Oualid TALHI Síntese e actividade biológica de híbridos ... · como flavonoides, cromonas e...

Universidade de Aveiro

2012

Departamento de Química

Oualid TALHI Síntese e actividade biológica de híbridos polifenólicos Synthesis and biological activities of polyphenolic hybrids

Universidade de Aveiro

2012

Departamento de Química

Oualid TALHI

Síntese e actividade biológica de híbridos polifenólicos Synthesis and biological activities of polyphenolic hybrids Avaliação da actividade anticancerígena, anti-inflamatória e antioxidante Evaluation of Anti-cancer, Anti-Inflammatory and Antioxidant activities

Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Química, realizada sob a orientação científica do Doutor Artur Manuel Soares da Silva, Professor Catedrático, e da Doutora Diana Cláudia Gouveia Alves Pinto, Professora Auxiliar, ambos do Departamento de Química da Universidade de Aveiro

The present dissertation is submitted to the University of Aveiro in purpose of fulfilling the requirements to obtain the degree of Doctor of philosophy (PhD) in Chemistry, which was realized under supervision of Dr. Artur Manuel Soares da Silva, Full Professor, and Dr. Diana Cláudia Gouveia Alves Pinto, Assistant Professor, both at the Department of Chemistry, University of Aveiro

Financial support of European Commission, Seventh Framework Programme (FP7/2007-20139] Marie Curie grant agreement nº 215009 RedCat Project

FCT and FEDER for funding the Organic Chemistry Research Unit

o júri

presidente Prof. Doutor António Carlos Matias Correia

Professor Catedrático do Departamento de Biologia da Universidade de Aveiro

Prof. Doutor Carlos Alberto Mateus Afonso Professor Catedrático da Faculdade de Farmácia da Universidade de Lisboa

Prof. Doutor José Abrunheiro da Silva Cavaleiro Professor Catedrático do Departamento de Química da Universidade de Aveiro

Prof. Doutor Artur Manuel Soares da Silva (Orientador/Supervisor) Professor Catedrático do Departamento de Química da Universidade de Aveiro

Doutora Paula Alexandra de Carvalho Gomes Professora Auxiliar com Agregação da Faculdade de Ciências da Universidade do Porto

Doutora Susana Paula Graça da Costa Professora Auxiliar da Escola de Ciências da Universidade do Minho

Doutoura Diana Cláudia Gouveia Alves Pinto (Co-Orientador/Co-Supervisor) Professora Auxiliar do Departamento de Química da Universidade de Aveiro

http://www.bio.ua.pt/pageperson.aspx?id=426

acknowledgments

I have taken efforts in achieving this study project. However, it would never be possible without the precious support of ALLAH the lord of all beings to whom I address all my best expressions of gratitude. Thanks to God “ALLAH” and may his peace and blessings be upon all his prophets for granting me the chance and the ability to successfully complete this study and then to many individuals and organizations for their kind help. I would like to extend my sincere thanks to all of them. I am highly indebted to my PhD supervisor Professor Artur Silva for his guidance and constant supervision as well as for providing necessary information regarding the PhD project and also for his support in completing the project. I will never forget his first advice to me “You are not obliged to love everyone but you are obliged to work with everyone”. I wish to express my deepest gratitude to my co-supervisor Dr. Diana Pinto for her valuable advice and guidance of this work, her continuous availability, services and smiling face. I also want to express my gratitude to the official referees of my dissertation work for devoting time and efforts to analyze and evaluate the scientific content. Special thanks are dedicated to Professor Gilbert Kirsch for his kind reception in his laboratory LIMBP in Metz-France and his availability in performing analysis the high resolution mass spectra of compounds. I am also grateful to my biologist collaborators, Lidia Brodziak-Jarosz, Jana Panning, François Gaascht, Emilie Bana, and all their supervisors and collaborators. I hope to keep our strong scientific relationship for the future. A word of gratitude is addressed to all staff of the Chemistry Department, University of Aveiro, especially to Dr. Hilário Tavares, thank you for your prompt contribution in obtaining NMR spectra. I would be very pleased to mention the precious efforts of Dr. Filipe Paz and Dr. José Fernandes who both have introduced me to the Single-Crystal X-ray technique and hardly contribute in achieving the structural solution of many complex compounds. Many thanks and hoping to go on with our collaboration in this field. Thanks are due to the University of Aveiro, FCT and FEDER for funding the Organic Chemistry Research Unit and the Portuguese National NMR Network (RNRMN). I should be very thankful to the (European Community’s) Seventh Framework Programme (FP7/2007-20139] under grant agreement nº 215009) for financial support of this PhD project. I would never forget the important role of Professor Maamar Hamdi in succeeding my personal aims of studies and life. Many thanks to you and all your family.

I would like to express my special gratitude and thanks to all ESRs and ERs involved in the RedCat Marie Curie ITN project and persons who were giving me attention and time. My thanks and appreciations also go to my colleagues in developing the project and people who have willingly helped me out of their abilities. Specials words of kindness and friendship go to my colleagues of the Organic Chemistry Research Group and all the staff of my laboratory, working with them was not only an usual experience but was a special period of sweetness in life. I would like to express my gratitude towards my parents, especially my ever beloved MAMA who is still bearing the long distance from her son. Many thanks to all members of my family Sofiane, Riad, Redouane, Meriem and their small families, your kind co-operation and encouragement helped me in completion of this project. Finally, I am particularly grateful to my wife, Lobna, for helping and assisting me in the last hard stages of this work. Thank you so much my dear.

palavras-chave

Híbridos polifenólicos; benzofuran-3-ona; benzopiran-(2 e 4)-ona; hidantoína; uracilo; adições conjugadas 1,4; reação em cascata; atividade biológica; atividade anticancerígena, anti-inflamatória e antioxidante; RMN 2D, difração de raios X.

resumo

Os compostos polifenólicos constituem uma classe de metabolitos secundários de plantas, mas existe também uma enorme quantidade de derivados sintéticos ou semi-sintéticos contendo múltiplas unidades fenólicas. Estes compostos apresentam importantes características biológicas, que dependem das suas estruturas básicas. Certos derivados desta família de compostos, tais como flavonoides, cromonas e cumarinas contribuem para os benefícios da dieta humana, e partilham o núcleo de benzopiran-(2 e 4)-ona ou benzofuran-3-ona. A presente dissertação inclui uma introdução geral e três capítulos que descrevem as novas rotas sintéticas estabelecidas para a preparação de novos híbridos de diversos compostos polifenólicos, assim como a sua elucidação estrutural e termina com a presentação dos resultados da avaliação biológica desses mesmos compostos. No segundo capítulo discute-se a preparação de híbridos de pirimidina- e imidazolidina-polifenóis, especialmente a síntese diastereoseletiva de novos híbridos benzofuran-3-ona-hidantoína e derivados de uracilo. A rota sintética envolve a ação de carbodiimidas sobre os ácidos cromona-(2- e 3)-carboxílicos num só passo ou em dois passos sequenciais, catalisada por uma base orgânica ou inorgânica. O terceiro capítulo descreve reações do tipo adições conjugadas 1,4 - hetero-ciclisações em cascata de compostos 1,3-dicarbonílicos em ácido cromona-3-carboxílico catalisadas por uma base orgânica, que originaram novas cromonas, cromanonas e flavonas polissubstituídas. As bispiranonas [bispiran-2 e 4)-onas] foram elaboradas numa reacção de acoplamento da 4-hidroxicumarina ou da lactona do ácido triacético com o ácido cromona-3-

carboxílico ou precursores formil-funcionalizados (ω-formil-2’-hydroxy

acetofenonas e cromona-3-carbaldeídos) utilizando organocatálise básica. Finalmente, alargou-se o estudo das adições conjugadas 1,4 para uma variedade de 4-hidroxipiran-2-onas e cetonas α,β-insaturadas para originar novos análogos de warfarina. Obteve-se uma variedade de estruturas complexas por hibridação das unidades de 4-hidroxicumarina ou da lactona do ácido triacético com os novos derivados de cromonas polissubstituídas.

Todos as reações foram executadas em condições suaves e ambientalmente favoráveis, utilizando a 4-pirrolidinopiridina como organocatalisador básico. As estruturas dos novos híbridos polifenólicos foram caracterizados por técnicas espectroscópicas de alta resolução, incluindo espectroscopia de ressonância magnética nuclear (1D e 2D) e por difractometria de raios-X, que nos permitiram resolver o complexidade estrutural dos compostos sintetizados.

O quarto capítulo apresenta os resultados da avaliação biológica obtidos com os híbridos polifenólicos sintetizados neste trabalho, mostrando a possibilidade de seu envolvimento na terapia do cancro. A maioria dos compostos foram avaliados quanto ao seu efeito sobre a citotoxicidade e proliferação de células leucémicas e ao seu envolvimento na regulação de via pró-inflamatória NF-kB, na qual, os híbridos de biscumarinas exibiram actividades elevadas (IC50 = 6-19 µM para inibição de NF-kB depois de 8 horas de incubação e IC50 = 15-39 µM para efeitos citotóxicos em células cancerosas, após 24 horas de incubação). Uma inibição moderada das enzimas HDAC e Cdc25 foi induzida pelos derivados de benzofuran-3-ona-hidantoína. Catorze dos novos derivados polifenólicos polissubstituídos, tendo como estrutura básica a benzopiran-4-ona, foram avaliados pela sua actividade quimiopreventiva do cancro mediada pela indução de sinalização citoprotectora Nrf2 (fator 2 relacionado com o fator nuclear da proteína E2) e capacidade para inibir a proliferação das células de cancro da mama. Os derivados da classe das cromanonas foram identificados como os indutores mais potentes da actividade Nrf2. As concentrações necessárias para aumentar a actividade de luciferase em 10 vezes (C10) foram de 2,8-21,3 µM. Todos os novos híbridos polifenólicos que apresentam atividade citotóxica e anti-proliferativa não afectam o crescimento de células saudáveis periféricas do sangue (PBMC) (IC50 > 50 µM), indicando a sua seletividade para as células cancerosas e sugerindo que alguns deles são estruturalmente interessantes para posteriores análises. A avaliação da atividade antioxidante utilizando os testes do radical livre DPPH e o poder redutor do ião férrico FRAP foram realizados em algumas estruturas híbridas polifenólicas. .

keywords

Polyphenolic hybrids; benzofuran-3-one; benzopyran-(2 and 4)-one; hydantoin, uracil; 1,4-conjugate addition; tandem reaction; biological activity; anticancer; anti-inflammatory; antioxidant; 2D NMR, X-ray diffractometry.

abstract

Polyphenolic compounds represent a class of secondary metabolites of plants, but there are also a great number of synthetic or semi-synthetic derivatives characterized by the presence of multiples phenol moieties. Polyphenolic compounds underlie a number of biological characteristics such as the metabolic and therapeutic properties which depends on their basic phenolic structure. Certain members of this class, like flavonoids, chromones and coumarins contribute to the therapeutic benefits of the human diet, they all share the benzopyran-(2 or 4)-one or benzofuran-3-one nucleus. The present dissertation includes a general introduction and three main chapters describing the new synthetic methodologies established for the production of new polyphenolic hybrids, their fine structural elucidation and their biological application in cancer therapy involving redox-regulation and inflammation pathways The second chapter discusses the preparation of pyrimidine- and imidazolidine- based polyphenolic hybrids, especially the diastereoselective synthesis of new benzofuran-3-one-hydantoin hybrids and uracil derivatives. The organic synthetic route starts by the organic/inorganic base-catalyzed action of carbodiimides on chromone-(2 and 3)-carboxylic acids in a one-pot reaction or sequential steps. The third chapter describes the application of basic organocatalysis in the 1,4-conjugate additions / heterocyclisations tandem processes of 1,3-dicarbonyls on chromone-3-carboxylic acid leading to novel polysubstituted- chromones, chromanones and flavones. The bispyranone scaffold [bispyran-(2 and 4)-ones] have been elaborated in a one-step coupling reaction of 4-hydroxycoumarin or triacetic acid lactone with chromone-3-carboxylic acid or formyl-functionalized precursors (ω-formyl-2’-hydroxyacetophenones and chromone-3-carbaldehydes). Finally, the application of the 1,4-conjugate addition approach is extended to a variety of 4-hydroxypyran-2-ones reacting with α,β-unsaturated ketones, including chalcones, to give the new warfarin-analogues. Also a variety of complex structures have been obtained by hybridizing 4-hydroxycoumarin or triacetic acid lactone units with the newly synthesized poly-substituted- chromones.

http://en.wikipedia.org/wiki/Synthetic_compound

http://en.wikipedia.org/wiki/Semisynthesis

http://en.wikipedia.org/wiki/Phenol

All the above organic reactions proceeds in mild and environmentally friendly conditions using 4-pyrrolidinopyridine as basic organocatalyst. The structures of the novel polyphenolic hybrids have been characterized by high resolution spectroscopic techniques including extensive 1D, 2D-NMR and single-crystal X-ray diffractometry which largely helped to solve the structural complexity. The fourth chapter briefly introduces the biological screenings performed on the novel synthesized polyphenolic hybrids showing their possible involvement in cancer therapy. Most of the newly obtained molecules have been evaluated for their effect on cytotoxicity and proliferation of leukemic cell lines and their involvement in regulation of NF-κB pro-inflammatory pathway, in which the biscoumarin hybrids exhibited high activities (IC50 = 6-19 µM for NF-κB inhibition after 8 hours of incubation and IC50 = 15-39 µM for cytotoxic effects on cancer cell after 24 hours of incubation). Moderate inhibitions of HDAC and Cdc25 enzymes are noticed for the previously mentioned benzofuran-3-one-hydantoin candidates. Fourteen polysubstituted benzopyran-4-one based polyphenolics were examined for their cancer chemopreventive activity mediated by induction of cytoprotective Nrf2 (nuclear factor E2-related protein 2) signalling and their ability to inhibit proliferation of breast cancer cells. Derivatives of the chromanone class were identified as the most potent inducers of Nrf2 activity. The concentrations required to increase luciferase activity by 10-fold (C10) were 2.8-21.3 μM. All the new cytotoxic and anti-proliferative polyphenolic hybrids did not affect the growth of healthy peripheral blood mononuclear cells (PBMC) (IC50 > 50 μM), indicating their selectivity for cancer cells, which make some of them interesting lead structure for further analyses. Antioxidant activity evaluations using DPPH free radical scavenging and ferric ion reducing FRAP tests have been carried out on some underlined polyphenolic hybrid structures.

vii

ABBREVIATIONS

2-CHCA Chromone-2-carboxylic acid

2D-NMR Bidimensional nuclear magnetic resonance spectroscopy

2-HPP 2’-Hydroxypropiophenone

2-STC 2-Styrylchromone based compound

3-CHCA Chromone-3-carboxylic acid

4-HCOM 4-Hydroxycoumarin

4-PPy 4-Pyrrolidinopyridine

BF Benzofuran-3-one

BV Baker-Venkataraman

Cdc25 Cell division cycle

CDK Cyclin-dependent-kinases

CHAL Chalcone

CHR Chromone based compound

CHRM Chromanone based compound

d Doublet

DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCC Dicyclohexylcarbodiimide

dd Double of doublet

ddd Double of doublet of doublet

DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

DEA Diethylamine

DMF N,N-Dimethylformamide

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

DPPH 2,2-Diphenyl-1-picrylhydrazyl

equiv Molar equivalente

ESI Electrospray ionization

viii

FLV Flavone based compound

HD Hydantoin

HDAC Histone deacetylases

HIV Human immunodeficiency virus

HMBC Heteronuclear multiple bond coherence (bidimensional NMR)

HOPO-1 (2-hydroxyphenyl)-3-oxoprop-1-enyl-

HOPO-2 (2-hydroxyphenyl)-3-oxoprop-1-yl-

HPLC High performance liquid chromatography

HRMS High resolution mass spectrometry

HSQC Heteronuclear single quantum coherence (bidimensional NMR)

IC50 Half maximal inhibitory concentration

IUPAC International Union of Pure and Applied Chemistry

J Coupling constant

K562 Human chronic myelogenous leukemia cell lines

m Multiplet

m/z Mass-to-charge ratio (HRMS parameter)

MAD Michael addition or 1,4-conjugate addition

MCF7 Breast cancer cell lines MCF-7 is the acronym of Michigan Cancer

Foundation - 7

NF-κB Transcription nuclear factor

13C NMR Carbon-13 nuclear magnetic resonance spectroscopy

NOESY Nuclear Overhauser effect spectroscopy (bidimensional NMR)

Nrf2 Nuclear factor E2-related protein 2

ORAC Oxygen radical absorbance capacity

PBMC Peripheral blood mononuclear cells

ppm Part per million

q Quartet

1H RMN Proton-1 nuclear magnetic resonance spectroscopy

RNA Ribonucleic acid

ROI Reactive oxygen intermediate

ix

ROS Reactive oxygen species

rt Room temperature

SAR Structure-activity-relationship

spet Septet

t Triplet

TAL Triacetic acid lactone or 4-hydroxy-6-methylpyran-2-one

TLC Thin layer chromatography

TNF-α Tumor necrosis factor-alpha

Tr Trolox

U Uracil

UV Ultraviolet

WRF Warfarin

XAN Xantohumol

Chemical shift parameter (ppm)

x

TABLE OF CONTENTS

Acknowledgments ................................................................................................................... i

Resumo (abstract in Portuguese version) ............................................................................ iii

Abstract (English version) ...................................................................................................... v

Abbreviations ........................................................................................................................ vii

Table of Content ..................................................................................................................... x

Chapter 1 – General Introdcution .......................................................................................3

1.1. Introduction .....................................................................................................................3

1.2. Project Plan .....................................................................................................................5

1.3. Overview .........................................................................................................................7

1.4. References ....................................................................................................................11

Chapter 2 - Synthesis of Hydantoin- and Uracil- Polyphenolic Hybrids .....................17

2.1. Benzofuran-3-one-hydantoin hybrids ...........................................................................17

2.1.1. Natural and synthetic organic hybrids .................................................................. 17

2.1.2. Benzofuran-3-ones and hydantoins based molecules ........................................ 19

2.1.3. Synthesis of benzofuran-3-one-hydantoin Hybrids ............................................. 23

2.1.3.1. Synthesis of 1’,3’-disubstituted spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones ........................................................................................................................ 24

2.1.3.2. Synthesis of 1’,3’-disubstituted 5-(3-oxo-2,3-dihydrobenzofuran-2-

yl)imidazolidine-2,4-dione .......................................................................................... 29

2.1.3.3. Synthesis of 1’,3’-disubstituted 5-[3-oxobenzofuran-2(3H)-ylidene]-

imidazolidine-2,4-dione .............................................................................................. 38

2.2. Polysubstituted uracil derivatives .................................................................................46

2.2.1. Uracil and nucleoside-based derivatives ............................................................. 46

2.2.2. Synthesis of 5-(hydroxybenzoyl)-1,3-disubstituted uracil derivatives ................. 47

2.3. References ....................................................................................................................50

xi

Chapter 3 - Synthesis of Benzopyran-(2 and 4)-ones Polyphenolic Hybrids ........... 59

3.1. Flavonoids, 2-styrylchromones and related benzopyran-4-one based compounds ... 59

3.1.1. Flavonoids, 2-styrylchromones: a word on their natural occurrence,

biological applications and chemistry ........................................................................ 59

3.1.1.1. Flavones ................................................................................................... 61

3.1.1.2. (E)-2-Styrylchromones ............................................................................. 62

3.1.1.3. Synthetic methods of flavones and 2-styrylchromones ........................... 63

3.1.2. Design and synthesis of benzopyran-4-one based polyphenolic hybrids ........... 65

3.1.2.1. Generalities on 1,4-conjugate addition (Michael additions) ......................... 68

3.1.2.2. Reactivity of chromone-3-carboxylic acid ..................................................... 69

3.1.2.3. Michael addition on chromone-3-carboxylic acid: a one-pot tandem

reaction towards novel polysubstituted -chromones, -flavones, and -

chromanones.............................................................................................................. 78

3.1.2.3.1. Synthesis of 3-substituted HOPO-1 -chromones, -flavones and -2-

styrylchromones .................................................................................................... 78

3.1.2.3.2. Synthesis of 2,3-disubstituted chromanones........................................ 83

3.1.2.3.3. Synthesis of 3-substituted HOPO-1 -2-(4-arylbuta-1,3-

dienyl)chromones .................................................................................................. 84

3.1.2.4. Michael addition on chromone-3-carboxylic acid: Mechanistic and

structural studies ........................................................................................................ 85

3.1.2.4.1. Mechanism, generalization and limits of Michael addition on 3-

CHCA ..................................................................................................................... 85

3.1.2.4.2. Structural characterization of the BP-4-based polyphenolic

compounds ............................................................................................................ 87

3.2. Hybrids of benzopyran-2-one and benzopyran-4-one ............................................... 103

3.2.1. Benzopyran-(2 and 4)-ones: The golden biologically active rings ..................... 103

3.2.1.1. Biscoumarins and bischromones as biologically active hybrids ................ 105

3.2.1.2. Warfarin: a benzopyran-2-one drug ........................................................... 106

3.2.1.3. Benzopyran-(2 and 4)-ones hybrids from nature ....................................... 107

3.2.2. Design and synthesis of benzopyran-(2 and 4)-ones hybrids ........................... 107

xii

3.2.2.1. Synthesis of bispyran-2-ones hybrids sharing HOPO-2 or BP-4 .............. 107

3.2.2.2. Michael addition of 4-hydroxypyran-2-ones (4-HCOM and TAL) on α,β

unsaturated ketone scaffolds ................................................................................... 119

3.2.2.2.1. Synthesis of warfarin-analogues ........................................................ 119

3.2.2.2.2. Synthesis of benzopyran-(2 and 4)-ones hybrids via Michael

addition ................................................................................................................ 128

3.3. References ................................................................................................................. 137

Chapter 4 – Biological screenings ................................................................................ 147

4.1. Chemistry, biology and medicine continuum ............................................................. 147

4.1.1. The transcription nuclear factor NF-κB .............................................................. 148

4.1.2. Leukemia cancer ................................................................................................ 149

4.1.3. Histone deacetylases (HDAC) ........................................................................... 150

4.1.4. Nuclear factor E2-related protein 2 - Nrf2: Keap1-Nrf2 activation pathway ...... 150

4.1.5. Cdc25 phosphatases.......................................................................................... 152

4.1.6. Antioxidant and radical scavenging capabilities ................................................ 153

4.2. Biological screenings of the new polyphenolic hybrids ............................................ 156

4.2.1. Cytotoxic and anti-proliferative effects of polyphenolic hybrids on K562 Cells . 156

4.2.1.1. Cytotoxic effects of benzofuran-3-one-hydantoin hybrids and

polysubstituted uracils ......................................................................................... 156

4.2.1.2. Cytotoxic and anti-proliferative effects of HOPO-1 and HOPO-2

substituted benzopyran-2-one and benzopyran-4-one based compounds ....... 158

4.2.2. Effect of polyphenolic hybrids on the NF-κB transactivation potential .............. 166

4.2.2.1. Effects of benzofuran-3-one-hydantoin on the NF-κB activation in

K562 cells ...................................................................................................... 166

4.2.2.2. Effects of HOPO-1 and HOPO-2 substituted benzopyran-2-one and

benzopyran-4-one based compounds on NF-κB activation in K562 cells ......... 167

4.2.2.3. Effect of the biscoumarin 16a on the NF-κB transactivation potential .. 168

4.2.2.3.1. Effect of the biscoumarin 16a on the NF-κB pathway in K562 cells .. 168

xiii

4.2.2.3.2. Effect of the biscoumarin 16a on the NF-κB pathway in jurkat cells .. 169

4.2.2.3.3. Effect of compound 16a on the NF-κB transactivation potential

analyzed by western blot ..................................................................................... 171

4.2.3. Viability and NF-κB pathway activation in K562 cells of 16a hybrid

substructures 2”-hydroxypropiophenone and 4-hydroxycoumarin .......................... 172

4.2.4. Biscoumarins cytotoxicity and NF-κB pathway inhibitory effect ........................ 176

4.2.5. Evaluation of HDAC inhibition of benzofuran-3-one-hydantoin hybrids ............ 181

4.2.6. Evaluation of Michael acceptor containing polyphenolic hybrids on the

activation of Keap1-Nrf2 pathway ............................................................................ 182

4.2.7. Evaluation of Cdc25 phosphatase inhibition of benzofuran-3-one-hydantoin

hybrids ...................................................................................................................... 184

4.2.8. Evaluation of the antioxidant activities ............................................................... 185

4.2.8.1. Ferric reducing antioxidant power (FRAP) assay .................................. 185

4.2.8.2. Free radical diphenylpicrylhydrazyl (DPPH) assay ……. ...................... 186

4.2.8.3. Antioxidant capabilities measurements ……. ........................................ 186

4.3. References ................................................................................................................. 188

Chapter 5 – Conclusion and Perspectives ................................................................... 193

5. Conclusion and perspectives ........................................................................................ 193

Chapter 6 – Experimental Part ............................................................................ 197

6.1. Materials ..................................................................................................................... 197

6.1.1. Analytical, chromatographic and biological techniques ..................................... 197

6.1.2. Chemicals and starting materials ....................................................................... 202

6.2. Experimental Methods ............................................................................................... 203

6.2.1. Synthesis of 1’,3’-disubstituted spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones 9a-c .............................................................................................................. 203

6.2.2. Synthesis of 1,3-disubstituted 5-(3-oxo-2,3-dihydrobenzofuran-2-

yl)imidazolidine-2,4-diones 10a-c ............................................................................ 204

xiv

6.2.3. Synthesis of 1,3-disubstituted 5-[3-oxobenzofuran-2(3H)-

ylidene]imidazolidine-2,4-dione 11a-c ..................................................................... 207

6.2.4. Synthesis of N,N-disubstituted-(carbamoyl)-4-oxo-4H-chromene-3-

carboxamide 12a(a) and 12b(a) ............................................................................. 210

6.2.5. Synthesis 1,3-disubstituted-5-(2-hydroxybenzoyl)pyrimidine-2,4(1H,3H)-

dione 12a-c ............................................................................................................. 211

6.2.6. Synthesis of the dimeric-product 13a via dimerisation of chromone-3-

carboxylic acid 2 ...................................................................................................... 213

6.2.7. Synthesis of 2-alkyloxychroman-4-ones 13a(b-d) ............................................. 214

6.2.8. Synthesis of enaminones 13a(e-g) .................................................................... 216

6.2.9. General procedure for Michael addition of 1,3-dicarbonyl compounds 4a-t on

the chromone-3-carboxylic acid 2: Synthesis of 3-(HOPO-1)-chromones,

-flavones, -2-styrylchromones 13a-q and 2,3-disubstituted chromanones 14q-t ... 216

6.2.10. General procedure for the base-catalyzed aldol-condensation of

cinnamaldehydes with (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-methyl-

4H-chromen-4-one 13f: Synthesis of 3-(HOPO-1)-2-(4-arylbuta-1,3-

dienyl)chromones 15a-c .......................................................................................... 226

6.2.11. General procedure for condensation of formyl precursors with 2-

hydroxypran-2-ones: Synthesis of bispyran-2-ones 16, 17, 18, 19 and 20 ............ 228

6.2.12. General procedure for Michael addition of 2-hydroxypyran-2-ones 6 and 7

on chalcones 8 and 3-(HOPO-1)-benzopyran-4-ones 13: synthesis of warfarin-

analogues 21 and benzopyran-(2 and 4)-one hybrids 22 ..................................... 235

6.3. References ................................................................................................................. 242

CHAPTER 1

- GENERAL INTRODUCTION -

Chapter 1 – General Introduction

3

1.1. INTRODUCTION

Over the past century and up-to-day, organic chemistry is considered as one of the

most important technological areas which have long served the humanity. A worth and

diversity of discoveries on natural and synthetic organic compounds exhibiting various

applications in medicine and agriculture, is known for the last decade. Several human-

threatening diseases are overcome when scientists have only used small organic

molecules obtained by developing ideas in laboratories or even accidentally discovered in

nature. Organic compounds are “living-matters” and are playing crucial functions in

human-body metabolism, indeed, human-beings (or animals) are continuously requiring

calorific energy from several natural sources of food (vegetal or animal) containing huge

amounts of organics like sugars, lipids and proteins which are necessary elements for

metabolic bio-reactions. At the same time, the human-body also needs other organic

molecules, not only for energetically purposes, but for important therapeutic aims, such as

a good metabolic functioning, health maintenance/protection and prevention from various

multifactorial diseases. Organic compounds exist in nature through which, the vegetal

kingdom has provided and still providing these multi-action components with biological

impacts like, vitamins, alkaloids, flavonoids, …etc. Most commonly, polyphenolic

compounds are found ubiquitously in plants. These polyvalent molecules are taking the

greatest part of the human diet acting as health protectors. Additionally, plants produce

polyphenolic compounds as secondary metabolites for self-protection purposes, for

instance, anti-fungal activities.

Humans understand that nature is the ever best manufacturer of these biologically

active molecules and even they know that nature always supplies them with the suitable

organic molecules for their health benefit, but humans’ curiosity is leading to extra

demands, since then, they started to defeat nature and effectively they did not stop

tracking and producing synthetic compounds for biological/medicinal purposes. Hopefully,

thanks to their creative and scientific abilities, humans have won the deal by creating

millions of organic structures capable to offer a variety of nutritional and biological actions

as the nature used to do so, or even better. The synthetic organic compounds have

greatly influence our lifestyles by surrounding us everywhere and in every simple example

like plastics, drugs, dyes, detergents, processed food …etc. This does not signify that

people can ignore the natural source of these interesting compound because, in fact,

nature is the starting point and the unique supplier of raw materials. The most important

point is that nature has initiated us to the key organic structures which are most useful for


4

our life, especially our biological needs. In this context, polyphenolic compounds have

gained a considerable attention persuading researches in the Organic and Medicinal

Chemistry fields. The beneficial effects inherent in their diverse basic structures, such as

the relevant benzopyranone and benzofuranone heteronucleus, give birth to the

categorization of some fascinating organic families like, chromones, flavonoids and

coumarins, the most studied natural-type of compound today.

The chemist rarely creates his molecules from his own imagination, indeed, he was, in

most of the time, copying from the mother nature structures and bringing to them from

slight to complex chemical modification in view of improving their biological effects and

reducing some associated drawbacks (toxicity, metabolic transformation). The chemist job

was accomplished thanks to the basis of synthetic organic chemistry science. In

particular, the tasks of an “organist/bioorganist” is searching for the best organic structure

(virtual or natural), easily synthetically accessed and structurally characterized, non-toxic

(no harmful side-effects) and metabolic resistant, being capable to respond to at least one

biological requirement for the human-body (or, in general, animals or plants). In the quest

for new potent anticancer, antioxidant, anti-inflammatory, antiviral and antifungal agents,

…etc, a huge scientific efforts have been conducted over the past century leading to a

considerable improvement in organic/bioorganic fields. Innumerous pharmacologically

active organic compounds were developed and typically inspired from natural models as

being the first key leader products in the drug discovery. In cancer therapy, natural

products are very important, since a large majority (79.8 %) of newly developed

anticancer drugs between 1981 and 2010 are of natural origin, the rest are organic

synthetic product which in fact are natural-type or inspired from nature models [1].

To fulfill the previously underlined tasks dedicated to a chemist, a platform/network

should be constructed within a scientific atmosphere involving several expertise of diverse

fields in organic chemistry and biology. As soon as the required tools are available, the

work is performed and the aims are achieved, all is based on the relevant Chemistry-

Biology-Medicine-Continuum. Therefore, the PhD dissertation presented herein, is a part

of a European project named RedCat (Redox Catalysts) established in 2009 and funded

by the European Community’s Seventh Framework Programme FP7/2007-20139 under

Marie Curie grant agreement nº 215009. RedCat is an Initial Training Network (ITN) for

early stage (PhD students) and experienced researchers (Post-Doc) (website:

http://www.redcat-itn.eu). RedCat provides research and training opportunities for 10 early

stage and 4 experienced researchers across Europe, with 10 partners from academic

http://www.redcat-itn.eu/


5

and/or industrial institutions and 8 associated partners in 5 European countries (Germany,

France, Luxembourg, Portugal, UK). RedCat conducts its own research, which addresses

highly significant, up-to-date research questions in the area of natural product research,

intracellular redox processes, drug development and green agriculture. Individual projects

run across scientific disciplines. They embrace chemistry, biochemistry, biology,

pharmacy, medicine and agricultural research. RedCat researchers receive extensive

training in research methods and research related subjects, including bioethics,

intellectual property and communication. Training is provided by experts in the field, at

excellent institutions with the most modern equipment and techniques.

The group of Professor Artur Silva, from the University of Aveiro (Portugal), is a

RedCat partner in charge of the molecular engineering and organic synthesis of natural-

type of compounds with applications in medicines and agriculture. In the last years,

Professor Silva and colleagues have conducted researches focusing on the synthesis of

new antioxidant and anti-inflammatory agents based on the benzopyranones (mainly

benzopyran-4-one) and the benzofuran-3-ones natural-type organic scaffolds. Several

successful and eco-friendly synthetic strategies were established aiming at the creation of

a molecular diversity. A range of biomimic polyhydroxylated 2-styrylchromones, flavones

and xanthones have been reported as showing potent antioxidant and anti-inflammatory

effects [2-21]. More recently, our research group have been working on the insertion of

the bio-activator moieties like prenyl- and glucosyl into the benzopyran-4-one nucleus

present in 2-styrylchromones, flavones and xanthones, in view of increasing their

hydrophilic or lipophilic character and increasing their biological potency [21-23].

1.2. PROJECT PLAN

The scientific project presented in this PhD dissertation, concerns the development of

simple and economic synthetic methodologies for the preparation of new organic

combinations of various heterocycles utilizing mainly the benzopyranone nucleus. The

project is particularly oriented to design and synthesize organic molecules which include a

biologically active natural product (like flavonoid, chromone, coumarin, pyrimidine,

imidazolidine, …etc) linked together or with selected organic and bioorganic

moieties/pharmacophores in a form of hybrid scaffolds.

Taking into account the initial information we gained about the important structural

features for a good antioxidant and anti-inflammatory agent involving the above-

mentioned skeletons, we have succeeded, in the present project, the development of new


6

generation of hybrid compounds based on biologically significant heterocycles. In the

quest for novel drug entities, the hybrid approach is a promising path to obtain new

molecular entities that can effectively target multifactorial diseases. The strategy is not so

common in medicinal chemistry, since the organic chemist (or biochemist) is often

tracking low molecular weight molecules with less structural complexity and potent

biological actions. But in fact, the covalent combination of distinct biological organic

entities in one molecule will certainly provide new electronic properties influencing the

whole molecule interactions with biological systems. It usually brings additive or even new

biological effects while the combined molecular weight of the constitutive organic

fragments could also be in favor of a reduced metabolic transformations and therefore an

increased bioavailability in the circulatory system (Fig. 1.1).

Figure 1. 1. Concept of organic molecules hybridizing and biological profile prediction

The present project is mainly divided into two parts: I) organic synthesis of the desired

hybrid compounds which has been carried out at the University of Aveiro within the

research unit QOPNA of the chemistry department, and II) biological evaluations, in

which, a set of screenings have been performed covering the applications of the obtained

compounds in developing drugs in the diverse field of anticancer agents, which represents

some of the major use of natural mimic products in medicinal chemistry. We also

contemplate their applications in other areas of biomedicine including antioxidant agents,

anti-inflammatory drugs in order to reach various therapeutic aims. All the aforementioned

biological studies have been conducted by early stage researcher-biologists from different

specialized institutions within the RedCat project including partners from, Epigenomics

and Cancer Risk Factors, Redox Regulation, DKFZ, Heidelberg (Germany); Laboratoire


7

de Biologie Moléculaire du Cancer, Hôpital Kirchberg, (Luxembourg) and Laboratoire

d’Ingénierie Moléculaire et Biochimie Pharmacologique, Metz, (France).

1.3. OVERVIEW

Preliminary results showed that the insertion of different organic moieties and/or

organic nucleus into the benzopyranone ring to form hybrid compounds, leads to a

hundred of novel structures with promising biological potential. In contrast, hybrids based

on benzofuran-3-one, hydantoin, uracil, chromone, flavone, 2-styrylchromone, 4-

hydroxycoumarin, triacetic acid lactone and chalcone, have been constructed and

effectively showed from moderate to potent biological properties, such as in the anti-

inflammation pathways as inhibitors of NF-κB activation, impact on cell growth and

proliferation, selective cytotoxicity to cancer cells, HDAC and Cdc25 enzymes inhibition,

cancer chemo-preventive effects through activation of trigger Keap1-Nrf2 pathway,

antioxidant capabilities and free radical scavenging [Talhi et al. results in publication,

2012]. The simplicity and efficiency of our synthetic strategy is mainly seen in the soft

execution using organo-base-catalysis to combine available starting materials like

chromone-2- 1 and chromone-3- 2 carboxylic acids (2-CHCA, 3-CHCA), carbodiimides 3,

1,3-dicarbonyls 4, chromone-3-carbaldehydes 5, 4-hydroxycoumarin 6 (4-HCOM),

triacetic acid lactone 7 (TAL ) and chalcones 8 (CHAL) (Fig. 1.2).

O

O

O

N C N

R

R

R

O

R

OH

OH

O O

OH

O

OH

O

O

O O

O

H

O

OH

O

O

R R

R

13

2

45

6 7 8

Figure 1. 2. Starting materials used in the synthesis of the polyphenolic hybrids

Some of the major focus is to evaluate the biological nucleus functions within the

hybrid molecule, although the study of its substitution influence have also been carried out

leading to structure-activity-relationship (SAR) considerations. Recent interest in

polyphenolic derivatives of benzopyranones have been stimulated by the potential health

benefits arising from their antioxidant and anti-inflammatory activities; more available data


8

have also demonstrated their high propensity to transfer electrons, to chelate ferrous ions,

to scavenge reactive oxygen species (ROS) as well as to inhibit lipoxygenases and

cyclooxygenases which were strongly associated with the presence of ortho-

dihydroxyphenyl (or catechol group) as the main structural feature of the polyphenolic

candidate. From our screening results, we have further deduced some SARs showing that

the ortho-hydroxybenzoyl substituent and the conjugated α,β-unsaturated-carbonyl

systems (like in chalcones, flavonoids and coumarins) and their dihydro- analogues (like

in chromanones, dihydrochalcones), are all essential elements for efficient antioxidant and

anti-inflammatory activities. These structural patterns are figuring in our molecular hybrid

models taking into account various of their stereochemical properties. Figure 1.3

represents the novel synthesized polyphenolic hybrids that are the subject of our

biological study.

The present dissertation includes three chapters describing the new synthetic

methodologies established for diverse production of the new polyphenolic hybrid

compounds, their fine structural elucidation and their biological properties which especially

undertakes their application as anticancer agents together with the involvement in further

medicinal utilities, such as redox-regulation and anti-inflammatory pathways. The second

chapter will discuss the elaboration of pyrimidine- and imidazolidine- based polyphenolic

hybrids 9-12, especially the new benzofuran-3-one-hydantoin (BF-HD) hybrids 10, 11 and

uracil (U) derivatives 12. The synthetic approaches starts by the action of carbodiimides 3

on 2-CHCA or 3-CHCA in a one-pot reaction or sequential steps using organic or

inorganic base catalysis. The third chapter describes various applications of organo-base

catalysis in promoting 1,4-conjugate additions/hetero-cyclisations tandem process toward

the synthesis of novel polysubstituted chromones (CHR), 2-styrylchromones (2-STC),

chromanones (CHRM), flavones (FLV) 13-15 from 3-CHCA and phenolic 1,3-dicarbonyls

4. The bispyranone scaffold (bispyran-2- and -4-one) 16-20 have been elaborated in a

one-step coupling reaction of 4-HCOM and TAL on 3-CHCA or formyl-functionalized

precursors (ω-formyl-2’-hydroxyacetophenones 4 and chromone-3-carbaldehydes 5)

using organobase catalysis. Finally, the application of the 1,4-conjugate addition,

commonly known as Michael addition (MAD) approach, is extended to a variety of 4-

hydroxypyran-2-one rings 6, 7 (4-HCOM, TAL) reacting on α,β-unsaturated ketone

systems including chalcones (CHAL) 8, to give the new warfarin-analogues 21. Also a

variety of complex structures 22 have been obtained by hybridizing 4-HCOM and TAL

units with the newly synthesized poly-substituted- CHRs, FLVs and 2-STCs 13 via MAD.


9

All the previously indicated organic reaction proceeds in mild and environmentally

friendly conditions using 4-pyrolidinopyridine (4-PPy) as organobase catalyst. The

reactions last from short to long time, up to 72 hours maximum time, and yields are in

general from moderate to good (>40%).

O

O

N

N

R

R

O

O

N

N

OH O

R

O

R

O

O

O

OOH

O

O

OOH

OO

OHOOH

O

OOH

OO

OHOOH

O

O

O

R

R

O

O

R

R

R

R

O

O

R

R

O

O

O

HO

OH

O

O

N

N

R

R

O

O

O

O

N

NO

O

R

R

O

O

O

O OH

O

O

HO

O

O

O

O OH

O

O

HO

O

O

O

OH

O

OOH

O

O

OO

OHO

HO

OO

OO

OHO

HO

9 10 11 12

13

15

O

O

R

16 1817

14

1920 21

22

O

O

OH

O

R

R

R

R

R

R

R

RR

R

R

O

HO

O

HO

O

HO

O

HO

Figure 1. 3. Novel polyphenolic hybrids for biological applications


10

The novel polyphenolic hybrid structures show a deep puzzling structural features

inducing various stereochemical aspects. A fine analytical characterization is established

on the basis of high resolution spectroscopic techniques including extensive mono (1H and

13C) and bidimensional nuclear magnetic resonance (2D-NMR) and single-crystal X-ray

diffractometry which largely helped to solve the structural complexity. The stereochemistry

presented in this work involves various aspects of the structural geometry basis, like the

presence of asymmetric (R/S) and diastereometric (E/Z) centers, spiro-structures,

tautomers and conformational forms which seems to affect the biological activity of the

studied entities.

The last fourth chapter will briefly introduce and describe the performed biological

screening tests on the recently synthesized polyphenolic hybrids explaining their possible

involvement in cancer therapy. Hence, most of the newly obtained molecules have been

evaluated for their effect on cytotoxicity and proliferation of leukemic cell lines as well as

their involvement in the regulation of NF-κB pro-inflammatory pathway. Within the context

of research for new targets of cancer therapy, HDAC and Cdc25 enzymes inhibition were

specifically tested on the previously mentioned benzofuran-3-one-hydantoin candidates. A

range of representatives polyphenolics from CHRs, CHRMs, FLVs and 2-STCs were

examined for their cancer chemopreventive activity mediated by induction of

cytoprotective Nrf2 (nuclear factor E2-related protein 2) signaling and their ability to inhibit

proliferation of breast cancer cells. Finally we cover the results with some antioxidant

activity evaluations using DPPH free radical scavenging test and FRAP ferric ion reducing

antioxidant power of some underlined structures.


11

1.4. REFERENCES

1. Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs over the 30

years from 1981 to 2010. J. Nat. Prod., 2012, 75, 311-335.

2. Filipe, P.; Silva, A.M.S.; Seixas, R.S.G.R.: Pinto, D.C.G.A.; Santos, A.; Patterson, L.K.;

Silva, J.N.; Cavaleiro, J.A.S.; Freitas, J.P.; Mazière, J.-C.; Santus, R.; Morlière, P. The

alkyl chain length of 3-alkyl-3’,4’,5,7-tetrahydroxyflavones modulates effective inhibition

of oxidative damage in biological systems: illustration with LDL, red blood cells and

human skin keratinocytes. Biochem. Pharmacol., 2009, 77, 957-964.

3. Gomes, A.; Neuwirth, O.; Freitas, M.; Couto, D.; Ribeiro, D.; Figueiredo, A.G.P.R.;

Silva, A.M.S.; Seixas, R.S.G.R.; Pinto, D.C.G.A.; Tomé, A.C.; Cavaleiro, J.A.S.;

Fernandes, E.; Lima, J.L.F.C. Synthesis and antioxidnt properties of new chromone

derivatives. Bioorg. Med. Chem., 2009, 17, 7218-7226;

4. Gomes, A.; Freitas, M.; Fernandes, E.; Lima, J.L. Biological activities of 2-

styrylchromones. Mini-Rev. Med. Chem., 2010, 10, 1-7.

5. Marinho, J.; Pedro, M.; Pinto, D.C.G.A.; Silva, A.M.S.; Cavaleiro, J.A.S.; Sunkel, C.E.;

Nascimento, M.S.J. 4’-Methoxy-2-styrylchromone a novel microtubule-stabilizing

antimitotic agent. Biochem. Pharmacol., 2008, 75, 826-835.

6. Gomes, A.; Fernandes, E.; Silva, A.M.S.; Pinto, D.C.G.A.; Cavaleiro, J.A.S.; Lima,

J.L.F.C. Anti-inflammatory potential of 2-styrylchromones regarding their interference

with arachidonic acid metabolic pathways. Biochem. Pharmacol., 2009, 78, 171-177.

7. Gomes, A.; Fernandes, E.; Silva, A.M.S.; Pinto, D.C.G.A.; Cavaleiro, J.A.S.; Lima,

J.L.F.C. 2-Styrylchromones: Novel strong scavengers of reactive oxygen and nitrogen

species. Bioorg. Med. Chem., 2007, 15, 6027-6036.

8. Gomes, A.; Fernandes, E.; Garcia, M.B.Q.; Silva, A.M.S.; Pinto, D.C.G.A.; Cavaleiro,

J.A.S.; Lima, J.L.F.C. Cyclic voltammetric analysis of 2-styrylchromones: Relationship

with the antioxidant activity. Bioorg. Med. Chem., 2008, 16, 7939-7943.

9. Rocha-Pereira, J.; Cunha, R.; Pinto, D.C.G.A.; Silva, A.M.S.; Nascimento, M.S.J. (E)-2-

Styrylchromones as potential anti-norovirus agents. Bioorg. Med. Chem., 2010, 18,

4195-4201.

10. Price, W.A.; Silva, A.M.S.; Cavaleiro, J.A.S. 2-Styrylchromones: Biological Action,

Synthesis, and Reactivity. Heterocycles, 1993, 36, 2601-2612.

11. Silva, A.M.S.; Pinto, D.C.G.A.; Cavaleiro, J.A.S.; Levai, A.; Patonay, T. Synthesis and

reactivity of styrylchromones. Arkivoc, 2004, vii, 106-123.


12

12. Pinto, D.C.G.A.; Silva, A.M.S.; Almeida, L.M.P.M.; Cavaleiro, J.A.S.; Levai, A.;

Patonay, T. Synthesis of 4-aryl-3-(2-chromonyl)-2-pyrazolines by the 1,3-cycloaddition

of 2-styrylchromones with diazomethane. J. Heterocycl. Chem., 1998, 35, 217-224.

13. Pinto, D.C.G.A.; Silva, A.M.S.; Cavaleiro, J.A.S.; Foces-Foces, C.; Llamas-Saiz, A.L.;

Jagerovic, N. Synthesis and molecular structure of 3-(2-benzyloxy-6-hydroxyphenyl)-5-

styryl-pyrazoles. Reaction of 2-styrylchromones and hydrazine hydrate. Tetrahedron,

1999, 55, 10187-10200.

14. Pinto, D.C.G.A.; Silva, A.M.S.; Cavaleiro, J.A.S. Synthesis of 3-(2-Benzyloxy-6-

hydroxyphenyl)-1-methylpyrazoles by the Reaction of Chromones with

Methylhydrazine. J. Heterocycl. Chem., 2000, 37, 1629-1634.

15. Barros, A.I.R.N.A.; Silva, A.M.S. Efficient Synthesis of Nitroflavones by

Cyclodehydrogenation of 2’-Hydroxychalcones and by the Baker-Venkataraman

Method. Monatsh. Chem., 2006, 137, 1505-1528.

16. Pinto, D.C.G.A.; Silva, A.M.S.; Cavaleiro, J.A.S. A convenient synthesis of new (E)-5-

hydroxy-2-styrylchromones by modifications of the Baker-Venkataraman method. New

J. Chem., 2000, 24, 85-92.

17. Barros, A.I.R.N.A.; Silva, A.M.S. Synthesis and structure elucidation of three series of

nitro-2-styrylchromones using 1D and 2D NMR spectroscopy. Magn. Reson. Chem.,

2009, 47, 885-896.

18. Silva, A.M.S.; Tavares, H.R.; Barros, A.I.N.R.A.; Cavaleiro, J.A.S. NMR and structural

and conformational features of 2’-hydroxichalcones and flavones. Spectrosc. Lett.,

1997, 30, 1655-1667.

19. Patonay, T.; Cavaleiro, J.A.S.; Lévai, A.; Silva, A.M.S. Dehydrogenation by Iodine-

Dimethylsulfoxide System: A General Route to Substituted Chromones and

Thiochromones. Heterocycl. Commun., 1997, 3, 223-229.

20. Silva, A.M.S.; Pinto, D.C.G.A.; Cavaleiro, J.A.S. 5-Hydroxy-2-(Phenyl or

Styryl)Chromones: One-pot Synthesis and C-6, C-8 13C NMR Assignments.

Tetrahedron Lett., 1994, 35, 5899-5902;

21. Silva, A.M.S.; Pinto, D.C.G.A.; Tavares, H.R.; Cavaleiro, J.A.S.; Jimeno, M.L.

Elguero, J. Novel (E)- and (Z)-2-Styrylchromones from (E,E)-2’-

Hydroxycinnamylideneacetophenones. Xanthones from Daylight Photooxidative

Cyclization of (E)-2-Styrylchromones. Eur. J. Org. Chem., 1998, 2031-2038.

22. Talhi, O.; Silva, A M.S. Advances in C-glycosylflavonoid Research. Curr. Org. Chem.,

2012, 16, 859-896.

http://www.ingentaconnect.com/content/ben/coc;jsessionid=53kb9elw66c0t.alice


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23. Talhi, O.; Silva, A M.S. C-prenylation of phenolics. Curr. Org. Chem., 2012, in

publication.


CHAPTER 2

- SYNTHESIS OF HYDANTOIN- AND URACIL-

POLYPHENOLIC HYBRIDS -

Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids

17

2.1. BENZOFURAN-3-ONE-HYDANTOIN HYBRIDS

2.1.1. Natural and synthetic organic hybrids

The structural diversity of natural and synthetic organic compounds has become key

topics in synthetic, bioorganic and medicinal chemistry. Recent investigations have been

directed to obtain increasing complexity of bioactive hybrid molecules constructed from

well-known organic families, like flavonoids, chromones, stilbenes, coumarins, alkaloids,

terpenoids, peptides and glycosides [1-9]. Indeed, the structural combination of two or

more of these interesting molecular models in one organic framework usually leads to

additive and/or new biological properties different from the parent constructive scaffolds.

Nature has utilized this strategy to construct several building block from various well-

known organic families which ends up with very interesting biological activities [10].

Tsogoeva et al. [11] have recently reviewed the progress in the development of synthetic

hybrids from both natural or unnatural bioactive compounds and their potential application

in medicinal chemistry. Literature rarely discusses how natural products can be hybridized

to generate compounds with unprecedented bioactivity enabling the development of novel

drugs and advanced materials. Thus, our bibliographic survey have only resulted in few

works describing the concept of hybrid in polyphenolic compounds from natural or

synthetic origins emphasizing their biological applications (Fig. 2.1).

Recent synthetic works have been dedicated to the flavonoid scaffolds bearing

glycosyl or prenyl moieties 23, 24, which demonstrate potent antioxidant and anti-

inflammatory activities. More and more scientific attentions are due to the C-glycosylation

and C-prenylation of phenolic compounds which is found to play a crucial biological

function by increasing both of their hydrophilic or lipophilic properties and facilitating their

circulation through biological cell tissues [12, 13]. Novel synthetic sugar-amino acid

hybrids 25 are hypothesized to possess similar structural features of β-amino acid

oligomers and the chemical and enzymatic resistance of C-glycosides to hydrolysis [14].

Moreover, supporting a carbohydrate moiety on quinoxaline system gave rise to new

compounds 26 with effective DNA cleavage and selective cytotoxicity to cancer cells by

photo-irradiation [15]. Interestingly, the flavone backbone has been tethered to

pyrrolobenzodiazepine through alkanedioxy spacers of varying lengths 27, the resulting

derivatives thereof exhibited significant DNA minor groove binding ability in comparison to

the naturally occurring DC-81 and appreciable in vitro cytotoxicity [16].


18

O

O

O

OHHOHO

OH O

O

O

HN O

O

OR

HN

NH

O

O

O

HOH3C

OO O

H3CHO

O O

N

N

N

N

O

O

OX

O

N

N

O

H

H3CO

O OH3CO

OHHO

O

OOH

HO

OH

O

O

OH

N

N

NH

N

O

O

23 2524

27

29

26

28 3031

HN

O

NH

O

Figure 2. 1. Synthetic and natural hybrid compounds

Belluti et al. [17] reported an important synthetic stilbene-coumarin hybrids 28 which

show anticancer activity especially when combining the 7-methoxycoumarin with the 3,5-

disubstitution pattern of the trans-vinylbenzene moiety; thus, promising structural features

have been achieved leading to excellent antitumor compounds endowed with a apoptosis-

inducing capability. Nature is always the perfect laboratory, where the structural diversity

is effectively expressed in various plant sources, as a subject of matter, it is necessary to

mention the new flavone-coumarin hybrid 29 extracted from the leaves and twigs of

Gnidia socotrana (Thymelaeaceae) [18]. Further new hybrid molecules containing

terpenoid fragments and plant alkaloids have been synthesized for biological interests

[19,20]. In addition, various hybrid molecules of caffeine and eudistomin D 30 were

elaborated exhibiting affinity and selectivity for adenosine receptors A1, A2A, and A3 [21].

Indirubin 31 is the active ingredient of Danggui Longhui Wan, a mixture of plants that is

used in traditional Chinese medicine to treat chronic diseases (antileukiaemia). Indirubin

and its analogues were identified as potent inhibitors of cyclin-dependent kinases (CDKs)

[22]. The structure of Indirubin comprises similar stereochemical features as those known

for our benzofuran-3-one-hydantoin hybrids 11 described in this chapter.


19

2.1.2. Benzofuran-3-ones and hydantoins based molecules

The titled heterocyclic systems, benzofuran-3-ones (BF) and hydantoins (HD), have

demonstrated an outstanding biological field of applications (Fig. 2.2) [23-42], however,

according to our best knowledge, no synthetic work has combined these organic

templates into a single hybrid.

BF HD

O

O

NN

O

O

R

R

R

RR

3

5

2

4

Figure 2. 2. Benzofuran-3-one (BF) and hydantoin (HD) heterocyclic systems

The BF heterocycle is particularly represented by aurones, a natural flavonoid-type

compound showing two possible diastereomers E and Z. The aurone molecule contains a

benzofuran-3-one ring linked to a benzylidene group at position 2. In general, aurones are

a chalcone-like molecules closed into a 5-membered ring rather than the 6-membered ring

which is more typical for the flavonoid skeleton (Fig. 2.2 structure BF, Fig. 2.3) [1].

O

O

O

O

Z E

Figure 2. 3. The two possible aurone diastereomers

Important biological manifestations of aurone derivatives have been well documented

in the literature. The ability of aurones to modulate the efflux activities of ABCG2 and

ABCB1 was investigated by Sim et al. [23]. Analgesic and anti-inflammatory effects of new

synthesized aurone-based derivatives were evaluated raising positive results [24]. Also, a

series of new aurone analogues 33 bearing a cyclic tertiary amine moiety were designed

and assayed for their antitumor activity against four kinds of human solid tumor cell lines

showing promising results [25]. The antimicrobial activities of aurone derivatives obtained

by extraction from natural sources or by synthesis have been proved, the natural

compounds from Ficus religiosa (Moraceae) inhibited glucan synthase, which may be

advantageously used as a dual acting antifungal agent [26]. In other fields, selected

aurone components have been utilized in various oil-in-water and water-in-oil types of

http://en.wikipedia.org/wiki/Flavonoid

http://en.wikipedia.org/wiki/Benzofuran

http://en.wikipedia.org/wiki/Benzylidene

http://en.wikipedia.org/wiki/Chalconoid

http://en.wikipedia.org/wiki/Flavonoid

http://en.wikipedia.org/wiki/Aurone#cite_note-0


20

formulations for either cosmetic or pharmaceutical applications, notably as dermatologic

agent, having a melanogenesis-inhibiting, a depigmenting, or an anti-tyrosinase activity

[27]. High throughput screening of a series of benzofuran-3-one-indole hybrids 36 has led

to the identification and optimization of new phosphatidylinositol-3-kinases (PI3K)

inhibitors, these compounds show activity in multiple cellular proliferation assays with

signaling through the PI3K pathway and confirmed via phospho-Akt inhibition in PC-3 cells

[28].

Only a handful of studies treating the HD heterocycle have been quoted, despite of its

high biological interest. In a more general sense, hydantoins are imidazolidine derived

nucleus sharing two carbonyl functions at positions 2 and 4 with substituents bonded via

carbon 5 and/or nitrogen 1 and 3 atoms (Fig. 2.2, structure HD) [29]. The hydantoin

hetero-nucleus was utilized in several anticonvulsant agents, such as ethotoin [30],

phenytoin [31], mephenytoin [32], fosphenytoin [33] (Fig. 2.4). Sutherland et al. [34]

established quantitative structure-activity relationships and classification models for

anticonvulsant activity of a large set of HD derivatives measured in mice and rats. HDs

have also shown muscle relaxant activities [35,36].

ethotoin

N

HNO

O

HN

HNO

O

phenytoin mephenytoin

N

HNO

O

fosphenytoin

N

HNO

OO

P

O

HOHO

Figure 2. 4. Anticonvulsant hydantoins

Regarding to the organic synthetic aspect, BF and HD rings have recently been aimed

by researchers and considered as important biological targets [24-26,28,37-42]. Several

papers were previously underlined concerning the synthesis of biological active aurone

analogues [24-26,28]. Similarly to what will be described in this chapter, the

rearrangement of the chromanone ring to benzofuran-3-one was achieved by the action of

amines on 3-bromochromone 32 in a one-pot synthesis of aurone analogues sharing

cyclic tertiary amine fragments 33 (Scheme 2.1). The synthetic protocol was found to

present many advantages, such as higher yields, shorter reaction time, mild condition,

and readily isolation of products [25]. The Knoevenagel condensation on the active

methylene of 4,6-dihydroxybenzofuran-3(2H)-one 34 with 3-formylindole derivatives 35

yielded the new conjugated benzofuran-3-one-indole hybrids 36 showing the

phosphatidylinositol-3-kinases inhibitory activity (Scheme 2.2) [28]. Pulina et al. [37]

http://en.wikipedia.org/wiki/Hydantoin#cite_note-0

http://en.wikipedia.org/wiki/Anticonvulsants

http://en.wikipedia.org/wiki/Ethotoin


http://en.wikipedia.org/wiki/Phenytoin


http://en.wikipedia.org/wiki/Mephenytoin


http://en.wikipedia.org/wiki/Fosphenytoin





21

described a simple regioselective Wittig-type reaction of 2,3-dihydro-2,3-

benzo[b]furandione 37 with the triphenylphosphazines of diazo compounds to afford

substituted 2-methylenehydrazono-2,3-dihydrobenzo[b]furan-3-ones 38, which have been

further modified by a sequence of reactions (Scheme 2.3).

O

O

Br

O

O

N

XR

t-BuO-K+, DMF

O

O

H

N

Br

XR

O-

O

Br

N

XR

32 33

HN

XR

Scheme 2. 1

O

O

OHCN

R

OH

HO

R1

R2

O

O

NR

OH

HO

R1

R2

+cat. HCl

EtOH / reflux

34 35 36

Scheme 2. 2

O

O

O Ph3P=N–N=CR1R2

– Ph3PO

O

O

N N

R2

R1

37 38

Scheme 2. 3

Earlier, 6-acetylspiro[benzofuran-2(3H),1'-cyclopropan]-3-one 40, the most potent

antiulcer compound in a series of spirocyclopropanes, was obtained by treating methyl

benzoate 39 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and NaCl in DMF at 150 °C

(Scheme 2.4) [38]. More recently, Filloux et al. [39] reported the development of a

multicatalytic, one-pot, asymmetric Michael/Stetter reaction of salicylaldehydes 41 with

electron deficient alkynes 42. The cascade reaction proceeds via amine-mediated Michael

addition followed by an N-heterocyclic carbene-promoted intramolecular Stetter reaction.

Several salicylaldehydes and doubly activated alkynes participate in a one-step or two-


22

step protocol (via the intermediates 43) to give a variety of BF products 44 in moderate to

good yields and good to excellent enantioselectivities. Terminal electrophilic allenes also

deliver a similar one-pot, two-step Michael/Stetter reaction with salicylaldehydes to afford

40 compounds. The origin of enantioselectivity in the reaction is explored; E∕Z geometry of

the reaction intermediate as well as the presence of catalytic amounts of catechol additive

are found to influence the reaction enantioselectivity (Scheme 2.5).

H3COC

O

O

O

O

OCH3O

O

DBU, NaCl

DMF / 150 °C

H3COC

39 40

Scheme 2. 4

O

OH

R

EWG1

EWG2

O

O

EWG1

EWG2

*

O

O

R

EWG1

EWG2

R

41

+

44

42

43

Multi-catalyst

Scheme 2. 5

In relation to the HD nucleus, target derivatives of 1,3,5-triphenylhydantoin 47 have

been obtained by an original pathway starting from 1,3-diphenylureas 45 and

phenylglyoxal derivatives 46 as it is detailed in Scheme 2.6. This reaction represents a

simple one-pot procedure to obtain, in optimal yields, some compounds with interesting

affinity and selectivity for the human CB1 cannabinoid receptor. The enantioselectivity (3:1

ratio for the R/S enantiomers) that correspond to the C-5 asymmetric carbon has been

revealed from crystallographic studies [40]. Also, the regioselective Knoevenagel-type

condensation has been reported in the synthesis of thioisatin-hydantoin hybrids 50.

Benzo[b]thiophene-2,3-dione 48 (commonly known as thioisatin) reacts with the hydantoin

active methylene 49 to afford selectively the isomer Z 50 (Scheme 2.7). The synthesized

products were screened for antimicrobial activity [41]. Electrophilic substitution of allantoin

(or 5-ureidohydantoin) in position 5 with 2-naphthol and 1-bromo-2-naphthol in the


23

presence of acid afforded the corresponding 5-(2'-hydroxy-1'-naphthyl)hydantoin and 5-

(5'-bromo-6'-hydroxy-2'-naphthyl)hydantoin in 82% and 52% yields, respectively [42].

HN

O

HN

O

O

H

N

N

O

O

R2

R1

R

R2

R1

R

CH3COOH-HCl (20:0.5)

Reflux, 6h

*

45

4647

+

Scheme 2. 6

S

O

OHN

NH

O

O

S

O

NH

NHO

O

KnoevenagelR

R

+Z

48 49 50

Scheme 2. 7

2.1.3. Synthesis of benzofuran-3-one-hydantoin hybrids

Because of their specific biological value, our first attentions were directed toward

designing benzofuran-3-one-hydantoin (BF-HD) hybrids 10, 11 bearing asymmetric or

diastereomeric centers (Scheme 2.8). We describe, herein, efficient synthetic methods for

these new hybrid compounds based on a reaction sequence composed of three steps: (A)

a one-pot coupling of the cheap chromone-2-carboxylic acid (2-CHCA) 1 with

carbodiimides 3a-c to afford 1’,3’-disubstituted spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones 9a-c. In the following step (B), the chromanone ring in compounds 9a-c was

transformed into the benzofuran-3-one nucleus giving rise to the new BF-HD hybrids 10a-

c, after treatment with sodium ethoxide, creating a second asymmetric center through a

diastereoselective rearrangement at the spiro-carbon, maintaining the hydantoin nucleus

unmodified. The final step (C) consists in the construction of rigid conjugated BF-HD

hybrids 11a-c through a diastereoselective dehydrogenation reaction of 10a-c using the I2

(catalytic)/DMSO system which has led to the major Z configuration of the resulting double

bond. The structure of the newly described hybrid compounds have been elucidated by

single-crystal X-ray diffraction and extensive 1D and 2D NMR analysis allowing to discuss

their stereochemistry and formation mechanism.


24

1 9a-c 10a-c 11a-c (Z)

3a-c

a. R= cyclohexylb. R= isopropylc. R= tolyl

O CO2H

O

O

O

N

NO

O

R

R

O

O

N

N

O

O

R

R

O

O

N

N

R

R

O

O

N C N

R

R ***

A

B C

Scheme 2. 8

2.1.3.1. Synthesis of 1’,3’-disubstituted spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones

Few investigations have been entertained regarding the reactivity of chromone-2-

carboxylic acid 1 that, under nucleophilic conditions, delivers a broad synthetic potential of

2-substituted chromones, especially chromone-2-carboxamides which were recently

reported to be potent biologically-active agents [43-48]. The action of carbodiimide 3a on

1 has been undertaken by Filliatre et al. [49], who have firstly isolated the 2-chromone-N-

acylurea intermediate 51 that was subsequently treated with ethanol to provide the

reported spiro[chroman-2,4'-imidazolidine]-2',4,5'-trione skeleton 9a. This two-step

procedure constitutes the first and sole report on spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones 9 synthesis (Scheme 2.9). More recently, Marcelli et al., [50] have reported a

density functional theory (DFT) study of a computational model reaction of carbodiimides

3 on activated α,β-unsaturated carboxylic acids. The results suggested the formation of N-

acyl ureas 52 and imino-oxazolidinones 53 as reaction intermediates to yield the final

hydantoin product 54 via two alternative pathways, (a) aza-Michael addition and imino-

oxazolidinone 53 rearrangement or (b) O→N acyl shift to form N-acyl ureas 52 and aza-

Michael addition (Scheme 2.10).

In this part, we describe a simple and efficient one-pot synthetic route (A) to produce

compounds 9a-c. The treatment of 1 with carbodiimides 3a-c, using catalytic amounts of

4-pyrrolidinopyridine (4-PPy) in dichloromethane at room temperature, has provided high

yields (66-88%) of spiro[chroman-2,4'-imidazolidine]-2',4,5'-triones 9a-c which are readily

isolated after recrystallization (Scheme 2.11).


25

O

O

N C N

COOH O

O

N

O

NH

O

O

O

N

NO

O+

1 3a 51 9a

EtOH

Scheme 2. 9

R4

R3

OH

O

NCR1

NR2

NN

O

O

R1

R2

R3R4

R4

R3

N

O

NH

O

R1 R2

523

53

54

R2

R1

O

O HN

N

R1

R2

CH2Cl2

(a)

(b)

ON

NR1

O

R2

R3R4

Computational model

Scheme 2. 10

2

9a-c3a-c


O COOH

O

O

O

N

NO

O

R

R

N C N

R

R

*+

A

A: 0.05 equiv 4-PPy, CH2Cl2, rt, overnight

1

3

1'

3'

Scheme 2. 11

The structure of compounds 9a, 9c was established by 1D and 2D NMR analysis. The

main feature in 1H NMR spectra of compounds 9a-c was the presence of two signals as

doublets at 2.89-3.14 ppm and 3.22-3.30 ppm with a large geminal protons coupling

constant (2J =16.7-16.9 Hz) assigned to the resonance of the diastereotopic chromanone

methylene protons involved in an AB spin system. The HSQC spectrum (Fig. 2.5) showed

that the methylene carbon C-3 (41.0-41.3 ppm) is bounded to both H-3 (2.89-3.14 and

3.22-3.30 ppm) protons, while the HMBC experiment (Fig. 2.6, 2.7) presents important


26

correlations of these methylene geminal protons with the neighboring carbons. Thus, H-3

(2.98-3.14 ppm) presents HMBC correlations with carbons C-10 (119.7-119.8 ppm), C-2

(88.5-89.2 ppm), C-4 (188.5-188.7 ppm; C=O) and C-5’ (167.1-168.7 ppm; C=O), while

the other H-3 (3.22-3.30 ppm) shows correlations with C-2 and two carbonyl groups, C-4

and C-5’. Additional HMBC correlations allowed differentiating between carbonyl groups

and N-substituent positions: C-4 (188.5-188.7 ppm) is confirmed as belonging to the

chromanone ring by correlating with H-5 (7.76-7.88 ppm) in all compounds 9a-c. In the

case of 9a, 9b H-1” (3.82 and 4.26 ppm) correlates with both carbonyls of the hydantoin

ring C-2’ (153.1-153.8 ppm) and C-5’ (167.1-168.7 ppm), while H-1”’ (3.33 and 3.77 ppm)

is only establishing correlation with the carbonyl C-2’, allowing to assign the 1-N- and 3-N-

cyclohexyl/isopropyl substituents on the hydantoin nucleus (Fig. 2.6, 2.7).

Figure 2. 5. HSQC spectrum of compound 9b (300 MHz)

H-3(AB)

C-3


27

O

O

N

NO

O

H

H

9a, 9b

O

O

N

NO

O

HA

HB

9a-c

H5

4 3

2 2'

5'

1"

1"'

Figure 2. 6. Main HMBC correlations in compounds 9a-c

Figure 2. 7. HMBC spectrum of compound 3b (300 MHz)

The collected crystals of compounds 9a, 9c, obtained from ethanol solutions at room

temperature, were analyzed by single-crystal X-ray diffractometry. We have therefore

established the 3D structure of compounds 9a-c after X-ray data analysis confirming that

these molecular units share both R and S configurations of the C-2 asymmetric spiro

carbon in a 1:1 ratio (Fig. 2.8). This fact is obvious and systematically deduced from the

centrosymmetric monoclinic crystal space groups P21/c and C2/c in which the compounds

9a and 9c crystallize, respectively (Fig. 2.9).

H-3(AB)

C-2"

C-2"’

H-1" H-1"’

C-2

C-10

C-2’ (C=O)

C-5’ (C=O)

C-4 (C=O)


28

The formation of compounds 9a-c can be rationalized by a mechanism based on three

steps: i) a nucleophilic addition of 2-CHCA 1 on the carbodiimide C=N double bond 3a-c,

followed by ii) an O→N acyl shift in the intermediate 55 to give the chromone-2-N-

acylurea 56 which was previously reported as an experimentally isolated product 51

[49,50]. Step iii) consists in an intramolecular cyclization (via aza-Michael addition) at

position 2 which has led to the creation of a spiro asymmetric carbon joining both of the

two chromanone and hydantoin rings (2:4’ positions) and generating a racemic mixture,

since the nucleophilic nitrogen attack at C-2 carbon has an equivalent probability (Scheme

2.12).

Figure 2. 8. Schematic representation of 9a, 9c molecular structures


29

Figure 2. 9. Crystal cell unit of compound 9a

O

O

O

O

H

C

N

N

R

R

O

O

O

O

NHN

R

R

O

O

N

NO

O

R

R

i) ii) iii)

O

O

N

O

NH O

R

R

1 9a-c

3a-c

55 56

Scheme 2. 12

2.1.3.2. Synthesis of 1’,3’-disubstituted 5-(3-oxo-2,3-dihydrobenzofuran-2-

yl)imidazolidine-2,4-dione

A very rare rearrangement of the chromanone ring into the benzofuran-3-one one was

observed while treating compounds 9a-c with an equivalent amount of sodium ethoxide in

ethanol yielding 5-(3-oxo-2,3-dihydrobenzofuran-2-yl)imidazolidine-2,4-diones 10a-c (47-

63%; Scheme 2.13). The dropwise addition of the sodium ethoxide solution in ethanol was

carried out at 0 °C and then the reaction is allowed to stirring at room temperature for one

hour. This process permits the creation of a new asymmetric center by opening the C-2

spiro-carbon and reclosing the ring at position 3 to get a benzofuran-3-one ring bounded

to the hydantoin one via a simple σ bond. Thus, new BF-HD hybrids 10a-c have been

elaborated sharing two asymmetric centers C-2’ and C-5.


30

9a-c 10a-c


O

O

N

NO

O

R

R

O

O

N

N

O

O

R

R

***B

B: 1 equiv EtONa/EtOH / 0 °C to rt, 1 hour

2'

59' 1

Scheme 2. 13

The 1H NMR spectra of 10a-c present two signals as doublets at 4.55-5.34 and 4.89-

4.93 ppm with low vicinal coupling constants (3J = 1.9-2.0 Hz) attributed to the proton

resonances of H-5 and H-2’, respectively. The aforementioned patterns are the sole

difference when comparing to the 1H NMR spectra of the starting materials 9a-c, while,

the aromatic proton signals are conserved despite some changes in the chemical shift

values (Fig. 2.10). The HSQC experiment revealed that both H-2’ and H-5 are bounded to

two different carbons C-2’ at 80.6-82.3 ppm and C-5 at 59.9-62.1 ppm, respectively (Fig.

2.11). The neighboring carbon resonances of H-2’ and H-5 are assigned from the main

HMBC correlations (Fig. 2.12, 2.13): i) H-5 correlates with three carbonyl groups C-2

(153.6-156.2 ppm) and C-4 (166.2-168.1 ppm) from the HD nucleus and C-3’ (196.8-

196.9 ppm) from the BF nucleus; and ii) H-2’ correlates with carbonyls C-4 and C-3’

together with C-8’ (172.5-176.6 ppm) (BF nucleus) and C-5 (HD nucleus) (Fig. 2.12, 2.13).

Further HMBC features enable to differentiate between the carbonyl groups. For instance

in all compounds 10a-c, carbonyl C-3’ is coupled to H-4’ (7.69-7.74 ppm) proving its

position in the BF ring. In the case of compounds 10a, 10b, H-1”’ (3.81 and 4.22 ppm)

correlates to both carbonyls of the HD ring C-2 and C-4, while H-1” shows only a weak

HMBC correlation with carbonyl C-2 and, therefore, allowing to assign the 1-N- and 3-N-

cyclohexyl/isopropyl substituents on the HD nucleus (Fig. 2.12, 2.13). Appreciable

information is gained from NOESY experiment of 10a-c, which shows strong NOE effects

between H-2’ (4.90 ppm) and H-5 (4.56 ppm) which is consistent with a gauche

conformation relatively to the σ bond C2’—C5 in solution (Fig. 2.14, 2.15).


31

Figure 2. 10. 1H NMR monitoring of benzopyran-4-one 9b to benzofuran-3-one 10b

rearrangement

O

O

N

NO

O

HHO

O

N

NO

O

HH

Geminal protons2J = 16.7-16.9 Hz

AB spin system

Vicinal protons3J = 1.9-2.0 Hz

H

H

H

H

9b 10b

10b

9b


32

Figure 2. 11. HSQC spectrum of compound 10a (300 MHz)

O

O

N

NO

O

HH

R

H

2' 5

9'

2

4'

O

O

N

NO

O

HH

H

H

2

1''

R

4

1'''

10a-c 10a, 10b

Figure 2. 12. Main connectivities observed in the HMBC spectra of compounds 10a-c

H-2’ H-5

C-5

C-2’


33

Figure 2. 13. HMBC spectrum of compound 10a (300 MHz)

O

O

N

N

O

O

HH

H

H

2' 5

1"

Figure 2. 14. Main NOE effects observed in the NOESY spectrum of 10a

H-2’ H-5

C-5

H-1"’ H-1"

C-8’

C-2 (C=O)

C-4 (C=O)

C-3’ (C=O)

H-4’


34

Figure 2. 15. Partial NOESY spectrum of compound 10a (300 MHz)

Starting from a racemic mixture of the reagents 9a-c, the resulting rearranged

products 10a-c can appear as a mixture of two diastereomers (each one as a pair of

enantiomers). In fact, we have determined that products 10a-c present a racemic

character, as it was clearly deduced from single-crystal X-ray diffraction studies for 10a

and 10c (Fig. 2.16). In these cases, the centrosymmetric space groups, triclinic Pī in 10a

and monoclinic P21/n in 10c, impose both of the two configurations R and S related to the

asymmetric carbons C-2’ and C-5 in 1:1 ratio. The synthesis of 10a-c has led to a unique

pair of enantiomers (R-2’,S-5) or (S-2’,R-5) present in the unit cell and, therefore, we

disclose a perfect diastereoselective synthetic route. An important remark which needs to

be mentioned is that two molecules of the crystallizing solvent (ethanol) are included in

the cell unit only in case of compound 10a (Fig. 2.17).

The crystallization conditions of compounds 10a and 10c in ethanol at 6 °C, yielded

crystals presenting a preferential enrichment of the gauche conformer relatively to the σ

bond C2’—C5 with dihedral angle (CH2’—CH5) of 64° as calculated from the

crystallographic studies. This data confirms that compound 10a and 10c exhibits a gauche

conformation both in solution and in crystallized forms. This conformer should have the

minimum energy concerning molecular stability which strongly depends on repulsion

H-2’ H-5

H-1"’ H-1"

H-2’

H-5

H-1"


35

forces between electronegative atoms and heterocyclic tensions of the whole molecule

(Fig. 2.18).

Figure 2. 16. Schematic representation of 10a and 10c molecular structures

Figure 2. 17. Crystalline unit cell of compound 10a


36

Figure 2. 18. Asymmetric unit of compound 10a showing the enantionmer (2’R, 5S) and

the corresponding Newman gauche projection

From a chemical stability point of view, compounds 9a-c present a high tension at the

spirocarbon connecting two heterocycles. This may favor a rearrangement or ring opening

to decrease these intramolecular interactions. Thus, under strong alkali conditions (using

sodium ethoxide), the formation of compounds 10a-c can be envisaged by an initial

deprotonation of the active C-3 methylene group in 9a-c compounds; followed by a

chromanone ring-opening of the intermediate 57 to 58 which undergoes a 5-membered

ring closure to afford the aurone-like form of 10a-c compounds as it is outlined in Scheme

2.14.

OH

HO

N

N

O

R

R

O

O

H

O

N

N

O-

R

R

O

O

O-

N

N

O

R

R

O

O

O

O

N

N

-O

R

O

R

O

O

N

N

O

R

O

R

EtOH

O

N

N

O

O

R

R

10c(a)

Chalcone-like form Z-configuration

EtO-

EtOH

Aurone-like form10a-c

9a-c 57 58

Scheme 2. 14

The reaction using derivative 9c (with tolyl substituent) was the unique case where it

was possible to isolate a small amount (>5%) of the opened chalcone-like form (Z)-1,3-

Newman projection Gauche

conformer


37

ditolyl-5-[2-(2-hydroxyphenyl)-2-oxoethylidene]imidazolidine-2,4-dione 10c(a) which was

identified by NMR analysis and analytical data, thus confirming the chromanone ring-

opening of the spiro-structures 9a-c (Scheme 2.14).

Regarding the stereochemical aspect, the chalcone-like intermediates 10c(a) should

normally present both E and Z isomers in 1:1 ratio since we started from a racemic

mixture of 9a-c [step (a), Scheme 2.15]. The nucleophilic attack of oxygen atom has an

equivalent probability to occur in both sides of the double bond plan in the chalcone-like

intermediate 10c(a). In this way, the resulting C-2’ asymmetric carbon of compounds 10a-

c should present a racemic character (R/S in a 1:1 ratio), imposing the C-5 asymmetric

carbon to have only one configuration (R or S) because the σ simple bond C5—H5 tends

to be formed in the opposite side of the oxygen attack relative to the molecule plan.

Statistically, starting from E and Z configurations of the chalcone-like intermediates

10c(a), two pairs of enantiomers should be generated, being (2’R,5S and 2’S,5R) and

(2’R,5R and 2’S,5S) in 1:1 ratio [step (b), Scheme 2.15]. However, the isolated

compounds 10a-c are identified as an unique pair of the referred enantiomers (2’R,5S and

2’S,5R). This means that the opening of compounds 9a-c selectively gives the Z

diastereoisomer of chalcone-like intermediate 10c(a). A fine 2D NMR analysis of the

isolated intermediate 10c(a) has confirmed its presence in the Z configuration, since the

NOESY spectrum does not show any NOE effect between the vinylic proton and the tolyl

ones. In conclusion, the origin of the reaction diastereoselectivity concerns the

chromanone ring-opening of 57 (Scheme 2.14), which provides a chalcone-like

intermediate in its more stable Z isomer, probably because in the E isomer, there are

electronic repulsions between carbonyls C-2’ and C-4.

O-

O NN

O

R

OR

H

O-

O

N

N

R

O

RO

H

ON

N

O

O

O

R

RH

H

S R

O

N

N

O

R

R

O

OH

H

S S

O

N

N

O

O

O

R

R

H

H

RS

O NN

O

R

R

O

O

H

H

RR

O N

N

O

R

R

O

O

ON

N

O

O

O

R

R

Z

E

9a-c

R

S

(a)(b)

Isolated products

10a-c

58

Scheme 2. 15


38

2.1.3.3. Synthesis of 1’,3’-disubstituted 5-[3-oxobenzofuran-2(3H)-ylidene]-

imidazolidine-2,4-dione

The final step (C) consists in the construction of a rigid double bond between the BF

and the HD rings. Accordingly, we have attempted the dehydrogenation of compounds

10a-c using catalytic amount of iodine I2 in refluxing DMSO. The desired 1,3-disubstituted-

5-[3-oxobenzofuran-2(3H)-ylidene]imidazolidine-2,4-diones 11a-c have been selectively

obtained as their Z diastereomer (35-62%, Scheme 2.16). Only in the case of the tolyl

derivative 10c, we were able to isolate (E)-11c in a lower yield (21%)

a R = cyclohexylb R = isopropylc R = tolyl

O

O

N

N

R

R

O

O

(Z)-11a-c

O

O

N

N

O

O

R

R

10a-c

(C)

C: 0.05 equiv I2, DMSO, reflux, 30 min

* *

O

O

N

N

(E)-11c

Z

O

O

tolyl

tolyl

E

Scheme 2. 16

This final dehydrogenation step was firstly monitored by NMR analysis. The 1H NMR

spectra of the resulting compounds 11a-c clearly shows the disappearance of both H-2’

and H-5 when comparing to those of the starting materials 10a-c. Thus, a double bond

bridged benzofuran-3-one-hydantoin structure should be formed which was supported by

high resolution mass spectra measurements indicating a loss of a hydrogen molecule in

11a-c compounds when compared with those of 10a-c. In fact, 2D NMR experiments are

nevertheless less informative for the study of the stereochemical aspect of this reaction, in

particular concerning the configuration (E or Z) of the double bond in the resulting olefins

11a-c, since there are no protons around the double bond. This structural feature can only

be unveiled by single-crystal X-ray diffraction studies. Therefore, the crystallization of

compounds 11a-c was not a trivial task. For instance, compound 11a was not isolated as

single-crystals with quality enough for a full structural elucidation even after using a

myriad of solvents and crystallization conditions. Compound 11c was, however,

successfully crystallized as long fine needles from toluene at 6 °C. Several attempts for

finding both isomers were performed, but only crystals of the Z-configured-product (Z)-11c

could be isolated (Fig. 2.19).


39

Figure 2. 19. Schematic representation of 11c molecular structure

A step of the utmost importance must be emphasized regarding the dehydrogenation

process of 10a-c. In the case of 10c reaction, the TLC monitoring shows the formation of

a minor amount of (E)-11c, which was isolated by preparative TLC, along with (Z)-11c.

This new compound shows similar features in the 1H NMR and high resolution mass

spectrum relative to (Z)-11c; thus, we assume that this compound must be the E

diastereoisomer (E)-11c. After several crystallization attempts of the isolated compound

(E)-11c (Scheme 2.16), no consisting results concerning the X-ray diffraction studies

could be attained and, therefore, the structure was elucidated on the basis of extensive

NMR data which effectively shows great differences in terms of proton and carbon

chemical shifts. In addition, melting points of compounds (Z)-11c and (E)-11c are largely

different (see experimental data). In this way, we have isolated two separable

diastereomers of derivative 11c (E/Z) obtained in 1:3 ratio which is calculated by NMR (in

CDCl3) proton integration and confirmed by HPLC analysis. Therefore, we disclose a

diastereoselective dehydrogenation mainly yielding the (Z)-11a-c isomers.

The diastereoselectivity of the last dehydrogenation step can be mechanistically

explained. The enolic form 59 of the benzofuran-3-ones 10a-c should probably be

transformed in the conformer 60 stabilized by intramolecular hydrogen bond. This

conformer suffers an α-iodination reaction giving 61 (in equilibrium with 62) which

underwent a E2 elimination of hydroiodic acid to yield the obtained Z configuration of

compounds 11a-c. The formed hydroiodic acid follows its redox cycle by reacting with

DMSO to regenerate the catalyst molecular iodine I2 and producing thioether (CH3)2S

which smells during the course of the reaction (Scheme 2.17). It is also important to

emphasize that the Z configuration is, indeed, the most stable because the E configuration

imposes a very close proximity between the BF’s carbonyl group and the neighboring

substituent, leading to a considerable steric hindrance. Thus, rotation around central bond


40

minimizes this interaction ultimately promoting the appearing of the thermodynamic most

stable Z product.

O

N

N

O

O

O

R

R

O

N

N

OH

O

O

R

R

O N

O

R

R

O

O

- HI

I2

Rotation

Z product

2 HI + CH3SOCH3 I2 + CH3SCH3 + H2O

H O N

OH

R

R

O

OH

O N

O

R

R

O

OH

IO N

O

R

R

O

HO

I

10a-c

11a-c

59 60

6162

Scheme 2. 17

The last dehydrogenation step has also known some drawbacks because of some by-

products formation during the course of the reaction which runs under energetically

oxidative conditions (I2/DMSO at reflux). As a result, we have isolated two compound

being 1,3-dicyclohexylparabanic acid 11a(a) (17% yield) and salicylic acid 11a(b) (by

preparative TLC) which resulted from an oxidative cleavage of the C—C bond linking the

two five-membered BF and HD rings of 1,3-dicyclohexyl-5-(3-oxo-2,3-dihydrobenzofuran-

2-yl)imidazolidine-2,4-dione 11a as a parasite reaction (Scheme 2.18).

O

O

N

N

O

O

R

R

O

N

N

O

O

R

R

OH

OH

O

+

11a(b)11a 11a(a)R = Cyclohexyl

I2 / DMSO

Reflux

Scheme 2. 18

We have succeeded, for the first time, the isolation of 11a(a) as single crystals which

have been accidently collected from the ethanolic recrystallization solution of the desired

compound 11a. The results of X-ray data collection has rather shown the structure of the

bilateral symmetrical 1,3-dicyclohexylparabanic acid 11a(a) which crystallizes into

different polymorphic networks [51, 52] (Fig. 2.20). The second molecular fragment

resulted from the oxidative cleavage in question, was identified as salicylic acid 11a(b)

which is confirmed according to 1H NMR data of the authentic sample.


41

Figure 2. 20. Schematic representation of 11a(a) molecular structure

Moreover, a crucial photochemical property has been discovered in compound 11c

(E/Z)-isomers. The (E/Z)-isomerization is an important issue in chemistry and biology

fields offering various industrial, technological and medicinal applications [53]. The (EZ)-

isomerism phenomenon is usually induced by photo- and/or thermal energy contribution

or even chemically catalyzed [54]. Regarding this research area, the selective photo-

dependent (EZ) and (ZE) isomerization equilibrium of (E/Z)-1,3-ditolyl-5-[3-

oxobenzofuran-2(3H)-ylidene]imidazolidine-2,4-dione 11c was accidently discovered

during the phase of preparative chromatographic TLC separation. The photochemical

process, which occurs spontaneously in solution when exposed to direct visible-light

(sunlight or classic electrical lamp), was investigated by 1H NMR spectroscopy and HPLC-

UV spectrophotometry [55]. Both diastereomers (E)-11c and (Z)-11c are stereochemically

stable in solid state, but show significant photo-sensibility in organic solutions. 1H NMR

analysis of CDCl3 solutions of pure (E)-11c and (Z)-11c in the dark displays remarkable

differences in their proton chemical shift values (Fig. 2.21, 2.22). Therefore, the (E/Z)-

photoisomerization equilibrium of 11c can simply be deduced from the 1H NMR profile

after visible-light irradiation (using electrical light) of NMR tube solutions of both (E)-11c

and (Z)-11c, indicating their presence in a 1:3 ratio (Fig. 2.23). The E/Z equilibrium ratio

depends on various parameters.


42

Figure 2. 21. 1H NMR spectrum of compound (Z)-11c (300 MHz)

Figure 2. 22. 1H NMR spectrum of compound (E)-11c (300 MHz)

1'

2'3'

5

1

5'

6'7'

8'

9' 3

2

44'

1''

1'''

O

O

N

NO

O

H-7’ H-5’

Tolyl protons

H-6’ H-4’

p-CH3

1'

2'3'

5

15'

6'7'

8'

9'

3

2

4

4'

1''

1'''O

O

N

N

O

O

H-7’ H-5’

Tolyl protons

H-4’ H-6’

p-CH3


43

Figure 2. 23. 1H NMR of (E/Z)-11c at the photoisomerization equilibrium (300 MHz)

Mechanistically, it is proposed that the photoisomerization process might pass by an

intermediate where the free rotation over the C=C double bond turns possible. Electronic

delocalizations are observed in the systems of O—C=C—C=O and/or N—C=C—C=O of

the BF-HD hybrid which can create a possible rotation over the central C—C σ bond in the

mesomeric forms O+=C—C=C—O- and/or N+=C—C=C—O-. Visible light promotes these

rotations generating the EZ and ZE photoisomerization transformations until the

equilibrium is reached (Scheme 2.19).

O+

O

N

N O

-O

O

O

N

N

O

O

(E)-11c (Z)-11c

O

O-

N+

N

O

O

O+

O

N

N

-O

O

O

O

N

N O

O

O

O-

N

N+ O

O

Transition state

Orhv hv

Scheme 2. 19

— (Z)-11c

— (E)-11c

H-7’ Tolyl protons

H-4’ H-6’

p-CH3

H-4’

O

O

N

N

O

O

(E)-11c (Z)-11c

O

O

N

N O

O

hv


44

The photoisomerization reaction was further monitored by HPLC-UV after separating

and isolating both isomers (E)-11c and (Z)-11c in a pure state using preparative-TLC

plates which is performed under strict lightless conditions (Fig 2.24, 2.25). We have

designed an homogeneous isotherm system (35°C) using electrical lamp as light source

of irradiations, providing the same photo-intensity during the whole period of study (see

experimental part). The results of the kinetic study reveals that (E Z)-equilibrium is

mainly photo-dependent being the transformation (EZ) proceeding faster than the

(ZE) in such circumstances (Fig 2.26). The final (E Z)-equilibrium ratio depends on

the used solvent (at 35°C, E/Z 1:3 in chloroform; 1:4 in dichloromethane), light source (at

25°C, E/Z 1:1.5 under sunlight irradiation in dichloromethane) and temperature (at 60°C,

E/Z 1:4 in chloroform). Thermal heating (up to 200°C) does not induce the isomerisation in

the absence of light; but has affected the kinetic rate of the photochemical process. Thus,

we demonstrated that both diastereomers (E)-11c and (Z)-11c are visible-light photo-

sensitive tending to co-exist together in equilibrium solutions at a determined ratio which

minimizes the thermodynamic energy of stability and increases the entropy of the system.

However, in all the studied conditions, the equilibrium was always shifting to the most

stable (Z)-product.

Figure 2. 24. Preparative TLC separation of (E)-11c (above) and (Z)-11c (below)

UV Visible


45

Figure 2. 25. HPLC separation of (E)-11c (10.4 min) and (Z)-11c (14.5 min)

Figure 2. 26. EZ (left) and ZE (right) kinetic studies of compound 11c in

dichloromethane (CH2Cl2) solution


46

2.2. POLYSUBSTITUTED URACIL DERIVATIVES

2.2.1. Uracil and nucleoside-based derivatives

One of the four nucleobases in the nucleic acid RNA is uracil (U), a naturally

occurring pyrimidine derivative which has been implicated in several pharmacologically

important molecules (Fig. 2.27). Synthetic 1,3-disubstituted uracils have thoroughly been

examined for their anti-HIV activity. Several reports of De Clercq and Maruyama have

generated a range of fully or partially substituted uracils showing potent anti-HIV activity

[56-60]. The uracil hetero-nucleus has found further biological applications, for instance,

anti-viral [61], anticancer [62], antimicrobial [63], cytotoxicity to cancer cell [64,65] and

various inhibitory activities including human deoxyuridine triphosphatase [66], thymidine

phosphorylase [67] and of Poly(ADP-ribose)polymerase-1 [68].

5-Fluorouracil 63 (Commercial brand: Adrucil, Carac, Efudix, Efudex and Fluoroplex) is

an uracil derived molecule used as drug in cancer treatment. It is one of the potential

thymidylate synthase inhibitors (GI50 = 17.1 µM) [69], which have created some hope for

the life of cancer patients (Fig. 2.27).

NH

HN O

O

NH

HN O

O

F

63U

1

2

345

6

Figure 2. 27. Uracil (U) and the derived drug 5-fluorouracil 63

Regarding the organic synthetic aspect, the literature has revealed several methods to

build the uracil nucleus and its polysubstituted derivatives [70-73]. A solid phase

condensation of resin-bound unsymmetrically substituted ureas with diketene in acetic

acid gave 6-methyl-1,3-disubstituted uracils compounds in good to excellent yields and

purities [70]. The Baylis-Hillman reaction have been applied in the synthesis of substituted

uracils via a multi-step procedure [71]. Interesting results have been reached by using

microwave activation as an alternative of conventional conditions for the metal carbene

complex chemistry. In particular, the synthesis of uracil derivatives 66 through reaction of

alkynyl alkoxy carbene complexes 64 with ureas 65 (Scheme 2.20) [72]. Most recently,

the synthesis of 1,3-disubstituted uracils was accomplished in good yields by the amino-

selenenylation of α,β-unsaturated esters and cyclization with isocyanates, followed by

oxidation-elimination of the phenylseleno group [73].

http://en.wikipedia.org/wiki/Nucleic_acid

http://en.wikipedia.org/wiki/Pyrimidine

http://en.wikipedia.org/wiki/Thymidylate_synthase


47

O

W(CO)5

NH

O

NH

RR

N

N

W(CO)5

O

R

R

N

N

O

O

R

R64 65 66

+

Scheme 2. 20

2.2.2. Synthesis of 5-(hydroxybenzoyl)-1,3-disubstituted uracil derivatives

Uracil derivatives exhibit a broad biological field of application in drug discovery and

development and therefore, improving organic routes of their synthesis is still a particular

deal. In this part, we wish to demonstrate a simple synthetic access to 5-

(hydroxybenzoyl)-1,3-disubstituted uracil compounds via ring transformation of chromone-

3-carboxylic acid (3-CHCA) 2 upon reacting with carbodiimides 3a-c. This greener

approach occurs in short time avoiding the multi-step procedures and the use of various

chemicals and metal-transfer, therefore, it overcomes the laborious purification steps.

Uncatalyzed nucleophilic addition of 3-CHCA 2 on the C=N double bond of

carbodiimide 3a, 3b followed by a Beckmann-like rearrangement, have led to the isolation

of 3-chromone-N-acylureas 12a(a), 12b(a) products. The reaction proceeds in refluxing

chloroform for 30 minutes (Scheme 2.21). Compounds 12a(a), 12a(b) are obtained in 74

and 80% yields, respectively, isolated in a pure state after recrystallization from an

appropriate solvent. This first rapid step is followed by N-cyclisation and parallel chromone

ring opening, leading to the uracil-based products 12a (64%), 12b (51%). The

transformation proceeds under organo-base catalysis using 4-pyrrolidinopyridine (4-PPy)

and under similar operating conditions as stated above. This second step is

recommended to be realized in situ by adding 4-PPy to directly transform 12a(a), 12b(a)

(after complete consumption of the starting material 2 monitored by TLC) to uracil

derivatives 12a, 12b without their isolation as intermediates. The use of 4-PPy catalyst in

the first step has rather led to the dimerisation of 3-CHCA 2 yielding predominantly

chromone-3-(2-hydroxyphenyl)-3-oxoprop-1-enyl which will be undertaken in detail in the

following chapter III. Therefore, the whole in situ two-step synthetic approach consists in a

ring transformation of chromone 2 into uracil 12a, 12b. The relevant procedure could be

abstracted in a one-pot uncatalyzed reaction only when reacting the carbodiimide 3c (with

tolyl group) on the acid 2, which has led to the direct production of the desired uracil

derivative 12c in green and environmentally friendly conditions without the formation of

any 3-chromone-N-acylurea or other intermediates. This case of reaction is accomplished


48

in refluxing chloroform after 30 minutes time affording 12c in 77% yield which is purely

obtained after recrystallization from ethanol (Scheme 2.21).

O

O

NC

NRR+

COOH

N

N

O

R

O

R

O

N

HN

O

R

O

R

O

O

Cat. 4-PPyCHCl3 reflux

30 min

R = Cyclohexyl, Isopropyl 12a(a), 12b(a)

3a-c

12a-c

2

OH

In situ two steps

One step

CH3Cl / reflux, 30 min

CH3Cl / reflux, 30 min

only when R = Tolyl

a. R = Cyclohexylb. R = Isopropylc. R = Tolyl

Scheme 2. 21

All compounds have been characterized by 1H and 13C NMR spectroscopy and further

supported by 2D-NMR experiments including, HSQC and HMBC. Preliminary, it was

possible to distinguish between the chromone and the uracil rings in compounds 12a(a),

12b(a) and 12a, 12b, respectively, only by referring to their proton NMR spectra. Both

vinylic protons H-2 (in chromones, 12a(a), 12b(a)) and H-6 (in uracils, 12a, 12b and 12c)

are characterized as singlet signals, however, it exists a clear chemical shift values

difference (H-2, δ = 8.07-8.08 ppm and H-6 δ = 7.69-7.94 ppm) explaining the neighboring

electronegative heteroatom-type influence (oxygen or nitrogen) (Fig. 2.27).

The ring transformation from the chromone 12a(a), 12b(a) to the uracil 12a, 12b is

also accompanied with proton transfer between nitrogen and oxygen atoms, this fact

could also be monitored by 1H NMR analysis, where the -NH proton, assigned as a broad

signal at 6.49-6.74 ppm in compounds 12a(a), 12b(a) [or it appears as a clear doublet d

coupled with the cyclohexyl tertiary proton in case of 12a(a)], is transformed into -OH

exchangeable proton (establishing a hydrogen bond with the carbonyl group) assigned at

11.71-11.79 ppm as singlet signal in 12a-c compounds (Fig. 2.27). HMBC correlations

play a great role in localizing all carbonyl groups and substituents’ relative positions.

Figure 2.28 represents the main HBMC correlations established between the vinylic

protons and carbonyl groups in both of the chromone and uracil scaffolds. Mass

spectrometry and elemental analysis supported the ring transformation event without any

chemical loss.


49

N

N

O

R

O

R

O

N

R

O O

O

b. R = Isopropyl

12b(a) 12b

6

O

N

O

R

HH

H

H2

Figure 2. 28. 1H NMR monitoring of chromone 12b(a) to uracil 12b ring transformation

N

N

O

O

OO

H

H

H

N

O O

O

HN

O

H

H

H

12a(a), 12b(a) 12a-c

2

3

6

5

Figure 2. 29. Main HMBC correlations in compounds 12a(a), 12b(a) and 12a-b

12b(a)

12b

-NH

H-2

-OH

H-6


50

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CHAPTER 3

- SYNTHESIS OF BENZOPYRAN-(2 AND 4)-ONES-

POLYPHENOLIC HYBRIDS -

Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids

59

3.1. FLAVONOIDS, 2-STYRYLCHROMONES AND RELATED BENZOPYRAN-4-

ONE BASED COMPOUNDS

3.1.1. Flavonoids, 2-styrylchromones: a word on their natural occurrence, biological

applications and chemistry

Flavonoids form a family of phenolic secondary metabolites of plants. All the classes of

this family of compounds share the same basic structure formed by two aromatic rings

attached together via a three carbon chain giving rise to a C6-C3-C6 system. Usually their

structure is closed into an oxygenated pyrano- or furano- heterocycle involving a ketone

function (pyranone or furanone) in the major part of cases. They constitute one of the

most numerous and widespread families of natural plant phyto-constituents with more

than 4000 structures identified, and categorized into several classes, namely, the six

member closed form (benzopyrano-) including flavones, flavanols (catechins), flavanones,

flavonols, isoflavones, anthocyanins and procyanidins; the five member closed form

(benzofurano-) is represented by aurones, in addition to the opened forms like chalcones

and dihydrochalcones (Fig. 3.1) [1,2].

C6 C3

C6

O

O

OO+

O

O

OH

OH

O

O

O

Aurone

Flavonol

Flavanol

Chalcone

Anthocyanin

Flavone

Figure 3. 1. Flavonoid main basic structures

Flavonoids are low-molecular-weight substances extracted from plants by various

methods [1,2]. They have primarily been identified as pigments responsible for the

autumnal shades (yellow, orange, and red) of many kinds of flowers and plants. Flavonols

(kaempferol 67, quercetin 68 and myricetin 69) and flavones (apigenin 70 and luteolin 71)

are the most common phenolic compounds in plant-based food. For instance, quercetin

68 is a predominant component of onions, apples, and berries [3]. Flavonoids like the


60

colorful anthocyanins are found in vegetables and fruits, such as red cabbage [4] and red

grapes [5], they also take part of the red wine phytochemicals as derived from red grapes

[6-8]. More colorful flavonoids are prominent components of citrus fruits and other food

sources. Flavanones, like naringin 72, are typically present in citrus fruits, and flavanols,

particularly catechins 73, are present as catechin gallate in beverages such as green or

black tea [9-12] and red wine [13] (Fig. 3.2).

O

O

O

O

OH

O

O

OH

O

OH

HO

R2

OH

OH

HO

R

OH

OH

O

OH

OH

HO

OH

OH

70. R = H71. R = OH

R1

67. R1, R2 = H

68. R1 = OH, R2 = H

69. R1, R2 = OH

OH

O

OH

HO

O

OH3CHO

OHOH

HO

72 73

Figure 3. 2. Predominant naturally occurring flavonoids

Flavonoids are not only giving to our food its fantastic colors, which mostly attract

consumers, but play a crucial protective role for human health. Consequently, many

structures are established as potential biologically active nutrients [1,2,14]. They have

also been credited with many diverse key functions in plant growth and development,

including stress protection, reproduction, signaling, and protection from insect and

mammalian consumption [1,2]. The daily intake of flavonoids in humans can reach an

approximate rate of 25 mg/day, an average amount which qualifies a pharmacological

worth to human-body fluids and tissues, guarantying a good absorption from the

gastrointestinal tract. In 1938, Szent-Gÿorgyi has first initiated the biological activity of

flavonoids, in his study on citrus peel flavonoids which provide an efficient activity in

preventing the capillary bleeding and fragility associated with scurvy [3]. Certain individual

members of the flavonoid family display a multiplicity of biological activities, and therefore

this most promising family of biologically active compounds becomes the key title of

several recent research work. Among the authors keen to the flavonoid compounds, we

state Morton et al. [15] who has published a review on distribution, bioavailability and

biological activity of the flavonoid compounds, suggesting that they may have a

physiological role as antioxidants.


61

Actually, it is accepted that natural flavonoids present in fruits and plant-derived-food

are relevant, not only for technological reasons and organoleptic properties, but also

because of their potential health-promoting effects, as suggested by the available

experimental and epidemiological data. Human trials on the antioxidant effects of

beverages rich in such polyphenolic compounds, like red wine, fruit juice and tea, have

been limited and results are, at present, inconclusive. This fact is particularly due to poor

inconvincible methodologies available to measure oxidative damage in vivo, and that is

why still further research efforts are being required. The use of appropriate biomarkers of

oxidant damage in vivo measure is primordial in order to prove that these compounds can

be conclusively considered as dietary antioxidants with nutritional benefit. In contrast, the

beneficial biological effects of these food components may be depicted by two of their

characteristic properties, their affinity for proteins and their antioxidant activity. Over the

last 15 years, numerous publications have demonstrated that besides their in vitro

antioxidant capacity (measured by DPPH, ORAC and other techniques) [16] and in vivo

evaluation [17], certain flavonoids, such as anthocyanins, catechins, proanthocyanidins

encountered in our daily food, may regulate different signaling pathways involved in cell

survival, growth and differentiation. These compounds are acting differently and selectivity

in various models as far as their antioxidant capacity is concerned, suggesting that multi-

models should be utilized in order to evaluate an antioxidant agent from natural sources

[18].

3.1.1.1. Flavones

Flavones (FLV, 2-arylchromones) are a group of flavonoids containing the

benzopyran-4-one (BP-4) nucleus. Over the last decade, FLVs gained a great attention

due to their potential biological and medicinal utilities. Reminding that a FLV backbone

respects the C6-C3-C6 skeleton system of the flavonoids family involving three aromatic

rings A, B and the heterocycle (pyranone) C as it is depicted in Figure 3.3 [1,2].These

compounds can be characterized as “privileged structures” for their ability to interact with

a number of different receptors in the body, thereby precipitating a wide range of

biological responses [19]. Among the naturally occurring FLVs and their synthetic

analogues, several derivatives display important biological properties, such as anticancer

[20], anti-inflammatory [21] and antioxidant [22] activities. These first promising results

explain the increasing interest in this class of the most abundant naturally occurring

compounds and the continuous isolation of new biologically active derivatives, such as

(7’’R)-8-[1-(4′-hydroxy-3′-methoxyphenyl)prop-2-en-1-yl]galangin 74 having a cytotoxic


62

activity to PANC-1 human pancreatic cancer cells [23]. Considerable attention was paid to

the synthesis of FLV derivatives especially those with biological activity predictions. For

instance, we underline some molecules like 3-alkyl-3’,4’,5,7-tetrahydroxyflavones 75

proved as potent active antioxidants evaluated in various biological systems, including in

vitro assays [24a,24b]. Flavopiridol 76 shows a cyclin-dependent kinase inhibitor effects

which is actually under phase II clinical trials for a number of different malignancies (Fig.

3.3) [24c-24h].

O

O

FLV

O

OOH

HOO

OOH

HOH

HO

H3CO

75

OH

OH

74

n

76

O

O

Cl

OH

HO

N

OH

R RA

B

C 2

35

6

78

3'4'

5'

6'

2'

4

Figure 3. 3. Molecular structures of the flavone backbone (FLV) and related bioactive

derivatives 74-76

3.1.1.2. (E)-2-Styrylchromones

2-Styrylchromones (2-STC) constitute a further important biological active BP-4-base-

structure, but very scarce as naturally occurring compounds [25]. Only four derivatives

have been isolated from nature, namely hormothamnione 77 and 6-

demethoxyhormothamnione 78 from the marine blue-green algae Chrysophaem taylori

[26], (E)-5-hydroxy-2-styrylchromone 79 from the rhizomes of Imperata cylindrical

(Poaceae) [27] and, more recently, (E)-6,4’-dihydroxy-3-methoxy-2-styrylchromone 80

isolated from the tree Aquilaria sinensis (Thymelaeaceae) [28] (Fig. 3.4). Natural 2-STC

derivatives have only demonstrated cytotoxic activity to leukemia cancer cells [26], while a

range of biological effects has been evidenced for synthetic derivatives, such as antiviral

[29], antitumor [30], antimitotic [31], anti-inflammatory [32] and antioxidant [24b,33]

activities. Some of the biologically active 2-STC synthetic derivatives present simple

structures; for example, the antimitotic (E)-4’-methoxy-2-styrylchromone 81 also

considered as a potent anti-norovirus agent [31], along with (E)-5-hydroxy-2-


63

styrylchromone 79 [34]. Moreover, 2-STC with a catechol pattern such as derivative 82,

present considerable anti-inflammatory and antioxidant activities [32] (Fig. 3.4).

O

O

77. R = OCH378. R = H

O

O

79

CH3

OCH3

OH

H3CO

R

OH

OH

O

O

OH

80

OH

HO

OCH3

82

O

O

81

O

O

OCH3

OH

OH

OH

O

O

R

R

2-STC

Figure 3. 4. Molecular structures of the (E)-2-styrylchromone backbone (2-STC) and

related bioactive derivatives 77-82

3.1.1.3. Synthetic methods of flavones and 2-styrylchromones

In light of the biological significance of the mentioned BP-4-based compounds, many

researchers dedicate their work to develop efficient synthetic methodologies for this type

of compounds. The most used synthetic routes for the synthesis of flavones and 2-

styrylchromones include the Baker-Venkataraman (BV) method and the

cyclodehydrogenation of 2’-hydroxychalcones and 2’-hydroxycinnamylidene-

acetophenones.

The Baker-Venkataraman (BV) route [35] is one of the oldest approaches drawn to the

synthesis of FLV derivatives and still being one of the most used efficient routes for 2-STC

production [36]. It involves a three-steps sequence where the final step consists in the

cyclization of β-diketones 84 (obtained from ester 83 via BV rearrangement), which exists

in equilibrium with its enolic form, into the corresponding BP-4 nucleus 85. Several

conditions can be employed to perform this cyclization in question, mostly under acidic

conditions. Extensive studies performed by Silva et al. indicate that molecular iodine


64

consists of a successful manner for FLVs [37] and (E)-2-STCs synthesis [38] (Scheme

3.1).

84

85

(i) KOH / DMSO

(ii) I2 / DMSO/ 90 -100 oC

O R

O

O

R

HOOH

(ii)

O

R

OOH R1

O

O

R1

RO

R2

(i)

R = or R2

R1

R1

83

R1 and R2 = OH, OCH3, NO2

Scheme 3. 1

As mentioned previously, the oxidative ring closure of the appropriate 2’-

hydroxychalcones and 2’-hydroxy-2-cinnamylideneacetophenones is another important

well-documented approach toward FLVs and (E)-2-STCs synthesis. Various reagent

systems are known for the oxidative cyclization of the 2’-hydroxychalcones 87 to the

corresponding flavones 90, namely disulfides [39], sodium periodate [40], hypervalent

iodine reagents [41], DDQ [42], oxalic acid [43], Wacker-Cook related oxidation [44], and

finally selenium dioxide [45]. Nevertheless, the use of molecular iodine/DMSO system

seems to be more flexible for its lower toxicity and cost, leading to better yields and

shorter reaction times. Further systematic studies have been conducted by Silva et al.

disclosing scopes and limits of this method [46]. Yet, the most important aspect of their

work was the successful application of this methodology to the synthesis of FLVs 90 and

(E)-2-STCs 92, via the oxidative BP-4 ring closure of the 2’-hydroxychalcones 87 and 2’-

hydroxy-2-cinnamylideneacetophenones 88 in 30 minutes. Also 5-hydroxyflavones 91 and

(E)-5-hydroxy-2-styrylchromones 92 have been similarly produced but lasting a longer

reaction time (Scheme 3.2) [47].


65

88 or 89

90 or 91

O R

O

OOH

30 min

2 h

92 or 93

O R

OOH

86

OOH

OHCR1

R3

+

87

(i) NaOH / MeOH, r.t.(ii) I2 / DMSO, reflux

or

OHC

R2

(i)

R

R1

R1

R3

R = or R3

R1 and R3 = OH, OCH3, NO2

R1

R2 R2

(ii)R2 = OBn

(ii)

R2 = H

For Compounds:88, 90, 91 89, 92, 93

Scheme 3. 2

3.1.2. Design and synthesis of benzopyran-4-one based polyphenolic hybrids

Still a huge amount of published data are considering the broad biological effects

inherent in the BP-4-based compounds including, flavones (FLV), chromones (CHR) and

chromanones (CHRM). The FLV structures have been well-documented in the literature

through various aspects emphasizing, for example, their high abundance in plants, fruits

and vegetables contributing as a part of human dietary source. They have majorly been

associated with several biological activities and nutritional benefits, being recognized as

anticancer, anti-inflammatory and antioxidant agents. In parallel, the similar chromone-

based compounds have received a great attention as ubiquitously found in the plant

kingdom showing various biological actions [48]. In particular, 2-STCs representing one of

the CHR-based structures, even less abundant in nature, but have generated a number of

synthetic reports underlining their biological potential, such as antiviral, antitumor,

antimitotic, anti-inflammatory and antioxidant activities. Besides, chromanones (CHRM)

are another representatives of BP-4-based scaffold, which are commonly encountered in

nature [48]; some novel CHRMs structures extracted from Calea uniflora (Asteraceae)

showed significant growth inhibition of Leishmania major parasitic promastigotes in the

micrograms per milliliter range [49].

Taking into account their first promising biological background, these important natural

BP-4-compounds have known a growing interest in organic synthetic and medicinal fields.

Different multi-stage methodologies have been entertained in regard of novel FLVs, CHRs

and CHRMs synthesis, by modifying substituents on their aromatic and heterocyclic rings


66

and/or adding organic/inorganic fragments which usually confers to the whole resulting

scaffold new biological profile and/or improvement of the initial bioactivity related to the

natural-parent-compound. However, further knowledge on structure-activity-relationship is

required for producing privileged BP-4-based frameworks suitable for in vivo and in vitro

biological interactions. Also, development of convenient synthetic pathways of these

compounds is actually a challenging issue but also of an urgent need in the medicinal

chemistry field. In this regard, a recent paper has reported new synthetic fully

phosphorylated-FLVs which are found to be potent pancreatic cholesterol

esterase inhibitors [50]. Ling et al. [51] have recently covered the advance in

the synthesis of 3-alkyl-FLVs due to their multiple-biological applications. In connection to

the organic synthetic aspect, Silva and co-workers have entirely devoted their interest on

3-substituted -FLVs and -2-STCs synthesis [31-33], for instance, 2-STCs 94 and 3-

cinnamoyl-2-styrylchromones 95 have been produced via a modified Baker–

Venkataraman pathway (Scheme 3.3) [38]. In other sides, a diastereoselective organo-

catalytic aldol/oxa-Michael reaction has been developed to deliver medicinally relevant

2,3-ring substituted chromanones [52]. A series of antioxidant 6-hydroxy-7-methoxy-4-

chromanones and chroman-2-carboxamides were synthesized via sequential reactions

[53].

OH

OH O

R OH

O

O

O O

O

OH O

RO

O

R

RO

O

O

O

O

O

R OH

O

2 x1 x

R'

R'

R'

R'

R =

R' = H, OH, OCH3, OBn, Cl

94 95

i

iiiii

iv

v

i: 1equiv DCC, 0.1eq, 4-PPy, CH2Cl2, rtii: KOH, DMSO under N2, rtiii: I2(cat.)/DMSO, 100°C or TsOH-DMSO 100°Civ: 2eq DCC, 0.2eq, 4-PPy, CH2Cl2, rtv: K2CO3, dry pyridine, under N2, 120°C

Scheme 3. 3

The insertion of the (2-hydroxyphenyl)-3-oxoprop-1-enyl- (HOPO-1) bioorganic

pharmacophore into the CHR structure was an initiative structural engineering toward a

probable increase of the antiallergic activity and decrease the toxicity of the whole


67

resulting chalcone-like derivatives, which have been synthesized in a sequence of several

steps [54]. Numerous pharmacological applications have been evidenced for natural

flavonoids sharing HOPO-1 moieties, as best example, xantohumol (2',4,4'-trihydroxy-6'-

methoxy-3'-C-prenylchalcone, XAN) is one of the most important biologically active

prenylated flavonoids extracted from hop (Humulus lupulus family: Cannabaceae)

delivering a multiplicity of biological effects, such as antioxidant activity, promotion for

nerve growth factor, induction of quinone reductase, acyltransferase inhibition, cancer

chemopreventive anticarcinogen and inhibition of human p450 enzymes [55].

(2-Hydroxyphenyl)-3-oxoprop-1-yl- (HOPO-2) is a bioorganic moiety (HOPO-2 is the

dihydro-derivative of HOPO-1) which have figured in several biological active compounds,

especially dihydrochalcones. Aspalathin 96 is a C-linked dihydrochalcone glucoside found

in rooibos tea (Aspalathus linearis, family, Fabaceae). Aspalathin contains the relevant

HOPO-2 fragment which could be the particular structural feature along with catechol

patterns associated with its antimutagenic and antioxidant properties [56] (Fig 3.5).

OOH

O

OCH3

OH

HO OH

XAN

OOH

HOPO-2HOPO-1

OH

OOH

HO

OH

OH

O

OHHO

HO

HO

96

Figure 3. 5. Naturally inspired biological pharmacophores HOPO-1 and HOPO-2

Accordingly, the addition of the HOPO-1 into the position 3 of CHRs, FLVs, 2-STCs

and higher conjugated homologues, has not been published yet and therefore, designing

of these new semi-hybrid compounds 13, 15 along with 2,3-disubtituted chromanone

building blocks 14 incorporating the HOPO-2 fragment (Fig. 3.6), should constitute a new

strategy with special biological and medicinal perspectives. All these novel BP-4-based

targets have been elaborated by a synthetic protocol starting from BV route to a one-step

Michael addition (MAD) tandem reaction of 1,3-dicarbonyls on the key material chromone-

3-carboxylic acid. The relevant method has led to a facile and efficient access to several

BP-4 scaffolds sharing the bioorganic fragments HOPO-1 and HOPO-2.

http://en.wikipedia.org/wiki/Carbon

http://en.wikipedia.org/wiki/Dihydrochalcone

http://en.wikipedia.org/wiki/Glucoside

http://en.wikipedia.org/wiki/Rooibos

http://en.wikipedia.org/wiki/Aspalathus_linearis

http://en.wikipedia.org/wiki/Fabaceae

http://en.wikipedia.org/wiki/Mutagen

http://en.wikipedia.org/wiki/Antioxidant


68

This part of the chapter is presented in a sequence of scientific discoveries on the

chromone-3-carboxylic acid reactivity towards various oxygen, nitrogen and carbon

nucleophiles. 3-CHCA is considered as a strong Michael acceptor delivering the BP-4-

based compounds incorporating the typical pharmacophores HOPO-1 and HOPO-2.

Several mechanistic events are experimentally confirmed and supported with data from

the literature. Conclusive results, recapitulation and a general comprehension of the whole

organic chemical aspect behind the key material 3-CHCA is well discussed.

O

O

R

R

O

O

R

R

R

R

O

O

R

R

13

14

O

O

O

O

O

HO

OH

R

15

HO

O

HO

O

HO

O

HO

O

Figure 3. 6. 3-(HOPO-1)-substituted -CHRs, -FLVs, -2-STCs 13, -(4-arylbuta-1,3-

dienyl)chromones 15, and 2,3-disubstituted-CHRMs 14 as new biological targets

3.1.2.1. Generalities on 1,4-conjugate addition (Michael additions)

Michael addition (MAD), or the actual designation 1,4-conjugate addition, is

the nucleophilic addition of carbanions/nucleophiles to an electron-deficient α,β-

unsaturated system which is usually catalyzed by organic or inorganic bases [57]. It has

long been the most useful methods for the efficient constructions of C-C bonds. Scheme

3.4 gives the general aspect of MAD reaction where the nucleophile Nu:, called the

Michael donor, can be a carbanion. Activated methylene are frequently utilized as Michael

donor, which can be transformed into reactive carbanion depending on the electron-

withdrawing strength of the neighboring groups, such as acyl, carboxyl, making their

acidic proton very labile and easily captured by organic/inorganic bases catalyst. The

http://en.wikipedia.org/wiki/Nucleophilic_addition

http://en.wikipedia.org/wiki/Carbanion

http://en.wikipedia.org/wiki/%CE%91,%CE%B2-unsaturated_carbonyl_compound


http://en.wikipedia.org/wiki/Nucleophile

http://en.wikipedia.org/wiki/Electron-withdrawing_group

http://en.wikipedia.org/wiki/Acyl

http://en.wikipedia.org/wiki/Carbon_acid


69

substituent R on the activated alkene (or α,β-unsaturated system) is called a Michael

acceptor, which is usually a ketone function (or nitro NO2). MAD reactions show

important advantages as an atom-economic and eco-friendly approach offering various

diastereoselective and enantioselective C-C bond formation. The MAD is further

generalized being applicable to several oxygen, nitrogen and sulfur nucleophiles, usually

the prefix oxa-, aza- and thia- are added to differentiate the nucleophilic type of the

Michael addition (Scheme 3.4).

RNu : + R

NuBase

O

R1

H

O

R2

B-

O

R1

O

R2_

R3

R4

O

O

R1

O

R2

R3

R4-OBH

O

R1

O

R2

R3

R4O

Scheme 3. 4

3.1.2.2. Reactivity of chromone-3-carboxylic acid

From the literature, a handful but few descriptions are reported on the organic

chemical background of chromone-3-carboxylic acid. 3-CHCA delivers various

transformations under nucleophilic conditions thanks to the high versatile reactivity of

C2—C3 double bond of the BP-4 system. The accentuated electron-withdrawing effects of

such groups, like carboxylic acid 3-COOH and 4-ketone functions, is found useful to

generate a good electrophilic site at the position 2 in favor of nucleophic attacks, for

instance, 1,4-conjugate additions (MADs). Only recent synthetic works have undertaken

the MAD on similar C-2 electron-deficient-chromones giving rise to the production of novel

functional polycyclic chromones [58] and functionalized 2-hydroxybenzophenones [59], via

tandem reaction processes. Peng et al. [60] have reported the synthesis of 4-substituted-

3,4-dihydrocoumarins 99 using a 1,4-conjugate addition/double decarboxylation cascade

reaction of β-ketocarboxylic acid 97 on 3-coumarin carboxylic acid 98. In this precise

case, the 3-COOH group sounds very utile in improving the C-4 coumarin Michael

acceptor character, since the simple coumarin did not allow this type of addition reactions

(Scheme 3.5).

http://en.wikipedia.org/wiki/Alkene


http://en.wikipedia.org/wiki/Ketone

http://en.wikipedia.org/wiki/Atom_economy

http://en.wikipedia.org/wiki/Enantioselective


70

HO

O

SPh

O

O O

COOH

O O

SPh

O

+TEA (20 mol%)

THF, rt / 6h

97 98 99

Scheme 3. 5

Further investigations are demonstrating a broad synthetic potential of 3-substituted

CHRs using the nucleophilic 3-carboxylic acid function of 3-CHCA. The action of ortho-

phenylenediamine on 3-CHCA gives benzimidazole derivatives [61]. In mimic conditions,

3-(3-alkyl-5-thioxo-1H-4,5-dihydro-1,2,4-triazol-4-yl)aminocarbonylchromones have been

prepared by treatment of 3-CHCA with 3-alkyl-4-amino-4,5-dihydro-1,2,4-triazole-5-(1H)-

thione in the presence of POCl3 [62].

The behavior of 3-CHCA towards primary and secondary amines, hydrazines,

cyanoacetohydrazide, cyanoacetamide and malononitrile in different media has mainly led

to ring transformation via pyranone-ring opening and decarboxylation [63]. In the referred

work, the author has accomplished the first synthesis of the target scaffold 3-[(2-

hydroxyphenyl)-3-oxoprop-1-enyl]chromone 13a [3-(HOPO-1)-chromone] (Scheme 3.6)

via intermolecular dimerisation and decarboxylation of 3-CHCA 2 in sodium hydroxide

solution. Nevertheless, the reaction was found to be strongly dependant on the base-

concentration and further precise operating conditions. Thus, treatment of 3-CHCA with

0.025 M sodium hydroxide solution in refluxing ethanol, gave rise to decarboxylation and

opening of the pyranone ring to afford ω-formyl-2’-hydroxyacetophenone. When the

reaction was carried out using 0.05 M aqueous sodium hydroxide solution at 70 ºC, the

dimeric-product 13a was obtained in 48% yield.

In this part, the behavior of 3-CHCA under organo-base catalysis is fully studied.

Initially, a catalytic amount of 4-pyrrolidinopyridine (4-PPy), as a tertiary amine organic

base, was sufficiently used to convert the acid 2 into 3-[(2-hydroxyphenyl)-3-oxoprop-1-

enyl]chromone 13a in optimal yield (67%). The protocol follows an intermolecular

dimerisation and decarboxylation. During the course of the reaction, a small quantity of

chromone 13a(a) is formed as a result of the decarboxylation process of 2. Compound

13a is readily isolated after recrystallisation from ethanol, while 13a(a) remains in ethanol-

solution and subsequently isolated by chromatography (Scheme 3.6). In mild conditions,

the organo-base-promoted dimerisation of 3-CHCA, using tertiary amines, shows better

results than the inorganic-base procedure (using NaOH in [63]), regardless the longer


71

reaction time (Table 3.1). Optimal conditions are revealed by the base-catalyzed

transformation using 4-pyrolidinopyridine (4PPy) at room temperature in dichloromethane

for 48 hours reaction time.

O

O

COOH

O

O

O

O

Base

Solvent, T °C

2 13a(a)

+

13a

General conditions: 2 (2.63 mmol, 0.5 g), base (0.13 mmol, 0.05 equiv) in solvent (10 mL)

O

OH

Scheme 3. 6

Table 3. 1. Dimerization of 3-CHCA under various organo-base-catalysis with amines

Entry Solvent Base Temp (°C) Time (h) Yield (%)

1 CH2Cl2 4-PPy rt 48 67 a,b

2 CHCl3 4-PPy Reflux 3 51 a,b

3 CH2Cl2 DBU rt 24 25 a,b,c

4 CH2Cl2 TEA rt 48 55 a,b,c

5 CH2Cl2 Pyridine rt > 48 - b,c

6 CH2Cl2 2,6-Lutidine rt > 48 - b,c a The isolated yield was calculated after recrystallisation.

b The formation of the by-product 13a(a) was confirmed by

analytical TLC using authentic sample, c The remaining starting material 2 was determined by TLC.

In order to compare our results to the literature, we have attempted the dimerisation of

2 in similar ethanol-refluxing conditions as reported in [63], by using catalytic amount of 4-

PPy (instead of sodium hydroxide). The dimerisation event was not observed, but 2-

ethoxychroman-4-one 13a(b) was produced in 92% yield. The discovered result was

further generalized by treating 2 with various aliphatic alcohols 13a(b-d), utilized as

solvent/reactant system or only reactant [like the case of 13a(d)] to produce some 2-

alkyloxychroman-4-one compounds 13a(b-d) sharing an asymmetric C-2 ketal centre

(Scheme 3.7). Compound 13a(d) was obtained in 64% yield, after column

chromatography purification and recrystallisation from hexane, accompanied with a small

amount of chromone 13a(a) as by-product which was recovered by column

chromatography.


72

O

O

COOH

O

O

O

O

Cat. 4-PPy

ROHReflux / 1 h

2

13a(b) 92%13a(c) 97%

13a(a)

+

OR

O

O

Cat. 4-PPy

CHCl3Reflux / 1 h

13a(d) 64%

O

OH

OCH3

b. R = ethylc. R = Isopropyl

d. R =

CH2

OH

OCH3

+ ROH

General conditions: 2 (2.63 mmol, 0.5 g), 4-PPy (0.13 mmol, 0.05 equiv, 0.02 g) in solvent ROH (10 mL) or vanillin alcohol (2.63 mmol, 0.4 g) in solvent (chloroform 10 mL)

Scheme 3. 7

We have also tried the dimerisation of 3-CHCA using alternatively a catalytic amount

of diethylamine (DEA), in the same optimized operating conditions (room temperature in

dichloromethane), but no reaction was revealed according to TLC monitoring, which

indicates the remaining starting material 3-CHCA. The use of an equivalent quantity of

diethylamine in refluxed chloroform has rather led to (E)-3-(diethylamino)-1-(2-

hydroxyphenyl)prop-2-en-1-one 13a(e) (yield 77%). This behavior of aliphatic amines

towards 3-CHCA has already been emphasized in the literature [63]. Our scientific

curiosity to a better understanding of 3-CHCA behavior toward amino-base-catalysis, has

led us to retake the subject of some primary RNH2 and secondary aliphatic amines RR’NH

actions on the acid 2. Hence, only few examples of enaminone products 13a(e-g) are

mentioned in this work (Scheme 3.8).

According to our preliminary results, a strong Michael acceptor character of chromone-

3-carboxylic acid 2 have been noticed under oxygen and nitrogen nucleophilic conditions.

In a general manner this key material 2 undergoes oxa- or aza-MAD/ decarboxylation

/ring-transformation or ring opening at the C-2 electron-deficient carbon. In addition, 3-

CHCA was demonstrated to undergo an intermolecular dimerisation affording the dimeric-

product 13a under type-independent base-catalysis conditions (either organic-type using

4-PPy as reported in this work, or inorganic-type using NaOH as reported in [63]).


73

.

O

O

COOH

O

CHCl3 / Reflux30 min

2

OH

N

OOH HN

HNOOH

DEA

Benzylamine

Ethylenediamine

OOH HN

13a(e) 77%

13a(f) 73%

13a(g) 88%

General conditions: 2 (2.63 mmol, 0.5 g), amine (2.63 mmol, 1 equiv), in chloroform (10 mL)

Scheme 3. 8

From a mechanistic point of view, 3-CHCA behaves similarly but following slightly

distinct reaction pathways with respect to the type of base-catalysis (organic or inorganic).

In case of the inorganic catalysis using sodium hydroxide, the formation of ω-formyl-2’-

hydroxyacetophenone 4a in ethanol/aqueous media is mechanistically justified as referred

in [63]. The action of hydroxide -OH on 2 is similar to that of an alkoxide (aliphatic

alcohols, ROH/RO-) which was undertaken in this work. In general, a base-promoted oxa-

MAD step is happened, followed by a decarboxylation process which results in the

chromanone ring. However, in case of alkali condition (-OH anion), the equilibrium is

totally shifted from the enolate-chromanone to the ring-opened-form yielding ω-formyl-2’-

hydroxyacetophenone 4a (Scheme 3.9) (Note: ω-formyl-2’-hydroxyacetophenone 4a

affords the corresponding 2-hydroxychroman-4-one 100 under acidic conditions, this

equilibrium depends strongly on the pH of the media). In a similar mechanistic fashion as

depicted in Scheme 3.9, aliphatic alcohol are added to the acid 2, but following a different

route at the decarboxylation stage in order to afford the Michael-adduct 2-

alkyloxychroman-4-ones 13a(b-d) accompanied with the parallel formation of small

quantity of the by-product chromone 13a(a) which is the result of the retro-Michael-

addition [related to the case of compound 13a(d) 2-vanillinoxychroman-4-one (Scheme

3.7)]. The obtained 2-alkoxychroman-4-ones 13a(b-d) present a C-2 chiral-centre; thus

interesting features are recorded from the 1H NMR profile where both protons of the C-3

methylene group of 13a(b-d) appears as doublet signals due to the existing asymmetric

environment. For instance, 2-vanillinoxychroman-4-one 13a(d) shows two distinct double

of doublets (dd) proton signals at 2.86 and 3.03 ppm with coupling constants J = 16.8 and


74

3.4 Hz attributable to the chromanone C-3 methylene protons H-3(AB) of the ABX spin

system. The ketal H-2(X) proton appears as a triplet (the sum of two dd) signal at 5.57

ppm (J = 3.4 Hz). In the same way, both protons of the benzylic methylene group

(assigned through HSQC and HMBC correlations), appear as an AB spin system with two

different doublet signals at 4.59 and 4.71 ppm (Fig. 3.7).

O

O

COOH

2

O

H

R

4-PPy

O

O

COOH-OH

O

O

COOH

O

O

OH

O

O-

O

- CO2

O

O-

OR

O

O

OR

O

O-

O

O

O

O

ORH2O

Retro-Michael-additionProduct

Michael-adduct

H2O ROH

4-PPyOH-

O

O-

O

OH

OH O

O-

O

OH

OR

- CO2

O

O-

OR

- CO2O

O-

OH

- ROH- 4PPy

O-

O

OH

or

- 4PPy

OH OH

H

Inorganic-base-catalysis Organic-base-catalysis

- OH-

4PPyH+4PPyH+

13a(b-d)13a(a)

4a

O

O

OH

100

Scheme 3. 9


75

Figure 3. 7. 1H NMR spectrum of 2-vanillinoxychroman-4-one 13a(d) (300 MHz)

We also propose a similar mechanism of reaction regarding the action of aliphatic

amines on 3-CHCA, in which the aza-Michael-addition is happened without the necessity

of base-catalysis since nitrogen nucleophilicity is strong enough to undergo this process

or may be considered as an autocatalytic reaction. Also, a decarboxylation event is

noticed followed by chromanone ring opening. In case of primary and secondary amines,

the reaction favors a ring opened form due to the structural stability of the resulting

products. According to 1H-NMR data, primary amines have led to Z-products 13a(f) (J(Z) =

6.1 Hz), 13a(g) (J(Z) = 7.7 Hz) imposed by intramolecular hydrogen bond established

between the carbonyl group and the amine’s proton, while secondary amines provide the

more thermodynamically stable E-product 13a(e) (J(E) = 12.3 Hz) in the absence of labile

protons on the amine function (Scheme 3.10)

Considering the dimerisation event of 3-CHCA, the mechanistic explanation may

depend on the type of the used base-catalysis. We have seen from the literature that 3-

CHCA affords ω-formyl-2’-hydroxyacetophenone 4a under certain -OH concentration

(0.025 M), whereas, this isolated compound 4a was expected to be an intermediate of the

reaction undergoing a self condensation in higher concentration of aqueous sodium

O

O O

H

H HH

H


76

hydroxide solution (0.05 M) to produce the dimeric-product 13a [63]. This mechanistic

proposition can be empirically falsified, since the isolated ω-formyl-2’-hydroxy-

acetophenone 4a failed to afford the dimeric-product 13a under the same conditions (0.05

M -OH aqueous media at 70 °C) as referred in [63].

O

O

COOH

O

O

O

O-

N+O

O-

O

O

N+

H

R

H

R'R

H

R'

RNHR'

- CO2

O

O-

N+

R

H

R'

O

N+

R

H

R'O-

OOH

NR'

R

OOH NH R

Primary amines (R' = H)Secondary amines

13a(e) (E configuration)

13a(f), 13a(g) (Z configuration)

2

Scheme 3. 10

According to the present study, the use of the organo-base-catalyst 4-PPy (or TEA,

DBU) allows the dimerisation of 2 in higher yields (up to 67%, Table 3.1). The mechanism

should therefore follow the pathway of primary and secondary amine attacks on 3-CHCA

(Scheme 3.10). Nevertheless, tertiary amines (4-PPy, TEA and DBU), presented in

catalytic amounts, are only able to create an enolic-chromanone form 101 after

decarboxylation upon reacting with the acid 2. The intermediate 101 will favor either the

elimination and regeneration of the base, justifying the formation of small to high

quantities of chromone 13a(a) as by-product (depending on the examined amines referred

in Table 3.1), or the condensation of 101 (through its carbanion) on another molecule of

acid 2 yielding predominantly the dimeric-product 13a (Scheme 3.11). Aromatic amines

such as pyridine and 2,6-lutidine did not allow the dimerisation event of 2, thus, we can

considered them as inactive catalysts.

In the case of inorganic-base-catalysis with -OH, the mechanism of 3-CHCA

dimerisation follows exactly the same steps drawn in Scheme 3.11 (replacing 4-PPy by


77

-OH) and therefore the production of the intermediate ω-formyl-2’-hydroxyacetophenone

4a (which replaces the intermediate 101 in Scheme 3.11) is perfectly confirmed and even

isolated under different conditions as referred in [63] (Note: according to the reference

[63], 4a was only isolated in low yields since the reaction mixture was not stoichiometric,

being 1 mmol of the acid 2 for 0.05 mmol of OH-, the reaction was quenched using water

which can cause participation of 4a in the media during a short period of time because

then the reaction was rapidly neutralized). Under such catalytic conditions, the enolate

chromanone-form of ω-formyl-2’-hydroxyacetophenone normally undergoes a subsequent

MAD on another molecule of the acid 2 rather than being self-condensed and therefore,

affording the dimeric-product 13a. Convincibly, our isolated 2-alkyloxychroman-4-ones

13a(b-d) do not dimerize under organo-base catalysis using 4-PPy which was also tested

in similar conditions (rt, CH2Cl2, Table 3.1) in order to support the achieved mechanistic

conclusion.

O

O

O

O

N+

H

-O

O

O

O

COOH

O

O

O

O-

N+O

O-

O

O

N+

H

O

O

N

N4-PPy =

- CO2

+O

O-

N+

O

HOOC

O

-O

O

O

O-

N+

O

O

N+

H

13a(a) By-product

2

101

- CO2

13a

O

OO

HO

R2

4-PPy_

Scheme 3. 11


78

3.1.2.3. Michael addition on chromone-3-carboxylic acid: a one-pot tandem reaction

towards novel polysubstituted -chromones, -flavones, and -chromanones

3.1.2.3.1. Synthesis of 3-substituted HOPO-1 -chromones, -flavones and -2-

styrylchromones

A better comprehension of the reaction mechanism related to 3-CHCA dimerisation

event, can lead to a general conclusion on its reactivity towards carbon nucleophilic

attacks. Therefore, since the resulting dimeric-product 13a is obtained via the described

pathway, which is based on the Michael addition of the ω-formyl-2’-hydroxyacetophenone

4a intermediate (or the intermediate 101) on 3-CHCA (Scheme 3.11), it is very fortunate

that any other 1,3-dicarbonyl reagent 4 can present a similar Michael donor behavior

towards 3-CHCA affording the desired compounds 13 which share the HOPO-1 moiety

(Scheme 3.12). We started progressively to study the C-2 Michael acceptor character of

3-CHCA under the action of carbon nucleophiles namely, 1,3-dicarbonyl compounds 4a-c.

The subject which has not been entertained yet in the literature and which mainly builds

our first biological perspective in finding suitable and appropriate organic pathways

towards the insertion of HOPO-1 as bioorganic activator.

O

O

COOHR1

O O

R2

OHR1

R2

O+

2 413

Michael addition

HO

O

Scheme 3. 12

The MAD procedure is carried out using a catalytic amount of 4-PPy as organo-base-

catalyst in refluxing chloroform. As a result, the action of ω-formyl-2’-hydroxy-

acetophenone 4a has effectively produced the dimeric-compound 13a in higher yield

(74% comparing to the dimerisation procedure of 3-CHCA which yielded 67% of 13a,

Table 3.1). This potential result absolutely affirms, as an empirical basic information, the

reaction mechanism of 3-CHCA dimerisation under organic (4-PPy in the present work) or

inorganic (NaOH in [63]) base conditions. Basically, the first MAD attempts have shown

sequential stationary steps of a tandem/cascade pathway by isolating different products

depending on the used 1,3-dicarbonyl derivatives 4a-c (R1COCH2COR2). Thus, the

tandem procedure has led, after the MAD step, to i) decarboxylation/pyranone-ring-


79

transformation giving rise to the CHRM nucleus 13b, then continuously to ii) the CHRM-

ring-opened structure 13c, and finally to iii) BP-4 ring closure affording 13a, being the

presence of an ortho-hydroxyphenyl group in the 1,3-dicarbonyl 4a, the inducer of the last

BP-4 ring closure in this tandem process (Scheme 3.13). Interestingly, the use of various

phenolic 1,3-dicarbonyl compounds (R1COCH2COR2) sharing mainly a 2-hydroxyphenyl

group (R1 = substituted-2-hydroxyphenyl) 4d-p synthesized via the Baker-Venkataraman

method (Scheme 3.1, see experimental part), is found to give the last additional

intramolecular-cyclisation event of the linked β-diketones portion into the BP-4 ring

producing a variety of C-3 supported HOPO-1 -chromones (R2 = H, CH3) 13a and 13e-g,

-flavones (R2 = substituted-phenyl) 13h-l and -2-styrylchromones (R2 = substituted-styryl)

11m-p, in a one-pot organo-base-catalyzed tandem reaction. The procedure is carried out

under conventional heating conditions using catalytic amount of 4-PPy in refluxing

chloroform which offers moderate to relatively high yields (Scheme 3.14, Table 3.2). As a

matter of fact, we have found that the relevant cyclization of β-diketones (or 1,3-

dicarbonyls) 4a and 4d-p into their corresponding BP-4 nucleus has failed to occur under

the operating condition of organo-base-catalysis (using 4-PPy) stated above in the

absence of 3-CHCA (Scheme 3.14). Moreover, the simple chromone does not behave as

a Michael acceptor when tested for this type of 1,4-conjugate addition reactions (Scheme

3.14). Hence, our new method of BP-4-based polyphenolic synthesis combines both the

BV pathway with MAD on 3-CHCA. The procedure is tentatively generalized on a certain

number of β-diketones (or 1,3-dicarbonyl compounds) templates with variable degree of

methoxy or methyl substitutions (Table 3.2).


80

O

O

COOHR1

O O

R2

O

O

OR1

R2

O

+

2 4a-c13b (56%)

13c (78%),

a. R1 = 2-hydroxyphenyl, R2 = H

b. R1 = ethoxy, R2 = methyl

c. R1 = R2 = methyl

Cat. 4PPy

CHCl3Reflux / 2 to 6 h

General conditions: 2 (5.26 mmol, 1 g), 4-PPy (0.26 mmol, 0.05 equiv, 0.04 g), 4a-c (5.26 mmol, 1 equiv) in chlorform (10 mL)

13a (74 %)

O

OO

HO

R2

OH

O

R1O

HO

R2

Scheme 3. 13

O

OO

HO

R2

General conditions: 2 (5.26 mmol, 1 g), 4-PPy (0.26 mmol, 0.05 equiv, 0.04 g), 4a, 4d-p (5.26 mmol, 1 equiv ) in chloroform (10 mL)

O

O

COOH

O O

R2+

OH

R

RCat. 4-PPy

CHCl3Reflux / time (h)

2 4a, 4d-p13d-p

R2 = H, methyl, aryl, styryl

O O

R2

OR2

O

OH

R

R

4a, 4d-p BP-4

O

OO

HO

R2

3-(HOPO-1)-BP-4

RO

O

Scheme 3. 14


81

Table 3. 2. Michael addition of ortho-hydroxyphenyl-1,3-dicarbonyl compounds 4a and

4d-p on 3-CHCA

Substrate Product Time (h) Yielda (%)

4a O

H

OOH

13a

O

OO

HO

6

74 b

4d O

H

OOH

H3CO

13d

O

OO

HO

OCH3

6

67 b

4e O

H

OOH

H3CO OCH3

13e

O

OO

HO

OCH3

OCH3

24

43 b,c,d

4f O OOH

13f

O

OO

HO

6

63 b

4g O OOH

13g

O

OO

HO

24

51 b,d

4h O OOH

OCH3

OCH3

H3CO

13h

O

OO

HO

OCH3

H3CO

OCH3

24

44 b,d


82

4i O OOH

OCH3

OCH3

13i

O

OO

HO

OCH3

H3CO

24

52 b,d

4j

O OOH

H3CO OCH3

13j

No product

> 72

- b,c

4k O OOH

H3CO OCH3 OCH3

OCH3

13k

No product

> 72

- b,c

4l O OOH

13l

O

OO

HO

24

75 b,d

4m O OOH

13m

O

OO

HO

24

43 b,d

4n O OOH

OCH3

OCH3

13n

O

OO

HO

H3CO

H3CO

24

68 b,d

4o O OOH

OCH3

13o

O

OO

HO

H3CO

24

72 b


83

4p O OOH

13p

O

OO

HO

24

46 b,d

a The isolated yield was calculated after recrystallisation.

b The formation of chromone 13a(a) was confirmed by analytical TLC

using authentic sample, c The formation of the dimeric product 13a was confirmed by TLC,

d The remaining starting materials 2

and 1,3-dicarbonyl 4a and 4d-p were determined by TLC.

3.1.2.3.2. Synthesis of 2,3-disubstituted chromanones

An exceptional circumstance was underlined regarding the reactivity of mono-, or di-

methoxy-2-hydroxyphenyl-1,3-dicarbonyl compounds 4q-t sharing a styryl fragment, in

which, the tested Michael addition on 3-CHCA has led to the selective production of the

novel 2,3-disubtituted chromanone-based compounds 14q-t incorporating the HOPO-2

moiety at position 2. The synthetic procedure is similar to the previously mentioned which

uses catalytic amount of 4-PPy in refluxing chloroform (Scheme 3.15, Table 3.3). In case

of 4q reaction on the acid 2, we have achieved the isolation of a minor quantity of the 3-

(HOPO-1)-substituted 2-styrylchromone 13q (yield 7%) along with the major product of

2,3-disubstituted chromanone 14q (yield 34%).

O

O

O

HO

OH

O

O

COOH

O O

+

OH

H3CO R H3CO

R

Cat. 4-PPy

CHCl3Reflux / time (h)

2 4q-t 14q-t

General conditions: 2 (5.26 mmol, 1 g), 4-PPy (0.26 mmol, 0.05 equiv, 0.04 g), 4q-t (5.26 mmol, 1 equiv) in chlorform (10 mL)

Scheme 3. 15


84

Table 3. 3. Michael addition of 1,3-dicarbonyls compounds 4q-t on 3-CHCA

Substrate Product Time (h) Yielda (%)

4q

O OOH

H3CO

13q

14q

O

OO

HO

OCH3

O

O

O

HO

OH

H3CO

24

24

7 b,c,d

35 b,c,d

4r O OOH

OCH3H3CO

14r O

O

O

HO

OHOCH3

H3CO

24

31 b,c,d

4s O OOH

H3CO OCH3

OCH3

14s

O

O

O

HO

OH

H3CO OCH3

OCH3

48

42 b,c,d

4t O OOH

OCH3H3CO OCH3

OCH3

14t

O

O

O

HO

OHOCH3

H3CO OCH3

OCH3

48

37 b,c,d

a The isolated yield was calculated after column chromatography purification and recrystallisation.

b The formation of chromone

13a(a) was confirmed by thin-layer chromatography (TLC), c The formation of the dimeric product 13a was confirmed by TLC,

d

The remaining starting materials 2 and 1,3-dicarbonyl 4q-t were determined by TLC.

3.1.2.3.3. Synthesis of 3-substituted HOPO-1 -2-(4-arylbuta-1,3-dienyl)chromones

In view of producing a molecular variety for our biological aims, we have envisaged the

synthesis of higher conjugated homologues of 3-substituted HOPO-1 -chromones,

therefore, the condensation of cinnamaldehyde derivatives on the activated 2-methyl

group of 3-[(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-methylchromone 13f has led to the


85

production of 3-substituted HOPO-1 -2-(4-arylbuta-1,3-dienyl)chromones 15a-c in

moderate to high yields. The procedure follows a classical aldol-condensation using

concentrated sodium ethoxide/ethanol solution at room temperature (Scheme 3.16).

+

CHO

O

O

Na

EtOH, rt/ 1 h

13f15a (67%)15b (73%)15c (81%)

a. R1 = R2 = H

b. R1 = H, R2 = methoxy

c. R1 = R2 = methoxy

R2

R1

R1

R2

General conditions: 13f (3.26 mmol, 1 g), cinnamldehydes (3.26 mmol, 1 equiv ), sodium (16.32 mmol, 5 equiv, 0.375g ) in solvent (ethanol 10 mL)

O

HO

O

OO

HO

Scheme 3. 16

3.1.2.4. Michael addition on chromone-3-carboxylic acid: Mechanistic and structural

studies

3.1.2.4.1. Mechanism, generalization and limits of Michael addition on 3-CHCA

With all these experimental findings in hand, the main idea of the combined Baker-

Venkataraman/Michael addition (BV-MAD) approach toward BP-4-based polyphenolic

synthesis was built and especially inspired from our interpretation of the dimerisation

mechanism of 3-CHCA under organic or inorganic base-catalysis. Taking into account the

possibility of ω-formyl-2’-hydroxyacetophenone 4a Michael addition on 3-CHCA, similar

1,3-dicarbonyl analogues 4a-t should provide a similar reactivity. Hence, on this basis, we

have firstly tried the direct action of ω-formyl-2’-hydroxyacetophenone 4a on 3-CHCA and

hopefully our objective was succeeded by isolating the dimeric-product 13a in better

yields. Various activated-methylene of 1,3-dicarbonyl reagents 4a-t have been tested

displaying different chemical behaviors towards 3-CHCA. A general conclusion on the

reactivity of 1,3-dicarbonyl compounds sharing an ortho-hydroxyphenyl group 4a, 4d-t

settles in a tandem/cascade reaction involving organo-based promoted

MAD/decarboxylation/BP-4-ring-opening (related to 3-CHCA reaction partner) followed by

either (A) CHR-ring-closure (related to the 1,3-dicarbonly compounds 4a and 4d-p

reaction partner) to produce compounds 13a and 13d-p (CHRs, FLVs) or (B) CHRM-ring-

closure to produce 14q-t (CHRMs) (Scheme 3.17).


86

We have previously mentioned that several methodologies can be employed to

perform the cyclization of β-diketones (or 1,3-dicarbonyl compounds) to BP-4, being the

most successful, the use of molecular iodine I2/DMSO system which is the up-dated

methodology developed by our research group [46] (Scheme 3.1). The new disclosed BV-

MAD offers several advantages, such as the use of catalytic amount of organo-base-

catalyst under mild conditions (in refluxing chloroform) which gives birth to a large

structural variety of new polysubstituted BP-4 (CHRs, CHRMs, FLVs, 2-STCs) generation

sharing HOPO-1 and HOPO-2 phenolic pharmacophores. Nevertheless, this method have

known some limits and exceptional cases, for instance, an absent reactivity is noticed for

derivatives 4j and 4k which could be a consequence of a lower acidity related to the

activated methylene protons and/or a structural hindrance due to the high level of

polymethoxy-substitution which is not in favor of a facile access of the tertiary amine 4-

PPy. In addition, the use of benzyl-OH-protected 1,3-dicarbonyl compounds has totally

failed to give the expected products (the dimeric product 13a and the chromone 13a(a)

by-product have been formed instead) justifying that the structural hindrance should play

a great role. At present, the unique catalyst-type studied herein is 4-PPy which is

considered as the most efficient tertiary amine base for the BV-MAD tandem approach.

Accordingly, 4-PPy has only allowed the formation of small quantities of the by-product

chromone 13a(a) during the course of the dimerisation reaction comparing to DBU and

TEA (Table 3.1). Besides, inconvenient issues are noticed in several cases of Michael

addition study of substituted 1,3-dicarbonyl compounds 4d-t on 3-CHCA, especially, the

formation of negligible to considerable quantities of the dimeric-product 13a and

chromone 13a(a) as by-products (indication are made in Table 3.2 and 3.3). Finally, this

type of 1,4-conjugate additions cannot be realized on simple chromones [13a(a) was the

unique case of study], because it is a less activated Michael acceptor and this fact clarifies

the crucial role played by the provisional “auxiliary” functional group 3-COOH promoting

nucleophilic MADs on the benzopyran-4-one ring, which is subsequently lost by

decarboxylation under mild conditions to give HOPO-1 or HOPO-2 pharmacophores. In

terms of mechanistic considerations, the tandem MAD reaction can be stopped at any of

the highlighted intermediate stages indicated in Scheme 3.17, but only in specific cases.

The isolation of products 13b and 13c was achieved in the absence of the ortho-

hydroxyphenyl group in the starting 1,3-dicarbonyl compounds 4b and 4c. These

experimental achievements are sufficiently informative about the mechanistic sequence of

the tandem MAD approach. Moreover, a very interesting mechanistic feature is observed

when using specifically mono- or di-methoxy- 2-hydroxyphenyl-1,3-dicarbonyl compounds


87

4q-t bearing a styryl fragment which have ended at a different intramolecular cyclisation

following an oxa-Michael addition to afford 2,3-disubstituted-CHRMs 14q-t incorporating a

HOPO-2 fragment at position 2 (Scheme 3.15). One exclusive case should be underlined

concerning the tandem reaction which can take both of the two mechanistic pathways A

and B, giving rise to parallel reactions, being the CHR-ring-closure to the 3-substituted

HOPO-1 2-stryryl)chromone 13q (isolated in low yield) in one hand and the CHRM-ring-

closure which has led to the 2,3-disubstituted chromanone 14q in the other hand (Scheme

3.17).

R1

O

R2

O

R1

O

R2

OH4-PPy

O

O-

O

O

HOOC

R1

R2

O

O

O

O-HOOC

R1

R2

O

O

R2

OHO

- CO2

R1 = 2-hydroxyphenyl

- H2O

O

O

R1

R2

O

O

13b13c

13a, 13d-p and 13q

2

4a-t

O

14q, 14s-t

Pathway BPathway A

O

HO

4-PPy

H

O

OO

HO

R2

OO

HO

R2

OH

OH

A

B

OH

O

R1O

HO

R2

Scheme 3. 17

3.1.2.4.2. Structural characterization of the BP-4-based polyphenolic compounds

The structural characterization of all the new BP-4-based polyphenolic products was

established on the basis of extensive NMR studies, including HSQC, HMBC and NOESY

spectra and further supported with exact mass measurements. 1H-NMR patterns are

putting in evidence several stereochemical features. In all the synthesized 3-substituted

(HOPO-1)-BP-4-polyphenolics including the dimeric-product 13a, CHRs 13d-f, FLVs 13g-


88

l, 2-STCs 13m-q and 2-(4-arylbuta-1,3-dienyl)chromones 15a-c, the 3-(2-hydroxyphenyl)-

3-oxoprop-1-enyl (HOPO-1) portion presents the E-configuration of the double bond as

deduced from the coupling constant values J(E) = 15.1-15.2 Hz. H-α and H-β of the double

bond (α,β-unsturated ketone HOPO-1) appear as doublet signals at different chemical

shifts, being those of H-β more affected by 2-substitutions on the BP-4 nucleus than those

of H-α. Thus, H-α appears at 8.84-8.95 ppm which is found more deshielded than H-β due

to anisotropic effects exerced by the BP-4 carbonyl function. H-β appears at 7.50-7.51

ppm for 2-unsubstituted BP-4s (13a, 13d, 13e); the corresponding chemical shift values

increase with respect to the degree of 2-substitution, thus δ = 7.70-7.78 ppm for 2-methyl-

and 2-aryl- substituted BP-4s (13f-l); 8.01-8.09 ppm for 2-styryl- and 2-(4-arylbuta-1,3-

dienyl)- substituted BP-4s (13m-q, 15a-c). These structural data can provide basic

information on the tri-dimensional arrangement of HOPO-1 fragment relative to the BP-4

core structure which means that only two possible ground state conformations exist for

such a conjugated diene, being s-trans and s-cis conformation as shown in Figure 3.8.

The cis or trans designation are based upon the relationship around the central C3—C1’

bond (Fig. 3.8) [64,65]. The s-trans conformation is therefore more thermodynamically

stable because the s-cis conformation has a steric repulsion between the inside

hydrogens (case of 2-unsubstituted BP-4s) or hydrogen-substituent (case of 2-substituted

BP-4s). However, both conformations can exist together at least to some extent. We have

seen that although the s-trans conformation is preponderant, the s-cis conformation is

extremely favored in some reactions as it has already been experienced in our laboratory

on the stereoselective synthesis of s-cis/s-trans 2-styryl -quinolones and –quinolines

which is found to depend on specific structural patterns [66].

OR

O

H

H

O

O

H

OR

OH

H

OO

H

s-trans s-cisSteric hindrance

Anisotropic effects

Substituent effects

31'

Figure 3. 8. s-trans and s-cis conformations of 3-(HOPO-1)-BP-4 conjugated diene


89

HSQC, HMBC correlation experiments along with NOE effects are such important to

solve the complex structures of the new synthesized benzopyran-4-one compounds.

Difficulties are found when treating FLVs, 2-STCs and higher homologues, requiring their

structural elucidation, a deep 2D-NMR interpretation in order to overcome the problem of

overlapped proton signals due to the presence of several polysubstituted

aromatic/phenolic rings.

In the case of the dimeric-compound 13a, the 1H NMR spectrum seems less

complicated (Fig. 3.9). Hα-2’ and Hβ-1’ of the HOPO-1 portion can furthermore be

differentiated through 2J and 3J HMBC correlations. It turns that, Hβ-1’ (7.53 ppm) shows

correlations with five carbons including, C-3 (119.9 ppm), C-2’ (124.4 ppm), C-2 (159.5

ppm) and both carbonyl groups C-4 (176.3 ppm), C-3’ (194.5 ppm); while Hα-2’ (8.84 ppm)

correlates only with C-3, C-1’ (136.1 ppm) and the nearest carbonyl group C-3’. The

HOPO-1 moiety is showing similar characteristic proton signals to those of the BP-4

nucleus, thus, with the aid of HSQC/HMBC correlations, it would be easier to differentiate

between them. For instance, we find that the main HMBC connectivities are established

between H-2 (8.23 ppm)/C-4 (176.3 ppm), H-2 (8.23 ppm)/C-9 (155.3 ppm) and H-5 (8.32

ppm)/C-4 (176.3 ppm), H-5 (8.32 ppm)/C-9 (155.3 ppm) related to the BP-4 ring, while in

HOPO-1 fragment, H-6” (8.05 ppm) correlates with the carbonyl C-3’ (194.5 ppm) and

more important are the correlations between the proton –OH (12.82 ppm) and C-1”(118.2

ppm), C-2” (163.5 ppm) and C-3” (118.3 ppm) (Fig. 3.10, 3.11).

Further structural data can be gained from NOESY experiments concerning the

conformational/stereochemical aspect of these new BP-4-based polyphenolic compounds.

As a result, compound 13a must exist in its C3—C1’ s-trans conformation according to

NOESY spectra (Fig. 3.12, 3.13), which clearly shows that Hβ-1’ is entertaining NOE

effects with H-2 (8.23 ppm), while Hα-2’ seems to be closer to H-6” (8.05 ppm) of the

ortho-hydroxyphenyl group in HOPO-1; this fact agrees with the E-configuration of the

C1’—C2’ double bond in HOPO-1.

The spectral characterization of CHRs 13d-f and FLVs 13g-l is made in a similar

fashion. For instance, the 2-aryl substituents of FLVs are characterized on the basis of

HMBC experiment which mainly shows the connectivities existing between their

characteristic protons and the C-2 168-169 ppm) carbon. FLVs 13g-l are also presenting

the most stable s-trans conformation; in case of compound 13h, Hα-2’ (8.90 ppm) shows

NOE effects with H-6’’’ (8.09 ppm) of the HOPO-1 fragment, while Hβ-1’ (7.77 ppm) is

evidenced to be closer to the 2-(3,4-dimethoxyphenyl) group by presenting, at the same

http://en.wikipedia.org/wiki/Geometric_isomerism

http://en.wikipedia.org/wiki/Geometric_isomerism


90

time, NOE effects with both H-2’ (7.22 ppm) and H-6’ (7.31 ppm), which is obviously due

to the continuous conformational rotation of this group (Fig. 3.13).

Figure 3. 9. 1H NMR spectra of compound 13a (300 MHz)

O

O

O

O

H

H

H

H

O

H

HO

O

OH

2

3

4 5

9

1'

2'3'

2"

3"

6"

1"

2

4

1'

2'3'

1"

R' R'

R

R = aryl

O

O

2

1'

R'H

H2'

6'

1"

Figure 3. 10. Main HMBC correlations in CHRs and FLVs compounds

H-3”

H-5”

H-4”

H-6

H-8

Hβ-1’

Hα-2’

H-7 H-6”

H-5

2”’-OH

H-2

O

O

12

34

56

78

9

101'

2'3'

2"

3"

4"5"

6"1"

O

HO


91

Figure 3. 11. Partial HMBC spectra of compound 13a (300 MHz)

Figure 3. 12. Partial NOESY spectrum of compound 13a (300 MHz)

Hβ-1’

Hα-2’

C-3

C-2’

C-2

C-4

C-3’

C-1’

Hβ-1’

Hα-2’

H-6”

H-2


92

O

O

O

O

H

H

H

2

1'

2'3'

6"

H

HO

H

HO

O

O

H

H3CO

OCH3

OCH3

H

H

H

2

1"2"

3"

6"'

2'

1'6'

13a 13h

Figure 3. 13. Main NOE effects observed in the NOESY spectra of compounds

13a and 13h

Finally after various crystallization attempts, compound 13a was, for the first time,

isolated as single crystals and subjected to X-ray diffraction studies. The data analysis

display the planar 3D structure of 13a (Fig. 3.14) in its s-trans conformation exactly as it

was expected/deduced through 1H NMR analysis and the extensive 2D-NMR studies. The

established structure also shows the hydrogen bridge in the ortho-hydroxyphenyl group

with C-3’ carbonyl group. Figure 3.15 represents the orthorhombic non-centrosymmetric

crystal cell unit (P n a 21) in which compound 13a crystallizes.

Figure 3. 14. Schematic representation of compound 13a molecular structure


93

Figure 3. 15. Crystal cell unit of compound 13a

Considering 13b and 13c as isolated intermediates of the MAD tandem approach,

these compounds have greatly attracted our attention since they have raised exciting 1H

NMR features. The chromanone-form 13b issued in the first step of MAD/decarboxylation

reaction, was found to deliver two diastereomers (2’R,2S and 2’S,2R) and (2’R,2R and

2’S,2S) in 1:1 ratio due to the presence of two asymmetric carbons. This event was

promptly deduced from the duplicated proton signals in the 1H and 13C NMR spectra. For

instance, a doublet signal is composed of two singlets, being each singlet attributable to

one methyl group (acyl) of an enantiomeric pair (2’R,2S and 2’S,2R) or (2’R,2R and

2’S,2S). Moreover, ABXY [for H-2(Y), H-2’(X) and H-3’(AB) protons] and AB (for

CH3CH2—O protons) spin systems need to be solved which are even found duplicated

due to the 1:1 diastereomeric ratio (Fig 3.16).

Compound 13c, which represents the CHRM-ring-opened form of 13b intermediate in

the following step of MAD tandem reaction, is therefore giving the most stable enolic form

(OH at 17.75 ppm) implicated in a high stabilized conjugated double bonds system (Fig

3.17). In this case, the carbonyls’ electron-withdrawing mesomeric effects play a crucial

role in making Hα-2 (7.03 ppm) less deshielded than Hβ-3 (7.88 ppm).


94

Figure 3. 16. ABXY and AB spin patterns in the chromanone 13b

Figure 3. 17. 1H NMR spectra of compound 13c

12 3 4

5

6

6'

5'

4'3'

2' 1'

O

O

O

OH

H

6-CH3/4-COCH3

H-5’

Hα-2

H-3’

H-4’

H-6’

Hβ-3

2’-OH

5-OH

H-3’(AB)

O

O

O

O

O

1

23

4

2'

3'4'10'

5'6'

7'8'

9'

1"

2"

1'

H-2"

H-4

H-2(Y)

H-1"(AB)

H-2’(X)

H-8’

H-6’

H-7’ H-5’


95

In a more complex structure, we state, as an example, compound 13n 3-(HOPO-1)-

substituted-3’,4’-dimethoxy-2-styrylchromone, in which, four vinylic protons are implicated

in high triene conjugated system. Hence, we have noticed similar HMBC correlations of

Hα-2”’ (8.94 ppm) and Hβ-1”’ (8.08 ppm) like in compound 13a for the HOPO-1 portion. In

what concerns the styryl double bond, we find Hα-1’(7.36 ppm) presenting mainly 2J

correlation with C-2 (163.3 ppm) and 3J with C-1” (127.9 ppm) of the 3’,4’-

dimethoxyphenyl group, while Hβ-2’ (7.76 ppm) can further establish J3 correlations with

C-2” (110.1 ppm) and C-6” (122.7 ppm) of the referred aromatic ring besides its

connections with C-2 (163.3 ppm) and C-1’ (113.9 ppm) (Fig. 3.18, 3.19, 3.22). In case of

2-STCs, the designations of α and β are attributed to proton positions of the styryl double

bond relative to the BP-4. Hα-1’ is found less deshielded than Hβ-2’ since this latter is

affected by mesomeric electron-withdrawing effects of the C-4 carbonyl in BP-4 or even

C-3”’ of the HOPO-1 moiety.

Figure 3. 18. 1H NMR spectrum of compound 13n (300 MHz)

3”,4”-OCH3

H-5’

H-5””

H-3””

H-8

H-4””

H-6

Hβ-1”’

H-6””

2””-OH

H-2’

H-6’

Hα-1’

Hα-2””

Hβ-2’

H-7 H-5

O

O

H3CO

OCH3

1

2

3 4

5

6

78

9

10

1'

2'

6"

5"

4" 3"2"

1"

1'"

2'"3'"

2""

3""

4""5""

6""1""

O

HO


96

Figure 3. 19. Partial HMBC spectrum of compound 13n (300 MHz)

In a general manner, the Hα and Hβ of the HOPO-1 portion are always found to exhibit

similar HMBC correlations in all the BP-4-polyphenolic scaffolds. In the case of compound

15a, the major difficulties can be encountered in protons attribution of the 2-(4-arylbuta-

1,3-dienyl) side-chain. These analytical odds are recovered by applying NOESY

experiment with help of the HMBC correlations. Thus, Hα-1’ (7.10-7.20 ppm) shows 2J

correlation with C-2 (163.0 ppm) and C-2’ (140.8 ppm) and 3J with C-3’ (127.3 ppm). In

the other side, Hβ-2’ (7.60-7.70 ppm) establishes 3J with both C-2 and C-4’ (141.1

ppm) and 2J with C-1’ (119.7 ppm); Hγ-3’ (7.05-7.01 ppm) correlates via J3 with the

neighboring carbons C-1’ and C-1’’(136.0 ppm) and finally Hδ-4’ (7.10-7.15 ppm) shows 3J

correlations with both ortho-carbons C-2’’/6’’ (128.9 ppm) of the phenyl group and C-2’

(140.8 ppm) (Fig. 3.20, 3.21, 3.22). Also, in the current case, Hβ-2’ is found directly

influenced by the mesomeric electron-withdrawing effects of the C-4 carbonyl group which

is even spread out to Hδ-4’ in less extent.

Hβ-2’

Hα-1’

C-2”

C-1’

C-1”

C-2

C-6”


97

Figure 3. 20. 1H NMR spectrum of compound 15a (300 MHz)

Figure 3. 21. Partial HMBC spectrum of compound 15c (300 MHz)

Hβ-2’

Hα-1’

C-3’

C-2

C-2’

C-4’

Hγ-3’

Hδ-4’

Hα-1’

H-4””

H-8

H-6

H-6””

2””-OH

H-7 Hα-2””

Hβ-1”’

H-5 Hβ-2’

H-4”

H-3”

H-2”

H-5””

H-3””

O

O

O

HO

1

2

34

56

78

9

10

1'

2'

2""

3""4""

5""

6""1""

1"'

2"'3"'

4"3"

2"

1"

3'

4'


98

The data obtained from the NOESY experiment supported our 1H NMR spectral

analysis study of these complicated polyene systems, like in 2-STCs 13m-p and 2-(4-

arylbuta-1,3-dienyl)chromones 15a-c. All the multiple double bond conjugated systems

are presenting the s-trans conformations relatively to i) the C1”’—C3 simple bond of the

HOPO-1 fragment in all the scaffolds, ii) C2—C1’ in the 2-styryl containing compounds

13m-p, iii) C2—C1’ and C2’—C3’ in the 2-(4-arylbuta-1,3-dienyl) containing compounds

15a-c. In the case of compound 13n, Hβ-1’” (8.08 ppm) shows NOE effects with Hα-1’

(7.36 ppm), while this latter along with Hβ-2’ (7.76 ppm) are confirmed to be close to the

3,4-dimethoxyphenyl group by entertaining NOE effects with both H-2” (7.16 ppm) and

H-6” (7.29 ppm) (continuous rotation of the 3,4-dimethoxyphenyl group). Compound 15c

is the best example to be treated in order to demonstrate that the 2-(4-arylbuta-1,3-dienyl)

portion exists in the s-trans conformation as proved via the NOE effects established

between Hα-1’/Hγ-3’, Hβ-2’/Hδ-4’ and both Hγ-3’, Hδ-4’ with both H-2”, H-6” of the rotating

phenyl group (Fig. 3.23).

2

3

4

1'

2'

1"'

2"'3"'

2"

1"

3'

4'

H

O

O

H3CO

OCH3

HO

HO

H

H

H

O

OH

O

HO

H

HH

H

13n 15a

2

3

4

1'

2'

1"'

2"'3"'

6"

2"

1"

Figure 3. 22. HMBC correlations observed for compounds 13n and 15a

The incorporated HOPO-2 fragment in the CHRMs compound 14q-t displays

interesting 1H NMR patterns, therefore, we have noticed an AMX spin system for the

alkylic protons since HOPO-2 is in the proximity of an asymmetric center. Each signal of

the AMX spin system appears as doublet of doublets (dd), and the coupling constants can

be extracted: H-1’(A), 2.99-3.00 (dd, J = 15.8, 3.7 Hz); H-1’(M), 3.86-3.95 (dd, J = 15.8,

9.2 Hz): H-2(X), 6.12-6.23 (dd, J = 9.2, 3.7 Hz, 1 H, H-2) (Figure 3.24). The aromatic

proton assignment are indicated in Figure 3.25.


99

1'2'

1"'

2"'

2"

3'

4'

1'

2'

1"'

2"'

6""

6"

2"

H

O

OH

O

HO

H

HH

H

H

H3CO

OCH3

H

H

H

O

O

H3CO

OCH3

HO

HO

H

H

H

H

H

6""

6"

13n 15c

Figure 3. 23. Main NOE effects observed in the NOESY spectra of

compounds 13n and 15c

Figure 3. 24. AMX spin system 1H-NMR patterns in asymmetric 2,3-disubstituted

chromanone (14q)

O

O

O

OH

M

A

X


100

The chromanone ketone function C-4 (182.1 ppm) and C-3 (105.0 ppm) carbons have

only been clearly located in case of compound 14q through HMBC correlation (Figure

2.26), in which H-2 (6.23 ppm) and H-5 (7.89 ppm) of the BP-4 ring show obvious

correlations with the BP-4 ketone function C-4. The C-3 (105.0 ppm) chemical shift is

assigned with help of HMBC correlations established with 1’’’-OH (16.28 ppm) and H-2

(6.23 ppm). Further HBMC correlations are prominent in the spectrum and most of them

are illustrated in Figure 3.27. Finally, the greatest challenge is faced when treating the 3D

spectral solution of the CHRMs building blocks 14q-t. Consequently, with the aid of the

NOESY experiment, we succeeded to modelize the 3D scaffold which consists in a planar

molecular structure due to the large sp2 carbons double bond sequence and an sp3 C-2

chiral core supporting perpendicularly the 2-(2-hydroxyphenyl)-2-oxoethyl side-chain

related to the HOPO-2 molecular fragment. For instance, the NOESY spectrum of

compound 14q has demonstrated the fact that 2-(2-hydroxyphenyl)-2-oxoethyl moiety

must be perpendicular to the horizontal plane of the molecule because both protons H-

1’(A,M) are not giving rise to any NOE effects with Hα-2’’’ (6.87 ppm) of the styryl double

bond; in contrary, this later is shows clear NOE effects with H-2 (6.23 ppm), which seems

to be much nearer (co-planar) to the molecule horizontal plane. This observation confirms

the sp3 character of the 2-C asymmetric carbon. Other NOESY effects are illustrated in

Figure 3.27 which finally resulted in modeling the whole 3D structure drawn in a correct

manner.

After the fine elucidation using 2D NMR techniques, X-ray crystallographic studies

unveils the 3D structure of the chromanone 14q and informs about further stereochemical

details. The single crystals of the compound have been harvested from light petroleum /

dichloromethane solution upon slow evaporation at room temperature. It ends up that the

MAD protocol under organobase catalysis does not allow an enantioselective production

of 2,3-disubstituted chromanones 14q-t, since the asymmetric unit of compound 14q

crystallizes in the triclinic P -1 space group containing an inversion center which

generates both R and S absolute configurations in a 1:1 ratio (Fig. 3.28).


101

Figure 3. 25. 1H NMR spectrum of compound 14q (300 MHz)

Figure 3. 26. HMBC spectrum of compound 14q (300 MHz)

H-1’(M) H-1’(A)

C-2

C-2’

C-3

C-4

H-2(X)

C-1”’

C-2”’

1”’-OH

H-1’(A)

12

34

5

6

78

9

10

2"3"

4"

5"

6"

1"

3'''

2'''1'''

2'

1'

1"" 2""

3""

4""O

O

O

HO

OH

H3CO

H-1’(M)

H-8

H-2(X)

H-6

H-5”

Hα-2”’

7-OCH3

H-2(X)

H-3”

H-4”

H-6”

H-3””

H-2””

H-4”” Hβ-3”’

H-5

2"-OH 1’"-OH


102

O

O

O

O

O

H3CO

H

HHH

HHH

2

34

7

2"3"

3'''

2'''1'''

2'

1'

2""

2

6"

3''' 2'''

1'

2""

2""

O

O

OCH3

OH

O

OH

HH

H

H

H

H

H

H

HMBC

NOESY

5

Figure 3. 27. HMBC connectivities and NOE effects observed in the appropriate 2D-NMR

spectra of the chromanone 14q

Figure 3. 28. Crystal cell unit of compound 14q

R

S


103

3.2. HYBRIDS OF BENZOPYRAN-2-ONE AND BENZOPYRAN-4-ONE

3.2.1. Benzopyran-(2 and 4)-ones: The golden biologically active rings

A priceless discussion has been previously brought up on the benzopyran-4-one (BP-

4) based compounds namely, flavonoids and 2-styrylchromones emphasizing their

curriculum of biological activities. Coumarins are benzopyran-2-one (BP-2) based

compounds which have also gained considerable attention as interesting class of oxygen

heterocycles being the sister family of BP-4 compounds, since they form the functional

isomer of chromones. Coumarins constitute an important category of natural products; to

date more than thousand coumarin derivatives have been described and mostly isolated

from above 800 plant species [67]. Coumarin derivatives exhibit useful pharmaceutical

activities, being also recognized as potent antioxidant, anti-inflammatory, anticancer and

molluscicidal active compounds [68]. A particular focus is due to 4-hydroxycoumarin (4-

HCOM) derived-compounds which have long been known as a specific class of vitamin K

antagonist, employed as anticoagulant drug molecules. These scaffolds are derived

from the BP-2 ring by adding a hydroxy group at the 4 position to obtain the 4-

hydroxycoumarin unit, which is coupled to a large aromatic/polyphenolic substituent at the

activated 3-C position (the active methylene group of the 1,3-dicarbonyl form of 4-HCOM)

to form a hybrid scaffold. The 3-substitution is such an important structural feature

required for anticoagulant activity. We cite some interesting examples from the literature

which are still being under development, like warfarin (WRF) [69a], phenprocoumon 102,

bromadiolone 103, coumafuryl 104 [69b] and the biscoumarin dicoumarol 105 [70]. These

target molecular models have mainly been produced via Michael additions (MAD) type of

reactions. In the other side, it is also significant to mention the preliminary screenings of

the triacetic acid lactone TAL (4-hydroxy-6-methypyran-2-one) derived-compounds on

human ovarian carcinoma (A2780) and human chronic myelogenous leukemia (K562) cell

lines, which demonstrated excellent results as potential anticancer agents. Many of other

pyran-2-one based scaffolds are attributed to a broad spectrum of antimicrobial activities

and thus making this hetero-nucleus a privileged basic structure [71] (Fig. 3.29).

In view of the natural occurrence and profitable range of biological activities associated

with coumarin scaffolds, several methods have been developed for their synthesis, being

one of the most widely used, the Pechmann reaction. The Pechmann condensation is

mainly utilized for the construction of benzopyran-2-ones. In particular, the synthesis of

coumarins and their 4-substituted derivatives 108, starts from a phenol precursor 106 and

http://en.wikipedia.org/wiki/Vitamin_K_antagonist

http://en.wikipedia.org/wiki/Vitamin_K_antagonist

http://en.wikipedia.org/wiki/Hydroxyl

http://en.wikipedia.org/wiki/Aromatic

http://en.wikipedia.org/wiki/Phenprocoumon

http://en.wikipedia.org/w/index.php?title=Coumafuryl&redirect=no

http://en.wikipedia.org/wiki/Dicoumarol

http://en.wikipedia.org/wiki/Coumarin

http://en.wikipedia.org/wiki/Phenol


104

a carboxylic acid or ester 107 containing a β-carbonyl group [72]. The condensation is

usually performed under Lewis acidic conditions. The mechanism involves an

esterification/transesterification followed by attachment of the activated carbonyl to the

ortho-position of the aromatic phenol ring generating an oxygen hetero-nucleus. The final

step is a dehydration, as it is known for an aldol condensation process (Scheme 3.18).

O

O O

O

O O

OH

2H-chromen-2-oneBP-2

4H-chromen-4-oneBP-4

TAL

O O

OH

O O

OH

O O

OH

OO

OH

O

O O

OH

4-HCOM

WRF 102

105

O O

OH

O 103

O

O O

OH OH

104

Br

Figure 3. 29. Benzopyran-2- and -4-one and pyran-2-one derivatives

OH OEt

O

O

O O+

OR

O

R

O+

AlCl3-

O O

HHO R

R

106 107108

AlCl3

RR R

Scheme 3. 18

We have seen from previous discussions that the pyranone ring (pyran-2-ones and

pyran-4-ones) is the finger-print of a number of biological active components most

widespread in natural or synthetic, like flavonoids, coumarins, …etc. Up-to-day, these

skeletons are the lead structures and responsible for the valuable anti-cancer, anti-

inflammatory and antioxidant effects. Such structural and biological significances

deserved our attention and therefore, we made further efforts in using both of the BP-2

http://en.wikipedia.org/wiki/Carboxylic_acid

http://en.wikipedia.org/wiki/Ester

http://en.wikipedia.org/wiki/Carbonyl

http://en.wikipedia.org/wiki/Pechmann_condensation#cite_note-0

http://en.wikipedia.org/wiki/Aldol_condensation


105

and BP-4 as the biologically golden rings for building new hybrid compounds in purpose of

getting the sum and/or new biological actions in one cocktail molecule.

In what follows, a short literature emphasis on natural occurrence, chemistry and the

biological profile of some specific target of BP-2 and BP-4 based scaffolds including

bispyran-(2 or 4)-ones, warfarin derivatives and hybrids of benzopyran-(2 and 4)-one. All

these polyphenolic templates are considered as one of our main research subjects and

applications in medicinal area.

3.2.1.1. Biscoumarins and bischromones as biologically active hybrids

Molecular hybridizing is a known natural concept; biscoumarins, for instance, are

naturally occurring hybrids of coumarins (4-hydroxcoumarin are the major examples). Few

reports are describing their synthesis, the most recent has shown both their elaboration

and application as urease inhibitors [73 and references therein]. Concerning the synthetic

aspect, biscoumarin scaffolds 109 are synthesized in high yields by condensing different

alkyl- and aryl- aldehydes with 4-hydroxycoumarin 6 at room temperature in the presence

of catalytic amount of piperidine (Scheme 3.19) [73]. Nature has also provided some

examples of bischromones, such as chrobisiamone A 110 (Fig 3.30), recently isolated

from the leaves of Cassia siamea (Fabaceae) and exhibiting antiplasmodial activity [74].

The elaboration of some important bioactive bischromones and bichromones scaffolds

have been reported [1,48,75]. Broader exploratory researches for biological aims have

resulted in a number of patented pharmaceutical compositions containing bischromones

[76,77].

O O

OH

RCHO

O O

OH

OO

OHR

Cat. Pipiridine

+2

6 109R = alkyl, aryl

Scheme 3. 19

O

O

O

O

OHHO

O O110

Figure 3. 30. Chrobisiamone A 110


106

3.2.1.2. Warfarin: a benzopyran-2-one based drug

Warfarin (WRF, Fig. 3.29) is a commercialized drug, known in the market under

various brands (Coumadin, Jantoven, Marevan, Lawarin, Waran and Warfant). It is

an anticoagulant normally used in the prevention of thrombosis and thromboembolism, the

formation of blood clots in the blood vessels and their migration elsewhere in the body

respectively. It was initially introduced in 1948 as a pesticide against rats and mice and is

still used for this aim. In the early 1950s, WRF was found to be effective and relatively

safe for preventing thrombosis and embolism (abnormal formation and migration of blood

clots) in many disorders. It was approved for use as a drug in 1954 and has remained

popular ever since; warfarin is the most widely prescribed oral anticoagulant drug in North

America [69a]. The sodium salt of WRF is one of the most widely prescribed

anticoagulants. WRF is also prescribed as a racemic mixture, however both enantiomers

exhibit different activity and metabolism [78].

WRF is one of the easiest synthetic target known in the medicinal chemistry field. Its

succinct synthesis starts by the elaboration of the 4-HCOM units which slightly differs from

the Pechmann pathway. The condensation of ortho-hydroxyacetophenone 111 with ethyl

carbonate 112 gives the β-ketoester 113 as the intermediate shown in the enol form. After

the oxygen hetero-cyclization (via transesterification) and formation of the 3-activated 4-

hydroxycoumarin 6, a subsequent Michael addition of the corresponding carbanion form

114 on the methyl styryl ketone 115 gives the desired Michael adduct and thus warfarin

(Scheme 3.20) [79]. Several methods using organocatalysis have been developed for the

preparation of enantiomerically enriched WRF [78 and reference therein].

OOH

EtO OEt

OOO-

EtO OH

OH

O O

OO

OH

O O

O

OO

+

+

EtO-

Base

WRF

111 112 1136

114115

_*

Scheme 3. 20

http://en.wikipedia.org/wiki/Anticoagulant

http://en.wikipedia.org/wiki/Thrombosis

http://en.wikipedia.org/wiki/Thromboembolism

http://en.wikipedia.org/wiki/Pesticide

http://en.wikipedia.org/wiki/Rat

http://en.wikipedia.org/wiki/Mouse

http://en.wikipedia.org/wiki/Prophylaxis

http://en.wikipedia.org/wiki/Thrombosis

http://en.wikipedia.org/wiki/Embolism

http://en.wikipedia.org/wiki/Warfarin#cite_note-Holbrook-0

http://en.wikipedia.org/wiki/Enol

http://en.wikipedia.org/wiki/Coumarin

http://en.wikipedia.org/wiki/Anion

http://en.wikipedia.org/wiki/Michael_reaction

http://en.wikipedia.org/wiki/Warfarin#cite_note-49


107

3.2.1.3. Benzopyran-(2 and 4)-ones hybrids from nature

Hybrids of benzopyran-4-ones and benzopyran-2-ones are very scare in nature, we

have seen in the previous chapter II, the structural complexity of hybrid compounds

expressed in a few plant sources. We re-take one of the fascinating models, the new

flavone-coumarin hybrid presented as a pair of positional isomers 29, 116 along with the

bicoumarin 117 identified in Gnidia socotrana (Thymelaeaceae) [80]. 3,4-Dihydro-

coumarins, which are considered as valuable building blocks, are discovered in a number

of important natural fused hybrids with the flavanone skeleton 118 [60 and reference

therein] (Fig. 3.31).

O

OOH

HO

OH

O

O

OH 29

O

OOH

HO

OH

116

O

O

OH

O OHOO

O

HO

117

O O

O

O

R1

R2

OH

118

Figure 3. 31. Some natural BP-2 and BP-4 hybrids

3.2.2. Design and synthesis of benzopyran-(2 and 4)-ones hybrids

3.2.2.1. Synthesis of bispyran-2-ones hybrids sharing HOPO-2 or BP-4

In this part, we have been oriented towards the biscoumarin and bischromones

scaffolds as promising biological active hybrids presenting a quite symmetrical geometry

and involving the relevant BP-2 and BP-4 rings. The bispyran-2-ones 16a (biscoumarin or

bis-4-hydroxycoumarin) and 17a [bis(4-hydroxy-6-methyl)pyran-2-one], discussed herein,

have first been elaborated by reacting two equivalents of the unit 4-hydroxycoumarin (4-

HCOM) 6 or 4-hydroxy-6-methypyrone (TAL) 7 with chromone-3-carboxylic acid 2 using

catalytic amount of 4-PPy in refluxing chloroform. The behavior of the acid 2 towards 1,3-

dicarbonyl compounds under organo-base catalysis, has already been clarified in previous

titles treating the BV-MAD tandem process (3.1.2.3). In this exclusive case, we have

discovered that the Michael addition tandem pathway was further extended to a second

1,4-conjugate addition (MAD) when using 4-hydroxypyran-2-ones (4-HCOM or TAL) as

cyclic 1,3-dicarbonyl attackers (Scheme 3.21). Mechanistically, the first MAD tandem

process has led to an HOPO-1 based intermediate 119 (considered as 4-HCOM analogue


108

of the CHR 13a) which suffers a subsequent classical Michael addition on the α,β-

unsaturated ketone system giving rise to the bispyran-2-ones 16a and 17a sharing

HOPO-2 group.

4-PPy

O

O

HOOC

- CO2

O

OH

O

O

O

O

O

O-HOOC

O

O

O O

O-

O

O

O

O

OH

O

O

OH

4PPy

O

OH

O

O- OH

OO

HOO

O

O

O OH

OO

HOO

HO

O

O

OH

O

= 4-HCOM, 16a (72%)

= TAL, 17a (23%)

2

6 or 7

119

Scheme 3. 21

On the other side, the new bispyran-4-one (or bischromone) 18 has been produced via

a new synthetic strategy based on the use of 3-CHCA as a unique precursor, thus three

equivalents of which are brought to react with one equivalent of 2-aminoethanol employed

as co-catalyst in the presence of a few amount of 4-PPy in refluxing chloroform. After the

required reaction time, the crude product obtained upon solvent evaporation, has been

recrystallized from ethanol to give bischromone 18 in 48% yield without any further

purification. The bi-functional role of 2-aminoethanol seems to be primordial because no

other type of amine (primary, secondary or tertiary) has afforded the desired product 18.

Moreover the reaction without 4-PPy as catalyst occurs only in very low yield after longer

reaction time (Scheme 3.22). We propose a mechanistic illustration based on the behavior

of 3-CHCA towards alcohols (ROH) and amines (RNH2) already studied in previous

chapters (Scheme 3.9, 3.10). Under organo-base catalysis (4-PPy), 2-aminoethanol

reacts through its two functions –OH and –NH2 with two molecules of 3-CHCA leading to

the predicted intermediate 120, which undergoes a sequence of intermolecular 1,4-

conjugate addition / decarboxylation in the presence of a third 3-CHCA molecule thus,


109

yielding the final bischromone 18 by intramolecular 1,4-conjugate addition and

eliminating/regenerating the 2-aminoethanol co-catalyst.

O

O

COOH

O

O

OOH

O

O

H2NOH +

Cat. 4-PPy

CHCl3Reflux / overnight

2 18 (48%)

3

O

O

OO

O

O

O NH

O

OOH

NHO

O

OO

H

HO

N

OH

OH

O

_ NH2CH2CH2OH

H

HO

O

O

COOH

120

Scheme 3. 22

A deep study of a reaction mechanism is usually fruitful and can lead to various

chemical reaction inspirations. Thus, if we give a small attention to the mechanism of 3-

CHCA behavior towards organic (4-PPy) or inorganic (-OH) base, an intermediate entity is

noticed which is the relevant aldehyde: ω-formyl-2’-hydroxyacetophenone (in equilibrium

with its chromanone tautomer) (Scheme 3.9, 3.11). The action of 4-hydroxycoumarin on

aliphatic and aromatic aldehydes has been reported in the literature delivering similar

biscoumarins scaffolds 109 [73 and references therein] (Scheme 3.19). In consequence,

our curiosity has guided us to attempt the reaction of 4-hydroxypyran-2-one precursors 6

and 7 with different ω-formyl-2’-hydroxyacetophenone derivatives 4a, 4a(5-Cl), 4d, 4e,

utilizing our optimized procedure (catalytic amount of 4-PPy in refluxing chloroform). This

procedure delivers the desired bispyran-2-ones (biscoumarins) 16a, 16a(5-Cl) 16a, 16e

and bis(4-hydroxy-6-methyl)pyran-2-one 17a in moderate to high yields, and most of the

products were promptly isolated in a pure state after solvent removal under vacuum and

direct recrystallisation from ethanol (Scheme 3.23, Table 3.4). Hence, we have

demonstrated the possible condensation of the activated methylene group present in the

4-hydroxypyran-2-one ring (4-HCOM and TAL) on the formyl group of different conjugated


110

1,3-dicarbonyl systems 4a, 4d, 4e, which has not been undertaken yet in the literature. By

this way, we generate the desired bispyran-2-ones biologically active scaffold linked to the

HOPO-2 bioorganic activator using an economic and eco-friendly procedure. The reaction

in question follows a succession of organo-base promoted double condensation of two 4-

HCOM or TAL units through their activated C-3 methylene gourp on the formyl function.

In a following work, we have further enlarged the application of this methodology to

design more complex hybrids by reacting chromone-3-carbaldehyde 5a-e derivatives

(synthesized via the Vilsmeier-Haack reaction, see experimental part) with 4-HCOM or

TAL in view of building a tetrahedral carbon supporting chromone-biscoumarins (CHR-

Bis-4-HCOM) 19a-e or chromone-bis(6-methyl-4-hydroxypyr-2-one) (CHR-Bis-TAL) 20a in

hybrid forms (Scheme 3.24, Table 3.4). Chromone-3-carbaldehyde 5a has recently been

used in the synthesis and biological evaluation of indole, pyrazole, chromone and

pyrimidine hybrids for tumor growth inhibitory activities and development of highly

efficacious cytotoxic agents [81].

O

OH

O

O OH

OO

HOO

HO

O

O

OH

O

= 4-HCOM, 16a, 16a(5-Cl), 16d, 16e

= TAL, 17a

2+

O OHOH

R2R1 Cat. 4-PPy

CHCl3Reflux / overnight

R1R2

6 or 7 4a, 4a(5-Cl), 4d, 4e

a. R1 , R2 = H or 5-Cl

d. R1 = methoxy, R2 = H

e. R1 , R2 = methoxy

Scheme 3. 23

O

OH

O

O

OH

O

= 4-HCOM, 19a-e

= TAL, 20a

6 or 7

a. R1 , R2, R3 = H

b. R1 , R3 = H, R2 = methyl

c. R1 , R3 = H, R2 = chloro

d. R1 , R2= H, R3 = methoxy

e. R1 = H, R2, R3 = methoxy

+Cat. 4-PPy

CHCl3Reflux / 3h

2

5a-e

O

OR3

R1

R2

O

O

O

R3

R1

R2

O

O

HO

HO

O O

Scheme 3. 24


111

Table 3. 4. Structure, chemical data and yields of bispyran-2-ones 16, 17, 19, 20

Product Formula MW (g/mol) Yield (%)

16a

OOH

OO

OHOOH

O

C27H18O8

470.43 83a

16d

OOH

OO

OHOOH

O

H3CO

C28H20O9

500.45 76a

16e

OOH

OO

OHOOH

O

H3COOCH3

C29H22O10

530.48 25a

16a(5-Cl)

OOH

OO

OHOOH

O

Cl

C27H17ClO6

504.87 68a

17a

OOH

OO

OHOOH

O

C21H18O8

398,36 55b

19a O

O

O

O

HO

HO

O O

C28H16O8

480,42 78a


112

19b O

O

O

O

HO

HO

O O

C29H18O8

494.44 69a

19c O

O

Cl

O

O

HO

HO

O O

C28H15ClO8

514.86 75a

19d O

O

OCH3

O

O

HO

HO

O O

C29H10O9

510.44 87b

19e O

O

OCH3H3CO

O

O

HO

HO

O O

C30H20O10

540.47 82b

20a O

O

O

O

HO

HO

O O

C22H16O8

408.35 58b

a Isolated yield after recrystallization.

b Isolated yield after column chromatography purification

The structure elucidation of compounds was made on the basis of extensive 1D and

2D NMR techniques. Regarding biscoumarins 16, the proton 1H NMR spectrum (CDCl3)

shows a pair of 4-hydroxycoumarin nucleus involved in the whole hybrid which was

deduced from proton signals integrations. The corresponding proton signals appearing in

the aromatic region are “superimposed” which seems to indicate a possible symmetrical

geometry of the constructed scaffold, however both exchangeable protons of hydroxyl


113

groups (4’/4”-OH) related to the 4-HCOM rings clearly present different chemical shift

values (11.45 and 12.10 ppm in 16a), which gives a contradictional information showing

that both 4-HCOM rings are not absolutely positioned in a symmetrical manner relative to

the sp3 medium plane of the tetrahedral carbon C-1 (25.3-27.0 ppm). Moreover, the

HOPO-2 alkylic protons exhibit a slight ABX spin system which could be deduced from the

1H NMR spectra of compound 16a (Fig. 3.32), thus H-2(AB) protons are assigned as

doublet of doublet (dd) at 4.03-4.25 ppm, whereas H-1(X) is attributed to a triplet (t is the

sum of two dd signals) at 5-10-5.44 ppm. The ABX patterns are resulted from the slight

asymmetric environment caused by the unsymmetrical relative position of 4-HCOM units.

With help of the NOESY experiment (Fig. 3.34), it is possible to illustrate a 3D

conformation of biscoumarins 16 as depicted in Figure 3.33. The main NOE effects are

observed in the HOPO-2 portion where H-6”’ correlates with the methylene protons H-2,

besides the 4’/4”-OH proton are showing NOE effects with both alkylic protons H-1 and H-

2. It is obviously seen that 2”’-OH proton of HOPO-2 could only establish NOE effects with

one of the two 4’/4”-OH protons proving that both coumarins units are not disposed in a

symmetric manner. Similar observations are made on the structural analogue bis(6-

methyl-4-hydroxypyran-2-one) 17a showing a double singlet (at 2.245 and 2.247 ppm)

signals both attributed to the 6-methy group of each TAL unit also disposed

unsymmetrically in the 3D space

In similar circumstances, the bischromone 18 has rather been structured in a possible

bilateral symmetry confirmed by the absence of the ABX spin patterns related to the

HOPO-2 alkylic protons in the 1H NMR spectra (Fig. 3.35). The referred protons are

assigned as doublet for H-1 (4.88 ppm) and a triplet for H-2 (4.06 ppm). The aromatic

proton signals of the CHR units are perfectly superimposed as observed in the spectrum

profile from chemical shift integration, thus, the symmetric conformation of bischromones

18 can be predicted as shown in the 3D image (Fig. 3.36).

The structures of CHR-Bis-4-HCOM 19a-e and CHR-Bis-TAL 20a hybrid compounds

were easily established from 1H NMR analysis, being the CHR, 4-HCOM and TAL units

clearly differentiated in the spectrum (Fig. 3.37). The main 1H NMR feature is due to the

allylic proton (5.94-6.03 ppm) of the tetrahedral carbon which is coupled with the H-2”

vinylic proton (7.96-8.03 ppm) of the CHR ring (4JCH=C—CH = 1.5-1.8 Hz). Besides, the

exchangeable 4’/4”-OH protons do not appear in case of DMSO-d6 solutions, the mostly

used solvent in analyzing CHR-Bis-4-HCOM 19a-e, but it was possible to locate them in


114

the case of 19d, 19e and CHR-Bis-TAL 20a since the 1H-NMR spectra of these

compounds are recorded in CDCl3.

Figure 3. 32. 1H NMR spectrum of compound 16a

Figure 3. 33. 3D prediction of the asymmetric conformation of the biscoumarins 16

according to NOE effects observed in the corresponding NOESY spectrum

OOH

OO

OHO

OH

O

1

23

3'

2' 1'

4'5'

6'

7'

8'9'

10'

1"'2"'

3"'

4"'5"'

6"'

2"

1"

3"

4"

5"

6"7"

8"9" 10"

H-2 (AB)

H-1 (X)

H-5"’ H-3"'

H-6’/6”,

H-8’/8”

H-4"’ H-7’/7” H-6"’

H-5’/5”

4’/4”-OH 4’/4”-OH

2”’-OH

sp3 medium plan

NOE effects


115

Figure 3. 34. Partial NOESY spectra of compound 16a

Figure 3. 35. 1H NMR spectrum of compound 18

O

O

OOH

O

O

1

23

3'

2'

1'

4' 5'

6'

7'

8'9'

10'

1"'2"'

3"'

4"'

5"'

6"'

2"

1"

3"4"

5"

6"

7"8"

9"10"

H-2

H-1

H-5"’ H-3"'

H-6’/6”,

H-8’/8”

H-4"’

H-7’/7”

H-6"’

H-5’/5” 2”’-OH

H-2’/2”

H-6’”

4’/4”-OH

4’/4”-OH

2’”-OH

2’”-OH

H-1(X)

H-2(AB)

H-6’”

H-1(X)

H-2(AB)

4’/4”-OH exchangeable protons / tautomeric equilibrium


116

Figure 3. 36. 3D prediction of the symmetric conformation of bischromone 18


Interestingly, with help of HMBC experiment (Fig 3.38), it was found that the proton H-

1 (5.10-5.44 ppm) shows important 2J and 3J HMBC correlations with C-3’/3” and all the

carbonyl groups of the whole bispyran-2-one molecules, including C-3 (200.5-202.9 ppm)

of the HOPO-2 fragment, C-2’/2” and C-4’/4 (at 164.1-169-5 ppm) of the 4-HCOM (or TAL)

units in biscoumarin 16 or bis(6-methyl-4-hydroxypyrone) 17 (Fig 3.41). In addition, H-2

sp3 medium plan

O

OO

OHOOH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"

5"

6"

10"

4"

21

4

5

67

8

910

2'

1'9' 8'

7'

6'5'

10'

H-6/6’

H-8/8’

H-6"

H-7/7’

H-8"

H-7"

H-5/5’

H-2"

H-5"

H-1


117

(4.03-4.25 ppm) shows weak HMBC correlations with C-3 and C-3’/3” enabling the

localization of this latter as double carbon signals at 105.3 and 106.0 ppm, each signal is

attributed to a C-3 carbon of a 4-HCOM unit involved in the unsymmetric conformation.

The bischromone 18 provides similar structural patterns as deduced from HMBC

correlations of proton H-1(4.88 ppm) with the surrounding carbons of the CHRs units,

which appears clearly in the HMBC/13C NMR spectrum (Fig 3.39, 3.41).

In relation to the new CHR-Bis-4-HCOM 19 and CHR-Bis-TAL 20, the O=C—C=C—

OH tautomeric system appears clearly and all carbons C-2/2’, C-3/3’ and C-4/4’ are

assigned at 163.4, 103.2 and 164.1 ppm, respectively (Fig 3.40). The main HMBC

correlations are seen between the H-1 allylic proton (5.94-6.03 ppm) and the tetrahedral

environment of C-3” (118.2-123.3 ppm), C-3/3’ (100.5-104.1 ppm) carbons connecting the

two 4-HCOMs (or TALs) and CHR skeletons as depicted in Figure 3.41.

Figure 3. 38. Partial HMBC spectrum of compound 16a (300 MHz)

H-2(AB)

C-2

C-3

C-3’/3”

C-4’/4”

H-1(X)

C-2’/2”


118

Figure 3. 39. Partial HMBC spectrum of compound 18 (300 MHz)

Figure 3. 40. HMBC spectrum of compound 19a (500 MHz)

H-2

C-2

C-3

C-3’

C-4’

H-1

C-2’

H-2”

C-3’/3”

C-4”

C-3”’

C-2/2’

H-1

C-2”

C-4/4’


119

O

O

OH

O H

H

2

3

3'

2'

4'1

OO

O

OO

OHO

OH

O

H

O H

H

23

3'

2'

4'1

18 16a

O

OO

OHO

OH

O

H

O3"'

3'

2'

4'1

H

H

H

H

19a

H

3

4

Figure 3. 41. Main HMBC connectivities found the HMBC spectra of

compounds 16a, 18 and 19a

3.2.2.2. Michael addition of 4-hydroxypyran-2-ones (4-HCOM and TAL) on α,β

unsaturated ketone scaffolds

3.2.2.2.1. Synthesis of warfarin-analogues

The MAD synthetic protocol of warfarin proceeds via the 4-HCOM reaction on

benzylideneacetone as first reported by Link in 1944 [82]. Several asymmetric versions of

this reaction exist using chiral catalysts [78 and reference therein]. In view of tracking new

biological potential of warfarin-analogues, we have employed another time the Michael

addition of 4-HCOM or TAL on various α,β-unsaturated ketone with complex structural

patterns. Therefore, our first attention was focused on the chalcone (CHAL) backbone

http://en.wikipedia.org/wiki/Warfarin

http://en.wikipedia.org/wiki/4-hydroxycoumarin

http://en.wikipedia.org/wiki/Benzylideneacetone

http://en.wikipedia.org/wiki/Michael_reaction#cite_note-16


120

which according to our best knowledge and up-to-date literature no report has succeeded

the synthesis of these Michael adducts.

The reaction idea has been inspired from the mechanistic study of bispyran-2-ones

synthesis, which has been detailed in the previous point (3.2.2.1, Scheme 3.21). Hence,

we have systematically realized that the double condensation of 4-hydroxypyran-2-one

unit (4-HCOM or TAL) on 3-CHCA passes through a reaction intermediate 119 resulted

from the first MAD tandem process of one 4-hydroxypyran-2-one unit creating an α,β-

unsaturated carbonyl system represented by the HOPO-1 group which is linked to the

coumarin nucleus. The second step of the pathway should be very close to a classical

Michael addition reaction of a second 4-hydroxypyran-2-one on the existing α,β-

unsaturated ketone (HOPO-1) giving rise to the desired bispyran-2-ones product linked

via HOPO-2 spacer (Scheme 3.21). By this way, we have applied the same procedure

under organo-base catalysis using 4-PPy (Scheme 3.25), to generate a new series of

WRF-analogues via typical MAD of 4-hydroxycoumarin 6 or triacetic acid lactone 7 on

chalcone derivatives 8 (CHALs 8 have been pre-synthesized via a classical aldol-

condensation, see experimental part). The new efficient method has offered a flexibility in

manipulating this type of organic reaction, various CHAL derivatives 8a-e have been used

for the purpose of producing a structural variety of warfarin-analogues 21a-e and

21b(TAL) obtained in moderate yields (Table 3.5). Some of the warfarin-analogues 21c-e

were designed to share the HOPO-2 moiety as a required structural feature for our

biological objectives.

O

R1

R2

Cat. 4-PPy

CHCl3 / Reflux, 24 hO

OH

O

O

OH

O

= 4-HCOM, 21a-e

= TAL, 21b(TAL)

excess of 6 or 78a-e

+

O

OH

O

O

R1

R2

a. R1 , R2 = H

b. R1 = methoxy, R2 = H

c. R1 = H, R2 = OH

d. R1 = methoxy, R2 =O H

e. R1 = methyl, R2 = OH

Scheme 3. 25

In terms of mechanistic aspects, the reaction follows exactly the typical Michael

addition pathway described in previous texts for the WRF synthesis (Scheme 3.20).

However, one crucial circumstance which deserves to be mentioned, is that the reaction


121

of 4-HCOM on chalcone 8f (resulted from an aldol-condensation of salicylaldehyde and

acetophenone) has led to the desired Michael adduct followed by an intramolecular

cyclisation steps to produce the hemiketal intermediate form and ending to the ketal

derivative 21f after water-elimination (Scheme 3.26). Accordingly, the hemiketal form has

also been reported in warfarin [84].

O

O

O

HO

H

HH

O

O

O

O

OH

O

OH

O

OH

H

HH

O

21f

O

Cat. 4-PPy

CHCl3 / Reflux, 24 hO

OH

O

excess of 68f

+

OH

_H2O

Michael adduct hemiketal

ketal

Scheme 3. 26

Table 3. 5. Michael addition of 4-HCOM 6 and TAL 7 on chalcone derivatives 8a-f

Product Formula MW (g/mol) Yield (%) a,b

21a

O

OH

O

O

C24H18O4 370.39 62

21b

O

OH

O

O

OCH3

C25H20O5 400.42 67

21b(TAL)

O

OH

O

O

OCH3

C22H20O5 364.39 >5


122

21c

O

OH

O

O OH

C24H18O5 386.39 34

21d

O

OH

O

O OH

OCH3

C25H20O6 416.42 54

21e

O

OH

O

O OH

C25H20O5 400.42 65

21f

O

O

O

O

C24H16O4 368.38 41

a The isolated yield was calculated after column chromatography purification.

b The remaining starting materials 6 or

7 and chalcones 8 were determined by TLC.

All the newly synthesized warfarin-analogues 21a-e, 21f and 21b(TAL) were

characterized on the basis of 1D and 2D NMR techniques. Once again, the HOPO-2 (with

or without the ortho-hydroxyphenyl function) side-chain is demonstrating the asymmetric

environment around carbon C-1’ (34.2-34.8 ppm) since the corresponding alkylic protons

appear as an AMX spin systems, being assigned as clear double of doublet (dd) signals to

H-2’(A) (3.64-3.84 ppm), H-2’(M) (4.46-4.52 ppm) and H-1’(X) (4.84-4.95 ppm) (Fig. 3.42

for compound 21e). Also, the HOPO-2 (with the ortho-hydroxyphenyl function) is mostly

characterized by its 2”’-OH (11.64-11.66 ppm) function in the scaffolds 21c-e. In all WRF-

analogues 21a-e, 21g and 21b(TAL), the 4-hydroxy groups appears at 8.75-8.80 ppm

being possible to be involved in a hydrogen bond with the carbonyl group C-3’ (206.7-

206.8 ppm).


123

Figure 3. 42. 1H NMR spectrum of compound 21e and AMX spin features (300 MHz)

Moreover, an important remark is drawn concerning a probable existence of a

tautomeric equilibrium of the new warfarin-analogues as it is proved for warfarin itself. X-

ray crystallographic studies of WRF show that it exists in two possible tautomeric forms,

acyclic and the cyclic hemiketal, which is formed from the 4-hydroxycoumarin and the

ketone of the C-3’ position [83] (Fig. 3.43). In the current study, we were able to detect the

cyclic hemiketal form in our new WRF-analogues by mean of 1H NMR (500 MHz) acquired

from an aged NMR tube solution of 21e sample (and other samples). Interestingly, the

obtained 1H-NMR spectrum displays small signals corresponding to another duplicated

AMX spin systems of the HOPO-2 fragment. The aromatic protons are also found

duplicated with different chemical shift regions. This explains the existence of the

cyclic hemiketal form as a pair of two diastereomers each one involves a racemic mixture

of (RS, SR) and (RR, SS) which is the consequence of having two asymmetric carbons at

C-1’ and C-3’ in the cyclic hemiketal structure comparing to the acyclic form that contains

only one (Fig. 3.44).

H-3"

O

OH

O

O OH

12

3

8

7

456

1"

2' 3' 3"'2"'1'

4"

1"'

H-2’(A) H-2’(M) H-1’(X)

H-3"’

H-5"’

H-2"

H-6

H-8

H-3"’

H-7

H-6"’

H-5

2"’-OH

4-OH

http://en.wikipedia.org/wiki/Tautomer

http://en.wikipedia.org/wiki/Hemiketal

http://en.wikipedia.org/wiki/Warfarin#cite_note-50





124

The existence of many 4-HCOM based anticoagulants (for example phenprocoumon

102) that do not possess C-3’ ketone group to create such a cyclic hemiketal structure,

suggests that the hemiketal must be hydrolyzed to the acyclic 4-hydroxy form as the most

stable in order to have warfarin derivatives in their activated form [84]. It is possible to

evaluate the tautomeric equilibrium ratio by 1H NMR integration, being 10:1 in favor of the

acyclic form in the case of compound 21e.

O O

O

ROH

R'

O O

O

R'

RHO

Figure 3. 43. Acyclic tautomer (left) and cyclic hemiketal tautomer (right)

Figure 3. 44. 1H NMR spectrum of compound 21e (500 MHz)

The bicyclic ketal 21f delivers an ABX spin system of another category as the whole

structure show two asymmetric centers at positions C-1’ and C-3’, thus the two double of

doublet (dd) of H-2’(AB) (2.42 and 2.48 ppm) are much closer in terms of chemical shift

values. In addition H-1’(X) (4.40 ppm) is transformed into a triplet (sum of two dd) as

http://en.wikipedia.org/wiki/Phenprocoumon


http://en.wikipedia.org/wiki/Warfarin#cite_note-pmid17691835-51


125

referred in the 1H NMR spectra (Fig. 3.45). The absence of the 4-hydroxy signal in the

spectrum (normally assigned at 8.70-8.80 ppm for WRF-analogues 21a-e) is also a proof

of being involved in the hemiketal intramolecular cyclization. Besides, exact mass

measurements [HRMS (ESI+): m/z calcd for [C24H16O4 + Na]+ 391.0946; found: 391.0937]

shows that compound 21f has lost a water-molecule (H2O) comparing to the rest of WRF-

analogues 21a-e, in which all their exact mass have been confirmed to be the sum of 4-

HCOM (or TAL) unit and the CHAL partner to form the Michael adduct. The loss of water

molecule in the bicyclic compound 21f is due to the production of the ketal form by

nucleophilic substitution at C-3’ position, thereby creating a second asymmetric center.

Two diastereomers must be generated as a pair of enantiomers (2’R,3’S and 2’S,3’R) and

(2’R,3’R and 2’S,3’S), however, according to the 1H NMR patterns, only one diastereomer

seems to be present.

Figure 3. 45. 1H NMR spectrum of the bicyclic ketal 21f (300 MHz)

The main HMBC correlations in WRF-analogues 21a-e are obvious over the

asymmetric centre in position C-1’, the connectivities established between H-1’ (4.84-4.95

ppm) and the surrounding carbons enable to assign especially the quaternary carbons,

such as the tautomeric system O=C—C=C—OH C-2 (162.0-162.4 ppm), C-4 (158.2-

160.7 ppm), C-3 (107.6-107.8 ppm) and C-1” (118.9-119.0 ppm) of the 1’-aryl substituent,

O

O

O

O

12

3

8

7

456

1"

2'

3'

3"'

2"'

1'

4"

1"'

3"

2"

4"'

5"6"

9

10

H-2’(AB)

H-1’(X)

H-4" H-2" H-6

H-8

H-3" H-7

H-1" H-4"’

H-3"’

H-2’"

H-5


126

all the main HMBC correlations are given in Figure 3.46. The structure of the bicyclic ketal

21f was unambiguously determined by mean of long-rang 2J and 3J HMBC correlations

established between H-1’ (4.40 ppm) and all carbons constructing the bicyclic skeleton

including the ketal carbon C-3’ (100.3 ppm) and the trapped enolic system O=C—C=C—

OH involving C-2 (161.6 ppm), C-3 (106.0 ppm) and C-4 (158.2 ppm); also H-1’ shows

connectivities with the quaternary carbon of the chromane substituent C-5” (151.3 ppm)

and C-6” (125.1 ppm) both involved in the bicyclic ketal form. Figure 3.46 indicates the

relevant HMBC connectivities which helped to elucidate the complex bicyclic structure of

21f.

O

O

O

O

H

H

H

H

H

2

3

45

1"

2'

3'

2"'

1'

5"6"

O

O

O

O O

2

3

9

45

1"

2'3' 3"'

2"'1'

1"'

H H

HH

H

H

H2"

6"'

21e 21f

Figure 3. 46. Main HMBC correlations of compounds 21e and 21f

Appreciable information are revealed from X-ray data collection of the first isolated

single crystals of the bicyclic ketal 21f, being its 3D spatial arrangement finely established.

Indeed, the first X-ray data refinement has led to the identification of a unique pair of

enantiomers (2’R,3’R and 2’S,3’S), thus a unique diastereomer is present as it was

suggested by the 1H-NMR patterns. The 2’R,3’R and 2’S,3’S are the typical absolute

configurations of asymmetric bicyclo[3.3.1]nonane-like compounds which are reported to

be usually presented in their most stable double-chair conformer [85], this geometric

feature is again observed in our bicylic ketal 21f (Fig. 3.47). Compound 21f crystallizes in

the orthorrhombic non-centrosymmetric system (space group P n a 21) containing mirror

plans which are the symmetry elements giving the origin of a racemic mixture (Fig. 3.48).

The NOESY experiment is such a useful technique in order to validate various structural

data obtained from crystallographic studies. For example, in the case of compound 21f,

we were able to determine some relevant NOE effects mainly established between

H-2’(AB) (2.42 and 2.48 ppm) and both H-2”’/6”’ (7.72-7.80 ppm) of the phenyl ortho-


127

positions. Further NOE effects are observed between H-1’(X) (4.40 ppm) and H-1” (7.53-

7.57 ppm), all these 2D-NMR date are consistent with the X-ray refined 3D structure of

compound 21f (Fig. 3.47).

Figure 3. 47. Schematic representation of compound 21f molecular structure and main

NOE effects observed in the corresponding NOESY spectrum

Figure 3. 48. Crystal cell unit of compound 21f

NOE effects


128

3.2.2.2.2. Synthesis of benzopyran-(2 and 4)-ones hybrids via Michael addition

In an ongoing work, the application of the 1,4-conjugate addition has been extended to

a more complex α,β-unsaturated carbonyl scaffolds, being the previously synthesized

benzopyran-4-one-based polyphenolics namely, CHRs 13a,d,f, FLVs 13g-i, and 2-STCs

13n,o which are harboring the typical Michael acceptor or chalcone-fragment HOPO-1 (2-

hydroxyphenyl)-3-oxoprop-1-enyl) at position 3. An increasing structural complexity has

been issued by hybridizing these scaffolds via the MAD with 2-hydroxypyran-2-one

nucleus (4-HCOM or TAL) resulting in fascinating hybrid compounds where it was

possible to marry the benzopyran-2-one (BP-2) with the benzopyran-4-one (BP-4) rings in

a single hybrid molecule 22. Up-to-day no investigation has reached such a structural

complexity, also the BP-2 and BP-4 hybrids have only been exemplified as natural

compounds with a very narrow distribution in the plant kingdom. The synthetic

methodology follows the procedure described in precedent chapters (Scheme 3.27).

Acceptable yields have been obtained when treating CHRs 13a, 13d, 13f and 2-STCs

13n, 13o with 4-HCOM 6 or TAL 7, resulting in 4-hydroxycoumarin(or triacetic acid

lactone)-chromone hybrids 4-HCOM-CHRs 22a, 22d, 22f, TAL-CHR 22f(TAL) and 4-

HCOM-2-STCs 22n, 22o. The TAL-2-STC hybrid 22o(TAL) has been isolated in low

yields (>5%) along with the major β-diketone as a by-product of the retro-Michael addition.

The flavones 13g-i did not show any reactivity and sometimes the reaction ends to the

unsubstituted flavone by-product issued from the parasite retro-Michael addition after long

reaction time (Table 3.6).

OO

O

O

HO

O

O

R

O

R1

HO

Cat. 4-PPy

CHCl3 / Reflux, timeO

OH

O

O

OH

O

22excess of 6 or 7

+

13

R'

R'

= 4-HCOM

= TAL

O

HO

Scheme 3. 27


129

Table 3. 6. Michael addition of 4-HCOM and TAL on BP-4-based polyphenolics 13

Substrate Product Time (h) Yield (%)a,b

13a O

O

OOH

22a

O

O

O

OH

O

OHO

24 39

13d O

O

OOH

OCH3

22d

O

O

O

OH

O

OHO

H3CO

24 43

13f O

O

OOH

22f

O

O

O

OH

O

OHO

24 51c

22f

TAL

O

O

O

OH

O

OHO

24 44

13g O

O

OOH

22g

No product

24

-c

13h O

O

OOH

H3CO

OCH3

OCH3

22h

No product

72

-c

13i

O

O

OOH

H3CO

OCH3

22i

No product

> 72 -c


130

13n

O

O

OOH

H3CO

H3CO

22n

O

O

OO

HO OOH

CH3O

OCH3

72 44

13o

O

O

OOH

H3CO

22o

O

O

OO

HO OOH

CH3O

72 49

22o

TAL

O

O

OO

HO OOH

CH3O

24 >5c

a The isolated yields are calculated after chromatographic purification.

b The remaining starting materials 6 or 7 and 13 were

determined by TLC. c The formation of uncharacterized or undesired by-products

1H NMR of compound 22a (Fig. 3.49) demonstrates the ABX spin system due to the

alkylic H-1’(X) (5.18 ppm), H-2’(A) (3.97 ppm) and H-2’(B) (4.31 ppm) of the HOPO-2

side-chain linking both of 4-HCOM and CHR units. The proton assignment of these units

are made through the 2D HMBC spectra analysis (Fig. 3.50). Mainly, H-1’ is establishing

3J long-range connections with all carbonyl groups of the hybrid skeleton, including

C-3’ (203.2 ppm) which belongs to the HOPO-2, C-4”’ (180.3 ppm) of the chromone and

the tautomeric system (O=C—C=C—OH) C-4 and C-2 (163.1 ppm) in the 4-

hydroxycoumarin unit. Further HMBC correlations of H-1’ enables the exact assignment of

C-3 (105.0 ppm) and C-3“’ (125.8-125.9 ppm) which both are joining the 4-HCOM and

CHR via the asymmetric carbon C-1’ (25.0 ppm), respectively (Fig. 3.51).

It is also important to indicate that these type of 4-HCOM-CHR hybrid scaffolds are

structurally similar to the previously reported WRF-analogues. Therefore, the hemiketal


131

form was also detected by 1H-NMR in case of compound 22f, the tautomeric equilibrium

ratio calculated by 1H NMR integration was 10:4 in favor of the acyclic form.

In 4-hydroxycoumarin-2-styrylchromone hybrid 22o, we find an increased complexity

in both structural and analytical aspects. From 1H NMR and HMBC spectra of compound

22o (Fig 3.52, 3.53, 3.54), we can characterize the styryl substituent in which the Hα-1”’

(8.50 ppm) and Hβ-2”’ (7.79 ppm) are exhibiting different chemical shifts values comparing

to those of the starting material 13o, Hα-1”’ (7.39 ppm), Hβ-2”’(7.79 ppm). Hα-1”’ is found

more deshielded than Hβ-2”’ probably because of the anisotropic effects of the 4-HCOM

carbonyls (enols) C-2 (164.9 ppm) and C-4 (164.1 ppm) which are somehow oriented

towards Hα-1”’ as it is imposed by the sp3 geometry of the central C-1’ (28.5 ppm) carbon.

A contrary case is deduced in the starting compound 13o and analogues, in which the Hα-

1”’ is found less deshielded than Hβ-2”’ due to mesomeric electron-withdrawing effects of

the HOPO-1 and/or BP-4 carbonyl groups (Fig 3.56).


O

O

O

OH

O

OHO

12

3

4 5

6

78

1'2'3'

1"

2"3"

4"

5"

6"7"

8"

9

10

9"

10"

1"'2"'

3"'

4"'

5"'6"'

H-2’(A)

H-2’(B)

H-3"’

H-5"’

H-1’(X)

H-6

H-8

H-4"’

H-7 H-6"

H-8"

H-7" H-2" H-5" H-5

H-6"’

2""-OH


132


O

O

O

OH

O

O

O

2

3

45

8

1'

2'3'

2"'3"'

4"'

5"'8"'

9

10

9"'10"'

1""

2""

3""

6""

HH

H

H

H

H

H

H

H

Figure 3. 51. Main HMBC correlations in compound 22a

H-2’

C-3

C-3’

C-4”

H-2” H-5” H-5

H-1

C-2/C-4

H-1’

C-3”

C-9”

C-9


133

Figure 3. 52. 1H NMR spectra compound 22o (300 MHz)


Hβ-2’"

H-2’(A) H-2’(B)

H-3b

H-5b

H-1’(X)

H-6

H-8

H-6"

H-4b

H-7"

Hα-1’"

H-5"’ H-5 H-6b

2b-OH

O

O

O

OH

O

OHO

12

3

4 56

78

1'2'

3'

1"

2"' 3"4"

5"

6"7"

8"

9

10

9"10"

1"'2"'

6b

H3CO

1b

2b3b

4b5b

1a

2a3a

4a

H-3a H-2

a

H-8"

H-7

4-OH

4a-OCH3

Hβ-2’"

C-2”

C-2”’

Hα-1’"

C-2a /6

a

C-1a

C-1””

C-3”


134

O

O

OH

O

OHO

2

3

41'

2'

3'

2"3"

4"

H

H

H3CO

O

O

O

OH

O

OO

2

4 5

8

3'

2" 4"

5"

8"

9

10

9"

10"

1b

2b3b

6b

H H

H

H

H

H

H3CO

H

H

2a

1a

2"'1'"

O

3"

Figure 3. 54. Main HMBC correlations in compound 22o

On the basis of the NOESY experiment (Fig 3.55) performed on compound 22o, we

could tentatively illustrate/predict the 3D spatial arrangement of 4-HCOM-2-STCs

scaffolds (Fig 3.56). In fact, no exceptional NOE effects are observed between the

benzenic protons of the 2-STC, 4-HCOM or HOPO-2 constructive fragments, which all

seem to be arranged far from each other in order to minimize the aromatic π-π

electrostatic interactions. However, informative data could be revealed from the NOESY

spectrum concerning the sp3 environment around the alkylic protons H-1’(X) and H-

2’(AB). Therefore, H-1’(X) (5.78 ppm) of the asymmetric center shows NOE effects with

Hα-1”’ of the styryl portion, this fact can initially inform about the proximity of Hα-1”’ to the

carbonyl group C-3” of the HOPO-2 portion, thus affected by its anisotropic effects. On the

other side, both H-6b (7.86 ppm) and 4-OH (14.05 ppm) show NOE effects with H-2’(AB)

(3.97 and 4.42 ppm) protons of the methylene group and since there are only weak NOE

effects established between H-1’(X) and H-2’(AB), the relative “anti-conformation” type of

the hydrogen bonds is appropriately predicted (torsion angle between ± 90° and 180°).

Further weak NOE effects are observed between 4-OH and both H-5 (8.00 ppm) and H-5”

(8.23 ppm) which demonstrate that the 4-HCOM and BP-4 rings are close to each other.


135

Figure 3. 55. Partial NOESY spectrum of compound 22o

Figure 3. 56. 3D geometry prediction of the 4-HCOM-2-STC 22o scaffold and possible

structural interactions

NOE effects

Anisotropic effects

Weak NOE effects

H-2’(B)

H-6b

H-6b

Hα-1’"

H-2’(A)

H-2’(B)

H-1’(X)

H-2’(A)

H-1’(X)

H-5 H-5"

Hα-1’"

2b-OH

4-OH


136

A deep observation of the predicted 3D geometry of 4-HCOM-2-STC hybrid (Fig 3.56)

can explain why the 4-HCOM-flavones hybrid analogues cannot be generated through the

Michael addition approach. It is strongly suggested that this fact is due to the occurrence

of a certain aromatic π-π structural interactions. In such a 4-HCOM-flavone structure, the

facial orientation (or face-to-face) of HOPO-2 (2-hydroxyphenyl group) and 2-aryl (FLV)

aromatic rings decreases the resulting structure stability which is due to the electrostatic

repulsion between these two negatively charged π-systems. The estimated distance

between the aromatic π−π faces is reported to be about 3.3−3.8 Å [86]. Nevertheless, in

the case of 4-HCOM-2-STC structure, the 2-styryl group is even bulky, but seems to be

more flexible since its β-aryl group is found much farther to avoid the facial π-π

interactions. Moreover, 2-methyl- or unsubstituted- chromones 13a, 13d and 13f are

susceptible to hybridize with 4-HCOM (or TAL) units because they present less steric

hindrance at position 2.


137

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CHAPTER 4

- BIOLOGICAL SCREENINGS -

Chapter 4 – Biological Screenings

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4.1. CHEMISTRY, BIOLOGY AND MEDICINE CONTINUUM

Medicinal chemistry is the discipline which gathers the complementary sciences

Chemistry and Biology which respects to a certain continuum (Fig 4.1). Our newly

synthesized polyphenolic hybrid compounds are mainly constructed by gathering

biomimetic organic scaffolds in one molecule. Therefore, we have accomplished our first

step of the project plan or of the Chemistry-Biology-Medicine continuum, which concerns

the chemical aspect of the new elaborated organic hybrids and their full spectral

elucidation. The following step in the continuum is the study of their interactions with

biological systems. Several of our new organic combinations of the biologically active

heterocycles BF, HD, U, BP-2 and BP-4 have been screened in various bio-systems

targeting one of the high morbidity/mortality diseases: “Cancer”. The hybrid approach can

be used to optimize certain biological properties like affinity and selectivity, which have

been studied in some specific pathways implicated in the pathogenesis of cancer

diseases, such as oxidation and oxidative stress, inflammation pathways, cytotoxicity, cell

viability and proliferation, enzyme/protein inhibition, cancer chemoprevention, redox

regulation. Besides, our main objective is tracking for a lead structure exhibiting this

multiplicity of biological actions.

Figure 4. 1. Chemistry-Biology-Medicine continuum

Cancer development and its therapy are in the top title of many researches. One major

pathway in inflammatory processes is the NF-κB pathway which is controlling the

expression of many genes involved in inflammation and cancerogenesis. The NF-κB

pathway can be activated in different manners and several steps have to be passed. Most

of the newly obtained hybrids have been evaluated for their effect on cytotoxicity and

proliferation of leukemic cell lines as well as their involvement in NF-κB regulation. HDAC

inhibition of the target benzofuran-3-one-hydantoin hybrids was undertaken and found to

depend on the isomerism factor. A range of the synthesized BP-4 based polyphenolic

compounds were examined by measuring their cancer chemopreventive activity mediated

by induction of cytoprotective Nrf2 (nuclear factor E2-related protein 2) signaling and their

ability to inhibit proliferation of breast cancer cells. The results of this study are further


148

supporting that compounds carrying Michael acceptor sites are usually considered as best

inducer of the Nrf2 activation, which is already brought up in the medicinal chemistry

literature. Cdc25 phosphatases inhibition was specifically tested on the benzofuran-3-one-

hydantoin candidates, being considered as structural-biomimics of Indirubin 31, the active

constituent of a Chinese antileukaemia medicine and the effective inhibitor of cyclin-

dependent kinases (CDKs). Finally, we cover the results with some antioxidant activity

evaluations using DPPH free radical scavenging test and FRAP ferric ion reducing

antioxidant power of some underlined structures.

In what follows a brief introduction/description of the biological systems utilized in the

screening part of this work, which have been performed on the our newly synthesized

polyphenolic hybrids.

4.1.1. The transcription nuclear factor NF-κB

Cancer is characterized by the uncontrolled growth and spread of cells. Smoking,

obesity, diet and physical inactivity are seen as main risk factors predispose to cancer

along with others resulting from a genetic level, for example, the activation of oncogenes

and the deactivation of tumor suppressor genes which lead to disabled regulatory circuits

[1-3]. Mantovani et al. [4] have well demonstrated that an inflammatory environment is

causative for cancer development. A central molecular mechanism involved in

inflammatory and innate immune response is the transcription nuclear factor NF-κB, the

family of which comprises five members including, relA (p65), relB, cRel, NFκB1 (p50)

and NFκB2 (p52) proteins mainly derived form homodimers and heterodimers. There are

four pathways leading to the activation of NF-κB, being the classical or canonical NF-κB-

activation pathway, the alternative or non-canonical NF-κB signaling pathway, the atypical

pathway and the UV-induced pathway [5-10]. The most common is the classical or

canonical NF-κB pathway activated by Toll-like-, TNF- or T-cell receptors [6,7,11]. RelA-

p50 dimers are associated with an inhibitory protein “Inhibitor of κB” (IκB) which remains

in an inactive form in the cytoplasm. After stimulation of cell surface receptors with pro-

inflammatory cytokines, IκB kinase (IKK) is activated and phosphorylates IκB. As a

consequence IκB is ubiquitinated and subsequently degraded by the proteasome. The

released NF-κB transcription factor then translocates to the nucleus where it activates the

transcription of many target genes involved in cancer initiation, promotion and

progression.


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4.1.2. Leukemia cancer

Actual challenges are faced with leukemia cancer (leukós: white and haïma: blood)

which is the malignancy of the blood-building system – the myeloid and lymphoid system.

It is an heterogeneous group of diseases divided into four main subtypes according to the

speed of evolution and the cell types involved: acute lymphoid leukemia (ALL), the acute

myeloid leukemia (AML), the chronic myeloid leukemia (CML) and the chronic lymphocytic

leukemia (CLL) [3]. Leukemic cells often show chromosomal abnormalities, like the

Philadelphia chromosome for chronic myelogenous leukemia (CML) [3,12], being caused

by chromosomal translocation. A part of the long arm (q) of chromosome 22 and of the

long arm (q) of chromosome 9 translocated mutually, this translocation leads to the

formation of a fusion gene on chromosome 22 named BCR-ABL. The activity of the

resulting “fusion kinase” leads to deregulated proliferation, inhibition of apoptosis and

arrest of differentiation [12].

Natural products have long being used as drugs in cancer therapy. The large majority

(79.8 %) of newly developed anticancer drugs between 1981 and 2010 are from a natural

origin [13]. The flavonoid family is a very prominent group of natural compounds showing

anti-proliferative effects; quercetin 68, for example, is able to inhibit the growth of several

tumor cell lines [14]; the chalcones 8 subclass of compounds have largely been reported

to be cytotoxic inducing cell death via apoptosis [15]. In parallel, coumarins (BP-2) show

anticancer effect and are cytotoxic compounds inducing apoptosis by cytochrome c

release and caspase activation [16]. Several other interesting bio-organic compounds

including polysulfides 121 from garlic [17], curcumin 122 from the rhizome of Curcuma

longa [18,19] and heteronemin 123 from the marine organism Hyrtios erecta [20], have

also shown anti-tumor, anti-inflammatory and anti-tumorigenic abilities based on the anti-

proliferative, pro-apoptotic character and/or cytotoxicity, leading to cell death by apoptosis

or autophagy depending on the cellular context (Fig. 4.2).

SS

S

SS

SS

OOH

OH

OCH3

HO

OCH3

OH

OAc

AcO

121

123

122

Figure 4. 2 Selective cytotoxic natural compounds to Leukemia cancer cells


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4.1.3. Histone deacetylases (HDAC)

Lysine acetylation is deeply implicated in the control of highly regulated biological

functions [21]. Thus, alteration of the acetylation status is involved in the development of

many diseases, including tumorigenesis [22,23]. The acetylation/deacetylation reactions

are catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs),

respectively. Since HDAC expression or activities are often deregulated in cancer, the

control of the acetylation status by modulating HDAC activities is a promising approach to

anticancer therapy. Numerous compounds from natural sources or synthetic derivatives

were identified as HDAC inhibitors and some of them are already in clinical trials for

anticancer therapy [21,24,25]. However, the search for new molecules that are more

potent with fewer side effects is still important within the field of epigenetic drug discovery.

In recent years, there has been an effort to develop HDAC inhibitors for cancer therapy

(Fig. 4.3) and vorinostat (SAHA) 126 has recently been approved for treatment of

cutaneous T cell lymphoma (CTCL). The exact mechanisms by which the compounds

may work are unclear, but epigenetic pathways are proposed [26] . In addition, a clinical

trial is studying valproic acid 127 effects on the latent pools of HIV in infected persons

[27]. HDAC inhibitors are currently being investigated as chemosensitizers for cytotoxic

chemotherapy or radiation therapy, or in association with DNA methylation inhibitors

based on in vitro synergy [28a]. HDAC inhibitors have also been shown to alter the activity

of many transcription factors, including NFκB [28b-c]. Research has shown that histone

deacetylase inhibitors may modulate the latency of some viruses, resulting in reactivation

[28d]. This has been shown to occur, for instance, with a latent human herpes virus-

6 infection.

HN

O

NH

O

OHHO

O

126 127

Figure 4. 3. HDAC inhibitors

4.1.4. Nuclear factor E2-related protein 2 - Nrf2: Keap1-Nrf2 activation pathway

The Keap1-Nrf2 pathway is triggered in cellular stress condition and play a crucial role

in recovery of cellular homeostasis. Dissociation of nuclear-E2–related factor 2 (Nrf2) from

its cytosolic partner - Keap1 (Kelch-like ECH-associated protein 1), is a key step in

activating Nrf2. The free Nrf2 translocates to the nucleus, heteromerizes with small Maf

(small musculoaponeurotic fibrosarcoma) proteins and binds to a cis-acting element

http://en.wikipedia.org/wiki/Vorinostat

http://en.wikipedia.org/wiki/Epigenetic

http://en.wikipedia.org/wiki/Histone_deacetylase#cite_note-21

http://en.wikipedia.org/wiki/Histone_deacetylase#cite_note-pmid19345475-22

http://en.wikipedia.org/wiki/Histone_deacetylase#cite_note-pmid19345475-22

http://en.wikipedia.org/wiki/Histone_deacetylase#cite_note-25

http://en.wikipedia.org/wiki/Human_herpesvirus-6

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known as antioxidant responsive element (ARE) or also as electrophile responsive

element (EpRE) within the regulatory regions of many genes. Studies using Nrf2-deficient

mice and microarray-based assays suggest that Nrf2 modulates transcription of almost

200 genes whose protein products function as antioxidants, phase II detoxification

enzymes, proteosomes, heat-shock proteins, and glutathione-synthesis enzymes. All

these proteins play a critical role in cellular defense against oxidative stress. This

elaborate network of highly inducible proteins protects aerobic cells against the cumulative

damaging effects of reactive oxygen intermediates and toxic electrophiles, which are the

major causes of neoplastic and chronic degenerative diseases. Genetic deletion of

transcription factor Nrf2 renders cells and animals much more sensitive to the damaging

effects of electrophiles, oxidants and inflammatory agents in comparison with their wild-

type counterparts [29].

The Keap1–Nrf2 pathway can be activated by various cellular stresses (e.g. oxidative

stress, shear stress, endoplasmic reticulum stress) and structurally diverse small

molecules (inducers) of endogenous (e.g., 15-deoxy-D12,14-prostaglandin J2, nitro oleic

acid, nitric oxide, hydrogen peroxide, hydrogen sulphide) as well as exogenous origin.

Such inducers chemically react with critical cysteine residues of the sensor protein Keap1,

leading to stabilisation and nuclear translocation of transcription factor Nrf2, and ultimately

to coordinate enhanced expression of genes coding for cytoprotective proteins allowing

survival and adaptation under various conditions of stress [29].

The development and use of a quantitative bioassay which evaluates the ability of

small molecules to induce the prototypic Nrf2 target NQO1 in Hepa1c1c7 murine

hepatoma cells led to the classification of inducers into ten distinct chemical classes:

oxidisable diphenols, phenylenediamines and quinones, Michael reaction acceptors

(olefins or acetylenes conjugated with electron-withdrawing groups), isothiocyanates and

sulfoxythiocarbamates, thiocarbamates, dithiolethiones, conjugated polyenes,

hydroperoxides, trivalent arsenicals, heavy metals and vicinal dimercaptans. Remarkably,

more than 20 years following its first application, at time long before Keap1 or Nrf2 had

been discovered, this assay remains a major screening tool for potential inducers and

arguably allows the most reliable comparisons of inducer potencies. Specific examples of

molecules that belong to three of the most prominent classes of inducers are given below

(Fig. 4.4). Many compounds have been discovered to induce cytoprotective enzymes

especially those bearing Michael acceptor group(s). The inducer potency correlation with


152

the reactivity in the Michael reaction was a critical milestone in the understanding of the

mechanism of action of inducers. The Michael acceptor functionality is essential for the

inducer activity of many natural products such as cinnamates 128, curcuminoids,

chalcones (xantohumol, 2,2”-dihyroxychalcone 129) (Fig. 4.4). Many inducers that belong

to different chemical classes have been shown to protect against chronic degenerative

diseases in various animal models of carcinogenesis, cardiovascular disease and

neurodegeneration. The double-Michael acceptor curcumin 122 inhibits tumour

development in animal models of oral, gastric, intestinal, colonic, hepatic and cutaneous

carcinogenesis. Sulforaphane 130 is an organosulfur compound that exhibits anti-cancer,

and antimicrobial properties in experimental models. It is obtained from cruciferous

vegetables such as broccoli, Brussels sprouts or cabbages [30].

OOH OH

H3CO

O

OCH3

OHS

NC

OS

128 129 130

Figure 4. 4. Chemical inducers of Keap1–Nrf2 pathway

4.1.5. Cdc25 phosphatases

The cell cycle represents the different stages in the life of a cell (birth, growth, division,

death), which are managed by different mechanisms of control and regulation. These

mechanisms involve different enzymes, whose interest in oncology has already been

shown in the literature. The control of each cell cycle phase requires the particular protein

family of Cyclin-dependent-kinases (CDKs), which participate in the transition between the

different stages of the cell cycle. CDKs are active when they are associated to a cyclin,

their regulating sub-unit. CDKs can be regulated by different manners: regulation by the

short transitory assembly with cyclins, post-transitional modifications like

phosphorylation/dephosphorylation, or transitory association with some protein inhibitors.

The most important regulation results from the phosphorylation/dephosphorylation

reactions which induce the activation or the inactivation of CDKs. For example, in its

inactivate form, CDK1 (one of the different CDK isoforms) is phosphorylated on specific

residues Thr14 and Tyr15. The dephosphorylation of these specific amino acids by a

phosphatase family called Cdc25(cell division cycle), and the phosphorylation of another

residue (Thr161) are needed for CDK1 activation. Cdc25s are double specificity

http://en.wikipedia.org/wiki/Organosulfur_compound

http://en.wikipedia.org/wiki/Cancer

http://en.wikipedia.org/wiki/Antimicrobial

http://en.wikipedia.org/wiki/Cruciferous_vegetables

http://en.wikipedia.org/wiki/Cruciferous_vegetables

http://en.wikipedia.org/wiki/Broccoli

http://en.wikipedia.org/wiki/Brussels_sprout

http://en.wikipedia.org/wiki/Cabbage


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phosphatases meaning that they will dephosphorylate two particular residues (Tyr/Thr) of

the CDKs. Three Cdc25 isoforms exist: A, B and C [31, 32].

Some chemical compounds have already been synthesized in order to inhibit Cdc25s

and about a hundred of molecules have been reported to interact with Cdc25 isoforms

such as quinonoid compounds, phosphate surrogates or electrophilic inhibitors. These

compounds are essentially synthetic ones, and only three compounds (BN82002,

BN82685 and IRC-083864) have been reported to inhibit the growth of human tumor

xenografts in nude mice. Among them, the most potent compound is BN82002 (Fig. 4.5)

[32] which is currently commercialized for research use.

N

OH

H3CON

NO2BN82002

Figure 4. 5. BN82002 a Cdc25 inhibitor

4.1.6. Antioxidant and radical scavenging capabilities

Nowadays, science becomes more immerged into daily life; who did not hear or at

least observe the word “antioxidant” which is frequently advertised on many sorts of

beverages and dairy products of the supermarket. Although most people ignore the real

significance of an antioxidant, many of them do use the “antioxidant” term while

discussing about nutrition, food and health; so far, the question must be posed “what is an

antioxidant ?”

In a general aspect, an antioxidant is a substance capable to prevent the oxidation

reaction mainly caused by oxygen. A concrete example is commonly observed in the well-

known phenomena of metals corrosion; iron is spontaneously oxidized by the oxygen of

air, while anticorrosive agents, which in fact are antioxidant substances, are usually used

to protect the metallic surfaces from corrosive damage. From a chemical point of view, the

oxidation reaction is a redox reaction involving electron transfer of a substance towards

the oxidant agent. This reaction can produce free radicals which are highly reactive

species that attack molecules by capturing electrons, thus modifying their chemical

structures. In other words, any substance that reduces the oxidative damage due to

oxygen (and/or free radicals) is called an “antioxidant”. It is therefore capable to stop the

destructive oxidation process by reacting with free radicals and inhaling their activity [33].

http://www.medterms.com/script/main/art.asp?articlekey=10690


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These antioxidant properties are encountered in specific families of natural and synthetic

organic compounds, the most relevant are phenolics.

In biology, despite of the crucial role of the oxidation reactions for metabolism and

organism functioning, they can also be highly destructive. A biological paradox is

eventually noticed for most of beings (animals and plants) requiring oxygen to insure their

life, while this molecule is extremely reactive and able to produce degradation for many

organisms. Under such circumstances, antioxidation systems are setup in a form of

antioxidant agents acting together with enzymes to prevent the formation of highly

reactive species or even to eliminate them just before they damage cell-components, like

DNA, lipids and proteins. Plants and animals utilize and produce various antioxidants to

protect themselves, such as glutathione, vitamin C and vitamin E, or enzymes like

catalase, peroxidase and superoxide dismutase. Similarly, our body itself produces, from

the amino acid cysteine, a powerful antioxidant, α-lipoic acid. A deficiency or total absence

of antioxidants cause oxidative stress that can damage or destroy cells. Oxidative stress

has been implicated in the pathogenesis of many human diseases, mainly cancer. So far,

the application of antioxidants in pharmacology has been studied in order to treat several

pathologies namely cardio, cerebral, vascular, atherosclerosis, neoplasia and

neurodegenerative diseases. However, it still remains unclear whether oxidative stress is

the cause or the consequence of these diseases [34].

A great attention was focused on the naturally occurring antioxidant phytochemicals as

potential therapy for cardiovascular diseases. These natural occurring components are

considered as important nutritional ingredients in food, acting as protectors for the body

health maintenance and preventing certain diseases, like cancer or heart failure. Although

studies suggest that nutritional antioxidants are beneficial to health, extensive clinical trials

did not reveal a very clear in vivo biological action on humans and have even suggested

that an excessive intake of these substances could sometimes have negatives effects [35-

37].

A huge amount of prominent organic compounds from both natural and synthetic

sources are recognized as potent antioxidant agents. Outstanding research works

reported a structural diversity of polyphenols with antioxidant properties, being the most

targeted those from the flavonoid family, widespread in the plant kingdom and accurately

associated with the antioxidant capability. A similar attention is due to the stilbene family

in which resveratrol 124, [38] the main grape skin and red wine active component, is the

chief leader of this family, displaying a broad spectrum of biological effects, also followed

by a queue of numerous biological active synthetic analogues.


155

A number of resveratrol 124 valuable properties have been attributed to its intrinsic

antioxidant capabilities, although their potential level in the circulation is likely not enough

to neutralize free radical scavenging. The brain and the heart are uniquely vulnerable to

hypoxic conditions and oxidative stress injuries. Increased heme oxygenase activity,

stimulated by 124 has led to significant protection against models of in vitro and in vivo

oxidative stress injury [39].

Resveratrol 124 acts as ROS inhibitor, which together with the accumulation of the

reactive oxygen intermediate (ROI) produced from cell antioxidant self-defence

(enzymes), are also responsible for cell tissue damage, aging and carcinogenesis. ROS

and ROI lead to oxidative stress phenomena responsible for the development of

cardiovascular diseases, cancer and oxidation of different macromolecules (DNA, lipids

and proteins) [40]. 124 is transformed itself into a stabilized free radical upon reacting with

DPPH radical leading to viniferin dimer 125 [41] (Scheme 4.1).

OH

OH

HO

OH

O

HO

OH

O

HO

OH

O

HO

OH

HO

O

OH

OH

OH

NO2

N

O2N

NO2

NPh

Ph

NO2

HN

O2N

NO2

NPh

Ph

+ +

Viniferin

Dimerisation

124

125

Scheme 4. 1


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4.2. BIOLOGICAL SCREENINGS OF THE NEW POLYPHENOLIC HYBRIDS

4.2.1. Cytotoxic and anti-proliferative effects of polyphenolic hybrids on K562 Cells

K562 cells were the first human immortalized myelogenous leukemia cell lines to be

established. K562 cells are of the erythroleukemia type, and the line is derived from a 53

year old female CML patient in blast crisis [42, 43]. The cells are non-adherent and

rounded, are positive for the bcr:abl fusion gene, and bear some proteomic resemblance

to both undifferentiated granulocytes [44] and erythrocytes [45]. As a first biological aim

toward tracking new anticancer agent, we have evaluated the cytotoxic effects of our

compounds on K562 cells by trypan blue assay. The impact of our polyphenolic hybrids

on the viability of K562 cells after 24, 48 and 72 h was analysed. The cells were incubated

at a concentration of 200 000 cells/mL with the tested compounds added at

concentrations between 1 and 100 μM as indicated. A positive control (CTRL+) with

DMSO was performed with the viability normalized to 100 % and the obtained average

number of cells being 550 000 cells/mL after 24 h, 1 170 000 cells/mL after 48 h and

1 900 000 cells/mL after 72 h. Results presented here are confirmed by mean of at least 3

independent repeated experiments.

4.2.1.1. Cytotoxic effects of benzofuran-3-one-hydantoin hybrids and

polysubstituted uracils

Preliminary results show that the new benzofuran-3-one-hydantoin 10a, 10c, 11a,

(E/Z)-11c have a significant cytotoxic effect, while the starting spiro-chromone-hydantoin

9a, 9c have failed to exhibit the cytotoxic activity to cancer cells (Fig. 4.6). Compounds

10a, and (Z)-11a decrease K562 cell viability after treatment with a concentration of 50 μM

during 48 hours, while 10c was inactive (Fig. 4.7, 4.8). Compounds (E/Z)-11c (1:3 ratio),

(E)-11c and (Z)-11c exert a cytotoxic effect after 24 hours on K562 treated cells with a

concentration of 5 μM (Fig. 4.8, 4.9). The isolated isomers (E)-11c and (Z)-11c

diastereoisomers have both shown similar activity to that of the original mixture (E/Z)-11c

(1:3 ratio), which means that, in such cytotoxic screenings, the geometry of the molecule

does not influence as much as the scaffold itself.

In general, polysubstituted-uracils 12a-c did not show remarkable results,

nevertheless, both 12a and 12a(a) which are the cyclohexyl substituted uracil and

chromone-N-acylurea based compounds, respectively, have interacted after 72 hours of

incubation with a concentration of 100 µM (Fig. 4.10).

http://en.wikipedia.org/wiki/Leukemia

http://en.wikipedia.org/wiki/Blast_crisis

http://en.wikipedia.org/wiki/Bcr:abl

http://en.wikipedia.org/wiki/Fusion_gene

http://en.wikipedia.org/wiki/Granulocytes

http://en.wikipedia.org/wiki/Erythrocytes


157

Figure 4. 6. Cytotoxic effect of compounds 9a (left) and 9c (right) on K562 Cells

Figure 4. 7. Cytotoxic effect of compounds 10a (left) and 10c (right) on K562 Cells

Figure 4. 8. Cytotoxic effect of compounds (Z)-11a (left) and (E/Z 1:3)-11c (right) on K562

Cells

0

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158

Figure 4. 9. Cytotoxic effect of compounds (Z)-11a (left) and (E)-11c (right) on K562 Cells

Figure 4. 10. Cytotoxic effect of compounds 12a(a) (left) and 12a (right) on K562 Cells

4.2.1.2. Cytotoxic and anti-proliferative effects of HOPO-1 and HOPO-2

substituted benzopyran-2-one and benzopyran-4-one based compounds

As a first candidate of analysis, we have started by the dimeric product 13a which is

the basic representative structure of BP-4 polyphenolic compounds sharing the HOPO-1

portion. Indeed, 13a has show modest cytotoxic effects only after 72 hours with the

concentration of 100 µM (Fig 4.11). After that, from the set of our synthesized BP-4-based

polyphenolics, a list of six compounds 13g-i, 13m-n, 14t along with one biscoumarin 16a

has been selected by the biologist in charge for analyzing their impact on viability and

proliferation of K562 cells. A Trypan Blue staining assay was used for this test. The IC50

values were calculated using a dose response curve (Fig 4.12-18). The IC50 values for all

compounds are listed below (Table 4.1).

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159

Figure 4. 11. Cytotoxic effect of the dimeric compound 13a

Table 4. 1. IC50 values for selected compounds after a treatment of 24, 48 and 72 h

Structure IC50 (μM)

24h 48h 72h

14t

O

O

O

HO

OHOCH3

H3CO OCH3

OCH3

12.5 7.5 7.7

13g

O

OO

HO

> 100 > 100 > 100

13h

O

O

H3CO

OCH3

OCH3

O

HO

> 100 > 100 > 100

0

20

40

60

80

100

120

0 5 10 50 100

Via

bil

ity (

%)

Concentration (mM)

24h

48h

72h


160

13i

O

O

H3CO

OCH3

O

HO

> 100 > 100 > 100

13m

O

OO

HO

7.8 3.9 4.5

13n

O

O

H3CO

H3CO

O

HO

> 100 > 100 > 100

16a

OOH

OO

OHOOH

O

27.0 21.8 26.8

For the viability assay over 24, 48 and 72 h, the CHRM 14t, 2-STC 13m and the

biscoumarin 16a showed activity. The most potent compound is 14t with a viability of 1.8

% when treated at a concentration of 100 μM after 72 h. 14t also showed a strong impact

at a concentration of 10 μM with a viability of 30 % after 72 h. Finally, an IC50 of 7.7 μM is

recorded for compound 14t after 72h. 13m also showed a strong effect when treated at a

concentration of 10 μM with a viability of 13 % after 72 h (IC50 = 4.5 μM). Compound 16a

shows a viability of 6 % at 100 μM after 72 h with an IC50 below 30 μM. As a result, the

chromanone 14t and the biscoumarin 16a are cytotoxic agents, while the 2-

styrylchromone 13m seems to be cytostatic.

From a structural point of view, among the BP-4 based structures, chromanone 14t

was found the most active, followed by 2-styrylchromone 13m and finally flavone 13g-i

which did not affect the cell proliferation or inducing death. Compound 14t presents

functional similarities to resveratrol 124 and curcumin 122, also the HOPO-2


161

pharmacophore could be a useful tool for the exerted cytotoxic and anti-proliferative

activities. Another newly discovered cytotoxic synthetic product is the biscoumarin 16a,

the 4-hydroxycoumarin and the HOPO-2 parts must be considered to have a positive

impact on the activity in question.

Figure 4. 12. Impact of compound 14t on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 14t was added in concentrations of 1 µM, 10 µM and 100

μM. Positive control (CTRL+) was performed with DMSO. **p < 0.01

Figure 4. 13. Impact of compound 13g on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 13g was added in concentrations of 10 µM and 100 μM.

Positive control (CTRL+) was performed with DMSO


162

Figure 4. 14. Impact of compound 13h on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 13h was added in concentrations of 10 µM and 100 μM.

Positive control (CTRL+) was performed with DMSO.

Figure 4. 15. Impact of compound 13i on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 13i was added in concentrations of 10 µM and 100 μM.



163

Figure 4. 16. Impact of compound 13m on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 13m was added in concentrations of 1 µM, 5 µM and 10 μM.

Positive control (CTRL+) was performed with DMSO. **p < 0.01, ***p < 0.001

Figure 4. 17. Impact of compound 13n on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 13n was added in concentrations of 10 µM and 100 μM.



164

Figure 4. 18. Impact of compound 16a on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 16a was added in concentrations of 10 µM, 50 µM and 100

μM. Positive control (CTRL+) was performed with DMSO. **p < 0.01

In parallel to a 72-hours-proliferation assay, an 8 h viability assay was performed. The

impact of the compounds was analyzed with and without TNF-α (tumor necrosis factor-

alpha) addition. The compounds were tested at concentrations varying between 1 and 100

μM as indicated. A positive control with DMSO was performed.

Incubated together with TNF-α, compound 14t decreased the viability down to 46 %.

Incubation with compound 13g-i leads to a viability of 81 %, 96 % and 92 %, respectively.

Compound 13m showed a slight effect with a viability of 78 % at a concentration of 10 μM

and 92 % at a concentration of 100 μM. Compound 13n had moderate effect with viability

of 86 %. Compound 16a did not have any impact on proliferation at a concentration of 1

μM and only a slight effect when increasing the concentration to 10 μM, with a viability of

100 % and 83 %, respectively. However at a concentration of 100 μM all cells died after 8

h (Fig. 4.19).

The tests performed without TNF-α addition showed similar results to those discussed

with TNF-α addition. Only compound 14t decreases viability to 60 %. Compound 13i, 13g,

13h and 13n showed comparable viabilities of 81 %, 88 %, 83 % and 84 % at 100 μM,

respectively. The viability of cells incubated with compound 13m at a concentration of 10

and 100 μM come to 86 % and 104 %. Also after incubation without TNF-α, compound

16a kills all cells at 100 μM (Fig. 4.20).


165

Figure 4. 19. Viability assay on K562 cells with TNF-α addition in the presence of

compound 13i-g, 13m-n, 14t and 16a

Viability of K562 cells was analyzed after 8 h. Compounds were analyzed in concentrations indicated. 20 ng/mL TNF-α were

added to the treated cell suspensions. Positive control (CTR+) was performed with DMSO and TNF-α. Culture medium was

used as negative control (CTR-). *p < 0.05, ***p < 0.001

Figure 4. 20. Viability assay on K562 cells without TNF-α addition in the presence of

compound 13i-g, 13m-n, 14t and 16a

Viability of K562 cells was analyzed after 8 h. Compounds were analyzed in concentrations indicated. Positive control

(CTR+) was performed with DMSO. Culture medium was used as negative control (CTR-). *p < 0.05; **p < 0.01, ***p <

0.001

14t 13i 13h 13g 13m 13n 16a

14t 13i 13h 13g 13m 13n 16a


166

4.2.2. Effect of polyphenolic hybrids on the NF-κB transactivation potential

4.2.2.1. Effects of benzofuran-3-one-hydantoin on the NF-κB activation in K562

cells

In a second time, we investigated the potential ability of some selected cytotoxic

compounds from the benzofuran-3-one-hydantoin hybrids to modulate inflammation by

analyzing their impact on the TNF-alpha induced NF-κB pathway. Transfected K562 cells

were pre-treated for 2 hours with the same concentrations of compound of interest used

for viability assay and challenged for 6 hours with 20 ng/mL of TNF-alpha. The effect of

compounds on the inhibition of TNF-alpha induced NF-κB activation was assessed by the

Dual-Glo™ Luciferase kit from Promega. The results are expressed as the ratio between

the luminescence of the Firefly luciferase plasmid and the luminescence of the Renilla

plasmid. Untreated cells were used as negative control (T-), cells treated with TNF-alpha

only as positive control (T+). Results presented are confirmed by mean of at least 3

independent repeated experiments (Fig. 4.21). The results show that compounds (Z)-11a,

(E/Z 1:3)-11c (100 μM) and (Z)-11c (≥ 50 μM) decrease the TNF-alpha induced NF-κB

pathway. Nevertheless, in this test, compound (E)-11c shows a moderate inhibitory effect

on the TNF-alpha induced NF-κB pathway, where we can consider the geometrical factor

as the consequence of a reduced activity.

Figure 4. 21. Inhibition of TNF-alpha induced NF-κB pathway activation by benzofuran-3-

one-hydantoin hybrids (Z)-11a, (E/Z 1:3)-11c, (E)-11c and (Z)-11c


167

4.2.2.2. Effects of HOPO-1 and HOPO-2 substituted benzopyran-2-one and

benzopyran-4-one based compounds on NF-κB activation in K562 cells

The impact of the BP-4-based polyphenolic compounds on the TNF-α induced NF-κB

pathway activation in K562 cells was analyzed with a luciferase assay. The analyses were

performed with TNF-α to show the inhibitory effect of the tested compounds. To ensure

that none of the compounds activated the pathway on its own, analyses without TNF-α

were performed. A positive control with DMSO is used. The chromanone 14t and flavones

13g-i have an increasing impact on the NF-κB pathway activation with an activation

percentage of 183 %, 179 %, 180 % and 172 %. Compound 13m is noticed more

impactful having a strong NF-κB activation effect of 265 % at 10 μM and 297 % at 100

μM. There might be a synergistic effect in combination with TNF-α as for the analysis

without TNF-α compound 13m did not show an activation of the NF-κB pathway on its

own. The biscoumarin 16a was the unique compound inhibiting the TNF-α induced NF-κB

pathway activation; at concentrations of 1, 10 and 100 μM, the inhibition compared to

TNF-α control reaches 22 %, 51 % and 99 %, respectively (Fig. 4.22). The analyses

without addition of TNF-α showed that none of the compounds activated the NF-κB

pathway on its own. All results obtained were below 1 % of NF-κB pathway activation (Fig.

4.23).

Figure 4. 22. NF-κB pathway inhibition assay on K562 cells with TNF-α addition by

benzopyran-4-one based polyphenolic compounds 13i-g, 13m-n, 14t and 16a

Luciferase activity was measured after 8 h. Compounds were analyzed in concentrations indicated. 20 ng/mL TNF-α were

added to the treated cell suspensions after 2 h of incubation. Positive control (CTR+) was performed with DMSO and TNF-

α. Negative control (CTR-) was untreated cell suspension.*p < 0.05; **p < 0.01

14t 13i 13h 13g 13m 13n 16a

http://www.google.pt/search?hl=pt-PT&tbo=d&biw=1366&bih=630&spell=1&q=synergistic&sa=X&ei=PSa_UP2LO8nQhAe-zICoBg&ved=0CCgQvwUoAA


168

Figure 4. 23. NF-κB pathway inhibition assay on K562 cells without TNF-α addition

addition by benzopyran-4-one based polyphenolic compounds 13i-g, 13m-n, 14t and 16a

Luciferase activity was measured after 8 h. Compounds were analyzed in concentrations indicated. Positive control (CTR+)

was performed with DMSO and TNF-α. Negative control (CTR-) was untreated cell suspension. ***p < 0.001 (compared to

CTR+)

4.2.2.3. Effect of the biscoumarin 16a on the NF-κB transactivation potential

4.2.2.3.1. Effect of the biscoumarin 16a on the NF-κB pathway in K562 cells

Here, we have selected the biscoumarin 16a as the most potent NF-κB inhibitor. The

impact on the TNF-α induced NF-κB pathway activation of compound 16a was analyzed

with concentrations varying between 5 and 100 μM. As it was already shown that 16a did

not activate the NF-κB pathway on its own, no separate test without TNF-α addition was

performed. Therefore, 16a reduces the activation of the NF-κB pathway in a dose-

dependent manner. Significant inhibition level of 24 % started at a concentration of 10 μM

compared to TNF-α control. Remarkable results were obtained with a concentration of 25

μM, whereas concentrations of 50 and 100 μM showed higher effects. Inhibition

percentages compared to TNF-α control were 73 %, 90 % and 100 %, respectively (Fig.

4.24). The IC50 is obtained at 15.7 μM as deduced from the dose-response-curve after 8

hours.

14t 13i 13h 13g 13m 13n 16a


169

Figure 4. 24. NF-κB pathway inhibition assay on K562 cells with TNF-α addition for

compound 16a

Luciferase activity was measured after 8 h. Compound 16a was analyzed in concentrations indicated. 20 ng/mL TNF-α were


α. Negative control (CTR-) was untreated cell suspension. *p < 0.05, **p < 0.01, ***p < 0.001

4.2.2.3.2. Effect of the biscoumarin 16a on the NF-κB pathway in jurkat cells

An NF-κB pathway inhibition assay was also performed on Jurkat cells. The analyses

were carried out with TNF-α to show the inhibitory effect of the compound. To ensure that

the compound did not activate the pathway on its own, analyses without TNF-α were

performed. A positive control with DMSO was accomplished. Compound 16a showed an

inhibition of the TNF-α induced NF-κB pathway activation at a concentration of 5 and 10

μM (Fig. 4.25). No inhibition could be detected for the analyses performed without TNF-α

addition (Fig. 4.26).


170

Figure 4. 25. NF-κB pathway inhibition assay on jurkat cells with TNF-α addition for

compound 16a

Luciferase activity was measured after 8 h. Compound 16a was analyzed in concentrations indicated. 20 ng/mL TNF-α were


α. Negative control (CTR-) was untreated cell suspension. **p < 0.01, ***p < 0.001

Figure 4. 26. NF-κB pathway inhibition assay on jurkat cells without TNF-α addition for

compound 16a

Luciferase activity was measured after 8 h. Compound 16a was analyzed in concentrations indicated. Positive control

(CTR+) was performed with DMSO and TNF-α. Negative control (CTR-) was untreated cell suspension. ***p < 0.001

(compared to CTR+)


171

4.2.2.3.3. Effect of compound 16a on the NF-κB transactivation potential

analyzed by western blot

The results obtained with the luciferase assay were further confirmed by Western Blot

analyses. IκBα phosphorylation and degradation as well as translocation of the NF-κB

subunits p50 and p65 from the cytoplasm to the nucleus were studied. A control with cells

in culture medium activated by TNF-α was performed first. The results showed that the

classical NF-κB pathway is activated through a phosphorylation of IκBα starting after 10

min and its degradation started at 15 min. After 90 min, IκBα was resynthetized and

appeared in the cytoplasm. A decrease of the subunits p50 and p65 in the cytoplasm

could not be detected. However an increase in the nucleus can clearly be observed after

30 min. The treatment with 16a prevents the degradation of IκBα in the cytoplasm. No

phosphorylation could be detected. A decrease of the subunits p50 and p65 in cytoplasm

could not be shown. With analyses of the nuclear fractions, it could be demonstrated that

the translocation to the nucleus was stopped as an increase of the p50 and p65 was

inhibited (Fig. 4.27).

Equal protein loading and extract purity were verified by analyses of α-tubulin and

lamin B in the cytoplasm and in the nucleus fractions. α-tubulin indicated an equal loading

while no lamin B could be detected in the cytoplasm, thus, the cytoplasmic extracted could

be assumed as pure. In conter-side; lamin B showed an equal loading but no α-tubulin

appeared in the nuclear fractions showing that the nuclear extract was pure (Fig. 4.27).


172

Figure 4. 27. Effect of 16a on the degradation of IκBα and the translocation of NF-κB

subunits p50 and p65 from cytoplasm to nucleus, analyzed by Western Blot

Jurkat cells were pre-treated with compound 16a (40 µM) for 2h followed by activation with TNF-α (20 ng/mL) for indicated

time periods. Cytoplasmic and nuclear extracts were tested for IκBα, p-IκBα, p50 and p65. Protein loading and purity of

extracts were verified by lamin B and α-tubulin Western blots.

4.2.3. Viability and NF-κB pathway activation in K562 cells of 16a hybrid

substructures 2”-hydroxypropiophenone and 4-hydroxycoumarin

Herein, we want to verify our hypothesis that hybrid molecules generate higher

bioactivity. Therefore the fragments/substructures of 16a, 2”-hydroxypropiophenone (2-

HPP) which is considered as the simplest HOPO-2 derivative and 4-hydroxycoumarin unit

(4-HCOM) (Fig. 4.28) were analyzed for their effect on viability and NF-κB pathway

activation in K562 cells. The viability NF-κB pathway activation in K562 cells experiences

have been retaken in similar conditions (concentration and time of incubation) (Fig. 4.29-

34). Compared to the results obtained for the analyses of the biscoumarin 16a, 2”-

hydroxypropiophenone and 4-hydroxycoumarin do not show any activity on the

proliferation of cancer cells or on the TNF-α induced activation of the NF-κB pathway.

Hence, the biological activities of the hybrid molecule 16a are completely new.


173

OOH

OO

OHOOH

O

OOH

OO

OH

+

16a 2-HPP 4-HCOM

Figure 4. 28. Chemical structures of 16a and its substructures

Figure 4. 29. Impact of 2”-hydroxypropiophenone on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. 2-HPP was added in concentrations of 1 µM, 10 µM and 100 μM.



174

Figure 4. 30. Impact of 4-hydroxycoumarin on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. 4-HCOM was added in concentrations of 1 µM, 10 µM and 100 μM.


Figure 4. 31. Viability assay on K562 cells for the substructures 2”-hydroxypropiophenone

and 4-hydroxycoumarin with TNF-α addition

Viability of K562 cells was analyzed after 8 h. Compounds were analyzed in concentrations indicated. 20 ng/mL of TNF-α

were added to the treated cell suspensions. Positive control (CTR+) was performed with DMSO and TNF-α. Culture medium

was used as negative control (CTR-).

2-HPP 4-HCOM


175

Figure 4. 32. Viability assay on K562 cells for substructures 2”-hydroxypropiophenone

and 4-hydroxycoumarin without TNF-α addition

Viability of K562 cells was analyzed after 8 h. Compounds were analyzed in concentrations indicated. Positive control

(CTR+) was performed with DMSO. Culture medium was used as negative control (CTR-).

Figure 4. 33. NF-κB pathway inhibition assay on K562 cells with TNF-α addition for


Luciferase activity was measured after 8 h. Compounds were analyzed in concentrations indicated. 20 ng/mL TNF-α were


α. Negative control (CTR-) was untreated cell suspension.

2-HPP 4-HCOM


176

Figure 4. 34. NF-κB pathway inhibition assay on K562 cells without TNF-α addition for


Luciferase activity was measured after 8 h. Compounds were analyzed in concentrations indicated. Positive control (CTR+)

was performed with DMSO and TNF-α. Negative control (CTR-) was untreated cell suspension. ***p < 0.001 (compared to

CTR+)

4.2.4. Biscoumarins cytotoxicity and NF-κB pathway inhibitory effect

According to the previous biotests, we were able to determine the dual biological

function associated with the biscoumarin 16a which exerts cytotoxicity to leukemia cancer

cells (IC50 = 27.0 µM, 24h) and anti-inflammatory activity through inhibition of the NF-κB

pathway (IC50 = 15.7 µM, 8h) at the same time. In this context, we also want to generalize

our conclusion on whether the biscoumarin scaffold itself is the responsible for the activity

or only the derivative 16a. For this purpose, the biologist has selected other three

biscoumarin derivatives 16d, 16e and 16a(5-Cl) sharing substituent on the HOPO-2

portion.

Cytotoxicity and proliferation effects of 16d, 16e and 16a(5-Cl) have been evaluated.

Compound 16d starts to exert its effects on proliferation of cell cancer at concentration of

10 µM, a considerable impact on cell growth is seen at 50 µM, and finally, its cytotoxic

activity is observed when using a concentration of 100 µM (Fig. 4.35). 16e shows a

moderate antiproliferative action at a concentration of 100 µM (Fig. 4.36). The chloro-

biscoumarin derivative 16a(5-Cl) shows the highest cytotoxic activity at a concentration of

50 µM (Fig. 4.37). Regarding the NF-κB inhibition potential (Fig. 4.39), 16a exerts the anti-

2-HPP 4-HCOM


177

inflammatory activity in a concentration dependant manner, their synthesized analogues

16d, 16e and 16a(5-Cl) undermine that the presence of two 4-hydroxycourmarin units and

HOPO-2 are necessary elements for the observed bioactivity. In fact, compounds 16d and

16a(Cl) exert higher NF-κB inhibition potential than product 16a. For both cytotoxic and

NF-κB inhibition effects, the lowest IC50 was recorded for the chloro-biscoumarin 16a(5-

Cl) which suggest that the substitution on HOPO-2 fragment reflects on the activity.

However, the presence of 4-hydroxcoumarin seem to be a crucial function. The IC50

values for the cytotoxic effects and NF-κB inhibition are given in the following Table 4.2.

Table 4. 2 IC50 values for biscoumarins 16 after treatment of 24h for the cell viability assay

and after 8h for NF-κB inhibition test

Structure IC50 (μM)

Cytotoxicity 24h NF-κB inhibition 8h

16a

OOH

OO

OHOOH

O

27.0 15.7

16d

OOH

OO

OHOOH

O

H3CO

39.0 13.6

16e

OOH

OO

OHOOH

O

H3COOCH3

>50 >50

16a(5-Cl)

OOH

OO

OHOOH

O

Cl

15.0 6.3

In order to validate our approach, we suggested to assess the effect of the cytotoxic

compounds including, the BP-4-based compounds, chromanone 14t (IC50 = 12.5 µM,

24h), the 2-styrylchromone 13m (IC50 = 7.8 µM, 24h) as showing the highest impact on

cancer cell growth and the BP-2 based biscoumarin hybrids 16, on the viability of healthy


178

peripheral blood mononuclear cells (PBMC) in order to determine differential toxicity of

these compounds and their selectivity to cancer cells. Hopefully, none of our compounds

affect the healthy cell growth (Fig 4.40). Recent reports reveal the selective cytotoxic

activity to cancer cells found in some coumarin derivatives, like 7,8-dihydroxy-4-

methylcoumarin, 7,8-diacetoxy-4-methylcoumarin [46] and 7-hydroxycoumarin [47]

showing antiproliferative effects to lung cancer cells but not to PBMCs. Our biscoumarins

are seen as new lead structures, consequently compound 16a(5-Cl) was the most active

with dual function as a cytotoxic agent (IC50 = 15.0 µM, 24h) and inhibitor of the relevant

NF-κB pathway (IC50 = 6.3 µM, 8h). The new biscoumarin structure is challenging the

antiproliferative cytostatic curcumin 122 which shows anti-inflammatory effects by

inhibiting NF-κB (IC50 = 17.8 µM, 8h) (Fig. 4.38, 4.39), the fascinating part of the results is

that compound 16a(5-Cl) was advantageously less impactful on healthy PBMCs than

curcumin 122 (Fig. 4.40).

Figure 4. 35. Impact of compound 16d on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 16d was added in concentrations of 1 µM, 10 µM and 100

μM. Positive control (CTRL+) was performed with DMSO


179

Figure 4. 36. Impact of compound 16e on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 16e was added in concentrations of 1 µM, 10 µM and 100


Figure 4. 37. Impact of compound 16a(5-Cl) on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 16a(5-Cl) was added in concentrations of 1 µM, 10 µM and

100 μM. Positive control (CTRL+) was performed with DMSO


180

Figure 4. 38. Impact of curcumin 122 on proliferation of K562 cells

Cell proliferation was analyzed for 24, 48 and 72 h. Compound 122 was added in concentrations of 1 µM, 10 µM and 100


Figure 4. 39. NF-κB pathway inhibition assay in K562 cells with TNF-α addition for the

biscoumarin compounds 16a, 16d, 16e and 16a(5-Cl)

Luciferase activity was measured after 8 h. Compounds 16a, 16d, 16e, 16a(5-Cl) and 122 were analyzed in concentrations

indicated. 20 ng/mL TNF-α were added to the treated cell suspensions after 2 h of incubation. Positive control (CTR+) was

performed with DMSO and TNF-α. Negative control (CTR-) was untreated cell suspension.


181

Figure 4. 40. Viability assay on healthy PBMC's of the new cytotoxic polyphenolic hybrids

14t,13m, 16a, 16d, 16e, 16a(5-Cl) and 122

4.2.5. Evaluation of HDAC inhibition of benzofuran-3-one-hydantoin hybrids

A range of concentrations of benzofuran-3-one-hydantoin hybrids were tested in

vitro on total HDAC activity. Only one molecule, (E/Z 1:3)-11c, displays a weak inhibitory

effect against total HDAC activities (30% inhibition at 100 µM). Since (E/Z 1:3)-11c are an

isomeric mixture, then we tested the two separated isomers, (E)-11c and (Z)-11c.

Interestingly, the (E)-isomer displayed an IC50 of 104 µM, whereas only 30% of inhibition

was observed at 100 µM with the (Z)-isomer. In order to determine if the inhibition

observed on total HDAC activity is due to a moderate inhibition of all HDAC isoforms or a

specific inhibition of one or some isoforms, in vitro assay using recombinant proteins were

performed with compounds (E)-11c and (Z)-11c. Both isomers were able to inhibit

isoforms of class I with IC50 between 30 and 69 µM. The (E)-isomer inhibits HDAC6 and

10 from class IIb with IC50 of 75 and 92 µM, respectively, whereas (Z)-isomer inhibits only

HDAC6 with IC50 of 41 µM (Table 4.3). Finally, (E)-isomer is not active against HDAC11,

but (Z)-isomer possesses an IC50 of 69 µM. These results reveal some differences in

inhibitory activity of the two diastereomers suggesting that the enzyme does not

accommodate these inhibitors in the same manner with a link between the 3D structure of

the molecules and their biological activities.


182

Table 4. 3. Effect of (E)-11c and (Z)-11c on the in vitro activity of HDAC isoforms

HDAC

IC50 (µM)

Class Isoform

(E)-11c (Z)-11c

Class I HDAC1

68 63

HDAC2

69 31

HDAC3

52 54

HDAC8

30 32

Class IIb HDAC6

75 41

HDAC10

92 Inactive

Class IV HDAC11

Inactive 69

Inactive means inhibition < 10% at 100 µM.

4.2.6. Evaluation of Michael acceptor containing polyphenolic hybrids on the

activation of Keap1- Nrf2 pathway

As most of our newly synthesized polyphenolic hybrid present a Michael acceptor

fragment, such as in the conjugated benzofuran-3-one-hydantoin (Z)-11a and (E/Z)-11c,

and in the benzopyran-4-one-based polyphenolics 13, 14, 15, we wish to evaluate their

potential on activation of Keap1-Nrf2 pathway. The most interesting results of this assay

are summarized in Figure 4.41.

Using sulforaphane SFN 130 as a positive control, the anti-carcinogenic potential was

examined by measuring the potential to induce the cytoprotective Nrf2 (nuclear factor E2-

related protein 2) signaling pathway and the ability of compounds to inhibit proliferation of

cancer cells. Utilizing MCF7 cells stably transfected with an ARE-dependent luciferase

reporter, we screened three conjugated benzofuran-3-one-hydantoin hybrids 11 and

fourteen derivatives of benzopyran-4-one-based compounds 13, 14 and 15. The

experiments are repeated three times. The benzofuran-3-one-hydantoin hybrids (Z)-11a

and isomers (E/Z)-11c show maximal fold induction of luciferase activity of 30-50±0.2 at

concentration of 12.5-25 µM which is comparable to SFN (32±2.2 -fold induction at 25

µM).


183

On the other side, the concentrations required to increase luciferase activity by 10-fold

(C10) were in the range of 2.9-23.8 μM for chromanone 14q-t, 3.9-45.1 μM for 2-

styryrchromone 13m-o, 8.9-48.2 μM for chromones 13a, 13d-f and > 100 μM for flavones

13g-i. C10 is the parameter enabling the comparison between compounds’ induction

potency, thus, the smaller is C10, the more potent is the compound. Maximal fold

induction of luciferase activity was achieved by stimulation with chromanones 14, among

which the most potent inducer 14t caused a 91.2±12.8-fold induction at a concentration of

12.5 µM (C10 = 4.1 µM), being almost 3 times more potent than sulforaphane SFN 130

(C10 = 8.9 µM). The chromanone 14q and the 2-styrylchromone 13m both demonstrate

comparable induction ability to that of the leader 14t but at higher concentrations (Maximal

fold induction 85±2.5, at optimal concentration of 25-50 µM) (Fig. 4.41).

Structure-activity relationship (SAR) analyses revealed that both the number and

position of methoxyl substituents significantly influences biological activity. In the

chromones class 13a and 13d-f, an increased activity is observed by the addition of -

OCH3 group(s) to ring A, however the 2-methylchromone 13f is found as most potent

among chromones. An inverse relationship was noticed in the 2-styrylchromone class

13m-o, since the addition of -OCH3 group(s) to the aromatic rings decreases the activity.

Flavones, 13g-i and 2-(4-arylbuta-1,3-diene)chromones 15a-c were inactive at a certain

extent. Finally, the most fascinating class is the chromanone one, in which the activity is

also increasing with respect to the degree of methoxy substitution but not in a linear

manner.

Interestingly, we have seen that several representatives of benzopyran-4-one-based

polyphenolics 13 and 14 have been additionally tested for effects on viability of leukemic

K562 cells by trypan blue assay. As mentioned previously the treatment with chromanone

14t for 24 h resulted in cytotoxic activity, with an IC50 of 12.5 μM, whereas growth of

peripheral blood mononuclear cells (PBMC) was not affected (IC50 > 50 μM). Similarly, the

2-styrylchromone 13m caused death of K562 cells (IC50 = 7.8, 24 h), without affecting

PBMC as well (IC50 > 50 μM). These data confirms the selectivity of our compounds for

cancer cells, making the chromanones and 2-styrylchromones an interesting lead

structure for further analyses.


184

Figure 4. 41. Activation of the cytoprotective Keap1-Nrf2 pathway by Michael acceptor

containing polyphenolic hybrids

4.2.7. Evaluation of Cdc25 phosphatase inhibition of benzofuran-3-one-hydantoin

hybrids

In vitro Cdc25 phosphatase activity was measured with a fluorimetric method using

purified human glutathione-Stranferase (GST)-Cdc25 recombinant enzymes. The

enzymatic activity was determined with 3-O-methylfluoresceinphosphate

dephosphorylation assay. The results are expressed in percent of residual Cdc25

phosphatase activity in presence of the tested benzofuran-3-one-hydantoin hybrids 10a,

10c, (Z)-11a and (E/Z 1:3)-11c. Lower is the percentage, stronger is the inhibition. The

activity of control (solvent DMSO alone) is used as reference for 100 % of phosphatase

activity. This study was made 3 times with 3 tests by assays (3 wells by plate on 3

different plates), it results in 9 values per compound per Cdc25 isoform tested. All

compounds are generally tested at 100 µM final concentration, except the reference

BN82002 which is tested at 10 µM. Considering global results, 10a, (Z)-11a and (E/Z

1:3)-11c seems to have slight inhibitory effects (Table 4.5, Fig. 4.42)


185

Table 4. 4. Evaluation of Cdc25 phosphatase inhibition in the presence of benzofuran-3-

one-hydantoin hybrids

CDC25 A CDC25 C

Assay residual

activity (%)

standard

deviation

residual

activity (%)

standard

deviation

DMSO 100,00 2,12 100,00 1,89

10a 59,67 1,29 57,81 9,65

10c 85,20 6,89 82,19 8,23

(Z)-11a** 79,53 5,73 23,70 4,66

(E/Z)-11c 58,40 4,80 23,90 0,75

BN82002* 10,28 5,63 3,48 8,02

*BN82002 was tested à 10µM, ** (Z)-11a was tested at 50µM (not soluble up to 5mM)

Figure 4. 42. Evaluation of Cdc25 phosphatase inhibition

4.2.8. Evaluation of the antioxidant activities

4.2.8.1. Ferric reducing antioxidant power (FRAP) assay

The ferric reducing antioxidant power (FRAP) assay depends on the reduction of a

ferric tripyridyltriazine (Fe3+–TPTZ) complex to the ferrous tripyridyltriazine (Fe2+–TPTZ)

by a reductant at low pH [48]. Fe2+–TPTZ has an intensive blue color and is usually

monitored at 593 nm. Experimentally, trolox (Tr) (6-hydroxy-2,5,7,8-tetramethyl-chroman-

2-carboxylic acid) a water soluble derivative of vitamin E, was used as a reference

antioxidant. The working FRAP reagent was prepared as required by mixing 25 ml acetate

buffer, 2.5 ml TPTZ solution, and 2.5 ml FeCl3.6H2O solution. Freshly prepared FRAP

reagent was warmed at 37 °C for 30 min and added to the samples every 30 s.


186

Absorbance values were read after 30 min every 30 s. Each molecule was tested two

times with three measures in each experience.

4.2.8.2. Free radical diphenylpicrylhydrazyl (DPPH) assay

DPPH (1,1-diphenyl-2-pycrilhydrazyl) is a stable radical with a violet intense color with

a maximum absorbance at 515 nm. The antioxidant power is expressed by the diminution

of the violet coloration caused by the neutralization of DPPH and it is expressed by a

absorbance decrease [49]. This change is proportional to the antioxidant capacity that is

measured with a spectrophotometer at 515 nm. The trolox was used as a reference

compound to make a range between 0 and 70 µM of Trolox. The range makes possible to

determinate the quantity of reduced DPPH in trolox equivalents. Trolox equivalents is a

trolox quantity [µM] corresponding to an optical density at 515 nm of the tested product.

In the experience, the stock solution of DPPH is prepared at the concentration of 2 mM

and the trolox solution at the concentration of 2.5 mM. Before measuring, the samples are

prepared with 5µL of the tested product (stock solution 10 µM), 935 µL of methanol and

60 µL of DPPH solution. The final volume is 1 mL. The optical density is read after

incubation in the dark during 30 minutes. Each molecule was tested two times with three

measures in each experience.

4.2.8.3. Antioxidant capabilities measurements

Both of the highlighted methods were used because each evaluates one different

parameter about the antioxidant power. In FRAP method (Fig. 4.43), the measure of the

antioxidant capacity is based on compound capacity to reduce ferric ions (Fe3+) to ferrous

ions (Fe2+). In the DPPH method (Fig. 4.44), the measure of the antioxidant power is

based in the capacity of inhaling the free radical DPPH. The objective of these tests was a

primary screening of most of our polyphenolic hybrids with purpose to find which of them

have the best antioxidant power. The concentrations of the used molecules was 25 µM for

FRAP and 10 µM for DPPH (a more sensitive method). In fact, the in vitro antioxidant

evaluation for the most active compound on cell culture did not correlate in perfect

manner, since for example the most active chromanone 14t did not show considerable

values on the FRAP test, while the DPPH gives an antioxidant capability of >20 µM of

trolox equivalent. The results of the antioxidant capabilities (very low on both FRAP and

DPPH tests) of the active bsicoumarin 16a also were not consistent with in vitro test on

cell culture. In addition, the dimeric compound 13a gives different values, up to 40 µM of

trolox equivalent in the DPPH test, however, a negligible value was achieved in the FRAP


187

test. Interestingly the 2-vanillinoxychromanone 13a(d) is a potent antioxidant according to

the FRAP test (2 nmol of Fe2+/nmol) which is comparable to that of trolox, but the DPPH

test gives a contradictional results.

Figure 4. 43. FRAP antioxidant activity evaluation of polyphenolic hybrids (25 µM)

Figure 4. 44. DPPH antioxidant activity evaluation of polyphenolic hybrids (10 µM)


188

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CHAPTER 5

- CONCLUSIONS AND PERSPECTIVES -

Chapter 5 – Conclusions & Perspectives

193

5. CONCLUSIONS AND PERSPECTIVES

By the end of the RedCat scientific project and the accomplishment of the Chemistry-

Biology-Medicine continuum, considerations and future perspectives are entertained

between the Chemist and Biologist which both have now established a strong relationship

based on continous collaboration, exchangeable experiences and skills transfer. The role

of the chemist in the Chemistry-Biology-Medicine continuum is obvious by the first

underlined word “Chemsitry”. In this regard, we have demonstrated in the presented PhD

dissertation, the facile manipulation of organic chemistry basis to creat from simple to

complex molecular structures fitting the biologist demands. This project has provided the

key to the gate of complex organic molecule synthesis through which tracking and

discovering new potential synthetic drugs is aimed in order to struggle against the most

actual threatening diseases “cancer”. Up-dating new green synthetic methodologies of

complex organic structures based on time saving and high scales of production have also

been considered. We have mentioned in the first chapter that these scientific purposes

can be reached by realizing several collaborations in which, the chemist and biologist

could work in the same atmosphere gathering all their expertise (organic and bio-organic

chemistry, biology, medicine) in a high developed platform (scientific research

infrastructure equipped with high technological facilities).

The referred work has allowed us to establish new synthetic methods for novel hybrid

polyphenolic compounds. In the second chapter, we have described synthetic approaches

leading to a facile hybridizing of biologically active heterocycles. σ simple bonded and π

double bonded benzofuran-3-one-hydantoin hybrids have been produced following

diastereoselective organic pathways, starting from cheap and commercially available

materials like chromone-2-carboxylic acid and carbodiimides. In depth spectroscopic

study, using the high analytical resolution of single-crystal X-ray diffraction and NMR

techniques have revealed various structural features regarding the stereochemical aspect

of the new benzofuran-3-one-hydantoin olefins. Moreover, important diastereoselective

patterns, conformational and geometrical observations have been discussed. Some of

these new hybrids present photochemical properties and modest biological actions. The

relevant uracil nucleus has been constructed via green routes, chemical and time savings.

A major focus was on the subject discussed in the third chapter, where we have

developed a new efficient reaction compilation of Baker-Venkataraman and Michael

addition tandem process for the synthesis of several benzopyran-4-one based


194

polyphenolic scaffolds. In particular, we have emphasized the new one-pot 1,4-conjugate

addition leading to various biologically target compounds, namely polysubstituted

chromones, chromanones and flavones starting from the commercially available

chromone-3-carboxylic acid and the easily prepared phenolic 1,3-dicarbonyl compounds.

The efficiency of the procedure is seen in its soft execution under mild conditions leading

to a structural diversity of polyphenols produced in moderate to high yields. The use of a

catalytic organic-base in the new developed Baker-Venkataraman-Michael addition

tandem sequence is also considered as an advantage, while other multistage procedures,

already reported in the literature, show high amounts of chemical use and time

consuming. Mechanistically, the presence of the α-carboxylic acid auxiliary function in the

α,β-unsaturated ketone of the chromone-3-one carboxylic acid is confirmed to play a great

role in promoting 1,4-conjugate additions on the benzopyran-4-one rings, which is

subsequently removed by decarboxylation, this concept is recently brought to the organic

synthetic literature. The presented mechanistic study has aimed some pedagogies in

understanding the organo-catalysis aspect of this type of Michael additions. From

analytical point of view, an in depth 2D-NMR analysis (HMBC and NOESY techniques)

was made to achieve a fine elucidation of complex structures, involving conformational,

chirality and other geometrical aspects. X-ray diffractommetry of organic compounds

becomes an indispensable tool which helped us to face the puzzling structure elucidation,

especially in the determination of the absolute asymmetric carbons configurations. Further

applications of 1,4-conjugate addition strategies have been entertained in view of

producing highly complex polyphenolic structures by introducing the benzopyran-2-one

moiety (mainly, 4-hydroxycoumarin). Thus, we have seen in a following work, various type

of synthetic chemistry on bispyranones, warfarin-analogues and the increasing complexity

of benzopyran-2-one-benzopyran-4-one hybrids which have been accomplished for the

first time. Half of the designed benzofuran-3-one, benzopyran-4-one and benzopyran-2-

one hybrid scaffolds prepared in this work have been screened for their activity in

preventing cancer at various stages, from the antioxidant protective effects of healthy cells

to anti-inflammatory pathways and ending to the direct actions on cell cancer influencing

their viability and proliferation. These are the main underlined stages involved in the

cancer therapy.

In terms of biological achievements, it could be shown that three classes of the newly

synthesized hybrid compounds are biological active. Firstly, the polysubstituted

chromanone structure 14t sharing the HOPO-2 bioactivator is the new lead structure

showing an impact on the viability and proliferation of K562 leukemic cancer cell lines.


195

More biological-data are collected by proving the cancer chemopreventive effects, the

anti-carcinogenic potential by inducing the cytoprotective Nrf2 signaling pathway and the

ability to inhibit proliferation of MCF7 breast cancer cells by the chromanone 14t.

Secondly, the 2-styrylchromone core structure sharing the HOPO-1 bioactivator has also

been characterized as cytotoxic class of compounds and the third class is the new

discovered biscoumarin hybrids (bis-4-hydroxycoumarins) exerting a dual action by

inhibiting the proliferation of K562 cancer cells and the strong impact on the activation of

the pro-inflammatory NF-κB pathway, where we can underline several lead structures

such as 16a, 16b and 16a(5-Cl). We have also rationalized the concept of the active

hybrid molecule built from inactive constructive fragments, in the case of the biscoumarin

16a, where the substructures of which (2-HPP and 4-HCOM) did not show any activity.

More convincible data are achieved when we discovered that all the newly elaborated

target compounds did not affect the growth of healthy peripheral blood mononuclear cells

(PBMC), thus proving their selectivity to cancer cells. The mechanism of apoptotic cell

death should be studied in order to validate our results. Additionally, some basic tests

have also been performed, like HDAC and Cdc25 enzyme inhibitions in view of

understanding the possible involvement of some of the hybrid scaffolds in the genetic

level and cell division cycle. Additionaly the in vitro antioxidant capabilities using FRAP

and DPPH techniques were performed but did not show compatibilities with the in vitro

test on cell culture. Summarizing these screening attempts, the creation of a new

generation of anticancer polyphenolic hybrid compounds was achieved and should be

pursued. Importantly, we should indicate that further interests are due to the rest of

benzopyran-(2 and 4)-one hybrids which still remain under biological assessments.

At the end, taking into consideration the ethical issues and human-rights related to

chemical and biological experiments, the project is seen as a successful step in

developments of different classes of synthetic hybrids mainly from natural biomimic

derived compounds, which remains very promising, as distinct features are implicated

such as structural organic complexity, the synthetic and analytical aspects and the

biological properties. Particular focus is put on the respective mode of action and the

corresponding rationale behind covalent combinations of various bioactive and/or inactive

agents to increase their therapeutic potential. Future human clinical trials may lead to the

development of our new polyphenolic hybrid as new potential drugs for cancer

therapy/prevention. This rather recent approach of hybrid natural compounds has already

found applications in the development of new drugs, hence, it deserves to be further

developed and explored.

CHAPTER 6

- EXPERIMENTAL PART -

Chapter 6 – Experimental Part

197

6.1. MATERIALS

6.1.1. Analytical, chromatographic and biological techniques

Melting points were measured on Buchi B-540 equipment (located in Universidade de

Aveiro – Portugal) and are uncorrected.

NMR spectra were recorded on Bruker Avance 300 or 500 spectrometers (300.13 or

500.13 for 1H and 75.47 and 125.77 MHz for 13C) (located in Universidade de Aveiro -

Portugal), with CDCl3 as solvent if not stated otherwise and the internal standard was

TMS. Chemical shifts (δ) are reported in ppm and coupling constants (J) in Hz which all

are calculated using MESTRENOVA 8 (Free Trail License) analytical chemistry software

suite for NMR, LC, GC and MS. The signals are described as s (singlet), d (doublet), dd

(doublet of doublet), ddd (double of doublet of doublet), t (triplet), tt (triplet of triplet), q

(quartet), sept (septet) and m (multiplet). Unequivocal 13C assignments were made with

the aid of 2D HSQC, HMBC experiments (delays for one bond and long-range JC/H

couplings were optimized for 145 and 7 Hz, respectively). 3D optimizations/predictions of

structures are made using ACD/3D viewer (Freeware Version 11.01) and further

supported with the empirical data from the 2D-NMR NOESY experiments.

Single crystals of compounds 9a, 9c, 10a, 10c, 13a, 14q and 21f were manually

harvested from the crystallization vials and immediately immersed in highly viscous

FOMBLIN Y perfluoropolyether vacuum oil (LVAC 140/13, Sigma-Aldrich) to avoid

degradation caused by evaporation of the solvent, and mounted on Hampton Research

CryoLoops [1]. Alternatively, (Z)-11c was immersed in silicone grease (Dow Corning) and

mounted in a glass fiber. All the crystals were mounted with the help of a Stemi 2000

stereomicroscope equipped with Carl Zeiss lenses. Data were collected on a Bruker X8

Kappa APEX II CCD area-detector diffractometer (Mo Kα graphite-monochromated

radiation, λ = 0.71073 Å) (located in Universidade de Aveiro – Portugal) controlled by the

APEX2 software package [2] and equipped with an Oxford Cryosystems Series 700

cryostream monitored remotely using the software interface Cryopad [3]. Images were

processed using the software package SAINT+ [4] and data were corrected for absorption

by the multiscan semi-empirical method implemented in SADABS [5].

Structures were solved using the direct method algorithm implemented in SHELXS-97,

[6] allowing the immediate location of most of the C, N and O atoms. The remaining non-

hydrogen atoms were located from difference Fourier maps calculated from successive


198

full-matrix least-squares refinement cycles on F2 using SHELXL-97 [6a, 7]. All non-

hydrogen atoms were successfully refined using anisotropic displacement parameters.

Hydrogen atoms bound to carbon were placed at their idealized positions using

appropriate HFIX instructions in SHELXL: 33 for the terminal –CH3 methyl groups; 23 for

the –CH2– groups; 13 for –CH aliphatic group and 43 for the –CH aromatic moieties. All

hydrogen atoms were included in subsequent refinement cycles in riding motion

approximation with isotropic thermal displacements parameters (Uiso) fixed at 1.5 (only for

–CH3) or 1.2×Ueq of the parent atoms. Information concerning crystallographic data

collection and structure refinement details of all the discussed crystalline compounds are

summarized in Table 6.1.

Most of the crystallographic data (including structure factors) for the structures reported

in this dissertation have been deposited with the Cambridge Crystallographic Data Centre

as supplementary publication No. CCDC-890663 (for 9a) and CCDC-890665 through

890668 (for 9c, 10a, 10c and (Z)-11c) and the rest are under process. Copies of the data

can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2

2EZ, U.K. FAX: (+44) 1223 336033. E-mail: [email protected].

Table 6. 1. Crystal data collection and structure refinement details for compounds 9a, 9c, 10a, 10c, (Z)-11c, 13a, 14q and 21f

9a 9c 10a

Formula C23H28N2O4 C25H20N2O4 C25H34N2O5

Formula weight 396.47 412.43 442.54

Temperature / K 180(2) 150(2) 150(2)

Crystal system Monoclinic Monoclinic Triclinic

Space group P21/c C2/c Pī

a / Å 9.943(3) 23.3081(17) 9.8622(6)

b / Å 6.542(2) 12.7857(9) 10.4905(7)

c / Å 31.932(10) 29.534(3) 2.8334(9)

α / ° 90 90 88.463(4)

β / ° 90.82(2) 110.140(3) 75.403(3)

γ / ° 90 90 64.273(3)

Volume / Å3 2077.0(11) 8263.3(11) 1152.31(13)

Z 4 16 2

Dc/g cm-3 1.268 1.326 1.275

mailto:[email protected]


199

(Mo-K) / mm-1 0.087 0.091 0.089

Crystal size / mm 0.20 × 0.08 × 0.02 0.10 × 0.08 × 0.07 0.18 × 0.17 × 0.13

Crystal type Colorless needle Yellow block Colorless block

θ range (º) 3.66 to 27.48 3.64 to 25.35 3.72 to 33.14

Index ranges

-12 ≤ h ≤ 12

-8 ≤ k ≤ 7

-38 ≤ l ≤ 41

-26 ≤ h ≤ 28

-9 ≤ k ≤ 15

-35 ≤ l ≤ 35

-15 ≤ h ≤ 14

-15 ≤ k ≤ 16

-19 ≤ l ≤ 19

Reflections collected 14463 21499 40081

Independent reflections 4719

[Rint = 0.0617]

7525

[Rint = 0.0598]

8547

[Rint = 0.0309]

Completeness 98.70%

to θ=27.48º

99.60%

to θ=25.35º

97.10%

to θ=33.14º

Final R indices [I>2σ(I)]

a,b

R1 = 0.0517

wR2 = 0.1043

R1 = 0.0496

wR2 = 0.1022

R1 = 0.0527

wR2 = 0.1381

Final R indices

(all data) a,b

R1 = 0.1421

wR2 = 0.1326

R1 = 0.1027

wR2 = 0.1223

R1 = 0.0738

wR2 = 0.1518

Weighting schemec m = 0.0545

n = 0.468

m = 0.0513

n = 1.0878

m = 0.0725

n = 0.3821

Largest diff. peak and

hole

0.222 and

-0.197 e.Å-3

0.275 and

-0.241 e.Å-3

0.804 and

-0.721 e.Å-3

CCDC no. 890663 890665 890666

Table 6.1. (Cont.)

10c (Z)-11c

Formula C25H20N2O4 C25H18N2O4

Formula weight 412.43 410.41

Temperature / K 150(2) 296(2)

Crystal system Monoclinic Monoclinic

Space group P21/n P21/n

a / Å 19.5879(8) 8.2827(5)

b / Å 16.8908(6) 7.9470(10)

c / Å 26.0908(11) 13.7810(6)

α / ° — —

β / ° 101.805(2) 93.712(3)

γ / ° — —


200

Volume / Å3 8449.7(6) 2044.24(19)

Z 16 4

Dc/g cm-3 1.297 1.334

(Mo-K) / mm-1 0.089 0.092

Crystal size / mm 0.12 × 0.08 × 0.05 0.10 × 0.10 × 0.10

Crystal type Colourless block Yellow block

θ range (º) 3.57 to 25.35 3.60 to 25.35

Index ranges

-23 ≤ h ≤ 23

-20 ≤ k ≤ 12

-31 ≤ l ≤ 31

-9 ≤ h ≤ 9

-21 ≤ k ≤ 19

-16 ≤ l ≤ 16

Reflections collected 45763 14509


[Rint = 0.0446]

3705

[Rint = 0.0406]

Completeness to θ=25.35º 99.10% 99.10%

Final R indices

[I>2σ(I)] a,b

R1 = 0.0499

wR2 = 0.1009

R1 = 0.0781

wR2 = 0.2411

Final R indices

(all data) a,b

R1 = 0.1143

wR2 = 0.1245

R1 = 0.1000

wR2 = 0.2561


n = 0.1225

m = 0.0072

n = 2.1663

Largest diff. peak and hole 0.332 and -0.221 e.Å-3 0.282 and -0.238 e.Å-3

CCDC no. 890667 890668

Table 6.1. (Cont.)

13a 21f 14t

Formula C18H12O4 C24H16O4 C27H22O6

Formula weight 292.28 368.37 442.45

Temperature / K 150(2) 150(2) 150(2)

Crystal system Orthorrhombic Orthorhombic Triclinic

Space group P n a 21 P n a 21 P -1

a / Å 23.420(3) 7.4992(3) 7.0483(2)

b / Å 4.9710(5) 19.5793(8) 12.0904(4)

c / Å 11.6351(13) 12.0443(5) 13.3284(10)

Volume / Å3 1354.6(2) 1768.45(12) 106.4800(10)

Z 4 4 2


201

Dc/g cm-3 1.433 1.384 1.371

(Mo-K) / mm-1 0.102 0.094 0.097

Crystal size / mm 0.12×0.10×0.02 0.09×0.07×0.03 0.13×0.08×0.06

Crystal type Yellow plate Colourless

block Yellow block

θ range (º) 1.74 to 29.60 1.99 to 29.13 2.73 to 29.13

Index ranges

-26 ≤ h ≤ 32

-6 ≤ k ≤ 6

-16 ≤ l ≤ 10

-10≤ h ≤ 10

-25 ≤ k ≤ 26

-16 ≤ l ≤ 16

-9 ≤ h ≤ 9

-16 ≤ k ≤ 16

-18 ≤ l ≤ 18

Reflections collected 8110 17037 18369


[Rint = 0.0897]

4730

[Rint = 0.0381]

5748 [Rint =

0.0231]

Completeness to θ=29.60º 99.2 % 100.0 % 99.5 %

Final R indices [I>2σ(I)] a,b R1 = 0.0495

wR2 = 0.0844

R1 = 0.443

wR2 = 0.0847

R1 = 0.0439 wR2 = 0.1126

Final R indices

(all data) a,b

R1 = 0.897

wR2 = 0.0968

R1 = 0.0693

wR2 = 0.0938

R1 = 0.0604

wR2 = 0.1228


n = 0.0000

m = 0.0424

n = 0.1543

m = 0.0536

n = 0.3800

Largest diff. peak and hole 0.315 and

-0.243 eÅ-3

0.272 and

-0.223 eÅ-3

0.372 and

-0.192 eÅ-3

a 1 /o c oR F F F

b 2 2

2 2 22 /o c owR w F F w F

c 22 21/ ow F mP nP

where 2 22 /3o cP F F

R1 = 0.0604 wR2 = 0.1228

m=0.0536

n=0.3800

0.372 and

-0.192 eÅ-3


202

Exact mass measurements were recorded on high resolution mass spectrometer

micrOTOF-Q (located in Université de Nancy - France) and elemental analysis on

Truspec 630-200-200 equipments (located in Universidad de Vigo - Spain).

HPLC analyses were performed on GILSON apparatus (located in Universidade de

Aveiro – Portugal) under conditions cited with the corresponding data of the analyzed

compound.

Column chromatography and preparative thin layer chromatography were performed

with Merck silica gel 60, 70-230 mesh. Analytical thin layer chromatography for reaction

monitoring were realized on pre-coated Merck silica gel plates.

Biological studies on cytotoxicity and proliferation of Leukemic cell lines (K562) using

trypan blue assay, NF-κB inhibition by luciferase reporter gene assay and Western Blot

and in vitro total HDAC activity (classes I, II and IV) measured using proteins extracted

from K-562 cells, have all been conducted by biologists from Laboratoire de Biologie

Moléculaire du Cancer, Hôpital Kirchberg, (Luxembourg) (website: http://www.rsl.lu).

The anti-carcinogenic potential examined by measuring the potential to induce the

cytoprotective Nrf2 (nuclear factor E2-related protein 2) signalling pathway and the ability

to inhibit proliferation of cancer cells utilizing MCF7 breast cancer cells stably transfected

with an ARE-dependent luciferase reporter, have been carried out by biologist from

Epigenomics and Cancer Risk Factors, Redox Regulation, DKFZ, Heidelberg (Germany)

(website: http://www.dkfz.de/en/index.html).

The Cdc25 phosphatases inhibition along with DPPH and FRAP antioxidant potentials

in vitro evaluations have been performed by biologists form Laboratoire d’Ingénierie

Moléculaire et Biochimie Pharmacologique, Metz, (France) (website: http://www.univ-

metz.fr/recherche/labos/limbp).

6.1.2. Chemicals and starting materials

Chromone-2-carboxylic acid 1, chromone-3-carboxylic acid 2, carbodiimides 3a-c, 4-

hydroxycoumarin 6, triacetic acid lactone 7 and 4-pyrrolidinopyridine (4-PPy) were

purchased from Sigma-Aldrich. All other chemicals and solvents used were purchased

from commercial sources. 1,3-Dicarbonyl compounds 4a-t have been prepared via the

Baker-Verkataraman method [8]. Chromone-3-carbaldehydes 5a-e have been prepared

according to the Vilsmeier-Haack reaction [9], Chalcones 8a-f have been prepared via the

aldol-condensation reaction [10].

http://www.rsl.lu/

http://www.dkfz.de/en/index.html

http://www.univ-metz.fr/recherche/labos/limbp)

http://www.univ-metz.fr/recherche/labos/limbp)


203

6.2. EXPERIMENTAL METHODS

6.2.1. Synthesis of 1’,3’-disubstituted spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones 9a-c.

Chromone-2-carboxylic acid 1 (2 g, 10.52 mmol) was added to a solution of

carbodiimides 3a-c (10.52 mmol, 1 equiv) in dichloromethane (20 mL), followed by the

addition of a catalytic amount of 4-PPy (0.52 mmol, 0.08 g, 0.05 equiv). The resulting

mixture was allowed to stirring overnight at room temperature. After that, the solvent is

evaporated to dryness and the resulting resinous solid was directly recrystallized from

ethanol to afford compounds 9a-c.

9a: (R/S)-1’,3’-dicyclohexylspiro[chroman-2,4'-imidazolidine]-2',4,5'-trione,

C23H28N2O4 (white crystalline solid, 3.32 g, yield 80%; Mp = 189-190°C, Mp(lit[11]) = 188°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.10-2.16 (m, 20H, -CH2-

cyclohexyl), 2.89 (d, J = 16.7 Hz, 1H, H-3(A)), 3.30 (d, J = 16.7

Hz, 1H, H-3(B)), 3.33 (tt, J = 12.2, 3.8 Hz, 1H, H-1”’), 3.83 (tt, J

= 12.2, 3.8 Hz, 1H, H-1”), 6.96 (dd, J = 8.3, 0.8 Hz, 1H, H-8),

7.05-7.11 (m, 1H, H-6), 7.50 (ddd, J = 8.3, 7.2, 1.6 Hz, 1H, H-

7), 7.88 (dd, J = 7.8, 1.6 Hz, 1H, H-5) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 24.9, 25.0, 25.68, 25.71, 26.15, 26.15, 29.29,

29.33, 30.8 and 31.0 (–CH2-, cyclohexyl), 41.3 (C-3), 51.6 (C-1”), 54.0 (C-1”’), 88.5 (C-2),

117.6 (C-8), 119.8 (C-10), 122.3 (C-6), 126.2 (C-5), 136.3 (C-7), 153.8 (C-2’), 158.4 (C-9),

168.7 (C-5’), 188.7 (C-4) ppm. HRMS (ESI+), m/z calcd for [C23H28N2O4+Na]+: 419.1947;

found: 419.1939. Anal Calcd for C23H28N2O4: C 69.67, H 7.12, N 7.07. Found: C 69.36, H

7.10, N 7.11%.

9b: (R/S)-1’,3’-diisoporpylspiro[chroman-2,4'-imidazolidine]-2',4,5'-trione,

C17H20N2O4 (white crystalline solid, 2.20 g, yield 66 %, Mp = 157-158°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.39 (d, J = 6.9 Hz, 6H, H-

2”), 1.46 (d, J = 6.9 Hz, 3H, H-2”’), 1.51 (d, J = 6.9 Hz, 3H, H-

2”’), 2.93 (d, J = 16.7 Hz, 1H, H-3(A)), 3.27 (d, J = 16.7 Hz, 1H,

H-3(B)), 3.77 (sept, J = 6.9 Hz, 1H, H-1”’), 4.26 (sept, J = 6.9

Hz, 1H, H-1”), 6.97 (dd J = 8.3, 0.9 Hz, 1H, H-8), 7.04-7.12 (m,

1H, H-6), 7.51 (ddd, J = 8.3, 7.3, 1.8 Hz, 1H, H-7), 7.88 (dd, J = 7.8, 1.8 Hz, 1H, H-5)

ppm. 13C NMR (75.47 MHz, CDCl3): δ = 19.57 and 19.63 (C-2”), 20.96 and 21.03 (C-2”’),

1

2

34

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78

9

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204

41.0 (C-3), 43.9 (C-1”), 45.9 (C-1”’), 88.6 (C-2), 117.5 (C-8), 119.7 (C-10), 122.3 (C-6),

126.3 (C-5), 136.4 (C-7), 153.6 (C-2’), 158.4 (C-9), 168.6 (C-5’), 188.5 (C-4) ppm. HRMS

(ESI+), m/z calcd for [C17H20N2O4+Na]+: 339.1321; found: 339.1324. Anal Calcd for

C17H20N2O4: C 64.54, H 6.37, N 8.86. Found: C 64.49, H 6.38, N 8.91%.

9c: (R/S)-1’,3’-ditolylspiro[chroman-2,4'-imidazolidine]-2',4,5'-trione, C25H20N2O4 (pale

yellow crystalline solid, 3.80 g, yield 88 %, Mp = 182-183°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.33 and 2.35 (s, 6H, 4”-

CH3 and 4”’-CH3), 3.14 (d, J = 16.9 Hz, 1H, H-3(A)), 3.22 (d,

J = 16.9 Hz, 1H, H-3(B)), 6.98-7.09 (m, 2H, H-6, H-8), 7.15-

7.38 (m, 8H, tolyl), 7.44-7.53 (m, 1H, H-7), 7.76 (dd, J = 7.8,

1.7 Hz, 1H, H-5) ppm. 13C NMR (75.47 MHz, CDCl3): δ =

21.06 and 21.08 (4”-CH3 and 4”’-CH3), 41.1 (C-3), 89.2 (C-

2), 117.4 (C-8), 119.8 (C-10), 122.4 (C-6), 125.5 and 128.4

(C-2” and C-2”’), 126.2 (C-5), 127.9 and 129.5 (C-1” and C-

1”’), 129.6 and 130.1 (C-3” and C-3”’), 136.3 (C-7), 138.4 and 139.1 (C-4” and C-4”’),

153.1 (C-2’), 158.0 (C-9), 167.1 (C-5’), 187.8 (C-4) ppm. HRMS (ESI+), m/z calcd for

[C25H20N2O4+Na]+: 435.1321; found: 435.1309. Anal Calcd for C25H20N2O4: C 72.80, H

4.89, N 6.79. Found: C 72.75, H 5.05, N 6.89%.

6.2.2. Synthesis of 1,3-disubstituted 5-(3-oxo-2,3-dihydrobenzofuran-2-

yl)imidazolidine-2,4-diones 10a-c

A solution of sodium (10.52 mmol, 0.242 g) in ethanol (5 mL) was added dropwise for

15 minutes to a solution of 1’,3’-disubstituted spiro[chroman-2,4'-imidazolidine]-2',4,5'-

triones 9a-c (10.52 mmol) in ethanol (20 mL), which was placed in a an ice-water bath (0

°C). The reaction is left for 1 hour to reach room temperature under stirring. After TLC

monitoring, the ethanolic solution is then poured in ice and water to be neutralized to pH 7

with diluted hydrochloric acid. A yellowish white precipitate appears which is purified by

silica gel column chromatography using a (1:1) of light petroleum:dichloromethane as

eluent. The resulting pure compounds were recrystallized from ethanol to afford

compounds 10a-c. In the case of 9c reaction, the chalcone-like compound 10c(a) was the

first eluted from the column chromatography, isolated and recrystallized from ethanol.

1

2

34

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205

10a: (2’R,5S)/(2’S,5R)-1,3-dicyclohexyl-5-(3-oxo-2,3-dihydrobenzofuran-2-

yl)imidazolidine-2,4-dione, C23H28N2O4 (white crystalline solid, 2.38 g, yield 57 %, Mp =

154-155°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.23-2.03 (m, 20H, -

CH2- cyclohexyl), 3.67 (tt, J = 12.0, 4.1 Hz, 1H, H-1”), 3.81

ppm (tt, J = 12.2, 3.8 Hz, 1H, H-1”’), 4.56 (d, J = 2.0 Hz,

1H, H-5), 4.90 (d, J = 2.0 Hz, 1H, H-2’), 7.07 (d, J = 8.5

Hz, 1H, H-7’), 7.11-7.17 (m, 1H, H-5’), 7.61 (ddd, J = 8.5,

7.3, 1.5 Hz, 1H, H-6’), 7.69-7.74 (m, 1H, H-4’) ppm. 13C

NMR (75.47 MHz, CDCl3): δ = 24.9, 25.4, 25.71, 25.74, 25.76, 25.82, 29.8, 29.2, 30.3 and

31.4 (-CH2-, cyclohexyl), 51.8 (C-1”’), 54.1 (C-1”), 60.1 (C-5), 82.3 (C-2’), 112.9 (C-7’),

121.7 (C-9’), 122.7 (C-5’), 124.3 (C-4’), 138.0 (C-6’), 156.2 (C-2), 168.1 (C-4), 172.5 (C-

8’), 196.9 (C-3’) ppm. HRMS (ESI+), m/z calcd for [C23H28N2O4+Na]+: 419.1947; found:

419.1947.

10b: (2’R,5S)/(2’S,5R)-1,3-diisopropyl-5-(3-oxo-2,3-dihydrobenzo-furan-2-

yl)imidazolidine-2,4-dione, C17H20N2O4 (white amorphous solid, 1.574 g, yield 47 %, Mp

= 122°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.30, 1.32 and 1.35 (d,

J = 7.0 Hz, 12H, H-2” and H-2”’), 4.10 ppm (sept, J = 7.0

Hz, 1H, H-1”), 4.22 ppm (sept, J = 7.0 Hz, 1H, H-1”’), 4.55

(d, J = 2.0 Hz, 1H, H-5), 4.89 ppm (d, J = 2.0 Hz, 1H, H-2’),

7.08 (dd, J = 8.4, 0.7 Hz, 1H, H-7’), 7.11-7.18 (m, 1H, H-

5’), 7.58-7.65 (m, 1H, H-6’), 7.70-7.74 (m, 1H, H-4’) ppm.

13C NMR (75.47 MHz, CDCl3): δ = 19.2, 19.5, 19.9 and 21.1 (C-2’ and C-2”’), 44.2 (C-1”’),

46.0 (C-1”), 59.9 (C-5), 82.2 (C-2’), 112.9 (C-7’), 121.7 (C-9’), 122.8 (C-5’), 124.4 (C-4’),

138.0 (C-6’), 156.0 (C-2), 168.0 (C-4), 172.5 (C-8’), 196.8 (C-3’) ppm. HRMS (ESI+), m/z

calcd for [C17H20N2O4+Na]+: 339.1324; found: 339.1318. Anal Calcd for C17H20N2O4: C

64.54, H 6.37, N 8.86. Found: C 64.49, H 6.38, N 8.91%.

1'

2'3' 5

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9' 3

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206

10c: (2’R,5S)/(2’S,5R)-1’,3’-ditolyl-5-(3-oxo-2,3-dihydrobenzofuran-2-

yl)imidazolidine-2,4-dione, C25H20N2O4 (white crystalline solid, 2.74 g, yield 63 %, Mp =

181-183°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.34 and 2.36 (s,

6H, 4’’-CH3, 4’’’-CH3 ), 4.93 (d, J = 1.9 Hz, 1H, H-2’),

5.34 (d, J = 1.9 Hz, 1H, H-5), 7.00 (d, J = 8.5 Hz, 1H,

H-7’), 7.06-7.13 (m, 1H, H-5’), 7.18-7.29 and 7.35-7.40

(m, 8H, tolyl), 7.55 (ddd, J = 8.5, 7.3, 1.5 Hz, 1H, H-6’),

7.69 (d, J = 7.7 Hz, 1H, H-4’) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 20.92 and 21.15 (4”-CH3, 4”’-CH3),

62.1 (C-5), 80.6 (C-2’), 113.0 (C-7’), 121.5 (C-9’),

122.7 (C-5’), 123.5 and 126.0 (C-2” and C-2”’), 124.3 (C-4’), 128.5 and 131.8 (C-1” and C-

1”’), 129.7 and 129.9 (C-3” and C-3”’), 136.6 and 138.5 (C-4” and C-4”’), 138.1 (C-6’),

153.6 (C-2), 166.2 (C-4), 172.6 (C-8’), 196.8 (C-3’) ppm. HRMS (ESI+), m/z calcd for

[C25H20N2O4+Na]+: 435.1321; found:. 435.1329.

10c(a): (Z)-1,3-ditolyl-5-[2-(2-hydroxyphenyl)-2-oxoethylidene]imidazolidine-2,4-

dione, C25H20N2O4 (yellow amorphous solid, 0.22 g, yield 5%, Mp = 223-224°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.31 and 2.41 (s, 6H,

4’-CH3 and 4”-CH3), 6.86-6.93 (m, 2H, H-3””, H-5””), 6.93

(s, 1H, H-1”’), 7.01-7.07, 7.30-7.35 and 7.37-7.41 (m, 8H,

tolyl), 7.44-7.48 (m, 1H, H-4””), 7.71 (dd, J = 8.0, 1.6 Hz,

1H, H-6””), 11.31 (s, 1H, 2””-OH) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 21.1 and 21.2 (4’-CH3 and 4”-CH3), 104.3 (C-1”’), 118.2 (C-3””), 119.0

(C-5””), 120.2 (C-1””), 125.7 and 126.2 (C-2’ and C-2”), 128.2 (C-1’ or C-1”), 129.7 and

129.9 (C-3’ and C-3”), 131.0 (C-6””, C-1” or C-1’), 135.1(C-5), 137.0 (C-4””), 138.8 (C-4’

and C-4”),152.9 (C-2), 161.7 (C-4), 162.3 (C-2””), 194.5 (C-2’’’) ppm. HRMS (ESI+), m/z

calcd for [C25H20N2O4+Na]+: 435.1321; found: 435.1329. Anal Calcd for C25H20N2O4: C

72.80, H 4.89, N, 6.79. Found: C 72.71, H 4.89, N 6.84%.

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207

6.2.3. Synthesis of 1,3-disubstituted 5-[3-oxobenzofuran-2(3H)-

ylidene]imidazolidine-2,4-dione 11a-c

Iodine (0.068 g dissolved in 1 mL of DMSO) was added to a solution of 1,3-

disubstituted 5-(3-oxo-2,3-dihydrobenzofuran-2-yl)imidazolidine-2,4-diones 10a-c (5.26

mmol) in DMSO (3 mL) and the reaction mixture was refluxed under nitrogen flow and

away from intense light for 30 minutes. After TLC monitoring, the reaction solution is

poured into ice (10 g) and water (20 mL), an intense yellow precipitate appears which is

filtrated and washed with water and then extracted with dichloromethane (3 x 50 mL). The

extract was washed again with a saturated solution of sodium thiosulfate (2 x 200 ml) and

finally purified by silica gel column chromatography using a (1:1) mixture of light petroleum

and dichloromethane as eluent. The resulting pure compound was recrystallized from

ethanol to give compounds 11a-c. In the case of 10a, the undesired compound 11a(a)

(1,3-dicyclohexyl parabanic acid and 11a(b) (salicylic acid which was compared to

authentic sample and 1H-NMR data) have been obtained through preparative TLC

separation along with (Z)-11a. In the case of 10c, two isomers (Z)-11c and (E)-11c were

jointly obtained in a 3:1 ratio (evaluated by NMR proton integration and confirmed by

HPLC analysis: Gilson HPLC conditions: Column: Silica Gel, Mobile phase: Hexane/THF

(80:20), Flow rate: 1 mL/min, UV-visible detection: λ = 254; Analysis: (E)-11c Retention

Time = 10.4 min, (Z)-11a Retention Time = 14.5 min.). Subsequently, the two isomers are

separated and isolated by preparative TLC using dichloromethane as eluent, being (E)-

11c the first eluted and recrystallized from ethanol, while (Z)-11c was recrystallized from

toluene. It is strongly recommended to work away from intense light since these

compounds are photosensitive.

(Z)-11a: (Z)-1,3-dicyclohexyl-5-[3-oxobenzofuran-2(3H)-ylidene]imidazolidine-2,4-

dione, C23H26N2O4 (yellow amorphous solid, 1.36 g, yield 65 %, Mp = 203-204°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.26-2.31 (m, 20H, -CH2-,

cyclohexyl), 4.02 (tt, J = 12.3, 3.5 Hz, 1H, H-1”), 4.63 (tt, J =

12.0, 3.6 Hz, 1H, H-1’”), 7.22-7.29 (m, 2H, H-5’, H-7’), 7.61-

7.68 (m, 1H, H-6’), 7.82-7.85 (m, 1H, H-4’) ppm. 13C NMR

(75.47 MHz, CDCl3): δ = 25.0, 25.1, 25.82, 25.85, 26.0, 26.3,

29.24, 29.29, 30.5 and 30.6 (-CH2-, cyclohexyl), 52.2 (C-1”’),

60.4 (C-1”), 113.2 (C-7’), 121.7 (C-2’), 123.5 (C-5’), 124 (C-9’), 124.3 (C-4’), 136.2 (C-5),

137.0 (C-6’), 153.3 (C-2), 161.6 (C-4), 165.9 (C-8’), 183.5 (C-3’) ppm. HRMS (ESI+), m/z

calcd for [C23H26N2O4+Na]+: 417.1790; found: 417.1792.

1'

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5

1

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6'7'

8'

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208

(Z)-11b: (Z)-1,3-diisopropyl-5-[3-oxobenzofuran-2(3H)-ylidene]imidazolidine-2,4-

dione, C17H18N2O4 (yellow amorphous solid, 0.58 g, yield 35 %, Mp = 135-137 °C).

1H NMR (300.13 MHz, CDCl3): δ = 1.45 and 1.58 (d, J = 6.9 Hz,

12H, H-2” and H-2”’), 4.44 ppm (sept, J = 6.9 Hz, 1H, H-1”’),

4.22 ppm (sept, J = 6.9 Hz, 1H, H-1”), 7.23-7.29 (m, 2H, H-7’,

H-5’), 7.62-7.68 (m, 1H, H-6’), 7.82-7.86 (m, 1H, H-4’) ppm. 13C

NMR (75.47 MHz, CDCl3): δ = 19.6 and 21.8 (C-2’ and C-2”’), 44.8 (C-1”’), 48.3 (C-1”),

112.3 (C-7’), 121.7 (C-5), 122.3 (C-9’), 124.0 (C-5’), 125.0 (C-4’), 135.0 (C-2’), 136.3 (C-

6’), 153.7 (C-2), 159.6 (C-4), 163.5 (C-8’), 180.5 (C-3’) ppm. HRMS (ESI+), m/z calcd for

[C17H18N2O4+Na]+: 337.1164; found: 337.1156.

(Z)-11c: (Z)-1,3-ditolyl-5-[3-oxobenzofuran-2(3H)-ylidene]imidazolidine-2,4-dione:

C25H18N2O4 (yellow crystalline solid, 1.35 g, yield 62 %, Mp = 232°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.39 and 2.46 (s, 6H, H-

4”-CH3 and 4”’-CH3,), 6.74 (dd, J = 8.9, 0.6 Hz, 1H, H-7’),

7.13-7.20 (m, 1H, H-5’), 7.24-7.29 and 7.37-7.42 (m, 8H,

tolyl), 7.46-7.54 (m, 1H, H-6’), 7.74-7.77 (m, 1H, H-4’) ppm.

13C NMR (75.47 MHz, CDCl3): δ = 21.1 and 21.2 (4”-CH3

and 4”’-CH3), 112.4 (C-7’), 119.7 (C-5), 121.7 (C-9’), 123.9

(C-5’), 124.7 (C-4’), 125.9 and 127.6 (C-2” and C-2”’), 128.3 and 131.4 (C-1” and C-1”’),

129.3 and 129.7 (C-3” and C-3”’), 136.40 (C-2’), 136.43 (C-6’), 138.5 and 138.9 (C-4” and

C-4”’),152.8 (C-2), 158.3 (C-4), 163.9 (C-8’), 180.5 (C-3’) ppm. HRMS (ESI+), m/z calcd

for [C25H18N2O4+Na]+: 433.1164; found:. 433.1171. Anal Calcd for C25H18N2O4: C 73.16, H

4.42, N 6.83. Found: C 73.23, H 4.52, N 6.70%.

(E)-11c: (E)-1,3-ditolyl-5-[3-oxobenzofuran-2(3H)-ylidene]imidazolidine-2,4-dione:

C25H18N2O4 (yellow amorphous solid, 0.46 g, yield 21 %, Mp = 208-210°C).


CH3 and 4”’-CH3), 7.15-7.21 (m, 1H, H-5’), 7.28-7.35 and

7.37-7.42 (2m, 9H, H-7’ and tolyl), 7.58-7.68 (m, 2H, H-6’,

H-4’) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 21.2 and 21.3

(4”-CH3 and 4”’-CH3), 112.4 (C-7’), 118.8 (C-5), 121.8 (C-9’),

124.0 (C-5’), 124.8 (C-4’), 126.0 and 127.7 (C-2” and C-2”’),

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1

5'

6'7'

8'

9' 3

2

44'

1''

1'''

O

O

N

NO

O

1'

2'3'

5

1

5'

6'7'

8'

9' 3

2

4

4'

1''

1'''

O

O

N

NO

O


209

128.3 and 131.5 (C-1” and C-1”’), 129.3 and 129.7 (C-3” and C-3”’), 136.5 (C-6’), 137.0

(C-2’), 138.6 and 139.0 (C-4” and C-4”’), 152.9 (C-2), 164.0 (C-8’), 180.6 (C-4), 191.3 (C-

3’) ppm. HRMS (ESI+), m/z calcd for [C25H18N2O4+Na]+: 433.1164; found:. 433.1171.

11a(a): 1,3-dicyclohexyl parabanic acid, C15H22N2O3 (white crytalline solid, MW: 278,35

g/mol, 0.249 g, yield 17 %, Mp = 175°C, Mp(Lit[12]) 174-175°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.14-1.26 and 1.63-1.71 (2 m, 4H, H-4′),

1.26-1.43 and 1.80-1.91 (2 m, 4H, H-3′), 1.73-1.77 and 1.97-2.19 (2 m, 4H,

H-2′), 4.00 (tt, J = 12.0 and 3.7 Hz, 2H, H-1′) ppm. 13C NMR (75.47 MHz,

CDCl3): δ = 24.7 (C-4′), 25.6 (C-3′), 29.5 (C-2′), 52.4 (C-1′), 153.4 (C-2),

156.4 (C-4,5) ppm. HRMS (ESI+): m/z Calcd for [C15H22N2O3 + Na]+:

301.1528; found 301.1523. Anal Calcd: C, 64.73; H, 7.97; N, 10.06; found:

C, 64.47; H, 7.96; N, 10.05.

Kinetic study of compound (E/Z)-11c photoisomerization: HPLC-UV detection was

utilized to study the EZ and ZE photoisomerization kinetics of compound 11c. The

evaluation of (E)-11c and (Z)-11c concentrations at various light exposure times (t ≠ 0) is

based on the chromatographic peak area of each isomer detected by UV absorption (at λ

= 254 nm). We assume that (E)-11c and (Z)-11c are isolated with enough purity, so that

their corresponding peak area at t = 0 corresponds to 100% pure isomer. [E]0 or [Z]0 are

the initial concentrations of (E)-11c or (Z)-11c solution prepared in dichloromethane at t =

0 (analyzed before exposed to light); [E]t or [Z]t are the concentrations of (E)-11c or (Z)-

11c at different time (t ≠ 0 when exposed to light) which could be calculated from the

percentage of transformation E (%) or Z (%) determined by AE or AZ peak areas ratio of

(E)-11c or (Z)-11c peaks in the chromatogram at different stage of transformation (Table

6.2, Fig. 2.25 of chapter II). After determination of molar extinction coefficient εE and εZ,

which are equal at λmax = 254 nm (εE= 11,651 mM-1 cm-1, εZ= 11,666 mM-1 cm-1), we have

therefore :

x 100 or

x 100

Sunlight was initially used as a source of irradiation, however it causes

nonhomogeneous system because of continuous variation of light intensity (clouds

shadow, sunlight orientation), the kinetic study of E/Z transformation was difficult to be

established under these conditions. For such systematic reasons, we designed an

N

N

O

O

O

1

23

4

5

1'2'

3'4'


210

homogeneous isotherm system (35 °C) using an electrical lamp as light source providing

the same photo-intensity during the whole period of study.

Table 6. 2. E/Z photoisomeric equilibrium kinetic study of 11c compound

ZE

Time (min) Z (%) E (%) [Z]t (g L-1

)

0 100,00 0,00 0,50 [Z]0

16 91,07 8,93 0,459

31 87,60 12,40 0,443

46 86,06 13,94 0,430

61 84,36 15,64 0,422

78 83,42 16,58 0,417

92 83,08 16,92 0,415

106 82,69 17,31 0,41

138 82,42 17,58 0,41

248 82,34 17,66 0,41

400 82,10 17,90 0,41

6.2.4. Synthesis of N,N-disubstituted-(carbamoyl)-4-oxo-4H-chromene-3-

carboxamide 12a(a) and 12b(a)

Chromone-3-carboxylic acid 2 (1 g, 5.26 mmol) was added to 3a-b (5.26 mmol, 1

equiv) in chloroform (20 mL) and the reaction mixture is brought to reflux for 30 minutes.

After that, the solvent is evaporated and the resulting resinous solid is directly

recrystallized from ethanol to afford compound 12a(a) and 12b(a). In case of 12b(a) the

recrystallization solvent is a mixture of light petroleum and ethyl acetate (5:1).

12a(a): N-cyclohexyl-N-(cyclohexylcarbamoyl)-4-oxo-4H-chromene-3-carboxamide,

C23H28N2O4 (white solid, MW = 396.48 g/mol, 1.550 g, yield 74 %, Mp = 185°C).

1H NMR (300.13 MHz, CDCl3): δ = 0.87-2.01 (m, 20H, -

CH2- cyclohexyl), 3.40-3.53 (m, 1H, H-1”’), 4.19 (tt, J =

11.7, 3.8 Hz, 1H, H-1”), 6.49 (d, J = 7.2 Hz, 1H, 4’-NH),

7.42-7.53 (m, 2H, H-6, H-8), 7.73 (ddd, J = 8.7, 7.1, 1.7

EZ

Time (min) Z (%) E (%) [E]t (g L-1

)

0 0 100 0,50

6 5,88 94,12 0,47

16 29,50 70,50 0,35

34 47,73 52,27 0,26

49 58,57 41,43 0,21

64 66,09 33,91 0,17

79 71,49 28,51 0,14

94 75,57 24,43 0,12

114 77,73 22,27 0,11

120 79,68 20,32 0,10

136 80,28 19,72 0,10

152 80,10 19,90 0,10

235 80,53 19,47 0,10

400 80,07 19,93 0,10

12

3

3'

2'1'

4'

9

10

4

5

6

7

8

N

O O

O

HN

O

1"

1"'


211

Hz, 1H, H-7), 8.07 (s, 1H, H-2), 8.22 (dd, J = 8.0, 1.7 Hz, 1H, H-5) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 24.4, 25.19, 25.26, 26.0, 30.7 and 32.1 (–CH2-, cyclohexyl), 49.7 (C-1”’),

55.7 (C-1”), 118.3 (C-8), 123.9 (C-3), 124.1 (C-10), 125.9 (C-6, C-5), 134.5 (C-7), 153.2

(C-3’), 154.2 (C-2), 156.1 (C-9), 162.3 (C-1’), 175.4 (C-4) ppm. MS (ESI+): m/z calcd for

[C23H28N2O4 + Na]+: 419.19; found: 419.2. Anal. Calcl: C, 69.67; H, 7.12; N, 7.07. Found:

C, 69.69; H, 7.14; N, 7.07.

12b(a): N-isopropyl-N-(isopropylcarbamoyl)-4-oxo-4H-chromene-3-carboxamide,

C17H20N2O4 (white solid, MW = 316.35 g/mol, 1.324 g, yield 80 %, Mp = 137-138°C).

1H NMR (300.13 MHz, CDCl3): δ = 0.99 (d, J = 6.6 Hz,

6H, H-2”’, -CH3), 1.42 (d, J = 6.8 Hz, 6H, H-2”, -CH3),

3.72-3.89 (m, 1H, H-1”’), 4.50 (sept, J = 6.8 Hz, 1H, H-1”),

6.74 (br, 1H, 4’-NH), 7.42-7.54 (m, 2H, H-6, H-8), 7.74

(ddd, J = 8.6, 7.1, 1.7 Hz, 1H, H-7), 8.08 (s, 1H, H-2), 8.23 (dd, J = 8.0, 1.7 Hz, 1H, H-5)

ppm. 13C NMR (75.47 MHz, CDCl3): δ = 20.7 (C-2”, -CH3), 21.9 (C-2”’, -CH3), 42.8 (C-1”’),

48.8 (C-1”), 118.3 (C-8), 124.1 (C-3), 124.2 (C-10), 126.0 (C-6, C-5), 134.5 (C-7), 153.2

(C-3’), 154.2 (C-2), 156.1 (C-9), 163.4 (C-1’), 175.1 (C-4) ppm. MS (ESI+): m/z calcd for

[C17H20N2O4 + Na]+: 339.13; found: 339.1. Anal. Calcl: C, 64.54; H, 6.37; N, 8.86. Found:

C, 64.51; H, 6.34; N, 8.84.

6.2.5. Synthesis of 1,3-disubstituted-5-(2-hydroxybenzoyl)pyrimidine-2,4(1H,3H)-

dione 12a-c

Chromone-3-carboxylic acid 2 (1 g, 5.26 mmol) was added to 3a-b (5.26 mmol, 1

equiv) in chloroform (20 mL). The reaction mixture is brought to reflux for 30 minutes.

After complete consumption of 2 and formation of 12a(a) and 12b(a) as controlled by TLC

analysis, 4-PPy is added (0.04 g, 0.05 equiv) to the reaction mixture which is allowed to

reflux for further 30 minutes. After TLC monitoring, the solvent is evaporated and the

resulting resinous solid is directly recrystallized from ethanol to afford compound 12a-b.

Note: The isolated 12a(a) and 12b(a) compounds have also been used to produce 12a-b

in similar conditions (catalytic 4-PPy in chloroform), however, the procedure occurs in

lower yields (10 to 20% loss) which is due to the isolation and purification

(recrystallization) steps.

12

33'

2'1'

4'

9

104

5

6

7

8

N

O O

O

HN

O

1"

1"'

2"'

2"'


212

12a: 1,3-dicyclohexyl-5-(2-hydroxybenzoyl)pyrimidine-2,4(1H,3H)-dione, C23H28N2O4

(white solid, MW = 396.48 g/mol, 1.342 g, yield 64 %, Mp = 80°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.07-2.04 and 2.35-2.48

(m, 20H, -CH2- cyclohexyl), 4.46-4.58 (m, 1H, H-1’), 4.84 (tt, J

= 12.2, 3.7 Hz, 1H, H-1”), 6.85-6.91 (m, 1H, H-5””), 6.97-7.02

(m, 1H, H-3””), 7.44-7.53 (m, 2H, H-4””, H-6””), 7.71 (s, 1H, H-

6), 11.77 (s, 1H, 2””-OH) ppm. 13C NMR (75.47 MHz, CDCl3):

δ = 24.9, 25.1, 25.5, 26.1, 28.2 and 32.0 (–CH2-, cyclohexyl),

54.7 (C-1”), 56.3 (C-1’), 113.0 (C-5), 118.0 (C-3””), 118.5 (C-5””), 119.3 (C-1””), 132.5 (C-

6””), 136.5 (C-4””), 143.0 (C-6), 150.3 and 159.8 (C-2, C-4), 162.4 (C-2””), 195.4 (C-1”’)

ppm. MS (ESI+): m/z calcd for [C23H28N2O4 + H]+: 397.21; found: 397.2. Anal. Calcl: C,

69.67; H, 7.12; N, 7.07. Found: C, 69.65; H, 7.11; N, 7.05.

12b: 1,3-diisopropyl-5-(2-hydroxybenzoyl)pyrimidine-2,4(1H,3H)-dione, C17H20N2O4

(white solid, MW = 316.35 g/mol, 0.846 g, yield 51 %, Mp = 128°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.39 (d, J = 6.8 Hz, 6H, H-2’,

-CH3), 1.51 (d, J = 6.9 Hz, 6H, H-2”, -CH3), 4.95 (sept, J = 6.8

Hz, 1H, H-1’), 5.24 (sept, J = 6.9 Hz, 1H, H-1”), 6.89 (ddd, J =

8.2, 7.2, 1.2 Hz, 1H, H-5””), 7.00-7.04 (m, 1H, H-3””), 7.47-7.54

(m, 2H, H-4””, H-6””), 7.69 (s, 1H, H-6), 11.79 (s, 1H, 2””-OH)

ppm. 13C NMR (75.47 MHz, CDCl3): δ = 19.1 (C-2”, -CH3), 21.5 (C-2’, -CH3), 46.6 (C-1”),

48.8 (C-1’), 113.5 (C-5), 118.3 (C-3””), 118.6 (C-5””), 119.4 (C-1””), 132.6 (C-6””), 136.8

(C-4””), 142.5 (C-6), 150.2 and 159.9 (C-2, C-4), 162.7 (C-2””), 195.5 (C-1”’) ppm. MS

(ESI+): m/z calcd for [C17H20N2O4 + Na]+: 339.13; found: 339.1. Anal. Calcl: C, 64.54; H,

6.37; N, 8.86. Found: C, 64.56; H, 6.40; N, 8.90.

Synthetic procedure for 1,3-ditolyl-5-(2-hydroxybenzoyl)pyrimidine-2,4(1H,3H)-dione

12c: Chromone-3-carboxylic acid 2 (1 g, 5.26 mmol) was added to 3c (5.26 mmol, 1

equiv) in chloroform (20 mL) and the reaction mixture is brought to reflux for 30 minutes.

After that, the solvent is evaporated and the resulting resinous solid is directly

recrystallized from ethanol to afford compound 12c.

1

2

33""

2"" 1"'

4"" 6""

1'

45

6

1"1""

5""

N

N

O

O

OOH

1

2

33""

2"" 1"'

4"" 6""

1'

45

6

1"1""

5""

N

N

O

O

OOH

2'

2"


213

12c: 1,3-ditolyl-5-(2-hydroxybenzoyl)pyrimidine-2,4(1H,3H)-dione, C25H20N2O4

(yellowish white solid, MW = 412.44 g/mol, 1.682 g, yield 77 %, Mp = 224°C).


4’-CH3, 4”-CH3 tolyl), 6.88 (ddd, J = 8.1, 7.2, 1.0 Hz, 1H,

H-5””), 7.00 (dd, J = 8.4, 1.0 Hz, 1H, H-3””), 7.17-7.20 (m,

8H, H-2’/2”, H-3’/3”), 7.48 (ddd, J = 8.4, 7.2, 1.6 Hz, 1H,

4””), 7.66 (dd, J = 8.1, 1.6 Hz, 1H, H-6””), 7.94 (s, 1H, H-

6), 11.71 (s, 1H, 2””-OH) ppm. 13C NMR (75.47 MHz,

CDCl3): δ = 21.1 and 21.2 (C-2’/2”, -CH3,), 114.3 (C-5),

118.1 (C-3””), 118.8 (C-5””), 119.3 (C-1””), 126.0 and 127.8 (C-2’/2”), 130.0 and 130.2 (C-

3’/3”), 131.6 (C-1”), 132.8 (C-6””), 135.8 (C-1’), 136.9 (C-4””), 139.0 and 139.6 (C-4’/4”),

147.4 (C-6), 150.3 and 160.0 (C-2, C-4), 162.7 (C-2””), 194.6 (C-1”’) ppm. MS (ESI+): m/z

calcd for [C25H20N2O4 + Na]+: 435.13; found: 435.1. Anal. Calcl: C, 72.80; H, 4.89; N, 6.79.

Found: C, 72.85; H, 4.91; N, 6.79.

6.2.6. Synthesis of the dimeric-product 13a via dimerisation of chromone-3-

carboxylic acid 2

Chromone-3-carboxylic acid 2 (2.63 mmol, 0.5 g) was added to a catalytic amount of

4-PPy (0.13 mmol, 0.02 g, 0.05 equiv) in dichloromethane (10 mL) and allowed to stirring

over 48 h, the solution colour instantly turns to yellow. The TLC monitoring (CH2Cl2

elution) shows two spots, the lower one was identified as by-product 13a(a) (chromone)

which was compared with commercial authentic sample. After that, the solvent is

evaporated and the resulting yellow solid is directly recrystallized from ethanol to afford

yellow needles of compound 13a. Compound 13a(a) was isolated from the ethanol

solution of recrystallisation by silica gel column chromatography using dichloromethane as

eluent and characterized by NMR (Mp 59 ºC; commercial sample, 59 ºC; 0.073 g, yield

19%).

13a: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-4H-chromen-4-one, C18H12O4 (MW

= 292.29 g/mol, 0.257 g, yield 67 %, pale yellow solid, Mp = 177°C, Mp(lit[13]) 173-174 ºC).

1H NMR (300.13 MHz, CDCl3): δ = 6.95 (ddd, J = 8.2, 7.2, 1.1 Hz, 1H, H-5”), 7.01 (dd, J =

8.4, 1.1 Hz, 1H, H-3”), 7.47-7.53 (m, 3H, H-4”, H-6, H-8), 7.53 (d, J = 15.2 Hz, 1H, H-1’),

7.74 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H, H-7), 8.05 (dd, J = 8.1, 1.6 Hz, 1H, H-6”), 8.23 (s, 1H,

H-2), 8.32 (dd, J = 7.9, 1.6 Hz, 1H, H-5), 8.84 (d, J = 15.2 Hz, 1H, H-2’), 12.82 (s, 1H, 2”-

OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 118.2 (C-8), 118.3 (C-3”), 118.9 (C-5”), 119.3

1

2

33""

2"" 1"'

4"" 6""

1'

45

6

1"1""

5""

N

N

O

O

OOH

2'

3'4'

2"3"

4"


214

(C-3), 119.9 (C-1”), 124.2 (C-10), 124.4 (C-2’), 126.1 (C-5),

126.2 (C-6), 130.3 (C-6“), 134.2 (C-7), 136.1 (C-1’), 136.5 (C-

4”), 155.3 (C-9), 159.5 (C-2), 163.5 (C-2”), 176.3 (C-4), 194.5

(C-3’) ppm. HRMS (ESI+): m/z calcd for [C18H12O4 + Na]+:

315.0633; found: 315.0612. Anal. Calcl: C, 73.97; H, 4.14.

Found: C, 73.76; H, 4.17.

6.2.7. Synthesis of 2-alkyloxychroman-4-ones 13a(b-d)

Chromone-3-carboxylic acid 2 (2.63 mmol, 0.5 g), was added to a catalytic amount of

4-PPy (0.13 mmol, 0.02 g, 0.05 equiv) and brought to reflux in ethanol (ROH b) or

isopropanol (ROH c) (10 mL, used as solvent/reactant system). After 1 h, the total

consumption of the starting material 2 was confirmed by TLC monitoring using CH2Cl2

elution, the reaction is then stopped and the solvent is evaporated. In case of ethanol b,

the resulting resinous solid is purified by silica gel column chromatography using

dichloromethane as eluent to afford compound 13a(b) which remains in a resinous state

(oil). In case of isopropanol c, the resinous solid obtained after solvent removal was

directly precipitated in petroleum ether to afford yellow solid of compound 13a(c).

13a(b): (R/S)-2-ethoxychroman-4-one, C11H12O3 (MW = 192.21 g/mol, 0.465 g, yield 92

%, oil).

1H NMR (300.13 MHz, CDCl3): δ = 1.15 (t, J = 7.1 Hz, 3H, H-3’),

2.84 (dd, J = 16.7, 3.4 Hz, 1H, H-3(A)), 3.01 (dd, J = 16.7, 3.5 Hz,

1H, H-3(B)), 3.64 (dq, J = 9.7, 7.1 Hz, 1H, H-2’(A)), 3.84 (dq, J =

9.7, 7.1 Hz, 1H, H-2’(B)), 5.52 (t, J = 3.4 Hz, 1H, H-2(X)), 6.97-

7.05 (m, 2H, H-6, H-8), 7.46-7.51 (m, 1H, H-7), 7.86-7.89 (m, 1H, H-5) ppm. 13C NMR

(75.47 MHz, CDCl3): δ = 14.7 (C-3’), 43.1 (C-3), 64.4 (C-2’), 99.8 (C-2), 118.0 (C-8), 121.4

(C-6 and C-10),126.2 (C-5), 135.9 (C-7), 157.5 (C-9), 190.2 (C-4) ppm. HRMS (ESI+): m/z

calcd for [C11H12O3 + Na]+: 215.0684; found: 215.0678. Anal. Calcl: C, 68.74; H, 6.29.

Found: C, 68.74; H, 6.37.

O O

O

12

34

56

78

9

10

1'

2'

3'

O

OO

HO

1

2

34

5

6

7

89

101'

2'3'

2"

3"

4"5"

6"1"


215

13a(c): (R/S)-2-isopropyloxychroman-4-one, C12H14O3 (MW = 206.24 g/mol, 0.528 g,

yield 97 %, yellow solid, Mp = 172-173°C).

1H NMR (300.13 MHz, CDCl3): δ = 1.08 and 1.18 (d, J = 6.2 Hz,

6H, H-3’(AB)), 2.80 (dd, J = 16.6, 3.5 Hz, 1H, H-3(A)), 2.99 (dd, J

= 16.6, 3.5 Hz, 1H, H-3(B)), 4.06 (sept, J = 6.2 Hz, 1H, H-2’(X)),

5.61 (t, J = 3.5 Hz, 1H, H-2(X)), 6.95-7.05 (m, 2H, H-6, H-8),

7.46-7.51 (m, 1H, H-7), 7.86-7.89 (m, 1H, H-5) ppm. 13C NMR (75.47 MHz, CDCl3): δ =

21.5 and 22.9 (C-3’), 43.6 (C-3), 71.0 (C-2’), 98.4 (C-2), 118.0 (C-8), 121.4 (C-6), 121.5

(C-10) 126.2 (C-5), 135.9 (C-7), 157.8 (C-9), 190.6 (C-4) ppm. MS (ESI+): m/z calcd for

[C12H14O3 + Na]+: 229.08; found: 229.1.

Synthetic procedure of 2-vanillinoxychroman-4-one 13a(d): Chromone-3-carboxylic

acid 2 (2.63 mmol, 0.5 g), was added to vanillin alcohol d (2.63 mmol, 0.4 g) and a

catalytic amount of 4-PPy (0.13 mmol, 0.02 g, 0.05 equiv), the reaction mixture was

brought to reflux in chloroform (10 mL). After 1 h, the total consumption of the starting

material 1 was confirmed by TLC monitoring using CH2Cl2 elution, the reaction is then

stopped and the solvent is evaporated. The resulting resinous solid is purified by silica gel

column chromatography using dichloromethane as eluent to afford compound 13a(d)

which was recrystallized from hexane as a white solid. Compound 13a(a) (chromone) was

subsequently eluted from the column chromatography and isolated as by-product which

was compared to authentic sample and further characterized by NMR (Mp 59 ºC;

commercial sample, 59 ºC; 0.113 g, yield 29 %)..

13a(d): (R/S)-2-vanillinoxychromanone, C17H16O5 (MW = 300.31 g/mol, 0.506 g, yield

64 %, white solid, Mp = 127-128°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.86, (dd, J = 16.8,

3.5 Hz, 1H, H-3(A)), 3.03 (dd, J = 16.8, 3.5 Hz, 1H, H-

3(B)), 3.78 (s, 3H, 3’-CH3O), 4.59 and 4.71 (d, J = 16.8

Hz, 2H, H-2’(AB)), 5.57 (t, J = 3.5 Hz, 1H, H-2(X)), 5.59

(s, 1H, 4”-OH), 6.69 (d, J = 1.8 Hz, 1H, H-2”), 6.77 (dd,

J = 8.0, 1.8 Hz, 1H, H-5”), 6.85 (d, J = 8.0 Hz, 1H, H-6”), 6.95-6.98 (m, 1H, H-8), 7.02-7.08

(m, 1H, H-6), 7.47-7.53 (m, 1H, H-7), 7.88-7.91 (m, 1H, H-5) ppm. 13C NMR (75.47 MHz,

CDCl3): δ = 43.0 (C-3), 55.6 (3”-OCH3), 70.1 (C-2’), 98.5 (C-2), 110.5 (C-2”), 114.0 (C-5”),

118.0 (C-8), 121.1 (C-6”), 121.6 (C-10), 121.7 (C-6), 126.2 (C-5), 128.3 (C-1”), 136.0 (C-

O O

O

12

34

56

78

9

10

1'2'

3'

OH

OCH3

O O

O

12

34

56

78

9

10

1'2' 3"

1"

4"

2"

5"6"


216

7), 145.4 (C-4”), 146.5 (C-3”), 157.4 (C-9), 190.2 (C-4) ppm. HRMS (ESI+): m/z calcd for

[C17H16O5 + Na]+: 323.0895; found: 323.0902.

6.2.8. Synthesis of enaminones 13a(e-g)

Chromone-3-carboxylic acid 2 (2.63 mmol, 0.5 g), was added to amines (2.63 mmol,

DEA, 0.28 mL or benzylamine, 0.285 g or diethylenediamine, 0.18 mL) and brought to

reflux in chloroform (10 mL). After 30 min, the total consumption of the starting material 2

confirmed by TLC monitoring, the reaction is then stopped and the solvent is evaporated.

In case of compound 13a(e), the resulting resinous solid is precipitated in light petroleum

as yellow solid, [Mp 77-78ºC, Mp(lit[13]) = 77-78 °C yield 0.446 g, 77%, the structure was

confirmed by NMR, MS and elemental analysis]. Compound 13a(f) was recrystallized from

ethanol as pale yellow crystals [Mp 137ºC, yield 0.487 g, 73%, the structure was

confirmed by NMR, MS and elemental analysis], also compound 13a(g) was obtained as

yellow crystals upon recrystallizing from toluene [Mp 140-141ºC, yield 0.409 g, 88%, the

structure was confirmed by NMR, MS and elemental analysis].

6.2.9. General procedure for Michael addition of 1,3-dicarbonyl compounds 4a-t on

chromone-3-carboxylic acid 2: Synthesis of 3-(HOPO-1)-chromones, -flavones,

-2-styrylchromones 13a-q and 2,3-disubstituted chromanones 14q-t

Chromone-3-carboxylic acid 2 (5.26 mmol, 1 g), was added to 4a-t (5.26 mmol) and a

catalytic amount of 4-PPy (0.26 mmol, 0.04 g, 0.05 equiv), the reaction mixture was

brought to reflux in chloroform (10 mL). During the required time of reaction (see Table 3.2

chapter III), the gradual consumption of the starting materials 2 and 4a-t was monitored by

TLC (using CH2Cl2 or CH2Cl2/light petroleum elution), which shows in most of the cases,

several spots, including the desired product accompanied with the formation of by-

products 13a and 13a(a) as verified by authentic samples and remaining starting materials

(especially, 1,3-dicarbonyl compounds 4a-t, observations are made in Table 3.2 chapter

III). The required reaction time is determined when there is no further change according to

TLC analysis. The reaction is then stopped and the solvent is removed to have a dark red

resinous solid which is either purified by silica gel column chromatography and then

recrystallized or directly recrystallized from an appropriate solvent to afford compounds

13a-q and 14a-t (indications are mentioned with each compound 13, 14). The by-products

13a (dimeric-product) and 13a(a) (chromone) (when formed, see Table 3.2 chapter III)

were only compared to authentic samples by TLC but never isolated, unless in case of 4j

and 4k reactions, where the dimeric-compound 13a was majorly produced.


217

13a: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-4H-chromen-4-one, was

synthesized via the Michael addition method which gives better yield (1.141 g, yield 74 %)

after direct recrystallisation from ethanol (see physical and spectral data above).

13b: (2R, 2’S)/(2S, 2’R) and (2R, 2’R)/(2S, 2’S)-ethyl 3-oxo-2-(4-oxochroman-2-

yl)butanoate was purified by preparative TLC plates using dichloromethane as eluent and

obtained as an oil after solvent removal. C15H16O5 (MW = 276.28 g/mol, 0.815 g, yield 56

%, oil).

1H NMR (300.13 MHz, CDCl3): δ = 1.30 and 1.32 (t, J = 7.8-

7.1 Hz, 3H, H-2”), 2.32 and 2.39 (s, 3H, H-4), 2.78 (dd, J =

16.8, 12.0, Hz, 1H, H-3’(A)), 2.92 (dd, J = 16.8, 3.3 Hz, 1H,

H-3’(B)), 3.97 and 4.00 (d, J = 4.4-3.6 Hz, 1H, H-2(Y)), 4.14-

4.36 (m, 2H, H-1”(AB)), 5.03-5.19 (m, 1H, H-2’(X)), 6.91-6.94

(m, 1H, H-8’), 7.02-7.06 (m, 1H, H-6), 7.45-7.51 (m, 1H, H-

7’), 7.87-7.91 (m, 1H, H-5’) ppm.13C NMR (75.47 MHz, CDCl3): δ = 13.9-14.0 (C-2”), 30.2

(C-4), 40.6-40.8 (C-3’), 61.9-62.0 (C-1”), 63.1-63.4 (C-2), 75.3-75.6 (C-2’), 117.73-

117.77(C-8’), 120.87-120.88 (C-10’), 121.8-121.9 (C-6’), 126.93-126.96 (C-5’), 136.0-

136.1 (C-7’), 160.4-160.6 (C-9’), 165.8-166.2 (C-1), 190.5-190.7 (C-4’), 199.4-199.7 (C-3)

ppm. HRMS (ESI+): m/z calcd for [C15H16O5 + Na]+: 299,0895; found: 299.0900.

13c: (E)-4-acetyl-1-(2-hydroxyphenyl)hex-2-ene-1,5-dione, was purified by column

chromatography and recrystallized from hexane to give pale yellow solid. C14H14O4 (MW =

246.26 g/mol, 1.015 g, yield 78 %, pale yellow solid, Mp = 137-138°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.43 (s, 6H, H-6), 6.93

(ddd, J = 8.2, 7.2, 1.2 Hz, 1H, H-5’), 7.03 (dd, J = 8.4, 1.2 Hz,

1H, H-3’), 7.03 (d, J = 15.4 Hz, 1H, Hα-2) 7.50 (ddd, J = 8.6,

7.2, 1.6 Hz, 1H, H-4’), 7.77 (dd, J = 8.0, 1.7 Hz, 1H, H-6’),

7.89 (d, J = 15.4 Hz, 1H, Hβ-3), 12.83 (s, 1H, 2’-OH), 17.74 (s,

1H, 5-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 25.2 (C-6), 110.4 (C-4), 118.7 abd

118.8 (C-5’, C-3’), 119.8 (C-1’), 120.5 (C-2), 129.2 (C-6’), 136.3 (C-4’), 140.2 (C-3), 163.6

(C-2’), 193.0 (C-1), 194.7 (C-5) ppm. HRMS (ESI+): m/z calcd for [C14H14O4 + Na]+:

269.0790; found: 269.0799. Anal. Calcl: C, 68.28; H, 5.73. Found: C, 68.43; H, 5.73.

O

O

O

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1

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218

13d: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-7-methoxy-4H-chromen-4-one,

was directly recrystallized from ethanol to give pale yellow solid. C19H14O5 (MW = 322.31

g/mol, 1.137 g, yield 67 %, pale yellow solid, Mp = 202-203°C ).

1H NMR (300.13 MHz, CDCl3): δ = 3.93 (s, 3H, 7-

OCH3), 6.89 (d, J = 2.4 Hz, 1H, H-8), 6.92-6.96 (m, 1H,

H-5”), 7.01 (dd, J = 8.4, 1.1 Hz, 1H, H-3”), 7.05 (dd, J =

8.9, 2.4 Hz, 1H, H-6), 7.46-7.52 (m, 1H, H-4”), 7.51 (d,

J =15.1 Hz, 1H, H-1’), 8.04 (dd, J = 8.1, 1.6 Hz, 1H, H-

6”), 8.15 (s, 1H, H-2), 8.21 (d, J = 8.9 Hz, 1H, H-5),

8.84 (d, J = 15.1 Hz, 1H, H-2’), 12.83 (s, 1H, 2”-OH)

ppm. 13C NMR (75.47 MHz, CDCl3): δ = 55.9 (7-OCH3), 100.3 (C-8), 115.3 (C-6), 118.0

(C-10), 118.3 (C-3”), 118.9 (C-5”), 119.2 (C-3), 119.9 (C-1”), 124.3 (C-2’), 127.6 (C-5),

130.3 (C-6”), 136.3 (C-1’), 136.4 (C-4”), 157.1 (C-9), 159.1 (C-2), 163.5 (C-7), 164.4 (C-

2”), 175.6 (C-4), 194.5 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C19H14O5 + H]+: 323.0919;

found: 323.0905.

13e: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-5,7-dimethoxy-4H-chromen-4-

one, was purified by column chromatography and subsequently recrystallized from

ethanol to give pale yellow solid. C20H16O6 (MW = 352.34 g/mol, 0.800 g, yield 43 %, pale

yellow solid, Mp = 213-214°C).


6H, 5-OCH3, 7-OCH3), 6.44 (d, J = 2.3 Hz, 1H, H-8),

6.49 (d, J = 2.3 Hz, 1H, H-6), 6.93 (ddd, J = 8.2, 7.2,

1.2 Hz, 1H, H-5”), 7.00 (dd, J = 8.4, 1.1 Hz, 1H, H-3”),

7.46 (d, J = 15.1 Hz, 1H, Hβ-1’), 7.44-7.54 (m, 1H, H-

4”), 8.01 (s, 1H, H-2), 8.08 (dd, J = 8.2, 1.7 Hz, 1H, H-

6”), 8.90 (d, J = 15.1 Hz, 1H, Hα-2’), 12.86 (s, 1H, 2”-

OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 55.9 and 56.6 (5/7-OCH3), 92.8 (C-8), 96.9

(C-6), 118.2 and 118.8 (C-3”, C-5”), 120.0-120.1 (C-10, C-3, C-1”), 124.2 (C-2’), 130.6 (C-

6), 136.4 (C-4”, C-1’), 157.6 (C-9), 159.0 (C-2), 163.5 (C-5, C-7), 164.4 (C-2”), 175.3 (C-

4), 194.8 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C20H16O6 + Na]+: 375.0845; found:

375.0847.

O

OO

HO

OCH3

1

2

34

5

6

78

9

101'

2'3'

2"

3"

4"5"

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OCH3

1

2

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101'

2'3'

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219

13f: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-methyl-4H-chromen-4-one, was

directly recrystallized from ethanol to give yellow solid C19H14O4 (MW = 306.31 g/mol,

1.017 g, yield 63 %, yellow solid, Mp = 187-188°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.72 (s, 3H, 2-CH3), 6.92-

6.97 (m, 1H, H-5”), 6.99-7.02 (m, 1H, H-3”), 7.42-7.52 (m, 3H,

H-4”, H-6, H-8), 7.66-7.71 (m, 1H, H-7), 7.78 (d, J = 15.1 Hz,

1H, H-1’), 8.04-8.08 (m, 1H, H-6”), 8.24-8.28 (m, 1H, H-5),

8.93 (d, J = 15.1 Hz, 1H, H-2’), 12.88 (s, 1H, 2”-OH) ppm. 13C

NMR (75.47 MHz, CDCl3): δ = 19.2 (2-CH3), 116.1 (C-3),

117.7 (C-8), 118.2 (C-3”), 118.9 (C-5”), 120.0 (C-10), 123.5

(C-1”), 124.5 (C-2’), 125.7 (C-6), 126.1 (C-5), 130.3 (C-6“), 133.8 (C-7), 135.2 (C-1’),

136.3 (C-4”), 154.9 (C-9), 163.5 (C-2”), 169.7 (C-2), 176.5 (C-4), 194.8 (C-3’) ppm. HRMS

(ESI+): m/z calcd for [C19H14O4 + Na]+: 329.0790; found: 329.0767. Anal. Calcl: C, 74.50;

H, 4.61. Found: C, 74.40; H, 4.63.

13g: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]flavone, was directly recrystallized

from ethanol to give yellow solid C24H16O4 (MW = 386.38 g/mol, 1.038 g, yield 51 %,

yellow solid, Mp = 230°C).

1H NMR (300.13 MHz, CDCl3): δ = 6.92-7.00 (m, 2H, H-3’’’,

H-5’’’), 7.45-7.75 (m, 10H, H-4’’’, H-6, H-8, H-7, H-1”, H-3’/5’,

H-2’/6’, H-4’), 8.07 (dd, J = 8.1, 1.6 Hz, 1H, H-6’’’), 8.31-8.34

(m, 1H, H-5), 8.93 (d, J = 15.2 Hz, 1H, H-2”), 12.82 (s, 1H,

2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 115.6 (C-3),

118.0 (C-8), 118.2 (C-3’’’), 118.8 (C-5’’’), 120.1 (C-1’’’), 123.4

(C-10), 124.8 (C-2”), 125.8 (C-6), 126.2 (C-5), 128.8 (C-3’/5’),

130.0 (C-2’/6’), 130.3 (C-6’’’), 131.7 (C-4’), 132 (C-1’), 134.1

(C-7), 136.2 (C-1”), 136.8 (C-4’’’), 155.2 (C-9), 163.4 (C-2’’’), 168.4 (C-2), 177.2 (C-4),

194.7 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C24H16O4 + Na]+: 391.0946; found:

391.0939.

O

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HO

12

34

5

6

78

9

101'

2'3'

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220

13h: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-3’,4’,7-trimethoxyflavone, was



1H NMR (300.13 MHz, CDCl3): δ = 3.93 (s, 3H, 7-

OCH3), 3.97 (s, 3H, 3’-OCH3), 4.00 (s, 3H, 4’-OCH3),

6.92-7.06 (m, 5H, H-8, H-5’’’, H-3’’’, H-6, H-5’), 7.22

(d, J = 2.1 Hz, 1H, H-2’), 7.31 (dd, J = 8.4, 2.1 Hz,

1H, H-6’), 7.48 (dd, J = 8.6, 7.2, 1.6 Hz, 1H, H-4”’),

7.77 (d, J = 15.2 Hz, 1H, H-1”), 8.09 (dd, J = 8.1, 1.6

Hz, 1H, H-6’’’), 8.22 (d, J = 8.9 Hz, 1H, H-5), 8.90 (d,

J = 15.2 Hz, 1H, H-2”), 12.90 (s, 1H, 2”’-OH) ppm.

13C NMR (75.47 MHz, CDCl3): δ = 55.9 (7-OCH3),

56.1 (3’-OCH3), 56.2 (4’-OCH3), 100.15 (C-8), 110.8 (C-5’), 112.7 (C-2’), 114.9 (C-

3), 115.0 (C-6), 117.2 (C-3’’’), 118.2 (C-5’’’), 118.8 (C-1’’’), 120.1 (C-10), 124.20 (C-6’ and

C-1’) 124.26 (C-2”), 127.6 (C-5), 130.3 (C-6’’’), 136.2 (C-4’’’), 137.5 (C-1”), 148.9 (C-3’),

152.0 (C-4’), 156.8 (C-9), 163.4 (C-2’’’), 164.3 (C-7), 167.7 (C-2), 176.5 (C-4), 194.9 (C-3’)

ppm. HRMS (ESI+): m/z calcd for [C27H22O7 + Na]+: 481.1263; found: 481.1269.

13i: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-3’,4’-dimethoxyflavone, was



1H NMR (300.13 MHz, CDCl3): δ = 3.97 (s, 3H, 3’-OCH3),

4.00 (s, 3H, 4’-OCH3), 6.93-7.02 (m, 2H, H-5’’’, H-3’’’), 7.06

(d, J = 8.4 Hz, 1H, H-5’), 7.24 (d, J = 2.1 Hz, 1H, H-2’),

7.34 (dd, J = 8.4, 2.1 Hz, 1H, H-6’), 7.46-7.56 (m, 3H, H-6,

H-4’’’, H-8), 7.70-7.73, (m, 1H, H-7), 7.78 (d, J = 15.2 Hz,

1H, H-1”), 8.10 (dd, J = 8.0, 1.6 Hz, 1H, H-6’’’), 8.30-8.33

(m, 1H, H-5), 8.90 (d, J = 15.2 Hz, 1H, H-2”), 12.89 (s, 1H,

2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 56.1 (4’-

OCH3), 56.2 (3’-OCH3), 110.8 (C-5’), 112.7 (C-2’), 115.0

(C-3), 117.9 (C-8), 118.2 (C-3’’’), 118.9 (C-5’’’), 120.1 (C-1’’’), 123.3 (C-10), 124.2 (C-1’),

124.3 (C-2”, C-6’), 125.7 (C-6), 126.2 (C-5), 130.3 (C-6’’’), 134.0 (C-7), 136.2 (C-4’’’),

137.4 (C-1”), 149.0 (C-3’), 152.1 (C-4’), 155.1 (C-9), 163.4 (C-2’’’), 168.0 (C-2), 177.1 (C-


451.1165.

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OCH3

H3CO

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221

13l: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-4’-methylflavone, was directly

recrystallized from ethanol to give yellow solid C25H18O4 (MW = 382.41 g/mol, 1.515 g,

yield 75 %, yellow solid, Mp = 229-230 °C).

1H NMR (300.13 MHz, CDCl3): δ = 2.49 (s, 3H, 4’-CH3), 6.92-

7.01 (m, 2H, H-5’’’, H-3’’’), 7.40 (dd, J = 8.4, 0.6 Hz, 2H, H-

3’/5’), 7.45-7.55 (m, 3H, H-6, H-4’’’, H-8), 7.59-7.62 (m, 2H,

H-2’/6’), 7.72 (d, J = 15.3 Hz, 1H, H-1”), 7.70-7.76 (m, 1H, H-

7), 8.09 (dd, J = 8.1, 1.6 Hz, 1H, H-6’’’), 8.31-8.34 (m, 1H, H-

5), 8.93 (d, J = 15.3 Hz, 1H, H-2”), 12.85 (s, 1H, 2”’-OH) ppm.

13C NMR (75.47 MHz, CDCl3): δ = 21.6 (4’-CH3), 115.4 (C-3),

118.0 (C-8), 118.2 (C-3’’’), 118.8 (C-5’’’), 120.1 (C-1’’’), 123.4

(C-10), 124.5 (C-2”), 125.7 (C-6), 126.2 (C-5), 129.1 (C-1’), 129.5 (C-3’/5’), 130.0 (C-2’/6’),

130.3 (C-6’’’), 134.0 (C-7), 136.2 (C-4’’’), 137.1 (C-1”), 142.5 (C-4’), 155.2 (C-9), 163.4 (C-

2’’’), 168.7 (C-2), 177.2 (C-4), 194.8 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C25H18O4 +

H]+: 383.1283; found: 383.1261.

13m: (E,E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-styryl-4H-chromen-4-one,

was directly recrystallized from ethanol to give yellow solid C26H18O4 (MW = 394.42 g/mol,

0.895 g, yield 43 %, yellow solid, Mp = 188°C).

1H NMR (300.13 MHz, CDCl3): δ = 6.96 (ddd, J = 8.2, 7.2 1.2

Hz, 1H, H-5””), 7.03 (dd, J = 8.4, 1.0 Hz, 1H, H-3””), 7.42-7.58

(m, 6H, H-6, H-8, H-4””, H-3”/5”, H-4”), 7.54 (d, J = 15.7 Hz,

1H, Hα-1’), 7.68-7.75 (m, 3H, H-2”/6”, H-7), 7.84 (d, J = 15.7

Hz, 1H, Hβ-2’), 8.08 (dd, J = 8.1, 1.6 Hz, 1H, H-6””), 8.08 (d, J

= 15.0 Hz, 1H, Hβ-1”’), 8.27 (dd, J = 9.0, 1.3 Hz, 1H, H-5),

8.95 (d, J = 15.0 Hz, 1H, Hα-2”’), 12.91 (s, 1H, 2””-OH) ppm.

13C NMR (75.47 MHz, CDCl3): δ = 115.1 (C-3), 116.3 (C-1’),

117.6 (C-8), 118.3 (C-3””), 118.9 (C-5””), 120.1 (C-1””), 123.3

(C-10), 125.4 (C-2”’), 125.5 (C-6), 126.2 (C-5), 128.2 (C-2”/6”), 129.1 (C-3”/5”), 130.4 (C-

6””), 130.6 (C-4”), 134.1 (C-7), 134.2 (C-1”’), 134.8 (C-1”), 136.4 (C-4””), 140.5 (C-2’),

154.6 (C-9), 162.8 (C-2), 163.5 (C-2””), 177.2 (C-4), 194.7 (C-3”’) ppm. HRMS (ESI+): m/z

calcd for [C26H18O4 + Na]+: 417.1103; found: 417.1094.

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222

13n: (E,E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl)-2-(3,4-dimethoxystyryl)-4H-

chromen-4-one, was directly recrystallized from ethanol to give orange solid. C28H22O6

(MW = 454.47 g/mol, 1.620 g, yield 68 %, orange solid, Mp = 207-208°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.96 (s, 3H, 4”-

OCH3), 4.00 (s, 3H, 3”-OCH3), 6.91-6.97 (m, 2H, H-5”,

H-5””), 7.02 (dd, J = 8.4, 1.1 Hz, 1H, H-3””), 7.16 (d, J =

2.0, Hz, 1H, H-2”), 7.29 (dd, J = 9.0, 2.0 Hz, 1H, H-6”),

7.36 (d, J = 15.6 Hz, 1H, Hα-1’), 7.43 (ddd, J = 9.0, 6.0,

1.1 Hz, 1H, H-6), 7.50 (ddd, J = 8.6, 5.1, 1.8 Hz, 1H, H-

4””), 7.53 (dd, J = 6.5, 1.1 Hz, 1H, H-8), 7.70 (ddd, J =

8.6, 7.2, 1.7 Hz, 1H, H-7), 7.76 (d, J = 15.6 Hz, 1H, Hβ-

2’), 8.08 (dd, J = 8.2, 1.5 Hz, 1H, H-6””), 8.08 (d, J =

15.0 Hz, 1H, Hβ-1”’), 8.25 (dd, J = 7.9, 1.7 Hz, 1H, H-5), 8.94 (d, J = 15.0 Hz, 1H, Hα-2”’),

12.95 (s, 1H, 2””-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 56.02 and 56.09 (3”/4”-

OCH3), 110.1 (C-2”), 111.2 (C-5”), 113.9 (C-1’), 114.4 (C-3), 117.4 (C-8), 118.2 (C-3””),

118.9 (C-5””), 120.1 (C-1””), 122.7 (C-6”), 123.3 (C-10), 124.9 (C-2”’), 125.3 (C-6), 126.1

(C-5), 127.9 (C-1”), 130.0 (C-6””), 133.9 (C-7), 134.5 (C-1”’), 136.3 (C-4””), 140.5 (C-2’),

149.3 (C-3”), 151.5 (C-4”), 154.5 (C-9), 163.3 (C-2), 163.5 (C-2””), 177.1 (C-4), 194.8 (C-

3”’) ppm. HRMS (ESI+): m/z calcd for [C28H22O6 + Na]+: 477.1314; found: 477.1325.

13o: (E,E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-(4-methoxystyryl)-4H-

chromen-4-one, was directly recrystallized from ethanol to give yellow solid. C27H20O5

(MW = 424.44 g/mol, 1.604 g, yield 72 %, yellow solid, Mp = 187-188°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.88 (s, 3H, 4”-OCH3),

6.91-6.99 (m, 1H, H-5””), 6.98 (d, J = 8.6 Hz, 2H, H-3”/5”),

7.02 (dd, J = 8.5, 1.1 Hz, 1H, H-3””), 7.39 (d, J = 15.5 Hz,

1H, Hα-1’), 7.39-7.47 (m, 1H, H-6), 7.47-7.54 (m, 1H, H-

4””), 7.51-7.57 (m, 1H, H-8), 7.65 (d, J = 8.6 Hz, 2H, H-

2”/6”), 7.71 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H, H-7), 7.79 (d, J

= 15.5 Hz, 1H, Hβ-2’), 8.09 (dd, J = 8.2, 1.7 Hz, 1H, H-6””),

8.09 (d, J = 15.0 Hz, 1H, Hβ-1”’), 8.26 (dd, J = 8.0, 1.7 Hz,

1H, H-5), 12.96 (s, 1H, 2””-OH) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 55.5 (4”-OCH3), 113.7 (C-1’), 114.4 (C-3), 114.6 (C-3”/5”), 117.5 (C-8),

118.3 (C-3””), 118.9 (C-5””), 120.2(C-1””), 123.4 (C-10), 124.9(C-2”’), 125.4(C-6), 126.2

(C-5), 127.7 (C-1”), 130.1 (C-2”/6”), 130.4(C-6””), 134.0(C-7), 134.6(C-1”’), 136.3, (C-4””),

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H3CO1

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140.3 (C-2’), 154.6 (C-9), 161.8 (C-4”) , 163.5 (C-2””, C-2), 177.2 (C-4), 194.8 (C-3”’) ppm.

HRMS (ESI+): m/z calcd for [C27H20O5 + Na]+: 447.1208; found: 447.1211.

13p: (E,E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-(4-methylstyryl)-6-methyl-4H-

chromen-4-one, was directly recrystallized from ethanol to give yellow solid. C28H22O4

(MW = 422.47 g/mol, 1.030 g, yield 46 %, yellow solid, Mp = 219-220°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.42 (s, 3H, 4”-CH3), 2.48

(s, 3H, 6”-CH3), 6.96 (ddd, J = 8.2, 7.2, 1.2 Hz, 1H, H-5””),

7.02 (dd, J = 8.4, 1.1 Hz, 1H, H-3””), 7.25-7-28 (m, 2H, H-

3”/5”), 7.43-7-54 (m, 3H, H-8, H-7, H-4””), 7.48 (d, J = 15.6

Hz, 1H, Hα-1’), 7.59 (d, J = 8.2 Hz, 2H, H-2”/6”), 7.79 (d, J =

15.6, Hz, 1H, Hβ-2’), 8.04 (dd, J = 1.5, 0.7 Hz, 1H, H-5), 8.09

(dd, J = 15.0 Hz, 1H, Hβ-1”’), 8.09 (dd, J = 8.1, 1.6 Hz, 1H,

H-6””), 8.95 (d, J = 15.0 Hz, 1H, Hα-2”’), 12.95 (s, 1H, 2””-

OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 21.0 (6”-CH3),

21.5 (4”-CH3), 114.6 (C-3), 115.2 (C-1’), 117.3 (C-8), 118.2 (C-3””), 118.9 (C-5””), 120.2

(C-1””), 123.0 (C-10), 125.0 (C-2”’), 125.5 (C-5), 128.2 (C-2”/6”), 129.8 (C-3”/5”), 130.4 (C-

6””), 132.2 (C-1”), 134.5 (C-1”’), 135.2 (C-7), 135.4 (C-2’), 136.3 (C-4””), 140.4 (C-2’),

141.2 (C-4”), 152.8 (C-9), 163.1 (C-2), 163.5 (C-2””), 177.3 (C-4), 194.8 (C-3”’) ppm.

HRMS (ESI+): m/z calcd for [C28H22O4 + H]+: 423.1596; found: 423.1579.

13q: (E,E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-7-methoxy-2-styryl-4H-

chromen-4-one was purified by column chromatography and subsequently recrystallized

from ethanol to give yellow solid. C27H20O5 (MW = 424.44 g/mol, 0.160 g, yield 7%, yellow

solid, Mp = 234-235°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.97 (s, 3H, 7-OCH3),

6.90-7.05 (m, 4H, H-5””, H-3””, H-6, H-8), 7.40-7.53 (m,

4H, H-3”/5”, H-4”, H-4””), 7.53 (d, J = 15.7 Hz, 1H, Hα-

1’), 7.66-7.73 (m, 2H, H-2”/6”), 7.80 (d, J = 15.7 Hz, 1H,

Hβ-2’), 8.07 (dd, J = 1.6, 0.6 Hz, 1H, H-6””), 8.07 (d, J =

15.0 Hz, 1H, Hβ-1”’), 8.18 (d, J = 8.8 Hz, 1H, H-5), 8.95

(d, J = 15.0 Hz, 1H, Hα-2”’), 12.93 (s, 1H, 2””-OH) ppm.

13C NMR (75.47 MHz, CDCl3): δ = 55.9 (7-OCH3), 99.9

(C-8), 114.7 (C-6), 115.0 (C-10), 116.4(C-1’), 117.3 (C-

3), 118.3 (C-3””), 118.9 (C-5””), 120.2 (C-1””), 125.4 (C-2”’), 127.6 (C-5), 128.2 (C-2”/6”),

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129.1(C-3”/5”), 130.4 and 130.5 (C-4”, C-6””), 134.4 (C-1"’), 134.9 (C-1”), 136.4 (C-4””),

139.9 (C-2’), 156.3 (C-9), 162.6 (C-2), 163.5 (C-7), 164.5 (C-2””), 176.6 (C-4), 194.8 (C-

3”’) ppm. HRMS (ESI+): m/z calcd for [C27H20O5 + Na]+: 447.1208; found: 447.1195.

14q: 2-[2-(2-hydroxyphenyl)-2-oxoethyl]-3-[(Z,E)-3-(phenyl)-1-hydroxyallylidene]-7-

methoxychroman-4-one, was purified by column chromatography and subsequently

recrystallized from ethanol to give yellow solid. C27H22O6 (MW = 442.46 g/mol, 0.800 g,

yield 35%, yellow solid, Mp = 234-235°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.99 (dd, J =

15.9, 3.7 Hz, 1H, H-1'(A)), 3.78 (s, 3H, 7-OCH3),

3.89 (dd, J = 15.9, 9.2 Hz, 1H, H-1’(M)), 6.23 (dd, J

= 9.2, 3.7 Hz, 1H, H-2(X)), 6.27 (d, J = 2.4 Hz, 1H,

H-8), 6.65 (dd, J = 8.8, 2.4 Hz, 1H, H-6), 6.76-6.85

(m, 1H, H-5”), 6.87 (d, J = 15.4 Hz, 1H, Hα-2’’’),

6.98 (dd, J = 8.3, 1.2 Hz, 1H, H-3”), 7.37-7.60 (m, 7H, H-4”, H-6”, H-3””/5””, H-2””/6””, H-

4””), 7.74 (d, J = 15.4 Hz, 1H, Hβ-3’’’), 7.89 (d, J = 8.8 Hz, 1H, H-5), 12.24 (s, 1H, 2”-OH),

16.28 (s, 1 H, 1’’’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 44.3 (C-1’), 55.6 (7-OCH3),

72.4 (C-2), 101.6 (C-8), 105.0 (C-3), 110.6 (C-6), 113.8 (C-10), 116.7 (C-2’’’), 118.7 (C-

3”), 118.9 (C-5”), 119.4 (C-1”), 128.2 (C-2””,6””, 5), 128.9 (C-3””,5””), 130.2 and 130.3 (C-

6”, C-4””), 134.9 (C-1””), 136.9 (C-4”), 141.5 (C-3’’’), 159.3 (C-9), 162.8 (C-2”), 166.0 (C-

7), 170.9 (C-1’’’), 182.1 (C-4), 202.6 (C-2’) ppm. HRMS (ESI+): m/z calcd for [C27H22O6 +

Na]+: 465.1314; found: 465.1310.

14r: 2-[2-(2-hydroxyphenyl)-2-oxoethyl]-3-[(Z,E)-3-(phenyl)-1-hydroxyallylidene]-5,7-

dimethoxychroman-4-one, was purified by column chromatography and subsequently

recrystallized from ethanol to give yellow solid. C28H24O7 (MW = 472.49 g/mol, 0.773 g,

yield 31%, yellow solid, Mp = 158-159°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.01 (dd, J =

16.0, 3.7 Hz, 1H, H-1'(A)), 3.75 (s, 3H, 7-OCH3),

3.89 (dd, J = 16.0, 9.3 Hz, 1H, H-1’(M)), 3.97 (s,

3H, 5-OCH3), 5.94 (d, J = 2.3 Hz, 1H, H-8), 6.15

(dd, J = 9.3, 3.8 Hz, 1H, H-2(X)), 6.15 (d, J = 2.3

Hz, 1H, H-6), 6.79-6.85 (m, 1H, H-5”), 6.88 (d, J =

15.5 Hz, 1H, Hα-2’’’), 6.98 (dd, J = 8.4, 0.9 Hz, 1H, H-3”), 7.36-7.59 (m, 7H, H-4”, H-6”, H-

3””/5””, H-2””/6””, H-4””), 7.72 (d, J = 15.5 Hz, 1H, Hβ-3’’’), 12.25 (s, 1H, 2”-OH), 16.70 (s,

1

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1H, 1’’’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 43.4 (C-1’), 55.6 (7-OCH3), 56.2 (5-

OCH3), 71.9 (C-2), 94.0 (C-6), 94.6 (C-8), 105.0 (C-3, C-10), 117.1 (C-2’’’), 118.6 (C-3”),

118.9 (C-5”), 119.4 (C-1”), 128.1 (C-6””/2””), 128.9 (C-3””/5””), 130.1 and 130.2 (C-6”, C-

4””), 135.1 (C-1’), 136.9 (C-4”) 140.8 (C-3’’’), 160.9 (C-9), 162.1 (C-5), 162.7 (C-2”), 166.2

(C-7), 170.1 (C-1’’’), 181.8 (C-4), 202.6 (C-2’) ppm. HRMS (ESI+): m/z calcd for [C28H24O7

+ Na]+: 495.1420; found: 495.1423.

14s: 2-[2-(2-hydroxyphenyl)-2-oxoethyl]-3-[(Z,E)-3-(3,4-dimethoxyphenyl)-1-

hydroxyallylidene)-7-methoxychroman-4-one, was purified by column chromatography

and subsequently recrystallized from ethanol to give yellow solid. C29H26O8 (MW = 502.51

g/mol, 1.110 g, yield 42%, yellow solid, Mp = 210-211°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.98 (dd, J

= 15.8, 3.5 Hz, 1H, H-1’(A)), 3.83 (m, 1H, dd,

J = 15.8, 9.1 Hz, 1H, H-1’(M)), 3.78 (s, 3H, 7-

OCH3), 3.94 and 3.97 (s, 6H, 3””-OCH3, 4””-

OCH3), 6.23 (dd, J = 9.1, 3.5 Hz, 1H, H-2(X)),

6.27 (d, J = 2.4 Hz, 1H, H-8), 6.65 (dd, J =

8.8, 2.4 Hz, 1H, H-6), 6.75 (d, J = 15.3 Hz, 1H, Hα-2”’), 6.82 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H,

H-5”), 6.90 (d, J = 8.3 Hz, 1H, H-5””), 6.98 (dd, J = 8.4, 1.1 Hz, 1H, 3”), 7.11 (d, J = 2.0 Hz,

1H, H-2’’), 7.18 (dd, J = 8.3, 2.0 Hz, 1H, H-6””), 7.44-7.51 (m, 1H, H-4””), 7.52 (dd, J = 8.1,

1.8 Hz, 1H, H-6”), 7.69 (d, J = 15.3 Hz, 1H, Hβ-3”’), 7.89 (d, J = 8.7 Hz, 1H, H-5), 12.27 (s,

1H, 2”-OH), 16.36 (s, 1 H, 1’’’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 44.4 (C-1’),

55.6, 55.7 and 55.9 (7-OCH3, 3””-OCH3, 4””-OCH3), 72.5 (C-2), 101.7 (C-8), 105.0 (C-3),

110.0 (C-2””), 110.4 (C-6), 111.1 (C-5””), 113.9 (C-10), 114.7(C-2’’’), 118.6(C-3”), 118.9(C-

5”), 119.5(C-1”), 122.7(C-6””), 128.0(C-5), 128.1 (C-1””), 130.3(C-6”), 137.0(C-4”),

141.6(C-3’’’), 149.2 (C-3””), 151.3 (C-4””), 159.2 (C-9), 162.8 (C-2”), 165.9 (C-7), 171.7 (C-

1”’), 181.5 (C-4), 202.9 (C-2’) ppm. HRMS (ESI+): m/z calcd for [C29H26O8 + Na]+:

525.1525; found: 525.1532.

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14t: 2-[2-(2-hydroxyphenyl)-2-oxoethyl]-3-[(Z,E)-3-(3,4-dimethoxyphenyl)-1-

hydroxyallylidene)-5,7-dimethoxychroman-4-one, was purified by column

chromatography and subsequently recrystallized from ethanol to give yellow solid.

C30H28O9 (MW = 532.54 g/mol, 1.041 g, yield 37 %, yellow solid, Mp = 292°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.00 (dd, J

= 16.0, 3.6 Hz, 1H, H-1'(A)), 3.76 (s, 3H, 7-

OCH3), 3.86-3.95 (m, 1H, H-1’(M)), 3.93, 3.95

and 3.96 (s, 9H, 5-OCH3, 3””-OCH3, 4””-

OCH3), 5.94 (d, J = 2.3 Hz, 1H, H-8), 6.12-

6.16 (m, 1H, H-2(X)), 6.15 (d, J = 2.3 Hz, 1H,

H-6), 6.75 (d, J = 15.4 Hz, 1H, Hα-2’’’), 6.81 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H, H-5”), 6.89 (d, J

= 8.3 Hz, 1H, H-5””), 6.98 (dd, J = 8.4, 1.1, Hz, 1H, H-3”), 7.10 (d, J = 2.0 Hz, 1H, H-2””),

7.16 (dd, J = 8.5, 2.0 Hz, 1 H, H-6””), 7.44-754 (m, 2H, H-4”, H-6”), 7.66 (d, J = 15.4 Hz, 1

H, Hβ-3’’’), 12.27 (s, 1H, 2”-OH), 16.77 (s, 1H, 1’’’-OH) ppm. 13C NMR (75.47 MHz, CDCl3):

δ = 43.5 (C-1’), 55.6 (7-OCH3), 55.92, 55.98 and 56.2 (5-OCH3, 3””-OCH3, 4””-OCH3), 72.0

(C-2), 93.9 (C-6), 94.6 (C-8), 105.0 (C-3, C-10), 110.0 (C-2””), 111.1 (C-5””), 115.0 (C-2’’’),

118.6 (C-3”), 118.9 (C-5”), 119.4 (C-1”), 122.4 (C-6””), 128.1 (C-1””), 130.3 (C-6”), 136.9

(C-4”), 140.8 (C-3’’’), 149.2 (C-3””), 151.0 (C-4””), 160.7 (C-9), 162.1 (C-5), 162.7 (C-2”),

166.0 (C-7), 170.8 (C-1’’’), 182.3 (C-4), 203.0 (C-2’) ppm. HRMS (ESI+): m/z calcd for

[C30H28O9 + Na]+: 555.1631; found: 555.1651.

6.2.10. General procedure for the base catalyzed aldol-condensation of

cinnamaldehydes with (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-methyl-

4H-chromen-4-one 13f: Synthesis of 3-(HOPO-1)-2-(4-arylbuta-1,3-

dienyl)chromones 15a-c

13f (3.26 mmol, 1g) was added to an ethanol (10 mL) solution of appropriate

cinnamaldehyde (RCH=CHCHO, 3.27 mmol), to which 5 mL of an ethanolic solution of

metallic sodium (16.32 mmol, 5 equiv, 0.375 g) is added dropwise under stirring. The

reaction mixture is allowed to stirring for 1 hour at room temperature. The TLC monitoring

shows a quasi-total consumption of the starting material 13f. After that, the whole

ethanolic solution is poured into ice (50 g) and water (100 mL) and brought to pH 5-6

using 10% HCl solution, a red-orange precipitate appears which is directly extracted with

2 x 100 mL of dichloromethane. The combined organic layers are 2 times washed with

brine and then distilled-water. After drying the dichloromethane solution on Na2SO4, the

solvent volume is reduced to 5 mL under low pressure. 5 mL of ethanol is added to the

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dichloromethane solution which is then eliminated by heating to 40 °C, subsequently, a

red-orange solid of compounds 15a-c precipitate in the ethanol solution which is allowed

to cool down. The obtained pure solid was filtrated, washed with cold ethanol and finally

dried under vacuum.

15a: (E,E,E)-3-[-3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-(4-phenyl)buta-1,3-dienyl)-

4H-chromen-4-one, C28H20O4 (MW = 420.46 g/mol, 0.920 g, yield 67%, yellow-orange

solid, Mp = 217-218°C).

1H NMR (300.13 MHz, CDCl3): δ = 6.96 (ddd, J = 8.2, 7.2,

1.2 Hz, 1H, H-5””), 7.02 (dd, J = 8.4, 1.1 Hz, 1H, H-3””),

7.04-7.19 (m, 3H, Hγ-3’, Hδ-4’, Hα-1’), 7.31-7.57 (m, 10H,

H-3”/5”, H-2”/6”, H-4”, H-6, H-8, H-4””), 7.64 (dd, J = 15.0,

10.2 Hz, 1H, Hβ-2’), 7.68-7.75 (m, 1H, H-7), 8.02 (d, J =

15.0 Hz, 1H, Hβ-1”’), 8.09 (dd, J = 8.2, 1.6 Hz, 1H, H-6””),

8.26 (dd, J = 7.9, 1.6 Hz, 1H, H-5), 8.95 (d, J = 15.0 Hz,

1H, Hα-2”’), 12.94 (s, 1H, 2””-OH) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 114.7 (C-3), 117.5 (C-8), 118.3 (C-3””),

118.9 (C-5””), 119.7 (C-1’), 120.2 (C-1””), 123.4 (C-10),

125.0 (C-2’”), 125.4 (C-6), 126.1 (C-5), 127.3 (C-3’, C-

3”/5”), 128.9 (C-2”/6”),129.4 (C-4”), 130.4 (C-6””), 134.0 (C-7), 134.3 (C-1”’), 136.0 (C-1”),

136.3 (C-4””), 140.8 (C-2’), 141.1 (C-4’), 154.6 (C-9), 163.0 (C-2), 163.5 (C-2””), 177.1 (C-

4), 194.8 (C-3”’) ppm. HRMS (ESI+): m/z calcd for [C28H20O4 + Na]+: 443.1259; found:

443.1246.

15b: (E,E,E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-

enyl]-2-[4-(4-methoxyphenyl)buta-1,3-dienyl]-4H-

chromen-4-one, C29H22O5 (MW = 450.48 g/mol, 1.067

g, yield 73%, red-orange solid, Mp = 235-236°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.86 (s, 3H, 4”-

OCH3), 6.91-7.05 (m, 6H, H-5””, H-3””, Hγ-3’, Hδ-4’, H-

3”/5”), 7.09 (d, J = 14.6 Hz, 1H, Hα-1’), 7.40-7.54 (m,

5H, H-6, H-8, H-4””, H-2”/6”), 7.59-7.74 (m, 2H, Hβ-2”),

8.03 (d, J = 15.0 Hz, 1H, Hβ-1”’), 8.09 (dd, J = 8.2, 1.6

Hz, 1H, H-6””), 8.26 (ddd, J = 7.9, 1.7 Hz, 1H, H-5),

8.95 (d, J = 15.0 Hz, 1H, Hα-2”’), 12.97 (s, 1H, 2””-OH) ppm. 13C NMR (75.47 MHz,

O

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H3CO


228

CDCl3): δ = 55.4 (4”-OCH3), 114.3 (C-3), 114.5 (C-3”/5”), 117.5 (C-8), 118.3 (C-3””), 118.4

(C-1’), 118.9 (C-5””), 120.2 (C-1””), 123.4 (C-10), 124.7 (C-2”’), 125.3 (C-6, C-3’), 126.2

(C-5), 127.7 (C-1”), 128.9 (C-2”/6”), 130.4 (C-6””), 133.9 (C-7), 134.6 (C-1”’), 136.3, (C-

4””), 141.0 (C-2’), 141.4 (C-4’), 154.6(C-9), 160.8 (C-4”) , 163.4 (C-2””), 163.6 (C-2), 177.2

(C-4), 194.9 (C-3”’) ppm. HRMS (ESI+): m/z calcd for [C29H22O5 + Na]+: 473.1365; found:

473.1364.

15c: (E,E,E)-3-[-3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-2-[4-(3,4-

dimethoxyphenyl)buta-1,3-dienyl]-4H-chromen-4-one, C30H24O6 (MW = 480.51 g/mol,

1.265 g, yield 81%, red-orange solid, Mp = 225-226°C).


6H, 3”-OCH3, 4”-OCH3), 6.87 (d, J = 8.0 Hz, 1H, H-5”),

6.91-7.11 (m, 7H, H-5””, H-3””, Hγ-3’, Hδ-4’, H-6”, H-2”,

Hα-1’), 7.41 (ddd, J = 8.0, 7.1, 1.1, Hz, 1H, H-6), 7.45-

7.52 (m, 2H, H-8, H-4””), 7.60 (ddd, J = 14.8, 7.2, 3.1

Hz, 1H, Hβ-2’), 7.68 (ddd, J = 8.7, 7.1, 1.6 Hz, 1H, H-

7), 8.01 (d, J = 15.0 Hz, 1H, Hβ-1”’), 8.09 (dd, J = 8.2,

1.6 Hz, 1H, H-6””), 8.23 (dd, J = 8.0, 1.6 Hz, 1H, H-5),

8.94 (d, J = 15.0 Hz, 1H, Hα-2”’), 12.95 (s, 1H, 2””-OH)

ppm. 13C NMR (75.47 MHz, CDCl3): δ = 55.87 and

55.97 (3”-OCH3, 4”-OCH3), 108.6 (C-2”), 111.1 (C-5”),

114.2 (C-3), 117.4 (C-8), 118.2 (C-3””), 118.5 (C-1’), 118.9 (C-5””), 120.2 (C-1””), 121.9

(C-6”), 123.3 (C-10), 124.7 (C-2”’), 125.3 (C-6), 125.5 (C-3’), 126.1 (C-5), 129.1 (C-1”),

130.4 (C-6””), 133.9 (C-7), 134.5 (C-1”’), 136.3 (C-4””), 141.12 and 141.16 (C-2’, C-4’),

149.3 (C-3”), 150.5 (C-4”), 154.5 (C-9), 163.3 (C-2), 163.5 (C-2””), 177.1 (C-4), 194.8 (C-

3”’) ppm. HRMS (ESI+): m/z calcd for [C30H24O6 + H]+: 481.1651; found: 481.1640.

6.2.11. General procedure for condensation of formyl precursors with 2-

hydroxypyran-2-ones: Synthesis of bispyran-2-ones 16, 17, 18, 19 and 20

4-hydroxypyran-2-one derivatives (6 [4-hydroxycoumarin] or 7 [triacetic acid lactone],

12.34 mmol) was added to formyl-functionalized precursors (4a, 4a(5-Cl), 4d, 4e [ω-

formyl-2’-hydroxyacetophenone] or 5a-e [3-chromone carbaldehydes], 6.17 mmol, 0.5

equiv) in chloroform (20 mL), a catalytic amount of 4-PPy (0.62 mmol, 0.09 g, 0.05 equiv)

is dropped into the solution which is allowed to stirring overnight in case of using ω-formyl-

2’-hydroxyacetophenone reagents, or for a maximum of 3 hours in case of using 3-

O

O

12

34

5

6

7

89

10

1'

2'

1"'

2"'

6"

5"

4" 3"2"

1"

3'

4'

O

HO 2""

3""

4""5""

6""1""

3"'

H3CO

OCH3


229

fromylchromone reagents. After TLC monitoring, the solvent is evaporated and the

resulting resinous solid is directly recrystallized from ethanol to afford the bispyran-2-ones

16, 17, 19 and 20. Compound 17a, 19d, 19e and 20 were isolated by precipitation in light

petroleum after a purification step by short plug silica gel column chromatography using

dichloromethane as eluent.

16a: 3,3'-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-2H-chromen-2-

one), C27H18O8 (yellowish white solid, MW = 470.43 g/mol, 2.410 g, yield 83 %, Mp = 223-

225°C).

1H NMR (300.13 MHz, CDCl3): δ = 4.21 (d, J = 6.7 Hz, 2H,

H-2(AB)), 5.44 (t, J = 6.7 Hz, 1H, H-1(X)), 6.86-6.97 (m,

2H, H-3”’, H-5’”), 7.30-7.41 (m, 4H, H-6’/6”, H-8’/8”), 7.42-

7.48 (m, 1H, H-4”’), 7.57-7.62 (m, 2H, H-7’/7”), 7.85-7.88

(m, 1H, H-6’”), 8.01 (br, 2H, H-5’/5”), 11.45 and 12.10 (br,

2H, 4’/4”-OH), 11.92 (s, 1H, 2”’-OH) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 26.7 (C-1), 37.8 (C-2), 105.3 and 106.0 (C-3’/3”), 116.6 (C-8’/8”), 118.5

(C-3”’), 119.0 (C-1”’), 119.1 (C-5”’), 124.0 (C-10’/10”), 124.4 (C-5’/5”), 125.0 (C-6’/6”),

129.6 (C-6”’), 132.8 (C-7’/7”), 136.7 (C-4”’), 152.2 (C-9’/9”), 162.2 (C-2’”), 164.1 and 165.3

(C2’/2”, C4’/4”), 202.6 (C-3) ppm. HRMS (ESI+): m/z calcd for [C27H18O8 + Na]+: 493.0899;

found: 493.0904. Anal. Calcl: C, 68.94; H, 3.86. Found: C, 68.96 ; H, 3.89.

16d: 3,3'-[3-(2-hydroxy-4-methoxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-2H-

chromen-2-one), C28H20O9 (pale yellow solid, MW = 500.45 g/mol, 2.352 g, yield 76 %,

Mp = 133-135°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.81 (s, 3H, 4”’-

OCH3), 4.08 (d, J = 6.8 Hz, 2H, H-2(AB)), 5.44 (t, J =

6.8 Hz, 1H, H-1(X)), 6.37 (d, J = 2.5 Hz, 1H, H-3”’),

6.45 (dd, J = 9.0, 2.5 Hz, 1H, H-5’”), 7.31-7.42 (m, 4H,

H-6’/6”, H-8’/8”), 7.56-7.61 (m, 2H, H-7’/7”), 7.76 (d, J =

9.0 Hz, 1H, H-6’”), 8.01 (br, 2H, H-5’/5”), 11.44 and

12.10 (br, 2H, 4’/4”-OH), 12.40 (s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ =

26.8 (C-1), 37.2 (C-2), 55.5 (4”’-OCH3), 100.9 (C-3”’), 105.4 and 105.9 (C-3’/3”), 107.9 (C-

5”’), 113.2 (C-1”’), 116.5 (C-8’/8”), 124.0 (C-10’/10”), 124.4 (C-5’/5”), 124.8 (C-6’/6”), 131.2

(C-6”’), 132.7 (C-7’/7”), 152.1 (C-9’/9”), 162.2 (C-2’”), 165.2 (C-4”’), 164.1 and 168.5

OOH

OO

OHO

OH

O

1

23

3'

2' 1'

4'5'

6'

7'

8'9'

10'

1"'2"'

3"'

4"'5"'

6"'

2"

1"

3"

4"

5"

6"7"

8"9" 10"

OOH

OO

OHO

OH

O 1

23

3'

2' 1'

4'

5'6'

7'

8'9'

10'

1"'2"'

3"'

4"'5"'

6"'

2"

1"

3"

4"

5"

6"7"

8"9" 10"

H3CO


230

(C2’/2”, C4’/4”), 200.5 (C-3) ppm. HRMS (ESI+): m/z calcd for [C28H20O9 + Na]+: 523.1005;

found: 523.0999. Anal. Calcl: C, 67.20; H, 4.03. Found: C, 67.19; H, 4.05.

16e: 3,3'-[3-(2-hydroxy-4,6-dimethoxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-

2H-chromen-2-one), C29H22O10 (pale yellow solid, MW = 530.48 g/mol, 0.820 g, yield 25

%, Mp = 138-141°C).


6H, 4”’-OCH3, 6”’-OCH3), 4.10-4.25 (m, 2H, H-

2(AB)), 5.39 (t, J = 6.8 Hz, 1H, H-1(X)), 5.92 (d, J =

2.4 Hz, 1H, H-4”’), 6.01 (d, J = 2.4 Hz, 1H, H-6”’),

7.34-7.38 (m, 4H, H-6’/6”, H-8’/8”), 7.56-7.60 (m 2H,

H-7’/7”), 8.02 (br, 2H, H-5’/5”), 11.40 and 12.03 (br,

2H, 4’/4”-OH), 13.63 (s, 1H, 2”’-OH). 13C NMR (75.47 MHz, CDCl3): δ = 27.04 (C-1), 43.3

(C-2), 55.5 and 55.7 (4”’-OCH3, 6”’-OCH3), 90.9 (C-3”’), 93.6 (C-5”’), 105.9 and 106.4 (C-

3’/3”), 105.5 (C-1”’), 116.5 (C-8’/8”), 124.0 (C-10’/10”), 124.3 (C-5’/5”), 124.6 (C-6’/6”),

132.5 (C-7’/7”), 152.0 (C-9’/9”), 162.7 (C-2’”), 166.1 (C-4”’, C-6”’), 163.8, 164.8 and 167.5

(C2’/2”, C4’/4”), 201.6 (C-3) ppm. HRMS (ESI+): m/z calcd for [C29H22O10 + Na]+:

553.1111; found: 553.1106. Anal. Calcl: C, 65.66; H, 4.18. Found: C, 65.68; H, 4.19 .

16a(5-Cl): 3,3'-[3-(5-chloro-2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-

2H-chromen-2-one), C27H17ClO6 (pale yellow solid, MW = 504.87 g/mol, 2.124 g, yield 68

%, Mp = 211-212°C).


H-2(AB)), 5.41 (t, J = 6.6 Hz, 1H, H-1(X)), 6.90 (d, J = 8.9

Hz, 1H, H-3”’), 7.33-7.43 (m, 5H, H-4”’, H-6’/6”, H-8’/8”),

7.58-7.63 (m, 2H, H-7’/7”), 7.81 (d, J = 2.5 Hz, 1H, H-6’”),

8.01 (br, 2H, H-5’/5”), 11.45 and 12.10 (br, 2H, 4’/4”-OH),

11.81 (s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3):

δ = 26.6 (C-1), 37.9 (C-2), 105.1 and 105.8 (C-3’/3”), 116.6 (C-8’/8”), 119.5 (C-1”’), 120.9

(C-3”’), 123.8 (C-5”’), 124.0 (C-10’/10”), 124.4 (C-5’/5”), 125.0 (C-6’/6”), 128.7 (C-6”’),

132.8 (C-7’/7”), 136.6 (C-4”’), 152.2 (C-9’/9”), 160.7 (C-2’”), 164.2 and 165.4 (C2’/2”,

C4’/4”), 201.9 (C-3) ppm. HRMS (ESI+): m/z calcd for [C27H17ClO6 + Na]+: 527.0510;

found: 527.0513. Anal. Calcl: C, 64.23; H, 3.39. Found: C, 64.26; H, 3.42.

OOH

OO

OHO

OH

O

1

23

3'

2' 1'

4'5'

6'

7'

8'9'

10'

1"'2"'

3"'

4"'5"'

6"' 2"

1"

3"

4"

5"

6"7"

8"9" 10"

H3CO OCH3

OOH

OO

OHO

OH

O

1

23

3'

2' 1'

4'5'

6'

7'

8'9'

10'

1"'2"'

3"'

4"'5"'

6"'

2"

1"

3"

4"

5"

6"7"

8"9" 10"

Cl


231

17a: 3,3'-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-6-methyl-2H-

chromen-2-one), C21H18O8 (pale yellow solid, MW = 398.36 g/mol, 1.356 g, yield 55 %,

Mp = 189-191°C).


6’/6”-CH3), 4.03 (br, 2H, H-2(AB)), 5.10 (t, J = 6.8 Hz, 1H, H-

1(X)), 6.02 (s, 2H, H-5’/5”), 6.85-6.99 (m, 2H, H-3”’, H-5”’),

7.46 (ddd, J = 8.5, 7.2, 1.5 Hz, 1H, H-4”’), 7.83 (dd, J = 8.1,

1.5 Hz, 1H, H-6”’), 11.03 and 11.36 (br, 2H, 4’/4”-OH), 12.00

(s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 19.5 (6’/6”-CH3), 25.3 (C-1), 37.2

(C-2), 103.2 (C-3’/3”), 104.3 (C-6’/6”), 118.4 (C-3”’), 119.0 (C-5”’, C-1”’), 129.6 (C-6”’),

136.5 (C-4”’), 161.4 (C-6’/6”), 162.1 (C-2”’), 169.0 and 169.5 (C2’/2”, C4’/4”), 202.9 (C-3)

ppm. HRMS (ESI+): m/z calcd for [C21H18O8 + H]+: 399.1080; found: 399.1086. Anal. Calcl:

C, 63.32; H, 4.55. Found: C, 63.28; H, 4.59.

General procedure for bispyran-4-one 18 (bischromones) synthesis: Chromone-3-

carboxylic acid 2 (0.67 g, 3.5 mmol) was added to 2-aminoethanol (0.21 mL, 3.5 mmol, 1

equiv) in chloroform (20 mL). A catalytic amount of 4-PPy (0.52 mmol, 0.08 g, 0.05 equiv)

is dropped into the solution which is allowed to reflux for one hour and then, further 2

equivalents of 2 (1.33 g, 7 mmol) are added to the reaction mixture which stays under

stirring and reflux overnight. After TLC monitoring, the solvent is evaporated and the

resulting resinous solid is directly recrystallized from ethanol to afford compound 18.

18: 3,3'-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4H-chromen-4-one),

C27H18O6 (white solid, MW = 438.43 g/mol, 0.742 g, yield 48 %, Mp = 270-272°C).

1H NMR (300.13 MHz, CDCl3): δ = 4.06 (d, J = 7.4 Hz, 2H, H-

2), 4.88 (t, J = 7.4 Hz, 1H, H-1), 6.87-6.97 (m, 2H, H-3’”, H-

5’”), 7.33-7.49 (m, 5H, H-4”’, H-6’/6”, H-8’/8”), 7.64 (ddd, J =

8.6, 7.1, 1.7 Hz, 2H, H-7’/7”), 7.39 (dd, J = 8.1, 1.6 Hz, 1H, H-

6”’), 8.17 (dd, J = 8.0, 1.7 Hz, 1H, H-5’/5”), 8.41 (s, 2H, H-

2’/2”), 12.1 (s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz,

CDCl3): δ = 30.44 (C-2), 38.47 (C-1), 118.19 (C-8’/8”), 118.46 (C-5”’), 119.05 (C-3”’),

119.38 (C-1”’), 122.50 (C-3’/3”), 124.09 (C-10’/10”), 125.08 (C-6’/6”), 125.61 (C-5’/5”),

129.94 (C-6”’), 133.56 (C-7’/7”), 136.50 (C-4”’), 155.28 (C-2’/2”), 156.13 (9/9”), 162.35 (C-

2”’), 177.37 (C-4’/4”), 203.82 (C-3) ppm. HRMS (ESI+): m/z calcd for [C27H18O6 + H]+:

439.1182; found: 439.1169. Anal. Calcl: C, 73.97; H, 4.14. Found: C, 73.92; H, 4.15.

O

O

OOH

O

O

1

23

3'

2'

1'

4' 5'

6'

7'

8'9'

10'

1"'2"'

3"'

4"'

5"'

6"'

2"

1"

3"4"

5"

6"

7"8"

9"10"

OOH

OO

OHO

OH

O

1

23

3'

2' 1'

4'5'

6'

1"'2"'

3"'

4"'5"'

6"'

2"

1"

3"

4"

5"6"


232

19a: 3,3'-[(4-oxo-4H-chromen-3-yl)methylene]bis(4-hydroxy-2H-chromen-2-one),

C28H16O8 (white yellowish solid, MW = 480.42 g/mol, 2.324 g, yield 78 %, Mp = 236-

238°C).

1H NMR (500.13 MHz, d6-DMSO): δ = 6.03 (d, J = 1.6 Hz, 1H,

H-1), 7.27-7.39 (m, 4H, H-6/6’, H-8/8’), 7.42-7.47 (m, 1H, H-

6”), 7.57 (ddd, J = 8.5, 7.3, 1.6 Hz, 2H, H-7/7’), 7.62 (d, J = 8.5

Hz, 1H, H-8”), 7.77 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H, H-7”), 7.89

(dd, J = 8.0, 1.6 Hz, 2H, H-5/5’), 7.99 (dd, J = 8.1, 1.8 Hz, 1H,

H-5”), 8.00 (d, J = 1.6 Hz, 1H, H-2”) ppm. 13C NMR (75.47

MHz, d6-DMSO): δ = 30.4 (C-1), 103.2 (C-3/3’), 115.9 (C-

8/8’), 117.9 (C-8”), 118.2 (C-10/10’), 122.5 (C-3”), 122.9(C-

10”), 123.5 (C-5/5’), 123.6 (6/6’), 125.1 (C-5” and C-6”), 131.5 (C-7/7’), 133.8 (C-7”), 152.2

(C-9/9’), 153.3 (C-2”), 155.8 (C-9”), 163.4 (C-2/2’), 164.1 (C-4/4’), 176.1 (C-4”) ppm.


19b: 3,3'-[(6-methyl-4-oxo-4H-chromen-3-yl)methylene]bis(4-hydroxy-2H-chromen-2-

one), C29H18O8 (white yellowish solid, MW = 494.44 g/mol, 2.091 g, yield 69 %, Mp = 244-

246°C).

1H NMR (500.13 MHz, d6-DMSO): δ = 2.39 (s, 3H, 6”-CH3),

6.02 (d, J = 1.5 Hz, 1H, H-1), 7.25-7.37(m, 4H, H-6/6’, H-8/8’),

7.51 (d, J = 8.6 Hz, 1H, H-8”), 7.54-7.61(m, 3H, H-7/7’, H-7”),

7.75 (d, J = 2.2 Hz, 1H, H-5”), 7.88 (dd, J = 7.9, 1.5 Hz, 2H,

H-5/5’), 7.96 (d, J = 1.5 Hz, 1H, H-2”) ppm. 13C NMR (75.47

MHz, d6-DMSO): δ = 20.4 (6”-CH3), 30.4 (C-1), 103.1 (C-

3/3’), 115.9 (C-8/8’), 117.9 (C-8”, C-10/10’), 122.2 (C-3”),

122.6 (C-10”), 123.4 and 123.6 (C-6/6’, C-5/5’), 124.3 (C-5”),

131.5 (C-7/7’), 134.5 (C-7”), 134.8 (C-6”), 152.2 (C-9/9’), 153.1 (C-2”), 154.1 (C-9”), 163.4

(C-2/2’), 164.1 (C-4/4’), 176.0 (C-4”) ppm. HRMS (ESI+): m/z calcd for [C29H18O8 + Na]+:

517.0899; found: 517.0871.

O

OO

OHO

OH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"

5"

6"

10"

4"

21

4

5

67

8

9

10

2'

1'9' 8'

7'

6'5'

10'

O

OO

OHO

OH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"

5"

6"

10"

4"

21

4

5

67

8

9

10

2'

1'9' 8'

7'

6'5'

10'


233

19c: 3,3'-[(6-chloro-4-oxo-4H-chromen-3-yl)methylene]bis(4-hydroxy-2H-chromen-2-

one), C28H15ClO8 (white yellowish solid, MW = 514.86 g/mol, 2.370 g, yield 75 %, Mp =

251-252°C).

1H NMR (500.13 MHz, d6-DMSO): δ = 6.01 (d, J = 1.6 Hz,

1H, H-1), 7.24-7.36 (m, 4H, H-6/6’, H-8/8’), 7.57 (ddd, J =

8.2, 7.2, 1.6 Hz, 2H, H-7/7’), 7.70 (d, J = 9.0 Hz, 1H, H-8”),

7.81 (dd, J = 9.0, 2.7 Hz, 1H, H-7”), 7.89 (dd, J = 7.9, 1.6 Hz,

2H, H-5/5’), 7.92 (d, J = 2.7 Hz, 1H, H-5”), 8.03 (d, J = 1.6

Hz, 1H, H-2”) ppm. 13C NMR (75.47 MHz, d6-DMSO): δ =

30.4 (C-1), 102.9 (C-3/3’), 115.9 (C-8/8’), 118.0 (C-10/10’),

120.8 (C-8”), 122.7 (C-3”), 123.5 and 123.6 (C-6/6’, C-5/5’),

123.9 (C-10”), 124.1 (C-5”), 129.6 (C-6), 131.5 (C-7/7’), 133.8

(C-7”), 152.2 (C-9/9’), 153.6 (C-2”), 154.4 (C-9”), 163.3 (C-2/2’), 164.3 (C-4/4’), 175.1 (C-

4”) ppm. HRMS (ESI+): m/z calcd for [C28H15ClO8 + Na]+: 537.0353; found: 537.0321.

19d: 3'-[(5-methoxy-4-oxo-4H-chromen-3-yl)methylene]bis(4-hydroxy-2H-chromen-2-

one), C29H18O9 (pale white solid, MW = 510.44 g/mol, 2.746 g, yield 87 %, Mp = 210-

213°C).

1H NMR (500.13 MHz, CDCl3): δ = 3.90 (s, 3H, 5”-OCH3), 5.97

(d, J = 1.8 Hz, 1H, H-2”), 6.77 (dd, J = 8.4, 1.0 Hz, 1H, H-8”),

7.00 (dd, J = 8.5, 1.0 Hz, 1H, H-6”), 7.33-7.40 (m, 4H, H-6/6’,

H-8/8’), 7.53 (t, J = 8.4 Hz, 1H, H-7”), 7.57-7.61 (m, 2H, H-

7/7’), 7.80 (d, J = 1.8 Hz, 1H, H-2”), 8.01 (dd, J = 8.3, 1.6 Hz,

2H, H-5/5’), 11.44 (s, 2H, 4/4’-OH) ppm. 13C NMR (75.47

MHz, CDCl3): δ = 30.2 (C-1), 56.4 (5”-OCH3), 104.1 (C-3/3’),

106.2(C-8”), 109.9 (C-6”), 114.0 (C-10”), 116.5 and 116.7 (C-

8/8’, C-10/10’), 119.9 (C-3”), 124.3 and 124.8 (C-6/6’, C-5/5’),

132.7 (C-7/7’), 133.7 (C-7”), 151.7 (C-2”), 152.2 (C-9/9’), 158.2 (C-9”), 159.9 (C-5”), 164.4

(C-4/4’), 168.1 (C-2/2’), 176.2 (C-4”) ppm. HRMS (ESI+): m/z calcd for [C29H18O9 + Na]+:

533.0849; found: 533.0831.

O

OO

OHO

OH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"5"

6"

10"

4"

214

5

67

8

910

2'

1'9' 8'

7'

6'5'

10'

OCH3

O

OO

OHO

OH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"

5"

6"

10"

4"

21

4

5

67

8

9

10

2'

1'9' 8'

7'

6'5'

10'

Cl


234

19e: 3'-[(5,6-dimethoxy-4-oxo-4H-chromen-3-yl)methylene]bis(4-hydroxy-2H-

chromen-2-one), C30H20O10 (pale white solid, MW = 540.47 g/mol, 2.721 g, yield 82%, Mp

= 162-163°C).


OCH3, 6”-OCH3), 5.99 (d, J = 1.9 Hz, 1H, H-1), 7.01 (d, J = 9.0

Hz, 1H, H-8”), 7.36-7.39 (m, 4H, H-6/6’, H-8/8’), 7.61 (ddd, J =

8.7, 7.3, 1.6 Hz, 2H, H-7/7’), 7.83 (d, J = 9.0 Hz, 1H, H-7”),

7.92 (d, J = 1.7 Hz, 1H, H-2”), 8.03 (dd, J = 8.2, 1.6 Hz, 2H, H-

5/5’), 11.50 (s, 2H, 4/4’-OH) ppm. 13C NMR (75.47 MHz,

CDCl3): δ = 30.4 (C-1), 56.4 and 61.6 (5”-OCH3, 6”-OCH3),

103.9 (C-3”), 110.2 (C-8”), 116.5 and 116.6 (C-8/8’, C-10/10’),

118.0 and 118.2 (C-3”, C-10”), 121.3 (C-7”), 124.3 and 124.8

(C-6/6’, C-5/5’), 132.8 (C-7/7’), 150.6 (C-9”), 152.3 (C-9/9’), 153.1 (C-2”), 156.4 (C-5”, C-

6”), 164.3 (C-4/4’), 168.1 (C-2/2’), 176.4 (C-4”) ppm. HRMS (ESI+): m/z calcd for

[C30H20O10 + H]+: 541.1135; found: 541.1126.

20a: 3,3'-[(4-oxo-4H-chromen-3-yl)methylene]bis(4-hydroxy-6-methyl-2H-pyran-2-

one), C22H16O8 (white yellowish solid, MW = 408.35 g/mol, 1.456 g, yield 58 %, Mp = 197-

199°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.14 (d, J = 1.0 Hz, 6H, 6/6’-

CH3), 5.45 (d, J = 1.4 Hz, 1H, H-1), 5.94 (d, J = 1.0 Hz, 2H, H-

5/5’), 7.45 (ddd, J = 8.0, 7.1, 1.0 Hz, 1H, H-6”), 7.60 (dd, J = 8.6,

1.0 Hz, 1H, H-8”), 7.68 (d, J = 1.4 Hz, 1H, H-2”), 7.77 (ddd, J =

8.6, 7.1, 1.7 Hz, 1H, H-7”), 8.01 (dd, J = 8.0, 1.7 Hz, 1H, H-5”),

11.19 (s, 2H, 4/4’-OH) ppm.13C NMR (75.47 MHz, CDCl3): δ =

19.2 (6/6’-CH3), 28.7 (C-1), 100.0 (C-5/5’), 100.5 (C-3/3’), 118.1

(C-8”), 122.7 (C-10”), 123.3 (C-3”), 125.0 and 125.1 (C-6”, C-5”), 133.7 (C-7”), 152.4 (C-

2”), 155.8 (C-9”), 160.2 (C-6/6’), 164.1 (C-4/4’), 166.1 (C-2/2’), 176.0 (C-4”) ppm. HRMS

(ESI+): m/z calcd for [C22H16O8 + Na]+: 431.0743; found: 431.0754.

O

OO

OHO

OH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"

5"

6"

10"

4"

2

1

4

56

2'

1'

6'

5'

O

OO

OHO

OH

O

O

3

3'

3"

4'1

2"1"

9"

8"

7"5"

6"

10"

4"

214

5

67

8

910

2'

1'9' 8'

7'

6'5'

10'

OCH3

OCH3


235

6.2.12. General procedure for Michael addition of 2-hydroxypyran-2-ones 6 and 7 on

chalcones 8 and 3-(HOPO-1)-benzopyran-4-ones 13: synthesis of warfarin-

analogues 21 and benzopyran-(2 and 4)-one hybrids 22

4-hydroxypyran-2-one derivatives (6 [4-hydroxycoumarin] or 7 [triacetic acid lactone],

12.34 mmol) was added in excess (2 equivalents) to chalcone 8a-f or 3-(HOPO-1)-

benzopyran-4-ones 13a, 13d, 13f, 13g-i, 13n, 13o (6.17 mmol, 1 equiv) in chloroform (20

mL). A catalytic amount of 4-PPy (0.62 mmol, 0.09 g, 0.05 equiv) is dropped into the

solution which is allowed to reflux for 24 hours, which is the required time in most of

reaction cases, except for 13f, 13g-i, 13n, 13o reactions with 4-hydroxycoumarin 6, where

a maximum of 72 hours time is reached. After TLC monitoring which does not indicate a

total consumption of the limitant reagent [chalcone 8, 3-(HOPO-1)-benzopyran-4-ones

13], the solvent is evaporated and the resulting resinous solid is purified by column

chromatography or preparative TLC plates using dichloromethane/light petroleum 1:2 as

eluent. Most of the resulting pure compounds of warfarin-analogues 21 and benzopyran-

(2 and -4)-one hybrids 22 are precipitated in light petroleum except compounds 22o and

22n which have been recrystallized from ethanol.

21a: 4-hydroxy-3-(3-oxo-1,3-diphenylpropyl)-2H-chromen-2-one, C24H18O4 (white

solid, MW = 370.39 g/mol, 1.410 g, yield 62 %, Mp = 160-161°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.80 (dd, J = 19.2,

2.4 Hz, 1H, H-2’(A)), 4.49 (dd, J = 19.2, 10.0 Hz, 1H, H-

2’(M)), 4.95 (dd, J = 10.0, 2.4 Hz, 1H, H-1’(X)), 7.18-7.55

(m, 10H, H-3”/5”, H-4”, H-8, H-6, H-3”’/5”’, H-4”’, H-2”/6”),

7.59-7.67(m, 1H, H-7), 7.99 (dd, J = 8.0, 1.6 Hz, 1H, H-

5), 8.04-8.14 (m, 2H, H-2”’/6”’), 9.87 (s, 1H, 4-OH) ppm.


O

OH

O

O

12

3

8

7

45

6

1"

2'3' 3"'

2"'1'

4"

1"'

3"

2"

4"'

5"'6"'

9

10

6"

5"


236

21b: 4-hydroxy-3-[3-(2-hydroxyphenyl)-1-(4-methoxyphenyl)-3-oxopropyl]-2H-

chromen-2-one, C25H20O5 (white solid, MW = 400.42 g/mol,1.657 g, yield 67 %, Mp =

125-126°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.71 (dd, J = 19.1, 2.5

Hz, 1H, H-2’(A)), 3.78 (s, 3H, 4”-OCH3), 4.46 (dd, J =

19.1, 10.0 Hz, 1H, H-2’(M)), 4.90 (dd, J = 10.0, 2.5 Hz,

1H, H-1’(X)), 6.84 (d, J = 8.7 Hz, 2H, H-3”/5”), 7.18-7.37

(m, 4H, H-2”/6”, H-8, H-6), 7.43-7.54 (m, 3H, H-3”’/5”’, H-

4”’), 7.57-7.67 (m, 1H, H-7), 7.97 (dd, J = 7.9, 1.6 Hz, 1H,

H-5), 8.03-8.12 (m, 2H, H-2”’/6”’), 9.82 (s, 1H, 4-OH) ppm. HRMS (ESI+): m/z calcd for

[C25H20O5 + Na]+: 423.1208; found: 423.1193.

21b(TAL): 4-hydroxy-3-[1-(4-methoxyphenyl)-3-oxo-3-phenylpropyl]-6-methyl-2H-

pyran-2-one, C27H18O6 (resinous solid, MW = 438.43 g/mol, yield >5%).

1H NMR (300.13 MHz, CDCl3): δ = 2.08 (d, J = 0.9 Hz, 1H, 6-

CH3), 3.64 (dd, J = 18.4, 4.2 Hz, 1H, H-2’(A)), 3.75 (s, 3H, 4”-

OCH3), 4.36 (dd, J = 18.4, 9.5 Hz, 1H, H-2’(B)), 4.84 (dd, J =

9.5, 4.1 Hz, 1H, H-1’(X)), 5.90 (d, J = 0.9 Hz, 1H, H-5), 6.81

(d, J = 8.7 Hz, 2H, H-3”/5”), 7.36 (d, J = 8.3 Hz, 1H, H-2”/6”),

7.41-7.50 (m, 2H, H-3”’/5”’), 7.52-7.62 (m, 1H, H-4”’), 7.98-

8.06 (m, 2H, H-2”’/6”’), 9.83 (s, 1H, 4-OH) ppm.

21c: 3-{3-(2-hydroxyphenyl)-3-oxo-1-phenylpropyl}-4-hydroxy-2H-chromen-2-one,



Hz, 1H, H-2’(A)), 4.52 (dd, J = 19.0, 9.7 Hz, 1H, H-2’(M)),

4.94 (dd, J = 9.7, 2.9 Hz, 1H, H-1’(X)), 6.93-7.02 (m, 2H,

H-3’”, H-5”’), 7.20-7.36 (m, 5H, H-3”/5”, H-4”, H-6, H-8),

7.39-7.45 (m, 2H, H-2”/6”), 7.46-7.57 (m, 2H, H-7, H-4”’),

7.90-8.02 (m, 2H, H-5, H-6”’), 8.87 (s, 1H, 4-OH), 11.64

(s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 34.8 (C-1’), 39.5 (C-2’), 107.6 (C-

3), 116.6 (C-8, C-10), 118.7 (C-3”’), 118.9 (C-1”’), 119.5 (C-5”’), 123.8 and 123.9 (C-6, C-

5), 126.9 (C-4”), 128.0 and 128.3 (C-2”/6”, C-3”/5”), 130.5 (C-6”’), 131.9 (C-7), 137.6 (C-

4”’), 139.5 (C-1”), 152.8 (C-9), 160.7 (C-4), 161.9 (C-2), 162.4 (C-2”’), 206.8 (C-3’) ppm.


O

OH

O

O

12

3

8

7

45

6

1"

2' 3' 3"'

2"'1'

4"OCH3

1"'

3"

2"

4"'

5"'6"'9

10

5"

6"

O

OH

O

O

12

3

45

6

1"

2'3' 3"'

2"'1'

4"OCH3

1"'

3"

2"

4"'

5"'6"'

5"

6"

O

OH

O

O OH

12

3

8

7

45

6

1"

2'3' 3"'

2"'1'

4"

1"'

3"

2"

4"'

5"'6"'

9

10

5"

6"


237

21d: 4-hydroxy-3-[3-(2-hydroxyphenyl)-1-(4-methoxyphenyl)-3-oxopropyl]-2H-

chromen-2-one, C25H20O6 (white solid, MW = 416.42 g/mol, 1.382 g, yield 54 %, Mp =

152-153°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.79 (s, 3H, 4”-

OCH3), 3.80 (dd, J = 19.0, 3.2 Hz, 1H, H-2’(A)), 4.48

(dd, J = 19.0, 9.6 Hz, 1H, H-2’(M)), 4.89 (dd, J = 9.6,

3.1 Hz, 1H, H-1’(X)), 6.86 (d, J = 8.7 Hz, 2H, H-3”/5”),

6.92-7.02 (m, 2H, H-3’”, H-5”’), 7.20-7.39 (m, 4H, H-8,

H-6, H-2”/6”), 7.44-7.56 (m, 2H, H-7, H-4”’), 7.93 (dd, J

= 7.9, 1.7 Hz, 1H, H-5), 7.97 (dd, J = 8.1, 1.7 Hz, 1H, H-6”’), 8.79 (s, 1H, 4-OH), 11.66 (s,

1H, 2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 34.2 (C-1’), 39.8 (C-2’), 55.2 (4”-

OCH3), 107.8 (C-3), 113.7 (C-3”/5”), 116.2 and 116.3 (C-8, C-10), 118.7 (C-3”’), 119.0 (C-

1”’), 119.5 (C-5”’), 123.7 and 123.9 (C-6, C-5), 129.1 (C-2”/6”), 130.5 (C-6”’), 131.5 (C-1”),

131.8 (C-7), 137.6 (C-4”’), 152.7 (C-9), 158.4 (C-4”), 160.5 (C-4), 162.0 (C-2), 162.4 (C-

2”’), 206.8 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C25H20O6 + Na]+: 439.1158; found:

439.1153.

21e: 3-[3-(2-hydroxyphenyl)-3-oxo-1-p-tolylpropyl]-4-hydroxy-2H-chromen-2-one,


1H NMR (300.13 MHz, CDCl3): δ = 2.32 (s, 3H, 4”-CH3),

3.82 (dd, J = 19.0, 3.2 Hz, 1H, H-2’(A)), 4.47 (dd, J =

19.0, 9.5 Hz, 1H, H-2’(M)), 4.91 (dd, J = 9.5, 3.2 Hz,

1H, H-1’(X)), 6.92-7.02 (m, 2H, H-5”’, H-3”’), 7.09-7.17

(m, 2H, H-3”/5”), 7.21-7.25 (m, 1H, H-8), 7.27-7.34 (m,

3H, H-6, H-2”/6”), 7.45-7.56 (m, 2H, H-7, H-4”’), 7.93

(dd, J = 8.0, 1.6 Hz, 1H, H-5), 7.97 (dd, J = 8.1, 1.6 Hz, 1H, H-6”’), 8.78 (s, 1H, 4-OH),

11.66 (s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 21.0 (4”-CH3), 34.5 (C-1’),

39.6 (C-2’), 107.7 (C-3), 116.2 (C-8), 116.3 (C-10), 118.7 (C-3”’), 119.0 (C-1”’), 119.5 (C-

5””), 123.7 and 123.8 (C-6, C-5), 127.8 (C-2”), 129.1 (C-3”), 130.4 (C-5), 131.8 (C-4”’),

136.4 (C-1”), 136.5 (C-4”), 137.5 (C-7), 152.7 (C-9), 160.6 (C-4), 162.2 (C-2”’), 162.4 (C-


423.1218.

O

OH

O

O OH

12

3

8

7

45

6

1"

2' 3' 3"'2"'1'

4"OCH3

1"'

3"

2"

4"'

5"

6"'9

10

5"'

6"

O

OH

O

O OH

12

3

8

7

45

6

1"

2' 3' 3"'2"'1'

4"

1"'

3"

2"

4"'

5"'6"'9

10

5"

6"


238

21f: 8-phenyl-8,14-methanobenzo[7,8][1,3]dioxocin-[5,4-c]chromen-1(14H)-one,

C24H16O4 (white solid, MW = 368.38 g/mol, 0.920 g, yield 41%, Mp = 244-245°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.42 (dd, J = 12.0, 3.2 Hz,

1H, H-2’(A)), 2.48 (dd, J =12.0, 3.0 Hz, 1H, H-2’(B)), 4.40 (t, J =

3.0 Hz, 1H, H-1’(X)), 6.95-7.09 (m, 2H, H-2”, H-4”), 7.17-7.33

(m, 3H, H-6, H-8, H-3”), 7.47-7.57 (m, 5H, H-4”’, H-3”’/5”’, H-7,

H-1”,), 7.72-7.80 (m, 2H, H-2”’/H-6”’), 7.89 (ddd, J = 7.9, 1.6, 0.5

Hz, 1H, H-5) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 27.1 (C-

1’), 32.9 (C-2’), 100.3 (C-3’, ketal), 106.0 (C-3), 115.1 (C-10),

116.3 (C-4”), 116.7 (C-8), 122.1 (C-2”), 122.7 (C-5), 124.0 (C-6), 125.1 (C-6”), 125.6 (C-

2”’), 128.2 (C-1”), 128.3 (C-3”), 128.6 (C-3”’), 129.4 (C-4”’), 132.0 (C-7), 139.7 (C-1),

151.3 (C-5”), 152.4 (C-9), 158.2 (C-4), 161.6 (C-2) ppm. HRMS (ESI+): m/z calcd for

[C25H20O5 + Na]+: 391.0946; found: 391.0937.

22a: 4-hydroxy-3-[3-(2-hydroxyphenyl)-3-oxo-1-(4-oxo-4H-chromen-3-yl)propyl]-2H-

chromen-2-one, C27H18O7 (white solid, MW = 454.43 g/mol, 1.090 g, yield 39 %, Mp =

166-167°C).


Hz, 1H, H-2’(A)), 4.31 (dd, J = 18.4, 8.6 Hz, 1H, H-2’(B)),

5.18 (dd, J = 8.6, 5.7 Hz, 1H, H-1’(X)), 6.87-6.98 (m, 2H,

H-3”’, H-5”’), 7.23-7.33 (m, 2H, H-6, H-8), 7.41-7.57 (m,

4H, H-8”, H-6”, H-7, H-4’”), 7.75 (ddd, J = 8.7, 7.1, 1.7 Hz,

1H, H-7”), 7.86-7.93 (m, 1H, H-6’”), 8.01 (ddd, J = 7.9,

1.6, 0.6 Hz, 1H, H-5), 8.28 (ddd, J = 8.1, 1.7, 0.5 Hz, 1H, H-5”), 8.52 (s, 1H, H-2”’), 12.01

(s, 1H, 2””-OH) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 25.0 (C-1’), 37.9 (C-2’), 105.0 (C-

3), 116.1 (C-8), 117.5 (C-10), 118.3 and 118.5 (C-8” and C-3’”), 119.1 (C-5’”) 119.2 (C-

1”’), 122.6 (C-10’’), 123.9 (C-6), 124.2 (C-5), 125.8-125.9 (C-3”, C-5”, C-6”) 129.7 (C-6”’),

131.9(C-7), 134.8 C-7”) 136.6 (C-4”’), 152.6 (C-9), 155.4 (C-2”), 156.5 (C-9”), 162.3 (C-

2”’), 163.1 (C-4 and C-2), 180.3 (C-4”), 203.2 (C-3’) ppm. HRMS (ESI+): m/z calcd for

[C27H18O7 + Na]+: 477.0950; found: 477.0934.

O

O

O

O

12

3

8

7

45

6

1"

2'

3'

3"'

2"'

1'

4"

1"'

3"

2"

4"'

5"6"

9

10

5"'

6"'

O

O

O

OH

O

OHO

1

2

3

4 5

6

7

8

1'2'3'

1"

2"3"

4"

5"

6"7"

8"

9

10

9"

10"

1"'2"'

3"'

4"'

5"'

6"'


239

22d: 4-hydroxy-3-[3-(2-hydroxyphenyl)-1-(7-methoxy-4-oxo-4H-chromen-3-yl)-3-

oxopropyl]-2H-chromen-2-one, C28H20O8 (white yellowish solid, MW = 484.45 g/mol,

1.276 g, yield 43 %, Mp = 125-126°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.91 (s, 1H, 7”-

OCH3), 3.93 (dd, J = 18.4, 5.7 Hz, 1H, H-2’(A)), 4.29 (dd,

J = 18.4, 8.6 Hz, 1H, H-2’(B)), 5.16 (dd, J = 8.6, 5.7 Hz,

1H, H-1’(X)), 6.86-6.97 (m, 3H, H-8”, H-3”’, H-5”’), 7.02

(dd, J = 9.0, 2.4 Hz, 1H, H-6”), 7.23-7.32 (m, 2H, H-8, H-

6), 7.43-7.53 (m, 2H, H-7, H-4”’), 7.86-7.93 (m, 1H, H-

6”’), 8.00 (dd, J = 7.9, 1.7 Hz, 1H, H-5), 8.16 (d, J = 9.0

Hz, 1H, H-5”), 8.42 (s, 1H, H-2”), 12.03 (s, 1H, 2”’-OH) ppm. 13C NMR (75.47 MHz,

CDCl3): δ = 26.0 (C-1’), 37.9 (C-2’), 56.0 (7”-OCH3), 99.8 (C-8”), 105.2 (C-3), 115.9 and

116.1 (C-6”, C-8, C-10, C-10”), 118.5 (C-3”’), 119.1 (C-5”’), 119.2 (C-1”’), 123.8 (C-6, C-

3”), 124.2 (C-5), 127.2 (C-5”), 129.7 (C-6”’), 131.9 (C-7), 136.5 (C-4”’), 152.6 (C-9), 156.3

(C-2”), 158.5 (C-9”), 162.3 (C-2”’), 163.2 (C-4, C-2), 165.0 (C-7”), 179.5 (C-4”), 203.3 (C-

3’) ppm. HRMS (ESI+): m/z calcd for [C28H20O8 + H]+: 485.1236; found: 485.1214.

22f: 4-hydroxy-3-[3-(2-hydroxyphenyl)-1-(2-methyl-4-oxo-4H-chromen-3-yl)-3-

oxopropyl]-2H-chromen-2-one, C28H20O7 (white solid, MW = 468.45 g/mol, 1.470 g, yield

51 %, Mp = 214-215°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.18 (s, 3H, 2”-CH3),

3.66 (dd, J = 18.5, 3.8 Hz, 1H, H2’(A)), 4.61 (dd, J =

18.5, 9.9 Hz, 1H, H-2’(B)), 5.47 (dd, J = 9.9, 3.8 Hz, 1H,

H-1’(X)), 6.84-6.97 (m, 2H, H-3”’, H-5”’), 7.23-7.32 (m,

2H, H-6, H-8), 7.36-7.58 (m, 4H, H-8”, H-6”, H-7, H-4’”),

7.71 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H, H-7”), 7.86 (dd, J =

8.0, 1.6 Hz, 1H, H-6”’), 8.01 (dd, J = 8.4, 1.7 Hz, 1H, H-5), 8.21 (dd, J = 8.1, 1.7 Hz, 1H,

H-5”), 12.07 (s, 1H, 2”’-OH), 13.78 (s, 1H, 4-OH) ppm. HRMS (ESI+): m/z calcd for

[C28H20O7 + Na]+: 491.1107; found: 491.1086.

O

O

O

OH

O

OHO

12

3

4 5

6

7

8

1'2'3'

1"

2"3"

4"

5"

6"7"

8"

9

10

9"

10"

1"'2"'

3"'

4"'

5"'6"'

H3CO

O

O

O

OH

O

OHO

12

3

4 5

6

7

8

1'2'3'

1"

2"3"

4"

5"

6"7"

8"

9

10

9"

10"

1"'2"'

3"'

4"'

5"'6"'


240

22f(TAL): 3-[1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-3-(2-hydroxyphenyl)-3-

oxopropyl]-2-methyl-4H-chromen-4-one, C25H20O7 (white yellowish solid, MW = 432.42

g/mol, 1.182 g, yield 44%, Mp = 105-106°C).


6-CH3), 3.12 (s, 3H, 2”-CH3), 3.59 (dd, J = 18.3, 4.0 Hz, 1H,

H2’(A)), 4.48 (dd, J = 18.4, 9.9 Hz, 1H, H-2’(B)), 5.30 (dd, J

= 9.9, 4.0 Hz, 1H, H-1’(X)), 5.85 (d, J = 0.9 Hz, 1H, H-5),

6.85-6.95 (m, 2H, H-3”’, H-5”’), 7.36-7.46 (m, 2H, H-4”’, H-

8”), 7.48 (dd, J = 8.6, 1.1 Hz, 1H, H-8”), 7.70 (ddd, J = 8.6,

7.1, 1.7 Hz, 1H, H-7”), 7.80-7.84(m, 1H, H-6”’), 8.18 (ddd, J = 8.1, 1.7, 0.5 Hz, 1H, H-5”),

12.08 (s, 1H, 2”’-OH), 13.10 (s, 1H, 4-OH) ppm. HRMS (ESI+): m/z calcd for [C25H20O7 +

Na]+: 455.1107; found: 455.1065.

22n: (E)-3-{1-[2-(3,4-dimethoxystyryl)-4-oxo-4H-chromen-3-yl]-3-(2-hydroxyphenyl)-

3-oxopropyl}-4-hydroxy-2H-chromen-2-one, C37H28O9 (orange solid, MW = 616.61

g/mol, 1.660 g, yield 44%, Mp = 236-238°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.95 (m, 1H, H-

2’(A)), 3.96 and 4.11 (s, 6H, 3a-OCH3, 4a-OCH3),

4.50 (dd, J = 18.3, 8.5 Hz, 1H, H-2’(B)), 5.78 (dd, J

= 8.5, 5.5 Hz, 1H, H-1’(X)), 6.84-6.91 (m, 2H, H-3b,

H-5b), 6.94 (d, J = 8.3 Hz, 1H, H-5a), 7.23-7.30 (m,

2H, H-6, H-8), 7.33-7.61 (m, 6H, H-6ª, H-2ª, H-6”,

H-4b, H-7, H-8”), 7.70-7.82 (m, 2H, H-7”, Hβ-2”’), 7.87 (dd, J = 8.1, 1.6 Hz, 1H, H-6b), 8.00

(dd, J = 8.2, 1.6 Hz, 1H, H-5), 8.23 (dd, J = 8.1, 1.6 Hz, 1H, H-5”), 8.49 (d, J = 15.5 Hz,

1H, Hα-1”’), 12.00 (s, 1H, 2b-OH), 13.99 (s, 1H, 4-OH) ppm. HRMS (ESI+): m/z calcd for

[C37H28O9 + H]+: 617.1812; found: 617.1813.

O

O

O

OH

O

OHO

1

2

3

45

6

1'2'3'

1"

2"3"

4"

5"

6"7"

8"

9"

10"

1"'2"'

3"'

4"'

5"'

6"'

O

O

O

OH

O

O

HO

12

34

5

6

78

1'

2'3'

1"

2"' 3"4"

5"

6"

7"

8"

9

10

9"

10"

1"'

2"'

6b

H3CO

1b2b3b

4b5b

1a

2a3a

4a

H3CO

6a5a


241

22o: (E)-4-hydroxy-3-{3-[2-hydroxyphenyl)-1-(2-(4-methoxystyryl)-4-oxo-4H-

chromen-3-yl]-3-oxopropyl}-2H-chromen-2-one C36H26O8 (yellow solid, MW = 586.59

g/mol, 1.766 g, yield 49 %, Mp = 213-214°C).

1H NMR (300.13 MHz, CDCl3): δ = 3.87 (s, 3H,

4ª-OCH3), 3.97 (dd, J = 18.3, 5.6 Hz, 1H, H-

2’(A)), 4.42 (dd, J = 18.3, 8.3 Hz, 1H, H-2’(B)),

5.78 (dd, J = 8.3, 5.6 Hz, 1H, H-1’(X)), 6.83-

6.91 (m, 2H, H-3b, H-5b), 6.99 (d, J = 8.8 Hz,

2H, H-3ª/5a), 7.23-7.31 (m, 2H, H-6, H-8), 7.36-

7.45 (m, 2H, H-6”, H-4b), 7.50 (ddd, J = 8.5,

7.2, 1.6 Hz, 1H, H-7), 7.58 (dd, J = 8.5, 1.1 Hz, 1H, H-8”), 7.74 (ddd, J = 8.6, 7.1, 1.6 Hz,

1H, H-7”), 7.79 (d, J = 15.7 Hz, 1H, Hβ-2”’), 7.82 (d, J = 8.8 Hz, 2H, H-2ª/6a), 7.86 (dd, J =

8.0, 1.6 Hz, 1H, H-6b), 8.00 (dd, J = 8.0, 1.6 Hz, 1H, H-5), 8.23 (dd, J = 8.1, 1.7 Hz, 1H, H-

5”), 8.50 (d, J = 15.7 Hz, 1H, Hα-1”’), 12.00 (s, 1H, 2b-OH), 14.05 (s, 1H, 4-OH) ppm. 13C

NMR (75.47 MHz, CDCl3): δ = 28.5 (C-1’), 38.5 (C-2’), 55.4 (4ª-OCH3), 105.4 (C-3), 114.5

(C-3a/5a), 116.0 (C-8), 116.5 (C-1”’), 117.4 (C-8’’), 117.5 (C-10), 118.4 and 118.9 (C-3b, C-

5b), 119.2 (C-1b), 119.6 (C-3”), 122.1 (C-10”), 123.7 (C-6), 124.1 (C-5), 125.3 (C-6’’),

125.7 (C-5’’), 128.4 (C-1ª), 129.7 (C-6b), 130.4 (C-2ª/6a), 131.9 (C-7), 134.6 (C-7”), 136.3

(C-4b), 140.5 (C-2”’), 152.5 (C-9), 155.3 (C-9”), 161.5 (C-4a), 162.1 (C-2b), 164.1 (C-4),

164.4 (C-2”), 164.9 (C-2), 181.1 (C-4’), 203.6 (C-3’) ppm. HRMS (ESI+): m/z calcd for

[C36H26O8 + Na]+: 609.1525; found: 609.1518.

22o(TAL): (E)-3-[1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-3-(2-hydroxyphenyl)-3-

oxopropyl)-2-(4-methoxystyryl)]-4H-chromen-4-one, C33H26O8 (white yellowish solid,

MW = 550.55 g/mol, yield >5% Mp = 278-280°C).

1H NMR (300.13 MHz, CDCl3): δ = 2.18 (d, J = 1.0 Hz,

3H, 6-CH3), 3.87 (s, 3H, 4ª-OCH3), 3.87 (m, H-2’(A)),

4.30 (dd, J = 18.2, 8.3 Hz, 1H, H-2’(B)), 5.59 (dd, J =

8.3, 5.8 Hz, 1H, H-1’(X)), 5.85 (d, J = 1.0 Hz, 1H, H-5),

6.84-6.93 (m, 2H, H-3b, H-5b), 6.99 (d, J = 8.7 Hz, 2H,

H-3ª/5a), 7.37-7.45 (m, 2H, H-6”, H-4b), 7.58 (d, J = 8.5

Hz, 1H, H-8”), 7.69-7.86 (m, 5H, H-7”, Hβ-2”’, H-2ª/6a, H-6b), 8.20 (dd, J = 8.0, 1.7 Hz, 1H,

H-5”), 8.45 (d, J = 15.6 Hz, 1H, Hα-1”’), 12.03 (s, 1H, 2b-OH), 13.29 (s, 1H, 4-OH) ppm.


O

O

O

OH

O

OHO

12

34

5

6

78

1'2'3'

1"

2"' 3"4"

5"

6"7"

8"

9

10

9"

10"

1"'

2"'

6b

H3CO

1b

2b3b

4b5b

1a

2a3a

4a

O

O

O

OH

O

OHO

12

3 45

6

1'2'

3'

1"

2"' 3"

4"

5"

6"7"

8"

9"

10"

1"'

2"'

6b

H3CO

1b

2b3b

4b5b

1a

2a3a

4a

6a5a


242

6.3. REFERENCES

[1] T. Kottke, D. Stalke, J. Appl. Crystallogr. 1993, 26, 615–619.

[2] APEX2, Data Collection Software, version 2.1-RC13; Bruker AXS: Delft, The

Netherlands, 2006.

[3] Cryopad, Remote monitoring and control, version 1.451; Oxford Cryosystems:

Oxford, U.K., 2006.

[4] SAINT+, Data Integration Engine, version 7.23a; Bruker AXS: Madison, WI, 2005.

[5] G. M. Sheldrick, SADABS, Bruker/Siemens Area Detector Absorption Correction

Program, version 2.01; Bruker AXS: Madison, WI, 1998.

[6] (a) G. M. Sheldrick, Acta Cryst. A. 2008, 64, 112-122; (b) G. M. Sheldrick, SHELXS-

97, Program for Crystal Structure Solution; University of Göttingen: Göttingen,

Germany, 1997.

[7] G. M. Sheldrick, SHELXL-97, Program for Crystal Structure Refinement; University of

Göttingen: Göttingen, Germany, 1997.

[8] a) Baker, W. Molecular rearrangement of some o-acyloxyacetophenones and the

mechanism of the production of 3-acylchromones. J. Chem. Soc., 1933, 1381-1389;

b) Mahal, H.S.; Venkataraman, K. Synthetical experiments in the chromone group.

Part XIV. The action of sodamide on 1-acyloxy-2-acetonaphthones. J. Chem. Soc.,

1934, 1767-1769.

[9] Hurd, C.D.; Webb, C.N. Vilsmeyer-Haack reaction of benzanilide and

dimethylaniline dimethylaniline. Org. Syn. Coll., 1941, 1, 217 and 1927, 7, 24.

[10] a) Pinto, D.C.G.A.; Silva, A.M.S.; Cavaleiro, J.A.S. Synthesis of 3-(2-Benzyloxy-6-


Methylhydrazine. J. Heterocycl. Chem., 2000, 37, 1629-1634; b) Barros, A.I.R.N.A.;

Silva, A.M.S. Efficient Synthesis of Nitroflavones by Cyclodehydrogenation of 2’-

Hydroxychalcones and by the Baker-Venkataraman Method. Monatsh. Chem., 2006,

137, 1505-1528.

[11] Filliatre, C.; Servens, C. Reaction of chromene-2-carboxylic acid with carbodiimides.

Synthesis of spiro(chroman-2:5'-hydantoin)-4-ones. J. Heterocycl. Chem., 1985, 22,

1009-10.

[12] Ulrichan, H. & Sayigh, D. A. A. R. J. The Reaction of oxalyl chloride with substituted

ureas and thioureas. J. Org. Chem., 1965, 30, 2781–2783.

[13] Ibrahim, M.A. Ring transformation of chromone-3-carboxylic acid under nucleophilic

conditions. Arkivoc, 2008, xvii, 192-204.





http://en.wikipedia.org/wiki/Dimethylaniline

http://en.wikipedia.org/wiki/Org._Syn.

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