University of Groningen Development and Application of Novel Scaffolds In

University of Groningen

Development and application of novel scaffolds in drug discovery Boltjes, André

DOI: 10.33612/diss.98161351

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Download date: 11-10-2021 DEVELOPMENT AND APPLICATION OF NOVEL SCAFFOLDS IN DRUG DISCOVERY The MCR approach

André Boltjes The research described in this thesis was carried out at the Department of Drug Design (Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands) and was financially supported by the University of Groningen.

Printing of this thesis was financially supported by the University Library and the Graduate School of Science, Faculty of Science and Engineering, University of Groningen, The Netherlands

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Development and application of novel scaffolds in Drug Discovery The MCR approach

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. C. Wijmenga en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

vrijdag 29 november 2019 om 14.30 uur

door

André Boltjes

geboren op 10 april 1984 te Wieringermeer Promotores Prof. dr. A.S.S. Dömling Prof. dr. F.J. Dekker

Beoordelingscommissie Prof. dr. R.V.A Orru Prof. dr. G.J. Poelarends Prof. dr. ir. A.J. Minnaard

Table of Contents

CHAPTER 1 GENERAL INTRODUCTION 1

CHAPTER 2 FRAGMENT BASED LIBRARY GENERATION FOR 11 THE DISCOVERY OF A PEPTIDOMIMETIC P53-MDM4 INHIBITOR

CHAPTER 3 UGI MULTICOMPONENT APPROACH TO 35 SYNTHESIZE SCHISTOSOMIASIS DRUG PRAZIQUANTEL

CHAPTER 4 UGI 4-CR SYNTHESIS OF γ- AND δ-LACTAMS 51 PROVIDING NEW ACCESS TO DIVERSE ENZYME INTERACTIONS, A PDB ANALYSIS

CHAPTER 5 Gd-TEMDO: DESIGN, SYNTHESIS AND MRI 75 APPLICATION

CHAPTER 6 THE GROEBKE-BLACKBURN-BIENAYMÉ 97 REACTION

CHAPTER 7 DIVERSE ONE-POT SYNTHESIS OF ADENINE 189 MIMETICS FOR BIOMEDICAL APPLICATIONS

CHAPTER 8 SUMMARY AND DISCUSSION 213

SAMENVATTING NEDERLANDSE SAMENVATTING 223

APPENDIX ACKNOWLEDGEMENTS 231 LIST OF PUBLICATIONS ABOUT THE AUTHOR

Chapter 1

Introduction

MCR introduction, Perspective and Outline Chapter 1

Multicomponent reactions

Multicomponent reactions (MCRs) are a special class of chemical transformations, which produce complex scaffolds in a single step by employing three or more starting materials, in which most of the atoms are present in the final product.1 This type of chemistry can be classified as a domino reaction. Similar to polymer chemistry, MCRs proceed through a cascade of intermediate reactions, however, yielding a complex but small molecule. The first example of a MCR reaction is the Strecker amino acid synthesis, developed almost 170 years ago in 1850. The Strecker reaction is a 3-component reaction (3-CR) between an aldehyde or ketone, ammonia and potassium cyanide and yields an α-aminonitrile, subsequent hydrolysis gives access to synthetic α-amino acids (Scheme 1).2 The high atom economy, efficiency, convergent nature and very high bond forming index of MCR reactions make this class extremely useful for drug exploration.3

O R + NH3 + KCN R H H2N COOH Scheme 1. The Strecker amino acid synthesis.

MCRs are often considered to be complex, because the formation of the final products proceed via a number of intermediate steps. In order to understand the complex nature of MCRs, a breakdown of the reaction into intermediate steps could aid in comprehension of the underlying mechanism. Difference in reactivity of each of the individual components is the driving force behind selective reactions, resulting in a predefined order in which each component will react. The most common studied and therefore well-developed early type of MCRs evolve around the reactivity of imine groups, usually obtained through reacting carbonyl compounds with amines. The resulting imines or Schiff bases are electrophiles and can react via electrophilic addition to for example α-methylene carbonyls in the Mannich 3-CR, to β-ketoesters in the Biginelli 3-CR, to boronates (from boronic acids) in the Petasis 3-CR and many other reactions, described in scheme 2.4-8 The formation of the Schiff base is performed in the presence of the third component, the electrophile, in a one-pot fashion, which in practice comes down to adding all the reactants together in a suitable solvent and stir the multicomponent reaction, often under ambient conditions. The selective order generally allows for a clean reaction with little byproducts and high conversion towards a single product.

1 Introduction

O R1 R2 NH Strecker + 2 + HCN R3 R3 N CN R1 R2 H 1

R3 R4 O O H O Mannich + N + R1 R R R5 N R6 R3 R4 1 2 R6 R2 R5

O R1 O O O O Hantsch + NH2 + 2 R2 R1 H N

O R O O 3 Staudinger + NH2 + R2 R3 N R1 H Cl R2 R1

R R O H 1 4 Petasis + N + B(OH)2 R H R2 R3 R4 N 1 R2 R3

O Ar O O O O EtO NH Biginelli + + Ar H H2N NH2 EtO H3C N O H Scheme 2. Overview of the most common MCR 3-CR of amines, carbonyl compounds and nucleophiles. In 1920, Passerini discovered the first isocyanide based MCR reaction (IMCR) and involves an isocyanide, an oxo component and a nucleophile.9 The carbene like properties of the isocyanide group allow for some very interesting transformations, as this functional group can behave as nucleophile and then as electrophile at the same atom, resulting to the so-called α-adduct.10 The IMCR subclass has complemented the field of MCR due to its versatile reactivity, variability and scaffolds and is currently the most widely used/applied type of MCR. Neverthe- less it took roughly 40 years until more developments were made in this field, which can be attributed to the obnoxious smell of the isocyanides. The smell is

2 Chapter 1 considered unpleasant, even in such a way that the US government investigated the use of isocyanides as non-lethal chemical weapons.11 The considered ‘constraint’ is limited to the liquid isocyanides, as higher molecular weight isocyanides are often solid and have little to no smell. Regardless of this fact, the result is poor commercial availability, which is not necessarily problematic as isocyanides can readily be prepared from primary amines or aldehydes in one or two steps.12-14

O O O R1 R2 H Passerini-3CR + + NC N R4 R3 O R4 1920 R1 R2 R3 OH O

O R R O O 1 2 H + NH2 + + NC N Ugi-4CR R3 R5 R4 N R5 1959 R1 R2 R4 OH R3 O

H R1 R2 O H NC N R4 Ugi-3CR + N + R N R R R5 5 1959 R1 R2 3 4 O R3

R1 R2 R5 O H + + + NC R3 N Ugi Tetrazole-4CR N HN3 N R R R5 N 1959 R1 R2 3 4 R4 N N

R O Ts 2 N N + NH + Van Leusen-3CR R 2 1977 R H 2 R NC 1 3 R1 R3

HN R2 Groebke Blackburn O 5- N + + NC N 6- R2 5- Bienaymè-3CR R1 R1 H NH2 6- 1998 N Scheme 3. Some of the well-known IMCRs

A considerable amount of pioneering work with IMCRs was performed by Ugi in the late 1950s. The Ugi-4CR is defined as the reaction of an amine with an aldehyde or ketone, an isocyanide and a carboxylic acid (Scheme 3).15 The limits of IMCRs were explored by Ivar Ugi and since the introduction of the Ugi 4-CR, many new IMCR reactions have found their way to the nowadays well accept-

3 Introduction ed field of MCR chemistry. The work described in this thesis is based on drug design, utilizing the Ugi reaction and several variations of the Ugi reaction to synthesize drug like compounds and medical probes. 1 Drug discovery and MCR. Identification of biochemical pathways related to a disease and the intervention of potential targets is the first step in the discovery of new medicines. Luckily more and more molecular targets are being identified, allowing for target vali- dation and development of small molecules. High throughput screening (HTS) is a common method in the pharmaceutical industry to discover new leads for molecular targets. Such screenings, however, are expensive, up to $10 million per screening, show low efficiency and have limited success rate. Therefore, -ac ademia aim at development of smarter design and selection criteria to enable screening of smaller focused compound collections to provide higher success rates and lower costs. Effective selection criteria can be obtained by analysis of the binding site in a co-crystal structure to identify a pharmacophore. This pharmacophore is used subsequently to design potential binders for screening. This approach relies heavily on the medicinal chemists knowledge of molecular interactions, more specifically the attractive interactions between two partner molecules in biological systems. Parallel synthesis is then applied to synthesize libraries of compounds for hit to lead optimization. In drug discovery, synthesis of compound libraries is necessary to optimize a lead. Determination of the biological activity on a specified target can identify which moieties are important and which need optimizing for increasing its activity, selectivity often via a structure activity analysis. Compiling a library of 20 compounds, where stepwise the individual parts of the target molecule have to be chemically connected, the number of reaction and purification steps quickly exceeds 100. This is first of all time consuming and second very resource demanding. Looking at the MCR approach, the philosophy is to obtain products in just a single step, where the product contains all the desired properties. With this in mind, the MCR methodology deems to be a competitive alternative to ordinary parallel multistep synthesis. To date there are many marketed drugs that can be prepared through a MCR (Figure 1). The local anesthetic lidocaine (Xylocaine®) is for example prepared by a Ugi-3CR in a single step by reacting formaldehyde, diethyl amine and 2,6-dimethyl-phenylisocyanide.16 This example is an early adaption of IMCR in commercial drug production. Other examples of MCR directed/assisted drug synthesis are shown in figure 1.

4 Chapter 1

Cl Ph N OH H * N N N H t-Bu O N O N O HO O H O O Crixivan® Mandipropamid U-4CR Passerini-3CR

F O H N O O O NH NH H O N N N N N F H O N N H2N N O Telaprevir KAF156 P-3CR and U-3CR GBB-3CR

NO2 H N O O N HN N O O O N S N H Lidocaine Olanzapine U-3CR Gewald-3CR Nifedipine Hantsch-3CR Figure 1. Various drug examples produced through the MCR methodology. The blue and purple color assigns the part of the molecule constructed with MCR chemistry. Aim of the research described in this thesis Drug design aims at the development of druglike compounds and probes which act as agonists or antagonists in biological processes. A pharmacophore model and complementing MCR methodologies can prove to be an invaluable asset in drug design. With the current state of the art in MCR chemistry, design and synthesis of vast libraries of compounds, targeting various identified biological targets is made possible. In turn, the MCR toolbox used for drug discovery is continuously expanded by the development of new scaffolds, simplification of existing chemistry with broader scope, better toleration towards sensitive functional groups and enables targeting of otherwise difficult to target binding sites such as flat surfaces. Noteworthy is the recent discovery of the catalytic enantioselective U-4CR, which proves that the rational design of MCR’s has never been so important and widely applicable in drug discovery.17-18 Development of new

5 Introduction probes and scaffolds using MCR chemistry and its application for the discovery of new drug like compounds is the main theme in this thesis. Thesis Outline 1 In Chapter 2 selective inhibitors of Mdm4 are presented. The Mdm4 protein is a closely related protein to Mdm2 and it also binds to the same epitope of p53. Both Mdm2 and Mdm4 (MdmX) were characterized as druggable by analysis of the Mdm2-p53 co-crystal structure and are considered as an important oncology target.19 All current known binders are highly specific for Mdm2. In a combinatorial synthetic approach, the U-4CR was applied to generate a library of peptidomimetic small molecules as potential Mdm2/4 binders. A selective Mdm4 binder was identified and subjected to subsequent hit-to-lead optimization. Chapter 3 describes the improvements made in the preexisting U-4CR assisted synthesis of the anti-helminthic drug praziquantel. There are multiple methods to prepare praziquantel on industrial scale. The U-4CR method was developed in 2009, but was not adopted for industrial production up to date, likely due to the requirement of isocyanide chemistry. With the introduction of the in situ isocyanide methodology by Neochoritis et al., this constraint could be overcome, which was demonstrated in a preparation procedure, published in Organic Syn- theses.20-21 Chapter 4 presents the post-MCR modification of ester containing UT-4CR products to obtain N-unsubstituted γ-and δ-lactams. The well-known UT-4CR allows for the introduction of the tetrazole moiety, which serves as a bio-isostere for carboxylic acids. In the experimental design tritylamine was used as a convertible amine component and an ester containing aldehyde as bi-functional building block, containing either 2 or 3 methylene groups. Removal of the tritylgroup results in compounds with both an amine and ester, which readily undergo aminolysis upon basic treatment, , resulting in intramolecular cyclization, thus formation of lactams. The peptidomimetic nature of the resulting tetrazolo-lactam motif can be applied as potent drugs attributed to the peptidomimetic proline cis-amide functionality. In chapter 5 a new generation of MRI contrast agents was introduced on the basis of the known metal chelator tetraxatan. In the current application of gadoteric acid, MRI contrast enhancement is achieved by the reduction of T1 relaxation times, more specifically by fast water exchange on the th9 coordination site of the gadolinium complex. Although the gadoteric acid complex is very stable with a stability constant (log Keq) of 25.8, swift clearance from the body is important to prevent severe nephrotoxic adverse effects due to release of gadolinium ions by metabolic degradation of the gadolinium complex. Patients with renal failure are therefore susceptible to these side effects. Replacement of the appendant carboxylate arms with tetrazole bio-isosteres would tackle this unfavored leaching and its subsequent toxicity. The tetrazole is introduced in a two-step synthetic strategy using the UT-4CR and a deprotection step. The stability constant of the

6 Chapter 1 complex was determined and the contract enhancing abilities were tested by in vivo experiments. Chapter 6 highlights the developments of the GBB-3CR since its discovery in 1998. During the course of two decades the GBB-3CR reaction has emerged as a very important MCR, resulting in over a hundred patents and a great number of publications in various fields of interest. To celebrate this event, we would like to present an overview of the developments in the GBB-3CR, including an analysis of each of the three starting material classes, solvents and catalysts described. Additionally, a list of patents and their applications and a more in-depth summary of the biological targets that were addressed, including structural biology analysis, is given. Chapter 7 focusses on the application of bis-amidines in the GBB-3CR. With multiple possible regioisomers, this amidine component could yield various unprecedented heterocyclic scaffolds. Pyrazine-2,3-diamine, however, is the only amidine affording products that did not show quick degradation at room temperature when exposed to air. The scaffold from this amidine, 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amines represent a similar shape to adenine and thus could be applied for development of novel kinase inhibitors, mimicking the adenosine part in the ATP binding site. To strengthen our hypothesis we performed comprehensive docking studies with multiple potential targets where our imidazo[1,2-a]pyrazin-8-amine scaffold can bind.

7 Introduction

References 1 I. Ugi, A. Dömling, W. Hörl, Endeavour 1994, 18, 115-122. 2 A. Strecker, Justus Liebigs Annalen der Chemie 1850, 75, 27-45. 1 3 L. F. Tietze, Chemical Reviews 1996, 96, 115-136. 4 N. A. Petasis, I. Akritopoulou, Tetrahedron Letters 1993, 34, 583-586. 5 A. Hantzsch, Berichte der deutschen chemischen Gesellschaft 1881, 14, 1637- 1638.

6 P. Biginelli, Berichte der deutschen chemischen Gesellschaft 1891, 24, 2962- 2967.

7 C. Mannich, W. Krösche, Archiv der Pharmazie 1912, 250, 647-667. 8 H. Staudinger, J. Meyer, Helvetica Chimica Acta 1919, 2, 635-646. 9 M. Passerini, L. Simone, Gazz. Chim. Ital. 1921, 51, 126-129. 10 A. Dömling, Chemical Reviews 2006, 106, 17-89. 11 M. C. Pirrung, S. Ghorai, Journal of the American Chemical Society 2006, 128, 11772-11773.

12 A. W. Hofmann, Ann. Chem. 1868, 146, 107. 13 I. Ugi, U. Fetzer, U. Eholzer, H. Knupfer, K. Offermann,Angewandte Che- mie International Edition in English 1965, 4, 472-484. 14 C. G. Neochoritis, T. Zarganes-Tzitzikas, S. Stotani, A. Domling, E. Herdt- weck, K. Khoury, A. Domling, Acs Comb Sci 2015, 17, 493-499. 15 I. Ugi, R. Meyr, U. Fetzer, C. Steinbrückner,Angew Chem Int Edit 1959, 71, 386-386.

16 A. Dömling, W. Wang, K. Wang, Chemical Reviews 2012, 112, 3083-3135. 17 S. Shaabani, A. Dömling, Angewandte Chemie International Edition 2018, 57, 16266-16268. 18 J. Zhang, P. Yu, S.-Y. Li, H. Sun, S.-H. Xiang, J. Wang, K. N. Houk, B. Tan, Science 2018, 361, eaas8707. 19 C. F. Cheok, C. S. Verma, J. Baselga, D. P. Lane, Nature Reviews Clinical Oncology 2010, 8, 25. 20 A. Boltjes, H. X. Liu, H. P. Liu, A. Dömling, Org Synth 2017, 94, 54-65. 21 C. G. Neochoritis, S. Stotani, B. Mishra, A. Dömling, Org Lett 2015, 17, 2002-2005.

Chapter 2

Fragment based Library Generation for the Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

André Boltjes, Yijun Huang, Rob van de Velde, Laurie Rijkee, Siglinde Wolf, James Gaugler, Katarzyna Lesniak, Katarzyna Guzik, Tad A. Holak,d Alexander Dömling

Published in: ACS Combinatorial Sciences, 2014, 16, 393-396 Chapter 2

Abstract Based on our recently resolved first co-crystal structure of Mdm4 in complex with a small molecule inhibitor (PDB ID: 3LBJ), we devised an approach for the generation of potential Mdm4 selective ligands. We performed the Ugi four-component reaction (Ugi-4CR) in 96-well plates with an indole fragment, which is specially designed to mimic Trp23, a key amino acid for the interaction between p53 and Mdm4. Generally the reaction yielded mostly precipitates collected by 96-well filter plates. The best hit compound was found to be active and selective for Mdm4 (Ki = 5 µM, 10 fold stronger than Mdm2). This initial hit may serve as the starting point for designing selective p53-Mdm4 inhibitors with higher affinity.

11 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

Introduction The protein-protein interaction between the transcription factor p53 and the negative regulator Mdm2 is an important recent oncology target.1 The interaction is crystallographically characterized and druggable and several compounds are in late preclinical and early clinical evaluation.2 The Mdm4 protein is a closely related protein to Mdm2 and it also binds to the same epitope of p53. The sequence homology, the shape, dimension and size is similar. Nevertheless all current compound scaffolds characterized by co-crystal structure analysis are highly specific for Mdm2 and show no or very little Mdm4 binding which, is surprising and not 2 well understood regarding the great similarity between the two proteins (Figure 1).3-4 Nutlin-3a 1, for example, binds to Mdm4 too but with a roughly 1000-fold lower affinity of about 25 µM.5 Several Novartis compounds 2-4 show weak low µM Mdm4 affinity while being very potent binders to Mdm2 (again ~1000-fold difference, Figure 2).6-7 Other described Mdm4 selective compounds are either covalent binders or not validated (5, 6).8-9 Surprisingly, pyrazolone compound6 5 loses activity to Mdm4 in the presence of a reducing reagent, dithiothreitol (DTT). Incubation of these compounds with Mdm4 under non-reducing conditions lead to a time dependent change of Mdm4 structure determined by NMR; concomitantly, the MS analysis showed the presence of covalent adducts of the compound with Mdm4. Additionally, we have found out, by 1H NMR, that the pyrazolone reacts with β-mercaptoethanol in chloroform.

Figure 1. Alignment of Mdm4 (green cartoon, PDB-3DAB) and Mdm2 (magenta cartoon PDB-1YCR) with the Mdm2 peptide (yellow cartoon and sticks) highlighting the similarities and differences of the two binding sites (the Mdm4 binding p53 peptide is omitted for clarity). The main differences between the two receptor p53 binding sites are in the p53L26 pocket. Due to the L>M and the H>P exchanges the Mdm4 pocket is more narrow and less deep. An additional difference is at the outer rim of the p53L26 pocket

12 Chapter 2 the M>V exchange. The key amino acids of Mdm2, Mdm4 and p53 are shown as sticks.

Selective Mdm4 antagonists are highly sought after since Mdm4 and Mdm2 proteins are differentially over-expressed in several cancers and both play a promi- nent but presumably different role in apoptosis induction.10 Herein, we describe the discovery of B1, a selective p53-Mdm4 inhibitor (with ~5 µM affinity to Mdm4 but 54 µM affinity to Mdm2) with reversed selectivity compared with most p53- Mdm2 inhibitors, which we believe is a good starting point to elaborate Mdm4 selective compounds.

Figure 2. Some known Mdm2 antagonists together with their Mdm4 activity.

13 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

Results and Discussion Based on our previously generated insight into the binding of small molecules into Mdm2 and Mdm4 and our recently developed Mdm2 and Mdm4 fluorescence polarization assay, we planned to synthesized libraries of potential Mdm2/4 binding compounds.5, 11-21 Thus, we generated a 96-member library of peptidomimetic small molecules via Ugi four-component reaction (Ugi-4CR) (Scheme 1). Each compound contains the indole or p-halobenzyl fragment to mimic the Trp23 “anchor”, which is the key anchor residue in the p53 Mdm2 and Mdm4 protein-protein interaction interface, respectively. Figure 3 illustrates 2 the structure of amine and isocyanide inputs, as well as the experimental setting in a 96-well microliter plate. Since the reaction products regularly precipitated, the compounds were collected by a 96-well filter plate, and washed with ether to remove unreacted starting materials. The yields of the isolated products were between low (6%) and good (56%) with an average of 28% over all 96 wells. In addition, the purities of the isolated materials were considered sufficient for an initial screening. The collected samples were dissolved as a 10 mM stock solution in DMSO for the screening.

2 R NH2 HCHO NH R2 O O N R1 N R1NC H N OH O H Scheme 1. Ugi-4CR for high throughput synthesis

Compound B1 was identified as a p53/Mdm4 inhibitor (Ki = 19 µM) via our recently reported fluorescence polarization assay. The hit compound was re-synthesized and purified by flash chromatography, which was further confirmed by the binding with Mdm4 (Ki = 5.5 µM), as shown in Figure 4.5 Although the p53-binding sites within the Mdm4 and Mdm2 proteins are closely related, known Mdm2 small-molecule inhibitors have been shown experimentally not or very poorly to bind to its homolog Mdm4. This hit compound may provide a new avenue for the design of potential selective inhibitors of the p53-Mdm4 interaction. Further studies are ongoing in our lab to uncover the puzzle of the Mdm2 and Mdm4 selectivity.

14 Chapter 2

A B

NH2 NH2 NH2 NH2 NC NC NC

Cl Cl F A B C 1 2 3 4 NC NC NC

NH2 NH2 NH2 NH2 CO2Me

5 6 7 8 D E F

NH2 NH2 NH2 NH2 NC NC

O N Ph Ph Cl Br Cl G H 9 10 11 12 Figure 3. Parallel synthesis of “anchor” biased compound library via Ugi- 4CR. Structures of the amine A and isocyanide B starting materials used.

A B C

OHN O N N H

Figure 4. Hit compound as p53/Mdm4 inhibitor. A: Structure of B1; B: Ki = 54 µM (Mdm2) C: Ki = 5.5 µM (Mdm4) as determined by FP.

For further optimizing purposes a second library was synthesized, that follows the structure of hit compound B1, yielding a total of 38 new compounds. Mi- nor changes were made in the indole moiety (from the carboxylic acid component) and different halogen substituted benzylamines were employed, keeping the cyclohexyl fragment intact, as shown in figure 5. This time a sequential approach was used, which made it possible to run 1 mmol scale reactions as opposed to 0.2 mmol scale in the 96-well plate. Increased yields up to 79% were observed, in average 46%, which confirms that larger scale Ugi reactions in general give better yields. Unfortunately, all the other compounds synthesized in Figure 5 showed worse (>50 µM) or no activity in the FP assay.

15 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

A B

H H N H H N 2 N N N COOH COOH COOH COOH Cl HO I J K L

CF3 H O 13 N O S COOH COOH COOH COOH N H2N M N O P 2

COOH COOH N COOH N COOH F 14 HO MeO N S T Q R H2N

Cl S COOH H COOH N COOH Cl Cl U V W 15

Figure 4. Starting materials for the second library of compounds with the structures of A the carboxylic acids and B the amines.

Conclusions In summary, this work demonstrates that the Ugi four-component reaction (Ugi- 4CR) can be used to address the requirements for efficient high-throughput synthesis of diverse compounds in a cost- and time-effective manner. Integrated with biochemical screening assays, a peptidomimetic p53-Mdm4 inhibitor B1 was identified from a 96-membered library generated via Ugi-4CR of an indole fragment. This approach provides an efficient strategy for the discovery of small molecule probes selectively targeting protein-protein interactions. Further optimization studies on B1 are ongoing and will be reported in due course.

16 Chapter 2

Experimental procedures and Spectral Data

All reagents were purchased from commercial sources and used without further purification. Proton and carbon NMR spectra were determined on Bruker Avance™ 600 MHz NMR spectrometer. Chemical shifts are reported as δ values in parts per million (ppm) as referenced to residual solvent. 1H NMR spectra are tabulated as follows: chemical shift, number of protons, multiplicity (s = singlet, br.s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and coupling constant. High Resolution Mass spectra were obtained at the University of Pittsburgh Mass Spectrometry facility. LC-MS analysis was performed on an SHIMADZU instrument (reverse-phase HPLC coupled to electrospray ionization-mass spectrometry), using an analytical C18 column (Dionex Acclaim 120 Å, 2.1 × 50 mm, 3.0 μm) coupled to an Applied Biosystems API2000 mass spectrometer (ESI-MS). Acetonitrile/water mixtures were used as the mobile phase for reverse-phase HPLC, the flow rate was maintained at 0.2 mL/min. The column temperature was kept at 40 °C. The UV detection was at 254 nm.

High throughput synthesis of a 96-member library. Add indole-2-carboxylic acid (200 µL, 1M methanol stock solution; 0.2 mmol), amine (200 µL, 1M methanol stock solution; 0.2 mmol), isocyanide (200 µL, 1M methanol stock solution; 0.2 mmol), formaldehyde (120 µL, 2M methanol stock solution; 0.24 mmol) into each well of 96 deep well plate with printed labeling (VWR D108839, 1.2 mL). The plated was sealed by aluminum foil sealing film and was sonicated for 1h, then stand overnight under RT. After the partial evaporation of the solvent, the product was collected by filtration and washed by ether (AcroPrepTM 96 filter plate from PALL (0.2 um GHP, 1 mL well)) was used, 96 deep well plate was used as the receiver). For the second library equal conditions were used, but performed in 4 mL vials. N-(4-chlorobenzyl)-N-(2-(cyclohexylamino)-2-oxoethyl)-1H-indole-2-carboxamide (B1)

Cl White solid, 20.6 mg; yield: 24%. HPLC/MS: tR = 11.61 + min; m/z = 424.2 [M+H] . HRMS: C24H26N3O2ClNa, 446.1611 (calcd.), 446.1634 (found). 1H NMR (600 MHz, 6 H d -DMSO): 1.18-1.27 (m, 5H), 1.53-1.74 (m, 4H), 3.58 (m, N 1H), 3.94 (m, 1H), 4.18 (m, 1H), 4.66 (m, 1H), 5.00 (m, N 1H), 6.53 (m, 1H), 6.75 (m, 1H), 7.03 (m, 1H), 7.18 (m, O O 1H), 7.37-7.56 (m, 4H), 7.84 (m, 1H), 8.05 (m, 1H), 11.70 NH (m, 1H). 13C NMR (150 MHz, d6-DMSO): 24.4, 25.1, 32.2, 47.5, 49.7, 51.2, 54.9, 103.9, 111.99, 112.04, 119.8, 121.4, 123.5, 126.8, 128.3, 129.8, 131.8, 135.9, 136.3, 163.8, 166.9. N-(tert-butyl)-N-(2-(cyclohexylamino)-2-oxoethyl)-1H-indole-2-carboxamide (B7):

17 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

O 1H NMR (600 MHz, DMSO): 1.13-1.21 (m, 3H), 1.27- H H N N 1.33 (m, 2H), 1.46 (s, 9H), 1.55-1.57 (m, 1H), 1.68-1.70 N (m, 2H), 1.77-1.79 (m, 2H), 3.63-3.66 (m, 1H), 4.21 (s, O 2H), 6.67 (s, 1H), 7.00-7.02 (t, 1H, J = 7.2Hz), 7.14-7.16 (t, 1H, J = 7.5 Hz), 7.37-7.38 (d, 2H, J = 8.4Hz), 7.53-7.54 (d, 1H, J = 8.4Hz), 7.93-7.94 (d, 1H, J = 7.8Hz), 11.52 (s, 1H). 13C NMR (150 MHz, DMSO): 24.9, 25.7, 28.0, 32.6, 48.0, 50.6, 58.0, 100.0, 103.3, 112.4, 120.0, 121.7, 123.4,

127.2, 133.2, 136.2, 166.0, 169.9. HRMS: C21H29N3O2Na, 378.2157 (calcd.), 378.2140 (found). N-(2-(benzylamino)-2-oxoethyl)-N-(tert-butyl)-1H-indole-2-carboxamide (C7): 2 1H NMR (600 MHz, DMSO): 1.47 (s, 9H), 4.32 (s, 2H), O 4.37-4.38 (d, 2H), 6.60 (s, 1H), 7.02 (m, 1H), 7.15 (m, N N N 1H), 7.26-7.39 (m, 5H), 7.37-7.39 (d, 1H, J = 8.4Hz), H H O 7.47-7.48 (d, 1H, J = 8.4Hz), 8.58-8.60 (t, 1H, J = 6.0Hz), 11.51 (s, 1H). 13C NMR (150 MHz, DMSO): 28.0, 42.8, 50.7, 58.0, 103.1, 112.4, 120.1, 121.7, 123.4, 127.2, 127.4, 127.9, 128.8, 133.0, 136.2,

139.8, 165.9, 170.9. HRMS: C22H25N3O2Na, 386.1844 (calcd.), 386.1812 (found). N-(tert-butyl)-N-(2-(mesitylamino)-2-oxoethyl)-1H-indole-2-carboxamide (E7):

O 1 H H H NMR (600 MHz, DMSO): 1.29 (s, 9H), 1.90 (s, 6H), N N N 2.00 (s, 3H), 4.27 (s, 2H), 6.52 (s, 1H), 6.67 (s, 2H), 6.78- 6.81 (t, 1H, J = 7.2Hz), 6.92-6.95 (t, 1H, J = 7.2Hz), 7.16- O 7.17 (d, 1H, J = 8.4Hz), 7.26-7.28 (d, 1H, J = 8.4Hz), 9.16 (s, 1H), 11.29 (s, 1H). 13C NMR (150 MHz, DMSO): 17.9, 20.5, 27.5, 57.5, 62.8, 102.7, 111.9, 119.7, 121.0, 123.0, 126.7, 128.6, 131.9, 132.6,

134.8, 135.6, 135.8, 165.5, 169.3. HRMS: C24H30N3O2, 392.2338 (calcd.), 392.2341 (found). N-(2-(cyclohexylamino)-2-oxoethyl)-N-phenethyl-1H-indole-2-carboxamide (B12): 1H NMR (600 MHz, DMSO): 1.13-1.29 (m, 6H), NH 1.55-1.57 (d, 1H, J = 12.6Hz), 1.68-1.70 (m, 2H), 1.75- O 1.77 (d, 2H, J = 12.0Hz), 2.92 (s, 2H), 3.03 (s, 1H), O 3.64 (s, 1H), 3.89 (m, 1H), 4.09 (s, 1H) 4.24 (s, 1H), N 6.68 (s, 1H) 7.03-7.05 (t, 1H, J = 7.32Hz), 7.18-7.26 N (m, 2H), 7.26-7.36 (m, 4H), 7.42-7.44 (d, 1H, J = H 8.4Hz), 7.57 (m, 1H), 8.11 (m, 1H), 11.64 (s, 1H). 13C NMR (150 MHz, DMSO): 24.5, 25.2, 32.3, 41.3, 47.6, 49.6, 52.0, 103.5, 112.0, 119.7, 121.3, 123.3, 126.2, 126.9, 128.5, 128.7, 135.8, 163.3,

167.4. HRMS: C25H30N3O2, 404.2338 (calcd.), 404.2328 (found).

18 Chapter 2

Table 1. The obtained U-4CR products.

Compound Structure Mass (mg) Yield (%) LC/MS

OHN O N N H tR = 11.61 min; A1 22.3 28 m/z = 398.3 [M+H]+ Cl

O NH tR = 11.61 min; H B1 N N 20.6 24 m/z = 424.2 + O [M+H]

OHN O t = 11.48 min; N N R C1 H 32.4 38 m/z = 432.3 [M+H]+

OHN t = 11.65 min; O R N N D1 H 15.6 18 m/z = 446.2 [M+H]+

OHN t = 12.01 min; O R N N E1 H 28.6 31 m/z = 460.2 [M+H]+

CO2Me OHN O N N tR = 10.75 min; F1 H 34.7 42 m/z = 414.3 [M+H]+ Cl

N OHN O t = 10.63 min; O R N N G1 H 30.8 34 m/z = 453.2 [M+H]+

19 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

Ph Ph OHN O tR = 12.18 min; N N H1 H 19.7 19 m/z = 508.2 [M+H]+ Cl

OHN O tR = 11.18 min; N N A2 H 25.4 35 m/z = 364.4 [M+H]+ 2

OHN t = 11.44 min; O R B2 N N 30.1 39 m/z = 390.4 H [M+H]+

OHN O tR = 11.10 min; N N C2 H 10.5 13 m/z = 398.4 [M+H]+

OHN tR = 11.32 min; O D2 N N 12.3 15 m/z = 412.3 H [M+H]+

OHN tR = 11.66 min; O E2 N N 12.8 15 m/z = 426.4 H [M+H]+

CO2Me OHN O tR = 10.31 min; N N F2 H 27.6 36 m/z = 380.2 [M+H]+

OHN O tR = 10.44 min; O G2 N N 34.2 41 m/z = 419.4 H [M+H]+

Ph Ph OHN O tR = 11.87 min; N N H2 H 19.1 20 m/z = 474.4 [M+H]+

20 Chapter 2

OHN O t = 11.78 min; N N R A3 H 41.6 51 m/z = 412.2 [M+H]+ Cl

OHN O tR = 11.98 min; N N B3 H 23 26 m/z = 438.2 [M+H]+

OHN O t = 11.65 min; N N R C3 H 5.5 6 m/z = 446.3 [M+H]+ Cl

OHN t = 11.82 min; O R N N D3 H 7.4 8 m/z = 460.3 [M+H]+

OHN t = 12.15 min; O R N N E3 H 17.2 18 m/z = 474.3 [M+H]+

CO2 OHN Me O t = 10.90 min; N N R F3 H 30.2 35 m/z = 428.3 + Cl [M+H]

OHN O t = 10.40 min; O R N N G3 H 37.7 40 m/z = 467.4 [M+H]+

Ph Ph OHN O tR = 12.33 min; N N H3 H 10.4 10 m/z = 522.3 [M+H]+ Cl

OHN O t = 11.24 min; N N R A4 H 23.9 31 m/z = 382.2 [M+H]+

21 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

OHN O tR = 11.48 min; N N B4 H 12.4 15 m/z = 408.4 [M+H]+

OHN O t = 11.16 min; N N R C4 H 21.2 26 m/z = 416.3 2 [M+H]+

OHN t = 11.38 min; O R N N D4 H 10.7 12 m/z = 430.1 [M+H]+

OHN t = 11.69 min; O R N N E4 H 49.2 56 m/z = 444.4 [M+H]+

CO2Me OHN O t = 10.39 min; N N R F4 H 30.5 38 m/z = 398.3 [M+H]+ F

OHN O t = 10.31 min; O R N N G4 H 19 22 m/z = 437.4 [M+H]+

Ph Ph OHN O tR = 11.88 min; N N H4 H 19.5 20 m/z = 492.3 [M+H]+ F

OHN O tR = 11.79 min; N N A5 H 21.1 29 m/z = 370.5 [M+H]+

22 Chapter 2

OHN t = 11.97 min; O R N N B5 H 10.6 13 m/z = 396.5 [M+H]+

OHN O tR = 11.62 min; N N C5 H 18.3 23 m/z = 404.3 [M+H]+

OHN tR = 11.84 min; O D5 N N 27.9 33 m/z = 418.2 H [M+H]+

OHN tR = 12.18 min; O E5 N N 27.4 32 m/z = 432.4 H [M+H]+

CO2Me OHN O tR = 10.82 min; N N F5 H 17.6 23 m/z = 386.3 [M+H]+

OHN O tR = 10.68 min; O G5 N N 14.5 17 m/z = 425.3 H [M+H]+

Ph Ph OHN t = 12.34 min; O R N N H5 H 26.3 27 m/z = 480.4 [M+H]+

O tR = 11.42 min; N N A6 H NH 26.9 38 m/z = 356.4 O [M+H]+

OHN tR = 11.63 min; O B6 N N 14.7 19 m/z = 382.2 H [M+H]+

OHN t = 11.28 min; O R N N C6 H 28.8 37 m/z = 390.3 [M+H]+

23 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

tR = 11.51 min; OHN D6 O 32.8 41 m/z = 404.2 N N H [M+H]+

t = 11.88 min; O R E6 H NH 33 40 m/z = 418.5 N N + O [M+H]

CO2Me OHN t = 10.43 min; 2 O R N N F6 H 23.7 32 m/z = 372.3 [M+H]+

N t = 10.34 min; OHN O R G6 O 21 26 m/z = 411.4 N N H [M+H]+

Ph Ph OHN tR = 12.07 min; O H6 N N 25.1 27 m/z = 466.5 H [M+H]+

OHN tR = 11.23 min; O A7 N N 7.2 11 m/z = 330.5 H [M+H]+

t = 11.44 min; OHN R B7 O 11.8 17 m/z = 356.2 N N H [M+H]+

OHN tR = 10.98 min; O C7 N N 15.7 22 m/z = 364.3 H [M+H]+

tR = 11.22 min; OHN D7 O 14.8 20 m/z = 378.4 N N + H [M+H]

tR = 11.58 min; OHN E7 O 28.8 37 m/z = 392.3 N N + H [M+H]

CO2Me OHN O tR = 9.95 min; m/z F7 N N 35 51 + H = 346.3 [M+H]

24 Chapter 2

OHN O tR = 9.90 min; m/z G7 O 22.1 29 + N N = 385.3 [M+H] H

Ph Ph OHN tR = 11.81 min; H7 O 16.6 19 m/z = 440.4 N N H [M+H]+

OHN t = 10.30 min; O R N N A8 H 8.3 13 m/z = 332.2 [M+H]+ MeO

OHN tR = 10.60 min; O B8 N N 13.5 19 m/z = 358.5 H [M+H]+ MeO

OHN t = 10.32 min; O R N N C8 H 9.6 13 m/z = 366.2 [M+H]+ MeO

t = 10.56 min; OHN R D8 O 14.9 20 m/z = 380.3 N N H [M+H]+ MeO

t = 10.92 min; OHN R E8 O 36.5 46 m/z = 394.3 N N H [M+H]+ MeO

CO2Me OHN O N N tR = 9.35 min; m/z F8 H 30.1 43 = 448.3 [M+H]+ MeO

N OHN O t = 9.28 min; m/z O R G8 N N 32.6 42 + H = 387.2 [M+H]

MeO

Ph Ph OHN tR = 11.48 min; O N N H8 H 19.1 22 m/z = 442.3 [M+H]+ MeO

25 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

OHN t = 10.80 min; O R N N A9 H 21.7 33 m/z = 328.4 [M+H]+

OHN tR = 11.03 min; O B9 N N 17.8 25 m/z = 354.3 H [M+H]+ 2

OHN t = 10.73 min; O R N N C9 H 13.8 19 m/z = 362.3 [M+H]+

t = 10.93 min; OHN R D9 O 9.4 13 m/z = 376.4 N N H [M+H]+

t = 11.32 min; OHN R E9 O 29.4 38 m/z = 390.4 N N H [M+H]+

CO2Me OHN O N N tR = 9.84 min; m/z F9 H 35.2 51 = 344.5 [M+H]+

N OHN O t = 9.75 min; m/z G9 O 25.8 34 R N N + H = 383.2 [M+H]

Ph Ph OHN tR = 11.58 min; O H9 N N 15.1 17 m/z = 438.4 H [M+H]+

OHN O t = 11.68 min; N N R A10 H 30.2 34 m/z = 442.1 [M+H]+

OHN O tR = 11.91 min; N N B10 H 29.1 31 m/z = 468.3 [M+H]+

26 Chapter 2

OHN O t = 11.58 min; N N R C10 H 44.9 47 m/z = 476.2 [M+H]+

OHN t = 11.76 min; O R N N D10 H 24.3 25 m/z = 490.2 [M+H]+

OHN t = 12.11 min; O R N N E10 H 28.7 29 m/z = 504.3 [M+H]+

CO2Me OHN O N N tR = 10.88 min; F10 H 28.3 31 m/z = 458.2 [M+H]+ Br

OHN O t = 10.73 min; O R N N G10 H 25.8 26 m/z = 497.1 [M+H]+

Ph Ph OHN O tR = 12.27 min; N N H10 H 17.9 16 m/z = 552.3 [M+H]+ Br

OHN O t = 11.93 min; N N R A11 H 12.1 14 m/z = 432.0 + Cl [M+H]

OHN O tR = 12.13 min; N N B11 H 51.4 56 m/z = 458.4 + Cl [M+H]

OHN O t = 11.80 min; N N R C11 H 17.8 19 m/z = 466.3 + Cl [M+H] Cl

27 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

OHN t = 11.98 min; O R N N D11 H 22 23 m/z = 480.3 + Cl [M+H]

OHN t = 12.28 min; O R N N E11 H 33.7 34 m/z = 494.2 2 [M+H]+ Cl

CO2Me OHN O t = 11.06 min; N N R F11 H 38 42 m/z = 448.1 Cl [M+H]+ Cl

OHN O t = 10.91 min; O R N N G11 H 30.2 31 m/z = 487.2 + Cl [M+H]

Ph Ph OHN O t = 12.47 min; N N R H11 H 22.4 21 m/z = 542.3 + Cl [M+H]

OHN O tR = 11.32 min; N N A12 H 28.9 38 m/z = 378.3 [M+H]+

OHN tR = 11.55 min; O N N B12 H 28.5 35 m/z = 404.3 [M+H]+

OHN O tR = 11.23 min; N N C12 H 14.2 17 m/z = 412.2 [M+H]+

OHN tR = 11.43 min; O D12 N N 41.7 49 m/z = 426.3 H [M+H]+

28 Chapter 2

OHN tR = 11.76 min; O E12 N N 48.2 55 m/z = 440.3 H [M+H]+

CO2Me OHN O tR = 10.48 min; N N F12 H 19.9 25 m/z = 394.2 [M+H]+

OHN O tR = 10.34 min; O G12 N N 21 24 m/z = 433.3 H [M+H]+

Ph Ph OHN t = 11.96 min; O R N N H12 H 31.3 32 m/z = 488.3 [M+H]+

29 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

References 1 C. F. Cheok, C. S. Verma, J. Baselga, D. P. Lane, Nat Rev Clin Oncol 2011, 8, 568-568. 2 K. Khoury, G. M. Popowicz, T. A. Holak, A. Domling, MedChemComm 2011, 2, 246-260. 3 G. M. Popowicz, A. Dömling, T. A. Holak, Angewandte Chemie Internatio- nal Edition 2011, 50, 2680-2688. 2 4 K. Khoury, G. M. Popowicz, T. A. Holak, A. Dömling, Medchemcomm 2011, 2, 246-260. 5 G. M. Popowicz, A. Czarna, U. Rothweiler, A. Szwagierczak, M. Krajew- ski, L. Weber, T. A. Holak, Cell Cycle 2007, 6, 2386-2392. 6 J. Berghausen, N. Buschmann, P. Furet, F. Gessier, L. J. Hergovich, P. Holzer, E. Jacoby, J. Kallen, K. Masuya, S. C. Pissot, H. Ren, S. Stutz, p. 448pp. 7 G. Bold, P. Furet, F. Gessier, J. Kallen, L. J. Hergovich, K. Masuya, A. Vaupel, p. 274pp. 8 D. Reed, Y. Shen, A. A. Shelat, L. A. Arnold, A. M. Ferreira, F. Zhu, N. Mills, D. C. Smithson, C. A. Regni, D. Bashford, S. A. Cicero, B. A. Schul- man, A. G. Jochemsen, R. K. Guy, M. A. Dyer, Journal of Biological Chemis- try 2010, 285, 10786-10796. 9 J. H. Lee, Q. Zhang, S. Jo, S. C. Chai, M. Oh, W. Im, H. Lu, H.-S. Lim, Journal of the American Chemical Society 2010, 133, 676-679. 10 J.-C. W. Marine, M. A. Dyer, A. G. Jochemsen, Journal of Cell Science 2007, 120, 371-378. 11 S. Srivastava, B. Beck, W. Wang, A. Czarna, T. A. Holak, A. Dömling, Journal of Combinatorial Chemistry 2009, 11, 631-639. 12 G. M. Popowicz, A. Czarna, S. Wolf, K. Wang, W. Wang, A. Dömling, T. A. Holak, Cell Cycle 2010, 9, 1104-1111. 13 Y. Huang, S. Wolf, M. Bista, L. Meireles, C. Camacho, T. A. Holak, A. Dömling, Chemical Biology & Drug Design 2010, 76, 116-129. 14 A. Czarna, B. Beck, S. Srivastava, G. M. Popowicz, S. Wolf, Y. Huang, M. Bista, T. A. Holak, A. Dömling, Angewandte Chemie-International Edition 2010, 49, 5352-5356. 15 M. Bista, K. Kowalska, W. Janczyk, A. Dömling, T. A. Holak, Journal of the American Chemical Society 2009, 131, 7500-7501.

30 Chapter 2

16 U. Rothweiler, A. Czarna, M. Krajewski, J. Ciombor, C. Kalinski, V. Kha- zak, G. Ross, N. Skobeleva, L. Weber, T. A. Holak, ChemMedChem 2008, 3, 1118-1128.

17 G. Popowicz, A. Czarna, T. Holak, Cell Cycle 2008, 7, 2441-2443. 18 U. Rothweiler, A. Czarna, L. Weber, G. M. Popowicz, K. Brongel, K. Ko- walska, M. Orth, O. Stemmann, T. A. Holak, Journal of Medicinal Chemis- try 2008, 51, 5035-5042. 19 A. Czarna, G. M. Popowicz, A. Pecak, S. Wolf, G. Dubin, T. A. Holak, Cell Cycle 2009, 8, 1176-1184. 20 Y. Huang, S. Wolf, D. Koes, G. M. Popowicz, C. J. Camacho, T. A. Holak, A. Dömling, ChemMedChem 2012, 7, 49-52. 21 D. Koes, K. Khoury, Y. Huang, W. Wang, M. Bista, G. M. Popowicz, S. Wolf, T. A. Holak, A. Dömling, C. J. Camacho, PLoS ONE 2012, 7, e32839.

31 Discovery of a Peptidomimetic p53-Mdm4 Inhibitor

Chapter 3

Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel André Boltjes, Haixia Liu, Haiping Liu and Alexander Dömling

Part of the research described in this chapter is published in: Org. Synth., 2017, 94, 54-65 Chapter 3

Abstract Praziquantel is currently the only effective drug for the treatment of schistosomiasis, a neglected tropical disease, affecting over 200 million people worldwide. The current production method to produce praziquantel is via the Merck process which consist of a five-step synthesis, in which the key step is a Reissert reaction requiring large excess of toxic KCN. The accompanying environmental treats of potential cyanide pollution cannot be ignored and therefore a more efficient three-step MCR Ugi-4CR was presented. The methodology incorporates the novel in situ formation of the phenethylisocyanide component. The procedure is described in a thorough detail oriented way to meet the requirements for publication in Organic Syntheses and was checked for reproducibility in the laboratory of a member of the Board of Editors.

35 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel

Introduction Schistosomiasis is a parasitic infection which affects over 200 million people worldwide and in particular sub-Saharan Africa.1-3 The infection is classified as a neglected tropical disease and manifests itself by flu-like symptoms due to egg deposition of schistosomes, into the tissues of the host, mostly in the intestine, liver and genitourinary system. Untreated infections will result in chronic schistosomiasis. The immune response of the host causes the formation of granulomas around the eggs ultimately leading to fibrosis in the affected tissues and calcifica- tion in the genitourinary tracts. It is currently one of the most common infectious diseases, which is efficiently treated by administering praziquantel. Praziquantel was initially developed as a potential tranquilizer by E. Merck in the 1970’s.4 It is, however, not uncommon that drugs are repurposed for other conditions such as sildenafil and thalidomine.5-6 Praziquantel’s anthelmintic properties were tested and proved by Bayer A.G. and initially marketed for the veterinary market and 3 then for the human market.7 In terms of drug safety, studies have shown that praziquantel is currently the saf- est drug available.8 Production of praziquantel is performed via a slight adaption of the original Merck process, developed in 1983 by Shin Poong Pharmaceutical company as depicted in scheme 1. This process is fairly cheap. Due to the safe use, amount of affected people, efficacy of treatment, praziquantel is placed on the WHO list of Essential medicines.9

KCN, CyCOCl H2/Ni 70 atm N N Cy NH O 90 oC 1 2 CN O 3 N Cy O H Cl Cl N O Et3N N O

HN Cl 4 5 N O Cy O

Scheme 1. The Merck industrial method is still the most widely applied. Isoquinoline (1) is converted to the Reissert compound (2) and then reduced by hydrogenation with nickel. N-alkylation with chloroacetyl chloride followed by a base assisted cyclisation yielding praziquantel (5). Current production sites are located in Asia (China, Korea) and in the process large quantities of KCN are used. Incidents with spills of cyanide waste have resulted in environmental pollution, killing life in rivers for many kilometers downstream. Efforts in finding alternative synthesis routes resulted in a MCR approach in 2010.10 The Ugi multicomponent reaction of amines, oxo components, isocyanides and inorganic or organic of acids leads to different scaffold types

36 Chapter 3 dependent on the nature of the acid component.11 For example carboxylic acids yield α-amino acylamides (Scheme 1). The Ugi multicomponent reaction is gain- ing increasing attention due to the rapid and convergent assembly of functional structures based on four classes of widely available starting material classes.12 In addition, our recent work on generating the isonitrile in situ is making the Ugi reaction even more accessible, as the use of the infamous isonitrile is cir- cumvented in this methodology.13 While several approaches towards α-amino acylamides are possible the Ugi approach is faster and impresses by a very large substrate scope.14-15 Here the schistosomiasis drug praziquantel (PQZ, 2-(cyclohexanecarbonyl)-2,3,6,7-tetrahydro-1H-pirazino[2,1-a]isoquinolin-4(11bH)-one) is prepared in just three steps using the key Ugi and Pictet-Spengler reactions.10 It comprises a short synthetic route from the readily available bulk starting materials, aminoacetaldehyde dimethylacetal (6), formaldehyde (7), 2-phenylethyl formamide (8) and cyclohexancarboxylic acid (9). Employing the high yielding formylation of phenethylamine with ethyl formate, N-phenethylformamide can be achieved in 99% yield. N-phenethylformamide reacts with triphosgene which in turn reacts with paraformaldehyde, aminoacetaldehyde dimethylacetal, and cyclohexylcarboxylic acid in a Ugi four component reaction to the advanced precursor 10 in 41% yield. In methanesulfonic acid at 70 °C for 6h, praziquantel 5 was afforded with 52% yield. These reactions were carried out under mild conditions and the sequence is atom economic, yielding only water and two equivalents of methanol as side products. O O H2N H H O Triphosgene, Et3N HN O 6 + 7 MeOH, DCM, -10 °C to r.t. O O N H N O O HO O 10

8 9

N O MSA N 70 °C O

5 Scheme 2. The Ugi-4CR with the in situ generated phenethylisocyanide from the formamide 8, followed by subsequent Pictet-Spengler reaction to yield praziquantel 5.

An Ugi-4CR reaction was used to construct the typical praziquantel functional groups, however, as the dipeptidic scaffold associated with the U-4CR. The

37 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel protected aldehyde moiety was subsequently deprotected and under the same conditions cyclized via a Pictet-Spengler reaction yielding praziquantel in only two steps (Scheme 2).16

Results and Discussion Here the schistosomiasis drug praziquantel (PQZ, 2-(cyclohexanecarbonyl)-2,3,6,7-tetrahydro-1H-pirazino[2,1-a]isoquinolin-4(11bH)-one) is prepared in just three steps using the key Ugi and Pictet-Spengler reactions.10, 17-18 It comprises a short synthetic route from the readily available bulk starting materials, 2-phenylethyl formamide, formaldehyde, cyclohexancarboxylic acid and aminoacetaldehyde dimethylacetal. Employing the high yielding formylation of phenethylamine with ethyl formate, N-phenethylformamide can be achieved in 99% yield. N-phenethylformamide reacts with triphosgene which in turn reacts 3 with paraformaldehyde, aminoacetaldehyde dimethylacetal, and cyclohexylcarboxylic acid in a Ugi four component reaction to the advanced precursor 2 in 41% yield. In methanesulfonic acid at 70 °C for 6h, praziquantel was afforded with 52% yield. These reactions were carried out under mild conditions and the sequence is atom economic, yielding only water and two equivalents of methanol as side products.

A. Preparation of N-phenethylformamide (8). A 500-mL single-necked round-bottom flask equipped with a 3 cm egg-shaped Teflon-coated stirring bar and a reflux condenser was charged with phenethylamine (95 mL, 0.75 mol) and ethyl formate (181 mL, 2.25 mol 1.8 eq.) (Note 1). The mixture was heated to reflux using an oil bath at 60° C for 20 hours until TLC indicates full consumption of the amine. The mixture was concentrated by rotary evaporation (40 °C, 10 mbar) to remove the excess of ethyl formate and ethanol byproduct (Note 2). N-phenethylformamide 111 g (99%) is afforded as pale yellow oil (Note 3).

B. Preparation of N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino)ethyl) cylcohexanecarboxamide (10). In a 500 mL 3-necked round-bottom flask equipped with a 7 cm Teflon blade overhead stirrer, a 100 mL pressure equalizing dropping funnel holding a nitrogen inlet and a temperature sensor (see photo below), a mixture of N-phenethylformamide (7.46g, 50 mmol) and triethylamine (16.8 mL, 120 mmol 2,4 eq.) in dichloromethane (50 mL) (Note 4) was cooled to -10 °C using an ethanol-ice bath (Note 5).

38 Chapter 3

Figure 1. Reaction Set-up for step B

While stirring (500 rpm) triphosgene (5.94 g, 20 mmol, 0,4 eq.) (Note 6) in dichloromethane (20 mL) was added dropwise via the dropping funnel over a period of 40 minutes (Note 7), resulting in a pale yellow solution. The reaction mixture was stirred at -10 °C for an additional 30 minutes (Note 8). In a separate 50 ml round bottom flask a mixture of aminoacetaldehyde dimethyl acetal (5.52 g, 52 mmol, 1.05 eq.) and paraformaldehyde (1.50 g, 50 mmol, 1 eq.) (Note 9) in 50 mL methanol was heated shortly with a heating gun until a clear solution developed; then cyclohexanecarboxylic acid (6.72 g, 52 mmol, 1.05 eq) was added; the resulting solution was then added together with another 50 mL MeOH to the in situ formed isocyanide reaction mixture at -10 °C and a slightly orange clear solution is formed. The reaction mixture was left to warm to room temperature. After stirred at room temperature for 48 h (Note 10), the mixture is concentrated (40 °C, 10 mbar) to remove the methanol. Then the mixture was redissolved in

50 mL CH2Cl2 and transferred to a 250 mL separatory funnel, washed with water

(3x 50mL), saturated NaHCO3 (3x 50mL), and dried over 5g MgSO4. The drying agent was removed by filtration over a P4 sintered glass filter and concentrated by rotary evaporation (40 °C, 10 mbar), yielding 9.05 gram crude product as an orange oil (Note 11), which crystalized on standing.

39 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel

Figure 2. Appearance of the Ugi product obtained in step B

Further trituration of the crude crystals with 10 mL cold Et2O, filtering through a

P4 glass filter and washing with another 10 mL cold Et2O yields after drying (0.5 mbar) 7.56 g (40%) pure N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino) ethyl)cylcohexanecarboxamide as pale yellow crystals (picture above) (Note 12). C. Preparation of 2-(cyclohexanecarbonyl)-2,3,6,7-tetrahydro-1H-pirazino[2,1-a]isoquinolin-4(11bH)-one (5).

In a 100 mL single-necked round-bottom flask under2 N atmosphere, N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino)ethyl)cyclohexanecarboxamide 2 (7.54 g, 20 mmol, 1 eq.) and 10 g activated molecular sieves (Note 13) was added at once to a solution of methanesulfonic acid (26.0 mL, 400 mmol, 20 eq.) at room temperature (Note 14). The mixture was heated to 70 °C (measured externally) in an oil bath for 6 h, then after cooling to room temperature the reaction mixture is gravity filtered through a slotted sieve filter into an 2L petri dish containing ice-water (200 mL) and NaHCO3 (40 g, 476 mmol) (Figure 3. Left).

40 Chapter 3

Figure 3. Left: Removal of the sieves and stirring. Right: Appearance of the crude Prazi- quantel obtained in step C.

200 mL Et2O is added and the mixture is stirred for 15 minutes using a 7 cm rod- shaped Teflon-coated stirring bar to dissolve most of the solids (Note 15). The solution is transferred to a 1L separation funnel and extracted with diethyl ether (3×100 mL) (Note 16). The combined organic layers are washed with brine (100 mL), dried over 10 g anhydrous magnesium sulfate and concentrated to dryness (40 °C, 600 =>10 mbar) to afford praziquantel (4.0 g, 64%) as orange oil, which crystalized on standing overnight (right picture above). The solid was triturated by adding 2x 10 mL 20% acetone in heptane, and filtered through a P4 sintered glass filter to obtain, after vacuum drying (0.5 mbar), 2.46 g pure praziquantel (picture below). The mother liquor was concentrated (1.5g orange oil) and purified by flash chromatography (Note 17). The fractions containing product were combined and yielded another 1.05 g praziquantel as an orange solid. Trituration with 5 mL Et2O yields 0.80 g pure praziquantel as a white solid. The combined yield is 3.51 g, 52% (Note 18) (Note 19).

Figure 4. Pure praziquantel obtained in step C after purification

41 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel

Notes 1. Phenethylamine (99%) and ethyl formate (>98%) were obtained from Sig- ma Aldrich and Acros Organics respectively and used as received. 2. Removal of the byproduct EtOH requires extended time (2 hours) on the rotary evaporator, 1H NMR is used to visualize traces of EtOH seen at 1.20 (triplet) and 3.66 (quartet) ppm. 3. N-phenethyl formamide

Rf = 0.45 (EtOAc), 1H NMR (500 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.37-7.24 (m, 2H), 7.26-7.02 (m, 3H), 6.34-5.88 (m, 1H), O N 3.52 (m, 2H), 2.89-2.70 (m, 2H) (only the major peaks of the H formamide rotamers were described); 13C NMR (125 MHz, Chloroform-d) δ 1, 161.4, 138.6, 137.7, 128.7, 128.6, 126.6, 39.2, 35.5 ppm. 3 4. The used solvents in step B and C: dichloromethane, ethyl acetate, petroleum ether 40-60 and methanol are technical grade and were purchased from Biosolve. 5. In a 1L dewar, 275 g ice and 150 g ethanol were used to obtain a temperature of at least -10 °C for the duration of the reaction. 6. The solid reagent triphosgene is a less hazardous substitute for highly toxic gaseous phosgene, however should be handled very carefully. The reaction should be performed in a well ventilated fume hood. 7. Faster addition or temperatures above 0 °C will reduce the yield dramatically. Color is a good indicator of proper addition speed: slightly yellow color is good, orange towards brown indicates too fast addition of triphosgene. 8. The isocyanide intermediate Rf = 0.74 (EtOAc) appears exclusively indicating full consumption of the formamide. 9. Not all reagents are fully consumed, but after 48 h no change in spot intensity, checked by TLC Merck silica gel 60 F254 plates (visualized with 254 nm UV lamp), was observed. 10. Reagents and solvents used in this preparation were commercially available and used without further purification, including triphosgene (98%) and aminoacetaldehyde dimethyl acetal (98%) from AK Scientific Inc., trimethylamine and paraformaldehyde (96%) from Fluka and cyclohexane carboxylic acid from Sigma Aldrich ( 98%). 11. The bulk of solvent should be removed. However, leaving a trace of solvent (~0.5 g) is actually beneficial in allowing the product to crystalize. The crystallization process takes some time (2 weeks) to complete.

42 Chapter 3

12. N-(2,2-dimethoxyethyl)-N-(2-oxo-2-(phenethylamino)ethyl) cylcohexanecarboxamide

mp = 86-88 °C Rf = 0.42 (EtOAc) 1H NMR (500 MHz, Chloro- HN O form-d) δ 7.36-7.25 (m, 2H), 7.25- 7.15 (m, 3H), 7.03 (t, J = 5.8 Hz, 0.5H), 6.51 (t, J = 6.0 Hz, 0.5H), 4.57 (t, J = 5.1 Hz, 0.5H), 4.39 (t, J = O N 5.2 Hz, 0.5H), 3.99 (d, J = 7.4 Hz, 2H), 3.55 (q, J = 6.8 Hz, 1H), 3.48 O (q, J = 6.8 Hz, 1H), 3.42 (dd, J = 7.0, 5.1 Hz, 2H), 3.37 (s, 3H), 3.33 O (s, 3H), 2.80 (dt, J = 21.9, 7.2 Hz, 2H), 2.58 (tt, J = 11.6, 3.4 Hz, 0.5H), 2.25 (tt, J = 11.5, 3.3 Hz, 0.5H), 1.85- 1.71 (m, 2H), 1.71-1.56 (m, 2H), 1.53-1.37 (m, 2H), 1.35- 1.15 (m, 3H); 13C NMR (125 MHz, Chloroform-d) δ 178.0, 177.8, 169.5, 169.2, 138.8, 138.6, 128.7, 128.7, 128.6, 126.6, 126.4, 103.5, 102.7, 77.2, 55.4, 55.1, 54.0, 52.1, 51.5, 50.3, 41.0, 40.7, 40.5, 40.3, 35.6, 35.6, 29.4,

29.3, 25.7, 25.7, 25.6 ppm; HRMS (ESI). [M + Li]+ calcd. for C22H32O4N2Li: 383.2517.

Found: 383.2514. Elemental Anal. calcd for C19H24N2O2: C, 66.99; H, 8.57; N, 7.44. Found: C, 66.89; H, 8.66; N, 7.43. The tertiary amide rotamers are clearly visible in the NMR spectrum and show a 1:1 ratio. 13. 3Å Molecular sieves from Sigma Aldrich were activated by washing the sieves with CH2Cl2, removing the majority of solvent, decantation, and drying by rotary evaporation (40 °C, 10 mbar) and oven for 2 hours at 120 °C. Next the molecular sieves were heated in a household microwave 3 times one minute at 600W with cooling periods (5 min each) in between with the microwave door opened. The sieves were left to cool in an evacuated desiccator and were used immediately. The activation was confirmed by putting a few sieves on the hand, adding a few drops of water and pressing with a finger on the sieves. The sieves should get hot. 14. Methanesulfonic acid is commercial available from Sigma Aldrich ( 99.5%.). 1H-NMR indicates a big peak at 3.33 ppm, likely water. Therefore it must be carefully dried before use, with activated 3Å molecular sieves (see note 10 for activation). Use of ‘wet’ methanesulfonic acid directly from the fresh bottle lead to a dramatic reduction of yield. 100 ml Methanesulfonic acid was dried with 10 gram molecular sieves for at least 1 month. 1H NMR analysis of proper dried methanesulfonic acid will only show 1 peak in the 3 ppm region (3.15 ppm). 15. Remaining solids consisting of powdered molecular sieves and praziquantel were collected by decanting and extracted by vigorously stirring with 20 mL 1:1 Et2O:water in a 50 mL round bottom flask for 10 minutes. After filtration through a P4 sintered glass filter, the filtrate was added to the main solution. 16. Diethyl ether is superior as an extraction solvent than ethyl acetate because the impurity is better soluble in ethyl acetate. 17. Flash chromatography was performed on a Grace Reveleris X2 using a Reveleris® Silica 12g column and elution with accomplished with a gradient of ethyl acetate/petroleum ether (bp 40-60 °C). The mixture was absorber on 2 grams silica. The gradient starts with 30% ethyl acetate and goes to 100% over 12

43 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel minutes in a total run time of 25 minutes. Only ELSD visible peaks are collected as fractions of ~15 mL. The desired product is collected in fraction 7-15 and concentrated by rotary evaporation (40 °C, 10 mmHg).

44 Chapter 3

18. Praziquantel

N O

O 4.04 0.69 2.13 0.72 0.21 0.71 0.22 0.22 1.96 1.72 0.95 4.98 2.01 3.07

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) 25.67 25.70 25.72 28.71 29.00 29.23 29.53 38.64 39.09 40.76 45.14 46.30 49.01 49.53 54.93 55.78 77.16 CDCl3 125.44 126.95 127.42 129.26 132.79 134.72 164.37 174.71

N O

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) Figure 5. Exemplary 1H and 13C NMR of the final product praziquantel.

45 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel

mp 136-138 °C; Rf = 0.46 (EtOAc); 1H NMR (500 MHz, Chloro- N O form-d) δ 7.43-7.05 (m, 4H), 5.15 (dd, J = 13.4, 2.4 Hz, 0.35H), 4.95-4.70 (m, 2H), 4.42 (dd, J = 44.4, 15.5 Hz, 1H), 3.97 (dd, J = N 111.7, 18.1 Hz, 1H), 3.26 (t, J = 12.2 Hz, 0.25H), 3.03-2.85 (m, 2H), 2.85-2.75 (m, 2H), 2.65- 2.41 (m, 1H), 1.95-1.65 (m, 5H), 1.65-1.44 O (m, 2H), 1.44-1.15 (m, 3H) ppm; 13C NMR (125 MHz, Chloro- form-d) δ 174.7, 164.4, 134.7, 132.8, 129.3, 127.4, 126.9, 125.4, 77.2, 55.8, 54.9, 49.5, 49.0, 46.3, 45.1, 40.8, 39.1, 38.6, 29.5, 29.2,

29.0, 28.7, 25.7, 25.7, 25.7 ppm; HRMS (ESI). [M + H]+ calcd. for C19H25O2N2: 313.19095. Found: 313.19105. Elemental Anal. calcd for C19H24N2O2: C, 73.05; H, 7.74; N, 8.97. Found: C, 72.79; H, 7.89; N, 8.95. 19. Two rotamers steaming from the presence of a tertiary amide group can be seen in the NMR spectra, resulting in minor and major peaks in the proton NMR and doublets in the carbon NMR, these were not assigned due to the com- plexity of the spectrum. Alignment of the 1H NMR of an analytical sample (Flu- 3 ka) of praziquantel with our sample proofs identity.

Conclusions In conclusion, an effective, shortest approach to synthesize praziquantel is described, starting with easily available materials. Based on the substrate scope of the Ugi reaction 30 praziquantel derivatives have been synthesized using the same reaction sequence in moderate to good yields (Table 1).

46 Chapter 3

References 1 K. J. Wilby, S. E. Gilchrist, M. H. H. Ensom, Clinical Pharmacokinetics 2013, 52, 647-656.

2 D. G. Colley, A. L. Bustinduy, W. E. Secor, C. H. King, The Lancet 2014, 383, 2253-2264. 3 M. Njoroge, N. M. Njuguna, P. Mutai, D. S. B. Ongarora, P. W. Smith, K. Chibale, Chemical Reviews 2014, 114, 11138-11163. 4 E. Groll, Advances in pharmacology and chemotherapy 1984, 20, 219-238. 5 J. B. Bartlett, K. Dredge, A. G. Dalgleish, Nature Reviews Cancer 2004, 4, 314-322. 6 I. Goldstein, A. L. Burnett, R. C. Rosen, P. W. Park, V. J. Stecher, Sexual Medicine Reviews 2019, 7, 115-128. 7 J. Seubert, R. Pohlke, F. Loebich, Experientia 1977, 33, 1036-1037. 8 D. Cioli, L. Pica-Mattoccia, A. Basso, A. Guidi, Molecular and Biochemical Parasitology 2014, 195, 23-29. 9 A. Dömling, K. Khoury, Chemmedchem 2010, 5, 1420-1434. 10 H. Cao, H. Liu, A. Dömling, Chemistry – A European Journal 2010, 16, 12296-12298.

11 I. Ugi, R. Meyr, U. Fetzer, C. Steinbrückner, Angew. Chem. 1959, 71, 386. 12 A. Dömling, Chemical Reviews 2006, 106, 17-89. 13 C. G. Neochoritis, S. Stotani, B. Mishra, A. Dömling, Org Lett 2015, 17, 2002-2005.

14 I. Ugi, C. Steinbrückner, Angew. Chem. Int. Ed. 1960, 72, 267. 15 S. Marcaccini, T. Torroba, Nature Protocols 2007, 2, 632. 16 J. H. Kim, Y. S. Lee, C. S. Kim, Heterocycles 1998, 48, 2279-2285. 17 A. Pictet, A. Gams, Berichte der deutschen chemischen Gesellschaft 1911, 44, 2480-2485. 18 H. Liu, S. William, E. Herdtweck, S. Botros, A. Dömling, Chemical Biology & Drug Design 2012, 79, 470-477.

47 Ugi multicomponent approach to synthesize schistosomiasis drug praziquantel

Chapter 4

Ugi 4-CR Synthesis of γ- and δ-Lactams providing new access to diverse enzyme interactions, a PDB analysis

André Boltjes, George P. Liao, Ting Zhao, Eberhardt Herdtweck, and Alexander Dömling

Published in: MedChemComm, 2014, 5, 949-952 Chapter 4

Abstract

A three step synthesis of N-unsubstituted tetrazolo γ- and δ-lactams involving a key Ugi-4CR is presented. The compounds, otherwise difficult to access, are conveniently synthesized in overall good yields by our route. PDB analysis of the N-unsubstituted γ- and δ-lactam fragment reveals a strongly tri-directional hydrogen bond donor acceptor interaction with the amino acids of the binding sites.

51 Ugi 4-CR Synthesis of γ- and δ-Lactams

Introduction

The γ-and δ-lactam moiety is an often found fragment in bioactive compounds. Examples of compounds with a lactam fragment include the anti-Parkinson drug oxotremorin1 or the anti-rhinoviral and -enteroviral rupintrivir.2 Generally, the lactam nitrogen can be either unsubstituted or substituted, which influences its hydrogen bonding profile in the receptor binding site. For example in the crystal structure of rupintrivir with the human rhinovirus 3C protease, the γ-lactam-N forms a short hydrogen bond to the Thr142 backbone carbonyl, whereas the δ-lactam-O forms two short contacts to side chain His161-NH and the Thr142-OH, respectively (Fig. 1).3 In our ongoing efforts in structure- and computational-based design of bioactive compounds we were interested in a short and versatile synthesis of N-unsubstituted γ- and δ-lactams with potential multiple hydrogen bond interactions.4 Combining the lactam functionality with a 1,5 disubstituted tetrazolo peptidomimetic could enhance the affinity of this tetrazole analog which is a well-known cis-amide bond mimic, which provides new synthetic probes for a series of biological targets.5-9 Multicomponent reactions (MCR) were found to be an excellent tool to rapidly access a large and versatile drug-like chemical space to address the corresponding biological space.5 A convenient and versatile synthesis of N-substituted γ- and δ-lactams using convergent Ugi-type MCR’s 4 was recently introduced by Marcos et al. and refined by others.10-12 However no MCR-based synthesis of N-unsubstituted γ- and δ-lactams has been described up to date. Clearly many synthetic approaches towards N-unsubstituted γ- nd δ-lactams are described, however these contain limitations regarding variability d length of the synthetic routes.13

52 Chapter 4

Figure 1.Trifurcated hydrogen bonding interactions (red dotted lines) of an N-unsubstituted γ-lactam (cyan sticks) with some protease amino acids in the example of rupintrivir (PDB ID 1CQQ).

Ammonia is one of the few amine components which does not regularly give convincing results in the Ugi MCRs.14-18 Therefore we recently introduced tritylamine as an ammonia surrogate in the Ugi tetrazole MCR.19 This reaction forms the core of our design of a synthetic pathway towards N-unsubstituted γ- and δ-lactams (Scheme 1). The first step comprises the Ugi tetrazole MCR using tritylamine, followed by cleavage of the trityl group. The formed primary amine should easily undergo cyclisation to form the target γ- and δ-lactam compounds.

53 Ugi 4-CR Synthesis of γ- and δ-Lactams

Scheme 1. Devised synthetic pathway to tetrazolo N-unsubstituted γ- and δ-lactams

Results and Discussion

The Ugi tetrazole synthesis was initially performed under Ugi azide conditions 4 with tritylamine (1), azidotrimethylsilane (2), phenylethylisonitrile and various aldehyde esters to check scope and limitations.20 The aliphatic aldehydes 3a-b were prepared according to previous described methods starting from the acid chloride (Scheme 2.).21 O O 2,6-Lutidine O

O n Cl Pd/C 10% 3 atm H2 O n O 3a, n=2 3b, n=3 Scheme 2. The two aldehydes utilized in the Ugi tetrazole synthesis

Due to the steric hindrance of the trityl moiety, the Schiff-base condensation step of the Ugi reaction only proceeds smoothly under microwave irradiation.19 More- over, it was found that only aliphatic aldehydes such as 3a and 3b gave good yields varying between 40 and 80 %. The used number of aldehydes was bigger with even n=4 and a cyclopropyl moiety in the chain. The Ugi tetrazole reaction was also successful for these aldehydes, however, the subsequent cyclisation gave problems, not yielding the desired lactams. Aromatic aldehydes were investigated and only in some reactions a moderate yield was observed.19 Aryl ester aldehydes did not yield any product at all.

Product diversity by using four other isonitriles (4) (Table 1.) showed yields comparable with phenylethylisocyanide. Deprotection conditions in order to cleave the trityl from the amine requires at least 2 equivalents of TFA. Full conversion into the amine is achieved within seconds monitored by TLC. Simple purifica-

54 Chapter 4 tion was performed by pouring the reaction mixture directly onto a 5 cm silica gel filter wetted with heptane:EtOAc (1:1) removing the impurities by washing with 50 mL heptane:EtOAc. Then the product was eluted from the filter using 50 mL CH2Cl2:MeOH (1:1) to obtain the pure amine. Subsequent cyclisation gave difficulties. Generally Et3N or potassium carbonate is sufficient to cyclize comparable molecules, however it was found that the resulting TFA salt needed to be liberated using a strong base prior to cyclisation. The main disadvantage of using a strong base is that this results in saponification of the initial ester and thus pre- venting cyclisation at all. Sodium hydride was found to be a suitable base giving in most cases only a minor conversion to amino acids and reasonable to very good yields for the γ- and δ-lactams (Table 1).

We could grow four crystals of γ- (6b, 6e) and δ-lactams (6f, 6j) suitable for single crystal structure determination (Fig. 2). Interestingly the lactam amide group in all cases show intermolecular hydrogen bonding involving the amide NH and the carbonyl CO. Five-membered γ-lactam 6b shows an intermolecular hydrogen bonding with a neighboring γ-lactam amide with a distance between the heavy atoms of 2.8 Å. Six-membered δ-lactam 6, however, exhibits a trifurcated hydrogen bonding network involving a neighboring δ-lactam and a co-crystallized water molecule. Thus the oxygen atom shows two hydrogen bonds along the lone pair electrons.

Table 1. Cyclization of the Ugi 4-CR to yield the isoindolone scaffold.

4 N R R4 N N N N N N NHTrityl N NH 5 COOMe 6 n n O

entry R(4) n Ugi (%) a entry cyclisation(%) b 5a 2 73 6a 55

5b 2 50 6b 89

5c 2 75 6c 32

5d 2 56 6d 72

55 Ugi 4-CR Synthesis of γ- and δ-Lactams

5e 2 40 6e 95

5f 3 78 6f 76

5g 3 62 6g 42

5h 3 43 6h 40

5i 3 68 6i 99

5j 3 41 6j 79 a TFA, CH Cl , RT. 1 min. b NaH, THF, RT, 4h 2 2 4

56 Chapter 4

Figure 2. Intermolecular hydrogen bond network of exemplary γ- and δ-lactams in the solid state. Molecule 6a shows a pair wise hydrogen bonding with a neighbour molecule, while 6f shows a bifurcated hydrogen bonding pattern including a neighbour molecule and a water molecule.

The γ- and δ-lactam moiety is present in many bioactive molecules and therefore we also decided to analyze the moieties in the protein data bank (PDB).1 Structure search on γ-lactams yielded a total of 817 structures containing this moiety; δ-lactams resulted in 37 structure hits. After removing structures with ≥ 95% structure similarity, the results were reduced to 281 structures and 27 structures respectively. Of those structures, only the N-unsubstituted lactam rings were included in the analysis. In addition, all lactam structures that were part of the protein and photosynthesis related structures were also excluded due to redundancy of the ligands. Although 37 and 11 structures, respectively for the γ- and δ-lactams, are not sufficient for a statistical analysis, itell w provides the basis for a trend. Key findings include: 1) for the majority of structures, the lactam amide group undergoes a trifurcated hydrogen bond network involving the carbonyl oxygen twice and the amide NH once. 2) the γ-lactams interact with the histidine side chains most frequently through the carbonyl oxygen. Analysis

57 Ugi 4-CR Synthesis of γ- and δ-Lactams of the structures show that the histidines are, all but one, part of viral proteas- es. In addition, the amide-nitrogen polar interactions are also similar for these structures. 3) the δ-lactams possess more hydrogen bonding interaction possibilities as many amino acid residues are found in close proximity to the lactam moiety. In figure 3, ten random structures (3D23, 3EWJ, 3QZR, 3RHK, 3TNT, 3UR9, 3DPM, 1H0V, 3JUC and 3Q3Y) are aligned showing the amino acid residues/molecules with which the γ-lactam moiety shows polar interactions.

Figure 3. Above: Alignment of several PDB structures showing the polar interactions for 10 γ-lactam containing ligands. The ligand γ-lactam moiety is shown as green sticks, the interacting receptor amino acids as colored lines and the hydrogen bonding as yellow dotted lines.

Conclusions We have designed a fast and reliable synthetic route to 5- and 6-membered unsubstituted tetrazololactams using a key azido-Ugi reaction, followed by a deprotection and cyclisation step. Analysis of the scaffold in the protein data bank indicates that the lactam-NH can undergo multiple and strong H-bonds. Moreover the scaffold is underused in medicinal chemistry and thus provides multiple chances for drug design.

58 Chapter 4

Experimental procedures and Spectral Data

All isonitriles were made in house by either performing the Hoffman or Ugi procedure. Other reagents were purchased from Sigma Aldrich, ABCR, Acros and AK Scientific and were used without further purification. All microwave irradiation reactions were carried out in a Biotage Initiator™ Microwave Synthesizer. The Ugi tetrazoles were purified by flash chromatography, on a Teledyne ISCO Rf 200, using RediSep Rf Normal-phase Silica Flash Columns (Silica Gel 60 Å, 230 - 400 mesh) unless otherwise noted. Column chromatography was performed with MP Ecochrom Silica Gel 32–63, 60 Å. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on a Bruker Avance DRX 500. Chemical shift values are 1 reported as part per million (δ) relative to residual solvent peaks (CDCl3, H δ = 7.26, 13C δ = 77.16 or TMS 1H δ = 0.00 ppm). The coupling constants (J) are reported in Hertz (Hz). Electrospray ionization mass spectra (ESI-MS) were recorded on a Waters Investigator Semi-prep 15 SFC-MS instrument.

Synthetic Procedures and characterization data for compounds 5a-j and 6a-j Synthetic procedure 1 Aldehyde (1 mmol), tritylamine (1 mmol) were mixed in methanol (1 mL) and subjected to microwave irradiation for 15 minutes. Subsequently azidotrimethylsilane (1 mmol) and isonitrile (1mmol) were added and the mixture was again subjected to microwave irradiation for 15 minutes at 100°C. The solvent was evaporated under reduced pressure and the residue was purified using flash chromatography to obtain the product.

Synthetic procedure 2 To a solution of Ugi tetrazole (0.5-1.0 mmol) in 3 mL was added TFA (150 µL, 2 mmol). After 1 minute the mixture was filtered through a silica bed washing with 50 mL Heptane:EtOAc 1:1 (v/v) to remove the trityl cation impurity. The amine was collected by washing the silica bed with 50 mL CH2Cl2:MeOH 1:1 (v/v). The mixture was concentrated under reduced pressure and redissolved in dry THF (3 mL). Sodium hydride (5 mmol) was washed with heptanes prior to addition. After 4 hours of stirring, EtOH was added to quench the reaction. The solvents were removed under reduced pressure, and the residue was purified by column chromatography using CH2Cl2:MeOH 20:1 (v/v) to afford the lactam.

59 Ugi 4-CR Synthesis of γ- and δ-Lactams

Methyl 4-(1-(tert-butyl)-1H-tetrazol-5-yl)-4-(tritylamino)butanoate (5a)

O O The product was obtained using procedure 1 starting from t-butyliso- 1 cyanide and 3a as a white solid (73%): Rf 0.50 (EtOAc:Hept 1:1). H Trt N N NMR ( 500MHz, CDCl ): δ 7.40 (d, J = 7.5, 6H), 7.19 (t, J = 7.5, 6H), 7.13 H N 3 N N (t, J = 7.2, 3H), 4.41 (t, J = 3.5 Hz, 1H), 3.63 (s, 3H), 2.80 – 2.67 (m, 1H), 2.49 – 2.34 (m, 1H), 2.28 – 2.22 (m, 1H), 1.87 – 1.80 (m, 1H), 1.42 (s, 9H) 13 ppm. C NMR (125 MHz, CDCl3) δ 173.67, 156.79, 145.62, 128.92, 127.89, 126.72, 71.71, 61.76, 51.76, 48.35, 32.29, 29.91, 28.34 ppm.

Methyl 4-(1-(2,4,4-trimethylpentan-2-yl)-1H-tetrazol-5-yl)-4-(tritylamino)butanoate (5b)

O O The product was obtained using procedure starting from t-octyliso- 1 cyanide and 3a as a white solid (50%): Rf 0.50 (EtOAc:Hept 1:1). H Trt N NMR (500 MHz, CDCl3) δ 7.39 (d, J = 7.5, 6H), 7.19 (t, J = 7.5, 6H), 7.14 N H N (t, J = 7.2, 3H), 4.50 – 4.40 (m, 1H), 3.62 (s, 3H), 3.54 (d, J = 9.0, 1H), 2.74 N N – 2.60 (m, 1H), 2.35 – 2.25 (m, 1H), 2.20 – 2.13 (m, 1H), 1.90 – 1.83 (m, 1H), 1.73 (m, 2H), 1.52 (s, 3H), 1.43 (s, 3H), 0.81 (s, 9H) ppm. 13C NMR 4 (125 MHz, CDCl3) δ 173.61, 156.98, 145.72, 128.99, 127.93, 126.76, 71.71, 66.13, 55.21, 51.69, 48.60, 32.18, 31.61, 31.11, 29.89, 28.45, 28.39 ppm.

Methyl 4-(1-phenethyl-1H-tetrazol-5-yl)-4-(tritylamino)butanoate (5c)

O O The product was obtained using procedure 1 starting from phene-

thylisocyanide and 3a as a white solid (75%): Rf 0.31 (EtOAc:Hept 1:2). Trt N 1 N ). H NMR (500 MHz, CDCl ) δ 7.35 (d, J = 7.5, 6H), 7.27 – 7.09 (m, H N 3 N N 12H), 7.00 (d, J = 7.2, 2H), 4.02 – 3.84 (m, 2H), 3.81 – 3.71 (m, 1H), 3.64 (s, 3H), 3.15 – 2.96 (m, 3H), 2.56 – 2.40 (m, 1H), 2.18 – 2.02 (m, 1H), 1.93 13 – 1.87 (m, 1H), 1.43 – 1.37 (m, 1H) ppm. C NMR (125 MHz, CDCl3) δ 173.33, 157.14, 145.15, 136.69, 128.96, 128.81, 128.62, 127.98, 127.32, 126.84, 71.59, 51.76, 48.56, 47.01, 35.43, 31.76, 29.01 ppm.

60 Chapter 4

Methyl 4-(1-benzyl-1H-tetrazol-5-yl)-4-(tritylamino)butanoate (5d)

O O The product was obtained using procedure 1 starting from benzyli- 1 socyanide and 3a as a white solid (56%): Rf 0.50 (EtOAc:Hept 1:1). H Trt N N NMR (500 MHz, CDCl3) δ 7.32 (d, J = 8.1, 6H), 7.24 – 7.27 (m, 3H), 7.20 H N N N – 7.11 (m, 9H), 7.05 (d, J = 6.7, 2H), 5.21 (d, J = 15.3, 1H), 4.76 (d, J = 15.3, 1H), 4.14 – 4.10 (m, 1H), 3.58 (s, 3H), 3.00 (d, J = 9.2, 1H), 2.47 – 2.41 (m, 1H), 2.20 – 2.07 (m, 1H), 1.96 – 1.89 (m, 1H), 1.59 – 1.53 (m, 13 1H), 1.33 – 1.24 (m, 1H) ppm. C NMR (125 MHz, CDCl3) δ 173.31, 157.08, 145.21, 133.28, 129.14, 128.92, 128.58, 128.03, 128.00, 126.83, 71.49, 51.73, 50.82, 47.06, 31.50, 28.86 ppm.

Methyl 4-(1-cyclohexyl-1H-tetrazol-5-yl)-4-(tritylamino)butanoate (5e)

O O The product was obtained using procedure 1 starting from cyclohex-

ylisocyanide and 3a as a white solid (40%): Rf 0.50 (EtOAc:Hept 1:1). 1 Trt H NMR (500 MHz, CDCl ) δ 7.40 (d, J = 8.1, 6H), 7.20 (t, J = 7.5, 6H), N N 3 H N 7.15 (t, J = 6.8, 3H), 4.16 – 4.02 (m, 1H), 3.78 – 3.68 (m, 1H), 3.61 (s, 3H), N N 3.12 (d, J = 8.3, 1H), 2.68 – 2.49 (m, 1H), 2.34 – 2.13 (m, 2H), 1.96 – 1.76 (m, 4H), 1.76 – 1.57 (m, 4H), 1.37 – 1.18 (m, 3H) ppm. 13C NMR (125

MHz, CDCl3) δ 173.33, 156.11, 145.39, 128.74, 127.97, 126.85, 71.80, 57.45, 51.77, 47.09, 33.59, 32.02, 31.99, 28.88, 25.34, 25.29, 24.80 ppm.

Methyl 5-(1-(tert-butyl)-1H-tetrazol-5-yl)-5-(tritylamino)pentanoate (5f)

O The product was obtained using procedure 1 starting from t-butyliso- O 1 cyanide and 3b as a white solid (78%): Rf 0.50 (EtOAc:Hept 1:1). H

NMR (500 MHz, CDCl3) δ 7.43 (d, J = 8.0, 6H), 7.20 (t, J = 7.5, 6H), 7.13 Trt N N H N (t, J = 7.0, 3H), 4.34 – 4.25 (m, 1H), 3.62 (s, 3H), 3.40 (d, J = 9.3, 1H), 2.25 N N (t, J = 6.9, 2H), 1.94 – 1.70 (m, 2H), 1.64 – 1.48 (m, 2H), 1.41 (s, 9H) ppm. 13 C NMR (125 MHz, CDCl3) δ 173.47, 157.18, 145.78, 128.95, 127.91, 126.69, 71.75, 61.64, 51.57, 49.33, 37.13, 33.91, 30.02, 19.88 ppm.

61 Ugi 4-CR Synthesis of γ- and δ-Lactams

Methyl 5-(1-(2,4,4-trimethylpentan-2-yl)-1H-tetrazol-5-yl)-5-(tritylamino)pentanoate (5g)

O The product was obtained using procedure 1 starting from t-octyliso- 1 O cyanide and 3b as a white solid (62%): Rf 0.50 (EtOAc:Hept 1:1). H

NMR (500 MHz, CDCl3) δ 7.43 (d, J = 7.5, 6H), 7.21 (t, J = 7.5, 6H), 7.14 Trt N N (t, J = 7.2, 3H), 4.42 – 4.25 (m, 1H), 3.61 (s, 2H), 3.39 (d, J = 8.9, 1H), 2.29 H N N N – 2.08 (m, 2H), 1.82 – 1.65 (m, 4H), 1.64 – 1.53 (m, 1H), 1.52 – 1.41 (m, 13 7H), 0.81 (s, 9H) ppm. C NMR (125 MHz, CDCl3) δ 173.42, 157.20, 145.87, 129.01, 127.94, 126.74, 71.72, 66.00, 55.22, 51.56, 49.55, 36.62, 33.89, 31.63, 31.14, 30.04, 28.53, 19.87 ppm.

Methyl 5-(1-phenethyl-1H-tetrazol-5-yl)-5-(tritylamino)pentanoate (5h)

O The product was obtained using procedure 1 starting from phene- O thylisocyanide and 3b as a white solid (43%): Rf 0.50 (EtOAc:Hept 1 1:1). H NMR (500 MHz, CDCl3) δ 7.35 (d, J = 7.6, 6H), 7.29 – 7.21 (m, Trt N N 3H), 7.19 (t, J = 7.4, 6H), 7.16 – 7.10 (m, 3H), 7.00 (d, J = 6.5, 2H), 4.04 H N N N – 3.92 (m, 1H), 3.85 – 3.69 (m, 2H), 3.65 (s, 3H), 3.13 – 2.99 (m, 2H), 4 2.93 (d, J = 8.3, 1H), 2.19 – 2.01 (m, 2H), 1.62 – 1.48 (m, 1H), 1.42 – 1.30 13 (m, 2H), 1.25 – 1.14 (m, 1H) ppm. C NMR (125 MHz, CDCl3) δ 173.32, 157.38, 145.23, 136.82, 128.99, 128.82, 128.63, 127.99, 127.34, 126.83, 71.67, 51.61, 48.67, 48.01, 36.44, 35.46, 33.61, 20.31 ppm.

Methyl 5-(1-benzyl-1H-tetrazol-5-yl)-5-(tritylamino)pentanoate (5i)

O The product was obtained using procedure 1 starting from benzyli- 1 O socyanide and 3b as a white solid (43%): Rf 0.50 (EtOAc:Hept 1:1). H

NMR (500 MHz, CDCl3) δ 7.36 (d, J = 7.4, 6H), 7.31 – 7.24 (m, 3H), 7.21 Trt N N (t, J = 7.3, 6H), 7.19 – 7.13 (m, 3H), 7.01 (d, J = 5.5, 2H), 5.29 (d, J = 15.4, H N N N 1H), 4.68 (d, J = 15.4, 1H), 4.02 – 3.91 (m, 1H), 3.58 (s, 3H), 2.87 (d, J = 8.3, 1H), 1.96 (t, J = 6.9, 2H), 1.54 – 1.43 (m, 1H), 1.32 – 1.16 (m, 3H) 13 ppm. C NMR (125 MHz, CDCl3) δ 173.12, 157.33, 145.25, 133.59, 129.11, 128.85, 128.59, 128.01, 127.74, 126.85, 71.62, 51.49, 50.87, 48.02, 36.09, 33.58, 20.23 ppm.

62 Chapter 4

Methyl 5-(1-cyclohexyl-1H-tetrazol-5-yl)-5-(tritylamino)pentanoate (5j)

O The product was obtained using procedure 1 starting from cyclohex-

O ylisocyanide and 3b as a white solid (43%): Rf 0.50 (EtOAc:Hept 1:1). 1 H NMR (500 MHz, CDCl3) δ 7.41 (d, J = 7.5, 6H), 7.21 (t, J = 7.5, 6H), Trt N N 7.15 (t, J = 7.2, 3H), 4.07 – 3.95 (m, 1H), 3.89 – 3.75 (m, 1H), 3.60 (s, 3H), H N N N 2.90 (d, J = 7.3, 1H), 2.22 – 2.06 (m, 2H), 1.96 – 1.62 (m, 8H), 1.58 – 1.45 13 (m, 1H), 1.43 – 1.19 (m, 5H)ppm. C NMR (125 MHz, CDCl3) δ 173.28, 156.28, 145.47, 128.75, 128.02, 126.87, 72.01, 57.61, 51.57, 48.12, 36.43, 33.58, 33.56, 32.27, 25.39, 24.90, 20.34 ppm.

5-(1-(tert-butyl)-1H-tetrazol-5-yl)pyrrolidin-2-one (6a)

O The product was obtained using procedure 2 starting from 5a as a N N 1 H N white solid (55%): H NMR (500 MHz, CDCl3) δ 7.69 (s, 1H), 5.40 – N N 5.13 (m, 1H), 2.75 – 2.57 (m, 2H), 2.43 – 2.31 (m, 2H), 1.80 – 1.75 (m, 13 9H) ppm. C NMR (125 MHz, CDCl3) δ 178.69, 155.80, 61.61, 49.12, 30.34, 29.32, 28.12 ppm. MS (ESI) (m/z) 210.1 [M+H]+.

5-(1-(2,4,4-trimethylpentan-2-yl)-1H-tetrazol-5-yl)pyrrolidin-2-one (6b)

O The product was obtained using procedure 2 starting from 5b as a N N 1 H N white solid (89%): H NMR (500 MHz, CDCl3) δ 7.75 (s, 1H), 5.35 – N N 5.20 (m, 1H), 2.78 – 2.56 (m, 2H), 2.46 – 2.31 (m, 2H), 1.97 (q, J = 15.3, 13 2H), 1.86 (d, J = 3.4, 6H), 0.79 (s, 9H) ppm. C NMR (125 MHz, CDCl3) δ 178.67, 156.33, 64.96, 54.34, 49.28, 31.84, 30.78, 30.53, 29.38, 28.20 ppm. MS (ESI) (m/z) 266.1 [M+H]+.

5-(1-phenethyl-1H-tetrazol-5-yl)pyrrolidin-2-one (6c)

O The product was obtained using procedure 2 starting from 5c as a N N 1 H Nwhite solid (32%): H NMR (500 MHz, CDCl3) δ 7.28 (d, J = 4.2, 3H), N N 7.06 – 6.91 (m, 2H), 6.82 (s, 1H), 4.71 – 4.46 (m, 2H), 4.44 – 4.25 (m, 1H), 3.37 – 3.11 (m, 2H), 2.54 – 2.37 (m, 1H), 2.33 – 2.08 (m, 2H), 2.08 – 1.87 13 (m, 1H) ppm. C NMR (125 MHz, CDCl3) δ 178.26, 155.80, 136.55, 129.32, 128.95, 127.76, 49.33, 47.19, 36.35, 29.26, 26.50 ppm. MS (ESI) (m/z) 258.1 [M+H]+.

63 Ugi 4-CR Synthesis of γ- and δ-Lactams

5-(1-benzyl-1H-tetrazol-5-yl)pyrrolidin-2-one (6d)

O The product was obtained using procedure 2 starting from 5d as a N N 1 H Nwhite solid (72%): H NMR (500 MHz, CDCl3) δ 7.83 (s, 1H), 7.37 (d, J N N = 6.3, 3H), 7.28 – 7.17 (m, 2H), 5.67 (dd, J = 47.8, 15.6, 2H), 5.02 – 4.88 (m, 1H), 2.44 – 2.33 (m, 1H), 2.33 – 2.18 (m, 2H), 2.05 – 1.90 (m, 1H) 13 ppm. C NMR (126 MHz, CDCl3) δ 178.70, 155.53, 133.33, 129.40, 129.17, 127.73, 51.31, 47.89, 29.24, 26.30 ppm. MS (ESI) (m/z) 244.1 [M+H]+.

5-(1-cyclohexyl-1H-tetrazol-5-yl)pyrrolidin-2-one (6e)

O The product was obtained using procedure 2 starting from 5e as a N N 1 H Nwhite solid (95%): H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 26.2, 1H), N N 5.18 – 4.93 (m, 1H), 4.37 – 4.14 (m, 1H), 2.74 – 2.55 (m, 2H), 2.53 – 2.33 (m, 2H), 2.13 – 1.91 (m, 6H), 1.78 (d, J = 12.7, 1H), 1.53 – 1.40 (m, 3H), 13 1.39 – 1.26 (m, 1H) ppm. C NMR (126 MHz, CDCl3) δ 178.73, 154.46, 58.17, 47.72, 33.45, 32.73, 29.48, 26.81, 25.19, 25.11, 24.75 ppm. MS (ESI) (m/z) 236.0 [M+H]+. 4

6-(1-(tert-butyl)-1H-tetrazol-5-yl)piperidin-2-one (6f)

The product was obtained using procedure 2 starting from 5f as a N 1 O N white solid (76%): H NMR (500 MHz, CDCl ) δ 7.48 (s, 1H), 5.22 – H N 3 N N 5.09 (m, 1H), 2.46 – 2.37 (m, 1H), 2.36 – 2.27 (m, 1H), 2.20 – 2.08 (m, 13 2H), 2.07 – 1.98 (m, 1H), 1.77 (s, 10H) ppm. C NMR (126 MHz, CDCl3) δ 172.08, 155.39, 61.63, 47.93, 30.90, 30.21, 28.03, 18.59 ppm. MS (ESI) (m/z) 224.1 [M+H]+.

6-(1-(2,4,4-trimethylpentan-2-yl)-1H-tetrazol-5-yl)piperidin-2-one (6g)

The product was obtained using procedure 2 starting from 5g as a N 1 O N white solid (42%): H NMR (500 MHz, CDCl3) δ 6.58 (d, J = 25.2, 1H), H N N N 5.15 (t, J = 6.1, 1H), 2.59 – 2.39 (m, 2H), 2.28 – 2.10 (m, 3H), 1.94 (q, J = 15.3, 2H), 1.86 (d, J = 12.9, 7H), 1.83 – 1.71 (m, 1H), 0.79 (s, 9H) ppm. 13 C NMR (126 MHz, CDCl3) δ 171.79, 155.62, 65.21, 54.45, 48.58, 31.86, 31.07, 30.83, 30.71, 30.65, 28.35, 19.01 ppm. MS (ESI) (m/z) 280.2 [M+H]+.

64 Chapter 4

6-(1-phenethyl-1H-tetrazol-5-yl)piperidin-2-one (6h)

The product was obtained using procedure 2 starting from 5h as a N 1 O N white solid (40%): H NMR (500 MHz, CDCl ) δ 7.27 (d, J = 6.7, 3H), H N 3 N N 7.06 – 6.91 (m, 3H), 4.65 – 4.46 (m, 2H), 4.28 (t, J = 5.8, 1H), 3.31 – 3.16 (m, 2H), 2.28 – 2.19 (m, 2H), 1.94 – 1.81 (m, 1H), 1.67 – 1.44 (m, 3H) 13 ppm. C NMR (126 MHz, CDCl3) δ 172.12, 155.36, 136.60, 129.23, 128.94, 127.66, 49.42, 46.67, 36.29, 31.01, 26.91, 18.83 ppm. MS (ESI) (m/z) 272.2 [M+H]+.

6-(1-benzyl-1H-tetrazol-5-yl)piperidin-2-one (6i)

The product was obtained using procedure 2 starting from 5i as a N 1 O N white solid (99%): H NMR (500 MHz, CDCl3) δ 7.44 – 7.33 (m, 3H), H N N N 7.24 (s, 1H), 7.23 – 7.17 (m, 2H), 5.66 (s, 2H), 4.91 – 4.81 (m, 1H), 2.37 – 2.21 (m, 2H), 1.98 – 1.82 (m, 2H), 1.77 – 1.59 (m, 2H). 13C NMR (125

MHz, CDCl3) δ 172.50, 155.12, 133.55, 129.47, 129.24, 127.67, 51.55, 47.37, 31.09, 26.95, 18.80 ppm. MS (ESI) (m/z) 258.1 [M+H]+.

6-(1-cyclohexyl-1H-tetrazol-5-yl)piperidin-2-one (6j)

The product was obtained using procedure 2 starting from 5j as a N 1 O N white solid (79%): H NMR (500 MHz, CDCl ) δ 7.50 (s, 1H), 4.95 (t, J H N 3 N N = 5.6, 1H), 4.38 – 4.19 (m, 1H), 2.48 – 2.32 (m, 2H), 2.23 – 1.91 (m, 10H), 1.91 – 1.82 (m, 1H), 1.82 – 1.70 (m, 1H), 1.51 – 1.39 (m, 2H), 1.39 – 1.31 13 (m, 1H) ppm. C NMR (126 MHz, CDCl3) δ 172.40, 154.11, 58.29, 46.90, 33.55, 32.87, 31.05, 27.48, 25.29, 25.26, 24.81, 18.86 ppm. MS (ESI) (m/z) 250.2 [M+H]+.

65 Ugi 4-CR Synthesis of γ- and δ-Lactams

Exemplary 1H NMR and 13C NMR for the UT-4CR and intramolecular cyclization product 5b and 6b. Compound 5b 1H NMR

O O 7.40 7.38 7.21 7.19 7.18 7.15 7.14 7.12 4.47 4.46 4.45 4.44 4.43 3.65 3.63 3.62 3.55 3.53 2.70 2.68 2.67 2.65 2.63 2.31 2.31 2.30 2.17 2.16 2.15 1.89 1.87 1.86 1.85 1.77 1.74 1.72 1.69 1.52 1.43 0.81 CH 8000 3

7500

7000 N NH N H C 6500 3 N H C N 3 6000 H C 3 H C 3 5500

CH 3 5000

4500

4000

3500

3000

2500

2000

1500

1000 500 4 0

-500 6.11 6.22 3.00 1.05 2.92 0.95 1.08 1.08 1.07 1.19 2.34 3.20 3.10 9.46

12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm)

Compound 5b 13C NMR

18000 173.61 156.98 145.72 128.99 127.93 126.76 71.71 66.13 55.21 51.69 48.60 32.18 31.61 31.11 29.89 28.45 28.39

17000 O O CH 3 16000

15000

N 14000 NH N H C 13000 3 N H C N 3 12000 H C 3 H C 11000 3

CH 10000 3

9000

8000

7000

6000

5000

4000

3000

2000

1000

-1000

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

66 Chapter 4

Compound 6b 1H NMR 6500 7.75 5.29 5.29 5.28 5.27 2.74 2.72 2.71 2.70 2.69 2.63 2.63 2.62 2.61 2.60 2.59 2.45 2.44 2.43 2.42 2.41 2.41 2.40 2.38 2.37 2.37 2.36 2.35 2.34 2.01 1.98 1.96 1.93 1.87 1.86 0.79 O N 6000 NH N H C 3 N 5500 H C N 3

H C 3 5000 H C 3

CH 3 4500

4000

3500

3000

2500

2000

1500

1000

500

-500 1.00 1.04 2.08 2.09 2.16 6.24 9.75

11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm)

Compound 6b 13C NMR

7500 28.20 29.38 30.53 30.78 31.84 49.28 54.34 64.96 156.33 178.67

7000

O N 6500 NH N H C 3 N 6000 H C N 3

H C 5500 3 H C 3 5000 CH 3 4500

4000

3500

3000

2500

2000

1500

1000

500

-500

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

67 Ugi 4-CR Synthesis of γ- and δ-Lactams

Single Crystal X-Ray Structure Determination

Crystallographic data were collected on an X-ray single crystal diffractometer equipped with a CCD detector (Bruker APEX II, κ-CCD), a fine focus sealed tube (Bruker AXS, D8) with MoKα radiation (λ= 0.71073 Å), and a graphite monochromator by using the SMART software package. The measurements were performed on single crystals coated with perfluorinated ether. The crystals were fixed on the top of a cactus prickle (Opuntia ficus-india) with perfluorinated ether and transferred to the diffractometer. The crystals were frozen under a stream of cold nitrogen. A matrix scan was used to determine the initial lattice parameters. Reflections were merged and corrected for Lorenz and polarization effects, scan speed, and background using SAINT. Absorption corrections, including odd and even ordered spherical harmonics were performed using SADABS. Space group assignments were based upon systematic absences, E statistics, and successful refinement of the structures. Structures were solved by direct methods with the aid of successive difference Fourier maps, and were refined against all data using WinGX23 based on SIR-92. If not mentioned otherwise, non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms could be located in the difference Fourier maps and were allowed to refine freely. Full-ma- 4 2 2 2 trix least-squares refinements were carried out by minimizing wΣ (Fo -Fc ) with SHELXL-97 weighting scheme. Neutral atom scattering factors for all atoms and anomalous dispersion corrections for the non-hydrogen atoms were taken from International Tables for Crystallography. Images of the crystal structures were generated by PLATON. CCDC 961190 (6b), CCDC 961191 (6e), CCDC 961188 (6f), and CCDC 961189 (6j) contains the supplementary crystallographic data for this compound. This data can be obtained free of charge from The Cambridge Crys- tallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif or via https:// www.ccdc.cam.ac.uk/services/structure_deposit/

68 Chapter 4

6b 6e

6f 6j . Figure 4. Ortep drawing drawings of compounds 6b, 6e, 6f and 6j with 50% ellipsoids.

Table 2. Data collection and refinement parameters of obtained crystals

Data Collection 6b 6e 6f 6j

Molecular For- C13H23N5O C11H17N5O C20H36N10O3 C12H19N5O mula

Molecular 265.36 g/mol 235.30 a.m.u. 464.59 a.m.u 249.32 a.m.u. Weight Crystal Color/ 0.20×0.25×0.43 0.28×0.41×0.51 0.10×0.30×0.36 0.25×0.25×0.28 mm size mm mm mm Space Group Monoclinic Triclinic Monoclinic Orthorhombic

P 21/c P1 C 2/c P bca

69 Ugi 4-CR Synthesis of γ- and δ-Lactams

Cell Constants a= 907.24 pm a = 654.62 pm a =2660.36 pm a =991.62(3) pm b= 1147.13pm α=65.9412° b =630.28 pm b =1193.89(3) pm β= 100.6508° β =109.7809° c =2152.52(7) pm c 1399.04 pm b = 925.70 pm c =1546.66 pm V = 2548.34·106 pm3 V= 1430.93∙106 β=81.5752° V = 2440.37·106 Z = 8 3 3 -3 pm pm Dcalc = 1.300 gcm Z = 4 c = 1055.29 pm Z = 4 γ=89.9391° Dcalc = 1.232 Dcalc = 1.265 g∙cm-3 gcm-3 V = 576.41·106 pm3

Z = 2

Dcalc= 1.356 gcm-3

F000: 576 252 1000 1072 Temperature (-173±1) °C (-173±1) °C (-150±1) °C (-150±1) °C Measurement 2.28° < θ < 25.46° 2.14 < θ < 25.38° 1.63° < θ < 2.79° < θ < 25.40° Range h: -10/10, h: -7/7, 25.55° h: -11/11 k: -13/13, k: -11/11 h: -32/32 k: -14/14 l: -16/16 l: -12/12 k: -7/7 l: -25/25 l: -18/18 Refinement 4 Refl. collected 48744 19536 35663 34576 Independent 2636 2111 2264 2338 reflections Rint: 0,020 Rint: 0,016 Rint: 0,026 Rint: 0,026 Extinction Cor- ε refined toε = No No No rection 0.0054 Goodness of fit 1.033 1.050 1.049 1.082 Resid. Electron +0.28 e/Å3 +0.26 e/Å3 +0.19 e/Å3 +0.24 e/Å3 Density -0.17 e/Å3 -0.23 e/Å3 -0.18 e/Å3 -0.24 e/Å3 R indices (all R1=0.0320 R1=0.0303 R1=0.0348 R1=0.0407 refelctions) wR2=0.0764 wR2=0.0774 wR2=0.0911 wR2=0.0994

70 Chapter 4

References 1 C. Tang, A. F. Castoldi, L. G. Costa, Biochem Mol Biol Int 1993, 29. 2 X. Zhang, Z. Song, B. Qin, X. Zhang, L. Chen, Y. Hu, Z. Yuan, Antiviral Research 2013, 97, 264-269. 3 D. A. Matthews, P. S. Dragovich, S. E. Webber, S. A. Fuhrman, A. K. Pa- tick, L. S. Zalman, T. F. Hendrickson, R. A. Love, T. J. Prins, J. T. Marako- vits, R. Zhou, J. Tikhe, C. E. Ford, J. W. Meador, R. A. Ferre, E. L. Brown, S. L. Binford, M. A. Brothers, D. M. DeLisle, S. T. Worland, Proceedings of the National Academy of Sciences 1999, 96, 11000. 4 D. Koes, K. Khoury, Y. Huang, W. Wang, M. Bista, G. M. Popowicz, S. Wolf, T. A. Holak, A. Dömling, C. J. Camacho, PloS one 2012, 7, e32839-e32839. 5 G. D. S. J. Zabrocki, J. B. Dunbar Jr., H. Iijima and G. R. Marshall, J. Am. Chem. Soc. 1988, 110, 5875. 6 S. Gunawan, C. Hulme, Organic & biomolecular chemistry 2013, 11, 6036- 6046. 7 S. Y. Kang, S.-H. Lee, H. J. Seo, M. E. Jung, K. Ahn, J. Kim, J. Lee, Bioorg Med Chem Lett 2008, 18, 2385-2389. 8 J. P. Alexander, B. F. Cravatt,Journal of the American Chemical Society 2006, 128, 9699-9704. 9 J. Li, S. Y. Chen, J. J. Li, H. Wang, A. S. Hernandez, S. Tao, C. M. Musial, F. Qu, S. Swartz, S. T. Chao, N. Flynn, B. J. Murphy, D. A. Slusarchyk, R. Seethala, M. Yan, P. Sleph, G. Grover, M. A. Smith, B. Beehler, L. Giuppo- ni, K. E. Dickinson, H. Zhang, W. G. Humphreys, B. P. Patel, M. Schwin- den, T. Stouch, P. T. W. Cheng, S. A. Biller, W. R. Ewing, D. Gordon, J. A. Robl, J. A. Tino, Journal of Medicinal Chemistry 2007, 50, 5890-5893. 10 A. Dömling, Chemical Reviews 2006, 106, 17-89. 11 C. F. Marcos, S. Marcaccini, G. Menchi, R. Pepino, T. Torroba, Tetrahedron Letters 2008, 49, 149-152. 12 S. Gunawan, J. Petit, C. Hulme, Acs Comb Sci 2012, 14, 160-163. 13 V. Y. Stolyarenko, A. A. Evdokimov, V. I. Shishkin, Mendeleev Communi- cations 2013, 23, 108-109. 14 A. K. Ghosh, S. Leshchenko-Yashchuk, D. D. Anderson, A. Baldridge, M. Noetzel, H. B. Miller, Y. Tie, Y.-F. Wang, Y. Koh, I. T. Weber, H. Mitsuya, Journal of Medicinal Chemistry 2009, 52, 3902-3914. 15 R. Pick, M. Bauer, U. Kazmaier, C. Hebach, Synlett2005 , 2005, 0757-0760.

71 Ugi 4-CR Synthesis of γ- and δ-Lactams

16 U. Kazmaier, C. Hebach, Synlett 2003, 2003, 1591-1594. 17 K. Sung, F.-L. Chen, P.-C. Huang, Synlett 2006, 2006, 2667-2669. 18 M. J. Thompson, B. Chen, The Journal of Organic Chemistry 2009, 74, 7084- 7093.

19 T. Zhao, A. Boltjes, E. Herdtweck, A. Domling, Org Lett2013 , 15, 639-641. 20 I. Ugi, R. Meyr, U. Fetzer, C. Steinbrückner,Angew Chem Int Edit 1959, 71, 386-386.

21 M. Treilhou, F. Couderc, Journal of Labelled Compounds and Radiopharma- ceuticals 2001, 44, 737-745. 22 J. W. H. M. Berman, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N.; Shindyalov, , P. E. Bourne, Nucleic Acids Res 2000, 28, 235. 23 L. Farrugia, J Appl Crystallogr 1999, 32, 837-838. 4

Chapter 5

Gd-TEMDO: Design, synthesis and MRI application

André Boltjes, Annadka Shrinidhi, Kees van de Kolk , Eberhardt Herdtweck, and Alexander Dömling

Published in: Chemistry a European journal., 2016, 22, 7352-7356 Chapter 5

Abstract A simple Ugi tetrazole multicomponent reaction allows the synthesis of a novel macrocyclic cyclen derivative with four appendant tetrazole arms in just two steps in excellent yields. This ligand, called TEMDO, turns out to be a very good chelator of lanthanoid metals. Here we describe the design, synthesis, solid state structure, binding constant and some MRI applications of the Gd-TEMDO complex as the first example of a congeneric family of oligo-amino tetrazoles.

75 Gd-TEMDO: Design, synthesis and MRI application

Introduction Since its discovery in 1971 magnetic resonance imaging (MRI) has evolved into a medical imaging technique of major importance and has rapidly found its entry into daily clinical diagnostics.1 Today more than 20.000 MRI scanners are operat- ing worldwide in hospitals and more than 50 million clinical MRI examinations are performed every year. The impact of this breakthrough technology for man- kind was honored by the Nobel Prize in Physiology or Medicine in 2003.2-3 As opposed to other imaging techniques, MRI is a non-ionizing radiation method and is therefore widely used in medical diagnosis and staging of disease. While MRI in principle does not require contrast agents, its use dramatically accelerates acquisition times and signal intensity. MRI contrast agents work by accelerating the relaxation of water protons in the surrounding tissues. Paramagnetic ions are suitable contrast agents and amongst them gadolinium (Gd3+) complexes are by far the most widely used MRI contrast agents due to its seven unpaired electrons, its slow electronic relaxation and its exquisite complex stability. Although clinically approved MRI contrast agents are generally considered as safe, a small number of fatalities were reported likely due to nephron- and neurotoxicity by Gd-leaking.4 In addition, not only patients with pre-existing renal failure are affected by Gd-leakage, but also patients with other conditions showed considerable neuronal tissue concentrations of Gd3+ after being subjected to Gd-based contrast agents (GBCA’s).5 Here we introduce the first example of the novel class of oligoamino tetrazoles as chelating agent useful in imaging: 1,4,7,10-tetrakis((1H-tetrazol-5-yl)meth- 5 yl)-1,4,7,10-tetraazacyclo-dodecane (TEMDO, 3) (scheme 1). We describe the design, synthesis, solid state structure, binding constant and some MRI applications of the Gd-TEMDO complex. The tetrazole is a known bioisostere of the carboxylic acid with often superior PKPD properties.6 For example the tetrazolate allows for a more wide delocalization of the negative charge and could thus facilitate better penetration into tissue. In the context of MRI a tetrazolate ligand could also help to increase proton relaxivity through changes in the metal-H distance by electron delocalization towards the ligand. Moreover through subtle changes in the ligand composition and geometry eventually higher tilt angles between the plane of the bound water and the metal–O bond could be induced by hydrogen bonding of the coordinated water to an appropriate side group of the chelate, which could potentially result in a significant decrease of the metal–proton distance. Thus we reasoned that oligoamino tetrazoles are suitable for MRI, exhibiting different and eventually better physicochemical and biological properties compared to their carboxylic acid analogues. Surprisingly oligoamino tetrazoles in general and specifically as MRI agents are unknown.7 Based on our longstanding expertise in multicomponent reaction (MCR) chemistry and its versatility, speed and ease-to-perform we choose MCR as a perfect tool to assemble this functional material class of MRI agents.8-9 As a first synthetic target of the class of the oligoamino tetrazoles (2) we chose the tetrazole analogue (3) of DOTA10-11 (1) (scheme 1).

76 Chapter 5

Scheme 1. DOTA, general formula of oligo-amino tetrazoles, TEMDO and retro synthesis thereof. Results and Discussion In our retrosynthesis we envisioned that TEMDO can be fast and convergently synthesized by applying a Ugi tetrazole reaction from available cyclen.12 The unprotected TEMDO ligand suitable for metal complexation has to be generated by cleavage of the isocyanide substituent. In principle several cleavable isocyanides such as Walborsky’s, benzyl- or tert-butyl are suitable, however we have chosen here β-cyanoethyl isocyanide 4 due to its cleavage under mild conditions.

77 Gd-TEMDO: Design, synthesis and MRI application

CN N H N N N N N N N NH N N HN CN NC CN MeOH, 12h N N N 5 H 4 CN RT, > 99% N N N N N N CH2O TMS-N3 8 N 6 7 N N NC

N NH N N N N N NaOH N NH ACN-H O, 4h 8 2 N RT, 86% N HN N N N N N 3 N HN N Scheme 2. Two step UT-4CR MCR synthesis of the TEMDO ligand 3 5 In fact after some optimization of the reaction conditions we could obtain the Ugi tetrazole product 8 in quantitative yields by reacting the commercially available starting materials cyclen 5, paraformaldehyde 6, and TMS azide 7, as a safe hydrogen azide source and β-cyanoethyl isocyanide 4 (scheme 2). The four β-cyanoethyl groups can be cleaved under mild conditions using NaOH in acetonitrile/ water at room temperature. The crude product was purified by precipitation at pH = 7.75, to give neutral TEMDO ligand (86%) in high purity. Next the chelating property of TEMDO towards the lanthanide element Gadolinium was assessed. The Gd3+ complex of TEMDO was prepared by either a) heating the 1:1.1 mixture of TEMDO ligand and GdCl3 in water at 70 °C for 7 days at pH 6.7, or by b) heating TEMDO (in acidic form), the approved excipient meglumin and GdCl3 1:1:1 in water for 7 days at 70 °C. The remaining free Gd3+ was removed using ion-exchange resin Chelex®-100 and the clean liquid was lyophilized to get pure Gd-TEMDO complex (>98%) as a white bench stable solid.

78 Chapter 5

N NH N N N N N N N N N N N N N N NH N 70 °C, 7d N 3+ N GdCl3 Gd N H2O N HN N N pH 6.7 N N N N N N N ~ 40% N N N (9) N HN N N N Scheme 3. Reaction scheme of the formation of Gd-TEMDO complex, the counter ion sodium or me- glumine is present depending on the method of preparation.

Then we investigated the complex-behaviour of TEMDO towards the lanthanide element Gadolinium by determining its solid state 3D structure using X-ray crystallography. The complex crystallized in small rhombic shapes from water and the solved structure is shown in figure 1. The side by side comparison of the obtained crystal structure of Gd-TEMDO with the Gd-DOTA complex shows a surprisingly high isosterism (figure 1).

Figure 1. A stick representation of the crystal structures (unbound crystal waters and counterions are omitted for clarity). Above row: top down view; below row: side on view; left; Gd-DOTA.

The central Gd3+ ion is surrounded by the four basal cyclen-N and apical by the four N1 of the appendant tetrazoles in a highly symmetrical twisted quadratic coordination sphere. In addition a water molecule is coordinated on top of the twisted cube in-between the four tetrazole ligands. The coordination number for

79 Gd-TEMDO: Design, synthesis and MRI application

Gd3+ is therefore 9 and the idealized complex belongs to the chiral point group

C4. In principle 9-fold coordinated Ln-complexes with C4 symmetry can present a square anti-prismatic (SAP) or twisted square antiprismatic (TSAP) geometry.

Moreover due to their membership to the chiral C4 point group Ln-TEMDO complexes can show the absolute chirality Λ (left-handed) or Δ (right-handed). The coordination geometry of the Gd-TEMDO and Eu-TEMDO complexes is square antiprismatic (SAP), whereas in the La-TEMDO we have found twisted square antiprismatic (TSAP) geometry. For example SAP geometry is the predominant form found for the Gd-DOTA complex in aqueous solution, besides some twisted square antiprism.7 Interestingly the X-ray structures of the Gd- and Eu-TEMDO reveal only one SAP enantiomer per crystal, while La-TEMDO is present in a TSAP geometry and both Λ and Δ enantiomers are present in one crystal (SI). Similarly in Ln-DOTA complexes SAP geometry is preferred over TSAP with increasing ionic lanthanide radius.13 The complex geometry is an important parameter in MRI active Ln complexes as the water exchange kinetics are linked to its geometry and relaxivity.14 Compared to TSAP, a SAP geometry will result in slower water exchange, but surprisingly a faster relaxation.15 The comparable size of the macrocyclic cavity of DOTA and TEMDO indicates that suitable metal ions will very well fit to form thermodynamically stable chelates.16 Qualitative assessment of free lanthanide ions with indicator xylenol orange showed that chelating generally occurs for the lanthanides. In table 1 we compare some key distances between Gd3+ with DOTA and TEMDO and its coordination towards N, and O as shown in solid state. The similarity of the alignment between Gd3+ and the both complexators DOTA and TEMDO expressed in distances gives an average difference of 0.09 Å RMSD. Such minor changes in bond length and an- gle, however, can have profound effects on how well the ion is held, the residence time of the water molecule and its exchange rate.17 5

Table 1. M-O, M-N and M-N’ bond lengths in the 9-coordinated Gd-DOTA and TEMDO complexes. Complex M-N (Å)[a] M-O (Å)[b] M-N’ (Å)[c] M-O’[d] − [Gd(TEMDO)(H2O)] 2,712 - 2,475 2.434 − [La(TEMDO)(H2O)] 2,780 - 2,616 2.507 − [Gd(DOTA)(H2O)] 2,648 2,377 - 2.458 [a] N represents the basal rim nitrogen’s. [b] Metal coordination to the four surrounding carboxylates [c] Metal coordination to the tetrazoles nitrogen [d] Average distance between the metal and water molecule..

The chelating properties of Gd-TEMDO were accessed by determination of the thermodynamic stability constant using colorimetry (table 1).18 The stability constant 16.6 confirms the strong chelating properties of the macrocyclic TEMDO, similar to Gd-(DTPA-BMA), a marketed MRI contrast agent (Omniscan, 16.8), however lower than Gd-DOTA. Studies on Gd3+ release revealed that not only thermodynamic stability, but also kinetic inertness and elimination rate of the complex plays an important role in the amount of free Gd3+ in vivo.15 Kinetic inertness expressed as dissociation rate is therefore important to predict toxicity through Gd3+ leakage (table 2). However, due to the general observed fast excre-

80 Chapter 5 tion of Gd-based contrast agents the complex is not long enough in the body to establish thermodynamic equilibrium. The lower stability constant of Gd-TEM- DO as compared to the DOTA complex is thus compensated by a two orders of magnitude slower release of Gd3+ from the TEMDO complex

Table 2. Binding constants and kinetic data of Gd3+ complexes. Metal Complex Log K [a] [b] − 3 -8 [Gd(TEMDO)(H2O)] 16.6 3.47 ± 0.5 x 10 1.20 ± 0.5 x 10 − [c] 3 -6 [Gd(DOTA)(H2O)] 24.1 5.16 ± 0.5 x 10 3.20 ± 0.5 x 10 [a] Formation rate, experimentally determined for both TEMDO and DOTA by UV/VIS spectrometry [b] Dissociation rate [c] Value under identical conditions determined as for TEMDO (lit. value for Gd-DOTA 24.719).

Finally we investigated the usefulness of Gd-TEMDO for magnetic resonance imaging. In a Bruker 9.4 T 400 MHz small bore MRI apparatus, we ran relaxation experiments resulting in phantom images of Gd-TEMDO (figure 2). From the -1 -1 phantom images we calculated a relaxivity r1 of 2.0 mM s which is in comparison with Gd-DOTA 6.0 mM-1 s-1 (measured under identical conditions at ambient temperature) somewhat lower, but being in the same range a promising starting point for further introduction of variations on TEMDO. The T1-weighted images as expected show, however a concentration dependent increase of the relaxation rate of the solvent water protons associated with Gd-based contrast agents. In-vivo assessment of Gd-TEMDO was performed via delayed contrast-enhanced MRI. Some Gd-based contrast agents such as Gd-DOTA accumulate in damaged myocardium tissues, diffusing from the intravascular into the interstitial space, unable to enter intact cells. Delay in measurement causes the majority of agent to wash out, and contrast agent absorbed in damaged tissue provides enhanced contrast.20 In a left-coronary-artery occlusion murine animal model we investigated Gd-TEMDO to image the myocardial infarcted tissue. This clearly shows that the TEMDO contrast agent is absorbed in damaged tissue and the enhanced contrast is shown in figure 2.

81 Gd-TEMDO: Design, synthesis and MRI application

Figure 2. Gd-TEMDO MRI. (a) T1-weighted MRI phantoms of Gd-TEMDO proving concentration dependent T1 shortening. (b) MRI obtained from isoflurane-anaesthetized mice, (c) taken 30 minutes after I.P. administration of Gd-TEMDO (0.6 mmol/kg). Left: the heart 5 fully visible; right: heart with reduced brightness, the damaged tissue remains visible due to absorbed Gd-TEMDO following the red line.

Conclusions In summary, we have described design, synthesis, X-ray structure, binding and some applications of the first example of a new class of Ln chelators with potential for use in MRI as Gd-based contrast agents. The macrocyclic TEMDO ligand is easy accessible by only two synthetic steps, in excellent yields employing a Ugi tetrazole multicomponent reaction. The thermodynamic stability constant of our first Gd3+ complex is comparable to clinically used Gd-(DTPA-BMA). So far we were able to grow diffractable crystals of the Gd(III), Eu(III) and La(III) complexes. Moreover we were able to show proof-of-principle, utilizing the Gd-TEMDO complex as contrast agent and visualizing myocardial infarcts in mice. Further analysis of the complex confirms comparable structural features as compared to marketed Gd-based contrast agents, a very good starting point for future development of this new class of oligotetrazolo-based metal complexes.

82 Chapter 5

Experimental procedures and Spectral Data

Instruments employed: Electrothermal digital melting point apparatus; Bruker Avance DRX 500 MHz NMR spectrometer (B AV-500) equipped with Bruker Au- tomatic Sample Changer (BACS 60). For MRI; Bruker 9.4T 89mm bore scanner equipped with 1500mT/m gradientset. Thar SFC-MS equipped with autosampler and autoinjector; pH meter pHenomenal® with Thermo Scientific Orion ROSS Ultra pH electrode; Jasco V-660 UV-VIS Spectrophotometer. High resolution mass spectra were recorded using a LTQ-Orbitrap-XL (Thermo) at a resolution of 60000@m/z400.

Chemicals required: DOTA, paraformaldehyde, sodium hydroxide, potassium chloride, potassium hydrogen phthalate, ethylene diamine tetraacetic acid, KOH concentrate (Sigma-Aldrich); Arsenazo III (TCI); 1,4,7,10-tetraazacyclododecane, azidotrimethylsilane, gadolinium chloride anhydrous (ABCR); phenolphthalein, xylenol orange (Fluka); acetic acid (Acros), sodium acetate (Merck); hydrochlo- ric acid, methanol, acetonitrile (Boomlab) were obtained commercially and used without any further purification. 3-Isocyanopropanenitrile was prepared in the laboratory from its corresponding formamide. Millipore water was used for the preparation of all solutions.

NMR spectra were recorded in CDCl3 (with 0.03% TMS), DMSO-d6 and D2O

(with 0.03% DSS) at either 500 MHz (δH) or 125 MHz (δC); the coupling constants (J) are in Hz. Abbreviations: mp (melting point), s (singlet); d (doublet); t (triplet); q (quartet); br (broad); dd (doublet of doublet), etc. The nomenclatures of all the compounds were derived by ChemDraw (CambridgeSoft).

83 Gd-TEMDO: Design, synthesis and MRI application

Synthetic procedures and characterization for compound 3, 4, 7, 8 and precursor 3,3’,3’’,3’’’-((1,4,7,10-tetrakis((1H-tetrazol-5-yl)methyl)-1,4,7,10-tetraazacyclododecane (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(methylene)) tetrakis(1H-tetrazole-5,1-diyl))tetrapropanenitrile (8)

CN In a 50 mL roundbottom flask was added parafor- N N N maldehyde (3.0 g, 100 mmol), 1,4,7,10-tetraazacyclo- N N N N dodecane (0,861 g, 5 mmol), azidotrimethylsilane N N (6,6 mL, 50 mmol) and 3-isocyanopropanenitrile (2,0 NC N g, 25,00 mmol) in methanol (15 mL) to give a yellow N CN N suspension. Stirring overnight, removing the sol- N N N vent by decantation, redissolving in 20 mL acetoni- N N N trile, filtering over Celite® and concentrating under N N reduced pressure yields NC 3,3’,3’’,3’’’-(5,5’,5’’,5’’’-((1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(methylene))tetrakis(1H-tetrazole-5,1-diyl))tet- 1 rapropanenitrile, (3.58 g, 100%) as an orange oil. H NMR (500 MHz, DMSO-d6) δ 4.75 (t, J = 6.4, 8H), 3.95 (s, 8H), 3.22 (t, J = 6.4, 8H), 2.69 (s, 16H) ppm. 13C NMR

(125 MHz, DMSO-d6) δ 152.9, 118.0, 50.9, 46.3, 42.5, 17.8 ppm. HRMS (ESI) m/z calculated [M+H]+: 713,39405; found [M+H]+: 713, 39435 1,4,7,10-tetrakis((1H-tetrazol-5-yl)methyl)-1,4,7,10-tetraazacyclododecane (3) 5 N NH In a 100 mL roundbottom flask was added N N N 3,3’,3’’,3’’’-(5,5’,5’’,5’’’-((1,4,7,10-tetraazacyclododec- N N N ane-1,4,7,10-tetrayl)tetrakis(methylene))tetrakis(1H- NH N tetrazole-5,1-diyl))tetrapropanenitrile (7) (2.0 g, 2,8 N mmol) and NaOH (1,80 g, 22,5 mmol) in acetonitrile-wa- HN N ter (5:1, 20 mL) to give a yellow solution. After stirring N N N N overnight at room temperature, the solvent was evapo- N rated from the reaction mixture and the solid mass was HN N dissolved in 75 mL water. The pH of solution was adjusted to 7.0 with aqueous HCl. This neutral solution was extracted with dichloromethane (2 x 25 mL) to remove organic impurities. The aqueous layer was lyophilized and redissolved in 100 mL methanol and undissolved solid mass was removed by filtration. The supernatant liquid was evaporated to dryness, redissolved in 5 mL water and the pH was adjusted 7.75 with aqueous NaOH. A white solid precipitated and was collected by filtration, washed with cold water and 1 dried to obtain 1.26 g (86%) of TEMDO. m.p.>300°C. H NMR (500 MHz, D2O) δ 13 4.20 (s, 8H), 2.87 (s, 16H). C NMR (125 MHz, D2O) δ 160.0, 52.4, 49.7 ppm. HRMS (ESI) m/z calculated [M+H]+: 501,28786; found [M+H]+: 501,28754

84 Chapter 5

Gadolinium(III) 1,4,7,10-tetrakis((1H-tetrazol-5-yl)methyl)-1,4,7,10-tetraazacyclododecane (9)

3+ N N The Gd complex of TEMDO was prepared by heating N the 1:1.1 mixture of TEMDO ligand (3) (1.0 g, 2.0 mmol) N N N N and GdCl ∙6H O (0.82 g, 2.2 mmol) in 20 mL Millipore N 3 2 N water at 70°C for 10 days at pH 6.8. The remaining free N Gd3+ Gd3+ was removed using Chelex 100 and the clean liquid N N was lyophilized to give pure Gd-TEMDO complex as a N N N white solid (>98%, 1.28g). Complexes for Eu and La N N N were prepared in the same manner. HRMS (ESI) m/z N N calculated [M+2H]+: 656,18848; found [M+2H]+: 656,18832

N-(2-cyanoethyl)formamide

H A solution of 3-aminopropionitrile (10g, 143 mmol) in ethyl formate N O NC (250 mL, 143 mmol) was refluxed for 5 hours. The mixture was concentrated in vacuo, yielding 13,9g, 99% as a yellow oil. The product 1 is obtained as a mixture of two amide rotamers. H NMR (500 MHz, DMSO-d6): δ = 8.22 (s, 1H, major), 6.97 (s, 1H, major), 3.57 (tt, J=9.0, 4.3, 2H), 2.79 – 2.61 (m, 13 2H) ppm. C NMR (125 MHz, CDCl3) δ 162.1, 118.2, 37.9, 34.3, 20.5, 18.4 ppm.

3-isocyanopropanenitrile (4)

NC NC To a cold (0 °C) solution of N-(2-cyanoethyl)formamide (8.65g, 88mmol) and triethylamine (61 mL, 441 mmol) in DCM was added drop wise over 60 min. POCl3 (8.2 mL, 88 mmol). After the addition the reaction was stirred at 0 °C for 2.5 hours. Then, carefully, 10% Na2CO3 was added followed by water (200 mL) to dissolve all solids. The organic layer was separated and the water layer extracted with DCM (3 × 25 mL). The combined organic layers were washed with brine (100 mL), dried over MgSO4, and concentrated in vacuo. The crude dark brown residue was purified by passing it through a plug of silica (100% DCM). After evaporation of all volatiles the product (4.2g, 59%) was obtained as a brown oil which solidified upon standing. 1H NMR (500 MHz, 13 CDCl3) δ 3.73 (t, J = 6.7 Hz, 2H), 2.81 (t, J = 6.7 Hz, 2H) ppm. C NMR (125 MHz,

CDCl3) δ 160.1, 115.8, 37.7, 37.6, 37.6, 18.8 ppm.

85 Gd-TEMDO: Design, synthesis and MRI application

1H-NMR and 13C-NMR of the protected UT-4CR product 8 and the deprotected tetrazoles of TEMDO 3. The simplicity of the spectra confirms the four-fold substitution of cyclen into a fully symmetric product. Compound 8 2.50 DMSO 2.69 4.76 4.75 4.74 3.95 3.33 HDO 3.23 3.22 3.21

N A (t) B (s) D (t) C (s) 4.75 3.95 3.22 2.69

N N N N N N N N N N

N N

N N N N N N N N N

N 8.26 7.95 8.21 16.00 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 f1 (ppm)

5 17.81 42.46 46.31 50.93 118.03 152.87

N N N N N N N N N N

N N

N N N N N N N N N

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

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Compound 3

87 Gd-TEMDO: Design, synthesis and MRI application

Crystallographic data Data were collected on an X-ray single crystal diffractometer equipped with a CCD detector (Bruker APEX II, κ-CCD), a fine-focus sealed tube (Bruker AXS,

D8) with MoKα radiation (λ = 0.71073 Å), and a graphite monochromator by using the SMART software package. The measurements were performed on a single crystal coated with perfluorinated ether. The crystal was fixed on the top of a cactus prickle (Opuntia ficus-india) and transferred to the diffractometer. The crystal was frozen under a stream of cold nitrogen. A matrix scan was used to determine the initial lattice parameters. Reflections were merged and corrected for Lorenz and polarization effects, scan speed, and background using SAINT. Ab- sorption corrections, including odd and even ordered spherical harmonics were performed using SADABS. Space group assignments were based upon systematic absences, E statistics, and successful refinement of the structures. Structures were solved by direct methods with the aid of successive difference Fourier maps, and were refined against all data using WinGX based on SIR-92 in conjunction with SHELXL-97.33-34 C–H atoms were placed in calculated positions and refined using a riding model, with C–H distances of 0.99 Å, and Uiso(H) = 1.2·Ueq(C). O–H atoms were placed in calculated positions, with O–H distances of 0.84 Å, and

Uiso(H) = 1.5·Ueq(O). Full-matrix least-squares refinements were carried out by mini- 2 2 2 mizing Σw(Fo -Fc ) with SHELXL-97 weighting scheme. Neutral atom scattering factors for all atoms and anomalous dispersion corrections for the non-hydrogen atoms were taken from International Tables for Crystallography. Images of the crystal structures were generated by PLATON. CCDC 1039908 (La-TEMDO) contains the supplementary crystallographic data for this compound. This data can be 5 obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif or via https://www.ccdc.cam.ac.uk/services/structure_deposit/

88 Chapter 5

Figure 3. – Ortep drawing of compound La-TEMDO with 50% ellipsoids. Full refinement was possible without running into problems. The atom O7 – crystal water was refined with an isotropic displacement parameter.

89 Gd-TEMDO: Design, synthesis and MRI application

Table 2. Data collection and refinement parameters of obtained crystals. The crystal quality of Gd-TEMDO and Eu-TEMDO was not sufficient for full refinement.

Data Collection La-TEMDO Gd-TEMDO (9) Eu-TEMDO

Molecular Formula C32H70La2N40Na2O11 C16H5GdN20NaO3+n C16H54EuN20NaO3+n Molecular Weight 1515.08 g/mol 946.79 g/mol 464.59 a.m.u Crystal Color/ size 0.13×0.15×0.25 mm 0.28×0.41×0.51 mm 0.10×0.30×0.36 mm Space Group Monoclinic Tetragonal Monoclinic

P 21/c P 4212 C 2/c Cell Constants a =11.1804 Å a =9.6950Å a =9.8064 Å b =17.6279 Å β = 91.9317° b =9.6950(4) Å b =3.71307 Å c =14.6590(4) Å c =20.3549(9) Å β=90.2319(10)° V = 2887.46(15) Å3 V = 1913.22(14) Å3 Z = 2 Z = 2 c =9.5809(2) Å -3 -3 Dcalc = 1.743 gcm Dcalc = 1.59 gcm V = 3488.55 Å; Z = 4

F000: 576 Temperature (-173±1) °C

Measurement Range 1.81° < θ < 25.43° h: -13/13 k: -21/21 l: -17/17 5 Refinement Refl. collected 84780

Independent reflec- 5322

tions Rint: 0,025 Extinction Correc- No tion Goodness of fit 1.087

Resid. Electron +1.01 e/Å3 Density -0.85 e/Å3 R indices (all reflec- R1=0.0289 tions) wR2=0.0693

90 Chapter 5

Figure 4. – Ortep drawing of compound Gd-TEMDO with 50% ellipsoids.

Figure 5. – Ortep drawing of compound Eu-TEMDO with 50% ellipsoids.

91 Gd-TEMDO: Design, synthesis and MRI application

References

1 L. Helm, A. E. Merbach, E. v. Tóth, The chemistry of contrast agents in medical magnetic resonance imaging, Second edition / ed., 2013. 2 P. C. Lauterbur, Angew. Chem. Int. Ed. 2005, 44, 1004-1011. 3 P. Mansfield,Angew. Chem. Int. Ed. 2004, 43, 5456-5464. 4 P. Hermann, J. Kotek, V. Kubicek, I. Lukes, Dalton Trans. 2008, 3027-3047. 5 R. J. McDonald, J. S. McDonald, D. F. Kallmes, M. E. Jentoft, D. L. Mur- ray, K. R. Thielen, E. E. Williamson, L. J. Eckel, Radiology 2015, 275, 772- 782.

6 R. J. Herr, Bioorgan Med Chem 2002, 10, 3379-3393. 7 S. Aime, M. Botta, M. Fasano, M. P. M. Marques, C. F. G. C. Geraldes, D. Pubanz, A. E. Merbach, Inorg. Chem. 1997, 36, 2059-2068. 8 A. Dömling, Chem. Rev. 2005, 106, 17-89. 9 A. Dömling, W. Wang, K. Wang, Chem. Rev. 2012, 112, 3083-3135. 10 H. Stetter, W. Frank, Angew. Chem. Int. Ed. 1976, 15, 686-686. 11 J. F. Desreux, Inorg. Chem. 1980, 19, 1319-1324. 5 12 I. Ugi, C. Steinbruckner, Chem. Ber. 1961, 94, 734-742. 13 M. Meyer, V. Dahaoui-Gindrey, C. Lecomte, L. Guilard, Coordin. Chem. Rev. 1998, 178, 1313-1405. 14 M. Milne, K. Chicas, A. Li, R. Bartha, R. H. E. Hudson, Org Biomol Chem 2012, 10, 287-292. 15 S. Avedano, M. Botta, J. S. Haigh, D. L. Longo, M. Woods, Inorg. Chem. 2013, 52, 8436-8450. 16 R. Delgado, V. Felix, L. M. P. Lima, D. W. Price, Dalton Trans. 2007, 2734- 2745.

17 W. P. Cacheris, S. K. Nickle, A. D. Sherry, Inorg. Chem. 1987, 26, 958-960. 18 Z. Baranyai, E. Gianolio, K. Ramalingam, R. Swenson, R. Ranganathan, E. Brucher, S. Aime, Contrast Media Mol. Imaging 2007, 2, 94-102. 19 M. Reková, P. Vañura, V. Jedináková-Křížová, The Open Inorganic Che- mistry Journal 2009, 3, 26-32. 20 S. Bohl, C. A. Lygate, H. Barnes, D. Medway, L. A. Stork, J. Schulz- Menger, S. Neubauer, J. E. Schneider, Am J Physiol-Heart C 2009, 296,

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H1200-H1208. 21 L. Burai, I. Fábián, R. Király, E. Szilágyi, E. Brücher, Journal of the Chemical Society, Dalton Transactions 1998, 243-248. 22 K. Kumar, M. F. Tweedle, Inorganic Chemistry 1993, 32, 4193-4199. 23 A. E. Merbach, É. Tóth, The chemistry of contrast agents in medical magnetic resonance imaging, Wiley Online Library, 2001, p191. 24 Z. Baranyai, E. Brücher, T. Iványi, R. Király, I. Lazar, L. Zékány, Helvetica chimica acta 2005, 88, 604-617. 25 A. Pasha, G. Tircsó, E. T. Benyó, E. Brücher, A. D. Sherry, European journal of inorganic chemistry 2007, 2007, 4340-4349. 26 X. Wang, T. Jin, V. Comblin, A. Lopez-Mut, E. Merciny, J. F. Desreux, Inorganic Chemistry 1992, 31, 1095-1099. 27 Marie Reková, P. Vaňura, V. Jedináková-Křížová, The Open Inorganic Che- mistry Journal 2009, 3, 26-32. 28 P. Brauner, L. G. Sillen, R. Whiteker, Ark. Kemi 1969, 31, 365-376. 29 S. Chaves, R. Delgado, J. J. R. F. Da Silva, Talanta 1992, 39, 249-254. 30 L. G. Sillen, B. Warnqvist, Ark. Kemi 1969, 31, 377-390. 31 W. P. Cacheris, S. K. Nickle, A. D. Sherry, Inorg. Chem. 1987, 26, 958-960. 32 E. T. Clarke, A. E. Martell, Inorganica Chimica Acta 1991, 190, 37-46. 33 L. Farrugia, J Appl Crystallogr 1999, 32, 837-838. 34 A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, M. C. Burla, G. Polidori, M. Camalli, J Appl Crystallogr 1994, 27, 435.

93 Gd-TEMDO: Design, synthesis and MRI application

Chapter 6

The Groebke-Blackburn-Bienaymé reaction

André Boltjes and Alexander Dömling

Published in: Eur. J. Org. Chem., 2019, In Press Chapter 6

Abstract Imidazo[1,2a]pyridine is a well-known scaffold in many marketed drugs, such as Zolpidem, Minodronic acid, Miroprofen and DS-1 and it also serves as a broadly applied pharmacophore in drug discovery. The scaffold revoked a wave of interest when Groebke, Blackburn and Bienaymé reported independently a new three component reaction resulting in compounds with the imidazo[1,2-a]-heterocycle as a core structure. During the course of two decades the Groebke Blackburn Bienaymé (GBB-3CR) reaction has emerged as a very important multicomponent reaction (MCR), resulting in over a hundred patents and a great number of publications in various fields of interest. Now two compounds derived from GBB-3CR chemistry received FDA approval. To celebrate the first 20 years of GBB-chemistry , we present an overview of the chemistry of the GBB-3CR, including an analysis of each of the three starting material classes, solvents and catalysts. Ad- ditionally, a list of patents and their applications and a more in-depth summary of the biological targets that were addressed, including structural biology analysis, is given.

97 The Groebke-Blackburn-Bienaymé reaction

Contents 1. Introduction ...... 101 1.1 Mechanism...... 105 2. Proceedings in the development of the GBB-3CR...... 107 3. Scope and limitations of the GBB-3CR...... 113 3.1 Cyclic Amidines in the GBB-3CR...... 114 3.2 Aldehydes...... 120 3.3. Isocyanides used in the GBB-3CR...... 130 4. Catalysts in the GBB-3CR...... 134 4.1 Base catalyzed GBB-3CR...... 138

5. Solvents used in the GBB-3CR...... 139 6. Biologically active compounds...... 142 7. Structural Biology...... 159 8. Patents...... 164 9. AnchorQuery...... 169 10. Conclusions...... 170 List of abbreviations...... 171 References...... 173 6

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1. Introduction: Medicinal chemistry and Multicomponent reactions Design and synthesis of biological active compounds are an important field of chemistry as to date there are still many conditions, lacking treatment possibilities. Introduction of novel drugs continuously improve human health despite dramatic increase in world population. The continuous discovery of new biological pathways and the protein targets involved are a great source for medicinal chemist to fill the void in drugs for unmet medical needs. Where the work of structural biologists ends, in elucidating the dynamics and crystallographic structures of large biological structures, medicinal chemists start by the design of agonists and antagonist for those proteins and enzymes. By mimicking the shape and electrostatics of a small natural ligand for example, small molecules can be used to influence those natural processes resulting in inhibition or activation of the associated pathways. Diversity oriented synthesis is often applied to discover small molecules and to optimize their binding affinity in receptor binding sites.1 Introduction of structural diversity is usually accomplished by the stepwise introduction of the individual moieties in the target molecule and each structural variation requires repetition of a part or even whole synthesis route. This divergent and sequential approach for the synthesis of compound libraries can be quite challenging and material and time consuming. A more convenient way would be to assemble a versatile scaffold and obtaining the variations in a single reaction step. With two component reactions the divergent synthesis of very large chemical space and size based on available building blocks is naturally limited. Multicomponent reactions (MCR), however, allow for the concomitant variation of three or more building blocks at the same time and consequently span a very large chemical space. Since the degree of the reaction enters the exponential, the chemical space is naturally much larger the higher the degree of the reaction. E.g. for 2-component and 5-component reaction based on 1.000 building blocks of each variable class, 1.000.000 and 1.000.000.000.000.000 products can be expected, respectively. This exponential explosion of chemical space is key to the philosophy of MCRs.2 MCRs allow for the introduction of various scaffold shapes introduced through several MCR variants such as Ugi 3-component reaction (U-3CR)3, Passerini (P-3CR)4, Gewald (G-3CR)5, Groebke-Blackburn-Bi- enaymé (GBB-3CR)6-8, Biginelli (B-3CR)9 and many other MCR name reactions. Decorating and derivatization of the scaffolds is simply done by choosing different starting materials (building blocks). For example, in the Ugi-4CR, with 100 different amines, aldehydes, carboxylic acids and isocyanides as reagents, 100 8 x 100 x 100 x 100 = 10 different compounds can be synthesized, all connected to the dipeptidic Ugi scaffold. Such information rich chemical space has found recently applications in advanced information technology such as molecular steg- anography.10 Clearly, the large chemical MCR space comprise an excellent playground for drug hunters. Of course there are more different starting materials available, and other MCR reactions, where bi-functional orthogonal starting materials could lead to even more complex (poly)cyclic structures in post modifications, such as UDC-procedures (Ugi-Deprotection-Cyclization) and Passerini-reaction-Amine-Deprotection-Acyl-Migration (PADAM) and giving access to a nearly unlimited number of compounds of most diverse shape and electrostatics and composition of 3D pharmacophores.11-12 The isocyanide-based multicomponent reactions (IMCR), U-3CR, P-3CR, van Leusen (vL-3CR)13, GBB-3CR and variations, are nowadays the most popular types of MCR because of the versatile be-

99 The Groebke-Blackburn-Bienaymé reaction havior of the isocyanides, carbene-type, α-anion and radical reactivity as well as the excellent atom economy. Examples of marketed drugs and lead compounds made by an MCR approach are Xylocaine (Ugi-3CR), Nifedipine (Hantzsch), Tel- aprevir (Passerini-3CR) and Crixivan (Ugi-4CR) (Scheme 1). It is estimated that approximately 5% of the currently marketed drugs can be advantageously as- sembled by an MCR. Thus MCR scaffolds are clearly ‘drug-like’.

Ph N OH H H N * N N N N t-Bu O O N O HO H Xylocaine U-3CR Crixivan® U-4CR

O H NO2 N O O O O O NH H O O O N N N N H N O H N Nifedipine Hantsch-3CR Telaprevir P-3CR and U-3CR Scheme 1. Drug synthesized using MCR reactions. The scaffold originating from the MCR is marked in blue or purple. The central piperazine element of the HIV protease inhibitor Crixivan was enantioselectively synthesized by U-4CR, the local anesthetic Xylocaine can be advantageously synthesized in one step using U-3CR, the cardiovascular blockbuster drug Nifedipin by a Hantzsch-3CR and the stereochemical complex HCV protease inhibitor Telaprevir by a combination of P-3CR (purple) and U-3CR (blue) thus reducing the total number of steps by half.14-17 6 Here we will focus on one of the youngest IMCRs, the GBB-3CR based on its enormous interest in applied chemistry. The development of the reaction started by Groebke et al. (Hoffman-La Roche), initially published as a side reaction of the U-4CR. While studying the effect of various amine components, it was found that amines with a cyclic H2N-C=N substructure (2-aminoazines or amidines), such as 2-aminopyridine, 2-aminopyrazine and 2-aminopyrimidine were yielding the corresponding 3-amino-substituted imidazo[1,2-a]-pyridines, -pyrazines and -pyrimidines respectively.6 In this report, they made the reference to Sugiura et al. and suggests that the very first report of the GBB-3CR methodology was made almost 30 years earlier by condensing 2-aminopyrazine with formaldehyde, the nitrile compound and sodium or potassium cyanide. This was in contrast to the conventional method to access imidazo[1,2a] annulated pyridines, pyrazines or pyrimidines whereby a heterocyclization reaction between α-haloketones with the corresponding amidines was performed.18-19

100 Chapter 6

Scheme 2. The discovery of the GBB-3CR: A, General description of the GBB-3CR using amidines, aldehydes and isocyanides. B, One of the examples of Bienaymé using 2-aminopyrimidine, benzaldehyde, t-butylisocyanide and perchloric acid as catalyst, the formation was verified by X-ray structure refinement (CCDC-101255). C, Blackburn et al. reporting scandium triflate as optional catalyst. D, One of the Groebke examples; with 2-aminopyrazine, catalyzed with acetic acid. At the very same time Blackburn et al. from Millennium Pharmaceuticals (USA) published their work. The additional value of this work is the use of scandium triflate as Lewis acid catalyst.20 It follows the finding of Groebke et al. that the presence of an Brønsted acid, such as acetic acid is resulting in higher yields. This pH dependency results from the requirement of proton-assisted activation of the Schiff base to enable attack of the isocyanide component. As a third inventor of the GBB-3CR, Bienaymé et al. (Rhône-Poulenc Technologies (France)) reported in search of new MCR chemistry an approach to apply two covalent bonded or bridged reagents and found a 3-component reaction of 2-aminopyridine or –pyrimidine with aldehydes and isocyanides in the presence of a catalytic amount of perchloric acid in methanol. This approach was applied to produce 31 examples with variation points in all three components with yields between 33 and 98%.8

101 The Groebke-Blackburn-Bienaymé reaction

Due to the nearly simultaneous reports on the discovery of this three component reaction the reaction was called later-on the Groebke-Blackburn-Bienaymé 3-component reaction or in short the GBB-3CR. Interestingly, since its initial description, it took ~8 years until an exponential increase in reports was observed using the GBB-3CR methodology broadly in chemistry and also leading to multiple patents (Figure 1).

30 15

25 15 4 3 2 11 20 4 10 13 15 4 6 9 23 21 21 22 10 18 3 15 16 12 12 5 9 9 0 6 7 0 1 3 2 0 0 1 0 2 1 0 0 1 1 1 1 1 1

Journal publications Patent applications

Figure 1. Development of the GBB-3CR over the last two decades as indicated by increasing number of publications and patents. The numbers were collected through a SciFinder search (April 2019) using GBB-3CR related keywords and structure search of 5- or 6 membered GBB-3CR products including all variations of hetero atoms of the amidine component, corrected for the non GBB-3CR originated 3-amino-substituted imidazo[1,2-a]-pyridines, -pyrazines and –pyrimidines. Quite impressively, so far, more than 200 publications and >100 patent applications have been reported, exploiting the GBB-3CR, with a clear trend of further 6 increasing interest. With this in mind a few reviews were published, dedicated to the GBB-3CR as an MCR approach to access 3-amino-substituted imidaz- o[1,2-a]-heterocycles. Singh et al. were the first to cover the reaction and addressed the used catalysts and the general chemistry behind the GBB-3CR.21 Abdel/Wa- hab et al. reported a more comprehensive review, highlighting biological targets the GBB scaffold could be used for, possible post-modifications and to a certain extend the scope and limitations were discussed with some examples of amidines, isocyanides, aldehydes used in the GBB-3CR.22 In addition Liu published an overview on the Asinger23 and GBB-3CR, in a historical fashion with emphasis on the possible post modifications and applications of both reactions.24 Also Liu dedicated a mini review solely on potent applications of the imidazo[1,2-a]-heterocycles.25 To our knowledge patents were never discussed and our review tries to give a complementary twist by giving insight into all the used reactants, all the catalysts and solvents used in the GBB-3CR, detailed discussion of the structural biology, in order to get more insight in how GBB scaffolds interact with therapeutically relevant targets.

102 Chapter 6

1.1 Mechanism Examples of isocyanide based MCR’s (IMCR’s) are the P-3CR, vL-3CR, U-4CR, Ugi-azide-4CR, U-3CR, and GBB-3CR (Scheme 3). Aside from the P-3CR and the vL-3CR, the latter 3 IMCR’s are mechanistically variations on the U-4CR. The acid component is decisive in how the iminium species will react towards other components, rearrange and ultimately is incorporated into the scaffold. O O O R1 R2 H Passerini-3CR + + NC N R4 R3 O R4 1920 R1 R2 R3 OH O

O R R O O 1 2 H + NH2 + + NC N Ugi-4CR R3 R5 R4 N R5 1959 R1 R2 R4 OH R3 O

H R1 R2 O H NC N R4 Ugi-3CR + N + R N R R R5 5 1959 R1 R2 3 4 O R3

R1 R2 R5 O H + + + NC R3 N Ugi Tetrazole-4CR N HN3 N R R R5 N 1959 R1 R2 3 4 R4 N N

R O Ts 2 N N + NH + Van Leusen-3CR R 2 1977 R H 2 R NC 1 3 R1 R3

HN R2 Groebke Blackburn O 5- N + + NC N Bienaymè-3CR 6- R2 5- R R1 H NH 1 1998 2 6- N Scheme 3. Overview of the most common IMCR’s. For example, the U-3CR is only taking the proton of specific acidic reagents (e.g. p-toluene sulfinic acid (pTSIA)) and eliminating the Mumm rearrangement that otherwise concludes the U-4CR. The Ugi azide-4CR reaction adheres to a similar mechanism as the U-3CR, hydrazoic acid is introduced by using sodium azide or TMSN3. In the GBB-3CR the additional reactivity of the endocyclic nitrogen in the amidine component allows for the formation of a different scaffold, whereas the acid component is not incorporated in the final product and serves only catalytic purposes instead. In the GBB-3CR, the imine intermediate is activated by a

103 The Groebke-Blackburn-Bienaymé reaction

Lewis or Brønsted acid, and follows a formal [4+1] cycloaddition sequence con- cluding with aromatization via a 1,3-H shift (Scheme 4 Pathway A) to form the imidazo[1,2-a]pyrimidine when 2-aminopyridine is used. The GBB-3CR could follow two possible pathways leading to regioisomers as indicated in scheme 4. Pathway A is the most common and is referred to as the GBB-3CR and pathway B as the ‘inverse’ GBB-3CR.

O H R2 Ugi 4CR N H R2 N R2 - R3 N R1 H C O R3 O R3 R1 C O N+ R1 - O N O + HN COO R H 4 R R4 R3 4

COOH Ugi 3CR R 3 R HN 2 H R2 R O pTSIA N HN 2 O - S S R1 R2 R H O O O O R1 H N 1 C R1 C HN + + R4 -H O N N 2 R H R R2 1 - R O 4 H2N N 4 H H N+ H R H R N 2 2 R2 H N N HN Ugi Azide 4CR - Acid N + N R1 H C N + R1 C N - R Catalysed N - N 1 N + N N N N N R4 R4 R4

+ R4 N GBB-3CR C N R4 HN R4 N [4+1] cycloaddition N H C 1,3-H shift H N H N Pathway A R N + R 1 N R1 N 1 N

H R1 H or

H R R R [4+1] 1 1 H 1 + R2 Pathway B N R1 cycloaddition + + N N N H N C N 1,3-H shift C NH N R N R NH NH 4 4

Scheme 4. Variations of the Ugi reaction and the Groebke Bienaymé Blackburn reaction. In the GBB mechanism one of the most commonly used amidines; 2-aminopyridine as R2- 6 group was used in the mechanistic overview to simplify the scheme.

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2-Aminopyrimidines tend to form both regioisomers as it was first described by Bradley et al., trying to explain the low yields of some of their reactions while the starting materials were fully consumed.26 Isolation of a second product having 1 nearly identical TLC-Rf values, mass and H NMR spectra gave reason to perform an X-ray structure analysis, uncovering the two regioisomers 1 and 2 depicted in scheme 5. Luckily, the normal GBB product is the major product for most cyclic amidine building blocks, thus rendering it a quite useful synthetic reaction. In fact, in most cases not even traces of inverse GBB products can be observed.

Scheme 5. Two possible regioisomers of the GBB-3CR. Left 1 (CCDC-189548) a typical GBB-3CR imidazo[1,2-a]pyrimidine and on the right 2 (CCDC-189549) its inverse GBB regioisomer.

2. Proceedings in the development of the GBB-3CR. Based on the pharmacological relevance of imidazo[1,2-a]heterocyclic compounds and their easy access through the GBB-3CR, many scientists explored the scope and limitations of this reaction, trying a large variety of catalysts, solvents and temperature conditions, resulting in a wide variety of publications with a confusing high number of different conditions. Herein, some of the more relevant reports are discussed, showing important developments, noteworthy applications and several reaction conditions used are described to fully compre- hend the versatile character of the GBB-3CR. For example, Varma et al. showed that a microwave (MW) assisted solvent-free method could be applied by the aid of montmorillonite K-10 clay.19 The conditions were quite unusual that time since microwave assisted procedures were not yet broadly applied in 1999. Here a household microwave was used with an unsealed test tube irradiated at 900 W for 3 minutes. Together with the clay catalyst and variations of simple aromatic aldehydes, various isocyanides and 2-amino-pyridine (3), pyrazine (4) or pyrimidine (5) yields of >80% were obtained (Scheme 6).

105 The Groebke-Blackburn-Bienaymé reaction

R2 R O 1 Montmorillonite K-10 NH N + clay Y N R1 X NH2 Y NC Microwave X N R2 X = Y = C 3 X = C, Y = N X = Y = C X = C, Y = N 4 X = N, Y = C 5 X = N, Y = C

NH NH NH NH N N N N N N N N N N N 86% 81% 64% 58%

Scheme 6. Montmorillonite K-10 clay-catalyzed and solvent free GBB-3CR. Whittaker et al. showed a very similar scandium triflate catalyzed reaction via a microwave assisted GBB-3CR in 10 minutes with yields between 33-93% and using substituted benzaldehydes and mostly aminopyridines, interestingly also a 5-membered aminothiazole substrate was introduced, giving lower yields mostly related to side products (6) formed by the addition of methanol to the intermediate Schiff base (Scheme 7).8, 27 The side product formation could, however be suppressed by the use trifluoroethanol as a less nucleophilic solvent.

R3 H+ N NH OMe R2 R N R 2 1 O NH N + 2 R2 R N R1 MeOH 1 H N 6 NC 6 R3 Scheme 7. Application of electron-poor cyclic amidines in the GBB-3CR results in poor conversion and side reactions such as addition of MeOH to the Schiff base intermediate. Hulme et al. showed TMSCN as an equivalent to the simplest isocyanide HNC, to directly access 3-aminoimidazo[1,2-a]pyridines 7-8, which otherwise would require an additional deprotection step when a convertible isocyanide such as the Walborski’s reagent (1,1,3,3-tetramethylbutylisonitrile) is used in the GBB- 3CR (Scheme 8).28

106 Chapter 6

HN NC N c R1 R2 a N

b TMS-CN NH2 N R1 R2 O N R1 R2 NH2 N a 27-73% HN d N NC R1 R2 N

NH2 NH2 N N N N R1 R2 N N N 7, 64% 8, 73% 9 Scheme 8. Access to N-exocyclic unsubstituted GBB products. A: Reagents and conditions: a) aminopyridine (1 equiv), R2CHO (1 equiv), 2 (1 equiv) or benzylisonitrile (1 equiv), Sc(OTf)3 (5 mol %), 16 h; b) Aminopyridine (1.2 equiv), R2CHO (1 equiv), TMSCN (1 equiv), Sc(OTf)3 (5 mol %), MeOH, microwave, 10 min, 140 °C. Followed by Si-trisamine (5 equiv); c) H2, EtOAc, 24 h; d) 10% TFA/CH2Cl2, 18 h B: Representative examples and their yields and the observed Schiff base as side product. Some side product was found to be the Schiff base9 , formation could be prevent- ed by using an excess of amidine. After the reaction the residual catalyst Sc(OTf)3 was removed using 5 equivalents of Si-trisamine, which is a powerful scavenger of electrophiles as well as an effective scavenger for transition metals. GBB-3CR products are often observed as being strongly fluorescent. Balakirev- et al. introduced “Flugis”; fluorescent Ugi (GBB) products as drug-like probes for the identification and visualization of potential targets.29 The compounds described as U-3CR compounds arise from the GBB-3CR and were used with the rationale to incorporate drug-like scaffolds into fluorescent molecules. The often fluorescent nature of GBB products should be kept in mind during biophysical receptor-ligand screens based on fluorescence principles! For example, in a recent screen for inhibitors of the NS3/4A serine protease of the hepatitis C virus, some of the compounds with the [1,2-a]pyridine scaffold were found to exhibit auto-fluorescence in UV that interfered in the enzymatic fluorescence detection assay.29 This led to their approach to incorporate this scaffold that is synthesized using the GBB- 3CR in their search of fluorophores in a 1600 compound containing microarray, resulting in fluorescent imidazo[1,2a]pyridine compounds, which are known to interact with the peripheral benzodiazepine receptor (known as the translocator

107 The Groebke-Blackburn-Bienaymé reaction

protein TPSO) and GABAA benzodiazepine receptors. The compounds were tested for their affinity with, and use as TPSO imaging probes. The introduction of a 18F-label to PET radiotracers via 18F-labelled prosthetic groups using MCR chemistry was discussed by Gouverneur et al.30 Labeling of 18F requires reaction conditions which might not always be compatible with the substrate. By using 18F-benzaldehydes in MCR assisted radiochemistry, mild conditions could easily be performed in the U-4CR, P-3CR, B-3CR and GBB-3CR while maintaining a swift reaction necessary for the rather short half-life time of 110 min for 18F.

N NH2 + NC Sc(OTf)3 NH 3-Methyl-1-butanol O 170 °C, 15 min N 18F 18F N Scheme 9. 18F introduction in GBB-3CR products. The ’hot’ GBB-3CR was performed in 3-methyl-1-butanol under conventional heating in most cases and some under MW conditions, where 150-170 °C was found to give the highest yields; 64-85% The use of hydrazines in the GBB-3CR has not been explored until 2014. A variation in which 2-hydrazinopyridine is used to obtain bicyclic pyridotriazines was introduced by Hulme et al.31 The proposed mechanism is quite similar to that of the GBB-3CR reaction, it involves a non-concerted [5+1]-cycloaddition of the Schiff base and isocyanide. Comparable to the example of ketones (Kumar et al. example in chapter 3.2) in the GBB-3CR discussed below, there is no rearomatization seen with both aldehydes and ketones, which otherwise concludes the GBB-3CR. The appendant moiety originating from the isocyanide component remains in the product as a stable imine (10). The reactive nature of the Schiff base 6 intermediate 11 allows introduction of not only isocyanide, but isocyanates, acyl chlorides and cyclic anhydrides as well (Scheme 10).

108 Chapter 6

N MeO MeO A NH N 2 H MeO 0.2 eq Sc(TfO) Cl + 3 N NH O rt, 20h N N NC DCM:MeOH 3:1 NH NH N N Cl 10 Cl

R3 R2 N O O R N N 2 N B R3 Cl NH N R3 N R3 NC R1 R2 NCO O O R N N R 3 N X N N 2 N HN R3 O H R2 11 O O N R3 R N O NCS 2 S N X N N OH N HN R3 Scheme 10. A: Hydrazines undergo a GBB-like [5+1] cycloaddition to furnish bicyclic triazines. This given example was obtained in 85% yield, aromatic aldehydes will not result in product formation B: The reactivity of the hydrazine derived Schiff base could be exploited for other transformations with various electrophiles.

Hulme et al. reported the use of acyl cyanide (14) as isocyanide replacement in the GBB-3CR. The resulting primary amine (15) subjected to the excess of aldehyde (4 eq.) and acyl cyanide (3 eq.) undergoes a domino/tandem acyl-Strecker reaction.32 A great example of a modified GBB-3CR: on one hand the acetyl cyanide serves as a less toxic replacement for TMSCN, on the other hand a great catalyst, acetic acid is freed during the course of this one-pot 3-step cascade reaction (Scheme 11).

Scheme 11. Acetyl cyanide assisted one-pot three-step GBB-Strecker cascade. In situ generation of amidines for use in the GBB-3CR was demonstrated by Mah- davi et al., using 2-bromopyridine 16, sodium azide, and aldehyde and isocyanide in a copper-catalyzed 4CR.33 The activated bromine is converted to amine 17 via a reductive amination as depicted in scheme 12, prior to take part in the GBB-3CR. The reaction sequence is described a one pot four component reaction, the reaction could as well be considered as a three-component reaction with in situ generated 2-aminopyridine.

109 The Groebke-Blackburn-Bienaymé reaction

ArCHO R R NC NH N Cu/L N Cu/L N N NaN NaBr N Ar Br 3 3 N2 NH2 DMSO 16 17 110 °C, 3h N Scheme 12. The scope of the GBB-3CR was expanded by the use of 2-bromopyridine, in situ converted into the essential amidine. Procedures in process chemistry are necessarily studied in depth for optimal conversion to the desired products. In this respect the report of Mathes et al. is very useful, as it investigates the driving forces of the GBB-3CR, introducing alternative purification methods that circumvent labor-intensive chromatography.34 Apart from the usual reagents, the Schiff base pre-formation was promoted by adding a catalytic amount of p-TSA. The following cycloaddition was in turn catalyzed by borontrifluoride-acetonitrile complex (BF3·MeCN) and two equivalents of trimethyl orthoformate as dehydrating agent was added to increase the rate of the reaction significantly, giving good yields in less than a day of reaction time (Scheme 13). Purification was done by adding 1,3 equivalents of sulfuric acid to precipitate the GBB-3CR products from i-PrOH as sulfate salts in high purity. From the optimized method, a large scale reaction was performed at 100 mmol scale, yielding the GBB product 18 in 82% yield, against 85% on 1 mmol scale, proving the scalability of the reaction.

F O N + 1. PTSA (1 mol%), toluene, reflux F N H NH2 2. trimethyl orthoformate (2 equiv) NH F BF3 MeCN (5 mol%), r.t. N F N F NC N 18, 85% F Scheme 13. The optimized one-pot two-step process of a BF ·MeCN catalyzed GBB-3CR. 3 6 Using bis- (19-20) or tris amidines in the GBB-3CR allows for multiple MCRs in a single transformation as demonstrated by Lavilla et al.35 The position of each amidine when applying asymmetric bis-amidine 19 determines the reactivity and therefore the selectivity of the first GBB-3CR and enables introduction of different aldehyde and isocyanide components in the second GBB-3CR. This regioselectivity is also seen in symmetric bis-amidines, but require stoichiometric amounts of the aldehyde and isocyanide components. Reactions with the tris-amidine melamine 21 exclusively yielded symmetric products. The fluorescence abilities of the synthesized compounds was highlighted and through introduction of EWG or EDG functionality at specific locations, the fluorescence emission wavelengths could be tuned. Additionally BODIPY (93) like fluorophores were made by reacting α-pyridyl GBB-3CR compounds with BF3OEt2, to create a BF2 bridge between the pyridine and imidazo rings. This bridged compound (23) showed a shift in the emission wavelength by 60 nm, a 40-fold emission increase and pH insensitivity as compared to unbridged (22) (Scheme 14).

110 Chapter 6

NH A. H N 2 H2N N NH2 2 N N N N N NH2 H2N N NH2 19 20 21

R4 R1 O R 3 O HN R3 N N NH2 B. NC NC H2N N NH2 R 2 R1 R4 N N N N N R NH 1 R2 N 19 NH R2

F -F N N B N+ N+ C. N N BF OEt HN 3 2 HN

22 23 Scheme 14. Bis-amidines in the GBB-3CR: A. The Bis- and Tris amidines reacted in the GBB-3CR. B. Asymmetric product formation using different aldehydes and isocyanides in two separate MCRs. C. Enhancement of fluorescent properties by introduction of a BF2 bridge.

3. Scope and limitations of the GBB-3CR. Immediately after the introduction of the GBB-3CR approach to access imidaz- o[1,2-a] heterocycles, the scope and limitations were elaborated. However, in each of the attempts only a small selection of variables was assessed. This went from solvent screening, varying reaction conditions, catalysts and reagent use to solid phase approaches. A good example is the use of glyoxylic acid to obtain an uncatalyzed formaldehyde-based product. In the general scope and limitation of the GBB-3CR it was found that the reaction is best performed at room temperature in MeOH, with a concentration ranging between 0.3-1.0 M, using arylalde- hydes, 2-aminopyridines, and aliphatic isocyanides in stoichiometric amounts, in the presence of a catalytic amount of 10 mol-% Sc(OTf)3. Difficulties in predicting reactivity were found with some combinations of starting materials and cannot always be attributed to electronic factors, as steric effects have a significant effect as well, illustrated in figure 2.[35] Aliphatic aldehydes give good yields when aromatic isocyanides are used, however, when both aldehyde and isocyanide are both electron rich, a reduction in yield was described recently.34 Although such findings are valuable starting point for better understanding the GBB-3CR, we’ve tried to illustrate the scope and limitations in a broader sense, including the effect of substituents on each of the reported starting materials. Therefore, a tabular summary of amidines, aldehydes, isocyanides and catalysts used in the GBB-3CR is presented in the following. Additionally, the tables act as reference guide to which components are suitable, including every report the component was successfully used in. The benefit here is a complete and simple access to the possible starting materials, grouped

111 The Groebke-Blackburn-Bienaymé reaction according to aromatic, hetero aromatic and aliphatic nature and electron donating or withdrawing substituents (EDG and EWG respectively) and bulkiness. Thus a quick look in the tables can reveal unambiguously which component has been used previously and will likely work in other instances.

Figure 2. Steric effects in the GBB-3CR. The bulk effect of t-butyl prevents the reaction from happening, the optimized geometry was calculated for compound 24, exchanging the t-But- for a cyclohexyl moiety, affording compound25 in a good yield. 6 3.1 Cyclic Amidines in the GBB-3CR The amidines are key to give the scaffold its typical imidazo[1,2-a] heterocyclic form, whereas the aldehyde and isocyanide components are more appendant substituents, sometimes called scaffold decoration. Nearly 90 different amidines were used in GBB-3CR, of which 22 are five membered and 66 are six- mem bered amidines. All of them were sorted in table 1 according to ring size, type and additional substituents. The most abundant amidine in the table, 2-aminopyridine is where the GBB-3CR was discovered with and is not surprisingly used in many model reactions to test other solvents and catalysts. Substituted 2-aminopyridines also show good reactivity, giving products in good yields. Electron withdrawing substituents, mostly halogens seem to increase the yield, with the exception of nitro-groups. Alkyl groups and other more EDG generally give lower yields. More electron deficient pyrimidines give lower yields in the GBB-3CR, both 2- and 4-aminopyrimidine with EWG substituents were reported as low yielding. Aminopyrazines are overall reported as good yielding. A broad range of 5-membered amidines appears often with other hetero atoms, such as

112 Chapter 6 oxazoles and thiazoles, and are not very reactive resulting in less good yields. 3-Amino-1,2,4-triazole, on the other hand, is reported as good yielding amidine. Nucleobase derived compounds are popular as they possess good hydrophilicity and solubility, which are therefore potentially therapeutically relevant heterocycles. Adenine 26, guanine 27 and cytosine 28 are nucleobases that bear an amidine moiety which was exploited in the GBB-3CR by Madaan et al. to furnish aminoimidazole-fused nucleobases.36 The polar nature of these amidines required a more polar solvent than methanol, as this solvent did not yield any product at all. The screened solvents PEG-400, ethylene glycol, DMSO, DMA and DMF all give product, with yields varying between 23 to 68% with ZrCl4 in DMSO as a catalyst (Scheme 15). Even the otherwise difficult to modify guanine is giving GBB-3CR products albeit in rather low yields.

Scheme 15. Synthesis of aminoimidazole-condensed nucleobases. Table 1. Amidines applied in the GBB-3CR. Those with R groups have multiple similar substitutions, used for producing derivatives. *This amidine has 10 more examples of N substituted piperazines.37-38

N NH N NH2 N NH2 2 N NH 2 8, 19, 30, 33, 38-122 28, 34, 57, 64, 68, 71, 79, 81, 87, 27-28, 41-42, 46, 51-52, 91, 93, 97, 101, 107, 110-112, 54-57, 60, 64-65, 68-69, 71, 41, 57, 63-64, 74-75, 90, 92, 95, 114-115, 123 75-76, 85-87, 96, 100-101, 101-103, 105, 107-110, 112-113, 103, 108-109, 116, 119, 122, 115-116, 122-123, 125, 130 124-129

N NH N NH2 2 N NH2 N NH2 59, 64, 68-70, 87, 95-97, 108, 28, 52 8 112, 115-116 42, 52, 117

113 The Groebke-Blackburn-Bienaymé reaction

N NH2 N NH2

N NH2 N NH2 64 8, 27, 104 58, 80 28, 42

Br Br

N NH2 Br N NH2 N NH2 R N NH2 64, 75, 98, 118, 120-121, 132-133 27, 51-52, 54-57, 59-60, 131 63-66, 68, 70, 73-76, 85, 94, 100, 117 97, 100, 102, 105, 108-110, 113-116, 119, 121-122, 124-

127, 130, 134

Br Br Br Br

N NH2 N NH2 N NH2 N NH2

66, 108-109, 115-116, 122-123 64, 87 69 87

Br Br Br Cl Cl

N NH N NH N NH2 2 2 N NH2 8, 30, 41, 52, 59, 63-64, 66, 123 8 68, 75, 86, 95, 102-103, 29, 66, 105, 112, 117 108-110, 112-113, 116, 119,

121-123, 125, 128, 130, 135

Cl F F

N NH F3C N NH2 6 N NH2 2 N NH2 86 108 64, 96, 107, 128 62

F3C CF3 F3C Cl

N NH2 N NH2 MeO N NH2

N NH2

75, 96, 103, 107, 130 110 64 107, 117

114 Chapter 6

MeO OMe OH

N NH2 O 102 N NH2 N NH2 N NH2 86 29 8, 40, 42, 52, 75, 93, 112, 118, 120

O H2NOC HOOC O HN B O N NH2 N NH2 N NH2 N NH2 8, 65, 96 58, 80, 86, 137-138

136 96

COOH MeOOC COOMe

N NH MeOOC N NH 2 2 N NH2 N NH2

139 29 29, 75, 86, 89, 102-103 29, 78, 96

COOMe O2N NH2

N NH N NH NC N NH 2 2 N NH2 2

29 65, 81, 92, 103, 110 73 64

NC CN COOMe N

N NH N NH2 R N 2 N NH2 N

64, 102-103, 125, 130 8, 19, 26-27, 29, 38-41, 43, 47, 49, NH 29, 117 2 N 58, 61, 63, 66, 73-74, 86, 97, 102- 103, 107, 112, 125, 141-143 140

Ph NH N N N N N

N NH2 N NH2 Ph N NH2 O N NH2 H

142 42, 142 144 36

N HN OMe N N N N NH2 N N NH N NH2 2 N NH2 29 145 36 145

115 The Groebke-Blackburn-Bienaymé reaction

Ph Ph N N Ph N N N N N N N O N NH2 N N H NH2 NH 2 NH2 36 147 146 146

OH N N Br N

N N NH2 N NH2 N NH2 HO N NH2

8, 19, 29, 34, 38-40, 42-43, 48, 42 94, 130 148 53-54, 56, 58-59, 61, 63, 66, 68, 72-75, 80, 86, 88-89, 94, 96-97, 101-103, 105, 108, 110, 112, 114,

123, 126, 130, 145, 149-151

N N Cl Cl N NHDBM

Cl N NH2 N NH2 N NH2 N NH

42, 64, 132-133 72-73, 152 153 N NH2

153

HO N R Cl

N Bn N N NH2 N N NH2 N NH2 N NH2 155 154 102-103, 156 66

O Ph N Cl N N N N N NH N N N Ph N NH2 N NH2 N N 6 N NH2 NH2 158 159 159 157

S H H R2 N N R1 N NH NH O N NH 2 2 NH 2 N N 2 MeOOC N 162-163 160-161 63, 114 164

CN H H Cl N N NH NH H NH2 2 2 N N N N N N N NH H 2 N N 8, 74, 166-167 167 165 167

116 Chapter 6

R S HOOC S NH H NH2 2 N N N NH N N 2 N N NH2 58 R N 58, 80, 105, 110, 114, 138, 167 160, 169-171 168

S S S BocHN S NH NH2 2 NH2 N MeO N N N NH2

112 8, 27, 29, 46, 59, 61, 74, 95, 48 160 105, 110, 171

S NC S R S O NH 2 NH NH2 N 2 N N EtO N S NH2 171 N N 8, 37, 170 42, 66 172

HN Ph O N NH N NH 2 S 2 S N NH2 N N 46 8 37-38 A feature which can be generally observed with many MCRs is their great functional group compatibility. Thus, heteroaromatic amidines can comprise all halogens, and pseudo halogens such as nitrile, nitro, methoxy, free carboxylic acids, esters, unprotected primary and secondary amines, unprotected phenolic and aliphatic hydroxyl, amides, alkynes, alkenes, and boronic acid esters. This great functional group compatibility is important for further reactivity of the initial GBB-3CR products and also for optimal interaction within a receptor pocket.

3.2 Aldehydes

Aldehydes are common building blocks which are cheap and have good commercial availability and with 180 different records they are very broadly applicable in the GBB-3CR reaction with great variability (Table 2). The diversity of aldehydes found is broad, from aromatic to aliphatic aldehydes with many different substituents, both electron withdrawing and donating and bulky and small groups and also aldehydes containing reactive to labile protective groups that allow secondary modifications. Aromatic aldehydes generally form stable Schiff bases, where an effective conjugation system is beneficial for its stability. Schiff bases from aliphatic aldehydes are found to be less stable and readily polymerize, which translates to reduced yields of subsequent transformations. Benzaldehyde is the mostly used aldehyde for reasons such as detectability on TLC for reaction monitoring and good reactivity. Substituents on benzaldehydes do affect their reactivity. Using substituted benzaldehydes bearing an electron donating group

117 The Groebke-Blackburn-Bienaymé reaction usually increases the yields, whereas withdrawing substituents reduce the yields. It would be an extensive exercise to discuss every variant in detail, therefore the more interesting examples were highlighted. The use of formaldehyde in order to obtain 2-unsubstituted-3-amino-imidazoheteocycles in the GBB-3CR was not reported until 2004 by Kercher et al.42 Successful preparations of the unsubstituted imidazole were actually scarce and the few reports that did, showed low yields in non-efficient synthetic routes.173-175 In this report formaldehyde (aqueous) and paraformaldehyde were used, resulting in poor yields of 36 and 44% respectively. Other formaldehyde substitutes were screened, showing that glyoxylic acid is giving good yields up to 71% with glyoxylic acid immobilized on macroporous polystyrene carbonate (MP-CO3) 29 in an uncatalyzed GBB-3CR reaction (Scheme 16). Kennedy et al. reported three examples with the successful use of paraformaldehyde with varying yields between 68-78% in a microwave assisted 136 GBB-3CR in MeOH, catalyzed with 4 mol-% MgCl2 at 160 °C in just 10 minutes. The first ketone example in a tetracyclic fused imidazo[1,2-a] pyridines from isatin 30 was reported by Che et al.119 The special multi- reactive nature of isatin is allowing for a formal [4+1] cycloaddition with different isocyanides and subsequent rearomatization via [1,5]- H shift through a retro-aza-ene reaction compound 31 (Scheme 17).

Scheme 16. Formaldehyde and formaldehyde surrogates in the GBB-3CR and their associated yields.

118 Chapter 6

Scheme 17. Two possible reaction pathways with the α-ketoamide isatin and some examples of the GBB-3CR ran with other ketones.

Using other ketones, Kumar et al. reports the synthesis of spiro-heterocycles 170 catalyzed with TiO2 nanoparticles in excellent yields. The quaternary spiro carbon lacks, however, the proton required for [1,3]-H shift, therefore rearomatization of the unstable [4+1] adduct could not take place. These adducts were reported as the products and also contains an example of an isatin containing product 32, wherein the structure was found to be surprisingly different from the isatin products described by Che119 etal. The conflicting structures are not likely to be attributed to the amidine and isocyanide components and the right structure is without appropriate structure elucidation such as X-ray diffraction spectroscopy difficult to confirm. The aldehyde pyridoxal 76 has a different outcome and will not result in the typical GBB-3CR scaffold when reacted with an amidine and isocyanide. The resulting product formed instead, a furo[2,3c]pyridine 78, was shown in scheme 32 and further discussed in the biologically active compounds section

Table 2. The large variety of aldehydes sorted by origin and substituents. O O O

8, 19, 26, 28, 33-34, 37-40, 43, 46-47, 50-51, 53-55, 58, 60, 63, 70-73, 96, 136 6, 26, 28, 40, 73, 83, 107 76, 79-80, 83-85, 89-90, 92-93, 96, 100, 104-107, 109, 111, 114, 117, 124, 126-127, 131-133, 136, 140, 143, 145, 148-150, 152, 155-156,

158, 162, 164-167, 172, 176

O O O O O

6, 19 29, 165 27, 114, 162 56, 81, 83, 160-161

119 The Groebke-Blackburn-Bienaymé reaction

O O O O

19-20, 33-34, 36-37, 43, 46, 51, 37, 144 29 90 54-55, 60, 76, 90, 100, 105, 109, 114, 124, 126-127, 132-133, 137,

139-140, 143, 164-165

O O O O TBDMS

166 37, 96 8 92-93, 141

O O O N O N N NC N H Et

61 59, 63, 70, 83, 162, 164, 169 29, 36, 63, 68, 169 29

O O O O R O OMe MeO

134 92-93, 133, 144, 148, 158, 6, 19, 29, 33-34, 36, 39, 43, 50, 72-73, 133, 152 167, 177 54-55, 59, 63, 68, 74, 77, 83, 90, 94, 100, 105-107, 114, 117, 126, 131-133, 136, 140, 143, 145-148, 150, 152-153, 155,

157-158, 166-167, 172

MeO MeO O O O O MeO OMe MeO MeO 6

29, 43, 63, 106, 158 29, 65, 69, 78, 89, 153, 158 50, 117, 141, 158, 166, 176 29

MeO EtO MeO BnO O O O O

N Cl N O MeO H OMe

130 43 68 8, 36, 59, 69, 74, 83, 141,

147, 158

BnO MeO O O O O

PhO BnO OBn BnO BnO

20, 137 65 65 6

120 Chapter 6

O O

O O O O

43, 117, 166 73, 83, 99 73

O O O O

MeO Rf8O2SO OMe OSO2Rf8

41 73 41 131

O Cl O O O O Rf8O2SO O OSO2Rf8 Cl 41 140, 162 41 162, 168

O O O O O O O O O O Cl O Br

56 156 130 29

HO MeO MeO O O O O HO O MeO HO HO OMe 134 134, 144, 158 85, 134, 142, 155, 169 142

COOMe O O N N O N Br Cl N N H N O R R 121 121

122 108

O Cl t-Bu O O O O S N H Cl Cl HO Cl t-Bu

88, 92-93, 109 68 96 142

121 The Groebke-Blackburn-Bienaymé reaction

HO O O O O OH HO HO

68, 72, 83, 92, 104-105 29, 68, 104, 117, 140, 142, 144, 69, 104, 134, 158 155, 158, 169 142

HO F O O O O O HO OH HO Cl O 65, 104 104, 117, 142 88 91

O O OR O O O O O N Cl O O O R' O 95, 118

155 113 91

O O O O MeO COOMe COOH O

59, 123, 163 52, 87, 106, 125, 159 20, 26, 38-39 106

HOOC OMe O O O HOOC MeO COOH O OMe MeO COOMe

58, 62-63, 80 58, 62, 80, 134 52 59 6 EtO Br F O O O O

HO HO HO F HO

117 117 117 117

Br O O O O

HO EtO OH Cl

47, 64, 67, 85, 135, 148, 167, 117 135 92-93 172

122 Chapter 6

O Cl Cl Cl O O O Cl Cl Cl

29, 33-34, 36, 43, 46-47, 51, 54, 83, 106, 139, 144, 167, 172 144-145 130, 146, 148 59-60, 63, 68-71, 74, 76-77, 79, 82-83, 85, 88, 90, 94, 100, 106, 111, 124, 132, 143, 146-147, 150, 152, 158, 167-168, 172,

176

F MeO Cl O O O

Cl Cl F O CN Cl OH 43, 156 156 102 106, 177

Br R O O O O

HO Br HO NO2

109 155 112 117

F O O O O HO OEt F F

117 140, 144, 147-148, 150, 169 30, 34, 37-38, 43, 47, 68, 85, 29, 96, 150 88-89, 94, 96, 105, 114, 141, 144-145, 147, 150, 158, 164-165,

168, 172

F F F3C O O O O

F F3CO

156 150, 165 156 156

O2N MeO O O O O

Cl O2N OH OH NHAc OMe 172 177 177 177

O O OMe O

NHSO2CH3 OH O F3C OEt MeO OMe 177 20, 107, 132-133, 136, 165 92 165

123 The Groebke-Blackburn-Bienaymé reaction

Br O O O O F Br Br OMe 43, 106 74, 105, 130, 133 29, 33, 36, 59, 69, 85, 90, 94, 156 109, 114, 117, 130, 133, 137

MeO OH O O O N MeO Br O N N OH

8, 27, 47, 63, 100, 105, 121, 131, 8, 28-29, 50, 81, 88, 96, 133, 130 72 133, 164 136, 145, 149, 158

O O2N O O O N NO2 O2N

51, 55, 68, 72, 106, 124, 126, 8, 105, 139-140, 158, 162 29, 33, 68-69, 71, 79, 83, 85, 51, 54-55, 60, 71, 79, 85, 88, 147, 158 88, 92-93, 111, 114, 140-141, 100, 111, 114, 124, 126-127, 145-148, 152-153, 158, 162, 164, 139, 144, 146, 148, 158

167, 172

R1O O O O O NO2 R2O N N O

29, 88 128 135, 171 86

F O O O O NC F3C F F

F CF3 88, 133, 168 165 133 165 6

O O CHO N Fe N O

57, 92, 104, 155 43, 83, 90, 105, 155, 167 132 120, 171

O Cl O O O O O

HO O O I

154 96 153 153

124 Chapter 6

O O O R O O O O O O O R' O

91 97-98 90 91

O OH O O N BocN O O Boc

O O O 96 48 110 171

O HO3S O Cl O O

O O O O

50, 92-93, 106, 114, 133, 140, 29 29 29, 81 149, 158

Br O O S O O S O O

135 8, 28-29, 58, 81, 85, 90, 92-93, 135, 143 135 105, 127, 133, 141, 144, 158

Br S S H N N O O O O

135 83, 135 81, 105, 133 29, 78

Boc O O O N R O 1 N N N Ac R2 48 135 29 117

O EtOOC Cl HN R HN N O N O N N O N R1 H

135 135 106, 135, 138, 158 R2

115-116

125 The Groebke-Blackburn-Bienaymé reaction

O Me SiO H O 3 N O Si O Me3SiO O CO2Me N O O CO2Me

101 45 29 45

O O Me3SiO Me3SiO

CO Me CO2Me 2 141, 149 38, 44, 47, 50, 59, 83, 92,

141, 176 45 45

O O O O

83, 96, 105, 133 19-20, 36, 47, 100 28, 40, 47, 107 8, 84, 107, 136, 165

O O O (CH2O)n H O 28, 69, 72, 131-132 83, 105 O 136

49, 149

H O O HO HO OH O O H HO OH O 6 66 42 66, 96

EtO O O S O O O Ph 83 75 70 8, 28, 34, 92, 132-133, 140-141 6

O O O O TMS

103 103 103 29

O O O O

BocHN SCH3 BocHN

29 48 105 48

126 Chapter 6

O BocHN O O O OtBu BocHN 48 48 170 170

O O O R R O O N N H H

170 119 170

Interestingly, 2-siloxycyclopropanecarboxylates were applied as aldehyde substitutes to obtain GBB-3CR compounds with a δ-amino acid backbone.45 The siloxanes go through a ring opening pathway and behave as the aldehyde component 33, together with 2-aminopyridine and p-methoxyphenyl isocyanide 34, catalyzed with acetic acid in MeOH to give methyl (3-aminoimidazo[1,2-a] pyridin-2-yl)propanoates in moderate to good yields (Scheme 18). Again the functional group compatibility of the GBB-3CR aldehyde component is amazing and comprises aromatic benzaldehydes include substitutions on all positions with alkyl, aryls, alkoxy groups, alcohols, acids, nitro groups, cyano groups, tertiary amines polyaromatics and fused heterocycles. Aliphatic aldehydes are also widely represented, including saturated and unsaturated alkyl groups (1°, 2° and 3°), substituted with alcohols, esters, adjacent carbonyls, thiols and cyclic alkyl groups. Thus, heteroaromatic amidines can comprise all halogens, and pseudo halogens such as nitrile, nitro, methoxy, free carboxylic acids, esters, unprotected primary and secondary amines, unprotected phenolic and aliphatic hydroxyl, amides, alkynes, alkenes, and boronic acid esters. This great functional group compatibility is important for further reactivity of the initial GBB-3CR products and also for optimal interaction within a receptor pocket.

Scheme 18. 2-Siloxycyclopropanecarboxylates as aldehyde substitutes in the GBB-3CR. One example of a GBB product 35 synthesized from 2-siloxycyclopropanecarboxylates and its resolved X-ray structure (CCDC-601760), in total 8 products were synthesized with variations on the amines and isocyanides with yields varying between 40-79%.

127 The Groebke-Blackburn-Bienaymé reaction

3.3 Isocyanides used in the GBB-3CR

The commercial availability of isocyanides is limited and those available are often expensive, likely because of the stability, applicability and the undesirable smell of most liquid isocyanides. Therefore only a few dozen of isocyanides are commonly used in many isocyanide-based MCRs. Due to the pricing and availability isocyanides are often prepared in house. Preparation of isocyanides from primary amines via the Hoffman- or Ugi route (formylation & dehydration) is most common and recently the substrate scope has been broadened by conversion of cheap and broadly available oxo-compounds (aldehydes and ketone) to isocyanides by the repurposed Leuckart-Wallach reaction as described by Dömling et al.178 Altogether an astonishing 100 different isocyanides were reported in the GBB- 3CR, proving that the isocyanide variation point is broadly exploited and is hardly to be considered as a limitation of variability in this three component reaction. Aromatic and aliphatic isocyanides participate with equal ease in the GBB-3CR, although it must be noted that combinations of bulk substituents affect the yield when bulky amidines and aldehydes are used simultaneously. Not surprisingly, there is again a great functional group compatibility. This includes aliphatic isocyanides widely substituted heterocycles, substituted with esters, alcohols and ethers. Also a broad selection of substituted phenyl isocyanides are compatible, with substitutions on all positions with alkyls, halogens, ethers, esters and thiols.

Table 3. Nearly 100 reported isocyanides used in the GBB-3CR and sorted by type and substituents. NC NC NC

29, 34, 52, 61, 73, 86, 128, 8, 19, 26, 29, 33, 36-38, 45-46, 49-51, 54-60, 62-63, 66, 69, 71-74, 76, 8, 19, 26, 28, 36, 42, 45-47, 53, 134, 140, 163 79-81, 85-86, 89, 91-93, 95, 97-98, 100-105, 109-116, 118-120, 122, 56, 63, 73, 76, 95, 97, 100, 102, 124, 126-127, 135-136, 139-140, 144, 146-147, 152-157, 159-162, 104-105, 110, 117, 127, 136, 164-165, 167, 169, 171-172, 176 143, 156, 160-161, 164-165 6

NC NC NC NC

145 41, 52, 61, 66, 73-74, 125, 134, 73, 128, 134, 152, 159, 162-163 66, 73, 131 156, 170

NC NC NC

176 37, 66, 128, 134, 140, 145 8, 19, 29-30, 33-34, 36, 41, 46, 49-52, 54-57, 60-61, 64-65, 67, 69-74, 76, 79-81, 85-86, 90-93, 95, 97-98, 100, 105-106, 111-112, 114, 117-121, 124, 126-128, 131, 136, 139-140, 144, 146-148, 150,

152-153, 156, 158-159, 162-165, 168, 170-172

128 Chapter 6

NC Ph NC Ph NC Ph NC

43 142 73, 117, 121 107

PhO MeO NC NC MeO NC O NC

42 48 169 169

TMSCN NC O N N NC NC O O N N 73, 102, 159 Ph 29, 50, 69, 74, 78, 89, 114, 159 8, 48, 77 8

MeO EtO O COOH NC NC NC O O tBu-O NC

42, 48, 52, 58, 78, 87, 89, 105, 27, 84, 86, 104, 112, 115-116, 8, 29, 86 40 108, 115-116, 122, 132, 165 121-122, 125, 131-132

Wang resin Wang resin NC BocHN t-BuOOC NC

O O HO O O 48 86

NC NC

40 40

AcO O S MeOOC NC NC AcO NC NC AcO OAc 87 34, 87 48 99

S NC O O NC

NC NC (rac)

143 87 43, 47, 77, 92, 105, 125, 145, 48 166, 176

NC NC NC NC

34, 43, 142, 145, 150 142 8, 42, 48, 50, 55, 57, 65-66, 97, 142 100-101, 124, 126-127, 136,

141-142, 156, 160-161, 172

129 The Groebke-Blackburn-Bienaymé reaction

NC NC NC MeO NC

OMe MeO 48, 145

43, 166 34, 36, 42, 45, 66, 72-73, 75, 86, 142 101, 118, 120, 130-131, 141-142,

145, 150, 159

MeO NC MeO NC O NC O NC

MeO MeO O O OMe

6, 141 48, 142, 150 142 77

NC F NC F NC F NC F F F

29, 43, 77, 134, 150, 166, 176 145, 150 150 150

F NC NC F NC NC F Cl F Cl

143 150 150 73

NC Cl NC NC NC

Cl Br COOMe

48 42, 61, 77, 125, 149, 168 49, 77 142

MeOOC NC NC NC NC O 6 O t-Bu SMe N

142 70, 123 8 141

NC NC NC NC N Boc N H N OH 150 66 42 149

NC NC PivO NC NC

O N F3C O 44 156 48 20, 141

130 Chapter 6

NC NC NC NC O2N

19-20, 27-29, 41-42, 45, 47-49, 132 77, 104, 129, 145 86 52, 58, 65-66, 70, 72-73, 77, 80, 86, 89, 97-98, 100, 112, 118, 120-121, 125, 129-130, 132, 136,

140, 153, 156, 159, 162-163, 171

NC Cl NC NC NC Cl

129 129 132 143

MeO OMe O NC NC NC MeO NC O MeO MeO 98, 129, 132, 168, 171, 177 87 87 87

NC NC MeO NC NC Ms MeO MeO MeO N

87 87 98, 120, 171 87

O O NC Boc NC N NC BocHN S NC Boc EtOOC N N H H 48 129 34, 88 96

NC Fe NC

104

+ N - N B F F

4. Catalysts in the GBB-3CR

From the moment the GBB-3CR was discovered, Lewis and Brønsted acid catalyst were used for an efficient transformation. Saying this, it is possible to run the GBB- 3CR without the aid of a catalyst, it generally depends on the nature of the reagents. Low electrophilic aldehydes such as various benzaldehydes are suspected to be unable to condensate with electron deficient amidines, whereas the addition of Lewis acids would increase the reactivity of the imine formation considerably.145

131 The Groebke-Blackburn-Bienaymé reaction

In figure 3 the occurrence of the catalysts used in the GBB-3CR are mapped. Clearly scandium triflate has the highest success rate followed by the Brønsted acids HClO4, p-TSA and acetic acid. However, when taking into account that early adaptors of the GBB-3CR were basically reproducing reaction conditions, the frequency of some of the applied catalysts is artificially higher and are possibly not the best catalysts in the given reactions. Examples of such catalysts are acetic acid and Montmorillonite k-10 clay which appear mostly in the first few years after the discovery of the GBB-3CR. Additionally some catalysts were reported multiple times, however from the same research groups, introducing a biased success rate.

25 20 15 24 10 15 14 13 5 10 8 7 6 5 4 4 4 3 0 2 2 2 2 2

Figure 3. Overview of GBB catalysts that performed best in reported catalyst screenings (Single hits were omitted for clarification).

Table 4. Every catalyst previously used in the GBB-3CR. Catalysts (formula) Occurrence Reference 8, 44, 66, 75, 78, 89, 96, 102-103, 6 119, 123, 150, 167, Perchloric acid (HClO4) 15 179-180

20, 26-30, 39, 41, 47-48, 58, 61-62, 67, 77, 86, 125, 137-138,

Scandium triflate (Sc(OTf)3) 24 140, 146, 149, 168, 177

105, 131-132, 153

Ytterbium triflate (Yb(OTf)3) 4 108-109, 122

Indium triflate (In(OTf)3) 3 113 Silver triflate (AgOTf) 1

132 Chapter 6

6, 45, 68, 110, 128-129, 142, 154,

Acetic acid (AcOH) 13 181-185

19, 64-65, 70, 121, Montmorillonite K-10 clay 6 186

34, 40, 55-56, 74, 85, 88, 97, 107, 130, 141,

p-toluenesulfonic acid, PTSA, p-TsOH 14 164, 187-188

43, 49, 54, 95, 144, 147, 156, 158, 166,

Ammonium chloride (NH4Cl) 10 189-190

124 Ionic liquid [bmim]Br 1 70 Ionic liquid [bmim]BF4 1 136

Aluminum chloride (AlCl3) 1 134, 136, 191-192

Magnesium chloride (MgCl2) 5 79

Ruthenium chloride (RuCl3) 1 81, 91

Bismuth chloride (BiCl3) 2 32

Calcium chloride (CaCl2) 1 92, 104

Lanthanum chloride (LaCl3.7H2O) 2 51 Cellulose sulfuric acid 1 193

Cellulose@Fe2O3 1 126, 148 Silica sulfuric acid 2 115

CuFe2O4@SiO2–SO3H 1 100 (MWCNTs) 1 170

TiO2 NPs (nanopowders) 1 116 ZnO NPs 1 57, 98, 122, 145

InCl3 4 37-38, 53, 152, 155, TMSCl 7 169, 176

52 Methanesulfonic acid 1 194 Zeolite HY 1

133 The Groebke-Blackburn-Bienaymé reaction

36, 55, 59, 63, 94, 99,

143, 195 ZrCl4 8

127

SnCl2.2H2O 1 60

Heteropolyacid H3PMo12O40 1 bromodimethylsulfonium bromide 69 1 (BDMS) 71

γ-Fe2O3@SiO2-OSO3H 1 72-73, 84, 117 HCl dioxane 4 cationic polyurethane dispersions 76 1 (CPUDs) 77 p-TsCl 1 162-163 Piperidine 2 135 Iodine 1 82, 165 Trifluoro acetic acid (TFA) 2 83 (TBBDA) 1 83 (PBBS) 1 90 nano-LaMnO3 1 111 NH2‐MIL‐53(Al) 1 33 CuI L-Proline 1 34

BF3·MeCN 1 120 6 2-chloroacetic acid (ClCH2COOH) 1

Table 4 summarizes the catalysts applied in the GBB-3CR, which give the highest yield in the described model reaction. Surprisingly not every catalyst screening includes the often excellent performing catalysts (Sc(OTf)3, HClO4, and p-TSA). Nearly 50 different catalysts were described as high yielding, indicating a great scope and freedom for selecting the catalyst of choice. There are some reports of catalyst free GBB-3CR’s, that run perfectly fine without any additional reagent. An uncatalyzed GBB-3CR was first reported by Lyon et. al. employing immobilized glyoxylic acid on macroporous polystyrene carbonate (MP-glyoxylate) as a formaldehyde equivalent. The decarboxylation leaves a 2-unsubstituted 3-amino-imidazo[1,2-a] heterocycle without use of any catalysts in a yield of 71% (Scheme 16).42 Further reports of catalyst free GBB-3CR’s employ bifunctional building blocks that hold a carboxylic acid which probably catalyzes the reaction. Truly catalyst free reactions are only recently reported by Sharma et al., but seem to work exclusively in a reaction using 2-aminothiazole under microwave conditions.160-161, 171-172, 196 In some of the catalyst-free reactions, a carboxylic acid functional group was found in one of the three components that facilitated the

134 Chapter 6 reaction in good yields, although it must be noted, that the use of carboxylic acids will show Passerini poisoning. This side reaction is driven by the presence of 3 components; aldehyde, isocyanide and carboxylic acid that allow for the P-3CR to happen (Scheme 19).

N R2 +

NH NH2 GBB-3CR N NC R1 R R O 2 N 1 O R P-3CR 1 H N R O R + O 3 2 O R3 OH

Scheme 19. The P-3CR is competing with the GBB-3CR when carboxylic acids are used as catalyst, consuming the aldehyde and isocyanide components that otherwise would be consumed by the GBB-3CR. As most Lewis acids rapidly decompose or get deactivated when they come in contact with water, anhydrous conditions are usually required when running a

Lewis acid catalyzed GBB-3CR. Sc(OTf)3 is, however, more stable and even applied in aqueous media as an Lewis acid.197 The GBB-3CR is a condensation reaction, the formation of a water molecule would explain why scandium triflate is such a successful catalyst in this reaction. The only disadvantage noteworthy is that scandium triflate has the ability to polymerize isocyanides, explaining the darkening of some reaction mixtures when this catalyst is applied.141 Phenyliso- cyanides are prone to polymerization and amongst them unsubstituted phenylisocyanide most.198-200 4.1 Base catalyzed GBB-3CR Sun et al. reported in 2013 the first application of piperidine (35) as a Brønst- ed base catalyst for the GBB-3CR, which was applied to overcome the need to run a deprotection-cyclization-alkylation sequence to obtain their tetracyclic compound. Later in 2013, Sun et al. reported the same strategy without the post modifications and therefore these base catalyzed conditions could be applied for the standard GBB-3CR.162-163 In the plausible mechanism, piperidine is initially involved in the formation of the Schiff base, then with the introduction of the isocyanide component, the [4+1] cycloaddition takes place, where piperidine fa- cilitates the 1,3-H shift resulting in rearomatization, giving the GBB-3CR product as depicted in scheme 20.

135 The Groebke-Blackburn-Bienaymé reaction

Scheme 20. Plausible mechanism for the piperidine catalyzed GBB-3CR projected on methyl 9-butyl-3-(tert-butylamino)-2-phenyl-9H-benzo[d]imidazo[1,2-a]imidazole-6- carboxylate (36) and its corresponding X-ray structure. Sun et al. reports the first Brønsted base catalyzed GBB-3CR of 2-aminobenzimidazoles 37, methyl 2-formylbenzoate 38 and isocyanides, piperidine was herein used as catalyst.163 In their search for a post modification on the MCR product, an intramolecular cyclization of the secondary amine with the methyl ester was performed. The secondary amine was arising from the isocyanide input as mostly cleavable isocyanides were utilized. They envisioned a tandem GBB-3CR and post modification sequence in a single step resulting in 20 compounds with a yield varying between 34 – 95% (scheme 21). The heterocyclic benzimidazole and dihydropyrimidine fragments combined are promising compounds as these are associated with PARP, topoisomerase I, INOS, PI3K and PrCP inhibition. 6

Scheme 21. Preparation of polyheterocyclic 39 unambiguously confirmed by X-ray structure analysis (CCDC 850793).

5. Solvents used in the GBB-3CR

Around 30 solvents and solvent mixtures used in the GBB-3CR were analyzed and discussed. Looking at the occurrence, methanol is by far the most popular solvent for the GBB-3CR reaction. This is largely attributed to the fact that the highest yields without many side products can be achieved with this solvent, the ease of removal and the ability to dissolve quite some polar catalysts and starting materials as well as more apolar reactants. Discussion of the solvents such as methanol trapping and methanol mediated intermediates rise the question on how broad the solvent range is and whether other solvents than methanol should

136 Chapter 6 be applied in the GBB-3CR. Furthermore, the stability of the intermediates from pathway A and B (Scheme 4) are predicted to have different stabilities that vary with the used solvent and therefore would yield either one of the two isomers or the trapped side product.43 While focusing on solvent screens and suitable solvents for the GBB-3CR, it is remarkable that the 2nd most frequent applied reaction conditions is actually solvent-less. The solvent-less reports show that neat reactions with simple stirring or mechanical mixing such as ball-milling will yield the GBB-3CR compounds as well, but often require more exotic catalysts such as zeolite HY, Montmorillonite K-10 clay and nano-magnetically modified sulfuric acid.19, 71, 194 The use of water in a catalyst free GBB-3CR is not only working, but also affords GBB-3CR products in high yields. The aforementioned mechanism should be different from the reaction usually following the concerted [4+1] cycloaddition through iminium ions but rather a non-concerted 5-exo-dig pathway. The protonation of the imine is in general performed with Brønsted acids, in the case of water without catalysts this is unlikely to happen since the pKa of water is higher than pKa’s found for iminium ions.171 Additionally the Woodward-Hoffmann rules which indicates that the uncatalyzed concerted [4+1] cycloaddition is in fact symmetry-forbidden. The GBB-3CR is, however, mostly performed with a catalyst, which disrupts the symmetry and therefore allowing the [4+1] cycloaddition.201 Considering the possibility of using water or a percentage of water in EtOH; it might be odd that several reports, indicate that anhydrous conditions (dry methanol, dry MeCN and so on) are required for the GBB-3CR while the individual starting materials and catalysts aren’t sensitive to water, with the exception of some Lewis acids catalysts. Water, however, has the downside to negatively affect the speed of the reaction, anhydrous solvents and the use of a dehydrating agent could greatly increase the reaction rate.34 The use of non-protic apolar solvents would suppress the intermediate in pathway B, yielding exclusively the product of pathway A; imidazo[1,2-a]pyrimidines. Toluene is such a solvent and is found quite often to be used together with ammonium chloride as catalyst with heating between 80 °C toward reflux temperatures, ruling out formation of any regiois omers getting exclusively the pathway A product (Scheme 4). Interestingly, while attempts were made to find conditions to control the regioselectivity towards pathway B, Stephenson et al. reported an alternative route, applying a base assisted Dimroth rearrangement giving access to the inverse GBB-3CR regioisomer with full conversion as depicted in scheme 22.47 The stability of the regioisomer is the driving force behind this rearrangement, resulting in a preferred single isomer.202

137 The Groebke-Blackburn-Bienaymé reaction

Cl A

NaOH, 80oC HN H O:MeOH 1:4 N N 2 NH Cl N N

HN OH HN O HN N N HN Cl Cl Cl N N N

Cl Cl

N N NH NH N N H

Scheme 22. The Dimroth rearrangement gives access to both regioisomers. A: The rearrangement is performed under basic conditions while heating. B: The proposed mechanism, driven by the stability of the product and properties of the initial starting material, such as aromaticity of the ring, bulkiness of substituents and solvent.

Solvents such as DMSO, DMF and PEG-400 are in general not the 6 solvents of choice because of their high boiling points and difficulties to remove during workup. They prove, however, to be quite useful when solubility issues with highly polar starting materials are present such as the aforementioned amidine containing nucleobases.36, 146

Table 5. All the solvents used in the GBB-3CR. Solvent References Occurence Solvent References Occurence 6, 8, 26-28, 30, 39, 45, 51, 54-56, 61, 66, 68, 74-75, 78, 87, 96-100, 103, 108, 110, 118, 120, 123, 125-126, 165, 180 128, 130, 136, 141-142, 150, 153- MeOH 51 1:1 EtOH:H2O 2 154, 156, 166-168, 177, 181-182, 184-185, 187, 193

19, 60, 71, 79, 81, 83, 85, 90, 92-93, 101, 105, 111-112, 127, 133, 140, 46, 76 No solvent 147-148, 161, 172, 186, 194 23 Water 2

43, 49, 52, 70, 95, 109, 144-145, 33, 36 Toluene 158-159, 163-164, 171, 183, 189-190 17 DMSO 2

138 Chapter 6

57, 84, 89, 104, 113, 115-116, 121-122, 131-132, 134-135, 139, 146, 180 EtOH 160, 191 16 DMF 2

53, 69, 72-73, 77, 117, 152, 155, 42 MeCN 169, 176, 188 9 MeOH:DCE 1

2:3 58, 62, 80, 137-138 67 5 TFE:DCE 1:1 1 MeOH:CH2Cl2 59, 94, 119, 192 162 n-BuOH 4 DCE 1 3:1 20, 39, 41 129 3 1:1 MeOH:CH2Cl2 1 MeOH:CH2Cl2 1:3 MeOH: 31, 86, 143 47 3 1:4 MeOH:CH2Cl2 1 CH2Cl2 Ionic liquid 106, 114, 124 48 3 1:2 MeOH:CH Cl 1 [bmim]Br; DES 2 2 50, 64-65 107

1,4-Dioxane 3 1:1 EtOH:CH2Cl2 1 37-38, 203 170

MEOH/MeCN 3 2:3 Ethanol:H2O 1 63, 195, 204 1:1:1 40 PEG-400 3 1 CHCl3:TMOF:MeOH 32, 180 29

TFE 2 DMSO:H2O 1:1 1 34, 102 88

CH2Cl2 2 Et2O 1

6. Biologically active compounds Quite often the imidazo[1,2-a]-heterocycles that arise from the GBB-3CR are linked to known drugs such as Minodronic acid (40), Saripidem (41) Zolpidem (42) and other members of the so-called Z-drug group of tranquilizers. There are however no reports that GBB-3CR compounds act in a similar way as Z-drugs on the GABAA receptor. The difference between Z-drugs and GBB-3CR compounds is found in the exocyclic secondary amine, originating from the isocyanide component. This amine enables ionic or hydrogen bonding, thus having its own influence on the binding affinity with various protein targets. As a whole it might be better to address real examples of the GBB-3CR scaffold and their associated biological activity. Examples of 3-aminoimidazo[1,2a]pyridines are shown in multiple pharmaceutical drugs or candidates 43-45 (Scheme 23).205 Most notably, the late stage autotaxin inhibitor GLPG-1690 (45) for the treatment of idiopathic pulmonary fibrosis (IPF) which has been discovered and developed whereby the GBB-3CR chemistry played a key role.183, 192

139 The Groebke-Blackburn-Bienaymé reaction

O O N HO P OH N O OH P N HO O N N Cl N N N 40 41 42 Minodronic acid Saripidem Zolpidem F

O NH O 2 Cl N N HO NC N N N N S N N N O N N N N N CN

43 44 45 GLPG-1690 (2014, Rigel Pharmaceuticals) (2005, Array biopharma Inc.) (2013, Galapagos NV) Scheme 23. Examples of imidazo[1,2a]pyridines 1-3 and 3-aminoimidazo[1,2a]pyridines, containing the GBB-3CR scaffolds (blue) reported as bioactive leads;43 anti-inflammatory 44 anticancer, 45 anti-fibrosis.

Only in 2009 the first biological target was addressed and published by using a true GBB scaffold as reported by Fraga et al.181 In their efforts to combine the structural features of (46), a selective p38 MAPK inhibitor, (47) celecoxib a PGHS- 2 inhibitor and (48) also a p38 MAPK inhibitor a GBB-3CR scaffold was used. The main target was to obtain polypharmacological 3-arylamine-imidazo[1,2-a]pyridine derivatives with anti-inflammatory and analgesic MOA (Scheme 24). This approach resulted in compound 49 which was identified as PGHS-2 inhibitor

(IC50 = 18.5 µM) and acts similar to the p38 MAPK inhibitor SB-203580, reverting capsaicin-induced thermal hyperalgesia, a pain model to assess analgesic prop- -1 erties. Compound 50 is a novel PGHS-2 inhibitor, with an ED50 = 22.7 µmol·kg , 10-fold more potent than celecoxib. 6 During a study on the TB targets M. tuberculosis glutamine synthetase (MtGS) inhibitory effect, a hit to lead exercise was performed on 3-amino-imidazo[1,2-a] pyridines as these were identified as MtGS inhibitors after a high-throughput screening.191 Two libraries were synthesized the first via a microwave assisted GBB-3CR sequence for 20-30 min at 160 °C and the second via a post-modification on the pyridine 6-position halogen under Suzuki coupling conditions to study the effect of hydrophobic groups such as aryl moieties 52 (Scheme 25).

Unfortunately, the Suzuki coupling did not lead to an improvement in IC50 value, leaving the aryl halides 51 themselves as potent inhibitors for this target. In a subsequent paper , lead optimization did result in a series of potent compounds, 134 53 was showing a five-fold improvement with an IC50 = 1,6µM. The mode of binding of 53 was discussed according to its co-crystalstructure with MtGS in the structural biology section.

140 Chapter 6

N CF3 H N N N O N Cl F S N N N N 47 F 46 Cl O O 48 F S O H2N

MeO

NH NH N N O S O N N N 49 50 Scheme 24. Synthesis of substituted imidazo[1,2-a]pyridine derivatives as anti-inflammatory and analgesic drugs.

Scheme 25. Synthesis and optimizing of MtGS inhibitors. Three years after their patent application, Djuric et al. published the work of 5-substituted indazoles as kinase inhibitors such as Gsk3β, Rock2, and Egfr,61 by using MCR methodologies such as the GBB-3CR with pyrimidines for imidaz- opyrimidines, thioamides for thiazolyl-indazoles and van Leusen TosMICs for imidazole substituents.

141 The Groebke-Blackburn-Bienaymé reaction

Scheme 26. 5-substituted indazoles as kinase inhibitors. Introduction of the indazole was achieved through the use of indazole-5-carbaldehyde as reactant in different multicomponent reactions and afforded compounds as 54-56. The imidazole[1,2-a]pyrimidine 54, GBB-3CR product, showed the highest inhibition against Gsk3β, imidazopyridine 55 was however, much less potent. Choosing different substituents on the amino group, coming from the isocyanide input (56), selective inhibition for Rock2 could be fine-tuned as well. Al-Tel et al described the synthesis of a series of imidazo[1,2-a]pyridine and imidazo[2,1-b][1,3]benzothiazole carrying quinolone and indole moieties introduced via the aldehyde component. Many of the synthesized compounds showed both antibacterial and antifungal activities. A very potent broad-spectrum antibiotic was identified as compound 57 depicted in scheme 27.138

F S Br N N O NH NH OMe Br H2N N NH N OBn F N N H N BnO MeO 57 58 59

O 6 CO Me 2 N NH NH O N NH N N OMe N N N N H MeO N N 60 61 62

HN N N N N

63 Scheme 27. Various GBB-3CR bioactive compounds. The effect of imidazo[1,2-a]pyridines was studied on colon cancer cell lines by

142 Chapter 6

Koning et al. by showing apoptosis inducing effects in HT-29 and Caco-2 cancer cells.65 Compounds 58 and 59 showed a cytotoxic effect towards the Huh7 cell line at low µM concentrations and interestingly minimal cytotoxicity against white blood cells. Schneider et al. performed a chemical advanced template search (CATS) for target profiling. CATS use topological descriptors generated from known binders and to these descriptors it assigns features such as lipophilic, aromatic, hydrogen-bond donors and hydrogen-bond acceptors to find new chemotypes. This virtual target screening yielded a library in which the starting point was the GBB-3CR. From the results, 3840 virtual products, the top 9 compounds were synthesized for PI3Ka inhibition.78 Four out of nine exhibited activity, with the most active compound 60 showing an IC50 = 131 µM confirming CATS usefulness as a tool to predict targets of virtually generated compounds. In addition, a set of 57 predicted compounds, with highest joint prediction scores for PI3K and DNA topoisomerases, were synthesized using a microwave assisted approach with perchloric acid as catalyst. No human DNA topoisomerase II inhibition was observed. Although no human DNA topoisomerase II inhibition was observed, four new compounds showed inhibitory effects on bacterial DNA gyrase such a compound 61, a bacterial type II topoisomerase. In silico screening was initiated by Brenk et al. to produce a library of novel kinase inhibitors derived from commercially available fragments.151 As a core structure they’ve chosen a ring system with heteroatom containing functional groups. Af- ter applying several selection steps, the number of hits was brought down from over two million compounds to 265 that yielded in 186 compounds being successfully synthesized. 17 of those were prepared using the GBB-3CR, starting from 3-amino-1,2,4-triazole, aldehydes and isocyanides, catalyzed with HClO4 in MeOH at room temperature. One of the obtained compounds 62 showed activity against the kinases EPH-B3 (158µM) and FGF-R1 (469 µM). Similar imidazo[2,1-c][1,2,4]triazoles derivatives were reported to possess anti- microbial and antioxidant activities by El Kaim et al. 168 The reaction conditions were different as compared to the method described by Akbarzadeh. A Sc(OTf)3 catalyzed reaction with substituted benzaldehydes, various isocyanides and substituted 5-amino-1,2,4-triazoles was performed in DMF (80 °C, 30h) to produce a small collection of compounds with yields between 54 and 72%, the best performing compound 63 possesses both antifungal and microbial activity. Another report from 2011 by Al-Tel et al. describes the identification of imidazopyridines derivatized with benzimidazole and/or arylimidazole as potent β-secretase inhibitors.62 In multiple rounds the GBB-3CR compound 64 gave a sub micro molar activity, post modification by removal of the t-butylamine group, a 40-fold increase in activity was achieved. The best compound 65 showed nanomolar potency for the BACE1 enzyme associated with Alzheimer´s disease.

143 The Groebke-Blackburn-Bienaymé reaction

NH N N N N N N 65 HN 64 HN 18 +/- 2 nm 312 +/- 34 nm IC50 IC50 F F Scheme 28. Highly selective β-Secretase inhibitors of the BACE1 enzyme. Baviskar et al. published in 2011 their work on novel topoisomerase IIα inhibitors as anticancer agents. With the aid of molecular docking studies they predicted bicyclic N-fused aminoimidazoles as potential human topoisomerase IIα (hTo- poIIα).63 Based on the previous reported topo II inhibitors in combination with known drugs containing an imidazo[1,2-a] scaffold, a library was synthesized using the GBB-3CR. The result is a GBB-3CR scaffold, by heating the corresponding heterocyclic amidine, aldehyde and isocyanide with a catalytic amount of ZrCl4 at 50 °C in PEG-400 or under MW conditions at 140 °C in n-butanol. In some examples the t-butyl group was cleaved using HBF4 yielding primary amines exhibiting similar activities, multiple compounds 65-70 shown higher potency anticancer activity as compared to 5-fluorouracil and etoposide in kidney cancer cells.

NH NH NH N N N N Cl COOH Cl N N N H 65 66 EtO O HN O 67 HN O NH2 NH2 S N O N H N N Cl NH Merbarone Cl N N N H 6 EtOOC Cl N

68 69 70 Scheme 29. Synthesis, screening and optimizing of hTopoIIα inhibitors. Introduction of C2-biaryl and C6-aryl substitutions on the same imidaz- o[1,2-a]-pyridine/pyrazine scaffold resulted in compound 69 which showed inhibitory effects in the catalytic cycle of hTopoIIα, additional studies suggests that its binds similar as compared to merbarone (a known catalytic inhibitor of hTo- poIIα) overlapping the etoposide binding domain. Callejo et al. reported a new cancer therapeutic approach by inhibition of the kinase B-Raf.149 In a virtual screening they identified imidazo[1,2-a]pyrazines as a binder of B-Raf, in a subsequent synthesis of a small library they found the derivative with an indanone oximesubstituent 71 to be highly potent with an IC50 of 4 nM. Exchange of the furfural moiety lead to compounds 72 and 73 showing improvement on B-Raf with an inhibition <2 and 0,2 nM respectively.

144 Chapter 6

Scheme 30. Imidazo[1,2-a]pyrazine based B-Raf inhibitors and its SAR assisted evolution towards sub-nanomolar inhibitors. Further SAR optimizing led to replacement of the imidazo[1,2-a]pyrazine moiety by thienopyridines 72 with improved inbibition (<2nM) and ultimately with fu- ropyridines 73 bearing superior sub-nanomolar activity (scheme 30). Chatterjee et al. described a screening campaign, using the public malaria database for the discovery of novel chemotypes that could inhibit Plasmodium falciparum, a selection was made of compounds that matched criteria such as good potency (IC50 of <1µM), good safety index, (>20-fold in a six-cell line toxicity pan- el) and easy synthesis. The imidazolopiperazine moiety was found to be an attractive hit, based on the aforementioned criteria.150

F Cl F Cl

NH 74 NH 75 N N F F N N H N N N 2 H H O Scheme 31. Post modifications of GBB-3CR product 74 lead to potent anti-malarial compound 75. The imidazolopiperazines 74 were accessed by performing a GBB-3CR in MeOH at RT catalyzed with HClO4, followed by reduction with PtO2 under hydrogen atmosphere. The most potent compound 75 exhibited an IC50 of 4 and 3 nM on the P. falciparum strains W2 and 3D7 respectively. As the GBB-3CR was discovered by using pyrimidines as a variation of the amine component in the U-4CR, David et al. reported a similar phenomenon that was observed when performing a GBB-3CR with pyridoxal 76 as aldehyde component.72 The expected imidazo[1,2-a]pyrazines and pyridines, from a reaction with various 2-aminopyrazines/pyridines, isocyanides and benzaldehydes were successfully obtained in a microwave assisted GBB-3CR and to act as Toll-like receptor 7/8-active agonists (TLR7/8).

145 The Groebke-Blackburn-Bienaymé reaction

NH CN N N N NH 2 + N 77 CHO OH HO OH HO N 76 HN N pyridoxal NH 78 N O

MeO

NH NH N OH N

N N 80 79

Scheme 32. Discovery of side reactions accompanied with use of pyridoxal as aldehyde component, compound 79 (CCDC-893566) confirmed by X-ray structure analysis. Surprisingly the use of pyridoxal 76 as benzaldehyde component did not yield imidazo[1,2-a]pyrazines/pyridine 77, but the unexpected furo[2,3c]pyridine scaffold 78. Along with this discovery, some analogues of the product exhibited activation of the TLR8-dependent NF-κB signaling. Beyond the scope of this paper, they proposed a mechanism and explored scope and limitation of this reaction. The use of anilines together with pyridoxal resulted in formation of the furo[2,3c] pyridine as well. Replacement of pyridoxal with the very similar salicyl alde- 6 hyde did however yield the usual GBB-3CR scaffold 79. The obtained imidaz- o[1,2-a]pyridin-3-amines were TLR7/8 inactive, but showed bacteriostatic activity against Gram-positive bacteria. SAR studies led to the synthesis of a library of 24 compounds with the emphasis on the size of aryl aldehydes, difference between 2-aminopyridines vs 2-aminopyrazines and aliphatic or aromatic isocyanides.73 The aim was to assess whether modifications were possible without loss of antibacterial activity, resulting in 80 as best compound. Anabasine 81 a natural product was used by Krasavin et al. as a template to prepare hydrazo-Ugi and GBB-3CR derivatives were evaluated as agonists and antagonists of nAChR. Targeting this family of neurotransmission receptors could lead to drugs for the treatment of associated with PD, AD, schizophrenia and depression.176

146 Chapter 6

Scheme 32. Discovery of side reactions accompanied with use of pyridoxal as aldehyde component, compound 79 (CCDC-893566) confirmed by X-ray structure analysis. Surprisingly the use of pyridoxal 76 as benzaldehyde component did not yield imidazo[1,2-a]pyrazines/pyridines 77, but the unexpected furo[2,3c]pyridine scaffold 78. Along with this discovery, some analogues of the product exhibited activation of the TLR8-dependent NF-κB signaling. Beyond the scope of this paper, they proposed a mechanism and explored scope and limitation of this reaction. The use of anilines together with pyridoxal resulted in formation of the furo[2,3c]pyridine as well. Replacement of pyridoxal with the very similar salicyl aldehyde did however yield the usual GBB-3CR scaffold 79. The obtained imidazo[1,2-a]pyridin-3-amines were TLR7/8 inactive, but showed bacteriostatic activity against Gram-positive bacteria. SAR studies led to the synthesis of a library of 24 compounds with the emphasis on the size of aryl aldehydes, difference between 2-aminopyridines vs 2-aminopyrazines and aliphatic or aromatic isocyanides.73 The aim was to assess whether modifications were possible without loss of antibacterial activity, resulting in 80 as best compound. Anabasine 81 a natural product was used by Krasavin et al. as a template to prepare hydrazo-Ugi and GBB-3CR derivatives were evaluated as agonists and antagonists of nAChR. Targeting this family of neurotransmission receptors could lead to drugs for the treatment of associated with PD, AD, schizophrenia and depression.176

147 The Groebke-Blackburn-Bienaymé reaction

Scheme 33. Anabasine derived GBB-3CR products as binders of nAChR. The five GBB-3CR products were synthesized in low to moderate yields 20-53%, employing various isocyanides, modified anabasine 2-aminopyridine and substituted benzaldehydes in MeCN, 70 °C using an stoichiometric amount of TMSCl as Lewis acid promoter (Scheme 33). The use of a alkynylbenzaldehydes 83 in the GBB-3CR allows for post modifications such as metal catalyzed intramolecular cyclization yielding a library of compounds, some with strong cytotoxicity against the HeLa cell line, due to binding affinity with the G-quadruplex DNA motif, thus exhibiting anti-prolif- erative activity as described by Shen et al.206 The cyclization to the tricyclic compounds, such as 84, was performed in a domino one pot approach of the Yb(OTf)3 catalyzed GBB-3CR between 2-aminopyridines, isocyanides and 2-alkynylbenz- aldehyde followed by a AgOTf catalyzed intramolecular cyclization (scheme 34).

NH NC NH N NH N 2 AgOTf N + N o Yb(OTf)3 55 C, 1h - N OHC OTf

84 83 6

Scheme 34. In total a library of 23 compounds was synthesized, in which 84 showed the best HeLa cell potency (IC50 = 0,58 µM). Proschak et al. reports hybrid imidazo[1,2-a]pyridine and an urea moiety as dual soluble epoxide hydrolase (sHE)/ 5-lipoxygenase (5-LOX) inhibitors.128 An eight step approach was used to introduce both the urea and imidazo[1,2-a]pyridine elements. In total 22 a library compounds were synthesized in an AcOH catalyzed GBB-3CR approach in MeOH in yields varying between 7-64% and showing good inhibition potential on recombinant enzyme on both targets. The best 5-LOX inhibitor 85 was not the best sHE inhibitor (86), and it is to be evaluated whether equipotent inhibition is favored over higher inhibition of one of the two targets.

148 Chapter 6

NH OMe NH Cl NH N O O N H 85 CF3

NH OMe NH NH N O O N H 86 OCF3

Scheme 35. Combined urea and GBB-3CR products synthesized in an 8-step sequence. In 2011 Al-Tell et al. described the application of various subsequent MCR’s.58 The carboxylic acid group attached to one of the three typical GBB-3CR starting materials would not participate in this MCR reaction, but is useful for follow up MCR chemistry via for example the U-4CR to synthesize antibiotics.80 Their 6 component reaction yielded 40 polyfunctionalized imidazopyridine, pyrimidine and pyrazine derivatives and were screened against multiple bacterial strains such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and screened against two human cancer cell lines; breast carcinoma (MCF7), A549 (lung cancer cell line) and melanoma (M8).

149 The Groebke-Blackburn-Bienaymé reaction

GBB-3CR U-4CR

O H N N 2 NH + H 87 Sc(OTf)3 N N NH2 + O MeOH/DCM, N N COOH rt, 45 min F C COOH 3 +

MeOOC NC NC

88 89 87 NH HN O NH N Ph Ph N N N NH N O N Bn N O N N O O HN O N Ph HN COOMe O O

F3C

NH 90 91 NH 92 N HN O N Ph S N N N O N NH N O O O O N HN HN COOMe N

O O Scheme 36. Application of a subsequent MCR approach to conveniently obtain complex compounds, exhibiting both antibacterial and anticancer properties.

Five of the synthesized compounds 87-91 were found to exhibit lower IC50 values 6 compared with the internal standard doxorubicin, against A549, M8 and MCF7 cell lines. Moreover, compound 92 exhibits a high potency against breast cancer. Vendrell et al. reported the synthesis of functionalized BODIPY probes to introduce fluorescence in otherwise difficult accessible structures.82 While BODIPY dyes were previously derivatized via BODIPY containing an amine or carboxylic acid substituent respectively, this paper focuses on the preparation of isocyanide functionalized BODIPY (93). The application of this dye was found through isocyanide-based MCR chemistry for the aid of in vivo imaging of phagocytic macro- phages. Different IMCR compounds were synthetized as the GBB-3R variant 94, and incubated with A549 cells and although all the adducts readily entered cells at concentrations in the nanomolar range, the Ugi adduct 95 exhibited the best coloured images (scheme 37).

150 Chapter 6

NC HN O H2N N F - N+ O F B H N Cl

+ N - N + B NH N - N B F F N Cl F F N 93 94 95

Scheme 37. BODIPY isocyanide 93 used as fluorescent tag. The GBB-3CR product 94 exhibits fluorescence in A549 cells depicted as image 4 on the right. The U-3CR products 95( ) image 7 was found to produce the best colored images. Reprinted with permission from reference 82. Copyright (2013) American Chemical Society. Glyoxylic acid based GBB was employed by Li el al. to obtain a series of mono-substituted imidazopyridines that target the RET kinase class, a known oncogene.207 In their first series, 100 MCR fragments were synthesized by a acetic acid catalyzed GBB-3CR using substituted 2 aminopyridines, isocyanides and glyoxylic acid. Screening resulted in a hit 96 which was subjected to computational binding analysis in the RET kinase. The phenyl moiety accesses a lyophilic pocket that could be extended from the allosteric pocket. Another non-MCR series of compounds were synthesized to engage this adjacent lipophilic pocket, resulting in an increase of inhibitory potency as shown by two compounds (97 = 0.21 µM and 98 = 0.75 µM) thus identified as novel RET kinase inhibitors (Scheme 38).

97 96 H N HN N O N O N N N

IC50 = 2 mM IC50 = 0.21 µM

N N 98 N O

IC50 = 0.75 µM NH O N O

Scheme 38. Initial GBB-3CR hit design towards novel non-MCR RET inhibitors.

151 The Groebke-Blackburn-Bienaymé reaction

Imidazo[1,2-a]pyridine-N-glycinylhydrazone derivatives were applied as inhibitors of tumor necrosis factor alpha (TNF-α) by Fraga et al.84 The design of the scaffold was performed by combining two earlier described TNF-α inhibitors wherein the N-phenyl-pyrazole nucleus 99 was replaced with the heterocyclic imidazo[1,2-a]pyridine isostere, easily accessible by the GBB-3CR.

O HN N

NH O N N

N H N H Cl N N O NH S O N N N 100 F 101 Scheme 39. Imidazo[1,2-a]pyridine-N-glycinylhydrazone derivatives as TNF-α inhibitors Via an acetic acid catalyzed reaction of benzaldehyde or pivaldehyde, 2-aminopyridine and ethyl 2-isocyanoacetate two GBB-3CR products were obtained with yields of 65 and 75% respectively. Post modification with hydrazine and subsequent hydrazone formation yielded 11 products that were screened in a TNF-α production assay, ultimately leading to the most active 100, equipotent with SB- 203580 (101) a known p38 MAPK inhibitor herein applied as anti TNF-α agent. Inhibition of the BET bromodomain is a promising approach as anti-cancer ther- apy. In a fragment based optimizing strategy, Bradner et al. used the dimethylisoxazole biasing element as a key fragment and screened on BRD4 and BRD4-de- 6 pendent lines, using different heterocyclic substituents to gather information on preferred regiochemistry.86 Bromo substituted benzaldehydes were employed in the MW assisted, Sc(OTf)3 catalyzed GBB-3CR to perform a follow-up Suzuki coupling in order to introduce the 3,5-dimethylisoxazole warhead. The best compound is built around the para-substituted phenyl-imidazo[1,2-a]pyrazine scaffold and a small library of 29 compounds was synthesized for SAR optimizing, resulting in lead compound 102.

NH N N N N O 102 values Found Kd BRD4 = 550 nM TAF1 = <1 µM Scheme 40. GBB-3CR compound 102 as potent BET bromodomain inhibitor. .

152 Chapter 6

In continuation of Shen et al.206 another HeLa cytotoxic series of 6H-pyri- do[2’,1’:2,3]imidazo[4,5-c]isoquinolin-5(6H)-ones was reported by the same research group87 In this report the tetracyclic profile was maintained, however the alkynyl moiety was replaced with a carboxylic acid (phthaldehydic acid) which direct reacts with the secondary amine, formed in the GBB-3CR (scheme 41).

OMe CHO OMe

COOH N NH2 Sc(TfO)3 = 13 nM 103 + O HeLa IC50 MeOH NHHO o N 55 C, 12h F N CN N N OMe N

104 105

Scheme 41. Application of the bi-functional phthaldehydic acid 103 giving access to tetracyclic heterocycles with example 104 exhibiting HeLa toxicity (IC50 = 1.8 µM). Shen et al. published, more work on cytotoxic compounds while abandoning the tetracyclic scaffold, switching to a bicyclic scaffold exhibiting intramolecular hydrogen bonding to form pseudo-tetracycles.177 A more straightforward methodology, as it only comprises a single GBB-3CR step. The library was step by step optimized with variations of all three components. The hydrogen bonding ability was most successful when an ortho substituted hydroxyl- or methoxybenzaldehyde component was used. A 100-fold activity increase over compound 104 was realized with compound 105. Foroumadi et al. reported novel anticancer agents based on N-fused aminoimidazole derivatives with triazine analogs.158 The basis of this compound were a combination of potent topoisomerase Iiα inhibitors63 and triazines208 to yield the N-cyclohexyl-3,4-diphenylimidazo[2,1-c][1,2,4]triazin-6-amine scaffold 109 (scheme 42). The aminotriazine 108 was prepared from condensation of benzil 106 with aminoguanidine 107. Noteworthy are the conditions that were found whilst optimizing the GBB model reaction. The reaction with toluene under reflux conditions gave the highest yield of 70% in the presence of 1 equivalent ammonium chloride as compared to 45% without.

O NH HN + ArCHO, CyNC NH2 N N H2N N Ar O H N N N NH2 N N 106 107 108 109

HN HN HN HN N N OMe N N N N N N N N N N N OH OH N N N N O 110 111 112 113 Scheme 42. A series of anticancer agents, through the clever condensation of benzyl 106 and aminoguanidine 107 and subsequent GBB-3CR.

153 The Groebke-Blackburn-Bienaymé reaction

A small library of 20 compounds was prepared and their cytotoxicity was assessed on HL60, MCF-7 and MOL-4 by the MTT reduction assay against cisplatin and doxorubicin. Results: The highest potency of compounds 110 and 111 was observed on MCF-7 and HL60 cells respectively. SAR optimizing showed that replacing the 7-aryl moiety with pyridyl or furan-2-yl (compounds 112 and 113) gave improvement of the potency against MOLT-4. In a substrate activity screening, Veken et al. decorated an imidazo-[1,2-a]pyridine scaffold with a library of fragments that bind to the urokinase plasminogen activator (uPA) S1 pocket.96 This target is a biomarker and therapeutic target for cancer types such as breast, ovarian and pancreatic cancer. In two rounds the imidazopyridine scaffold was optimized. The first round focused on mono-substitution with fragments on the 3 position of the imidazopyridine scaffold using a GBB-3CR to identify fragments that bind to the S1 pocket. In a second round structural optimization was performed by introducing substituents through the aminopyridine and aldehyde component. This resulted from an IC50 (uPA) value (114) of 9.04 µM towards 0.097 µM (115) improvement between the first and second round respectively (Scheme 43).

HN HN N N NH H NH N N N N N H NH H NH2 O 2 114 115

Scheme 43. Binders of the urokinase Plasminogen Activator (uPA) S1 pocket, round 1 and 2 top performing products. The imidazo[1,2-a]pyrazine scaffold is found in Heat shock protein 90 (Hsp90) inhibitor PU-H71, using the Zolpidem rationale that absence of the amine moiety in the 3 position wouldn’t affect the inhibitory effects, Shen et al. synthesized a -li brary of substituted 3,8-diaminoimidazo[1,2-a]pyrazines to target this protein.153 The biggest challenge in these compounds was the incorporation of the adenine 6 motif with an amine in the 8 position, which will in unprotected form participate in the GBB-3CR. Introduction of a chlorine at position 8 followed by aminolysis as well as protected amines were tried by the authors. The resulting lead compound 116 exhibits moderate Hsp90 inhibition as compared to PU-H71 (117), however, the co-crystallization of the reported 3,8-diaminoimidazo[1,2-a]pyrazines have shown to bind in the same way as the known inhibitor (Scheme 44).

154 Chapter 6

Scheme 44. Development of novel imidazo[1,2-a]pyrazine based Hsp90 inhibitors. Coelenterazione 118 is a bioluminescent imidazopyrazine and can be found in several marine organisms. The properties such as bioluminescent and antioxidant are useful and utilized as optical signaling agents for cancer behavior studies. Vuocolo et al. reports the synthesis of 3 deoxy- derivatives of coelenterazine to improve some of its properties, such as its instability.154 The 3 deoxy approach brings room to apply the GBB-3CR with substituents on the C-3 position.

HO HO O HO NH N N N HN N N N N N

OH OH OH 118 119 120 Scheme 45. 3 deoxy- derivatives of coelenterazine as bioluminescent and antioxidant In this report only t-butylisocyanide was used in an acetic acid catalyzed GBB- 3CR under dry conditions (3Å molecular sieves) yielding compound 119 in 64% and in follow up reactions a deprotection and reductive deamination (NaNO2/ H2SO4) routine was used to obtain compound 120. Liu et al. describes a library of imidazopyridine and quinoxaline derived compounds and introduces ferrocene incorporation via the aldehyde or isocyanide component through the GBB-3CR, with the aim of developing new antioxidants to reduce oxidative stress/ DNA damage due to reactive oxygen species.104

155 The Groebke-Blackburn-Bienaymé reaction

Fe Fe

NH NH N N N Fe N N Fe 121 122

HN HN N N N N N Fe N Fe

123 124 OMe O O

Scheme 46. Ferrocene based anti-oxidants.

The LaCl3.7H2O has proven to be an excellent catalyst with yields >84% at a reaction temperature of 60 °C in EtOH for 2h. Compounds 121 and 122, the ferrocene bearing compounds, were the best in inhibiting DNA oxidation. More improvement was made by derivatization of the imidazo[1,2-a]quinoxaline scaffold.155 Decoration with ferrocenyl and p-methoxyphenyl (123) or flavonyl (124) groups on either the 2 or 4 position boosted the radical suppression/inhibition of DNA oxidation. A hybrid structure between imidazo[1,2-a]pyridine and coumarins were synthesized by Shah et al. as potential Hepatitis C virus replicator inhibitor.110 The 20 compounds were synthesized using a AcOH (20 mol-%) catalyzed GBB-3CR in MeOH at 70 °C for 2h in yields between 67-92%. Molecular docking studies indi- 6 cate good binding interactions of compound 125 with nonstructural protein 5B.

156 Chapter 6

Scheme 47. Coumarine based GBB-3CR products, compound 128 was crystalized and the structure was unambiguously verified by X-ray analysis (CCDC-1053723).

Trivedi et al. combined the structural properties of anti-cancer agents that commonly have a coumarin structure and anti-osteoporotic agents holding pyridine or imidazole heterocycles into coumarin–imidazo[1,2-a]pyridine hybrids for the treatment of cancer induced osteoporosis.113 18 GBB-3CR products were obtainevd using ethanol reflux conditions and 20 mol-% AgOTf with excellent yields (71-88%) including compound 126. Compound 127 was found to possess anti-breast cancer activity (MDA-MB-231 IC50 = 14.12 +/- 3.69 µM) as well as disrupting the differentiation of osteoblast and osteoclast cells and therefore indicating the compound as promising hybrid for both aforementioned diseases.

The first-in-class autotaxin (ATX) inhibitor GLPG1690 was described by Heckmann et al. and was discovered and optimized through the synthesis of a 2,3,6 trisubstituted imidazo[1,2a]pyridine series, identified by HTS.183, 192 Approximately 100.000 compounds were subjected to screening in which ATX activity is promoting release of the fluorophore FS-3, an HTS suitable substitute of LPC, thus easily identifying inhibitors by a fluorescence detection assay. The N-benzyl-2-ethyl-N-methyl-imidazo[1,2-a]pyridin-3-amine hit series 128 displayed a IC50 around 30 nM in the FS-3 assay (Scheme 48).

157 The Groebke-Blackburn-Bienaymé reaction

Scheme 48. Autotaxin inhibitors; From hit to lead.

The metabolic stability of this series was however poor and a second series with improved pharmacokinetic properties was developed, resulting in an early lead compound 129 (IC50 = 122 nM (FS-3 assay). The reduced potency gave reason to continue SAR analysis and synthesis. Furthermore the ATX activity could not be reproduced with biochemical assays; using the natural substrate LPC displayed only micromolar activity. Extensive optimizing on all substituents, assisted by analysis of the binding mode using compound 129, resulted in lead compound 130 with a potency of 27 nM in the biochemical assay and 22 nM in a rat plasma assay. The improved potency was achieved through the 6 position by extending the substituent and the addition of a basic amine and an 6 H-bond donor or acceptor. The addition of a nitrile group on the thiazole moiety pushes out a high energy water by full occupation by the ligand, increasing the potency even more. In a second report Heckmann et al. describes improvement of lead compound 130, focusing on ADMET (hERG and CYP3A4) and PK properties (clearance and bioavailability), ultimately resulting in the clinical candidate GLPG1690 (131) (IC50 = 131 nm). The five-fold loss in activity of 131 was outweighed by the absence of CYP3A4 TDI and the compound is overall the best combination of activity, pharmacokinetic, ADMET and PK properties. The ATX inhibitor was further discussed in the structural biology section.

7. Structural Biology

Farnesoid X receptors (FXRs) are nuclear hormone receptors expressed in high concentration in tissues that participate in bilirubin metabolism including the kidneys, intestines, and liver. In animal models of nonalcoholic fatty liver disease (NAFLD), FXR activation protects against fatty liver injury and nonalcoholic steatohepatitis (NASH), and improved hyperlipidemia, glucose

158 Chapter 6 intolerance, and insulin sensitivity. Thus, FXR agonists could potentially be used as drugs to treat nonalcoholic fatty and cholestatic liver diseases. A GBB product 132 was described in a cocrystal structure bound to FXR (Figure 4 A).209 Remarkably, 132 fits tightly in the pocket undergoing a range of hydrogen, pi- stacking and van der Waals interactions (Figure 4. C-E).209 The 2,6-dimethyl phenyl moiety (derived from the isocyanide) ensures an out-of-plane orientation of the two phenyl substituents with regard to the imidazopyridine bicycle through sterically repulsion which can help to increase binding affinity through preorganisation of the binding conformation (Figure 4.B).

H4 A B C F

D E

F3 W4

Figure 4. GBB product 132 bound to farnesoid X receptor (FXR, PDB ID 5Q12). A) 2D structure of 132; B) scorpion score (14.0) and its heavy atom distribution on 132 (color code: red strong to purple weak contribution, grey minor contribution); C) the imidazole-N of 132 acts as a hydrogen bond acceptor towards the His451 (2.8 Å); D) 132 forms two pi-stacking interactions between Phe333 and the 2,6-difluorophenyl moiety and the Trp458 and the 2-methyl pyridine moiety; E) multiple hydrophobic interactions can be found between all parts of 132 and the surrounding hydrophobic receptor amino acids; specifically to mention is AF (red in B) which makes multiple contacts with His298, Met332 and Ala295.

The protein bromodomain (BD) serves the recognition of acetylated lysine as in histones and is composed of approximately 110 amino acids. In the context of signal transduction, the bromodomain function as a “reader” of the lysine acetylation. For a family of bromodomains, the BET (bromodomain and extra terminal domain) family, several compounds are in clinical evaluation. Members of the BET family are targets in both human cancer and multiple sclerosis. The bromodomain has also been investigated as antifungal target. Screening a library of 80,000 chemically diverse compounds by a HTRF inhibition assay revealed GBB 133.210 Compound 133 inhibits the CaBdf1 BD2 with high selectivity: HTRF and∼ ITC assays yielded IC50 and Kd values of 1.1 and 2.1 μM, respectively, whereas human Brd4 BD2 and CaBdf1 BD1 were only weakly inhibited (IC50≥40 μM). Moreover, no significant inhibition of any human BDs was observed for compound

159 The Groebke-Blackburn-Bienaymé reaction

133 by BROMOscan profiling and by an ITC assay on the two most sensitive BDs identified by this screen. Only mild cytotoxicity was observed towards mammalian cells (EC50 60 μM) for compound 133. A cocrystal structure of 133 in Candida albicans Bdf1 reveals shape complementarity to the receptor and a network of hydrogen bonding,∼ pi-stacking, van der Waals interactions (Figure 5).

A B C

D E F G Y42 5 N46 8 P46 7 N46 8 P41 V46 0 Figure 5. Second0 Bromodomain (BD2) from Candida albicans Bdf1 binding to an GBB imidazopyridine (PDB 5N18). A) 2D structure of 133; B) scorpion score (5.6) and its heavy atom distribution on 133 (color code: red strong to purple weak contribution, grey minor contribution); ; C) overall GBB receptor binding site featuring multiple electrostatic interactions and a string of four signature structural waters (green, yellow and red balls) ; D) two water mediated hydrogen bondings including Pro410 carbonyl-H2O-N imidazole and Val460 carbonyl-H2O-p-hydroxyphenyl and Asn468 side chain amide-CO and exocyclic NH of isocyanide moiety; E) T-shaped pi-pi interactions between Phe467 and Tyr425 and the phenyl groups of the isocyanide and aldehyde components; F) multiple van der Waals 6 interactions involving Phe467, Val415, Ph411, Val474 and Leu420; G) a H-bond donor- pi interaction involving the side chain amide NH of Asn468 and the aldehyde phenyl.

Autotaxin (ATX) is a secreted lysophophospholipase D that converts lysophosphatidyl choline (LPC) into the bioactive phospholipid derivative lysophosphatidic acid (LPA). LPA exerts its biological activities through activation of the LPA receptors: LPA1−6. The ATX/LPA axis is involved in numerous physiological and pathophysiological processes. Autotaxin inhibitors have been proposed to be useful in various pathologies such as cancer, pain, and cholestatic pruritus, as well as fibrotic, inflammatory, and cardiovascular diseases. Appropriate structural modifications of the initial GBB hit lead to 134 with improved ADMET (hERG and CYP3A4 TDI) and PK properties (bioavailability and clearance).183 Compound 134 was able to decrease LPA levels in mouse plasma. Additionally, compound 134 demonstrated significant activity in a mouse fibrosis model at doses of 10 and 30 mg/kg twice a day, with an efficacy comparable or superior to that of the reference compound pirfenidone. Compound 134 is currently being evaluated in an exploratory phase 2a study in idiopathic

160 Chapter 6 pulmonary fibrosis patients. Compound134 was also cocrystallized with its target to better understand the molecular basis of its disease modifying activity (Figure 6). A B C

A B C

D F13 E

8 L4 D E F 8 F21 1

W26 S5 D9 1 2 3 F27 F27 H25 Figure 7. GBB compound 135 binding to HSP-90 (PDB ID 4R3M). A) 2D structure of 135; B) scorpion score (14.8) and its heavy atom distribution on (color code: red strong to 5 4 2 135 purple weak contribution, grey minor contribution); C) overall receptor ligand interaction Figure 6. GBB Imidazo[1,2-a]pyridine derivatives with potent autotaxin/ENPP2 involving multiple van der Waals, stacking and hydrogen bonding interactions; D) a parallel inhibitor activity (PDB ID 5MHP). A) 2D structure of 134; B) scorpion score (10.6) and pi-pi stacking interaction between Phe138 and the moiety 1,2- methylenedioxybenzene its heavy atom distribution on 134 (color code: red strong to purple weak contribution, moiety is a strong affinity contributor; E) the adenine-NH2 forms a direct hydrogen bond grey minor contribution); C) overall topology of the interaction of 134 with its receptor towards Asp93 carboxylate and a tripartite hydrogen bond Leu48 backbone carbonyl and featuring multiple hydrophobic, hydrogen bonding and stacking interactions; D) Ser52-OH mediated by two structural water molecules accounting for the adenine-NH2 x is embedded in a aromatic cage including Phe’s211, 275, 274, Trp261 and His252; importance for binding. Phe274 carbonyl forms a pi-pi interaction with the thiazole sulfur; E) two H bond donor pi interactions involve His252 and a structural water molecule; F) a water Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and mediated hydrogen bond involves the azetidine carbonyl and the NH of Trp261. ammonia under hydrolysis of adenosine 5’-triphosphate (ATP). The enzyme is involved in nitrogen metabolism and cell wall biosynthesis in pathogenic Heat shock protein 90 (HSP-90) is a so-called chaperone protein that assists mycobacteria and thus comprise a potential anti tuberculosis target. ATP- proteins to fold properly, stabilizes proteins against heat stress, and aids competitive MtGS inhibitors were identified in a high-throughput screening in protein degradation. It has shown to stabilize proteins during cancer (HTS). One hit compound was a GBB 3-amino-imidazo[1,2-a]pyridines. This development. The natural product geldanamycin and derivatives and totally was an ideal candidate class for structure–activity relationship (SAR) studies, synthetic compounds show promising antitumor activity in clinical trials and because diverse analogs can easily be obtained in one step via GBB-3CR.134 The therefore HSP-90 is a validated cancer target. The GBB compound 135 was best compound showed an IC50 of 0.6 µM. Co-crystallization of one of the best synthesized based on the observation of ATP-competitive Hsp90 inhibitors inhibitors with MtGS 136, helped to rationalize the observed SAR (Figure 8). containing the 8,9-disubstituted adenine motif, which might be replaced with the 2,3-disubstituted 8-aminoimidazo[1,2-a]pyrazine scaffold (GBB).153 Additionally, computational scaffold hopping exercises suggested that a similarly substituted 8-aminoimidazo[1,2-a]pyrazine might possess Hsp90 inhibitory activity due to the remarkable resemblance to PU-H71, an Hsp90 inhibitor in Phase I clinical trial. The hypothesis proved to be trough and multiple GBB compounds with this adenosine mimicking patterns showed competitive binding to HSP-90. 135 has been cocrystallized with HSP-90 (Figure 7) and shows multiple contacts to the ATP binding site including hydrogen bonding, pi-stacking of the piperonyl moiety and van der Waals interactions.

161 The Groebke-Blackburn-Bienaymé reaction

A B C

D F13 E

8 L4 8

S5 D9

2 3 Figure 7. GBB compound 135 binding to HSP-90 (PDB ID 4R3M). A) 2D structure of 135; B) scorpion score (14.8) and its heavy atom distribution on 135 (color code: red strong to purple weak contribution, grey minor contribution); C) overall receptor ligand interaction involving multiple van der Waals, stacking and hydrogen bonding interactions; D) a parallel pi-pi stacking interaction between Phe138 and the moiety 1,2- methylenedioxybenzene moiety is a strong affinity contributor; E) the adenine-NH2 forms a direct hydrogen bond towards Asp93 carboxylate and a tripartite hydrogen bond Leu48 backbone carbonyl and

Ser52-OH mediated by two structural water molecules accounting for the adenine-NH2 importance for binding.

Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and ammonia under hydrolysis of adenosine 5’-triphosphate (ATP). The enzyme is involved in nitrogen metabolism and cell wall biosynthesis in pathogenic mycobacteria and thus comprise a potential anti tuberculosis target. ATP- 6 competitive MtGS inhibitors were identified in a high-throughput screening (HTS). One hit compound was a GBB 3-amino-imidazo[1,2-a]pyridines. This was an ideal candidate class for structure–activity relationship (SAR) studies, because diverse analogs can easily be obtained in one step via GBB-3CR.134 The best compound showed an IC50 of 0.6 µM. Co-crystallization of one of the best inhibitors with MtGS 136, helped to rationalize the observed SAR (Figure 8).

162 Chapter 6

W28 A B C D 2 R36 H27 Y12 4 8 9 F23 2 W28

2L36 1 S28 E F G R36 H 4 0 F23 2 W28 2 L36 L36 1 1 Figure 8. Crystal structure of Mycobacterium tuberculosis glutamine synthetase in complex with GBB 136 (PDB ID 4ACF). A) 2D structure of X; B) scorpion score (10.3) and its heavy atom distribution on 136 (color code: red strong to purple weak contribution, grey minor contribution); C) 136 is embedded in a hydrophobic site involving Arg364, Phe232, Lys361, Trp282 and Tyr129; D) a halogen bonding between Hi278 backbone carbonyl and the Br is identified; E) a parallel pi-pi stacking interaction between Phe231 and the pyridine central part of GBB backbone is formed: F) a ionic interaction is spotted between the ligands carboxy group and the Lys361; G) a cation-pi interaction is formed between Arg364 and the pyridine part of the GBB backbone; H) the aminobutyl side chain- NH is involved in a direct hydrogen bonding with Lys361 carbonyl and a tripartite water mediated network with Trp282 indole NH and Ser280-OH.

8. Patents The first appearance of the GBB-3CR in patents took roughly 3 years when a nitric oxide synthase inhibitor was reported by Gruenenthal GmbH.211 About 100 patents were filed in the course of 18 years, in which some had the imidazo[1,2-a]pyridine/ pyrazine as main decorated scaffold obtained using the GBB methodology, while others reported the scaffold either by sequential synthesis and as one of many different claimed structures. The GBB-3CR was found in the pharmaceutical industry and shortly after its discovery patents concerning the [1,2a]imidazo scaffold appeared in patent applications, either directly or indirectly as seen in the 2004 Patent from Merck & Co. Inc.212 where the double bonds in the originating pyrazine were reduced to obtain a piperazine. The drug was with various structural modifications brought to the market as Sitagliptin, also known under the tradename Januvia. As many patents were reported in the last decades, not many true GBB-3CR scaffolds were marketed as actual drugs. Recently compound KAF156in development by Novartis AG, completed phase 2 clinical trials (NCT01753323), being effective against thePlasmodium falciparum and Plasmodium vivax forms of the malaria parasite with clearance of the parasites within 2 days.213- 214 More examples of patents that leverage the GBB scaffold were listed in table 6.

163 The Groebke-Blackburn-Bienaymé reaction

Table 6. The structures found in the patent were found as GBB products, whereas the listed structures comprise the in general most active compound, shown as the active component of interest. Assignee Year Structure Target/Condition Class Patent number Gruenenthal 2002 Ac CNS, Ischemia, 211 + WO2002030428 GmbH N Brain disease, N Shock, Septicemia

Ac - Cl

Merck & Co., Inc 2004 F Diabetes Melitus DPP-4 WO 2004058266212 NH2 O type II F N N COOEt N

Esbatech A.-G 2005 RTK Kinases 215 R4 WO2005120513A1 N N R N 4 R5 R R1 2

Array 2006 RAF Kinases WO2006125101A2216 R2 BioPharma Inc. N N R N 1 R3 X R4 R6

217 Britol-Myers 2006 X N R GLPP-1(7-36) Peptidase WO2006071752A1 Squib company Diabetes II A N NH2 X n Y

Merck Patent 2007 O SGLT I, II (Diabetes WO2007147478A1218 G.m.b.H CH2 Y I, II) n ' X N R4 R " 1 N R2 R4 R ' 1 N ' " R2 R1 R NH R2

R3 R' ' R3

Astex 2008 H FGFR3, PDGFR Kinases WO 2008078091219 Therapeutics N Limited, UK O 6

N N

Merck Patent 2008 SGLT1, Diabetes Transport DE 102006048728220 N GmbH, Germany S proteins

N N NH

S bio Pte. Ltd., 2008 MeO HDAC Epigenetics US 20080085896221

Singapore MeO NH H O OH N N MeO H H Ph N

O N 222 Array 2008 MeO PDGFR Class 3 and 5 WO 2008121687 BioPharma Inc., N Kinases USA NH N

164 Chapter 6

Cytokinetics, 2008 SKM Myofibril Skeletal WO2008016648A2223 Incorporated, HN AC1.4 muscle myosin USA modulators N OH N

ACADIA 2008 O CB2 receptors Cannabinoid WO2008141239A1224 Pharmaceuticals CB2 receptors N Inc., USA O

NH F N N Cl

Schering 2008 N JNK1, ERK Kinases WO2008082490A2225 H2N H Corporation, S N O USA N N S

NH O

Merck Sharp & 2008 GABAA GABAA GB2448808A226 Dohme Ltd., UK HN N N

227 Fuji Film RI 2009 I amyloid β (1-40) Alzheimer’s JP2009007348A Pharma Co., N N disease, Down’s Ltd., Japan; syndrome, N O Daiichi Sankyo Co., Ltd. Universidade 2007 N MAPK p38 and Anti- BR 2007011519 Federal do Rio PGHS-2 inflammatory 228 N de Janeiro - N and analgesic UFRJ, Brazil NH activity

Bristol-Myers 2009 O CK2 Protein kinase, WO2009100375229 Squibb N cell proliferation Company, USA H HN

N NH2 N N N N H N H O

230 Merck Patent 2009 N SGLT1 Sodium- WO2009049731A1 GmbH, Germany dependent N N NH glucose O 2 NH transporter O Et

Oncalis A.-G., 2010 N Ser,Thr,Tyr kinases; Protein kinase WO2010083617A1231 Switz. OH c-src, EphB2/4 HER- inhibitors 1, PDGF, RET

NH N N N O

University of the 2010 MeO Caco-2, HT-29 Anticancer WO2010116302A1232 Witwatersrand, N (Colon cancer) activity Johannesburg, S. Afr N Br NH OMe

165 The Groebke-Blackburn-Bienaymé reaction

Ortho-McNeil- 2010 O OMe (AD) Gamma Gamma WO2010089292A1233 Janssen N secretase complex secretase Pharmaceuticals, inhibitors Inc, USA NH N N

Shanghai 2010 MeO HeLa Mitosis CN101717397A234 Institute of inhibitors Materia Medica, Chinese O Academy of Sciences, Peop. N Rep. China N N

IRM LLC 2011 O Antimalarial WO2011006143A2213 (Novartis), H2N activity N N Bermuda N F HN F

EQUISnZAROO 2011 Cl H1N1, H3N2 Influenza virus KR2011097448A235 Co., Ltd., S. N Korea OH N NH Cl

Centro 2011 O PI3 Kinase WO2011036461A1236 Nacional de inhibitory Investigaciones N activity Oncologicas N (CNIO), Spain HO N N OEt O

Schering 2012 Edwarsiella spp Microbial WO2012018662A2237 Corporation, N infections USA; Merck Cl Sharp & Dohme N N Corp N HN N Cl N

Supergen, Inc., 2012 N Pim-1 Kinases US20120058997A1238 USA N N N 6 H N

OCF3

GlaxoSmithKline 2013 HCV Innate immune WO2013059559A2239 CF3 LLC, USA responses N N F3C N N N O

National 2011 Topoisomerase IIα Anticancer IN2011DE00091A240 R1 Institute of NH agents Pharmaceutical Education N and Research R2 (Niper), India N

F. Hoffmann- 2014 h-TRPA1 Respiratory WO2014076021A1241 La Roche disorders AG, Switz.; NH Cl Hoffmann-La Br Roche Inc N N

166 Chapter 6

UCB Pharma 2014 N TNFα TNFα WO2014009295A1242 S.A., Belg. N Cl HN N Cl

Galapagos NV, 2014 F Human LPC Prophylaxis WO2014202458A1243 Belg. hENPP2 N O N N N CN N N S N Et N

Kyowa Hakko 2014 CaV3.2 T-type Ca2+ Pruritus WO2014021383A1244 R3 W1 N Kirin Co., Ltd., channel Japan R1 W2 N

Q R2

Bristol-Myers 2014 NH CKIε, CKIδ Kinase I WO2014100533A1245 Squibb Company, USA N O N N N N NH N

Heesung 2015 Non medical Organic light- KR2015075169A246 Material Ltd., S. emitting device Korea materials

N N N

HDI Foundation, 2016 Imaging Huntington WO2016033440A1247 Inc., USA protein L2 R N 1 R 3 m L1 N

Bristol-Myers 2016 TNFα WO2016149439A1248 Squibb MeO N Company, USA N N N

249 Janssen 2016 NH2 pAkt-S473, pAkt_ PI3Kβ kinase WO2016097347A1 Pharmaceutica N OH Thr308 inhibitors NV, Belg. N N N O

CF3

Janssen 2016 O PI3Kα, PI3Kδ, PI3Kβ kinase WO2016097359A1250 Pharmaceutica N S O PI3Kγ and MTOR inhibitors NV, Belg. HN

N N N N N O

CF3

167 The Groebke-Blackburn-Bienaymé reaction

NEOMED 2017 N Bromodomain WO2017066876A1251 Institute, Can. inhibitors N N Ph O

252 University of the 2017 R4 Anticancer WO2017178992A1 Witwatersrand, R2 NH activity Johannesburg, R 1 N S. Afr. R3 N N

Albert Einstein 2017 Pak1 inhibitors Kinases WO2017192228A1253 O College of O Medicine, Inc., USA.

NH N S N

Dana-Farber 2017 Anticancer WO2018058029A1254 Cancer Institute, activity Inc., USA. O NHHO OH O N OH N

Dana-Farber 2018 O TRIM33 E3 ubiquitin- WO2018200988A1255 Cancer Institute, protein ligase Inc., USA. N

O NH O N N N N H

10. AnchorQuery

The scaffold of the GBB-3CR is one of the 27 MCR scaffolds that is included in the AnchorQuery library which was recently introduced as powerful tool for rational structure-based design of protein-protein interaction (PPI) 6 inhibitors.256 The often deeply buried amino acids comprise a major energetic hotspot which is leveraged in a pharmacophore model search that rapidly screens libraries yielding a possible 31 million synthesizable compounds, each containing an anchor motif that is bioisosteric to amino acid residues, including D, W, Y, F, V and E anchor side chains. Important examples of the power of AnchorQuery are demonstrated through the role it played in the development of novel p53-MDM2 inhibitors and an allosteric inhibitor.257-259

In the virtual screening the suggested scaffolds are amenable in one to maximal three reaction steps. More specifically, the screening platform contains xyz GBB in xyz different conformations. Using AnchorQuery as a tool is freeof charge, accessible via http://anchorquery.csb.pitt.edu and will provide all the tools necessary to interactively perform online virtual screening, rapidly going through millions of compounds with pharmacophore elucidation and enrichment analysis queries. A great benefit of the screening platform is obviously thata binding hypothesis can be immediately tested by resynthesizing the virtual hit.

168 Chapter 6

Figure 9. Workflow of the online virtual screening platform ANCHOR.QUERY.

Conclusions

The GBB-3CR has been described the first-time 20 years ago and is currently one of the mostly used MCR reactions. According to the definition of the Ugi reaction as an α-addition of a Schiff base and a suitable acid component and a rearrangement reaction it belongs formally to the same class of reactions. Similar to the classical Ugi MCR the GBB-3CR also shows a great scope in terms of diversity of starting materials. For mostly any combination between aldehydes, isocyanides and heterocyclic amidines, with the help of a suitable catalyst, the reaction is giving the expected products. Not surprisingly the GBB scaffold is described in multiple publications and patents. The first derivatives of GBB products have entered clinical trials. This is very well in line with the saying that after 15-20 years since the discovery of a new reaction first products based on new reactions will enter the market. It can be predicted that several more bioactive compounds will enter the market in the future. An interesting freely accessible virtual screening platform will be useful in the discovery of tool compounds against difficult to drug targets. Currently still underdeveloped are materials science application of the GBB-3CRs, such as functional polymers or organic conductors or light to energy transformers. Clearly the GBB scaffold has the potential that many interesting electronic and optical applications will be described in the future.

List of abbreviations

AcOH Acetic acid MW Microwave Nuclear magnetic AD Alzheimers disease NMR resonance Absorption, distribution, Passerini 3-component ADMET P-3CR metabolism, and excretion reaction Passerini-reaction- ATX Autotaxin PADAM Amine-Deprotection- Acyl-Migration 3CR 3-component reaction PD Parkinsons Disease Platelet-derived growth BODIPY Boron-dipyrromethene PDGFR factor receptor CB2 Cannabinoid receptor type 2 PEG Polyethylene glycol Positron emmision CH Cl Dichloromethane PET 2 2 tomography

169 The Groebke-Blackburn-Bienaymé reaction

Prostaglandin- CK2 Casein Kinase II PGHS-2 endoperoxide synthase 2 Phosphoinositide DMA Dimethylaceetamide PI3 3-kinase Proto-oncogene serine/ DMF Dimethylformamide Pim-1 threonine-protein kinase DMSO Dimethylsulfoxide PK Pharmacokinetic Extracellular signal–regulated ERK PTSA para-Toluenesulfonic acid kinases FDA Food and Drug Administration p-TSIA para-Toluenesulfinic acid Fibroblast growth factor Rearranged FGFR3 RET receptor 3 during transfection Structure-activity GABA Gamma aminobutyric acid SAR relationship

GLP-1 Glucagon-like peptide-1 Sc(OTf)3 Scandium triflate Transient receptor potential h- Sodium-glucose transport cation channel, subfamily A, SGLT TRPA1 proteins member 1

HClO4 Perchloric acid SKM Leukemic cell line Time-dependent HCV Hepatitis C TDI inhibition HDAC Histone deacetylase Tf Triflate Ectonucleotide pyrophosphatase/ hENPP2 TFE Trifluoroethanol phosphodiesterase family member 2 (ATX) HTS High throughput screening TLC Trimethyl orthoformate Isocyanide-based IMCR TMOF Trimethyl orthoformate multicomponent reactions

JNK1 See MAPK8 TMSN3 Trimethylsilyl azide Tumor necrosis factor 6 LPC Lysophosphatidylcholine TNFa alpha Mitogen-activated protein MAPK8 TPSO Translocator protein kinase 8 Tripartite motif- MCR Multicomponent reactions TRIM33 containing 33 MDM2 Murine double minute 2 U-3CR Ugi 3-component reaction

MeCN Acetonitrile U-4CR Ugi 4 component reaction

MeOH Methanol UDC Ugi-deprotection-cyclize Macroporous polystyrene van Leusen 3 component MP-CO vL-3CR 3 carbonate reaction MTOR Mammalian target of rapamycin

170 Chapter 6

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R. Zhao, T. T. Diagana, P. Pertel, F. J. Leong, New Engl J Med 2016, 375, 1152- 1160. 215 C. Berset, S. Audetat, J. Tietz, T. Gunde, A. Barberis, A. Schumacher, P. Traxler, Esbatech A.-G., Switz. . 2005, p. 44 pp. 216 E. Laird, G. Topalov, J. P. Lyssikatos, M. Welch, J. Grina, J. Hansen, B. Newhouse, Array BioPharma Inc., USA . 2006, p. 103 pp. 217 W. Meng, L. G. Hamann, R. P. Brigance, Bristol-Myers Squibb Company, USA . 2006, p. 180 pp. 218 W. Mederski, N. Beier, B. Cezanne, R. Gericke, M. Klein, C. Tsaklakidis, Merck Patent G.m.b.H., Germany . 2007, p. 113pp. 219 V. Berdini, G. E. Besong, O. Callaghan, M. G. Carr, M. S. Congreve, A. L. Gill, C. M. Griffiths-Jones, A. Madin, C. W. Murray, R. K. Nijjar, M. A. O‘Brien, A. Pike, G. Saxty, R. D. Taylor, E. Vickerstaffe, Astex Therapeutics Limited, UK . 2008, p. 404pp. 220 M. Klein, R. Gericke, N. Beier, B. Cezanne, C. Tsaklakidis, W. Mederski, Merck Patent GmbH, Germany . 2008, p. 41pp. 221 K. C. Lee, E. T. Sun, H. Wang, S bio Pte. Ltd., Singapore . 2008, pp. 129 pp., Cont.-in-part of Appl. No. PCT/SG2006/000064. 222 F. P. Marmsaeter, M. C. Munson, J. P. Rizzi, J. E. Robinson, S. T. Schlachter, G. T. Topalov, Q. Zhao, J. P. Lyssikatos, Array BioPharma Inc., USA . 2008, p. 107 pp. 223 A. Muci, J. T. Finer, B. P. Morgan, A. J. Russell, D. J. Morgans, Jr., Cytokinetics, Incorporated, USA . 2008, p. 75 pp. 224 R. Olsson, E. Burstein, A. E. Knapp, J. Eskildsen, J. Castillo, ACADIA Pharmaceuticals Inc., USA . 2008, p. 95 pp. 225 P. A. P. Reddy, A. M. Siddiqui, P. K. Tadikonda, U. F. Mansoor, G. W. Shipps, Jr., D. B. Belanger, L. Zhao, Schering Corporation, USA . 2008, p. 362pp. 226 M. B. Van Niel, A. Miah, Merck Sharp & Dohme Ltd., UK . 2008, p. 28pp. 227 K. Bando, K. Taguchi, Fuji Film RI Pharma Co., Ltd., Japan; Daiichi Sankyo Co., Ltd. . 2009, p. 70 pp. 228 E. J. d. L. Barreiro, C. A. M. Fraga, A. L. P. Miranda, C. K. Freitas, L. Louback, R. B. Lacerda, Universidade Federal do Rio de Janeiro - UFRJ, Brazil . 2009, p. 95pp. 229 B. E. Fink, L. Chen, P. Chen, D. S. Dodd, A. V. Gavai, S.-H. Kim, W. Vaccaro, L. H. Zhang, Y. Zhao, Bristol-Myers Squibb Company, USA . 2009, p. 433pp.

183 The Groebke-Blackburn-Bienaymé reaction

230 M. Klein, W. Mederski, C. Tsaklakidis, N. Beier, Merck Patent GmbH, Germany . 2009, p. 75pp.; Chemical Indexing Equivalent to 150:472736 (DE). 231 H.-G. Capraro, Oncalis A.-G., Switz. . 2010, p. 81pp. 232 E. D. Farkas, H. Davids, C. Langley, C. B. De Koning, University of the Witwatersrand, S. Afr. . 2010, p. 36pp. 233 H. J. M. Gijsen, A. I. Velter, G. J. MacDonald, F. P. Bischoff, T. Wu, S. F. A. Van Brandt, M. Surkyn, M. Zaja, S. M. A. Pieters, D. J.-C. Berthelot, M. A. J. De Cleyn, D. Oehlrich, Ortho-Mcneil-Janssen Pharmaceuticals, Inc, USA . 2010, p. 164pp. 234 J. Shen, T. Meng, Z. Zhang, Z. Miao, J. Ding, L. Ma, D. Hu, Y. Zhang, Y. Jin, X. Wang, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Peop. Rep. China . 2010, p. 30pp. 235 C. G. Han, J. H. Yoon, N. D. Kim, G. J. Park, H. M. Kim, B. R. Sung, U. J. Shin, H. M. Lee, G. T. Noh, G. Y. Nam, S. H. Kim, EQUISnZAROO Co., Ltd., S. Korea . 2011, p. 22pp. 236 J. Pastor Fernandez, S. Martinez Gonzalez, R. M. Alvarez Escobar, S. Rodriguez Aristegui, E. Gonzales Cantalapiedra, A. I. Hernandez Higueras, C. Varela Busto, Centro Nacional de Investigaciones Oncologicas CNIO , Spain . 2011, p. 150pp. 237 T.-Y. Chan, H. M. Vaccaro, Schering Corporation, USA; Merck Sharp & Dohme Corp. . 2012, p. 82 pp. 238 Y. Xu, B. G. Brenning, S. G. Kultgen, X. Liu, M. Saunders, K.-K. Ho, Supergen, Inc., USA . 2012, pp. 106pp., Cont.-in-part of U.S. Ser. No. 785,322. 239 A. L. Banka, J. Botyanszki, E. G. Burroughs, J. G. Catalano, W. H. Chern, H. D. 6 Dickson, M. J. Gartland, R. Hamatake, H. Hofland, J. D. Keicher, C. B. Moore, J. B. Shotwell, M. D. Tallant, J.-P. Therrien, S. You, GlaxoSmithKline LLC, USA . 2013, p. 224pp. 240 S. K. Guchhait, C. N. Kundu, U. C. Banerjee, A. Baviskar, C. Madaan, A. Agarwal, R. Preet, P. Mohapatra, National Institute of Pharmaceutical Education and Research Niper , India . 2013, p. 53pp. 241 S. Bachmann, S. D. Erickson, D. I. Laine, Y. Qian, F. Hoffmann-La Roche AG, Switz.; Hoffmann-La Roche Inc. . 2014, p. 34pp. 242 J. M. Bentley, D. C. Brookings, J. A. Brown, T. P. Cain, P. T. Chovatia, A. M. Foley, E. O. Gallimore, L. J. Gleave, A. Heifetz, H. T. Horsley, M. C. Hutchings, V. E. Jackson, J. A. Johnson, C. Johnstone, B. Kroeplien, F. C. Lecomte, D. Leigh, M. A. Lowe, J. Madden, J. R. Porter, J. R. Quincey, L. C. Reed, J. T. Reuberson, A. J. Richardson, S. E. Richardson, M. D. Selby, M. A. Shaw, Z. Zhu, UCB Pharma S.A., Belg. . 2014, p. 458pp.

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243 N. Desroy, B. Heckmann, R. C. X. Brys, A. M. Joncour, C. Peixoto, X. M. Bock, C. G. Housseman, Galapagos NV, Belg. . 2014, p. 262pp. 244 T. Furuta, T. Sawada, T. Danjo, T. Nakajima, N. Uesaka, Kyowa Hakko Kirin Co., Ltd., Japan . 2014, p. 251pp. 245 U. Velaparthi, C. P. Darne, D. S. Dodd, A. J. Sampognaro, M. D. Wittman, S. Kumaravel, D. Mullick, C. Reddy R, P. Liu, Bristol-Myers Squibb Company, USA . 2014, p. 379pp. 246 S. H. Jang, Y. S. Noh, D. J. Kim, H. G. Jang, S. J. Eum, J. D. Lee, Heesung Material Ltd., S. Korea . 2015, p. 69pp. 247 C. Dominguez, J. Wityak, J. Bard, A. Kiselyov, C. J. Brown, M. E. Prime, P. D. Johnson, D. Clark-Frew, S. Schaertl, F. Herrmann, S. K. Grimm, J. D. Kahmann, C. Scheich, CHDI Foundation, Inc., USA . 2016, p. 105pp. 248 L. R. Marcin, S. T. Wrobleski, T. G. M. Dhar, Bristol-Myers Squibb Company, USA . 2016, p. 93pp. 249 L. A. Mevellec, L. Meerpoel, S. Coupa, V. S. Poncelet, I. N. C. Pilatte, E. T. J. Pasquier, D. J.-C. Berthelot, O. A. G. Querolle, C. Meyer, P. R. Angibaud, C. G. M. Demestre, G. J. M. Mercey, Janssen Pharmaceutica NV, Belg. . 2016, p. 302pp. 250 L. A. Mevellec, L. Meerpoel, S. Coupa, V. S. Poncelet, I. N. C. Pilatte, E. T. J. Pasquier, D. J.-C. Berthelot, O. A. G. Querolle, C. Meyer, P. R. Angibaud, C. G. M. Demestre, G. J. M. Mercey, Janssen Pharmaceutica NV, Belg. . 2016, p. 203pp. 251 M. Pourashraf, M.-A. Beaulieu, S. Claridge, M. Bayrakdarian, S. Johnstone, J. S. Albert, A. Griffin, NEOMED Institute, Can. .2017 , p. 135pp. 252 C. B. De Koning, L. Harmse, J. Dam, University of the Witwatersrand, Johannesburg, S. Afr. . 2017, p. 46pp. 253 U. Steidl, A. M. Eckel, R. F. Stanley, B. Rogovoy, I. Okun, Albert Einstein College of Medicine, Inc., USA . 2017, p. 81pp. 254 J. E. Bradner, J. Qi, A. Federation, Z. Jacobson, A. Varca, Dana-Farber Cancer Institute, Inc., USA . 2018, p. 125pp. 255 J. Qi, C.-K. Pei, Dana-Farber Cancer Institute, Inc., USA . 2018, p. 128pp. 256 D. R. Koes, A. Dömling, C. J. Camacho, Protein Sci 2018, 27, 229-232. 257 E. Surmiak, C. G. Neochoritis, B. Musielak, A. Twarda-Clapa, K. Kurpiewska, G. Dubin, C. Camacho, T. A. Holak, A. Dömling, Eur J Med Chem 2017, 126, 384-407. 258 S. Shaabani, C. G. Neochoritis, A. Twarda-Clapa, B. Musielak, T. A. Holak, A.

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Dömling, Medchemcomm 2017, 8, 1046-1052. 259 E. Kroon, J. O. Schulze, E. Suss, C. J. Camacho, R. M. Biondi, A. Dömling, Angew Chem Int Ed Engl 2015, 54, 13933-13936.

186

Chapter 7

Diverse One-Pot Synthesis of Adenine Mimetics for Biomedical Applications

André Boltjes, Markella Konstantinidou, Angel J. Ruiz-Moreno, Li Gao, John de Boer, Marco A. Velasco-Velazquez and Alexander Dömling

Manuscript in preparation Chapter 7

Abstract

A series of unprecedented imidazo[1,2-a]pyrazin-8-amines are synthesized from simple and readily available building blocks. This scaffold is of high interest due to its similarity to adenine. Multiple di-aminopyrazines, -pyrimidines and –pyridazines were subjected to Groebke-Blackburn- Bienaymè reaction conditions to afford imidazo[1,2-a]pyrazines, pyrimidines and pyridazines respectively. Pyrazine-2,3-diamine, as amidine component furnished a clean and selective reaction resulting in an adenine mimetic library of over 20 compounds in a single step under mild conditions.

189 Diverse one-pot synthesis of adenine mimetics for biomedical applications

In classical drug discovery a protein target which is believed to have a positive influence on a disease course is pursued and antagonists or agonist are searched for. Small molecules against this drug target can be found for example by high throughput screening of large libraries. This is the most popular approach in drug discovery and leads to more or less hits depending on the target class. Often no hits are resulting from so called undruggable targets. However, from the initial hit to a useful lead compound to probe the biology of a target protein is a long and stony way involving much time and efforts to improve the hit compounds affinity, selectivity, stability etc. and finally biological activity. Chemists on the other hand often find a new and versatile reaction pathway to a novel class of scaffold. However, a priori there is no knowledge what biological space could correspond to this particular chemical space. Traditionally, examples of the new scaffolds could be screened in HTS to eventually find an active hit for a given target. In the human proteome, however, there are hundreds of thousands of potentially targetable proteins and thus it is unlikely that a target will be found for a given compound in a reasonable time even in a pharma company which runs multiple HTS per year. A solution to this dilemma is reverse or inverse docking which aims to find a target for a given small molecule based on computational screening of the full or part of the PDB.1-2

Figure 1. Adenine and a new synthetic pathway towards mimics. Left: Zoom into the adenine substructure of substructure of nicotineamide-adenine-dinucleotide binding pocket of the enoyl-ACP reductase of Mycobacterium tuberculosis InhA (PDB ID 4TRO). Right: Previous and current work using GBB-3CR towards adenine derivatives.

One of the most successful target classes in modern medicine are kinases and more than 30 small molecule inhibitors were introduced into clinical practice.3 Kinases catalyze the phosphorylation of Ser, Thr, His and Tyr and use as cofactor 7 ATP. Small molecule kinase inhibitor drugs mimic the adenosine part of ATP and fit into a deep and narrow binding site and form in the so-called hinge region a bifurcated hydrogen bond between a diglycine and the adenine-N1 and C6-NH2. Besides kinases many more protein use adenine as a cofactor. In figure 1 a typical interaction of an adenine substructure is shown forming the signature hydrogen bonding network.4 In the current version of the protein data bank (PDB) more than 22,000 structures can be found containing the adenine substructure.5 Thus, adenine mimics are highly sought-after substructures and synthesis of substituted heterocycles mimicking A are promising potentially bioactive compounds. Most current synthetic pathways for adenine mimicking small molecules suffer from a lengthy multistep synthesis.6-8 In 2011 Pirali described a 2-step route towards adenine derivatives, involving a Groebke-Blackburn-Bienaymè three

190 Chapter 7

component reaction (GBB-3CR) of 2-amino-3-chloropyrazine followed by a SnAr with ammonia.9-13 A similar approach reported by Ren et al. in 2015 is showing a side by side comparison between the 2-amino-3-chloropyrazine substrate and N-2,4-dimethoxybenzyl (DMB) protected diaminopyrazine.14 Although the yields improved by using DMB protected diaminopyrazine, probably due to the better nucleophilicity, again a two-step methodology was highlighted. These GBB-3CR amidine substrates are quite unreactive and need considerable activation by high temperature. The subsequent SnAr with ammonia also requires harsh conditions and both reactions are not compatible with many delicate substituents. Moreover, only substituted benzaldehydes and aryl carbaldehydes were reported to be used in this limited methodology. Therefore, we want to report here our design of a one-pot general, middle and high yielding synthesis to 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amine as adenine mimics.

Amidine containing aromatic 5- and 6-membered heterocycles are interesting building blocks in bioactive molecules, able to form bifurcated hydrogen bondings to -COOH and -OH of Asp, Glu and Tyr. Thus, we were investigating the behavior of corresponding diamine substituted pyrazines and pyridazines in the GBB-3CR with the target to find conditions for a selective mono reaction (Scheme 1).

R2 NH N NH2 N R N 1 N NH2 N

NH2 NH2 GBB-3CR R2 NH N Degradation N N N NH2 R1 N H2N R2 R2 R1 NH Degradation N N NH R + HN 2 H2N N HN H2N NH2 N N R N 1 R N 1 N Scheme 1. Pyrazines, pyrimidines and pyridazines in the GBB-3CR. Although these three amidines give yield to the expected products (confirmed by MS), the products from pyridazine-3,6-diamine degrade upon exposure to air during purification. Furthermore, the pyridazine-3,6-diamine product is low yielding, shows degradation and yields an additional bis-GBB-3CR product which degrades within a short amount of time as well, probably due to the easy oxidizable bridging double bond. We found that the GBB-3CR products from pyrazine-2,3-diamine; 2,3-disubstituted imidazo[1,2-a]pyrazin-8-amines do not show degradation, therefore this amidine together with benzaldehyde and 2,6-dimethylphenyl isocyanide was used in an optimizing sequence in which various reaction conditions were evaluated as depicted in table 1.

191 Diverse one-pot synthesis of adenine mimetics for biomedical applications

Table 1. Optimizing conditions applied on the most promising amidine.

NH2 N NC N N NH2 O N + + Catalyst NH N NH2 MeOH 1 2a 3a 4a

Reaction r.t.a 5% Sc(OTf) b 20% Sc(OTf) b 10% HClO b 10% PTSAb Time 60 °C MW (100 °C) 3 3 4

10 min -- -- 62% ------

4 h 37% 42% -- 20% 39% 16% 14%

8 h 49% 59% -- 31% 61% 33% 27%

24 h 82% 64% -- 62% 75% 46% 44%

48 h 82% 75% -- 68% 75% 82% 67% a Standard protocol: amidine 1 (0.25 mmol, 1 eq.) and aldehyde 2 (0.25 mmol, 1 eq.) were dissolved in 1 mL MeOH, after 5 minutes isocyanide 3 (0.25 mmol, 1 eq.) and Sc(OTf)3 (0.025 mmol, 0.1 eq.) were added and stirred at room temperature. b The percentages described are mol-%. Analysis: 10 µL reaction mixture was diluted 20.000 times in MeOH. Each sample was analyzed via MS flow injection analysis, collecting signal only from the products [M+1]+ channel. The reported percentages are conversion, corrected to the actual yield after column chromatography. The reaction progress was monitored by quantitative MS analysis and 10 µL aliquots of reaction mixture were taken over time while varying temperature and catalysts. As expected at elevated temperature the rate of product formation increases, the amount of product in the room temperature reaction however, surpassed that of the heated reactions after 24 hours of stirring. Furthermore, TLC analysis is showing a cleaner reaction mixture in the rt experiment, explaining the higher conversion towards product. Assessment of three catalysts; scandium triflate (Sc(OTf)3, p-toluenesulfonic acid (PTSA) and HClO4(aq) and additionally catalyst load of scandium triflate; 5, 10 and 20 mol-% resulted in

Sc(OTf)3 (10 mol-%) and perchloric acid HClO4 (10 mol-%) as good candidates, giving comparable conversion. Ease of handling and shorter reaction time made that we chose Sc(OTf) as optimal catalyst. Purification afforded N3-(2,6- 3 7 dimethylphenyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4) in a yield of 44%. The concentration of the screening reaction was 0.25 M.

With respect to the two amino groups present in the amidine, the aldehyde was hypothesized to be scavenged through double Schiff-base formation. Two more dilute reactions C = 0.1 M were run, a 30 minute incubation of the amidine with aldehyde and slow addition of the aldehyde to the amidine respectively. Proper dilution generally results in mono substitution and to enforce our hypothesis, a third, higher concentrated reaction was performed at C = 0.5 M with a small excess of aldehyde (1.1 equiv.) and isocyanide (1,3 equiv.). Surprisingly dilution and slow addition of aldehyde lead to lower yields as compared to the more concentrated reaction with yields of 45, 24 and 82% respectively. When

192 Chapter 7 benzaldehyde and 2,6-dimethylphenyl isocyanide were used, precipitation of the product was observed, resulting in a slurry in the 0.5 M concentration. To ensure proper mixing, the concentration was not further increased and kept at 0.5 M for all described reactions.

Figure 2. Top picture of fluorescent compound4b spotted on a TLC plate. Bottom picture showing a selection of pure products in solution <1 mg/mL in MeOH. Scope and limitations for a large variety of substituents was assessed (Table 2). Both the aldehyde and isocyanide components give room to test compatibility of aromatic and aliphatic substituents. Moreover, additional functional groups attached to 1R or R2 such as esters, boronic acids, ethers and Boc protected amines were used to demonstrate the versatile character of this reaction. Interestingly the majority of the products exhibited fluorescence as observed while taking TLCs (figure 2). Overall there was no significant difference observed between the used aliphatic and aromatic isocyanides, while keeping the aldehyde (benzaldehyde) component constant. We were able to grow a crystal from compound 4e, confirming the proposed structure and not the possible inverse GBB-3CR product (Figure 2).

193 Diverse one-pot synthesis of adenine mimetics for biomedical applications

3.5

3.5 3.2

Figure 2. Crystal structures of some GBB-3CR products. Left: Crystal structure of 4a. Right: X-ray structure refinement unambiguously confirms formation of4e and not the inverse GBB- 3CR product 5. Hydrogen bonding of the exocyclic amine on C-3 underlines the importance of the adenine mimetic structure.

The amino acid derived isocyanides (Gly and β-Ala) worked well, the additional carbon gave some improvement in yield of 60 and 78% for 4r and 4d respectively. Examples 4b and 4c with variations on the aldehyde component, having electron withdrawing groups gave significant lower yields. The yield was, however, observed to be higher when an aromatic isocyanide such as 2,6-dimethylphenyl isocyanide (3a) was used, indicated in example 4t. Glyoxylic acid was used as formaldehyde component and gave a poor yield of only 35% (4k). The Schiff base precipitated, which could explain the low yield. Heating would typically dissolve the Schiff base and should result in a better conversion. The reaction was therefore repeated in a microwave reactor at 100 °C for 10 minutes, resulting in a clear reaction mixture, but similar poor yield of 30% after purification, indicates that glyoxylic acid in combination with the pyrazine substrate (1) under these conditions is not optimal.

194 Chapter 7

Table 2. Substrate scope for aldehydes and isocyanides.

N NH2 R2 R1 O NH N NH2 MeOH, Sc(OTf)3 1 + 2 N 24h, rt R1 NC N N R2 4a-t 3 NH2

BocHN O O NH NH NH NH N N N NO N Cl N 2 N N N N N N N NH NH 2 2 NH2 NH2 4a (82%) 4b (20%) 4c (40%) 4d (78%) BocHN

NH NH NH NH N N OMe N N N N N N N N N N NH2 NH2 NH2 NH 4e (96%) 4f (66%) 4g (75%) 2 4h (68%)

NH NH NH NH N NH S N N N BPin N N N N N N N N NH2 NH2 NH2 NH2 4i (54%) 4j (91%) 4k (35%) 4l (78%)

HO NH NH NH B NH OH N N N N N N N N N N N N NH2 NH2 NH2 4m (96%) 4n (81%) NH2 4o (78%) 4p (81%)

O O NH NH NHBoc NH NH N N N Cl N N N N N N N N N NH2 NH NH 2 2 4t (75%) 4q (60%) NH2 4r (60%) 4s (90%) O O NH NH N N OMe N N N N NH NH 2 4u (61%) 2 4v (72%)

Functional groups such as esters (4n, 4s), Boc-groups (4h, 4s), boronic acids,

195 Diverse one-pot synthesis of adenine mimetics for biomedical applications unprotected (4p) and its pinacol ester (4l) were well tolerated, with yields between 68 and 90%. During purification of reactions that produced 4e and 4u, minor bis product formation was observed. The electron donating groups on the used aromatic aldehydes for these products seem to slightly activate the remaining amidine in the mono product, thus allowing for a second GBB-3CR. About less than half of the performed reactions showed precipitation of the product. The reported yields of these reactions were yields after column chromatography. Repetition of these reactions and filtration of the products gave slightly lower to similar yields. The lower yield could be explained by the slight solubility of the product in the mother liquor, which is otherwise retrieved by column chromatography. To highlight the simplicity of this reaction we ran a multigram reaction of 2,3-diaminopyrazine, 2,6-dimethylphenylisocyanide and benzaldehyde to afford 1.97 g ( 6.0 mmol, 60% ) 4a after filtration in a single step as a brown solid.

Molecular docking

An adenine substructure-based search was performed in the PDB database in order to define structures that can bind adenine-related ligands. We selected only X-ray protein structures corresponding to the biological assembly 1 for the experiments, resulting in 13.864 structures. Structures retrieved from PDB were prepared for molecular docking experiments by removing water, ions, and solvent molecules using Mdtraj (26488642). Co-crystallized ligands containing adenine or adenine- substructures (adenine-scaffold) were saved separately from proteins to serve as references. For some proteins, more than one co-crystallized ligand was found, because of that, the number of references was 23.267. For proteins, hydrogens, missing atoms, and residues were added, and nonstandard residues were converted to their standard forms using PDBfixer https:// ( github.com/pandegroup/pdbfixer). Prepared proteins were saved as receptors. Nine adenine-mimetic ligands were designed through our original GBB one-pot synthesis of adenine-mimetic molecules. Ligands contain 2,3-dimethylimidazo[1,2-a]pyrazin-8-amine plus aliphatic, and/or aromatic modifications in R1 and/or R2 groups (see scheme at the top). Structures of designed ligands can be found in figure 3. For each ligand, hydrogens, charges and 3D coordinates were added to the molecules by OpenBabel (21982300). 7

196 Chapter 7

NH NH NH N N N N N N N N N

NH2 NH2 NH2 6 7 8

NH NH NH N N N N N N N N N

NH2 NH2 NH2 9 10 11

NH NH NH N N N N N N N N N

NH2 NH2 NH2 12 13 14 Figure 3. Ligands based on the synthesized GBB-3CR products used for docking. With variations on the R1 and R2 positions from the aldehyde and isocyanide respectively.

The identification of potential targets for the designed ligands was performed by implementing an inverse docking approach considering the co-crystallized references. For dockings, the 3D coordinates of reference were used to define a docking cavity in the receptor. The cavity was established by selecting the receptor residues 8 Å far from the reference. Then, ligand and reference were aligned by an adenine-scaffold atom map pairing within the receptor cavity implementing the open source cheminformatics library RDKit (http://www.rdkit.org). After that, molecular docking between each ligand and receptor was performed utilizing GOLD from the Cambridge Crystallography Data Center (9126849). GOLD docking experiments were evaluated by ChemPLP score considering a penalization whether during docking adenine-mimetic from ligand moved more than 1.5 Å of the adenine-scaffold in reference. For the docking results, best pose of each docking was kept for further analysis. Nevertheless, poses with negative score values were not considered for analysis due they correspond to null predicted affinity between the ligand and the receptor or were highly penalized for lack of match between adenine-mimetic and adenine-scaffold.

197 Diverse one-pot synthesis of adenine mimetics for biomedical applications

Conclusions

In conclusion, we have developed a mild method to conveniently synthesize in a single step 2,3-di-substituted imidazo[1,2-a]pyrazin-8-amines through the GBB- 3CR in overall good to excellent yields. This method is superior to previously reported methods in terms of number of steps and generality. The mild reaction conditions allowed a large variety of substituents, including functional groups, such as ester and BOC groups that would otherwise degrade or react (4b, 4d, 4h, 4l, 4r and 4s) when extended elevated temperatures would have been applied as previously described. Functional group compatibility beyond very simple model substrates is key for an early industrial adaption and avoiding a synthetic bias in medicinal chemistry programs toward making molecules with less drug-like properties.15 In total 22 reactions were performed and those reactions without exception were found to give the expected products. We are currently investigating the reaction scaffold to discovery novel adenine mimetics with biological activity, which will be reported in due course.

Experimental procedures and Spectral Data Instruments employed: Grace Reveleris X2 flash chromatography apparatus, Advion CMS ExpressionL. Mass spectrometer, Bruker Avance DRX 500 MHz NMR spectrometer (B AV-500) equipped with Bruker Automatic Sample Changer (B ACS 60); High resolution mass spectra were recorded using a LTQ-Orbitrap- XL (Thermo) at a resolution of 60000@m/z400.NMR spectra were recorded in

CDCl3 (with 0.03% TMS), DMSO-d6 at either 500 MHz (δH) or 125 MHz (δC); the coupling constants (J) are in Hz. Abbreviations: s (singlet); d (doublet); t (triplet); q (quartet); br (broad); dd (doublet of doublet), etc. The nomenclatures of all the compounds were derived by ChemDraw (CambridgeSoft). Flash chromatography separations were performed on Flashpure Normal-phase Silica Flash Columns (Silica Gel 40µm irregular) unless otherwise noted. Reagents were commercial grades and were used without any purification unless otherwise noted. Reagents were commercial grades and were used without any purification unless otherwise noted. Methanol, dichloromethane, ethyl acetate, petroleum ether 40 – 60, were purchased from commercial sources. 7 Synthetic procedures and characterization for compound 4a-4v

General procedure for all GBB-3CR reactions: In a 4 mL vial Sc(OTf)3 (24.6 mg, 0.05 mmol) and 2,3-diaminopyrazine (55 mg, 0.5 mmol, 1 equiv.), were dissolved in methanol (1 mL). Then aldehyde (0.55 mmol, 1.1 equiv.) and isocyanide (0.65 mmol, 1,3 equiv.) were added and the reaction mixture was stirred for 48 h at room temperature. The solvent was removed under reduced pressure and the remainder was purified by flash chromatography with either 0 - 5% methanol in dichloromethane (method A) or 0 - 100% ethyl acetate in petroleum ether 40-60 (method B) to afford the product.

198 Chapter 7

N3-(2,6-dimethylphenyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4a)

Using benzaldehyde, 2,6-dimethylphenyl isocyanide and purification method B; 135 mg, 82% product was obtained as a NH 1 pale yellow solid. H NMR (500 MHz, DMSO-d6) δ 8.02 – 7.95 (m, N 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.26 – 7.19 (m, 2H), 7.16 (d, J = 4.6 Hz, N N 1H), 7.03 (d, J = 4.7 Hz, 1H), 6.95 (s, 2H), 6.89 (d, J = 7.5 Hz, 2H), 13 NH2 6.69 (t, J = 7.5 Hz, 1H), 1.90 (s, 6H), 1.22 (s, 1H). C NMR (126 MHz, DMSO) δ 150.1, 140.7, 134.9, 133.6, 129.4, 129.3, 128.1, 128.1, 127.7, 127.0, 126.7, 126.6, 126.2, 124.4, 120.7, 120.6, 106.8, 18.5, 18.4. HRMS (ESI) + + m/z calculated for C20H20N5 [M+H] : 330,17132; found [M+H] : 330,17130 tert-Butyl (2-((8-amino-2-(4-chlorophenyl)imidazo[1,2-a]pyrazin-3-yl)amino) ethyl)carbamate (4b)

BocHN Using 4-chlorobenzaldehyde, tert-butyl (2-isocyanoethyl) carbamate and purification method A; 40.5 mg, 20% NH product was obtained as a yellow solid. 1H NMR (500 N MHz, DMSO-d6) δ 8.16 – 8.09 (m, 2H), 7.59 (d, J = 4.3 Hz, Cl N N 1H), 7.51 – 7.46 (m, 2H), 7.21 (s, 1H), 6.93 – 6.77 (m, 3H), 4.95 (t, J = 6.3 Hz, 1H), 3.04 (q, J = 6.3 Hz, 2H), 2.96 (q, J = NH2 6.8, 6.4 Hz, 2H), 1.34 (s, 9H). 13C NMR (126 MHz,

DMSO-d6) δ 155.6, 150.1, 144.1, 133.0, 131.7, 131.4, 129.1, 128.4, 128.4, 128.1, 128.0, 127.8, 107.1, 107.0, 78.3, 77.7, 47.5, 40.7, 28.3, 28.2, 28.2. HRMS (ESI) m/z calculated + + for C19H24N6O2Cl [M+H] : 403,16438; found [M+H] : 403,16479

N3-(tert-butyl)-2-(4-nitrophenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4c)

Using p-nitro-benzaldehyde, t-butyl isocyanide and NH purification method B; 66 mg, 40% product was obtained 1 N as an orange solid. H NMR (500 MHz, DMSO-d6) δ 8.48 NO N 2 (d, J = 9.0 Hz, 2H), 8.29 (d, J = 9.0 Hz, 2H), 7.67 (d, J = 4.6 N Hz, 1H), 7.23 (d, J = 4.6 Hz, 1H), 6.91 (s, 2H), 4.84 (s, 1H), NH 2 1.03 (s, 9H). 13C NMR (126 MHz, DMSO) δ 150.3, 145.9, 141.8, 134.8, 130.7, 128.2, 128.0, 123.9, 123.4, 108.1, 56.2, 30.0. HRMS (ESI) m/z + + calculated for C16H19O2N6 [M+H] : 327,1564; found [M+H] : 327,15628

199 Diverse one-pot synthesis of adenine mimetics for biomedical applications

Methyl 3-((8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)amino)propanoate (4d)

O Using benzaldehyde, methyl 3-isocyanopropanoate, O purification method A; 121mg, 78% product was obtained 1 NH as a brown solid. H NMR (500 MHz, Chloroform-d) δ 7.92 (d, J = 6.8 Hz, 2H), 7.50 – 7.41 (m, 3H), 7.36 – 7.27 (m, 2H), N N 5.83 (s, 2H), 3.82 (t, J = 6.9 Hz, 1H), 3.67 (s, 3H), 3.29 (q, J = N 6.1 Hz, 2H), 2.54 (t, J = 5.8 Hz, 2H). 13C NMR (126 MHz, NH2 Chloroform-d) 13C NMR (126 MHz, Chloroform-d) δ 173.0, 149.9, 135.7, 133.7, 128.9, 128.2, 127.8, 127.6, 127.2, 107.9, 52.0, 43.7, 34.5. HRMS + + (ESI) m/z calculated for C16H18N5O2 [M+H] : 312,14550; found [M+H] : 312,14532 N3-(tert-butyl)-2-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4e)

Using anisaldehyde, t-butyl isocyanide and purification NH method A; 148,8 mg, 96% product was obtained as an 1 N off-white solid. H NMR (500 MHz, DMSO-d6) δ 8.15 – OMe N N 7.99 (m, 2H), 7.62 (d, J = 4.6 Hz, 1H), 7.18 (d, J = 5.1 Hz,

NH2 1H), 7.02 – 6.92 (m, 2H), 6.75 (s, 2H), 4.56 (s, 1H), 3.78 (s, 13 3H), 0.98 (s, 9H). C NMR (126 MHz, DMSO-d6) δ 158.4, 149.9, 137.5, 129.1, 128.9, 128.9, 128.7, 128.5, 127.5, 127.2, 125.5, 113.4, 113.3, 113.3,

108.2, 108.1, 55.6, 30.1, 30.1 ppm. HRMS (ESI) m/z calculated for C17H22N5O [M+H]+: 312,18237; found [M+H]+: 312,18189 2-Benzyl-N3-(tert-butyl)imidazo[1,2-a]pyrazine-3,8-diamine (4f)

Using 2-phenyl-acetaldehyde, t-butyl isocyanide and NH purification method B; 97 mg, 66% product was obtained as a brown solid.1H NMR (500 MHz, Chloroform-d) δ 7.47 N (d, J = 4.2 Hz, 1H), 7.26 – 7.20 (m, 2H), 7.21 – 7.13 (m, 4H), N N 13 4.14 (s, 2H), 1.14 (s, 9H). C NMR (126 MHz, CDCl3) δ 149.1, NH2 139.4, 139.0, 128.5, 128.4, 127.3, 126.2, 125.7, 108.8, 55.8, 33.7, + 30.4. HRMS (ESI) m/z calculated for C17H22N5 [M+H] : 296,18697; found [M+H]+: 296,18759

N3-(2,6-dimethylphenyl)-2-isobutylimidazo[1,2-a]pyrazine-3,8-diamine (4g) 7

Using isovaleraldehyde, 2,6-dimethylphenyl isocyanide and purification method A; 115.3 mg, 75% product was obtained NH 1 as an off-white solid. H NMR (500 MHz, DMSO-d6) δ 7.43 (d, N J = 4.7 Hz, 1H), 7.21 (d, J = 4.7 Hz, 1H), 6.97 (d, J = 7.4 Hz, 2H), N N 6.78 (t, J = 7.5 Hz, 1H), 6.76 – 6.67 (m, 3H), 2.03 (d, J = 7.1 Hz, 2H), 1.92 (s, 6H), 1.85 – 1.74 (m, 1H), 0.71 (d, J = 6.6 Hz, 6H). NH2 13 C NMR (126 MHz, DMSO-d6) δ 144.6, 138.2, 133.3, 131.5, 130.9, 129.7, 125.9, 122.9, 118.2, 109.8, 109.6, 34.7, 28.6, 28.5, 23.1, 23.0, 19.3, 19.2. + + HRMS (ESI) m/z calculated for C18H24N5 [M+H] : 310,20262; found [M+H] :

200 Chapter 7

310,20236 tert-Butyl (2-((8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)amino)ethyl) carbamate (4h)

BocHN Using benzaldehyde, tert-butyl (2-isocyanoethyl)carbamate, purification method A; 126 mg, 68% product was obtained NH as a yellow solid. 1H NMR (500 MHz, Chloroform-d) δ 7.84 N (d, J = 7.6 Hz, 2H), 7.39 (t, J = 7.7 Hz, 2H), 7.35 – 7.25 (m, 2H), N N 7.18 (s, 1H), 6.09 (s, 2H), 5.13 (s, 1H), 3.60 (s, 1H), 3.22 – 3.00 (m, 4H), 1.42 (s, 9H). 13C NMR (126 MHz, Chloroform-d) δ NH2 156.5, 149.9, 135.5, 133.6, 128.8, 128.8, 128.6, 127.7, 127.5,

127.2, 107.6, 79.7, 48.4, 41.0, 28.5. HRMS (ESI) m/z calculated for C19H25N6O2 [M+H]+: 369,20335; found [M+H]+: 369,20334

N3-(2,6-dimethylphenyl)-2-(thiophen-2-yl)imidazo[1,2-a]pyrazine-3,8-diamine (4i)

Using thiophene-2-carbaldehyde, 2,6-dimethylphenyl isocyanide and purification method B; 91 mg, 54% product was obtained as a grey solid. 1H NMR (500 MHz, DMSO-d ) δ NH 6 S 7.44 (dd, J = 5.1, 1.0 Hz, 1H), 7.19 (dd, J = 3.6, 1.1 Hz, 1H), 7.16 N (d, J = 4.8 Hz, 2H), 7.06 (d, J = 4.6 Hz, 1H), 7.00 (dd, J = 5.0, 3.6 N N Hz, 1H), 6.93 (d, J = 7.5 Hz, 2H), 6.85 (s, 2H), 6.73 (t, J = 7.5 Hz, 13 NH2 1H), 1.90 (s, 6H). C NMR (126 MHz, DMSO) δ 149.8, 140.5, 136.6, 131.5, 129.3, 128.4, 127.6, 127.5, 126.3, 125.3, 123.9, 123.0, + 120.8, 106.7, 18. 4, 18. 3. HRMS (ESI) m/z calculated for C18H18N5S [M+H] : 336,12774; found [M+H]+: 336,12781

N3-(tert-butyl)-2-(1H-indol-3-yl)imidazo[1,2-a]pyrazine-3,8-diamine (4j)

Using indole-3-carboxaldehyde, t-butyl isocyanide and

NH purification method A; 146,2 mg, 91% product was obtained as an off-white solid.1 H NMR (500 MHz, DMSO-d ) δ 11.20 N NH 6 N (d, J = 2.5 Hz, 1H), 8.52 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 2.6 Hz, N 1H), 7.64 (d, J = 4.7 Hz, 1H), 7.42 – 7.37 (m, 1H), 7.19 (d, J = NH2 4.7 Hz, 1H), 7.16 – 7.09 (m, 1H), 7.09 – 7.03 (m, 1H), 6.78 (s, 13 2H), 4.58 (s, 1H), 1.03 (s, 9H). C NMR (126 MHz, DMSO-d6) δ 149.4, 135.9, 135.9, 128.4, 126.6, 126.6, 126.4, 124.9, 124.5, 124.5, 122.2, 122.2, 121.2, 119.0, 111.2, 109.3, 108.2, 108.1, 55.7, 30.2, 30.1 ppm. HRMS (ESI) m/z + + calculated for C18H21N6 [M+H] : 397,23465; found [M+H] : 397,23444

201 Diverse one-pot synthesis of adenine mimetics for biomedical applications

N3-(2,6-dimethylphenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4k)

Using glyoxylic acid, 2,6-dimethylphenyl isocyanide and purification method A; 43.8 mg, 35% product was obtained as an NH 1 orange solid. H NMR (500 MHz, DMSO-d6) δ 7.53 (d, J = 4.7 Hz, N 1H), 7.20 (d, J = 4.6 Hz, 1H), 7.08 – 7.02 (m, 3H), 6.93 (t, J = 7.5 Hz, 13 N N 1H), 6.77 (s, 2H), 6.50 (s, 1H), 2.06 (s, 6H). C NMR (126 MHz,

NH2 DMSO-d6) δ 150.3, 140.0, 131.5, 131.4, 128.7, 127.7, 127.4, 123.7, 118.3, 118.2, 107.1, 106.9, 18.0, 17.9. HRMS (ESI) m/z calculated for + + C14H16N5 [M+H] : 254,14002; found [M+H] : 254,13988

N3-(tert-butyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl) imidazo[1,2-a]pyrazine-3,8-diamine (4l)

Using 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) NH benzaldehyde, t-butyl isocyanide and purification N method B; 160 mg, 78% product was obtained as a white BPin N 1 N solid. H NMR (500 MHz, DMSO-d6) δ 8.20 (d, J = 8.2 Hz,

NH2 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 4.7 Hz, 1H), 7.19 (d, J = 4.7 Hz, 1H), 6.80 (s, 2H), 4.66 (s, 1H), 1.30 (s, 12H), 13 0.99 (s, 9H). C NMR (126 MHz, CDCl3) δ 150.1, 137.9, 136.8, 134.2, 134.0, 128.9, 127.5, 126.9, 126.8, 108.1, 108.0, 83.6, 55.9, 30.0, 24.8, 24.7. HRMS (ESI) m/z + + calculated for C22H31O2N5B [M+H] : 408,25653; found [M+H] : 408,25641

N3-(tert-butyl)-2-(naphthalen-1-yl)imidazo[1,2-a]pyrazine-3,8-diamine (4m)

Using 1-naphtaldehyde, t-butyl isocyanide and purification NH method B; 160 mg, 96% product was obtained as a brown 1 N solid. H NMR (500 MHz, DMSO-d6) δ 8.22 (dd, J = 7.9, 1.6 Hz, N N 1H), 8.00 – 7.89 (m, 2H), 7.77 (dd, J = 7.1, 1.2 Hz, 2H), 7.64 – 7.53

NH2 (m, 1H), 7.50 (td, J = 8.0, 1.4 Hz, 3H), 7.20 (s, 2H), 4.40 (s, 1H), 0.73 (s, 9H).13C NMR (126 MHz, DMSO) δ 133.4, 132.2, 131.7, 128.4, 128.3, 128.1, 128.03, 128.0, 126.5, 126.0, 125.7, 125.2, 125.1, 54.9, 29.6. HRMS (ESI) m/z calculated for C H N [M+H]+: 332,18697; found [M+H]+: 332,18698 20 22 5 7

202 Chapter 7

N3-pentyl-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4n)

Using benzaldehyde, 1-pentyl isocyanide, purification NH method A; 119 mg, 81% product was obtained as a brown N solid. 1H NMR (500 MHz, Chloroform-d) δ 7.87 (d, J = 6.9 N N Hz, 2H), 7.45 (t, J = 7.6 Hz, 2H), 7.40 (d, J = 4.4 Hz, 1H), 7.34

NH2 (t, J = 7.4 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 6.00 (s, 2H), 3.25 (s, 1H), 3.04 (t, J = 7.1 Hz, 2H), 1.56 (p, J = 7.2 Hz, 2H), 1.37 – 1.28 (m, 4H), 0.87 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) δ 149.5, 135.5, 133.7, 129.6, 128.9, 128.2, 127.9, 127.2, 126.2, 108.1, 48.7, 30.5, 29.2, 22.6, 14.1. HRMS + + (ESI) m/z calculated for C17H22N5 [M+H] : 296,18697; found [M+H] : 296,18680 N3-benzyl-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4o)

Using benzaldehyde, benzyl isocyanide, purification method A; 123 mg, 78% product was obtained as a brown solid. 1H NH NMR (500 MHz, Chloroform-d) δ 7.86 (d, J = 7.2 Hz, 2H), 7.42 N (t, J = 7.7 Hz, 2H), 7.35 – 7.25 (m, 7H), 7.19 (d, J = 4.6 Hz, 1H), N N 6.10 (s, 2H), 4.17 (d, J = 4.4 Hz, 2H), 3.60 (s, 1H). 13C NMR (126 NH2 MHz, Chloroform-d) δ 150.0, 138.8, 135.6, 133.7, 128.8, 128.8, 128.7, 128.2, 128.1, 127.8, 127.7, 127.1, 107.7, 52.5. HRMS (ESI) + + m/z calculated for C19H18N5 [M+H] : 316,15567; found [M+H] : 316,15555 (3-(8-Amino-3-(tert-butylamino)imidazo[1,2-a]pyrazin-2-yl)phenyl)boronic acid (4p)

Using tert-butyl (3-formylphenyl)carbamate, t-butyl HO NH B OH isocyanide and purification method A, adapted for the N polar character of the boronic acid moiety, by adding 5 N minutes to the run going up to 10% MeOH in DCM; N 132,2 mg, 81% product was obtained as a pale yellow NH 2 1 solid. H NMR (500 MHz, DMSO-d6) δ 8.49 (s, 1H), 8.19 – 8.13 (m, 1H), 8.04 (s, 2H), 7.74 – 7.69 (m, 1H), 7.67 (d, J = 4.8 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 4.8 Hz, 1H), 7.04 (s, 2H), 4.63 (s, 1H), 0.97 (s, 9H). 13C NMR

(126 MHz, DMSO-d6) δ 149.4, 138.4, 133.9, 133.8, 133.7, 133.7, 133.1, 133.0, 129.5, 128.4, 127.1, 127.0, 126.8, 125.7, 108.4, 108.2, 55.8, 30.0, 30.0, 30.0 ppm. HRMS (ESI) + + m/z calculated for C16H21BN5O2 [M+H] : 326,17828; found [M+H] : 326,17838

203 Diverse one-pot synthesis of adenine mimetics for biomedical applications

N3-(tert-butyl)-2-phenylimidazo[1,2-a]pyrazine-3,8-diamine (4q)

Using benzaldehyde, t-butyl isocyanide, purification method 1 NH B; 84 mg, 60% product was obtained as an off-white solid. H N NMR (500 MHz, DMSO-d6) δ 8.16 – 8.11 (m, 2H), 7.63 (d, J = 4.7 Hz, 1H), 7.40 (t, J = 7.7 Hz, 2H), 7.32 – 7.24 (m, 1H), 7.19 (d, N N J = 4.7 Hz, 1H), 6.76 (s, 2H), 4.62 (s, 1H), 0.98 (s, 9H). 13C NMR NH 2 (126 MHz, DMSO-d6) δ 150.0, 137.4, 135.0, 128.7, 127.9, 127.6,

127.4, 127.0, 126.2, 108.0, 55.7, 30.0. HRMS (ESI) m/z calculated for C16H20N5 [M+H]+: 282,17132; found [M+H]+: 282,17093

Methyl (8-amino-2-phenylimidazo[1,2-a]pyrazin-3-yl)glycinate (4r)

Using benzaldehyde, methyl 2-isocyanoacetate and purification O O method B; 90 mg, 60% product was obtained as a grey solid. 1H

NH NMR (500 MHz, DMSO-d6) δ 8.05 (d, J = 7.1 Hz, 2H), 7.71 (d, J N = 4.7 Hz, 1H), 7.44 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 7.19 N N (d, J = 4.7 Hz, 1H), 6.75 (s, 2H), 5.50 (t, J = 6.4 Hz, 1H), 3.78 (d, J 13 NH2 = 6.4 Hz, 2H), 3.52 (s, 3H). C NMR (126 MHz, DMSO) δ 171.9, 150.0, 134.1, 131.9, 128.4, 127.8, 127.2, 126.9, 126.4, 107.8, 51.6, + + 48.2. HRMS (ESI) m/z calculated for C15H16N5O2 [M+H] : 298,12985; found [M+H] : 298,12955 tert-butyl (3-(8-amino-3-(tert-butylamino)imidazo[1,2-a]pyrazin-2-yl)phenyl) carbamate (4s)

Using tert-butyl (3-formylphenyl)carbamate, t-butyl

NH NHBoc isocyanide and purification method B; 180 mg, 90% 1 N product was obtained as a brown solid. H NMR (500 N MHz, Chloroform-d) δ 7.94 (b, 1H), 7.56 (b, 1H), 7.52 (d, N J = 7.0 Hz, 1H), 7.36 – 7.30 (m, 2H), 6.66 (b, 1H), 6.06 (s, NH 2 1H), 3.15 (b, 3H), 1.53 (s, 9H), 1.04 (s, 9H).13C NMR (126

MHz, CDCl3) δ 152.8, 149.5, 139.4, 138.7, 135.0, 129.0, 126.8, 125.9, 122.5, 118.1, 117.7, 108.9, 80.5, 56.6, 30.2, 28.3. HRMS (ESI) m/z calculated for C H O N [M+H]+: 397,23465; found [M+H]+: 21 29 2 6 7 397,23444

204 Chapter 7

2-(4-Chlorophenyl)-N3-(2,6-dimethylphenyl)imidazo[1,2-a]pyrazine-3,8- diamine (4t)

Using 4-chlorobenzaldehyde, 2,6-Dimethylphenyl isocyanide, purification method A; 1mmol scale, 221mg, NH 61% product was obtained as solid. 1H NMR (500 MHz, N Cl DMSO-d6) δ 7.99 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.6 Hz, 2H), N N 7.25 (s, 1H), 7.14 (d, J = 4.6 Hz, 1H), 6.96 (d, J = 4.7 Hz, 1H), NH2 6.94 – 6.88 (m, 4H), 6.71 (t, J = 7.4 Hz, 1H), 1.88 (s, 6H). 13C

NMR (126 MHz, DMSO-d6) δ 150.2, 140.5, 133.5, 132.5, 131.6, 129.3, 128.4, 128.3, 128.2, 127.8, 126.4, 124.7, 120.9, 106.7, 18.5, 18.3. HRMS + + (ESI) m/z calculated for C20H19ClN5[ M+H] : 364,13235; found [M+H] : 364,13220 N3-(2,6-dimethylphenyl)-2-(4-methoxyphenyl)imidazo[1,2-a]pyrazine-3,8- diamine (4u)

Using 4-methoxybenzaldehyde, 2,6-dimethylphenyl isocyanide, purification method A; 1mmol scale 270mg, NH 75 % product was obtained as solid. 1H NMR (500 MHz, N OMe Chloroform-d) δ 7.99 (d, J = 8.9 Hz, 2H), 7.15 (d, J = 4.7 Hz, N N 1H), 6.99 (d, J = 7.5 Hz, 2H), 6.94 – 6.88 (m, 3H), 6.83 (t, J = NH 2 7.5 Hz, 1H), 5.71 (s, 2H), 3.82 (s, 3H), 3.48 (s, 1H), 2.02 (s, 6H). 13C NMR (126 MHz, Chloroform-d) δ 159.4, 149.5, 140.0, 136.9, 129.9, 128.4, 128.2, 127.9, 126.3, 126.0, 123.0, 121.9, 114.1, 108.1, 55.4, 18.6. HRMS (ESI) m/z + + calculated for C21H22N5O [M+H] : 360,18189; found [M+H] : 360,18146 N3-(benzo[d][1,3]dioxol-5-ylmethyl)-2-phenylimidazo[1,2-a]pyrazine-3,8- diamine (4v)

O Using benzaldehyde, piperonyl isocyanide, purification O method A; 130 mg, 72.2% product was obtained as a yellow NH solid. 1H NMR (500 MHz, Chloroform-d) δ 7.82 (d, J = 7.1 N Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.35 – 7.27 (m, 2H), 7.20 (d, N N J = 4.8 Hz, 1H), 6.74 (s, 1H), 6.67 (t, J = 6.4 Hz, 2H), 6.08 (s, 13 NH2 2H), 5.90 (s, 2H), 4.04 (s, 2H), 3.57 (s, 1H), 3.44 (s, 1H). C NMR (126 MHz, Chloroform-d) δ 149.9, 147.9, 147.1, 135.7, 133.7, 132.6, 128.8, 128.1, 127.7, 127.5, 127.1, 121.5, 108.6, 108.3, 107.7, 101.1, 52.3. + + HRMS (ESI) m/z calculated for C20H18N5O2 [M+H] : 360,14550; found [M+H] : 360,14542

205 Diverse one-pot synthesis of adenine mimetics for biomedical applications

Exemplary 1H-NMR and 13C-NMR of typical GBB-3CR products. 2-Benzyl-N3-(tert-butyl)imidazo[1,2-a]pyrazine-3,8-diamine (4f)

10000 7.26 CDCl3 7.26 7.25 7.24 7.23 7.22 7.22 7.21 7.21 7.21 7.20 7.20 7.19 7.18 5.74 4.17 2.56 1.16 7.27 7.27 7.28 7.28 7.28 7.29 7.50 7.49 7.29 7.29

9000 CH 3 CH 3 H C 3 8000 NH

N 7000 N N

NH 6000 2

5000

4000

3000

2000

1000

0 1.03 6.18 2.20 2.00 0.66 8.74

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) 90000

85000

80000 149.44 139.42 139.34 129.18 128.71 128.71 128.63 127.22 127.08 126.37 109.16 77.41 CDCl3 77.16 CDCl3 76.91 CDCl3 55.89 34.04 30.57

CH 3 75000 CH 3 H C 3 70000 NH

65000 N

N 60000 N

55000 NH 2 50000

45000

40000 35000 7 30000

25000

20000

15000

10000

5000

-5000

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

206 Chapter 7

N3-(tert-butyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)phenyl)imidazo[1,2-a]pyrazine-3,8-diamine (4l) 3400 6.80 8.21 8.19 7.71 7.69 7.64 7.63 7.19 7.18 4.66 3.33 2.50 2.50 2.50 2.49 2.49 1.30 0.99 3200

3000

2800

2600

2400

2200

2000

F (d) C (s) 7.63 0.99 1800

D (d) E (d) G (d) H (s) A (s) B (s) 1600 8.20 7.70 7.19 6.80 4.66 1.30

1400

1200

1000

800

600

400

200

-200 1.98 2.04 0.96 0.99 1.91 1.00 12.34 9.10 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm)

26000 24.71 24.79 30.04 30.07 39.02 39.19 39.35 39.44 39.52 39.61 39.69 39.78 39.85 39.94 40.02 40.11 55.85 83.58 108.03 108.11 126.80 126.85 127.52 128.87 134.03 134.15 136.82 137.92 150.11

24000

22000

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm)

207 Diverse one-pot synthesis of adenine mimetics for biomedical applications

Single Crystal X-Ray Structure Determination

Crystallographic data were collected on the«Xcalibur-3» diffractometer

(graphite monochromated MoKα radiation, CCD detector, ω-scanning,

2Θmax = 60°). The structure was solved by direct method using SHELXTL package.16 Positions of the hydrogen atoms were located from electron density difference maps and refined by “riding” model with Uiso = nUeq (n= 1.5 for methyl groups and n=1.2 for other hydrogen atoms) of the carrier atom.

Figure 4. Molecular structure of compound 4e according to X-ray diffraction data. Thermal ellipsoids are shown at the 50 % probability level.

The colourless crystals of 4e (C17H21N5O) are triclinic. At 293 K a = 6.0032(5), b = 12.429(2), c = 12.840(2) Å, α = 118.34(2)°, β = 90.432(9)°, γ = 103.477(9)°, V = 3 1 3 812.2(2) Ǻ , Mr = 311.39, Z = 2, space group P , dcalc= 1.273 g/сm , µm(MoKαa) = 0.084 mm-1, F(000) = 332. Intensities of 8230 reflections (4732 independent, 2 7 Rint=0.068) Full-matrix least-squares refinement against F in anisotropic approximation for non-hydrogen atoms using 4585 reflections was converged to wR2 = 0.154 (R1 = 0.068 for 1638 reflections with F>4σ(F), S = 0.813).

208 Chapter 7

Figure 5. Molecular structure of compound 4a according to X-ray diffraction data. Thermal ellipsoids are shown at the 50 % probability level.

The colourless crystals of 4a (C20H19N5) are monoclinic. At 293 K a = 20.174(3), 3 b = 5.9755(8), c = 29.439(4) Å, β = 104.96(1)°, V = 3428.7(8) Ǻ , Mr = 329.40, Z 3 -1 = 8, space group C2/c, dcalc= 1.276 g/сm , µm(MoKαa) = 0.079 mm , F(000)

= 1392. Intensities of 11965 reflections (3010 independent,int R =0.112) were measured on the«Xcalibur-3» diffractometer (graphite monochromated α MoK radiation, CCD detector, ω-scanning, 2Θmax = 50°). The structure was solved by direct method using SHELXTL package. Positions of the hydrogen atoms were located from electron density difference maps and refined by “riding” model with Uiso = nUeq (n= 1.5 for methyl group and n=1.2 for other hydrogen atoms) of the carrier atom. Full-matrix least-squares refinement against F2 in anisotropic approximation for non-hydrogen atoms using 2995 reflections was converged to wR2 = 0.141 (R1 = 0.066 for 1453 reflections with F>4σ(F), S = 0.943).

209 Diverse one-pot synthesis of adenine mimetics for biomedical applications

References

1 P. Muller, G. Lena, E. Boilard, S. Bezzine, G. Lambeau, G. Guichard, D. Rognan, Journal of Medicinal Chemistry 2006, 49, 6768-6778. 2 X. Xu, M. Huang, X. Zou, Biophysics Reports 2018, 4, 1-16. 3 F. M. Ferguson, N. S. Gray, Nature Reviews Drug Discovery 2018, 17, 353. 4 A. Chollet, L. Mourey, C. Lherbet, A. Delbot, S. Julien, M. Baltas, J. Bernadou, G. Pratviel, L. Maveyraud, V. Bernardes-Genisson, J Struct Biol 2015, 190, 328- 337. 5 H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res 2000, 28, 235-242. 6 D. Poli, M. Falsini, F. Varano, M. Betti, K. Varani, F. Vincenzi, A. M. Pugliese, F. Pedata, D. Dal Ben, A. Thomas, I. Palchetti, F. Bettazzi, D. Catarzi, V. Colotta, European Journal of Medicinal Chemistry 2017, 125, 611-628. 7 R. Garamvolgyi, J. Dobos, A. Sipos, S. Boros, E. Illyes, F. Baska, L. Kekesi, I. Szabadkai, C. Szantai-Kis, G. Keri, L. Orfi, European Journal of Medicinal Chemistry 2016, 108, 623-643. 8 J. R. Sayer, K. Wallden, T. Pesnot, F. Campbell, P. J. Gane, M. Simone, H. Koss, F. Buelens, T. P. Boyle, D. L. Selwood, G. Waksman, A. B. Tabor, Bioorgan Med Chem 2014, 22, 6459-6470. 9 H. Bienayme, K. Bouzid, Angew. Chem., Int. Ed. 1998, 37, 2234-2237. 10 C. Blackburn, B. Guan, P. Fleming, K. Shiosaki, S. Tsai, Tetrahedron Letters 1998, 39, 3635-3638. 11 K. Groebke, L. Weber, F. Mehlin, Synlett 1998, 661-663.

12 M. Guasconi, X. Y. Lu, A. Massarotti, A. Caldarelli, E. Ciraolo, G. C. Tron, E. Hirsch, G. Sorba, T. Pirali, Organic & Biomolecular Chemistry 2011, 9, 4144- 4149. 7 13 D. B. Salunke, E. Yoo, N. M. Shukla, R. Balakrishna, S. S. Malladi, K. J. Serafin, V. W. Day, X. K. Wang, S. A. David, Journal of Medicinal Chemistry 2012, 55, 8137-8151. 14 J. Ren, M. Yang, H. C. Liu, D. Y. Cao, D. Q. Chen, J. Li, L. Tang, J. H. He, Y. L. Chen, M. Y. Geng, B. Xiong, J. K. Shen, Organic & Biomolecular Chemistry 2015, 13, 1531-1535. 15 S. W. Krska, D. A. DiRocco, S. D. Dreher, M. Shevlin, Accounts Chem Res 2017, 50, 2976-2985. 16 G. Sheldrick, Acta Crystallographica Section A 2008, 64, 112-122.

210

Chapter 8

Summary and Discussion Chapter 8

The research described in this thesis covers the application of MCR methodologies aimed at expansion of the scaffold library and implementation of these scaffolds for the development of pharmaceutical relevant, drug-like compounds and diagnostic probes. Scaffold design was achieved either by clever application of bifunctional MCR components, or implementation of underexplored starting materials. Applicability of the scaffolds was found to be quite broad, covering biological targets such as anti-cancer and kinases, application as a new class of MRI contrast agents and peptidomimetics for yet to be discovered targets. More- over, the thorough review of one the youngest MCRs, the GBB-3CR, revealed affinity of the associated scaffold for a wide variety of biological targets. This scaffold is still underexplored, thus exhibits high potential for drug discovery. Improvement of IMCRs by addressing limitations in availability and/or problematic application of isocyanides was also shown. The praziquantel example covers an important process of both an MCR approach to synthesize a marketed drug in less steps into a more optimized reaction, as well as in situ formation of the isocyanide compound through readily available formamides. Overall the MCR scaffold design described in this thesis was successfully linked to new potential targets and the resulting compounds were applied as drug-like compounds and introduced beyond the scope of drug discovery.

In Chapter 2 the protein-protein interaction of p53 and Mdm2 was dissected into anchor points and linked to chemical mimics of the corresponding amino acid residues. Analysis of direct contacts between the protein binding site and interacting ligand residues allowed us to develop small molecule inhibitors.1 The protein databank PDB is continuously updated with newly identified crystal structures.2 Apoprotein crystal structures are often a good starting point, but co-crystal structures will deliver more information on how the ligand binds. A useful tool assisting in finding good binders is AnchorQuery (anchorquery.ccbb. pitt.edu/), recently published.3 By loading the receptor protein and defined pharmacophore points, a query can be run. The results are small molecule structures, containing a narrow variation of the predefined pharmacophores, connected intramolecular by MCR scaffolds. By docking a hit set, more structural information can be retrieved, and by removal of any structure either sticking out of the binding site, bearing structural features of PAINS or having ADMET compromising features, the hit set is reduced to a small library of possible binders. Due to the efficient and convergent nature of MCR chemistry, all synthesizable by a single step. The Ugi 4-component reaction is used to synthesize in a single step selective Mdm4 inhibitors. Although Mdm4 does not act as E3 ubiquitin ligase, the protein inhibits p53 by binding to the proteins transcriptional activation domain.4 The three anchor points for this interaction were Phe18, Trp23 and Leu26 and were chemically mimicked by various benzyl amines, carboxylic acids and isocyanides respectively. Paraformaldehyde, the fourth component serves as backbone of the dipeptidic U-4CR scaffold. By using indole like carboxylic acids in combination with bulky isocyanides and amines, many products precipitated from the reaction mixture in methanol. Therefore reactions were carried out in a 96-well filter plate, allowing high throughput synthesis of peptidomimetic molecules, wherein

213 Summary and Discussion the products were collected by filtration. The resulting compounds were found to be pure enough to be screened without the use of the often required, time consuming column chromatography purification step. Screening was performed by a fluorescence polarization assay, identifying hit compound B1 as a selective p53/

Mdm4 inhibitor (Ki = 5.5 µM). Exploration of structure-activity relationships was performed on the basis of this hit compound and a second library of compounds was synthesized. The structural features of B1; an indole and p-chlorobenzyl moiety originating from the carboxylic acid and amine respectively were slightly modified, while keeping the cyclohexyl group from the isocyanide intact. Despite variations on the benzylamine component, introducing various halogens (p-flu- oro-, p-CF3-, o,p-dichloro) and on the carboxylic acid component using substituted indoles and similar bicyclic heterocycle substitutions, no improvement on the Mdm4 inhibitory activity was observed in this library. Either way a selective Mdm4 inhibitor was developed, showing a 10-fold higher activity over Mdm2. Where further optimization will be presented in due course. Drugs going generic allows for innovation in the production process and once a generic drug enters the market, competition on the market could reduce the price significantly. It isn’t a surprise that praziquantel (PZQ), listed as essential medicine by the WHO, is affordable. The production of praziquantel is to date, however, unchanged since 1983. A process that is in current standards considered to be an environmental burden. With the introduction of the MCR methodology for preparing praziquantel in 2010, a big step was made towards a more green process, as the number of reaction steps was reduced from four to two, and no excess of highly toxic potassium cyanide is required in the synthesis.5 The use of isocyanides in the U-4CR might have hampered the adaptation of this green method. Chapter 3 introduced a way to circumvent the requirement of handling isocyanides by in situ formation of this reactant.6 With a variety of pre-existing work on the use of isocyanide precursors with in situ conversion in a one pot reaction, the isocyanide is only shortly present in the reaction mixture. Such methods are conversion of halides, using AgCN, from epoxides and formamide conversion with diphosgene. None of these methods were found to be broadly applicable with random starting materials and a method allowing more diverse product types was developed. The basis of this method is the dehydration of formamides. Al- though formamides are readily available, synthesis from aldehydes and amines by reductive amidation and formylation respectively, adds up to the diversity at lower costs. The methodology of in situ formation of phenethylisocyanide, required for the U-4CR was accomplished by using phenethylformamide dissolved in dichloromethane, in the presence of 2,4 equivalents of trimethylamine and slow addition of 0,4 equivalents of triphosgene at -10 °C. This temperature was later found to be stretched to even lower temperatures, enabling faster addition 8 of triphosgene, without formation of unwanted side products. Subsequent dilution of methanol is needed for the U-4CR and by the addition of paraformaldehyde, aminoacetaldehyde dimethyl acetal and cyclohexanecarboxylic acid, this reaction is run at room temperature for 48 hours. For the scale the reaction was performed on, we focused on application of only simple purification methods

214 Chapter 8 as crystallization and trituration. This approach gave a yield of 40% at 50 mmol scale for the Ugi reaction, yields are expected to increase at larger scale reactions. The Pictet Spengler reaction was unaltered from previously described methods and resulted in praziquantel in 50% yield. The challenge in this step, that should be noted, is the unreactive unsubstituted phenyl, taking part in this intramolecular cyclisation reaction. Where the reaction would readily run under mild acidic conditions, usually with indoles or methoxy- substituted phenyl rings, harsh conditions, using anhydrous methane sulfonic acid neat and heating at 70 °C was needed to accomplish the Pictet Spengler reaction for praziquantel. The next step in improvement in the production of praziquantel should be directed towards the enantioselective synthesis of the drug. Currently the racemic mixture is used to treat patients, in which the inactive enantiomer adds up to the dose size (30-60 mg/kg) for average adult of a staggering 2,400 mg, furthermore the bitter taste of the (S)-PZQ enantiomer hinders administration to infants.7-8 Asymmetric synthesis of the active (R)-PZQ would overcome these issues. A major challenge is maintaining the low cost of praziquantel production and is not possible with known resolution methods.9 Synthesis of amino tetrazoles as amino acid bio-isosteres has been developed using the Ugi azide 4-CR, using trityl amine as convertible amine component.10 Cleaving the trityl moiety yields a primary amine, which was found to open op- portunities such as secondary modifications. Additionally, the use of bifunctional starting materials allows for post modifications too and by aligning reactivity of a bifunctional reactant with a functional group in the same molecule, rearrange- ments or intramolecular transformations could take place. In this respect amines and esters would react to lactams. The combination of a tetrazole and a lactam connected to the same α-carbon would introduce a cis amide bond, of interest in biological systems as this type of bond is responsible for introduction of loop structures in proteins frequently through proline and glycine residues. In Chap- ter 4 the synthesis of 1,5 disubstituted tetrazolo lactam peptidomimetics was accomplished by reacting trityl amine, TMS azide, various isocyanides with linear aliphatic ester aldehydes.11 The trityl group on the UT-4CR was cleaved using TFA and subsequent cyclization was performed using sodium hydride. Through this procedure γ- and δ-lactams were synthesized with yields between 32-99%. Syn- thesis of compounds with longer chain length for the ε-lactam and aliphatic rings in the chain did yield the expected UT-4CR products. Nonetheless, cyclisation conditions as used for the γ- and δ-lactams was unsuccessful and rather yielded carboxylic acids as saponification products. Both X-ray structure analysis of some of the obtained products and analysis of the lactam moiety in the protein databank confirmed the importance of the N-unsubstituted lactam ring towards biological interactions. The key feature is the intermolecular hydrogen bond network the lactam-NH could undergo, manifesting in a bifurcated or trifurcated hydrogen bonding pattern with neighboring residues and/or water molecules, which is otherwise impossible with previous reported methods yielding exclusively N-substituted lactams. Exchange of the carboxylate with a bioisostere, by a 1H-tetrazole, on gadoter-

215 Summary and Discussion ic acid, it is believed that the stability of the compound would improve. This compound is used for MRI contrast enhancement and the gadolinium containing Dotarem agent, although being one of the best chelators of lanthanide metals, slowly releases the metal in the body. Normally fast clearance by the kidneys would remove the complex efficiently, and the concentration of free gadolinium would remain below toxic levels. Renal failure disrupts the fast clearance and consequently nephron- and neurotoxic effects will have a severe effect on the patients’ health. Attempts to functionalize the EDTA like macrocycles showed multistep routes and never a 3- or 4-fold substitution of the carboxylic acid with tetrazoles. Retrosynthetic analysis of the desired tetra tetrazole substitution indicated the UT-4CR as a suitable way to synthesize the tetra tetrazole compound and was described in Chapter 5.12 In this MCR, isocyanides will introduce a substituent incorporated on the tetrazole ring, thus the use of a convertible isocyanide is required to obtain the 1H-tetrazole after cleavage of the substituent. Usually t-butyl, t-octyl or benzylisocyanides are used due to their proven ease of removal by either acidic treatment or hydrogenation for the aliphatic or benzyl substituents respectively. During initial experiments however, the use of Walborsky’s reagent (t-octylisocyanide) resulted in formation of many sideproducts which were difficult to separate. Benzylisocyanide in the UT-4CR with cyclen and formaldehyde did yield the desired product and was subsequently subjected to hydrogenation conditions, using H2 (3 bar) and 40 mol-% Pd/C catalyst. Product was found in trace amounts and it was speculated that either the intermediate or deprotected product would form a complex with the catalyst, resulting in catalyst poisoning. Addition of a stoichiometric amount of catalyst, relative to the envisioned qua- druple deprotection gave a better conversion, visualized by 1H-NMR, but still incomplete and therefore difficult to purify, as the mono-, di- and tri-deprotected products exhibit a similar elution profile as compared to the fully deprotected compound in column chromatography. Aime et al. demonstrated in similar compounds that the β-cyanoethyl protecting group, often used as phosphotriester protecting group in solid-phase oligonucleotide synthesis, could efficiently be cleaved from tetrazoles under mildly basic conditions.13-14 This led to the development and application of the corresponding cleavable β‑cyanoethyl isocyanide.15 The reaction of this isocyanide resulted in quantitative conversion to the UT-4CR product, forming a sticky precipitate. Decanting the solvent together with unreacted TMSN3 and isocyanide, followed by dissolving the precipitate in ACN allowed us to remove the unreacted paraformaldehyde by filtration. Removal of the β-cyanoethyl protecting group went smooth within 2 hours in quantitative yields. Initially the deprotected product was purified by ion exchange chromatography, however by adjusting the pH of the compound in aqueous media to the isoelectric point (pH = 7,75) precipitated the neutral 1,4,7,10-tetrakis((1H- 8 tetrazol-5-yl)methyl)-1,4,7,10-tetraazacyclododecane (TEMDO). A key benefit in this method is the elimination of counter ions that otherwise would have been introduced by ion exchange chromatography. Preparation of the gadolinium complex did not pose any difficulties. Determination of the stability constant was, however, not that straightforward. Conventional titration methods such as

ITC and NMR rely on short equilibrium times and could not be used as the Kon

216 Chapter 8 kinetics of Gd3+-TEMDO were rather slow. Therefore a colorimetric method was used, with individual titrations of Gd-Arsenazo III (a color complex) with addition of TEMDO chelator aliquots. The series of individual mixtures in sealed vials were incubated for 6 weeks at 65 °C and from the absorbances measured by UV-VIS spectroscopy, the kinetics were determined. Although the stability constant was relatively high and in the range of other known contrast agents (Omniscan), the identical core structure to Dotarem reveals that tetrazoles are not the best ligands for lanthanide metal binding, supported by the seven orders of magnitude better stability constant of this marketed contrast agent. Stability parameters are generally performed under controlled conditions, nevertheless the high stability constant of Dotarem does not prevent Gd3+ leakage upon in vivo exposure, more specifically by degradation factors such as metabolism and transmetalation by endogenous metal ions, e.g. Cu(II), Ca(II) and Zn(II). Proof of concept was demonstrated by the delayed contrast-enhanced MRI application of Gd-TEMDO in vivo. Myocardial tissue damage, induced by occlusion of the left coronary artery, was studied in an animal model and by our contrast agent successfully visualized. The current applied gadolinium based contrast agents are based on chelators having appendant carboxylate ligand sites. Introduction of unprecedented tetrazoles as bioisosteric carboxylate replacements has proven to form a highly stable gadolinium complex, applicable as MRI contrast agent and the complex is suspected to exhibit improved stability in vivo. Such studies as well as introduction of functionalized TEMDO chelators are anticipated. One of the youngest MCRs, the Groebke-Blackburn-Bienaymè reaction is continuously explored and new amidines, aldehydes and isocyanides are tested for their scope and limitations in this reaction. The developments, highlights, scope and limitations of this reaction and expert opinion were discussed in Chapter 6. Based on the statement of the possibilities arising by introduction of bifunctional starting materials, the underexplored heterocyclic bis-amidine was investigated in Chapter 7. Bis-amidines can be arranged in symmetric and asymmetric pyrazines, pyrimidines and pyridazines. In the experimental design, these amidines were selected upon commercial availability and by this pyridazine-3,6-diamine, pyrazine-2,3-diamine and pyrimidine-4,6-diamine were subjected to typical GBB-3CR conditions using benzaldehyde, 2,6-dimethylphenylisocyande, with scandium triflate as catalyst. Reactions with pyridazine-3,6-diamine and pyrimidine-4,6-diamine both gave yield to the mono and bis GBB-3CR products, however found to degrade quickly when exposed to air. Unlike the aforementioned amidines, pyrazine-2,3-diamine exclusively yield the mono GBB-3CR product and does not exhibit stability issues. The imidazo[1,2-a]pyrazin-8-amine motif from this product was found to resemble an important part of the features responsible for binding of adenine/adenosine containing ligands. Moreover, the majority of small molecule kinase inhibitor drugs mimic the hydrogen bonds interactions formed through the adenosine part of ATP.16 This key feature of GBB- 3CR compounds as adenine mimics is a promising starting point, thus scope and limitations were explored for this reaction. First this one step procedure was optimized by variations in temperature, catalysts, solvents and catalyst load. Al-

217 Summary and Discussion though elevated temperature indicated a faster reaction course, the completeness was lacking as compared to the room temperature reaction. Heating of MCR reactions is often accompanied by formation of side products and could explain the observation of lower conversion, ultimately reducing yields. The least side products were observed in methanol as solvent, 10 mol-% Sc(OTf)3 while stirring at room temperature for 24 hours. The subsequent synthesized library of 22 compounds is a result of in total 22 performed reactions, although not every combination of isocyanide and aldehyde was high yielding, not a single reaction failed and an average yield of 70% was observed. Imidazo[1,2-a]pyrazin-8-amines were previously described in multistep reactions and involve an aromatic nucleophilic substitution from a halide to amine by treatment with ammonia, for the introduction of the amino group on the 8-position. This conversion is described to require harsh conditions, thus significantly reducing the substituent scope in regard to delicate substituents. In the synthesized library esters, Boc PGs, boronic acids and the associated pinacol esters were well tolerated, proving the mildness of this reaction. Application of synthesized adenine mimics and their biological activities will be investigated, where inhibition of Epac1/2, a protein involved in cAMP signaling, would be a good starting point. Conclusion Scaffold design is more relevant than ever as the lack of novel druglike structures hampers the development of drugs for newly discovered biological targets. The MCR methodologies included in this thesis have proven to efficiently introduce features such as selective high affinity binders and mimics of otherwise metabolic unstable moieties. Moreover, shortening the reaction routes of multiple existing preparations of drug and druglike compounds was successfully demonstrated. The Ugi 4-CR was used to combine the Leu-Trp-Phe pharmacophore features towards selective p53/Mdm4 inhibitor. This approach, resulted in a Mdm4 inhibitor, an anti-cancer target, exhibiting a 10-fold selectivity over Mdm2. Synthesis of amino acid derived tetrazole analogues has proven to be a valuable step towards discovery of new applications. Tetrazolo lactams as cis amido bond peptidomimetics and tetrazole based chelators for MRI application have been developed using the Ugi Tetrazole 4-CR. Exploration of applicability of bis-amidines in the Groebke-Blackburn-Bienaymè 3-CR led to the discovery of a one-step procedure for the preparation of imidazo[1,2-a]pyrazin-8-amines. Analysis of the PDB revealed interesting binding possibilities of this scaffold, as adenine mimetic is useful for targeting a wide variety of kinase/phosphorylation related pathways. The GBB-3CR scaffold is still underexplored, thus exhibits high potential for drug discovery. Improvement of IMCRs by addressing limitations in availability and/ or problematic application of isocyanides was also shown. The importance of 8 such an improvement, complemented by the developed scaffolds and mimics paved the way for broader application of in general IMCRs and therefore easy access to complex structures in a single reaction step.

218 Chapter 8

References 1 A. Boltjes, Y. J. Huang, R. van de Velde, L. Rijkee, S. Wolf, J. Gaugler, K. Lesniak, K. Guzik, T. A. Holak, A. Domling, Acs Comb Sci 2014, 16, 393- 396. 2 H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res 2000, 28, 235-242. 3 D. R. Koes, A. Domling, C. J. Camacho, Protein Science 2018, 27, 229-232. 4 S. Sane, K. Rezvani, International Journal of Molecular Sciences 2017, 18, 442.

5 H. P. Cao, H. X. Liu, A. Domling, Chem-Eur J 2010, 16, 12296-12298. 6 A. Boltjes, H. X. Liu, H. P. Liu, A. Domling, Org Synth 2017, 94, 54-65. 7 C. A. Palha De Sousa, T. Brigham, B. Chasekwa, M. N. N. Mbuya, J. M. Tielsch, J. H. Humphrey, A. J. Prendergast, The American Journal of Tropi- cal Medicine and Hygiene 2014, 90, 634-637. 8 J. Reinhard-Rupp, K. Klohe, Infectious Diseases of Poverty 2017, 6, 122. 9 M. Woelfle, J.-P. Seerden, J. de Gooijer, K. Pouwer, P. Olliaro, M.H. Todd, PLOS Neglected Tropical Diseases 2011, 5, e1260. 10 T. Zhao, A. Boltjes, E. Herdtweck, A. Domling, Org Lett2013 , 15, 639-641. 11 A. Boltjes, G. P. Liao, T. Zhao, E. Herdtweck, A. Domling, Medchemcomm 2014, 5, 949-952. 12 A. Boltjes, A. Shrinidhi, K. van de Kolk, E. Herdtweck, A. Domling, Chem-Eur J 2016, 22, 7352-7356. 13 S. Aime, G. Cravotto, S. G. Crich, G. B. Giovenzana, M. Ferrari, G. Palmi- sano, M. Sisti, Tetrahedron Letters 2002, 43, 783-786. 14 R. L. Letsinger, K. K. Ogilvie, Journal of the American Chemical Society 1969, 91, 3350-3355. 15 E. Kroon, K. Kurpiewska, J. Kalinowska-Tluscik, A. Domling, Org Lett 2016, 18, 4762-4765. 16 S. Gross, R. Rahal, N. Stransky, C. Lengauer, K. P. Hoeflich,The Journal of clinical investigation 2015, 125, 1780-1789.

219 Summary and Discussion

220

Samenvatting Ontwikkeling van geneesmiddelen begint met het achterhalen van de biologische processen die verantwoordelijk zijn voor specifieke ziektebeelden. Ontrege- ling van een enzym of eiwit activiteit in het lichaam is vaak de reden dat een ziekte zich kan manifesteren. Door dit soort enzymen of eiwitten te remmen of juist te activeren kan de biochemische balans hersteld worden en zo de ziekte genezen. De werking van eiwitten kan vergeleken worden met een stuk gereed- schap wat in staat is om lichaamseigen stoffen te herkennen en eventueel aan te passen. Welke stof herkend wordt, verschilt per eiwit en zo heeft het lichaam een groot scala aan eiwitten en enzymen, elk met een eigen functie op verschillende plekken in het lichaam. Sommige eiwitten communiceren met andere eiwitten en geven zo signalen door in het lichaam, terwijl andere eiwitten, zoals de enzymen, stoffen kunnen afbreken of opbouwen. De specificiteit van eiwitten is toe te wijden aan het zogenoemde sleutel-slot model. De vorm van een eiwit, de receptor, maakt dat alleen stoffen (chemische structuren/moleculen) met diezelfde vorm, het substraat, erin passen en dus interactie kunnen aangaan. Bij een onbal- ans in eiwit activiteit kan door middel van het namaken van het substraat in het lab een enzym geremd worden. Remming of inhibitie van eiwitten en enzymen is een belangrijk thema binnen geneesmiddelonderzoek. Binnen het vakgebied drug design wordt de receptor van eiwitten onderzocht, vaak met computer software en worden moleculen ontwikkeld die zowel de juiste vorm als affiniteit (aantrekkende krachten) hebben om te kunnen binden aan deze receptoren. De kunst is om een dusdanige affiniteit te krijgen dat de moleculen zeer makkelijk kunnen binden, maar ook selectiviteit om te voorkomen dat de verkeerde eiwitten worden geremd. Daarnaast is het belangrijk dat het lichaam de moleculen op de juiste plek kan krijgen, zonder dat deze door aller- lei metabole processen uit het lichaam worden verwijderd. Dit soort parameters bepalen hoeveel van de verbinding nodig is om een effect te geven, oftewel hoe groot de dosis moet zijn. Een dosis van 100 gram van een medicijn is onpraktisch en snelle afbraak maakt dat er vaker een dosis toegediend moet worden. Het doel is dus om potente verbindingen te ontwikkelen met goede bio-beschikbaarheid en geschikte ADMET (Administration (toediening), Distributie, Metabolisme, Excretie, Toxiciteit) eigenschappen. Het maken van medicijnen, de actieve verbindingen, wordt veelal in een chemisch lab gedaan. Een verbinding is een molecuul en moleculen worden gemengd met andere geschikte moleculen om deze met elkaar te laten reageren tot een nieuw molecuul, met nieuwe eigenschappen. Het opbouwen of synthetiseren van moleculen kan stap voor stap gedaan worden, zoals een lego-bouwset, tot- dat de verbinding alle eigenschappen bezit van een ‘drug-like’ verbinding. Elke synthese stap kost tijd en omdat niet elke chemische reactie volledig verloopt is chemische opzuivering vereist wat bij elke stap tot verlies lijdt. In plaats van een dergelijke sequentiële synthese kunnen ook alle stappen verwerkt worden in één reactie. Multi Component Reactie (MCR) chemie kan worden omschreven als het laten reageren van drie of meer componenten/moleculen tot één nieuw molecuul. In één reactie stap worden alle benodigde eigenschappen gecombineerd en door slimme keuze van de begincomponenten kunnen ‘drug-like’ verbindingen zeer efficiënt gesynthetiseerd worden. Er bestaan verschillende soorten MCR reacties, elk met de mogelijkheid om bep- aalde eigenschappen te kunnen combineren. Voor een brede toepasbaarheid van MCR chemie is het belangrijk om nieuwe MCR varianten te ontwikkelen. Het

223 Nederlandse Samenvatting werk beschreven in dit proefschrift omvat de ontwikkeling van een aantal nieuwe MCR variaties en hun toepassing als eiwit-remmers en diagnostische probes.

2 R NH2 HCHO NH R2 O O N R1 N R1NC H N OH O H Schema 1. Ugi-4CR voor high throughput synthese. In Hoofdstuk 2 wordt een remmer ontwikkeld tegen de eiwit-eiwit interactie van p53 met Mdm4. P53 is een eiwit dat verantwoordelijk is voor het stilleggen van de celdeling, wat bijvoorbeeld nodig is om schade aan het DNA te kunnen repareren. Verder zal p53 de cel laten sterven (apoptose) als de cel ongezond is. Mdm4 is net als Mdm2 verantwoordelijk voor de afbraak van p53, nadat de reparatie werkzaamheden zijn voltooid. Dit herstelt het vermogen om de cellen op een gezonde manier verder te laten delen. In het geval van kankercellen wordt dit reparatie mechanisme verstoord door overactiviteit van Mdm2/Mdm4 of door mutaties van het p53-gen, waardoor deze cellen zich blijven delen. Door de synthese van MdmX remmers wordt de functie van p53 hersteld met apoptose tot gevolg. Dit is een effectieve manier om kanker te bestrijden. Door middel van de Ugi 4-component reactie (Ugi-4CR) (schema 1) is een bibliotheek van ruim 100 verbindingen gesynthetiseerd. Hierbij is een nieuwe selectieve Mdm4-p53 inhibitor ontdekt. De selectiviteit is 10 keer zo hoog ten opzichte van Mdm2, met een Ki = 5,5 µM.

O O H2N H H O Triphosgene, Et3N HN O 6 + 7 MeOH, DCM, -10 °C to r.t. O O N H N O O HO O 10

8 9

N O MSA N 70 °C O

5 Schema 2. De Ugi-4CR met de in situ gegenereerde phenethylisocyanide vanuit het formamide 8, gevolgd door de Pictet-Spengler reactie waarbij uiteindelijk praziquantel (5) wordt verkregen.

224 In Hoofdstuk 3 wordt de synthese van praziquantel geoptimaliseerd. Praziquan- tel is een geneesmiddel dat gebruikt wordt ter behandeling van Schistosomiasis, een parasitaire ziekte welke wereldwijd meer dan 200 miljoen mensen besmet heeft. De synthese omvat een vier-staps-synthese, waarbij in de éérste stap grote hoeveelheden kalium cyanide wordt gebruikt. Door incidenten tijdens deze pro- ductie methode is in besmet rivierwater al het leven kilometers stroomafwaarts gedood. De nieuwe MCR methode omvat een Ugi-4CR, waarbij één van de stink- ende bestandsdelen in situ wordt gevormd (schema 2). Zodoende wordt middels ‘groene’ chemie in slechts twee synthese stappen praziquantel gemaakt, waarbij het direct hanteren van isocyanides vermeden wordt.

Schema 3. Beoogde route voor de synthese van tetrazolo N-ongesubstitueerde γ- en δ-lactams. Hoofdstuk 4 omvat de combinatie van een aminotetrazool- en een lactam- verbinding. De aminotetrazoolverbinding fungeert als een bio-isostere verbinding van een aminozuur. Lactams hebben een brede toepassing, zoals in antibioti- ca en als HRV protease inhibitors. De genoemde combinatie zou de affiniteit van dit soort verbindingen kunnen verhogen en eventueel fungeren als probe voor de ontwikkeling van nieuwe medicijnen. Door het toepassen van een ester bev- attend bi-functioneel component in de Ugi-Tetrazool-4CR, wordt eerst het amino tetrazool gedeelte geïntroduceerd. In een tweede reactie laten we de estergroep in basisch milieu intramoleculair reageren met het aanwezige amine tot N-ongesubstitueerde γ- en δ-lactams (schema 3). In totaal zijn er 11 verbindingen gesynthetiseerd, met variaties in lactam ringgrootte en tetrazool substituenten.

225 Nederlandse Samenvatting

Schema 4. De structuur van DOTA (1) DOTA, algemene oligotetrazolen, TEMDO en de retrosynthese hiervan.

De ontwikkelde aminotetrazool chemie wordt in Hoofdstuk 5 aangepast voor de ontwikkeling van een nieuwe generatie MRI contrast middelen. Moment- eel worden voor diagnostische MRI metingen, contrast middelen, zoals DOTA op basis van gadolinium toegepast. Door de toxiciteit van dit metaal is het van essentieel belang dat deze uitsluitend als het niet toxische gadoliniumcomplex wordt toegepast. De complexerende werking berust op coördinatie van gadolinium aan meerdere amino- en carbonzuurgroepen van het complexerende molecuul. Door metabolisme in het lichaam wordt dit complex afgebroken, met het lekken van gadolinium tot gevolg. Door het vervangen van de carbonzuren met tetrazolen wordt gepoogd de metabolische afbraak en daarmee lekken tegen te gaan. De verbinding TEMDO wordt middels de Ugi-Tetrazool 4CR gesynthetiseerd, gecomplexeerd met gadolinium en toegepast als MRI contrast middel, waarbij de contrast verbetering succesvol aangetoond is (schema 4).

In Hoofdstuk 6 wordt een overzicht gegeven van de ontwikkeling van de Groe- bke-Blackburn-Bienaymè 3CR (GBB-3CR). Deze reactie is in 1998 ontdekt als een variatie van de Ugi 4CR, waarbij amidines als amine component een ander product gaven dan in eerste instantie verwacht werd. Dit product bevat een imidazo[1,2a]heterocyclische verbinding als basisstructuur en heeft een veelbelovende toepassing als bioactieve verbinding. In de afgelopen twintig jaar is de reactie doorontwikkeld tot belangrijke MCR en in het overzicht worden de mogelijke variaties van de drie componenten, belangrijke ontwikkelingen van de reac- tiecondities en de biologische toepassing van de GBB-3CR

226 R2 NH N NH2 N R N 1 N NH2 N

NH2 NH2 GBB-3CR R2 NH N Degradation N N N NH2 R1 N H2N R2 R2 R1 NH Degradation N N NH R + HN 2 H2N N HN H2N NH2 N N R N 1 R N 1 N . Schema 5. Pyrazines, pyrimidines en pyridazines in de GBB-3CR.

In het in hoofdstuk 6 beschreven overzicht blijkt dat het gebruik van bis-amidines ondervertegenwoordigd is. Zodoende wordt in hoofdstuk 7 de toepasbaarheid van bis-amidines onderzocht. Het bleek dat diverse amidines weliswaar product geven, alsmede producten van een dubbele GBB-3CR, maar vervolgens de- graderen binnen een periode van minuten wanneer deze bloot gesteld worden aan lucht. Het bis-amidine pyrazine-2,3-diamine gaf echter stabiele producten en resulteerde daarnaast uitsluitend het mono GBB-3CR product. Het hierbij verkregen imidazo[1,2-a]pyrazin-8-amine motief heeft een vrij exocyclisch amine, wat mogelijkheden geeft tot waterstofbrug vorming en daarnaast grote gelijkenis vertoont met adenine, een onderdeel van adenosine. Het merendeel van klein moleculaire geneesmiddelen die werken als remmers op kinases, bootsen de gevormde waterstofbruggen na van het adenosine deel van ATP. Deze eigenschappen maken de GBB-3CR producten veelbelovend als adenine ‘mimetic’ met potentiele toepassing als kinase inhibitoren.

227 Nederlandse Samenvatting

228

Appendix

Acknowledgements List of Publications About the Author 231 Appendix

Acknowledgements Roughly four year have passed since I started with my own research projects resulting in several publications and ultimately the opportunity to pursue a PhD. Although it was not a typical trajectory I believe the sense of achievement must be comparable and I feel a sincere gratitude towards the many people I’ve met over the years, either giving inspiration or the support required to get this far and to finish this thesis. Dear Alex, your vision, dedication and drive have never stopped to amaze me. The trust you have given me over the years and the support to pursue my goals leading to this moment were absolutely invaluable. I am truly thankful for ev- erything you have done for me and showing the pragmatic approach to so many things and of course for being my promotor. Also an important part of this journey is to be attributed to my second promotor Prof. Frank Dekker. We started working more or less at the same time at the University of Groningen and after the medicinal chemistry group ceased to exist, you adopted me into your group until a new chair of drug design was found. During this period you have guided me through multiple challenging synthesis projects and taught me how even the littlest details matter. Thank you for your dedication during this period, your suggestions, discussions and the help on fi- nalizing my thesis. I would like to thank the assessment committee: Prof. Gerrit Poelarends, Prof. Adri Minnaard and Prof. Romano Orru for being so kind to invest their time to read and evaluate my thesis. Special thanks to the crystallization core of the Drug Design group, the first few that defined the methods and main structure, currently followed by the current group members: The first PhD student I was heavily involved in guiding; Ting, thank you for being my roommate and input in the initial synthesis of amino- tetrazoles. The Greek duo Dinos and Tryfon, thank you for all your knowledge and fun during activities in- and outside the lab. Edwin for your down to earth view on a lot of things, help as ‘sheriff’ of the SFC and the not to be underestimat- ed Dutch contribution to the group. Patil your ability to get chemistry projects to work in a matter of days are unmet, your directness towards others is extremely Dutch and your willingness to help and contribute wherever you can, deserves great respect and gratitude. My collaborators Eberhardt Herdtweck, Katarzyna Kurpiewska, Justyna Ka- linowska-Tłuścik and Svitlana Shishkina, thank you for the measurement and refinement of the various single crystal X-ray structures described in this thesis. Kees van de Kolk, I am grateful for your help with MRI measurements, the animal studies and supplying the proof of concept for our MRI contrast agent. I would like to thank Marcel, Annie and Margot for their help in performing both MS and HRMS analysis. Annadka Shrinidhi, thank you for giving insights in anorganic chemistry and from this perspective, your fresh view on performing organic chemistry and your help in the difficult task to determine the stability constant and all the associated calculations that came with it. I would like to thank my students; Rob, Laurie, Leander, George, Harmen, Benno, Iris, Kees and John for taking their part in the synthesis projects.

232 As the Drug Design group has grown tremendously in the past 8 years and many students, post-docs and visiting scientists came and left, it is easy to forget someone. I will try my best to acknowledge all of you. First of all the current direct colleague’s; Angelina, Bidong, Zefeng, Ruixue, Fandi, Maryam, Shabnam, Angel, Jingyao, Marta, Anne-1, Anne-2, Sara, Hylke, Kumchok and Francesco, thank you for all the help in the lab. Roberto, thank you for your assistance with ‘Ordnung’ in the lab and teaching me the secret recipe for making delicious pas- ta Bolognese. Li and Markella, thank you for your help in the adenine project and all the nice discussions we’ve had. Qian, the stereotype of a Chinese person does not apply to you, the way you ‘get to the point’ and our interactions made a smile to my face every time, thanks! Robin, don’t underestimate the part you have played in relieving me from several lab tasks, making my daily job as lab manager easier and allowing me to spend time on research. Also the humor and filling the Dutch void from when Edwin left, was much appreciated. Thank you! Much gratitude goes out to the structural biology subgroup of Drug Design, led by Prof. Matthew Groves and his students; Kai, Marleen, Juliana, Paul, Rick, Fer- nando, Jan Marten, Atilio, Ran, Chao and Wenjia. Without you guys, understanding of actual biological binding would not have been possible. The PhD students and post-docs that recently left the group shouldn’t be forgotten either; Many thanks to Natalia, Eman, Ajay, Chary, Naveen, Bhupendra, Kareem, Yuanze, Al- anod, Alaa, Eswar, Soraya, Niels, Santosh, Daniel, Wei and Silvia. The interdepartmental scientific interactions were mostly found in the Chemical and Pharmaceutical Biology group. I would like to acknowledge a few of my CFB colleagues; Martijn, Haigen, Linda, Hao, Nick and of course Pieter. Next I would like to thank my ‘other’ colleagues, not directly involved in the scientific processes. First and foremost, I would like to express my sincere gratitude to our secretary Jolanda, who is always there for the administrative aid, as well as being our tower of strength within the group. JP, although your position as ‘Tech- niker einz’ has my highest regard, I appreciate our conversations regarding our shared interests most, generally not related to work. Barbara, the list of things you've helped me with is endless. Thank you so much for everthing. Thanks to the guys from the instrument workshop, indirectly involved in taking care of the labs equipment and manufacturing all sorts of handy tools; Rick, Robert, Wolter, Frans, Roland, Erik, Hans, Jeroen and Evert. Special thanks to Anne Lexmond for the important role you have played of setting the official part of my PhD trajectory in motion. Without your intervention, the short period between admittance to the graduate school GSSE and ultimately the defense of this thesis, would not have been possible. My best friend Jelle, the way you approached your PhD and the years after, in- spired me to push through and finish my thesis. Thank you for your involvement in the smaller details of my PhD, the necessary moments of ‘epibreren’ and for being my paranymph. Jey, the first of the dark side, thank you for being my second paranymph. Over the years we started to appreciate each other’s fields more and more and nowadays are good friends through more than work only and both appreciate a proper ‘bakkie’ at any given time.

233 Appendix

Bij deze wil ik ook graag mijn vrienden bedanken, met name Sven, Arjan, Ju- riaan, Roy, Ronnie en Thanas, voor de broodnodige afleiding en ontspanning buiten werk om. Mijn familie, Pap, Mam, bedankt voor jullie steun tijdens elke fase van mijn opleiding en alle keuzes die onderweg gemaakt zijn. Het is dan eindelijk toch zover gekomen en zover gekomen ben ik. Daniëlle, mijn zus, ik vind het prachtig hoe je mijn uitleg over de chemie altijd wist te counteren met je kennis over de tandheelkunde. Het heeft me geholpen om te leren denken vanuit een breder perspectief dan uitsluitend mijn eigen vakgebied. Roos, Marit en Amber, mijn drie prachtige dochters. Ik wil jullie graag bedanken voor jullie enthousiasme voor alles wat te maken heeft met natuur en techniek. Het heeft me de drive gegeven om met hetzelfde enthousiasme te blijven ontdekken en nieuwe dingen te leren. Josina, mijn lieve vrouw, bedankt voor dat je dit avontuur samen met mij bent aangegaan en me gesteund hebt op moeilijke momenten. Zonder jouw liefde, jouw zorgzame karakter, support en de geschonken vrijheid, was promoveren nog een veel moeilijkere opgave geweest. Om de beurt maken we een stap voorwaarts, dit was mijn stap, nu mag jij weer.

234 List of Publications . A. Boltjes, A. Dömling, The Groebke-Blackburn-Bienaymé Reaction. Eur. J. Org. Chem. 2019, (In Press, 10.1002/ejoc.201901124). . A. Boltjes, H. X. Liu, H. P. Liu, A. Dömling, Ugi Multicomponent Reac- tion. Org. Synth. 2017, 94, 54-56. . A. Boltjes, A. Shrinidhi, K. van de Kolk, E. Herdtweck, A. Dömling, Gd-TEMDO: Design, Synthesis, and MRI Application. Chem-Eur J. 2016, 22, 7352-7356. . A. Boltjes, G. P. Liao, T. Zhao, E. Herdtweck, A. Domling, Ugi 4-CR synthesis of gamma- and delta-lactams providing new access to diverse enzyme interactions, a PDB analysis. Med. Chem. Comm. 2014, 5, 949-952. . A. Boltjes, Y. J. Huang, R. van de Velde, L. Rijkee, S. Wolf, J. Gaugler, K. Lesniak, K. Guzik, T. A. Holak, A. Dömling, Fragment-Based Library Generation for the Discovery of a Peptidomimetic p53-Mdm4 Inhibitor. Acs Comb. Sci. 2014, 16, 393-396. . S. Yerande, K. Newase, B. Singh, A. Boltjes, A. Dömling, Application of cyclic ketones in MCR: Ugi/amide coupling based synthesis of fused tetrazolo[1,5-a][1,4]benzodiazepines. Med. Chem. Comm. 2014, 55, 3263- 3266. . T. Zhao, A. Boltjes, E. Herdtweck, A. Dömling, Tritylamine as an Ammo- nia Surrogate in the Ugi Tetrazole Synthesis. Org. Lett. 2013, 15, 639-641. . R. Wisastra, M. Ghizzoni, A. Boltjes, H. J. Haisma, F. J. Dekker, Anacard- ic acid derived salicylates are inhibitors or activators of lipoxygenases. Bioorgan. Med. Chem. 2012, 20, 5027-5032. . M. Ghizzoni, A. Boltjes, C. de Graaf, H. J. Haisma, F. J. Dekker, Im- proved inhibition of the histone acetyltransferase PCAF by an anacardic acid derivative. Bioorgan. Med. Chem. 2010, 18, 5826-5834.

235 Appendix

Conferences . A. Boltjes, A. Dömling. The Groebke-Blackburn-Bienaymé reaction: Two decades later. Poster presentation: 7th International MCR 2018 Conference; 2018 Aug 26-31, Düsseldorf, Germany . A. Boltjes, H. X. Liu, H. P. Liu, A. Dömling. New approaches to employ the Ugi reaction, demonstrated through the synthesis of praziquantel. Poster session presented: NWO CHAINS Conference; 2017 Dec 5-7, Veldhoven, The Netherlands . A. Boltjes, A. Shrinidhi, K. van de Kolk, E. Herdtweck, A. Dömling. The next generation of tetrazole based MRI Contrast Agents. Orally presented at the MCB lecture series. 2014 Nov 27, Groningen, The Netherlands . A. Boltjes. K. van de Kolk, E. Herdtweck, A. Dömling. Development of a novel tetrazole based MRI contrast agent via the Ugi tetrazole reaction. Orally presented at the annual congress; Figon Dutch Medicine Days; 2014 Oct 4-6; Ede, The Netherlands . A. Boltjes, K. van de Kolk, E. Herdtweck, A. Dömling. Gadolinium(III) based chelators by multi component reactions: a new class of potential MRI contrast agents. Poster session presented: MCB2014 Conference; 2014 Aug 25-26, Groningen, The Netherlands . A. Boltjes, G. P. Liao, T. Zhao, E. Herdtweck, A. Dömling. Ugi 4-CR synthesis of gamma and delta lactams providing new acces to diverse enzyme interactions, a PDB analysis. Poster session presented: MCB2014 Conference; 2014 Aug 25-26, Groningen, The Netherlands

236 About the Author André Boltjes was born on the 10th of April 1984 in Wieringerwerf, The Netherlands. In 2007 he obtained his Bachelor degree in Organic and Analytical Chemistry at the Hanze University of Applied Sciences. For his graduation he worked on the development of new materials (hotmelts, coatings, foams, micro-encapsulation, etc.) in the field of polymer chemistry. After graduating, he started working at the University of Groningen as a technician for the Medicinal Chemistry group within the Groningen Research Institute of Pharmacy. In the initial years the focus was on the development of CNS drugs and prodrugs against conditions such as Parkinsons disease. Later, together with Prof. Frank Dekker, this focus moved to epigenetics and the synthesis of HAT and HDAC inhibitors. Finally, with the appointment of Prof. Alexander Dömling in 2011, his position developed into senior research technician/laboratory manager and was introduced to MCR chemistry. In 2014, under the supervision of Prof. Alexander Dömling, he proceeded with the basis of his doctorate work with attention toDevelopment and application of novel scaffolds in drug discovery. The research resulted in multiple publications in peer reviewed journals which are described in this thesis.

237