyl)tetrazoles (2.16a-g) by the Mannich reaction. ... 32

2-3 Alternative method to the Mannich products 2.18a,b. ........................................ 33

2-4 1H, 13C 15N chemical shifts (δ, ppm, DMSO-d6) of 2.18a and 2.18b. .................. 37

2-5 Ratio of N1 and N2 isomers in different solvents. ............................................... 38

3-1 Preparation of of N-Cbz-protected(α-aminoxy)carboxylic acids (3.18a-d, 3.18c+c’). ........................................................................................................... 54

3-2 Preparation of N-Cbz-protected(α-aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’). ........................................................................................................... 55

3-3 Preparation of O-(protected--aminoxyacyl)steroids 3.20a-d and O-

(protected--aminoxyacyl)terpenes 3.20e-h. ...................................................... 56

3-4 Preparation of N-(protected--aminoxyacyl)nucleosides 3.26a,b. ...................... 61

4-1 1H and 13C NMR chemical shifts, ppm of some pyridazine derivatives 4.2 – 4.5. ..................................................................................................................... 86

4-2 15N NMR chemical shifts, ppm of pyridazines 4.2 – 4.5. ..................................... 87

5-1 Preparation of 1,2,4-oxadizaoles 5.63a-g. ........................................................ 103

5-2 Preparation of 1,2,4-oxadizaoles 5.63h,i. ......................................................... 103

5-3 Ring fragmentation products for 1,2,4-oxadiazoles 5.63a-i. ............................. 106

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LIST OF FIGURES

Figures page 2-1 1H-15N gHMBC-CIGAR experiment for 2.18aN1+ 2.18aN2. ............................... 35

2-2 1H-13C gHMBC experiment for 2.18aN1+ 2.18aN2. ............................................ 35

2-3 1H- 15N CIGAR-gHMBC experiment for 2.18bN1+ 2.18bN2. .............................. 36

2-4 1H-13C gHMBC experiment for 2.18bN1+ 2.18bN2. ........................................... 36

2-5 VT NMR for 2.18aN1+ 2.18aN2. ........................................................................ 39

3-1 1H-13C gHMBC experiment of 3.24. .................................................................... 58

3-2 1H-1H dQCOSY of 3.24. ...................................................................................... 59

3-3 1H-1H dQCOSY expansion for sugar fragment of 3.24. ...................................... 59

3-4 1H and 13C chemical shifts assignments of 3.24. ................................................ 60

4-1 VT NMR spectrum of Cbz-L-Phe-N-galactopyranose (4.1). ............................... 81

4-2 Selective decoupling experiment of Cbz-L-Phe-N-galactopyranose in CDCl3. ... 82

4-3 1H-1H dQCOSY of Cbz-L-Phe-N-galactopyranose (4.1) amide fragment. ......... 83

4-4 1H-1H dQCOSY of Cbz-L-Phe-N-galactopyranose (4.1) sugar part expansion. .. 84

4-5 1H-13C gHMQC of 3,6-dimethyl-4-(methylthio)pyridazine (4.4). .......................... 88

4-6 1H-13C gHMBC of 3,6-dimethyl-4-(methylthio)pyridazine (4.4)............................ 88

4-7 1H-15N CIGAR-gHMBC of 3,6-dimethyl-4-(methylthio)pyridazine (4.4). .............. 89

4-8 Total assignment of 2-ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6). ........................ 89

4-9 1H-15N CIGAR-gHMBC of 2-ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6). ............... 90

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LIST OF SCHEMES Schemes page 1-1 Example of a tautomeric equilibrium of N-(α-aminoalkyl)tetrazoles. ................... 20

1-2 Esterification versus amidation reaction. ............................................................ 21

1-3 Carboxylic acid activation and coupling step. ..................................................... 22

1-4 1H-Benzotriazole as synthetic auxiliary. ............................................................. 23

1-5 New rearrangements of 1,2,4-oxadiazoles. ........................................................ 26

2-1 The tautomeric structures of 1H-tetrazole and 2H-tetrazole. .............................. 27

2-2 Applications of tetrazole as pharmacophore and as anion scavenger. ............... 28

2-3 Examples of biologically active pharmacophores containing N-(α-aminoalkyl)tetrazoles. ......................................................................................... 28

2-4 Tautomeric equilibrium between 1- and 2-substituted benzotriazoles. ............... 29

2-5 N-(α-Dialkylaminomethyl)benzotriazole tautomers. ............................................ 30

2-6 The rearrangement mechanism of N-(α-dialkylaminomethyl)benzotriazole. ....... 30

2-7 Tetrazole substrates for the tautomerism studies. .............................................. 31

2-8 Preparation of tetrazoles (2.14a-f). ..................................................................... 32

2-9 Preparation of N-(α-aminoalkyl)tetrazoles (2.16a-g) by the Mannich reaction. ... 32

2-10 Preparation of N-(α-aminoalkyl)tetrazoles (2.18a,b) by an alternative method. .. 33

2-11 Cross-over experiment of N-(α-aminoalkyl)tetrazoles 2.16a,g. .......................... 39

2-12 Cross-over experiment of 2.16h,i ....................................................................... 40

2-13 Dissociation-recombination mechanism of N-(α-aminoalkyl)tetrazoles............... 41

2-14 Preparation of Mannich product 2.18a. ............................................................... 46

2-15 Preparation of Mannich product 2.18b. .............................................................. 48

3-1 Applications of aminoxy acids in molecular design. ............................................ 50

3-2 Some applications of acylation in drug design. ................................................... 51

3-3 Preparation methods for N-protected aminoxy acids. ......................................... 52

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3-4 Synthesis of N-protected-α-aminoxy acids conjugates. ...................................... 53

3-5 Synthesis of N-Cbz-protected(α-aminoxy)carboxylic acids (3.18a-d, 3.18c+c’). ........................................................................................................... 54

3-6 Synthesis of N-Cbz-protected(α-aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’). ........................................................................................................... 55

3-7 Synthesis of O-(protected--aminoxyacyl)steroids 3.20a-d and O-(protected-

-aminoxyacyl)terpenes 3.20e-h. ....................................................................... 56

3-8 Preparation of O-(protected-α-aminoxyacyl)sugar (3.24). .................................. 57

3-9 Synthesis of (protected--aminoxyacyl)nucleosides 3.26a,b. ............................ 60

4-1 The structures of the investigated compounds ................................................... 75

4-2 Examples of biologically active aminosugars...................................................... 76

4-3 Syn- and anti- periplanar conformations for Z and E isomers. ........................... 77

4-4 The structures of compounds (4.2 – 4.6). ........................................................... 78

4-5 Some pyridazine derivatives previously characterized by 1H-15N CIGAR-gHMBC. .............................................................................................................. 79

4-6 Possible rotamers of Cbz-L-Phe-N-galactopyranose (4.1). ................................ 80

4-7 Total proton assignment of Cbz-L-Phe-N-galactopyranose. ............................... 85

5-1 General reaction scheme of the Boulton-Katritzky rearrangement. .................... 93

5-2 Applications of 1,2,4-oxadiazoles. ...................................................................... 94

5-3 Preparative methods of 1,2,4-oxadiazoles. ........................................................ 95

5-4 Types of ANRORC rearrangements. .................................................................. 96

5-5 ANRORC degenerative rearrangements of 1,2,4-oxadiazoles. .......................... 97

5-6 ANRORC [2+3] rearrangements of 1.2.4-oxadiazoles. ....................................... 97

5-7 5-Perfluoroalkyl-1,2,4-oxadiazoles reactions with hydrazines. ........................... 98

5-8 5-Perfluoroalkyl-1,2,4-oxadiazoles reactions with methyl hydrazine. .................. 99

5-9 Some examples of the Boulton-Katritzky rearrangement of 1,2,4-oxadiazoles with pivotal nucleophile at C(3). ........................................................................ 100

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5-10 Photochemical transformations of 1,2,4-oxadiazoles. ...................................... 101

5-11 Synthesis of N-imidoyl-aziridines. ..................................................................... 101

5-12 Photochemical rearrangements of 1,2,4-oxadiazoles with pivotal nucleophile at C(3). ............................................................................................................. 102

5-13 Addition of strong nucleophiles to 1,2,4-oxadiazole ring at low temperature. ... 102

5-14 Preparation of 1,2,4 oxadiazoles 5.63a-g. ........................................................ 103

5-15 Preparation of 1,2,4 oxadiazoles 5.63h,i. ......................................................... 103

5-16 Possible 1,2,4-oxadiazole BKR rearrangements with pivotal nucleophile at C(3) or C(5). ..................................................................................................... 104

5-17 Possible rearrangements of 1,2,4-oxadiazoles using a pivotal nucleophile at position C(5). .................................................................................................... 104

5-18 The direct rearrangement of 2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenol (5.63). . 105

5-19 Novel rearrangements of 1,2,4-oxadiazoles. .................................................... 106

5-20 Ring fragmentation of 1,2,4 oxadiazoles 5.63a-i............................................... 106

5-21 Possible rotamers of 2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenol (5.61a). ............ 108

5-22 1,2,4-Oxadiazole rearrangements in the presence of n-butyllithium. ................ 109

5-23 Preparation of 2-(3-phenyl-1,2,4-oxadiazol-5-yl)benzenthiol (5.63c). ............... 115

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LIST OF ABBREVIATIONS [α]21

D refractive index at 21 oC

Ac Acetyl (CH3C=O)

Ala Alanine

Anal. Analysis

ANRORC Addition of Nucleophile, Ring-Opening and Ring-Closure

Ar Aryl

br Broad signal (spectral)

Bn Benzyl

BKR Boulton Katritzky Rearrangement

BOP-Cl Bis(2-oxo-3-oxazolidinyl)phosphinic chloride

bs broad signal (spectra)

BtH Benzotriazol

Bu Butyl

oC Degree Celsius

Calcd. Calculated

Cbz Carbobenzyloxy (BnOC=O)

CDI Carbonyldiimidazole

CIGAR-HMBC Constant time inverse-detection gradient accordion rescaled heteronuclear multiple bond correlation spectroscopy (NMR technique)

d Doublet (spectral)

D (10 point) Dextrorotatory (right)

D (12 point) Dipole moment (Debyes)

DCC N,N’-Dicyclohehxlcarbodiimine

DCM Methylene chloride

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DIC Diisopropylcarbodiimide

DIPEA Diisopropylethylamine

DMAP 4-Dimethylaminopyridine

DMSO-d6 Dimethylsulfoxide (solvent)

DMF Dimethylformamide (solvent)

E Entgegen (opposite, trans)

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

eq. Equivalent(s)

et al. and others

EtOAc Ethyl Acetate (solvent)

g Gram(s)

gDQCOSY Gradient Double Quantum Correlation Spectroscopy (NMR technique)

gHMQC Gradient Heteronuclear Multiple Quantum Coherence (NMR technique)

gHMBC Gradient Heteronuclear Multiple Bond Correlation (NMR technique)

Gly Glycine

HOBt N-Hydroxybenzotriazole

HBTU O-benzotriazole-N’N’N’N’-tetramethyluroniumhexafluorophosphate

HRMS High resolution mass spectroscopy

Hz Hertz (spectral)

i-Pr Isopropyl

J Coupling constant

L (10 point) Levorotatory (left)

Leu Leucine

m Multiplet

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m Meta locant

Me Methyl

min. Minute(s)

mL Mililiter

mol Mole(s)

MW Microwave

m.p. Melting point

m/z Mass-to-charge ratio

NMR Nuclear Magnetic Resonance

NOE Nuclear Overhauser Effect (NMR technique)

Nu Nucleophile

o Ortho locant

p Para locant

Ph Phenyl

Phe Phenylalanine

ppm Part per million

q Quartet (NMR technique)

R (10 point) Rectus (right)

Ref. Reference

TLC Thin layer chromatography

r.t. Room temperature

s Singlet (spectral)

S Siister (left)

sx Sextet (spectral)

t Triplet (NMR technique)

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t Tertiary

TBTU O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

THF Tetrahydrofuran (solvent)

TMS Tetramethylsilane

UV Ultraviolet

W Watt(s)

wt% Weight percent

Z Zusammen (together, cis)

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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

NMR AND SYNTHETIC STUDIES OF HETEROCYCLES

By

Bogdan Draghici

December 2011

Chair: Alan R. Katritzky Major: Chemistry

The theme of this thesis is to explore new synthetic methodologies with

applications in the field of heterocyclic chemistry. Chapter 1 presents a general

introduction to the subsequent chapters and a brief discussion of the importance of

benzotriazole methodology in the organic synthesis. Chapter 2 describes the tautomeric

equilibrium of a new series of N-(α-aminoalkyl)tetrazoles, factors that influence the

equilibrium between the two tautomers and some applications in medicinal and

supramolecular chemistry. Chapter 3 presents the applications of benzotriazole

methodology in the synthesis of various aminoxy acid bioconjugates. In this chapter, we

have investigated the reactivity of the aminoxy acids activated as N-Cbz-protected(α-

aminoacyl)benzotriazoles in the presence of a variety of nucleophiles such as terpenes,

sterols, nucleosides and unprotected sugars.

The coupling reactions take place under mild conditions; the retention of chirality

was confirmed by 1H NMR. We found this coupling reaction to be selective, efficient and

convenient; this proves the utility of this method.

Chapter 4 focuses on the 2D NMR characterization of a variety of heterocyclic

systems. In this chapter we have investigated the conformational preference in solution

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of a protected aminosugar, the 1H, 13C, 15N chemical shifts of some pyridazines and of a

nitrated furan.

Chapter 5 gives an overview of thermal and photochemical transformations of

some 1,2,4-oxadiazoles and presents a new approach to quinazolines and 1,3-

benzothiazines via 1,2,4-oxadiazoles rearrangements with pivotal nucleophiles at C(5).

These transformations take place by a modified version of ANRORC (Addition of a

Nucleophile Ring Opening Ring Closure) mechanism, in which we have utilized n-BuLi

as base and as nucleophile to generate the corresponding rearranged products.

A summary of the achievements is presented in Chapter 6.

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CHAPTER 1 GENERAL INTRODUCTION

Heterocyclic chemistry is an important field of organic chemistry which focuses on

cyclic organic compounds containing at least one heteroatom besides carbon, such as

oxygen, nitrogen, or sulfur inside the cycle. Heterocyclic compounds are important

structural motifs because they present significant biological and physical properties, and

because they are used as additives in various fields of industry, such as

pharmaceutical, cosmetic and food industry.

This thesis studies important compounds from the field of heterocyclic chemistry

for example N-(α-aminoalkyl)tetrazoles found applications in medicinal chemistry as

modified protein-formation inhibitors. They are useful in the prevention and treatment of

diseases associated with diabetes and hypertension [2007WP051930].

A good drug is a target-specific drug with high efficiency and low side effects.

Carbohydrates, nucleosides, sterols are important templates of anti-cancer and anti-

viral drugs; however, their bioavailability is low. In order to overcome these limitations,

they are administered in their acylated form; for example acyclovir, a well known anti-

viral drug, is used in clinical treatment as valine ester (Valacyclovir) [1989D233].

Bioavailability is described as the fraction of an administrated dose of unchanged

drug that reaches the systemic circulation. In an effort to overcome these limitations,

chemists have developed new strategies – peptidomimetics foldamers as unnatural

oligomeric molecules able to fold into more rigid secondary structures, mimicking the

structures and biological functions. These structures increase the efficiency of drug

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delivery into the biological systems. From this perspective, new and efficient preparative

methods are desired.

Inspired by nature, chemists design new molecular systems that are able to mimic

the ones found in the biological systems. For this reason, the vast majority of

pharmaceutical molecules contain heteroaromatic systems with 4-, 5- membered rings

capable to selectively link with a target host.

Many of these molecular systems exist as two or more tautomeric structures that

usually involve migration of a fragment from one site to another within the molecule.

A tautomeric equilibrium is one between two or more isomeric structures of a

single compound that are interconverted by the movement of an atom (usually

hydrogen) or a group of atoms in the molecular structure. Isomers, like tautomers,

possess the same atomic composition but in general do not interconvert easily. There is

no clear border between tautomerism and isomerism; tautomers are considered

isomers that interconvert below 20 kcal·mol-1 [2010JCAMD475].

Tautomeric equilibrium is profoundly dependent on the dielectric constant of the

medium. This translates into the ability of solvents to interact with each tautomer; the

more polar solvent favors the more polar tautomer. Molecular structure and solvent

polarity influence the equilibrium position of a tautomeric system.

The thesis is structured in 6 chapters, each of which is briefly described in what if

follows.

Chapter 2 presents the synthesis and some tautomerism studies of a new series

of N-(α-aminoalkyl)tetrazoles. In this particular chapter we are trying to rationalize the

factors that influence the equilibrium between N1- to N2- substituted N-(α-

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aminoalkyl)tetrazoles (Scheme 1-1). For structure designation see Chapter 2, results

and discussion section.

Scheme 1-1. Example of a tautomeric equilibrium of N-(α-aminoalkyl)tetrazoles.

Nuclear Magnetic Resonance (NMR) is an appropriate technique for investigating

the equilibrium between different populations because it does not interfere with the

reaction media, and because the tautomer population ratio can be monitored at different

temperatures / activation energies and in various solvents. For example, we have used

15N CIGAR-HMBC experiment to discriminate between the two tautomers and to

rationalize the chemical shift pattern of each of them.

NMR is a powerful technique for the structure elucidation, as it reveals the

correlations through bonds (based on scalar couplings) and correlations through space

(based on dipolar couplings). NMR can be used to elucidate the structure of complex

molecules. Chapter 4 focuses on the 1H, 13C, 15N NMR analysis of a series of

heterocycles such as pyridazines and a nitrated furan. It presents also the total 1H

assignment of a rotamer mixture of a protected acylated aminosugar.

Azoles and their benzoanellated derivatives have attracted considerable interest

during the last decades because of their theoretical and synthetic value. Particularly, the

reactivity of benzotriazole was intensively studied in various chemical transformations.

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Chapter 3 focuses on efficient and economically convenient methods of amidation

and esterification of a series of protected aminoxy carboxylic acids.

Amide or ester bond formations between an acid (1.2) and, respectively, an amine

(1.3) or an alcohol (1.6) are formally condensations. The usual esterification is an

equilibrium reaction whereas mixing an amine with a carboxylic acid is an acid-base

reaction. In other words, the amide bond formation has to overcome the adverse

thermodynamics, the direct condensation of the salt (1.4), to give the corresponding

amide (1.5). This transformation can be achieved at high temperatures (usually 160-180

oC) [1993SC2761, 2005T10827], which is usually quite incompatible with the presence

of other functional groups within the molecular structure (Scheme 1-2).

Scheme 1-2. Esterification versus amidation reaction.

In order to overcome these limitations, new and efficient strategies have been

developed. These strategies are based on the activation of the carboxylic acid by

attachment of a leaving group at the acyl carbon to allow the attack of the nucleophile

(Scheme 1-3).

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Scheme 1-3. Carboxylic acid activation and coupling step.

Carboxyl components may be activated as acyl halides, acyl azides, acylimidazoles,

anhydrides, esters, etc. There are different ways of coupling reactive carboxyl

derivatives with a nucleophile:

- Using an intermediate acylating agent formed and isolated, then subjected to the

coupling reaction

- Using a reactive acylating agent generated in situ, followed by immediate

treatment with the nucleophile.

Benzotriazole (1.10) is a valuable synthetic auxiliary, because it can act as:

i) leaving group (1.11), ii) proton activator (1.12), iii) cation stabilizer (1.13), iv)

radical precursor, v) anion precursor or iv) ligand for metal catalysis (1.15a,b)

[2007TL4207, 2009EJIC3094]. Moreover benzotriazole is an inexpensive, stable

compound that is soluble in common organic solvents such as ethanol, benzene, THF,

chloroform, and DMF.

As another aspect of a good auxiliary, benzotriazole (BtH) can act as a weak base

(pKa= 1.6) or weak acid (pKa = 8.3) [1948JCS2240, 1991T2683]; this facilitates the

easy removal of benzotriazole under acidic or basic conditions.

N-Acylbenzotriazoles (1.14) are useful acylating reagents because they are stable,

mostly crystalline, easily prepared and handled at laboratory scale.

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Scheme 1-4. 1H-Benzotriazole as synthetic auxiliary.

N-Acylbenzotriazoles can be prepared directly from carboxylic acids and 1H-

benzotriazole in the presence of thionyl chloride, or using 1-methanesulfonyl-1H-1,2,3-

benzotriazole in the presence of triethylamine. N-acylbenzotriazoles are advantageous

for N-, O-, C-, S- acylation [2000JOC8210, 2003JOC5720, 2005SL1656, 2005S397,

2006S411, 2006S3231, 2008OBC2400], especially when the corresponding acid

chlorides are difficult to prepare, unstable or toxic.

Benzotriazole derivatives are important synthetic auxiliaries and they have found

applications in a vast series of synthetic transformations such as: alkylation

[1994CSR363], acylation [20034932, 2003JOC5720, 2005S1656], imine acylation

[2000S2029], and imidoylation [1997T6771, 1999OL977, 2002JOC4667]. In addition,

the benzotriazole derivatives have been used in Mannich reactions [1994JHC917] and

Grignard reactions [2007S3141].

24

N-Acyl benzotriazoles can react with L-cysteine to give exclusively S-acylated, or

N- acylated products in the presence of triethylamine (Scheme 1-5). S-Acyl and N-

benzyloxycarbonyl cysteine are useful potential intermediates for the synthesis of

cysteine and oxytocin-like peptides. Moreover, S-acylcysteine 1.17 can be converted to

N-acylcysteine 1.18 by a native chemical ligation (NCL) reaction by using a phosphate

buffer solution pH = 8.0 [1994S776, 1999JA11684]. Chemical ligation is a useful tool for

creating long peptide chains from smaller unprotected peptides. This reaction takes

place in aqueous media and is chemoselective. This protocol expands the utility of the

benzotriazole methodology developed by our group. The reaction described in Scheme

1-5, takes place via a 5-membered ring transition state but larger ring transition states

are under current investigation within the group.

Scheme 1-5. Selective synthesis of S-acyl and N-acylcysteines

In addition, Chapter 3 presents an extension of the benzotriazole methodology; we

have investigated the reactivity of N-acylbenzotriazoles in the presence of hindered

alcohols such as sterols, terpenes, and unprotected sugars and nucleosides. Our

results indicate that in the case of multiple nucleophilic centers, the reaction is selective

and this proves the utility of this methodology. The corresponding acylated compounds

25

were prepared in moderate to good yields, and under mild reaction conditions. The

original chirality was preserved as evidenced by NMR. The coupling reactions can be

accelerated by the microwave irradiation, with the reactions being completed within 45

min; additionally, we have investigated the acylation position of an unprotected sugar by

1H-1H dQOCSY and 1H-13C gHMBC experiments, these results are described in detail in

Chapter 3.

Chapter 5 presents a new rearrangement of 1,2,4-oxadiazole ring with a pivotal

nucleophile at C(5) in the presence of strong nucleophiles such as n-butyllithium. The

proposed rearrangement takes place at low temperature, the reaction is selective and

the nucleophilic attack / rearrangement can be controlled by the reaction conditions.

In this chapter we are trying to rationalize the reaction mechanism and the

substitution effect on the rearrangement of 1,2,4-oxadiazoles (Scheme 1-5). We have

adapted the method developed earlier by Srivastava. We are exploring its utility and its

limitations in the design of various heterocyclic systems such as quinazolines 1.20 and

benzothiazines 1.22.

Chapter 5 gives also an overview of the rich chemistry of heterocyclic

transformations of 1,2,4-oxadiazoles, including the Boulton – Katritzky rearrangement

(BKR) and the ANRORC rearrangement. These methodologies are valuable synthetic

tools used in the ring transformations of various heterocyclic systems.

Vivona et al. [2006JOC8106, 2009ARK235] have used these approaches to

prepare a variety of heterocycles, including 1,2,4-triazoles, 1,2,4-oxadiazoles, 1,2,4-

triazines, 1,2,4-oxadiazoles and indazoles starting from activated 1,2,4-oxadiazole

derivatives.

26

Scheme 1-5. New rearrangements of 1,2,4-oxadiazoles.

Chapter 6 presents a summary of the achievements together with final remarks.

27

CHAPTER 2 RELATIVE STABILITIES OF N-(Α-AMINOALKYL)TETRAZOLES1

2.1 Background

This chapter investigates the relative equilibrium in solution of a new series of N-(α

-aminoalkyl)tetrazoles. The tetrazole ring has wide applications in chemistry,

supramolecular chemistry and drug design [1996CHC1, 2010JCAMD475]. The tetrazole

ring can serve as a metabolically stable analogue for the carboxyl group [1980PMC151,

1977AHC323] and this may confer useful biological properties. Tetrazoles may exist in

solution as a mixture of 1H-tautomer (2.1a) and 2H-tautomer (2.1b). (Scheme 2-1)

Scheme 2-1. The tautomeric structures of 1H-tetrazole and 2H-tetrazole.

Tetrazoles bind anions tightly in polar solution, as opposed to the corresponding

carboxylic acids; in fact tetrazole receptors bind 50,000 times stronger than the

corresponding carboxylic hosts [2008OL4653].

This remarkable difference in binding strength can be rationalized by considering

the fast tautomeric equilibrium in tetrazoles between 1H-tetrazole and 2H-tetrazole and

the conformational preferences in the carboxylic acids; the tetrazole 1H-tautomer is

energetically more favored than the 2H-tautomer by 3 kcal·mol-1 in polar solvents and it

resembles an anti conformation of the carboxylic acid which is energetically disfavored

(-6 kcal·mol-1) (Scheme 2-2).

1Reproduced in part with the permission from J. Org. Chem. 6468-6476, 2010. Copyright ©

American Chemical Society 2010

28

The tetrazole fragment was found to have applications as pharmacophore, being

used as a cardiovascular drug or as an Angiotensin II receptors antagonist [2007CHC1].

A few examples include Losartan (2.2), Diovan (2.3) and Avapro (2.4) [2010JCAMD475]

(Scheme 2-2).

Scheme 2-2. Applications of tetrazole as pharmacophore and as anion scavenger.

Recently, N-(α-aminoalkyl)tetrazoles have been used as modified protein-

formation inhibitors in the prevention and treatment of diseases associated with AGEs

(advanced glycation end) and ALEs (advanced lipoxidation end products) (Scheme 2-3)

[2007WOP051930].

Scheme 2-3. Examples of biologically active pharmacophores containing N-(α-aminoalkyl)tetrazoles.

29

Previous studies on the relative stabilities of 1- and 2-substituted benzotriazoles

[1975JCSPT(1)1181, 1987JCSPT(1)2673, 1989JA7348] showed exchange between

two tautomeric species 2.10a and 2.10b. (Scheme 2-4)

Scheme 2-4. Tautomeric equilibrium between 1- and 2-substituted benzotriazoles.

Tautomeric equilibrium is one between two or more isomeric structures of a

single compound that are interconverted by movement of an atom (usually hydrogen) or

a fragment from one side to another within the molecule.

If the substituent is hydrogen, a rapid tautomeric exchange takes place; N1-

substituted benzotriazole is found exclusively in the solid state and in polar solvents, but

N2- substituted benzotriazoles are found in the gas phase and non-polar solvents

(Scheme 2-5). The stability of 1H-benzotriazole in solution can be explained by a

greater dipole moment which favors interactions with itself (solid state) or with the

solvent (in solution) and also aromaticity [1989JA7348].

Similarly, N-(α-dialkylaminomethyl)benzotriazoles exist as the N1 isomer in the

crystalline phase and as a mixture of N1 and N2 in solution and vapor phase (Scheme

2-5).

Empirical methods such as MP 6-31G*//6-31G show a difference of 2.71 kcal·mol-1

between N1 and N2 benzotriazole tautomers and respectively 0.47 kcal·mol-1 for N-(α-

dimethylamino)methyl-benzotriazole tautomers [1987JCSPT(1)2673].

30

These results are explained by the greater aromaticity of N1 benzotriazole, and

the fact that in the media of low dielectric constant, the higher dipole moment of

1-substituted benzotriazole (µ = 4.65 D) compared to 2-substituted benzotriazole (µ =

0.77 D) pushes the equilibrium toward the 2-substituted benzotriazole [1994JOC2799].

Scheme 2-5. N-(α-Dialkylaminomethyl)benzotriazole tautomers.

The interconversion between the two tautomeric species takes place via a

dissociation recombination mechanism, involving an iminium cation and a benzotriazole

anion facilitated by the cleavage of the C-N bond. This type of mechanism is known as

cationotropy because it is the cation that moves from one position to another within the

molecule (Scheme 2-6).

Scheme 2-6. The rearrangement mechanism of N-(α-dialkylaminomethyl)benzotriazole.

31

The 1H NMR analysis of N-(α-dialkylaminomethyl)benzotriazoles shows the

existence of two tautomeric species, two singlets in the region 5.50 – 5.40 ppm

corresponding to N-CH2-N protons, indicating the presence of both the benzotriazol-1yl

and 2-yl isomers.

Similar behavior has been observed for N-substituted tetrazoles. Non-empirical

quantum calculations (MP2/6-31G* and MP2/6-31G*//HF/6-31G*) show that N2-

substituted tetrazole is more stable than N1-substituted tetrazole in the gas phase and

non-polar solvents, which is in agreement with the reported data for N-substituted

triazoles. [2003RJGC275]

The ratio between the two tautomers may be explained by the difference in the

dipole moments, for instance 1-methyl-5-phenyltetrazole (µ = 5.88 D) is more polar than

2-methyl-5-phenyltetrazole (µ = 2.52 D) [1984ZOK2464].

2.2 The Proposed N-(α-Aminoalkyl)Tetrazole Substrates

A series of N-(α-aminoalkyl)tetrazoles with a substituted aromatic ring at position

C(5) and electron rich or electron poor amino alkyl substituents was synthesized in

order to study the substitution effect on the tautomer stability (Scheme 2-7).

Scheme 2-7. Tetrazole substrates for the tautomerism studies.

32

The tetrazoles 2.14a-f were prepared from commercially available nitriles

(Table 2-1) in 10-75% yield, except 1H-tetrazole and 5-phenyl-1H-tetrazole which were

obtained from commercial sources. The resulting tetrazoles were converted into their

corresponding Mannich products 2.16a-g by the Bechmann - Heisey method

[1946JA2496] (Table 2-2), whereas compounds 2.18a,b were prepared by a different

method because the Bechmann - Heisey procedure failed (Table 2-3) [1993JOC917].

Scheme 2-8. Preparation of tetrazoles (2.14a-f).

Table 2-1. Synthesis of tetrazoles (2.14a-f). Entry R3 Yield Time Conditions References

2.14a Me- 10 40h AlCl3, THF, reflux 1987CJC166 2.14b p-MeO-C6H4- 68 48h ZnBr2, H2O, reflux 1984JCSPT2-721 2.14c p-Cl-C6H4- 75 48h ZnBr2, H2O, reflux 1976ZC17 2.14d o,o-(F,F)C6H3- 71 24h ZnBr2, H2O, reflux novel 2.14e CH3CH=CH- 12 24h ZnBr2, H2O, reflux novel 2.14f p-N(Me)2-C6H4- 75 70h ZnBr2, H2O, reflux 2003M3457

Scheme 2-9. Preparation of N-(α-aminoalkyl)tetrazoles (2.16a-g) by the Mannich

reaction.

Table 2-2. Preparation of N-(α-aminoalkyl)tetrazoles (2.16a-g) by the Mannich reaction. Entry R3 R1 R2 Major Tautomer Yielda(%)

2.16a Me- -(CH2)2-O-(CH2)2- N2 60

2.16b p-MeO-C6H4- -(CH2)2-O-(CH2)2- N2 55

2.16c p-Cl-C6H4- -(CH2)2-O-(CH2)2- N2 80

2.16d o,o-F,F-C6H3- -(CH2)2-O-(CH2)2- nr -

2.16e CH3CH=CH- -(CH2)2-O-(CH2)2- nr -

2.16f p-N(Me)2-C6H4- -(CH2)2-O-(CH2)2- N2 70

2.16g Ph- CH3- CH3- N2 70

nr – no reaction a-Isolated yields

33

Scheme 2-10. Preparation of N-(α-aminoalkyl)tetrazoles (2.18a,b) by an alternative

method.

Table 2-3. Alternative method to the Mannich products 2.18a,b.

Entry 2.19a,b Major Tautomer Yield %

2.18a

N2

64

2.18b

N2

74

2.4 Results and Discussion

The tautomeric equilibrium between N1 and N2 isomers was investigated by 1H

NMR using a variety of polar and non-polar solvents to determinate which tautomer

predominates in solution. In the case of triazole, the substitution pattern can be easily

obtained by the signal multiplicity of the 1H NMR spectra. However, in the case of

tetrazole derivatives supplementary data is required. Compounds 2.18a,b were

characterized by 2D NMR techniques such as 1H-1H gDQCOSY, 1H-13C gHMBC, 1H-13C

gHMQC, and 1H-15N CIGAR-gHMBC. The N2 isomer is predominant in both cases

2.18a,b in DMSO-d6.

1H NMR experiments for 2.16a-g and 2.18a,b were conducted in order to indentify

the predominant isomer. Polar solvents are expected to stabilize the N1 isomer due to

its higher dipole moment, but the N2 isomer should be stabilized by non-polar solvents.

34

1H NMR presents the ratio between the two isomers, but it offers no information about

the substitution position on the tetrazole ring. In order to predict the substitution pattern

on the tetrazole ring, 2D NMR experiments (1H-1H gDQCOSY, 1H-13C gHMBC, 1H- 13C

gHMQC, and 1H-15N CIGAR-gHMBC) were carried out for 1-((1H-tetrazol-1-

yl)methyl)pyrrolidine-2,5-dione (2.18a) and 2-((1H-tetrazol-yl)methyl)isoindoline-1,3-

dione (2.18b). In both cases the N2 isomer predominates, and both compounds present

the following characteristic patterns: N(CH2)N- protons are more shielded in N1

tautomer that in N2, and –N-CH=N proton is more deshielded in the N1 isomer, also the

tetrazole carbon is more deshielded in N2 than in N1 as previously reported

[1988MRC134].

15N Chemical shifts were assigned based on the following correlations seen in 1H-

15N CIGAR gHMBC experiment: For 2.18aN1, the methylene protons (6.02 ppm) show

two bond correlation with N-1 (239.3 ppm) and the pyrrolidine-2,5-dione nitrogen N1''

(180.4 ppm) and three bond correlation with N-2 (370.0 ppm). H-5 (9.42 ppm) shows

two bond correlation to both N-1 (239.3 ppm) and N-4 (395.0 ppm). For 2.18aN2, the

methylene protons H-1' (6.20 ppm) show two bond correlations to N-2 (307.9 ppm) and

pyrrolidine-2,5-dione nitrogen N1'' (180.0 ppm) and three bond correlations to both N-1

(383.0 ppm) and N-3 (284.9 ppm). Moreover, N-1 and N-2 showed correlation to H-5

(9.01 ppm) by cross peaks with N-1 (383.0 ppm) and small coupling in N-2 (307.9 ppm)

(Figure 2-1).

The 13C chemical shifts of 2.18a(N1) and 2.18a(N2) were obtained from 1H-13C

gHMBC (Figure 2-2) and are presented in Table 2-4.

35

Figure 2-1. 1H-15N gHMBC-CIGAR experiment for 2.18aN1+ 2.18aN2.

Figure 2-2. 1H-13C gHMBC experiment for 2.18aN1+ 2.18aN2.

For 2.18b(N1), the methylene protons H-1' (6.27 ppm) showed two bond

correlation to N-1 (240.7 ppm) and phthalimide nitrogen N-2'' (163.0 ppm) and three

bond correlation to N-2 (369.2 ppm). H-5 (9.54 ppm) showed two bond correlation to N-

1 (240.7 ppm) and N-5 (395.7 ppm).

36

For 2.18b(N2), the methylene protons H-1' (6.46 ppm) showed two bond

correlation to N-1 (307.9 ppm) and phthalimide nitrogen N-2'' (161.7 ppm) and three

bond correlation to N-1 (382.5 ppm) and N-3 (287.8 ppm) (Figure 2-3).

Figure 2-3. 1H- 15N CIGAR-gHMBC experiment for 2.18bN1+ 2.18bN2.

Figure 2-4. 1H-13C gHMBC experiment for 2.18bN1+ 2.18bN2.

37

Table 2-4. 1H, 13C 15N chemical shifts (δ, ppm, DMSO-d6) of 2.18a and 2.18b.

1H NMR

H-5 H-1' H-2'' H-3'' Other

2.18a N-1 9.42 6.02 - 2.69

N-2 9.01 6.20 - 2.74

2.18b

N-1 9.54 6.27 - - H-4'' (7.91), H-5'' (7.95), H-6'' (7.95), H-7'' (7.91)

N-2 9.02 6.46 - - H-4'' (7.97), H-5'' (7.88), H-6'' (7.88), H-7'' (7.97)

13C NMR

C-5 C-1' C-2'' C-3'' Other

2.18a N-1 146.4 50.0 178.4 30.0

N-2 155.3 54.5 178.0 29.9

2.18b N-1 145.1 48.9 - 167.1 C-1'' (167.1), C-4a'' (131.9), C-4'' (135.7), C-5'' (124.5).

N-2 154.2 53.3 - 166.8 C-1'' (166.8), C-4a'' (131.9), C-4'' (124.3), C-5'' (135.7).

15N NMR

N-1 N-2 N-3 N-4 Other

2.18a N-1 239.3 370.0 383.0 395.0 N-1'' (180.4)

N-2 383.0 307.9 284.9 333.9 N-1'' (180.0)

2.18b N-1 240.7 369.2 nm 395.7 N-2'' (163.0)

N2 382.5 307.1 287.8 nm N-2'' (161.7)

nm – not measured

38

The ratio of the two tautomers is given by the 1H NMR integral ratio of the signals

–N-CH2-N-, and the results are presented in Table 2-5.

Table 2-5. Ratio of N1 and N2 isomers in different solvents.

Comp D2O (CD3)2SO CD3CN CD3OD (CD3)2CO CDCl3 C6D6

2.16a 63/37 60/40 64/36 Reacts 55/45 18/78 20/80

2.16b ns 1/99 1/99 Reacts Reacts 1/99 1/99

2.16c ns 1/99 1/99 Reacts 1/99 1/99 1/99

2.16f ns 1/99 1/99 1/99 1/99 1/99 1/99

2.16g 1/99 1/99 1/99 1/99 1/99 1/99 1/99

2.18a 40/60 40/60 40/60 40/60 40/60 NS 20/80

2.18b ns 15/85 15/85 Reacts 15/85 15/85 15/85

ns- not soluble

Preliminary studies on the tautomeric composition of N-(α-aminoalkyl)tetrazoles

reported that 4-(1H-tetrazol-1-ylmethyl)morpholine exists predominately as the N2

isomer in CDBr3 with ΔG0 = -1.03 kcal/mol and ΔG≠ = 17.6 kcal/mol. The N2 isomer was

predominant in less polar solvents such as toluene-d8 but the equilibrium was

substantially shifted toward the N1 isomer in CD3NO2. Similarly for 4-((5-methyl-1H-

tetrazol-1-yl)methyl)morpholine the N2 isomer was predominant in CDBr3 with ΔG≠ =

20.0 kcal/mol for the interconversion of tautomers. However electron poor amines do

not favor isomerisation, as the VT NMR experiments of 2.18a and 2.18b show no

39

exchange / interconversion between N1 and N2 isomers. Moreover, they are thermally

stable, since no decomposition was observed at 150oC in DMSO-d6 (Figure 2-5).

Figure 2-5. VT NMR for 2.18aN1+ 2.18aN2.

2.5 Cross-over Experiments

The interconversion mechanism between N1 and N2 tautomers was investigated

using equimolar mixtures of N-(α-aminoalkyl)tetrazoles (2.16g) and (2.16a). Preliminary

studies show no interconversion even in polar solvents such as DMSO-d6 (Scheme 2-

11).

Scheme 2-11. Cross-over experiment of N-(α-aminoalkyl)tetrazoles 2.16a,g.

40

The 1H NMR analysis shows no formation of 2.20a,g suggesting that the N-(α-

aminoalkyl)tetrazoles with aromatic substituents at position C(5) do not dissociate in

solution. However, a cross-over experiment of a mixture of N-(α-aminoalkyl)tetrazoles

2.16h,i in toluene-d8 displayed for N-CH2-N and benzylic protons twelve signals in the

region 4.9-3.7 ppm corresponding to eight possible isomers resulted from the cross-

over process (Scheme 2-12).

Scheme 2-12. Cross-over experiment of 2.16h,i

In addition, the activation parameters for tautomer conversion 2.20hN1 to 2.20hN2

(measured in CD3CN) gave Ea = 16.5 kcal·mol-1, ΔH≠ = 15.9 kcal·mol-1 and ΔS≠=-4.5

e.u and respectively Ea = 18.5 kcal·mol-1, ΔH≠ = 17.9 kcal·mol-1 and ΔS≠=-6.0 e.u for

2.20hN2 to 2.20hN1 interconversion. The low entropies of activation in both directions

suggest that the isomerisation process takes place via a unimolecular dissociation

recombination mechanism involving a tight ion pair mechanism [2010JOC6468].

. Compounds 2.16h,i and 2.20h,i were prepared and characterized by Dr. Bahaa

El-Dien El-Gendy as part of a collaborative project [2010JOC6468].

41

2.6 Conclusions

N-(α-Aminoalkyl)tetrazoles with aliphatic substituent at C(5) exist in solution as a

mixture of N1 and N2 tautomers. Aromatic 5-substituted tetrazoles exist exclusively as

the N2 tautomer and the equilibrium between N1 and N2 tautomer is shifted toward N2

by the steric effect. In the case of 4-((5-methyl-1H-tetrazol-1-yl)methyl)morpholine

(2.16a) the N2 isomer is major in non-polar solvents such as benzene and chloroform or

bromoform, but N1 isomer predominates in polar solvents such as acetonitrile or

dimethyl sulfoxide. This result is consistent with the dissociation-recombination

mechanism.

The N-(α-aminoalkyl)tetrazole products with electron deficient substituents are

thermally stable, since heating of 2.18a,b in DMSO-d6 at 150oC does not produce any

decomposition; they do not dissociate in solution, these results are in the agreement

with the dissociation recombination mechanism (Scheme 2-13).

Scheme 2-13. Dissociation-recombination mechanism of N-(α-aminoalkyl)tetrazoles.

Cross-over experiments of equimolar mixtures of N-(α-aminoalkyl)tetrazoles have

showed interconversion between N1 and N2 tautomers when non-aromatic

substituents are present at the position C(5) of the tetrazole ring.

42

The isomerisation of N-(α-aminoalkyl)tetrazoles takes place at low entropies ΔS≠=

-6.0 e.u. suggesting that the interconversion mechanism takes place via a unimolecular

dissociation-recombination mechanism involving a tight ion pair.

2.7 Experimental Section

Melting points were determined on a capillary point apparatus equipped with a

digital thermometer. NMR spectra were recorded in CDCl3 or DMSO-d6 on Gemini or

Varian NMR operating at 300 MHz for 1H and 75 MHz for 13C with TMS as internal

standard and the chemical shifts δ are given in ppm. The 2D NMR experiments were

recorded on Inova 500 equipped with indirect detection probe operating at 500 MHz for

1H and 125 MHz for 13C and 50 MHz for 15N. Elemental analyses were performed on a

Carlo Erba-1106 instrument.

5-Methyl-1H-tetrazole (2.14a). Sodium azide (4.88 g, 75 mmol) was added in

small portions to a stirred solution of acetonitrile (1.23 g, 30 mmol) in fresh distilled THF

(10 mL) to give a suspension, which was then stirred at r.t. for 10 min; AlCl3 (3.47 g, 26

mmol) was dissolved in THF (20 mL) and the resulting mixture was poured over the

acetonitrile suspension. The reaction mixture was heated under reflux for 40 h, and the

addition of THF facilitated the suspension to break. The reaction mixture was allowed to

cool to r.t.; HCl (4N) was added dropwise (pH =2) under nitrogen and stirring was

continued for 8 h. The solvent was removed in vacuo to give a white powder. The

product was then extracted via a Soxhlet using chloroform. The solvent was removed in

vacuo to give the crude product as yellow oil. Addition of fresh chloroform facilitated

isolation of the product as white needles (0.25g, 10%); m.p. 145.0 -147.0 oC, (lit. m.p.

145.0-147.0 oC [1987CJC166]); 1H NMR (Acetone-d6) δ 2.56 (s, 3H), 8.7 (s, 1H).13C

NMR (Acetone-d6) δ 8.50, 153.4.

43

Compounds 2.2b-f were prepared by Sharpless method [2001JOC7945].

5(4-Methoxyphenyl)-1H-tetrazole (2.14b). 4-Methoxybenzonitrile (1.33 g, 10

mmol) was poured into a stirring solution of ZnBr2 (2.25 g, 10.0 mmol) in H2O (25 mL).

The solution was stirred at r.t. for 10 min and sodium azide (0.72 g, 11.0 mmol) was

added in small portions. The reaction mixture was heated under reflux for 48 h.

Vigorous stirring is essential, because as the reaction proceeds, the viscosity increases

as a result of the formation of Zn coordination products; then HCl (10 mL, 4N) was

added dropwise under nitrogen flow. The crude tetrazole was extracted with EtOAc (50

mL), the solvent was then removed in vacuo to give a white residue which was

redissolved in NaOH (10 mL solution 0.5 N), and the resulting precipitate Zn(OH)2 was

filtered off. The filtrate was treated with HCl (4 N) (pH=1) to give the product as white

needles (1.28g, 68%); m.p. 234.1-239.0 oC, (lit. m.p. 233.0-235.0 oC[1984JCSPT1972]);

1H NMR (300 MHz, DMSO-d6) δ 7.99 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 8.7 Hz, 2H), 3.86

(s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 161.5, 128.7, 116.3, 114.9, 99.6, 55.5. Anal.

Calcd. for C8H8N4(160.07) required: C, 54.54; H, 4.58; N, 31.80. Found: C, 54.56; H,

4.42; N, 31.73.

5-(4-Chlorophenyl)-1H-tetrazole (2.14c). White microcrystals, (1.35 g, 75 %);

(m.p. 246-250 oC, lit. m.p. 256.0-257.0 oC [2001JOC7945]); 1H NMR (300 MHz, DMSO-

d6) δ 8.02 (d, J = 6.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 2H), 5.75 (br s, 1H). 13C NMR (75

MHz, DMSO-d6) δ 154.9, 135.9, 129.2, 128.4, 123.2. Anal. Calcd. for C7H5ClN4 (180.60)

required: C, 46.55; H, 2.79; N, 31.02. Found: C, 46.94; H, 2.80; N, 30.88.

5-(2,6-Difluorophenyl)-1H-tetrazole (2.14d). White microcrystals, (1.30 g, 71 %);

m.p. 151.0 – 152.0 oC; 1H NMR (300 MHz, DMSO-d6) δ 7.41(t, J = 8.5 Hz, 2H), 7.81-

44

7.71 (m, 1H). 13C NMR (75 MHz, DMSO-d6) δ 159.8 (d, J = 247.5 Hz), 154.5 (t, J = 6.8

Hz), 134.1 (t, J =8.4 Hz), 113.0-112.4 (m). Anal. Calcd. for C7H4F2N4(182.13) required:

C, 46.16; H, 2.21; N, 30.76. Found C, 45.95; H, 2.20; N, 30.42.

(E, Z)-5-(Prop-1-enyl)-1H-tetrazole (2.14e). Brown crystals, (0.13 g, 12 %); m.p.

125.2-125.6 oC; 1H NMR (Acetone-d6) δ 6.98-6.86 (m, 1H), 6.59-6.56 (m, 1H), 1.98-1.93

(m, 3H). 13C NMR (Acetone-d6) δ 155.2, 138.2, 115.1, 18.6. Anal. Calcd. for C4H6N4

(110.12) required: C, 43.63; H, 5.49; N, 50.88. Found: C, 43.67; H, 5.57; N,50.62.

N,N-Dimethyl-4-(1H-tetrazol-5-yl)aniline (2.14f). Yellow microcrystals (0.25 g, 63

%); m.p. 78.0-80.0 oC (lit. m.p. 81.0-83.0 oC [2003M3457]); 1H NMR (300 MHz,

Acetone-d6) δ 7.94 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 3.05 (s, 6H). 13C NMR

(75 MHz, acetone-d6) δ 153.2, 129.1, 128.7, 112.9, 40.2.

4-((5-Methyl-2H-tetrazol-1-yl)methyl)morpholine

(2.16a). Morpholine (0.1 mL, 0.087g, 1.00 mmol) was

added in small portions to a stirring solution of 5-methyl-

tetrazole (0.084 g, 1.00 mmol) in water (5 mL). The

resulting mixture was stirred at r.t. for 5 min until it

become clear. Formalin (37 % solution) was then added in small portions and the

resulting reaction mixture was stirred overnight. The solvent was removed in vacuo to

give a colorless oil which was recrystallized from chloroform: hexane (3:1) to give 4-((5-

methyl-1H-tetrazol-1-yl)methyl)morpholine (0.11 g, 60 %) as white needles; m.p. 75.0 -

77.0 oC; 1H NMR (300 MHz, CDCl3) δ 5.37 (s, 1.6H from A), 5.05 (0.4H, from B), 3.67 (t,

J = 4.4 Hz, 4H), 2.62 (t, J = 4.6 Hz, 4H), 2.53 (s, 3H).13C NMR (75 MHz, CDCl3) δ

162.9, 73.8, 68.6, 66.8, 66.6, 50.4, 50.0, 31.0, 11.0, 9.26. Anal. Calcd. for C7H13N5O

45

(183.21) required: C, 45.89; H, 7.15; N,38.23. Found: C, 46.33; H, 6.78; N, 37.75. The

major tautomer is N2.

4-((5-(4-Methoxyphenyl)-2H-tetrazol-1-yl)methyl)morpholine (2.16b). The

compound was recrystallized from toluene to give white microcrystals (0.30 g, 55 %);

m.p. 147.0 – 148.0 oC; 1H NMR (300 MHz, CDCl3) δ 8.10 (d, J = 8.6 Hz, 2H), 7.01 (d, J

= 8.7 Hz, 2H), 5.48 (s, 2H), 3.88 (s, 3H), 3.72 (t, J = 4.6 Hz, 4H), 2.72 (t, J = 4.7 Hz,

4H), 13C NMR (300 MHz, CDCl3) δ 165.1, 161.5, 128.6, 120.2, 114.5, 74.1, 66.9, 55.6,

50.1, 46.7. Anal. Calcd. for C13H17N5O2 (275.31) required: C, 56.72; H, 6.22; N, 25.44.

Found: C, 56.34; H, 6.19; N, 25.04. The major tautomer is N2.

4-((5-Chlorophenyl)-2H-tetrazol-1-yl)metyl)morpholine (2.16c). The compound

was recrystallized from toluene to give white microcrystals (0.23 g, 80 %); m.p. 112.0 –

113.0 oC; 1H NMR (300 MHz, CDCl3) δ 8.11 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.5 Hz,

2H), 5.51 (s, 2H), 3.72 (t, J = 4.6 Hz, 4H), 2.71 (t, J = 4.5 Hz, 4H). 13C NMR (75 MHz,

CDCl3) δ 179.0, 164.2, 136.4, 129.2, 128.2, 125.9, 74.2, 66.6, 49.8. Anal. Calcd. for

C12H14ClN5O (279.73) required: C, 51.53; H, 5.04; N, 25.04. Found: C, 51.93; H, 5.07;

N, 24.64. The major tautomer is N2.

N,N-Dimethyl-4-(1-morpholinomethyl)-2H-tetrazol-5-yl)aniline (2.16f). The

compound was recrystallized from toluene to give yellow microcrystals (0.20 g, 70 %);

m.p. 146.0 – 147.0 oC; 1H NMR (300 MHz, Acetone-d6) δ 7.97 (d, J = 8.7Hz, 2H), 6.86

(d, J = 8.7Hz, 2H), 5.55 (s, 2H), 3.63 (t, J = 4.5Hz, 4H), 3.04 (s, 6H), 2.67 (t, J = 4.3Hz,

4H). 13C NMR (300 MHz, Acetone-d6) δ 152.8, 128.4, 116.1, 112.7, 74.4, 67.1, 50.7,

40.0. Anal. Calcd. for C14H20N6O (288.36) required: C, 58.31; H, 6.99; N, 29.14. found:

C, 57.97; H, 6.70; N, 28.97. The major tautomer is N2.

46

N,N-Dimethyl-1-(5-phenyl-2H-tetrazol-2-yl)methanamine (2.16g). The compound

was recrystallized from diethyl ether to give white microcrystals (0.28 g, 70 %); m.p.

50.0 – 51.0 oC; 1H NMR (300 MHz, DMSO-d6) δ 8.06 - 8.10 (m, 2H), 7.59-7.53 (m, 3H),

5.58 (s, 2H), 2.35 (s, 6H). 13C NMR (75 MHz, DMSO-d6) δ 162.9, 130.4, 129.2, 127.0,

126.4, 75.1, 41.3. Anal. Calcd. for C10H13N5 (203.25) required: C, 59.09; H, 6.45; N,

34.46. Found: C, 58.94; H, 6.34; N, 34.60. The major tautomer is N2

Scheme 2-14. Preparation of Mannich product 2.18a.

1-(Hydroxymethyl)pyrrolidine-2,5-dione (2.21a). Paraformaldehyde (0.66 g, 22.0

mmol) was added to a stirring solution of succinimide (1.98 g, 20 mmol) in ethanol (15

mL) in the presence of NaOH (0.01% mol). The resulting reaction mixture was heated

under reflux for 1 h until it became clear. The solvent was removed under vacuo to give

a white precipitate. The precipitate was recrystallized from EtOAc to give 2.21a as white

prisms (12.49g, 97%); m.p. 59.0 -60.0 oC. (lit. m.p. 66.0 oC [1925HCA567]); 1H NMR

(300 MHz, DMSO-d6) δ 6.30 (br s, 1H), 4.72 (s, 2H), 3.35 (s, 4H). 13C NMR (75 MHz,

DMSO-d6) δ 177.2, 60.4, 28.0. Anal. Calcd. for C5H7NO3 (129.12) required: C, 46.51; H,

5.46; N, 10.85. Found: C, 46.24; H, 5.31; N, 10.66.

1-(Chloromethyl) pyrrolidine-2,5-dione (2.22a). Phosphorus trichloride (0.70 g,

0.06 mL, 0.70 mmol) was added dropwise to a stirring solution of 1-

(hydroxymethyl)pyrrolidine-2,5-dione (0.26 g, 2.00 mmol) in dichloromethane (10 mL).

The resulting reaction mixture was stirred under argon atmosphere for 2 h. The reaction

47

mixture was washed with a NaHCO3 solution (10 mL, 2 %), the organic layer was

separated and dried over MgSO4. The solvent was then removed in vacuo to give 1-

(chloromethyl) pyrrolidine-2,5-dione (0.2g, 70%) as a colorless oil that crystallized in

vacuo; m.p. 54.0-56.0 oC, (lit. m.p. 58.0 oC. [1925HCA567]); 1H NMR (300 MHz, CDCl3)

δ 5.23 (s, 2H), 2.79 (s, 4H). 13C NMR (75 MHz, CDCl3) δ 174.7, 44.4, 28.1.

1-((2H-Tetrazol-1-yl)methyl)pyrrolidine-2,5-

dione (2.18a).

1-(Chloromethyl)pyrrolidine-2,5-dione (0.20g,

1.4 mmol) was added in small portions to a stirring

solution of 5H-tetrazole solution (4.44 mL 0.45 M in

acetonitrile), Na2CO3 (0.15 g, 1.4 mmol) and NaI (0.03 g, 0.2 mmol). The resulting

reaction mixture was stirred overnight at r.t. under argon atmosphere. The solution was

then filtered through celite, the white residue was washed with dry acetone (10 mL), and

the solvent was removed under vacuo to give a pale brown oil which was recrystallized

from toluene: ethanol (1:2) to give the product as white prisms (0.16g, 64%); m.p. 142.0

-143.0 oC; 1H NMR (300 MHz, Acetone-d6) δ 9.13 (s, 0.4H from A), 8.74 (s, 0.5H from

B), 6.27 (s, 1.2H from B), 6.13 (s, 0.8H from A), 2.84 (s, 2.4H from B), 2.80 (s, 1.6H

from A). 13C NMR (75 MHz, Acetone-d6) δ 177.0, 176.5, 154.0, 145.1, 53.6, 49.1, 28.9,

28.9. Anal. Calcd. for C6H7N5O2 (181.15) required: C, 39.78; H, 3.89; N, 38.66. Found:

C, 39.52; H, 4.23; N, 37.90.

48

Scheme 2-15. Preparation of Mannich product 2.18b.

2-(Hydroxymethyl)-1H-isoindoline-1,3-dione (2.21b). The procedure is similar to

2.21a, where the product was recrystallized from EtOAc to give white prisms (1.68 g, 95

%); m.p. 152.0-158.0 oC (lit m.p. 146.0-148.0 oC [1922JA817]); 1H NMR (300 MHz,

DMSO-d6) δ 7.81-7.92 (m, 4H), 6.41 (t, J = 7.0Hz, 1H), 4.96 (d, J = 6.8Hz, 2H). 13C

NMR (75 MHz, DMSO-d6) δ 167.4, 134.7, 131.5, 123.3, 60.1. Anal. Calcd. for C9H7NO3

(177.16) required: C, 61.02; H, 3.98; N, 7.91. Found: C, 60.89; H, 4.00; N, 7.83.

2-((1H-Tetrazol-1-yl)methyl)isoindoline-1,3-

dione (2.18b).

The procedure is similar with 2.18a. The product

was recrystallized from toluene to give pale-yellow

prisms (0.28 g, 60 %); m.p. 98.0 – 99.0 oC; 1H NMR

(300MHz, CDCl3) δ, 9.29 (s, 0.2H from A), 8.76 (s,

0.8H from B), 7.97-7.92 (m, 6H), 6.54 (s, 2H from B), 6.40(s, 1.5H from A). 13C NMR (75

MHz, CDCl3) δ, 167.4, 167.0, 154.2, 144.9, 136.0, 135.8, 132.7, 132.6, 124.7, 124.6,

53.5, 49.0. Anal. Calcd. for C10H7N5O2 (229.20) required: C, 52.40; H, 3.08; N, 30.56.

Found: C, 52.65; H, 3.30; N, 30.02. N2 is the major tautomer.

49

CHAPTER 3 EFFICIENT SYNTHESIS OF PROTECTED Α-AMINOXYACYL CONJUGATES

3.1 Background

We present a new preparative method for protected α-aminoxyacid conjugates,

which are good substitutes for peptides, using the benzotriazole methodology. In nature

peptides are important components of enzymes, hormones, neurotransmitters and

immunomodulators, they found applications in various physiological processes such as

metabolism, digestion, pain sensitivity and immune response. Due to their

conformational flexibility and low bioavailability, however, these peptides have limited

applications. On the other hand, α-aminoxy acids RCH(ONH2)CO2H are highly resistant

to enzymatic degradation and are thus of interest as structurally modified α-amino acids

and analogs of β-aminoacids [2003ACIE4395].

α-Aminoxy acids have found applications as building blocks of hybrid peptides and

peptidomimetics. Hybrid peptides such as β-peptides and γ-peptides, as well as α-

peptides, can adopt discrete secondary structures, such as helices [2003JA8539,

2003JA5559], turns and sheets [2002JA7324, 2003ACIE2402] similar to proteins and

peptides found in the biological systems.

Yang et. al. [2010CEJ577] found that peptides including α-aminoxy acid units can

adopt rigid secondary foldamer structures of considerable interest as novel analogs of

γ-peptides (3.1). Repulsion between lone pair electrons on the N and O atoms form

torsional characteristics that are distinctive for the N-O bond in aminoxy acids; in

particular, the backbone of aminoxy peptides is more rigid than that of the natural

peptides. For D-α-aminoxy peptides, 1.88 helices independent of side chains were

observed in peptides that contain as few as two residues [1996JA9794]. Theoretical

50

calculations on aminoxypeptide foldamers show that the most favorable conformation is

the rigid eight member ring hydrogen bonded structure 3.1b and the distance between

O and H-N is 2.07 Å, suggesting a hydrogen bond interaction [2006CC3367].

α-Aminoxy peptides have also facilitated the construction of anion receptors and

channels 3.2. (Scheme 3.1) [2002JA12410].

Scheme 3-1. Applications of aminoxy acids in molecular design.

The clinical development of biologically active compounds is often diminished by

undesirable biopharmaceutical properties such as low water solubility, stability and

permeability through biological membranes. These drawbacks can be overcome by

derivatization of the biomolecules by preparation of bioconjugates. Linkage of

aminoacids with hydroxylic terpenes affords effective medical agents in the therapy of

atherosclerosis. [WO2654338] Some N4 amino acid substituted derivatives of the

antitumor active nucleoside analogue 1--D-arabinofuranosylcytosine (3.3) showed

superior biological activity and bioavailability in comparison with the parent drug

[2009EJMC3596].

51

Sugar modified 2,6-diaminopurine such as 3’-fluoro-2,6-diaminopurine-2’,3’-

dideoxyinosine depsipeptide derivative (FddDAPR) (3.4) presents biological activity

against human immunodeficiency virus (HIV) [1988MP243]. Other antiviral drugs

include Valacyclovir (3.5) as valine ester of acyclovir that has increased solubility in

water and oral bioavailability relative to acyclovir (Scheme 3-2) [2008MRR929].

Scheme 3-2. Some applications of acylation in drug design.

3.1.1 General preparative methods of N-Protected-α-Aminoxy acids

N-Protected-α-aminoxy acids have been prepared from their corresponding

aminoacids 3.3a-e. A reported literature method for the preparation of N-protected

aminoxy acid includes the coupling of N-hydroxyphthalimide with protected lactic acid

derivatives at the carboxylic site, under Mitsunobu conditions; this protocol requires six

steps, N-hydroxyphthalimide can be used as protective group because carbamates

such as benzyl hydroxycarbamate 3.17 can cause side reactions (Entry 1 Scheme 3.3)

[1993JA11010]. On the other hand, the α-bromination method requires only two steps

[2009JOC8690].

An alternative method is the asymmetric synthesis using an equimolar mixture of

aldehyde 3.11 and alkyne 3.12 in the presence of Zn(OTf)2 and N-methyl ephedrine as

a chiral template (Entry 2 Scheme 3-3) [2009SL3159].

52

Scheme 3-3. Preparation methods for N-protected aminoxy acids.

3.1.2 Literature Methods of Acylation

Recently, we utilized N-protected-(α-aminoxyacyl)benzotriazoles as a mild general

method for the preparation of aminoxyacyl amides and aminoxy hybrid peptides

[2009JOC8690], showing advantages over the existing literature methods which use (i)

coupling reagents including Bop-HOBt-NEM, HBTU-HOBt-NEM, DIC-HOBt

53

[2003SL325], EDCI-HOBt/HOAt [2008JOC9443], TBTU/HOBt/DIEA [2000TL2361],

DIC/HOBt [2002OL869], (ii) active esters [2007T11952] or (iii) α -aminoxy diazoketones

[2004JOC7577]. All these methods require reaction times of 8-10 hours, multiple steps

and sometimes give low yields.

N-Acylbenzotriazoles are stable, crystalline, easy to handle, advantageous for N-,

O-, C- and S-acylation, especially when the corresponding acid chlorides are unstable,

toxic or difficult to prepare [2009SL2392]. The synthesis of chiral di-, tri- and

tetrapeptides from natural amino acids using N-(protected-α-aminoacyl)benzotriazoles

in solution occurs with complete retention of the chirality [2009JOC8690, 2009S1708].

A new preparative method for aminoxyacid conjugates with sterically hindered

nucleophiles such as steroids, terpenes and nucleosides using benzotriazole is

described.

3.2 Results and Discussion

3.2.1 Synthesis of N-Protected-α-Aminoxy acids Conjugates

A new series of N-protected-α-aminoxy acids conjugates was synthesized using

benzyl-1-(1H-benzotriazol-1-yl)aminoxycarbamate acid derivatives (3.14) with various

nucleophiles (NuH) such as terpenes, sterols, sugars, and nucleosides. We have also

investigated the acylation position in the presence of multiple nucleophilic centers such

as partially unprotected sugars (Scheme 3-4).

Scheme 3-4. Synthesis of N-protected-α-aminoxy acids conjugates.

54

3.2.2 Synthesis of N-Cbz-Protected α-Aminoxy acids (3.18 a-d, 3.18c+c’)

The N-Cbz-protected-α-aminoxy acids (3.18a-d, 3.18c+c’) were prepared by first

transforming the amino acids (3.15a-d, 3.15c+c’) into the corresponding α-

bromocarboxylic acids (3.16a-d, 3.16c+c’) (40-80% yield). Then, without further

purification, (3.16 a-d, 3.16c+c’) were reacted with benzyl hydroxycarbamate (3.17) in

the presence of sodium hydride to produce the corresponding N-protected α-aminoxy

acids (3.18a-d, 3.18c+c’) (53-88% yield) (Scheme 3-5).

Scheme 3-5. Synthesis of N-Cbz-protected(α-aminoxy)carboxylic acids (3.18a-d, 3.18c+c’).

Table 3-1. Preparation of of N-Cbz-protected(α-aminoxy)carboxylic acids (3.18a-d, 3.18c+c’).

3.15 a-d 3.16a-d 3.18

yielda (%) m.p. (◦C) yielda (%) m.p. (◦C)

Gly 3.16a b b 3.18a 53 140.0-140.0 Ala(L) 3.16b 80 oil 3.18b 60 92.0-94.0 Phe(L) 3.16c 45 oil 3.18c 68 oil

Phe(DL) 3.16c+c’ 40 oil 3.18c+c’ 62 oil Leu(L) 3.16d 70 oil 3.18d 88 oil

b-commercially available; a- isolated yields

3.2.3 Synthesis of N-Cbz-Protected(α-Aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’)

The N-Cbz-protected(α-aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’) were

prepared in 76-88% yield from the corresponding N-protected aminoxy acids 3.18a-d,

3.18c+c’ according to our previously published method (Scheme 3.6) [2009JOC8690].

55

Scheme 3-6. Synthesis of N-Cbz-protected(α-aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’).

Table 3-2. Preparation of N-Cbz-protected(α-aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’).

Entry R 3.14

yielda (%) m.p. (◦C)

3.14a H- 76 88.0-90.0

3.14b CH3(D)- 78 86.0-88.0

3.14c Ph-CH2(D)- 88 90.0-91.0

3.14c+c’ Ph-CH2(DL)- 82 90.0-91.0

3.14d (CH3)2CHCH2(D)- 86 oil

3.2.4 Synthesis of O-(Protected-α-Aminoxyacyl)steroids 3.20a-e and O-(protected-α-aminoxyacyl)terpenes 3.20e-h

The preparation of the O-(-protected-aminoxyacyl)sterols (3.20a-f, 3.20b+b’) was

investigated first. In the initial experiments the reaction of the N-Cbz-protected(α-

aminoxyacyl)benzotriazoles (3.14b) with stigmasterol (3.19a) was not completed after

24 h at 20 °C. Any attempts to optimize the reaction conditions were unsuccessful.

However, under microwave irradiation, the synthesis of the sterol conjugates was

accomplished within 45-60 min. Thus, the coupling of the N-Cbz-protected (α-

aminoxyacyl)benzotriazoles (3.14b-d, 3.14c+c’) with various nucleophiles was

investigated under microwave irradiation in the presence of a catalytic amount of 4-

(N,N-dimethylamino)pyridine (0.1 eq.). In all cases, the completion of the reaction was

monitored by TLC (Scheme 3-7).

56

Scheme 3-7. Synthesis of O-(protected--aminoxyacyl)steroids 3.20a-d and O-

(protected--aminoxyacyl)terpenes 3.20e-h.

Table 3-3. Preparation of O-(protected--aminoxyacyl)steroids 3.20a-d and O-

(protected--aminoxyacyl)terpenes 3.20e-h.

N-(Cbz-Protected aminoxyacyl)-benzotriazoles

NuH Optimized reaction

conditions 3.20

yield (%) 3.20

m.p. (oC)

Cbz-AO-Ala(D)-Bt, 3.14b

3.19c 50 °C, 45 W,

50 min. 3.20a

22 102.0 – 103.0

Cbz-AO-Phe(D)-Bt 3.14c

3.19b 50 °C, 45 W,

50 min. 3.20b

25 114.3 – 115.2

Cbz-AO-Phe(DL)-Bt 3.14c+c’

3.19b 50 °C, 45 W,

50 min. 3.20b+b’

25 112.0 – 114.0

Cbz-AO-Phe(D)-Bt 3.14c

3.19c 50 °C, 45 W,

50 min. 3.20c

26 112.2 – 113.5

Cbz-AO-Leu(D)-Bt, 3.14d

3.19a 50 °C, 45 W,

50 min. 3.20d

20 120.1-122.0

Cbz-AO-Ala(D)-Bt, 3.14b

3.19e 50 °C, 45 W,

30 min. 3.20e

60 Oil

Cbz-AO-Phe(D)-Bt 3.14c

3.19d 50 °C, 45 W,

30 min. 3.20f

55 Oil

Cbz-AO-Phe(DL)-Bt, 3.14c+c’

3.19d 50 °C, 45 W,

30 min. 3.20g+g’

54 Oil

Cbz-AO-Leu(D)-Bt, 3.14d

3.19e 50 °C, 45 W,

30 min. 3.20h

50 Oil

Compounds designated as 3.14c+c‘, 3.20b+b‘, 3.20g+g‘ – represent racemates

57

3.2.5 Synthesis of O-(Protected-α-Aminoxyacyl)sugar (3.24)

The acylation position of a partially unprotected sugar 1,2-O-isopropylidene-α-D-

glucofuranose 3.23 was investigated in the presence of DMAP (0.1 eq.) (Scheme 3-8).

The reaction mixture was subjected to microwave irradiation (50 W, 30 min at 60 °C)

and the product 3.24 was obtained after purification by column chromatography in a

yield of 62%.

Scheme 3-8. Preparation of O-(protected-α-aminoxyacyl)sugar (3.24).

The acylation position of the partially unprotected sugar 3.23 was determined by

the 1H-13C gHMBC experiment. The diastereotopic protons, CH2 at 4.00 and 4.30 ppm

showed three bond correlation with ester carbon at 171.8 ppm (Figure 3-1).

The proton connectivity for the sugar fragment was obtained by the 1H-1H COSY

experiment (Figure 3-2). The expansion for the sugar fragment (Figure 3-3) displayed

the cross peaks of anomeric proton at δ = 5.79 ppm with vicinal proton at 4.39 ppm, the

other proton chemical shifts on the sugar fragment being assigned by their cross peaks.

The total assignment of 3.24 is presented in Figure 3-4.

The compound 3.24 was prepared by Dr. Finn Hansen as part of a collaborative

project.

58

Figure 3-1. 1H-13C gHMBC experiment of 3.24.

59

Figure 3-2. 1H-1H dQCOSY of 3.24.

Figure 3-3. 1H-1H dQCOSY expansion for sugar fragment of 3.24.

60

Figure 3-4. 1H and 13C chemical shifts assignments of 3.24.

3.2.6 Synthesis of N-(Protected-α-Aminoxyacyl)nucleosides 3.26a,b

Finally, we have utilized the Cbz-(protected-α-aminoxyacyl)benzotriazoles

(3.14c,d) for the synthesis of N-(protected--aminoxyacyl)nucleosides 3.26a,b. The

attempted synthesis of 3.26a,b under microwave irradiation provided diacylated

products. Consequently, we repeated these experiments at room temperature and the

reaction of 3.14d with cytidine (3.25a) and 3.14c with adenosine (3.25b) in DMF at r.t.

provided the corresponding N-acylated products 3.26a,b having a yield of 21% and

54%, respectively (Scheme 3-9).

Scheme 3-9. Synthesis of (protected--aminoxyacyl)nucleosides 3.26a,b.

61

Table 3-4. Preparation of N-(protected--aminoxyacyl)nucleosides 3.26a,b.

N-(Cbz-Protected

aminoxyacyl)-

benzotriazole, 3.14

NuH,

3.25

Optimized reaction

conditions

3.26,

yield (%)

3.26,

m.p. (oC)

Cbz-AO-Leu(D)-Bt,

3.14d 3.25a r.t., 24h 3.26a, 21 107.0 -108.0

Cbz-AO-Phe(D)-Bt,

3.14c 3.25b r.t., 24h 3.26b, 54 110.0-113.0

3.3 Conclusions

Aminoxyacyl bioconjugates are important scaffolds for pharmaceuticals because

they present higher resistance against proteolysis.

Cbz-protected-(α-aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’) are stable,

usually crystalline and readily available reagents, which were utilized for the convenient

preparation of aminoxy acid conjugates with sugars, terpenes, steroids and

nucleosides. Microwave-assisted preparation provided a series of Cbz-protected

aminoxy acid conjugates with moderate to good yields and short reaction times without

detectable racemization.

3.4 Experimental Section

Melting points were determined on a capillary point apparatus equipped with a

digital thermometer and are uncorrected. NMR spectra were recorded in CDCl3 or

DMSO-d6 on a Gemini or Mercury NMR operating at 300 MHz for 1H and 75 MHz for

13C with TMS as an internal standard. The 2D NMR experiments were recorded on

Inova 500 equipped with an indirect detection probe operating at 500 MHz for 1H and

125 MHz for 13C. Elemental analyses were performed on a Carlo Erba-1106 instrument.

All microwave assisted reactions were carried out with a single mode cavity Discover

Microwave Synthesizer (CEM Corporation, NC). The reaction mixtures were transferred

62

into a 10 mL glass pressure microwave tube equipped with a magnetic stirrer bar. The

tube was closed with a silicon septum and the reaction mixture was subjected to

microwave irradiation (Discover mode; run time: 60 sec.; PowerMax-cooling mode).

Benzyl hydroxycarbamate (3.17). A solution of benzyl chloroformate (5.70 mL, 40

mmol) in CH2Cl2 (30 mL) was added dropwise to a stirred mixture of hydroxylamine

hydrochloride (3.48 g, 50.0 mmol) and sodium bicarbonate (11.30 g, 142 mmol) in THF:

water (80 mL, 5:1), at -4 oC over 30 min. The reaction mixture was stirred overnight, and

then most of the solvent was removed in vacuo and the obtained residue was

redissolved in water (10 mL). The aqueous phase was acidified with HCl (3N, pH=4)

then extracted with ether (3 x 30 mL). The organic phase was dried over MgSO4, then

the solvent was removed under reduced pressure to give the crude product.

Recrystallization from EtOAc: hexanes (1:5) gave benzyl hydroxycarbamate (3.17) as

white microcrystals (5.90 g, 90 %); m.p. 66.0 - 67.0 oC. (lit. m.p. 68 - 69.0 oC.

[1960JCS299]); 1H NMR (300 MHz, CDCl3) δ 7.44 (s, 1H), 7.37-7.35 (m, 5H), 5.16 (s,

2H). 13C NMR (75 MHz, CDCl3) δ 159.3, 135.5, 128.7, 128.6, 128.5, 68.0.

3.4.1 General Procedure for (L)-2-Bromo-carboxylic acids synthesis (3.8a-d).

A solution of sodium nitrate (1.4 eq.) in water (4 mL) was added dropwise at -17oC

over 2 h to a stirred solution of (L)-aminoacid (1 eq.) and potassium bromide (3.3 eq.) in

sulfuric acid (3 M, 5 mL). The reaction mixture was stirred for an additional 6 h at r.t.,

then the mixture was extracted with diethyl ether (3 x 30 mL). The combined organic

layers were washed with water (2 x 50 mL), dried over MgSO4, and then the solvent

was removed under reduced pressure to give the corresponding 2-bromo-carboxylic

acid.

63

(L)-2-Bromopropanoic acid (3.16b). [1985JA7072] Yellow oil, (80%); 1H NMR (300

MHz, CDCl3) δ 10.29 (s, 1H), 4.40 (q, J = 6.9 Hz, 1H), 1.84 (d, J = 6.9 Hz, 3H).13C NMR

(75 MHz, CDCl3) δ 176.4, 39.5, 21.5. The compound was used in the next step without

any further purification.

(L)-2-Bromo-3-phenylpropanoic acid (3.16c).[2009JOC4242] Yellow oil, (45%);

1H NMR (300 MHz, CDCl3) δ 9.83 (s, 1H), 7.20-7.30 (m, 5H), 4.41(dd, J = 7.3, 7.9 Hz,

1H), 3.45 (dd, J = 14.2, 7.9 Hz, 1H), 3.23 (dd, J = 14.2, 7.3 Hz, 1H).13C NMR (75 MHz,

CDCl3) δ 175.3, 136.4, 129.3, 128.9, 127.6, 45.0, 40.9. The compound was used in the

next step without any further purification.

(DL)-2-Bromo-3-phenylpropanoic acid (3.16c+c’). [1907BDCG3996] Yellow oil,

(62%); 1H NMR (300 MHz, CDCl3) δ 10.67 (br s, 1H), 7.30 – 7.15 (m, 5H), 4.38 (dd, J =

8.0, 7.3 Hz, 1H), 3.41 (dd, J = 14.2, 8.0 Hz, 1H), 3.19 (dd, J = 14.2, 7.3 Hz, 1H). 13C

NMR (75 MHz, CDCl3) δ 175.2, 136.1, 129.0, 128.6, 127.3, 44.8, 40.5. The compound

was used in the next step without any further purification.

(L)-2-Bromo-4-methylpentanoic acid (3.16d). [1990JMC263] Yellow oil, (70%); 1H

NMR (300 MHz, CDCl3) δ 9.60 (s, 1H), 4.29 (t, J = 7.7 Hz, 1H), 1.92 (t, J = 7.6 Hz, 2H),

1.76-1.90 (m, 1H), 0.97 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz,

CDCl3) δ 176.2, 44.1, 43.3, 26.5, 23.3, 22.5, 21.7. The compound was used in the next

step without any further purification.

3.4.2 General Synthesis of α-2-(Benzyloxycarbonylaminooxy)carboxylic acid (3.18a-d, 3.18c+c’)

NaH (2.2 eq. 60% in mineral oil) was added portionwise to a stirring solution of

benzyl hydroxycarbamate 3.17 (1 eq.) in THF (10 mL) under argon atmosphere at -4 0C.

The resulting reaction mixture was stirred for 15 min., then 2-bromo-carboxylic acid (1

64

eq.) was added in small portions, and the reaction mixture was stirred at r.t. for 8 h

under argon. The solvent was removed under reduced pressure, giving a pale yellow

precipitate, which was then washed with diethyl ether: hexane (1:1, 20 mL) yielding a

fine powder, which was redissolved in water and washed with hexane (10 mL). The

aqueous layer was acidified with HCl (3N pH = 3) and extracted with ether (3 x 20 mL).

The combined organic layers were then dried over MgSO4 and the solvent was at last

removed under reduced pressure to give the corresponding products (3.18a-d,

3.18c+c’).

2-(Benzyloxycarbonylaminooxy)acetic acid (3.18a). [2009JOC8690] Yellow oil,

(53%); 1H NMR (300 MHz, CDCl3) δ 8.58(s, 1H), 7.53 (s, 1H), 7.26-7.53 (m, 5H), 5.19

(d, J = 9.3 Hz, 1H), 5.16 (d, J = 9.6 Hz, 1H), 4.55 (s, 0.5H), 4.45 (s, 1.5H). The

compound was used in the next step without any further purification.

(D)-2-(Benzyloxy)carbonylaminooxy)propanoic acid (3.18b). White microcrystals,

(60%); m.p. 92.0 - 94.0 oC; 1H NMR (300 MHz, CDCl3) δ 9.14 (s, 1H), 8.35 (s, 1H), 7.40

– 7.32 (m, 5H), 5.19 (s, 2H), 4.51 (q, J = 7.1 Hz, 1H), 1.49 (d, J = 7.1 Hz, 3H). 13C NMR

(75 MHz, CDCl3) δ 175.8, 158.2, 135.0, 128.6, 128.6, 128.3, 80.2, 68.2, 16.2. Anal.

Calcd. For C11H13N1O5 (239.08) required: C, 55.23; H, 5.48; N, 5.85. Found C, 55.32, H,

5.60; N, 5.63.

(D)-2-(Benzyloxycarbonylaminooxy)-3-phenylpropanoic acid (3.18c).

[1975GB1394170] Yellow oil, (68%); 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 7.30-

7.35 (m, 5H), 7.24-7.30 (m, 5H), 5.13 (s, 2H), 4.61 (dd, J = 8.9, 3.7 Hz, 1H), 3.26 (dd, J

= 14.7, 3.8 Hz, 1H), 3.04 (dd, J = 14.7, 8.9 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 176.8,

65

158.8, 136.1, 134.8, 129.4, 129.1, 128.8, 128.7, 128.6, 127.1, 86.0, 68.6, 37.3. The

compound was used without any further purification.

(DL)-2-(Benzyloxycarbonylaminooxy)-3-phenylpropanoic acid (3.18c+c’).

[1975GB1394170] Yellow oil, (53%); 1H NMR (300 MHz, CDCl3) δ 8.18 (br s, 1H), 7.41

– 7.20 (m, 10H), 5.13 (s, 2H), 4.61 (dd, J = 8.9, 3.7 Hz, 1H), 3.26 (dd, J = 14.7, 3.8 Hz,

1H), 3.04 (dd, J = 14.7, 8.9 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 173.9, 158.8, 147.6,

129.5, 129.2, 128.9, 128.7, 128.6, 127.2, 86.1, 68.7, 37.4.

(D)-2-(Benzyloxycarbonylaminooxy)-4-methylpentanoic acid (3.18d). Pale yellow

oil, (88%); 1H NMR (300 MHz, CDCl3) δ 8.64(s, 1H), 7.34 (s, 5H), 6.78 (s, 1H), 5.19 (d,

J = 12.1 Hz, 1H), 5.14 (d, J = 12.1 Hz, 1H), 1.86-1.90 (m, 1H), 1.65-1.75 (m, 1H), 1.50-

1.59 (m, 1H), 1.20-1.24 (m, 1H), 0.89-0.98 (m, 6H). 13C NMR (75 MHz, CDCl3) (mixture

of rotamers) δ 179.9, 176.3, 159.5, 158.1, 135.4, 135.2, 128.7, 128.6, 128.5, 128.4,

127.2, 82.9, 69.0, 68.1, 43.3, 39.8, 24.7, 24.6, 23.3, 23.2, 21.5. Anal. Calcd. For

C14H19N1O5 (281.13) required: C, 59.78; H, 6.81; N, 4.98. Found: C, 59.47; H, 7.22; N,

5.10.

3.4.3 General Synthesis of N-Cbz-Protected(α-Aminoxyacyl)benzotriazoles (3.14a-d, 3.14c+c’)

(D)-2-(Benzyloxycarbonylaminoxy)carboxylic acid (1 eq.) was added portionwise to

a stirring solution of benzotriazole (3 eq.) and thionyl chloride (1 eq.) in THF (10 mL).

The resulting reaction mixture was stirred at r.t. for 2 h. The precipitate was filtered off,

the solvent was removed under reduced pressure to give an oil which was then

redissolved in diethyl ether (20 mL) and washed first with water (20 mL) and then with

Na2CO3 (10 %, 2 x 20 mL). The resulting colorless oil was then recrystallized from

66

diethylether: hexane (1:2) to give the corresponding benzyl 1-(1H-benzotriazol-1-yl)-1–

yloxycarbamate acid derivatives.

2-(2-(1H-Benzotriazol-1-yl)-2-oxoethoxy)isoindoline-1,3-dione (3.14a). White

prisms (77%); m.p. 88.0 – 90.0 oC, (lit m.p. 86.0 – 87.0 oC [2009JOC8690]); 1H NMR

(300 MHz, CDCl3) δ 8.32 (ddd, J = 8.3, 0.9, 0.9 Hz, 1H), 8.15 (ddd, J = 8.3, 0.9, 0.9 Hz,

1H), 7.90 – 7.86 (m, 2H), 7.81 – 7.73 (m, 2H), 7.71 (ddd, J = 8.2, 7.2, 1.0 Hz, 1H), 7.56

(ddd, J = 8.3, 7.1, 1.0 Hz, 1H), 5.93 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 165.1, 162.9,

145.9, 134.8, 131.0, 130.7, 128.8, 126.7, 123.9, 120.4, 114.1, 74.3., Anal. Calcd. For

C16H14N4O4(326.10) required: C, 58.89; H, 4.32; N, 17.17. Found: C, 59.93; H, 4.36; N,

17.07.

(D)-Benzyl 1-(1H-benzotriazol-1-yl)-1-oxopropan-2-yloxycarbamate (3.14b), White

prisms (78%); m.p. 86.0 - 88.0 oC; 1H NMR (300 MHz, CDCl3) δ 8.23 (dd, J = 8.3,

1.0Hz, 1H), 8.15 (dd, J = 8.3, 1.0 Hz, 1H), 7.91 (s, 1H), 7.70 (ddd, J = 8.2, 7.1, 1.0 Hz,

1H), 7.55 (ddd, J = 8.2, 7.1, 1.0 Hz, 1H), 7.26-7.36 (m, 5H), 5.94 (q, J = 7.0 Hz, 1H),

5.19 (s, 1H), 1.77 (d, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 171.0, 157.2, 145.8,

135.2, 131.0, 130.9, 128.6, 128.5, 128.3, 126.7, 120.4, 114.3, 80.6, 67.9, 17.0. Anal.

Calcd. For C17H16N4O4(548.04) required: C, 60.00; H, 4.74; N, 16.46. Found: C, 59.93;

H, 4.54; N, 16.36.

(D)-N-(1-(1H-Benzotriazol-1-yl)-1-oxo-3-phenylpropan-2-yloxy)-2-phenylacetamide

(3.14c). White microcrystals (88%); m.p. 90.0 - 91.0 oC; 1H NMR (300 MHz, CDCl3) δ

8.28 (ddd, J = 8.2, 0.8, 0.8 Hz, 1H), 8.14 (ddd, J = 8.2, 0.8, 0.8 Hz, 1H), 7.79 (s, 1H),

7.68 (ddd, J = 8.2, 7.5, 0.8 Hz, 1H), 7.54 (ddd, J = 8.2, 7.5, 0.8 Hz, 1H), 7.32-7.19 (m,

10H), 6.14 (dd, J = 7.8, 4.3 Hz, 1H), 5.11 (s, 2H), 3.47 (dd, J = 14.7, 4.3 Hz, 1H), 3.36

67

(dd, J = 14.7, 7.8 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 169.7, 157.2, 146.0, 135.3,

135.3, 131.0, 129.5, 128.7, 128.6, 128.6, 128.4, 127.3, 126.8, 120.5, 114.34 85.1 68.0,

37.8. Anal. Calcd for C23H20N4O4 (416.15) required: C, 66.34; H, 4.84; N, 13.45. Found:

C, 66.44; H, 5.06; N, 12.98.

(DL)-N-(1-(1H-Benzotriazol-1-yl)-1-oxo-3-phenylpropan-2-yloxy)-2-

phenylacetamide (3.14c+c’). Yellow crystals (86%); m.p. 91.0 -92.0 oC; 1H NMR (300

MHz, CDCl3) δ 8.28 (ddd, J = 8.2, 0.8, 0.8 Hz, 1H), 8.14 (ddd, J = 8.2, 0.8, 0.8 Hz, 1H),

7.79 (s, 1H), 7.68 (ddd, J = 8.2, 7.5, 0.8 Hz, 1H), 7.54 (ddd, J = 8.2, 7.5, 0.8 Hz, 1H),

7.32-7.19 (m, 10H), 6.14 (dd, J = 7.8, 4.3 Hz, 1H), 5.11 (s, 2H), 3.47 (dd, J = 14.7, 4.3

Hz, 1H), 3.36 (dd, J = 14.7, 7.8 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 169.7, 157.2,

146.0, 135.3, 135.3, 131.0, 129.5, 128.7, 128.6, 128.6, 128.4, 127.3, 126.8, 120.5,

114.34 85.1 68.0, 37.8. Anal. Cald for C23H20N4O4(416.15) required: C, 66.34; H, 4.84;

N, 13.45. Found: C, 66.50; H, 4.88; N, 13.67.

(D)-Benzyl-1-(1H-benzotriazol-1-yl)-4-methyl-1-oxopentan-2-yloxycarbamate

(3.14d). Colorless oil (86%); 1H NMR (300 MHz, CDCl3) δ 8.28 (dd, J = 8.2, 1.0 Hz, 1H),

8.13 (dd, J = 8.13, 1.1 Hz, 1H), 8.10 (s, 1H), 7.66 (ddd, J = 8.2, 7.2, 1.1 Hz, 1H), 7.53

(ddd, J = 8.3, 7.2, 1.1 Hz, 1H), 7.50 – 7.30 (m, 5H), 5.94 (dd, J = 9.8, 3.1 Hz, 1H), 5.18

(s, 2H), 2.08 – 2.18 (m, 1H), 1.80-1.94 (m, 2H), 1.10 (d, J = 6.7 Hz, 3H), 0.97 (d, J =

6.7 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 171.3, 157.4, 146.0, 135.3, 131.2, 131.0,

128.7, 128.7, 128.6, 126.57 120.35 114.5, 83.5, 68.0, 40.5, 25.02 23.4, 21.4. Anal.

Calcd. for C20H22N4O4(382.16) required: C, 62.82; H, 5.80; N, 14.65. Found: C, 62.63;

H, 5.90; N, 14.18.

68

3.4.4 General Synthesis of O-(Protected-α-Aminoxyacyl)steroids (3.20a-d), and O-(Protected-α-Aminoxyacyl)terpenes (3.20e-h)

The respective N-Cbz-protected (α-aminoxyacyl)benzotriazoles (3.14b-d,

3.14c+c’) (1eq.) were added portionwise to a stirring solution of steroid 3.19a-c or

terpene 3.19d,e (1 eq.) in THF (1 mL) in the presence of a catalytical amount of DMAP

(0.1 eq.). The reaction mixture was subjected to microwave irradiation (power,

temperature and hold time as indicated in Table 3-3) and then allowed to cool to room

temperature, transferred to a round bottomed flask, the solvent removed under reduced

pressure.

The compounds were purified as follows:

The crude steroid conjugates were purified by column chromatography using

hexanes: EtOAc (95: 5) as eluent to afford the O-(-protected-aminoxyacyl)steroids

(3.20a-d) as analytical pure products.

The crude terpene conjugates 3.20e-h were redissolved in EtOAc (10 mL), and

the solutions were washed with Na2CO3 (2 x 10 mL 10% v/w) and dried over MgSO4 to

give 3.20a-d.

(2R)-(10R,13R)-17-((2R,5R)-5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl

2-((((benzyloxy)carbonyl)amino)oxy)propanoate (3.20a). White mycrocrystals (22%);

m.p. 102.0 – 103.0°C; [α]21D = +25.31 (c 0.23, CH2Cl2);

1H NMR (300 MHz, CDCl3) δ

7.88 (s, 1H), 7.37 – 7.35 (m, 5H), 5.38 (d, J = 3.8 Hz, 1H), 5.20 (d, J = 12.1 Hz, 1H),

5.15 (d, J = 12.1 Hz, 1H), 4.70 – 4.67 (m, 1H), 4.47 (q, J = 7.0 Hz, 1H), 2.33 (d, J = 7.7

Hz, 2H), 2.02 – 1.95 (m, 2H), 1.95 – 1.61 (m, 2H), 1.61 - 0.76 (m, 41H), 0.67 (s, 3H).

13C NMR (75 MHz, CDCl3) δ 171.6, 157.0, 139.4, 135.6, 128.7, 128.6, 128.4, 123.2,

69

79.2, 75.2, 67.7, 56.8, 56.2, 50.1, 46.0, 42.5, 39.9, 39.0, 38.4, 37.0, 36.7, 36.3, 36.0,

34.1, 33.9, 32.6, 32.1, 32.0, 30.4, 29.3, 28.4, 27.9, 26.2, 24.4, 23.2, 21.2, 20.4, 20.0,

19.5, 19.2, 18.9, 18.9, 18.4, 16.4, 15.5, 12.2, 12.0. Anal. Calcd for C40H61NO5(635.45)

required: C, 75.55; H, 9.67; N, 2.20. Found: C, 75.36; H, 10.04; N, 2.13.

(2R)-(10R,13R)-10,13-Dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl

2-((((benzyloxy)carbonyl)amino)oxy)-3-phenylpropanoate (3.20b). White microcrystals

(25%); m.p. 114.0 – 115.0 °C; [α]21D = +20.35 (c 0.21, CH2Cl2);

1H NMR (300 MHz,

CDCl3) δ 7.79 (s, 1H), 7.36 – 7.31 (m, 5H), 7.28 – 7.21 (m, 5H), 5.36 (d, J = 4.7 Hz, 1H),

5.16 (d, J = 12.1 Hz, 1H), 5.10 (d, J = 12.1 Hz, 1H), 4.64 (dd, J = 6.8, 5.4 Hz, 1H), 4.59

(dd, J = 8.4, 4.8 Hz, 1H), 3.13 (s, 1H), 3.10 (d, J = 1.6 Hz, 1H), 2.25 (d, J = 7.7 Hz,

2H), 2.07 – 1.90 (m, 2H), 1.90 – 1.69 (m, 3H), 1.62 – 1.10 (m, 17H), 1.00 – 0.82 (m,

13H), 0.67 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 170.4, 157.1, 139.4, 135.9, 135.6,

129.6, 128.7, 128.6, 128.4, 128.4, 127.0, 123.1, 84.5, 75.3, 67.7, 56.8, 56.3, 50.1, 42.4,

39.8, 39.7, 38.1, 37.3, 37.0, 36.7, 36.3, 35.9, 32.0, 32.0, 28.4, 28.2, 27.7, 24.4, 24.0,

23.0, 22.7, 21.2, 19.4, 18.9, 12.0. Anal. Calcd for C44H61NO5(683.45) required: C, 77.27;

H, 8.99; N, 2.05. Found: C, 76.92; H, 9.33; N, 1.96.

(10R,13R)-10,13Dimethyl-17-((R)-6-methylheptan-2-yl)-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl

2-((((benzyloxy)carbonyl)amino)oxy)-3-phenylpropanoate (3.20b+b’). White

microcrystals (25%); m.p. 112.0 – 114.0°C; [α]21D = -14.63 (c 0.19, CH2Cl2);

1H NMR

(300 MHz, CDCl3) δ 7.76 (s, 1H), 7.37 – 7.30 (m, 5H), 7.28 – 7.21 (m, 5H), 5.36 – 5.34

(m, 1H), 5.16 (d, J = 12.1 Hz, 1H), 5.10 (d, J = 12.2 Hz, 1H), 4.63 (t, J = 6.2 Hz, 1H),

70

4.60 -4.57 (m, 1H), 3.12 (d, J = 6.5 Hz, 2H), 2.28 – 1.67 (m, 7H), 1.57 – 0 85 (m, 33H)

, 0.67 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 170.4, 157.1, 139.4, 135.9, 135.6, 129.6,

128.6, 128.4, 128.4, 127.0, 123.1, 84.5, 75.4, 67.7, 56.8, 56.3, 50.1, 42.5, 39.9, 39.7,

38.1, 38.0, 37.3, 37.0, 36.7, 36.3, 35.9, 32.0, 31.2, 28.4, 28.2, 27.8, 27.7, 24.4, 24.0,

23.0, 22.7, 21.2, 19.4, 18.9, 12.0. Anal. Calcd for C44H61NO5(683.45) required: C, 77.27;

H, 8.99; N, 2.05. Found: C, 77.04; H, 9.33; N, 2.13.

(2R)-(10R,13R)-17-((2R,5R)-5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl

2-((((benzyloxy)carbonyl)amino)oxy)-3-phenylpropanoate (3.20c). White microcrystals

(26%); m.p. 112.0 – 113.0 °C; [α]21D = +12.91 (c 0.22, CH2Cl2);

1H NMR (300 MHz,

CDCl3) δ 7.78 (s, 1H), 7.36 – 7.29 (m, 5H), 7.29 – 7.24 (m, 5H), 5.36 (d, J = 4.8 Hz, 1H),

5.20-5.16 (m, 1H), 5.16 (d, J = 12.4 Hz, 1H), 5.11 (d, J = 12.3 Hz, 1H), 5.01 (dd, J =

15.3, 8.7 Hz, 1H), 4.65 (dd, J = 6.7 , 5.8 Hz, 1H), 4.62 – 4.58 (m, 1 H), 3.13 (s, 1H),

3.11 (d, J = 1.8 Hz, 1H), 2.25 (d, J = 7.8 Hz, 2H), 2.23 – 1.85 (m, 3H), 1.85 – 1.62 (m,

3H), 1.59 – 1.38 (m, 8H), 1.38 - 1.13 (m, 7H), 1.03 - 0.99 (m, 8H), 0.98 – 0.90 (m, 2H),

0.85 – 0.79 (m, 9H), 0.69 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 170.4, 157.1, 139.4,

138.5, 135.9, 135.6, 129.6, 129.5, 128.8, 128.6, 128.5, 127.1, 123.2, 84.5, 75.4, 67.8,

57.0, 56.1, 51.5, 50.2, 42.4, 40.7, 39.8, 38.2, 37.3, 37.1, 36.8, 32.1, 29.1, 27.8, 25.6,

24.6, 21.5, 21.3, 21.2, 19.5, 19.2, 12.5, 12.3. Anal. Calcd for C46H65NO5(711.48)

required: C, 77.60; H, 9.20; N, 1.97. Found: C, 77.73; H, 9.29; N, 1.91.

(2R)-(10R,13R)-17-((2R,5S,E)-5-Ethyl-6-methylhept-3-en-2-yl)-10,13-dimethyl-

2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl

2-((((benzyloxy)carbonyl)amino)oxy)-4-methylpentanoate (3.20d). White microcrystals

71

(20%); m.p. 120.1-122.0 °C; [α]21D = +25.89 (c 0.10, CH2Cl2);

1H NMR (300 MHz, CDCl3)

δ 7.79 (s, 1H), 7.36 – 7.34 (m, 5H), 5.38 (d, J = 5.0 Hz, 1H), 5.20 (d, J = 12.1 Hz, 1H),

5.14 (d, J = 12.1 Hz, 1H), 5.01 (dd, J = 15.2, 8.5 Hz, 1H), 4.69 – 4.67 (m, 1H), 4.40 (dd,

J = 9.8, 3.9 Hz, 1H), 2.33 (d, J = 7.7 Hz, 2H), 2.11 – 1.82 (m, 6H), 1.78 – 1.38 (m, 14H),

1.38 – 1.08 (m, 5H), 1.03 – 0.98 (m, 8H), 0.95-0.93 (m, 6H), 0.88 – 0.78 (m, 9H), 0.70

(s, 3H). 13C NMR (75 MHz, CDCl3) δ 171.7, 157.0, 139.4, 138.4, 135.6, 129.4, 128.7,

128.6, 128.5, 123.1, 82.8, 75.1, 67.8, 64.3, 56.9, 56.1, 51.4, 50.2, 42.4, 40.7, 39.9, 39.8,

38.2, 37.1, 36.7, 32.0, 29.1, 27.9, 25.6, 24.7, 24.5, 23.3, 21.7, 21.4, 21.3, 19.5, 19.2,

12.4, 12.2. M/z calc for [C43H65NO5+Na]+=698.4755. Found [M+23]+=698.4774.

(R,Z)-3,7-Dimethylocta-2,6-dien-1-yl2-((((benzyloxy)carbonyl)amino)oxy)

propanoate (3.20e). Colorless oil (60%); [α]21D = +90.97 (c 0.22, CH2Cl2);

1H NMR (300

MHz, CDCl3) δ 8.11 (s, 1H), 7.35 – 7. 33 (m, 5H), 5.34 (t, J = 7.7 Hz, 1H), 5.58 (d, J =

12.3 Hz, 1H), 5.13 (d ,J = 12.3 Hz, 1H), 5.10 – 5. 06 (m, 1H), 4.64 (d, J = 7.6 Hz, 2H),

4.50 (q, J = 7.0 Hz, 1H), 2.10 – 2.05 (m, 4H), 1.76 (s, 3H), 1.67 (s, 3H), 1.59 (s, 3H),

1.45 (d, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 172.2, 157.1, 143.4, 135.6, 132.3,

128.6, 128.4, 128.3, 123.5, 118.6, 79.8, 67.5, 61.9, 32.2, 26.6, 25.7, 23.5, 17.7, 16.3.

Anal. Calcd for C21H29NO5(375.20): C 67.18; H 7.78; N 3.73. Found: C 67.21; H 7.81; N

4.42. m/z calc for [C21H29NO5+Na]+= 398.1938. Found = 398.1949.

(R,E)-3,7-Dimethylocta-2,6-dien-1-yl-2-((((benzyloxy)carbonyl)amino)oxy)-3-

phenylpropanoate (3.20f). Yellow oil (55%); [α]21D = +42.47 (c 0.23, CH2Cl2);

1H NMR

(300 MHz, CDCl3) δ 7.86 (s, 1H), 7.35-7.31 (m, 5H), 7.29 -7.22 (m, 5H), 5.24 (t, J = 7.0

Hz, 1H), 5.14 (d, J = 12.3 Hz, 1H), 5.09 (d, J = 12.3 Hz, 1H), 5.09 – 5.06 (m, 1H), 4.67

(dd, J = 6.7, 5.3 Hz, 1H), 4.61 (d, J = 7.3 Hz, 2H), 3.13 – 3.10 (m, 2H), 2.07 – 2.03 (m,

72

4H), 1.67 (s, 3H), 1.66 (s, 3H), 1.59 (d, J = 0.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ

170.9, 157.0, 143.3, 135.8, 135.5, 132.0, 129.5, 128.7, 128.5, 128.4, 128.4, 127.0,

123.8, 117.6, 84.4, 67.7, 62.2, 39.6, 37.2, 26.4, 25.8, 17.9, 16.6. Anal. Calcd for

C27H33NO5(451.23) required: C, 71.82; H, 7.37; N, 3.10. Found: C, 71.70; H, 7.51; N,

3.37.

(R,S)-(E)-3,7-Dimethylocta-2,6-dien-1-yl-2-((((benzyloxy)carbonyl)amino)oxy)-3-

phenylpropanoate (3.20g+g’). Colorless oil (54%); [α]21D = +0.96 (c 0.30, CH2Cl2);

1H

NMR (300 MHz, CDCl3) δ 7.78 (s, 1H), 7.37 – 7. 28 (m, 5H), 7.28 – 7.18 (m, 5H), 5.24

(t, J = 7.2 Hz, 1H), 5.15 (d, J = 12.1 Hz. 1H), 5.10 (d, J = 12.1 Hz, 1H), 5.10 – 5.04 (m,

1H), 4.67 (dd, J = 7.0, 5.2 Hz, 1H), 4.62 (d, J = 7.2 Hz, 2H), 3.16 (dd, J = 12.1, 4.9 Hz,

1H), 3.09 (dd, J = 12.5, 5.1 Hz, 1H), 2.10 – 2.03 (m, 4H), 1.68 (s, 3H), 1.66 (s, 3H), 1.60

(s, 3H). 13C NMR (75 MHz, CDCl3) δ 170.9, 157.0, 143.3, 135.8, 135.5, 132.1, 129.5,

128.7, 128.5, 128.4, 127.0, 123.7, 117.6, 84.5, 67.7, 62.3, 39.7, 37.3, 26.4, 25.9, 17.9,

16.7. Anal. Calcd for C27H33NO5(451.23) required: C, 71.82; H, 7.37; N, 3.10. Found: C,

72.15; H, 7.75; N, 3.25.

(R,E)-2,7-Dimethyloctal-2,6-dien-1-yl-2-(((benzyloxy)carbonyl)amino)oxy)-4-

metylpentanoate (3.20h). Colorless oil (50%); [α]21D = +56.78 (c 0.29, CH2Cl2);

1H NMR

(300 MHz, CDCl3) δ 8.24 (br s, 1H), 7.32 (s, 5H), 5.34 (t, J = 7.2 Hz, 1H), 5.18 (d, J =

12.1 Hz, 1H), 5.12 (d, J = 12.3 Hz, 1H), 5.09 – 5.08 (m, 1H), 4.63 (d, J = 7.3 Hz, 2H),

4.44 (dd, J = 9.8, 3.8 Hz, 1H), 2.11 – 2.10 (m, 6H), 1.99 – 1.88 (m, 1H), 1.76 (s, 3H),

1.67 (s, 3H), 1.59 (s, 3H), 0.93 (d, J = 5.7 Hz, 3H), 0.91 (d, J = 5.1 Hz, 3H). 13C NMR

(75 MHz, CDCl3) δ 172.3, 157.3, 143.2, 135.7, 132.2, 128.6, 128.4, 123.6, 118.7, 82.7,

67.6, 61.8, 61.2, 39.9, 32.2, 26.7, 25.8, 24.6, 23.6, 23.2, 21.6, 17.7. Anal. Calcd for

73

C24H35NO5(417.25) required: C, 69.04; H, 8.45; N, 3.35. Found: C, 68.70; H, 8.87; N,

3.22.

3.4.5. Preparation of D-(D-2-Hydroxy-2-((3aR,5R,6S,6aR)-6-hydroxy-2,2-dimethyltetrahydrofuro[3,2-d][1,3]dioxol-5-yl)ethyl)2 benzyloxycarbonyl-aminooxy)propanoate (3.24)

(D)-Benzyl (1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-yl)oxycarbamate

(3.14b), (1eq.) was added portionwise to a stirring solution of 1,2-O-isopropylidene-α-D-

glucofuranose 3.23 (1.4 eq.) in THF (1 mL) and a catalytical amount of DMAP (0.1 eq.).

The reaction mixture was subjected to microwave irradiation (50 W, 60oC, 30 min) and

subsequently allowed to cool to room temperature, then transferred to a round bottomed

flask and the solvent removed under reduced pressure. The product was purified by

column chromatography, using EtOAc:Hexanes: (1:1) to afford 3.24 as white

microcrystals (62%). m.p. 112.0 – 115.0 °C; [α]21D = +43.3 (c 0.13, CH2Cl2);

1H NMR

(300 MHz, DMSO-d6) δ 10.50 (s, 1H), 7.40-7.30 (m, 5H), 5.79 (d, J = 3.5 Hz, 1H), 5.27

(d, J = 4.5 Hz, 1H), 5.14-5.04 (m, 3H), 4.43-4.26 (m, 3H), 4.07-3.84 (m, 4H), 1.36 (s,

3H), 1.33 (d, J = 6.9 Hz, 3H), 1.22 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.1, 157.1,

136.2, 128.4, 128.0, 127.9, 110.6, 104.5, 84.6, 80.2, 79.1, 72.8, 67.3, 66.1, 65.1, 26.7,

26.2, 16.3. Anal. Calcd for C20H27NO10(441.16) required: C, 54.42; H, 6.16; N, 3.17.

Found: C, 54.46; H, 6.22; N, 3.00.

3.4.6 General Synthesis of O-(Protected-α-aminoxyacyl)nucleosides (3.26a,b).

N-Cbz-protected(α-aminoxyacyl)benzotriazoles (3.14c,d) (1eq.) were added

portionwise to a stirring solution of nucleoside 3.25a,b in DMF (3 mL). The reaction

mixture was stirred at r.t. for 24 h, and then the solvent was removed under vacuo to

give a yellow oil. The product was purified by column chromatography CH2Cl2: MeOH

(5:1) to give the corresponding O-(protected-α-aminoxyacyl)nucleosides (3.26a,b).

74

Benzyl ((2R)-1-((1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-

1,2-dihydropyrimidin-4-yl)amino)-4-methyl-1-oxopentan-2-yl)oxycarbamate (3.26a).

White microcrystals (21%); m.p. 107.0 -108.0°C; [α]21D = +25.89 (c 0.10, CH2Cl2);

1H

NMR (300 MHz, CD3OD) δ 8.59 (d, J = 7.5 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.32 –

7.28 (m, 5H), 5.88 (s, 1H), 5.15 (s, 2H), 4.42 (dd, J = 9.3, 4.2 Hz, 1H), 4.18 – 4.15 (m,

2H), 4.11 – 4.09 (m, 1H), 3.98 (dd, J = 12.6, 2.1 Hz, 1H), 3.80 (dd, J = 12.3, 2.7 Hz,

1H), 2.90 – 1.85 (m, 1H), 1.80 – 1.67 (m, 1H), 1.57 – 1.50 (m, 1H), 0.98 (d, J = 4.8 Hz,

3H), 0.94 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz, CD3OD) δ 174.4, 164.0, 160.0, 158.0,

147.1, 137.4, 129.6, 129.5, 129.4, 98.0, 93.3, 86.7, 86.0, 76.7, 70.1, 68.6, 61.4, 41.5,

25.8, 23.7, 22.3. Anal. Calcd for C23H30N4O9(506.20) required: C, 54.54; H, 5.97; N,

11.06. Found: C, 54.23; H, 6.13; N, 10.77.

Benzyl ((R)-1-((9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-

2-yl)-9H-purin-6-yl)amino)-1-oxo-3-phenylpropan-2-yl)oxycarbamate (3.26b). White

microcrystals (54%); m.p. 109.0 -112.0 °C; [α]21D = +25.89 (c 0.10, CH2Cl2);

1H NMR

(300 MHz, CD3OD) δ 10.54 (br s, 1H), 8.34 (s, 1H), 8.35 (s, 1H), 7.43-7.20 (m, 12H),

7.00 – 6.87 (m, 1H), 5.96 – 5.92 (m, 1H), 5.73 – 5.71 (m, 1H), 5.36 (dd, J = 5.1, 1.2 Hz,

1H), 5.08 (s, 2H), 4.99 – 4.95 (m, 1H), 4.73 (dd, J = 7.8, 4.5 Hz, 1H), 4.10 – 4.09 (m,

1H), 3.70 – 3.57 (m, 2H), 3.21 (dd, J = 15.3, 4.5 Hz, 1H), 3.03 (dd, J = 15.0, 7.8 Hz,

1H). 13C NMR (75 MHz, CD3OD) δ 169.4, 156.9, 156.2, 152.4, 149.0, 139.9, 136.2,

129.2, 129.0, 128.4, 128.1, 128.0, 127.8, 126.5, 119.4, 87.5, 83.5, 83.2, 74.3, 71.6,

66.1, 61.6, 36.6. Anal. Calcd for C27H28N6O8(564.20) C, 57.44; H, 5.00; N, 14.89.

Found: C, 57.17; H, 5.01; N, 14.27. m/z calc for [C27H28N6O8+Na]+= 587.1861. found =

587.1859.

75

CHAPTER 4 MULTINUCLEAR NMR ANALYSIS OF A VARIETY OF MOLECULAR STRUCTURES

4.1 Background

This chapter discusses the multinuclear NMR spectra of a series of compounds

including a dipeptide derivative of galactopyranose (4.1), a series of pyridazine

derivatives (4.2-4.5) and a trinitroderivative of furan (4.6).(Scheme 4-1)

Scheme 4-1. The structures of the investigated compounds

4.1.1 Applications of Amino Sugars in drug design

Synthetic studies on carbohydrates originated with the early investigations of

Emil Fischer during 1890 and they have been extensively reviewed and applied ever

since [1890BDCG799]. Sugars are able to mediate many biological processes and

interactions between proteins and the carbohydrates present on the external surface of

cell membranes [2004GEN48]. Amino sugars present biological activity; a few examples

include Doxorubicin (4.7) [1988JNCI1152], Dannorubicin (4.8) [2006C333]. These drugs

present cytotoxic activity; they are able to bind to nucleic acids, presumably by a

specific interaction of the planar anthracycline moiety with the DNA double helix.

Monosacharides found applications as cancer chemotherapeutic agents – Licomycin

(4.9) [2006CCD2] or antibacterials – Tobramycin (4.10) (Scheme 4-2)

[2011JAMPDD175].

76

Scheme 4-2. Examples of biologically active aminosugars

The field of antibiotics is extremely challenging because some pathogens such

as Candida (single cell fungi yeasts) acquire rapidly decreased susceptibility to

antifungal antibiotics and other pharmacophores such as Amphotericin B [1982CAR59].

Some Candida species are even able to survive medicamentation, from this point of

view, new and more effective antifungal drugs have to be designed. Biological

properties of organic compounds are structurally and conformationally dependent and

therefore the correct assignment is crucial in the drug design. Many natural products

and biologically active compounds have an N-acyl group joined by a sugar moiety.

These structural motifs appear in antibiotics such as N-acetylcalicheamicins [1990TL21,

1992JA985], istamycines [1982CAR33], glycocinnamoylspermidines [1978JA2515] and

streptomycins [1982CAR59]. In these cases signals for individual conformers were not

identified and the conformational preference in solution was not investigated in detail,

although the syn- , anti- and E/Z isomerisation of the amides has been intensively

studied [1979COC373, 1979COC957]. The possible syn-, anti- periplanar

77

conformations for Z and E isomers of a series of protected amino sugars have been

investigated recently (Scheme 4-3) [1992JCSPT(2)2005].

Scheme 4-3. Syn- and anti- periplanar conformations for Z and E isomers.

For unambiguous structure elucidation and conformation preference in solution,

NMR is the best available method because it does not interfere with the equilibrium

between the possible conformers. In this chapter, phase sensitive experiments such as

1H-1H dQCOSY, selective decoupling (NOE), and variable temperature (VT NMR)

experiments were used to get the proton connectivity of the peptide chain and sugar

fragment of Cbz-L-Phe-N-galactopyranose (4.1).

4.1.2 Applications of 15N in Structural Elucidation

Nitrogen is one of the most important elements in organic chemistry and

biochemistry. Nitrogen can be found in all living organisms; it is a structural motif of

nucleic acids, proteins and alkaloids. The large diversity of nitrogen containing

molecules made 15N NMR one of the major investigated nuclei beside 1H and 13C in

solving structural problems in organic and bioorganic molecules.

The second part of this chapter focuses on the structural elucidation using 15N

NMR of several heterocyclic systems including some pyridazine derivatives (4.2 – 4.5)

78

and a high energy nitrated furan derivative (4.6). Complete assignment, including 1H,

13C, 15N chemical shifts were obtained by using multiple bond (long range) correlation

experiments such as 1H-13C gHMBC, 1H-15N CIGAR-gHMBC (Scheme 4-4).

Scheme 4-4. The structures of compounds (4.2 – 4.6).

Pyrazines are important biological scaffolds; they have found applications as:

antibacterial [1994EP579059], antidepressant [1974EJMC644], anti hypertensive

[1990EP327800], analgesic [1996JCPB980], nephrotropic [JP9071535], anti-

inflammatory [1974EJMC644] anticancer [2002JMC563], and cardiotonic

pharmaceuticals [EP327800, 1987JMC1157].

The 1H, 13C, 15N chemical shifts of several pyrazines were obtained from 1H-13C

gHMQC, 1H-13C gHMBC and 1H-15N CIGAR-gHMBC experiments. 1H-13C gHMQC gives

one bond correlation between 1H and 13C; 1H-13C gHMBC gives long range correlations

between 1H and 13C; 1H-15N CIGAR-gHMBC, a pulse sequence experiment optimized

for 15N, gives long range couplings between 1H and 15N.

15N NMR has been used for structural elucidation of several heterocyclic systems

containing pyrazine fragment. Holzer et al. have reported the complete assignment of all

nitrogens in a series of isoxazolo[3,4-d]pyridazin-7(6H)-ones (4.11). The observed 15N

chemical shifts of these pyridazinones nitrogens vary from 307.1 to 324.0 ppm for N-5,

and respectively 188.2 to 215.7 ppm for N-6 [2005MRC240].

79

Pazderski and co-workers reported the full assignment of 5-methyl-7-phenyl-2,5-

dihydro[1,2,3]triazolo[4,5-d]pyridazin-4-one (4.12) and the corresponding N methylated

products 4.13a,b (Scheme 4-5) [2002JMS73].

Scheme 4-5. Some pyridazine derivatives previously characterized by 1H-15N CIGAR-gHMBC.

1H-15N CIGAR-gHMBC studies for 4.12, 4.13a,b revealed the cross peak

correlations for the two tautomeric two isomers. The 15N chemical shift for N-5 varies

from 192.4-195.5 ppm, and chemical shifts for N-6 vary from 323.6-383.8 ppm. The

electronic environment influences the chemical shifts; for example, N-2 resonates at

383.8 ppm for 4.13a and 264.5 ppm for 4.13b, N-6 resonates at 323.6 ppm (4.13a) and

respectively 327.2 ppm for 4.13b [2002JMS73].

Dinitro-derivatives of five-membered heterocycles show considerable biological

activity, for example 2,4-dinitroimidazole derivatives increase the sensitivity of hypoxic

cells toward irradiation in cancer radiotherapy [1979JMC583]; they are useful

intermediates for the conversion of dinitrofuran into various polysubstituted phenols

80

[2001ARK29]; they are of potential interest as energetic materials and blowing agents

[2008S699].

4.2 Results and Discussion

4.2.1 Proton Correlations of Cbz-L-Phe-N-Galactopyranose

β-N-Glycoaminoacids were prepared by Dr. Tamari Narindoshvili by coupling of N-

Z-α-aminoacyl)benzotriazoles with 2,3,4,6-tetra-O-pivaloyl-β-D-galactopyranosylamines

using the benzotriazole methodology [2008JOC511].

Nα-Carbobenzyloxy-Nδ-(2,3,4,6-tetra-O-pivaloyl-β-D-galactopyranosyl)-L-

phenylalanine (Cbz-L-Phe-N-galactopyranose) exists in solution as a mixture of two

rotamers (Scheme 4-6).

The 1H chemical shifts of Cbz-L-Phe-N-galactopyranose for each rotamer were

assigned using several NMR techniques including 2D NMR dQCOSY, selective

decoupling and VT NMR.

Scheme 4-6. Possible rotamers of Cbz-L-Phe-N-galactopyranose (4.1).

The VT NMR experiment (Figure 4-1) confirms the existence of two rotamers

(70:30 in DMSO-d6) that coalesce on heating. For example, HC,c gives two sets of

quartets at 4.30 and 4.32 ppm with different intensities; the Z-anti conformer (4.1a) is

favored due to steric effects. Variable temperature 1H NMR data in DMSO-d6 over the

81

range of 25-115oC using 10 oC increments supported the assumption that the complex

peaks at 20 oC were due to restricted rotation of the amide bond. The two rotamers

coalesced at 55 – 65 oC. The multiplet at 2.64 - 2.89 ppm coalesced at 105 oC, and

saccharine signals at 3.9 - 4.42 and 5.26 - 5.46 ppm coalesced at 55 oC; these changes

were reversible on heating-cooling sequences (Figure 4-1).

Figure 4-1. VT NMR spectrum of Cbz-L-Phe-N-galactopyranose (4.1).

Preliminary studies showed that anomeric proton Hf,F is overlapped in both

rotamers by other proton signals resulting in a multiplet at 5.10-5.40 ppm. Therefore, the

stereochemistry of the anomeric proton and thus the orientation of the (HF,f) glycosidic

bond was investigated by a selective decoupling experiment.

Selective decoupling (NOE) was achieved by irradiation at the appropriate

frequency of the amide proton NHE,e (6.48 ppm in CDCl3). As a result, the coupling JHf-Hg

was found to be 9.7 Hz which indicates that both HG,g and HF,f are either E or Z-anti

(Figure 4-2).

82

Figure 4-2. Selective decoupling experiment of Cbz-L-Phe-N-galactopyranose in CDCl3.

Anti- and/ or syn- periplanar conformations of the amiosugar have been previously

investigated, although Karplus equation [1975T2177,1969T493], is not able to

discriminate between the syn- and anti- conformation. The previous observed values for

JNHCH (8.0 – 10.9 Hz) are considered as antiperiplanar disposition [1989JCSPT(1)1923,

1971CI96, 1972ABC1071, 1974CAR233, 1987CAR71, 1992JCSPT(2)2205]. In most of

the cases Z –anti predominates in solution due to steric effect.

Our results fit the previous reported data (JNHCH = 9.7 Hz) suggesting an anti-

conformer preference [2008JOC511].

The 1H chemical shifts for each rotamer were assigned based on the cross peak

correlation observed in 1H-1H dQCOSY (DMSO-d6) experiment followed by integration

of 1H NMR to establish the rotamer ratio. The dQCOSY experiment confirms the

existence of two species. For example, the amide proton NHB (7.47 ppm) correlates

with HC(4.30 ppm) and NHE (9.03 ppm) correlates with HF (5.46 ppm) of the major

rotamer. The NHb (7.55ppm) correlates with Hc (4.32ppm) and NHe (8.99 ppm)

correlates with Hf (5.46 ppm). HC (4.30 ppm) correlates with HD1,2 (2.88 and 2.68 ppm)

and Hc correlates with Hd1,2 (2.85 and 2.64 ppm). The preliminary results for major

83

rotamer and minor rotamer of the expanded region of dQCOSY are presented in Figure

4-3.

Figure 4-3. 1H-1H dQCOSY of Cbz-L-Phe-N-galactopyranose (4.1) amide fragment.

The protons of both rotamers of the sugar unit were assigned by their cross peaks

with HF,f. For instance HF (5.46 ppm) correlates with HG (5.15 ppm) for major rotamer

and Hf (5.46 ppm) correlates Hg (5.14 ppm) of the minor rotamer. Furthermore HG (5.15

ppm) correlates HH (5.38ppm) of major rotamer and Hg (5.14 ppm) correlates Hh

(5.42ppm) for minor rotamer; HH (5.38 ppm) correlates with HI (5.29 ppm) and

respectively Hh (5.42 ppm) with Hi (5.31 ppm) (Figure 4-4).

84

Figure 4-4. 1H-1H dQCOSY of Cbz-L-Phe-N-galactopyranose (4.1) sugar part expansion.

The 1H NMR shows the presence of two rotamers, the ratio between them was

estimated by the integral ratio of HA and Ha (70:30 in DMSO-d6 at 25 oC).

85

The aromatic protons appear at 7.27-7.38 ppm as multiplets and their assignment

was not possible.

The final proton assignment of the Cbz-L-Phe-N-galactopyranose is presented in

Scheme 4-7.

Scheme 4-7. Total proton assignment of Cbz-L-Phe-N-galactopyranose.1

1H NMR (500 MHz, DMSO-d6) δ 9.03 (d, J = 9.7 Hz, HE), 8.99 (d, J = 9.7 Hz, He),

7.55 (d, J = 9.3 Hz, Hb), 7.47 (d, J = 9.2 Hz, HB), 7.38 - 7.27 (m, 6H), 7.20 (d, J = 7.2 Hz,

2H), 7.17 (d, J = 7.5 Hz, 2H), 5.46 (t, J = 9.6 Hz, HF), 5.46 (t, J = 9.4 Hz, Hf), 5.42 (dd, J

= 10.1, 3.8 Hz, Hh), 5.38 (dd, J = 10.3, 3.2 Hz, HH), 5.31 (d, J = 3.3 Hz, Hi), 5.29 (d, J =

3.4 Hz, HI), 5.15 (t, J = 9.7 Hz, HG), 5.14 (t, J = 9.5 Hz, Hg), 4.91 (s, Ha), 4.89 (d, J =

13.2 Hz, HA1), 4.85 (d, J = 13.2 Hz, Ha1), 4.44 (t, J = 7.4 Hz, HJ), 4.42 (t, J = 7.2 Hz,

Hj), 4.32 (t, J = 9.9 Hz, HC), 4.30 (t, J = 9.9 Hz, Hc), 4.09 (dd, J = 10.5, 6.5 Hz, Hk1), 4.06

(dd, J = 10.8, 6.8 Hz, HK1), 3.90 (dd, J = 10.3, 7.8 Hz, Hk2), 3.90 (dd, J = 10.8, 7.6 Hz,

HK2), 2.88 (dd, J = 13.2, 2.7 Hz, HD), 2.85 (dd, J = 13.1, 2.4 Hz, Hd1), 2.68 (dd, J = 13.1,

11.5 Hz, HD2), 2.64 (dd, J = 13.4, 11.5 Hz, Hd2), 1.24 (s), 1.23 (s), 1.10 (s), 1.10 (s),

1.05 (s), 1.04 (s), 1.00 (s).

Reproduced in part with the permission from J. Org. Chem., 2008, 73(2), 511. Copyright © American Chemical Society

2008.

86

4.2.2 1H 13C 15N Chemical shifts of some Pyrazine derivatives 2

The chemical 1H, 13C, 15N shifts of pyridazines 4.2- 4.5 were obtained by long

range quantum correlation 2D NMR techniques using field gradients and indirect

detection. The results are presented in the Table 4.1 for 1H and 13C and in the Table 4.2

for 15N.

Table 4-1. 1H and 13C NMR chemical shifts, ppm of some pyridazine derivatives 4.2 –

4.5. No. R3 R6 1H

H-4 H-5 Other H

4.2 H H 7.65 (t, J = 3.5 Hz)

7.59 (m)

7.65 (t, J =3.5 Hz)

7.61 (m)

9.23 (t, J = 3.5 Hz, H-3,6)

4.3 9.76 (s, H-1, H-4), 8.22 (m, H-5, H-8), 8.08 (m, H-6, H-7)

4.4 CH3 CH3 - 7.24 (s) 2.42 (s, CH3-3’), 2.48 (s, CH3S-

4’), 2.50 (s, CH3-6’)

4.5 - - 7.02 (d, J = 9.8 Hz) 7.17 (d, J = 10 Hz) 7.57 (ddd, J =7.9, 1.2, 1.2 Hz),

7.46 (t, J = 7.8 Hz),

7.36 (ddd, J = 7.5, 1.1, 1.1 Hz)

No.

R3 R6 13C

C-3,

C-3’ a

C-4,

C-4’ a

C-5 C-6,

C-6’a

4.2 H H 152.5(H-4) 127.4 (H-3, H-6) 127.4 (H-3, H-6) 152.5 (H-5)

4.3 151.7 (C-1, C-4), 126.5 (C-4a, C-8a (H-1, H-4, H-5, H-6, H-7, H-8)), 127.0 (C-5, C-8 (H-6,

H-7)), 133.5 (C-6, C-7 (H-5, H-8))

4.4 CH3 CH3 154.1 (H-3’, H-5),

20.2 (H-3’)a

142.6 (H-3’, H-4’,

H-5),

13.1(H-4’)a

120.6 (H-6’) 157.4 (H-6’),

22.0 (H-6’)a

4.5 O OH 157.7b 134.6 128.3 126.2,

129.1,

128.0,

133.9b a – Chemical shifts form 1H-13C gHMQC, b- Uncorrelated values

87

Table 4-2. 15N NMR chemical shifts, ppm of pyridazines 4.2 – 4.5. No. R3 R6 15N

N-1 N-2

4.2 H H 402.7 (H-3, H-4, H-5, H-6)

4.3 372.4 (N-2, N3 (H1,H4))

4.4 CH3 CH3 373.0 (H-5, H-6’) 387.2 (H-3’)

4.5 O OH 301.5 (H-5) 197.4 (H-4)

An example of the total assignment of 3,6-dimethyl-4-(methylthio)pyridazine (4.4)

is presented in Figures 4-5, 4-6, 4-7. The 1H-13C gHMQC experiment gives one bond

correlation between 1H and 13C (Figure 4-5). The H3’ proton (2.50 ppm) correlates with

C-3’ (22.0 ppm); H-4’ (2.48 ppm) gives one bond correlation with C-4’ (13.1 ppm); H5

(7.24ppm) correlates with C-5 (120.6 ppm) and H-6’ (2.42 ppm) correlates with (20.2

ppm).

The 1H-13C gHMBC gives long range correlation (Figure 4-6). This experiment is

useful for quaternary carbon assignments, for example H-3 shows three bond

correlation with quaternary C-3 (157.4 ppm). Furthermore, H-4’ (2.84 ppm) shows

correlation with C-3 (157.4 ppm) and with C-5 (142.6 ppm). Moreover H-5’ (2.48 ppm)

shows correlations with C-5 (142.6 ppm); H-6’ (2.42 ppm) shows correlation with C-6

(154.1 ppm) and C-5 (142.6 ppm) (Figure 4-5).

1H-15N CIGAR-gHMBC (Figure 4-7) gives the long range coupling of 1H and 15N.

For example H-6’ (2.42 ppm) correlates with N-1 (387.2) and H-3’ (2.50 ppm) and H-4

(7.24 ppm) both correlates with N-2 (387.2 ppm).

2Reproduced in part with the permission from Magn. Reson. Chem., 2010, 48(5), 397 Copyright © Royal Chemical Society 2010

88

Figure 4-5. 1H-13C gHMQC of 3,6-dimethyl-4-(methylthio)pyridazine (4.4).

Figure 4-6. 1H-13C gHMBC of 3,6-dimethyl-4-(methylthio)pyridazine (4.4).

89

Figure 4-7. 1H-15N CIGAR-gHMBC of 3,6-dimethyl-4-(methylthio)pyridazine (4.4).

4.2.3 Total correlation of 2-Ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6)

The compound 2-ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6) was characterized by

1H-15N CIGAR-gHMBC and 1H-13C gHMBC. Nitromethane (CH3NO2) was used as

internal standard (0 ppm for 15N scale). The 1H, 13C and 15N chemical shifts of 4.6 are

presented in Figure 4-8.

Figure 4-8. Total assignment of 2-ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6).

1H-15N gHMBC-CIGAR Experiment shows the three bond correlations of N-2a (-

12.2 ppm) and N-2a’ (-10.9 ppm) with H-3 (6.70ppm). N-5a (2.3 ppm) shows correlation

with H-6a (2.43 ppm) and H-6b (2.59 ppm) (Figure 4-9).

90

Figure 4-9. 1H-15N CIGAR-gHMBC of 2-ethyl-2,5,5-trinitro-2,5-dihydrofuran (4.6).

4.3 Conclusions

dQCOSY Experiments together with selective decoupling and variable

temperature NMR afford the total proton assignment of chemical shifts and coupling

constants of glycopeptides (4.1).

The NOE decoupling experiment was carried out in CDCl3 because the amide

protons NHE and respectively NHe (6.48 ppm) signals are separated from the aromatic

region; but the proton separation of sugar fragment protons was not acceptable. A

better signal separation of the sugar fragment protons was obtained in DMSO-d6,

therefore, the proton connectivity was investigated in DMSO-d6.

91

The 1H, 13C and 15N chemical shifts of pyridazines (4.2-4.5) and of a high energy

nitrated furan (4.6) were successfully obtained from long range heteronuclear

correlation experiments.

4.4 Experimental Section

The 1H-1H dQCOSY experiment was recorded on a Varian Inova 500. The

selective decoupling experiments were recorded on a Varian Mercury 300 while the VT

NMR was recorded on Varian Mercury 300BB. The phase sensitive experiment

dQCOSY affords the proton connectivity of Cbz-L-Phe-N-galactopyranose (4.1).

The 1H-13C gHMBC, gHMQC and 1H-15N CIGAR-gHMBC experiments were

recorded on a Varian Inova instrument equipped with a three channel 5 mm indirect

detection probe and with z-axis gradients, operating at 500 MHz for 1H, 125 MHz for 13C

and 50 MHz for 15N. The experiments were recorded in DMSO-d6, at 25 °C, unless

otherwise specified. The chemical shifts for 1H and 13C were referenced to the residual

solvent signal, 2.50 ppm for 1H and 39.5 ppm for 13C (DMSO-d6) , and respectively, 7.27

ppm for 1H and 77.16 ppm for 13C (CDCl3) on the tetramethylsilane scale. The chemical

shifts for 15N were referenced to Ξ = 10.1328898, corresponding to 0 for neat ammonia.

On the Ξ scale the frequency of protons in tetramethylsilane is 100.0000000 MHz. For

conversion to the neat nitromethane scale, subtract 381.7 ppm [2002COC35]. Typically,

1H-13C gHMBC spectra were acquired in 2048 points in f2, on a spectral window from 0

to 11 ppm, and 1 s relaxation delay.

1H-15N CIGAR-gHMBC spectra were acquired with a pulse sequence optimized

for 15N, as described earlier [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

92

FID was zero-filled twice prior to Fourier transform. The experiments were completed in

most of the cases within 2 h.

Melting points were determined on a capillary point apparatus equipped with

digital thermometer and are uncorrected.

Nα-Carbobenzyloxy-Nδ-(2,3,4,6-tetra-O-pivaloyl-β-D-galactopyranosyl)-L

phenylalanine (Cbz-L-Phe-N-galactopyranose) (4.1) was prepared by former group

member Tamari Narindoshvili [2008JOC511]; pyridazine (4.2) and phthalazine (4.3)

were obtained from Acros Chemical Company and were used without any further

purification.

3,6-Dimethyl-4-(metylthio)pyridazine (4.4) [2010MRC397], m.p. 90.0 - 91.0 oC.

Anal. Calcd. For C7H10N2S(154.05) required: C, 54.51; H, 6.53; N, 18.16. Found C,

54.48; H, 6.42; N, 18.15.

6-Hydroxy-2-phenylpyridazin-3(2H)-one (4.5) m.p. 270.0 -272.0 oC, lit. m.p. 273.0

-274.0 oC [1977JOC1367].

2-Ethyl-2,5,5-trinitro-2,5dihydrofuran (4.6) was prepared by former group

member Dr. Anatoliy V. Vakulenko. [2008S699].

93

CHAPTER 5 SYNTHESIS OF 2,4-DISUBSTITUTED QUINAZOLINES, 4H-BENZO[E][1,3]OXAZINE

AND 4H-BENZO[E][1,3]THIAZINE BY ANRORC REARRANGEMENTS OF 1,2,4-OXADIAZOLES

5.1 Background

In this chapter we have applied the procedure developed by Srivastava et al.

[2000H191] to prepare a series of 4H-benzo[e][1,3]oxazoline, and, after gaining some

insight about the reaction, we then extended the methodology to the synthesis of 2,4-

disubstituted quinazolines and 4H-benzo[e][1,3]thiazines. We have found that these

transformations take place via a modified version of the ANRORC (Addition of

Nucleophile, Ring Opening Ring Closure) mechanism [2003JOC605].

This chapter gives also a literature overview of the main transformations of the

1,2,4-oxadiazole ring under thermal and photochemical conditions.

Among the most investigated ring-transformations is the Boulton-Katritzky

rearrangement (BKR) which is an interconversion between five membered heterocycles

where a three-atom side chain and a pivotal annular nitrogen are involved. This

methodology is widely used in the literature for the construction of a variety of

heterocyclic systems (Scheme 5-1) [1964ACIE693, 1967JCS2005].

Scheme 5-1. General reaction scheme of the Boulton-Katritzky rearrangement.

94

5.1.1 The importance of 1,2,4-Oxadiazoles

Synthetically, 1,2,4-oxadiazoles are important structural motifs because of their

high reactivity and great tendency to undergo molecular rearrangements to give more

stable heterocycles. This is mainly because the 1,2,4-oxadiazole system is considered

one of the least aromatic five-membered heterocyclic systems, with an index of

aromaticity I5 = 39 or IA = 48 [1985T1409, 1992T335].

1,2,4-Oxadiazole is a versatile ring system which has found applications in

medicinal chemistry as analgesic, anti-inflamatory and antirhinoviral [1994JMC2421,

1990JMC1128], as a component in drug molecules, including Perebron

[1963GB924608] and Libexin [1999W04822], and in material science in formulation of

ionic liquid crystals and OLEDs. [2000WO096043]

Scheme 5-2. Applications of 1,2,4-oxadiazoles.

5.1.2 Preparation of 1,2,4–Oxadiazoles

The most common pathways to 1,2,4-oxadiazoles involve the couplings of

amidoximes with: (i) activated carboxylic acid derivatives such as acid chlorides

[2001JMC619, 2003S899], fluorides [1999TL9359], anhydrides [1996TL6627,

95

1995JOC3112], or active esters [1999BMC2359, 1999JMC4088]; (ii) carboxylic acids in

the presence of coupling reagents including dicyclohexylcarbodiimide (DCC)

[1995JOC3112, 2004S1589], 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC)

[2001JMC619, 1996TL6627], 2-dimethylamino)isopropyl chloride (DIC)/HOBt

[1999TL8547, 2003TL6079], bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl)

[1995JOC3112], 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

(TBTU) [2001TL1495, 2003TL9337], or 1,1’-carbonyldiimidazole (CDI) [1999BMC209].

Other methods to obtain 1,2,4-oxadiazoles include reactions of amidoximes with

(iii) aryl halides using palladium catalysts [1998TL3931], or with (iv) aldehydes followed

by oxidation (Scheme 5-3) [2003BMC1821].

1,2,4-Oxadiazoles can be prepared from amidoximes and Meldrum acids under

microwave irradiation [2006SL1765].

Scheme 5-3. Preparative methods of 1,2,4-oxadiazoles.

96

5.1.3 ANRORC rearrangements of 1,2,4-Oxadiazoles

Heterocyclic rearrangements allow the synthesis of new heterocyclic structures,

which may be difficult to achieve or may require multiple steps; in this context,

ANRORC represents a useful synthetic method for the preparation of new heterocyclic

systems. Buscemi et al. have investigated the rearrangements of 5-perfluoroalkyl-1,2,4-

oxadiazoles in the presence of bidentate nucleophiles such as hydroxylamine,

hydrazine or methylhydrazines. In this case ANRORC rearrangements take place by

[3+2] or [4+2] bidentate attack (Scheme 5-4) [2003JOC605].

Scheme 5-4. Types of ANRORC rearrangements.

The ANRORC [3+2] rearrangement of activated 1,2,4-oxadiazoles, such as 5-

perfluoro-1,2,4-oxadiazoles, allows the synthesis of the corresponding isomer of 1,2,4-

oxadiazole in one step if hydroxyl amine is used as a bidentate nucleophile (Scheme 5-

5) or 1,2,4 triazoles 5.24 if hydrazine is used as nucleophile (Scheme 5-6); the resulting

hydroxyl amine can react further to give the ring-degenerative oxadiazole

[2003JOC605, 2004EJOC974, 2006H307, 2006JOC8106, 2009ARK235].

97

Scheme 5-5 ANRORC degenerative rearrangements of 1,2,4-oxadiazoles.

Scheme 5-6. ANRORC [2+3] rearrangements of 1.2.4-oxadiazoles.

The ANRORC [4+2] is a useful method for conversion of 5-perfluoroalkyl-1,2,4-

oxadiazles into 1,2,4-triazin-5(2H)-ones (5.31) via a stable triazinone oxime (5.30) (Path

A, Scheme 5-7); however, triazole is formed as a side product (5%) (Path B, Scheme

5.7) [2006JOC8106].

98

Scheme 5-7. 5-Perfluoroalkyl-1,2,4-oxadiazoles reactions with hydrazines.

The ratio between 5.31 and 5.33 is influenced by the steric bulkiness of RF; for

example, if RF is C7F15 then the reaction takes place via a BKR mechanism; on the

other hand, if RF = CF3 the reaction gives exclusively 5.31.

The reaction with 5-perfluoroalkyl-1,2,4-oxadiazoles 5.27a-c with asymmetric

nucleophiles such as N-methylhydrazine gives exclusively the corresponding triazole

product 5.34a-c. This confirms that only the NH2 end of the bidentate nucleophile is

involved in the first attack at C(5); however, if a leaving group is present at C(3), the

corresponding triazinones 5.38 are obtained (Scheme 5-8) [2005JOC3288,

2006JOC8106].

99

Scheme 5-8. 5-Perfluoroalkyl-1,2,4-oxadiazoles reactions with methyl hydrazine.

The ANRORC mechanism takes place by a bidentate nucleophile attack at C(5) of

an activated 1,2,4-oxadiazole ring, followed by a ring-opening and then by a ring closure

to give the corresponding rearranged product. The carbonyl group at C(3) favors the

[4+2] pathway. Only hydrazine and hydroxyl amine were investigated as bidentate

nucleophiles [2009T1472].

5.1.4 The Boulton-Katritzky rearrangements of 1,2,4-Oxadiazoles

The 1,2,4-oxadiazoles ring system can be opened then closed under thermal

conditions to give new heterocyclic systems such as 1,3-benzoxazoles (5.40)

[1998HC1551], benzimidazoles (5.42) [1988HC1551, 1996JOC8397], indazoles

[1996JOC8397], 1,3-oxazoles (5.45) [2007JOC7656], these transformations taking

place via the Boulton-Katritzky rearrangement (Scheme 5-9).

100

Scheme 5-9. Some examples of the Boulton-Katritzky rearrangement of 1,2,4-oxadiazoles with pivotal nucleophile at C(3).

5.1.5 Photochemical rearrangement of 1,2,4-Oxadiazoles

1,2,4-Oxadiazoles are able to give a series of rearrangements under

photochemical irradiation. The reaction involves the initial cleavage of the O-N bond to

give a zwitterion (5.47) or a nitrene (5.49) that may react with internal or external

nucleophiles (Scheme 5-10) [2005JOC2322].

101

Scheme 5-10. Photochemical transformations of 1,2,4-oxadiazoles.

The formation of nitrene intermediate 5.54 was investigated by the reaction of 5.46

with 2,3-dimethyl-2-butene (5.53) under UV irradiation giving N-imidoyl-aziridines (5.55)

(Scheme 5-11) [2007H1529].

Scheme 5-11. Synthesis of N-imidoyl-aziridines.

Photochemical irradiation of 3-(o-aminophenyl)-1,2,4-oxadiazoles (5.41a,c) or 3-

[O-(methylamino)phenyl]-1,2,4-oxadiazoles (5.41b) produce a mixture of 1,2

benzimidazoles (5.58a-c) and 1,3-benzimidazoles (5.59a-c) [1996JOC8397].

102

Scheme 5-12. Photochemical rearrangements of 1,2,4-oxadiazoles with pivotal nucleophile at C(3).

5.1.6 1,2,4-Oxadiazoles rearrangements using Strong Nucleophiles

Recently, Srivastava et al. [2000H191] have reported that 1,2,4-oxadiazoles (5.60)

can undergo ring opening – ring closing reactions at low temperature (-78 oC) in the

presence of n-BuLi to give 4,5-dihydro-1,2,4-oxadiazoles (5.61) and 4,4-di-n-butyl-2-

phenylbenzo-1,3-oxazine (5.62) (Scheme 5-13). We have used this reaction as a

template for our study.

Scheme 5-13. Addition of strong nucleophiles to 1,2,4-oxadiazole ring at low temperature.

103

5.2 Results and Discussion

5.2.1 Preparation of 1,2,4-Oxadiazoles (5.63a-j)

We prepared 1,2,4-oxadiazoles (5.63a-g) from their corresponding N-

acylbenzotriazoles (5.64a-e) and (Z)-N'-hydroxybenzimidamides (5.65a-c) in good to

excellent yields (Table 5-1), following the procedure reported earlier [2005ARK36].

Scheme 5-14. Preparation of 1,2,4 oxadiazoles 5.63a-g.

Table 5-1. Preparation of 1,2,4-oxadizaoles 5.63a-g. Entry R1 R2 5.63a-g

Yield (%)

5.63a 2-OH-C6H4 Ph 88 5.63b 2-OH-C6H4 4-CH3-C6H4 88 5.63c 2-(S-C6H4-) Ph 63 5.63d 3-OH-2-naphthyl Ph 82 5.63e 2-OH-1-naphthyl Ph 79 5.63f Ph Ph 88 5.63g 2-OH-C6H4 4-O2N-C6H4 92

2-(3-Substituted-1,2,4-oxadiazol-5-yl)anilines (5.63h,i) are readily available from

isatoic anhydride (5.66) and (Z)-N’-hydroxybenzimidamides (5.65b,c) (Table 5-2),

following the procedure of Nagahara [1975CPB3178].

Scheme 5-15. Preparation of 1,2,4 oxadiazoles 5.63h,i.

Table 5-2. Preparation of 1,2,4-oxadizaoles 5.63h,i. Entry R2 5.63h,i

Yield (%)

5.63h Ph 65 5.63i 4-CH3-C6H4 62

104

5.2.2 Substrate Design

1,2,4-Oxadiazoles are able to undergo the Boulton-Katritzky rearrangement when

the pivotal nucleophile is placed at the position C(3) [2007JOC7656]. A nucleophile at

C(5) will generate the intermediates 5.71 and 5.72 (Scheme 5-16).

Scheme 5-16. Possible 1,2,4-oxadiazole BKR rearrangements with pivotal nucleophile at C(3) or C(5).

All 1,2,4-oxadiazole fragmentations take place by the initial cleavage of the N-O

bond, thus the BKR requires the presence of the pivotal nucleophile at C(3) (Scheme 5-

17). To the best of our knowledge, no BKR rearrangement of the 1,2,4-oxadiazole has

been reported for pivotal nucleophile at position C(5).

Scheme 5-17. Possible rearrangements of 1,2,4-oxadiazoles using a pivotal nucleophile at position C(5).

105

First, we investigated the rearrangement of 2-(3-phenyl-1,2,4-oxadiazol-5-

yl)phenol (5.63) in the presence of a variety of bases. In all cases, 5.63 was

quantitatively recovered after work-up (Scheme 5.18).

Scheme 5-18. The direct rearrangement of 2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenol (5.63).

Second, we investigated the ring opening-ring closure of the 1,2,4 oxadiazole ring

by using an excess of strong nucleophiles, such as n-butyllithium or Grignard reagents.

n-Butyllithium acts as a base and as a nucleophile at position C(5) of the 1,2,4-

oxadiazole ring; two equivalents of n-butyllithium give monoaddition, while 3 or 4

equivalents of n-butyllithium give the ANRORC rearrangement. This result is in

agreement with earlier studies of Srivastava [2000H191]. Srivastava et al. concluded

that 4,4-di-n-butyl-2-phenyl-1.3-oxazine are obtained from 4,5-dihydro-1,2,4-oxadiazole

by using n-BuLi (4 eq.) (Scheme 5-13). We found that in the case of HX =NH2,

monoaddition of n-butyllithium takes place at C(5) by the ANRORC mechanism giving

quinazolines (5.77h,i) (Scheme 5.19); moreover, this methodology can be expanded for

the preparation of a variety of benzoxazines (5.77a,d) (HX=HO) and benzothiazines

(5.77c) (HX=HS). The reaction proceeds through a lithium enolate transition state that

activates the 1,2,4-oxadiazole ring at N(4) (Scheme 5-19). This activation favors the

106

monoaddition of the n-butyl nucleophile at C(5). The monoaddition products 5.76b,f (for

HX=OH) can be isolated if 2 equivalents of n-BuLi are used (Scheme 5-20, Table 5-3).

Scheme 5-19. Novel rearrangements of 1,2,4-oxadiazoles.

Grignard reagents do not open the 1,2,4-oxadiazole ring; this suggests that

lithium enolates favor the transition state.

Scheme 5-20. Ring fragmentation of 1,2,4 oxadiazoles 5.63a-i.

Table 5-3. Ring fragmentation products for 1,2,4-oxadiazoles 5.63a-i.

Entry 5.63, Conditions Products, Yield (%)

1

5.63a

4 eq. n-BuLi THF

-78oC to r.t. 40 min

5.77a

40

107

Table 5-3 Continued

Entry 5.63, Conditions Products, Yield (%)

2

5.63b

2 eq. n-BuLi THF

-78oC to r.t. 40 min

5.76b

67

3

5.63c

4 eq. n-BuLi THF

-78oC to r.t. 40 min

5.77c

20

4

5.63d

4 eq. n-BuLi THF

-78oC to r.t. 40 min

5.77d

45

5

5.63e

4 eq. n-BuLi THF

-78oC to r.t. 40 min

Decomposition

-

6

5.63f

2 eq. n-BuLi THF

-78oC to r.t. 40 min

5.76f

72

7

5.63g

2 eq. n-BuLi THF

-78oC to r.t. 40 min

Decomposition

-

8

5.63h

4 eq. n-BuLi THF

-78oC to r.t. 40 min

5.77h

75

9

5.63i

4 eq. n-BuLi THF

-78oC to r.t. 40 min

5.77i

25

Compound 5.77a is the same as 5.62a previously reported by Srivastava et al.

108

5.2.3 Rearrangement Results

Srivastava et al. showed by computational methods, such as PM3, AM1 and HF/6-

31G, that 5.63a exists as two coplanar rotamers 5.63a(A) and 5.63a(B), the hydrogen

atom of the –OH group (H9) being in the close proximity of O(1) from 1,2,4-oxadiazole

ring. The distance between O(1) and H(9) has been found to be 1.90 Å, suggesting

hydrogen bonding. Moreover, the rotational barrier of the phenyl ring at C(3) is 22.05

kJ/mol, whereas the rotational barrier of the 2-hydroxyphenyl group is 49.67 kJ/mol,

indicating that the hydrogen bond with N(4) is more stable. Computational results show

that 5.63a(B) is 13.98 kJ/mol more stable than 5.63a(A) (Scheme 5-21) [2003JMS49].

Scheme 5-21. Possible rotamers of 2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenol (5.61a).

Similar results were observed in our case; the 13C NMR in DMSO-d6 at 120oC of

an analytically pure sample of 5.63g displays extra aromatic peaks while 1H NMR

shows broad signals. Further investigation in solution was limited due to low solubility of

5.63g in organic solvents (the NMR spectra were recorded in DMSO-d6 at 130 oC).

Srivastava’s computational results partially explain the 1,2,4-oxadiazole

rearrangement in the case of n-BuLi as nucleophile. The radius of Li+ is 90 pm while the

radius of Mg2+ is 150 pm. The lithium cation does stabilize the six member ring transition

state (5.78) better than Mg2+ concomitantly with the activation of C(5) for the nucleophile

109

attack. The activated 1,2,4-oxadiazole ring can now initiate the ANRORC cascade

(Scheme 5-22).

Scheme 5-22. 1,2,4-Oxadiazole rearrangements in the presence of n-butyllithium.

Srivastava suggested initially the mechanism for the O- pivotal nucleophile 5.63a

when the 5.77a was isolated and characterized. Our results expand the utility of the

method for S- and N- pivotal nucleophiles. The reaction follows the Srivastava pathway

for S- pivotal, where the corresponding benzothiazines are formed. In the case of N-

110

pivotal, the rearrangement gives quinazolines. The rearranged products can be isolated

after a slow quenching with CO2.

5.3 Conclusions

Heterocyclic rearrangements are usually carried out under harsh thermal

conditions or photochemical irradiation. In this study, the 1,2,4-oxadiazoles with pivotal

nucleophile at position C(5) were activated using n-BuLi at low temperature.

It is worth mentioning that activation with Grignard reagents, such as methyl

magnesium chloride, did not produce the expected rearranged products. This reaction

was investigated at -78oC, r.t. and reflux in THF. In all of these cases, the starting

material was completely recovered.

1-(3-Phenyl-1,2,4-oxadiazol-5-yl)naphthalen-2-ol (5.63e) did not give the

corresponding rearranged product; this can be explained by the steric hindrance

generated by naphthyl fragment at C(5). However, its isomer 3-(3-phenyl-1,2,4-

oxadiazol-5-yl)naphthalen-2-ol (5.63d) gave the corresponding rearranged product

(5.77d).

The treatment of 1,2,4-oxadiazoles 5.63a,f with 2 equivalents of n-BuLi generated

the corresponding monoaddition products 5.77b,f. This suggests that the

rearrangement takes place via the ANRORC mechanism.

2-(3-(4-Nitrophenyl)-1,2,4-oxadiazol-5-yl)phenol (5.63g) gave mostly

decomposition in the reaction with n-BuLi at -78 oC. This particular reaction was also

investigated at -94 oC (methanol: liquid nitrogen, 50:50) but no reaction took place.

When warmed up to -78 oC, decomposition occurred prior to the rearrangement. This

can be explained by the presence of an electron withdrawing group (4-NO2-C6H4) that

activates position C(3) of the oxadiazole ring.

111

5.4 Experimental Section

Melting points were determined on a capillary point apparatus equipped with a

digital thermometer and are uncorrected. The 1H and 13C NMR spectra were recorded

on a Varian Gemini instrument, operating at 300 MHz for 1H and 75 MHz for 13C with

TMS as internal standard. The chemical shifts δ are given in ppm. Elemental analyses

were performed on a Carlo Erba-1106 instrument.

5.4.1 General procedure for the preparation of N-Acylbenzotriazoles (5.64a-e)

Thionyl chloride (20 mmol) was added to a solution of benzotriazole (80 mmol) in

THF (30 mL) and the reaction mixture was stirred under argon flow for 45 min. The

corresponding hydroxyacid (20 mmol) was then added portionwise, and the resulting

reaction mixture was stirred for an additional 2 h; the formed precipitate was then

filtered off and the solvent was removed under reduced pressure giving N-

acylbenzotriazoles 5.64a,c-e. The preparation of 5.64b was similar to that of 5.64a,c-e;

thionyl chloride (40mmol) was added to a solution of benzotriazole (160 mmol) in THF

(30mL), then 2,2'-disulfanediyldibenzoic acid (20 mmol) was added portionwise; the

resulting reaction mixture was stirred for 4 h.

1H-Benzo[d][1,2,3]triazol-1-yl)(2-hydroxyphenyl)methanone (5.64a). White

microcrystals (80%); m.p. 113.0 – 114.0 oC (Lit. m.p. 115.0 – 116.0 oC [2006JOC3364]);

1H NMR (300 MHz, CDCl3) δ 10.8 (s, 1H), 8.61 (ddd, J = 8.4, 1.6, 0.4 Hz, 1H), 8.33

(ddd, J = 8.3, 1.0, 1.0 Hz, 1H), 8.19 (ddd, J = 8.2, 1.0, 1.0 Hz, 1H), 7.72 (ddd, J = 8.3,

7.2, 1.2 Hz, 1H), 7.62 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.57 (ddd, J = 8.2, 7.2, 1.0 Hz,

1H), 7.14 (ddd, J = 8.5, 1.2, 0.4 Hz, 1H), 7.06 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H). 13C NMR

(75 MHz, CDCl3) δ 169.4, 163.8, 145.7, 137.4, 134.1, 132.7, 130.8, 126.7, 120.6, 119.9,

118.6, 115.1, 113.7.

112

Disulfanediylbis(2,1-phenylene))bis((1H-benzo[d][1,2,3]triazol-1-yl)methanone

(5.64b) Yellow microcrystals (68%), m.p. 153.0 – 154.0 oC; 1H NMR (300 MHz, CDCl3)

δ 8.38 (ddd, J = 8.3, 1.0, 1.0 Hz, 2H), 8.15 (ddd, J = 8.2, 1.0, 1.0 Hz, 2H), 7.88 – 7.83

(m, 4H), 7.73 (ddd, J = 8.2, 1.0, 1.0 Hz, 2H), 7.57 (ddd, J = 8.0, 7.2, 1.0 Hz, 2H), 7.54 –

7.50 (m, 2H), 7.39 (td, J = 7.5, 1.1 Hz, 2H). 13C NMR (75 MHz, CDCl3) δ 166.3, 146.3,

139.2, 133.2, 132.0, 131.9, 130.9, 129.3, 127.0, 126.8, 120.5, 114.9. Anal. Calcd. For

C26H16N6O2S2(508.58) required: C, 61.40; H, 3.17; N, 16.52. Found: C, 61.14; H, 3.06;

N, 16.21.

(1H-Benzo[d][1,2,3]triazol -1-yl)(3-hydroxynaphthalen-2-yl)methanone (5.64c).

Orange microcrystals (73%); m.p. 156.0 -158.0 oC (Lit m.p. 157.0 – 158.0 oC

[2006JOC3364]); 1H NMR (300 MHz, CDCl3) δ 9.94 (s, 1H), 9.15 (s, 1H), 8.36 (d, J =

7.8 Hz, 1H), 8.21 (d, J = 7.7 Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.74- 7.72 (m, 2H), 7.59 –

7.57 (m, 2H), 7.43 (s, 1H), 7.37 (t, J = 7.2 Hz, 1H). 13C NMR (75MHz, CDCl3) δ 169.1,

156.8, 145.7, 138.2, 137.8, 132.6, 130.9, 130.5, 130.4, 127.2, 126.8, 126.4, 120.6,

115.6, 115.1, 112.7. Anal. Calcd for C17H11N3O2 (289.20) required: C, 70.58; H, 3.83; N,

14.52. Found: C, 70.17; H, 3.87; N, 14.41.

(1H-Benzo[d][1,2,3]triazol-1-yl)(2-hydroxynaphthalen-1-yl)methanone (5.64d).

White microcrystals (80%); m.p. 140.0 – 141.0 oC (Lit m.p 138.0 – 140.0 oC

[2006JOC3364]); 1H NMR (300 MHz, CDCl3) δ 10.67 (s, 1H), 8.41 (d, J = 8.2 Hz, 1H),

8.29 (d, J = 8.3 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.88 (ddd, J =

8.1, 7.3, 0.8 Hz, 1H), 7.69 (ddd, J = 8.3, 7.5, 0.8 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.48

(ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 7.40 (ddd, J = 8.0, 7.1, 1.0 Hz, 1H), 7.33 (d, J = 9.1 Hz,

113

1H). 13C NMR (75 MHz, CDCl3) δ 167.1, 154.0, 145.6, 132.8, 131.3, 131.0, 130.6,

128.4, 128.0, 127.3, 126.8, 123.6, 122.6, 120.1, 118.0, 114.0, 113.7.

1-Benzoyl-1H-benzotriazole (5.64e). Colorless microcrystals. m.p. 110.0 -111.0 oC

(Lit m.p 112.0 -113.0 oC [2000JOC3679]); 1H NMR (300 MHz, CDCl3) δ 8.40 (ddd, J =

8.3, 1.0, 1.0 Hz, 1H), 8.25 -8.21 (m, 2H), 8.18 (ddd, J = 8.4, 1.0, 1.0 Hz, 1H), 7.74 -7.58

(m, 2 H), 7.56 – 7.53 (m, 3H). 13C NMR (75 MHz, CDCl3) δ 116.9, 145.9, 133.8, 132.5,

131.9, 131.6, 130.5, 128.5, 126.5, 120.3, 114.9.

5.4.2 Synthesis of N-Hydroxybenzimidamide (5.65a-c)

Hydroxyl amine 50% wt (0.15 mol) was added to a solution of the corresponding

benzonitrile (0.1 mol) in ethanol (10 mL) and the mixture was heated under reflux for 8

h. After cooling, the solvent was removed in vacuo and the product was obtained in a

pure form after crystallization from ethanol.

(Z)-N’-hydroxybenzimidamide (5.65a). Colorless needles (67%); m.p. 69.0 – 71.0

oC (Lit. m.p. 70.0 – 71.0 oC [1986JMC2174]); 1H NMR (300 MHz, CDCl3) δ 9.16 (br s,

1H), 7.64 – 7.62 (m, 2H), 7.42 – 7.36 (m, 3H), 4.97 (br s, 2H). 13C NMR (75 MHz,

CDCl3) δ 152.8, 132.5, 130.0, 128.7, 126.0. Anal. Calcd. For C7H8N2O(136.15) required:

C, 61.75; H, 5.92; N, 20.58. Found C, 61.83; H, 5.94; N, 20.71.

(Z)-N’-hydroxy-4-methylbenzimidamide (5.65b). Colorless crystals (98%); m.p.

142.0 – 143.0 oC (Lit. m.p. 147.0 [1954JCS4251]) 1H NMR (300 MHz, CDCl3) δ 7.52 (d,

J = 6.4 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 4.88 (s, 2H), 2.37 (s, 3H). 13C NMR (75 MHz,

CDCl3) δ 152.8, 140.2, 129.8, 129.5, 125.9, 21.6. Anal. Calcd. for C8H10N2O (150.08)

required: C, 63.98; H, 6.71; N, 18.65. Found: C, 64.06; H, 6.78; N, 18.68.

(Z)-N’-hydroxy-4-nitrobenzimidamide (5.65c). Yellow microcrystals (91%); m.p.

183.0 – 185.0 oC 1H NMR (300 MHz, CDCl3) δ 10.13 (s, 1H), 8.23 (d, J = 9.1 Hz, 2H),

114

7.95 (d, J = 9.1 Hz, 2H), 6.07 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 149.3, 147.4, 139.5,

126.3, 123.3. Anal Calcd. for C7H7N3O3(181.15) required: C, 46.41; H, 3.89; N, 23.20.

Found C, 46.61; H, 3.70; N, 22.58.

5.4.3 Preparation of 1,2,4-Oxadiazoles (5.63a-f)

A mixture of (Z)-N’-hydroxybenzimidamide derivative (10 mmol), N-

acylbenzotriazole (10 mmol), and triethylamine (20 mmol) in DMF (10 mL) was heated

under reflux for 6h. The reaction was cooled to r.t., the solvent was then removed under

reduced pressure giving a brown residue which was then recrystallized from ethanol to

giving the products {5.63a,b,d-f}.

2-(3-Phenyl-1,2,4-oxadiazol-5-yl)phenol (5.63a). Colorless needles (76%); m.p.

159.0 – 160.0 oC (lit. m.p 160.0 -161.0 oC [1999ICA1]); 1H NMR (300 MHz, CDCl3) δ

10.61 (s, 1H), 8.11 (dd, J = 7.9, 1.8 Hz, 2H), 8.01 (dd, J = 7.9, 1.4 Hz, 1H), 7.62-7.60

(m, 3H), 7.54 (ddd, J = 8.7, 7.1, 1.5 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 7.05 (t, J = 7.6 Hz,

1H). 13C NMR (75 MHz, CDCl3) δ 175.0, 167.2, 157.2, 134.8, 131.6, 130.0, 129.2,

127.2, 126.1, 119.7, 117.4, 109.8. Anal. Calcd. For C14H10N2O2(238.25) required: C,

70.58; H, 4.23; N, 11.76. Found C, 70.47; H, 4.17; N, 11.86.

2-(3-(p-Tolyl)-1,2,4-oxadiazol-5-yl)phenol (5.63b). Colorless needles (88%); m.p.

160.0 -162.0 oC; 1H NMR (300 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.02 (d, J = 8.1 Hz,

2H), 8.02 – 8.00 (m, 1H), 7.52 (ddd, J = 8.7, 7.3, 1.8 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H),

7.15 (dd, J = 8.5, 0.9 Hz, 1H), 7.04 (ddd, J = 8.1, 7.3, 1.1 Hz, 1H), 2.44 (s, 3H). 13C-

NMR (75 MHz, DMSO-d6) δ 174.3, 167.3, 158.3, 142.3, 135.4, 129.9, 128.0, 127.7,

123.1, 120.3, 118.0, 108.4, 21.9 . Anal. Calcd. For C15H12N2O2 (252.28) required: C,

71.41; H, 4.79; N, 11.10. Found C, 71.18; H, 4.72; N, 11.49.

115

2-(3-Phenyl-1,2,4-oxadiazol-5-yl)benzenethiol (5.63c) was prepared from 1,2-

bis(2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenyl)disulfane (5.86c) by using NaBH4 (1. eq.) in

THF (20 mL) for 4h at r.t. (Scheme 5-23) The product was purified by column

chromatography using Hexanes:EtOAc (5:1) to give 2-(3-Phenyl-1,2,4-oxadiazol-5-

yl)benzenthiol (5.63c).

Scheme 5-23. Preparation of 2-(3-phenyl-1,2,4-oxadiazol-5-yl)benzenthiol (5.63c).

1,2-Bis(2-(3-phenyl-1,2,4-oxadiazol-5-yl)phenyl)disulfane (5.86c) was prepared

according to the procedure for {5.63a,b,d-f}. Yellow microcrystals (90%); m.p 182.0 –

184.0 oC; 1H NMR (300 MHz, DMSO-d6) δ 8.25 (d, J =7.6 Hz, 2H), 8.23 -8.15 (m, 4H),

7.90 (d, J = 8.1 Hz, 2H), 7.70 (t, J = 7.7 Hz, 2 H), 7.65-7.63 (m, 6H), 7.54 (t, J = 7.7 Hz,

2H); 13C NMR (75 MHz, DMSO-d6) δ 173.6, 167.9, 136.7, 133.7, 131.9, 130.8, 129.4,

127.3, 127.2, 126.7, 125.8, 121.1. Anal. Calcd. for C28H18N4O2S2 (506.61) required: C,

66.38; H, 3.58; N, 11.06. Found: C, 66.01; H, 3.55; N, 10.94.

2-(3-Phenyl-1,2,4-oxadiazol-5-yl)benzenethiol (5.63c). Yellow microcrystals (70%);

m.p. 121.0 – 122.0 oC; 1H NMR (300 MHz, CDCl3) δ 8.19 – 8.17 (m, 3H), 7.55 – 7.53

(m, 5H), 7.40 (t, J = 7.9 Hz, 1H), 7.28 (t, J = 7.9 Hz, 1H), 6.62 (br s, 1H); 13C NMR (75

MHz, CDCl3) δ 175.0, 168.3, 136.9, 132.3, 131.5, 130.8, 130.1, 129.1, 127.7, 126.7,

125.2, 120.3.

116

3-(3-Phenyl-1,2,4-oxadiazol-5-yl)naphthalen-2-ol (5.63d). White needless (82%);

m.p. 238.0 – 239.0 oC; 1H NMR (300 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.73 (s, 1H), 8.16

– 8.15 (m, 2H), 8.05 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.65 – 7.63 (m, 3H),

7.57 (t, J = 7.5 Hz, 1H), 7.46 (s, 1H), 7.41 (t, J = 7.5 Hz, 1H); 13C NMR (75 MHz,

DMSO-d6) δ 175.0, 167.5, 153.0, 136.5, 132.0, 131.7, 129.3, 128.9, 127.2, 127.0,

126.1, 126.0, 124.1, 112.8, 111.0. Anal. Calcd. for C18H12N2O2 (288.31) required: C,

74.99; H, 4.20; N, 9.72. Found: C, 74.82; H, 4.12; N, 9.92.

1-(3-Phenyl-1,2,4-oxadiazol-5-yl)naphthalen-2-ol (5.63e). White needles (79%);

m.p. 145.0-146.0 oC; 1H NMR (300 MHz, CDCl3) δ 11.11(s, 1H), 8.17 – 8.15 (m, 2H),

8.12 (d, J = 9.0 Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.66 – 7.60

(m, 3H), 7.58 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.44 (ddd, J = 8.0, 7.0, 0.9 Hz, 1H), 7.38

(d, J = 9.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 174.6, 167.5, 156.9, 134.5, 131.7,

131.6, 129.3, 128.6, 128.4, 127.5, 127.2, 126.2, 123.8, 123.1, 118.3, 102.9. Anal.

Calcd. for C18H12N2O2 (288.31) required: C, 74.99; H, 4.20; N, 9.79. Found: C, 74.59; H,

4.11; N, 9.79.

3,5-Diphenyl-1,2,4-oxadiazole (5.63f). White microcrystals (86%); m.p. 111.0 –

112.0 oC; 1H NMR (300 MHz, CDCl3) δ 8.19 – 8.07 (m, 4H), 7.73 – 7.60 (m, 6H); 13C

NMR (75 MHz, CDCl3) δ, 175.4, 168.2, 133.3, 131.6, 129.5, 129.2, 127.9, 127.1, 126.1,

123.3. Anal. Calcd. for C14H10N2O (222.25) required: C, 75.66; H, 4.54; N, 12.60.

Found: C, 75.63; H, 4.51; N, 12.94.

2-(3-(4-Nitrophenyl)-1,2,4-oxadiazol-5-yl)phenol (5.63g). Yellow microcrystals

(81%); m.p. 248.0 – 250.0 oC; 1H NMR (500 MHz, DMSO-d6) δ, 10.70 (s, 1H), 8.43 (d, J

= 8.9 Hz, 2H), 8.36 (d, J = 8.9 Hz, 2H), 8.03 (dd, J = 8.0, 1.6 Hz, 1H), 7.55 (ddd, J = 8.8,

117

7.4, 1.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H); 13C NMR (125 MHz,

DMSO-d6) δ, 176.3, 166.9, 158.1, 135.5, 132.8, 130.7, 130.5, 129.5, 129.4, 129.3,

125.1, 125.0, 124.9, 120.9, 120.3, 118.1, 117.9, 110.5. Anal. Calcd. for C14H9N3O4

(283.25) required: C, 59.37; H, 3.20; N, 14.82. Found: C, 59.53; H, 3.11; N, 15.07.

5.4.4 Preparation of 1,2,4-Oxadiazoles (5.63h,i)

A mixture of (Z)-N’-hydroxybenzimidamide derivative (10 mmol), isatoic anhydride

(10 mmol) and triethylamine (20 mmol) in DMF (10 mL) was heated under reflux for 6h.

The reaction was cooled to r.t. and the solvent was then removed under reduced

pressure to obtain a brown residue. The product was then recrystallized from ethanol to

obtain (5.63h), respectively (5.63i).

2-(3-Phenyl-1,2,4-oxadiazol-5-yl)aniline (5.63h). Brown microcrystals (65%); m.p.

130.0 – 132.0 oC (Lit. m.p. [1984JHC949] 130.0 -132.0 oC) 1H NMR (500 MHz, DMSO-

d6) δ 8.19 -8.13 (m, 2H), 7.85 (d, J = 8.2 Hz, 1H), 7.62 (br s, 3H), 7.36 (t, J = 7.6 Hz,

1H), 7.01 (s, 2H), 6.99 (t, J = 8.5 Hz, 1H), 6.70 (t, J = 7.5 Hz, 1H). 13C NMR (125 MHz,

DMSO-d6) δ 174.7, 167.2, 148.8, 134.0, 131.6, 129.2, 128.4, 127.2, 126.2, 116.6,

115.6, 103.5.

2-(3-(p-Tolyl)-1,2,4-oxadiazol-5-yl)aniline (5.63i). Brown crystals (62%); m.p. 148.0

– 150.0 oC (Lit. mp 152.0 – 153.0 oC [1979H239]); 1H NMR (300 MHz, DMSO-d6) δ

8.04 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.34 (t, J =

7.6 Hz, 1H), 6.99 (s, 2H), 6.95 (d, J = 8.6 Hz, 1H), 6.69 (t, J = 7.5 Hz, 1H), 2.40 (s, 3H);

13C NMR (75 MHz, DMSO-d6) δ 174.5, 167.2, 148.8, 141.5, 134.0, 129.7, 128.4, 127.1,

123.4, 116.5, 115.6, 103.5, 21.1.

118

5.4.5 Synthesis and characterization data of the addition products 5.76 and rearranged products 5.77

The corresponding 1,2,4-oxadiazole derivative (0.5 mmol) was dissolved in THF (2

mL). The resulting solution was cooled to -78oC, followed by the dropwise addition of n-

butyllithium; the reaction mixture was then stirred for an additional 30 min at -78 oC,

after which it was allowed to warm up to r.t. . The reaction was monitored by TLC and

was completed within 2 h.

n-Butyllithium was added as follows: 1 mmol of n-butyllithium (1.6 M in hexanes)

for the preparation of monoaddition products {5.76b,f}, and 2 mmol of n-butyllithium (1.6

M in hexanes) for the preparation of rearranged products {5.77a,c,d,h,i}.

The monoaddition products 5.76b,f were purified by column chromatography

EtOAc:Hexanes (1:5). The rearranged products {5.77a,c,d,h,i} were purified by column

chromatography EtOAc:Hexanes (1:10).

4,4-Dibutyl-2-phenyl-4H-benzo[e][1,3]oxazine (5.77a). Colorless oil (40%); 1H

NMR (300 MHz, CDCl3) δ 8.10 (dd, J = 7.5, 1.7 Hz, 2H), 7.48 – 7.42 (m, 3H), 7.21 –

7.18 (m, 1H), 7.15 – 7.12 (m, 1H), 7.12 – 7.10 (m, 1H), 7.00 (dt, J = 7.8, 0.8 Hz, 1H),

1.87 – 1.81 (m, 4H), 1.27 – 1.16 (m, 7H), 0.99 – 0.85 (m, 2H), 0.78 (t, J = 7.0 Hz, 6H).

13C NMR (75 MHz, CDCl3) δ 149.7, 132.8, 130.8, 128.9, 128.3, 127.6, 127.5, 125.7,

124.7, 124.6, 115.3, 59.3, 44.5, 26.8, 23.1, 14.2, 14.1. Anal. Calcd. For C22H27NO

(321.46) required: C, 82.20; H, 8.47; N, 4.36. Found C, 81.95; H, 8.83; N, 4.21.

2-(5-Butyl-3-(p-tolyl)-4,5-dihydro-1,2,4-oxadiazol-5-yl)phenol (5.76b). Colorless

microcrystals (67%); m.p. 166.0 – 168.0 oC; 1H NMR (300 MHz, CDCl3) δ 7.57 (d, J =

8.1 Hz, 2H), 7.39 (dd, J = 7.9, 1.4 Hz, 1H), 7.19 (d, J = 8.1 Hz, 2H), 6.92 (t, J = 7.5 Hz,

1H), 6.85 (d, J =8.1 Hz, 1H), 5.44 (s, 1H), 2.37 (s, 3H), 2.20 – 2.16 (m, 2H), 1.55 – 1.28

119

(m, 4H), 0.88 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 156.5, 153.3, 141.6,

129.7, 129.6, 128.0, 126.6, 126.5, 122.5, 120.5, 117.3, 101.3, 40.3, 25.8, 22.8, 21.7,

14.2. Anal. Calcd. For C19H22N2O2 (310.39) required: C, 73.52; H, 7.14; N, 9.02. Found

C, 73.34; H, 7.08; N, 9.11.

4,4-Dibutyl-2-phenyl-4H-benzo[e][1,3]thiazine (5.77c). Yellow oil (20%). 1H NMR

(300 MHz, CD2Cl2) δ 8.08 – 8.05 (m, 2H), 8.03 – 7.51 (m, 4H), 7.49 – 7.20 (m, 3H),

5.80 (t, J = 7.6 Hz, 1H), 2.57 (q, J = 7.4 Hz, 2H), 1.56 (sx, J = 7.5 Hz, 4H), 0.97 (t, J =

7.4 Hz, 6H). 13C NMR (75 MHz CD2Cl2) δ 141.2, 138.1, 131.7, 129.6, 129.1, 128.7,

128.3, 127.7, 127.2, 126.8, 125.5, 40.7, 29.4, 23.7, 23.5, 14.3. HRMS (MALDI-TOF) m/z

calculated for C22H27NS [M+H]+ 338.1898. Found 338.1903.

4,4-Dibutyl-2-phenyl-4H-naphtho[2,3-e][1,3]oxazine (5.77d). Brown microcrystals

(50%); m.p. 76.0 – 77.0 oC;1H NMR (300 MHz, CDCl3) δ 8.17 – 8.14(m, 2H), 7.80 –

7.43 (m, 2H), 7.40 (s, 1H), 7.37 -7.22 (m, 7H), 2.00 – 1.92 (m, 4H), 1.32 – 1.17 (m, 6H),

1.15 – 0.93 (m, 2H), 0.72 (t, J = 3.8 Hz, 6H). 13C NMR (75MHz, CDCl3) δ 150.4, 148.2,

133.1, 132.9, 131.4, 130.8, 128.3, 127.9, 127.6, 127.1, 126.6, 126.3, 124.8, 124.7,

110.7, 59.7, 45.1, 26.7, 23.1, 14.2. Anal. Calcd. For C26H29NO (371.52) required: C,

84.05; H, 7.87; N, 3.77. Found C, 84.35; H, 8.17; N, 3.45.

5-Butyl-3,5-diphenyl-4,5-dihydro-1,2,4-oxadiazole (5.76f). Colorless microcrystals

(72%); m.p. 121.0-122.0 oC (Lit. m.p. 122.0-124.0 oC [2000H191]); 1H NMR (300 MHz,

CDCl3) δ 7.70(br d, J = 6.5 Hz, 2H), 7.56 (br d, J = 7.6 Hz, 2H), 7.41-7.31 (m, 6H), 4.75

(m, 1H), 2.16 – 2.13 (m, 2H), 1.56 – 1.36 (m, 4H), 0.91 (t, J = 6.5 Hz, 3H). 13C NMR (75

MHz, CDCl3) δ 155.1, 143.5, 130.9, 128.8, 128.7, 128.3, 126.6, 126.0, 124.9, 100.4,

40.6, 25.8, 22.9, 14.1.

120

4-Butyl-2-(p-tolyl)quinazoline (5.77h). Green microcrystals (75%); m.p. 73.0 – 74.0

oC; 1H NMR (300 MHz, CD2Cl2) δ 8.44 (d, J = 8.2 Hz, 2H), 8.04 (ddt, J = 8.4, 1.4, 0.7

Hz, 1H), 7.92 (ddt, J = 8.4, 1.2, 0.6 Hz, 1H), 7.75 (ddd, J = 8.5, 7.0, 1,6 Hz, 1H), 7.47

(ddd, J = 8.1, 6.8, 1,1 Hz, 1H), 7.24 (d, J = 8.2 Hz, 2H), 3.25 – 3.20 (m, 2H), 2.35 (s,

3H), 1.91 – 1.81 (m, 2H), 1.44 (sx, J = 7.4 Hz, 2H), 0.93 (t, J = 7.5 Hz, 3H). 13C NMR

(75 MHz, CD2Cl2) δ 172.0, 151.2, 141.2, 136.3, 133.8, 129.7, 129.6, 128.9, 127.0,

125.3, 123.0, 34.8, 31.2, 23.3, 21.8, 14.3. Anal.Calcd. for C19H20N2 (276.38) required: C,

82.57; H, 7.29; N, 10.14. Found C, 83.39; H, 7.40; N, 9.94.

4-Butyl-2-phenylquinazoline (5.77i). Yellow oil (25%); 1H NMR (300 MHz, CD2Cl2)

δ, 8.57 – 8.54 (m, 2H), 8.07 (ddd, J = 8.3, 1.4, 0.7 Hz, 1H), 7.96 (ddd, J = 8.3, 1.4, 0.7

Hz, 1H), 7.78 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.51 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.46 –

7.29 (m, 3H), 3.26 (t, J = 7.9 Hz, 2H), 1.91 – 1.81 (m, 2H), 1.46 (sx, J = 7.3 Hz, 4H),

0.94 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CD2Cl2) δ, 172.2, 160.2, 151.2, 139.0,

133.9, 130.8, 129.7, 129.0, 127.3, 125.3, 123.1, 34.8, 31.2, 23.3, 14.4. HRMS (MALDI-

TOF) m/z calculated for C18H18N2 [M+H]+ 263.1543. Found 263.1557.

121

CHAPTER 6 FINAL CONCLUSIONS AND ACHIEVEMENTS

This thesis is divided into four distinct parts; Chapter 2 investigates the

tautomerism of some N-(α-aminoalkyl)tetrazoles, Chapter 3 describes the preparation of

different aminoxyacyl conjugates by using the benzotriazole methodology developed by

our group, Chapter 4 presents some NMR correlations of a variety of molecular

structures, and the Chapter 5 displays a new preparative method of quinazolines and

4H-benzothiazines via 1,2,4-oxadiazoles rearrangements.

The general design, structures, synthesis, and interconversion mechanism of

some N-(α-aminoalkyl)tetrazole tautomers are described in Chapter 2. Molecular

structure and solvent polarity influences the equilibrium position and the population

ratio. The existence of two tautomeric structures was confirmed by 1H NMR. We have

used 1H-15N CIGAR gHMBC to discriminate between the two possible tautomers. We

found that polar solvents favor the N-1 tautomer while steric effect favors the N-2

tautomer. These results are in agreement with previous studies of N-(α-

dialkylaminomethyl)benzotriazoles.

N-(α-Aminoalkyl)tetrazoles found applications as pharmacophores; they have

been used as modified protein-formation inhibitors, used in prevention and treatment of

diseases associated with diabetes, hypertension and Meniere’s disease.

I synthesized some N-( α-aminoalkyl)tetrazoles and studied their tautomeric

behavior by NMR. The equilibrium between the N-1 and N-2 tautomers of N-( α-

aminoalkyl)tetrazoles is influenced by solvent polarity and substitution. Non-polar

solvents favor the N-2 tautomer; polar solvents favor the N-1 tautomer. Bulky

substituents in the 5-position of the tetrazole ring favor the N-2 tautomer.

122

Many important aspects of the benzotriazole chemistry have been explored over

the last 30 years in the Katritzky Group. My graduate studies aimed to further apply the

benzotriazole methodology to the synthesis of different heterocyclic compounds with

potential biological properties. To summarize, Chapter 3 presents an efficient

methodology for the preparation of aminoxyacids conjugates. We have investigated the

reactivity of the corresponding N-acylbenzotriazole derivatives, such as N-Cbz-

protected(α-aminoxyacyl)benzotriazoles, with hindered nucleophiles (sterols and

terpenes) and nucleophiles with multiple nucleophilic centers (partially unprotected

sugars and nucleosides). In the case of unprotected sugars, the steric effect influences

the acylation position. The reaction is regiospecific at the least hindered nucleophilic site

for unprotected sugars, while in the case of nucleosides, the reaction is N-selective. We

found this protocol efficient, convenient and economically advantageous; most N-Cbz-

protected(α-aminoxyacyl)benzotriazoles are crystalline, bench stable and readily

available from non-expensive starting materials. The corresponding acylated products

were prepared in moderate to good yields. The acylation position was confirmed by 1H-

13C gHMBC experiment.

Chapter 4 presents the NMR characterization of a variety of molecular structures

including a protected acylated amino sugar, some pyridazines and a nitrated furan.

These compounds were characterized by 1H, 13C, 15N NMR correlation experiments

including 1H-1H COSY, 1H-13C gHMBC, 1H-13C gHMQC and 1H-15N CIGAR –gHMBC.

Chapter 5 gives an overview of thermal and photochemical transformations of

some 1,2,4-oxadiazoles and describes a new approach to quinazolines and 1,3-

benzothiazines via 1,2,4-oxadiazoles rearrangements. These reactions take place by a

123

modified version of ANRORC (Addition of a Nucleophile Ring Opening Ring Closure)

mechanism, in which we have used n-BuLi as base and as nucleophile to generate the

corresponding rearranged products. The reaction mechanism is described in detail

within the Chapter 5; for structure designations, see the results and discussion section.

By this work, I was able to extend the rich chemistry of 1,2,4-oxadiazoles

rearrangements.

124

LIST OF REFERENCES

The reference citation system employed throughout this research report is from

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number indicating the year followed by the letter code of the journal and the page

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Additional notes to this reference system are as follows:

1) Each reference code is followed by conventional literature citation in the ACS

style.

2) Journals which are published in more than one part including in the abbreviation

cited the appropriate part.

3) Less commonly used books and journals are still abbreviated as using initials of

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4) Patents are given by their application number.

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140

BIOGRAPHICAL SKETCH

Bogdan Draghici was born in September 1982, in Bucharest Romania. Bogdan

attended the University “Politehnica” of Bucharest where he received his BS in 2006.

During this time, Bogdan had worked under the supervision of prof. Florea Dumitrascu

at Center of Organic Chemistry in Bucharest, where he was involved in the synthesis

and NMR characterization of various molecular structures. Upon graduation he joined

University of Florida as an adjunct assistant in the chemistry under the supervision of

Prof. Alan R. Katritzky. Since August 2008 he had joined the graduate program at the

University of Florida under the supervision of Prof. Alan R. Katritzky. His research

interest focuses on the synthesis and NMR characterization of various heterocyclic

systems.