“Synthesis and Biological Evaluation of the Antioxidant Properties of New

Università degli Studi di Palermo Westälische Wilhelms-Universität Mϋnster Dottorato in Ph.D in Scienze Molecolari e Biomolecolari Pharmaceutical and Medicinal Chemistry Dipartimento STEBICEF Department of S.S.D. -- CHIM/08 Pharmaceutical and Medicinal Chemistry

“Synthesis and biological evaluation of the antioxidant properties of new analogues of natural products with piperidine based structure and evaluation of the affinity and activity towards σ-receptors and ĸ-receptors”

PhD STUDENT COORDINATOR DANIELE AIELLO PROF. PATRIZIA DIANA

TUTOR TUTOR PROF. PATRIZIA DIANA PROF. DR. BERNHARD WÜNSCH PROF. DR. MATTHIAS LEHR

CICLO XXX 2019 Pharmaceutical and Medicinal Chemistry Pharmazeutische und Medizinische Chemie

Synthesis and biological evaluation of the antioxidant properties of new analogues of natural products with piperidine based structure and evaluation of the affinity and

activity towards σ-receptors and ĸ-receptors

Inaugural-Dissertation

To obtain the Doctoral degree in “Scienze Molecolari e Biomolecolari” from the Faculty of Pharmacy from Università degli Studi di Palermo

and

zur Erlangung des Doktorgrades

der Naturwissenschaften im Fachbereich Chemie und Pharmazie

der Mathematisch-Naturwissenschaftlichen Fakultät

der Westfälischen Wilhelms-Universität Münster

vorgelegt von

Daniele Aiello

Aus Palermo, Italy

2019

For the University of Palermo

Rector: Prof. Fabrizio Micari First supervisor (expert): Prof. Patrizia Diana

For the Westfälischen Wilhelms-Universität

Dekan: Prof. Dr. Uwe Karst

Erster Gutachter: Prof. Dr. Bernhard Wünsch

Zweiter Gutachter: Prof. Dr. Matthias Lehr

Tag der mündlichen Prüfung:

Tag der Promotion:

I have completed this work at the Institut für Pharmazeutische und Medizinische Chemie, Westfälischen Wilhelms-Universität Münster and at the department of Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Università degli Studi di Palermo under the supervision of

Herrn Prof. Dr. Bernhard Wünsch

I would like to extend my sincere gratitude for accepting me and let me be part of your research group. Without your patience, constant motivation and support this work would not have been achievable. Your wisdom, knowledge in the field of organic and medicinal chemistry and enthusiasm showed me how a good scientist and more importantly a wise leader must be. Your willingness to share knowledge, kindness and optimism inspired me, both professionally and personally. For all of this, I will be forever grateful to you.

Prof. Patrizia Diana

I would like to extend my earnest gratitude to Prof. Diana for accepting me in her research group. Her support, understanding, commitment and patience contribute immensely to my personal development as a researcher. I thank her for providing me with an interesting research topic, tremendous academic support and intellectual freedom in my work. Without her guidance this work would not have been achievable.

Acknowledgments:

I would like to thank:

● Prof. Dr. Matthias Lehr for being my mentors and for fruitful discussions in my progress reports. ● Prof. Girolamo Cirrincione for accepting me in his research group, for the advices and for the support. ● Dr. Anna Carbone, for her patience, support and guide on my road to be a good researcher. For the chats, small talks and for your help in everyday problems both in the lab and outside. For this and for a lot more, especially for enduring me during my bad days, I will always be grateful. ● Prof. Paola Barraja, Prof. Alessandra Montalbano and Dr. Virginia Spanò for your constant support, advices, the small talks and jokes in good days and for enduring me during the bad days. ● Dr. Barbara Parrino, Dr. Stella Cascioferro for the help and support. ● Prof. Luisa Tesoriese for the biological testing and for your support, advices and your kindness. ● Dr. Anna Junker for our talks, your advices, support and constant motivation. ● Dr. Dirk Schepmann and Bastian Frehland for the biological testing and for your kindness. ● Dr. Jens Köhler, Claudia Thier and Thomas Meiners for your support with NMR measuraments. ● Dr. Jörg Fabian and Thomas Meiners for the mass spectra recording. I also thank Frederik for the help with the LC-MS. ● Kirstin Lehmkuhl for your constant support with the HPLC measurements, orders of chemicals and your friendship during my stay in Mϋnster. ● Dott. Vincenzo Delia, Giuseppe Impeduglia and Paolo Bonomo, for your help in everyday problems in the lab, with the orders and with tech. problems. ● Dr. Peter Dziemba, Laura Prause and Sarah Berling for your organization of the institute and for your constant help with bureaucratic problems. ● Ulrich Heczko and Alfred Gerber for their help with chemicals and lab supplies. ● Thomas Gottschlich, Michael Janning, Vanessa Gulczynski, Annemarie Erdt, Karsten Suwelack and Heribert Wennemer for their constant work. ● My “students”, Chiara, Alessandra, Federica, Maria Grazia, Hendrik and Kevin for their help in the lab and their friendship. ● My colleagues of lab 337: Paul, Susi, Giovi, Cathy, Elena and Jonathan and Hendrik. For the friendship, help, that you are still giving to me, your jokes, your music and our delicious dinners. Thanks guys without you my PhD life in Mϋnster would have been much more difficult. ● The all AK Wünsch, Oriana Agoglitta, Dr. Samuel Asare, Kim Bachmann, Dr. Sören Baumeister, Elena Bechthold, Dr. Melanie Bergkemper, Sarah Berling, Frederik Börgel, Kathrin Brömmel, Dr. Paul Bunse, Charlotte Doering, Clemens Dobelmann, Dr. Sebastian Elfering, Dr. Siong Wan Foo, Bastian Frehland, Dr. Fabian Galla, Dr. Magdalena Galster, Donglin Gao, Dr. Dominik Heimann, Dr. Elisabeth Heinrichs, Christian Heselmeyer, Dr. Jonathan Hoffmann, Dr. Ralph Holl, Denise Ilari, Dr. Anna Junker, Dr. Dmitrii Kalinin, Max Kock, Dr. Jens Köhler, Dr. Lukas Kröger, Anja Langenstroer, Kirstin Lehmkuhl, Jan Lemmerhirt, Dr. Marius Löppenberg, Dr. Ines Lütnant, Benedikt Martin, Dr. Kengo Miyata, Dr. Hannes Müller, Laura Prause, Remya Rajan, Dr. Dirk Schepmann, Dr. Claudia Schmithals, Dr. Adrian Schulte, Julian Schreiber, Martha Sieg, Dr. Janine Stefanowitz, Dr. Ann-Katrin Strunz, Dr. Marina Szermerski, Dr. Louisa Temme, Dr. Stefanie Theiler, Dr. Simone Thum, Tullio Torregrossa, Karen Olejua Torres, Dr. Héctor Torres Goméz, Jan Uth, Dr. Frauke Weber und Dr. Christian Wittig. Thanks for your help, friendship and for creating an amazing atmosphere in the group. ● My “German teachers” Steffi, Susi, Paul, Cathy and Giovi and Hendrik. Thanks for your patience and you constant support. If I know something more than “ein Bier, bitte” is thank to you. ● My friends from CiM: Abhiyan, Anto, Denise and Jan, Giovanni, Guangxia, Tamara, Zahra and Mohammad. Thanks for your support, friendship and for the nice evening spent together. ● My friends from Italy: Antonietta, Andrea, Cristian, Domenico, Enza, Ilaria, Lorenzo, Paolo e Sandro for the support and the good time spent together during my Ph.D. ● My best friend from Italy Antonio, even from far away you were always able to let me feel your support. ● My friend Timo Wenning, without your support, help and friendship and your hamburgers my stay in Mϋnster would not have been the same, thanks for all. ● My sister Annamaria and especially my brother Luca, who endured my bad mood and took all over my duties during the worst periods of my PhD. ● My sincere gratitude is extended to my family, my parents, parents-in-law for their support and understanding. ● I want to thank also anyone else I forgot to mention here….

I would also like to give a special thanks to Hendrik Jonas. I could not have done it without your help and support. You made me able to be in two places at the same time. For this I will always be grateful to you and I will always be there if you will need help. Thank you so much.

THANKS!!!

To my family who always encourages and supports me to pursue my dreams

Table of Contents

1. Introduction……………………………………………………………………………………1

1.1. Piperidine and its derivatives……………………………………………………………1

1.2. Betalains…………………………………………………………………………………...3

1.2.1. Introduction………………………………………………………………………….3

1.2.2. Biosynthesis of Betalains…………………………………………………………..4

1.2.3. Betalains: use and activity………………………………………………………….8

1.2.4. Synthesis of betalamic acid………………………………………………………..9

1.3. Opioid Receptors…………………………………………………………………………13

1.3.1. Introduction………………………………………………………………………...13

1.3.2. Structural features of opioid receptors…………………………………………..14

1.3.3. ĸ-Opioid Receptor (KOR)…………………………………………………………16

1.3.4. KOR agonists and antagonists…………………………………………………..16

1.4. σ-Receptors………………………………………………………………………………18

1.4.1. Introduction………………………………………………………………………...18

1.4.2. σ1-Receptor: structure…………………………………………………………….20

1.4.3. Expression and physiological functions of σ1-receptor………………………..22

1.4.4. σ2-Receptor……………………………………………………………………….. 24

2. Aim of the project……………………………………………………………………………25

3. Ideas and synthesis plans…………………………………………………………………27

3.1. Idea and plan for the synthesis of new betaxanthin derivatives…………………….27

3.2. Idea and plan for the synthesis of conformationally restricted KOR agonists……..28

3.3. Idea and plan for the synthesis of 1,2,3 triazole derivatives with potential antitumor activity and σ-receptors affinity…………………………………………………………30

4. Synthesis of new betaxanthin derivatives………………………………………………32 I

5. Synthesis of conformationally restricted KOR agonists…………………………….55

6. Synthesis of new 1,2,3 triazole derivatives with potential antitumor activity and σ- receptors affinity…………………………………………………………………………….62

7. Pharmacological characterization………………………………………………………..69

7.1. Affinity towards σ receptors……………………………………………………………..69

7.1.1. General principle…………………………………………………………………..69

7.1.2. σ1 and σ2 binding assays………………………………………………………….71

7.2. σ-Receptors affinity of newly synthetized 1,2,3 triazole derivatives…………………72

7.3. Folin-Ciocalteu assay…………………………………………………………………… 73

8. Summary……………………………………………………………………………………...74

8.1. Part I: synthesis of new analogues of indicaxanthin………………………………….74

8.2. Part II: synthesis of advanced intermediates to obtain bicyclic KOR agonists……………………………………………………………………………………75

8.3. Part III: synthesis of new 1,2,3 triazole derivatives and evaluation of their σ-receptors affinity……………………………………………………………………………………...76

9. Experimental Part……………………………………………………………………………78

9.1. Synthesis and Analytical characterization……………………………………………..78

9.1.1. General procedures and material………………………………………………..78

9.1.1.1. General Remarks………………………………………………………….78

9.1.1.2. Solvent……………………………………………………………………...78

9.1.1.3. Thin Layer Chromatography……………………………………………...79

9.1.1.4. Flash column chromatography…………………………………………...79

9.1.1.5. Melting points………………………………………………………………79

9.1.1.6. HPLC……………………………………………………………………….79

9.1.1.7. NMR spectroscopy………………………………………………………..80

9.1.1.8. IR spectroscopy……………………………………………………………81 II

9.1.1.9. Mass Spectrometry………………………………………………………..81

9.2. Synthetic procedures and analytical data………………………………………………82

9.3. Receptors binding studies……………………………………………………………..114

9.3.1. Materials………………………………………………………………………….114

9.3.2. Preparation of membrane homogenates from guinea pig brain……………114

9.3.3. Preparation of membrane homogenates from rat liver………………………114

9.3.4. Protein determination……………………………………………………………115

9.3.5. General procedures for binding assays……………………………………….115

9.3.6. Performance of the binding assays……………………………………………116

9.4. Folin-Ciocalteu assay…………………………………………………………………..117

9.4.1. Preliminary procedures………………………………………………………….117

9.4.2. General procedure……………………………………………………………….117

9.4.3. Determinantion of the TPC of compounds 88a, 88c, 109, 110, 114…………117

10. Bibliography………………………………………………………………………………...118

11. Appendices………………………………………………………………………………….144

11.1. List of acronyms………………………………………………………………….144

11.2. List of compounds………………………………………………………………..147

12. Curriculum Vitae……………………………………………………………………………151

III

1. Introduction

1.1. Piperidine and its derivatives

In 1819, Hans Christian Ørsted isolated from the fruits of Piper nigrum a new alkaloid named piperine (1), which was responsible for the pungency and spicy taste of the black pepper.1 In 1850 and in 1852, during their work on the alkaloid piperine T. Anderson and A. Cahours isolated independently a new base that was called piperidine (2) (Figure 1).2-4

Figure 1: Piperine (1) and Piperidine (2).

Piperidine (2) is a heterocycle that can be found widely in both natural and synthetic compounds (Figure 2). Several studies have shown the wide range of biological activities of this scaffold including antimicrobial, antitumor, antioxidant and analgesic effects. Chemists have synthetized several piperidine analogues like the natural product coniine (3), the strong analgesic fentanyl (4) and the antidepressant paroxetine (5).5-8

Figure 2: (S)-Coniine (3), Fentanyl (4) and Paroxetine (5).

In 2014, the structures of 640 drugs approved by the Federal Drugs Administration (FDA) were analysed. It was found that piperidine represents the most often heterocycle in these drugs followed by pyridine (Figure 3).9 1

Figure 3: Classification of the structural diversity of 640 drugs approved by the FDA.9 The coloured legend indicates the number of atoms of the heterocycle and if it is aromatic or non-aromatic, fused system or bicyclic.

1.2. Betalains

1.2.1. Introduction

Various species displaying a great variety of colours from red/purple to yellow/orange belong to the order of Caryophyllales. In this order plants of the family Amaranthaceae like Beta vulgaris, commonly known as red beet, and of the family of Cactaceae, which includes plants like Opuntia ficus indica are found. These plants produce colours in the range from red to purple and from purple to yellow and orange, respectively. Colours are caused by betalains, hydrophilic pigments containing nitrogen.10-14 At first, these pigments were associated with the family of anthocyanins, another class of molecules related to flavonoids that give plants colours from purple to blue. Later, it was found that the key enzymes for the production of anthocyanins are not present in the betalain-producing plants.15,16 Betalains are divided in two subclasses: betaxanthins like indicaxanthin (6) and betacyanins like betanin (7). The common element of both classes is their biosynthetic precursor betalamic acid (8) (Figure 4).

Figure 4: Indicaxanthin (6), Betanin (7), Betalamic acid (8) and cyclo-DOPA (9).

Bethaxanthins are iminium derivatives of betalamic acid (8) with different amines or amino acids. These molecules present a yellow colour independently from the amino acid or the amine condensed with betalamic acid. Betaxanthins possess an absorption maximum at a wavelength of 480 nm. Betacyanins are violet compounds derived from condensation of betalamic acid (8) with cyclo-dihydroxyphenylalanine (cyclo-DOPA 9), with an absorption maximum at 536 nm. Moreover, a sugar moiety is connected to one of the hydroxy moieties of the cyclo-DOPA part of betacyanin.10,17,18,

1.2.2. Biosynthesis of Betalains

Studies of betalains started to grow in the second half of the 20th century. The development of new spectroscopic techniques and of new extraction methods allowed the isolation, structural characterization and identification of betalains. In plants, betalains are secondary metabolites.

Several studies were performed to elucidate the enzymatic pathways leading to these compounds. Experiments with radioactively labelled L-tyrosine demonstrated its incorporation into betalains.19 The biosynthesis leading to betalains begins with the formation of betalamic acid (8) (Scheme 1).

Scheme 1: Biosynthesis of betalamic acid (8) and indicaxanthin (6).10

The first enzyme involved in this pathway promotes the hydroxylation of L-tyrosine (10) to L-DOPA (11) through the monophenolase activity of the enzyme tyrosinase. This enzyme was isolated and characterised from several betalain-forming plants: Portulaca grandiflora, Beta vulgaris and Suaeda salsa; this enzyme was also purified from a fungus belonging to the family of Amanita muscaria, which seems to produce betalain-related compounds.20-24 L-DOPA (11) produced in the first step of the biosynthetic pathway is the substrate for the second enzyme involved in the formation of betalamic acid: 4,5-DOPA-extradiol- dioxygenase. This enzyme catalyses the cleavage of L-DOPA (11) to form intermediate 4,5- seco-DOPA (12). This enzyme was first found in A. muscaria extracts25 and successively in P. grandiflora, Mirabilis jalapa and B. vulgaris.26-28 Spontaneous intramolecular condensation between the amino group of the 4,5-seco-DOPA (12) and the enzymatically produced enol provided betalamic acid (8). Betalamic acid is formed as 95:5 mixture of (S)- and (R)-configured enantiomers. This ratio of enantiomers is often found in betalamic acid isolated from plants and pigments derived from betalamic acid (8). In an in vitro experiment 4,5-DOPA-extradiol-dioxygenase converted enantiomerically pure L-DOPA (11) into the same 95:5 mixture of (S)- and (R)-configured betalamic acid.27,28

All betalains described in literature contain the betalamic acid (8) scaffold, which represents the chromogenic electron push-pull system. Depending on the nature of the condensed amine or amino acid, different spectroscopic properties can be obtained.29 In plants, the yellow betaxanthins are obtained by condensation of betalamic acid with amines or amino acids. It was assumed that the final step for the formation of betaxanthin could be a spontaneous reaction between the formyl group of betalamic acid (8) and the amino group of amines or amino acids to form the corresponding imines (Scheme 1).30,31

The biosynthesis of betacyanins like betanin (7) is more complicated and it is not yet completely elucidated. Whereas the biosynthesis of indicaxanthin (6) requires the proteinogenic amino acid proline, the modified amino acid cyclo-DOPA (9) is necessary for the biosynthesis of betanin (7). Several pathways have been proposed to explain the formation of betacyanins (Scheme 2).

Scheme 2: Biosynthesis of betacyanins. Abbreviations: AA, ascorbic acid; DAA dehydroascorbic acid; cyt P-450, cytochrome P-450.10

Betacyanins derived from the condensation of betalamic acid (8) with cyclo-DOPA (9). L- DOPA (11) is also the substrate for tyrosinase. The enzyme catalyses the oxidation of L- DOPA to o-DOPA-quinone (13). The amino group of the o-DOPA-quinone (13) is able to add Intramolecularly to the quinone system leading to the intermediate leuko-DOPA-chrome 6

(9, cyclo-DOPA).32-34 In B. vulgaris, it has been shown that cyclo-DOPA was formed by oxidation with cytochrome P-450.35 Once formed, cyclo-DOPA (9) reacts with betalamic acid (8) leading to betanidin (15), which is the key intermediate for the synthesis of different betacyanins.36 The last step of the biosynthesis is catalysed by the enzyme betanidin-5-O- glucosyltransferase. This enzyme catalyses the transfer of glucose to the 5-hydroxy moiety producing betanin (7).37

The proposed mechanism for the biosynthesis of betacyanins is unlikely to occur at biological level for several reasons. First of all, cyclo-DOPA (9) undergoes spontaneous oxidation to form DOPA-chrome (14). A reducing agent like ascorbic acid (AA, scheme 2) is necessary to avoid formation of this compound that polymerises leading to a brown polymer. Moreover, the concentration of L-DOPA (11) is constant during the biosynthesis. L-Tyrosine is the only metabolite existing in different concentrations during the biosynthesis.38 Therefore, another plausible mechanism was proposed for the biosynthesis of betanidin (15) (Scheme 3). The mechanism involves the formation of a betaxanthin by condensation of betalamic acid (8) with L-tyrosine (10) leading to portulacaxanthin II (16). Betaxanthin 16 can be a substrate for the enzyme L-tyrosinase that catalayses the formation of dopaxanthin (17).39 Dopaxanthin (17) is also a substrate for L-tyrosinase, which is able to oxidize it to dopaxanthin-quinone 18. In plants, where dopaxanthin (17) is produced, studies have shown that high levels of ascorbic acids are present.39 When the concentration of ascorbic acid is low, dopaxanthin-quinone 18 can undergo spontaneous cyclization leading to betanidin (15).40 Betanidin-5-O-glucosyltransferase catalyses the transfer of a glucose molecule to the hydroxy group in 5-position of the aromatic ring leading to betanin (7).

Scheme 3: Alternative mechanism for the biosynthesis of betanin (7).

1.2.3. Betalains: use and biological activity

During the last years, industry became interested in betalains as food colourants. 41,42 Furthermore, betalains reveal antioxidant and radical scavenging properties. 29,43-51Further studies showed antiproliferative and chemoprotective properties.52-55 It was also demonstrated that these molecules work synergistically with other natural antioxidants to protect LDL and red blood cells from oxidation.56-59 Moreover, betalains showed also anti- inflammatory properties. These molecules are able to reduce the production of pro- inflammatory factors such as interleukin-8 and to enhance the production of factors like interleukin-10, which inhibits the production of pro-inflammatory cytokines.60-63

Betalains used for biological studies derived from pigments extracted directly from plants using a solid-liquid extraction. Vegetables were typically macerated to make the diffusion of 8 the compounds easier even though during the destruction of the tissue several additional cellular components were released requiring further purification steps. In case of betalains, the material containing the pigments was extracted with water, although the use of methanol or ethanol could facilitate the extraction. Unfortunately, this method required long extraction times, further purification steps and resulted in low yields. Therefore, novel extraction techniques were applied to improve the efficiency of betalain isolation. The techniques include diffusion extraction, ultrafiltration, reverse osmosis and cryogenic freezing.51,64-67 Another major problem during the extraction and purification of these pigments is their chemical instability towards acid and base, light exposure and heat. These factors influence the efficiency of the extraction and purification processes.68 Strategies to improve the stability of the molecules have been used especially in the food industry, where these pigments are widely used. 69

1.2.4. Synthesis of betalamic acid

Betalamic acid (8) is the key intermediate for both subclasses of betalains: betacyanins and betaxanthins. It is produced predominantly by extraction of the pigments followed by basic hydrolysis, although the yields are low and condensation reactions took place during the process. Other techniques have been developed such as the use of modified cell lines for the in vitro production of the pigments. Alternatively, a key enzyme of the biosynthesis such as 4,5-DOPA-extradiol-dioxygenase can be used in a bioreactor for the production of betalamic acid (8).70,71

In literature, two syntheses of racemic betalamic acid derivatives are reported. The first synthesis developed by Dreiding et al.72,73 started with chelidamic acid (19). Hydrogenation of 19 in the presence of rhodium on activated alumina afforded an all-cis-configured piperidine derivative, which was converted into the dimethyl ester 20 upon treatment with methanol and HCl. Oxidation of the secondary alcohol led to the piperidin-4-one 21. To avoid overoxidation to the corresponding pyridine derivative, a polymeric carbodiimide was used for the Pfitzner-Moffatt oxidation and the transformation was carefully monitored. For the introduction of the side chain, a fully methyl-protected semicarbazide was employed as Horner-Wittig reagent. This reagent led to the formation of hydrazone 22 as pure (E)- configured diastereomer (C=N-bond). Dehydrogenation of 22 with t-butyl hypochlorite and triethylamine (NEt3) provided dihydropyridine 23 as 7:3 mixture of (E)- and (Z)-configured

9 diastereomers. In this case (E)- and (Z)-configuration refers to the exocyclic C=C-double bond whereas the C=N-double bond is still (E)-configured. Recrystallization from t-butanol provided pure (E,E)-configured betalamic acid derivative (E,E)-23. (Scheme 4).72-75

Scheme 4: Synthesis of betalamic acid derivative 23 according to Dreiding et al.72,73.

In Scheme 5 the second strategy for the synthesis of betalamic acid (8) developed by Bϋchi et al.76 is displayed. This approach started from benzylnorteleoidine 24 obtained by a Robinson-Schӧpf condensation, following a procedure developed for the synthesis of teloidinone.77 The first reaction includes a protection of the diol by formation of a cyclic ortho ester. Hydrogenolytic cleavage of the N-benzyl protective group provided the secondary amine 25. Reaction of the aminoketone 25 with allyl magnesium chloride yielded the tertiary alcohol 26 with high diastereoselectivity. The secondary amine 26 was then converted into

O-benzoylhydroxylamine 27. First, amine 26 was neutralized with K2CO3 and reacted with dibenzoyl peroxide in DMF leading to the protected amine. Subsequently, acetylation of the alcohol provided O-benzoylhydroxylamine 27. Next, the ortho ester was cleaved with oxalic 10 acid in water to obtain diol 28. This latter compound was then oxidized with N- chlorosuccinimide (NCS) and dimethylsulfide to achieve the diketone 29. Ozonolysis of 29 led to the aldehyde 30. Treatment of 30 with lead tetraacetate in benzene and methanol converted the diketone moiety into an unstable dicarboxylic acid which upon loss of HOAc and BzOH yielded (±)-betalamic acid (8) as a mixture of (E)- and (Z)-configured diastereomers after silica gel chromatography.74

Scheme 5: Synthesis of betalamic acid (8) according to Bϋchi et al.76

Despite the fact that two methods for the synthesis of betalamic acid (8) and its derivatives have been reported in literature, most of the studies using betalamic acid derivatives were conducted using compounds extracted from plants.This might be explained by the rather low overall yields, which were obtained by both strategies. Therefore, an efficient method to obtain betalamic acid (8) and its derivatives in large amounts is highly desired to proceed with careful analysis of the biological properties of this type of compounds.

1.3. Opioid Receptors

1.3.1 Introduction

Opium, the dried latex extract from Papaver sonniferum, contains a mixture of alkaloids; (Figure 5) the major one is morphine (31), a compound well known for its analgesic properties. Other alkaloids present in opium are codeine (32) and thebaine (33) and non- analgesic alkaloids like papaverine (34) and noscapine (35). The pharmacological effects of opium are known since prehistory and described in many important documents. Theophrastus cited opium use around 300 BC. Sumerian, Assyrian, Egyptian, Indian, Minoan, Greek, Roman, Persian and Arab Empires made widespread use of opium, which was the most potent form of pain relief available. Almost forgotten during Middle Ages, it was reintroduced by Paracelsus (1500 AD). Opium spread through Europe during 17th century. In China, opium abuse generated a conflict between China and England (1839- 1842). In 1806, the pharmacist Friederich Sertϋrner isolated for the first time morphine (31), Sertϋrner’s discovery opened the way for the use of morphine and the other analgesic alkaloids isolated by Robiquet (codeine 32, 1832 AD) and Merck (papaverine 34, 1832 AD) in the medical field.78

Figure 5: Major alkaloids extracted from Papaver sonniferum: morphine (31), codeine (32), thebaine (33), papaverine (34) and noscapine (35).

During the second half of the 20th century, the first studies appeared regarding the receptors for these alkaloids. The presence of these receptors in the mammalian brain was demonstrated by binding assays with radiolabeled compounds. In 1971, the use of [3H]levorphanol as radioligand for the study of these receptors was reported by Goldstein et al.79 In 1973, Candace B. Pert and Solomon H. Snyder published the first detailed binding study using [3H]naloxone that led to the discovery of the µ-opioid receptor (MOR).80 To date, four opioid receptor subtypes have been isolated and characterized: the MOR, the ĸ-opioid (KOR) and the δ-opioid receptor (DOR), which are considered as “classical” opioid receptors. In 1994, a fourth subtype was discovered, isolated and named nociceptin/orphanin FQ receptor (NOR). The opioid receptors got their names from the compounds binding to them (e.g. µ for morphine, ĸ for ketocyclazocine) or from the tissue where high density was observed (δ from mouse vas deferens).81 The endogenous agonists for all of the four opioid receptors have been identified: endorphin for MOR, enkephalins for DOR, dynorphins for KOR and nociceptins for NOR.82-84

1.3.2. Structural features of opioid receptors

Opioid receptors belong to the class A (rhodopsin-like) family, which is coupled with the 85,86 inhibitory Gi/Go protein. The binding of an endogenous or exogenous agonist to these receptors induces multiple intracellular effects such as inhibition of the adenylyl cyclase and voltage-gated Ca+2 channels. Furthermore, K+ channels are opened and phospholipase Cβ (PLCβ) is activated.87-89 Opioid receptors consist of seven transmembrane domains (7TM) connected by three extracellular and three intracellular loops, an N-terminal extracellular domain and a C-terminal intracellular domain (Figure 6).85 The transmembrane domains of all opioid receptors show a homology of more than 60 %. Due to this homology, many agonists can interact unselectively with more than one opioid receptor leading to analgesia but also to other side effects.90-92 Opioid receptors are found both in the CNS and, except for NOR, in peripheral tissues. The activation in different tissues leads to different effects (Table 1).85,93

Figure 6: Serpentine model of opioid receptors. Transmembrane helices are labeled with roman numbers. Intracellular (IL) and extracellular loops (EL) are labelled with arabic numbers. White empty circles are nonconserved amino acids among OR. White circles with a letter represent identical amino acids among all four OR. Violet circles represent identical amino acids among MOR, DOR, and KOR. Green circles are highly conserved fingerprint residues of class A receptors.85

Table 1: Distribution of OR in the body and response on activation.85

Receptor Location Response on activation

brain, peripheral sensory analgesia, physical dependence, DOR neurons antidepressant effects, seizures brain, spinal cord, analgesia, immunomodulation, sedation, KOR peripheral sensory diuresis, dysphoria neurons brain, spinal cord, analgesia, physical dependence, respiratory MOR peripheral sensory depression, euphoria, reduced neurons, intestinal tract gastrointestinal motility, obstipation anxiety, pain, depression, increased NOR brain, spinal cord appetite

1.3.3. ĸ-Opioid Receptor (KOR)

KOR is found both in the central nervous system and in the periphery. KOR is a Gi/Go protein coupled receptor. Activation of KOR leads to reduction of neurotransmitter release with concomitant inhibition of signal transmission of neurons. Only in 2012, Stevens and coworkers were able to obtain the first crystal structure of KOR in complex with the highly selective KOR antagonist JDTic94 (36) (Figure 7).95,96 The crystal structure of KOR/JTDic represents the inactive form of the receptor whilst in 2018, Roth et al. published the crystal structure of the active form of KOR i.e. KOR in complex with the agonist, MP1104 (37) (Figure 7).97,98

Figure 7: Structures of KOR antagonist JDTic (36) and KOR agonist MP1104 (37) used for crystallization of KOR.

1.3.4. KOR agonists and antagonists

Activation of KOR in the central nervous system leads to strong analgesia, which is associated with centrally mediated dysphoria, sedation and diuresis.99,100 KOR agonists, which are not able to penetrate into the central nervous system, activate selectively KORs located in the periphery. Compounds of this type can be used for the treatment of inflammatory visceral and neuropathic pain in the periphery without causing the above mentioned centrally mediated side effects.101,102 Moreover, peripherally acting KOR agonists are useful for the treatment of itching skin diseases, such as atopic dermatitis and psoriasis.103,104 On the contrary, antagonists of centrally located KORs show antidepressant and anti-addictive effects. Thus, KOR antagonists are beneficial for the treatment of withdrawal symptoms caused by abuse of ethanol, cocaine or methamphetamine.105

Four classes of KOR agonists are known: peptides, morphinans and benzomorphans, terpenoids and ethylenediamines. Morphinans and benzomorphans represent the first

16 detected class of KOR agonists and are morphine derivatives. Some of these compounds show high affinity towards opioid receptors, but poor selectivity toward a particular opioid receptor subtype. Nalfurafine (38)106,107 is a morphinan derivative that was approved as KOR-selective drug for clinical treatment of uremic pruritus in patients subjected to hemodialysis.108 Ketocyclazocine (39)109 belongs to the class of benzomorphans and is a partial agonist of KOR.

Synthetic peptides such as difelikefalin (40) and CR665 (41) represent the second class of KOR agonists. Their hydrophilicity and size allow only interactions with KOR in the periphery, because they are not able to cross the blood-brain barrier avoiding centrally mediated side effects.110,111

Terpenoids, e.g. salvinorin A (42), belong to the third class of KOR agonists. This compound lacks a basic amino group, which had been considered essential for KOR activation for a long time. Several studies were done to understand the interaction between salvinorin A (42) and KOR, which is still not completely elucidated.112

U-50,488 (43) represents the prototypical member of the ethylenediamine class of KOR agonists (Figure 8).

Figure 8: Examples of the four classes of KOR agonist: nalfurafine (38), ketocyclazocine (39), difelikefalin (40), CR665 (41), salvinorin A (42) and U-50,488 (43).

1.4. σ-receptors

1.4.1. Introduction

In 1976, Martin et al. postulated the existence of three opioid receptors (µ, ĸ, and σ receptors) during animal studies with morphine and different benzomorphans. Morphine (31) is the prototype agonist of MOR. Activation of MOR causes miosis, bradycardia, hypothermia depression of nociceptive responses and indifference towards environmental stimuli. Ketocyclazocine (39) activates KOR and produces sedation without altering pulse rate markedly. In contrast, the  agonist (±)SKF-10,047 (44) causes mydriasis, tachypnea, tachycardia and mania (Figure 9).113

Figure 9: Morphine (31), ketocyclazocine (39), SKF-10.047 ((+)-44, (-)-44) and phencyclidine (45).

After the first hypothesis on the existence of σ receptors, several efforts were undertaken to identify this receptor and its endogenous ligands. (±)-SKF-10,047 ((±)-44) was used as σ receptor agonist in the above mentioned animal studies. However, this compound presents a complex pharmacological profile. In benzomorphan derivatives, stereochemistry plays a crucial role for the interaction with receptors. The laevorotatory enantiomer (-)-SKF-10.047 ((-)-44) has higher affinity for MOR and KOR, whilst the dextrorotatory enantiomer (+)-SKF- 10,047 ((+)-44) shows high σ affinity.114 Later, it was observed that (+)-SKF-10,047 binds to two different binding sites. At first, it was assumed that both binding sites were constituted by the same receptor. However, the use of the radioligands [3H]SKF-10.047 and [3H]PCP ([3H]45) demonstrated that the identified binding sites belong to two different proteins, i.e. the  receptor and the NMDA receptor.

In 1990, a study showed the existence of two σ receptor subtypes while looking for the presence of σ receptors in pheochromocytoma (PC12) cells.115 Hellewell and Bowen used (R)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((R)-3-PPP 46) and 1,3-di-o-tolylguanidine (DTG 47) to avoid the cross-binding to the “PCP receptor” of (+)-SKF-10,047 (Figure 10). Both compounds are highly selective σ ligands. Binding sites for [3H](R)-3-PPP and [3H]DTG

19 in PC12 cell line show similarities with the σ receptors present in guinea pig brain like the high affinity binding of (R)-3-PPP (46), DTG (47) and haloperidol (48).

Figure 10: (R)-3-PPP (46), dit-o-olylguanidine (47) and haloperidol (48).

However, the identified binding sites in PC12 cells showed considerable differences in binding of (+)-SKF 10,047, (+)-pentazocine and other (+)-benzomorphans and (+)- morphinans receptor. PC12 cells binding site exhibited low to negligible affinity for (+)- benzomorphans and (+)-morphinans and higher affinity for (-)-benzomorphans. In contrast, guinea pig brain σ receptors exhibited a strong preference for (+)-benzomorphans over (-)- benzomorphans. As a result, the binding sites in PC12 cells and guinea pig brains have opposite stereoselectivty. Thus, [3H]DTG/[3H](R)-3-PPP binding sites in PC12 cells were

115 termed σ2 receptors and the receptor in guinea pig brain tissues was termed σ1 receptor. Up to date, the σ receptor is accepted as a particular receptor class with two subtypes, the 116 σ1 and the σ2 receptor.

1.4.2. σ1 Receptor: structure

In 1996, Hanner et al. were able to isolate, purify and clone the σ1 receptor from guinea pig 117 liver. In the same year, Kekuda et al. were able to clone the human σ1 receptor from human placental choriocarcinoma cells for the first time. The protein consists of 223 amino acids and has a mass of 25.3 kDa with a single putative transmembrane domain. The amino acid sequence of human σ1 receptors shows a significant homology to the guinea pig receptor (93 % identity, 96 % similarity) but does not show any homology to any other mammalian protein. However, the σ1 receptor reveals a 30 % identity, a 67 % homology, and a similar ligand binding profile as the Δ8/Δ7-sterol isomerase of yeasts.118-120 In 2002,

Aydar et al. proposed a first model of the topology of the σ1 receptor. The main features of this model were two transmembrane domains, a ~50 amino acids extracellular loop, a short

(~10 amino acids) intracellular N-terminal end and a long (~125 amino acids) C-terminal 121 end. Hydrophobicity analyses suggest a topological similarity between the σ1 receptor and the fungal sterol isomerase, which implies the presence of three hydrophobic regions. It was proposed that two of these three regions are similar to steroid binding domains. Using photoaffinity labelling, the presence of these two hydrophobic regions was confirmed. Later studies demonstrated that the ligand binding site is close to these two hydrophobic regions. (Figure 11).122-125

Figure 11: Model of the human σ1 receptor; amino acids of the SBDL I and SBDL II are colored in blus; amino acids important for haloperidol (48) binding are labeled in red; amino acids important for (+)- pentazocine binding are shown in yellow; green are the amino acids important for cholesterol binding.125

In 2011, a 3D homology model of the σ1 receptor was proposed. Four different proteins (PDB codes 3CIA, 1I24, 2Z2Z, and 2Q8I) sharing ≥30 % identity with the corresponding 126 parts of the σ1 receptor were used as templates for the construction of the model. Only small parts of the receptor had to be built up de novo. The model was validated using data obtained from photoaffinity labeling studies, mutagenesis experiments, structure−affinity relationships, pharmacophore models, and ligand docking/molecular dynamics (MD) calculations.125,127 In 2016, two crystal structures of the protein were published in complex with two different ligands, PD144418 (49) and 4-IBP (50) (Figure 12).128

Figure 12: Ligands in complex with crystallized σ1 receptor: PD14418 (49), 4-IBP (50).

1.4.3. Expression and physiological functions of σ1 receptors

It was shown that σ1 receptors are found both in the CNS and in peripheral tissues. The

σ1 receptor is present in liver, heart, kidney, endocrine organs and eye. Furthermore, it was also found in proliferating tumor cells and in leukocytes.129-141 The exact physiological role of the σ1 receptor is still not completely clear. Many studies have shown the 142-149 involvement in neurotransmission and modulation of ion channels. Moreover, σ1 receptors play a role in important human diseases including schizophrenia,150,151 depression,152,153 Alzheimer’s disease154 and drug/alcohol dependence155,156. Antagonists enhance the pain-relieving effect of opioid analgesics indicating that these receptors are involved in the antinociceptive system.157

To date, the endogenous ligand of σ1 receptor is unknown. Some hypotheses proposed 158 neurosteroids like progesterone and pregnenolone as natural σ1 receptor ligands. 159 Moreover, σ1 receptors are able to bind cholesterol. Recently, N,N-dimethyltriptamine was supposed to be the endogenous ligand.160

Ligands for σ1 receptor with high affinity and selectivity have different structural features. Generally, dextrorotatory benzomorphans are considered agonists whilst drugs like haloperidol are antagonists of this receptor. Moreover, several clinically used drugs such as psychostimulants (e.g. cocaine),161 fungicides (e.g., fenpropimorph),120 anxiolytics (e.g. opipramol),162 anti-Alzheimer drugs (e.g. donepezil),163 centrally active antitussives (e.g. dextromethorphan)164 as well as antidepressants (e.g. sertraline, fluoxetine)165 are endowed with high to moderate σ1 affinity. Since σ1 receptor ligands are quite different, it was important to find general structural features to design new ligands with high affinity and selectivity. A key feature of all σ1 receptor ligands is a basic amino moiety. All σ1 ligands (exception neurosteroids) share this common structural element. The basic amino 22 moiety seems to be pivotal for the interactions with the σ1 receptor. In 1993, Glennon et al. proposed a model based on phenylalkylamine derivatives (51) (Figure 13).166

Figure 13: Lead compound used for the pharmacophore model of Glennon et al.166

In this study, it was observed that secondary and tertiary amines are well tolerated. Cyclic amines including pyrrolidine and piperidine derivatives have high affinity, but quaternary ammonium compounds are not accepted by the σ1 receptor. Configuration of the chiral center adjacent to the amino moiety appears to play a minimal role. When the phenethylamines possess an α-methyl group. In case of larger substituents, a greater effect of the configuration on the σ1 receptor binding was observed. Removal of the methyl group has little to no effect on σ1 affinity. The two hydrophobic groups A and B can be aromatic or aliphatic. The distance between group A and the amino moiety should be in the range 2.9- 3.9 Å. The size of group A has a greater influence than size of group B (Figure 14).166

166 Figure 14: Pharmacophore model for σ1 receptor ligands developed by Glennon et al.

1.4.4. σ2 Receptor

115 The σ2 receptor was identified by Hellewell and Bowen in PC12 tumor cells. The σ2 receptor has a molecular weight of ~21 kDa. In contrast to the σ1 receptor, which was cloned firstly in 1996 and crystallized 20 years later by Kruse and coworkers,128 the knowledge about the structure of the σ2 receptor was only limited for a long time. Very recently, the identity of the σ2 receptor and the transmembrane protein TMEM97 located in the endoplasmic reticulum was proven. Its structure shows four transmembrane helices with both the C- and N-terminus located on the cytosolic side. Mutagenesis experiments revealed 167 the importance of Asp29 and Asp56 for the binding of basic σ2 receptor ligands. the σ2 receptor was validated as biomarker for tumor cell proliferation and as target for 168 chemotherapy. The density of σ2 receptors in proliferative breast cancer cells is 169 considerably higher than in the quiescent cells. The expression of σ2 receptor is up- regulated during the transition from quiescent to proliferative status and down-regulated 170 during the opposite development. The high σ2 receptor density can be used as diagnostic tool to establish the proliferative status of solid tumors171 and stimulates the development of 172-174 new σ2 receptor addressing drugs to treat solid tumors. Development of σ2 receptor radioligands can be used to image tumors by positron emission tomography (PET) or single emission computed tomography (SPECT)175-179

The potential application of novel drugs addressing σ2 receptors is very promising.

Unfortunately, the mechanism of action is not known yet. Studies have revealed that σ2 receptors play a crucial role in the regulation of tumor cell proliferation and that ligands are able to induce apoptosis. An example is represented by Kashiwagi and coworkers. They 180 showed that σ2 receptor ligands stimulated the activity of caspase-3. Another mechanism involves cytosolic calcium that can be caspase- and p53- independent. Down-regulation of P-gp, a key player in multidrug resistence was also observed.181

2. Aim of the project

The aim of this project is the development of a novel synthesis of betalamic acid derivatives starting from piperidine derivatives.

Scheme 6: Development of new betalamic acid derivatives.

Intermediates of the synthesis will be used for the synthesis of conformationally restricted KOR agonists.

Scheme 7: Development of conformationally restricted KOR agonists.

Finally, the side chain in 4-position of the piperidine ring will be exploited to introduce pharmacophoric structural elements characteristic for 1 and 2 ligands, such as a triazole moiety. Ligands designed for the interaction with 1 and 2 receptors will be tested in receptor binding studies and relationships between the structure and the 1 and 2 affinity will be analysed. Furthermore, the antitumor activity of selected compounds will be recorded.

Scheme 8: Development of new σ1 and σ2 ligands.

3. Ideas and synthesis plans

3.1. Idea and plan for the synthesis of new betaxanthin derivatives

The first part of the project was dedicated to the synthesis of new betaxanthin derivatives using indicaxanthin (6) as lead compound. At first the carboxy moieties in 2- and 6- position should be removed (compound 61) in order to analyse their effects on the antioxidant activity. To get access to the proline derivative 61 the aldehyde 52 should be synthesized allowing the introduction of various imino moieties in the side chain (Scheme 9).

Scheme 9: Idea for the development of new decarboxylated derivatives 61 of betaxanthin.

In Scheme 10 the plan for the synthesis of 61 is displayed. Starting from piperidin-4-one (62) oxidation and Wittig reaction should lead to the conjugated system of 63 important for the antioxidant activity. The α,β-unsaturated ester 63 will be reduced to the α,β-unsaturated aldehyde 52. The key intermediate 52 will be used to introduce imino or iminium moieties to obtain 61 as analog of betaxanthin 6.

Scheme 10: Plan for the synthesis of betaxanthin analogue 61.

3.2. Idea and plan for the synthesis of conformationally restricted KOR agonists

In this part of the project novel conformationally restricted KOR agonists will be developed, which are derived from the potent piperazine-based KOR agonist 64 (Ki = 0.31 nM) (Scheme 11).182 Conformational restriction can be achieved by systematic connection of the terminal methyl moiety of the side chain of 64 with different positions of the piperazine ring. Some derivatives have already been prepared and pharmacologically evaluated (Scheme 11).183- 188

Scheme 11: Development of conformationally restricted KOR agonists derived from the piperazine derivative 64.

The KOR affinity of 67 is rather low (Ki = 73 nM). This can be due to an unfavorable dihedral angle of the KOR pharmacophoric ethylenediamine structure NpyrC-C-Namide of 58.3° according to X-ray crystal structure analysis. Alternatively, the additional basic N-atom at the bridgehead position (1-position) could be responsible for the reduced KOR affinity. In order to analyse the reason for the reduced KOR affinity, the second project is devoted to the synthesis and pharmacological evaluation of azabicyclic compounds 55, which represent analogues of 64 without the bridgehead N-atom. The length of the bridge will be modified in order to change the dihedral angle of the KOR pharmacophore.

The synthesis of 2-azabicyclooctanes 55a (n = 0), -nonanes 55b (n = 1) and –decanes 55c (n = 2) will start with the piperidine derivative 58. The ketone in 4-position of the piperidine ring is used to install the unsaturated side chain of 57. The preferred reaction for this transformation is the Wittig reaction using homologous Wittig reagents

Ph3PCH(CH2)nCO2CH3 (n = 0-2) to obtain the homologous esters 57a-c (Scheme 12).

Scheme 12: Plan for the synthesis of new conformationally restricted KOR agonists.

The allyl strain induced by the large Boc group at the piperidine N-atom forces the adjacent ester moiety to adopt the axial orientation. Thus, the axially oriented ester moiety will shield the upper side of the double bond directing the subsequent hydrogenation of the double bond of 57 to the backside giving predominantly cis-configured piperidine

29 derivatives 70. In the next step, the bridge will be established by a Dieckmann cyclization of the cis-configured diesters 70 providing the β-ketoesters 56. Saponification and decarboxylation of β-ketoesters 56 will lead to the ketones 71, which represent the key intermediates in this project as they allow the diastereoselective introduction of exo- and endo-configured amino groups, e.g. a pyrrolidinyl moiety. At the end of the synthesis the Boc-group is cleaved off and the resulting secondary amines are acylated with various acyl chlorides. The preferred acyl moiety is the 3,4-dichlorophenylacetyl group as shown for compounds 55. In addition to the ring size, diversity is achieved by introduction of various amino and acyl moieties at the end of the synthesis. Moreover, the ester moiety of the intermediate β-ketoesters 56 can be used to introduce various substituents, in particular polar groups (CH2OH, CH2NR2, etc.) The stereochemistry represents a further dimension of diversity in this compound class.

The KOR affinity of all final compounds 55 and some precursors will be tested in receptor binding assays using the radioligand [3H]U-69.593 and guinea pig brain membrane preparations. The affinity towards MOR and DOR will also be determined (receptor binding assays) in order to check the selectivity of the KOR ligands.

3.3. Idea and plan for the synthesis of 1,2,3 triazole derivatives with potential antitumor activity and σ receptors affinity.

The 1,2,3-triazole ring is an aromatic five-membered heterocycle containing three adjacent N-atoms. The triazole moiety is able to form various non-covalent interactions with proteins (enzymes and receptors) such as hydrogen bonds, ion-dipole, cation-π, and π-π interactions.188 Moreover, the 1,2,3-triazole is able to mimic different functional groups like amides and esters making this heterocycle useful for structural modifications and the synthesis of new biologically active compounds.190,191 In some cases the substitution of an amide group with a 1,2,3-triazole moiety led to compounds with anticancer activity higher than natural products towards several human tumor cell lines.192-199

It has been shown that the natural product indicaxanthin (6) extracted from Opuntia ficus- indica exerts interesting anti-proliferative and pro-apoptotic activities in human colonrectal carcinoma (Caco-2) cells.54,55 Therefore, it was planned to introduce the 1,2,3-triazole moiety into this natural product and evaluate the anticancer activity of the resulting 1,4 30 disubstituted 1,2,3-triazole derivatives. Furthermore, the σ receptor affinity of the triazoles should be investigated.

For the synthesis of novel triazole derivatives 72 the allylic alcohol 74 will be used as starting material. The hydroxy group of 74 will be transformed into an azido group to obtain compound 73, which represents the key intermediate of the third part of this thesis. Various alkynes will be selected to perform Click reactions with the azide 73 to obtain various triazole derivatives 72. The σ receptor affinity and antitumor activity of triazoles 72 will be investigated (Scheme 13).

Scheme 13: Plan for the synthesis of new 1,2,3-triazole deruvatives 72.

4. Synthesis of new betaxanthin derivatives

The main goal of the first part of the project was the synthesis of aldehyde 52, an analogue of betalamic acid (8) without the carboxy moieties in 2- and 6-position of the piperidine ring. In 2011, Chang et al. reported the synthesis of the α,β-unsaturated ester 63a (Scheme 14).200

Scheme 14: Approach of the synthesis of of ester 63a according to Chang et al..200

The α,β-unsaturated ester 63a represents the protected precursor of aldehyde 52, key intermediate for the synthesis of the new betaxanthin analogues. Unfortunately, Chang’s approach required the synthesis of the non-commercially available sulfonamide 75a that is used as starting material. To obtain sulfonamide 75a, the commercially available piperidin-

4-one hydrochloride monohydrate (62) was neutralized with triethylamine (NEt3) and reacted with benzenesulfonyl chloride to provide the sulfonamide 75a in 98 % yield (Scheme 15).201

Scheme 15: Synthesis of the sulfonamide 75a200 and α-bromination.199

The second step of the synthesis involved an α-bromination of ketone 75a (Scheme 15).200 The reaction described in literature200 was performed with freshly recrystallized N- bromosuccinimide (NBS) as source of bromine and acetic acid as solvent. The resulting product mixture had to be purified, because of the presence of vinyl bromide 78. The desired -bromoketone 77 could be isolated by flash column chromatography (fc). However, the

32 yield of 77 was only 35 % which was attributed to an instability of the α-bromoketone 77 during fc purification.

Treatment of the α-bromoketone 77 with NaOMe dissolved in a mixture of methanol and THF led to the dimethyl ketal 76 (Scheme 16).200

200 Scheme 16: Reaction of -bromoketone 77 with NaOCH3.

In this reaction, formation of a ketal with concomitant substitution of the bromide by a hydroxy moiety was observed. Chang et al. postulated the following reaction mechanism for this transformation (Scheme 17).200

Scheme 17: Possible mechanism for the formation of ketal 76.200

At first, nucleophilic attack of methanolate at the carbonyl moiety leads to the hemiketal anion I. Intramolecular nucleophilic attack leads to the formation of the epoxide II with the removal of bromide. The epoxide II undergoes ring-opening by methanolate producing the ketal 76 with a yield of 26 %.

In the next step, compound 76 was treated with trimethyl orthoformate and (ethoxycarbonylmethylene)triphenylphosphorane (79a) in the presence of p- toluenesulfonic acid (p-TsOH). With this reaction, the simultaneous formation of the side chain and the conjugated system of double bonds was planned to give the desired compound 63a (Scheme 18).

Scheme 18: Reaction of ketal 76 with orthoester and Wittig reagent 79a.199

For this reaction, the mechanism proposed by Chang et al.200 involves first the formation of intermediate II under acidic condition. Subsequently, stereoselective olefination of II generated intermediate (E)-III, which underwent dehydration promoted by trimethyl orthoformate via the intermediate IV to give the diene 63a (Scheme 19).200 Unfortunately, despite several attempts it was not possible to isolate the desired compound 63a using these experimental conditions.

Scheme 19: Mechanism proposed by Chang et al.200 for the formation of compound 63a.

Since the transformation of ketal 76 into the desired α,β-unsaturated ester failed, another approach to obtain 63a was pursued (Scheme 20).

Scheme 20: Second approach for the synthesis of intermediates of betaxanthin derivatives.

In this second approach the double bond in 2/3-position should be introduced before the formation of the α,β-unsaturated ester or aldehyde. For this purpose three piperidin-4-ones 75a, 75b and 75c with different N-protective groups were selected. At first, the dehydrogenation of the N-benzyl protected ketone 75b was carried out using mercury (II) acetate (Hg(OAc)2) in the presence of ethylenediaminetetracetic acid (EDTA) in a mixture of water and ethanol (Scheme 21).202

201 Scheme 21: Dehydrogenation of various piperidin-4-ones 75a, 75b and 75c with Hg(OAc)2.

These reaction conditions provided the dihydropyridine 80b in 36 % yield. Different conditions were investigated to increase the yield of 80b. It was observed that in the presence of acid EDTA the yield was always lower than 36 %. However, when employing

Na2EDTA the yield was increased to 70 %. The reaction was also tried with Boc protected piperidin-4-one 75c and with sulfonyl protected piperidine-4-one 75a. However, in case of carbamate 75c and sulfonamide 75a a transformation could not be observed, only

35 piperidines 75c and 75a were reisolated (Table 2). This observation can be nicely explained by the reaction mechanism, which starts with a coordination of Hg2+ with the basic amino moiety of 75b (see Scheme 22). Acetate is now able to remove a proton in α-position to the N-atom leading to the formation of a cyclic imine, which tautomeizes to give the dihydropyridone 80b. 202,203

Scheme 22: Mechanism for the formation of dihydropyridone 80b.

Table 2: Experimental conditions used to oxidize the piperidine-4-ones 75a, 75b, 75c.

compd. R temperature time Hg(OAc)2/EDTA solvent yield

75b Bn 80 °C 2 h 1.05 eq.,1.05 eq. acid H2O:EtOH 36

1.05 eq., 1.05 eq. 75b Bn 80 °C 2 h H2O:EtOH 70 Na2EDTA 1.05 eq., 1.05 eq. 75c Boc 80 °C 2 h H2O:EtOH - acid 1.05 eq., 1.05 eq. 75c Boc 80 °C 2 h H2O:EtOH - Na2EDTA 1.05 eq., 1.05 eq. 75a SO2Ph 80 °C 2 h H2O:EtOH - Na2EDTA 4.0 eq., 4.0 eq. 75a SO2Ph 90 °C 2 h AcOH 5 % - Na2EDTA 4.0 eq., 4.0 eq. 75a SO2Ph 90 °C 16 h AcOH 5% - Na2EDTA

In the next step, dihydropyridone 80b should be converted into the α,β-unsaturated aldehyde 86b using the method of Valenta et al.204 The reaction involves hydroboration of ethoxyethyne (81) with BH3·SMe2 to generate the tris(vinyl) borane 82. Transmetalation from boron to zinc at -78 °C leads to zinc intermediate 83. Addition of aldehyde or ketone generates the zinc alcoholate 84, which readily undergoes hydrolysis and elimination on work-up with 2 M HCl (Scheme 23).204,205

Scheme 23: Synthesis of α,β-unsaturated aldehyde 85 according to Valenta et al.204

Unfortunately, reaction of the (ethoxyvinyl)zinc intermediate 83 with the dihydropyridone 80b did not lead to the desired aldehyde 86b (Scheme 24).

Scheme 24: Attempt to synthesize aldehyde 86b.

Valenta’s approach did not provide the desired aldehyde 86b probably because the nucleophilicity of the zinc intermediate 83 was too low to attack the carbonyl moiety of dihydropyridone 80b. Therefore, more reactive Wittig and Horner-Wittig reagents should be employed to initiate the condensation reaction. This strategy involves the Wittig reagent 79a and the triethyl phosphonoacetate 79b deprotonated by potassium tert-butoxide (KOtBu) in DMF (Scheme 25).206-208

Scheme 25: Attempted synthesis of 63b using the Wittig and Wittig-Horner reaction.

Several experimental conditions were applied for the olefination of ketone 80b (Table 3). However, all reaction conditions using both reagents 79a and 79b failed to afford the - unsaturated ester 63b, the ketone 80b was always reisolated.

Table 3: Experimental conditions used for the olefination of ketone 80b.

reagent temperature time solvent yield Ph3PCHCO2Et 40 °C 5 h CH2Cl2 - (79a) Ph3PCHCO2Et 40 °C 24 h CH2Cl2 - (79a) Ph3PCHCO2Et 40 °C 48 h Toluene - (79a) (EtO)2POCH2CO2Et 120 °C 48 h DMF - (79b)

It was assumed that the carbonyl activity of the ketone 80b was too low for the nucleophilic attack of the Wittig reagents due to its conjugation with the N-atom in the dihydropyridine ring (vinylogous amide). Therefore, the double bond in 2/3-position of the dihydropyridine 38 ring should be introduced after olefination of the ketone. The sulfonamide 75a and the N- benzyl protected piperidine-4-one 75b were selected to react with triethyl phosphonoacetate (79b) in the olefination reaction (Scheme 26).208

Scheme 26: Olefination reactions of ketones 75a and 75b.

Deprotonation of 79b with KOtBu provided the -unsaturated esters 87a and 87b in 83 % and 66 % yield, respectively. In the next step, the synthesis required the introduction of the double bond in 2/3-position to complete the conjugated system. This dehydrogenation was tried with Hg(OAc)2, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and m- chloroperoxybenzoic acid (m-CPBA) as oxidizing agents (Scheme 27, Table 4).202,209-211

Scheme 27: Attempted oxidation of compounds 87a and 87b.

Table 4: Experimental conditions for the oxidation of compounds 87a and 87b.

oxidizing compd. R solvent temperature time yield agent

87a SO2Ph DDQ 1,4 dioxane reflux 3 h -

87a SO2Ph DDQ 1,4 dioxane reflux 16 h - -15 °C 30 min 87a SO2Ph m-CPBA CH2Cl2 - 0 °C 1 h Hg(OAc)2 H2O/EtOH 87b Bn 80 °C 2 h - 2:1 m-CPBA -10 °C 10 min 87b Bn CH2Cl2 - 0 °C 1 h 87b Bn DDQ 1,4 dioxane RT 16 h - DDQ RT 3 h 87b Bn 1,4 dioxane - reflux 16 h

Unfortunately, all experiments to oxidize the -unsaturated esters 87a and 87b with these oxidizing agents did not lead to the desired dihydropyridines 63a and 63b, only starting material was reisolated.

Scheme 25 showed that the dihydropyridone 80b could not be converted into the - unsaturated ester 63b. On the other hand, the introduction of the double bound after establishing the -unsaturated ester also failed (Scheme 27). Therefore, a new strategy for the synthesis of the fully conjugated systems 88 and 52 was planned. According to the new strategy a bromine atom should be introduced into the saturated piperidine ring (77), which allows the formation of the double bound by -elinination after olefination of the ketone, The conjugated ester 63 should give access to the key intermediate 52 (Scheme 28).

Scheme 28: Plan for the synthesis of key intermediate 52.

The new plan involves the use of sulfonamide 75a as starting material for the α-bromination. Bromination of sulfonamide 75a was carried out according to the method of Chang et al. using NBS.200 Unfortunately, this method led to the bromo derivative 77a in only low yields, since some side products were formed. For this reason, the bromination was performed with 212 Br2 in CH2Cl2. This reaction provided the α-bromoketone 77a in 99 % yield. α- Bromoketone 77a reacted with the Wittig reagent 79a providing the -unsaturated ester 89a in 94 % yield (Scheme 29).206,208

Scheme 29: α-Bromination and olefination of sulfonamide 75a.

The 1H NMR spectrum confirms the introduction of the side chain in 4-position. The spectrum shows a triplet at 1.25 ppm and a quartet at 4.13 ppm confirming the presence of an ethyl ester. The singlet at 5.96 ppm is caused by the methine proton of the exocyclic double bond (Figure 15).

Figure 15: 1H NMR spectra of compounds 77a (top) and 89a (bottom).

The next step involves the introduction of the double bond in 2/3-position by dehydrohalogenation of the α,β-unsaturated ester 89a. In order to find the best reaction conditions several bases and solvents were tried for the envisaged β-elimination (Scheme 30, Table 5).212-214

Scheme 30: Synthesis of ester 63a.

Table 5: Optimization of the dehydrohalogenation of ester 89a.

base eq. solvent temperature time yield of 63a KOtBu 3 t-BuOH 80 °C 1 h - RT 2 h Et3N 3 CH2Cl2 30 40 °C 3 h Et3N 13 Toluene 110 °C 16 h 38 Et3N 13 THF 66 °C 16 h 42 Et3N 20 Toluene 110 °C 16 h 34 DBU 1.2 CH2Cl2 40 °C 16 h 34 KOtBu 3 DMF RT 1 h - Li2CO3 6 DMF 130 °C 1 h 78 LiCl 6 Li2CO3 6 DMF 130 °C 1 h 86 LiBr 6

According to the results displayed in Table 15, Li2CO3/LiBr/DMF represent the best reaction conditions to induce the elimination reaction. Reactions performed with these conditions led to the conjugated ester 63a with 86 % yield. Moreover, the 1H NMR spectrum shows the presence of a mixture of conjugated esters 63a.

Reduction of the conjugated ester to an aldehyde represents the next step of the synthesis. At first, reduction of 63a with diisobutylaluminium hydride (DIBAL-H) and morpholine in THF was tried.216 The mechanism of this reaction involves the formation of a DIBAL-H morpholine adduct 90, which is able to transfer one hydride to an ester under mild reaction conditions. (Scheme 31).216

Scheme 31: Mechanism for the reduction of an ester with DIBAL-H in the presence of morpholine.

Unfortunately, this method failed to afford the aldehyde 88a. Furthermore, the allylic alcohol 74a could not be isolated as well probably because all the equivalents of DIBAL-H were used to form adduct 90 (Scheme 32).

Scheme 32: Attempt to reduce the ester 63a to form the aldehyde 88a.

Thus, a two-step synthesis of the aldehyde 88a was envisaged. At first, the ester 63a was reduced with LiAlH4 to provide the allylic alcohol 74a in 32 % yield. Therefore, careful optimization of this reduction was performed (Scheme 33, Table 6).217,218

Scheme 33: Reduction of conjugated ester 63a.

Table 6: Experimental conditions for the reduction of conjugated ester 63a to give allylic alcohol 74a.

reducing eq. solvent temperature time yield of 74a agent LiAlH4 2 THF 0 °C 1 h 32 LiAlH4 1 THF RT 1.5 h 38 LiAlH4 2 THF -10 °C 1 h 82* DIBAL-H 3 Toluene -78 °C 40 min 94 DIBAL-H 3 THF -78 °C 40 min 94 * During the work-up potassium sodium tartrate was added to complex Al3+.

The yield after reduction of ester 63a with LiAlH4 could be raised to 82 % upon addition of potassium sodium tartrate during the work-up procedure. However, fc purification was still

44 necessary. Reduction with DIBAL-H at -78 °C in toluene or THF led to the allylic alcohol 74a with 94 % yield without the need of fc purification. Initially, it was planned to produce the aldehyde 88a by reduction of the ester 63a with DIBAL-H. However, allylic alcohol 74a was the only product that could be isolated. The NMR spectra confirmed the formation of the alcohol 74a. The 1H NMR spectrum shows a triplet for the OH moiety at 4.57 ppm. After addition of D2O this triplet disappeared indicating this proton to be connected to a heteroatom. The triplet at 3.93 ppm was caused by the methylene moiety connected to the OH group (Figure 16). In addition, the IR spectrum confirmed the presence of the hydroxy group showing a broad band at 3356 cm-1.

1 Figure 16: H NMR spectra of allylic alcohol 74a without (top) and with (bottom) addition of D2O.

Since the direct reduction of the ester 63a did not lead to the aldehyde 88a, the oxidation of the allylic alxohol 74a was envisaged (Scheme 34).219-221

Scheme 34: Oxidation of allylic alcohol 74a.

At first, the oxidation was performed with Dess–Martin periodinane (DMP, 91), a reagent able to oxidize primary alcohols to aldehydes. The oxidizing mechanism is shown in Scheme 35.222

Scheme 35: Oxidation mechanism of primary alcohols with Dess-Martin periodinane (91).222

The oxidation of alcohol 74a with DMP led to the aldehyde 88a in 20 % yield. The byproducts formed during this reaction did not allow the complete purification of the aldehyde 88a. To improve the purification arising with DMP oxidation, 2,2,6,6-tetramethylpiperidine- 1-oxyl (TEMPO, 92) was selected as oxidizing agent. The reaction involves a catalytic cycle, where a single-electron oxidation of the nitroxide radical produces a highly electrophilic oxoammonium species, which serves as the active oxidizing agent (Scheme 36).223

Scheme 36: Catalytic cycle of TEMPO oxidation.223

In Table 7 several experiments are summarized with the aim to increase the yield of the aldehyde 88a.

Table 7: Optimization of the oxidation of allylic alcohol 74a

reagent eq. solvent temperature time yield of 88a

DMP 1.4 CH2Cl2 RT 1.5 h 20

DMP 2.0 CH2Cl2 RT 4 h -

DMP 2.2 CH2Cl2 RT 1.5 h 30 -10 °C 30 min, DMP 2.0 CH2Cl2 - RT 1 h TEMPO 0.1 CH2Cl2 RT 1 h - BAIB 1.1 TEMPO 0.1 DMF RT 16 h 76 CuCl 0.1

Finally, it was found that the reaction of allylic alcohol 74a with TEMPO (92) and CuCl provided the aldehyde 88a in 76 % yield. The presence of the formyl group was confirmed by 1H NMR and IR spectra. In the 1H NMR spectrum two doublets are present at 9.84 ppm and 9.94 ppm for the formyl proton, which indicates the presence of two diastereomeric aldehydes 88a in the ratio of 75:25.

Finally, the aldehyde 88a with a protecting group at the ring N-atom should be transformed into imines or iminium ions. (S)-Proline, (S)-aspartate and phenylethylamine were selected for these experiments (Scheme 37, Table 8).

Scheme 37: Attempts to produce iminium ions of imines from aldehyde 88a.

Table 8: Experimental conditions for the planned formation of imines or iminium ions.

yield of imine or amine eq. solvent temperature time iminium ion (S)-Proline 1 CH3OH RT 30 min - (S)-Proline 2 EtOH RT 1 h - (S)-Proline 1 CH3OH RT 1 h - CF3SO3H phenylethyl 1 CH3OH RT 3 h - amine (S)-Aspartate 10 EtOH RT 3 h -

Unfortunately, all experiments failed to give the desired imines or iminium ions. It was hypothesized that the electron withdrawing sulfonyl group at the N-atom was responsible for the failure. Therefore, it was planned to remove the sulfonyl group of aldehyde 88a before reaction with primary and secondary amines.

Scheme 38: General deprotection reaction of aldehyde 88a.

Table 9: Attempts to remove the phenylsulfonyl moiety from 88a

reagent eq. solvent temperature time yield of 52 KOH 1 EtOH RT 20 min - KOH 1 EtOH RT 1 h - KOH 3 EtOH RT 1 h - KOH 10 EtOH RT 2.5 h - KOH 100 EtOH RT 10 min - KOH 3 EtOH Reflux 15 min - TBAF 1 THF RT 2 h - TBAF 5 THF RT 1.5 h - TBAF 20 THF RT 1 h - TBAF 1 THF Reflux 1 h - Mg 10 MeOH 0 °C 1.5 h - NaOH 1 DMF RT 1.5 h - H2O NaOH 1 THF RT 26 h - H2O RT 2 h NaOH 1 THF - 50 °C 2 h Zn 20 THF Reflux 16 h - TiCl4 3 HCl - H2O Reflux 16 h - HOAc Zn 20 THF RT 24 h - NH4Cl Cs2CO3 1 MeCN RT 36 h - PhSH 1.1 SmI2, Et3N, 1 THF RT 30 min - H2O SmI2 3 THF 0 °C 30 min -

Table 9 summarizes all experiments performed to remove the phenylsulfonyl group from the N-atom of 88a. Unfortunately, all experiments failed to give the dihydropyridine derivative

52. Only unwanted side reactions were observed (Scheme 38, Table 9).224-228 TLC analysis and NMR spectra showed that aldehyde 88a reacts with the used reagents leading to new spots on TLC plates. After isolation and purification, it became obvious that these compounds were not the free amine 52.

Due to problems with the removal of the phenylsulfonyl protective group of 88a it was decided to start the synthesis with another protective group, which should be easier removed. For this purpose, the Boc-protected piperidin-4one 75c was selected as starting material. Firstly, carbamate 75c was reacted with Br2 to afford the 3-bromo derivative 77c. Whereas this reaction worked perfectly with sulfonamide 75a, carbamate 77c was isolated in only low yields. It was postulated that during the reaction with Br2 considerable amounts of HBr were formed, which led to cleavage of the Boc-group. To avoid cleavage of the Boc group various reaction conditions were tried (Table 10). However, the yield of the α-bromo ketone 77c did not exceed 46 % (Scheme 39).229-331

Scheme 39: -Bromination of carbamate 75c.

Table 10: Experimental conditions for the α-bromination of carbamate 75c.

reagent solvent temperature time Yield of 77c Br2 CH2C2 0 °C  RT 5 min  2 h 30 Br2 CHCl3 0 °C  RT 30 min  12 h 26 Br2, AlCl3 THF: Et2O 0 °C 18 h 46

According to table 10, the highest yield was obtained using Br2 and AlCl3 in a mixture of THF and Et2O.

Scheme 40: Synthesis of aldehyde 88c.

The next steps of the synthesis are shown in Scheme 40. Wittig reaction of α-bromo ketone 77c led to the α,β-unsaturated ester 89c, which underwent elimination of HBr upon treatment with LiBr and Li2CO3. DIBAL-H reduction of the α,β-unsaturated ester 63c provided the primary alcohol 74c, which was oxidized with TEMPO and CuCl to end up with the α,β-unsaturated aldehyde 88c. The α,β-unsaturated aldehyde 88c was isolate in an overall yield of 81 %. It should be mentioned that the reaction conditions of the N- phenylsulfonyl protected compounds (a-series) had to be optimized for the N-Boc-protected derivatives (c-series), respectively.207,213,221

Scheme 41: Cleavage of the Boc group.

In the next step, the Boc group of 88c should be cleaved off (Scheme 41).232-235 Trifluoroacetic acid is a common acid used for the cleavage of the Boc group. Unfortunately, the Boc group of 88c could not be removed upon treatment with TFA, the dihydropyridine 52 could not be isolated. Further experiments to remove the Boc protective group of 88c are summarized in Table 11.

Table 11: Attempts to remove the Boc group of 88c.

reagent solvent temperature time yield of 52 TFA CH2Cl2 0 °C  RT 30 min  6 h - HCl CH3OH RT 16 h - TBAF THF RT  reflux 2 h  6 h - / H2O:1,4 dioxane 90 °C 2 h 5/10* TFA/Et3SiH CH2Cl2 RT 2 h 0 *The exact yield could not be determined due to the instability of the product.

Cleavage of Boc was achieved when aldehyde 88c was heated to 90 °C in a mixture of water and 1,4-dioxane without any catalyst. 232,236 Recently, Houk et al. proposed a theory about water catalysis based on molecular dynamics studies. They predicted that ester hydrolysis in water is catalyzed by a water molecule acting as a dual acid/base catalyst.237,238 When water temperature rises, the c(H+) and c(OH-) increase due to enhanced water dissociation (-logKw at 100 °C is 12, whilst -logKw at 25 °C is 14). According to this theory, a mechanism was proposed (Scheme 42). Firstly, carbamate is activated by protonation of the carbonyl O-atom by a hydronium ion. OH- from dissociated water attacks the carboxyl moiety providing a tetrahedral intermediate, which may expel an amine (path A) or tert- butanol (path B). Cleavage of the Boc group on aromatic and aliphatic amines should go through route (B). This mechanism was also proposed by Coudert et al., when using Bu4NF as an N-Boc deprotection reagent (Scheme 42).236,239

Scheme 42: Mechanism proposed by Houk et al. for the cleavage of a Boc group.236 52

The aldehyde 52, an analogue of betalamic acid (8) without the carboxylic acids in 2- and 6- position, was isolated, purified and characterized by 1H NMR and 13C NMR spectroscopy (Figure 17). In the 1H NMR two doublets are present at 9.79 ppm and at 9.93 ppm for the formyl proton, which indicate the presence of two diastereomeric aldehydes in the ratio of 60:40. At 5.19 and 5.29 ppm the two doublets are caused by the methine proton of the exocyclic double bond, whilst the multiplet at 4.74-4.81 ppm is caused by the proton of the secondary amine.

Figure 17: 1H NMR spectrum of key intermediate 52.

During purification of aldehyde 52 two problems arose. Firstly, aldehyde 52 started to decompose during work-up and fc, probably due to its high reactivity. Secondly, aldehyde 52 presents a secondary amine and an aldehyde in the same molecule, which could react with each other intermolecularly. Thus, polymerization of the aldehyde 52 is possible. For these two reasons, production of larger amounts of intermediate 52 was not possible. Therefore, this route was no longer pursued.

In this first part of this project, a new synthetic strategy was developed for the synthesis of novel betaxanthin analogues. The key intermediate 52 was obtained and characterized as novel betaxanthin analog, but the instability of aldehyde 52 did not allow to continue further with the synthesis of additional new compounds.

Nevertheless, the antioxidant properties of compounds 88a and 88c were tested using the Folin-Ciocalteu method240. This method determines the total polyphenols content (TPC) and it is based on reducing properties of the phenolic function. The Folin-Ciocalteu reagent, used in this assay, is formed by two hexavalent phosphomolybdic/phosphotungstic acid yellow complexes with the following structures in solution:

3H2O•P2O5•13WO3•5MoO3•10H2O

3H2O•P2O5•14WO3•4MoO3•10H2O

Upon oxidation of phenolic functions, the yellow solution turns blue because of the formation of the molybdenum and tungsten oxides. The optical density, linearly proportional to the content of polyphenols in solution, is measured at a wavelength of 765 nm.

Unfortunately, during the assay the solution did not turn blue and the optical density could not be measured. The results indicate that protected aldehydes 88a and 88c do not present any relevant antioxidant activity. Two hypotheses have been postulated to explain the lack of antioxidant activity in compounds 88a and 88c. The first hypothesis considers the structure of betalamic acid (8). For this compound, two resonance structures can be drawn: 8a, with a formyl group and secondary amine, and 8b, which presents an enol and a tertiary amine. The structure that possesses the antioxidant activity could be a hybrid derived by the contribution of both structures. With compound 88a and 88c, the second structure presents an enol and a quaternary amine positively charged. This quaternary amine could be the reason for the lack of antioxidant activity. The second hypothesis considers the presence of the two carboxylic functions in 2/6-position of betalamic acid (8). According with the obtained results, these groups seem to be important for the antioxidant activity.

Scheme 43: Possible resonance structures of betalamic acid (8) and aldehydes 88a and 88c.

5. Synthesis of conformationally restricted KOR agonists

The second part of the project was devoted to the synthesis of conformationally restricted KOR agonists. At the beginning, the synthesis of the ketone 58 must be done.

Figure 18: Starting material for the synthesis of conformationally restricted KOR agonists.

This ketone is commercially available as pure (R) and (S) enantiomers, but both the high price and the low availability from the suppliers don’t allow the use of the commercial source. For this reason, it was necessary to synthesize this ketone.

Scheme 44: Synthesis of ketone 101 described by Bousquet et al..241

In literature, a total synthesis of the acid of this compound as racemate is available as shown in Scheme 44.241,242 In this synthesis, methyl (S)-aspartate hydrochloride (97) was neutralized with NEt3 and reacted with methyl acrylate. In situ N-protection with Boc2O led to diester 98. Sodium methanolate was used for Dieckmann cyclization. The mixture of enolized β-ketoesters was decarboxylated in situ by addition of water and heating. Subsequent acidification and addition of tert-butylamine led to precipitation of 100. Addition of diluited HCl led to acid 101.

Scheme 45: Michael addition and Boc protection of amino acid 97.

According to the reported synthesis of Bousquet et al.241 aspartate 97 was added to methyl acrylate and the product was reacted with Boc2O. This reaction led to the diester 98 in 21 % yield (Scheme 45). The next step involves the ring-closing reaction of the diester 98 with sodium methanolate to form the piperidine ring (Scheme 46). However, the reaction conditions reported by Bousquet et al. did not provide the ketone 101.

Scheme 46: Attempt of the Dieckmann condensation and decarboxylation of diester 98.

For this reason and furthermore to replace the toxic methyl acrylate, a new plan for the synthesis of ketone 58 was designed. In the first step a conjugate addition (aza-Michael addition) of β-alanine methyl ester hydrochloride (102) at diethyl fumarate 103 was performed (Scheme 47).241-245

Scheme 47: Conjugate addition (aza-Michael addition) for the synthesis of triester 104.

In table 12 the experimental conditions applied for the aza-Michael addition are showed.

Table 12: Experimental conditions used for the aza-Michael addition.

yield base eq. catalyst eq. solvent temperature time of 104 0 °C 10 min Et3N 2.5 / / H2O - RT 20 h Et3N 1 / THF 0  50 °C 20 h - DABCO 1 TBAI 0.5 / 100 °C 16 h 15 NaHCO3 1 / / MeCN 90 °C 16 h 12 K2CO3 2 LiBr 1 DMF 100 °C 16 h 54 K2CO3 2 LiBr 1 DMF 120 °C 16 h 34 K2CO3 2 LiBr 1 DMF RT 3 d 79

Firstly, the reaction was performed using the same conditions as reported by Bousquat et al.. However, these conditions did not lead to the triester 104. A yield of 15 % of triester 104 was obtained using 1,4-diazabicyclo[2.2.2]octane (DABCO) without solvent. Using DABCO, triester 104 could be isolated and characterized (Figure 19). In 1H NMR spectrum, two triplets at 1.23 and 1.26 ppm and two quartets at 4.13 and 4.19 ppm indicate the presence of the ethyl ester. The singlet at 3.66 ppm is caused by the protons of methyl ester.

Figure 19: 1H NMR spectrum of triester 104.

In order to increase the yield of the triester 104, further bases were tested. The best results were obtained using K2CO3 in DMF with LiBr as catalyst. In these experiments, it was observed that temperature determined the final yield of the 104 (Table 12). High temperatures led to lower yields. In contrast, reactions at ambient temperature led to higher yields, but required longer reaction times.

The next step involved the protection of the secondary amine of triester 104. After several attempts, Boc2O and K2CO3 were used in DMF to provide N-protected amine in 88 % yield (Scheme 48, Table 13).

Scheme 48: Protection of amino group in triester 104.

Table 13: Experimental conditions for Boc protection of compound 104.

base solvent temperature time yield of 105 0 °C RT 10 min Et3N CH2Cl2 70 RT 16 h

Et3N THF RT 2 d 65

K2CO3 DMF RT 2 d 88

After protection of the secondary amine, the next step was a Dieckmann cyclization to form the piperdine ring. For this reaction, different solvents and bases were tested (Scheme 49, Table 14).241,246-248

Scheme 49: Attempted Dieckman condensation of triester 105.

Table 14: Conditons tried to obtain 106.

base solvent temperature time yield of 106 NaOMe 30 % THF reflux 4 h - in CH3OH NaH DMSO RT 5 h - NaOMe 30 % Toluene reflux 16 h - in CH3OH Na/CH3OH THF RT 16 h - Na EtOH RT 16 h - Na/CH3OH Toluene reflux 16 h -

Different experimental conditions were tried to obtain the mixture of constitutional isomers 106. The reaction performed with sodium methanolate in methanol showed the complete consumption of the starting material. However, the residue isolated could not be purified by fc. Reaction with Na in methanol provided a new compound, but both NMR and mass spectrometry confirmed that it was not the mixture of isomers 106. When Na and EtOH were

59 used, it was possible to isolate a mixture of compounds. Recorded NMR spectra of the compound mixture indicated the presence of a five-member ring rather than a piperidine ring.

Since Dieckmann cyclization did not lead to the desired β-ketoester mixture 106, it was decided to use the commercially available starting material (R)-1-Boc-4-oxopiperidine-2- carboxylate (58).

For the synthesis of KOR agonists a side chain should be introduced in 4-position of the piperidine 58. For this purpose ketone 58 was reacted with the Wittig reagent 79a in toluene (Scheme 50). Repetition of this experiment showed, that a low concentration of the Wittig reagent gave lower yields of the -unsaturated ester 57a. Finally, the Wittig reaction provided to the α,β-unsaturated ester 57a in 96 % yield. NMR spectroscopy and mass spectrometry confirmed the formation of the α,β-unsaturated ester 57a.

Scheme 50: Wittig reaction of ketone 58 to give α,β-unsaturated ester 57a.

Hydrogenation of the exocyclic double bond of 57a was performed with H2 in the presence of Pd/C as catalyst (Scheme 51). Complete hydrogenation of the α,β-unsaturated ester 57a was observed after 16 h. Purification by fc provided the diester 70a in 98 % yield (Scheme 51).

Scheme 51: Hydrogenation of α,β-unsaturated ester 57a.

The key step in the synthesis of the designed KOR agonists was a Dieckmann cyclization of diester 70a. After several experiments, the reaction of diester 70a with NaHMDS in THF249,250 led to the azabicyclo[3.2.1]octane 56a in 5 % yield (Scheme 52).

Scheme 52: Dieckmann cyclization of diester 70a.

The identity of the formed -ketoester 56a was determined by NMR and IR spectroscopy and mass spectrometry. In the 1H NMR spectrum three multiplets caused by methine protons were observed at 2.94-2.98 ppm for proton in 8-position, 3.00-3.05 ppm for proton in 4-position and at 4.34-4.46 ppm for proton in 2-position. These signals confirmed the formation of the ring. Moreover, only one triplet at 1.27 ppm and a quartet at 4.17 ppm were observed indicating the presence of only one ethyl ester. Furthermore in 13C NMR spectrum a signal at 208.6 ppm indicates the presence of a ketone, whilst the signal at 167.8 ppm shows the presence carbonyl of the ethyl ester.

To increase the yield of -ketoester 70a several optimization experiments should be performed. However, due to the limited time and low available amount of diester 70a these experiments could not be performed. Moreover, ketone 58 was no longer available from the supplier. For this reason, this part of the project was stopped at this stage.

6. Synthesis of new 1,2,3 triazole derivatives with potential antitumor activity and σ receptors affinity.

The third part of the project is devoted to the synthesis of new compounds with a 1,2,3 triazole ring in the structure (Scheme 53). Furthermore, the antitumor activity and the affinity toward σ receptors should be evaluated. The idea behind this part of the project resides in 167 the role of the σ2 receptor as biomarker and as target for chemotherapy. In addition, studies reported interesting anti-proliferative and pro-apoptotic activities in human 54,55 colonrectal carcinoma (Caco-2) cells of indicaxanthin (6). The link between σ2-receptor ligands and indicaxanthin (6) is represented by the 1,2,3 triazole moiety. Studies reported that in several natural compounds active as inhibitors of heat shock protein 90 (Hsp90) the substitution of the amide bond with the 1,2,3 triazole ring led to new compounds with good and in some cases higher activity than natural products.189,191-198

Scheme 53: Development of new triazoles derivatives.

For the synthesis of triazoles the allylic alcohols 74 prepared as intermediates for the betaxanthin synthesis served as starting material. The phenylsulfonyl and Boc protected allylic alcohols 74a and 74c reacted under Mitsunobu conditions with Ph3P, diisopropyl azodicarboxylate (DIAD) and a Zn(N3)2-2Py to form the azides 73a and 73c, respectively (Scheme 54). The azides 74a and 74c were isolated in 38 % and 25 % yield, respectively.

Scheme 54: Synthesis of the azides 74a and 74c.

The presence of the azide group was confirmed by IR spectroscopy. The band at 2095 cm-1 is characteristic for the azide moiety. Furthermore, NMR spectra and mass spectrometry showed the formation of the azides 73a and 73c. In 1H NMR spectrum, two doublets are present at 3.87 ppm and 3.97 ppm caused by the methylene protons connected to the azide indicating the presence of a mixture of azides with a ratio of 53:47.

In order to establish the triazole moiety a Cu+ catalyzed azide-alkyne cycloaddition (Click reaction), should be performed.

The postulated mechanism of the Cu+ catalyzed 1,3-dipolar cycloaddition is depicted in Scheme 55. At first Cu+ is coordinated at the π-system of the alkyne (A). Subsequently, a second Cu+ ion interacts with the alkyne moiety generating a covalent C-Cu-σ-bond (B). This intermediate is now able to react with an azide (C) leading to a six-membered copper metallacycle (D). The catalytic cycle is closed by elimination of Cu+ resulting in ring contraction of the metallacycle to form a triazolyl-copper derivative (E). Protonation generates the triazole (F).251

Scheme 55: First proposed mechanism of the cu+ catalyzed 1,3-dipolar cycloaddition.251

The copper(I) source is a major factor to influence the efficiency of this 1,3-dipolar cycloaddition.252-254 CuI is often used in organic reactions, but CuI alone is inefficient in this 1,3-dipolar cycloaddition. Combination of CuI with tertiary amines gave good results, as the amines are able to disaggregate the stable polymers of CuI by coordination. Thus, Cu+ coordinated at the tertiary amine represents the “active” Cu(I) species for the formation of copper(I) acetylide (species B in the catalytic cycle of Scheme 55). In addition to coordinate Cu+, the tertiary amine works as base to remove the proton from terminal alkyne. 255 Sharpless has observed that byproducts were formed when NEt3 or DIPEA were used as additives. To date, the combination of CuSO4 and sodium ascorbate (NaAsc) in aqueous t- BuOH represents most common catalytic system for the 1,3-dipolar cycloaddition rather than the CuI/NR3 system. However, CuI/NR3 is highly beneficial to be used in non-aqueous or non-protonic solvents, which is practically very important, since various substrates do not tolerate water or protic solvents.256

At first, azide 73c was reacted with phenylacetylene (107), CuSO4 and NaAsc in a mixture of water and t-butanol to produce the new triazoles (Scheme 56).

Scheme 56: First experiment for the synthesis of triazoles.

The first reaction led to a mixture of the desired triazole 109 and the saturated triazole 108. For both compounds, the azide band at 2096 cm-1 had disapperared in the IR spectrum. In the 1H NMR spectrum a singlet at 7.66 ppm indicates the presence of the proton in 5-position of the triazole ring. The presence of three multiplets at 2.25, 2.37 and 2.97 ppm indicates the formation of a mixture of compounds 108 and 109 in a ratio of 66:34. The presence of the saturated triazole 108 was also confirmed by mass spectrometry.

Different solvents were tested to avoid the reduction of the double bond to form the saturated system 108, but in all the experiments formation of multiple compounds was observed. Therefore, another strategy for the synthesis of triazole containing test compounds was pursued. In 2007, Straub257 reported that the C-Cu bond in the final intermediate (species E in Scheme 55) could be protonated by HOAc within a few minutes (Scheme 57).

Scheme 57: Mechanism for the 1,3-dipolar cycloaddition with CuI, DIPEA and HOAc.256

The results reported by Straub strongly imply that CuI/NR3-catalyzed 1,3-dipolar cycloadditions can be enhanced by addition of HOAc. Therefore, the 1,3-dipolar cycloaddition was performed using CuI as source of copper(I), DIPEA to disaggregate the CuI polymer and HOAc. The reaction of azides 73a and 73c with different terminal alkynes using these components in CH2Cl2 led to the new 1,2,3-triazole derivatives 109-114 (Scheme 58, Table 15).

Scheme 58: Synthesis of new triazoles 109-114.

Table 15: New 1,2,3 triazole derivatives 109-114.

compd. R R1 yield

109 SO2Ph 68

110 SO2Ph 63

111 SO2Ph 75

112 SO2Ph 85

113 SO2Ph 71

114 Boc 93

IR and 1H NMR spectra confirm the successful formation of the triazole ring. In the IR spectrum, the intense band at 2096 cm-1 typical for the azide moiety is no longer present. In 1H NMR spectrum, the formation of the triazole ring is confirmed by two singlets at 7.66 and 7.68 ppm. The two signals represent two diastereomers along the exocyclic double bond. The ratio of diastereomers is 54:46 (Figure 20).

Figure 20: 1H NMR spectrum of triazole derivative 109.

7. Pharmacological characterization

The affinity towards σ receptors and the antitumor activity of the synthesized triazole derivatives 109-114 should be investigated. The σ receptor affinity was recorded in Münster by radioligand binding studies. The antitumor activity will be tested at the National Cancer Institute of Bethesda. The triazoles 109-114 will be included into the NCI-60 Human Tumor Cell Lines screen. Furthermore, the antioxidant activity of triazoles 109, 110 and 114 and of betalamic acid analogues 88a and 88c was tested.

7.1. Affinity towards  receptors

7.1.1. General principle

The selective binding of a biologically active compound at a specific receptor site generating a biological response is the basis of ligand receptor-binding assays.258 This interaction has to be specific, reversible and saturable and can be expressed by the following equation:

The ligand L interacts with the receptor R to generate a receptor-ligand complex RL. k1 and k2 represent the association and dissociation constants, respectively. After some time, the concentrations of the free and bond ligand reach an equilibrium. The ratio of the two rate constants k1 and k2 at the equilibrium state gives the equilibrium dissociation constant Kd and it is inversely proportional to the affinity of the ligand to the receptor.

푘2 푐(푅) ∙ 푐(퐿) 퐾푑 = = 푘1 푐(푅퐿)

Equation 1: Dissociation costant Kd; c(R) = concentration of receptors; c(L) = concentration of the ligand; c(RL) = concentration of the receptor-ligand complexes.

In radioligand binding assays, the affinity of new ligands is determined by competition with a radiolabeled ligand L*. The radioligand L* is a well-known compound with high affinity and selectivity for the receptor of interest. Radioligands are often labeled with tritium [3H] due to its long half-life (12.3 years) and to its low penetrating power. The introduction of the radioligand L* generates a new equilibrium with two different ligand-receptor complexes:

During competition experiments, the molar concentrations of the receptor c(R) and the radiolabeled ligand c(L*) in each well are constant, whereas the concentration of the unlabeled novel ligand c(L) is increasing. A high concentration of L shifts the equilibrium towards the ligand-receptor complex RL, inhibiting the binding of radioligand L* to the receptor. The displaced radioligand L* is then removed by filtration generating different levels of radioactivity on the filter, which can be measured by a scintillation analyzer. Plotting the radioactivity trapped on the filter versus the concentration of test compound c(L) leads to a sigmoidal curve (Figure 21).

Figure 21: Example of a sigmoidal curve obtained by a competitive binding assay.

The concentration of the unlabeled ligand L able to displace 50 % of the radiolabeled ligand

L* is called IC50 value. The IC50 value is strongly dependent on the concentration of the receptor c(R), the radioligand c(L*) and the affinity of the radioligand. For this reason, it is difficult to compare IC50 values obtained in different assays. To obtain a more independent value the IC50 value is converted into the inhibition constant Ki, using the Cheng-Prusoff equation.259

퐼퐶50 퐾푖 = ∗ 1 + 푐(퐿 ) 퐾푑

Equation 2: Cheng-Prusoff equation: Ki = inhibition costant; c(L*) = concentration of the radioligand; Kd = dissociation costant of the radioligand. 70

7.1.2. σ1 and σ2 binding assays

In the σ1 assay, guinea pig brain membrane preparations were used as source of the receptor and [3H]-(+)-pentazocine (115, Figure 22) served as the radioligand. Unlabeled (+)-pentazocine was used for the determination of non-specific binding.

3 In the σ2 assay, rat liver homogenates were used as source of the receptor and [ H]-di-o- tolylguanidine ([3H]DTG, 47, Figure 22) was employed as radioligand. Since [3H]DTG has high affinity for both σ receptor subtypes, σ1 receptors were masked with (+)-pentazocine

(100 nM). Unlabeled DTG was used for the determination of non-specific binding. The Ki values of reference compounds (+)-pentazocine, haloperidol and DTG were measured and compared with literature values.

Figure 22 - Radioligands in the σ1 and σ2 binding assays.

Table 16: Comparison of recorded and reported Ki values of reference compounds.

σ1 σ2 Reference Ki ± SEM [nM] Ki ± SEM [nM] Ki ± SEM [nM] Ki ± SEM [nM] compound (literature) (experimental) (literature) (experimental)

(+)-pentazocine 2.1 ± 0.1260 5.4 ± 0.5 - -

haloperidol 1.8 ± 0.1261 6.6 ± 0.9 22 ± 8.5261 125 ± 33

DTG 107 ± 21260 71 ± 8 40 ± 2.6260 54 ± 8

7.2. σ Receptor affinity of newly synthetized 1,2,3 triazole derivatives 109 - 114

Table 17: Affinity of 1,2,3 triazole derivatives 109 – 114 towards 1 and 2 receptors.

Inhibition of radioligand binding (in %) after addition of the test compound in a concentration of a 1 µM 1 Compd. R R 1 2 [3H]-(+)- [3H]DTG pentazocine

109 SO2Ph 0 0

110 SO2Ph 2 0

111 SO2Ph 0 0

112 SO2Ph 0 0

113 SO2Ph 0 0

114 Boc 0 0

Up to a concentration of 1 µM, the triazoles 109-114 did not inhibit the radioligand binding to the  receptors. This result can be explained by the low basicity of the triazoles 109-114. Due to protection of the piperidine N-atom by a phenylsulfonyl moiety or a Boc group, this position is no longer basic. On the contrary, the basicity of the triazole ring is extremely low (Scheme 59). According to all pharmacophore models a basic functional group is required to achieve high affinity towards 1 and 2 receptors. Thus, it is postulated that the low basicity is responsible for the low 1 and 2 affinity of the triazoles 109-114.

Scheme 59: Tautomerism of 1,2,3 triazole and pka values.

7.3. Folin-Ciocalteu assay

The Folin-Ciocalteu method240 is used to determine the antioxidant activity through the evaluation of the Total Polyphenols Content (TPC). The Folin-Ciocalteu reagent (FCR), used in this assay, is formed by two hexavalent phosphomolybdic/phosphotungstic acid yellow complexes with the following structures in solution:

3H2O•P2O5•13WO3•5MoO3•10H2O

3H2O•P2O5•14WO3•4MoO3•10H2O

The reaction between the FCR and phenols is conducted in basic conditions (pH ~ 10, obtained with the use of Na2CO3 solutions) to increase the reaction rate. Upon oxidation of phenolic functions, the yellow solution turns blue because of the formation of the molybdenum and tungsten oxides. The optical density, linearly proportional to the content of polyphenols in solution, is measured at a wavelength of 765 nm. The results are expressed in mg of Gallic acid equivalents (GAE)/g of the sample.

Betalamic acid (8) analogues 88a and 88c and 1,2,3 triazoles 109, 110, 114 were tested with the Folin-Ciocalteu method using gallic acid as standard in basic solution pH ~ 10. Two aliquots of each sample have been used: 1 µL and 100 µL. Reaction of the samples with the FCR did not lead to a blue solution indicating the presence of antioxidant activity.

8. Summary

The present work deals with the design, synthesis and biological evaluation of new analogues of natural products and structurally related compounds. The project was divided in three parts: (1) design, synthesis and antioxidant activity of analogues of the natural product indicaxanthin (6); (2) design and synthesis of novel azabicyclo[3.2.1]octanes, which will be further developed into new conformationally restricted KOR agonists; (3) synthesis of new 1,2,3-triazoles and evaluation of their affinity towards σ receptors. The antitumor activity of these compounds will be investigated at the NCI.

8.1. Part I: synthesis of new analogues of indicaxanthin

The first part of the project was devoted to the synthesis of new analogs of natural products belonging to the class of betaxanthins. Several experiments were necessary to find the synthetic strategy providing the aldehyde 52 (Scheme 60).

Scheme 60: Synthesis of aldehyde 52 as novel betaxanthin analog.

The synthesis of aldehyde 52 started with sulfonamide 75a, and the successful synthesis was then transferred to the carbamate 75c. At first, α-bromination of ketone 75a with Br2 was performed. The reaction with sulfonamide 75a worked perfectly, whereas the reaction

74 with the Boc derivative 75c caused some problems because of the sensitivity of the Boc group towards acidic conditions. Nevertheless, compounds 77a and 77c were obtained in 100 % and 46 % yields. A Wittig reaction of the ketones 77a and 77c with the Wittig reagent 79a provided the α,β-unsaturated esters 89a and 89c. Base-catalyzed dehydrobromination of 89a and 89c led to the dihydropyridines 63a and 63b. At this point, the conjugated system, important for the antioxidant activity, was completed. Reduction of the esters 63a and 63b with DIBAL-H followed by oxidation using TEMPO and CuCl led to the N-protected analogues of betalamic acid without the carboxy moieties in 2- and 6-position. Finally, aldehyde 88a was used to perform Schiff bases with (S)-proline, (S)-aspartate and phenylethylamine. Unfortunately, these reactions did not result in the desired products probably due to the presence of the protecting group on the N-atom in the piperidine ring. Several attempts were performed to remove the N-protective groups. However, only the Boc group could be cleaved off by heating the Boc-derivative 88c in neutral water leading to the betalamic acid analogue 52. Unfortunately, due to the high reactivity of the aldehyde 52, it was not possible to improve its yield and to go further with this part of the project.

The antioxidant activity of the protected aldehydes 88a and 88c was tested using the Folin- Ciocalteau assay240. However, none of the compounds submitted to the antioxidant assay showed any antioxidant activity.

8.2. Part 2: synthesis of advanced intermediates to obtain bicyclic KOR agonists

The second part of the project was devoted to the synthesis of new conformationally restricted intermediates, which can be used to develop KOR agonists and antagonists with a rigid scaffold. A major problem of this project arose from the starting ketone 58. At the beginning of the work the ketone 58 was commercially available, but later it could not be obtained anymore from suppliers. Furthermore, it was sold as pure enantiomer and was rather expensive. For this reason, it was decided to synthesize the ketone 58 as racemic mixture. Despite several attempts, the ketone 58 could not be obtained by synthesis. To start the synthesis of rigid KOR agonists, the commercially available ketone 58 was bought. At first a Wittig reaction of ketone 58 with the Wittig reagent 79a was performed to produce the -unsaturated ester 57a. Hydrogenation of the double bond of 57a led to the diester

70a in 98 % yield. The final Dieckmann cyclization of diester 70a resulted in the bicyclic - keotester 56a in only 5 % yield (Scheme 61).

Scheme 61: Synthesis of the advanced intermediate 56a, which will be exploited to obtain conformationally restricted KOR agonists.

Unfortunately, the yield of the β-ketoester 56a could not be increased to obtain considerable amounts of 56a to proceed further in the synthesis. Furthermore, the starting material 58 was not available any more from the supplier. For this reason, it was not possible to optimize the ring-closing reaction and make further progress in the project.

8.3. Part 3: synthesis of new 1,2,3 triazole derivatives and evaluation of their σ receptor affinity

The third part of the project involved the synthesis of new 1,2,3 triazole derivatives, which were designed to interact with 1 and 2 receptors and inhibit tumor growth. For the synthesis of triazoles the allylic alcohols 74a and 74c prepared during the synthesis of aldehyde 52 were used as starting material. Allylic alcohols 74a and 74c represented the precursor for the key intermediate of this third part of the project. In scheme 62 the synthesis of the new triazoles is showed.

Scheme 60: Synthesis of new 1,2,3-triazoles 109-114.

For the synthesis of triazoles, allylic alcohols 74a and 74c, were converted into allylic azides

73a and 73c by a Mitsunobu reaction using Zn(N3)22Py as nucleophile. The azides 73a and 73c underwent a Cu+ catalyzed 1,3-dipolar cycloaddition to yield the 1,2,3-triazoles 109- 114.

The prepared 1,2,3-triazoles 109-114 were characterized chemically and spectroscopically. Subsequently, the affinity towards both σ receptor subtypes was recorded. However, up to a concentration of 1 µM triazoles 109-114 did not show any competition with the radioligands indicating very low affinity toward both  receptor subtypes. It is postulated that the low basicity of triazoles 109-114 is responsible for the low  affinity. Furthermore, triazoles 109, 110 and 114 were tested for their antioxidant activity. However, an antioxidant activity was not detected.

All the synthetized 1,2,3-triazoles 109-114 were accepted by the NCI for the NCI-60 Human Tumor Cell Lines screen. They have been submitted to the NCI and will be tested there for their antitumor activity. These tests are in progress.

9. Experimental Part

9.1. Synthesis and Analytical Characterization

9.1.1. General procedures and materials

9.1.1.1. General remarks

Oxygen and moisture sensitive reactions were carried out under nitrogen, dried with silica gel with moisture indicator (orange gel, Merck) and in dry glassware (Schlenk flask or Schlenk tube). Reaction mixtures were stirred with magnetic stirrer MR 3001 K (Heidolph) or RCT CL (IKA).

Temperatures were controlled with dry ice/acetone (-78 °C), ice/water (0 °C), Cryostat (Julabo FT 901 or Huber TC100E-F), magnetic stirrer MR 3001 K (Heidolph) or RCT CL (IKA®), together with temperature controller EKT HeiCon (Heidolph) or VT-5 (VWR) and PEG or silicone bath.

The intermediates 46, 47, 57, 85 and 86 are commercially available, though no synthetic procedure is reported in the literature.

Chemical structures were generated by ChemDraw (v15.0.0.106).

9.1.1.2. Solvents

All solvents were of analytical grade quality. Demineralized water was used. Water free solvents were freshly distilled under N2 atmosphere prior to use or dried with molecular sieves.

CH3CN: dried with molecular sieves (3 Å)

CH2Cl2: distilled from calcium hydride

DMF: dried with molecular sieves (4 Å)

Ethanol abs.: dried with molecular sieves (3 Å)

Ethyl acetate: dried with molecular sieves (4 Å)

Methanol: distilled from magnesium methanolate

Pyridine: dried with molecular sieves (3 Å) 78

THF: distilled from sodium/benzophenone

Toluene: dried with molecular sieves (4 Å)

9.1.1.3. Thin layer chromatography

Thin layer chromatography was conducted with TLC silica gel 60 F254 on aluminum sheets (Merck) as stationary phase in a saturated chamber at ambient temperature. Spots were visualized with UV light (254 nm or 365 nm). Compositions of the mobile phase and retention factor (Rf) of the compounds are given in the description of the synthetic procedures. As the retention factor (Rf) values strongly depend on the exact ratio of components of the eluent and some of these are highly volatile, the given retention factor values represent just approximate values.

9.1.1.4. Flash column chromatography

Flash column chromatography was performed with silica gel 60 (40 - 63 μm, Macherey-Nagel) as stationary phase. Pressure was applied with compressed air.

9.1.1.5. Melting points

Melting points were determined with melting point system MP50 (Mettler Toledo) using an open capillary. The values are uncorrected.

9.1.1.6. HPLC

HPLC was used to determine the purity of the synthesized compounds and was carried out at room temperature.

Equipment 1: Pump: L-7100, degasser: L-7614, autosampler: L-7200, UV detector: L-7400, interface: D-7000, data transfer: D-line, data acquisition: HSM-Software (all from LaChrom, Merck Hitachi).

Equipment 2: Pump: LPG-3400SD, degasser: DG-1210, autosampler: ACC-3000T, UV- detector: VWD-3400RS, interface: DIONEX UltiMate 3000, data acquisition: Chromeleon 7 (Thermo Fisher Scientific).

Column: LiChropher® 60 RP-select B (5 µm), LiChroCART® 250-4 mm cartridge

Guard column: LiChropher® 60 RP-select B (5 µm), LiChroCART® 4-4 mm cartridge (No.: 1.50963.0001), manu-CART® NT cartridge holder

Solvent A: demineralized water + 0.05 % (V/V) trifluoroacetic acid

Solvent B: acetonitrile with 0.05 % (V/V) trifluoroacetic acid

Gradient elution (% A): 0 - 4 min: 90 %; 4 - 29 min: gradient from 90 % to 0 %; 29 - 31 min: 0 %; 31 - 31.5 min: gradient from 0 % to 90 %; 31.5 - 40 min: 90 %.

Flow rate: 1.0 mL/min

Injection volume: 5.0 µL; method: cut lead and rear

Detection wavelength: 210 nm

Stop time: 30.0 min

Calculation: manual integration, calculation method: area %, use of blank substraction from the same series

9.1.1.7. NMR spectroscopy

NMR spectra were recorded on Agilent DD2 400 MHz and 600 MHz spectrometers and on Bruker Avance II series 200 MHz spectrometer. The frequencies are given in the descriptions of the synthetic procedures. MestReNova software (version 10.0.0 - 14381, © 2014 by Mestrelab Research S.L.) was used for analyzing NMR spectra. Abbreviations for the multiplicities of the signal: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, sept = septet, m = multiplet, dd = doublet of doublet, etc. The multiplicities are reported as observed in the spectra. Chemical shifts (δ) are reported in parts per million (ppm) against the reference substance tetramethylsilane (TMS) and calculated using the solvent residual peak of the undeuterated solvent.

1H NMR spectroscopy (NMR frequency: 200 MHz, 400 MHz and 600 MHz)

δ (TMS) = δ (CDCl3) - 7.26

δ (TMS) = δ (DMSO-D6) - 2.54

13C NMR spectroscopy (NMR frequency: 50 MHz, 101 MHz and 150 MHz)

δ (TMS) = δ (CDCl3) - 77.16 ppm

δ (TMS) = δ (DMSO-D6) - 39.52 ppm

Two-dimensional (2D) NMR spectroscopy

If necessary, 1H and 13C NMR assignments were supported by the following two- dimensional NMR spectroscopy techniques:

COSY (1H, 1H correlation spectroscopy)

gHSQC (gradient heteronuclear single quantum coherence)

gHMBC (gradient heteronuclear multiple bond correlation)

9.1.1.8. IR spectroscopy

IR spectra were recorded on a FT/IR IRAffinity-1 IR spectrometer (Shimadzu) using ATR technique or with a Shimadzu FT / IR 8400S spectrophotometer. Samples were applied to the device without solvent and were directly measured or dissolved in bromoform. Absorption bands are characterized by their wave numbers ṽ [cm-1].

9.1.1.9. Mass spectrometry

All samples were measured in the positive ion mode, so all specified molecules, fragments or adducts represent positively charged ions.

Exact mass spectra of synthesized compounds were recorded as follows:

Exact Mass (APCI): Atmospheric pressure chemical ionization (APCI) mass spectra were recorded with a MicroTOFQII mass spectrometer (Bruker Daltonics). Deviations of the found

81 exact masses from the calculated exact masses were 5 mDa or less, unless otherwise stated. The data were analyzed with DataAnalysis (Bruker).

9.2. Synthetic procedures and analytical data

1-(Phenylsulfonyl)piperidin-4-one (75a)

TEA (15.2 mL, 109 mmol, 3.0 eq.) was added to a solution of piperidin-4-one monohydrate hydrochloride 62 (5.0 g, 37 mmol, 1.0 eq.) in THF (166 mL). Benzenesulfonyl chloride (5.1 mL, 40 mmol, 1.1 eq.) was added dropwise to the mixture and the solution was stirred at room temperature for 18 h. Afterwards, the solution was diluted with water and extracted with ethyl acetate (3x). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography

(cyclohexane/ethyl acetate 9/1  4/6, Rf = 0.23 (cyclohexane/ethyl acetate 7:3)). Colourless solid, mp 123-124 °C, yield 8.7 g (36 mmol, 98 %).

Purity by HPLC, method 1: 99.5 %, tR = 14.8 min.

+ Exact mass (APCI): m/z = 240.0680 (calcd. 240.0694 for C11H14NO3S [M+H ]).

1 H NMR (400 MHz, CDCl3): δ (ppm) = 2.52 (t, J = 6.2 Hz, 4H, CO(CH2)2), 3.39 (t, J = 6.2

Hz, 4H, N(CH2)2), 7.49 – 7.58 (m, 2H, 3-Harom., 5-Harom.), 7.58 – 7.66 (m, 1H, 4-Harom.), 7.75

– 7.83 (m, 2H, 2-Harom., 6-Harom.).

13 C NMR (101 MHz, CDCl3): δ (ppm) = 40.7 (2C, CO(CH2)2), 45.8 (2C, N(CH2)2), 127.5 (2C,

C-2arom., C-6arom.), 129.3 (2C, C-3arom., C-5arom.), 133.2 (1C, C-4arom.), 136.5 (1C, C-1arom.), 205.4 (1C, C=O).

FT-IR (neat): ṽ (cm-1) = 2966, 2916 (C-H, aliph.), 1708 (C=O), 1165 (S=O).

3-Bromo-1-(phenylsulfonyl)piperidin-4-one (77a)

In a round-bottom flask, ketone 75a (1.0 g, 4.2 mmol, 1.0 eq.) was dissolved in dry CH2Cl2 (26 mL) and the solution was cooled to -5 °C. At this temperature bromine (0.21 mL, 4.2 mmol, 1.0 eq.) dissolved in CH2Cl2 (4.1 mL) was added dropwise and the reaction mixture was stirred for 1 h at -5 °C until decolouration of the mixture. Afterwards, a saturated solution of NaHCO3 (26 mL) was added to the mixture and extracted with CH2Cl2 (3x). The organic layers were collected, dried over Na2SO4, filtered and the solvent removed under reduced pressure. The obtained solid 107 was used without further purification. Light pink solid (Rf = 0.29, cyclohexane/ethyl acetate 7:3), mp 114-115 °C, yield 1.29 g (4 mmol, 97 %).

Purity by HPLC, method 1: 99.9 %, tR = 14.8 min.

+ Exact mass (APCI): m/z = 317.9785 (calcd 317.9800 for C11H13BrNO3S [M+H ]).

1 H NMR (400 MHz, CDCl3): δ (ppm) = 2.59 – 2.72 (m, 1H, 5-CH2), 2.91 – 3.03 (m, 1H, 5-

CH2), 3.26 – 3.35 (m, 1H, 6-CH2), 3.37 – 3.44 (m, 1H, r2-CH2), 3.56 – 3.70 (m, 1H, 6-CH2),

3.93 – 4.03 (m, 1H, 2-CH2), 4.48 – 4.59 (m, 1H, 3-CH), 7.51 – 7.62 (m, 2H, 3-Harom., 5-Harom.),

7.58 – 7.69 (m, 1H, 4-Harom.), 7.75 – 7.88 (m, 2H, 2-Harom., 6-Harom.).

13 C NMR (101 MHz, CDCl3): δ (ppm) = 39.0 (1C, C-5), 46.1 (1C, C-6), 49.5 (1C, C-3), 53.4

(1C, C-2), 127.5 (2C, C-2arom., C-6arom.), 129.6 (2C, C-3arom., C-5arom.), 133.6 (1C, C-4arom.),

136.9 (1C, C-1arom.), 197.7 (1C, C=O).

FT-IR (neat): ṽ (cm-1) = 3062, 3005, 2873 (C-H, aliph.), 1728 (C=O),1165 (S=O), 690 (C- Br).

Ethyl (Z)- and (E)-2-[3-bromo-1-(phenylsulfonyl)piperidin-4-ylidene]acetate (89a)

In a round-bottom flask, α-bromoketone 77a (2.00 g, 6.3 mmol, 1.0 eq.) was dissolved in

CH2Cl2 (157 mL) and to the solution was added (ethoxycarbonylmethylene)triphenylphosphorane (2.40 g, 6.9 mmol, 1.1 eq.). The mixture was heated to 40 °C for 1 h and then cooling to room temperature. Afterwards, all volatiles were removed in vacuo and the residue was purified by flash column chromatography

(cyclohexane/ethyl acetate 9/1  7/3, Rf = 0.49 (cyclohexane/ethyl acetate 7:3)). Colourless solid, mp 121 °C, yield 2.29 g (6 mmol, 94 %).

Purity by HPLC, method 1: 99.4 %, tR = 20.9 min.

+ Exact mass (APCI): m/z = 388.0193 (calcd. 388.0218 for C15H19BrNO4S [M+H ]).

1 H NMR (400 MHz, CDCl3): δ (ppm) = 1.23 (t, J = 7.1 Hz, 3H, OCH2CH3), 2.74 – 2.85 (m,

1H, 6-CH2), 3.13 – 3.27 (m, 1H, 5-CH2, 1H, 2-CH2), 3.29 – 3.41 (m, 1H, 5-CH2), 3.47 – 3.58

(m, 1H, 6-CH2), 3.70 (m,, 1.6 Hz, 1H, 2-CH2), 4.11 (q, J = 7.1 Hz, 2H, OCH2CH3), 4.61 - 4.63

(m, 1H, 3-CH), 5.95 (s, 1H, R2C=CH), 7.46 – 7.57 (m, 2H, 3-Harom., 5-Harom.), 7.53 – 7.64 (m,

1H, 4-Harom.), 7.69 – 7.81 (m, 2H, 2-Harom., 6-Harom.).

13 C NMR (101 MHz, CDCl3): δ (ppm) = 14.1 (1C, OCH2CH3), 25.3 (1C, C-5), 46.2 (1C, C-

6), 49.5 (C-3), 53.8 (1C, C-2), 60.5 (1C, OCH2CH3), 118.2 (1C, R2C=CH), 127.5 (2C, C-

2arom., C-6arom.), 129.2 (2C, C-3arom., C-5arom.), 133.1 (1C, C-4arom.), 136.6 (1C, C-1arom.), 151.9

(1C, R2C=CH), 165.4 (1C, CO2Et).

Only one set of signals can be seen.

FT-IR (neat): ṽ (cm-1) = 3020 (C-H aliph.), 1728 (C=O), 1165 (S=O), 690 (C-Br).

Ethyl (Z)- and (E)-2-[1-(phenylsulfonyl)-2,3-dihydropyridin-4(1H)-ylidene]acetate (63a)

In a round-bottom flask, ester 89a (0.30 g, 0.77 mmol, 1.0 eq.) was dissolved in dry DMF

(7.7 mL). Li2CO3 (0.34 g, 4.6 mmol, 6.0 eq.) and LiBr (0.40 g, 4.6 mmol, 6.0 eq.) were added and the mixture was heated to 130 °C under vigorous stirring for 1 h. Afterwards, the mixture was cooled to room temperature and filtered. The solid was washed with DMF and the organic layer was poured into water and ice. The solid was then filtered, washed with water and dried. The solid obtained 63a was used without further purification. Colourless solid (Rf = 0.55, cyclohexane/ethyl acetate 7:3), mp 104-105 °C, yield 0.22 g (0.72 mmol, 94 %).

Purity by HPLC, method 1: 85.7 %, tR = 21.1 min.

+ Exact mass (APCI): m/z = 308.0935 (calcd.308.0957 for C15H18NO4S [M+H ]).

1 H NMR (600 MHz, DMSO-d6): δ (ppm) = 1.13 (t, J = 7.1 Hz 3H, OCH2CH3), 2.96 - 3.00 (m,

2H, 5-CH2), 3.34-3.37 (m, 2H, 6-CH2) , 4.00 (m, 2H, OCH2CH3), 5.57 (s, 0.8H, R2C=CH),

5.64 (d, J = 8.1 Hz, 0.8H, 3-CH), 5.67 (s, 0.2H, R2C=CH), 6.71 (d, J = 8.4 Hz, 0.2H, 3-CH),

7.05 (d, J = 8.1 Hz, 0.8H, 2-CH), 7.11 (d, J = 8.4, 0.2H, 2-CH), 7.59 – 7.76 (m, 3H, 3-Harom.,

4-Harom., 5-Harom.), 7.79 – 7.86 (m, 2H, 2-Harom., 6-Harom.).

13 C NMR (151 MHz, DMSO-d6): δ (ppm) = 17.3 (1C, -OCH2CH3), 27.7 (0.8C, C-5), 31.1

(0.2C, C-5), 45.7 (0.8C, C-6), 46.3 (0.2C, C-6), 62.3 (1C, -OCH2CH3), 108.9 (0.2C, C-3),

113.9 (0.8C, C-3), 115.2 (0.8C, R2C=CH), 118.4 (0.2C, R2C=CH), 130.0 (2C, C-2arom., C-

6arom.), 132.9 (2C, C-3arom., C-5arom.), 135.0 (0.8C, C-2), 135.6 (0.2C, C-2) 137.1 (1C, C-

4arom.), 139.5 (1C, C-1arom.), 148.6 (0.2C, C-4), 150.1 (0.8C, C-4) , 169.0 (1C, CO2Et).

Compound 63a was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 8:2 is observed.

FT-IR (neat): ṽ (cm-1) = 3066, 2981 (C-H aliph.), 1705 (C=O), 1616 (C=C), 1585 (C=C cyclic), 1161 (S=O).

(Z) and (E) 2-[1-(phenylsulfonyl)-2,3-dihydropyridin-4(1H)-ylidene]ethan-1-ol (74a)

Procedure 1

Under N2, 63a (500 mg, 1.6 mmol, 1.0 eq.) was dissolved in dry toluene (4 mL). The solution was cooled to -78 °C in a dry ice/acetone bath, DIBAL-H (1 M solution in hexane, 6.5 mL, 6.5 mmol, 3.0 eq.) was added dropwise in 15 min. The reaction mixture was stirred at -78 °C for 40 min. After complete consumption of the starting material, checked by TLC analysis, methanol (2 mL) was added carefully to the mixture at -78 °C and the mixture was warmed warm to room temperature. Afterwards, the mixture was filtered, the solid was washed with ethyl acetate (3x) and the volatiles were removed under reduced pressure. The compound was used as racemic mixture without further purification. Yellow oil, yield 406 mg (1.5 mmol, 94 %).

Procedure 2

Under N2, 63a (300 mg, 0.98 mmol, 1.0 eq.) was dissolved in dry THF (2 mL) and the solution was cooled to -10 °C. LiAlH4 (74 mg, 1.9 mmol, 2.0 eq.) was added and the mixture was stirred for 1 h at -10 °C. A saturated solution of potassium sodium tartrate in water (5 mL) was added and the mixture extracted with ethyl acetate (3x). The organic layer was dried

86 with Na2SO4 and volatiles were removed under reduced pressure. The residue was purified by flash column chromatography (cyclohexane/ethyl acetate 9/1  5/5, Rf = 0.49 (petroleum ethert/ethyl acetate 5:5)). Yellow oil, yield 0.21 g (0.80 mmol, 82 %).

Purity by HPLC, method 1: 94.3 %, tR = 16.1 min.

+ Exact mass (APCI): m/z = 266.0784 (calcd.266.0851 for C13H16NO3S [M+H ]).

1 H NMR (600 MHz, DMSO-d6) δ (ppm) 2.27 – 2.33 (m, 2H, 5-CH2), 3.27 – 3.35 (m, 2H, 6-

CH2), 3.91 (dd, J = 6.7, 5.4 Hz, 1.1H, CH2OH), 3.97 (dd, J = 6.7, 5.4 Hz, 0.9H, CH2OH), 4.53

(t, J = 5.4 Hz, 0.55H, OH), 4.55 (t, J = 5.4 Hz, 0.45H, OH), 5.13 (m, 0.45H, R2C=CH), 5.31

– 5.37 (m, 0.55H, R2C=CH), 5.52 (d, J = 8.2 Hz, 0.55H, 3-CH), 5.77 (d, J = 8.4 Hz, 0.45H, 3-CH), 6.62 (d, J = 8.1 Hz, 0.55H, 2-CH), 6.71 (d, J = 8.4 Hz, 0.45H, 2-CH), 7.59 – 7.68 (m,

2H, 3-Harom., 5-Harom.), 7.68 – 7.76 (m, 1H, 4-Harom.), 7.78 – 7.85 (m, 2H, 2-Harom., 6-Harom.).

13 C NMR (151 MHz, DMSO-d6) δ (ppm) 23.2 (0.55C, C-5), 29.2 (0.45C, C-5), 43.2 (0.55C,

C-6), 44.0 (0.45C, C-6), 56.4 (0.45C, CH2OH), 56.7 (0.55C, CH2OH), 106.6 (0.45C, C-3),

113.0 (0.55C, C-3), 124.6 (0.55C, C-2), 125.3 (0.45C, R2C=CH), 125.9 (0.45C, C-2), 126.35

(0.55C, R2C=CH), 126.81 (2C, C-2arom., C-6arom.), 127.3 (0.55C, C-4), 128.6 (2C, C-3arom., C-

5arom.), 129.3 (0.45C, C-4), 133.16 (1C, C-4arom.), 136.6 (1C, C-1arom.).

Compound 73a was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5.5:4.5 is observed.

FT-IR (neat): ṽ (cm-1) = 3545, 3356 (O-H), 2920, 2870 (C-H aliph.), 1643 (C=C), 1608 (C=C, cyclic), 1165 (S=O).

(E) and (Z)-2-[1-(phenylsulfonyl)-2,3-dihydropyridin-4(1H)-ylidene]acetaldehyde (88a)

A racemic mixture of allylic alcohol 73a (320 mg, 1.2 mmol, 1.0 eq.) was dissolved in dry DMF (3 mL). CuCl (12 mg, 0.12 mmol, 0.1 eq.) and TEMPO (19 mg, 0.12 mmol, 0.1 eq.) were added and the solution was stirred at room temperature for 16 h. Afterwards, the 87 solution was poured into water/ice slowly and the mixture was warmed to room temperature. The solid was filtered off, washed with water and dried. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  5/5, Rf = 0.45 (cyclohexane/ethyl acetate 6:4)). Pale yellow solid, mp 97-100 °C, yield 240 mg (0.91 mmol, 76 %).

Purity by HPLC, method 1: 80.5 %, tR = 17.3 min.

+ Exact mass (APCI): m/z = 264.0668 (calcd.264.0694 for C13H14NO3S [M+H ]).

1 H NMR (200 MHz, DMSO-d6): δ (ppm) = 2.60 (t, 7.4 Hz, 0.5H, 5-CH2) 3.03 (t, J = 7.8 Hz,

1.5H, 5-CH2), 3.44 (m, 2H, 6-CH2), 5.45 – 5.61 (m, 0.25H, R2C=CH) 5.72-5.76 (m, 0.75H,

3-CH, 0.75H, R2C=CH), 6.57 (d, J = 8.4, 0.25H, 3-CH), 7.21 (d, J = 8.1 Hz, 0.75H, 2-CH),

7.47 – 8.06 (m, 0.25H, 2-CH, 5H, 2-Harom., 3-Harom., 4-Harom., 5-Harom., 6-Harom.), 9.88 (d, J = 8.2 Hz, 0.75H, CH=O), 9.94 (d, J = 7.9 Hz, 0.25H, CH=O).

13 C NMR (50 MHz, DMSO-d6): δ (ppm) = 23.6 (0.75C, C-5), 29.4 (0.25C, C-5), 42.6 (0.75C,

C-6), 42.8 (0.25C, C-6), 104.0 (0.25C, C-3), 110.5 (0.75C, C-3), 122.0 (0.25C, R2C=CH)

122.5 (0.75C, R2C=CH), 127.0 (2C, C-2arom., C-6arom.), 129.9 (2C, C-3arom., C-5arom.),133.0

(0.25C, C-2), 133.5 (0.75C, C-2), 134.1 (1C, C-4arom.), 136.2 (1C, C-1arom.),149.1 (0.25C, C- 4) 149.6 (0.75C, C-4), 190.3 (0.25C, CH=O) 190.7 (0.75C, CH=O).

Compound 88a was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 7.5:2.5 is observed.

FT-IR (neat): ṽ (cm-1) = 3066, 2970, 2920, 2850 (C-H aliph.), 1732 (C=O), 1660 (C=C), 1593 (C=C, cycle), 1165 (S=O).

88 tert-Butyl 4-oxopiperidine-1-carboxylate (75c)

Piperidin-4-one monohydrate hydrochloride 62 (5.0 g, 32.5 mmol, 1.0 eq.) was dissolved in a 1:1 mixture of THF:H2O (100 mL) at room temperature. NaHCO3 (5.47 g, 65 mmol, 2.0 eq.) was added and the mixture as stirred for 15 min at rt. Afterwards, Boc2O (8.52 g, 39 mmol, 1.2 eq.) was added and the mixture was stirred for 16 h at room temperature. The mixture was diluted with Et2O (50 mL) and washed with aqueous solution of KHSO4 5 % (3 x 50 mL), H2O (3 x 50 mL) and brine (3 x 50 mL). The organic layer was collected and volatiles were removed under reduced pressure. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  4/4, Rf = 0.34 (cyclohexane/ethyl acetate 7:3)). Colourless solid, mp 73-76 °C, yield 6.22 g (31 mmol, 96 %).

+ Exact mass (APCI): m/z = 200.1279 (calcd.200.1287 for C10H18NO3 [M+H ]).

1 H NMR (600 MHz, DMSO-d6): δ (ppm) = 1.42-1.44 (m, 9H, -C(CH3)3), 2.34 (t, J = 6.2 Hz,

4H, 3-(CH2)2), 3.60 (t, J = 6.2 Hz, 4H, 2-(CH2)2).

13 C NMR (151 MHz, DMSO-d6): δ (ppm) = 28.0 (3C, -C(CH3)3), 40.0 (2C, C-3), 40.3 (2C, C-

2), 79.2 (1C, OC(CH3)3), 153.8 (1C, C(=O)OC(CH3)3), 207.4 (1C, R2C=O).

-1 FT-IR (neat): ṽ (cm ) = 2985, 2870 (C-H, aliph.), 1724 (C=Ocarb.), 1678 (C=Oketone), 1161 (C- O).

89 tert-Butyl 3-bromo-4-oxopiperidine-1-carboxylate (77c)

1-Boc-piperidin-4-one 75c (10 g, 50 mmol, 1.0 eq.) was dissolved in THF (30 mL) and Et2O

(30 mL). AlCl3 (0.67 g, 5.0 mmol, 0.1 eq.) was added and the reaction mixture was then cooled to 0 °C. Br2 (2.6 mL, 50 mmol, 1.0 eq.) was added slowly over a period of 30 min. Afterwards, the solution was stirred at 0 °C for 18 h. After completion of transformation, the formed solid was filtered off and washed with Et2O diethyl ether. The organic layer was dried

(Na2SO4), concentrated in vacuo and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  5/5, Rf = 0.41 (cyclohexane/ethyl acetate 7:3)). Colourless solid, mp 90-93 °C, yield 6.42 g (23 mmol, 46 %).

+ Exact mass (APCI): m/z = 278.0329 (calcd.278.0392 for C10H17BrNO3 [M+H ]).

1 H NMR (200 MHz, DMSO-d6): δ (ppm) = 1.44-1.46 (m, 9H, -C(CH3)3), 2.40-2.50 (m, 1H,

5-H), 2.74 (m, 1H, 5-H), 3.66-3.70 (m, 3H, 6-CH2, 2-CH2 (1H)), 3.97 – 4.23 (m, 1H, 2-CH2), 4.78 (s, 1H, 3-CH).

13 C NMR (50 MHz, DMSO-d6): δ (ppm) = 27.9 (3C, C(CH3)3), 35.8 (1C, C-5), 42.7 (1C, C-

6), 47.7 (1C, C-2), 49.0 (1C, C-3), 79.8 (1C, OC(CH3)3), 153.8 (1C, C(=O)-OC(CH3)3), 199.7

(1C, R2C=Oketone).

-1 FT-IR (neat): ṽ (cm ) = 2978, 2931 (C-H. aliph.), 1724 (C=Oketone), 1674 (C=Ocarbamate), 1157 (C-O), 648 (C-Br).

90 tert-Butyl (E)- and (Z)-3-bromo-4-(ethoxycarbonylmethylene)piperidine-1-carboxylate (89c)

tert-Butyl 3-Bromo-4-oxopiperidine-1-carboxylate 77c (5.03 g, 18 mmol, 1.0 eq.) was dissolved in CH2Cl2 (450 mL). (ethoxycarbonylmethylene)triphenylphosphorane (6.9 g, 20 mmol, 1.1 eq.) was added and the reaction mixture was stirred at 39 °C for 2 h. After completion of the transformation, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3, Rf = 0.57 (cyclohexane/ethyl acetate 7:3)). Colourless solid, mp 114-115 °C, yield 5.98 g (17 mmol, 95 %).

+ Exact mass (APCI): m/z = 348.0777 (calcd.348.0810 for C14H23BrNO4 [M+H ]).

1 H NMR (200 MHz, DMSO-d6): δ (ppm) = 1.21 (t, J = 7.1 Hz, 3H, -OCH2CH3), 1.42 (s, 9H,

C(CH3)3), 2.56 - 2.88 (m, 2H, 5-CH2 (1H), 6-CH2 (1H)), 3.34 - 3.51 (m, 2H, 5-CH2 (1H), 2-

CH2 (1H)), 4.01 – 4.29 (m, 4H, OCH2CH3, 6-CH2 (1H), 2-CH2 (1H)), 5.04 - 5.12 (m, 1H, 3-

CH), 6.12 (s, 1H, R2C=CH).

13 C NMR (50 MHz, DMSO-d6): δ (ppm) = 14.0 (1C, OCH2CH3), 24.1 (1C, C-5), 27.9 (3C, -

C(CH3)3), 42.5 (1C, C-6), 51.0 (1C, C-2), 53.3 (1C, C-3), 59.9 (1C, OCH2CH3), 79.2 (1C, -

C(CH3)3), 113.8 (1C, R2C=CH), 153.7 (1C, -C(=O)OC(CH3)3), 154.0 (1C, C-4), 165.1 (1C,

CO2Et).

Only one set of signals can be seen in the spectra.

FT-IR (neat): ṽ (cm-1) = 2985, 2920 (C-H, aliph.), 1712 (C=O), 1670 (C=O), 1654 (C=C), 1161 (C-O), 641 (C-Br).

91 tert-Butyl (E)- and (Z)-4-(ethoxycarbonylmethylene)-3,4-dihydropyridine-1(2H)- carboxylate (63c)

Ester 89c (5.74 g, 16 mmol, 1.0 eq) was dissolved in dry DMF (165 mL). LiBr (8.6 g, 99 mmol, 6.0 eq.) and Li2CO3 (7.31 g, 99 mmol, 6.0 eq.) were added and the solution was stirred at 75 °C for 3 h. After completion of the transformation, the mixture was cooled to room temperature and extracted with ethyl acetate (3 x 100 mL). The organic layer was collected and the volatiles were removed under vacuo. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3, Rf = 0.72 (cyclohexane/ethyl acetate 7:3)). Yellow oil, yield 3.88 g (14 mmol, 88 %).

+ Exact mass (APCI): m/z = 268.1497 (calcd.268.1549 for C14H22NO4 [M+H ]).

1 H NMR (600 MHz, DMSO-d6): δ (ppm) = 1.19 (t, J = 7.1 Hz, 3H, OCH2CH3), 1.46 (s, 9H,

C(CH3)3), 2.49 – 2.53 (m, 0.2H, 5-CH2), 3.00 - 3.07 (m, 1.8H, 5-CH2), 3.52 - 3.60 (m , 2H,

6-CH2,), 4.06 (q, J = 7.1 Hz, 2H, OCH2CH3), 5.32 - 5.34 (m, 0.1H, R2C=CH), 5.47 - 5.53 (m,

0.9H, 3-CH) 5.55 - 5.59 (m, 0.9H, R2C=CH), 6.54 - 6.61 (m, 0.1H, 3-CH) 7.00 - 7.13 (m, 0.9H, 2-CH), 7.14 - 7.16 (m, 0.1H, 2-CH).

13 C NMR (151 MHz, DMSO-d6): δ (ppm) = 14.2 (1C, OCH2CH3), 24.8 (0.9C, C-5), 27.7 (3C,

-C(CH3)3), 30.0 (0.1C, C-5), 40.0 (1C, C-6), 59.0 (1C, OCH2CH3), 81.5 (1C, -C(CH3)3), 103.4

(0.1C, C-3), 108.1 (0.9C, C-3), 109.6 (0.1C, R2C=CH) 110.6 (0.9C, R2C=CH), 132.5 (0.9C,

C-2), 133.0 (0.1C, C-2), 147.5 (1C, C(=O)OC(CH3)3), 147.9 (0.1C, R2C=CH) 149.1 (0.9C,

R2C=CH), 166.0 (1C, CO2Et).

Compound 63c was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 9:1 is observed.

FT-IR (neat): ṽ (cm-1) = 2978, 2931, 2900 (C-H, aliph.), 1708 (C=O), 1700 (C=O), 1608 (C=C), 1145 (C-O), 1111 (C-O).

92 tert-Butyl (E)- and (Z)-4-(2-hydroxyethylidene)-3,4-dihydropyridine-1(2H)-carboxylate (74c)

Procedure 1

Under N2, 63c (2.80 g, 10.5 mmol, 1.0 eq.) was dissolved in dry toluene (25 mL). The solution was cooled to -78 °C in a dry ice/acetone bath, DIBAL-H (1 M solution in hexane, 31.5 mL, 31.5 mmol, 3.0 eq.) was added dropwise within 15 min, and the reaction mixture was stirred at -78 °C for 40 min. After complete transformation, methanol (2 mL) was added carefully at -78 °C and the mixture was warmed to room temperature. The mixture was filtered, the solid was washed with ethyl acetate (3 x30 mL) and the organic layer was dried under reduced pressure. Yellow oil, yield 2.22 g (9.9 mmol, 94 %).

Procedure 2

Under N2, 63c (570 mg, 2.1 mmol, 1.0 eq.) was dissolved in dry THF (5 mL) and the solution was cooled to -10 °C. At this temperature LiAlH4 (160 mg, 4.3 mmol, 2.0 eq.) was added and the mixture was stirred for 40 min. A saturated solution of potassium sodium tartrate in water (5 mL) was added, the mixture was extracted with ethyl acetate (3 x15 mL) and the organic layer was dried with Na2SO4. After filtration the solution was concentrated in vacuo and the residue was purified by flash column chromatography (cyclohexane/ethyl acetate 9/1  6/4, Rf = 0.45 (petroleum ether/ethyl acetate 7:3)). Yellow oil, yield 0.33 g (1.5 mmol, 70 %).

+ Exact mass (APCI): m/z = 226.1383 (calcd.226.1443 for C12H20NO3 [M+H ]).

1 H NMR (600 MHz, DMSO-d6) δ (ppm) = 1.44 (s, 9H, C(CH3)3), 2.36 – 2.44 (m, 2H, 5-CH2),

3.48 – 3.55 (m, 2H, 6-CH2), 3.96 – 4.01 (m, 2H, CH2OH), 4.53 – 4.58 (m, 1H, OH), 5.13 (t,

J = 6.8 Hz, 0.15H, R2C=CH), 5.35 (t, J = 6.8 Hz, 0.85H, R2C=CH), 5.40 – 5.46 (m, 1H, 3- CH), 6.65 – 6.78 (m, 1H, 2-CH).

13 C NMR (151 MHz, DMSO-d6) δ (ppm) = 26.9 (1C, 5-CH2), 30.9 (3C, -C(CH3)3), 43.1 (1C,

6-CH2), 59.9 (1C, CH2OH), 83.6 (1C, -C(CH3)3),, 112.7 (1C, 3-CH), 127.8 (1C, R2C=CH),

128.4 (1C, 2-CH), 133.2 (1C, R2C=CH), 154.3 (1C, C(=O)OC(CH3)3).

Compound 73c was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 8.5:1.5 is observed. Peaks of minor isomer are not all clearly visible in the spectra.

FT-IR (neat): ṽ (cm-1) = 3398 (O-H), 2974, 2931, 2873 (C-H, aliph.), 1701 (C=O), 1643 (C=C), 1612 (C=C), 1161, 1141 (C-O).

tert-Butyl (E)- and (Z)-4-(formylmethylene)-3,4-dihydropyridine-1(2H)-carboxylate (88c)

CuCl (11 mg, 0.12 mmol, 0.1 eq.) and TEMPO (17 mg, 0.12 mmol, 0.1 eq.) were added to a solution of racemic mixture of allylic alcohol 73c (270 mg, 1.12 mmol, 1.0 eq.) in dry DMF (3 mL). The solution was stirred at room temperature for 16 h. Afterwards, the solution was poured into water/ice slowly and the mixture was warmed to room temperature. The solid was filtered, washed with water and dried. Purification by flash column chromatography

(petroleum ether/ethyl acetate 9/1  7/3 Rfa = 0.38, Rfb= 0.30 (cyclohexane/ethyl acetate 7:3)). Yellow oil, yield 240 mg (0.9 mmol, 76 %).

+ Exact mass (APCI): m/z = 447.3508 (calcd.447.2495 for C24H35N2O6 [M+H ]).

1 H NMR (600 MHz, CDCl3) δ (ppm) 1.52 (s, 3.6H, C(CH3)3), 1.53 (s, 5.4H, C(CH3)3), 2.64 (t,

J = 6.8 Hz, 0.8H, 5-CH2), 2.91 – 3.14 (m, 1.2H, 5-CH2), 3.74 (t, J = 6.8 Hz, 2H, 6-CH2), 5.44

– 5.53 (m, 0.6H, 3-CH), 5.57 (d, J = 7.8 Hz, 0.4H, R2C=CH), 5.78 (d, J = 7.7 Hz, 0.6H,

R2C=CH), 6.23 – 6.36 (m, 0.4H, 3-CH), 7.13 – 7.19 (m, 0.6H, 2-CH), 7.27 – 7.41 (m, 0.4H, 2-CH), 9.94 (d, J = 7.9 Hz, 0.6H, CHO), 10.05 (d, J = 7.9 Hz, 0.4H, CHO).

13 C NMR (151 MHz, CDCl3) δ (ppm) 24.7 (0.6C, C-5), 28.2 (3C, C(CH3)3), 30.9 (0.4C, C-5),

40.4 (1C, C-6), 82.8 (1C, -C(CH3)3), 101.2 (0.4C, C-3), 108.1 (0.6C, C-3), 121.5 (1C,

R2C=CH), 134.0 (0.6C, C-2), 134.5 (0.4C, C-2), 151.1 (1C, R2C=CH), 151.3 (1C,

C(=O)OC(CH3)3), 189.6 (0.4C, CHO), 190.0 (0.6C, CHO).

Compound 88c was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 6:4 is observed. Peaks of minor isomer are not all clearly visible in the spectra.

FT-IR (neat): ṽ (cm-1) = 3062, 2966, 2877 (C-H, aliph.), 1712 (C=O), 1647 (C=C), 1593 (C=C), 1141, 1122 (C-O).

(E)- and (Z)-2-[2,3-dihydropyridin-4(1H)-ylidene]acetaldehyde (52)

A solution of 88c (110 mg, 0.49 mmol, 1.0 eq.) in water (9 mL) and 1,4-dioxane (1 mL) was heated to 85 °C for 2 h. After completion of the transformation, the solution was cooled to room temperature and the mixture was extracted with ethyl acetate (6 x 15 mL). The organic layer was dried with Na2SO4 and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  1/9 Rfa = 0.15 (ethyl acetate)). Yellow/orange oil, yield 12 mg (0.10 mmol, 20 %).

The compound is highly unstable and decomposed fast during purification.

1 H NMR (200 MHz, CDCl3) δ (ppm) = 2.61 (t, J = 7.0 Hz, 0.8H, 5-CH2), 3.00 – 3.12 (m, 1.2H,

5-CH2), 3.31 – 3.46 (m, 2H, 6-CH2), 4.7- 4.81 (m, 1H, NH), 5.19 (d, J = 7.2 Hz, 0.6H,

R2C=CH), 5.29 (d, J = 7.9 Hz, 1H, R2C=CH), 5.60 (d, J = 8.3 Hz, 0.6H, 3-CH), 6.00 (d, J = 7.5 Hz, 0.4H, 3-CH), 6.66 - 6.77 (m, 1H, 2-CH), 9.79 (d, J = 8.4 Hz, 0.6H, CHO), 9.93 (d, J = 8.0 Hz, 0.4H, CHO).

13 C NMR (50 MHz, CDCl3): δ (ppm) = 25.3 (0.6C, C-5), 31.75 (0.4C, C-5), 40.5 (0.6C, C-6),

40.9 (1C, C-6), 93.9 (0.4C, C-3), 100.3 (0.6C, C-3), 116.2 (0.4C, R2C=CH), 117.1 (0.6C,

R2C=CH), 142.6 (1C, C-2), 143.4 (1C, C-2), 155.3 (0.4C, R2C=CH), 171.3 (0.6C, R2C=CH), 189.2 (0.4C, CHO), 189.3 (0.6C, CHO).

Compound 52 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 6:4 is observed. Peaks of minor isomer are not all clearly visible in the spectra.

Dimethyl (S)aspartate HCl (97)

(S)-Aspartic acid (5 g, 37 mmol, 1.0 eq.) was added to a solution of SOCl2 (2.74 mL, 37 mmol, 1.0 eq.) in CH3OH (25 mL) at 0 °C. After stirring for 30 min at 4 °C, the solution was concentrated under reduced pressure at 40 °C and the residue was dissolved in a mixture of methanol (5 mL) and ethyl acetate (20 mL) and left overnight at 5° C. The crystals formed were collected by filtration, washed with ethyl acetate and dried at 90 °C to obtain dimethyl (S)-aspartate HCl (97). Colourless solid, mp 191-193 °C, yield 4.62 g (25 mmol, 67 %).

1 H NMR (400 MHz, DMSO-d6): δ (ppm) = 2.98 (d, J = 5.7, 2H, 3-CH2), 3.64 (s, 3H, OCH3), 4.16 (t, J = 5.7 Hz, 1H, 2-CH).

13 C NMR (101 MHz, DMSO-d6): δ (ppm) = 34.0 (1C, C-3), 48.4 (1C, C-2), 52.0 (1C, OCH3),

169.6 (1C, C(=O)OCH3), 169.7 (1C, C(=O)OH).

Diethyl N-[2-(methoxycarbonyl)ethyl]aspartate (104)

Methyl-3-aminopropionatae HCl 102 (2.00 g, 14 mmol, 1.0 eq.) was dissolved in DMF (60 mL). K2CO3 (3.96 g, 28 mmol, 2.0 eq.), LiBr (1.24 g, 14 mmol, 1.0 eq.) and diethyl fumarate (2.34 mL, 14 mmol, 1.0 eq.) were added and the mixture was stirred for 3 d at room 96 temperature. Afterwards, the mixture was diluted with water (60 mL) and extracted with ethyl acetate (3 x100 mL). The organic layers were collected, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography

(cyclohexane/ethyl acetate 9/1  7/3 Rf = 0.45 (cyclohexane/ethyl acetate 6:4)). Colourless oil, yield 3.11 g (11 mmol, 79 %).

+ Exact mass (APCI): m/z = 276.1440 (calcd.276.1447 for C12H22NO6 [M+H ]).

1 H NMR (400 MHz, CDCl3): δ (ppm) = 1.23 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.26 (t, J = 7.1

Hz, 3H, OCH2CH3), 2.03 (s, 1H, NH), 2.47 (t, J = 6.6 Hz, 2H, CH2CH2NH), 2.60 (dd, J =

15.8/7.0 Hz, 1H, CH2CO2Et), 2.70 (dd, J = 15.8/6.0 Hz, 1H, CH2CO2Et), 2.78 (dt, J = 11.8/6.6

Hz, 1H, CH2CH2NH), 2.99 (dt, J = 11.8/6.7 Hz, 1H, CH2CH2NH), 3.62 (dd, J = 7.0/6.0 Hz,

1H, CHCH2), 3.66 (s, 3H, OCH3), 4.13 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.19 (q, J = 7.1 Hz,

2H, OCH2CH3).

13 C NMR (101 MHz, CDCl3) δ (ppm) = 14.2 (1C, OCH2CH3), 14.3 (1C, OCH2CH3), 34.9 (1C,

CH2CH2NH), 38.2 (1C, CH2CO2Et), 43.5 (1C, CH2NH), 51.7 (1C, CH3O), 57.9 (1C, NHCH),

60.8 (1C, OCH2CH3), 61.2 (1C, OCH2CH3), 170.9 (1C, CH-CO2Et), 172.9 (1C, CH2CO2Et),

173.5 (1C, CH3O2C).

Diethyl N-(tert-butoxycarbonyl)-N-[2-(methoxycarbonyl)ethyl]aspartate (105)

Triester 104 (2.00 g, 7.2 mmol, 1.0 eq.) was dissolved in a mixture of water (20 mL) and

THF (20 mL). K2CO3 (2.00 g, 14.5 mmol, 2.0 eq.) (Boc)2O (1.90 g, 8.7 mmol, 1.2 eq.) were added and the mixture was stirred at room temperature for 16 h. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 x30 mL). The organic layers were collected, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified

97 by flash column chromatography (cyclohexane/ethyl acetate 9/1  7/3 Rf = 0.75 (cyclohexane/ethyl acetate 7:3)). Colourless oil, yield 2.59 g (7 mmol, 95 %).

+ Exact mass (APCI): m/z = 376.1991 (calcd.376.1971 for C17H30NO8 [M+H ]).

1 H NMR (600 MHz, CDCl3-d): δ (ppm) = 1.16 – 1.32 (m, 6H, OCH2CH3), 1.41 (s, , 5.4H,

C(CH3)3), 1.45 (s, 3.6H, C(CH3)3), 2.58 – 2.73 (m, 2H, MeO2CCH2), 2.76 (dd, J = 16.6, 7.5

Hz, 0.6H, CH2CO2Et), 2.85 (dd, J = 16.8/8.0 Hz 0.4H, CH2CO2Et), 3.12 – 3.23 (m, 1H,

CH2CO2Et), 3.45 – 3.57 (m, 1H, CH2CH2N ), 3.62 – 3.65 (m, 0.4H, CH2CH2N), 3.66 (s, 1.8H,

CH3O) 3.68 (s, 1.2H, CH3O), 3.71 – 3.80 (m, 0.6H, CH2CH2N), 4.04 – 4.26 (m, 4H,

OCH2CH3), 4.38 – 4.48 (m, 1H, NCHCH2).

13 C NMR (151 MHz, CDCl3): δ (ppm) = 14.1 (1C, CH2CO2CH2CH3), 14.3 (1C,

CHCO2CH2CH3), 28.3 (1.8C, O-C(CH3)3), 28.4 (1.2C, O-C(CH3)3), 33.5 (0.6C, CH2CH2N),

34.1 (0.4C, CH2CH2N), 35.6 (0.4C, CH-CH2CO2Et), 36.6 (0.6C, CHCH2CO2Et), 45.4 (0.6C,

CH2CH2N), 45.8 (0.6C, CH2CH2N), 51.7 (0.6C, CH3O), 51.8 (0.4C, CH3O), 58.7 (0.6C,

NCHCH2), 58.8 (0.4C, NCHCH2), 60.9 (0.4C, CH2CO2CH2CH3) 61.0 (0.6C,

CH2CO2CH2CH3), 61.6 (0.4C, CHCO2CH2CH3), 61.7 (0.6C, CH2CO2CH2CH3), 80.9 (0.4C,

C(CH3)3), 81.2 (0.6C, C(CH3)3), 154.3 (0.6C, N-C=O), 154.8 (0.4C, N-C=O), 170.5 (0.4C,

CH2CO2Et) 170.6 (0.6C, CH2CO2Et), 171.3 (0.6C, CHCO2Et), 171.6 (0.4C, CHCO2Et),

172.6 (0.4C, MeO2CCH2), 172.6 (0.6C, MeO2CCH2).

Two rotamers exist in the ratio of 60:40.

1-(tert-Butyl) 2-ethyl (E)- and (Z)-4-(ethoxycarbonylmethylene)piperidine-1,2- dicarboxylate (57a)

(Ethoxycarbonylmethylene)triphenylphosphorane 79 (1.76 g, 5.0 mmol, 2.75 eq.) was suspended in toluene (2.35 mL). E (R)-1-Boc-4-oxopiperidine-2-carboxylate 58 (450 mg, 1.65 mmol, 1.0 eq.) was added to the suspension and the mixture was heated to 115 °C for 3 h. The mixture was concentrated in vacuo and the residue was purified by flash column chromatography (cyclohexane/ethyl acetate 9/1  7/3 Rf = 0.75 (cyclohexane/ethyl acetate 6:4)). Colourless oil, yield 506 mg (1.5 mmol, 90 %).

+ Exact mass (APCI): m/z = 342.1931 (calcd.342.1917 for C17H28NO6 [M+H ]).

1 H NMR (600 MHz, CDCl3) δ (ppm) = 1.19 – 1.27 (m, 6H, OCH2CH3), 1.36 (m, 4.5H,

C(CH3)3) – 1.41 (m, 4.5H, C(CH3)3), 2.19 – 2.79 (m, 2H, 5-CH2, 1H, 3-CH2), 3.06 – 3.35 (m,

1H, 6-CH2), 3.35 – 3.62 (m, 1H, 3-CH2), 3.87 – 4.05 (m, 1H, 6-CH2), 4.07 – 4.26 (m, 4H,

OCH2CH3), 4.58 – 5.11 (m, 1H, 2-CH), 5.74 (s, 0.5H, R2C=CH), 5.80 (s, 0.5H, R2C=CH).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 14.4 (1C, -OCH2CH3), 14.4 (1C, --OCH2CH3), 27.8

(1.5C, -C(CH3)3), 27.9 (1.5C, -C(CH3)3), 28.9 (1C, C-3), 36.8 (1C, C-5), 40.4 (1C, C-6), 55.2

(1C, C-2), 61.2 (1C, OCH2CH3), 61.3 (1C, OCH2CH3), 80.6 (1C, -C(CH3)3), 116.6 (1C,

R2C=CH), 154.4 (1C, N-C=O), 166.3 (C=CH-CO2Et), 170.7 (1C, NCHCO2Et).

Compound 57a was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5:5 is observed. Some peaks are not clearly visible in the spectra.

FT-IR (neat): ṽ (cm-1) = 2985, 2920 (C-H, aliph.), 1710 (C=O), 1675 (C=O), 1161 (C-O).

1-(tert-Butyl) 2-ethyl cis- and trans-4-(ethoxycarbonylmethyl)piperidine-1,2- dicarboxylate (70a)

Under N2 atmosphere, Pd/C 10 % (17 mg, 0.16 mmol, 0.1 eq.) was added to a solution of compound 57a (550 mg, 1.6 mmol, 1.0 eq.) in dry CH3OH (33 mL). The suspension was ® stirred for 16 h under H2 atmosphere (balloon). After filtration through Celite , the solvent was removed under reduced pressure and the residue purified by flash column chromatography (cyclohexane/ethyl acetate 9/1  7/3 Rf = 0.68 (cyclohexane/ethyl acetate 6:4, stain with Dragendorff reagent)). Colourless oil, yield 530 mg, (1.5 mmol, 96 %).

+ Exact mass (APCI): m/z = 344.2096 (calcd.344.2073 for C17H30NO6 [M+H ]).

1 H NMR (400 MHz, DMSO-d6) δ (ppm) = 1.24 (t, 6.0 Hz, 3H, -OCH2CH3), 1.26 (t, 6.2 Hz, 3H

-OCH2CH3), 1.38 – 1.44 (s, 9H, -C(CH3)3), 1.68 – 1.81 (m, 2H, 5-CH2, 1H, 3-CH2), 1.95 –

2.07 (m, 1H, 3-CH2), 2.05 – 2.13 (m, 1H, 4-CH), 2.27 – 2.33 (m, 2H, R2CH-CH2), 3.24 – 3.36

(m, 1H, 6-CH2), 3.45 – 3.57 (m, 1H, 6-CH2), , 4.08 – 4.19 (m, 4H, -OCH2CH3), 4.26 (t, J = 6.2 Hz, 1H, 2-CH).

13 C NMR (101 MHz, DMSO-d6) δ (ppm) = 13.1 (1C, -OCH2CH3), 13.2 (1C, -OCH2CH3), 27.2

(1C, C-4), 27.4 (3C, -C(CH3)3), 27.8 (1C, C-5), 29.8 (1C, C-3), 37.2 (1C, R2CH-CH2), 38.3

(1C, C-6), 53.6 (1C, C-2), 59.7 (1C, OCH2CH3), 59.8 (1C, OCH2CH3), 78.7 (1C, -C(CH3)3),

154.2 (1C, NC=O), 171.2 (1C, CH2CO2Et), 171.4 (1C, CHCOOEt).

FT-IR (neat): ṽ (cm-1) = 2985, 2920 (C-H, aliph.), 1718 (C=O), 1683 (C=O), 1158 (C-O).

100

2-(tert-Butyl) 6-ethyl (1RS,5SR)-7-oxo-2-azabicyclo[3.2.1]octane-2,6-dicarboxylate (56a)

Under N2, a solution NaHMDS (1 M) in THF (0.58 mL, 0.58 mmol, 2.0 eq.) was added dropwise to an ice-cooled solution of ester 70a (100 mg, 0.29 mmol, 1.0 eq.) in THF (2 mL) and the reaction mixture was stirred for 2 h at 0 °C. The mixture was allowed to warm at ambient temperature and was stirred for additional 14 h at room temperature. Afterwards,

HOAc (33.5 µL) and water were added and the mixture was extracted with CH2Cl2 (3 x 5 mL). The organic layers were collected, dried over Na2SO4 filtered and concentrated in vacuo. The residue was purified by flash column chromatography (cyclohexane/ethyl acetate 9/1  5/5 Rf = 0.38 (cyclohexane/ethyl acetate 6:4, stain with Dragendorff reagent)). Light yellow oil, yield 20 mg (0.07 mmol, 23 %).

+ Exact mass (APCI): m/z = 298.1636 (calcd.298.1654 for C15H24NO5 [M+H ]).

1 H NMR (600 MHz, CDCl3): δ (ppm) = 1.27 (t, 7.2 Hz, 3H, OCH2CH3), 1.45 (s, 9H, -C(CH3)3),

1.63 – 1.74 (m, 1H, 5-CH2), 1.77 – 1.88 (m, 1H, 3-CH2), 1.89 – 2.04 (m, 1H, 5-CH2), 2.18 –

2.31 (m, 1H, 3-CH2), 2.91 (m, 1H, 6-CH2), 2.94 – 2.98 (m, 1H, 8-CH), 3.00 – 3.05 (m, 1H, 4-

CH), 3.87 – 4.04 (m, 1H, 6-CH2), 4.17 (q, J = 7.2 Hz, 2H, O-CH2CH3), 4.34 – 4.46 (m, 1H, 2-CH).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 14.2 (1C, -OCH2CH3), 28.4 (3C, -C(CH3)3, 29.7 (1C, C-5), 34.6 (1C, C-3), 35.3 (1C, C-8), 38.3 (1C, C-6), 57.1 (1C, (1C, C-4), 58.8 (1C, C-2),

61.8 (1C, OCH2CH3), 80.8 (1C, -C(CH3)3), 154.3 (1C, NCO2C(CH3)3), 167.8 (1C, COOEt),

208.6 (1C, C=Oketone).

-1 FT-IR (neat): ṽ (cm ) = 3456 (OHenol), 2962, 2860 (C-H, aliph.), 1766 (C=O, ketone), 1724 (C=O, ester) 1670 (C=O, Boc), 1165 (C-O).

101

(E)- and (Z)-4-(2-azidoethylidene)-1-(phenylsulfonyl)-1,2,3,4-tetrahydropyridine (73a)

PPh3 (6.37 g, 24.3 mmol, 2.1 eq.) and Zn(N3)22Py (2.73 g, 8.9 mmol, 0.75 eq.) were dissolved in toluene (200 mL). A solution of allylic alcohol 73a (3.10 g, 11.7 mmol, 1.0 eq.) in toluene (4 mL) was added slowly. The reaction mixture was cooled with an ice-bath to 0 °C and at this temperature DIAD (4.6 mL, 23.4 mmol, 2.0 eq.) was added dropwise. After complete addition of DIAD, the ice bath was removed and the mixture was stirred for additional 16 h at room temperature. After completion of the transformation, the mixture was concentrated in vacuo and the residue was purified by flash column chromatography

(petroleum ether/ethyl acetate 9/1  7/3 Rf = 0.37 (petroleum ether/ethyl acetate 9:1)). Light yellow oil, yield 1.3 g (4.5 mmol, 38 %).

+ Exact mass (APCI): m/z = 291.0919 (calcd.291.0916 for C13H15N4O2S [M+H ]).

1 H NMR (600 MHz, DMSO-d6) δ (ppm) = 2.37 – 2.40 (m, 0.94H, 5-CH2), 2.40 – 2.43 (m,

1.06H, 5-CH2), 3.30 – 3.34 (m, 1.06H, 6-CH2), 3.34 – 3.38 (m, 0.94H, 6-CH2), 3.87 (d, J =

7.6 Hz, 0.53H, CH2N3), 3.92 (d, J = 7.7 Hz, 0.47H, CH2N3), 5.16 (t, J = 7.7 Hz, 0.47H,

R2C=CH), 5.36 – 5.41 (m, 0.53H, R2C=CH), 5.61 (d, J = 8.1 Hz, 0.53H, 3-CH), 5.87 (d, J = 8.4 Hz, 0.47H, 3-CH), 6.73 (d, J = 8.1 Hz, 0.53H, 2-CH), 6.82 (dd, J = 8.4 Hz, 0.47H, 2-CH),

7.63 – 7.68 (m, 2H, 3-Harom., 5-Harom.), 7.70 – 7.76 (m, 1H, 4-Harom.), 7.80 – 7.86 (m, 2H, 2-

Harom., 6-Harom.).

13 C NMR (151 MHz, DMSO-d6) δ (ppm) = 23.3 (0.53C, C-5), 29.1 (0.47C, C-5), 43.2 (0.53C,

C-6), 43.9 (0.47C, C-6), 46.3 (0.47C, CH2N3), 46.9, (0.53C, CH2N3), 105.4 (0.47C, C-3),

111.8 (0.53C, C-3), 116.7 (0.47C, R2C=CH), 117.7 (0.53C, R2C=CH), 126.2 (0.53C, C-2),

102

126.8 (2C, C-2arom., C-6arom.), 127.5 (0.47C, C-2), 129.7 (2C, C-3arom., C-5arom.), , 132.8 (1C,

R2C=CH), 133.7 (1C, C-1arom.), 133.7 (1C, R2C=CH), 136.6 (1C, C-4arom.).

Compound 74a was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5.3:4.7 is observed.

FT-IR (neat): ṽ (cm-1) = 3062 (C-H, aromatic), 2951, 2924 (C-H, aliph.), 2094 (N=N=N), 1643 (C=C), 1600 (C=C), 1165 (S=O).

tert-Butyl (E)- and (Z)-4-(2-azidoethylidene)-3,4-dihydropyridine-1(2H)-carboxylate (73c)

PPh3 (2.94 g, 11.2 mmol, 2.1 eq.) and Zn(N3)2 2Py (1.26 g, 4.0 mmol, 0.75 eq.) were dissolved in toluene (90 mL). A solution of allylic alcohol 73c (1.21 g, 5.4 mmol, 1.0 eq.) in toluene (4 mL) was added slowly. The reaction mixture was cooled with an ice-bath to 0 °C and at this temperature DIAD (2.12 mL, 10.8 mmol, 2 eq.) was added dropwise. After complete addition of DIAD, the ice-water bath was removed and the mixture was astirredfor additional 16 h at room temperature. After completion of the transformation, the mixture was concentrated in vacup and the residue was purified by flash column chromatography

(petroleum ether/ethyl acetate 9/1  8/2 Rf = 0.69 (petroleum ether/ethyl acetate 9:1)). Light yellow oil, yield 350 mg (1.4 mmol, 25 %).

+ Exact mass (APCI): m/z = 251.1519 (calcd.251.1508 for C12H19N4O2 [M+H ]).

1 H NMR (200 MHz,CDCl3): δ (ppm) = 1.42 (s, 9H, C(CH3)3), 2.37 – 2.42 (m, 2H, 5-CH2),

3.51 (m, 2H, 6-CH2), 3.91 (d, 6.0 Hz, 1H, CH2N3), 3.97 (d, 6.3 Hz, 1H, CH2N3), 5.17 (t, J =

103

7.8 Hz, 0.5H, R2C=CH) 5.37 (t, J = 7.8 Hz, 0.5H, R2C=CH), 5.42 – 5.53 (m, 0.5H, , 3-CH),

5.70 – 5.82 (m, 0.5H, 3-CH), 6.79 – 6.94 (m, 1H, , 2-CH).

13 C NMR (50 MHz,CDCl3): δ (ppm) = 26.9 (1C, -C-5), 30.9 (3C, -C(CH3)3), 43.1 (1C, C-6),

59.9 (1C, CH2N3), 83.6 (1C, -C(CH3)3), 112.7 (1C, C-3), 127.8 (1C, R2C=CH), 128.4 (1C, C- 2), 133.2 (1C, C-4), 154.3 (1C, N-C(=O)).

Compound 74c was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5.3:4.7 is observed. Peaks for minor isomer are not clearly visible.

FT-IR (neat): ṽ (cm-1) = 2951, 2924 (C-H, aliph.), 2104 (N=N=N), 1711 (C=O), 1643 (C=C), 1600 (C=C), 1165 (C-O).

(E)- and (Z)-4-[2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethylidene]-1-(phenylsulfonyl)-1,2,3,4- tetrahydropyridine (109)

Procedure 1

Azide 74a (250 mg, 0.86 mmol, 1.0 eq.) and phenylacetylene (94 µL, 0.86 mmol, 1.0 eq.) were dissolved in a mixture of tert-butanol (4 mL) and water (4 mL). CuSO4*5H2O (11 mg, 0.04 mmol, 0.05 eq.) and sodium ascorbate (17 mg, 0.09 mmol, 0.1 eq.) were added and the solution stirred at ambient temperature for 16 h. After complete transformation, the solution was diluted with water and extracted with ethyl acetate (3x), the organic layers were collected, dried with Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.38, RfB = 0.31 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 121-124 °C, yield 141 mg (0.36 mmol, 42 %).

104

Procedure 2

CuI (2 mg, 0.01 mmol, 0.02 eq.), N,N-diisopropylethylamine (3.6 µL, 0.02 mmol, 0.04 eq.), glacial acetic acid (1.2 µL, 0.02 mmol, 0.04 eq.) and phenylacetylene (57 µL, 0.52 mmol, 1.0 eq.) were added in sequence to a solution of azide 74a (150 mg, 0.52 mmol, 1.0 eq.) in

CH2Cl2 (1 mL). The solution was stirred at ambient temperature for 16 h. Afterwards, the mixture was concentrated in vacuo and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.38, RfB = 0.31 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 121-124 °C, yield 141 mg (0.36 mmol, 68 %).

Purity by HPLC, method 1: 99.9 %, tR = 21.5 min.

+ Exact mass (APCI): m/z = 393.1411 (calcd.393.1385 for C21H21N4O2S [M+H ]).

1 H NMR (600 MHz, CDCl3) δ (ppm) = 2.47 (t, J = 5.7 Hz, 1.1H, 5-CH2), 2.52 – 2.61 (m, 0.9H,

5-CH2), 3.47 (t, J = 7.3 Hz, 1.1H, 6-CH2), 3.50 (t, J = 6.4 Hz, 0.9H, 6-CH2), 5.00 (d, J = 7.4

Hz, 0.9H, CH2Ntriazole), 5.06 (d, J = 7.4 Hz, 1.1H, CH2Ntriazole), 5.26 (t, J = 7.3 Hz, 0.55H,

R2C=CH), 5.47 (t, J = 7.5 Hz, 0.45H, R2C=CH), 5.50 (d, J = 8.3 Hz, 0.45H, 3-CH), 5.78 (d, J = 8.4 Hz, 0.55H, 3-CH), 6.79 (d, J = 8.1 Hz, 0.45H, 2-CH), 6.93 (d, J = 8.4 Hz, 0.55H, 2-

CH), 7.29 – 7.36 (m, 1H, 4-Harom.), 7.37 – 7.44 (m, 2H, 3-Harom., 5-Harom.), 7.48 – 7.58 (m,

2H, 3’-Harom., 5’-Harom.), 7.58 – 7.65 (m, 1H, 4’-Harom.), 7.66 (s, 0.45H, CHtriazole), 7.68 (s,

0.55H, 5-CHtriazole), 7.80 (m, 4H, 2-Harom., 6-Harom., 2’-Harom., 6’-Harom.).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 24.2 (0.45C, C-5), 30.1 (0.55C, C-5), 43.2 (0.45C, C-

6), 43.8 (0.55C, C-6), 47.0 (0.55C, CH2Ntriazole), 47.3 (0.45C, CH2Ntriazole), 103.8 (0.55C, C-

3), 110.8 (0.45C, C-3), 115.6 (0.55C, R2C=CH), 116.4 (0.45C, R2C=CH), 119.1 (0.45C, C-

5triazole), 119.2 (0.55C, C-5triazole), 125.8 (2C, C-2arom., C-6arom.), 127.1 (2C, C-2’arom., C-6’arom.),

127.4 (0.45C, C-2), 128.3 (1C, C-4arom.), 128.98 (2C, C-3arom., C-5arom.), 129.0 (0.55C, C-2),

129.5 (2C, C-3’arom., C-5’arom.), 130.5 (1C, C-1arom.), 133.5 (1C, C-4’arom.), 133.6 (0.55C

R2C=CH), 134.8 (0.45C, R2C=CH) , 137.6 (1C, C-1’arom.), 148.0 (0.55C, C-4triazole), 148.1

(0.45C, C-4triazole).

Compound 109 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5.5:4.5 is observed.

FT-IR (neat): ṽ (cm-1) = 2924, 2856 (C-H, aliph.), 1647 (C=C), 1612 (C=N, triazole ring), 1350 and 1168 (S=O).

105

(E)- and (Z)-4-{2-[4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl]ethylidene}-1- (phenylsulfonyl)-1,2,3,4-tetrahydropyridine (110)

CuI (2 mg, 0.01 mmol, 0.02 eq.), N,N-diisopropylethylamine (3.6 µL, 0.02 mmol, 0.04 eq.), glacial acetic acid (1.2 µL, 0.02 mmol, 0.04 eq.) and (4-methoxyphenyl)acetylene (68 mg, 0.52 mmol, 1.0 eq.) were added in sequence to a solution of azide 74a (150 mg, 0.52 mmol,

1.0 eq.) in CH2Cl2 (1 mL) and the mixture was stirred at room temperature for 16 h..Afterwards, the mixture was concentrated in vacuo and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.43, RfB = 0.39 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 154 °C yield 140 mg (0.33 mmol, 63 %).

Purity by HPLC, method 1: tR = 21.4 min, purity 95.8%.

+ Exact mass (APCI): m/z = 423.1457 (calcd.423.1491 for C22H23N4O3S [M+H ]).

1 H NMR (600 MHz, CDCl3) δ (ppm) = 2.46 (t, J = 6.1 Hz, 1H, 5-CH2), 2.54 – 2.60 (m, 1H, 5-

CH2), 3.46 (t, J = 7.5Hz, 1H, 6-CH2), 3.49 (t, 6.0 Hz, 1H, 6-CH2), 3.84 (s, 1.5H, -OCH3), 3.85

(s, 1.5H, OCH3), 4.99 (d, J = 7.4 Hz, 1H, CH2Ntriazole), 5.04 (d, J = 7.3 Hz, 1H, CH2Ntriazole),

5.22 – 5.32 (m, 0.5H, R2C=CH), 5.47 (t, J = 7.3 Hz, 0.5H, R2C=CH), 5.50 (d, J = 8.3 Hz, 0.5H, 3-CH), 5.77 (d, J = 8.4 Hz, 0.5H, 3-CH), 6.79 (d, J = 8.2 Hz, 0.5H, 2-CH), 6.90 – 6.97

(m, 0.5H, 2-CH, 2H, 3’-Harom.’, 5’-Harom.), 7.55 (m, 2H, 2-Harom., 6-Harom.), 7.59-7.66 (m, 1H,

5-CHtriazole, 1H, 4-Harom.), 7.69 – 7.76 (m, 2H, 2’-Harom., 6’-Harom.), 7.77 – 7.84 (m, 2H, 3-Harom.,

5-Harom.).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 24.2 (0.5C, C-5), 30.1 (0.5C, C-5), 43.2 (0.5C, C-6),

43.8 (0.5C, C-6), 47.1 (0.5C, CH2Ntriazole), 47.3 (0.5C, CH2Ntriazole), 55.4 (0.5C, -OCH3), 55.5

(0.5C, OCH3), 103.8 (0.5C, C-3), 110.8 (0.5C, C-3), 114.4 (2C, C-2’arom., C-4’arom.), 115.6

106

(0.5C, R2C=CH), 116.4 (1C, R2C=CH), 118.4 (0.5C, C-5triazole), 118.5 (0.5C, C-5triazole), 123.0

(1C, C-1’arom.), 127.18 (2C, C-3arom., C-5arom.), 127.2 (2C, C-2’arom., C-6’arom.), 127.2 (1C, C-

2), 129.0 (1C, C-2), 129.4 (2C, C-2arom., C-6arom.), 133.4 (1C, C-4arom.), 133.5 (1C, C-1arom.),

137.6 (0.5C, R2C=CH), 137.7 (0.5C, R2C=CH), 147.8 (1C, C-4triazole), 147.8 (0.5C, C-4triazole),

159.8 (1C, C-4’arom.).

Compound 110 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5:5 is observed.

FT-IR (neat): ṽ (cm-1) = 3151 (C-H, alkene), 3078, 3005, 2962 (C-H, aliph.), 1647 (C=C), 1612 (C=N, triazole ring), 1558, 1492, 1446 (C=C, aromatic ring), 1350, 1195 (S=O), 1300, 999 (C-O).

(E)- and (Z)-4-{2-[4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl]ethylidene}-1- (phenylsulfonyl)-1,2,3,4-tetrahydropyridine (111)

Procedure 2

CuI (2 mg, 0.01 mmol, 0.02 eq.), N,N-diisopropylethylamine (3.6 µL, 0.02 mmol, 0.04 eq.), glacial acetic acid (1.2 µL, 0.02 mmol, 0.04 eq.) and (4-fluorophenyl)acetylene (59 µL, 0.52 mmol, 1.0 eq.) were added in sequence to a solution of azide 74a (150 mg, 0.52 mmol, 1.0 eq.) in CH2Cl2 (1 mL) and the mixture was stirred at ambient temperature for 16 h. Afterwards, the mixture was concentrated in vacuo and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.56, RfB = 0.50 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 133-135 °C, yield 160 mg (0.39 mmol, 75 %).

107

Purity by HPLC, method 1: 99.6 %, tR = 21.8 min.

+ Exact mass (APCI): m/z = 411.1327 (calcd.411.1291 for C21H20FN4O2S [M+H ]).

1 H NMR (600 MHz, CDCl3): δ (ppm) = 2.43 – 2.50 (m, 1.1H, 5-CH2), 2.52 – 2.62 (m, 0.9H,

5-CH2), 3.48 (m, 2H, 6-CH2), 5.00 (d, J = 7.5 Hz, 0.9H, CH2Ntriazole), 5.05 (d, J = 7.4 Hz,

1.1H, CH2Ntriazole), 5.25 (t, J = 7.3, Hz, 0.55H, R2C=CH), 5.44 – 5.49 (m, 0.45H, R2C=CH), 5.50 (d, J = 8.3 Hz, 0.45H, 3-CH), 5.77 (d, J = 8.4, Hz, 0.55H, 3-CH), 6.80 (d, J = 8.1 Hz,

1H, 2-CH), 6.93 (d, J = 8.4, Hz, 0.55H, 2-CH), 7.08 – 7.12 (m, 2H, 3’-Harom., 5’-Harom.), 7.50

– 7.59 (m, 2H, 3-Harom., 5-Harom.), 7.59 – 7.68 (m, 2H, 4-Harom., 5-CHtriazole), 7.70 – 7.87 (m,

4H, 2-Harom., 6-Harom., 2’-Harom.’, 6’-Harom.).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 24.3 (0.45C, C-5), 30.1 (0.55C, C-5), 43.1 (0.45C, C-

6), 43.8 (0.55C, C-6), 47.1 (0.55C, CH2Ntriazole), 47.3 (0.45C, CH2Ntriazole), 103.7 (0.55C, C-

3), 110.7 (0.45C, C-3), 115.5 (0.55C, R2C=CH2), 115.99 (d, J = 21.8 Hz, 2C, C-3’arom., C-

5’arom.), 116.2 (0.45C, R2C=CH), 118.9 (0.45C, C-5triazole), 119.0 (0.55C, C-5triazole), 126.7 (t,

J = 3.3 Hz, 1C, C-1’arom.), 127.1 (0.45, C-2), 127.5 (2C, C-2arom., C-6arom.), 127.6 (dd, J = 8.1,

2.8 Hz, 2C, C-2’arom., C-6’arom.), 129.1 (0.55C, C-2), 129.5 (d, J = 4.6 Hz, 2C, C-3arom., C-

5arom.), 133.5 (d, J = 7.8 Hz, 1C, C-1arom.), 133.7 (0.45C, R2C=CH), 134.9 (0.55C, R2C=CH),

137.6 (d, J = 4.8 Hz, 1C, C-4arom.), 147.2 (0.55C, C-4triazole), 147.2 (1C, C-4triazole) 162.8 (d, J

= 247.5 Hz, 1C, C-4’arom.).

Compound 111 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5.5:4.5 is observed.

FT-IR (neat): ṽ (cm-1) = 3136 (C-H, alkene), 3062, 2870 (C-H, aliph.), 1643 (C=C, alkene), 1600 (C=N, triazole ring), 1554, 1492, 1462, 1450 (C=C, arom.), 1377, 1195 (S=O), 1346 (C-F).

108

(E)- and (Z)-4-{2-[4-(4-bromophenyl)-1H-1,2,3-triazol-1-yl]ethylidene}-1- (phenylsulfonyl)-1,2,3,4-tetrahydropyridine (112)

Procedure 2

CuI (2 mg, 0.01 mmol, 0.02 eq.), N,N-diisopropylethylamine (3.6 µL, 0.02 mmol, 0.04 eq.), glacial acetic acid (1.2 µL, 0.02 mmol, 0.04 eq.) and (4-bromophenyl)acetylene (94 mg, 0.52 mmol, 1.0 eq.) were added in sequence to a solution of azide 74a (150 mg, 0.52 mmol, 1.0 eq.in CH2Cl2(1 mL) and the mixture was stirred at ambient temperature for 16 h. Afterwards, the mixture was concentrated in vacuo and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.39, RfB = 0.33 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 168-169 °C, yield 210 mg (0.44 mmol, 85 %).

Purity by HPLC, method 1: 99.0 %, tR = 22.8 min.

+ Exact mass (APCI): m/z = 471.0488 (calcd.471.0490 for C21H20BrN4O2S [M+H ]).

1 H NMR (600 MHz, CDCl3) δ (ppm) = 2.43 – 2.50 (m, 1.2H, 5-CH2), 2.53 – 2.60 (m, 0.8H,

5-CH2), 3.43 – 3.51 (m, 2H, 6-CH2), 5.00 (d, J = 7.5 Hz, 0.8H, CH2-Ntriazole), 5.05 (d, J = 7.4

Hz, 1.2H, CH2-Ntriazole), 5.25 (t, J = 7.4, , 0.6H, R2C=CH), 5.47 (d, J = 8.5, Hz, 0.4H,

R2C=CH), 5.50 (d, J = 8.2 Hz, 0.4H, 3-CH), 5.77 (d, J = 8.4, Hz, 0.6H, 3-CH), 6.80 (d, J = 8.1 Hz, 0.4H, 2-CH), 6.93 (d, J = 8.4 Hz, 0.6H, 2-CH), 7.52 – 7.57 (m, 4H, 3’-H, 5’-H, 3-H,

5-H), 7.59 – 7.65 (m, 1H, 4-H), 7.65 – 7.69 (m, 3H, 5-CHtriazole, 2’-H, 6’-H), 7.77 – 7.83 (m, 2H, 2-H, 6-H).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 24.3 (0.4C, C-5), 30.1 (0.6C, C-5), 43.1 (0.4C, C-6),

43.8 (0.6C, C-6), 47.0 (0.4C, CH2Ntriazole), 47.3 (0.6C, CH2Ntriazole), 103.7 (0.6C, C-3), 110.7

(0.4C, C-3), 115.5 (0.6C, R2C=CH), 116.2 (0.4C, R2C=CH), 119.2 (0.4C, C-5triazole), 119.3

(0.6C, C-5triazole), 122.2 (1C, C-4’arom.), 127.1 (2C, C-2’arom., C-6’arom.), 127.3 (0.4C, C-2),

109

127.5 (2C, C-2arom., C-6arom.), 129.1 (0.6C, C-2), 129.5 (2C, C-3arom., C-5arom.), 132.1 (2C, C-

3’arom., C-5’arom.), , 133.4 (1C, C-4arom.), 133.5 (1C, C-1arom.), 133.7 (0.6C, R2C=CH), 134.9

(0.4C, R2C=CH), 137.6 (1C, C-1’arom.), 147.1 (1C, C-4triazole).

Compound 112 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 6:4 is observed.

FT-IR (neat): ṽ (cm-1) = 3097 (C-H alkene), 2947, 2916 (C-H, aliph.), 1643 (C=C), 1612 (C=N, triazole ring), 1589, 1543, 1477 (C=C, arom.), 1354 (S=O), 655 (C-Br).

(E)- and (Z)-1-(Phenylsulfonyl)-4-{2-[4-(p-tolyl)-1H-1,2,3-triazol-1-yl]ethylidene}- 1,2,3,4-tetrahydropyridine (113)

Procedure 2

CuI (2 mg, 0.01 mmol, 0.02 eq.), N,N-diisopropylethylamine (3.6 µL, 0.02 mmol, 0.04 eq.), glacial acetic acid (1.2 µL, 0.02 mmol, 0.04 eq.) and (4-methylphenyl)acetylene (66 µL, 0.52 mmol, 1.0 eq.) were added in sequence to a solution of azide 74a (150 mg, 0.52 mmol, 1.0 eq.) in CH2Cl2 (1 mL) and and the mixture was stirred at room temperature for 16 h. Afterwards, the mixture was concentrated in vacuo and the residue was purified by flash column chromatography (petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.36, RfB = 0.30 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 145-146 °C, yield 150 mg (0.37 mmol, 71 %).

Purity by HPLC, method 1: 99.9 %, tR = 22.3 min.

+ Exact mass (APCI): m/z = 407.1579 (calcd.407.1542 for C22H23N4O2S [M+H ]).

1 H NMR (600 MHz, CDCl3): δ (ppm) = 2.37 (s, 3H, aryl-CH3), 2.41 – 2.54 (m, 1.1H, 5-CH2),

2.55 - 2.62 (m, 0.9H, 5-CH2), 3.44-3.52 (m, 2H, 6-CH2), 4.89 - 5.09 (m, 2H, CH2Ntriazole), 5.19 110

- 5.34 (m, 0.55H, R2C=CH), 5.46 – 5.53 (m, 0.45H, 3-CH, 0.45H, R2C=CH), 5.78 (d, J = 7.4 Hz, 0.55H, 3-CH), 6.79 (d, J = 7.8 Hz, 0.45H, 6-CH), 6.89 – 6.97 (m, 0.55H, 6-CH), 7.22 (m,

2H, 3’-Harom., 5’-Harom.), 7.49 – 7.58 (m, 2H, 3-H, 5-H), 7.58 – 7.65 (m, 1H, 4-Harom.), 7.66 –

7.73 (m, 2H, 2’-Harom., 6’-Harom.), 7.74 – 7.85 (m, 2H, 2-Harom., 6-Harom.).

A singlet for the CH group of the triazole ring for both (E)- and (Z)-isomers is not visible in the 1H NMR spectrum even though signal in the 13C NMR spectrum is present for the corresponding C-atom.

13 C NMR (151 MHz, CDCl3): δ (ppm) = 21.4 (1C, aryl-CH3), 24.4 (0.45C, C-5), 30.1 (0.55C,

C-5), 43.3 (0.45C, C-6), 43.9 (0.55C, C-6), 47.2 (0.55C, CH2Ntriazole), 47.6 (0.45C,

CH2Ntriazole), 103.9 (0.55C, C-3), 110.9 (0.45C, C-3), 115.9 (0.55C, R2C=CH), 116.5 (0.45C,

R2C=CH), 119.2 (1C, C-5triazole), 125.1 (2C, C-2’arom., C-6’arom.), 127.1 (2C, C-2arom., C-6arom.),

127.3 (0.45C, C-2), 127.7 (1C, C-1’arom), 129.0 (0.55C, C-2), 129.4 (2C, C-3arom., C-5arom.),

129.6 (2C, C-3’arom., C-5’arom.), 133.4 (1C, C-4arom.), 134.7 ((1C, R2C=CH), 137.6 (1C, C-

1arom.), 138.2 (1C, C-4’arom.), 148.2 (1C, C-4triazole).

Compound 113 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 5.5:4.5 is observed.

FT-IR (neat): ṽ (cm-1) = 3136 (C-H, alkene), 2927 (C-H, aliph.), 1651 (C=C), 1612 (C=N), 1597, 1492, 1446 (C=C, arom.), 1357 (S=O).

111 tert-Butyl (E)- and (Z)-4-[2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethylidene]-3,4- dihydropyridine-1(2H)-carboxylate (114)

CuI (3 mg, 0.01 mmol, 0.02 eq.), N,N-diisopropylethylamine (5.3 µL, 0.03 mmol, 0.04 eq.), glacial acetic acid (1.7 µL, 0.03 mmol, 0.04 eq.) and phenylacetylene (83 µL, 0.76 mmol, 1.0 eq.) were added in sequence to a solution of azide 74c (190 mg, 0.76 mmol, 1.0 eq.) in

CH2Cl2 (1 mL) and the mixture was stirred at ambient temperature for 16 h. The mixture was concentrated in vacuo and the residue was purified by flash column chromatography

(petroleum ether/ethyl acetate 9/1  7/3 RfA = 0.46, RfB = 0.42 (cyclohexane/ethyl acetate 5:5)). Colourless solid, mp 123 °C, yield 250 mg (0.70 mmol, 93 %).

Purity by HPLC, method 1: 98.9 %, tR = 22.4 min.

t Exact mass (APCI): m/z = 252.1375 (calcd.252.174 for C15H17N4 [M – CO2 Bu]).

1 H NMR (600 MHz, CDCl3) δ (ppm) = 1.51 (s, 3.6H, -C(CH3)3), 1.51 (s, 5.4H, -C(CH3)3), 2.53

(t, J = 6.4 Hz, 1.2H, 5-CH2), 2.63 (t, J = 7.0 Hz, 0.8H, 5-CH2), 3.63 - 3.74 (m, 2H, 6-CH2),

5.07 (d, J = 7.5 Hz, 0.8H, CH2Ntriazole), 5.11 (d, J = 7.5 Hz, 1.2H, CH2Ntriazole), 5.28 (t, J = 7.7

Hz, 0.6H, R2C=CH), 5.34- 5.48 (m, 0.4H, 3-CH), 5.50 (t, J = 7.6 Hz, 0.4H, R2C=CH), 5.56 – 5.77 (m, 0.6H, 3-CH), 6.80 - 7.02 (m, 0.4H, 2-CH), 7.03 – 7.20 (m, 0.6H,2-CH), 7.28 – 7.37

(m, 1H, 4-Harom.), 7.41 (m, 2H, 3-Harom., 5-Harom.), 7.71 (s, 0.4H, 5-CHtriazole), 7.72 (s, 0.6H,

5-CHtriazole), 7.81 (m, 2H, 2-Harom., 6-Harom.).

13 C NMR (151 MHz, CDCl3) δ (ppm) = 24.6 (0.4C, C-5), 28.3 (1.8C, -C(CH3)3), 28.4 (1.2C,

-C(CH3)3), 30.5 (0.6C, C-5), 40.6 (0.6C, C-6), 41.6 (0.4C, C-6), 47.1 (0.6C, CH2Ntriazole), 47.5

(0.4C, CH2Ntriazole), 81.6 (0.6C, -C(CH3)3), 81.7 (0.4C, -C(CH3)3), 101.2 (0.6C, C-3), 108.3

(0.4C, C-3), 114.1 (0.6C, R2C=CH), 115.1 (0.4C, R2C=CH), 119.1(0.4C, C-5triazole), 119.1

(06C, C-5triazole), 125.8 (2C, C-2arom., C-6arom.), 128.2 (0.6C, C-2), 128.2 (1C, C-4arom.), 128.9

(2C, C-3arom., C-5arom.), 129.8 (1C, C-2), 130.8 (1C, C-1arom.), 133.9 (1C, R2C=CH), 148.1

(1C, C-4triazole), 165.4 (1C, N-C(=O)OC(CH3)3). 112

Compound 114 was isolated as a mixture of ((E):(Z)) isomers. In the NMR spectra a ratio of 6:4 is observed.

FT-IR (neat): ṽ (cm-1) = 3128 (C-H, alkene), 2981, 2924 (C-, aliph.), 1693 (C=O), 1651 (C=C), 1612 (C=N triazole ring), 1477, 1462, 1415 (C=C, arom.), 1165 (C-O).

113

9.3. Receptor binding studies 9.3.1. Materials

Guinea pig brains and rat livers were commercially available (Harlan-Winkelmann, Borchen, Germany). Homogenizers: Elvehjem Potter (B. Braun Biotech International, Melsungen, Germany) and Soniprep® 150, MSE, London, UK). Centrifuges: Cooling centrifuge Eppendorf 5427R (Eppendorf, Hamburg, Germany) and High-speed cooling centrifuge model Sorvall® RC-5C plus (Thermo Fisher Scientific, Langenselbold, Germany). Multiplates: standard 96 well multiplates (Diagonal, Muenster, Germany). Shaker: self-made device with adjustable temperature and tumbling speed (scientific workshop of the institute). Harvester: MicroBeta® FilterMate 96 Harvester. Filter: Printed Filtermat Typ A and B. Scintillator: Meltilex® (Typ A or B) solid state scintillator. Scintillation analyzer: MicroBeta® Trilux (all Perkin Elmer LAS, Rodgau-Jügesheim, Germany).

9.3.2. Preparation of membrane homogenates from guinea pig brain

5 guinea pig brains were homogenized with the potter (500-800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 23,500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23,500 x g (20 min, 4 °C). This procedure was repeated twice. The final pellet was resuspended in 5-6 volumes of buffer and frozen (-80 °C) in 1.5 mL portions containing about 1.5 mg protein/mL.

9.3.3. Preparation of membrane homogenates from rat liver

Two rat livers were cut into small pieces and homogenized with the potter (500-800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 31,000 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 8.0) and incubated at RT for 30 min. After the incubation, the suspension was centrifuged again at 31,000 x g for 20 min at 4 °C. The final pellet was resuspended in 5-6 volumes of buffer and stored at -80 °C in 1.5 mL portions containing about 2 mg protein/mL.

114

9.3.4. Protein determination

The protein concentration was determined by the method of Bradford,262 modified by Stoscheck.263 The Bradford solution was prepared by dissolving 5 mg of Coomassie Brilliant

Blue G 250 in 2.5 mL of EtOH (95 %, v/v). 10 mL deionized H2O and 5 mL phosphoric acid (85 %, m/v) were added to this solution, the mixture was stirred and filled to a total volume of 50 mL with deionized water. The calibration was carried out using bovine serum albumin as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 mg /mL). In a 96 well standard multiplate, 10 µL of the calibration solution or 10 µL of the membrane receptor preparation were mixed with 190 µL of the Bradford solution, respectively. After 5 min, the UV absorption of the protein-dye complex at  = 595 nm was measured with a plate reader (Tecan Genios®, Tecan, Crailsheim, Germany).

9.3.5. General procedures for the binding assays

The test compound solutions were prepared by dissolving approximately 10 µmol (usually 2-4 mg) of test compound in DMSO so that a 10 mM stock solution was obtained. To obtain the required test solutions for the assay, the DMSO stock solution was diluted with the respective assay buffer. The filtermats were presoaked in 0.5 % aqueous polyethylenimine solution for 2 h at rt before use. All binding experiments were carried out in duplicates in the 96 well multiplates. The concentrations given are the final concentration in the assay. Generally, the assays were performed by addition of 50 µL of the respective assay buffer, 50 µL of test compound solution in various concentrations (10-5, 10-6, 10-7, 10-8, 10-9 and 10- 10 mol/L), 50 µL of the corresponding radioligand solution and 50 µL of the respective receptor preparation into each well of the multiplate (total volume 200 µL). The receptor preparation was always added last. During the incubation, the multiplates were shaken at a speed of 500-600 rpm at the specified temperature. Unless otherwise noted, the assays were terminated after 120 min by rapid filtration using the harvester. During the filtration, each well was washed five times with 300 µL of water. Subsequently, the filtermats were dried at 95 °C. The solid scintillator was melted on the dried filtermats at a temperature of 95 °C for 5 min. After solidifying of the scintillator at rt, the trapped radioactivity in the filtermats was measured with the scintillation analyzer. Each position on the filtermat corresponding to one well of the multiplate was measured for 5 min with the [3H]-counting protocol. The overall counting efficiency was 20 %. The IC50 values were calculated with the

115 program GraphPad Prism® 3.0 (GraphPad Software, San Diego, CA, USA) by non-linear regression analysis. Subsequently, the IC50 values were transformed into Ki values using 259 the equation of Cheng and Prusoff. The Ki values are given as mean value ± SEM from three independent experiments.

9.3.6. Performance of the binding assays

σ1 receptor

The assay was performed with the radioligand [3H]-(+)-pentazocine (22.0 Ci/mmol; Perkin Elmer). The thawed membrane preparation of guinea pig brain cortex (about 100 μg of the protein) was incubated with various concentrations of test compounds, 2 nM [3H]-(+)-pentazocine, and TRIS buffer (50 mM, pH 7.4) at 37 °C. The non-specific binding was determined with 10 μM unlabeled (+)-pentazocine. The Kd value of (+)-pentazocine is 2.9 nM.260

σ2 receptor

The assays were performed with the radioligand [3H]di-o-tolylguanidine (specific activity 50 Ci/mmol; ARC, St. Louis, MO, USA). The thawed rat liver membrane preparation (about 100 µg protein) was incubated with various concentrations of the test compound, 3 nM [3H]di-o-tolylguanidine and buffer containing (+)-pentazocine (500 nM (+)-pentazocine in TRIS buffer (50 mM TRIS, pH 8.0)) at rt. The non-specific binding was determined with 261 10 μM non-labeled di-o-tolylguanidine. The Kd value of di-o-tolylguanidine is 17.9 nM.

116

9.4. Folin-Ciocalteu assay 9.4.1. Preliminary procedures

 Prepare a solution of Na2CO3 2 % (w/v) by weighting 2 g of Na2CO3 and dissolve in 100 mL of distilled water.  Dilute FCR with distilled water to obtain a solution 50 % (v/v).  Prepare a solution of Gallic acid (standard) 6 mM by weighting 1 mg of gallic acid (PM = 170,12) and solubilize in 1 mL of distilled water.

The quantities of solution of gallic acid 6 nM used are:

- 10µL (10µg) + 90 µL di H2O

- 30µL (30µg) + 70 µL di H2O

- 50µL (50µg) + 50 µL di H2O

- 70µL (70µg) + 30 µL di H2O

The final volumes of the sample and standard must be equal to 100 µL.

9.4.2. General procedure  In each tube add the exact volume of distilled water to reach 100 µL then add sample or standard.  Prepare the standard with 100 µL.

 Add 3 mL of 2% Na2CO3 (w/v) solution.  Add 100 µLof FCR solution 50 % (v/v).  Stirring and incubation at RT in dark place for 60 min  Measure the optical density with an UV-spectrophotometer at 765 nm.  Create the curve for data analysis  Express the results in mg GAE/g sample.

9.4.3. Determination of the TPC of compounds 88a, 88c, 109, 110, 114 The samples were solubilized in DMSO to obtain a 100 mM solution. For each sample two aliquots of 1 µL and 100 µL were used and the volumes were adjust to 100 µL

with distilled water. To the samples 3 mL of 2% Na2CO3 (w/v) solution were added followed by addition of 100 µL of FCR. The samples were incubated for 60 min in a dark place at RT. After 60 min no color change was observed indicanting the absence of antioxidant activity.

117

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[8] Sankar, C.; Pandiarajan, K. Synthesis and anti-tubercular and antimicrobial activities of some 2r, 4c diaryl-3-azabicyclo[3.3.1]nonan-9-one N-isonicotinoylhydrazone derivatives. Eur. J. Med. Chem. 2010, 45 (11), 5480-5485.

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11. Appendices 11.1. List of acronyms ∅ Diameter δ Chemical shift υ Frequency abs. Absolute APCI Atmospheric pressure chemical ionization arom Aromatic

Asp Aspartate BBB Blood-brain-barrier ca. Circa calcd. Calculated cAMP Cyclic adenosine monophosphate

CDCl3 Deutereted chloroform CNS Central nervous sistem

cpm Counts per minute CuAAC Copper(I)-catalyzed azide-alkyne cycloaddtion DABCO 1,4-Diazabicyclo[2.2.2]octane DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone DIAD Diisopropyl azodicarboxylate DIBAL-H Diisobutylaluminium hydride DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide

DMP Dess-Martin periodinane

EC50 Half maximal effective concentration EDTA Ethylenediaminetetraacetic acid eq. Equivalent

et al. And others fc Flash chromatography

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FCR Folin-Ciocalteu reagent

FT-IR Fourier transform infrared (-Spectroscopy) gHMBC Gradient heteronuclear multiple bond correlation gHSQC Gradient heteronuclear single quantum coherence GPCR G-Protein-coupled receptor HPLC High performance liquid chromatography

HRMS High resolution mass spectrometry

IC50 Concentration oft he test substance needed to inhibit 50 % oft he biological target IL Interleukin J Coupling constant

Kd Dissociation constant

Ki Inhibition constant l Lenght of the stationary phase L Ligand

L* Radioligand M Molarity m-CPBA m-Chloroperoxybenzoic acid mp Melting point MS Multiple sclerosis m/z Masse to charge ratio n Number of experiments NaHMDS Sodium bis(trimethylsilyl)amide

NBS N-Bromosuccinimide NMR Nuclear magnetic resonance NOESY Nuclear Overhauser effect spectroscopy ppm Parts per million PET Positron-Emission-Tomography R Receptor

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RL Ligand-Receptor-Complex RL* Radioligand-Receptor-Complex

Rf Retention factor RT Room temperature SEM Standard error of the mean

SN2 Bimolecular nucleophilic substitution Tab. Table TBAF Tetrabutylammoniumfluorid

TEMPO 2,2,6,6-Tetramethylpiperidinnyloxyl THF Tetrahydrofuran TLC Thin layer chromatography TMS Tetramethylsilane TOF Time-of-flight Tos Tosyl TPC Toal polyphenols content Tyr Tyrosin tR Retention time UV Ultraviolet

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11.2. Compound list The following is the list of the compounds discussed in this work. The compounds are classify under:

Number: compound number

Page number (general section and experimental section)

If possible, test compounds are denoted as WMS79-XX

75a: 32, 82 77a:32, 83

89a: 41, 84 63a: 32, 85

74a: 44, 86 88a: 46, 87

75c: 35, 89 77c: 50, 90

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89c: 51, 91 63c: 51, 92

74c: 51, 93 88c: 51, 94

52: 27, 95 97: 55, 96

104: 57, 96 105: 58, 97

57a: 60, 99 70a: 60, 100

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56a: 61, 101 73a: 63, 102

73c: 63, 103 109: 66, 104 (WMS79-01)

110: 66, 106 (WMS79-03) 111: 66, 107 (WMS79-05)

112: 66, 109 (WMS79-06) 113: 66, 110 (WMS79-07)

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114: 66, 112 (WMS79-10)

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12. Curriculum vitae

Personal Data Name Daniele Aiello Date of birth 18.10.1987 Place of birth Palermo, Italy Nationality Italian

Education

School From 9/2001 to 7/2006 Classical High School Diploma Liceo classico “F. Scaduto”, Bagheria, Italy

University From 9/2006 to 10/2013 Master Degree in Pharmaceutical chemistry and technology University of Palermo, School of Pharmacy, Italy.

From 5/2012 to 1/2013 Internship in pharmacy

12/2013 State exam in pharmacy University of Palermo, Italy

From 03/2014 to 09/2014 Internship Laboratorio di analisi delle acque e degli alimenti, Dott. Rosalia Balistreri Bagheria, Italy

From 01/2014 to 07/2014 Internship With Dott. Vincenzo Nicolì as chemist Bagheria, Italy

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From 01/2015 to 02/2019 Joint PhD Student in Medicinal Chemistry Dipt. Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Università degli Studi di Palermo Palermo, Italy Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Germany. Supervisors: Prof. Patrizia Diana Prof. Dr. Bernhard Wünsch

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