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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
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.
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.
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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
Chapter 1 – General Introduction
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
Chapter 1 – General Introduction
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
Chapter 1 – General Introduction
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
Chapter 1 – General Introduction
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
Chapter 1 – General Introduction
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.
Chapter 1 – General Introduction
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
Chapter 1 – General Introduction
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.
Chapter 1 – General Introduction
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.
Chapter 1 – General Introduction
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.
Chapter 1 – General Introduction
13
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].
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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.
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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]
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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-
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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.
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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).
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
a. R= cyclohexylb. R= isopropylc. R= tolyl
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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)
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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.
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
30
9a-c 10a-c
a. R= cyclohexylb. R= isopropylc. R= tolyl
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).
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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’
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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’
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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"
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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).
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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.
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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.
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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].
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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.
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 2 – Synthesis of Hydantoin- & Uracil- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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.
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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-
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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].
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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.
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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.
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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]).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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-
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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-
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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’
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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”
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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'
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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.
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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,
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
116
Figure 3. 36. 3D prediction of the symmetric conformation of bischromone 18
Figure 3. 37. 1H NMR spectrum of compound 19a (500 MHz)
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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”
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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’
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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-
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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).
Figure 3. 49. 1H NMR spectrum of compound 22a (300 MHz)
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
132
Figure 3. 50. Partial HMBC spectrum of compound 22a (300 MHz)
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
133
Figure 3. 52. 1H NMR spectra compound 22o (300 MHz)
Figure 3. 53. Partial HMBC spectrum of compound 22a (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”
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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.
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
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.
Chapter 3 – Synthesis of Benzopyran-(2 & 4)-ones- Polyphenolic Hybrids
137
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CHAPTER 4
- BIOLOGICAL SCREENINGS -
Chapter 4 – Biological Screenings
147
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
Chapter 4 – Biological Screenings
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.
Chapter 4 – Biological Screenings
149
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
Chapter 4 – Biological Screenings
150
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
Chapter 4 – Biological Screenings
151
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
Chapter 4 – Biological Screenings
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
Chapter 4 – Biological Screenings
153
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].
Chapter 4 – Biological Screenings
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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.
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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).
Chapter 4 – Biological Screenings
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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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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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Chapter 4 – Biological Screenings
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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
Chapter 4 – Biological Screenings
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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
Chapter 4 – Biological Screenings
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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
Chapter 4 – Biological Screenings
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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.
Positive control (CTRL+) was performed with DMSO.
Chapter 4 – Biological Screenings
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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.
Positive control (CTRL+) was performed with DMSO.
Chapter 4 – Biological Screenings
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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).
Chapter 4 – Biological Screenings
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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
Chapter 4 – Biological Screenings
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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
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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
Chapter 4 – Biological Screenings
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
Chapter 4 – Biological Screenings
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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
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, ***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).
Chapter 4 – Biological Screenings
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
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 < 0.01, ***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+)
Chapter 4 – Biological Screenings
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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).
Chapter 4 – Biological Screenings
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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.
Chapter 4 – Biological Screenings
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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.
Positive control (CTRL+) was performed with DMSO.
Chapter 4 – Biological Screenings
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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.
Positive control (CTRL+) was performed with DMSO.
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
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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
substructures 2”-hydroxypropiophenone and 4-hydroxycoumarin
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.
2-HPP 4-HCOM
Chapter 4 – Biological Screenings
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Figure 4. 34. NF-κB pathway inhibition assay on K562 cells without TNF-α addition for
substructures 2”-hydroxypropiophenone and 4-hydroxycoumarin
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
Chapter 4 – Biological Screenings
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
Chapter 4 – Biological Screenings
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
Chapter 4 – Biological Screenings
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
μM. Positive control (CTRL+) was performed with DMSO
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
Chapter 4 – Biological Screenings
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
μM. Positive control (CTRL+) was performed with DMSO
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.
Chapter 4 – Biological Screenings
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.
Chapter 4 – Biological Screenings
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).
Chapter 4 – Biological Screenings
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.
Chapter 4 – Biological Screenings
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)
Chapter 4 – Biological Screenings
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.
Chapter 4 – Biological Screenings
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
Chapter 4 – Biological Screenings
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)
Chapter 4 – Biological Screenings
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
Chapter 5 – Conclusions & Perspectives
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.
Chapter 5 – Conclusions & Perspectives
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
Chapter 6 – Experimental Part
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: deposit@ccdc.cam.ac.uk.
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
Chapter 6 – Experimental Part
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 / ° — —
Chapter 6 – Experimental Part
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
Independent reflections 15362
[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
Weighting schemec m = 0.0530
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
Chapter 6 – Experimental Part
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
Independent reflections 2929
[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
Weighting schemec m = 0.0404
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
Chapter 6 – Experimental Part
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].
Chapter 6 – Experimental Part
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
1056
78
9
3'
2'
1'
5'
1''
1'''
O
O
N
NO
O
1
2
34
1056
78
9
3'
2'
1'
5'
1''
1'''
O
O
N
NO
O
Chapter 6 – Experimental Part
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
1056
78
9
3'
2'
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5'
1''
1'''
O
O
N
NO
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3'''
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2''
3''
4''
Chapter 6 – Experimental Part
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
1
5'
6'
7' 8'
9' 3
2
4
4' 1'''
O
O
N
NO
O
HH
1''
1'
2'3' 5
1
5'
6'
7' 8'
9' 3
2
4
4' 1'''
O
O
N
NO
O
HH
1''
Chapter 6 – Experimental Part
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%.
1''
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OH 12
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Chapter 6 – Experimental Part
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'
2'3'
5
1
5'
6'7'
8'
9' 3
2
44'
1''
1'''
O
O
N
NO
O
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 2.42 and 2.45 (s, 6H, 4”-
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”’),
1'
2'3'
5
15'
6'7'
8'
9'
3
2
4
4'
1''
1'''O
O
N
N
O
O
1'
2'3'
5
1
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6'7'
8'
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2
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O
O
N
NO
O
1'
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5
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Chapter 6 – Experimental Part
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'
Chapter 6 – Experimental Part
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
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O O
O
HN
O
1"
1"'
Chapter 6 – Experimental Part
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"'
Chapter 6 – Experimental Part
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"
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 2.39 and 2.40 (s, 6H,
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"
Chapter 6 – Experimental Part
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"
Chapter 6 – Experimental Part
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"
Chapter 6 – Experimental Part
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.
Chapter 6 – Experimental Part
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
O
O
1
23
4
2'
3'4'10'
5'
6'
7'8'
9'
1"
2"
1'
12 3 4
5
6
6'
5'
4'
3'
2' 1'
O
O
O
OH
H
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 3.91 and 4.01 (s,
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.
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Chapter 6 – Experimental Part
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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.
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HO
12
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Chapter 6 – Experimental Part
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13h: (E)-3-[3-(2-hydroxyphenyl)-3-oxoprop-1-enyl]-3’,4’,7-trimethoxyflavone, was
directly recrystallized from ethanol to give yellow solid C27H22O7 (MW = 458.46 g/mol,
1.063 g, yield 44 %, yellow solid, Mp = 237-238°C).
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
directly recrystallized from ethanol to give yellow solid C26H20O6 (MW = 428.43 g/mol,
1.174 g, yield 52 %, yellow solid, Mp = 229-230°C).
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-
4), 194.8 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C26H20O6 + Na]+: 451.1158; found:
451.1165.
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H3CO
OCH3
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Chapter 6 – Experimental Part
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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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Chapter 6 – Experimental Part
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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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H3CO
H3CO1
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H3CO
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Chapter 6 – Experimental Part
223
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”),
O
OO
HO
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Chapter 6 – Experimental Part
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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,
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Chapter 6 – Experimental Part
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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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Chapter 6 – Experimental Part
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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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Chapter 6 – Experimental Part
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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
O
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6"5"
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O
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Chapter 6 – Experimental Part
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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).
1H NMR (300.13 MHz, CDCl3): δ = 3.92 and 3.96 (s,
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
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Chapter 6 – Experimental Part
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
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 3.79 and 3.90 (s,
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).
1H NMR (300.13 MHz, CDCl3): δ = 4.17 (d, J = 6.6 Hz, 2H,
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
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 2.245 and 2.247 (s, 2H,
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"
Chapter 6 – Experimental Part
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.
HRMS (ESI+): m/z calcd for [C28H16O8 + Na]+: 503.0743; found: 503.0727.
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'
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Chapter 6 – Experimental Part
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
Chapter 6 – Experimental Part
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).
1H NMR (500.13 MHz, CDCl3): δ = 3.97 and 3.98 (s, 6H, 5”-
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
Chapter 6 – Experimental Part
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.
HRMS (ESI+): m/z calcd for [C24H18O4 + Na]+: 393.1103; found: 393.1085.
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"
Chapter 6 – Experimental Part
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,
C24H18O5 (white solid, MW = 386.39 g/mol, 0.805 g, yield 34 %, Mp = 195-196°C).
1H NMR (300.13 MHz, CDCl3): δ = 3.84 (dd, J = 19.0, 3.0
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.
HRMS (ESI+): m/z calcd for [C24H18O5 + Na]+: 409.1052; found: 409.1067.
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"
Chapter 6 – Experimental Part
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,
C25H20O5 (white solid, MW = 400.42 g/mol, 1.611 g, yield 65 %, Mp = 181-183°C).
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-
2), 206.7 (C-3’) ppm. HRMS (ESI+): m/z calcd for [C25H20O5 + Na]+: 423.1208; found:
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"
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 3.97 (dd, J = 18.4, 5.7
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"'
Chapter 6 – Experimental Part
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"'
Chapter 6 – Experimental Part
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).
1H NMR (300.13 MHz, CDCl3): δ = 2.17 (d, J = 0.9 Hz, 3H,
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
Chapter 6 – Experimental Part
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.
HRMS (ESI+): m/z calcd for [C33H26O8 + Na]+: 573.1525; found: 573.1485.
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
Chapter 6 – Experimental Part
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-
hydroxyphenyl)-1-methylpyrazoles by the Reaction of Chromones with
Methylhydrazine. J. Heterocycl. Chem., 2000, 37, 1629-1634; b) Barros, A.I.R.N.A.;
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1009-10.
[12] Ulrichan, H. & Sayigh, D. A. A. R. J. The Reaction of oxalyl chloride with substituted
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