Promising Caspases Modulators With Flavonoid Scaffold

Joana Patrícia Martins Moreira Promising caspases modulators with flavonoid scaffold Dissertation presented to the Faculdade de Farmácia da Universidade do Porto, to obtain the degree of Master in Pharmaceutical Chemistry Work developed under the scientific supervision of Professor Honorina Maria de Matos Cidade and Professor Lucília Helena Ataíde Saraiva. July 2015 ii De acordo com a legislação em vigor, não é permitida a reprodução de qualquer parte desta dissertação. iii This work was developed in the Centro de Química Medicinal da Universidade do Porto-CEQUIMED-UP, Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia da Universidade do Porto, and Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto. This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020 and the Project Pest-OE/SAU/UI4040/2014 (FCT) and REQUIMTE-Pest-C/EQB/LA0006/2013. iv v Some results presented in this dissertation are part of the following scientific poster communications: 4. Moreira J.*, Salazar S., Pinto M., Leão M., Saraiva L., Cidade H. Baicalein derivatives with caspase modulatory activity. 10th Yes Meeting - Young European Scientist Meeting, Porto, Portugal, 17-20 September 2015 (submitted). 3. Moreira J.*, Pinto M., Saraiva L., Cidade H. Synthesis of alkylated derivatives of chrysin with caspase modulatory activity. 8th IJUP - Encontro Investigaçao Jovem da Universidade do Porto, Porto, Portugal, 13-15 May 2015 (P-423). 2. Mokra E.*, Moreira J., Pinto M., Saraiva L., Cidade H. Synthesis of alkylated derivatives of 3,7-dihydroxyflavone with potential antitumor activity. 8th IJUP - Encontro Investigação Jovem da Universidade do Porto, Porto, Portugal, 13-15 May 2015 (P-422). 1. Moreira J.*, Pinto M., Saraiva L., Cidade H. Synthesis of caspases modulators with flavonic scaffold. XX Encontro Luso-Galego de Química, Porto, Portugal, 26-28 November 2014 (P-377). * Presenting author vi vii Acknowledgements É um prazer agradecer a todos aqueles que fizeram com que esta tese fosse possível, não só para o apoio científico, mas também pela sua amizade. No entanto, gostaria de expressar a minha mais profunda gratidão particularmente à: Professora Doutora Honorina Maria de Matos Cidade, orientadora desta tese, pelos valiosos ensinamentos, espirito crítico notável, todo o incentivo e amizade demonstrados ao longo do trabalho. Um especial obrigada por toda a disponibilidade e, sobretudo, por ter despertado em mim “este bichinho” pela investigação. Professora Doutora Lucília Helena Ataíde Saraiva, co-orientadora desta tese, pela orientação científica e revisão crítica desta tese, pelos ensinamentos e pela sua prontidão de auxílio. Professora Madalena Maria de Magalhães Pinto, responsável do Laboratório de Química Orgânica e Farmacêutica, por toda a simpatia e disponibilidade. Por todas as conversas reconfortantes nos momentos menos bons. Doutora Andreia Palmeira, pela colaboração e ensinamentos no estudo de Docking, por toda a disponibilidade e apoio. O seu contributo foi essencial para o enriquecimento deste trabalho. Dra Sara Cravo, pelos conhecimentos transmitidos, colaboração científica e, sobretudo, por toda a ajuda prestada na síntese em micro-ondas, assim como no desenvolvimento do trabalho em HPLC. Os seus conhecimentos e apoio foram sem dúvida um fator importante para o trabalho. Gisela Adriano pelo suporte técnico. À minha querida Eva Mokrá, pela ajuda em algumas das sínteses, pelo empenho, pelo interesse, pela amizade e sobretudo pela experiência de um auxílio mútuo. Sofia Salazar, pela realização dos estudos de avaliação da atividade moduladora de caspases. Todos os docentes e colegas deste laboratório, em particular ao Pedro Brandrão pelo auxílio nos primeiros tempos no laboratório, à Ana Oliveira, Ana Rita Neves, Inês Cruz e Leticia Carraro por todo o apoio, conversas e debates de “tudo e de nada”. viii Aos meus amigos de sempre e aos que não são de sempre mas serão para sempre e à família Castro, por compreenderem as ausências, pelo carinho, preocupação e apoio. Aos meus queridos pais, pelo apoio, carinho e, sobretudo, compreensão. Às minhas irmãs, de sangue e coração, e cunhados, que mesmo longe tentam estar sempre perto. Aos quatro rebentos desta família, Miguel, Margarida, Rodrigo e Leonardo e à minha afilhada Constança, que trazem alegria à minha vida. Por fim, a ti Simão Castro, pelo cuidado constante, pelo amor, carinho e compreensão. “Se tiver o hábito de fazer as coisas com alegria, raramente encontrará situações difíceis” Baden Powell ix Abstract The apoptosis is a highly regulated programmed cell death that is important for maintaining the integrity and homeostasis of multicellular organisms. Conversely, deregulation of apoptosis, resulting in either less or excessive cell death, is associated with several pathologies including cancer. The intracellular machinery responsible for apoptosis is dependent on a family of proteases that have a cystein at their active site and cleave their target proteins at specific aspartic acids, called caspases. Thus, modulators of these proteins may represent a promising therapeutic strategy against apoptosisrelated diseases. Flavonoids represent an interesting class of naturally occurring compounds that have been attracting attention of the scientific community because of their wide range of biological properties, being the antitumor activity one of the most studied. The antitumor activity of flavonoids is associated, at least in part, to their ability to induce apoptosis by affecting the expression or activity of a wide variety of molecules involved in apoptosis pathways. Additionally, this class of secondary metabolites has shown to be activators of caspases. In order to improve the antitumor effects and to study the influence of alkylation of the flavonoid scaffolds on this activity, we have proposed to develop alkylated flavonoids with enhanced antitumor activity. Accordingly, in this work we described the synthesis of twenty-six alkylated derivatives (eighteen flavones and eight flavonols) of three natural flavonoids (baicalein, 3,7-dihydroxyflavone and chrysin). On the basis of our current state of knowledge sixteen from these twenty-six alkylated flavonoids are described for the first time. The synthesis of alkylated flavonoids was carried out by reacting the building blocks with alkylating agents, in alkaline medium using microwave-assisted organic synthesis (MAOS). Previously, the suitable amount of alkylating agent to obtain the intended alkylated derivatives was determined by a HPLC-DAD study. The structure elucidation of the synthesized compounds was established on the basis of IR and NMR techniques. The ability of three synthesized alkylated flavonoids (Biso1, Brop1 and Bali1) to modulate procaspases-3 and -7 activity was evaluated using yeast-based assays. Results xvi xvii List of Figures Figure 1: Chemical structures of prenylflavonoids identified as procaspase-7 activators. ............... 4 Figure 2: A schematic representation of structural features of human procaspases (C represent the active site residue). ............................................................................................................................ 8 Figure 3: Schematic representation of the domain organization of human procaspases from different subfamilies. .......................................................................................................................... 9 Figure 4: Mechanisms of initiator caspases activation. ................................................................... 10 Figure 5: Mechanisms of effector caspases activation. .................................................................. 10 Figure 6: A schematic representation of active caspases structure (C represent the active site residues). .......................................................................................................................................... 11 Figure 7: Mechanisms of inflammatory caspases activation.. ......................................................... 12 Figure 8: The caspases pathways: extrinsic and intrinsic. .............................................................. 13 Figure 9 : Activation of the extrinsic pathway. ................................................................................. 14 Figure 10: Anti-apoptotic and pro-apoptotic BCL-2 family.. ............................................................ 16 Figure 11: Activation of the intrinsic pathway. ................................................................................. 17 Figure 12: Activation of effector caspases. ..................................................................................... 18 Figure 13: Structure illustrating substrate/inhibitor residues (P) and protease binding sites (S). Prime and non-prime indications distinguish respectively between the Cand N-side of the cleavage site. ................................................................................................................................... 20 Figure 14 Structure of the active-site inhibitor IDN-6556 (15). ........................................................ 22 Figure 15: Structure of peptide-derived caspase inhibitors VX-765 (16) and VRT-043198 (17). ... 22 Figure 16: Structure of cyanopropanate-containing small molecules based on peptidic scaffold of VX-765 as inhibitors of caspase-1 (NCGC00185682 (18), NCGC00183434 (19) and NCGC00183681 (20)). ..................................................................................................................... 23 Figure 17: Structures of the isatin sulfonamides caspase inhibitors 21-25. .................................... 24 Figure 18: Structures of 1,4benzodioxane derivatives 26 and 27. ................................................ 24 Figure 19: Caspase inhibitors with a pyrrolo[3,4-c]quinoline-1,3-dione scaffold (28) and selected caspases-3 inhibitor (29). ................................................................................................................. 25 Figure 20: Model of allosteric inhibition. .......................................................................................... 26 Figure 21: Model of disulfide trapping. ............................................................................................ 26 Figure 22: Structures of the allosteric inhibitors of caspases-3 and -7 DICA (30) and FICA (31). . 27 Figure 23: Structure of the caspase-1 allosteric inhibitor, compound 32. ....................................... 27 Figure 24: Structures of XIAP inhibitors, compounds 33 e 34. ....................................................... 28 Figure 25: Structures of PAC-1 (35) and its close analog PAC-1B (36). ........................................ 29 Figure 26: The proposed mechanism for PAC-1-induced activation of procaspase-3 in vitro. ....... 30 Figure 27: Derivatives of PAC-1 (37, 38, 39 and 40) with enhanced metabolic stability in liver microsomes, and improved tolerability in mice. ............................................................................... 31 Figure 28: Structures of PAC-1 analogues (41, 42 and 43). ........................................................... 31 xviii Figure 29: Structures of 1541 (44) and its derivatives1541B (45), 1541C (46) and 1541D (47)..... 32 Figure 30: The proposed mechanism of activation-assisted procaspase-3 self-activation.. ........... 33 Figure 31: Basic skeletons and numbering pattern of structural derivatives of 1,1-, 1,2and 1,3diphenylpropane (47, 49 and 51 respectively). ................................................................................ 34 Figure 32: Basic skeletons of several subclasses of flavonoids and numbering of skeletons ........ 35 Figure 33: Biosynthesis of flavonoids.. ............................................................................................ 36 Figure 34: Examples of natural flavonoids that have entered in the late phase of clinical trials for various cancers. ................................................................................................................................ 38 Figure 35: Multistage model of carcinogenesis and potential effects of flavonoids on cancer progression.. ..................................................................................................................................... 38 Figure 36: Flavonoids may induce apoptosis by affecting the expression or activity of a wide range of molecules involved in apoptosis pathways. .................................................................................. 40 Figure 37: Natural flavonoids used as building blocks for molecular modification. ......................... 61 Figure 38: Synthesis of alkylated derivatives of Baicalein (B). ........................................................ 63 Figure 39: HPLC-DAD chromatogram of a mixture of baicalein (B) 5,6-dihydroxy-7-(isopentyloxy)- 2-phenyl-4H-chromen-4-one (Biso1) and 5-hydroxy-6,7-bis(isopentyloxy)-2-phenyl-4H-chromen-4one (Biso2) at 275 nm with MeOH:H2O:MeCO2H (95:5:1 v/v/v) as mobile phase. ......................... 63 Figure 40: HPLC-DAD chromatograms at 275 nm of the reaction mixture with isopentyl iodide, with (A) MeOH:H2O:MeCO2H (95:5:1 v/v/v) and (B) MeOH:H2O:MeCO2H (50:50:1 v/v/v) as mobile phase. ............................................................................................................................................... 64 Figure 41: Main connectivities found in the HMBC of derivatives Biso1 and Biso2....................... 75 Figure 42: Main connectivities found in the HMBC of derivatives Butil1 and Butil2. ..................... 75 Figure 43: Main connectivities found in the HMBC of derivatives Brop1 and Brop2. .................... 76 Figure 44: Main connectivities found in the HMBC of derivatives Bali1 and Bali1'. ....................... 77 Figure 45: Main connectivities found in the HMBC spectrum of derivative Betil1. ......................... 77 Figure 46: Main connectivities found in the HMBC of derivatives Bemet1 and Bemet2. ............... 78 Figure 47: 1H and 13C NMR data for the alkyl groups of derivatives Hiso (A), Hutil1/2 (B), Hrop (C), Hali (D), Hetil (E), Hemet (F) and prenyl group of derivative Hrep (G). ................................... 84 Figure 48: Main connectivities found in the HMBC of alkylated derivatives of 3,7-dihydroxyflavone (Hiso (A), Hutil1 (B),Hutil2 (C), Hrop (D), Hali (E), Hetil (F), Hemet (G) and Hrep (H)). .............. 85 Figure 49:1H and 13C NMR data for the alkyl groups of derivatives Ciso (A), Cutil (B), Crop (C), Cali (D), Cetil (E), Cemet (F) and prenyl group of derivative Crep (G). .......................................... 89 Figure 50: Main connectivities found in the HMBC of alkylated derivatives of chrysin (Ciso (A), Cutil (B), Crop (C), Cali (D), Cetil (E), Cemet (F) and Crep (G)). .................................................. 90 Figure 51: Concentration-response curves for the effects of flavonoids Biso1, Brop1 and Bali1 on the growth of yeast expressing human procaspase-3 (A) or -7 (B), for 24 hours treatment. . ......... 95 Figure 52: Structures of the positive controls used in the docking study. ....................................... 97 Figure 53: Compound 122 (A), 116 (B) and 44 (1541) (C) docked into procaspase-3. .................. 99 Figure 54: compound 115 (A), 117 (B) and 116 (C) docked into procaspase-7............................ 100 xix Figure 55: compound 118 (A), 119 (B), 120 (C), 121 (D) and 44 (1541) (E) docked into procaspase-6. ................................................................................................................................. 100 Figure 56: Interactions of Biso1 (A) Brop1 (B) and Bali1 (C) (blue sticks) with residues in the allosteric site of procaspase-3. ....................................................................................................... 101 Figure 57: Interactions of Biso1 (A) Brop1 (B) and Bali1 (C) (blue sticks) with residues in the allosteric site of procaspase-7.. ...................................................................................................... 102 Figure 58: Interactions of Biso1 (A) Brop1 (B) and Bali1 (C) (blue sticks) with residues in the allosteric site of procaspase-6. ....................................................................................................... 103 xx xxi List of Tables Table 1: Azidomethylene-based peptidomimetics inhibitors of caspase-1.12 .................................. 20 Table 2: Benzylamine inhibitors of caspases-1.13 ............................................................................ 21 Table 3: Some flavones and flavonols reported since 2000 as inducers of apoptosis by modulating different key targets involved in apoptotic pathways, including caspase family proteins reported. . 41 Table 4: Alkylation of baicalein (B) with isopentyl iodide with different reaction times. .................. 65 Table 5: Alkylation of baicalein with different quantities of isopentyl iodide. ................................... 66 Table 6: General conditions for the synthesis of alkylated derivatives of baicalein (B). ................. 67 Table 7: General conditions for the synthesis of alkylated derivatives of 3,7-dihydroxyflavone (H). .......................................................................................................................................................... 70 Table 8: General conditions for the synthesis of alkylated derivatives of Chrysin (C). ................... 72 Table 9: IR data of baicalein (B) and its alkylated derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1', Betil1, Bemet1 and Bemet2)........................................................................ 73 Table 10: 1H NMR data of baicalein (B) and its alkylated derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1’, Betil1, Bemet1 and Bemet2). .......................................................... 79 Table 11: 13C NMR data of baicalein (B) and its alkylated derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1’, Betil1, Bemet1 and Bemet2). .......................................................... 81 Table 12: IR data of 3,7-dihydroxyflavone (H) and its alkylated derivatives (Hiso, Hutil1, Hutil2, Hrop, Hali, Hetil, Hemet and Hrep). ............................................................................................... 83 Table 13: 1H NMR data of 3,7-dihydroxyflavone (H) and its alkylated derivatives (Hiso, Hutil, Hrop, Hali, Hetil, Hemet and Hrep). ......................................................................................................... 86 Table 14: 13C NMR data of 3,7-dihydroxyflavone (H) and its alkylated derivatives (Hiso, Hutil1, Hutil2, Hrop, Hali, Hetil, Hemet and Hrep). ................................................................................... 87 Table 15: IR data of chrysin (C) and its alkylated derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep). ........................................................................................................................................ 88 Table 16: 1H NMR data of chrysin (C) and its alkylated derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep). ............................................................................................................................ 91 Table 17: 13C NMR data of Chrysin (C) and its alkylated derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep). ............................................................................................................................ 92 Table 18: Purity values of the alkylated derivatives of baicalein (Biso1, Brop1 and Bali1). .......... 93 Table 19: GI25 values obtained for PAC-1 and flavonoids Biso1, Brop1 and Bali1. ...................... 96 Table 20: Docking scores (Kcal.mol-1) for procaspase activators described in the literature and derivatives Biso1, Brop1 and Bali1. ............................................................................................... 98 Table 21: Residues of procaspases-3, -7, and -6 involved in polar interactions with alkylated derivatives (Biso1, Brop1 and Bali1). ........................................................................................... 104 Table 22: Docking scores (Kcal.mol-1) for modulators described in the literature and alkylated derivatives of baicalein (Biso1), 3,7-dihydroxyflavone (Hiso) and chrysin (Ciso). ....................... 105 Table 23: Structures of the synthesized flavonoids and their precursors. ..................................... 151 xxii xxiii Abbreviations and Symbols 13C NMR NMR Carbon nuclear magnetic resonance 1H NMR NMR proton nuclear magnetic resonance ANS Anthocyanidin synthase Apaf-1 Apoptotic protease-activating factor-1 ASK-1 Apoptosis signal regulating kinase 1 Bad Bcl-2-associated death promoter BAK Bcl-2 antagonist/ Killer BAX Bcl-2 associated X protein BcL-2 B cell lymphoma 2 BcL-XL B cell lymphoma –extra large BH3-only Bcl-2 homology domain 3 only Bid BH3-interacting domain death agonist BIR Baculoviral IAP repeats Brs Broad singlet CARD Caspases-recruitment domain CDKs Cyclin-dependent kinases CEQUIMED-UP Centro de Química Medicinal da Universidade do Porto c.f.u. Colony-forming units CHI Chalcones isomerase CHS Chalcones synthase Ciimar Interdisciplinary Centre of Marine and Environmental Research Cit c Cytochrome c CrmA Cytokine response modifier C4H Cinnamic acid 4-hydroxylase DED Death effector domain d doublet dd double doublet DD Death domain DFR Dihydroflanonol-4-reductase DISC Death-induced signaling complexes DMSO dimethylformamide DR Death receptor DR3 Death receptor 3 EGFRs Epidermal growth factor receptors ERK Extracellular-regulated kinase EtOAc Ethyl acetate FADD Fas-associated death domain xxiv FHT Flavanone 3β-hydroxylase FLS Flavonol synthase FNS I Flavone synthase Ι GI50 Concentration of compound that inhibited 50% of the net cell growth HMBC Heteronuclear multiple bond correlation HPLC High performance liquid chromatography HPLC-DAD High performance liquid chromatography with Diode Array Detector HSQC Heteronuclear single quantum coherence HTS High-throughput screening IAP Inhibitor apoptosis protein IC50 Concentration of compound that causes 50% growth inhibition IMAs Isatin Michael acceptors IR Infrared spectroscopy J coupling constant JNK C-Jun N-terminal kinase Ki Values of inhibition constant LRR Leucine-rich repeats m multiplet MAOS Microwave-assisted organc synthesis MARK Mitogen-activated protein kinases Mcl-1 Anti-apoptotic BCL-2 protein Me2CO Acetone MOMP Mitochondrial outer membrane permeabilization MW Microwave PAC-1 Procaspase-activating compound -1 PAL Phenylalanine ammonia lyase PARP Poly (ADP-ribose) polymerase PDGFRs Platelet derived growth factor receptors PKs Protein-Kinases PYP Pyrin domains q Quadruplet ROS Reactive oxygen species Rt Retention time s singlet SAR Structure-activity relationship t triplet TLC Thin layer chromatography TNF Tumor necrosis factor TNFR Tumor necrosis factor receptor TNFR1 Tumor necrosis factor receptor-1 xxv TRAIL-R1 TNF-related apoptosis-inducing ligand receptor-1 TRAIL-R2 TNF-related apoptosis-inducing ligand receptor-2 VEGFRs Vascular endothelial growth factor receptors XIAP X chromosome-linked inhibitor of apoptosis protein WHO World Health Organization 4CL 4-Coumarate: Coenzyme A ligase ∆Ψm Variation of the membrane potential δ chemical shift δc Carbon chemical shift δH Proton chemical shift ʋ Wavenumver (cm-1) Chapter 2: Introduction 7 2.1. Caspases 2.1.1. Structure and classification Caspases form a cysteine protease/peptidases family that uses the cysteine residue as catalytic nucleophile. They share a high specificity to break target proteins in sites after aspartic acid residues. The name "caspase" is derived from this characteristic molecular function: cysteine-aspartic-acid-proteases.1 The caspases family consists in fifteen different proteins, being eleven expressed in human cells (caspases-1 to -10 and -14).2 They are grouped into two families: the inflammatory and apoptotic. However, these proteins are usually classified into three subfamilies because apoptotic caspases are subclassified according to their mechanism of action.3 Thus, according with their physiologic function, substrate specificity and sequencing similarities, they are grouped into three subfamilies: inflammatory (Group I), initiator of apoptosis (Group II) and effector/executioner of apoptosis (Group III) caspases.2 The inflammatory caspases are involved in the inflammatory response of the cytokines activation, while the initiator and effector/executioner caspases are involved in the apoptotic cascade.4 The initiator caspases are the first to be activated in a particular death pathway and are the first step for the executioner caspases activation.1 All the caspases can be found in cells in their inactive form, as zymogen (procaspase) precursor. The initiator caspases are present in the cell as zymogen monomers and the executioner caspases exist as inactive zymogen dimers.4 These procaspases are simple strand proteins, with a variable size N-terminal prodomain, a central large subunit, with the cysteine residue active site, and a C-terminal small subunit (p10; ~10 KDa). The catalytic domain includes the large subunit (p20; ~20 KDa) and the small subunit, which has essential residues for caspases activity (Figure 2).2 Chapter 2: Introduction 8 The Group I, including procaspases-1, -4 and -5, presents a large N-terminal prodomain that has a caspases–recruitment domain (CARD), crucial for the formation of protein complex that lead to caspases activation (Figure 3). The Group II, including procaspases-2, -8, -9 and -10, has a large N-terminal prodomian (>90 amino acids), containing a CARD (procaspases -2 and -9) or a death effector domain (DED; procaspases-8 and -10) that is important in protein–protein interactions with other adaptive proteins (Figure 3). The Group III, including the effector procaspases-3, -6 and - 7, has only a short prodomain of 20-30 amino acids (Figure 3).2 catalytic domain Cleavage site Aps-x Cleavage site Aps-x N-terminus C-terminus Prodomain (variable size) Large subunit (p20) small subunit (p10) (~10KDa) Catalytic Cysteine conserved site C Figure 2: A schematic representation of structural features of human procaspases (C represent the active site residue). Chapter 2: Introduction 9 2.1.2. Caspases activation Biochemical and structural analysis revealed an underlying conservation of the caspases activation process. Additionally, the stabilization process of the proenzyme latent state is also conserved.1,5 The activation of initiator caspases is mediated by dimerization, through “induced proximity”, when adaptor proteins interact with the prodomains. However, interdomain cleavage, promoted by initiator caspases, is involved in the activation of executioner caspases.5,6 Studies revealed that active initiator caspases are dimers with identical catalytic units, where each catalytic unit has one active site. The subunits are derived of only one molecular precursor (procaspase) through an internal cleavage in subunits marked sites. In fact, it has been demonstrated that this cleavage is neither necessary nor sufficient for the initiator and inflammatory caspases activation.4 It is required a dimerization of the zymogen monomers to assume an active conformation. The dimerization process occurs in a multiprotein activation complex that recruits procaspase through the N-terminal recruitment domain (Figure 4).1 Inflammatory caspases Caspases-1, -4 and -5 Initiator caspases Caspases-2 and -9 Caspases-8 and -10 Executioner caspases Caspases-3, -6 and -7 CARD DED Large subunit Small subunit Prodomain Figure 3: Schematic representation of the domain organization of human procaspases from different subfamilies. Chapter 2: Introduction 10 Concerning executioner caspases, their precursor procaspases are found in the cytosol in the inactive dimer form.4 These are activated through limited proteolysis inside the interdomain linker, by initiator caspases or occasionally by other proteases in specific circumstances (Figure 5).1 Effector caspase (inactive dimer-procaspase ) Cleavage by Initiator Caspase Active Effector Caspase Initiator or inflammatory caspase (inactive monomer-procaspase) Proximity Induced Dimerization Autocleavage Active Initiator Caspase Figure 4: Mechanisms of initiator caspases activation. Dark Blue indicates small subunit, clear blue indicates large subunit, green represent prodomain that contains CARD represented by the yellow color. Figure 5: Mechanisms of effector caspases activation. Dark Blue indicates small subunit, clear blue indicates large subunit and the prodomain is represented in green. Chapter 2: Introduction 11 p20 p20 p10 p10 C C Studies of X-ray crystalline structure of some caspases have suggested that caspases are present in the dimers form, with two large and two small subunits, like heterodimers (p202-p102) (Figure 6).5,7 Each heterodimer subunits are formed from hydrophobic interactions that bend into a compact cylinder, dominated by a central strand of six β-sheets. The heterodimers interact through a twelve β-sheets which are surrounded by five α-helices, distributed in the opposite side of the β-sheets formed site. This unique protease structure is almost caspase exclusive. On the caspases heterodimers, two of these cylinders align in a head-tail configuration getting the two active sites on opposite sides.5 Regarding the activation of inflammatory caspases-1 and -5, it occurs in a complex named inflammasome (Figure 7). The inflammasome includes procaspases-1 and -5, which contains CARD, as well as the NALP-1 protein. This complex formation results in the processing and activation of IL-1β and IL-18 cytokines that has a central role in the immunity response to microbial pathogens.5 Figure 6: A schematic representation of active caspases structure (C represent the active site residues). Dark Blue indicates small subunit and clear blue indicate large subunit. Chapter 2: Introduction 12 ASC Procaspases -1/-5 CARD PYD NACH LRR FIND procaspase -1 Procaspase -5 IL-1β IL-18 Pro IL-1β Pro IL-18 Inflammasome Caspase -1 Figure 7: Mechanisms of inflammatory caspases activation. The inflammasome is formed with proteins (such as NALP-1 protein) and procasases-1 and -5. The NLR family is characterized by the presence of a central nucleotide-binding and oligomerization (NACHT) domain, which is commonly flanked by C-terminal leucinerich repeats (LRRs) and N-terminal caspase recruitment (CARD) or pyrin (PYD) domains. Procaspase-1 binds to CARD in ASC that which in turn have PYD that is responsible for recruitment with PYD of NLRL-1, and procaspase-5 binds to the CARD. Active caspases are released and allows activation of pro IL-11β and pro IL-18 in IL-1β and IL-18 cytokines. Chapter 2: Introduction 13 Intrinsic pathway Apoptosome Procaspase-9 Caspase-9 Cytochrome c Extrinsic pathway DISC Procaspase-8 Procaspase-10 Caspase-8 Caspase-10 Procaspase-3 Procaspase-6 Procaspase-7 Caspase-3 Caspase-6 Caspase-7 APOPTOSIS 2.1.2.1. The caspase pathways The signal transduction for caspase activation may occur by two main distinct pathways: extrinsic, which is a receptor-mediated pathway, and intrinsic, a mitochondrialmediated pathway (Figure 8). 5 Figure 8: The caspases pathways: extrinsic and intrinsic. The extrinsic pathway, formed DISC enables the activation of caspase-8 and -10, which will activate procaspases-3, -6 and -7. The intrinsic pathway is in apoptosome that allows the activation of procaspase-9, which will also activate procaspases-3, -6 and -7. Thereby apoptosis is activated. Chapter 2: Introduction 14 2.1.2.1.1. Extrinsic pathway The process is initiated by extracellular signals delivered in the form of ligands binding to death receptors (DRs).3 DRs are the transmembrane proteins containing an extracellular ligand-binding domain and an intracellular death domain (DD), which is required for receptors to recruit downstream apoptotic proteins.8 They are members of the tumor necrosis factor (TNF) superfamily and include TNF receptor-1 (TNFR1), the FAS (also known as CD95 or APO-1), death receptor 3 (DR3), and TNF-related apoptosisinducing ligand receptor-1/2 (TRAIL-R1/R2).3,5 The bounding of TNF and other ligands with the transmembrane death receptors leads to the recruitment of intracellular adaptor proteins (Fas-associated death domain, FADD), which in turn recruit the initiator procaspases-8 and 10, forming death-induced signaling complexes (DISC).6 It is inside this DISC that the initiator procaspases-8 and -10 are activated through induced proximity dimerization (Figure 9).6 Ligands FADD DRs (TNFR) Procaspase-8 or Procaspase-10 Caspase-8/-10 Extrinsic pathway TNFR1 FAS (CD95 or APO-1) DR3 TRAIL-R1/R2 DISC Figure 9 : Activation of the extrinsic pathway. Chapter 2: Introduction 15 The DISC molecules interactions are based in homotypic contacts. The DD of the receptor interacts with the DD of the FADD, the DED of the FADD interacts with the DED of the procaspases N-terminal and cell inhibitor proteins.8 The procaspase-8 processing includes two cleavages. The first cleavage occurs between the protease domains, where the subunits p43/41 and p10 are formed, remaining both the products bounded to the DISC (p43/41 through DED interactions and p10 by interactions with proteases large domain). The second cleavage occurs between the prodomain and the large subunit. As a result of this cleavage, p43/41 is processed to the p26/p24 prodomain and p18.5 Therefore, after the dimerization, the N-terminal DED are proteolytically removed and the mature caspase-8 is released to the cytosol, triggering the apoptosis process. The same happens with caspase-10.1 After this, caspase-8 and 10 subsequently activates the downstream executioner procaspases to induce apoptosis. 2.1.2.1.2. Intrinsic pathway In the intrinsic pathway, several stimuli like oxidative stress, thermal shock, DNA damage, among others, trigger the release of mitochondria cytochrome c to the cytoplasm, because the mitochondrial outer membrane permeabilization (MOMP) is altered.4 The MOMP is highly controlled primarily through interactions between proand antiapoptotic members of the BCL-2 protein family. BCL-2 protein family is divided into three groups based on their BCL-2 homology domain organization: anti-apoptotic (e.g. BCL-2 and BCL-XL), pro-apoptocic effectors (e.g. BAX and BAK), and pro-apoptotic BH3-only proteins (e.g. BID).9 Activation of BAX and BAK is essential for MOMP.4 The antiapoptotic BCL-2/Bcl-xL inhibit BAX and BAK and the BH3-only proteins help in the regulation of the balance between the proand antiapoptotic proteins (Figure 10).6 Chapter 2: Introduction 22 Several peptidomimetic caspase inhibitors were developed by pharmaceutical companies and have entered in clinical trials.2 One example is the broad-spectrum caspase inhibitor IDN-6556 (15, Figure 14) that is a potent and irreversible inhibitor of Fas-induced apoptosis in vitro.14 IDN-6556 (15) entered in clinical trials for treatment of liver diseases and in patients undergoing liver transplantation.14 Figure 14 Structure of the active-site inhibitor IDN-6556 (15). In addition to IDN-6556, other inhibitor VX-765 (16, Figure 15) entered in clinical trials. VX-765 is a second generation reversible peptide-derived inhibitor that has been examined in clinical trials for inflammatory diseases.15 It is a prodrug that requires esterase cleavage of the 5-ethoxydihydrofuran-2(3H)-one moiety to yield the aldehyde functionality of the drug VRT-043198 (17, Figure 15). This moiety covalently interacts with the nucleophilic Cys of the active site and, therefore, inhibits caspases.16 Figure 15: Structure of peptide-derived caspase inhibitors VX-765 (16) and VRT-043198 (17). The syntheses of several cyanopropanate-containing small molecules based on the peptidic scaffold of prodrug VX-765 were also accomplished (Figure 16). These Esterases Chapter 2: Introduction 23 compounds were found to be selective and potent inhibitors of caspase-1 (IC50 values ≤1 nM).15 Figure 16: Structure of cyanopropanate-containing small molecules based on peptidic scaffold of VX-765 as inhibitors of caspase-1 (NCGC00185682 (18), NCGC00183434 (19) and NCGC00183681 (20)). 2.1.3.1.2. Natural caspase inhibitors The first natural caspase inhibitor found was a cytokine response modifier (CrmA), a product of cowpox virus. It was responsible for the effective inhibition of the interleukin-1β activation in chorioallantoic membranes of chicken embryos. The inhibitor interacts with caspase-1 active site, due to the pseudosubstrate Leu – Val – Ala – Asp sequence.17 2.1.3.1.3. Nonpeptidic inhibitors Simultaneously to the development of peptidomimetic caspase inhibitors, other scaffolds were identified through high-throughput screening (HTS) of chemical libraries. 5Nitroisatin (21, Figure 17) was first discovered as a nonpeptide caspase-3 inhibitor by using a HTS of the Smith Kline Beechan compound collection.18 The modification of this compound led to the development of a series of potent isatin analog inhibitors of caspases-3, -7, including compounds 22 and 23 (Figure 17).19 These isatin sulfonamide compounds showed to be potent and selective caspase inhibitors.19 However, their use may be limited because of the highly reactive nature of their ketone carbonyl groups toward nucleophiles. So, a new class of isatin sulfonamide analog compounds has been achieved called isatin Michael acceptors (IMAs), like compound 24. This class has comparable potency against caspases-3 and -7, but reduced selectivity over caspases-1, -6 and -8.20 However, when pyrrolidine was substituted for a thiomorpholinyl residue (compound 25), an increased caspase-6 inhibition and moderate selectivity over Esterases Chapter 2: Introduction 24 caspases-3 and -7 was observed, representing a new class of small molecules caspase-6 inhibitors which could serve as lead compounds for the development of a second generation inhibitors.21 Figure 17: Structures of the isatin sulfonamides caspase inhibitors 21-25. Another class of caspase inhibitors includes those possessing a 1,4 – benzodioxane scaffold, like compounds 26 and 27 (Figure 18). Compound BI-7E7 (26) was identified as an inhibitor of caspase-8-mediated peptide cleavage. Optimization of the thiazole ring through docking studies resulted in compound BI-9B12 (27) with an improved inhibitory activity on caspases-3, -7 and -8.22 Figure 18: Structures of 1,4benzodioxane derivatives 26 and 27. In 2005, other class of caspase inhibitors was introduced based on the 2,3-dihydro1H-pyrrolo[3,4-c]quinoline-1,3-dione scaffold (28, Figure 19).23 The mechanism of Chapter 2: Introduction 25 caspase inhibition of these compounds is mediated by the nucleophilic attack of catalytic cysteine to one of the carbonyl functions at the fused pyrrol ring, leading to the reversible covalent inhibition of the targeted caspases.2 Many compounds were described based in this scaffold, differing at the substituents R1, R2, and R3. According to Kravchenko et al. (2005), caspase-3 inhibitory activity was highly dependent on substitutions on the core scaffold, especially at the 8-position. The compound 29 (Figure 19) with a morpholinesulfonyl moiety at the 8-position and a 1,3,5-trimethyl-1-H-pyrazol-4-yl group at the 2-position was the most potent (IC50 = 4 nM).24 Figure 19: Caspase inhibitors with a pyrrolo[3,4-c]quinoline-1,3-dione scaffold (28) and selected caspases-3 inhibitor (29). 2.1.3.2. Allosteric inhibitors of caspases Allostery is a basic principle of control of enzymatic activities based on the interaction of a protein or a small molecule with a site of an enzyme distinct from the active one. Allosteric modulators represent an alternative approach to the design and synthesis of small molecule activators or inhibitors of proteases and are therefore of wide interest for Medicinal Chemistry.25 Allosteric regulation involves the understanding of some concepts. The concept of conformational selection proposes that a protein exists in equilibrium of multiple conformations, some of them catalytically active, with others being inactive. With the bond of a preferential molecule, there is conformation stabilization (Figure 20). The conformational selection can arise in two ways: or a molecule bonds to the recognized orthosteric site through an endogenous ligand or it bonds to an allosteric site.2 Chapter 2: Introduction 26 (Adapted) 25. Many new allosteric interactions with caspases were revealed by disulfide trapping. A disulfide reversible ligation is formed between cysteine residues at the target protein surface with a small molecule containing sulfhydryl. If a small molecule shows affinity to the protein, the dissulfite is stabilized and a possible modulation of the biological function is measured (Figure 21).26 (Adapted) 27 On-state Off-state Substrate Allosteric inhibitor Active site Allosteric site Substrate Allosteric inhibitor SH S S Off-state HS On-state HS Figure 20: Model of allosteric inhibition. A dynamic protein exists in equilibrium of several low-energy conformations, which are active (on-state) or inactive (off-state). Reversible binding to or irreversible trapping of a distinct conformation shifts this equilibrium, inducing an alteration of enzyme activity. Figure 21: Model of disulfide trapping. A dynamic protein exists in equilibrium of several low-energy conformations, which are active (on-state) or inactive (off-state). Protein has cysteine residues near the allosteric site that interact with small molecule that also contain cysteine residue, preventing the binding of substrate. The caspases is shown as monomer, for simplicity. Chapter 2: Introduction 27 The first successful utilization of disulfide trapping for the caspases allosteric modulation was reported in 2004.28 The screening of a thiol-containing compound database for caspase-3 binding resulted in the discovery of DICA (30, Figure 22) and FICA (31, Figure 22) as new inhibitors of caspase-3. DICA and FICA were found to trap the enzyme in a zymogen-like conformation 29, binding selectively to Cys264 located in a central cavity at the dimerization interface.25 In the caspase-7, different binding modes of DICA and FICA in the allosteric site were demonstrated, but an identical conformational rearrangement in the protease domain was observed. Both compounds induce a switch of active dimerized caspase-7 conformation into a procaspase-7-like conformation by irreversibly trapping the zymogenlike conformation.25 Figure 22: Structures of the allosteric inhibitors of caspases-3 and -7 DICA (30) and FICA (31). The disulfide trapping approach was also applied to the discovery of new inflammatory caspase-1 inhibitors. Despite the low rate of homology between caspases3/7 and caspase-1, small molecule inhibitors could be identified which interfere with a comparable located Cys331 residue at the dimerization interface. Using this approach, compound 32 (Figure 23) was identified as an inhibitor of caspase-1.27 The identification of functional allosteric sites in C1, C3 and C7 served as source of inspiration for further efforts to identify allosteric modulators of these and other caspases.29 Figure 23: Structure of the caspase-1 allosteric inhibitor, compound 32. Chapter 2: Introduction 28 In addition to the small molecule allosteric inhibitors of caspases, some macromolecular inhibitors are also known. For example, AR-F8 is one of the caspase-2 specific macromolecular ligands that significantly inhibits its activity. For this peptidic inhibitor, a high selectivity for caspase-2 over caspases-3, -7, -8, and -9 was observed. Contrary to compounds 30 and 31, which caspase inhibition lead to zymogen-like structures, AR-F8 induced only minor conformational changes within caspase-2. The strong inhibitory effect is due to a slight misalignment of the active cysteine site and the disruption of hydrogen-bond interactions between the substrate and the active site.2 Another example of macromolecular inhibitors is the endogenous inhibitor XIAP, a member of inhibitor apoptosis proteins (IAPs). IAPs are a cell protein family that includes eight members, being six of them identified in humans. This, in general, are expressed at high levels in the majority of human cancers.30 XIAP protein is a caspase inhibitor of caspases-3, -7 and -9 that can interact not only with the allosteric site, but also with the active binding site, depending on the caspase. Caspase-3 and -7 are inhibited by prevention of substrate access to the active site whereas caspase-9 is inhibited allosterically.25 XIAP is composed by three baculoviral IAP repeats (BIR) with distinct binding properties. For caspases-3 and -7, the second BIR domain is the responsible for preventing the substrate bonding. On the other hand, the third BIR domain is responsible for XIAP-mediated inhibition of caspase-9. BIR3 bonds to the N-terminal region of the small subunit of caspase-9.2,5 IAPs do not bind nor inhibit caspase-8 (extrinsic pathway), however they bind and inhibit procaspase-3 substrate, stopping the cascade and protecting the caspase-8 induced apoptosis. The interaction between XIAP and caspases can be disrupted by several small molecules. One example is the compound 33 (Figure 24) that interferes with XIAP’s ability to inhibit caspase-3 31, and compound 34 (Figure 24) that interacts with de BIR3 domain of XIAP, avoiding its bond to caspase-9.32 With this approach, the inhibitory effect of XIAP on caspases activity will be blocked and caspases activity will no longer be inhibited. Figure 24: Structures of XIAP inhibitors, compounds 33 e 34. Chapter 2: Introduction 29 2.1.3.3. Small molecule activators of caspases Activation, instead of inhibition, of caspase activity with synthetic small molecules has been less investigated. Such small molecules can reveal key aspects in the activation of these enzymes and provide a direct effect on important physiological processes. Only in 2006, it was published the first report about caspase activators, when approximately 20,500 structurally diverse small molecules were screened for their ability to activate procaspase-3 in vitro.33 The evaluation of the ability of a group of structural diverse smallmolecules to activate procaspase-3 resulted in the identification of the procaspase-3 activator compound, PAC-1 (35, Figure 25). Additionally, further PAC-1 derivatives were synthesized and evaluated in order to determine their effects on procaspase-3. Consequently, a PAC-1 analogue (PAC-1B or de-allyl PAC-1, 36) was identified, with similar potency than PAC-1 (EC50 value 0.43 µM, PAC-1B value 0.22 µM).33 Figure 25: Structures of PAC-1 (35) and its close analog PAC-1B (36). SAR studies allowed concluding that the presence of the N-benzylpiperazinyl group and the aromatic hydroxyl moieties are crucial for activation and the allyl group is dispensable for the biological activity.34 Peterson et al. (2009), demonstrated that zinc inhibits the enzymatic activity of procaspase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc 34. In fact, it was demonstrated that PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM.34 Moreover, the presence of the N-benzylpiperazinyl group and the aromatic hydroxyl moieties in PAC-1 seems to be fundamental for the ability of PAC-1 to bind zinc ions from the active site, allowing the proenzyme to autoactivate itself to caspase-3 (Figure 26).35 Although, PAC-1 was the first compound to be considered as direct Chapter 2: Introduction 30 activator of caspases, its direct activating ability was questioned, particularly by Denault et al, who demonstrated that PAC-1 and related compounds were not activating executioner caspases directly, reinforcing the idea that the mechanism of action is due to the zinc chelatio9n.36 (Adapted) 34 The relatively short in vivo half-live of PAC-1 (2.1 ± 0.3 h in dogs)37 following i.v. administration is a major challenge for using this compound in vivo studies. In this sense, very recently, Roth et al. (2015) designed a series of PAC-1 analogues containing modifications that systematically block sites of metabolic vulnerability. Evaluation of the library of compounds identified four potentially superior candidates (37-40, Figure 27) with comparable anticancer activity in cell culture, enhanced metabolic stability in liver microsomes, and improved tolerability in mice.37 PAC-1 Procaspase-3 Procaspase-3 Zn2+ Zn2+ PAC-1 Procaspase-3 Caspase-3 Zn2+ Figure 26: The proposed mechanism for PAC-1-induced activation of procaspase-3 in vitro. In the presence of PAC-1, the zinc ion is removed by giving up the caspase-3 activation to function as an enzyme and proteolytically cleave another molecule of procaspase-3. When the zinc ion is not removed, there is an impediment to activation of procaspase-3. Chapter 2: Introduction 31 Figure 27: Derivatives of PAC-1 (37, 38, 39 and 40) with enhanced metabolic stability in liver microsomes, and improved tolerability in mice. In 2014, several benzothiazole derivatives emerged as procaspase-3 activators, from a series of benzothiazole derivatives bearing the ortho-hydroxy-N-acylhydrazone moiety.37,38 Among those compounds, benzothiazoles 41-43 (Figure 28) revealed to be potent inhibitors of human tumor cell lines and more potent than PAC-1 as procaspase-3 activators. SAR studies demonstrated that the phenyl group on the 2-hydroxyphenyl ring was critical for the in vitro pharmacological activity.39 and that the introduction of a lipophilic group at the 4-position of the 2-hydroxy phenyl ring was beneficial to the antitumor activity.38 Figure 28: Structures of PAC-1 analogues (41, 42 and 43). Chapter 2: Introduction 38 Figure 34: Examples of natural flavonoids that have entered in the late phase of clinical trials for various cancers. Given the importance of the antitumor activity of flavonoids, several studies have been conducted in order to get some insights into the cellular and molecular mechanisms of action of these compounds. Flavonoids display a vast array of cellular effects, affecting the overall process of carcinogenesis in different stages (initiation, promotion and progression) by several mechanisms (Figure 35)47, including inhibition of DNA topoisomeraseI/II activity70,71, regulation of reactive oxygen species72, cell cycle arrest57, modulation of proliferation pathways73, and interference with the apoptotic cascade.74,75 Figure 35: Multistage model of carcinogenesis and potential effects of flavonoids on cancer progression. Red arrows indicate the inhibited systems and the green arrows indicate the activated systems by flavonoids. Topoisomerase I/II Apoptosis Cell cycle arrest Angiogenesis PKs Angiogenesis Receptors (EGFRs, VEGFRs PDGFRs) Normal cell Initiated cell Preneoplasia Tumor Metastasis Initiation Promotion Progression on Chapter 2: Introduction 39 Additionally, flavonoids have been reported to modulate several protein kinases. Protein-kinases (PKs) are enzymes that catalyse the phosphorylation of different cellular substrates. Deregulation of PKs can lead to alterations in phosphorylation that result in several abnormalities, namely in uncontrolled cell division and inhibition of apoptosis, being these effects closely linked to various cancers.76 So, inhibition of PKs is an important aspect in cancer chemoprevention by the flavonoids. Some examples of the protein kinases that are inhibited by flavonoids include serine/threonine kinases, phosphatidylinositol kinase and cyclin-dependent kinases.51 Flavonoids are also capable of interact with several receptors which play important roles in cancer pathology 77, namely the epidermal growth factor receptors (EGFRs)78-80, platelet derived growth factor receptors (PDGFRs), vascular endothelial growth factor receptors (VEGFRs)81,82 and cyclin-dependent kinases (CDKs).83 2.2.1.2. Flavonoids as pro-apoptotic agents in cancer Activation of apoptosis is a key molecular mechanism responsible for the anticancer activities of several currently studied potential anticancer agents, including flavonoids.84 Several reports indicate that these compounds may induce apoptosis by affecting the expression or activity of a wide range of molecules involved in apoptosis pathways (Figure 36). In fact, the induction of apoptosis by flavonoids can be related to the activation of caspases, decrease of variation of the membrane potential (∆Ψm), release of cytochrome c from mitochondria, regulation of Bcl-2 family proteins members, and regulation of IAP by release of Smac/DIABLO from mitochondria, cleavage of poly (ADPribose) polymerase (PARP), increase levels of reactive oxygen species (ROS) and activation of death receptors (Figure 36). Chapter 2: Introduction 40 Bax, Bak intrinsic pathway Apoptosome Procaspase-9 Caspase-9 Cytochrome c extrinsic pathway DISC Procaspase-8 Caspase-8 Procaspase-3 Procaspase-6 Procaspase-7 Caspase-3 Caspase-6 Caspase-7 v APOPTOSIS Bcl-2,Bcl-x Bid IAP v v Smac/ DIABLO v FLAVONOID Direct stimulatory modification Indirect stimulatory modification Direct inhibitory modification Figure 36: Flavonoids may induce apoptosis by affecting the expression or activity of a wide range of molecules involved in apoptosis pathways. Chapter 2: Introduction 41 Some examples of flavonoids that induce apoptosis by modulating different key targets involved in apoptotic pathways, including caspase family proteins are listed in Table 3. Despite few structure-activity relatioship studies (SAR) have been conducted regarding antitumor activity of flavonoids, some researchers suggest that flavones and flavonols are the most potent flavonoids.85 So, the flavonoids listed in Table 3 are restricted to these two classes of flavonoids. Table 3: Some flavones and flavonols reported since 2000 as inducers of apoptosis by modulating different key targets involved in apoptotic pathways, including caspase family proteins reported. Compound Target or affected process References Natural flavones and flavonols Methoxyflavones and flavonols 56: nobiletin -Decrease Bcl-2 expression -Increase Bax expression -Increase caspase-3 expression, in HCC cell line SMMC-7721 and HCC cell line H22 -Induction of cell cycle arrest in G0/G1 phase -Inhibition ERK -Activation of p38 -Activation of caspase-8,-9 and - 3, in HL-60 AML cells -Cleavage of PARP -Increase caspase-3 activity, in HUVEC -Cleavage of PARP -Decrease Bcl-2 expression -Increase p21 expression -Increase caspase-3,-7 and -9 activities 86-89 57:5-Hydroxy-3,6,7,8,3',4'- hexamethoxyflavone (5-OH-HxMF) -Decrease ∆Ψm -Increase levels of ROS -Increase caspase-2, -3, -6 and -7 activities -Cleavage of PARP -Increase Bad expression, in HL-60 cells 90 Chapter 2: Introduction 42 58: R=OCH3, tangeretin 59: R=OH, 5-DT -Decrease ∆Ψm -Increase caspases-3, -8 and -9 activities -Increase Bax expression -Increase Bid expression -Increase tBid expression -Increase Fas expression -Increase FasL expression -Increase p53 expression, in AGS cells -Arrest cell cycle in G2/M phase -Increase caspases-3, -7 and -9 activities -Decrease Mcl-1 expression -Decrease Bcl-Xl expression, in K562 cells -Increase p53 expression -Increase Bax expression -Decrease cdc-2 (cdk1) and cyclin B1 expression -Increase p21 expression -Cleavage of PARP, in human nonsmall cell lung cancer (H460,H1299 and A549) cells 91-93 60: R3,R4´,R5´=H; R5,R7=OCH3,DMF 61: R3,R5´=H; R5,R7,R4´=OCH3, TMF 62: R3,R5,R7,R5´,R4´=OCH3, PMF 63: R3,R7,R2´R4´,R5´=H; R5=OCH3, 5-MF 64: R3,R5,R7,R4´,R5´=H R2´=OCH3, 2´-MF -Increase DRs expression -Decrease cFLIP expression -Decrease Mcl-1 expression -Increase Bax expression -Decrease Bid expression -Activation of caspase-3 and -8 -Increase oxidative stress, in human leukemic MOLT-4 cells 5-MF: -Increase caspases-3 and -7 activation -Cleavage of PARP ,in HCT116 colo cancer cells 94,95 Hydroxyflavones and flavonols Chapter 2: Introduction 43 65: luteolin -Decrease ∆Ψm -Increase cit c release -Cleavage of PARP -Increase Bad expression -Increase Bax expression -Decrease Bcl-2 expression -Decrease Bcl-Xl expression -Induce and increase caspase-3 and -9 activities, in HL-60 cells -Inhibit Bcl-2 expression -Increase Bax expression -Increase caspase-3 activity, in MG-63 cells -increased Fas expression -Activation of caspases-8 and - 3, in MDA-MB-231 96-98 66: apigenin -Increase levels of ROS -Induction of cell cycle arrest in G2/M phase, in SCC25 and A431 cells -Increase TNF-R expression -Increase TRAIL-R expression -Decrease Bcl-2 expression -Activation of caspase-3, in SCC25 cells -Increase levels of ROS -Decrease ∆Ψm -Increase cit c release -Increase Bax expression -Decrease Bcl-2 expression -Activation caspases-3 and -9, in A375 and A549 cells -Increase cit c release -Increase caspase-3 activity, in breast cancer cells (with HER2/neu-over-expressing) -Increase caspase-3 and -9 activities -Increase levels of ROS -Activation of ERK and p38, in THP-1 cells -Increase caspase-3 activity -Cleavage of PARP -Increase Bax expression, in NUB-7 cells 99-103 Chapter 2: Introduction 44 -Increase levels of ROS -Decrease ∆Ψm -Increase cit c release -Increase Bax expression -Decrease Bcl-2 expression -Decrease Bcl-xl expression -Cleavage of PARP -Increase caspse-3 and -9 activities, in 22Rv1 cells 67: 7,8-DHF -Increase Fas expression -Increase FasL expression -Increase DR4 expression -Increase TRAIL expression -Activate caspase-3,-8 and -9 -Cleavage of PARP -Increase Bax expression -Increase cit c release -Activation ERK and JNK, in U937 cells 104 68: morin -Decrease ∆Ψm -Increase cit c release -Decrease Bcl-2 expression -Increase Bax expression -Cleavage of PARP -Increase caspase-3, -8 and -9 activities, in U937 cells 105 69: kaempferol -Decrease Bcl-xL expression -Increase p53 expresson -Increase Bad expression -Increase Bax expression -increase caspase-3 and -7 activities, in Ovarian cancer cell lines (OVCAR-3, A2780/CP70, and A2780/wt), - Decrease Bcl-xL expression -Increase Bad expression -Cleavage of PARP -Increase caspase-3, -7, -8 and -9 activity in HT-29 Human Colon Cancer Cells -decrease Bcl-2 expression -Increase Bax expression 106-108 Chapter 2: Introduction 45 -Cleavage of PARP -increase caspase-3 and -9 activities, in gastric cancer 70: quercetin -Decrease ∆Ψm -Increase Bax expression -Decrease Bad expression -increase caspase-3,-8 and -9 activities, in MDA-MB-231 cells -Decrease Bcl-xL expression -Increase Bax expression -Increase caspase-3 and -9 activities, in HepG2 cells 109,110 Methoxyand-Hydroxyflavones and Flavonols 71: R1 =OCH3; R2,R3,R4,R5= H, wogonin 72: R1, R2= H; R3,R4,R5=OH, fisetin 73: R1=OH; R2,R3,R4,R5=H, norwogonin wogonin : -Increase levels of ROS -Decrease ∆Ψm -Increase caspases-3,-9 and -8 activities -Increase Bax expression -Increase Bad expression -Increase cit c release -Increase Fas/CD95 ,In U-2 OS -increase TRAIL-R2 expression, in ATL cells -Increase levels of ROS -Cleavage of PARP -Increase caspase-3 activity, in HL-60 cells -Cleavage of PARP -Increase caspase-3 activity, in SK-HEP-1 cells norwogonin: -Increase levels of ROS -Cleavage of PARP -Increase caspase-3 activity, in HL-60 cells Fisetin: Cleavage of PARP -Increase caspase-3 activity, in SK-HEP-1 cells 111-114 Chapter 2: Introduction 46 74: acacetin -Induce caspases-3,-8 and -9 activities -Increase FADD expression -Increase FAF1 expression -Increase Apaf-1 expression -Increase cit c expression -Increase Bax expression -Decrease Bcl-2 expression, in human T cell leukemia Jurkat cells -Decrease Bcl-2 expression -Decrease ∆Ψm -Increase levels of ROS -Activation of the SAPK/JNK1/2 and c-JUN -Activation of caspase-7 and -8, in MCF-7 cells -Cleavage of PARP -Decrease ∆Ψm -Increase levels of ROS -Increase cit c release -Increase Bax expression -Increase p53 expression -Increase Fas expression -Increase Bad expression -Decrease Bcl-2 expression -Increase caspase-3 activity, in AGS cells 115 116,117 75: eupatilin -Decrese Bcl-2 expression -Increase Bax expression -Cleavage of PARP -Cleavage of caspase-3 -Decrease ∆Ψm , in AGS cells 118 76: Skullcapflavone I -Cleavage of PARP -Increase caspases-3 and -9 activities, in T-HSC/Cl-6 cells 119 Chapter 2: Introduction 47 77: jaceosidin -Increase levels of ROS -Cleavage of PARP -Decrease Bcl-2 expression -Increase Bax expression -Increase p53 expression -Inhibition of the ERK pathway, in MCF10A-ras cells 120 78: cirsilineol -Decrease ∆Ψm -Increase cit c release -Cleavage of PARP -Increase caspase-3 and -9 activities, in CAov-3 cells 121 79:R1=H,R2=OMe,R3=OMe, 5HPMF 80: R1,R2,R3=OMe, 5HHMF 81: R1=H,R2=H,R3=OMe, 5HTMF -5HPMF induces cell cycle arrest in G2/M in HCT116 and HT29 cells -Cell cycle arrest in G0/G1 phase -Decrease Mcl-1 expression -Cleavage of PARP -Decrease iNOS expression -Decrease COX-2 expression, in H1299 cells -5HTMF induces cell cycle arrest in G0/G1 phase -Decrease Mcl-1 level, in HCT116, HT29 and H1299 cells -Decrease Mcl-1 expression -Cleavage of PARP -Decrease iNOS expression -Decrease COX-2 expression, in H1299 cells - 5HTMF induces cell cycle arrest in G2/M phase -Increase p53 expression -Increase Bax ecpression -Decrease cdc-2 (cdk1) and cyclin B1 expression -Increase p21 expression -Cleavage of PARP, in human nonsmall cell lung cancer (H460,H1299 and A549) cells -Increase Bax expression, in H460 and A549 cells 122-124 Chapter 2: Introduction 54 106: ayanin diacetate -Decrease ∆Ψm -Increase cit c release -Cleavage of PARP -Increase caspase-8 and -9 activities -Decrease Bcl-XL expression -Cleavage of Bid, in leukemia cells 158 107: 8-bromo-7-methoxychrysin -Arrest cell cycle in G1 phase, in HepG2 and Bel-7402 cells -Increase caspase-3 activity -Increase ROS level, in HepG2 cells 159 108:WYCO2-9 -Increase levels of ROS -Decrease ∆Ψm -Activation of caspases-3 and -9 -Cleavage of PARP ,in DU145 prostate cancer cells 160 109: R=Cl, methyl 2-(2chlorophenyl)-6,7-dimethyl-4-oxo4H-chromene-8-carboxylate 110: R=CH3, 1 methyl 6,7-dimethyl4-oxo-2-(o-tolyl)-4H-chromene-8carboxylate -Cleavage of PARP -Increase cit c realese -Activation of the ERK pathway, in HL-60 cells 161 111: 4’-bromoflavonol -Increase caspase-3,-6,-7 -9 and -8 activities -Increase TRAIL expression -Increase DR4 expression -Increase DR5 expression -Decrease Bid expression -Arrest cell cycle in S phase, in HL-60 cells -Decrease Bid expression, in 162 Chapter 2: Introduction 55 Molt-3 cells -Increase caspase-3,-6-7,-8 and -9 activities -Increase TRAIL expression -Increase DR4 expression -Increase DR5 expression -Processing of Bid -Arrest cell cycle in S phase, in U937 cells 112:6-chloro-2-(3,5dimethoxyphenyl)-4H-chromen-4one -Increase caspases-8 and -9 activities -Increase Bax expression -Decrease bcl-2 expression, in HepG-2 cells 163 113: 2´-Nitroflavone -Increase TRAIL expression -Increase DR5 expression -Increase Bax expression -Increase cit c release -Induction cell cycle arrest in G2/M phase -Activation of p38 and JNK, -Decrease ERK, in HL-60 cells -Increase Bax expression -Increase Fas expression -Increase caspase-3, -8 and-9 activities -Increase cit c release -Increase Bax expression -Decrease Bcl-2 expression -Decrease Bcl-Xl expression, in LM3 murine mammary adenocarcinoma cells -Decrease Bcl-xl expression -Increase Bax expression -Increase Fas expression -Increase Fas-L expression -Increase cit c release -Increase caspase-3,-8 and -9 activities, in HeLa human cervical carcinoma cells 164-166 Chapter 2: Introduction 56 114:diethyl flavon-7-yl phosphate (FP) -Cleavage of PARP -Increase caspase-3 activity, in HeLa cells 167 (PARP= poly (ADP-ribose) polymerase; ASK-1=apoptosis signal regulating kinase 1; ROS= reactive oxygen species; cit c=cytochrome c; ∆Ψm= variation of the membrane potential; MARK=mitogen-activated protein kinases; ERK=extracellular-regulated kinase; JNK=c-Jun Nterminal kinase; NF-R= tumor necrosis factor receptor; TRAIL-R=TNF-related apoptosis-inducing ligand receptor; Mcl-1=anti-apoptotic BCL-2 protein) As described in Table 3, most flavonoids that significantly induce apoptosis in tumor cells are not direct activators of caspase family proteins, but interfere with other targets related to the intrinsic or extrinsic apoptosis pathways and consequently, increase the caspases expression or levels in tumor cell lines. In fact, the reports about the direct activation of caspases by flavonoids are rare. Recently, in 2014 two activators of caspase7 (1 and 2, Figure 1, pag 4) were discovered by Pereira et al. (2014). Compounds 1 and 2 are two prenylated derivatives of baicalein (B, Figure 37) and 3,7-dihydroxyflavone (H, Figure 37), two natural flavonoids that have shown to be potent inhibitors of tumor cell lines, being this effect attributed at least in part to an apoptotic response.168-170 Moreover, it was demonstrated that the introduction of prenyl side chains on baicalein (B) and 3,7dihydroxyflavone (H) was associated with an increase of their human tumor cell lines growth inhibitory activity.171 In this work, innovative assays using Saccharomyces cerevisiae individually expressing human caspases-3, -7 or procaspases -3, -7 were developed for screening assays. The validation of this model was accomplished by testing the effect of the known activator of caspase-3 and -7, PAC-1 (35, pag 29).172 For development of yeast growth-inhibition assays, initially it has been found that the expression of human procaspases-3 and -7 have no effect on cell growth. While the expression of the cleavaged caspases-3 and -7 have a marked effect on cell growth. The relation between this effect and the expression of these human proteins was confirmed by Western Blot analysis.173,174 It was also found that PAC-1 induced growth inhibition in Table 3(contd) Chapter 2: Introduction 57 yeast cells expressing procaspases-3 and -7 and caspases-3 and -7. These results indicated that PAC-1 was able to activate these caspases, as well as their inactive zymogens, in this model. Using the same model, flavonoids 1 and 2 also induced procaspase-7-mediated growth inhibition and increase the caspase-7-induced growth inhibition. Nevertheless, no effect on yeast expressing procaspase-3 and caspase-3 were observed with these compounds. These results suggested that, in opposition to PAC-1, flavonoids 1 and 2 were selective activators of caspase-7. In vitro processing assays confirmed that both flavonoids 1 and 2 directly processed procaspase-7 to the active caspase-7.175 The effect of these flavonoids was also evaluated in HL-60 and MCF-7 human tumor cell lines. HL-60 is an acute promyelocytic leukemia cell line expressing both caspases-3 and -7, while MCF-7 is a breast adenocarcinoma cell line that only expresses caspase-7. The results reveled that both flavonoids inhibit the growth of the two cell lines with a similar GI50, confirming an effect independent of caspase-3. Considering the structure of flavonoids 1 and 2, and their building blocks, as no activity was observed for compound 2, it was suggested that the presence of the 7-prenyl group of flavonoid 1 seems to be associated with selectivity of this flavonoid to caspase-7, and the presence of the 7-geranyl of flavonoid 2 seems to be associated with the caspase-7 activation. Chapter 2: Introduction 58 Chapter 3: Results and Discussion Chapter 3: Results and Discussion 61 3.1. Chemistry Analogues of the prenylflavonoids FP2 (1) and FP11 (2) were synthesized by molecular modification of three natural flavonoids: baicalein (B), 3,7-dihydroxyflavone (H) and chrysin (C) (Figure 37). (B) (H) (C) Figure 37: Natural flavonoids used as building blocks for molecular modification. Since baicalein (B) and 3,7-dihydroxyflavone (H) were the precursors of FP2 and FP11, both natural flavones were chosen as building blocks. Additionally, these natural compounds have already demonstrated to have antitumor activity in human tumor cell lines, which seems to be related to the interference with caspases-dependent pathways.168,176,177 Similarly, the antitumor activity of chrysin (C) has also been described178, as well as its capacity to induce apoptosis through caspases-dependent pathways84,179 and the interference with other molecules involved in the apoptotic cascade.180 Moreover, considering the similarity of the substitution pattern on A ring between, baicalein (B), 3,7-dihydroxyflavone (H), and chrysin (C), the molecular modification of these three building blocks and the study of their caspase modulatory activity will allow to study the influence of the ring-A substitution pattern, namely the hydroxylation pattern, on the caspase modulatory activity. The molecular modification follow the alkylation of the natural flavonoids using alkylating agents possessing 1-5 carbon atoms, including linear (methyl, ethyl, propyl, allyl, butyl) and branched chains (isopentyl and prenyl) with or without unsaturation, aiming to allow SAR Studies. The synthesis of the alkylated derivatives followed nucleophilic substitution reactions of the building blocks with alkyl halide in alkaline medium. Chapter 3: Results and Discussion 62 3.1.1. Synthesis 3.1.1.1. Optimization of baicalein alkylation Previous studies revealed that the direct alkylation with prenyl and geranyl bromide of baicalein (B) gave a mixture of the monoand dialkylated derivatives, as well as the building block.171 In addition, it has been demonstrated that the 7-monoprenylated derivatives were potent inhibitors of human tumor cell lines, in opposite to the 6,7diprenylated derivatives, being this effect related, at least in part, to the activation of caspase-7.175 Although no antiproliferative activity have been found for these 6,7diprenylated derivatives, other 6,7modified baicalein derivatives have reveal interesting antitumor activity.181,182 Moreover, several structure related flavones possessing a similar substitution pattern in A ring (hydroxyl group in C-5 and alkyloxy groups in C-6 and C-7) revealed to be potent inhibitors of human tumor cell lines. For example, among the flavones listed in Table 3 (pag 46, 50 and 51), compounds 78, 92, 95, 97 and 98 showed pro-apoptotic effect by modulating different key targets involved in apoptotic pathways, including caspase family proteins. Therefore, the direct alkylation of this building block was investigated by HPLC-DAD to determine the more suitable amount of alkyl halide necessary to obtain mainly the monoalkylated derivative but also some amount of the dialkylated derivative, to be used in the biological assays. Since it would be difficult to perform this study for all alkylating agents, it was decided to optimize the conditions for the longest and branched chain without unsaturation (isopentyl iodide). 3.1.1.1.1. Optimization of chromatographic conditions Baicalein mono- (Biso1) and diisopentyl- (Biso2) derivatives were synthesized as described in Figure 38 and after their purification and characterization by IR and NMR, were used as standards for the HPLC-DAD study. Chapter 3: Results and Discussion 63 Figure 38: Synthesis of alkylated derivatives of Baicalein (B). Firstly, the chromatographic conditions to quantify Biso1 and Biso2 as well as baicalein in the reaction mixture were established. To fullfil this, a mixture of these compounds was analized by HPLC-DAD on C18 HPLC column using several mixtures of MeOH:H2O:MeCO2H as mobile phase. As shown in Figure 39 the mixture MeOH:H2O:MeCO2H (95:5:1 v/v/v) allowed to obtain the suitable chromatographic conditions to quantify the major constituents of the reaction with a good resolution (Figure 39). Figure 39: HPLC-DAD chromatogram of a mixture of baicalein (B) 5,6-dihydroxy-7-(isopentyloxy)-2-phenyl4H-chromen-4-one (Biso1) and 5-hydroxy-6,7-bis(isopentyloxy)-2-phenyl-4H-chromen-4-one (Biso2) at 275 nm with MeOH:H2O:MeCO2H (95:5:1 v/v/v) as mobile phase. Chapter 3: Results and Discussion 70 Table 7: General conditions for the synthesis of alkylated derivatives of 3,7-dihydroxyflavone (H). Building block Reaction conditions Product Yield H isopentyl iodide, K2CO3, Me2CO, 2 h Hiso 12.9% butyl iodide, K2CO3, Me2CO, 2 h Hutil 1 9.7% Hutil 2 7.1% propyl iodide, K2CO3, Me2CO, 2 h Hrop 3.7% allyl iodide, K2CO3, Me2CO, 2 h Hali 9.3% ethyl iodide, K2CO3, Me2CO, 2 h Hetil 1.7% methyl iodide, K2CO3, Me2CO, 2 h Hemet 9.2% prenyl bromide, KI, K2CO3, Me2CO, 2 h Hrep 7.4% Chapter 3: Results and Discussion 71 With this building block only the monoalkylated derivatives (Hiso, Hutil1, Hrop, Hali, Hetil, Hemet and Hrep) have been isolated, except for the reaction with butyl iodide, for which the dialkylated derivative (Hutil2) was also obtained. In fact, for all reactions although two derivatives were detected by TLC in the crude product, successive attempts of purification of the dialkylated derivatives by several methods (flash column chromatography, thin layer chromatography and crystallization) have failed to isolate the dialkylated derivatives. As already referred for the reactions with baicalein, the yield obtained for the monoalkylated derivative Hutil1 was slightly higher than the yield obtained for the dialkylated derivative Hutil2, suggesting that the hydroxyl group on the para position to the carbonyl group (C7) is the first to be alkylated. Incomplete reactions associated with the time consuming work-up procedure which involved column and preparative thin layer chromatography followed by crystallization justify the low yields of these reactions (1.7-12.9%). 3.2.1.4. Derivatives of chrysin The synthetic approach used for the synthesis of alkylated derivatives was based on the reaction of chrysin (C) with 1.6 eq. of the isopentyl, butyl, propyl, allyl, ethyl, and methyl iodide or 2 eq. of the prenyl bromide in presence of anhydrous potassium carbonate using MAOS methodology. Table 8 summarizes the reaction conditions and the results obtained in the synthesis of chrysin alkyl-derivatives. Chapter 3: Results and Discussion 72 Table 8: General conditions for the synthesis of alkylated derivatives of Chrysin (C). Building block Reaction conditions Product Yield C isopentyl iodide, K2CO3, Me2CO, 1.5 h Ciso 30% butyl iodide, K2CO3, Me2CO, 1.5 h Cutil 29% propyl iodide, K2CO3, Me2CO, 1.5 h Crop 22% allyl iodide, K2CO3, Me2CO, 1.5 h Cali 31% ethyl iodide, K2CO3, Me2CO, 1.5 h Cetil 32% methyl iodide, K2CO3, Me2CO, 1.5 h Cemet 44% prenyl bromide, KI, K2CO3, Me2CO, 1.5 h Crep 67 % Unlike reactions with other building blocks, as expected, only the monoalkylated derivatives possessing the alkyloxy group linked to C-7 (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep) were obtained for reactions with chrysin, being this reflected in the higher yield of these reactions (22-67%). Chapter 3: Results and Discussion 73 3.1.2. Structure elucidation The structure elucidation of all alkylated flavonoids was established on the basis of IR, and NMR techniques. 13C NMR assignments were determined by 2D heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments. The numbering concerning the NMR assignments of alkyl groups is presented in Table 23 and the numbering relative to the NMR assignments of flavonoid scaffolds is presented in Figure 37. 3.1.2.1. Derivatives of baicalein The IR data of all baicalein derivatives were in accordance with the performed molecular modification (Table 9). Accordingly, this spectra revealed the presence of at least one free hydroxyl group, attending that a large band of stretching vibration between 3600–3300 cm-1 (hydroxyl groups) was visualized for all synthesized compounds, suggesting that the hydroxyl groups were not fully replaced. In addition, the observation of bands at 2961-2851 cm-1 (aliphatic C-H) on the IR spectra of baicalein derivatives, in contrast to their starting material, suggested the presence of alkyl groups. Table 9: IR data of baicalein (B) and its alkylated derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1', Betil1, Bemet1 and Bemet2). Groups ʋ (cm-1) B Biso1 Biso2 Butil1 Butil2 Brop1 OH 35003300 36003300 36003300 36003300 36003300 36003300 Aliphatic C-H - 2955 2921 2854 2957 2922 2851 2956 2923 2873 2961 2925 2851 2927 2922 2851 C=O 1651 1657 1653 1655 1657 1617 Aromatic C=C 1609 1573 1498 1462 1489 1477 1450 1559 1497 1457 1419 1502 1584 1464 1451 1497 1457 1419 1559 1489 1473 1459 C-O 1287 1115 1120 1191 1121 1125 Chapter 3: Results and Discussion 74 Table 9 (contd.): IR data of baicalein (B) and its alkylated derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1', Betil1, Betil2, Bemet1 and Bemet2). Groups ʋ (cm-1) Brop2 Bali1 Bali1’ Betil1 Bemet1 Bemet2 OH 36003300 36003300 36003300 36003300 36003300 36003300 Aliphatic C-H 2965 2921 2851 2925 2923 2854 2925 2923 2854 2952 2922 2922 2922 C=O 1657 1663 1664 1653 1631 1634 Aromatic C=C 1499 1470 1458 1576 1489 1449 1585 1489 1451 1559 1507 1457 1467 1457 C-O 1122 1252 1115 1189 1131 1129 The 1H and 13C NMR data of baicalein (B) and alkyl-derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1’, Betil1, Bemet1 and Bemet2) are reported in Table 10 and Table 11, respectively. The 1H NMR spectra of baicalein (B) derivatives showed that all of them have a non-substituted B-ring (H-2’, 6’: δH 7.91-7.87 m; H-3’, 4’, 5’: δH 7.58-7.49 m) as the precursor B. Like the flavone B, 1H NMR spectra of all derivatives showed a signal at δH 6.69-6.67 s corresponding to H-3, and a signal at δH 6.63-6.55 s corresponding to H-8. For most derivatives, namely Biso2, Brop1, Brop2, Bali1, Bali1’, Betil1, and Bemet1 a hydrogen-bonded hydroxyl group at C-5 (δH 13.04-12.50 s) was also observed, like for the starting material B. Besides these, for derivatives Biso1, Brop1, Betil1 and Bemet1 the signal of the hydroxyl group at C6 (δH 5.37-5.35 s) was also observed. These data confirms that neither of these positions is substituted. Considering Biso1 and Biso2 the 1H NMR and 13C NMR spectra showed characteristic signals of one or two isopentyl groups (δH: 4.18-4.06 t, 1.93-1.65 m, 1.961.23 m, 1.01-0.97 d; δC: 71.8-67.5, 38.9-37.5, 29.8-29.3, 22.7-22.6, 22.6-22.5) respectively (Table 10 and Table 11). Although, the signal of the hydroxyl group at C-6 was observed for the flavone Biso1, the correspondent signal was not observed for the derivative Biso2, suggesting that the second isopentyl group should be linked to the hydroxyl group at position 6. For Biso1 the position of the isopentyl side chain on flavone Chapter 3: Results and Discussion 75 skeleton was confirmed by the correlations observed on the HMBC spectra between the proton signals of the oxymethylene group (δH 4.18 t) and the carbon signal of C-7 (δC 152.3). For Biso2 a similar correlation was observed for the proton signals of the oxymethylene groups (δH: 4.12 t and 4.06 t) and the carbon signals of C-7 (δC 158.9) and C-6 (δC 131.9), respectively (Figure 41). Figure 41: Main connectivities found in the HMBC of derivatives Biso1 and Biso2. For derivatives Butil1 and Butil2, the 1H and 13C NMR spectra put in evidence the presence of one or two butyl side chains at positions 6 or 7 (Table 10 and Table 11). This group was evidenced by the signals of one oxymethylene group (δH 4.16-4.05 t, δC 73.169.3), two methylenic groups (δH 1.94-1.73 m and δH 1.62-1.48 m, δC 32.2-31.0 and δC 19.2) and one methylic group (δH 1.02-0.97 t, δC 13.9-13.8). As Butil1 was the major product of the nucleophilic substitution reaction it was proposed that this compound should result from the substitution at position 7. On the contrary Butil2 should result from the substitution at position 6. This hypothesis was confirmed by the correlation observed in the HMBC spectrum of Butil1 and Butil2 (Figure 42). Figure 42: Main connectivities found in the HMBC of derivatives Butil1 and Butil2. Chapter 3: Results and Discussion 76 Regarding Brop1 the 1H NMR spectra displayed two triplets at δH 4.12 (2H) and δH 1.10 (3H), and one multiplet at δH 2.05-1.98 m (2H), suggesting the presence of a propyloxy side chain at C-7. Instead, for Brop2 the 1H NMR spectra showed four triplets at δH 4.05 (2H), δH 4.01 (2H), δH 1.10 (3H), and δH 1.06 (3H) and two multiplets at δH 1.961.89 (2H) and δH 1.85-1.78 (2H), revealing the presence of two propyloxy groups linked to flavone scaffold. Additionally, the signal of the hydroxyl group at C-6 was observed for the flavone Brop1. However, the correspondent signal was not observed for the derivative Brop2, suggesting that the second propyloxy group should be at position 6. For both propyl derivatives, the linkage of propyloxy groups to C-7 was confirmed by the correlations observed on the HMBC spectra between the proton signals of the oxymethylene group at δH 4.12 and δH 4.05 and the carbon signals of C-7 δC 152.3 and δC 158.8, respectively) (Figure 43) The linkage of propyloxy group to C-6 for compound Brop2 was also confirmed by the correlation between the proton signals at δH 4.01 t and the carbon signal of C-6 (δC 132.0) (Figure 43). Figure 43: Main connectivities found in the HMBC of derivatives Brop1 and Brop2. For derivatives Bali1 and Bali1’, the 1H and 13C NMR spectra showed signals that put in evidence the presence of one allyloxy group at positions 6 or 7 (Table 10 and Table 11). This group was evidenced by the signals of one oxymethylene group (δH 4.77-4.74 d, δC 73.7-70.2), one olephynic proton at δH 6.18 -6.01 m linked to a carbon at δC 133.0131.8, other olephinic carbon at δC 120.0-119.1 which is linked to two olephynic protons (δH 5.51-5.28 m). As Bali1 was the major product of the nucleophilic substitution reaction it was proposed that this compound should result from the substitution at position 7. On the contrary Bali1’ should result from the substitution at position 6. This hypothesis was confirmed by the correlation observed in the HMBC spectrum of Bali1 and Bali1’ (Figure 44). Chapter 3: Results and Discussion 77 Figure 44: Main connectivities found in the HMBC of derivatives Bali1 and Bali1'. Considering Betil1 the 1H NMR and 13C NMR spectra showed characteristic signals of one ethyl group (δH 4.24 q, 1.55 t; δC 65.2, 14.6) (Table 10 and Table 11). The correlations observed on the HMBC spectra between the proton signals of the oxymethylene group (δH 4.24 q) and the carbon signal of C-7 (δC 152.2) confirmed the position of this side chain on the flavone skeleton (Figure 45). Figure 45: Main connectivities found in the HMBC spectrum of derivative Betil1. Regarding Bemet1 the 1H and 13C NMR spectra displayed the presence of signals characteristic of one methoxyl group (δH 3.94 s, δC 60.9) confirming the methylation of one hydroxyl group of the flavone scaffold. Instead, for Bemet2 the 1H and 13C NMR spectra showed signals of two methoxyl groups (δH 3.93-3.98 s, δC 56.4-66.9) revealing the methylation of both hydroxyl groups at C-6 and C-7. For both methyl derivatives, the linkage of methoxyl groups to C-7 was confirmed by the correlations observed on the HMBC spectra between the proton signals of the methoxyl group δH 3.94 and δH 3.98 and the carbon signals of C-7 (δC 153.1 and δC 159.0) (Figure 46). The linkage of methoxyl group to C-6 for compound Bemet2 was also confirmed by the correlation between the proton signals at δH 3.93 s and the carbon signal of C-6 (δC 132.7) (Figure 46). Chapter 3: Results and Discussion 78 Figure 46: Main connectivities found in the HMBC of derivatives Bemet1 and Bemet2. For derivatives Brop2, Butil2, Bali1, Bemet1 and Bemet2 the 1H and 13C NMR data are in accordance to the published data. 183-185 Chapter 3: Results and Discussion 79 Table 10: 1H NMR data of baicalein (B) and its alkylated derivatives (Biso1, Biso2, Butil1, Butil2, Brop1, Brop2, Bali1, Bali1’, Betil1, Bemet1 and Bemet2). B Biso1 Biso2 Butil1 Butil2 Brop1 H-2’,6’ 7.92-7.88 (m) 7.91-7.88 (m) 7.91-7.88 (m) 7.91-7.87 (m) 7.91-7.88 (m) 7.91-7.88 (m) H-3’,4’,5’ 7.60-7.52 (m) 7.54-7-52 (m) 7.54-7.52 (m) 7.54-7.52 (m) 7.54-7.52 (m) 7.55-7.52 (m) H-3 6.64 (s) 6.69 (s) 6.67 (s) 6.68 (s) 6.67 (s) 6.69 (s) H-5 12.72 (OH, s) n.o 12.61 (OH,s) n.o n.o. 12.50 (OH,s) H-6 n.o. 5.35 (OH,s) - n.o - 5.37 (OH,s) H-7 n.o. - - - - - H-8 6.60 (s) 6.62 (s) 6.56 (s) 6.61 (s) 6.55 (s) 6.61 (s) H-1’’ — 4.18 (t, J=6.6) 4.12 (t, J=6.5) 4.16 (t, J=6.6) 4.09 (t, J=6.5) 4.12 (t, J=6.9) H-2’’ — 1.93-1.77 (m) 1.82-1.75 (m) 1.94-1.85 (m) 1.93-1.83 (m) 2.05-1.98 (m) H-3’’ — 1.29-1.23 (m) 1.96-1.84 (m) 1.61-1.48 (m) 1.62-1.48 (m) 1.10 (t, J=7.4) H-4’’ — 1.01 (d, J=6.3) 1.00 (d, J= 6.5) 1.02 (t, J=7.4) 1.02 (t, J=7.4) - H-5’’ — - - - H-1’’’ — 4.06 (t, J=6.9) - 4.05 (t, J=6.6) - H-2’’’ — 1.72-1.65 (m) - 1.82-1.73 (m) - H-3’’’ — 1.96-1.84 (m) - 1.62-1.48 (m) - H-4’’’ — 0.97 (d, J=6.6) - 0,97 (t, J=7.4) - H-5’’’ — - - - n.o. not observed; Values in parts per million (δH). Measured in CDCl3 at 300.13 MHz. J values (Hz) are presented in parentheses. Chapter 3: Results and Discussion 86 Table 13: 1H NMR data of 3,7-dihydroxyflavone (H) and its alkylated derivatives (Hiso, Hutil1, Hutil2, Hrop, Hali, Hetil, Hemet and Hrep). H Hiso Hutil1 Hutil2 Hrop Hali Hetil Hemet Hrep H-2’,6’ 8.24-8.21 (m) 8.258.23 (m) 8.25-8.22 (m) 8.10-8.07 (m) 8.25-8.22 (m) 8.25-8.22 (m) 8.25-8.22 (m) 8.24-8.21 (m) 8.25-8.22 (m) H-3’,4’,5’ 7.53-7.43 (m) 7.56-7.42 (m) 7.56-7.43 (m) 7.52-7.49 (m) 7.61-7.43 (m) 7.61-7.43 (m) 7.56-7.43 (m) 7.55-7.42 (m) 7.56-7.43 (m) H-3 n.o. n.o. n.o. - n.o. n.o. n.o. n.o. n.o. H-5 8.04 (d, J=8.0) 8.12 (d, J=8.9) 8.13 (d, J=8.6) 8.14 (d, J=8.9) 8.13 (d, J=8.9) 8.15 (d, J= 8.9) 8.13 (d, J= 8,9) 8.13 (d, J=8.9) 8.13 (d, J=8.8) H-6 6.96 (m) 6.98 (dd, J=8.9, 2.3) 7.00 (dd, J=8.8, 2.3) 6.96 (dd, J=8.9, 2.3) 7.00 (dd, J=10.4, 2.3) 7.03 ( dd, J= 8.6, 2.3) 6.99 (dd, J=8.9, 2.1) 6.99 (dd, J=8.9, 2.3) 7.00 (dd, J=8.8, 2.2) H-7 - - - - - - - - H-8 6.93 (m) 6.94 (d, J=2.2) 6.95 (d, J=2.3) 6.90 (d, J=2.3) 6.97 (d, J=2.3) 6.97 (d, J=2.3) 6.95 (d, J=2.1) 6.94 (d, J=2.3) 6.97 (d, J=2.2) H-1’’ - 4.11 (t, J=6.6) 4.09 (t, J=6.5) 4,06 (t, J=6.5) 4.05 (t, J=6.6) 4.67 (dt, J=5.3, 1.5) 4.17 (q, 6.9) 3.93 (s) 4.64 (d, J=6.8) H-2’’ — 1.78-1.72 (m) 1.89-1.80 (m) 1.87-1.78 (m) 1.95-1.83 (m) 6.16-6.03 (m) 1.50 (t, J=6.9) - 5.53 (t, J=6.8, 2.0) H-3’’ — 1.92-1.81 (m) 1.60-1.47 (m) 1.59-1.49 (m) 1.09 (t, J=7.4) 5.52-5.44 (m) 5.40-5.31 (m) - - - H-4’’ — 1.00 (d, J= 6.5) 1.01 (t, J=7.4) 1.00 (t, J=7.4) - - - - 1.84 (s) H-5’’ — - - - - - - 1.80 (s) H-1’’’ — - - 4.02 (t, J=6.5) - - - - - H-2’’’ — - - 1.73-1.63 (m) - - - - - H-3’’’ — - - 1.42-1.32 (m) - - - - - H-4’’’ — - - 0.87 (t, J=7.4) - - - - - H-5’’’ _ - - - - - - - - n.o. not observed; Values in parts per million (δH). Measured in CDCl3 at 300.13 MHz. J values (Hz) are presented in parentheses. Chapter 3: Results and Discussion 87 Table 14: 13C NMR data of 3,7-dihydroxyflavone (H) and its alkylated derivatives (Hiso, Hutil1, Hutil2, Hrop, Hali, Hetil, Hemet and Hrep). H Hiso Hutil1 Hutil2 Hrop Hali Hetil Hemet Hrep C1’ 131.0 131.3 131.3 131.3 131.3 131.2 131.3 131.2 131.3 C2’ 127.0 127.5 127.5 128.5 127.5 127.5 127.5 127.5 127.5 C3’ 127.9 128.6 128.9 128.3 128.6 128.6 128.8 128.6 128.6 C4’ 129.1 129.9 130.2 130.4 129.9 129.9 129.9 129.9 129.9 C5’ 127.9 128.6 128.9 128.3 128.6 128.6 128.8 128.6 128.6 C6’ 127.0 127.5 127.5 128.5 127.5 127.5 127.5 127.5 127.5 C2 143.5 144.1 143.1 155.3 144.1 144.1 144.1 144.2 144.2 C3 137.7 138.1 137.1 140.5 138.1 138.1 138.1 138.1 139.5 C4 172.4 172.9 206.0 174.8 172.9 172.8 172.9 172.8 207.0 C4a 113.5 115.1 113.4 118.0 114.4 114.7 114.4 114,6 114.7 C5 126.2 126.7 126.5 127.1 126.7 126.8 126.7 126.8 126.7 C6 114.7 115.3 114.2 114.8 115.3 115.2 115.2 114.9 115.4 C7 162.4 163.9 162.9 163.6 163.9 163.2 163.7 164.3 163.6 C8 101.9 100.3 99.3 100.3 100.3 100.8 100.3 99.8 100.6 C8a 156.7 157.4 156.4 157.1 157.4 157.3 157.4 157.4 157.4 C1’’ - 67.2 67.4 68.4 70.3 69.6 64.3 53.9 65.5 C2’’ - 37.6 29.9 31.0 22.4 131.9 14.6 - 118.5 C3’’ - 25.1 18.2 19.1 10.5 118.6 - - 31.0 C4’’ - 22.6 12.8 13.8 - - - - 25.9 C5’’ - - - - - - - 18.3 C1’’’ - - 72.6 - - - - - C2’’’ - - 32.1 - - - - - C3’’’ - - 19.2 - - - - - C4’’’ - - 13.8 - - - - - C5’’’ - - - - - - - - Values in parts per million (δC). Measured in CDCl3 at 75.47 MHz. Chapter 3: Results and Discussion 88 3.1.2.3. Derivatives of chrysin The IR data of chrysin (C) as well as chrysin derivates (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep) are presented in Table 15. The observation of bands at 29852851 cm -1 (aliphatic C-H), suggested the presence of alkyl groups. The presence of a large band of stretching vibration at 3451-3441 cm -1 corresponding to hydroxyl groups is an indicator that the alkylation did not occur at both hydroxyl groups of the precursor (C). Table 15: IR data of chrysin (C) and its alkylated derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep). Groups ʋ(cm-1) C Ciso Cutil Crop Cali Cetil Cemet Crep OH 36003400 36003400 36003400 36003400 36003400 36003400 36003400 36003400 Aliphatic C-H - 2956 2921 2851 2952 2933 2868 2964 2920 2874 2961 2920 2851 2985 2924 2852 2923 2854 2967 2917 2853 C=O 1652 1662 1664 1661 1665 1663 1668 1659 Aromatic C=C 1577 1555 1499 1588 1452 1589 1569 1450 1442 1585 1569 1507 1451 1586 1506 1451 1586 1509 1454 1588 1495 1452 1436 1588 1504 1449 C-O 1168 1169 1170 1173 1170 1173 1160 1178 The 1H and 13C NMR data of chrysin (C) and its alkyl-derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep) are reported in Table 16 andTable 17. The 1H NMR spectra of chrysin (C) derivatives indicated that all of them have a nonsubstituted B-ring (H-2’,6’ δH 7.91-7.82 m; H-3’,4’,5’ δH 7.58-7.48 m) as the precursor C. Like for chrysin (C), the 1H NMR spectra showed signals for two A-ring aromatic protons (H-6: δH 6.40-6.33 d, H-8: δH 6.53-6.46 d) and a signal at δH 6.68-6.61 s corresponding to H-3. In addition, the signal of the hydroxyl group at C-5 (δH 12.73-12.69 s) was also observed for all derivatives, except for Cutil and Cetil. Besides these signals, the 1H and 13C NMR spectra of chrysin (C) derivatives showed characteristic signals of one prenyl (Crep), isopentyl (Ciso), butyl (Cutil), propyl (Crop), allyl (Cali), ethyl (Cetil), or methyl (Cemet) side chain, as illustrated in Figure 49. Chapter 3: Results and Discussion 89 Figure 49:1H and 13C NMR data for the alkyl groups of derivatives Ciso (A), Cutil (B), Crop (C), Cali (D), Cetil (E), Cemet (F) and prenyl group of derivative Crep (G). The position of these side chains was evidenced by the correlations found in the HMBC as indicated in Figure 50. Chapter 3: Results and Discussion 90 Figure 50: Main connectivities found in the HMBC of alkylated derivatives of chrysin (Ciso (A), Cutil (B), Crop (C), Cali (D), Cetil (E), Cemet (F) and Crep (G)). For derivatives Cali, Cetil and Cemet, the 1H and 13C NMR data obtained are in accordance to the published data. 186-188 Chapter 3: Results and Discussion 91 Table 16: 1H NMR data of chrysin (C) and its alkylated derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep). C Ciso Cutil Crop Cali Cetil Cemet Crep H-2’,6’ 7.90-7.87 (m) 7.90-7.88 m 7.87-7.84 (m) 7.90-7.87 (m) 7.90-7.88 (m) 7.89-7.86 (m) 7.91-7.88 (m) 7.86-7.82 (m) H-3’,4’,5’ 7.55-7.52 (m) 7.55-7.52 (m) 7.52-7.50 (m) 7.56-7.49 (m) 7.58-7.51 (m) 7.53-7.51 (m) 7.55-7.52 (m) 7.51-7.48 (m) H-3 6.67 (s) 6.67 (s) 6.63 (s) 6.66 (s) 6.68 (s) 6.65 (s) 6.67 (s) 6.61 (s) H-5 12.70 (OH, s) 12.71 (OH,s) n.o. 12.70 (OH,s) 12.72 (OH,s) n.o. 12.73 (OH,s) 12.69 (OH,s) H-6 6.30 (d, J=2.2) 6.37 (d, J=2.2) 6.34 (d, J=2.2) 6.37 (d, J=2.2) 6.40 (d, J=2.2) 6.35 (d, J=2.2) 6.38 (d, J=2.3) 6.33 (d, J=2.2) H-7 6.14 (OH,s) - - - - - - - H-8 6.48 (d, J=2.2) 6.50 (d, J=2.2) 6.47 (d, J=2.2) 6.50 (d, J=2.2) 6.53 (d, J=2.2) 6.48 (d, J=2.2) 6.51 (d, J=2.3) 6.46 (d, J=2.2) H-1’’ 4.07 (t, J=6.6) 4.02 (t, J=6.5) 4.00(t, J=6.6) 4.63 (dt, J=5.2, 1.4) 4.10 (q, J=7.0) 3.89 (s) 4.56 (d, J=6.9) H-2’’ 1.75-1.68 (m) 1.84-1.75 (m) 1.90-1.79 (m) 6.10-6.02 (m) 1.45 (t, J=7.0) - 5.48 (t, J=6.7) H-3’’ 1.92-1.78 (m) 1.56-1.44 (m) 1.06 (t, J= 7.4) 5.48-5.44 (m) 5.37-5.34 (m) - - - H-4’’ 0.98 (d, J=6.5) 0.99 (t, J=7.4) - - - 1.81 (s) H-5’’ - - - - - 1.76 (s) n.o. not observed; Values in parts per million (δH). Measured in CDCl3 at 300.13 MHz or 500 MHz. J values (Hz) are presented in parentheses. Chapter 3: Results and Discussion 92 Table 17: 13C NMR data of Chrysin (C) and its alkylated derivatives (Ciso, Cutil, Crop, Cali, Cetil, Cemet and Crep). C Ciso Cutil Crop Cali Cetil Cemet Crep C1’ 131.9 131.8 131.3 131.4 131.4 131.3 131.3 131.2 C2’ 126.3 126.3 126.2 126.3 126.3 126.3 126.3 126.2 C3’ 129.1 129.1 129.1 129.1 129.1 129.1 129.1 129.0 C4’ 131.9 131.8 131.8 131.8 131.9 131.8 131.9 131.8 C5’ 129.1 129.1 129.1 129.1 129.1 129.1 129.1 129.0 C6’ 126.3 126.3 126.2 126.3 126.3 126.3 126.3 126.2 C2 163.9 163.9 163.8 163.9 164.0 163.9 164.0 163.8 C3 105.9 105.9 105.8 105.9 105.9 105.8 105.9 105.7 C4 182.4 182.5 182.4 182.5 182.5 182.5 182.5 182.4 C4a 105.6 105.6 105.5 105.6 105.8 105.6 105.7 105.6 C5 162.0 162.1 162.1 162.1 162.2 162.1 162.2 162.0 C6 99.5 98.6 98.6 98.6 98.8 98.6 98.2 98.8 C7 165.0 168.2 165.2 165.2 164.6 165.0 165.6 164.9 C8 94.2 93.1 93.0 93.1 93.4 93.1 92.7 93.2 C8a 157.8 158.0 157.8 157.8 157.8 157.8 157.8 157.7 C1’’ - 67.1 68.4 70.2 69.3 64.2 55.8 65.5 C2’’ - 25.0 31.0 22.3 132.1 14.6 - 118.6 C3’’ - 37.6 19.2 10.4 118.5 - - 139.2 C4’’ - 22.5 13.8 - - - - 25.8 C5’’ - - - - - - 18.3 Values in parts per million (δC). Measured in CDCl3 at 75.47 MHz or 125.77 MHz Chapter 3: Results and Discussion 93 3.2. Peck purity For baicalein (B) derivatives Biso1, Brop1 and Bali1, prior to their biological activity evaluation, their purity was evaluated by HPLC-DAD as described in chapter 5. A percentage of purity greater than 95% was found for all derivatives (Table 18). Table 18: Purity values of the alkylated derivatives of baicalein (Biso1, Brop1 and Bali1). Compound Peak purity Biso1 99.7% Brop1 99.7% Bali1 99.8% Chapter 3: Results and Discussion 94 3.3. Biological activity 3.3.1. Modulation of procaspase-3 and -7 activity * In this section it is discussed the results obtained in the evaluation of the procaspases-3 and -7 modulatory activity of some synthesized flavonoids (the alkylated derivatives Biso1, Brop1 and Bali1). The effect of the derivatives Biso1, Brop1 and Bali1 on the activity of human procaspases-3 and -7 was evaluated using yeast cell assays. In this yeast assay, the expression of procaspases-3 and -7 does not affect the yeast growth, but activators of procaspases-3 and -7, such as PAC-1 (positive control33) induce yeast growth inhibition. Using this approach, the effect of 0.1-50 µM PAC-1 and 0.1-15 µM flavonoids on the growth of yeast individually expressing procaspases-3 or -7 was evaluated and the percentage of drug-induced growth inhibition was estimated, considering 100% growth the number of colony-forming units (c.f.u.) obtained with DMSO only. The results are summarized in Figure 51. * Work developed by the Master student Sofia Salazar under the supervision of Professor Lucília Saraiva (FFUP) – ongoing studies. Chapter 3: Results and Discussion 95 Figure 51: Concentration-response curves for the effects of flavonoids Biso1, Brop1 and Bali1 on the growth of yeast expressing human procaspase-3 (A) or -7 (B), for 24 hours treatment. The percentage of druginduced growth inhibition was estimated considered 100% growth the number of CFU obtained with DMSO only. Data are mean ± SEM of six independent experiments; values significantly different from DMSO are indicated (*p=0.01, **p=0.001; unpaired student t’ test). The results indicate that compounds Biso1, Brop1 and Bali1, induced growth inhibition in yeast cells expressing procaspase-7 at 0.1 - 15 µM (Figure 51, B), with the maximal effect achieved at 10 µM for Brop1 and Bali1 and 1 µM for Biso1, without interfering with the growth of control yeast (data not shown). Interestingly, the effect of these compounds was less pronounced in yeast cells expressing procaspase-3 (Figure 51, A). The comparison of the concentration-response curves for the effect of flavonoid Biso1 here reported with those reported for the baicalein derivative FP2 175, revealed that at the concentration of 1 µM (minimum concentration for which the maximal effect was achieved for Biso) the percentage of inhibition for Biso1 (about 30 %) was higher than the percentage of inhibition obtained for FP2 (about 8 %), suggesting that Biso1 was more potent than FP2. For procaspase-7, the GI25 values was obtained from concentration–response curves (Table 19). A B Chapter 3: Results and Discussion 102 A Figure 57: Interactions of Biso1 (A) Brop1 (B) and Bali1 (C) (blue sticks) with residues in the allosteric site of procaspase-7. Polar interactions are represented as yellow broken lines. Carbon, oxygen, nitrogen and sulphur atoms are represented in green, red, blue, and yellow, respectively. B C Chapter 3: Results and Discussion 103 A B C Figure 58: Interactions of Biso1 (A) Brop1 (B) and Bali1 (C) (blue sticks) with residues in the allosteric site of procaspase-6. Polar interactions are represented as yellow broken lines. Carbon, oxygen, nitrogen and sulphur atoms are represented in green, red, blue, and yellow, respectively. Chapter 3: Results and Discussion 104 All alkylated derivatives (Biso1, Brop1 and Bali1) of baicalein (B) establish polar interactions with procaspase-3, -7, and -6 allosteric sites. The different residues involved in polar interaction are listed in Table 21. Table 21: Residues of procaspases-3, -7, and -6 involved in polar interactions with alkylated derivatives (Biso1, Brop1 and Bali1). Tested compounds Procaspase-3 Procaspase-7 Procaspase-6 Biso1 GLU 124 ILE 886 TYR 199 Brop1 GLU 124 TYP 197 ARG 164 ILE 886 GLU 114 THP 199 Bali1 TYR 197 THP 333 ARG 318 ARG 266 GLU 214 THP 199 Biso1 is the most active procaspase activator both in vitro and in silico. Despite not containing the largest number of polar interactions in silico. Nonetheless, it is necessary not to disregard the non-polar interactions. Besides establishing polar interactions, the isopentoxyl group allows a more favorable orientation and a deeper insertion of the molecule into the allosteric groove of the target, as well as the establishment of additional van der Waals interactions with the hydrophobic cavity. Once Biso1 showed to be the most active derivative, we decided to make an in silico docking study to predict the procaspases modulatory activity of other synthesized derivatives possessing a 7-isopentyloxy group (derivatives Hiso and Ciso) (Table 22). Chapter 3: Results and Discussion 105 Table 22: Docking scores (Kcal.mol-1) for modulators described in the literature and alkylated derivatives of baicalein (Biso1), 3,7-dihydroxyflavone (Hiso) and chrysin (Ciso). Procaspase-3 Procaspase-7 Procaspase-6 Controls 115* -6.1 116* -7.6 -6.1 117* -6.4 118* -9 119* -9 44* -9 -9.9 120* -11.4 121* -11.4 122* -7.1 Tested compounds Biso1 -7.2 -7.7 -8.7 Hiso -6.8 -9.6 -7.6 Ciso -7.1 -9.3 -6.3 *used as positive controls for one, two, or three of the tested procaspases, according to the activation capacity previously described in the literature for those targets189,190,190. By analysis of the results obtained for procaspase-3, it was found that Hiso and Ciso had docking scores similar with Biso1. However, for procaspase-6 the results of Hiso and Ciso were worse than Biso1. Curiously, in procaspase-7 the compound Hiso had the best result and Ciso had a similar result. These results suggest that the hydroxyl group in position 6 may be detrimental to the interaction with procaspase-7. Concerning procaspase-3, the results suggest that the hydroxyl group in position 5 is important for interaction with procaspase-3. Furthermore, concerning modulation of procaspase-6, the presence of two hydroxyl groups in C-5 and C-6 in the flavone scaffold seems to be associated to a higher modulatory activity than the presence of just one hydroxyl group in C-5. Chapter 3: Results and Discussion 106 Chapter 4: Conclusions Chapter 4: Conclusions 109 Cancer is an increasing concern of our aging society, being responsible for a high mortality rate. Thus, cancer has been the focus of a great concern in general society, leading to numerous research studies by the scientific community. Considering that the resistance to apoptosis is one of the hallmarks of cancer, the search for compounds that induce apoptosis may be a key to the discovery of new antitumor agents. Caspases are involved in a cascade cellular of events that results in activation of initiator caspases and consequently in the activation of effector caspases triggering the apoptosis. Thus, the search for modulators of caspases with the ability to promote their activation might be an effective strategy to the activation of apoptosis in tumor cells. Flavonoids have a wide range of biological activities including antitumor activity. Many flavonoids and in particular prenyland alkyl-flavonoids were described as inducers of apoptosis by affecting the expression or activity of a wide range of molecules involved in apoptosis pathways, namely caspases. However, the reports about the direct activation of caspases by flavonoids are scarce and recent. The current work allowed the synthesis and identification of twenty-six flavonoids, being sixteen described for the first time, using MAOS methodologies. For three synthesized alkylated flavonoids (Biso1, Brop1 and Bali1) their ability to modulate the activity of procaspases-3 and -7 was evaaluated, using yeast-based assays. For bioactive compounds were performed docking studies with procaspases-3,-6 and -7. Considering the aims proposed for this dissertation, it is possible to conclude that the synthesis of alkylflavonoids by molecular modification of three naturally occurring compounds using non-classic methodologies (MAOS) were successfully accomplished. However other strategies to obtain these derivatives with a higher yield should be investigated in the future. All tested compounds revealed to be procaspase-7 activators, particularly Biso1, which showed a lower GI25 value (1.2 ± 2.26 µM) than the positive control, PAC-1 (24.5 ± 4.82 µM). Futher studies must be perform in order to evaluate their potency and selectivity for procaspases-3, -6 and -7, particularly in comparison to that obtained with their starting materials. The results presented in this dissertation also suggest that the suitable alkylation of flavonoid scaffolds may result in interesting derivatives with the ability to modulate the procaspases activity, which deserve further studies in order to confirm the results obtained in the yeast cell model. In the future it would be also important to investigate the Chapter 4: Conclusions 110 procaspases modulatory activity of other synthesized compounds using yeast cell assays and assays with tumor cell lines to establish structure-activity relationship (SAR). Chapter 5: Experimental Procedures Chapter 5: Experimental Procedures 118 6-(allyloxy)-5,7-dihydroxy-2-phenyl-4H-chromen-4-one (Bali1’). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9:1) followed by preparative TLC (SiO2; nhexane: EtOAc, 8:2). Yield: 1.1%; 166-168 ºC; IR (kBr) ʋmax: 3600-3300, 2925, 2923, 2854, 1664, 1585, 1489, 1451, 1115 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 13.04 (OH, s, H-5), 7.90-7.87 (2H, m, H-2’, 6’), 7.56-7.52 (3H, m, H-3’, 4’, 5’), 6.67 (1H, s, H-3), 6.62 (1H, s, H-8), 6.14-6.01 (1H, m, H-2’’),5.44-5.37 (1H, m, H-3’’a), 5.32-5.28 (1H, m, H-3’’b), 4.77 (2H, dt, J=3.3,1.2, H-1’’); 13C NMR (CDCl3, 75.47 MHz) δ= 183.0 (C4), 164.1 (C2), 155.5 (C7), 153.3 (C8a), 152.1 (C5), 133.0 (C2’’), 131.9 (C4’), 131.3 (C1’), 129.1 (C3’, 5’), 128.7 (C6), 126.3 (C2’, 6’), 120.0 (C3’’), 106.1 (C4a), 105.3 (C3), 93.4 (C8), 73.7 (C1’’). 7-ethoxy-5,6-dihydroxy-2-phenyl-4H-chromen-4-one (Betil1). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9.5:0.5). Yield: 4.5 %; mp 159-161 ºC; IR (kBr) ʋmax: 3600-3300, 2952, 2922, 1653, 1559, 1507, 1457, 1189 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 12.55 (OH, s, H-5), 7.91-7.88 (2H, m, H-2’,6’), 7.55-7.53 (3H, m, H3’,4’,5’), 6.69 (1H, s, H-3), 6.61 (1H, s, H-8), 5.37 (OH, brs, H-6), 4.24 (2H, q, J=14.0, 7.0, H-1’’), 1.55 (3H, t, J=7.0, H-2’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.7 (C4), 164.1 (C2), 152.2 (C7), 150.7 (C8a), 145.7 (C5), 131.8 (C4’), 131.5 (C1’), 129.7 (C6), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 106.0 (C4a), 105.5 (C3), 91.1 (C8), 65.2 (C1’’), 14.6 (C2’’). 5,6-dihydroxy-7-methoxy-2-phenyl-4H-chromen-4-one (Bemet1). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9:1) followed by flash chromatography (SiO2; n-hexane). Yield: 15 x%; mp 203-205 ºC; IR(kBr) ʋmax: 3600-3300, 2922, 1631, 1467 cm-1; 1H NMR (CDCl3, 300.13MHz) δ= 12.69 (OH, s, H-5), 7.92-7.89 (2H, m, H-2’, 6’), 7.55-7.53 (3H, m, H-3’, 4’, 5’), 6.69 (1H, s, H-3), 6.58 (1H, s, H-8), 5.35 (OH, brs, H-6), 3.94 (3H, s, H-1’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.8 (C4), 164.0 (C2), 153.1 (C7), 151.1 (C8a), 145.5 (C5), 131.9 (C4’), 131.3 (C1’), 129.7 (C6), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 106.1 (C4a), 105.7 (C3), 90.7 (C8), 60.9 (C1’’). 5-hydroxy-6,7-dimethoxy-2-phenyl-4H-chromen-4-one (Bemet2). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9.5:0.5) followed by flash chromatography (SiO2; n-hexane). Yield: 1.5x%; mp 150-152 ºC; IR(kBr) ʋmax: 3600-3300, 2922, 1634, 1505, 1457, 1413 cm-1; 1H NMR (CDCl3, 300.13MHz) δ= 12.69 (OH, s, H-5), 7.92-7.85 (2H, m, H-2’, 6’), 7.57-7.52 (3H, m, H-3’, 4’, 5’), 6.68 (1H, s, H-3), 6.58 (1H, s, H-8), 3.98 (3H, s, H-1’’), 3.93 (3H, s, H-1’’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.8 (C4), 164.0 (C2), Chapter 5: Experimental Procedures 119 159.0 (C7), 153.4 (C8a), 145.8 (C5), 131.9 (C4’), 131.3 (C1’), 132.7 (C6), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 106.1 (C4a), 105.7 (C3), 90.7 (C8), 66.9 (C1’’’), 56.4 (C1’’). 5.1.1.4. Derivatives of 3,7-dihydroxyflavone 5.1.1.4.1. Alkylated derivatives of 3,7-dihydroxyflavone A mixture of 3,7-dihydroxyflavone (H) (0.19g, 0.74mmol), isopentyl / butyl / propyl / allyl / ethyl / methyl iodide (1.18 mmol) and anhydrous K2CO3 (0.55 g, 3.7 mmol) in anhydrous acetone (60 mL) was submitted to successive 30 min of microwave irradiation at 200 W of potency. Total irradiation time was 2h and the final temperature was 60C. After cooling, the solid was filtered and the solvent removed under reduced pressure to afford the crude product. The yellow-green solid obtained was dissolved in acetone and purified as described below. 3-hydroxy-7-(isopentyloxy)-2-phenyl-4H-chromen-4-one (Hiso). Purified by flash chromatography (SiO2; petroleum ether: EtOAc, 9.5:0.5) followed by preparative TLC (SiO2; n-hexane: EtOAc, 8:2) and crystallization (chloroform: n-hexane). Yield: 12.9 x %; mp = 181-184ºC; IR (kBr) ʋmax: 3600-3300, 2958, 2921, 2854, 1605, 1504, 1467, 1452, 1410, 1260 cm-1; δ= 8.25-8.22 (2H, m, H-2’, 6’), 8.12 (1H, d, J=8.9, H-5), 7.56-7.42 (3H, m, H-3’, 4’, 5’), 6.98 (1H, dd, J=8.9, 2.3, H-6), 6.94 (1H, d, J= 2.3, H-8), 4.11 (2H, t, J= 6.6, H-1’’), 1.92-1.81 (1H, m, H-3’’), 1.78-1.72 (2H, m, H-2’’), 1.00 (6H, d, J=6.5, H-4’’, 5’’). 13C NMR (CDCl3, 75.47 MHz) δ= 172.9 (C4), 163.9 (C7), 157.4 (C8a), 144.1 (C2), 138.1 (C3), 131.3 (C1’), 129.6 (C4’), 128.6 (C3’, 5’), 127.5 (C2’, 6’), 126.7 (C5), 115.3 (C6), 115.1 (C4a), 100.3 (C8), 67.2 (C1’’), 37.6 (C2’’), 25.1 (C3’’), 22.6 (C4’’, 5’’). 7-butoxy-3-hydroxy-2-phenyl-4H-chromen-4-one (Hutil1). Purified by flash chromatography (SiO2; n-hexane : EtOAc, 9.5;0.5) followed by preparative TLC (SiO2; nhexane: EtOAc, 8:2) and crystallization (chloroform: n-hexane) Yield: 9.7 x %; mp= 149151ºC ; IR (kBr) ʋmax: 3600-3300, 2958, 2922, 2854, 1604, 1566, 1462, 1452, 1419, 1250 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.25-8.22 (2H, m, H-2’, 6’), 8.13 (1H, d, J=8.6, H-5), 7.56-7.43 (3H, m, H-3’, 4’, 5’), 7.00 (1H, dd, J=8.8, 2.3, H-6), 6.95 (1H, d, J= 2.3, H-8), 4.09 (2H, t, J= 6.5, H-1’’), 1.89-1.80 (2H, m, H-2’’), 1.60-1.47 (2H, m, H-3’’), 1.01 Chapter 5: Experimental Procedures 120 (3H, t, J=7.4, H-4’’). 13C NMR (CDCl3, 75.47 MHz) δ= 206.0 (C4), 162.9 (C7), 156.4 (C8a), 143.1 (C2), 137.1 (C3), 131.3 (C1’), 130.2 (C4’), 128.9 (C3’, 5’), 127.5 (C2’, 6’), 126.5 (C5), 114.2 (C6), 113.4 (C4a), 99.3 (C8), 67.4 (C1’’), 29.9 (C2’’), 18.2 (C3’’), 12.8 (C4’’). 3,7-dibutoxy-2-phenyl-4H-chromen-4-one (Hutil2). Purified by flash chromatography (SiO2; n-hexane : EtOAc, 9.75:0.25). Yield: 7.1 x%; mp= 190-193ºC ; IR(kBr) ʋmax: 3600-3300, 2936, 1873, 1623, 1499, 1466, 1447, 1260 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.10-8.07 (2H, m, H-2’, 6’), 8.14 (1H, d, J=8.9, H-5), 7.52-7.49 (3H, m, H-3’, 4’, 5’), 6.96 (1H, dd, J=8.9, 2.3, H-6), 6.90 (1H, d, J= 2.3, H-8), 4.06 (2H, t, J=6.5, H-1’’), 4.02 (2H, t, J=6.5, H-1’’’), 1.87-1.78 (2H, m, H-2’’), 1.73-1.63 (2H, m, H-2’’’), 1.59-1.49 (2H, m, H-3’’), 1.42-1.32 (2H, m, H-3’’’), 1.00 (3H, t, J=7.4, H-4’’), 0.87 (3H, t, J=7.4, H-4’’’). 13C NMR (CDCl3, 75.47 MHz) δ= 174.8 (C4), 163.6 (C7), 157.1 (C8a), 155.3 (C2), 140.5 (C3), 131.3 (C1’), 130.4 (C4’), 128.3 (C2’, 6’), 128.7 (C3’. C5’), 127.1 (C5), 114.8 (C6), 113.6 (C4a), 100.3 (C8), 72.6 (C1’’’), 68.4 (C1’’), 32.1 (C2’’’), 31.0 (C2’’), 19.2 (C3’’’), 19.1 (C3’’), 13.8 (C4’’, 4’’’). 3-hydroxy-2-phenyl-7-propoxy-4H-chromen-4-one (Hrop). Purified by flash chromatography (SiO2; petroleum ether: EtOAc, 95:5) followed by preparative TLC (SiO2; n-hexane: EtOAc, 8:2) and crystallization (chloroform: n-hexane). Yield: 3.7 x% as yellow crystals; mp= 173-174ºC; IR (kBr) ʋmax: 3600-3300, 2970, 1920, 1603, 1576, 1504, 1432, 1412, 1259 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.25-8.22 (2H, m, H-2’, 6’), 8.13 (1H, d, J=8.9, H-5), 7.61-7.43 (3H, m, H-3’, 4’, 5’). 7.00 (1H, dd, J= 10.4, 2.3, H-6), 6.97 (1H, d, J=2.3, H-8), 4.05 (2H, t, J=6.6, H-1’’), 1.95-1.83 (2H, m, H-2’’), 1.09 (3H, t, J=7.4, H-3’’). 13C NMR (CDCl3,75.47 MHz) δ= 172.9 (C4), 163.9 (C7), 157.4 (C8a), 144.1 (C2), 138.1 (C3), 131.3 (C1’),129.9 (C4’), 128.6 (C3’, 5’), 127.5 (C2’, 6’), 126.7 (C5), 115.3 (C6), 113.6 (C4a), 100.3 (C8), 70.3(C1’’), 22.4 (C2’’), 10.5 (C3’’). 7-(allyloxy)-3-hydroxy-2-phenyl-4H-chromen-4-one (Hali). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9.75:0.25) followed by preparative TLC (SiO2; n-hexane: EtOAc, 8:2) and crystallization (chloroform: n-hexane). Yield: 9.3 x%; mp= 144146ºC; IR(kBr) ʋmax: 3600-3300, 2999, 2964, 2921, 2847, 1615, 1564, 1503, 1473, 1453, 1260 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.25-8.22 (2H, m, H-2’, 6’), 8.15 (1H, d, J=8.9, H-5), 7.03 (1H, dd, J= 8.6, 2.3, H-6), 6.97 (1H, d, J=2.3, H-8), 6.16-6.03 (1H, m, H2’’), 5.52-5.44 (2H, m, H-3’’a), 5.40-5.31 (2H, m, H-3’’b), 4.67 (2H, dt, J=5.3, 1.5, H-1’’). 13C NMR (CDCl3, 75.47 MHz) δ= 172.8 (C4), 163.2 (C7), 157.3 (C8a), 144.1 (C2), 138.1 Chapter 5: Experimental Procedures 121 (C3),131.9 (C2’’) 131.2 (C1’), 129.9 (C4’), 128.6 (C3’, 5’), 127.5 (C2’, 6’), 126.8 (C5),118.6 (C3’’), 115.2 (C6), 100.3 (C8), 69.6 (C1’’). 7-ethoxy-3-hydroxy-2-phenyl-4H-chromen-4-one (Hetil). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9.75:0.25) followed by preparative TLC (SiO2; n-hexane: EtOAc, 8:2) and crystallization (chloroform: n-hexane). Yield: 1.7 %; mp=195197ºC; IR(kBr) ʋmax: 3600-3300, 2926, 1614, 1504, 1457, 1408, 1262 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.25-8.22 (2H, m, H-2’, 6’), 8.13 (1H, d, J=8.9, H-5),7.56-7.43 (3H, m, H-3’, 4’, 5’), 6.99 (1H, dd, J= 8.9, 2.1, H-6), 6.95 (1H, d, J=2.4, H-8), 4.17 (2H, q, J= 6.9, H-1’’), 1.50 (3H, t, J=6.9, H-2’’). 13C NMR (CDCl3, 75.47 MHz) δ= 172.9 (C4), 163.7 (C7), 157.4 (C8a), 144.1 (C2), 138.1 (C3), 131.3 (C1’), 129.9 (C4’), 128.8 (C3’, 5’), 127.5 (C2’, 6’), 126.7 (C5), 115.2 (C6), 114.4 (C4a), 100.3 (C8), 64.3 (C1’’), 14.6 (C2’’). 3-hydroxy-7-methoxy-2-phenyl-4H-chromen-4-one (Hemet). Purified by flash chromatography (SiO2; n-hexane: EtOAc, 9.75:0.25) followed by preparative TLC (SiO2; n-hexane: EtOAc, 8:2) and crystallization (chloroform: n-hexane). Yield: 9.2x%; mp 180182 ºC; IR(kBr) ʋmax: 3600-3300, 2922, 1622, 1508, 1456, 1401, 1262 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.24-8.21 (2H, m, H-2’, 6’), 8.13 (1H, d, J=8.9, H-5), 7.55-7.42 (3H, m, H-3’, 4’, 5’), 6.99 (1H, dd, J= 8.9, 2.3, H-6), 6.94 (1H, d, J=2.3, H-8), 3.93 (3H, s, H-1’’). 13C NMR (CDCl3, 75.47 MHz) δ= 172.8 (C4), 164.3 (C7), 157.4 (C8a), 144.2 (C2), 138.1 (C3), 131.2 (C1’), 129.9 (C4’), 128.6 (C3’, 5’), 127.5 (C2’, 6’), 126.8 (C5), 115.2 (C6), 114.9 (C4a), 99.8 (C8), 53.9 (C1’’). 5.1.1.4.2. Prenylated derivative of 3,7-dihydroxyflavone A mixture of 3,7-dihydroxyflavone (H) (0.188 g, 0.74 mmol), prenyl bromide (171µl, 1.48 mmol), potassium iodide (0.012 g, 0.074 mmol) and anhydrous K2CO3 (0.55g, 3.7mmol,) in anhydrous acetone (60ml) was submitted to successive 15 min of microwave irradiation at 200 W of potency. Total irradiation time was 45 min and the final temperature was 60 °C. After cooling, the solid was filtered and the solvent removed under reduced pressure to afford the crude product. The solid obtained was dissolved in acetone and purified by flash chromatography (SiO2; petroleum ether: EtOAc, 9.75:0.25) and then by preparative TLC (SiO2; n-hexane: EtOAc, 7:3) and crystallization (chloroform: n-hexane). One compound was obtained (Hrep). Chapter 5: Experimental Procedures 122 3-hydroxy-7-((3-methylbut-2-en-1-yl)oxy)-2-phenyl-4H-chromen-4-one (Hrep). Yield: 7.4 x%; mp= 174-176ºC; IR (kBr) ʋmax: 3600-3300, 2974, 2920, 2854, 1613, 1566, 1463, 1451, 1408, 1258 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 8.25-8.22 (2H, m, H-2’ H6’), 8.13 (1H, d, J=8.7, H-5), 761-7.44 (3H, m, H-3’ H-4’ H-5’), 7.02 (1H, d, J=2.3, H-6) ou 6.98 (1H, dd, J= 6.2, 2.2, H-6), 7.01 (1H, dd, J=8.8, 2.3, H-8) ou 6.97 (1H, d, J=2.2, H-8), 5.56-5.51 (1H, m, H-2’), 4.64 (2H, d, J=6.7, H-1’’), 1.84 (3H, s, H-4’’), 1.80 (3H, s, H-5’’). 13C NMR (CDCl3, 75.47 MHz) δ= 207.0 (C4), 163.6 (C7), 157.4 (C8a), 144.2 (C2), 139.5 (C3), 131.3 (C1’), 129.9 (C4’), 128.6 (C3’, 5’), 127.5 (C2’, 6’), 126.7 (C5), 118.5 (C2’’), 115.4 (C6), 114.7 (C4a), 100.6 (C8), 65.5 (C1’’), 31.0 (C3’’), 25.9 (C4’’’), 18.3 (C5’’). 5.1.1.5. Derivatives of chrysin 5.1.1.5.1. Alkylated derivatives of chrysin A mixture of chrysin (C) (0.188g, 0.74mmol), isopentyl / butyl / propyl / allyl / ethyl / methyl iodide (1.18 mmol) and anhydrous K2CO3 (0.55 g, 3.7 mmol) in anhydrous acetone (60 mL) was submitted to successive 30 min of microwave irradiation at 200 W of potency. Total irradiation time was 90 min and the final temperature was 60 °C. After cooling, the solid was filtered and the solvent removed under reduced pressure to afford the crude product. The solid obtained was dissolved in acetone and purified by flash chromatography (SiO2; n-hexane: EtOAc; 9:1). 5-hydroxy-7-(isopentyloxy)-2-phenyl-4H-chromen-4-one (Ciso). Yield: 30%; mp 129-132 ºC; IR (kBr) ʋmax: 3600-3400, 2956, 2921, 1851, 1662, 1588, 1452, 1169 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 12.71 (OH, s, H-5), 7.90-7.87 (2H, m, H-2’, 6’), 7.557.52 (3H, m, H-3’, 4’, 5’), 6.67 (1H, s, H-3), 6.50 (1H, d, J=2.2, H-8), 6.37 (1H, d, J=2.2, H6), 4.06 (2H, t, J=6.6, H-1’’), 1.92-1.78 (1H, m, H-3’’), 1.75-1.68 (2H, m, H-2’’), 0.98 (6H, d, J=6.5, H-4’’, 5’’). 13C NMR (CDCl3, 75.47 MHz) δ= 182.5 (C4), 168.2 (C7), 163.9 (C2), 162.1 (C5), 131.8 (C1’), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 105.9 (C3), 98.6 (C6), 93.1 (C8), 67.1 (C1’’), 37.6 (C2’’), 25.0 (C3’’), 22.5 (C4’’, 5’’). 5-hydroxy-2-phenyl-7-propoxy-4H-chromen-4-one (Cutil). Yield: 29%; mp 145148 ºC; IR (kBr) ʋmax: 3600-3400, 2952,2933, 2868, 1664, 1589, 1569, 1450, 1442, Chapter 5: Experimental Procedures 123 1274, 1170 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 7.87-7.84 (2H, m, H-2’, 6’), 7.52-7.50 (3H, m, H-3’, 4’, 5’), 6.63 (1H, s, H-3), 6.47 (1H, d, J=2.2, H-8), 6.34 (1H, d, J=2.2, H-6), 4.02 (2H, t, J=6.5, H-1’’), 1.84-1.75 (2H, m, H-2’’), 1.56-1.44 (2H, m, H-3’’), 0.99 (3H, t, J=7.4, H-4’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.4 (C4), 165.2 (C7), 163.8 (C2), 162.1 (C5), 157.8 (C8a), 131.8 (C4’), 131.3 (C1’), 129.1 (C3’, 5’), 126.2 (C2’, 6’), 105.8 (C3), 105.5 (C4a), 98.6 (C6), 93.0 (C8), 68.4 (C1’’), 31.0 (C2’’), 19.2 (C3’’), 13.8 (C4’’). 5-hydroxy-2-phenyl-7-propoxy-4H-chromen-4-one (Crop). Yield: 22%; mp 132135 ºC; IR (kBr) ʋmax: 3600-3400, 2964, 2920, 2874, 1661, 1585, 1569, 1507, 1451, 1173 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 12.70 (OH, s, H-5), 7.90-7.87 (2H, m, H-2’, 6’), 7.56-7.49 (3H, m, H-3’, 4’, 5’), 6.66 (1H, s, H-3), 6.50 (1H, d, J=2.2, H-8), 6.37 (1H, d, J=2.2, H-6), 4.00 (2H, t, J=6.6, H-1’’), 1.90-1.79 (2H, m, H-2’’), 1.06 (3H, t, J=7.4, H-3’’). 13C NMR (CDCl3,75.47 MHz) δ= 182.5 (C4), 165.2 (C7), 163.9 (C2), 162.1 (C5), 157.8 (C8a), 131.8 (C4’), 131.4 (C1’), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 105.9 (C3), 105.6 (C4a), 98.6 (C6), 93.1 (C8), 70.2 (C1’’), 22.3 (C2’’), 10.4 (C3’’). 7-(allyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (Cali). Yield: 31%; mp 118120 ºC; IR (kBr) ʋmax: 3600-3400, 2961, 2920, 2851, 1665, 1586, 1506, 1451, 1203, 1170 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 12.72 (OH, s, H-5), 7.90-7.88 (2H, m, H-2, 6’), 7.58-7.51 (3H, m, H-3’, 4’, 5’), 6.68 (1H, s, H-3), 6.53 (1H, d, J=2.3, H-8), 6.40 (1H, d, J=2.3, H-6), 6.10-6.02 (1H, m, H-2’’) 5.48-5.44 m (1H, m, H-3’’a), 5.37-5.34 (1H, m, H3’’b), 4.63 (2H, dt, J=5.2, 1.4, H-1’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.5 (C4), 164.6 (C7), 164.0 (C2), 162.2 (C5), 157.8 (C8a), 132.1 (C2’’), 131.9 (C4’), 131.4 (C1’), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 118.5 (C3’’’), 105.9 (C3), 105.8 (C4a), 98.8 (C6), 93.4 (C8), 69.3 (C1’’). 7-ethoxy-5-hydroxy-2-phenyl-4H-chromen-4-one (Cetil). Yield: 32%; mp 153-154 ºC; IR (kBr ) ʋmax: 3600-3400, 2985, 2924, 2852, 1663, 1586, 1509, 1454, 1204, 1173 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 7.89-7.86 (2H, m, H-2’,6’), 7.53-7.51 (3H, m, H-3’, 4’, 5’), 6.65 (1H, s, H-3), 6.48 (1H, d, J=2.2, H-8), 6.35 (1H, d, J=2.2, H-6), 4.10 (2H, q, J=7.0, H-1’’), 1.45 (3H, t, J=7.0, H-2’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.5 (C4), 165.0 (C7), 163.9 (C2), 162.1 (C5), 157.8 (C8a), 131.8 (C4’), 131.3 (C1’), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 105.8 (C3), 105.6 (C4a), 98.6 (C6), 93.1 (C8), 64.2 (C1’’), 14.6 (C2’’). Chapter 5: Experimental Procedures 124 5-hydroxy-7-methoxy-2-phenyl-4H-chromen-4-one (Cemet). Yield: 44%; mp 165168 ºC; IR(kBr) ʋmax: 3600-3400, 2923, 2854, 1668, 1588, 1495, 1452, 1436, 1202, 1160 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 12.73 (OH, s, H-5), 7.91-7.88 (2H, m, H-2’, 6’), 7.55-7.52 (3H, m, H-3’, 4’, 5’), 6.67 (1H, s, H-3), 6.51 (1H, d, J=2.2, H-8), 6.38 (1H, d, J=2.2, H-6), 3.89 (3H, s, H-1’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.5 (C4), 165.6 (C7), 164.0 (C2), 162.2 (C5), 157.8 (C8a), 131.9 (C4’), 131.3 (C1’), 129.1 (C3’, 5’), 126.3 (C2’, 6’), 105.9 (C3), 105.7 (C4a), 98.2 (C6), 92.7 (C8), 55.8 (C1’’). 5.1.1.5.2. Prenylated derivative of chrysin A mixture of Chrysin (C) (0.188 g, 0.74 mmol), prenyl bromide (171 µL, 1.48 mmol), potassium iodide (0.012 g, 0.074 mmol) and anhydrous K2CO3 (0.55 g, 3.7 mmol) in anhydrous acetone (60 mL) was submitted to successive 30 min of microwave irradiation at 200 W of potency. Total irradiation time was 90 min and the final temperature was 60 °C. After cooling, the solid was filtered and the solvent removed under reduced pressure to afford the crude product. The yellow-orange solid obtained was dissolved in acetone and purified by flash chromatography (SiO2; n-hexane: EtOAc; 9:1). One compound was isolated (Crep). 5-hydroxy-7-((3-methylbut-2-en-1-yl)oxy)-2-phenyl-4H-chromen-4-one (Crep): Yield: 67%; mp 104-107 ºC; IR (kBr) ʋmax: 3600-3400, 2967, 2917, 2853, 1659, 1588, 1504, 1449, 1178 cm-1; 1H NMR (CDCl3, 300.13 MHz) δ= 12.69 (OH, s, H-5), 7.86-7.82 (2H, m, H-2’, 6’), 7.51-7.48 (3H, m, H-3’, 4’, 5’), 6.61 (1H, s, H-3), 6.46 (1H, d, J=2.2, H-8), 6.33 (1H, d, J=2.2, H-6), 5.48 (1H, t, J=6.7, H-2’’), 4.56 (2H, d, J=6.9, H-1’’), 1.81 (3H, s, H-4’’), 1.76 ( 3H, s, H-5’’); 13C NMR (CDCl3, 75.47 MHz) δ= 182.4 (C4), 164.9 (C7), 163.8 (C2), 162.0 (C5), 157.7 (C8a), 139.2 (C3’’), 131.8 (C4’), 131.2 (C1’), 129.0 (C3’, 5’), 126.2 (C2’, 6’), 118.6 (C2’’), 105.7 (C3), 105.6 (C4a), 98.8 (C6), 93.2 (C8), 65.5 (C1’’), 25.8 (C4’’), 18.3 (C5’’). Chapter 5: Experimental Procedures 125 5.2 Peak purity Analytical HPLC-DAD analyses were performed on a SpectraSYSTEM (Thermo Fisher Scientific, Inc, USA) equipped with a P4000 pump, a AS3000 autosampler and a diode array detector UV8000. The separation was carried out on a 250 x 4.6 mm i.d. FortisBIO C18 (5 µm) (FortisTM Technologies Ltd, Cheshire, UK). LC analysis was performed by isocratic elution using a mixture of MeOH:H2O:MeCO2H (70:30:1 v/v/v) as mobile phase and the flow rate was set at 1 mL/ min. The injected volume was 20 µL and the eluent was monitored at 275 nm. The detector was set at a wavelength range of 190–800 nm with a spectral resolution of 1 nm. The purity parameters included a 95 % active peak region and a scan threshold of 5 mAU. ChromQuest 5.0 (version 3.2.1) software (Thermo Fisher Scientific Inc.) managed chromatographic data. Methanol (HPLC grade) was obtained from Carlo Erba Reagents (Val de Reuil, Italy), acetic acid (HPLC grade) was obtained from Romil Pure Chemistry (Cambridge, UK) and HPLC grade water obtained from a Simplicity® UV Ultrapure Water System, Millipore Corporation, USA. Prior to use, mobile phase solvents were degassed in an ultrasonic bath for 15 min. Chapter 5: Experimental Procedures 126 5.3. Biological activity 5.3.1. Reagents and stock solutions of compounds Procaspase-activating compound-1 (PAC-1) were purchased from Calbiochem. Solutions of compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at -20 ºC. Appropriate dilutions of the compounds were freshly prepared with culture medium prior to the assays. Glucose, galactose and glycerol were from SigmaAldrich (Sintra, Portugal). Yeast nitrogen base without amino acids from Difco (Quilaban, Sintra, Portugal). Sabouraud Dextrose Agar (SDA) from Liofilchem (Frilabo, Porto, Portugal). 5.3.2. Yeast procaspase-3 and -7 assay † 5.3.2.1. Compounds Details concerning the synthesis of tested flavonoids Biso1, Brop1 and Bali1 are described in the chapter III and V. 5.3.2.2. Plasmids For expression in yeast of procaspase-3 and -7 the expression vectors pGALL- (LEU2) encoding each human protein under a GAL1-10 promoter were used. 5.3.2.3. Yeast strain, transformation and growth conditions † Work developed by the Master student Sofia Salazar under the supervision of Professor Lucília Saraiva (FFUP) – ongoing studies. Chapter 5: Experimental Procedures 127 Saccharomyces cerevisiae (strain CG379 Mata, ade5, his2, leu2-112, trp1-289, ura3-52) was granted by Yeast Genetic Center, University of California, USA. Saccharomyces cerevisiae was transformed using the standard lithium acetate method 175. For the selection of transformed yeast, cells were grown in glucose minimal selective medium with 2% (w/v) glucose, 0.67% (w/v) yeast nitrogen base without amino acids and all the amino acids required for yeast growth (50 µg/ml) except leucine, and incubated at 30 ºC, under continuous orbital shaking (170 r.p.m.). To induce the expression of human proteins, yeast cultures were diluted to 0.05 optical density at 600 nm (OD600) in induction selective medium containing 2% (w/v) galactose and 1% glycerol and grown at 30 ºC under continuous shaking. The effects of procaspase-3 and -7 on yeast growth were analyzed as previously described for the active caspase-3 192. Briefly, yeast cells expressing the human protein and control yeast (transformed with the empty vector, pGALL) were grown in induction selective medium for up to 24 h for growth curves experiments. Yeast growth was analyzed by counting the number of colony-forming units per ml (CFU/ml) after 2 days incubation at 30 ºC on Sabouraud Dextrose Agar plates. 5.3.2.4. Effects of the compounds on yeast cell growth In yeast assays with procaspase-3 and -7, the known activator of caspase-3 and -7, PAC-1 33, was used as positive control. To analyze the effect of compounds on yeast cell growth, transformed cells were incubated in induction selective medium in the presence of 0.1–15 µM (for procaspases) compounds, 0.1-50 µM of PAC-1, or DMSO only (0.1%). Cells were incubated to approximately 0.3 OD600 (achieved with each transformant incubated with DMSO only), and the cell growth was analyzed as described above. For each culture, the percentage of drug-induced growth inhibition was estimated considering 100% growth the number of CFU obtained with yeast incubated with DMSO only. 5.3.2.5. Statistical analysis For yeast-based assays results were analyzed statistically using the SigmaPlot 12.00 software and differences between means were tested for significance using the unpaired student´s t-test (p < 0.01 or p < 0.001).