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Danny Lenstra

Design, Synthesis and Evaluation of Histone Lysine Methyltransferase Inhibitors

Daniël Cornelis Lenstra Cover: Evie Lelieveldt - [email protected] Lay-out: Ilse Modder - www.ilsemodder.nl Press: Gildeprint, Enschede

All rights reserved. No part of this book may be reproduced, distributed, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author. Design, Synthesis and Evaluation of Histone Lysine Methyltransferase Inhibitors

Proefschrift

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. J.H.J.M. van Krieken, volgens besluit van het college van decanen in het openbaar te verdedigen op vrijdag 28 februari 2020 om 10.30 uur precies

door

Daniël Cornelis Lenstra geboren op 24 december 1990 te Vlissingen Promotor Prof. dr. F. P. J. T. Rutjes

Copromotor Dr. J. Mecinović

Manuscriptcommissie Prof. dr. D. A. Wilson Prof. dr. H. Ovaa (Leids Universitair Medisch Centrum) Dr. E. Spruijt

Paranimfen Dr. R. Hammink M. J. H. Spijker TABLE OF CONTENTS

Chapter 1 - Introduction 15 1.1. A general introduction to epigenetics 16 1.2. DNA methylation 17 1.3. Posttranslational modifications on histones 19 1.4. Histone methylation 21 1.5. SETD7 catalysed methylation of H3K4 27 1.6. G9a and GLP catalysed methylation of H3K9 31 1.7. Labile zinc fingers as drug targets 35 1.8. Outline of this thesis 38 1.9. References 39

Chapter 2 - Structure-activity relationship studies onR ( )-PFI-2 analogues as 47 inhibitors of histone lysine methyltransferase SETD7 2.1. Introduction 49 2.2. Results and discussion 51 2.3. Conclusion 56 2.4. Supporting information 56 2.5. References 89

Chapter 3 - An investigation ofR ( )-PFI-2 analogues with a dual purpose: 93 substrates and inhibitors of histone lysine methyltransferase SETD7 3.1. Introduction 95 3.2. Results and discussion 96 3.3. Conclusion 103 3.4. Supporting information 104 3.5. References 135

Chapter 4 - Bioisosteric replacement of the sulfonamide moiety of (R)- 139 PFI-2: Towards the development of novel inhibitors of histone lysine methyltransferase SETD7 4.1. Introduction 141 4.2. Results and discussion 143 4.3. Conclusion 148 4.4. Supporting information 148 4.5. References 172 Chapter 5 - Inhibition of histone lysine methyltransferases G9a and GLP by 175 ejection of structural Zn(II) 5.1. Introduction 177 5.2. Results and discussion 179 5.3. Conclusion 183 5.4. Supporting information 184 5.5. References 192

Chapter 6 - Discussion and future perspectives 195 6.1. Introducing novel bioisosteres for the sulfonamide core of (R)-PFI-2 197 6.2. SETD7 as a catalyst for azide-alkyne click chemistry 198 6.3. Dynamic combinatorial chemistry for accelerated drug discovery 201 6.4. Development of irreversible covalent inhibitors targeting SETD7 203 6.5. Conclusion 206 6.6. Supporting information 206 6.7. References 214

Chapter 7 217 Summary 218 Nederlandse Samenvatting 222 Dankwoord 226 Curriculum Vitae 232 List of publications 234

LIST OF ABBREVIATIONS AND SYMBOLS

α Alpha b Beta δ Chemical shift e Epsilon 2-OG 2-Oxoglutarate 5caC 5-Carboxylcytosine 5fC 5-Formylcytosine 5hmC 5-Hydroxymethylcytosine 5mC C5-Methylcytosine aDMA Asymmetric dimethyl arginine Asp, D Aspartic acid atm Atmosphere Boc tert-Butoxycarbonyl Calcd Calculated Cbz Benzyloxycarbonyl CD Circular dichroïsm Cys, C Cysteine DAST Diethylamino sulfur trifluoride DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCC Dynamic combinatorial chemistry DCL Dynamic combinatorial library DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF N,N-Dimethylformamide DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide equiv Molecular equivalents ERα Estrogen receptor α ESI Electrospray ionisation

Et3N Triethylamine EtOAc Ethyl acetate ETP Epidithiodiketopiperazine FAD Flavin adenine dinucleotide Fmoc Fluorenylmethyloxycarbonyl FZ-3 FluoZinTM-3 G9a Euchromatic histone lysine N-methyltransferase 2 GLP Euchromatic histone lysine N-methyltransferase 1

10 h Hours HDAC Histone deacetylase His, H Histidine HKMT Histone lysine methyltransferase HOBt Hydroxybenzotriazole HPLC High-performance liquid chromatography HRMS High resolution mass spectrometry HTS High-throughput screening Hz Hertz

IC50 Half maximum inhibitory concentration J Coupling constant JmjC Jumonji C KDM Histone demethylase LCMS Liquid chromatography mass spectrometry LRMS Low resolution mass spectrometry LSD Lysine-specific histone demethylases Lys, K Lysine m Meta MALDI-TOF Matrix-assisted laser desorption/ionisation time-of-flight Me Methyl MeCN Acetonitrile MeOH Methanol min Minutes MMA Monomethyl arginine MOE Molecular operating environment MS Mass spectrometry NCp Nucleocapsid NMR Nuclear magnetic resonance o Ortho p Para PDB ID Protein databank identifier Ph Phenyl Phe, F Phenylalanine ppm Parts per million PRC2 Polycomb repressive complex 2 PRMT Protein arginine methyltransferase PTM Posttranslational modification RNA Ribonucleic acid rt Room temperature

11 SAH S-Adenosylhomocysteine SAM S-Adenosylmethionine SAR Structure-activity relationship Satd Saturated sDMA Symmetric dimethyl arginine SET Suppressor of variegation 3–9, enhancer of zeste and trithorax SGC Structural genomics consortium SUMO Small ubiquitin-like modifier SUV39H1 Suppressor of variegation 3-9 homolog 1 tBu tert-Butyl TET Ten eleven translocation TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography TMS Tetramethylsilane TosMIC Toluenesulfonylmethyl isocyanide Tyr, Y Tyrosine WT Wild type ZF Zinc finger Zn Zinc

12 13 14 1

Introduction

15 1.1. A general introduction to epigenetics

1 One of the largest known molecules in biological systems is the double-stranded deoxyribonucleic acid (DNA) helix found in eukaryotic organisms: if completely stretched, it can have a length of up to two meters (consisting of approximately 6 × 109 base pairs).[1] The entire DNA has to fit in the cell nucleus, which is in comparison very small; for eukaryotic nuclei the radius is in the range of 0.5 – 3.0 µm. In comparison, this is similar to fitting a 150 km long thread inside a regular size 5 football, but then in a highly organised manner, assuring the DNA remains functional. So in order to make the DNA fit into the nucleus of a cell, it has to be compacted and it is therefore wrapped around histone proteins. The histone core around which the DNA is wrapped is an octameric assembly and consists of 8 histone proteins, which are called histones H2A, H2B, H3 and H4, each two times.[2] On average, 145-147 base pairs of DNA are wrapped around each histone core, together forming what is called a nucleosome core particle (Figure 1).[3] Adjacent nucleosomes are connected by short strands (20-90 base pairs) of linker DNA, and through short-range interactions the nucleosomes assemble into chromatin fibres.[4] Subsequent interactions between chromatin fibres lead to the formation of higher-order chromatin structures, known as chromosomes.[5]

Figure 1. Crystal structure of the human nucleosome core particle (PDB id: 1kx5). DNA double helix (cyan) wrapped around the histone octamer, which consists of histone H2A (blue), H2B (orange), H3 (green), and H4 (red), each two times, and histone tails protruding from the nucleosome.[6]

16 The human genome contains approximately 20,000 genes, which encode for proteins, and all cells are genetically identical, meaning that the DNA containing these genes is present in every single cell in the human body.[7] Nevertheless, the phenotypes of cells 1 within the same organism can be very different, for instance, liver cells have distinct cellular functions and thus require and express other proteins than muscle cells. The underlying mechanisms of how cells with a similar genotype can differentiate into cells with a distinct phenotype, without changing the underlying DNA sequence, are called epigenetics (derived from the words epi, meaning ‘outside’, and genetics).[8] These mechanisms include DNA methylation and posttranslational modifications (PTMs) on histones, such as acetylation of lysine residues, phosphorylation of serine groups, and methylation of arginine and lysine residues.[9, 10] Ultimately, these epigenetic mechanisms determine the identify and fate of cells.

1.2. DNA methylation

In eukaryotic organisms the DNA contains bases that are methylated such asC5- methylcytosine (5mC), N4-methylcytosine (4mC), and N6-methyladenine (6mA). The transfer of the methyl group from S‑adenosylmethionine (SAM, also known as AdoMet) to DNA is catalysed by members of the DNA methyltransferase (DNMT) family.[11] To date, five different DNMTs have been identified in humans: DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. From these, only DNMT1, DNMT3A and DNMT3B were found to possess catalytic methyltransferase activity. The most prevalent DNA methylation mark in eukaryotic organisms is 5mC typically found in CpG regions of the genome. The methylation of cytosine proceeds via a covalent mechanism (Scheme 1a).[12] First, a cysteine residue performs nucleophilic addition to C6 on the cytosine ring. The intermediate is stabilised by a neighbouring glutamic acid residue. Subsequently, a nucleophilic attack of the cytosine C5 on the electrophilic methyl group of SAM (Scheme 1b), followed by deprotonation at C5 and elimination of the cysteine residue, leads to the formation of 5mC and S-adenosylhomocysteine (Scheme 1c, SAH, also known as AdoHcy).

The methylation of DNA is reversible, and demethylation is catalysed by members of the ten eleven translocation (TET) enzymes.[13] The enzymatic activity of the TET enzyme family was first described in 2009.[14] Three TET enzymes have been identified till now, called TET1, TET2 and TET3, all of which are iron Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. TET enzymes consecutively catalyse the oxidation of 5mC to 5‑hydroyxmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5‑carboxylcytosine

17 (5caC) in DNA (Scheme 1d). These three oxidised methyl cytosine analogues are all stable, and can be found in varying amounts in eukaryotic cells, with 5hmC being the 1 most abundant.[15] The demethylation of 5mC can be completed via several pathways starting from either 5hmC, 5fC or 5caC. Several research groups have shown that both 5fC and 5caC can be enzymatically excised and replaced with unmodified cytosine through base excision repair by thymine DNA glycosylase.[16] Furthermore, it has also been shown that in vitro dehydoxymethylation of 5hmC or decarboxylation of 5caC is catalysed by C5-MTases, e.g. DNMT3A, under reducing conditions.[17, 18] However, at this time it is unclear whether these reactions also occur in vivo.

Scheme 1. a) Mechanism of the SAM-dependent methylation of cytosine catalysed by DNA methyltransferases; b) structure of S-adenosylmethionine (SAM); c) structure of S-adenosylhomocysteine (SAH) and d) reaction cascade for the demethylation of 5mC catalysed by the ten eleven translocation (TET) enzymes.

In addition to DNMTs and TET enzymes that catalyse the (de)-methylation ofDNA, readers of the various DNA modifications have also been identified. For instance, 5mC is recognised by methyl CpG binding protein 2 (MeCP2) and several members of the 5-methylcytosine binding domain (MBD1‑4) proteins.[19, 20] Several zinc finger containing proteins, including the Kaiso family, are known to recognise both methylated and unmethylated DNA.[21] The third family of readers of 5mC is the Set and RING- associated (SRA) domain proteins, and consists of two members UHRF1 (ubiquitin-like, containing PHD and RING finger domains) and UHRF2.[22] Several readers of the oxidised

18 5mC products 5hmC, 5fC, and 5caC have also been reported.[23, 24] In turn, these reader proteins of 5mC and oxidised analogues are known to recruit transcriptional activators or/and repressors and thereby promote or shutdown DNA transcription, depending on 1 what is required in the cell.[25]

Since the discovery it has become evident that the dynamic methylation of DNA by DNMTs and demethylation by TET proteins play a pivotal role in the regulation of numerous cellular processes, and are essential for the normal functioning of organisms. Processes that are, at least in part, controlled by DNA methylation include regulation of gene expression, genomic imprinting, embryonic development, ageing, and chromatin modification.[26, 27] Dysregulation of DNMTs or/and TET proteins may lead to altered DNA methylation patterns, which have been observed in numerous types of cancer, e.g. breast, liver, colon, and several types of bone cancer.[28] Notably, DNMT inhibitors 5‑aza‑2’‑deoxycytidine and 5-azacytidine are clinically approved for the treatment of myelodysplastic syndrome, a type of cancer in which blood cells do not properly mature, and which may lead to acute myeloid leukaemia. Other commonly occurring human diseases in which alterations of DNA methylation patterns have been observed are rheumatoid arthritis,[29] multiple sclerosis,[30] and diabetes.[31]

1.3. Posttranslational modifications on histones

The overall structure of chromatin is not inert, on the contrary, it continuously changes states, dynamically regulated by external stimuli. Two states of chromatin can be distinguished: a highly condensed heterochromatin, in which transcription ofthe underlying genome is repressed, and a loosely packed euchromatin, in which the DNA is accessible for transcription. The interchange between hetero- and euchromatin is regulated by histone PTMs. These PTMs mainly take place on the unstructured histone tails, which protrude from the nucleosome core particle (Figure 1). The proteins/ enzymes that regulate the covalent modifications on histone proteins can be divided into three categories: i) ‘writers’, which are responsible for introducing the various chemical entities to the side chains of amino acids residues;ii ) ‘readers’ which recognise specific posttranslational modifications and subsequently initiate or affect downstream processes, and iii) ‘erasers’, which are responsible for removal of chemical groups.[32]

19 1

Figure 2. Amino acid sequence of the N-terminal tails of histones H2A, H2B, H3, and H4 with known common posttranslational modifications: methylation (Me, red), acetylation (Ac, purple), phosphorylation (P, orange), and citrullination (Ci, blue).

The protruding histone tails are highly alkaline and rich in lysine and arginine residues, which can undergo extensive modifications by epigenetic writers. To date, a wide range of histone PTMs have been identified, for example, lysine residues may have small moieties attached like methyl or acetyl, but also larger modifications are introduced such as ubiquitin or small ubiquitin-like modifier (SUMO) proteins. Other known PTMs on histones are phosphorylation, citrullination, crotonylation, and glycosylation. A schematic overview of the most important histone modifications is depicted in Figure 2. Taken together, these covalent alterations on the histone tails affect the interactions between adjacent nucleosomes and also with the DNA that is wrapped around each histone core. For instance, lysine acetylation neutralises the positive charge that is present under physiological conditions on unmodified lysine, thereby altering the electronic interactions with either negatively charged DNA, or between adjacent nucleosomes.[33] As a result the higher order arrangement of chromatin fibres is affected, and this eventually changes the structure and state of chromatin.[34] Indeed, acetylation of lysine is associated with the formation of euchromatin.

Histone PTMs would not be dynamic without the existence of enzymes opposing the function of writers. The removal of chemical modifications on histones is catalysed by

20 erasers. Histone deacetylases (HDACs) are responsible for the removal of acetyl groups from the ε–amino group of lysine residues. Since their first discovery, which dates back to 1969,[35] HDACs have been widely studied and implicated in several diseases including 1 several types of cancer, Alzheimer’s disease, and Huntington’s disease.[36-38] To date, few HDAC inhibitors have been approved for clinical use by the food and drug administration (FDA). For example Vorinostat, approved in 2004 for the treatment of T‑cell lymphoma.[39] Other enzymes belonging to the eraser family include histone phosphatases for the removal of phosphoryl groups on serine, threonine and tyrosine residues, and histone demethylases (KDMs) for the removal of one or more methyl groups from arginine and lysine residues. Besides writers and erasers of the various histone PTMs, reader enzymes are responsible for recognising specific PTMs and subsequently recruiting other proteins that initiate downstream cellular processes. To date, several families of reader proteins have been described in literature, which are discussed in Section 1.4 of this thesis.

Often, enzymes or multisubunit protein complexes associated with chromatin remodelling contain multiple domains with several functions, i.e. both writing, reading or/and erasing. It has been hypothesised that there is an interdependence between histone modifications, a specific PTM on one position may be recognised by reader domain, which in turn recruits other writers or/and erasers that affect PTMs on neighbouring amino acids.[40] For instance G9a and GLP, two histone lysine methyltransferases that are generally considered as writers of H3K9me1/2, have an Ankyrin repeat domain for binding of mono and dimethyllysine residues on histone 3, showing that their function is not limited to writing, but also reading.[41] Another example of a protein complex associated to chromatin remodelling that has multiple functions is PRC2 (polycomb repressive complex 2), which consists of four major subunits: Suppressor of Zeste 12 (SUZ12), Retinoblastoma Suppressor Associated Protein 46/48 (RbAp46/48), Enhancer of Zeste Homolog 2 (EZH2), and embryonic ectoderm development (EED) domain. In PRC2, EED is a reader domain that recruits monomethylated lysine 27 on histone 3 (H3K27me), which is subsequently di- and trimethylated by the EZH2 catalytic domain to form H3K27me2/3. Furthermore, SUZ12 is important for the assembly and stability of the PRC2 complex, whereas RbAp46/48 plays an essential role in regulating the chromatin structure.[42]

1.4. Histone methylation

Methylation of lysine and arginine residues is one of the most prevalent histone PTMs. Methylation is catalysed by histone methyltransferases (HMTs), a family of enzymes

21 that catalyse the transfer of the methyl group from SAM to the ε-amino group of lysine or guanidinium group of arginine residues. HMTs possess high specificity for their 1 substrates and for the degree of methylation. For example, SETD8 (also known as SET8, Pr-SET7, KMT5A) is a methyltransferase that specifically methylates Lys 20 on histone H4.[43] SETD8 catalyses the addition of one, but not two or three, methyl groups to H4K20. Demethylation of lysine and arginine is catalysed by Fe(II)/2OG- or/and flavin adenine dinucleotide (FAD)‑dependent histone demethylases. In the following section, the enzyme families that are involved in introduction and removal of methyl groups to arginine and lysine on histones will be described.

Histone arginine methylation Arginine methylation is catalysed by one of the nine members of the protein arginine methyltransferase (PRMT) family. PRMTs catalyse the transfer of one or two methyl group(s) from SAM to the guanidinium nitrogen groups of arginine residues on the various histone tails.[44] In eukaryotes, there are three forms of methylarginine: monomethyl arginine (MMA), asymmetric dimethylarginine (aDMA) and symmetric dimethylarginine (sDMA) (Scheme 2). The nine PRMTs that have been discovered so far can be divided into three categories according to their function. Type I (PRMT1, PRMT2, PRMT3, PRMT6, and PRMT8) and type II (PRMT5, PRMT9) arginine methyltransferases catalyse first the monomethylation of arginine and subsequently the formation of aDMA (type I) or sDMA (type II). Only PRMT7 belongs to the type III category, and catalyses only the formation of MMA, but not further to either aDMA or sDMA.

22 Me N NH Me 2 NH 1

N Me H H N NH O 2 2 HN NH2 PRMT Type I aDMA NH NH SAM SAH

SAM SAH N PRMTs N Me Me H H O Type I, II, III O HN NH

Arginine MMA PRMT NH Type II

N H O sDMA Scheme 2. Arginine methylation catalysed by protein arginine methyltransferases (PRMTs): type I catalyse the formation of monomethyl arginine (MMA), followed by addition of a second methyl group toform asymmetric dimethyl arginine (aDMA). Type II PRMTs catalyse the formation of symmetric dimethyl arginine (sDMA), and type III PRMTs only catalyse the transfer of one methyl group to form MMA.

The PRMT family of enzymes is involved in regulation of various important biological functions, for instance DNA repair, ribonucleic acid (RNA) metabolism, epigenetic regulation, and modulation of signalling pathways and transcription factors.[45-48] Consequently, aberrant activity of PRMTs has been linked with various diseases, such as cancer, cardiovascular diseases, and multiple sclerosis.[47, 49, 50] The discovery and development of potent and selective inhibitors of PRMTs is therefore highly desired, and significant effort has been made by both academic groups and pharmaceutical companies.[51-53] Most notably, a joint effort by scientists from Epizyme and GlaxoSmithKline has led to the development of GSK3326595 (Figure 3), a PRMT5 inhibitor, which inhibits tumour cell growth in animal models. GSK3326595 has recently entered the phase-1 clinical trials for the treatment of Non-Hodgkin lymphoma and acute myeloid leukaemia.[54, 55]

N N N OH H N N N H O GSK3326595 Figure 3. Structure of PRMT5 inhibitor GSK3326595.

23 Due to contradicting reports, the existence of arginine demethylases (RDMs) was controversial for many years: in 2007 Chang and colleagues reported that JMJD6, an 1 Fe(II) and 2OG‑dependent dioxygenase, catalyses the demethylation of H3R2me1/2 and H4R3me1/2.[56] However, subsequent studies were unable to reproduce these results.[57] Only recently, an elaborate study showed that some, but not all, members of the Jumonji C (JmjC) lysine demethylase (KDM) family were able to catalyse the demethylation of arginine residues in synthetic histone peptide mimics.[58] It was found that KDM3A, KDM4E, KDM5C, and KDM6B were able to demethylate arginine residues in vitro. However, at this point there is limited evidence that the JmjC proteins also possess an ability to demethylate arginine in vivo.

Histone lysine methylation Histone lysine methyltransferases (HKMTs) transfer the methyl group from SAM to the ε-amino group of lysine residues on the N-terminal tails of histones. Up to three methyl groups can be transferred, and as a result four possible methylation states exist on lysine: it can be either unmodified (K), monomethylated (Kme), dimethylated (Kme2) or trimethylated (Kme3) (Scheme 3).[59] For several methyltransferases it has been shown that the methylation state that is achieved, i.e. the product specificity, is determined by a specific amino acid in the active site. If this residue in the active site is phenylalanine (Phe), typically di- and trimethylation can occur, whereas a tyrosine (Tyr) residue restricts the catalysis to mono- and dimethylated products. For example, replacement of Phe1205 in G9a to Tyr highly affected the product specificity, as the F1205Y mutant was only able to catalyse monomethylation of H3K9 rather than the usual di- and trimethylation catalysed by wild-type (WT) G9a.[60]

Where lysine acetylation is usually associated with the formation of heterochromatin, i.e. repression of transcription, lysine methylation can have very distinct outcomes on the state of chromatin. Methylation does not alter the positive charge that is present on lysine residues, however it does affect the ability to form hydrogen bonds, thereby changing the interactions between adjacent nucleosomes. In general, methylation of H3K4, H3K36, and H3K79 is associated with transcriptional activation, whereas methylation of H3K9, H3K27, and H4K20 leads to suppression of transcription.[61] However, H3K9 methylation has also been observed in transcriptionally active regions of chromatin, highlighting the complexity of histone PTMs and their consequences.[62]

24 Me Me Me Me Me NH2 NH N N Me

HKMT HKMT HKMT 1 N KDM N KDM N KDM N H H H H O O O O K Kme Kme2 Kme3 Lysine Monomethyllysine Dimethyllysine Trimethyllysine Scheme 3. HKMT catalysed transfer of the methyl group from SAM to the ε-amino group of lysine residues on histone and non-histone proteins leads to the formation of mono-, di- and trimethyllysine (Kme, Kme2, and Kme3, respectively). Demethylation is catalysed by the KDM family of erasers, which utilise either Flavin Adenine Dinucleotide (FAD) or Fe(II), 2OG, and molecular oxygen.

HKMTs are a class of enzymes which all, with the exception of DOT1L, have a conserved SET (Suppressor of variegation 3–9, Enhancer of zeste and Trithorax) catalytic domain, that consists of approximately 130 amino acids.[63] The first SET-domain containing protein that possesses HKMTase activity was reported in 2000, and was named suppressor of variegation 3-9 homolog 1 (SUV39H1).[64] SUV39H1 selectively methylates lysine 9 on histone 3. The ability to catalyse the transfer of a methyl group from SAM to lysine residues on histone proteins does not solely rely on the SET domain itself, but also on adjacent domains that are responsible for stability and binding of both histone and SAM substrates.[65] Just before the SET domain on the N-terminal side, a pre-SET (or nSET) domain is found. It appears that the main function of the pre-SET is to achieve structural stability of the SET domain itself.[66] On the other hand, recruitment of the histone protein is largely performed by the I-SET and post-SET (also known as C-SET) domains, which are located directly after the SET domain, on the C‑terminal side. Besides histone binding, both the I-SET and post-SET domain contribute to binding of cofactor SAM. The composition and shape of these domains varies largely between different methyltransferases. Notably, the importance of the role of domains surrounding the SET domain in recognising and recruiting specific histone proteins was emphasised by the observation that phosphorylation of serine 10 on histone 3, completely blocks SUV39H1’s methyltransferase activity on adjacent unmodified lysine 9.[64]

Histone lysine demethylation The methylation of lysine residues on histones is reversible, and demethylation is catalysed by histone lysine demethylases (KDMs).[67] Two functional KDM families can be distinguished based on the mechanism of the catalytic demethylation. Thefirst family consists of the lysine-specific histone demethylases (LSD), which utilise FAD to oxidise mono- or dimethyllysine to the corresponding imine, followed by (non- enzymatic) hydrolysis to form formaldehyde and the demethylated amine.[68] Because

25 a lone pair is required on the nitrogen for the initial reaction with FAD, only mono- and dimethyllysine, but not trimethyllysine, are substrates of the LSD enzymes. The second 1 and largest family of KDMs consist of the JmjC domain containing enzymes. The JmjC demethylases use 2OG, Fe(II) and molecular oxygen to oxidise methylated lysine residues to hydroxymethyllysine, which is unstable and spontaneously releases formaldehyde and lysine. In contrast to the LSD proteins, JmjC domain containing demethylases can remove the methyl group from mono-, di-, and trimethylated lysine residues.

Readers of methyllysine The PTMs introduced on histone proteins can serve as a recognition site for binding domains of other proteins or protein complexes, thereby invoking subsequent enzymatic cascades, for example to alter the transcription of the underlying genes. Over the past two decades, several evolutionary conserved binding (reader) domains that recognise specific PTMs have been discovered: chromodomains (recognition of a range of Kme1/2/3 residues on H3 and H4),[69] bromodomains (acetylated histones),[70] plant homeodomain (PHD) fingers (recognition of unmodified H3K4, H3K4me3, H3K9me3, H3R2 and H3K14Ac),[71] proline–tryptophan–tryptophan–proline (PWWP) domains (5mC, H3K36me3, H3K79me3, H4K20me1/3),[72] TUDOR domains (various methylated lysine and arginine residues), Ankyrin repeat (Kme1/2 on H3),[41] WD40 repeat (Kme1/2/3 on H3 and H4),[73] and the malignant brain tumour (MBT) domains (Kme1/2 residues on H3 and H4).[74] For binding of methylated histones, these domains are typically comprised of a conserved aromatic cage consisting of two up to four phenylalanine, tryptophan or tyrosine residues. For example, bromodomain and PHD domain transcription factor (BPTF) binds H3K4me3 in an aromatic cage that is comprised of three tyrosine and one tryptophan residue (Figure 4).[75] The architecture and composition of the cage usually determines the selectivity between different Kme1/2/3 methylation states, whereas specificity for the histone peptide sequence that is to be recognised by the reader is achieved by amino acid residues adjacent to the aromatic cage. Reader domains that do not rely on binding by an aromatic cage have also been reported.[76]

Since the discovery of SUV39H1, over 50 different HKMTs have been identified,[77] all of which play important roles in diverse biological processes such as regulation of chromatin structure, transcription, DNA repair, and modulation of the activity of non-histone proteins through methylation.[78] Due to the vast amount of histone lysine methyltransferases and inhibitors for these enzymes that have been discovered over the past decades, only SETD7, G9a and GLP with their known inhibitors are described in this introduction and later in thesis.

26 1

Figure 4. Crystal structure (PDB id: 2F6J) of the bromodomain and PHD domain transcription factor (BPTF) methyllysine reader in complex with a 15-mer H3K4me3 histone peptide mimic. a) The surface of the H3K4me binding pocket of BPTF is depicted in grey, the aromatic cage residues in orange, and the histone peptide in yellow; b) The aromatic cage of BPTF is comprised of three tyrosine and one tryptophan residue.

1.5. SETD7 catalysed methylation of H3K4

The discovery of histone lysine methyltransferase SETD7 (SET domain containing lysine methyltransferase 7, also known as SET7/9, or KMT7) was first reported in 2001.[79] SETD7’s function was initially identified as a methyltransferase of lysine 4 on histone 3 (H3K4), the resulting methylation mark, H3K4me, correlating to transcriptional activation in humans.[80] SETD7 has two substrate binding pockets, which are located on opposite surfaces of the enzyme, one for binding of the histone protein, and the other for cosubstrate SAM. The two pockets are connected through a narrow hydrophobic channel that runs through the core of the enzyme. Inside this channel the methyl transfer takes place: the first step in SETD7-catalysed methylation of H3K4 is based on the binding of SAM, followed by histone binding.[81] Notably, the histone protein cannot bind to SETD7 before SAM. The reaction then proceeds according to the following mechanism (Scheme 4): Tyr335 is first deprotonated by bulk solvent upon entering the lysine binding pocket, then the protonated side chain of lysine 4 is first desolvated followed by deprotonation by Tyr335. Through H‑bonding with Tyr245, Tyr305 and a water molecule, the ε‑amino group is oriented in the correct way towards the methyl group of SAM. The reaction takes place in an SN2 fashion to form H3K4me and SAH and the positive charge that forms on the ε-amino group during methylation is stabilised by Tyr245 and Tyr305.[82, 83] The methyl transfer is followed by dissociation of H3K4 and SAH from SETD7.

Unlike many other methyltransferases, SETD7 is only able to install one methyl group on its substrates, due to the presence of Tyr305. As mentioned before, the product specificity of HKMTs is determined by a key residue (Phe or Tyr) in the active site, for

27 SETD7 it has been shown that Tyr/Phe switch mutant Y305F alters the specificity of SETD7 from a monomethyltransferase to a dimethyltransferase.[84] Furthermore, SETD7 1 was found to be highly specific towards modifications of natural L-lysine 4 on a synthetic peptide mimic of histone 3: no methylation was observed when the stereochemistry was changed from L-Lys to D-Lys.[85] Histone 3 peptide mimics in which Lys4 was replaced by lysine analogues bearing shorter or longer side chains, i.e. ornithine (chain length 3 instead of 4) or homolysine (chain length 5 instead of 4), were not accepted as substrates by SETD7.[86] These observations clearly show that SETD7 is highly specific for lysine, and that the underlying stereochemistry and side chain length importantly contribute to an efficient methylation reaction.

Scheme 4. Mechanism of SETD7 catalysed methylation of H3K4. Hydrogen bonds are dashed. Scheme is adapted from literature.[82, 83] Tyr335 is first deprotonated by bulk solvent. Subsequently, Tyr335 is able to abstract a proton from protonated H3K4. After correct orientation of the H3K4 ε-amino group mediated by

Tyr245 and Tyr305, the methyl transfer takes place in an SN2-type reaction forming H3K4me and SAH.

Since its discovery, a variety of non-histone substrates of SETD7 have been identified. For instance, tumour suppressor p53 is methylated at K372 by SETD7, and this methylation was found to be essential for p53 stability and activity.[87] Other known substrates include several transcription factors and initiators (estrogen receptor α (ERα),[88] p65,[89] FoxO3,[90] TAF10[91]), DNA methyltransferase 1 (DNMT1),[92] and acetyltransferase p300/ CBP associated factor (PCAF).[93] Methylation of these substrates can have significantly different outcomes, for example DNMT1 methylation at K142 makes it more susceptible

28 to proteasome-mediated degradation, whereas methylation of ERα at K302 is stabilising and essential for activation of downstream processes. 1 The broad set of substrates that are subjected to methylation by SETD7 signifies its involvement in many important cellular pathways. As a result, an aberrant activity or/and malfunctioning of SETD7 has been linked to a variety of disorders. Depending on the disease and underlying mechanisms it may be beneficial to either inhibit, or enhance SETD7’s methyltransferase activity. Thus, the development of potent and selective chemical probes targeting SETD7 is highly desired. In the following sections, the known inhibitors of SETD7 are described.

Inhibitors targeting histone lysine methyltransferase SETD7 The first compound that showed inhibitory activity against human SETD7 was antifungal metabolite sinefungin (Figure 5). Unfortunately, sinefungin is not selective in inhibiting SETD7, as it inhibits a plethora of other SAM-dependent methyltransferases. In 2010, inhibitors derived from the structure of sinefungin were reported by Mori and co- workers.[94] In their work, a library of SAM mimics, named AzaAdoMet analogues, in which the sulfur atom is replaced by a nitrogen atom with various ethylene linked alkylamino substituents were synthesised. No IC50 values were determined for these analogues, but the percentage inhibition at fixed concentrations was evaluated and compared to the inhibitory activity of sinefungin. It was found that DAAM‑3 (Figure 5) was the most potent, showing approximately 50% inhibition at 10 µM concentration, similar to the inhibition of SETD7 by sinefungin. A crystal structure of the SETD7- DAAM-3 complex was reported in 2013 by the same group, confirming the binding mode of DAAM-3 in the SAM binding pocket of SETD7.[95]

The first SAM competitive inhibitors, which displayed selectivity for SETD7 over other methyltransferases, were reported by Meng and colleagues in 2015.[96] They performed a virtual screening to identify SAM-competitive inhibitor DC-S100 (structure not shown, IC50 = 30 µM) as a hit compound that inhibits SETD7. Subsequent structure- activity relationship (SAR) studies led to the discovery of DC-S238 (IC50 = 4.88 µM) and

DC-S239 (IC50 = 4.59 µM). The selectivity of these inhibitors was evaluated against six other methyltransferases, including DNMT1, DOT1L, EZH2, NSD1, SETD8, and G9a. It was found that both DC-S238 and DC-S239 are selective towards SETD7, as no significant inhibition of these other methyltransferases was observed at 100 µM inhibitor concentration. Furthermore, the antiproliferative activity of DC-S329 was evaluated against several cell lines, and it was found that proliferation of MCF7 (breast cancer), HL60 (human leukaemia) and MV4-11 (human leukaemia) cells was inhibited

29 in a dose-dependent manner.[97] Additional SAR studies on DC-S328 and DC-S329 led to the discovery of DC-S303, a novel analogue with an approximate 4-fold improved [98] 1 potency of IC50 = 1.1 µM.

Figure 5. Known inhibitors of histone lysine methyltransferase SETD7. IC50 values strongly depend on the type of assay and conditions employed.

The first inhibitor that competes with the histone binding site of SETD7 was reported in 2014 by the Structural Genomics Consortium (SGC). Barsyte-Lovejoy and co- workers identified (R)‑PFI‑2 (Figure 5) by a high-throughput screening (HTS) of a library containing 150,000 compounds, followed by optimisation of the initial hit.[99] (R)-PFI-2

inhibits SETD7’s methyltransferase activity with an excellent IC50 of 2.0 nM, whereas its [100] (S)-enantiomer was found to be ~500 times less potent (IC50 = 1.0 µM), highlighting the importance of the stereochemistry. Though (R)‑PFI‑2 is histone competitive, its binding to SETD7 is SAM-dependent, i.e. if SAM is not present, (R)‑PFI‑2 does not bind, an observation similar to the histone binding process. Furthermore, inhibition by (R)‑PFI‑2 was found to be selective for SETD7 over 18 human methyltransferases, including G9a/GLP, SETD8, and EZH2. Since its discovery, (R)-PFI-2 has been successfully applied for SETD7 knockdown in various cell-based studies.[101-103]

In 2016, Takemoto and co-workers performed a HTS and identified cyproheptadine as a novel inhibitor of SETD7. Cyproheptadine, which is approved for clinical use as

30 an anti-allergy agent targeting histamine receptor H1, inhibits SETD7 in a histone competitive manner with an IC50 of 3.4 µM. Importantly, cyproheptadine is cell active and decreased ERα expression and activity in MCF7 breast cancer cells, resulting in 1 inhibition of estrogen-dependent tumour growth. Subsequent SAR explorations on cyproheptadine and its analogues led to the discovery of 2‑hydroxycyproheptadine, [104, 105] which has an improved ability to inhibit SETD7 with an IC50 value of 0.41 µM. The co-crystal structure of SETD7 in complex with 2-hydroxycyproheptadine revealed that the increased affinity is a result of hydrogen bonding interactions of the 2-hydroxy group with Asp338.

1.6. G9a and GLP catalysed methylation of H3K9

The mono-, di- and trimethylation of H3K9 is mainly catalysed by HKMTs G9a (Euchromatic Histone Lysine N-methyltransferase-2 (EHMT2)) and closely related homologue GLP (G9a-like protein, also known as EHMT1).[106, 107] G9a and GLP are both SET-domain containing lysine methyltransferases and share about 80% sequence identity in their SET domains, and though they have the same substrate specificity, both enzymes independently methylate their substrates.[106, 108] Knockdown of either G9a or GLP results in severely reduced levels of H3K9me and H3K9me2, and double knockdown of both enzymes results in formation of similarly reduced methylation levels, indicating that knockdown of either of the methyltransferases cannot be compensated by the other.[108] Methylation of H3K9 in euchromatin leads to formation of heterochromatin, i.e. repression of transcription.

Inhibitors targeting G9a and GLP histone methyltransferases Six years after the discovery of G9a, Kubicek and co-workers reported BIX-01294, the first selective inhibitor of G9a (Figure 6).[109] BIX-01294 was identified by a HTS from a chemical library consisting of approximately 125,000 compounds. BIX-01294 inhibits

G9a’s methyltransferase activity with IC50 = 1.7 µM. The selectivity of BIX-01294 was evaluated against several methyltransferases, including SETD7 (H3K4), PMRT1 (H4R3me2), SETDB1 (H3K9me2/3), SUV39H1 (H3K9me3), and GLP. Besides G9a, no significant inhibition was observed for the other methyltransferases, except for GLP, for which only moderate inhibition was observed (IC50 = 38 µM) in the initial report. It was found later that oversaturating conditions were employed during the assessment of the IC50 of BIX‑01294 against GLP, thereby mainly generating H3K9me3, which was [110] not detected in the assay. The correct IC50 for GLP inhibition by BIX-01294 turned out to be 0.7 µM, making it an even better inhibitor of GLP than G9a. Similar to inhibition

31 of SETD7 by (R)-PFI-2,[99] BIX-01294 inhibits G9a in a histone competitive way, but only binds to the G9a or GLP and SAM complex. In the same work the discovery of BIX-01338 1 (structure not shown) was also revealed, however it was found to inhibit several other human methyltransferases in a selectivity test. Kinetic analysis revealed that BIX‑01338 is a SAM‑competitive inhibitor. The structure of BIX-01338 was later used in order to [111] develop a more potent and selective G9a/GLP inhibitor BRD4770 (IC50 = 6.3 µM).

Figure 6. Known inhibitors of histone lysine methyltransferases G9a and GLP. IC50 values strongly depend on the type of assay and conditions employed.

32 In multiple successive studies, the structure of BIX‑01294 has served as the starting point for the development of G9a and GLP inhibitors with improved potency, selectivity, and cellular activity. In 2009, the first preliminary SAR exploration led to the discovery 1 of UNC0224, which is approximately seven times more potent (IC50 = 15 nM) than BIX- [112] 01294 (IC50 = 106 nM), evaluated in a ThioGlio assay against G9a. The differences in measured IC50 values for BIX-01294 are a result of a different assay that is employed. Binding studies performed by isothermal titration calorimetry studies confirmed the higher affinity of UNC0224 (Kd = 23 nM) for G9a with respect to BIX-01294 (Kd = 130 nM). In 2010, UNC0224 was further optimised in a more elaborate SAR study, which revealed UNC0321 with an excellent ability to inhibit G9a (IC50 = 43-57 nM) and GLP [113] (IC50 = 50-58 nM). Unfortunately, though UNC0321 was more potent in in vitro biochemical assays, it was less potent than BIX-01294 in cellular assays. In the same year, Chang and co-workers found that by adding lysine mimicking groups to BIX‑01294, [114] its potency as GLP inhibitor could be significantly improved (E72, IC50 = 100 nM). The first BIX-01294 derived G9a/GLP inhibitor with high potency, selectivity, excellent cellular activity, and low toxicity was reported in 2011 by the SGC; UNC0638, which selectively inhibits G9a (IC50 = 15 nM) and GLP (IC50 = 19 nM) over 17 other enzymes involved in epigenetic regulation.[115] Furthermore, at 250 nM UNC0638 concentration in cells, an approximate 60-80% reduction in H3K9me2 levels could be observed.

The discovery of A-366 by Sweis and co-workers revealed the first G9a and GLP inhibitor with a core different from the quinazoline present in BIX-01294, UNC0638, and related [116] structures. An initial hit with IC50 = 153 nM (G9a) was identified by a chemical diversity screen of an in-house compound library, and subsequent structure guided optimisation afforded A-366 which inhibits G9a in a histone‑competitive manner with IC50 = 3 nM.

With the exception of GLP (IC50 = 38 nM), A-366 displays excellent selectivity: A-366 did not significantly inhibit 21 other human methyltransferases. Significant reduction of H3K9 methylation was achieved in human prostate cancer PC3 cells in the presence of A‑366, highlighting its applicability as a chemical tool for in vitro studies involving G9a and GLP. Another structure with a distinct pharmacophore is DCG066 (IC50 = 6.5 µM), which was found through a structure based virtual screening of approximately 90,000 compounds, followed by synthesis and biological evaluation.[61]

As a result of the high degree of similarity in sequence and function, inhibitors designed to target G9a often also inhibit GLP, andvice versa. However, G9a and GLP have distinct physiological functions, and selective inhibitors would therefore be valuable tools in order to study the biological role of the individual enzymes. Only recently in 2017, the first inhibitor displaying significant selectivity (>140‑fold) for GLP over G9a was reported

33 by the SGC.[117] A screening of a quinazoline collection, and subsequent optimisation led to the discovery of MS012 (Figure 6). Though crystal structure analysis reveals that the 1 binding mode of MS012 is identical to both enzymes, G9a is only moderately inhibited

(IC50 = 992 nM), whereas GLP very effectively (IC50 = 7 nM). The excellent selectivity was not only observed for GLP over G9a, as MS012 was also unable to inhibit 29 different human lysine, arginine, DNA, and RNA methyltransferases. A subsequent elaborate

SAR study on MS012 reported two additional GLP selective inhibitors: MS3748 (IC50 = 5

nM, >40-fold selectivity) and MS3745 (IC50 = 4 nM, >45-fold selectivity (structures not shown).[118] The most recently reported inhibitor of G9a and GLP is EML741, an inhibitor with a novel chemotype containing a nonquinazoline core rather than a quinazoline [119] core. EML741 inhibited G9a with IC50 = 23 nM.

Finally, it was found that epidithiodiketopiperazine (ETP) alkaloid chaetocin (Figure 7), which is produced by a genus of fungi called Chaetomium Minutum, is also able [120] to inhibit G9a (IC50 = 2.4 µM). Besides G9a, Greiner and co-workers showed that chaetocin has a significant ability to inhibit related H3K9 methyltransferase SUV39H1, [121] with IC50 = 0.6 µM. In their work, it is reported that SUV39H1 is inhibited by chaetocin in a SAM-competitive manner, and that the disulfide warhead is not essential for inhibition. However, a subsequent report by the Fuchter group contradicts these findings and shows that there is no competition between SAM and chaetocin, and also that a chaetocin analogue that lacks the disulfide warhead is unable to inhibit SUV39H1, indicating that this is an essential contributor to chaetocin’s potency.[122] In addition, the Fuchter group reported that the inhibition of HKMTs G9a and SUV39H1 by chaetocin proceeds via a covalent mechanism targeting reactive cysteine residues which are involved in binding of structural zinc in so-called zinc fingers (ZFs), and ultimately leads to enzyme denaturation.[123] It was found that related ETP’s chetomin and gliotoxin

were also able to inhibit G9a (IC50 = 0.17 and 0.53 µM, respectively) and SUV39H1

(IC50 = 0.07 and 0.26 µM, respectively) through a similar covalent mechanism, but not methyltransferase SETD7. Bisdethiobis(methylthio)acetylgliotoxin, a gliotoxin analogue that lacks the disulfide warhead, was unable to inhibit G9a, SUV39H1, and SETD7.[124] Though to date there has been no report in literature with irrefutable evidence, it is likely that these ETPs target one or more reactive cysteine residues in structural ZFs, which results in ejection of Zn(II), followed by protein denaturation, and eventually a loss of catalytic function. A concise introduction to targeting ZFs with electrophilic small molecules, which results in the loss of Zn(II) and consequently function of a particular enzyme, is given in the next Section 1.7.

34 Me O O HO H N N S S N S Me N S HO N O Me 1 O N O O S N Me N S N N N S S N Me H OH H H O O OH Chaetocin Chetomin

Me O S O

N S S N Me N N Me H H OH Me Et O OH O S OH O Me

Gliotoxin Bisdethiobis(methylthio)- acetylgliotoxin Figure 7. Epidithiodiketopiperazine (ETP) alkaloids chaetocin, chetomin and gliotoxin which possess an ability to inhibit G9a and SUV39H1 methyltransferases through a reactive disulfide warhead, and bisdethiobis(methylthio)acetylgliotoxin analogue which is not an inhibitor of G9a and SUV39H1.

1.7. Labile zinc fingers as drug targets

Besides copper and iron, zinc is one of the most abundant metal ions found in biological systems. Zinc is essential for normal functioning of organisms, and it can be found in many proteins where it is required for catalytic activity or/and stabilisation of structure. Zinc ions are fixed in so-called zinc fingers, small protein domains of which several types exist. Catalytic zinc is commonly bound to histidine residues and water molecules in the catalytic site, thereby zinc retains its Zn(II) character, and thus its ability to serve as a Lewis acid for catalysis. On the other hand, structural zinc is found in ZFs with up to 2 histidine and at least 2 cysteine residues, with the most common motifs being Cys4, [125] - Cys3His, Cys2His2, and Cys6. The cysteine’s thiolate (S ) transfers its charge to the Zn ion, thereby making it unable to act as a Lewis acid for catalysis.[126] Both catalytic and structural ZFs are potential targets for the development of novel therapeutic agents.

It has been shown that the sulfur atom of cysteine residues in labile ZFs can be covalently modified with electrophilic, thiol modifying small molecules. Compounds that can react with labile ZFs and induce a release of Zn(II), are also known as zinc- ejectors. There are several factors playing an important role in whether a ZF is labile, i.e. whether it reacts with an electrophilic molecule, or whether it does not. First of all the ZF has to be accessible to react with the electrophile; if the ZF is located inside the protein, it is unlikely to react.[127] Secondly, the reactivity of the ZF depends on

35 the charge of the cysteine’s thiolate: decreasing its negative charge, for instance by H-bonding, makes it less prone to react with an electrophile. Finally, the Zn’s positive 1 charge may also be an important factor. The more positively charged the character of the Zn ion is, the better it can compete with electrophilic compounds. This positive charge may be affected by the type of ZF the Zn is in, i.e. the more His residues the ZF contains, the more positively charged Zn will be, whereas with increasing negatively charged Cys residues, the positive charge on Zn will decrease.[125]

Cys Cys Cys Cys Cys Cys S Cys S S Cys -Zn2+ SH HS Cys S 2+ Zn2+ Zn S S S S S E SH Cys Cys i Cys E E+

2+ -E+ -Zn E+ ii iii iv

Cys Cys Cys Cys Cys Cys SH HS Cys S HS Cys SH HS Cys S S S S S S Cys Cys E Cys E E

+ Scheme 5. Proposed mechanism for zinc ejection from a Cys4-type zinc finger by electrophile E , adapted from literature reference.[131]

A proposed mechanism for zinc ejection by electrophiles is shown in Scheme 5. One of the cysteine thiolates (R‑S‑) reacts with the electrophilic part of a zinc ejector E+, leading to covalent modification of the ZF that could result in i) direct ejection of Zn(II) due to reduced binding affinity, or ii) disulfide formation and subsequent ejection of Zn(II). After zinc ejection, the ZF can iii) react to form one or more internal disulfide bonds, or iv) react with another equivalent of zinc ejector E+. As a result of the loss of zinc, the native structure of the protein is lost, and in case of an enzyme, consequently also its catalytic function. Electrophilic moieties commonly found in small molecule zinc ejectors include platinum complexes, nitroso compounds, disulfides, organoselenium compounds, and various others.[127-130]

A strategy for inhibition by zinc ejection was first employed in 1993 in the search for novel therapies for the treatment of AIDS, which is caused by human immunodeficiency virus (HIV). Two ZF motifs are present in HIV-1 nucleocapsid (NCp) protein, which are

36 absolutely conserved among all known retroviruses.[132] In their work, Rice and co- workers reported 3-nitrosobenzamide (NOBA), which targets the ZF of HIV‑1 NCp.[133] 1H NMR spectroscopy was used to show that NOBA ejects structural Zn(II) from an 1

18‑mer synthetic peptide mimic of the Cys3His N-terminal ZF of HIV-1 NCp. Since the discovery of NOBA, various compounds have been reported that inhibit NCp, such as azidocarbonamide, several dithiobis[benzamides], pyridinoalkanoylthioesters, and multiple pentathiepin derivatives (Figure 8a).[134-137]

An approach in which reactive cysteine residues are targeted by small molecules for inhibition has also been applied to other targets. For instance, disulfiram (Figure 8b, known as Antabus or Refusal for commercial use) is a drug to treat alcoholism, it was first approved by the FDA in 1951.[138] Disulfiram targets aldehyde dehydrogenase (ALDH), thereby blocking the metabolism of alcohol.[139] After the consumption of alcohol patients on disulfiram quickly (5-10 min) experience adverse effects such as headaches and nausea. Disulfiram was also shown to eject zinc from histone lysine demethylase JMJD2A,[131] and hepatitis C virus NS5A protein.[127] The inhibition of hypoxia inducible transcription factor (HIF) and transcriptional coactivators p300/CBP by indandione and benzoquinone derivatives was also shown to proceed via a zinc ejection mechanism (Figure 8c).[130] Other examples include the ejection of structural zinc from metallothionein by organoselenium compound ebselen (Figure 8d).[140]

a) R O R NH S S O O S S O O N O S Ph O NH H N N S 2 2 N NH S N S 2 N O O S Me Ph HN R

3-Nitrosobenzamide Azidocarbonamide Dithiobis[benzamides] Benzisothiazolines Pentathiepin

b) c) O d) O S OH O N S NH S N R N Se S O O O

Disulfiram Indandiones Naphthoquinone Ebselen

Figure 8. Structures of commonly used inhibitors targeting reactive cysteine residues for a) human immunodeficiency virus (HIV) nucleocapsid (NCp) protein; b) aldehyde dehydrogenase (ALDH), histone lysine demethylase JMJD2A, and hepatitis C virus NS5A protein; c) hypoxia inducible transcription factor (HIF) and transcriptional coactivators p300/CBP and d) metallothionein.

37 1.8. Outline of this thesis

1 The goal of the work described in this thesis is to develop and evaluate novel inhibitors for histone lysine methyltransferases. The majority of the work is focussed on SETD7, a monomethyltransferase of lysine 4 on histone 3, which is implicated in several diseases. (R)-PFI-2, a potent and selective inhibitor reported by the SGC, is used as a template for the development of novel highly potent inhibitors in this thesis. Evaluation is performed by in vitro biochemical assays targeting recombinantly expressed human SETD7. In addition, a new approach towards the inhibition of G9a and GLP is presented.

Chapter 2 describes the structure-activity relationship studies on (R)-PFI-2, a potent and selective inhibitor of histone lysine methyltransferase SETD7. A library of29 analogues is synthesised and the ability of each compound to inhibit human SETD7’s methyltransferase activity is evaluatedin vitro using a matrix-assisted laser desorption- ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay.

In Chapter 3, building on the SAR exploration described in the previous chapter, our efforts towards the synthesis of novel (R)‑PFI‑2 analogues containing nucleophilic moieties are described. We introduced various nitrogen, oxygen, sulfur and carbon nucleophiles, and these analogues are subsequently tested for inhibitory activity against SETD7-catalysed methylation, but also as potential novel small-molecule substrates for methyltransferase catalysis.

Our efforts towards the replacement of the sulfonamide core of (R)-PFI-2 are described in Chapter 4. In this work we have used computation tools for scaffold hopping to identify potential bioisosteres of the sulfonamide. Using the van Leusen 3-component reaction, we have synthesised 56 (R)-PFI-2 analogues bearing a 1,5-disubstituted imidazole core. Each compound is tested for inhibition against SETD7.

Chapter 5 addresses the discovery and evaluation of inhibitors of histone lysine methyltransferases G9a and GLP. These inhibitors, in particular clinically approved ebselen, disulfiram, and cisplatin, covalently modify cysteine residues involved in zinc binding. Structural Zn(II) is ejected from G9a or GLP, and as a result, the secondary and tertiary structure is altered, resulting in a loss of methyltransferase activity.

Finally, in Chapter 6, the work described in Chapters 2-5 of this thesis is discussed, and perspectives for the future development of SETD7 inhibitors are described.

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44 1

45 46 2

Structure-activity relationship studies on (R)-PFI-2 analogues as inhibitors of histone lysine methyltransferase SETD7

This chapter has been published as: D. C. Lenstra, E. Damen, R. G. G. Leenders, R. H. Blaauw, F. P. J. T. Rutjes, A. Wegert and J. Mecinović, ChemMedChem. 2018, 13, 1405-1413.

47 Abstract

SETD7 is a histone H3K4 lysine methyltransferase involved in human gene regulation. Aberrant expression of SETD7 has been associated with various diseases, including cancer, therefore SETD7 is considered as a target for the development of new epigenetic drugs. To date, few selective small molecule inhibitors have been reported that target SETD7, the most potent being (R)-PFI-2. Here we report structure-activity relationship studies on (R)-PFI-2 and its analogues. A library of 29 structural analogues 2 of (R)-PFI-2 was synthesised and evaluated for inhibition of recombinantly expressed human SETD7. The key interactions were found to be a salt-bridge and a hydrogen bond + formed between (R)‑PFI-2’s NH2 and SETD7’s Asp256 and His252, respectively.

48 2.1. Introduction

Covalent posttranslational modifications on the unstructured N-terminal histone tails play an important role in regulating the activity of human genes.[1] These modifications are introduced by ‘writers’ and, among others, include methylation, acetylation, phosphorylation, citrullination, and ubiquitination.[2] Methylation is one of the most abundant and well-studied modifications on histones.[3] The transfer of a methyl group from cosubstrate S-adenosylmethionine (SAM) to lysine residues is catalysed by histone 2 lysine methyltransferases (HKMTs).[4] HKMTs can catalyse the addition of up to three methyl groups to the ε-amino group of lysine residues, affording methyllysine (Kme), dimethyllysine (Kme2) and trimethyllysine (Kme3).[5] With the exception of DOT1L, an H3K79 methyltransferase, all HKMTs contain a highly conserved SET (Su(var)3-9, enchanter-of-zeste, thritorax) domain, which is responsible for their catalytic activity.[6] SET domain containing lysine methyltransferase 7 (SETD7, also known as SET7/9) was originally identified as a monomethylase of lysine 4 on histone 3 (H3K4).[7] Unlike many other methyltransferases, SETD7 only monomethylates H3K4, but does not catalyse di- or trimethylation of lysine. This methylation mark (H3K4me) has a functional effect on the chromatin state resulting in activation of transcription. SETD7 has also been found to catalyse methylation of a range of non-histone proteins, including estrogen receptor α (ERα)[8], DNA methyltransferases 1 (DNMT1)[9], TAF10[10], FoxO3[11], transcription factors YY1 and Pdx1[12], androgen receptor[13], ARTD1[14], β‑catenin[15], and tumour suppressor p53[16]. Recent biomedical studies revealed that SETD7 is involved in multiple signalling pathways, and its aberrant activity has been linked to various types of cancer[17, 18] and vascular dysfunction in patients with type 2 diabetes.[19] The development of SETD7 small molecule inhibitors as chemical probes for studying their biological activity in cells is therefore highly desired and may lead to the development of therapeutic agents for the treatment of human diseases.[20-23]

To date, only a few selective SETD7 inhibitors have been identified. Clinically approved antihistaminic cyproheptadine was found to inhibit SETD7-mediated methylation of

H3K4 (IC50 of 3.4 µM). The substrate in this study was ERα, and inhibition led to blocking of estrogen-dependent breast cancer cell growth in MCF7 cells.[24] A structure-activity relationship study was performed, however, none of the analogues proved to be more potent than cyproheptadine.[25] Several SAM derivatives have also been reported to [26] inhibit SETD7 methyltransferase activity; for instance, DAAM-3 (IC50 10 µM) and DC- [27] S239 (IC50 4.59 µM). Unfortunately, SAM-competitive inhibitors usually have poor selectivity amongst other methyltransferases due to the highly conserved SAM binding domain in the methyltransferase family. The most potent known SETD7 inhibitor (IC50

49 = 2.0 ± 0.2 nM) is (R)‑PFI‑2 (Figure 1b-c), while its enantiomer (S)-PFI-2 proved to be a very weak SETD7 inhibitor, highlighting that the stereochemistry of the amino acid side chain defines its potency.[28] (R)‑PFI‑2 is a histone competitive inhibitor, and exhibits an inhibition potency in vitro and in cellular assays.[28] Comparative studies demonstrated that (R)-PFI-2 is selective for SETD7 over 18 other human methyltransferases. Although the structure of (R)-PFI-2 in complex with SETD7 has been solved (Figure 1b), no further analogues have been reported. To investigate which structural elements of (R)‑PFI‑2 are essential for effective inhibition of SETD7, we carried out a structure-activity 2 relationship (SAR) study. We modified three distinctive moieties that have a potential to crucially contribute to the potency of (R)-PFI-2: i) The amino acid side chain (Figure 1c, part A); ii) the pyrrolidine amide (Figure 1c, part B); and iii) the tetrahydroisoquinoline moiety (Figure 1c, part C).

Figure 1. a) SETD7-catalysed methylation of H3K4; b) Crystal structure of SETD7 (grey) in complex with (R)- PFI-2 (cyan) (PDB ID: 4JLG); c) Structure of (R)-PFI-2, with sites A, B, and C for SAR exploration.

50 2.2. Results and discussion

We initiated our investigations with the synthesis of (R)-PFI-2 (1) and its 29 structural analogues (Scheme 1). In general, the synthesis was adapted from literature,[28] and starts from an unnatural D‑amino acid; typically Boc-protected D-phenylalanine derivatives were coupled to the requisite amines using EDC, HOBt, and DIPEA. Then the Boc group was removed with TFA in DCM, followed by a basic work-up to afford the free amine. The sulfonyl part of the inhibitor was synthesised from various aryl 2 bromides (Scheme 1). These aryl bromides were first reacted with benzylmercaptan in the presence of Pd2(dba)3 and Xantphos to afford aryl benzyl sulfides, followed by oxidation with trichloroisocyanuric acid to yield the corresponding sulfonyl chlorides.

The sulfonyl chlorides were then coupled to the free amines in the presence of Et3N in DCM to afford the desired structural analogues of (R)-PFI-2.

Scheme 1. Synthesis of (R)-PFI-2 and its analogues 1-28. Reagents and conditions:i ) Boc2O, and NaOH in H2O/ dioxane (1:1 v/v), rt, 20 h; ii) EDC, HOBt, DIPEA, and R2NH in DCM, rt, 20 h; iii) TFA/DCM (1:1 v/v), rt, 2-4 h; iv) Pd2(dba)3, Xantphos, DIPEA, and benzylmercaptan in dioxane, 100 °C, 6 h; v) Benzyltrimethylammonium chloride, trichloroisocyanuric acid, and Na2CO3 in H2O/MeCN, 0 °C to rt, 1 h; vi) Et3N in DCM, rt, 4 h. Where applicable, Boc-protected compounds were deprotected using TFA/DCM (1:1 v/v).

Initially, all synthesised compounds were tested for their inhibitory activity usingan established matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay, monitoring the monomethylation of a synthetic 21-mer histone 3 peptide mimic containing a lysine at position 4.[29, 30] In a typical assay, 200 nM of SETD7, 10 µM of H3K4 histone peptide, 16 µM of SAM and 10 or 100 µM of compounds 1-30 were incubated for 1 h at 37 °C. For compounds showing >50% inhibition at 100 µM,

IC50 values were determined. In cases where <50% inhibition at 100 µM was observed,

IC50 values were not determined and this data is presented as IC50 >100 µM. Enzymatic

51 activity of these compounds at 100 µM concentration can be found in FigureS1in Section 2.4. Representative MALDI data of selected compounds at 10 µM are shown in Figure 2. In a control experiment without inhibitor (5% v/v of DMSO) nearly quantitative methylation was observed in the presence of human SETD7 (m/z = 2269.5) (Figure 2a). Under standard conditions, but in the presence of 10 µM of (R)-PFI-2 (compound 1), no methylation was observed in the MALDI spectrum (Figure 2b).

Figure 2. a) Representative MALDI-TOF MS data for 200 nM SETD7, 21-mer H3K4 peptide (10 μM), SAM (16 μM) in 50 mM glycine pH 8.8 containing 5% DMSO (v/v) after 1 h at 37 °C; b) 10 μM of (R)-PFI-2 (compound 1); c) 10 μM compound 9; d) 10 μM compound 14; e) 10 μM compound 27. Y-axis represents relative abundance (Rel abun).

We further examined the potency of (R)-PFI-2 against recombinantly expressed human

SETD7. Under our assay conditions, an IC50 value of 138 nM was obtained for (R)-PFI-2 (Figure 3, compound 1), indicating very effective inhibition of SETD7 methyltransferase [28, 30] activity. Previously reported IC50 values for (R)-PFI-2 were 2.0 and 55 nM, respectively;

the observed difference with our measured IC50 value is due to differences in the employed assay and assay conditions. Representative inhibition curves can be found

in Figure S2. Two isomers of 1, with ortho- (compound 2) and para-CF3 (compound

3) substituents also displayed excellent inhibitory activity with an IC50 of 203 and 173 nM, respectively. The potencies of analogues 4-6 bearing a methyl-, chloro-, or bromo functionality at the meta-position were evaluated next. All compounds appeared

excellent inhibitors of SETD7 with IC50 values ranging from 132 nM to 178 nM. Also compound 7, which does not contain any substituent on the phenyl ring, was a

52 good SETD7 inhibitor with an IC50 of 266 nM. Adding an extra fused ring, as in the naphthalene-containing compound 8, gave an IC50 of 145 nM, indicating that there is sufficient space available for additional functionalisation. Notably, removing the entire side chain (as in glycine-based compound 9) led to rather poor inhibition with an IC50 of 79 µM, implying that the aromatic ring is crucial for potency.

Figure 3. A library of (R)-PFI-2 (compound 1) and its analogues 2-30, showing the maximum half inhibitory concentrations (IC50 values).

Next, the effect of modifications in the pyrrolidine amide side chain was investigated (Figure 1c, part B). The analysis of the crystal structure of (R)-PFI-2 as cocrystal with SETD7 and SAM (PDB ID: 4JLG) illustrates that the pyrrolidine part of the inhibitor is

53 positioned inside the lysine channel pointing towards SAM (Figure 1b). Based on this structural information, it is clear that space in this pocket is limited and that a significant increase in the size of the pyrrolidine moiety would presumably not be tolerated. Indeed, 1-adamantyl substituted compound 10 did not show any inhibitory activity

against SETD7. No significant difference in IC50 was observed when the 5-membered ring of the pyrrolidine amide (compound 7, 266 nM) was replaced by a 4-membered

ring: an IC50 of 290 nM was obtained for azetidine derived analogue 11. By slightly increasing the ring size to piperidine or morpholine amide, as in analogues 12 and 13, 2 IC50 values of 1.3 and 1.9 µM were observed, respectively. This result shows that a slight increase in ring size from 5- to 6-membered already leads to a ~5-fold drop in potency (compound 7 vs. 12). Replacement of the 5-membered ring with acyclic diethylamine derivative 14, resulted in a higher degree of rotational freedom, which could lead to a steric clash with SETD7’s pocket; this proposition is in line with a surprisingly higher

IC50 value of 20 µM. An almost 10-fold lower IC50 of 2.6 µM was obtained by decreasing the size and rotational freedom in dimethylamine derivative 15. Removal of the entire pyrrolidine amide side chain (as in compound 16), resulted in the formation of an achiral compound. This significant structural change caused that 16 is no longer an inhibitor of SETD7 within the limits of detection.

Finally, the importance of the tetrahydroisoquinoline rings was investigated (Figure 1c, part C). The simplest analogues 17-19, consisting of a phenyl ring, either unsubstituted or with fluorine on the meta- or para-position, did not significantly inhibit SETD7 methyltransferase activity, indicating the essential role of the tetrahydroisoquinoline

scaffold of (R)-PFI-2 in inhibition of SETD7. IC50 values of >100 µM were observed for several heteroatom-substituted analogues 20-22, containing for instance a planar or nonplanar heterocyclic system. To investigate the importance of the fluorine

substituent, we synthesised analogue 23, which has an IC50 value of 532 nM; this is

approximately a two-fold increase with respect to fluorine-containing compound7 (IC50 = 266 nM). Approximately 10% inhibition of methyltransferase activity was observed in the presence of 100 µM of compound 24, an analogue of 7 that has the positively charged quaternary ammonium group (under physiological conditions) replaced by a neutral methylene group of equal size (Figure S1). This result demonstrates that the + positively charged NH2 of (R)-PFI-2 is involved in two interactions with SETD7, namely as a salt-bridge with Asp252 and a hydrogen bond with His252. Notably, when this nitrogen was translocated one position, as in tetrahydroquinoline25 , again no inhibition

was observed, whereas for compound 26, only a poor inhibition with an IC50 value of 20 µM was seen. Based on our examination of compounds 23-26, it is evident that the most important contributor to the excellent potency of (R)-PFI-2 is the nitrogen-

54 containing tetrahydroisoquinoline functionality. Having a benzylamine moiety instead of the cyclic structure, as in compound 27, a moderately active compound is obtained

(IC50 = 2.7 µM). With compound 28, a formation of a salt-bridge is no longer possible after changing the free amine to an amide functionality, and consequently no inhibition was observed. The two individual fragments of which the (R)-PFI-2 scaffold consists also showed no inhibitory activity (Figure 3, compounds 29 and 30).

Figure 4. Docking studies on various (R)-PFI-2 analogues (cyan) with SETD7 (cartoon, grey) with SAM (grey). SETD7 residues that form key interactions are shown in purple: a) Redocked structure of (R)-PFI-2 (compound 1), RMSD = 0.638; b) docking of compound 8, containing a naphthalene side chain; c) docking of compound 12, containing a piperidine amide functionality; d) docking of carbon analogue 24; e) docking of benzylamine-type compound 27; f) superimposition of structures shown in Figures 4a-e, with SETD7 surface visualisation (grey).

Docking studies were then carried out to obtain more detailed information about the potential binding modes of various (R)-PFI-2 analogues (Figures 4a-f). Initially, to confirm the correct docking mode, (R)-PFI-2 was successfully redocked into the structure of SETD7 (RMSD = 0.638 Å, Figure 4a). The ligand interaction diagram for SETD7-(R)-PFI-2 is shown in Figure S3. Compound 8 was docked into SETD7 and the binding mode of 8 was found to be similar to that of (R)-PFI-2 (Figure 4b). In the docked structure of 12, the piperidine group occupies the narrow lysine-binding channel; although it is somewhat larger than (R)-PFI-2’s pyrrolidine group, 12 does not noticeably clash with the protein (Figure 4c). Finally, two structures with the tetrahydroisoquinoline core

55 were docked. Despite being a very poor inhibitor of SETD7 (10% inhibition at 100 µM, Figure S1), docking of the carbon analogue 24 was successful and the apparent binding mode seems similar to that of (R)-PFI-2, in spite of the absence of a salt-bridge with Asp256 (Figure 4d). In contrast, the benzylamine-derived compound 27 does have a potential to form a salt-bridge with Asp256 under physiological conditions, and docking of this molecule into SETD7 confirmed the existence of the energetically favourable + salt-bridge between the NH3 of 27 and Asp256 from SETD7 (Figure 4e).

2 2.3. Conclusion

A library of 29 (R)-PFI-2 analogues was synthesised and evaluated as SETD7 inhibitors with the aim to determine which structural features contribute to the excellent potency of (R)-PFI-2. Our results demonstrate that small perturbations in the pyrrolidine amide side chain, i.e. piperidine amide 12 or dimethylamide 15 were tolerated relatively well, whereas substituents with more rotational freedom (e.g. diethylamide 14) or with increased size (e.g. adamantane 10) were not tolerated. Substitutions on the aromatic side chain led to highly potent (R)-PFI-2 analogues. The key interactions between SETD7 and (R)-PFI-2 were identified to be a salt-bridge and a hydrogen bond that are + formed between SETD7’s Asp256/His252 and the tetrahydroisoquinoline’s NH2 of (R)-PFI-2. Removal or repositioning of this ammonium group resulted in analogues that were inactive (compounds 24 and 25) or only poorly active (compound 26), whereas a replacement of the tetrahydroisoquinoline moiety by benzylamine (as in compound 27) resulted in a ~10 times less potent inhibitor. It is envisioned that this study will importantly contribute to the growing field of epigenetic drug discovery.

2.4. Supporting information

2.4.1. General experimental All chemicals were purchased from Sigma-Aldrich Chemicals, Acros Organics, Alfa Aesar, Fluorochem, and used without further purification. Histone lysine methyltransferase SETD7 was obtained as previously described. Synthetic 21-mer histone 3 peptide mimic (ARTKQTARKSTGGKAPRKQLA) was obtained as previously described by our group. Chemical reactions were carried out under constant magnetic stirring and under an inert atmosphere of argon where applicable. Standard syringe techniques were applied for the transfer of dry solvents and air- or moisture- sensitive reagents. Flash column chromatography was carried out on a Biotage Isolera ONE system using Silicycle SiliaSEP prepacked columns

56 with the indicated solvents. Thin layer chromatography (TLC) analyses was performed on glass backed silica sheets (Merck Silica Gel 60 F254) and visualised by UV fluorescence

(254 nm) and/or staining with potassium permanganate (KMnO4) or ninhydrin. Nuclear Mangetic Resonance (NMR) data were recorded at ambient temperature on a Bruker Avance III 400 MHz or Bruker Avance III 500 MHz spectrometer in the indicated deuterated solvents. 1H NMR chemical shifts are reported as δ in units of parts per million (ppm) relative to tetramethylsilane (TMS, δ 0.00 ppm) as the internal standard. 13C chemical shifts are reported as δ in units of parts per million (ppm) relative to CHCl (δ, 77.0 ppm). 3 2 Multiplicities are given as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), app (apparent). Coupling constants are given in Hz. Low resolution mass spectra (LRMS) were recorded on a Thermo Finnigan LCQ Advantage Max electrospray ion-trap mass spectrometer (ESI). High resolution mass spectra (HRMS) were recorded on a JEOL AccuTOF CS JMS-T100CS mass spectrometer. High resolution values were calculated to five decimal places from the molecular formula, all found within a tolerance of 5 ppm. Methylation of lysine 4 on a synthetic 21-mer peptide mimic (ARTKQTARKSTGGKAPRKQLA) of histone 3 was monitored on a Microflex LRF matrix- assisted laser desorption-ionisation time of flight (MALDI-TOF) mass spectrometer. m/zAll values are given in Daltons (Da). Purity of final compounds was assessed on a Shimadzu HPLC system containing a reverse phase C18 Prodigy ODS3 column (Phenomenex). All final compounds were found to be of at least 95% purity.

2.4.2. MALDI-TOF MS in vitro inhibition assay

For determination of maximum half inhibitory concentrations (IC50 values) a MALDI-TOF MS based assay monitoring methylation of a 21-mer histone 3 peptide mimic containing a lysine at position 4 was used. Briefly, SETD7 (200 nM final conc.) was preincubated with compound (100% DMSO stock solution at appropriate concentration) for 5 min at 37 °C in 18 µL of 50 mM glycine pH 8.8 as assay buffer. After this, 2 µL of a SAM (16 µM final concentration) and 21-mer peptide (10 µM final concentration) premixture was added to initiate the enzymatic reaction, thereby obtaining a final reaction volume of 20 µL containing 5% DMSO (v/v). The reaction was left to incubate for an additional 55 min at 37 °C after which it was quenched by the addition of 20 µL of MeOH. Each experiment was performed in triplicate.

For MALDI-TOF MS analysis, a 2 µL aliquot from the quenched reaction mixture was mixed with 8 µL of saturated HCCA matrix (α-cyano-4-hydroxycinnamic acid, Sigma- Aldrich). From this mixture 1 µL was deposited on the MALDI plate for crystallization. Methyltransferase activity was calculated by taking the peak areas of each methylation state (including all isotopes and adducts), and is expressed relative to the reaction

57 in which no compound was present (control experiment in which just 5% DMSO was present). The calculated activities were plotted against the corresponding compound concentrations and fitted to the Hill-equation using Origin Pro, representative inhibition curves are shown in Figure S2.

Compound 30 Compound 29 2 Compound 28 Compound 26 Compound 25 Compound 22 Compound 21 Compound 20 Compound 19 Compound 18 Compound 17 Compound 16 Compound 10

0 20 40 60 80 100 120 % Activity compared to dmso

Figure S1. SETD7 enzymatic activity at 100 µM compound concentrations for compounds that have IC50 > 100 µM

Figure S2. Representative inhibition curves for compounds2 (IC50 = 203 ± 10 nM), 12 (IC50 = 1.3 ± 0.2 µM), 14

(IC50 = 20 ± 0.3 µM), and 27 (IC50 = 2.7 ± 0.2 µM).

58 2.4.3. Molecular docking The crystal structure of SETD7 in complex with (R)-PFI-2 and S-adenosylmethionine (SAM) was downloaded from the Protein Data Bank (PDB ID: 4JLG). The protein-ligand complex (chain A) was prepared for docking using the QuickPrep wizard in Molecular Operating Environment (MOE).[31] All compounds to be docked were prepared using the default settings of the Wash wizard as implemented in MOE. Molecular docking was performed using Molegro Virtual Docker v6.0.[32] The active binding site region was defined as a spherical region, which encompasses all protein within 10.0 Åof 2 bound (R)-PFI-2. Both the MolDock Score [GRID] and PLANTS Score [GRID] with a grid resolution of 0.2 Å were evaluated to score and rank the 5 best docking poses for each compound. MolDock SE was selected as search algorithm using its default settings. All figures we prepared with PyMOL visualization software.[33]

Figure S3. Ligand interaction diagram of (R)-PFI-2 and SETD7 (PDB ID: 4JLG), prepared with Molecular Operating Environment (MOE).

59 2.4.5. Characterisation of compounds N-(4-Bromo-2-fluorobenzyl)-2,2-dimethoxyethan-1-amine (S4): Prepared according to the procedure described in literature.[28] A 500 mL round bottom flask was charged with 4-bromo-2- fluorobenzaldehyde (S1, 10.2 g, 50 mmol, 1.0 equiv) and toluene (200 mL) followed by the addition of aminoacetaldehyde dimethyl acetal (S2, 7.99 g, 75 mmol, 1.5 equiv). The reaction mixture was refluxed for 16 h using a Dean Stark apparatus after which the solvent was evaporated. The 2 crude imine S3 was redissolved in MeOH (300 mL) and NaBH4 (2.08 g, 55 mmol, 1.1 equiv) was added whilst cooling on an ice-water bath. The reaction mixture was left to stir at rt for 4 h. Subsequently the reaction mixture was quenched with ice-water and the solvent was evaporated. The crude mixture was taken in EtOAc (200 mL) and

washed with brine (200 mL), dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash column chromatography (50-80% EtOAc in n-heptane) to afford 12.7 g (87% isolated yield) of N-(4-bromo-2-fluorobenzyl)-2,2- 1 dimethoxyethanamine S4 as a yellow liquid. TLC Rf = 0.3 (n-heptane/EtOAc 1:2). H

NMR (CDCl3, 400 MHz) δ 7.30 – 7.18 (m, 3H), 4.47 (t, J = 5.5 Hz, 1H), 3.81 (s, 2H), 3.37 13 (s, 6H), 2.72 (d, J = 5.5 Hz, 2H), 1.62 (bs, 1H); C NMR (100 MHz, CDCl3) δ 162.1, 159.7, 131.4, 131.4, 127.4, 127.4, 126.3, 126.2, 120.9, 120.8, 119.1, 118.8, 103.8, 54.0, 50.4, 19 + 46.7, 46.6; F NMR (377 MHz, CDCl3) δ –116.50; MS (ESI) m/z 291.9 [M+H] .

N-(4-Bromo-2-fluorobenzyl)-N-(2,2-dimethoxyethyl)-4-methylbenzenesulfonamide (S5): Prepared according to the procedure described in literature.[28] N-(4- bromo-2-fluorobenzyl)-2,2-dimethoxyethanamine (S4, 12.5 g, 42.8 mmol, 1.0 equiv) was dissolved in DCM (143 mL, 0.3 M) followed by

the addition of DMAP (260 mg, 2.14 mmol, 0.05 equiv), Et3N (8.7 g, 85.6 mmol, 2.0 equiv), and p-toluenesulfonyl chloride (9.8 g, 51.4 mmol, 1.2 equiv) whilst cooling on an ice-water bath. The reaction mixture was stirred at rt for 4 h after which it was quenched by dropwise addition of ice-water. The crude mixture was then diluted with DCM (150 mL), washed with water (150 mL), brine (150

mL) and dried over Na2SO4, filtered, and concentrated in vacuo. The crude product wash purified by flash column chromatography (5-15% EtOAc in n-heptane) to afford 14.9 g (78% isolated yield) of N-(4-bromo-2-fluorobenzyl)-N-(2,2-dimethoxyethyl)-4-

methylbenzenesulfonamide S5 as a colourless oil which crystallised over time. TLC Rf = 1 0.6 (n-heptane: EtOAc 1:4). H NMR (CDCl3, 400 MHz) δ 7.67 (d, J = 8.3 Hz, 2H), 7.35 – 7.22 (m, 4H), 7.15 (dd, J = 1.9, 9.5 Hz, 1H), 4.45 (bs, 2H), 4.38 (t, J = 5.3 Hz, 1H), 3.27 – 13 3.23 (m, 8H), 2.43 (s, 3H); C NMR (100 MHz, CDCl3) δ 161.7, 159.2, 143.6, 136.8, 131.8, 131.7, 129.7, 127.5, 127.5, 127.2, 123.2, 123.1, 121.6, 121.5, 118.9, 118.6, 104.0, 54.7,

60 19 + 50.3, 46.3, 46.2, 21.5; F NMR (377 MHz, CDCl3) δ –115.57; MS (ESI) m/z 470.1 [M+Na] .

6-Bromo-8-fluoroisoquinoline (S6): Prepared according to the procedure described in in literature.[28] A

Schlenk flask was charged with AlCl3 (17.3 g, 130 mmol, 4.0 equiv) and DCM (120 mL) and whilst cooling on an ice-water bath N‑(4‑bromo-2- fluorobenzyl)-N-(2,2-dimethoxyethyl)-4-methylbenzenesulfonamide (S5, 14.5 g, 32.5 mmol, 1.0 equiv) in DCM (60 mL) was added under protected 2 atmosphere. The reaction was left to stir overnight at rt. Subsequently the reaction mixture was quenched by dropwise addition of an aqueous 10% HCl solution, diluted with DCM (100 mL), washed with satd aq NaHCO3 (2 × 250 mL), and brine (250 mL). The organic layer was dried over Na2SO4, filtered and dried in vacuo. The crude product was purified by flash column chromatography (5 - 15% EtOAc in n-heptane) to afford 3.0 g

(41% isolated yield) of 6-bromo-8-fluoroisoquinoline S6 as a yellow solid. TLC Rf = 0.3 1 (n-heptane/EtOAc 3:1); H NMR (CDCl3, 400 MHz) δ 9.50 (s, 1H), 8.63 (d, J = 5.8 Hz, 1H), 7.85 – 7.79 (m, 1H), 7.62 – 7.55 (m, 1H), 7.40 (dd, J = 1.6, 9.5 Hz, 1H); 13C NMR (126 MHz,

CDCl3) δ 159.9, 157.8, 146.3, 146.2, 144.8, 137.6, 137.5, 124.9, 124.9, 124.5, 124.4, 119.0, 19 118.9, 117.9, 117.8, 115.7, 115.5.; F NMR (377 MHz, CDCl3) δ –120.15; MS (ESI) m/z 226.2 + [M+H] .

6-Bromo-8-fluoro-1,2,3,4-tetrahydroisoquinoline (S7): Prepared according to the procedure described in literature.[28] 6-Bromo-8- fluoroisoquinoline (S6, 3.0 g, 13.3 mmol, 1.0 equiv) was dissolved in a

THF:acetic acid mixture (50 mL, 2:1, 0.25 M) and NaBH4 (1.5 g, 39.8 mmol, 3.0 equiv) was added whilst cooling on an ice-water bath. The reaction mixture was stirred rt for 4 h, after which it was quenched with a satd aq NaHCO3 solution. The product was extracted with EtOAc (3 × 40 mL), the combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated in vacuo to 1 obtain a crude yellow solid containing compound S7. TLC Rf = 0.1 (DCM/MeOH 9:1); H

NMR (CDCl3, 400 MHz) δ 7.05 (bs, 1H), 7.01 (dd, J = 1.9, 8.9 Hz, 1H), 3.95 (s, 2H), 3.09 (t, J = 5.9 Hz, 2H), 2.76 (t, J = 5.9 Hz, 2H); MS (ESI) m/z 365.9 [M+K]+.

General procedure I (Boc protection of aryl bromides) tert-Butyl 6-bromo-8-fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate (S8): The crude 6-bromo-8-fluoro-1,2,3,4,-tetrahydroisoquinoline (S7) was dissolved in THF (90 mL) followed by the addition of DIPEA (2.6 g, 20

mmol, 1.5 equiv) and (Boc)2O (3.5 g, 16 mmol, 1.2 equiv). The reaction mixture was stirred at rt overnight after which the solvent was removed

61 in vacuo. The crude product was redissolved in EtOAc (150 mL) and washed with water

(2 × 150 mL), brine (150 mL), dried over Na2SO4, filtered and concentratedin vacuo. The crude product was purified by flash column chromatography (0-10% EtOAc inn -heptane) to afford 3.64 g (83%, over 2 steps from S6) of tert-butyl 6-bromo-8-fluoro-1,2,3,4,- tetrahydroisoquinoline-2(1H)-carboxylate (S8) as a slightly yellow oil. 1H NMR (400

MHz, CDCl3) δ 7.13 – 7.03 (m, 2H), 4.51 (bs, 2H), 3.63 (t, J = 5.8 Hz, 2H), 2.81 (t, J = 5.8 13 Hz, 2H), 1.49 (s, 9H); C NMR (126 MHz, CDCl3) δ 160.2, 158.2, 154.7, 138.8, 127.3, 120.6, 119.7, 119.6, 116.3, 116.2, 80.3, 41.2, 40.5, 40.0, 28.5, 28.5, 28.4; 19F NMR (377 2 MHz, CDCl3) δ –117.15 (d, J = 241.6 Hz).

tert-Butyl 6-bromo-3,4-dihydroisoquinoline-2(1H)-carboxylate (S9): 6-Bromo-1,2,3,4-tetrahydroisoquinoline (400 mg, 1.9 mmol) was protected according to general procedure I. After column chromatography (0-5% EtOAc in n-heptane) 570 mg (97% isolated 1 yield) of compound S9 was obtained as a colourless oil. H NMR (400 MHz, CDCl3) δ 7.32 – 7.27 (m, 2H), 7.00 – 6.94 (m, 1H), 4.51 (s, 2H), 3.62 (t, J = 5.8 Hz, 2H), 2.80 (t, J = 13 5.8 Hz, 2H), 1.49 (s, 9H); C NMR (101 MHz, CDCl3) δ 154.8, 137.0, 131.5, 129.3, 128.0, 119.9, 85.2, 80.0, 28.8, 28.5, 27.4.

tert-Butyl 6-bromo-3,4-dihydroquinoline-1(2H)-carboxylate (S10): 6-Bromo-1,2,3,4-tetrahydroquinoline (500 mg, 2.2 mmol) was protected according to general procedure I. After column chromatography (0-5% EtOAc in n-heptane) 508 mg (60% isolated yield) of compound S10 was 1 obtained as a white solid. H NMR (400 MHz, CDCl3) δ 7.58 – 7.53 (m, 1H), 7.25 – 7.19 (m, 2H), 3.73 – 3.62 (m, 2H), 2.76 – 2.69 (t, J = 6.6 Hz, 2H), 1.94 – 1.86 13 (m, 2H), 1.52 – 1.50 (s, 9H); C NMR (101 MHz, CDCl3) δ 153.7, 146.7, 137.7, 132.0, 131.1, 128.7, 125.7, 115.9, 85.2, 81.1, 44.6, 28.4, 27.4, 27.4, 23.2.

tert-Butyl (4-bromo-2-fluorobenzyl)carbamate (S11): 4-Bromo-2-fluorobenzylamine (500 mg, 2.5 mmol) was protected according to general procedure I. After column chromatography (0-5% EtOAc in n-heptane) 672 mg (97% isolated yield) of compound S11 was 1 obtained as an off-white solid; H NMR (400 MHz, CDCl3) δ 7.33 – 7.15 13 (m, 3H), 4.92 (bs, 1H), 4.30 (d, J = 6.3 Hz, 2H), 1.44 (s, 9H; C NMR (101 MHz, CDCl3) δ 161.9, 159.4, 155.7, 131.0, 130.9, 127.5, 127.5, 125.4, 125.2, 121.3, 121.2, 119.1, 19 118.9, 79.8, 38.2, 28.4, 25.2; F NMR (377 MHz, CDCl3) δ –116.5.

62 General procedure II (Synthesis of compounds S12-S15) tert-Butyl 6-(benzylthio)-8-fluoro-1,2,3,4‑tetrahydroisoquinoline-2(1H)-carboxylate (S12): Prepared according to the procedure described in literature.[28] A round bottom flask was charged with 6-bromo-8-fluoro-1,2,3,4,- tetrahydroisoquinoline-2(1H)-carboxylate (S8, 1.55 g, 4.7 mmol, 1.0 equiv) and to this dioxane (15 mL) and DIPEA (1.21 g, 9.4 mmol, 2.0 equiv) were added. The mixture was degassed with argon. Subsequently, Xantphos (272 mg, 0.47 mmol, 0.1 equiv), Pd (dba) (215 mg, 0.24 mmol, 0.05 equiv), and 2 3 2 benzylmercaptan (0.7 g, 5.6 mmol, 1.2 equiv) were added and the reaction mixture was refluxed for 6 h. The solvent was removed in vacuo and the crude product redissolved in EtOAc (50 mL) followed by filtration over celite. The mixture was washed with 2.0 M aqueous NaOH (2 × 50 mL), water (50 mL), brine (50 mL), dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (5-15% EtOAc in n-heptane) to afford 1.5 g (86% isolated yield) oftert - butyl 6-(benzylthio)-8-fluoro-1,2,3,4,-tetrahydroisoquinoline-2(1H)-carboxylate S12 as 1 a yellow oil. TLC Rf = 0.5 (EtOAc/n-heptane 1:4). H NMR (CDCl3, 400 MHz) δ 7.34 – 7.20 (m, 5H), 6.93 – 6.83 (m, 2H), 4.52 (bs, 2H), 4.09 (s, 2H), 3.61 (t, J = 5.8 Hz, 2H), 2.75 (t, 19 J = 5.8 Hz, 2H), 1.49 (s, 9H); F NMR (377 MHz, CDCl3) δ –119.07 (d, J = 237.13 Hz). tert-Butyl 6-(benzylthio)-3,4-dihydroisoquinoline-2(1H)-carboxylate (S13): Following general procedure II, 540 mg (95% isolated yield) of S13 was 1 obtained as a yellow oil. H NMR (500 MHz, CDCl3) δ 7.32 – 7.19 (m, 5H), 7.13 (dd, J = 8.0, 1.9 Hz, 1H), 7.07 (bs, 1H), 6.99 (d, J = 8.0 Hz, 1H), 4.52 (s, 2H), 4.09 (s, 2H), 3.61 (t, J = 5.9 Hz, 2H), 2.75 (t, J = 5.9 Hz, 2H), 1.49 (s, 9H). tert-Butyl 6-(benzylthio)-3,4-dihydroquinoline-2(1H)-carboxylate (S14): Following general procedure II, 429 mg (94% isolated yield) of S14 was 1 obtained as a yellow solid. H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 8.7 Hz, 1H), 7.30 – 7.19 (m, 5H), 7.09 (ddt, J = 8.7, 2.1, 0.7 Hz, 1H), 7.01 (dt, J = 2.1, 0.7 Hz, 1H), 4.04 (s, 2H), 3.71 – 3.63 (m, 2H), 2.67 (t, J = 6.6 Hz, 2H), 1.94 – 1.82 (m, 2H), 1.51 (s, 9H). tert-Butyl (4-(benzylthio)-2-trifluorobenzyl)carbamate (S15): Following general procedure II, 457 mg (82% isolated yield) of S15 1 was obtained as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.32 – 7.18 (m, 5H), 7.03 (dd, J = 8.0, 1.8 Hz, 1H), 6.97 (dd, J = 10.5, 1.8 Hz, 1H), 4.86 (bs, 1H), 4.29 (d, J = 6.1 Hz, 2H), 4.11 (s, 2H), 1.44 (s, 9H); 19F

NMR (377 MHz, CDCl3) δ –118.4.

63 General procedure III (Synthesis of sulfonyl chlorides S16-S19) tert-Butyl 6-(chlorosulfonyl)-8-fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate (S16): Sulfonyl chlorides were synthesised according to the procedure described in literature.[28] In short, benzyltrimethylammonium chloride (2.0 g, 9 mmol, 3.4 equiv) was dissolved in 6 mL of water. To this was added trichloroisocyanuric acid (0.9 g, 4 mmol, 1.5 equiv) dissolved in 10 mL of MeCN and the mixture was left to stir for 0.5 h at rt. Next, whilst cooling on an ice-water bath, the mixture was added drop-wise to a solution of compound S12 (1.0 g, 2 3 mmol, 1.0 equiv) in MeCN (20 mL) followed by the addition of 1.0 sodium carbonate (3 mL, 3 mmol). The reaction mixture was left to stir at 0 °C for 1.0 h after which it was

quenched by the addition of satd aq NaHCO3 (20 mL). The reaction mixture was filtered to remove any precipitate. The mixture was extracted with EtOAc (2 × 20 mL) and the

combined organic layers were washed with brine (40 mL), dried over Na2SO4, and concentrated in vacuo. After column chromatography (10-20% EtOAc in n-heptane) 522 mg (60% isolated yield) of tert-butyl 6-(chlorosulfonyl)-8-fluoro-3,4-dihydroisoquinoline- 2(1H)-carboxylate (S16) was obtained as a colourless oil which crystallised to form a 1 white solid over time. H NMR (500 MHz, CDCl3) δ 7.70 – 7.65 (bs, 1H), 7.63 – 7.57 (dd, J = 8.2, 1.8 Hz, 1H), 4.68 (bs, 2H), 3.71 (t, J = 5.8 Hz, 2H), 2.96 (t, J = 5.8 Hz, 2H), 1.51 (s, 9H); 13 C NMR (126 MHz, CDCl3) δ 159.9, 157.9, 154.4, 142.9, 142.8, 139.6 (bs), 130.0 (bs), 19 123.0, 111.5, 111.3, 80.8, 65.4, 40.9 (bs), 39.6 (bs), 28.9, 28.4; F NMR (377 MHz, CDCl3) δ –114.82 (d, J = 222.7 Hz).

tert-Butyl 6-(chlorosulfonyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (S17): Following general procedure III, 253 mg (54% isolated yield) of S17 1 was obtained as a white solid. H NMR (500 MHz, CDCl3) δ 7.89 – 7.82 (m, 2H), 7.37 (d, J = 8.1 Hz, 1H), 4.70 (s, 2H), 3.72 (t, J = 5.7 Hz, 2H), 13 2.97 (t, J = 5.7 Hz, 2H), 1.52 (s, 9H); C NMR (126 MHz, CDCl3) δ 154.5, 142.3, 137.0 (bs), 127.8 (bs), 127.4 (bs), 124.6, 80.5, 46.2 (bs), 45.4 (bs), 41.1 (bs), 39.9 (bs), 29.0, 28.4.

tert-Butyl 6-(chlorosulfonyl)-3,4-dihydroquinoline-1(2H)-carboxylate (S18): Following general procedure III, 100 mg (54% isolated yield) of S18 was 1 obtained as a white solid. H NMR (500 MHz, CDCl3) δ 8.04 (d, J = 9.0 Hz, 1H), 7.81 – 7.76 (d, J = 9.0 Hz, 1H), 7.74 (bs, 1H), 3.77 (t, J = 6.2 Hz, 2H), 2.85 (t, J = 6.3 Hz, 2H), 1.98 (quint, J = 6.3 Hz, 2H); 13C NMR (126 MHz,

CDCl3) δ 153.3, 145.2, 137.4, 130.5, 127.5, 125.1, 123.9, 82.4, 45.4, 28.3, 28.0, 22.6.

64 tert-Butyl (4-(chlorosulfonyl)-2-fluorobenzyl)carbamate (S19): Following general procedure III, 72 mg (45% isolated yield) of S19 1 was obtained as a white solid. H NMR (400 MHz, CDCl3) δ 7.85 – 7.81 (dd, J = 8.2, 1.9 Hz, 1H), 7.75 – 7.69 (dd, J = 8.7, 1.9 Hz, 1H), 7.67 – 7.61 (t, J = 7.5 Hz, 1H), 5.06 (bs, 1H), 4.48 – 4.44 (d, J = 6.3 Hz, 13 2H), 1.47 – 1.45 (s, 9H); C NMR (101 MHz, CDCl3) δ 161.2, 158.7, 155.7, 144.2, 144.1, 134.9, 134.7, 130.6, 123.0, 123.0, 114.5, 114.2, 80.4, 38.4, 28.3; 19F NMR (377 MHz, CDCl ) δ –113.5. 3 2

General procedure IV (Synthesis of compounds S20-S22) (tert-Butoxycarbonyl)-D-phenylalanine (S20): D-Phenylalanine (5.0 g, 30.3 mmol, 1.0 equiv) was dissolved in 60 mL of water and dioxane (1:1 v/v), followed by the addition of 1.0 M NaOH (30

mL, 1.0 equiv), and Boc2O (7.3 g, 33.3 mmol, 1.1 equiv). The reaction mixture was stirred at rt overnight. Dioxane was evaporated and subsequently the pH was adjusted to 2–4 by adding aqueous 1.0 M HCl. The product was extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with brine (75 mL) and dried over Na2SO4, filtered and concentrated in vacuo to afford N-Boc-D-phenylalanine S20 as a sticky colourless oil. 1H NMR (400 MHz,

DMSO-d6) δ 7.27 – 7.12 (m, 5H), 6.98 (d, J = 8.2 Hz, 1H), 4.03 (td, J = 10.1, 8.2, 4.5 Hz, 1H), 2.97 (dd, J = 13.8, 4.5 Hz, 1H), 2.78 (dd, J = 13.8, 10.1 Hz, 1H), 1.28 (s, 9H); MS (ESI) m/z 264.1 [M-H]-. Compound S20 was used without further purification in the next reaction.

(R)-2-((tert-Butoxycarbonyl)amino)-3-(4-(trifluoromethyl)phenyl)propanoic acid (S21): Crude compound S21 was obtained as a white solid according to general procedure IV starting from (R)-2-amino-3-(4- trifluoromethylphenyl)propanoic acid (200 mg, 0.86 mmol). 1H NMR

(400 MHz, CD3OD) δ 7.59 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 7.9 Hz, 2H), 4.43 (dd, J = 9.4, 5.1 Hz, 1H), 3.28 (dd, J = 13.8, 5.1 Hz, 1H), 3.00 (dd, J = 13.8, 9.35 Hz, 1H), 1.38 (s, 9H); MS (ESI) m/z 231.9 [M-H]-. Compound S21 was used without further purification in the next reaction.

(R)-2-((tert-Butoxycarbonyl)amino)-3-(naphthalen-2-yl)propanoic acid (S22): Crude compound S22 was obtained as an off white solid according to general procedure IV starting from (R)-2-amino-3-(naphthalen-2-yl) 1 propanoic acid (200 mg, 0.86 mmol). H NMR (400 MHz, CD3OD) δ 7.70 – 7.64 (m, 3H), 7.57 (s, 1H), 7.37 – 7.23 (m, 3H), 4.37 (dd, J = 9.1, 5.0 Hz,

65 1H), 3.26 – 3.18 (m, 1H), 2.97 (dd, J = 13.8, 9.1 Hz, 1H), 1.21 (s, 9H); MS (ESI) m/z 313.9 - [M-H] . Compound S22 was used without further purification in the next reaction.

General procedure V (Synthesis of amides S23-S37) tert-Butyl (R)-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (S23): N-Boc-D-phenylalanine S20 (1.5 g, 5.7 mmol, 1.0 equiv), HOBt (1.2 g, 7.6 mmol, 1.35 equiv), and EDC (1.5 g, 7.6 mmol, 1.35 equiv) were dissolved in DCM (20 mL), followed by the addition of DIPEA (2.2 g, 17 mmol, 3.0 2 equiv), and pyrrolidine (0.48 g, 6.8 mmol, 1.2 equiv). The reaction mixture was stirred overnight at rt. The reaction mixture was diluted with DCM (20 mL), and subsequently washed with 1.0 M NaOH (40 mL), 1.0 M HCl

(40 mL), and brine (40 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product, which purified using column chromatography (20-50% EtOAc in n-heptane) to affordtert -butyl (R)-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl) 1 propan-2-yl)carbamate (S23, 1.6 g, 87% isolated yield). H NMR (400 MHz, CDCl3) δ 7.30 – 7.18 (m, 5H), 5.40 (d, J = 8.8 Hz, 1H), 4.58 (td, J = 8.8, 6.0 Hz, 1H), 3.48 – 3.22 (m, 4H), 3.02 – 2.90 (m, 2H), 2.60 – 2.51 (m, 1H), 1.77 – 1.49 (m, 4H), 1.42 (s, 9H); MS (ESI) m/z 318.9 [M+H]+.

tert-Butyl (R)-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl) carbamate (S24): Compound S24 (308 mg, 80% isolated yield) was prepared according to general procedure V starting from (R)-2-(tert-butoxycarbonyl)amino)-3- (3-(trifluoromethyl)phenyl)propanoic acid (333 mg, 1.0 mmol, 1.0 equiv) and pyrrolidine (85.3 mg, 1.2 mmol, 1.2 equiv). 1H NMR (400 MHz,

CDCl3) δ 7.54 – 7.36 (m, 4H), 5.51 – 5.45 (d, J = 8.8 Hz, 1H), 4.66 – 4.54 (q, J = 7.7 Hz, 1H), 3.49 – 3.37 (m, 2H), 3.37 – 3.26 (m, 1H), 3.07 – 3.00 (d, J = 7.3 Hz, 2H), 2.67 – 2.60 (m, 1H), 1.86 – 1.54 (m, 4H), 1.43 – 1.38 (s, 9H); MS (ESI) m/z 386.9 [M+H]+.

tert-Butyl (R)-(1-oxo-1-(pyrrolidin-1-yl)-3-(2-(trifluoromethyl)phenyl)propan-2-yl) carbamate (S25): Compound S25 (292 mg, 76% isolated yield) was prepared according to general procedure V starting from (R)-2-(tert-butoxycarbonyl)amino)-3- (2-(trifluoromethyl)phenyl)propanoic acid (333 mg, 1.0 mmol, 1.0 equiv) 1 and pyrrolidine (85.3 mg, 1.2 mmol, 1.2 equiv). H NMR (500 MHz, CDCl3) δ 7.69 – 7.65 (m, 1H), 7.51 – 7.46 (m, 1H), 7.39 – 7.34 (m, 2H), 3.83 (t, J = 7.4 Hz, 1H), 3.53 – 3.46 (m, 1H), 3.43 – 3.34 (m, 2H), 3.12 (dd, J = 13.6, 7.4

66 Hz, 1H), 3.06 (dd, J = 13.6, 7.4 Hz, 1H), 2.77 – 2.70 (m, 1H), 1.87 – 1.62 (m, 6H); MS (ESI) m/z 386.9 [M+H]+. tert-Butyl (R)-(1-oxo-1-(pyrrolidin-1-yl)-3-(4-(trifluoromethyl)phenyl)propan-2-yl) carbamate (S26): Compound S26 (332 mg, 86% isolated yield) was prepared according to general procedure V starting from compound S21 (333 mg, 1.0 mmol, 1.0 equiv) and pyrrolidine (85.3 mg, 1.2 mmol, 1.2 equiv). 1H 2 NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 5.31 (d, J = 8.8 Hz, 1H), 4.55 (q, J = 7.6 Hz, 1H), 3.44 – 3.31 (m, 2H), 3.31 – 3.18 (m, 1H), 3.00 – 2.90 (m, 2H), 2.73 – 2.62 (m, 1H), 1.83 – 1.51 (m, 4H), 1.33 (s, 9H); MS (ESI) m/z 386.9 [M+H]+. tert-Butyl (R)-(1-oxo-1-(pyrrolidin-1-yl)-3-(m-tolyl)propan-2-yl)carbamate (S27): Compound S27 (310 mg, 93% isolated yield) was prepared according to general procedure V starting from (R)-2-(tert-butoxycarbonyl)amino)-3- (3-tolyl)propanoic acid (279 mg, 1.0 mmol, 1.0 equiv) and pyrrolidine 1 (85.3 mg, 1.2 mmol, 1.2 equiv). H NMR (400 MHz, CDCl3) δ 7.15 (t, J = 7.7 Hz, 1H), 7.05 – 6.97 (m, 3H), 5.43 (d, J = 8.7 Hz, 1H), 4.56 (q, J = 8.7, 5.9 Hz, 1H), 3.50 – 3.26 (m, 3H), 3.00 – 2.85 (m, 2H), 2.59 – 2.50 (m, 1H), 1.79 – 1.48 (m, 4H), 1.42 (s, 9H); MS (ESI) m/z 332.9 [M+H]+. tert-Butyl (R)-(3-(3-chlorophenyl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (S28): Compound S28 (301 mg,85% isolated yield) was prepared according to general procedure V starting from (R)-2-(tert-butoxycarbonyl)amino)-3- (3-chlorophenyl)propanoic acid (300 mg, 1.0 mmol, 1.0 equiv) and 1 pyrrolidine (85.3 mg, 1.2 mmol, 1.2 equiv). H NMR (400 MHz, CDCl3) δ 7.20 (d, J = 3.5 Hz, 3H), 7.14 – 7.08 (m, 1H), 5.46 (d, J = 8.8 Hz, 1H), 4.57 (q, J = 7.8 Hz, 1H), 3.51 – 3.29 (m, 3H), 2.95 (d, J = 7.3 Hz, 2H), 2.73 – 2.64 (m, 1H), 1.86 – 1.59 (m, 4H), 1.41 (s, 9H); MS (ESI) m/z 353.9 [M+H]+. tert-Butyl (R)-(3-(3-bromophenyl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (S29): Compound S29 (361 mg, 91% isolated yield) was prepared according to general procedure V starting from (R)-2-(tert-butoxycarbonyl)amino)-3- (3-bromophenyl)propanoic acid (344 mg, 1.0 mmol, 1.0 equiv) and 1 pyrrolidine (85.3 mg, 1.2 mmol, 1.2 equiv). H NMR (400 MHz, CDCl3) δ 7.39 – 7.33 (m, 2H), 7.18 – 7.12 (m, 2H), 5.42 (d, J = 8.8, 1H), 4.56 (q, J = 8.1, 1H), 3.49 – 3.30 (m, 3H), 2.96 – 2.90 (m, 2H), 2.69 – 2.59 (m, 1H),

67 1.84 – 1.58 (m, 4H), 1.42 (s, 9H); MS (ESI) m/z 396.8 [M+H]+.

tert-Butyl (R)-(3-(naphthalen-2-yl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (S30): Compound S30 (304 mg, 83% isolated yield) was prepared according to general procedure V starting from compound S22 (315 mg, 1.0 mmol, 1.0 equiv) and pyrrolidine (85.3 mg, 1.2 mmol, 1.2 equiv). 1H NMR (400

MHz, CDCl3) δ 7.82 – 7.73 (m, 3H), 7.68 – 7.65 (m, 1H), 7.49 – 7.40 (m, 2H), 7.35 (dd, J = 8.4, 1.8 Hz, 1H), 5.45 (d, J = 8.8 Hz, 1H), 4.68 (td, J = 2 8.8, 6.3 Hz, 1H), 3.50 – 3.25 (m, 3H), 3.18 – 3.06 (m, 2H), 2.56 – 2.45 (m, 1H), 1.73 – 1.46 (m, 3H), 1.41 (s, 9H); MS (ESI) m/z 368.9 [M+H]+.

tert-Butyl (2-oxo-2-(pyrrolidin-1-yl)ethyl)carbamate (S31): Compound S31 (423 mg, 93% isolated yield) was prepared according to general procedure V starting from (tert-butoxycarbonyl)glycine (350 mg, 2.0 mmol, 2.0 equiv) and pyrrolidine (170 mg, 2.4 mmol, 1.2 equiv). 1H NMR

(400 MHz, CDCl3) δ 5.56 – 5.42 (bs, 1H), 3.93 – 3.85 (d, J = 4.4 Hz, 2H), 3.54 – 3.45 (m, 2H), 3.40 – 3.32 (t, J = 6.8 Hz, 2H), 2.04 – 1.93 (m, 2H), 1.91 – 1.80 (m, 2H), 1.50 – 1.38 (s, 9H); MS (ESI) m/z 228.9 [M+H]+.

tert-Butyl ((R)-1-(((3S,5S,7S)-adamantan-1-yl)amino)-1-oxo-3-phenylpropan-2-yl) carbamate (S32): Compound S32 (319 mg, 80% isolated yield) was prepared according to general procedure V starting compoundS20 (265 mg, 1.0 mmol, 1.0 equiv) and 1-adamantylamine (181 mg, 1.2 mmol, 1.2 equiv). 1H NMR (400 MHz,

CDCl3) δ 7.34 – 7.20 (m, 5H), 5.20 (bs, 1H), 4.15 (bs, 1H), 3.09 (dd, J = 13.3, 5.9 Hz, 1H), 2.91 (t, J = 13.3, 8.3 Hz, 1H), 2.02 (bs, 2H), 1.85 – 1.81 (m, 6H), 1.69 – 1.56 (m, 6H), 1.43 (s, 9H); MS (ESI) m/z 399.0 [M+H]+.

tert-Butyl (R)-(1-(azetidin-1-yl)-1-oxo-3-phenylpropan-2-yl)carbamate (S33): Compound S33 (324 mg, 74% isolated yield) was prepared according to general procedure V starting compound S20 (380 mg, 1.4 mmol, 1.0 equiv) and azetidine hydrochloride salt (161 mg, 1.7 mmol, 1.2 equiv).1 H

NMR (400 MHz, CDCl3) δ 7.36 – 7.17 (m, 5H), 5.37 (d, J = 8.6 Hz, 1H), 4.29 (td, J = 9.0, 5.6 Hz, 1H), 4.06 – 3.90 (m, 2H), 3.84 (td, J = 9.7, 6.1 Hz, 1H), 3.10 (td, J = 8.9, 6.1 Hz, 1H), 2.98 (dd, J = 12.9, 5.6 Hz, 1H), 2.90 (dd, J = 12.9, 9.0 Hz, 1H), 2.17 – 2.02 (m, 1H), 1.99 – 1.85 (m, 1H), 1.42 (s, 9H); MS (ESI) m/z 327.1 [M+Na]+.

68 tert-Butyl (R)-(1-oxo-3-phenyl-1-(piperidin-1-yl)propan-2-yl)carbamate (S34): Compound S34 (274 mg, 80% isolated yield) was prepared according to general procedure V starting compound S20 (265 mg, 1.0 mmol, 1.0 equiv) and piperidine (102 mg, 1.2 mmol, 1.2 equiv). 1H NMR (400 MHz,

CDCl3) δ 7.31 – 7.15 (m, 5H), 5.48 (d, J = 8.7 Hz, 1H), 4.85 (q, J = 7.5 Hz, 1H), 3.53 – 3.37 (m, 3H), 3.28 – 3.20 (m, 1H), 3.07 – 2.99 (m, 1H), 2.95 (d, J = 7.1 Hz, 2H), 1.73 – 1.44 (m, 5H), 1.41 (s, 9H), 1.38 – 1.29 (m, 1H); MS (ESI) m/z 332.9 [M+H]+. 2 tert-Butyl (R)-(1-morpholino-1-oxo-3-phenylpropan-2-yl)carbamate (S35): Compound S35 (274 mg, 82% isolated yield) was prepared according to general procedure V starting compoundS20 (265 mg, 1.0 mmol, 1.0 equiv) 1 and morpholine (105 mg, 1.2 mmol, 1.2 equiv). H NMR (400 MHz, CDCl3) δ 7.32 – 7.17 (m, 5H), 5.45 – 5.38 (d, J = 8.7 Hz, 1H), 4.84 – 4.74 (q, J = 8.3 Hz, 1H), 3.65 – 3.36 (m, 5H), 3.32 – 3.22 (m, 1H), 3.07 – 2.81 (m, 4H), 1.46 – 1.38 (s, 9H); MS (ESI) m/z 233.1 [M+H]+. tert-Butyl (R)-(1-(diethylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (S36): Compound S36 (261 mg, 82% isolated yield) was prepared according to general procedure V starting compound S20 (200 mg, 0.7 mmol, 1.0 equiv) and diethylamine (121 mg, 1.7 mmol, 2.2 equiv). 1H NMR (400

MHz, CDCl3) δ 7.31 – 7.14 (m, 5H), 5.38 (d, J = 9.1 Hz, 1H), 4.80 – 4.67 (m, 1H), 3.56 – 3.46 (m, 1H), 3.15 – 2.85 (m, 5H), 1.41 (s, 9H), 1.03 (t, J = 7.2 Hz, 3H), 0.96 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 320.9 [M+H]+. tert-Butyl (R)-(1-(dimethylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (S37): Compound S37 (281 mg, 96% isolated yield) was prepared according to general procedure V starting compound S20 (265 mg, 1.0 mmol, 1.0 equiv) and dimethylamine (2.0M solution in THF, 0.8 mL, 2.2 mmol, 2.2 1 equiv). H NMR (400 MHz, CDCl3) δ 7.31 – 7.16 (m, 5H), 5.42 (d, J = 8.8 Hz, 1H), 4.87 – 4.77 (m, 1H), 3.01 – 2.90 (m, 1H), 2.85 (s, 3H), 2.61 (s, 3H), 1.41 (s, 9H); MS (ESI) m/z 292.8 [M+H]+.

69 General procedure VI (deprotection of S23-S37 to afford compounds 30, S38-S51) (R)-2-Amino-3-phenyl-1-(pyrrolidin-1-yl)propan-1-one (30): tert-Butyl (R)-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (S23, 1.7 g, 5.4 mmol, 1.0 equiv) was dissolved in 20 mL of TFA:DCM (1:1 v/v) and stirred at rt for 2 h. When TLC analysis (EtOAc:n-heptane 1:1) showed that the reaction was complete, the solvent was removed in vacuo. The crude mixture was redissolved in DCM (25 mL), and washed with satd aq NaHCO (2 × 25 mL), and brine (50 mL). The organic layer 2 3 was dried over Na2SO4, filtered, and evaporated to give the crude product, which was purified by column chromatography (0-5% MeOH in DCM) to give 943 mg (80% isolated yield) of D-phenylalanine pyrrolidine amide 30 as a yellow solid. 1H NMR (400 MHz,

CDCl3) δ 7.32 – 7.26 (m, 2H), 7.25 – 7.18 (m, 3H), 3.71 (t, J = 7.2 Hz, 1H), 3.52 – 3.43 (m, 1H), 3.42 – 3.29 (m, 2H), 2.95 (dd, J = 13.1, 7.2 Hz, 1H), 2.84 – 2.75 (m, 2H), 1.86 – 1.60 13 (m, 6H); C NMR (126 MHz, CDCl3) δ 173.1, 137.9, 129.3, 128.4, 126.7, 55.2, 46.0, 45.8, 43.0, 25.9, 24.0; MS (ESI) m/z 219.0 [M+H]+; HRMS (ESI) found m/z 241.13149 [M+Na]+, + calcd for C13H18N2ONa m/z 241.13168.

(R)-2-Amino-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-1-one (S38): Starting from compoundS24 (308 mg, 0.8 mmol, 1.0 equiv) and following general procedure VI, 197 mg (86% isolated yield) of compound S38 was 1 obtained as a yellow solid. H NMR (500 MHz, CDCl3) δ 7.48 – 7.35 (m, 4H), 3.69 (t, J = 7.3 Hz, 1H), 3.47 – 3.40 (m, 1H), 3.37 – 3.30 (m, 2H), 2.98 (dd, J = 13.2, 7.6 Hz, 1H), 2.84 (dd, J = 13.2, 7.6 Hz, 1H), 2.77 – 2.71 (m, 13 1H), 1.84 – 1.58 (m, 6H); C NMR (126 MHz, CDCl3) δ 172.6, 138.9, 132.8, 131.0, 130.8, 130.5, 130.3, 128.8, 127.3, 125.9, 125.8, 125.8, 125.8, 125.1, 123.5, 123.4, 123.4, 123.4, 123.0, 120.8, 54.8, 46.0, 45.8, 42.5, 25.8, 23.9; 19F + NMR (377 MHz, CDCl3) δ –62.6; MS (ESI) m/z 287.1 [M+H] .

(R)-2-Amino-1-(pyrrolidin-1-yl)-3-(2-(trifluoromethyl)phenyl)propan-1-one (S39): Starting from compoundS25 (300 mg, 0.8 mmol, 1.0 equiv) and following general procedure VI, 210 mg (95% isolated yield) of compound S39 was 1 obtained as an off-white solid. H NMR (500 MHz, CDCl3) δ 7.69 – 7.65 (m, 1H), 7.51 – 7.46 (m, 1H), 7.39 – 7.34 (m, 2H), 3.83 (t, J = 7.4 Hz, 1H), 3.53 – 3.46 (m, 1H), 3.43 – 3.34 (m, 2H), 3.12 (dd, J = 13.3, 7.4 Hz, 1H), 3.06 (dd, J = 13.3, 7.4 Hz, 1H), 2.77 – 2.70 (m, 1H), 1.87 – 1.62 (m, 6H); 13C NMR (126

MHz, CDCl3) δ 172.8, 136.3, 132.7, 131.5, 129.3, 129.0, 128.8, 128.6, 127.9, 126.9, 126.2, 126.1, 126.1, 126.0, 125.7, 123.5, 121.3, 54.1, 45.9, 45.8, 39.7, 25.9, 24.0; 19F + NMR (377 MHz, CDCl3) δ –59.20; MS (ESI) m/z 287.1 [M+H] .

70 (R)-2-Amino-1-(pyrrolidin-1-yl)-3-(4-(trifluoromethyl)phenyl)propan-1-one (S40): Starting from compound S26 (300 mg, 0.8 mmol, 1.0 equiv) and following general procedure VI, 185 mg (83% isolated yield) of compound S40 was obtained as a pale yellow oil which crystallised 1 over time. H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 7.7 Hz, 2H), 7.33 (d, J = 7.7 Hz, 2H), 3.72 (t, J = 7.2 Hz, 1H), 3.54 – 3.44 (m, 1H), 3.43 – 3.33 (m, 2H), 3.01 (dd, J = 13.2, 7.2 Hz, 1H), 2.93 – 2.81 (m, 2H), 1.90 – 1.62 (m, 6H); 13C NMR (101 MHz, CDCl ) δ 172.7, 142.2, 142.2, 142.2, 142.1, 129.7, 129.5, 3 2 129.2, 128.9, 128.6, 128.2, 125.5, 125.4, 125.3, 125.3, 125.2, 122.8, 120.1, 54.9, 46.1, 19 + 45.9, 42.4, 25.9, 24.0; F NMR (377 MHz, CDCl3) δ –62.48; MS (ESI) m/z 287.1 [M+H] .

(R)-2-Amino-1-(pyrrolidin-1-yl)-3-(m-tolyl)propan-1-one (S41): Starting from compoundS27 (300 mg, 0.9 mmol, 1.0 equiv) and following general procedure VI, 180 mg (86% isolated yield) of compound S41 was 1 obtained as a yellow solid. H NMR (500 MHz, CDCl3) δ 7.23 – 7.15 (m, 1H), 7.08 – 6.99 (m, 3H), 3.72 (t, J = 7.3 Hz, 1H), 3.54 – 3.44 (m, 1H), 3.45 – 3.33 (m, 2H), 2.93 (dd, J = 7.3, 13.1 Hz, 1H), 2.84 (m, 1H), 2.75 (dd, J = 7.3, 13.1 13 Hz, 1H), 2.34 (s, 3H), 1.88 – 1.64 (m, 6H); C NMR (126 MHz, CDCl3) δ 173.1, 138.0, 137.7, 130.0, 128.3, 127.4, 126.3, 55.1, 46.1, 45.8, 42.9, 26.0, 24.1, 21.4; MS (ESI) m/z 233.1 [M+H]+.

(R)-2-Amino-3-(3-chlorophenyl)-1-(pyrrolidin-1-yl)propan-1-one (S42): Starting from compound S28 (300 mg, 0.85 mmol, 1.0 equiv) and following general procedure VI, 161 mg (77% isolated yield) of compound S42 was 1 obtained as yellow oil. H NMR (500 MHz, CDCl3) δ 7.26 – 7.19 (m, 3H), 7.13 – 7.09 (m, 1H), 3.71 (t, J = 7.2 Hz, 1H), 3.53 – 3.35 (m, 3H), 2.94 (dd, J = 7.2, 13.2 Hz, 1H), 2.88 – 2.82 (m, 1H), 2.78 (dd, J = 7.2, 13.2 Hz, 1H), 1.89 – 1.68 13 (m, 6H); C NMR (126 MHz, CDCl3) δ 172.8, 140.0, 134.2, 129.7, 129.3, 127.6, 126.9, 54.9, 46.1, 45.9, 42.4, 26.0, 24.1; MS (ESI) m/z 252.1 [M+H]+.

(R)-2-Amino-3-(3-bromophenyl)-1-(pyrrolidin-1-yl)propan-1-one (S43): Starting from compound S29 (250 mg, 0.6 mmol, 1.0 equiv) and following general procedure VI, 153 mg (82% isolated yield) of compound S43 was 1 obtained as a brown solid; H NMR (400 MHz, CDCl3) δ 7.40 – 7.34 (m, 2H), 7.19 – 7.14 (m, 2H), 3.75 (t, J = 7.3 Hz, 1H), 3.52 – 3.34 (m, 3H), 2.94 (dd, J = 13.2, 7.3 Hz, 1H), 2.86 – 2.72 (m, 2H), 2.38 (s, 2H), 1.91 – 1.64 (m, 4H); 13C

NMR (101 MHz, CDCl3) δ 172.3, 140.0, 132.2, 130.0, 129.9, 128.1, 122.5, 54.7, 46.2, 45.9, 41.9, 25.9, 24.0; MS (ESI) m/z 297.0 [M+H]+.

71 (R)-2-Amino-3-(naphthalen-2-yl)-1-(pyrrolidin-1-yl)propan-1-one (S44): Starting from compoundS30 (300 mg, 0.8 mmol, 1.0 equiv) and following general procedure VI, 164 mg (75% isolated yield) of compound S44 was 1 obtained as yellow solid. H NMR (400 MHz, CDCl3) δ 7.81 – 7.73 (m, 3H), 7.65 (bs, J = 1.2, 1H), 7.49 – 7.40 (m, 2H), 7.33 (dd, J = 8.4, 1.8 Hz, 1H), 3.81 (t, J = 7.2 Hz, 1H), 3.52 – 3.31 (m, 3H), 3.13 (dd, J = 13.1, 7.2 Hz, 1H), 2.94 (dd, J = 13.1, 7.2 Hz, 1H), 2.84 – 2.76 (m, 1H), 1.79 – 1.46 (m, 6H); 13C NMR (101 MHz, CDCl ) δ 173.0, 135.4, 133.4, 132.3, 128.0, 127.8, 2 3 127.6, 127.6, 127.5, 126.1, 125.5, 55.1, 46.0, 45.8, 43.1, 25.8, 24.0; MS (ESI) m/z 269.0 [M+H]+.

2-Amino-1-(pyrrolidin-1-yl)ethan-1-one (S45): Starting from compound S31 (300 mg, 1.3 mmol, 1.0 equiv) and following general procedure VI, 121 mg (72% isolated yield) of compound S45 was 1 obtained as yellow oil. H NMR (400 MHz, CDCl3) δ 3.82 (s, 2H), 3.44 (t, J = 6.9 Hz, 1H), 3.36 (t, J = 6.9 Hz, 1H), 1.99 (quint, J = 6.9 Hz, 1H), 1.88 (quint, 13 J = 6.9 Hz, 1H); C NMR (101 MHz, CDCl3) δ 46.3, 45.6, 40.6, 25.7, 23.9; MS (ESI) m/z 129.1 [M+H]+.

(R)-N-((3S,5S,7S)-Adamantan-1-yl)-2-amino-3-phenylpropanamide (S46): Starting from compound S32 (200 mg, 0.5 mmol, 1.0 equiv) and following general procedure VI, 138 mg (92% isolated yield) of compound S46 was 1 obtained as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.36 – 7.29 (m, 2H), 7.27 – 7.21 (m, 3H), 6.88 (bs, 1H), 3.46 (dd, J = 8.9, 4.4 Hz, 1H), 3.20 (dd, J = 13.7, 4.4 Hz, 1H), 2.71 (dd, J = 13.7, 8.9 Hz, 1H), 2.07 (s, 3H), 1.98 13 (s, 6H), 1.68 (s, 6H); C NMR (126 MHz, CDCl3) δ 173.1, 138.2, 129.4, 128.6, + 126.7, 56.9, 51.1, 41.5, 41.1, 36.4, 29.4; MS (ESI) m/z 299.1 [M+H] .

(R)-2-Amino-1-(azetidin-1-yl)-3-phenylpropan-1-one (S47): Starting from compound S33 (300 mg, 1.0 mmol, 1.0 equiv) and following general procedure VI, 110 mg (55% isolated yield) of compound S47 was 1 obtained as a white solid. H NMR (400 MHz, CDCl3) δ 7.31 – 7.08 (m, 5H), 3.94 – 3.78 (m, 3H), 3.39 (dd, J = 7.9, 6.7 Hz, 1H), 3.26 (td, J = 8.8, 6.1 Hz, 1H), 2.83 (dd, J = 13.0, 7.9 Hz, 1H), 2.72 (dd, J = 13.0, 6.7 Hz, 1H), 2.13 – 13 2.01 (m, 1H), 1.97 – 1.85 (m, 1H), 1.72 (bs, 2H); C NMR (101 MHz, CDCl3) δ 173.9, 137.7, 129.2, 128.4, 126.7, 52.6, 49.6, 47.6, 42.6, 15.2; MS (ESI) m/z 205.1 [M+H]+.

72 (R)-2-Amino-3-phenyl-1-(piperidin-1-yl)propan-1-one (S48): Starting from compound S34 (250 mg, 0.8 mmol, 1.0 equiv) and following general procedure VI, 159 mg (91% isolated yield) of compound S48 was 1 obtained as a yellow oil. H NMR (400 MHz, CDCl3) δ 7.38 – 7.11 (m, 5H), 3.96 (t, J = 7.1 Hz, 1H), 3.62 – 3.44 (m, 2H), 3.35 – 3.23 (m, 1H), 3.18 – 3.07 (m, 1H), 2.94 (dd, J = 13.3, 6.7 Hz, 1H), 2.77 (dd, J = 13.3, 7.5 Hz, 1H), 1.82 13 (bs, 2H), 1.62 – 1.36 (m, 5H), 1.17 – 1.10 (m, 1H); C NMR (101 MHz, CDCl3) δ 173.0, 138.0, 129.5, 128.7, 128.6, 126.8, 52.5, 46.4, 43.2, 43.0, 26.2, 25.6, 24.6. 2

(R)-2-Amino-1-morpholino-3-phenylpropan-1-one (S49): Starting from compound S35 (300 mg, 0.9 mmol, 1.0 equiv) and following general procedure VI, 163 mg (78% isolated yield) of compound S49 was 1 obtained as a yellow solid. H NMR (400 MHz, CDCl3) δ 7.34 – 7.16 (m, 4H), 3.94 – 3.89 (t, J = 7.4 Hz, 1H), 3.70 – 3.59 (m, 2H), 3.53 – 3.44 (m, 3H), 3.37 – 3.24 13 (m, 1H), 3.09 – 2.79 (m, 4H), 1.80 – 1.65 (bs, 2H); C NMR (126 MHz, CDCl3) δ 173.4, 137.5, 129.4, 128.7, 127.0, 66.6, 66.1, 52.3, 45.7, 43.3, 42.2; MS (ESI) m/z 325.0 [M+H]+.

(R)-2-Amino-N,N-diethyl-3-phenylpropanamide (S50): Starting from compound S36 (250 mg, 0.8 mmol, 1.0 equiv) and following general procedure VI, 101 mg (59% isolated yield) of compound S50 was 1 obtained as yellow oil. H NMR (400 MHz, CDCl3) δ 7.32 – 7.26 (m, 2H), 7.25 – 7.17 (m, 3H), 3.80 (t, J = 7.1 Hz, 1H), 3.57 – 3.46 (m, 1H), 3.23 – 2.94 (m, 4H), 2.77 (dd, J = 13.2, 7.1 Hz, 1H), 1.67 (bs, 2H), 1.06 (dt, J = 15.2, 7.1 Hz, 13 6H); C NMR (101 MHz, CDCl3) δ 174.0, 137.9, 129.4, 129.4, 128.5, 126.7, 43.2, 41.3, 40.5, 14.5, 12.9; MS (ESI) m/z 221.0 [M+H]+.

(R)-2-Amino-N,N-dimethyl-3-phenylpropanamide (S51): Starting from compound S37 (250 mg, 0.9 mmol, 1.0 equiv) and following general procedure VI, 129 mg (79% isolated yield) of compound S51 was 1 obtained as a yellow solid. H NMR (400 MHz, CDCl3) δ 7.32 – 7.17 (m, 5H), 3.93 (t, J = 7.1 Hz, 1H), 2.99 – 2.91 (m, 1H), 2.91 (s, 3H), 2.82 – 2.73 (m, 1H), 13 2.74 (s, 3H), 1.65 (bs, 2H); C NMR (101 MHz, CDCl3) δ 174.7, 137.8, 129.3, 128.5, 126.7, 42.9, 36.6, 35.7; MS (ESI) m/z 193.0 [M+H]+.

73 General procedure VII (Synthesis and deprotection of sulfonamides 1-29) (R)-8-Fluoro-N-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-1,2,3,4- tetrahydroisoquinoline-6-sulfonamide (7): Sulfonyl chloride S16 (50 mg, 0.14 mmol,) and D-phenylalanine pyrrolidine amide 30 (37 mg, 0.17 mmol, 1.2 equivalents) were

dissolved in 2 mL of DCM. To this was added Et3N (43 mg, 3.0 equiv) and the reaction was left to stir at rt until consumption of the sulfonyl chloride was observed by TLC (approximately 2-4 h, 2 TLC in EtOAc:n‑heptane 1:1). After completion, the reaction mixture was diluted by the addition of 10 mL of DCM and was washed with 1.0 M HCl

(2 × 10 mL), followed by brine (10 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude mixture was purified using flash column chromatography to afford 60 mg (79% isolated yield) of compoundS52 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.28 (bs, J = 1.2 Hz, 1H), 7.21 – 7.10 (m, 4H), 7.08 – 7.00 (m, 2H), 5.83 (d, J = 9.7 Hz, 1H), 4.51 (bs, 2H), 4.11 (app. q, J = 8.3 Hz, 1H), 3.58 (bs, J = 10.8 Hz, 2H), 3.17 – 2.67 (m, 7H), 2.39 (bs, 1H), 1.65 – 1.46 (m, 4H), 1.42 (s, 9H); MS (ESI) m/z 531.9 [M+H]+. Next, 25 mg of compound S52 was dissolved in 2 mL DCM:TFA (1:1 v/v) and left to stir at rt until TLC (MeOH:DCM 1:9) showed the reaction was complete (approximately 2 h). The solvent was removed in vacuo, and the crude

mixture was redissolved in DCM (20 mL), washed with satd aq NaHCO3 (2 × 20 mL),

brine (20 mL), dried over Na2SO4, and concentrated in vacuo. The crude product was purified by flash column chromatography to obtain 13 mg (64% isolated yield) of the 1 desired compound 7 as a white solid. H NMR (500 MHz, CDCl3) δ 7.33 (bs, 1H), 7.25 – 7.18 (m, 4H), 7.14 – 7.09 (m, 2H), 4.16 (dd, J = 8.9, 6.2 Hz, 1H), 4.02 (bs, 2H), 3.17 – 2.87 (m, 7H), 2.85 – 2.73 (m, 2H), 2.44 – 2.39 (m, 1H), 1.67 – 1.56 (m, 3H), 1.52 – 1.45 (m, 13 1H) 1.25 (bs, 1H); C NMR (126 MHz, CDCl3) δ 168.8, 160.1, 158.1, 139.2, 139.2, 139.1, 135.9, 129.8, 129.1, 128.9, 128.8, 127.5, 123.8, 123.8, 111.4, 111.2, 56.5, 46.4, 46.0, 19 43.2, 42.8, 42.7, 40.9, 29.4, 29.3, 26.0, 24.2; F NMR (377 MHz, CDCl3) δ –117.3; MS (ESI) m/z 432.1 [M+H]+; HRMS (ESI) found m/z 454.15748 [M+Na]+, calcd for + 21 C22H26FN3O3SNa m/z 545.15766; [α]D = –67.1° (DCM).

(R)-8-Fluoro-N-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl)- 1,2,3,4-tetrahydro-isoquinoline-6-sulfonamide (1): Compound 1 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (45 mg, 0.13 mmol) and compound S38 (49 mg, 0.17 mmol. 1.2 equiv) to afford 77 mg (78% isolated yield) of S53 as an off-white solid. 1H NMR (400

MHz, CDCl3) δ 7.50 – 7.44 (m, 1H), 7.39 – 7.31 (m, 4H), 7.26 –

74 7.21 (dd, J = 8.8, 1.8 Hz, 1H), 6.04 – 5.99 (d, J = 9.5 Hz, 1H), 4.62 – 4.55 (bs, 2H), 4.25 – 4.17 (q, J = 8.0 Hz, 1H), 3.71 – 3.59 (bs, 2H), 3.28 – 3.09 (m, 3H), 3.02 – 2.98 (d, J = 7.5 Hz, 2H), 2.91 – 2.76 (m, 2H), 2.63 – 2.50 (bs, 1H), 1.79 – 1.54 (m, 4H), 1.51 – 1.47 (s, 9H); MS (ESI) m/z 599.7 [M+H]+. Compound S53 (45 mg, 0.08 mmol) was deprotected and after column chromatography 25 mg (75% isolated yield) of compound1 was obtained 1 as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.51 – 7.46 (m, 1H), 7.40 – 7.35 (m, 2H), 7.33 – 7.31 (m, 2H), 7.19 (d, J = 8.7, 1H), 4.19 (t, J = 7.5, 1H), 4.03 (bs, 2H), 3.22 – 3.06 (m, 5H), 2.99 (d, J = 7.5, 2H), 2.86 – 2.75 (m, 2H), 2.59 – 2.47 (m, 1H), 1.76 – 1.52 2 13 (m, 4H), 1.25 (bs, 1H); C NMR (101 MHz, CDCl3) δ 168.3, 160.0, 157.5, 139.0, 138.9, 138.8, 138.7, 136.7, 133.0, 133.0, 131.3, 131.0, 130.7, 130.3, 129.0, 128.5, 128.3, 126.1, 126.1, 126.0, 126.0, 125.2, 124.1, 124.0, 124.0, 123.9, 123.4, 123.3, 122.5, 111.0, 110.8, 110.0, 55.8, 46.2, 45.8, 42.6, 42.1, 42.1, 39.9, 28.8, 28.8, 25.7, 23.8; 19F + NMR (377 MHz, CDCl3) δ –62.6, –116.9; MS (ESI) m/z 500.0 [M+H] ; HRMS (ESI) found + + 21 m/z 500.16332 [M+H] , calcd for C23H26F4N3O3S m/z 500.16310; [α]D = –73.2° (DCM).

(R)-8-Fluoro-N-(1-oxo-1-(pyrrolidin-1-yl)-3-(2-(trifluoromethyl)phenyl)propan-2-yl)- 1,2,3,4-tetrahydro-isoquinoline-6-sulfonamide (2): Compound 2 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and compound S39 (50 mg, 0.17 mmol, 1.2 equiv) to afford 80 mg (93% isolated yield) of S54 as a white solid. 1H NMR (400 MHz,

CDCl3) δ 7.58 – 7.52 (m, 1H), 7.41 – 7.17 (m, 5H), 6.25 (d, J = 9.7 Hz, 1H), 4.58 (bs, 2H), 4.39 – 4.26 (m, 1H), 3.64 (bs, 2H), 3.37 – 3.21 (m, 3H), 3.05 (d, J = 7.7 Hz, 2H), 2.83 (q, J = 5.4 Hz, 2H), 2.74 – 2.66 (m, 1H), 1.81 – 1.61 (m, 4H), 1.51 (s, 9H); MS (ESI) m/z 599.9 [M+H]+. Compound S54 (45 mg, 0.08 mmol) was deprotected and after column chromatography 31 mg (83% isolated yield) 1 of compound 2 was obtained as a white solid. H NMR (400 MHz, CDCl3) δ 7.53 – 7.48 (m, 2H), 7.33 (bs, 1H), 7.28 – 7.24 (m, 3H), 7.20 (dd, J = 1.7, 8.7 Hz, 1H), 4.20 (t, J = 7.3 Hz, 1H), 4.02 (s, 2H), 3.24 – 3.06 (m, 5H), 2.99 (d, J = 7.3 Hz, 2H), 2.88 – 2.70 (m, 2H), 13 2.67 – 2.56 (m, 1H), 1.79 – 1.52 (m, 4H), 1.26 (bs, 1H); C NMR (100 MHz, CDCl3) δ 168.1, 160.0, 157.5, 139.8, 139.8, 139.8, 139.8, 139.0, 138.9, 138.8, 138.7, 129.9, 129.7, 129.4, 128.9, 128.7, 125.4, 125.3, 125.3, 125.3, 123.4, 123.4, 122.7, 111.0, 110.7, 55.7, 46.2, 45.8, 42.8, 42.4, 42.3, 39.9, 29.0, 29.0, 25.7, 23.9; 19F NMR (377 MHz, + CDCl3) δ –62.5, –117.1; MS (ESI) m/z 500.1 [M+H] ; HRMS (ESI) found m/z 500.16353 + + 21 [M+H] , calcd for C23H26F4N3O3S m/z 500.16310; [α]D = –91.3° (DCM).

75 (R)-8-Fluoro-N-(1-oxo-1-(pyrrolidin-1-yl)-3-(4-(trifluoromethyl)phenyl)propan-2-yl)- 1,2,3,4-tetrahydro-isoquinoline-6-sulfonamide (3): Compound 3 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, mmol) and compound S40 (49 mg, 0.17 mmol. 1.2 equiv) to afford 70 mg (81% isolated yield) of S55 as a white solid. 1H NMR (400

MHz, CDCl3) δ 7.50 (d, J = 8.2 Hz, 2H), 7.37 (bs, 1H), 7.30 – 7.23 (m, 3H), 6.02 (bs, 1H), 4.58 (bs, 2H), 4.22 (q, J = 8.0 Hz, 2 1H), 3.64 (bs, 2H), 3.26 – 3.08 (m, 3H), 3.00 (d, J = 8.0 Hz, 2H), 2.90 – 2.76 (m, 2H), 2.65 (bs, 1H), 1.74 – 1.56 (m, 4H), 1.49 (s, 9H); MS (ESI) m/z 599.9 [M+H]+. S55 (46 mg, 0.08 mmol) was deprotected and after column chromatography 35 mg (91% isolated yield) 1 of compound 3 was obtained as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.7 Hz, 1H), 7.43 – 7.28 (m, 4H), 7.18 – 7.13 (m, 1H), 4.30 (t, J = 7.7 Hz, 1H), 4.02 (s, 2H), 3.23 (t, J = 6.3 Hz, 3H), 3.18 – 3.00 (m, 4H), 2.84 – 2.73 (m, 2H), 2.68 – 2.61 (m, 1H), 13 1.81 – 1.57 (m, 4H), 1.24 (bs, 1H); C NMR (100 MHz, CDCl3) δ 168.7, 160.0, 157.5, 139.0, 138.9, 138.7, 138.7, 134.1, 133.3, 133.2, 131.6, 129.0, 128.7, 128.6, 128.5, 127.3, 126.0, 125.9, 125.8, 123.4, 123.4, 123.1, 111.0, 110.8, 55.2, 46.0, 45.9, 42.8, 19 42.4, 42.4, 36.7, 30.9, 29.0, 25.8, 23.9; F NMR (377 MHz, CDCl3) δ –62.6, –117.1; MS (ESI) m/z 500.1 [M+H]+; HRMS (ESI) found m/z 500.16319 [M+H]+, calcd for + 21 C23H26F4N3O3S m/z 500.16310; [α]D = –74.0° (DCM).

(R)-8-Fluoro-N-(1-oxo-1-(pyrrolidin-1-yl)-3-(m-tolyl)propan-2-yl)-1,2,3,4- tetrahydroisoquinoline-6-sulfonamide (4): Compound 4 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (75 mg, 0.21 mmol) and compound S41 (60 mg, 1.2 equiv) to afford 81 mg (69% isolated 1 yield) of S56 as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.34 (bs, 1H), 7.26 – 7.22 (m, 1H), 7.13 – 7.06 (m, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.93 – 6.87 (m, 2H), 5.71 (bs, 1H), 4.58 (bs, 2H), 4.15 (t, J = 6.9 Hz, 1H), 3.64 (bs, 2H), 3.25 – 3.00 (m, 3H), 2.94 – 2.77 (m, 4H), 2.46 (bs, 1H), 2.26 (s, 3H), 1.70 – 1.53 (m, 4H), 1.50 (s, 9H); MS (ESI) m/z 546.0 [M+H]+. S56 (70 mg, 0.13 mmol) was deprotected and after column chromatography 47 mg (81% isolated yield) of compound 4 was obtained as an off- 1 white solid. H NMR (400 MHz, CDCl3) δ 7.34 – 7.31 (m, 1H), 7.20 (dd, J = 1.8, 8.8 Hz, 1H), 7.11 (t, J = 7.9 Hz, 1H), 7.03 – 6.98 (m, 1H), 6.94 – 6.88 (m, 2H), 4.16 (dd, J = 6.7, 8.5 Hz, 1H), 4.03 (bs, 2H), 3.22 – 3.01 (m, 5H), 2.94 – 2.73 (m, 4H), 2.49 – 2.38 (m, 1H), 13 2.27 (s, 3H), 1.71 – 1.45 (m, 4H), 1.26 (bs, 1H); C NMR (100 MHz, CDCl3) δ 168.7, 160.0, 157.5, 139.1, 139.1, 138.7, 138.6, 138.0, 135.5, 130.1, 128.4, 128.3, 128.2,

76 127.8, 126.4, 123.4, 123.4, 111.1, 110.8, 56.2, 46.1, 45.7, 42.7, 42.2, 42.2, 40.4, 28.9, 19 + 25.7, 23.9, 21.3; F NMR (377 MHz, CDCl3) δ –117.2; MS (ESI) m/z 446.1 [M+H] ; HRMS + + 21 (ESI) found m/z 446.19004 [M+H] , calcd for C23H29FN3O3S m/z 446.19006; [α]D = –91.1° (DCM).

(R)-N-(3-(3-Chlorophenyl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)-8-fluoro-1,2,3,4- tetrahydroisoquino-line-6-sulfonamide (5): Compound 5 was synthesised according to general procedure 2 VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and compound S42 (44 mg, 0.17 mmol, 1.2 equiv) to afford 70 mg (87% isolated yield) of S57 as a white solid. 1H NMR (400 MHz,

CDCl3) δ 7.36 – 7.29 (m, 1H), 7.23 (dd, J = 8.8, 1.7 Hz, 1H), 7.18 – 7.13 (m, 2H), 7.08 – 6.99 (m, 2H), 6.06 (d, J = 9.1 Hz, 1H), 4.59 (s, 2H), 4.17 (q, J = 7.8 Hz, 1H), 3.65 (t, J = 5.9 Hz, 2H), 3.31 – 3.08 (m, 3H), 2.96 – 2.77 (m, 4H), 2.69 (bs, 1H), 1.76 – 1.58 (m, 3H), 1.50 (s, 9H); MS (ESI) m/z 565.8 [M+H]+. S57 (21 mg, 0.04 mmol) was deprotected and after column chromatography 14 mg (81% isolated yield) of compound 5 was obtained as an off- 1 white solid. H NMR (400 MHz, CDCl3) δ 7.32 – 7.29 (bs, 1H), 7.22 – 7.14 (m, 3H), 7.08 – 7.00 (m, 2H), 4.19 – 4.13 (t, J = 7.4 Hz, 1H), 4.02 (s, 2H), 3.28 – 3.09 (m, 5H), 2.91 (d, J = 7.4 Hz, 2H), 2.85 – 2.76 (q, J = 6.1 Hz, 2H), 2.66 – 2.56 (m, 1H), 1.79 – 1.58 (m, 4H), 13 1.26 (bs, 1H); C NMR (126 MHz, CDCl3) δ 168.4, 159.8, 157.8, 138.8, 138.8, 138.8, 137.7, 134.2, 129.7, 129.4, 128.6, 128.5, 127.7, 127.3, 123.4, 123.4, 111.0, 110.8, 55.9, 19 46.2, 45.9, 42.8, 42.3, 42.3, 39.9, 28.9, 28.9, 25.8, 23.9; F NMR (377 MHz, CDCl3) δ –117.0; MS (ESI) m/z 466.1 [M+H]+; HRMS (ESI) found m/z 466.13652 [M+H]+, calcd for + 21 C22H26ClFN3O3S m/z 466.13674; [α]D = –88.9° (DCM).

(R)-N-(3-(3-Bromophenyl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)-8-fluoro-1,2,3,4- tetrahydroisoquino-line-6-sulfonamide (6): Compound 6 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (75 mg, 0.14 mmol) and compound S43 (77 mg, 0.25 mmol, 1.2 equiv) to afford 40 mg (31% isolated yield) of S58 as a white solid. 1H NMR (400 MHz,

CDCl3) δ 7.34 – 7.30 (m, 2H), 7.25 – 7.19 (m, 2H), 7.13 – 7.06 (m, 2H), 4.59 (bs, 2H), 4.17 (t, J = 7.5 Hz, 1H), 3.65 (t, J = 5.9 Hz, 2H), 3.49 (s, 1H), 3.32 – 3.10 (m, 3H), 2.92 – 2.79 (m, 4H), 2.66 (bs, 1H), 1.81 – 1.62 (m, 4H), 1.50 (s, 9H); MS (ESI) m/z 609.8 [M+H]+. S58 (30 mg, 0.05 mmol) was deprotected and after column chromatography 18 mg (72% isolated yield) 1 of compound 6 was obtained as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.35 –

77 7.29 (m, 2H), 7.24 – 7.16 (m, 2H), 7.14 – 7.06 (m, 2H), 4.16 (t, J = 7.5 Hz, 1H), 4.05 (s, 2H), 3.28 – 3.10 (m, 5H), 2.88 (d, J = 7.5 Hz, 2H), 2.85 – 2.78 (m, 2H), 2.66 – 2.55 (m, 1H), 13 1.79 – 1.58 (m, 4H), 1.26 (bs, 1H); C NMR (126 MHz, CDCl3) δ 168.4, 159.7, 157.8, 138.9, 138.9, 138.7, 138.6, 138.0, 132.3, 130.2, 130.0, 128.8, 128.3, 128.2, 128.2, 123.3, 123.3, 122.4, 111.0, 110.8, 55.9, 46.2, 45.9, 42.7, 42.2, 42.2, 39.8, 39.7, 29.7, 19 + 29.3, 28.7, 25.8, 23.9; F NMR (377 MHz, CDCl3) δ –117.1; MS (ESI) m/z 511.9 [M+H] ; + + HRMS (ESI) found m/z 510.08574 [M+H] , calcd for C22H26BrFN3O3S m/z 510.08523; [α] 21 = –50.8° (DCM). 2 D

(R)-8-Fluoro-N-(3-(naphthalen-2-yl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)-1,2,3,4- tetrahydroisoquino-line-6-sulfonamide (8): Compound 8 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and compound S44 (46 mg, 0.17 mmol, 1.2 equiv) to afford crude S59. The crude mixture containing compound S59 was used for deprotection without further purification to afford 25 mg (85% isolated yield) of compound 8 as an off-white solid. 1H NMR

(500 MHz, CDCl3) δ 7.81 – 7.76 (m, 1H), 7.73 – 7.68 (m, 2H), 7.54 (bs, 1H), 7.48 – 7.42 (m, 2H), 7.25 – 7.21 (m, 2H), 7.16 (dd, J = 8.7, 1.7 Hz, 1H), 4.30 (t, J = 7.5 Hz, 1H), 3.93 – 3.86 (m, 2H), 3.26 – 3.14 (m, 3H), 3.07 (dd, J = 7.5, 1.8 Hz, 2H), 3.01 (t, J = 5.9 Hz, 2H), 2.71 – 2.55 (m, 3H), 1.70 – 1.50 (m, 3H), 1.45 – 1.38 (m, 1H), 1.25 13 (bs, 1H); C NMR (126 MHz, CDCl3) δ 168.8, 159.6, 157.6, 138.9, 138.9, 138.7, 138.6, 133.3, 133.2, 132.3, 128.5, 128.4, 128.2, 128.0, 127.6, 127.5, 127.4, 126.2, 125.8, 123.2, 123.2, 110.9, 110.7, 56.2, 46.1, 45.8, 42.7, 42.2, 42.2, 40.4, 28.7, 28.7, 25.7, 19 + 23.9; F NMR (377 MHz, CDCl3) δ –117.4; MS (ESI) m/z 482.1 [M+H] ; HRMS (ESI) found + + 21 m/z 482.19176 [M+H] , calcd for C26H29FN3O3S m/z 482.19136; [α]D = –97.1° (DCM).

8-Fluoro-N-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-1,2,3,4-tetrahydroisoquinoline-6- sulfonamide (9): Compound 9 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and compound S45 (22 mg, 0.17 mmol, 1.2 equiv) to afford 34 mg (54% isolated yield) of S60 as a white solid. 1H NMR (500 MHz,

CDCl3) δ 7.49 (bs, 1H), 7.42 (d, J = 8.8 Hz, 1H), 5.89 (t, J = 4.3 Hz, 1H), 4.63 (bs, 2H), 3.73 – 3.65 (m, 4H), 3.44 (t, J = 6.7 Hz, 2H), 3.31 (t, J = 6.7 Hz, 2H), 2.90 (t, J = 5.8 Hz, 2H), 1.98 (quint, J = 6.7 Hz, 2H), 1.87 (quint, J = 6.7 Hz, 2H), 1.51 (s, + 9H); MS (ESI) m/z 441.8 [M+H] . S60 (30 mg, 0.07 mmol) was deprotected and after column chromatography 18 mg (75% isolated yield) of compound 9 was obtained as an

78 1 off-white solid. H NMR (400 MHz, CDCl3) δ 7.43 (bs, 1H), 7.34 (dd, J = 8.7, 1.7 Hz, 1H), 4.05 (s, 2H), 3.67 (s, 2H), 3.43 (t, J = 6.8 Hz, 2H), 3.29 (t, J = 6.8 Hz, 2H), 3.13 (t, J = 5.9 Hz, 2H), 2.85 (t, J = 5.9 Hz, 2H), 2.01 – 1.92 (m, 2H), 1.90 – 1.81 (m, 2H), 1.35 (t, J = 7.3 13 Hz, 1H); C NMR (101 MHz, CDCl3) δ 164.7, 160.3, 157.8, 139.1, 139.1, 137.8, 137.8, 129.0, 128.8, 123.5, 123.5, 111.3, 111.0, 46.2, 45.9, 45.4, 44.3, 42.8, 42.5, 42.4, k 29.0, 19 + 25.8, 24.1; F NMR (377 MHz, CDCl3) δ –117.0; MS (ESI) m/z 342.0 [M+H] ; HRMS (ESI) + + found m/z 342.12923 [M+H] , calcd for C15H22N3O3S m/z 342.12876.

2 (R)-N-((3S,5S,7S)-Adamantan-1-yl)-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6- sulfonamido)-3-phenylpropanamide (10): Compound 10 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and compound S46 (51 mg, 0.17 mmol, 1.2 equiv) to afford 75 mg (86% isolated yield) of S61 as a white solid. 1H NMR (400 MHz,

CDCl3) δ 7.31 – 7.29 (m, 1H), 7.24 – 7.15 (m, 4H), 7.08 – 7.02 (m, 2H), 5.37 (bs, 1H), 4.59 (bs, 2H), 3.74 (t, J = 7.1 Hz, 1H), 3.64 (t, J = 5.8 Hz, 2H), 2.95 (dd, J = 7.1, 1.7 Hz, 2H), 2.86 – 2.79 (m, 2H), 2.04 – 1.97 (m, 3H), 1.80 – 1.69 (m, 6H), 1.68 – 1.56 (m, 6H), 1.51 (s, 9H); MS (ESI) m/z 612.9 [M+H]+. S61 (65 mg, 0.11 mmol) was deprotected and after column chromatography 42 mg (77% isolated yield) of compound 10 was obtained as an off-white solid. 1H NMR (400 MHz,

CDCl3) δ 7.29 – 7.26 (m, 1H), 7.25 – 7.19 (m, 3H), 7.13 (dd, J = 8.6, 1.8 Hz, 1H), 7.09 – 7.02 (m, 2H), 5.45 (bs, 1H), 4.03 (s, 2H), 3.77 (t, J = 7.0 Hz, 1H), 3.12 (t, J = 5.9 Hz, 2H), 2.95 (qd, J = 13.8, 7.0 Hz, 2H), 2.83 – 2.76 (m, 2H), 2.04 – 1.97 (m, 3H), 1.80 – 1.71 (m, 13 6H), 1.67 – 1.55 (m, 6H), 1.26 (bs, 1H); C NMR (101 MHz, CDCl3) δ 168.5, 160.2, 157.7, 138.9, 138.8, 138.2, 138.1, 135.8, 129.3, 128.8, 128.7, 128.7, 128.5, 127.2, 123.5, 123.5, 111.1, 110.9, 58.6, 52.1, 42.7, 42.3, 42.3, 41.1, 39.4, 36.2, 29.3, 28.8, 28.8; 19F + NMR (377 MHz, CDCl3) δ –116.6; MS (ESI) m/z 512.1 [M+H] ; HRMS (ESI) found m/z + + 21 512.23804 [M+H] , calcd for C28H35FN3O3S m/z 512.23831; [α]D = –19.1° (DCM).

(R)-N-(1-(Azetidin-1-yl)-1-oxo-3-phenylpropan-2-yl)-8-fluoro-1,2,3,4- tetrahydroisoquinoline-6-sulfonamide (11): Compound 11 was synthesised according to general procedure VII starting from sulfonyl chloride S47 (50 mg, 0.14 mmol) and compound S47 (38 mg, 0.19 mmol, 1.3 equiv) to afford 72 mg (97% isolated yield) of S62 as a white solid. 1H NMR (400 MHz,

CDCl3) δ 7.36 (bs, J = 1.4 Hz, 1H), 7.30 – 7.22 (m, 4H), 7.14 – 7.10 (m, 2H), 5.48 (d, J = 9.4 Hz, 1H), 4.61 (bs, 2H), 3.91 (q, J = 8.2 Hz, 1H), 3.80 – 3.71 (m, 2H), 3.70 – 3.60 (m, 3H), 3.09 (q, J = 8.3 Hz, 1H), 2.96 – 2.80 (m, 4H),

79 2.08 – 1.97 (m, 1H), 1.96 – 1.86 (m, 1H), 1.49 (s, 9H); MS (ESI) m/z 517.9 [M+H]+. S62 (60 mg, 0.12 mmol) was deprotected and after column chromatography 39 mg (81% 1 isolated yield) of compound 11 was obtained as a white solid. H NMR (400 MHz, CDCl3) δ 7.35 (bs, J = 1.6 Hz, 1H), 7.30 – 7.22 (m, 4H), 7.15 – 7.10 (m, 2H), 4.05 (s, 2H), 3.90 (dd, J = 9.0, 6.1 Hz, 1H), 3.82 – 3.64 (m, 3H), 3.13 (t, J = 5.9 Hz, 2H), 3.07 (td, J = 8.9, 6.2 Hz, 1H), 2.95 – 2.76 (m, 4H), 2.08 – 1.99 (m, 1H), 1.97 – 1.83 (m, 1H), 1.30 – 1.22 (m, 1H); 13 C NMR (101 MHz, CDCl3) δ 169.2, 160.1, 157.6, 139.1, 139.0, 138.9, 138.8, 135.7, 129.4, 128.7, 128.5, 128.5, 127.2, 123.4, 123.3, 111.0, 110.8, 53.7, 49.7, 47.6, 42.8, 2 19 42.4, 42.3, 40.2, 29.0, 28.9, 15.2; F NMR (377 MHz, CDCl3) δ –117.2; MS (ESI) m/z 418.2 + + + [M+H] ; HRMS (ESI) found m/z 418.16006 [M+H] , calcd for C21H25FN3O3S m/z 21 418.15965; [α]D = –87.0° (DCM).

(R)-8-Fluoro-N-(1-oxo-3-phenyl-1-(piperidin-1-yl)propan-2-yl)-1,2,3,4- tetrahydroisoquinoline-6-sulfonamide (12): Compound 12 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and compound S48 (40 mg, 0.17 mmol, 1.2 equiv) to afford 73 mg (94% isolated yield) of S63 as an off-white solid. 1H NMR (400

MHz, CDCl3) δ 7.34 (bs, 1H), 7.27 – 7.20 (m, 4H), 7.14 – 7.09 (m, 2H), 5.77 (d, J = 8.9 Hz, 1H), 4.58 (bs, 2H), 4.40 (q, J = 7.7 Hz, 1H), 3.64 (d, J = 8.9 Hz, 2H), 3.41 – 3.29 (m, 1H), 3.21 – 3.11 (m, 1H), 3.05 – 2.69 (m, 6H), 1.49 (s, 9H), 1.46 – 1.06 (m, 6H); MS (ESI) m/z 546.1 [M+H]+. S63 (60 mg, 0.11 mmol) was deprotected and after column chromatography 47 mg (96% isolated yield) of 1 compound 12 was obtained as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.34 – 7.31 (m, 1H), 7.28 – 7.18 (m, 4H), 7.16 – 7.09 (m, 2H), 4.41 (dd, J = 7.8, 6.6 Hz, 1H), 4.02 (s, 2H), 3.36 – 3.26 (m, 1H), 3.26 – 3.17 (m, 1H), 3.10 (t, J = 5.9 Hz, 2H), 3.04 – 2.72 (m, 13 6H), 1.51 – 1.07 (m, 6H), 0.99 – 0.80 (m, 1H); C NMR (101 MHz, CDCl3) δ 168.3, 160.0, 157.6, 138.9, 138.8, 138.8, 138.7, 135.5, 129.6, 128.6, 128.5, 128.5, 127.2, 123.5, 123.5, 111.2, 111.0, 53.6, 46.3, 43.1, 42.8, 42.4, 42.3, 40.9, 29.0, 29.0, 25.6, 25.1, 24.0; 19 + F NMR (377 MHz, CDCl3) δ –117.2; MS (ESI) m/z 446.1 [M+H] ; HRMS (ESI) found m/z + + 21 446.19111 [M+H] , calcd for C23H29FN3O3S m/z 446.19136; [α]D = –92.0° (DCM).

(R)-8-Fluoro-N-(1-morpholino-1-oxo-3-phenylpropan-2-yl)-1,2,3,4- tetrahydroisoquinoline-6-sulfonamide (13): Compound 13 was synthesised according to general procedure VII starting from sulfonyl chlorideS16 (50 mg, 0.14 mmol, mmol) and compound S49 (40 mg, 0.17 mmol, 1.2 equiv) to afford 75 mg (96% isolated yield) of S64 as a white solid. 1H NMR (400 MHz,

80 CDCl3) δ 7.37 (s, 1H), 7.31 – 7.20 (m, 4H), 7.16 – 7.09 (m, 2H), 6.05 (d, J = 9.5 Hz, 1H), 4.60 (bs, 2H), 4.42 (td, J = 9. 5, 6.1 Hz, 1H), 3.65 (t, J = 5.7 Hz, 2H), 3.49 – 3.31 (m, 3H), 3.30 – 3.14 (m, 2H), 3.09 – 2.70 (m, 7H), 1.50 (s, 9H); MS (ESI) m/z 547.8 [M+H]+. S64 (50 mg, 0.09 mmol) was deprotected and after column chromatography 37 mg (91% isolated 1 yield) of compound 13 was obtained as a slightly yellow solid. H NMR (400 MHz, CDCl3) δ 7.34 (s, J = 7.8 Hz, 1H), 7.30 – 7.20 (m, 4H), 7.16 – 7.09 (m, 2H), 4.39 (dd, J = 9.4, 5.8 Hz, 1H), 4.03 (s, 2H), 3.47 – 3.28 (m, 3H), 3.28 – 3.07 (m, 4H), 3.04 – 2.64 (m, 7H), 1.25 (bs, 1H); 13C NMR (126 MHz, CDCl ) δ 169.0, 159.8, 157.9, 139.0, 139.0, 138.9, 138.8, 135.36, 3 2 129.6, 129.0, 128.8, 128.7, 127.5, 123.4, 123.4, 111.1, 110.9, 66.2, 65.6, 53.3, 45.7, 42.8, 19 42.4, 42.4, 42.2, 41.0, 29.1, 29.0; F NMR (377 MHz, CDCl3) δ –117.3; MS (ESI) m/z 448.1 + + + [M+H] ; HRMS (ESI) found m/z 448.17145 [M+H] , calcd for C22H27FN3O4S m/z 448.17063; 21 [α]D = –86.2° (DCM).

(R)-N,N-Diethyl-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6-sulfonamido)-3- phenylpropanamide (14): Compound 14 was synthesised according to general procedure VII starting from sulfonyl chlorideS16 (50 mg, 0.14 mmol, mmol) and compound S49 (38 mg, 0.17 mmol, 1.2 equiv) to afford 70 mg (92% isolated yield) of S65 as a white solid. 1H NMR (400

MHz, CDCl3) δ 7.34 (bs, 1H), 7.30 – 7.18 (m, 4H), 7.15 – 7.08 (m, 2H), 6.02 (d, J = 9.4 Hz, 1H), 4.57 (s, 2H), 4.35 (q, J = 9.4, 7.1 Hz, 1H), 3.63 (t, J = 5.8 Hz, 2H), 3.26 (dq, J = 14.1, 7.1 Hz, 1H), 3.06 (dq, J = 14.1, 7.1 Hz, 1H), 2.99 – 2.74 (m, 6H), 1.50 (s, 9H), 0.92 (t, J = 7.1 Hz, 3H), 0.87 (t, J = 7.1 Hz, 2H); MS (ESI) m/z 534.9 [M+H]+. S65 (50 mg, 0.09 mmol) was deprotected and after column chromatography 41 mg (85% isolated yield) of compound 14 was obtained as a white 1 solid. H NMR (400 MHz, CDCl3) δ 7.34 – 7.30 (m, 1H), 7.26 – 7.19 (m, 4H), 7.15 – 7.09 (m, 2H), 4.34 (dd, J = 7.7, 6.5 Hz, 1H), 4.01 (s, 2H), 3.31 – 3.20 (m, 1H), 3.13 – 3.00 (m, 3H), 2.99 – 2.82 (m, 4H), 2.78 (q, J = 5.2 Hz, 2H), 1.25 (bs, 1H), 0.92 (t, J = 7.1 Hz, 3H), 13 0.84 (t, J = 7.1 Hz, 3H); C NMR (126 MHz, CDCl3) δ 169.5, 159.8, 157.9, 139.3, 139.2, 138.8, 138.7, 135.6, 129.5, 128.6, 128.5, 128.5, 127.2, 123.3, 123.3, 111.1, 110.9, 54.1, 19 42.8, 42.3, 42.3, 41.5, 40.9, 40.6, 29.0, 28.9, 13.7, 12.6; F NMR (377 MHz, CDCl3) δ –117.3; MS (ESI) m/z 434.1 [M+H]+; HRMS (ESI) found m/z 434.19037 [M+H]+, calcd for + 21 C22H29FN3O3S m/z 434.19082; [α]D = –64.5° (DCM).

(R)-N,N-Dimethyl-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6-sulfonamido)-3- phenylpropanamide (15): Compound 15 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and

81 compound S50 (33 mg, 0.17 mmol, 1.2 equiv) to afford 68 mg (94% isolated yield) of 1 S66 as a white solid. H NMR (400 MHz, CDCl3) δ 7.33 (bs, 1H), 7.26 – 7.19 (m, 4H), 7.13 – 7.06 (m, 2H), 5.91 (bs, 1H), 4.58 (bs, 2H), 4.43 (t, J = 5.8 Hz, 1H), 3.64 (t, J = 5.8 Hz, 2H), 2.96 – 2.76 (m, 4H), 2.68 (s, 3H), 2.51 (bs, 3H), 1.50 (s, 9H); MS (ESI) m/z 505.7 [M+H]+. S66 (50 mg, 0.10 mmol) was deprotected and after column chromatography 38 mg (95% isolated yield) of compound 15 was obtained as an off-white solid. 1H NMR (400

MHz, CDCl3) δ 7.31 (bs, 1H), 7.26 – 7.18 (m, 4H), 7.13 – 7.08 (m, 2H), 4.42 (dd, J = 7.9, 6.8 Hz, 1H), 4.03 (s, 2H), 3.11 (t, J = 5.9 Hz, 2H), 2.91 (d, J = 8.4 Hz, 2H), 2.80 (q, J = 5.7 2 13 Hz, 2H), 2.67 (s, 3H), 2.48 (s, 3H), 1.24 (bs, 1H); C NMR (101 MHz, CDCl3) δ 170.3, 160.0, 157.6, 139.0, 139.0, 138.8, 138.7, 135.6, 129.3, 128.6, 128.5, 128.4, 127.2, 123.3, 123.3, 111.0, 110.7, 54.0, 42.8, 42.3, 42.3, 40.5, 36.5, 35.5, 28.9, 28.9; 19F NMR + (377 MHz, CDCl3) δ –117.3; MS (ESI) m/z 406.1 [M+H] ; HRMS (ESI) found m/z 406.16060 + + 21 [M+H] , calcd for C20H25FN3O3S m/z 406.16006; [α]D = –91.1° (DCM).

8-Fluoro-N-phenethyl-1,2,3,4-tetrahydroisoquinoline-6-sulfonamide (16): Compound 16 was synthesised according to general procedure VII starting from sulfonyl chloride S16 (50 mg, 0.14 mmol) and phenethylamine (21 mg, 0.17 mmol, 1.2 equiv) to afford 60 mg (97% isolated yield) of S67 as a white solid. 1H NMR (500 MHz,

CDCl3) δ 7.40 (bs, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.30 – 7.20 (m, 3H), 7.13 – 7.09 (m, 2H), 4.80 (t, J = 6.2 Hz, 1H), 4.63 (s, 2H), 3.68 (t, J = 5.8 Hz, 2H), 3.25 (q, J = 6.8 Hz, 2H), 2.88 (t, J = 5.8 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 1.53 (s, 9H); MS (ESI) m/z 457.2 [M+Na]+. S67 (50 mg, 0.12 mmol) was deprotected and after column chromatography 31 mg (81% isolated yield) of compound 16 was obtained as 1 an off-white solid. H NMR (400 MHz, CDCl3) δ 7.34 (bs, 1H), 7.30 – 7.19 (m, 4H), 7.12 – 7.07 (m, 2H), 4.04 (s, 1H), 3.23 (t, J = 6.9 Hz, 1H), 3.11 (t, J = 5.9 Hz, 1H), 2.86 – 2.75 13 (m, 4H), 2.06 (bs, 1H), 1.25 (bs, 1H); C NMR (101 MHz, CDCl3) δ 160.3, 157.8, 138.9, 138.9, 138.7, 138.6, 137.6, 128.8, 128.8, 128.7, 128.7, 128.5, 126.9, 123.3, 123.3, 19 111.1, 110.8, 44.2, 42.7, 42.3, 42.3, 35.8, 28.9, 28.9; F NMR (377 MHz, CDCl3) δ –116.9; MS (ESI) m/z 335.1 [M+H]+; HRMS (ESI) found m/z 335.12305 [M+H]+, calcd for + C17H20FN2O2S m/z 335.12295.

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)benzenesulfonamide (17): Compound 17 was synthesised according to general procedure VII starting from benzenesulfonyl chloride (50 mg, 0.28 mmol) and D-phenylalanine pyrrolidine amide 30 (74 mg, 0.34 mmol, 1.2 equiv) to afford 88 mg (88% isolated yield) of compound17 as a white solid. 1 H NMR (500 MHz, CDCl3) δ 7.84 – 7.80 (m, 2H), 7.56 – 7.51 (m, 1H),

82 7.49 – 7.43 (m, 2H), 7.27 – 7.19 (m, 3H), 7.17 – 7.12 (m, 2H), 5.95 – 5.90 (d, J = 9.5 Hz, 1H), 4.17 – 4.05 (td, J = 9.5, 5.9 Hz, 1H), 3.14 – 3.06 (m, 1H), 3.03 – 2.89 (m, 3H), 2.87 13 – 2.80 (m, 1H), 2.32 – 2.24 (m, 1H), 1.57 – 1.36 (m, 4H); C NMR (126 MHz, CDCl3) δ 168.4, 139.9, 135.7, 132.6, 129.4, 128.8, 128.4, 127.2, 127.2, 56.0, 45.9, 45.5, 40.7, 25.6, 23.7; MS (ESI) m/z 359.0 [M+H]+; HRMS (ESI) found m/z 381.12487 [M+Na]+, calcd + 21 for C19H22N2O3SNa m/z 381.12433; [α]D = –125.8° (DCM).

(R)-3-Fluoro-N-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)benzenesulfonamide (18): 2 Compound 18 was synthesised according to general procedure VII starting from 3-fluorophenylsulfonyl chloride (50 mg, 0.26 mmol) and D-phenylalanine pyrrolidine amide 30 (67 mg, 0.31 mmol, 1.2 equiv) to afford 94 mg (94% isolated yield) of compound18 as a white solid. 1 H NMR (500 MHz, CDCl3) δ 7.62 (dt, J = 8.0, 1.7, 1.0 Hz, 1H), 7.51 (dt, J = 8.0, 2.5, 1.7 Hz, 1H), 7.45 (td, J = 8.0, 5.2 Hz, 1H), 7.28 – 7.20 (m, 4H), 7.17 – 7.13 (m, 2H), 6.27 (d, J = 9.7 Hz, 1H), 4.20 (ddd, J = 9.7, 8.4, 6.7 Hz, 1H), 3.23 – 3.11 (m, 1H), 3.07 – 2.94 (m, 4H), 2.45 – 2.36 (m, 1H), 1.68 – 1.42 (m, 4H); 13C NMR

(126 MHz, CDCl3) δ 168.3, 163.1, 161.1, 142.4, 142.3, 135.6, 130.7, 130.7, 129.4, 128.5, 127.2, 123.0, 122.9, 119.7, 119.6, 114.5, 114.3, 56.2, 46.0, 45.7, 40.5, 25.7, 23.8; 19F + NMR (377 MHz, CDCl3) δ –109.8; MS (ESI) m/z 376.9 [M+H] ; HRMS (ESI) found m/z + + 21 399.11732 [M+Na] , calcd for C19H21FN2O3SNa m/z 399.11546; Optical rotation [α]D = –116.4° (DCM).

(R)-4-fluoro-N-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)benzenesulfonamide (19): Compound 19 was synthesised according to general procedure VII starting from 3-fluorophenylsulfonyl chloride (50 mg, 0.26 mmol) and D-phenylalanine pyrrolidine amide 30 (67 mg, 0.31 mmol, 1.2 equiv) to afford 78 mg (78% isolated yield) of compound 19 as a 1 white solid. H NMR (500 MHz, CDCl3) δ 7.83 – 7.77 (m, 2H), 7.27 – 7.18 (m, 3H), 7.15 – 7.06 (m, 4H), 6.15 (d, J = 9.8 Hz, 1H), 4.19 – 4.09 (m, 1H), 3.18 – 3.08 (m, 1H), 3.05 – 2.89 (m, 4H), 2.44 – 2.35 (m, 1H), 1.63 – 1.51 13 (m, 3H), 1.50 – 1.41 (m, 1H); C NMR (126 MHz, CDCl3) δ 168.6, 166.0, 163.9, 136.2, 136.2, 135.6, 130.0, 129.9, 129.4, 128.5, 127.2, 116.0, 115.8, 56.0, 46.0, 45.7, 40.4, 19 + 25.6, 23.8; F NMR (377 MHz, CDCl3) δ –105.4; MS (ESI) m/z 377.1 [M+H] ; HRMS (ESI) + + 21 found m/z 399.11607 [M+Na] , calcd for C19H21FN2O3SNa m/z 399.11546; [α]D = –96.7° (DCM).

83 (R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-2,3-dihydrobenzo[b][1,4]dioxine- 6-sulfonamide (20): Compound 20 was synthesised according to general procedure VII starting from 2,3-dihydro-benzo[1,4]dioxine-6-sulfonyl chloride (50 mg, 0.21 mmol) and D-phenylalanine pyrrolidine amide 30 (56 mg, 0.26 mmol, 1.2 equiv) to afford 88 mg (99% isolated yield) of compound 20 as a white solid. 1H NMR (500 MHz, CDCl ) δ 7.32 (d, J = 2.3 Hz, 1H), 7.30 – 7.19 (m, 4H), 7.17 2 3 – 7.11 (m, 2H), 6.89 (d, J = 8.5 Hz, 1H), 5.81 (d, J = 9.8 Hz, 1H), 4.33 – 4.21 (m, 4H), 4.13 (td, J = 5.9, 9.55 Hz, 1H), 3.18 – 3.03 (m, 2H), 3.01 – 2.89 (m, 3H), 2.38 – 2.29 (m, 1H), 13 1.67 – 1.53 (m, 3H), 1.50 – 1.39 (m, 1H); C NMR (126 MHz, CDCl3) δ 168.6, 147.2, 143.2, 135.8, 132.5, 129.4, 128.4, 127.1, 120.9, 117.5, 116.8, 64.5, 64.1, 56.0, 45.9, 45.6, 40.6, 25.7, 23.8; MS (ESI) m/z 417.0 [M+H]+; HRMS (ESI) found m/z 439.13019 + + 21 [M+Na] , calcd for C21H24N2O5SNa m/z 439.13036. [α]D = –106.4° (DCM).

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)benzo[d]thiazole-6-sulfonamide (21): Compound 21 was synthesised according to general procedure VII starting from 6-benzothiazolesulfonyl chloride (50 mg, mmol) and D-phenylalanine pyrrolidine amide 30 (56 mg, 0.26 mmol, 1.2 equiv) to afford 87 mg (98% isolated yield) of compound 21 as a 1 white solid. H NMR (500 MHz, CDCl3) δ 9.20 (s, 1H), 8.44 (dd, J = 0.6, 1.90 Hz, 1H), 8.18 (dd, J = 0.6, 8.6 Hz, 1H), 7.92 (dd, J = 1.9, 8.6 Hz, 1H), 7.23 – 7.14 (m, 3H), 7.14 – 7.09 (m, 2H), 6.21 (bs, 1H), 4.19 (dd, J = 6.4, 8.7 Hz, 1H), 3.10 – 2.93 (m, 3H), 2.93 – 2.77 (m, 2H), 2.42 – 2.33 (m, 1H), 1.51 – 1.36 (m, 3H), 13 1.35 – 1.24 (m, 1H); C NMR (126 MHz, CDCl3) δ 168.4, 157.9, 155.4, 137.4, 135.6, 133.8, 129.4, 128.4, 127.2, 125.0, 123.9, 122.0, 56.2, 45.9, 45.6, 40.5, 25.6, 23.7; MS (ESI) m/z 416.1 [M+H]+; HRMS (ESI) found m/z 438.09165 [M+Na]+, calcd for + 21 C20H21N3O3S2Na m/z 438.09220. [α]D = –107.9° (DCM).

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-2,3-dihydrobenzofuran-5- sulfonamide (22): Compound 22 was synthesised according to general procedure VII starting from 2,3-dihydrobenzofuran-5-sulfonyl chloride (50 mg, 0.23 mmol) and D-phenylalanine pyrrolidine amide 30 (60 mg, 0.27 mmol, 1.2 equiv) to afford 89 mg (97% isolated yield) of 1 compound 22 as a white solid. H NMR (500 MHz, CDCl3) δ 7.62 – 7.55 (m, 2H), 7.25 – 7.17 (m, 3H), 7.15 – 7.10 (m, 2H), 6.74 (d, J = 8.3 Hz, 1H), 5.91 (d, J = 9.9 Hz, 1H), 4.63 (t, J = 8.8 Hz, 2H), 4.16 – 4.06 (m, 1H), 3.29 –

84 3.09 (m, 3H), 3.05 – 2.88 (m, 4H), 2.38 – 2.30 (m, 1H), 1.60 – 1.50 (m, 3H), 1.47 – 1.40 13 (m, 1H); C NMR (126 MHz, CDCl3) δ 168.8, 163.7, 135.8, 131.5, 129.4, 128.7, 128.4, 128.2, 127.1, 124.6, 108.8, 72.3, 55.9, 45.9, 45.6, 40.5, 29.0, 25.6, 23.8; MS (ESI) m/z 401.0 [M+H]+; HRMS (ESI) found m/z 423.13553 [M+Na]+, calcd for + 21 C21H24N2O4SNa m/z 423.13553. [α]D = –124.2° (DCM).

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-1,2,3,4-tetrahydroisoquinoline- 6-sulfonamide (23): 2 Compound 23 was synthesised according to general procedure VII starting from sulfonyl chloride S17 (50 mg, 0.15 mmol) and D-phenylalanine pyrrolidine amide 30 (39 mg, 0.18 mmol, 1.2 equiv) to afford 75 mg (97% isolated yield) ofS68 as an off-white 1 solid. H NMR (400 MHz, CDCl3) δ 7.60 – 7.54 (m, 2H), 7.25 – 7.18 (m, 3H), 7.17 – 7.14 (m, 1H), 7.13 – 7.09 (m, 2H), 5.80 (d, J = 9.7 Hz, 1H), 4.58 (bs, 2H), 4.24 – 4.12 (m, 1H), 3.62 (t, J = 5.5 Hz, 3H), 3.15 – 3.06 (m, 1H), 3.03 – 2.75 (m, 6H), 2.34 (bs, 1H), 1.57 – 1.39 (m, 13H); MS (ESI) m/z 536.0 [M+Na]+. S68 (70 mg, 0.14 mmol) was deprotected and after column chromatography 56 mg (99% isolated yield) of compound 23 was obtained as an off-white solid. 1H NMR (400

MHz, CDCl3) δ 7.56 – 7.48 (m, 2H), 7.26 – 7.16 (m, 3H), 7.15 – 7.04 (m, 3H), 4.13 (dd, J = 8.9, 6.2 Hz, 1H), 4.07 (s, 2H), 3.21 – 3.06 (m, 3H), 3.06 – 2.71 (m, 7H), 2.40 – 2.26 (m, 13 1H), 1.64 – 1.48 (m, 3H), 1.50 – 1.36 (m, 1H), 1.23 (bs, 1H); C NMR (101 MHz, CDCl3) δ 168.7, 140.1, 137.8, 135.8, 135.6, 129.4, 128.4, 128.0, 127.1, 126.8, 124.5, 56.0, 47.8, 45.9, 45.6, 43.2, 40.5, 28.8, 25.7, 23.8; MS (ESI) m/z 414.1 [M+H]+; HRMS (ESI) found + + 21 m/z 414.18487 [M+H] , calcd for C22H28N3O3S m/z 414.18514. [α]D = –96.9° (DCM).

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-5,6,7,8-tetrahydronaphthalene- 2-sulfonamide (24): Compound 24 was synthesised according to general procedure VII starting from 5,6,7,8-tetrahydronaphthalene-2-sulfonyl chloride (100 mg, 0.43 mmol) and D-phenylalanine pyrrolidine amide 30 (114 mg, 0.52 mmol, 1.2 equiv) to afford 153 mg (86% isolated yield) of compound 24 as a colourless oil. 1H NMR (400 MHz,

CDCl3) δ 7.51 – 7.45 (m, 2H), 7.25 – 7.17 (m, 3H), 7.15 – 7.08 (m, 3H), 5.77 (d, J = 9.6 Hz, 1H), 4.08 (td, J = 9.6, 5.9 Hz, 1H), 3.14 – 3.03 (m, 1H), 3.02 – 2.85 (m, 3H), 2.85 – 2.66 (m, 5H), 2.31 – 2.19 (m, 1H), 1.78 (quint, 13 J = 3.3 Hz, 4H), 1.58 – 1.32 (m, 4H); C NMR (126 MHz, CDCl3) δ 168.6, 142.6, 137.9, 136.7, 135.8, 129.4, 129.4, 128.4, 127.8, 127.1, 124.2, 55.9, 45.8, 45.4, 40.7, 29.5, 29.3, 25.6, 23.8, 22.7, 22.7; MS (ESI) m/z 413.1 [M+H]+; HRMS (ESI) found m/z 435.17130

85 + + 21 [M+Na] , calcd for C23H28O3N2SNa m/z 435.17183. [α]D = –92.7° (DCM).

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-1,2,3,4-tetrahydroquinoline-6- sulfonamide (25): Compound 25 was synthesised according to general procedure VII starting from sulfonyl chloride S18 (50 mg, 0.15 mmol) and D-phenylalanine pyrrolidine amide 30 (39 mg, 0.18 mmol, 1.2 equiv) to afford 69 mg (89% isolated yield) of S69 as an off-white 2 1 solid. H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 8.8 Hz, 1H), 7.55 (dd, J = 8.8, 2.3 Hz, 1H), 7.50 (d, J = 2.3 Hz, 1H), 7.27 – 7.17 (m, 3H), 7.16 – 7.10 (m, 2H), 5.80 (d, J = 9.8 Hz, 1H), 3.75 – 3.65 (m, 2H), 3.14 – 2.69 (m, 7H), 2.33 - 2.27 (m, 1H), 1.97 – 1.83 (m, 2H), 1.61 – 1.48 (m, 12H), 1.44 – 1.35 (m, 1H); MS (ESI) m/z 514.0 [M+H]+. S69 (50 mg, 0.10 mmol) was deprotected and after column chromatography 32 mg (79% isolated yield) of compound 25 was 1 obtained as a slightly yellow solid. H NMR (400 MHz, CDCl3); δ 7.30 – 7.23 (m, 2H), 7.19 – 7.09 (m, 3H), 7.09 – 7.02 (m, 2H), 6.27 (d, J = 8.5 Hz, 1H), 5.49 (d, J = 9.8 Hz, 1H), 3.94 (td, J = 9.8, 5.7 Hz, 1H), 3.25 (dd, J = 6.1, 5.0 Hz, 2H), 3.06 – 2.87 (m, 3H), 2.87 – 2.51 (m, 4H), 2.20 – 2.06 (m, 1H), 1.85 – 1.75 (m, 2H), 1.51 – 1.38 (m, 3H), 1.35 – 1.23 (m, 1H); 13 C NMR (101 MHz, CDCl3) δ 168.1, 147.4, 135.0, 128.4, 127.9, 127.3, 126.0, 125.9, 124.1, 119.0, 111.2, 54.8, 44.8, 44.5, 40.5, 39.7, 25.9, 24.6, 22.8, 20.1; MS (ESI) m/z 414.0 + + + [M+H] ; HRMS (ESI) found m/z 436.16680 [M+Na] , calcd for C22H27N3O3SNa m/z 21 436.16708; [α]D = –134.3° (DCM).

(R)-N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)-1,2,3,4-tetrahydroisoquinoline- 7-sulfonamide (26): Compound 26 was synthesised according to general procedure VII starting from 2-(2,2,2-trifluoroacetyl)-1,2,3,4- tetrahydroisoquinoline-7-sulfonyl chloride (50 mg, 0.15 mmol) and D-phenylalanine pyrrolidine amide 30 (40 mg, 0.18 mmol, 1.2 equiv) to afford 59 mg (76% isolated yield) of S70 as a white 1 solid. H NMR (400 MHz, CDCl3) δ 7.65 – 7.59 (m, 1H), 7.55 (bs, 1H), 7.28 – 7.16 (m, 4H), 7.14 – 7.08 (m, 2H), 6.06 (dd, J = 9.7, 4.8, 1H), 4.77 (q, J = 17.2, 2H), 4.24 – 4.12 (m, 1H), 3.96 – 3.78 (m, 2H), 3.20 – 2.89 (m, 7H), 2.52 – 2.41 (m, 1H), 1.68 – 1.44 (m, 4H); MS (ESI) m/z 510.0 [M+H]+. For deprotection, S70 (80 mg, 0.16 mmol) was dissolved in 0.5 mL MeOH/water (5:1 v/v), and to this was added potassium carbonate (22 mg, 0.16 mmol). The reaction was stirred at rt for 1 h after which it was diluted with water (10 mL) and extracted with EtOAc (2 × 10 mL). The combined organic

fractions were washed with brine (20 mL), dried over Na2SO4, and concentrated in

86 vacuo. The crude product was purified by column chromatography to afford 62 mg (95% isolated yield) of compound 26 was obtained as an off-white solid. 1H NMR (500

MHz, CDCl3) δ 7.53 (dd, J = 8.0, 2.0 Hz, 1H), 7.46 (bs, 1H), 7.26 – 7.18 (m, 3H), 7.17 – 7.09 (m, 3H), 4.12 (dd, J = 9.2, 6.0 Hz, 1H), 4.02 (q, J = 16.2 Hz, 2H), 3.20 – 3.08 (m, 3H), 3.06 – 2.88 (m, 4H), 2.83 (t, J = 6.0, 2H), 2.38 – 2.29 (m, 1H), 1.64 – 1.35 (m, 4H); 13C

NMR (126 MHz, CDCl3) δ 168.6, 140.2, 137.3, 136.5, 135.7, 129.7, 129.4, 128.4, 127.1, 125.2, 124.7, 56.0, 47.9, 45.9, 45.5, 43.2, 40.6, 29.2, 25.6, 23.8; MS (ESI) m/z 414.1 [M+H]+; HRMS (ESI) found m/z 414.18545 [M+H]+, calcd for C H N O S+ m/z 414.18514; 22 28 3 3 2 21 [α]D = –107.9° (DCM).

(R)-4-(aminomethyl)-3-fluoro-N-(1-oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl) benzenesulfonamide (27): Compound 27 was synthesised according to general procedure VII starting from sulfonyl chloride S19 (50 mg, 0.15 mmol) and D-phenylalanine pyrrolidine amide 30 (40 mg, 0.19 mmol, 1.2 equiv) to afford 69 mg (88% isolated yield) of S71 as a white 1 solid. H NMR (400 MHz, CDCl3) δ 7.56 – 7.50 (m, 1H), 7.46 – 7.38 (m, 2H), 7.25 – 7.18 (m, 3H), 7.15 – 7.09 (m, 2H), 6.06 (d, J = 9.7 Hz, 1H), 5.10 – 5.01 (m, 1H), 4.36 (d, J = 6.4 Hz, 2H), 4.17 (q, J = 8.4 Hz, 1H), 3.15 (dt, J = 12.6, 6.4 Hz, 1H), 3.02 (quint, J = 6.7 Hz, 2H), 2.97 – 2.89 (m, 2H), 2.46 – 2.37 (m, 1H), 1.68 – 1.49 (m, 4H), 1.44 (s, 9H); MS (ESI) m/z 505.9 [M+H]+. S71 (50 mg, 0.10 mmol) was deprotected and after column chromatography 35 mg (87% isolated yield) 1 of compound 27 was obtained as a slightly yellow solid. H NMR (400 MHz, CDCl3) δ 7.56 (dd, J = 8.0, 1.8 Hz, 1H), 7.51 – 7.46 (m, 1H), 7.42 (dd, J = 9.4, 1.8 Hz, 1H), 7.26 – 7.17 (m, 3H), 7.16 – 7.08 (m, 2H), 4.19 (dd, J = 8.5, 6.6 Hz, 1H), 3.94 (s, 2H), 3.14 (dd, J = 13.0, 6.1 Hz, 1H), 3.10 – 2.99 (m, 2H), 3.00 – 2.84 (m, 2H), 2.50 – 2.36 (m, 1H), 1.71 13 – 1.54 (m, 3H), 1.54 – 1.41 (m, 1H); C NMR (126 MHz, CDCl3) δ 168.4, 161.0, 159.0, 140.5, 140.5, 135.6, 135.2, 135.1, 129.5, 129.5, 129.4, 128.5, 127.2, 123.1, 123.0, 19 114.3, 114.1, 56.2, 46.1, 45.7, 40.5, 40.0, 39.9, 25.7, 23.8; F NMR (377 MHz, CDCl3) δ –116.5; MS (ESI) m/z 406.1 [M+H]+; HRMS (ESI) found m/z 406.16057 [M+H]+, calcd for + 21 C20H25FN3O3S m/z 406.16006; [α]D = –82.2° (DCM).

(R)-4-(N-(1-Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)sulfamoyl)benzamide (28): Compound 28 was synthesised according to general procedure VII starting from 4-(aminocarbonyl)benzenesulfonyl chloride (50 mg, 0.23 mmol) and D-phenylalanine pyrrolidine amide 30 (60 mg, 0.27 mmol, 1.2 equiv) to afford 69 mg (75% isolated yield) of compound 28 as a white solid. 1H NMR (500 MHz,

87 DMSO-d6) δ 8.39 (s, 1H), 8.14 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.5 Hz, 2H), 7.58 (s, 1H), 7.28 – 7.09 (m, 4H), 4.10 (t, J = 7.6 Hz, 1H), 3.24 – 3.15 (m, 1H), 2.92 (dt, J = 6.9, 11.5 Hz, 1H), 2.88 – 2.66 (m, 4H), 1.71 – 1.60 (m, 1H), 1.52 - 1.45 (m, 3H); 13C NMR

(126 MHz, DMSO-d6) δ 168.0, 167.1, 143.5, 137.8, 137.0, 129.7, 128.6, 128.3, 127.1, 126.7, 56.1, 46.0, 45.6, 38.6, 25.2, 23.8; MS (ESI) m/z 402.0 [M+H]+; HRMS (ESI) found + + 21 m/z 424.13147 [M+Na] , calcd for C20H23N3O4SNa m/z 424.13070; [α]D = –91.7° (DCM).

8-fluoro-6-sulfamoyl-1,2,3,4-tetrahydroisoquinolin-2-ium trifluoroacetate (29): 2 Compound 29 was prepared starting from starting from sulfonyl chloride S16 (50 mg, 0.14 mmol, 1.0 equiv), which was dissolved in 1.0 mL of tetrahydrofuran. Whilst stirring, ammonium hydroxide (72 mg, 0.57 mmol, 3.0 equiv) was added, and the reaction mixture was left to stir for 1 h. The reaction was diluted with water (10 mL), and extracted with EtOAc (3 × 10mL). The combined organic layers were

washed with brine (30mL), dried over MgSO4 and concentrated in vacuo. The crude product was purified using column chromatography (10-25% EtOAc in n-heptane) to afford 40 mg (85% isolated yield) of compoundS72 as a white solid. 1H NMR (400 MHz,

CDCl3) δ 7.54 (bs, 1H), 7.44 (dd, J = 8.8, 1.7 Hz, 1H), 5.25 (s, 2H), 4.61 (s, 2H), 3.66 (t, J = 5.8 Hz, 2H), 2.89 (t, J = 5.8 Hz, 2H), 1.50 (s, 9H). S72 (20 mg, 60 µmol) was deprotected by stirring at rt for 1 h in TFA/DCM (1:3) to afford 21 mg (>99% isolated yield)of 1 trifluoroacetate salt29 as a white solid. H NMR (500 MHz, CD3OD) δ 7.67 (d, J = 1.5 Hz, 1H), 7.57 (dd, J = 9.2, 1.6 Hz, 1H), 4.47 (s, 2H), 3.57 (t, J = 6.3 Hz, 2H), 3.23 (t, J = 6.3 Hz, 13 2H); C NMR (126 MHz, CD3OD) δ 160.0, 158.0, 145.1, 145.0, 135.4, 135.3, 122.0, 122.0, 120.3, 120.1, 110.9, 110.7, 40.7, 39.3, 39.2, 24.5, 24.5; 19F NMR (377 MHz, - + CD3OD) δ –77.0 (CF3COO ), –118.1; MS (ESI) m/z 231.1 [M+H] ; HRMS (ESI) found m/z + + 231.06035 [M+H] , calcd for C9H12FN2O2S m/z 231.05922.

88 2.5. References

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89 Ogawa, M. Tariq, N. Nishino, S. Dan, H. Kagechika, T. Yamori, S. Yokoyama, M. Yoshida, J. Med. Chem. 2016, 59, 3650-3660. 25. T. Fujiwara, K. Ohira, K. Urushibara, A. Ito, M. Yoshida, M. Kanai, A. Tanatani, H. Kagechika, T. Hirano, Bioorg. Med. Chem. 2016, 24, 4318-4323. 26. H. Niwa, N. Handa, Y. Tomabechi, K. Honda, M. Toyama, N. Ohsawa, M. Shirouzu, H. Kagechika, T. Hirano, T. Umehara, S. Yokoyama, Acta Crystallogr. D Biol. Crystallogr. 2013, 69, 595-602. 27. F. Meng, S. Cheng, H. Ding, S. Liu, Y. Liu, K. Zhu, S. Chen, J. Lu, Y. Xie, L. Li, R. Liu, Z. Shi, Y. Zhou, Y. C. Liu, M. Zheng, H. Jiang, W. Lu, H. Liu, C. Luo, J. Med. Chem. 2015, 58, 8166-8181. 2 28. D. Barsyte-Lovejoy, F. Li, M. J. Oudhoff, J. H. Tatlock, A. Dong, H. Zeng, H. Wu, S. A. Freeman, M. Schapira, G. A. Senisterra, E. Kuznetsova, R. Marcellus, A. Allali-Hassani, S. Kennedy, J. P. Lambert, A. L. Couzens, A. Aman, A. C. Gingras, R. Al-Awar, P. V. Fish, B. S. Gerstenberger, L. Roberts, C. L. Benn, R. L. Grimley, M. J. Braam, F. M. Rossi, M. Sudol, P. J. Brown, M. E. Bunnage, D. R. Owen, C. Zaph, M. Vedadi, C. H. Arrowsmith, Proc. Natl. Acad. Sci. USA 2014, 111, 12853-12858. 29. D. C. Lenstra, A. H. K. Al Temimi, J. Mecinović, Bioorg. Med. Chem. Lett. 2018, 28, 1234-1238. 30. K. Guitot, T. Drujon, F. Burlina, S. Sagan, S. Beaupierre, O. Pamlard, R. H. Dodd, C. Guillou, G. Bolbach, E. Sachon, D. Guianvarc’h, Anal. Bioanal. Chem. 2017, 409, 3767-3777. 31. Chemical Computing Group Molecular Operating Environment (MOE), https://www.chemcomp.com/. 32. Qiagen Molegro Virtual Docker v6.0, https://www.qiagenbioinformatics.com/. 33. Schrödinger PyMOL, https://pymol.org/.

90 2

91 92 3

An investigation of (R)-PFI-2 analogues with a dual purpose: substrates and inhibitors of histone lysine methyltransferase SETD7

93 Abstract

In this chapter the synthesis and biological evaluation of 20 novel (R)-PFI-2 analogues that are modified at the pyrrolidine amide moiety is described. With the aim to develop inhibitors of human histone lysine monomethyltransferase SETD7, which can also act as small molecule substrates, we replaced the pyrrolidine ring of (R)-PFI-2 with several side chains bearing nucleophilic functional groups. Initially, we explored the inhibitory activity of these compounds, and found that from this set the most potent analogue

has a hydroxyethyl side chain, with IC50 = 0.96 µM. Then, SETD7’s ability to catalyse methylation of various analogues was evaluated by mass spectrometric assays, and we observed efficient methylation of analogues bearing lysine mimicking nucleophilic amines in the side chain. The optimal side chain was found to be an aminoethyl group, which was surprisingly also dimethylated by SETD7. 3

94 3.1. Introduction

In eukaryotes the DNA is highly organised and packed into chromosomes. The chromatin fibre of chromosomes exists in two forms, a more compact heterochromatin anda more loose euchromatin state.[1] Heterochromatin typically results in gene silencing, whereas the euchromatin form leads to transcriptional activation. The interconversion between euchromatin and heterochromatin is a dynamic process that is regulated by posttranslational modifications (PTMs) on histone proteins.[2] Histone PTMs, such as methylation, acetylation, phosphorylation, and ubiquitination are mainly introduced to amino acid side chains on different N‑terminal histone tails, which protrude from the nucleosome core particle.[3]

a) b) CF3 N HO N HO N OH N OH 3 H N Me O H N O 2 + 2 N S N S N O N O O O NH2 NH2 S O N H HN N

SAM SAH F Me NH3 H2N

O O SETD7 S O N N N H H H O O HN N R1 R2 H3K4 H3K4me1 F

Figure 1. a) SETD7-catalysed transfer of the methyl group from S-adenosylmethionine (SAM) to histone 3 lysine 4 (H3K4); b) The structure of (R)-PFI-2 (top) and analogues (bottom) that are evaluated in this work, where the pyrrolidine amide is replaced by side chains with different nucleophilic functionalities.

SET domain-containing protein 7 (SETD7, also known as SET7/9 and KMT7) is a histone lysine methyltransferase (HKMT), which catalyses the transfer of one methyl group from S‑adenosylmethionine (SAM) to the ε-amino group of lysine 4 on histone tail 3 (H3K4, Figure 1a).[4, 5] Recent studies have demonstrated that SETD7 regulates a variety of important cellular processes such as cell growth and apoptosis, through methylation of histones, but also numerous non-histone proteins, including estrogen receptor α (ERα), DNA methyltransferase 1 (DNMT1), FOXO3, NFkB, TAF10, and PCAF.[6-11] Dysfunction of SETD7’s methyltransferase activity is linked with several diseases, including vascular dysfunction in patients with type 2 diabetes[12] and several types of cancer.[13-15] To investigate SETD7’s functions, selective and potent chemical probes are highly desired.[16] For this purpose, several SETD7 inhibitors have been recently developed, the most

95 [17] potent being (R)-PFI-2, with an IC50 of 2.0 ± 0.2 nM (Figure 1b). (R)‑PFI‑2 is a histone competitive inhibitor, which was found to be selective for SETD7 over 18other human methyltransferases. Other inhibitors of SETD7 that have been identified since

the discovery of (R)-PFI-2 are cyproheptadine (IC50 = 3.4 µM) and its analogues with [18-20] improved potency, for instance 2-hydroxcyproheptadine (IC50 = 0.41 µM).

A structure-activity relationship (SAR) study on (R)-PFI-2 was recently reported by our group and described in chapter 2 of this thesis.[21] We evaluated a library of 29 analogues bearing variations in (R)-PFI-2 at three distinct parts, namely the tetrahydroisoquinoline moiety, the D-phenylalanine side chain and the pyrrolidine amide of (R)‑PFI‑2. We + found that the tetrahydroisoquinoline’s NH2 was essential for effective inhibition of human SETD7. This SAR exploration inspired us to design and develop a series of (R)-PFI-2 analogues with a variety of nucleophilic functionalities on the pyrrolidine 3 amide moiety that occupies the lysine-binding pocket of SETD7 and makes a direct contact with cosubstrate SAM. We hypothesised that a replacement of the pyrrolidine amide by a lysine mimic in (R)‑PFI‑2 might lead to inhibitors with an increased potency, and that such compounds might also act as SETD7 substrates (Figure 1c). In biological assays, a small molecule substrate of SETD7 may be used to modulate the activity of SETD7 and the local levels of SAM cosubstrate. To this purpose, we introduced several different side chains with various lengths, bearing different nucleophilic moieties, such as amines, alcohol, thiol, alkene, and an alkyne.

3.2. Results and discussion

We started our investigations with the synthesis of 20 structural analogues of (R)-PFI-2 bearing different functional groups substituting the pyrrolidine amide (Figure 1b). Depending on the nature of the functional group that was introduced, the synthesis started from either Cbz, Fmoc, or Boc protected D-phenylalanine, which was coupled to the respective amine in the presence of EDC, HOBt and DIPEA to produce intermediates S1-S19 (Scheme 1). Subsequently, the protecting group was removed to obtain compounds S20‑S37: Cbz was deprotected by catalytic hydrogenation,

employing 10% Pd on carbon and H2 gas (1 atm), Fmoc was removed in the presence of DBU in DCM, and Boc under acidic conditions with TFA/DCM. The free amine was then coupled to sulfonyl chloride S38, which was synthesised as previously described.[17, 21]

After Boc deprotection of compounds S39-S58, the final compounds 1-17 were obtained. Analogues 18 and 19 were obtained using similar chemistry, however

96 employing 3‑fluorosulfonyl chloride rather than S38 in the synthesis of 18, and Cbz- Gly-OH (S18) instead of D‑phenylalanine derivatives for the synthesis of 19. Fragment 20 was synthesised directly from D‑phenylalanine derivative S19.

3 Scheme 1. Reagents and conditions: i) PG-D-Phe-OH (1.2 equiv, PG = Fmoc, Boc, or Cbz), amine (1.0 equiv),

EDC (1.5 equiv), HOBt (1.8 equiv), Et3N (2.0 equiv), DCM, 0 °C to rt, 20 h; ii) 10% Pd/C, H2 (1 atm), MeOH, rt,

16 h; or TFA/DCM (1:1 v/v), rt, 2 h; or DBU, DCM, rt, 1 h; iii) Et3N (3.0 equiv), DCM, rt, 20 h; iv) TFA/DCM (1:1 v/v), rt, 2 h.

After successful synthesis of the library of (R)-PFI-2 analogues, we set out to explore the level by which compounds 1-20 inhibit human SETD7-catalysed methylation of H3K4. A matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) based assay was used to determine the half maximum inhibitory [22, 23] concentrations (IC50 values) for all compounds (Figure 2). In short, recombinantly expressed human SETD7 (200 nM) was pre-incubated at 37 °C for 5 min with compounds 1-20 at various concentrations in 50 mM glycine pH 8.8 as assay buffer, after which the enzymatic reaction was initiated by the addition of a premixture of synthetic histone peptide (10 µM) consisting of the 21 amino acids of the N-terminal tail of histone 3 (residues 1-21) and cosubstrate SAM (16 µM), and stirring for an additional 55 min at

37 °C. For comparison, an IC50 of 138 nM was observed for (R)‑PFI‑2 in previous work employing this assay.[21] For compound 1, which possesses the shortest side chain, an

IC50 value of 4.71 µM was observed (Figure 2). By increasing the length of the side chain, the potency of the respective analogues dropped significantly: for compound 2 we observed IC50 = 17.8 µM; for compound 3, IC50 = 36.4 µM; and compound 4, with a side chain consisting of 5 carbons between the amine and amide functionalities, IC50 = 84.9 µM (Figure 2). As binding of (R)‑PFI‑2 and analogues is SAM-dependent, meaning that SAM has to bind first before the inhibitor can bind, it is likely that with increasing the length of the side chain, the longer substrates cannot bind optimally, leading to

97 lower IC50 values. Piperazine 5 inhibited SETD7 with an approximately similar degree

to that of compound 3, with an IC50 value of 36.9 µM (Figure 2). The increased size and reduced rotational freedom of the aniline group in compound 6 made it no longer an

inhibitor of SETD7 (IC50 > 100 µM, Figure 2).

Figure 2. Library of (R)-PFI-2 analogues 1-20, showing the maximum half inhibitory concentrations (IC50 values).

Next, we evaluated compounds 7-12, which possess the same chain length and different nucleophilic functional groups, as inhibitors of SETD7. Both the alcohol

and thiol containing analogues were found to be good SETD7 inhibitors with IC50 values of 0.96 and 2.25 µM, respectively (Figure 2, 7 and 8). Amide 9, was found to

98 inhibit SETD7 with IC50 = 3.17 µM, whereas the carboxylic acid analogue 10 did not significantly inhibit SETD7 (IC50 > 100 µM). Compounds 11 and 12 bearing alkene and alkyne functionality were found to be good SETD7 inhibitors with IC50 values of 1.76 and 4.03 µM, respectively. The introduction of one or two N-methyl groups on the side chain of compound 1, as in compounds 13 and 14, surprisingly resulted in analogues that were unable to inhibit SETD7 (IC50 > 100 µM). Compound 15, the carbon analogue of compound 1, was found to be a good SETD7 inhibitor with IC50 = 1.38 µM. Based on the observation that compound 16, which is the carbon analogue of compound 13, is still able to inhibit SETD7 with IC50 = 4.19 µM, we hypothesised that the polar NH group of compound 13 makes an unfavourable interaction with the hydrophobic channel of

SETD7, and is thus not able to inhibit SETD7. Comparing compound 17 (IC50 > 100 µM) with compound 14, shows that the dimethyl group of 14, is likely too big to enter the narrow channel of SETD7. In line with our expectations, we observed IC50 values greater than 100 µM for analogues 18-20. 3

We then moved on to explore SETD7’s ability to catalyse methylation of (R)-PFI-2 analogues that possess substitutions at the pyrrolidine site using a liquid chromatography-mass spectrometry (LCMS) based assay. To this purpose, SETD7 (200 nM) was incubated with SAM (50 µM), and compounds 1-20 (10 µM) in 50 mM glycine pH 8.8 as assay buffer for 2-20 h at 37 °C (Figure 3). We started with testing of the methylation of analogues 1‑4, bearing lysine‑mimicking side chains with various lengths. We were pleased to find that the shortest analogue 1 was efficiently methylated by SETD7; we observed significant formation of the monomethylated (m/z = 435.1 Da) analogue within 2 h (Figure 3a), whereas after 20 h, compound 1 was completely monomethylated and also dimethylation (m/z = 449.0 Da) started to occur (Figure 3b and S1). This observation is interesting, because SETD7 is known to only monomethylate lysine residues in natural lysine containing substrates.[24, 25] Notably, in all cases where we observed methylation of one of the analogues, both the starting compound and its methylated analogue possessed the same retention time on LCMS; therefore, we were unable to separate and thus quantify the degree of methylation using this technique. Controls in the absence of SETD7 showed no methylation of compound 1 (Figure 3c). The degree of methylation decreased with an increasing length of the side chain: for analogue 2 we observed only traces of monomethylation (m/z = 449.0 Da) after 2 h, which significantly increased upon prolonged 20 h incubation (Figure 3d and S2). For compound3 we observed only traces of methylation (m/z = 463.1 Da) even after 20 h incubation (Figure 3e and S3). By increasing the length even further, i.e. for compound 4, we no longer observed any methylation within detection limits (Figure S4). A limited ability of SETD7 to catalyse methylation of analogues with an increasing length of the side chain is attributed to additional conformations and

99 likely steric clash with SAM, which results in that these analogues are no longer capable of optimal binding in the histone binding pocket of SETD7. Nevertheless, SETD7 is much more tolerant to variations in chain length in these synthetic substrates in comparison to lysine 4 on histone 3, as a previous study on the chain length of lysine showed that only natural L-lysine in histone peptides is methylated by human SETD7, whereas shorter or longer lysine analogues were not methylated by SETD7.[26] Piperazine-derived analogue 5 was also accepted as a substrate for SETD7-catalysed methylation, forming a tertiary amine (m/z = 461.0 Da, Figure 3f and S5). However, no methylation was observed for analogue 6 (Figure S6), likely due to the reduced nucleophilicity of the aromatic amine, an increased steric hindrance, and lack of rotational freedom (i.e. the amine points in the

wrong direction for an NS 2 reaction to occur).

Figure 3. Mass spectrometric data showing the SETD7-catalysed methylation of 1-3, and 5. Conditions: SETD7 (200 nM), SAM (50 µM), compound (10 µM) in 50 mM glycine pH 8.8 at 37 °C, measured by LCMS. a) compound 1 (m/z = 421.1 Da) after 2 h incubation showing almost complete monomethylation (m/z = 435.1 Da); b) compound 1 after 20 h, complete conversion and a small degree of dimethylation; c) compound 1 without SETD7 showing no methylation; d) compound 2 (m/z = 435.0 Da) after 20 h; e) compound 3 (m/z = 449.0 Da) after 20 h; f) compound 5 (m/z = 447.0 Da) after 20 h.

Having identified the optimal chain length for SETD7-catalysed methylation of (R)-PFI-2 analogues, we tested whether other analogues could also undergo enzymatic

100 methylation. No methylation of alcohol 7, which is less nucleophilic than amine 1, was observed even upon prolonged incubation (20 h) (Figure S7). On the other hand, we hypothesised that thiol 8 could be methylated, as thiols are generally more nucleophilic than amines; however we did not observe any methylation of compound 8 within detection limits, demonstrating that SETD7 specifically catalyses methylation of nucleophilic amines, but not thiols or alcohols. With compound 8, we did observe significant disulfide formation after 20 h (m/z = 873.0 Da, Figure S8). For amide and carboxylic acid derivatives 9 and 10 no methylation was observed within limits of detection (Figure S9 and S10). Furthermore, two carbon nucleophiles containing either a terminal alkene (11) or alkyne (12) functionality were also not methylated in the presence of SETD7 (Figure S11 and S12). These results show that SETD7 is highly specific for the methylation of amines.

The products of the methylation reaction of 1 in the presence of SETD7 and SAM, 3 compounds 13 and 14, were synthesised in order to investigate whether it would be possible to increase the methylation state of either a mono (13) or dimethyl (14) starting material. For compound 13, we observed traces of additional methylation to form the dimethylated analogue 14 after 20 h (Figure S13). Compound 14 itself, however, was not further methylated (Figure S14). In compounds 1‑6, two amine functionalities are present; namely the amine in the side chain, but also the tetrahydroisoquinoline’s + NH2 , and both could in principle be methylated by SETD7, although the structural + analyses indicate that the site of methylation is not tetrahydroisoquinoline NH2 . As a control experiment we also tested whether carbon analogues 15-17 would be methylated by SETD7. However, no methylation was observed (Figures S15-S17), which indirectly shows that methylation is indeed taking place on the side chain’s amino group rather than on the tetrahydroisoquinoline’s nitrogen. Compound 18, lacking the tetrahydroisoquinoline’s nitrogen that is crucial for binding to SETD7, was not a substrate of SETD7 (Figure S18). Surprisingly, though having IC50 > 100 µM, compound 19 was found to be moderately methylated by SETD7 (Figure S19). Finally, we did not observe any methylation of 20 (Figure S20).

Having shown that compound 1 is the best substrate for methylation by SETD7 in the presence of SAM, we were interested to know whether it is also possible to introduce other alkyl-groups such as ethyl or allyl. Therefore we incubated SETD7 and compound 1 with various simple SAM analogues: AdoEth, bearing an ethyl group; AdoSeEth, in which the sulfur is replaced by a larger selenium atom; and AdoAllyl, a SAM analogue bearing an allyl group instead of methyl. After 2 h, we observed little ethylation (+28 Da) in the presence of AdoEth (Figure S21), however, when more reactive AdoSeEth

101 was used, we observed a significant level of ethylation within 2 h (Figure S22). Also the introduction of an allyl group to compound 1 was observed, however only moderate conversion (+40 Da) was achieved (Figure S23).

Figure 4. Time course for the SETD7-catalysed methylation of 1. Unmodified 1 is depicted in green,

monomethylated (1-Me1) in purple, and dimethylated product (1-Me2) in blue. Conditions: SETD7 (8.0 µM), SAM (2.0 mM), compound 1 (400 µM) in 50 mM glycine pH 8.8 at 37 °C, measured by HPLC.

Unfortunately, we were unable to separate the different methylation states of 1 on LCMS, so in order to quantify the degree of methylation we performed experiments using the same stoichiometry as for the LCMS experiments mentioned above, however at 40× higher concentration (i.e. 8.0 µM SETD7, 2.0 mM SAM, and 400 µM compound 1), and performed analysis using high-performance liquid chromatography (HPLC). A time course of the methylation of 1 is depicted in Figure 4. Using HPLC, we found that after 2 h, 100% of compound 1 was enzymatically converted to the monomethylated

product (1-Me1). With longer incubation times, also the dimethylated product (1-Me2) of compound 1 started to form: after 20 h we observed 26% dimethylation, whereas after 2 days the dimethylation increased to 42%. Upon prolonged incubation, we did not observe a further increase of dimethylation of compound 1, likely due to lost activity of SETD7 (Figure S24).

Finally, to gain an insight in the possible binding mode of compound 1, we performed docking studies using Molecular Operating Environment (MOE).[27] We used the crystal

102 structure of the ternary complex between SETD7, (R)-PFI-2 and SAM for docking (PDB ID: 4JLG, Figure 5a). The proposed binding mode of compound 1 is depicted in Figure 5b, and its binding mode is highly similar to that of (R)‑PFI‑2. The primary amino-group of compound 1 is pointed directly towards the methyl group of SAM cosubstrate, enabling an efficient methyl transfer reaction. The distances between SAM and 1 in Figure 5b was measured 4.3 Å for N-S and 3.2 Å for N-C (measurement shown in Figure S25). In comparison, the crystal structure of the ternary complex between SETD7, S‑adenosylhomocysteine (SAH) and a methylated H3K4 peptide histone mimic is depicted in Figure 5c. Here, the distances were measured at 3.7 Å for C-S and 5.2 Å for N-S (Figure S26), confirming an optimal length of the side chain of1 for methyltransferase catalysis. a) b) c)

Figure 5. a) Crystal structure (PDB ID: 4JLG) of the complex between SETD7 (cartoon, grey), SAM (green), and (R)-PFI-2 (cyan); b) proposed binding mode of compound 1 (cyan), docked in the crystal structure of SETD7 (PDB ID: 4JLG); c) crystal structure (PDB ID: 1O9S) of the complex between SETD7 (cartoon, grey) with S-adenosylhomocysteine (SAH, green) and monomethylated H3K4 histone peptide mimic (H3K4me, cyan).

3.3. Conclusion

In summary, we have designed, synthesised and evaluated novel (R)-PFI-2 analogues, with some of them acting as substrates and inhibitors of human histone lysine methyltransferase SETD7. The SAR of the amide part of (R)‑PFI‑2 was explored and it was found that only minor modifications are tolerated, whereas the introduction of functional groups with increased size or polarity leads to compounds that are no longer able to inhibit SETD7. From this panel of analogues, compound 7 was the most potent

SETD7 inhibitor with an IC50 of 0.96 µM. Furthermore, it was found that compounds 1-3, 5, and 13, bearing a side chain with a nucleophilic amine functionality that mimics the lysine side chain in H3K4, were methylated by SETD7. Our observations show that SETD7 is highly specific for methylation of amines, but not for other nucleophilic functional groups, including thiols, alcohols, amides, carboxylates, alkenes and alkynes. Also, different alkylating agents were tolerated well, and allowed for the introduction

103 of larger ethyl or allyl groups. These or other unnatural SAM analogues might be used for specific labelling of inhibitors targeting SETD7. We envision that, after additional optimisation and testing, these synthetic substrates of SETD7 might act as suitable candidates to modulate the activity of SETD7 and the level of SAM cosubstrate in in vitro as well as in vivo studies related to SAM-dependent methyltransferases.

3.4. Supporting information

3.4.1. General experimental For general experimental details, see Section 2.4.

3.4.2. MALDI-TOF MS inhibition assay 3 Inhibition studies to determine the half maximum inhibitory concentrations (IC50 values) were performed as previously described, see Section 2.4.[21, 22]

3.4.3. Alkylation assay Alkylation experiments were performed as follows: compound (10 µM final conc. was combined with SETD7 (200 nM final conc.) and SAM (50 µM final conc.) in 50mM glycine pH 8.8 as assay buffer in a final volume of 60 µL. The enzymatic reaction was incubated at 37 °C, after which it was quenched by the addition of an equal volume of MeOH. The quenched mixture was incubated at 4 °C for 4 h, and subsequently centrifuged at 12.000 rpm for 10 min. The supernatant was taken for analysis by liquid- chromatography mass-spectrometry (LCMS) (Figures S1-S20, S22-S23)

For quantitative analysis of the methylation of compound 1, the experiment described above was performed, however 40× more concentrated, i.e. 1 (400 µM final conc.) was combined with SETD7 (8.0 µM final conc.) and SAM (2.0 mM final conc.). Analysis was performed using HPLC, with 99.9% MQ + 0.1% TFA as solvent A and 99.9% MeCN + 0.1% TFA as solvent B. In order to separate the different methylation states of 1, a gradient of 10-90% solvent B in A was employed over 12 min. The data was quantified using the peak areas of each methylation state (Figure S24).

104 3.4.4. Supporting figures

Figure S1. SETD7-catalysed methylation of 1 (m/z = 421.1 Da) after top) 2 h; bottom) 20 h of incubation. Conversion (-14 Da) of SAM (m/z = 398.9 Da) to SAH (m/z = 384.9 Da) is also observed. 3

Figure S2. SETD7-catalysed methylation of 2 (m/z = 435.1 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S3. SETD7-catalysed methylation of 3 (m/z = 449.1 Da) after top) 2 h; bottom) 20 h of incubation.

105 Figure S4. SETD7-catalysed methylation of 4 (m/z = 463.2 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S5. SETD7-catalysed methylation of 5 (m/z = 447.0 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S6. SETD7-catalysed methylation of 6 (m/z = 469.0 Da) after top) 2 h; bottom) 20 h of incubation.

106 Figure S7. SETD7-catalysed methylation of 7 (m/z = 422.0 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S8. SETD7-catalysed methylation of 8 (m/z = 437.9 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S9. SETD7-catalysed methylation of 9 (m/z = 434.9 Da) after top) 2 h; bottom) 20 h of incubation.

107 Figure S10. SETD7-catalysed methylation of 10 (m/z = 436.0 Da) after a) 2 h; b) 20 h of incubation.

Figure S11. SETD7-catalysed methylation of 11 (m/z = 417.9 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S12. SETD7-catalysed methylation of 12 (m/z = 415.9 Da) after top) 2 h; bottom) 20 h of incubation.

108 Figure S13. SETD7-catalysed methylation of 13 (m/z = 435.1 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S14. SETD7-catalysed methylation of 14 (m/z = 449.1 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S15. SETD7-catalysed methylation of 15 (m/z = 420.0 Da) after top) 2 h; bottom) 20 h of incubation.

109 Figure S16. SETD7-catalysed methylation of 16 (m/z = 434.0 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S17. SETD7-catalysed methylation of 17 (m/z = 448.0 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S18. SETD7-catalysed methylation of 18 (m/z = 366.0 Da) after top) 2 h; bottom) 20 h of incubation.

110 Figure S19. SETD7-catalysed methylation of 19 (m/z = 331.12 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S20. SETD7-catalysed methylation of 20 (m/z = 208.1 Da) after top) 2 h; bottom) 20 h of incubation.

Figure S21. SETD7-catalysed alkylation of 1 with AdoEth after top) 2 h; bottom) 20 h of incubation.

111 Figure S22. SETD7-catalysed alkylation of 1 with AdoSeEth after top) 2 h; bottom) 20 h of incubation.

Figure S23. SETD7-catalysed alkylation of 1 with AdoAllyl after top) 2 h; bottom) 20 h of incubation.

100

1 1-Me1 50 1-Me2 Abundance (%)Abundance

0 0 2 4 6 Time (days) Figure S24. Time course of SETD7-catalysed methylation of 1.

112 Figure S25. Docked structure of compound 1 (cyan) in complex with SETD7 (grey) and SAM (green). Distance N-S (4.3 Å) and N-C (3.2 Å) measured with PyMOL. 3

Figure S26. Docked structure of methylated histone peptide (cyan) in complex with SETD7 (grey) and SAH (green). Distance C-S (3.7 Å) and N‑S (5.2 Å) measured with PyMOL.

3.4.5. Characterisation of compounds General procedure I (GPI): Amide coupling To a mixture of Cbz, Boc or Fmoc protected D-Phe-OH (1.2 equiv) was added the respective amine (1.0 equiv) in DCM (5 mL). Whilst stirring at 0 °C (ice-water bath), Et3N (2.0 equiv), HOBt (1.8 equiv), and EDC (1.5 equiv) were added. The mixture was allowed to warm to rt and stirred overnight. The reaction mixture was concentrated, redissolved in EtOAc (20 mL), washed with brine (2 × 30 mL) and satd aq NaHCO3 (30 mL). Unless stated otherwise, the crude product was purified by column chromatography in the indicated solvents.

113 Benzyl (R)-(1-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-1-oxo-3-phenylpropan-2- yl)carbamate (S1): Synthesised according to GPI starting from Cbz-D-Phe-OH (499 mg, 1.5 mmol, 1.2 equiv) and N‑boc‑ethylenediamine (199 µL, 1.3 mmol, 1.0 equiv). The crude product was purified by column chromatography (10-40% EtOAc in n-heptane) to afford 279 mg 1 (42% isolated yield) of S1. H NMR (500 MHz, CDCl3) δ 7.40 - 7.30 (m, 7H), 7.28 – 7.25 (m, 1H). 7.23 - 7.19 (m, 2H), 6.24 (bs, 1H), 5.37 (bs, 1H), 5.15 – 5.06 (m, 2H), 4.70 (bs, 1H), 4.43 – 4.33 (m, 1H), 3.35 – 3.21 (m, 2H), 3.21 – 3.09 (m, 3H), 3.04 13 (s, 1H), 1.44 (s, 9H); C NMR (126 MHz, CDCl3) δ 171.3, 136.6, 136.2, 129.4, 128.9, 128.7, 128.4, 128.2, 127.3, 79.8, 67.3, 56.6, 40.6, 40.2, 38.9, 31.1, 28.5; MS (ESI) m/z 441.9 [M+H]+.

3 Benzyl (R)-(1-((3-((tert-butoxycarbonyl)amino)propyl)amino)-1-oxo-3-phenylpropan-2- yl)carbamate (S2): Synthesised according to GPI starting from Cbz-D-Phe-OH (299 mg, 1.0 mmol) and N-Boc-1,3-propanediamine (147 µL, 0.84

mmol). The crude mixture was poured into satd aq NaHCO3 (40 mL). The resulting precipitate was collected by filtration to afford crude S2, which was used without further purification in the next 1 reaction. H NMR (400 MHz, CDCl3) δ 7.42 – 7.11 (m, 10H), 6.47 (bs, 1H), 5.44 (bs, 1H), 5.16 – 5.02 (m, 2H), 4.81 (s, 1H), 4.39 (t, J = 8.1 Hz, 1H), 3.25 – 3.00 (m, 4H), 2.93 (q, J = 6.4 Hz, 2H), 1.42 (s, 9H); MS (ESI) m/z 455.8 [M+H]+.

Benzyl (R)-(1-((4-((tert-butoxycarbonyl)amino)butyl)amino)-1-oxo-3-phenylpropan-2- yl)carbamate (S3): Synthesised according to GPI starting from Cbz-D-Phe-OH (299 mg, 1.0 mmol) and N-Boc-1,4-butanediamine (161 µL, 0.84

mmol). The crude mixture was poured into satd aq NaHCO3 (40 mL). The resulting precipitate was collected by filtration to afford crudeS3 , which was used without further purification in 1 the next reaction. H NMR (400 MHz, CDCl3) δ 7.43 (m, 1H), 7.48 – 7.10 (m, 10H), 6.73 (t, J = 5.7 Hz, 1H), 4.97 – 4.83 (m, 3H), 4.18 – 4.02 (m, 1H), 3.05 – 2.85 (m, 8H), 2.78 – 2.56 (m, 2H), 1.33 (s, 9H); MS (ESI) m/z 469.9 [M+H]+.

114 Benzyl (R)-(1-((5-((tert-butoxycarbonyl)amino)pentyl)amino)-1-oxo-3-phenylpropan-2- yl)carbamate (S4): Synthesised according to GPI starting from Cbz‑D‑Phe‑OH (302 mg, 1.0 mmol) and N-Boc-cadaverine (175 µL, 0.84 mmol). Crude S4 was used without further purification in the 1 next reaction. H NMR (500 MHz, CDCl3) δ 7.38 – 7.21 (m, 8H), 7.19 (d, J = 7.3 Hz, 2H), 5.68 (t, J = 5.9 Hz, 1H), 5.42 (bs, 1H), 5.08 (s, 2H), 4.56 (bs, 1H), 4.33 (q, J = 7.6 Hz, 1H), 3.17 – 2.97 (m, 4H), 1.67 (bs, 2H), 1.44 (s, 9H), 1.42 – 1.30 (m, 4H), 1.19 – 1.13 (m, 2H); MS (ESI) m/z 506.1 [M+Na]+. tert-Butyl 4-(((benzyloxy)carbonyl)-D-phenylalanyl)piperazine-1-carboxylate (S5): Synthesised according to GPI starting from Cbz-D-Phe-OH (299 mg, 1.0 mmol) and N‑Boc-piperazine (156 mg, 0.84 mmol). Crude S5 was used without further purification in the next reaction. 1H NMR (400 MHz, 3

CDCl3) δ 7.48 – 7.07 (m, 10H), 6.15 (d, J = 8.6 Hz, 1H), 5.06 (q, J = 12.3 Hz, 2H), 4.88 (td, J = 8.7, 6.1 Hz, 1H), 3.62 – 2.86 (m, 9H), 2.74 – 2.57 (m, 1H), 1.43 (s, 9H); MS (ESI) m/z 467.8 [M+H]+.

Benzyl (R)-(1-((3-((tert-butoxycarbonyl)amino)phenyl)amino)-1-oxo-3-phenylpropan-2- yl)carbamate (S6): Synthesised according to GPI starting from Cbz-D-Phe-OH (299 mg, 1.0 mmol) and N-Boc-m-phenylenediamine (351 mg, 1.7 mmol). Crude S6 was used without further purification in the 1 next reaction. H NMR (500 MHz, CDCl3) δ 7.89 (bs, 1H), 7.52 (s, 1H), 7.26 (m, 11H), 7.01 (m, 1H), 6.62 (s, 1H), 5.58 (m, 1H), 5.11 (m, 2H), 4.57 (m, 1H), 3.15 (d, J = 6.97 Hz, 2H), 1.73 (bs, 1H), 1.53 (s, 9H); MS (ESI) m/z 512.1 [M+Na]+. tert-Butyl (R)-(1-((2-hydroxyethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (S7): Synthesised according to GPI starting from Boc-D-Phe-OH (398 mg, 1.5 mmol) and aminoethanol (76 µL, 1.26 mmol). The crude product was purified by column chromatography (30-60% EtOAc inn -heptane) 1 to afford 228 mg (49% isolated yield) ofS7 . H NMR (500 MHz, CDCl3) δ 7.24 (m, 5H), 6.79 (s, 1H), 5.45 (d, J = 8.13 Hz, 1H), 4.36 (q, J = 7.37 13 Hz, 1H), 3.58 (m, 2H), 3.28 (m, 3H), 3.03 (m, 2H), 1.38 (s, 9H); C NMR (126 MHz, CDCl3) δ 172.5, 155.8, 136.9, 129.4, 128.7, 127.0, 80.3, 61.5, 56.2, 42.3, 39.0, 28.4; MS (ESI) m/z 308.9 [M+H]+.

115 2-(Tritylthio)ethan-1-amine (S8): Cysteamine hydrochloride (400 mg, 3.5 mmol) was dissolved in 20 mL DCM/DMF (1:1 v/v) and to this was added trityl chloride (1.5 g, 5.3 mmol, 1.5 equiv) and the reaction was stirred at rt for 2h. Then, the mixture was concentrated in vacuo and subsequently co-evaporated with toluene (3 × 30 mL). The crude product was dissolved in DCM (100 mL) and washed with satd aq

NaHCO3 (50 mL) and brine (50 mL). The organic phase was dried over anhydrous MgSO4, filtered and concentration in vacuo. The crude product was purified by column chromatography (5-10% MeOH in DCM) to afford 2-tritylsulfanyl-ethylamine (875 mg, 1 78% isolated yield) as an off-white solid. H NMR (400 MHz, CDCl3) δ 7.45 – 7.40 (m, 6H), 7.30 – 7.25 (m, 7H), 7.23 – 7.18 (m, 3H), 2.60 (t, J = 6.5 Hz, 2H), 2.32 (t, J = 6.5 Hz, 2H), 1.36 (bs, 2H); MS (ESI) m/z 320.0 [M+H]+.

3 (9H-Fluoren-9-yl)methyl (R)-(1-oxo-3-phenyl-1-((2-(tritylthio)ethyl)amino)propan-2-yl) carbamate (S9): Synthesised according to GPI starting from Fmoc-D-Phe-OH (300 mg, 1.13 mmol) and 2-(tritylthio)ethan-1-amine S8 (479 mg, 1.5 mmol, 1.3 equiv). The crude product was purified by column chromatography (5-25% EtOAc in n-heptane) to afford 771 mg (99% 1 isolated yield) of S9 as a white solid. H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.55 – 7.47 (m, 2H), 7.43 – 7.32 (m, 8H), 7.32 – 7.06 (m, 16H), 5.55 (bs, 1H), 5.34 (bs, 1H), 4.47 – 4.20 (m, 3H), 4.16 (t, J = 6.8 Hz, 1H), 3.14 – 2.80 (m, 13 4H), 2.41 – 2.14 (m, 2H); C NMR (126 MHz, CDCl3) δ 170.3, 155.8, 144.5, 143.7, 143.7, 141.3, 136.4, 129.5, 129.2, 128.8, 128.0, 127.7, 127.1, 127.0, 126.8, 125.0, 120.0, 67.0, 66.9, 56.2, 47.2, 38.9, 38.3, 31.4; MS (ESI) m/z 712.0 [M+H]+.

Benzyl (R)-(1-((2-amino-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (S10): Synthesised according to GPI starting from Cbz-D-Phe-OH (599 mg, 2.0 mmol) and 2-aminoacetamide (186 mg, 1.7 mmol). The crude product was purified by column chromatography (10-50% EtOAc in n-heptane) to afford 285 mg (40% isolated yield) ofS10 . 1H NMR (400

MHz, CD3OD) δ 7.26 – 7.26 (m, 10H), 5.03 – 5.02 (m, 2H), 4.34 (dd, J = 9.0, 6.0 Hz, 1H), 3.87 (d, J = 17.1 Hz, 1H), 3.70 (d, J = 17.1 Hz, 1H), 3.15 (dd, J = 13.8, 13 6.0 Hz, 1H), 2.91 (dd, J = 13.8, 9.0 Hz, 1H); C NMR (101 MHz, CD3OD) δ 174.6, 174.2, 158.5, 138.4, 138.0, 130.3, 129.5, 129.4, 129.0, 128.7, 127.8, 67.7, 58.2, 43.2, 38.6; MS (ESI) m/z 378.1 [M+Na]+.

116 tert-Butyl ((benzyloxy)carbonyl)-D-phenylalanylglycinate (S11): Synthesised according to GPI starting from Cbz-D-Phe-OH (597 mg, 2.0 mmol) and tert-butyl glycinate HCl (436 mg , 2.6 mmol, 1.3 equiv). The crude product was purified by column chromatography (20-40% EtOAc in n-heptane) to afford 758 mg (89% isolated yield) 1 of S11. H NMR (400 MHz, CDCl3) δ 7.43 – 7.10 (m, 10H), 6.31 (bs, 1H), 5.33 (bs, 1H), 5.07 (d, J = 2.1 Hz, 2H), 4.52 – 4.39 (m, 1H), 3.91 (dd, J = 18.3, 5.2 Hz, 1H), 3.81 (dd, J = 18.3, 4.9 Hz, 1H), 3.22 – 2.95 (m, 2H), 1.45 (s, 9H); 13C NMR (101 MHz,

CDCl3) δ 170.8, 168.4, 136.3, 136.1, 129.3, 128.7, 128.5, 128.2, 128.0, 127.0, 82.4, 67.1, + 56.1, 42.0, 38.4, 29.0, 28.0; MS (ESI) m/z 413.3 [M+H] . tert-Butyl (R)-(1-(allylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (S12): Synthesised according to GPI starting from Boc-D-Phe-OH (267 mg, 1.0 mmol) and allylamine (63 µL, 0.84 mmol). Crude S12 was used directly 3 in the next reaction without further purification. 1H NMR (400 MHz,

CDCl3) δ 7.33 – 7.18 (m, 5H), 6.19 (t, J = 4.9 Hz, 1H), 5.61 (ddt, J = 16.1, 10.8, 5.5 Hz, 1H), 5.28 – 5.18 (m, 1H), 5.10 – 5.00 (m, 2H), 4.43 – 4.33 (m, 1H), 3.72 (s, 2H), 2.98 (d, J = 7.2 Hz, 2H), 1.32 (s, 9H); MS (ESI) m/z 304.7 [M+H]+. tert-Butyl (R)-(1-oxo-3-phenyl-1-(prop-2-yn-1-ylamino)propan-2-yl)carbamate (S13): Synthesised according to GPI starting from Boc-D-Phe-OH (267 mg, 1.0 mmol) and propargylamine (54 µL, 0.84 mmol). Crude S13 was used without further purification in the next reaction. 1H NMR (400 MHz,

CDCl3) δ 7.38 – 7.16 (m, 5H), 6.04 (bs, 1H), 4.99 (bs, 1H), 4.42 – 4.26 (m, 1H), 4.09 – 3.91 (m, 2H), 3.09 (d, J = 6.9 Hz, 2H), 2.21 (t, J = 2.6 Hz, 1H), 1.43 (s, 9H); MS (ESI) m/z 302.9 [M+H]+. tert-Butyl (R)-(2-(2-(((benzyloxy)carbonyl)amino)-3-phenylpropanamido)ethyl)(methyl) carbamate (S14): Synthesised according to GPI starting from Cbz-D-Phe-OH (302 mg, 1.0 mmol) and N-Boc-N-methylenediamine (150 µL, 0.84 mmol). Crude S14 was used without further purification in the next 1 reaction. H NMR (500 MHz, CDCl3) δ 7.40 – 7.22 (m, 8H), 7.21 – 7.14 (m, 2H), 6.69 (bs, 1H), 5.39 – 5.27 (m, 1H), 5.15 – 5.02 (m, 2H), 4.48 – 4.31 (m, 1H), 3.40 (bs, 1H), 3.36 – 3.24 (m, 3H), 3.13 – 3.05 (m, 2H), 2.83 (bs, 3H), 1.44 (s, 9H); MS (ESI) m/z 478.2 [M+Na]+.

117 Benzyl (R)-(1-((2-(dimethylamino)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamate (S15): Synthesised according to GPI starting from Cbz-D-Phe-OH (299 mg, 1.0 mmol) and dimethylethane-1,2-diamine (93 µL, 0.84 mmol). Crude S15 was used without further purification in the next reaction. 1 H NMR (500 MHz, CDCl3) δ 7.40 – 7.08 (m, 12H), 6.29 (bs, 1H), 5.52 (d, J = 8.2 Hz, 1H), 5.08 (s, 2H), 4.37 (q, J = 7.9 Hz, 1H), 3.30 – 3.06 (m, 3H), 2.99 (dd, J = 13.6, 7.9 Hz, 1H), 2.11 (s, 6H); MS (ESI) m/z 370.1 [M+H]+.

tert-Butyl (R)-(1-(butylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (S16): Synthesised according to GPI starting from Boc-D-Phe-OH (300 mg, 1.13 mmol) and n-butylamine (146 µL, 1.5 mmol). The crude product 3 was purified by column chromatography (5-25% EtOAc in n-heptane) to afford 222 mg (61% isolated yield) of S16 as a white solid. 1H NMR

(400 MHz, CDCl3) δ 7.35 – 7.14 (m, 5H), 5.62 (bs, 1H), 5.07 (bs, 1H), 4.25 (q, J = 7.5 Hz, 1H), 3.21 – 2.95 (m, 4H), 1.42 (s, 9H), 1.38 – 1.13 (m, 4H), 0.86 (t, J = 13 7.3 Hz, 3H); C NMR (126 MHz, CDCl3) δ 171.0, 155.4, 136.9, 129.3, 128.6, 126.8, 80.0, 56.1, 39.1, 38.8, 31.4, 28.3, 19.9, 13.7; MS (ESI) m/z 342.0 [M+Na]+.

tert-Butyl (R)-(1-(isopentylamino)-1-oxo-3-phenylpropan-2-yl)carbamate (S17): Synthesised according to GPI starting from Boc-D-Phe-OH (300 mg, 1.13 mmol) and 3-methylbutan-1-amine (171 µL, 1.5 mmol). The crude product was purified by column chromatography (5-25% EtOAc in n‑heptane) to afford 243 mg (65% isolated yield) ofS17 as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.40 – 7.06 (m, 5H), 5.60 (bs, 1H), 5.07 (bs, 1H), 4.25 (q, J = 7.5 Hz, 1H), 3.27 – 2.91 (m, 4H), 1.41 (s, 9H), 1.33 – 1.17 (m, 3H), 0.85 (dd, 13 J = 6.6, 2.3 Hz, 6H); C NMR (126 MHz, CDCl3) δ 170.9, 155.4, 136.9, 129.3, 128.6, 126.9, 80.1, 56.1, 38.8, 38.2, 37.7, 28.3, 25.6, 22.4, 22.3; MS (ESI) m/z 357.0 [M+Na]+.

Benzyl (2-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-oxoethyl)carbamate (S18): Synthesised according to GPI starting from Cbz-Gly-OH (314 mg, 1.5 mmol) and N-Boc-ethylenediamine (202 mg, 1.26 mmol). The crude product was purified by column chromatography (0-5% 1 MeOH in DCM) to afford 514 mg (59% isolated yield) ofS18 . H NMR (500 MHz, CD3OD) δ 7.40 – 7.26 (m, 5H), 5.11 (s, 2H), 3.76 (s, 2H), 3.27 (t, J = 6.1 Hz, 2H), 3.15 (t, J = 6.2 13 Hz, 2H), 1.43 (s, 9H); C NMR (126 MHz, CD3OD) δ 172.5, 159.1, 138.0, 129.5, 129.0, 128.9, 111.4, 80.2, 67.9, 45.0, 40.8, 40.7, 28.7; MS (ESI) m/z 351.8 [M+H]+.

118 tert-Butyl (R)-(1-((2-((tert-butoxycarbonyl)amino)ethyl)amino)-1-oxo-3-phenylpropan- 2-yl)carbamate (S19): Synthesised according to GPI starting from Boc-D-Phe-OH (100 mg, 0.38 mmol) and N-Boc-ethylenediamine (51 mg, 0.32 mmol). The crude product was purified by column chromatography (40-80% EtOAc in n-heptane) to afford 78 mg (51% isolated yield) of S19. 1H

NMR (500 MHz, CDCl3) δ 7.33 – 7.18 (m, 5H), 6.35 (bs, 1H), 5.12 (bs, 1H), 4.80 (bs, 1H), 4.34 – 4.25 (m, 1H), 3.34 – 3.20 (m, 2H), 3.19 – 3.08 (m, 2H), 3.08 – 3.00 13 (m, 2H), 1.43 (s, 9H), 1.40 (s, 9H); C NMR (126 MHz, CDCl3) δ 156.5, 155.5, 136.9, 129.4, 128.8, 127.1, 80.3, 79.6, 56.2, 40.3, 38.9, 32.0, 28.5, 28.4, 22.8; MS (ESI) m/z 430 [M+Na]+.

General procedure II (GPII) for Cbz-deprotection: Cbz-protected amino acids were deprotected by catalytic hydrogenation: the respective compound was dissolved in MeOH (10 mL), and to this was added 10% Pd/C. The reaction was stirred under an 3 atmosphere of H2 (balloon) at rt overnight. The reaction was filtered over celite and the crude product purified by column chromatography (0-10% MeOH in DCM).

General procedure III (GPIIII) for Boc-deprotection: Boc-protected amino acids were deprotected under acidic conditions: the respective compound was dissolved in 5 mL of TFA/DCM (1:1 v/v) and stirred at rt for 3 h. The reaction was concentratedin vacuo and redissolved in DCM (20 mL), washed with satd aq NaHCO3 (20 mL), dried over Na2SO4, and concentrated in vacuo. The crude product purified by column chromatography (0- 10% MeOH in DCM). tert-Butyl (R)-(2-(2-amino-3-phenylpropanamido)ethyl)carbamate (S20): Starting fromS1 and following GPII, 42 mg (22% isolated yield) of S20 1 was obtained. H NMR (500 MHz, CDCl3) δ 7.51 (bs, 1H), 7.34 – 7.18 (m, 5H), 4.97 – 4.86 (m, 1H), 3.67 – 3.56 (m, 1H), 3.40 – 3.29 (m, 2H), 3.28 – 3.17 (m, 3H), 2.72 (dd, J = 13.7, 9.1 Hz, 1H), 1.61 (bs, 2H), 1.43 13 (s, 9H); C NMR (126 MHz, CDCl3) δ 175.2, 156.5, 137.9, 129.4, 128.8, 127.0, 79.5, 56.6, 41.2, 40.7, 39.8, 28.5; MS (ESI) m/z 307.9 [M+H]+. tert-Butyl (R)-(3-(2-amino-3-phenylpropanamido)propyl)carbamate (S21): Starting from compound S2 and following GPII, 150 mg (47% 1 isolated yield) of S21 was obtained. H NMR (400 MHz, CDCl3) δ 7.51 (bs, 1H), 7.34 – 7.28 (m, 2H), 7.27 – 7.19 (m, 3H), 5.21 (bs, 1H), 3.60 (dd, J = 9.1, 4.3, 1H), 3.30 (q, J = 6.4, 2H), 3.23 (dd, J = 13.6, 4.3, 1H), 3.08 (q, J = 6.4, 2H), 2.72 (dd, J = 13.7, 9.1 Hz, 1H),

119 13 1.62 (q, J = 6.4 Hz, 2H), 1.55 – 1.39 (m, 11H); C NMR (126 MHz, CDCl3) δ 174.7, 156.3, 137.9, 129.3, 128.7, 126.8, 79.1, 56.5, 41.1, 37.2, 35.9, 30.1, 28.4; MS (ESI) m/z 321.9 [M+H]+.

tert-Butyl (R)-(4-(2-amino-3-phenylpropanamido)butyl)carbamate (S22): Starting from compound S3 and following GPII, 55 mg (26% 1 isolated yield) of S22 was obtained. H NMR (500 MHz, CDCl3) δ 7.38 – 7.18 (m, 6H), 4.64 (bs, 1H), 3.61 (bs, J = 7.8 Hz, 1H), 3.31 – 3.19 (m, 3H), 3.12 (q, J = 6.4 Hz, 2H), 2.72 (dd, J = 13.7, 9.1 Hz, 13 1H), 1.70 (bs, 2H), 1.57 – 1.39 (m, 13H); C NMR (126 MHz, CDCl3) δ 174.2, 156.1, 138.0, 129.4, 128.8, 126.9, 79.2, 56.5, 41.1, 40.3, 38.8, 28.5, 27.6, 27.0; MS (ESI) m/z 336.1 [M+H]+.

3 tert-Butyl (R)-(5-(2-amino-3-phenylpropanamido)pentyl)carbamate (S23): Starting from compound S4 and following GPII, 220 mg (65% 1 isolated yield) of S23 was obtained. H NMR (400 MHz, CDCl3) δ 7.35 – 7.19 (m, 5H), 4.83 (bs, 1H), 3.59 (dd, J = 9.1, 4.2 Hz, 1H), 3.29 – 3.19 (m, 3H), 3.10 (q, J = 6.7 Hz, 2H), 2.72 (dd, J = 13.7, 9.1 Hz, 1H), 1.56 – 1.40 (m, 14H), 1.41 – 1.25 (m, 5H); 13C NMR

(101 MHz, CDCl3) δ 175.3, 156.0, 137.9, 129.2, 128.6, 126.7, 78.8, 56.4, 41.0, 40.3, 38.8, 29.6, 29.2, 28.4, 24.0; MS (ESI) m/z 350.0 [M+H]+.

tert-Butyl 4-(D-phenylalanyl)piperazine-1-carboxylate (S24): Starting from compound S5 and following GPII, 58 mg (27% isolated yield) 1 of S24 was obtained. H NMR (400 MHz, CDCl3) δ 7.35 – 7.15 (m, 5H), 3.94 (bs, 1H), 3.68 – 3.56 (m, 1H), 3.53 – 3.35 (m, 2H), 3.33 – 3.15 (m, 3H), 3.09 – 2.98 (m, 1H), 2.97 – 2.88 (m, 2H), 2.79 – 2.70 (m, 1H), 1.79 (bs, 2H), 1.45 13 (s, 9H); C NMR (101 MHz, CDCl3) δ 175.1, 156.3, 137.9, 129.3, 128.6, 126.8, 79.3, 56.5, 41.0, 40.6, 39.7, 28.4; MS (ESI) m/z 334.0 [M+H]+.

tert-Butyl (R)-(3-(2-amino-3-phenylpropanamido)phenyl)carbamate (S25): Starting from compound S6 and following GPII, 158 mg (50% 1 isolated yield) of S25 was obtained. H NMR (500 MHz, CDCl3) δ 9.84 (bs, 1H), 7.59 (s, 1H), 7.30 – 7.00 (m, 9H), 4.91 (bs, 1H), 4.49 (bs, 1H), 3.30 – 3.22 (m, 1H), 3.19 – 3.11 (m, 1H), 3.02 (bs, 1H), 1.47 13 (s, 9H); C NMR (126 MHz, CDCl3) δ 168.5, 153.2, 139.3, 137.8, 129.8, 129.5, 128.9, 127.5, 115.1, 114.8, 110.6, 80.7, 55.8, 52.8, 38.4, 28.5; MS (ESI) m/z 356.9 [M+H]+.

120 (R)-2-amino-N-(2-hydroxyethyl)-3-phenylpropanamide (S26): Starting from compound S7 and following GPIII, 158 mg (50% isolated 1 yield) of S26 was obtained. H NMR (400 MHz, CD3OD) δ 7.36 – 7.22 (m, 5H), 3.79 (t, J = 7.1 Hz, 1H), 3.59 – 3.46 (m, 3H), 3.30 – 3.24 (m, 2H), 3.10 (dd, J = 13.6, 6.5 Hz, 1H), 2.93 (dd, J = 13.6, 7.6 Hz, 1H); 13C NMR

(126 MHz, CD3OD) δ 173.4, 137.3, 130.4, 129.8, 128.3, 61.3, 56.8, 42.8, 40.5; MS (ESI) m/z 209.0 [M+H]+.

(R)-2-amino-3-phenyl-N-(2-(tritylthio)ethyl)propanamide (S27): Compound S9 (300 mg, 0.44 mmol, 1.0 equiv) was dissolved in DCM (1.5 mL) and to this was added DBU (72 µL, 0.48 mmol, 1.1. equiv). The reaction was stirred at rt for 15 min, after which the solvent was removed in vacuo. The crude product was purified by column 3 chromatography (0-5% MeOH in DCM) to afford 182 mg (90% isolated 1 yield) of S27 as a white solid. H NMR (400 MHz, CDCl3) δ 7.43 – 7.38 (m, 6H), 7.31 – 7.16 (m, 14H), 3.53 (dd, J = 9.4, 4.0 Hz, 1H), 3.22 (dd, J = 13.7, 4.0 Hz, 1H), 3.11 (q, J = 6.4 Hz, 2H), 2.63 (dd, J = 13.7, 9.4 Hz, 1H), 2.37 (t, J = 6.4 Hz, 2H), 1.26 (bs, 2H); 13C NMR

(126 MHz, CDCl3) δ 174.0, 144.6, 137.9, 129.5, 129.2, 128.7, 127.9, 126.7, 126.7, 66.7, 56.4, 41.0, 37.8, 32.0; MS (ESI) m/z 467.3 [M+H]+.

(R)-2-amino-N-(2-amino-2-oxoethyl)-3-phenylpropanamide (S28): Starting from compound S10 and following GPII, 89 mg (51% isolated 1 yield) of S28 was obtained. H NMR (500 MHz, CDCl3) δ 8.06 (t, J = 5.1 Hz, 1H), 7.32 – 7.17 (m, 5H), 6.62 (s, 1H), 6.12 (s, 1H), 3.90 (d, J = 5.1 Hz, 2H), 3.68 (dd, J = 9.3, 4.3 Hz, 1H), 3.20 (dd, J = 13.7, 4.3 Hz, 1H), 13 2.73 (dd, J = 13.7, 9.3 Hz, 1H), 2.09 (bs, 2H); C NMR (126 MHz, CDCl3) δ 175.1, 171.9, 137.5, 129.3, 128.8, 127.0, 56.3, 42.7, 40.8; MS (ESI) m/z 222.1 [M+H]+. tert-Butyl D-phenylalanylglycinate (S29): Starting from compoundS11 and following GPII, 112 mg (40% isolated 1 yield) of compound S29 was obtained. H NMR (400 MHz, CDCl3) δ 7.75 (bs, 1H), 7.36 – 7.29 (m, 2H), 7.28 – 7.19 (m, 3H), 4.06 – 3.87 (m, 2H), 3.65 (dd, J = 9.9, 3.8 Hz, 1H), 3.32 (dd, J = 13.8, 3.8 Hz, 1H), 2.67 13 (dd, J = 13.8, 9.9 Hz, 1H), 1.48 (s, 9H); C NMR (101 MHz, CDCl3) δ 174.5, 169.1, 138.0, 129.3, 128.7, 126.8, 82.2, 56.5, 41.7, 41.0, 28.1; MS (ESI) m/z 278.9 + [M+H] .

121 (R)-N-Allyl-2-amino-3-phenylpropanamide (S30): Starting from compound S12 and following GPIII, 140 mg (67% isolated 1 yield) of S30 was obtained. H NMR (400 MHz, CDCl3) δ 7.44 (bs, 1H), 7.37 – 7.18 (m, 5H), 5.84 (ddt, J = 17.1, 10.2, 5.6 Hz, 1H), 5.21 – 5.09 (m, 2H), 3.93 – 3.87 (m, 2H), 3.64 (dd, J = 9.3, 4.1 Hz, 1H), 3.29 (dd, J = 13.7, 4.1 Hz, 1H), 2.74 (dd, J = 13.7, 9.3 Hz, 1H), 1.49 (bs, 2H); 13C NMR (101

MHz, CDCl3) δ 174.1, 137.9, 134.3, 129.3, 128.7, 126.8, 116.1, 56.5, 41.4, 41.1 ; MS (ESI) m/z 205.2 [M+H]+.

(R)-N-Propargyl-2-amino-3-phenylpropanamide (S31): Starting from compound S13 and following GPIII, 99 mg (48% isolated 1 yield) of S31 was obtained. H NMR (500 MHz, CDCl3) δ 7.55 (bs, 1H), 7.35 – 7.12 (m, 5H), 4.07 – 4.03 (m, 2H), 3.65 (dd, J = 9.3, 4.1 Hz, 1H), 3.28 (dd, 3 J = 13.8, 4.1 Hz, 1H), 2.71 (dd, J = 13.8, 9.3 Hz, 1H), 2.22 (t, J = 2.6 Hz, 1H), 13 1.86 (bs, 2H); C NMR (126 MHz, CDCl3) δ 173.9, 137.6, 129.3, 128.8, 126.9, 79.6, 71.4, 56.2, 40.7, 28.8; MS (ESI) m/z 202.9 [M+H]+.

tert-Butyl (R)-(2-(2-amino-3-phenylpropanamido)ethyl)(methyl)carbamate (S32): Starting from compoundS14 and following GPII, 106 mg (39% isolated 1 yield) of S32 was obtained as a yellow oil. H NMR (500 MHz, CDCl3) δ 7.65 – 7.44 (m, 1H), 7.36 – 7.18 (m, 5H), 3.59 (bs, 1H), 3.47 – 3.34 (m, 4H), 3.28 (d, J = 13.1 Hz, 1H), 2.87 (s, 3H), 2.71 – 2.61 (m, 1H), 1.46 (s, 13 9H), 1.34 (bs, 2H); C NMR (126 MHz, CDCl3) δ 174.8, 156.6, 138.1, 129.3, 128.7, 126.8, 79.7, 56.6, 41.0, 37.7, 34.6, 29.7, 28.4; MS (ESI) m/z 321.9 [M+H]+.

(R)-2-Amino-N-(2-(dimethylamino)ethyl)-3-phenylpropanamide (S33): Starting from compound S15 and following GPII, 128 mg (52% 1 isolated yield) of S33 was obtained. H NMR (400 MHz, CDCl3) δ 7.54 (s, 1H), 7.34 – 7.27 (m, 2H), 7.23 (d, J = 7.7 Hz, 3H), 3.61 (dd, J = 9.1, 4.5 Hz, 1H), 3.38 (td, J = 6.1, 3.2 Hz, 2H), 3.23 (dd, J = 13.6, 4.5 Hz, 1H), 3.08 (bs, 2H), 2.73 (dd, J = 13.6, 9.1 Hz, 1H), 2.49 (t, J = 6.1 Hz, 13 2H), 2.29 (s, 6H); C NMR (101 MHz, CDCl3) δ 174.4, 138.0, 129.3, 128.6, 126.7, 57.9, 56.7, 45.0, 41.2, 36.3; MS (ESI) m/z 236.1 [M+H]+.

122 (R)-2-Amino-3-phenyl-N-propylpropanamide (S34): Starting from compound S12 and following GPII, 67 mg (36% isolated 1 yield) of S34 was obtained. H NMR (500 MHz, CDCl3) δ 7.37 – 7.20 (m, 6H), 3.60 (dd, J = 9.3, 4.1 Hz, 1H), 3.28 (dd, J = 13.8, 4.1 Hz, 1H), 3.22 (tdd, J = 7.0, 5.9, 4.4 Hz, 2H), 2.70 (dd, J = 13.7, 9.3 Hz, 1H), 1.52 (h, J = 7.3 Hz, 2H), 1.36 (bs, 2H), 0.91 (t, J = 7.4 Hz, 3H); 13C NMR (126 MHz,

CDCl3) δ 174.2, 138.1, 129.4, 128.8, 126.9, 56.6, 41.2, 40.9, 22.9, 11.5; MS (ESI) m/z 207.0 [M+H]+.

(R)-1-(Butylamino)-1-oxo-3-phenylpropan-2-aminium 2,2,2-trifluoroacetate (S35): Compound S16 (150 mg, 0.47 mmol) was dissolved in 5 mL DCM/ TFA (1:1 v/v) and stirred at rt until TLC analysis (EtOAc:n-heptane 1:3) showed the reaction was complete. The solvent was evaporated 3 and the crude product co-evaporated with Et2O (3 × 10 mL). Crude S35 was used without further purification. 1H NMR (400 MHz,

CD3OD) δ 7.44 – 7.17 (m, 5H), 4.00 (t, J = 7.5 Hz, 1H), 3.26 – 3.02 (m, 4H), 1.44 – 1.33 13 (m, 2H), 1.29 – 1.20 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); C NMR (126 MHz, CD3OD) δ 171.0, 136.9, 129.3, 128.6, 126.8, 39.1, 38.8, 31.4, 28.3, 19.9, 13.7; MS (ESI) m/z 221.4 [M+H]+.

(R)-1-(Isopentylamino)-1-oxo-3-phenylpropan-2-aminium 2,2,2-trifluoroacetate (S36): Compound S17 (150 mg, 0.47 mmol) was dissolved in 5 mL DCM/ TFA (1:1 v/v) and stirred at rt until TLC analysis (EtOAc:n-heptane 1:3) showed the reaction was complete. The solvent was evaporated

and the crude product co-evaporated with Et2O (3 × 10 mL). Crude S36 was used without further purification. 1H NMR (400 MHz,

CD3OD) δ 7.44 – 7.21 (m, 5H), 3.99 (t, J = 7.5 Hz, 1H), 3.25 (dt, J = 13.4, 7.5 Hz, 1H), 3.19 – 3.03 (m, 3H), 1.53 – 1.38 (m, 1H), 1.35 – 1.24 (m, 2H), 0.89 (dd, 13 J = 6.6, 1.6 Hz, 6H); C NMR (126 MHz, CD3OD) δ 169.2, 135.7, 130.5, 130.0, 128.8, 55.9, 38.9, 38.7, 26.6, 22.7, 22.7; MS (ESI) m/z 235.5 [M+H]+. tert-Butyl (2-(2-aminoacetamido)ethyl)carbamate (S37): Starting from compound S18 and following GPII, crude S37 was obtained, which was used without further purification in the next 1 reaction. H NMR (400 MHz, CDCl3) δ 7.55 (bs, 1H), 5.05 (bs, 1H), 3.37 (q, J = 5.7 Hz, 2H), 3.33 (s, 2H), 3.31 – 3.20 (m, 2H), 1.68 (s, 2H), 1.42 (s, 9H).

123 tert-Butyl 6-(chlorosulfonyl)-8-fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate (S38): S38 was synthesised as described in Section 2.4.

General procedure IV (GPIV): Synthesis of analogues 1-19. (R)-6-(N-(1-((2-ammonioethyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8-fluoro- 1,2,3,4-tetra-hydroisoquinolin-2-ium di(2,2,2-trifluoroacetate) (1):

O CF3COO O 2 S O O Cl O O + S O S O BocN O N N H2N H H BocN HN H2N HN F HN NHBoc NH3 NHBoc F F 3 S38 S20 S39 1

To a solution of compound S20 (53 mg, 0.17 mmol, 1.2 equiv) and tert-butyl 6-(chlorosulfonyl)-8-fluoro-3,4-dihydroisoquinoline-2(1H)-carboxylate (S38, 50 mg,

0.14 mmol, 1.0 equiv) in 5 mL of DCM was added Et3N (60 µL, 0.43 mmol, 3.0 equiv). The mixture was stirred at rt for 2h until TLC analysis (MeOH/DCM = 1:10) showed the reaction was complete. The reaction was quenched with water and concentrated in vacuo. The crude product was redissolved in DCM (15 mL) and washed with water

(2 × 10 mL). The organic layer was dried over Na2SO4, concentrated in vacuo, and purified by column chromatography to afford 49 mg (56% isolated yield) of compound 1 S39. H NMR (400 MHz, CDCl3) δ 7.23 – 6.92 (m, 8H), 5.69 (d, J = 6.5 Hz, 1H), 5.01 (bs, 1H), 4.57 (s, 2H), 3.85 (q, J = 9.0, 6.9, 5.2 Hz, 1H), 3.70 – 3.57 (m, 2H), 3.39 – 3.25 (m, 2H), 3.24 – 3.13 (m, 2H), 3.09 (dd, J = 14.0, 5.2 Hz, 1H), 2.85 – 2.72 (m, 3H), 1.51 (s, 9H), 1.43 (s, 9H); MS (ESI) m/z 643.1 [M+Na]+. Next, S39 was dissolved in 6 mL TFA/DCM (1:1 v/v) and stirred at rt for 3 h. The reaction mixture was concentrated in vacuo, and co-

evaporated with Et2O (5 × 20 mL) to afford 26 mg (50% isolated yield) of 1 an off-white 1 solid. H NMR (400 MHz, CD3OD) δ 7.33 (bs, 1H), 7.21 – 7.11 (m, 4H), 7.10 – 7.03 (m, 2H), 4.41 (s, 2H), 3.93 (dd, J = 9.6, 5.4 Hz, 1H), 3.52 (t, J = 6.3 Hz, 2H), 3.39 (td, J = 6.3, 1.7 Hz, 2H), 3.10 (q, J = 5.8 Hz, 2H), 3.01 (dd, J = 13.8, 5.4 Hz, 1H), 2.97 (td, J = 6.4, 1.7 13 Hz, 2H), 2.72 (dd, J = 13.8, 9.6 Hz, 1H); C NMR (101 MHz, CD3OD) δ 174.7, 160.1 (d, J

124 = 250.8 Hz), 142.8 (d, J = 7.4 Hz), 137.8, 136.5 (d, J = 3.7 Hz), 130.3, 129.4, 127.8, 124.2 (d, J = 3.4 Hz), 122.2 (d, J = 16.2 Hz), 112.9 (d, J = 24.0 Hz), 60.1, 42.0, 40.5 (d, J = 6.5 Hz), 19 40.4, 39.3, 38.1, 25.8 (d, J = 2.2 Hz), 24.2 ; F NMR (377 MHz, CD3OD) δ –77.2, –117.3; + + + MS (ESI) m/z 421.1 [M+H] ; HRMS (ESI) found m/z 421.17238 [M+H] , C20H26FN4O3S requires m/z 421.17096.

(R)-6-(N-(1-((3-ammoniopropyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8- fluoro-1,2,3,4-tetra-hydroisoquinolin-2-ium di(2,2,2-trifluoroacetate) (2): Starting from compound S21 (55 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 68 mg (75% isolated yield) of 1 compound S40 was obtained. H NMR (400 MHz, CDCl3) δ 7.23 (bs, 1H), 7.19 – 6.95 (m, 7H), 5.64 (d, J = 7.0 Hz, 1H), 4.95 (bs, 1H), 4.58 (s, 2H), 3.88 (q, J = 7.1 Hz, 1H), 3.65 (t, J = 6.0 Hz, 2H), 3.23 (d, J = 6.3 Hz, 2H), 3.09 (dd, 3 J = 14.0, 5.5 Hz, 1H), 3.02 (q, J = 6.4 Hz, 2H), 2.88 – 2.74 (m, 3H), 1.52 (s, 9H), 1.44 (s, 9H); MS (ESI) m/z 657.2 [M+Na]+. Next, compound S40 was deprotected and purified by preparative HPLC to afford 7 mg (15% isolated yield) of2 as a white solid. 1H NMR (400

MHz, CD3OD) δ 7.32 (bs, 1H), 7.24 – 7.12 (m, 4H), 7.09 – 7.03 (m, 2H), 4.41 (s, 2H), 3.92 (dd, J = 9.5, 5.7 Hz, 1H), 3.53 (t, J = 6.3 Hz, 2H), 3.28 – 3.20 (m, 2H), 3.14 – 3.06 (m, 2H), 2.99 (dd, J = 13.7, 5.7 Hz, 1H), 2.93 – 2.78 (m, 2H), 2.70 (dd, J = 13.7, 9.5 Hz, 1H), 1.80 13 (p, J = 6.9 Hz, 2H); C NMR (126 MHz, CD3OD) δ 174.5, 160.1 (d, J = 250.5 Hz), 143.0 (d, J = 7.3 Hz), 137.8, 136.5 (d, J = 3.6 Hz), 130.3, 129.4, 127.8, 124.1 (d, J = 3.4 Hz), 122.1 (d, J = 16.2 Hz), 112.8 (d, J = 23.9 Hz), 60.2, 42.0, 40.6 (d, J = 6.5 Hz), 39.4, 38.0, 36.8, 19 + 28.6, 25.9 ; F NMR (377 MHz, CD3OD) δ –117.5; MS (ESI) m/z 435.1 [M+H] ; HRMS + + (ESI) found m/z 435.18796 [M+H] , C21H28FN4O3S requires m/z 435.18661.

(R)-6-(N-(1-((4-ammoniobutyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8-fluoro- 1,2,3,4-tetra-hydroisoquinolin-2-ium di(2,2,2-trifluoroacetate) (3): Starting from compound S22 (58 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 44 mg (47% isolated yield) of compound S41 was obtained. 1H NMR (400 MHz,

CDCl3) δ 7.23 – 6.92 (m, 7H); 6.62 (bs, 1H), 4.70 – 4.62 (m, 1H), 4.59 (bs, 2H), 3.86 (q, J = 6.6 Hz, 1H), 3.70 – 3.60 (m, 2H), 3.21 (d, J = 5.6 Hz, 2H), 3.13 – 3.00 (m, 3H), 2.88 – 2.74 (m, 3H), 1.52 (s, 9H), 1.45 (s, 9H); MS (ESI) m/z 671.2 [M+Na]+. Next, compound S41 was deprotected to afford 23 mg (50% isolated yield) of 3 as a brown 1 solid. H NMR (400 MHz, CD3OD) δ 7.33 (s, 1H), 7.19 (dd, J = 9.0, 1.6 Hz, 1H), 7.17 – 7.13 (m, 3H), 7.10 – 7.04 (m, 2H), 4.40 (s, 2H), 3.94 (dd, J = 9.5, 5.6 Hz, 1H), 3.52 (t, J = 6.3

125 Hz, 2H), 3.23 – 3.06 (m, 4H), 2.98 (dd, J = 13.7, 5.6 Hz, 1H), 2.91 (t, J = 7.4 Hz, 2H), 2.69 13 (dd, J = 13.8, 9.5 Hz, 1H), 1.67 – 1.45 (m, 4H); C NMR (101 MHz, CD3OD) δ 173.6, 160.1 (d, J = 250.3 Hz), 143.1 (d, J = 7.3 Hz), 137.9, 136.5 (d, J = 3.6 Hz), 130.3, 129.3, 127.7, 124.1 (d, J = 3.5 Hz), 122.0 (d, J = 16.2 Hz), 112.8 (d, J = 23.9 Hz), 60.2, 42.0, 40.5 (d, J = 19 6.2 Hz), 40.3, 39.6, 39.4, 27.2, 25.9 (d, J = 2.1 Hz), 25.7; F NMR (377 MHz, CD3OD) δ –77.1, –117.5; MS (ESI) m/z 449.2 [M+H+]; HRMS (ESI) found m/z 449.20324 [M+H]+, + C22H30FN4O3S requires m/z 449.20226.

(R)-6-(N-(1-((5-ammoniopentyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8- fluoro-1,2,3,4-tetra-hydroisoquinolin-2-ium di(2,2,2-trifluoroacetate) (4): Starting from compound S23 (60 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 73 mg (77% isolated yield) of compound S42 was obtained. 1H NMR (500 MHz, 3 CDCl3) δ 7.23 – 7.11 (m, 4H), 7.04 (bs, 1H), 6.97 – 6.92 (m, 2H), 6.39 (bs, 1H), 5.14 (d, J = 6.0 Hz, 1H), 4.70 – 4.62 (m, 1H), 4.58 (bs, 2H), 3.86 – 3.76 (m, 1H), 3.66 (bs, 2H), 3.27 – 3.14 (m, 2H), 3.12 – 3.03 (m, 3H), 2.87 – 2.74 (m, 3H), 1.52 (s, 9H), 1.44 (s, 9H); MS (ESI) m/z 662.7 [M+H]+. Next, compound S42 was deprotected to afford 59 1 mg (79% isolated yield) of 4 as a yellow solid. H NMR (400 MHz, CD3OD) δ 7.35 (bs, 1H), 7.22 (dd, J = 9.1, 1.7 Hz, 1H), 7.18 – 7.13 (m, 3H), 7.10 – 7.05 (m, 2H), 4.40 (s, 2H), 3.95 (dd, J = 9.1, 6.1 Hz, 1H), 3.52 (t, J = 6.5 Hz, 2H), 3.16 – 3.01 (m, 4H), 2.96 (dd, J = 13.7, 6.1 Hz, 1H), 2.92 – 2.85 (m, 2H), 2.71 (dd, J = 13.7, 9.1 Hz, 1H), 1.70 – 1.56 (m, 2H), 1.49 13 – 1.38 (m, 2H), 1.36 – 1.26 (m, 2H); C NMR (101 MHz, CD3OD) δ 173.2, 160.1 (d, J = 250.5 Hz), 143.2 (d, J = 7.6 Hz), 137.9, 136.5 (d, J = 3.6 Hz), 130.4, 129.3, 127.7, 124.2 (d, J = 3.4 Hz), 122.0 (d, J = 16.1 Hz), 112.8 (d, J = 23.9 Hz), 60.1, 42.0, 40.6, 40.5 (d, J = 19 6.4 Hz), 39.8, 39.8, 29.6, 28.1, 25.9 (d, J = 2.1 Hz), 24.4; F NMR (377 MHz, CD3OD) δ –77.0, –117.5; MS (ESI) m/z 463.40 [M+H]+; HRMS (ESI) found m/z 463.21895 [M+H]+, + C23H32FN4O3S requires m/z 463.21791.

(R)-8-fluoro-6-(N-(1-oxo-3-phenyl-1-(piperazin-1-ium-1-yl)propan-2-yl)sulfamoyl)- 1,2,3,4-tetrahydro-isoquinolin-2-ium di(2,2,2-trifluoroacetate) (5): Starting from compound S24 (57 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 48 mg (52% isolated yield) of compound 1 S43 was obtained. H NMR (400 MHz, CDCl3) δ : 7.35 (bs, 1H), 7.28 – 7.20 (m, 5H), 7.14 – 7.06 (m, 2H), 5.75 (d, J = 9.5 Hz, 1H), 4.58 (bs, 2H), 4.47 – 4.38 (m, 1H), 3.71 – 3.55 (m, 2H), 3.43 – 3.32 (m, 1H), 3.27 – 2.75 (m, 10H), 2.67 – 2.56 (m, 1H), 1.50 (s, 9H), 1.44 (s, 9H); MS (ESI) m/z 647.4 [M+H]+. Next,

126 compound S44 was deprotected to afford 36 mg (71% isolated yield) of 5 as a brown 1 solid. H NMR (400 MHz, CD3OD) δ 7.51 (bs, 1H), 7.41 (dd, J = 9.1, 1.7 Hz, 1H), 7.32 – 7.22 (m, 3H), 7.20 – 7.15 (m, 2H), 4.57 (dd, J = 8.8, 6.9 Hz, 1H), 4.44 (s, 2H), 3.81 – 3.69 (m, 1H), 3.59 – 3.47 (m, 4H), 3.40 – 3.33 (m, 1H), 3.18 (t, J = 6.3 Hz, 2H), 3.12 – 2.83 (m, 13 4H), 2.82 – 2.72 (m, 1H), 2.18 – 2.08 (m, 1H); C NMR (101 MHz, CD3OD) δ 171.2, 160.2 (d, J = 250.8 Hz), 143.6 (d, J = 7.4 Hz), 137.3, 136.8 (d, J = 3.4 Hz), 130.8, 129.8, 128.4, 124.2 (d, J = 3.5 Hz), 122.3 (d, J = 16.2 Hz), 112.9 (d, J = 23.8 Hz), 54.4, 44.1, 43.6, 42.0, 19 40.5 (d, J = 6.5 Hz), 40.4, 39.7, 25.9 (d, J = 2.1 Hz); F NMR (377 MHz, CD3OD) δ –77.1, –117.4; MS (ESI) m/z 447.5 [M+H]+; HRMS (ESI) found m/z 447.18742 [M+H]+, + C22H28FN4O3S requires m/z 447.18661.

(R)-N-(3-aminophenyl)-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6-sulfonamido)-3- phenylpropan-amide (6): Starting from compoundS25 (61 mg, 0.17 mmol, 1.2 equiv) 3 and following GPIV, 63 mg (66% isolated yield) of compound 1 S45 was obtained. H NMR (500 MHz, CDCl3) δ 7.95 (s, 1H), 7.55 (s, 1H), 7.25 – 6.98 (m, 10H), 6.59 (s, 1H), 5.35 (bs, 1H), 4.53 (s, 2H), 3.98 (d, J = 14.3 Hz, 1H), 3.64 – 3.51 (m, 2H), 3.14 (dd, J = 14.0, 5.9 Hz, 1H), 2.93 (dd, J = 14.1, 8.3 + Hz, 1H), 2.74 (qt, J = 16.5, 5.8 Hz, 2H), 1.51 (s, 18H); MS (ESI) m/z 686.1 [M+NH4] . Next, compound S45 was deprotected and purified by column chromatography (0‑10% MeOH in DCM) to afford 33 mg (68% isolated yield) of6 as a yellow solid. 1H NMR (500

MHz, CDCl3) δ 7.3 (s, 1H), 7.2 – 7.2 (m, 6H), 7.0 (t, J = 8.0 Hz, 1H), 6.7 (t, J = 2.0 Hz, 1H), 6.5 (dd, J = 8.0, 2.0 Hz, 1H), 6.4 (dd, J = 8.0, 2.0 Hz, 1H), 4.1 (t, J = 7.6 Hz, 1H), 3.8 (s, 2H), 3.0 (dd, J = 13.7, 7.1 Hz, 1H), 2.9 – 2.9 (m, 3H), 2.7 (dt, J = 17.0, 5.8 Hz, 1H), 2.5 (dt, J = 13 17.0, 5.8 Hz, 1H); C NMR (126 MHz, CDCl3) δ 170.4, 160.2 (d, J = 247.8 Hz), 149.4, 140.8 (d, J = 7.5 Hz), 139.9, 139.4 (d, J = 4.7 Hz), 137.7, 130.4, 130.2, 129.3, 128.4 (d, J = 17.2 Hz), 127.8, 124.7 (d, J = 3.1 Hz), 112.4, 112.2 (d, J = 24.8 Hz), 110.4, 107.7, 60.3, 19 42.8, 42.2 (d, J = 5.1 Hz), 40.2, 28.5 (d, J = 2.3 Hz); F NMR (377 MHz, CD3OD) δ –119.5; + + + MS (ESI) m/z 469.1 [M+H] ; HRMS (ESI) found m/z 469.17200 [M+H] , C24H26FN4O3S requires m/z 469.17096.

(R)-8-fluoro-6-(N-(1-((2-hydroxyethyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)- 1,2,3,4-tetrahyd-roisoquinolin-2-ium 2,2,2-trifluoroacetate (7): Starting from compound S26 (36 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 18 mg (25% isolated yield) of compound 1 S46 was obtained. H NMR (500 MHz, CDCl3) δ 7.33 – 6.92 (m, 7H), 5.67 (bs, 1H), 4.64 – 4.52 (m, 2H), 3.87 (dd, J = 9.2,

127 5.2 Hz, 1H), 3.70 – 3.60 (m, 4H), 3.46 – 3.38 (m, 1H), 3.38 – 3.30 (m, 1H), 3.12 (dd, J = 14.1, 5.2 Hz, 1H), 2.83 – 2.75 (m, 3H), 1.51 (s, 9H); MS (ESI) m/z 521.9 [M+H]. Next, compound S46 was deprotected to afford 26 mg (70% isolated yield) of 7 as a yellow 1 sticky solid. H NMR (500 MHz, CD3OD) δ 7.38 (bs, 1H), 7.25 (dd, J = 9.0, 1.6 Hz, 1H), 7.18 – 7.17 (m, 3H), 7.11 – 7.11 (m, 2H), 4.40 (bs, 2H), 4.00 (dd, J = 9.1, 5.9 Hz, 1H), 3.52 (t, J = 6.5 Hz, 2H), 3.43 – 3.42 (m, 2H), 3.13 – 3.11 (m, 4H), 2.99 (dd, J = 13.7, 5.9 Hz, 1H), 2.75 13 (dd, J = 13.7, 9.1 Hz, 1H); C NMR (126 MHz, CD3OD) δ 173.2, 160.1 (d, J = 250.4 Hz), 143.2 (d, J = 7.5 Hz), 137.9, 136.4 (d, J = 3.5 Hz), 130.4, 129.3, 127.7, 124.3 (d, J = 3.4 Hz), 121.9 (d, J = 16.0 Hz), 112.9 (d, J = 23.9 Hz), 61.3, 60.0, 42.8, 42.0, 40.6 (d, J = 6.5 Hz), 19 + 40.0, 25.9; F NMR (377 MHz, CD3OD) δ –77.1, –117.7; MS (ESI) m/z 422.5 [M+H] ; + + HRMS (ESI) found m/z 422.15585 [M+H] , C20H25FN3O4S requires m/z 422.15498.

(R)-8-fluoro-6-(N-(1-((2-mercaptoethyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)- 3 1,2,3,4-tetra-hydroisoquinolin-2-ium 2,2,2-trifluoroacetate (8): Starting from compound S27 (40 mg, 86 µmol, 1.2 equiv) and following GPIV, 21 mg (38% isolated yield) of compound 1 S47 was obtained. H NMR (400 MHz, CDCl3) δ 7.43 – 7.35 (m, 6H), 7.34 – 7.15 (m, 11H), 7.16 – 6.98 (m, 3H), 6.95 – 6.87 (m, 2H), 6.11 (s, 1H), 5.08 (d, J = 7.0 Hz, 1H), 4.58 (s, 2H), 3.75 (q, J = 7.0 Hz, 1H), 3.64 (s, 2H), 3.02 – 2.86 (m, 3H), 2.85 – 2.72 (m, 3H), 2.39 – 2.23 (m, 2H), 1.51 (s, 9H); MS (ESI) m/z 802.2 [M+H]+. Next, compound S47 was deprotected and purified by preparative HPLC to afford 1.7 mg (11% isolated yield) 7of 1 as a white solid. H NMR (400 MHz, CD3OD) δ 7.31 (s, 1H), 7.19 (dd, J = 9.1, 1.7 Hz, 1H), 7.13 – 7.06 (m, 3H), 7.02 (dd, J = 7.2, 2.5 Hz, 2H), 4.31 (s, 2H), 3.90 (dd, J = 8.7, 6.5 Hz, 1H), 3.43 (t, J = 6.4 Hz, 2H), 3.13 – 2.93 (m, 4H), 2.88 (dd, J = 13.6, 6.5 Hz, 1H), 2.68 (dd, 19 J = 13.6, 8.7 Hz, 1H), 2.37 – 2.20 (m, 2H); F NMR (377 MHz, CD3OD) δ –77.1, –117.6; + + + MS (ESI) m/z 438.1 [M+H] ; HRMS (ESI) found m/z 438.13226 [M+H] , C20H25FN3O3S requires m/z 438.13214.

(R)-6-(N-(1-((2-amino-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8- fluoro-1,2,3,4-tetr-ahydroisoquinolin-2-ium 2,2,2-trifluoroacetate (9): Starting from compoundS28 (38 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 46 mg (60% isolated yield) of compound 1 S48 was obtained. H NMR (500 MHz, CDCl3) δ 7.81 (bs, 1H), 7.04 (s, 7H), 6.79 (bs, 1H), 6.61 (bs, 1H), 6.30 (bs, 1H), 4.54 (bs, 2H), 4.10 (bs, 1H), 3.96 (bs, 1H), 3.91 (dd, J = 10.6, 4.2 Hz, 1H), 3.67 – 3.57 (m, 2H), 3.18 (dd, J = 14.0, 3.9 Hz, 1H), 2.79 – 2.69 (m, 3H), 1.52 (s, 9H); MS (ESI) m/z 557.2 [M+Na]+. Next, compound S48 was deprotected to afford 41

128 1 mg (>99% isolated yield) of 9 a yellow solid. H NMR (500 MHz, CD3OD) δ 7.3 (bs, 1H), 7.2 – 7.2 (m, 4H), 7.1 – 7.1 (m, 2H), 4.4 (s, 2H), 4.0 (dd, J = 9.7, 5.2 Hz, 1H), 3.7 (d, J = 4.8 Hz, 2H), 3.5 (t, J = 6.4 Hz, 2H), 3.1 – 3.1 (m, 2H), 3.0 (dd, J = 13.9, 5.2 Hz, 1H), 2.8 (dd, J 13 = 13.9, 9.7 Hz, 1H); C NMR (126 MHz, CD3OD) δ 173.8, 173.7, 160.1 (d, J = 250.6 Hz), 142.5 (d, J = 7.4 Hz), 137.8, 136.6 (d, J = 3.5 Hz), 130.3, 129.3, 127.8, 124.3 (d, J = 3.3 Hz), 122.2 (d, J = 16.0 Hz), 112.8 (d, J = 23.8 Hz), 60.0, 43.1, 42.0, 40.5 (d, J = 6.4 Hz), 19 + 39.4, 25.8; F NMR (377 MHz, CD3OD) δ –77.0, –117.3; MS (ESI) m/z 435.5 [M+H] ; + + HRMS (ESI) found m/z 435.15190 [M+H] , C20H24FN4O4S requires m/z 435.15023.

(R)-6-(N-(1-((carboxymethyl)amino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8-fluoro- 1,2,3,4-tetra-hydroisoquinolin-2-ium 2,2,2-trifluoroacetate (10): Starting from compound S29 (25 mg, 86 µmol, 1.2 equiv) and following GPIV, 17.5 mg (43% isolated yield) of compound S49 1 3 was obtained. H NMR (400 MHz, CDCl3) δ 7.24 – 7.10 (m, 4H), 7.05 (d, J = 8.6 Hz, 1H), 7.00 – 6.92 (m, 2H), 6.74 (app t, J = 5.1 Hz, 1H), 5.03 (d, J = 6.7 Hz, 1H), 4.59 (bs, 2H), 3.97 (dd, J = 18.2, 5.6 Hz, 1H), 3.93 – 3.87 (m, 1H), 3.83 (dd, J = 18.2, 4.7 Hz, 1H), 3.66 (bs, 2H), 3.13 (dd, J = 14.1, 5.3 Hz, 1H), 2.91 – 2.75 (m, 3H), 1.52 (s, 9H), 1.48 (s, 9H); MS (ESI) m/z 614.1 [M+H]+. Next, compound S49 was deprotected to afford 16.2 mg (99% isolated yield) of 10 white 1 solid. H NMR (400 MHz, CD3OD) δ 7.38 (bs, 1H), 7.28 – 7.12 (m, 6H), 4.40 (s, 2H), 4.10 (dd, J = 9.6, 5.0 Hz, 1H), 3.85 – 3.67 (m, 2H), 3.53 (t, J = 6.3 Hz, 2H), 3.13 (t, J = 6.3 Hz, 2H), 13 3.08 (dd, J = 13.9, 5.0 Hz, 1H), 2.78 (dd, J = 13.9, 9.6 Hz, 1H); C NMR (101 MHz, CD3OD) δ 171.9, 171.5, 160.0, 157.5, 141.8, 141.7, 136.6, 135.1, 135.1, 129.0, 127.9, 126.3, 122.9, 122.8, 120.7, 120.5, 111.6, 111.3, 65.5, 58.4, 40.7, 39.3, 39.2, 38.6, 24.5, 24.4, 14.0; 19F

NMR (377 MHz, CD3OD) δ –76.95, –117.65; MS (ESI) m/z 436.1; HRMS (ESI) found m/z + + 436.13425 [M+H] , C20H23FN3O5S requires m/z 436.13424.

(R)-N-allyl-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6-sulfonamido)-3- phenylpropanamide (11) Starting from compound S30 (35 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 42 mg (57% isolated yield) of compound 1 S50 was obtained. H NMR (400 MHz, CDCl3) δ 7.24 – 6.93 (m, 7H), 6.50 (t, J = 5.9 Hz, 1H), 5.73 (ddt, J = 17.2, 10.3, 5.5 Hz, 1H), 5.36 (d, J = 6.8 Hz, 1H), 5.17 – 5.07 (m, 2H), 4.58 (bs, 2H), 3.93 – 3.75 (m, 3H), 3.65 (t, J = 5.9 Hz, 2H), 3.10 (dd, J = 14.0, 5.4 Hz, 1H), 2.87 – 2.76 (m, 3H), 1.52 (s, 9H); MS (ESI) m/z 540.1 [M+Na]+. Next, compound S50 was deprotected and purified by column chromatography (0-10% MeOH in DCM) to afford 31 mg (93% isolated yield) of 11 a yellow solid. 1H NMR (400

129 MHz, CD3OD) δ 7.24 (bs, 1H), 7.11 – 7.10 (m, 6H), 5.65 (ddt, J = 15.9, 9.7, 5.3 Hz, 1H), 5.09 – 4.97 (m, 2H), 3.99 – 3.93 (m, 3H), 3.62 (qdt, J = 15.9, 5.3, 1.6 Hz, 2H), 3.06 (t, J = 5.7 Hz, 2H), 2.97 (dd, J = 13.6, 6.2 Hz, 1H), 2.85 – 2.75 (m, 2H), 2.70 (dd, J = 13.5, 4.6 Hz, 13 1H); C NMR (101 MHz, CD3OD) δ 172.9, 160.1 (d, J = 247.5 Hz), 140.9 (d, J = 7.7 Hz), 139.7 (d, J = 4.8 Hz), 137.8, 134.8, 130.3, 129.3, 128.7 (d, J = 17.2 Hz), 127.7, 124.4 (d, J = 3.2 Hz), 116.3, 111.8 (d, J = 24.9 Hz), 59.9, 43.3, 42.6, 42.5, 40.0, 29.1; 19F NMR (377 + MHz, CD3OD) d –76.9, –119.6; MS (ESI) m/z 418.1 [M+H] ; HRMS (ESI) found m/z + + 418.16111 [M+H] , C21H24FN3O3S requires m/z 418.16283.

(R)-8-fluoro-6-(N-(1-oxo-3-phenyl-1-(prop-2-yn-1-ylamino)propan-2-yl)sulfamoyl)- 1,2,3,4-tetrahydro isoquinolin-2-ium 2,2,2-trifluoroacetate (12): Starting from compound S31 (35 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 32 mg (43% isolated yield) of compound 3 1 S51 was obtained. H NMR (400 MHz, CD3OD) δ 7.24 (bs, 1H), 7.16 – 7.03 (m, 6H), 4.58 (bs, 2H), 3.94 (dd, J = 9.3, 5.5 Hz, 1H), 3.78 (d, J = 2.6 Hz, 2H), 3.66 (t, J = 5.7 Hz, 2H), 2.96 (dd, J = 13.8, 5.5 Hz, 1H), 2.82 (t, J = 5.9 Hz, 2H), 2.70 (dd, J = 13.8, 9.3 Hz, 1H), 2.57 (t, J = 2.6 Hz, 1H), 1.52 (s, 9H); MS (ESI) m/z 538.1 [M+Na]+. Next, compound S51 was deprotected 1 to afford 24 mg (93% isolated yield) of 12 a yellow solid. H NMR (500 MHz, CD3OD) δ 7.25 (bs, 1H), 7.12 – 7.11 (m, 7H), 4.01 (bs, 2H), 3.96 (dd, J = 8.9, 6.0 Hz, 1H), 3.76 (d, J = 6.7 Hz, 2H), 3.12 (t, J = 6.0 Hz, 2H), 2.96 (dd, J = 13.7, 6.0 Hz, 1H), 2.84 (t, J = 6.0 Hz, 13 2H), 2.72 (dd, J = 13.7, 8.9 Hz, 1H); C NMR (126 MHz, CD3OD) δ 172.7, 160.2 (d, J = 247.8 Hz), 141.2 (d, J = 7.5 Hz), 139.3 (d, J = 4.6 Hz), 137.7, 130.3, 129.3, 127.7, 124.4 (d, J = 3.1 Hz), 112.0 (d, J = 24.7 Hz), 79.9, 72.6, 59.7, 43.1, 42.3 (d, J = 5.2 Hz), 40.0, 19 + 29.5, 28.7, 23.7; F NMR (377 MHz, CD3OD) δ –76.9, –119.3; MS (ESI) m/z 416.0 [M+H] ; + + HRMS (ESI) found m/z 416.14730 [M+H] , C21H23FN3O3S requires m/z 416.14441.

(R)-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6-sulfonamido)-N-(2-(methylamino) ethyl)-3-phenyl propanamide (13): Starting from compound S32 (55 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 60 mg (67% isolated yield) of 1 compound S52 was obtained. H NMR (500 MHz, CDCl3) δ 7.28 (bs, 1H), 7.21 – 7.06 (m, 6H), 4.61 (s, 2H), 4.39 (q, J = 7.1 Hz, 1H), 3.86 (bs, 1H), 3.68 (bs, 2H), 3.45 – 3.25 (m, 5H), 3.09 (dd, J = 14.1, 5.3 Hz, 1H), 2.88 (s, 3H), 2.87 – 2.77 (m, 3H), 1.54 (s, 9H), 1.48 (s, 9H); MS (ESI) m/z 657.3 [M+Na]+. Next, compound S52 was deprotected to afford 16 mg (39% isolated yield) of13 a yellow sticky solid.1 H NMR

(400 MHz, CD3OD) δ 7.25 (bs, 1H), 7.12 – 7.12 (m, 6H), 3.95 (s, 2H), 3.89 (dd, J = 8.6, 6.6

130 Hz, 1H), 3.21 (dt, J = 13.7, 6.3 Hz, 1H), 3.08 – 3.07 (m, 3H), 2.96 (dd, J = 13.6, 6.6 Hz, 1H), 2.82 – 2.79 (m, 2H), 2.74 (dd, J = 13.6, 8.6 Hz, 1H), 2.53 (t, J = 6.3 Hz, 2H), 2.33 (s, 3H); 13 C NMR (101 MHz, CD3OD) δ 173.5, 160.2 (d, J = 247.5 Hz), 140.7 (d, J = 7.7 Hz), 139.9 (d, J = 5.1 Hz), 137.8, 130.3, 129.4, 129.0 (d, J = 17.2 Hz), 127.8, 124.5 (d, J = 3.2 Hz), 111.8 (d, J = 24.9 Hz), 60.0, 50.9, 43.3, 42.6 (d, J = 5.1 Hz), 39.8, 39.1, 35.5, 29.2 (d, J = 19 + 2.4 Hz); F NMR (377 MHz, CD3OD) δ –76.9, –119.6; MS (ESI) m/z 435.3 [M+H] ; HRMS + + (ESI) found m/z 435.18792 [M+H] , C21H28FN4O3S requires m/z 435.18661.

((R)-6-(N-(1-((2-(dimethylammonio)ethyl)amino)-1-oxo-3-phenylpropan-2-yl) sulfamoyl)-8-fluoro-1,2,3,4-tetrahydroisoquinolin-2-ium 2,2,2-trifluoroacetate (14): Starting from compound S33 (20 mg, 86 µmol, 1.2 equiv) and following GPIV, 30 mg (77% isolated yield) of 1 compound S53 was obtained. H NMR (400 MHz, CDCl3) δ 7.22 (bs, 1H), 7.19 – 7.06 (m, 5H), 7.05 – 6.98 (m, 2H), 4.59 3 (bs, 2H), 3.94 (dd, J = 8.8, 5.6 Hz, 1H), 3.71 – 3.60 (m, 2H), 3.52 – 3.42 (m, 1H), 3.34 – 3.23 (m, 1H), 3.10 (dd, J = 13.9, 5.6 Hz, 1H), 3.00 – 2.85 (m, 2H), 2.81 (d, J = 6.4 Hz, 2H), 2.59 (t, J = 5.8 Hz, 2H), 2.38 (s, 6H), 1.54 (s, 9H); MS (ESI) m/z 459.1 [M+H]+. Next, compound S53 was deprotected to 1 afford 22 mg (57% isolated yield) of14 as an off-white solid. H NMR (400 MHz, CD3OD) δ 7.24 (bs, 1H), 7.12 – 7.11 (m, 6H), 3.99 (bs, 2H), 3.91 (dd, J = 8.9, 6.1 Hz, 1H), 3.17 – 3.17 (m, 4H), 2.98 (dd, J = 13.7, 6.1 Hz, 1H), 2.84 – 2.80 (m, 2H), 2.72 (dd, J = 13.7, 8.9 13 Hz, 1H), 2.39 – 2.37 (m, 2H), 2.32 (s, 6H); C NMR (101 MHz, CD3OD) δ 173.4, 158.9 (d, J = 247.6 Hz), 141.0 (d, J = 7.6 Hz), 139.6 (d, J = 4.7 Hz), 137.8, 130.3, 129.3, 127.7, 124.4 (d, J = 3.2 Hz), 111.9 (d, J = 24.7 Hz), 60.0, 58.7, 49.9, 45.3, 43.2, 42.4 (d, J = 4.5 Hz), 19 + 39.8, 37.7, 28.9; F NMR (377 MHz, CD3OD) δ -76.9, -119.4; MS (ESI) m/z 449.6 [M+H] ; + + HRMS (ESI) found m/z 449.20268 [M+H] , C22H30FN4O3S requires m/z 449.20226.

(R)-2-((8-fluoro-1,2,3,4-tetrahydroisoquinoline)-6-sulfonamido)-3-phenyl-N- propylpropanamide (15): Starting from compound S34 (35 mg, 0.17 mmol, 1.2 equiv) and following GPIV, 75 mg (100% isolated yield) of compound 1 S54 was obtained. H NMR (400 MHz, CDCl3) δ 7.24 – 7.20 (m, 1H), 7.15 (s, 3H), 7.06 (d, J = 8.3 Hz, 1H), 7.01 – 6.93 (m, 2H), 6.34 (t, J = 5.9 Hz, 1H), 5.39 (d, J = 4.9 Hz, 1H), 4.58 (bs, 2H), 3.85 (d, J = 6.7 Hz, 1H), 3.65 (t, J = 6.0 Hz, 2H), 3.24 – 2.94 (m, 3H), 2.89 – 2.67 (m, 3H), 1.51 (s, 9H), 1.50 – 1.36 (m, 2H), 0.85 (t, J = 7.4 Hz, 3H); MS (ESI) m/z 542.0 [M+Na]+. Next, compound S54 was deprotected and purified by column chromatography (0-10% MeOH in DCM) to afford 22 mg (37% isolated yield) of15 as a yellow solid. 1H NMR (400

131 MHz, CD3OD) δ 7.3 (s, 1H), 7.2 – 7.0 (m, 6H), 4.0 (s, 2H), 4.0 (dd, J = 8.7, 6.5 Hz, 1H), 3.1 (t, J = 6.0 Hz, 2H), 3.0 – 2.9 (m, 3H), 2.9 – 2.8 (m, 2H), 2.7 (dd, J = 13.6, 8.7 Hz, 1H), 1.4 13 – 1.3 (m, 2H), 0.8 (t, J = 7.4 Hz, 3H); C NMR (101 MHz, CD3OD) δ 172.8, 160.1 (d, J = 250.3 Hz), 143.3 (d, J = 7.3 Hz), 136.4 (d, J = 3.6 Hz), 136.2, 130.4, 129.3, 127.7, 124.2 (d, J = 3.4 Hz), 121.9 (d, J = 16.2 Hz), 112.9 (d, J = 23.9 Hz), 60.0, 42.1, 42.0, 40.5 (d, J = 19 6.5 Hz), 40.0, 25.8 (d, J = 2.2 Hz), 23.3, 11.6; F NMR (377 MHz, CD3OD) δ –119.6; MS + + + (ESI) m/z 420.1 [M+H] ; HRMS (ESI) found m/z 420.17667 [M+H] , C21H27FN3O3S requires m/z 420.17571.

(R)-6-(N-(1-(butylamino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-8-fluoro-1,2,3,4- tetrahydroisoquino-lin-2-ium 2,2,2-trifluoroacetate (16): Starting from compound S35 (31 mg, 93 µmol, 1.2 equiv) and following GPIV, 32 mg (84% isolated yield) of compound 3 1 S55 was obtained. H NMR (400 MHz, CDCl3) δ 7.23 (bs, 1H), 7.20 – 7.12 (m, 3H), 7.08 (d, J = 8.5 Hz, 1H), 7.00 – 6.93 (m, 2H), 6.23 (t, J = 5.9 Hz, 1H), 5.23 (d, J = 6.8 Hz, 1H), 4.60 (bs, 2H), 3.89 – 3.76 (m, 1H), 3.65 (d, J = 6.2 Hz, 2H), 3.27 – 3.10 (m, 2H), 3.06 (dd, J = 14.0, 5.9 Hz, 1H), 2.90 – 2.76 (m, 3H), 1.52 (s, 9H), 1.45 – 1.35 (m, 2H), 1.32 – 1.20 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H); MS (ESI) m/z 534.1 [M+H]+. Next, compound S55 was deprotected to afford 33.8 mg (>99% isolated yield) of 16 as an off-white solid. 1H NMR (400 MHz,

CD3OD) δ 7.29 (s, 1H), 7.16 (dd, J = 9.2, 1.7 Hz, 1H), 7.07 (dd, J = 5.1, 1.9 Hz, 3H), 7.00 (hept, J = 3.7, 3.2 Hz, 2H), 4.30 (s, 2H), 3.89 (dd, J = 8.6, 6.5 Hz, 1H), 3.42 (t, J = 6.3 Hz, 2H), 3.09 – 2.74 (m, 5H), 2.65 (dd, J = 13.6, 8.7 Hz, 1H), 1.28 – 1.15 (m, 3H), 1.17 – 1.02 13 (m, 2H), 0.77 (t, J = 7.3 Hz, 3H); C NMR (126 MHz, CD3OD) δ 171.3, 159.7, 157.7, 142.0, 142.0, 136.4, 135.1, 135.0, 129.0, 127.9, 126.3, 122.9, 122.8, 120.6, 120.4, 111.6, 111.4, 58.6, 40.6, 39.2, 39.1, 38.7, 38.6, 30.8, 24.5, 24.5, 19.5, 12.7; 19F NMR (377 MHz, + CD3OD) δ –77.2, –117.7; MS (ESI) m/z 434.5 [M+H] ; HRMS (ESI) found m/z 434.19090 + + [M+H] , C22H29FN3O3S requires m/z 434.19136.

(R)-8-fluoro-6-(N-(1-(isopentylamino)-1-oxo-3-phenylpropan-2-yl)sulfamoyl)-1,2,3,4- tetrahydroisoqui-nolin-2-ium 2,2,2-trifluoroacetate (17): Starting from compound S36 (32 mg, 93 µmol, 1.2 equiv) and following GPIV, 21 mg (54% isolated yield) of compound 1 S56 was obtained. H NMR (400 MHz, CDCl3) δ 7.23 (bs, 1H), 7.22 – 7.12 (m, 3H), 7.08 (d, J = 8.5 Hz, 1H), 7.01 – 6.93 (m, 2H), 6.17 (t, J = 5.9 Hz, 1H), 5.17 (d, J = 6.7 Hz, 1H), 4.60 (bs, 2H), 3.87 – 3.76 (m, 1H), 3.66 (t, J = 6.0 Hz, 2H), 3.28 – 3.10 (m, 2H), 3.05 (dd, J = 14.0, 5.9 Hz, 1H), 2.93 – 2.70 (m, 3H), 1.52 (s, 9H), 1.38 – 1.26 (m,

132 3H), 0.88 (d, J = 6.6 Hz, 6H); MS (ESI) m/z 547.8 [M+H]+. Next, compound S56 was deprotected to afford 21.5 mg (>99% isolated yield) of17 as an off-white solid.1 H NMR

(400 MHz, CD3OD) δ 7.31 (bs, 1H), 7.18 (dd, J = 9.1, 1.7 Hz, 1H), 7.11 – 7.06 (m, 3H), 7.05 – 6.98 (m, 2H), 4.31 (s, 2H), 3.90 (dd, J = 8.6, 6.6 Hz, 1H), 3.44 (td, J = 6.4, 1.5 Hz, 2H), 3.09 – 2.94 (m, 3H), 2.92 – 2.78 (m, 2H), 2.66 (dd, J = 13.6, 8.6 Hz, 1H), 1.41 – 1.28 (m, 13 1H), 1.21 – 1.07 (m, 3H), 0.77 (dd, J = 6.6, 3.3 Hz, 6H); C NMR (126 MHz, CD3OD) δ 171.2, 159.7, 157.7, 142.1, 142.0, 136.4, 135.0, 135.0, 129.0, 127.9, 126.3, 122.9, 122.8, 120.5, 120.4, 111.6, 111.4, 58.6, 40.7, 39.2, 39.2, 38.6, 37.6, 37.1, 25.2, 24.5, 19 + 24.5, 21.3; F NMR (377 MHz, CD3OD) δ –77.2, –117.7; MS (ESI) m/z 526.1 [M+H] ; + + HRMS (ESI) found m/z 448.20671 [M+H] , C23H31FN3O3S requires m/z 448.20701.

(R)-2-(2-((3-fluorophenyl)sulfonamido)-3-phenylpropanamido)ethan-1-aminium 2,2,2-trifluoroacetate (18): Starting from compound S20 (41 mg, 0.17 mmol, 1.2 equiv) and 3 3-fluorobenzenesulfonyl chloride (27 mg, 0.14 mmol, 1.0 equiv) and following GPIV, 30 mg (58% isolated yield) of compound S57 1 was obtained. H NMR (500 MHz, CDCl3) δ 7.46 – 7.41 (m, 1H), 7.37 (td, J = 8.0, 5.1 Hz, 1H), 7.29 – 7.15 (m, 5H), 7.01 – 6.95 (m, 2H), 6.82 – 6.77 (m, 1H), 5.55 (bs, 1H), 4.89 (bs, 1H), 3.87 (dd, J = 8.2, 5.9 Hz, 1H), 3.32 – 3.07 (m, 4H), 3.03 (dd, J = 13.9, 5.9 Hz, 1H), 2.87 (dd, J = 14.0, 8.2 Hz, 1H), 1.44 (s, 9H); MS (ESI) m/z 465.7 [M+H]+. Next, compound S57 was deprotected to afford 29 mg (96% isolated yield) of 18 as an off-white solid. 1H NMR

(500 MHz, CD3OD) δ 7.45 – 7.45 (m, 2H), 7.30 – 7.29 (m, 2H), 7.15 – 7.15 (m, 3H), 7.05 – 7.05 (m, 2H), 3.90 (dd, J = 8.9, 6.2 Hz, 1H), 3.29 – 3.27 (m, 2H), 2.97 (dd, J = 13.7, 6.2 Hz, 1H), 2.88 (hept, J = 6.4 Hz, 2H), 2.74 (dd, J = 13.7, 8.9 Hz, 1H); 13C NMR (126 MHz,

CD3OD) δ 174.6, 163.6 (d, J = 249.4 Hz), 143.6 (d, J = 6.9 Hz), 137.5, 132.1 (d, J = 7.9 Hz), 130.2, 129.5, 128.0, 123.9 (d, J = 3.2 Hz), 120.7 (d, J = 21.6 Hz), 115.1 (d, J = 24.6 Hz), 19 + 59.9, 40.4, 39.5, 38.0; F NMR (377 MHz, CD3OD) δ –112.2; MS (ESI) m/z 366.0 [M+H] ; + + HRMS (ESI) found m/z 366.12922 [M+H] , C17H21FN3O3S requires m/z 366.12876.

6-(N-(2-((2-ammonioethyl)amino)-2-oxoethyl)sulfamoyl)-8-fluoro-1,2,3,4- tetrahydroisoquinolin-2-ium di(2,2,2-trifluoroacetate) (19): Starting from compound S37 (27 mg, 0.12 mmol) and following GPIV, 52 mg (95% isolated yield) of compound 1 S58. H NMR (500 MHz, CDCl3) δ 7.44 (d, J = 1.7 Hz, 1H), 7.37 (dd, J = 8.5, 1.7 Hz, 1H), 7.17 (bs, 1H), 6.43 (bs, 1H), 5.18 (bs, 1H), 4.60 (s, 2H), 3.65 (t, J = 5.8 Hz, 2H), 3.56 (s, 2H), 3.33 (q, J = 5.7 Hz, 2H), 3.21 (q, J = 5.9 Hz, 2H), 2.87 (t,

133 J = 5.7 Hz, 2H), 1.48 (s, 9H), 1.40 (s, 9H); MS (ESI) m/z 530.9 [M+H]+. Next, compound S58 was deprotected to afford 49 mg (93% isolated yield) of 19 as a yellow solid. 1H

NMR (500 MHz, CD3OD) δ 7.62 (d, J = 1.5 Hz, 1H), 7.54 (dd, J = 9.1, 1.6 Hz, 1H), 4.46 (s, 2H), 3.58 (s, 2H), 3.55 (t, J = 6.3 Hz, 2H), 3.47 (t, J = 6.0 Hz, 2H), 3.22 (t, J = 6.3 Hz, 2H), 13 3.05 (t, J = 6.0 Hz, 2H); C NMR (126 MHz, CD3OD) δ 172.2, 160.5 (d, J = 251.0 Hz), 142.3 (d, J = 7.3 Hz), 137.2 (d, J = 3.5 Hz), 124.5 (d, J = 3.4 Hz), 122.7 (d, J = 16.3 Hz), 113.0 (d, 19 J = 23.9 Hz), 46.5, 42.0, 40.7, 40.6 (d, J = 6.5 Hz), 38.1, 25.9; F NMR (377 MHz, CD3OD) δ –76.8, –115.3; MS (ESI) m/z 331.0 [M+H]+; HRMS (ESI) found m/z 331.12417 [M+H]+, + C13H20FN4O3S requires m/z 331.12401.

(R)-1-((2-ammonioethyl)amino)-1-oxo-3-phenylpropan-2-aminium di(2,2,2- trifluoroacetate) (20): Starting from compound S19, 52 mg (>99% isolated yield) of 20 3 1 was synthesised according to GPIII. H NMR (400 MHz, CD3OD) δ 7.44 – 7.25 (m, 5H), 3.63 – 3.47 (m, 1H), 3.42 – 3.34 (m, 1H), 3.21 (dd, J = 13.8, 7.1 Hz, 1H), 3.10 (dd, J = 13.3, 7.1 Hz, 2H), 3.09 – 3.00 (m, 1H), 2.96 (ddd, J = 13.1, 7.2, 5.8 Hz, 1H); 13C NMR (101

MHz, CD3OD) δ 171.6, 134.4, 129.1, 128.7, 127.4, 60.2, 54.5, 38.9, 36.7; MS (ESI) m/z 208.0 [M+H]+; HRMS (ESI) found m/z 230.12599 [M+H]+, + C11H18N3O requires m/z 208.14499.

134 3.5. References

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135 Bioorg. Med. Chem. 2016, 24, 4318-4323. 21. D. C. Lenstra, E. Damen, R. G. G. Leenders, R. H. Blaauw, F. P. J. T. Rutjes, A. Wegert, J. Mecinović, ChemMedChem. 2018, 13, 1405-1413. 22. K. Guitot, T. Drujon, F. Burlina, S. Sagan, S. Beaupierre, O. Pamlard, R. H. Dodd, C. Guillou, G. Bolbach, E. Sachon, D. Guianvarc’h, Anal. Bioanal. Chem. 2017, 409, 3767-3777. 23. D. C. Lenstra, A. H. K. Al Temimi, J. Mecinović, Bioorg. Med. Chem. 2018, 28, 1234-1238. 24. J. R. Wilson, C. Jing, P. A. Walker, S. R. Martin, S. A. Howell, G. M. Blackburn, S. J. Gamblin, B. Xiao, Cell 2002, 111, 105-115. 25. B. Xiao, C. Jing, J. R. Wilson, P. A. Walker, N. Vasisht, G. Kelly, S. Howell, I. A. Taylor, G. M. Blackburn, S. J. Gamblin, Nature 2003, 421, 652-656. 26. A. H. K. A. Temimi, Y. V. Reddy, P. B. White, H. Guo, P. Qian, J. Mecinović, Sci. Rep. 2017, 7, 16148. 27. Chemical Computing Group Molecular Operating Environment (MOE), https://www.chemcomp.com/.

136 3

137 138 4

Bioisosteric replacement of the sulfonamide moiety of (R)-PFI-2: Towards the development of novel inhibitors of histone lysine methyltransferase SETD7

139 Abstract

Building on the structure-activity relationship studies performed in chapters 2 and 3, we aimed to replace the sulfonamide core of (R)-PFI-2 to develop new inhibitors of histone lysine methyltransferase SETD7. Computational tools for scaffold hopping were used to identify 1,5‑disubstituted imidazole as a potential bioisostere for the sulfonamide moiety. A library of 56 imidazole (R)-PFI-2 analogues was synthesised using the van Leusen 3-component reaction. The ability of each compound to inhibit SETD7 was evaluated using a MALDI-TOF MS based assay monitoring the methylation of a synthetic histone H3 peptide.

140 4.1. Introduction

SET domain containing lysine methyltransferase 7 (SETD7), also known as SET7, SET7/9 or KMT7, is a histone lysine methyltransferase (HKMT) involved in human gene regulation. SETD7’s main function was initially identified to catalyse the transfer of the methyl group from S‑adenosylmethionine (SAM) to lysine 4 on histone tail 3 (H3K4, Scheme 1a).[1] The ε‑amino group of the lysine side chain can accommodate up to three methyl groups, however SETD7 is known to only monomethylate H3K4.[2] The resulting methylation mark (H3K4me1) prevents the condensation of chromatin, thereby activating transcription.[3] However, SETD7’s methyltransferase activity is not limited to the methylation of histones. Since its discovery, a range of non-histone substrates have been identified, many of which are involved in important cellular processes. For example, transcription factor FOX03 is methylated by SETD7 at lysine 271, modulating its stability and activity.[4] Other non-histone substrates include tumour suppressor p53,[5] TAF10,[6] and many others.[7-9] As a result, aberrant activity of SETD7 has been linked to the development and maintenance of several types of cancer, and vascular dysfunction in patients with type 2 diabetes.[10, 11]

a) b) H 4 H H A CF3 N H H H Me CF3 N N SETD7 F SAM O O S O N O H N N N HN N H H N N O O C F B H3K4 H3K4me1 (R)-PFI-2

Scheme 1. a) SETD7-catalysed monomethylation of lysine 4 on histone tail 3; b)(R)‑PFI‑2 (left) and its 1,5-disubstituted imidazole analogue (right).

For this reason, it is of significant importance that selective inhibitors targeting SETD7 are continuously developed and improved.[12] These compounds can be used as chemical probes to study the methyltransferase activity invitro as well as in vivo, and may serve as a starting point for the development of novel therapeutic agents targeting diseases or disorders related to abnormal SETD7 activity.[13] In 2014, (R)‑PFI‑2, the first potent and highly selective inhibitor of SETD7, was reported by the structural genomics consortium (SGC) (Scheme 1b).[14] (R)-PFI-2 was identified by a high-throughput screening ofa compound library containing approximately 150,000 drug-like molecules, and was found to be a histone competitive inhibitor of SETD7 with an excellent IC50 of 2.0 nM.

141 Furthermore, the (S)‑enantiomer is 5000-fold less potent, and(R)-PFI-2 is selective for SETD7 over 18 other human methyltransferases. Two years later, Takemodo et al. reported cyproheptadine, also known as dibenzosuberene, to be a SETD7 inhibitor with [15] an IC50 of 1.0 µM. The substrate in this study was estrogen receptor (ER) α, which is monomethylated by SETD7 at position K302, resulting in activation of its transcriptional activities. It was shown that cyproheptadine is also active in cellular assays, blocking tumour growth in estrogen-dependent breast cancer cells. In subsequent studies, the same group performed structure-activity studies on cyproheptadine and its analogues,

and found an increased ability to inhibit SETD7 for 2‑hydroxycyproheptadine (IC50 = 0.41 µM).[16, 17] Besides (R)‑PFI‑2 and cyproheptadine and analogues, which are all inhibitors that compete with the histone binding site, also several inhibitors targeting the SAM binding site have been reported in literature. These include SAM analogues in which the sulfur atom is replaced by several alkylamine functionalities,[18] and various

other small molecule inhibitors such as DC-S238 (IC50 = 4.88 µM), DC-S239 (IC50 = 4.59 [19, 20] µM), and DC-S303 (IC50 = 1.1 µM).

In chapters 2 and 3 of this thesis we carried out structure-activity relationship (SAR) studies on (R)‑PFI‑2 and its analogues.[21] In these chapters variations in three distinct parts of (R)-PFI-2 were introduced: i) the amino acid side chain (Scheme 1b, part A), 4 ii) the pyrrolidine amide part (Scheme 1b, part B), and iii) the tetrahydroisoquinoline + moiety (Scheme 1b, part C). In chapter 2 it was found that the NH2 of the tetrahydroisoquinoline part was a very important contributor to (R)‑PFI‑2’s excellent potency, due to a salt bridge formation with Asp256 and a hydrogen bonding with His252 of SETD7. In chapter 3, extensive modifications on the pyrrolidine amide part (Scheme 1b, part B) were investigated, however, none of the alterations improved the

potency. Only the sulfonamide core of (R)‑PFI‑2 (-SO2NH-), depicted in red in Scheme 1b, was left unmodified in the preceding chapters. In this chapter, we explore whether a replacement of the sulfonamide moiety by a 1,5‑disubstituted imidazole bioisostere leads to a series of novel (R)-PFI-2 analogues that act as SETD7 inhibitors.

Computational chemistry can provide valuable support in the different stages of drug discovery. For example, structure-guided virtual screenings have been extensively used for identification of new hits, optimisation of a hit to improve binding affinity and selectivity, and also subsequent optimisation of properties of a lead compound such as solubility and cell-permeability.[20, 22] This type of modelling is also referred to as computer-aided drug design (CADD). In this chapter, we have used Cresset’s SPARK and TORCH, software designed for computational scaffold hopping, to identify novel (R)-PFI-2 analogues in which the sulfonamide moiety is replaced.[23]

142 4.2. Results and discussion

We started our investigations with a ligand-based similarity screening of(R)-PFI-2’s sulfonamide moiety using Cresset’s SPARK and TORCH.[24] The sulfonamide core of the (R)-PFI-2 scaffold that is selected for replacement is depicted in red in Scheme 1b. SPARK software assigns field points based on the steric and electronic nature of the original ligand. The field point representation of (R)‑PFI-2 is depicted in Figure 1a. SPARK then searches in several databases for functional groups, which resemble the initial scaffold in terms of these field points. The search was performed in SPARK internal databases: ‘Commercial’ (Commercially available compounds and reagents), ‘ChEMBL’ (reported in literature), ‘VEHICLe’ (theoretical rings), and include ‘VeryCommon’ (fragments appearing in more than 650 molecules) up to ‘ExteremelyRare’ (fragments which appear in 3-4 molecules). The SPARK run returned 500 candidate bioisosteres; based on internal scoring functions, careful visual inspection, evaluation of synthetic accessibility, and docking of potential bioisosteres in SETD7’s crystal structure (PDB id: 4JLG) using MOE software (Figures 3d-f),[25, 26] we decided to synthesise analogues in which the sulfonamide core is replaced by a 1,5-disubstituted imidazole functionality (Scheme 1b). The field point representations of (R)‑PFI‑2 imidazole analogue and an overlay with the original (R)‑PFI‑2 are depicted in Figures 1b and c. 4

Figure 1. Field representation of a) (R)-PFI-2; b) an imidazole analogue; c) an overlay of (R)-PFI-2 and its imidazole analogue: blue field points represent negative charges, red field points positive charges, yellow field points represent van der Waals minima, and orange field points represent hydrophobicity. The size of the points is related to the strength of the interaction; d) crystal structure of SETD7 in complex with (R)- PFI-2; e) docked representation of imidazole analogue; f) overlay of d) and e); Oxygen atoms are shown in red, nitrogen in blue, and sulfur in yellow. Figures d-f were prepared with PyMOL visualisation software.[24]

143 Table 1. Optimisation of the van Leusen reaction for the synthesis of (R)-PFI-2 imidazole analogues.a

O TosMIC, Base + O O O H H N N N 2 Solvent, Solvent, conditions N rt, 2h N N N

b Entry Solvent ([M]) Equiv TosMIC Base (equiv) T2 (°C) t2 (h) Conversion (%)

1 DMF (0.5) 1.2 K2CO3 (1.5) rt 20 3

2 DMF (0.5) 1.2 K2CO3 (1.5) 50 20 7

3 THF (0.5) 1.2 K2CO3 (1.5) 50 20 1

4 MeOH (0.5) 1.2 K2CO3 (1.5) 50 20 2

5 EtOH (0.5) 1.2 K2CO3 (1.5) 50 20 10 6 EtOH (0.5) 1.2 KOtBu (1.5) 50 20 - t 7 EtOH (0.5) 1.2 BuNH2 (1.5) 50 20 -

8 EtOH (0.5) 1.2 Et3N (1.5) 50 20 - 9 EtOH (0.5) 1.2 piperazine (1.5) 50 20 < 1 c 10 EtOH (0.5) 3.0 K2CO3 (3.0) 50 20 35

11 EtOH (0.5) 3.0 K2CO3 (3.0) 50 20 47

12 EtOH (0.5) 3.0 K2CO3 (3.0) 75 20 54

13 EtOH (0.25) 3.0 K2CO3 (3.0) 75 20 45

14 EtOH (0.5) 3.0 K2CO3 (1.0) 75 20 100 a) Conditions:i ) aldehyde (0.125 mmol, 1.0 equiv), amine (Compound S2, 0.15 mmol, 1.2 equiv), solvent ([M]), rt, 2 h, then ii) TosMIC, base, T , t ; b) Conversion of imine to desired imidazole product, determined by LCMS; 4 2 2 c) TosMIC added in two portions.

A straightforward approach to the synthesis of 1,5-disubsituted imidazoles is based on the van Leusen imidazole synthesis, also known as the van Leusen three-component reaction (vL-3CR, see scheme in Table 1).[27] This reaction allows for the synthesis of imidazoles starting from tosylmethyl isocyanide (TosMIC) and aldimines. The latter can be prepared in situ by condensation of aldehydes and amines. In literature, the van Leusen reaction typically proceeds well at rt in polar solvents such as DMF or MeOH in t [28, 29] the presence of K2CO3 or BuNH2 as base. Our model system for the optimisation of the van Leusen reaction consists of benzaldehyde and D‑phenylalanine pyrrolidine amide (Table 1). When we applied conditions commonly found in literature to our substrates, we only observed the formation of traces (3% conversion with respect to the imine intermediate) of the desired product (Table 1, entry 1). A slight increase of product formation was observed when the temperature of the second step of the reaction was increased to 50 °C (Table 1, Entry 2). We then tested several solvents, and found that neither THF nor MeOH were suitable (Table 1, entries 3 and 4), however, when the reaction was performed in EtOH, 10% conversion into the desired product was observed (Table 1, entry 5). It is known that the stability of TosMIC strongly depends on

144 the counterion of the base that is used when it is deprotonated.[27] We therefore tested several bases, and found that with a stronger base, such as KOtBu, the reaction led to no product (Table 1, entry 6). Also, when amine bases were used, no product formation was observed (Table 1, entries 7‑9). With the instability of the K2CO3-TosMIC salt in mind, we performed an experiment in which TosMIC was added in 2 portions (2 × 1.5 equiv), and we were pleased to find that this led to 35% conversion into the desired imidazole product (Table 1, entry 10). We then performed the same experiment but added an excess (3.0 equiv) of TosMIC from the start, thereby increasing the conversion to 47% (Table 1, entry 11). The temperature of the second step in the reaction was then increased to 70 °C, resulting in a slightly higher 54% conversion (Table 1, entry 12). It was observed that in particular with the formation of the intermediate imine the reaction mixture almost solidified, therefore we attempted to lower the concentration to 0.25 M; unfortunately, this led to a decreased conversion of 45% (Table 1, entry 13).

However, we were very pleased to find that when the amount of K2CO3 was decreased to 1.0 equiv, the imine was fully consumed (Table 1, entry 14). Notably, under the optimal conditions in Table 1, entry 14, all imine intermediate had reacted, however, several unidentified side-products were also formed. With the optimal conditions in hand (Table 1, entry 14), a library of 56 analogues was synthesised in a parallel fashion. A broad range of aldehydes were employed in the reaction to obtain different 4 substitutions on the 5 position of the imidazole. An overview of the compounds that were synthesised is given in Scheme 2.

To investigate whether the synthesised imidazoles have an ability to inhibit SETD7’s methyltransferase activity, the obtained compounds were initially screened for inhibitory activity at a fixed concentration of 500 µM. This was done as described previously by our group,[21, 30] using matrix-assisted laser desorption/ionisation time- of-flight (MALDI-TOF) mass spectrometry. In short, SETD7‑mediated methylation of a synthetic histone peptide mimic consisting of the 21 amino acids of the N-terminal tail of histone 3 (H3K4 peptide with m/z 2256.0 Da) was monitored by MALDI-TOF MS. In the absence of compound, i.e. just SETD7 (200 nM), H3K4 histone peptide (10 μM), and methyl donor SAM (16 μM) in 50 mM glycine pH 8.8 as assay buffer, a mass increase of +14 Da corresponding to monomethylation was observed (Figure 2a, H3K4me1, m/z 2270.0 Da). Inhibition could be observed as a decrease or complete disappearance of the H3K4me1 peak in the MALDI-TOF MS spectrum.

145 4

Scheme 2. Library of 1,5-disubstituted imidazole analogues of (R)-PFI-2. Conditions: i) aldehyde (0.125 mmol, 1.0 equiv), amine (S4, 0.15 mmol, 1.2 equiv) in EtOH (250 µL), rt, 2 h, then ii) TosMIC (0.375 mmol, 3.0 equiv),

K2CO3 (0.125 mmol, 1.0 equiv), 75 °C, 20 h.

146 Figure 2. Representative MALDI-TOF MS data showing H3K4 methylation in the presence of 200 nM SETD7, 21-mer H3K4 peptide (10 μM), SAM (16 μM) in 50 mM glycine pH 8.8 containing 5% DMSO (v/v) after 1 h at 37 °C; a) no compound present; b) 500 μM of compound 1; c) 500 μM compound 6; d) 500 μM compound 42.

It was expected that in particular those analogues that contain the imidazole core, and thus closely resemble (R)‑PFI‑2, would have an ability to inhibit SETD7 to a certain extent. Unfortunately, we found that none of the analogues 1–17, which were predicted to form similar interactions with SETD7 as (R)‑PFI‑2, i.e. a salt bridge with Asp256 and/ or H-bonding with His252, did not inhibit SETD7 within detection limits. Representative MALDI-TOF MS data for compounds 1 and 6 can be seen in Figures 2b-c: no decrease of the H3K4me1 (m/z = 2270 Da) methylation mark was observed. Various analogues bearing (heteroatom-containing) bicyclic R-groups were also unable to inhibit SETD7 (Scheme 2, 18‑29). Analogues with (hetero)aromatic R-groups, including pyridines and pyrimidines, bearing different substitutions, also showed no inhibition of SETD7’s methyltransferase activity (Scheme 2,30 –37). Furthermore, no inhibition was observed for those compounds where the R-group is a (methyl-substituted) pyrrole, pyrazole, or imidazole (Scheme 2, 38–47). For example in the MALDI-TOF MS data for compound 42, no inhibition was observed (Figure 2d). Unfortunately, other compounds with a variety of substitutions yielded no active SETD7 inhibitors (Scheme 2, 48–56).

147 4.3. Conclusion

In summary, we used a computational approach to identify potential bioisosteres of the sulfonamide core of (R)-PFI-2. This was achieved using SPARK software tool for scaffold hopping, developed by Cresset. Based on the visual inspection, docking studies, and evaluation of synthetic accessibility, the 1,5-disubstituted imidazole was chosen from the obtained SPARK results. To synthesise the desired library of structurally diverse (R)‑PFI‑2 imidazole analogues from readily available starting materials, the van Leusen 3-component reaction was employed. The van Leusen reaction uses amines and aldehydes, which are condensed in situ to the corresponding aldimine, and subsequent

addition of TosMIC and K2CO3 as a base results in the formation of 1,5-disubstituded imidazoles. Initially, the van Leusen reaction was optimised for the substrates used in this work, and it was found that the reaction proceeded well in EtOH at 75 °C. Furthermore, it proved to be important to use an excess of TosMIC reagent and only

one equivalent of K2CO3. Using the optimised van Leusen synthesis a library of 56 novel analogues was synthesised. These were evaluated for in vitro inhibitory activity against human SETD7 using a MALDI-TOF based mass spectrometry assay. Unfortunately, none of the compounds inhibited SETD7’s methyltransferase activity. In future studies, other potential bioisosteres that were generated by SPARK could be synthesised and 4 evaluated as inhibitors of histone lysine methyltransferase SETD7.

4.4. Supporting information

4.4.1. General remarks All solvents and reagents were purchased from commercial suppliers and used without further purification. Standard syringe techniques were applied for the transfer of dry solvents. Flash chromatography was carried out using a Reveleris flash chromatography system in the indicated solvents. Thin layer chromatography (TLC) analysis was

performed on glass backed silica sheets (Merck TLC Silica 60 F254) and visualised by UV

light at 254 nm and/or staining with potassium permanganate (KMnO4) or ninhydrin. 1H and 13C Nuclear magnetic resonance (NMR) spectroscopy data were recorded at

ambient temperature on Bruker DMX-400 and AV-400 instruments (400 MHz) in CDCl3 1 or CD3OD solutions. H NMR chemical shifts are reported as δ in units of parts per million (ppm) relative to tetramethylsilane (TMS, δ 0.00 ppm) as the internal standard. Multiplicities are given as: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), app (apparent). Coupling constants are reported asJ -values in Hertz (Hz). LCMS (MS, m/z) were recorded on either i) an Agilent 1260 LC system containing a

148 Waters XSelectTM CSH C18 (30x2.1mm, 3.5 m) column using a 5 to 98% gradient of

MeCN/10 mM NH4HCO3 in water (pH = 9.5) at a flowrate of 1.0 mL/min at 25 °C, and masses detected with an Agilent G6130B LCMS system (ESI), or ii) a Waters ACQUITY IClass UPLC system containing a Waters XSelect CSH C18 (50x2.1mm, 2.5m) column using a 5 to 98% gradient of MeCN/10 mM NH4HCO3 in water (pH = 9.5) at a flowrate of 0.6 mL/min at 40 °C, and masses detected with a Waters SQD2 MS system (ESI). Preparative HPLC/MS was carried out on an Agilent Technologies 1200 preparative LC system containing a preparative Waters XSelect (C18, 100x30mm, 10µ) column using a gradient of MeCN/10 mM NH4HCO3 in water (pH = 9.5), with an Agilent Technologies G6130B Quadrupole LC-MS mass detector. Fractions were collected based on MS (ESI+) and DAD (220-320nm).

4.4.2. Characterisation of compounds Boc-D-phenylalanine pyrrolidine amide (S1): Boc-D-phenylalanine (5.31 g, 20.0 mmol) was dissolved in DMF (dry, 50 mL), to this was added DIPEA (4.2 mL, 24.0 mmol) and HATU (6.7 g, 20.0 mmol) and the reaction was stirred at rt for 1 h. Subsequently, pyrrolidine (2.0 mL, 24.0 mmol) was added and the reaction was stirred at rt for an additional 19 h. The reaction mixture was poured onto water (250 mL) 4 and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with satd aq NaHCO3 (250 mL), water (250 mL), and brine (250 mL), the organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified using EtOAc/n-heptane (10-20%) and afforded Boc-D- phenylalanine pyrrolidine amide (S1, 5.35 g, 84% isolated yield) as a yellow solid. 1H

NMR (400 MHz, CDCl3) δ 7.30 – 7.16 (m, 5H), 5.42 (d, J = 8.8 Hz, 1H), 4.59 (td, J = 8.8, 5.9 Hz, 1H), 3.49 – 3.22 (m, 3H), 3.06 – 2.88 (m, 2H), 2.62 – 2.49 (m, 1H), 1.83 – 1.47 (m, 4H), 1.42 (s, 9H); LCMS (ESI) m/z 319.2 [M+H]+. Data are in accordance to that previously reported.[21]

D-Phenylalanine pyrrolidine amide (S2): Boc-D-phenylalanine pyrrolidine amide (Compound S1, 5.35 g, 16.8 mmol) was dissolved in DCM (40 mL) and to this was added TFA (10 mL). The reaction mixture was stirred at rt until TLC analysis (EtOAc:n-heptane = 1:1) showed that the reaction was complete (1.5 h). The solvent was evaporated and the crude product redissolved in DCM (150 mL), washed

with satd aq NaHCO3 (150 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford crude afford D‑phenylalanine pyrrolidine amide (S2, 3.09 g, 14.15 mmol, 84% yield) as a pale orange oil which crystallised over time. The

149 1 crude amine was used without further purification. H NMR (400 MHz, CDCl3) δ 7.32 – 7.17 (m, 5H), 3.77 (t, J = 7.3 Hz, 1H), 3.51 – 3.42 (m, 1H), 3.42 – 3.29 (m, 2H), 2.96 (dd, J = 13.1, 7.5 Hz, 1H), 2.83 (dd, J = 13.1, 7.0 Hz, 1H), 2.80 – 2.70 (m, 1H), 2.30 (d, J = 16.7 Hz, 2H), 1.84 – 1.56 (m, 6H). ; LCMS (ESI) m/z 219.1 [M+H]+. Data are in accordance to that previously reported.[21]

Boc-3-(trifluoromethyl)-D-phenylalanine pyrrolidine amide (S3): Boc-3-(trifluoromethyl)-D-phenylalanine (5.00 g, 15 mmol) was dissolved in DMF (dry, 37.5 mL), to this was added DIPEA (3.14 mL, 18.0 mmol) and HATU (5.70 g, 15.0 mmol) and the reaction was stirred at rt for 1 h. Subsequently, pyrrolidine (1.5 mL, 18.00 mmol) was added and the reaction was stirred at rt for an additional 19 h. The reaction mixture was poured onto water (250 mL) and extracted with EtOAc (3 × 100 mL).

The combined organic layers were washed with satd aq NaHCO3 (250 mL), water (250 mL), and brine (250 mL), the organic layer was dried over anhydrous

Na2SO4 and concentrated in vacuo. The crude product was purified using EtOAc/n-heptane (10-20%) and afforded Boc-3-(trifluoromethyl)-D-phenylalanine pyrrolidine amide (S3, 4.8 g, 12.3 mmol, 82 % yield) as a white solid. 1H NMR (400 MHz, CDCl ) δ 7.56 – 7.36 (m, 4H), 5.42 (d, J = 8.8 Hz, 1H), 4.60 (q, J = 7.6 Hz, 1H), 3.52 – 3.25 4 3 (m, 3H), 3.04 (d, J = 7.3 Hz, 2H), 2.62 (dt, J = 9.7, 6.8 Hz, 1H), 1.85 – 1.59 (m, 4H), 1.41 (s, 9H); LCMS (ESI) m/z 387.1 [M+H]+. Data are in accordance to that previously reported.[21]

3-(Trifluoromethyl)-D-phenylalanine pyrrolidine amide (S4): Boc-3-(trifluoromethyl)-D-phenylalanine pyrrolidine amide (S3, 4.76 g, 12.32 mmol) was dissolved in DCM (40 mL) and to this was added TFA (10 mL). The reaction mixture was stirred at rt until TLC analysis (EtOAc:n- heptane = 1:1) showed the reaction was complete (1.5 h). The solvent was evaporated and the crude product redissolved in DCM (150 mL), washed

with satd aq NaHCO3 (150 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford crude 3-(trifluoromethyl)-D-phenylalanine pyrrolidine amide (S4, 3.52 g, 12.29 mmol, >99% yield), which was used without further 1 purification. H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 1.5 Hz, 1H), 7.45 (bs, 1H), 7.43 – 7.39 (m, 2H), 3.72 (app t, J = 7.3 Hz, 1H), 3.55 – 3.42 (m, 1H), 3.42 – 3.31 (m, 1H), 3.01 (dd, J = 13.2, 7.6 Hz, 1H), 2.87 (dd, J = 13.2, 6.9 Hz, 1H), 2.81 – 2.72 (m, 1H), 1.91 – 1.58 (m, 6H); LCMS (ESI) m/z 287.1 [M+H]+, purity 96.4%. Data are in accordance to that previously reported.[21]

150 General Procedure: van Leusen synthesis of imidazoles

An LCMS vial (1.5 mL) equipped with a magnetic stir bar was charged with aldehyde (0.125 mmol, 1.0 equiv) and to this was added 250 µL of a 0.6 M stock solution of compound S4 (0.150 mmol of total amine, 1.2 equiv). The reaction was stirred at rt for 2 h, after which LCMS showed whether the imine has formed. Subsequently, 1-((isocyanomethyl)sulfonyl)-4-methylbenzene (TosMIC, 73.2 mg, 0.375 mmol, 3.0 equiv) and anhydrous potassium carbonate (17.3 mg, 0.125 mmol, 1.0 equiv) were added and the reaction was left to stir at 75 °C for an additional 16 h. The solvent was evaporated and the crude product was purified by preparative HPLC.

(R)-1-(pyrrolidin-1-yl)-2-(5-(1,2,3,4-tetrahydroisoquinolin-6-yl)-1H-imidazol-1-yl)-3-(3- (trifluorometh-yl)phenyl)propan-1-one (1): 4 Prepared according to the general procedure using tert-butyl 6-formyl- 3,4-dihydroisoquinoline-2(1H)-carboxylate (33 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC affordedS5 (11.6 mg, 16% isolated yield). LCMS (ESI) m/z 569.2 [M+H]+, purity 54%. Compound S5 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 3.2 mg 1 (74% isolated yield from S5) of the desired imidazole 1. H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 1.1 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.25 (s, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 6.97 (d, J = 1.1 Hz, 1H), 6.83 (dd, J = 7.9, 1.8 Hz, 1H), 6.73 (d, J = 1.8 Hz, 1H), 4.90 (dd, J = 8.1, 6.9 Hz, 1H), 4.05 (s, 2H), 3.55 (dd, J = 13.6, 8.1 Hz, 1H), 3.48 – 3.34 (m, 2H), 3.26 (dd, J = 13.6, 6.9 Hz, 1H), 3.16 (t, J = 6.0 Hz, 2H), 2.84 – 2.63 (m, 4H), 1.80 – 1.60 (m, 4H); LCMS (ESI) m/z 443.1 [M+H]+, purity 98.4%.

151 (R)-1-(pyrrolidin-1-yl)-2-(5-(1,2,3,4-tetrahydroisoquinolin-7-yl)-1H-imidazol-1-yl)-3-(3- (trifluorometh-yl)phenyl)propan-1-one (2): Prepared according to the general procedure using tert-butyl 7-formyl- 1,2,3,4-tetrahydroisoquinoline-2-carboxylate (32.7 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded S6 (5.2 mg, 7% isolated yield). 1H NMR

(400 MHz, CDCl3) δ 8.06 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.22 – 7.17 (m, 2H), 7.14 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 1.1 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H), 4.89 (t, J = 7.5 Hz, 1H), 4.51 (s, 2H), 3.75 – 3.59 (m, 2H), 3.53 (dd, J = 13.5, 7.8 Hz, 1H), 3.48 – 3.36 (m, 2H), 3.32 – 3.19 (m, 1H), 2.86 (t, J = 5.9 Hz, 2H), 2.81 – 2.69 (m, 2H), 1.85 – 1.60 (m, 4H), 1.50 (s, 9H); LCMS (ESI) m/z 458.1 [M+H]+, purity 94.1%. Compound S6 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 2.4 mg (56% isolated yield from S6) of the desired 1 imidazole 2. H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 1.1 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.25 (s, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 6.97 (d, J = 1.1 Hz, 1H), 6.82 (dd, J = 7.8, 1.8 Hz, 1H), 6.65 (d, J = 1.8 Hz, 1H), 4.90 (dd, J = 8.1, 6.9 Hz, 1H), 3.95 (d, J = 3.8 Hz, 2H), 3.55 (dd, J = 13.6, 8.1 Hz, 1H), 3.46 – 3.37 (m, 2H), 3.26 (dd, J = 13.6, 6.9 Hz, 1H), 3.15 (t, J = 6.0 Hz, 2H), 2.83 (t, J = 6.0 Hz, 2H), 2.79 – 2.67 4 (m, 2H), 1.78 – 1.68 (m, 4H); LCMS (ESI) m/z 469.1 [M+H]+, purity 99.4%.

(R)-2-(5-(1H-indol-5-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1one (3): Prepared according to the general procedure using indole-5- carboxaldehyde (18.1 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 28.6 mg (51% isolated yield) of the desired imidazole 3. 1H

NMR (400 MHz, CDCl3) δ 9.37 (s, 1H), 8.10 (d, J = 1.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.37 – 7.28 (m, 4H), 7.23 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 1.0 Hz, 1H), 6.89 (dd, J = 8.3, 1.6 Hz, 1H), 6.57 – 6.48 (m, 1H), 4.97 (dd, J = 8.6, 6.5 Hz, 1H), 3.58 (dd, J = 13.4, 8.6 Hz, 1H), 3.44 – 3.28 (m, 3H), 2.66 – 2.56 (m, 1H), 2.56 – 2.38 (m, 1H), 1.76 – 1.43 (m, 4H); LCMS (ESI) m/z 453.1 [M+H]+, purity 97.5%.

(R)-2-(5-(1H-indol-6-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (4): Prepared according to the general procedure using 1H-indole-6-carbaldehyde (18.0 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by

152 preparative HPLC afforded the 26.2 mg (46% isolated yield) of desired 1 imidazole 4. H NMR (400 MHz, CDCl3) δ 8.95 (bs, 1H), 8.11 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.39 – 7.29 (m, 3H), 7.21 (d, J = 7.8 Hz, 1H), 7.00 (d, J = 11.6 Hz, 2H), 6.82 (dd, J = 8.1, 1.5 Hz, 1H), 6.61 – 6.55 (m, 1H), 4.98 (dd, J = 8.5, 6.7 Hz, 1H), 3.55 (dd, J = 13.4, 8.5 Hz, 1H), 3.41 – 3.27 (m, 3H), 2.63 – 2.55 (m, 1H), 2.54 – 2.40 (m, 1H), 1.75 – 1.43 (m, 4H); LCMS (ESI) m/z 453.1 [M+H]+, purity 98.8%.

(R)-2-(5-(4-(2-aminoethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl) propan-1-one (5): Prepared according to the general procedure using tert-butyl 4-formylphenethylcarbamate (31.2 mg, 0125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded S7 (8.4 mg, 12% isolated yield). 1H NMR

(400 MHz, CDCl3) δ 8.07 (d, J = 1.0 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.26 (s, 1H), 7.23 – 7.16 (m, 3H), 7.00 – 6.93 (m, 2H), 4.88 (dd, J = 8.1, 6.9 Hz, 1H), 4.58 (s, 1H), 3.54 (dd, J = 13.5, 8.1 Hz, 1H), 3.45 – 3.32 (m, 5H), 3.26 (dd, J = 13.5, 6.9 Hz, 1H), 2.83 (t, J = 7.2 Hz, 2H), 2.71 – 2.63 (m, 2H), 1.83 – 1.62 (m, 4H), 1.44 (s, 9H); LCMS (ESI) m/z 457.2 [M+H]+, 4 purity 84.5%. Compound S7 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 4.6 mg (67% isolated yield from S7) the desired imidazole 5. 1H NMR (400

MHz, CDCl3) δ 8.06 (d, J = 1.0 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.25 – 7.17 (m, 3H), 7.00 – 6.94 (m, 3H), 4.88 (dd, J = 8.2, 6.8 Hz, 1H), 3.55 (dd, J = 13.5, 8.2 Hz, 1H), 3.44 – 3.35 (m, 2H), 3.26 (dd, J = 13.5, 6.8 Hz, 1H), 3.00 (t, J = 7.0 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H), 2.73 – 2.62 (m, 2H), 1.76 – 1.66 (m, 4H); LCMS (ESI) m/z 457.1 [M+H]+, purity 98.9%.

(R)-2-(5-(3-(aminomethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl) propan-1-one (6): Prepared according to the general procedure using tert-butyl 3-formylbenzylcarbamate (29.4 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC affordedS8 (7 mg, 10% isolated yield). LCMS (ESI) m/z 543.1 [M+H]+, purity 58.4%. Compound S8 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 2.1 mg (64% isolated yield from S8) of the desired imidazole 6. 1H NMR (400 MHz,

153 CDCl3) δ 8.09 (d, J = 1.1 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.40 – 7.32 (m, 3H), 7.28 (s, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.07 (s, 1H), 7.02 (d, J = 1.1 Hz, 1H), 6.98 – 6.93 (m, 1H), 4.93 (dd, J = 8.4, 6.7 Hz, 1H), 3.88 (s, 2H), 3.57 (dd, J = 13.5, 8.4 Hz, 1H), 3.47 – 3.33 (m, 2H), 3.27 (dd, J = 13.5, 6.7 Hz, 1H), 2.78 – 2.60 (m, 2H), 1.73 – 1.59 (m, 4H); LCMS (ESI) m/z 443.1 [M+H]+, purity 98.4%.

(R)-2-(5-(4-(aminomethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl) propan-1-one (7): Prepared according to the general procedure using tert-butyl N-(4- formylbenzyl)carbamate (29.4 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC 1 afforded S9 (7.4 mg, 11% isolated yield). H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.29 (d, J = 8.0 Hz, 2H), 7.25 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.01 – 6.97 (m, 3H), 4.94 (bs, 1H), 4.88 (dd, J = 8.1, 6.9 Hz, 1H), 4.35 (d, J = 6.2 Hz, 2H), 3.54 (dd, J = 13.5, 8.1 Hz, 1H), 3.47 – 3.34 (m, 2H), 3.26 (dd, J = 13.5, 6.9 Hz, 1H), 2.75 – 2.61 (m, 2H), 1.80 – 1.61 (m, 4H), 1.47 (s, 9H); LCMS (ESI) m/z 543.0 [M+H]+, purity 96.1%. Compound S9 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 4.2 4 1 mg (70% isolated yield from S9) of the desired imidazole 7. H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.39 – 7.31 (m, 3H), 7.24 (s, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.04 – 6.98 (m, 3H), 4.89 (t, J = 7.5 Hz, 1H), 3.93 (s, 2H), 3.55 (dd, J = 13.4, 8.1 Hz, 1H), 3.46 – 3.35 (m, 2H), 3.25 (dd, J = 13.4, 6.8 Hz, 1H), 2.78 – 2.63 (m, 2H), 1.78 – 1.66 (m, 4H); LCMS (ESI) m/z 443.1 [M+H]+, purity 98.4%.

(R)-2-(5-(3-aminophenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (8): Prepared according to the general procedure using (3-formyl- phenyl)-carbamic acid tert-butyl ester (27.7 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded S10 (8.3 mg, 13% isolated yield). LCMS (ESI) m/z 529.1 [M+H]+, purity 58.2%. Compound S10 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 1.4 mg (36% isolated yield from S10) of the desired imidazole 8. 1H

NMR (400 MHz, CDCl3) δ 8.09 (d, J = 1.1 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 1.1 Hz, 1H), 6.71 (ddd, J = 7.8, 2.4, 1.0 Hz, 1H), 6.48 (dt, J = 7.8, 1.6, 1.0 Hz, 1H), 6.30 (t, J = 2.4,

154 1.6 Hz, 1H), 4.98 (dd, J = 8.3, 6.8 Hz, 1H), 3.58 (dd, J = 13.5, 8.3 Hz, 1H), 3.46 – 3.35 (m, 2H), 3.30 (dd, J = 13.5, 6.8 Hz, 1H), 2.83 – 2.62 (m, 2H), 1.83 – 1.50 (m, 4H); LCMS (ESI) m/z 429.3 [M+H]+, purity 98.8%.

(R)-2-(5-(4-aminophenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (9): Prepared according to the general procedure using (4-Formyl-phenyl)- carbamic acid tert-butyl ester (27.7 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded S11 (20.2 mg, 30.6% isolated yield). 1H

NMR (400 MHz, CDCl3) δ 8.10 (d, J = 1.0 Hz, 1H), 7.50 (d, J = 7.7 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.28 (s, 1H), 7.21 (d, J = 7.7 Hz, 1H), 6.97 (d, J = 1.0 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 6.88 (s, 1H), 4.89 (dd, J = 8.1, 6.9 Hz, 1H), 3.55 (dd, J = 13.5, 8.1 Hz, 1H), 3.44 – 3.38 (m, 2H), 3.27 (dd, J = 13.5, 6.9 Hz, 1H), 2.83 – 2.70 (m, 1H), 2.71 – 2.62 (m, 1H), 1.79 – 1.61 (m, 4H), 1.54 (s, 9H); LCMS (ESI) m/z 529.1 [M+H]+, purity >99.9%. Compound S11 was dissolved in 1.0 mL DCM/TFA (4:1 v/v) and the reaction was left to stir at rt for 2 h. The crude product was purified by preparative HPLC and afforded 9.5 mg (60% isolated yield from S11) of the desired imidazole 9. 1H NMR (400 MHz, CDCl ) δ 8.06 (s, 1H), 7.50 (d, J = 7.7 3 4 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 6.94 (s, 1H), 6.80 (d, J = 8.3 Hz, 2H), 6.67 (d, J = 8.3 Hz, 2H), 4.86 (dd, J = 8.4, 6.7 Hz, 1H), 3.55 (dd, J = 13.4, 8.4 Hz, 1H), 3.44 – 3.33 (m, 2H), 3.25 (dd, J = 13.4, 6.7 Hz, 1H), 2.79 – 2.56 (m, 2H), 1.77 – 1.55 (m, 4H); LCMS (ESI) m/z 429.1 [M+H]+, purity 98.4%.

(R)-2-(5-(4-(dimethylamino)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)propan-1-one (10): Prepared according to the general procedure using 4-(dimethylamino) benzaldehyde (19 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 20.6 mg (35% isolated yield) of the desired imidazole 10. 1H

NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.31 – 7.22 (m, 2H), 6.95 – 6.87 (m, 3H), 6.73 – 6.67 (m, 2H), 4.87 (dd, J = 8.6, 6.3 Hz, 1H), 3.57 (dd, J = 13.4, 8.6 Hz, 1H), 3.46 – 3.31 (m, 2H), 3.23 (dd, J = 13.4, 6.3 Hz, 1H), 2.99 (s, 6H), 2.84 – 2.62 (m, 2H), 1.80 – 1.58 (m, 4H); LCMS (ESI) m/z 457.1 [M+H]+, purity 97.9%.

155 (R)-2-(5-(4-(diethylamino)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)propan-1-one (11): Prepared according to the general procedure using 4-(diethylamino) benzaldehyde (22.2 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 36.1 mg (59.6% isolated yield) of the desired imidazole 11. 1 H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.25 (d, J = 7.8 Hz, 1H), 6.93 (s, 1H), 6.88 (d, J = 8.7 Hz, 2H), 6.64 (d, J = 8.7 Hz, 2H), 4.88 (dd, J = 8.8, 6.1 Hz, 1H), 3.58 (dd, J = 13.4, 8.8 Hz, 1H), 3.49 – 3.28 (m, 6H), 3.23 (dd, J = 13.4, 6.1 Hz, 1H), 2.81 – 2.63 (m, 2H), 1.80 – 1.56 (m, 4H), 1.18 (t, J = 7.0 Hz, 6H); LCMS (ESI) m/z 485.2 [M+H]+, purity 95.5%.

(R)-2-(5-(3-hydroxyphenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (12): Prepared according to the general procedure using 3-hydroxybenzaldehyde (15.3 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 3.4 mg (6.3% isolated yield) of the desired imidazole 4 1 12. H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 1.0 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.31 (bs, 1H), 7.23 (d, J = 7.7 Hz, 2H), 6.97 (d, J = 1.0 Hz, 1H), 6.95 – 6.89 (m, 1H), 6.68 – 6.64 (m, 1H), 6.53 (dt, J = 7.7, 1.1 Hz, 1H), 4.99 (dd, J = 8.9, 6.4 Hz, 1H), 3.53 (dd, J = 13.2, 8.9 Hz, 1H), 3.34 (h, J = 6.0 Hz, 1H), 3.25 (dd, J = 13.2, 6.4 Hz, 1H), 2.78 – 2.64 (m, 1H), 2.54 – 2.43 (m, 1H), 1.80 – 1.49 (m, 4H); LCMS (ESI) m/z 430.3 [M+H]+, purity 99.9%.

(R)-2-(5-(4-hydroxyphenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (13): Prepared according to the general procedure using 4-hydroxybenzaldehyde (12.41 µL, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 31.6 mg (59% isolated yield) of the desired imidazole 13. 1H

NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.31 (s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.00 – 6.93 (m, 3H), 6.91 – 6.86 (m, 2H), 4.94 (app. t, J = 7.7 Hz, 1H), 3.53 (dd, J = 13.3, 8.4 Hz, 1H), 3.43 – 3.34 (m, 2H), 3.29 (dd, J = 13.4, 6.8 Hz, 1H), 2.80 – 2.69 (m, 1H), 2.62 – 2.53 (m, 1H), 2.01 (s, 1H), 1.80 – 1.54 (m, 4H); LCMS (ESI) m/z 430.1 [M+H]+, purity 99.5%.

156 (R)-2-(5-(3-(hydroxymethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)propan-1-one (14): Prepared according to the general procedure using 3-(hydroxymethyl) benzaldehyde (17.0 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 1.3 mg (2.3% isolated yield) of the desired imidazole14 . 1H

NMR (400 MHz, CDCl3) δ 8.12 (d, J = 1.0 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 – 7.34 (m, 3H), 7.29 (bs, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.16 – 7.14 (m, 1H), 7.06 – 6.99 (m, 2H), 4.96 (dd, J = 8.3, 6.8 Hz, 1H), 4.74 (d, J = 0.7 Hz, 2H), 3.58 (dd, J = 13.4, 8.3 Hz, 1H), 3.50 – 3.34 (m, 2H), 3.29 (dd, J = 13.4, 6.8 Hz, 1H), 2.84 – 2.63 (m, 2H), 1.81 – 1.65 (m, 4H); LCMS (ESI) m/z 444.3[M+H]+, purity 99.0%.

(R)-2-(5-(4-(hydroxymethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)propan-1-one (15): Prepared according to the general procedure using 4-(hydroxymethyl) benzaldehyde (17.0 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 3.0 mg (5% isolated yield) of the desired imidazole 15. 1H 4 NMR (400 MHz, CDCl3) δ 8.08 (d, J = 1.0 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.35 (t, J = 7.8 Hz, 1H), 7.24 (s, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 8.1 Hz, 2H), 6.97 (d, J = 1.0 Hz, 1H), 4.89 (dd, J = 8.1, 6.9 Hz, 1H), 4.76 (s, 2H), 3.54 (dd, J = 13.5, 8.1 Hz, 1H), 3.46 – 3.34 (m, 2H), 3.26 (dd, J = 13.5, 6.9 Hz, 1H), 2.80 – 2.61 (m, 2H), 1.79 – 1.59 (m, 4H); LCMS (ESI) m/z 444.1 [M+H]+, purity 99.0%.

(R)-2-(5-(3-(methoxymethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)propan-1-one (16): Prepared according to the general procedure using 3-(methoxymethyl)benzaldehyde (18.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.4 mg (4.2% isolated yield) of the 1 desired imidazole 16. H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 1.1 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.39 – 7.31 (m, 3H), 7.27 (s, 1H), 7.22 (d, J = 7.7 Hz, 1H), 7.13 – 7.09 (m, 1H), 7.02 (d, J = 1.1 Hz, 1H), 6.98 (dt, J = 6.6, 2.1 Hz, 1H), 4.92 (dd, J = 8.4, 6.7 Hz, 1H), 4.44 (s, 2H), 3.55 (dd, J = 13.4, 8.4 Hz, 1H), 3.47 – 3.31 (m, 5H), 3.27 (dd, J = 13.4, 6.7 Hz, 1H), 2.81 – 2.70 (m, 1H), 2.68 – 2.56 (m, 1H), 1.77 – 1.62 (m, 4H); LCMS (ESI) m/z 458.1 [M+H]+, purity 94.9%.

157 (R)-2-(5-(4-(methoxymethyl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (17): Prepared according to the general procedure using 4-(methoxymethyl)benzaldehyde (18.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.7 mg (4.7% isolated yield) ofthe 1 desired imidazole 17. H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 1.0 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.29 (s, 2H), 7.22 (d, J = 7.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 1.0 Hz, 1H), 4.92 (dd, J = 8.2, 6.8 Hz, 1H), 4.51 (s, 2H), 3.58 (dd, J = 13.4, 8.2 Hz, 1H), 3.45 (s, 5H), 3.29 (dd, J = 13.4, 6.8 Hz, 1H), 2.77 – 2.60 (m, 2H), 1.78 – 1.59 (m, 4H); LCMS (ESI) m/z 458.3 [M+H]+, purity 97.3%.

(R)-2-(5-(1H-indol-4-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (18): Prepared according to the general procedure using indole-4- carboxaldehyde (18.1 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 8.3 mg (14.7% isolated yield) of the desired imidazole 18. 4 1 H NMR (400 MHz, CDCl3) δ 8.70 (bs, 1H), 8.19 (d, J = 1.1 Hz, 1H), 7.47 (dt, J = 8.3, 1.0 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.35 – 7.27 (m, 2H), 7.24 – 7.15 (m, 3H), 7.14 (d, J = 1.1 Hz, 1H), 6.79 (dd, J = 7.2, 0.9 Hz, 1H), 6.31 (ddd, J = 3.1, 2.0, 0.9 Hz, 1H), 4.84 (dd, J = 9.3, 6.0 Hz, 1H), 3.56 (dd, J = 13.1, 9.3 Hz, 1H), 3.36 – 3.19 (m, 3H), 2.16 (t, J = 6.8 Hz, 2H), 1.62 – 1.32 (m, 4H); LCMS (ESI) m/z 453.3 [M+H]+, purity 94.0%.

(R)-2-(5-(1H-indol-3-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (19): Prepared according to the general procedure using indole-3- carboxaldehyde (18.1 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 17.0 mg (30% isolated yield) of the desired imidazole 19. 1H

NMR (400 MHz, CDCl3) δ 9.30 (bs, 1H), 8.18 (d, J = 1.0 Hz, 1H), 7.47 – 7.43 (m, 2H), 7.35 – 7.24 (m, 3H), 7.22 (bs, 1H), 7.20 – 7.06 (m, 2H), 7.08 (d, J = 1.0 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H), 4.87 (dd, J = 8.3, 7.0 Hz, 1H), 3.53 (dd, J = 13.3, 8.3 Hz, 1H), 3.38 – 3.22 (m, 3H), 2.34 – 2.15 (m, 2H), 1.66 – 1.47 (m, 2H), 1.44 – 1.33 (m, 2H); LCMS (ESI) m/z 453.1 [M+H]+, purity 93.3%.

158 (R)-2-(5-(1H-indol-7-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (20): Prepared according to the general procedure using 1H-indole-7- carbaldehyde (21.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 4.1 mg (7.3% isolated yield) of the desired imidazole 20. 1H

NMR (400 MHz, CDCl3) δ 8.70 (s, 1H), 8.16 (s, 1H), 7.68 (dt, J = 7.9, 0.9 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.24 – 7.06 (m, 5H), 6.85 (dd, J = 7.3, 1.1 Hz, 1H), 6.57 (dd, J = 3.2, 2.0 Hz, 1H), 4.74 (dd, J = 9.0, 6.5 Hz, 1H), 3.50 (dd, J = 13.2, 9.0 Hz, 1H), 3.30 (dd, J = 13.2, 6.5 Hz, 1H), 3.25 – 3.03 (m, 2H), 2.15 – 1.97 (m, 1H), 1.90 – 1.73 (m, 1H), 1.47 (quint, J = 6.9 Hz, 2H), 1.30 (quint, J = 6.9 Hz, 2H); LCMS (ESI) m/z 453.3 [M+H]+, purity 91.0%.

(R)-2-(5-(1-methyl-1H-indazol-5-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)-propan-1-one (21): Prepared according to the general procedure using 1-methyl-1H- indazole-5-carbaldehyde (24.0 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 8.7 mg (15% isolated yield) of the desired imidazole 4 1 21. H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 1.1 Hz, 1H), 7.97 (d, J = 0.9 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.42 – 7.32 (m, 3H), 7.24 (s, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 1.1 Hz, 1H), 7.02 (dd, J = 8.6, 1.6 Hz, 1H), 4.86 (dd, J = 7.9, 7.1 Hz, 1H), 4.12 (s, 3H), 3.54 (dd, J = 13.5, 7.9 Hz, 1H), 3.47 – 3.34 (m, 2H), 3.31 (dd, J = 13.5, 7.1 Hz, 1H), 2.69 – 2.54 (m, 2H), 1.78 – 1.53 (m, 4H); LCMS (ESI) m/z 468.3 [M+H]+, purity 90.5%.

(R)-2-(5-(1-methyl-1H-indol-6-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)-propan-1-one (22): Prepared according to the general procedure using 1-methylindole-6- carbaldehyde(23.3 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 27.4 mg (47% isolated yield) of the desired imidazole 22. 1H

NMR (400 MHz, CDCl3) δ 8.10 (d, J = 1.0 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 7.28 (s, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.13 (d, J = 3.1 Hz, 1H), 7.04 (s, 1H), 7.03 (d, J = 1.0 Hz, 1H), 6.81 (dd, J = 8.1, 1.4 Hz, 1H), 6.52 (dd, J = 3.1, 0.8 Hz, 1H), 5.01 (dd, J = 8.4, 6.7 Hz, 1H), 3.75 (s, 3H), 3.57 (dd, J = 13.5, 8.4 Hz, 1H), 3.45 – 3.28 (m, 3H), 2.70 – 2.60 (m, 1H), 2.58 – 2.47 (m, 1H), 1.73 – 1.48 (m, 4H); LCMS (ESI) m/z 467.1 [M+H]+, purity 97.2%.

159 (R)-2-(5-(5-methoxy-1H-indol-3-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)-propan-1-one (23): Prepared according to the general procedure using 5-methoxy- 1H-indole-3-carbaldehyde (21.9 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 14.8 mg (25% isolated yield) of the 1 desired imidazole 23. H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.17 (d, J = 1.0 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.20 (s, 1H), 7.15 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 1.0 Hz, 1H), 6.89 (dd, J = 8.9, 2.5 Hz, 1H), 6.76 (dd, J = 5.5, 2.5 Hz, 2H), 4.83 (dd, J = 8.2, 7.1 Hz, 1H), 3.74 (s, 3H), 3.51 (dd, J = 13.4, 8.2 Hz, 1H), 3.37 – 3.27 (m, 3H), 2.41 – 2.15 (m, 2H), 1.66 – 1.47 (m, 2H), 1.48 – 1.34 (m, 2H); LCMS (ESI) m/z 483.1 [M+H]+, purity 95.9%.

(R)-2-(5-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (24): Prepared according to the general procedure using 7-azaindole-3- carboxaldehyde (18.3 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 13.7 mg (24% isolated yield) of the desired imidazole 24. 4 1 H NMR (400 MHz, CDCl3) δ 11.29 (bs, 1H), 8.39 (dd, J = 4.8, 1.5 Hz, 1H), 8.18 (d, J = 1.0 Hz, 1H), 7.70 (dd, J = 7.9, 1.5 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.25 (bs, 1H), 7.18 – 7.11 (m, 2H), 7.10 (d, J = 1.0 Hz, 1H), 6.92 (s, 1H), 4.87 (t, J = 7.6 Hz, 1H), 3.54 (dd, J = 13.5, 7.6 Hz, 1H), 3.40 – 3.30 (m, 3H), 2.50 – 2.40 (m, 1H), 2.39 – 2.30 (m, 1H), 1.61 (quint, J = 6.8 Hz, 2H), 1.45 (quint, J = 6.8 Hz, 2H); LCMS (ESI) m/z 454.1 [M+H]+, purity 96.5%.

(R)-2-(5-(naphthalen-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (25): Prepared according to the general procedure using 2-napthaldehyde (19.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 3.6 mg (6.2% 1 isolated yield) of the desired imidazole 25. H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 1.0 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.81 – 7.74 (m, 1H), 7.60 – 7.49 (m, 4H), 7.36 (t, J = 7.8 Hz, 1H), 7.33 (bs, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.19 (dd, J = 8.4, 1.7 Hz, 1H), 7.13 (d, J = 1.0 Hz, 1H), 5.00 (dd, J = 8.1, 7.0 Hz, 1H), 3.61 (dd, J = 13.6, 8.1 Hz, 1H), 3.50 – 3.32 (m, 3H), 2.72 – 2.61 (m, 1H), 2.61 – 2.51 (m, 1H), 1.76 – 1.49 (m, 4H); LCMS (ESI) m/z 464.1 [M+H]+, purity 98.5%.

160 (R)-2-(5-(6-methoxynaphthalen-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (26): Prepared according to the general procedure using 6-methoxy-2- naphthaldehyde (23.3 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 10.3 mg (17% isolated yield) of the desired imidazole 26. 1H

NMR (400 MHz, CDCl3) δ 8.13 (d, J = 1.1 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.40 (d, J = 1.7 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.24 – 7.18 (m, 2H), 7.17 – 7.14 (m, 1H), 7.11 (dd, J = 8.4, 1.7 Hz, 1H), 7.07 (d, J = 1.1 Hz, 1H), 4.95 (dd, J = 8.2, 6.9 Hz, 1H), 3.95 (s, 3H), 3.58 (dd, J = 13.5, 8.2 Hz, 1H), 3.43 – 3.31 (m, 3H), 1.71 – 1.48 (m, 4H); LCMS (ESI) m/z 494.1 [M+H]+, purity 90.8%.

(R)-2-(5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3- (3-(trifluoromet-hyl)phenyl)propan-1-one (27): Prepared according to the general procedure using 1,4-benzodioxan-6- carboxaldehyde (20.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 10.0 mg (17% isolated yield) of the desired imidazole 27. 1H 4 NMR (400 MHz, CDCl3) δ 8.06 (d, J = 1.1 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.31 – 7.27 (m, 1H), 7.22 (d, J = 7.7 Hz, 1H), 6.97 (d, J = 1.1 Hz, 1H), 6.87 (d, J = 8.7 Hz, 1H), 6.55 (dq, J = 4.4, 2.1 Hz, 2H), 4.89 (dd, J = 8.5, 6.5 Hz, 1H), 4.33 – 4.21 (m, 4H), 3.56 (dd, J = 13.4, 8.5 Hz, 1H), 3.47 – 3.31 (m, 2H), 3.23 (dd, J = 13.4, 6.5 Hz, 1H), 2.82 – 2.74 (m, 1H), 2.73 – 2.63 (m, 1H), 1.81 – 1.57 (m, 4H); LCMS (ESI) m/z 472.3 [M+H]+, purity 93.0%.

(R)-2-(5-(2,3-dihydrobenzofuran-6-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phen-yl)propan-1-one (28): Prepared according to the general procedure using 2,3‑dihydrobenzofuran‑5‑carbaldehyde (18.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 22.9 mg (40% isolated yield) of the desired 1 imidazole 28. H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 1.1 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.28 (s, 1H), 7.20 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 1.1 Hz, 1H), 6.84 – 6.72 (m, 3H), 4.87 (d, J = 7.4 Hz, 1H), 4.62 (t, J = 8.7 Hz, 2H), 3.54 (dd, J = 13.5, 8.0 Hz, 1H), 3.45 – 3.36 (m, 2H), 3.27 (dd, J = 13.5, 7.0 Hz, 1H), 3.19 (td, J = 8.7, 3.2 Hz, 1H), 1.80 – 1.61 (m, 4H); LCMS (ESI) m/z 456.1 [M+H]+, purity 94.7%.

161 (R)-2-(5-(benzo[d][1,3]dioxol-4-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)-propan-1-one (29): Prepared according to the general procedure using 2,3‑(methylenedioxy)benzaldehyde (18.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.4 mg (4.2% isolated yield) ofthe 1 desired imidazole 29. H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 1.1 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.34 (bs, 1H), 7.32 – 7.28 (m, 1H), 7.11 (d, J = 1.1 Hz, 1H), 6.95 – 6.85 (m, 2H), 6.62 (dd, J = 7.0, 2.1 Hz, 1H), 6.01 (d, J = 1.2 Hz, 1H), 5.96 (d, J = 1.2 Hz, 1H), 4.97 (dd, J = 9.6, 5.3 Hz, 1H), 3.67 (dd, J = 13.1, 9.6 Hz, 1H), 3.43 – 3.33 (m, 2H), 3.26 (dd, J = 13.1, 5.3 Hz, 1H), 2.80 – 2.59 (m, 2H), 1.73 – 1.56 (m, 4H); LCMS (ESI) m/z 458.1 [M+H]+, purity 99.2%.

(R)-3-(1-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl)-1H- imidazol-5-yl)benzo-nitrile (30): Prepared according to the general procedure using 3-cyanobenzaldehyde (16.9 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative 4 HPLC afforded 4.4 mg (8.0% isolated yield) of the desired imidazole 1 30. H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 1.0 Hz, 1H), 7.65 (dt, J = 7.8, 1.4 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.23 (t, J = 1.4 Hz, 1H), 7.20 – 7.12 (m, 3H), 7.04 (d, J = 1.0 Hz, 1H), 4.80 (t, J = 7.6 Hz, 1H), 3.54 – 3.39 (m, 3H), 3.31 (dd, J = 13.7, 8.0 Hz, 1H), 2.85 – 2.74 (m, 2H), 1.89 – 1.64 (m, 4H); LCMS (ESI) m/z 438.2 [M+H]+, purity 97.0%.

(R)-4-(1-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl)-1H- imidazol-5-yl)benzo-nitrile (31): Prepared according to the general procedure using 4-cyanobenzaldehyde (16.4 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.5 mg (4.6% 1 isolated yield) of the desired imidazole 31. H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 8.7 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 1.3 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.47 (d, J = 1.3 Hz, 1H), 7.41 – 7.35 (m, 2H), 7.21 (d, J = 7.7 Hz, 1H), 4.92 (t, J = 7.6 Hz, 1H), 3.59 – 3.44 (m, 3H), 3.37 – 3.13 (m, 3H), 1.94 – 1.73 (m, 4H); LCMS (ESI) m/z 439.3 [M+H]+, purity 91.5%.

162 (R)-N-(4-(1-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl)-1H- imidazol-5-yl)phe-nyl)acetamide (32): Prepared according to the general procedure using 4-acetamidobenzaldehyde (24.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 13.1 mg (22% isolated yield) of the 1 desired imidazole 32. H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.99 (s, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.26 (s, 1H), 7.20 (d, J = 7.7 Hz, 1H), 7.04 – 6.90 (m, 3H), 4.90 (app t, J = 7.6 Hz, 1H), 3.53 (dd, J = 13.4, 8.1 Hz, 1H), 3.47 – 3.34 (m, 2H), 3.28 (dd, J = 13.4, 7.0 Hz, 1H), 2.87 – 2.60 (m, 2H), 2.21 (s, 3H), 1.79 – 1.56 (m, 4H); LCMS (ESI) m/z 471.1 [M+H]+, purity 98.0%.

(R)-5-(1-(1-oxo-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)propan-2-yl)-1H- imidazol-5-yl)pyridin-2(1H)-one (33): Prepared according to the general procedure using 6-oxo-1,6-dihydro- pyridine-3-carbaldehyde (15.4 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 8.4 mg (16% isolated yield) of the desired imidazole33 . 4 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.0 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.29 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 6.99 – 6.91 (m, 3H), 6.55 (d, J = 10.3 Hz, 1H), 4.75 (app t, J = 7.6 Hz, 1H), 3.56 – 3.40 (m, 3H), 3.31 (dd, J = 13.6, 8.0 Hz, 1H), 2.99 – 2.82 (m, 2H), 1.88 – 1.68 (m, 4H); LCMS (ESI) m/z 431.1 [M+H]+, purity 93.8%.

(R)-2-(5-(pyridin-4-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (34): Prepared according to the general procedure using isonicotinaldehyde (13 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.2 mg (4.3% 1 isolated yield) of the desired imidazole 34. H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 6.1 Hz, 2H), 8.23 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.17 (d, J = 7.6 Hz, 1H), 7.11 (s, 1H), 6.91 (d, J = 6.1 Hz, 1H), 4.95 (app. t, J = 7.5 Hz, 1H), 3.54 (dd, J = 13.7, 7.4 Hz, 1H), 3.43 (t, J = 6.5 Hz, 2H), 3.34 (dd, J = 13.7, 7.9 Hz, 1H), 2.87 – 2.72 (m, 2H), 1.83 – 1.69 (m, 4H); LCMS (ESI) m/z 415.1 [M+H]+, purity 96.4%.

163 (R)-2-(5-(2-(dimethylamino)pyrimidin-5-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl-)phenyl)propan-1-one (35): Prepared according to the general procedure using 2-(dimethylamino) pyrimidine-5-carbaldehyde (18.9 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 23.3 mg (41% isolated yield) of the 1 desired imidazole 35. H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 1.1 Hz, 1H), 7.93 (s, 2H), 7.51 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.25 (s, 1H), 7.18 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 1.1 Hz, 1H), 4.76 (t, J = 7.5 Hz, 1H), 3.53 (dd, J = 13.5, 7.5 Hz, 1H), 3.48 – 3.38 (m, 2H), 3.28 – 3.16 (m, 7H), 2.92 – 2.77 (m, 2H), 1.83 – 1.66 (m, 4H); LCMS (ESI) m/z 459.1 [M+H]+, purity 95.6%.

(R)-2-(5-(pyrazin-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (36): Prepared according to the general procedure using pyrimidine-4- carboxaldehyde (13.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.9 mg (5.6% isolated yield) of the desired imidazole 36. 1H NMR (400 MHz, CDCl ) δ 9.09 (d, J = 1.4 Hz, 1H), 8.61 (d, J = 5.4 Hz, 1H), 4 3 8.21 (d, J = 1.0 Hz, 1H), 7.62 (d, J = 1.0 Hz, 1H), 7.43 – 7.37 (m, 1H), 7.36 – 7.32 (m, 2H), 7.28 – 7.23 (m, 2H), 6.97 (t, J = 7.6 Hz, 1H), 3.59 – 3.43 (m, 3H), 3.36 – 3.19 (m, 3H), 1.87 – 1.75 (m, 4H); LCMS (ESI) m/z 416.1 [M+H]+, purity 99.0%.

(R)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl)-2-(5-(5-(trifluoromethyl)pyridin-2- yl)-1H-imidazol-1-yl)propan-1-one (37): Prepared according to the general procedure using 6-(trifluoromethyl) pyridine-3-carboxaldehyde (21.9 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.1 mg (3.5% isolated yield) of the desired imidazole 1 37. H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 2.2 Hz, 1H), 8.29 (d, J = 1.0 Hz, 1H), 7.66 (dd, J = 8.1, 0.9 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.42 – 7.31 (m, 2H), 7.20 (s, 1H), 7.16 – 7.10 (m, 2H), 4.86 (dd, J = 8.2, 7.0 Hz, 1H), 3.59 – 3.43 (m, 3H), 3.34 (dd, J = 13.7, 8.2 Hz, 1H), 2.89 (t, J = 6.6 Hz, 2H), 1.89 – 1.69 (m, 4H); LCMS (ESI) m/z 483.2 [M+H]+, purity 97.3%.

164 (R)-2-(5-(1H-pyrrol-3-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (38): Prepared according to the general procedure using 1H-pyrrole-3- carbaldehyde (11.9 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 29.8 mg (59% isolated yield) of the desired imidazole 38. 1H

NMR (400 MHz, CDCl3) δ 9.40 (bs, 1H), 7.99 (d, J = 1.1 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.30 (s, 1H), 7.21 (d, J = 7.7 Hz, 1H), 6.94 (d, J = 1.1 Hz, 1H), 6.85 (q, J = 2.4 Hz, 1H), 6.54 (q, J = 2.0 Hz, 1H), 5.98 (q, J = 2.4 Hz, 1H), 5.08 (t, J = 8.2, 6.7 Hz, 1H), 3.53 (dd, J = 13.4, 8.2 Hz, 1H), 3.40 (t, J = 6.3 Hz, 2H), 3.26 (dd, J = 13.4, 6.7 Hz, 1H), 2.89 – 2.73 (m, 2H), 1.80 – 1.63 (m, 4H); LCMS (ESI) m/z 403.1 [M+H]+, purity 96.4%.

(R)-2-(5-(1H-pyrrol-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (39): Prepared according to the general procedure using 2-pyrrole- carboxaldehyde (11.9 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 18 mg (36% isolated yield) of the desired imidazole 39. 1H 4 NMR (400 MHz, CDCl3) δ 9.37 (bs, 1H), 8.10 (d, J = 1.1 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.33 – 7.30 (m, 1H), 7.21 (d, J = 7.7 Hz, 1H), 6.99 (d, J = 1.1 Hz, 1H), 6.86 (td, J = 2.6, 1.4 Hz, 1H), 6.25 (q, J = 3.5, 2.6 Hz, 1H), 6.06 (td, J = 3.5, 2.6, 1.4 Hz, 1H), 5.09 (t, J = 7.7 Hz, 1H), 3.44 (dd, J = 13.4, 8.1 Hz, 1H), 3.36 (t, J = 6.1 Hz, 2H), 3.26 (dd, J = 13.4, 7.3 Hz, 1H), 2.94 – 2.79 (m, 1H), 2.74 – 2.65 (m, 1H), 1.82 – 1.55 (m, 4H); LCMS (ESI) m/z 403.1 [M+H]+, purity 91.8%.

(R)-2-(5-(5-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)- propan-1-one (40): Prepared according to the general procedure using 3-methyl-1H- pyrazole-4-carbaldehyde (13.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 28.6 (55% isolated yield) of the desired imidazole 40. 1H NMR

(400 MHz, CDCl3) δ 8.12 (d, J = 1.0 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.29 (s, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.06 (s, 1H), 6.95 (d, J = 1.0 Hz, 1H), 4.82 (t, J = 7.6 Hz, 1H), 3.53 (dd, J = 13.5, 8.0 Hz, 1H), 3.45 – 3.37 (m, 2H), 3.27 (dd, J = 13.5, 7.1 Hz, 1H), 2.81 – 2.62 (m, 2H), 2.12 (s, 3H), 1.78 – 1.62 (m, 4H); LCMS (ESI) m/z 418.1 [M+H]+, purity 98.8%.

165 (R)-2-(5-(1-methyl-1H-pyrazol-5-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)- propan-1-one (41): Prepared according to the general procedure using 1-methyl-1H- pyrazole-5-carboxaldehyde (13.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.6 mg (5.0% isolated yield) of the desired imidazole 1 41. H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 0.9 Hz, 1H), 7.56 – 7.52 (m, 2H), 7.39 (t, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 0.9 Hz, 1H), 6.04 (d, J = 1.9 Hz, 1H), 4.81 (dd, J = 8.5, 7.0 Hz, 1H), 3.58 (s, 3H), 3.53 (dd, J = 13.4, 8.5 Hz, 1H), 3.41 – 3.33 (m, 2H), 3.28 (dd, J = 13.3, 7.0 Hz, 1H), 2.68 – 2.59 (m, 1H), 2.54 – 2.45 (m, 1H), 1.79 – 1.54 (m, 4H); LCMS (ESI) m/z 417.2 [M+H]+, purity >99.9%.

(R)-2-(5-(1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (42): Prepared according to the general procedure using pyrazole-4- carboxaldehyde (14.4 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 18.8 mg (37% isolated yield) of the desired imidazole 42. 1H 4 NMR (400 MHz, CDCl3) δ 8.10 (d, J = 1.1 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.29 – 7.20 (m, 3H), 7.14 (d, J = 7.7 Hz, 1H), 6.98 (d, J = 1.1 Hz, 1H), 4.87 (app t, J = 7.5 Hz, 1H), 3.50 (dd, J = 13.8, 7.1 Hz, 1H), 3.46 – 3.40 (m, 2H), 3.33 (dd, J = 13.7, 7.9 Hz, 1H), 2.93 – 2.76 (m, 2H), 1.85 – 1.63 (m, 4H); LCMS (ESI) m/z 404.0 [M+H]+, purity 96.8%.

(R)-2-(5-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)-propan-1-one (43): Prepared according to the general procedure using 1-methyl-1H- pyrazole-4-carbaldehyde (13.7 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 26.8 mg (51% isolated yield) of the desired imidazole 43. 1H

NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.26 (s, 1H), 7.17 (s, 1H), 7.07 (d, J = 7.8 Hz, 1H), 6.92 (s, 1H), 6.67 (s, 1H), 4.82 (t, J = 8.5, 6.4 Hz, 1H), 3.85 (s, 2H), 3.51 – 3.41 (m, 3H), 3.32 (dd, J = 13.8, 8.5 Hz, 1H), 3.00 – 2.89 (m, 1H), 2.89 – 2.79 (m, 1H), 1.85 – 1.69 (m, 4H); LCMS (ESI) m/z 418.1 [M+H]+, purity 99.6%.

166 (R)-2-(5-(1-methyl-1H-pyrazol-3-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl)-propan-1-one (44): Prepared according to the general procedure using 1-methyl-1H- pyrazole-3-carbaldehyde (13.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 6.6 mg (13% isolated yield) of the desired imidazole 44. 1H

NMR (400 MHz, CDCl3) δ 7.92 (d, J = 1.1 Hz, 1H), 7.54 (bs, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.34 – 7.27 (m, 3H), 7.20 (d, J = 1.1 Hz, 1H), 6.40 (dd, J = 8.7, 6.1 Hz, 1H), 6.25 (d, J = 2.3 Hz, 1H), 3.93 (s, 3H), 3.54 (dd, J = 13.2, 8.7 Hz, 1H), 3.47 – 3.38 (m, 2H), 3.25 – 3.15 (m, 2H), 3.10 – 3.02 (m, 1H), 1.84 – 1.64 (m, 4H); LCMS (ESI) m/z 418.3 [M+H]+, purity 95.3%.

(R)-2-(3H,3’H-[4,4’-biimidazol]-3-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl) propan-1-one (45): Prepared according to the general procedure using 1H-imidazole-5- carbaldehyde (12.01 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 27.9 mg (55% isolated yield) of the desired imidazole 45. 1H NMR (400 MHz, CDCl ) δ 8.01 (s, 1H), 7.67 (s, 1H), 7.43 – 7.36 (m, 2H), 3 4 7.33 – 7.22 (m, 2H), 7.04 (s, 1H), 6.94 (s, 1H), 6.36 (s, 1H), 3.60 – 3.36 (m, 3H), 3.34 – 3.14 (m, 3H), 2.01 (s, 1H), 1.81 – 1.68 (m, 4H); LCMS (ESI) m/z 404.1 [M+H]+, purity 99.8%.

(R)-2-(3’-methyl-3H,3’H-[4,4’-biimidazol]-3-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (46): Prepared according to the general procedure using 1-methyl-1H- imidazole-5-carboxaldehyde (13.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.7 mg (5.2% isolated yield) of the desired 1 imidazole 46. H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 1.0 Hz, 1H), 7.55 (s, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.28 (s, 1H), 7.24 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 0.9 Hz, 1H), 6.90 (d, J = 1.0 Hz, 1H), 4.91 (t, J = 7.8 Hz, 1H), 3.51 (dd, J = 13.3, 8.2 Hz, 1H), 3.40 – 3.36 (m, 2H), 3.29 (dd, J = 13.3, 7.3 Hz, 1H), 3.25 (s, 3H), 2.81 – 2.72 (m, 1H), 2.66 – 2.55 (m, 1H), 1.80 – 1.67 (m, 4H); LCMS (ESI) m/z 418.1 [M+H]+, purity 91.2%.

167 (R)-2-(1-methyl-1H,3’H-[4,4’-biimidazol]-3’-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (47): Prepared according to the general procedure using 1-methyl-1H- imidazole-4-carbaldehyde (13.8 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 33.4 mg (64% isolated yield) of the desired imidazole 47. 1H

NMR (400 MHz, CDCl3) δ 7.88 (d, J = 1.0 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.33 (s, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 1.0 Hz, 1H), 6.68 (d, J = 1.5 Hz, 1H), 6.37 (t, J = 7.5 Hz, 1H), 3.65 (s, 3H), 3.50 – 3.36 (m, 4H), 3.23 (dd, J = 13.4, 7.7 Hz, 1H), 3.18 – 3.09 (m, 1H), 1.83 – 1.70 (m, 4H); LCMS (ESI) m/z 418.1 [M+H]+, purity 99.6%.

(R)-2-(5-(furan-3-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl)phenyl) propan-1-one (48): Prepared according to the general procedure using furan-3- carbaldehyde (12.0 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 14.6 mg (29% isolated yield) of the desired imidazole 48. 1H NMR (400 MHz, CDCl ) δ 8.05 (d, J = 1.0 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 4 3 7.49 – 7.44 (m, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.12 (s, 1H), 6.99 (d, J = 1.0 Hz, 1H), 6.07 (d, J = 1.8 Hz, 1H), 4.89 (app t, J = 7.5 Hz, 1H), 3.51 (dd, J = 13.6, 7.3 Hz, 1H), 3.46 – 3.40 (m, 2H), 3.29 (dd, J = 13.6, 7.7 Hz, 1H), 2.91 – 2.82 (m, 2H), 1.84 – 1.67 (m, 4H); LCMS (ESI) m/z 404.1 [M+H]+, purity 92.2%.

(R,E)-2-(5-(1-(furan-2-yl)prop-1-en-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (49): Prepared according to the general procedure using (E)-3-(furan-2-yl)-2- methylacrylaldehyde (17.0 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 12.8 mg (23% isolated yield) of the desired imidazole 49. 1H

NMR (400 MHz, CDCl3) δ 8.03 (d, J = 1.0 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 1.9 Hz, 1H), 7.42 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 6.96 (d, J = 1.0 Hz, 1H), 6.46 (dd, J = 3.4, 1.9 Hz, 1H), 6.33 (d, J = 3.4 Hz, 1H), 6.03 (d, J = 1.4 Hz, 1H), 5.09 (dd, J = 8.4, 6.6 Hz, 1H), 3.61 (dd, J = 13.5, 8.4 Hz, 1H), 3.43 (q, J = 6.5 Hz, 2H), 3.28 (dd, J = 13.5, 6.7 Hz, 1H), 3.11 (dt, J = 9.4, 6.6 Hz, 1H), 2.92 (dt, J = 8.9, 6.2 Hz, 1H), 2.20 (d, J = 1.2 Hz, 3H), 1.84 – 1.68 (m, 4H); LCMS (ESI) m/z 444.1 [M+H]+, purity 91.7%.

168 (R)-2-(5-(5-morpholinothiophen-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (50): Prepared according to the general procedure using 5-morpholinothiophene-2-carbaldehyde (24.7 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 26.6 mg (42% isolated 1 yield) the desired imidazole 50. H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 1.0 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.40 – 7.31 (m, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 1.0 Hz, 1H), 6.44 (d, J = 3.9 Hz, 1H), 6.04 (d, J = 3.9 Hz, 1H), 5.06 (dd, J = 8.1, 6.8 Hz, 1H), 3.88 – 3.82 (m, 4H), 3.55 (dd, J = 13.5, 8.1 Hz, 1H), 3.25 (dd, J = 13.5, 6.8 Hz, 1H), 3.14 – 3.08 (m, 4H), 2.96 – 2.83 (m, 2H), 1.73 (qd, J = 9.5, 8.2, 4.7 Hz, 4H); LCMS (ESI) m/z 505.1 [M+H]+, purity 97.9%.

(R)-2-(5-(1-(dimethylamino)-2-methylpropan-2-yl)-1H-imidazol-1-yl)-1-(pyrrolidin-1- yl)-3-(3-(trifluoro-methyl)phenyl)propan-1-one (51): Prepared according to the general procedure using 3-(dimethylamino)- 2,2-dimethylpropanal (13.2 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 10.1 mg (19% isolated yield) of the desired imidazole 51. 1H 4 NMR (400 MHz, CDCl3) δ 8.32 (d, J = 1.2 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.47 – 7.39 (m, 2H), 7.30 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 1.2 Hz, 1H), 5.60 (dd, J = 8.6, 6.9 Hz, 1H), 3.54 – 3.38 (m, 5H), 3.31 (dd, J = 13.3, 6.9 Hz, 1H), 2.75 – 2.63 (m, 1H), 2.34 (s, 2H), 2.07 (s, 6H), 1.87 – 1.57 (m, 4H), 1.27 (s, 3H), 1.20 (s, 3H); LCMS (ESI) m/z 437.3 [M+H]+, purity 94.4%.

(R)-2-(5-(3-fluorophenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (52): Prepared according to the general procedure using 3-fluorobenzaldehyde (15.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 1.3 mg (2.4% isolated yield) of the desired imidazole 1 52. H NMR (400 MHz, CDCl3) δ 8.15 (d, J = 1.1 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.42 – 7.33 (m, 2H), 7.21 (d, J = 7.7 Hz, 1H), 7.11 (tdd, J = 8.4, 2.6, 1.0 Hz, 1H), 7.05 (d, J = 1.1 Hz, 1H), 6.83 (ddd, J = 7.6, 1.6, 1.0 Hz, 1H), 6.73 (ddd, J = 9.4, 2.6, 1.6 Hz, 1H), 4.92 (t, J = 7.6 Hz, 1H), 3.56 (dd, J = 13.5, 7.9 Hz, 1H), 3.51 – 3.39 (m, 2H), 3.31 (dd, J = 13.5, 7.2 Hz, 1H), 2.81 – 2.64 (m, 2H), 1.82 – 1.67 (m, 4H); LCMS (ESI) m/z 432.3 [M+H]+, purity 99.9%.

169 (R)-2-(5-(4-fluorophenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3-(trifluoromethyl) phenyl)propan-1-one (53): Prepared according to the general procedure using 4-fluorobenzaldehyde (15.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 2.7 mg (5.0% 1 isolated yield) of the desired imidazole 53. H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.1 Hz, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.28 (bs, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.09 (t, J = 8.6 Hz, 2H), 7.02 – 6.94 (m, 3H), 4.83 (app t, J = 7.5 Hz, 1H), 3.54 (dd, J = 13.5, 7.8 Hz, 1H), 3.47 – 3.38 (m, 2H), 3.30 (dd, J = 13.5, 7.3 Hz, 1H), 2.76 – 2.67 (m, 2H), 1.78 – 1.62 (m, 4H); LCMS (ESI) m/z 432.2 [M+H]+, purity 88.6%.

(R)-2-(5-(4-(1H-pyrazol-1-yl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)phenyl-)propan-1-one (54): Prepared according to the general procedure using 4-(1H-pyrazol-1-yl) benzaldehyde (21.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 3.5 mg (5.8% isolated yield) of the desired imidazole 54. 1H NMR (400 MHz, CDCl ) δ 8.17 (d, J = 1.1 Hz, 1H), 7.96 (dd, J = 2.5, 0.6 4 3 Hz, 1H), 7.76 (dd, J = 1.8, 0.6 Hz, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.29 (s, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H), 7.03 (d, J = 1.1 Hz, 1H), 6.52 (dd, J = 2.5, 1.8 Hz, 1H), 4.91 (t, J = 7.6 Hz, 1H), 3.54 (dd, J = 13.5, 7.7 Hz, 1H), 3.46 – 3.37 (m, 1H), 3.32 (dd, J = 13.5, 7.4 Hz, 1H), 2.83 – 2.75 (m, 1H), 2.74 – 2.66 (m, 1H), 1.77 – 1.59 (m, 4H); LCMS (ESI) m/z 480.3 [M+H]+, purity 90.0%.

(R)-2-(5-(4-(1H-imidazol-1-yl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (55): Prepared according to the general procedure using 4-(1H-imidazol-1- yl)benzaldehyde (21.5 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 4.5 mg (7.5% isolated yield) of the desired imidazole 55. 1H

NMR (400 MHz, CDCl3) δ 8.16 (d, J = 1.0 Hz, 1H), 7.89 (d, J = 1.1 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.46 – 7.33 (m, 3H), 7.33 – 7.29 (m, 1H), 7.26 – 7.24 (m, 2H), 7.17 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 8.5 Hz, 2H), 7.03 (d, J = 1.0 Hz, 1H), 4.89 (t, J = 7.5 Hz, 1H), 3.53 (dd, J = 13.7, 7.3 Hz, 1H), 3.49 – 3.37 (m, 2H), 3.32 (dd, J = 13.7, 7.8 Hz, 1H), 2.89 – 2.75 (m, 2H), 1.87 – 1.49 (m, 4H); LCMS (ESI) m/z 480.1 [M+H]+, purity 93.3%.

170 (R)-2-(5-(4-(1H-1,2,4-triazol-1-yl)phenyl)-1H-imidazol-1-yl)-1-(pyrrolidin-1-yl)-3-(3- (trifluoromethyl)-phenyl)propan-1-one (56): Prepared according to the general procedure using 4-(1H-1,2,4- triazol-1-yl)benzaldehyde (21.6 mg, 0.125 mmol) and amine S4 (250 µL, 0.6 M solution in EtOH, 0.150 mmol). Purification by preparative HPLC afforded 4.6 mg (7.7% isolated yield) of the desired imidazole 1 56. H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 8.19 (d, J = 1.1 Hz, 1H), 8.14 (s, 1H), 7.70 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 7.05 (d, J = 1.1 Hz, 1H), 4.90 (app t, J = 7.5 Hz, 1H), 3.54 (dd, J = 13.7, 7.4 Hz, 1H), 3.48 – 3.38 (m, 2H), 3.33 (dd, J = 13.7, 7.7 Hz, 1H), 2.91 – 2.71 (m, 2H), 1.84 – 1.61 (m, 4H); LCMS (ESI) m/z 481.2 [M+H]+, purity 98.0%.

171 4.5. References

1. H. Wang, R. Cao, L. Xia, H. Erdjument-Bromage, C. Borchers, P. Tempst, Y. Zhang, Mol. Cell 2001, 8, 1207-1217. 2. K. Nishioka, S. Chuikov, K. Sarma, H. Erdjument-Bromage, C. D. Allis, P. Tempst, D. Reinberg, Genes Dev. 2002, 16, 479-489. 3. C. D. Allis, S. L. Berger, J. Cote, S. Dent, T. Jenuwien, T. Kouzarides, L. Pillus, D. Reinberg, Y. Shi, R. Shiekhattar, A. Shilatifard, J. Workman, Y. Zhang, Cell 2007, 131, 633-636. 4. D. R. Calnan, A. E. Webb, J. L. White, T. R. Stowe, T. Goswami, X. Shi, A. Espejo, M. T. Bedford, O. Gozani, S. P. Gygi, A. Brunet, Aging-Us 2012, 4, 462-479. 5. S. Chuikov, J. K. Kurash, J. R. Wilson, B. Xiao, N. Justin, G. S. Ivanov, K. McKinney, P. Tempst, C. Prives, S. J. Gamblin, N. A. Barlev, D. Reinberg, Nature 2004, 432, 353-360. 6. A. Kouskouti, E. Scheer, A. Staub, L. Tora, I. Talianidis, Mol. Cell 2004, 14, 175-182. 7. C. K. Ea, D. Baltimore, Proc. Natl. Acad. Sci. USA 2009, 106, 18972-18977. 8. T. Masatsugu, K. Yamamoto, Biochem. Biophys. Res. Commun. 2009, 381, 22-26. 9. K. Subramanian, J. Da, P. Kapoor-Vazirani, D. R. Powell, R. E. Collins, D. Sharma, J. Peng, X. Cheng, P. M. Vertino, Mol. Cell 2008, 30, 336-347. 10. F. Paneni, S. Costantino, R. Battista, L. Castello, G. Capretti, S. Chiandotto, G. Scavone, A. Villano, D. Pitocco, G. Lanza, M. Volpe, T. F. Luescher, F. Cosentino, Circ. Cardiovasc. Genet. 2015, 8, 150-U304. 4 11. I. A. A. Batista, L. A. Helguero, Signal Transduct. Target. Ther. 2018, 3, 19. 12. H. U. Kaniskan, J. Jin, ACS Chem. Biol. 2015, 10, 40-50. 13. M. Luo, Chem. Rev. 2018, 118, 6656-6705. 14. D. Barsyte-Lovejoy, F. Li, M. J. Oudhoff, J. H. Tatlock, A. Dong, H. Zeng, H. Wu, S. A. Freeman, M. Schapira, G. A. Senisterra, E. Kuznetsova, R. Marcellus, A. Allali-Hassani, S. Kennedy, J.-P. Lambert, A. L. Couzens, A. Aman, A.-C. Gingras, R. Al-Awar, P. V. Fish, B. S. Gerstenberger, L. Roberts, C. L. Benn, R. L. Grimley, M. J. S. Braam, F. M. V. Rossi, M. Sudol, P. J. Brown, M. E. Bunnage, D. R. Owen, C. Zaph, M. Vedadi, C. H. Arrowsmith, Proc. Natl. Acad. Sci. USA 2014, 111, 12853-12858. 15. Y. Takemoto, A. Ito, H. Niwa, M. Okamura, T. Fujiwara, T. Hirano, N. Handa, T. Umehara, T. Sonoda, K. Ogawa, M. Tariq, N. Nishino, S. Dan, H. Kagechika, T. Yamori, S. Yokoyama, M. Yoshida, J. Med. Chem. 2016, 59, 3650-3660. 16. T. Fujiwara, K. Ohira, K. Urushibara, A. Ito, M. Yoshida, M. Kanai, A. Tanatani, H. Kagechika, T. Hirano, Bioorg. Med. Chem. 2016, 24, 4318-4323. 17. T. Hirano, T. Fujiwara, H. Niwa, M. Hirano, K. Ohira, Y. Okazaki, S. Sato, T. Umehara, Y. Maemoto, A. Ito, M. Yoshida, H. Kagechika, ChemMedChem. 2018, 13, 1530-1540. 18. S. Mori, K. Iwase, N. Iwanami, Y. Tanaka, H. Kagechika, T. Hirano, Bioorg. Med. Chem. 2010, 18, 8158- 8166. 19. H. Ding, W. C. Lu, J. C. Hu, Y. C. Liu, C. H. Zhang, F. L. Lian, N. X. Zhang, F. W. Meng, C. Luo, K. X. Chen, Molecules 2018, 23.

172 20. F. Meng, S. Cheng, H. Ding, S. Liu, Y. Liu, K. Zhu, S. Chen, J. Lu, Y. Xie, L. Li, R. Liu, Z. Shi, Y. Zhou, Y. C. Liu, M. Zheng, H. Jiang, W. Lu, H. Liu, C. Luo, J. Med. Chem. 2015, 58, 8166-8181. 21. D. C. Lenstra, E. Damen, R. G. G. Leenders, R. H. Blaauw, F. P. J. T. Rutjes, A. Wegert, J. Mecinović, ChemMedChem. 2018, 13, 1405-1413. 22. J. Lyu, S. Wang, T. E. Balius, I. Singh, A. Levit, Y. S. Moroz, M. J. O’Meara, T. Che, E. Algaa, K. Tolmachova, A. A. Tolmachev, B. K. Shoichet, B. L. Roth, J. J. Irwin, Nature 2019, 566, 224-229. 23. B. R. Bellenie, N. P. Barton, A. J. Emmons, J. P. Heer, C. Salvagno, Bioorg. Med. Chem. Lett.2009 , 19, 990- 994. 24. Cresset SPARK and TORCH, https://www.cresset-group.com. 25. Chemical Computing Group Molecular Operating Environment (MOE), https://www.chemcomp.com/. 26. Schrödinger PyMOL, https://pymol.org/. 27. A. M. Van Leusen, J. Wildeman, O. H. Oldenziel, J. Org. Chem. 1977, 42, 1153-1159. 28. V. Gracias, A. F. Gasiecki, S. W. Djuric, Org. Lett. 2005, 7, 3183-3186. 29. J. Sisko, A. J. Kassick, M. Mellinger, J. J. Filan, A. Allen, M. A. Olsen, J. Org. Chem. 2000, 65, 1516-1524. 30. D. C. Lenstra, A. H. K. Al Temimi, J. Mecinović, Bioorg. Med. Chem. 2018, 28, 1234-1238.

173 174 5

Inhibition of histone lysine methyltransferases G9a and GLP by ejection of structural Zn(II)

This chapter has been published as: D. C. Lenstra, A. H. K. Al Temimi and J. Mecinović, Bioorg. Med. Chem. Lett., 2018, 28, 1234-1238.

175 Abstract

Histone lysine methyltransferases G9a and GLP are validated targets for the development of new epigenetic drugs. Most, if not all, inhibitors of G9a and GLP target the histone substrate binding site or/and the S-adenosylmethionine cosubstrate binding site. Here, we report an alternative approach for inhibiting the methyltransferase activity of G9a and GLP. For proper folding and enzymatic activity, G9a and GLP contain structural zinc fingers, one of them being adjacent to the S‑adenosylmethionine binding site. Our work demonstrates that targeting these labile zinc fingers with electrophilic small molecules results in ejection of structural zinc ions, and consequently inhibition of the methyltransferase activity. Very effective Zn(II) ejection and inhibition of G9a and GLP was observed with clinically used ebselen, disulfiram and cisplatin.

176 5.1. Introduction

Histone posttranslational modifications (PTMs), including methylation, acetylation, phosphorylation and many others, play an important role in human gene regulation.[1] Methylation of lysine residues is catalysed by members of histone lysine methyltransferases (KMTs) that transfer the methyl group from S-adenosylmethionine (SAM) to lysine residues on histone N-terminal tails, core histones and non-histone proteins. Recent structural, mutagenesis and molecular modelling studies provided basic mechanistic insight into histone methyltransferase catalysis.[2-5] SET domain- containing proteins G9a and its highly related homologue G9a-like protein (GLP) (also known as EHMT2 and EHMT1, respectively) catalyse mono-, di- and trimethylation of lysine 9 on histone 3 (H3K9me1/2/3, Figure 1a) and several other proteins.[6] The highest methylation mark (H3K9me3) results in formation of heterochromatin, i.e. the transcriptionally inactive form of chromatin, and has been linked to the development and maintenance of various types of cancer.[7] For instance, recent work has shown that increased expression of G9a in aggressive lung cancer cells is associated with greater mortality in patients.[8] Therefore G9a and GLP have been recognised as validated targets for development of small molecule inhibitors for therapies against a variety of diseases, including cancer.[9]

Figure 1. a) G9a- and GLP-catalysed methylation of H3K9 (n = 1, 2, 3); b) Crystal structure of GLP in complex with histone 3 peptide (yellow) and S-adenosylhomocysteine (SAH, light cyan); a zoomed view on the Cys residues (magenta) involved in the chelation of structural Zn(II) (orange) (PDB id: 2RFI).

177 Recent medicinal chemistry studies have demonstrated that inhibition of G9a and GLP (and other HKMTs) can be achieved by small molecules that act as histone-competitive or/and SAM‑competitive inhibitors.[10-12] BIX-01294, the first known selective inhibitor of G9a, was reported in 2007.[13] This small molecule inhibitor, which was identified by high-throughput screening, targets the histone binding site. Since its discovery, the structure of BIX-01294 has been used in various structure-activity relationship (SAR) explorations and structure-based design studies, which led to the development of inhibitors with an improved potency and selectivity, and reduced toxicity to cells, such as UNC0638,[14] A-366,[15] E72,[16] and DCG066.[17] Few SAM-competitive inhibitors of G9a and GLP have also been recently reported, including BIX-01338 and BRD4770.[13, 18] However, SAM-competitive inhibitors are often unselective due to high homology in SAM-binding sites between different methyltransferases. Most known inhibitors have similar inhibitory activity against G9a and GLP, and developing selective inhibitors for either one is considered challenging due to their high protein homology and similarity of the histone and SAM binding sites (~80%).[19] Nonetheless, recent work has shown that a high degree of selective inhibition can be achieved; MS012 and related structures have up to 140-fold selectivity for GLP over G9a.[20]

In order to establish proper folding and enzymatic activity, G9a and GLP methyltransferases each contain four structural zinc ions. Both enzymes contain two distinguishable types of zinc fingers; three Zn(II) ions are chelated in a triangular cluster by 9 cysteines (Figure 1b, top left), whereas one Zn(II) ion is chelated by 4 cysteines in [21] a Cys4-type zinc finger (Figure 1b, top right). The latter zinc finger is adjacent to the SAM-binding site. Recent studies have highlighted that significant efforts have been 5 made in developing strategies for targeting labile Zn-fingers with electrophilic small molecules, most notably by ebselen and disulfiram.[22, 23] Inhibition of important biological processes by the release of structural zinc ions has been shown for a variety of proteins, including nucleocapsid 7,[24] p300,[25] γ-butyrobetaine,[26] and histone lysine demethylase JMJD2A.[27] We hypothesised that it would be possible to inhibit G9a and GLP methyltransferases by small molecule-mediated ejection of structural zinc ions.

It was envisioned that the ejection of Zn(II) from the Cys4-Zn finger, which is located adjacent to the SAM-binding site (Figure 1b, top right), would lead to a loss of the methyltransferase activity of G9a and GLP.

178 5.2. Results and discussion

We initiated our investigations by testing whether 20 known and potential zinc ejectors, including clinically used ebselen, disulfiram and cisplatin, have an ability to inhibit the G9a and GLP methyltransferase activity (Table 1). The chosen examples include: selenium-based compounds 1–7, sulfur-based compounds 8–15, and various other potential Zn(II) ejectors 16–20. Initially, all compounds were tested at a concentration of 10 µM against both G9a and GLP. Therefore, methylation of a synthetic 15-mer peptide (residues 1–15) mimic of the N-terminal histone 3 tail containing a lysine at position 9 (H3K9) was monitored using matrix-assisted laser desorption-ionisation time-of- flight (MALDI-TOF) mass spectrometry.[28] Representative inhibition data for GLP- catalysed methylation of H3K9 can be found in Figure 2. Molecules that did not show significant inhibition at 10 µM were also tested at 100 µM. Inhibition data at 10 and 100 µM for all other compounds can be found in Figures S1–S4. For those compounds that showed >50% inhibition at a concentration of 100 µM, half maximum inhibitory concentrations (IC50) were obtained using a MALDI-TOF based assay using 200 nM enzyme concentrations (Table 1).

Figure 2. Representative MALDI-TOF MS data of a) GLP-catalysed methylation of H3K9 peptide (m/z = 1561.0); b) with 10 µM ebselen; c) with 10 µM disulfiram; d) with 10 µM cisplatin.

179 In the absence of inhibitor, 15-mer H3K9 histone mimic underwent near quantitative trimethylation (m/z = 1603.1 Da, Figure 2a); this result is consistent with our recent studies on HKMT-catalysed methylation of lysine.[29] For ebselen 1, which is known to inhibit various zinc finger containing proteins, such as metallothionein,[30] histone lysine [27] [26] demethylase JMJD2A, and γ-butyrobetaine hydroxylase, submicromolar IC50 values were obtained (0.40 µM for G9a and 0.73 µM for GLP), demonstrating very effective inhibition. Notably, at 10 µM concentration of ebselen, only unmethylated peptide was observed in MALDI-TOF spectrum (Figure 2b, m/z = 1560.9). Related seleno compounds

2–5 were also observed to be excellent inhibitors of G9a and GLP; IC50 values were found to be 0.45–4.4 µM for G9a, and 1.0–4.9 µM for GLP. Sodium selenate 6 only showed ~10% inhibition for G9a and GLP at 100 µM concentration (Figures S2 and S4). Diphenyl

diselenide 7 inhibited G9a and GLP with IC50 = 0.55 μM and 0.86 μM, respectively.

a Table 1. Inhibition IC50 data.

a) Half maximum inhibitory concentration (IC50) obtained at 200 nM enzyme concentration; b) IC50 for G9a;

c) IC50 for GLP.

180 Having shown that selenium-based compounds act as good inhibitors of G9a and GLP, we examined related sulfur-based small molecules as potential inhibitors for these two methyltransferases. Dithiocarbamates disulfiram 8 (Figure 2c) and thiram 9 inhibited the activity of G9a with submicromolar IC50 values of 0.60 and 0.55 µM, whereas for

GLP the observed IC50 values are 1.6 and 1.1 µM, respectively. Inhibition by clinically safe disulfiram is particularly important, as it has been used for decades for treatment of alcoholism by targeting acetaldehyde dehydrogenase (ADH), thereby causing instantaneous nausea upon consumption of alcohol.[31] Besides ADH, disulfiram is also known to inhibit hepatitis C viral replication by targeting a labile zinc finger,[32] and to induce apoptosis in a number of human cancer cell lines or inhibit cancer cell growth.[33-35] Sodium diethyldithiocarbamate 10, the reduced form of disulfiram, was found to only poorly inhibit GLP. This result is consistent with observations on inhibition of HIV-1 nucleocapsid protein.[36]

2,2’-Dithiodipyridine 11, also known as aldrithiol, is a known zinc ejector, targeting for instance the zinc finger in nucleocapsid protein of human immunodeficiency [37] virus type 1. Compound 11 inhibited G9a activity with IC50 = 0.65 µM and GLP with

IC50 = 2.6 µM. We also tested disulfide cysteamine dihydrochloride salt 12, but its IC50 value was found to be above 100 µM for GLP, whereas for G9a an IC50 value of 15 µM was obtained. Glutathione, in both the reduced (13) and oxidised (14) form, did not inhibit G9a and GLP. Also, no inhibition was observed for cystamine 15, the reduced form of cysteamine dihydrochloride. Azidocarbonamide 16 showed very high inhibition activity against both G9a and GLP (IC50 = 0.50 and 1.7 µM, respectively). Naphthoquinone 17 and ninhydrin 18, both known to eject zinc from p300,[25] were also tested against G9a 5 and GLP. Naphthoquinone 17 was observed to be a potent inhibitor of G9a with an IC50 value of 2.0 µM, whereas it inhibited GLP with IC50 = 14 µM. Ninhydrin 18 only poorly inhibited GLP (~40% inhibition at 100 µM, Figure S4) and an IC50 value of 54 µM was observed for G9a. In addition, anthraquinone 19, a structurally related analogue of naphthoquinone, exhibited rather poor inhibition activity (~20% for both G9a and GLP) at 100 µM. Finally, inhibitory activity of cisplatin20 was evaluated. Cisplatin is a potent chemotherapeutic drug and is known to be highly reactive towards Cys4 or Cys3His type [38] Zn-fingers. Importantly, we found that cisplatin inhibits G9a and GLP with similar IC50 values of 1.4 and 1.7 µM, respectively.

181 Figure 3. a–c) Zinc ejection data for GLP (2 µM) in the presence of various concentrations of ebselen, disulfiram, and cisplatin, measured in the presence of Zn(II)-selective fluorophore FluoZinTM-3; d-f) Dose- response curves after 1 h incubation in the presence of ebselen, disulfiram, and cisplatin.

The observations that several Se- and S-based electrophiles, including clinically used ebselen and disulfiram, possess excellent inhibition activity against G9a and GLP prompted us to examine their mode of action. We envisioned that they inhibit G9a and 5 GLP by the release of structural zinc ions, as a result of covalent modification of cysteine residues. The release of zinc from G9a and GLP was therefore monitored using the Zn(II)- selective indicator FluoZin™-3. In line with observed MALDI-TOF data, we found that zinc was released from both methyltransferases in the presence of those compounds that showed inhibitory activity (Figures S5-S10). Because of their current use in clinics, we were particularly interested in inhibition of G9a/GLP by ebselen, disulfiram and cisplatin. Therefore, zinc release from G9a and GLP in the presence of various concentrations of ebselen, disulfiram and cisplatin was monitored over time in the presence of FluoZin-3 (Figures 3a–c). We observed that ebselen ejects Zn(II) very rapidly (within minutes) for both G9a and GLP; disulfiram and cisplatin also have the ability to eject zinc ions from G9a/GLP, but require somewhat longer times to achieve it (Figures 3b–c).

Based on a calibration curve with known Zn(II) concentrations, a dose-response curve for the amount of zinc released from each methyltransferase was plotted

182 (GLP: Figures 3d–f, G9a: Figure S11). Each methyltransferase contains in total four structural zinc ions, one of which is very close to the active site, and three at a more distant location (Figure 1). In the presence of ebselen (>25 μM), all four zinc ions were released from G9a and GLP, whereas higher concentrations of disulfiram (>50 µM) were required for complete zinc ejection. On average only~ 2.5 Zn(II) ions are removed after 1 h in the presence of 100 µM cisplatin, although it is possible that all four zinc ions can be released after prolonged time, and/or at even higher concentrations of cisplatin. We also tested whether UNC0638,[14] a highly potent histone competitive inhibitor for G9a and GLP, was able to eject structural Zn(II); as expected, no zinc release was observed (Figures S9 and S10).

Having shown that ebselen and disulfiram effectively inhibit G9a and GLP via a zinc ejection mechanism, we next explored whether the folding of these two enzymes has been affected in the presence of ebselen and disulfiram. In order to investigate potential changes in the secondary and/or tertiary structure of these methyltransferases, we employed circular dichroism (CD), a well-established tool in protein/peptide research.[39] Upon the addition of 100 µM of ebselen or disulfiram to either 2 µM G9a or GLP, we observed significant distortions in the CD spectrum (Figures S12 and S13), implying that both enzymes became partially unfolded.

5.3. Conclusion

In conclusion, we have demonstrated that the ejection of structural zinc ions from G9a and GLP in the presence of selenium- and sulfur-containing electrophilic small 5 molecules leads to inhibition of these two biomedically important epigenetic enzymes. Our work demonstrates that clinically used ebselen, disulfiram and cisplatin act as very effective inhibitors of G9a and GLP with submicromolar or low micromolar IC50 values. It is possible that the observed physiological effects of these molecules may in part arise as a result of their ability to affect epigenetic processes regulated by G9a and GLP methyltransferases. Inhibition of biomedically important epigenetic processes is currently a subject of intensive investigations, therefore we envision that future studies will lead to important advances in design and development of specific inhibitors of the therapeutic potential. Although it is unlikely that highly electrophilic compounds studied here, most notably ebselen,[22] act as specific inhibitors of endogenous proteins, it might be possible that exploring a broader chemical space via substitutions on the ebselen and disulfiram scaffolds may lead to a higher degree of specificity for certain protein targets. Detailed structure-activity relationships studies on small molecules

183 that target structural zinc fingers may direct a design of novel type of inhibitors with an improved selectivity. Towards this aim, our work highlights that targeting zinc finger sites of histone lysine methyltransferases is an alternative strategy to commonly used approaches that target histone substrate and SAM cosubstrate binding sites; this strategy leads to efficient inhibition of G9a and GLP histone lysine methyltransferases that possess structural zinc ions.

5.4. Supporting information

5.4.1. General remarks All reagents and compounds were purchased from commercial suppliers and used without further purification. The synthetic 15-mer histone peptide mimic containing a lysine at position 9 was synthesised as previously described by our group.[29]

5.4.2. Expression and purification of histone lysine methyltransferases[21] G9a expression: Wild-type G9a (EHMT-2) histone lysine methyltransferase (Homo sapiens, residues 913-1193) was expressed in terrific broth (TB), using E. coli Rosetta BL21

DE3 PlysS as expression host. Bacteria were cultured to OD600 ≈ 0.6 at 37 °C, 200 rpm after which the cultures were induced using 0.1 mM isopropyl β-D-1-thiogalactopyranoside

(IPTG, final concentration) and 0.1 mM ZnCl2 (final concentration). Cultures were then incubated at 16 °C for 16 h. Cells were harvested and lysed by sonication in 25 mM NaCl, 2 mM b-mercaptoethanol, phosphate buffer saline pH 7.4, 5% glycerol, and 0.1% Triton X-100. Lysate was centrifuged and the supernatant purified using Ni-NTA beads. 5 Proteins were washed with 20 mM TRIS pH 8.0, 250 mM NaCl, 50 mM imidazole, and 5% glycerol prior to elution with 20 mM TRIS pH 8.0, 250 mM NaCl, 250 mM imidazole, and 5% glycerol. Size exclusion chromatography, using a superdex 75 column, was employed as a final purification step with 20 mM TRIS pH 8.0, 150 mM NaCl as eluent. Protein concentration was determined by UV/Vis spectroscopy at 280 nm.

GLP expression: Wild-type GLP (EHMT-1) histone lysine methyltransferase (Homo sapiens, residues 951-1235) was expressed in terrific broth (TB), using E. coli Rosetta

BL21 DE3 PlysS as expression host. Bacteria were cultured to OD600 ≈ 0.6 at 37 °C, 200 rpm after which the cultures were induced using 1.0 mM IPTG (final concentration)

and 0.1 mM ZnCl2 (final concentration). Cultures were then incubated at 16 °C for 16 h. Cells were harvested and lysed by sonication in 25 mM NaCl, 2 mM b-mercaptoethanol, phosphate buffer saline pH 7.4, 5% glycerol, and 0.1% Triton X-100. The lysate was centrifuged and the supernatant purified using Ni-NTA beads. Proteins were washed

184 with 20 mM TRIS pH 8.0, 250 mM NaCl, 50 mM imidazole, and 5% glycerol prior to elution with 20 mM TRIS pH 8.0, 250 mM NaCl, 250 mM imidazole, and 5% glycerol. Size exclusion chromatography, using a superdex 75 column, was employed as a final purification step with 20 mM TRIS pH 8.0, 150 mM NaCl as eluent. Protein concentration was determined by UV/Vis spectroscopy at 280 nm.

5.4.3. MALDI-TOF MS based inhibition studies A MALDI-TOF MS based assay monitoring the mono-, di-, and trimethylation of a 15-mer histone H3 derived peptide (ARTKQTARKSTGGKA) was performed.[28] Briefly, recombinant enzyme (G9a or GLP, at 200 nM final concentration) and inhibitor (100% DMSO stock solution at appropriate concentration) were combined in assay buffer (50 mM glycine pH 9.8, 10% glycerol). The mixture of enzyme and inhibitor was preincubated for 5 min at 37 °C. Next, 5 mL of a premixture of peptide (final concentration 10 mM) and SAM (final concentration 60 mM) were added to obtain a final reaction volume of 50 mL, containing 5% DMSO (v/v). The mixture was incubated for an additional 60 min at 37 °C, after which it was quenched by the addition of an equal volume of MeOH. Each experiment was performed in triplicate.

For MALDI-TOF MS analysis, a 5 mL aliquot of the quenched reaction mixture was mixed with a saturated solution of matrix α-cyano-4-hydroxycinnamic acid (CHCA, in

MeCN:H2O (1:1)) (4:1 (v/v)) and from this 1 mL was deposited onto the MALDI plate for crystallisation. MALDI-TOF MS data were recorded on a Bruker Microflex LRF MALDI-TOF system. Total peak area of each methylation state, including all isotopes and adducts ([M+H]+ and [M+Na]+) were used for determination of enzymatic activity in the presence of inhibitors relative to the activity in the absence of inhibitors (i.e. 5 only with 5% DMSO). The relative activity (%) was plotted against the logarithm of the inhibitor concentrations and fitted to the Hill equation with variable Hill coefficient using OriginPro software to obtain IC50 values.

185 5.4.4. Initial screen of compounds at 10 µM and 100 µM

N-(Phenylseleno)phthalimide Phenylselenyl chloride Phenylselenyl bromide Benzene selenic acid Sodium selenate Diphenyldiselenide Disulfiram Thiram Sodium diethyldithiocarbamate Aldrithiol Cystamine dihydrochloride Cysteamine Oxidized glutathione Reduced glutathione Azidocarbonamide Naphthoquinone Ninhydrin Anthraquinone Cisplatin 0 20 40 60 80 100 Activity (%) Figure S1. G9a activity in the presence of 10 µM of compounds.

Sodium selenate

Cystamine dihydrochloride

Oxidized glutathione

Reduced glutathione

Cysteamine

Ninhydrin

Anthraquinone

0 20 40 60 80 100 5 Activity (%)

Figure S2. G9a activity in the presence of 100 µM of compounds.

Ebselen N-(Phenylseleno)phthalimide Phenylselenyl chloride Phenylselenyl bromide Benzene selenic acid Sodium selenate Diphenyldiselenide Disulfiram Thiram Sodium diethyldithiocarbamate Aldrithiol Cystamine dihydrochloride Cysteamine Oxidized glutathione Reduced glutathione Azidocarbonamide Naphthoquinone Ninhydrin Anthraquinone Cisplatin 0 20 40 60 80 100 GLP activity (%) Figure S3. GLP activity in the presence of 10 µM of compounds.

186 Sodium selenate

Cystamine dihydrochloride

Oxidized glutathione

Reduced glutathione

Cysteamine

Ninhydrin

Anthraquinone

0 20 40 60 80 100 GLP activity (%) Figure S4. GLP activity in the presence of 100 µM of compounds.

5.4.5. Monitoring of Zn(II) ejection with FluozinTM-3 Release of Zn(II) from G9a and GLP methyltransferases was monitored using the zinc specific fluorophore FluoZinTM-3 (FZ-3, tetrapotassium salt, cell-impermeant, InvitrogenTM). The assay was performed as previously described.[27] All experiments were performed in 50 mM TRIS pH 8.0 assay buffer. FZ-3 was diluted in assay buffer to obtain a stock concentration of 100 µM, which was aliquoted and stored at -20 °C. Prior to each experiment a calibration curve was obtained: the fluorescence intensity of various concentrations of Zn(II) (ranging between 0 – 10 µM ZnCl2 dissolved in MQ) in the presence of 5 µM FZ-3 was recorded. For Zn(II) ejection experiments, a 25 µL enzyme mix in assay buffer was prepared containing 4 µM enzyme and 10 µM FZ-3. A 25 µL inhibitor mix was prepared by mixing 2.5 µL of inhibitor (dissolved in 100% dmso 5 at appropriate concentration) with 22.5 µL assay buffer. The enzyme and inhibitor mix were combined on a 96-well plate prior to insertion into the plate reader to obtain a final volume of 50 µL, containing final concentrations of 2 µM enzyme, 5 µM FluozinTM-3 and 5% dmso (v/v). Fluorescence intensity was recorded on a Tecan Infinite Pro M200 plate reader at 37 °C for 120 cycles of 30 seconds per cycle, with 5 seconds shaking after each measurement. All experiments were performed in duplicate.

Zn(II) ejection with all compounds at 100 µM All compounds were tested at a concentration of 100 µM in the presence of 2 µM of enzyme. Zn(II) ejection was monitored in the presence of 5 µM FluoZin-3 as described in section S4. The fluorescence intensity was normalised and plotted against time.

187 1,0

0,8 dmso N-(phenylseleno)-phtalamide 0,6 Diphenyldiselenide Benzeneselenic acid PhSeCl PhSeBr 0,4 Sodium Selenate Ebselen Normalized Fluorescence 0,2

0,0 0 500 1000 1500 2000 2500 3000 3500 Time (s) Figure S5. Zn(II) ejection at 2 µM G9a concentration in the presence of 100 µM compound.

1,0

0,8 dmso Diphenyldiselenide 0,6 Benzeneselenic acid PhSeCl PhSeBr 0,4 Sodium Selenate Ebselen N-(phenylseleno)-phtalamide Normalized Fluorescence 0,2

0,0 0 500 1000 1500 2000 2500 3000 3500 Time (s) Figure S6. Zn(II) ejection at 2 µM GLP concentration in the presence of 100 µM compound. 5

1,0

0,8 dmso Sodium diethyldithiocarbamate 0,6 Cystamine dihydrochloride Disulfiram Reduced glutathione 0,4 Thiram Cysteamine Aldrithiol Normalized Fluorescence 0,2

0,0 0 500 1000 1500 2000 2500 3000 3500 Time (s) Figure S7. Zn(II) ejection at 2 µM G9a concentration in the presence of 100 µM compound.

188 1,0

0,8 dmso Cystamine dihydrochloride 0,6 Cysteamine Aldrithiol Sodium diethyldithiocarbamate 0,4 Disulfiram Thiram Reduced glutathione Normalized Fluorescence 0,2

0,0 0 500 1000 1500 2000 2500 3000 3500 Time (s) Figure S8. Zn(II) ejection at 2 µM GLP concentration in the presence of 100 µM compound.

1,0

0,8 dmso Azidocarbonamide 0,6 Naphthoquinone Cisplatin Ninhydrin 0,4 Anthraquinone UNC0638 Normalized Fluorescence 0,2

0,0 0 500 1000 1500 2000 2500 3000 3500 Time (s)

Figure S9. Zn(II) ejection at 2 µM G9a concentration in the presence of 100 µM compound. 5

1,0

0,8 dmso Ninhydrin 0,6 UNC0638 Cisplatin Azidocarbonamide 0,4 Napthaquinone Anthraquinone Normalized Fluorescence 0,2

0,0 0 500 1000 1500 2000 2500 3000 3500 Time (s)

Figure S10. Zn(II) ejection at 2 µM GLP concentration in the presence of 100 µM compound.

189 Figure S11. a–c) Zinc(II) ejection data for G9a in the presence of various concentrations of ebselen, disulfiram, and cisplatin, measured in the presence ofZn (II)-selective fluorophore FluoZinTM-3.; d-f) Dose-response curves after 1 h incubation in the presence of ebselen, disulfiram, and cisplatin.

5.4.6. Circular dichroism (CD) analyses of enzymes treated with ebselen and disulfiram CD measurements were conducted using a J-815 circular dichroism spectropolarimeter 5 (JASCO) at 0.1 mg ml-1 protein concentration in a 1 mm cuvette using 10 mM

NaH2PO4 pH 7.5 as buffer. Ebselen and disulfiram were dissolved in MeCN at appropriate concentration, so that the percentage of MeCN was 1% (v/v) and a final concentration of 50 µM compound. The wavelength range scanned was 195 – 249 nm using a bandwidth of 1 nm at a time constant of 0.5 second and scan rate of 50 nm sec-1. Obtained spectra are averages of 10 measurements and have been smoothed using a Savitzky-Golay filter with a convolution width of 10.

190 G9a 6 G9a + Ebselen G9a + Disulfiram 4

0 200 210 220 230 240 250

CD (mdeg) -2 Wavelength (nm)

-4

-6

Figure S12. CD data for GLP (2 µM) in the presence of 100 µM ebselen (green line) or disulfiram (blue line).

GLP 4 GLP + Ebselen GLP + Disulfiram 2

0 200 210 220 230 240 250 -2 Wavelength (nm)

CD (mdeg) -4

-6

-8 5

-10

Figure S13. CD data for GLP (2 µM) in the presence of 100 µM ebselen (blue line) or disulfiram (green line).

191 5.5. References

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193 194 6

Discussion and future perspectives

195 Abstract

In the work described in the preceding chapters of this thesis we have synthesised, characterised and evaluated >100 analogues of SETD7 inhibitor (R)-PFI-2, however none of the analogues proved to be more potent than the original scaffold. These findings emphasise the difficulty in developing new highly potent inhibitors for biomedically important SETD7, but also extends to other methyltransferases. This observation is also supported by the fact that over the past years, no literature has emerged in which highly potent and selective SETD7 inhibitors with a novel chemotype are described. In this chapter, future perspectives with respect to this research are described. In order to move to inhibitors with a novel chemotype, additional bioisosteres could be synthesised. The development of (R)-PFI-2 analogues as potential covalent irreversible inhibitors is also described. Furthermore, to avoid a laborious SAR study to identify new inhibitors, two approaches based on fragment based drug discovery (FBDD) are proposed.

196 6.1. Introducing novel bioisosteres for the sulfonamide core of (R)-PFI-2

In Chapter 4 of this thesis computational tools were used to identify potential bioisosteres of the sulfonamide core of (R)-PFI-2. Because this work was performed at Mercachem B.V. in the department of parallel chemistry, one bioisostere which could be synthesised from readily available starting materials was chosen. We then aimed to synthesise a library of (R)-PFI-2 analogues containing this specific bioisostere, the 1,5-disubstituted imidazole, which was synthesised from various aldehydes and one D‑phenylalanine derived amine (for more details on the synthesis of the different analogues, see Chapter 4). Unfortunately, none of the analogues inhibited SETD7- catalysed methylation of H3K4. However, a large panel of other potential bioisosteres was identified, and future work on this topic could focus on the synthesis of (R)-PFI-2 analogues bearing these bioisosteres. Examples are given in Figure 1.

Pyrazoles: 1,2,4-triazoles:

2 2 R1 R R1 R 1 R2 1 R2 N R 2 R N N R N N N N N N N CF Me 3 Me R1 2 1 R 2 4-methylimidazole: R R1 R N O N O 1 R2 N R N S O N H2N F N H HN N Me N F 1,2,3-triazoles: tetrazole: (R)-PFI-2 2 2 2 R1 R R1 R R1 R N N N N N N N N Cl N N Figure 1. Potential bioisosteres of the sulfonamide core of(R)-PFI-2 (depicted in blue), generated using Cresset’s SPARK software.[1] 6

Several substituted pyrazoles were generated by Cresset’s SPARK as candidate bioisosteres. These pyrazoles could be synthesised using the Knorr pyrazole synthesis, i.e. imine formation between an R2‑substituted hydrazine and a 1,3-dicarbonyl which is substituted with R1 (Scheme 1, top).[2, 3] Starting from the same hydrazine, a 1,2-4-triazole substituted with R2 can be synthesised upon treatment with formamide, as described by Shelke et al. (Scheme 2, bottom).[4] Cu(I)-catalysed functionalisation

197 at the 5‑position of the triazole with R1-Br or R1-I should then afford the desired 1,5-disubstituted 1,2,4‑triazole.[5] The 1,2,3-triazoles are easily access through a Ruthenium-catalysed Azide Alkyne Cycloaddition (RuAAC) reaction from R1‑alkyne and 2 [6] R -N3, to afford a 1,5-disubstituted 1,2,3,-triazole.

CF3

O O 1 R1 H R O N N N CF3 O NBoc CF3 1) EtO2C CO2Et 2) TFA / DCM O H2N O H2N N H N N

CF3 CF3 O R1-Br, H NH 2 Cu(I) + ligand R1 O O N N N N N N N N

Scheme 1. Proposed preparation of the pyrazole and 1,2,4-tetrazole bioisosteres. The first step is hydrazine formation as is described in reference [3]. R1 is depicted in Figure 1.

6.2. SETD7 as a catalyst for azide-alkyne click chemistry

Though proven as a successful approach in medicinal chemistry, a structure-activity relationship (SAR) study is a demanding endeavour in terms of time and resources. In particular the synthesis of large libraries of compounds can take several years, and still fail to improve the potency or/and selectivity of an initial HTS hit. Over the past decades, several approaches have been developed in order to accelerate the drug discovery process. One such approach is protein templated click chemistry, a fragment based drug discovery (FBDD) approach in which a fragment with a low binding affinity 6 for a particular protein is grown/linked inside the receptor of the protein of interest.[7] This fragment with poor affinity contains either an azide or alkyne functionality, which can, under the right circumstances, undergo a 1,3-dipolar cycloaddition (also known as a ‘click reaction’) to form a 1,4- or 1,5-disubstituted 1,2,3-triazole. Typically, an initial fragment that is known to bind to the receptor with low affinity is used in combination with a collection of complementary building blocks. The enzyme is used as the template, and if azide and alkyne are properly aligned inside the receptor/ protein, the protein serves as a catalyst for the click reaction. Analysis is as simple as

198 identifying whether click product has formed.[8] The generated 1,2,3‑triazole product better occupies the receptor, and therefore usually has a much higher binding affinity. This target-generated approach can significantly reduce the time and effort required for identifying binders with high potency.

Scheme 2. Synthesis of azide and alkyne modified (R)-PFI-2 analogues. Reagents and conditions:i ) pyrrolidine (1.3 equiv), EDC (1.35 equiv), HOBt (1.35 equiv), DIPEA (3.0 equiv) in DCM, overnight, rt; ii) 10% Pd/C (1.1 equiv), H2 (1 atm), EtOH, overnight, rt; iii) MsCl (1.2 equiv), Et3N (1.5 equiv) in anhydrous DCM, 4 h, -30 °C to rt, then NaN3 (2.0 equiv) in anhydrous DMF, 50 °C, overnight; iv) TFA/DCM (1:1 v/v), 1 h, rt; v) Et3N (4.0 equiv), DCM, 2.5 h, rt, then TFA/DCM (1:1 v/v), 2 h, rt.

We have initiated the exploration of the part of the histone binding pocket of SETD7 that is occupied by the 3‑(trifluoromethyl)phenyl ring of R( )-PFI-2 (Figure 1) using a FBDD approach. We aimed to explore this part of the scaffold using protein templated 6 click chemistry. To this purpose, we synthesised two (R)-PFI-2 analogues bearing an azide (compound 7) or an alkyne (compound 11) moiety (Scheme 2). The synthesis of compound 7 starts from commercially available Boc‑D‑Ser(Bn)‑OH, which is coupled to pyrrolidine with EDC and HOBt. The benzyl group of 2 is deprotected under hydrogenation conditions (Pd/C, 1 atm H2) to obtain 71% of the free alcohol 3, which is subsequently converted to the corresponding mesyl alcohol before substitution with

NaN3 to afford azide 4 in a moderate 55% yield. After removal of the Boc-protecting group, fragment 5 is coupled to sulfonyl chloride 6, which again is Boc-deprotected

199 to afford the final azide 7 in 60% isolated yield. Alkyne 11 is obtained using similar chemistry starting from D-propargylglycine. In line with our expectations, the inhibitory

activity of both azide 7 (IC50 = 16.2 µM) and alkyne 11 (IC50 = 13.9 µM) towards SETD7 proved to be quite moderate (Figure S1).

With the azide and alkyne templates in hand, we performed a variety of experiments under conditions adapted from literature.[9-11] Azide 7 or alkyne 11 (50‑200 µM final conc.) were incubated with multiple alkynes (200‑500 µM final conc.) or azides (200‑500 µM final conc.) and SETD7 (2‑10 µM final conc.) in 50 mM TRIS pH 8.0 as assay buffer. An overview of the alkynes and azides is given in Figure S2. Aliquots were taken at different time points and analysis performed by LCMS in order to detect if any 1,2,3‑triazole had formed due to a SETD7-templated click reaction. Unfortunately, with the conditions described, we did not observe any formation of triazole, indicating that SETD7 is not catalysing the click reaction between azides and alkynes under these conditions. In order to confirm that the potency improved when a 1,2,3-triazole is formed, we performed a Cu(I) catalysed click reaction between Boc‑protected alkyne 11 and benzyl azide to form compound 12 (Scheme 3). Indeed, when we evaluated

the inhibitory activity, we found IC50 = 1.39 µM for triazole 12 (Figure S1), a significant 10‑fold improvement with respect to 11. Future work within this project could lead to the discovery of potent SETD7 inhibitors, and should include testing of additional alkynes and azides, a screening of buffers, pH, temperature, time, and stoichiometry between alkyne, azide, and SETD7.

6 Scheme 3. Formation of the 1,4-disubstituted 1,2,3-triazole (R)-PFI-2 analogue 12. Reagents and conditions:

i) alkyne Boc-11 (1.0 equiv), BnN3 (1.0 equiv), CuSO4.5H2O (0.4 equiv), sodium ascorbate (1.2 equiv) in H2O/ EtOH/DCM (1:1:1 v/v), rt, 2 days; ii) TFA/DCM (1:1 v/v), 2 h, rt.

200 6.3. Dynamic combinatorial chemistry for accelerated drug discovery

An alternative approach within FBDD is dynamic combinatorial chemistry (DCC), a technique which is based on the formation of a dynamic combinatorial library (DCL) through reversible reactions.[12] Examples of reversible reactions employed in DCC are imine formation between amines and aldehydes or ketones,[13] boronate ester formation between boronic acids and diols,[14] and disulfide formation between thiols.[15-17] Two approaches towards the formation of a DCL are known, the first is in the presence of the target enzyme. The ligand from the DCL that binds best can be identified through comparison with a DCL to which no target enzyme is added. A second approach is regularly applied in cases where the target enzyme is unstable under the DCL conditions; here the DCL is first allowed to reach equilibrium before the addition of the enzyme. Comparison of the DCL before and directly after the addition of the target will allow for the identification of the best binding ligand. Several analytical techniques have been applied for identification of the best-binding fragment combination from a DCL, for instance HPLC,[18] LCMS,[17] (non-denaturing protein) MS,[13, 14] and NMR.[19]

We hypothesised that using DCC might also be an efficient approach towards the exploration of the SAR for the 3‑(trifluoromethyl)phenyl side chain of (R)-PFI-2. As Fmoc-D-Cys(Trt)-OH is commercially available, we considered a dynamic library based on the thiol-disulfide exchange as a sensible starting point. The synthesis is similar to related analogues (Scheme 4): amide coupling between Fmoc‑D‑Cys(Trt)‑OH and pyrrolidine afforded amide13 in moderate 40% isolated yield. Fmoc deprotection with DBU proceeded well and amine 14 was obtained in 73% isolated yield. Sulfonamide formation between amine 14 and sulfonyl chloride 6, and subsequent deprotection of the Boc-, and Trt-protecting groups in one pot under acidic conditions, afforded (R)-PFI-2 thiol analogue 16 in 44% isolated yield after purification by preparative HPLC.

It was found that 16 inhibits SETD7 with IC50 = 7.5 µM (Figure S1).

6 We then set out to explore the appropriate conditions for obtaining a dynamic equilibrium, i.e. conditions where interconversion between different disulfides occurs. The thiol-disulfide exchange highly depends on the pH of the assay buffer, and typically proceeds well under basic conditions, pH range 7-9.[20] Using LCMS as analytical technique, we evaluated three buffer systems (50 mM TRIS, NH4OAc, or glycine buffer) each at different pH (7.5, 8.0, and 8.5), and found that after approximately 10 days in

50 mM NH4OAc at any of the tested pH, equilibrium is reached.

201 Scheme 4. Synthesis of (R)-PFI-2 thiol analogue 16 for dynamic combinatorial chemistry. Reagents and conditions: i) pyrrolidine (1.3 equiv), EDC (1.35 equiv), HOBt (1.35 equiv), DIPEA (3.0 equiv) in DCM,

overnight, rt; ii) DBU (1.1 equiv), DCM, 1 h, rt; iii) Et3N (4.0 equiv), DCM, 2.5 h, rt; iv) TIPS (1.1 equiv), TFA/ DCM (1:1 v/v), 2 h, rt.

With these conditions (50 mM NH4OAc pH 7.5), we started with the formation of a small dynamic library (Scheme 5) consisting of the template16 (400 µM final conc.) and 11 thiols R1-11 (each at 50 µM final conc.) with different properties (i.e. hydrophobic/ hydrophilic or aromatic/aliphatic). After 10 days the equilibrium was ‘frozen’ by acidification with 0.1 % TFA in MQ. The DCL was measured by LCMS, and the different disulfides were assigned to each peak in the chromatogram using the corresponding

MS data. Besides the formation of the disulfide of 16 with itself (assigned as (16)2), we observed the different disulfides 16-R1-11 (Figure 2a). Unfortunately, when we performed an identical experiment but now adding SETD7 (25 µM final conc.) from the start, and performed analysis by LCMS after 10 days, we obtained data which was not suitable for interpretation (Figure 2b). Though we attempted to precipitate SETD7 from the DCL before LCMS analysis, it appears that the enzyme is still present and prevents proper analysis of the constituents of the library. In order to solve this problem, SETD7 has to be removed from the DCL mixture. This could be achieved by size-exclusion chromatography.[13] Alternatively, different analytical techniques could be applied, for 6 instance HPLC or NMR spectroscopy. After identification of an appropriate (protocol for an) analytical technique, the amount of thiols in the library can be expanded. Once established, a similar approach based on the disulfide exchange, can be used to rapidly explore the SAR of other parts of (R)‑PFI‑2.

202 R1-11 S SH HS-R1-11 S O O O O S O (50 µM each) S O N N H H HN N HN N 50 mM NH4OAc F pH 7.5 F

16 (400 µM) 16-R1-11

F CF3 OMe Et N NH N F Et 2

R1 R2 R3 R4 R5 R6

OH OH O O OH OH

R7 R8 R9 R10 R11 Scheme 5. Formation of a dynamic combinatorial library between compound (R)-PFI-2 thiol analogue 16 and several thiols R1-11SH.

a) b)

Figure 2. LCMS analysis of a) dynamic combinatorial library in the absence of SETD7 at t = 10 days showing the different disulfides 16-R1-11; b) dynamic combinatorial library in the presence of SETD7 at t = 10 days.

6.4. Development of irreversible covalent inhibitors targeting 6 SETD7

The use of covalent inhibitors has been controversial for many years because it was long believed that inhibitors bearing reactive functional groups (also known as warheads) have a risk of forming reactive metabolites, and thus potentially have severe toxic side effects.[21] However, many clinically used drugs, after using them for many years, were found to act via covalent inhibition mechanisms; for instance antibiotic penicillin, gastric-acid inhibitor omeprazole, and aspirin.[22] As a result, recently interest in the

203 development and clinical evaluation of covalent inhibitors has significantly increased. A similar approach could be employed to develop inhibitors of SETD7 with an improved potency. Installing a reactive warhead on (R)-PFI-2 could allow for irreversible covalent inhibition. In particular three tyrosine residues (i.e. Y245, Y305, and Y335), which are present in the active site, are of interest.[23] An analysis of the crystal structure of the ternary complex between SETD7, SAM and (R)‑PFI‑2 (PDB id: 4JLG) shows that a warhead is best introduced near the pyrrolidine ring. Examples of warheads that have been used in literature to target tyrosine residues in active sites include sulfonyl fluorides[24, 25] and a dichlorotriazine.[26] Examples of potential (R)-PFI-2 analogues bearing a reactive warhead and possible synthetic routes are depicted in Scheme 6.

Scheme 6. Proposed synthesis of covalent inhibitors based on (R)-PFI-2.

The synthesis of an (R)-PFI-2 analogue with a sulfonyl fluoride warhead could be achieved as follows:[27] oxidation of compound 17 (synthesis described in Chapter 3) with hydrogen peroxide followed by treatment with NaOAc and Pd/C will afford the

204 sodium sulfonate intermediate. Fluorination with diethylamino sulfur trifluoride (DAST) and Boc-deprotection with HCl in dioxane will generate the desired sulfonyl fluoride analogue 18 hydrochloric acid salt. Compound 20, containing the dichlorotriazine warhead, is also accessible from a previously prepared compound 19 (for a description of the synthesis see Chapter 3 of this dissertation). Treatment of 19 with cyanuric chloride followed by Boc‑deprotection will yield 20 in two steps.[26]

In order to investigate whether these proposed electrophilic warheads fit inside the narrow channel of SETD7, and if yes, to investigate with which tyrosine they are likely to react, we performed molecular docking studies. Docking studies were performed on compounds bearing both tyrosine-reactive warheads, with different lengths of the side chain (n = 2, 3, and 4) using Molecular Operating Environment (MOE).[28] Several tyrosine residues are present in the active site of SETD7 near (R)‑PFI‑2. We attempted covalent docking using an internal reaction template in MOE, which is based on sulfonic ester formation between tyrosine and sulfonyl halides. We initially attempted a covalent modification on all of the tyrosine residues that are present in the active site of SETD7, and observed that modification of Tyr335 with sulfonyl fluoride 18 (and also analogues with a longer side chain) resulted in a covalent SETD7‑inhibitor complex that resembles the binding of (R)-PFI-2 to SETD7 (Figure 3). We did not observe any good docking results with dichlorotriazine containing compound 20, likely due to the size of the warhead. a) b) SAM

Y335

R-SO2F 6

Figure 3. a) Non-covalent docking results of compound 18 (R-SO2F, yellow) in the active site of SETD7 (grey, cartoon) (PDB id: 4JLG); b) Results of covalent docking to Y335 (orange), forming a covalent adduct between 18 and SETD7. SAM is depicted in purple.

205 6.5. Conclusion

The development and improvement of potent and selective inhibitors targeting histone lysine methyltransferase SETD7 is an arduous task. Extensive SAR explorations require considerable time, effort, and resources. A fragment based drug discovery approach could be applied in order to speed up the progress of laborious SAR studies. In this chapter we have described our initial efforts towards the SAR exploration of (R)-PFI-2 using two FBDD approaches; protein templated click chemistry and dynamic combinatorial chemistry. After additional optimisation, these FBDD approaches might be used to rapidly explore the SAR of particular parts of (R)‑PFI‑2. Furthermore, additional potential bioisosteres of the sulfonamide core of(R)-PFI-2 that were identified by Cresset’s SPARK are described. These analogues could be synthesised as proposed, and evaluated against SETD7, and might lead to the discovery of inhibitors with a distinct chemotype. Finally, a novel approach towards the inhibition of SETD7 is proposed. Here, SETD7 is targeted with an (R)‑PFI‑2 analogue bearing a reactive sulfonyl fluoride warhead. This sulfonyl fluoride could be used to form a covalent adduct with SETD7’s Tyr335. Taken together, the approaches described in this chapter may serve as a starting point for the development of novel potent and selective inhibitors of SETD7.

6.6. Supporting information

For general experimental considerations see the experimental section of Chapter 2 (Section 2.4).

6.6.1. Characterisation of compounds tert-Butyl (R)-(3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (2): N-Boc-D-Ser(Bn)-OH (618 mg, 2.0 mmol, 1.0 equiv), HOBt (413 mg, 2.7 mmol, 1.35 equiv), and EDC (517 mg, 2.7 mmol, 1.35 equiv) were dissolved in DCM (10 mL), followed by the addition of DIPEA (775 mg, 6.0 mmol, 3.0 6 equiv), and pyrrolidine (185 mg, 2.6 mmol, 1.3 equiv). The reaction mixture was stirred overnight at rt. The reaction mixture was diluted with DCM (20 mL), and subsequently washed with 1.0 M NaOH (40 mL), 1.0 M HCl (40 mL), and brine (40

mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product, which was purified using column chromatography (15-25% EtOAc inn -heptane) 1 to afford 390 mg (54% isolated yield) of2 . TLC Rf = 0.35 (n-heptane/EtOAc 1:1); H NMR

(500 MHz, CDCl3) δ 7.36 – 7.23 (m, 5H), 5.57 (d, J = 8.6 Hz, 1H), 4.70 (q, J = 7.4 Hz, 1H), 4.55 – 4.47 (m, 2H), 3.70 – 3.42 (m, 4H), 1.97 – 1.76 (m, 4H), 1.43 (s, 9H); 13C NMR (126

206 MHz, CDCl3) δ 169.7, 155.4, 137.7, 128.4, 127.8, 127.5, 80.1, 73.3, 70.3, 51.9, 46.9, 46.4, 28.3, 25.9, 24.1; MS (ESI) m/z 349.5 [M+H]+. tert-Butyl (R)-(3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (3): Compound 2 (300 mg, 0.9 mmol, 1.0 equiv) was dissolved in EtOH (5.0 mL) and treated with 10% Pd/C under an argon atmosphere. The reaction

mixture was evacuated and backfilled with H2 gas three times, and then

stirred under an H2 atmosphere (1 atm, balloon) at rt overnight. The crude reaction mixture was filtered over celite and concentrated in vacuo to afford 158 mg

(71% isolated yield) of crude 3 as an off-white solid. TLC Rf = 0.30 (n-heptane/EtOAc 1 1:1); H NMR (400 MHz, CDCl3) δ 4.53 (bs, 1H), 3.94 – 3.37 (m, 6H), 2.04 – 1.81 (m, 4H), 13 1.44 (s, 9H); C NMR (101 MHz, CDCl3) δ 156.0, 80.2, 64.1, 53.5, 46.8, 46.3, 28.5, 26.1, 24.3; MS (ESI) m/z 259.3 [M+H]+. tert-Butyl (R)-(3-azido-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)carbamate (4): Crude compound 3 (150 mg, 0.58 mmol, 1.0 equiv) was dissolved in anhydrous DCM (5.8 mL) and cooled to –30 °C, followed by the addition of

methanesulfonyl chloride (80 mg, 0.70 mmol, 1.2 equiv) and Et3N (88.2 mg, 0.87 mmol, 1.5 equiv). The reaction was stirred at -30 °C for 3 h and then allowed to warm to rt. After 1 h at rt, the solvent was removed in vacuo, and the mixture redissolved in anhydrous DMF (5.8 mL) followed by the addition of NaN3 (76 mg, 1.2 mmol, 2.0 equiv). After additional stirring at 50 °C for 18 h, diluted with water (25 mL), and extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with brine (75 mL), dried over anhydrous MgSO4, and filtered. The solvent was removed in vacuo and purified using column chromatography (25-50% EtOAc in n-heptane) to afford 91 mg (51% isolated yield) of 4 as a colourless oil. TLC Rf = 0.40 1 (n-heptane/EtOAc 1:1); H NMR (400 MHz, CDCl3) δ 5.50 (d, J = 8.4 Hz, 1H), 4.66 – 4.56 13 (m, 1H), 3.67 – 3.43 (m, 6H), 2.07 – 1.81 (m, 4H), 1.44 (s, 9H); C NMR (101 MHz, CDCl3) δ 167.9, 155.0, 80.2, 52.6, 51.7, 46.7, 46.3, 28.3, 26.0, 24.1; MS (ESI) m/z 283.9 [M+H]+.

6 (R)-3-Azido-1-oxo-1-(pyrrolidin-1-yl)propan-2-aminium trifluoroacetate (5): Compound 4 (50 mg, 0.18 mmol, 1.0 equiv) was dissolved in 2 mL of TFA:DCM (1:1 v/v) and stirred at rt for 1 h. When TLC analysis (EtOAc:n- heptane 1:1) showed the reaction was complete, the solvent was removed in vacuo. Residual TFA was removed by co-evaporation with

Et2O (5 × 10 mL). Without further purification 52 mg (>99% yield) of 5 was obtained as 1 a colourless oil. H NMR (400 MHz, CD3OD) δ 4.36 (dd, J = 7.1, 4.6 Hz, 1H), 3.90 (dd, J = 13.5, 4.6 Hz, 1H), 3.80 (dd, J = 13.5, 7.1 Hz, 1H), 3.68 – 3.50 (m, 3H), 3.49 – 3.40 (m, 1H),

207 2.12 – 1.88 (m, 4H); MS (ESI) m/z 184.1 [M+H]+.

(R)-6-(N-(3-Azido-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)sulfamoyl)-8-fluoro-1,2,3,4- tetrahydroisoquin-oline trifluoroacetate (7): Sulfonyl chloride 6 (25 mg, 71 µmol, 1.0 equiv) and azide 6 (26 mg, 86 µmol, 1.2 equiv) were dissolved in 1 mL of DCM.

To this was added Et3N (29 mg, 29 µmol, 4.0 equiv) and the reaction was left to stir at rt for 2.5 h. After completion, the reaction mixture was diluted by the addition of DCM (10 mL) and was washed with 1.0 M HCl (2 × 10 mL), followed by brine (10 mL). The organic layer was dried over anhydrous

MgSO4 and concentrated in vacuo. The crude mixture was purified using flash column chromatography (25-40% EtOAc in n-heptane) to afford 17.3 mg (50% isolated yield) of 1 compound Boc-7 as a white solid. H NMR (400 MHz, CDCl3) δ 7.46 (bs, 1H), 7.37 (d, J = 8.7 Hz, 1H), 5.97 (d, J = 9.1 Hz, 1H), 4.62 (bs, 2H), 4.18 (q, J = 6.7 Hz, 1H), 3.66 (bs, 2H), 3.52 – 3.40 (m, 3H), 3.41 – 3.29 (m, 2H), 3.29 – 3.19 (m, 1H), 2.96 – 2.81 (m, 2H), 1.86 (dddd, J = 42.9, 29.8, 13.2, 6.5 Hz, 4H), 1.50 (s, 6H); MS (ESI) m/z 519.1 [M+H]+. Next, Boc-7 was dissolved in 1 mL TFA:DCM (1:1 v/v) and left to stir at rt until TLC (MeOH:DCM 1:9) showed the reaction was complete (approximately 2 h). Residual TFA was removed

by co-evaporation with Et2O (5 × 10 mL). Without further purification, 16.8 mg (96% 1 yield) of 7 was obtained as a white solid. H NMR (400 MHz, CD3OD) δ 7.62 (d, J = 1.6 Hz, 1H), 7.54 (dd, J = 9.1, 1.6 Hz, 1H), 4.47 (s, 2H), 4.35 (t, J = 6.9 Hz, 1H), 3.66 – 3.54 (m, 4H), 3.51 (dd, J = 12.5, 6.7 Hz, 1H), 3.43 (dd, J = 12.5, 7.1 Hz, 1H), 3.34 – 3.13 (m, 3H), 13 3.13 – 3.03 (m, 1H), 2.03 – 1.74 (m, 4H); C NMR (101 MHz, CD3OD) δ 167.1, 160.1, 157.6, 142.3, 142.2, 135.6, 135.6, 122.9, 122.9, 121.1, 120.9, 111.6, 111.4, 54.1, 51.6, 46.5, 45.9, 40.6, 39.2, 39.1, 25.5, 24.6, 24.6, 23.6; MS (ESI) m/z 397.1 [M+H]+.

(R)-2-((tert-Butoxycarbonyl)amino)pent-4-ynoic acid (8): D-Propargylglycine (500 mg, 4.42 mmol, 1.0 equiv) was dissolved in 18 mL water/dioxane (1:1 v/v) followed by the addition of 5.0 mL 1.0 M NaOH. Next, Boc O (1.06 g, 4.86 mmol, 1.1 equiv) was added and the reaction was 6 2 stirred at rt for 18 h. The reaction mixture was acidified to pH ~3 with 1.0 M HCl, extracted with EtOAc (3 × 20 mL). The combined organic layers were dried over

anhydrous MgSO4, filtered and concentrated in vacuo to afford 0.85 g (90% isolated yield) of crude 8 as a pale yellow oil, which was used without further purification. 1H

NMR (500 MHz, CD3OD) δ 4.29 (app t, J = 6.1, 1H), 2.73 (ddd, J = 16.9, 5.3, 2.7, 1H), 2.67 (ddd, J = 16.9, 6.9, 2.7, 1H), 2.36 (t, J = 2.7, 1H), 1.48 (s, 9H).

208 tert-Butyl (R)-(1-oxo-1-(pyrrolidin-1-yl)pent-4-yn-2-yl)carbamate (9): Compound 9 (278 mg, 70% isolated yield, colourless oil) was prepared analogous to compound 2, starting from8 (320 mg, 1.5 mmol, 1.0 equiv). 1 TLC Rf = 0.40 (n‑heptane/EtOAc 1:1); H NMR (400 MHz, CDCl3) δ 5.40 (d, J = 8.8 Hz, 1H), 4.63 (q, J = 7.3 Hz, 1H), 3.74 – 3.62 (m, 2H), 3.61 – 3.40 (m, 2H), 2.67 – 2.61 (m, 2H), 2.01 – 1.86 (m, 5H), 1.41 (s, 9H); MS (ESI) m/z 267.4 [M+H]+.

(R)-1-oxo-1-(pyrrolidin-1-yl)pent-4-yn-2-aminium trifluoroacetate (10): Compound 10 (262 mg, 100% yield) was prepared analogous to compound 5, starting from 9 (250 mg, 0.94 mmol, 1.0 equiv). 1H NMR

(400 MHz, CD3OD) δ 4.37 (t, J = 6.6 Hz, 1H), 3.69 – 3.60 (m, 2H), 3.59 – 3.42 (m, 2H), 2.87 (ddd, J = 17.3, 6.6, 2.7 Hz, 1H), 2.80 (ddd, J = 17.3, 6.6, 2.7 Hz, 1H), 2.68 (t, J = 2.7 Hz, 1H), 2.10 – 1.90 (m, 4H); 13C NMR (101

MHz, CD3OD) δ 166.9, 77.3, 74.6, 51.5, 47.9, 47.6, 26.9, 25.0, 21.4; MS (ESI) m/z 245.1 [M+H]+.

(R)-8-Fluoro-6-(N-(1-oxo-1-(pyrrolidin-1-yl)pent-4-yn-2-yl)sulfamoyl)-1,2,3,4- tetrahydroisoquinolin-2-ium trifluoroacetate (11): Compound 11 was prepared analogous to compound 7; starting from sulfonyl chloride 6 (25 mg, 71 µmol, 1.0 equiv) and amine 10 (24 mg, 86 µmol, 1.2 equiv) to obtain first Boc-

11 (23 mg, 68% isolated yield). TLC Rf = 0.20 (n-heptane/ 1 EtOAc 1:1); H NMR (400 MHz, CDCl3) δ 7.45 (bs, 1H), 7.37 (dd, J = 8.7, 1.7 Hz, 1H), 5.92 (d, J = 9.5 Hz, 1H), 4.61 (bs, 2H), 4.24 (q, J = 7.9 Hz, 1H), 3.66 (bs, 2H), 3.56 (dt, J = 9.8, 6.5 Hz, 1H), 3.39 – 3.26 (m, 2H), 3.25 – 3.13 (m, 1H), 2.97 – 2.79 (m, 2H), 2.64 – 2.49 (m, 2H), 2.00 (t, J = 2.7 Hz, 1H), 1.97 – 1.64 (m, 4H), 1.50 (s, 9H); MS (ESI) m/z 480.4 [M+H]+. Boc-11 (20 mg, 42 µmol) was then deprotected to afford 21 mg (>99% isolated yield) of 1 compound 11 as a white solid. TLC Rf = 0.20 (n-heptane/EtOAc 1:1); H NMR (400 MHz, CD OD) δ 7.62 – 7.58 (m, 1H), 7.52 (dd, J = 9.1, 1.7 Hz, 1H), 4.46 (d, J = 1.2 Hz, 2H), 4.38 3 6 (dd, J = 8.5, 6.5 Hz, 1H), 3.71 – 3.59 (m, 2H), 3.56 (t, J = 6.3 Hz, 2H), 3.30 – 3.14 (m, 3H), 3.09 – 2.98 (m, 1H), 2.59 (ddd, J = 16.5, 8.5, 2.7 Hz, 1H), 2.48 (ddd, J = 16.5, 6.5, 2.7 Hz, 1H), 2.40 (t, J = 2.7 Hz, 1H), 2.05 – 1.91 (m, 2H), 1.90 – 1.76 (m, 2H), 1.31 (bs, 1H); 13C

NMR (126 MHz, CD3OD) δ 168.2, 158.8 (d, J = 250.6 Hz), 142.3 (d, J = 7.4 Hz), 135.4 (d, J = 3.6 Hz), 122.9 (d, J = 3.5 Hz), 120.8 (d, J = 16.0 Hz), 111.5 (d, J = 24.0 Hz), 78.0, 71.0, 53.2, 46.8, 45.7, 40.6, 39.2 (d, J = 6.5 Hz), 25.5, 24.6 (d, J = 2.2 Hz), 23.7, 22.0; 19F NMR + (377 MHz, CD3OD) δ –77.0, –117.8; MS (ESI) m/z 380.4 [M+H] ; HRMS (ESI) found m/z + + 380.14698 [M+H] , C18H23FN3O3S requires m/z 380.14441.

209 (R)-N-(3-(1-benzyl-1H-1,2,3-triazol-4-yl)-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)-8-fluoro- 1,2,3,4-tetra-hydroisoquinoline-6-sulfonamide (12): Compound 12 was synthesised from Boc-11 (16 mg, 33 µmol),

which was dissolved in 1.0 mL of H2O/EtOH/DCM (1:1:1 v/v). To

this was added benzyl azide (4.4 mg, 1.0 equiv), CuSO4.5H2O (3.3 mg, 0.4 equiv), and sodium ascorbate (7.8 mg, 1.2 equiv). The reaction was stirred at rt for 2 days until all the starting material was consumed as monitored by TLC. The product was purified by column chromatography (20-40% EtOAc in n-heptane) to afford 13.4 mg (66% isolated yield) of 1 Boc‑12. H NMR (500 MHz, CDCl3) δ 7.43 (bs, 1H), 7.40 (bs, J = 1.6 Hz, 1H), 7.38 – 7.32 (m, 3H), 7.29 (d, J = 8.1 Hz, 1H), 7.26 – 7.23 (m, 2H), 5.99 (d, J = 9.3 Hz, 1H), 5.51 (d, J = 14.8 Hz, 1H), 5.41 (d, J = 14.8 Hz, 1H), 4.60 (bs, 2H), 4.32 – 4.23 (m, 1H), 3.64 (bs, 2H), 3.23 (t, J = 6.8 Hz, 2H), 3.07 (t, J = 6.9 Hz, 2H), 3.03 – 2.79 (m, 4H), 1.89 – 1.54 (m, 4H), 1.49 (s, 9H); MS (ESI) m/z 613.1 [M+H]+. Boc-12 (12 mg, 20 µmol) was dissolved in 1.0 mL of TFA/DCM (4:1 v/v) and stirred at rt until TLC showed the reaction was complete.

The reaction mixture was diluted with DCM (10 mL) and washed with satd aq NaHCO3

(10 mL) and brine (10 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude mixture was purified using flash column chromatography (0-10% MeOH in DCM) to afford 7.3 mg (73% isolated yield) of the 1 desired triazole 12. TLC Rf = 0.15 (10% MeOH in DCM); H NMR (400 MHz, CDCl3) δ 7.44 (s, 1H), 7.41 – 7.34 (m, 4H), 7.28 – 7.22 (m, 3H), 5.54 (d, J = 14.8 Hz, 1H), 5.44 (d, J = 14.8 Hz, 1H), 4.28 (dd, J = 7.6, 6.3 Hz, 1H), 4.06 (s, 2H), 3.24 (t, J = 6.6 Hz, 2H), 3.17 – 3.05 (m, 5H), 3.00 (t, J = 7.5 Hz, 2H), 2.85 (dt, J = 12.1, 5.9 Hz, 2H), 1.86 – 1.58 (m, 4H); 13 C NMR (126 MHz, CDCl3) δ 168.2, 158.9 (d, J = 249.3 Hz), 142.7, 138.9 (d, J = 4.9 Hz), 138.6 (d, J = 7.4 Hz), 134.7, 129.1, 128.7, 128.0, 123.6 (d, J = 3.3 Hz), 122.5, 110.9 (d, J = 24.7 Hz), 54.5, 54.1, 46.4, 45.9, 42.7, 42.3 (d, J = 3.6 Hz), 30.0, 28.9, 25.8, 23.9; 19F + NMR (377 MHz, CDCl3) δ –117.0; MS (ESI) m/z 513.1 [M+H] .

(9H-Fluoren-9-yl)methyl (S)-(1-oxo-1-(pyrrolidin-1-yl)-3-(tritylthio)propan-2-yl) carbamate (13): 6 Compound 13 (187 mg, 15% isolated yield, white solid) was prepared analogous to compound 2 starting from Fmoc-D-Cys(Trt)-OH (1.17 g, 2.0 1 mmol, 1.0 equiv). TLC Rf = 0.30 (n-heptane/EtOAc 1:1); H NMR (400 MHz,

CDCl3) δ 7.74 (ddd, J = 7.5, 2.1, 1.1 Hz, 2H, Ar-H), 7.59 (d, J = 6.8 Hz, 1H, Ar-H), 7.44 – 7.34 (m, 9H, Ar-H), 7.30 – 7.24 (m, 9H, Ar-H), 7.23 – 7.17 (m, 3H, Ar-H), 5.62 (d, J = 8.8 Hz, 1H, CH), 4.46 – 4.25 (m, 3H), 4.20 (t, J = 7.2 Hz, 1H), 3.49 – 3.29 (m, 2H), 2.96 (q, J = 8.3, 6.8 Hz, 1H), 2.56 (d, J = 6.7 Hz, 2H), 1.87 – 1.71 (m, 5H); 13C NMR

(101 MHz, CDCl3) δ 168.7, 155.7, 144.5, 143.9, 143.8, 141.3, 141.2, 129.7, 128.0, 127.7,

210 127.1, 127.1, 126.8, 125.2, 119.9, 67.1, 53.4, 51.9, 50.8, 47.1, 46.4, 46.0, 34.5, 25.9, 24.1; MS (ESI) m/z 639.6 [M+H]+.

(S)-2-Amino-1-(pyrrolidin-1-yl)-3-(tritylthio)propan-1-one (14): Compound 15 (100 mg, 0.16 mmol, 1.0 equiv) was dissolved in 1.0 mL of DCM, followed by the dropwise addition of DBU (26.2 mg, 0.17 mmol, 1.1 equiv) dissolved in 0.25 mL of DCM. The reaction was stirred at rt for 1 h, and the solvent removed in vacuo. Purification by column chromatography (0-5% MeOH in DCM) afforded 59 mg (91% isolated yield) of 14 as an off-white solid. 1 TLC Rf = 0.40 (MeOH/DCM 1:9); H NMR (400 MHz, CDCl3) δ 7.45 – 7.40 (m, 6H), 7.31 – 7.24 (m, 6H), 7.23 – 7.18 (m, 3H), 3.45 – 3.29 (m, 2H), 3.22 – 3.10 (m, 2H), 2.84 – 2.75 (m, 1H), 2.51 (dd, J = 12.7, 8.6, 1H), 2.46 (dd, J = 12.7, 5.2, 1H), 1.88 – 1.72 (m, 4H), 1.60 13 (bs, 2H); C NMR (126 MHz, CDCl3) δ 171.6, 144.8, 129.7, 127.9, 126.7, 67.1, 46.0, 45.9, 37.7, 25.9, 24.0; MS (ESI) m/z 417.3 [M+H]+. tert-Butyl (S)-8-fluoro-6-(N-(1-oxo-1-(pyrrolidin-1-yl)-3-(tritylthio)propan-2-yl) sulfamoyl)-3,4-dihydro-isoquinoline-2(1H)-carboxylate (15): Compound 15 (45.2 mg, 87% isolated yield, white solid) was prepared analogous to compound Boc-7, starting from sulfonyl chloride 6 (25 mg, 71 µmol, 1.0 equiv) and amine 14 (36 mg, 86 1 µmol, 1.2 equiv). TLC Rf = 0.25 (n‑heptane/EtOAc 1:1); H NMR

(500 MHz, CDCl3) δ 7.41 (d, J = 1.5 Hz, 1H), 7.33 – 7.18 (m, 18H), 5.56 (d, J = 9.4 Hz, 1H), 4.54 (s, 2H), 3.84 (d, J = 7.8 Hz, 1H), 3.57 (s, 2H), 3.22 (dt, J = 12.7, 6.8 Hz, 1H), 3.13 (s, 1H), 2.99 (s, 1H), 2.83 – 2.65 (m, 3H), 2.52 (dd, J = 12.8, 8.3 Hz, 1H), 2.39 – 2.32 (m, 1H), 1.80 – 1.64 (m, 4H), 1.50 (s, 9H); MS (ESI) m/z 752.7 [M+Na]+.

(S)-8-fluoro-6-(N-(3-mercapto-1-oxo-1-(pyrrolidin-1-yl)propan-2-yl)sulfamoyl)-1,2,3,4- tetrahydroiso-quinolin-2-ium trifluoroacetate (16): Compound 15 (38 mg, 50 µmol, 1.0 equiv) was dissolved in 1.0 mL of DCM, followed by the addition of triisopropylsilane 6 (TIPS, 12 µL, 60 µmol, 1.1 equiv) and TFA (0.25 µL). The mixture was left to stir at rt for 2 h. Next the solvent was removed in vacuo and the crude was purified by preparative HPLC to afford 11.5 mg (44 1 % isolated yield) of 16 as a white solid. H NMR (500 MHz, CD3OD) δ 7.58 (d, J = 1.4 Hz, 1H), 7.50 (dd, J = 9.1, 1.7 Hz, 1H), 4.48 – 4.40 (m, 2H), 4.27 (dd, J = 8.1, 6.4 Hz, 1H), 3.61 (qt, J = 10.3, 6.8 Hz, 2H), 3.54 (t, J = 6.3 Hz, 2H), 3.29 – 3.25 (m, 1H), 3.23 – 3.17 (m, 2H), 3.02 (dt, J = 12.0, 7.0 Hz, 1H), 2.74 (dd, J = 13.6, 8.0 Hz, 1H), 2.63 (dd, J = 13.6, 6.4 Hz, 13 1H), 2.01 – 1.74 (m, 5H); C NMR (126 MHz, CD3OD) δ 168.1, 158.9 (d, J = 251 Hz),

211 142.4 (d, J = 7.3 Hz), 135.5 (d, J = 3.5 Hz), 122.9 (d, J = 3.4 Hz), 120.9 (d, J = 16.2 Hz), 111.5 (d, J = 23.9 Hz), 56.9, 45.7, 40.6, 39.1 (d, J = 6.5 Hz), 25.5 (d, J = 5.6 Hz), 24.6 (d, J 19 + = 2.1 Hz), 23.7; F NMR (377 MHz, CD3OD) δ –77.1, –117.7; MS (ESI) m/z 398.2 [M+H] .

6.6.3. Inhibition curves for compounds 7, 11, 12, and 16 For a description of the experimental details on the inhibition measurements see the experimental section of Chapter 2 (Section 2.4.).

Compound 7 Compound 11

IC50 = 13.9 ± 1.4 µM 100 IC50 = 16.2 ± 0.5 µM 100

50 50 Activity (%)

0 0 1 2 3 4 5 2 3 4 5 6 Log [11] (nM) Log [7] (nM)

Compound 12 Compound 16

IC50 = 8.52 ± 1.1 µM 100 IC50 = 1.39 ± 0.2 µM 100

50 50 Activity (%) Activity (%)

0 0 2 4 6 1 2 3 4 5 6 Log [12] (nM) Log [16] (nM)

Figure S1. Inhibition curves for compounds 7, 11, 12, and 16.

212 6.6.2. Azides and alkynes used in the templated-click approach

Figure S2. Azides and alkynes that were evaluated in the SETD7 templated click approach towards the identification of novel inhibitors of SETD7 based on (R)-PFI-2.

213 6.7. References

1. Cresset SPARK and TORCH, https://www.cresset-group.com. 2. J. J. Li, in Name Reactions: A Collection of Detailed Mechanisms and Synthetic Applications Fifth Edition, Springer International Publishing, Cham, 2014, 347-348. 3. A. Armstrong, L. H. Jones, J. D. Knight, R. D. Kelsey, Org. Lett. 2005, 7, 713-716. 4. A. Kumar, G. Shelke, V. Rao, M. Jha, T. Cameron, Synlett. 2015, 26, 404-407. 5. H. Q. Do, R. M. Khan, O. Daugulis, J. Am. Chem. Soc. 2008, 130, 15185-15192. 6. J. R. Johansson, T. Beke-Somfai, A. Said Stalsmeden, N. Kann, Chem. Rev. 2016, 116, 14726-14768. 7. M. Mondal, M. Y. Unver, A. Pal, M. Bakker, S. P. Berrier, A. K. Hirsch, Chem. Eur. J. 2016, 22, 14826-14830. 8. R. Manetsch, A. Krasinski, Z. Radic, J. Raushel, P. Taylor, K. B. Sharpless, H. C. Kolb, J. Am. Chem. Soc. 2004, 126, 12809-12818. 9. I. Glassford, C. N. Teijaro, S. S. Daher, A. Weil, M. C. Small, S. K. Redhu, D. J. Colussi, M. A. Jacobson, W. E. Childers, B. Buttaro, A. W. Nicholson, A. D. MacKerell, Jr., B. S. Cooperman, R. B. Andrade, J. Am. Chem. Soc. 2016, 138, 3136-3144. 10. V. P. Mocharla, B. Colasson, L. V. Lee, S. Roper, K. B. Sharpless, C. H. Wong, H. C. Kolb, Angew. Chem. Int. Ed. 2004, 44, 116-120. 11. A. Bhardwaj, J. Kaur, M. Wuest, F. Wuest, Nat. Commun. 2017, 8, 1. 12. M. Mondal, A. K. Hirsch, Chem. Soc. Rev. 2015, 44, 2455-2488. 13. Z. Fang, W. He, X. Li, Z. Li, B. Chen, P. Ouyang, K. Guo, Bioorg. Med. Chem. Lett. 2013, 23, 5174-5177. 14. M. Demetriades, I. K. Leung, R. Chowdhury, M. C. Chan, M. A. McDonough, K. K. Yeoh, Y. M. Tian, T. D. Claridge, P. J. Ratcliffe, E. C. Woon, C. J. Schofield, Angew. Chem. Int. Ed. 2012, 51, 6672-6675. 15. B. Rasmussen, A. Sorensen, H. Gotfredsen, M. Pittelkow, Chem. Commun. 2014, 50, 3716-3718. 16. S. Otto, R. L. Furlan, J. K. Sanders, Science 2002, 297, 590-593. 17. O. Ramström, J.-M. Lehn, ChemBioChem. 2000, 1, 41-48. 18. S. Zameo, B. Vauzeilles, J.-M. Beau, Eur. J. Org. Chem. 2006, 2006, 5441-5444. 19. G. Joshi, E. V. Anslyn, Org. Lett. 2012, 14, 4714-4717. 20. S. Sobczak, W. Drozdz, G. I. Lampronti, A. M. Belenguer, A. Katrusiak, A. R. Stefankiewicz, Chem. Eur. J. 2018, 24, 8769-8773. 21. R. Mah, J. R. Thomas, C. M. Shafer, Bioorg. Med. Chem. Lett.2014 , 24, 33-39. 6 22. J. G. Robertson, Biochemistry 2005, 44, 5561-5571. 23. H. B. Guo, H. Guo, Proc. Natl. Acad. Sci. USA 2007, 104, 8797-8802. 24. J. M. Hatcher, G. Wu, C. Zeng, J. Zhu, F. Meng, S. Patel, W. Wang, S. B. Ficarro, A. L. Leggett, C. E. Powell, J. A. Marto, K. Zhang, J. C. Ki Ngo, X. D. Fu, T. Zhang, N. S. Gray, Cell Chem. Biol. 2018, 25, 460-470. 25. E. C. Hett, H. Xu, K. F. Geoghegan, A. Gopalsamy, R. E. Kyne, Jr., C. A. Menard, A. Narayanan, M. D. Parikh, S. Liu, L. Roberts, R. P. Robinson, M. A. Tones, L. H. Jones, ACS Chem. Biol. 2015, 10, 1094-1098. 26. L. A. Crawford, E. Weerapana, Mol. Biosyst. 2016, 12, 1768-1771. 27. A. J. Brouwer, T. Ceylan, A. M. Jonker, T. van der Linden, R. M. Liskamp, Bioorg. Med. Chem. 2011, 19,

214 2397-2406. 28. Chemical Computing Group Molecular Operating Environment (MOE), https://www.chemcomp.com/.

215 216 7

Summary, Nederlandse samenvatting, Dankwoord, Curriculum Vitae & List of Publications

217 Summary

218 Over the past decade it has become evident that histone lysine methyltransferases (HKMTs) play an essential role in the normal functioning of cells, and an aberrant activity of HKMTs has been linked to a variety of diseases, including cancer. Consequently, the development of selective and potent small molecule inhibitors targeting HKMTs involved in human epigenetic regulation has attracted significant interest from both industry and academic research groups. The work described in this dissertation is mainly focused on the development of inhibitors targeting SETD7, a HKMT that was first described as a monomethyltransferase of lysine 4 on histone 3.

In Chapter 2 of this thesis we have explored the structure-activity relationship of (R)‑PFI‑2, the most potent SETD7 inhibitor known to date (Figure 1). In total, 29 analogues were synthesised bearing modifications on the tetrahydroisoquinoline moiety, the pyrrolidine amide part, and the amino acid side chain. The ability to inhibit recombinantly expressed human SETD7 was evaluated using a MALDI-TOF MS based assay monitoring the methylation of a synthetic histone 3 peptide mimic containing a lysine at position 4. It was found that on the pyrrolidine amide part, small modifications were tolerated well, however with increasing size of the substituent, the potency of the corresponding analogues decreased significantly. Analogues bearing various substituents on the phenyl ring were all found the be highly potent, with IC50 values similar to that of (R)-PFI-2. We concluded that the most important contributor to the + excellent potency of (R)-PFI-2 was the tetrahydroisoquinoline’s NH2 . Replacement of this nitrogen almost completely abolished the ability to inhibit SETD7.

CF3

O O S O N H HN N

Figure 1. Structure of (R)-PFI-2, inhibitor of histone lysine methyltransferase SETD7.

Chapter 3 describes the synthesis and evaluation of 20 novel (R)-PFI-2 analogues which were designed to act as inhibitors and small molecule substrates of SETD7. In this work, (R)-PFI-2’s pyrrolidine amide was replaced with several nucleophilic moieties, such as amines, thiol, alcohol, carboxylic acid, amide, alkene or alkyne functionality. The 7 most potent inhibitor was found to possess a hydroxyethyl side chain, with IC50 = 0.96 µM. Notably, we observed that various amine containing side chains were efficiently

219 methylated by human SETD7, however the optimal length was found to be two carbons between the amide and amine (Figure 2). This analogue was fully monomethylated by SETD7 within 2 h, and upon prolonged incubation (>20 h) also dimethylation started to occur, an unusual observation because SETD7 is known to only monomethylate its natural lysine containing substrates. These findings also show that SETD7 is highly selective towards the methylation of amines, but not other functional groups. After additional optimisation and evaluation, these or related analogues might be used to modulate the levels of SAM present in in vitro as well as in vivo studies of SETD7 and related methyltransferases.

O O O SETD7 O S O S O N SAM N H H HN HN HN HN Me NH2 N F F Me

Figure 2. SETD7-catalysed mono- and dimethylation of a synthetic substrate.

The work described in Chapter 4 addresses the use of computational tools to identify potential replacements for the sulfonamide core of (R)-PFI-2. Cresset’s scaffold-hopping and fragment replacement tool SPARK was applied to generate a library of possible bioisosteres. From this collection the 1,5-disubstituted imidazole was selected based on visual inspection, docking studies and evaluation of synthetic accessibility (Figure 3). Initially, the synthesis was explored using the van Leusen three component reaction. With this reaction 1,5-disubstituted imidazoles can be accessed directly in two steps in a one-pot reaction from readily available aldehydes, amines and tosylmethyl isocyanide (TosMIC). After identification of the optimal conditions, a library of 56 analogues was synthesised and the ability of each imidazole to inhibit SETD7 was evaluated. Unfortunately, none of the analogues inhibited SETD7 within limits of detection.

H CF3 N CF3 F O O S O N O H N HN N N N 7 F

Figure 3. Bioisosteric replacement of the sulfonamide core of (R)-PFI-2 with a 1,5-disubstituded imidazole core.

220 In the Chapter 5, we reported the discovery of novel type inhibitors of histone 3 lysine 9 methyltransferases G9a and GLP. These inhibitors, in particular ebselen, cisplatin and disulfiram, target reactive cysteine residues of the zinc fingers that are present in both enzymes. With the use of FluoZin‑3, a selective fluorescent indicator for the detection of free Zn(II) in solution, we demonstrated that zinc ions are rapidly ejected in the presence of these inhibitors. Furthermore, CD analysis revealed that the secondary or/ and tertiary structure of G9a and GLP is significantly altered after exposure to ebselen, cisplatin or disulfiram.

221 Nederlandse Samenvatting

222 In het afgelopen decennium is het duidelijk geworden dat histon lysine methyltransferases (HKMTs) een essentiële rol spelen in het normaal functioneren van cellen en dat afwijkende activiteit van deze enzymen gelinkt is aan verschillende ziektebeelden, waaronder kanker. Als gevolg heeft de ontwikkeling van selectieve en potente inhibitoren voor deze HKMTs, die betrokken zijn bij epigenetische regulatie, de interesse en aandacht getrokken van onderzoeksgroepen op universiteiten en in de farmaceutische industrie. In dit proefschrift ligt de nadruk op de ontwikkeling van inhibitoren van SETD7; een HKMT die aanvankelijk geïdentificeerd is als mono methyltransferase van lysine 4 op histon 3.

In hoofdstuk 2 van dit proefschrift is onderzoek gedaan naar de structuur- activiteitsrelatie van (R)-PFI-2, de meest potente inhibitor van SETD7, tot nu toe is beschreven in de literatuur (Figuur 1). In totaal zijn er 29 analogen gesynthetiseerd met verschillende modificaties op de tetrahydroisoquinoline groep, het pyrrolidine amide en de aminozuur zijketen. Het vermogen om recombinant menselijk SETD7 te inhiberen is geëvalueerd met behulp van een op MALDI-TOF MS gebaseerde assay, waarbij de methylering van een synthetisch peptide dat histon 3 met een lysine op positie 4 imiteert wordt gemonitord. Wij constateerden dat op het pyrrolidine amide kleine variaties getolereerd worden, echter naarmate de grootte van de substituent toeneemt, was er een significante afname in de potentie waarneembaar. Analogen met verschillende substituenten op de fenyl ring waren allen zeer potent, met IC50 waarden + vergelijkbaar met die van (R)-PFI-2. Wij zijn tot de conclusie gekomen dat de NH2 van het tetrahydroisoquinoline de meest belangrijke bijdrage levert aan de excellente potentie van (R)-PFI-2. Vervanging van deze stikstof in verschillende analogen resulteerde in bijna compleet onvermogen om SETD7 te inhiberen.

CF3

O O S O N H HN N

F Figuur 1. Structuur van (R)-PFI-2, inhibitor van histon lysine methyltransferase SETD7.

Hoofdstuk 3 beschrijft de synthese en evaluatie van 20 nieuwe (R)-PFI-2 analogen 7 die ontworpen zijn om zowel als inhibitor als substraat van SETD7 te fungeren. In dit hoofdstuk is het pyrrolidine amide in (R)-PFI-2 vervangen met verschillende nucleofiele

223 groepen, waaronder verschillende amines, thiol, alcohol, carbonzuur, amide, alkeen of alkyn functionaliteit. De meest potente inhibitor van deze set analogen bevatte een

hydroxyethyl zijketen, met een IC50 waarde van 0.96 µM. Daarnaast constateerden we dat verschillende analogen met een amine in de zijketen efficiënt gemethyleerd worden door menselijk SETD7. De optimale lengte van de zijketen bevatte twee koolstofatomen tussen het amide en het amine (Figuur 2). Dit analoog werd volledig gemonomethyleerd door SETD7 in twee uur tijd en bij langere incubatietijd (twintig uur of meer) trad ook dimethylering op. Dit laatste is een ongebruikelijke observering, omdat SETD7 bekend staat als een monomethylase van natuurlijke lysine bevattende substraten. Deze bevindingen laten ook zien dat SETD7 zeer selectief is in de methylering van amines ten opzichte van andere functionele groepen. Na aanvullende optimalisatie en experimenten zouden deze analogen eventueel gebruikt kunnen worden om de hoeveelheid SAM te moduleren in zowel in vitro als in vivo studies aan SETD7 en gerelateerde methyltransferases.

O O O SETD7 O S O S O N SAM N H H HN HN HN HN Me NH2 N F F Me

Figuur 2. SETD7 gekatalyseerde mono- en dimethylering van een synthetisch substraat.

In hoofdstuk 4 zijn computationele tools gebruikt om potentiële bioisosterische vervangingen van de sulfonamide kern van (R)-PFI-2 te identificeren. Cresset’s SPARK, een programma geschikt voor ‘scaffold-hopping’ en het vervangen van fragmenten binnen een molecuul, is gebruikt om een aantal reeks bioisosteren te genereren. Op basis van visuele inspectie, docking studies en evaluatie van synthetische toegankelijkheid, is uit deze collectie bioisosteren het 1,5-digesubstitueerde imidazool gekozen. In eerste instantie is de chemie verkend, waarbij gebruik werd gemaakt van de van Leusen 3-componentenreactie. Met deze reactie worden 1,5-digesubstitueerde imidazolen in slechts twee stappen gemaakt van aldehyden, amines en tosylmethyl isocyanide. Na het vaststellen van de optimale condities is een collectie van 56 verbindingen 7 gesynthetiseerd en het vermogen om SETD7 te inhiberen geëvalueerd. Helaas was geen van de verbindingen in staat om SETD7 de inhiberen binnen de limieten van wat meetbaar is.

224 H CF3 N CF3 F O O S O N O H N HN N N N F

Figuur 3. Bioisosterische vervanging van de sulfonamide kern van (R)-PFI-2.

In hoofdstuk 5 beschrijven we het onderzoek naar nieuwe typen inhibitoren van histon 3 lysine 9 methyltransferases G9a en GLP. Deze inhibitoren, met name ebselen, cisplatina en disulfiram, richten zich op reactieve cysteïne residuen in de zink vingers die in beide enzymen aanwezig zijn. Met behulp van FluoZin-3, een selectieve fluorescente indicator voor de detectie van vrij Zn(II) in oplossing, hebben we laten zien dat zink- ionen snel verwijderd worden bij de aanwezigheid van deze inhibitoren. Verder hebben we met behulp van CD analyse aangetoond dat de secundaire en/of tertiaire structuur van zowel G9a als GLP significant veranderd na blootstellig aan ebselen, cisplatina of disulfiram.

225 Dankwoord

226 Dan zijn we nu aangekomen bij het enige hoofdstuk dat door iedereen gelezen zal worden. Wellicht ook het belangrijkste hoofdstuk, want hoewel ik dit proefschrift zelf geschreven heb, was dit zonder de steun en hulp van familie, vrienden en collega’s nooit gelukt. Hierbij dus een kleine ode aan iedereen die direct of indirect heeft bijgedragen aan het tot stand komen van dit proefschrift.

Dear Jasmin, first of all I would like to thank you for offering me the opportunity to work with you in the exciting field of epigenetics. I am very grateful for everything you taught me, your daily guidance and supervision. I very much enjoyed our frequent discussions about all the projects we were working on, science in general, and occasionally football. You are a great mentor, and I am very proud of what we have achieved. I wish you all the best in your future career, of which I am sure will bring you many highly cited publications in excellent journals.

Beste Floris, bedankt voor alle tijd die je de afgelopen jaren in mijn promotietraject hebt gestoken en voor de uitstekende begeleiding. Met name het laatste jaar, toen Jasmin vertrokken was naar Denemarken, heb ik veel gehad aan onze meetings, jouw hulp met de afronding van mijn promotie en het advies met betrekking tot de volgende stap in mijn carrière.

De manuscriptcommissie: Daniela, Huib en Evan, bedankt voor jullie tijd voor het zorgvuldig lezen van het manuscript van mijn proefschrift.

Dr. Roel, jij hebt een belangrijke rol gespeeld tijdens mijn promotie, waarvoor ik je zeer dankbaar ben. Allereerst omdat je mijn lunchbuddy was, maar des te meer omdat je altijd beschikbaar was om me te adviseren of te helpen met verschillende experimenten, het uitleggen van technieken die mij onbekend waren, of het analyseren en uitwerken van data. Uiteraard ook bedankt dat je mijn paranimf bent, ik ben er trots op dat ik op deze manier mijn tijd als promovendus met jou aan mijn zijde kan afsluiten. Ik wens je heel veel succes in de toekomst, ik denk dat je een fantastisch wetenschapper bent, dus Veni of geen Veni, ik twijfel er niet aan dat je een mooie carrière tegemoet gaat. En als laatste; ik mis onze wandelingen naar de koffieautomaat (misschien wel het meest van allemaal).

Mark, ik kan heel veel lovende woorden opschrijven over jou als mijn maat, maar het feit dat als mensen mij vragen waar mijn wederhelft is en ze dan jou bedoelen in plaats 7 van Anne, zegt wat mij betreft genoeg over onze vriendschap. Bedankt dat je er altijd voor me bent en natuurlijk ook omdat je paranimf bent bij mijn promotie!

227 Meneer Boer (what would you do if you were a molecule?): bedankt voor de inspirerende scheikundelessen op de middelbare school, mede dankzij uw passie voor het vak, die u op mij in ieder geval goed heeft overgedragen, heb ik het punt kunnen bereiken waar ik mij op dit moment bevind.

Zoals te zien is op de publicatielijst heb ik het werk niet alleen gedaan. In tegendeel, het werk dat beschreven staat in deze publicaties zou nooit mogelijk geweest zijn zonder de studenten waarmee ik heb mogen samenwerken gedurende deze vier jaar: Tjeu, Roderick, Daan, Peter, Joris, Maryam, Remi, Miriam, Cynthia, Rens, Zach en Tobias, bedankt voor jullie inzet en voor de gezelligheid zowel in het lab als daarbuiten. Ik heb veel van jullie geleerd, ik hoop jullie ook iets van mij. Heel veel succes in jullie huidige en toekomstige carrières!

Dear Abbas, one of the best things about the past four years is that I got to work with you in the lab every day (though, for me ‘every day’ was Monday to Friday, you usually added two extra days). You are truly the most helpful, kind, and good-hearted person I know. I am very grateful for all the times that you helped me out with all kinds of experiments and very proud of what we achieved in our joint projects. I wish you all the best in your future career, and hope we will work together again at some point. We waren een super-team ;)

All my former colleagues in the Mecinović group: Bas, bedankt voor het tot expressie brengen van de eiwitten die we nodig hadden voor onze projecten en des te meer voor alle dingen die je aan mij en Abbas geleerd hebt, zodat we na jouw vertrek nog steeds aan enzymen konden komen om ons werk voort te zetten. Vijay, it was an honour to work with you, and I very much appreciate that you were always willing to help me with both practical as well as theoretical problems, mostly related to organic chemistry. I will always remember the wonderful Diwali festival celebration! Giordano, I really enjoyed having you as a friend and colleague, I wish you all the best with your PhD in Denmark! Yali, I am very grateful for all the work and help you performed/provided on the biological aspects of our projects, and for always being the most happy person in the entire building, if not all of Nijmegen. I hope we will meet again in the future! Miriam, ik vond het erg leuk en leerzaam om jou te begeleiden tijdens jouw bachelor- en masterstage, je hebt in deze periode erg goed en mooi werk verricht wat terug te vinden is in hoofdstuk 3 van dit proefschrift, hiervoor ben ik je eeuwig dankbaar! Dit 7 hoofdstuk gaat zonder twijfel een mooie publicatie opleveren. Jona, hoewel jij je vaak in vleugel 1 verstopte en later zelfs permanent verhuisde, vond ik het altijd erg gezellig als je wat peptides kwam maken bij ons op het lab.

228 Vleugel 4 was jarenlang mijn tweede thuis. Gelukkig zat ik hier niet alleen, maar heb ik door de jaren heen deze plek kunnen delen met heel veel leuke, gezellige en vriendelijke collega’s en studenten van over de hele wereld. In willekeurige volgorde; Sjoerd R., Jaw, Sjoerd van der G., Dion, Renate, Nicole, Vu, Ruben, Romano, Christian, Aline, Marijn, Arthur, Zhaobao, Jan, Mariarosa, Guilherme, Erik, Rafael, Noël, Vera, Esther, Joyce, Emma, Lena en Emilia: bedankt voor de mooie tijd!

Ook in de andere vleugels van het Huygensgebouw had ik fantastische collega’s: Rens & Lianne (ik hoop dat jullie de tijd van jullie leven hebben aan de andere kant van de planeet, zorg maar wel dat jullie op tijd terug zijn voor Drift!), Floris, Iris, Selma, Lise, Bastiaan, Fleur, Emiel, Hidde, Victor, Ivan, Alejandra, Torben, Bob, Yvonne, Sam, Lorenzo, Peter, Claudia, Antoine, Max, Luuk, Freek, Laura, Jordi, Albert, Pieter, Jeroen, Melek, Maaruthy, Oliver, Aleksandr, Ilia, Pim en Daniël. Bedankt allemaal voor de mooie tijd!

All professors and staff members of the molecular cluster; Wilhelm, Roeland, Jana, Daniela, Dennis, Paul, Hans, Peter, Evan, Thomas, Kim, Dani, Martin. Thank you for your valuable input during all the group- and cluster meetings.

Professor Pruijn, bedankt voor het beschikbaar stellen van ruimte in uw lab, zodat Abbas en ik hier de eiwit expressies konden doen tijdens het eerste gedeelte van onze promoties. En natuurlijk Wilma en Ilmar voor hun hulp bij het uitvoeren van deze experimenten, zonder jullie hadden wij ongetwijfeld meerdere keren met lege epjes (in ieder geval zonder eiwitten erin) gestaan.

Marieke en Jacky, bedankt voor alle dingen die jullie voor mij geregeld hebben de afgelopen jaren! Ik vond het altijd erg gezellig om bij jullie op kantoor even een praatje te maken! Uiteraard ook mijn dank aan Desiree en Paula voor het waarnemen van jullie taken als jullie er niet waren.

Alle technici op de afdelingen: Peter van D., Peter van G., Ad, Paul, Helene, Jan, Theo, Frank, bedankt voor alle support: het voorzien van alle benodigdheden voor het labwerk, allerhande reparaties, introducties voor apparaten en hulp met het opzetten van experimenten. Zonder jullie zou de gemiddelde PhD niet vier maar minstens acht jaar duren. 7 Verder wil ik Ricardo, Christel, Geert-Jan, Gerben, Lars, Jeroen, Mirjam, Rene, Serge, Stefan en Victor, mijn collega’s van de piketploeg bedanken voor de leuke tijd. Een

229 oproep op de pieper kwam niet altijd even goed uit, maar was altijd leerzaam en een welkome afwisseling van het labwerk.

The people at Mercachem who have helped me greatly during my PhD: Anita, Richard, Eddy, and Ruben for their valuable input and contributions to the projects described in this thesis. And those who have helped me in some way or the other during the 2.5 months I spent in the department of parallel chemistry: Brian (voor de uitstekende dagelijkse begeleiding), Jorg, Robin, Erwin, Erik, Martijn, Marcel, Dennis, Sergio, Otto, Bram, Eef & Pascal (voor het preppen van al mijn compounds!). Ik ben blij dat ik jullie sinds oktober 2019 officieel collega’s kan noemen.

Buiten het werk om zijn er een aantal mensen die ik wil bedanken, hoewel jullie vaak geen idee hadden van wat ik precies uitspookte op de universiteit, zorgden jullie er met zijn alle voor dat ik een goede balans kon bewaren tussen werk en ontspanning. Olivier, Dennis, Tom, Frank, Wouter, Valmir, Jack, Robert, Rens, Rinaldo, Joris; we wonen allemaal verspreid door het land en zien elkaar niet altijd even vaak, desondanks ben ik jullie erg dankbaar voor jullie vriendschap en geniet ik er altijd van als ik jullie weer zie op feestjes, verjaardagen, weekendjes weg, festivals, of om wat voor reden dan ook. Michiel (Buur, tijd voor een kopje koffie?!), mijn sportmaatje en goede vriend; met jou even de beest uithangen bij CrossFit Waalfront is altijd gezellig en een perfecte uitlaatklep na een dag werken & ons tripje naar Vietnam was fantastisch en zal ik nooit vergeten! Edward en Renee, een hapje eten, drankje doen, of een festival met jullie doet me altijd goed! De Jongens van Holland: Nick, Mark, Daan, Wouter, Rens, Bas, Koen, Lars, Maarten, Mario, Ronnie Rover, Pim, Michiel, op vrijdagavond met jullie de werkweek afsluiten met een potje zaalvoetbal is voor mij (bijna) elke week weer een genot. Berend, ik ben enorm dankbaar dat wij ooit collega’s zijn geworden bij Bijlesnetwerk en dat hier zo’n mooie vriendschap uit voort is gekomen!

Martin, Ellen, Tom en Karlijn, of het nou in Kerkrade, Amsterdam of in Nijmegen is, ik kijk altijd uit naar een weekend vol gezelligheid met jullie! Bedankt voor alle leuke momenten en natuurlijk jullie support door de jaren heen.

Mijn lieve Oma de Reu en Opa Klaas, omdat jullie altijd zo goed voor ons zijn en voor jullie oprechte interesse in alles wat ik doe.

7 Lieve Robin, Annique, Jeroen, Eelco, Donna, het is altijd fijn thuiskomen in Zeeland als jullie er zijn, bedankt dat jullie altijd klaar staan om me te helpen!

230 Lieve Pap en Mam, bedankt voor alles: voor jullie onvoorwaardelijke steun en hulp bij alles wat ik doe. Ik ben enorm trots op wat jullie bereikt hebben en dankbaar dat jullie altijd voor ons klaar staan. Zonder jullie was dit nooit gelukt!

Lieve Anne, zelfs na al die jaren (12.5!) dat we samen zijn ben ik nog steeds gelukkig dat ik mijn leven met jou kan delen. Ik wil je bedanken voor al de mooie momenten die we samen hebben mogen meemaken en voor je steun op de momenten dat het wat minder ging. We gaan samen een prachtige toekomst tegemoet, ik hou van jou!

231 Curriculum Vitae

232 Daniël Cornelis Lenstra was born on the 24th of December 1990 in Vlissingen, the Netherlands. He attended high school at the SSG Nehalennia in Middelburg, where he received his VWO diploma in 2009. He then moved to Nijmegen to study chemistry at the Radboud University, where he obtained his Bachelor’s degree in 2013 and his Master’s degree in 2015 (Cum laude). His studies included two research internships. The first internship was conducted in the Synthetic Organic chemistry group at the Radboud University under the supervision of Dr. Jasmin Mecinović, where he worked on the development of novel catalytic methodologies for the synthesis of amide bonds. The second internship was performed at the University of Oxford under the supervision of Prof. Dr. Michael C. Willis, where he worked on developing novel strategies towards the synthesis of sulfoxides. After returning to the Netherlands he started his PhD research at the Radboud University in August 2015 under the supervision of Dr. Jasmin Mecinović and Prof. Dr. Floris P. J. T. Rutjes. The results of this research are described in this thesis. After his PhD he joined Mercachem as a Senior Scientist in Medicinal Chemistry.

233 List of publications

234 D. C. Lenstra, R. Vellinga, Z. Wijfjes, C. den Harder, T. Maas and J. Mecinović, “Development of an organophosphorus-catalysed Appel reaction”, Manuscript in preparation.

M. P. Porzberg,* D. C. Lenstra,* E. Damen, R. H. Blaauw, F. P. J. T. Rutjes, A. Wegert and J. Mecinović, “(R)-PFI-2 analogues as substrates and inhibitors of histone lysine methyltransferase SETD7”, Manuscript in preparation.

A. H. K. Al Temimi, Q. Meng, F. Gularek, D. C. Lenstra, H. Guo, E. Weinhold, P. Qian, and

J. Mecinović, “Histone lysine allylation proceeds via the SN2’ mechanism”, Manuscript in preparation.

A. H. K. Al Temimi, V. Tran, R. Teeuwen, A. J. Altunc, H. I. V. Amatdjais-Groenen, P. B. White, D. C. Lenstra, G. Proietti, Y. Wang, A. Wegert, R. Blaauw, P. Qian, W. Ren, H. Guo, and J. Mecinović, “Examining sterically demanding lysine analogues for histone lysine methyltransferase catalysis”, Accepted for publication in Sci. Rep.

A. H. K. Al Temimi, M. Martin, Q. Meng,D. C. Lenstra, P. Qian, H. Guo, E. Weinhold and J. Mecinović, “Lysine ethylation by histone lysine methyltransferases”, ChemBioChem, 2019, DOI: 10.1002/cbic.201900359

A. H. K. Al Temimi, R. Teeuwen, V. Tran, A. J. Altunc, D. C. Lenstra, W. Ren, P. Qian, H. Guo and J. Mecinović, “Importance of the main chain of lysine for histone lysine methyltransferase catalysis”, Org. Biomol. Chem., 2019, 17, 5693-5697.

R. Leenders, R. Zijlmans, B. L. van Bree, M. van de Sande, F. Trivarellia, E. T. Damen, A. Wegert, D. Müller, J. E. Ehlert, D. Feger, C. Heidemann-Dinger, M. Kubbutat, C. Schächtele, D. C. Lenstra, J. Mecinović and G. Müller, “Novel SAR for quinazoline inhibitors of EHMT1 and EHMT2”, Bioorg. Med. Chem. Lett., 2019, 29, 2516-2524.

D. C. Lenstra, J. J. Wolf and Jasmin Mecinović, “Catalytic Staudinger reduction at room temperature”, J. Org. Chem., 2019, 84, 6536-6545.

Y. Wang, Y. V. Reddy, A. H. K. Al Temimi, H. Venselaar, F. H. T. Nelissen, D. C. Lenstra, and J. Mecinović, “Investigating the active site of human trimethyllysine hydroxylase”, Biochem. J., 2019, 476, 1109-1119. 7

235 D. C. Lenstra, P. E. Lenting and J. Mecinović, “Sustainable organophosphorus-catalysed Staudinger reduction”, Green Chem., 2018, 20, 4418-4422.

D. C. Lenstra, E. Damen, R. G. G. Leenders, R. H. Blaauw, F. P. J. T. Rutjes, A. Wegert and J. Mecinović, “Structure-Activity Relationship Studies on (R)-PFI-2 Analogues as Inhibitors of Histone Lysine Methyltransferase SETD7”, Chem. Med. Chem., 2018, 13, 1405-1413.

D. C. Lenstra, A. H. K. Al Temimi and J. Mecinović, “Inhibition of histone lysine methyltransferases G9a and GLP by ejection of structural Zn(II)”, Bioorg. Med. Chem. Lett., 2018, 28, 1234-1238.

D. F. J. Hamstra, D. C. Lenstra, T. J. Koenders, F. P. J. T. Rutjes and J. Mecinović, “Poly(methylhydrosiloxane) as a green reducing agent in organophosphorus-catalysed amide bond formation”, Org. Biomol. Chem., 2017, 15, 6426-6432.

D. C. Lenstra, F. P. J. T. Rutjes, “Organic Synthesis in Flow: Toward Higher Levels of Sustainability” in “Sustainable Flow Chemistry: Methods and Applications”, edited by L. Vaccaro, Wiley‑VCH, 2017, 103-134.

D. C. Lenstra, V. Vedovato, E. Ferrer Flegeau, J. Maydom and M. C. Willis, “One-Pot Sulfoxide Synthesis Exploiting a Sulfinyl-Dication Equivalent Generated from a DABSO/ Trimethylsilyl Chloride Sequence”, Org. Lett., 2016, 18, 2086-2089.

T. Nguyen, D. C. Lenstra and J. Mecinović, “Calcium-Catalysed Amidation of Carboxylic Esters”, RSC Adv. 2015, 5, 77658-77661.

D. C. Lenstra, D. T. Nguyen and J. Mecinović, “Zirconium-Catalysed Direct Amide Bond Formation Between Carboxylic Esters and Amines”, Tetrahedron, 2015, 71, 5547-5553.

D. C. Lenstra, F. P. J. T. Rutjes and J. Mecinović, “Triphenylphosphine-catalysed amide bond formation between carboxylic acids and amines”, Chem. Commun. 2014, 50, 5763-5766. * Authors contributed equally

236 7

237 15,13mm

Danny Lenstra