Malaria chemotherapy and prevention: towards the development of dual action drugs and the understanding of vaccine-induced antibody responses against P. falciparum

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Malaria chemotherapy and prevention: towards the development of dual action drugs and the understanding of vaccine-induced antibody responses against P. falciparum Bianca Celidet Pérez de Lucani PhD in Chemistry Departamento de Química e Bioquímica 2013 Adviser Paula Alexandra de Carvalho Gomes, Assistant Professor, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto Co-adviser Cátia Andreia Silva Teixeira, Centro de Investigação em Cerâmicas e Materiais Compósitos, Universidade de Aveiro ! ! ! Detection of antibody response against malaria Homology modeling and docking studies against a malaria-like disease Design, synthesis, characterization, and evaluation of potential dual-action antimalarials D Acknowledgements | iii Acknowledgements To my advisers, Dr. Cátia Teixeira and Prof. Dr. Paula Gomes, for their patience, caring, support, and guidance during the PhD journey and for helping me to continue to grow professionally and personally and to become a more independent researcher. I love our meetings together, they were highly motivating. To my family, specially, to my husband, for his support and motivation that helped me keep a hard working rhythm during the entire PhD program. Dr. Daniel E. Lucani, you’re my researcher idol. To my friends, specially, to Ana Gomes, for all the help provided for the development of the project and for being a vivid example of a true friend in the difficult and wonderful moments of my life since the beginning of the PhD program. To P. falciparum CRESIB’s team, distinctively, to Joseph Campo and Dr. Carlota Dobaño, for introducing me to the immunology world for vaccine development and to Joseph Campo for the statistical analysis of the data. It was a pleasure to work in such highly motivating research environment. To Rosenthal’s and Prudêncio’s research team, for the antiplasmodial assays carried out for the development of the project and to A. Domingos, N. Mateus, and M. S. Gomes research team for the in vitro assays against babesipain-1, malign cells, and Leishmania, respectively. To Mariana Andrade and Zélia Azevedo for the NMR spectra and ESI-MS studies, respectively, and to Prof. Dr. Jose E. R. Borges for always having his door open to offer help when I needed it. Also, to Dr. Jose R. B. Gomes for providing the means to carried out the computational side of the PhD work. To my colleagues for their laboratory help, specially, to Sofia Santos for the help provided for the synthesis of HECINs. To the Alves and the Gomes family for treating me as a loved member of their family and making me feel at home in Portugal. Thanks to Prof. Freddy Castillo, Dr. Helen Restrepo, Prof. Dr. Michael Pollastri, and Prof. Dr. Simón López who through their teaching made me fall in love with chemistry and encouraged me to follow this career. Thanks to the jury members for taking the time to read the thesis and be present in my defense. Last, to the Portuguese Foundation of Science (FCT-Fundação para a Ciência e a Tecnologia) for the Doctoral research grant (SFRH/BD/86166/2012) and the Research grant (PTDC/QUI-QUI/116864/2010) and to the ERASMUS program for the grant to cover the living expenses of my 3-month research experience in Barcelona. Abstract | v Abstract Malaria is an infectious tropical disease which mostly affects the world’s poorest populations and causes above a half million deaths per year. Its eradication is yet an unmet goal, mostly, due to the emergence of parasite resistance to classical antimalarials and the lack of a commercially available malaria vaccine to prevent the disease. In this context, the doctoral project herein reported comprised two main research lines for malaria eradication. The first and major work of the project centered on the development of dual-action antimalarial drugs. The second research line was dedicated to the study of the influence of vaccination with RTS,S, the most clinically advanced malaria vaccine candidate, on the immune response of African children exposed to the disease. This doctoral project initially focused on the development of dual-action drugs potentially capable of inhibiting two processes related to the host’s hemoglobin degradation by the intraerythrocytic malaria parasite: i) globin degradation by parasite’s cysteine proteases, falcipains (FP), and ii) heme detoxification through hemozoin formation (H-f). First generation of compounds, HEDICINs, included a cinnamoyl moiety linked to the 4amino-7-chloroquinolyl group, the chloroquine (CQ) core, through a dipeptide chain. HEDICINs resulted active in the micromolar range (IC50 = 0.83-11 µM) against intraerythrocytic CQ-resistant strain W2 of Plasmodium falciparum (Pf), but such activity did not correlate with inhibition of FP or H-f. Removal of the hydrophobic dipeptide spacer or substitution of the chloroquinoline core by a non-aromatic ring was detrimental for the antiplasmodial activity. HEDICINs were further evaluated against another protozoal cysteine protease, babesipain-1 (bbspn-1) from Babesia bigemina, whose three-dimensional (3D) structure is yet unavailable. Hence, in silico studies were carried to build the corresponding 3D structure by homology modeling in order to evaluate the interaction of the HEDICINs with bbspn-1. For rationalizing in vitro data at the molecular level, a comparative analysis of the compounds’ behavior against FP and bbspn-1 was also performed as HEDICINs demonstrated to be more efficient inhibitors of bbspn-1. Second generation of compounds, HEFLECINs, were designed as HEDICINs analogues where the dipeptide spacer was replaced by a more flexible hydrophobic spacer, an alkyl chain. Compounds displayed activities in the nanomolar range (IC50 = 11.0-46.6 nM) better than CQ (IC50 = 138 nM), the reference drug against blood-stage Pf CQ-resistant strain W2. These compounds were also highly potent (IC50 = 1.1-4.1 µM) in vitro against liver-stage P. berghei malaria parasites, and superior to primaquine (IC50 = 7.5 µM), the reference drug against the latter stage, thus representing dual-stage antimalarial leads. Furthermore, CQ core was respectively replaced by others heteroaromatic core from vi | Abstract known antimalarials. Analysis of the structure-activity relationships demonstrated the relevance of i) the butyl chain, ii) the CQ core, and iii) the amide functionality, for optimal dual-stage antimalarial activity. Two HEFLECINs were further confirmed to be active in vivo against blood stage malaria in rodents. Moreover, HEFLECINs were evaluated in vitro against the promastigote and amastigote forms of Leishmania infantum, and consequently, established as promising anti-leishmanial agents. The mechanism of action of these second-generation compounds is yet to be elucidated, as their antiplasmodial activities did not correlate with the inhibition of H-f or FP. The second part of this doctoral project consisted in the measurement of the antibody responses to pre-erythrocytic and erythrocytic Pf antigens in African children vaccinated with RTS,S or control vaccine and naturally exposed to malaria, and subsequent analysis of the data. According to the results obtained, there seems to be a difference in IgG levels, between the RTS,S and vaccine control groups, that drops over time. The results of this study contribute to the understanding of a pre-erythrocytic malaria vaccine intervention in children. Keywords: Acridine, antimalarial, babesiosis, chloroquine, cinnamic acid, dual-action, leishmaniasis, malaria, Plasmodium, primaquine, vaccine, RTS,S. Resumo | vii Resumo A malária é uma doença infeciosa que afecta maioritariamente as populações mais pobres do mundo e causa mais de meio milhão de mortes por ano. Embora a malária já tenha sido erradicada em diversos países, a doença continua presente em cerca de cem países, principalmente devido ao aparecimento de estirpes do parasita resistentes aos antimaláricos clássicos e, à falta de uma vacina contra a malária comercialmente disponível. Neste contexto, o projeto de doutoramento aqui apresentado engloba duas linhas de investigação principais contribuindo para a erradicação da malária. O primeiro, e principal trabalho do projeto, centrou-se no desenvolvimento de antimaláricos de ação dual. O segundo trabalho de investigação dedicou-se ao estudo da influência da vacinação com RTS,S, o candidato a vacina da malária clinicamente mais avançado, na resposta imune de crianças africanas expostas à doença. A primeira parte do projeto iniciou-se com o desenvolvimento de fármacos potencialmente capazes de atuar contra dois processos intra-eritrocitários, relacionados com a degradação da hemoglobina do hospedeiro pelo parasita da malária: i) degradação de globlina pelas falcipaínas (FP), proteases de cisteína do parasita, e ii) desintoxicação do heme por formação de hemozoína (H-f). A primeira geração de compostos, HEDICINs, consistiu num grupo cinamoílo ligado ao grupo 4-amino-7cloroquinolinoílo, o núcleo da cloroquina (CQ), através de uma cadeia dipeptídica. Estes compostos resultaram ativos, no ordem do micromolar (IC50 = 0.83-11 µM), contra a estirpe resistente à CQ de Plasmodium falciparum (Pf) W2, mas não se verificou qualquer correlação entre a atividade antiplasmodial e a inibição de H-f ou FP. A remoção do espaçador dipeptídico ou a substituição do núcleo cloroquinolina por um anel não aromático demonstrou ser prejudicial à atividade antiplasmodial. Adicionalmente, os compostos HEDICINs foram avaliados contra a babesipaína-1 (bbspn-1), uma protease de cisteína do protozoário Babesia bigemina, cuja estrutura tridimensional (3D) não está disponível. Assim, usaram-se técnicas de modelação por homologia para construir um modelo 3D da bbspn-1, a fim de avaliar a sua interação com os HEDICINs. Com o objetivo de se avançar com uma explicação a nível molecular dos resultados in vitro, comparou-se a interação dos compostos com a FP e bbspn-1, respectivamente, já que os HEDICINs mostraram ser inibidores mais eficazes da bbspn1. A segunda geração de compostos, HEFLECINs, corresponde a análogos dos HEDICINs, em que a cadeia dipeptídica foi substituída por um espaçador mais flexível e hidrofóbico, uma cadeia alquílica. Os compostos apresentaram atividade antiplasmodial contra a estirpe de Pf resistente à CQ, W2, na ordem do nanomolar (IC50 = 11.0- viii | Resumo 46.6 nM), atividades superiores às da CQ (IC50 = 138 nM), o farmáco de referência contra a fase sanguínea do parasita Pf. Adicionalmente, estes compostos revelaram-se mais ativos in vitro (IC50 = 1.1-4.1 µM), contra o parasita P. berghei da fase hepática da malária, quando comparados com a primaquina (IC50 = 7.5 µM), o farmáco de referência contra esta fase, demonstrando o potencial dos HEFLECINs como antimaláricos de ação dual. Posteriormente, substituiu-se o núcleo da CQ por outros núcleos heteroaromáticos de antimaláricos conhecidos e investigou-se a influência da natureza do espaçador. Este estudo de relação estrutura-atividade demonstrou a relevância i) da cadeia de butilica, ii) do núcleo da CQ, e iii) da funcionalidade amida para as ótimas atividades dos composto contra ambas fases da malária acima indicadas. Além disso, dois HEFLECINs foram confirmados ativos contra a infecção da malária na fase sanguínea em roedores. Os HEFLECINs foram ainda testados em culturas de promastigotas e amastigotas de Leishmania infantum, revelando-se agentes promissores contra da leishmaniose visceral. Uma vez que a atividade antiplasmodial dos HEFLECINs não se correlacionou com a inibição de H-F ou FP, o mecanismo de ação dos compostos permanece por elucidar. A segunda parte do projeto de doutoramento consistiu na medição da resposta de anticorpos a antígenos pré-eritrocitários e eritrocitários do Pf em crianças africanas vacinados com a vacina RTS,S ou controle e naturalmente expostos à malária, e subsequente análise dos dados. Com base nos resultados obtidos, observou-se uma diferença nos níveis de IgG, entre o grupo da vacina controle e do RTS,S, que diminui ao longo do tempo. Os resultados deste estudo contribuem para a compreensão da intervenção da vacinação pré-eritrocitária contra a malária em crianças. Palavras-chave: Ácido cinâmico, ação dual, acridina, antimalárico, babesiose, cloroquina, leishmaniose, malária, Plasmódio, primaquina, vacina, RTS,S. ! Table of content | ix Table of content! ABBREVIATIONS ......................................................................................................... XIX FOREWORD ................................................................................................................... 23 LIST OF PUBLICATIONS .............................................................................................. 27 CHAPTER 1 .................................................................................................................... 31 1.0. INTRODUCTION ....................................................................................................... 33 1.1. TARGETING INHIBITION OF GLOBIN DEGRADATION BY FALCIPAINS ............................. 35 1.2. TARGETING INHIBITION OF HEMOZOIN FORMATION ................................................... 39 1.3. THE COVALENT BITHERAPY APPROACH AGAINST PF MALARIA ................................... 41 1.4. REFERENCES ......................................................................................................... 52 CHAPTER 2 .................................................................................................................... 59 2.0. PREAMBLE ............................................................................................................. 61 2.1. HE[DI]CINS ........................................................................................................... 62 2.1.1. Synthesis of HEDICINs and HECINs ............................................................ 63 2.1.2. Characterization of HEDICINs and HECINs ................................................. 69 2.1.3. Assessment of cinnamoylated derivatives .................................................... 74 2.2. EXPERIMENTAL SECTION ........................................................................................ 81 2.2.1. Chemistry ...................................................................................................... 81 2.3. REFERENCES ......................................................................................................... 99 CHAPTER 3 .................................................................................................................. 101 3.0. PREAMBLE ........................................................................................................... 103 3.1. BABESIOSIS AND BABESIA PARASITES – BRIEF OVERVIEW ...................................... 103 3.2. IN VITRO EVALUATION OF HE[DI]CINS AS BBSPN-1 INHIBITORS .............................. 106 3.3. IN SILICO STUDY OF BBSPN-1 INHIBITION BY HE[DI]CINS ....................................... 107 3.3.1. Generation and validation of the bbspn-1 model structure by homology modeling…………………………………………………………………… .................. 107 3.3.2. Docking studies ........................................................................................... 123 3.4. IN SILICO PROTOCOL ............................................................................................ 126 3.5. REFERENCES ....................................................................................................... 127 CHAPTER 4 .................................................................................................................. 131 4.0. RATIONALE .......................................................................................................... 133 4.1. CHLOROQUINOLINE-BASED HEFLECINS .............................................................. 134 4.1.1. Synthesis and characterization of HEFLECINs ........................................... 134 4.1.2. In vitro assays of HEFLECINs ..................................................................... 140 xvi | List of figures Figure 54. Luminex assays to evaluate the IgG response of each selected child. PBSBN: 1% BSA/0.05% sodium azide in PBS. ................................................................... 217! Figure 55. Ab responses of positive hyperimmune controls: In the left are all initial assays Ab responses. In the right corner are the results once the four assays were repeated. ....................................................................................................................... 218! Figure 56. Geometric mean of anti-BSA Ab units of both control (C) and RTS,S (R) group. ............................................................................................................................ 218! Figure 57. Ab breadth representation by age. .............................................................. 219! Figure 58. Anti-AMA-1 (left) and anti-MSP-142 (Right) Ab responses against both strains 3D7 in the “Y” axis and FVO in the “X” axis, respectively. Dash lines defines the anti-BSA Ab units + 2SD. ............................................................................................................. 220! Figure 59. Parasitemia, shown as proportion of children with Pf infection (left), and antiAMA-1 3D7 Ab units (right). The red color indicated the highest prevalence or Ab responses and the yellow the lowest. ........................................................................... 221! Figure 60. RTS,S (R) and control (C) groups difference in terms of parasitemia (left) and anti-CSP Ab response (right). ....................................................................................... 222! Figure 61. Anti-AMA-1 Ab response (3D7 and FVO) versus time since last exposure (in days) to a malaria episode or parasitemia by lowess smoothing analysis. Dot line represents the seropositivity threshold. ........................................................................ 224! Figure 62. Anti-AMA-1 and anti-MSP-1 ab responses, respectively, vs time since last exposure (in days). Control (C) and RTS,S (R) group. ................................................. 225! Figure 63. Lattice plot of individuals’ antibody levels over time. Each subplot represents a randomly selected individual. The title box contains the subject ID for this study, his/her age in years, and which vaccination group the individual belongs to (RTS,S or Control). The Ab response against specific antigens is represented in colored lines: AMA-1 3D7, black; AMA-1 FVO, maroon; MSP-142 3D7, navy; MSP-142 FVO, green; EBA-175, orange; LSA-1, purple; SSP2, olive; CelTOS, red; DBL-alpha, blue. Each point represents a cross-sectional survey sampling. White diamonds represent documented clinical episodes of malaria, and black diamonds represent parasitemia at crosssectional timepoints. The x-axis scale is in days, ranging from 1st June 2003 to 1st June 2007 which includes time points from D0 to M45. ........................................................ 226! Figure 64. General structure of the cinnamoyl derivatives including an additional protonable amine. ......................................................................................................... 240! List of tables | xvii List of tables Table 1. Reaction conditions for the coupling of Boc-D-hPhe-OH to obtain 4. ............... 66! Table 2. Amide bond formation for HECINs. .................................................................. 68! Table 3. In vitro data of HEDICINs (1 and 8) and HECINs (2). included. ....................... 75! Table 4. In vitro results against β-H for compounds 3, 5, and 7. .................................... 78! Table 5. In vitro data on test compounds, HEDICINs 1 and HECINs 2, as bbspn-1 inhibitors. Compounds’ activity as FP2 inhibitors (determined as described in chapter 2) is also displayed, for better comparison. ..................................................... 107! Table 6. Homolog sequences identified with BLAST according to the established search criteria. ............................................................................................................... 110! Table 7. Sequence similarity against the active site of bbspn-1. .................................. 111! Table 8. Homology modeling validation scores obtained by Modeller 9.11. ................. 115! Table 9. Additional scoring parameters of models built and selected according to Modeller 9.11 Z-DOPE score. ....................................................................................... 115! Table 10. ProSA Z-score of experimental structure and build model. .......................... 118! Table 11. Active site of bbspn-1 and FP2 cysteine proteases. ..................................... 123! Table 12. In vitro data of HEFLECINs against Pf W2 and Pf 3D7. clog P of the series of compounds 13 are also shown. ...................................................................... 141! Table 13. In vitro assays against β-hematin formation. ................................................ 147! Table 14. In vitro assays of N-cinnamoylated derivatives 13-14, 20-21, and 24-25 against P. berghei liver stage malaria. .......................................................................... 153! Table 15. Antiplamosdial activity of compounds against liver stage malaria. ............... 159! Table 16. In vitro efficiency of acridine derivatives 29 against CQ-resistant Pf W2 and IC50 of compound 29d against liver stage parasite. ............................................... 165! Table 17. In vitro data of PQ-based HEFLECINs against L. infantum promastigotes (PM) .............................................................................................................................. 169! Table 18. In vitro data of CQ-based HEFLECINs against L. infantum promastigotes (PM) and amastigotes (AM) and cytotoxicity results on mouse bone marrow-derived macrophages (MP). ....................................................................................................... 170! Table 19. Statistically significant difference found (p values lower than 0.05). ............ 223! Table 20. Microsphere coupling. .................................................................................. 228! ! ! ! Abbreviations | xix Abbreviations ab antibody ACT Artemisinin-combination therapies AMA-1 Apical membrane antigen 1 AQ Acridine AMBER Assisted Model Building with Energy Refinement ART Artemisinin B. Babesia β-H β-hematin Boc tert-Butoxycarbonyl BSA Bovine serum albumin Caco-2 Human colorectal adenocarcinoma cell line Cbz Carboxylbenzyl CelTOS Cell-traversal protein for ookinetes and sporozoites CI Confidence Interval COMU (1-Cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylaminomorpholino-carbenium hexafluorophosphate CQ Chloroquine CRESIB Barcelona Center for International Health Research CSP Circumsporozoite protein d doublet DBL Duffy binding-like DCM Dichloromethane dd doublet of doublets DIEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide DMSO Dimethylsulfoxide DMSO-d6 Hexadeuterated dimethylsulfoxide DPAP1 Dipeptidyl Amino Peptidase 1 xx | Abbreviations EBA-175 Erythrocyte binding antigen 175 EDC 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide Eq Molar equivalents ESI-IT MS Electrospray ionization-ion trap mass spectrometry E64 1-[N-[(L-3-trans-carboxyoxirane-2carbonyl)-L-leucyl]amino]-4guanidinobutane E64C [(2S,3S)-3-carboxyoxirane-2-carbonyl]-LLeucine (3-Methylbutyl)amide L-trans-epoxysuccinyl-Leu-3methylbutylamide Fe(III)PPIX Ferriprotoporphyrin IX Fig. Figure FP Falcipain FV Food Vacuole GOLD Genetic optimization for ligand docking HeLa Henrietta Lacks (human cervical carcinoma cell line) H-f Hemozoin formation HFF-1 Human foreskin fibroblast cell line HPLC High-performance liquid chromatography HOBT 1-Hydroxybenzotriazole IC50 Half maximal inhibitory concentration IFAT Immunofluorescence antibody test ITN Insecticide-treated-net hPhe Homophenylalanine J Coupling constant (in Hz) Leu Leucine LSA-1 Liver stage antigen-1 m multiplet MCF-7 Michigan Cancer Foundation-7 (human breast adenocarcinoma cell line) MKN-28 Human gastric epithelial carcinoma cell line Abbreviations | xxi mp Melting point MSP Merozoite surface protein Mu Morpholine ND Not determined NMR Nuclear magnetic resonance NPP New permeability pathways P. Plasmodium PCD Passive Case Detection PDB Protein data bank Pf Plasmodium falciparum PfCRT Plasmodium falciparum chloroquine resistant transporter. PQ Primaquine PSAC Plasmodium Surface Anion Channel p-TsOH p-Toluenesulfonic acid Py Pyridine PyAOP (7-Azabenzotriazol-1yloxy)tripyrrolidinophosphonium hexafluorophosphate PyBOP Benzotriazolyloxy-tris[pyrrolidino]- phosphonium hexafluorophosphate PyBrop Bromo-tris-pyrrolidino phosphoniumhexafluorophosphate PyClock 6-Chloro-benzotriazole-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate PyOxim [Ethyl cyano(hydroxyimino)acetato-O2]tri1-pyrrolidinylphosphonium hexafluorophosphate RBC Red Blood Cell RESP Restrained electrostatic potential RMSD Root mean standard deviation rt Room temperature s singlet SAR Structure-activity relationships SNAr Nuclephilic aromatic substitution xxii | Abbreviations SSP2 Second sporozoite surface protein Sulfo-NHS N-hydroxysulfosuccinimide t triplet TBAF Tetra-n-butylammonium fluoride TBDMSCl tert-Butyldimethylsilyl chloride TBTU O-(Benzotriazol-1-yl)-N,N,N’,N’- tetramethyluronium tetrafluoroborate TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin Layer Chromatography Uniprot Universal Protein Resource WHO World Health Organization Foreword | 23 Foreword Malaria is an infectious disease responsible for about a half million deaths every year, mostly affecting developing countries in tropical areas.1, 2 There are five species of the genus Plasmodium which cause malaria in humans, P. malarie, P. ovale, P. knowlesi, P. vivax, and P. falciparum (Pf).3 The latter is the most deadly species, mainly among children under five years of age, and is predominant in Africa. Malaria is transmitted by female Anopheles mosquitoes. Once an infected mosquito bites the human skin, it injects sporozoites which penetrate lymph nodes and capillaries (Fig. 1). The sporozoites that enter the lymph nodes are believed to contribute to the immune response. On the other hand, the sporozoites which go into the capillaries travel to the liver, where they invade hepatocytes to form a schizont, inside which asexual reproduction takes place to produce thousands of merozoites. Subsequently, these merozoites are released into the blood stream to invade red blood cells (RBC) and there reproduce asexually into more merozoites. When the infected RBC collapse, released merozoites are ready to infect other blood cells, thus perpetuating the disease in the human host. Occasionally, some intraerythrocytic parasites differentiate into male and female gametocytes. The life cycle continues when a mosquito bites the infected human and ingests such gametocytes. The sexual reproduction begins within the mosquito gut to end with formation of sporozoites. These sporozoites migrate into the salivary glands of the mosquito that becomes able to infect other human hosts.4 ! ! Figure 1. Life cycle of malaria parasites in humans.(Retrieved from Hill Philos Trans R Soc Lond B Biol Sci 2011) 24 | Foreword The Malaria Global Plan from 2011 set the challenge of having, by the end of 2015, i) reduced by 75% malaria cases incidence compared to levels in 2000 ii) lowered malaria deaths proximate to zero, and iii) eliminated the disease in ten new countries taking into account the countries where malaria have been eradicated since 2008.1 Although there have been significant advances in malaria containment to meet this global target, such progress seems to have slowed down recently making it insufficient to meet worldwide needs in this area. Besides, in spite of the fact that the percent of households protected with insecticide-treated-net (ITN) and indoor residual spread (IRS) have increased,1 parasite resistance to ITN continues to emerge. Additionally, concerns have been arisen regarding the use of bed nets, since by protecting infants from exposure to the malaria parasite, these vector control strategies might be impairing the respective acquirement of natural immunity and consequently, the burden of the disease might be shifting to older age children resulting in little or none long term benefit for vector control.6 In addition, though the number of diagnostic tests and treatments is increasing, millions of people continue to lack rapid diagnostics and efficient therapies against malaria.1 Furthermore, the malaria parasite has started to show resistance to artemisinins, the key components of artemisinin-combination therapies (ACT), which are the current first-line treatments against chloroquine (CQ)-resistant malaria. For instance, parasite resistance against artemisinins has already been detected in four countries of Southeast Asia.1 Besides political, social, and economic factors which contribute to the malaria burden on the world,7 malaria eradication is yet an unmet goal due to three main reasons: i) lack of a large scale ecofriendly way to control the vector, and consequently prevent transmission of the disease, ii) widespread parasite resistance to classical antimalarial drugs and emerging resistance to current first-line ones, and iii) lack of an effective and commercially available antimalarial vaccine to prevent infection.7 Therefore, in order to reduce the malaria incidence and death rates, all these boundaries need to be eliminated. In this dissertation, the work focused on two of the main and complementary approaches towards malaria eradication, namely, malaria chemotherapy and prevention. The first and major work of the doctoral research, conducted in the area of malaria chemotherapy, centered on the development of economically affordable dual action antimalarials, drugs capable of acting against two different processes in the malaria life cycle to improve efficacy and hinder emergence of parasite resistance. In parallel, work carried in the area of malaria prevention contributed to the understanding of whether the low exposure to Pf blood stage antigens could influence the immune response of vaccinated children against malaria in the long term, by obtaining and partially analyzing the antibody (Ab) Foreword | 25 response induced by the most clinically advanced pre-erythrocytic antimalarial vaccine, RTS,S.8 Chapter 1 | 33 1.0. Introduction Nowadays, there are drugs targeting each stage of the malaria asexual reproduction cycle within the human host. Chloroquine (CQ, I) and artemisinin (II) both target the erythrocytic stage1-3 while primaquine (III) targets the liver stage parasite.4, 5 However, most malaria control strategies are in jeopardy due to the emergence of parasite resistance. Pf parasites have displayed widespread resistance to one of the safer and most affordable drugs, CQ.6, 7 Furthermore, there is growing evidence that Pf is developing resistance to the key compounds of the first-line antimalarial medicines for CQ-resistant malaria, the artemisinin-combination therapies (ACT), 8 despite the fact that ACT have been designed to improve efficacy and hinder emergence of resistance. Therefore, there is an increasing need to develop new effective and affordable antimalarials capable of delaying parasite resistance. The erythrocytic stage is one of the most relevant stages of the malaria life cycle since the symptoms of malaria appear in this stage. The symptoms include fever, chill, prostration, and anemia and in more severe cases, delirium, metabolic acidosis, cerebral malaria, and multi-organ system failure. Therefore, most antimalarial aim to target this stage of the malarial life cycle. In this stage, the host’s hemoglobin is endocytosed to the parasitophorous vacuole (PV) formed inside the infected RBC, and transported to the parasite’s food vacuole (FV) where it is degraded into amino acids required for the parasite growth and maturation.9 In addition, hemoglobin degradation 34 | Chapter 1 allows the modulation of the osmotic stability within the host cell.10 This degradation process is believed to occur in a semi-ordered pathway (Fig. 2), where aspartyl proteases make the initial cleavage of the hemoglobin. Namely, plasmepsins I (PlmI) and II (PlmII) catalyze this first step by breaking down hemoglobin between Phe33 and Leu34 of the α-chain to produce globin and free heme (Fig. 2).11 On one hand, globin is degraded into small peptide chains by cysteine proteases known as falcipains.12 Once falcipains have degraded globin into small peptides, these are following broken into oligopeptides of 5 to 10 amino acids by falcilysin.13 Subsequently, dipeptidyl aminopeptidase 1 (DPAP1) cleaves these oligopeptides into dipeptides.14 Finally, these dipeptides are transported outside the FV into the PV’s cytoplasm where they are degraded into amino acids by exopeptidases.15 A comprehensive review regarding recent advance towards the development of antimalarial inhibitors of the proteases, which participate in the hemoglobin degradation, has been recently published (See appendix A). Figure 2. Proteolytic semi-ordered pathway of hemoglobin degradation into amino acids. Parasitophorous!vacuole! ! Food!Vacuole! ! Hemoglobin! Falcipain s! Plasmepsins! Falcilysin s! Small!Peptides! ! Small!peptides! Exopeptidase s! Amino!Acids! ! Heme! DPAP1! Globin! Chapter 1 | 35 On the other hand, heme is readily oxidized to ferriprotoporphyrin IX [Fe(III)PPIX]16 which is toxic to the parasite. [Fe(III)PPIX] is known to cause lipid peroxidation and changes in membrane’s permeability.17 The oxidation of membrane components caused by free heme promotes cell lysis and subsequent death of the parasite,16 which can’t degrade [Fe(III)PPIX] as mammals do (using the enzyme oxygenase) because it lacks a mechanism to sequester the free Fe(III) generated.17 Therefore, the main detoxification process of [Fe(III)PPIX] involves formation of hemozoin (β-hematin), also known as the malaria pigment. Hemozoin is a dark insoluble biocrystal that contains solely monomers of [Fe(III)PPIX] and is released into the host RBC every time a parasite completes a cycle. Inhibition of hemozoin formation is one of the antimalarial strategies.18 1.1. Targeting inhibition of globin degradation by falcipains Other potential target to impair the malaria parasite development constitutes inhibition of falcipains, which are one of the most studied families of Pf proteases.19 According to the analysis of the Pf genome, there are four cysteine proteases, falcipain 1 (FP1), falcipain 2 (FP2), falcipain 2’ (FP2’), and falcipain 3 (FP3).20 FP1 has a low sequence identity compared to the other falcipains and its role in the malaria life cycle is yet to be elucidated. Still reported studies suggest that this protease might participate in the generation of oocysts during parasite development in the mosquito midgut.21 FP2, FP2’, and FP3 show high sequence identity. For instance, FP2 has been demonstrated to be 93% similar to FP2’ which is thought to arise by gene duplication.22 FP2 and FP3 present a sequence identity of 68% and contribute more or less equally in the digestion of globin. In addition, FP2 and FP3 require acidic pH and reducing environment for optimal proteolytic activity. Although FP2 concentration in trophozoites is 1.8 times higher than that of FP3, the latter cleaves globin two times faster than FP2. Today there are several reports in the literature concerning the development of falcipain inhibitors.20 Inhibitors of falcipains can be generally classified in peptide-based inhibitors and non-peptidic inhibitors. Some of the most active compounds of each family are further below presented. The main catalytic residues of falcipains constituted Cys and His, whose side chains form an ion pair (thiolate/imidazolium) correctly orientated due to influence of a neighboring Asn residue. Molecules bearing electrophilic warheads are generally used to inhibit these enzymes because they form irreversible or reversible covalent bond with the active site cysteine. Examples of such warhead are α-ketoamide,23 fluoromethyl ketone,24 epoxysuccinyl25 or azirine derivatives,25 and the most used, Michael acceptors. The crystal structures of FP2 (PDB code = 3BPF) and FP3 (PDB code = 3BPM) reported 36 | Chapter 1 in complex with known inhibitors of cysteine proteases, such as E64 (IV) and leupeptin (V), have facilitated the design of new falcipains inhibitors.12 Accordingly, the substituents in the position P1, P2, and P3 of the ligands, E64 and leupeptin, respectively interact with residues in the S1, S2, and S3 sites of the active cavity of falcipains (Fig. 3). The S2 subsite seems to govern specificity towards FP2 and/or FP3, and to prefer substrates with a leucine residue in the corresponding P2 site. In addition, the catalytic residues of the FP2 and FP3, cysteines, form a covalent irreversible bond with the epoxide moiety of E64 and a covalent reversible bond with the carbonyl motif of leupeptin, respectively. ! Figure 3. E-64 and leupeptin as inhibitors of falcipains. One of the most successful examples of the use of Michael acceptor is that of peptidylvinyl sulfones which have to shown to inhibit FP2 in the nanomolar range.26 Inhibition of falcipains by such vinyl sulfones has been shown to correlate with the impairment of Pf development.19 These compounds include a dipeptide segment for better recognition by the parasite’s protease, whereas the vinyl sulfone moiety acts as a Michael acceptor to irreversibly alkylate the catalytic Cys residue of the protease (Fig. 4).27 Chapter 1 | 37 ! Figure 4. Example of irreversible alkylation of the cysteine residue of falcipains. Accordingly to Rosenthal’s studies, the Leucine-homoPhenylalanine (Leu-hPhe) dipeptide, has been demonstrated to confer the highest FP inhibitory activity of the respective vinyl sulfones. Further studies reported the structural activity relationship (SAR) of these and related cysteine inhibitors (Fig. 5).26 In these series, the potency of the compounds against FP2 and FP3, respectively, is not an ideal predictor of the antiplasmodial activity since SAR against falcipain does not completely correlated with the SAR found against the parasite development. For instance, vinyl sulfonamide and vinyl sulfonyl derivatives demonstrated to be more potent than vinyl sulfonate ester against the parasite, contrary to what was found against FP2. In addition, SAR regarding antiplasmodial activity showed that there is a clear preference for -OMe over -H over -F substitution in aromatic ring of the vinyl sulfonate esters, opposite to results found against falcipains. ! Figure 5. Structure of vinyl sulfone inhibitors of FP2/3 developed by Rosenthal and co-workers.26 In addition, other peptide-based inhibitors have been reported to reversibly bind to falcipains and inhibit FP2 and FP3 in the nanomolar level, for instance, compounds reported by O’Neill and co-workers.28 These compounds possessed the Leu-hPhe 38 | Chapter 1 dipeptide segment, as the best vinyl sulfone inhibitors reported by Rosenthal’s group, but did not include a Michael acceptor moiety; instead, an aldehyde moiety was used in the place of the vinyl sulfone warhead. Compounds (VII) and (VIII) displayed IC50 of 1.4 nM and 16.3 nM against FP2 and 198.9 nM and 214.6 nM against FP3, correspondingly. Also, these compounds (VII) and (VIII) did inhibit the CQ sensitive Pf strain 3D7 with IC50 of 27.1 nM and 9.3 nM, respectively, and were found to fit well inside the enzyme’s active pocket according to docking studies. Apart from peptidyl irreversible and reversible inhibitors of FP, non-peptidic compounds, known to not be prone to proteolytic degradation, have also shown to inhibit FP in the nanomolar range, for instance, compounds reported by Fiandor’s team.29 The most potent inhibitors of the series were the 2-cyanopyrimidine derivatives including the 2cyano-5-chloropyrimidines and 2-cyano-5-bromopyrimidines. Although these compounds were nanomolar inhibitors of FP2 and FP3, they presented antiparasitic activity in the micromolar range. Furthermore, Fiandor and co-workers found that introduction of a protonable amine read to a significant increase in the antiplasmodial activity. For instance, one of the most active compound (IX), which contains a protonable amine, displayed an IC50 below 0.5 nM against FP2 and FP3 and an IC50 of 1 nM against the CQ-resistant parasite. Chapter 1 | 39 1.2. Targeting inhibition of hemozoin formation As mentioned above, hemozoin formation is a unique process adopted by Plasmodium parasites to detoxify free heme. It is a validated target for most of the well-known existing antimalarial drugs and considered to be suitable target to developed new antimalarials.30 There are several families of compounds which impair parasite growth through inhibition of hemozoin formation (Fig. 6). The major families are quinolines (X), azoles (XI), isonitriles (XII), methylene blue (XIII), and xanthones (XIV).18 For instance, xanthone and derivatives inhibit hemozoin formation through the generation of a soluble complex with heme. Xanthones presenting a single hydroxyl group displayed IC50 > 50µM against Pf clone D6. However, xanthone with multiple hydroxyl substituents, especially on position 4and 5-, demonstrated to be more active (IC50 = 0.1-40 µM) against Pf D6.31 In fact, the antimalarial activity demonstrated to be positively correlated with the number of hydroxyl groups on the compounds. In terms of inhibition of hemozoin formation, xanthones with hydroxyl groups in positions 4and 5showed the highest activity of the series. Further reports in the literature showed that xanthones with an N,N-diethylaminoalkyl side chain of 5 or 6 carbons presented significantly higher antimalarial activity against Pf strain D6 (IC50 = 70-100 nM) exhibiting strong heme affinity. ! Figure 6. Known inhibitors of hemozoin formation: amodiaquine (X), clotrimazole (XI), diterpene isonitriles derivative (XII), methylene blue (XIII), and xanthone derivative (XIV),against CQ-sensitive Pf. Other major family of compounds whose SAR against hemozoin formation has been well established constitute quinolines and derivatives such as amodiaquine (X) and the 40 | Chapter 1 widely used CQ. CQ prevents hemozoin growth by capping the growth of the biocrystal, thus leading to accumulation of free heme to levels that cause parasite death (Fig. 7).32, 33 The chlorine atom in position seven of the quinoline ring in CQ has been found essential for the drug’s activity against hemozoin formation (Fig. 8).34, 35 In terms of optimal antiparasitic activity of antimalarial 4-aminoquinolines, like CQ, i) a halogen in position seven (-Cl, -Br, or -I but not -F), ii) a protonable amine at position 1 of the quinoline ring, and iii) a dialkylaminoalkylamino side chain are all key structural features.36 In addition, a dialkylaminoalkylamino side chain of 2 to 12 carbons leads to low nanomolar activities against the CQ-sensitive Pf strain. Moreover, it has been show that the protonable amine at position 1 of the quinoline ring and the terminal amine in the dialkylaminoalkylamino side chain are important for the accumulation of the compounds in the food vacuole and consequent inhibition of its target.37 ! Figure 7. CQ-heme complex incorporate in the forming biocrystal and inhibits hemozoin formation.32 ! Figure 8. Main characteristics of CQ for the optimal inhibition of hemozoin formation.38 Chapter 1 | 41 Additionally, CQ can accumulate in the FV and subsequently inhibit its target because it is a weak base that can diffuse through the parasite’s membrane. Once the drug is inside the FV, it is protonated and consequently, trapped in the acidic FV (pH trapping), which promotes the inhibition of hemozoin. However, the excessive use of CQ due to its high efficacy, low cost, and limited host toxicity led to a gradual decline of the parasite sensitivity to this drug,39 despite it aims at a non-proteic (i.e., non-genetically coded) target. It has been found that CQ concentrates substantially less in the FV of CQresistant Pf strains. It is widely believed that this lower accumulation is due to a mutant transmembrane protein, PfCRT, which promotes CQ efflux from the parasite’s FV, where the drug should exert its action.40 In view of the wide spread parasite resistance to CQ, especially in Africa, nowadays the first line of antimalarial treatment for CQ-resistance malaria is based on ACT, where the potent artemisinins are combined with other antimalarial to hinder the parasite resistance usually associated to monotherapy. However, there has been growing evidence of decreasing sensitivity of plasmodial to ACT. Malaria parasite has started to show resistance to other antimalarials such as artemisinins, the basis compounds of ACT.41 New approaches to delay parasite emergence resistance consist on the development of dual-action drugs, compounds capable of acting against two processes in the Pf malaria life cycle. Hybrid compounds have displayed potent antimalarial activity and no toxicity.42 Based on their high efficacy and low toxicity, dual-action drugs may constitute the next generation of antimalarials.42 1.3. The covalent bitherapy approach against Pf malaria The covalent bitherapy approach against malaria refers to the development of hybrid drugs that covalently link two antimalarial moieties acting against different processes which are essential for parasite survival.43 One of the advantages of hybrid drugs, as opposed to multicomponent ones, is that they might diminish the risk of drug-drug adverse interactions. An example of hybrid antimalarials is that of trioxaquines, reported by Meunier and co-workers, which combine a trioxolane moiety (mimic of the artemisinin’s active core) with the CQ’s 4-amino-7-chloroquinoline core.44 The most promising trioxaquine (XV) of the series was synthesized from two precursors: i) 4-(Naminoethyl)amino-7-chloroquinoline obtained from nucleophilic substitution of ethane1,2-diamine on 4,7-dichloroquinoline, and ii) a trioxane-ketone synthesized from αterpinene, through photo-oxygenation and subsequent condensation with cyclohexane1,4-dione (Scheme 1).45 Trioxaquine (XV) was active in the nanomolar range against the asexual intraerythrocytic stage (IC50 = 28 nM) and young (IC50 = 69 nM) or old 48 | Chapter 1 ! ! Scheme 5. a) Synthetic pathway developed by Chibale and co-workers in 2007.50 b) Most active compound of the synthesized series. Following, the same group reported a series of γand δ-lactams linked to 4-amino-7chloroquinoline through an alkyl chain (XXII) .51 The synthetic pathway involved, first, the nucleophilic substitution on 4,7-dichloroquinoline with the relevant alkanediamine. Posteriorly, the resulted 4-(N-aminoalkyl)amino-7-chloroquinoline was reacted with levulinic acid or 4-acetylbutyric acid and the relevant isocyanide, through a one-pot Ugi reaction (Scheme 6). The most active compound (XXIIa) displayed activity in the nanomolar range against Pf CQ-resistant W2 strain (IC50 = 96 nM) that did not correlate with its ability to inhibit FP2 action (IC50 = 17.6 µM). The lack of correlation between antiplasmodial and FP-inhibitory activity and the fact that the compounds presented lower IC50 compared to CQ (IC50 = 240 nM) may suggest that these molecules might be eluding efflux from the FV. Chapter 1 | 49 ! Scheme 6. One-pot Ugi reaction in methanol at room temperature (rt) carried out by Chibale and coworkers.51 Furthermore, Chibale’s team synthesized a new series of compounds (XXIII) that joined chloroquinoline to chalcone through a triazole linker aiming at inhibition of both hemozoin formation and FP2 action.52, 53 The synthesis pathway globally involved the previous obtainment of suitable precursors, namely, chalcones and 4-azido-7-chloroquinoline, followed by an azide-alkyne coupling (Huisgen’s cycloaddition) to yield 1,2,3-triazoles (Scheme 7). The most active compound of series (XXIIIa) presented activity (IC50 = 40 nM) comparable to CQ (IC50 = 17 nM) against the Pf W2 strain. Although the compounds showed to inhibit hemozoin formation as CQ did, they only displayed activity in the micromolar range against FP2, suggesting that the main mechanism of these series was inhibition of hemozoin formation. ! Scheme 7. Synthethic pathway developed by Chibale’s team in 2010.53 More recently, Gomes and co-workers developed potential dual-action antimalarials (XXIV), expectedly able to also inhibit hemozoin formation and falcipain activity. These compounds included the 4-amino-7-chloroquinoline core, meant to impair hemozoin formation, linked to an α,β-unsaturated carbonyl moiety expected to act as a Michael 50 | Chapter 1 ! acceptor, and thus alkylate the catalytic Cys in falcipains.54 The authors believed those two moieties should be linked through the Leu-hPhe dipeptide, previously described by Rosenthal to enhance inhibitor recognition by falcipains. However, in order to prevent premature proteolytic degradation of the dipeptide spacer, the authors used the corresponding retro-inverso dipeptide, using the respective D-amino acids (Fig. 10).55, 56 ! Figure 10. General structure of hybrid compounds (XXIV) designed by Gomes’s team.54 Most promising compound (XXIVa) synthesized. Gomes’s team chose cinnamic acid derivatives motivated by cinnamic acid potential ability to act as inhibitor of enzyme catalytic Cys residues,57 due to the α,β-unsaturated carbonyl moiety, and by previous reports on the antimalarial properties of cinnamoyl derivatives.58-60 One such report, by Schlitzer and co-workers, suggested that the use of 4-alkoxy-substituted cinnamic acids improved the antiplasmodial activity of identified antimalarial leads (Fig. 11).58 Specifically, replacement of the 3-phenylpropionyl moiety of the lead structure (XXV) by a 4-propoxycinnamoyl functionality (XXVI) resulted in higher activity (IC50 (XXV)= 2.7 µM vs IC50 (XXVI) = 0.20 µM) against the CQ-resistance Pf strain Dd2. In addition, they also demonstrated that substitution of the 3phenylpropionyl moiety by the 4-phenylcinnamoyl functionality (XXVII) led to significantly lower cytotoxicity (IC50 (XXV) < 36.9 µM vs IC50 (XXVII) >180 µM against HeLa) and 5fold increase on activity (IC50 (XXV) = 2.7 µM vs IC50 (XXVII) = 0.65 µM) against Pf Dd2.59 Chapter 1 | 51 ! Figure 11. Cinnamic acid derivatives found by Schlitzer research team.58, 59 Gomes et al’s in vitro results demonstrated two of the synthesized derivatives to be active in the micromolar range, with the best compound (XXIVa) displaying an IC50 of 1.4 µM against Pf strain W2. Although the approach showed promising results, further studies were required to improve the antimalarial activity of these cinnamic derivatives. 52 | Chapter 1 ! 1.4. References 1. Eckstein-Ludwig, U.; Webb, R. J.; Van Goethem, I. D.; East, J. M.; Lee, A. G.; Kimura, M.; O'Neill, P. M.; Bray, P. G.; Ward, S. A.; Krishna, S. 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Benoit-Vical, F.; Lelièvre, J.; Berry, A.; Deymier, C.; Dechy-Cabaret, O.; Cazelles, J.; Loup, C.; Robert, A.; Magnaval, J.-F.; Meunier, B. Trioxaquines Are New Antimalarial Agents Active on All Erythrocytic Forms, Including Gametocytes. Antimicrob Agents Ch 2007, 51, 1463-1472. 45. Dechy-Cabaret, O.; Benoit-Vical, F.; Loup, C.; Robert, A.; Gornitzka, H.; Bonhoure, A.; Vial, H.; Magnaval, J. F.; Seguela, J. P.; Meunier, B. Synthesis and antimalarial activity of trioxaquine derivatives. Chemistry 2004, 10, 1625-36. 46. Walsh, J. J.; Coughlan, D.; Heneghan, N.; Gaynor, C.; Bell, A. A novel artemisinin-quinine hybrid with potent antimalarial activity. Bioorg Med Chem Lett 2007, 17, 3599-602. 47. Capela, R.; Oliveira, R.; Goncalves, L. M.; Domingos, A.; Gut, J.; Rosenthal, P. J.; Lopes, F.; Moreira, R. Artemisinin-dipeptidyl vinyl sulfone hybrid molecules: design, synthesis and preliminary SAR for antiplasmodial activity and falcipain-2 inhibition. Bioorg Med Chem Lett 2009, 19, 3229-32. 56 | Chapter 1 ! 48. Capela, R.; Cabal, G. G.; Rosenthal, P. J.; Gut, J.; Mota, M. M.; Moreira, R.; Lopes, F.; Prudêncio, M. Design and evaluation of primaquine-artemisinin hybrids as a multistage antimalarial strategy. Antimicrob Agents Ch 2011, 55, 4698-706. 49. Chiyanzu, I.; Clarkson, C.; Smith, P. J.; Lehman, J.; Gut, J.; Rosenthal, P. J.; Chibale, K. Design, synthesis and anti-plasmodial evaluation in vitro of new 4aminoquinoline isatin derivatives. Bioorg Med Chem 2005, 13, 3249-61. 50. Chipeleme, A.; Gut, J.; Rosenthal, P. J.; Chibale, K. Synthesis and biological evaluation of phenolic Mannich bases of benzaldehyde and (thio)semicarbazone derivatives against the cysteine protease falcipain-2 and a chloroquine resistant strain of Plasmodium falciparum. Bioorg Med Chem 2007, 15, 273-82. 51. Musonda, C. C.; Gut, J.; Rosenthal, P. J.; Yardley, V.; Carvalho de Souza, R. C.; Chibale, K. Application of multicomponent reactions to antimalarial drug discovery. Part 2: New antiplasmodial and antitrypanosomal 4-aminoquinoline gammaand delta-lactams via a 'catch and release' protocol. Bioorg Med Chem 2006, 14, 5605-15. 52. Guantai, E. M.; Ncokazi, K.; Egan, T. J.; Gut, J.; Rosenthal, P. J.; Smith, P. J.; Chibale, K. Design, synthesis and in vitro antimalarial evaluation of triazole-linked chalcone and dienone hybrid compounds. Bioorg Med Chem 2010, 18, 8243-56. 53. Guantai, E. M.; Ncokazi, K.; Egan, T. J.; Gut, J.; Rosenthal, P. J.; Bhampidipati, R.; Kopinathan, A.; Smith, P. J.; Chibale, K. Enone– and Chalcone– Chloroquinoline Hybrid Analogues: In Silico Guided Design, Synthesis, Antiplasmodial Activity, in Vitro Metabolism, and Mechanistic Studies. J Med Chem 2011, 54, 3637-3649. 54. Figueiras, M. Potenciais Fármacos de Acção Dual Contra Fase Sanguínea da Malária. Master Thesis, Faculdade de Ciências da Universidade do Porto 2010. 55. Hong, S. Y.; Oh, J. E.; Lee, K.-H. Effect of d-amino acid substitution on the stability, the secondary structure, and the activity of membrane-active peptide. Biochem Pharmacol 1999, 58, 1775-1780. 56. Müller-Schiffmann, A.; Petsch, B.; Leliveld, S. R.; Muyrers, J.; Salwierz, A.; Mangels, C.; Schwarzinger, S.; Riesner, D.; Stitz, L.; Korth, C. Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity. Mol Immunol 2009, 46, 532-540. 57. Kouassi, Y. A. O.; Shelef, L. A. INHIBITION OF LISTERIA MONOCYTOGENES BY CINNAMIC ACID: POSSIBLE INTERACTION OF THE ACID WITH CYSTEINYL RESIDUES. J Food Safety 1998, 18, 231-242. Chapter 1 | 57 58. Wiesner, J.; Mitsch, A.; Wissner, P.; Jomaa, H.; Schlitzer, M. Structure-activity relationships of novel anti-malarial agents. Part 2: cinnamic acid derivatives. Bioorg Med Chem Lett 2001, 11, 423-4. 59. Wiesner, J.; Wissner, P.; Dahse, H. M.; Jomaa, H.; Schlitzer, M. Discovery of a novel lead structure for anti-malarials. Bioorg Med Chem 2001, 9, 785-92. 60. Herrin, T. R.; Pauvlik, J. M.; Schuber, E. V.; Geiszler, A. O. Antimalarials. Synthesis and antimalarial activity of 1-(4-methoxycinnamoyl)-4-(5-phenyl-4-oxo2-oxazolin-2-yl)piperazine and derivatives. J Med Chem 1975, 18, 1216-23. ! 64 | Chapter 2 ! ! Scheme 8. Synthesis pathway to obtain HEDICINs 1. The yield-limiting step in the synthesis pathway to HEDICINs was the coupling of 3 to Boc-D-hPhe-OH, probably because of the low nucleophilicity of the amino group in 3. In fact, the nitrogen atom at position 1 of 4-amino-7-chloroquinoline 3 withdraws electron density from the amino substituent at position 4, making the latter less prone to attack an electrophile. Hence, looking for the appropriate reaction conditions to improve yield, highly efficient in situ amide coupling reagent were used as: PyBOP, ethyl cyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOXIM), (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholinocarbenium hexafluorophosphate (COMU), 6-chloro-benzotriazole-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate (PyClock), bromo-tris-pyrrolidino phosphonium hexafluorophosphate (PyBrop), and (7-azabenzotriazol-1yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) (Fig. 14), still the highest yield obtained was 35% (Table 1). The coupling reagent that resulted the best yield in this step was TBTU.4 This was an unexpected observation, since PyAOP has been described to significantly improve coupling yields when using poor nucleophiles and hindered amino acids.4, 5 In addition, increasing the temperature of the reaction up to 50 Chapter 2 | 65 oC and the amount of DIEA base up to 3.3 molar equivalents (eq), even risking the possibility of racemization of the desired product,6 did not increase the yield achieved in the reaction step. ! ! Figure 14. Coupling reagents used to promote in situ coupling of 3 with Boc-hPhe-OH. 66 | Chapter 2 ! Table 1. Reaction conditions for the coupling of Boc-D-hPhe-OH to obtain 4. Coupling Reagent DIEA Day(s) Temperature (oC) Solvent % Yield PyBOP 2.2 2 rt DMF:DMSO (1:1) 8 2.2 2 50 6 3.3 2 50 9 2.2 2 rt DCM + Drops of DMF 21 2.2 1 rt 22 2.2 1 rt DMF 7 COMU 2.2 2 rt DMF 14 PyOXIM 2.2 2 rt DMF 14 PyBrop 2.2 2 rt DMF 29 3 rt DMF 31 PyClock 2.2 2 rt DMF 8 PyAOP 2.2 2 rt DMF 20 TBTU 2 1 rt DMF 35 Table 1 also shows that when increasing temperature or reaction time, no significant difference was observed among the yields obtained. However, a decrease of the amount of DMF used as a solvent when using PyBOP as a coupling reagent led to approximately two-fold yield increment. This could suggest that the DMF used to synthesize the desired compounds possessed water, which could be solvating the nucleophile, and impeding its subsequent reaction with the corresponding activated cinnamic acid. Moreover, in order to evaluate the influence of the configuration of the amino acid in the bioactivity of HEDICINs, an additional HEDICIN (1a’) was synthesized using L-amino acids instead of D-amino acids following the synthetic procedure presented in scheme 8. Furthermore, to evaluate the effect of chloroquinoline ring on the antiplasmodial activity of the compounds, cinnamoylated derivative 8 was also synthesized. Compound 8 was obtained following similar procedure to that used for HEDICINs but using as a starting material morpholine instead of 4-amino-7-chloroquinoline (Scheme 9). As expected the coupling of h-Phe was carried in good yields which confirmed that in the synthetic pathway of HEDICINs 1, the coupling of Boc-D-hPhe-OH to obtain compound 4 is limited by the poor nucleophilicity of 4-amino-7-choloroquinoline 3. Morpholine was selected Chapter 2 | 67 based on the structure of the vinyl sulfone reported by Rosenthal’s team and its flexibility. The latter could promote a better fit of the ligand into falcipain catalytic site although the inhibition of hemozoin formation would be disregarded. ! Scheme 9. Synthetic pathway to obtain morpholine derivative 8. ! ! b) HECINs In parallel to the synthesis of HEDICINs, HECINs 2a-k were obtained by coupling different cinnamic acids, or the respective acyl chlorides, to compound 3 (Scheme 10), previously obtained by ammonolysis of 4,7-dichloroquinoline. Initially, PyBOP was used as the coupling reagent to couple the respective cinnamic acids. However, the latter reaction conditions led to low yields, which encouraged us to try different reaction conditions to improve the yields obtained. In this context, the respective acyl chloride, when available, and K2CO3 and 18-crown-6 (catalytic amounts) were used to obtain the desired HECINs. Accordingly, this reaction conditions provided better results. For instance, in the case of the p-metoxy cinnamoyl derivative 2d, when using PyBOP as coupling reagents and DMF as solvent, the desired product 2d was obtained with 1% yield. However, when using trans-4-methoxycinnamoyl chloride, the desired product was achieved with an 18% yield (Table 2); a significant increase that facilitates the characterization of the desired compound 2d. Additional experimental procedures carried out to improve the yields obtained for HECINs demonstrated that, when using PyBOP as a coupling reagent with DCM and some drops of DMF instead of exclusively DMF as a solvent, the yields increased but not higher than 20% which is in agreement 68 | Chapter 2 with results obtained for HEDICINs when coupling compound 3 to Boc-D-hPhe-OH and using the same reaction conditions. ! Scheme 10. Synthesis pathways to obtain HECINs. Table 2. Amide bond formation for HECINs. X= -Cl, -OH Reaction conditions PyBOP, DIEA, DMF, rt Respective cinnamic acid K2CO3, 18-Crown-6, THF, rt % Yield % Yield R= H 3 18 R= -OMe 1 18 R= -NO2 Not Obtained 5 ! ! ! ! ! Chapter 2 | 69 2.1.2. Characterization of HEDICINs and HECINs All HEDICINs 1a’, 1a-l, and 8 and HECINs 2a-k were characterized by electrospray ionization-ion trap mass spectrometry (ESI-IT MS) as well as by proton (1H-) and carbon (13C-) nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC). Results were in agreement with the desired structures and are provided in the experimental section at the end of this chapter. The HPLC results displayed a peak for the relevant sample with a percent of purity that allow further in vitro evaluation. In addition, the characteristic signals of the corresponding samples in the ESI-IT MS and NMR were observed. As an example for HEDICINs, the relevant NMR and MS spectra of the p-methoxycinnamic acid derivative 1d is provided in figures 15−17. 70 | Chapter 2 ! ! Figure 15. 1H-NMR (400 MHz, DMSO-d6) of HEDICIN 1d above 6 ppm (Upper corner) and below 5 ppm (bottom corner). d d e e a a b c b-c f f Chapter 2 | 71 ! Figure 16. 13C-NMR (100 MHz, DMSO-d6) spectrum of the HEDICIN 1d. ! Figure 17. ES-IT MS of HEDICINs 1d. BP-148_110322085607 #2RT: 0,03 AV: 1NL: 3,93E7 T: + p ESI Full ms [ 50,00-2000,00] 500 1000 1500 2000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 613,73 1274,80 1226,47 1347,33 130,20 1043,13 870,20 258,80 721,93 274,47 1839,73 546,60 1475,07 a b c d d a-c 72 | Chapter 2 ! Fig. 15 shows the protons downfield and upfield in the 1H-NMR spectrum of 1d. The downfield region displays the vinyl protons of the α,β-unsaturated carbonyl moiety, the proton of the Cα at 6.63 ppm and the proton of the Cβ at 7.38 ppm with coupling constant J = 16 Hz which is characteristic of trans-vicinal vinyl protons. Also, the 1HNMR spectrum displays the signal of the amide bond attached to the quinoline ring at 10.34 ppm. In the upfield region (Fig. 15), the 1H-NMR spectrum shows the multiplets of the protons of the Cα to the amide functionalities, respectively, at 4.70 ppm and 4.56 ppm. The singlet at 3.79 ppm represents the three deshielded protons of the methoxy substituent in the molecule. In addition, the six shielded protons of the methyl group of the leucine isobutyl side chain are also observed. In fig. 16, the 13C-NMR spectrum displayed the signals in agreement with the structure of compound 1d, for instance, the deshielded signals of the carbonyl groups above 166 ppm and the signal of the methoxy group at 55.2 ppm. Please refer to the experimental section of this chapter for a more detailed analysis of the spectra of HE[DI]CINs. Moreover, the mass spectrum (Fig. 17) shows an intense peak at 613.73 ppm which corresponds to the quasi-molecular ion of 1d. Spectroscopy data of HECINs also correspond with the desired compounds. For example, for HECIN 2d, in the upfield region, apart from the respective signal of DMSO and water, the signal of protons of the methoxy substituent in the cinnamoyl moiety at 3.82 ppm (Fig. 18) is displayed. The 1H-NMR spectrum also displays a singlet at 10.34 ppm that corresponds to the proton of the amide bond and a doublet with a small coupling constant of 2 Hz at 8.05 ppm that belongs to the proton of the carbon at position 8 of the quinoline ring. In addition, a doublet with a coupling constant of 15.6 Hz at 7.12 ppm was observed, which agrees with the proton of the Cα of α,β-unsaturated carbonyl. In fig. 18, the 13C-NMR spectrum displays the number of signal in agreement with the desired compounds 2d, for instance, the upfield region shows one signal which represents the methoxy group at 55.3 ppm and 16 signals in the downfield region, from 111 ppm to 165.1 ppm. The most deshielded signal of the spectrum (δC = 165.1 ppm) belongs to the carbonyl. In addition, the mass spectrum (Fig. 19) displays the peak corresponding to the quasi-molecular ion of 2d at 339.33 and also the corresponding dimer at 676.93. Chapter 2 | 73 ! Figure 18. 1H-NMR (400 MHz, DMSO-d6) spectrum of HECIN 2d (Top corner) and 13C-NMR (100 MHz, DMSO-d6) spectrum of HECIN 2d (Bottom corner). b a c a b d d d c 80 | Chapter 2 ! c) Antiplasmodial assays According to antiplasmodial results, all HEDICINs displayed activity in the micromolar range against the parasite (IC50 = 0.83-10.8 µM), contrary to HECINs which were not active against the parasite (Table 3). Interesting, the most active compound of the series 1c was the most lipophilic, demonstrating that this property plays a important role in antiplasmodial activity as found by Miller and Lin.16 This could also explain the lack of activity of HECINs, since HEDICINs are more lipophilic than HECINs due to dipeptide spacer use to join the cinnamoyl moiety and the chloroquinoline ring. In addition, there was no correlation regarding the activity of the compounds and the electro-donating properties of the substituents in the cinnamoyl moiety. Contrary, results suggest that as the electronegativity of the para substituent increases, the activity decreases. For instance, the p-Br derivative 1i (IC50 = 2.55 µM) and the p-Cl 1h (IC50 = 2.89 µM) were more active than the p-F 1g (IC50 = 4.67 µM). Also, para substituent seems to be preferable over the meta substituents as demonstrated by the p-F (IC50 = 4.67 µM) vs the m-F (IC50 = 5.43 µM) derivatives and the p-NO2 (IC50 = 1.23 µM) vs the m-NO2 derivative (IC50 = 2.10µM). Results demonstrated the relevance of the chloroquinoline moiety in the antiplasmodial activity of the compounds since compound 8 was not active against the malaria parasite (IC50 > 10 µM). Moreover, as in the case of inhibition of falcipain action, D-amino acids, in the case of HEDICINs, seems to be promoting impairment of parasite growth. Results demonstrated that compounds ability to inhibit of hemozoin formation or falcipain action does not correlate with compounds ability to impair parasite growth. However, HEDICINs displaying antiplasmodial activity below 2µM which were also active against β-H formation, for instance, the p-Me derivative 1b (IC50 = 1.96 µM) and the p-NO2 derivative 1k (IC50 = 1.23 µM), suggests that inhibition of hemozoin formation might be one of the mechanism through which these derivatives exert their activity. In addition, the fact that, for instance, the m-NO2 derivative 2j, the most active of HECINs against FP2 (IC50 = 14.2µM) was not active against the Pf parasite (IC50 > 10 µM), might be suggesting that HECINs may not be reaching its corresponding target. The latter could be explained by the low basicity of the HECINs which could be impeding the necessary accumulation of the molecules in the FV to inhibit the respective target. Overall, the relevance of the hydrophobic link and chloroquine moiety to impair parasite growth are demonstrated. Chapter 2 | 81 2.2. Experimental Section ! 2.2.1. Chemistry 2.2.1.1. Chemicals and instrumentation All solvents and common chemicals were from Sigma Aldrich (Spain), whereas Bocprotected amino acids were from NovaBiochem (VWR International, Portugal) and TBTU was from Bachem (Switzerland). NMR spectra were acquired on a Bruker Avance III 400 spectrometer from solutions in either deuterated chloroform or deuterated dimethylsulfoxide (DMSO-d6) containing tetramethylsilane as internal reference. MS spectra were obtained on a Thermo Finnigan LCQ Deca XP Max LC/MSn instrument operating with electrospray ionization and ion-trap (ESI-IT) quadrupole detection. HPLC analyses were run for target compounds (1-2 and 8) using the following conditions: 30100% of B in A (A =H2O with 0.05% of trifluoroacetic acid; B =acetonitrile) in 22 min with a flow rate of 1 mL/min on a MerckeHitachi Lachrom Elite instrument equipped with a diode-array detector (DAD) and thermostated (Peltier effect) autosampler, using a Purospher STAR RP-18e column (150 4.0 mm; particle size, 5 µM). 2.2.1.2. Synthesis of compounds 3-7 ! 4-amino-7-chloroquinoline (3). An earlier method by Reitsema and co-workers was followed:3 4,7-dichloroquinoline (6 g, 0.03 mol) and phenol (30 g, 0.3 mol) were mixed at room temperature in a round bottom flask. After increasing the temperature to 150oC, ammonia was bubbled into the solution for 2 hours. After cooling the mixture back to room temperature, a solution of glacial acetic acid (7.5 mL) in water (15 mL) was added with stirring, followed by addition of ice-cold ethyl ether (30 mL). The white precipitate formed was collected by suction filtration and dissolved in hot water. The pH of the resulting solution was adjusted to 11 with 1M NaOH (aq) and, after cooling the mixture, a white solid precipitated and was collected, dried and identified as the target 4-amino-7chloroquinoline, 2 (4.8g, 89%); mp 110-112 oC; RF (DCM/MeOH 20:1) 0.59; δH (400 MHz, DMSO-d6) 8.31 (d, J= 5.2 Hz, 1H, Ha), 8.19 (d, J=9.2 Hz, 1H, He), 7.76 (d, J= 2 Hz, 1H, Hh), 7.40 (dd, J= 8.5 Hz, J= 2 Hz, 1H, Hf), 6.93 (s, 2H, -NH2), 6.55 (d, J= 5.2 Hz, 1H, Hb); δC (100 MHz, DMSO-d6) 151.6, 151,5, 149.4 (Ca, Cc, Ci), 133.4, 127.3, 124.6, 123.7 (Ce-Ch), 117.0 (Cd), 102.6 (Cb); ESI-IT MS: m/z (M+H+) 179.33 (C9H7ClN23 requires 178.03). 82 | Chapter 2 ! 4-[(2R)-2-(tert-butoxycarbonyl)amino-4-phenylbutanoyl]amino-7-chloroquinoline (4) Boc-D-homophenylalanine (0.43 g, 1.5 mmol), TBTU (0.49 g, 1.54 mmol), DIEA (0.36 mL, 2.8 mmol) and dimethylformamide (DMF, 7 mL) were mixed in a round bottom flask at 0 ºC. After 10 minutes, a solution of 4-amino-7-chloroquinoline 3 in DMF (7 mL) was added and the reaction was allowed to proceed for one day at room temperature. Following, water was added to the reaction mixture and the product was extracted with dichloromethane (3 times); the organic layers were pooled and sequentially washed with 1% aq. HCl (3 times) and 5% aq. Na2CO3 (3 times). Finally, the organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by column liquid chromatography on silica, using dichloromethane:acetone (DCM/Me2CO) 6:1 v/v as eluent. The resulting product was obtained as a white solid and corresponded to the target compound 4 (218 mg, 35%); mp 150-152oC; RF (DCM/Me2CO 6:1) 0.57; δH (400 MHz, CDCl3) 9.72 (s, 1H, -NH-QN), 8.8 (d, J= 5.2 Hz, 1H, Ha), 8.29 (d, J= 5.2 Hz, 1H, Hb), 8.05 (d, J= Hz, 1H, Hh), 7.95 (d, J= 8.8 Hz, 1H, He), 7.47 (dd, J= 8 Hz, J= 2 Hz, Hf), 7.33-7.29 (m, 2H), 7.24-7.15 (m, 4H) (-NH-hPhe, Ho-Hq), 4.26 (q, J= 7.3 Hz, 1H, Hk), 2.83-2.79 (m, 2H, Hm), 1.50 (s, 9H, Ht), 1.45-1.44 (m, 2H, Hl); δC (100 MHz, CDCl3); 170.8 (Cj), 157.2, 152.3, 149.3, 141.0, 140.3, 135.3, 129.1, 128.7, 128.4, 127.2, 126.4, 121.5 (Ca, Cc, Ce-Ci, Cn-r), 118.3 (Cd), 110.4 (Cb), 81.7 (Cs), 54.7 (Ck), 32.0, 31.3, 28.3 (Cm, Ct, Cl); ESI-IT MS: m/z (M+H+) 440.20 (C24H26ClN3O3 requires 439.17). 4-[(2R)-2-amino-4-phenylbutanoyl]amino-7-chloroquinoline (5) Compound 4 (0.13 g, 0.3 mmol) was reacted with neat trifluoroacetic acid (TFA, 1 mL) for 2 at room temperature, under constant stirring. Then, the pH was adjusted to 12 with 30% Na2CO3 (aq) and the solution extracted with DCM. The organic layer was dried with anhydrous Na2SO4 and evaporated to dryness under reduced pressure. Compound 5 was thus obtained as a yellow oil (85.3 mg, 85%); RF (DCM/Me2CO 6:1) 0.26; δH (400 MHz, CDCl3) 10.89 (s, 1H, -NH-QN), 8.81 (d, J= 5.2 Hz, 1H, Ha), 8.38 (d, J= 5.2 Hz, 1H, Hb), 8.08 (d, J= 2 Hz, 1H, Hh), 7.77 (d, J=9.2Hz, 1H, He), 7.48 (dd, J= 9Hz, J= 2Hz, 1H, Hf), 7.33-7.18 (m, 7H, Ho-Hq, -NH2), 3.63-3.59 (m, 1H, Hk), 2.90-2.75 (m, 2H, Hm), 2.011.85 (m, 2H, Hl); δC (100 MHz, CDCl3) 173.3 (Cj), 152.2, 148.9, 140.1, 139.9, 134.8, 128.9, 128.3, 128.0, 126.8, 126.0, 120.5 (Ca, Cc, Ce- Chapter 2 | 83 Ci, Cn-Cq), 118.0 (Cd), 109.3 (Cb), 55.1 (Ck), 35,7, 31.8 (Cl-Cm); ESI-IT MS: m/z (M+H+) 340.33 (C19H18ClN3O requires 339.11). 4-{(2R)-2-[(2R)-2-(tert-butoxycarbonyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (6) Boc-D-leucine (0.13 g, 0.38 mmol), PyBOP (0.22 g, 0.42 mmol), DIEA (0.162 mL, 1.25 mmol) and DCM (2 mL) were mixed in a round bottom flask at 0 ºC and put under stirring for 20 minutes. Then, a solution of 5 in DCM (2 mL) was added and the reaction allowed to proceed for one day at room temperature. Following, the reaction mixture was diluted with 14 mL of DCM and sequentially washed with 1% aq. HCl (3×18 mL) and 5% aq. Na2CO3 (3×18 mL). Finally, the organic layer was dried with anhydrous Na2SO4, filtered, and evaporated to dryness. The crude product was purified by column liquid chromatography on silica, using DCM/Me2CO 6:1 v/v as eluent. The target compound, 6, was isolated as a yellowish oil (122 mg, 58%); RF (DCM/Me2CO 6:1) 0.53; δH (400 MHz, CDCl3); 9.75 (s, 1H, -NH-QN), 8.79 (d, J= 5.2 Hz, 1H, Ha), 8.22 (d, J= 4.8 Hz, 1H, Hb), 8.07 (d, J= 2 Hz, 1H, Hh), 7.95 (d, J= 9.2 Hz, 1H, He), 7.49 (dd, J= 8.8 Hz, J= 2 Hz, 1H, Hf), 7.30-7.26 (m, 2H, -NH-Leu, -NH-hPhe), 7.24-7.17 (m, 5H, Ho-Hq), 4.60 (q, J= 7.2 Hz, 1H, Hk), 4.16 (b, 1H, Hs), 2.83-2.74 (m, 2H, Hm), 2.22-2.11 (m, 2H, Hl), 1.81-1.71 (m, 2H, Ht), 1.65-1.55 (m, 1H, Hu), 1.4 (s, 9H, Hy), 0.9 (d, J= 6.8 Hz, 6H, Hv); δC (100 MHz, CDCl3) 174.5 (Cj), 170.1 (Cr), 155.9 (Cw), 152,1, 149.3, 141.2, 140.4, 135.3, 129.0, 128.7, 128.4, 127.3, 126.4, 121.9 (Ca, Cc, Ce-Ci, Cn-Cq), 118.5 (Cd), 110.9 (Cb), 69.7 (Cx), 53.9, 53.1 (Ck, Cs), 40.3, 30.8, 28.2, 24.7, 22.7, 18.8 (Cl-Cm, Ct-Cv, Cy); ESI-IT MS: m/z (M+H+) 553.27 (C30H37ClN4O4 requires 552.25). 4-{(2R)-2-[(2R)-2-amino-4-methylpentanoyl]amino-4-phenylbutanoyl}amino-7chloroquinoline (7) Compound 6 (0.122 g, 0.22 mmol) was reacted with neat trifluoroacetic acid (TFA, 1 mL) for 2 at room temperature, under constant stirring. Then, the pH was adjusted to 12 with 30% aq. Na2CO3 and the desired compound was extracted with DCM. The organic layer was dried with anhydrous Na2SO4 and evaporated to dryness under reduced pressure. Compound 6 was thus obtained as a yellowish oil (72 mg, 87%); RF (DCM/Me2CO 6:1) 0.16; δH (400 MHz, CDCl3) 10.15 (s, 1H, -NH-QN), 8.77 (d, J=5.2Hz, 1H, Ha), 8.28 (b, 84 | Chapter 2 ! 1H, Hb), 8.11-8.01 (m, 3H, -NH-hPhe, He, Hh), 7.51 (d, J=8.8Hz, 1H, Hf), 7.31-7.13 (m, 7H, -NH2, Ho-Hq), 4.57 (b, 1H, Hk), 3.52-3.36 (m, 1H, Hs), 2.81-2.78 (m, 2H, Hm), 2.041.84 (m, 2H, Hl), 1.44-1.29 (m, 3H, Hu-Ht), 1.03-0.87 (m, 6H, Hv); δC (100 MHz, CDCl3) 178.8 (Cj), 171.3 (Cr), 153.1, 150,2, 142.2, 141.4, 136.2, 129.9, 129.6, 129.3, 128.3, 127.3, 122.9 (Ca, Cc, Ce-Ci, Cn-Cq), 119.3 (Cd), 111.3 (Cb), 54.6, 54.1 (Ck, Cs), 44.5, 33.0, 25.8, 24.3, 22.2 (Cm-Cl, Ct-Cv); ESI-IT MS: m/z (M+H+) 453.27 (C25H29ClN4O2 requires 452.20). 2.2.1.3. Synthesis of compounds 1a-l and 1a’ (HEDICINs) The relevant cinnamic acid (1.1 eq), PyBOP (1.1 eq), DIEA (2 eq.) and DCM (2 mL) were mixed in a round bottom flask and put under stirring for 20 min. Then, a solution of 7 in DCM (2 mL) was added and the reaction allowed to proceed for three days. Precipitation was observed and the precipitate was collected by suction filtration, washed with ice-cold DCM, dried and identified as the pure target compound, except in the case of 1l (pnitrocinnamic acid derivative), which was further submitted to liquid chromatography on silica, using DCM/Me2CO 6:1 (v/v) as eluent. In the particular case of compound 1e, derived from p-aminocinnamic acid, the N-Boc-protected precursor of this cinnamic acid derivative was coupled to 7 as described, and the resulting Boc-protected compound (1e’) was treated with neat TFA as described for the synthesis of 5 and 7, to give the final unprotected compound, 1e. Analytical and spectroscopic data for HEDICINs are given below. 4-{(2R)-2-[(2R)-2-(cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1a) White solid (29.5 mg, 66%); mp 241-245 oC; RF (DCM/Me2CO 6:1) 0.36; δH (400 MHz, DMSO-d6) 10.36 (s, 1H, -NH-QN), 8.84 (d, J=5.2 Hz, 1H, Ha), 8.59 (d, J=7.6 Hz, 1H, He), 8.37-8.32 (m, 2H, -NH-Leu, -NHhPhe), 8.05-8.04 (m, 2H, Hb, Hh), 7.67 (dd, J= 9.28 Hz, J= 2 Hz, 1H, Hf), 7.56 (d, J=7.2 Hz, 2H), 7.46-7.34 (m, 4H) (Ha’-Hc’, Hy), 7.29-7.15 (m, 5H, Ho-Hq); 6.79 (d, J= 15.6 Hz, 1H, Hx); 4.72-4.67 (m, 1H), 4.58 (q, J= 7.6 Hz, 1H) (Hk, Hs), 2.85-2.60 (m, 2H, Hm), 2.19-2.01 (m, 2H, Hl), 1.72-1.61 (m, 1H, Hu), 1.61-1.51 (m, 2H, Ht), 0.90 (dd, J= 16 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6); 172.7, 171.8, 164.9 (Cj, Cr, Cw), 152.2, 149.1, 141.5, 141.1, 139.0, 134.8, 134.2, 129.5, 128.9, 128.3, 128.3, 128.0, 127.5, 126.4, 125.9, 124.5, 121.9, 119.4, 112.4 (Ca-Ci, Cn-Cq, Cx-Cc’), 53.5, 51.1 (Ck, Cs), Chapter 2 | 85 40.9, 33.0, 31.5, 24.2, 23.0, 21.6 (Cl-Cm, Ct-Cv); ESI-IT MS: m/z (M+H+) 583.47 (C34H35ClN4O3 requires 582.24); HPLC-DAD: rt = 12.4 min (% Area = 96%). 4-{(2R)-2-[(2R)-2-((p-methyl)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1b) White solid (30.1 mg, 76%); mp 230-235 oC; RF (DCM/Me2CO 6:1) 0.24; δH (400 MHz, DMSO-d6) 10.35 (s, 1H, -NH-QN), 8.83 (d, J= 5.2 Hz, 1H, Ha), 8.55 (d, J= 7.6 Hz, 1H, He), 8.36-8.26 (m, 2H, -NHLeu, -NH-hPhe), 8.06-8.04 (m, 2H, Hb, Hh), 7.66 (dd, J= 9 Hz, J= 2 Hz, 1H, Hf), 7.46-7.35 (m, 3H, Ha’,Hy), 7.31-7.10 (m, 7H, Ho-q, Hb’), 6.75 (d, J= 16 Hz, 1H, Hx), 4.72-4.67 (m, 1H), 4.57 (q, J= 7.7, 1H) (Hk, Hs), 2.81-2.62 (m, 2H, Hm), 2.32 (s, 3H, Hd’), 2.20-2.00 (m, 2H, Hl), 1.74-1.50 (m, 3H, Ht-u), 0.90 (dd, J= 16 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.7, 171.7, 165.0 (Cj, Cr, Cw), 152.2, 149.0, 141.4, 141.0, 139.2, 134.9, 134.1, 132.0, 129.5, 128.3, 128.2, 127.9, 127.4, 126.3, 125.8, 124.5, 120.8, 119.3, 112.4 (Ca-Ci, Cn-Cq, Cx-Cc’), 53.4, 51.6 (Ck, Cs), 40.8, 32.9, 31.4, 24.8, 22.9, 21.6, 20.9 (Cl-Cm, Ct-Cv, Cd’); ESI-IT MS: m/z (M+H+) 597.60 (C35H37ClN4O3 requires 596.26); HPLC-DAD: rt = 18.3 min (% Area = 98%). 4-{(2R)-2-[(2R)-2-((p-isopropyl)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1c) White solid (23 mg, 47%); mp 238-240 oC; RF (DCM/Me2CO 6:1) 0.44; δH (400 MHz, DMSO-d6) 10.34 (s, 1H, -NH-QN), 8.83 (d. J= 5.2 Hz, 1H, Ha), 8.56 (d, J= 6.8 Hz, 1H, He), 8.36-8.26 (m, 2H, -NHLeu, -NH-hPhe), 8.06-8.04 (m, 2H, Hb, Hh), 7.67 (d, J= 8.8 Hz, 1H, Hf), 7.507.38 (m, 4H, Ha’-b’), 7.30-7.23 (m, 6H, Ho-q, Hy), 6.73 (d, J= 16 Hz, 1H, Hx), 4.72-4.67 (m, 2H, Hk, Hs), 2.97-2.62 (m, 3H, Hm, Hd’), 2.20-2.00 (m, 2H, Hl), 1.77-1.51 (m, 3H, Htu), 1.20 (d, J= 6.8 Hz, 6H, He’), 0.90 (dd, J= 16.6 Hz, J= 6.4 Hz, 6H); δC (100 MHz, DMSO-d6) 172.7, 171.7, 165.0 (Cj, Cr, Cw), 152.1, 150.0, 149.0, 141.4, 141.0, 138.9, 134.1, 132.4, 128.3, 128.2, 127.9, 127.5, 126.8, 126.3, 125.8, 124.5, 120.9, 119.36, 112.4 (Ca-Ci, Cn-Cq, Cx-Cc’), 53.4, 51.0 (Ck, Cs), 40.8, 33.2, 33.0, 31.4, 24.8, 23.6, 86 | Chapter 2 ! 22.9, 21.6 (Cl-Cm, Ct-Cv, Cd’-Ce’); ESI-IT MS: m/z (M+H+) 625.36 (C37H41ClN4O3 requires 624.29); HPLC-DAD: rt = 14.5 min (% Area = 93%). 4-{(2R)-2-[(2R)-2-((p-methoxy)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1d) White solid (38 mg, 47%); mp 223-241 oC; RF (DCM/Me2CO 6:1) 0.35; δH (400 MHz, DMSO-d6) 10.34 (s, 1H, -NH-QN), 8.83 (d, J= 5.2 Hz, 1H, Ha), 8.54 (d, J= 7.6 Hz, 1H, He), 8.32 (d, J= 8.8 Hz, 1H), 8.24 (d, J= 8 Hz, 1H) (-NH-Leu, -NH-hPhe), 8.068.04 (m, 2H, Hb, Hh), 7.66 (d, J= 9.2 Hz, J=2 Hz, 1H, Hf), 7.50 (d, J= 8.8 Hz, 2H, Ha’), 7.38 (d, J= 16 Hz, 1H, Hy), 7.29-7.15 (m, 5H, Ho-q), 6.98 (d, J= 8.8 Hz, 2H, Hb’), 6.63 (d, J= 15.6 Hz, 1H, Hx), 4.72-4.68 (m, 1H), 4.61-4.52 (m, 1H) (Hk, Hs), 3.79 (s, 3H, OCH3), 2.85-2.62 (m, 2H, Hm), 2.20-2.00 (m, 2H, Hl), 1.77-1.51 (m, 3H, Ht-u), 0.90 (dd, J= 16.8 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.8, 171.7, 165.2 (Cj, Cr, Cw), 160.3, 152.1, 149.0, 141.4, 141.0, 138.7, 134.1, 129.0, 128.3, 128.2, 127.9, 127.3, 126.3, 125.8, 124.5, 120.4, 119.4, 114.3, 112.4 (Ca-Ci, Cn-Cq, Cx-Cc’), 55.2, 53.4, 51.0 (Ck, Cs, Cd’), 40.8, 33.0, 31.4, 24.2, 22.9, 21.6 (Cl-Cm, Ct-Cv); ESI-IT MS: m/z (M+H+) 613.73 (C35H37ClN4O4 requires 612.25); HPLCDAD: rt = 12.3 min (% Area = 91%). 4-{(2R)-2-[(2R)-2-((p-(tert-butoxycarbonyl)amino)cinnamoyl)amino-4methylpentanoyl]amino-4-phenylbutanoyl}amino-7-chloroquinoline (1e’) Yellow solid (10 mg, 13%); mp (dec.) 238 oC; RF (DCM/Me2CO 6:1) 0.35; δH (400 MHz, DMSOd6) 10.33 (s, 1H, -NH-QN), 9.55 (s, 1H, -NHBoc), 8.83 (d, J= 4.8 Hz, 1H, Ha), 8.53 (d, J= 7.2 Hz, 1H, He), 8.32 (d, J= 9.2 Hz, 1H), 8.26 (d, J= 8 Hz, 1H) (-NHLeu, -NH-hPhe), 8.06-8.04 (m, 2H, Hb, Hh), 7.66 (dd, J= 9.2 Hz, J= 2 Hz, 1H, Hf), 7.507.38 (m, 4H, Ha’-Hb’), 7.34 (d, J= 16 Hz, 1H, Hy), 7.23 (m, 5H, Ho-q), 6.65 (d, J= 15.6 Hz, 1H, Hx), 4.72-4.68 (m, 1H), 4.61-4.52 (m, 1H) (Hk, Hs), 2.85-2.62 (m, 2H, Hm), 2.202.00 (m, 2H, Hl), 1.77-1.51 (m, 3H, Ht-u), 1.48 (s, 9H, Hf’), 0.90 (dd, J= 17.2 Hz, J= 6.4 Hz, 6H, Hv’); δC (100 MHz, DMSOd6) 172.8, 171.8, 165.2 (Cj, Cr, Cw), 152.5, 152.2, Chapter 2 | 87 149.0, 141.4, 141.0, 140.8, 138.7, 134.1, 128.5, 128.3, 128.2, 128.1, 127.1, 126.4, 125.9, 124.5, 119.7, 119.4, 118.0, 112.0 (Ca-Ci, Cn-Cq, Cx-Cd’), 79.3 (Ce’), 53.4, 51.1 (Ck, Cs), 40.7, 33.0, 31.4, 28.0, 24.2, 22.9, 21.6 (Cl-Cm, Ct-Cv, Cf’); ESIIT MS: m/z (M+H+) 698.47 (C39H44ClN5O5 requires 697.30); HPLCDAD: rt = 16.5 min (% Area = 97%). 4-{(2R)-2-[(2R)-2-((p-amino)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1e) Yellow solid (12 mg, 78%); mp 240242 oC; RF (DCM/Me2CO 6:1) 0.11; δH (400 MHz, DMSO-d6) 10.32 (s, 1H, - NH-QN), 8.83 (d, J= 5.2 Hz, 1H, Ha), 8.49 (d, J= 7.6 Hz, 1H, He), 8.32 (d, J= 9.2 Hz, 1H), 8.08 (d, J= 8 Hz, 1H) (-NH-Leu, -NH-hPhe), 8.06-8.04 (m, 2H, Hb, Hh), 7.66 (dd, J= 9 Hz, J=2 Hz, 1H, Hf), 7.29-7.16 (m, 8H, Ho-q, Hy, Ha’), 6.56 (d, J= 8.4 Hz, 2H, Hb’), 6.43 (d, J= 16 Hz, 1H, Hx), 5.57 (s, 2H, -NH2), 4.71-4.65 (m, 1H), 4.55-4.49 (m, 1H) (Hk, Hs), 2.80-2.59 (m, 2H, Hm), 2.20-2.00 (m, 2H, Hl), 1.75-1.50 (m, 3H, Ht-u), 0.90 (dd, J= 18.4 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.9, 171.8, 165.9 (Cj, Cr, Cw), 152.1, 150.5, 149.0, 141.4, 141.1, 139.9, 134.1, 129.0, 128.3, 128.2, 127.9, 126.4, 125.8, 124.5, 122.0, 119.4, 115.4, 113.6, 112.4 (Ca-Ci, Cn-Cq, Cx-Ca’, Cc’-Cd’), 53.4, 51.2 (Ck, Cs), 40.7, 33.0, 31.4, 24.2, 22.9, 21.6 (Cl-Cm, Ct-Cv); ESI-IT MS: m/z (M+H+) 598.30 (C34H36ClN5O3 requires 597.25); HPLC-DAD: rt =16.5 min (% Area = 97%). 4-{(2R)-2-[(2R)-2-((m-fluoro)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1f) White solid (28 mg, 56%); mp 249-254 oC; RF (DCM/Me2CO 6:1) 0.35; δH (400 MHz, DMSO-d6) 10.36 (s, 1H, -NHQN), 8.83 (d, J= 4.8 Hz, 1H, Ha), 8.58 (d, J= 7.2 Hz, 1H, He), 8.37-8.30 (m, 2H, -NH-Leu, -NH-hPhe), 8.06-8.05 (m, 2H, Hb, Hh), 7.67 (d, J= 9.2 Hz, J= 2 Hz 1H, Hf), 7.50-7.38 (m, 4H, Ha’- d’), 7.31-7.15 (m, 6H, Ho-q, Hy), 6.83 (d, J= 16 Hz, 1H, Hx), 4.73-4.68 (m, 1H), 4.63-4.56 (m, 1H) (Hs, Hk), 2.82-2.63 (m, 2H, Hm), 2.20-2.00 (m, 2H, Hl), 1.75-1.52 (m, 3H, Ht-u), 88 | Chapter 2 ! 0.90 (dd, J= 15.4 Hz, J= 6.8 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.6, 171.7, 164.5, (Cj, Cr, Cw), 163.6, 161.1, 152.2, 149.0, 141.4, 141.0, 137.6, 137.4, 134.1, 130.9, 128.3, 128.2, 127.9, 126.3, 125.8, 124.5, 123.5, 119.4, 116.1, 113.8, 112.4 (Ca-Ci, Cn-Cq, CxCe’), 53.5, 51.1 (Ck, Cs), 40.8, 33.0, 31.5, 24.2, 22.9, 21.6 (Cl-Cm, Ct-Cv); ESI-IT MS: m/z (M+H+) 601.40 (C34H34ClFN4O3 requires 600.23); HPLC-DAD: rt = 11.2 min (% Area = 100%). 4-{(2R)-2-[(2R)-2-((p-fluoro)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1g) White solid (39 mg, 78%); mp 246-249 oC; RF (DCM/Me2CO 6:1) 0.35; δH (400 MHz, DMSOd6) 10.35 (s, 1H, -NH-QN), 8.83 (d, J= 5.2 Hz, 1H, Ha), 8.56 (d, J= 7.2 Hz, 1H, He), 8.32 (d, J= 9.2 Hz, 2H, -NH-Leu, -NHhPhe), 8.06-8.04 (m, 2H, Hb, Hh), 7.67-7.60 (m, 3H, Ha’, Hf), 7.44 (d, J= 15.6 Hz, 1H, Hy), 7.29-7.14 (m, 7H, Ho-q, Hb’), 6.73 (d, J= 15.6 Hz, 1H, Hx), 4.73-4.68 (m, 1H), 4.61-4.55 (m, 1H) (Hk, Hs), 2.822.62 (m, 2H, Hm), 2.20-2.00 (m, 2H, Hl), 1.75-1.52 (m, 3H, Ht-u), 0.90 (dd, J= 16 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.7, 171.7, 164.8 (Cj, Cr, Cw), 152.2, 149.0, 141.4, 141.0, 137.8, 134.1, 131.4, 129.6, 128.3, 128.2, 127.9, 126.3, 125.8, 124.5, 121.8, 119.4, 116.0, 115.7, 112.4 (Ca-Ci, Cn-Cq, Cx-Cc’), 53.5, 51.0 (Ck, Cs), 40.8, 33.0, 31.5, 24.2, 22.9, 21.6 (Cl-Cm, Ct-Cv); ESI-IT MS: m/z (M+H+) 601.47 (C34H34ClFN4O3 requires 600.23); HPLC-DAD: rt= 12.5 min (% Area = 85%). 4-{(2R)-2-[(2R)-2-((p-chloro)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1h) White solid (25 mg, 59%); mp 249-260 oC; RF (DCM/Me2CO 6:1) 0.35; δH (400 MHz, DMSO-d6) 10.35 (s, 1H, -NHQN), 8.83 (d, J= 4.8 Hz, 1H, Ha), 8.57 (d, J= 7.6 Hz, 1H, He), 8.34-8.30 (m, 2H, -NHLeu, -NH-hPhe), 8.06-8.04 (m, 2H, Hb, Hh), 7.66 (dd, J= 9 Hz, J=2, 1H, Hf), 7.58 (d, J= 8.8 Hz, 2H), 7.48 (d, J= 8.4 Hz, Chapter 2 | 89 2H) (Ha’-b’), 7.42 (d, J= 16 Hz, 1H, Hy), 7.22 (m, 5H, Ho-q), 6.79 (d, J= 15.6 Hz, 1H, Hx), 4.73-4.68 (m, 1H), 4.61-4.55 (m, 1H) (Hk, Hs), 2.82-2.62 (m, 2H, Hm), 2.20-2.00 (m, 2H, Hl), 1.75-1.52 (m, 3H, Ht-u), 0.90 (dd, J= 16 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.6, 171.7, 164.7 (Cj, Cr, Cw), 152.2, 149.0, 141.4, 141.0, 137.6, 134.1, 133.8, 133.7, 129.1, 128.9, 128.3, 128.2, 127.9, 126.3, 125.8, 124.5, 122.7, 119.4, 112.4 (Ca-Ci, Cn-Cq, Cx-Cc’), 53.5, 51.1 (Ck, Cs), 40.8, 33.0, 31.5, 24.2, 22.9, 21.6 (Cl-Cm, Ct-Cv); ESI-IT MS: m/z (M+H+) 617.23 (C34H34Cl2N4O3 requires 616.20); HPLC-DAD: rt = 18.5 min (% Area = 100%) 4-{(2R)-2-[(2R)-2-((p-bromo)cinnamoyl)amino-4-methylpentanoyl]amino-4phenylbutanoyl}amino-7-chloroquinoline (1i) White solid (31 mg, 70%); mp 240-246 oC; RF (DCM/Me2CO 6:1) 0.35; δH (400 MHz, DMSOd6) 10.35 (s, 1H, -NH-QN), 8.83 (d, J= 4.8 Hz, 1H, Ha), 8.57 (d, J= 7.2 Hz, 1H, He), 8.39-8.31 (m, 2H, -NH-Leu, -NH-hPhe), 8.068.04 (m, 2H, Hb, Hh), 7.71-7.61 (m, 3H), 7.51 (d, J= 8.4 Hz, 2H) (Ha’-b’, Hf), 7.40 (d, J= 15.6 Hz, 1H, Hy), 7.31-7.14 (m, 5H, Ho-q), 6.80 (d, J= 16 Hz, 1H, Hx), 4.73-4.68 (m, 1H), 4.61-4.55 (m, 1H) (Hk, Hs), 2.82-2.62 (m, 2H, Hm), 2.20-2.00 (m, 2H, Hl), 1.74-1.49 (m, 3H, Ht-v), 0.90 (dd, J= 16 Hz, J= 6.4 Hz, 6H, Hv); δC (100 MHz, DMSO-d6) 172.6, 171.7, 164.6 (Cj, Cr, Cw), 152.2, 149.0, 141.4, 141.0, 137.7, 134.1, 131.8, 129.4, 128.3, 128.2, 127.9, 126.3, 125.8, 124.5, 122.8, 122.6, 119.4, 112.4 (Ca-Ci, Cn-Cq, CxCc’), 53.4, 51.1 (Ck, Cs), 40.8, 33.0, 31.4, 24.2, 22.9, 21.6 (Ct-Cv); ESI-IT MS: m/z (M+H+) 663.40 (C34H34BrClN4O3 requires 660.15); HPLC-DAD: rt = 18.6 min (% Area = 98%). 96 | Chapter 2 ! 4-[(p-chloro)cinnamoyl]amino-7-chloroquinoline (2g) White solid (44 mg, 21%); mp 240-250 oC; RF (DCM/Me2CO 3:1) 0.56; δH (400 MHz, DMSO-d6) 10.44 (s, 1H, -NH-QN), 8.85 (d, J= 5.2 Hz, 1H, Ha), 8.47 (d, J= 9.2 Hz, 1H, He), 8.32 (d, J= 4.8 Hz, 1H, Hb), 8.06 (d, J= 2 Hz, 1H, Hh), 7.72 (m, 4H, Hf, Hl, Hn), 7.55 (d, J= 8.4 Hz, 2H, Ho), 7.26 (d, J= 16 Hz, 1H, Hk); δC (100 MHz, DMSO-d6) 164.6 (Cj), 152.2, 149.1, 141.7, 140.5, 134.5, 134.1, 133.5, 129.5, 129.1, 128.0, 126.3, 124.3, 122.3, 119.0, 111.6 (Ca-Ci, Ck-Cp); ESI-IT MS: m/z (M+H+) 343.33 (C18H12Cl2N2O requires 342.03); HPLC-DAD: rt = 8.30 min (% Area = 96%). 4-[(p-bromo)cinnamoyl]amino-7-chloroquinoline (2h) Green solid (38 mg, 7%); mp (dec.) 190 oC; RF (DCM/Me2CO 3:1) 0.56; δH (400 MHz, DMSO-d6) 10.44 (s, 1H, -NH-QN), 8.85 (d, J= 5.2 Hz, 1H, Ha), 8.47 (d, J= 8.8Hz, 1H, He), 8.32 (d, J=4.8 Hz, 1H, Hb), 8.08 (d, J= 2.4 Hz,1H, Hh), 7.74-7.70 (m, 6H, Hf, Hl, Hn-o), 7.27 (d, J= 16 Hz, 1H, Hk); δC (100 MHz, DMSOd6) 164.6 (Cj), 152.2, 149.1, 141.7, 140.5, 134.1, 133.8, 132.0, 129.8, 128.0, 126.3, 124.3, 123.3, 122.4, 118.9, 111.6 (Ca-Ci, Ck-Cp); ESI-IT MS: m/z (M+H+) 387.27 (C18H12BrClN2O requires 385.98); HPLC-DAD: rt = 8.60 min (% Area = 97%). 4-[(o-nitro)cinnamoyl]amino-7-chloroquinoline (2i) Yellow solid (36 mg, 7%); mp 168-200 oC; RF (DCM/Me2CO 3:1) 0.56; δH (400MHz,DMSO-d6) 10.56 (s, 1H, -NH-QN), 8.87 (d, J= 5.2 Hz, 1H, Ha), 8.47 (d, J= 8.8 Hz, 1H, He), 8.32 (d, J= 4.8 Hz, 1H, Hb), 8.12 (d, J= 8 Hz, 1H, Hq), 8.07 (d, J= 2 Hz, 1H, Hh), 7.99 (d, J= 15.6 Hz, 1H, Hl), 7.887.86 (m, 2H), 7.76-7.69 (m, 2H, Hf, Hn-p), 7.25 (d, J= 15.6 Hz, 1H, Hk); δC (100 MHz, DMSO-d6) 163.9 (Cj), 152.2, 149.0, 148.2, 141.4, 137.0, 134.1, 133.9, 130.7, 129.8, 128.8, 128.0, 126.4, 126.1, 124.8, 124.3, 119.0, 111.7 (Ca-Ci, Ck-Cr); ESI-IT MS: m/z Chapter 2 | 97 (M+H+) 354.33 (C18H12ClN3O3 requires 353.06); HPLC-DAD: trt = 7.90 min (% Area= 97%). 4-[(m-nitro)cinnamoyl]amino-7-chloroquinoline (2j) White solid (11 mg, 2%); mp (dec.) 240-247 oC; RF (DCM/Me2CO 3:1) 0.56; δH (400 MHz, DMSO-d6) 10.49 (s, 1H, -NH-QN), 8.86 (d, J= 4.8 Hz, 1H, Ha), 8.53 (m, 1H, Hr), 8.49 (d, J= 9.2 Hz, He), 8.34 (d, J= 5.2Hz, 1H, Hb), 8.27 (dd, J= 8.2 Hz, J= 1.2 Hz, 1H, Hp), 8.11 (d, J= 8 Hz, 1H, Hn), 8.07 (d, J= 2 Hz, 1H, Hh), 7.84 (d, J= 16 Hz, 1H, Hl), 7.80-7.73 (m, 2H, Hf, Ho), 7.46 (d, J= 16 Hz, 1H, Hk); δC (100 MHz, DMSO-d6) 164.2 (Cj), 152.2, 149.1, 148.2, 141.5, 139.4, 136.3, 134.5, 134.2, 130.6, 128.0, 126.4, 124.4, 124.3, 121.5, 118.9, 111.5 (Ca-Ci, Ck-Cr); ESIIT MS: m/z (M+H+) 354.53 (C18H12ClN3O3 requires 353.06); HPLC-DAD: rt = 7.20 min (% Area = 100%). 4-[(p-nitro)cinnamoyl]amino-7-chloroquinoline (2k) Beige solid (9 mg, 5%); mp desc. 205 oC; RF (DCM/Me2CO 3:1) 0.56; δH (400 MHz, DMSO-d6) 10.58 (s, 1H, -NH-QN), 8.87 (d, J= 5.2 Hz, 1H, Ha), 8.48 (d, J= 9.2 Hz, 1H, He), 8.35-8.32 (m, 3H, Hb, Ho), 8.07 (d, J= 2.4 Hz, 1H, Hh), 7.94 (d, J= 8.8 Hz, 2H, Hn), 7.83 (d, J= 16 Hz, 1H, Hl), 7.74 (dd, J= 9 Hz, J= 2 Hz, 1H, Hf), 7.44 (d, J= 15.6 Hz, 1H, Hk); δC (100 MHz, DMSO-d6) 164.1 (Cj), 152.2, 149.1, 147.8, 141.5, 141.0, 139.3, 134.2, 128.9, 128.0, 126.4, 125.8, 124.3, 124.2, 119.0, 111.7 (Ca-CI, Ck-Cp); ESI-IT MS: m/z (M+H+) 354.27 (C18H12ClN3O3 requires 353.06); HPLC-DAD: rt = 7.20 min (% Area= 92%). ! 98 | Chapter 2 ! 2.2.2. In vitro assessment of inhibition of β-H formation The inhibition of β-H formation assay was performed as previously described.7, 8 Briefly, in a 96-microwell plate it was respectively added i) 50 µL of different concentrations (0.11 mM) of test compounds dissolved in DMSO, in triplicate, and ii) 50 µL hemin chloride dissolved in DMSO (5.2 mg/mL). Controls contained equal volumes of water or DMSO. β-H formation was initiated by the addition of acetate buffer 0.2 M (100 µL, pH 4.4), following plates were incubated at 37 oC for 48 h, and subsequently, they were centrifuged at 3000 rpm for 15 min (SIGMA 3-30K). After discarding the supernatant, the pellet was washed four times with DMSO (3×200 µL), and finally dissolved in 0.2 M aq. NaOH (200 µL). The solubilized aggregates were further diluted 1:6 with 0.1 M aq. NaOH and absorbances recorded at 405 nm on a Biotek Powerwave XS with software Gen5 1.07. Chapter 2 | 99 2.3. References ! 1. Hong, S. Y.; Oh, J. E.; Lee, K.-H. Effect of d-amino acid substitution on the stability, the secondary structure, and the activity of membrane-active peptide. Biochem Pharmacol 1999, 58, 1775-1780. 2. Müller-Schiffmann, A.; Petsch, B.; Leliveld, S. R.; Muyrers, J.; Salwierz, A.; Mangels, C.; Schwarzinger, S.; Riesner, D.; Stitz, L.; Korth, C. Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity. Mol Immunol 2009, 46, 532-540. 3. Price, C. C.; Leonard, N. J.; Peel, E. W.; Reitsema, R. H. Some 4-Amino-7chloroquinoline Derivatives1. J Am Chem Soc 1946, 68, 1807-1808. 4. Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.; Kates, S. A. On the Use of PyAOP, a phosphonium salt derived from HOAt, in Solid-Phase Peptide Synthesis. Tetrahedron Lett 1997, 38, 4853-4856. 5. Han, S.-Y.; Kim, Y.-A. Recent development of peptide coupling reagents in organic synthesis. Tetrahedron 2004, 60, 2447-2467. 6. Lassen, M. J. K. Evolution of amide bond formation. ARKIVOC 2010, 8. 7. Baelmans, R.; Deharo, E.; Munoz, V.; Sauvain, M.; Ginsburg, H. Experimental conditions for testing the inhibitory activity of chloroquine on the formation of beta-hematin. Exp Parasitol 2000, 96, 243-8. 8. Barazarte, A.; Lobo, G.; Gamboa, N.; Rodrigues, J. R.; Capparelli, M. V.; AlvarezLarena, A.; Lopez, S. E.; Charris, J. E. Synthesis and antimalarial activity of pyrazolo and pyrimido benzothiazine dioxide derivatives. Eur J Med Chem 2009, 44, 1303-10. 9. Shenai, B. R.; Sijwali, P. S.; Singh, A.; Rosenthal, P. J. Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum. J Biol Chem 2000, 275, 29000-10. 10. Sijwali, P. S.; Shenai, B. R.; Gut, J.; Singh, A.; Rosenthal, P. J. Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3. Biochem J 2001, 360, 481-9. 11. Vale, N.; Prudêncio, M.; Marques, C. A.; Collins, M. S.; Gut, J.; Nogueira, F.; Matos, J.; Rosenthal, P. J.; Cushion, M. T.; do Rosário, V. E.; Mota, M. M.; Moreira, R.; Gomes, P. Imidazoquines as antimalarial and antipneumocystis agents. J Med Chem 2009, 52, 7800-7. 12. ChemAxon. MarvinSketch 5.2.2. http://www.chemaxon.com. 2009. 100 | Chapter 2 ! 13. Lavrado, J.; Gani, K.; Nobre, P. A.; Santos, S. A.; Figueiredo, P.; Lopes, D.; Rosário, V.; Gut, J.; Rosenthal, P. J.; Moreira, R.; Paulo, A. Bis-alkylamine quindolone derivatives as new antimalarial leads. Bioorg Med Chem Lett 2010, 20, 5634-7. 14. Sandlin, R. D.; Carter, M. D.; Lee, P. J.; Auschwitz, J. M.; Leed, S. E.; Johnson, J. D.; Wright, D. W. Use of the NP-40 detergent-mediated assay in discovery of inhibitors of beta-hematin crystallization. Antimicrob Agents Ch 2011, 55, 3363-9. 15. Sabnis, Y. A.; Desai, P. V.; Rosenthal, P. J.; Avery, M. A. Probing the structure of falcipain-3, a cysteine protease from Plasmodium falciparum: comparative protein modeling and docking studies. Protein Sci 2003, 12, 501-9. 16. Lin, A. J.; Miller, R. E. Antimalarial Activity of New Dihydroartemisinin Derivatives. 6. .alpha.-Alkylbenzylic Ethers. J Med Chem 1995, 38, 764-770. Chapter 3 HE[DI]CINs against a malaria-like disease Chapter 3 | 103 3.0. Preamble HEDICINs and HECINs were also evaluated against another parasitic cysteine protease, babesipain-1 from Babesia bigemina, one of the causing agents of babesiosis, a malarialike disease that mainly affects cattle and represents a huge economic burden in the cattle industry.1, 2 In vitro evaluation of babesipain-1 inhibition by HEDICINs and HECINs was carried out by Dr. Ana Domingos’s team at the Instituto de Higiene e Medicina Tropical – Universidade Nova de Lisboa. The doctoral candidate performed a parallel study in silico, in order to evaluate the interaction of HEDICINs and HECINs with babesipain-1 at the atomic level bbspn-1. As such, and given that there was no threedimensional (3D) structure available for babesipain-1, a homology model was built and validated, followed by the respective molecular docking of the ligands. 3.1. Babesiosis and Babesia parasites – brief overview Babesiosis is a worldwide distributed disease caused by protozoans of the genus Babesia, which is transmitted by Rhipicephalus (Boophilus) sp. ticks.2 The symptoms of babesiosis included fever, anemia, hemoglobinuria, malaise, and anorexia.2 There are several species which cause babesiosis, but the two species which cause more economical impact are Babesia bovis and Babesia bigemina.2 The reproductive life cycle of Babesia is similar to that of Plasmodia parasites. The sporogony and the gamogony takes places in the invertebrate host while merogony, the asexual dividing stage, occurs within the erythrocyte of the human host (Fig. 22).3, 4 Vaccines containing live attenuated parasites are the main strategy to control this disease but failures of protective immunity provided by vaccination have been observed since these vaccines do not offer cross protection against the different variants in the field.5, 6 Moreover, chemoprophylaxis is used as short-term protection during epidemics when cattle are being relocated, or to treat pregnant cows. Some examples of drugs used to treat babesiosis are diminazene aceturate (XXVIII) or imidocarb (XXIX), and in clinical cases clindamycin (XXX) and quinine (XXXI), the latter two are also known to treat against malaria.4 Nonetheless, there is the need for a more specific, fast acting, and safe chemotherapy to combat the disease.7 104 | Chapter 3 ! Figure 22. Babesia life cycle: sporozoites (Sz) differentiate into trophozoites (T) when they enter into the vertebrate host’s blood stream. Later, T asexually divide into merozoites (M) which exit the erythrocyte and invade other cells and continue to replicate. Some M stop division and transform into pre-gametocytes (G). When G are taken into the invertebrate host by a tick feeding, they differentiate into Strahlenkörper (Sk); Sk fuse and form zigotes (Z), which undergo meiosis given rises to kinetes (Ts and To). To represents kinetes which go into the tick’s ovaries and eggs, whereas Ts represents kinetes which transform in the salivary glands (Sg) of the tick into Sz ready to infect the next vertebrate host.2 Chapter 3 | 105 Like Plasmodia, B. bigemina is a protozoan of the phylum Apicomplexa.4 The sequencing of the genome of B. bigemina is a project in active progress8 and the information obtained to this point has revealed potential targets that may be useful for the development of new chemotherapy strategies against babesiosis. For instance, three cysteine proteases from B. bigemina, BbiCPL1 to BbiCPL3, have been identified which belong to the same cysteine protease family and share many features with papain.9 These features include: i)!20–23 amino acid putative transmembrane domain, ii) the ERFNIN and GNFD pro-sequence motifs typical of cathepsin L-like cysteine proteases, iii) the main catalytic residues, for instance, histidine, cysteine and asparagine, and iv) six cysteine residues predicted to form disulfide bonds in the mature domain of the protease.9 BbiCPL1 (bbspn-1) is the first cysteine protease from a piroplasm described to possess proteolytic activity.9 It is believed to act in the cytosol at an optimum acidic pH of 5.5, and it is inhibited by leupeptin and E64,7, 9 known inhibitors of cysteine proteases. In this context and based on the fact that i) the roles of cysteine proteases in protozoan parasites are often associated with cell invasion and rupture10, and ii) it has been found that modest falcipain inhibitors11 are more potent inhibitors of bbspn-1 than of FP2,1 some HEDICINs and HECINs were further evaluated against bbspn-1. 112 | Chapter 3 ! ! Figure 24. Multiple sequence alignment performed with PSI-coffee mode of T-Coffee of the amino acid sequence of bbspn-1 and the selected templates. The boxes indicate the Cys25, His155, and Asn177 catalytic regions, correspondingly. The arrows show the catalytic residues glutamine (Gln19), cysteine (Cys25), histidine (His155) and asparagine (Asn177), respectively. Chapter 3 | 113 All these described characteristics make the selected 3D structures suitable templates to build the 3D model structure of bbspn-1. Moreover, bbspn-1 will present a higher probability to fold in the same structure of the selected template if it has a percent sequence identity above ~30% and the length of the templates sequence, individually, fall below 250 as compared to the homolog protein (See figure 25).14 In other words, the higher the sequence similarity between the selected 3D template obtained from the PDB and bbspn-1, the more reliable will be the obtained results. However, since BLAST search only resulted proteins with identical residues to bbspn-1 closed to the limit but within the safe zone (Fig. 25), building the 3D structure of bbspn-1 through homology modeling represented a major challenge. ! Figure 25. The safe zone defined for homology modeling.14 iii) Sequence alignments and homology models generation Homology models were built by two different strategies: a) single template: using one available 3D structure homolog to bbspn-1, and b) multiple templates: combining up to three 3D structures homolog to bbspn-1. Selected templates were carefully aligned with bbspn-1 using PSI-coffee.16 Results demonstrated alignment scores, reflecting the expected accuracy of the alignment, up to 93%. Homology modeling calculations were done using Modeller v.9.11 (http://salilab.org/modeller/about_modeller.html) and PSIcoffee alignments. One hundred models were generated for each template combination. As previous studies reported in the literature, Modeller performed best when using combinations of two or three templates as compared to a single template since it can utilize information from multiple templates.19 ! 508 HOMOLOGY MODELING 100 90 80 70 60 50 40 30 20 10 00 50 100 150 Number of aligned residues Safe homology modeling zone Twilight zone Percentage of identical residues 200 250 Figure 25.1. The two zones of sequence alignments. Two sequences are practically guaranteed to fold into the same structure if their length and percentage sequence identity fall into the region marked as ‘‘safe.’’ An example of two sequences with 150 amino acids, 50% of which are identical,isshown(graycross). Imagine that we want to know the structure of sequence A (150 amino acids long, Figure 25.2, steps 1 and 2). We compare sequence A to all the sequences of known structures stored in the PDB (using, for example, BLAST), and luckily find asequenceB(300aminoacidslong)containingaregionof150aminoacidsthat match sequence A with 50% identical residues. As this match (alignment) clearly falls in the safe zone (Fig. 25.1), we can simply take the known structure of sequence B (the template), cut out the fragment corresponding to the aligned region, mutate those amino acids that differ between sequences A and B, and finally arrive at our model for structure A. Structure A is called the target and is of course not known at the time of modeling. In practice, homology modeling is a multistep process that can be summarized in seven steps: 1. Template recognition and initial alignment 2. Alignment correction 3. Backbone generation 4. Loop modeling 5. Side-chain modeling 6. Model optimization 7. Model validation At almost all the steps choices have to be made. The modeler can never be sure to make the best ones, and thus a large part of the modeling process consists of serious thought about how to gamble between multiple seemingly similar choices. A lot of research has been spent on teaching the computer how to make these decisions, so that homology models can be built fully automatically. Currently, this allows modelers to construct models for about 25% of the amino acids in a genome, thereby supplementing the efforts of structural genomics projects (Sanchez and Sali, 1999, Peitsch, Schwede, and Guex, 2000). This average value of 25% differs significantly 114 | Chapter 3 ! iv) Validating the built 3D structure of bbspn-1 The quality assessment basically predicts substantial errors in the built model analyzing whether energy or structural parameters of the built models are comparable to those already reported experimental 3D structures.20 Multiple validations tools were used to assess the built 3D structures of babesipain-1 and enhance the probability to find possible errors non-detected by a single validation tool. For instance, ProSA measurement of model quality highly depends on protein size; the higher the protein size, the lowest the ProSA accuracy. Therefore, using multiple validation scores will suffice for the lack of accuracy of some validation tools. In this work, resulted homology models were assessed not only by Modeller 9.11 Z-score, but also by PROCHECK,21 ProQ,22 QMEAN,20 and ProSA17, 18 (See tables 8-9) and visualization of the biomolecular system of the different models built. In addition, all the structures presenting a Modeller Z-DOPE >-1.00 were disregarded. Accordingly, the best models were expected to present i) >90 % of the amino acids in the most favorable region of the Ramachandran plot as determined by PROCHECK; ii) a Prosa Z-score which agrees with the scores obtained for the PDB structures of the templates used to derive the corresponding model; iii) an LG score > 5 and a Maxsub score > 0.5 as determined by ProQ; and iv) a QMEAN score > 0.75. These validation score restrictions will help in the selection of the most accurate model to evaluate the interaction of HEDICINs and HECINs against bbspn-1. Furthermore, docking calculations of a well-known inhibitor of cysteine protease, namely E64-C, were ran to evaluate whether resulted ligand conformations would allow a possible alkylation of the cysteine residue in the active site of bbspn-1 and the different functionalities of the ligands, respectively, were to place in the corresponding reported subsite of the cavity. Detailed analysis of the results of each validation tool is provided below. Chapter 3 | 115 Table 8. Homology modeling validation scores obtained by Modeller 9.11. Model Alignment Alignment score[a] Modeller (Z-DOPE) 1 1EWM-1S4V-2BDZ_42 91 -1.06 2 1S4V-2BDZ_18 93 -1.06 3 1S4V-2BDZ_54 93 -1.05 4 1EWM-1S4V-2BDZ_51 91 -1.04 5 1EWM-1S4V-2BDZ_76 91 -1.04 6 2BDZ-2P7U_77 89 -1.03 7 1S4V-2BDZ_51 93 -1.01 8 1S4V-2P7U_13 89 -1.01 9 1S4V-2P7U_68 89 -1.00 [a] Alignment score by Psi-coffee. Table 9. Additional scoring parameters of models built and selected according to Modeller 9.11 Z-DOPE score. Model ProQ QMEAN Score PROCHECK (%)[a] RMSD (Å)[b] LGscore MaxSub 1 5.068 0.536 0.773 88.7; 10.2; 1.1; 0.0 1.14; 0.80; 0.64 2 5.118 0.555 0.742 91.4; 7.0;0.0;1.6 0.91; 0.65 3 5.318 0.560 0.706 91.4; 7.5; 0.0; 1.1 0.94; 0.67 4 5.091 0.508 0.720 87.6; 11.3; 0.5; 0.5 1.11; 0.74; 0.61 5 5.358 0.547 0.718 89.8; 8.1; 2.2; 0.0 1.15; 0.80; 0.67 6 5.394 0.575 0.703 82.3; 14.5; 1.6; 1.6 1.08; 0.87 7 5.384 0.565 0.751 90.8; 7.0; 1.1; 1.1; 0.79; 0.71 8 5.391 0.543 0.677 88.7; 10.2; 1.1; 0.0 0.82; 1.10 9 5.297 0.538 0.684 90.3; 8.1; 1.6; 0.0 0.61; 1.10 [a] Correspond to the percentage of residues that are located in the most favorable, additionally allowed, generously allowed, and disallowed regions, respectively according to the Ramachandran Plot. [b] RMSD values of babesipain-1 model compared to the 3D structures of the templates used for the alignment 116 | Chapter 3 ! VMD visualization All models (1-9) built for bbspn-1 were aligned and visualized using the molecular visualization program VMD. Accordingly, most structures were quite similar since the RMSD between the built models oscillated between 0.37 and 1.20. As expected the main differences observed in the binding cavity of bbspn-1 resulted from the phenylalanine residues, specifically, Phe132, Phe67, Phe137. Generally, the rest of the residues in the main cavity displayed a significantly similar conformation. PROCHECK According to PROCHECK results, model 7 has more than 90% of the amino acids in the most favored regions (Table 9) and there are only two amino acids in the disallowed region (Asp199, Arg102) of the Ramachandran diagram (Fig. 26). Analysis of the 3D structure in VMD shows that these amino acids are outside of the binding pocket of bbspn-1. In fact, the closest amino acid, Asp199, is located at ~20 Å of the α−carbon of the catalytic cysteine, falling completely out of the region used for docking HEDICINs and HECINs against the enzyme. Therefore, these amino acids should not have impact on further docking results. Model 5 and model 9 also present ≥ 90% of the amino acids in the most favorable regions but a difference from model 7, they do not present any amino acid in the disallowed regions. However, model 7 was derived from the alignment with the highest PSI-coffee alignment score and it presents the lowest RMSD compared to the original structures 1S4V and 2BDZ, which increases the probability that the sequence of bbspn-1 folds correctly on the template combination used to build the structure. Chapter 3 | 117 ! Figure 26. Ramachandran plot of built 3D structure of bbspn-1 for model 7. The different colored areas indicate ‘‘disallowed’’ (beige), ‘‘generously allowed’’ (yellow), ‘‘additional allowed’’ (brown), and ‘‘most favored’’ (red) regions. ProSA Additionally, ProSA, a diagnostic tool that is based on the statistical analysis of all available PDB structures, was used to reveal whether the built structures presented misfolded areas. This validation score specifically assesses whether the interactions of each residue in the built structure are favorable.23, 24 Accordingly, ProSA Z-score resulted for each experimental structure used are presented in Table 10. Comparison of ProSA Z-scores of experimental results with those obtained for each built model indicates overall good quality of the models since they Z-score are comparable to those obtained for the corresponding used templates. The best results are obtained for models 4, 6, 7, and 9. As example, the overall model quality of model 7 is displayed in fig. 27. Zscores are -7.98, -6.48 and –6.85 for 1S4V (A), model 7 of bbspn-1 (B) and 2BDZ (C), respectively. Results demonstrated that although the Z-score of model 7 is slightly lower than that of template 1S4V, it is in the same range of template 2BDZ and it is a perfect fit within the structures in PDB. 118 | Chapter 3 ! Table 10. ProSA Z-score of experimental structure and build model. Model ProSA[a] Model 1EWM(A) 1S4V(A) 2BDZ(C) 2P7U(A) 1 -6.43 -6.85 -7.98 -6.85 -7.14 2 -6.31 3 -6.20 4 -6.58 5 -6.25 6 -6.60 7 -6.48 8 -6.39 9 -6.64 [a] ProSA determined using ProSA-web. https://prosa.services.came.sbg.ac.at/prosa.php ! Figure 27. ProSA analysis for the model structure of bbspn-1 (B) the template structures, 1S4V (A) and 2BDZ (C). ProQ Models were also validated by ProQ which help to separate correct models from less correct models by finding similar fragments between a built model and a native structure. In other words, ProQ score allows to determine whether protein folds are not compatible with a protein sequence.25 The quality is quantified taking into account indexes LGscore22 and MaxSub.26 A model can be qualified as appropriate if LGscore > 1.5 and MaxSub > 0.1, good if LGscore > 3 and MaxSub > 0.5, and very good if LGscore > 5 and MaxSub > 0.8.27 Accordingly, model 6 and 7 presented the best ProQ Chapter 3 | 119 scores (Table 9). For instance, model 7 was evaluated as very good and good according to the LG score (5.384) and the MaxSub index (0.565), respectively. QMEAN QMEAN score was also used to validate the model. This score corresponds to the global score of the whole model, on the basis of a linear combination of six structural descriptors, reflecting the predicted model reliability ranging from 0 to 1 with higher scores for reliable models.20 In contrast to other validation tools such as ProSA, QMEAN’s quality score is not dependable on protein size. Accordingly, model 1 and 7 presents the best reliability, compared to the rest of the built models, with a global QMEAN score of 0.773 and 0.751, respectively (Table 9). Moreover, the quality of the model can be further compared to reference structures of high resolution obtained from X-ray crystallography analysis through QMEAN Z-score, where a value of 0 is the average value for a good model.20 According to Benkert et al, QMEAN Z-score provides an estimation of the “degrees of nativeness” of the structural features observed in a model and indicates if the model has a quality comparable to experimental structures.20 Based on results, QMEAN Z-score for babesipain-1 model 7 is -0.22 (Fig. 28), which reinforces the good quality of this derived model for babesipain-1. ! Figure 28. Graphical representation of the estimation of the absolute quality of bbspn-1 model 7 assessed by QMEAN Z-score. Good models are generally located in the dark zone. The red cross represents the positioning of model 7. ! Docking known cysteine proteases inhibitors Docking studies were carried using the docking algorithm GOLD28 and the ligand E64-C, a known inhibitor of cysteine proteases. An active site radius of 12 Å centered in the 120 | Chapter 3 ! thiolate moiety of Cys25 was established. Docking calculation were run with flexible ligands and results obtained were compared with crystallographic structure already reported for papain (1PPP).29 Except for results found for model 7 (Fig. 29), most docking conformations resulted for each built model located the electrophilic warhead far away from Cys25, out of the binding site and into S2 and S3 pocket, or the oxygen of the epoxide of E64-C facing the thiolate moiety of Cys25. In the case of model 7, the most frequent ligand conformations place the electrophilic warhead of E64-C, respectively, at 3.7 Å and right over the catalytic cysteine, a distance which could allow a cysteine alkylation to take place. Moreover, the docking conformation found for E64-C against model 7 significantly match the typical binding mode of the ligand against other cysteine protease such as papain (1PPP). Based on all above, model 7 is suitable to evaluate the interaction of HEDICINs and HECINs with bbspn-1 since it was the model that presented the best generally validation score according to PROCHECK, ProSA, ProQ, Qmean, and a known inhibitor of cysteine proteases, E64-C, docked well within the active pocket the corresponding built model. Therefore, the respective docking studies of HEDICINs and HECINs were carried using the 3D structure of model 7. ! ! Figure 29. Most frequent docking conformation observed for E64-C against model 7, respectively. All within 15 Å of Cys25 is represented in MSMS, E64-C is represented in Licorice, and the rest of the protein in NewRibbons (cyan). Detailed description of model 7! The overall topology of bbspn-1 structure is similar to that of other family members as it presents typical papain-like cysteine protease features (Fig. 30-31): Chapter 3 | 121 i) it displays two domains, an α-helix-rich (L) domain and a β-sheet-rich (R) domain, separated by the active site of the protein (Figure 30 (A, C)); the L domain is composed of four helices and the R domain is formed by six antiparallel β-sheets;30 ii) it presents six cysteine residues forming disulfide bonds (Cys22-Cys63, Cys56-Cys95 and Cys148-Cys201) and one cysteine residue (Cys25) in the active site; iii) it shows Cys25 close to His155 and Asn177 which may facilitate the appropriate orientation for the thiolate/imidazolium ion pair (Fig. 30 (B)); iv) it locates Gln19 and Trp179 as to allow the possibility of the formation of the “oxyanion hole” that stabilizes the tetrahedral adduct during the nucleophilic attack of the thiolate anion to the electrophilic warhead of the substrate (Figure 30 (A));18 v) it also demonstrates the presence of a glycine-rich region, comprising mainly of Gly65 and Gly66, that has been found to provide additional stability to the complex in other papain-like cysteine proteases, by forming a constellation of hydrogen bonds with the substrates.31 vi) the binding cavity of papain-like cysteine proteases are generally constituted by four pockets, S1, S1’, S2 and S3, as shown in Figure 31 for bbspn-1. S1 vii) and S1’ contain Gln19 and Trp179, respectively, the amino acids which form the “oxyanion hole,” S2 pocket governs ligand specificity, and S3 pocket contains the glycine rich region of the binding site. 128 | Chapter 3 ! 16. Notredame, C.; Higgins, D. G.; Heringa, J. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 2000, 302, 205-17. 17. Rawlings, N. D.; Barrett, A. J.; Bateman, A. MEROPS: the peptidase database. Nucleic Acids Res 2010, 38, D227-33. 18. Sajid, M.; McKerrow, J. H. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 2002, 120, 1-21. 19. Larsson, P.; Wallner, B.; Lindahl, E.; Elofsson, A. Using multiple templates to improve quality of homology models in automated homology modeling. Protein Sci 2008, 17, 990-1002. 20. Benkert, P.; Biasini, M.; Schwede, T. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 2011, 27, 343-50. 21. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 1993, 26, 283-291. 22. Cristobal, S.; Zemla, A.; Fischer, D.; Rychlewski, L.; Elofsson, A. A study of quality measures for protein threading models. BMC Bioinformatics 2001, 2, 5. 23. Sippl, M. J. Recognition of errors in three-dimensional structures of proteins. Proteins 1993, 17, 355-62. 24. Wiederstein, M.; Sippl, M. J. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 2007, 35, W407-10. 25. Wallner, B.; Elofsson, A. Can correct protein models be identified? Protein Science 2003, 12, 1073-1086. 26. Siew, N.; Elofsson, A.; Rychlewski, L.; Fischer, D. MaxSub: an automated measure for the assessment of protein structure prediction quality. Bioinformatics 2000, 16, 776-85. 27. SBC Stockholm Bioinformatics Center. ProQ - Protein Quality Predictor. http://www.sbc.su.se/~bjornw/ProQ/ProQ.html 28. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and validation of a genetic algorithm for flexible docking. J Mol Biol 1997, 267, 72748. 29. Kim, M. J.; Yamamoto, D.; Matsumoto, K.; Inoue, M.; Ishida, T.; Mizuno, H.; Sumiya, S.; Kitamura, K. Crystal structure of papain-E64-c complex. Binding diversity of E64-c to papain S2 and S3 subsites. Biochem J 1992, 287 ( Pt 3), 797-803. 30. Drenth, J.; Jansonius, J. N.; Koekoek, R.; Swen, H. M.; Wolthers, B. G. Structure of papain. Nature 1968, 218, 929-32. Chapter 3 | 129 31. Brinen, L. S.; Hansell, E.; Cheng, J.; Roush, W. R.; McKerrow, J. H.; Fletterick, R. J. A target within the target: probing cruzain's P1' site to define structural determinants for the Chagas' disease protease. Structure 2000, 8, 831-40. 32. Di Tommaso, P.; Moretti, S.; Xenarios, I.; Orobitg, M.; Montanyola, A.; Chang, J. M.; Taly, J. F.; Notredame, C. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res 2011, 39, W13-7. 33. Sali, A.; Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 1993, 234, 779-815. 34. Case DA, D. T., Cheatham TE, Simmerling CLI, Wang J, Duke RE, Luo R, Crowley M, Walker RC, Zhang W, Merz KM, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry KF, Wong KF, Paesani F, Vanicek F, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Mathews DH, Seetin MG, Sagui C, Babin V, Kollman PA. AMBER 10, University of California, San Francisco; 2008. 35. Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J Comput Chem 2003, 24, 1999-2012. ! Chapter 4 HEFLECINs Second generation of heterocyclic-cinnamic acid conjugates Chapter 4 | 133 4.0. Rationale Following synthesis, in vitro and in silico evaluation of HEDICINs and HECINs, as described in the previous chapters, a second generation of potential dual-action antimalarials was envisioned. Second generation compounds, called HEFLECINs (HEterocyclic-FLExible spacer-CINnamic acid conjugates), were designed to also join in the same molecular construct a heterocyclic core from a classical antimalarial drug and a cinnamoyl moiety, but this time linked together through a flexible alkyl chain (Fig. 34). The choice of this spacer was based on earlier reports on the favorable role of alkyl chains linked to the heterocyclic core of antimalarial aminoquinolines.1 The substitution of the rigid dipeptide spacer in HEDICINs by the flexible and more lipophilic ones in HEFLECINs might improve antimalarial properties of the corresponding compounds. Finally, HEFLECINs were predicted to have a basicity closer to that of reference antimalarial drug chloroquine (pKa~7) than HEDICINs (pKa~4),2 which might favor their accumulation in the FV of intraerythrocytic Pf . ! ! Figure 34. General structure of HEFLECINs. As in the case of HE[DI]CINs the doctoral candidate was in charge of the chemical synthesis and characterization of HEFLECINs and their evaluation as inhibitors of β-H formation, whereas assessment of their inhibitory activity against both falcipains (in vitro) and development of blood-stage Pf parasites (in vitro and in vivo) was carried out by Prof. Philip J. Rosenthal’s team. The experimental research work was developed alongside an in silico study carried out by the thesis co-supervisor, Dr. Cátia Teixeira. Further evaluation of HEFLECINs against liver-stage P. berghei parasites and Leishmania was carried out by Dr. Miguel Prudêncio’s team at Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa and Dr. M. S. Gomes’s team 134 | Chapter 4 in IBMC (Instituto de Biologia Molecular e Celular) in Universidade do Porto, respectively. 4.1. Chloroquinoline-based HEFLECINs According to previous results found for HEDICINs, N-alkylcinnamoylated/chloroquine analogues were synthesized to evaluate the influence of the alkyl chain instead of the dipeptide chain in the antimalarial properties of the chloroquinoline derivatives here reported. 4.1.1. Synthesis and characterization of HEFLECINs Synthesis of HEFLECINs 13-15, 20-21, and 24-25 Chloroquinoline-based HEFLECINs 13-15 were synthesized in two steps, starting with an SNAr reaction between the appropriate alkanediamine and 4,7-dichloroquinoline, which was followed by condensation of the resulting 4-(N-aminoalkyl)amino-7chloroquinoline with the relevant cinnamic acid (Scheme 11). In order to form the amide bond, the respective carboxylic acid was activated using TBTU and DIEA as coupling reagents, and the amine 16-18 was added to reaction. The main challenge found in the synthetic procedure was that most cinnamoyl/chloroquinoline derivatives were only partially soluble in DCM turning difficult the extraction of the desired product from reaction mixture. The same solubility issue appeared when using other polar organic solvent as ethyl acetate, known to extract electron donor solutes.3 Nonetheless, all reactions afforded the cinnamoyl derivatives from low to good yields (8-67%). Chapter 4 | 135 ! Scheme 11. Synthetic pathway of HEFLECINs (13-15). As in HEDICINs and HECINs, the p-NO2 derivative of series 13 presented significantly lower yield than the other compounds of the series. However, experimental observations suggested that there was no difference behavior between the reactions. For instance, for each cinnamic acid, once its activation started, a solid began to appear and stopped forming at around half the activation time suggesting that this activation step was straightforward. Moreover, this solid disappeared when the corresponding amine was added to the reaction to obtain the desired amide bond. Calculation of the atomic Fukui indices (f+), derived from Natural Bond Order population analysis of the neutral compound and its respective anion,4 was done to quantify the electrophilicity of the carbonyl moiety for the activated unsubstituted cinnamic acid 19a and p-nitrocinnamic acid 19b, which correspond to the best (%yield = 52) and the worst (%yield = 8) yields for compounds 13, respectively. The Fukui indices obtained (f+19a = 0.08; f+19b = 0.04) suggested that the greater the magnitude of f+, the greater the change in electron density near the atoms of interest, and thus the higher reactivity of a molecule at that atomic site towards nucleophilic attack. The latter explains the higher yield obtained for the generation of the unsubstituted compound as compared to the p-nitro substituted derivative. 136 | Chapter 4 Furthermore, compounds 20-21 were synthesized, following similar procedures as the above described (Scheme 12), to assess the influence of the ester bond and chlorine substituent in position seven of the quinoline ring on the antimalarial activity. Briefly, the convenient amine 22 and alcohol 23 derivatives were synthesized. The amine 22 was further coupled to cinnamic acids using TBTU and DIEA, whereas the cinnamoylated product 21 was obtained through reaction of alcohol 23 with the adequate cinnamoyl chloride, in the presence of Et3N. Accordingly, compounds 20-21 were obtained in low yields (11-21%). ! Scheme 12. Synthetic pathway to obtain cinnamoylated derivatives 20-21. Moreover, in order to assess the influence of the quinoline moiety on the antimalarial activity, two additional N-cinnamoylated compounds were synthesized, the pyridine derivative 24 and the morpholine derivative 25. Compound 24 was synthesized following the procedure previously used to achieve the CQ analogues 13-15. Initially, it was obtained the corresponding amine 26 that was later coupled to p-methoxy cinnamic acid (Scheme 13). To yield the morpholine derivative 24, first an N-alkylation with Nbromobutylphthalimide was carried, followed by the removal of the phthalimide protecting group; subsequently, p-isopropyl cinnamic acid was coupled to the resulting amine 27 using TBTU and DIEA (Scheme 13). Both compounds 24 and 25 were obtained in fairly good yields (30-61%). Chapter 4 | 137 ! Scheme 13. Synthetic route to yield cinnamoylated derivatives 24-25. Characterization of HEFLECINs 13-15 and 20-21 and 24-25 All compounds 13a-l, 14a-l, 15a-c, 20, 21, 24 and 25 were characterized by 1H-NMR, 13C-NMR, ESI-IT MS, and HPLC, and the data obtained agreed with the desired compounds. As an example, the NMR spectra of compound 13i is shown (Fig. 35-36). Accordingly, figure 35 displays the characteristic signals of compound 13i, for instance, the shielded region below 4 ppm shows the 4 protons corresponding to the methylene groups at 3.36 ppm overlapped with the protons of water from the use of DMSO-d6 as a solvent to run the NMR spectra, and the 4 protons of the most shielded methylene groups in 13i at 1.64 ppm. The α-vinyl proton is observed at 6.64 ppm with J= 15.6 Hz, a coupling characteristic of a trans-vinyl proton. The deshielded aromatic protons are also visible. For example, in the downfield region, the 1H-NMR spectrum shows the characteristic signal of the proton at position 3 of the quinoline ring at 6.47 ppm, the protons of the position 8 in the quinoline ring at 7.77 ppm with a small coupling constant J= 2 Hz, and the triplet of one of the proton bound to a nitrogen atom at 8.17 ppm. The 13C-NMR (Fig. 36) displays the number of signals in agreement with the structure of the desired compounds: 16 signals in the downfield region and four signals in the upfield region, the most deshielded signal corresponding to the carbonyl functionality. For more detailed analysis of the NMR data, please refer to the experimental section of this chapter. In the mass spectrum, two signals were expected since bromine atom has two isotopes approximately equally abundant. Interestingly, instead of displaying two peaks associated to two different molecules, containing different isotope 79Br and 81Br, respectively, the m/z peak showed only one peak corresponding to the compound containing the isotope of 81Br (Fig. 37). 144 | Chapter 4 lipophilic (logP ≈ 5). In addition, compounds’ in vivo activities were significantly lower compared to the in vitro result, this could be explained by: i) the compounds’ limited bioavailability, ii) biological difference between P. berghei and Pf, and/or iii) extensive binding to plasma proteins. Further studies need to be carried out to assess the bioavailability properties of the compounds to proceed with any needed structural modifications in order to improve the correlation between the in vitro and in vivo displayed activities. Chapter 4 | 145 ! Figure 39. Survival curves for P.berghei-infected mice treated with compounds 13c, 13d and 13h, and CQ. 13c 13d 13h Control 13c 13d 13h CQ Control 13c 13d 13h CQ Control 146 | Chapter 4 4.1.4. Mechanism of action of N-cinnamoylated derivatives Inhibition of falcipain activity and of β -hematin formation Docking calculations showed that CQ-derived HEFLECINs could fit well into the active pocket of falcipain-2, making electrostatic and Van der Waals interactions with the residues in the cavity of the protease. However, further MD demonstrated that the ligand moves away from the binding pocket. Possibly, the non-covalent interactions that the ligand establishes with the residues of the active site are not strong enough to keep it within the cavity. These last results suggest that the α,β-unsaturated portion of the ligand will not be able to alkylate the catalytic Cys. However, since the docked conformations of compounds 13-14 place the vinyl within ~3.5 Å from the thiolate, we still envisaged that, if favoured by its kinetics, the reaction could occur and the ligand could form a covalent interaction with Cys before being expulse of the active cavity of falcipain. In this context, compounds 13-14 were evaluated as inhibitors of falcipain-2 by Rosenthal’s team.10 In addition, in vitro assays against β-H formation (Table 13) were carried following the procedure described in the experimental section of chapter 2. Although none of the compounds exhibited activity against FP2, most of them showed to be active in vitro against β-H formation. Accordingly, results suggest that inhibition of the β-H formation seems to be one of the mechanisms contributing to the antiplasmodial activities of the compounds since variation of inhibition of β-H formation follows the same trend of activities against CQ-resistant strain Pf W2, as shown by compounds 13c vs 14c vs 15a. Also, the chlorine in position 7 of the chloroquinoline aromatic ring is once again shown to be essential for the activity of CQ and its analogues against the β-H formation.11, 12 However, since the most active compounds inhibiting the parasite development, namely, the p-iPr and p-Cl derivatives 13c (IC50 = 11.0 nM) and 13h (IC50 = 11.6 nM), were not the most active inhibitors of β-H formation, results suggest the existence of an additional mechanism through which HEFLECINs exert their activities. Chapter 4 | 147 Table 13. In vitro assays against β-hematin formation. Compound R1 n X R2 β-H[a] 13a Cl 3 N H - 13b p-Me - 13c p-iPr[c] + 13d p-OMe[c] + 13e p-NH2 - 13f m-F + 13g p-F + 13h p-Cl[d] + 13i p-Br ++ 13j o-NO2 + 13k m-NO2 - 13l p-NO2 ++ 13m p-NMe2 + 13n m,p-diOMe - 14a 2 N H - 14b p-Me - 14c p-iPr - 14d p-OMe - 14e p-NH2 - 14f m-F - 14g p-F - 14h p-Cl + 14i p-Br - 14j o-NO2 + 14k m-NO2 + 14l p-NO2 + 15a 4 N p-iPr + 15b p-OMe - 15c p-Cl - 20 3 O p-OMe + 21 H 3 N p-iPr - CQ ++ [a] β-H inhibition was determined and ranked as shown in chapter 2; test compounds were ranked as follows: <50%, not active (-); between 50 and 75%, moderately active (+); ≥75%, highly active (++). 148 | Chapter 4 Inhibition of New Permeability Pathways in Pf-infected erythrocytes One possible additional mechanism underlying the activity of HEFLECINs against bloodstage Pf could be inhibition of intraerythrocytic parasite’s nourishment: the growth of Pf within RBC relies on the uptake of certain nutrients from the extracellular medium; during the intraerythrocytic development of the parasite, the RBC show increased permeability to a wide range of structurally unrelated low molecular weight solutes, through the socalled “New Permeability Pathways” (NPP), whose nature is still elusive and may comprehend one or several host-encoded channels, or a single parasite-encoded channel also known as plasmodial surface anion channel (PSAC).13, 14 Some inhibitors of these NPP include furosemide (XXXII), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, XXXIII)15 and dantrolene (XXXIV).13 The latter nitro-substituted compound (XXXIV) was found not to have any measurable effect on other anion channels previously identified, consequently, addressing the concern of designing inhibitors with specificity towards this relatively new antimalarial target.13 Remarkably, the nitro substituted HEFLECINs are highly active as compared with most of the other substituted derivatives. Additionally, Kanaani and co-workers found that cinnamic acid derivatives inhibit NPP.16 Interestingly, they found that the antiplasmodial activity correlated with the hydrophobic character of the molecules evaluated. In addition, Kirk’s team found, through the study of a series of arylaminobenzoates,17 analogues of the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), that the antimalarial activity of the derivatives increase with the length and lipophilicity of the hydrophobic tail. These findings are comparable to the results obtained for HEFLECINs, which might suggest that NPP could be the additional mechanism of action through Chapter 2 | 149 which the cinnamoyl derivatives here presented exert their activity. In order to test this hypothesis, preliminary NPP-inhibition assays were carried out by sorbitol-induced hemolysis assays, as following described. The erythrocyte membrane is normally impermeant to sorbitol, but NPP appearance in the infected RBC membrane allows the passage of sorbitol and, when mature parasitized erythrocytes are suspended in an isosmotic sorbitol solution, there is a net uptake of sorbitol and water into the erythrocyte, resulting in cell swelling and hemolysis.18 The rate of hemolysis can be correlated with the influx of sorbitol, hence, can be used as a good indicator of NPP inhibitory activity. In this context, the rate of hemolysis of p-isopropyl cinnamic derivatives 13c and 1c was evaluated since the pisopropyl derivatives were the compounds which generally presented the highest activity among the series (Fig. 40). These in vitro assays were performed by Egée’s team at Université Pierre et Marie Curie, France. Compound 13c was shown to display (at 1 µM) significant NPP-inhibition activity as it markedly delayed sorbitol-induced hemolysis in Pf infected RBC, whereas 1c did not. These results correlate with in vitro antiplasmodial data since HEFLECIN 13c (IC50 = 11 nM) was more active than HEDICIN 1c (IC50 = 830 nM) against Pf W2. However, these are only preliminary results and additional experiments are already scheduled to test all HEFLECINs using the patch-clamp technique, which is today the most precise way to decipher subtle changes in channel activity.19 ! Figure 40. Half-haemolysis time of p-isopropyl cinnamic derivatives 13c and 1c. Half-haemolysis time Compounds concentration 1µM Control 13c 1c 150 | Chapter 4 Although the results here presented reinforce the idea that HEFLECINs might exert their antimalarial action through inhibition of NPP, other MOA cannot be ruled out. For instance, inhibition of plasmodial cytosolic proteases20, 21 like aminopeptidases PfA-M1 and PfA-M1722 or CQ-chemosensitization.23 It has been shown that resistance to chloroquine is due to mutations in a putative transmembrane chloroquine transporter, PfCRT, which seems to allow the drug to rapidly efflux from the parasite digestive vacuole, preventing sufficient inhibition of heme polymerization to block parasite development.24 Chloroquine chemosensitizers are thought to interact with PfCRT in such a way that drug efflux is impeded.23 HEFLECINs described here match relevant structural factors of chloroquine-chemosensiziting agents recently reported by Kelly25 and Lavrado26 such as a hydrogen bond acceptor (nitrogen) or aminobutyl chains between the heterocyclic core of the compound and its N-substituted terminal amine. Therefore, we cannot rule out that HEFLECINs might (also) through this mechanism, and future investigations should be undertaken to pursue this hypothesis. 4.1.5. Antiplasmodial assays against liver-stage P. berghei parasites Motivated by the fact that preliminary studies suggest HEFLECINs to be capable of inhibiting NPP (see previous section), and since similar anion channel enhanced activity has been described for P. berghei-infected liver cells,27 we decided to further evaluate the in vitro activity of HEFLECINs against liver stage malaria. In vitro assays were carried out following procedure previously reported.28 Remarkably, all CQ-derived HEFLECINs were active against liver stage parasites (Fig. 41 and Table 14), displaying activity in the low micromolar range (IC50 = 1.1-6.5 µM). These are unprecedented results since CQ has been reported to be inactive against liver stage malaria29 and, to the best of our knowledge, HEFLECINs were the first CQ analogues with dual-stage antimalarial activity. As shown in table 14, compounds present IC50 5 to 15 times lower than CQ and 3-7 times lower than those of the reference drug for liver stage malaria, PQ. Some preliminary SAR could be devised from the liver stage inhibition results: • As observed for blood stage activity, the butyl spacer is preferred over the propyl and pentyl spacers, for instance, activity displayed by 13c-d,h vs 14c-d,h and 15a-c. • Contrary to results against blood stage malaria, substitution on the cinnamoyl moiety leads to a decrease in the activity, as the most active compound was the unsubstituted derivative 13a (IC50 = 1.1 µM). Chapter 4 | 151 • The presence of electro-withdrawing groups in the para position of the cinnamoyl ring leads to a slight increase on the activity, as demonstrated by 13b-d (IC50 = 2.4-2.9 µM) and 13g-i (IC50 = 1.1-1.4 µM). • Similar to blood stage results, removal of the chlorine in position 7 of the quinoline ring or substitution of the amide bond by an ester functionality lead to a decrease in the activity (Fig. 41). • Substitution of the chloroquinoline core by a non-aromatic ring or pyridine is also detrimental for liver-stage activity, as shown by compound 13c (IC50 = 2.5 µM) vs 25 (IC50 > 10 µM) and 13d (IC50 = 2.9 µM) vs 24 (IC50 = >10 µM). The mechanism of HEFLECINs against liver stage Plasmodia remains to be elucidated, and work is currently in course that aims at investigating whether the compounds are also able to inhibit anion channel activity in P. berghei-infected hepatocytes. Based on the antiplasmodial results against both liver and blood stage parasite, compound 13d and 13h were evaluated in vivo against the liver stage form of malaria. In vivo assays were carried out by Prudêncio’s team at Universidade de Lisboa. However, none of the compounds displayed in vivo activity against this stage. The latter might be explained by the fact that the liver is responsible for most drug metabolism, since the liver has the most drug metabolising enzymes relative to other organs.30, 31 Thus HEFLECINs might be metabolized into inactive compounds. Nonetheless, compounds here presented represent new leads towards the discovery of multistage antimalarials. 152 | Chapter 4 ! Figure 41. Anti-infective activity vs toxicity to hepatoma cells plot. PQ and CQ were included for comparison. The black, red, and blue circles represent results for the tested compounds at 10, 5, and 1 µM, respectively. Infection loads of Huh7 cells, from a human hepatoma cell line, were determined by bioluminescence measurements of cell lysates 48 h after infection with luciferase-expressing P. berghei parasites by Prudêncio’s team. Chapter 4 | 153 Table 14. In vitro assays of N-cinnamoylated derivatives 13-14, 20-21, and 24-25 against P. berghei liver stage malaria. Compound HET n X R2 Liver Stage IC50 (µM)[a] 13a Cq R1= Cl 3 N H 1.1 ± 0.1 13b p-Me 2.4 ± 0.5 13c p-iPr[c] 2.5 ± 0.2 13d p-OMe[c] 2.9 ± 0.4 13e p-NH2 ND 13f m-F 1.1 ± 0.2 13g p-F 1.4 ± 0.2 13h p-Cl[d] 1.4 ± 0.3 13i p-Br 2.3 ± 0.6 13j o-NO2 1.1 ± 0.2 13k m-NO2 ND 13l p-NO2 13m p-NMe2 13n m,p-diOMe 4.1 ± 0.2 14a Cq R1= Cl 2 N H ND 14b p-Me 14c p-iPr 14d p-OMe 4.0 ± 0.6 14e p-NH2 ND 14f m-F 14g p-F 14h p-Cl 14i p-Br 14j o-NO2 14k m-NO2 14l p-NO2 15a Cq R1= Cl 4 N p-iPr 15b p-OMe 2.3 ± 0.6 15c p-Cl 1.6 ± 0.1 20 Cq R1= Cl 3 O p-OMe 6.5 ± 0.9 21 Cq R1= H 3 N p-iPr ND 24 Py 3 N p-OMe >10 25 Mu 3 N p-iPr >10 ART ND CQ 15.9 PQ 7.5 [a] IC50 of the most active compounds against liver stage.