SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS

SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS By BAHAA EL-DIEN M. EL-GENDY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF ...
Author: Guest
3 downloads 0 Views 5MB Size
SYNTHESIS AND NMR STUDIES OF HETEROCYCLIC SYSTEMS

By BAHAA EL-DIEN M. EL-GENDY

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1

© 2010 Bahaa El-Dien M. El-Gendy

2

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

3

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

4

TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF SCHEMES......................................................................................................................13 LIST OF ABBREVIATIONS ........................................................................................................14 ABSTRACT ...................................................................................................................................18 CHAPTER 1

GENERAL INTRODUCTION ..............................................................................................20

2

(-AMINOACYL)AMINO-SUBSTITUTED HETEROCYCLES AND RELATED COMPOUNDS .......................................................................................................................23 2.1 Introduction.......................................................................................................................23 2.2 Results and Discussion .....................................................................................................25 2.2.1 Preparation of Acylamino-Thiazoles, -Benzothiazoles, -Benzimidazoles and Thiadiazoles .................................................................................................................25 2.2.2 Acylation of Aminopyrimidones ............................................................................28 2.2.3 Acylation of Aminopyrazoles.................................................................................28 2.2.4 Acylation of Aminopyridines .................................................................................29 2.3 Conclusions.......................................................................................................................32 2.4 Experimental .....................................................................................................................32 2.4.1 General Methods ....................................................................................................32 2.4.2 General Procedure for the Preparation of 2.6a-j, 2.6a'-c' .......................................32 2.4.3 General Procedure for the Preparation of N-Substituted Amides 2.8a-d, 2.8a'c', 2.14a-b, 2.14a', 2.16a-b, 2.18a-g, 2.18d' and Dipeptide Amides 2.12a,b ................32

3

TAUTOMERISM OF 2-HYDRAZONO-3-PHENYLQUINAZOLIN-4(3H)-ONES STUDIED BY 15N NMR ........................................................................................................41 3.1 Introduction.......................................................................................................................41 3.2 Results and Discussion .....................................................................................................46 3.2.1 Syntheses ................................................................................................................46 3.2.2 Tautomerism and NMR ..........................................................................................48 3.2.3 Stereochemistry of the C=N Bonds ........................................................................57 3.3 Conclusions.......................................................................................................................58 3.4 Experimental .....................................................................................................................59 3.4.1 General Methods ....................................................................................................59 3.4.2 Preparation of 3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (3.21)..........60 3.4.3 Preparation of 2-hydrazino-3-phenylquinazolin-4(3H)-one (3.22) ........................60 3.4.4 General Procedure for Preparing Compounds 3.11a-i ...........................................61 3.4.5 Preparation of 4-[(2-(1H-benzimidazol-2-yl)hydrazono)methyl]-N,Ndimethylaniline (3.12) ..................................................................................................64 5

3.4.6 Preparation of 3-[2-(4,6-dimethylpyrimidin-2-yl)hydrazono]-1-methylindolin2-one (3.13) ..................................................................................................................64 3.4.7 Preparation of 2-(methylsulfanyl)-3-phenyl-4(3H)-quinazolinone (3.24) .............65 3.4.8 Preparation of 3-amino-2-anilino-4(3H)-quinazolinone (3.25) ..............................65 4

NMR STUDY OF THE TAUTOMERIC BEHAVIOUR OF N-(-AMINOALKYL)TETRAZOLES .......................................................................................................................66 4.1 Introduction.......................................................................................................................66 4.2 Results and Discussion .....................................................................................................69 4.2.1 Preparation of 5-Substituted Tetrazoles .................................................................69 4.2.2 Preparation of N-(α-Aminoalkyl)Tetrazoles ..........................................................70 4.2.3 NMR Characterization and Solvent Effects on Tautomeric Equilibrium of 1and 2-Substituted Tetrazoles ........................................................................................70 4.2.4 Thermodynamic and Kinetic Parameters ...............................................................75 4.2.5 Cross-Over Experiment ..........................................................................................78 4.3 Conclusions.......................................................................................................................81 4.4 Experimental .....................................................................................................................81 4.4.1 General Methods ....................................................................................................81 4.4.2 General Procedure for Preparation of Compound 6.4b (Method A) .....................82 4.4.3 General Procedure for Preparation of Compounds 4.9c and 4.9e (Method B) .....83 4.4.4 General Procedure for Preparation of Compounds 4.11a, 4.11c-h ........................84 4.4.5 Preparation of N-hydroxymethylsaccharin (4.12). .................................................88 4.4.6 Preparation of N-chloromethylsaccharin (4.13). ....................................................88 4.4.7 Preparation of N-((1H-tetrazol-1-yl)methyl)-1,2-benzisothiazole-3(2H)-one1,1-dioxide (4.11b).......................................................................................................88

5

CONFORMATIONAL EQUILIBRIA AND BARRIERS TO ROTATION IN SOME NOVEL NITROSO DERIVATIVES OF INDOLIZINES AND 3- AND 5AZAINDOLIZINES - AN NMR AND MOLECULAR MODELLING STUDY .................90 5.1 Introduction.......................................................................................................................90 5.2 Results and Disscussion ....................................................................................................93 5.2.1 NMR Spectroscopy ................................................................................................93 5.2.1.1 2,3-Dimethyl-1-nitrosoindolizine (5.1) ........................................................93 5.2.1.2 2,6,7-Trimethyl-5-nitrosopyrrolo[1,2-b]pyridazine (5.5) ............................95 5.2.1.3 Methyl 2-methyl-1-nitrosoindolizine-3-carboxylate (5.2) ...........................96 5.2.1.4 Ethyl 2-(methylamino)-1-nitrosoindolizine-3-carboxylate (5.3) .................97 5.2.1.5 4-Methoxy-3-nitrosopyrazolo[1,5-a]pyridine (5.6) ...................................100 5.2.1.6 2-Methyl-3-nitrosoindolizine (5.4).............................................................101 5.2.2 15N Chemical Shifts ..............................................................................................102 5.2.3 Barriers to Rotation ..............................................................................................103 5.2.4 Molecular Modelling ............................................................................................103 5.3 Conclusions.....................................................................................................................112 5.4 Experimental ...................................................................................................................114 5.4.1 General Methods ..................................................................................................114 5.4.2 Characterization of Compounds 5.1-5.6 ...............................................................115

6

6

1

H, 13C, AND 15N NMR SPECTRA OF SOME PYRIDAZINE DERIVATIVES ..............116

6.1 Introduction.....................................................................................................................116 6.2 Results and Discussion ...................................................................................................118 6.2.1 1H NMR Spectra ...................................................................................................118 6.2.2 13C NMR Spectra ..................................................................................................119 6.2.3 15N NMR Spectra .................................................................................................119 6.3 Conclusions.....................................................................................................................125 6.4 Experimental ...................................................................................................................126 6.4.1 General Methods ..................................................................................................126 6.4.2 Characterization of Compounds 6.7-6.20 .............................................................127 7

DIVERSE APPLICATIONS OF 15N NMR IN STRUCTURAL ANALYSIS ....................129 7.1 Introduction.....................................................................................................................129 7.1.1 Literature Background to Structural Elucidation by 15N NMR ............................130 7.1.2 Literature Background to Protonation Studies by 15N NMR................................131 7.2 Results and Discussion ...................................................................................................135 7.2.1 Structural Elucidation of S- and N-Acylcysteines ................................................135 7.2.1.1 Structural elucidation of S-(4-methoxybenzoyl)-L-cysteine (7.13a) ..........135 7.2.1.2 Structural elucidation of S-(2-naphthoyl)-L-cysteine (7.13b) ....................138 7.2.1.3 Structural elucidation of N-(4-methoxybenzoyl)-L-cysteine (7.14a) .........138 7.2.1.4 Structural elucidation of N-(2-naphthoyl)-L-cysteine (7.14b) ....................140 7.2.2 1H, 13C, and 15N NMR Chemical Shift Assignments and Protonation Study of pH-Sensitive GFP Chromophore Analogues .............................................................141 7.2.2.1 1H, 13C, and 15N NMR of 2-phenyl-4-(thiophen-2-ylmethylene)oxazol5(4H)-one (7.16a) ...............................................................................................143 7.2.2.2 1H, 13C, and 15N NMR and protonation study of 1-{2(dimethylamino)ethyl)-4-(5-methylfuran-2-yl)methylene}-2-phenyl-1Himidazol-5(4H)-one (7.17a) ................................................................................143 7.2.2.3 1H, 13C, and 15N NMR and protonation study of 4-{(1H-pyrrol-2-yl)methylene}-1-(2-dimethylaminoethyl)}-2-phenyl-1H-imidazol-5(4H)-one (7.17b) .................................................................................................................145 7.3 Conclusions.....................................................................................................................146 7.4 Experimental ...................................................................................................................148 7.4.1 General Methods ..................................................................................................148 7.4.2 Characterization of Compounds 7.13a,b, 7.14a,b, 7.16a. and 7.17a.b .................149

8

CONCLUSIONS AND SUMMARY OF ACHIEVEMENTS .............................................152

LIST OF REFERENCES .............................................................................................................155 BIOGRAPHICAL SKETCH .......................................................................................................174

7

LIST OF TABLES Table

page

2-1

Preparation of acylamino-thiazoles, -benzothiazoles, -benzimidazoles and thiadiazoles ........................................................................................................................27

2-2

Preparation of (acylamino)pyridines from N-acyl and N-(aminoacyl)-benzotriazoles......31

3-1

1

3-2

13

3-3

15

3-4

1

3-5

13

3-6

15

H chemical shifts (ppm) in compounds 3.11a-f, 3.12, 3.21, 3.22 and 3.24 .....................54 C chemical shifts (ppm) in compounds 3.11a-f, 3.12, 3.21, 3.22 and 3.24 ....................55

N chemical shifts (ppm) in compounds 3.11a-f, 3.12, 3.21, 3.22 and 3.24. Protons which couple to a 15N are given in parentheses .................................................................55 H chemical shifts (ppm) in compounds 3.11g-i and 3.13 .................................................56 C chemical shifts (ppm) in compounds 3.11g-i, 3.13 and 3.29a-c .................................57

15

N chemical shifts (ppm) in compounds 3.11g-i and 3.13. Protons which couple to a N are given in parentheses ..............................................................................................58

4-1

Synthesis of Tetrazoles 4.9b,c,e ........................................................................................69

4-2

Synthesis of N-(α-Aminoalkyl)tetrazoles 4.11a-h .............................................................70

4-3

Percentage of N1 Isomer Observed in Different Solvents .................................................72

4-4

1

4-5

Coalescence Temperatures (Tc), Equilibrium Constants (K), Chemical Shift Differences (∆v), Free Energy Barriers (∆G≠), and Natural Logarithm of Exchange Rate Constant (kr) from the 1H and 13C NMR Spectra of 4.11c (Acetonitrile-d3 as Solvent) ..............................................................................................................................77

4-6

Signal Assignments and Molar Percentage Ratios of Compounds 4.11a, 4.11d, 4.11e, and 4.11i in Cross-Over Experiment .......................................................................80

5-1

1

5-2

13

5-3

1

5-4

Calculated energy differences (kcal/mol) and molar fractions, at -65 °C, for conformers of 5.1 .............................................................................................................105

5-5

Calculated energy differences (kcal/mol) and molar fractions, at -65 °C, for conformers of 5.5 .............................................................................................................106

H, 13C, and 15N Chemical shift assignments in 4.11b,c....................................................73

H chemical shifts in compounds 5.1-5.9 ..........................................................................95 C chemical shifts in compounds 5.1-5.9 .........................................................................98

H chemical shifts in compounds 5.1-5.7 and 5.9............................................................102

8

5-6

Calculated energy differences (kcal/mol) and molar fractions, at -65 °C, for conformers of 5.2 .............................................................................................................106

5-7

Calculated energy differences (kcal/mol) and molar fractions, at -65 °C, for conformers of 5.3 .............................................................................................................108

5-8

Calculated energy differences (kcal/mol) and molar fractions, at -65 °C, for conformers of 5.6 .............................................................................................................109

5-9

Calculated energy differences (kcal/mol) and molar fractions, at 25 °C, for conformers of 5.4 .............................................................................................................110

5-10

Calculated energy differences (kcal/mol) for conformers of model compounds 5.105.12...................................................................................................................................111

5-11

Calculated stabilization energy and ∆E (trans-cis) (kcal/mol) for the donor-acceptor interaction of interest in model compounds 5.10-5.12. ...................................................111

5-12

Distances (Å) for some bonds at the lowest (0°) and highest point of the rotation (90°) in model compounds 5.10-5.12 ..............................................................................111

5-13

Electron Donor-Acceptor interactions and N4 occupancy at the lowest (0°) and highest point of the rotation (90°) in model compounds 5.10-5.12 .................................112

6-1

1

6-2

13

6-3

15

7-1

1

H NMR (ppm) chemical shifts (DMSO-d6) ...................................................................122 C NMR (ppm) chemical shifts (DMSO-d6)..................................................................123 N NMR (ppm) chemical shifts (DMSO-d6) .................................................................125

H, 13C, 15N NMR chemical shifts of 7.16a, 7.17a, and 7.17b ........................................144

9

LIST OF FIGURES Figure

page

2-1

Biologically active (-aminoacyl)amino-substituted heterocycles ...................................23

3-1

Dominant tautomeric forms of amino-, hydroxy-, mercapto-, and methyl-pyridines .......41

3-2

Compounds investigated in this thesis ...............................................................................42

3-3

Tautomers A and B of 2-hydrazino-3-phenylquinazolin-4(3H)-ones 3.11a-i and their common cations C and D ...................................................................................................43

3-4

Tautomers of hydrazinopyrimidin-4(3H)-ones ..................................................................44

3-5

Tautomers of disubstituted guanidines ..............................................................................44

3-6

Tautomers of 2-pyrimidinamine and of isocytosine ..........................................................45

3-7

Tautomeric forms of 2-quinolylhydrazones 3.18...............................................................46

3-8

Expansions of the 1H-13C gHMBC spectrum of compound 3.11b ....................................49

3-9

Expansions of the 1H-15N CIGAR spectrum of compound 3.11b .....................................50

3-10

X-ray structure of 3.11b .....................................................................................................50

3-11

15

3-12

Isomers/rotamers of compound 3.13..................................................................................56

4-1

Some examples of bioactive compounds containing N-(α-aminoalkyl)tetrazole scaffolds .............................................................................................................................67

4-2

X-ray structure of 4.11f......................................................................................................75

4-3

1

4-4

Plot of ln kr vs 1/ Tc in case of inter-conversion of A

4-5

Different isomers expected from crossover experiment between 4.11a and 4.11e ...........78

4-6

NOESY spectrum of crossover experiment between 4.11a and 4.11e in toluene-d8 at -10 ˚C. ................................................................................................................................80

5-1

C-Nitroso derivatives of indolizines and 3- and 5-azaindolizines taken into study ..........90

5-2

Monomer-dimer equilibria in heteroaromatic nitroso compounds ....................................90

5-3

Rotamer equilibrium in 5.1 ................................................................................................91

N chemical shifts in related heterocycles from ref.[1995MRC389] ...............................51

H spectra of 4.11c in acetonitrile-d3 from 0 ˚C to 70 ˚C in steps of 5 ˚C ........................76

10

B and B

A. .............................78

5-4

Possible conformations for 5.3, relevant proton chemical shifts, and nOes ......................99

5-5

Expansion of the NOESY spectrum of 5.3 ......................................................................100

5-6

Parent compounds for 5.5 and 5.6 ...................................................................................101

5-7

Parent compound of 5.4 ...................................................................................................102

5-8

Conformers of 5.1 ............................................................................................................105

5-9

Conformers of 5.5 ............................................................................................................105

5-10

Conformers of 5.2 ............................................................................................................106

5-11

Conformers of 5.3 ............................................................................................................108

5-12

Conformers of 5.6 ............................................................................................................109

5-13

Conformers of 5.4 ............................................................................................................109

5-14

Model compounds for the s-cis vs. s-trans conjugation ..................................................110

6-1

Isoxazolo[3,4-d]pyridazin-7(6H)-ones 6.1, tetrazolopyridazines 6.2, and triazolopyridazines 6.3 .................................................................................................................117

6-2

Compounds investigated in this thesis .............................................................................118

6-3

1

6-4

The 1H-13C gHMBC spectrum of 6.17 in DMSO-d6.......................................................120

6-5

The 1H-15N CIGAR-HMBC spectrum of 6.10 with expansion in DMSO-d6 .................121

6-6

The 1H-15N CIGAR-HMBC spectrum of 6.17 with expansion in DMSO-d6 .................121

7-1

Spiro[pyrrolidine-2,3'-oxindoles] (7.5a,b,c) ....................................................................131

7-2

Nitrogen chemical shifts of 1-pheny1-3-methyl-5-N-benzylideneaminopyrazole (7.6) in CDCl3 and in TFA-d ....................................................................................................132

7-3

Ethenoadenosine (7.7) in the neutral form (asterisks are used to show the rotation of the outer imidazole ring with respect to the inner one) ...................................................132

7-4

Equilibrium between [5,6]pinene-bpy (7.8) and its monoprotonated form (7.8H+) ........133

7-5

[4,5]CHIRAGEN[0] (7.9) AND [4,5]CHIRAGEN[0] (7.10)..........................................134

7-6

1

H NMR of 3-diethylamino-1-ethyl-6-iodopyridazin-1-ium iodide (6.17) .....................119

H, 13C, and 15N chemical shifts of 7.13a ........................................................................136

11

7-7

1

H-1H gDQCOSY spectrum of compound 7.13a ............................................................136

7-8

1

H-13C gHMBC spectrum of 7.13a ..................................................................................137

7-9

1

H-15N CIGAR-gHMBC spectrum of 7.13a ....................................................................137

7-10

1

H, 13C, and 15N chemical shifts of 7.13b ........................................................................138

7-11

1

H, 13C, and 15N chemical shift assingments of 7.14a .....................................................139

7-12

1

H-13C gHMBC spectrum of 7.14a ..................................................................................139

7-13

1

H-15N CIGAR-gHMBC spectrum of 7.14a with expansion...........................................140

7-14

1

H, 13C, and 15N chemical shift assignments of 7.14b .....................................................140

7-15

1

H-13C gHMBC spectrum of 7.14b..................................................................................141

7-16

1

H-15N CIGAR-gHMBC spectrum of 7.14b with expansion ..........................................142

7-17

Compounds 7.16a, 7.17a, and 7.17b with numbering .....................................................143

7-18

1

H-15N CIGAR-gHMBC spectrum of 7.17a in CDCl3 ....................................................145

7-19

1

H-15N CIGAR-gHMBC spectrum of 7.17a in TFA-d ....................................................145

7-20

1

H-15N CIGAR-gHMBC spectrum of 7.17b in CDCl3 with expansion...........................147

7-21

1

H-15N CIGAR-gHMBC spectrum of 7.17b in TFA-d ....................................................147

7-22

Expansions of 1H-15N CIGAR-gHMBC spectrum of 7.17b in TFA-d ............................148

12

LIST OF SCHEMES Scheme

page

2-1

Preparation of N-substituted amides 2.8a-d and 2.8a'-c' ..................................................25

2-2

Preparation of dipeptidoyl amides 2.12a,b ........................................................................28

2-3

Preparation of (acylamino)pyrimidones 2.14a,b and 2.14a' .............................................28

2-4

Preparation of (acylamino)pyrazoles 2.16a,b ....................................................................29

2-5

Preparation of acylamino pyridines 2.18a-g and 2.18d' ....................................................30

3-1

Reagents and conditions: a) EtOH, reflux 2 h; b) n-BuOH, N2H4.H2O, reflux 2 h. ..........47

3-2

Reagents and conditions: a) (CH3O)2SO2, 2 % Ethanolic sodium hydroxide, rt, 1 h. ; b) N2H4.H2O, EtOH, reflux, 20 h.; c) EtOH, 3.23d, reflux, 7 h. .......................................48

4-1

N-1 to N-2 Substituent isomerization of (N, N-disubstituted-aminomethyl) benzotriazoles ....................................................................................................................68

4-2

(i) N1-, N2-, and N4-Substituted isomers of N-(α-aminoalkyl)-1,2,4-triazoles. (ii) N1- and N2-Substituted isomers on N-(α-aminoalkyl)-1,2,3-triazoles..............................69

4-3

N-1 to N-2 Substituent isomerization of 4.11c ..................................................................77

7-1

..........................................................................................................................................130

7-2

Selective synthesis of S- and N-acyl-L-cysteines 7.8a,b and 7.9a,b ................................135

7-3

Synthesis of compounds 7.16a, 7.17a, and 7.17b ...........................................................142

7-4

..........................................................................................................................................146

13

LIST OF ABBREVIATIONS α

Alpha locant

[α]D

Specific rotation

Ala

Alanine

Ar

Aryl



Beta locant

Bn

Benzyl

Boc

t-Butoxycarbonyl

BOP

Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate

br

Broad

Bt

Benzotriazol-1-yl

C

Carbon

˚C

Degree Celcius

Calcd

Calculated

Cbz

Carbobenzyloxy

CDCl3

Deuterated chloroform

CIGAR-HMBC

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

Cys

Cysteine

δ

Chemical shift in parts per million downfield from tetramethylsilane

d

Douplet

D

Dextrorotatory (right)

DCC

N,N'-Dicyclohexylcarbodiimide

DCM

Dichloromethane

DMF

Dimethylformamide

14

DMSO

Dimethylsulfoxide

D2O

Deuterium oxide

EDC

1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (stands as an abbreviation for EDAC and EDCI as well)

Et

Ethyl

et al.

And others

Et3N

Triethylamine

EtOAc

Ethyl acetate

g

Gram(s)

gDQCOSY

Gradient double quantum correlation spectroscopy (NMR technique)

GFP

Green fluorescent protein

gHMQC

Gradient heteronuclear multiple quantum coherence (NMR technique)

gHMBC

Gradient heteronuclear multiple bond coherence (NMR technique)

Gly

Glycine

h

Hour

H

Hydrogen

HBTU

O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate

HOBt

1-Hydroxybenzotriazole

HPLC

High performance liquid chromatography

HRMS

High resolution mass spectrometry

Hz

Hertz

IR

Infrared

J

Coupling constant

L (10

point)

Levorotatory (left)

Lit

Literature

m

Multiplet 15

M

Molar

Me

Methyl

MeCN

Acetonitrile

MeOH

Methanol

Met

Methionine

min

Minute(s)

MgSO4

Magnesium sulfate

mol

Mole(s)

mp

Melting point

MW

Microwave

m/z

Mass-to-charge ratio

N

Nitrogen

Na2CO3

Sodium carbonate

NaOH

Sodium hydroxide

NMR

Nuclear magnetic resonance

NOE

Nuclear Overhauser effect

NOESY

Nuclear Overhauser effect spectroscopy

o

Ortho locant

O

Oxygen

OEt

Ethoxy

OH

Hydroxyl group

OMe

methoxy

p

Para locant

Ph

Phenyl

Phe

Phenylalanine

16

ppm

Part per million

Pro

Proline

Py

Pyridine

q

Quartet

R

Rectus (right)

ref.

Reference

rt

Room temperature

s

Singlet

S

Siister (left)

SCS

Substituent chemical shift

SOCl2

Thionyl chloride

t

Triplet

t

Tertiary

TFA

Trifluoroacetic acid

TLC

Thin-layer chromatography

TMS

Trimethylsilane

TOCSY

Total Correlation Spectroscopy (NMR technique)

Trp

Tryptophan

Val

Valine

W

Watt(s)

17

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

18

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

19

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

20

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

21

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

22

CHAPTER 2 (-AMINOACYL)AMINO-SUBSTITUTED HETEROCYCLES AND RELATED COMPOUNDS1 2.1 Introduction N-Substituted heterocycles show anti-inflammatory [1998WOP9822475], antiproliferative [20006USP009457], antithrombotic [1998USP5780590], antifungal [1982FA450] and antineurological biological activities [2007USP066613]. Such units occur in diverse pharmacologically active molecules including cell adhesion inhibitors [2001WOP012183], platelet-activating factor (RAF) or angiotensin II antagonists [1993WOP9314069], mitogenactivated protein (MAP) kinase [2003WOP035638] and mitotic kinesin KSP inhibitors [2003WOP103575]. (-Aminoacyl)amino-substituted heterocycles are useful synthetic intermediates (Figure 2-1) for endomorphin-2 (EM-2) analogues (2.1) [2004JME3591], bacterial RND efflux pump inhibitors (EPIs) such as MC-04,124 (2.2) [2003BMC4241] and MC-02,595 (2.3) [2003BMC2755], γ-secretase inhibitor LY411575 (2.4) [2004TL2323], and inhibitors of tumor necrosis factor-α converting enzyme (TACE) GW 3333 (2.5) [2001JME4252]. O

O

H2 N

O

H N

O HN R

R = Pyr, Qln, Isq

N H

H2 N

H2 N

O

H N

N H

O

N

N

MC-02,595 H2 N

2.3

2.2

OH F O F

O

H N

MC-04,124

EM-2 2.1

HO

Ph

Ph

Ph N

H N

O N Me H

OH N

H

H N

O

NH Me

O

O

LY411575 2.4

O

N H

N

GW 3333 2.5

Figure 2-1. Biologically active (-aminoacyl)amino-substituted heterocycles 1

Reproduced in part with permission from The Journal of Organic Chemistry, 2008, 73, 5442-5445. Copyright © 2008 American Chemical Society

23

N-Acylbenzotriazoles [2005SL1656] have been employed for: (i) N-acylation [2007S3673] in the preparation of primary, secondary, tertiary [2000JOC8210], and Weinreb amides [2002ARK39]; (ii) C-acylation for the preparation of -ketosulfones [2003JOC1443], primary and secondary -cyanonitriles [2003JOC4932], -nitroketones [2005JOC9211], ketones [2006JOC9861], and -ketoazines [2005ARK329]; and (iii) O-acylation of aldehydes [1999JHC777] and of steroids [2006ST660] to give esters. N-(Boc-aminoacyl)benzotriazoles and chiral amines give N-(Boc--amino)amides with no detectable racemization [2002ARK134]. Numerous N-(protected--aminoacyl)benzotriazoles couple with unprotected amino acids in mixed organic/aqueous solution with complete preservation of the original chirality [2007JOC5794, 2004S2645]. In continuation of this considerable research in our group, I now report the synthesis of N-substituted amides 2.8a-d, 2.8a'-c', 2.9a-b, 2.14a', 2.16a-b, 2.18a-g, 2.18d' and N-protected dipeptidoyl amides 2.12a,b by treatment of the corresponding N-(protected-aminoacyl)benzotriazoles 2.6a-e,g,i,j, 2.6a'-c', Nacylbenzotriazoles 2.6f,h or N-(protected-peptidoyl)benzotriazoles 2.6a,b with heterocyclic amines under microwave irradiation. Microwave irradiation is known to accelerate the reaction rates and shorten the reaction times substantially [2002ACR717]. This acceleration is mainly due to thermal effects but it could also be due to some microwave effects. Thermal effects are more generally accepted by the scientific community to be the main reason for drastic enhancement of reaction rates by microwave. In microwave organic assisted reactions, heating process is rapid and produces heat profiles that can not be obtained through conventional heating [2001T9225]. The energy transfer to reactants is instantaneous in case of microwave while it takes place via classical conduction in 

Compound numbers written with primes represent racemates, e.g., 2.6a'-c', 2.8a'-c', and 2.18d'.

24

conventional heating. Moreover, heaing process is highly controlled, energy efficient, smooth, and homogeneous and that is could be the reason why it leads to formation of products of higher purity and higher yields. 2.2 Results and Discussion 2.2.1 Preparation of Acylamino-Thiazoles, -Benzothiazoles, -Benzimidazoles and Thiadiazoles The starting N-(protected-aminoacyl)benzotriazoles 2.6a-d, 2.6a', 2.6b', 2.6c' were prepared from N-protected amino acids following a published one-step procedure [2003S2795, 2006S411]. Treatment of 2-aminothiazole (2.7a), 2-amino-6-methoxybenzothiazole (2.7b), N-benzyl2-aminobenzimidazole (2.7c) and 5-amino-3-methoxy-1,2,4,-thiadizole (2.7d) and N-(protectedα-aminoacyl)benzotriazoles 2.6a-d, 2.6a'-c' under microwave irradiation at 70 °C for 30 min (150 min for 2.7d with 2.6d) gave the N-substituted amides 2.8a-d and 2.8a'-c' in 50-98% yields (Scheme 2-1 and Table 2-1).

Scheme 2-1. Preparation of N-substituted amides 2.8a-d and 2.8a'-c' The enantiopurity of compounds 2.8a-c was confirmed by HPLC analysis. As expected, HPLC analysis of enantiopure 2.8a-c gave a single peak for each compound. In contrast, two peaks were observed for the corresponding racemic N-substituted heterocycles 2.8a', 2.8b' and 2.8c' (Table 2-1).

25

As a further application of this synthetic approach, Cbz-L-Met-L-Trp-OH (2.10a) (prepared as reported [2004S2645] by coupling of Cbz-L-Met-Bt (2.6e) with unprotected L-Ala (2.9a) in aqueous acetonitrile) was treated with benzotriazole and SOCl2 to provide the N-(protecteddipeptidoyl)benzotriazole Cbz-L-Met-L-Trp-Bt (2.11a). Compound 2.11a was reacted with 2.7a under microwave irradiation (100 W) in DMF at 70 °C for 30 minutes to give dipeptidoyl amide 2.12a in 60% yield (Scheme 2-2). Dipeptidoyl amide 2.12b was prepared in 52 % by coupling 6methoxybenzothiazol-2-amine (2.7b) with Cbz-L-Phe-L-Ala-Bt (2.11b) as described above (Scheme 2-2).

Scheme 2-2. Preparation of dipeptidoyl amides 2.12a,b Few literature reports describe the preparation of α-aminoacyl derivatives of heterocyclic amines, i.e., carboxamides of type 2.8. Kraus et al. [2004JCO695, 2005OBC612] investigated the coupling reaction between amino acids and weakly nucleophilic heteroaromatic amines including substituted 2-aminothiazole and substituted 2-aminobenzothiazole using four different coupling reagents such as (i) DCC/HOBt (ii) EDC, (iii) HBTU (iii) Benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and (iv) Phosphorus oxychloride

26

POCl3/pyridine. Use of the uronium coupling reagent (HBTU) failed. The best literature yields (41-93%) were achieved with POCl3 in pyridine. The authors conclude “the N-acylation of weakly nucleophilic heterocyclic amines by protected amino acids is not a straight forward reaction which could be achieved under any standard coupling conditions” [2004JCO695]. Other literature methods utilizing DCC/HOBt [2003BMC5529] or EDC [2001JMC4252] as coupling reagents reported yields of 27-36% and reaction times of 4-16 h. Table 2-1. Preparation of acylamino-thiazoles, -benzothiazoles, -benzimidazoles and thiadiazoles Yielda R.T. Entry Reactant Product [α]25D (%) (min) 1

Cbz-L-Trp-Bt (2.6a)

81

-39.8

3.57

66

racemic

3.52 and 5.36

98

-49.8

3.41

78

racemic

3.46 and 4.01

98

-44.6

3.39

82

racemic

2.97 and 3.56

50 (27)b

-63.9

NMc

2.8a 2

Cbz-DL-Trp-Bt (2.6a') 2.8a'

3

Cbz-L-Ala-Bt (2.6b) 2.8b

4

Cbz-DL-Ala-Bt (2.6b') 2.8b'

5

Cbz-L-Val-Bt (2.6c) 2.8c

6

Cbz-DL-Val-Bt (2.6c') 2.8c'

7

Cbz-L-Phe-Bt (2.6d) 2.8d

a

b

c

Isolated yield. [2003BMC5529]. Not measured.

27

2.2.2 Acylation of Aminopyrimidones Procedures similar to those of Section 2.2.1 above coupled Cbz-L-Ala-Bt (2.6b), Cbz-DLAla-Bt (2.6b') and 4-ClPhCOBt (2.6f), with 4-amino-1-benzylpyrimidin-2-one (2.13a) under microwave irradiation to give novel 2.14a,b and 2.14a' in 76-98% yields (Scheme 2-3). The structures of compounds 2.14a,b and 2.14a' were supported by spectroscopic data together with microanalyses. The 13C NMR and 1H NMR spectra of N-substituted amides 2.14a,b and 2.14a' showed characteristic signals in the regions 165.9-175.2 ppm and 10.94-11.31 ppm which were assigned to the N-heteroaryl amide carbonyl carbon and the proton of the NH respectively.

Scheme 2-3. Preparation of (acylamino)pyrimidones 2.14a,b and 2.14a' Previous preparations of N-substituted aminopyrimidones reported yields of 19 to 79% and reaction times of 17-40 h using carbodiimide based reagents such as DCC [1995EJM789], 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) [2000BMC539] or (1-ethyl-3-(3'dimethylaminopropyl)carbodiimide (EDCI) [1992JME3344] in the presence of HOBt. Kenner et al.[1955JCS855] acylated 3-methylcytosine with benzoyl chloride in pyridine at 100 C (1.5 h) in 65% yield. 2.2.3 Acylation of Aminopyrazoles N-Substituted pyrazoles were prepared in yields of 23 to 89% and reaction times of 5-10 h. from activated aromatic acids and N-protected amino acids via isolated intermediates including acyl chlorides [1996EJM461, 1998EJM375] or N-protected-aminoacyl chlorides [1982FA450],

28

that are not easily storable and sensitive to degradation and racemization [1990JA9651, 1991TL1303]. Literature couplings without isolation of intermediates include activation by HCTU/HATU, EDC/HOBt or phosponate anhydrides (T3P) in yields ranging of up to 42% in reaction times up to 16 h [2004JOC5168]. Coupling of 4-ClPhCOBt 2.6f and Cbz-Gly-Bt 2.6g with 5-amino-3-methyl-1-phenylpyrazole 2.15a was achieved in DMF under microwave irradiation (100 W, 70 °C) during 30 min (Scheme 2-4) to obtain 2.16a,b (40% and 75%, respectively). N-(Aminoacyl)benzotriazoles are stable, easy to handle reagents and can be stored at 20 C for months.

Scheme 2-4. Preparation of (acylamino)pyrazoles 2.16a,b 2.2.4 Acylation of Aminopyridines Microwave irradiation of 2.6f and 2-aminopyridine (2.17a) at 70 °C for 30 minutes gave N(4-chloropyridin-2-yl)benzamide (2.18a) in 94% yield (heating 2.6f and 2.17a in DMF at 100 °C for 6 h gave 2.18a in 75%). The microwave conditions were applied to the reactions of Nacylbenzotriazoles 2.6d,e,g-j and 2.6d' with 2-aminopyridine (2.17a), 2-amino-4-methylpyridine (2.17b) and 2-amino-4,6-dimethylpyridine (2.17c), thus providing 55-98% of the corresponding heteroaryl carboxamides 2.18a-g and 2.18d' (Scheme 2-5 and Table 2-2). The absence of racemization was confirmed for 2.18d by HPLC analysis, which showed a single peak at 3.66 min, while two peaks of equal intensity at retention times 3.63 min and 5.74 min were observed for racemic Cbz-DL-Phe-NHPy-2 (2.18d').

29

Scheme 2-5. Preparation of acylamino pyridines 2.18a-g and 2.18d' This methodology is advantageous compared to several recent approaches to (-aminoacyl)amino-substituted pyridines that was previously prepared in yields from unreported to 77% and reaction times from unreported to 54 h using: (i) N,N`-Dicyclohexyl-carbodiimide (DCC) and 1-hydroxybenzotriazole HOBt [2001TL4799], (ii) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and HOBt [1998BMCL1359], (iii) 1,1`-Carbonyldiimidazole (CDI) [1998EJM635], (iv) Ethyl chloroformate [2006AXEo3947], (v) Phosphorus trichloride (PCl3).[2004JME3591] and (vi) Acid chloride method [2004EJO3254]. Our approach provides known compounds 2.18a,b,d,f,g in better or comparable yields to those reported in the literature (Table 2-2) and afforded previously unreported N-substituted amides 2.18c, 2.18d', 2.18e and 2.12b in isolated yields of 5298%. The previously reported method [1998EJMC635] i.e. activating the corresponding N-protected-amino acids with CDI followed by treatment with 2-amino-4,6-dimethylpyridine is advantageous for N-substituted amides from 2-amino-4,6-dimethylpyridine, and 2.18f was prepared (68%, cf 77%) by this procedure. However, similar treatment of 2-amino-4-methylpyridine failed to yield compound 2.18g, which was prepared in high yield (98%) by our alternative methodology, demonstrating the wide scope of benzotriazole approach.

30

Table 2-2. Preparation of (acylamino)pyridines from N-acyl and N-(aminoacyl)benzotriazoles Yielda Entry Reactant Product Mp (oC) [α]25D (%) O Bt

1 Cl

94 (30d)

130-131

Non chiral

82 (66e)

76-78

Non chiral

82

122-123

Non chiral

55 (60f)

129-131

-18.3

70

51-53

Racemic

68

oil

-24.0

76 (77g)

108-110

Non chiral

2.18ai

2.6f O Bt

2 2.6h

3

Boc--Ala-Bt 2.6i

2.18b O Boc

N H

N H

N

2.18ci 4

5

6

7

Cbz-L-Phe-Bt 2.6d Cbz-DL-Phe-Bt 2.6d' Cbz-L-Met-Bt 2.6e

2.18d

b

2.18d'

c

2.18e

Cbz-Gly-Bt 2.6g 2.18f

8

Cbz-L-Pro-Bt 2.6j

98 (N/Ah) 125-126

-92.5

2.18gh a

Isolated yield, bHPLC for 2.18d: 3.66 min; cHPLC for 2.18d': 3.63 and 5.74 min.dRef. [1958JOC1909], eRef. [2004EJO3254], fRef. [2004JME3591], gRef. [1998EJM635], hno yield stated Ref. [2006AXEo3947], iCompounds 2.18a and 2.18c were prepared by colleague.

31

2.3 Conclusions In summary, a general and convenient route for the preparation of N-substituted amides derived from diverse heterocyclic amines and carboxylic acids under simple reaction conditions have been developed. 2.4 Experimental 2.4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl3 or DMSO-d6 with TMS for 1H (300 MHz) and 13C (75 MHz) as an internal reference. N-Cbz-Amino acids and amino acids were used without further purification. All the reactions were carried out under microwave irradiation with a single mode cavity microwave synthesizer producing a continuous irradiation at 2450 MHz (with infrared temperature control system). Optical rotation values were measured using the sodium D line. Column chromatography was performed on silica gel (200-425 mesh). HPLC analyses were performed using Chirobiotic T column (4.6–1250 mm), detection at 254 nm, flow rate 1 mL/min, and methanol as solvent. 2.4.2 General Procedure for the Preparation of 2.6a-j, 2.6a'-c' N-Aroyl-(2.6f,h), N-(Boc--aminoacyl)-, and N-(Cbz--aminoacyl)-benzotriazoles (2.6ae,g,i,j, 2.6a'-c') and dipeptidoyl benzotriazoles (2.11a,b) were prepared according to literature procedures [2003S2795, 2006S411]. 2.4.3 General Procedure for the Preparation of N-Substituted Amides 2.8a-d, 2.8a'-c', 2.14a-b, 2.14a', 2.16a-b, 2.18a-g, 2.18d' and Dipeptide Amides 2.12a,b A dried heavy-walled Pyrex tube containing a small stir bar was charged with benzotriazole adduct (0.25 mmol) and aminoheterocycle 2.6 (0.25 mmol) dissolved in DMF (1 mL). The reaction mixture was exposed to microwave irradiation (100 W) for 30 minutes at a

32

temperature of 70 °C. The mixture was allowed to cool through an inbuilt system until the temperature had fallen below 30 °C (ca. 10 min). The reaction mixture was quenched with water and extracted with EtOAc (3 × 25 mL). The extracts were washed with (10%) Na2CO3 (3 × 50 mL), water (3 × 50mL), and were dried over MgSO4. The solvent was removed under reduced pressure and the residue was subjected to silica-gel column using EtOAc/Hexane (1:1) as an eluent to give the corresponding N-substituted amide. Benzyl N-[(1S)–1-(1H–indol–3-ylmethyl)–2–oxo–2-(1,3–thiazol–2-ylamino) ethyl]carbamate (2.8a). White microcrystals (81%); mp 94.0–96.0 °C; [α]D25 = -39.8 (c 1.7, CHCl3). 1

H NMR (300 MHz, CDCl3)  4.84-4.86 (m, 1H), 3.21–3.35 (m, 2H). 5.06 (d, J = 12.1 Hz, 1H),

5.11 (d, J = 12.5 Hz, 1H), 5.90 (d, J = 8.0 Hz, 1H), 6.76 (s, 2H), 6.95 (t, J = 7.1 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 7.19–7.32 (m, 7H), 7.45 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 11.73 (s, 1H). 13C NMR (75 MHz, CDCl3)  29.0, 55.6, 67.2, 109.5, 111.2, 113.7, 118.4, 119.7, 122.3, 123.0, 127.1, 128.0, 128.2, 128.5, 135.9, 136.9, 156.1, 158.3, 170.0. Anal. Calcd for C22H20N4O3S: C, 62.84; H, 4.79; N, 13.32. Found: C, 62.65; H, 4.74; N, 13.14. Benzyl N-[1-(1H–indol–3-ylmethyl)–2–oxo–2-(1,3-thiazol-2-ylamino)ethyl]carbamate (2.8a'). White solid (66%); mp 188.0-190.0 °C. 1H NMR (300 MHz, DMSO-d6)  3.00-3.04 (m, 1H), 3.16-3.19 (m, 1H), 4.60 (br s, 1H), 4.95 (s, 2H), 6.97-7.04 (m, 3H), 7.20-7.31 (m, 8H), 7.49 (s, 1H), 7.70-7.77 (m, 1H), 10.85 (s, 1H), 12.49 (s, 1H). 13C NMR (75 MHz, DMSO-d6)  27.6, 55.4, 65.5, 109.4, 111.3, 113.7, 118.2, 118.8, 120.9, 124.3, 127.2, 127.7, 127.8, 128.4, 136.1, 136.9, 137.8, 155.9, 157.9, 171.2. Anal. Calcd for C22H20N4O3S: C, 62.84; H, 4.79; N, 13.32. Found: C, 62.61; H, 4.91; N, 12.95. Benzyl N-{(1S)–2-[(6–methoxy-1,3-benzothiazol-2-yl)amino]-1-methyl-2-oxoethyl}carbamate (2.8b). White microcrystals (98%); mp 90.0–92.0 °C; [α]D25 = -49.8 (c 2.1, CHCl3).

33

1

H NMR (300 MHz, CDCl3)  1.50 (d, J = 7.0 Hz, 3H), 3.85 (s, 3H), 4.78 (qui, J = 7.0 Hz, 1H),

5.13 (d, J = 12.2 Hz, 1H), 5.22 (d, J = 12.2 Hz, 1H), 6.19 (d, J = 7.7 Hz, 1H), 6.98 (dd, J = 8.9, 2.5 Hz, 1H), 7.29 (d, J = 2.5 Hz, 1H), 7.30–7.33 (m, 5H), 7.56 (d, J = 8.9 Hz, 1H), 11.06 (s, 1H). C NMR (75 MHz, CDCl3)  18.4, 50.7, 55.7, 67.4, 104.1, 115.3, 121.6, 128.1, 128.2,128.5,

13

133.3, 135.9, 142.3, 155.9, 156.3, 156.8, 171.6. Anal. Calcd for C19H19N3O4S: C, 59.21; H, 4.97; N, 10.90. Found: C, 58.86; H, 4.97; N, 10.63. Benzyl N-2-[(6-methoxy-1,3-benzothiazol-2-yl)amino]-1-methyl-2-oxoethylcarbamate (2.8b'). Colorless microcrystals (78%); mp 83.0-85.0 °C. 1H NMR (300 MHz, CDCl3)  1.50 (d, J = 7.1 Hz, 3H), 3.86 (s, 3H), 4.75-4.81(m, 1H), 5.18 (dd, J = 29.1, 12.3 Hz, 2H), 5.92 (br s, 1H), 6.99 (dd, J = 8.8, 2.5 Hz, 1H), 7.26-7.34 (m, 6H), 7.65 (d, J = 8.8 Hz, 1H), 10.74 (br s, 1H). 13C NMR (75 MHz, CDCl3)  18.5, 51.1, 67.9, 56.1, 104.5, 115.7, 121.9, 128.5, 128.7, 128.9, 133.7, 136.1, 142.8, 156.1, 156.7, 157.2, 171.7. Anal. Calcd for C19H19N3O4S: C, 59.21; H, 4.97; N, 10.90. Found: C, 59.55; H, 5.18; N, 10.55. Benzyl N-((1S)–1-[(1-benzyl-1H–benzimidazol-2-yl)amino]carbonyl-2-methylprop-yl)carbamate (2.8c). Colorless prisms (98%); mp 70.0–72.0 °C; [α]D25 = -44.6 (c 2.2, CHCl3). 1H NMR  0.9 (d, J = 6.7 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H), 2.33-2.39 (m, 1H), 4.38 (dd, J = 8.7, 4.3 Hz, 1H), 5.12 (t, J = 12.8 Hz, 2H), 5.31 (s, 2H), 5.76 (d, J = 8.7 Hz, 1H), 7.18–7.36 (m, 14H), 12.04 (s, 1H). 13C NMR  17.4, 19.5, 32.2, 45.7, 62.5, 66.6, 108.1, 109.9, 111.2, 123.3, 127.7, 127.9, 128.0, 128.1, 128.4, 128.8, 129.1, 135.1, 135.4, 136.8, 153.4, 156.5, 182.7. Anal. Calcd for C27H28N4O3: C, 71.03; H, 6.18; N, 12.27. Found: C, 71.06; H, 5.92; N, 11.89. Benzyl N-(1-[(1-benzyl-1H-benzimidazol-2-yl)amino]carbonyl-2-methylpropyl)carbamate (2.8c') Colorless prisms (82%); mp 131.0–133.0 °C. 1H NMR (300 MHz, CDCl3)  0.90 (d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H), 2.33-2.38 (m, 1H), 4.38 (dd, J = 8.8, 4.1 Hz, 1H), 5.12-

34

5.18 (m, 2H), 5.30 (s, 2H), 5.77 (d, J = 8.5 Hz, 1H), 7.17-7.35 (m, 14H), 12.05 (br s, 1H). 13C NMR (75 MHz, CDCl3)  17.8, 19.9, 32.5, 46.0, 62.8, 66.9, 110.2, 111.6, 123.6, 128.0, 128.3, 128.4, 128.8, 129.2, 129.5, 135.8, 137.1, 153.8, 156.9, 183.0. Anal. Calcd for C27H28N4O3: C, 71.03; H, 6.18; N, 12.27. Found: C, 71.20; H, 6.44; N, 11.94. Benzyl N-(1S)-1-benzyl-2-[(3-methoxy-1,2,4-thiadiazol-5-yl)amino]-2-oxoethylcarbamate (2.8d). White microcrystalls (50%); mp 150.0-152.0 °C (lit. mp not reported) [2003BMC5529], [α]D25 = -63.9 (c 0.18, CHCl3). 1H NMR (300 MHz, CDCl3)  3.12-3.14 (m, 2H), 4.00-4.04 (m, 3H), 5.04-5.17 (m, 3H), 5.81-5.93 (dd, J = 25.2, 8.06 Hz, 1H), 7.02-7.32 (m, 10H), 12.45 (s, 1H). C NMR (75 MHz, CDCl3)  39.6, 55.6, 56.8, 67.2, 127.4, 128.1, 128.4, 128.6, 128.7, 129.3,

13

135.2, 136.1, 155.6, 166.5, 172.4, 176.3. Anal. Calcd for C20H20N4O4S: C, 58.24; H, 4.89; N, 13.58. Found: C, 58.34; H, 4.90; N, 13.33. Benzyl (S)-1-((S)-3-(1H-indol-3-yl)-1-oxo-1-(thiazol-2-ylamino)propan-2-ylamino)-4(methylthio)-1-oxobutan-2-ylcarbamate (2.12a). White microcrystals (60%); mp 145.0–147.0 °C; [α]D25 = -20.7 (c 1.9, CHCl3). 1H NMR (300 MHz, CDCl3)  0.88–0.90 (m, 1H), 1.25-1.26 (m, 1H), 1.94 (s, 3H), 2.31–2.46 (m, 2H), 3.13 (dd, J = 14.3, 6.4, 1H), 3.24 (dd, J = 14.7, 7.2, Hz, 1H), 4.54–4.58 (m, 1H), 5.09 (d, J = 12.9 Hz, 1H), 5.15 (d, J = 12.9 Hz, 1H), 5.32–5.34 (m, 1H), 6.82–6.85 (m, 1H), 6.86–6.94 (m, 1H), 7.00 (t, J = 7.4 Hz, 2H), 7.12–7.18 (m, 1H), 7.21–7.31 (m, 6H), 7.36-7.41 (m, 2H), 7.76 (d, J = 8.2, 1H), 8.12 (s, 1H), 11.80 (s, 1H). 13C NMR (75 MHz, CDCl3)  15.1, 28.5, 30.0, 31.5, 31.9, 53.6, 53.8, 67.0, 109.2, 111.1, 113.7, 118.3, 119.4, 121.9, 123.4, 127.1, 127.9, 128.1, 128.5, 135.8, 136.4, 137.3, 156.6, 158.2, 169.9, 170.0, 172.4. Anal. Calcd for C27H29N5O4S2: C, 58.78; H, 5.30. Found: C, 59.13; H, 5.68. m/z (TOF.MS): 551.1661 [M+ + H] 552.1739.

35

Benzyl {(S)-1-[(S)-1-(6-Methoxy-benzothiazol-2-ylcarbamoyl)ethylcarbamoyl]-2-phenylethyl}carbamate (2.12b). White prisms (52%); mp 190.0–192.0 °C; [α]D25 = -68.8 (c 2.3, CHCl3). 1

H NMR  12.05 (br s, 1H), 8.15 (d, J = 8.9 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.8

Hz, 1H), 7.27–7.23 (m, 6H), 7.03–7.00 (m, 5H), 6.86 (dd, J = 8.9, 2.3 Hz, 1H), 5.42–5.35 (m, 2H), 5.12 (d, J = 12.6 Hz, 1H), 5.03 (dd, J = 16.2, 8.4 Hz, 1H), 3.85 (s, 3H), 3.13–2.98 (m, 2H), 1.49 (d, J = 6.6 Hz, 3H). 13C NMR  172.8, 170.7, 156.8, 156.7, 156.0, 142.5, 136.6, 136.0, 133.2, 129.3, 128.3, 127.8, 127.7, 126.9, 121.9, 115.1, 103.9, 103.3, 66.9, 56.5, 55.7, 48.7, 39.9, 19.2. Anal. Calcd for C28H28N4O5S: C, 63.14; H, 5.30; N, 10.52. Found: C, 62.77; H, 5.34; N, 10.34. Benzyl N-(1S)-2-[(1-benzyl-2-oxo-1,2-dihydro-4-pyrimidinyl)amino]-1-methyl-2-oxoethylcarbamate (2.14a). Colorless needles (97%); mp 152.0–153.0 °C; [α]D25 = +10.7 (c 0.15, DMF). 1H NMR (300 MHz, DMSO-d6)  1.26 (d, J = 7.0 Hz, 3H), 4.23-4.26 (m, 1H), 5.02 (s, 4H), 7.19 (d, J = 7.1 Hz, 1H), 7.31-7.35 (m, 10H), 7.69 (d, J = 6.7 Hz, 1H), 8.26 (d, J = 7.1 Hz, 1H), 10.94 (s, 1H). 13C NMR (75 MHz, DMSO-d6)  17.3, 50.9, 52.3, 65.5, 95.4, 127.6, 127.7, 127.8, 128.4, 128.6, 136.8, 150.6, 155.2, 155.8, 162.5, 174.2. Anal. Calcd for C22H22N4O4: C, 65.01; H, 5.46; N, 13.78. Found: C, 64.62; H, 5.50; N, 13.48. Benzyl N–2-[(1-benzyl-2-oxo-1,2-dihydro-4-pyrimidinyl)amino]-1-methyl-2-oxoethylcarbamate (2.14a'). Colorles needles (76%); mp 194.0–196.0 °C. 1H NMR (300 MHz, DMSOd6)  1.26 (d, J = 7.0 Hz, 3H), 4.27-4.23 (m, 1H), 5.03 (s, 4H), 7.18-7.15 (m, 1H), 7.36-7.32 (m, 10H), 7.70 (d, J = 6.7 Hz, 1H), 8.26 (d, J = 7.0 Hz, 1H), 10.95 (s, 1H). 13C NMR (75 MHz, DMSO-d6)  17.3, 50.9, 52.3, 65.5, 95.5, 127.7, 127.8, 127.9, 128.4, 128.6, 136.8, 136.9, 150.6, 155.2, 155.9, 162.5, 174.3. Anal. Calcd for C22H22N4O4: C, 65.01; H, 5.46; N, 13.78. Found: C, 64.86; H, 5.37; N, 13.63.

36

N-(1-Benzyl-2-oxo-1,2-dihydro-4-pyrimidinyl)-4-chlorobenzamide (2.14b). White needles (98%); mp 253.0-255.0 °C. 1H NMR (300 MHz, DMSO-d6)  5.05 (s, 2H), 7.31-7.40 (m, 6H), 7.59 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 8.5 Hz, 2H), 8.31 (d, J = 7.0 Hz, 1H), 11.31 (s, 1H). 13C NMR (75 MHz, DMSO-d6)  45.5, 52.3, 96.3, 127.6, 127.8, 128.5, 128.6, 128.8, 130.4, 131.2, 132.1, 136.7, 137.6, 150.3, 163.0, 166.5. Anal. Calcd for C18H14ClN3O2: C, 63.63; H, 4.15; N, 12.37. Found: C, 63.46; H, 4.01; N, 12.20. 4-Chloro-N-(3-methyl-1-phenyl-1H-pyrazol-5-yl)benzamide (2.16a). White microcrystals (75%); mp 151.0–153.0 °C (lit mp 156.0-157.0 °C) [1996EJMC461]. 1H NMR (300 MHz, CDCl3)  2.35 (s, 3H), 6.63 (s, 1H), 7.39-7.56 (m, 7H), 7.66 (d, J = 8.5 Hz, 2H), 7.96 (br s, 1H). C NMR (75 MHz, CDCl3)  14.0, 98.8, 124.5, 128.4, 128.5, 129.2, 129.6, 129.9, 131.6, 135.6,

13

137.9, 138.8, 149.8, 162.7. Anal. Calcd for C17H12ClN3O: C, 65.49; H, 4.53; N, 13.48. Found: C, 65.24; H, 4.44; N, 13.31. Benzyl N-2-[(3-methyl-1-phenyl-1H-pyrazol-5-yl)amino]-2-oxoethylcarbamate (2.16b). Colorless microcrystals (40%); mp 152.0–153.0 °C (lit. mp 153.0-155.0 °C) [1982FA450]. 1H NMR (300 MHz, CDCl3)  2.30 (s, 3H), 3.88 (d, J = 5.6 Hz, 2H), 5.03 (s, 2H), 5.46 (br s, 1H), 6.48 (s, 1H), 7.20-7.44 (m, 10H), 8.31 (br s, 1H). 13C NMR (75 MHz, CDCl3)  13.9, 45.3, 67.5, 86.3, 98.6, 100.2, 124.6, 128.1, 128.5, 128.6, 129.7, 135.2, 135.6, 137.7, 149.6, 166.1, 169.9. Anal. Calcd for C20H20N4O3: C, 65.92; H, 5.53; N, 15.37. Found: C, 66.16; H, 5.67; N, 15.33. 4-Chloro-N-(2-pyridinyl)benzamide (2.18a). Yellowish needles (94%); mp 130.0–131.0 °C (lit. mp. 139.0°C) [1958JOC1909]. 1H NMR (300 MHz, CDCl3)  7.04-7.08 (m, 1H), 7.46 (d, J = 8.7 Hz, 2H), 7.73–7.79 (m, 1H), 7.87 (d, J = 8.4 Hz, 2H), 8.19 (d, J = 4.5 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H),  9.02 (br s, 1H); 13C NMR (75 MHz, CDCl3)  114.3, 120.1, 128.7, 129.0, 132.7,

37

138.5, 147.8, 151.4, 164.8. Anal. Calcd For C12H9ClN2O: C, 61.95; H, 3.90; N, 12.04. Found: C, 61.98; H, 3.83; N, 12.12. N-(2-Pyridinyl)benzamide (2.18b). White needles (82%); mp 76.0–78.0 °C (lit. mp. 82.0– 84.0 °C) [2004EJO3254]. 1H NMR (300 MHz, CDCl3)  7.02-7.06 (m, 1H), 7.46-7.59 (m, 3H), 7.73-7.78 (m, 1H), 7.94 (d, J = 7.3 Hz, 2H), 8.19 (d, J = 4.3 Hz, 1H), 8.41 (d, J = 8.4 Hz, 1H), 9.06 (s, 1H). 13C NMR (75 MHz, CDCl3)  114.3, 119.9, 127.3, 128.7, 132.2, 134.3, 138.5, 147.8, 151.6, 165.9. Anal. Calcd For C12H10N2O: C, 72.71; H, 5.08; N, 14.13. Found: C, 72.48; H, 5.03; N, 13.97. 3-(tert-Butoxyamino)-N-(2-pyridinyl)propanamide (2.18c). White microcrystals (82%); mp 122.0–123.0 °C. 1H NMR (300 MHz, CDCl3)  1.43 (s, 9H), 2.63-2.66 (m, 2H), 3.47-3.53 (m, 2H), 5.30 (br s, 1H), 7.03-7.07 (m, 1H), 7.69-7.74 (m, 1H), 8.19 (d, J = 8.1 Hz, 1H), 8.28 (d, J = 3.6 Hz, 1H), 8.98 (br s, 1H). 13C NMR (75 MHz, CDCl3)  28.4, 36.2, 37.2, 79.4, 114.3, 119.8, 138.5, 147.7, 151.3, 156.0, 170.5. Anal. Calcd For C13H19N3O3: C, 58.85; H, 7.22; N, 15.84. Found: C, 58.51; H, 7.42; N, 15.79. Benzyl N-[(1S)-1-benzyl-2-oxo-2-(2-pyridinylamino)ethyl]carbamate (2.18d). White needles (55%); mp 129.0–131.0 °C (lit. mp. 137-139.0 °C) [2004JME3591]; [α]D25 = -18.3 (c 0.3, CHCl3). 1H NMR (300 MHz, CDCl3)  3.08 (dd, J = 13.2, 7.0 Hz, 1H), 3.17 (dd, J = 13.9, 6.2 Hz, 1H), 4.73-4.76 (m, 1H), 5.05 (d, J = 12.3 Hz, 1H), 5.12 (dd, J = 20.7, 12.2 Hz, 1H), 5.81 (d, J = 7.6 Hz, 1H), 6.99-7.04 (m, 1H), 7.09–7.12 (m, 2H), 7.18–7.21 (m, 3H), 7.23–7.29 (m, 5H), 7.66–7.71 (m, 1H), 8.20–8.25 (m, 2H), 9.15 (s, 1H). 13C NMR (75 MHz, CDCl3)  38.6, 56.8, 67.2, 114.3, 120.1, 127.1, 128.0, 128.1, 128.5, 128.6, 129.2, 135.9, 136.0, 138.5, 147.8, 150.9, 156.1, 170.2. Anal. Calcd For C22H21N3O3: C, 70.38; H, 5.64; N, 11.19. Found: C, 70.07; H, 5.78; N, 11.04.

38

Benzyl N-[1-benzyl-2-oxo-2-(2-pyridinylamino)ethyl]carbamate (2.18d'). White needles (70%); mp 51.0–53.0 °C. 1H NMR (300 MHz, CDCl3)  3.11-3.13 (m, 1H), 3.19 (dd, J = 13.9, 6.3 Hz, 1H), 4.69-4.71 (m, 1H), 5.10 (dd, J = 20.6, 12.2 Hz, 1H), 5.65 (br s, 1H), 7.01–7.05 (m, 1H), 7.13–7.30 (m, 10H), 7.67–7.72 (m, 1H), 8.20–8.25 (m, 2H), 8.96 (s, 1H). 13C NMR (75 MHz, CDCl3)  38.5, 56.8, 67.2, 114.2, 120.1, 127.1, 128.0, 128.2, 128.5, 128.7, 129.2, 135.8, 138.5, 147.8, 150.8, 156.0, 169.9, 170.0. Anal. Calcd For C22H21N3O3: C, 70.38; H, 5.64; N, 11.19. Found: C, 70.00; H, 5.84; N, 11.18. Benzyl N-{(1S)-3-(methylsulfanyl)-1-[(2-pyridylamino)carbonyl]propyl}carbamate (2.18e). Colorless oil (68%); [α]D25 = -24.0 (c 2.8, CHCl3). 1H NMR (300 MHz, CDCl3)  2.002.05 (m, 1H). 2.07 (s, 3H), 2.17–2.24 (m, 1H), 2.54–2.60 (m, 2H), 4.59-4.69 (m, 1H), 5.12 (d, J = 12.2 Hz, 1H), 5.18 (d, J = 12.2 Hz, 1H), 5.82 (br s, 1H), 7.35–7.62 (m, 5H), 7.04–7.08 (m, 1H), 7.69–7.74 (m, 1H), 8.20 (d, J = 8.1 Hz, 1H), 8.30-8.31 (m, 1H), 9.02 (br s, 1H). 13C NMR (75 MHz, CDCl3)  15.2, 30.0, 32.1, 54.8, 67.2, 114.4, 120.2, 128.0, 128.1, 128.5, 136.0, 138.5, 147.8, 150.9, 156.2, 170.6. Anal. Calcd For C18H21N3O3S: C, 60.15; H, 5.89; N, 11.69. Found: C, 59.79; H, 6.02; N, 11.69. Benzyl N-2-[(4,6-dimethyl-2-pyridinyl)amino]-2-oxoethylcarbamate (2.18f). White microcrystals (76%); mp 108.0–110.0 °C (lit. mp. 103.0 °C) [1998EJM635]. 1H NMR (300 MHz, CDCl3)  2.26 ( s, 3H), 2.34 (s, 3H), 4.00 (d, J = 4.8 Hz, 2H), 5.08 (s, 2H), 5.67 (s, 1H), 6.69 (s, 1H), 7.28 (s, 5H), 7.80 (s, 1H), 8.89 (s, 1H). 13C NMR (75 MHz, CDCl3)  21.4, 23.1, 45.3, 67.3, 111.9, 120.8, 128.2, 128.5, 136.0, 149.6, 151.5, 155.3, 156.6, 167.8. Anal. Calcd For C17H19N3O3: C, 65.16; H, 6.11; N, 13.41. Found: C, 65.00; H, 6.03; N, 13.32. Benzyl (2S)-2-[(4-methyl-2-pyridinyl)amino]carbonyltetrahydro-1H-pyrrole-1-carboxylate (2.18g). White microcrystals (98%); mp 125.0–126.0 °C (lit. mp. not reported)

39

[2006AXEo3947]; [α]D25 = -92.5 (c 2.4, CHCl3). 1H NMR (300 MHz, CDCl3) [two rotamers]  1.94-2.20 (m, 3H), 2.21-2.27 (m, 1H), 2.35 (s, 3H), 3.50–3.60 (m, 2H), 4.39-4.52 (m, 1H), 5.15– 5.72 (m, 2H), 6.78 (s, 1H), 7.18–7.37 (m, 5H), 8.05 (s, 1H), 8.31 (s, 1H), 8.49 (s, 0.4H), 9.22 (s, 0.6H). 13C NMR (75 MHz, CDCl3)  [two rotamers] 21.3, 23.8, 24.5, 28.6, 31.3, 47.2, 47.6, 61.5, 67.5, 114.5, 121.0, 128.0, 128.4, 136.2, 147.4, 149.7, 151.2, 156.3, 169.9, 170.3. Anal. Calcd for C19H21N3O3: C, 67.24; H, 6.24; N, 12.38. Found: C, 66.93; H, 6.43; N, 12.62.

40

CHAPTER 3 TAUTOMERISM OF 2-HYDRAZONO-3-PHENYLQUINAZOLIN-4(3H)-ONES STUDIED BY 15N NMR1 3.1 Introduction The tautomeric equilibria of heterocycles are extremely important for understanding the function of numerous biologically important components of living systems [1997MI1]. Thus the genetic code could only be deciphered after the dominant structures of the nucleotide bases were correctly represented. Moreover, it is now realized that the whole basis of evolution depends on the occurrence of minor proportions of the less stable nucleotide base tautomers which cause advantageous genetic mistakes [1953MI2].

Figure 3-1. Dominant tautomeric forms of amino-, hydroxy-, mercapto-, and methyl-pyridines The tautomeric equilibria of heterocycles have been investigated extensively [1963AHC1, 1976AHS1, 2000AHC1, 2006AHC1] and the following major trends are now clear (Figure 3-1). (i) Most aminohetero-aromatic compounds exist predominantly in the amino form cf. 3.1 (and not the imino form cf. 3.2) under normal conditions (aqueous solution or the crystalline state). (ii) Under these conditions most (although not all) hydroxyheteroaromatic compounds exist predominantly in the tautomeric carbonyl form (for example 2-hydroxypyridine 3.3 and 4hydroxypyridine 3.5 exist as 2-pyridone 3.4 and 4-pyridone 3.6 respectively). (iii) Mercapto 1

Org. Biomol. Chem., 2009, 7, 4110-4119. Reproduced in part by permission of the Royal Society of Chemistry.

41

derivatives of 6-membered heteroaromatic tend to follow their hydroxy analogs and exist as the thione, e.g. 3.8, and not as mercapto form, e.g., 3.7. In contrast mercapto derivatives of fivemembered heterocyclic rings exist as mercaptans (following the amino analogs). (iv) Methyl groups and most substituted methyl groups exist largely as the methyl tautomer, e.g. 3.9, and not the possible methylene tautomer, e.g., 3.10 which is far less stable. Knowledge of the tautomerism of potential drugs is relevant to the modeling of their interaction with a receptor since different tautomers have different affinities for the receptor. It is also important in the tuning of a desired pharmacological activity.

Figure 3-2. Compounds investigated in this thesis The present chapter discusses the tautomeric equilibrium of a set of 2-hydrazono-3-phenyl3H-quinazolin-4-ones 3.11a-i, all representatives of a group of compounds of intense current interest in the development of commercial drugs for analgesic and anti-inflammatory activity [2007BMC235, 2007CPB76, 2007AP41, 2008AF174]. The related 2-hydrazinobenzimidazole 3.12, and 2-hydrazinopyrimidine 3.13 derivatives (Figure 3-2) were also included in the study. Tautomeric equilibria can depend significantly on the nature of the medium. There are two major factors. Firstly, the dielectric constant of the medium is important and of course this can

42

range from very low in the vapor phase and nonpolar solvents (favoring the tautomeric form with the lowest dipole moment) to very high for certain polar solvents (favoring the form with the highest dipole moment). The second major effect of medium is its hydrogen bonding donor and acceptor ability, which can lead to different interactions of the tautomeric forms. In the present chapter we deal with equilibrium positions in DMSO as a solvent. Given the poor solubility of these compounds in water, this is probably the best medium to use for comparisons with biological systems.

Figure 3-3. Tautomers A and B of 2-hydrazino-3-phenylquinazolin-4(3H)-ones 3.11a-i and their common cations C and D Compounds 3.11a-i, 3.12 and 3.13 could exist in two tautomeric forms, amino forms A or imino forms B, as shown in Figure 3-3 for 3.11a-f. Apparently there has been no previous discussion published of the tautomerism of 2-hydrazinoquinazolin-4(3H)-ones 3.11. The 404 hits for substructure 3.11 in a Beilstein search are depicted in the presumed amino form 3.11A and none in the imino form 3.11B. However, no supporting evidence such as interatomic distances or

43

angles from solid state crystallographic data has been provided for their existence in the amino form A.

Figure 3-4. Tautomers of hydrazinopyrimidin-4(3H)-ones The tautomerism of 2-hydrazinopyrimidin-4(3H)-ones 3.14 (Figure 3-4) has also not been studied. Derivatives of 3.14 are usually represented in the amino form 3.14A (308 Beilstein hits). The 18 hits for the alternative imino form 3.14B were perhaps due to the fact that they represented compounds synthesized from uracil.

Figure 3-5. Tautomers of disubstituted guanidines The tautomerism of compounds 3.11a-i, 3.12, and 3.13 each involves a guanidine moiety 3.15. In substituted guanidines 3.15a-c (Figure 3-5), the most stable tautomer is 3.15a with the double bond attached to the nitrogen carrying the most electron-attracting substituent (e.g. R = NO2, CN, -SO2-C6H4-NH2) [1994JCS(P2)1849]. This can be rationalized by realizing that, of the two exchangeable protons in the common cation (3.11a-f C and 3.11a-f D in Figure 3-3), the hydrogen attached to the most electronegative nitrogen is the most acidic. The experimental evidence for such tautomerism includes 15N NMR [1995MRC383], and crystal structures [1965AXA47]. When two of the guanidine nitrogens are part of an aromatic system, the ‘most aromatic’ tautomer is usually favored. The tautomerism of 2-aminopyrimidines has been studied extensively; both experimental data (low temperature IR

44

spectroscopy) [1987JST275] and semiempirical and ab initio calculations [1995MI3, 1996JST375] confirm the 2-amino tautomer as the most stable. Spectroscopic studies (UV, IR and 1H-NMR) of N-substituted-2-pyrimidinamine 3.16 (R =H, NO2) show that neither the phenyl [1980BCJ717] nor the 2,4,6-trinitrophenyl substituents [1992JHC1461] are electronegative enough to shift the equilibrium from the amino form 3.16a to the imino form 3.16b.

Figure 3-6. Tautomers of 2-pyrimidinamine and of isocytosine 2-Amino-3H-pyrimidin-4-one 3.17 (isocytosine), which was studied extensively, both experimentally [1993T7627, 1993T595, 1980BCJ3073] and computationally [2007MI4], exists in aqueous solution mainly as 2-amino-3H-pyrimidin-4-one 3.17a with some 2-amino-1Hpyrimidin-4-one 3.17b. Calculations suggest the 2-imino form 3.17c, not detected experimentally, is 5.6 kcal/mol less stable in aqueous solution than the 2-amino form 3.17b. The tautomerism of 2-hydrazinopyrimidine has apparently not been studied. 2Quinolylhydrazones 3.18 (Figure 3-7) exist in solution predominantly in the amino form 3.18a, although the imino form was also detected [1976JOC2491, 1975JOC2512, 1975JCS(P1)2036]. In solid state, compounds 3.18 were found in the amino form when Ar = thienyl or 5chlorothienyl and in the imino form when Ar = 5-bromo-2-thienyl [1997AXC973].

45

Figure 3-7. Tautomeric forms of 2-quinolylhydrazones 3.18 The present work aims to identify the tautomeric preferences of the title compounds 3.11ai, 3.12, 3.13. The literature data presented above support both the ‘most aromatic’ amino form A and the imino form B which has the double bond on the most electronegative nitrogen. Since the two tautomeric forms differ in the protonation of two different nitrogens, 15N NMR is the method of choice since a large difference in the chemical shifts of the alternative structures is expected. Measurement of the 15N chemical shifts at natural abundance is now facile with the advent of indirect detection and pulsed field gradients [2002COR35, 2000JNP543]. 3.2 Results and Discussion 3.2.1 Syntheses Products 3.11a-i were prepared by the literature procedure described in Scheme 3-1 [1985JHC1535]. Compounds 3.12 and 3.13 were prepared by condensing 2-hydrazino-1Hbenzimidazole and 2-hydrazino-4,6-dimethyl-pyrimidine with 4-(dimethylamino)- benzaldehyde and 1-methyl-1H-indole-2,3-dione respectively. Our initial attempt to prepare compound 3.22 following the previously reported procedure [2007BMC235, 2007AP41, 2005BML1877, 2005BPB1531, 2006AF834, 2006JPP1249, 2008JHC709] described in Scheme 3-2 gave 3-amino-2-anilino-4(3H)-quinazolinone 3.25 instead. There is a single literature report [1985JHC1535], in which 3.25 was characterized only by melting point and elemental analysis.

46

Scheme 3-1. Reagents and conditions: a) EtOH, reflux 2 h; b) n-BuOH, N2H4.H2O, reflux 2 h. Our proof for the structure of compound 3.25 is based on NMR data. The nitrogen which couples with the ortho protons of the phenyl ring was at 102.2 ppm and bears the proton at 9.36 ppm. This proton couples with carbons C1' and C5a', and with three nitrogens at 187.5, 165.5, and 64.3. The nitrogen at 187.5 ppm was assigned to N1, because it couples with H8. The nitrogen at 64.3 has the chemical shift of an amino group and it carries two protons at 5.71. These latter protons couple with the nitrogen at 165.5 (N3 in 3.25) and with two carbons, C4 and another one assigned as C2 (see experimental section for atom labeling). Compound 3.25 reportedly condensed with benzaldehydes to give the corresponding Schiff‟s base 3.26 (Scheme 3-2) [1985JHC1535]. In our case 3.25 with p-nitrobenzaldehyde

47

(3.23d), gave compound 3.11d instead of the corresponding 3.26. The reaction was much slower than in the case of 3.22.

Scheme 3-2. Reagents and conditions: a) (CH3O)2SO2, 2 % Ethanolic sodium hydroxide, rt, 1 h. ; b) N2H4.H2O, EtOH, reflux, 20 h; c) EtOH, 3.23d, reflux, 7 h. 3.2.2 Tautomerism and NMR 1

1

H and 13C chemical shifts were assigned based on 1H-1H, and one-bond and long-range

H-13C couplings, seen in the gDQCOSY, gHMQC and gHMBC spectra. They are presented in

Tables 3-1, 3-2, 3-4 and 3-5. Position numbering is given at the top of the tables. A typical assignment started with identifying the sequence H5-H8 in the gDQCOSY spectrum. H5 and C4 were then assigned by their cross-peak in the gHMBC spectrum. The 1H13

C gHMBC spectrum of 3.11b is presented in Fig. 3-8. Cross-peaks of C4a with H6 and H8, and

of C8a with H5 and H7 reveal these quaternary carbons. The phenyl protons, H1'-H3' can be assigned from their intensity and coupling pattern and C5a' couples with H2'. In 2-hydrazono-3-phenyl-3H-quinazolin-4-ones, H3'', the singlet on a carbon at ca. 150 ppm couples with C1''' and C5a'''. Other cross-peaks in the gHMBC spectrum were then used to complete the assignments on the substituted benzylidene moiety C1'''-C5a'''.

48

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

1.09 13.2

7.48 129.6 7.40 128.6

3.37 44.5

7.31 130.0

O 161.3

N

137.3

N

N

150.6

149.3 111.4 6.64 130.3 7.68

N

122.6 154.5 7.87

7.88 128.1 7.11 122.1

114.9 140.8

N

135.7 7.63 116.2 7.64

H

10.41

Figure 3-8. Expansions of the 1H-13C gHMBC spectrum of compound 3.11b

49

1.09 13.2

7.48 129.6

78.8

7.31 130.0

7.40 128.6

3.37 44.5

O

7.88 128.1

161.3

N

137.3

149.3

N

151.3

111.4 6.64 130.3 7.68

N

122.6 154.5 7.87

7.11 122.1

114.9 140.8

N

150.6

329.1 247.2

N

99.7

135.7 7.63 116.2 7.64

H

10.41

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

Figure 3-10. X-ray structure of 3.11b 50

There is one more long-range cross-peak of the exchangeable proton in the 1H-15N CIGAR spectrum of 3.11b, with one of the nitrogens three bonds away, which was assigned as N3 (151.3 ppm), because it also couples with H1'. N1'' and N2'' both couple with H3'' only, therefore, they could not be distinguished based on their couplings with protons. Their chemical shifts at 329.1 and 247.2 ppm, are different enough, however, to allow assignment based on chemical shifts seen in related compounds. A particularly interesting example is presented in Figure 3-11 [1995MRC389], which has the same sequence of nitrogen atoms as in amino-guanidines, in the amino form in 3.27, and in the imino form in 3.28. Based on the 15N chemical shifts in compound 3.28, the signal at 247.2 ppm was assigned to N1'', and the signal at 329.1 ppm to N2''. The heterocycles in Figure 3-11 demonstrate that the chemical shifts of N1 and N1'' are good reporters of the amino-imino tautomerism of 2-hydrazono-3-phenyl-3H-quinazolin-4-ones, as a difference of ca. 100 ppm is to be expected in the two tautomers. The value of 99.7 ppm for N1 in 3.11b is in the range found for N1 in a series of 2,3-dihydro-1H-quinazolin-4-ones, 92-100 ppm; in a series of quinazolin-4(3H)-ones N1 was at 253-270 ppm. In both series, N3 was at 140-190 ppm [2000JHC831].

Figure 3-11.

15

N chemical shifts in related heterocycles from ref.[1995MRC389]

Of all the compounds in this study, 3.11a and 3.11b were the only ones in which the signal of the exchangeable proton was sharp enough to afford cross-peaks in the 1H-13C gHMBC spectrum. Cross-peaks with C4a, C8 and C8a identified the imino tautomer. A cross-peak with

51

C3''' would have been a proof for the amino tautomer. For all of the other compounds, exchange with water or between tautomeric forms broadens the signal of the exchangeable proton too much for it to display any couplings with carbons or with nitrogens. In all of these cases, the tautomerism was assigned based solely on 15N chemical shifts. In both 2-hydrazono-3-phenyl-3H-quinazolin-4-ones 3.11a-f and in the 2-[N'-(2-oxo-1,2dihydro-indol-3-ylidene)-hydrazono]-3-phenyl-3H-quinazolin-4-ones 3.11g-i, N1 displayed a cross-peak with H8 in the CIGAR spectrum. The range of chemical shifts for N1 was 100-114 ppm. In the first series, the chemical shift of N1'' was detected through the coupling between N1'' and H3'' and the values for N1'' chemical shifts were 247-251 ppm. These chemical shifts identify the imino form as the predominant tautomer in solution for compounds 3.11a-i. The N1 chemical shift is expected to depend mainly on the position of the tautomeric equilibrium, and could be used to estimate this position, if values of the two tautomers are known. When the guanidine unit is part of a more aromatic structure, the amino form prevails. This is illustrated by compounds 3.12 and 3.13. In 3.12 where rapid exchange of the proton between N1 and N3 (numbering relative to the aminoguanidine moiety, as in 3.11a-i) produced an AA'XX' pattern for the H5-H8 signals. The NH protons were in fast exchange with the residual water in DMSO-d6, and did not produce a separate signal. The chemical shift of N1, N3 was revealed by coupling with H5, H8; cross-peaks with H3'' revealed N1'' and N2''. The chemical shifts in the pair N1'', N2'' (142.9, 305.1) were closer to the values in 3.27 (133.0, 328.0) than to those in 3.28 (238.0, 384.0), indicating that compound 3.12 is present in DMSO solution predominantly in the amino form. The chemical shift of the azole nitrogen cannot be used for the assignment of the tautomerism, at least in the presence of fast exchange. The value in 3.12, 136.3 ppm, is closer to the value for N1 in 1-methyl-2-aminobenzimidazole (134.6 ppm), than to their

52

average (N3 is at 191.9 ppm), which suggests that 3.12 is in the imino form. However, in 1methylbenzimidazole N1 and N3 are at 143.8 and 243.9 ppm, correspondingly, while in benzimidazole, the equivalent nitrogens are at 143.2 ppm [1997MRC35]. Compound 3.13 did not dissolve in DMSO-d6 at room temperature, but did so at 70 °C. About 30 minutes after dissolution, the proton spectrum of 3.13 displayed the signals of two compounds, in a ratio 2:1. 1H-13C correlations indicated that both compounds contain the 4,6dimethylpyrimidine and the 1,3-dihydroindole-2-one moieties. After one day, the sample consisted entirely of what was initially the minor compound. Correlations to the methyl protons identified the pyrimidine nitrogens at 254.2 and 252.4 ppm in the initially major and minor compounds, respectively. These values are comparable to the value in 2-amino-4,6dimethylpyrimidine, at 242 ppm [1981OMR106], indicating that both isomers are in the amino form. Further evidence comes from the initial minor compound, which displayed a sharp signal for the exchangeable proton at 12.80 ppm. This proton is attached to the nitrogen at 164.3, and displays long-range couplings with the nitrogens at 252.4 and 340.0 ppm. Since these are amino tautomers, they must differ in the configuration of the C3'''=N2'' double bond. The sharp, deshielded signal of the NH proton in the initially minor compound suggests an intramolecular hydrogen bond, possible only in the Z isomer. Some of the signals in the other isomer, E, are broadened, particularly H4''' and the pyrimidine CH probably due to restricted rotation. The bonds to be considered as having partial double bond character are C2-N1'' and N1''-N2''. Restricted rotation about C2-N1'' would produce broadening of the signals of the methyl groups in the 4,6-dimethylpyrimidine moiety, but not of the pyrimidine CH or of H4'''. Broadening of these latter signals is due to restricted rotation about the N1''-N2'' bond (Figure 3-12). The assignment of the Z and E isomers was also confirmed by 13C chemical shifts, as described later.

53

Discrimination between compounds 3.22 and 3.25 (Scheme 3-2) was based on the 15N NMR data. The 1H-15N correlations are presented in the experimental part for 3.25 and in Table 3-3 for 3.22. N1 was identified in both compounds by its cross-peak with H8 in the CIGARgHMBC spectrum. Chemical shift values for N1 of 183.9 ppm in 3.22 and 188.0 ppm in 3.25 demonstrate that these compounds are both present in solution predominantly as the amino tautomer. This is to be expected for 3.25, in which the exocyclic guanidine nitrogen does not carry nitrogen. The preference for the amino tautomer of 3.22 is surprising, considering that its derivatives 3.11a-i prefer the imino form, but can be explained by the greater electronegativity of N2'' in the latter compounds. Table 3-1. 1H chemical shifts (ppm) in compounds 3.11a-f, 3.12, 3.21, 3.22 and 3.24

\Position 1 Compd.\ 3.11a 3.11b

5

6

7

8

1'

2'

3'

3''

1'''

2'''

3'''

4'''

10.61 7.91 7.16 7.69 7.69 7.34 7.49 7.40 8.07 7.93 7.41 7.38 10.41 7.88 7.11 7.63 7.64 7.31 7.48 7.40 7.87 7.68 6.64 3.37,1.09

3.11c

NM

3.11d

NM

b

5'''

7.41 7.93 a

6.64 7.68

8.00 7.37 7.83 8.04 7.49 7.61 7.59 8.59 3.80 6.60 3.83

6.65 8.38

7.97 7.22 7.72 7.72 7.36 7.52 7.44 8.19 8.16 8.21 -

8.21 8.16

3.11e

10.83 7.96 7.22 7.72 7.72 7.36 7.52 7.44 8.16 8.11 7.86 -

7.86 8.11

3.11f

10.68 7.94 7.18 7.70 7.70 7.35 7.50 7.42 8.12 9.09 -

8.55

7.43 8.31

3.12

NM

8.19 7.68 6.74 2.96

6.74 7.68

3.21

13.03 7.96 7.35 7.78 7.46 7.29 7.50 7.42 c

7.40 7.16 7.16 7.40 d

-

-

-

-

-

-

-

-

-

-

-

-

3.22

8.87

8.50 7.29 7.79 7.88 7.53 7.43 7.29 -

-

-

3.24

2.50e 8.10 7.49 7.84 7.65 7.47 7.58 7.58 -

-

-

a

b

c

d

e

CH2 and CH3, correspondingly. Not measured. Measured in pyridine-d5 at -30 °C. H''. CH3S in position 2.

54

Table 3-2. 13C chemical shifts (ppm) in compounds 3.11a-f, 3.12, 3.21, 3.22 and 3.24 \Position 2 4 4a 5 6 7 8 8a 1' 2' 3' 5a' Compd.\ 152.2 161.3 115.1 128.0 122.6 135.8 116.4 140.6 129.9 129.5 128.7 137.1 3.11a 150.6 161.3 114.9 128.1 122.1 135.7 116.2 140.8 130.0 129.6 128.6 137.3 3.11b NM 160.4 116.0 128.0 124.9 136.4 118.0 139.4 130.0 130.4 130.4 134.2 3.11c NM 161.2 115.9 128.2 123.0 135.8 116.8 140.3 129.9 129.5 128.6 137.0 3.11d NM 161.3 115.4 128.1 122.9 135.8 116.7 140.4 129.9 129.5 128.7 136.9 3.11e NM 161.4 115.3 128.1 122.7 135.8 116.4 140.5 129.9 129.5 128.6 137.0 3.11f NM 133.5 112.6 122.7 122.7 112.6 133.5 3.12 NM 160.5 116.9 128.1 125.0 136.3 116.4 140.3 129.7 129.6 128.8 140.0 3.21 a 3.22 NM 163.1 118.7 128.0 122.9 135.4 125.8 150.9 130.2 130.8 130.0 136.0 158.7 161.5 120.3 127.3 126.5 135.6 126.8 148.1 130.1 130.7 130.0 137.0 3.24 3'' 1''' 2''' 3''' 4''' 5''' 5a''' other 153.9 128.5 129.0 130.3 129.0 128.5 135.9 3.11a 154.5 130.3 111.4 149.3 111.4 130.3 122.6 44.4 (CH2), 13.2 (CH3) 3.11b 149.7 160.5 98.6 164.0 107.3 129.4 114.9 56.4 (CH3O-1'''); 56.3 (CH3O-3''') 3.11c 151.5 129.2 124.2 148.4 124.2 129.2 142.3 3.11d 152.0 128.9 132.9 112.1 132.9 128.9 140.4 119.5 (CN) 3.11e 151.0 149.8 150.7 124.3 135.1 131.7 3.11f 147.0 129.1 112.3 152.1 112.3 129.1 122.2 40.5 (CH3) 3.12 15.7 (CH3) 3.24 a

Measured in pyridine-d5 at -30 °C.

Table 3-3. \Position Compd.\ 3.11a 3.11b 3.11ca 3.11d 3.11ea

15

N chemical shifts (ppm) in compounds 3.11a-f, 3.12, 3.21, 3.22 and 3.24. Protons which couple to a 15N are given in parentheses

1

3

1''

2''

other

100.4 (H8) 99.7 (H1, H8) 110.5 (H8) 113.9 (H8) NM

151.9 (H1') 151.3 (H1'') 156.3 (H1') 157.2 (H1') 157.1 (H1')

247.0 (H3'') 247.2 (H3'') Nm 251.7 (H3'') 251.1 (H3'')

346.0 (H3'') 329.1 (H3'') 318.5 (H3'') 369.2 (H3'') 361.2 (H3'')

3.11f

104.4 (H8)

156.4 (H1')

251.2 (H3'')

354.7 (H3'')

3.12 3.21 3.22b 3.24

136.3 (H7,H8) 150.9 (H1, H8) 183.9 (H8, H1'') 230.6 (H8)

136.3 (H4,H5) 191.1(H1, H1') 161.4 (H1') 180.6 (H1')

142.9 (H3'') 106.5 (H1'') -

305.1 (H3'', H1''') 62.7 (H1'') -

78.8 (H2''', CH2) 372.9 (H2''') 318.2 (H1''', H3''', H4''') 55.5 (H2''', CH3) -

a

Measured at 70 °C. bMeasured in pyridine-d5 at -30 °C.

55

The substituents on N1'' could be used to control the tautomeric equilibrium of 2hydrazino-3-substitutedquinazolin-4(3H)-ones and related compounds, in order to fine tune their pharmacological and optical properties.

Figure 3-12. Isomers/rotamers of compound 3.13 Table 3-4. 1H chemical shifts (ppm) in compounds 3.11g-i and 3.13

\Position Compd.\

1

5

3.11g

11.70

7.99 7.27 7.75 7.75 7.45 7.60 7.53 10.42 6.81 1.97a 6.93 6.63

3.11h

11.76

8.01 7.33 7.77 7.87 7.45 7.60 7.55 3.14a 7.11 -

3.11i

11.80

8.03 7.29 7.75 7.86 7.45 7.60 7.55

3.13 (E)c

10.59d 6.86 (CH), 2.40 (CH3)

-

-

-

3.19a 8.06 7.10 7.40 7.04

3.13 (Z)c

12.80d 6.85 (CH), 2.38 (CH3)

-

-

-

3.24a 7.58 7.12 7.37 7.08

a

6

7

8

1'

2'

3'

1'''

b

4'''

5'''

7'''

7.39 6.90

6.94 6.59 7.19 6.85

CH3. b4.34 (CH2α), 5.83 (CH), 5.09 (CH2γ, H-cis to H), 5.12 (CH2γ, H-trans to H). cAt 70 °C. dH1''

56

6'''

3.2.3 Stereochemistry of the C=N Bonds The barrier to rotation about a C=N bond is lower than that about a C=C bond, and decreases with the electronegativity of the substituents on the N atom. In hydrazones, often the E and Z forms equilibrate in a matter of hours or days [1984JCS(P1)2109]. NOe experiments when both forms were available identified the isomers and demonstrated that 13C chemical shifts can be diagnostic for the stereochemistry. In particularly, carbons alpha to the C=N carbons are shifted upfield when syn to the vicinal nitrogen, relative to the situation when they are anti. This is the gamma effect and it is due to steric compression. Table 3-5. 13C chemical shifts (ppm) in compounds 3.11g-i, 3.13 and 3.29a-c \Position 2 4 4a 5 6 7 8 8a 1' 2' Compd.\

3'

5a'

3.11g

NMa 161.2 116.1 128.0 123.8 136.1 117.2 140.0

129.6 129.9 129.3 137.3

3.11h

NM

161.2 116.3 128.0 124.1 136.0 117.8 139.8

129.2 129.9 129.9 136.9

3.11i

NM

161.2 116.3 128.1 123.9 138.0 117.6 140.0

129.6 130.0 128.8 137.0

3.13 (E)b

NM

168.6 (q), 115.4 (CH), 24.1 (CH3)

-

-

-

-

3.13 (Z)b

NM

168.8 (q), 115.4 (CH), 24.0 (CH3)

-

-

-

-

2'''

3'''

3a'''

4'''

5'''

6'''

7'''

7a'''

other

3.29a (E)c 166.8 134.6 118.9 125.5 120.8 128.1 109.0 140.7 3.29a (Z)c 158.9 132.2 124.3 118.5 120.6 127.1 109.0 139.6 3.29b (E)c 165.1 133.8 118.0 125.2 121.3 128.0 107.5 141.9 3.29b (Z)c 156.9 130.8 123.4 118.0 120.9 126.8 107.4 140.7 3.29c (E)c 166.8 134.8 118.9 126.1 129.4 128.4 108.6 138.5 3.11g

166.8 144.8 118.2 128.0 131.0 131.9 110.1 141.0

21.3 (CH3)

3.11h

164.1 142.2 114.6 129.0 118.8 133.6 110.7 143.2

25.9 (CH3)

3.11i

164.9 NM

117.6 127.7 122.3 131.1 109.5 143.5

41.9 (Cα), 132.8 (C), 117.2 (Cγ)

164.6 NM

116.2 125.5 122.7 131.7 109.4 144.5

-

161.9 131.7 120.7 120.1 123.4 130.4 110.1 143.0

-

3.13 (E)b b

3.13 (Z) a

b

c

Not measured. At 70 °C. From ref.[1996JHC675]

57

Table 3-6.

15 15

N chemical shifts (ppm) in compounds 3.11g-i and 3.13. Protons which couple to a N are given in parentheses

\Position 1 Compd.\

3

104.2 (H8) 156.7(H1') NMa 109.4 (H8) 161.8 (H1') NM 105.1 (H8) 157.5 (H1') NM

3.11g 3.11h 3.11i

3.13 (E)b 254.2 (CH3) 3.13 (Z)b 252.4 (CH3,CH,NH ) a

1''

NM 164.3 (NH, CH)

2''

1'''

377.6 (H1''') NM NM

133.0 (H1''', H7''') 131.8 (CH3, H7''')

136.2 (CH2α, CH, H7''') NM NM 340.0 (NH, H7''') 133.1 (CH3, H7''', H6''')

Not measured. bAt 70 °C

The E-Z pairs of the related isatin guanylhydrazones [1996JHC675] 3.29a and 3.29b (Table 3-4) display 13C chemical shift differences of ca. 6 ppm in positions 2''', 3a''' and 4'''. The 13

C chemical shifts of these carbons in 3.11g-i demonstrate the E configuration for the C3'''=N2''

double bond in these compounds. The chemical shifts of the same positions in 3.13 (E) and 3.13 (Z) confirm the assignment. The X-ray structure of 3.11b (Figure 3-10) shows the E configuration of the C3'''=N2'' double bond. Since this is the expected configuration of hydrazones of aldehydes [1984JCS(P1)2109] it is reasonable to assume the same all of the 3.11a-f. The C2=N1'' double bond is in the E configuration in 3.11b. The same configuration is expected for all of 3.11a-i, because the 2-hydrazono-3-phenyl-3H-quinazolin-4-one moiety is common to all of these compounds. The Z configuration would be higher in energy due to the steric hindrance between the phenyl in position 3 and N2''. There is also a hydrogen bond between H1 and N2'', which stabilizes the E form. 3.3 Conclusions 15

N NMR is a powerful technique for the elucidation of tautomerism involving protonation

of a nitrogen atom, since a large chemical shift, ca. 100 ppm, is expected for the nitrogen of the two tautomeric forms. 15N chemical shifts can be measured by indirect detection, through 58

coupling of the nitrogen reporter of tautomerism with non-exchangeable protons 2 or 3 bonds away. With typical samples of 15-30 mg, at natural abundance of 15N, the total experiment time was ca. 2 hours. 2-(2-Substituted-methylenehydrazinyl)-3-phenylquinazolin-4(3H)-ones (3.11a-i) were found to be predominantly in the imino form in DMSO solution, following the tautomeric preferences of the aminoguanidines. 2-Hydrazino-3-phenyl-3H-quinazolin-4-one (3.22) itself is in the amino form, demonstrating that the terminal nitrogen in the hydrazine moiety has to be involved in a double bond for the imino form to prevail. When the 3,4-dihydro-4-oxo-3-phenylquinazolin-2-yl moiety of 3.11a-i is replaced by a more aromatic one, as in benzimidazol-2-yl in 3.12 or 4,6-dimethylpyrimidin-2-yl in 3.13, the amino tautomer dominates the equilibrium in DMSO solution. 3.4 Experimental 3.4.1 General Methods Melting points were determined on a capillary point apparatus equipped with a digital thermometer. The NMR spectra were recorded on a Varian Inova instrument, operating at 500 MHz for 1

H, 125 MHz for 13C and 50 MHz for 15N, equipped with a three channel, 5 mm, indirect

detection probe, with z-axis gradients. The solvent was DMSO-d6, and the temperature was 25 °C, unless specified otherwise. The chemical shifts for 1H and 13C were reference to the residual solvent signal, 2.50 ppm for 1H and 39.5 ppm for 13C, 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 [2002COR35]. 59

1

H spectra were acquired in one transient, with a 90° pulse, no relaxation delay and an

acquisition time of 5 s, over a spectral window from 16 to -2 ppm. The FID was zero-filled to 131072 points prior to Fourier transform. Typically, 1H-13C gHMBC spectra were acquired in 2048 points in f2, on a spectral window from 6.5 to 11 ppm, and 1 s relaxation delay. In f1, 256 increments were acquired in 1 transient over a spectral window from 110 to 170 ppm, and then the corresponding FID’s were zero-filled twice prior to the second Fourier transform. 1

H-15N CIGAR-gHMBC spectra were acquired with a pulse sequence optimized for 15N, as

described in ref. [2003MRC307]. 2048 points were acquired in f2, over a spectral window typically from 6.5 to 11 ppm, with 1 s relaxation delay. 1024 increments were acquired in f1, on a spectral window from 0 to 400 ppm, and the corresponding FID was zero-filled twice prior to Fourier transform. The accordion delay was optimized for a value of 1H-15N coupling constants between 3 and 10 Hz. The number of transients per increment was between 4 and 64, depending on the concentration of the sample. Total experiment time was in most cases, ca. 2 h. 3.4.2 Preparation of 3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (3.21) Anthranilic acid (1.37 g, 0.01 mol) and phenylisothiocyanate (1.35 g, 0.01 mol) were heated under reflux in 50 mL ethanol for 2 h.The solid obtained was filtered off and purified by crystallization from DMF: white microcrystals (2.03 g, 80%); mp 313-315 ˚C [Lit. mp 300 ˚C] [1985JHC1535]. Anal. Calcd. for C14H10N2OS (254.31): C, 66.12; H, 3.96; N, 11.02. Found: C, 66.11; H, 3.78; N, 10.94. 3.4.3 Preparation of 2-hydrazino-3-phenylquinazolin-4(3H)-one (3.22) A mixture of 3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one 3.21 (0.25 g, 1 mmol) and hydrazine hydrate (99 %, 0.05 g, 10 mmol) was heated under reflux in n-butanol for 3 h. After the reaction was cooled down, a white precipitate separated that was recrystallized from n60

butanol to give the desired product in 79% yield (0.20 g, 0.8 mmol). White needles; mp 193-195 ˚C [Lit. mp 202-203 ˚C] [1985JHC1535]. Anal. Calcd. for C14H12N4O (252.28): C, 66.65; H, 4.79; N, 22.21. Found: C, 66.27; H, 4.70; N, 21.96. 3.4.4 General Procedure for Preparing Compounds 3.11a-i 2-Hydrazinyl-3-phenylquinazolin-4(3H)-one 3.22 (0.25 g, 1 mmol) was heated under reflux in ethanol (25 mL) for 15 min. to 1 h with 1 mmol of the corresponding aldehyde or ketone 3.23a-i. The precipitate formed was collected and crystallized from the appropriate solvent to give the desired products in quantitative yields. (E)-2-((E)-Benzylidenehydrazono)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one (3.11a). The product was crystallized from EtOH to give white needles (98%); mp 215-217 ˚C [Lit. mp 218 ˚C] [1985JHC1535]. Anal. Calcd. for C21H16N4O (340.39): C, 74.10; H, 4.74; N, 16.46. Found: C, 74.21; H, 4.63; N, 16.46. (E)-2-((E)-(4-(Diethylamino)benzylidene)hydrazono)-3-phenyl-2,3-dihydro-quinazolin4(1H)-one (3.11b). The solid obtained was crystallized from DCM/hexanes to give the desired product as yellow needles (97%); mp 202-204 ˚C. Anal. Calcd. for C25H25N5O (411.51): C, 72.97; H, 6.12; N, 17.02. Found: C, 72.81; H, 6.12; N, 17.11. X-ray experimental for 3.11b Data were collected with a APEX II CCD area detector, using graphite monochromatised Mo Kα radiation (λ = 0.71073 Å). The structures were solved by direct methods using SHELXS [1990AXA467], and refined on F2 using all data by full-matrix least-squares procedures with SHELXL-97 [G. M. Sheldrick, SHELXL-97, University of Göttingen, 1997]. Hydrogen atoms were included in calculated positions with isotropic displacement parameters 1.3 times the isotropic equivalent of their carrier atoms.

61

Crystal data and structure refinement for 3.11b Identification code

pjs

Empirical formula

C25 H25 N5 O

Formula weight

411.50

Temperature

153(2) K

Wavelength

0.71073 Å

Crystal system

Orthorhombic

Space group

P 21 21 21

Unit cell dimensions

a = 5.1252(10) Å

α= 90°.

b = 17.913(4) Å

= 90°.

c = 23.305(5) Å

γ = 90°.

Volume

2139.6(7) Å3

Z

4

Density (calculated)

1.277 Mg/m3

Absorption coefficient

0.081 mm-1

F(000)

872

Crystal size

0.20 x 0.08 x 0.05 mm3

Theta range for data collection

3.47 to 25.10°.

Index ranges

-5

Suggest Documents