Thuc Dinh Ngoc FACULTY OF SCIENCE DEPARTMENT OF CHEMISTRY MOLECULAR DESIGN AND SYNTHESIS DIVISION Celestijinenlaan 200F box 2404 B-3001 HEVERLEE, BELGIUM tel + 32 16 32 00800 thuc.dinhngoc@chem.kuleuven.be www.website.kuleuven.be ARENBERG DOCTORAL SCHOOL FACULTY OF SCIENCE HETEROCYCLIC TRITERPENE NATURAL PRODUCT ANALOGUES AS POTETIALLY BIOLOGICALLY ACTIVE MOLECULES HETEROCYCLIC TRITERPENE NATURAL PRODUCT ANALOGUES AS POTENTIALLY BIOLOGICALLY ACTIVE MOLECULES Thuc Dinh Ngoc May 2014 Supervisor: Prof Wim Dehaen Dissertation presented in partial fulfilment of the requirements for the degree of Doctor in Science May 2014 HETEROCYCLIC TRITERPENE NATURAL PRODUCT ANALOGUES AS POTENTIALLY BIOLOGICALLY ACTIVE MOLECULES Thuc DINH NGOC Supervisor: Prof Dr Wim Dehaen Dissertation presented in partial fulfillment of the requirements for the degree of Doctor in Science Members of the Examination Committee: Prof Dr Luc Van Meervelt Prof Dr Wim De Borggraeve Prof Dr Christophe Pannecouque Prof Dr Milan Urban Prof Dr Mario Smet May 2014 © 2014 KU Leuven, Science, Engineering & Technology Uitgegeven in eigen beheer, Thuc Dinh Ngoc, Thanh Hoa, Vietnam Alle rechten voorbehouden Niets uit deze uitgave mag worden vermenigvuldigd en/of openbaar gemaakt worden door middel van druk, fotokopie, microfilm, elektronisch of op welke andere wijze ook zonder voorafgaandelijke schriftelijke toestemming van de uitgever All rights reserved No part of the publication may be reproduced in any form by print, photoprint, microfilm, electronic or any other means without written permission from the publisher ISBN 978-90-8649-713-3 D/2014/10.705/22 ACKNOWLEDGEMENT I would like to take this opportunity to express my deepest gratitude and sincere appreciation to everyone who has helped me in different ways in order to accomplish my doctoral work First and foremost, I would like to express my gratitude to my supervisor, Prof Wim Dehaen for his patient guidance and enthusiastic encouragement during my PhD course Without his guidance and persistent help this dissertation would not have been possible Next, I would like to thank the members of the jury: Prof Luc Van Meervelt, Prof Wim De Borggraeve, Prof Christophe Pannecouque, Prof Milan Urban and Prof Mario Smet for correction and giving essential suggestions on my thesis In addition, many thanks to Prof Dang Thi Oanh, Prof Le Thi Anh Dao and Prof Vu Quoc Trung at the Hanoi National University of Education in Vietnam for introducing me to the University of Leuven where I have a chance to my research A special thank to Prof Minh Tho Nguyen at the University of Leuven for his valuable advices during my research Many thanks I would like to send to Mr Karel Duerinckx and Mr Anh Hung Mai for NMR spectra measurement; Mr Bert Demarsin for mass spectra measurement; Mr Reinhoud Bomans for taking care all of chemicals and glassware ordering; Mr Jeroen Jacobs for high quality X-ray crystallographic data; Dr Nico Moons for his guidance of the first time when I started doing research and Mrs Ann Petré for help with all administrative procedures during period of my PhD I would like to thank everyone from LOSA, LOSH and LOMAC divisions for being supportive through the course of my PhD; Dr Youngju Kim for correction manuscript; Mr Stijn Boodts for fluorescence measurement and other people for their valuable advices Many thanks to the Vietnamese Student Association in Leuven and the Vinakul Football Club in Leuven for enjoying and sharing living experience with me in order to avoid lonely when I has been living far from home I would like to thank a Belgian family in Leuven, Mr & Mrs Dirk Witters and Monique Swart for peaceful place to live and friendly social communication Living beside them makes me feel pretty comfortable I am deeply grateful to the Vietnamese Government and the University of Leuven for financial support Without these sponsors, I could not have a chance to finish my thesis I also would like to thank the Hong Duc University in Vietnam where I have been working for about all convenient conditions that spends for me on studying abroad Lastly but the most important, I am grateful to my family: my parents, my parents-in-law, my wife and my children Their blessing, love and patience encourage me to overcome all of the difficult circumstances during period my PhD Leuven – Belgium, May 2014 Thuc Dinh Ngoc (Đinh Ngọc Thức) General information Table of contents Table of contents ………………………………………………… Summary…………………………………………………………… Samenvatting……………………………………………………… List of abbreviations……………………………………………… INTRODUCTION 1.1 General introduction………………………………………… 1.2 Betulin – allobetulin and their derivatives…………………… 1.3 Betulinic acid – betulonic acid and their derivatives.………… 1.4 Biological properties of betulin, allobetulin, betulinic acid and betulonic acid derivatives…………………………………… OBJECTIVES……………………………………………………… RESULTS AND DISCUSSIONS…………………………………… 3.1 Fused heterocyclic derivatives of allobetulone and betulonic acid ………………………………………………………… 3.1.1 Unsuccessful classical reactions ….…………………… 3.1.2 Beckmann rearrangement and fragmentation reactions 3.1.3 Fischer-indole synthesis ….…………………………… 3.1.4 Synthesis of triterpenoid triazine derivatives ………… 3.1.5 Aldol condensation …………………………………… 3.1.6 Synthesis of 1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives 3.1.7 Synthesis of triterpenoid BODIPYs ….……………… 3.1.8 Biological activities …………………………………… 3.1.9 Fluorescent properties ………………………………… 3.1.10 Conclusion …………………………………………… 3.2 Regioselectivity of functionalization reactions of 2-oxoallobetulin……………………………………… 3.2.1 Fischer indolization…………………………………… 3.2.2 Claisen rearrangement………………………………… 3.2.3 Bromination of 2-oxoallobetulin ……………………… 3.2.4 Baeyer-Villiger reaction ……………………………… 3.2.5 Beckman rearrangement……………………………… 3.2.6 Synthesis of 1,2,3-thiadiazole and 1,2,3-selenadiazole derivatives ………………………………………… 3.2.7 Biological activities …………………………………… 3.2.8 Conclusions…………………………………………… 3.3 Direct reaction of allobetulone, 2-oxoallobetulin and further reaction to fused heterocyclic compounds…………………… 3.3.1 Grignard reaction ……………………………………… 3.3.2 Click reaction ………………………………………… 3.3.3 Biological activities …………………………………… i iii iv vi 1 18 27 29 31 31 32 36 38 39 43 46 47 50 53 55 57 57 60 62 67 69 72 76 78 79 79 81 89 i General information 3.3.4 Conclusions …………………………………………… GENERAL CONCLUSIONS AND OUTLOOK………………… EXPERIMENTAL DATA………………………………………… 5.1 General remarks……………………………………………… 5.2 Experimental data 3.1………………………………………… 5.3 Experimental data 3.2………………………………………… 5.4 Experimental data 3.3………………………………………… 5.5 X-ray crystallographic data…………………………………… 5.6 Biological activity assays …………………………………… LIST OF PUBLICATIONS AND ATTENDED CONFERENCES CURRICULUM VITAE ……………………………………………… References……………………………………………………………… ii 90 91 93 93 97 117 126 133 135 137 138 139 General information Summary Terpenoids are at present the oldest and the most widespread group of natural products which also possess promising potential biological activities Among them, the triterpenoids are abundant in nature with more than 200 different triterpene skeletons known They are also having a role as precursors for synthesis of drugs inducing anti-inflammatory, anti-arthritic responses, inhibition of tumor growth in animal cells and especially as tools in cancer therapy The triterpenoids betulin and betulinic acid are very abundant in nature and currently are commercially available The investigation of (allo)betulin and betulinic acid themselves and their derivatives was started as early as the nineteenth century and is continuously being further developed The synthetic transformation and modification of natural products with the aim to improve the biological properties is an area of current interest In this thesis, a series of novel allobetulin and betulinic acid derivatives were synthesized and two BODIPY dyes were obtained In the first part of this study, novel 2,3-fused heterocyclic derivatives of allobetulin and betulonic acid were obtained The compounds include pyrrole, 1,2,3-thiadiazole, 1,2,3-selenadiazole, 1,2,4triazine, thienopyridine and BODIPY fused heterocyclic derivatives These products were tested for their biological activities and the BODIPY dyes were investigated for their fluorescent properties In the second part, the regioselectivity of the functionalization of 2oxoallobetulin was investigated by using a number of classical reactions From the results, it can be concluded that the most reactive position is at C-3 Thus, a series of new heterocyclic products was obtained and tested for biological activities Finally, we investigated the Grignard addition reaction at the ketone group of both allobetulone and 2-oxoallobetulin (isomers) and further synthesis of new conjugated heterocyclic products A series of new 1,2,3triazole derivatives was obtained and tested for biological properties The structure of all of the new compounds was confirmed by advanced techniques such as X-ray crystallography, 2D-NMR spectroscopy iii General information Samenvatting Terpenoiden zijn de oudste en meest verspreide groep natuurproducten, en ze bezitten daarenboven veelbelovende biologische activiteiten De triterpenoiden vormen hiervan een belangrijk onderdeel met meer dan 200 verschillende gekende basisstructuren Deaze natuurproducten kunnen een rol vervullen als beginproduct voor de synthese van geneesmiddelen met anti-inflammatoire of anti-arthritische werking, inhibitie van tumorgroei in dierlijke cellen en in het bijzonder voor kankertherapie De triterpenoiden betuline en betulinezuur komen in grote hoeveelheid voor in de natuur en zijn commercieel beschikbaar Onderzoek naar (allo)betuline, betulinezuur en hun derivaten werd reeds gestart in de negentiende eeuw en is nog steeds actueel De synthetische omzetting en modificatie van natuurproducten gericht op het verbeteren van de biologische eigenschappen staat momenteel in de belangstelling In het kader van dit thesisonderzoek werden een reeks nieuwe derivaten van allobetuline en betulinezuur, en verder nog twee hiervan afgeleide BODIPY-kleurstoffen, bekomen In het eerste deel van deze studie werden nieuwe 2,3-gefusioneerde heterocyclische derivatives van allobetuline en betulonzuur aangemaakt Het ging hierbij over pyrrool, 1,2,3-thiadiazool, 1,2,3-selenadiazool, 1,2,4-triazine, thienopyridine en BODIPY-gefusioneerde heterocyclische verbindingen Deze producten werden getest op hun biologische activiteit en het fluorescentiegedrag van de BODIPY-kleurstoffen werd nagegaan In het tweede gedeelte werd de regioselectiviteit van de functionalizatie van 2-oxoallobetuline onderzocht door middel van een aantal klassieke reacties De resultaten laten toe te concluderen dat de reactiefste positie op C-3 gelokaliseerd is In deze contezt werden een reeks heterocyclische producten bekomen en hun biologische eigenschappen werden getest Tenslotte hebben we de Grignard-additiereactie onderzocht op de ketongroep zowel van allobetulone als 2-oxoallobetuline (isomeren) en werd verdere synthese uitgevoerd met de vorming van nieuwe geconjugeerde heterocyclische producten Een reeks nieuwe 1,2,3iv General information triazoolderivaten werd bekomen en getest voor hun biologische eigenschappen De structuur van alle nieuwe verbindingen werd bevestigd door geadvanceerde technieken zoals X-stralen kristallografische analyse en 2D-NMR spectroscopie v Experimental data 5.6 Biological Activity Assays 5.6.1 Cytostatic Assays All assays were performed in 96-well microtiter plates To each well were added (5−7.5) x 104 tumor cells and a given amount of the test compound The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37°C in a humidified CO2-controlled atmosphere At the end of the incubation period, the cells were counted in a Coulter counter The IC50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited tumor cell proliferation by 50% 5.6.2 Antiviral Activity Assays The compounds were evaluated against the following viruses: herpes simplex virus type (HSV-1) strain KOS, thymidine kinase-deficient (TK−) HSV-1 KOS strain resistant to ACV (ACVr), herpes simplex virus type (HSV-2) strain G, varicella-zoster virus (VZV) strain Oka, TK− VZV strain 07−1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, parainfluenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, Sindbis, reovirus-1, Punta Toro, human immunodeficiency virus type strain IIIB and human immunodeficiency virus type strain ROD The antiviral, other than anti-HIV, assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK) Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 or 100 plaque forming units (PFU) (VZV or HCMV) in the presence of varying concentrations of the test compounds Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds Antiviral activity was expressed as the EC50 or compound concentration required reducing virus-induced cytopathogenicity or viral plaque formation by 50% 135 Experimental data 5.6.3 Anti-HIV Activity Assays The anti-HIV-1 and anti-HIV-2 activity of each compound in CD4+ MT-4 cells was determined by tetrazolium-based (MTS) colourimetric assay (Ref: R Pauwels, J Balzarini, M Baba, R Snoeck, D Schols, P Herdewijn, J Desmyter, E De Clercq Rapid and automated tetrazoliumbased colorimetric assay for the detection of anti-HIV compounds J Virol Methods 1988, 20, 309-321) Briefly, 3-fold annulations of various rest compounds were added in a 96-well plate and pre incubated for 20 minutes at 37oC with MT-4 cells (1x106 cells/ml) Next, various concentration of virus (HIV-1 strain NL4.3 and HIV-2 strain ROD) were given depending on the TCID50 (50% tissue culture infectious close) of viral stock After days, cytopathic effects (CPE) were scored microscopically and antiviral activity was measured by MTS/PES using a Spectromax 96-well plate reactor (Molecular Devices) as previously described 136 List of publications and attended conferences LIST OF PUBLICATIONS CONFERENCES AND ATTENDED 6.1 List of publications Thuc Dinh Ngoc and Wim Dehaen Selective funtionalization of 2oxoallobetulin derivatives Tetrahedron 2014, 70, 1836-1840 Thuc Dinh Ngoc, Nico Moons, Youngju Kim, Wim De Borggraeve, Anastassiya Mashentseva, Graciela Andrei, Robert Snoeck, Jan Balzarini and Wim Dehaen Synthesis of triterpenoid triazine derivatives from allobetulone and betulonic acid with biological activities Bioorg Med Chem 2014, DOI: http://dx.doi.org/10.1016/j.bmc.2014.04.061 Thuc Dinh Ngoc, Stijn Boodts, Youngju Kim and Wim Dehaen Synthesis of triterpenoid BODIPY dyes from allobetulin with fluorescent properties Manuscript in preparation 6.2 List of attended conferences 14th Sigma-Aldrich Organic Synthesis Meeting (December 2-3, 2010, Spa, Belgium) Participation without presentation 15th Sigma-Aldrich Organic Synthesis Meeting (December 1-2, 2011, Spa, Belgium) Participation without presentation 16th Sigma-Aldrich Organic Synthesis Meeting (December 6-7, 2011, Spa, Belgium) Participation without presentation International Congress of Young Chemists 2013 (October 9-13, 2013, Poznan, Poland) Poster presentation: Selective funtionalization of 2-oxoallobetulin derivatives 17th Sigma-Aldrich Organic Synthesis Meeting (December 5-6, 2013, Blankenberge, Belgium) Poster presentation: Synthesis of triazine derivatives from allobetulone and betulonic acid 137 Curriculum vitae CURRICULUM VITAE Personal information Full name: Thuc Dinh Ngoc (Đinh Ngọc Thức) First name: Thuc and Surname: Dinh Ngoc Date of birth: April 20th 1977 Nationality: Vietnam Marital status: Married Email: thuc.dinhngoc@yahoo.com dinhngocthuc@hdu.edu.vn Professional Career and Education 138 1996-2000 BSc in Chemistry Faculty of Chemistry - Ha Noi National University of Education - Vietnam 2001-2003 MSc in Organic Chemistry Faculty of Chemistry Ha Noi National University of Education - Vietnam 2004-2009 Lecturer at Faculty of Natural Science – Hong Duc University – Thanh Hoa city – Vietnam 2010-2014 PhD student at Department of Chemistry – Faculty of Science – University of Leuven – Belgium References References Saleem, M.; Kweon, M H.; Yun, J M.; Adhami, V M; Khan, N.; Syed D N.; Mukhtar, H Cancer Res 2005, 65, 11203-11213 Paul, M Dewick Medicinal 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