Department of Pharmaceutical Sciences PhD Course in Pharmaceutical Sciences (XXIX Cycle) Synthesis of New Sulfurated Derivatives of Natural and Synthetic Systems as Multitarget Anticancer Agents and Development of New Drug Discovery Methodologies Supervisor: Prof. Anna SPARATORE Coordinator: Prof. Marco DE AMICI PhD Thesis of: Elena Gabriele R10658 Academic year 2015/2016 “I don’t believe there would be any science at all without intuition.” Rita Levi Montalcini A mio marito e ai miei genitori Preface This thesis is divided in two parts. The former has been carried out at the Universitá degli Studi di Milano, department of Pharmaceutical Sciences, in the research group of Professor Anna Sparatore. This project treats the synthesis of new sulfurated compounds with the aim of obtaining anticancer agents acting through a multitarget mechanism. This part, entitled "design and synthesis of new derivatives of natural and synthetic systems endowed with anticancer activity, through a multitarget mechanism" is discussed from page 1 to page 149. The latter has been developed at Swiss Federal Institute of Technology in Zurich (ETH Zurich), Institute of Pharmaceutical Sciences (IPW), under the supervision and in the research group of Professor Dario Neri. This project explains the advantages of using DNA-encoded chemical libraries (DECLs) in drug discovery process. In particular, since in the majority of cases DECLs require at least one-step of amide bond formation between amino modified DNA and a carboxylic acid, we optimized a new methodology of synthesis in order to facilitate the construction of single-pharmacophore libraries (DECLs). This part, entitled "optimized reaction conditions for amide bond formation in DNA-encoded combinatorial libraries" is discussed from page 151 to page 187. TABLE OF CONTENTS CHAPTER I Design and Synthesis of New Derivatives of Natural and Synthetic Systems Endowed With Anticancer Activity, through a Multitarget Mechanism ABSTRACT 1. INTRODUCTION……………………………………………………………………………………………………….…3. 1.1 Cancer: general aspects…………………………………………………………………………………………...3 1.2 STAT3…………………….……………………………………………………………………………….……………..…5 1.2.1 Signal Transducer and Activator of Transcription proteins…………………….……..….5 1.2.2 STATs signalling pathway……………………………………………………………………………..….6 1.2.3 Negative regulators of STATs signalling………………………………………………………..….7 1.2.4 Influence of STAT3 on biological functions……………………………………………………... 8 1.2.5 STAT3 as a novel anticancer drug target…………………………………………………………10 1.2.6 STAT3 inhibitors classification……………………………………………………………………….…10 1.3 NFkB………………………………………………………………………………………………………………………..13 1.3.1 Nuclear factor kappa B…………………………………………………………………………………….13 1.3.2 NfkB signalling pathway…………………………………………………………………………………..14 1.3.3 Negative regulators of NfkB signalling………………………………………………………..….16 1.3.4 Influence of NfkB on biological functions………………………………………………………..16 1.3.5 NfkB as a novel anticancer drug target…………………………………………………………..17 1.3.6 NfkB inhibitors classification……………………………………………………………………………17 1.4 Sulfurated compounds……………………………………………………………………………….………..….19 1.5 Natural compounds………………………………………………………………………………………….…….19 1.5.1 Curcumin and cinnamic acids………………………………………………………………………….22 1.5.2 Phenolic acids…………………………………………………………………………………………….…. 24 1.5.3 Celastrol…………………………………………………………………………….…………………………. 27 1.5.4 Folic Acid…………………………………………………………………………………………..………..….28 1.6 Semi-synthetic compounds: rosmaricine and derivatives………………………....………… 30 1.7 Synthetic systems………………………………………………………………………………………………….. 32 1.7.1 S3I-201 and derivatives………………………………………………………………………….…….….32 1.7.2 Heterocyclic compounds……………………………………………………………………….…….….33 I 2. AIMS OF THE RESEARCH PROJECT…………………………….……………………………………………3 4 2.1 Sulfurated intermediates…………………………………………….……………………………………..…….34 2.2 S3I-201 and related compounds………………………………….……………………………………………36 2.3 Curcumin and cinnamic acids derivatives………………………………………………….……………37.. 2.4 Celastrol hybrids………………………………………………………………………………………………………39 2.5 Rosmaricine and derivatives……………………………………….……………………………………………40 2.6 Heterocyclic derivatives………………………………………………..…………………………..…………….41 3. CHEMISTRY………………………………………………………………………..……………………………..…….……43 3.1 Sulfurated intermediates………………………………………………..……………………………………….43 3.2 S3I-201 and related compounds………………………………………..……………………………………..46 3.3 Curcumin and cinnamic acids derivatives………………………….…………………………………….48 3.3.1 Curcumin derivatives and thio-analogue…………………….……………………………………48 3.3.2 Ferulic acid derivatives……………………………………………….……………………………….……49 3.3.3 Caffeic acid derivatives……………………………………………….……………………………….……50 3.3.4 Cinnamic acid derivatives…………………………………………….………………….………………..52 3.4 Celastrol hybrids………………………………………………………………….……………………………..…….52 3.5 Rosmaricine and derivatives…………………………………………………………………………….………56 3.6 Heterocyclic derivatives……………………………………………………………………………………………57 4. EXPERIMENTAL……………………………………………………………………………………………………….....60 4.1 General……………………………………………………………………………………………………………………60. 4.2 Sulfurated intermediates………………………………………………………………………….…………..….61 4.3 S3I-201 and related compounds……………………………………………………………….………..…….75 4.4 Curcumin and cinnamic acids derivatives…………………………………………………………………83 4.4.1 Curcumin derivatives and thio-analogue……………………………………………….…….……83 4.4.2 Ferulic acid derivatives…………………………………………………………………………………….91 4.4.3 Caffeic acid derivatives……………………………………………………………………..…………… 100. 4.4.4 Cinnamic acid derivatives…………………………………………………………………..……………. 108 4.5 Celastrol hybrids ……………………………………………………………………………………..…………..….111 4.6 Rosmaricine and derivatives…………………………………………………………………………………….122 4.7 Heterocyclic derivatives………………………………………………………………………………………….124 5. BIOLOGICAL ASSAYS……………………………………………………………………………………………..….133 5.1 AlphaScreen-based assay…………………………………………………………………………….………….133 II 5.2 Cytotoxicity (MTT) on HCT-116 cell line…………………………………………………………..……….134 5.3 NfkB Luciferase Reporter promoter activity assay………………………………………..…..……135 5.4 STAT3 Luciferase Reporter gene assay……………………………………………………………..… 136 6. RESULTS AND DISCUSSION……………………………………………………………………………….…….137 6.1 S3I-201 and related compounds………………………………………………………………..…….…. 137 6.2 Curcumin and cinnamic acids derivatives…………………………………………………………… 138 6.3 Celastrol hybrids………………………………………………………………………………………………..…….142. 6.4 Rosmaricine and derivatives…………………………………………………………………………………….144 6.5 Heterocyclic derivatives…………………………………………………………………………….………… 146 7. CONCLUSIONS…………………………………………………………………………………………………………….148. CHAPTER II Optimized Reaction Conditions for Amide Bond Formation in DNA-Encoded Combinatorial Libraries ABSTRACT 1. INTRODUCTION…………………………………………………………………………………………..…..…..…..153. 1.1 Technologies for Drug discovery……………………………………………………………..………..…….153 1.2 DNA-encoded chemical libraries …………………………………………………………………………….156 1.2.1 Single-pharmacophore-DNA-encoded libraries…………………………………………………157 1.2.2 Dual-pharmacophore-DNA-encoded libraries………………………………………..………158... 1.3 Library selection and decoding…………………………………………………………….……………..160. 1.3.1 High throughput DNA sequencing technologies…………..……………………………….…161 1.3.2 Hit validation………………………………………………………………………………………..…….….162 1.4 DNA compatible chemistry…………………………………………………………….……………..…….164 2. AIM OF THE RESEARCH PROJECT…………………………………………………………………….……. 165 3. RESULTS AND DISCUSSION……………………………………………………………………………….…….166 4. EXPERIMENTAL…………………………………………………………………………………………………...…….174 5. CONCLUSIONS…………………………………………………………………………………………………………….187 III CHAPTER III Bibliography and Acknowledgements 1. REFERENCES………………………………………………………………………………………………………………..190. 2. ACKNOWLEDGEMENTS………………………………………………………………………….………….………210 IV CHAPTER I Design and Synthesis of New Derivatives of Natural and Synthetic Systems Endowed with Anticancer Activity, through a Multitarget Mechanism Università degli Studi di Milano Department of Pharmaceutical Sciences 1 ABSTRACT The scientific research on cancer therapy has made important progresses during last years, but mortality for some types of cancer keeps increasing. Cancer chemotherapy can not be considered completely satisfactory and, due to the extreme toxicity of anticancer drugs, it becomes essential to identify new compounds more potent and less toxic. It is well known that inflammatory conditions in selected organs increase the risk of cancer. Compounds of the inflammatory tumour microenvironment include leukocytes, cytokines, complement components and are orchestrated by transcription factors, such as STAT3 and NFkB. Therefore, drugs able to inhibit one or both transcription factors could be useful tools to treat cancer disease. Based on these considerations, the aim of the present thesis was the synthesis of different novel sets of sulfureted hybrid molecules, potentially candidates for the treatment of cancer. On one hand, we explored the potentialities of new sulfurated chemical scaffolds, such as dithiolethiones, methanethiosulfonates and allyldisulfides to evaluate their antiproliferative activity, as well as their ability to inhibit STAT3 and NfkB, and to design new anticancer agents. On the other hand, we evaluated the possibility of creating hybrid molecules, combining moieties with different mechanism of action, which could carry out a synergistic antitumor effect. In particular, the sulfurated compounds synthetized have been combined with different STAT3 and/or NfkB inhibiting structures such as natural products (curcumin, phenolic acids and celastrol), semi-synthetic and synthetic systems (rosmaricine and heterocyclic