University of Groningen

Development of novel anticancer agents for protein targetsEstrada Ortiz, Natalia

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DEVELOPMENT OF NOVEL ANTICANCER AGENTS

FOR PROTEIN TARGETS

NATALIA ESTRADA ORTIZ 2017

Paranimphs:

Constantinos Neochoritis

Viktoriia Starokozhko

Cover design: Felipe Uribe Morales

Layout design: Natalia Estrada Ortiz

Printed by: Ipskamp printing

The research presented in this thesis was financially supported by the Department of Science, Technology and Innovation of the Colombian Government (Colciencias). Printing of this thesis was supported by the University of Groningen, Faculty of Science and Engineering and the University Library.

ISBN (printed version): 978-94-034-0142-3

ISBN (digital version): 978-94-034-0141-6

No parts of this thesis may be reproduced or transmitted in any form or any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system, without permission of the author

Development of Novel Anticancer Agents for Protein Targets

PhD thesis

to obtain the degree of doctor at theUniversity of Groningen

under the authority of therector Magnificus Prof. Dr. E. Sterken

and in accordance withthe decision by the College of Deans.

The public defense will take place on

Friday 20 October 2017 at 16.15 hours

by

Natalia Estrada Ortiz

born August 17, 1985in Medellin, Colombia

SupervisorsProf. A.S.S. DömlingProf. G.M.M. GroothuisProf. A. Casini

Assessment CommitteeProf. F.J. DekkerProf. P. OlingaProf. R.J. Pieters

CHAPTER 1

GENERAL INTRODUCTION

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CANCER

Cancer is a group of diseases involving abnormal cell growth with the potential to spread to other parts of the body, affecting populations in all countries and all regions. In 2012 there were an estimated 14.1 million new cases of cancer diagnosed worldwide (excluding non-melanoma skin cancer) and 8.2 million estimated deaths from neoplastic conditions.1,2 Specifically, cancer is a disease of abnormal gene expression, comprising more than 100 types of malignant neoplasms affecting humans and its increased incidence is often associated with genetic predisposition, factors as gender, ethnicity and age. Additionally, lifestyle choices as smoking, reduced physical activity, poor diet, and environmental exposure to toxicants, are known to be linked to the occurrence of a multitude of neoplastic diseases (Figure 1).3

Figure 1. Risk factors for development of cancer

In the last decades the main interest of various pharmaceutical companies is the development and discovery of potential anticancer drugs and treatments.4,5 For several years the research was focused on the discovery and development of conventional cytotoxic compounds. However, this approach showed a major liability, adverse effects due to general toxicity, due to poor selectivity of the drug candidates.

In fact, in the last 20 years, a great number of new experimental chemotherapeutic compounds have not been approved by the FDA (U.S Food and Drug Administration) and EMA (European Medicines Agency) due to their undesirable toxicity and cross reactivity observed in clinical trials.6 Recently, thanks to the advances in genomics and proteomics, our understanding of cancer has grown considerably, and enables the design of targeted therapies, leading to changes in the paradigms of anticancer drug discovery towards molecularly targeted therapeutics.7

The hallmarks of the events leading to neoplastic growth shared by most of the human cancers are depicted in Figure 2 and include: self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis,8 deregulation of cellular energetics, avoiding immune destruction, genome instability and mutation, and tumor-promoting inflammation.9 These characteristics comprise several pathways that

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can be targeted to develop anticancer drugs (small molecules, antibodies, etc.), hopefully with reduced side effects.

Figure 2. The hallmarks of cancer and therapeutic targeting strategies. Adapted from: hallmarks of cancer new generation, Hanahan, D.; Weinberg, R. A, 2011.

CHEMOTHERAPY STRATEGIES:

Most of the anticancer chemotherapeutic agents have severe side effects due to possible interactions with various biological targets, low tissue and cell specificity (i.e. no discrimination between healthy and cancer cells). Another drawback is the development of drug resistance particularly in very advanced tumors.10 In general, the mechanism of action of the classical anticancer drugs involves interference with pathways of cellular division, synthesis of nucleic acids and/or cell cycle proteins. This alteration can affect rapidly dividing healthy cells, leading to adverse responses, including induction of new tumors as a response to the genotoxicity of the treatment itself.11

The main targets of most of the new molecular-targeting drugs can be listed as receptors, membrane phospholipids, integrins, adhesion molecules, membrane-anchored receptors, enzymes, signaling proteins, RNA, microRNA, and DNA. They are typically dysregulated in tumor cells, compared to normal tissue and the novel anti-cancer drugs are designed to specifically modulate these targets.7,12 This dysregulation can occur as specific mutations or changes in the expression profile of certain proteins or transcription factors, to allow the tumor cells to grow rapidly avoiding the cell cycle check points that in normal conditions should lead to apoptosis or cell death. Therefore, targeting the specific dysregulated pathways should reduce the side effects observed in the traditional anticancer drugs. About 60% of the compounds available for chemotherapy are from natural origin 13–15 including taxol obtained from T. brevifolia, vincristine derived from the plant V. rosea, doxorubicin and bleomycin (figure 3), fermentation products of bacteria from the Streptomyces genus, among others.4,15,16 Analogues of natural compounds have been synthesized to improve their efficacy or toxicity profiles.13

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Figure 3. Examples of anticancer natural drugs

As an alternative to small molecules as anticancer agents, antibody-based therapy is one of the most valuable strategy to treat hematological malignancies and solid tumors.17,18 When the T cells of the immune system recognize cancer cells as abnormal, they generate a population of cytotoxic T lymphocytes (CTLs), that are able to locate and infiltrate malignancies, binding with them and subsequently killing the cancer tissue. CTLs, under normal physiological conditions require a balance to avoid destroying healthy tissue surrounding the malignancies.17,19–22 However, this response, is often inefficient, since tumors can actively highjack immunity, upregulating negative signals through cell surfaces, thereby inhibiting T-cell activation.18,19,22,23 The most common antibody therapies include targeting epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), lymphocyte-associated antigens CTLA4, CD20, CD30 and CD52.18 Recently, an extra target came to play an important role in immunotherapy against cancer, programmed death-1 (PD-1) protein, several antibodies based drugs targeting the PD-1 pathway are currently in clinical trials21 and a few had been approved by the FDA and the EMA.24,25 Additionally, the antibody-targeted therapy includes antibody drug conjugates, using antibodies and cytotoxic drugs (cisplatin, doxorubicin, etc.) in a synergistic manner to boost the cancer elimination and survival rate in the clinic.26 Some examples of the most successful antibodies based drugs in the market to treat cancer are cetuximab, bevacizumab and rituximab targeting EGFR, VEGF and CD20 antigens, respectively 27,28

Small molecules (organic and metal-based) are an essential element in the arsenal of drugs to fight cancer. Their main targets include: DNA, tyrosine kinases, protein-protein interactions (p53/mdm2, Bcl-xL/BH3, XIAP/Smac, etc), and modulation of protein synthesis.7 In the following section, the two different approaches with organic and metal containing compounds that were studied in this thesis are described.

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1. TARGETING PROTEIN-PROTEIN INTERACTIONS WITH SMALL MOLECULES In human cells, there are more than 300,000 Protein-Protein Interactions (PPIs)29 and this number is greatly larger than the number of single proteins. PPIs are of utmost importance and are implicated in almost all biological processes. Proteins fulfill their roles by interacting with many other cellular components, and the various interaction patterns of a protein are at least as important as the biochemical activity of the protein itself. Therefore, specific and potent modulation of PPIs by small, drug-like molecules would facilitate novel ways of drug discovery. 29–32 Although the biological relevance of PPIs is evident, this target class has been problematic in the design of new drugs.33,34 Crystal structures produced during the last 30 years showed that PPI interfaces are generally flat and large (roughly 1,000–2,000 Å2), making the design and development of small molecules that could target those regions troublesome.33,35 However, in the last decades, a huge progress has been made in developing synthetic compounds that modulate PPIs, using several strategies, including peptide mimetics, virtual screening, structural based design or screening of natural products or synthetic libraries.31 Currently, they are about 40 PPIs described as potential targets and several of these PPI inhibitors are currently in clinical trials.34,36 Herein, we present some examples of the most relevant PPIs as oncology targets for small molecules.

1.1. P53/MDM2(X) The protein p53, described for the first time in 1979, is considered to be the cellular gatekeeper or guardian for cell division and growth37,38 and was the first tumor-suppressor gene to be identified.39 P53 stimulates the expression of a set of downstream target genes that can induce apoptosis, facilitate DNA repair or activate cell cycle arrest upon cellular stress signals induced by DNA damage, oncogene activation and hypoxia.40–43 P53 is mutated in over 50% of human cancers and these mutations are related to the DNA-binding domain and preclude p53 from acting as a transcription factor.44 In other cases, the activity of p53 is inhibited either by binding to viral proteins or by alterations in other genes that code for proteins interacting with or linked to the function of p53 such as mouse double minute MDM2 and MDMX.45 The level of p53 in cells is subject to tight control, and its main non-redundant regulators are MDM2 and MDMX (MDM2 and MDM4 in mice, HDM2 and HDMX in humans), which cooperate with each other as part of a feed-back loop to regulate p53 activity in a different fashion .46 Therefore, blocking the interaction between wild-type p53 and its negative regulators MDM2 and MDMX has become an important approach in oncology to restore p53s antitumor activity. Interestingly, based on the multiple disclosed compound classes and co-crystal structures, p53–MDM2 is perhaps the most studied and targeted protein–protein interaction in the last decade.47–49 Furthermore, several compounds have proceeded into phase I, II and III clinical trials (Figure 4).30,49–51 Additional information about the current state of development of inhibitors of this important protein-protein interaction can be found in chapter 3.

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Figure 4. Main pathways with interesting PPIs to target with novel anticancer therapeutics. Adapted from: Protein–protein interactions and cancer: small Molecules going in for the kill, M. Arkin, 2005

1.2. BCL-XL/BH3 Evasion of apoptosis is one of the hallmarks of cancer cells, and the Bcl-2 family proteins play a crucial part.52,53 The Bcl-2 family of proteins consists of three main classes: the pro-apoptotic members Bax and Bak stimulate apoptosis by releasing cytochrome c from the mitochondria, while the anti-apoptotic members Bcl-2 and Bcl-xL inhibit Bax and Bak, and the BH3-only proteins (Bcl-2 homologue) promote apoptosis by inhibiting Bcl-2 and Bcl-xL proteins.54 Up-regulation of Bcl-2 or Bcl-xL is a common phenomenon in cancer and is usually related to drug resistance. Therefore, inhibitors of the of Bcl-2 or Bcl-xL, by using BH3 analogues to mimic Bcl-2/BH3 or Bcl-xL/BH3 protein-protein interaction, could induce apoptosis or work together with other know anticancer therapies.32,55 So far no dual inhibitor for both Bcl-2 and Bcl-xL made it to the clinic, but in 2016 a Bcl2 inhibitor, venetoclax (Figure 4), was approved by the FDA and the EMA for the treatment of chronic lymphocytic leukaemia.56

1.3. XIAP/SMAC Inhibitors-of-apoptosis proteins (IAPs) are negative regulators of apoptosis. X-linked inhibitor of apoptosis protein (XIAP), also known as inhibitor of apoptosis protein 3 (IAP3), is a protein that stops apoptotic cell death. XIAP is up-regulated in many cancers, therefore it has been an interesting oncological target for drug discovery. IAPs bind and inactivate caspases 3, 7 and 9.57–59 The pro-apoptotic protein Smac, a mitochondrial protein and negative regulator of XIAP, binds to IAPs, and induces the activation of caspases thereby re-activating the apoptosis cascade.7,31 Several compounds have been developed to inhibit XIAP to modulate its activity, and currently there are various compounds in pre-clinical and clinical trials (Figure 4).57,60–64

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Figure 5. Small molecules in clinical trials as modulators of p53/MDM2 (5-6), BCL-XL/BH3 (7) and XIAP/SMAC (8-9) protein-protein interactions.

2. METAL-BASED COMPOUNDS Metals have been used in medicine for 5000 years:65 in 3000 BC the Egyptians were using copper to sterilize water, to treat headaches, burns, and itching.66,67 Thereafter, in 2500 BC the Chinese were treating furuncles, smallpox and skin ulcers with pure gold.68 During the Renaissance mercurous chloride was used as diuretic agent and at the beginning of last century, arsenic (arsphenamine) was used to treat syphilis and gold cyanide was used for tuberculosis treatment.69 In 1912, antimony compounds were introduced to treat leishmaniasis, and in 1929 gold compounds were officially introduced as a therapeutic option for rheumatoid arthritis treatment.65,67,69

In the late 60’s, platinum drugs emerged as an alternative to treat cancer, and nowadays, they still appear in more chemotherapy regimens than any other class of anticancer agents.67,70,71 Since then, a multitude of experimental metal complexes have been developed showing different mechanisms of action and applications.65,67,72–74

2.1. CISPLATIN AND ANALOGUES Like numerous cytotoxic anticancer compounds, cisplatin was accidentally discovered by Barnett Rosenberg while he was studying the effect of electric fields on bacterial growth.75,76 E. coli cells were grown in medium containing ammonium chloride buffer, and an electrical current was applied to the bacteria through platinum electrodes submerged in the solution. and inhibition of the bacterial cell

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division was observed in the treated cultures.76,77 After a thorough investigation, it was found that the inhibition was induced by the hydrolysis products from the platinum electrodes which behave as cytostatic agents.78 Following these initial experiments, cisplatin was tested against panels of human cancer cell lines and found to be a potent cytotoxic compound. The mechanism of action of cisplatin is generally thought to be its interaction with DNA to form DNA-cisplatin adducts, primarily 1,2-intrastrand cross-links with purine bases (Figure 6). The damaged DNA elicits DNA repair mechanisms, which in turn activate apoptosis through several signal transduction pathways, including ATR, p53, p73, and MAPK, leading finally to cell death.79–81

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Figure 6. Molecular mechanism of cisplatin toxicity.

The potency of cisplatin was demonstrated in clinical studies, and it is currently used to treat different types of cancers including sarcomas, cancers of soft tissue, bones, and muscles.80 Unfortunately the side effects associated with cisplatin treatment in the clinic include severe toxicity to the kidney and the nervous system, hearing difficulties, nausea, and vomiting, among others. Subsequently, several studies were performed to optimize the properties of cisplatin, and to reduce its side effects, leading to the development and approval of carboplatin and oxaliplatin (Figure 7). Both these compounds became alternatives for clinical use for cancer types with acquired resistance to cisplatin and they are included along with cisplatin in the list of essential drugs of the world health organization.27,81–83

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Figure 7. Cisplatin and derivatives.

2.2. OTHER METALS The discovery of cisplatin and its platinum (II) analogues, suggested that other metal complexes might have similar antitumor activity, and probably could display a diverse pattern of cancer cells toxicity and selectivity.65 Starting from the 80’s, numerous metal containing compounds have been studied as potential anticancer agents, including ruthenium, palladium, copper, tin, and gold, among others.70 Metal-based compounds are known to bind and interact with a diversity of proteins with different roles, including transporters, antioxidants, electron transfer proteins, and DNA-repair proteins. 70,84–87 Among the possible pharmacological targets, several proteases are inhibited by Pt(II), Ru(II), Re(IV), Cu(II) and Co(III) complexes74,88–90. Several studies reported on the proteasome inhibition by anticancer Au(III),91,92 Ga(III), Zn(II) and Cu(II) compounds.93 Additionally, anti-diabetic vanadium complexes (Figure 8) have been studied as protein tyrosine phosphatase (PTP) inhibitors.94

Remarkable examples of metal complexes that made it into clinical trials (besides platinum derivatives) to tackle cancer are ruthenium coordination complexes. NAMI-A (Figure 8), imidazolium

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trans-tetrachloro-(dimethylsulfoxide)imidazole-ruthenate(III), and KP1019 were the first Ruthenium compounds to enter into clinical trials for anticancer therapy.67 Ruthenium complexes showed a different mechanism of action compared to the known platinum drugs, with reduced cytotoxicity and without inhibition of primary tumor growth, nonetheless, the complex was able to decrease the spread of metastasis.95,96

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Figure 8. Examples of metal containing drugs studied for the treatment of diabetes and cancer

2.3. GOLD COMPLEXES Gold-based complexes are particularly interesting due to their different possible oxidation states (e.g. Au(I) and Au(III)), stability and ligand exchange reactions, which confer them different mechanisms of activity compared to cisplatin.97–99 Preliminary studies on the anticancer activity of the Au(I) complex auranofin ([Au(I)(2,3,4,6-tetra-O-acetyl-1-(thio- S)- -D-glucopyranosato)(triethylphosphine)]), presently used in the clinic to treat severe rheumatoid arthritis, revealed cytotoxic activity levels similar to cisplatin on cancer cells in vitro (Figure 9).100 Auranofin is currently undergoing clinical trials to treat cancer, HIV, amoebiasis and tuberculosis.101 These findings subsequently led to a large number of Au(I) complexes being currently evaluated and developed as cytotoxic anticancer agents.98

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Figure 9. Gold complexes with interesting anticancer activity

Among the possible targets for cytotoxic Au(I) complexes, the seleno-enzyme thioredoxin reductase (TrxR) is among the most studied protein targets.70,74 TrxR belongs to the thioredoxin system involved in H2O2 detoxification and it is overexpressed in a number of cancer types.70,102 The thioredoxin system comprises the small redox protein thioredoxin (Trx), reduced nicotinamide adenine dinucleotide phosphate (NADPH), thioredoxin reductase (TrxR) and peroxiredoxin (Prx) (Figure 10). Thioredoxins are proteins that act as antioxidants by facilitating the reduction of other proteins by cysteine thiol-disulfide exchange, they are found in nearly all known organisms. Mitochondria are considered the most important cellular sources of reactive oxygen species (ROS). Superoxide anion can originate from the respiratory chain and is converted by superoxide dismutase to hydrogen peroxide, which is subsequently inactivated by the glutathione and thioredoxin systems leading to a steady state between formation and consumption of hydrogen peroxide.70,102 Gold is known for its high affinity towards thiol groups and consequently it is hypothesized that Cys-Se-Cys moiety present in C-terminal redox active site, could be the target of the gold containing compounds70,103

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Figure 10. Thioredoxin system scheme leading to intracellular redox balance by conversion of H2O2. From: Thioredoxin reductase: A target for gold compounds acting as potential anticancer drugs. Adapted from: A. Bindolini, et al., 2009.

The inhibition of thioredoxin reductase by gold(I/III) complexes, makes it unable to reduce back oxidized thioredoxin that accumulates together with hydrogen peroxide and both act on different intramitochondrial targets leading to the opening of the mitochondrial permeability transition pore and/or to an increase of the permeability of the outer membrane. Hydrogen peroxide is released to the cytosol where causes oxidation of Trx1, that, similarly, to mitochondrial thioredoxin (Trx2), cannot be reduced back by the gold(I/III) complexes-inhibited thioredoxin reductase. Oxidized thioredoxin stimulates the MAP kinases pathways leading to cell death. (Figure 11).

Figure 11. Mechanism of thioredoxin system by gold(I/III) complexes leading to cellular death. Adapted from: Thioredoxin reductase: A target for gold compounds acting as potential anticancer drugs, A. Bindolini, et al., 2009.

One of the most representative family of gold(I) compounds tested for their anticancer effects, is the organometallic gold(I) N-heterocyclic carbene (NHC) complexes featuring anticancer activity in the micromolar or sub-micromolar range in vitro.104–109 For example, compound 16 (Figure 9) and its derivatives displayed cytotoxic activity in the low micromolar range towards a small panel of cancer cell lines, comparable with cisplatin activity, and showed no interactions with model proteins such as lysozyme and cytochrome c. Conversely, they formed adducts with Atox-1 and it may imply that these gold compounds are able to inhibit the copper trafficking system with all the cellular consequences.107

Specifically, gold (I) NHC derivatives exert their effects via several pathways besides the inhibition of the seleno-enzyme thioredoxin reductase described above.70,110,111 Mitochondrial damage is common for cationic gold (I) biscarbene complexes, which behave as delocalized lipophilic cations that can

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accumulate selectively inside the mitochondria of cancer cells due to their higher mitochondrial membrane potential),109,112–114 . Furthermore, inhibition of protein tyrosine phosphatases (PTP) was also reported for certain anticancer Au(I) NHC complexes,115 and even stabilization of DNA G-quadruplexes (Figure 9, compound 17).98,116,117

CONCLUDING REMARKS

Over the last decades, the discovery and development of cancer therapeutics has been a rapidly growing area, with different approaches and applications to improve the current therapies used in the clinic nowadays.

However, adverse side effects are still a main concern in the drug development process. Therefore, it is important to improve the methodologies and the screening systems to determine potential adverse effects of drug candidates. Targeted therapies are designed to tackle cancer cells on their weak spots, specific miss-regulated pathways, but it is still necessary to carefully study possible off-targets to avoid side effects.

There is a growing need to improve the efficiency of developing new drug-like compounds. A new compound entering phase 1 clinical trial for any indication has about 8%4 chance to make it to the next clinical phase.

Drugs with different chemical profile, such us metal complexes, besides cisplatin, offer a new opportunity to discover new targets and ways to modulate toxicity directed to cancer cells due to the specific redox balance and microenvironment of such cellular group. Additionally, classic organic compounds, can be produced in mass to discover new lead hits. A way to reduce time and optimize the chemical synthesis is the use of multicomponent reactions (MCRs) to produced libraries of compounds that can be tested for biological activity on High Throughput Screening (HTS) systems to validated Structure Activity Relationship (SAR) and select lead compounds for further optimization.

In this thesis, we present the design and evaluation of new p53-MDM2/X inhibitors based on previously described compounds, to increase their potency and explore a different region on the p53-MDM2/X interphase as drug target. Additionally, a series of gold complexes were studied to understand their possible mechanism of action compared with cisplatin, including cancer cell based studies and healthy tissue toxicity evaluation using rat Precision Cut Tissue Slices (PCTS) to unravel uptake, pathways involved in the toxicity and possible selectivity of the compounds towards cancer cells.

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CHAPTER 2

AIMS AND OUTLINE OF THE THESIS

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This thesis is divided in two sections, exploring two different approaches to develop and validate potential anticancer agents, organic compounds and experimental metal complexes, to target different proteins and unravel or gain insights on the mechanism of action.

AIMS:

PART A: INHIBITORS OF P53/MDM2 INTERACTION:

To design and synthetize potent inhibitors of the p53/MDM2 interaction. To establish a structure activity relationship and validate their affinity towards the receptor, comparing the new series of compounds with previously described inhibitors.

PART B: BIOLOGICAL ACTIVITY OF VARIOUS FAMILIES OF METAL COMPLEXES

To evaluate in vitro the toxicity of the different families of metal complexes in human cancer cell lines to determine their potential as anticancer agents To evaluate the toxicity of potential anticancer metallodrugs in healthy tissue using rat precision cut liver and kidney slices. To study the mechanisms of transport of well-known and new metal based drugs using rat precision cut kidney slices.

OUTLINE

In chapter 1, an introduction about the main topics disclosed in this thesis is given, including basic background about cancer, currently used chemotherapeutic strategies and different kinds of targets, like protein families and DNA. A main focus is directed to small molecule inhibitors of protein-protein interactions with oncological importance, such as p53/MDM2, BCL-XL/BH3, XIAP/SMAC interactions. Additionally, an overview of the use of metals and metal complexes through history, and metallodrugs used currently in the clinic, their mechanism of action, with emphasis on cisplatin and experimental gold complexes.

Chapter 3 is a systematic review of p53/MDM2 interaction inhibitors, with emphasis on crystallographic structures and observed binding modes for different types of scaffold, their properties as well as preclinical and clinical studies of these small molecules and peptides. General guidelines for design of inhibitors based of the structural data, including conserved water molecules are given.

Chapters 4 and 5, describe the chemical synthesis of a new class of p53/MDM2 inhibitors, designed based on previously described compounds. For this we used our pharmacophore based virtual screening platform ANCHOR.QUERY as a tool to obtain multicomponent reaction scaffolds to antagonize p53-MDM2, binding specifically to the deep hydrophobic pockets of MDM2 on which the p53 protein binds as a helix. Chapter 4 describes the design and biochemical evaluation of the affinity of macrocyclic compounds that contain the same anchoring moiety predicted by our in-house

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software and an aliphatic handle to cover a large hydrophobic surface area formed by MDM2Tyr67, MDM2Gln72, MDM2His73 MDM2Val93 and MDM2Lys94, increasing the affinity to the receptor. Chapter 5 presents the synthesis and biochemical evaluation of the affinity of a new class of 1,2,3-trisubstituted bis(indoles) heterocycles derivatives designed to mimic the three key p53’s aminoacids for the binding with MDM2, and an extra interaction over MDM2Val93.

Chapter 6 examines the toxicity in cancer cells and in healthy tissue of a series of gold compounds synthetized as bifunctional compounds to act as chimeric compounds combining the cytotoxicity of gold and the drug-resistance reduction of a lansoprazole moiety through the decrease of the acidic microenvironment in cancer cells. The toxicity assessment was performed in an ex-vivo model, using rat precision cut kidney and liver slices, to determine the toxicity profile and was compared with the formerly reported toxicity in cancer cells. Classical pathways involved in cellular stress were studied to get insight in the mechanism of action.

Chapter 7 studies the mechanisms of uptake as well as toxicity of cisplatin in comparison to a cytotoxic cyclometallated gold(III) compound in precision cut kidney slices. The involvement of the Organic Cation Transporters (OCTs) in the uptake mechanism of cisplatin was studied using specific OCT inhibitors.

In chapter 8, a series of organometallic N-heterocyclic carbene (NHC) complexes was synthesized and characterized. The cytotoxic activities of the compounds were tested in 4 human cancer cell lines and their toxicity in healthy tissue was determined using rat precision cut kidney slices as a tool to determine the potential selectivity towards cancer cells.

Finally, the outcome of the work presented in this thesis is summarised and discussed in chapter 9. Additionally, future perspectives are included for the development and study of potential anticancer compounds, including the use of Precision Cut Tissue Slices to assess the possible undesirable side effects and toxicity in healthy tissue.

PART A

INHIBITORS OF P53/MDM2 INTERACTION

CHAPTER 3

HOW TO DESIGN A SUCCESSFUL P53-MDM2/X

INHIBITOR: A THOROUGH OVERVIEW BASED ON

CRYSTAL STRUCTURES

Natalia Estrada-Ortiz, Constantinos G. Neochoritis and Alexander Dömling*

Department of Drug Design, University of Groningen Antonius Deusinglaan 1, 9700 AD Groningen, The Netherlands.

Published in ChemMedChem 2016, 11, 757–772.

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Abstract: A recent therapeutic strategy in oncology is based on blocking the protein-protein interaction MDM2/X-p53. Inhibiting the binding between wild-type p53 and its negative regulators MDM2 and/or MDMX has become an important target in oncology to restore the anti-tumor activity of p53, the so-called guardian of our genome. Interestingly, based on the multiple disclosed compound classes and structural analysis of small molecule-MDM2 adducts, the p53-MDM2 complex is perhaps the best studied and most targeted protein-protein interaction. Several classes of small molecules have been identified as potent, selective and efficient inhibitors and many co-crystal structures with the protein are available. In this report we are describing properties, preclinical and clinical studies of the small molecules and peptides classified in categories of scaffolds. A special focus is on crystal structures and the binding mode of the compounds including conserved waters.

Keywords: -

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1. INTRODUCTION

Protein-protein interactions are of outmost importance and implicated in almost all biological processes. Proteins should not be considered to function as single, isolated entities but display their roles by interacting with other cellular components and the different interaction patterns of a protein is at least as important as the intrinsic biochemical activity of the protein itself. Therefore, the biological role of a protein is heavily based on the protein-protein interactions. Especially for diseases, the majority of cases ultimately relies on regulating the PPIs. Identifying and successfully targeting the PPIs by finding inhibitors or activators is the basis of the drug discovery.1

P53 is the principal regulator of the cell division and growth,2,3 being able to control genes implicated in cell cycle control, apoptosis, angiogenesis metabolism, senescence and autophagia.4 Mutations in this protein are present in about 50% of human cancers; altering the DNA binding domain ceasing p53’s activity as a transcription factor.5 The remaining tumors, p53 pathway is inactivated by up-regulation of p53 inhibitors, such as the mouse double minute proteins (MDM2 and MDMX, -also known as MDM4- HDM2 and HDMX in humans), or by down-regulation of p53 cooperators, such as ARF.6,7

The MDM2 gene was found to be upregulated in approximately 7% of tumors, with increased transcript levels and enhanced translation. Mutation of p53 and upregulation of MDM2 do not usually occur within the same tumor, indicating that MDM2 over-expression is an effective path for inactivation of p53 function in tumorgenesis.8 MDM2 functions as an inhibitor of the N-terminal trans-activation domain (TAD) of p53, and promotes p53 degradation through the ubiquitin-proteasome system (E3 ligase activity).9,10 On the other hand, MDMX can downregulate p53 via inhibition of the TAD domain, and it can upregulate MDM2.11

Moreover, it does not have E3 ligase activity, but its binding with MDM2 increases the rates of ubiquitinylation of p53 by MDM2.11,12 The C-terminal RING domains of both MDM2 and MDMX, is involved in dimerization; MDM RING domains can form homodimers, the heterodimers can be form by a reduced autoubiquitylation of MDM2 and increased p53 ubiquitylation.13 Consequently, MDM2 is stabilized by MDMX, keeping the levels of p53 low in healthy cells.13–15 The use of dual action MDM2/MDMX antagonists in cancer cells expressing wild type p53 should activate p53 more significantly than agents that only inhibit MDM2, resulting in more effective anti-tumor activity.16–18

The p53-MDM2 interaction is well druggable by small molecules based on a buried surface area of ~700 Å2, a well-structured deep and hydrophobic binding site of similar dimension than small molecules. For comparison the immune-oncology target protein-protein interaction PD1-PD1L has a buried surface area of ~2000 Å2, and is flat and featureless (figure 1). 19,20

The binding site of p53 in MDM2 is formed by 14 residues: Leu57, Met62, Tyr67, Gln72, Val75, Phe86, Phe91, Val93, His96, Ile99 and Tyr100. The cleft at the surface of MDMX is highly similar, 4 out of 14 residues are however different: MDM2Leu54>MDMXMet53, MDM2Phe86>MDMXLeu85, MDM2His96> MDMXPro95 and MDM2Ile99>MDMXLeu98.18 MDM2 and MDMX have a deep hydrophobic pocket on which the p53 protein binds as an alpha helix. Three p53 amino acids are deeply buried in the MDM2 and MDMX clefts: Phe19, Trp23, and Leu26 and the interaction is mostly governed by hydrophobicity (PDB: 1YCR, figure 1).21,22

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Figure 1. Two important protein-protein interaction targets: left p53 (green surface) with MDM2 (grey surface), PDB: 1YCR; below: footprint of p53 on MDM2 shown as blue surface. Right: PD1 (green surface) with PD1L (grey surface, PDB: 4ZQK); below: footprint of PD1 on PD1L shown as blue surface

Figure 2. p53 key amino acids Phe19, Trp23 and Leu26 bound to MDM2 (PDB: 1YCR). Red dotted lines indicate the polar contacts

Additionally, the Trp23 indole-NH forms a hydrogen bond with the backbone carbonyl of MDM2Leu54. In MDMX, Met53 and Tyr99 are bulged into the hydrophobic surface groove making it smaller and slightly different in shape (PDBs: 3DAB, 3DAC).21 The known three finger pharmacophore model for p53-MDM2 is now widely accepted to be responsible for the binding of small molecules and peptides to the MDM2.23–25 Recently an extended four finger model was proposed taking into account the intrinsically disordered MDM2 N-terminus which can be ordered by certain small molecules as shown by co-crystallization.26,27

Due to the importance of p53-MDM2/X protein-protein interaction, several reviews have been published in the last years.28–31 This review is giving a broad recent overview of inhibitors of the p53-MDM2/MDMX protein-protein interaction, mostly focusing on the available structural data. Additionally, some insights into the rational design and optimization of MDM2/X binder, their activities in vitro, in vivo and clinical trials are given. Moreover, a preliminary water analysis of the current crystal structures is presented.

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2. INHIBITORS OF THE P53-MDM2/MDMX INTERACTIONS

2.1. NUTLIN-TYPE COMPOUNDS The first class of highly potent, specific and orally active MDM2 inhibitors, was disclosed in 2004 by scientists from Hoffmann-La Roche,32 cis-diphenyl substituted imidazolines, known as Nutlins (1). Modifications and optimizations resulted in derivatives named Nutlin 1 (1a), Nutilin 2 (1b) and Nutlin 3a (1c) (figure 3). Nutlin compounds show inhibitory concentration values (IC50) of 260, 140 and 90 nM, respectively. These compounds displaced recombinant p53 fragment (corresponding to residues 1-312 of human p53) from its complex with MDM2 using surface plasmon resonance in a competition assay (SPR).32 Nutlin 2 complexed with MDM2 co-crystal structure (PDB: 1RV1, figure 4) led to the elucidation, for the first time of a non-peptide, small molecule structural information of the interaction.32 Superimposition of the co-crystal structures MDM2-Nutlin 2 and MDM2-p53 (PDB: 1YCR), showed that the two bromo-substituted phenyl rings and the ethoxyl group of Nutlin 2 mimic the Trp23, Leu26 and Phe19, respectively, the key hydrophobic binding residues of the p53 peptide (figure 1).32 Nutlin 3a bound to MDM2 (PDB: 4J3E)33 shows that both 4-chlorophenyl groups perfectly fill the Leu26 and Trp23 pockets, while the isopropoxy group reaches deep into the Phe19 pocket. It activates wild-type p53 and selectively kills cancer cells, with IC50 of 1-2 μM in the SJSA-1, HCT116 and RKO cell lines (osteosarcoma, colorectal and colon carcinoma, respectively) and showed 10-fold selectivity compared with p53 mutated cell lines MDA-MB-435 and SW480 (melanoma and colorectal adenocarcinoma, respectively).32 Furthermore, in vivo studies demonstrate the capability of Nutlin 3a to reduce tumor growth by 90% in SJSA-1 in mice xenograft model.32

In 2008, compound 2 (RG7112) based on Nutlins (PDB: 4IPF),34 completed phase I clinical trials against advanced solid and soft tissue tumors and hematological malignancies.35,36 Four key modifications were made to enhance the binding activity to MDM2, cellular growth inhibition and improve pharmacokinetic properties: Two methyl groups were introduced in the imidazole ring to protect from metabolism, the isopropyl ether was replaced to ethyl ether to reduce the molecular weight maintaining the hydrophobic interactions, the methoxy group was changed by a tert-butyl moiety to decrease the metabolic liability and lastly, methyl sulphonyl group was added onto the piperidine ring to increase the binding affinity and improve the pharmacokinetics via reduced logD. After these modifications, compound 2, displayed enhanced binding activity towards MDM2 with a Kd 10.7 nM and cellular growth inhibition 3-fold more potent compared with Nutlin 3a.33 The main drawback shown during the clinical trials in patients with liposarcoma, was thrombocytopenia, but the data obtained from biopsies in this preliminary trial suggested that the p53 pathway can be reactivated despite the presence of excess of MDM2, resulting in cytostatic and possibly apoptotic effects in tumor cells (figure 3).35,37

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ON

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Figure 3. Nutlins and imidazole, imidazothiazole derivatives

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Figure 4. Crystal structure of Nutlin-2 bound to MDM2 (PDB: 1RV1) Nutlin-2 (green sticks) receptor (grey sticks) interactions are indicated by colored dotted lines: yellow; -cation dipolar, chocolate; halogen, red; dipolar interactions. The receptor and compound are aligned with the p53 hot spot amino acids (magenta lines, PDB: 1YCR) (color code and the alignment maintained throughout the remaining figures). The 3-(4- -Leu54; the 2-(4-bromophenyl) residue is making multiple hydrophobic contacts to Leu54, Gly58, Ile61 and Val93.

2.2. IMIDAZOLES AND IMIDAZOTHIAZOLES Tri-substituted imidazole compounds 3-5 with a 6-chloroindole moiety as tryptophan mimic were discovered by a virtual Trp23-anchor based pharmacophore screening approach and validated as MDM2 inhibitors (figure 3).38 The side chain of p53-Trp23 is embedded in a deep hydrophobic pocket formed by the MDM2 residues Leu57, Phe86 and Ile99 and it was noted early on that at the bottom of the indole-Trp23 unfilled hydrophobic space is left which could be filled by a suitable hydrophobe.39 Thus substitution of the indole in the 6-position by a methyl, alkynyl group or halogen atom can enhance the binding affinity by a factor up to 50. In addition, two other fragments were introduced to mimic leucine and phenylalanine residues, leading to 3 (WK23), co-crystalized with MDM2 (PDB: 3LBK) with a binding affinity 916 nM and 36 μM for MDM2 and MDMX, respectively. Its analogue 4 (WK298) with a binding affinity 109 nM and 11 μM for MDM2 and MDMX, respectively, comprises the first co-crystal structure of a small molecule bound to MDMX (PDB: 3LBJ).40 These crystal structures clearly reveal that 1,3,5-trisubstituted 5-indolo imidazoles compounds can bind both to MDM2 and MDMX and thus provide a starting point for the design of dual action MDM2/X antagonists. Compound 3 binds to MDM2 by filling its Trp23 subpocket with the 6-chloroindole group, the 4-phenyl group is located in the Phe19 and 1-(4-chlorobenzyl) group in the Leu26 pockets. A hydrogen bond is formed between the indole of 3 and the Leu54 carbonyl oxygen of MDM2 similar to the Trp23-Leu54 hydrogen bond found in the endogenous p53-MDM2 interaction (figures 3 and 5).40 The inhibitor 4 binds to MDMX in a similar way, despite the different shapes of the p53 binding sites in MDM2 and MDMX. The His96-Tyr100 region has the most pronounced differences in the shape of the Leu26 pocket, but the position of 1-(4-chlorobenzyl) is not altered. Two hydrogen bonds with MDMXMet53 and MDMXHis54 are formed, whereas the N,N-dimethylpropylamine part of 4 folds over MDMXGly57 and MDMXMet61, forming additional hydrophobic protection of the binding cleft, where MDMXTyr99 closes the p53Leu26 subpocket.40

Furthermore, the 3-imidazolyl indole inhibitor 5 (PDB: 4DIJ) was reported based on the central valine concept of Novartis by which a planar aromatic ring (imidazole) was placed in Van der Waals contact with the side chain of Val93, occupying a central position in the upper part of the MDM2 pocket and providing different type of substitutions.41 -between MDM2 residue His96 and the benzylic chlorophenyl ring of the compound was observed.

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Attempts to optimize compound 5 either by promoting the formation of hydrogen bond with His96 and/or improving the cellular activity, led to the tetra-substituted imidazoles 6 (PDB: 4OQ3) and 7. Modeling showed that the plane of the inhibitor core imidazole ring and that of its chlorophenyl ring are nearly perpendicular, with an angle of 80o (figure 3).41,42

Figure 5. Crystal structure of compound 3 (WK23) bound to MDM2 (PDB: 3LBK): A hydrogen bond between the indole N-H of 3 and the MDM2Leu54 carbonyl oxygen is depicted. The 1-(4-chlorobenzyl) group undergoes a T- -stacking to His96. The compound is making multiple hydrophobic interactions with Leu57, Gli58, Ile61, Met62, Val93, His96 Ile99 and Tyr100.

developed as potent inhibitors of the p53-MDM2 interaction. As an initial lead, scientists from Daiichi-Sankyo reported compound 8.[22] Further optimization by using a methyl group onto the C-6 position to avoid oxidation, and by modifying the C-2 moiety of the additional proline motif, led to compound 9 (PDB: 3VZV) with an IC50 of 8.3 nM in a homogeneous time-resolved fluorescence assay (HTRF).43,44 The pyrrolidine moiety at the C-2 position induced another hydrophobic interaction site with MDM2 protein (Met50, Tyr67), as the co-crystal structure analysis revealed (figure 6). Moreover, solubility was improved by introducing an alkyl group into the pyrrolidine at the C-2 substituent and modifying the terminal substituent of the proline motif. Compound 10 (PDB: 3W69) with an IC50 of 59 nM (HTRF) exhibited also good pharmacokinetic profile and significant antitumor efficacy via oral administration on mice xenograft model using MV4-11 cells bearing wild type p53 (figure 3).43,44

Figure 6. Crystal structure of compound 9 bound to MDM2 (PDB: 3VZV): The pyrrolidine moiety induced a new hydrophobic interaction site with Met50 and Tyr67. Multiple hydrophobic interactions are formed with Thr26, Leu54, Ile61, Met62, Val93, Ile99 and Tyr100.

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2.3. INDOLES AND SPIROOXINDOLES Using the pharmacophore search software ANCHOR.QUERY,45 a series of 6-chloro-indole derivatives were discovered, based on Ugi multicomponent reaction chemistry (figure 8).46

A novel class of compounds derived from an Ugi multi component reaction (Ugi four component-five centers, U-5C-4CR) showed potent binding towards MDM2 and MDMX, via the classical three finger pharmacophore model, mimicking the hot spot amino acids Phe19, Trp23 and Leu26. The most potent compounds are 11 (PDB: 3TJ2), 12 (PDB: 4MDQ) and 13 (PDB: 4MDN) with Ki of 0.4, 1.2 and 0.6 μM, respectively in a fluorescence polarization assay (FP), which was verified by heteronuclear single quantum coherence (HSQC) experiment where 15N labeled MDM2 is used and thus the ligand-induced perturbations in NMR chemical shifts are observed.26,46 Compound 13 exhibited a novel fourth pharmacophore binding motif in MDM2, that is directly connected to the Leu26 sub-pocket forming a deep binding spot around the fourth pharmacophore point of the molecule. A hydrogen bond is formed again between the indole of 13 and the Leu54 carbonyl oxygen of MDM2 mimicking the Trp23-Leu54 interaction. Furthermore, a halogen bond between the Cl of the p-chlorobenzyl moiety and the carbonyl groups of Glu23 is possibly formed (figure 7).26,46,47

Researchers from Hoffmann-La Roche, identified a series of indolyl hydantoin compounds as potent, dual MDM2/MDMX inhibitors. The most representative compound of the series, 14 (RO-2443, PDB: 3VBG) showed an outstanding inhibitory activity against both MDM2 and MDMX with IC50 for the binding to p53 of 33 nM and 41 nM respectively. Additionally, RO-2443 binds to the p53 pocket of MDMX and crystallographic and biochemical investigations suggested protein homodimerization and heterodimerization as mechanism of action (PDB: 3U15).48 Due to poor water solubility it was initially not possible to assess cellular activity; thus after supplementary optimization, compound 15 (RO-5963) was derived which inhibited the binding of p53 to MDM2 and MDMX and showed strong antiproliferative activity in cancer cell lines overexpressing MDMX (figure 8).48 These dimerizer molecules comprise a Michael acceptor system and thus the observed antiproliferative effects observed cannot be clearly assigned to one mode-of-action.

Figure 7. Crystal structure of compound 13 bound to MDM2 (PDB: 4MDN): A hydrogen bond is formed again between the indole NH of 13 and the carbonyl oxygen of Leu54. The compound is making hydrophobic interactions with Glu23, Met50, Leu54, Phe55, Gly58, Gln59 Ile61, Met62, Val93, Ile99 and Tyr100.

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Figure 8. Indoles and spirooxindoles derivatives as inhibitors of p53-MDM2/X interaction.

Recently, by performing high-throughput virtual screening, a new class of pyrido[b]indole derivatives were discovered, with the most active compound of this class compound 16 (SP-141), possessing a Ki value of 28 nM for the binding with MDM2 in a FP based assay. Significant inhibition of breast cancer cell growth, and decreased growth of tumors in two different breast cancer xenograft models were observed (figure 7).49 However the binding mode of 16 is unclear and does not fit the 3-point pharmacophore model shown by the rest of the compound discussed here.

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Another privileged structure for the p53-MDM2 inhibition is the spirooxindole. Based on the insight that an oxindole group can mimic the Trp23 moiety, spirooxindole-containing natural products were identified and docked to MDM2. A series of compounds e.g. 17 (PDB: 4JVR), discovered via high-throughput screening assays based on spirooxindoles, were developed with favorable results in HTRF assays showing IC50 of 9.4 nM.50 Compound 18 (MI-63) was also developed having a Ki of 36 nM and 55 uM on MDM2 and MDMX respectively.51 Analyzing the crystal structure of a MI-63 analogue (PDB: 3LBL), the compound binds to MDM2 also by nesting the chlorophenyl substructure of the 6-chlorooxindole into the Trp23 subpocket. The Leu26 subpocket is filled by the 2-fluoro-3-chlorophenyl ring which is located as in compounds 3 (WK23) and 4 (WK298) but its plane is rotated to allow the phenyl substituent atoms to fill the bottom of the MDM2 pocket. The neopentyl fragment fills the Phe19 pocket and causes a substantial induced-fit reshaping of the binding cleft, the Tyr67 side-chain is rotated to close the binding region and the whole Tyr67-His73 region acquires a different fold to allow the Tyr67 movement. The compound forms two hydrogen bonds with Leu54 and His96. The ethyl-morpholino part of the compound is not taking part in the binding and is not seen in the electron density (figure 7).40,50

Thorough optimization of the scaffold led to compounds with Ki values in the low μM and nM range (compounds 19-21), giving promising results in vitro and in vivo assays with compounds 20 (MI-219) and 21 (MI-888) showing Ki values of 8.5 μM and 5 nM (HTRF), respectively.22,50 An analogue of compounds 20 and 21 which has been advanced into phase I clinical trials, is compound 22 (MI773, SAR405838). This compound exhibits Ki of 0.88 nM (10-fold more potent than MI-219). As the co-crystal structure reveals the inhibitor 22 mimics the three important amino acids, captures additional interactions that were not observed in the p53-MDM2 complex and induces refolding of the unstructured N-terminal region of MDM2 to achieve its high affinity. Furthermore, 22 activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis (figure 7).52

Based on MI-series of compounds, Hoffman la Roche scientists developed modified spirooxindole small molecules using an additional phenyl moiety to fit into the Phe19 pocket and an isopropyl group to mimic Leu26 (23, RO-8994) with an IC50 of 5 nM (HTRF).53,54 As expected, the 6-chloro-1,3-dihydro-2H-pyrrolo[3,2-c]pyridin-2-one moiety in compound 23 is buried into the Trp23 pocket, and its NH moiety forms a hydrogen bond with a backbone carbonyl of MDM2 for enhanced binding affinity. Since the interaction in the Trp23 pocket appears to be the most critical, further exploration of bioisosteric replacements on the phenyl moiety of 6-chlorooxindole, while preserving other important architectural features in RO8994 for optimal binding and pharmacological properties led to 24 (RO-2468) and 25 (RO-5353, PDB: 4LWV) with an IC50 of 6 and 7 nM (HTRF) respectively. In the latter, the 2-chlorothienyl[3,2-b]pyrrol-5-one group is buried into the Trp23 pocket, whereas the 3-chloro-2-fluorophenyl group occupies the Leu26 pocket (figure 9).53 Examples 23-25 nicely show that bioisosterc replacement of the abundant p-halogen substituted phenyl groups in the Trp23 pocket by heteroraromatic rings is possible and can be potentially used to improve the overall very hydrophobic properties of MDM2 antagonists.

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Figure 9. Crystal structure of compound 25 (RO-5353) bound to MDM2 (PDB: 4LWV): Hydrogen bonds are observed both between the N-H group of the pyrrole and Leu54 and between the carbonyl of the amide and His96. Additionally

- -methoxy-4-carbamoyl amide aromatic ring, and His96 with the 2-fluoro-3-chloro benzene ring. Hydrophobic interactions are observed with Leu54, Leu57, Ile61, Met62, Phe86, Val93, Ile99 and Tyr100.

2.4. PYRROLIDINES After the discovery of compounds 2 (RG7112) and 23 (MI-219), novel pyrrolidine derivatives were also developed as p53-MDM2/X inhibitors by Roche scientists. The prototype compounds 26 and 27 (PDBs: 4JRG and 4JSC) showed good potency (IC50 = 196 nM and 74 nM respectively) in the HTRF assay.54 The 4-chlorophenyl ring of compound 26 is buried in the Trp23 pocket, the 3-chloro-phenyl occupies the Leu26 pocket and the neopentyl binds to the Phe19 pocket (figure 10).

Figure 10. Crystal structure of compound 26 -depicted. Additionally the pyrrolidine carbonyl forms a hydrogen bond with NH of His96. Several hydrophobic interactions are observed with Leu54, Leu57, Ile61, Met62, Phe86, Phe91, Val93, Lys94, Ile99 and Tyr100.

Further optimization led to the development of the compound 28 with an IC50 of 6 nM (RG7388), which effectively activates the p53 pathway, leading to cell cycle arrest and/or apoptosis in cell lines

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expressing wild-type p53 and tumor growth inhibition or regression of osteosarcoma xenografts in nude mice. RG7388 is undergoing clinical investigation in solid and hematological tumors (figure 11).54

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Figure 11. Pyrrolidine derivatives screened for the suppression on p53-MDM2.

2.5. ISOQUINOLINES, PIPERIDINONES AND MORPHOLINONES In 2012 Novartis scientists described substituted isoquinolines and piperidinones as inhibitors of MDM2 and MDMX with the most potent compound 29 having IC50 values of 0.8 and 2.1 μM, respectively for both proteins in TR-FRET assay.55 Two additional patents reported the cyclohexylisoquinoline compound 30 and hydroxyl isoquinoline 31 which are also capable of inhibiting the interaction between p53-MDM2/X with IC50

56,57 Recently, Novartis described a new series of dihydro-isoquinolinone derivatives derived from a virtual screening exercise using 2D and 3D presentations of ~50.000 compounds from the Novartis compound collection and compound 32 was identified with an IC50 -FRET biochemical assay. The binding mode of compound 32 in the MDM2 pocket obtained by docking, adjusts to the central valine concept.41 Further optimization yielded compound 33 (PDB: 4ZYC) with an IC50 -FRET assay. Remarkably, the bicyclic core makes hydrophobic contacts with residues Ile54, Phe55 and Gly58 of MDM2 instead of the proposed interaction with Val93.58 The development of this class of compounds led to compound 34 (NVP-CGM097, PDB: 4ZYI) with IC50 = 1.7 nM with MDM2 and strong antiproliferative effect in the HCT116 p53WT cell line compared to an isogenic HCT116 knockout for the expression of p53, showing 35-fold selectivity. Additionally, it was determined a 58-fold selectivity between SJSA-1 cell line and p53-null osteosarcoma SAOS-2 cell line.59 Presently, NVP-CGM097 is going through phase 1 clinical trials (figure 12).59,60

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Figure 12. Various isoquinolines, piperidinones and morpholinones as potent inhibitors.

Scientists from Amgen described the structure-based design of new piperidinone compounds, based on yet another scaffold class.61 In compound 35, with an IC50 of 34 nM (PDB: 2LZG), the p-chloro substituted phenyl ring occupies the Trp23 pocket, the m-chloro substituted phenyl ring sits in the Leu26 pocket and the cyclopropyl group fills the Phe19 pocket (figure 13). The carboxylic acid moiety forms a hydrogen bond with NH of His96 (figure 13).61,62

A chiral tert-butyl 2-butanoate replaced the cyclopropyl group in compound 35 and led to piperidinone 36, 8-times more potent than its predecessor towards MDM2 (PDB: 4ERE).61 Further modification of the tert-butyl ester of 36 derived into compound 37 (PDB: 4HBM), which after

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optimization led to the compound 38 (AM-8553, PDB: 4ERF), with an IC50 of 1.1 nM towards MDM2 (figure 12), showing satisfactory tumor regression in mice SJSA-1 tumor xenograft model, demonstrating its strong antitumor activity.63 Supplementary development of AM-8553, with the introduction of the sulfonamide moiety in order to search interactions between glycine in a shallow, underutilized cleft of MDM2 surface, yielded to compound 39 (AM-232, PDB: 4OAS) with Ki = 0.045 nM, IC50 = 9.4, 11.3 and 23.8 nM in SJSA-1, HCT116 and ACHN (renal adenocarcinoma derived cell line), respectively.64,65 Additional evaluations in seven p53 mutant cell lines, showed no significant effect on cell growth and in studies of SJSA-1, HCT116, A375 (malignant melanoma) and NCI-H460 (large cell lung cancer) xenograft models it were observed significant tumor regression.64 The evaluation of AM-232 in preclinical trials, in rats, monkeys and dogs, showed differences in the pharmacokinetics with a good correlation between in vivo and in vitro studies. This methodology led to a prediction of low human plasma clearance and long half-life in human for compound 39.66 Currently, the compound is undergoing clinical trials (figure 12). 64–67

Figure 13. Crystal structure of compound 35 bound to MDM2 (PDB: 2LZG): The 3- chlorophenyl ring has a - stacking interaction with His96; additionally the carboxylic acid moiety forms a hydrogen bond with NH of His96. Numerous hydrophobic interactions with Val14, Leu54, Gly58, Ile61, Tyr67 and Val93 are shown.

In 2014, additional efforts by Amgen workers to enhance the biochemical and cellular potency, using pyridine or thiazole as isosteres of the carboxylic acid moiety of AM-232 led to potent piperidinone inhibitors, among them compound 40 (AM-6761, PDB: 4ODE), a thiazolyl-containing inhibitor with a HTRF IC50 = 0.1 nM, SJSA-1 IC50 = 16 nM, antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg and promising pharmacokinetic properties.68 Furthermore, an additional modification of AMG-232, replacing the carboxylic acid with a 4-amidobenzoic acid, led to compound 41 (AM-7209, PDB: 4WT2), with a Kd value of 38 pM, SJSA-1, IC50 = 1.6 nM, in vivo antitumor activity in SJSA-1 and HCT116 xenograft model and outstanding pharmacokinetic properties.69 Using the knowledge gained from the piperidinone series, changing to a morpholinone core had a significant impact on potency and metabolic stability. Morpholinone inhibitors are 5- to 10-fold less potent than their piperidinone counterparts, however they are more stable in hepatocytes, a feature can compensate the reduction in potency. Compound 42 (AM-8735, PDB: 4OBA), the most representative compound of this series showed an IC50 = 25 nM in SJSA-1 cells, remarkable pharmacokinetic properties and in vivo efficacy in a SJSA-1 osteosarcoma xenograft model (ED50 = 41 mg/kg).68,70

40

2.6. BENZODIAZEPINES In 2005 Johnson & Johnson reported a class of benzodiazepine compounds as MDM2 inhibitors (figure 14).71,72 With an initial high-throughput screening, two lead compounds were identified with a binding affinity with MDM2 between 15 and 30 μM, after further optimization of the substituents on the three phenyl rings resulted in compound 43 with IC50 Kd = 80 nM.73

NH

NIO

CO2H

O

Cl

Cl

43

N

N

I

Cl

Cl

NH2

O

O

O ONH

NO

CO2H

S

Cl

Cl

Leu-Thr-Phe-HH2N

O O

Glu-Tyr-HNO

Ala-Gln

NH

HN

O

O

Ser-Ala-Ala-NH2

46 (ATSP-7041)

44 45

Figure 14. Benzodiazepines and a staple peptide showing potency as MDM2/X inhibitors

Co-crystal structure of MDM2 and 43 S,S-isomer at a resolution of 2.7 Å (PDB: 1T4E) showed that that the binding resembles the three key p53 residues for interaction with MDM2.73 The Phe19 pocket was filled by the iodobenzene ring, the Trp23 pocket was occupied by the p-chlorophenyl and the Leu26 pocket was filled with other p- -position corresponding to the carboxylic acid group (figure 15). The iodo substituent of the benzodiazepinedione scaffold forms a strong halogen bonding with the backbone carbonyl-O of Gln72. As expected a study reduction of affinity is observed be switching to lower halogens.71

Additional optimization resulted in compound 44 by reducing epimerization issues comparing with the -carbonyl position of 43 and 44 introduced a hydrogen bond with Val93

with IC50 74 Any of this class of MDM2 inhibitors progressed into clinical development due to their unsatisfactory antitumor activity in human cancer cells, low cell permeability, rapid plasma clearance and low bioavailability shown in vitro and in animal models.28,29,74 In 2012, a 2-thiobenzodiazepine derivative (compound 45) was described in a patent as a MDM2 inhibitor, with an IC50 of 3.18 μM/mL in Saos-2 p53 null and 6.54 μM/mL in U-2-os wtp53 (Osteosrcoma cell lines)(figure 14).75

41

3

4

5

6

7

8

9

Figure 15. Crystal structure of compound 43 bound to MDM2 (PDB: 1T4E): A halogen bond between the iodine and Gln72 is observed. Hydrogen bond between Ser17 and the carboxyl c acid moiety and a - stacking interaction with His96 and the p-chlorophenyl. Furthermore, various hydrophobic interactions with Leu54, Gly58, Ile61, Tyr67, Phe91, Val93, Ile99 and Tyr100 are shown.

2.7. PEPTIDES Peptides and derivatives also can be designed to become potent p53-MDM2/X inhibitors. In 2007, a 12-residue peptide (pDI) was identified using phage display that selects for maximal inhibitory activity against MDM2 and MDMX, with IC50 = 0.01 and 0.1 μM for the binding in ELISA assay for MDM2 and MDMX, respectively.76 The co-crystal structures of the pDI with MDMX (PDB: 3JZO) and a single mutant derivative (pDI6W, PDBs: 3JZP, 3JZR) bound to human MDMX and MDM2, served as template to design of 11 diverse pDI-derivative peptides that were tested for inhibitory potential. The best derivative (pDIQ, PDB: 3JZQ) exhibited a 5-fold increase in potency over the parental peptide with IC50 for the binding with MDM2 and MDMX of 8 and 110 nM, respectively.77

While peptidic inhibitors based on the modified p53 sequence offer very high affinity toward MDM2, they suffer from low cell permeability and are proteolytically unstable. A successful attempt to overcome these problems has been made by designing cyclic peptides that are closed by an all-hydrocarbon “staple”. The staple stabilizes the helical structure of the peptide, a feature which likely contributes to the enhanced affinity of the peptide for MDM2 relative to the wild-type peptide. The peptide SAH-p53-86 (PDB: 3V3B), which interestingly also targets MDMX, is the most effective stapled-peptide MDM2 inhibitor using its Phe19, Trp23, and Leu26 to fill the binding site in a manner similar to the native p53 peptide. The Trp23 indole ring is bound to Leu54 by a hydrogen bond. The aliphatic staple protects this hydrogen bond and forms an extended hydrophobic network with Leu54, Phe55, Gly58, and Met62 of MDM2 (figure 16).78

42

Figure 16. Crystal structure of the peptide SAH-p53-86 bound to MDM2 (PDB: 3V3B): The staple is indicated in pink. The hydrogen bonds are the same present in the p53-MDM2 interaction, the NH in SAH-p53-86Trp23 and the carbonyl in MDM2Leu54, and between the NH in SAH-p53-86Phe19 and MDM2Gln72. Additionally, several hydrophobic interactions are depicted.

The PMI peptide (PDB: 3EQS bound to MDM2 and PDB: 3EQY bound to MDMX) a 12-mer peptide selected from a phage displayed peptide library, is highly soluble in aqueous solutions and has better affinity (compared to pDI) for MDM2 and MDMX with Kd values of 3.3 and 8.9 nM, respectively.79 Aided by mirror image phage display and native chemical ligation, several proteolysis-resistant duodecimal D-peptide antagonists of MDM2, termed DPMI- 80 The prototypic D-peptide inhibitor DPMI-pathway in tumor cells in vitro and inhibits tumor growth in vivo. Furthermore, the design of a superactive D-peptide antagonist of MDM2 was reported, named DPMI-affinity for MDM2 improved over DPMI- d = 220 pM).81

In 2013, the stapled peptide 46 (ATSP-7041, PDB: 4N5T, co-crystallized with MDMX) was synthesized which activates the p53 pathway in tumors in vitro and in vivo, with Ki of 0.9 and 6.8 nM for MDM2 and MDMX, respectively. This stapled peptide has shown improved bioavailability compared to previously described peptides and induces p53-dependent apoptosis and inhibits cell proliferation in multiple MDM2 and MDMX overexpressing tumors in cell based models. Interestingly, beyond the well-known triad of Phe19, Trp2, and Leu26, ATSP-7041 demonstrated additional interactions between Tyr22 and the staple moiety itself with the MDMX protein. The Tyr22 interacts with MDMX binding pocket through Van der Waals contact with Gln66, Arg67, Gln68, His69, Val89, and Lys90 as well as through water-mediated hydrogen bonds with N of the Lys90 side-side-chain. An extensive binding pocket for the staple exists on the MDMX protein, and a number of van der Waals contacts (to Lys47, Met50, His51, Gly54, Gln55, and Met58) were also observed (figure 14).82

The MDM2-binding stapled peptide, M06, showed high affinity for the binding with mutant MDM2 (Nutlin resistant). The M06 stapled peptide forms interactions very similar to those made by the SAH-8 peptide, including the reorientation of the Leu26 side chain that appears to be associated with increased helicity (PDB: 4UMN). Apart from attenuating the binding of Nutlin, the M62A mutation in MDM2 also causes a significant change in the conformation of the aliphatic staple. In the SAH-8 structure the hydrocarbon chain packs predominantly against Leu54, Phe55, Gly58 and Met62. The absence of the methionine side chain in MDM2-Met62A causes the conformation of the staple to

43

3

4

5

6

7

8

9

change as it packs more closely against Gly58. The plasticity of the new designed stapled peptide enables it to respond to the M62A mutation by making the compensatory contacts.83

3. CONCLUSIONS AND PERSPECTIVES

Recently, plenty novel small molecules with promising activity have been published as MDM2 inhibitors, including a new synthetic approach to obtain stapled peptides,84 chlorofusin inspired class of analogs,85 fluorescent triazolylpurines,86 sulfamide and triazole benzodiazepines,87 oxazoloisoindolinones,88 dispiro-indolinones,89 a camptothecin analogue (FL118),90 and an inauhzin analogue,91 among others. Very recently, a 2,3 -bis(1 -indole) scaffold was published by our group showing additional hydrophobic interactions with the p53Val93 as indicated by 2D NMR and modelling studies.92 These compounds possess often modest affinities to MDM2 or MDMX in the micromolar range. All potent MDM2 antagonistic scaffolds currently undergoing early clinical trials have been structurally characterized and SBDD played an important role in their optimization. Thorough analysis of the available structure and SAR can provide some guiding principles for the design of novel and potent p53-MDM2 antagonists. The endogenous interaction of p53 with MDM2 and MDMX reveals a clear 3-point pharmacophore model formed by the side chains of p53 Phe19-Trp23-Leu26, which competitive inhibitors have to mimic. Multiple scaffolds have been designed capable of doing so and have been discussed in the previous sections. All structurally characterized MDM2 binder have a T-shaped topology where the ends of the T comprise the three moieties addressing the pharmacophore points. The central scaffold is mostly a heterocyclic ring, annulated rings or in some cases acyclic linear. Amongst the three buried hydrophobic amino acids side chains Trp23 contributes most to the interaction energy and thus must be closely mimicked in terms of hydrophobicity and shape. This has been accomplished by phenyl groups, indoles, oxindoles or 2-oxo benzimidazoles. The choice of the right anchor residue mimicking Trp23 might be important in terms of compounds selectivity. A considerable increase in binding affinity can be reached by the suitable introduction of a hydrophobic halogen at the bottom of the Trp23 binding pocket. A per-p-halophenyl substituted inhibitor scaffold might potentially bind into other similar -helix mediated protein-protein interactions, whereas an anchor residue closely mimicking the Trp23-Leu54 hydrogen bond might be more selective and able to differentiate between similar PPI binding interfaces. Additional interactions to receptor amino acids sees in several cocrystal structures involve a hydrogen bonding or - interaction to His96, hydrogen bonding to Ser17, halogen bonding or hydrogen bonding to backbone or sidechain Glu72, respectively, and dipolar interaction with His73. In general the MDM2 crystal structures show a high degree of crystallographic water presence but a water on top of the indole ring of p53Trp23 seems to be highly conserved in many structures. In 17 out of 25 MDM2 structures (~70%) a highly conserved water molecule can be observed (figure 17). This water is involved in a tight network of interactions between a polar inhibitor substituent on top of the central scaffold and the main chain Phe55 carbonyl-O in the MDM2 receptor. The inclusion of this water network could be an important aspect to consider into future design of MDM2 inhibitors.

Dual activity both in MDM2 and MDMX is essential for further substantial development. Current small molecules do however not address the duality nor are potent MDMX inhibitors known. In contrast peptidic inhibitors can be designed with dual potency for MDM2 and MDMX. In addition the intrinsically disordered protein states of MDM2 should be taken into account and the resulting 4th pharmacophore point, for discovery of novel scaffolds as shown in previous studies.26,27

Drug combination, and cyclotherapy had been studied as supplementary opportunities to treat cancer, exerting synergistic effects or protect selectively normal tissues.29 Representatives are the evaluation of Nutlin with actinomycin D, gemcitabine, vincristine, roscovitine and doxorubicin in cell based models.93–98 Another highly interesting and complementary p53 reactivation approach not discussed here is based on small molecule restauration of certain p53 mutants. 99,100

44

The research efforts during the last decade led to several compounds currently in clinical trials for the treatment of different types of cancer and also in combination with other classes of chemotherapeutics (table 1). The available structural information and knowledge gather during the last years helped to design better compounds with greater affinity towards MDM2, but not in the same degree for MDMX, improved pharmacokinetic profile, oral bioavailability and selectivity. During the studies with animals as well as in clinical trials is imperative the determination of proper doses, schedules and possible combination therapies to circumvent possible acquired resistance to MDM2 inhibitors and side effects.

Figure 17. Highly conserved surface water in ligand-MDM2 structures. Above: Superimposition of the co-crystal structures including the waters, main cluster (shown in cyan) located in the groove formed by Phe55, Tyr56, Lys57, Gly58 and Gln59, Below: Examples of the interaction of the small molecules, the water and Phe55 of MDM2. First row: Compounds 1c (Nutlin 3, PDB: 4J3E) and 3 (WK23, PDB: 3LBK). Second row: Compounds 11 (PDB: 3TJ2) and 40 (AM-6761, PDB: 4ODE). Third row: Compounds 34 (NVP-CGM097, PDB: 4ZYI) and 43 (PDB: 1T4E).

45

3

4

5

6

7

8

9

Table 1. MDM2/X inhibitors in clinical trials

Drug Condition Phase Sponsor Ref

RG7112

(RO5045337)

Solid Tumors, advanced solid tumors, Leukemia, hem,atological neoplasms, liposarcomas

1 (completed) Hoffmann-La Roche 35–37,101

RG7112

(RO5045337) with Doxorubicin

Soft Tissue Sarcoma 1 (completed) Hoffmann-La Roche 102

RG7112

(RO5045337) with Cytarabine

Acute Myelogenous Leukemia 1 (completed) Hoffmann-La Roche 103

RG7775

(RO6839921)

Advanced Cancers, Including Acute Myeloid Leukemia

1 (Recruiting) Hoffmann-La Roche 104

RO5503781 Advanced Malignancies, Except Leukemia

1 (Completed) Hoffmann-La Roche 105

RO5503781 with Posaconazole Solid Tumors 1 (Completed) Hoffmann-La Roche 106

RO5503781 with Cytarabine

Acute Myelogenous Leukemia 1 (Recruiting) Hoffmann-La Roche 107

RG7388 (Alone and with Pegasys)

Polycythemia Vera and Thrombocythemia

1 (Recruiting) Hoffmann-La Roche 108

MK-8242 Advanced Solid Tumors 1 (Completed) Merck Sharp & Dohme

Corp. 109

MK-8242 -Cytarabine Acute Myelogenous Leukemia 1 (Completed) Merck Sharp & Dohme

Corp. 110

46

Drug Condition Phase Sponsor Ref

DS-3032b

Hematological Malignancies, advanced tumors or lymphomas

1 (Recruiting) Daiichi Sankyo Inc. 111

HDM201 Advanced Tumors TP53wt 1 (Recruiting) Novartis 112

HDM201 with LEE011 Liposarcoma 1 (Recruiting) Novartis 113

CGM097 Solid Tumor With p53 Wild Type Status 1 (Recruiting) Novartis 60,114

SAR405838 (MI-888 analogue) Neoplasm Malignant 1 (Active) Sanofi 115

SAR405838 (MI-888 analogue) with pimasertib Neoplasm Malignant 1 (Recruiting) Sanofi 116

AMG232 Advanced Solid Tumors or Multiple Myeloma

1

(Recruiting) Amgen 67

AMG232 Metastatic Melanoma

1b/2a

(Recruiting) Amgen 117

AMG232 with Trametinib Acute Myeloid Leukemia

1b

(Recruiting) Amgen 118

47

3

4

5

6

7

8

9

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(101) Clinicaltrials.Gov Identifiers for RG7112: NCT01164033, NCT01677780, NCT01143740, NCT00623870 and NCT00559533.

(102) Clinicaltrials.Gov Identifier for RG7112 with Doxorubicin: NCT01605526.

(103) Clinicaltrials.Gov Identifier for RG7112 with Cytarabine: NCT01605526.

(104) Clinicaltrials.Gov Identifiers for RG7775: NCT02098967.

(105) Clinicaltrials.Gov Identifier for RO5503781: NCT01462175.

(106) Clinicaltrials.Gov Identifier for RO5503781 with Posaconazole: NCT01901172.

(107) Clinicaltrials.Gov Identifier for RO5503781 with Cytarabine: NCT01773408.

(108) Clinicaltrials.Gov Identifier for RG7388 with Pegasys: NCT02407080.

(109) Clinicaltrials.Gov Identifier for MK-8242: NCT01463696.

(110) Clinicaltrials.Gov Identifier for MK-8242 with Cytarabine: NCT01451437.

(111) Clinicaltrials.Gov Identifier for DS-3032b: NCT02319369 and NCT01877382.

(112) Clinicaltrials.Gov Identifier for HDM201: NCT02143635.

(113) Clinicaltrials.Gov Identifier for HDM201 with LEE011: NCT02343172.

(114) Clinicaltrials.Gov Identifier for CGM097: NCT01760525.

(115) Clinicaltrials.Gov Identifier for SAR405838: NCT01636479.

(116) Clinicaltrials.Gov Identifier for SAR405838 with Pimasertib: NCT01985191.

(117) Clinicaltrials.Gov Identifier for AMG232: NCT02110355.

(118) Clinicaltrials.Gov Identifier for AMG232 with Trametinib: NCT02016729.

CHAPTER 4

ARTIFICIAL MACROCYCLES AS POTENT P53-MDM2

INHIBITORS

Natalia Estrada-Ortiz,a, * Constantinos G. Neochoritis,a, * Aleksandra Twarda-Clapa,b, c Bogdan Musielak,d Tad A. Holak,b, d Alexander Dömlinga

a Department of Drug Design, University of Groningen, A. Deusinglaan 1, Groningen 9700AV, The Netherlands. b Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. c Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland. d Department of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland. * Shared first authors

Published in: ACS Med. Chem. Lett. 2017. doi: 10.1021/acsmedchemlett.7b00219

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Abstract: ABSTRACT: Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53-MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73 Val93 and Lys94 yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence polarization (FP) assay and 1H-15N 2D HSQC NMR experiments.

Keywords: Macrocycles, Ugi reaction, p53, MDM2, protein-protein interaction, cancer

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In nature, macrocycles are not uncommon and often exhibit interesting biological activities.1 Often macrocyclic compounds have distinct advantages over their open chain analogous including higher affinity and selectivity,2 preferable entropic signature, better membrane permeation and oral bioavailability or higher stability.1,2 Thus, macrocycles can increase affinity and selectivity for a specific target.1 In spite of their potential, macrocycles pose considerable synthesis problems and also the accurate prediction of their conformation makes it difficult to predict activity.1,2

The tumor suppressor protein p53 is involved in controlling pathways of cell cycle, apoptosis, angiogenesis metabolism, senescence and autophagia.3,4 TP53 gene is one of the most frequently mutated gene in a multitude of human cancer.5 Additionally, in multiple cases where TP53 is intact, p53’s function is impaired by its negative regulators: MDM2 and MDMX, due to amplification or enhanced expression of their coding genes.3-5 Multiple potent and selective compound classes to inhibit the p53-MDM2 interaction have been discovered, described and evaluated in early clinical trials.6 However, the pharmacokinetic and pharmacodynamics properties of the studied scaffolds could still be optimized to minimize the side effects. Thus, the discovery of new p53-MDM2 inhibitors with diverse structures to improve their properties is still of importance.6-8 The three finger pharmacophore model for p53-MDM2 is recognized as responsible for the binding of small molecules and peptides to the MDM2.9,10 We already described several series of potent p53-MDM2 antagonists, proposing an extended four finger model; the intrinsically disordered MDM2 N-terminus is ordered by certain small molecules, which can be obtained by multicomponent reaction chemistry,11,12 as shown by co-crystallization.13,14 Recently, several macrocyclic stapled peptides have been described with great affinity towards MDM2 and MDMX.6 ALRN-6924 (Aileron Therapeutics) is currently undergoing phase I and II clinical trials in patients suffering of solid tumors, lymphoma and myeloid leukemias (ClinicalTrials.gov ID: NCT02264613 and NCT02909972).

Here we propose a novel series of non peptidic artificial macrocyclic compounds that inhibit the p53-MDM2 interaction, which might have a different activity profile from the currently available scaffolds. Our synthesis strategy is shown in Figure 1. Based on the Ugi scaffold, we introduced two terminal ene-functionalities, via the carboxylic acid and the isocyanide component and we were able to cyclize the compounds by ring closing metathesis (RCM, Figure 1).15, 16

Figure 1. Macrocyclization strategy to inhibit the p53-MDM2 interaction.

Our previously introduced three and four-point phar-macophore models were the basis of the discovery and development of the current inhibitors. Thus, we used as the starting point our formerly

-aminoacylamide (YH300, shown in cyan sticks, Figure 2) with a Ki of 600 nM and its crystal structure in complex with MDM2 recep-tor (PDB ID 4MDN).13 Accordingly, the 6-chloroindole-2-carboxylic acid was used as an ‘anchor’ in order to mimic the Trp23 amino acid and constrain the position of other substituents and three additional binding sites were defined, Phe19, Leu26 and the induced Leu26 subpocket

58

.

Figure 2. Four and three-point pharmacophore modelling of macrocyclic compounds; Left: YH300 -PDB ID: 4MDN- (cyan lines), Tyr67, Gln72, His73 Val93 and Lys94 (blue sticks), proposed macrocycle to explore the four-pharmacophore point (pink sticks). Right: SAH-p53-8 stapled-peptide -PDB ID: 3V3B- (cyan lines), Tyr67, Gln72, His73, Val93 and Lys94 (blue sticks), proposed macrocycle to explore the three-pharmacophore point (pink sticks).

covering a large hydrophobic surface area formed by Tyr67, Gln72, His73, Val93 and Lys94 (Figure 2) and potentially increasing the affinity to the receptor. Moreover, we reasoned that the addition of a methyl group on the ring could mimic the tert-butyl group of YH300,13 likely leading to increased affinity.

The retrosynthetic plan of the designed macrocycle 1 foresees (Scheme 1), a ring closing metathesis reaction (RCM), followed by a classical Ugi four-component (U-4CR). The Ugi adduct is formed of the anchoring 6-chloro-3-carboxaldehyde 6, suitably substituted benzylamines 5 and the long-chained aliphatic carboxylic acids 8 along with the isocyanides 7 incorporating terminal double bonds.

Aldehyde 6 was synthesized from the 6-chloro-indole derivative using the Vilsmeier-Haack formylation reaction.17,18 For the preliminary SAR analysis of the Leu26 and the induced pockets, the benzylamines 5 were used as commercially available (5b-h) or obtained via Williamson ether synthesis (5a). Probing both the Phe19 pocket and the larger hydrophobic surface area formed by Tyr67, Gln72, His73, Val93 and Lys94, the isocyanides 7a,b were synthesized from the corresponding formamides, whereas the carboxylic acids are commercially available. In particular, the amine 5a with the oxygen linker that designed to probe the induced pocket was synthesized through a Williamson ether synthesis of the protected 4-hydroxybenzylamine with the 3,4-benzyl chloride under basic conditions (Supporting information, Scheme 1). The isocyanides 7a,b were synthesized from the formamides via the revised Leuckart-Wallach reaction of the corresponding carbonyl compounds (SI, Scheme 2).19

59

4

5

6

7

8

9

Scheme 1. Retrosynthetic plan based on a U-4CR and a ring clousing metathesis

NNH

OO

R1X

n

m

NH

CO2HCl

NNH

OO

R1X

n

NH

CO2EtCl

CO2H

R1

NC

NH

CHO

CO2EtCl

+

NH2X

m n

NNH

OO

R1X

n

NH

CO2EtCl

disconnection point

U-4CR

m m

1 3 4

5 6

7 8

RCM

Next, we proceeded in the Ugi four-component reaction. Equimolar mixture of the substituted benzylamine 5, aldehyde 6, isocyanide 7 and carboxylic acid 8 in TFE was irradiated at 120 oC for 1 h in a microwave oven yielding compounds 4 (Scheme 2). Afterwards, we successfully performed the ring closing metathesis with the 2nd generation of Grubbs catalystTM affording compounds 3 as mixture of isomers (E and Z). Due to the fact that the existence of the double bond gives rise to two possible isomers and most importantly reduces the flexibility of the macrocycle, we decided to subject the mixture to hydrogenation on Pd/C isolating compounds 2. Last step was the ester hydrolysis obtaining the final screening compounds 1 (Scheme 2). Performing a preliminary SAR, we built a small library of macrocycles of various ring sizes (12, 13, 18, 19 and 24 number of atoms) targeting the hydrophobic region around Tyr67, Gln72, His73 Val93 and Lys94. We maintained the anchoring indole group for the Trp23 and the phenyl group for Leu26 and further explored the induced pocket with the extended dichlorobenzyloxy moiety.

Scheme 2. Synthesis of the macrocycle library based on the U-4CR/RCM strategy

CO2H

R1

NC

NH

CHO

CO2EtCl

+N

NH

OO

R1X

nNH2X

m n

NH

CO2EtCl

NNH

OO

R1X

n

NH

CO2EtCl

NNH

OO

R1X

n

NH

CO2EtCl

Grubbs catalyst,2nd generation (8-10%)

DCM, reflux,48 h

H2, Pd/C,DCM, 1 h

MW, 120 oC, 1h

TFE

NNH

OO

R1X

n

NH

CO2HCl

m m

m m

EtOH-H2O 1:1, rt, 3 d

LiOH

5a-g 6

7a,b 8a-c4a-k (15-60%) 3a-k (10-96%)

2a-k (70-99%)1a-k (11-70%)

5a: X= 4-OCH2-3,4-di-Cl-C6H35b: X= 4-F5c: X=4-Cl5d: X= 2,4-di-Cl5e: X= 4-OMe5f: X= 3-OMe5g: X= 3,4-di-Cl5h: X= 3,4,5-tri-F

7a: R1= Me, m= 17b: R1= H, m= 7

8a: n= 18b: n= 28c: n= 7

60

NNH

OO

NH

CO2HCl

O

ClCl

1a (d.r. 1:1)

N

NH

O

O

NHCl

CO2H

O

ClCl

NNH

OO

F

NHCl

CO2H

1d

NNH

OO

Cl

NHCl

CO2H

NNH

OO

O

NHCl

CO2H

1g

NNH

OO

Cl

NHCl

CO2H

1i

NNH

OO

NHCl

CO2H

ONH

NO

O

NHCl

CO2H

F

FF

1j

N

NH

O

O

O

NH

ClCO2H

Cl

Cl

1k

Cl

NNH

OO

Cl

NHCl

CO2H

1f

Cl

1b (d.r. 1:1) 1c (d.r 1:1) 1e

1h

NNH

OO

O

NHCl

CO2HClCl

NH

NO

O

NHCl

CO2H

F

FF

1ja

NH

NO

O

NHCl

CO2H

F

FF

1jb

NH

NO

O

NHCl

CO2H

F

FF

3j

N

NH

O

O

O

NH

ClCO2H

Cl

Cl

3k

NNH

OO

NH

CO2EtCl

O

ClCl

2a

N

NH

O

O

O

NH

ClCO2Et

Cl

Cl

2k

HN

NO

O

NH

Cl CO2H

F

F F

9

NNH

OO

NH

Cl

10

NNH

OO

Cl

Cl11

Two complementary assays based on independent physicochemical principles, fluorescence polarization (FP) and 1H-15N 2D HSQC NMR were used to measure affinity and to exclude false positive hits. Fluorescence polarization (FP) assay was employed to determine the inhibitory affini-ties (Ki) of the macrocycles against MDM2 as previously described20 and the results are presented in Table 1.

Examining both the three and four-finger pharmaco-phore model, it seems that most of the obtained macrocy-cles are active towards MDM2, many of them demonstrat-ing an affinity below 1 μM. Although it is a preliminary SAR study, we can conclude that the ideal ring size for the four-finger model, seems to be around 18 (entry 3). The affinity improves while increasing the size from 12 (1a, entry 1) to 13 (1b, entry 2) and eventually 18 (1c, entry 3) atom ring size. Moreover, compound 1c has

-membered macrocycle 1k M).

In addition, we focused on the three-finger-pharmacophore model, which characterizes the vast ma-jority of the currently available small-molecule MDM2 inhibitors.6 We synthesized various macrocycles with a different substitution pattern. The position of halogens on the phenyl group seems to play a significant role since the para fluoro (1d, entry 4) or chloro substituted (1f, entry 6) derivatives are only slightly or not active at all. On the contrary, the addition of a second chlorine in o- or m-position influenced the binding mode; the 2,4-dichloro derivative (1e) showed activity of 5.25

-dichloro compound 1h (entry 8) displayed an activity of 80 nM. Whereas placing the donor group -OMe in compound 1g (entry 7), improved the affin-ity with

-OMe substitution keeping the same ring size of 18 (1i, entry 9), did not show

were able to synthe-size fluorinated phenyls as compound 9 with Ki up to 100 nM, we employed the 3,4,5-trifluorobenzylamine and we synthesized compound 1j (entry 10) which exhibited an interesting affinity as a racemic mixture of 140 nM. Enan-tiomeric separation of the racemic mixture via chiral SFC

61

4

5

6

7

8

9

provided the enantiomers (+)-1ja and (-)-was expected, the separated enantiomers showed a significant increase of the activity compared to the racemic mixture.

The existence of the double bond in the macrocycles 3j and 3k, as anticipated, reduced significantly -pared with the corresponding

esponding esters, 2a and 2k (entries 14, 15), are orders of magnitude less reactive or inactive comparing with the acids in ac-cordance with our previous experience (entries 1, 11).11,12,22-25 Interestingly, the acyclic Ugi-adduct 4j was also proven to be practically inactive both in the ester and acid forms (SI, Table S1, entry 15). Moreover, changing the anchor to a non-substituted indole moiety (compound 10) or to the 3- or 4-phenyl moiety (compound 11), resulted in nearly no activity. The expected ligand-induced perturbations in 1H-15N 2D HSQC NMR spectra were indeed observed (Figure 3). The 15N-labeled MDM2 was titrated with increasing concentration of the compound. Since all cross peaks in the MDM2 spectrum were assigned to particular amino acid residues,26 it was possible to analyze the interaction within the MDM2/1j complex. Particularly, Val93 is clearly involved in the interaction, as its cross peak shifted between titration steps for MDM2:1j molar ratios equal to 2:1 and 1:1. After 1:1 step the peak remained in the same position. NMR titration also confirmed the tight binding of 1j, as e.g. for Arg29 NMR signal splitting was observed (Figure 3), which indicated strong interaction with MDM2 at Kd

Table 1. Synthesized MDM2 inhibitors

Entry Compound Ring size Ki (μM)

1 1a 12 0.35

2 1b 13 0.32

3 1c 18 0.104 1d 18 N/A5 1e 18 5.25

6 1f 18 N/A

7 1g 18 1.26

8 1h 18 0.08

9 1i 18 1.78

10 1j 190.14 (rac-1j)

0.09 {(+)-1ja}0.70 {(-)-1jb}

11 1k 24 1.91

12 3j 19 0.34

13 3k 24 2.53

14 2a 12 2.98

15 2k 24 N/A

N/A: not active

62

Figure 3. Spectrum of the 15N-labeled MDM2 (blue) superimposed with spectrum after addition of 1j in MDM2/1j molar ratio equal to 2:1 (red) and 1:1 (green). The close-up view shows selected peaks assigned to Val93 and Arg29. For Arg29, NMR signal splitting indicates strong interaction at Kd

Three of the macrocyclic compounds (1c, 1h and 1j) obtained, demonstrated improved binding affinities (Ki ~100 nM) over the lead acyclic molecule, YH300 (Ki = 600 nM). In order to rationalize the tight receptor ligand interaction, we exploit modelling studies using MOLOC27 based on the HSQC binding data having as template a known co-crystal structure (PDB ID: 3TU1)21 and the small network analysis using Scorpion software (Figure 4).28 It revealed the existence of van der Waals interactions of the aliphatic handle with Tyr67 and His73, the expected alignment of the 6-chloro-indole moiety of the designed compounds with the p53Trp23 pocket, whereas the 3,4,5-trifluorophenyl ring occupied the p53 - -trifluorophenyl fragment and several van der Waals interactions with Leu54, Ile61, Phe86, Phe91, Val93, His96 and Tyr100 are depicted. These findings support our initial hypothesis of the divergent hydrophobic handle position compared to staple peptides shown before,29-34 suggesting a new approach to improve and diversify the extensive collection of MDM2/X inhibitors.

To analyze and compare the physicochemical properties of our newly synthesized macrocycles with the orally available macrocycle drugs,35 we plotted molecular weight, clogP, TPSA, number of HBDs and HBAs as well as the number of rotatable bonds (Figure 5). Interestingly, the values of the properties in most of our macrocycles are set in the appropriate range, demonstrating the significance of this new strategy to develop potentially oral bioavailable macrocycles targeting p53-MDM2 interaction. In the case of 1c and 1k, clogP, goes off the limits as expected since we were targeting a very lipophilic surface. However, this will be overcome in the future with a strategy to incorporate heteroatoms (oxygens) on both the acid and isocyanide linker. After this initial SAR study, we will in the future synthesize libraries of novel macrocycles as potent p53-MDM2 inhibitors with higher diversity and complexity.

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4

5

6

7

8

9

Figure 4. Small network analysis of 1j -and van der Waals interactions are shown in orange and yellow dotted lines, respectively.

Figure 5. Physicochemical properties of the synthesized macrocycles, compared with the oral macrocycle marketed drugs on a hexagon radar graph. The dark gray area contains the low limits of the oral macrocycle drugs, whereas the light gray the highest limits.

SUPPLEMENTARY DATA Experimental procedures for the synthesis of compounds, characterization of compounds, crystal data, as well FP assay and NMR HSQC are provided in the supporting information

64

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(14) Bauer, M. R.; Boeckler, F. M. Hitting a Moving Target: Targeting Transient Protein States. Structure 2013, 21 (12), 2095–2097.

(15) Beck, B.; Larbig, G.; Mejat, B.; Magnin-Lachaux, M.; Picard, A.; Herdtweck, E.; Dömling, A. Short and Diverse Route toward Complex Natural Product-like Macrocycles. Org. Lett. 2003, 5 (7), 1047–1050.

(16) Wessjohann, L. A.; Rivera, D. G.; Vercillo, O. E. Multiple Multicomponent Macrocyclizations (MiBs): A Strategic Development toward Macrocycle Diversity. Chem. Rev. 2009, 109 (2), 796–814.

(17) Dömling, A. P53-mdm2 Antagonists. Patent Application WO 2012/033525 A3, 2012.

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Wallach Route Toward Isocyanides and Some Applications. ACS Comb. Sci. 2015, 17 (9), 493–499.

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(21) Huang, Y.; Wolf, S.; Koes, D.; Popowicz, G. M.; Camacho, C. J.; Holak, T. A.; Dömling, A. Exhaustive Fluorine Scanning toward Potent p53–Mdm2 Antagonists. ChemMedChem 2012, 7 (1), 49–52.

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66

SUPPORTING INFORMATION

1. EXPERIMENTAL MATERIALS AND METHODS

1.1. SYNTHESIS AND ANALYSIS All the reagents and solvents were purchased from Sigma-Aldrich, AK Scientific, Fluorochem, Abcr GmbH, Acros and were used without further purification. All microwave irradiation reactions were carried out in a Biotage Initiator™ Microwave Synthesizer. Thin layer chromatography was performed on Millipore precoated silica gel plates (0.20 mm thick, particle size 500 or 600 spectrometers {1 13

Chemical shifts for 1H NMR were reported as values and coupling constants were in hertz e following abbreviations were used for spin multiplicity: s = singlet, br s = broad

singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd = double of doublets, ddd = double doublet of doublets, m = multiplet. Chemical shifts for 13C NMR were reported in ppm relative to the solvent peak. Flash chromatography was performed on a Reveleris® X2 Flash

measured on a Waters Investigator Supercritical Fluid Chromatograph with a 3100 MS 2 on a Viridis silica gel column (4.6 x

-ethyl pyridine column (4.6 x 250 mm, 5 μm particle on a Reprosil Chiral-IC column (4.6 x 250 mm, 5 μm

-preparative SFC was performed with stacked injector (250 μL -

MeOH/CO2 as mobile phase. High resolution mass spectra were recorded using a LTQ-Orbitrap-

1.2. PROTEIN EXPRESSION AND PURIFICATION Fragment of the N-terminal domain of human MDM2 (residues 1-pET- - -RIL as described previously.1 In brief, cells were cultured at 37 °C. Protein expression was induced with 1 mM IPTG at OD600 of 0.8 and cultured for additional 5 h at 37 °C. Cells were collected by centrifugation and lysed by sonication. Inclusion bodies were collected by centrifugation,

-X100 and subsequently solubilized in 6 M guanidine hydrochloride in 100 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 10 mM -mercaptoethanol. The protein was dialyzed against 4 M guanidine hydrochloride pH 3.5 supplemented with 10 mM -mercaptoethanol. Following, the protein was refolded by dropwise addition into 10 mM Tris-HCl, pH 7.0, containing 1 mM EDTA and 10 mM -mercaptoethanol and slow mixing overnight at 4 °C. Ammonium sulfate was added to the

67

4

5

6

7

8

9

final concentration of 1.5 M and the refolded protein was recovered on Butyl Sepharose 4 -HCl pH 7.2 containing 5

-mercaptoethanol and further purified by gel filtration on HiLoad 16/600 Superdex75 ffer pH 7.4 containing 150 mM NaCl and 5 mM DTT

1.3. FLUORESCENCE POLARIZATION ASSAY All FP measurements were performed using Tecan Infinite® 200 PRO plate reader. The assay was conducted in 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA contaidetermine the optimal concentration of the protein for the competition binding assay, the effective concentration of MDM2 (1-apparent Kd towards 5’FAM- assay was performed by contacting serial dilutions of tested compounds with 10 nM P2 at protein concentration yielding f0 = 0.8. Fluorescence polarization was determined at 485 nm excitation and 535 nm emission 15 min after mixing all assay components. All tests were performed using Corning black 96-well NBS assay plates at room temperature.

1.4. NMR EXPERIMENTS All NMR spectra were acquired at 300 K using a Bruker Avance 600 MHz spectrometer. Uniform 15N isotope labelling was achieved by expression of the protein in the M9 minimal medium containing 15NH4Cl as the sole nitrogen source. MDM2(1-

2O was added to the samples to provide lock signal. Water suppression was carried out using the WATERGATE sequence.2 Stock solutions of inhibitors used for titration were prepared in d6-DMSO. Several titrperformed in order to assess the binding. The spectra were processed with TopSpin 3.2 software. 1H-15N heteronuclear correlations were obtained using the fast HSQC pulse sequence.3 Assignment of the amide groups of MDM2 was obtained as previously reported.4

2. SYNTHETIC PROCEDURES AND ANALYTICAL DATA

2.1. PROCEDURE AND ANALYTICAL DATA FOR AMINE 5A

NHBoc NH2

OH O

3,4-benzylchloride

NH2

OH

(Boc)2O, NaHCO3

MeOH

Cl

Cl

9 5a

K2CO3, MeCN

Scheme 1.

68

2.1.1. Tert-butyl 4-

4- 2

NaHCO3 and methanol

was evaporated. To the resulting slurry, water and dichloromethane were added and the aqueous phase was

extracted with dichloromethane. Organic layers were collected, washed with water, dried over anhydrous MgSO4 and evaporated, giving crude product as brown oil. The crude product

compound 9 1H NMR (600 MHz, CDCl3 [ppm] 7.10 (d, J = 7.3 J = 13C NMR (151

MHz, CDCl3 [ppm] 156.3, 155.5, 130.5, 129.0, 115.6, 79.9, 44.3, 28.6. LC- R = 5.31 min, Calcd for C12H17NO3 -H]- 222.26, found: [M-H]- 222.10.

2.1.2. (4-((3,4-

Tert-butyl 4-hydroxybenzylcarbamate 9 -2CO3

reaction was cooled to rt and the solvent was evaporated. To the resulting solid, water and

dichloromethane were added and the aqueous phase was extracted with dichloromethane. Organic layers were collected washed with water, dried over anhydrous MgSO4 and evaporated, giving crude product as white-yellowish powder. Afterwards the amine was

was stirred at rt for 1.5 h. Water was added to stop the reaction and stirred for 5 min more, followed by washing with 2 times with DCM and once with water, to obtain the free amine in the aqueous phase. Further addition of NaOH 1 N and DCM to extract the amine, the organic layer were collected, washed with water, dried over anhydrous MgSO4 and evaporated, giving the final product 5a 1H NMR (500 MHz, CDCl3 [ppm] 7.54 (d, J =

J = – 7.22 (m, 3 – 13C NMR (126 MHz, CDCl3 [ppm] 137.5, 136.4, 130.7, 129.3, 128.5, 126.6, 115.0,

68.7, 46.0.

2.2. PROCEDURE AND ANALYTICAL DATA FOR ETHYL 6-CHLORO-3-FORMYL-1H-INDOLE-2-CARBOXYLATE (6)

The 6-chloro-1H-indole-2--bottom flask equipped

with CaCl2 tube. Then, POCl3 an

NH

O

O

HO

OCl NH2

Cl

NH

CO2Et

CHO

Cl

69

4

5

6

7

8

9

Afterwards, the reaction was cooled to rt, quenched with saturated NaHCO3 solution and extracted with ethyl acetate. Organic layer was collected, washed with water and brine, dried over anhydrous MgSO4 and evaporated. The crude product was washed with diethyl ether giving compound 6;5 1H NMR (600 MHz, DMSO-d6 [ppm]

J = J = J = 8.6, .46 (q, J = J = 13C (151 MHz, DMSO-d6 [ppm]

187.5, 159.9, 136.1, 133.5, 130.4, 124.0, 123.9, 123.4, 118.2, 112.6, 62.0, 14.1; LC-MS R = 2.97 min, Calcd for C12H10ClNO3 -H]- 250.03, [M+2-H]- 250.02, found:

[M-H]- 250.05, [M+2-H]- 252.05.

2.3. PROCEDURE AND ANALYTICAL DATA FOR ISOCYANIDES 7

OmNH2CHO

R1

R1 = H, Mem = 1,7

NHCHOm

R1

HCO2H

POCl3, Et3N

DCMNCm

R1

10a,b 7a,b

Scheme 2.

2.3.1. General procedure of the modified Leuckart Wallach formamide synthesis

The 10-undecenal or 5-hexen-2-

extracted with DCM, organic layer was collected, washed with water, dried over anhydrous MgSO4 and evaporated. The products were obtained without further purification.

N-(hex-5-en-2-

mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl3

– – J = 14.0, 6.8 – – J = 13C NMR (126 MHz,

CDCl3 [ppm] 160.7, 137.7, 115.0, 43.7, 35.8, 30.0, 20.8.

N-(undec-10-en-1-

mixture of rotamers is observed, major rotamer is given; 1H NMR (500

MHz, CDCl3 – – J = 6.6 Hz, J = J = J = 13C NMR (126

MHz, CDCl3 [ppm] 161.4, 139.1, 114.1, 41.9, 38.2, 33.8, 29.44, 29.36, 29.2, 29.05, 28.9, 26.83.

NHCHO

NHCHO

70

2.3.2. General procedure of the isocyanide synthesis

The corresponding formamides 10a and 10b were put together in a round-bottom flask and cooled to 0 °C. Then POCl3 added dropwise and the reaction was stirred at rt for 3-4 h. After the reaction was completed, the products were poured into ice cold solution of NaHCO3 and left to reach rt. The precipitated solid was filtered off and washed with DCM. The remaining water phase was extracted with DCM. Organic layers were collected, washed with water, dried over anhydrous MgSO4 and evaporated. The resulting oils were purified on silica pad with copious amount of DCM, which was then collected and evaporated, giving the pure isocyanides 7a,b.

5-isocyanohex-1-

mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl3 [ppm] 5.77 (ddt, J = 17.0, 10.2, 6.7 Hz,

– J = J = – – 13C NMR (126 MHz,

CDCl3 [ppm] 154.6, 136.4, 116.1, 49.6, 46.0, 35.8, 29.8, 21.6, 8.6.

11-isocyanoundec-1-

mixture of rotamers is observed, major rotamer is given; 1H NMR (500 MHz, CDCl3

[ppm] 5.90 – – 4.89 (m, 2H J = J = J = – 13C NMR (126 MHz,

CDCl3 [ppm] 154.4, 136.5, 116.1, 49.6, 45.9, 35.8, 31.6, 29.9, 21.6, 8.6.

2.4. GENERAL PROCEDURE OF THE UGI FOUR-COMPONENT REACTION (UT-4CR)

The corresponding amine 5 6 7 acid 8 -vial which irradiated at 12crude mixture was purified by flash chromatography (hexane-corresponding compounds 4 (yields 15-

NC

NC

71

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(N-(4-((3,4- -4- -2-(hex-5-en-2--2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 J = 7.48 – – J = 14.5, 8.6 Hz,

– J = J = J = – 4.90

– – J = 13.5, J = J =

J = J = 7.3 Hz, .99 – J = J = 12.5,

J = – J = 13C NMR (126 MHz, CDCl3 [ppm] 173.5, 172.2, 169.3, 162.8, 160.9, 157.6, 156.8, 137.8, 137.5, 137.0, 135.8, 132.6, 131.8, 131.6, 131.2, 130.8, 130.5, 130.3, 130.0, 129.2, 129.2, 129.0, 128.0, 127.2, 126.5, 126.4, 126.3, 125.7, 122.9, 122.4, 115.7, 115.3, 115.0, 114.7, 114.3, 111.8, 100.0, 77.3, 77.1, 76.8, 68.5, 68.4, 61.6, 54.4, 50.2, 49.1, 45.5, 43.9, 43.0, 35.8, 35.5, 33.1, 30.2, 29.9, 29.6, 29.4, 20.5, 14.4; LC- R = 3.22 min, Calcd for C38H40Cl3N3O5

-H]- 722.20, found: [M-H]- 722.13.

Ethyl 6-chloro-3-(1-(N-(4-((3,4- -5- -2-(hex-5-en-2-ylami -2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 J = 7.58 – – – J = 8.6 – – J = – – 4.63 (m,

– – – 2.37 – – J = 14.3, 7.3 Hz,

– – – 1.28 – J = J = 6.5 Hz,

13C NMR (126 MHz, CDCl3 [ppm] 190.8, 177.3, 174.1, 172.8, 169.3, 161.0, 157.7, 156.8, 138.1, 138.0, 137.8, 137.7, 137.3, 137.2, 135.9, 132.7, 132.6, 132.1, 131.9, 131.8, 131.7, 131.2, 130.9, 130.7, 130.6, 130.5, 129.3, 129.2, 129.0, 127.1, 126.5, 126.4, 126.3, 125.8, 124.7, 123.0, 122.8, 122.4, 115.4, 115.4, 115.1, 115.0, 114.9, 114.7, 114.2, 112.2, 111.8, 77.3, 77.1, 76.8, 68.7, 68.5, 68.4, 61.7, 54.4, 49.1, 45.5, 45.4, 43.9, 43.0, 35.9, 35.4, 33.3, 33.2, 33.1, 33.0, 33.0, 30.2, 29.9, 24.8, 24.5, 24.0, 20.6, 20.5, 14.4; LC- R = 3.21 min, Calcd for C39H42Cl3N3O5 -H]- 736.22, found: [M-H]- 736.15.

N

O

HN

Cl

O

HN

O

EtO2C

Cl Cl

N

O

HN

Cl

O

HN

O

EtO2C

Cl Cl

72

Ethyl 6-chloro-3-(1-(N-(4-((3,4- -10- -2-(hex-5-en-2--2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 [ppm] 9.64 (d, J = –

– – J = J = – –

– J = 15.4 – J =

(t, J = – – – 2.39 (m, J = J = 2.02 (dq, J = J = –

– – J = 13C NMR (126 MHz, CDCl3 [ppm] 177.5, 174.4, 174.4, 173.3, 169.5, 169.4, 161.1, 160.2, 157.6, 156.8, 139.2, 139.2, 139.1, 138.0, 137.8, 137.3, 137.2, 136.0, 132.7, 132.6, 131.9, 131.8, 131.5, 131.3, 130.9, 130.5, 130.5, 129.2, 129.1, 129.0, 128.1, 127.7, 127.2, 126.5, 126.4, 126.3, 125.7, 122.9, 122.8, 122.3, 121.7, 115.3, 115.1, 115.0, 115.0, 114.9, 114.7, 114.6, 114.2, 114.2, 114.2, 114.1, 113.9, 112.3, 111.9, 77.4, 77.1, 76.9, 68.5, 68.4, 61.6, 61.3, 57.2, 56.9, 54.5, 54.3, 53.5, 49.2, 49.1, 46.3, 45.7, 45.5, 45.4, 43.0, 36.8, 35.9, 35.4, 34.1, 33.8, 30.2, 29.9, 29.6, 29.4, 29.4, 29.3, 29.3, 29.2, 29.2, 29.1, 28.9, 25.8, 25.4, 24.9, 20.6, 20.5, 14.3; LC-

R = 3.14 min, Calcd for C44H52Cl3N3O5 -H]- 806.30, found: [M-H]- 806.32.

Ethyl 6-chloro-3-(1-(N-(4- -4- -2-oxo-2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.50 (d, J = 8.5 J =

J = J = (d, J = J = J = (dt, J = J = 5.08 – J = J = 14.3, 7.2

J = J = J = .21 (dt, J = J = 17.0, 10.8,

J = J = 2.02 (q, J = J = J = J = 13C NMR (126 MHz, CDCl3 [ppm] 173.4, 169.8, 160.8, 160.4, 139.2, 137.4, 135.8, 133.6, 131.7, 127.6, 127.2, 126.5, 126.5, 125.7, 122.6, 121.6, 115.5, 115.3, 114.7, 114.6, 114.1, 113.9, 112.3, 111.9, 77.3, 77.1, 76.8, 61.7, 61.4, 56.4, 54.1, 49.1, 46.8, 40.0, 33.8, 33.1, 32.5, 29.4, 29.4, 29.2, 29.1, 28.9, 26.9, 14.4; LC- R = 2.82 min, Calcd for C36H45ClFN3O4 -H]- 636.31, found: [M-H]- 636.31.

N

O

NH

Cl

O

HN

O

CO2Et

Cl Cl

N

HN

Cl

O

HN

O

EtO2C

F

73

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(N-(2,4- -4- -2-oxo-2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J = 8.6

– – J = 12.4 Hz, J = J = 5.5 Hz,

– 5.02 – – 4.32 J = J =

3.10 – – – – J = J = 22.7, 15.1, 7.4 Hz,

J = 13C NMR (126 MHz, CDCl3 [ppm] 173.5, 173.5, 169.4, 169.4, 160.9, 160.9, 139.2, 139.2, 137.2, 137.2, 137.1, 137.1, 136.8, 135.8, 135.8, 133.8, 133.8, 132.8, 132.8, 132.3,

132.3, 132.0, 132.0, 131.1, 131.1, 129.3, 129.3, 128.8, 128.8, 128.0, 128.0, 127.5, 127.5, 127.3, 127.3, 126.9, 126.9, 126.4, 126.4, 125.8, 125.8, 125.5, 125.5, 122.8, 122.8, 121.8, 121.8, 115.8, 115.8, 115.5, 115.5, 115.3, 115.3, 115.2, 115.2, 114.3, 114.3, 114.1, 114.1, 112.1, 112.1, 111.9, 111.9, 77.3, 77.3, 77.0, 77.0, 76.8, 76.8, 61.9, 61.9, 61.6, 61.6, 56.3, 54.2, 54.2, 47.3, 47.3, 45.3, 40.9, 40.9, 40.3, 40.3, 40.1, 40.1, 35.7, 35.7, 33.8, 33.8, 32.8, 32.8, 32.4, 32.4, 29.5, 29.5, 29.4, 29.4, 29.4, 29.4, 29.2, 29.2, 29.2, 29.2, 29.1, 29.1, 28.9, 28.9, 27.0, 27.0, 26.9, 26.9, 23.4, 23.4, 14.5, 14.5, 14.3, 14.3; LC- R = 2.86 min, Calcd for C36H44Cl3N3O4 -H]- 686.24, found: [M-H]- 686.23.

Ethyl 6-chloro-3-(1-(N-(4- -4- -2-oxo-2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 (d, J = J = 8.6 Hz,

– J = J = J = J = J = 7.7

J = J = 17.4 J = J = J

= J = J = 23.8, 19.8, 11.5, J = – 3.34 (m J =

J = J = 14.4, 6.5 Hz, – J = – J = 7.5,

– J = 13C NMR (126 MHz, CDCl3 [ppm] 173.9, 173.4, 169.9, 160.8, 139.2, 137.6, 137.3, 136.5, 135.9, 132.2, 131.7, 129.0, 127.9, 127.4, 127.4, 127.2, 126.4, 125.6, 124.9, 122.6, 122.5, 121.5, 115.4, 115.1, 114.4, 114.1, 112.4, 112.0, 77.3, 77.1, 76.8, 61.7, 61.4, 56.4, 54.2, 51.6, 49.1, 46.9, 40.2, 40.0, 33.8, 33.1, 32.5, 29.4, 29.4, 29.2, 29.1, 28.9, 27.1, 27.0, 26.9, 23.6, 14.4; LC-MS

R = 2.89 min, Calcd for C36H45Cl2N3O4 -H]- 652.28, found: [M-H]- 652.31.

N

HN

Cl

O

HN

O

EtO2C

Cl

Cl

N

HN

Cl

O

HN

O

EtO2C

Cl

74

Ethyl 6-chloro-3-(1-(N-(4- -4- -2-oxo-2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

(d, J = J = J = 8.3 J = – –

– J = – 4.35 J = J = 13.2, 6.7

– 3. J = – 2.43 J = J = –

1.31 (m, J = J = 13C NMR (126 MHz, CDCl3 [ppm] 169.9, 139.2, 139.2, 137.5, 137.5, 135.8,

135.8, 131.6, 131.6, 129.9, 129.2, 129.2, 126.2, 126.2, 122.8, 122.8, 122.5, 122.5, 115.5, 115.2, 115.2, 114.1, 114.1, 114.1, 114.1, 113.3, 113.3, 111.8, 111.8, 77.3, 77.3, 77.0, 77.0, 76.8, 76.8, 61.7, 61.7, 55.2, 55.2, 54.3, 54.3, 49.1, 49.1, 43.1, 40.0, 40.0, 33.8, 33.8, 33.1, 33.1, 29.4, 29.4, 29.4, 29.4, 29.2, 29.2, 29.1, 29.1, 28.9, 28.9, 26.9, 26.9, 14.4, 14.4; LC-MS

R = 3.03 min, Calcd for C37H48ClN3O5 -H]- 648.33, found: [M-H]- 648.28.

Ethyl 6-chloro-3-(1-(N-(3,4- -4- -2-oxo-2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = – 7.27 (m,

J = – 6.44 (d, J = J = J = 16.7, 10.2

– J = J = 17.4 – – J

= J = – J = J = J =

J = J = 12.8, – J = 58.9, 36.3, 16.5, 9.6 Hz,

– J = J = 23.2, 16.5, 9.4, – – – 13C NMR (126 MHz,

CDCl3 [ppm] 173.4, 169.9, 160.8, 139.2, 139.2, 138.5, 137.2, 137.2, 131.8, 131.8, 129.7, 129.7, 129.1, 129.1, 127.8, 127.8, 127.0, 127.0, 125.4, 125.4, 124.4, 124.4, 122.7, 122.7, 122.2, 122.2, 115.6, 115.6, 115.3, 115.3, 114.1, 114.1, 112.5, 112.5, 112.2, 112.2, 77.3, 77.3, 77.1, 77.1, 76.8, 76.8, 61.9, 61.9, 61.6, 61.6, 56.1, 54.1, 54.1, 48.8, 48.8, 46.7, 40.1, 40.1, 33.8, 33.8, 33.0, 33.0, 32.4, 32.4, 29.4, 29.4, 29.1, 29.1, 28.9, 28.9, 26.9, 26.9, 14.3; LC-MS

R = 2.94 min, Calcd for C36H44Cl3N3O4 -H]- 686.24, found: [M-H]-686.23.

N

HN

Cl

O

HN

O

EtO2C

O

N

HN

Cl

O

HN

O

EtO2C

Cl

Cl

75

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(N-(3- -4- -2-oxo-2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

J = J = 6.51 – J = J = 12.4

J = – J = J = J =

17. – J = J = J = J =

J = J = (ddd, J = 22.3, 16.2 J = – J =

J = J = 13C NMR (126 MHz, CDCl3 [ppm] 173.7, 170.1, 161.1, 159.3, 139.7, 139.2, 137.4,

136.8, 136.1, 131.5, 128.9, 127.4, 125.9, 122.5, 122.4, 117.4, 115.4, 115.3, 114.3, 114.1, 112.5, 112.1, 110.3, 77.4, 77.1, 76.9, 61.7, 56.8, 55.2, 54.8, 54.3, 49.7, 47.4, 40.0, 35.8, 33.8, 33.1, 29.4, 29.4, 29.3, 29.2, 29.1, 28.9, 28.8, 26.9, 21.0, 14.2. LC- R = 4.55 min, Calcd for C37H48ClN3O5 -H]- 648.33, found: [M-H]- 648.39.

Ethyl 6-chloro-3-(2-oxo-1-(N-(3,4,5- -5- -2-(undec-10-en-1--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

(d, J = J = J = 21.2, 8.4 Hz, –

J = J = – J = J =

– J = – 2.34 J = –

1.95 – – 13C NMR (126 MHz, CDCl3 [ppm] 174.4, 173.8, 169.7, 169.5, 160.7, 139.2, 138.2, 137.9, 135.8,

132.1, 127.1, 125.5, 124.8, 123.0, 123.0, 122.3, 121.4, 115.4, 115.3, 115.1, 114.1, 112.3, 112.0, 109.8, 109.6, 109.1, 109.0, 77.3, 77.0, 76.8, 62.0, 61.7, 55.9, 54.0, 48.7, 46.8, 40.2, 40.1, 33.8, 33.3, 33.0, 33.0, 32.8, 32.6, 32.2, 29.5, 29.4, 29.4, 29.2, 29.1, 28.9, 26.9, 26.8, 24.2, 23.9, 23.8, 14.3; LC- R = 3.47 min, Calcd for C37H45ClF3N3O4 -H]- 686.31, found: [M-H]- 686.10.

N

HN

Cl

O

HN

O

EtO2C

O

N

HN

Cl

O

HN

O

EtO2C

FF F

76

Ethyl 6-chloro-3-(1-(N-(4-((3,4- -10- -2-oxo-2-(undec-10-en-1- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 (d, J = – 7.40

– J = 6.53 (d, J = – J = 14.7,

– 4.71 (d, J = – – 3.25 (dt, J = J = J =

– – (dq, J = – – 1.04

13C NMR (126 MHz, CDCl3 [ppm] 174.2, 169.9, 160.9, 156.7, 139.2, 139.2, 137.3, 135.8, 132.7, 131.9, 131.7, 130.9, 130.6, 129.3, 129.0, 127.6, 127.1, 126.4, 126.3, 125.8, 122.9,

122.5, 121.8, 115.1, 115.0, 114.9, 114.2, 114.1, 113.9, 112.1, 111.7, 77.3, 77.0, 76.8, 68.6, 68.4, 61.7, 61.3, 56.7, 54.1, 49.1, 46.6, 43.0, 40.0, 33.8, 33.8, 33.3, 29.4, 29.4, 29.3, 29.2, 29.1, 28.9, 26.9, 25.4, 24.9, 24.8, 14.4, 14.2; LC- R = 3.41 min, Calcd for C49H62Cl3N3O5 -H]- 876.38, found: [M-H]- 876.27.

2.5. GENERAL PROCEDURE FOR THE RING-CLOSURE METATHESIS (RCM) REACTION

The corresponding UT-4CR compound 4

2 atmosphere. The reaction mixture was refluxed for 3 d. The solvent was evaporated and the crude mixture was purified by flash chromatography (hexane- 3 (yields 10- -brown powders.

Ethyl 6-chloro-3-(1-(4-((3,4- -5-methyl-3,12-dioxo-1,4-diazacyclododec-8-en-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.71 (d, J = 7.57 – – – 7.29 – – – 7.04 – J = J = 8.6 Hz,

J = J = J = J = J =

4.35 – – J = – J = – J =

NNH

OO

NH

CO2Et

Cl

O

ClCl

N

HN

Cl

O

HN

O

EtO2C

O

ClCl

77

4

5

6

7

8

9

1.42 – J = J = J = 10.6, 8.6 Hz, 13C NMR (126 MHz, CDCl3 [ppm] 176.4, 173.6, 169.5, 168.9, 160.3, 160.1, 157.2,

156.9, 139.0, 137.3, 137.1, 135.9, 135.1, 132.6, 132.5, 131.9, 131.1, 130.6, 130.5, 130.3, 129.5, 129.1, 129.1, 127.7, 127.0, 126.9, 126.5, 125.8, 125.7, 124.9, 124.5, 124.0, 121.5, 121.4, 121.1, 115.6, 114.4, 114.2, 114.1, 113.6, 112.7, 112.3, 77.3, 77.1, 76.8, 68.4, 61.3, 61.2, 58.8, 54.6, 48.2, 46.8, 46.2, 45.7, 34.5, 33.6, 33.1, 32.8, 31.9, 31.4, 31.3, 30.2, 29.8, 29.7, 29.5, 29.4, 27.1, 26.7, 22.7, 21.8, 20.5, 18.2, 14.3, 14.2; LC- R = 5.52 min, Calcd for C36H36Cl3N3O5 -H]- 694.17, found: [M-H]- 694.15.

Ethyl 6-chloro-3-(1-(4-((3,4- -5-methyl 3,13-dioxo-1,4-diazacyclododec-8-en-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 – 7.42 (m,

– J = J = (d, J = J = – J = 4.99 (q, J = J =

J = J = J = 16.8 Hz, J = 34.3, – J = – 1.56

J = J = J = 13C NMR (126 MHz, CDCl3 [ppm] 174.1, 171.2, 169.7, 160.0, 157.3, 137.3, 135.9, 132.5, 131.9, 131.4, 130.6, 130.5, 129.7, 129.3, 129.2, 129.1, 126.7, 126.5, 124.9, 121.9, 121.5, 115.8, 115.3, 115.0, 114.5, 112.6, 77.3, 77.0, 76.8, 68.6, 68.4, 61.2, 60.4, 57.8, 48.6, 45.0, 43.0, 35.9, 34.8, 33.1, 30.8, 30.2, 30.0, 24.7, 22.5, 22.2, 21.1, 14.3, 14.2; LC- R = 3.50 min, Calcd for C37H38Cl3N3O5 -H]- 708.19, found: [M-H]- 708.30.

Ethyl 6-chloro-3-(1-(4-((3,4- -5-methyl-3,18-dioxo-1,4-diazacyclooctadec-8-en-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.98 (d, J = –

– – J = J = J =

– – J = – 4.34 (m, 1H – J = – 1.97

– – J = 22.1, 13C NMR (126 MHz, CDCl3 [ppm] 174.4, 169.6, 161.1, 157.1, 156.7, 137.3,

135.9, 132.6, 131.8, 131.6, 131.5, 130.8, 130.5, 130.2, 129.4, 129.1, 129.0, 128.6, 127.2, 126.4, 126.4, 125.9, 123.3, 122.3, 122.0, 114.9, 114.2, 113.6, 111.8, 77.3, 77.1, 76.8, 68.4, 61.6, 61.3, 54.5, 49.2, 45.8, 45.2, 44.1, 38.0, 36.9, 33.6, 33.2, 31.8, 30.9, 30.2, 29.7, 29.4, 28.8, 28.5, 28.4, 28.1, 27.5, 27.2, 26.9, 26.5, 25.8, 25.6, 24.5, 20.7, 20.6, 19.5, 14.4; LC-MS

R = 3.53 min, Calcd for C42H48Cl3N3O5 -H]- 778.27, found: [M-H]- 781.52.

N

NH

O

O

NHCl

CO2Et

O

Cl

Cl

NNH

OO

O

NHCl

CO2EtCl

Cl

78

Ethyl 6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 9.23 (d, J = J =

7.10 (dd, J = J = – 6.46 – J = – 5.43 (m, J = – 4 J = 15.5

J = J = (tt, J = – – J = 17.0, 16.6, 8.3

– – J = 1.51 – – 13C NMR (126 MHz, CDCl3 [ppm] 174.0, 170.2, 170.1, 162.2, 160.2, 160.0, 135.8, 134.8, 131.7, 131.5, 130.2, 129.4, 128.7, 127.6, 127.6, 127.0, 126.7, 125.0, 122.8, 122.7, 122.4, 121.4, 114.9, 114.6, 114.0, 113.9, 112.3, 112.0, 77.4, 77.1, 76.8, 61.3, 56.1, 55.9, 54.6, 49.1, 46.9, 40.0, 39.7, 39.5, 34.3, 33.7, 33.4, 31.7, 31.5, 31.3, 29.8, 29.4, 28.6, 28.5, 28.1, 28.0, 27.9, 27.8, 27.5, 27.1, 27.0, 26.8, 26.7, 26.2, 25.9, 25.8, 24.9, 14.4; LC- R = 3.32 min, Calcd for C34H41ClFN3O4 -H]- 608.28, found: [M-H]- 608.28.

Ethyl 6-chloro-3-(1-(2,4- -3,18-dioxo-1,4-diazacyclooctadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 10.51 – J = 26.7, 8.8 Hz,

J = J = , J = 8.8 – J = –

J = J = 36.3, 30.0 Hz, J = – – 4.98

– – J = J = 12.3, – – J = J

= 9.8 – J = – 1.56 J = – 13C NMR (126 MHz,

CDCl3 [ppm] 174.0, 170.1, 136.1, 134.7, 132.2, 131.5, 131.2, 129.5, 128.4, 127.8, 126.7, 125.7, 124.7, 122.6, 121.7, 114.0, 112.3, 77.4, 77.1, 76.9, 61.3, 56.0, 45.4, 40.0, 33.6, 31.7, 29.2, 28.3, 28.1, 27.8, 27.7, 27.6, 27.5, 27.0, 26.2, 24.9, 14.6; LC- R = 3.47 min, Calcd for C34H40Cl3N3O4 -H]- 658.21, found: [M-H]- 658.44.

NNH

OO

F

NHCl

CO2Et

NNH

OO

Cl

NHCl

CO2EtCl

79

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

– – J = J = –

J = – J = 15.6 J = J =

– J = 12.5, 4.9 Hz, 1H J = 31.4, 15.8, – J = J =

– – – 13C NMR (126 MHz, CDCl3 [ppm] 173.9, 170.1, 159.9, 137.7, 135.6, 131.9, 131.6, 131.5, 129.4, 128.1, 127.4, 127.3, 127.2, 126.9, 126.5, 125.0, 122.8, 121.5, 114.7, 112.2, 77.3, 77.0, 76.8, 61.4, 56.0, 47.0, 40.0, 33.6, 31.7, 29.8, 29.3, 28.6, 28.1, 28.0, 27.9, 27.7, 27.4, 27.0, 26.1, 25.8, 14.4; LC-MS R = 3.41 min, Calcd for C34H41Cl2N3O4 -H]- 624.25, found: [M-H]- 624.47.

Ethyl 6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = 7.08 (dd, J = J = J =

53 (d, J = J = 14.7, 7.5 Hz, J = J = J =

J = – – J = J = 12.6, 6.2 H –

(td, J = J = – – – – 13C NMR (126 MHz, CDCl3 [ppm]

174.0, 170.4, 160.1, 158.0, 135.8, 131.5, 131.5, 131.2, 129.4, 128.8, 127.8, 127.6, 127.0, 126.5, 125.0, 122.8, 122.3, 122.2, 121.5, 114.7, 113.4, 113.0, 112.9, 112.3, 112.0, 77.3, 77.1, 76.8, 61.5, 61.2, 56.6, 55.2, 54.8, 49.1, 46.7, 39.9, 39.4, 33.7, 33.5, 31.7, 31.2, 29.4, 29.2, 28.4, 27.9, 27.7, 27.4, 27.3, 27.0, 26.9, 26.7, 26.5, 26.1, 25.8, 24.9, 23.6, 14.4; LC-MS

R = 3.50 min, Calcd for C35H44ClN3O5 -H]- 620.30, found: [M-H]- 620.42.

Ethyl 6-chloro-3-(1-(3,4-dichlorobenz -3,18-dioxo-1,4-diazacyclooctadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

7.18 – J = J = – J = J =

NNH

OO

O

NHCl

CO2Et

NNH

OO

Cl

NHCl

CO2Et

NNH

OO

Cl

NHCl

CO2Et

Cl

80

– J = 12.2, 10.6, 5.4 Hz, J = 8.3, 5.0 Hz, – – J = J =

– J = – 1.97 J = 21.6, – J = 15.1, 7.2 Hz,

13C NMR (126 MHz, CDCl3 [ppm] 173.9, 170.0, 160.0, 139.6, 135.7, 131.9, 131.4, 131.1, 129.6, 129.4, 129.0, 127.8, 126.9, 125.5, 124.9, 123.0, 121.3, 114.5, 112.4, 77.3, 77.1, 76.8, 70.6, 70.0, 69.1, 63.7, 61.6, 55.8, 46.8, 40.1, 33.6, 31.7, 29.3, 28.7, 28.2, 28.0, 27.8, 27.7, 27.5, 27.1, 26.2, 14.4; LC- R = 3.5 min, Calcd for C34H40Cl3N3O4 -H]- 658.21, found: [M-H]- 658.17.

Ethyl 6-chloro-3-(1-(3- -3,18-dioxo-1,4-diazacyclooctadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 9.14 – J = J = 8.7

J = – J = 9.1 J = J = J = – J = J =

J = J = 16.9, 6.3 Hz, J = J = J = J

= – J = – (dd, J = – J = J = 13.0,

J = – J = 2.63 – 2.4 – J = J =

J = – – 13C NMR (126 MHz, CDCl3 [ppm] 173.9, 170.2, 160.0, 158.8, 140.6, 135.7,

131.6, 131.5, 131.3, 129.5, 129.0, 128.7, 128.4, 127.2, 125.2, 123.0, 122.6, 122.4, 121.7, 118.5, 117.5, 114.8, 112.5, 112.4, 112.1, 111.8, 110.9, 110.6, 77.3, 77.0, 76.8, 61.7, 61.4, 56.3, 54.8, 54.7, 49.7, 47.4, 39.9, 39.4, 33.7, 33.4, 31.7, 31.3, 29.4, 28.4, 28.0, 27.9, 27.7, 27.4, 27.1, 26.9, 26.7, 26.1, 25.8, 24.9, 14.4, 14.3; LC- R = 3.47 min, Calcd for C35H44ClN3O5 -H]- 620.30, found: [M-H]- 620.26.

Ethyl 6-chloro-3-(3,19-dioxo-1-(3,4,5- -1,4-diazacyclononadec-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

7.25 – J = J = 5.85 (d, J = – J = 13.0, 6.5

J = – – J = –

– – dd, J = J = (d, J = J = – – –

13C NMR (126 MHz, CDCl3 [ppm] 174.7, 169.8, 169.5, 159.9, 135.5, 132.2, 131.7, 131.6, 131.4, 130.0, 129.5, 129.4, 129.1, 126.9, 124.9, 123.2, 121.3, 112.2, 109.7, 77.3,

NNH

OO

NHCl

CO2Et

O

NH

N

OO

NHCl

CO2Et

F

FF

81

4

5

6

7

8

9

77.2, 77.0, 76.8, 61.8, 56.0, 46.8, 46.7, 40.0, 39.8, 33.4, 31.9, 31.6, 31.4, 29.3, 28.8, 28.4, 28.3, 28.0, 27.7, 27.4, 27.3, 27.0, 26.9, 26.6, 26.3, 26.2, 25.5, 25.2, 25.1, 25.0, 24.8, 14.3; LC-

R = 3.25 min, Calcd for C35H43ClF3N3O4 -H]- 660.29, found: [M-H]- 660.24.

Ethyl 6-chloro-3-(1-(4-((3,4- -3,24-dioxo-1,4-diazacyclotetracos-14-en-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 8.95 (d, J = J = 14.1 Hz,

J = J = J = J = –

J = J = –

4.67 (d, J = 4.44 – J = J = J = J = – –

– J = 13C NMR (126 MHz, CDCl3 [ppm] 174.9, 170.1, 162.5, 159.9, 156.9, 156.6, 137.5, 135.7, 132.7, 131.6, 130.8, 130.6, 130.4, 130.0, 129.3, 129.0, 127.7, 127.0, 126.4, 126.3, 125.0, 123.0, 122.4, 122.2, 121.6, 115.0, 114.3, 114.0, 112.1, 111.7, 77.3, 77.2, 77.0, 76.8, 68.3, 61.5, 61.3, 60.4, 57.1, 54.6, 50.9, 49.1, 46.4, 39.9, 33.9, 33.8, 33.6, 32.2, 32.0, 29.7, 29.6, 29.5, 29.4, 29.2, 29.2, 29.0, 28.9, 28.7, 28.3, 28.1, 27.9, 27.2, 27.0, 26.9, 26.1, 25.5, 14.4, 14.2. LC- R = 3.53 min, Calcd for C47H58Cl3N3O5 -H]- 848.34, found: [M+Na+H]+ 873.55.

2.6. GENERAL PROCEDURE FOR THE HYDROGENATION REACTIONS

The corresponding RCM adduct 3 -neck flask with a H2 and N2 supply. The solution was bubbled with N2

N2 was removed and the H2 was released and let flow for 1 h through the mixture. The crude reaction was filtrated over celite, the solvent was evaporated, giving the corresponding compounds 2 (yields 70-

Ethyl 6-chloro-3-(1-(4-((3,4- -5-methyl-3,12-dioxo-1,4-diazacyclododecan-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.53 – (d, J = J = J = 27. J = J = J = J =

– J = 40.5

NNH

OO

NH

CO2Et

Cl

O

ClCl

N

NH

O

O

O

NHCl

CO2Et

Cl

Cl

82

– – J = – 1.09 (m, J = – J =

J = J = 9.9 J = J = J = J =

– – J = 15.1 Hz, – J = J = 17.6, 9.3, 6.1 Hz,

13C NMR (126 MHz, CDCl3 [ppm] 175.8, 169.2, 160.0, 157.2, 137.3, 135.7, 131.7, 130.6, 129.1, 126.4, 122.1, 115.5, 114.5, 112.2, 77.3, 77.0, 76.8, 68.4, 61.3, 58.4, 47.0, 45.7, 32.4, 31.9, 29.8, 29.7, 29.4, 26.9, 25.1, 24.4, 24.2, 22.9, 22.7, 14.5, 14.1; LC- R = 3.48 min, Calcd for C36H38Cl3N3O5 -H]- 696.19, found: [M-H]- 696.35.

Ethyl 6-chloro-3-(1-(4-((3,4- -5-methyl-3,13-dioxo-1,4-diazacyclotridecan-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 –

– J = 40.8 4.95 (d, J =

J = – J = J =

13C NMR (126 MHz, CDCl3 [ppm] 175.0, 170.3, 160.0, 157.5, 137.2, 136.0, 132.1, 131.3, 130.6, 129.6, 129.2, 129.2, 129.1, 126.5, 121.7, 121.4, 115.0, 114.7, 114.5, 112.7, 77.3, 77.1, 76.8, 68.5, 68.4, 61.3, 58.5, 45.4, 44.4, 43.0, 36.8, 32.8, 31.6, 31.5, 29.7, 26.0, 25.5, 24.5, 23.4, 22.4, 19.9, 19.0, 14.3; LC- R = 3.42 min, Calcd for C37H40Cl3N3O5 -H]- 710.20, found: [M-H]- 710.14.

Ethyl 6-chloro-3-(1-(4-((3,4- -5-methyl-3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.61 (d, J = J = J = J = 14.4,

J = J = 21.2 Hz, J = – J =

– J = 3.03 (t, J = – J = (dt, J = 13C NMR (126 MHz, CDCl3 [ppm] 174.4, 169.6, 161.0, 156.7, 137.3, 136.5, 135.8, 131.8, 131.5, 131.4, 130.5, 129.0, 127.4, 127.0, 126.4, 126.3, 125.9, 123.0, 122.6, 122.4, 122.1, 119.4, 118.8, 114.5, 114.4, 114.2, 113.6, 111.7, 111.3, 77.3, 77.1, 76.8, 68.4, 62.6, 61.6, 57.7, 54.2, 49.2, 45.9, 45.0, 37.1, 36.7, 33.8, 33.5, 29.7, 28.8, 28.1, 28.0, 27.6, 27.5, 27.4, 27.2, 27.0, 26.8, 26.4, 26.1, 25.9, 25.5, 25.3, 24.9, 24.4, 22.7, 20.9, 20.6, 14.4, 14.1; LC- R = 3.44 min, Calcd for C42H50Cl3N3O5 -H]- 780.28, found: [M-H]- 780.27.

N

NH

O

O

NHCl

CO2Et

O

Cl

Cl

NNH

OO

O

NHCl

CO2EtCl

Cl

83

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

J = 03 – (d, J = J = J =

J = J = 17.3 J = – J = 15.4

3.68 (ddd, J = J = J = J = J = – –

– – – – 0.83 (m, 13 [ppm]. 174.8, 174.2, 170.3, 170.0, 160.0, 135.8, 134.5,

133.6, 131.5, 127.7, 127.0, 126.6, 124.9, 122.8, 122.3, 121.2, 114.8, 114.6, 114.1, 114.0, 112.4, 112.0, 100.0, 77.3, 77.1, 76.8, 61.5, 61.2, 56.6, 54.9, 49.2, 46.6, 40.1, 39.5, 33.5, 33.4, 29.6, 29.0, 28.5, 28.0, 27.8, 27.6, 27.4, 27.1, 27.0, 26.7, 26.4, 26.2, 26.1, 25.5, 25.1, 24.4, 14.3; LC- R = 3.18 min, Calcd for C34H43ClFN3O4 -H]- 610.29, found: [M-H]- 610.24.

Ethyl 6-chloro-3-(1-(2,4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 9.34 (d, J = J = 7.88 (d, J = J = – J = J = 7.14 (d, J = J = –

J = – 4.96 (m J = – 4.22 (m,

J = – – – 2.14 (dt, J = – – –

13 [ppm] 174.1, 169.5, 160.9, 160.3, 136.0, 135.7, 133.8, 133.0, 132.3, 132.1, 131.9, 131.5, 129.4, 128.8, 128.2, 128.0, 127.8, 126.9, 126.7, 125.8, 125.3, 124.7, 123.3, 122.8, 122.6, 121.5, 114.8, 112.1, 111.9, 77.3, 77.1, 76.8, 61.7, 61.5, 56.5, 55.1, 47.5, 45.2, 45.1, 40.2, 40.1, 39.6, 33.7, 33.2, 33.1, 31.7, 29.7, 29.6, 29.4, 29.0, 28.6, 28.2, 28.0, 27.9, 27.8, 27.5, 27.5, 27.4, 27.2, 27.2, 27.1, 27.0, 26.9, 26.7, 26.4, 26.2, 26.1, 25.6, 24.8, 24.3, 14.5, 14.2; LC- R = 4.42 min, Calcd for C34H42Cl3N3O4

-H]- 660.22, found: [M-H]- 660.01.

NNH

OO

F

NHCl

CO2Et

NNH

OO

Cl

NHCl

CO2EtCl

84

Ethyl 6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 10.60 (d, J = J = 8.7 Hz,

J = – J = 8.7 Hz, – J = J = 14.2

J = J = 6.47 (d, J = J =

(d, J = J = – J = 19.8, 6.5, J = J = J = 15.1, 6.4

J = – – 1.75 ( – – 13 [ppm] 174.4, 173.9, 170.3,

170.1, 161.0, 160.3, 138.0, 137.0, 136.2, 132.0, 131.2, 131.0, 127.9, 127.5, 127.3, 127.1, 127.1, 126.6, 125.4, 124.9, 122.7, 122.2, 122.0, 121.3, 114.3, 114.1, 112.5, 112.2, 77.5, 77.2, 77.0, 61.2, 61.0, 56.2, 56.0, 54.8, 49.2, 47.1, 47.0, 40.1, 40.0, 40.0, 39.8, 39.5, 39.3, 33.3, 33.1, 32.4, 29.6, 29.4, 28.9, 28.6, 28.0, 27.7, 27.5, 27.4, 27.3, 27.2, 27.1, 27.0, 26.9, 26.7, 26.4, 26.1, 26.1, 25.5, 25.3, 25.0, 24.4, 23.7, 14.5, 14.3; LC- R = 3.26 min, Calcd for C34H43Cl2N3O4 -H]- 626.26 found: [M-H]- 626.42.

Ethyl 6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J = 8.2

– J = J = J = J = 17.6

– – – –

– 13 [ppm] 174.8, 174.3, 170.5, 170.1, 160.9, 158.3, 135.9, 131.4, 129.8, 127.9, 127.1, 126.4, 125.7, 123.1, 122.1, 121.5, 114.9, 113.4, 113.1, 112.4, 111.9, 77.3, 77.1, 76.8, 61.5, 61.2, 57.1, 55.2, 55.0, 49.2, 46.3, 40.0, 39.5, 33.6, 29.4, 29.0, 28.6, 28.0, 27.8, 27.6, 27.5, 27.3, 27.0, 26.9, 26.6, 26.4, 26.2, 26.1, 25.6, 25.1, 24.5, 14.3; LC- R = 3.33 min, Calcd for C35H46ClN3O5

[M-H]- 622.31, found: [M-H]- 622.35.

NNH

OO

O

NHCl

CO2Et

NNH

OO

Cl

NHCl

CO2Et

85

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(3,4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

J = J = J = J =

J = 15.7 Hz, J = – 4.38 J = J = J = 13.1, 6.3 Hz,

J = – J = (dq, J = 19.3, – – –

– J = J = 13C [ppm] 174.9, 170.1, 160.0, 139.5, 135.8, 132.1, 131.3, 130.0, 129.3,

128.0, 127.3, 125.8, 125.1, 124.7, 123.2, 122.9, 121.5, 115.0, 112.5, 112.1, 77.5, 77.2, 77.0, 70.8, 70.2, 69.3, 63.9, 62.0, 61.8, 56.4, 54.8, 49.1, 46.8, 40.4, 39.7, 33.6, 33.4, 31.9, 29.9, 29.7, 29.2, 28.8, 28.2, 28.0, 27.8, 27.5, 27.4, 27.3, 27.1, 26.9, 26.7, 26.4, 26.3, 25.6, 25.3, 24.6, 14.6. LC- R = 3.34 min, Calcd for C34H42Cl3N3O4 -H]- 660.22, found: [M-H]- 660.15.

Ethyl 6-chloro-3-(1-(3- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 9.27 – J = J =

J = J = J = J = –

J = J = 6.08 (d, J = J =

J = J = – J = – J = – J =

– 3.07 (dd, J = – – (dd, J = – J = 13C NMR (126 MHz, CDCl3 [ppm] 174.3, 170.0, 160.9, 159.4, 139.7, 135.9, 131.4, 129.0, 128.6, 123.1, 122.3, 118.7, 117.5, 114.8, 112.7, 112.4, 111.8, 111.1, 110.5, 77.3, 77.1, 76.8, 61.6, 61.3, 56.9, 54.9, 54.7, 49.8, 47.1, 40.0, 39.5, 33.6, 29.4, 29.0, 28.6, 28.0, 27.8, 27.6, 27.4, 27.1, 27.0, 26.6, 26.4, 26.2, 26.1, 25.5, 25.1, 24.6, 14.4, 14.3; LC- R = 3.31 min, Calcd for C35H46ClN3O5 -H]- 622.31, found: [M-H]- 622.17.

NNH

OO

Cl

NHCl

CO2Et

Cl

NNH

OO

NHCl

CO2Et

O

86

Ethyl 6-chloro-3-(3,19-dioxo-1-(3,4,5- -1,4-diazacyclononadecan-2- -1H-indole-2-

mixture of rotamers observed; 1 [ppm] 9.27 – J = J = 19.7, 8.6

J = J = 6.91 (t, J = J = – (dd, J = J = J = 7.2 Hz,

J = .74 (d, J = (d, J = – J = – J = – J = – (dd, J = 2.92 – – J = 14.7, 6.3 Hz,

– J = 13C NMR (126 MHz, [ppm] 174.6, 169.7, 160.0, 135.5, 132.2, 126.9, 124.9, 123.2, 121.3, 114.8, 112.2,

109.9, 109.7, 77.0, 76.8, 61.7, 55.9, 54.7, 46.7, 40.2, 33.1, 29.4, 28.4, 28.2, 27.8, 27.6, 26.9, 26.8, 26.6, 26.2, 26.2, 25.8, 24.6, 14.3; LC- R = 3.16 min, Calcd for C35H43ClF3N3O4 -H]- 662.29, found: [M+2-H]- 662.29.

(+ -ethyl 6-chloro-3-(3,19-dioxo-1-(3,4,5- -2- -1H-indole-2-

Chiral column tR = 12.84 min, HRMS: Calcd for C35H43ClF3N3O4 -H]- 662.2942, found: [M+2-H]- 662.2967. [ ]D20= + 31.5° (c = 0.67, MeOH)

(- -ethyl 6-chloro-3-(3,19-dioxo-1-(3,4,5- -2- -1H-indole-2-

Chiral column tR = 17.83 min, HRMS: Calcd for C35H43ClF3N3O4 [M+2-H]- 662.2942, found: [M+2-H]- 662.2968. [ ]D20= - 32.9° (c = 1.47, MeOH)

NH

N

OO

NHCl

CO2Et

F

FF

N

OO

NHCl

CO2Et

F

FF

N

OO

NHCl

CO2Et

F

FF

87

4

5

6

7

8

9

Ethyl 6-chloro-3-(1-(4-((3,4- -3,24-dioxo-1,4-diazacyclotetracosan-2--1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.89 (d, J = J = 24.4, 8.8 Hz,

– J = J = J = –

7.15 – J = – 6.87 J = –

– J = 21.3 J = 15.2 Hz, – J = J = –

J = J = – 0.87 (q, J = 13C NMR (126 MHz, CDCl3 [ppm] 136.2, 130.8, 130.5, 130.0, 129.2, 129.0, 129.0, 127.7, 126.5, 126.4, 126.3, 124.7, 124.5, 124.0, 123.2, 123.2, 122.4, 115.1, 114.3, 113.9, 112.4, 112.1, 111.7, 77.3, 77.3, 77.0, 76.8, 68.6, 68.4, 61.3, 49.1, 40.0, 32.0, 31.9, 31.4, 30.2, 29.7, 29.7, 29.7, 29.6, 29.5, 29.4, 29.4, 28.9, 28.3, 28.1, 27.9, 27.2, 26.8, 26.7, 26.3, 26.1, 25.5, 22.7, 14.4, 14.1; LC- R = 3.46 min, HRMS Calcd for C47H60Cl3N3O5 -H]- 850.36, found: [M-H]- 850.51.

2.7. GENERAL PROCEDURE FOR THE HYDROLYSIS REACTION

To a stirred solution of the corresponding compound 2 - for 3 days. Afterwards, pH

was adjusted to approximately 6 with the addition of 1 N HCl and then the reaction mixture extracted with DCM. The organic layer was separated, washed with water, dried over anhydrous MgSO4 and evaporated, affording the corresponding compounds 1 (yields 11-as a yellow-brown oils.

6-chloro-3-(1-(4-((3,4- -5-methyl-3,12-dioxo-1,4-diazacyclododecan-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.49 (d, J = –

J = 4.51 – .58 (d, J = J = 42.1 Hz,

J = 13C NMR (126 MHz, CDCl3 [ppm] 169.3, 169.2, 160.0, 130.6, 129.1, 126.4, 122.1, 114.5, 112.2, 77.3, 77.0, 76.8, 68.4, 61.3, 47.0, 45.7, 29.7, 26.9, 24.2, 22.9, 14.5; LC- R = 4.83 min, HRMS: Calcd for C34H34Cl3N3O5 -H]- 670.1643, found: [M-H]- 670.1637.

NNH

OO

NH

CO2H

Cl

O

ClCl

N

NH

O

O

O

NHCl

CO2Et

Cl

Cl

88

6-chloro-3-(1-(4-((3,4- -5-methyl-3,13-dioxo-1,4-diazacyclotridecan-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 J = 9.4 Hz,

J = – – 7.02 J = J =

6.69 (dd, J = 18.2, 8. J = J = J = J = 16.7, 7.0

– – – – 1.15 – , J = J = 13C NMR

(126 MHz, CDCl3 [ppm] 175.0, 170.3, 160.0, 157.5, 137.2, 136.0, 132.8, 132.1, 132.0, 131.5, 130.6, 129.6, 129.3, 129.2, 129.1, 127.9, 127.0, 126.5, 124.8, 121.9, 121.6, 115.0, 114.9, 114.7, 112.6, 77.3, 77.1, 76.8, 68.6, 68.4, 61.3, 58.4, 45.4, 44.4, 43.0, 36.8, 32.8, 31.6, 31.5, 29.7, 26.1, 25.5, 24.5, 23.4, 22.4, 19.9, 19.0, 14.3, 14.0; LC- R = 4.27 min, HRMS: Calcd for C35H36Cl3N3O5 -H]- 684.1798, found: [M-H]- 684.1793.

6-chloro-3-(1-(4-((3,4- -5-methyl-3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers and diastereomers observed; 1H NMR (500 MHz, CDCl3 J = J = – 7.33

– J = (d, J = – J =

4.79 (d, J = J = J = 3.91 (t, J = J = – (q, J =

J = – 13C NMR (126 MHz, [ppm] 175.9, 156.9, 137.1, 136.5, 135.8, 131.5, 130.5, 129.1, 127.4, 126.4, 126.3,

122.6, 122.4, 122.1, 119.4, 118.8, 114.4, 112.1, 111.9, 111.3, 77.3, 77.0, 76.8, 68.4, 62.6, 60.5, 54.6, 49.5, 36.4, 33.6, 29.7, 28.6, 28.1, 27.9, 27.4, 27.3, 27.1, 26.8, 26.3, 25.3, 24.9, 20.7, 14.2; LC- R = 3.69 min, HRMS: Calcd for C40H46Cl3N3O5 -H]- 754.2583, found: [M-H]- 754.2576

6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-carboxylic

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 – –

(d, J = – J = (s, 1H J = J =

13C NMR (126 MHz, CDCl3 [ppm]. 173.4, 134.7, 129.3, 115.3, 115.2, 77.4, 77.2, 76.9, 42.5, 40.4, 40.2,

N

NH

O

O

NHCl

CO2H

O

Cl

Cl

NNH

OO

O

NHCl

CO2HCl

Cl

NNH

OO

F

NHCl

CO2H

89

4

5

6

7

8

9

40.0, 39.9, 39.7, 36.5, 34.2, 30.3, 29.4, 29.3, 26.9, 25.8, 24.9, 15.0; LC- R = 4.03 min, Calcd for C32H39ClFN3O4 -H]- 583.26 found: [M-H]- 583.40.

6-chloro-3-(1-(2,4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 J = J =

J = J = – J =

J = J = J = J =

13C NMR (126 MHz, CDCl3 [ppm]. 163.6, 137.5, 133.2, 130.3, 129.0, 127.1, 125.8, 123.1, 121.1, 116.0, 115.7, 112.1, 108.2, 77.5, 77.3, 77.0, 68.0, 40.5, 40.1, 40.0, 39.8, 39.6, 39.4, 36.3, 31.8, 29.6, 29.4, 29.2, 25.7, 14.1; LC- R = 4.87 min, Calcd for C32H38Cl3N3O4 -H]- 632.19, found: [M-H]- 632.10.

6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-carboxylic

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 8.14 (d, J = J = J = 20.2

J = 8 (dd, J = 7.08 (dt, J = J = – 4.37 (m,

J = J = J = 6.1 Hz, J = J = J = – 1.98 – – – –

13C NMR (126 MHz, CDCl3 [ppm] 170.2, 158.9, 137.1, 133.4, 130.9, 129.0, 128.8, 128.7, 128.7, 128.6, 123.4, 121.5, 116.6, 115.8, 111.9, 110.5, 108.8, 77.3, 77.3, 77.1, 76.8, 43.6, 42.8, 42.7, 40.7, 40.3, 38.7, 36.7, 36.6, 34.1, 30.0, 29.7, 29.4, 29.3, 29.3, 29.2, 29.1, 29.0, 28.8, 26.8, 26.7, 25.8, 24.9, 24.8, 15.2, 15.0; LC- R = 4.10 min, Calcd for C32H39Cl2N3O4 (m/z -H]- 599.23, found: [M-H]- 599.31

6-chloro-3-(1-(4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-carboxylic acid

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.86 – J = J = 26.8

J = – – 6.88 (m, – –

4.40 – J = 2.35 (dd, J = J = – 1.02 (m,

NNH

OO

O

NHCl

CO2H

NNH

OO

Cl

NHCl

CO2HCl

NNH

OO

Cl

NHCl

CO2H

90

J = 13C NMR (126 MHz, CDCl3 [ppm] 176.0, 173.9, 171.6, 159.4, 158.7, 152.1, 152.1, 145.2, 143.7, 141.1, 141.1, 133.9, 130.7, 129.6, 129.6, 128.2, 126.9, 123.4, 122.6, 117.2, 116.4, 114.5, 114.5, 114.0, 111.1, 109.1, 77.7, 77.7, 77.5, 77.2, 55.7, 55.6, 49.9, 43.6, 40.3, 37.2, 37.2, 34.1, 32.4, 30.1, 30.1, 29.9, 29.7, 29.7, 29.5, 28.5, 28.4, 28.3, 27.8, 27.4, 27.2, 26.8, 26.2, 26.0, 25.3, 23.1, 21.2, 14.6; LC- tR = 4.79 min, HRMS: Calcd for C33H42ClN3O5 -H]- 596.2889, found: [M-H]- 596.2886.

6-chloro-3-(1-(3,4- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 – J = 29.2

J = – –

– 13C NMR (126 MHz, CDCl3 [ppm] 143.2, 130.6, 129.4, 127.0, 77.3, 77.0, 76.8, 68.0, 42.4, 36.6, 31.9,

29.7, 29.4, 29.2, 27.4, 26.9, 25.7, 25.6, 14.9, 14.1; LC- R = 4.77 min,HRMS: Calcd for C32H38Cl3N3O4 -H]- 634.2003, found: [M-H]- 634.2001

6-chloro-3-(1-(3- -3,18-dioxo-1,4-diazacyclooctadecan-2- -1H-indole-2-

mixture of rotamers observed; 1H NMR (500 MHz, CDCl3 [ppm] 7.82 (d, J = J = – J = J = 8.2, 2.6

(d, J = – J = 6.7 J = J = J

= – – J = J = J =

3.65 (dd, J = – J = (t, J = J = – J = –

– J = – J = 13C NMR (126 MHz, CDCl3 [ppm] 159.9, 133.5,

129.7, 120.0, 116.8, 116.0, 113.4, 112.9, 77.3, 77.0, 76.8, 55.2, 43.6, 39.6, 36.8, 34.0, 29.7, 29.4, 29.3, 29.1, 29.0, 26.9, 25.8, 24.7, 15.2; LC- R = 4.78 min, HRMS: Calcd for C33H42ClN3O5 -H]- 596.2887, found: [M-H]- 596.2886.

NNH

OO

Cl

NHCl

CO2H

Cl

NNH

OO

NHCl

CO2H

O

91

4

5

6

7

8

9

6-chloro-3-(3,19-dioxo-1-(3,4,5-trifluoroben -1,4-diazacyclononadecan-2- -1H-indole-2-carboxylic acid

Racemic mixture 1H NMR (500 MHz DMSO-d6

11.83 (d, J = J = J = J = –

– 6.1 J = J = 4.35 (p, J = J = – 2.95 (m,

J = – – J = –

13C NMR (126 MHz, DMSO-d6 [ppm] 174.9, 173.4, 173.0, 170.3, 170.0, 164.0, 162.6, 160.8, 153.0, 150.5, 148.5, 142.6, 137.8, 137.4, 136.5, 136.4, 136.3, 131.2, 130.0, 129.4, 128.7, 128.6, 127.5, 126.0, 125.8, 125.3, 125.1, 124.9, 122.2, 121.8, 114.8, 112.4, 111.9, 111.7, 110.7, 110.0, 90.7, 68.5, 61.4, 55.5, 47.2, 41.4, 40.5, 40.4, 40.3, 40.2, 40.2, 40.1, 40.0, 39.9, 39.8, 39.7, 39.6, 39.4, 39.3, 38.5, 35.7, 35.6, 35.5, 34.1, 34.1, 32.8, 31.2, 29.9, 29.5, 29.2, 28.7, 28.5, 28.3, 27.7, 27.4, 26.9, 26.2, 25.6, 24.8, 18.4; LC-MS (DAD/ES R = 3.96 min, HRMS: Calcd for C33H39ClF3N3O4 -H]- 634.2629, found: [M-H]- 634.2654.

(+ -6-chloro-3-(3,19-dioxo-1-(3,4,5- -2- -1H-indole-2-

33H39ClF3N3O4 -H+2]- 632.2629, found: [M-H+2]- 634.26563.

[ ]D20= + 36.6° (c = 1.00, MeOH) (- -6-chloro-3-(3,19-dioxo-1-(3,4,5- -

2- -1H-indole-2-carboxylic acid

33H39ClF3N3O4 -H+2]- 632.2629, found: [M-H+2]- 634.26563.

[ ]D20= - 29.62° (c = 1.08, MeOH)

6-chloro-3-(1-(4-((3,4- -3,24-dioxo-1,4-diazacyclotetracosan-2- -1H-indole-2-

1H NMR (500 MHz, CDCl3 J = J = 81.5, 42.4 Hz,

J = J = 58.1 Hz, J = 4 J =

J = – 13C NMR (126 MHz, CDCl3 [ppm] 170.2, 160.0, 157.6, 156.6, 137.2,

NH

N

OO

NHCl

CO2H

F

FF

N

NH

O

O

O

NHCl

CO2H

Cl

Cl

N

OO

NHCl

CO2H

F

FF

N

OO

NHCl

CO2H

F

FF

92

135.7, 130.8, 130.5, 129.2, 129.0, 128.4, 126.4, 124.5, 115.0, 114.3, 113.9, 77.5, 77.3, 77.3, 77.0, 76.8, 68.5, 68.4, 61.3, 57.1, 52.1, 49.2, 46.5, 43.0, 40.0, 36.8, 33.8, 32.5, 32.0, 31.4, 30.2, 29.7, 29.6, 29.4, 28.9, 28.1, 27.2, 26.9, 26.8, 26.2, 25.6, 24.9, 22.7, 14.4, 14.1; LC-MS

R = 4.92 min, Calcd for C45H56Cl3N3O5 -H]- 822.23, found: [M-H]- 822.32.

3. EXEMPLARY COPIES OF NMR AND MS DATA OF FINAL COMPOUNDS

N

NH

Cl

OHN O

CO2Et

F

F

F

4j

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)

33. 4

9

8.54

2.19

1.67

1.57

0.69

3.74

0.67

1.15

4.80

0.90

7.13

1.15

0.65

1.00

4.90

1.43

1.76

0.62

1.00

1.77

1.74

0.45

0.87

1.00

0.45

0.77

0.99

0.41

0.39

1.12

1.22

1.35

1.36

1.38

1.40

1.41

1.47

1.70

1.76

1.99

2.08

2.34

2.37

2.85

3.20

3.26

3.28

3.64

3.67

4.04

4.35

4.42

4.49

4.66

4.89

4.98

5.24

5.54

5.70

5.97

6.30

6.50

6.96

7.12

7.14

7.44

7.74

7.76

7.79

8.36

8.37

8.81

8.83

8.99

9.07

9.70

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

14. 3

0

23. 8

324

.21

26. 9

329

. 07

29. 3

529

. 45

33.7

740

. 08

40.2

2

46. 7

8

54. 0

055

. 95

61. 7

361

. 98

76. 7

877

. 03

77. 2

9

108.

9610

9.11

109.

6410

9.79

112.

0011

2.29

114.

1211

5.13

115.

3212

1.38

122.

3312

2.95

123.

04

132.

11

135.

7913

7.91

138.

1813

9.19

160.

67

169.

5416

9.75

173.

8317

4.44

93

4

5

6

7

8

9

94

NH

N

OO

NHCl

CO2Et

F

FF

3j

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

1.42

27. 8

6

4.65

11. 3

7

2.54

2.68

3.32

1.15

3.13

0.95

2.61

1.00

1.48

1.13

1.39

1.21

1.26

1.27

1.28

1.32

1.33

1.35

1.38

1.38

1.39

1.40

1.54

1.55

1.65

2.00

2.33

2.55

2.74

2.95

3.04

3.17

3.52

3.58

3.59

3.64

4.35

4.37

4.38

5.21

5.27

5.41

5.43

5.51

5.74

5.83

5.88

5.98

6.49

6.52

7.17

7.19

7.26

7.30

7.50

7.52

8.94

0102030405060708090100110120130140150160170180f1 (ppm)

14. 3

2

24. 7

925

. 10

25. 5

426

. 29

26. 8

727

. 35

27.7

1

31. 3

639

. 78

39. 9

8

46. 6

746

. 84

55. 9

7

61. 7

5

76. 7

677

. 01

77.2

277

.27

109.

6611

2.22

121.

2512

3.17

124.

8612

6.94

129.

0612

9.38

129.

48

131.

7013

5.51

159.

89

169.

4816

9.76

174.

71

95

4

5

6

7

8

9

96

NH

N

OO

NHCl

CO2Et

F

FF

2j

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)

37. 2

6

2.82

1.83

2.41

1.24

0.94

1.05

2.52

2.62

1.10

0.94

3.01

0.84

1.75

1.31

0.90

1.00

1.25

1.35

1.36

1.38

1.39

1.43

1.44

1.46

1.47

1.49

1.54

1.68

1.81

2.38

2.40

2.42

2.61

2.83

2.86

3.17

3.18

3.19

3.59

3.61

3.62

3.63

3.65

4.33

4.35

4.39

4.42

4.43

5.04

5.24

5.27

5.93

5.97

5.98

6.18

6.50

6.94

7.10

7.12

7.19

7.52

7.54

8.93

0102030405060708090100110120130140150160170180190200f1 (ppm)

14. 3

0

24. 5

626

. 15

26. 5

926

. 94

28.4

133

.14

40.1

7

46. 7

5

54. 6

555

. 93

61.7

4

76.7

777

. 03

109.

7310

9.91

112.

2111

4.80

121.

3112

3.19

124.

9112

6.91

132.

22

135.

54

159.

95

169.

66

174.

55

97

4

5

6

7

8

9

98

99

4

5

6

7

8

9

ENANTIOSEPARATION OF COMPOUND 2J

Enantiomeric fractions of the racemic mixture 2j were separated in a semi-preparative SFC -

MeOH/CO2 as mobile phase. Each fraction was evaporated, affording the corresponding pure enantiomers 2ja and 2jb. Afterwards, each enantiomer was subjected to hydrolysis giving the corresponding acids 1ja and 1jb. The optical rotation of all the four adducts was measured to determine the purity of the enantiomers.

NH

NO

O

NHCl

CO2Et

F

FF

NH

NO

O

NHCl

CO2H

F

FF

1. SFC chiral separation

2. LiOH, EtOH-H2O, rt

NH

NO

O

NHCl

CO2H

F

FF

+

1:12j (+)-1ja: (-)-1jb

100

c:\xcalibur\...\2ul\17mdv053-ne82mix 4/12/2017 8:35:21 PM

400 450 500 550 600 650 700 750 800m/z

0

50

1000

50

1000

50

100

Rel

ativ

e Ab

unda

nce

0

50

100 684.2786

662.2970430.9136

721.3704566.8887

790.3574446.8876 648.2814499.2629684.2788

430.9137 662.2972

721.3705566.8887 648.2815446.8876 790.3574499.2632662.2967

684.2783

721.3702430.9136

566.8887 648.2816 735.3860494.2273662.2968

684.2785

721.3704430.9136 648.2818566.8887 735.3862494.2000

NL: 5.17E517mdv053-ne82mix#576 RT: 12.95 AV: 1 T: FTMS + p ESI Full ms [385.00-800.00]

NL: 4.32E517mdv053-ne82mix#792 RT: 17.82 AV: 1 T: FTMS + p ESI Full ms [385.00-800.00]

NL: 1.27E617mdv053-ne82a#571 RT: 12.80 AV: 1 T: FTMS + p ESI Full ms [385.00-800.00]

NL: 2.26E617mdv053-ne82b#803 RT: 17.90 AV: 1 T: FTMS + p ESI Full ms [385.00-800.00]

101

4

5

6

7

8

9

NH

N

OO

NHCl

CO2H

F

FF

1j

012345678910111213141516f1 (ppm)

39. 4

3

4.72

2.41

2.23

1.18

2.02

1.49

2.17

0.43

0.83

8.16

2.98

1.95

0.94

2.27

0.39

1.70

0.91

1.00

0.71

1.22

1.25

1.28

1.30

1.31

1.33

2.12

2.14

2.19

4.23

4.24

4.32

4.34

4.35

4.36

4.45

5.01

5.04

5.08

6.11

6.14

6.21

6.35

6.89

7.08

7.20

7.37

7.92

8.22

8.23

8.27

8.40

11. 8

211

. 93

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

18. 3

626

. 25

27. 3

628

. 67

31.2

135

. 49

39. 3

239

. 82

40. 1

6

47.2

1

55. 5

2

61. 3

5

68.4

7

90.7

4

109.

9711

0.72

111.

7311

1.88

112.

4111

4.75

121.

7712

5.14

126.

0412

8.69

136.

28

142.

63

148.

5415

0.51

153.

04

160.

7716

2.63

164.

01

170.

0417

0.29

173.

0217

3.44

174.

94

102

4. ACTIVITIES OF MACROCYCLIC INHIBITORS OF P53-MDM2 INTERACTION

Table S1. Results of the evaluation of inhibitory activity (Ki MDM2 using FP assay.

Entry Number Structure Ki MDM2

[μM] Plot (MDM

1 1a NNH

OO

NH

CO2HCl

O

ClCl

0.354

3 1b N

NH

O

O

NHCl

CO2H

O

ClCl

0.320

4 1c NNH

OO

O

NHCl

CO2HCl

Cl

0.098

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

103

4

5

6

7

8

9

5 1d

NNH

OO

F

NHCl

CO2H

not active -

6 1e N

NH

OO

Cl

NHCl

CO2HCl

5.250

7 1f

NNH

OO

Cl

NHCl

CO2H

not active -

8 1g N

NH

OO

O

NHCl

CO2H

1.260

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

104

9 1h N

NH

OO

NHCl

CO2H

ClCl

0.082

10 1i N

NH

OO

NHCl

CO2H

O

1.780

11 1j NH

NO

O

NHCl

CO2H

F

FF

0.139

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

105

4

5

6

7

8

9

12 1ja NH

NO

O

NHCl

CO2H

F

FF

0.093

13 1jb NH

NO

O

NHCl

CO2H

F

FF

0.700

14 1k N

NH

O

O

O

NH

ClCO2H

Cl

Cl

1.910

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

106

15 3j NH

N

OO

NHCl

CO2H

F

FF

0.340

16 3k N

NH

O

O

O

NH

ClCO2H

Cl

Cl

2.53

17 2a NNH

OO

NH

CO2EtCl

O

ClCl

2.98

18 2k N

NH

O

O

O

NH

ClCO2Et

Cl

Cl

not active -

19

12

NNH

OO

NH

Cl

not active -

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

0,000

0,100

0,200

0,300

0,400

0,500

0,600

0,700

0,800

0,900

1,000

1,100

1,200

1,300

0,0001 0,0010 0,0100 0,1000 1,0000 10,0000 100,0000

Frac

tion

of b

ound

rep

orte

r pe

ptid

e

Concentration of the inhibitor [μM]

107

4

5

6

7

8

9

20 13 N

NH

OO

Cl

Cl

not active -

21 14 NNH

OO

Cl

Cl

not active -

22 15 NH

NO

O

Cl

Cl

not active -

23 11 HN

NO

O

NH

Cl CO2H

F

F F

0.100

REFERENCES Czarna, A.; Popowicz, G. M.; Pecak, A.;

Wolf, S.; Dubin, G.; Holak, T. A. High Affinity Interaction of the P53 Peptide-Analogue with Human Mdm2 and Mdmx. Cell Cycle Georget. Tex 2009, 8

–1184. Piotto, M.; Saudek, V.; Sklenár, V.

Gradient-Tailored Excitation for Single-Quantum NMR Spectroscopy of Aqueous Solutions. J. Biomol. NMR 1992, 2 –665.

Mori, S.; Abeygunawardana, C.; Johnson, M. O.; Vanzijl, P. C. M. Improved Sensitivity of HSQC Spectra of Exchanging Protons at Short Interscan Delays Using a New Fast

Avoids Water Saturation. J. Magn. Reson. B 1995, 108 –98.

Stoll, R.; Renner, C.; Hansen, S.; Palme, S.; Klein, C.; Belling, A.; Zeslawski, W.; Kamionka, M.; Rehm, T.; Mühlhahn, P.; Schumacher, R.; Hesse, F.; Kaluza, B.; Voelter, W.; Engh, R. A.; Holak, T. A. Chalcone Derivatives Antagonize Interactions between the Human

Oncoprotein MDM2 and P53. Biochemistry (Mosc.) 2001, 40 336–344.

Dömling, A.; Holak, T. Novel P53-Mdm2/P53-Mdm4 Antagonists to Treat Proliferative Disease. WO2011106650 A3, January 19, 2012.

CHAPTER

2,3'-BIS(1'H-INDOLE) HETEROCYCLES: NEW

P53/MDM2/MDMX ANTAGONISTS

Constantinos G. Neochoritis,a Kan Wang,b Natalia Estrada-Ortiz,a Eberhardt Herdtweck,c Katarzyna Kubica,d Aleksandra Twarda,e, f Krzysztof M. Zak,e Tad A. Holak,d, e and Alexander Dömlinga, b*

aDepartment of Drug Design, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands. bDepartment of Chemistry, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA. cDepartment Chemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching bei München, Germany. dDepartment of Organic Chemistry, Jagellonian University, Ingardena 3, 30-060 Krakow, Poland. eMalopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Krakow, Poland fFaculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland.

Published in Bioorg Med Chem Lett. 2015 December 15; 25(24): 5661–5666

110

Abstract: The protein-protein interaction of p53 and MDM2/X is a promising non genotoxic anticancer target. A rapid and efficient methodology was developed to synthesize the 2,3'-bis(1'H-indole) heterocyclic scaffold 2 as ester, acid and amide derivatives. Their binding affinity with MDM2 was evaluated using both fluorescence polarization (FP) assay and HSQC experiments, indicating good inhibition and a perfect starting point for further optimizations.

Keywords: - cancer

111

5

6

7

8

9

The transcription factor and tumor suppressor p53 has multiple roles in stabilizing the genome by preventing mutations.1,2 However p53, our genome guardian, is the most mutated protein in human cancer with >50% of cancers not showing functional p53.3 The remainder group of cancers also exhibit reduced p53 pathway activity due to the negative regulation through the protein-protein interaction (PPI) with inhibitors as MDM2/X or viral proteins. MDM2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. MDM2 inhibits the N-terminal trans-activation domain (TAD) of p53, and promotes p53 degradation through the ubiquitin-proteasome system (E3 ligase activity).4,5 Similarly to MDM2, MDMX (90% homology with MDM2) its negative regulation of p53 is via inhibition of the TAD domain and forming heterodimers with MDM2 can increase the rates of ubiquitinylation of p53 by MDM2.6,7 Therefore, blocking the interaction between wild-type p53 and its negative regulators MDM2 and MDMX has become an important target in oncology to restore the anti-tumor activity of p53.8–10

The discovery of novel p53-MDM2/X inhibitors was one of the highlights in anti-tumor agents.11 Since the disclosure of Nutlin-3,12 many scaffolds have been prepared and evaluated, including indo-imidazole, imidazoline, benzodiazepinedione and spirooxindole scaffolds.13 Thus, antagonizing the p53- trategy where several compounds presently undergo early clinical evaluations.14–17 However, the discovery of new p53/MDM2/MDMX scaffolds is still of high interest due to low single agent activity currently seen in clinical trials and insufficient PKPD properties.

A three finger pharmacophore model is now widely accepted to be responsible for the binding of small molecules to the MDM2 and recently an extended four finger model was experimentally shown by co-crystallization.18,19 The corresponding amino acid residues in p53 are tryptophan (Trp23), leucine (Leu26) and phenylalanine (Phe19). Initially, indole fragment was taken as starting point to mimic tryptophan residue, where an important hydrogen bond was observed. Studies by Garcia-Echeverria et al. on a p53-derived linear octapeptide showed that a Trp23 to (6-Cl) Trp substitution gave rise to a 63-fold increase in affinity for MDM2.20 The starting point for our antagonist discoveries was the anchoring side chain of tryptophan embedded in a deep hydrophobic pocket formed by the residues Leu57, Phe86 and Ile99 using our pharmacophore based virtual screening platform ANCHOR.QUERY.10,18,21–25 This had led to several scaffolds potently antagonizing p53-MDM2. Amongst them we could also solve representative co-crystal structures of the imidazoloindole derivative 1 binding to MDM2 (PDB ID: 3LBK) and the close relative MDMX (PDB ID: 3LBJ) confirming the initial binding hypothesis.21,22 Based on these findings, a new class of 1,2,3-trisubstituted bis(indoles) heterocycles (Figure 1) was designed. In this paper, we report the novel synthesis and preliminary biophysical evaluation of this first of its kind antagonists bisindoles 2. It should be noted that the class of p53-MDM2 antagonistic imidazoloindole has also discovered independently by another group.21

Figure 1. Protein- -helix with sticks) with MDM2 (grey surface) (PDB ID 1YCR). Key amino acids Leu22 (marine sticks), Phe19 (green sticks), Trp23 (blue sticks) and Leu26 (red sticks) are

-helix. Leu26 is embedded into the hydrophobic receptor amino acids Ile99, Ile103,

112

Leu54 and Val93. The Trp23 pocket is formed by Leu57, Phe86 and Ile99. The Phe19 pocket consists of Val93, Gln72, Val75, Tyr67, Met62, Il261 and Gly58. Val93 undergoes hydrophobic interactions with p53Leu22.

Our hypothesis is that the additional phenyl annulated ring in scaffold 2 will make additional hydrophobic interactions with the mdm2Val93,13 mimicking p53Leu22 (Figure 2). To test this speculation we developed a short synthesis towards scaffold 2.

NN

NH

Cl

YN

NH

Y

Cl

Leu22

1 2

Figure 2. Conversion of central imidazole scaffold 1 into indole scaffold 2 to address p53Leu22.

Our retrosynthetic plan of the designed derivatives 2 is based on the indole acetyl derivative 3 and hydrazine 4 which can react in a Fischer indole synthesis (Scheme 1).

Scheme 1. Retrosynthesis of scaffold 2

3 4

NH

YCl

O

NNH2

+

N

NH

YCl

2

Fischer indole

After some optimization, derivative 3a was easily synthesized by selective acylation at the 3-position of the 6-chloroindole derivative 5a in high yields in the presence of Lewis acids, such as SnCl4 (Scheme 2).26–28 Compound 4a was derived by the direct alkylation of the secondary amine of the phenylhydrazine 7a, with no need of protecting groups.29,30 Finally, refluxing derivatives 3a and 4a in acetic acid, gave the targeted bisindole 2a in good to very good yields in a just 3-step sequence.

Scheme 2. 3-step sequence for preparing 2,3'-bis(1'H-indole) heterocycles.

N

NH

OOEt

Cl

NNH2

NH

NH2 +

Br

Et3N, toluene

NH

OEt

OCl NH

OEt

OCl

PhCl

O

O Ph

SnCl4, CH2Cl2/MeNO2

5a

6a

3a

7a 8a4a 2a

AcOH

The synthesis of bisindoles 2, offers many possibilities for SARs since both of the main components (3 and 4) could easily be modified (Figure 3). Different halogens as R1, R2 and R3 and different sizes in the linkers of the two aromatic groups were tested. Moreover, the importance of carboxylate moiety was taken into account, as found in previous MDM2 binders.18,19,21,22 Using the ethyl esters, the corresponding acids and various amides could be synthesized in order to optimize some physicochemical properties (Figure 3).

113

5

6

7

8

9

N

NH

Y

Cl

2

R3

R1R2

Figure 3. SAR potential of bisindoles.

The 3-alkylation of the 6-chloroindole derivatives proceeded smoothly affording the corresponding compounds in excellent yields as shown in table 1.28 The crystal structure obtained of the indole derivative 3c, is described in supporting information (CCDC 981828).

Table 1. 3-Acylation of 6-chloroindole derivatives

NHCl N

H

Y

Cl

XCl

O

OX

SnCl4,CH2Cl2/MeNO2

5

6

3a-e

Y

Entry Y X Compound

(yield %)

1 -CO2Et Ph 3a (99)

2 -CO2Et 4-Cl-C6H4 3b (97)

3 -CO2Et 4-F-C6H4 3c (95)

4 -CO2Et 4-Cl-C6H4CH2 3d (95)

5 H Ph 3e (99)

The hydrazine derivatives 4a-c were synthesized by the regioselective alkylation of the secondary amine, thus different hydrazines and benzyl halides were utilized in order to initially explore the aforementioned interactions in the MDM2 pocket (Table 2).

114

Table 2. Alkylation of the secondary amine of hydrazines 7.

NNH2

NH

NH2+

Br

78 4a-c

tolueneEt3N

R1

R2

R1

R2

N

NH2

4d

Entry R1 R2 Compound (yield %)

1 H H 4a (40)

2 H Cl 4b (60)

3 Cl Cl 4c (42)

Next, the synthesis of the desired bisindoles 2a-h was performed. Combining the two moieties, the indole derivatives 3 and hydrazines 4 into a Fischer indole synthesis produced a series of active compounds (Scheme 3). All the combinations and compounds that were synthesized are shown below.

Scheme 3. Library of the indole derivatives 2

NX

R2

NH

Y

Cl3 4

R1

NH

YCl

O X

NNH2

R2

R1

+

2

AcOH

reflux

NHCl

NPh

O

OEt

Cl

2a (64%)

NHCl

NPh

O

OEt

Ph

2b (65%)

NHCl

NPh

Cl

2c (70%)

NHCl

N

O

OEt

ClCl

2d (61%)

NHCl

N

O

OEt

ClF

2e (66%)

NHCl

NPh

O

OEt

Cl

Cl

2f (45%)mixture of isomers 1:1

NHCl

NPhO

OEt

Cl

2g (50%)

NHCl

NPhO

OEt

F

2h (56%)

The structures of the above compounds were unambiguously characterized and confirmed by the crystal structures of the compounds 2a and 2h (SI, CCDCs 981829 and 981827 respectively). Due to the fact that ester groups are readily cleaved in cells by esterases and are sometimes metabolically unstable, we hydrolyzed, under basic conditions specific derivatives 2 into the corresponding acids 9 in

115

5

6

7

8

9

order to compare and evaluate the activity. Additionally we know from our previous studies on scaffold with the indole anchor, that the free –COOH is always 1-2 orders of magnitude more potent towards MDM2 protein.10,18–20,25,31

Scheme 4. Hydrolysis of derivatives 2 to the corresponding acids 9.

NX

R2

NH

CO2Et

Cl

R1

2

NX

R2

NH

CO2HCl

R1

LiOH

EtOH/H2O

9

NHCl

NPh

O

OH

Cl

NHCl

NPh

O

OH NHCl

N

O

OH

ClCl

9a (61%) 9b (45%) 9c (52%)

NHCl

N

O

OH

ClF

9d (55%)

NHCl

NPhO

OH

Cl

9e (58%)

Apart from poor absorption-distribution-metabolism-excretion-toxicology (ADMET) properties, insufficiently water-soluble compounds often lead to poor reproducibility and unreliable results or even false positive hits during in vitro screening. In order to potentially improve the properties of our compounds, we converted the ester group of the derivatives 2 into the corresponding better water soluble amides 10 with a one-step TBD-catalyzed amidation procedure (Scheme 5).32 In our previous studies, amidation of the indole-2-carboxylate gave often improved water solubility.31

Scheme 5. TBD-catalyzed amidation of the bisindoles 2

NX

R2

NH

CO2Et

Cl

R1

2

NHR4R5

TBD, THF

NX

R2

NHCl

R1

O

NR4R5

10

NHCl

NPh

O

N

Cl

H

N

O

10a (45%)

NHCl

N

O

N

Cl

H

N

OCl

10b (46%)

NHCl

NPh

O

N

Cl

H

OH

10c (50%)

NHCl

NPh

O

N

Cl

H

N O

10d (70%)

NHCl

NPh

O

HN

Cl

O

OH

10e (35%)

NHCl

NPh

O

N

Cl

O10f (46%)

Binding affinities were determined by the fluorescence polarization method (FP) towards MDM2 and MDMX (SI).25 Representatives from each class compounds, esters, acids and an amide, were evaluated as inhibitors of the PPI. The results are summarized in the table 3. Noticeably, the acids 9a and 9d comparing with the corresponding esters 2a and 2e were found highly active towards both MDM2 and MDMX as previously reported by the indole anchor.10,18–20,25,31 In addition, compounds 9a,d provided some initial dual activity (MDM2/X) which is an interesting factor that most of the current inhibitors

116

fail to combine.17 Moreover comparing the binding affinities of scaffold 1 (WK23)22 9, is more potent towards MDMX

without significant loss of activity in MDM2 (9a) or even slight improvements (9d).

Table 3. Results of the evaluation of inhibitory activity of compounds 2,3'-bis(1'H-indole) Heterocycles towards MDM2/MDMX as determined by FP.

Ki (μM)

Compound MDM2 MDMX

2a 17 NA

2c 10 NA

2d >22 NA

2e 6.7 5.0

2f NA NA

9a 1.8 0.2

9d 0.7 1.5

10d NA 6,09

WK23 (1) 0.92 36

NA: not active, Ki > 60 μM.

FP-based screening of protein-protein interactions often gives a high fraction of false positives especially with hydrophobic molecules and therefore it is advisable to run a second orthogonal biophysical assay. As a second, orthogonal screening system we performed the well-known heteronuclear single quantum coherence (HSQC) experiment where compound the 15N labeled MDM2 is titrated with compound 9d.The expected ligand-induced perturbations in NMR chemical shifts are indeed observed (Figure 4).33 Since all cross peaks in the MDM2 spectrum were assigned to particular amino acid residues before 34–36 it is possible to analyze the way of interaction in the MDM2/9d complex. For example, the interaction of 9d with the MDM2 tryptophan subpocket is expressed by the movement of the peak assigned to Mdm2Val93 (Figure 4). Chemical shift changes of T101 and M62 indicate interaction with the leucine and phenylalanine subpocket, respectively. Both the FP assay and HSQC test indicate that the acid derivatives are the active species as p53-MDM2/X inhibitors.

NN

NH

Cl

CO2H

Cl

WK23

117

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8

9

Figure 4. Superposition of NMR HSQC spectra of the 15N-labeled MDM2 titrated against 9d. The spectrum of free MDM2 is shown in blue. The spectrum of 9d-MDM2 (ratio 1:1, respectively) is shown in red.

Our modelling (Figure 5) based on the HSQC binding data and known co-crystal structures and using MOLOC software37 revealed the nice alignment of the 6-chloro-indole moiety of compound 9d with the anchoring p53Trp23, whereas the two phenyl rings occupy the hydrophobic pockets mimicking p53Phe19 and p53Leu26. Moreover, the additionally introduced phenyl-annulated ring of the second indole moiety is predicted to cover mdm2Val93 through hydrophobic interactions. Then we rationalized the tight receptor ligand interaction using a small world network approach using Scorpion software (Figure 5).31 His96 is forming a pi-pi interaction with the p-Cl phenyl fragment and a hydrogen bond is formed between the Leu57 backbone carbonyl and the indole NH. Multiple van der Waals interactions can be used to rationalize the tight interactions. Amongst the major contributors of the interaction, shown as red balls, are F37-Tyr67, F27-Met62, C20-Met62, N7-Leu57. Cl10 contributes to an extended network including Phe86, Ile99, Ile103 and Leu57. Less strong contributors are C13-Val93, C3-, C1-, C6- of the buried indole moiety and C31-Leu57 and Cl36-Ile99. Interestingly small world network analysis of WK23 (PDB ID 3LBK) reveals no direct contribution of the scaffold imidazole to the tight interaction, whereas in 9d C13 interacts with Val93, supporting our hypothesis.

118

Figure 5. above: Modeled binding pose of the most potent compound 9d (green sticks) in the MDM2 pocket (grey surface presentation, PDB ID 1YCR) and alignment with the hot-spot triade F19W23L26 (blue sticks). L57 and V93 are shown as grey sticks; below: Small network analysis of 9d in the MDM2 receptor. - , hydrogen bonding and van der Waals interactions are shown in red, blue and magenta dotted lines, respectively. Each ligand non-H atom is shown as colored ball according to its importance of contribution to the network (descending importance: red, purple, grey). Atom numbering of 9d is given in the right corner.

In conclusion, the designed 2,3'-bis(1'H-indole) scaffold 9 is active as dual action inhibitor of the p53-MDM2/X interactions with initial sub-μM affinities. Our hypothesis, that the extra phenyl ring in scaffold 2 makes additional hydrophobic interactions with the Mdm2Val93 comparing with the derivatives 1, is suggested by 2D NMR and modeling studies. Further studies are ongoing to introduce more ‘drug-like’ properties into this scaffold and to investigate cellular mechanism-based anti-cancer behaviors.

SUPPLEMENTARY DATA Experimental procedures for the synthesis of compounds, characterization of compounds, crystal data, as well FP assay and NMR HSQC are provided in the supporting information.

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9

SUPPORTING INFORMATION

GENERAL METHODS

All reactions were performed under air atmosphere. All other reagents and solvents are purchased without further purification. Analytical thin-layer chromatography (TLC) was performed on SiO2 plates on Alumina available from Whatman. Visualization was accomplished by UV irradiation at 254 nm, or by staining with any one of the following reagents: iodine, ninhydrin (0.3% w/v in glacial acetic acid/n-butyl alcohol 3:97), Vaughn’s reagent (4.8 g of (NH4)6Mo7O24

.4H2O and 0.2 g of Ce(SO4)2.4H2O in 10 mL of conc. H2SO4 and

90 mL of H2O). Flash column chromatography was performed using SiO2 60 (particle size 0.040-0.055 mm, 230-400 mesh, EM science distributed by Bioman). Preparative TLC was conducted using preparative silica gel TLC plates NMR spectra were obtained on Bruker Avance™ 600 MHz NMR spectrometer. Chemical shifts

1H NMR spectra are tabulated as follows: chemical shift, multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd = double of doublets, ddd = double of doublet of doublets, m = multiplet), coupling constant(s) and number of protons. High Resolution Mass spectra were obtained at the University of Pittsburgh Mass Spectrometry facility. LC-MS analysis was performed on an SHIMADZU instrument, using an analytical C18

PROCEDURE AND ANALYTICAL DATA OF INDOLE DERIVATIVES 3

To a stirred solution of the corresponding indole derivatives (5.0 mmol) in DCM (10 mL), SnCl4 (1.0 M in DCM, 7.5 mmol) was added. The reaction mixture stirred for 30 min at rt and then, the corresponding phenyl acetyl chloride (6.0 mmol) was added slowly following the addition of MeNO2 (10 mL). After stirring overnight at rt, the reaction was quenched with ice/water, extracted with ethyl acetate, washed by 1.0 M NaOH solution three times and one time by brine. The solvent was removed and the product was generated and used in the next step without further purification.

ethyl 6-chloro-3-(2-phenylacetyl)-1H-indole-2-carboxylate (3a)

yellow solid, 99% yield; 1H NMR (CDCl3, 600 MHz): 9.49 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.36 (s, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.20-7.27 (m, 3H), 7.15 (d, J = 8.4 Hz, 1H), 4.48 (q, J = 6.6 Hz, 2H), 4.45 (s, 2H), N

H

OEt

OCl

OPh

122

1.43 (t, J = 6.6 Hz, 3H) ppm; 13C NMR (CDCl3, 150 MHz): 198.3, 160.5, 135.2, 134.7, 133.2, 129.6, 128.5, 126.8, 126.4, 125.4, 123.6, 123.5, 121.5, 111.6, 62.2, 50.5, 14.3 ppm. ESL-TOF for C19H16ClNO3 (M+) found: m/z: 341.0818; Calc. Mass: 341.0819.

ethyl 6-chloro-3-(2-(4-chlorophenyl)acetyl)-1H-indole-2-carboxylate (3b)

yellow solid, 97% yield; 1H NMR (DMSO-d6, 600 MHz): 12.5 (br s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 4.44 (q, J = 6.6 Hz, 2H), 4.37 (s, 2H), 1.36 (t, J = 6.6 Hz, 3H) ppm; 13C NMR (DMSO-d6, 150 MHz): 197.1, 161.1, 136.2, 134.8, 132.0, 131.7, 130.4, 128.8, 128.6, 125.1,

123.7, 123.2, 120.1, 112.7, 62.3, 48.9, 14.5 ppm. ESL-TOF for C19H16ClNO3Na (M+) found: m/z: 398.0329; Calc. Mass: 398.0327.

ethyl 6-chloro-3-(2-(4-fluorophenyl)acetyl)-1H-indole-2-carboxylate (3c)

yellow solid, 95% yield; 1H NMR (600 MHz, CDCl3): 9.20 (s, 1H), 7.81 (d, 1H, J = 8.0 Hz), 7.40 (s, 1H), 7.18-7.23 (m, 3H), 6.98-7.01 (m, 2H), 4.50 (q, 2H, J = 7.2 Hz), 4.43 (s, 2H), 1.46 (t, 3H, J = 7.2 Hz); 13C NMR (150 MHz, CDCl3): 14.3, 49.4, 62.3, 111.5, 115.3, 115.4, 121.5, 123.7, 123.9, 125.5, 126.4, 131.2, 132.4, 135.1, 160.3, 161.1, 162.7,

197.9 ppm. ESL-TOF for C19H15ClFNO3 (M+) found: m/z: 359.0729; Calc. Mass: 359.0725.

ethyl 6-chloro-3-(3-(4-chlorophenyl)propanoyl)-1H-indole-2-carboxylate (3d)

yellow solid, 95% yield; 1H NMR (DMSO-d6, 600 MHz): 7.90 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 7.27 (dd, J = 7.8, 2.4 Hz,

1H), 4.43 (q, J = 7.2 Hz, 2H), 3.38 (t, J = 7.2 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 1.37 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (DMSO-d6, 150 MHz): 198.0, 160.8, 140.3, 136.0, 130.4, 130.2, 129.6, 128.1, 124.6, 123.3, 122.6, 119.3, 112.3, 61.7, 43.9, 29.3, 13.9 ppm; ESL-TOF for C20H17Cl2NO3 (M+) found: m/z: 269.0610; Calc. Mass: 269.0607.

1-(6-chloro-1H-indol-3-yl)-2-phenylethanone (3e)

yellow solid, 99% yield; 1H NMR (CDCl3, 600 MHz): 12.06 (s, 1H), 8.44 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.25-7.29 (m, 4H), 7.14-7.19 (m, 2H), 4.10 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 193.7, 137.6, 136.4, 135.9, 129.7, 128.8, 128.1, 126.9, 124.6, 123.0, 122.7,

NHCl

OEt

O

O

F

NHCl

OEt

O

O

Cl

NHCl

OEt

O

O

Cl

NHCl

OPh

123

5

6

7

8

9

116.3, 112.4, 46.1 ppm. ESL-TOF for C16H12ClNO (M+) found: m/z: 269.0612; Calc. Mass: 269.0607.

PROCEDURE AND ANALYTICAL DATA OF HYDRAZINE DERIVATIVES 4

To a stirred solution of the corresponding benzyl chloride (10.0 mmol) in toluene (10 mL), the corresponding phenyl hydrazine (10.0 mmol) and triethylamine (10.0 mmol) were added and the reaction mixture was refluxed overnight. Afterwards, the solvent was evaporated and the crude product purified with flash chromatography on silica gel, eluted with hexane-ethyl acetate (1:1).

1-benzyl-1-phenylhydrazine (4a)1

yellow oil, 40% yield; 1H NMR (CDCl3, 400 MHz,): 7.39-7.27 (m, 7H), 7.11-7.15 (m, 2H), 6.87-6.82 (m, 1H), 4.62 (s, 2H), 3.58 (s, 2H) ppm; 13C NMR (CDCl3, 100 MHz): 148.1, 139.4, 129.2, 128.6, 127.5, 127.2, 117.5, 112.8, 48.3 ppm.

1-(4-chlorobenzyl)-1-phenylhydrazine (4b)2

yellow oil, 60% yield; 1H NMR (CDCl3, 600 MHz): 7.32-7.22 (m, 6H), 7.05 (d, J = 7.8 Hz, 2H), 6.83 (t, J = 7.2 Hz, 1H), 4.56 (s, 2H), 3.58 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 151.5, 136.2, 133.1, 129.17, 129.15, 128.8, 118.8, 113.5, 59.8 ppm. ESL-TOF for C13H13ClN2 (M+)

found: m/z: 232.0769; Calc. Mass: 232.0767.

1-(4-chlorobenzyl)-1-(3-chlorophenyl)hydrazine (4c)

yellow oil, 42% yield; 1H NMR (CDCl3, 600 MHz): 7.33 (d, J = 7.2 Hz, 2H), 7.22 (d, J = 7.2 Hz, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.11 (s, 1H), 6.90 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 7.2 Hz, 1H), 4.58 (s, 2H), 3.59 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 152.5, 135.5, 135.1, 133.4,

130.1, 129.1, 129.0, 118.4, 111.4, 111.3, 59.2 ppm. ESL-TOF for C13H12Cl2N2 (M+) found: m/z: 266.0376; Calc. Mass: 266.0378.

PROCEDURE AND ANALYTICAL DATA OF BIS-INDOLE DERIVATIVES 2

To a stirred solution of the corresponding compounds 3a-e (4.0 mmol) in acetic acid (2 mL), compounds 4a-d (4.0 mmol) were added and the reaction mixture refluxed for 30 min. The

NNH2

Cl

NNH2

Cl

Cl

NNH2

124

solvent was removed and the crude product purified with flash chromatography on silica gel eluted with hexane-ethyl acetate (4:1).

ethyl 6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylate (2a)

yellow solid, yield 64%; 1H NMR (CDCl3, 600 MHz): 9.03 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.28-7.15 (m, 7H), 7.10 (t, J= 7.2 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 9.0 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 5.23

(d, J = 16.8 Hz, 1H), 5.07 (d, J = 16.8 Hz, 1H), 4.05 (qd, J = 7.2, 2.4 Hz, 2H), 1.03 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (CDCl3, 150 MHz): 160.9, 137.0, 136.3, 135.7, 135.1, 132.9, 132.0, 128.8, 128.5, 128.4, 128.1, 127.8, 127.5, 127.3, 126.9, 125.7, 122.8, 122.5, 122.4, 120.2, 119.9, 118.2, 112.5, 111.8, 110.1, 61.2, 47.0, 14.0 ppm. ESL-TOF for C32H24Cl2N2O2 (M+) found: m/z: 538.1215; Calc. Mass: 538.1215.

ethyl 1-benzyl-6'-chloro-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylate (2b)

yellow solid, yield 65%; 1H NMR (CDCl3, 600 MHz): 8.95 (s, 1H), 7.78 (d, J = 1.8 Hz, 1H), 7.35 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.21-7.15 (m, 4H), 7.14-7.08 (m, 3H), 7.04-7.00 (m, 4H), 6.97 (d, J = 9.0 Hz, 1H), 6.80-6.75 (m, 2H), 5.20 (d, J = 16.2 Hz, 1H), 5.02 (d, J =

16.2 Hz, 1H), 3.95 (t, J = 7.8 Hz, 2H), 0.93 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (CDCl3, 150 MHz): 161.0, 137.8, 137.1, 135.7, 135.3, 131.9, 128.8, 128.7, 128.3, 128.1, 127.6, 127.2, 127.1, 127.0, 126.5, 125.6, 122.7, 122.6, 122.2, 120.1, 119.8, 118.0, 112.6, 111.8, 110.3, 61.1, 47.7, 14.0 ppm. ESL-TOF for C32H25ClN2O2 (M+) found: m/z: 504.1607; Calc. Mass: 504.1605.

6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-2,3'-biindole (2c)

white solid, yield 70%; 1H NMR (CDCl3, 600 MHz): 8.22 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.36 (s, 2H), 7.35 (s, 1H), 7.28-7.10 (m, 9H), 6.98 (dd, J = 7.8, 1.8 Hz, 2H), 6.85 (d, J = 7.8 Hz, 2H), 5.24 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 137.0,

136.8, 136.1, 135.3, 132.9, 130.4, 129.3, 128.8, 128.6, 128.2, 127.5, 126.2, 126.0, 125.6, 122.3, 121.4, 120.8, 120.4, 119.7, 117.2, 111.3, 110.2, 107.4, 46.9 ppm. ESL-TOF for C29H20Cl2N2 (M+) found: m/z: 466.0998; Calc. Mass: 466.1004.

ethyl 6'-chloro-1-(4-chlorobenzyl)-3-(4-chloro phenyl)-1H,1'H-[2,3'-biindole]-2'-carboxylate (2d)

NHCl

NPh

O

OEt

Cl

NHCl

NPh

O

OEt

Ph

NHCl

NPh

Cl

NHCl

N

O

OEt

ClCl

125

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6

7

8

9

yellow solid, yield 61%; 1H NMR (CDCl3, 600 MHz): 9.10 (s, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.44 (s, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.29-7.10 (m, 7H), 7.03-7.09 (m, 3H), 6.76 (d, J = 8.4 Hz, 2H), 5.22 (d, J = 16.8 Hz, 1H), 5.05 (d, J = 16.8 Hz, 1H), 4.07 (q, J = 6.6 Hz, 2H), 1.04 (t, J = 6.6 Hz, 3H) ppm; 13C NMR (CDCl3, 150 MHz): 137.0, 136.1, 135.7, 133.7, 133.0, 132.2, 131.4, 130.0, 128.7, 128.5, 128.4, 127.8, 127.3, 127.0, 126.9, 122.9, 122.6, 122.3, 120.5, 119.6, 117.0, 112.2, 112.0, 110.2, 61.3, 47.1, 14.0 ppm. ESL-TOF for C32H23Cl3N2O2 (M+) found: m/z: 572.0823; Calc. Mass: 572.0825.

ethyl 6'-chloro-1-(4-chlorobenzyl)-3-(4-fluoro phenyl)-1H,1'H-[2,3'-biindole]-2'-carboxylate (2e)

yellow solid, yield 66%; 1H NMR (DMSO/CDCl3, 600 MHz): 12.20 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.08-7.22 (m, 5H), 7.04 (d, J = 7.8 Hz, 2H), 6.94-6.86 (m, 3H), 6.79 (d, J = 6.6 Hz, 2H), 5.23 (d, J = 16.2

Hz, 1H), 5.10 (d, J = 16.2 Hz, 1H), 4.11-3.99 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (DMSO/CDCl3, 150 MHz): 160.7 (t, J = 243 Hz), 160.7, 136.9 (d, J = 9 Hz), 136.8, 132.3, 131.6, 130.5, 130.3 (d, J = 8 Hz), 129.7, 128.4, 128.3, 127.6, 127.0, 126.8, 122.4, 122.1, 122.0, 120.4, 119.1, 116.0, 115.3, 115.2, 112.8, 111.2, 110.7, 60.8, 46.8, 14.3 ppm. ESL-TOF for C32H23Cl2FN2O2 (M+) found: m/z: 556.1200; Calc. Mass: 556.1121.

ethyl 6,6'-dichloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylate,

ethyl 4,6'-dichloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylate (2f)

NHCl

NPh

O

OEt

Cl

Cl

NHCl

NPh

O

OEt

Cl

Cl

Crude proton NMR indicated a mixture of two isomers in a 1:1 ratio

Yellow solid, yield 45%, Isomer 1: 1H NMR (CDCl3, 600 MHz): 8.97 (s, 1H), 7.35 (d, J = 1.8 Hz, 1H), 7.19-7.23 (m, 4H), 7.14 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.12-7.05 (m, 5H), 7.04 (dd, J = 8.4, 1.8 Hz, 1H), 6.80 (d, J = 8.4 Hz, 2H), 5.18 (d, J = 16.8 Hz, 1H), 5.06 (d, J = 16.8 Hz, 1H), 4.10-4.18 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H) ppm; Isomer 2: 1H NMR (CDCl3, 600 MHz): 9.06 (s, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.66 (s, 1H), 7.65 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 1.2 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 1.8 Hz, 1H), 7.22-6.88 (m, 3H), 6.83 (d, J = 7.8 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 5.16 (d, J = 16.2 Hz, 1H), 5.03 (d, J = 16.2 Hz, 1H), 4.02-4.09 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H) ppm. ESL-TOF for C32H23Cl3N2O2 (M+) found: m/z: 572.0823; Calc. Mass: 572.0825. 13C NMR (CDCl3, 150 MHz): 160.6, 137.8, 135.8, 135.5, 134.6, 133.1, 131.9, 131.3, 130.8, 128.6, 127.8, 127.5, 127.1, 126.8, 126.6, 126.2, 124.2, 122.7, 122.5, 122.2,

NHCl

N

O

OEt

ClF

126

121.3, 118.5, 111.9, 111.8, 108.8, 61.3, 47.4, 14.1 ppm; ESL-TOF for C32H23Cl3N2O2 (M+) found: m/z: 572.0823; Calc. Mass: 572.0825.

ethyl 6'-chloro-3-(4-chlorobenzyl)-1-phenyl-1H,1'H-2,3'-biindole-2'-carboxylate (2g)

yellow solid, 50%; 1H NMR (CDCl3, 600 MHz): 8.93 (s, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.40 (s, 1H), 7.40-7.37 (m, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.24-7.28 (m, 3H), 7.20 (t, J = 7.2 Hz, 3H), 7.17-7.04 (m, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 7.2 Hz, 1H), 4.13 (dq, J = 11.4, 7.2 Hz, 1H), 4.06 (d, J = 16.2 Hz, 1H), 4.04 (dq, J = 11.4, 7.2 Hz, 1H), 3.88 (d, J = 16.2 Hz, 1H), 1.05 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (CDCl3,

150 MHz): 160.7, 139.7, 138.1, 137.8, 135.6, 131.8, 131.2, 129.6, 129.5, 128.7, 128.0, 127.9, 127.5, 126.9, 126.7, 122.6, 122.4, 120.0, 119.3, 115.1, 112.9, 111.8, 110.5, 61.0, 30.4, 14.1 ppm. ESL-TOF for C32H24Cl2N2O2 (M+) found: m/z: 5638.1210; Calc. Mass: 538.1215.

ethyl 6'-chloro-3-(4-fluorophenyl)-1-phenyl-1H,1'H-2,3'-biindole-2'-carboxylate (2h)

yellow solid, 56%; 1H NMR (CDCl3, 600 MHz): 8.87 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.33 (s, 1H), 7.30 (t, J = 7.8 Hz, 2H), 7.27-7.18 (m, 6H), 7.12 (s, 2H), 6.99 (d, J = 8.4 Hz, 1H), 6.91 (t, J = 8.4 Hz, 2H), 4.20-4.04 (m, 2H), 1.06 (t, J = 7.8 Hz, 3H) ppm; 13C NMR (CDCl3, 150 MHz): 161.0, 140.1, 139.1,

138.2, 136.3, 130.4, 130.2, 129.4, 129.3, 129.1, 128.0, 127.7, 126.5, 125.9, 125.7, 122.4, 121.4, 120.0, 119.3, 114.8, 113.9, 112.8, 110.5, 30.2, 14.5. ESL-TOF for C31H22ClFN2O2 (M+) found: m/z: 508.1360; Calc. Mass: 508.1354;

PROCEDURE AND ANALYTICAL DATA OF THE BIS-INDOLE DERIVATIVES 9

To a stirred solution of the corresponding compounds 2 (1.0 mmol) in EtOH-water (1:1), LiOH (10.0 mmol) was added and the reaction mixture refluxed overnight. Then, pH was adjusted to approximately 6 with the addition of 1 N HCl and the reaction mixture extracted with DCM (3x20 mL). The organic layer was separated, washed with water, dried with magnesium sulfate, filtered and concentrated in vacuo, affording the product as solid.

6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylic acid (9a)

white solid, yield 61%; 1H NMR (MeOH-d4, 600 MHz): 7.78 (d, J = 7.8 Hz, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 7.8 Hz, 2H), 7.21 (t, J = 7.2 Hz, 1H), 7.17 (t, J =

7.8 Hz, 2H), 7.15 (t, J = 7.8 Hz, 1H), 7.07 (t, J = 7.2 Hz, 1H), 7.03 (d, J = 8.4 Hz, 2H), 7.00 (d, J =

NHCl

NPh

O

OH

Cl

NHCl

NPhO

OEt

Cl

NHCl

NPhO

OEt

F

127

5

6

7

8

9

8.4 Hz, 1H), 6.87 (dd, J = 8.4, 1.8 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 5.24 (s, 2H) ppm; 13C NMR (MeOH-d4, 150 MHz): 162.3, 137.2, 136.9, 136.4, 135.5, 132.3, 130.7, 129.3, 128.7, 127.85, 127.84, 127.6, 127.2, 126.9, 125.2, 121.8, 121.7, 121.3, 119.6, 118.9, 117.3, 112.2, 111.6, 109.8, 46.4 ppm. ESL-TOF for C30H20Cl2N2O2 (M+) found: m/z: 510.0904; Calc. Mass: 510.0902.

1-benzyl-6'-chloro-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxylic acid (9b)

yellow solid, yield 45%; 1H NMR (MeOH-(d, J = 7.8 Hz, 1H), 7.30 (d, J = 1.8 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.18 (d, J = 7.2 Hz, 2H), 7.05-6.97 (m, 4H), 6.91 (t, J = 7.8 Hz, 1H), 6.89-6.86 (m, 4H), 6.87-6.91 (m, 3H), 5.10 (s, 2H) ppm;

13C NMR (MeOH-d4, 150 MHz): .6, 130.6, 129.5, 128.7, 127.82, 127.80, 127.6, 127.2, 127.0, 126.5, 126.2, 125.1, 122.0, 121.6, 121.1, 119.5, 118.8, 117.1, 112.5, 111.6, 110.1, 47.1 ppm. HRMS ESL-TOF for C30H21ClN2O2 (M+) found: m/z: 476.1295; Calc. Mass 476.1292.

6'-chloro-1-(4-chlorobenzyl)-3-(4-chlorophenyl)-1H,1'H-[2,3'-biindole]-2'-carboxylic acid (9c)

white solid, yield 52%; 1H NMR (MeOH-(d, J = 7.8 Hz, 1H), 7.35 (s, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.10 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 7.8 Hz, 3H), 6.90 (d, J = 7.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.79 (d,

J = 9.0 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H), 5.12 (s, 2H) ppm; 13C NMR (MeOH-d4, 150 MHz): 163.0, 138.6, 138.2, 137.9, 135.7, 133.7, 132.3, 132.2, 131.4, 131.1, 129.27, 129.25, 129.2, 128.3, 128.2, 123.3, 123.1, 122.8, 121.3, 120.1, 117.5, 113.1, 111.4, 47.8 ppm. HRMS ESL-TOF for C30H19Cl3N2O2 (M+) found: m/z: XX; Calc. Mass 544.0512.

6'-chloro-1-(4-chlorobenzyl)-3-(4-fluorophenyl)-1H,1'H-[2,3'-biindole]-2'-carboxylic acid (9d)

yellow solid, yield 55%; 1H NMR (DMSO-8.12 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 8.4, 6.0 Hz, 2H), 7.17 (t, J = 7.8 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H),

7.04 (d, J = 8.4 Hz, 1H), 6.94 (t, J = 8.4 Hz, 2H), 6.87 (t, J = 6.6 Hz, 3H), 5.21 (s, 2H) ppm; 13C NMR (DMSO-d6, 150 MHz): J = 243 Hz), 141.7, 141.4 (d, J = 15 Hz), 136.9, 136.5 (d, J = 3 Hz), 135.2 (d, J = 8 Hz), 134.8 (d, J = 5 Hz), 133.5, 133.2 (d, J = 8 Hz), 131.8, 131.6, 127.0, 126.8, 126.3, 125.1, 123.8 (d, J = 12 Hz), 120.5, 120.1, 119.9, 117.4, 115.6, 115.6, 47.7 ppm. ESL-TOF for C30H19Cl2FN2O2 (M+) found: m/z: 528.0810; Calc. Mass: 528.0808.

NHCl

N

O

OH

ClF

NHCl

NPh

O

OH

NHCl

N

O

OH

ClCl

128

6'-Chloro-3-(4-chlorobenzyl)-1-phenyl-1H,1'H-2,3'-biindole-2'-carboxylic acid (9e)

yellow solid, yield 58%; 1H NMR (MeOH-7.50 (d, J = 7.8 Hz, 1H), 7.40 (s, 1H), 7.37 (t, J = 8.4 Hz, 2H), 7.31 (t, J = 7.2 Hz, 1H), 7.25-7.10 (m, 7H), 7.07 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 4.08 (d, J = 16.2 Hz, 1H), 3.94 (d, J = 16.2 Hz, 1H) ppm; 13C NMR (MeOH-d4, 150 MHz):

129.7, 129.5, 129.0, 128.8, 128.1, 127.9, 127.4, 127.2, 126.9, 122.9, 122.59, 122.65, 120.2, 119.5, 115.4, 115.0, 111.9, 110.6, 30.4 ppm. ESL-TOF for C30H20Cl2N2O2 (M+) found: m/z: 510.0910; Calc. Mass: 510.0902.

PROCEDURE AND ANALYTICAL DATA OF THE INDOLE AMIDE DERIVATIVES 10

To a stirred solution of the corresponding compounds 2 (1.0 mmol) in THF, triazabicyclodecene (TBD, 1.0 mmol) and the corresponding amine (10.0 mmol) were added and the reaction mixture refluxed for 12 h. Afterwards, the solvent was evaporated and the crude product purified with flash chromatography on silica gel eluted with ethyl acetate with 1% triethylamine.

6'-chloro-1-(4-chlorobenzyl)-N-(3-morpholino propyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxamide (10a)

white solid, yield 45%; 1H NMR (DMSO-d6, 600 MHz): 9.88 (s, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 1.2 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.24-7.30 (H), 7.22 (t, J = 7.8 Hz, 2H), 7.17 (t, J = 5.4 Hz, 1H), 7.11 (dd, J = 8.4, 1.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.73

(d, J = 8.4 Hz, 2H), 6.25 (t, J = 6.0 Hz, 1H), 5.20 (t, J = 15.6 Hz, 1H), 4.99 (d, J = 15.6 Hz, 1H), 3.51-3.60 (m, 4H), 3.05-3.10 (m, 2H), 1.97-2.07 (m, 4H), 1.82-1.91 (m, 2H), 1.27-1.40 (m, 2H) ppm; 13C NMR (DMSO-d6, 150 MHz): 160.2, 137.6, 135.6, 135.3, 133.6, 133.4, 131.1, 130.1, 128.73, 128.72, 128.6, 128.1, 127.8, 126.9, 126.7, 123.5, 122.6, 121.6, 121.1, 120.3, 118.7, 112.3, 110.5, 106.0, 66.9, 55.4, 53.3, 47.1, 37.1, 26.0 ppm. HRMS ESL-TOF for C37H34Cl2N4O2 (M+) found: m/z: 636.2072; Calc. Mass 636.2059.

6'-chloro-1-(4-chlorobenzyl)-3-(4-chlorophenyl)-N-(3-morpholino propyl)-1H,1'H-[2,3'-biindole]-2'-carboxamide (10b)

white solid, yield 46%; 1H NMR (CDCl3, 600 MHz): 10.50 (br s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.47 (d, J =

NHCl

NPh

O

N

Cl

H

N

O

NHCl

N

O

N

Cl

H

N

OCl

NHCl

NPhO

OH

Cl

129

5

6

7

8

9

8.4 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.18-7.22 (m, 5H), 7.10 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 7.8 Hz, 2H), 6.22 (t, J = 5.4 Hz, 1H), 5.20 (d, J = 16.2 Hz, 1H), 4.99 (d, J = 16.2 Hz, 1H), 3.50-3.60 (m, 4H), 3.11 (q, J = 6.0 Hz, 2H), 1.97-2.10 (m, 4H), 1.83-1.92 (m, 2H), 1.30-1.42 (m, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 160.3, 137.6, 135.6, 135.5, 133.5, 132.4, 132.2, 131.1, 130.1, 129.8, 129.0, 128.7, 128.2, 128.1, 127.6, 126.6, 123.7, 122.7, 121.4, 121.3, 120.0, 117.4, 112.6, 110.7, 105.6, 66.9, 55.4, 53.3, 47.1, 37.2, 26.0 ppm. HRMS ESL-TOF for C37H33Cl3N4O2 (M+) found: m/z: 670.1660; Calc. Mass 670.1669.

6'-chloro-1-(4-chlorobenzyl)-N-(2-hydroxyethyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxamide (10c)

yellow solid, yield 50%; 1H NMR (CDCl3, 600 MHz): 10.11 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 1.2 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.26-7.30 (m, H), 7.23 (t, J = 7.8 Hz, 2H), 7.17 (t, J = 7.8 Hz, 1H), 7.12 (dd, J = 9.0, 1.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.4 Hz, 2H), 6.56 (t, J =

5.4 Hz, 1H), 3.40-3.46 (m, 1H), 3.35-3.40 (m, 1H), 3.19-3.25 (m, 1H), 3.08-3.15 (m, 1H), 1.75 (s, 1H) ppm; 13C NMR (CDCl3, 150 MHz): 161.2, 137.7, 135.8, 135.5, 133.7, 133.4, 131.2, 129.5, 128.72, 128.67, 128.6, 127.9, 127.8, 126.9, 126.7, 123.5, 122.7, 121.6, 121.0, 120.3, 118.9, 112.4, 110.5, 106.5, 61.6, 47.1, 42.2 ppm. HRMS ESL-TOF for C32H25Cl2N3O2 (M+) found: m/z: 553.1321; Calc. Mass 553.1324.

6'-chloro-1-(4-chlorobenzyl)-N-(2-morpholino ethyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-carboxamide (10d)

yellow solid, yield 70%; 1H NMR (CDCl3, 600 MHz): 9.80 (s, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.34 (t, J = 7.2 Hz, 1H), 7.28 (d, J = 7.2 Hz, 2H), 7.20 (t, J = 7.8 Hz, 2H), 7.15 (t, J = 3.6 Hz, 1H), 7.14 (d, J

= 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.04 (dd, J = 8.4, 1.8 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H), 6.63 (d, J = 4.2 Hz, 1H), 5.18 (d, J = 16.2 Hz, 1H), 5.04 (d, J = 16.2 Hz, 1H), 3.18-3.31 (m, 2H), 2.88-3.10 (m, 4H), 2.22 (ddd, J = 12.0, 7.6, 4.8 Hz, 1H), 2.02-2.12 (m, 3H), 1.88-1.93 (m, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 160.6, 137.6, 135.7, 135.5, 134.0, 130.2, 128.7, 128.6, 128.5, 128.3, 127.6, 127.5, 127.2, 126.5, 123.5, 122.4, 121.6, 121.0, 120.3, 119.0, 112.3, 110.4, 106.5, 66.1, 56.7, 53.2, 47.1, 35.9 ppm. HRMS ESL-TOF for C36H32Cl2N4O2 (M+) found: m/z: 622.1899; Calc. Mass 622.1902.

6'-chloro-1-(4-chlorobenzyl)-N-(2-(2-hydroxyethoxy)ethyl)-3-phenyl-1H,1'H-[2,3'-biindole]-2'-

carboxamide (10e)

white solid, yield 35%; 1H NMR (CDCl3, 600 MHz): 10.40 (s, 1H), 7.92 (s, 1H), 7.48 (s, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.25-7.30 (m, 4H), 7.22 (t, J = 7.2

NHCl

NPh

O

N

Cl

H

OH

NHCl

NPh

O

N

Cl

H

N O

NHCl

NPh

O

HN

Cl

O

OH

130

Hz, 2H), 7.10 (d, J = 7.2 Hz, 1H), 7.02-7.12 (m, 3H), 6.75 (t, J = 7.2 Hz, 2H), 6.57 (s, 1H), 5.22 (d, J = 16.2 Hz, 1H), 5.04 (d, J = 16.2 Hz, 1H), 3.35 (s, 3H), 3.26 (s, 2 ), 3.14 (s, 1H), 3.06-3.10 (m, 3H) ppm; 13C NMR (CDCl3, 150 MHz): 160.4, 137.6, 135.6, 135.5, 133.7, 133.3, 131.0, 129.8, 128.7, 128.62, 128.59, 128.0, 127.8, 127.7, 127.1, 126.6, 123.4, 122.5, 121.5, 120.9, 120.1, 118.9, 112.4, 110.6, 106.3, 72.1, 69.0, 61.3, 47.0, 39.1 ppm. HRMS ESL-TOF for C34H29Cl2N3O3 (M+) found: m/z: 597.1581; Calc. Mass 597.1586.

(6'-chloro-1-(4-chlorobenzyl)-3-phenyl-1H,1'H-[2,3'-biindol]-2'-yl)(morpholino)methanone (10f)

yellow solid, yield 46%; 1H NMR (CDCl3, 600 MHz): 9.96 (s, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.40 (s, 1H), 7.39 9d, J = 9.0 Hz, 1H), 7.31 (t, J = 7.2 Hz, 1H), 7.22-7.28 (m, H), 7.19 (t, J = 8.4 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 6.6 Hz, 2H), 5.28 (d, J = 16.2 Hz, 1H), 5.23 (d, J = 16.2 Hz,

1H),3.70 (s, 2H), 2.85-3.60 (m, 4H), 2.89 (s, 2H) ppm; 13C NMR (CDCl3, 150 MHz): 162.4, 137.5, 136.3, 135.7, 134.4, 133.3, 130.6, 129.9, 128.7, 128.63, 128.58, 128.55, 128.2, 127.2, 126.5, 126.2, 123.1, 122.3, 122.0, 120.8, 120.2, 117.8, 112.0, 110.5, 107.2, 68.2, 66.3, 47.1, 46.5 ppm. HRMS ESL-TOF for C34H27Cl2N3O2 (M+) found: m/z: 579.1476; Calc. Mass 579.1480.

SINGLE CRYSTAL X-RAY STRUCTURE DETERMINATION

GENERAL: Data were collected on an X-ray single crystal diffractometer equipped with a CCD detector (Bruker APEX II, CCD), a rotating anode (Bruker AXS, FR591) with MoK radiation ( = 0.71073 Å), and a graphite monochromator by using the SMART software package (compounds 2h, 3c). Data were collected on an X-ray single crystal diffractometer equipped

with a CCD detector (APEX II, CCD) at the window of a fine-focused sealed tube with MoK

radiation ( = 0.71073 Å) and a graphite monochromator by using the SMART software package (compound 2a).3 The measurements were performed on single crystals coated with perfluorinated ether. Each crystal was fixed on the top of a glass fiber 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.4 Absorption corrections, including odd and even ordered spherical harmonics were performed using SADABS.4 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 WinGX5

based on SIR-92.6 If not mentioned otherwise, non-hydrogen atoms were refined with anisotropic displacement parameters. Compound 2a: All hydrogen atom positions were found in the difference map calculated from the model containing all non-hydrogen atoms. The

NHCl

NPh

O

N

Cl

O

131

5

6

7

8

9

hydrogen positions were refined with individual isotropic displacement parameters. Compound 2h: In the difference map(s) calculated from the model containing all non-hydrogen atoms, not all of the hydrogen positions could be determined from the highest peaks. For this reason, the hydrogen atoms were placed in calculated positions (dC-H = 95, 98, 99 pm; dN-H = 88 pm). Isotropic displacement parameters were calculated from the parent carbon atom (UH = 1.2/1.5 UC; UH = 1.2 UN). The hydrogen atoms were included in the structure factor calculations but not refined. Compound 3c: The hydrogen atom bound to the nitrogen atom was found in the difference Fourier. The hydrogen position was refined with an individual isotropic displacement parameter. All other hydrogen atoms were placed in calculated positions (dC-H = 95, 98, 99 pm). Isotropic displacement parameters were calculated from the parent carbon atom (UH = 1.2/1.5 UC). The hydrogen atoms were included in the structure factor calculations but not refined. Methyl hydrogen atoms were refined as part of rigid rotating groups. Full-matrix least-squares refinements were carried out by minimizing

w (Fo2-Fc

2)2 with SHELXL-977 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.8 Images of the crystal structures were generated by PLATON.9 CCDC 981829 (2a), CCDC 981827 (2h), and CCDC 981828 (3c) contains the supplementary crystallographic data for this compound. This data can be 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/

Special:

2a: Small extinction effects were corrected with the SHELXL-977 procedure. refined to = 0.021(2)

2h: Problems with unresolvable solvent molecules were cured by using the calc squeeze procedure.8

Compound 2a

132

Figure 1. Ortep drawing of compound 2a with 50% ellipsoids. 8

Operator: *** Herdtweck *** Molecular Formula: C32 H24 Cl2 N2 O2 Crystal Color / Shape Pale yellow fragment Crystal Size Approximate size of crystal fragment used for data collection: 0.36 0.51 0.76 mm Molecular Weight: 539.43 a.m.u. F000: 560 Systematic Absences: none

Space Group: Triclinic P 1 (I.T.-No.: 2) Cell Constants: Least-squares refinement of 9841 reflections with the programs

"APEX suite" and "SAINT" [1,2]; theta range 1.74° < < 25.48°;

Mo(K ); l = 71.073 pm a = 1031.81(5) pm a = 115.551(2)° b = 1196.13(5) pm b = 90.435(2)° c = 1327.37(6) pm g = 113.632(2)° V = 1321.37(11)· 106 pm3; Z = 2; Dcalc = 1.356 g cm-3; Mos. = 0.95 Diffractometer: Kappa APEX II (Area Diffraction System; BRUKER AXS); sealed

tube; graphite monochromator; 50 kV; 30 mA;

= 71.073 pm; Mo(K ) Temperature: (20±1) °C; (293±1) K Measurement Range: 1.74° < < 25.48°; h: -12/12, k: -14/14, l: -16/16 Measurement Time: 2 7.5 s per film Measurement Mode: measured: 8 runs; 1526 films / scaled: 8 runs; 1526 films

133

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7

8

9

j- and w-movement; Increment: Dj/Dw = 1.00°; dx = 40.0 mm

LP - Correction: Yes [2] Intensity Correction No/Yes; during scaling4 Absorption Correction: Multi-scan; during scaling; m = 0.279 mm-1 4 Correction Factors: Tmin = 0.6931 Tmax = 0.7452 Reflection Data: 35612 reflections were integrated and scaled 35612 reflections to be merged 4868 independent reflections 0.020 Rint: (basis Fo

2) 4868 independent reflections (all) were used in refinements 4139 independent reflections with Io > 2s(Io) 99.0 % completeness of the data set 440 parameter full-matrix refinement 11.1 reflections per parameter Solution: Direct Methods [3]; Difference Fourier syntheses Refinement Parameters: In the asymmetric unit: 38 Non-hydrogen atoms with anisotropic displacement parameters 24 Hydrogen atoms with isotropic displacement parameters Hydrogen Atoms: All hydrogen atom positions were found in the difference map

calculated from the model containing all non-hydrogen atoms. The hydrogen positions were refined with individual isotropic displacement parameters.

Atomic Form Factors: For neutral atoms and anomalous dispersion [4]

Extinction Correction: Fc (korr) = kFc[1+ 0.001 · e · Fc2· l3/sin(2Q)]-1/4 SHELXL-97 [5]; e

refined to e = 0.021(2) Weighting Scheme: w-1 = s2(Fo

2)+(a*P)2+b*P

with a: 0.0427; b: 0.6518; P: [Maximum(0 or Fo2)+2*Fc

2]/3

Shift/Err: Less than 0.001 in the last cycle of refinement:

Resid. Electron Density: +0.37 e0- /Å3; -0.43 e 0

- /Å3

R1: S(||Fo|-|Fc||)/S|Fo| [Fo > 4s(Fo); N=4139]: = 0.0386 [all reflctns; N=4868]: = 0.0480 wR2: [Sw(Fo

2-Fc2)2/Sw(Fo

2)2]1/2 [Fo > 4s(Fo); N=4139]: = 0.0947 [all reflctns; N=4868]: = 0.1049 Goodness of fit: [Sw(Fo

2-Fc2)2/(NO-NV)]1/2 = 1.060

Remarks: Refinement expression Sw(Fo2-Fc

2)2

134

Compound 2h

Figure 2. Ortep drawing of compound 2h with 50% ellipsoids.8

Operator: *** Herdtweck *** Molecular Formula: C31 H22 Cl F N2 O2 Crystal Color / Shape Colorless fragment Crystal Size Approximate size of crystal fragment used for data collection: 0.46 0.46 0.56 mm Molecular Weight: 508.96 a.m.u. F000: 528 Systematic Absences: none

Space Group: Triclinic P 1 (I.T.-No.: 2) Cell Constants: Least-squares refinement of 9834 reflections with the programs

"APEX suite" and "SAINT" [1,2]; theta range 1.74° < < 25.21°;

Mo(K ); l = 71.073 pm a = 1104.86(6) pm a = 114.043(2)° b = 1269.09(7) pm b = 97.343(2)° c = 1318.92(6) pm g = 102.677(3)° V = 1598.01(15)· 106 pm3; Z = 2; Dcalc = 1.058 g cm-3; Mos. = 0.60 Diffractometer: Kappa APEX II (Area Diffraction System; BRUKER AXS); rotating

anode; graphite monochromator; 50 kV; 40 mA; = 71.073 pm;

Mo(K ) Temperature: (-150±1) °C; (123±1) K Measurement Range: 1.74° < < 25.21°; h: -13/13, k: -15/15, l: -15/15

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9

Measurement Time: 2 10 s per film Measurement Mode: measured: 8 runs; 3052 films / scaled: 8 runs; 3052 films j- and w-movement; Increment: Dj/Dw = 0.50°;

dx = 35.0 mm LP - Correction: Yes [2] Intensity Correction No/Yes; during scaling [2] Absorption Correction: Multi-scan; during scaling; m = 0.151 mm-1 [2] Correction Factors: Tmin = 0.6836 Tmax = 0.7452 Reflection Data: 49951 reflections were integrated and scaled 49951 reflections to be merged 5722 independent reflections 0.042 Rint: (basis Fo

2) 5722 independent reflections (all) were used in refinements 4768 independent reflections with Io > 2s(Io) 99.4 % completeness of the data set 346 parameter full-matrix refinement 16.5 reflections per parameter Solution: Direct Methods [3]; Difference Fourier syntheses Refinement Parameters: In the asymmetric unit: 38 Non-hydrogen atoms with anisotropic displacement parameters Hydrogen Atoms: In the difference map(s) calculated from the model containing all

non-hydrogen atoms, not all of the hydrogen positions could be determined from the highest peaks. For this reason, the hydrogen atoms were placed in calculated positions (dC-H = 95, 98, 99 pm; dN-H = 88 pm). Isotropic displacement parameters were calculated from the parent carbon atom (UH = 1.2/1.5 UC; UH = 1.2 UN). The hydrogen atoms were included in the structure factor calculations but not refined.

Atomic Form Factors: For neutral atoms and anomalous dispersion [4] Extinction Correction: no Weighting Scheme: w-1 = s2(Fo

2)+(a*P)2+b*P

with a: 0.0737; b: 0.5130; P: [Maximum(0 or Fo2)+2*Fc

2]/3

Shift/Err: Less than 0.001 in the last cycle of refinement:

Resid. Electron Density: +0.63 e0- /Å3; -0.37 e 0

- /Å3

R1: S(||Fo|-|Fc||)/S|Fo| [Fo > 4s(Fo); N=4768]: = 0.0477 [all reflctns; N=5722]: = 0.0559 wR2: [Sw(Fo

2-Fc2)2/Sw(Fo

2)2]1/2 [Fo > 4s(Fo); N=4768]: = 0.1354 [all reflctns; N=5722]: = 0.1399 Goodness of fit: [Sw(Fo

2-Fc2)2/(NO-NV)]1/2 = 1.092

136

Remarks: Refinement expression Sw(Fo2-Fc

2)2

Compound 3c

Figure 3. Ortep drawing of compound 3c with 10% ellipsoids.8

Operator: *** Herdtweck *** Molecular Formula: C20 H17 Cl2 N O3 Crystal Color / Shape Colorless fragment Crystal Size Approximate size of crystal fragment used for data collection: 0.15 0.20 0.64 mm Molecular Weight: 390.25 a.m.u. F000: 404 Systematic Absences: none

Space Group: Triclinic P 1 (I.T.-No.: 2) Cell Constants: Least-squares refinement of 9972 reflections with the programs

"APEX suite" and "SAINT" [1,2]; theta range 1.52° < < 25.33°;

Mo(K ); l = 71.073 pm a = 544.89(2) pm a = 85.0829(13)° b = 1268.09(4) pm b = 78.8518(13)° c = 1366.51(4) pm g = 86.7201(15)° V = 922.19(5)· 106 pm3; Z = 2; Dcalc = 1.405 g cm-3;

Mos. = 0.70 Diffractometer: Kappa APEX II (Area Diffraction System; BRUKER AXS); rotating

anode; graphite monochromator; 50 kV; 40 mA;

= 71.073 pm; Mo(K ) Temperature: (-150±1) °C; (123±1) K Measurement Range: 1.52° < < 25.33°; h: -6/6, k: -15/15, l: -16/16

137

5

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8

9

Measurement Time: 2 5 s per film Measurement Mode: measured: 11 runs; 4279 films / scaled: 5 runs; 2150 films j- and w-movement; Increment: Dj/Dw = 0.50°;

dx = 35.0 mm LP - Correction: Yes [2] Intensity Correction No/Yes; during scaling [2] Absorption Correction: Multi-scan; during scaling; m = 0.372 mm-1 [2] Correction Factors: Tmin = 0.6421 Tmax = 0.7452 Reflection Data: 14074 reflections were integrated and scaled 1 obvious wrong intensity and rejected 14073 reflections to be merged 3352 independent reflections 0.024 Rint: (basis Fo

2) 3352 independent reflections (all) were used in refinements 3159 independent reflections with Io > 2 (Io) 99.1 % completeness of the data set 240 parameter full-matrix refinement 14.0 reflections per parameter Solution: Direct Methods [3]; Difference Fourier syntheses Refinement Parameters: In the asymmetric unit: 28 Non-hydrogen atoms with anisotropic Displacement parameters 1 Hydrogen atoms with isotropic displacement parameters Hydrogen Atoms: The hydrogen atom bound to the nitrogen atom was found in

the difference Fourier. The hydrogen positions was refined with an individual isotropic displacement parameter.

Hydrogen Atoms: All other hydrogen atoms were placed in calculated positions (dC-

H = 95, 98, 99 pm). Isotropic displacement parameters were calculated from the parent carbon atom (UH = 1.2/1.5 UC). The hydrogen atoms were included in the structure factor calculations but not refined.

Atomic Form Factors: For neutral atoms and anomalous dispersion [4] Extinction Correction: no Weighting Scheme: w-1 = s2(Fo

2)+(a*P)2+b*P

with a: 0.0395; b: 0.6428; P: [Maximum(0 or Fo2)+2*Fc

2]/3

Shift/Err: Less than 0.001 in the last cycle of refinement:

Resid. Electron Density: +0.50 e0- /Å3; -0.47 e 0

- /Å3

R1: S(||Fo|-|Fc||)/S|Fo| [Fo > 4s(Fo); N=3159]: = 0.0359 [all reflctns; N=3352]: = 0.0379 wR2: [Sw(Fo

2-Fc2)2/Sw(Fo

2)2]1/2

138

[Fo > 4s(Fo); N=3159]: = 0.0905 [all reflctns; N=3352]: = 0.0925 Goodness of fit: [Sw(Fo

2-Fc2)2/(NO-NV)]1/2 = 1.037

Remarks: Refinement expression Sw(Fo2-Fc

2)2

FLUORESCENCE POLARIZATION BINDING ASSAYS

All fluorescence experiments were performed as described by Czarna et al.10 Briefly, the fluorescence polarization experiments were read on an Ultra Evolution 384-well plate reader (Tecan) with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities parallel (Intparallel) and perpendicular (Intperpedicular) to the plane of excitation were measured in black 384-well NBS assay plates (Corning) at room temperature (~20°C). The background fluorescence intensities of blank samples containing the references buffer were subtracted and steady-state fluorescence polarization was calculated using the equation: P = (Intparallel – GIntperpedicular)/ (Intparallel + GIntperpedicular), and the correction factor G (G = 0.998 determined empirically) was introduced to eliminate differences in the transmission of vertically and horizontally polarized light. All fluorescence polarization values were expressed in millipolarization units (mP). The binding affinities of the fluorescent p53-derived peptide of Hu at al.11 (the P4 peptide in Czarna et al.) towards MDM2 and MDMX proteins were determined in the buffer which contained 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 10% DMSO. Each sample contained 10 nM of the fluorescent P4 peptide and MDM2 (the MDM2 concentration used, from 0 to 1 M and MDMX, from 0 to 10 M) in a final volume of 50 l. Competition binding assays were performed using the 10 nM fluorescent P4 peptide, 15 nM MDM2 or 120 nM MDMX. Plates were read at 30 min after mixing all assay components. Binding constant and inhibition curves were fitted using the SigmaPlot (SPSS Science Software). Fluorescein labeled p53 mimicking peptide TSFAEYWNLLSP described previously was used.12

Entry Compound Plot (MDM2/MDMX) Ki (uM) MDM2 MDMX

1 2a

17 ND

2 2c

10 ND

3 2d >22 ND

139

5

6

7

8

9

4 2e

6.7

5.0

5 2f ND ND

6 9a

0.01 0.1 1 100.2

0.4

0.6

0.8

1.0

norm

alize

d va

lue

of F

P

compound [

1.5

0.18

7 9d

0.7

1.5

8 10d

0.1 1 10 1000.2

0.4

0.6

0.8

1.0

1.2

norm

alize

d va

lue

of F

P

compound [

ND

6.09

140

2D-NMR MEASUREMENTS

Uniform 15N isotope labeling was achieved by expression of the protein in the M9 minimal media containing 15NH4Cl as the sole nitrogen source. Final step of purification of MDM2 for NMR consisted of gel filtration into the NMR buffer (50 mM phosphate buffer pH 7.4 containing 150 mM NaCl, 5 mM DTT). 10% (v/v) of D2O was added to samples to provide lock signal. All the spectra were recorded at 300 K using a Bruker Avance 600 MHz spectrometer. 1H-15N heteronuclear correlations were obtained using the fast HSQC pulse sequence.13 Assignment of the amide groups of MDM2 was obtained after Stoll et al.14

ADMET PROPERTIES OF SELECTED COMPOUNDS

Compound logP PSA (pH = 7.4) Solubility (logS, pH = 7.4)

2a 7.82 47.02 -10.75 2c 7.61 20.72 -10.05 2d 8.38 47.02 -11.40 2e 7.98 47.02 -10.98 2f 8.38 47.02 -11.40 9a 7.22 60.85 -7.87 9d 7.38 60.85 -8.11

10d 6.4 62.29 -10.00

REFERENCES(1) Zhu, M.; Zheng, N. Photoinduced

Cleavage of N-N Bonds of Aromatic Hydrazines and Hydrazides by Visible Light. Synthesis 2011, 2011 (14), 2223–2236.

(2) Nara, S.; Sakamoto, T.; Miyazawa, E.; Kikugawa, Y. A Convenient Synthesis of 1-Alkyl-1-Phenylhydrazines from N -Aminophthalimide. Synth. Commun. 2003, 33 (1), 87–98.

(3) APEX Suite of Crystallographic Software. APEX 2 Version 2008.4. Bruker AXS Inc., Madison, Wisconsin, USA (2008).

(4) SAINT, Version 7.56a and SADABS Version 2008/1. Bruker AXS Inc., Madison, Wisconsin, USA (2008).

(5) Farrugia, L. J. WinGX Suite for Small-Molecule Single-Crystal Crystallography. J. Appl. Crystallogr. 1999, 32 (4), 837–838.

(6) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A.; Burla, M.

C.; Polidori, G.; Camalli, M. SIR92 – a Program for Automatic Solution of Crystal Structures by Direct Methods. J. Appl. Crystallogr. 1994, 27 (3), 435–435.

(7) Sheldrick, G. M. “SHELXL-97”, University of Göttingen, Göttingen, Germany, (1998).

(8) International Tables for Crystallography, Vol. C, Tables 6.1.1.4 (Pp. 500-502), 4.2.6.8 (Pp. 219-222), and 4.2.4.2 (Pp. 193-199), Wilson, A. J. C., Ed., Kluwer Academic Publishers, Dordrecht, The Netherlands, 1992.

(9) A. L. Spek, “PLATON”, A Multipurpose Crystallographic Tool, Utrecht University, Utrecht, The Netherlands, (2010).

(10) Czarna, A.; Popowicz, G. M.; Pecak, A.; Wolf, S.; Dubin, G.; Holak, T. A. High Affinity Interaction of the P53 Peptide-Analogue with Human Mdm2 and

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9

Mdmx. Cell Cycle Georget. Tex 2009, 8 (8), 1176–1184.

(11) Hu, B.; Gilkes, D. M.; Chen, J. Efficient P53 Activation and Apoptosis by Simultaneous Disruption of Binding to MDM2 and MDMX. Cancer Res. 2007, 67 (18), 8810–8817.

(12) Mori, S.; Abeygunawardana, C.; Johnson, M. O.; Vanzijl, P. C. M. Improved Sensitivity of HSQC Spectra of Exchanging Protons at Short Interscan Delays Using a New Fast HSQC (FHSQC) Detection Scheme That Avoids Water Saturation. J. Magn. Reson. B 1995, 108 (1), 94–98.

(13) Pazgier, M.; Liu, M.; Zou, G.; Yuan, W.; Li, C.; Li, C.; Li, J.; Monbo, J.; Zella, D.; Tarasov, S. G.; Lu, W. Structural Basis for High-Affinity Peptide Inhibition of P53 Interactions with MDM2 and MDMX. Proc. Natl. Acad. Sci. 2009, 106 (12), 4665–4670.

(14) Stoll, R.; Renner, C.; Hansen, S.; Palme, S.; Klein, C.; Belling, A.; Zeslawski, W.; Kamionka, M.; Rehm, T.; Mühlhahn, P.; Schumacher, R.; Hesse, F.; Kaluza, B.; Voelter, W.; Engh, R. A.; Holak, T. A. Chalcone Derivatives Antagonize Interactions between the Human Oncoprotein MDM2 and P53. Biochemistry (Mosc.) 2001, 40 (2), 336–344.

PART B

BIOLOGICAL ACTIVITY OF VARIOUS FAMILIES OF METAL COMPLEXES

CHAPTER 6

GOLD(I) COMPLEXES WITH LANSOPRAZOLE-TYPE LIGANDS: EX VIVO TOXICOLOGICAL EVALUATION

N. Estrada-Ortiz,a E. Lopez-Gonzales,a E. Post, I. A. M.a de Graaf,a G. M. M. Groothuis,a A. Casinia,b*

a Dept. Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy. University of Groningen. A. Deusinglaan 1, 9713AV Groningen, The Netherlands. b School of Chemistry. Cardiff University. Main Building, Park Place, CF103AT Cardiff, United Kingdom.

Manuscript in preparation

146

ABSTRACT

In previous studies, bifunctional metallocompounds were synthetized and characterized, to target specifically tumor cells and act as chimeric compounds combining the cytotoxicity of gold and a lansoprazole moiety, which decreases the acidic microenvironment in cancer tissue, thereby increasing the efficacy of the basic drugs. The cytotoxic activity of the compounds was evaluated previously in a small panel of cancer cells, including cell lines sensitive and resistant to cisplatin, and a non-cancerous cell line. These series of compounds showed to be more cytotoxic to the cancer cell lines than in the non-cancerous cell line, suggesting a potential selectivity towards cancer cells. In the present study, the potential selectivity of these compounds was studied in an ex-vivo model, using rat precision cut kidney and liver slices (PCKS and PCLS), to determine to which extent these compounds are toxic to healthy tissue. The results obtained showed a different toxicity profile for the tested compounds, but the stress responses seem to be similar for both evaluated organs. The obtained results open new perspectives towards the design of bifunctional gold complexes for chemotherapeutic applications with reduced toxicity in healthy tissues.

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INTRODUCTION

In the drug development process in addition to animal experiments the prediction of toxicity in human tissue and organs is crucial to anticipate possible side effects before starting clinical trials of potential drug-like compounds. 2-D models are by far the most commonly used models to predict effectivity and toxicity for humans. The advantage of these models is the variety of cell types available to study, including primary cells, cell lines, stem cells and cancer cells among others. The main disadvantage is the absence of the complexity of a tissue, with its multitude of cell types playing different roles and secreting different signaling molecules, and the absence of a proper extra cellular matrix to maintain and regulate the function and activities of the specific tissue.1 Therefore animal models are widely used, where the complexity of a whole organism is intact. However, the use of animals for preclinical studies expose two important problems: the large number of animals used is an ethical problem and the translation of such studies from any species (even primates) to the human situation is not always accurate and presents a risk to the patients entering to the first rounds of clinical trials.2–5

However, in 1923 Otto Warburg and later in 1933 HA Krebs used liver tissue slices. They were produced manually, leading to reduced viability and reproducibility.6,7 After the introduction of the Krumdieck slicer, the tissue slices technique was greatly improved, offering the opportunity to produce slices with precise thickness and sufficiently thin to allow oxygen and substrate supply to all cell layers.8 The technique is known as precision cut tissue slices (PCTS) and it became a powerful technique, which can be applied to many organs. PCTS contain all cell types of the tissue in their natural environment, with intercellular and cell-matrix interactions remaining intact, making the technic a powerful in vitro tool to serve as a model for human diseases, such as fibrosis and cirrhosis. Additionally, the PCTS technique offers the opportunity to test the activity, metabolism, transport and toxicity of new drug candidates, including comparison among species and organs.6,9–15 PCTS is an FDA-approved model for drug toxicity and metabolism studies and offers an opportunity of reducing the number of animals used in pre-clinical studies.6,9,16

In a previous study, Au(I) compounds with ligands were synthetized (Figure 1), featuring lansoprazole as ligand, and studied for their anticancer effects in human cancer cells in vitro.17 Lansoprazole is a drug currently in use for the treatment of ulcers and gastroesophageal reflux disease.18,19 The clinical efficacy of lansoprazole has been studied in the treatment of duodenal and gastric ulcers, reflux oesophagitis, and eradication of H. pylori in combination with clarithromycin and amoxicillin.18 The postulated mechanism of action is to selectively inhibit the membrane enzyme H+/K+ATPase in gastric parietal cells. The enzyme H+/K+ATPase is a proton pump located in the apical membrane of parietal cells and is responsible for gastric acid secretion. Proton pump inhibitors (PPIs) exert their effects by blocking the translocation of H+ to form HCl. Thus, lansoprazole prevents acid formation in the stomach.18,20

It has been proposed that PPIs, such as lansoprazole, can modify the acidic microenvironment present in most solid tumors and help to sensitize them to cytotoxic anticancer drugs.21–24 In tumors the low extracellular pH is a major cause of tumor unresponsiveness to most of the cytotoxic drugs; the H+ rich tumor microenvironment leads to protonation of the therapeutic agent causing its neutralization and stopping the compound to reach its targets inside the cells.19,25–28 Within this context, in the last decade, several studies have shown a potential application in cancer research, using proton pump

148

inhibitors to revert chemoresistance and increase chemosensitivity of different human tumor cells in combination with other cytotoxic drugs.17,28 Furthermore, lansoprazole and derivatives showed antituberculosis activity targeting cytochrome bc1 and displayed activity against different strains of the mycobacterium.29

The structure of lansoprazole includes a benzimidazole ring and a pyridine ring connected through a sulfinyl linkage. The compound is relatively unstable and can be modified in enzymatic and non-enzymatic reactions.29,30 The compound's metabolic activation occurs by protonation of the nitrogens on both sides of the sulfinyl group. The protonation on the benzimidazole ring causes rearrangement of the sulfinyl into a sulfenic acid or a sulfonamide. The latter can react with two cysteine residues of the H+K+-ATPase to form one or two disulfide bonds, which results in complete inhibition of basal and induced acid secretion when two inhibitors are bound per molecule of enzyme.18,20

Based on preliminary promising results in our group on the possible application of the Au(I)-lansoprazole derivatives (Figure 1) as potential cytotoxic agents 17 (Table 1) or as antibiotics (data not shown), we decided to investigate the toxicity in healthy tissue, using rat PCTS of liver and kidney. These studies are useful to be able to propose the compounds as good drug candidates for further preclinical investigation.

Figure 1. Lansoprazole and derivatives evaluated in this study.

Recently, we have used PCTS to study the toxic effects of experimental anticancer organometallic compounds,31–34 aminoferrocene-containing pro-drugs,35 ruthenium-based kinase inhibitors,36 as well as supramolecular metallocages as possible drug delivery systems.37 Thus, we report here the use of this methodology to investigate the possible toxic effects of the above mentioned Au(I) lansoprazole derivatives. Specifically, assessment of the ATP content and histomorphological studies were conducted on PCTS from rat liver and kidney treated with the Au(I) compounds in comparison to lansoprazole. Finally, to get more insights about the possible mechanism of the toxicological action, the mRNA expression of specific stress markers was assessed in slices, including expression of genes coding for proteins that play important roles in the pathways of oxidative stress, apoptosis and hypoxia.

N

OCH2CF3

S

N

HN

O

N

OCH2CF3

S

N

HN

1

AuPh3P

[BF4]

ON

OCH2CF3

S

N

N

2

Au

O

Lansoprazole

PN

N N

N

OCH2CF3

S

N

N

3

AuPh3P

[BF4]

OAu

PPh3

149

6

7

8

9

RESULTS AND DISCUSSION

VIABILITY AND TC50 DETERMINATION Complexes 1-3 were tested for their possible toxicity in healthy rat kidney and liver PCTS.6,9 Kidney and liver slices were incubated with various concentrations of each gold complex and after 24 h the viability of the tissues was determined measuring the ATP content (Figure 2). Lansoprazole, cisplatin and auranofin were also tested for comparison.

Figure 2. Viability of rat PCKS and PCLS relative to the controls (untreated slices) after treatment with compounds 1-3, lanzoprazole, cisplatin and auranofin for 24 h. The error bars show the standard deviation of at least three

independent experiments.

All the evaluated compounds, including cisplatin and auranofin, displayed a concentration dependent toxicity profile, with complex 3 and auranofin as the most toxic, with TC50 below 10 μM. The results obtained in this ex vivo model were compared with the cytotoxicity observed towards the cancer cells (Table 1). For compounds 2 and 3 the safety margin for toxicity was poor with a ratio of TC50 PCKS/IC50 cells between 1.7 and 4.1. Whereas, for compound 1 the TC50 PCKS/IC50 cells ratio cells was 20 for kidney slices and 8.9 for liver slices indicating selective toxicity towards cancer cells compared to healthy tissue. Notably, no significant differences were found between toxicity of 2 and 3 compounds in kidney and liver slices.

Table 1. Toxicity of Au(I) complexes in PCKS and PCLS (TC50 values) and their comparison with the IC50 of the antiproliferative effects in cancer cell lines.

Compound IC50

a (μM) IC50 (μM) (average)

TC50a (μM) TC50/IC50

A2780 A2780R kidney liver kidney liver 1 1.1 ± 0.3 0.7 ± 0.1 0.9 18 ± 2 8 ± 3 20 8.9 2 16.2 ± 1.1 13.2 ±4 .6 14.7 27 ± 5 25 ± 3 1.8 1.7 3 1.5±0.3 0.9±0.4 1.2 5 ± 2 4 ± 1 4.1 3.3

Lansoprazole 45.6 ± 2.6 59.0 ± 15.2 52.3 > 50 > 50 0.9 0.9 Cisplatin 2.4 ± 0.6 35.0 ± 7.0 18.7 16 ± 1 24 ± 1 0.9 1.3

Auranofin ND ND NA 2.9 ± 1 4 ± 1 ND ND a The reported values are the mean ± SD of at least three independent experiments.

ND: Not determined

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HISTOMORPHOLOGY In order to confirm the decrease in viability observed by ATP, further morphological analysis of the PCTS was performed. The characteristic toxic effects on kidney and liver slices of the compounds 1-3, cisplatin and auranofin were evaluated at a concentration close to the calculated TC50 for each compound (25 μM for compounds 1, 2 and cisplatin; 10 μM for compound 3 and 5 μM for auranofin). Periodic acid-Schiff staining (PAS) was used to evaluate kidney slices integrity and particularly to visualize the basement membranes and epithelial brush border in the proximal tubule cells, as reported in the experimental section. Furthermore, haematoxylin and eosin staining was used to visualize the integrity of the hepatocytes present in the liver slices. After 24 h incubation, the untreated kidney slices show minor morphological changes, indicated by pyknosis and swelling of some of the tubular cells (Figure 3A). Pronounced toxic effects were observed upon treatment with complexes 1, 2 and 3, which induced dilatation of Bowman’s space in the glomerulus and necrosis of the distal tubule cells, as well as discontinuation of the brush border in some of the proximal tubule cells (Figure 3B, 3C and 3D). In contrast, exposure of slices to cisplatin (Figure 3E) showed injury to the proximal tubular cells with loss of nuclei and more distinct damage of the brush border; additionally, damage of the distal tubule is evident as previously reported in the literature.11 Interestingly, the morphological characteristics of the samples treated with auranofin present similar damaged as the evaluated Au complexes, with a more pronounced damage to the distal tubular cells. In the case of liver slices, after 24 h incubation, the hepatocytes present normal large nuclei and defined shape (Figure 4A) that are indicative of healthy tissue. However, the morphological changes observed in liver slices exposed to the evaluated compounds showed similar toxic effect of compounds 1 and 3 (figure 4B and 4D), with evidence of pyknosis, necrosis and loss of nuclei. However, complex 2 seems to induce slightly less toxicity at the morphological level (Figure 4C) showing some pyknosis and necrosis, but occasional viable cells are observed. Liver slices treated with cisplatin and auranofin showed extensive damage, and pyknosis and necrosis were induced by all tested metal complexes (Figure 4E and 4F). The tested concentrations were too high to observe differential damage in different cell types. Currently, more experiments are in progress using concentrations around the TC25 value for each compound.

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Figure 3. Morphology of rat kidney slices. A: 24 h control incubation; B: compound 1 (25 μM); C: compound 2 (25 μM); D: compound 3 (10 μM); E: cisplatin (25 μM) and F: auranofin (5 μM). PT: proximal tubule, DT: distal tubule, G: glomerulus. Scale bar indicates 50 μm.

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Figure 4. Morphology of rat liver slices. A: 24 h control incubation; B: compound 1 (25 μM); C: compound 2 (25 μM); D: compound 3 (10 μM); E: cisplatin (25 μM) and F: auranofin (5 μM). Arrows indicate necrotic cells. Scale bar indicates 50 μm.

DETERMINATION OF STRESS MARKERS EXPRESSION To get insights of the specific type of stress that the Au complexes evaluated in this study, we selected specific genes that code for proteins that belong to pathways that are activated under hypoxia (Hif1a),38 oxidative stress (Nrf2)39 and DNA damage (p53)40. Based on the work of Limonciel, et al., 2015,41 we chose two or three genes related with the mentioned pathways that displayed significant up or down regulation after treating human and rat hepatocytes, and RPTEC/TERT1 cells (human renal proximal tubule cell line transfected with human telomerase) with several known toxicants.41 All the selected bio-markers are expressed by kidney and liver cells. Thus, liver and kidney slices were treated with the compounds at concentrations below and close to the calculated TC50 values (1 and 10 μM for compound 1 and 3, 5 and 25 μM for compound 2, 50 and 75 μM for lansoprazole) during 24 h.

(hypoxia- pathway, we selected to measure the expression levels of ALDOA that codes for the Fructose-Bisphosphate Aldolase A enzyme, ENO2 that codes for enolase 2 and SLC2A1 that codes for the glucose transporter protein type 1 (GLUT1). All these genes promote survival of the cells in hypoxic conditions by inducing glycolysis. In the case of PCKS significant up or down regulation of any of the selected genes was not observed (Figure 5), indicating that the compounds do not promote a hypoxic environment as a toxicity mechanism. In the case of PCLS, the

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responses are more diverse, where exposure of PCLS to compound 1 seems to induce an upregulation of SLC2A1 gene at the highest concentration only. Moreover, treatment with compound 3 at 10 μM induces upregulation about 3-fold of ENO2 compared with the untreated samples. These results suggest some activation of the hypoxia pathway in the liver slices. Lansoprazole did not induce any change in the expression of the tested genes. More experiments are needed to fully understand if hypoxia is the toxicity mechanism specifically in the case of liver tissue using more bio-markers that are modulated in response to hypoxia, such and FABP3 (fatty acid binding protein 3).

Figure 5. Gene expression of ALDOA (A), ENO2 (B) and SLC2A1 (C) in kidney and liver slices exposed to compounds 1, 2, 3 and lansoprazole for 24 h, in comparison to untreated slices set as 1. The error bars show the standard deviation of at least three independent experiments.

The selected genes from the Nrf2 pathway include GCLM and HMOX-1. GCLM codes for Glutamate-Cysteine Ligase Modifier Subunit, part of the Glutamate-cysteine ligase and is the first enzyme of the glutathione biosynthetic pathway. HMOX-1 codes for Heme Oxygenase 1 which is an essential enzyme in heme catabolism and plays an important role as antioxidant under oxidative stress conditions. Both genes are over expressed under oxidative stress conditions in kidney and liver tissue to compensate the excessive production of free radicals.42–44 GLCM expression is significantly upregulated by the higher concentration of compound 3 in kidney slices, whereas in liver slices the effect is not evident. HMOX-1 expression is significantly upregulated by the higher concentration of compound 3 in kidney and liver slices. HMOX-1 displays a trend towards upregulation after treatment of PCKS and PCLS with compounds 1 and 2, even though the differences are not significant in all the cases, an analysis of individual experiments revealed a clear trend to upregulation of both genes (Figure 6). Lansoprazole did not induce any change in the expression of the tested genes. These findings suggest oxidative stress as the possible mechanism of toxicity in kidney and liver slices being more pronounced in kidney. More experiments evaluating the glutathione and thioredoxin redox balance assays, could lead to confirmation of our findings

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Figure 6. Gene expression of GCLM (A) and HMOX-1 (B) in kidney and liver slices exposed to compounds 1, 2, 3 and lansoprazole for 24 h, in comparison to untreated slices set as 1. The error bars show the standard deviation of at least

three independent experiments.

The p53 pathway was explored by determining the expression levels of protein p53 that stimulates the expression of a set of downstream target genes that can induce apoptosis, facilitate DNA repair or activate cell cycle arrest upon cellular stress signals induced by DNA damage, oncogene activation and hypoxia.45–48 BAX that codes for the Bcl-2-associated X protein that plays an important role in apoptosis.49 Additionally SULF-2 gene was evaluated, it codes for sulfatase 2 enzyme, which is upregulated upon activation of p53 due to DNA damage, thereby affecting the cell cycle.50 Neither p53, BAX or SULF-2 showed major regulation changes upon treatment of kidney and liver slices (Figure 7). These findings are in line with results obtained to assess the caspase 3 and 7 activation in PCKS (results not shown), where after treatment with the compounds no evidence of caspase activation was found, indicating that apoptosis is not the mechanism of cell death in these tissue slices.

Figure 7. Gene expression of p53 (A), BAX (B) and SULF-2 (C) in kidney and liver slices exposed to compounds 1, 2, 3 and lansoprazole for 24 h, in comparison to untreated slices set as 1. The error bars show the standard deviation of at least three independent experiments.

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CONCLUSIONS

The potential therapeutic application of lansoprazole Au(I) derivatives with antiproliferative effect17 and as antituberculosis agents, prompted us to study the toxicity in healthy tissue using kidney and liver slices aiming to get insights about the possible side effects if the compounds are administered in vivo. We assessed the toxicity by ATP content and histomorphology in PCKS and PCLS. Additionally, mRNA expression of specific stress markers was assessed in slices, including expression of genes coding for proteins that play important roles in pathways of oxidative stress, apoptosis and hypoxia.

The obtained ATP results showed a different toxicity profile for the tested compounds. Compound 2 shows the lowest toxicity in both liver and kidney slices, also lower than cisplatin, whereas compounds 1 and 3 are more toxic in the liver slices. Notably, both compounds bear phosphine ligands, known to be an intrinsically toxic ligand. However, in kidney slices compound 1 is less toxic than compound 3, and is equal to compound 2. Compound 3 is the most toxic in both organs. The presence of a second Au(I) center in 3, may be responsible for the higher toxicity in healthy tissue compared to 1 and 2.

However, not the intrinsic toxicity but the selectivity of toxicity in healthy organs versus cancer cells is the most relevant parameter. This is estimated by calculating the ratio of the TC50 in slices to the IC50 in the cancer cells. Using this ratio as the potential selectivity of the complexes, compound 1 presents the best ratio of toxicity in healthy tissue to anticancer efficacy, which is much higher than that for cisplatin, which may indicate that this compound can lead to a better drug candidate for further development. However, it should be stressed that these ratios do not represent the absolute values of selectivity, because the experimental methods in cell lines and in slices are different. For instance, the slices are incubated for 24 h and the cancer cells are investigated after 72 h. Moreover, the medium composition is different, and the potential effect of protein binding could be very different in the two systems. Nevertheless, the calculated ratios can be used to compare the different compounds with standard drugs like cisplatin. In vivo animal experiments or experiments with healthy tissue slices and tumor tissue slices, preferably from human origin, could give a better estimation of the selectivity of the drugs for the cancer cells.

Notably, the histomorphology evaluation shows similarities for all three Au(I) complexes with respect to the specific kidney cell types that suffer the most extensive damage, where they seem to have a preferent toxicity towards the distal tubular cells. In contrast to cisplatin which is known to show toxicity in the proximal tubular cells as described in previous reports.11,34 Regarding the liver histomorphological evaluation, all the tested compounds including cisplatin and auranofin induce extensive hepatocellular necrosis.

Interestingly, of all the stress pathways evaluated, the clearest impact was on the Nrf2 pathway, indicating oxidative stress as a possible mechanism of toxicity. This result is in line with the previously reported data, which showed the effects of Au(I) complexes on the inhibition of the seleno-enzyme thioredoxin reductase (TrxR) involved in the maintenance of the intracellular redox balance.51,52 For future studies, it might be relevant to include specific markers for distinct cell types in the kidney and the liver to get more information about the cell-specific toxicity of the compounds evaluated in this study.

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The broad spectrum of applications of the PCTS technology allows us to study in detail the mechanism of the toxicity of the potential drug candidates, using a reduced number of animals with minimal suffering. Using PCTS it is possible to obtain valuable knowledge on the structure-toxicity relationship of experimental compounds, enabling further optimization and selection of better candidates with improved properties and reduced toxicity in healthy tissue.

The obtained results open new perspectives towards the understanding of the selectivity and mechanism of toxicity of the evaluated chimeric compounds and prompt us to continue with the design of more families of bifunctional gold complexes for medical applications and diverse targets with reduced toxicity in healthy tissues.

EXPERIMENTAL SECTION

PREPARATION OF RAT PRECISION-CUT LIVER AND KIDNEY SLICES (PCLS-PCKS) Male Wistar rats (Charles River, France) of 250-300 g were housed under a 12 h dark/light cycle at constant humidity and temperature. Animals were permitted ad libitum access to tap water and standard lab chow. All experiments were approved by the committee for care and use of laboratory animals of the University of Groningen and were performed according to strict governmental and international guidelines.

Kidneys were harvested (from rats anesthetized with isoflurane) and immediately placed in University

lengthwise using a scalpel, and cortex cores of 5 mm diameter were made from each half perpendicular to the cut surface using disposable Biopsy Punches (KAI medical, Japan). PCKS were made as described by de Graaf et al.6,9 The cores were sliced with a Krumdieck tissue slicer (Alabama R&D, Munford, AL, USA) in ice-cold Krebs-Henseleit buffer, pH 7.4 saturated with carbogen (95% O2 and 5% CO2). Liver slices (5 mg (3 mg, incubated individually in 12-well plates (Greiner bio-one GmbH, Frickenhausen, Austria), at 37°C in 1.3 mL Williams’ medium E (WME, Gibco by Life Technologies, UK) with glutamax-1, supplemented with 25 mM D-glucose (Gibco) and streptomycin(Gibco) (PCLS) ciprofloxacin HCl (PCKS) (10 μg/mL, Sigma-Aldrich, Steinheim, Germany) in an incubator (Panasonic biomedical) in an atmosphere of 80% O2 and 5% CO2 with shaking (90 times/min). Slices were pre-incubated 1 h and then transferred to plates with fresh medium with the tested compounds to remove debris and dead cells. Stock solutions of compounds 1 to 3, auranofin were prepared by diluting a stock solution (10-2 M in DMSO, ethanol in the case of auranofin; 10-3 M in water for cisplatin). The final concentration of DMSO and ethanol during the PCLS and PCKS incubation was always below 1 and 0.025 %, respectively to exclude solvent toxicity. For each concentration, three slices were incubated individually for one hour in WME and subsequently, different dilutions of the compounds were added to the wells, to obtain a final concentration from 1 up to 75

VIABILITY AND TC50 DETERMINATION After the incubation, slices were collected for ATP and protein determination, by snap freezing them in 1 ml of ethanol (70% v/v) containing 2 mM EDTA with pH=10.9. After thawing the slices were homogenized using a mini bead beater and centrifuged. The supernatant was used for the ATP essay and the pellet was dissolved in 5N NaOH for the protein essay. The viability of PCKS was determined by measuring the ATP using the ATP Bioluminescence Assay kit CLS II (Roche, Mannheim, Germany) as

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described previously.9 The ATP content was corrected by the protein amount of each slice and -Rad DC

Protein Assay (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA, Sigma-Aldrich, Steinheim, Germany) for the calibration curve. The TC50 value was calculated as the concentration reducing the viability of the slices by 50%, in terms of ATP content corrected by the protein amount of each slice and relative to the slices without any treatment using a nonlinear fitting of log(concentration compound) vs response and is presented as a mean (± SD) of at least three independent experiments.

HISTOMORPHOLOGY After incubation, liver and kidney slices were fixated in 4% formalin for 24 hours and stored in 70%

staining was used for histopathological evaluation. Afterwards, the glass slides were deparaffinised with xylene and ethanol 100%. For the H&E staining the glass slides were hydrated in 50% ethanol, followed by hematoxylin staining for 5 minutes, then rinsed with tap water and treated with acid and basic solutions of ethanol, later the glass slides were stained with eosin for 2 minutes and washed with ethanol 100% and xylene. For the PAS staining the glass slides were washed with distilled water, followed by treatment with a 1% aqueous solution of periodic acid for 20 minutes and Schiff reagent for 20 minutes, the slides were rinsed with tap water, finally, a counterstain with Mayer’s hematoxylin for 5 minutes was used to visualize the nuclei.

DETERMINATION OF STRESS MARKERS EXPRESSION RNA isolation Three precision cut kidney slices from each treatment group were snap-frozen in RNase free Eppendorf’s. RNA was isolated with the Maxwell® 16 simplyRNA Tissue Kit (Promega, Leiden, the Netherlands). Slices were homogenised in homogenisation buffer using a minibead beater. The homogenate was diluted 1:1 with lysis buffer. The mixture was processed according to the manufacturer’s protocol using the Maxwell machine. RNA concentration was quantified on a NanoDrop One UV-Vis Spectrophotometer (Thermoscientific, Wilmington, US) right before conversion to cDNA.

cDNA generation RNA samples were diluted to using random primers with TaqMan Reverse Transcription Reagents Kits (Applied Biosystems, Foster

--

primers. cDNA was generated in the Eppendorf mastercycler (Hamburg, Germany) with a gradient of 20°C for 10 min, 42°C for 30 min, 20°C for 12 min, 99°C for 5 min and finally, 20°C for 5 min.

qPCR Real-time quantitative PCR was used to determine relative mRNA levels of a set of specific genes involved in toxicity pathways. PCR Was performed using SensiMixTM SYBR Low-ROX kit (Bioline, London, UK) with the QuantStudio 7 Flex Real-Time PCR System (Thermoscientific, Wilmington, US) with 1 cycle of 10 min at 95°C, 40 cycles of 15 sec at 95°C and 25 sec at 60°C, with a final dissociation stage of 15 sec at 95°C, 1 min at 60°C and 15 sec at 95°C

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l and measured in triplicate. All primers were purchased from Sigma-Aldrich. Fold induction of each gene was calculated using the housekeeping gene GAPDH.

Primer sequences used in qPCR:

- - -CCGGAGCTACAATTCGGTGA- - - -TCGTATTTGCCATCGCGGTA-

- - -AGAAACCCATAAGCACGGCA- - - -CGCCAGGGAGGTACTCAAAC-

HMOX- - - -AAGGCGGTCTTAGCCTCTTC- - CCCCTGAAGACTGGATAAC- -AACTCTGCAACATCCTGGGG-

BAX: -ACAGGGGCCTTTTTGTTACAG- -GGGGAGTCCGTGTCCACGTCA- ; SULF2: -CGTGTGTGTTTAGAGGCGAGC - -AGCCTCTTTCCGCTTTTTGGT-

-CGCTGG - -CTGTGGTCATGAGCCCTTCC-

STATISTICS A minimum of three independent experiments were performed using slices in triplicates from each rat kidney or liver. Statistical testing was performed with one-way ANOVA with each individual experiment as random effect. We performed a Tukey HSD post-hoc test for pairwise comparisons. A p-

deviation (SD) are shown.

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CHAPTER 7

NEW INSIGHTS INTO THE TOXICITY AND TRANSPORT

MECHANISMS OF CISPLATIN IN KIDNEY IN

COMPARISON TO A GOLD-BASED ANTICANCER AGENT

N. Estrada-Ortiz,a* Sarah Spreckelmeyer,a* Gerian Prins,a Margot van der Zee,a Stefan Stürup,b Inge A. M. de Graaf,a Geny Groothuis,a Angela Casinia,c

a Dept. Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy. University of Groningen. A. Deusinglaan 1, 9713AV Groningen, The Netherlands. b Dept. of Biology/ Dept. of Pharmacy, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark. c School of Chemistry. Cardiff University. Main Building, Park Place, CF103AT Cardiff, United Kingdom. *Shared 1st authors

Submitted

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ABSTRACT

Mechanisms of toxicity and cellular transport of anticancer metallodrugs, including platinum-based agents, have not yet been fully elucidated. Here, we studied the toxic effects and accumulation mechanisms of cisplatin in healthy rat kidneys ex vivo, using the Precision Cut Tissue Slices (PCTS) method. In addition, for the first time, we investigated the nephrotoxic effects of an experimental anticancer cyclometallated complex [Au(pyb-H)(PTA)Cl]PF6 (PTA = 1,3,5-triazaphosphaadamantane). The viability of the kidney slices after metallodrug treatment was evaluated by ATP content determination and histomorphology analysis. A concentration dependent decrease in viability of PCKS was observed after exposure to cisplatin or the Au(III) complex, which correlated with the increase in slice content of Pt and Au, respectively. Metal accumulation in kidney slices was analysed by ICP-MS. The involvement of OCTs and MATE transporters in the accumulation of both metal compounds in kidneys was evaluated co-incubating the tissues with cimitedine, inhibitor of OCT and MATE. Studies of mRNA expression of the markers KIM-1, villin, p53 and Bax showed that cisplatin damages proximal tubules, whereas the Au(III) complex preferentially affects the distal tubules. However, no effect of cimetidine on the toxicity or accumulation of cisplatin and the Au(III) complex was observed. The effect of temperature on metallodrug accumulation in kidneys suggests the involvement of a carrier-mediated uptake process, other than OCT2, for cisplatin; while carrier-mediated excretion was suggested in the cases of the Au(III) complex.

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INTRODUCTION Cisplatin (cis-diamminedichloridoplatinum(II), Figure 1) is an antineoplastic drug used in the treatment of many solid tumours, including those of the head, neck, lung, and testis. Unfortunately, severe side effects following cisplatin treatment may occur, including ototoxicity and myelosuppression,1 with the main dose-limiting side effect being nephrotoxicity.2 The pathophysiological basis of cisplatin nephrotoxicity has been studied for the last four decades, and the emerging picture is that the exposure of tubular cells to cisplatin activates complex signalling pathways, leading to tubular cell injury and death via both apoptosis and necrosis.3 Studies in rats and mice suggest that the drug undergoes metabolic activation in the kidney to a more potent toxin, a process possibly involving glutathione and mediated by gluthathione-S-transferase.4 Activation of cisplatin to its highly reactive and toxic metabolites includes spontaneous intracellular aquation reactions, which involve the substitution of the chlorido ligands with water/hydroxide molecules.5,6 Previously reported studies using kidney slices,7 cultured renal epithelial cells8 and isolated perfused proximal tubule segments9 have provided evidence for basolateral carrier-mediated uptake of cisplatin. Moreover, it was found that cisplatin concentration within the kidneys exceeds the concentration in blood by at least five-fold, suggesting accumulation of the drug by renal parenchymal cells.7 At a molecular level, experimental evidence has led to the conclusion that cisplatin enters cells via two main pathways: (i) passive diffusion and (ii) facilitated uptake by a number of transport proteins,6,10 including copper transporters (CTR) and organic cation transporters (OCT).6,11 Pabla et al demonstrated that CTR1 is mainly expressed in both proximal and distal tubular cells in mouse kidneys, whereas cisplatin toxicity has been observed mainly in the proximal tubular cells.11 In the same study, it was shown that down-regulation of CTR1 in human embryonic kidney cells (HEK293), by small interfering RNA or copper (Cu(I)) pre-treatment, resulted in decreased cisplatin uptake. Various studies, demonstrating that cisplatin can be transported by OCTs in cells, are based on competition experiments with other established OCTs substrates such as tetraethylammonium (TEA) and inhibitors such as cimetidine.12–14 OCTs belong to the solute carrier SLC22A family consisting of three sub-categories: the electrogenic transporter (OCT1-3), electroneutral organic cation/carnitine transporter (OCTN1-3) and the organic anion transporter (OATs and urate transporters, URAT-1).15 Many transporters of the SLC22A family are found in secretory organs such as the liver and the kidneys, as well as the intestine, where they play pivotal roles in drug adsorption and excretion.16 Moreover, different OCTs show species and tissue-specific distribution. For example, the human OCT1 is highly expressed in the sinusoidal membrane of the liver and in the apical membrane of the jejunum17 but not in the kidney. Instead, human OCT2 is mainly expressed in the basolateral side of renal proximal tubule cells, and in the dopaminergic brain regions.18 In order to correctly interpret translational studies, it is important to note that in rodents, both OCT1 and OCT2 show a high renal expression in the basolateral membrane of proximal tubule cells. 19–21 hOCT3 shows a much broader tissue distribution, including skeletal muscle, heart, brain, and placenta, but the distribution in the membrane and physiological role of OCT3 are not yet clearly understood.16 The interaction of cisplatin with hOCT2 in the kidney, or hOCT1 in the liver, was investigated with the fluorescent cation 4-[4-(dimethyl-amino)styril]-methylpyridinium (ASP) in stably transfected HEK293 cells overexpressing these transporters, and in cells physiologically expressing them, such as human proximal tubules and human hepatocyte couplets.22 Notably, cisplatin inhibited ASP transport in hOCT2-HEK293 but not in hOCT1-HEK293. Furthermore, incubation with cisplatin induced apoptosis in hOCT2-HEK293 cells; a process that was completely suppressed by simultaneous incubation with the hOCT2 inhibitor

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cimetidine. Moreover, in isolated human proximal tubules, cisplatin competed with basolateral organic cation transport, whereas it had no effect in human hepatocytes.22 Overall, these findings support the idea of the interaction of cisplatin with hOCT2 in renal proximal tubules, but not with hOCT1, possibly explaining its organ-specific toxicity. In 2010, the functional effects of cisplatin treatment on kidney and hearing were studied in vivo in wild-type and OCT1/2 double-knockout mice.23 No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice, while cisplatin accumulation in the kidneys was reduced.23 Co-medication of wild-type mice with cisplatin and the organic cation cimetidine resulted in protection against ototoxicity and partly against nephrotoxicity.23 Moreover, experiments in rats showed that treatment with both cisplatin and cimetidine did not interfere with the antitumoral activity of the Pt drug.24 Based on these studies and others, hOCT2 has been proposed as a target for protective therapeutic interventions in cisplatin chemotherapy. Furthermore, membrane transporters are also involved in carrier-mediated Pt efflux pathways, including the ATP-binding cassette (ABC) multidrug transporters6,25 and the multidrug and toxin extrusion proteins (MATEs).26–28 MATEs belong to the SLC47 family and are also part of organic cation homeostasis. Specifically, MATEs act as H+/organic cation antiporters, transporting protons from the extracellular side to the cytoplasm in concomitance with organic cations export to the lumen of the proximal tubule. Two isoforms are known, SLC47A1 (MATE1) and SLC47A2 (MATE2-K). Both in human and in rat, MATE1 is primarily expressed in the liver and kidney, while MATE2-K exhibits a kidney-specific expression at the brush border membrane of the tubular cells. 29 Several studies have confirmed cisplatin transport by MATEs.13,14 Overall, these studies suggest that, in humans, the interplay between OCT2 and MATE is responsible for the net renal secretion of cisplatin, and possibly also for the net accumulation of cisplatin in the tubular cells, but further investigation is essential to fully elucidate the complex pathways of cisplatin transport and related side-effects.30 Within this framework, the lack of conclusive information is at least partly due to the lack of suitable models to study transport mechanisms in renal tissues. In vitro models, generally 2D cell cultures, have been applied to study the mechanisms of action, metabolism and transport of metallodrugs.30 These 2D cultured cells usually are characterized by low level of differentiation and mostly consist of one cell type, thereby lacking interactions between the different cell types as in a tissue. Therefore, a model including all cell types in their natural environment is indispensable for studying complex, multicellular organ functions and the pharmacological and toxicological response to drugs, as well as for the identification of the transport mechanisms. The precision cut tissue slices (PCTS) is such a technique, where the original cell-cell and cell-matrix contacts stay unaltered and as such is a useful technique for drug testing ex vivo.31 In a PCTS model, the tissue can remain viable during culture with physiological expression and localization of enzymes and transporters. Thus, the PCTS system is uniquely suited to examine molecular responses to toxicant exposures and compare species differences, and is nowadays a FDA-approved technology. 32 Recently, our group has successfully used the PCTS technique to study the toxic effects of experimental anticancer organometallic compounds,33–36 aminoferrocene-containing pro-drugs,37 ruthenium-based kinase inhibitors,38 as well as supramolecular metallacages as possible drug delivery systems.39 Interestingly, nephrotoxic side effects induced by cisplatin were already investigated in human and rat kidney slices and characterized morphologically, as well as in terms of gene expression and functional changes, providing evidence for the mechanisms of apoptosis induction.40–42 Furthermore, the acute nephrosis of tubular epithelium induced by cisplatin in vivo was reproduced in both human and rat kidney slices ex vivo, while the glomerulus appeared unaffected even at high drug concentration (80

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μM).40 However, further studies are necessary to evaluate possible transport mechanisms using the precision-cut kidney slices (PCKS) ex vivo model. Here, we report on the toxicity and mechanisms of accumulation and transport of cisplatin studied in rat kidney using the PCKS technique. Tissues viability was assessed by three different methods, including ATP content, histomorphology, and mRNA determination of different biomarkers. Moreover, intracellular metal accumulation was determined by inductively coupled plasma mass spectrometry (ICP-MS). In addition, the involvement of carrier–mediated transport was investigated performing experiments in the presence of cimetidine, an inhibitor of both rat OCTs and MATE transporters, as well as varying the temperature of tissues incubation. Furthermore, we also studied the toxicity, accumulation and transport mechanisms of another metallodrug, the previously reported experimental cytotoxic cyclometallated (C^N) Au(III) complex [Au(pyb-H)(PTA)Cl]PF6 (PTA = 1,3,5-triazaphosphaadamantane, Figure 1) featuring a relatively stable Au(III) centre.43 Interestingly, this Au(III) complex showed promising antiproliferative effects against several cancer cell lines and inhibits the zinc-finger enzyme PARP-1 in nM concentrations.43 For most of these new generation experimental metal-based compounds with cytotoxicity towards cancer cells, the mechanisms leading to their pharmacological and toxicological profiles are still not fully elucidated and different biological targets and transport systems have been proposed which still need validation.30

NAu

Cl

N

PNN

PF6

PtCl

H3N Cl

H3N

cisplatin [Au(pyb-H)(PTA)Cl]PF6

Figure 1. Structure of the anticancer metal complexes evaluated in this study.

RESULTS AND DISCUSSION

TOXICITY EVALUATION ATP content determination.

Initially, to determine the toxicity of the evaluated compounds, PCKS were incubated with different concentrations of cisplatin and Au(III) complex for 24 h. In addition, another set of kidney slices of the same rat were co-incubated with 100 μM cimetidine to assess its effect on the toxicity of cisplatin and Au(III) complex in the PCKS. It is hypothesized that this inhibitor for OCTs and MATE transporters might reduce the accumulation of cisplatin and thereby protects against toxicity. The viability of the kidney slices was determined by measuring the ATP/protein content. The obtained results are presented in Figure 2. Both compounds show concentration-dependent reduction of viability. The Au(III) complex showed a higher toxicity than cisplatin, with TC50 values of 4.3 ± 0.2 μM and 17 ± 2.0 μM, respectively (Table 1). Co-incubation of cisplatin or Au(III) complex with 100 μM cimetidine did not result in any significant change of toxicity in the rat kidney slices. These results are in contrast with

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previously reported studies in in vitro cellular models. 22,23 It should be noted that cimetidine inhibits not only the uptake transporter OCT2, which is supposed to reduce cellular accumulation, but also the active efflux transporter MATE, which may result in higher accumulation in the slices and thereby increasing toxicity.44

Thus, our toxicity evaluation results suggest that cisplatin and Au(III) complex accumulation is not dependent on rat OCTs or MATEs.

Table 1. TC50 values for PCKS treated with cisplatin or Au(III) complex, in the absence and presence of cimetidine, for 24 h.

Compound TC50

No cimetidine + 100 μM cimetidine

cisplatin 17.0 ± 2.0 14.7 ± 3.5

Au(III) complex 4.3 ± 0.2 4.4 ± 0.9

Figure 2. Viability of PCKS treated for 24 h with different concentrations of cisplatin (top) and of Au(III) complex (bottom), without cimetidine (black bars) and co-incubated with cimetidine (grey bars, indicated as: + CIM). The error bars show the standard deviation of at least three independent experiments.

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HISTOMORPHOLOGY

The differential effects of cisplatin and Au(III) complex on PCKS viability, in the absence or presence of cimetidine, were further assessed by histomorphology. Specifically, Periodic Acid-Schiff staining (PAS) was used to evaluate slice integrity and particularly to visualize the basal membranes and epithelial brush border in the proximal tubule. After 24 h incubation, the untreated kidney slices show minor morphological changes, indicated by occasional pyknosis and swelling of tubular cells (Figure 3A). The kidney slices co-incubated with cimetidine (100 μM) showed similar characteristics of integrity as the untreated controls (Figure 3A and 3B). However, the cell swelling is more evident in the samples treated with cimetidine, including slight Bowman’s space dilatation (Figure 3B). As expected, exposure of PCKS to cisplatin (Figure 3) and to the Au(III) complex (Figure 4) results in damage in several of the cellular structures in the cortex. In the case of cisplatin, at 5 μM concentration (Figure 3C-D) there is evidence of damage on the distal and proximal tubular cells as well as some dilatation of the Bowman’s space in the glomerulus, which seems more prominent in the samples treated with cimetidine. With the increase of the concentration of cisplatin to 10 and 25 μM (Figure 3E-H) the damage intensifies and disruption of the brush borders of the proximal tubules becomes more evident. Furthermore, cimetidine treatment did not reduce the kidney damage induced by cisplatin, and slices resulted to be equally affected by the metallodrug as those incubated without cimetidine. The tubular damage found in our study for cisplatin is in line with the results of Vickers et al. in rat PCKS. 40 However, in our study we observed that cisplatin also affects the glomerulus structure. This difference in toxicity profiles might be caused by the different culture media used, especially the presence of serum in the study of Vickers might be the cause of the difference between these results. In conclusion, our data shows tubular damage by cisplatin, which is not influenced by cimetidine, in line with the ATP viability data. For the samples exposed to the Au(III) complex, the increase in drug concentration does not produce any major differences with respect to the lower tested concentration. In fact, in all cases, extensive damage is observed mainly in the distal tubular cells, with the structure of the brush border in the proximal tubule almost intact (Figure 4 C-H). No difference was observed in the absence (Fig. 4, left column) or presence (Fig. 4, right column) of cimetidine. While cisplatin generates more damage towards the glomeruli and the proximal tubular cells, the Au(III) complex displayed selective damage of the distal tubular cells, which was also previously described for anticancer Au(I) complexes.36 Overall, the presented histomorphology and ATP results show that there is no evidence of reduced toxicity in the slices exposed to either cisplatin or Au(III) complex co-incubated with cimetidine (Figures 3 and 4, right columns).

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Figure 3. Morphology of rat precision cut kidney slices exposed to different concentrations of cisplatin for 24 h. Left column: absence of cimetidine; right column: co-incubation with cimetidine. A and B: 24 h control incubation; C and D: 5 μM; D and E: 10 μM; F and G: 25 μM h. PT: proximal tubule, DT: distal tubule, G: glomerulus, BSD: Bowman’s space dilatation, N: Necrotic areas. Scale bar indicates 50 μm.

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Figure 4. Morphology of rat precision kidney slices exposed to different concentrations of Au(III) complex for 24 h. Left column: absence of cimetidine; right column: co-incubation with cimetidine. A and B: 24 h control incubation; C and D: 1 μM; E and F: 2.5 μM; G and H: 5 μM. PT: proximal tubule, DT: distal tubule, G: glomerulus, BSD: Bowman’s space dilatation, N: Necrotic areas. Scale bar indicates 50 μm.

EXPRESSION OF KIDNEY-INJURY MOLECULE-1 (KIM-1), VILLIN, P53 AND BAX.

The variation of the expression levels of different markers were evaluated in PCKS after incubation with the Pt(II) and the Au(III) metallodrugs. Specifically, kidney injury molecule-1 (KIM-1) and villin were chosen as proximal tubule damage specific biomarkers. An increase of KIM-1 fold expression is expected in the presence of tubular damage, 45,46 whereas decrease in the expression levels of villin is

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considered a sign of brush border damage.47–49 Additionally, p53 and Bax were studied to determine the role of apoptosis in the toxicity of PCKS upon exposure to cisplatin and to the Au(III) complex. In fact, it has been proposed that cisplatin induced nephrotoxicity consists of activation of multiple stress pathways, including p53 mediated responses and intrinsic and extrinsic apoptosis pathways (with Bax playing an important role in the intrinsic ones).50–53 Thus, KIM-1, villin, p53 and Bax mRNA expression was evaluated in PCKS after 3 h, 5 h and 24 h exposure with cisplatin and Au(III) complex at concentrations close to their TC25 and TC50. The obtained results are shown in Figure 5.

In the untreated control samples, the KIM-1 expression showed a tendency to increase over time compared with the 0 h controls reaching a peak after 24 h, ca. 100-fold increase, (Figure 5A); these results suggest the tubular cells are per se undergoing damage by the slicing and/or culturing process. Interestingly, KIM-1 expression decreased with increasing cisplatin concentration compared to the time-matched controls; even the lowest concentration of cisplatin and the shortest period of

– 3 h) resulted in a decreased KIM-1 expression compared to untreated controls. However, due to the high variation, this decrease reached significance only after 24 h exposure to 15 μM cisplatin. Interestingly, upon exposure to the Au(III) complex, no change in KIM-1 expression was observed. From these findings, it can be hypothesized that when slices are exposed to high concentrations of cisplatin, the cellular machinery of the proximal tubular cells is too damaged to be able to produce KIM-1 mRNA.

As shown by the morphological studies, the gold compound seems to target more specifically the distal tubular cells, with reduced damage to the proximal tubular cells, explaining the lack of effect on KIM-1. Concerning villin mRNA expression (Figure 5B), a reduction up to 40% was observed in the controls after incubation for 24 h, indicating some damage of the brush border of the proximal tubular cells. However, no dose-dependent effect of cisplatin was observed, while the Au(III) compound slightly reduced the villin expression, which only reached significance after 5 h at 2 μM.

The assessment of the expression patterns of p53 (Figure 5C) displayed a slight, but not significant increase of the p53 expression during incubation of the control slices. Conversely, upon treatment with cisplatin after 5 h at 15 μM, a decrease of p53 expression is observed, as well as 24 h at 7.5 and 15 μM compared to the untreated samples at each time point. These results differ from the findings in the previously mentioned study of Vickers et al,40 where the expression of p53 increased in rat PCKS after treatment with cisplatin at 20 and 40 μM for 24 and 48 h, respectively.40 Exposure to the Au(III) complex had no effect on p53 expression (Figure 5C, right panel).

Finally, no significant difference in Bax expression levels was observed during incubation of the control samples (Figure 5D). Bax expression increased slightly but significantly after 24 h exposure to the highest concentration of both compounds. These results indicate possible activation of the intrinsic apoptotic pathway depending mainly on the mitochondrial integrity. This finding is in line with previous reports that indicate the possibility of induction of apoptosis independent of p53 after treatment with cisplatin of human and mouse cancer cell lines.53–58

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Figure 5. Fold change of KIM-1 (A), villin (B) p53 (C) and Bax (D) after exposure to cisplatin (left) and to the Au(III) complex (right) during 3 h, 5 h and 24 h. The untreated control (0 h) was set as 1 to calculate the relative fold induction (not shown). The error bars show the standard deviation of at least three independent experiments. Statistical significance was determined by repeated measures ANOVA and Bonferroni as post hoc test to compare the treated samples with the untreated controls for each time point (*: p<0.05, **: p<0.01, ***: p<0.001).

UPTAKE STUDIES Metal content determination by ICP-MS In order to assess the intracellular accumulation of the tested compounds and to evaluate the relation between toxicity and intracellular metal content, we determined the Pt and Au content of PCKS exposed to cisplatin and to the Au(III) complex by ICP-MS. Thus, PCKS were incubated for 24 h in the same conditions as for the ATP determination. The concentrations of cisplatin and Au(III) complex used were below or around their TC50. As can be seen in Figure 6, the Pt and Au contents increase as a function of the compounds’ initial concentration (up to 110.3 ng Pt per slice in the case of slices treated with 10 M cisplatin, and up to 84 ng Au per slice treated with 5 M of Au(III) complex). However, the obtained results did not show a significant difference between the samples treated with cimetidine or without it, which is in line with the viability and histomorphology studies above, suggesting that OCT and MATE are not involved in the transport of the compounds at the tested concentrations. Moreover, it is worth mentioning that the Au(III) complex appears to be more efficiently accumulated into PCKS than cisplatin: the amount of Au and Pt after 24 h of incubation is approximately the same (~28 ng) for the samples treated with cisplatin at 3 μM concentration or Au(III) complex at 1 μM. Additionally, from the obtained results, it can be calculated that the

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accumulation of cisplatin and of the Au(III) complex results in approximately 20-fold and 30-fold increased concentration of the metals in the slices compared to the medium, respectively. This is in line with the reported accumulation of cisplatin in kidneys in vivo,40 indicating either high binding or metabolism in the cells, or the involvement of active uptake transporters.

Figure 6. Total metal content determined by ICP-MS in PCKS treated with cisplatin or with the Au(III) complex at different concentrations, without and with cimetidine (indicated as: + CIM), for 24 h. The error bars show the standard deviation of at least three independent experiments.

Effect of temperature on PCKS uptake To evaluate if the uptake of cisplatin and of the Au(III) complex is by passive diffusion or carrier-mediated transport, PCKS were incubated with the selected compounds at three different concentrations at 4°C or 37 °C over a period of 60 min. Slices were collected after 0, 10, 30 and 60 min incubation with the metallodrugs, washed with ice-cold Krebs Henseleit buffer and their metal content was evaluated by ICP-MS to assess the effect of different temperatures on the uptake of the drugs.

Slices treated with cisplatin at 5 μM and 25 μM incubated at 4°C or 37°C showed an initial rapid uptake phase followed by a slower accumulation, indicating sequestration by excretion. No significant differences in the Pt content were seen between the two temperatures (Figure 7), suggesting that only passive uptake mechanisms play a role at these tested concentrations. However, slices treated with cisplatin at 100 μM showed significant differences at 30 and 60 min, with a lower Pt content in the slices incubated at 4°C compared to 37°C, indicating that carrier-mediated uptake mechanisms are implicated in cisplatin accumulation in cells at this high concentration. Apparently, both passive and carrier-mediated mechanisms are involved with cisplatin uptake at low concentrations, while carrier-mediated transport is only significantly involved in Pt accumulation at higher concentrations, indicating a low affinity for the transporter.6,26,30

Figure 7. Pt content (ng per slice) in PCKS treated with cisplatin at 5 μM, 25 μM, 100 μM. Incubated at 37°C and 4°C and collected at three different time points (10, 30 and 60 min). t=0 value for the lowest concentration was not determined due to limitations in the amount of tissue, but is estimated to be 0.8 ng Pt/slice based on the values found

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for the two higher concentrations. The error bars show the standard deviation of at least three independent experiments.

Remarkably, evaluation of the Au content in rat kidney slices upon treatment with the Au(III) complex showed a significant higher Au accumulation at 4°C compared to 37°C at all concentrations and time points (Figure 8). A fast initial uptake rate is followed by a slower uptake rate, which is observed for all concentrations during 60 min. The higher Au content found in the slices incubated at 4°C could be due to the inhibition of active excretion mechanisms such as an efflux transporter, other than MATE, at this low temperature.

Figure 8. Au content (ng per slice) in PCKS treated with 1 at 2 μM (A), 5 μM (B), 10 μM (C). Incubated at 37°C and 4°C and collected at three different time points (10, 30 and 60 minutes). t=0 value for the lowest concentration was not determined due to limitations in the amount of tissue, but is estimated to be 0.5 ng Au/slice. The error bars show the standard deviation of at least three independent experiments.

CONCLUSIONS

In the past decades, several studies have been carried out to elucidate the mechanisms of uptake and efflux of cisplatin on kidney cells, related to the nephrotoxic effects of this extensively used anticancer drug, and various in vitro assays were conducted. Nonetheless, such transport mechanisms are not yet fully understood.30 Moreover, the mechanisms leading to toxicity and accumulation of new generation anticancer gold complexes have not been fully elucidated. Even less is known on the transport of organometallic gold complexes in the kidney. Therefore, we investigated and compared the toxicity and the accumulation of cisplatin and a cytotoxic experimental organometallic Au(III) complex in rat kidney tissues using the PCKS technology. Additionally, we evaluated the involvement of rOCTs and rMATE transporters using their inhibitor cimetidine, in competition experiments. Furthermore, passive or active transport mechanisms were assessed by measuring metal uptake by ICP-MS in PCKS at 37°C or 4°C, respectively. As expected, a concentration dependent decrease in viability of PCKS was observed after 24 h exposure to both compounds, which correlated with the increase in PCKS content of platinum and gold after treatment. The gold complex seems to be more toxic for the kidney slices than cisplatin based on the TC50’s being 4.3 ± 0.2 μM and 17 ± 2.0 μM respectively. Interestingly, the histomorphological changes after treatment suggest that the Au(III) compound exerts its toxicity towards different target cells than cisplatin, showing extensive damage of the distal tubular cells, whereas cisplatin is more toxic towards the proximal tubular cells. The latter results are in line with previously reported studies.36,40 However, at variance with other studies present in the literature using cell cultures or isolated human tubuli,22–24 in our ex vivo model no effect of co-incubation with cimetidine on the toxicity or accumulation of cisplatin and Au(III) complex was found. Based on these results we conclude that rOCTs and rMATE transporters do not play a prominent role in cisplatin or

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Au(III) complex accumulation in the rat kidney slices at the tested cimetidine concentration. Alternatively, cimetidine may inhibit MATE transporters with higher efficacy than OCT, thereby reducing its renoprotective effect. Furthermore, KIM-1 and villin mRNA expression were studied as markers of proximal tubular damage. KIM-1 expression decreased with increasing cisplatin concentrations, whereas the Au(III) complex induced no change in KIM-1 expression at increasing concentrations. These findings are in agreement with the different localization of the damage in the tissue. In contrast, villin expression was not affected by cisplatin, but was slightly reduced by the Au(III) complex. It could be important to evaluate other markers of damage including markers of structures other than proximal tubular cells to assess kidney injury induced by metallodrugs in a more comprehensive way. Surprisingly, p53 overexpression was not induced in PCKS exposed to cisplatin as previously reported.40 However, as mentioned before, the culture conditions, and specifically the serum protein content of the medium, and the concentrations of cisplatin used were different and these circumstances can lead to substantial differences in the obtained results. Moreover, Bax mRNA expression increased over time in control slices indicating possible activation of intrinsic apoptotic pathways. Nevertheless, the increment is higher when PCKS were treated during 24 h with the highest concentration of cisplatin or gold compound as evidence of further stress compared to the controls. To get insight into the specific toxic mechanisms activated after treatment with cisplatin or other metallodrugs, it is imperative to consider the species and tissue distinct gene expression profiles during incubation without and with drugs. Specifically, cisplatin is known to activate several stress pathways, but it is dependent on the concentration, cell type and culture conditions whether the cells die by apoptosis, necrosis or both. Our ex vivo studies to evaluate the passive or active character of the transport of cisplatin and the Au(III) complex revealed that both passive and active processes might play a role. Moreover, the uptake of cisplatin is achieved by both passive and active mechanisms but this becomes evident only at higher concentrations, indicating a low affinity for the active transporters. On the other hand, the results obtained for the Au(III) complex suggested an important role of carrier-mediated excretion, shown by the increased Au content in the slices incubated at 4°C compared to 37°C. Further studies to explore the role of different transporters are needed to better understand the concentration dependent and organ-specific toxicity of our metallodrugs, which is valuable to design new experimental metallodrugs with reduced side effects in specific tissues. Certainly, the use of PCKS offers good opportunities to evaluate toxicity, uptake and accumulation of metallodrugs in different organs and species, and finally to get insight into the effect in human tissues derived from patients. However, optimization of the experimental set-up to reduce the damage of the PCKS induced by culturing is still necessary to exclude possible interference on the obtained results. Furthermore, new advanced approaches, such as the CRISPR-Cas9 genome editing, should be applied to validate both transport and intracellular trafficking mechanisms for metallodrugs. This technology has been recently applied to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS in cells, in vitro.59 The obtained results suggest that these proteins are not essential for the mechanism by which cisplatin enters human embryonic kidney cells (HEK293T) and ovarian carcinoma OVCAR8 cell lines and is transported to the nucleus, contradicting numerous previously reported studies in the field. Overall, new investigational efforts should be spent to elucidate the complex mechanism of toxicity of metallodrugs and the role of different transport pathways in tissues.

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EXPERIMENTAL

MATERIALS Cisplatin and cimetidine were purchased from Sigma Aldrich, and the Au(III) complex was synthesized according to the protocol previously reported.43

PCKS Male Wistar rats (Charles River, France) of 250-300 g were housed under a 12 h dark/light cycle at constant humidity and temperature. Animals were permitted ad libitum access to tap water and standard lab chow. All experiments were approved by the committee for care and use of laboratory animals of the University of Groningen and were performed according to strict governmental and international guidelines.

Kidneys were harvested (from rats anesthetized with isoflurane) and immediately placed in University olengthwise using a scalpel, and cortex cores of 5 mm diameter were made from each half perpendicular to the cut surface using disposable Biopsy Punches (KAI medical, Japan). PCKS were made as described by de Graaf et al..31,60 The cores were sliced with a Krumdieck tissue slicer (Alabama R&D, Munford, AL, USA) in ice-cold Krebs-Henseleit buffer, pH 7.4 saturated with carbogen (95% O2 and 5% CO2). Kidney slices weighing about 3 mg (~150 m thickness), were incubated individually in 12-well plates (Greiner bio-one GmbH, Frickenhausen, Austria), at 37°C in culture medium, Williams’ medium E (WME, Gibco by Life Technologies, UK) with glutamax-1, supplemented with 25 mM D-glucose (Gibco) and ciprofloxacin HCl (10 μg/mL, Sigma-Aldrich, Steinheim, Germany) in an incubator (Panasonic biomedical) in an atmosphere of 80% O2 and 5% CO2 with shaking (90 times/min).

Before the start of all experiments, PCKS were pre-incubated for 1 h in culture medium and then the samples were transferred to new plates containing fresh medium to remove debris and dead cells.

EVALUATION OF ATP CONTENT After 30 min incubation, different concentrations of cisplatin and Au(III) complex were added to the wells and the slices were incubated for 10 min, 30 min, 60 min or 24 h. After the incubation time, slices were collected for ATP and protein determination, by snap freezing in 1 ml of ethanol (70% v/v) containing 2 mM EDTA with pH=10.9. After thawing, the slices were homogenized using a mini bead beater and centrifuged. The supernatant was used for the ATP essay and the pellet was dissolved in 5N NaOH for the protein essay. ATP was measured using the ATP Bioluminescence Assay kit CLS II (Roche, Mannheim, Germany) as described previously. The ATP content was corrected by the protein

determined by the Bio-Rad DC Protein Assay (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA, Sigma-Aldrich, Steinheim, Germany) for the calibration curve. The TC50 value was calculated as the concentration reducing the viability of the slices by 50%, in terms of ATP content corrected by the protein amount of each slice and relative to the slices without any treatment using a nonlinear fitting of log(concentration compound) vs response and is presented as a mean (± SD) of at least three independent experiments.

EVALUATION OF OCT2/MATE DRUG TRANSPORTER

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To evaluate the involvement of rOCT2 as uptake transporter and rMATE as efflux transporter, the slices were first incubated for 30 min with a non-toxic concentration of 100 μM cimetidine. Afterwards, cisplatin or Au(III) complex were added in the selected concentrations. Each condition is evaluated in triplicates after 24h incubation. Control slices were taken directly after slicing, after pre-incubation and after 24h incubation.

HISTOMORPHOLOGY Kidney slices we

sections were made, which were mounted on glass slides and PAS staining was used for histopathological evaluation. Briefly, the glass slides were deparaffinised, washed with distilled water, followed by treatment with a 1% aqueous solution of periodic acid for 20 minutes and subsequently Schiff reagent for 20 minutes. Then, the slides were rinsed with tap water; finally, a counterstain with Mayer’s haematoxylin (5 min) was used to visualize the nuclei.

EXPRESSION DETERMINATION OF KIDNEY-INJURY MOLECULE-1 (KIM-1), VILLIN, P53 AND BAX. RNA isolation Three precision cut kidney slices from each treatment group were pooled and snap-frozen in RNase free Eppendorf tubes. RNA was isolated with the Maxwell® 16 simplyRNA Tissue Kit (Promega, Leiden, the Netherlands). Slices were homogenised in homogenisation buffer using a minibead beater. The homogenate was diluted 1:1 with lysis buffer. The mixture was processed according to the manufacturer’s protocol using the Maxwell machine. RNA concentration was quantified on a NanoDrop One UV-Vis Spectrophotometer (Thermoscientific, Wilmington, US) right before conversion to cDNA.

cDNA generation

random primers with TaqMan Reverse Transcription Reagents Kits (Applied Biosystems, Foster City, CA). To each samp -

-

qPCR Real-time quantitative PCR was used to determine relative mRNA levels of KIM-1, villin, p53 and Bax. PCR was performed using SensiMixTM SYBR Low-ROX kit (Bioline, London, UK) with the QuantStudio 7 Flex Real-Time PCR System (Thermoscientific, Wilmington, US) with 1 cycle of 10 min at 95°C, 40 cycles of 15 sec at 95°C and 25 sec at 60°C, with a final dissociation stage of 15 sec at 95°C, 1 min at

primers were purchased from Sigma-Aldrich. Fold induction of each gene was calculated using the housekeeping gene GAPDH.

The primer sequences used in qPCR were: KIM- -GTGAGTGGACAAGGCACAC- -AATCCCTTGATCCATTGTTT-

- GCTCTTTGAGTGCTCCAACC- - - CCCCTGAAGACTGGATAAC- -AACTCTGCAACATCCTGGGG- - ACAGGGGCCTTTTTGTTACAG- -GGGGAGTCCGTGTCCACGTCA-

GAPHD: 5’-CGCTGGTGCTGAGTATGTCG-3’ (forward) and 5’-CTGTGGTCATGAGCCCTTCC-3’ (reverse).

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All primers were purchased from Sigma-Aldrich. Fold induction of the evaluated genes was calculated using as reference the housekeeping gene GAPDH.

ICP-MS After incubation with different concentrations of cisplatin or the Au(III) compound, PCKS were washed with ice-cold Krebs-Henseleit buffer and snap-frozen and stored at -80°C until the analysis.

Sample preparation The tissue samples were digested with 100 μL nitric acid overnight, all samples were completely dissolved. 100 μL hydrochloric acid and 800 μL milliQ were added to produce a volume of 1 mL. Prior to analysis the samples were diluted 20 times with 0.56% HNO3/0.1% HCl.

ICP-MS analysis The Pt and Au contents were quantitated applying a Perkin Elmer (Waltham, MA, USA) Sciex Elan 6100 DRC-e ICP-MS instrument, equipped with a Cetac ASX-110FR autosampler, a 0.2 mL min-1 MicroMist U-series pneumatic concentric nebulizer (Glass Expansion, West Melbourne Vic, Australia) and a PC3 cyclonic spray chamber (Elemental Scientific Inc., Omaha, NE, USA). ICP-MS RF power, lens voltage and nebulizer gas and flow were optimized on a daily basis and other settings were: 1 sweep/reading, 25 readings/replicate, 5 replicates, 50 ms dwell time. 197Au+, 195Pt+, and 194Pt+ isotopes were monitored. Pt and Au concentrations were determined by external calibration (0-20 ppb Pt and Au). LODs were 0.1 and 0.2 μg L-1 for Pt and Au, (3*SD on blank, n=10) and the spike recovery were 102% and 99% for Pt and Au, respectively. Pt and Au single element PlasmaCAL standards (SCP Science, Quebéc, Canada) were used and the standards were prepared in a mixture of 0.1% HCl and 0.65% subboiled HNO3 in MilliQ water. This mixture was furthermore used to dilute samples after digestion and as blank solution.

Temperature dependency For the evaluation of temperature dependency, after the preincubation at 37°C for 1 h, rat kidney slices were incubated for 10, 30 and 60 min with the metal complexes at 37°C or 4°C, and were subsequently washed with ice-cold Krebs-Henseleit buffer and snap-frozen as described above.

STATISTICS A minimum of three independent experiments were performed using slices in triplicates from each rat kidney. The TC50 values were calculated as the concentration reducing the viability of the slices by 50%, relative to the untreated samples using a nonlinear fitting of log(concentration compound) vs response and is presented as a mean (± SD) of at least three independent experiments. Statistical testing was performed with repeated measures ANOVA and Bonferroni as post hoc test to compare the treated samples with the untreated controls. A p-In all graphs and tables, the mean values and standard deviation (SD) are shown.

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CHAPTER 8

ANTICANCER GOLD N-HETEROCYCLIC CARBENE

COMPLEXES: A COMPARATIVE IN VITRO AND EX VIVO

STUDY

Natalia Estrada-Ortiz,a Federica Guarra,b Inge A. M. de Graaf,a Lorella Marchetti,b Marina H. de Jager,a Geny M. M. Groothuis,a Chiara Gabbiani b and Angela Casini*a,c

a Dept. Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy. University of Groningen. A. Deusinglaan 1, 9713AV Groningen, The Netherlands. b Department of Chemistry and Industrial Chemistry. University of Pisa. Via Moruzzi, 3, 56124 Pisa, Italy. c School of Chemistry. Cardiff University. Main Building, Park Place, CF103AT Cardiff, United Kingdom.

Published in: ChemMedChem 2017, 12 (17), 1429–1435

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Abstract: A series of organometallic Au(I) N-heterocyclic carbene (NHC) complexes was synthesized and characterized on anticancer activity in four human cancer cell lines. The compounds’ toxicity in healthy tissue was determined using precision cut kidney slices (PCKS) as a tool to determine the potential selectivity of the Au complexes ex vivo. All evaluated compounds presented cytotoxic activity towards the cancer cells in the nano- or low micromolar range. The mixed Au(I) NHC complex - (ter-butylethynyl)-1,3-bis-(2,6-diisopropylphenyl)-imidazol-2-ylidene gold(I), bearing an alkynyl moiety as ancillary ligand, showed high cytotoxicity in cancer cells in vitro, while being barely toxic in healthy rat kidney tissues. The obtained results open new perspectives towards the design of mixed NHC-alkynyl gold complexes for cancer therapy

Keywords:

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INTRODUCTION

Metals and metal complexes have been used for medicinal applications since ancient times. For example, copper was the first metal described as sterilizing agent between 2600 and 2200 B.C. (Smith Papyrus). Furthermore, the Ebers Papyrus from 1500 B.C. describes the use of copper to reduce inflammation and the use of iron to treat anaemia, while approximately at the same time, zinc was being used to heal wounds.1,2 In recent years, medicinal inorganic chemistry has become a rapidly growing field with a broad range of medical applications for inorganic and metal-based compounds, including mineral supplements (Fe, Zn, Cu, Se), diagnostics (Gd, Mn, Ba, I), antimicrobial (Ag), anticancer (Pt), antiulcer (Bi) and antiarthritic (Au) agents, among others.3,4

The interest in developing new metal-containing therapeutic compounds increased largely after the success of cisplatin in the treatment of solid malignancies.5 Unfortunately, platinum(II)-containing compounds present several limitations such as toxicity in healthy tissues, restricted spectrum of activity and development of resistance.5,6 Subsequently, an extensive number of metal-containing compounds were described with interesting cytotoxic activities, displaying diverse mechanisms of action and pharmacological profiles.7–10 Within this framework, gold-based complexes are particularly interesting due to their different possible oxidation states (e.g. Au(I) and Au(III)), stability and ligand exchange reactions, which confer them different mechanisms of activity compared to cisplatin. 4,11,12

Early studies on the anticancer activity of the Au(I) complex auranofin ([Au(I)(2,3,4,6-tetra-O-acetyl-1-(thio- S)- -D-glucopyranosato)(triethylphosphine)]), presently used in the clinic to treat severe rheumatoid arthritis, revealed activity levels similar to cisplatin in vitro,13 which subsequently led to a large number of Au(I) complexes being evaluated for antiproliferative effects. Interestingly, most of the anticancer gold complexes reported so far, appear to exert their activity via the interaction with protein targets,12–14 and only in a few cases DNA binding has emerged as a likely route to cancer cell death.15,16 Among the various families of gold compounds tested for their anticancer effects in the last decade, a variety of organometallic gold(I/III) N-heterocyclic carbene (NHC) complexes were designed, featuring anticancer activity in the micromolar or sub-micromolar range in vitro.17–21 Specifically, gold(I) NHC derivatives exert their effects via different pathways, including: (i) mitochondrial damage (common for cationic gold(I) biscarbene complexes, which behave as delocalized lipophilic cations (DLC) being able to accumulate selectively inside the mitochondria of cancer cells due to their higher mitochondrial membrane potential), 21–24 (ii) inhibition of the seleno-enzyme thioredoxin reductase,26–

28 (iii) inhibition of protein tyrosine phosphatases (PTP)29 and (iv) stabilization of DNA G-quadruplexes.30–32

Moreover, gold(I) NHC complexes are appealing from a synthetic point of view due to their high stability with respect to ligand exchange reactions, relatively easy synthetic procedures, possibility of functionalization leading to increased structural diversity, as well as tuneable lipophilic-hydrophilic properties to enhance biological activity.11,19,33,34

Therefore, herein we report the synthesis of four Au(I) NHC complexes (1-4) (Figure 1) including: mono- (1) and bis-NHC (2) compounds featuring a 1-butyl-3-methyl-imidazol-2-yilidene ligand. This ligand was chosen to obtain gold complexes possessing favourable lipophilicity, in line with previous studies.33 Based on the same NHC scaffold, an auranofin-type complex (3) was obtained, where the

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NHC moiety replaces the phosphane ligand. Moreover, a mixed NHC-alkynyl complex (4) was synthesized, with a 1,3-bis-(2,6-diisopropylphenyl)-imidazol-2-ylidene ligand. The latter was chosen since it was impossible to stabilize the mixed NHC-alkynyl metal complexes using the 1-butyl-3-methyl-imidazol-2-yilidene ligand. It should be noted that, recently, Au(I) complexes of the type (alkynyl)Au(I)(phosphane), containing an anionic alkynyl group as well as a neutral phosphane ligand, have been shown to possess interesting anticancer effects in vitro and in vivo. 35,36 However, to the best of our knowledge the anticancer properties of mixed NHC-alkynyl Au(I) complexes have not been reported so far.

Finally, for comparison purposes and to evaluate the effect of the metal ion on the biological activity, the Pt(II) (5) analogue of 2 was also synthesized. The biological activity of our series of Au(I) NHC complexes was evaluated both in vitro and ex vivo. Specifically, the cytotoxicity of the compounds was tested in four cancer cell lines, namely a p53 wild-type and a p53 null variant of HCT 116 (colorectal carcinoma), MCF-7 (breast adenocarcinoma) and A375 (malignant melanoma). Additionally, the new compounds were tested for their toxicity on healthy rat kidney tissue ex vivo using precision cut kidney slices (PCKS).37

37,38. This technique is an FDA-approved model for drug toxicity and metabolism studies.37,38 Recently, we have successfully used the precision cut tissue slices technique to study the toxic effects of experimental anticancer organometallic compounds,31,39,40 aminoferrocene-containing pro-drugs,41 ruthenium-based kinase inhibitors,42 as well as supramolecular metallacages as possible drug delivery systems.43

AuN

N

S O

OO

O

O

O

O O

OAuN

N

Cl

AuN

N

Au N

N

N

N

PF6

Pt N

N

N

N

Pd N

N

N

N

Cl

Cl

Cl

Cl

1 2 3

4 5 6

Figure 1. Structure of the reported metal NHC complexes.

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SYNTHESIS AND CHARACTERIZATION

The carbene (1-butyl-3-methyl-imidazol-2-yilidene)gold(I)chlorido Au(BMIm)Cl (1) and the bis-carbene bis(1-butyl-3-methyl-imidazole-2-yilidene)gold(I) hexafluorophosphate [Au(BMIm)2]PF6 (2) were synthetized according to previously reported procedures,34 and adapted established protocols.33,44

Thus, synthesis of compound 1 was achieved by a transmetalating route from the correspondent Ag(I) carbene. Compound 2 was synthetized by reaction of Au(SMe2)Cl with 2 equiv. of 1-methyl-3-butyl-2-ylidene, prepared in situ by deprotonation of the correspondent imidazolium salt with LiHMDS.

Complex 3 is a new analogue of auranofin bearing thio- -glucose-tetraacetate as ancillary ligand while replacing the phosphine with the NHC carbene ligand. The compound was obtained in high yields by adapting a procedure previously published by Baker et al.45 that allows the substitution of a chlorido ligand using K2CO3 in CH2Cl2. The compound was found to be soluble in chlorinated solvents (dichloromethane and chloroform), in acetone and in DMSO. This new gold(I) complex was characterized in solution by 1H and 13C NMR in CDCl3 (Figures S1 and S2 in the Supplementary material). The signals are consistent with the ones reported for similar compounds, confirming the proposed structure.33,44 The most diagnostic feature in the 13C NMR spectrum of compound 3 (Figure S2) is the carbene carbon signal at 183.7 ppm, which shows a downfield shift compared with the signal at 171.9 ppm of the corresponding precursor 1 with chlorido as ancillary ligand, most likely due to the better donating ability of the thiolate ligand.44 The signal of the thiol carbon (C1’) shows a downfield shift from 78.9 ppm to 83.0 ppm as a consequence of the coordination to the gold(I) centre. In the 1H NMR spectrum (Figure S1) the coordination of the thiolate is confirmed by the absence of the S-H resonance. In FT-IR spectrum the most diagnostic feature is the band of the carbonyls at = 1744 cm-1. (Figure S3).

Preparation of the gold(I) alkynyl compound 4 was performed according to already established procedures,46,47 by reacting 3,3-dimethyl-1-butyne with the precursor gold(I) NHC carbene (Au(IPr)Cl) in presence of a strong base (ButOK) in MeOH. The compound was characterized in solution by 1H and 13C NMR in CDCl3 (Figure S4 and S5). The 13C NMR spectrum is featured by the signal of the carbene

carbons of the alkynyl moiety are

66.5 ppm) owing to the coordination to gold(I). In the 1H NMR he signal of the four isopropyl protons is a diagnostic feature, featuring a septet at 2.60 ppm as well as the corresponding doublets of the methyl groups at 1.34 and 1.18 ppm. Another characteristic feature of the spectrum is the singlet of the three methyl groups of the alkynyl moiety at 1.10 ppm. In the FT-IR spectrum the weak band of the triple bond at = 2116 cm-1 can be recognized (Figure S6).

Compound 5 is a platinum(II) derivative bearing the same NHC ligand of compounds 1, 2, 3. It was synthesized by transmetalating routes from the correspondent Ag(I) carbene by adapting previously reported procedures.48 Thus, 5 was obtained by refluxing Ag(BMIm)Cl with 0,5 eq of K2PtCl4 in CH2Cl2 for 4 days. The 1H NMR spectrum of 5 in CDCl3 (Figure S7) shows the superposition of two sets of

195Pt NMR spectrum confirms the presence of two complexes in which the metal nucleus resona -3177.8; -3179.0 ppm) (Figure S8). The nature of the isomers can be better elucidated through 13C NMR spectrum (Figure S9). It suggests the presence of the two rotamers trans-syn and trans-anti as

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frequently observed for nickel(II) and palladium(II) complexes of two unsymmetrical NHC ligands.49–51 As a matter of fact, only one carbenic carbon signal at chemical shift typical of trans-bis(NHCs)PtX2

48 The formation of the cis isomer can therefore be excluded, since it would have given a carbenic carbon signal at significantly upfield shifted chemical shift as reported fo -150 ppm).52,53

The stability of gold compounds 3 and 4 was monitored by 1H NMR spectroscopy of mixtures of water and DMSO-d6 during 7 days at room temperature. The complexes were found to be stable in solution in these conditions (Figures S10-S11). Moreover, the two complexes were reacted with 1.5 equivalents of DL-homocysteine, as an intracellular model nucleophile, in CD3OD. 1H NMR spectra revealed that no reaction with 3 occurred after 24 h. Indeed, neither the signals of the complex nor of homocysteine showed any variation (Figures S12-14).

On the contrary, 4 was found to react with homocysteine with the formation of a new species that could be identified as the product of the substitution of the alkyne with the thiol. Indeed, a series of 1H NMR spectra registered during 24 h shows the progressive conversion of 4 into a new complex (Figure S15), most likely involving coordination of the amino acid to the gold centre with substitution of the

7.55, 7.50, 7.36 p

relevant feature is the simultaneous progressive disappearance of the signal of the methyl groups of

of a dissociated 3,3-dimethyl-butyne moiety (Figure S16-S18). Furthermore, the integration of the signals of coordinated homocysteine with the ones of the NHC moiety let us suppose that only the alkyne was substituted by the amino acid nucleophile in these conditions (Figure S19). This hypothesis was further confirmed by 13C NMR spectrum of the mixture after 24 h, where the carbenic carbon

IN VITRO CELL VIABILITY ASSAYS

The antiproliferative properties of the Au(I) NHC complexes and of cisplatin and auranofin as comparison, were assessed using the MTT assay in the human cancer cell lines HCT116 p53 wt, HCT116 p53 null, MCF-7 and A375.

All the tested Au(I) complexes presented antiproliferative effects in the evaluated cell lines in the low M range (Table 1). Instead, the Pt(II) complex 5 was scarcely active (EC50 result is in accordance with other studies on Pt(II) bis-NHC complexes with chlorido ligands (see ref 20 and citations therein) featuring moderate cytotoxic effects in vitro. Notably, the compounds 1, 2 and 3

of the four cell lines, A375 being the exception. Overall, no significant differences could be observed between the EC50 values for the mono-carbene (1), bis-carbene (2), and auranofin-analogue (3) in the different cancer cell lines. Interestingly, the alkynyl derivative (4) was effective although ca. 4-10 fold less potent compared to the other gold NHC complexes. However, in general 4 was markedly more active or as active as cisplatin, except in the A375 cell line. Additionally, the lack of differences

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observed between the HCT116 p53 wt and p53 null cells treated with the gold complexes indicates that their toxicity mechanism is independent of p53 activity, in contrast to cisplatin. The latter is known to induce rapid p53-dependent apoptosis and, as a secondary effect, p53-independent cell cycle arrest.54,55

Table 1. EC50 values of Au(I) NHC complexes in various human cancer cell lines, in comparison to auranofin and cisplatin, after 72 h incubation.

EC50a (μM)

Compound HCT116

p53wt HCT116 p53null MCF-7 A375

1 0,5 ± 0,3 0,6 ± 0,2 0,8 ± 0,4 2 ± 1

2 0,6 ± 0,4 0,24 ± 0,09 1,1 ± 0,3 1,0 ± 0,6

3 0,8 ± 0,2 1 ± 0,4 2 ± 0,8 1,2 ± 0,2

4 9 ± 3 8,8 ± 0,9 6 ± 2 10 ± 1

5 > 50 > 50 > 50 ND

auranofin 5,1 ± 0,7 7,10 ± 0,01 7 ± 2 1,3 ± 0,8

cisplatin 11 ± 3 20,6 ± 0,9 12 ± 2 3,7 ± 0,9

a The reported values are the mean ± SD of at least three independent experiments. ND: not determined.

EX VIVO TOXICITY EVALUATION

Due to their potent cytotoxic effects in cancer cells, complexes 1-4 were tested for their possible toxicity in an ex vivo model in healthy rat kidney tissue using the PCKS technology.37,38

Kidney was selected since cisplatin is well known to induce severe nephrotoxicity in patients.56

Hence, kidney slices were incubated with various concentrations of each gold complex, and after 24 h the viability of the tissues was determined measuring the ATP content (Table 2 and Figure 2). Auranofin and cisplatin were also tested for comparison.

The gold complexes, including auranofin, displayed a concentration dependent toxicity profile, with complexes 1 and 2 as the most toxic, with TC50 below 1 μM. Interestingly, compounds 1-3 were more toxic than cisplatin (ca. 6-12-fold). Remarkably, complex 4 showed no toxicity up to 50 μM. For compounds 1-3 the safety margin for toxicity was small to absent with a ratio of TC50 PCKS/EC50 cells between 0,4 and 3,3. However, for compound 4 the TC50 PCKS/EC50 cells ratio cells was higher than 5,0 to 8,3 indicating selective toxicity towards cancer cells compared to healthy kidney tissue.

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The differential effects of complexes 1 and 4 (5 μM concentration) on kidney slices were further assessed by histomorphology using Periodic acid-Schiff staining (PAS) to evaluate slice integrity and particularly to visualize the basement membranes and epithelial brush border in the proximal tubule cells, as reported in the experimental section. After 24 h incubation, the untreated kidney slices show minor morphological changes, indicated by pyknosis and swelling of some of the tubular cells (Figure 3A). Marked effects were observed upon treatment with complex 1, which induced dilatation of Bowman’s space in the glomerulus and necrosis of the distal tubule cells, as well as discontinuation of the brush border in the proximal tubule cells at some sites (Figure 3B). In contrast, exposure of slices to complex 4 (Figure 3C) did not induce significant morphological changes compared to controls. Auranofin treatment led to damage of the distal tubule cells and loss of nuclei from the proximal tubule cells. Moreover, cisplatin treatment (25 M corresponding to 2-fold the TC50, Figure 3E) showed damage to the proximal tubular cells with loss of nuclei and discontinuation of the brush border; additionally, damage of the distal tubule is evident as previously reported in the literature.57

Table 2. Toxicity of Au(I) NHC complexes (TC50 values) in PCKS in comparison to auranofin and cisplatin.

Compound TC50a (μM)

TC50 (PCKS)/

EC50 (cells)

1 0,8 ± 0,3 0,4 - 1,6

2 0,8 ± 0,7 1,3 - 3,3

3 2,1 ± 0,4 1,1 - 2,6

4 > 50 >5,0 - 8,3

auranofin 2,9 ± 1,4 0,4 - 2,2

cisplatin 12 ± 6 0,6 - 3,2

a The reported values are the mean ± SD of at least three independent experiments.

0 0,5 1 2 5 100

50

100

Auranofin1234

[ M]

Viab

ility

(%)

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Figure 2. Viability of rat PCKS) relative to the controls (untreated slices) after treatment with complexes 1-4 and auranofin for 24 h. The error bars show the standard deviation of at least three independent experiments.

Figure 3. Morphology of rat kidney slices. A: 24 h control incubation; B: complex 1; C: complex 4; D: auranofin; E: cisplatin. The Au(I) complexes were incubated at 5 μM concentration for 24 h, while cisplatin at 25 μM for 24 h. PT: proximal tubule, DT: distal tubule, G: glomerulus. Scale bar indicates 50 μm.

CONCLUSIONS

The broad spectrum and synthetic possibilities in organometallic chemistry allowed us to develop and study different NHC ligands “fine-tuning” the physico-chemical properties of the resulting Au(I) complexes, and, possibly, achieving selectivity towards cancer tissue.

Herein, we reported the synthesis of complexes 3 and 4, as a continuation of our previous work where we described the synthesis and very preliminary biological evaluation of complexes 1 and 2.34 In this study, complexes 1-4 showed interesting cytotoxic activity against HCT116 p53 wt and p53 null, MCF-7 and A357, in the low M range. Instead, the Pt(II) complex 5 was poorly cytotoxic, indicating the essential role of Au(I) ions in the biological activity. However, complexes 1-3 and auranofin displayed severe toxicity in healthy kidney tissue (PCKS), even higher than cisplatin, indicating that these compounds, when administered in vivo, may also induce severe nephrotoxicity. Conversely, the mixed NHC and alkynyl complex 4 appears to be at least 5-fold less toxic in healthy tissue, while maintaining antiproliferative effects at the low micromolar concentration. The reduced bioactivity of this

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compound may be partly due to its lower stability in the presence of nucleophiles, such as sulfur donor ligands. However, the selectivity of 4 for cancer cells with respect to healthy tissues is still promising in comparison to the other tested Au(I) NHC complexes. This initial result prompts us to develop new mixed organometallics with enhanced selectivity against cancer cells. Moreover, ongoing studies in our labs are aimed at further investigating the mechanisms of action of toxicity in cancer cells and kidney slices. So far, pilot studies using mass spectrometry (MS) analysis of complexes 1-4 showed no reactivity towards model protein targets (cytochrome c and lysozyme), while 1 and 2 could bind to the copper chaperon protein Atox-1 upon complete ligand loss.34

Overall, we believe that it is important that toxicology studies, as those presented here using our ex vivo model, should be conducted as early as possible on new experimental metallodrugs to select the optimal chemical scaffolds and to orient the drug design at its early stages.

EXPERIMENTAL SECTION

GENERAL Unless stated otherwise the reactions were performed under inert atmosphere of nitrogen in anhydrous conditions. Solvents and reagents were used without prior treatments. NMR spectra were recorded on a Varian Gemini 200 BB instrument (1H, 200 MHz; 13C, 50.3 MHz, 195Pt, 42.8 MH) at room temperature; frequencies are referenced to the residual resonances of the deuterated solvent. UV-visible spectra were recorded on an Agilent Cary 60 spectrophotometer. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on a Spectrum One Perkin Elmer instrument equipped with an UATR unit.

SYNTHETIC PROCEDURES The carbenes (1-butyl-3-methyl-imidazol-2-yilidene)silver(I)chlorido Ag(BMIm)Cl, (1-butyl-3-methyl-imidazol-2-yilidene)gold(I)chlorido Au(BMIm)Cl (1) and the bis-carbene bis(1-butyl-3-methyl-imidazole-2-yilidene)gold(I) hexafluorophosphate [Au(BMIm)2]PF6 (2) were synthetized according to procedures previously reported by us34 and their purity was confirmed by elemental analysis, and resulted to be > 98 %.

Synthesis of thio- -D-glucose-tetraacetate-(1-butyl-3-methyl)-imidazol-2-ylidene-gold(I) (3)

Precursor 1 was reacted with thio- -D-glucose-tetraacetate to prepare compound 3. 0.1 mmol of 1 was dissolved in 15 ml of CH2Cl2, 1 mmol of K2CO3 and 0,1 mmol of thio- -glucose-tetracetate (tgt) were added to the solution. The suspension was stirred at room temperature for 10 min in the dark. The mixture was filtered and the solution was concentrated by evaporation to about 2 ml. The product was precipitated with hexane and washed to obtain a light brown solid (yield > 99%).

1H NMR (CDCl3, 4 and H5); 5.12- 5.03 (m, 3H, tgt); 4.25-4.12 (m, 2H, H6, 3H tgt); 3.83 (s, 3H, H10); 3.76-3.68 (m, 1H, H5’); 2.08 (s, 3H, OAc); 2.02 (s, 3H, OAc); 1.99 (s, 3H, OAc); 1.96 (s, 3H, OAc); 1.83 (apparent quintet J= 7.4 Hz, 2H, H7a-b); 1.68 (s, H2O); 1.37 (apparent sextet J= 7.4 Hz, 2H, H8a-b) ; 1.26 (hexane); 0.96 (t J= 7.4 Hz, 3H, H9); 0.86 (hexane).

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13

121.4 (C4 or C5); 120.3 (C4 or C5); 83.3 (C1’); 77.9 (C2’); 75.8 (C5’); 74.5 (C3’); 69.2 (C4’); 63.2 (C6’); 50.9 (C6); 36.1 (C10); 33.4 (C7);31.4 (hexane); 22.8 (hexane) 21.4 (OAc); 21.0 (OAc); 20.9 (2OAc); 19.8 (C8); 14.1 (hexane); 13.9 (C9).

IR (solid state): (cm-1) = 2962 (w-m); 2916 (w); 2870 (vw); 1744 (m, CO); 1567 (vw); 1469 (w); 1412(w); 1258 (s); 1220 (m); 1086 (s); 1016 (vs); 911 (w-m); 865 (w-m); 795 (vs); 734 (w-m); 680 (w-m). Anal. Calc. (%) for C22H33AuN2O9S: C 37.70, H 4.75, N 4.00; found: C 37.64, H 4.65, N 3.98.

Synthesis of (ter-butylethynyl)-1,3-bis-(2,6-diisopropylphenyl) imidazol-2-ylidene -gold(I) (4)

The compound was prepared according to previously reported procedures.46,47 The colourless solid was obtained with 80% yield.

1H NMR (CDCl3, -aromatic); 7.28 (d, J=7.4 Hz, 4H, CH-aromatic); 7.06 (s, 2H, CH-imidazole); 2.61 (sept, J= 6.9 Hz, 4H, CH(CH3)2 isopropyl); 1.35 (d, J= 6.8 Hz, 12H, CH(CH3)2 isopropyl); 1.18 (d, J= 6.8 Hz, 12H, CH(CH3)2 isopropyl); 1.10 (s, 9H, C(CH3)3 ter-butylethynyl).

13C NMR (CDCl3, CN imidazol); 145.7 (CH aromatic); 134.7 (C aromatic); 130.4 (CH aromatic); 124.2 (CH aromatic); 123.2 (CH imidazole); 114.05 (Au-CC); 112.51 (Au-CC); 32.6 (C(CH3)3 ter-butylethynyl); 28.9 (CH(CH3)2 isopropyl); 28.3 (C(CH3)3 ter-butylethynyl); 24.5 (CH(CH3)2 isopropyl); 24.3 (CH(CH3)2 isopropyl).

IR (solid state): ): (cm-1) = 3150 (w); 2966 (m/s); 2923 (m, sh); 2864 (w/m); 2116 (vw, CC); 1956 (vw); 1890 (vw); 1825 (vw); 1593 (w); 1720 (vw); 1677 (vw); 1650 (vw); 1558 (w); 1458 (s); 1410 (m); 1387 (w/m); 1364 (m); 1354 (w/m); 1343 (w/m); 1329 (w/m); 1275 (w); 1249 (m); 1208 (w/m); 1208 (w/m); 1181 (w/m); 1104 (w/m); 1082 (w/m); 1062 (w/m); 1032 (w); 981 (w); 944 (m); 805 (s); 764 (s); 731 (s); 697 (s); 561 (w). ESI-MS (DMSO-MeOH), positive mode exact mass for [C33H46N2Au]+ (667.3321): measured m/z 667.3322 [M]+. Anal. Calc. (%) for C33H45AuN2: C 59.45, H 6.80, N 4.20; found: C 59.39, H 6.58, N 4.18.

Synthesis of trans-dichlorido-bis(1-butyl-3-methyl-imidazole-2-yilidene)platinum(II) (5)

161 mg of K2PtCl4 (0,388 mmol) were suspendend in 15ml of CH2Cl2, afterwards 218 mg of Ag(BMIm)Cl (0,775 mmol) were added. The mixture was refluxed for 4 days and then filtered on celite. Solvent was removed under reduced pressure and the product was obtained with 86% yield.

1H NMR (CDCl3, 293K), syn and anti -4.46 (m, 8H, NCH2CH2CH2CH3); 4.11, 4.08 (s+ s, 12H, NCH3); 2.14-1.96 (m, 8H, NCH2CH2CH2CH3); 1.63 (H2O); 146-

AuN

N

S O

OO

O

O

O

O O

O

25

4

6

10

7

89

1'

7'

8'

9'10'

11' 12'

6'

5' 4'

3'2'

13'14'

194

1.43 (m, 8H, NCH2CH2CH2CH3); 1.00 (t J2 = 7.3Hz; 6H, NCH2CH2CH2CH3); 0.99 (t J2= 7.3 Hz, 6H, NCH2CH2CH2CH3)

13C NMR (CDCl3, 293K), syn and anti Pt-C= 23.3 Hz, CH imidazole); 120.1 (s+d, JPt-C= 24.5 Hz, CH imidazole); 50.0 (s+d, JPt-C= 18.9 Hz, NCH2CH2CH2CH3); 37.1 (NCH3); 37.0 (NCH3); 33.2 (NCH2CH2CH2CH3); 20.2 (NCH2CH2CH2CH3); 14.0 (NCH2CH2CH2CH3); 13.9 (NCH2CH2CH2CH3).

195Pt NMR (CDCl3, 293K), syn and anti -3177.8; -3179.0.

NMR STABILITY STUDIES IN AQUEOUS MEDIA AND REACTIVITY WITH HOMOCYSTEINE. 4·10-3 mmol of 3 were dissolved in 0.4 ml of DMSO-d6/H2O 60:40, while 6·10-3 mmol f 4 were dissolved in 0.4ml of DMSO-d6/H2O 80:20. 1H NMR spectra were registered at various time intervals (0h, 24h, 1 week). Afterwards, 5.0·10-3 mmol of 3 or 4.2·10-3 mmol of 4 were reacted with 1.5 equivalents of DL-homocysteine in 0.4 ml of CD3OD. Reactions were monitored through 1H NMR (0h, 6h, 24 h) and 13C NMR.

CELL LINES The human colorectal carcinoma HCT 116 p53 null and HCT 116 wt p53 variants (kindly provided by Dr. Götz Hartleben, University of Groningen), the human breast adenocarcinoma MCF-7 (Leibniz-Institut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), and human malignant melanoma A375 (kindly provided by Prof. Sylvestre Bonnet, Leiden University) were cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing glutamax supplemented with 10% FBS and 1% penicillin/streptomycin (all from Invitrogen), at 37 °C in an incubator (Thermo Fisher Scientific, US) with humidified atmosphere of 95% of air and 5% CO2.

IN VITRO CELL VIABILITY ASSAYS Cells in an exponential growth rate were seeded (8000 cells per well) in 96-wells plates (Costar 3595) grown for 24 h in complete medium. Solutions of the gold compounds were prepared by diluting a stock solution (10-2 M in DMSO, ethanol in the case of auranofin) in culture medium (DMSO or ethanol in the culture medium never exceeded 1%). Subsequently, different dilutions of the compounds were

-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the cells at a final concentration of 0.50 mg/ml in PBS (phosphate buffered saline solution, pH 7.4) and incubated for 2.5 h. The incubation medium was removed and the violet formazan crystals in the cells were dissolved in DMSO, and the optical density of each well was quantified at 550 nm, using a multi-well plate reader (ThermoMax microplate reader, Molecular devices, US). The percentage of surviving cells was calculated from the ratio of absorbance between treated and untreated cells. Each treatment was performed in quadruplate. The EC50 value was calculated as the concentration causing 50% decrease in cell number and is presented as a mean (± SD) of at least three independent experiments.

PREPARATION OF RAT PRECISION-CUT KIDNEY SLICES (PCKS) AND TOXICITY STUDIES EX VIVO Male Wistar rats (Charles River, France) of 250-300 g were housed under a 12 h dark/light cycle at constant humidity and temperature. Animals were permitted ad libitum access to tap water and standard lab chow. All experiments were approved by the committee for care and use of laboratory animals of the University of Groningen and were performed according to strict governmental and

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international guidelines. Kidneys were harvested (from rats anesthetized with isoflurane) and immediately placed in University of Wisconsin removing fat, kidneys were cut in half lengthwise using a scalpel, and cortex cores of 5 mm diameter were made from each half perpendicular to the cut surface using disposable Biopsy Punches (KAI medical, Japan). PCKS were made as described by de Graaf et al..37,38 The cores were sliced with a Krumdieck tissue slicer (Alabama R&D, Munford, AL, USA) in ice-cold Krebs-Henseleit buffer, pH 7.4 saturated with carbogen (95% O2 and 5% CO2

thickness), were incubated individually in 12-well plates (Greiner bio-one GmbH, Frickenhausen, Austria), at 37°C in Williams’ medium E (WME, Gibco by Life Technologies, UK) with glutamax-1, supplemented with 25 mM D-glucose (Gibco) and ciprofloxacin HCl (10 μg/mL, Sigma-Aldrich, Steinheim, Germany) in an incubator (Panasonic biomedical) in an atmosphere of 80% O2 and 5% CO2 with shaking (90 times/min). Stock solutions of compounds 1 to 4, auranofin and cisplatin were prepared as for the studies on cell lines. The final concentration of DMSO and ethanol during the PCKS incubation was always below 0.5% to exclude solvent toxicity. For each concentration, three slices were incubated individually for one hour in WME and subsequently, different dilutions of the

PCKS were incubated for 24 h. After the incubation, slices were collected for ATP and protein determination, by snap freezing them in 1 ml of ethanol (70% v/v) containing 2 mM EDTA with pH=10.9. After thawing the slices were homogenized using a mini bead beater and centrifuged. The supernatant was used for the ATP essay and the pellet was dissolved in 5N NaOH for the protein essay. The viability of PCKS was determined by measuring the ATP using the ATP Bioluminescence Assay kit CLS II (Roche, Mannheim, Germany) as described previously.37 The ATP content was corrected by the

rotein content of the PCKS was determined by the Bio-Rad DC Protein Assay (Bio-Rad, Munich, Germany) using bovine serum albumin (BSA, Sigma-Aldrich, Steinheim, Germany) for the calibration curve. The TC50 value was calculated as the concentration reducing the viability of the slices by 50%, in terms of ATP content corrected by the protein amount of each slice and relative to the slices without any treatment, and is presented as a mean (± SD) of at least three independent experiments.

HISTOMORPHOLOGY Kidney

sections were made, which were mounted on glass slides and PAS staining was used for histopathological evaluation. Briefly, the glass slides were deparaffinised, washed with distilled water, followed by treatment with a 1% aqueous solution of periodic acid for 20 minutes and Schiff reagent for 20 minutes, the slides were rinsed with tap water, finally, a counterstain with Mayer’s haematoxylin (5 min) was used to visualize the nuclei.

STATISTICS A minimum of three independent experiments were performed with the cells, with 4 replicas for each condition. PCKS were prepared from 3 rats and in each experiment slices were exposed in triplicate. Statistical testing was performed with one way ANOVA with each individual experiment as random effect. We performed a Tukey HSD post-hoc test for pairwise comparisons. In all graphs and tables the mean values and standard deviation (SD) are shown.

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(16) Patel, M. N.; Bhatt, B. S.; Dosi, P. A. DNA Binding, Cytotoxicity and DNA Cleavage Promoted by Gold(III) Complexes. Inorg. Chem. Commun. 2013, 29, 190–193.

(17) Ott, I. On the Medicinal Chemistry of Gold Complexes as Anticancer Drugs. Coord. Chem. Rev. 2009, 253 (11–12), 1670–1681.

(18) Berners-Price, S. J.; Filipovska, A. Gold Compounds as Therapeutic Agents for Human Diseases. Met. Integr. Biometal Sci. 2011, 3 (9), 863–873.

(19) Oehninger, L.; Rubbiani, R.; Ott, I. N-Heterocyclic Carbene Metal Complexes in Medicinal Chemistry. Dalton Trans. 2013, 42 (10), 3269–3284.

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(20) Liu, W.; Gust, R. Update on Metal N-Heterocyclic Carbene Complexes as Potential Anti-Tumor Metallodrugs. Coord. Chem. Rev. 2016, 329, 191–213.

(21) Cinellu, M. A.; Ott, I.; Casini, A. Gold Organometallics with Biological Properties. In Bioorganometallic Chemistry; Jaouen, G., Salmain, M., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA, 2014; pp 117–140.

(22) Barnard, P. J.; Baker, M. V.; Berners-Price, S. J.; Day, D. A. Mitochondrial Permeability Transition Induced by Dinuclear Gold(I)–carbene Complexes: Potential New Antimitochondrial Antitumour Agents. J. Inorg. Biochem. 2004, 98 (10), 1642–1647.

(23) Barnard, P. J.; Berners-Price, S. J. Targeting the Mitochondrial Cell Death Pathway with Gold Compounds. Coord. Chem. Rev. 2007, 251 (13–14), 1889–1902.

(24) Hickey, J. L.; Ruhayel, R. A.; Barnard, P. J.; Baker, M. V.; Berners-Price, S. J.; Filipovska, A. Mitochondria-Targeted Chemotherapeutics: The Rational Design of Gold(I) N-Heterocyclic Carbene Complexes That Are Selectively Toxic to Cancer Cells and Target Protein Selenols in Preference to Thiols. J. Am. Chem. Soc. 2008, 130 (38), 12570–12571.

(25) Sundelacruz, S.; Levin, M.; Kaplan, D. L. Role of Membrane Potential in the Regulation of Cell Proliferation and Differentiation. Stem Cell Rev. 2009, 5 (3), 231–246.

(26) Bindoli, A.; Rigobello, M. P.; Scutari, G.; Gabbiani, C.; Casini, A.; Messori, L. Thioredoxin Reductase: A Target for Gold Compounds Acting as Potential Anticancer Drugs. Coord. Chem. Rev. 2009, 253 (11–12), 1692–1707.

(27) Rubbiani, R.; Kitanovic, I.; Alborzinia, H.; Can, S.; Kitanovic, A.; Onambele, L. A.; Stefanopoulou, M.; Geldmacher, Y.; Sheldrick, W. S.; Wolber, G.; Prokop, A.; Wölfl, S.; Ott, I. Benzimidazol-2-Ylidene Gold(I) Complexes Are Thioredoxin Reductase Inhibitors with

Multiple Antitumor Properties. J. Med. Chem. 2010, 53 (24), 8608–8618.

(28) Citta, A.; Schuh, E.; Mohr, F.; Folda, A.; Massimino, M. L.; Bindoli, A.; Casini, A.; Rigobello, M. P. Fluorescent Silver(I) and Gold(I)-N-Heterocyclic Carbene Complexes with Cytotoxic Properties: Mechanistic Insights. Metallomics 2013, 5 (8), 1006–1015.

(29) Krishnamurthy, D.; Karver, M. R.; Fiorillo, E.; Orrú, V.; Stanford, S. M.; Bottini, N.; Barrios, A. M. Gold(I)-Mediated Inhibition of Protein Tyrosine Phosphatases: A Detailed in Vitro and Cellular Study. J. Med. Chem. 2008, 51 (15), 4790–4795.

(30) Stefan, L.; Bertrand, B.; Richard, P.; Le Gendre, P.; Denat, F.; Picquet, M.; Monchaud, D. Assessing the Differential Affinity of Small Molecules for Noncanonical DNA Structures. ChemBioChem 2012, 13 (13), 1905–1912.

(31) Bertrand, B.; Stefan, L.; Pirrotta, M.; Monchaud, D.; Bodio, E.; Richard, P.; Le Gendre, P.; Warmerdam, E.; de Jager, M. H.; Groothuis, G. M. M.; Picquet, M.; Casini, A. Caffeine-Based Gold(I) N-Heterocyclic Carbenes as Possible Anticancer Agents: Synthesis and Biological Properties. Inorg. Chem. 2014, 53 (4), 2296–2303.

(32) Bazzicalupi, C.; Ferraroni, M.; Papi, F.; Massai, L.; Bertrand, B.; Messori, L.; Gratteri, P.; Casini, A. Determinants for Tight and Selective Binding of a Medicinal Dicarbene Gold(I) Complex to a Telomeric DNA G-Quadruplex: A Joint ESI MS and XRD Investigation. Angew. Chem. Int. Ed Engl. 2016, 55 (13), 4256–4259.

(33) Baker, M. V.; Barnard, P. J.; Berners-Price, S. J.; Brayshaw, S. K.; Hickey, J. L.; Skelton, B. W.; White, A. H. Cationic, Linear Au(I) N-Heterocyclic Carbene Complexes: Synthesis, Structure and Anti-Mitochondrial Activity. Dalton Trans. 2006, No. 30, 3708–3715.

(34) Messori, L.; Marchetti, L.; Massai, L.; Scaletti, F.; Guerri, A.; Landini, I.; Nobili, S.; Perrone, G.; Mini, E.; Leoni,

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P.; Pasquali, M.; Gabbiani, C. Chemistry and Biology of Two Novel Gold(I) Carbene Complexes as Prospective Anticancer Agents. Inorg. Chem. 2014, 53 (5), 2396–2403.

(35) Meyer, A.; Bagowski, C. P.; Kokoschka, M.; Stefanopoulou, M.; Alborzinia, H.; Can, S.; Vlecken, D. H.; Sheldrick, W. S.; Wölfl, S.; Ott, I. On the Biological Properties of Alkynyl Phosphine Gold(I) Complexes. Angew. Chem. Int. Ed. 2012, 51 (35), 8895–8899.

(36) Andermark, V.; Göke, K.; Kokoschka, M.; Abu el Maaty, M. A.; Lum, C. T.; Zou, T.; Sun, R. W.-Y.; Aguiló, E.; Oehninger, L.; Rodríguez, L.; Bunjes, H.; Wölfl, S.; Che, C.-M.; Ott, I. Alkynyl Gold(I) Phosphane Complexes: Evaluation of Structure–activity-Relationships for the Phosphane Ligands, Effects on Key Signaling Proteins and Preliminary in-Vivo Studies with a Nanoformulated Complex. J. Inorg. Biochem. 2016, 160, 140–148.

(37) de Graaf, I. A. M.; Olinga, P.; de Jager, M. H.; Merema, M. T.; de Kanter, R.; van de Kerkhof, E. G.; Groothuis, G. M. M. Preparation and Incubation of Precision-Cut Liver and Intestinal Slices for Application in Drug Metabolism and Toxicity Studies. Nat. Protoc. 2010, 5 (9), 1540–1551.

(38) de Graaf, I. A. M.; Groothuis, G. M.; Olinga, P. Precision-Cut Tissue Slices as a Tool to Predict Metabolism of Novel Drugs. Expert Opin. Drug Metab. Toxicol. 2007, 3 (6), 879–898.

(39) Bertrand, B.; Citta, A.; Franken, I. L.; Picquet, M.; Folda, A.; Scalcon, V.; Rigobello, M. P.; Le Gendre, P.; Casini, A.; Bodio, E. Gold(I) NHC-Based Homo- and Heterobimetallic Complexes: Synthesis, Characterization and Evaluation as Potential Anticancer Agents. J. Biol. Inorg. Chem. JBIC Publ. Soc. Biol. Inorg. Chem. 2015, 20 (6), 1005–1020.

(40) Muenzner, J. K.; Rehm, T.; Biersack, B.; Casini, A.; de Graaf, I. A. M.; Worawutputtapong, P.; Noor, A.; Kempe, R.; Brabec, V.; Kasparkova, J.;

Schobert, R. Adjusting the DNA Interaction and Anticancer Activity of Pt(II) N-Heterocyclic Carbene Complexes by Steric Shielding of the Trans Leaving Group. J. Med. Chem. 2015, 58 (15), 6283–6292.

(41) Daum, S.; Chekhun, V. F.; Todor, I. N.; Lukianova, N. Y.; Shvets, Y. V.; Sellner, L.; Putzker, K.; Lewis, J.; Zenz, T.; de Graaf, I. A. M.; Groothuis, G. M. M.; Casini, A.; Zozulia, O.; Hampel, F.; Mokhir, A. Improved Synthesis of N-Benzylaminoferrocene-Based Prodrugs and Evaluation of Their Toxicity and Antileukemic Activity. J. Med. Chem. 2015, 58 (4), 2015–2024.

(42) Rajaratnam, R.; Martin, E. K.; Dörr, M.; Harms, K.; Casini, A.; Meggers, E. Correlation between the Stereochemistry and Bioactivity in Octahedral Rhodium Prolinato Complexes. Inorg. Chem. 2015, 54 (16), 8111–8120.

(43) Schmidt, A.; Molano, V.; Hollering, M.; Pöthig, A.; Casini, A.; Kühn, F. E. Evaluation of New Palladium Cages as Potential Delivery Systems for the Anticancer Drug Cisplatin. Chem. Weinh. Bergstr. Ger. 2016, 22 (7), 2253–2256.

(44) Baker, M. V.; Barnard, P. J.; Brayshaw, S. K.; Hickey, J. L.; Skelton, B. W.; White, A. H. Synthetic, Structural and Spectroscopic Studies of (Pseudo)Halo(1,3-Di-Tert-Butylimidazol-2-Ylidine)Gold Complexes. Dalton Trans. 2005, No. 1, 37–43.

(45) Baker, M. V.; Barnard, P. J.; Berners-Price, S. J.; Brayshaw, S. K.; Hickey, J. L.; Skelton, B. W.; White, A. H. Synthesis and Structural Characterisation of Linear Au(I) N-Heterocyclic Carbene Complexes: New Analogues of the Au(I) Phosphine Drug Auranofin. J. Organomet. Chem. 2005, 690 (24–25), 5625–5635.

(46) Gao, L.; Partyka, D. V.; Updegraff, J. B.; Deligonul, N.; Gray, T. G. Synthesis, Structures, and Excited-State Geometries of Alkynylgold(I)

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Complexes. Eur. J. Inorg. Chem. 2009, 2009 (18), 2711–2719.

(47) Laitar, D. S.; Müller, P.; Gray, T. G.; Sadighi, J. P. A Carbene-Stabilized

Organometallics 2005, 24 (19), 4503–4505.

(48) Newman, C. P.; Deeth, R. J.; Clarkson, G. J.; Rourke, J. P. Synthesis of Mixed NHC/L PlRestricted Rotation of the NHC Group. Organometallics 2007, 26 (25), 6225–6233.

(49) Shibata, T.; Ito, S.; Doe, M.; Tanaka, R.; Hashimoto, H.; Kinoshita, I.; Yano, S.; Nishioka, T. Dynamic Behaviour Attributed to Chiral Carbohydrate Substituents of N-Heterocyclic Carbene Ligands in Square Planar Nickel Complexes. Dalton Trans. 2011, 40 (25), 6778–6784.

(50) Bernhammer, J. C.; Huynh, H. V. Benzimidazolin-2-Ylidene Complexes of Palladium(II) Featuring a Thioether Moiety: Synthesis, Characterization, Molecular Dynamics, and Catalytic Activities. Organometallics 2014, 33 (5), 1266–1275.

(51) Yu, K.-H.; Wang, C.-C.; Chang, I.-H.; Liu, Y.-H.; Wang, Y.; Elsevier, C. J.; Liu, S.-T.; Chen, J.-T. Coordination Chemistry of Highly Hemilabile Bidentate Sulfoxide N-Heterocyclic Carbenes with Palladium(II). Chem. – Asian J. 2014, 9 (12), 3498–3510.

(52) Rehm, T.; Rothemund, M.; Muenzner, J. K.; Noor, A.; Kempe, R.; Schobert, R. Novel Cis-[(NHC)1(NHC)2(L)Cl]Platinum(II) Complexes – Synthesis, Structures, and Anticancer Activities. Dalton Trans. 2016, 45 (39), 15390–15398.

(53) Ahrens, S.; Herdtweck, E.; Goutal, S.; Strassner, T. Synthesis, Structure and Stability of New PtII–Bis(N-Heterocyclic Carbene) Complexes. Eur. J. Inorg. Chem. 2006, 2006 (6), 1268–1274.

(54) Zamble, D. B.; Jacks, T.; Lippard, S. J. P53-Dependent and -Independent Responses to Cisplatin in Mouse Testicular Teratocarcinoma Cells. Proc.

Natl. Acad. Sci. 1998, 95 (11), 6163–6168.

(55) di Pietro, A.; Koster, R.; Boersma-van Eck, W.; Dam, W. A.; Mulder, N. H.; Gietema, J. A.; de Vries, E. G. E.; de Jong, S. Pro- and Anti-Apoptotic Effects of P53 in Cisplatin-Treated Human Testicular Cancer Are Cell Context-Dependent. Cell Cycle 2012, 11 (24), 4552–4562.

(56) Hall, M. D.; Okabe, M.; Shen, D.-W.; Liang, X.-J.; Gottesman, M. M. The Role of Cellular Accumulation in Determining Sensitivity to Platinum-Based Chemotherapy. Annu. Rev. Pharmacol. Toxicol. 2008, 48, 495–535.

(57) Vickers, A. E. M.; Rose, K.; Fisher, R.; Saulnier, M.; Sahota, P.; Bentley, P. Kidney Slices of Human and Rat to Characterize Cisplatin-Induced Injury on Cellular Pathways and Morphology. Toxicol. Pathol. 2004, 32 (5), 577–590.

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SUPPORTING INFORMATION

1. ANALYTICAL DATA OF COMPOUNDS 3, 4 AND 5

Compound 3: thio- -D-glucose-tetraacetate-(1-butyl-3-methyl)-imidazol-2-ylidene-gold(I)

AuN

N

S O

OO

O

O

O

O O

O

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Figure S2. 13C NMR of compound 3 in CDCl3.

Figure S3. FT-IR of compound 3 in the solid state.

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Compound 4: (ter-butylethynyl)-1,3-bis-(2,6-diisopropylphenyl) imidazol-2-ylidene -gold(I)

AuN

N

Figure S4. 1H NMR of compound 4 in CDCl3.

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Figure S5. 13C NMR of compound 4 in CDCl3.

Figure S6. FT-IR spectrum of compound 4 in the solid state.

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Compound 5: trans-dichlorido-bis(1-butyl-3-methyl-imidazole-2-yilidene)platinum(II)

Pt N

N

N

N

Cl

Cl

Figure S7. 1H NMR of compound 5 in CDCl3.

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Figure S8. 195Pt NMR of compound 5 in CDCl3.

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2. NMR STABILITY STUDIES IN AQUEOUS MEDIA AND REACTIVITY WITH

HOMOCYSTEINE.

Figure S10. 1H NMR spectra of 3 in 60% DMSO d6 40% H2O.

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Figure S11.1H NMR spectra of 4 in 80% DMSO d6 20% H2O.

208

Figure S12. 1H NMR spectra of DL homocysteine in CD3OD.

Figure S13. 1H NMR spectrum of 3 in CD3OD.

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Figure S14. 1H NMR spectra of the reaction between 3 and homocysteine (1: 1.6) after 0, 6, 24 h.

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Figure S15. 1H NMR spectra of the reaction between 4 and homocysteine (1: 1.3) after 0, 6, 24 h.

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Figure S16. Comparison of 1H NMR spectra in CD3OD of 4 in presence of free 3,3-dimethylbutyne and of the reaction between 4 and homocysteine (t=10 min).

H2O

212

Figure S17. 1H NMR spectra of 4 + 3,3 dimethylbutyne in CD3OD.

Figure S18. 1H NMR spectra of 4 in CD3OD.

H2O

H2O

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Figure S19. 1H NMR spectra in CD3OD of the reaction between 4 and homocysteine after 24 h.

Figure S20. 13C NMR spectra of the reaction between 4 and homocysteine after 24 h.

AuN

N

S

NH2

COOH

H2O

CHAPTER 9

SUMMARY AND DISCUSSION

(NEDERLANDSE SAMENVATTING)

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SUMMARY AND DISCUSSION

The research described in this these was aimed to discover and evaluate new anticancer drugs. Two different approaches were undertaken. In the first part, the design and evaluation of new p53-MDM2/X inhibitors based on previously described compounds are presented (Part A), in an attempt to increase their potency and explore different regions on the p53-MDM2/X interphase. In the second part, another class of compounds, metal complexes (Part B), were synthetized aiming to interact with several protein targets and act as anticancer agents. Several series of metal complexes were studied to determine their potential cytotoxic activity against cancer cell lines and to unravel their possible mechanism of action compared to known metal containing drugs such as cisplatin and auranofin. Furthermore, evaluation of their toxicity was performed in healthy tissue using rat Precision Cut Tissue Slices (PCTS) to unravel uptake, pathways involved in the toxicity and possible selectivity of the compounds towards cancer cells.

PART A In this section, we described the design of novel p53/MDM2 inhibitors. The discovery of p53/MDM2 inhibitors is still a hot topic in cancer research.1–3 This targeted strategy aims to be selective for cancer cells with overexpression of MDM2. Since the disclosure of Nutlin-3,4 many scaffolds have been synthesized and evaluated, including indo-imidazole, imidazoline, benzodiazepinedione, spirooxindole, among others.5,6 Several compounds presently undergo early clinical evaluations.7–10 However, the discovery of new p53/MDM2/MDMX scaffolds is still of high interest due to low single agent activity currently seen in clinical trials and insufficient PKPD properties.

Chapter 3 presented in a systematic review the current state of the art of different classes of inhibitors of p53/MDM2 and their associated co-crystal structures, with a special focus on the binding mode of the compounds including the importance of the highly conserved water molecules have for the design of new inhibitors.

An important discussion point is the lack of dual action compounds targeting MDM2 and MDMX to assure fully restored p53. So far the few compounds that address the duality are not as potent as inhibitors of MDMX.10 However, peptidic inhibitors possess dual inhibitory activity towards MDM2 and MDMX. Recently, several stapled peptides have been described6,10 with great affinity towards MDM2 and MDMX. ALRN-6924 (Aileron Therapeutics) is currently undergoing phase I and II clinical trials in patients suffering of solid tumors, lymphoma and myeloid leukemias.11

Important common features present in p53/MDM2 inhibitors are:

- 3-point pharmacophore model formed by the side chains of p53 Phe19-Trp23-Leu26, which competitive inhibitors have to mimic.

- MDM2 binders have a T-shaped topology where the ends of the T comprise the three moieties addressing the pharmacophore points.

- The central scaffold is mostly a heterocyclic ring, annulated rings or in some cases acyclic linear.

- P53Trp23 contributes most to the interaction energy in the binding of P53 and MDM2 and thus must be closely mimicked by inhibitors in terms of hydrophobicity and shape (e.g. phenyl

218

groups, indoles, oxindoles or 2-oxo benzimidazoles). A considerable increase in binding affinity can be reached by the suitable introduction of a hydrophobic halogen at the bottom of the Trp23 binding pocket.

- His96, hydrogen bonding to Ser17, halogen bonding or hydrogen bonding to backbone or sidechain Glu72, respectively, and dipolar interaction with His73. In this regard, the presence of crystal water molecules is of importance which act as potential stabilization points though hydrogen bonding In chapter 3, we discovered that in general the MDM2 crystal structures show a high degree of crystallographic water presence and that a water on top of the indole ring of p53Trp23 seems to be highly conserved in many structures. Later studies led to unravel 2 new positions where conserved waters are present (data not published). The water molecule on top of the indole ring of p53Trp23 was found to be present in about 60% of 66 evaluated co-crystal structures. Additionally, waters close to MDM2Lys55, and in the area covered by MDM2Val93 and MDM2His96 seems to be common in 45 and 41% of the co-crystals, respectively (figure 1). These findings show potential spots to consider as target for the design of MDM2 inhibitors because it could be important to include possible hydrogen bond formation with the mentioned waters as a strategy to stabilize the binding of the inhibitors and increase the potency of new series of compounds targeting p53/MDM2 interactions.

Figure 1. Regions of conserved crystallographic waters in ligand-MDM2 structures. Cyan: water clusters; light pink: MDM2 residues interacting with the water clusters and hot pink: p53 key amino acids Phe19, Trp23 and Leu26.

Chapter 4 focusses on the development of artificial macrocycles as potent p53-MDM2 inhibitors, aiming to target the hydrophobic area formed by Tyr67, Gln72, His73 Val93 and Lys94, not widely explored with different classes of inhibitors. By the introduction of this hydrophobic handle, we expected to observe an increment of the affinity to the receptor as well as improvement in cellular permeation. The novelty of this series of compounds besides the different interaction area, is the method for synthesis with the use of Ugi multicomponent reaction followed by a ring closing metathesis (RCM) as an alternative to access diverse ring sizes and conformations. This approach furnished a series of compounds with affinity to the target in the low μM range (figure 2). One

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example of macrocycle was purified to obtain its enantiomers, resulting in a difference in affinity of 7-fold compared to the racemic compound.

Figure 2. Macrocycles with higher affinity towards MDM2/X.

Furthermore, chapter 5 describes the design and synthesis of 2,3'-Bis(1'H-indole) heterocycles, which offered an easy and effective way to prepare new p53/MDM2/MDMX inhibitors. This series of derivatives possess a phenyl annulated ring that makes additional hydrophobic interactions with the mdm2Val93, mimicking p53Leu22 and helping to stabilize the binding and increase the potency of previously described indole derivatives.12 The best derivative from this family achieved a Ki of 1.8 and 0.2 μM for MDM2 and MDMX, respectively (Figure 3). The obtained results open new synthetic opportunities to develop derivatives offering three points of diversity along with linkers of different lengths that can help to establish a structure activity relationship and producing more compounds with higher affinity and more drug-like properties.

Figure 3. 2,3'-Bis(1'H-indole) derivative with the higher affinity towards MDM2

A few families of MDM2 inhibitors13–15 (figure 4) were screened in cell based assays (data not published), leading to moderate cytotoxic activity (IC50 40-100 μM) assessed with a small panel of cancer cell lines. Furthermore, we encountered solubility problems of the macrocyclic derivatives in aqueous media. Therefore, more investigation is ongoing to optimize the chemical synthesis strategy to obtain compounds with better solubility, PKPD properties, more diversity and hopefully higher affinity to the receptor. Our group is currently working in two different synthetic approaches to develop of macrocycles (Figure 5):

NNH

OO

Cl

NHCl

CO2HNH

NO

O

NHCl

CO2H

F

FF

Cl

NNH

OO

O

NHCl

CO2HClCl

1. (d.r 1:1)Ki= 0.098μM

2Ki= 0.082μM

3Ki Rac= 0.139μMKi A= 0.093μMKi B= 0.7μM

NHCl

N

O

OH

ClF

4Ki MDM2= 1.8μMKi MDMX= 0.2μM

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1. Assembly of macrocycles, using as linkers isocyano carboxylic acids and the subsequent ring closure by Ugi reaction with a primary amine and oxo component.16 To obtain p53/MDM2 inhibitors the oxo component should be our anchor indole used in the synthesis of macrocycles and 2,3'-bis(1'H-indole) heterocycles described in this thesis.

2. Using an artificial amino acid linker formed as a reaction between diamines and anhydrides, which undergo Ugi cyclization reaction with oxo component (anchor indole) and isocyanide.

Figure 4. Different scaffolds screened in cell based assays. 5. Tetrazoles14; 6 -lactams13; 7. Macrocycles (chapter 4); 8. 2,3'-Bis(1'H-indole) Heterocycles 15 (chapter5)

Figure 5. Examples of different synthetic routes to access artificial macrocycles as p53/MDM2 inhibitors

Due to the importance of p53-MDM2/X protein-protein interaction, several research groups and companies have been working on the development of effective inhibitors. Recently, new compounds entered clinical trials, however the preliminary results showed limited PKPD properties and severe side effects in some cases leading to gastrointestinal and hematological toxicity.17,18 Therefore there is still room for the development of more active and selective compounds, with improved metabolic profile and dual targeting function of MDM2 and MDMX.

PART B In this section, the study of the toxicity of different families of gold compounds in cancer cells (in vitro) and in healthy tissues (ex vivo) is described. Metals and metal containing compounds have been used for therapeutic purposes since 5000 years.19 Nowadays, the medical applications of metal-based compounds are well known in various areas, including as anticancer agents, as diagnostics and for diabetes treatment, among others. Currently platinum(II) complexes are widely used in the clinic and

NN

NNHNR1

R2

NH

CO2HCl

NNH

OO

R1X

n

m

NH

CO2HClN

HCl

NO

X

O

NH

R

CO2H

5 6 7

NX

R2

NH

CO2HCl

R1

9

Cl

H2N

NH

CO2Et

CHO

Cl

HONC

O

N HN

O

OCl

NH

CO2EtCl

BHN

CO2Et

CHO NH

CO2Et

NH

NC

HN

SO

OH

OH2N NH

N S

O

O

Cl

Cl

O

A N HN

O

OCl

NH

CO2HCl

NH

CO2H

NH

NH

N S

O

O

Cl

O

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9

are in the list of essential medicines from the world health organization.20 However, the leading compound cisplatin is highly toxic displaying a large range of side effects, most prominently nephrotoxicity and ototoxicity.21,22 Therefore, different chemical strategies have been developed over the last years, to achieve new alternatives and elucidate new potential targets for improved anticancer therapeutics. In this context, gold-based complexes are interesting due to their different redox chemistry (Au(I) and Au(III) as possible oxidation states) and ligand exchange reactions, which offer different mechanisms of activity compared to cisplatin.23–25

In chapter 6 the toxicity of a series of bifunctional Au(I)-based compounds was studied using precision cut liver and kidney slices26,27 to understand tissue specific toxicity and elucidate a possible mechanism of action. The described derivatives were designed to act as chimeric compounds combining the cytotoxicity of gold ions and the proton pump inhibition properties of lansoprazole as ligand. In a previous study from Casini and coworkers, the cytotoxic activity of these compounds was evaluated in a small panel of cancer cells, including cell lines sensitive and resistant to cisplatin, and a non-cancerous cell line.28 These series of compounds showed to be more cytotoxic to the cancer cell lines (ca. 5 fold) than in the non-cancerous cell line, suggesting potential selective pharmacological effects.28

In our study, we selected representative compounds (Figure 6) of this series and assessed them for their effects on PCTS ex vivo after 24 h incubation in comparison to lansoprazole. The complexes displayed toxicity in the μM range in kidney and liver slices, with complex 11 as the least toxic derivative in healthy tissue with a TC50 values of ca. 27 and 25 μM in kidney and liver respectively, and a TC50 PCKS/IC50 ratio of 1.8 and 1.7 for kidney and liver slices, respectively (where the IC50 for A2780 and A2780R cells was taken from Serratice et al.28) indicating lack of cancer cell specificity. However, for compound 10 the TC50 PCKS/IC50 cells ratio was 20 for kidney slices and 8.9 for liver slices, indicating possible selective toxicity towards cancer cells compared to healthy tissue. Furthermore, a slightly higher toxicity is observed in the liver slices compared to kidney slices for compounds that bear a phosphine ligand, 10 and 12, as demonstrated by both the ATP and histomorphology data. Additionally, after analysing the results of RNA expression profiles of pathways activated under hypoxia (HIF1a),29 oxidative stress (Nrf2)30 and DNA damage (p53)31 we observed that the clearest impact was on the Nrf2 pathway, indicating oxidative stress as a possible mechanism of toxicity. This is in line with the known redox chemistry of Au(I) complexes which often induce intracellular redox damage. More studies are necessary to confirm oxidative stress as the toxicity mechanism, including inhibition studies of enzymes involved in the maintenance of the intracellular redox homeostasis, such as those involved in the thioredoxin and glutathione systems.

Figure 6. Lansoprazole derivatives evaluated in this study.

N

OCH2CF3

S

N

HN

10

Au

Ph3P

[BF4]

ON

OCH2CF3

S

N

N

11

Au

O

PN

N N

N

OCH2CF3

S

N

N

12

AuPh3P

[BF4]

OAu

PPh3

222

As previously mentioned, cytotoxic chemotherapeutic metallodrugs, such as cisplatin, show adverse drug reactions including nephrotoxicity.32 Unfortunately, there is still lack of knowledge about its mechanism of accumulation in cells and tissues. Several studies have been carried out to elucidate the mechanisms of uptake and efflux of cisplatin on different cancer cell lines and various in vitro assays with kidney cells or isolated tubules were used. Nonetheless, those mechanisms are not yet fully understood.33 Thus, in chapter 7, we studied the toxicity and accumulation mechanisms of cisplatin using the PCKS technique in comparison to a previously reported cytotoxic cyclometallated Au(III) complex (13, Figure 7) with improved stability in aqueous environment compared with similar complexes. 34 We evaluated the involvement of OCT and MATE transporters in cisplatin and Au(III) complex to compare the uptake and accumulation in rat kidney slices in absence and presence of the “unselective” OCTs and MATEs inhibitor cimetidine and its effect on cisplatin and Au(III)complex 13 toxicity in healthy rat PCKS. The viability of the PCKS after treatment was determined using ATP concentration and histomorphology analysis. Moreover, accumulation of platinum and gold in PCKS was assessed using ICP-MS. As expected, a concentration dependent decrease in viability of PCKS, which correlated with the increase in slice content of platinum or gold, was observed after exposure to the metal complexes. However, no effect of cimetidine on the toxicity or accumulation of both compounds was found. Based on these results we conclude that OCTs and MATEs do not play a prominent role in cisplatin or Au(III) complex uptake and accumulation. Further studies to explore the role of different transporters are needed. Noteworthy, all pre-clinical in vivo/ex vivo techniques, including this PCTS model, bear the same problem of interspecies differences, rat kidneys express OCT1, OCT2 and MATE1, whereas human kidneys express additionally MATE2-K and OCT 2 but no OCT1.35 Several attempts to use mRNA expression of KIM1 and villin as cell specific biomarkers to assess kidney toxicity were as yet unsuccessful. The use of PCTS for uptake and accumulation studies of metallodrugs offers good opportunities for future application on human tissue to obtain human-specific data. However, the use of more specific inhibitors, other than cimetidine, should be preferable to exclude overlapping inhibitory effects of different transporters.

Figure 7. Au complexes evaluated in chapter 7 and 8.

Chapter 8 focusses on the synthesis and evaluation of the biological activity of Au(I)-N-heterocyclic carbene complexes as anticancer agents in vitro and ex vivo using PCKS. The antiproliferative effects of the Au(I) complexes were tested in four cancer cell lines, namely a p53 wild-type and a p53 null variant of HCT 116 (colorectal carcinoma), MCF-7 (breast adenocarcinoma) and A375 (malignant melanoma). The selection of the p53 wild type and null variants cell lines was based on the known mechanism of cisplatin toxicity of induction of rapid p53-dependent apoptosis and, as a secondary effect, p53-independent cell cycle arrest.36,37 The MCF-7 cell line was chosen because of its resistance to cisplatin treatment compared to other breast cancer cell lines.38 Thus, these cell lines offered the opportunity

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to compare the toxicity mechanism of cisplatin with the Au(I) complexes and the involvement of p53. The last cell line used in our study was chosen due to the scarce information available on metal complexes studied on melanoma derived cell lines. Our results suggest that the toxicity mechanism of the Au(I) complexes is independent of p53 activity and they exert higher toxicity in the MCF-7 cell line. Most of the compounds and auranofin in three of the four cell lines, A375 being the exception. However, compound 14 (Figure 7) was markedly more active in HCT116 p53 null and MCF-7 cell lines or as active as cisplatin in HCT116 p53 wild-type cell line but much less toxic in the A375 cell line.

Due to their potent cytotoxic effects in cancer cells, Au(I) complexes were tested for their possible toxicity using PCKS. Most of the derivatives displayed high toxicity on the PCKS, with TC50s in the low μM range. Remarkably, complex 14 showed no toxicity up to 50 μM as evaluated by ATP content and histomorphology. The selectivity of complex 14 for cancer cells compared to healthy tissues is still promising and prompt us to develop new derivatives of this compound with enhanced anticancer properties and selectivity against cancer cells.

In addition to the work presented in the previous chapters of this thesis, we reported for the first time Ru(II) complexes conjugated to cyc(RGDfK) peptide39 as a way to target the specifically integrin

.40 The binding affinities of the bioconjugates were in the nM range and showed high selectivity to

compared to However, the cytotoxicity of all the reported bioconjugates was low in cancer cells with different integrin receptors expression levels (SKOV-3 and A549).41 Further studies are needed to develop targeted metallodrugs with intrinsically higher cytotoxic potency.

FINAL REMARKS Overall, in this thesis two different approaches were explored to design potential anticancer drugs, targeting a specific protein-protein interaction with high oncological relevance and the design of metal complexes with a broad spectrum of activities. In both cases, there are still open questions regarding the compound specificity, stability, transport, metabolic transformations and capability of reaching the specific cellular target.

Further studies are needed to give answer to such questions, including more extensive cell based studies to test the efficacy of the compounds as anticancer agents, and the use of different models to get more information regarding the specific mechanisms of action. The use of PCTS could be one of the most suitable options to evaluate different parameters to understand the mechanisms of action to a larger extent. Additionally, using this technique would allow us to have information regarding the toxicity in healthy tissue, useful to improve the design of the compounds to decrease the possibility of unfavourable side effects before administration in vivo. In addition, the application of the PCTS model favours the reduction and refinement of the use of experimental animals, due to the large variety of tests and experiments that are possible to perform in just one animal instead of sacrificing a large number of them. Furthermore, with this technique, it is possible to obtain human specific data, when PCTS are prepared from human organ tissue. This will provide translational data and will result in a better safety of the drugs that are tested in phase 1 clinical studies.

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NEDERLANDSE SAMENVATTING

Het onderzoek beschreven in dit proefschrift is gericht op het ontdekken van nieuwe anti-kanker geneesmiddelen. Twee verschillende benaderingen zijn hierbij toegepast. In het eerste deel wordt de ontwikkeling en evaluatie beschreven van nieuwe p53-MDM2/X remmers, op basis van eerder ontdekte remmers (Deel A), met als doel meer potente stoffen te ontwikkelen en het effect van binding aan de verschillende regio’s in de p53-MDM2/X interface beter te kunnen begrijpen. In het tweede deel wordt de synthese van een andere klasse van verbindingen, metaalcomplexen (Deel B), beschreven, die zijn gericht op interactie met verschillende eiwit targets, met als doel om als anti-kanker geneesmiddel te fungeren. Enkele reeksen van metaalcomplexen werden onderzocht om hun potentiële cytotoxiciteit voor kankercellijnen vast te stellen en om het mogelijke werkingsmechanisme te ontrafelen in vergelijking met bekende metaal-bevattende medicijnen, zoals cisplatina en auranofine. Bovendien werd de toxiciteit van deze metaalcomplexen onderzocht in gezond weefsel, gebruik makend van ‘Precision Cut Tissue Slices’ (PCTS) om de opname, de mechanismen betrokken bij de toxiciteit en de mogelijke selectiviteit van de verbindingen voor kanker cellen te bestuderen.

DEEL A In dit deel beschrijven we de ontwikkeling van nieuwe p53-MDM2 en p53-MDMX remmers als geneesmiddel tegen kanker. De ontdekking van p53-MDM2/X interactie is nog steeds een hot topic in het kanker onderzoek.1–3 Remmers van deze interactie veroorzaken het vrijkomen van p53 en bevorderen zo de apoptose in kankercellen. Het is de bedoeling dat deze nieuwe remmers selectief zijn voor kankercellen die MDM2/X tot overexpressie brengen. Sinds de ontdekking van Nutlin-34 als p53-MDM2 remmer, zijn meerdere structuren gesynthetiseerd en geëvalueerd, waaronder indo-imidazool, imidazoline, benzodiazepinedion en spiroindool verbindingen.5,6 Verschillende van deze verbindingen worden op dit moment onderzocht in klinische studies.7–10 De ontdekking van nieuwe p53-MDM2/X remmers is echter nog steeds van groot belang vanwege de lage activiteit en ontoereikende PKPD eigenschappen die tijdens de huidige klinische onderzoeken van deze verbindingen worden waargenomen.

Hoofdstuk 3 presenteert in een systematisch overzicht de huidige ‘state of the art’ van verschillende klassen p53-MDM2/X remmers en hun co-kristalstructuren, met een bijzondere focus op het mechanisme van de binding van de remmers, en met name de invloed die de geconserveerde watermoleculen hebben voor het ontwerpen van nieuwe remmers.

Een belangrijke discussiepunt is het ontbreken van de zogenaamde ‘dual action’ van de remmers, namelijk binding aan zowel MDM2 als MDMX om het volledige vrijkomen van p53 te verzekeren. Tot nu toe blijken de verbindingen die deze ‘dual action’ bezitten geen erg potente inhibitors van MDMX te zijn.10 Er zijn echter peptide remmers bekend die actieve remming veroorzaken van zowel MDM2 als MDMX. Recentelijk zijn enkele ‘stapled’ peptiden (peptiden waarvan de 3D structuur middels een linker wordt vastgezet) beschreven met grote affiniteit voor MDM2 en MDMX. ALRN-6924 (Aileron Therapeutics) is zo’n stapled peptide en is momenteel in fase I en II klinische studies voor patiënten die lijden aan solide tumoren, lymfomen en myeloïde leukemie.11

Belangrijke eigenschappen van goede p53/MDM2/X remmers zijn:

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- Als competitieve remmers bootsen ze het 3-punts farmacofoor model na dat wordt gevormd door de zijketens van Phe19, Trp23 en Leu26 van p53.

- MDM2 binders hebben een T-vormige topologie waar de einden van de T de drie delen omvatten die overeen komen met de farmacofoor punten.

- De centrale structuur is meestal een heterocyclische of geannuleerde cyclische structuur of een acyclisch lineaire structuur.

- Het aminozuur P53Trp23 draagt het meest bij aan de interactie energie in de binding van p53 en MDM2 en zodoende is het belangrijk dat dit deel goed nagebootst wordt wat betreft hydrofobiciteit en vorm (bijv. fenyl groepen, indolen, oxindolen of 2-oxo benzimidazolen). Een aanzienlijke toename in bindingsaffiniteit kan bewerkstelligd worden door het introduceren van een geschikt hydrofoob halogeen onder in de Trp23 bindingspocket.

- ctie met His96, een waterstofbrug met Ser17, een halogeenbinding of waterstofbrug met Glu72 en een dipool interactie met His73. In dit verband is de aanwezigheid van kristalwatermoleculen van belang die fungeren als mogelijke stabilisatiepunten door vorming van een waterstofbrug. In hoofdstuk 3 hebben we ontdekt dat in het algemeen de MDM2 kristalstructuren een hoge graad van aanwezig kristalwater laten zien en dat een water boven de indoolring van p53Trp23 behouden blijft bij binding van de inhibitoren van de MDM2-p53 interactie aan MDM2.. Vervolgstudies hebben geleid tot het ontrafelen van twee nieuwe locaties waar geconserveerde watermoleculen aanwezig zijn. Het watermolecuul boven de indoolring van p53Trp23 bleek aanwezig te zijn in ongeveer 60% van de 66 geëvalueerde co-kristalstructuren. Verder lijkt het erop dat watermoleculen in de buurt van MDM2Lys55 en in het gebied begrensd door MDM2Val93 and MDM2His96 voorkomen in respectievelijk 45% en 41% van de co-kristallen (figuur 1). Deze bevindingen laten zien welke plaatsen in het eiwit MDM2 mogelijk beschouwd kunnen worden als target voor remmers. Het kan namelijk van belang zijn om de mogelijke vorming van waterstofbruggen te betrekken bij de strategie om de binding van remmers te stabiliseren en de remmingspotentie van de nieuwe reeks verbindingen gericht op p53/MDM2 interacties te vergroten.

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Figuur 1. Regio’s van geconserveerde kristalwatermoleculen in inhibitor-MDM2 structuren. Cyaan: clusters van watermoleculen; licht roze: MDM2 residuen die interactie hebben met de waterclusters; donker roze: de belangrijke p53 aminozuren Phe19, Trp23 en Leu26.

Hoofdstuk 4 legt de focus op de ontwikkeling van macrocyclische verbindingen als potente p53-MDM2 remmers, die gericht zijn op het hydrofobe gebied, gevormd door Tyr67, Gln72, His73, Val93 en Lys94, welke over het algemeen geen bindingsplaats is van de remmers van de andere klassen van inhibitoren. Door gebruik te maken van dit hydrofobe handvat verwachten we een verbetering van de affiniteit te zien van de remmer voor het MDM2 eiwit, alsmede een verbetering in permeabiliteit van de celmembraan. Het vernieuwende aan deze series van verbindingen, naast het afwijkende gebied van interactie, is de methode van synthese, die gebruik maakt van de Ugi reactie, gevolgd door een ringsluiting metathese (RCM), hetgeen de vorming van verschillende formaten en conformaties van de ringstructuren mogelijk maakt. Deze aanpak heeft geresulteerd in een reeks van verbindingen met affiniteit voor de target in de lagere μM range (figuur 2). Eén van de macrocycles (verbinding 3 in figuur 2) kon worden opgezuiverd en gescheiden in zijn enantiomeren, hetgeen resulteerde in een 7-voudig toename in affiniteit vergeleken met het racemische mengsel.

Figuur 2. Macrocyclische verbindingen met hogere affiniteit voor MDM2/X

Hoofdstuk 5 beschrijft de ontwikkeling en synthese van 2,3'-Bis(1'H-indole) heterocycles, die een eenvoudige en effectieve basis vormen voor de synthese van nieuwe p53/MDM2/X remmers. De derivaten in deze serie bevatten een geannuleerde fenylring, die een additionele hydrofobe interactie met de mdm2Val93 aangaat en die p53Leu22 nabootst en helpt bij het stabiliseren van de binding alsmede de potentie verhoogt van eerder beschreven indool derivaten.12 Het beste derivaat van deze familie bereikte een Ki van 1.8 en 0.2 μM voor respectievelijk MDM2 en MDMX (figuur 3). De verkregen resultaten bieden nieuwe kansen voor de synthese en ontwikkeling van nieuwe derivaten die drie punten van mogelijke variatie in het molecuul bieden, alsmede linkers van verschillende lengtes, welke kunnen bijdragen aan het vaststellen van een structuur-activiteitrelatie en aan het produceren van meer verbindingen met hogere affiniteit en betere ‘drug-like’ eigenschappen.

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3Ki Rac= 0.139μMKi A= 0.093μMKi B= 0.7μM

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Figuur 3. 2,3'-Bis(1'H-indool) derivaat met een hogere affiniteit voor MDM2

Een paar families van MDM2 remmers13-15 (figuur 4) zijn gescreend in celkweken en vertoonden middelmatige cytotoxische activiteit (IC50 40-100 μM) op basis van onderzoek in een kleine groep kanker cellijnen. Verder bleken deze macrocyclische derivaten slecht oplosbaar te zijn in waterig medium. Daarom wordt er nu verder onderzocht hoe de strategie van chemische synthese geoptimaliseerd kan worden om verbindingen te krijgen met verbeterde oplosbaarheid, PKPD eigenschappen, meer diversiteit en hopelijk hogere affiniteit voor de receptor. Onze onderzoeksgroep is momenteel bezig met twee verschillende synthetische benaderingen om macrocycles te ontwikkelen (Figuur 5):

1. Synthese van macrocyles gebruik makend van linkers zoals isocyano-carbonzuren en de daarop volgende ringsluiting middels de Ugi reactie met een primair amine en een oxo component.16 Voor het verkrijgen van p53/MDM2 remmers moet de oxo component het indool-ankerpunt zijn in de synthese van macrocycles en 2,3'-bis(1'H-indool) heterocycles zoals beschreven in dit proefschrift.

2. Het gebruik maken van een kunstmatige aminozuur-linker, die wordt gesynthetiseerd in een reactie tussen diamines en anhydrides, welke een Ugi cyclisatie ondergaan met de oxo component (indool) en isocyanide.

Figuur 4. Verschillende structuren welke gescreend zijn in celkweken. 5. Tetrazolen14; 6. -lactams13; 7. Macrocycles (hoofdstuk 4); 8. 2,3'-Bis(1'H-indole) Heterocycles 15 (hoofdstuk 5)

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Figuur 5. Voorbeelden van verschillende syntheseroutes voor kunstmatige macrocycles als p53/MDM2 remmers

Vanwege het belang van de p53-MDM2/X eiwit-eiwit interactie werken verscheidene onderzoeksgroepen en bedrijven aan de ontwikkeling van effectieve remmers. Recentelijk zijn nieuwe verbindingen getest in klinische onderzoeken, maar helaas laten de eerste resultaten slechts beperkte PKPD eigenschappen en ernstige bijwerkingen zien, die in sommige gevallen leiden tot gastro-intestinale en hematologische toxiciteit.17,18 Er is daarom dus nog steeds ruimte voor ontwikkeling van actievere en selectievere verbindingen, met een verbeterd metabool profiel en het gelijktijdig targeten (dual action) van de functie van MDM2 en MDMX.

DEEL B In dit deel wordt de studie van de toxiciteit van verschillende families van goud verbindingen in kankercellen (in vitro) en in gezonde weefsels (ex vivo) beschreven. Metalen en metaal-bevattende verbindingen worden al 5000 jaar gebruikt voor therapeutische doeleinden.19 Tegenwoordig zijn meerdere medische toepassingen van metaal-bevattende verbindingen bekend, onder anderen als een middel tegen kanker en voor het diagnosticeren en behandelen van diabetes. In de kliniek worden momenteel platinum (II) complexen veel gebruikt en deze staan in de lijst van essentiële geneesmiddelen van de wereldgezondheidsorganisatie.20 De meest toegepaste verbinding, cisplatina, is echter zeer giftig en toont een groot aantal bijwerkingen, voornamelijk nefrotoxiciteit en ototoxiciteit.21,22 Daarom zijn er in de afgelopen jaren verschillende chemische strategieën ontwikkeld om tot nieuwe alternatieven te komen en om nieuwe potentiële targets te ontdekken voor verbeterde geneesmiddelen tegen kanker. In deze context zijn op goud gebaseerde complexen interessant vanwege hun verschillende redox chemie (met Au(I) en Au(III) als mogelijke oxidatiestatus) en ligand uitwisselingsreacties, die werkingsmechanismen hebben die verschillen van die van cisplatina.23–25

In hoofdstuk 6 is de toxiciteit in gezond weefsel van een reeks bifunctionele Au(I)-verbindingen bestudeerd door gebruik te maken van precies-gesneden lever- en nierplakjes26,27 om de weefsel-specifieke toxiciteit te begrijpen en om een mogelijk werkingsmechanisme te ontrafelen. De beschreven stoffen werden ontworpen om te fungeren als chimere verbindingen die de cytotoxiciteit van goud ionen combineert met de protonpomp-remmende eigenschappen van lansoprazol. In een eerdere studie van Casini en collega's werd de cytotoxische activiteit van deze verbindingen

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bestudeerd in een klein panel van kankercellen, waaronder cellijnen die gevoelig zijn voor cisplatina, cellijnen die resistent zijn tegen cisplatina en een niet-kankercellijn.28 Deze reeksen verbindingen bleken meer cytotoxisch te zijn voor de kankercellijnen (ongeveer 5 keer) dan voor de niet-kankercellijn, wat potentiëel selectieve farmacologische effecten suggereert.28

In onze studie hebben we enkele representatieve verbindingen (Figuur 6) van deze serie beoordeeld op hun effecten op precies-gesneden weefsel plakjes (PCTS) ex vivo na 24 uur incubatie in vergelijking met lansoprazol. De verbindingen toonden toxiciteit in het μM concentratiegebied op de nier-en leverplakjes met complex 11 als de minst toxische derivaat in gezond weefsel met een TC50 waarde van ca. 27 en 25 μM voor de nierplakjes (PCKS) en leverplakjes (PCLS) respectievelijk. Er werd een TC50 (PCKS)/IC50(kankercel) ratio van 1.8 en 1.7 voor de nierplakjes en leverplakjes respectievelijk berekend, waarin de IC50 voor de A2780 en A2780R cellen uit Serratice et al.28 werd genomen. Dit geeft aan dat de specficiteit voor kankercellen gering is. Echter, voor complex 10 bedroeg de TC50 (PCKS)/IC50 (kankercel) ratio 20 in nierplakjes en 8.9 in leverplakjes, wat mogelijk een selectieve toxiciteit voor kankercellen vergeleken met gezonde weefsel aangeeft. Bovendien werd middels het ATP gehalte en histomorfologische data een iets hogere toxiciteit waargenomen in de leverpreparaten in vergelijking met nierpreparaten voor de verbindingen 10 en 12, die een fosfineligand hebben. Daarnaast hebben we, na het analyseren van de resultaten van RNA-expressieprofielen van pathways geactiveerd door hypoxie (HIF1a), 29 oxidatieve stress (Nrf2) 30 en DNA-schade (p53) 31 geconstateerd dat de Nrf2-pathway het meest wordt beïnvloed, wat aangeeft dat oxidatieve stress mogelijk het mechanisme van toxiciteit is. Dit komt overeen met de bekende redoxchemie van Au (I) complexen die vaak intracellulaire redox schade veroorzaken. Meer studies zijn noodzakelijk om oxidatieve stress te bevestigen als het toxiciteitsmechanisme, inclusief remmingsstudies van enzymen die betrokken zijn bij het behoud van de intracellulaire redoxhomeostase, zoals enzymen die betrokken zijn bij de thioredoxine- en glutathione-huishouding.

Figuur 6. De lanzoprazole derivaten die in dit proefschrift zijn bestudeerd.

Zoals eerder vermeld, vertonen cytotoxische chemotherapeutische metallodrugs, zoals cisplatina, bijwerkingen, met name nefrotoxiciteit.32 Helaas is er nog gebrek aan kennis over het mechanisme van opname en accumulatie in cellen en weefsels. In het verleden zijn verscheidene studies uitgevoerd om de mechanismen van opname en eliminatie van cisplatina in verschillende kankercellijnen te verduidelijken, waarbij diverse in vitro analyses met niercellen of geïsoleerde niertubuli werden gebruikt. Deze mechanismen worden helaas nog steeds niet volledig begrepen.33

Daarom hebben we in hoofdstuk 7 de mechanismen van toxiciteit en accumulatie van cisplatina bestudeerd met behulp van de PCKS-techniek en vergeleken met een eerder vermeld cytotoxisch

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cyclometallo- Au (III) complex (13, Figuur 7) dat een verbeterde stabiliteit in een waterig medium heeft ten opzichte van vergelijkbare complexen.34 We hebben de betrokkenheid van OCT- en MATE-transporters in cisplatina- en Au (III) -complex opname bestudeerd door de opname en accumulatie in PCKS van ratten in afwezigheid en aanwezigheid van de OCT en MATE inhibitor cimetidine te vergelijken en het effect ervan op de toxiciteit van cisplatina en Au (III ) complex 13 te bestuderen. De levensvatbaarheid van de PCKS na de behandeling werd bepaald met behulp van ATP concentratie en histomorfologie. Bovendien werd accumulatie van platina en goud in PCKS gemeten met behulp van ICP-MS. Zoals verwacht werd na de blootstelling aan de metaalcomplexen een concentratieafhankelijke afname in levensvatbaarheid van PCKS waargenomen, die correleerde met de stijging van de hoeveelheid platina of goud in het weefsel. Er werd echter geen effect gevonden van cimetidine op de toxiciteit of accumulatie van beide verbindingen. Op basis van deze resultaten concluderen we dat OCT's en MATE's geen prominente rol spelen bij de opname en accumulatie cisplatin of Au (III) complex. Er zijn nieuwe studies nodig om de eventuele rol van transporters te onderzoeken. Het is duidelijk is dat alle preklinische in vivo/ex vivo technieken, inclusief dit PCTS-model, te maken hebben met specifieke verschillen tussen soorten, zoals de expressie van OCT1, OCT2 en MATE1 in de nieren van ratten, terwijl MATE2-K en OCT2, maar niet OCT1,35 tot expressie komen in menselijke nieren. De pogingen om de mRNA-expressie van KIM1 en villin als cel-specifieke biomarkers te gebruiken om niertoxiciteit te beoordelen waren nog niet succesvol. Het gebruik van PCTS voor opname- en accumulatie studies van metallodrugs biedt goede kansen voor toekomstige toepassing op menselijk weefsel om menselijke specifieke gegevens te verkrijgen. Het gebruik van meerdere specifieke remmers naast cimetidine is nodig om overlappende remmende effecten van verschillende transporters uit te sluiten.

Figuur 7. De Au complexen die in dit proefschrift zijn bestudeerd.

Hoofdstuk 8 richt zich op de synthese en evaluatie van de biologische activiteit van Au (I) -N-heterocyclische carbene complexen als middel tegen kanker in vitro en ex-vivo met behulp van PCKS. De antiproliferatieve effecten van de Au (I) complexen werden getest in vier kankercellijnen, namelijk een p53 wildtype en een p53 nulvariant van HCT 116 (colorectaal carcinoom), MCF-7 (borst adenocarcinoom) en A375 (maligne melanoma cellen). De selectie van de p53 wild type en nulvariant cellijnen was gebaseerd op het bekende mechanisme van cisplatina toxiciteit van inductie van snelle p53-afhankelijke apoptose en, als een secundair effect, p53-onafhankelijke remming van de celcyclus.36,37 De MCF-7 cellijn werd gekozen vanwege zijn resistentie tegen cisplatina behandeling in vergelijking met andere borstkankercellijnen.38 Zo bieden deze cellijnen de mogelijkheid om het toxiciteitsmechanisme van cisplatina te vergelijken met de Au (I) complexen en de betrokkenheid van p53 te onderzoeken. De laatste cellijn die in onze studie werd gebruikt, werd gekozen vanwege de schaarse informatie die beschikbaar is over het effect van metaal complexen op melanoom-afgeleide

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cellijnen. Onze resultaten suggereren dat het toxiciteitsmechanisme van de Au (I) complexen onafhankelijk is van p53-activiteit en dat ze een hogere toxiciteit in de MCF-7 cellijn uitoefenen. De

cisplatina en auranofine in drie van de vier cellijnen, met uitzondering van de A375 cellijn. Verbinding 14 (Figuur 7) was echter sterker actief in HCT116 p53 nul- en MCF-7 cellijnen en even actief als cisplatina in HCT116 p53 wildtype cellijn maar veel minder giftig in de A375 cellijn.

Door hun sterke cytotoxische effecten in kankercellen werden de Au (I) complexen getest op hun mogelijke toxiciteit met behulp van PCKS. De meeste derivaten vertoonden hoge toxiciteit op de PCKS, met TC50 14 geen toxiciteit vertoonde tot een

14 voor kankercellen in vergelijking met gezonde weefsels is nog steeds veelbelovend en stimuleert ons om nieuwe derivaten van deze verbinding te ontwikkelen met verbeterde anti-kanker eigenschappen en selectiviteit tegen kankercellen.

Naast het werk dat in de hoofdstukken van dit proefschrift is gepresenteerd, hebben we voor het eerst Ru(II) complexen geconjugeerd aan cyclisch (RGDfK) peptide39 als een manier om specifiek te targeten naar de -vorming.40

-bereik -receptor. De cytotoxiciteit van alle

gerapporteerde bioconjugaten was echter laag in kankercellen met verschillende niveaus van integrine receptor expressie (SKOV-3 en A549).41 Additionele studies zijn nodig om metallodrugs te ontwikkelen die specifieke targeting combineren met intrinsiek hogere cytotoxiciteit.

SLOTOPMERKINGEN Samenvattend, er werden in dit proefschrift twee verschillende benaderingen onderzocht om potentiële geneesmiddelen tegen kanker te ontwerpen, enerzijds gericht op een specifieke eiwit-eiwit interactie met hoge oncologische relevantie en anderzijds gericht op het ontwerp van metaalcomplexen met een breed spectrum aan anti-kankeractiviteiten. In beide gevallen zijn er nog open vragen over de specificiteit, stabiliteit, transport en metabole omzettingen van de ontwikkelde verbindingen en hun vermogen om het specifieke doel te bereiken.

Verdere studies zijn nodig om antwoorden op dergelijke vragen te geven, inclusief uitgebreidere studies in cellijnen om de werkzaamheid van de verbindingen te testen als middel tegen kanker en het gebruik van verschillende modellen om meer informatie te krijgen over de specifieke werkingsmechanismen. Het gebruik van PCTS kan een van de meest geschikte opties zijn om verschillende parameters te evalueren en om de mechanismen beter te begrijpen. Met behulp van deze techniek kunnen we ook informatie verkrijgen over de toxiciteit in gezond weefsel, wat nuttig is om het ontwerp van de verbindingen te verbeteren en om de mogelijkheid van ongunstige bijwerkingen te bestuderen voordat ze in vivo worden toegediend. Daarnaast bevordert het gebruik van PCTS de reductie en verfijning van proefdiergebruik tijdens preklinisch onderzoek doordat een grote verscheidenheid aan tests en experimenten met weefsel uit één dier mogelijk is. Hierdoor is het niet nodig om een groot aantal dieren op te offeren. Bovendien is het met deze techniek mogelijk om mens-specifieke gegevens te verkrijgen wanneer PCTS worden gemaakt uit menselijk orgaanweefsel.

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Dit zal translationele gegevens verschaffen en resulteren in een betere veiligheid van de geneesmiddelen die worden getest in fase 1 klinische studies.

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APPENDIX

ACKNOWLEDGEMENTS ABOUT THE AUTHOR

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ACKNOWLEDGEMENTS Finally, after 4 years and a lot of interesting projects, I would like to write a few words in order to express my gratitude to those that helped me reach to this point.

Starting first by thanking the Administrative Department of Science, Technology and Innovation from the Colombian Government (Colciencias) for funding this PhD project.

At the beginning of my PhD, I started working at the Drug Design group, where I had the opportunity to learn about medicinal chemistry. I would like to thank Alex for taking me into his group and give me the chance to work on something completely new for me: organic and medicinal chemistry. Additionally, I want to thank you for the fun cooking afternoons and your world’s famous lasagne.

In parallel to my work in the chemistry lab, I started working at the Pharmacokinetics, Toxicology and Targeting group under the supervision of Angela Casini and Geny Groothuis as part of my PhD project. First, I would like to thank Angela, you were my daily supervisor for little bit more than 2 years, you are a very bright scientist, energetic, funny and with great ideas. Thank you very much for all your guidance and help during these 4 years, also I want to thank you for arranging dinners and gatherings with the other members of the group, it was always fun discussing about different topics besides work. I wish you great success in your growing career!

Dear Geny, at the beginning of my PhD you were not directly involved in my projects, but during the last two years we had very fruitful discussions and I got the opportunity to get to know you as a scientist and as a person. You always have great ideas and were available for questions, discussions and even paper work. I really appreciate your help, especially during the last months of my PhD!

I would like to thank the members of the assessment committee: Prof. Frank Dekker, Prof. Peter Olinga and Prof. Ronald Pieters for taking their time to evaluate my thesis.

During these 4 years as a PhD student, I had an extra supervisor at the Drug Design group Dinos whom I consider a great friend and scientist (by the way also my paranymph). Thanks for all the training in the lab, the discussions (not just scientific) and all the fun during these years. I wish you all the best and I hope we can keep in touch, we have to arrange your trip to Colombia!

I will like to thank all the staff from both research groups, for the nice atmosphere and help in the labs. I want to thank specially to our lab technicians for all the help, training and support. Andre, you were always available to answer my questions (or complains about the mess in the lab), it was fun sharing the office with you, Edwin and Robin, I wish you all the best and success. Marina and Marjolijn, I’m grateful for all your help and training with the PCTS. Eduard and Catharina, thanks for all your help at the cell culture lab, you were always available to solve my doubts about reagents and protocols (and scanning a few sections). Jan Visser, thanks for the technical support, the lab tour and your dedication to keep the labs in order. Gillian, I will miss you a lot, besides being a great secretary, you are an amazing person, always happy and willing to make fun of everyone!

Additionally, I would like to extent my acknowledgements to Prof. Klaas Poelstra, Prof. Barbro Melgert, Dr. Anna Salvaty, Dr. Leonie Beljaars, Dr Sylvia Notenboom, Dr Matthew Grooves and Dr. Inge de Graaf, thank you all for the productive discussions during the group meetings, I wish you great success with your groups and students. Inge, I want to thank you for your help and guidance during the last part of my PhD, I wish you all the best!

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I would like to thank all my colleagues from both groups for the great time, a few special acknowledgments to Daphne, Roberta and Laura, it was great sharing the office with you during the last months of my PhD, you are really sweet girls, I wish you great success with your PhDs and future life. Viktoriia, my paranymph (I hope you don’t miss the opportunity to see the northern lights because of me), I want to thank you for your help with new experiments and the nice discussions, you are a remarkable woman and I know that you will achieve great things in the future. Andreia and Sarah: my “little” sisters, it was a great experience working together, and having fun in and outside of the lab. Sarah, I wish you the best of luck finishing your thesis and your future defense and Andreia, I wish you great success in your career. I would also like to thank my students Federica, Marina and Elena, it was a great experience working with you and developing our projects, good luck for all your future efforts.

I would like to thank my friends Nick, Georgia, Sergio and Juliana for their support and funny times, it was very helpful to relax after stressful days of work!!

And of course, Tryf! Thanks a lot for being there in the happy and stressful times, you were a great support and partner in a personal level but also at work. I admire your patience and your disposition to help everyone, I’m sure that you will be truly successful!

Por último quiero agradecer a mi familia, con su apoyo este proceso fue un poco más fácil. Mis padres y hermano siempre atentos y presentes desde la distancia durante estos 4 años. Mi sobrina Luna que me sacó más de una sonrisa en los momentos más difíciles. Erika, gracias por ser mi “guía de fe” y siempre estar disponible para mí. Muchísimas gracias a mi familia Colombo-Holandesa por apoyarme, ayudarme y acompañarme durante este tiempo. Quiero extender mis agradecimientos a mis amigos de Colombia que a pesar de la distancia siguen presentes: Lina, Andrea, David Ahmedt, Vero y Jhon Fernando.

Natalia

11/09/2017

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ABOUT THE AUTHOR Natalia was born on the 17th of August 1985 in Medellín, Colombia. In 2009, she graduated as a Biological Engineer from the National University from Colombia, where she continued with her Master studies in Biotechnology. Her master research was focused on the evaluation and screening of natural products obtained from plants and marine sponges with anticancer properties, her Bachelor and master’s theses were carried out under the supervision of Prof. J. B. López Ortiz and Prof. M. E. Márquez Fernández. In 2013, she obtained a scholarship from the Administrative Department of Science, Technology and Innovation form the Colombian Government (Colciencias) which allowed her to move to Groningen to start her PhD studies at the University of Groningen under the supervision of Prof. A. S. S Domling, Prof. G. M. M. Groothuis and Prof. A. Casini in the groups of Drug Design and Pharmacokinetics, Toxicology and Targeting at the Groningen Research Institute for Pharmacy. Her research focused on the design, screening and toxicity studies of organic and metallorganic compounds as anticancer agents. Currently she is a research associate at Vedas Research and Innovation, a Colombian nonprofit organization focused on applied and basic research, formed by several Colombian researchers from diverse areas of knowledge to create a network to exchange knowledge, ideas and collaboration between the Colombian diaspora and international young researchers.

AWARDS 2013: Beca Colciencias Doctorado en el exterior (Colciencias’ Scholarship for PhD abroad)

2010: Jovenes investigadores e innovadores (Young Researchers and Innovators), Research grant. Colciencias.

LIST OF PUBLICATIONS:

- Estrada-Ortiz, N.;* Neochoritis, C. G.;* Twarda-Clapa, A.; Musielak, B.; Holak, T. A.; Dömling, A. Artificial Macrocycles as Potent P53-MDM2 Inhibitors. ACS Med. Chem. Lett. 2017. doi: 10.1021/acsmedchemlett.7b00219.

- Estrada-Ortiz, N.; Guarra, F.; de Graaf, I. A. M.; Marchetti, L.; de Jager, M. H.; Groothuis, G. M. M.; Gabbiani, C.; Casini, A. Anticancer Gold N-Heterocyclic Carbene Complexes: A Comparative in Vitro and Ex Vivo Study. ChemMedChem 2017, 12 (17), 1429–1435.

- Jürgens, S., Scalcon, V., Estrada-Ortiz, N., Folda, A., Tonolo, F., Jandl, C., Browne, D.L., Rigobello, M.P., Kühn, F.E., Casini, A., 2017. Exploring the C^N^C theme: Synthesis and biological properties of tridentate cyclometalated gold(III) complexes. Bioorg. Med. Chem. doi:10.1016/j.bmc.2017.08.001.

- Estrada-Ortiz, N.; Lopez Gonzales, E.; de Graaf, I. A. M.; Post, E.; Groothuis, G. M. M.; Casini, A. Gold (I) Complexes with Lansoprazole-Type Ligands: Ex Vivo Toxicological Evaluation. (Manuscript in preparation).

- Estrada-Ortiz, N.;* Spreckelmeyer, S.;* Prins, G.; Van der Zee, M.; Post, E.; de Graaf, I. A. M.; Stürup, S.; Groothuis, G. M. M.; Orvig, C.; Casini, A. Ex Vivo Studies on Anticancer Drugs Toxicity and Transport Mechanism in Kidney: Cisplatin vs New Generation Gold-Based Compound. (Manuscript in preparation).

242

- Hahn, E. M.; Estrada-Ortiz, N.; Han, J.; Ferreira, V. F. C.; Kapp, T. G.; Correia, J. D. G.; Casini, A.; Kühn, F. E. Functionalization of Ruthenium(II) Terpyridine Complexes with Cyclic RGD Peptides to Target Integrin Receptors in Cancer Cells. Eur. J. Inorg. Chem. 2017, (12), 1667–1672.

- Estrada-Ortiz, N.; Neochoritis, C. G.; Dömling, A. How to Design a Successful p53-MDM2/X Interaction Inhibitor: A Thorough Overview Based on Crystal Structures. ChemMedChem 2016, 11 (8), 757–772.

- Neochoritis, C. G.; Wang, K.; Estrada-Ortiz, N.; Herdtweck, E.; Kubica, K.; Twarda, A.; Zak, K. M.; Holak, T. A.; Dömling, A. 2,3’-Bis(1’H-Indole) Heterocycles: New p53/MDM2/MDMX Antagonists. Bioorg. Med. Chem. Lett. 2015, 25 (24), 5661–5666.

- Neochoritis, C.; Estrada-Ortiz, N.; Khoury, K.; Dömling, A. Chapter Twelve: p53–MDM2 and MDMX Antagonists. In Annual Reports in Medicinal Chemistry; Desai, M. C., Ed.; Elsevier, 2014; Vol. 49, pp 167–187.

- Estrada-Ortiz, N.; Fernández, D. M. M.; Ortiz, J. B. L.; Fernández, M. E. M. Licania arborea fraction bioactive potential assessment in jurkat and cho-k1 cell lines. Rev. Cuba. Farm. 2016, 50 (4).

- Blandón, L.; Estrada-Ortiz, N.; López, J.; Márquez, M. Esponjas marinas: ¿producción biotecnlogica sostenible?. Rev. Fac. Cienc. 2014, 3 (2), 11–29.

- Estrada-Ortiz, N.; Ortiz, J. B. L.; Márquez, D. M.; Martinez, A.; Fernández, M. E. M. Evaluación citotóxica de fracciones de esponjas marinas del Caribe Colombiano. Rev. Fac. Cienc. 2013, 2 (1), 35–51.