Oualid TALHI
Síntese e actividade biológica de híbridos
polifenólicos
Synthesis and biological activities of polyphenolic
hybrids
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)
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.
vii
2-CHCA Chromone-2-carboxylic acid2D-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
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
13
C 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
1
H RMN Proton-1 nuclear magnetic resonance spectroscopy
RNA Ribonucleic acid
ix
SAR Structure-activity-relationshipspet 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
x
T
ABLE OFC
ONTENTSAcknowledgments ... 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
xi
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
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
xiii
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
... 19
7 6.1. Materials ... 1976.1.1. 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
C
HAPTER
1
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1.1. I
NTRODUCTIONOver the past century and up-to-day, organic chemistry is considered as one of the most important technological areas which have long served the humanity. A worth and diversity of discoveries on natural and synthetic organic compounds exhibiting various applications in medicine and agriculture, is known for the last decade. Several human-threatening diseases are overcome when scientists have only used small organic molecules obtained by developing ideas in laboratories or even accidentally discovered in nature. Organic compounds are “living-matters” and are playing crucial functions in human-body metabolism, indeed, human-beings (or animals) are continuously requiring calorific energy from several natural sources of food (vegetal or animal) containing huge amounts of organics like sugars, lipids and proteins which are necessary elements for metabolic bio-reactions. At the same time, the human-body also needs other organic molecules, not only for energetically purposes, but for important therapeutic aims, such as a good metabolic functioning, health maintenance/protection and prevention from various multifactorial diseases. Organic compounds exist in nature through which, the vegetal kingdom has provided and still providing these multi-action components with biological impacts like, vitamins, alkaloids, flavonoids, …etc. Most commonly, polyphenolic compounds are found ubiquitously in plants. These polyvalent molecules are taking the greatest part of the human diet acting as health protectors. Additionally, plants produce polyphenolic compounds as secondary metabolites for self-protection purposes, for instance, anti-fungal activities.
Humans understand that nature is the ever best manufacturer of these biologically active molecules and even they know that nature always supplies them with the suitable organic molecules for their health benefit, but humans’ curiosity is leading to extra demands, since then, they started to defeat nature and effectively they did not stop tracking and producing synthetic compounds for biological/medicinal purposes. Hopefully, thanks to their creative and scientific abilities, humans have won the deal by creating millions of organic structures capable to offer a variety of nutritional and biological actions as the nature used to do so, or even better. The synthetic organic compounds have greatly influence our lifestyles by surrounding us everywhere and in every simple example like plastics, drugs, dyes, detergents, processed food …etc. This does not signify that people can ignore the natural source of these interesting compound because, in fact, nature is the starting point and the unique supplier of raw materials. The most important point is that nature has initiated us to the key organic structures which are most useful for
4
our life, especially our biological needs. In this context, polyphenolic compounds have gained a considerable attention persuading researches in the Organic and Medicinal Chemistry fields. The beneficial effects inherent in their diverse basic structures, such as the relevant benzopyranone and benzofuranone heteronucleus, give birth to the categorization of some fascinating organic families like, chromones, flavonoids and coumarins, the most studied natural-type of compound today.
The chemist rarely creates his molecules from his own imagination, indeed, he was, in most of the time, copying from the mother nature structures and bringing to them from slight to complex chemical modification in view of improving their biological effects and reducing some associated drawbacks (toxicity, metabolic transformation). The chemist job was accomplished thanks to the basis of synthetic organic chemistry science. In particular, the tasks of an “organist/bioorganist” is searching for the best organic structure (virtual or natural), easily synthetically accessed and structurally characterized, non-toxic (no harmful side-effects) and metabolic resistant, being capable to respond to at least one biological requirement for the human-body (or, in general, animals or plants). In the quest for new potent anticancer, antioxidant, anti-inflammatory, antiviral and antifungal agents, …etc, a huge scientific efforts have been conducted over the past century leading to a considerable improvement in organic/bioorganic fields. Innumerous pharmacologically active organic compounds were developed and typically inspired from natural models as being the first key leader products in the drug discovery. In cancer therapy, natural products are very important, since a large majority (79.8 %) of newly developed anticancer drugs between 1981 and 2010 are of natural origin, the rest are organic synthetic product which in fact are natural-type or inspired from nature models [1].
To fulfill the previously underlined tasks dedicated to a chemist, a platform/network should be constructed within a scientific atmosphere involving several expertise of diverse fields in organic chemistry and biology. As soon as the required tools are available, the work is performed and the aims are achieved, all is based on the relevant
Chemistry-Biology-Medicine-Continuum. Therefore, the PhD dissertation presented herein, is a part
of a European project named RedCat (Redox Catalysts) established in 2009 and funded
by the European Community’s Seventh Framework Programme FP7/2007-20139 under
Marie Curie grant agreement nº 215009. RedCat is an Initial Training Network (ITN) for early stage (PhD students) and experienced researchers (Post-Doc) (website: http://www.redcat-itn.eu). RedCat provides research and training opportunities for 10 early stage and 4 experienced researchers across Europe, with 10 partners from academic
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. P
ROJECTP
LANThe scientific project presented in this PhD dissertation, concerns the development of simple and economic synthetic methodologies for the preparation of new organic combinations of various heterocycles utilizing mainly the benzopyranone nucleus. The project is particularly oriented to design and synthesize organic molecules which include a biologically active natural product (like flavonoid, chromone, coumarin, pyrimidine, imidazolidine, …etc) linked together or with selected organic and bioorganic moieties/pharmacophores in a form of hybrid scaffolds.
Taking into account the initial information we gained about the important structural features for a good antioxidant and anti-inflammatory agent involving the above-mentioned skeletons, we have succeeded, in the present project, the development of new
6
generation of hybrid compounds based on biologically significant heterocycles. In the quest for novel drug entities, the hybrid approach is a promising path to obtain new molecular entities that can effectively target multifactorial diseases. The strategy is not so common in medicinal chemistry, since the organic chemist (or biochemist) is often tracking low molecular weight molecules with less structural complexity and potent biological actions. But in fact, the covalent combination of distinct biological organic entities in one molecule will certainly provide new electronic properties influencing the whole molecule interactions with biological systems. It usually brings additive or even new biological effects while the combined molecular weight of the constitutive organic fragments could also be in favor of a reduced metabolic transformations and therefore an increased bioavailability in the circulatory system (Fig. 1.1).
Figure 1. 1. Concept of organic molecules hybridizing and biological profile prediction The present project is mainly divided into two parts: I) organic synthesis of the desired hybrid compounds which has been carried out at the University of Aveiro within the research unit QOPNA of the chemistry department, and II) biological evaluations, in which, a set of screenings have been performed covering the applications of the obtained compounds in developing drugs in the diverse field of anticancer agents, which represents some of the major use of natural mimic products in medicinal chemistry. We also contemplate their applications in other areas of biomedicine including antioxidant agents, anti-inflammatory drugs in order to reach various therapeutic aims. All the aforementioned biological studies have been conducted by early stage researcher-biologists from different specialized institutions within the RedCat project including partners from, Epigenomics
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de Biologie Moléculaire du Cancer, Hôpital Kirchberg, (Luxembourg) and Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Metz, (France).
1.3. O
VERVIEWPreliminary 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 1 3 2 4 5 6 7 8
Figure 1. 2. Starting materials used in the synthesis of the polyphenolic hybrids Some of the major focus is to evaluate the biological nucleus functions within the hybrid molecule, although the study of its substitution influence have also been carried out leading to structure-activity-relationship (SAR) considerations. Recent interest in polyphenolic derivatives of benzopyranones have been stimulated by the potential health benefits arising from their antioxidant and anti-inflammatory activities; more available data
8
have also demonstrated their high propensity to transfer electrons, to chelate ferrous ions, to scavenge reactive oxygen species (ROS) as well as to inhibit lipoxygenases and cyclooxygenases which were strongly associated with the presence of ortho-dihydroxyphenyl (or catechol group) as the main structural feature of the polyphenolic candidate. From our screening results, we have further deduced some SARs showing that the ortho-hydroxybenzoyl substituent and the conjugated α,β-unsaturated-carbonyl systems (like in chalcones, flavonoids and coumarins) and their dihydro- analogues (like in chromanones, dihydrochalcones), are all essential elements for efficient antioxidant and anti-inflammatory activities. These structural patterns are figuring in our molecular hybrid models taking into account various of their stereochemical properties. Figure 1.3 represents the novel synthesized polyphenolic hybrids that are the subject of our biological study.
The present dissertation includes three chapters describing the new synthetic methodologies established for diverse production of the new polyphenolic hybrid compounds, their fine structural elucidation and their biological properties which especially undertakes their application as anticancer agents together with the involvement in further medicinal utilities, such as redox-regulation and anti-inflammatory pathways. The second chapter will discuss the elaboration of pyrimidine- and imidazolidine- based polyphenolic hybrids 9-12, especially the new benzofuran-3-one-hydantoin (BF-HD) hybrids 10, 11 and uracil (U) derivatives 12. The synthetic approaches starts by the action of carbodiimides 3 on 2-CHCA or 3-CHCA in a one-pot reaction or sequential steps using organic or inorganic base catalysis. The third chapter describes various applications of organo-base catalysis in promoting 1,4-conjugate additions/hetero-cyclisations tandem process toward the synthesis of novel polysubstituted chromones (CHR), 2-styrylchromones (2-STC), chromanones (CHRM), flavones (FLV) 13-15 from 3-CHCA and phenolic 1,3-dicarbonyls 4. The bispyranone scaffold (bispyran-2- and -4-one) 16-20 have been elaborated in a one-step coupling reaction of 4-HCOM and TAL on 3-CHCA or formyl-functionalized precursors (ω-formyl-2’-hydroxyacetophenones 4 and chromone-3-carbaldehydes 5) using organobase catalysis. Finally, the application of the 1,4-conjugate addition, commonly known as Michael addition (MAD) approach, is extended to a variety of 4-hydroxypyran-2-one rings 6, 7 (4-HCOM, TAL) reacting on α,β-unsaturated ketone systems including chalcones (CHAL) 8, to give the new warfarin-analogues 21. Also a variety of complex structures 22 have been obtained by hybridizing 4-HCOM and TAL units with the newly synthesized poly-substituted- CHRs, FLVs and 2-STCs 13 via MAD.
9
All the previously indicated organic reaction proceeds in mild and environmentally friendly conditions using 4-pyrolidinopyridine (4-PPy) as organobase catalyst. The reactions last from short to long time, up to 72 hours maximum time, and yields are in general from moderate to good (>40%).
O O N N R R O O N N OH O R O R O O O O OH O O O OH O O OH O OH O O OH O O OH O OH 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 N O O R R O O O O OH O O HO O O O O OH O O HO O O O OH O O OH O O O O OH O HO O O O O OH O HO 9 10 11 12 13 15 O O R 16 17 18 14 19 20 21 22 O O OH O R R R R R R R R R R R O HO O HO O HO O HO
10
The novel polyphenolic hybrid structures show a deep puzzling structural features inducing various stereochemical aspects. A fine analytical characterization is established on the basis of high resolution spectroscopic techniques including extensive mono (1H and 13C) and bidimensional nuclear magnetic resonance (2D-NMR) and single-crystal X-ray diffractometry which largely helped to solve the structural complexity. The stereochemistry presented in this work involves various aspects of the structural geometry basis, like the presence of asymmetric (R/S) and diastereometric (E/Z) centers, spiro-structures, tautomers and conformational forms which seems to affect the biological activity of the studied entities.
The last fourth chapter will briefly introduce and describe the performed biological screening tests on the recently synthesized polyphenolic hybrids explaining their possible involvement in cancer therapy. Hence, most of the newly obtained molecules have been evaluated for their effect on cytotoxicity and proliferation of leukemic cell lines as well as their involvement in the regulation of NF-κB pro-inflammatory pathway. Within the context of research for new targets of cancer therapy, HDAC and Cdc25 enzymes inhibition were specifically tested on the previously mentioned benzofuran-3-one-hydantoin candidates. A range of representatives polyphenolics from CHRs, CHRMs, FLVs and 2-STCs were examined for their cancer chemopreventive activity mediated by induction of cytoprotective Nrf2 (nuclear factor E2-related protein 2) signaling and their ability to inhibit proliferation of breast cancer cells. Finally we cover the results with some antioxidant activity evaluations using DPPH free radical scavenging test and FRAP ferric ion reducing antioxidant power of some underlined structures.
11
1.4. R
EFERENCES1. 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.
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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.
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23. Talhi, O.; Silva, A M.S. C-prenylation of phenolics. Curr. Org. Chem., 2012, in
C
HAPTER
2
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S
YNTHESIS OF
H
YDANTOIN
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AND
U
RACIL
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2.1. B
ENZOFURAN-3-
ONE-
HYDANTOIN HYBRIDS2.1.1. 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].
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O O O OH HO HO OH O O O HN O O OR H N N H O O O HOH 3C O O O H3C HO O O N N N N O O O X O N N O H H3CO O O H3CO OH HO O O OH HO OH O O OH N N N H N O O 23 24 25 27 29 26 28 30 31 H N O NH OFigure 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.
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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 N N O O R R R R R 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
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 H N O O HN H N O O phenytoin mephenytoin N H N O O fosphenytoin N H N O O O 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]
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 X R t-BuO-K+, DMF O O H N Br X R O -O Br N X R 32 33 HN X R Scheme 2. 1 O O OHC N R OH HO R1 R2 O O N R OH HO R1 R2 + cat. HCl EtOH / reflux 34 35 36 Scheme 2. 2 O O O Ph3P=N–N=CR1R2 – Ph3PO O O N N R2 R1 37 38 Scheme 2. 3
Earlier, 6-acetylspiro[benzofuran-2(3H),1'-cyclopropan]-3-one 40, the most potent antiulcer compound in a series of spirocyclopropanes, was obtained by treating methyl benzoate 39 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and NaCl in DMF at 150 °C (Scheme 2.4) [38]. More recently, Filloux et al. [39] reported the development of a multicatalytic, one-pot, asymmetric Michael/Stetter reaction of salicylaldehydes 41 with electron deficient alkynes 42. The cascade reaction proceeds via amine-mediated Michael addition followed by an N-heterocyclic carbene-promoted intramolecular Stetter reaction. Several salicylaldehydes and doubly activated alkynes participate in a one-step or
two-22
step protocol (via the intermediates 43) to give a variety of BF products 44 in moderate to good yields and good to excellent enantioselectivities. Terminal electrophilic allenes also deliver a similar one-pot, two-step Michael/Stetter reaction with salicylaldehydes to afford 40 compounds. The origin of enantioselectivity in the reaction is explored; E∕Z geometry of the reaction intermediate as well as the presence of catalytic amounts of catechol additive are found to influence the reaction enantioselectivity (Scheme 2.5).
H3COC O O O O OCH3 O O DBU, NaCl DMF / 150 °C H3COC 39 40 Scheme 2. 4 O OH R EWG1 EWG2 O O EWG1 EWG2 * O O R EWG1 EWG2 R 41 + 44 42 43 Multi-catalyst Scheme 2. 5
In relation to the HD nucleus, target derivatives of 1,3,5-triphenylhydantoin 47 have been obtained by an original pathway starting from 1,3-diphenylureas 45 and phenylglyoxal derivatives 46 as it is detailed in Scheme 2.6. This reaction represents a simple one-pot procedure to obtain, in optimal yields, some compounds with interesting affinity and selectivity for the human CB1 cannabinoid receptor. The enantioselectivity (3:1 ratio for the R/S enantiomers) that correspond to the C-5 asymmetric carbon has been revealed from crystallographic studies [40]. Also, the regioselective Knoevenagel-type condensation has been reported in the synthesis of thioisatin-hydantoin hybrids 50. Benzo[b]thiophene-2,3-dione 48 (commonly known as thioisatin) reacts with the hydantoin active methylene 49 to afford selectively the isomer Z 50 (Scheme 2.7). The synthesized products were screened for antimicrobial activity [41]. Electrophilic substitution of allantoin (or 5-ureidohydantoin) in position 5 with 2-naphthol and 1-bromo-2-naphthol in the
23
presence of acid afforded the corresponding (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 46 47 + Scheme 2. 6 S O O HN NH O O S O N H NH O O Knoevenagel R 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.
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1 9a-c 10a-c 11a-c (Z)
3a-c a. R= cyclohexyl b. R= isopropyl c. R= tolyl O CO2H O O O N N O 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).
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O O N C N COOH O O N O N H O O O N N O O + 1 3a 51 9a EtOH Scheme 2. 9 R4 R3 OH O N C R1 N R2 N N O O R1 R2 R3 R 4 R4 R3 N O NH O R1 R2 52 3 53 54 R2 R1 O O HN N R1 R2 CH2Cl2 (a) (b) O N NR1 O R2 R3 R 4 Computational model Scheme 2. 10 2 9a-c 3a-c a. R= cyclohexyl b. R= isopropyl c. R= tolyl O COOH O O O N N O O R R N C N R R * + AA: 0.05 equiv 4-PPy, CH2Cl2, rt, overnight 1
3 1' 3'
Scheme 2. 11
The structure of compounds 9a, 9c was established by 1D and 2D NMR analysis. The main feature in 1H NMR spectra of compounds 9a-c was the presence of two signals as doublets at 2.89-3.14 ppm and 3.22-3.30 ppm with a large geminal protons coupling constant (2J =16.7-16.9 Hz) assigned to the resonance of the diastereotopic chromanone
methylene protons involved in an AB spin system. The HSQC spectrum (Fig. 2.5) showed that the methylene carbon C-3 (41.0-41.3 ppm) is bounded to both H-3 (2.89-3.14 and 3.22-3.30 ppm) protons, while the HMBC experiment (Fig. 2.6, 2.7) presents important
26
correlations of these methylene geminal protons with the neighboring carbons. Thus, H-3 (2.98-3.14 ppm) presents HMBC correlations with carbons C-10 (119.7-119.8 ppm), C-2 (88.5-89.2 ppm), C-4 (188.5-188.7 ppm; C=O) and C-5’ (167.1-168.7 ppm; C=O), while the other H-3 (3.22-3.30 ppm) shows correlations with C-2 and two carbonyl groups, C-4 and C-5’. Additional HMBC correlations allowed differentiating between carbonyl groups and N-substituent positions: C-4 (188.5-188.7 ppm) is confirmed as belonging to the chromanone ring by correlating with H-5 (7.76-7.88 ppm) in all compounds 9a-c. In the case of 9a, 9b H-1” (3.82 and 4.26 ppm) correlates with both carbonyls of the hydantoin ring C-2’ (153.1-153.8 ppm) and C-5’ (167.1-168.7 ppm), while H-1”’ (3.33 and 3.77 ppm) is only establishing correlation with the carbonyl C-2’, allowing to assign the 1-N- and 3-N-cyclohexyl/isopropyl substituents on the hydantoin nucleus (Fig. 2.6, 2.7).
Figure 2. 5. HSQC spectrum of compound 9b (300 MHz) H-3(AB)
