Synthesis of new nampt inhibitors as potential anti-cancer drugs. An approach to antibody-drug conjugates

DEPARTAMENTO DE QUÍMICA ORGÁNICA FACULTAD DE QUÍMICA UNIVERSIDAD DE SEVILLA SYNTHESIS OF NEW NAMPT INHIBITORS AS POTENTIAL ANTI-CANCER DRUGS. AN APPROACH TO ANTIBODY-DRUG CONJUGATES SIMONE FRATTA SEVILLA 2022 2 3 DEPARTAMENTO DE QUÍMICA ORGÁNICA FACULTAD DE QUÍMICA UNIVERSIDAD DE SEVILLA SYNTHESIS OF NEW NAMPT INHIBITORS AS POTENTIAL ANTI-CANCER DRUGS. AN APPROACH TO ANTIBODY-DRUG CONJUGATES Memoria presentada por el Licenciado Simone Fratta para optar al grado de Doctor en Química Sevilla 2022 4 5 DEPARTAMENTO DE QUÍMICA ORGÁNICA FACULTAD DE QUÍMICA UNIVERSIDAD DE SEVILLA SYNTHESIS OF NEW NAMPT INHIBITORS AS POTENTIAL ANTI-CANCER DRUGS. AN APPROACH TO ANTIBODY-DRUG CONJUGATES Vº Bº de los Directores de la Tesis Fdo. Dra. Inmaculada Robina Ramírez Universidad de Sevilla Fdo. Dra. Ana Teresa Carmona Asenjo Universidad de Sevilla Fdo. Dr. Antonio J. Moreno Vargas Universidad de Sevilla 6 7 AKNOWLEDGMENTS I would like to thank Prof. Inmaculada Robina, Prof. Ana T. Carmona, and Prof. Antonio J. Moreno Vargas for supporting and supervising me during my PhD. I would like to extend my gratitude to the European Union, specifically to European Community's H2020-MSCA-ITN-2018 call. Action, MSCA-ITN-ETN. (European Training Networks) for funding the project INTEGRATA (Project: H2020-813284). The INTEGRATA project (“Integrating Chemical and Biological approaches to target NAD production and signaling in cancer integrating chemical and biological approaches to target NAD production and signaling in cancer”) is a training network that has gathered an excellent multidisciplinary team which has been very helpful in my formation. I thank, Dr. Caffa, Dr. Piacente, Dr. Benzi, Dr. Del Rio, Dr. Hechler, Prof. Aimable, Prof. Duchosal, Prof. Nencioni and Prof. Bruzzone for their mentorship during my secondments. I also thank the other ITN fellows, Jorge, Paulina, and Pablo. I would like to thank my lab mates, Enrique, Macarena, Carlos, and Alex at the Department of Organic Chemistry for a cherished time spent together. My appreciation also goes out to my mother, my brother, my family, and my friends Matteo, Amedeo, Carlotta, Alice and Federico for their encouragement and support throughout my studies. A special thanks to Irene, for being my home and my inspiring muse. Finally, I would like to dedicate this PhD thesis and the rest of my career to my lovely dad. 8 9 TABLE OF CONTENTS ABBREVIATIONS ................................................................................................ 15 SUMMARY ......................................................................................................... 20 1. SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIP OF NEW NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT) INHIBITORS WITH ANTITUMOR ACTIVITY ON SOLID AND HAEMATOLOGICAL CANCER. ................... 26 1.1. INTRODUCTION .................................................................................... 28 1.1.1. The Role of Nicotinamide Phosphoribosyl Transferase in cancer cell growth and proliferation .................................................................................... 28 1.1.2. NAMPT: Enzymatic reaction and crystal structure .............................. 30 1.1.3. NAMPT inhibitors in clinical trials ........................................................ 33 1.1.4. Medicinal chemistry of NAMPT inhibitors ........................................... 35 1.1.4.1. Previous results of our research group………………………………………….36 1.1.4.2. Chemical modification of the Cap group .......................................... 37 1.1.4.3. Chemical modification of the connecting unit ................................. 38 1.1.4.4. Miscellaneous modifications of the spacer and the tail group ........ 39 1.1.4.5. Nonsubstrate NAMPT inhibitors ...................................................... 40 1.2. OBJECTIVES .......................................................................................... 42 1.3. RESULTS AND DISCUSSION ................................................................... 43 1.3.1. CHEMISTRY ........................................................................................... 43 16 DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EC50 Half maximal effective concentration EtOAc Ethyl acetate ESI-LRMS ElectroSpray Ionization Low Resolution Mass Spectrometry ESI-HRMS ElectroSpray Ionization High Resolution Mass Spectrometry Et2O Diethyl ether EtOH Ethanol Fab Antigen binding fragment FDA Food and Drug Administration Fmoc Fluorenylmethyloxycarbonyl GSH Glutathione h Hour His Histidine HOBt N-Hydroxybenzotriazole HOMO Highest energy Occupied Molecular Orbital HPLC High Performance Liquid Chromatography HSQC Heteronuclear Single Quantum Correlation iEDDA inverse Electron Demand Diels-Alder i.p. Intraperitoneal IUPAC International Union of Pure and Applied Chemistry i.v. Intravenous J Coupling constant 17 kd Dissociation constant LC-MS Liquid Chromatography-Mass Spectrometry LUMO Lowest energy Unoccupied Molecular Orbital Lys Lysine m Multiplet mAb Monoclonal antibody Me Methyl MeOH Methanol min Minute MMAE Monomethyl Auristatin E NBS N-Bromosuccinimide NCS N- Chlorosuccinimide NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser Effect OND Oxanorbornadiene PABA p-Aminobenzyl alcohol PBS Phosphate-buffered solution PDB Pyrrolobenzodiazepine Ph Phenyl PK Pharmacokinetics ppm parts-per-million PTSA p-toluensulfonic acid q quartet 18 quant quantitative rDA retro Diels-Alder RP-HPLC Reversed Phase-High Perfomance Liquid Chromatography r.t. Room temperature s singlet SAR Structure-Activity Relationship SDS-PAGE Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis SEC-HPLC Size Exclusion-High Performance Liquid Chromatography SNVσ Nucleophilic vinylic substitution t triplet t1/2 half-life TBAF Tetrabutylammonium fluoride TBDMSCl tert-Butyldimethylsilyl chloride tBu tert-Butyl TCEP Tris(2-carboxyethyl)phoshine TFA Trifluoroacetic acid THF Tetrahydrofuran TME tumor microenvironment TMRM Tetramethylrhodamine methyl ester perchlorate TMS Trimethylsilyl Ts Tosyl Tz Tetrazine _____________________________________________________________Summary 20 SUMMARY Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the biosynthesis of Nicotinamide Adenine Dinucleotide (NAD+), a cofactor essential for several cellular processes. NAD+ is involved in important biological processes such as energy metabolism, sirtuin function, DNA repair machinery and intracellular oxidative stress. Cancer cells are highly dependent on NAMPT activity for proliferation due to an increased metabolism and a high activity of NAD+-consuming enzymes. NAMPT represents an interesting target for the development of anticancer drugs given that this enzyme is overexpressed in different types of tumours and that NAMPT expression appears to be related with tumour progression. The reference compound in the field of NAMPT inhibition is FK866. Despite its potent antiproliferative activity both in vitro and in vivo, it failed in human cancer trials (phase II clinical trial) because of the lack of antitumor benefit and the presence of side effects. In the last two decades several analogues have been described and, recently, some of them have also reached clinical phase trials. In the first chapter of this thesis, we present the synthesis and biological evaluation of a library of new NAMPT inhibitors (Figure 1). A structure-guided design was used for the preparation of the new inhibitors. For this goal, we made use of significant data from reported structure activity relationships of known NAMPT inhibitors and of crystallographic data of known complexes enzyme-inhibitors. Chemical structural variations were made at the cap group, connecting unit, linker, and tail group of FK866 (Figure 1). A furan moiety that could mimic the benzamide of FK866 while increase the polarity of the molecule and thus the water solubility, was incorporated in most of the new compounds. Additionally, furan could act as a handle for bioconjugation strategies that may be used for the generation of antibody-drug- conjugates (ADCs, Chapter 2). The new inhibitors prepared in this chapter showed excellent in vitro anticancer activity in MIA PaCa-2 pancreatic cancer cells and in several haematological cancer cell lines. Some candidates exhibited a much higher _____________________________________________________________Summary 21 cytotoxicity than that showed by reference compound FK866. However, when the lead compounds were assayed in in vivo xenograft mouse models, unfortunately, there was not a good correlation between the in vitro results and the in vivo activity. Figure 1. Structural motifs for the new inhibitors prepared in this Thesis. The second chapter of the Thesis deals with the preparation of antibody-drug- conjugates (ADCs). An ADC consists of a potent cytotoxic compound (payload) that is bioconjugated to a tumour-targeted antibody by a chemical linker. This type of conjugates can deliver the cytotoxic payload directly to tumour cells while sparing healthy cells. Several drugs of this type have been developed for the treatment of several cancers that do not respond to other therapies. Due to the lack of activity of the most cytotoxic compounds prepared in chapter 1 in the in vivo biological evaluation, we accomplished structural modifications of the most promising candidates to provide these molecules with a chemical function that could act as a handle for bioconjugation purposes. With these molecules in hand, we focused on the preparation of NAMPT inhibitor-based ADCs which have been scarcely explored in the field of selective drug delivery up to now. _____________________________________________________________Summary 22 Two different approaches have been followed: (1) preparation of ADCs using classical peptide linkers; (2) development of a new strategy of drug-release based on the chemistry of oxanorbornadienes (ONDs) that could be used for the preparation of novel ADCs. The first approach allowed the preparation of six new ADCs, by combination of the new modified NAMPT inhibitors with several classical enzymatically cleavable linkers (peptide linkers). The bioconjugation of the resulting inhibitor-linker ensembles with the monoclonal antibody CD138 afforded the final ADCs (Figure 2). Biological evaluation of the new ADCs is underway and is not included in this Thesis. Figure 2. New NAMPT inhibitor-based ADCs. _____________________________________________________________Summary 23 The second strategy is based on the reactivity of heteronorbornadiene systems. The high reactivity of these compounds makes them useful as linkers for bioconjugation reactions, where fast reactions with nucleophilic amino acids of the proteins are needed. As most of the inhibitors prepared in chapter 1 contains a furan tail, the Diels Alder (DA) reaction between the furan moiety of the inhibitor and an activated alkyne would afford bioconjugable oxanorbornadienic NAMPT inhibitors. Our hypothesis is based on the ability of these OND-based bioconjugable inhibitors to be attached to an antibody by cysteine-selective bioconjugation (Scheme 1), through a process that was previously studied in our group with azanorbornadiene analogues. A cleavable mechanism should liberate the drug from the antibody inside the cells following a strategy of glutathione (GSH)-triggered release. Scheme 1. ONDs as linkers in drug delivery systems. In order to validate the strategy of glutathione-triggered release of cytotoxic drugs in our OND systems, we first explored the reactivity of simple models of oxanorbornadienic thiovinyl sulfones in the presence of an excess of a thiol derivative (Scheme 2). We have demonstrated that these OND systems can suffer a fragmentation reaction via a two-reaction sequence: a conjugate addition followed by a spontaneous retro-Diels-Alder (rDA) reaction. Therefore, these ONDs could be used as thiol-sensitive linkers for drug delivery strategies. _____________________________________________________________Summary 24 Scheme 2. Fragmentation of ONDs in the presence of thiols. We then assayed the DA reaction of some of the furan-tailed NAMPT inhibitors with the adequate activated alkyne for the preparation of bioconjugable oxanorbornadienic inhibitors. The first reaction attempts revealed that the nucleophilic pyridine moiety of the inhibitor gave side reactions with the alkyne/oxanorbornadiene system, hence not allowing the formation of the desired OND system. To overcome this problem, we accomplished the preparation and biological evaluation of a small library of 2-fluoropyridine analogues, where the nucleophilicity of the pyridinic N is clearly decreased. Although the biological evaluation of these new analogues showed lower cytotoxicity than the nonfluorinated ones, the level of cytotoxicity is still enough for ADC applications. Attempts to transform these new 2-fluoropyridine-based NAMPT inhibitors into oxanorbornadienic bioconjugable inhibitors worked as it was expected (Scheme 3). This strategy represents a preliminary approach for the use of oxanorbornadienes as innovative linkers in the field of selective drug delivery. Scheme 3. Incorporation of furan-containg NAMPT inhibitors into bioconjugable electrophilic oxanorbornadienes. Chapter 1__________________________________________________Introduction 32 Figure 3. Nicotinamide mononucleotide (NMN, in yellow) complexed in the active site of NAMPT.16 The crystal structure of FK866 (compound 1) in complex with NAMPT is shown in Figure 4. The pyridine moiety of FK866 is sandwiched between the side chain of Phe193 and Tyr18, similarly to nicotinamide (NAM) in complex with NAMPT, making a π-π stacking interaction. The oxygen atom of acrylamide makes a hydrogen bond with Ser275 while nitrogen uses a molecule of water to bridge and stabilize the interactions with Ser245, Asp219, and Val242. The alkyl chain does not add any affinity in the hydrophobic tunnel (Val242, Ile309, Ile351, and His191), but it is mandatory since it serves as a linker between the acrylamide and the benzoyl group. The solvent-exposed part of the molecule, the benzoyl-piperidine group, can be considered as a tail group that anchors the molecule and increases the affinity toward the enzyme. Chapter 1__________________________________________________Introduction 33 Figure 4. Binding pose of compound 1 complexed in the active site of NAMPT. Furthermore, it has been found that an autophosphorylation at His247 residue stabilizes the interactions of the two monomers increasing the affinity for its substrate PRPP by 10-fold.17 Even if it is transient and represents only an intermediate of the reaction, this post-translational modification on NAMPT enhances the affinity for NAM by 160,000-fold and increases its enzymatic activity by 1125-fold.18 1.1.3 NAMPT inhibitors in clinical trials NAMPT enzyme plays an important role in the proliferation and growth of cancer cells, therefore represents an important therapeutic target for the development of anti-cancer drugs. The disclosure of the crystal structure of 1 (FK866) in complex with the enzyme,15 encouraged and boosted the synthesis of new NAMPT inhibitors in the last two decades (Figure 5). Chapter 1__________________________________________________Introduction 34 Figure 5. Timeline of NAMPT inhibitors.3 Over the past years, several compounds have been identified as potent NAMPT inhibitors with strong anti-cancer activity. Compound FK86619 (compound 1, Figure 6) was the first identified selective NAMPT inhibitor, with cytotoxicity at nM concentrations in different types of cancers. Although this molecule entered clinical trials in patients with advanced solid tumors, the results were disappointing due to the lack of antitumor efficacy and secondary side effects (e.g., thrombocytopenia). Similar results were obtained in patients treated with Teglarinad, a prodrug of the known NAMPT inhibitor CHS82820 (compound 2, Figure 6), which was found to induce thrombocytopenia and gastrointestinal symptoms, without beneficial clinical effects. However, inhibition of NAMPT still remains a promising strategy for cancer therapy, as it has been demonstrated with the entry of new compounds in human clinical phase. For instance, KPT-927421 (compound 3, Figure 6) a dual inhibitor of NAMPT and of the serine/threonine protein kinase PAK4, was successfully transited in phase I from solid tumors to acute myeloid leukemia. Compound OT-8222 (compound 4, Figure 6) was also recently identified as a new NAMPT inhibitor with marked efficacy against hematopoietic malignancies such as leukemia, lymphoma, and myeloma and is also being currently evaluated in clinical studies. Chapter 1__________________________________________________Introduction 35 Figure 6. Representative examples of known NAMPT inhibitors with relevant anti-cancer activity. The limitations of FK866, CH828, and KPT9274 in the clinical phase for solid tumors and the off-target toxicities such as thrombocytopenia may endorse the development of NAMPT inhibitors towards haematological cancers.22 1.1.4 Medicinal chemistry of NAMPT inhibitors The crystal structures of NAMPT in complex with different inhibitors revealed a common pharmacophore based on the nature of the pocket of the active site (Figure 7). This model contains a NAM mimic group that is constituted of a pyridine or any other nitrogen-containing heterocycle and a connecting unit able to form hydrogen bonds with Ser275 and Asp219. Besides, a hydrophobic tunnel binder serves as spacer group between the connecting unit and the tail group that is important to make interactions in the solvent-exposed region of the enzyme. Chapter 1__________________________________________________Introduction 36 Figure 7. NAMPT inhibitors pharmacophoric model. Structure-Activity Relationship (SAR) studies performed in the last 20 years by academic or industrial research groups are remarkable. As a result, triazole-, urea-, thiourea-, amide-, and cyanoguanidine derivatives were identified as potent NAMPT inhibitors showing similar or improved NAMPT inhibition and/or cytotoxicity with respect to FK866 and CHS828.1,9,23 1.1.4.1 Previous results of our research group Very recently, our group published a research work about the synthesis and biological evaluation of more than fifty FK866 analogues.24 The modifications were focused on the phenyl ring of the piperidine benzamide group of FK866, and on the central alkyl chain. Several compounds showed low nanomolar or sub-nanomolar cell growth inhibitory activity towards pancreatic cell lines (Figure 8). It is worth to highlight that this work also demonstrated that the N-oxidation of the pyridine cap group led to a completely non-active compound. Although compound 7 showed a similar cytotoxicity than FK866 in MIA PaCa-2 cell line, it may be less prone to N-oxidation, and therefore, could be more stable and less toxic upon blood circulation. Chapter 1__________________________________________________Introduction 37 Figure 8. In vitro anti-proliferative activity of FK866 and its analogues 5, 6 and 7 toward three pancreatic cancer cell lines (IC50s calculated after 72 h).24 1.1.4.2 Chemical modification of the Cap group The short half-life of FK866 during phase 1 of clinical trial was found to be approximately 8 h and the N-oxidation of the pyridine was detected as the main chemical modification during metabolism.19 This suggested that the rapid clearance was in part due to the formation of N-oxide metabolites, which are non-active compounds towards NAMPT.25 Moreover, replacement of the pyridine moiety by the phenyl group led to an inactive compound26 and therefore chemical efforts were focused to find new heterocyclic moieties as pyridine mimetic moieties (Figure 9). Compound 827 depicted in Figure 9 is one of the rare examples in literature where the IC50 was comparable to FK866, even though the cap group was replaced with a heterocycle different from pyridine. In most of the cases, the presence of a nitrogenated heterocycle in the compound is mandatory to have NAMPT inhibitors with potent cytotoxic activity (compound 9, Figure 9). Chapter 1__________________________________________________Introduction 38 Figure 9. Replacement of the pyridine with heterocycles as cap group. MCF7= Breast cancer; A2780= Ovarian cancer. 1.1.4.3 Chemical modification of the connecting unit Replacement of the acrylamide of FK866 was investigated by different research groups both in academia and pharmaceutical companies with the aim to find a moiety that could bind the active site of NAMPT as the vinylogous amide group does.28,29 It is worth to point out that the substitution of acrylamide by a sulphonamide moiety resulted in a completely inactive compound, while replacement with a 1,2,3-triazole led to a cytotoxic compound (compounds 10 and 11, Figure 10).30,31 Several SAR studies indicated that a hydrogen bond acceptor should be at a 3-carbon distance from the cap group in order to maintain strong binding interactions within the active site.1 Chapter 1__________________________________________________Introduction 39 Figure 10. Modifications of the acrylamide connecting unit of FK866. SH-SY5Y= Neuroblastoma; n.d.= not determined, N.A.= not active. 1.1.4.4 Miscellaneous modifications of the spacer and the tail group The N-benzoyl 4-butylpiperidine moiety of FK866 was also modified with the aim of increasing the affinity towards the hydrophobic tunnel that connects the cap group with the solvent-exposed area of the active site of NAMPT. The result is an extensive variety of spacer groups such as hexyl, heptyl, and octyl chains or benzene rings (Figure 11). Modification of the tail group was important to anchor the molecule to the external part of the enzyme, increasing the possibility for the molecule to be retained in the pocket.23,28 Chapter 1__________________________________________________Introduction 40 Figure 11. NAMPT inhibitors with modifications of the spacer and tail groups. W1L2= Limphoblastoid; Calu-8= Lung cancer; n.d.= not determined. 1.1.4.5 Nonsubstrate NAMPT inhibitors The pyridine moiety is present in the most potent NAMPT inhibitors reported so far, which could be explained considering that NAM, a pyridine-containg molecule, is the substrate of NAMPT. As previously commented, crystalographic data showed that pyridine ring of FK866 is sandwiched between Tyr18 and Phe193 of the active site, by a π–π stacking interaction mimicking the position of NAM and establishing a hydrogen bond with the OH group of Ser275 and the N of the pyridine ring.14 In vitro metabolic stability studies on previously reported pyridine-based NAMPT inhibitors have shown that pyridine nitrogen is prone to microsomal oxidation, giving the corresponding N-oxide metabolite whose cytotoxicity is reduced with respect to the Chapter 1__________________________________________________Introduction 41 non-oxidized precursor.25 This fact could be one of the reasons for the lack of efficacy of inhibitors in in vivo xenograft models. Moreover, a recent published paper about nonsubstrate NAMPT inhibitor A1293201 (Figure 12), demonstrated that phosphoribosylation of the pyridine nitrogen of NAMPT inhibitors is not required for in vivo antitumor efficacy32 as it was previously postulated.33 In this work, Wilsbacher et al. indientified a series of substrate and nonsubstrate NAMPT inhibitors and proved that both types of compounds effectively deplete intracellular level of NAD+ and ATP leading cancer cells to death by NAMPT inhibition. This evidence led to explore nonsubstrate compounds as possible NAMPT inhibitors.9 Figure 12. Nonsubstrate NAMPT inhibitor A1293201. Chapter 1__________________________________________Results and discussion 48 good yields. Compound 50 was obtained by reaction of 30 with 46a under the same standard coupling conditions. Scheme 9. Synthesis of (pyridin-4-yl)cyanoguanidine-based inhibitors 47a, 47d, 48a-b, 48d, 49a-e and 50. Reaction of indole derivatives 25 and 37 with phenyl N-cyano-N’-(pyridin-3- yl)carbamimidate 46b, easily prepared from commercial diphenyl cyanocarbonimidate and 3-aminopyridine, afforded indole (pyridin-3-yl) cyanoguanidine derivatives 52 and 53 in moderate-to-good yields (Scheme 10). Chapter 1__________________________________________Results and discussion 49 Scheme 10. Synthesis of (pyridin-3-yl)cyanoguanidine-based inhibitors 52 and 53. As most compounds of these two subfamilies showed excellent results in cytotoxicity assays (see Table 2, pag. 57), two new analogues (compounds 57 and 58) that present a triazole moiety linking the tunnel binder and the furan tail were prepared (Scheme 11). Thus, reaction of 1,6-dibromohexane with an excess of sodium azide gave the corresponding diazido derivative, which was further subjected to a controlled Staudinger reduction in biphasic media affording the corresponding monoamino derivative. Subsequent coupling with 46a gave azido derivative 56 with a moderate overall yield (46%, 3 steps). CuAAC reactions of 56 and terminal alkynes 54 and 55, previously synthesized by N-alkylation of 37d and 37a with propargyl bromide, gave final compounds 57 and 58 in good-to-excellent yields, respectively. Chapter 1__________________________________________Results and discussion 50 Scheme 11. Synthesis of (pyridin-4-yl)cyanoguanidine-based inhibitors 57 and 58. 1.3.1.4 Synthesis of nonsubstrate compounds It has been recently demonstrated that phosphoribosylation of the pyridine nitrogen of NAMPT inhibitors is not required for in vivo antitumor efficacy32 as it has been previously postulated.33 Therefore, we decided to synthesize non pyridine-based structures as possible NAMPT inhibitors. First, we focused on the preparation of isoindoline-based derivatives inspired by the interesting results previously obtained with NAMPT inhibitor A1293201 (Figure 12, pag. 41).32 aqui Thus, the reaction between commercial isoindoline and ethyl 4-isocyanobenzoate gave urea 60 (Scheme 12). Basic hydrolysis of 60 followed by standard amide coupling between the resulting acid 61 and furfurylamine provided final compound 62 (the furyl analogue of 59). A similar strategy was followed for the preparation of 63, except that N-cyclopentylfurfurylamine 37a was used in the final coupling. Chapter 1__________________________________________Results and discussion 51 Scheme 12. Synthesis of isoindoline-based NAMPT inhibitors 62 and 63. Then, we decided to synthesize the isoindoline analogues (compounds 65a, 65b and 65d, Scheme 13) of three of the most cytotoxic cyanoguanidine derivatives (49a, 49b, 49d, (Scheme 9, pag. 48; Table 2, pag. 57). Thus, acidic deprotection of 38a, 38b and 38d followed by reaction with phenyl N-cyanoisoindoline-2-carbimidate 64, previously synthesized by coupling of isoindoline with diphenyl cyanocarbonimidate, afforded 65a, 65b and 65d respectively, in moderate yield. Chapter 1__________________________________________Results and discussion 52 Scheme 13. Synthesis of isoindoline-based inhibitors 65a, 65b and 65d. Finally, we decided to replace the pyridin-4-yl moiety by a phenyl group in two highly cytotoxic cyanoguanidines, sulfonamide 47a and its amide analogue 49a. The synthesis of these new non-pyridine analogues (compounds 67 and 68a-c) is depicted in Scheme 14. Acidic deprotection of compound 38a followed by coupling reaction with phenyl N-cyano-N’-phenylcarbamimidate 66a afforded derivative 67 in good yield. Similarly, reaction of amine 41a with carbamimidates 66a-c yielded final compounds 68a-c. (p-Substituted) phenyl N-cyano-N’-phenylcarbamimidates 66a-c were prepared by reaction of diphenyl cyanocarbonimidate with p-substituted anilines under the same conditions previously used for the preparation of analogue 64. Chapter 1__________________________________________Results and discussion 53 Scheme 14. Synthesis of phenyl-based inhibitors 67, and 68a-c. 1.3.2 BIOLOGICAL EVALUATION 1.3.2.1 Cytotoxicity on MIA PaCa-2 cells, effect on intracellular NAD+ concentration and NAMPT inhibition. The new compounds were tested in vitro for their anti-proliferative effects towards MIA PaCa-2 cells in Dr. Alessio Nencioni’s group at the University of Genova (Table 2). The human pancreatic cancer cell line MIA PaCa-2 was used as a model in the cytotoxicity assays as it is known that this cell line is sensitive to NAMPT inhibitors.38 Triazoles 19-23 were much less active than FK866 (Table 2, entries 2-6) towards MIA PaCa-2, with IC50 values in the micromolar range. Compounds 22 and 23 were approximately 300-fold less cytotoxic than FK866, what indicates that the substitution of the acrylamide group by a triazole and the benzamido group by a more hydrophilic indole substituent was detrimental for the biological activity. A similar modification was introduced in compounds 35 (Table 2, entry 10), and 53 (Table 2, Chapter 1__________________________________________Results and discussion 54 entry 25), with the exception that a thiourea and a cyanoguanidine groups were incorporated, respectively, instead of the triazole moiety of 22. Cyanoguanidine 53 (Table 2, entry 25) showed a cytotoxic effect 6.4-fold greater than triazole 22 while the cytotoxicity of thiourea 35 (Table 2, entry 10) was even lower than that presented by compound 22. Other pyridine-3-yl thioureas were also prepared using a more flexible tunnel binder (C6 or C7 alkyl chain) to connect the thiourea and tail groups. One of the members of this family, sulfonamide 40, showed a high cytotoxicity (IC50 = 16.4 nM, Table 2, entry 12). The comparison of the cytotoxicity of 40 and its amide analogue 39 (IC50 = 153 nM, Table 2, entry 11) clearly showed the benefit of having a sulfonamide group to connect the tail group (a furan moiety in both cases). Additionally, the sulfonamide can behave as a very good hydrogen bond acceptor for the interaction within the binding site of NAMPT. The analysis of the cytotoxicity of (pyridin-3/4-yl)cyanoguanidine derivatives 47a, 47d, 48a, 48b, 48d, 49a-49e, 52 and 53 afforded several conclusions. Most cyanoguanidines (Table 2, entries 13-22) belong to a general structure which contains: 1) a flexible alkyl C5 or C6 carbon chain as a tunnel binder connecting cyanoguanidine and tail groups through a tertiary amide/sulfonamide function and 2) a furan as a tail group. The cytotoxicity of these compounds was excellent with IC50 < 16 nM. In fact, in many cases, the IC50 values were lower than those obtained for FK866 in this cell line (2.4 nM). The C5 alkyl chain was always found to lead to slightly higher IC50 values (less cytotoxicity) than the C6 alkyl chain in different analogues (entry 15 vs 18, entry 16 vs 19, entry 17 vs 21). In the series of tertiary amides, different N-substituents together with the 2-furfuryl group were used, but no high differences in cytotoxicity were observed, being the compounds with N-cyclopentyl group slightly better cytotoxic agents than the rest of molecules (entry 15 vs entries 16,17; entry 18 vs entries 19-22). As was already observed for the family of pyridine-3-yl thioureas, the substitution of the amide function in N-cyclopentylamide 49a with a N-cyclopentylsulfonamide led to a great improvement of the cytotoxic activity (entry 18 vs 13), yielding the most potent compound of this study (compound 47a, IC50 = 5 ρM). Another example where the Chapter 1__________________________________________Results and discussion 55 bioisosterism led to a compound with improved potency is represented by the nonsubstrate inhibitor 68a (Table 2, entry 34). Compared to the amide analogue 67 (entry 34 vs 33), the sulfonamide 68a showed an increase in potency of 40-fold. However, amide 49d and sulfonamide 47d (Table 2, entries 21 and 14), presented similar cytotoxicity. The NMR spectra of 47d showed signals corresponding to a mixture of rotamers, in contrast with the other sulfonamide derivatives where only one set of signals was observed in the 1H and 13C NMR spectra. Our hypothesis to explain the lower cytotoxicity of 47d compared to other sulfonamide derivatives is that one of the rotamers could fit better than the other at the active site of NAMPT due to a more favorable binding pose, leading to a decrease in the global binding efficacy of the compound. In addition to amide and sulfonamide connecting groups, the triazole moiety was also explored in some of the cyanoguanidine family of compounds. The biological evaluation of 57 and 58 showed a decrease in their cytotoxicity compared to amides 49d and 49a or sulfonamides 47d and 47a, respectively (entry 26 vs entries 21,14; entry 27 vs entries 18 and 13). Finally, the activity of the series of non-pyridine- based compounds was also analysed. Replacement of pyridine with an isoindoline group was detrimental to the antitumor activity of the compounds in all cases (entries 28-32 vs. entries 19-21). Compounds 62 and 63 (Table 2, entries 28, 29), which have a 2-furyl moiety instead of the 3-tetrahydrofuryl group of A1293201 (compound 59, Scheme 12, pag. 51), showed cytotoxicity in the µM range (entries 28 and 29) as in the case of the other isoindoline analogues 65a, 65b and 65d (entries 30-32). Replacement of the pyridine-4-yl moiety in the highly cytotoxic sulfonamide 47a with a phenyl or 4-substituted phenyl moieties (compounds 67 and 68a-c) was detrimental in terms of cytotoxicity (entry 13 vs. entries 33-36). Compound 68a was the most cytotoxic compound in this family of non-pyridine-based inhibitors with an IC50= 176 nM. Chapter 1__________________________________________Results and discussion 56 Next, we evaluated intracellular NAD+ concentration (iNAD+) in MIA PaCa-2 cells in response to the most cytotoxic compounds, to confirm that the observed antitumor effect was associated with NAD+ depletion, which would be in line with these compounds being NAMPT inhibitors. Consistent with this hypothesis, all the compounds tested reduced the concentration of NAD+ in cells (Table 2). Some of the compounds (40, 47a, 47d, 48a, 48b, 49a-c) reduced iNAD+ to similar, or even greater extent, compared to FK866. Compound 68a, a non-pyridine-based compound, reduced iNAD+ when added in the nM range. Compounds 40, 47a, 49a and 58, which all effectively lowered iNAD+ levels, were also assayed as NAMPT inhibitors on the recombinant enzyme. All these compounds exerted a strong inhibition of NAMPT activity (IC50 < 70 nM). Again, compound 47a showed the highest potency (ie, IC50 = 3.5 nM), which was similar to the potency of the reference compound, FK866 (IC50 = 3.3 nM). Interestingly, the potency of 47a in terms of inhibition of recombinant NAMPT did not exactly match its cytotoxic activity in MIA PaCa-2 cells, the latter being already clearly detectable in the low ρM range. For the other compounds, 40, 49a and 58, inhibiting NAMPT activity in the sub-nanomolar range, no such discrepancy was observed. Therefore, these results suggest that the exceptional cytotoxic activity of 47a may also reflect other mechanisms in addition to NAMPT inhibition in MIA PaCa-2 cells. Indeed, in MDA-MB-231 (triple-negative breast cancer cell line), the IC50s obtained with 47a for iNAD+ reduction and for cell toxicity were similar (0.56 ± 0.15 and 0.47 ± 0.64 nM, respectively). By comparison, in these cells, the FK866 IC50s for iNAD+ depletion and cell toxicity were 0.54 ± 0.12 and 3.47 ± 0.41 nM, respectively, being similar values to those obtained in MIA PaCa-2 cells. The timedependent decrease in NAD+ and ATP levels in MIA PaCa-2 cells demonstrated that the high cytotoxic activity observed in the presence of 47a is not due to a NAD+- unrelated intracellular ATP depletion, since NAD+ decrease preceded the fall in ATP levels (Figure 14A). Cell death induced by FK866 analogues could be due to the triggering of apoptotic processes, as confirmed by the detection of PARP cleavage after 48 hours of treatment with compound 47a and 58 (Figure 14B). Chapter 1__________________________________________Results and discussion 57 Table 2. Evaluation of the cytotoxicity and iNAD+ depletion on MIA PaCa-2 cells for the new compounds. NAMPT inhibition assays. NA = not available. a) Data are mean ± SD, n ≥ 3. Entry R Compound Viability MIA PaCa-2 cells (IC50 in nM, except when indicated) iNAD+ depletion 24h on MIA PaCa-2 cells (IC50 in nM) NAMPT inhibition assay (IC50 in nM) 1 1 (FK866) 2.4±0.5 0.34±0.08 3.27±0.38 (Pyridin-3-yl)triazoles 2 19 494±143 NA NA 3 20 > 1000 NA NA 4 21 549±101 NA NA 5 22 727±187 NA NA 6 23 860±191 NA NA (Pyridin-3-yl)thioureas 7 32 854±188.5 NA NA Chapter 1__________________________________________Results and discussion 64 Table 4. Evaluation of iNAD+ depletion after 24h of drug treatment on different haematological cancer cell lines for compounds FK866, 47a, and 58 depicted as IC50 [nM]. Data are mean ± SD, n ≥ 3. Compound ML2 JRKT RPMI8226 1 (FK866) 0.11±0.03 0.37±0.003 0.1±0.005 47a 0.05±0.03 0.09±0.009 0.05±0.01 58 0.09±0.01 0.21±0.2 0.06±0.04 The intracellular content of NAD(P)H/NAD(P)+ plays a crucial role in oxidative stress and thus, the depletion of NAD+ upon exposure to NAMPT inhibitors is expected to result in an increased ROS production.40 To test this issue, mitochondrial (mO2-) and cytosolic (cO2-) superoxide anions, as well as intracellular hydrogen peroxide (H2O2) were monitored in leukemic cells treated with FK866, 47a, 49a and 58 using MitoSOX, DHE, and carboxy-H2DCFDA probes, respectively. The novel compounds caused a tremendous increase in ROS levels production in all treated cell types (Figure 16A-F). Figure 16. NAMPT inhibitors induce ROS production upon cell death in malignant cells. ML2 (A, B, C) and Jurkat (D, E, F) cells were treated with FK866, 47a, 49a and 58 for 72 h. Mitochondrial (A, D), cytosolic (B, E) superoxide anions, and hydrogen peroxide (C, F) were detected with MitoSOX, DHE and H2DCFDA probes, respectively, using flow cytometry. The percentage of positive cells is proportional to the amount of superoxide anions and hydrogen peroxide produced. Data are ± SD, n=3, **P<0.005, ***P<0.001, ****P<0.0001. Chapter 1__________________________________________Results and discussion 65 Excess ROS generation is known to lead to mitochondrial damage. Therefore, we evaluated whether these compounds could affect the mitochondrial membrane potential (MMP). Different hematopoietic malignant cells were exposed to NAMPT inhibitors and a time-dependent analysis of the MMP loss was measured using a fluorescent dye (TMRM) and flow cytometry analysis. As shown in Figure 17A – C, treatment with the new NAMPT inhibitors (47a, 49a and 58) induced a potent, timedependent mitochondrial membrane depolarization at 72 hours, but not at earlier time point (24 hours; Figure 17A – C). The timing of MMP loss correlates with that of cell death (Figure 17D – F). Figure 17. NAMPT inhibitors induce the loss of MMP and cell death over time. ML2 (A, D), Jurkat (B, E) and RPMI8226 (C, F) cells were incubated with FK866, 47a, 49a and 58 for 96 hours. MMP (A, B, C) and cell death (D, E, F) were measured every day over a period of 96 hours. MMP was measured using TMRM staining, and cell death is depicted here as a total cell death consisting of annexin V (ANXN) and 7AAD stainings. Both were assessed with flow cytometry. Data are ± SD, n=3. *P<0.05, **P<0.005, ***P<0.001, ****P<0.0001. To provide evidence that NAD+ depletion is the primary cause of cell death in malignant cells treated with the new NAMPT inhibitors 47a, 49a and 58, various hematopoietic malignant cells were incubated with the inhibitors and with an excess of NAD+ precursors (NAM, nicotinic acid [NA]) or NAD+ itself. Cell death was monitored as described above. Indeed, supplementation with NAM, NA, or NAD+ fully Chapter 1__________________________________________Results and discussion 66 blunted the killing effect of all tested NAMPT inhibitors (Figure 18A – C), supporting that NAD+ depletion was responsible for cell death. Figure 18. The addition of NAD, NAM, and NA can reverse the cytotoxic effect of NAMPT inhibitors. ML2 (A), Jurkat (B) and RPMI8226 (C) cells were incubated for 96 hours with FK866, 47a, 49a and 58 without or with NAD (0.5 mM), NAM (1 mM) or NA (10 µM). Cell death was assessed with flow cytometry by double staining with annexin V (ANXN) and 7AAD after 96 hours of treatment. The percentages of early apoptotic cells (ANXN+ 7AAD-) are shown in white and the percentage of late apoptotic and dead cells (7AAD+) are shown in black. Data are ± SD, n=3. ****P<0.0001 (inhibitors treated vs. NAD, NAM, and NA groups). Duchosal et al previously reported that treatment with FK866 leads to the decrease in catalase (CAT), a powerful ROS scavenger enzyme, resulting in ROS accumulation, what ultimately leads to cell death. Therefore, we tested the ability of CAT to prevent the cell death mediated by the new NAMPT inhibitors. As shown in Figure 19 the extracellular addition of CAT before 47a, 49a and 58 fully abrogated their cytotoxic effects, which is in line with previous studies.41,42 Chapter 1__________________________________________Results and discussion 67 Figure 19. Supplementation of catalase abrogates the killing effect of FK866, 49a, 58 and 47a in tested cell lines. Catalase was added 1 hour before the inhibitors. The cell death at 96 hours was assessed on ML2 (A), Jurkat (B) and RPMI8226 (C). Cell death was determined as in Figure 18 and is depicted here as total cell death. Data are ± SD, n=3. ****P<0.0001 (inhibitors treated vs. catalase treated groups). Altogether, these results indicate that the new NAMPT inhibitors (47a, 49a and 58) could be promising antitumor agents. Mechanistically, these data demonstrate that, similarly to FK866, they deplete NAD+ cell content, which induces ROS accumulation that damage mitochondria, leading to ATP loss, and ultimately to cell death. NAD+ depletion was indeed responsible for cell death. 1.3.2.3 In vivo antitumor activity Compounds 47a and 58 were selected to further evaluate their in vivo efficacy using a xenograft mouse model of human acute myeloid leukemia. Both compounds had shown similar or increased potency in cytotoxicity, iNAD+ depletion and NAMPT inhibition compared to reference compound 1 (FK866). The compounds (10 mg/Kg) were administered intraperitoneally twice a day for 4 days in a row for 3 weeks. In terms of safety, this dose was well tolerated, without signs of toxicity including no premature deaths, loss of body weight (Figure 20), lethargy or rough coat. Chapter 1__________________________________________Results and discussion 68 Figure 20. Mean mice weight after treatment with 47a and 58; nvehicle=3, n47a,58=4. Regarding the in vivo efficacy, mice treated with compound 58 compared to vehicle (untreated), significantly delayed the tumor growth and prolonged mouse survival. On the other hand, only one tumor treated with 47a decreased in volume up to two weeks and then increased again after the second cycle; the other tumors essentially grew similarly as in untreated group (Figure 21). Figure 21. Mean tumor volume after treatment with 47a and 58. Treatment days are indicated in grey; nvehicle=3, n47a,58=4 with SD **P < 0.05. Although FK866 significantly prolonged the life of the treated mice compared to 47a and 58 (Figure 22), it is interesting to note that, especially in the latter group, the tumors relapsed at different time points. Chapter 1__________________________________________Results and discussion 69 Figure 22. Mouse survival graph. 1.3.3 MOLECULAR MODELLING Docking analysis (Glide) was performed to investigate the potential role of the furan of 47a in the binding site of NAMPT (Crystal structure, PDB: 2GVJ). The binding pose of 1 (FK866) was mainly stabilized in the active site by a π−π stacking interaction of the pyridine ring with Phe193 and Tyr18’ and H-bonding of the carbonyl group with Ser275 (Figure 23A). On the other hand, the docked structure of compound 47a (Figure 23B) showed the π−π stacking interaction of the pyridine ring with Phe193, two hydrogen bonds between the cyanoguanidine moiety and Asp219 and a π-π stacking interaction of the furan tail group with Tyr188. As showed in recent publications, interactions of the tail group of NAMPT inhibitors with the enzyme have been reported to strongly enhance the inhibitory potency.9 Chapter 1__________________________________________Results and discussion 70 Figure 23. Compounds 1 (FK866) (A) and 47a (B) docked in the active site of NAMPT (Crystal structure, PDB: 2GVJ). The cyanoguanidine moiety of compound 47a clearly forms two hydrogen bonds with Asp219 while the furan group is stabilized by a π-π stacking interaction with Tyr188. A B Chapter 1______________________________________________Experimental part 71 1.4 EXPERIMENTAL PART Scheme 15. Synthesis of (pyridin-3-yl)triazole-based inhibitors 19 and 20. 8-Azidooctanoic acid (16)43 Sodium azide (1.0 g, 16 mmol) and NaI (100 mg, 900 µmol) in DMF (50 mL) were mixed. The mixture was stirred to form a heterogeneous suspension and 8- bromooctanoic acid (2.0 g, 9 mmol) was added. The reaction mixture was stirred at r.t. for 16 h, EtOAc was added, and the solution was cooled to 0 °C. To the resulting solution, 1 M HCl was added dropwise until the suspension became clear. The reaction mixture was extracted with EtOAc (3x). The organic layer was washed with brine (3x), dried over anh. NaSO4, filtered, and concentrated in vacuo resulting in 16 as a yellow oil. The crude product was used in the next step without further purification.1H NMR (300 MHz, CDCl3, δ ppm) 10.40 (s, 1H), 3.23 (t, J = 6.9 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.69 – 1.52 (m, 4H), 1.43 – 1.23 (m, 6H). NMR data match with those previously reported for this compound. 8-Azido-1-(1H-pyrrol-1-yl)octan-1-one (17) A mixture of 16 (1.6 g, 8.4 mmol) and 1,1’-carbonyldiimidazole (1.4 g, 8.6 mmol) was dissolved in dry THF (20 mL) and the reaction was stirred under argon at r.t. for 16 h. Pyrrole (0.7 mL, 9.5 mmol) was taken up in dry THF (5 mL) and the solution was cooled to -78 °C. n-Butyl lithium (2.5 M in hexanes; 5 mL, 13 mmol) was added and the Chapter 1______________________________________________Experimental part 72 mixture was stirred at -78 °C for 3 h under argon. Then, the activated compound was added quickly via syringe and the reaction was slowly warmed to r.t. and stirred overnight. A sat. aq. soln. of NH4Cl was added and the mixture extracted with EtOAc (3x). The organic phases were dried over anh. NaSO4, filtered, and concentrated in vacuo. Purification was performed by chromatography column on silica gel (EtOAc:Cy, 1:15) to yield 17 (800 mg, 38%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 7.37 – 7.25 (m, 2H, pyrrole), 6.33 – 6.24 (m, 2H, pyrrole), 3.26 (t, J = 6.9 Hz, 2H, CH2-N3), 2.82 (t, J = 7.4 Hz, 2H, CH2-CO), 1.84 – 1.73 (m, 2H, CH2), 1.67 – 1.54 (m, 2H, CH2), 1.43 – 1.35 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 170.6 (C=O), 119.1 (2 CH, pyrrole), 113.1 (2 CH, pyrrole), 51.5 (CH2-N3), 34.6 (CH2-CO), 29.1 (CH2), 29.0 (CH2), 28.9 (CH2), 26.6 (CH2), 24.5 (CH2). ESI-HRMS m/z calcd for C12H18N4ONa [M+Na]+, 257.1373; found, 257.1373. 8-(4-(Pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-1-(1H-pyrrol-1-yl)octan-1-one (19) To a solution of 17 (50 mg, 210 µmol) in toluene (3 mL), 3-ethynylpyridine (45 mg, 430 µmol), DIPEA (140 µL, 810 µmol) and CuI (8 mg, 40 µmol) were added, and the solution was stirred at 60°C for 24 h. Quadrasil® resin (150 mg) was added and the mixture stirred for 1 h at r.t. Then, it was filtrated through a celite pad, and the solvent evaporated. The resulting residue was dissolved in EtOAc and a sat. aq. soln. of NaHCO3 was added. The aq. phase was extracted with EtOAc (x3) and the organic layers were dried over anh. Na2SO4, filtered and evaporated. The resulting residue was purified by chromatography column on silica gel (EtOAc:Cy, 7:1) to obtain 19 (50 mg, 70%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 9.00 (br s, 1H, Py), 8.56 (br s, 1H, Py), 8.25 – 8.14 (m, 1H, Py), 7.84 (s, 1H, triazole), 7.41 – 7.32 (m, 1H, Py), 7.31 – 7.25 (m, 2H, pyrrole), 6.32 – 6.22 (m, 2H, pyrrole), 4.41 (t, J = 7.2 Hz, 2H, CH2- triazole), 2.80 (t, J = 7.3 Hz, 2H, CH2-CO), 2.03 – 1.90 (m, 2H, CH2), 1.83 – 1.69 (m, 2H, CH2), 1.44 – 1.34 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 170.5 (C=O), 149.2 (qC, Py), 147.1 (CH, Py), 144.8 (qC, triazole), 133.1 (CH, Py), 127.0 (CH, Py), 123.9 (2 CH, pyrrole), 119.9 (CH, Py), 119.1 (CH, triazole), 113.2 (2 CH, pyrrole), 50.6 (CH2- Chapter 1______________________________________________Experimental part 73 triazole), 34.5 (CH2-CO), 30.3 (CH2), 28.9 (CH2), 28.8 (CH2), 26.4 (CH2), 24.4 (CH2). ESIHRMS m/z calcd for C19H24N5O [M+H]+, 338.1975; found, 338.1975. 8-Azido-N-(furan-2-ylmethyl)octanamide (18) A solution of 16 (1.7 g, 9.0 mmol), furfurylamine (1.2 mL, 13.0 mmol) and PyBOP (7.9 g, 15.0 mmol) in DMF (36 mL) was cooled to 0 °C. DIPEA (7.5 mL, 43.0 mmol) was slowly added at 0 °C, and the reaction mixture was stirred at r.t. for 16 h. Then, the solvent was evaporated and the resulting crude diluted with EtOAc (50 mL), washed with 1 M HCl (x3) and brine (x3), dried over anh. Na2SO4, filtered, and concentrated in vacuo. The crude was purified by chromatography column on silica gel (EtOAc:Cy 1:2) to yield 18 (1.9 g, 78%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 7.37 – 7.29 (m, 1H, furan), 6.30 (dd, J = 3.1, 1.9 Hz, 1H, furan), 6.20 (d, J = 3.2 Hz, 1H, furan), 5.92 (br s, 1H, NH-CO), 4.41 (d, J = 5.3 Hz, 2H, CH2-NH), 3.23 (t, J = 6.9 Hz, 2H, CH2-N3), 2.17 (t, J = 7.2 Hz, 2H, CH2-CO), 1.69 – 1.50 (m, 4H, 2 CH2), 1.39 – 1.26 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 172.9 (C=O), 151.5 (qC, furan), 142.3 (CH, furan), 110.6 (CH, furan), 107.5 (CH, furan), 51.5 (CH2-N3), 36.6 (2 C, CH2-NH, CH2-CO), 29.1 (CH2), 28.9 (CH2), 28.8 (CH2), 26.6 (CH2), 25.6 (CH2). ESI-HRMS m/z calcd. for C13H20N4O2Na [M+Na]+, 287.1479; found, 287.1478. N-(Furan-2-ylmethyl)-8-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)octanamide (20) To a solution of 18 (50 mg, 210 µmol) in toluene (3 mL), 3-ethynylpyridine (40 mg, 400 µmol), DIPEA (130 µL, 720 µmol) and CuI (7 mg, 40 µmol) were added, and the solution was stirred at 60 °C for 24 h. Quadrasil® resin (150 mg) was added and the mixture was stirred at r.t. for 1 h. Then, it was filtrated through a celite pad, and the solvent evaporated. The resulting residue was dissolved in CH2Cl2 and a sat. aq. soln. of NaHCO3 was added. The aq. phase was extracted with CH2Cl2 (x2), and the organic layers were dried over anh. Na2SO4, filtered, and evaporated. The resulting residue was purified by chromatography column on silica gel (MeOH:EtOAc:CH2Cl2, 0.4:5:1) Chapter 1______________________________________________Experimental part 80 Scheme 18. Synthesis of (pyridin-3-yl)thiourea based inhibitor 32. 1-(8-Oxo-8-(1H-pyrrol-1-yl)octyl)-3-(pyridin-3-yl)thiourea (32) To a solution of 17 (0.5 g, 2.1 mmol) in pyridine (15 mL), H2S was bubbled. After stirring at r.t. overnight the mixture was evaporated under vacuum. The crude product 29 was used directly in the next step without any further purification. ESIHRMS m/z calcd for C12H21N2O [M+H]+, 209.1647; found, 209.1648. To a solution of 29 (60 mg, 300 µmol) in dry CH2Cl2 (5 mL), 3-pyridyl isothiocyanate (40 µL, 400 µmol) was added. After stirring at r.t. for 6 h, the solvent was removed under vacuum and the resulting residue was purified by chromatography column on silica gel (EtOAc:Cy 4:1) to give 32 (25 mg, 28%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 8.58 – 8.49 (m, 1H, Py), 8.48 – 8.40 (m, 1H, Py), 8.32 (br s, 1H, NH-Py), 7.89 – 7.80 (m, 1H, Py), 7.42 – 7.34 (m, 1H, Py), 7.29 (br s, 2H, pyrrole), 6.44 – 6.31 (m, 1H, NH-CH2), 6.31 – 6.24 (m, 2H, pyrrole), 3.60 (q, J = 6.4 Hz, 2H, CH2-NH), 2.81 (t, J = 7.3 Hz, 2H, CH2- CO), 1.82 – 1.68 (m, 2H, CH2), 1.68 – 1.52 (m, 2H, CH2), 1.44 – 1.30 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 181.3 (C=S), 170.8 (C=O), 147.4 (qC, Py), 146.1 (CH, Py), 134.2 (CH, Py) 132.6 (CH, Py), 124.3 (CH, Py), 119.1 (2CH, pyrrole), 113.2 (2CH, pyrrole), 45.5 (CH2-NH), 35.5 (CH2-CO), 29.8 (CH2), 29.0 (CH2), 28.8 (CH2), 26.6 (CH2), 24.4 (CH2). ESI-HRMS m/z calcd for C18H25N4OS [M+H]+, 345.1737; found, 345.1744. Chapter 1______________________________________________Experimental part 81 Scheme 19. Synthesis of (pyridin-3-yl)thiourea based inhibitor 33. 8-Amino-N-(furan-2-ylmethyl)octanamide (30) To a solution of 18 (100 mg, 380 µmol) in THF (1 mL), H2O (40 µL) and Ph3P (150 mg, 600 µmol) were added. The reaction mixture was stirred at reflux for 7 h, evaporated under vacuum and the residue purified by chromatography column on silica gel (NH4OH:MeOH:CH2Cl2, 0.1:1:10) to yield 30 (85 mg, 94%) as a white solid. 1H NMR (500 MHz, CDCl3, δ ppm) 7.33 (dd, J = 1.8, 0.7 Hz, 1H, furan), 6.30 (dd, J = 3.2, 1.9 Hz, 1H, furan), 6.20 (dd, J = 3.2, 0.6 Hz, 1H, furan), 5.87 (br s, 1H, NH-C=O), 4.42 (d, J = 5.5 Hz, 2H, CH2-NH), 2.67 (br s, 2H, NH2), 2.17 (t, J = 7.5 Hz, 2H, CH2-C=O), 1.68 – 1.57 (m, 2H, CH2-(CH2-CO), 1.49 – 1.36 (m, 4H, CH2-NH2, CH2), 1.35 – 1.24 (m, 6H, 3 CH2). 13C NMR (125.7 MHz, CDCl3, δ ppm) 172.9 (C=O), 151.6 (qC, furan), 142.3 (CH, furan), 110.6 (CH, furan), 107.5 (CH, furan), 42.0 (CH2-NH2), 36.7 (CH2-NH), 36.6 (CH2-CO), 33.7 (CH2), 29.3 (CH2), 28.8 (CH2), 26.8 (CH2), 25.7 (CH2). ESI-HRMS m/z calcd for C13H23N2O2 [M+H]+, 239.1753; found, 239.1754. N-(Furan-2-ylmethyl)-8-(3-(pyridin-3-yl)thioureido)octanamide (33) Reaction of 30 (70 mg, 300 µmol) and 3-pyridyl isothiocyanate (80 µL, 800 µmol) was performed according to the same procedure as that used to prepare 32. Chromatography column (MeOH:EtOAc:CH2Cl2, 0.4:5:1). Yield: 91%, white solid. 1H NMR (300 MHz, CD3OD, δ ppm) 8.57 (dd, J = 2.5, 0.6 Hz, 1H, Py), 8.28 (dd, J = 4.8, 1.3 Hz, 1H, Py), 8.10 – 7.89 (m, 1H, Py), 7.44 – 7.36 (m, 2H, 1H Py, 1H furan), 6.33 (dd, J = 3.2, 1.9 Hz, 1H, furan), 6.22 (dd, J = 3.2, 0.7 Hz, 1H, furan), 4.34 (s, 2H, CH2-NH), 3.68 – 3.44 (m, 2H, CH2-thiourea), 2.21 (t, J = 7.4 Hz, 2H, CH2-CO), 1.71 – 1.54 (m, 4H, 2 CH2), 1.44 – 1.29 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CD3OD, δ ppm) 183.1 (C=S), Chapter 1______________________________________________Experimental part 82 176.1 (C=O), 153.2 (qC, furan), 145.8 (2CH, Py), 143.3 (CH, furan), 138.3 (qC, Py), 133.3 (CH, Py), 124.9 (CH, Py), 111.3 (CH, furan), 108.0 (CH, furan), 45.6 (CH2- thiourea), 37.1 (CH2-NH), 36.9 (CH2-CO), 30.04 (CH2), 30.02 (CH2), 27.8 (CH2), 26.8 (CH2), 25.7 (CH2). ESI-HRMS m/z calcd for C19H27N4O2S [M+H]+, 375.1845; found, 375.1849. Scheme 20. Synthesis of (pyridin-3-yl)thiourea based inhibitor 34. N-(8-Aminooctyl)-1H-indole-2-carboxamide (31) To a solution of 25 (100 mg, 32 mmol) in THF (1 mL), H2O (30 µL) and Ph3P (126 mg, 500 µmol) were added. The reaction mixture was stirred at reflux for 30 h, evaporated under vacuum and the residue purified by chromatography column on silica gel (NH4OH:MeOH:CH2Cl2, 0.1:1:10) to yield 31 (45 mg, 50%) as a white solid. 1H NMR (300 MHz, DMSO-d6, δ ppm) 11.42 (br s, 1H, NH indole), 8.48 – 8.33 (m, 1H, NH- C=O), 7.59 (d, J = 7.8 Hz, 1H, indole), 7.42 (d, J = 8.3 Hz, 1H, indole), 7.16 (t, J = 7.4 Hz, 1H, indole), 7.09 (s, 1H, indole), 7.02 (t, J = 7.4 Hz, 1H, indole), 3.27 (q, J = 6.7 Hz, 2H, CH2-NH), 2.62 – 2.37 (m, 2H, CH2-NH2), 1.61 – 1.45 (m, 2H, CH2-(CH2-NH)), 1.41 – 1.17 (m, 10H, 5 CH2). 13C NMR (75.4 MHz, DMSO-d6, δ ppm) 160.9 (C=O), 136.3 (qC, indole), 131.9 (qC, indole), 127.1 (qC, indole), 123.1 (CH, indole), 121.4 (CH, indole), 119.6 (CH, indole), 112.2 (CH, indole), 102.1 (CH, indole), 41.5 (CH2-NH2), 39.1 (CH2- (NH-CO)), 33.1 (CH2), 29.3 (CH2), 29.0 (CH2), 28.8 (CH2), 26.5 (CH2), 26.4 (CH2). ESIHRMS m/z calcd for C17H26N3O [M+H]+, 288.2070; found, 288.2070. N-(8-(3-(Pyridin-3-yl)thioureido)octyl)-1H-indole-2-carboxamide (34) Chapter 1______________________________________________Experimental part 83 Reaction of 21 (37 mg, 0.1 mmol) and 3-pyridyl isothiocyanate (36 µL, 300 µmol) was performed according to the procedure used to prepare 32. Chromatography column (MeOH:EtOAc:CH2Cl2, 0.4:5:1). Yield: 80%, white solid. 1H NMR (300 MHz, DMSO-d6, δ ppm) 11.52 (s, 1H, NH indole), 9.53 (s, 1H, NH-Py), 8.55 (t, J = 2.2 Hz, 1H, Py), 8.43 (t, J = 5.7 Hz, 1H, NH-C=O), 8.28 (dd, J = 4.7, 1.4 Hz, 1H, Py), 8.03 – 7.86 (m, 1H, Py), 7.60 (d, J = 7.9 Hz, 1H, indole), 7.42 (dd, J = 8.2, 0.7 Hz, 1H, indole), 7.33 (dd, J = 8.2, 4.7 Hz, 1H, Py), 7.20 – 7.12 (m, 1H, indole), 7.09 (s, 1H, indole), 7.07 – 6.98 (m, 1H, indole), 3.51 – 3.39 (m, 2H, CH2-thiourea), 3.28 (q, J = 6.4 Hz, 2H, CH2-amide), 1.60 – 1.49 (m, 4H, 2 CH2), 1.40 – 1.26 (m, 8H, 4 CH2). 13C NMR (75.4 MHz, DMSO-d6, δ ppm) 180.9 (C=S), 161.0 (C=O), 144.6 (CH, Py), 144.4 (CH, Py), 136.3 (qC, indole), 131.9 (qC, indole), 131.8 (qC, Py), 130.2 (CH, Py), 127.1 (qC, indole), 123.1 (2CH, Py, indole), 121.4 (CH, indole), 119.6 (CH, indole), 112.2 (CH, indole), 102.1 (CH, indole), 43.9 (CH2-thiourea), 38.7 (CH2-amide), 29.3 (CH2), 28.8 (2(CH2)), 28.4 (CH2), 26.5 (CH2), 26.4 (CH2). ESI-HRMS m/z calcd for C23H30N5OS [M+H]+, 424.2162; found, 424.2166. Scheme 21. Synthesis of (pyridin-3-yl)thiourea based inhibitor 35. 4-(1-(1H-Indole-2-carbonyl)piperidin-4-yl)butyl 4-methylbenzenesulfonate (36) Compound 26 (483 mg, 1.2 mmol) was dissolved in 20 % TFA/CH2Cl2 (9 mL) and the mixture was stirred at r.t. for 4.5 h. The mixture was evaporated under vacuum and the crude used directly in the next step. This compound (257 mg, 600 µmol), 1H- Chapter 1______________________________________________Experimental part 84 indole-2-carboxylic acid (146 mg, 910 µmol) and PyBOP (534 mg, 1 mmol) in DMF (3.5 mL) were mixed and cooled to 0 °C. DIPEA (0.51 mL, 2.90 mmol) was slowly added at 0 °C, and the reaction mixture was stirred at r.t. for 16 h. The reaction mixture was diluted with EtOAc (35 mL) and washed with 1 M HCl (x3) and brine (x3), dried over Na2SO4, filtered, and concentrated in vacuo. The crude was purified by chromatography column on silica gel (EtOAc:Toluene, 1:2) to yield 36 (193 mg, 70%) as a white solid. 1H NMR (300 MHz, DMSO-d6, δ ppm) 11.53 (s, 1H, NH indole), 8.07 (d, J = 8.4 Hz, 1H, indole), 7.82 (d, J = 8.3 Hz, 1H, indole), 7.68 – 7.55 (m, 2H, Ts), 7.51 – 7.37 (m, 2H, Ts), 7.20 – 7.11 (m, 1H, indole), 7.10 – 6.98 (m, 1H, indole), 6.73 (d, J = 1.4 Hz, 1H, indole), 4.56 (t, J = 6.4 Hz, 2H, CH2-OTs), 4.50 – 4.37 (br d, 2H, piperidine), 3.00 (br s, 2H, piperidine), 2.50 (s, 3H, CH3 Ts), 1.84 – 1.70 (m, 4H, piperidine), 1.65 – 1.47 (m, 1H, CH piperidine), 1.38 – 1.24 (m, 2H, CH2), 1.24 – 1.01 (m, 2H, CH2). 13C NMR (75.4 MHz, DMSO-d6, δ ppm) 161.8, 142.8, 135.8, 130.3, 128.4, 127.0, 126.8, 125.0, 123.0, 121.2, 119.7, 119.6, 112.0, 109.2, 103.4, 80.8, 40.0, 35.4, 35.3, 32.5, 32.1, 27.7, 22.1. (4-(4-Aminobutyl)piperidin-1-yl)(1H-indol-2-yl)methanone (37) Compound 36 (170 mg, 370 µmol) was dissolved in DMF (2.5 mL), NaN3 (73 mg, 1.1 mmol) was added, and the mixture was stirred at 120 °C for 32 h. The mixture was evaporated under vacuum and the residue dissolved in CH2Cl2. The organic layer was washed with water and brine, dried over anh. Na2SO4, filtered, and concentrated in vacuo. The residue obtained (113 mg, 350 µmol) was dissolved in MeOH (10 mL), Pd/C 10% (50 mg) was added, and the reaction mixture was stirred under H2 (1 atm) at r.t. overnight. The mixture was filtered through a celite pad and concentrated in vacuo. The crude product was used in the next step without further purification. 1-(4-(1-(1H-Indole-2-carbonyl)piperidin-4-yl)butyl)-3-(pyridin-3-yl)thiourea (35) Chapter 1______________________________________________Experimental part 85 Reaction of 37 (22 mg, 100 µmol) and 3-pyridyl isothiocyanate (18 µL, 200 µmol) was performed according to the same procedure as that used to prepare 32. Chromatography column (MeOH:EtOAc:CH2Cl2, 0:5:1 to 1:5:1:). Yield: 91%, white solid. 1H NMR (300 MHz, Acetone-d6, δ ppm) 10.63 (s, 1H, NH indole), 8.91 (s, 1H, NH- Py), 8.65 – 8.53 (m, 1H, Py), 8.31 (dd, J = 4.7, 1.3 Hz, 1H, Py), 8.07 – 7.94 (m, 1H, Py), 7.68 – 7.58 (m, 1H, indole), 7.57 – 7.39 (m, 2H, 1H indole, NH-CH2), 7.31 (dd, J = 8.1, 4.6 Hz, 1H, Py), 7.27 – 7.15 (m, 1H, indole), 7.15 – 7.00 (m, 1H, indole), 6.80 (d, J = 1.3 Hz, 1H, indole), 4.71 – 4.51 (m, 2H, piperidine), 3.72 – 3.54 (m, 2H, CH2-NH), 3.00 (br s, 2H, piperidine), 1.90 – 1.75 (m, 2H, piperidine), 1.75 – 1.55 (m, 3H, 1H piperidine, CH2), 1.52 – 1.10 (m, 2H piperidine, 2 CH2). 13C NMR (75.4 MHz, Acetone-d6, δ ppm) 181.8, 161.8, 145.3, 136.1, 136.0, 130.4, 127.5, 123.3, 122.9, 121.3, 119.8, 111.8, 111.7, 103.8, 103.7, 44.9, 44.2, 35.9, 32.4, 28.8, 23.6. ESI-HRMS m/z calcd for C24H30N5OS [M+H]+, 436.2158; found, 436.2166. Scheme 22. Synthesis of (pyridin-3-yl)thiourea-based inhibitor 39. N-(Furan-2-ylmethyl)cyclopentanamine (37a)46 A solution of furfural (6.9 mL, 83.0 mmol) and cyclopentanamine (8.2 mL, 83.0 mmol) was stirred in dry CH2Cl2 (200 mL) over 3 Å MS at r.t. for 24 h. Then the reaction mixture was concentrated in vacuo and the residue was dissolved in MeOH (200 mL). NaBH4 (6.3 g, 0.2 mol) was added in small portions to the mixture at 0 °C. The reaction was warmed to r.t. and stirred for 16 h. The solvent was removed under reduce Chapter 1______________________________________________Experimental part 86 pressure and the residue was dissolved in water and extracted with EtOAc (3x). The organic phases were extracted with 3% HCl (3x), and the pH of the obtained acidic aqueous solution was raised to pH 10 with sat. aq. soln. of NaOH. The aqueous phase was extracted with EtOAc (3x), and the organic phases were washed with water, brine, and dried over anh. Na2SO4. The solvent was evaporated in vacuo to give 37a (13 g, 91%) as a yellow oil. 1H NMR (300 MHz, CDCl3, δ ppm) 7.34 (dd, J = 1.8, 0.8 Hz, 1H), 6.29 (dd, J = 3.1, 1.9 Hz, 1H), 6.16 (dd, J = 3.2, 0.7 Hz, 1H), 3.76 (s, 2H), 3.08 (quint, J = 6.6 Hz, 1H), 1.91 – 1.76 (m, 2H), 1.76 – 1.60 (m, 3H), 1.60 – 1.44 (m, 2H), 1.44 – 1.26 (m, 2H). NMR data match with those previously reported for this compound.46 7-((tert-Butoxycarbonyl)amino)heptanoic acid (42)37 7-Aminoheptanoic acid (2 g, 14 mmol) and NaOH (550 mg, 14 mmol) were dissolved in dioxane-H2O (2:1, 60 mL). This mixture was cooled to 0 °C and Boc2O (3.3 g, 15.2 mmol) was added in small portions. The reaction mixture was stirred at r.t. for 16 h, and then was concentrated under reduced pressure. The basic residue was redissolved in H2O and washed with EtOAc (2x). The aqueous phase was acidified to pH 1-2 with aqueous 1M HCl and extracted with EtOAc (3x). The combined organic extracts were dried over anh. Na2SO4, filtered, and concentrated under reduced pressure to afford 42 as colourless oil, which slowly became a white solid. The crude product was used in the next step without further purification. ESI-HRMS m/z calcd for C12H23NO4Na [M+Na]+, 268.1523; found, 268.1519. tert-Butyl (7-(cyclopentyl(furan-2-ylmethyl)amino)-7-oxoheptyl)carbamate (38a) A solution of 37a (80 mg, 480 µmol), 42 (178 mg, 730 µmol) and PyBOP (428 mg, 820 µmol) in DMF (2.5 mL) was cooled to 0 °C. DIPEA (0.21 mL, 1.21 mmol) was slowly added at 0 °C, and the reaction mixture was stirred at r.t. for 16 h. Then, it was concentrated in vacuo, diluted with EtOAc and washed with 1 M HCl (x3) and brine (x3), dried over anh. Na2SO4 and concentrated in vacuo. The crude was purified by Chapter 1______________________________________________Experimental part 87 chromatography column on silica gel (EtOAc:Cy, 1:4-→1:1) to yield 38a (170 mg, 89%) as a colourless oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.46 – 7.10 (m, 2H, furan), 6.39 – 6.23 (m, 2H, furan), 6.23 – 6.07 (m, 2H, furan), 4.86 – 4.63 (m, 1H, CH ciclopentyl rotamer A), 4.58 – 4.28 (m, 6H, 2 CH2-furan, 2 NH), 4.28 – 4.10 (m, 1H, CH ciclopentyl rotamer B), 3.08 (br s, 4H, 2 CH2-NH), 2.50 – 2.26 (m, 4H, 2 CH2- C=O), 1.91 – 1.21 (m, 50H, 8 CH2, 8 CH2 ciclopentyl, 2 C(CH3)3). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.9, 173.2, 156.2, 152.3, 152.3, 142.1, 141.0, 110.6, 107.6, 107.0, 79.2, 77.6, 77.2, 76.7, 60.5, 58.8, 56.2, 41.9, 38.4, 30.1, 29.8, 29.2, 28.6, 26.7, 25.4, 23.9, 14.3. ESI-HRMS m/z calcd for C22H36N2O4Na[M+Na]+, 415.2564; found, 493.2567. N-Cyclopentyl-N-(furan-2-ylmethyl)-7-(3-(pyridin-3-yl)thioureido)heptanamide (39) Compound 38a (156 mg, 400 µmol) was dissolved in 20 % TFA/CH2Cl2 (2.5 mL) at 0 °C and the mixture was stirred at r.t. for 3 h. The solvent was evaporated under vacuum and the residue was azeotropically dried with toluene (2x). Finally, the residue was dried under high vacuum to yield a colourless oil that was used in the next step without further purification. To a solution of this compound (110 mg, 270 µmol) in dry CH2Cl2 (6 mL), 3-pyridyl isothiocyanate (75 µL, 700 µmol) was added. After stirring at r.t. for 3 h, the solvent was removed under vacuum and the resulting residue was purified by chromatography column on silica gel (MeOH:EtOAc, 1:9) to give 39 (80 mg, 70%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.92 – 8.72 (m, 2H, Py), 8.55 – 8.41 (m, 2H, Py), 8.41 – 8.26 (m, 2H, Py), 8.18 – 8.00 (m, 2H, Py), 7.41 – 7.07 (m, 6h, 2H furan, 2 NH-Py, 2 NH-CH2), 6.40 – 6.16 (m, 2H, furan), 6.18 – 6.02 (m, 2H, furan), 4.69 (quint, J = 8.3 Hz, 1H, CH cyclopentyl rotamer A), 4.40 (br d, 4H, 2 CH2-furan), 4.29 – 4.09 (m, 1H, CH cyclopentyl rotamer B), 3.72 – 3.51 (m, 4H, 2 CH2-NH), 2.67 – 2.31 (m, 4H, 2 CH2-C=O), 1.94 – 1.23 (m, 32H, 8 CH2 cyclopentyl, 8 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 181.8, 174.5, 173.9, 152.0, 151.6, 145.8, 145.6, 145.0, 142.3, 141.4, 136.0, 132.2, 123.6, 123.6, 110.6, Chapter 1______________________________________________Experimental part 88 107.3, 77.6, 77.2, 76.7, 59.0, 56.5, 45.2, 44.9, 41.8, 38.8, 33.6, 33.3, 30.0, 29.1, 28.3, 27.6, 27.1, 26.8, 24.6, 24.4, 23.9, 23.8. ESI-HRMS m/z calcd for C23H33N4O2S [M+H]+, 429.2316; found, 429.2319. Scheme 23. Synthesis of intermediate 45. 2-(6-Bromohexyl)isoindoline-1,3-dione (43)47 Potassium phthalimide (12.0 g, 65 mmol) was suspended in acetone (200 mL) and 1,6-dibromohexane (25 mL, 200 mmol) was added. The suspension was refluxed for 48 h and the solvent was evaporated under vacuum. The residue was dissolved in water and extracted with EtOAc (x2). The combined organic layers were dried over anh. Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (EtOAc:Cy, 1:5) to provide 43 (17.0 g, 84%) as white crystals. 1H NMR (300 MHz, CDCl3, δ ppm) 7.84 (dd, J = 5.5, 3.1 Hz, 2H), 7.70 (dd, J = 5.5, 3.1 Hz, 2H), 3.68 (t, J = 7.2 Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H), 1.93 – 1.78 (m, 2H), 1.70 (quint, J = 7.5 Hz, 2H), 1.56 – 1.31 (m, 4H). NMR data match with those previously reported for this compound. S-(6-(1,3-Dioxoisoindolin-2-yl)hexyl) ethanethioate (44)48 To a stirred solution of 43 (3.9 g, 12.5 mmol) in THF (150 mL) was added KSAc (4.3 g, 37.5 mmol) in small portions during 20 min and the mixture was refluxed for 3 h. The solvent was evaporated in vacuo, water was added to the residue and extracted with EtOAc (3x). The combined organic phases were washed with water and brine, dried over anh. Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (EtOAc:Cy, 1:2) to give 44 (3.4 g, 89%) as light brown Chapter 1______________________________________________Experimental part 89 crystals. 1H NMR (300 MHz, CDCl3, δ ppm) 7.91 – 7.77 (m, 2H), 7.76 – 7.64 (m, 2H), 3.74 – 3.59 (m, 2H), 2.85 (t, J = 7.2 Hz, 2H), 2.30 (s, 3H), 1.74 – 1.50 (m, 4H), 1.50 – 1.27 (m, 4H). NMR data match with those previously reported for this compound. 6-(1,3-Dioxoisoindolin-2-yl)hexane-1-sulfonyl chloride (45)28 2N HCl (2.4 mL) was added to a stirred solution of 44 (1.3 g, 4.3 mmol) in MeCN (24 mL) at 0 °C. NCS (2.4 g, 18.3 mmol) was added in small portions over 30 min and the reaction mixture was warmed to r.t. and stirred for 1 h. Then, it was quenched with ice-cold water and extracted with Et2O (x3). The combined organic layers were washed with sat. aq. soln. of NaHCO3, water and brine, dried over anh. Na2SO4 and concentrated to afford 45 (1.35 g, 96%) as a white solid that was used in the next step without further purification. 1H NMR (300 MHz, CDCl3, δ ppm) 7.84 (dd, J = 5.4, 3.1 Hz, 2H), 7.71 (dd, J = 5.4, 3.1 Hz, 2H), 3.77 – 3.59 (m, 4H), 2.14 – 1.96 (m, 2H), 1.72 (quint, J = 7.2 Hz, 2H), 1.66 – 1.49 (m, 2H), 1.49 – 1.33 (m, 2H). ESI-HRMS m/z calcd for C14H16ClNO4SNa [M+Na]+, 352.0378; found, 352.0381. NMR data match with those previously reported for this compound. Scheme 24. Synthesis of (pyridin-3-yl)thiourea-based inhibitor 40. 6-Amino-N-cyclopentyl-N-(furan-2-ylmethyl)hexane-1-sulfonamide (41a) To a suspension of 37a (1.1 g, 6.7 mmol) in dry CH2Cl2 (38 mL), Et3N (2.6 mL, 19.0 mmol) was added under argon at 0 °C and stirred over 3 Å MS. Compound 45 (5.3 g, 16.0 mmol) in dry CH2Cl2 (40 mL) was added over 1 h and the resulting mixture was stirred for 3 h at 0 °C. Then, it was quenched with water and extracted with CH2Cl2 Chapter 1______________________________________________Experimental part 96 1.67 – 1.50 (m, 4H, 2 CH2), 1.50 – 1.29 (m, 8H, 4 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 169.5, 157.6, 150.8, 150.1, 150.0, 145.0, 143.0, 143.0, 134.9, 130.4, 128.4, 115.8, 110.8, 110.7, 110.0, 109.9, 67.0, 66.9, 55.7, 55.7, 53.7, 52.2, 52.0, 50.9, 45.4, 45.3, 43.2, 42.4, 40.1, 29.2, 28.9, 28.1, 27.8, 26.5, 26.1, 25.5, 25.4, 23.3. ESI-HRMS m/z calcd for C25H38N7O4S [M+H]+, 532.2693; found, 532.2700. Scheme 28. Synthesis of intermediates 38b-e, 51a, 51b and 51d. tert-Butyl-(7-((4-chlorobenzyl)(furan-2-ylmethyl)amino)-7-oxoheptyl)carbamate (38b) To a solution of 37b (80 mg, 360 µmol) and 7-((tert-butoxycarbonyl)amino)heptanoic acid51 (106 mg, 430 µmol) in CH2Cl2 (2 mL), DMAP (4 mg, 40 µmol) and DCC (112 mg, 540 µmol) in CH2Cl2 (1 mL) were added at 0 °C. The reaction mixture was stirred overnight at r.t., filtered through celite, washed with EtOAc (20 mL) and concentrated under vacuum, and the residue was purified by chromatography column on silica gel (EtOAc:Cy, 1:2→1:1) to obtain 38b (130 mg, 80%) as a colourless oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.40 – 7.20 (m, 6H, 2H furan, 4H Ph), 7.19 – 6.98 (m, 4H, Ph), 6.36 – 6.22 (m, 2H, furan), 6.22- 6.08 (m, 2H, furan), 4.98 – 3.99 (m, 10H, 2 NH, 2 CH2-furan, 2 CH2-Ph), 3.18 – 2.96 (m, 4H, 2 CH2-NH), 2.60 – 2.22 (m, 4H, 2 CH2-C=O), 2.00 – 1.53 (m, 8H, 4 CH2), 1.53 – 1.19 (m, 26H, 2 C(CH3)3, 4 CH2). 13C NMR Chapter 1______________________________________________Experimental part 97 (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.7, 173.3, 156.1, 154.2, 151.0, 150.1, 142.8, 142.3, 136.1, 135.4, 133.4, 133.2, 129.7, 129.1, 128.8, 127.8, 110.5, 108.9, 108.3, 79.1, 77.6, 77.2, 76.7, 56.0, 50.0, 49.8, 47.5, 43.7, 41.5, 35.7, 33.2, 33.1, 32.8, 31.0, 30.0, 30.0, 29.1, 29.0, 28.9, 28.5, 26.7, 26.6, 26.4, 25.6, 25.4, 25.2, 25.1, 24.8. ESI-HRMS m/z calcd for C24H33ClN2O4Na [M+Na]+, 471.2016; found, 471.2021. tert-Butyl-(7-((furan-2-ylmethyl)(4-nitrobenzyl)amino)-7-oxoheptyl)carbamate (38c) Compound 38c was prepared according to the same procedure that was employed for 38b, except that 37c was used as starting material. Chromatography column (EtOAc:Cy, 1:2→1:1). Yield: 76%, yellow oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.23 – 7.94 (m, 4H, Ph), 7.35 – 7.14 (m, 6H, 2H furan, 4H Ph), 6.29 – 6.18 (m, 2H, furan), 6.18 – 6.06 (m, 2H, furan), 4.69 – 4.22 (m, 10H, 2 NH, 2 CH2-furan, 2 CH2-Ph), 3.13 – 2.88 (m, 4H, 2 CH2-NH), 2.60 – 2.15 (m, 4H, 2 CH2-C=O), 1.93 – 1.49 (m, 6H, 3 CH2), 1.49 – 0.96 (m, 28H, 2 C(CH3)3, 5 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.4, 173.1, 156.0, 154.1, 150.5, 149.6, 147.5, 147.2, 145.3, 144.5, 142.9, 142.3, 128.6, 127.0, 124.1, 123.7, 110.5, 110.4, 109.2, 108.6, 79.0, 77.5, 77.1, 76.7, 50.3, 49.3, 48.2, 44.4, 41.8, 35.6, 33.7, 33.1, 33.0, 32.7, 30.9, 29.9, 29.0, 28.9, 28.8, 28.4, 26.6, 26.5, 26.3, 25.6, 25.5, 25.3, 25.0, 25.0, 24.9, 24.7. ESI-HRMS m/z calcd for C24H33N3O6Na [M+Na]+, 482.2259; found, 482.2262. tert-Butyl-(7-((furan-2-ylmethyl)(3-morpholinopropyl)amino)-7- oxoheptyl)carbamate (38d) Compound 38d was prepared according to the same procedure that was employed for 38b, except that 37d was used as starting material. Chromatography column (MeOH:EtOAc, 1:10→1:6). Yield: 67%, colourless oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.36 – 7.31 (m, 1H, furan rotamer A), 7.31 – 7.27 (m, 1H, furan rotamer B), 6.36 – 6.23 (m, 2H, furan), 6.23 – 6.10 (m, 2H, furan), 4.68 – 4.45 (m, 4H, Chapter 1______________________________________________Experimental part 98 CH2-furan rotamer A, 2 NH), 4.40 (s, 2H, CH2-furan rotamer B), 3.76 – 3.54 (m, 8H, 4 CH2), 3.46 – 3.22 (m, 4H, 2 CH2), 3.13 – 2.92 (m, 4H, 2 CH2), 2.53 – 2.12 (m, 16H, morpholine), 1.77 – 1.54 (m, 8H, 4 CH2), 1.54 – 1.21 (m, 30H, 2 C(CH3)3, 2 NH, 5 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.0, 172.9, 156.1, 151.6, 150.7, 142.6, 141.9, 110.5, 110.4, 108.4, 107.8, 79.0, 77.6, 77.2, 76.7, 67.0, 67.0, 56.2, 55.5, 53.7, 53.6, 45.2, 45.1, 44.4, 41.4, 33.2, 33.0, 30.0, 29.7, 29.2, 29.1, 28.5, 26.7, 26.6, 25.5, 25.3, 25.1, 24.5. tert-Butyl-(7-((furan-2-ylmethyl)(3-(pyrrolidin-1-yl)propyl)amino)-7- oxoheptyl)carbamate (38e) Compound 38e was prepared according to the same procedure that was employed for 38b, except that 37e was used as starting material. Chromatography column (MeOH:DCM:NH4OH, 1:20:0.1→1:10:0.1). Yield: 44%, colourless oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.36 – 7.21 (m, 2H, furan), 6.31 – 6.20 (m, 2H, furan), 6.20 – 6.11 (m, 2H, furan), 4.59 (s, 2H, 2 NH), 4.49 (s, 2H, CH2-furan rotamer A), 4.39 (s, 2H, CH2-furan rotamer B), 3.44 – 3.22 (m, 4H, 2 CH2-NH), 3.09 – 2.97 (m, 4H, CH2-N-C=O), 2.84 – 2.70 (m, 4H, 2 CH2-N), 2.69 – 2.22 (m, 12H, 4 CH2 pyrrolidine, 2 CH2-C=O), 1.92 – 1.77 (m, 6H, 2 CH2 pyrrolidine, CH2), 1.77 – 1.65 (m, 4H, 2 CH2 pyrrolidine), 1.65 – 1.49 (m, 4H, 2 CH2), 1.49 – 1.11 (m, 30H, 2 C(CH3)3, 6 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.4, 172.9, 156.0, 151.5, 150.3, 142.6, 141.8, 110.4, 110.4, 108.3, 108.2, 78.9, 77.6, 77.2, 76.7, 54.0, 53.8, 53.5, 53.0, 45.4, 44.8, 43.6, 41.4, 33.0, 32.8, 30.1, 29.9, 29.6, 29.3, 29.0, 28.9, 28.4, 27.6, 26.6, 26.5, 25.7, 25.2, 25.0, 23.3. ESI-HRMS m/z calcd for C24H42N3O4 [M+H]+, 436.3165; found, 436.3170. tert-Butyl (6-(cyclopentyl(furan-2-ylmethyl)amino)-6-oxohexyl)carbamate (51a) A solution of 37a (80 mg, 500 µmol), 6-((tert-butoxycarbonyl)amino)hexanoic acid51 (168 mg, 730 µmol) and PyBOP (428 mg, 820 µmol) in DMF (2.5 mL) was cooled to 0 Chapter 1______________________________________________Experimental part 99 °C. DIPEA (0.41 mL, 2.32 mmol) was slowly added at 0 °C, and the reaction mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated in vacuo and diluted with EtOAc and washed with 1 M HCl (x3) and brine (x3), dried over Na2SO4 and concentrated in vacuo. The crude was purified by chromatography column on silica gel (EtOAc:Cy, 1:1) to yield 51a (166 mg, 90%) as a yellow oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.40 – 7.19 (m, 2H, furan), 6.38 – 6.23 (m, 2H, furan), 6.23 – 6.05 (m, 2H, furan), 4.88 – 4.30 (m, 7H, 2 CH2-furan, CH ciclopentyl rotamer A, 2 NH), 4.29 – 4.03 (m, 1H, CH ciclopentyl rotamer B), 3.22 – 2.94 (m, 4H, 2 CH2-NH), 2.49 – 2.26 (m, 4H, 2 CH2-C=O), 1.88 – 1.17 (m, 46H, 6 CH2, 8 CH2 ciclopentyl, 2 C(CH3)3). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.1, 156.1, 152.7, 152.3, 142.1, 141.0, 110.6, 107.6, 107.0, 79.2, 58.7, 56.2, 41.9, 40.7, 38.4, 33.8, 33.6, 30.0, 29.2, 28.6, 26.7, 25.1, 23.9. ESI-HRMS m/z calcd for C21H34N2O4Na [M+Na]+, 401.2407; found, 401.2411. tert-Butyl(6-((4-chlorobenzyl)(furan-2-ylmethyl)amino)-6-oxohexyl)carbamate (51b) Reaction of 37b (80 mg, 360 µmol), 6-((tert-butoxycarbonyl)amino)hexanoic acid51 (125 mg, 540 µmol) was performed according to the same procedure used to prepare 51a. Chromatography column (EtOAc:Cy, 1:2→1:1). Yield: 83%, colourless oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.45 – 7.20 (m, 6H, 2H furan, 4H Ph), 7.20 – 7.00 (m, 4H, Ph), 6.38 – 6.23 (m, 2H, furan), 6.23 – 6.09 (m, 2H, furan), 4.60 – 4.29 (m, 10H, 2 NH, 2 CH2-furan, 2 CH2-Ph), 3.21 – 2.99 (m, 4H, 2 CH2-NH), 2.53 (t, J = 7.6 Hz, 2H, CH2-C=O rotamer A), 2.32 (t, J = 7.3 Hz, 2H, CH2-C=O rotamer B) 1.80 – 1.60 (m, 4H, 2 CH2), 1.57 – 1.20 (m, 26H, 2 C(CH3)3, 4 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.2, 156.2, 151.0, 150.1, 142.9, 142.4, 136.1, 135.4, 133.5, 133.3, 129.8, 129.2, 128.8, 127.8, 110.5, 109.0, 108.4, 79.1, 50.1, 47.6, 43.7, 41.6, 40.7, 33.2, 30.0, 28.6, 26.7, 25.0. ESI-HRMS m/z calcd for C23H31ClN2O4Na [M+Na]+, 457.1860; found, 457.1865. Chapter 1______________________________________________Experimental part 100 tert-Butyl(6-((furan-2-ylmethyl)(3-morpholinopropyl)amino)-6- oxohexyl)carbamate (51d) A solution of 37d (80 mg, 480 µmol), 6-((tert-butoxycarbonyl)amino)hexanoic acid51 (124 mg, 540 µmol) was performed according to the same procedure used to prepare 51a. Chromatography column (MeOH:EtOAc, 1:12→1:6). Yield: 80%, yellow oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.40 – 7.33 (m, 1H, furan rotamer A), 7.34 – 7.28 (m, 1H, furan rotamer B), 6.35 – 6.25 (m, 2H, furan), 6.25 – 6.15 (m, 2H, furan), 4.65 – 4.47 (m, 4H, CH2-furan rotamer A, 2 NH), 4.43 (s, 2H, CH2-furan rotamer B), 3.82 – 3.62 (m, 8H, morpholine), 3.49 – 3.24 (m, 4H, 2 CH2), 3.13 – 3.05 (m, 4H, 2 CH2), 2.61 – 2.21 (m, 16H, 8H morpholine, 4 CH2), 1.76 – 1.56 (m, 12H, 6 CH2), 1.56 – 1.26 (m, 24H, 2 C(CH3)3, 2 NH, 2 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.2, 172.9, 156.1, 151.6, 150.6, 142.7, 142.0, 110.6, 110.5, 108.5, 108.1, 79.1, 77.6, 77.2, 76.7, 66.9, 66.4, 56.2, 55.6, 53.7, 53.4, 45.3, 45.1, 44.2, 41.5, 34.4, 33.2, 33.0, 30.0, 29.8, 28.5, 26.7, 25.5, 25.0, 24.9, 24.1. ESI-HRMS m/z calcd for C23H40N3O5 [M+H]+, 438.2959; found, 438.2962. Scheme 29. Synthesis of (pyridin-3/4-yl)cyanoguanidine-based inhibitors 48a,b,d, 49a-e and 50. Chapter 1______________________________________________Experimental part 101 (E)-6-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-cyclopentyl-N-(furan-2- ylmethyl)hexanamide (48a) Compound 51a (144 mg, 380 µmol) was dissolved in 20% TFA/CH2Cl2 (500 µL TFA, 2 mL CH2Cl2) at 0 °C and the mixture was stirred at r.t. for 2.5 h. The solvent was evaporated in vacuo and the residue was azeotropically dried with toluene (2 × 5 mL). Finally, the residue was dried under high vacuum to yield a colourless oil that was used in the next step without further purification. This compound (149 mg, 380 µmol) was dissolved in dry MeCN:DMF (4 mL, 3:1) and 46a (82 mg, 350 µmol), Et3N (0.23 mL, 1.70 mmol) and DMAP (9 mg, 70 µmol) were added, and the mixture was heated and stirred at 80 °C under argon for 16 h. The reaction mixture was concentrated under vacuum and the residue was purified by chromatography column on silica gel (MeOH:EtOAc:CH2Cl2, 0.8:5:1) to give 48a (113 mg, 78%) as a white sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.50 – 8.30 (m, 4H, Py), 7.40 – 7.20 (m, 4H, 2H Py, 2H furan), 6.49 (br s, 2H, 2 NH-Py), 6.39 – 6.21 (m, 2H, furan), 6.15 – 6.03 (m, 2H, furan), 4.72 – 4.53 (m, 1H, CH ciclopentyl rotamer A), 4.38 (s, 4H, 2 CH2-furan), 4.28 – 4.09 (m, 1H, CH ciclopentyl rotamer B), 3.52 – 3.40 (m, 4H, 2 CH2- NH), 2.52 – 2.31 (m, 4H, 2 CH2-C=O), 1.91 – 1.21 (m, 30H, 6 CH2, 8 CH2 ciclopentyl, 2 NH-CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.6, 157.7, 152.1, 151.6, 150.4, 150.2, 145.7, 142.3, 141.4, 117.0, 115.5, 115.3, 110.7, 107.2, 58.9, 56.3, 42.2, 41.6, 38.8, 33.4, 33.1, 30.0, 29.8, 29.1, 28.3, 28.1, 26.0, 25.8, 23.8. ESI-HRMS m/z calcd for C23H31N6O2 [M+H]+, 423.2497; found, 423.2503. (E)-N-(4-Chlorobenzyl)-6-(2-cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2- ylmethyl)hexanamide (48b) Compound 48b was prepared according to the same procedure that was employed for 48a, except that 51b was used as starting material. Chromatography column (NH4OH:MeOH:EtOAc, 0.1:0.5:10). Yield: 70%, colourless sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.51 – 8.25 (m, 4H, Py), 7.43 – 7.18 (m, 10H, Chapter 1______________________________________________Experimental part 102 2H furan, 4H Ph, 4H Py), 7.16 – 6.94 (m, 4H, Ph), 6.51 – 6.36 (m, 2H, 2 NH-Py), 6.36 – 6.24 (m, 2H, furan), 6.19 – 6.10 (m, 2H, furan), 4.55 – 4.28 (m, 8H, 2 CH2-Ph, 2 CH2- furan), 3.51 – 3.41 (m, 4H, 2 CH2-NH), 2.58 (t, J = 6.7 Hz, 2H, CH2-C=O rotamer A), 2.35 (t, J = 6.7 Hz, 2H, CH2-C=O rotamer B), 1.80 – 1.30 (m, 14H, 6 CH2, 2 NH-CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.6, 157.7, 150.4, 150.2, 149.6, 145.7, 145.7, 143.0, 142.6, 135.6, 134.9, 133.7, 133.4, 129.3, 128.9, 127.8, 117.1, 115.6, 110.6, 109.1, 108.8, 50.8, 50.1, 47.9, 43.9, 42.2, 41.8, 32.8, 32.7, 28.5, 28.4, 26.2, 26.0, 23.5, 23.4. ESI-HRMS m/z calcd for C25H28ClN6O2 [M+H]+, 479.1949; found, 479.1957. (E)-6-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2-ylmethyl)-N-(3- morpholinopropyl)hexanamide (48d) Compound 48d was prepared according to the same procedure that was employed for 48a, except that 51d was used as starting material. Chromatography column (MeOH:Acetone:CH2Cl2, 0.4:1:1). Yield: 57%, white sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.43 – 8.31 (m, 4H, Py), 7.40 – 7.18 (m, 6H, 4H Py, 2H furan), 6.62 – 6.42 (m, 2H, 2 NH-Py), 6.36 – 6.22 (m, 2H, furan), 6.22 – 6.10 (m, 2H, furan), 4.49 – 4.37 (m, 4H, 2 CH2-furan), 3.70 – 3.57 (m, 8H, 4 CH2 morpholine), 3.48 – 3.26 (m, 8H, 4 CH2), 2.61 – 2.12 (m, 18H, 2 NH, 4 CH2 morpholine, 4 CH2), 1.72 – 1.50 (m, 12H, 6 CH2), 1.45 – 1.28 (m, 4H, 2 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.1, 157.7, 150.3, 150.3 145.5, 142.7, 142.1, 117.1, 115.4, 110.5, 108.4, 108.1, 66.9, 66.8, 55.0, 55.3, 53.6, 53.5, 45.3, 45.1, 44.5, 42.2, 42.0, 41.7, 32.7, 32.4, 28.5, 28.4, 26.1, 26.0, 25.3, 24.4, 23.6, 23.5. ESI-HRMS m/z calcd for C25H36N7O3[M+H]+, 482.2868; found, 482.2874. (E)-7-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-cyclopentyl-N-(furan-2- ylmethyl)heptanamide (49a) Chapter 1______________________________________________Experimental part 103 Compound 49a was prepared according to the same procedure that was employed for 48a, except that 38a was used as starting material. Chromatography column (MeOH:EtOAc:DCM, 0.8:5:1). Yield: 76%, white sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.50 – 8.30 (m, 4H, Py), 7.40 – 7.20 (m, 6H, 4H Py, 2H furan), 6.49 (br s, 2H, NH-Py), 6.39 – 6.21 (m, 2H, furan), 6.15 – 6.03 (m, 2H, furan), 4.82 – 4.64 (m, 1H, CH ciclopentyl rotamer A), 4.53 – 4.30 (m, 4H, 2 CH2-furan), 4.26 – 4.08 (m, 1H, CH ciclopentyl rotamer B), 3.52 – 3.40 (m, 4H, 2 CH2-NH), 2.52 – 2.31 (m, 4H, 2 CH2-C=O), 1.91 – 1.21 (m, 32H, 8 CH2, 8 CH2 ciclopentyl, 2 NH-CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 174.3, 173.6, 157.7, 152.1, 151.6, 150.4, 150.2, 145.7, 142.3, 141.4, 117.0, 115.5, 115.3, 110.7, 110.7, 107.2, 77.6, 77.2, 76.7, 58.9, 56.3, 42.2, 41.6, 38.8, 33.4, 33.0, 30.0, 29.8, 29.1, 28.3, 26.1, 26.0, 23.8, 23.3. ESI-HRMS m/z calcd for C24H33N6O2 [M+H]+, 437.2656; found, 437.2660. (E)-N-(4-Chlorobenzyl)-7-(2-cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2- ylmethyl)heptanamide (49b) Compound 49b was prepared according to the same procedure that was employed for 48a, except that 38b was used as starting material. Chromatography column (MeOH:EtOAc:DCM, 0.8:5:1). Yield: 64%, white sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.46 – 8.30 (m, 4H, Py), 7.42 – 7.20 (m, 10H, 2H furan, 4H Ph, 4H Py), 7.16 – 6.99 (m, 4H, Ph), 6.37 – 6.23 (m, 4H, 2H furan, 2 NH-Py), 6.21 – 6.09 (m, 2H, furan), 4.58 – 4.30 (m, 8H, 2 CH2-Ph, 2 CH2-furan), 3.49 – 3.35 (m, 4H, 2 CH2-NH), 2.57 (t, J = 6.9 Hz, 2H, CH2-C=O rotamer A), 2.35 (t, J = 6.9 Hz, 2H, CH2-C=O rotamer B), 1.77 – 1.23 (m, 18H, 8 CH2, 2 NH-CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.6, 157.5, 150.5, 149.9, 149.7, 145.9, 143.0, 142.5, 135.7, 135.0, 133.4, 129.4, 129.3, 128.9, 127.8, 116.9, 115.6, 110.6, 109.1, 108.7, 77.6, 77.2, 76.7, 50.1, 47.9, 43.9, 42.4, 41.8, 32.9, 28.6, 28.5, 28.3, 28.1, 26.1, 24.3. ESI-HRMS m/z calcd for C26H30ClN6O2 [M+H]+, 493.2107; found, 493.2113. Chapter 1______________________________________________Experimental part 104 (E)-7-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2-ylmethyl)-N-(4- nitrobenzyl)heptanamide (49c) Compound 49c was prepared according to the same procedure that was employed for 48a, except that 38c was used as starting material. Chromatography column (MeOH:EtOAc:DCM, 0.8:5:1). Yield: 96%, yellow sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.44 – 8.33 (m, 4H, Py), 8.24 – 8.04 (m, 4H, Py), 7.40 – 7.18 (m, 8H, 2H furan, 4H Ph), 6.41 – 6.23 (m, 4H, 2H furan, 2 NH-Py), 6.23 – 6.11 (m, 2H, furan), 4.75 – 4.35 (m, 8H, 2 CH2-Ph, 2 CH2-furan), 3.45 – 3.31 (m, 4H, 2 CH2-NH), 2.69 – 2.25 (m, 4H, 2 CH2-C=O), 1.82 – 1.29 (m, 18H, 8 CH2, 2 NH-CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.9, 173.7, 157.5, 150.1, 149.9, 149.3, 147.7, 147.4, 146.0, 145.0, 143.1, 142.7, 128.5, 127.1, 124.3, 123.9, 116.9, 116.8, 115.7, 110.7, 109.5, 109.1, 77.6, 77.4, 77.2, 76.7, 50.9, 48.5, 44.7, 42.5, 42.2, 33.0, 28.7, 28.5, 28.2, 26.2, 24.5. ESI-HRMS m/z calcd for C26H30N7O4 [M+H]+, 504.2346; found, 504.2354. (E)-7-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2-ylmethyl)-N-(3- morpholinopropyl)heptanamide (49d) Compound 49d was prepared according to the same procedure that was employed for 48a, except that 38d was used as starting material. Chromatography column (MeOH:EtOAc:NH4OH, 1:10:0.1→1:6:0.1). Yield: 61%, white sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.49 – 8.34 (m, 4H, Py), 7.42 – 7.22 (m, 6H, 4H Py, 2H furan), 6.36 – 6.26 (m, 2H, furan), 6.26 – 6.12 (m, 4H, furan, 2 NH-Py), 4.53 (s, 2H, CH2-furan rotamer A), 4.45 (s, 2H, CH2-furan rotamer B), 3.75 – 3.58 (m, 8H, 4 CH2 morpholine), 3.52 – 3.28 (m, 8H, 4 CH2), 2.53 – 2.22 (m, 16H, 4 CH2 morpholine, 4 CH2), 1.77 – 1.52 (m, 12H, 6 CH2), 1.52 – 1.22 (m, 10H, 2 NH, 4 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.4, 173.4, 157.6, 151.0, 150.6, 150.5, 150.3, 145.4, 142.8, 142.3, 117.0, 115.6, 115.6, 110.6, 110.6, 108.5, 108.2, 77.6, 77.2, 76.7, 67.0, 66.8, 56.1, 55.5, 53.8, 53.6, 50.8, 45.5, 45.2, 44.5, 42.4, 42.3, 41.9, 32.9, Chapter 1______________________________________________Experimental part 105 32.6, 28.5, 28.2, 28.0, 26.0, 25.9, 25.5, 24.4, 24.2, 24.0. ESI-HRMS m/z calcd for C26H38N7O3 [M+H]+, 496.3021; found, 496.3031. (E)-7-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2-ylmethyl)-N-(3-(pyrrolidin-1- yl)propyl)heptanamide (49e) Compound 49e was prepared according to the same procedure that was employed for 48a, except that 38e was used as starting material. Chromatography column (MeOH:DCM:NH4OH, 1:10:0.1→1:6:0.1). Yield: 82%, white sticky solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 8.36 – 8.23 (m, 4H, Py), 7.36 – 7.24 (m, 6H, 4H Py, 2H furan), 6.50 (br s, 2H, 2 NH-Py), 6.32 – 6.28 (m, 2H, furan), 6.24 – 6.20 (m, 2H, furan), 4.54 – 4.33 (m, 4H, 2 CH2-furan), 3.51 – 3.28 (m, 8H, 2 CH2-NH, 2 CH2-N- C=O) , 3.12 – 2.59 (m, 12H, 2 CH2-N, 4 CH2 pyrrolidine), 2.52 – 2.24 (m, 4H, 2 CH2- C=O), 2.04 – 1.89 (m, 6H, 3 CH2), 1.89 – 1.70 (m, 6H, 3 CH2), 1.69 – 1.47 (m, 8H, 4 CH2), 1.42 – 1.20 (m, 8H, 4 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 174.3, 173.2, 162.7, 157.2, 150.2, 149.7, 145.8, 142.9, 142.1, 116.9, 114.9, 110.6, 108.7, 77.5, 77.0, 76.6, 54.0, 53.8, 53.1, 52.8, 45.1, 43.2, 42.3, 41.8, 32.7, 31.5, 30.1, 29.7, 29.2, 28.8, 28.2, 26.0, 24.9, 24.4, 23.4, 23.2, 22.7, 14.1. ESI-HRMS m/z calcd for C26H38N7O2 [M+H]+, 480.3075; found, 480.3081. (E)-8-(2-Cyano-3-(pyridin-4-yl)guanidino)-N-(furan-2-ylmethyl)octanamide (50) The preparation of 50 from 46a (64 mg, 270 µmol) and 30 (70 mg, 300 µmol) followed the same procedure described previously for 47a (pag. 95). Chromatography column (MeOH:EtOAc, 1:10→1:5). Yield: 91%, white solid. 1H NMR (300 MHz, CD3OD, δ ppm) 8.43 – 8.35 (m, 2H, Py), 7.46 – 7.39 (m, 1H, furan), 7.39 – 7.31 (m, 2H, Py), 6.33 (dd, J = 3.1, 1.9 Hz, 1H, furan), 6.28 – 6.21 (m, 1H, furan), 4.34 (s, 2H, CH2-furan), 3.38 (t, J = 7.2 Hz, 2H, CH2-NH-C=N), 2.21 (t, J = 7.4 Hz, 2H, CH2-C=O), 1.70 – 1.53 (m, 4H, 2 CH2), 1.43 – 1.30 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CD3OD, δ ppm) 176.0 (C=O), 159.2 (C=N), 153.2 (qC, Py), 150.6 (2CH, Py), 148.4 (qC-furan), 143.3 (CH, furan), 117.8 Chapter 1______________________________________________Experimental part 112 Scheme 33. Synthesis of isoindoline-based inhibitors 62 and 63. Ethyl 4-(isoindoline-2-carboxamide)benzoate (60)54 Isoindoline (60 μL, 530 µmol) was dissolved in THF (5 mL) and cooled to 0 °C. Then, methyl 4-isocyanatobenzoate (84 mg, 440 µmol) was added and the resulting mixture was warmed to r.t. and stirred for 16h. The solution was concentrated, Et2O was added, and the resulting precipitate was filtered to provide 60 (119 mg, 88%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 7.98 (d, J = 8.3 Hz, 2H, Ph), 7.57 (d, J = 8.4 Hz, 2H, Ph), 7.35 – 7.23 (m, 4H, CH isoindoline), 6.65 (s, 1H, NH), 4.84 (s, 4H, 2 CH2, isoindonline), 4.34 (q, J = 7.1 Hz, 2H, OCH2CH3) 1.38 (t, J = 7.1 Hz, 3H, OCH2CH3). 13C NMR (75 MHz, CDCl3, δ ppm) 166.5 (C=O), 153.4 (C=O, urea), 143.5 (2 qC, isoindoline), 136.3 (qC, Ph), 130.9 (2 CH, Ph), 127.9 (2 CH, isoindoline), 124.8 (qC, Ph), 122.9 (2 CH, isoindoline), 118.4 (2 CH, Ph), 60.8 (OCH2CH3), 52.3 (2 CH2, isoindoline), 14.5 (OCH2CH3). NMR data match with those previously reported for this compound. 4-(Isoindoline-2-carboxamido)benzoic acid (61)32 Chapter 1______________________________________________Experimental part 113 Compound 60 (90 mg, 320 μmol) was dissolved in THF (2 mL), EtOH (1 mL) and H2O (0.5 mL), and LiOH (30 mg, 1.3 mmol) were added. The mixture was stirred at 70 °C for 2h and then the solvent was removed in vacuo. The obtained residue was dissolved in water, the pH was adjusted to 3 with HCl 2M and the resulting precipitate was filtered and washed with water to provide 61 (73 mg, 82%) as a white solid. 1H NMR (300 MHz, DMSO-d6, δ ppm) 12.53 (br s, OH), 8.68 (s, 1H, NH), 7.86 (d, J = 9.0 Hz, 2H, Ph), 7.73 (d, J = 9.0 Hz, 2H, Ph), 7.43 – 7.26 (m, 4H, CH isoindoline), 4.80 (s, 4H, CH2, isoindoline). 13C NMR (75.4 MHz, DMSO-d6, δ ppm) 167.6 (C=O), 154.0 (C=O, urea), 145.3 (2 qC, isoindoline), 137.1 (qC, Ph), 130.6 (2 CH, Ph), 127.8 (2 CH, isoindoline), 124.0 (qC, Ph), 123.3 (2 CH, isoindoline), 118.7 (2 CH, Ph), 52.4 (2 CH2, isoindoline). NMR data match with those previously reported for this compound. N-(4-((Furan-2-ylmethyl)carbamoyl)phenyl)isoindoline-2-carboxamide (62) Compound 61 (45 mg, 160 μmol), HOBt (32 mg, 240 μmol), furfurylamine (19 mg, 190 μmol) and DIPEA (110 μL, 640 μmol) were suspended in DMF (0.5 mL) and treated with EDCI (46 mg, 240 μmol). The mixture was stirred at r.t. overnight to give a solution that was diluted with 5 mL of ice water. The resulting turbid mixture was stirred at 0 oC for 1 h, then filtered and concentrated to yield 62 (52 mg, 90%) as an off-white solid.1H NMR (300 MHz, DMSO-d6, δ ppm) δ 8.76 (t, J = 5.8 Hz, 1H, NH amide), 8.58 (s, 1H, NH urea), 7.81 (d, J = 8.4 Hz, 2H, Ph), 7.67 (d, J = 8.4 Hz, 2H, Ph), 7.56 (d, J = 1.8 Hz, 1H, furan), 7.44 – 7.23 (m, 4H, isoindoline), 6.39 (br t, 1H, furan), 6.26 (br d, 1H, furan), 4.79 (s, 4H, CH2, isoindoline), 4.45 (d, J = 5.6 Hz, CH2-furan).13C NMR (75.4 MHz, DMSO-d6, δ ppm) δ 165.7 (C=O, amide), 153.6 (C=O, urea), 152.7 (qC, furan), 143.3 (qC, isoindoline), 141.9 (CH, furan), 136.7 (qC, Ph), 127.8 (2 CH, Ph), 127.3 (CH, isoindoline), 127.0 (qC, Ph), 122.7 (2 CH, isoindoline), 118.2 (2 CH, Ph), 110.4 (CH, furan) 106.7 (CH, furan), 51.9 (CH2, isoindoline), 35.9 (N-CH2). ESI-HRMS m/z calcd for C21H19N3O3Na [M+Na]+: 384.1324; found, 384.1313. Chapter 1______________________________________________Experimental part 114 N-(4-(Cyclopentyl(furan-2-ylmethyl)carbamoyl)phenyl)isoindoline-2-carboxamide (63) Compound 63 was prepared according to the same procedure that was employed for 62, except that 37a was used as starting material. Yield: 81%, off-white solid. 1H NMR (300 MHz, DMSO-d6, δ ppm) δ 8.52 (s, 1H, NH, urea), 7.65 (d, J = 8.2 Hz, 2H, Ph), 7.57 (d, J = 1.9 Hz, 1H, furan), 7.42 – 7.26 (m, 6H, Ph, isoindoline), 6.40 (t, J = 2.7 Hz, 1H, furan), 6.26 (d, J = 3.2 Hz, 1H, furan), 4.79 (s, 4H, CH2 Isoindoline), 4.52 (s, 2H, CH2- furan), 4.21 (br s, 1H, CH Cyclopentyl), 1.80 - 1.32 (m, 8H, 4 CH2 Cyclopentyl). 13C NMR (75.4 MHz, DMSO-d6, δ ppm) δ 171.4 (C=O, amide), 154.3 (C=O, urea), 152.9 (CH, furan), 142.2 (qC, Ph), 142.0 (CH, furan), 137.2 (qC, isoindoline), 130.6 (qC, Ph), 127.8 (CH, isoindoline), 127.4 (2 CH, Ph), 123.3 (CH, isoindoline), 119.2 (2 CH, Ph), 111.1 (CH, furan), 107.4 (CH, furan), 59.6 (CH, Cyclopentyl), 52.4 (CH2, Isoindoline), 40.4 (N- CH2), 29.5 (2 CH2, Cyclopentyl), 24.0 (2 CH2, Cyclopentyl). ESI-HRMS m/z calcd for C26H27N3O3Na [M+Na]+: 452.1950; found, 452.1940. Scheme 34. Synthesis of isoindoline-based inhibitors 65a, 65b and 65d. Phenyl (E)-N-cyanoisoindoline-2-carbimidate (64) Isoindoline (60 μL, 530 μmol) was added to a solution of diphenylcyanocarbonimidate (130 mg, 530 μmol) in propan-2-ol (1.2 mL). The reaction mixture was stirred under Chapter 1______________________________________________Experimental part 115 nitrogen for 3 h at r.t. The precipitate was filtered, washed with propan-2-ol and dried to yield 64 (102 mg, 73%) as a white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 7.48 – 7.12 (m, 9H, 5H Ph, 4H isoindoline), 5.20 (s, 2H, isoindoline), 5.01 (s, 2H, isoindoline). 13C NMR (75.4 MHz, CDCl3, δ ppm) 157.0 (qC, Ph), 151.8 (C=N), 134.9 (2 qC, isoindoline), 130.0 (2 CH, Ph), 128.4 (2 CH, isoindoline), 126.5 (CH, Ph), 122.8 (2 CH, isoindoline), 120.4 (2 CH, Ph), 113.7 (C≡N), 54.3 (2 CH2 isoindoline). ESI-HRMS m/z calcd for C16H13N3ONa [M+Na]+, 286.0951; found, 286.0951. (E)-7-(N'-Cyanoisoindoline-2-carboximidamido)-N-cyclopentyl-N-(furan-2- ylmethyl)heptanamide (65a) Compound 65a was prepared according to the same procedure that was employed for 48a (pag. 101), except that 38a and 64 were used as starting materials. Chromatography column (EtOAc:Cy, 2:1). Yield: 36%, white solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.42 – 7.17 (m, 10H, 8H isoindoline, 2H furan), 6.29 (br d, 2H, furan), 6.14 (br s, 2H, furan), 5.36 (br s, 1H, NH-CH2 rotamer A), 5.25 (br s, 1H, NH-CH2 rotamer B), 4.90 (s, 8H, 4 CH2 isoindoline), 4.81 – 4.61 (m, 1H, CH cyclopentyl rotamer A), 4.48 – 4.33 (m, 4H, 2 CH2-furan), 4.27 – 4.11 (m, 1H, CH cyclopentyl rotamer B), 3.62 – 3.44 (m, 4H, 2 CH2-NH), 2.49 – 2.28 (m, 4H, 2 CH2-C=O), 1.91 – 1.28 (m, 32H, 8 CH2 cyclopentyl, 8 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.8, 173.2, 156.0, 152.7, 152.1, 142.1, 141.1, 135.5, 128.0, 122.7, 118.0, 110.6, 107.3, 107.1, 103.3, 77.6, 77.2, 76.7, 58.8, 56.3, 53.7, 43.0, 42.7, 41.9, 38.5, 33.7, 33.5, 30.1, 29.9, 29.1, 28.7, 28.4, 26.4, 26.0, 25.1, 23.9. ESI-HRMS m/z calcd for C27H36N5O2 [M+H]+, 462.2860; found, 462.2864. (E)-N-(4-Chlorobenzyl)-7-(N'-cyanoisoindoline-2-carboximidamido)-N-(furan-2- ylmethyl)heptanamide (65b) Compound 65b was prepared according to the same procedure that was employed for 48a (pag. 101), except that 38b and 64 were used as starting materials. Chapter 1______________________________________________Experimental part 116 Chromatography column (EtOAc:Cy, 2:1). Yield: 42%, white solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.40 – 7.02 (m, 18H, 8H Ph, 8H isoindoline, 2H furan), 6.41 – 6.25 (m, 2H, furan), 6.25 – 6.11 (m, 2H, furan), 5.17 – 5.01 (m, 2H, NHCH2), 4.89 (s, 8H, 4 CH2 isoindoline), 4.58 – 4.29 (m, 8H, 2 CH2-Ph, 2 CH2-furan), 3.66 – 3.44 (m, 4H, CH2-NH), 2.55 (t, J = 7.3 Hz, 2H, CH2-C=O rotamer A), 2.35 (t, J = 7.3 Hz, 2H, CH2-C=O rotamer B), 1.83 – 1.27 (m, 16H, 8 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.4, 156.0, 150.9, 150.0, 142.9, 142.4, 136.1, 135.4, 135.3, 133.6, 133.3, 129.6, 129.2, 128.9, 128.1, 127.9, 122.7, 117.9, 110.5, 108.9, 108.5, 77.6, 77.2, 76.7, 53.7, 50.1, 47.7, 43.8, 43.1, 41.6, 33.0, 30.0, 28.8, 28.6, 26.4, 26.3, 25.0. ESI-HRMS m/z calcd for C29H33ClN5O2 [M+H]+, 518.2314; found, 518.2317. (E)-7-(N'-Cyanoisoindoline-2-carboximidamido)-N-(furan-2-ylmethyl)-N-(3)- morpholinopropyl)heptanamide (65d) Compound 65d was prepared according to the same procedure that was employed for 48a (pag. 101), except that 38d and 64 were used as starting materials. Chromatography column (NH4OH:MeOH:EtOAc, 0.1:1:10). Yield: 52%, colourless oil. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.42 – 7.22 (m, 10H, 8H isoindoline, 2H furan), 6.36 – 6.26 (m, 2H, furan), 6.26 – 6.15 (m, 2H, furan), 5.15 – 4.99 (m, 2H, 2 NH-CH2), 4.90 (s, 8H, isoindoline), 4.56 (s, 2H, CH2-furan rotamer A), 4.45 (s, 2H, CH2-furan rotamer B), 3.77 – 3.64 (m, 8H, 4 CH2 morpholine), 3.62 – 3.49 (m, 4H, 2 CH2-NH), 3.44 – 3.29 (m, 4H, 2 CH2), 2.54 – 2.23 (m, 16H, 4 CH2 morpholine, 4 CH2), 1.78 – 1.53 (m, 8H, 4 CH2), 1.52 – 1.30 (m, 8H, 4 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.2, 173.0, 156.0, 151.5, 150.7, 142.7, 142.1, 135.4, 128.1, 122.8, 117.8, 110.6, 108.4, 108.1, 77.6, 77.2, 76.7, 67.1, 67.0, 56.3, 55.6, 53.8, 53.7, 50.9, 45.4, 45.2, 44.5, 43.1, 43.0, 41.6, 33.1, 32.9, 30.0, 29.9, 28.7, 26.3, 26.3, 25.6, 25.1, 25.0, 24.5. ESI-HRMS m/z calcd for C29H41N6O3 [M+H]+, 521.3239; found, 521.3235. Chapter 1______________________________________________Experimental part 117 Scheme 35. Phenyl-based inhibitors 67, 68a-c. Phenyl (Z)-N'-cyano-N-phenylcarbamimidate (66a)55 Aniline (0.2 mL, 2.2 mmol) was added to a solution of diphenyl N-cyanocarbonimidate (524 mg, 2.20 mmol) in propan-2-ol (5 mL). The reaction mixture was stirred under nitrogen for 2 h at r.t. The precipitate was filtered, washed with propan-2-ol and dried to yield 66a (450 mg, 75%) as a white solid. 1H NMR (300 MHz, DMSO-d6, δ ppm) 10.84 (s, 1H), 7.55-7.36 (m, 6H), 7.35 – 7.21 (m, 4H). NMR data match with those previously reported for this compound. Ethyl (Z)-4-(((cyanoimino)(phenoxy)methyl)amino)benzoate (66b) Compound 66b was prepared according to the same procedure that was employed for 66a, except that ethyl 4-aminobenzoate was used as starting material. Yield: 35%, white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 9.11 (s, 1H, NH), 8.05 (d, J = 8.6 Hz, 2H, Ph), 7.53 – 7.28 (m, 5H, OPh), 7.15 (d, J = 8.6 Hz, 2H, Ph), 4.37 (q, J = 7.1 Hz, 2H, CH2CH3), 1.39 (t, J = 7.1 Hz, 3H, CH3CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 165.9 (C=O), 151.0 (C=N), 139.6 (CN), 130.9 (2CH, Ph), 130.0 (2CH, OPh), 128.18 (qC, Ph), 127.22 (2CH, OPh), 122.01 (2C, qC, CH OPh), 121.45 (2CH, Ph), 61.26 (CH2CH3), 14.45 Chapter 1______________________________________________Experimental part 118 (CH2CH3). ESI-HRMS m/z calcd for C17H15N3O3Na [M+Na]+, 332.1004; found, 332.1006. Phenyl (Z)-N-(4-azidophenyl)-N'-cyanocarbamimidate (66c) Compound 66c was prepared according to the same procedure that was employed for 66a, except that ethyl 4-azidoaniline hydrochloride was used as starting material. Yield: 67%, light-brown solid. 1H NMR (300 MHz, CDCl3, δ ppm) 8.59 (s, 1H, NH), 7.50 – 7.28 (m, 5H, OPh), 7.13 (d, J = 7.8 Hz, 2H, Ph), 7.04 (d, J = 8.8 Hz, 2H, Ph). 13C NMR (75.4 MHz, CDCl3, δ ppm) 151.0 (2C, C=N, qC OPh), 138.6 (C≡N), 132.1 (2qC, Ph), 129.9 (2CH, OPh), 127.2 (CH, OPh), 124.9 (2CH, OPh), 121.5 (2CH, Ph), 119.9 (CH, Ph). ESIHRMS m/z calcd for C14H10N6ONa [M+Na]+, 301.0809; found, 301.0808. (E)-7-(2-Cyano-3-phenylguanidino)-N-cyclopentyl-N-(furan-2- ylmethyl)heptanamide (67) Compound 67 was prepared according to the same procedure that was employed for 48a (pag. 101), except that and 66a was used as starting material. Chromatography column (EtOAc:Cy, 1:1→2:1). Yield: 80%, white solid. 1H NMR (300 MHz, CDCl3, δ ppm, mixture of rotamers) 7.60 – 7.16 (m, 14H, 10H Ph, 2H furan, 2 NH-Ph), 6.29 (br d, 2H, furan), 6.13 (br s, 2H, furan), 5.02 (s, 2H, 2 NH-CH2), 4.80 – 4.60 (m, 1H, CH cyclopentyl rotamer A), 4.38 (br d, 4H, 2 CH2-furan), 4.28 – 4.06 (m, 1H, CH cyclopentyl rotamer B), 3.33 – 3.17 (m, 4H, 2 CH2-NH), 2.47 – 2.23 (m, 4H, 2 CH2-C=O), 1.89 – 1.15 (m, 32H, 8 CH2 cyclopentyl, 8 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm, mixture of rotamers) 173.7, 173.0, 158.8, 152.7, 152.2, 142.1, 141.0, 135.6, 130.2, 127.4, 125.5, 118.0, 110.6, 107.5, 107.0, 77.6, 77.2, 76.7, 58.7, 56.2, 41.9, 41.8, 38.4, 33.7, 33.5, 30.0, 29.1, 29.0, 28.7, 26.4, 25.0, 23.9. ESI-HRMS m/z calcd for C25H34N5O2 [M+H]+, 436.2707; found, 436.2707. (E)-6-(2-Cyano-3-phenylguanidino)-N-cyclopentyl-N-(furan-2-ylmethyl)hexane-1- sulfonamide (68a) Chapter 1______________________________________________Experimental part 119 Compound 68a was prepared according to the same procedure that was employed for 47a (pag. 95), except that 66a was used as starting material. Chromatography column (EtOAc:Cy, 1:1→2:1). Yield: 96%, white solid. 1H NMR (300 MHz, CDCl3, δ ppm) 7.52 – 7.16 (m, 7H, 5H Ph, 1H furan, NH-Py), 6.41 – 6.21 (m, 2H, furan), 4.89 (t, J = 5.8 Hz, 1H, NH-CH2), 4.35 (s, 2H, CH2-furan), 4.13 (quint, J = 8.3 Hz, 1H, CH cyclopentyl), 3.31 – 3.18 (m, 2H, CH2-NH), 2.80 – 2.67 (m, 2H, CH2SO2), 1.95 – 1.41 (m, 12H, 8H cyclopentyl, 2 CH2), 1.41 – 1.17 (m, 4H, 2 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 158.9 (C=N), 151.5 (qC, furan), 142.2 (CH, furan), 135.4 (qC, Ph), 130.3 (2CH, Ph), 127.8 (CH, Ph), 125.8 (2CH, Ph), 118.0 (CN), 110.8 (CH, furan), 109.2 (CH, furan), 59.3 (CH, cyclopentyl), 53.3 (CH2SO2), 41.8 (CH2-NH), 40.0 (CH2-furan), 30.0 (2 CH2, cyclopentyl), 29.1 (CH2), 27.9 (CH2), 26.2 (CH2), 23.6 (2 CH2, cyclopentyl), 23.3 (CH2). ESI-HRMS m/z calcd for C24H33N5O3SNa [M+Na]+, 494.2196; found, 494.2196. Ethyl(E)-4-(2-cyano-3-(6-(N-cyclopentyl-N-(furan-2- ylmethyl)sulfamoyl)hexyl)guanidino)benzoate (68b) Compound 68b was prepared according to the same procedure that was employed for 47a (pag. 95), except that 66b was used as starting material. Chromatography column (EtOAc:Cy, 1:2→1:1). Yield: 95%, pale-yellow solid. 1H NMR (300 MHz, CDCl3, δ ppm) 8.06 (d, J = 8.6 Hz, 2H, Ph), 7.88 (s, 1H, NH-Ph), 7.38 – 7.34 (m, 1H, furan), 7.30 (d, J = 8.6 Hz, 2H, Ph), 6.36 – 6.25 (m, 2H, furan), 5.24 (br s, 1H, NH-CH2), 4.43 – 4.30 (m, 4H, CH2CH3, CH2-furan), 4.13 (quint, J = 8.4 Hz, 1H, CH cyclopentyl), 3.30 (q, J = 6.8 Hz, 2H, CH2-NH), 2.79 – 2.70 (m, 2H, CH2SO2), 1.92 – 1.45 (m, 12H, 8H cyclopentyl, 2 CH2), 1.45 – 1.20 (m, 7H, CH3CH2, 2 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 165.7 (C=O), 158.2 (C=N), 151.4 (qC, furan), 142.2 (CH, furan), 140.2 (qC, Ph) 131.5 (2CH, Ph), 128.5 (qC, Ph), 123.5 (2CH, Ph), 110.8 (CH, furan), 109.2 (CH, furan), 61.4 (CH2CH3), 59.3 (CH, cyclopentyl), 53.2 (CH2SO2), 42.1 (CH2-NH), 40.0 (CH2-furan), 30.0 (2CH2, cyclopentyl), 29.0 (CH2), 27.8 (CH2), 26.2 (CH2), 23.6 (2CH2, cyclopentyl), 23.3(CH2), 14.4 (CH3CH2). ESI-HRMS m/z calcd for C27H37N5O3SNa [M+Na]+, 566.2400; found, 566.2408. Chapter 1______________________________________________Experimental part 120 (E)-6-(3-(4-Azidophenyl)-2-cyanoguanidino)-N-cyclopentyl-N-(furan-2- ylmethyl)hexane-1-sulfonamide (68c) Compound 68c was prepared according to the same procedure that was employed for 47a (pag. 95), except that 66c was used as starting material. Chromatography column (EtOAc:Cy, 1:2→1:1). Yield: 84%, light-red solid. 1H NMR (300 MHz, CDCl3, δ ppm) 7.64 (s, 1H, NH-Ph), 7.35 (dd, J = 1.9, 0.9 Hz, 1H, furan), 7.23 (d, J = 8.7 Hz, 2H, Ph), 7.06 (d, J = 8.7 Hz, 2H, Ph) 6.40 – 6.16 (m, 2H, furan), 4.89 (br s, 1H, NH-CH2), 4.35 (s, 2H, CH2-furan), 4.13 (quint, J = 8.5 Hz, 1H, CH, cyclopentyl), 3.24 (q, J = 6.8 Hz, 2H, CH2-NH), 2.80 – 2.69 (m, 2H, CH2SO2), 1.95 – 1.41 (m, 12H, 8H cyclopentyl, CH2), 1.41 – 1.08 (m, 6H, 3 CH2). 13C NMR (75.4 MHz, CDCl3, δ ppm) 159.0 (C=N), 151.5 (qC, furan), 142.2 (CH, furan), 139.6 (C≡N), 132.1 (qC, Ph), 127.4 (2CH, Ph), 120.7 (2CH, Ph), 118.0 (qC, Ph), 110.8 (CH, furan), 109.2 (CH, furan), 59.3 (CH, cyclopentyl), 53.2 (CH2SO2), 41.9 (CH2-NH), 40.0 (CH2-furan), 30.0 (2CH2, cyclopentyl), 29.8 (CH2), 29.1 (CH2), 27.9 (CH2), 26.2 (CH2, cyclopentyl), 23.6 (CH2, cyclopentyl), 23.3 (CH2). ESIHRMS m/z calcd for C24H32N8O3SNa [M+Na]+, 535.2209; found, 535.2210. ____________________________________________________________Chapter 2 121 2. SYNTHESIS OF NEW INNOVATIVE PAYLOADS AND GLUTATHIONE-SENSITIVE LINKERS FOR ANTIBODY-DRUG CONJUGATES (ADCS) Chapter 2__________________________________________________Introduction 128 Mylotarg® and Besponsa®, reached the market in 2000 and 2017 respectively, with the first one being withdrawn (2010) because of an increase in treatment-related deaths compared to the control group receiving normal chemotherapy. 2.1.5.2.1.2 Glutathione-sensitive disulfide linkers ADCs that present disulfide-linkers undergo nucleophilic attack by thiol-containing molecules such as glutathione (GSH) (Figure 28A), which concentration is much higher in the cytosol (ca. 10 mmol/L) than in the extracellular environment (ca. 10 µmol/L).68 This difference between the intracellular and blood (extracellular) concentration of GSH, as well as the oxidative stress, contributes to the preferential release of the drug inside cancer cells. The most representative payload associated with disulfide linkers is Maytansine, a tubulin disruptor (Figure 28B). The steric hindrance on carbon atoms adjacent to the disulfide bond has been reported to impact the overall resistance to the reduction and thus the plasma stability.69 Compound SAR-3419 (Figure 28B), an ADC containing maytansine as payload and a disulfide linker with gem-dimethyl groups, presented the perfect balance of plasma stability, high rate of in situ drug release, and cytotoxic activity.70,71 Figure 28. A) Schematic structure reactivity of glutathione-sensitive linkers. B) Chemical structure of SAR-3419 (R= H, R1=R2= CH3), an ADC containing maytansine as payload and a disulfide linker. Chapter 2__________________________________________________Introduction 129 2.1.5.2.2 Enzymatically cleavable linkers To effectively release the drug in the cancer cell, the proteome of the lysosome has been targeted to exploit the high concentration of the enzymes able to cleave the linkers and release the drug. These peptide-based linkers can be divided into Cathepsin B-, β-Glucuronidase-, Phosphatase-, Pyrophosphatase-, β-Galactosidase- and Sulfatase-sensitive linkers. 2.1.5.2.2.1 Cathepsin B-sensitive linker Cathepsin-B is a lysosomal cysteine protease that has been found to be overexpressed in several human cancers. The very first use of this strategy was the incorporation of highly cytotoxic anti-cancer agents such as Doxorubicin (DOX), Paclitaxel and Mitomycin C (MCC) conjugated to PABC (p-aminobenzyl carbamate) and a dipeptide linker (Scheme 36).72 The PABC self-immolative group serves as linker between the drug and the cleavable amide group. The faster drug release combination was given when there was a protonable/hydrophilic residue in AA1 and a hydrophobic residue in AA2 position. Scheme 36. Doxorubicin-released mechanism via Cathepsin B-sensitive dipeptide linker. The most widely used dipeptide linker is Val-Cit with five FDA-approved ADCs on the market and 25 in clinical trials in 2021.63 With nine ADCs in clinical trials, Val-Ala has Chapter 2__________________________________________________Introduction 130 also drawn attention recently due to the first FDA approval of Val-Ala-based ADC Zynlonta® in 2021. Numerous comparisons have been described in the literature between Val-Cit and Val-Ala based ADCs.60,73,74 Most reports highlight the benefits of using Val-Ala over Val-Cit, mainly due to the lower ADC aggregation (non-specific interactions arising from attached drug and linker moieties on the mAb).75,76 2.1.5.2.2.2 β-Glucuronidase-sensitive linker β-glucuronidases are hydrolytic enzymes that have been found mainly in the lysosome, with an increased activity in tumour tissues respect to healthy ones.77 Remarkably, these enzymes are inactive (10% of activity) at physiological pH and present their optimal activity at pH 4, which is the pH of the lysosome.78 The main driving forces that have led chemists to design efficient β-glucuronidase cleavable linkers for prodrugs and ADCs are their abundance in the lysosome and in the tumor microenvironment as well as their strong plasma stability.79,80 The hydrophilic nature of the β-glucuronide linker is ideal for highly hydrophobic payloads, which suffer from ADC aggregation. Due to the hydrophilicity, the lower aggregation phenomena, better clearance and solubility than dipeptide-based linkers, the use of βglucuronidase-sensitive linkers is considered an excellent strategy for target drug delivery (Scheme 37).81,82 Scheme 37. Enzymatic hydrolysis of the glycosidic bond of β-glucuronidase-sensitive linker is followed by the spontaneous decomposition of the spacer group, leading to the release of the active payload. Chapter 2__________________________________________________Introduction 131 2.1.6 Cytotoxic Payloads Two types of cytotoxic molecules have been used for the synthesis of clinically approved ADCs, microtubule-disrupting drugs and DNA damaging agents. Recently, another class of cytotoxic agents are emerging as new payloads for ADCs, such as apoptosis inducer molecules like amatoxins and NAMPT inhibitors.83 As several studies pointed out that only 1-2% of injected ADCs reached the tumor site,84,85 more potent payloads (IC50 of 10-10-10-12 M) are needed when designing an ADC. Payloads should also have low molecular weight to reduce immunogenicity and should be synthetically accessible to allow derivatization with the linker while retaining their potency. Furthermore, an adequate balance between aqueous solubility and stability as conjugate in aqueous formulations are important physico-chemical requirements for a payload in order to avoid antibody aggregation.56 2.1.6.1 Microtubule-disrupting drugs Tubulin inhibitors constitute more than 70% of the payloads in clinical ADCs, being auristatins and maytansinoids the two main families of compounds. 2.1.6.1.1 Auristatins Auristatins are synthetic analogues of linear pentapeptide Dolastatin 10 (Figure 29), isolated from marine mollusc Dolabella auricularia.86 Dolastatin 10 strongly inhibits the microtubule polymerization and tubulin-dependent GTP hydrolysis, which causes cell cycle arrest in the G2/M phase and apoptosis.87,88 Chapter 2__________________________________________________Introduction 132 Figure 29. Structure of Dolastatin 10 and its key building blocks. Although Dolastatin 10 was advanced into human clinical trials due to its high cytotoxicity in several human cancer cell lines, no significant therapeutic effect was achieved mainly due to its toxicity at maximum tolerated dose (MTD). Several research groups, both in academia and industry performed Structure-Activity Relationship (SAR) studies for the use of these compounds as potent anti-cancer agents.89 Miyazaky et al. determined some of the structural requirements that have been used as key points for synthetic modifications of Dolastatin 10 (Figure 30).90 Noteworthy are the two analogues of auristatins (MMAE and MMAF)91,92 developed by Seattle Genetics (Doronina et al.), which are currently being employed as payloads in six commercially-available ADCs in the market and more than twenty in clinical studies.57,58,93 Chapter 2__________________________________________________Introduction 133 Figure 30. Chemical structures of key auristatin analogues.94 Adcetris® (SGN-35 or Brentuximab vedotin, Figure 31), the first auristatin-based FDA- approved ADC in 2011, incorporates MMAE to a maleimidocaproyl-Val-Cit-PABC linker that is bioconjugated to a chimeric IgG1 anti-CD-30 mAb through a cysteine residue.95 After ADC internalization, cathepsin B triggers the release of the PABCMMAE intermediate that immediately release the drug by a spontaneous 1,6- elimination. The released MMAE kills the targeted cells and diffuses across the cell membrane to reach and destroy other CD30-positive as well as CD30-negative cancer cells, a phenomenon called the bystander killing effect. As a result, cancer cells that do not express the targeted antigen will also be killed.96–98 Figure 31. Chemical structure of commercially approved anti-CD30 Brentuximab vedotin (Adcetris®). Chapter 2__________________________________________________Introduction 134 2.1.6.1.2 Maytansinoid derivatives Maytansine and its derivatives (Maytansinoids, Figure 32A) are benzoansamacrolide antibiotics that were originally isolated from the bark of the Ethiopian shrubs Maytenus ovatus and Maytenus serrata.99 Like auristatins, they share the same tubulin binding site and mechanism of action as vinca alkaloids by depolymerizing microtubules and arresting cells in the mitosis stage (G2/M phase). As maytansine does not present any suitable chemical handle for linker conjugation, several sulfhydryl-containing analogues were prepared.100 DM1 (Mertansine or Emtansine) and DM4 (Ravtansine or Soravtansine) are examples of new synthetic maytansinoids that have been employed as payloads in numerous ADCs (i.e. Kadcyla®, Figure 32B). At present, there are ca. ten maytansinoid-containing ADCs in clinical trials, with seven of them in phase II/III.101–106 Figure 32. A) Chemical structures of Maytansinoids; B) Chemical structure of commercially approved Kadcyla®. Chapter 2__________________________________________________Introduction 135 2.1.6.2 DNA damaging agents 2.1.6.2.1 Calicheamicin Calicheamicins are natural compounds originally isolated from the fermentation broth of a soil microorganism called Micromonosporta echinospora.107 The first generation of ADCs represented by Mylotarg® and Besponsa® (Figure 33A) are the only examples using Calicheamicin as payload. After ADC internalization, the payload release proceeds in two steps: 1) the hydrazone bond is cleaved at endosomal acidic pH, 2) intracellular glutathione-promoted disulfide reduction releases the drug. The thiolate Calicheamicin analogue that is released undergoes a Bergman cyclization,108,109 generates a highly reactive diradical compound that induces doublestrand DNA cleavage, leading cancer cells to death (Figure 33B).110 Figure 33. A) Schematic representation of the 1st generation FDA-approved ADCs, anti-CD33 Mylotarg®, and anti-CD22 Besponsa®; B) Bergman cyclization followed by DNA damage mechanism. Chapter 2__________________________________________________Introduction 136 2.1.6.2.2 Camptothecins Camptothecin (CPT) is a Topoisomerase I inhibitor that functions as a roadblock for the replication fork resulting in DNA double-strand cleavage, which leads to cell death.111,112 Structure-Activity Relationship studies indicated that the carboxylic acid of the CPT presented less antitumor activity than its lactone form (Figure 34A). The inability to resolve the water-insolubility of CPT lactone limited its use as anti-cancer agent and clinical studies were discontinued. However, the water-soluble form of CPT, Irinotecan (Figure 34B) was approved for metastatic colorectal cancer. Its active metabolite, SN38 is generated in vivo through the cleavage of the carbamate bond by human liver carboxylesterase. Figure 34. A) Chemical structure of Camptothecin in its lactone active form in equilibrium with CPT in its carboxylate inactive form; B) in vivo transformation of Irinotecan into SN-38. Two different ADC strategies were adopted towards the stabilization of the lactone form of CTP analogues. The first one led to the approval of Enhertu® in 2019 that incorporated Exathecan (10-fold more potent against ovarian cancer than SN-38) as payload.113,114 The cyclohexane ring that bridges the 7 and 9 position confers the necessary rigidity to favour the equilibrium towards the lactone form, while the amino group contributes to increase the overall water solubility. The payload is Chapter 2__________________________________________________Introduction 137 connected to a tetrapeptide enzyme-sensitive linker through an amino methylene self-immolative spacer. Once the ADC is internalized, the cleavage of the linker releases a hemiaminal intermediate that rapidly gives the toxin and methylene imine as product (Figure 35).115 Figure 35. Enhertu® is composed of an anti-HER2 Trastuzumab conjugated to Exathecan via a tetrapeptide protease-sensitive linker (Gly-Gly-Phe-Gly). The second strategy implied the attachment of metabolite SN-38 to the linker through a cleavable carbonate bond. Conjugation of anti-TROP-2 antibody afforded ADC IMMU-132, approved by the FDA in 2020 and commercialized as Trodelvy®.116 Scientists at Immunomedics envisioned two different linker attachments on the active metabolite of Irinotecan, SN-38 and came out with an idea based on a previous study.117 IMMU132 is a result of the attempt of stabilising the lactone form of SN-38 by connecting the C-20 hydroxyl group through a carbonate bond, thus leaving the lactone ring intact while coupled to the linker (Figure 36).118,119 Chapter 2__________________________________________________Introduction 144 Currently, in the case of Cys-modification of proteins, maleimides are preferred as chemical handles in the linker because of their unique reactivity in thiol-Michael additions.144 The two electron-withdrawing groups and the strain of the double bond decrease the energy of the LUMO (lowest unoccupied molecular orbital) synergistically resulting in a potent electrophile towards nucleophilic attack. As a result, maleimides are more reactive Michael acceptors than vinyl sulfones or acrylates.145–148 Nevertheless, maleimide-based conjugation faces its own challenges. The resulting thiosuccinimide thioether (ADC A) is in slow equilibrium with the corresponding free maleimide-payload (B) (Scheme 39).149 An exogenous thiol in plasma, such as the Cys-34 residue of the abundant albumin, can react with B leading to the new bioconjugate C. As a consequence, the original ADC A loses the efficacy because of the partial release of cytotoxic drug before reaching the target cell, which increases off-target toxicities and jeopardizes the overall ADC efficacy.150 Scheme 39. Loss of efficacy of maleimide-based ADCs.149 2.1.7.3 New linker technologies Due to the limitations shown by maleimide, there is an urgent need to find innovative linkers that are more stable in plasma during circulation, thus positively impacting the overall ADC pharmacokinetic profile. Here below, several new linkers are summarized. Chapter 2__________________________________________________Introduction 145 2.1.7.3.1 Next-Generation Maleimides (NGM) 2.1.7.3.1.1 N-Aryl maleimides Dimasi et al.151 demonstrated that substitution of the alkyl chain of a maleimide moiety with an aryl group increased thiol addition and thiosuccinimide hydrolysis by increasing the electrophilicity of the maleimide. The resonance structures of the aryl and alkyl thiosuccinimides highlight the character of the partial double bond that shifts from the amide to the aryl moiety, thus increasing the electrophilicity of the carbonyl group. As a result, the aryl thiosuccinimide moiety is more prone to hydrolysis, yielding a more stable intermediate without loss of drug (Scheme 40). Scheme 40. Hydrolysis of N-alkyl vs N-aryl thiosuccinimide derivatives. 2.1.7.3.1.2 -amino maleimides A strategy based on the incorporation of an amino group close to the carbonyl maleimide moiety to promote an intramolecular-catalysed hydrolysis of the imide function (Figure 42A) has been developed at Genentech company. This catalysis leads to the fast formation of the stable succinamic acid thioether. An ADC prepared with such linker and monomethyl auristatin E (MMAE) as payload, led to improved ADC efficacy and better tolerability in in vivo studies (Figure 42B).152 Chapter 2__________________________________________________Introduction 146 Figure 42. Impact of intramolecular base catalysis in the hydrolysis of the thiosuccinimide moiety. 2.1.7.3.2 Non-classic bioconjugation systems Although, the next generation maleimides are tackling the major drawbacks of classic maleimides, new site-specific bioconjugation reactions are needed to improve the stability and integrity of the ADC. Recently, several approaches using heteronorbornadiene-based linkers have been described and are summarized in the next section. 2.1.7.3.2.1 Oxanorbornadienes (ONDs) Finn and co-workers described the selective conjugate addition of 2-mercaptoethan- 1-ol to a 7-oxanorbornadienedicarboxylate (OND) to generate an oxanorbornenic adduct that suffered a spontaneous retro Diels-Alder reaction (rDA) reaction to give furan derivatives and thiomaleates (Scheme 41).153 Chapter 2__________________________________________________Introduction 147 Scheme 41. Conjugate addition of thiol to ONDs and subsequent rDA reaction. To verify whether the ONDs might have an application in the field of thiol-sensitive linkers for targeted drug delivery, functionalized electron-deficient oxanorbornadiene reagents were synthesized to study their ability to release the cargo once they were bioconjugated to circulating albumin.154 Albumin, which accounts for 90% of plasma thiols in mammals, is an attractive carrier that binds covalently small molecules in vivo. In this regard, Finn et al. described the first covalent attachment of ONDs to circulating albumin in rodents to modulate the kinetic of the release of gadolinium cargos. The substituents on the bicyclic system (R and R’) helped tuning the rate at which the drug was released into the blood (Figure 43). Figure 43. Release of a Gadolinium derivative from an OND conjugated to albumin. Chapter 2__________________________________________________Introduction 148 2.1.7.3.2.2 Azanorbornadienic vinyl sulfones Recently, our research group has developed several [2.2.1]azabicyclic vinyl sulfones as versatile reagents for dual bioorthogonal labelling of proteins.155 These systems present the two essential requirements for this purpose: 1) they are Michael acceptors for the bioconjugation with cysteine residues and, 2) they present an anchor point (N bridge) for the incorporation of an affinity probe. First, the authors used the modified ubiquitin protein (Ub-K63C), which contains a free cysteine residue, to make it react with a biotinylated azabicyclic vinyl sulfone. It was observed that, when the cysteine residue of Ub-K63C is bioconjugated to the azanorbornadiene and forms the corresponding azanorbornene, a spontanous rDA reaction takes place with the release of the Biotin-containing pyrrole (Scheme 42). Scheme 42. Bioconjugation of Ub-K63C to an azanorbornadienic vinyl sulfone and subsequent spontaneous rDA reaction. In this work, our research group also carried out the reaction of the C2Am protein with a biotin-containing azanorbornadiene (Scheme 43). The azanorborneneconjugate formed was stable enough to react with fluorogenic tetrazines via a bioorthogonal inverse Electron Demand Diels-Alder (iEDDA) reaction. As a result, a stable compound was formed in which the spontaneous rDA reaction did not occur. Therefore, this azanorbornadienic system can be considered as a versatile tool for dual chemical labelling, allowing the incorporation of two biologically relevant molecules, an affinity probe (biotin) and a fluorogenic probe (Cyanine, Cy3).155 Chapter 2__________________________________________________Introduction 149 Scheme 43. Dual labelling of protein C2Am by using azanorbornadienic vinyl sulfones as bioconjugation tool. The above mentioned Oxanorbornadienes (ONDs) and azanorbornadienes (ZNDs) are not suitable for the development of ADCs because they are prone to a spontaneous reaction of rDA after protein bioconjugation, what would lead to partial release of the drug into the blood stream before reaching the tumor site.156 2.1.7.3.2.3 Azanorbornadienic bromovinyl sulfones Recently, a new generation of azanorbornadienic systems has been developed in our group. The new compounds contain the functionality bromovinyl sulfone and have proved to be selective for cysteine bioconjugation (Scheme 44).157 These bicyclic compounds are able to react with the thiol group of Cys residues through a nucleophilic vinylic substitution (SNVσ) with bromide as leaving group.158–161 Scheme 44. Competition between Cys and Lys residue for conjugation with [2.2.1]azabicyclic bromovinyl sulfone. In a further experiment, the bioconjugation between protein Ub-K63C and the biotinylated azanorbornadienic bromovinyl sulfone was performed (Scheme 45). The Chapter 2__________________________________________________Introduction 150 resulting Ub-K63C/azanorbornadiene conjugate was shown to be stable in blood plasma after incubation for 24 h at 37 °C. However, incubation of this conjugate with an excess of glutathione for 1 h resulted in a thioketal intermediate, which evolved to give the pyrrole derivative by a spontaneous rDA. Therefore, the glutathionesensitive character of these bicyclic systems could find applications in the field of ADCs. Scheme 45. Stability studies of Ub-K63C-containing azanorbornadienic reagents. Chapter 2____________________________________________________Objectives 151 2.2 OBJECTIVES In order to improve the in vivo cytotoxic activity of the NAMPT inhibitors synthesized in Chapter 1, an approach to the preparation of ADCs will be performed. For this purpose, we plan to develop conveniently functionalized new inhibitors based on the structure of inhibitor 47a, to be used as payloads (Figure 44). As the conjugation of the payload to the linker needs a chemical handle in the molecule, we envisaged the introduction of an amino group (furfurylamine, pyrrolidine/pyrrolidine) in the inhibitor. The amino group can be easily involved in the formation of carbamates and is the most common handle used in ADCs. Figure 44. Possible chemical modification to make 47a a bioconjugable compound for ADC. Once the best cytotoxic compounds are identified, we envisage the synthesis of ADCs. For that purpose, two different strategies are planned: 1) Incorporation of the payloads into CD138 antibody through an enzymatically cleavable linker following a classical approach. 2) Development of a new OND-based linker strategy for the preparation of ADCs that could be used for the drug delivery of furan-tail NAMPT inhibitors. The corresponding NAMPT inhibitor could be incorporated into the OND through a Diels- Chapter 2____________________________________________________Objectives 152 Alder reaction with the appropriate alkyne (Scheme 46). The bromovinyl functionality of these systems would allow the incorporation of the payload-linker system into a mAb through site-selective bioconjugation. Scheme 46. Diels Alder (DA) cycloaddition between activated alkynes and furan-tailed NAMPT inhibitors. Since the use of the OND-linker strategy is based in the ability of these bicyclic systems to the fragmentation in the presence of glutathione (via rDA reaction), we planned to carry out a preliminary study of the fragmentation reaction of differently substituted ONDs in the presence of a model of a biological thiol. For a future application in ADCs, a cytotoxic furan-containing drug (e.g., NAMPT inhibitors) would be released (Scheme 47). Scheme 47. Proposed study of the thiol-promoted OND fragmentation. Chapter 2__________________________________________Results and discussion 153 2.3 RESULTS AND DISCUSSION 2.3.1 CHEMISTRY 2.3.1.1 Preparation of new bioconjugable NAMPT inhibitors To study the influence of the conformational flexibility of the linker in compound 47a (the most cytotoxic compound towards MIA Paca-2 cells), we addressed the preparation of compounds 74 and 75 (Scheme 48). The synthesis of 74 started by coupling commercially available 4-cyanobenzenesulfonyl chloride with N- cyclopentylfurfurylamine 37a, to yield sulfonamide 70. Reduction of 70 with LiAlH4 afforded the corresponding amino compound 72, which after coupling with phenyl N-cyano-N’-(pyridin-4-yl)carbamimidate 46a yielded compound 74 (Scheme 48). In a similar way, reaction of 37a with sulfonyl chloride 69 afforded sulfonamide 71. 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