Keywords: Antibody-Drug-Conjugate Adsorption, distribution, metabolism, excretion Accelerator Mass Spectroscopy Arginine Aspartate Atmosphere Gold Area under curve Positron Electron isotopes Symbol Protons Neutrons Atomic mass Radioactivity Discovery Hydrogen

de thèse -Français Fonctionnalisation de liaisons C-H par catalyse à l'iridium : Développement de nouvelles méthodes pour l'échange d'isotopes d'hydrogène sur des petites molécules et des composés biothérapeutiques Mégane Valero Le développement de nouvelles méthodes, efficaces, rapides et facile à mettre en oeuvre pour le marquage avec du deutérium ou du tritium de composés organiques tels que des médicaments est indispensable dans les secteurs industriels comme académique. Ces composés isotopiquement marqués sont essentiels lors des études précliniques. En effet, ces méthodes de marquages permettent d'obtenir plus rapidement les données nécessaires concernant la métabolisation et le profil de sécurité d'un candidat médicament, ce qui permet de réduire le temps investit dans cette recherche. Les méthodes classiques de marquages sont basées sur la synthèse d'un précurseur qui est ensuite marqué isotopiquement puis modifié jusqu'à l'obtention de la structure finale du candidat médicament. Ces anciennes méthodes, coûteuses en temps et en argent et génératrices d'effluents radioactifs, peuvent être évitées par en utilisant directement le composé d'intérêt, en particulier par échange d'isotopes d'hydrogène (HIE). Dans un premier temps, nous avons utilisé une nouvelle génération de catalyseur d'iridium avec ligand P,N-bidentate. Ce catalyseur élaboré par l'équipe du professeur Tamm, a montré son efficacité pour la réaction d'échange d'isotopes d'hydrogène et sur divers substrats. Son utilisationa été étendue par l'évaluation de son activité dans les réactions d'échange d'isotopes d'hydrogène avec les sulphonamides (1), les N-oxydes (2) et les phosphonamides (3) qui sont des motifs présents dans des molécules d'intérêt pharmaceutique. Après optimisation des conditions de réaction, la réaction HIE a pu être réalisée dans des conditions très douces, et a fourni une très bonne incorporation de deutérium dans les positions ortho des sulphonamides DG1 jusqu'à 92% D, des phosphonamides DG2 jusqu'à 99% D et des Noxydes DG3 jusqu'à 100% D (schéma 1). 1 Schéma 5: Réaction d'échange d'isotopes d'hydrogène sur des Carbones(sp3)-H à l'aide de catalyse à l'iridium 5 R1 N H

My first thanks go to Christophe. Thank you for your guidance and advise even though being far from each other. In particular during the heavy task of writing up the PhD, these months were pretty intense, and this actual manuscript would have not been possible without your constant help. I am very thankful I had the opportunity to become part of the ISOTOPICS network. It was great three years meeting all together in different countries. This whole came to life because before being my PhD director, you were the coordinator of the project, so I want to thank you for that too.

I would like to thank the four jury members who have accepted to take the huge responsibility of evaluating my work at the end of these intense three years! Prof. Giang Vo-Thanh, Dr. Joanna Wencel-Delord, Prof. Michael Schnürch and Prof. Luc Demange, I hope you will enjoy reading this heavy thesis.

Of course, thanks to my German bodyguards. So many anecdotes, already from the first time we have met for the interview. Apparently, I had put on too much perfume… Thank you for the supervision over the 3 years, for all these group meeting where I had so much fun and learned a lot. I think from the "you have to change office" to today, a lot have been achieved.

Jens, first of all thank you for picking me up for this position. I still remember the phone call in October 2016 and how happy I was. No beginning is easy, and definitely not the one of my PhD (three slides in 2 weeks!), but what an adventure! You have been always very kind and available for any help. Thank you for all the scientific discussions, training, input over these three years. When you have changed position, this was hard to accept for me, but I am thankful you have not just left. Indeed, you have stayed around and involved in my PhD when other would have stopped caring. I have enjoyed a lot our Wednesday lunch as well, always waiting for you because YOU were the one late, yes! Seeing you eating salad for three years and discussing about science, dog, cat, holidays, German/French fun facts, people we don´t like, Game of Thrones etc. We also had good time during the different Isotopics meeting, I will never forget the driving to Liege sitting at the back like a little kid! I am also glad I met someone as competitive as me haha I hope life will allow us to meet again in the future! Volker, I don´t even know where to start! You are an incredible person. I have no problem to write down here how much I feared you in the beginning. Such a cold and tough German man. I was even exchanging with your former PhD student, asking for advice and if my situation would improve or stay as it was for three years... Luckily Romain was right: "He will make your first months very tough, but it is for the best you will see, it will get better". I did not believe a word at that time! Six month later (yes that was relatively long) indeed it started to improve. Your training to make me become a scientist finally worked out, you stopped putting up this horrible "face-to-face Friday meeting" just before leaving for the weekend, we started to talk more, and this enabled me to become more self-confident. Thank you for always be available anytime I have entered your office to present you new results, exchange new ideas, ask for help (PhD related or German Bürokratie related…) or just complain about everything, but more specifically the LCMS machine which does not work properly! "I can´t work like this!". In any circumstances you always had the ability to calm me down when I was stressed, so be prepared to get phone call in the future if I am not able to do it myself wherever I will be. You made me grow up so much during these years, professionally and personally. I have enjoyed and learned so much during our long discussions about life at coffee breaks and during our Tuesday lunch. I needed time in the beginning to notice and understand that everything you have done or advised me to change/do was for myself. I hope I brought you enough unexpected fun over these three years, you don´t know it yet, but you are going to miss the Easter and Christmas decorations in the lab, I know it! The blinking lights on the windows and the Christmas balls on your plant! I count on you to put at least your red Pompom on your lab coat, I will wait for a proof picture every 1 st of December! Overall, I feel now ready to live the rest of my life, and this is mainly thanks to you. There are not enough words to express how much I am thankful that life has mixed our routes.

Remo, to work with a colleague like you, I could have not wish for better! Even busy with tritiation you were always there to help me, answering chemistry questions, training me with the manifolds etc. You made things so easy, never change that. I know anytime a new student came in the lab, you always said to Volker this was not a good choice, even with me, "The Queen of France". But I heard I am the last one for who you would say it was not a good choice, as you always ended up being wrong, which I am happy about. Thank you for your kindness and patience, in particular in moments I was overstressed. Of course, I am also happy about the different memories we had, going eating with the team in restaurant and playing bowling! How would I have survived my PhD without you Helena? From our first meeting thanks to Werngard, where I was impressed by you (god knows why...) to our first lunch asking already too private questions! I would not be the person I am today if I have not met you. You have always been present, being so patient, dealing with my moods when everything was wrong. I will never thank you enough for that. I don´t think I have ever met someone as patient, as kind, as flexible, as available, as good listener, as funny, as spontaneous, as positive, as….everything! People like you are not that common out there. Thank you for all the hours you kept listening to my drama, because there were indeed so many! Nevertheless, and for the best, there were also plenty of happy stories! You initiated me to travelling (and trained me to pack light!): from Sheffield (yes that one was the first, your beloved University…and country!) to Barcelona, from Dubrovnik to Paris (thanks again for visiting me during my secondment), from Corfu to Koblenz (yes sometimes there is no need to go far). Travelling with you was amazing, but simple things can be as good: there is no best partner than you for a "Netflix and chill" day (official…or not…). Laughing out loud with Grace and Frankie, getting depressed with The Crown, (you) crying watching what I call "funny" movie, getting Christmas mood watching Love Actually for the 467th times and so many others! Thank you for always feeding me with good food and filling up my glass of wine (even though during writing I was filling it up myself...). I am enjoying right now our last month's together until I move for a new adventure but even after I am not worried, we just need to decide which new country we want to visit (Island…)! ¡T'estimo! Marion Champignon! You have been my lovely French bubble here in Germany. I am so glad you were around when I started my PhD. We have so many funny stories about my beginning with my huge need of sleeping and you, Helena and Prisca keeping asking me for a social life! I am happy I have stopped (reduced....) being stubborn as a donkey and started hanging out with you. So many nice memories we have built together, all these nights at your place talking and eating Aroma like piggies! All these Sunday brunch at Hollycross getting food for free and about to get my statue until we changed for Sugar Mama! And these delicious cooking from you which I miss (no one on earth can make the Rhubapaaa pie as good as you do!). Yes....most of these memories are food related....and??! Is that so bad?! But that is not true! There were also great trips around Europe. Konstance and my Easter enigma, Warsaw and Nemo following us everywhere! Thank you for all this great time together over the last 3 years! Thank you as well for keeping contact even when you decided to leave to speak Swissy. I am looking forward to our next trip! Also with Guillhem, the very tall patata! It is always fun to be with you guys! Marta, my polish lady! I can just smile now when I remember Marion informing us about her new roommate. I did not want to meet you or even worse, like you, I did not want a new person in our girls' group…I needed just one dinner to start asking you (not directly of course) to join anytime. Thank you for your support, always listening to this Chemistry which sounded like coming from another world. So many dinners, so many trips as well! Our guide to Warsaw, taking us for Vodka shot from 17h on, moving from one bar to another because indeed, we had to test all of them! What is life for if not, right? It feels always so warm to discuss with you, you are so generous, giving anything you can from yourself. Dziękuję Ci! Prisca, even though our shared time was short, thank you for being by my side! Another Frenchie in Town, I enjoyed spending time with you, and I am happy even after 3 years we keep contact in the group and still share any good new or complain about any annoying thing! Schéma 1: Réaction d'échange d'isotopes d'hydrogène à l'aide d'un catalyseur d'iridium Exemples sélectionnés:

[85]

[96]

[96]

[99] 1 2 3

Au cours de ces travaux de thèse, nous avons développé la première méthode de marquage catalytique à l'iridium dans des conditions douces (température ambiante) sur les motifs dérivés d'acides phenylacetiques tels que esters et amides, motifs également très présents dans les structures pharmacologiques. Cette méthode a permis d'introduire du deutérium ou du tritium en position ortho du groupement directeur de façon sélective avec des rendements satisfaisants à très bons (schéma 2). Cette méthode a été appliquée avec sucès au marquage de plusieurs composés pharmaceutiques (schéma 2) 2 L'ensemble des travaux réalisés pendant cette thèse a permis le marquage au tritium de composés médicamenteux tels que le DM4, le Benalaxyl, ou encore le pharmacophore du Volixibat.

En conlusion, l'introduction de deutérium ou de tritium dans la molécule finale a été effectuée en utilisant la catalyse de l'iridium homogène et hétérogène et nous avons vu au cours des différentes parties de la thèse comment ces méthodes ont été développées d'abord sur de petites molécules modèles et appliquées avec succès ensuite sur des composés médicamenteux.

Sur la base des travaux élaborés dans la thèse, il reste quelques défis à relever et certains projets scientifiques qui n'ont pas pu être achevés par manque de temps :

• Nous avons développé une nouvelle méthode de marquage C-(sp3) des petits peptides (jusqu'à quatre acides aminés). Cependant, la méthode ne fonctionne qu'avec des groupes protecteurs sur les fonctions azotées ou oxygénées. Il serait utile de développer une méthode sélective pour des peptides plus gros sans avoir besoin de groupes protecteurs. Par conséquent, de nouveaux catalyseurs avec une stabilité élevée et une meilleure solubilité dans les solvants polaires sont nécessaires.

• Nous avons découvert une nouvelle façon de prédire l'influence des différents groupements directeur dans les réactions d'échange d'isotopes d'hydrogène en situation de compétition. C'est le cas normal d'une approche de fonctionnalisation à un stade avancé. Il serait donc intéressant de rapprocher les observations obtenues avec d'autres réactions de couplage catalysées par les métaux de transition C-C et C-X.

• Nous avons étudié les IrNPts dans les réactions d'HIE. C'est la première fois que ces nanoparticules sont utilisées en catalyse et il serait très intéressant d'élargir leur champ d'application et d'étudier ces nanoparticules dans d'autres réactions comme dans les hydrogénations ou les réductions.

• Le dernier sujet à traiter serait basé sur les conjugués d'anticorps monoclonaux, comme discuté dans la partie sur les ADC. Il serait important non seulement de pouvoir marquer radioactivement le médicament, mais également de développer des méthodes permettant de marquer l'anticorps et de suivre sa distribution.

Table of content

Part I Introduction 1

I.1 Drug Discovery and Development

The ultimate target of pharmaceutical companies is to develop new drugs and to improve the life of patients. This is of great ethical importance and a high benefit for human society in general as diseases are generating costs in billions. Mostly big pharmaceutical companies are investing high levels of time and money in drug research. Indeed, the Drug Discovery and Development (DDD) process is extremely risky because most of the drug candidates fail. When looking at the number of compounds (figure I.1), the first screenings involve thousands of molecules which are evaluated in each step of the DDD process. This procedure can be considered to work in principle as a funnel and will provide finally one drug compound. The overall DDD process takes between 15 and 20 years, and cost an average between 1 to 2.5 billion €, sometimes more than 5-6 billion € in some cases.

Figure I. 1: Drug discovery and Development process

In a cost and time driven efficient organization, it is a constant duty to further optimize the whole process towards operational excellence. Therefore, there is a continuous need to identify and develop new methods to make the process faster and less elaborated. One of these methods is the faster supply and easier application of isotopically labelled compounds in the DDD process. It is envisioned that with these new achievements in isotope chemistry, better drug candidates can be identified in a much shorter time frame.

The Drug Discovery & Development (DDD) process involves globally seven major steps (figure I.1). The first one is the identification of the disease or disorder the company wants to cure or treat, in connection with the related target which needs to be identified. It can be for example a membrane, a membrane receptor, an enzyme, an ion channel, or a proteinprotein interaction. The second step is the identification of one to three lead molecules by biological assay evaluation with high-throughput screening (HTS). The numbers of tested molecules are differing strongly depending of the project and disease model, but can reach millions of compounds in the primary assay (activity related). In the secondary assay, which is more selectivity based normally, numbers up to 10000 (see Figure I.1) are tested. During this optimization process, the different lead series are optimized in iterative cycles to improve several parameters. These steps are called Drug Discovery and are ending with the nomination of a drug candidate. After this important decision there is no further change of the chemical structure of the compound possible and the Drug Development is starting. The third step concerns the preclinical studies of the identified drug candidate. Different pathways to synthesize and analyze the compound are explored to end up with the most efficient and easy-to-handle synthesis. The pharmacokinetic (PK) parameters of the compound is evaluated by profiling absorption, distribution, metabolism and excretion (ADME) studies as well as toxicology evaluation. The preclinical studies are done in vitro (e.g. hepatocytes, S9 fractions) and in vivo (in animal models). When all collected and analyzed pharmocokinetic and toxicologic data allow the progression of the drug candidate, the fourth step is starting: the clinical studies in humans which involve four phases. The Phase 0, first try in healthy volunteers (10-15 subjects) on a very short duration (≤7 days), consists of collecting the first PK-PD data (e.g.: bioavailability, clearance time. etc.) prior the Phase 1. It can be done via conventional methods by extrapolation from animal models or by microdosing studies using radiolabeled isotopes. The analysis by Accelerator Mass Spectroscopy will mostly employ 14 C whereas Positron Emission Tomography will mostly use 11 C. The Phase 1 enables to determine the safety, dosing and formulation. The drug candidate is tested in total on 60 to 90 healthy human volunteers, by single dose followed by repeated dose in order to assess safety and side-effects and to find the correct drug dosage. The Phase 2 is the first try in patients to demonstrate the effectiveness of the new treatment and to continue safety studies. This clinical evaluation which shows how the drug works lasts several years and is done with cohorts of 100 to 500 patients. The Phase 2 represents the highest risk of failure in the whole DDD process with almost 70% of drug attrition (even more for molecules targeting the central nervous system). This partly results from insufficient preliminary PK/PD evaluation, a problem which could be overcome by an early extensive use of (radio)labeled drug candidates.

It is followed by the Phase 3, which consists of testing the drug candidate on a higher number of patients, up to 10000 in various conditions including different populations, different dosages, sex/age effects and drug combinations. The effect, the tolerance and the side effects will be further investigated during this phase and usually permits to detect potential drug interactions. From the start of the Phase 3, the probability of success is increasing. Finally, the last step consists of the constitution of the Marketing Authorization dossier for the approval from the authorities in the different countries, e.g. Food and Drug Administration (FDA) or European Medicines Evaluation Agency (EMEA). 1 Phase 4 clinical trial is the post-marketing follow-up of the drug in order to track long-term drug side-effects and drug interaction on a real population after prolonged time of use. This phase may also lead to the withdrawal of the drug from the market in case serious issues and drawbacks are detected.

I.2 Limitations of Drug Discovery and Development

The pharmaceutical industry is facing huge challenges like reduced Research and Development (R&D) efficiency, stagnant success rate, patent expirations for key drugs, price competition from generics, sanitary scandals, high regulatory hurdles, and the industry's perceived tarnished image in the public. 1 Despite major scientific and technical advances in DDD and unprecedented investment in pharmaceutical R&D, 2 the number of new drugs approved by the EMEA or FDA was dramatically low in the years 2005-2010. 3 Figure I. 2: Novel FDA approvals since 1993. Annual numbers of new molecular entities (NMEs) and biologics license applications (BLAs) approved by the Center for Drug Evaluation and Research (CDER) 5 By comparison to 1995-2000, it took at least twice more drug candidates coming out of preclinical trials for one product to reach final launch. 4 However, in 2018 the FDA registered a record of novel drug approvals (59) indicating that the changes in R&D management in the last years are making impact (figure I.2). 5 This is in part due to the emerging of new methods for (radio)labeling drug candidates.

The regulatory authorities only accept non-altering (radio)labelling such as deuterium, tritium, carbon-14, carbon-11, fluorine-18 etc., except in the case of large biologics for which slight modifications are accepted. However, it is known that their common way of synthesis (labelling of the first building block and carrying the radioactivity over the whole synthesis providing the drug structure) can be time-consuming, expensive and far from trivial especially for short half-life isotopes. Indeed, there is a clear lack of effective methods for isotopic labelling of drug molecules and for this reason, we need to development innovative labelling strategies, applicable to large sets of small molecules, biomolecules and drug candidates, without affecting their molecular properties. 3

I.3. Isotopically labelled compounds in DDD

The choice of isotope label which is being used in an application of a labelled compound depends on several parameters. There are on one hand commercial reasons, like availability or costs. Another general point is time-dependent, either how fast potential isotopically labelled precursors can be delivered or how fast these compounds can be synthetically introduced into the final labelled target molecule. In this context, it is always beneficial if direct labelling methods, e.g. hydrogen or carbon isotope exchange, are known and can be applied immediately. In all applications, the isotope label must be stable under the used experiment conditions, the use as internal MS standard under aqueous conditions with low pH or metabolic or chemical stability in in-vivo experiments with long-or short-lived radioisotopes (e.g. 14 C or 11 C).

For radioactive isotopes, there are additional parameters to be considered which can differ depending on the followed experiment. Mostly the isotope label should not change the chemical and physical properties of the target molecule, and therefore for most organic drug candidates labelling with isotopes of hydrogen, carbon, nitrogen, sulphur and phosphorus are beneficial. Based on the parameters mentioned above and the half-life, specific activity or emitted energy of the isotope, the best isotope label for the experiment is chosen. Finally, all chemical methods used should be handled safely without any risk to the operating chemist or the environment.

In Figure I.3, the applications of isotopically labelled compounds in DDD is described. For early research mainly tritium ( 3 H) is used for e.g. protein binding, receptor occupancy or receptor identification in-vitro studies. This is mostly due to the low cost, minimal compound needs and limited synthesis efforts. As the 3 H label can be in principle metabolically cleaved, generally 14 C labelling is preferred for in-vivo studies, like tissue distribution or animal PK studies. Short-lived isotopes like fluor-18 or carbon-11 with a half-life of just 110 and 20 min, respectively, can be applied in non-invasive imaging experiments by Positron Emission Tomography (PET). Stable labelled compounds (e.g. labelled with deuterium, carbon-13 or nitrogen-15) are mostly applied to generate isotopically labelled MS standards mainly during the preclinical and clinical time frame. Recently they are also applied in deuterated drugs, which is discussed later.

Figure I. 3: Applications of isotopically compounds in DDD

Herein the connection with whole ISOTOPICS consortium can be easily identified bridging the needs for new and innovative methods in all discussed areas of isotopically stable-, longand short-lived (radio)isotopes.

I.4 Hydrogen isotopes in DDD and life sciences

The popularity of hydrogen isotopes in the life sciences is due to their ability to be incorporated into the target molecule without changing its chemical structure, physical properties or biological activity. 6 Therefore, hydrogen isotopes are unique tools for identifying and understanding biological and chemical processes (figure I.4). They have many properties of ideal tracer nuclides and can be detected with very high sensitivity. In comparison to 13 C or 14 C, hydrogen isotope labelling is easier, quicker and much cheaper. 7 However a disadvantage can be the lack of metabolic stability prediction of deuterated and tritiated compounds. The main risk with tritium is to form in vivo tritiated water 3 H2O which can be distributed in the whole body and due to the radioactive emission may cause cancer. 8,6 After incorporation tritated water stays inside the human body for 7-14 days. 9 Liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) has become the most efficient bioanalytical tool for the investigation of samples coming from environmental, animal, and human studies. 11 Internal standards are essential for a quantitative LC-MS/MS analysis of an analyte in complex solutions (e.g. blood, urine, or bile). Deuterium labelled internal standards are advantageous due to their close physical and chemical properties with the analyte and its standard and the suppression of the matrix effect (figure I.5). 6,14 The compound of interest can be then measured relatively to the added internal standard by correlating a response ratio with a concentration ratio. 12

b) Kinetic Isotope Effect and deuterated drugs

After the administration of the drug in the body, it has to face metabolic transformation to facilitate elimination by increased hydrosolubility. 13 The main metabolizing enzymes in human are from the cytochrome P450 enzyme family, which provided them much attention by the pharmaceutical industry. 14 Significant kinetic isotope effects (KIE) were observed for several P450-catalyzed reactions: specific deuteration incorporation has the potential to impact the rate of metabolic transformations, provided that the rate-limiting step involves cleavage of a C-H bond (figure I.6). 15,6

Figure I. 6: Kinetic isotope effects of C-H and C-D bonds cleavage 6

Depending on the enzymes involved and, therefore, the mechanism of the rate-determining step, another consequence can be a different metabolic turnover of drug molecules when

Stable Isotope labeled internal standards need to have a sufficient mass difference (typically, for small molecules without chlorine, bromine or sulfurcontaining functionalities, an incorporation of 3 to 5 deuterium atoms) to avoid cross signal overlapping and negligible remaining M0.

replacing hydrogen for deuterium. 16 Thus, the alteration of ADME properties by selective deuterium incorporation has recently received much attention. 17,6 The kinetic isotope effect lowers the rates of metabolism reactions and thus notably prolongs the biologic half-life of deuterated drugs. Thanks to this property, we can obtain a reduced systemic clearance and higher systemic exposure, as well as reduced formation of toxic or reactive metabolites, while retaining the potency of the original drug (figure I.7).

Consequently, potential drug benefits could include a smaller dosage, a lower risk for drugdrug interactions, and fewer side effects, resulting overall in an improved PK and safety profile, and an optimized effectiveness. 6

Figure I. 7: Potential pharmacological effects of specific drug deuteration 6

Significant clinical progress has been made in recent years regarding the use of deuterated drugs. 18 The FDA has accepted the New Drug Application (NDA) for Tetrabenazine 1 (Austedo ® ), 19 for the treatment of chorea associated with Huntington disease, based on successful Phase 3 clinical trials. 20 Other examples of deuterated drugs, such as CTP-499 2 (deuterated pentoxifylline) 21 or AVP-786 3 (deuterated dextromethorphane), 22 are currently in advanced clinical testing 23 or have demonstrated improved in vivo PK properties (scheme I.1). 24,6 These deuterium properties could also be an opportunity to reconsider old drugs which were abandoned due to side-effects related to chemical or metabolic instability. This is true for tetrabenazine which was metabolically more stable with deuterium, and deuterated thalidomide which was chemically more stable. The isotope 18 F is preferably employed in PET due to its longer half-life (109.8 min) compare to 11 C (20.4 min). However, the insertion of deuterium in the tracer in plus of the 18 F had showed to increase metabolic stability. Indeed, 18 F-D4-Fluoromethylcholine 4 has proven to be a better PET tracer, with a favorable radiation dosimetry profile for clinical imaging compared to the non-deuterated 18 F -Fluoromethylcholine (scheme I. A convenient in vivo imaging method is the Quantitative Whole Body Audioradiography (QWBA). This experiment provides qualitative and quantitative information on drug distribution and PK, by measuring drug-related radioactivity from body slices or organ sections (figure I.8). 26 These data are important to assess whether the drug reaches the target organ or accumulates in tissues associated with potential safety issues. QWBA studies are routinely conducted for small molecule drug candidates, to determine the maximum radioactive dose that can be administered to humans in the following radiolabelled human In relation with tritium, the short path length of the emitted β --particles provides a high spatial resolution and tissue distribution information at the cellular level. 29,6

I.4.2.b) Tritium in scintillation proximity assay

Tritium allows radioligand binding assays or scintillation proximity assay (figure I.9), which are widely used to characterize receptors, to study receptor dynamics and localization, to monitor enzyme reactions, to identify novel chemical structures that interact with receptors, and to define ligand activity and selectivity in normal and diseased tissues. 30 The selection of the radioligand is based on the stability, selectivity, and a high specific activity, in the range of 50-100 Ci/mmol which typically requires the introduction of 2-4 tritium atoms (considering the 3 H specific radioactivity of 29 Ci/mmol). In addition to this, radioligands should have a high affinity and low non-specific binding. 6

I.4.2.c) Tritium in plasma protein binding

Besides receptor binding, another important PK/PD parameter that can impact the efficiency of a drug is Plasma Protein Binding (PPB). 31 Plasma proteins prevent membrane insertion of hydrophobic drugs, help in drug transport and protect drugs against metabolization.

Interspecies differences in PPB may affect drug-safety margins and thus, a careful assessment of unbound drug concentration in human and animal plasma is fairly requested by authorities before starting any clinical trials. Due to their relatively low costs and more straightforward access, tritium labelled compounds are able to provide a valuable tool which allows early assessment of reactive metabolite formation 32,6 (figure I.10).

Hydrogen isotopes labelling methods

The incorporation of deuterium or tritium into an organic molecule can be achieved by two main routes: either by a conventional multistep synthesis with a radioactive building block or by a late-stage functionalization (LSF) approach. A classical synthesis starting from appropriate commercially available labelled precursor is less and less considered, as it can be very time and resource consuming, depending on the complexity of the chemistry, the chemical structure of the target molecule, or the labelling position. Therefore, in the recent years, the development of methods for fast and convenient LSF with deuterium or tritium into organic molecules was highly investigated. 33 However, all the traditional LSF pathways need a sometimes long-lasting precursor synthesis. 34 As described in the 3 H-synthesis of galantamin 6, the brominated precursor 5 was needed to perform the reaction (scheme I.3, A). The halogen/tritium exchange was carried out with palladium on charcoal as catalyst at room temperature to yield 3 H-galantamin 6. 35 In the example of 3 H-mecillinam 8 synthesis, the unsaturated precursor 7 was synthesized in 6 steps and finally hydrogenated with tritium gas and palladium on charcoal catalysis (scheme I.3, B). 36

Scheme I. 3:

Traditional approaches to introduce tritium into complex molecules 34,35,36,37 Another very common method is based on the use of metal tritides, which need to be synthesized freshly from tritium gas prior use in the reduction of carbonyl or imine moieties.

S O O N N O O N N H N N O S O O N N O N N H N N O H 3 H 3 N N S O OH O H N N S O OH O H H 3 H 3 O N Br O O H O N H 3 O O H (C) [ 3 H]-Vardenafil (B) Pd/C (10% Pd) H 2 O, rt, 2h
In the example(scheme I.3, C) 3 H-vardenafil 10 was synthesised by reduction of the amide carbonyl function with lithium aluminium tritide. 37 While all these methods have proven their usefulness in many reported examples, the precursor synthesis remains the key to a fast delivery of the tritiated compound. Another approach to apply the principles of chemical modification of complex molecules at the latest possible time point (LSF) is the hydrogen isotope exchange (HIE). It enables a selective installation of C-D 38 and C-T 39 bonds in the target molecule by exchange of a C-H bond. 40 Especially for the synthesis of tritium labelled substances, a labelling procedure at the latest possible reaction step is minimizing radioactive waste and decreases the handling time with the radioactive products. Numerous HIE protocols utilizing homogeneous or heterogeneous catalysts have already been described. 41 In scheme I.4, the tritiation of valsartan 11 under classical HIE conditions with an iridium catalyst is described. 42 Furthermore, with the HIE method only a minimum amount of starting material is necessary, which is especially for early projects a great advantage for the fast compound supply. The principles of this reaction will be discussed in more detail below.

Scheme I. 4: typical HIE reaction with iridium catalysis of valsartan

Ir PR 3 N N Mes Mes + PF 6 - NH N N N N O Bu O OH NH N N N N O Bu O OH H 3 Kerr B 15 Ci/mmol 3 H 2 CH 2 Cl 2 , rt, 12h [ 3 H]-Valsartan

11

HIE reactions can be performed under acid/base, heterogeneous and homogeneous catalysis (scheme I.5). Typically, heterogeneous metal-catalyzed HIE generally results in relatively unspecific incorporation of numerous deuterium atoms into a molecular substrate (scheme I. 5, left). Accordingly, heterogeneous metal-catalyzed H/D exchange is typically the method of choice for preparation of stable isotopically-labeled internal standards for LC-MS/MS investigations. In contrast, homogeneous metal catalysed HIE methods are typically much more selective incorporating deuterium only at specific positions in the molecule, e.g. next to a directing group (scheme I.5, middle). Therefore, these methods are of particular importance for tritium incorporation via H/T exchange. 6 Acid/base-mediated labeling methods are still used, even though they are among the oldest methods, there are still new examples being reported. The selectivity of these methods largely depends on innate electronic activation within the target molecule (scheme I.5, right). Na2CO3/D2O 47 or even near-critical D2O 48 are convenient reagents for introducing the hydrogen isotope into aliphatic CH-positions. Due to the fact that the isotope source is used in high excess there are only a few acid/base-catalyzed protocols known to introduce tritium. 6 As peptides have become more and more important in pharmaceutical research, a significant increase of reported methods to prepare deuterium-labeled amino acids or peptides has been observed. For example, Hashimoto et al. reported the deuteration of αamino acids derivatives by triflic acid (trifluoromethanesulfonic acid) with high deuterium content (scheme I. 6, 12,13 =>14,15 ). 49 In one example, a pentapeptide was deuterated at room temperature in 9 hours with up to 8 deuterium atoms introduced at the aromatic positions. 50 Chen and Yin et. al. demonstrated, by Brønsted acid-catalyzed deuteration at the methyl group of N-heteroarylmethanes, the deuteration of complex molecules like tropicamide 17 or papaverine 19, under mild reaction conditions (scheme I.7). 51 Quinolines, pyridines, benzo [d]thiazoles, indoles and imines were all deuterated at the methyl groups with high (>90%D) deuterium incorporation.

Scheme I. 5: Illustration of various selectivities available by distinct HIE methods.

Scheme I.7:

Bronsted acid-assisted labeling in benzylic position of heteroarenes. [99]

[99]

[99]

[99]

[99]

[99]

[90]

[90]

[99/<5]

[99] Xie and Chen et al. developed a highly effective method using NaOH as a catalyst and D2O as the deuterium source under microwave irradiation at 180 °C (scheme I.9). 53 High degrees of both, regioselectivity and deuterium incorporation, for the ortho-and/or para-hydrogens relative to the oxygen atom was achieved. The method showed a relatively high functional group tolerance and allowed the deuteration of complex pharmaceutically interesting substrates, such as Desvenlafaxine 34.

Scheme I.9: Base-medated HIE with Phenols

Selected Examples:

[96]

[96]

[96]

[98]

[95] Finally, it can be summarized that the field of acid/base-catalyzed H/D exchange is slowly progressing with some very convenient new approaches and methods emerging.

I.5.3. Hydrogen isotope exchange with heterogeneous metal catalysis

One of the technical advantages of heterogeneous catalysis is the possibility to remove the catalyst by simple filtration on reaction completion. Moreover, in exchange processes that occur without side reactions or decomposition, no further purification step is necessary. Catalysts are also often more stable and much less expensive than homogeneous catalysts. However, due to the generally low levels of selectivity there is always the possibility of unwanted dehalogenation, hydrogenation, hydrolysis, or, under more harsh conditions, racemization. High catalytic activity for H/D exchange by heterogeneous approaches has been found with palladium, platinum, rhodium, nickel, cobalt, and, more recently, ruthenium catalysts. On the other hand, no particular exchange activity has been observed in heterogeneous reaction procedures with either iridium or iron, which are used with success in homogeneous catalysis (see next section). Regarding the isotope source, gaseous deuterium or tritium, deuterium-or tritium-oxide, and deuterated protic solvents that transfer their labile deuterium to the substrate have all been used as isotopic hydrogen sources. Error! Bookmark not defined.,Error! Bookmark not defined. A selection of the most recent method developments and trends of heterogeneously catalyzed HIE reactions is described below.

I.5.3.a) Transition metal-catalyzed HIE reactions

Palladium is amongst the most common transition metals applied in C-H functionalization, and there are many recent applications of such heterogeneously catalyzed HIE reactions reported. 8e,54,55,56,57,58,59,60 In principle, Pd/C is one of the most widely used heterogeneous catalyst in organic synthesis, as typically applied in hydrogenation reactions. Many different catalyst variations are commercially available, generating a complex portfolio of hydrogenation reactivity measured in turn-over-numbers of different model reactions. Pd/C has also proven its usefulness in HIE reactions.

Pd/C has been widely applied to introduce tritium into molecules. 15,58,59 In addition to palladium, platinum also plays an appreciable role in heterogeneous catalyzed HIE reactions. Platinum has especially proven to be very efficient for aromatic C-H positions, with much higher deuterium incorporation compared to aliphatic C-H positions. Recently, a method for deuteration of several arenes under Pt/C-i PrOH-D2O conditions was reported (scheme I.10).

Remarkably, the activation of the metal surface was performed by in situ generated hydrogen/deuterium through transfer hydrogenation from iso-propanol. This developed external hydrogen gas-free method could, therefore, be used on process scale or for substrates where undesired Pt/H2-reductions were otherwise likely. One of the major improvements in this general field over the past 10 years has been the application of ruthenium in the heterogeneous catalyzed HIE reaction. While ruthenium catalysts have been used in selective homogeneous HIE reactions for many years, heterogeneous ruthenium catalysts on support have been neglected. In a first example, a regioselective deuteration of carbinol carbons (38-40, scheme I.11) was achieved by the combination of ruthenium on carbon (Ru/C), hydrogen gas, and deuterium oxide (D2O). The reaction proceeded with high deuterium efficiency and regioselectivity at the α-position relative to the hydroxy unit, with hydrogen exchange of either one deuterium atom for secondary alcohols or two deuterium atoms for primary alcohols, respectively. (41-43). With this method, benzylic and aromatic positions were deuterated very effectively (scheme I.12). 63 In another example reported by Derdau and Atzrodt, the synergistic effect was proven after NaBD4-activation of the catalyst mixture resulting in significantly higher deuterium incorporations compared to those obtained with the single catalysts induced H/D exchange of heterocycles (scheme I.13). 64 More specifically, a significant synergistic effect could be observed for quinoline-2-carboxylic acid 44, aminonaphthalene 45, and 5-aminobenzothiophene 46. For 8-methylquinoline 47, an almost complete deuteration was achieved with the Pd/Pt/C catalyst mixture and, in contrast to Pd/C alone, a representative mass peak at M+9 could also be identified and, thus, the material could be used as precursor for internal MS standard preparation.

Scheme

Scheme I.13: Synergistic effect in the NaBD4 activated microwave-induced H/D exchange.

I.5.3.c) HIE reactions with nanoparticles

S N H 2 N N NH 2 CO 2 H
Another cutting-edge catalyst application is the use of nanoparticles. 65 These heterogeneous particles are fully dispersed in an aqueous or organic solvent and invisible to the human eye, and may be considered homogeneous in contrast with heterogeneous catalysts which are made of metal nanoparticles supported on an inorganic matrix. Therefore, the critical reaction parameter continues to be the surface of the activated catalyst with an exceptionally huge surface/volume ratio.

Nevertheless, nanoparticles have shown very interesting reactivities and selectivities in a number of HIE reactions. For example, Pieters et al. reported an efficient H/D exchange in the presence of ruthenium nanoparticles of pyridines, quinolines, indoles, and alkyl amines allowing selective deuteration α to the nitrogen atom position (48→49, scheme I.14). At stereogenic centers the reaction proceeded under full retention of stereo information. 18 Under the described reaction conditions there was no difference in reported C(sp 2 )-or C(sp 3 )-labeling selectivity as long as a stereoscopic proximity to a nitrogen atom was present.

Scheme I.14: Ru-nanoparticle-mediated HIE of amines.

N O H R NH(R) R NH(R) D D N H N H O NH 2 N H N H O O O F N N [99] [50] [62] [40]
Selected examples:

[99] [95] [53] [96] RuNP@PVP (3mol%), 55°C D 2 (1-2 bar), THF, 36h R = H, C [70]
[50]

[95]

49

The same Ru nanoparticles were applied in the selective deuteration of amino acids and peptides (50→51, scheme I.15). 66 An enantiospecific CH-activation reaction followed by deuterium incorporation at stereogenic centers was demonstrated. Deeper insights into the mechanism of the process suggested that the key intermediate is probably a four membered dimetallacycle which is triggering the selectivity in the HIE reaction for the α-position of the directing heteroatom. These results clearly demonstrate the potential of nanoparticles for the effective catalysis of CH bond activation and pave the way for new enantiospecific CH-functionalization reactions.

Scheme I.15:

Ru-nanoparticle-mediated HIE of amino acids and small peptides.

NH 2 R O N H R H NH 2 R O N H R D O N H O OH N H 2 O N H O O N H 2 N H N H NH 2 O OH O OH OH N H 2 O N H NH 2 O N N H O NH O OH NH 2 N H O N H OH O [81]
RuNP@PVP ( 5 Generally, significant improvements have been made by applying the synergistic effect of catalyst mixtures to deliver higher deuterium contents. As a new and emerging field, nanoparticles are promising for further improvements in deuteration efficiency and selectivity.

I.5.4. Hydrogen isotope exchange with homogeneous metal catalysis

Homogeneous catalysts generally constitute a transition metal core and different surrounding organic ligands. As these can be conveniently modified the catalytic properties of the catalyst can be tuned easily. Furthermore, homogeneous catalysis can be performed under very mild, widely applicable reaction conditions. Therefore, parallel to advances in pHmediated HIE and heterogeneous catalysis, the homogeneous catalysed HIE reaction dominates modern isotope labelling. Schnürch et. al. reported in 2012 the regioselective deuteration of N-heterocycles using the Ru3(CO)12 and t BuOD as the deuterium source (scheme I.17). 70 In some substrates, deuteration occurred at aromatic and benzylic positions, postulated to react via a nitrogen coordination directed intermediate 61, favoring the C(sp 3 )-carbon for H/D exchange when both were available.

Scheme I.17: Ru-catalyzed directed HIE with N-heterocycles

N X Ph N X D D Ph R R D N Ph N N N H Ph N N H Ph t BuO (5 eq.),  or MW D N X [Ru] D Ph R Ru 3 (CO)
12 (5 mol%) Selected examples :

[42]

[87]

[88]

[89]

[51] While the underlying mechanism is complex due to the photochemical activation and the following redox process, the observed exchange regioselectivities and deuterium/tritium values were remarkably high. However, a more preferred tritium source is tritium gas as it is both less toxic than T2O, routinely handled with modern manifold systems and higher specific activities can be more easily achieved. 71 Homogeneous metal-catalyzed HIE dominates current efforts to achieve labeling in complex molecules by late-stage C-H functionalization. Indeed, a broad range of directing groups and functionalities are now amenable to these selective labeling methods, and often by more than one class of catalyst or metal. Associated with this, the most attractive methods remain those where conditions are mild and most practicable, and where catalysts are readily handled within routine preparative reaction protocols. Accordingly, as researchers strive to establish more advanced methods, air and moisture stable techniques for broad adoption should be the target of future developments in the field. For directed labeling techniques, broadening the drug-like functionalities that can be used as directing groups should remain an additional and prominent focus. Among all transition metals employed in homogeneous HIE methods, iridium is arguably the most widely studied. Due to the increasing significance of the HIE reactions, there are recently published methods utilizing metals like iron 72 , cobalt 73 or ruthenium 74,75,76 which were optimized for this reaction type. However, the state-of-the-art-procedure for HIE reactions remains dominated by iridium catalysts such as those explored by Crabtree A 77,78 , Kerr Ba (counter anion PF6-), Bb (counter anion BArF24-) and C (chlorine instead of PPh3), 79,80 Pfaltz D 81 , Burgess E 82 , Tamm F 83 or Ding G 84 (scheme I.19). Most of these catalysts are commercially available and are nowadays applied regularly in HIE reactions within industry laboratories. 33 The iridium catalyst by coordinating to a directing group (DG) followed by a CHfunctionalization process is able to catalyze the exchange of the hydrogen with its isotopes, deuterium or tritium. In the aromatic HIE reactions on sp 2 -carbons the two ortho-positions of the DG are affected, while in the directed aliphatic exchange only the two hydrogens at one sp 3 -carbon are exchanged (scheme I.20). In both cases, the general isotope source is deuterium or tritium gas. While reaction types look quite similar, the activation energies and geometries in the transition states of the HIE reaction are quite different and are catalystdependent which will be outlined and explored in the later chapters of the thesis. The first mechanism proposal of the iridium-catalyzed HIE reaction was done by Heys et al. in 1996, 85 and further improved by Kerr et al. in 2014. 79c Following the exposure to deuterium gas, the iridium complex Ba loses the hydrogenated COD ligand as D4-cyclooctane (scheme I.21). The resulting Ir specie needs to be stabilized by coordination of the substrate as the Ir is coordinately unsaturated. In this example, the substrate 70 is coordinating to the Ir catalyst and delivers to 71. Next, the metal inserts into the CH-bond by a sigma-complex assisted metathesis process, and the hydrogen atom is bound in the coordination sphere of the iridium(I) metal (oxidative addition, 72a). In the following step, there are several equilibria wherein H-D and D-D bonds are broken in the ligand sphere of the iridium metal, and in equilibrium with the gaseous deuterium atmosphere (72a↔72b). When the deuterium atom from the Ir complex is inserting into the Ir-C bond, the H/D exchange occurs (72c). Finally, the product 73 is eliminated from the iridium complex by reductive elimination.

I.6. Directed iridium-catalyzed HIE reactions

Scheme I.20: Principle of directed-iridium catalysed HIE reaction

Scheme I.21: Proposed mechanism of directed-iridium catalysed HIE reaction

A broad range of directing groups and functionalities are now described for these selective labeling methods, and often by more than one class of catalyst. Accordingly, air and moisture stable catalysts should be the target of future developments in the field. Also, broadening the chemical functionalities usable as directing groups should remain an additional and prominent focus, in particular for functional groups frequently found in drug molecules. 33 HIE on carbon-sp 2 centers are already for some cases described in the literature, using Nevertheless, some key functionalities present in drug compounds are still not applicable for known HIE reactions, or the conditions are not fulfilling the requirements of the tritium chemistry. Furthermore, sometimes the labelling positions are not metabolically or chemically stable or the obtained specific activity is not suitable for the followed biological experiment and request other positions of the molecule to be radiolabelled.

I.7. CH functionalization

Generally, the overall process CH functionalization can be understood as catalytic replacement of a C-H bond (mainly by transition metals) followed by a substitution either by carbon, halogen, oxygen or nitrogen atoms, or hydrogen isotopes (scheme I.24). Therefore, the HIE reaction can be considered as the most fundamental of all CH functionalization processes. The catalytic activation of C-H bonds has been in the spotlight of research for several decades. 89,90 There are numerous investigations on CH functionalization towards C-C, 91 C-X, 92 C-N, 93 or C-O 94 bond formation, wherein in some cases also HIE reactions were studied to get insights into the mechanism. Nevertheless, it should be stated that there is a strong connection between the HIE reaction and CH functionalization reactions to form C-C, C-N or C-X bonds.

Scheme I.24: A principle method of CH functionalization to form C-D/T or C-X (C, N, O) bonds.

Consequently, CH activation, in the broad context of late stage functionalization, has become a strong tool in lead optimization of bioactive molecules in the life science academia and industry. 95

I.8. Objectives of the thesis

The objective of this PhD thesis is to develop efficient, selective, fast and safe methods for the labelling of pharmaceutical relevant molecules by HIE applying iridium catalysis. Even though there has been a renaissance in HIE method development in the last ten years, there are still some synthetic challenges which involve both biological and small molecules. In the biologicals field particularly the labelling of compound classes such as peptides, proteins and sugars proved to be highly challenging. In spite of recent progress, known methods for directed aromatic ortho-selective HIE reactions usually do not give reasonable incorporation levels for some specific molecule classes such as sulphonamides (e.g. sildenafil 89), phenylacetic acid derivatives (e.g. diclofenac 90), amino acids (e.g. lisinopril diester 91), alkaloids (e.g. DM4 92), anilines (e.g. volixibat pharmacophore 93), which is why the HIE labelling of these drugs still poses significant challenges. These methods should allow the direct deuteration or tritiation of the desired target molecule, at a selective and hopefully metabolically stable position in a single reaction step. Deuterated compounds with a high deuterium content at a specific position are especially interesting from the perspective of deuterated drugs, where the metabolism at one specific position should be blocked or at least minimized. For tritiations, the reaction conditions and results have to comply with specific requirements: I. 0.5-2.0 tritium atoms added into the target molecule II.

Scheme I.25: Challenging drug structures

5-20 mol% catalyst III.

Room temperature (if possible) IV. Reduced pressure of tritium gas T2 (50-200 mbar, 1-3 Ci T2 gas) V. Can be used safely on the tritium manifold VI. Safe waste management: easy and safe workup without volatile radioactive sideproducts These specifications allow on the one hand to generate radioactive material which fulfils the requirements for the prior discussed biological experiments, like protein binding, QWBA studies or RIA assays, on the other hand allow the safe and efficient supply of these compounds in a cost-and resource-driven industry environment.

We intended to develop new methods by using deuterium (1 atmosphere). As a stable isotope, deuterium can be handled under standard laboratory conditions without special permissions, handling licenses, or radiation safety measures. We were particularly interested to get the deuterated molecules and to test them later in biologic assays or in hepatocyte metabolism studies. Furthermore, we wanted to select the catalysts and procedures based on the list of specifications compatible with tritium labelling (see above).

However, due to the high kinetic isotope effect H > D > T and the necessary reduced pressure it was planned to add a final adaption of every optimized deuteration method to a tritium application. Furthermore we were interested to get more insights in the parameters driving selectivity and reactivity of HIE reactions with complex structures, which occurs for most drug compounds.

In the first project of the PhD it was envisioned to study sulphonamides (e.g. sildenafil 89, scheme I.25) which are still a challenging DG for HIE. This project should also include two other not reported DG in this context: phosphonamides and N-oxides (Part II-Chapter 1).

The following chapter will be devoted to an old challenge in HIE which are the phenylacetic acid derivatives often present in drug compounds, like diclofenac 90 (scheme I.25), and still not available as a DG in mild and easy to handle conditions (Part II-Chapter 2).

In the third part, we want to look experimentally and theoretically at the influence of the reaction condition parameters on the selectivity and reactivity of HIE reactions (Part III-Chapter 1 and 2).

The fourth part of the manuscript will be focusing on HIE on carbon-C(sp 3 ) centers, describing a new method for the labelling of amino acids and small peptides (e.g. lisinopril diester 91 , this being included in an Antibody-Drug-Conjugate (ADC) Sanofi project involving DM4 92 (Part IV).

As this previous parts were describing only homogeneous iridium catalysis, we will present in the fifth part a heterogeneous iridium catalysis method using newly reported iridium nanoparticles, applied in HIE on anilines such as Volixibat pharmacophore 93 (Part V).

Finally we will conclude this PhD work and have a look on our future prospects and perspectives (Part VI).

As working in a strong scientific network is one of the major parameters in The highly air-and moisture-sensitive, non-commercial catalysts D with phosphine-oxazoline P,N ligands, originally developed by Pfaltz for asymmetric hydrogenation of olefins, proved efficiency also in the HIE reaction of weakly coordinating substrates such as secondary sulphonamide [H]-8 (scheme II.1.3, A). 8 However this single example was the only one reported so far showing good deuterium incorporation. Based on a comprehensive screening of readily available Ir-catalysts, Derdau and Atzrodt et al have identified another hydrogenation catalyst which has also an appreciable HIE capacity: the commercial air-stable Burgess catalyst E. 9 With this catalyst, they have developed the first practical HIE protocol for selective ortho-deuteration of various secondary and tertiary sulphonamides [H]-9, the best results being obtained on tertiary sulphonamides (scheme II. 1.3, B). 7 They have also showed a similar reactivity by using monodentate Kerr catalyst C, 10 which proved to be even more efficient in the HIE reaction of secondary sulphonamides [H]-10 (scheme II. 1.3, C). 7 They also applied their method to sulpha drugs and even adapted to the special conditions required for selective tritium labelling (5-10 eq. of T2 gas, low pressure). However, the need of elevated temperatures (100-120°C) to obtain reasonable deuterium incorporation (up to 78%), still remain an important limitation of this method. 7

Scheme II.1. 3: Previous established HIE methods for selective ortho-deuteration of secondary and tertiary sulphonamides 7,8 D 2 (1 atm) An evaluation of N-oxides as substrates for the directed HIE reaction with catalyst F was also of our interest. Heterocyclic N-oxides have emerged as potent compounds with anticancer, antibacterial, antihypertensive, antiparasitic, anti-HIV, anti-inflammatory, herbicidal, neuroprotective, and precognitive properties. 11 They can also be employed as sedative medication such as Chloradiazepoxide 11 and anticoagulant such as Otamixaban 12 (scheme II. 1.4). 12 N-Oxides have rarely been described as directing groups in HIE reactions, 13 which would in principle enable the labeling of pyridines or quinolines after reductive cleavage of the N-O bond. This HIE approach can be convenient if direct labeling in the ortho-positions of the heterocyclic nitrogen atom (Rh black, D2O 14 or Rh black, THF-D2O 15 ) cannot be accomplished or if higher specific activities in the case of tritium introduction are required. Furthermore, we have examined phosphonamides as they have never been applied in orthodirected HIE reactions before. Phosphorus-containing drugs are still very rarely approved.

S O O NR 1 R 2 D D R Ir + Cl N N S NR 1 R 2 H O O H S NR 1 R 2 D D O O D 2 (1 atm) N N N O Ir Ad Ar + BArF 24 - R R S N H O O D D S N H O O H H D (1 bar) 2 Ir N O O R 2 P Ph S O O NR 1 R 2 H H R A B C PhCl,
One actual example is the phosphoric ester Sofosbufir 13, used against hepatitis C (scheme II. 1.5). However due to the limited chemical stability of phosphoric esters, it was decided to explore the family of phosphonamides first. • HIE reaction on the parallel synthesizer: the substrate is reacted under several conditions such as different solvents, temperatures, reaction times and catalyst amounts. All reactions are first analyzed by LC-MS and only if the success of the HIE reaction can be stated by shift of the M0 peak, the reaction mixture is worked up and analyzed by 1 H NMR. The comparison of the 1 H NMR of the starting material and the product provides the following information: position of the labelling, degree of deuterium incorporation and overall purity.

• HIE reaction on the deuterium manifold: after identification of the optimum reaction conditions with the parallel synthesizer, the substrate is run at lower pressure of D2. The reaction is analyzed by LC-MS, and the conditions are further optimized as long as no acceptable deuterium incorporation is observed (at least 70% D) with as reduced D2 pressure as possible.

• HIE reaction on the tritium manifold: after successful HIE reaction on the deuterium manifold, the same conditions are applied on the tritium manifold. After workup, an aliquot is transferred and analysed by LC-MS. If the reaction is successful, purification and isolation via HPLC are done to provide the tritiated product which is then analysed by radio-HPLC, 1 H/ 3 H NMR and MS.

II.1.3. Optimization of the HIE reaction conditions on the model sulphonamide compound

We started to evaluate Tamm's catalyst F for the HIE reaction of tertiary sulphonamide In the next step, we tried to find optimized conditions for a subsequent broader substrate screening (table II .1.2). We modified catalyst content and reaction time and found that generally 10 mol% catalyst F are needed to obtain high deuterium introduction for substrate [H]-14 within two hours (entry 9). Even though higher excess of deuterium was used in the optimization reactions, the deuteration reached a plateau after 30 min (entry 7) not proceeding significantly by longer reaction times up to 6h (entry 10).

II.1.4. Application of the optimized conditions on arylsulphonamides, N-oxides and aryl-phosphonamides

We then studied the HIE reaction of various sulphonamides under the optimized reaction conditions (scheme II. 1.7). Even though the labelling was not significantly higher after 120 min than after 30 min, we chose a reaction time of 2h for all further substrate screening. The catalyst revealed high reactivity and deuterium incorporation in the HIE reactions of primary, secondary and even tertiary sulphonamides. In the case of the primary sulphonamide 15, catalyst F at room temperature showed similar amount of deuterium incorporation as compared to the results reported by Kerr with catalyst C done at higher temperature (120°C). 10 Interestingly, with catalyst F, secondary (16-18) and tertiary (14, 19-21) sulphonamides underwent successful HIE reactions at room temperature as well, whereas the deuteration only proceeded at elevated temperatures (> 80-120°C) with catalysts C and E. 7 A temperature sensitive Sanofi drug candidate was therefore deuterated and later on tritiated, a reaction which was completely out of reach with the former reaction protocol. In our study, the deuteration of the quinolone moiety in substrates 22-27 proceeded with moderate to very good and selective incorporation, independently of other aromatic substituents (scheme II. 1.8). The 2-and 8-positions of 3-methylquinoline N-oxide 22 were deuterated in 73% and 100%, respectively. Similar results were obtained for 3-bromoquinoline N-oxide 23 and 6-methoxyquinoline N-oxide 24 with deuterium incorporation in these positions higher than 80%. Interestingly, the level of deuterium introduction dropped to approx. 50% at the 3-position in isoquinoline N-oxide 25 and in simple pyridine N-oxides 26 and 27 Since position 1 of isoquinoline N-oxide 25 was still exchanged by deuterium in 93%, a significant electronic effect can be considered as the steric differences between the 1-and 3-position are comparatively small. Indeed, as 1-position 1 of isoquinoline N-oxide 25 is a benzylic position, it is electronically more enriched than the quinoline 3-position. Concerning the HIE reactions of aryl-phosphonamides with catalyst F, we have found good to excellent H/D exchange in the reaction of primary phosphonamide 28 (82%D), secondary phosphonamide 39 (70%D), and tertiary phosphonamides 30-33 (60-99%D) (scheme II. 1.9). Unfortunately, the application of these reaction conditions in HIE reactions with Sildenafil 6, Otamixaban 12 and Sofosbuvir 13 did not enable the incorporation of deuterium.

II.1.5. Conclusion

In this scientific project, we have extended the use of the newly discovered catalyst F by evaluation of its activity in HIE reaction with aryl-sulphonamides, N-oxides and arylphosphonamides. After optimization we were able to run the HIE reaction under very mild reaction conditions which permitted very high deuterium incorporation (up to 99%D). The results emphasize the usefulness of this air-stable catalyst and, in the case of the sulphonamides, catalyst F seems to be more active than other commercially available iridium catalysts.

During this preliminary phase of my PhD, I became familiar of standard HIE procedures and associated analysis. I also became aware of the limits of model compounds we are working on, as observed when transferring our developed method on drug molecules. General procedure for the synthesis of the phosphonamides 29-33 (Organic letters, 2013, 15(17), 4504-4507) To a solution of the corresponding amine (40.0 mmol, 2 eq.) and a catalytic amount of DMAP (0.2 mmol, 0.01 eq.) in dichloromethane (180 mL) was added dropwise a solution of dichlorophenylphosphine (20.0 mmol, 1 eq.) and trimethylamine (40.0 mmol, 2 eq.) in dichloromethane at 0°C. The mixture was stirred at 0°C for 1h, allowed to warm at RT and stirred overnight. The solvent was evaporated and diethylether (200 mL) to yield a salt. The salt was removed by filtration and washed with diethylether. After removal of the solvent the residue was purified by silica gel chromatography (DCM/Acetone 1/1).

Materials and

➢ Phosphonic diamide, P-phenyl-N,N'-dipropyl (29)

Propylamine (2.365 g, 40.0 mmol, 2 eq.), DMAP (25.0 mg, 0.2 mmol, 0.01 eq), dichlorophenylphosphine (2.6 mL, 20.0 mmol, 1 eq.), trimethylamine (5.6 mL, 40.0 mmol, 2 eq.). Yield=60% 1 H-NMR (CDCl3; 300MHz): (δ ppm) 7.83 (m, 2H, H4), 7.59 -7.41 (massif, 3H, H5 and H6), 2.95 (q, J = 7.0 Hz, 4H, H3), 2.38 (s large, 2H, NH), 1.50 (sx, J = 7.0 Hz, 4H, H2), 0.92 (t, , J = 7.0 Hz, 6H, H1). 13 C-NMR (CDCl3; 300MHz): (δ ppm) 133.14 (C6), 131.34 (C4), 128.38 (C5), 42.55 (C3), 25.27 (C2), 11.24 (C1).

➢ Phosphonic diamide, P-phenyl N,N,N',N'-tetraethyl (30) Diethylamine (2.900 g, 40.0 mmol, 2 eq), DMAP (25.0 mg, 0.2 mmol, 0.01 eq), dichlorophenylphosphine (2.6 mL, 20.0 mmol, 1 eq.), trimethylamine (5.6 mL, 40.0 mmol, 2 eq.).

➢ Piperidine, 1,1'-(phenylphosphinylidene)bis (31)

Piperidine (3.410 g, 40.0 mmol, 2 eq), DMAP (25.0 mg, 0.2 mmol, 0.01 eq), dichlorophenylphosphine (2.6 mL, 20.0 mmol, 1 eq.), trimethylamine (5.6 mL, 40.0 mmol, 2 eq.). Yield=65% 1 H-NMR (DMSO-d6; 300MHz): (δ ppm) 7.67 (m, 2H, H4), 7.57 -7.41 (massif, 3H, H5 and H6), 2.90 (m, 8H, H3), 1.58-1.29 (m, 12H, H1 and H2).

➢ Morpholine, 4,4'-(phenylphosphinylidene)bis (32)

Morpholine (3.490 g, 40.0 mmol, 2 eq), DMAP (25.0 mg, 0.2 mmol, 0.01 eq), dichlorophenylphosphine (2.6 mL, 20.0 mmol, 1 eq.), trimethylamine (5.6 mL, 40.0 mmol, 2 eq.). Yield=61% 1 H-NMR (DMSO-d6; 300MHz): (δ ppm) 7.70 (m, 2H, H3), 7.64 -7.47 (massif, 3H, H4 and H5), 3.65-3.45 (massif, 8H, H1), 3.10-2.87 (massif, 8H, H2). NMR 13 C (DMSO-d6; 300MHz): (δ ppm) 132.12 (C3), 131.80 (C5), 128.62 (C4), 67.08 (C1), 44.45 (C2).

➢ Phosphonic diamide, P-phenyl N,N'-methoxy, N,N'-methyl (33)

Methanamine-N-methoxy,hydrochloride (3.900 g, 40.0 mmol, 2 eq), DMAP (25.0 mg, 0.2 mmol, 0.01 eq), dichlorophenylphosphine (2.6 mL, 20.0 mmol, 1 eq.), trimethylamine (5.6 mL, 40.0 mmol, 2 eq.). 300 MHz,3 J = 8.0 Hz,2H,7.40 (d,3 J = 8 Yield: 80%.

HIE reactions of N-oxides 22-27 catalyzed by F (scheme II.1.8)

Conditions: substrate (5.0 μmol, 1 eq.), catalyst F (0.8 mg, 0.5 µmol, 10 mol%, 0.1 eq.), PhCl (2 mL), D2 (1 atm), rt, 2 h.

HIE reactions of phosphonamides 28-33 catalyzed by F (scheme II.1.9)

Conditions: substrate (10.0 μmol, 1 eq.), catalyst F (1.5 mg, 1.0 µmol, 10 mol%, 0.1 eq.), PhCl (1 mL), D2 (1 atm), rt, 2 h. Determined against integral at δ 2.05 (blue arrow).

Yield: 75%. 300 MHz,3 J = 8.6 Hz,2H,7.55 (m,3H, Yield: 87%. 300 MHz,3 J = 7.9 Hz,2H,7.65 (d,3 J = 7 Conditions: substrate (5 μmol, 1 eq.), catalyst F (0.8 mg, 0.5 µmol, 10 mol%, 0.1 eq.), PhCl (2 mL), D2 (1 atm), rt, 2 h.

N-oxide

HIE reactions of phosphonamides 28-33 catalyzed by F (scheme II.1.9)

Conditions: substrate (10 μmol, 1 eq.), catalyst F (1.5 mg, 1.0 µmol, 10 mol%, 0.1 eq.), PhCl (1 mL), D2 (1 atm), rt, 2 h.

II.2.1.2. Literature background of HIE reactions on phenylacetic acid derivatives

In 1996, Heys et al. studied the HIE performance of several iridium(I) complexes such as Crabtree's catalyst A, [Ir(COD) (PPh3)2]BF4 18 and[Ir(COD)(dppe)]BF4 19. A very low deuterium incorporation of only up to ca. 7% (11) was found for ethyl phenylacetate [H]-11 (scheme II.2.2, A). 1 Sajiki et al. developed an unselective method based on heterogeneous catalysis for labeling of all aromatic protons (12) with palladium and platinum on charcoal at 180 °C in D2O (scheme II.2.2, B). 2

Scheme II.2. 2: Known aromatic HIE reactions for CH-activation/deuteration in phenylacetic acid derivatives

69

Even though the deuterium incorporation was high (up to 99 %D), the harsh conditions at 180°C, the low selectivity and the large excess of D2O were limiting the general usefulness of the method. The high excess of the isotope source (D2O) made the transfer to tritium chemistry impractical. 3 The first ortho-selective HIE method was reported by Yu et al. in 2014. They discovered a palladium-catalyzed HIE reaction of phenylacetic acids [H]-13 with deuterated acetic acid as deuterium source and ortho-deuteration up to 99 %D (scheme II.2.2, C). 4 However high temperature and low pH limit the applicability of the method. Moreover, due to the high excess of the deuterium source (used as solvent), the method was not transferable to the tritium requirements.

Recently, the approach was adapted to tritium chemistry by Hoover et al. 5 They generated substrate-palladium complexes [H]-14, which were isolated and purified, and then in a second step, were reduced subsequently by deuterium or tritium (scheme II.2.2, D). However, Hoover's protocol is non-catalytic and requires a potentially troublesome precursor synthesis, together with tedious removal of excess palladium and ligand.

In order to circumvent these shortcomings, we became interested in developing an improved catalytic HIE method under very mild reaction conditions with D2/T2 as hydrogen isotope source.

II.2.1.3. What makes phenylacetic acid derivatives such a challenge?

As described above, phenylacetic acid derivatives have remained a challenge in direct HIE methodology for tritium applications. For iridium catalyzed HIE reactions, the comparison of the activation energies H for different aromatic substrates As the P,N-ligated iridium catalyst F had demonstrated unique reactivity features and an unprecedented HIE transition-state flexibility involving 5-, 6-and even 4-membered rings, 7 we wanted to investigate its activity towards phenylacetic acid derivatives.

II.2.2. Optimization of the model compound

II.2.2.1. Catalyst screening

The HIE study was initiated by examining the deuteration of methyl phenylacetate [H]-12 as model substrate under 1 bar of D2 with different commercially available iridium(I) catalyst (globally noted Ir-cat) and catalyst F. Catalyst F can be synthesized in five reaction steps according the procedure described by Tamm et al. 7 The reaction was run at room temperature in chlorobenzene for 2h (table II

II.2.2.3. Solvent and temperature screening

II.2.2.4. Catalyst loading screening

We have also investigated the catalyst loading of F (table II .2.3) in order to optimize the labelling conditions. A loading of 5 mol% resulted in 1.5 added deuterium atoms in the substrate (entry 1) whereas with 10-15-20 mol%, as before, the same maximum isotopic enrichment was 1.80 D/molecule. Consequently, we used 10 mol% of catalyst F for the project.

Table II.2. 3 By varying the reaction parameters, such as catalyst amount, solvent, temperature and time, optimal conditions were identified to be 10 mol% catalyst F in chlorobenzene at room temperature for 2 h. Under these conditions, we have pursued our study on a series of phenylacetic acid derivatives.

II.2.3. Scope and limitation of catalyst F in HIE of phenylacetic acid derivatives II.2.3.1. HIE reactions with catalyst F on phenylacetic ester derivatives

Catalyst F has promoted ortho-directed deuteration of a variety of phenylacetic acid esters (11, 12, 18-27) with high yields, good selectivity and good to high deuterium incorporation of up to 95 %D (scheme II.2.3).

Scheme II.2. 3: HIE reactions of phenylacetic acid esters 11, 12, 21-37

with catalyst F a,b O O O O O O O O O O O O O O O H O O O H O O OH O O X O O X O O N N O O N OH O OH O N O O OH O N O O N O OH O [87] 12, 83% [68] [49]
11, 78% 21, 81%

[85]

22, 87%

[55]

23, 91%

[45]

[94]

24, 82%

[82]

25, 89%

[95]

26, 91%

[76]

[52]

27, 91%

[26]

[87] 28a (X=F), 32%

[86]

[53] 28b (X=Cl), 90% 28c (X=Br), 88%

[77] [16]

[0] [0] [0] [0] [0] [0] [0]
a Conditions: substrate (1 eq.), catalyst F (10 mol%), chlorobenzene (1 mL), D2 (1atm), rt, 2 h. b Position and percentage of deuterium incorporation determined by 1 H NMR.

Interestingly, the efficiency of the HIE reaction depended strongly on the size of the ester function, resulting in decreasing deuterium incorporation levels in the order methyl (12, 87%D) > ethyl (11, 68%D) > isopropyl (21, 49%D). Additionally, the HIE reaction was somewhat sensitive towards the substitution pattern at the aryl ring. While metasubstitution in 3-tolylacetic acid methyl ester 23 gave 45 %D and 55 %D, respectively, the corresponding para-(22) or ortho-substituted (24) analogues gave better deuterium incorporation (85 %D and 94 %D). This was also observed in the halogenated substrates 28 and 29. Interestingly, this trend was not observed for the hydroxyl-substituted esters 25-27, indicating that not only steric but also electronic factors, like electron density or mesomeric resonance structures, exert influence on the outcome of the HIE reaction.

Even though the kinetic study shown previously had demonstrated that after 2 h, the reaction reached its maximum of deuterium incorporation, the reaction time for compounds with lower deuterium incorporation like 21, 23 or 27 was thus increased to 24 h. However, this had no positive effect on the final deuteration results. Probably this is due to the inactivation of the catalyst as no decomposition was detected and still large excess of D2 remained in the reaction system. Unfortunately, the HIE reactions on free phenylacetic acids like 31-34 were not successful at all. A possible reason could be that the acidic proton of the carboxylic acid is coordinating to the iridium(I) metal and inhibiting any further activity. In addition, compounds containing nitrile function directly on the phenyl ring (36) or in the benzylic position (37) did not work at all either. The triple C-N bond being extremely rich in electrons and the iridium(I) being a transition metal looking for stabilization of his electronic environment, the iridium might directly coordinate with the N-atom of the cyanide, and therefore due to the rigidity too distant from the aromatic ring to catalyse a successful HIE reaction. Moreover, the absence of reactivity of 37 can also be due to the steric environment at the benzylic position.

The influence of the pyridine functionalities was investigated, first with the free phenylacetic acid (34) but as we already observed with compounds 31-33, free acids are not providing successful results. Consequently, the ester derivatives 34, 35 containing the pyridine were also run under our reaction conditions. However, no deuterium incorporation has been observed. This is probably due to the N-heteroatom in the pyridine moiety as a competing directing group. As the pyridine is a much better metal ligand in comparison to the carboxylic acid functional group (see Part III Chapter 2) the resulting coordinated catalyst is unable to perform the HIE reaction.

II.2.3.2. HIE reactions with catalyst F on phenylacetic amide derivatives

Under the general reaction conditions, ortho-directed deuteration of a variety of phenylacetic amides (38-49) was also achieved with high yields, good selectivity and high to excellent deuterium incorporation up to 99%D (scheme II. 2.4). In nearly all reactions of the N-methyl amides 38, 39, 41, 42, 44 and 45, we observed additional deuteration at the N-methyl group with 13-33 %D (39, 63 %D). Interestingly, these aliphatic labelling side reactions could be completely avoided by introduction of larger N-substituents like in amides 40, 43, 46 and 47, indicating that excellent selectivity and deuterium incorporation can be achieved. Unfortunately, we observed no HIE reaction with primary amides like 48 and 49 under these reaction conditions. We propose that the primary amide chelates to the iridium(I) atom similar to the carboxylates and therefore a more stable complex is being formed making a HIE reaction impossible. [33]

Scheme II.2. 4: HIE reaction on various phenylacetic amides 38-49

with catalyst F. a,b N H O O O Cl O N H O O N H O N N N H O O N O O O N H N N O O O N O N O NH 2 NH 2 O O 38, 88% 39
[92]

[22]

[95]

[13]

[98]

[29]

[29]

[96]

43, 96%

[98]

[63]

[63]

[99] 40, 97%

[79]

[13]

[13] 49 48 [0] [0]

a Conditions: substrate (1 eq.), catalyst F (10 mol%), chlorobenzene (1 mL), D2 (1atm), rt, 2 h. b Position and percentage of deuterium incorporation determined by 1 H NMR.

II.2.3.3. Application on phenylacetic acid derivatives drug molecules

II.2.3.3.a) HIE reactions on drugs with excess of deuterium gas

We further applied our method to more complex and multi-functionalized drug-like molecules bearing a phenyl acetyl moiety 5, 10, 50-53 at atmospheric deuterium pressure (scheme II.2.5). Naproxen ester For the ibuprofen derivatives 50a-e, no HIE product has been observed for the primary amide 50a, thus confirming the negative impact of this chemical function on the iridiumcatalyzed HIE. However, good to excellent deuterium introduction were obtained for secondary and tertiary amides 50b-d (81-96 %D). The corresponding ester 50e (32 %D) afforded lower deuteration efficiency compared to the amides 50b-d. A remarkable selectivity and deuterium incorporation of 99%D was achieved for drug-like compound 48 8 without side labelling of the other aromatic ring systems. For both naproxen esters 52a (75 %D) and 52b (76 %D), deuteration results are identical, but interestingly, only the 3-position has reacted. Ester 52b was also converted into the corresponding acid (naproxen 7) under basic conditions without significant loss of deuterium. Surprisingly, next to the expected aromatic labeling (72 %D), we have also observed deuteration in the benzylic position ( 64

Scheme

R = Me R = Et R = H R = Me [72] [31] [64] [0] Camylofine Ibuprofen derivatives 51, 87% 52a, 79% 52b, 100% 5, 85% 53, 79% 10, 90% [0] [81] [96] [88] [32] X=N R1 = R2 = H R1 = H, R2 = Pr R1 = R2 = Et R1 = R2 = tBu X=O R1 = Me, R2 = none 50a 50b, 98% 50c, 100% 50d, 95% 50e, 97%
[0] [0] [0] [0] [0] [0] [0] [0] [0]
a Conditions: substrate (1 eq.), catalyst F (10 mol%), chlorobenzene (1 mL), D2 (1atm), rt, 2 h. Even though we had success in the HIE reactions of drugs 5, 10, 50-53, we obtained no deuteration for the compounds presented in scheme II.2.6. Fexofenadine 6, either as a free acid (6) or as the methyl ester (54), Penicillin G 3 and Diclofenac 58, both of them investigated as free acid (3 and 58) and as methylated ester (57 and 59) did not react at all.

As expected, the free acid Ketoprofen 60 was not successful either in the HIE reaction. Also, Atropine 55 did not provide a good result. Finally, atenolol 56 containing a primary amide group could not be labelled with deuterium.

II.2.3.3.b) Transfer of the procedure to the deuterium manifold

Finally, we intended to test our developed method for tritiation of drug-like compounds. This implies that the reaction procedure is preliminarily adapted to the deuterium manifold. Camylofine 5 and Benalaxyl 10 have been selected to investigate conditions at lower deuterium pressures (<200 mbar) (table II .2.4).

For comparison, we run the HIE reaction of Camylofine 5 in the manifold under atmospheric deuterium pressure first and obtained the deuterated product with 1.5 D/molecule (entry 1).

In the HIE reaction with 10 (entry 2), 20 (entry 3) and 30 (entry 4) equivalents of D2, rather similar deuterium introduction was observed (1.1 -1.3). As Benalaxyl 10 was deuterated with 96 %D at 1 bar of D2 gas (entry 5), the equivalents window was in this case reduced down to 2.5 to 10 eq. of D2. A very good result of 1.7 D/molecule (85 %D) was obtained with 5 eq. of D2 (entry 7), the same as with 10 eq. of D2 (entry 8).

II.2.3.3.c) Transfer of the procedure to the tritium manifold

Finally, we performed the HIE reactions of Camylofine 5 and Benalaxyl 10 under the optimized conditions in the tritium manifold (scheme II.2. 7). We observed that the HIE reaction of Camylofine 5, performed with 10 eq. of tritium, led to a 26 %T incorporation in the ortho-position and 11 %T incorporation in the benzylic position. In total, we obtained a specific activity of 32 Ci/mmol for this molecule.

Benalaxyl 10 was deuterated selectively in the ortho-position with 96 %D as we have seen earlier. Despite an anticipated kinetic isotope effect (D vs. T) and a much lower tritium pressure (60 mbar, 7 equivalents vs. atmospheric deuterium pressure), a specific activity of 14 Ci/mmol (25 %T) was obtained in 10, which was more than sufficient for the planned invitro studies with 3 H-benalaxyl. Therefore, the profile for the C-H activation step (IN1→TS1→IN2) was also calculated for these substrates. This has revealed the expected lower activation barriers and significant stabilization of the corresponding intermediates, especially for the acetamide (NH) derivative, which can be ascribed to conjugation within the NH-and O-bridged metallacycles. Interestingly, attempts to perform HIE on substrate [H]-12 in the presence of complex 61 in chlorobenzene at room temperature afforded only marginal deuterium incorporation, indicating that this complex is not part of the catalytic cycle, but represents a deactivated form or an overly stabilized resting-state, respectively. 12

II.2.5. Conclusion

In conclusion, in collaboration with the research group of Prof. Tamm, it was possible to solve a longstanding challenge in direct labelling of phenylacetic acid derivatives. While former methods have utilised non-catalytic reaction pathways or large excess of isotope source, the first efficient catalytic protocol for ortho-selective hydrogen isotope exchange of By direct tritium labelling of Benalaxyl 10 and Camylofine 5, it was proven that the method was fully adaptable to the specific requirements of tritium chemistry mentioned in the objectives of this thesis (Part I). DFT calculations and kinetic studies performed in collaboration with Braunschweig, indicated why the Tamm catalyst F is so successful for phenylacetic acid derivatives via a six-membered ring metallacycle transition-state. Therefore, one of the longstanding challenges in iridium-catalyzed C-H functionalization and HIE has been solved, and we believe that our findings will stimulate further applications of F and related catalysts in this field. Nevertheless, modifications in the catalyst structure needs to be performed to overcome the steric constraints identified in benzylic substituted phenylacetic acid derivative drugs 54-60 which have not been successful so far. 1 H NMR (300 MHz, CDCl3): δ 7.17 (m, 1H, CH meta-phenyl), 6.82 (m, 1H, CH ortho-phenyl), 6.77 (s, 1H, CH ortho-phenyl), 6.75 (m, 1H, CH para-phenyl), 3.72 (s, 3H, CH3), 3.59 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 6.82 and 6.77 (red arrow). Determined against integral at δ 3.72 (blue arrow).

Supporting information

General method: 10.1 mg (61.0 µmol) 26; 9.3 mg (6.1 µmol) catalyst F, 2h. Yield: a) 8.9 mg, 54.0 µmol, 88%; 77% D for δ 6.82 and 96% D for δ 6.77. b) 9.5 mg, 57.0 µmol, 94%; 75% D for δ 6.82 and 93% D for δ 6.77. Yield: a) 10.9 mg, 42.0 µmol, 100%; 75% D for δ 7.17.

b) 9.9 mg, 38.0 µmol, 95%; 75% D for δ 7.17.

Saponification of deuterated naproxen ethyl ester 52b

The deuterated naproxen ethyl ester 52b (5.0 mg, 19 µmol, 1 eq.) was dissolved in 2 mL of ethanol. A solution of 20 mol% KOH was added (200 µL, 0.1 eq.) and the reaction was heated at 80°C for 2 h in the microwave. The tritiation of Camylofine 5 was carried out using a standard Tritec ® tritium manifold. 3.2 mg (9980 µmol) of Camylofine was dissolved in 0.35 mL dry chlorobenzene followed by addition of a solution of 1.5 mg Tamm-catalyst F in 0.35 mL dry chlorobenzene. The 1 mL reaction flask was adapted to a tritium manifold and the solution was frozen in liquid nitrogen. The flask was evacuated, charged with tritium (2.5 Ci, 49.9 mmol, 5. eq). The reaction mixture was then allowed to warm to room temperature before stirring at rt for 2 hours. After removal of solvent in vacuo the labile tritium was exchanged and removed by addition of methanol and a freeze-drying process (three times repeated). The solid residue was dissolved in 3 mL methanol. Purification and isolation via HPLC (Phenomenex Gemini NX C18 10x150 mm, 5 mm, 5 mL/min flow, ACN/water gradient program) gave 5 (1,5 mg, 5513 MBq, 5,9 % RCY, 1184 GBq/mmol (32 Ci/mmol) in>98% purity (HPLC, UV/RD). The tritiation of Benalaxyl 10 was carried out using a standard Tritec ® tritium manifold. 2.50 mg (7680 µmol) Benalaxyl was dissolved in 0.35 mL dry chlorobenzene followed by addition of a solution of 1.2 mg Tamm-catalyst F in 0.35 mL dry chlorobenzene. The 1 mL reaction flask was adapted to a tritium manifold and the solution was frozen in liquid nitrogen. The flask was evacuated, charged with tritium (2.5 Ci, 42.2 mmol, 5.5 eq). The reaction mixture was then allowed to warm to room temperature before stirring at rt for 2.3 hours. After removal of solvent in vacuo the labile tritium was exchanged and removed by addition of methanol and a freeze-drying process (three times repeated). The solid residue was dissolved in 3 mL methanol. Purification and isolation via HPLC (Phenomenex Gemini NX C18 10x150 mm, 5 mm, 5 mL/min flow, ACN/water gradient program) gave 10 (2 mg, 3188 MBq, 3,5 % RCY, 518GBq/mmol (14 Ci/mmol) in 98% purity (HPLC, UV and RD). The flask was evacuated and filled with hydrogen gas three times. The mixture was then stirred over night until the color changed to a dark blue. The solvent was removed under vacuum and the blue precipitate was washed two times with 0.5 mL toluene each and three times with 0.5 mL hexane each. The product 61 was isolated as a dark blue solid.

HRMS (positive ESI

HRMS (positive ESI

Crystals for X-Ray diffraction analysis were generated by slow diffusion of hexane into a saturated solution of 61 in dichloromethane.

Yield: 63.0 mg, 0.022 mmol, 88 %.

Crystallographic details

Crystal data and refinement parameters for 61 are shown in Table 5. Suitable single crystals were mounted on MiTeGen mounts in per-fluorinated inert oil. Intensity measurements were performed at 100K using Rigaku Oxford Diffraction XtaLAB Synergy S diffractometers and HyPix-6000HE Hybrid Photon Counting (HPC) detector with mirror-focussed MoK radiation. The diffractometer software CrysAlisPRO was employed. [1] Absorption correction was based on multi-scans in combination with face indexation and gaussian absorption correction. The structure was refined anisotropically on F 2 using the program SHELXL-2018. [2] The compound was crystallized from a partially deuterated sample. Because of a deuteration grade of approximately only 30 %, all H atoms were treated as protium atoms rather than deuterium atoms. One hydrogen atom on each iridium atom could not be found and refined freely and was therefore omitted. The sum formula and all derived parameters are based on the refined structure without this second set of hydrogen atoms. The bridging hydrogen atom H1 was refined freely. All other H atoms were placed in idealised positions and refined using a riding model, with a common U restrained to be 1.2 (1.5 for methyl groups) times the equivalent isotropic displacement parameter of the parent atom. Ordered methyl groups were treated as rigid bodies and allowed to rotate around the E-CH3 bond.

Crystallographic data for 61 have been deposited with the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge CB21EZ, UK. Copies of the data can be obtained free of charge on quoting the depository number CCDC-1886537 (Fax: +44-1223-336-033; E-Mail: deposit@ccdc.cam.ac.uk, http://www.ccdc.cam.ac.uk). Scheme 3: Backward reaction (hydrogenation).

A 100 mL 3-neck round bottomed flask, equipped with a stopcock valve and a suba seal was charged with the substrate (0.533 mmol; Phenylacetic acid methyl ester for the forward reaction or phenylacetic acid methyl ester-d2 for the backwards reaction), catalyst F (8 mg, 1 mol%) and DCM (15 mL). The flask was sealed with open connection to the gas inlet and then evacuated until slight bubbling of the solution and then refilled with deuterium (or hydrogen) gas from a balloon for three times. The mixture was then stirred vigorously at room temperature. 0.3 mL aliquots were taken from the reaction mixture after 5 min, 7 min, 9 min, 11 min and 13 min via syringe. The samples were purified as described in the general procedure for HIE.

To determine the kinetic isotope, effect the reaction progress was plotted over time (Figure 5). Dividing the rate of the forward reaction (𝑘 𝐷 ) by the rate of the backward reaction (𝑘 𝐻 ) leads to the kinetic isotope effect

Computational Details

All computations to calculate the gas-phase electronic structures and energies for all species involved in H/D exchange reactions were performed using the density functional method M06 [3] as implemented in the Gaussian09 program. [4] For all main group elements (C, H, D, N, P, and O) the all-electron triple-ζ basis set 6-311G(d,p) [5] was used and a Stuttgart RSC [6,7] effective core potential basis set for the 5d transition metal iridium. The participating transition states (TS) are located at the same level of theory. Harmonic vibrational frequencies are calculated to characterize respective minima (reactants, intermediates, and products with no imaginary frequency) and first order saddle points (transition states with one imaginary frequency).

Calculation of theoretical Kinetic Isotope Effect (KIE)

Having calculated the thermal properties of the CH activation process for the deuterium incorporation, similar calculations were carried out started from the analogous aryl 2,6-dideuterated product and working in reverse. The relative enthalpy (ΔH) values for the forward and backward reactions are shown on Figure S6. Subsequently, the theoretical KIE value can be obtained from Equation 1:

𝑘 𝐷 𝑘 𝐻 = 𝑒 (Δ𝐻 𝐻 ‡ -Δ𝐻 𝐷 ‡ ) 𝑅𝑇 (𝐄𝐪𝐮𝐚𝐭𝐢𝐨𝐧 𝟏)
𝑘 𝐷 = rate of forward reaction -"deuteration" 𝑘 𝐻 = rate of backward reaction -"hydration" Δ𝐻 𝐷 ‡ = transition state enthalpy of forward reaction (J mol -1 )

Δ𝐻 𝐻 ‡ = transition state enthalpy of backward reaction (J mol -1 ) R = gas constant (8.314 J mol -1 K -1 ) T = temperature (298.15 K)

. Introduction

As mentioned in Part I, an introduction of a radioactive label in a metabolically stable position is essential for the application of tritiated compounds for in-vivo experiments. This implies that chemists have access to a complete toolbox of catalysts and HIE reactions in order to specifically and selectively label particular positions into drug molecules. For this purpose, it is necessary to understand the influence of the different directing groups (e.g. DG1 versus DG2, scheme III.1.1) on HIE in complex molecules. One parameter which is known to influence reactivity and selectivity in HIE reactions is the reaction temperature. Even though many details on the optimisation of reactions conditions in iridium-catalysed HIE are already reported, 1 there is still a lack of head to head comparison. For this reason, we have concluded that a general comparison study with the most commonly applied catalysts A-G could help to use the optimized approach, for either high selectivity or maximum deuterium incorporation. III. 1.1). Interestingly, depending on the used catalyst, we observed strong differences in reactivities and selectivities of the HIE reaction leading to the deuterated equivalents of compound 8. At -80°C only catalysts A (entry 6) showed reasonable reactivity (> 25%D incorporation), however with only low selectivity in favour of position B (ketone directing group). At lower temperatures, the other catalysts had increasing reactivity in the order of F > Ba = E > G > D.

Table III.1. 2: Evaluations of iridium(I) catalysts

For catalysts Ba (entry 10), E (entry 20) and G (entry 25), a complete selectivity in favour of position B was observed at -30°C. It is further noted that for catalysts A, E, G and F there are no big differences in the outcome of the HIE reaction of 25 or 0°C. These reactions clearly indicate that HIE reactions at lower temperature have a chance to change the reactions outcome and should be studied more intensively in the future. As a prove of concept, we were interested to demonstrate how this knowledge of the labelling specificity and selectivity can be applied to industry and influence the efficient isotope chemistry planning. Indeed, any labelling request is expected to be delivered as soon as possible. Therefore, speed is the crucial parameter in industry and academic research. The time and difficulty for labelling a drug candidate strongly depends on knowledge of the catalysts and their reactivities. Improved prediction of potential HIE outcomes, or in other words to pick the best catalyst and reactions conditions, is increasing the probability of success for a fast delivery of the target compound. Therefore, we selected five more complex molecules in order to validate our strategy.

III.1.3. Application on complex drug-like compounds

One interesting example for the different reactivity of iridium catalysts is described in scheme III. 1.4. With catalyst F at 25°C, we have obtained selectively deuterium incorporation in the aromatic position A of phenylacetic amide 11. 5 Interestingly, with catalyst Ba we observed no deuterium introduction at all at 25°C. However, when we increased the temperature to 80°C, the deuteration in both positions A and B was obtained, indicating that by increasing the temperature both activation energies H for the transitions states of the pathways for either C(sp 2 )-or C(sp 3 )-carbons are sufficient. The labelling of glycine C(sp 3 )positions with catalyst Ba has been demonstrated by us already (see Part IV). 6 With catalyst F, we obtained only traces of deuterated product, probably due to the prior described inactivation of the catalyst at elevated temperatures (80°C). Unfortunately, we haven't found a catalyst and/or conditions to selectively introduce deuterium at the C(sp

Scheme III.1. 6: HIE reaction of celecoxib 13 with three different iridium catalysts and temperatures

Next, we performed HIE reactions with glibenclamide 14 (scheme III.1.7), an inhibitor of the ATP-sensitive potassium channels (KATP). With catalyst G at 100°C selectively only position A was deuterated, while with catalyst E, as reported before, 7a both positions A and B (59%D and 83%D) with favoring of deuterated position B was obtained. Both catalysts (E, G) showed no reactivity in reactions at 25°C.

In an internal Sanofi research project, a HIE reaction with compound 16 in the presence of an alkyne moiety was tried. Unfortunately, no reaction conditions could be found to perform a successful HIE exchange without reduction of the triple bond. Triple bonds are generally easily reduced by platinum, palladium and nickel. Considering the lower reactivity of iridium in hydrogenation processes, it was anticipated that iridium catalyst could be inactive enough towards the unwanted hydrogenation side-reaction. However, in first experiments at room temperature, we obtained the deuterated product with up to 8-10 additional mass units indicating that the triple bond was mainly reduced by deuterium. Therefore, we intended to overcome this hydrogenation side reaction by performing the HIE reaction at low temperatures (0 to -30°C, scheme III.1.9). Unfortunately, even though we tested all catalyst A-G, we couldn't circumvent the triple bond reduction. As the triple bond was crucial to proceed with the research project, the decision was taken to stop. The idea to find a more suitable application for HIE reactions with catalysts A-F at low temperatures (<0°C) was not further followed due to other priorities.

Scheme III.1. 9: HIE reaction of building block 16 with iridium catalysts A-G at low temperatures

III.1.4. Conclusions

We have demonstrated that, by using the optimized combination of iridium catalysts A-G and varying reaction temperatures (-60 to 130°C), different HIE reaction outcomes can be achieved in terms of efficiency, specificity and selectivity. Notably the temperature window for HIE reactions with most studied iridium catalysts A-G was surprisingly broad. To use the right catalyst with the ideal reaction conditions, is the trigger to increase the selectivity or the deuterium incorporation. Rising the reaction temperature to a maximum prior facing catalyst deactivation is the key for maximum deuterium incorporation into the target molecule. On the other hand, decreasing the temperature can circumvent unwanted sidereactions (hydrogenation) or substrate decomposition. Therefore, it should be possible to tune either regioselectivity and catalyst HIE activity. However, more studies in this context need to be done in the future. Even though these iridium complex catalysts seem to be remarkably efficient to perform HIE reactions at higher temperatures methodological studies at elevated temperature are still few in number. 6,7,8 Furthermore to our knowledge there is only one study on HIE reactions at -20°C. 9 Based on our results, we suggest to start the HIE optimization experiments with catalysts Ba, E and G at room temperature and at 80°C in parallel.

Supporting information Part III -Chapter 1

Materials: see Annex 1

General deuteration procedure (Annex 1):

-Method A (table III.1.1): substrate 8 (1 eq.), catalyst A-G (5 mol%, 0.05 eq), DCM (3 mL), D2 (1 atm), 4 h, indicated temperature from -60 °C to 25 °C. After four hours the reaction was stopped by evacuation of the flask and evaporation of DCM. The products were analyzed by LC-MS and 1 H-NMR without purification needed. -Method B (table III.1.2): substrate 8 (1 eq.), catalyst A-G (5 mol%, 0.05 eq), chlorobenzene-D5 (3 mL), D2 (1 atm), 4 h, indicated temperature from 25 °C to 130 °C.

After four hours the reactions were stopped by evacuation of the flask and the products were analysed directly by 1 H-NMR and LC-MS.

-

Deuteration results (tables):

See Annex 1 for the details about the deuterium incorporation calculations showed in the tables III.

Introduction

As previously described in Part I-II, numerous HIE protocols utilizing homogeneous (or heterogeneous) catalysts have already been described. 1,2 Even though a new generation of bidentate iridium catalyst systems such as those from Pfaltz D, 3 Burgess E, 4 and Tamm F, 5 have extended the scope of the aromatic ortho-directing HIE reactions, the commercially available Crabtree's A 6 and Kerr's catalyst Ba 7,8 stay the most regularly applied iridium catalysts in radiochemistry laboratories today. Nevertheless, while there are numerous publications on the use of different directing groups such as ketones, esters, amides, carboxylic acids, nitro groups, sulfones, sulfonamides etc., there is still a lack of understanding and prediction for how the different directing groups enable HIE if more than one directing group is present in the molecule (scheme III.2.1). This is especially important as the introduction of a radioactive label in a metabolically stable position is essential for the application of tritiated compounds for in vivo experiments. 9 In comparison to 14 C-labelling, where a loss of the radioactive marker is less frequent, the loss of tritium can occur by simple metabolic enzymatic hydroxylations (see Part I). 9 Therefore, it would be useful for synthetic chemists to be able to predict the labelling position in complex molecules, instead of performing multiple try and error experiments.

Scheme III.2. 1: Concept of prediction about iridium-catalyzed ortho-directed aromatic HIE reactions

How: [H]-1 1

Experiments and DFT calculations

To evaluate the potential scope of such a prediction, we were motivated to perform a comprehensive analysis of the most commonly occurring directing groups in the top 200 most prescribed drugs in 2016, in particular those containing aromatic rings as reported by Njardarson et al. (figure III.2.1). 10 We have identified several functional groups, for example, imidazole (N-heterocycle), ketones, esters, amides, carbamates, acids, nitro groups, sulfones, sulfonamides, phenols, etc. repeatedly being present in aromatic drug molecules.

Based on these outcomes, we have studied the influence of the previously mentioned functional groups as a directing group in a standard HIE reaction with deuterium gas as the isotope source. 11

III.2.2. Competition HIE reactions study

To achieve our aim, initial experiments have been run under standard HIE conditions applying the commercial Kerr catalyst Ba (1 atm D2, rt, dichloromethane, 2h) to make our results as comparable as possible with literature results. 12 We conducted a series of competition experiments, 13 employing two simple mono-substituted aromatic substrates in one reaction flask, along with catalyst Ba, to examine which of the two directing groups is giving a better result in deuteration in the HIE reaction (scheme III.2.2). Applying this methodology to aromatic compounds 2-10, we have obtained the results presented in table III.2.1.

Scheme III.2. 2:

Competition HIE experiment of aromatic compounds with catalysts Ba a a) Conditions: substrate X, Y (1 eq.), catalyst Ba (5 mol%, 0.05 eq.), DCM (6 mL), D2 (1 atm), rt, 2h. a) Conditions: substrate X,Y (1 eq.), catalyst Ba (5 mol%, 0.05 eq.), DCM (6 mL), D2 (1 atm), rt, 2h. b)

The analysis was done either by mass spectrometry (if substrate ionizable and no overlapping between the two substrates) or by 1 H-NMR.

The competition HIE experiment between substrates 4 and 10 for example, showed 91%D incorporated in substrate 4 and only 7%D incorporated in substrate 10. As a conclusion, the DG of 4 is predominant compared to 10 regards to its reactivity in HIE reaction with catalyst Ba. The difference of deuterium incorporation in substrates 5 and 6, 62%D and 69%D respectively, is very small so these two DGs can be considered as equal in their capacity to direct HIE with catalyst Ba.

0%D 0%D 0%D 0%D 0%D 5%D 0%D 2%D 2 82%D 89%D 89%D 82%D 80%D 85%D 78%D 85%D 89%D 21%D 22%D 15%D 10%D 8%D 11%D 3 87%D 73%D 78%D 86%D 66%D 81%D 89%D 69%D 56%D 50%D 12%D 25%D 7%D 4 82%D 87%D 77%D 91%D 91%D 91%D 69%D 25%D 21%D 9%D 9%D 5 62%D 31%D 44%D 88%D 92%D 28%D 16%D 6%D 2%D 6 44%D 77%D 77%D 78%D 10%D 3%D 13%D 7 34%D 33%D 20%D 11%D 12%D 8 39%D 59%D 10%D 9 8%D
The interpretation of the data provided in table III

III.2.3. DFT calculations and energy profiles for insights into the competition study

The interpretation of the previous results can allow an estimation on the prediction of the labelling positions with catalyst Ba, in more complex molecules containing these directing groups. However, to understand the theoretical background of our proposal, density functional theory (DFT) calculations, 15 have been done by Dr. Stefan Güssregen from the Computational Drug Design group in Sanofi. These calculations were following the approach applied by Kerr et al. 16,17,18 based on the mechanism suggested by Heys. 19 It is important to remember that the Gibbs free energy G is defined by the following relation: G=H-TS (H=enthalpy and S=entropy). The more the G value is negative, the easier the reaction is. The enthalpy H represents the activation energy needed to form the transition state 11b from the coordination complex 11a (see figure III.2.2). Based on the Gibbs free energy equation, we can see that the temperature T is also playing an important role and this will be discussed below. The calculated G values are given in table III.2.2 and sorted by their Grel energy values for their formation of coordination complex 11a (table III .2.2). For comparison, we also added the activation energy H for the CH-activation step (11a=>11b), showing that there are significant differences between these two parameters.

Based on the data from table III.2.2, the free energy profiles have been constructed (with catalyst Ba), and shown in figure III.2.2, for the first leg step of the HIE reaction. The activation energy H (11a => 11b) is known to be the main parameter for explaining formation of the deuterated products. 18 The theoretical G values given in table III.2.2 are in adequacy with the experimental order found in scheme III.2.3, except for the DG nitro 9 interestingly. In these competition experiments, which take place at room temperature, the relative populations of the coordination complex 11a (G) is strongly influencing the outcome of the overall reaction. Once the coordination complex 11a has been formed, the reaction will proceed to completion, via transition-state 11b (H) and insertion product 11c. This is reflected in the order of the calculated relative energies of the coordination complex 11a, which matches very well the order experimentally observed. For those similar coordinating directing groups where the coordination complexes are populated almost equally, relative energies of the transition-states become dominating again. This can be seen nicely for compound pairs 4 vs 6, 7 vs 8 and 9 vs 10. This is a significant contribution to the understanding of CH activation mechanism processes with complex substrates. In competition cases it is not the activation barrier 20,17,18 between the coordination complex 11a and transition state 11b responsible for the HIE reaction outcome, but the lower energy of the initially formed coordination complex 11a. Overall, table III.2.2 summarizes most of the common directing groups in Drug Discovery and therefore should be very helpful for synthesis planning.

III.2.4. Prediction and experimental prove in HIE reactions of disubstituted molecules

After the success of the competition experiments, we sought to obtain more insight into the role of competing directing groups in the same molecule. We studied aromatic compounds 12-22 with at least two different substituents. Due to the higher complexity of 1 H-NMR analysis in 1,2-or 1,3-disubstituted benzenes, we focused on 1,4-disubstituted ones (scheme III.2.4). Furthermore, the H values of each of the two directing groups have been calculated. These H values are shown below the respective substrates in scheme III.2.4. The higher the difference in H energy between each of the two directing groups is, the higher the difference on deuterium incorporation is expected to be. It is highlighted by a yellow star in the scheme which of the two directing groups to be the predominant one. This is calculated by subtraction of the coordination complex 11a energies H of both DGs. By comparing the experimental results with the predicted energy difference in the coordination complex, we were hoping to obtain a rational correlation.

All predicted results of the deuteration of 12-22 support the reported order obtained in the competition experiments (table III.2.1 and scheme III.2.3). However, by this method we are able to predict the main product but not the final deuteration result in %D. Indeed, if we look at compound 18, the H value (6.2 kcal/mmol -1 ) is high, therefore we expected a high difference between the deuterium incorporation of both DG, with a higher incorporation in ortho-position of the aldehyde. This is true as more deuterium has been incorporated in this position (52%D against 19%D in ortho-position of the sulfonamide). Nevertheless, the deuteration ratio between the two positions is not dramatically high. For comparison, compound 19 with a lower H value (2.2 kcal/mmol -1 ) than for compound 18 (6.2 kcal/mmol -1 ), shows a higher ratio (97:22) in deuterium incorporation between the two ortho-positions.

Compounds 14 and 15 have been run at 0°C to see if the prediction would correlate better with the experiment. Interestingly, it has shown than by lowering the reaction temperature to 0°C, the selectivity of deuteration was shifted in favor of the ketone as the main directing group. This strongly suggests that differences in regioselectivities in the HIE reactions play a much bigger role at temperatures below 0°C.

III.2.5. Prediction and experimental prove in HIE reactions of complex molecules and drugs

Finally, we have performed HIE reactions catalyst Ba on complex molecules 23-31 where at least two different directing groups were present (scheme III.2.5). The red arrow is showing the predicted main deuteration position based on our order from table III.2.1 and scheme III.2.3. The deuterium incorporation in blue is the experimental result. In all cases shown, we were able to predict the correct labelling position. However, as this is a thermodynamic model there are many other factors such as solubility, salts and impurities can which can influence the HIE outcome.

Scheme III.2. 5: HIE experiment of drugs 23-31 catalysts Ba a

a) Conditions: substrate (1 eq.), catalyst Ba (5 mol%, 0.05 eq.), D2 (1 atm), DCM (3 mL), rt, 2 h.

III.2.6. Conclusion

The work presented in this chapter have enabled us to elaborate a clear order of directing group influence on HIE reactions with catalyst Ba. With DFT calculations, we have identified the iridium-substrate coordination complex to be the main trigger for reactivity and selectivity, in the competition situation with two and more directing groups. Only if the energies of two competing coordination complexes are very similar, the formation of the CH activation transition-state became the most demanding parameter.

These results are not only allowing predictions in regioselective HIE reactions of complex molecules, but also give an outreach to develop the concept of directing group-induced latestage functionalization in general. Understanding the influence of the directing groups could allow a much better planning and successful prediction of late-stage functionalization pathways. Such a prediction methodology could save time and resources in drug discovery research or could even broaden the applications of tritiated molecules to new fields of the life sciences. Most generally this would make iridium catalysed HIE reactions a significantly improved tool in isotope chemistry.

In summary, this finding could be a general principle to understand competition situations of different directing groups in one molecule and can be probably transferred to further transition metal catalyzed CH functionalization reactions. Other research groups have found empirically similar regioselectivities in directed CH functionalization competition reactions for C-C or C-X bond formations in complex molecules, 21 either applying catalysts like iridium, 22 rhodium, 23 palladium, 24 cobalt, 25 or ruthenium 26 metals. There is furthermore a standing interest to establish convenient prediction models. 27

Result:

1) 86% D for compound 2, 1% D for compound 6.

2) 78% D for compound 2, 0% D for compound 6.

Result:

1) 83% D for compound 2, 8% D for compound 8.

2) 87% D for compound 2, 3% D for compound 8.

Result:

1) 92% D for compound 4, 4% D for compound 10.

2) 90% D for compound 4, 9% D for compound 10.

Result:

1) 81% D for compound 3, 11% D for compound 9.

2) 82% D for compound 3, 5% D for compound 9.

Result:

1) 90% D for compound 3, 11% D for compound 10.

2) 88% D for compound 3, 2% D for compound 10.

Result:

1) 22% D for compound 5, 21% D for compound 7.

2) 39% D for compound 5, 30% D for compound 7.

Result:

1) 82% D for compound 4, 69% D for compound 5.

Average: 82% D for 4, 69% D for 5.

Result:

1) 77% D for compound 6, 11% D for compound 8.

2) 77% D for compound 6, 14% D for compound 8.

Result:

1) 76% D for compound 6, 6% D for compound 9.

2) 78% D for compound 6, 7% D for compound 9.

Average: 77% D for 6, 6% D for 9.

Result:

1) 43% D for compound 7, 10% D for compound 8. Average: y=97%, 96% D at position a, 96% D at position b.

Method B (-15 °C): 10 mg, 56 µmol 14, 2.9 mg, 2.8 µmol catalyst Ba.

LC-MS (positive ESI): m/z 178.2 [M+H] + (31%), 179.2 [M(1D)+H] + (58%), 180.1 [M(2D)+H] + (11%).

Yield: 1) 9.9 mg, 56 µmol, 99%; 27% D for δ 7.90, 2% D for δ 7.69.

2) 9.1 mg, 51 µmol, 91%; 37% D for δ 7.90, 9% D for δ 7.69. H NMR (300 MHz, DMSO-d6: δ 8.08 (d,3 J= 8.3 Hz,2H,Ha),7.86 (d,3 J= 8.3 Hz,2H),7.80 (d,3 J= 8.7 Hz,2H),7.30 (d,3 J= 8.7 Hz, 2H, Hb), 3.92 (s, 3H), 2.34 (s, 3H) ppm. Incorporation expected at δ 8.08 and/or δ 7.30 ppm (red arrow). Determined against reference-integral at δ 2.34 ppm (blue arrow).

Method B: 10 mg, 37 µmol 16, 1.9 mg, 1.8 µmol catalyst Ba. Yield: 1) 10 mg, 37 µmol, 100%; 55% D for δ 8.08.

LC-MS (positive ESI

2) 8.3 mg, 31 µmol, 83%; 42% D for δ 8.08. 1 H NMR (300 MHz, DMSO-d6): δ 10.33 (s,1H), 9.85 (s,1H),7.84 (d,3 J= 8.7 Hz,2H,Ha),7.78 (d,3 J= 8.7 Hz, 2H, Hb), 2.09 (s, 3H) ppm. Incorporation expected at δ 7.84 and/or δ 7.78 ppm (red arrow). Determined against reference-integral at δ 2.09 ppm (blue arrow). Yield: 1) 9.8 mg, 60 µmol, 98%; 58% D for δ 7.84, 78% for δ 7.78.

2) 9.9 mg, 60 µmol, 99%; 59% D for δ 7.84, 79% for δ 7.78.

Average: y=99%, 58% D at position a, 78% D at position b. (d,3 J= 8.6 Hz,2H,Ha),7.59 (d,3 J= 8.6 Hz,2H,Hb) 1 H NMR (300 MHz, MeOD): δ 8.78 (d,3 J= 8.6,1H,CH(d) or CH(g)), 8.44 (s, 1H, CH(c)), 8.19 (d,3 J= 8.6 Hz,1H,CH(d) or CH(g)), 8.09 (d,3 J= 8.5,2H,2 CH(b)), 7.84 (m, 1H, CH(e) or CH(f)), 7.67 (m, 1H, CH(e) or CH(f)), 7.40 (d,3 J= 8.5,2H,2 CH(a)), 2.45 (s, 3H, CH3) ppm. Incorporation expected at δ 8.09 ppm (red arrow). Determined against reference-integral at δ 2.45 ppm (blue arrow). Yield: 1) 4.9 mg, 12.0 µmol, 98%; 70%D for δ 7.81 and 67%D for δ 7.56.

Part IV Homogeneous iridium catalysis on C(sp 3 )-H centers

IV.1. Isotopic labeling of the drug payload of antibody-drug conjugates used for cancer treatment

As shown in the previous Part II-III, we were able to develop new efficient, fast and simple methods of HIE on C(sp2)-H carbon centers using iridium catalysis. Interestingly the hydrogen isotope labelling methods on C(sp3)-H carbons is in comparison to aromatic C(sp2)-H carbons still rarely described. 1 Within a Sanofi research project there was a specific request for labelling a maytansinoid DM4 1a involved in a serie of antibody drug conjugates (ADCs) (figure IV.1).

Figure IV. 1:

Structure of DM4 1a-c and DM1 2.

N O O O O Cl N O O N H O O OH O H H S R N O O O O Cl N O O N H O O OH O H H SH DM1 2 DM4 1 R = H R = Me R = SMe 1a 1b 1c

IV.1.1. Application of ADC for treatment in oncology

Antibody-drug conjugates have become promising anticancer treatment agents that combine the selectivity of targeted treatment with the cytotoxic potency of chemotherapy drugs. 2,3 They are composed of a monoclonal antibody linked to a biologically active cytotoxic drug. The monoclonal antibody targets the antigen present in high quantity at the surface of the tumor cells whereas its expression is limited on healthy cells. The linker is stable in blood circulation and able to release the active drug into the tumor cells due to an internalization process (endocytosis). The drug with a highly potent cytotoxic activity is able to trigger the apoptosis process of the cell (figure IV. 2, left). By combination of the unique targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, ADCs allow unlike standard chemotherapy, the sensitive discrimination between healthy and cancer tissue. This huge advantage compare to unselective treatments, which are damaging healthy cells around, allows the injection of a higher dose of the complex which gives a larger window of action (figure IV.2, right).

After injection of the ADC in the blood circulation (figure IV.3, step 1), the antigen binding site of the antibody will bind specifically the antigen present on the tumor cell (step 2). The endocytosis will allow the ADC to be internalized in the cell via the endosome pathway (step 3), which will maturate as a lysosome during the trafficking of the vesicle (step 4). The acidic media present in the lysosome will enable to release the cytotoxic drug in the cytoplasm (step 5) and trigger the apoptosis of the cancer cell (step 6).

Figure IV. 3: Mode of action of the antibody drug conjugate

IV.1.2. The Maytansine derivatives as ADCs

Maytansine, originally isolated from the bark of the Ethiopian shrub Maytenus ovatus, 4 displayed highly potent cytotoxic activity with IC50 values in the picomolar range resulting from its ability to disrupt microtubule polymerization. 5 Maytansine was extensively evaluated in human clinical trials, but failed to demonstrate a therapeutic benefit at tolerable doses. 6 However, maytansine analogues (maytansinoides) modified by ImmunoGen Inc. for conjugation with antibodies through disulfide-containing linkers enabled the development of antibody-maytansinoid conjugates. 7 9,10,13 IV.1.

Former approach for the synthesis of 3 H-DM4

With regards to the isotope labelling of DM1 2, DM4 1a and the subsequent antibody conjugation, tritium is the label of choice because of the approx. 500 times higher specific activity (28.8 Ci/mmol) achievable as compared to a single carbon-14 label (62.4 mCi/mmol). 12 Thus, several reported studies utilized tritium tracer with a tritium label at the C-20 methoxy group of the maytansinoid, which proved to be a metabolically stable position. 13 However, the synthesis of [methoxy-3 H]DM1 or [methoxy-3 H]DM4 is quite tedious, comprising at least 5 partly low yielding radioactive steps as well as a fermentative demethylation. Although utilized and referenced several times, no detailed procedures have been described in literature to date. 12 Consequently, we became interested in developing an alternative synthesis approach for the preparation of 3 H labelled DM4 1 for decreasing precursor costs and synthetic efforts.

Typically, the preparation of maytansinoid analogues labelled with tritium at the C-20 position starts from ansamytocin P-3 3 produced by fermentation of the bacterial actinomycetes strain Actinosynnema pretiosum (scheme IV.1). 14 First the methyl ether of the methoxy group in ansamytocin P-3 3 was removed in 30% yield applying Streptomyces platensis, another actinobacteria strain followed by extensive preparative HPLC purification.

The resulting phenol moiety of PDM-3 4 is then re-alkylated with 3 H-methylnosylate (specific activity >80 Ci/mmol) to obtain 3 H-ansamitocin P3 5 in 71% yield. Depending on study requirements, at this stage the specific activity can be adjusted (e.g. to <1 Ci/mmol) by dilution with unlabeled ansamitocin P3 to increase the scale for subsequent reaction steps following previously described pathways for DM1 2 and/or DM4 1a synthesis. 15 Hydrolysis of the C3 isobutyrate ester is achieved under reductive conditions using DIBALH to obtain 3 Hmaytansiol 6 in 92% yield.

A significantly improved diastereoselectivity for the acylation of maytansinol 6 is achieved by applying a two-step procedure for the installation of the side chain. Firstly 3 H-maytansinol 6 is acylated with the N-carboxyanhydride (NCA, 7), generated from Boc-NMe-Ala-OH with PCl3, followed by coupling with an activated ester of 4-methyl-4-(methyldithio) pentanoic acid 9. However, the acylation of the maytansinol 6 with N-carboxy anhydride (NCA, 7) of Nmethylalanine proved to be crucial due to moisture sensitivity and sometimes incomplete conversion. The small amount of the undesired diasteromer formed in this step was separated by preparative HPLC to obtain pure L-DM4-SMe 1c in 35% yield. Finally, cleavage of the disulfide with Cleland's reagent 16 (dithiothreitol, DTT) in phosphate buffer at pH 7.5 and subsequent preparative HPLC purification afforded 3 H-DM4 1a with an overall yield of 7% in five radioactive steps. Depending on the anticipated DAR values (drug antibody ratio) the specific activity may have to be further adjusted prior to conjugation to the respective antibody. According to the approach described above, the Sanofi team had prepared several 3 H-DM4conjugated ADCs for in-vivo metabolism studies. Throughout these ADME studies, it appeared that the stability of 3 H-DM4 1a, particularly at higher specific activity, was quite limited and that the compound completely decomposed after three months. Thus, a resynthesis was needed from time to time. However, revisiting the lengthy and costly synthesis of 3 H-DM4 1a, we evaluated potential alternative options such as hydrogen isotope exchange. 17

Scheme IV. 1:

Conventional Synthesis of 3 H-DM4 1a O Cl N O O O N O O OH O H H O H 3 H 3 H 3 O H Cl N O OH O N H O O OH O H H H 3 H 3 H 3 H 3 H 3 H 3 N O O O O Cl N O O N H O O OH O H H S S H 3 H 3 H 3 N O O O O Cl N O O N H O O OH O H H SH O H Cl N O O O N O O OH O H H O O Cl N O O O N O O OH O H H O O N O O O S S O N O O H 3 H 3 H 3 NH O O O Cl N O O N H O O OH O H H 3 H-MeONOS

IV.2. Iridium-catalyzed hydrogen isotope exchange on L-DM4

During this project we have worked mainly with the derivative DM4 (-SMe) 1c which was chemically more stable than DM4 (-H) 1a and was neither prompt to uncontrolled oxidation nor side-reaction with the catalyst through metal chelation by the thiol moieties. We have also worked with DM4 (-Me) 1b which is chemically more stable but as the following step after isotope labelling is the conjugation, the disulfide bond S-S has to be deprotected easily to provide the free S-H.

To investigate conditions for direct HIE on DM4 1c, a catalyst screening had been done previously, 18 using commonly available iridium catalysts known to show success in HIE (table IV.1). Table IV. 1: Condition screening on DM4 1c in HIE reaction a,b. 28 From the catalyst screening, no success was achieved (table IV.1, entry 1-5) except with catalyst Kerr Barf Bb where a slight reactivity started to be observed (entry 6). Keeping the catalyst Bb, the solvent has been changed and whereas THF (entry 7) and chlorobenzene (entry 8) gave no deuterium incorporation at all, isopropyl acetate allowed a very moderate incorporation up to 0.2 D/molecule (entry 9). When heating up the reaction to 50 °C, a remarkable deuterium incorporation reaching 2.0 D/molecule has been found (entry 10). A similar incorporation was obtained by running the reaction at room temperature but for 20 h (entry 11). The very high catalyst loading chosen for the initial experiment reflects the urgent need in labelled LMD4 for the implementation of a Sanofi project.

N O O S O O Cl N O O N H O O OH O H H S N O O S O O Cl N O O N H O O OH O H H S D D 2 (1 atm) LDM4-SMe D x -LDM4-SMe 1c D x -1c
With regards to the functionalities present in DM4 1c, the labelling was expected to occur on the aromatic ring in ortho-position of the acetanilide, even though the nitrogen was methylated. Surprisingly, after deuteration, the aromatic proton signals in the 1 H-NMR spectrum of 1c appeared unchanged. However, a clear assignment of the labelling position was not possible, because of a highly complex aliphatic region in the 1 H-NMR spectrum of 1c. Therefore, to run a tritium experiment under the same reaction conditions to enable 3 H-NMR for determination of the labelling position was decided (figure IV.5). Interestingly, a very selective tritium incorporation was found into the side chain of 1c with a 94 to 6 ratio in favor of the -carbonyl position. This is particularly interesting because iridium catalysed HIE usually results in highly ortho-selective exchange of aromatic positions. H/T exchange of sp 3 -centers has been observed only occasionally, often with low selectivity or specific activity. 19 In our case, a specific activity of 31 Ci/mmol was obtained which was more than sufficient for the planned in-vivo studies with 3 H-DM4 and corresponding 3 H-ADCs.

The identification of the labelling position through analysis of the tritium experiment prompted us to apply the optimum conditions described above on the three DM4 1a-c derivatives. The HIE reaction with deuterium ran on these three compounds have given very good results in LC-MS up to 2.0 D/molecule (figure IV.6). Motivated by these exciting initial results and impressed by the high regioselectivity, we became interested whether we could develop a real methodology of hydrogen isotope labelling on C(sp3)-H centers.

IV.3. Methodology development of Hydrogen isotope Exchange on sp 3 -carbon centers

IV.3.1. Methodology development on the sulfur side chain

We started our investigation using model substrate [H]-10 containing the sulfur side-chain and a benzyl group which was making the compound UV-visible. Moreover, ionization on the nitrogen renders possible the LC-MS monitoring of [H]-10 with regards to the deuterium incorporation.

The conditions developed previously have shown to be the best for the labelling of DM4 1a. Nevertheless, we still evaluated these conditions in a more general way on model substrate [H]-10.

IV.3.1.a) Conditions screening of [H]-10 in HIE reaction

The conditions screening started by evaluation of the iridium catalysts activity towards the HIE of [H]-10 (table IV .2). We observed no deuterium incorporation at all with catalysts F (entry 5) and G (entry 6), and between 0.5 and 0.8 D/molecule with catalysts A (entry 1), Ba (entry 2) and E (entry 4). The best result was obtained with catalyst Bb (10 mol%) which enabled deuterium incorporation up to 1.9 D/molecule at 50 °C (entry 3) and up to 2.0 D/molecule at 80°C (entry 7). Keeping a temperature of 80 °C, the reaction run with 5 mol% of catalyst Bb gave only 0.6 D/molecule as maximum deuterium exchange (entry 8). We studied the influence of the solvent (table IV.3) through a comparison between chloroform (entry 1), MTBE (entry 2), ethanol (entry 3), MeTHF (entry 4), and chlorobenzene (entry 6). Finally, isopropyl acetate (entry 5) provided the highest deuterium incorporation in 10.

After identification of the optimum conditions (10 mol% of catalyst Bb reaction at 80 °C in isopropyl acetate), we have applied them to diverse DM4 side chain precursors.

IV.3.1.b) Application of the developed methodology on DM4 side chain precursors

Our optimized conditions have been applied first to side chain precursors 11 and 12 (scheme IV.2). We obtained complete deuterium incorporation of 95-99% for these simple amide derivatives. The reaction is very selective for the -amide carbon with no deuterium incorporation into the aromatic or benzyl-position in 10. We were interested to apply our conditions to the Ir-catalyzed HIE on a precursor which looks even more like the original DM4 1c containing and amino acyl moiety. Three precursors have been tested: the alanine derivative 13, the serine derivative 14 and the glycine derivative 15 (scheme IV.3). During the screening of these DM4 side-chain model substrates, an interesting observation attracted our attention. The very high -selectivity still observed for 13 and 14 (99 %D and 95%D respectively) was lost in the glycine containing substrate 15. In this specific case, two positions were reactive with a clear preference for the -glycine protons (84%D) in comparison to the -amide position (36%D).

Scheme

By going further with our investigation, steric and electronically modified derivatives 16 and 17 have been tested (scheme IV.3). A complete change in selectivity in favor of the α-amino acid protons was observed in the glycine conjugates 16 and 17. In both cases excellent deuterium incorporation in the glycine position was observed while the -amide position was not labelled at all. Obviously even small steric changes in the substrates may have a strong effect on the selectivity and outcome of the HIE which triggered us to investigate this reaction on amino acid derivatives in more detail. a) Conditions: substrate (1 eq.), catalyst Bb (10 mol%), D2 (1 atm), isopropylacetate (1 mL), 80°C, 3h.

Scheme IV. 3: HIE reaction of DM4 side chain precursors 13-17 with catalyst

IV.3.2. Investigation of the small amino acids reactivity in HIE

IV.3.2.a) Influence of the N-terminal protecting group

The influence of the protecting group in glycine methyl ester 18 has been investigated (table IV .4). Unfortunately, high deuterium incorporation was observed only for acetyl (18a), while more synthetically versatile protecting groups such as Cbz 18b or Fmoc 18c resulted only in moderate deuteration. For Boc 18d or Bn 18e no deuterium incorporation was observed under these conditions. More insights are given from the DFT calculations (see section IV. 3.4). a) Conditions: substrate (1 eq.), catalyst Bb (10 mol%), D2 (1 atm), isopropylacetate (1 mL), 80°C, 3h.

Table IV. 4: Influence of the N-protecting group on the deuterium incorporation in HIE of

IV.3.2.b) Analysis of the stereocontrol in HIE reaction of alanine

As the stereo information in amino acids is crucial and must be conserved during the reaction, we tested our HIE method on the protected L-alanine 19a and D-alanine 19b. On top of analyzing the deuterium incorporation, we have looked by enantioselective HPLCseparation if the original stereo information was kept in both cases.

IV.3.2.c) Application of the HIE method on sterically more demanding amino acids

Sterically more demanding amino acids such as Ac-Leu-OMe 20 or Ac-Phe-OMe 21 displayed no deuteration at all (scheme IV.4). In contrast Ac-Gly-OMe 22 gave a high deuterium incorporation (91%D) similar to what was observed on glycine 18a. We have noticed from these results that our method was working mainly on the glycine moiety. This prompted us to investigate larger glycine-containing peptides.

Scheme

IV.3.3. Application of the optimized method in larger peptides

We started by applying our conditions on dipeptides with N-terminal glycine moiety (scheme IV.5). The dipeptide (L)-Ac-Gly-Phe-OMe 23 was selectively labelled at the glycine position with 55%D. We looked at the HIE on the dipeptides (L)-Gly-Phe methyl ester, as N-Cbz (24), N-Fmoc (25) and N-Boc (25) protected. In accordance to what we have observed already on the Ac-Gly-OMe 18a-e (table IV.4), the deuterium incorporation dropped to 14%D in 24 (N-Cbz) and there was no deuteration at all for 25 (N-Fmoc) and 26 (N-Boc). By using the acetyl protection group, which had demonstrated to be the best one for deuterium incorporation, we have obtained up to 93%D in (L)-Ac-Gly-Tyr(OtBu)-OtBu 27. From these examples, we have noticed that the steric demand of the C-terminal amino acid does not influence strongly the deuterium incorporation. The high selectivity for glycine brought us to test the deuteration of the Ac-Gly-Gly-OMe dipeptide 28 and in this case the deuterium was incorporated on both glycine with 56%D (N-Terminal) and 37%D (C-Terminal).

Considering this result, we thought to make one of the method limitations a strength. Indeed, we have seen that the Boc protecting group was inhibiting the deuterium labelling on the N-terminal glycine (18d and 26), and with the result on 28, compound 29 has been investigated. Successfully, 29 has been labelled with up to 52%D selectively at the C-terminal glycine.

The results obtained in the different dipeptides 23-29 showed that the regioselectivity of the deuterium labelling can be tuned by the choice of protecting group. We became interested to look at more dipeptides containing a C-terminal glycine. As expected, the selectivity of the HIE reaction for the C-terminal glycine was confirmed on dipeptides 30-34 when applying our reaction conditions (scheme IV.6). This effect is even more pronounced for more sterically demanding N-terminal amino acids, resulting in a good deuterium incorporation for Boc-Ala-Gly-OMe 30 (60%D) to very good deuterium incorporations for Boc-Leu-Gly-OMe 31 (74%D) and Boc-Phe-Gly-OMe 32 (92%D). In line with previous findings (22), protons in both the -glycine and -amide positions of Boc-Gln(NEt)-Gly-OMe 33 were exchanged. Interestingly in Boc-Pro-Gly-OMe 34, the deuterium labelling happened at the n+1 amino acid position relative to glycine (93%D) and to proline (36%D). Even though the N-terminal proline was Boc-protected, the deuterium labelling in the n+1 position occurred. It is known that the proline is giving a different 3-Dimensional geometry to a peptide chain. We postulate that the proline geometry and the connected amide or carbamate bonds cis:trans effect (20:80; higher cis amide bond isomer compared to other amino acids where it is 5:95) enables the -carbon of the proline to be in closer interaction with the iridium catalyst, and by allowing deuterium incorporation in this position. 20 To our great delight, the method can be extended to longer peptides as demonstrated by the excellent D incorporation obtained for tripeptide Cbz-Tyr(OtBu)-Gly-Leu-OtBu 35 (94%D) and tetrapeptide Boc-Trp-Gly-Ala-Phe-OMe 36 (95%D) (scheme IV.7). Like for 33, the amide protons in 36 also exchanged well. A slight deuterium incorporation was observed in dipeptide 37 (23%D) which is aligned with the values obtained in table IV.4 on the Fmoc protected glycine 18c.

Scheme

Unexpectedly, selective HIE reaction have occurred in the -carbon of the methionine in dipeptide 38 (95%D). Unfortunately, we observed no deuterium incorporation in the -carbon of the serine or the tyrosine in dipeptides 39 and 40, respectively.

Scheme IV. 7: HIE reaction on peptides

IV.3.4. Insights in the mechanism

DFT calculations (M06 functional) have been done by Dr. Stefan Güssregen from the Computational Drug Design group in Sanofi, following the approach successfully applied by Kerr et al. 21 based on the mechanism suggested by Heys. 22 Our purpose was to better understand the pronounced glycine selectivity of the HIE reaction. 23 The constructed free energy profile with catalyst Bb and compound 18a is shown in figure IV.8. After initial iridium coordination (40), the insertion product 42a is reached via transition-state 41. The equilibrium between 42a and 42b is going through a low barrier transition-state. The deuterated iridium adduct 44 is formed via the reductive elimination transition-state 43. We found that the relative energies of the transition-states for the oxidative addition step 41 (+23.8 kcal mol -1 ) and the reductive elimination step 43 (+24.1 kcal mol -1 ) are significantly higher than those calculated for HIE reactions with catalyst Bb at aromatic C(sp 2 )-carbons like benzaldehydes (+18.6 kcal mol -1 ). 21a This difference in energy might explain why elevated temperatures are needed (in our cases).

As we have seen in section IV.3.2.a), the Boc protecting group totally inhibits the HIE on glycine 18d, in comparison to the acetyl protecting group in glycine 18a. A comparison of the relative transition-state energies (ΔΔGtrans) for the reaction with catalyst Bb revealed +7.3 kcal mol -1 for Boc-Gly-OMe 18d compared to Ac-Gly-OMe 18a (figure IV.9). Also, a comparison of the relative transition-state energies (ΔΔGtrans) has been done for Ac-Ala-OMe 19a and revealed a +4.3 kcal mol -1 compared to Ac-Gly-OMe 18a. These results are fully consistent with the experimental findings and explain the different deuterium incorporation for 18a (93%D), 18d (0%D) and 19a (50%D).

Furthermore, sterically more demanding amino acids increase the ΔΔGtrans values in thetates in the following order: Gly (18a) < Ala (19) < Phe (21). Finally, we found that the proposed mechanism is well in line with the experimental observations. Our results show that the observed energetic differences of C(sp 3 )-and C(sp 2 )-labelling can be attributed to increased steric demands of the substrate. 24

IV.4. Conclusion

A new method for highly selective hydrogen isotope exchange of C(sp 3 )-centers in aliphatic amides has been developed. Starting from commercially available Kerr-catalyst Bb, the HIE reaction on a series of common linker side chains of antibody-drug-conjugates proceeded with high chemical yields, high regioselectivity and with deuterium incorporations up to 99%D. The scope of the method was further extended to alpha amino acids, especially successful for glycine and alanine (up to 93%D). In di-and tripeptides, a very interesting protecting group-dependent selectivity was observed. This suggest new options for selective labelling approaches by varying the N-protecting group. While with N-acetyl-protecting groups the labelling at the -carbon was observed, this could be completely circumvented by using Boc or Fmoc protecting groups. Furthermore, DFT calculations gave insights into the mechanism and steric requirements of the HIE reaction with glycine (18) and alanine ( 19) derivatives, and explained the observed selectivity and the influence of the amino acid Nprotecting groups. The method is fully translatable to the specific requirements of tritium chemistry discussed in the objectives of the thesis. This latter point was demonstrated through tritium labelling of the maytansine DM4 drug, suggesting that the developed method is a suitable and highly valuable new tool in isotope research.

Ac-Gly-OMe 18a

Boc-Gly-OMe 18d

Catalyst B a Catalyst B a Deuteration of DM4 derivatives 1a-c

N O O S O O Cl N O O N H O O OH O H H R R = H, Me, SMe 1a 1b 1c
Tritiation of DM4-SMe 1c (reaction 1; 7 eq tritium)

The tritiation of DM4-SMe 1c was carried out using a standard Tritec ® tritium manifold. DM4-SMe (4.0 mg, 4.8 µmol) 1c, and catalyst Bb (2.1 mg, 1.2 µmol, 25 mol%) were dissolved in isopropyl acetate (0.7 mL) in a 1.0 mL reaction flask before being connected to the manifold. The solution was frozen in liquid nitrogen and the flask was evacuated, then charged with tritium (1.9 Ci, 61 mbar, 7eq.). The reaction mixture was then allowed to warm to room temperature over 15 min before stirring at r.t. for a further 30 min (168mbar pressure). The reaction mixture was then heated to 50 °C (245 mbar pressure) and stirred for 180 min before cooling again to r.t. The solvents were evaporated; methanol added and the solvent evaporated into a waste ampule. This procedure was repeated three times in total. The residue was dissolved in methanol and filtered. Purification and isolation via HPLC gave the tritiated product (3.1 mg, 3.7 µmol 1c, 96.4% radiochemical purity, 1374 MBq, 0.37 TBq/mmol = 10 Ci/mmol).

MS (positive ESI

Conditions screening (table IV.2 and IV.3)

For the screening of different solvents, catalysts, catalysts loading, temperature and time, the following conditions have been applied on model substrate [H]-10. 4-[(4-methyl)dithio]-4-methyl-N-(benzyl)-pentanamide [H]-10 (14 μmol, 500 μL, 1 eq.) and the catalyst of choice (5 to 10 mol%) were dissolved in 1 mL of the solvent of choice. All reactions were performed according to the general HIE protocol at the temperature and time of choice.

The data presented in the tables have been determined by 1 H-NMR for the labelling position and by LC-MS for the percentage of deuterium incorporation. L-Alanine, N- [4-methyl-4-(methyldithio) .

1 H NMR (300 MHz, CDCl3): δ 6.73 (br s, 1H, NH), 4.65-4.58 (m, 1H), 4.25 (q, 3 J=7.5 Hz, 2H, OCH2CH3), 3.96 (dd, 3 J=9.6 Hz, 3 J=3.9 Hz, 2H, CH2OH), 2.39 (s, 3H, SCH3), 2.37-2.33 (m, 2H), 1.96-1.90 (m, 2H), 1.29-1.25 (m, 9H) Glycine, N- [4-methyl-4-methoxy-1-oxopentyl] 1 H NMR (300 MHz, CDCl3): δ 6.21 (br s, 1H, NH), 3.99 (dd, 3 J=9.6 Hz, 3 J=7.5 Hz, 2H, CH2NH), 3.69 (s, 3H, COOCH3), 3.33-3.29 (m, 1H), 3.26 (s, 3H, OCH3) 1 H NMR (300 MHz, CDCl3): δ 6.50 (br s, 1H, NH), 4.11 (dt, 3 J= 6.9 Hz, 3 J= 6.9 Hz, 2H), 3.46 (q, 3 J= 6.9 Hz, 2H, OCH2CH3), 2.48 (t, 3 J= 6.9 Hz, 2H), 1.94 (s, 3H, COCH3), 1.21 (t, 3 J= 6.9 Hz, 3H, OCH2CH3) ppm. Incorporation expected at 3.46 δ (red arrow). Determined against integral at δ 1.89 (blue arrow). The PES for (D/L) Ac-Ala-OMe was calculated in the analogous to Ac-Gly-OMe. The results are summarized in Figure S2. Although the overall reaction enthalpy remains the same, the energies of the transition states increase compared to Gly. This is in line with the experimental finding that the reaction reaches a plateau. very efficient for H/D exchange. 1 However, these particles are sometimes very efficacious for arene hydrogenation which is undesired in the HIE reaction. Indeed, our preliminary experiments showed that attempts at H/D exchange on few tested substrates using RuICyNps as catalyst lead to complete reduction of the aromatic ring. In order to circumvent this problem, we proposed that iridium could be a good substitute to ruthenium since iridium complexes are very efficient for H/D exchange, presumably because of a low barrier to C-H activation. However, they are generally less effective as hydrogenation catalysts than their ruthenium equivalents. Therefore, the project has focused on the synthesis of novel iridium nanoparticles, stabilized by N-heterocyclic carbenes (NHCs) as already done with RuNps. These ligands have demonstrated their strong binding to various transition metal like Au, 3 Pd, 4 Ni, 5 and Ru 6 nanoparticles. Also, to the best of our knowledge, there is only one report on the stabilization of Iridium NPs (IrNPs) using imidazolium-based ionic liquids. 7 In our joined project, Donia has synthetized and characterized the nanoparticles we have used (scheme V.

V.2. Literature background -HIE reactions of anilines in the past

Lautens et al. have described an unselective aniline labelling with deuterium (scheme V.3, A), utilizing strong acids like DCl in large excess at temperatures above 100°C giving access to 99%D incorporated (12). 8 Jung et al. have also developed a similar method, using in their case TFA instead of DCl. 9 Junk et al. described complete exchange (99%D) to generate [D7]aniline 13 in D2O/NaOD at 400°C (600 bar) over 10h (scheme V.3, B). 10 Recently Bagley reported an unselective deuteration of aniline and aminopyridines in neutral D2O at 190°C (very high pressure) with up to 25%D for all CH-aniline positions in 14 (scheme V.3, C). 11 Another approach reported by Garnett et al. started from nitrobenzene 15, which was reacted over raney-nickel (alternatively also platinum or palladium were applied) with D2 or D2O (or T2 or HTO respectively) at 175°C over 4 days, to obtain the corresponding deuterated/tritiated anilines 16, but with low D/T incorporation when the isotope source was not used in large excess (scheme V.3, D). 12 Unfortunately, all these methods, with some others, 13 are difficult to transfer to tritium chemistry, yielding mainly a low specific activity. 14 Moreoever, only a few functional groups tolerate these harsh conditions. Scheme V. 3: Known aromatic HIE reactions on anilines 8,10,11,12 As showed above, even though there are already some examples published, there is still no simple and general method for the tritiation of complex anilines. This motivated us to search for an easy to handle, mild and generally applicable HIE method for isotope exchange on anilines, because mostly harsh conditions (A-D, scheme V.4) methods have been developed for over the past 50 years.

V.3. Evaluation of the iridium nanoparticles

The synthesis of IrNps has provided three different batches 3a-c, with NHC-ligand ratios going from 0.05 to 0.25 equivalents of ligands regards to the metal iridium (scheme V. Finally, the homogeneity between the different batches (figure V.5) appeared crucial to be evaluated, since nanoparticles 3b have been synthetized in Toulouse throughout different synthesis. In total, five batches prepared at different dates were evaluated with our model substrate 17 before using them in HIE reactions with various anilines. Successfully, the synthesis of these nanoparticles was able to provide homogeneous batches of 3b, with an HIE around 1.87 D/molecule in 17. This demonstrates a sufficient homogeneity of the different IrCy 3b batches.

It is important to state that these nanoparticles are air-stable since they were handled in the open-air without any precautions and used in HIE reactions without the need of a glovebox. This particularity suggests that it should be possible developing "easy to handle" methods for HIE reactions.

V.4. Application of IrNPts in HIE of small aniline molecules

The IrICy nanoparticles 3b have been applied to HIE on anilines 17-30 and gave good to excellent deuterium substitution (55-99%D) for simple anilines 17-21 (scheme V.4). Either electron-withdrawing functional groups (18,20,21) nor electron-donating ones (17,19) seemed to have a significant influence on the outcome of the HIE reaction. In contrast, steric interactions were identified as the major driver of the IrNPs-catalyzed HIE reactions. For example, no deuteration at all in ortho-position (20,21) of CF3-groups were found. In the HIE reaction of naphthalenes 22-26, different %D were observed for positions 2 and 8 in 1amino-naphthalenes 22-24. Interestingly, the C(sp 2 )-H is significantly more reactive than the C(sp 3 )-H as can be seen by comparison of 1-amino-naphthalenes 22 and 24, probably due to a higher activation energy barrier. Furthermore, an evaluation of this HIE reaction with more complex anilines 26-30 containing other functional groups was done and deuterium incorporation was going from 26%D to 92%D. The reaction conditions tolerate halogens (18,28), phenols (19), ester (26,30) and ketones ( 27) without any influence on selectivity or side-reactions. Up to now there is no scientific explanation for the decreased deuterium incorporation in aniline 30. This observation needs to be investigated in the future. Nevertheless, we observed for a group of anilines 31-42 that the prior optimized conditions yield to low or nonexistent deuterium incorporation (scheme V.5). We postulated that due to the high polarity of the amino, a protic co-solvent like D2O could be beneficial. Furthermore, as the boiling point of D2O (101°C) is higher than for THF (66°C), we choose to use a 1:1 mixture of both, to be able to increase the reaction temperature.

Scheme V. 5: HIE reaction with various anilines

N H 2 OH O N H boc N N H N H 2 N H 2 N H Cl N H 2 S NH O O NH 2 O N N N H 2 N H 2 S NH 2 O O N H 2 OH O O N H 2 OH OH O N H 2 S O F F F O N H 2 NH 2 O O O N H 2 O N H 2 O O NH 2 F 3 C O N H 2 O O N
Performing the HIE exchange with a THF/D2O (1:1) solvent mixture (80°C) permitted to achieve successful HIE reactions with high deuterium uptake and regioselectivity. While in the HIE reaction of 30 there was only 26%D incorporation obtained with method A (THF, 55°C, blue bracket), with method B (THF/D2O, 80°C, green bracket) the deuterium incorporation increased to 97%D. For 21 the deuterium incorporation increased from 55%D to 99%D. We observed similar improvements for anilines 35, 36, 39 and 42. Whereas no deuteration at all was observed for anilines 31-34 and 41 with method A, conversely application of conditions B improved the HIE reaction outcome up to 81%D. While we obtained in most examples a high selectivity on the ortho-position relative to the aniline function, in the case of N-heterocycles an additional labelling was determined in orthoposition of the aromatic nitrogen atom (29, 31, 32). Furthermore, we have observed the labelling in the meta-position of anilines 35, 36 and 42. As the use of D2O increased the deuterium incorporation, we were interested to understand its role in the HIE reaction.

V.5. The role of D 2 O in IrNPts catalyzed HIE reactions

We investigated the influence of D2O in the HIE reaction catalyzed with the IrNPs. For this purpose, deuteration of substrate 21 was performed for 3h, RT, 1 bar of gas (if any) as previously reported. If the reaction was carried out in THF under D2 and in the absence of D2O (scheme V.6, A), we observed 55% of isotopic enrichment, whereas if the reaction was performed in THF/D2O in the absence of H2 or D2, no deuteration was found (scheme V.6, B). However, if the deuteration was carried out in the presence of D2 and in a THF/D2O mixture (scheme V.6, C), the deuterium introduction jumped to 99%D. Thus, we hypothesized that upon fast equilibrium for HIE between gas and aqueous phase, D2O may serve as kind of a reservoir to replenish the consumed D2 in the gas phase. In order to verify this assumption, we planned two complementary experiments in THF/H2O under D2 (scheme V.6, D) and in THF/D2O under H2 (scheme V.6, E) where 0 and 99 %D deuteration were obtained, respectively. 15 Interestingly these experiments demonstrate that this catalytic system does not require an additional supply of D2 gas but can conveniently be carried out under H2 in D2O/THF mixtures. And it is likely to be extended to other D2O organic solvent mixtures.

Scheme V. 7: Understanding the role of D2O in the HIE reaction of aniline 21 with IrICy

3b a,b,c O N H 2 F 3 C O N H 2 F 3 C O N H 2 F 3 C O N H 2 F 3 C O N H 2 F 3 C O N H 2

V.6. Applications of IrNPts in HIE reactions of complex molecules and drugs

The IrICy nanoparticles 3b have been used for the HIE reaction on more complex structures, like the drugs sulfadimethoxine 5, aminoglutethimide 8, sulfamoxole 10, methyl anthranilate 11 and the saccharose 4-aminophenyl-beta-D-galactopyranoside 7 (scheme V.7). In all these examples, a good HIE efficacy was achieved providing deuterium incorporation from 50%D and up to 96%D. For the drug menthyl anthranilate 11 and the saccharose 7, a side labelling in the meta-position with regards to the aniline DG has been observed, as this happened also for substrates 35, 36 and 42. Unfortunately, no deuteration was obtained for drugs 4, 6 and 9.

Scheme V. 8: HIE reaction with aniline drugs

4-11 a,b N H 2 N H O O N H 2 S N H O O N N O O N H 2 S O N O O N H 2 O O O O OH OH OH OH N H 2 O N H 2 S N O O IPr N H O O O OH H H N H 2 S N O O NH 2 NH 2 N H 2 O Cl O N H N [ 99
The late(last)-stage tritiation of a complex pharmaceutical, which was one of the major issues of this PhD work, has been realized on most target molecules using this novel iridium NPts. The deuterium and tritium HIE reaction conditions have been optimized using the aniline 43, which is a pharmacophore of the drug Volixibat (scheme V.8). 16 This complex molecule is a medication under development as for cholestatic liver disease. As the use of tritiated water T2O was not practicable, an optimization of the reaction conditions in THF had to be done. The deuteration of 43 was done by using 4 mol% of the IrICy 3b, in THF at 55°C (as used so far). These conditions led to a selective deuterium incorporation of 43 up to 70%D (scheme V.8, blue bracket). Nevertheless, as it is expected to get a lower tritium incorporation due the KIE between H, D and T, the conditions were further optimized to get more deuterium incorporated. Consequently, the reaction was run with a higher catalyst loading reaching 80 mol% of IrICy 3b, still at 55°C in THF. In this case, both ortho-positions were deuterated with 41%D and 87%D in Volixibat precursor 43 (scheme V.8, purple bracket). Finally, the catalytic loading of 80 mol% of IrICy 3b was used for the tritiation of Volixibat pharmacophore 43. The tritium reaction was run using subatmospheric pressure of tritium gas (p= 0.8 bar, 10 Ci) within a relatively short reaction time (3 h). In terms of regioselectivity, the labelling of the less sterically encumbered orthoposition of the NH2 group was mainly observed in 43 with 86%T incorporated (scheme V.8, red bracket). Usually, a specific activity between 10-20 Ci/mmol is considered as suitable for the use of a tritiated molecules in the context of ADME studies. The high specific activity of 25 Ci/mmol obtained here clearly demonstrates the high potential of this novel method in the context of the late-stage labelling of complex pharmaceuticals containing aniline substructures.

V.7. Conclusion

For the first time air-stable and easy-to-handle iridium NHC-ligated nanoparticles can be used in HIE reactions. These catalysts showed a unique catalytic activity allowing a selective and efficient hydrogen isotope incorporation on anilines using D2 or T2 as isotopic source. The usefulness of this transformation has been demonstrated by the deuterium and tritium labelling of diverse complex pharmaceuticals. The method was fully adaptable to the specific requirements of tritium chemistry, which was demonstrated by direct tritium labelling of the volixibat pharmacophore 43 with high selectivity and specific radioactivity of 25 Ci/mmol (theoretical specific radioactivity of 29 Ci/mmol). This data is clearly fulfilling all requirements mentioned in the objectives of the thesis for the development of late-stage tritium labeling of drugable compounds. Yield: 16.9 mg, 137.4 µmol, 92%; 87% D for δ 6.59.

Other conditions (Scheme V.6):

-THF/H2O (1:1) (3 mL), 80 °C, 3h: no deuterium incorporation.

-THF/D2O (1:1) (3 mL), 80 °C, 3h, no gas: no deuterium incorporation. 3)), 6.56 (m, 2H, CH(2) and CH( 4)), 3.52 (br s, 2H, NH2), 2.77 (m, 2H, CH2( 6)), 2.48 (m, 2H, CH2( 9)), 1.86 (m, 4H, CH2(8) and CH2( 7)) ppm. Incorporation expected at δ 6.56 (red arrow). Determined against integral at δ 2.48 (blue arrow). Yield: 21.9 mg, 127.5 µmol, 85%; 81% D for δ 6.70.

General method A: no deuterium incorporation. Yield: 18.7 mg, 112.6 µmol, 75%; 16% D for δ 6.76 and 99% D for δ 6.33.

Iriridium nanoparticles data

This work was done by Donia Bouzouita in the LPCNO laboratory in Toulouse (France), within our collaboration with Prof. Chaudret et Dr. Tricard.

Synthesis of IrICy (0.05 eq) nanoparticles 3a

In the glove box, 10 mg of 1,3-dicyclohexylimidazolium chloride (0.037 mmol) and 5 mg KO t Bu (0.044 mmol) were dispersed in THF (15 mL) in a Schlenk flask. The mixture was let to stir at room temperature for 15 hours to give a yellowish solution containing the free carbene. Then, 200.0 mg of [Ir(OMe)(COD)]2 (0.3 mmol) was dispersed in THF (15 mL) in a Fischer Porter bottle. The carbene solution was filtered through celite to remove the KCl formed during the deprotonation of the imidazolium salt and was transferred dropwise into the Fischer Porter containing the [Ir(OMe)(COD)]2 solution. The mixture was pressurized with H2 (5 bar) under stirring and heated at room temperature overnight. At the end of the reaction, the NPs were isolated as a brown solid after evaporation of the solvent under vacuum. TGA analysis: Ir, 91 wt%.

Synthesis of IrICy (0.125eq) nanoparticles 3b

In the glove box, 20 mg of 1,3-dicyclohexylimidazolium chloride (0.07 mmol) and 9 mg KO t Bu (0.077 mmol, 1.1 eg.) were dispersed in THF (15 mL) in a Schlenk flask. The mixture was let to stir at room temperature for 15 hours to give a yellowish solution containing the free carbene. Then, 200.0 mg of [Ir(OMe)(COD)]2 (0.3 mmol) was dispersed in THF (15 mL) in a Fischer Porter bottle. The carbene solution was filtered through celite to remove the KCl formed during the deprotonation of the imidazolium salt and was transferred dropwise into the Fischer Porter containing the [Ir(OMe)(COD)]2 solution. The mixture was pressurized with H2 (5 bar) under stirring and heated at room temperature overnight. At the end of the reaction, the NPs were isolated as a brown solid after evaporation of the solvent under vacuum. Yield: 100 mg, 66%. TGA analysis: Ir, 77 wt%.

Synthesis of IrICy (0.25eq) nanoparticles 3c

In the glove box, 40 mg of 1,3-dicyclohexylimidazolium chloride (0.14 mmol) and 20 mg KO t Bu (0.18 mmol) were dispersed in THF (15 mL) in a Schlenk flask. The mixture was let to stir at room temperature for 15 hours to give a yellowish solution containing the free carbene. Then, 200.0 mg of [Ir(OMe)(COD)]2 (0.3 mmol) was dispersed in THF (15 mL) in a Fischer Porter bottle. The carbene solution was filtered through celite to remove the KCl formed during the deprotonation of the imidazolium salt and was transferred dropwise into the Fischer Porter containing the [Ir(OMe)(COD)]2 solution. The mixture was pressurized with H2 (5 bar) under stirring and heated at room temperature overnight. At the end of the reaction, the NPs were isolated as a brown solid after evaporation of the solvent under vacuum. TGA analysis: Ir, 70 wt%. In the Part II-Chapter 1, the use of a newly discovered Tamm catalyst (F) has been extended by evaluation of its activity in HIE reactions with sulfonamides (1), N-oxides (2) and phosphonamides (3). After optimization of the reaction conditions, the HIE reaction was able to be run under very mild conditions and provided very good deuterium incorporation in ortho-positions of sulphonamides DG1 up to 92%D, phosphonamides DG2 up to 99%D and Noxides DG3 up to 100%D (scheme VI. Selected examples:

[85]

[96]

[96]

[99] 1 2 3

In collaboration with the working group of Prof. Tamm, a long standing challenge in direct labelling of phenylacetic acid derivatives was solved (Part II-Chapter 2). While former methods have used non-catalytic reaction pathways or large excess of isotope source, the first efficient catalytic protocol for ortho-selective HIE of pharmacologically important phenylacetic acid esters and amides with D2/T2 gas under very mild reaction conditions was developed. The HIE reaction on 19 different phenylacetic esters has provided deuterium enrichment up to 91%D. The HIE reaction on 10 different phenylacetic amides has given deuterium incorporation up to 99%D. Furthermore, it was proven that the method was fully adaptable to the specific requirements of tritium chemistry mentioned in the thesis objectives. Indeed, a direct tritium labeling of Benalaxyl (25%T, 14 Ci/mmol) 5 and Camylofine (26%T, 32 Ci/mmol) 6 was achieved (Scheme VI.3). In Chapter 1 -Part III we have demonstrated that by applying the optimized combination of iridium catalysts A-G and reaction temperatures (-60 to 130°C), different HIE reaction outcomes in selectivity and reactivity can be achieved. Notably the temperature window for most studied iridium catalysts A-G were surprisingly broad. To use the right catalyst with the ideal reaction conditions is the trigger to either increase the selectivity and/or the deuterium incorporation (scheme VI.4). Based on our results, it is suggested to start the HIE optimization experiments with catalysts Ba, E and G at room temperature and at 80°C in parallel. While rising the reaction temperature to a maximum prior catalyst decomposition is the key for maximum deuterium incorporation into the target molecule and was demonstarted for drugs like Celecoxib 7, Vismodegib 8 and Glibenclamide 9. We believe that evaluating selectivity and reactivity of different catalysts in a comparison study of a reaction class can make the prediction of CH-functionalization reaction more reliable and understandable. This could enable a greater probability of success with HIE reactions to increase effectiveness and reduce the number of reactions needed and will therefore help to speed up drug discovery. One of the key features in planning a synthesis is the predictivity and probability of success of the chosen approach. In Chapter 2-Part III, we described the result of experimental competition experiments to obtain an order of directing group influence in HIE reactions with catalyst Ba. This was followed by DFT calculations, where we tried to get a theoretical understanding to explain the deuteration outcome (scheme VI.5). Interestingly, we have identified the iridium-substrate coordination complex to be the main trigger for reactivity and selectivity in the competition situation with two and more directing groups. This is remarkable as the general understanding of product formation is principally based on the deuterium incorporation up 95%D in the glycine moiety. Furthermore, DFT calculations gave insights into the mechanism and steric requirements of the HIE reaction, explaining the observed selectivities and the influence of the amino acid N-protecting groups. 5 Finally, from homogeneous iridium catalysis we moved to heterogeneous iridium catalysis. In the Part V, air-stable and easy-to-handle iridium NHC-ligated nanoparticles were developed for the first time and used in HIE reactions. These nanocatalysts showed a unique catalytic activity allowing a selective and efficient hydrogen isotope incorporation on anilines 17-21 using D2 or T2 as isotopic source (scheme VI.7). The application of the HIE reaction conditions was demonstrated by the deuterium and tritium labelling of diverse complex pharmaceuticals. Furthermore, the method was fully adaptable to the specific requirements of tritium chemistry, which was demonstrated by direct tritium labelling of the volixibat pharmacophore 21 with high selectivity and specific activity of 25 Ci/mmol. Introduction of deuterium or tritium into the final molecule has been done using homogeneous and heterogenous iridium catalysis and we have seen over the different Part II-V how these methods have been developed on small molecules first and successfully applied after on drug compounds.

Scheme VI. 6: Homogeneous iridium catalysis on C(sp3)-H centers (Part IV)

Based on the work elaborated in the thesis, there are some challenges remaining and some scientific side projects which could not be followed due to limited time:

• In Part IV, we have developed a new method for C(sp 3 )-labelling of small peptides (up to four amino acids). However, the method is only working with protecting groups on the N,O-atoms. It would be highly needed to develop a selective method for bigger peptides without the need of any protecting groups. Therefore, new catalysts with a high stability and a better solubility in polar solvents are needed.

• In Part III -Chapter 2, we discovered a new way to predict the strength of different directing groups in HIE reactions in a competition situation. This is the normal case for a late-stage functionalization approach. It is therefore of great interest to bridge the obtained observations with other transition metal-catalyzed C-C and C-X coupling reactions.

• In Part V, we have studied IrNPts in HIE reactions. This has been the first time that these nanoparticles have been used in catalysis. It would be very interesting to follow a broader application scope and to study these IrNPts in further HIE reactions as in hydrogenations or reductions.

• The last topic necessary to address would be based on monoclonal antibody conjugates, like discussed in Part IV for the ADC. It would be of high value not only to be able to put a radioactive label on the drug, but also to develop methods that allow to visualize the pathway and distribution of the antibody.

Since the beginning of the PhD, highly interesting new HIE methods have been developed, especially from the ISOTOPICS groups. General deuteration procedure: To a carousel flask was added the substrate of choice (1 eq., unless otherwise stated) in solution (500 µL), unless otherwise stated, and iridium(I) catalyst (10 mol%, unless otherwise stated) in stock solution (500 µL), unless otherwise stated, under air. The desired solvent was added unless otherwise stated, rinsing the inner walls of the tube. The tube was sealed with the Teflon cap. The flask was evacuated and flushed with deuterium (1 atm) via a balloon three times. After sealing the flask, the reaction was started. The reaction mixture was stirred (500 rpm) for the indicated time at the indicated temperature. After the reaction, the mixture was analyzed by LC-MS. If the reaction was successful, the solvent was evaporated in vacuo. The residue was purified by chromatography with the indicated eluant. The product was analyzed by 1 H-NMR. The degree of deuterium incorporation and the regioselectivity of the labelling in the substrate were determined by 1 H-NMR. The integrals were calibrated against a peak corresponding to a position not expected to be labelled.

The first action is to choose an adapted model substrate which is, in plus of being related to the chosen topic, easy to get and easy to analyse. Also, the model compound must be ionisable and UV detectable to be easily seen in the mass spectrometry detector (LC-MS). In Sanofi there are automatic samplers connected with the LC-MS systems as standard analysis instruments, which provide a very quick and efficient analysis of the reaction, giving you immediately the answer if the HIE reaction was successful or not. In addition, the 1 H-NMR of the model compound needs to be clean enough to be analysed. In our case, 1 H-NMR was the only method to identify the labelling position. Therefore, it is important that there is no overlapping of the peaks characterising the molecule and the deuterated solvent. This is especially important when it comes to the evaluation of complex molecules.

• The parallel synthesizer

Having the model substrate chosen, the reaction was run on a Radley parallel synthesizer, in the following called "the carousel" (figure 2), under several conditions as different solvents, temperatures, times and catalyst ratios. However, the constant parameter on the carousel is the D2 gas pressure, D2 will always be 1013 mbar (= always set at 1 atm).

Figure 2: The carousel Radleys®

The carousel is composed of 12 glass flasks, which are tightened by locking the Teflon cap on top. The system is connected to a vacuum pump and to a balloon reservoir filled with D2. The vacuum pump is evacuating the reaction flask before being filled with deuterium. This step is repeated at least three times. The vacuum pressure in the flask is the most critical parameter as any leakage in the system would cause false reaction outcomes due to the limited D2 equivalents. There are other parameters which can negatively influence the reaction like decomposed catalyst or impurities in the solvent or substrates which can poison the catalyst.

For this reason, it is always important to run a standard test reaction with the same conditions (solvent, temperature, time, catalyst) but with a substrate, which is known to work in the HIE reaction very well. Consequently, when 12 reactions are run on the carousel, 11 are test reactions and the last one is the control (blank) reaction. Finally, reactions are always run at least twice to ensure the reproducibility of the method. All values calculated are the mean (average) of at least two performed reactions.

• The deuterium manifold

After identification of the optimized deuteration conditions of a drug compound using the carousel with 1 bar D2, the reaction needs to be adapted to the requirements of tritium chemistry. The deuterium manifold allows controlling the pressure of D2, and thus the number of equivalents of D2 in the reaction (figure 3). At 1 atm of D2 gas on the carousel, a large excess of D2 equivalent is used whereas 5 to 10 equivalents of tritium are generally used for the tritiation reaction. The pressure value depends of the molecular mass of the substrate and the number of equivalents used for the reaction. At the maximum we work in Sanofi with 500 mbar in our tritium manifold. b) of tritium is much higher than for deuterium, so it is expected that the isotopic enrichment is lower with tritium than with deuterium. For this reason, the deuterium incorporation obtained on the

Step 1

Step 2

Step 3

Step 4

Flask 1 Flask 2 Flask 3

Step 5 367 deuterium manifold should be significant (above 70%D) that even with transfer to the tritium chemistry it is still allowing a successful HIE reaction.

The deuterium manifold system consists of vacuum-lines made of stainless steel, which are leak tested down to less than 10 -9 mbar l/s. The reaction flask 1 is connected at the bottom part and tightly attached (figure 3, step 1). The flask 1 is frozen in liquid nitrogen. The vacuum valve is opened to evacuate the flask but also the whole line system (figure 3, step 2). Then all valves are closed and the lines are filled with D2 from the balloon (figure 3, step 3). As we want to control the number of equivalents, we carefully open the vacuum valve to evacuate D2 until the wanted pressure value is reached. The pressure in the lines is seen on the blue screen on the top right of the manifold (figure 3, step 4). When we obtain the desired pressure, the vacuum valve is closed and the one controlling the opening of the reaction flask 1 is opened to let the gas get in. The diffusion of the D2 between the flask 1 and the lines is made by equilibrium, the pressure in the flask 1 can be seen on the blue screen on top aligned with the respective flask and it must be noted carefully (figure 3, step 5). In the end, all valves are closed, the liquid nitrogen is removed and the reaction flask is enabled to warm up at room temperature within 15 min. The stirring and the reaction time are started. When the reaction is over, the flask is unplugged from the manifold and the reaction analysed by LC-MS.

• The tritium manifold

After a successful HIE reaction on the deuterium manifold, the same conditions are applied on the tritium manifold (figure 4). The technology is similar than those of the deuterium manifold, however with a scrubber system attached to minimize tritium in the exhaust air after the reaction. The tritium gas is stored on unranium beds as uranium tritide (UT3) and is released at the required pressure by heating up higher than 450°C. The biggest advantage of the manifold is the reversible formation of UT3 after completion of the reaction.

The reaction flask is tightly connected to the manifold and frozen in liquid nitrogen. By opening and closing accordingly the different valves, the pipes are evacuated, and the flask is charged with tritium. The reaction mixture is then allowed to warm up to room temperature over 15 min and is run under the optimized deuterium conditions. In some cases, the reaction time is prolonged to take account of the KIE for tritium. After the reaction the solvent is evaporated. The flask is unplugged, methanol is added and the solvent mixture is evaporated again into a waste ampule. This procedure is repeated three times in total to get rid of volatile tritium atoms. The reaction mixture is then dissolved in methanol and filtered.

A probe is transferred and analysed by LC-MS. If this was a success, purification and isolation via HPLC will be done and provide the tritiated product which will be analysed by 1 H and 3 H NMR.

General Work-up

After the HIE reaction, a probe is run in LC-MS. If the reaction was successful, the solvent is evaporated and the dry mixture is purified by chromatography on silica gel to separate the product from the catalyst. As the reactions were done on small scale (around 10 mg), the chromatography was done in a Pasteur pipette filled with cotton at the bottom and silica gel.

In the ideal case, the product was eluted first and the catalyst was kept on the silica. When the substrate was too polar, the catalyst was eluted first with a medium polar solvent and then the polarity of the mobile phase was slightly increased to isolate the product. In these cases, the purity of the isolated compounds was always decreased which was seen in the 1 H-NMR spectra. After purification, the solution is evaporated and the dry product is diluted in the deuterated solvent. Degree of deuterium incorporation and position are identified by 1 H-NMR.

Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Abstract : The development of new efficient, fast and easy to handle methods to label drug compounds with deuterium or tritium in one single step is of great importance in academia and industry. These labelled compounds are an essential part in drug discovery and help to speed up the generation of the safety profile of a drug candidate. Classical isotope labeling mainly relies on the transformation of precursors which require multistep synthesis, a drawback which may be overcome by late-stage functionalization. We reported the first efficient catalytic protocol for ortho-selective iridium(I)-catalyzed Hydrogen Isotope Exchange (HIE) reactions of pharmacologically important phenylacetic acid esters and amides with D2 or T2 under very mild reaction conditions (room temperature). We have demonstrated that by applying the optimized combination of different iridium catalysts and reaction temperatures (-60 to 130°C), different HIE reaction outcomes in selectivity and reactivity can be achieved.

Together with DFT calculations, we have postulated a guideline for directing group strength of several functional groups, to predict the outcome of HIE reactions in the competition situation of complex molecules. Starting from a complex tritium labelling of the maytansine DM4 drug, the HIE reaction on a series of common linker side chains of antibody-drug-conjugates proceeded with high chemical yields, high regioselectivity and with deuterium incorporations up to 99%. The scope of the method was further extended to amino acids, di-and tripeptides, with deuterium incorporation up 95%D in the glycine moiety. Finally, in collaboration with CEA-Saclay and CNRS, airstable and easy-to-handle iridium NHC-ligated nanoparticles were developed for the first time and used in HIE reactions of complex anilines. The usefulness of the methods developed all along the PhD was demonstrated by tritiation of DM4, Camylofine, Benalaxyl and the Volixibat pharmacophore.

1 D

 1 sur des composés contenant les groupements directeurs suivant : sulphonamide, phosphonamide et N-oxydes

Schéma 2 :6 3 Schéma 3 : 4 Schéma 4 : 6 Schéma 6 :

 2334466 Réaction d'échange d'isotopes d'hydrogène sur des dérivés d'acides phénylacétiques avec un catalyseur à l'iridium 2 Lors d'un autre projet, nous avons mis en évidence que la position de marquage au sein d'une molécule avec plusieurs groupements directeurs peut être modulée en fonction du catalyseur d'iridium utilisé ainsi que de la température réactionnelle (de -60°C à +130°C) (schéma 3). Contrôle de la régio-sélectivité de la réaction d'échange d'isotopes d'hydrogène en fonction de la température et du catalyseur utilisé 3 L'utilisation de calculs de DFT nous a permis de postuler un ordre de préférence classifiant différents groupements directeurs par rapport à leur réactivité vis-à-vis de la réaction d'HIE en situation de compétition (deux substrats et un catalyseur d'iridium). Ces données théoriques ont été confirmées par les données expérimentales (schéma 4) sur des molécules modèles mais aussi sur des médicaments. Prédiction de la régio-selectivité de la réaction d'échange d'isotopes d'hydrogène en situation de compétition entre deux différents groupements directeurs au sein d'un même substrat 4 Lors d'un autre projet, le marquage sélectif au tritium d'une structure complexe tel qu'un médicament cytotoxique (maytansine DM4) a débouché sur le marquage de différentes chaînes latérales employées dans des méthodes de conjugaison aux anticorps. Ces données ont permis d'étendre cette méthodologie au marquage au deutérium d'acides aminés et de di-tri et tétra-peptide, sélectivement sur le carbone alpha d'un résidu glycine (schéma 5collaboration avec le CEA-Saclay et le CNRS, nous avons développé les premières nanoparticules d'iridium stables à l'air et actives vis-à-vis de l'échange d'isotopes d'hydrogène sur des anilines (schéma 6). Réaction d'échange d'isotopes d'hydrogène sur des composés organiques avec un groupement directeur aniline à l'aide de nanoparticules d'iridium stabilisées par des ligands

Figure I. 4 :I. 4 . 1 . 10 I

 44110 Figure I. 4: Applications of hydrogen isotopes in life sciences 6

Figure I. 5 :I

 5 Figure I. 5: LC/MS of the internal standard6

A: 1 :

 1 Scheme I. 1: Selected deuterated clinical drug development candidates and correspondingunlabeled original drugs6

  Scheme I. 2: Structure of the 18F-D4-Fluoromethylcholine

Figure I. 9 : 6 Figure I. 8 :

 968 Figure I. 9: Principle of the scintillation proximity assay 6

Figure I. 10 :

 10 Figure I. 10: Plasma Protein Binding (PPB) assay based on equilibrium dialysis6

Scheme I. 6 :

 6 Scheme I.6: Acid-mediated labeling of amino acid derivatives

2 .

 2 Scheme I.8: Base-mediated labeling of benzylamines

  applications of nanoparticles show different reactivity or selectivity compared to the already known homogeneous or heterogeneous HIE reaction systems. It is, therefore, anticipated that advances in material science will also enrich the armory of HIE reaction methods in the future.Currently known techniques for heterogeneous labeling with deuterium/tritium atoms use the following representative combinations of catalyst and hydrogen isotope sources: Pd/C-D2 or Pd/C-T2, Pd/C-(H2/D2)-D2O(DCl), Pd/C-Et3SiD-iPrOH-D8, Pd/C(en)-D2, Pd/PVP-D2, Pt/C-D2, PtO2-D2-D2O, Rh/C, or Rh black-D2, Ru/C-D2, and, more rarely, cobalt or nickel metal.

  Scheme I.18: Photo redox-mediated hydrogen isotope exchange with D2O/T2O

a

  Scheme I.19: Homogeneous iridium(I) catalysts

  Scheme II.1. 4: N-oxide containing drugs

Scheme II. 1 . 5 :

 15 Scheme II.1. 5: Phosphorus-containing drugs -Sofosbuvir 13

Scheme II. 1 . 7 :

 17 Scheme II.1. 7: HIE reactions of aryl-sulphonamides 14-21 catalyzed by F.a,b

Scheme II. 1 . 8 :

 18 Scheme II.1. 8: HIE reactions of N-oxides 22-27 catalyzed by F.a,b

Scheme II. 1 . 9 :a

 19 Scheme II.1. 9: HIE reactions of aryl-phosphonamides 28-33 catalyzed by F.a,b

  (DMSO-d6; 300MHz): (δ ppm) 7.68 (m, 2H, H3), 7.59 -7.41 (massif, 3H, H4 and H5), 2.95 (m, 8H, H2), 0.95 (t, J = 7.0 Hz,12H, H1). 13 C-NMR (DMSO-d6; 300MHz): (δ ppm) 131.94 (C3), 130.85 (C5), 128.11 (C4), 38.36 (C2), 13.62 (C1).

  13 C-NMR (DMSO-d6; 300MHz): (δ ppm) 131.83 (C4), 130.97 (C6), 128.34 (C5), 45.03 (C3), 26.13 (C2), 24.75 (C1).

  300MHz): (δ ppm) 7.92 (m, 2H, H3),7.55 -7.48 (massif, 3H, H4 and H5), 3.45 (s, 6H, H1), 2.95 (ds, 6H, H2). 13 C-NMR (CDCl3; 300MHz): (δ ppm) 131.40 (C5), 130.92 (C3), 128.20 (C4), 61.47 (C1), 35.39 (C2).52HIE reactions of sulphonamides 14-21catalyzed by F (scheme II.1.7)Conditions: substrate (22.0 μmol, 1 eq.), catalyst F (3.4 mg, 2.2 µmol, 10 mol%, 0.1 eq.), PhCl (2 mL), D2 (1 atm), rt,

  .0 Hz, 2H, 2 CH ortho-CH3), 7.33 (s large, 1H, NH), 2.38 (s, 3H, CH3) ppm. Incorporation expected at δ 7.65 (red arrow). Determined against integral at δ 2.38 (blue arrow).

1 H

 1 , DMSO-d6): δ 7.69 (d, 3 J = 8.5 Hz, 2H, 2 CH ortho-SO2NH2), 7.42 (s large, 1H, NH), 7.38 (d, 3 J = 8.5 Hz, 2H, 2 CH ortho-CH3), 2.80 (m, 2H, CH2), 2.40 (s, 3H, CH3), 2.25 (m, 2H, CH2), 2.05 (s, 6H, 2 CH3) ppm. Incorporation expected at δ 7.69 (red arrow).

1 H

 1

  3 CH phenyl), 7.20 (m, 2H, 2 CH phenyl), 7.08 (d, 3 J = 8.6 Hz, 2H, 2 CH meta-SO2NH2), 7.00 (m, 1H, CH phenyl) ppm. Incorporation expected at δ 7.75 (red arrow). Determined against integral at δ 7.00 (blue arrow). , DMSO-d6): δ 7.62 (d, 3 J = 8.2 Hz, 2H, 2 CH ortho-SO2NH2), 7.45 (d, 3 J = 8.2 Hz, 2H, 2 CH ortho-CH3), 2.85 (m, 4H, 2 CH2), 2.40 (s, 3H, CH3), 2.35 (m, 4H, 2 CH2), 2.40 (s, 3H, CH3) ppm. Incorporation expected at δ 7.62 (red arrow). Determined against integral at δ 2.85 (blue arrow).

1 H

 1 , DMSO-d6): δ 7.95 (m, 3H, 3 CH), 7.80 (m, 1H, CH), 7.67 (m, 1H, CH), 7.59 (m, 3H, 3 CH), 7.35 (m, 1H, CH), 7.25 (m, 1H, CH), 6.85 (m, 1H, CH) ppm. Incorporation expected at δ 7.95 and/or 7.80 (red arrow). Determined against integral at δ 7

  .9 Hz, 2H, 2 CH ortho-Cl), 3.15 (m, 4H, 2 CH2), 1.02 (m, 6H, 2 CH3) ppm. Incorporation expected at δ 7.82 (red arrow). Determined against integral at δ 1.02 (blue arrow).

  1 H-NMR described previously pages 53-54.

1 H 1 . 3 , 4 , 5 ,

 11345 Scheme II.2. 1: Phenylacetic acid derivative drugs

  Scheme II.2. 2: Known aromatic HIE reactions for CH-activation/deuteration in phenylaceticacid derivatives [H]-11-141,2,4,5

Figure II. 2 . 1 :

 21 Figure II.2. 1: Potential energy profile for ortho-deuteration of 12, 15-17 with iridiumcatalyst F scaled to Gibbs free energy (ΔG 0 ).

Figure II. 2 . 2 : 2 F

 222 Figure II.2. 2: Kinetic study of the HIE reaction of methylphenylacetate ester [H]-12 withcatalyst F.a,b

:a

  Evaluation of catalyst F loading in the HIE reaction of [H]-12. Conditions: substrate [H]-12 (1.5 mg, 10 µmol, 1 eq.), catalyst F (1.5 mg, 1.0 μmol, 10 mol%), chlorobenzene (1 mL), D2 (1atm), rt, 2 h. b Position determined by 1H NMR and percentage of deuterium incorporation analysed by LC-MS.

II. 2 . 5 :

 25 Scheme II.2. 5: HIE reaction with various drug-like phenylacetic acid esters and amides.a,b

3 1 2 a

 32 Conditions: substrate (1 eq.), catalyst F (10 mol%), chlorobenzene (1 mL), D2 (1atm), rt, 2 h. b Position and percentage of deuterium incorporation determined by 1H NMR.

6 :

 6 Scheme II.2. 6: Unsuccesful drug structures used in HIE reactions with catalyst F.a,b

b

  Deuterium incorporation analysed by LC-MS.

Scheme II. 2 . 8 : 10 II. 2 . 4 .

 281024 Scheme II.2. 8: Tritiation of drug compounds with catalyst F

  12

  a) All theoretical calculations were performed according to the Density Functional Theory (DFT) method M06/6-311G(d,p) for all main group elements and a "Stuttgart 1997 ECP" -double-ξ basis set with an effective core potential -for the 5d transition metal iridium. For the stationary points, all hydrogen atoms, except for those involved in the reaction pathway, were omitted for clarity, and only a rudimentary N-C-C-P-C2 skeleton of the P,N ligand is shown.

Figure II. 2 . 3 :

 23 Figure II.2. 3: Potential energy profile for ortho-deuteration of phenyl acetic methylester ([H]-12) with iridium catalyst F, scaled to Gibbs free energy (ΔG 0 ). a

Figure II. 2 . 4 :

 24 Figure II.2. 4: ORTEP diagram of the dication in the complex [(P,N)2Ir2H4][BArF24]2 (61) a

  acid esters and amides with gaseous D2/T2 under very mild reaction conditions was developed.

  (300 MHz, CDCl3): δ 7.33 (br s, 5H, CH phenyl), 3.72 (s, 3H, OCH3), 3.66 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.33 (red arrow). Determined against integral at δ 3.69 (blue arrow). General method: 10.0 mg (67.0 µmol) 12; 10.2 mg (6.7 µmol) catalyst F, 2h. Yield: a) 8.7 mg, 58.0 µmol, 87%; 90% D for δ 7.33. b) 7.9 mg, 53.0 µmol, 79%; 84% D for δ 7.33. (300 MHz, CDCl3): 7.32 (m, 5H, CH phenyl), 4.18 (q, 3 J = 7.0 Hz, 2H, CH2 ethyl), 3.63 (s, 2H, CH2 benzylic), 1.27 (t, 3 J = 7.0 Hz, 3H, CH3 ethyl) ppm. Incorporation expected at δ 7.32 (red arrow). Determined against integral at δ 4.18 (blue arrow). General method: 9.8 mg (60.0 µmol) 11; 9.2 mg (6.0 µmol) catalyst F, 2h. Yield: a) 7.5 mg, 45.0 µmol, 76%; 66% D for δ 7.32. c) 7.9 mg, 48.0 µmol, 80%; 70% D for δ 7.32. (300 MHz, CDCl3): 7.33 (m, 5H, CH phenyl), 5.06 (m,1H, CH isopropyl), 3.63 (s, 2H, CH2 benzylic), 1.25 (m, 6H, 2CH3 isopropyl) ppm. Incorporation expected at δ 7.33 (red arrow). Determined against integral at δ 1.25 (blue arrow). General method: 10.0 mg (56.0 µmol) 21; 8.5 mg (5.6 µmol) catalyst F, 2h. Yield: a) 8.8 mg, 49.0 µmol, 88%; 54% D for δ 7.33. b) 7.4 mg, 42.0 µmol, 74%; 44% D for δ 7.33. (300 MHz, CDCl3): δ 7.19 (d, 3 J=8.5 Hz, 2H, CH2 meta-phenyl), 7.14 (d, 3 J=8.5 Hz, 2H, CH2 ortho-phenyl), 3.70 (s, 3H, OCH3), 3.60 (s, 2H, CH2 benzylic), 2.34 (s, 3H, CH3 para-phenyl) ppm. Incorporation expected at δ 7.14 (red arrow). Determined against integral at δ 3.70 (blue arrow). General method: 10.0 mg (61.0 µmol) 22; 9.3 mg (6.1 µmol) catalyst F, 2h. Yield: a) 8.1 mg, 49.0 µmol, 81%; 79%D for δ 1.91-1.85. c) 9.2 mg, 56.0 µmol, 92%; 91% D for δ 91-1.85. (300 MHz, CDCl3): δ 7.23 (m, 1H, CH2 meta-phenyl), 7.11 (s, 1H, Me-C-CH-C-R), 7.09 (m, 2H, ortho/para-CH), 3.72 (s, 3H, OCH3), 3.61 (s, 2H, CH2 benzylic), 2.37 (s, 3H, CH3 metaphenyl) ppm. Incorporation expected at δ 7.11 and δ 7.09 (red arrow). Determined against integral at δ 3.72 (blue arrow). General method: 9.9 mg (60.0 µmol) 23; 9.0 mg (6.0 µmol) catalyst F, 2h. Yield: a) 8.7 mg, 53.0 µmol, 88%; 53%D for δ 7.09 and 40%D for δ 7.11. b) 9.2 mg, 56.0 µmol, 93%; 57% D for δ 7.09 and 51%D for δ 7.11. (300 MHz, CDCl3): δ 7.20 (m, 4H, aromatic), 3.70 (s, 3H, OCH3), 3.67 (s, 2H, CH2 benzylic), 2.32 (s, 3H, CH3 ortho-phenyl) ppm. Incorporation expected at δ 7.20 (red arrow). Determined against integral at δ 3.70 (blue arrow). General method: 9.9 mg (60.0 µmol) 24; 9.0 mg (6.0 µmol) catalyst F, 2h. Yield: a) 7.7 mg, 47.0 µmol, 78%; 95% D for δ 7.10. b) 8.4 mg, 51.0 µmol, 85%; 93% D for δ 7.10. (300 MHz, CDCl3): δ 7.14 (d, 3 J=8.5 Hz, 2H, CH2 ortho-phenyl), 6.77 (d, 3 J=8.5 Hz, 2H, CH2 meta-phenyl), 5.15 (s, 1H, OH), 3.71 (s, 3H, OCH3), 3.56 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.14 (red arrow). Determined against integral at δ 3.71 (blue arrow). General method: 10.0 mg (60.0 µmol) 25; 9.2 mg (6.0 µmol) catalyst F, 2h. Yield: a) 7.8 mg, 47.0 µmol, 78%; 87% D for δ 7.14. b) 10.8 mg, 65.0 µmol, 100%; 77% D for δ 7.14.

  26

  (300 MHz, CDCl3): δ 7.23 (s, 1H, OH), 7.12 (m, 1H, CH para-phenyl), 7.02 (s, 1H, CH ortho-phenyl), 6.83 (m, 2H, CH meta-phenyl), 3.67 (s, 3H, CH3), 3.61 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.02 and 6.83 (red arrow). Determined against integral at δ 3.67 (blue arrow). General method: 10.0 mg (60.0 µmol) 27; 9.2 mg (6.0 µmol) catalyst F, 2h. Yield: a) 9.4 mg, 57.0 µmol, 94%; 48%D for δ 7.02 and 20% D for δ 6.83. b) 8.8 mg, 53.0 µmol, 88%; 56%D for δ 7.02 and 33% D for δ 6.83. (300 MHz, CDCl3): δ 7.25 (d, 3 J=8.5 Hz, 2H, CH2 ortho-phenyl), 7.02 (d, 3 J=8.5 Hz, 2H, CH2 meta-phenyl), 3,70 (s, 3H, CH3), 3.60 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.25 (red arrow). Determined against integral at δ 3.70 (blue arrow). General method: 10,2 mg (61.0 µmol) 28a; 9.3 mg (6.1 µmol) catalyst F, 2h. Yield: a) 3.4 mg, 20.2 µmol, 33%; 82% D for δ 7.25 and 14% D for δ 7.02. b) 3.0 mg, 18.0 µmol, 29%; 91% D for δ 7.25 and 5% D for δ 7.02.

  (red arrow). Determined against integral at δ 3.71 (blue arrow).General method: 11.2 mg (61.0 µmol) 29b; 9.3 mg (6.1 µmol) catalyst F, 2h. Yield: a) 10.1 mg, 55.0 µmol, 90%; 37% D for δ 7.29 and 73% D for δ 7.17. b) 9.8 mg, 53.0 µmol, 88%; 44% D for δ 7.29 and 72% D for δ 7.17

  .

  (300 MHz, CDCl3): δ 7.43 (m, 2H, CH ortho-Br), 7.21 (m, 2H, CH meta/para-Br), 3.72 (s, 3H, CH3), 3.61 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.43 and 7.21 (red arrow). Determined against integral at δ 3.72 (blue arrow). General method: 13.8 mg (60.0 µmol) 29c; 9.0 mg (6.0 µmol) catalyst F, 2h. Yield: a) 12.6 mg, 55.0 µmol, 91%; 21% D for δ 7.43 and 68% D for δ 7.21. b) 13.7 mg, 60.0 µmol, 99%; 24%D for δ 7.43 and 75% D for δ 7.21.

  Yield: a) 11.0 mg, 43.0 µmol, 100%; 99% D for δ 7.22. b) 8.9 mg, 35.0 µmol, 87%; 99% D for δ 7.22. (300 MHz, CDCl3): δ 7.21 (d, 3 J = 8.5 Hz, 2H, CH2 ortho-phenyl), 7.09 (d, 3 J = 8.5 Hz, 2H, CH2 meta-phenyl), 3.69 (q, 3 J = 7.0 Hz, 1H, CH benzylic), 2.43 (d, 3 J = 7.0 Hz, 2H, CH2 iPr), 1.84 (sept, 3 J = 7.0 Hz, 1H, CH iPr), 1.49 (d, 3 J = 7.0 Hz, 3H, CH3), 0.89 (d, 3 J = 7.0 Hz, 6H, CH3 iPr) ppm. Incorporation expected at δ 7.21 (red arrow). Determined against integral at δ 2.43. General method: 10.3 mg (50.0 µmol) 50a; 7.6 mg (5.0 µmol) catalyst F, 2h. Yield: a) 10.0 mg, 49.0 µmol, 97%; 0% D for δ 7.21. (300 MHz, CDCl3): δ 7.19 (d, 3 J = 8.0 Hz, 2H, CH2 ortho-phenyl), 7.11 (d, 3 J = 8.0 Hz, 2H, CH2 meta-phenyl), 5.43 (bs, 1H, NH), 3.53 (q, 3 J = 7.0 Hz, 1H, CH benzylic), 3.14 (m, 2H, CH2 propyl), 2.45 (d, 3 J = 6.9 Hz, 2H, CH2 iPr), 1.85 (sept, 3 J = 6.9 Hz, 1H, CH iPr), 1.51 (d, 3 J = 7.0 Hz, 3H, CH3), 1.41 (sext, 3 J = 7.5 Hz, 2H, CH2 propyl), 0.89 (d, 3 J = 6.9 Hz, 6H, CH3 iPr), 0.80 (t, 3 J = 7.5 Hz, 3H, CH3 propyl) ppm. Incorporation expected at δ 7.19 (red arrow). Determined against integral at δ 2.45. General method: 11.1 mg (45.0 µmol) 50b; 6.9 mg (4.5 µmol) catalyst F, 2h. Yield: a) 10.5 mg, 43.0 µmol, 95%; 80% D for δ 7.19. b) 11.9 mg, 48.0 µmol, 100%; 82% D for δ 7.19. (300 MHz, CDCl3): δ 7.16 (d, 3 J = 8.0 Hz, 2H, CH2 ortho-phenyl), 7.06 (d, 3 J = 8.0 Hz, 2H, CH2 meta-phenyl), 3.79 (q, 3 J = 7.0 Hz, 1H, CH benzylic), 3.28 (m, 4H, CH2 ethyl), 2.43 (d, 3 J = 7.0 Hz, 2H, CH2 iPr), 1.83 (sept, 3 J = 7.0 Hz, 1H, CH iPr), 1.42 (d, 3 J = 7.0 Hz, 3H, CH3), 1.09 (t, 3 J = 7.0 Hz, 3H, CH3 ethyl), 0.94 (t, 3 J = 7.0 Hz, 3H, CH3 ethyl), 0.88 (d, 3 J = 7.0 Hz, 6H, CH3 iPr) ppm. Incorporation expected at δ 7.16 (red arrow). Determined against integral at δ 2.43. General method: 11.7 mg (45.0 µmol) 50c; 6.9 mg (4.5 µmol) catalyst F, 2h. Yield: a) 11.7 mg, 45.0 µmol, 100%; 96% D for δ 7.16. b) 12.5 mg, 48.0 µmol, 100%; 96% D for δ 7.16.

1 H-NMR Reference 50e 1 H 1 H

 111 -NMR HIE reaction 50e Average: y=97%, 32% D δ 7.19 2-[2,4-Bis(trifluoromethyl)phenyl]-N-[(R)-2-oxo-5-phenyl-1-(2,2,2-trifluoro-ethyl)-2,3dihydro-1H-1,4-benzodiazepin-3-yl]-acetamide (51) δ 7.93 (s, 1H, CH aromatic), 7.80 (d, 3 J = 8.0 Hz, 1H, CH metaphenylacetamide), 7.71 (d, 3 J = 8.0 Hz, 1H, CH ortho-phenylacetamide), 7.61 (m, 1H, CH aromatic), 7.55 (m, 1H, CH aromatic), 7.53 (m, 1H, CH aromatic), 7.48 (m, 1H, CH aromatic), 7.42 (m, 1H, CH aromatic), 7.39 (m, 2H, CH2 aromatic), 7.32 (m, 2H, CH2 aromatic), 5.60 (d, 3 J = 8.0 Hz, 1H, CH-NH), 5.20 (sext, 3 J = 8.0 Hz, 1H, CH2-CF3), 4.16 (sext, 3 J = 8.0 Hz, 1H, CH2-CF3), 3.96 (s, 2H, CH2 benzylic). Incorporation expected at δ 7.71 (red arrow). Determined against integral at δ 3.96. General method: 11.7 mg (20.0 µmol) 51; 3.1 mg (2.0 µmol) catalyst F, 2h. Yield: a) 8.7 mg, 15.0 µmol, 74%; 95% D for δ 7.71. b) 12.8 mg, 22.0 µmol, 100%; 81% D for δ 7.71. δ 7.69 (m, 3H, CH aromatic), 7.40 (m, 1H, CH aromatic), 7.13 (m, 2H, CH aromatic), 3.91 (s, 3H, OCH3), 3.86 (q, 3 J = 7.2 Hz, 1H, CH benzylic), 3.67 (s, 3H, CH3 ester), 1.58 (d, 3 J = 7.3 Hz, 3H, CH3 benzylic) ppm. Incorporation expected at δ 7.40 (red arrow). Determined against integral at δ 3.68 (blue arrow). General method: 10.0 mg (39.0 µmol) 52a; 6.0 mg (3.9 µmol) catalyst F, 2h. Yield: a) 7.6 mg, 29.6 µmol, 76%; 76% D for δ 7.40. b) 8.1 mg, 31.6 µmol, 81%; 74% D for δ 7.40. δ 7.70 (m, 3H, CH aromatic),7.42 (m, 1H, CH aromatic), 7.14 (m, 2H, CH aromatic), 4.14 (m, 2H, CH2 ethyl), 3.92 (s, 3H, OCH3), 3.84 (q, 3 J = 7.2 Hz, 1H, CH benzylic), 1.57 (d, 3 J = 7.2 Hz, 3H, CH3), 1.21 (t, 3 J = 7.0 Hz, 3H, CH3 ethyl) ppm. Incorporation expected at δ 7.42 (red arrow). Determined against integral at δ 4.14.General method: 10.0 mg (39.0 µmol) 52b; 6.0 mg (3.9 µmol) catalyst F, 2h. Yield: a) 10.3 mg, 40.0 µmol, 100%; 77% D for δ 7.42. b) 10.6 mg, 41.0 µmol, 100%; 74% D for δ 7.42.

  An aliquote was analyzed by LC-MS showing quantitative conversion to the corresponding acid. , CD2Cl2): δ 7.25 (m, 5H, CH aromatic), 4.27 (s, 1H, CH benzylic), 3.91 (m, 2H, CO(=O)CH2 ), 2.38 (m, 9H, 4 x N-CH2; NH), 1.37 (m, 3H, CH2-CH aliphatic), 0.83 (br m, 12H, 4x CH3). Incorporation expected at δ 7.25 and δ 4.27 (red arrow). Determined against integral at δ 0.83 (blue arrow). General method: 10.0 mg (31.0 µmol) 5; 4.7 mg (3.1 µmol) catalyst F, 2h. Yield: a) 8.7 mg, 26.9 µmol, 87%; 71% D for δ 7.25 and 63% D for δ 4.27. b) 8.2 mg, 25.4 µmol, 82%; 72% D for δ 7.25 and 64% D for δ 4.27. , CDCl3): δ 7.32 (m, 5H, CH aromatic), 4.24 (s, 1H, CH benzylic), 4.02 (m, 2H, CO(=O)CH2 ), 2.62 (m, 8H, N-CH2), 2.30 (s, 3H, N-CH3), 1.51 (m, 3H, CH2-CH aliphatic), 1.06 (t 3 J = 7.1 Hz, 6H, CH3 ethyl), 0.82 (m, 6H, CH3-C-CH3). Incorporation expected at δ 7.32 (red arrow). Determined against integral at δ 2.30 (blue arrow). General method: 10.0 mg (29.9 µmol) 53; 4.40 mg (2.9 µmol) catalyst F, 2h. Yield: a) 7.6 mg, 22.7 µmol, 76%; 31% D for δ 7.32. b) 8.1 mg, 24.2 µmol, 81%; 30% D for δ 7.32.

  ): m/z 321.3 [M +H] + , 323.3 [M(T)+H] + ; 325.3 [M(T2)+H] + 1 H NMR (300 MHz, CD2Cl2): δ 7.25 (m, 5H, CH aromatic), 4.27 (s, 1H, CH benzylic), 3.91 (m, 2H, CO(=O)CH2 ), 2.38 (m, 9H, 4 x N-CH2; NH), 1.37 (m, 3H, CH2-CH aliphatic), 0.83 (br m, δ 7.20 (m, 5H, 2CH meta-phenyl, 2CH' meta-aniline, CH paraphenyl), 7.05 (d, 3 J = 7.0 Hz, 1H, CH para-aniline), 6.95 (m, 2H, CH ortho-phenyl), 4.45 (q, 3 J = 7.5 Hz, 1H, CH), 3.79 (s, 3H, OCH3), 3.36 (d, 3 J = 14.5 Hz, 1H, CH benzylic), 3.22 (d, 3 J = 14.5 Hz, 1H, CH benzylic), 2.41 (s, 3H, CH3 ortho-aniline), 1.89 (s, 3H, CH3 ortho-aniline),1.01 (d, 3 J = 7.5 Hz, 3H, CH3) ppm. Incorporation expected at δ 6.95 (red arrow). Determined against integral at δ 3.79. General method: 12.7 mg (39.0 µmol) 10; 6.0 mg (3.9 µmol) catalyst F, 2h. Yield: a) 11.6 mg, 36.0 µmol, 91%; 96% D for δ 6.95. b) 11.3 mg, 35.0 µmol, 89%; 96% D for δ 6.95.

Scheme 1 .

 1 Scheme 1. Synthesis of 61 F (76.2 mg, 0.05 mmol) was dissolved in 5 mL of a 1:1 mixture of dichloromethane and toluene. The flask was evacuated and filled with hydrogen gas three times. The mixture was then stirred over night until the color changed to a dark blue. The solvent was removed under vacuum and the blue precipitate was washed two times with 0.5 mL toluene each and three times with 0.5 mL hexane each. The product 61 was isolated as a dark blue solid.

  δ 161.9(d, 2 JC,P = 15 Hz, C-1), 162.1 (q, 1 JC,B = 49 Hz, BArF-i-C), 147.2 (NCN), 135.2 (br s, BArF-o-CH), 134.2(d, 4 JC,P = 2 Hz, C-5), 134.1 (C-3), 125.0 (q, 1 JC,F = 272 Hz,BArF-CF3), 121.7 (CCH3), 123.9 (d, 1 JC,P = 25 Hz, C-2), 120.7(d, 3 JC,P = 7 Hz, C-4), 117.9(m, BArF-p-CH), 114.1(d, 3 JC,P = 10 Hz, C-6), 38.6 (d, 1 JC,P = 30 Hz, . for C106H96B2F48Ir2N6P2: C 44.93, H 3.41, N 2.97. Found: C 44.62, H 3.18, N 2.86 (average of three measurements).IR (CH2Cl2):νIr-H = 2307 cm -1 .H NMR spectrum of 61 in CD2Cl2 at 296 K. H{ 31 P} NMR spectrum of 61 in CD2Cl2 at 296 K. P NMR spectrum of 61 in CD2Cl2 at 296 K.

Figure 2 . 1 H

 21 Figure 2. 1 H NMR spectrum of 61 at various temperatures from 296 K to 183 K.

Figure 3 .

 3 Figure 3. IR-Spectra of 61 in CH2Cl2.

Figure 4 .

 4 Figure 4. ORTEP plot of the molecular structure of the dication of 61 with thermal ellipsoids set at 50% probability level. Two BArF24 counteranions and all hydrogens, except the bridging ones, are omitted for clarity. Ir1i, H1i and the corresponding ligand moiety is generated via a crystallographic centre of inversion situated between the two iridium atoms.

Figure 5 .

 5 Figure 5. Reaction progress plotted over reaction time of forward reaction (deuteration, blue) and backward reaction (hydrogenation, red).

Figure 6 .

 6 Figure 6. Relative enthalpy values for forward and backward CH activation processes.

Scheme III. 1 . 1 : 1 .

 111 Scheme III.1. 1: Modulation of the reactivity and selectivity in directed HIE reactions by change of temperature

  A-G in a HIE reaction of 4-acetamidoacetophenone 8 at temperatures from -80°C to 25°C in dichloromethane a)Conditions: substrate 8 (1 eq.), catalyst (5 mol%), D2 (1 atm), dichloromethane (3 mL), 4 h. b)Determined by 1 H-NMR, b determined by LC-MS.

  a)Conditions: substrate 8 (1 eq.), catalyst (5 mol%), D2 (1 atm), C6D5Cl (3 mL), 4 h b) Determined by 1 H NMR, b determined by LC-MS.

Figure III. 1 . 1 :

 11 Figure III.1. 1: Graphical overview in which temperature window the catalysts A-G are efficiently working (threshold 25%D) for HIE reactions with compound 8. aRemarkably, no H/D exchange at the C(sp 3 )-positions was observed, neither on the ketone or acetanilide methyl-groups. Catalysts D showed generally a moderate activity in the HIE reaction of compound 8. Catalyst F was the most sensitive one in relation to heat. Above 50°C the deuterium introduction decreased significantly, indicating that the HIE activity with these catalysts decreased due to their deactivation. To prove this, the active catalyst of F was formed with D2 (without substrate), and then the mixture was heated to 50°C for one hour. After, substrate 8 was added via syringe and the HIE reaction was continued at 50°C for an additional hour. LC-MS analysis of the reaction mixture showed only compound 8 without any deuterium incorporation indicating that the catalyst activity was completely lost while heating in the first hour.

4 : 6 Scheme III. 1 . 5 :

 4615 Scheme III.1. 4: HIE reaction of phenylacetic glycine amide derivative 11 with two iridium catalysts (5 mol%). No deuterated product with catalyst Ba at 25°C and catalyst F at 80°C

  δ 7.35(d, 3 J = 8.5 Hz, 2H, CH2 ortho-chloro), 7.27(d, 3 J = 8.5 Hz, 2H, CH2 ortho-phenyl), 4.06 (q, 3 J = 7.1 Hz, 2H, CH2 ethyl), 3.81 (d, 3 J = 5.9 Hz, 2H, CH2 glycine),3.48 (s, 2H, CH2 benzylic), 1.15 (t, 3 J = 7.1 Hz, 3H, CH3 ethyl) ppm. Deuterium incorporation notified by the coloured dots. Determined against integral at δ 3.48 (blue arrow). Conditions: 10.2 mg (40.0 µmol) 11, 5 mol% cat., PhCl (3 mL), 4h. a) 3.1 mg (2.0 µmol) catalyst F, RT. b) 3.5 mg (2.0 µmol) catalyst Bb, 80°C. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yields: a) 10.2 mg, 40.0 µmol, 100%; 85% D for δ 7.27. b) 10.0 mg, 39.0 µmol, 98%; 80% D for δ 7.27 and 77% D for δ 3.81.

a) 1 1 H

 11 .9 mg (1.2 µmol) catalyst G, 100°C. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yields: a) 6.8 mg, 22 µmol, 92%, 75% D for δ 7.22. δ 7.90 (d, 3 J = 8.3 Hz, 2H, CH2 aromatic ortho S), 7.47 (d, 3 J = 8.3 Hz, 2H, CH2 aromatic ortho N), 7.18 (d, 3 J = 8.0 Hz, 2H, CH2 aromatic), 7.11 (d, 3 J = 8.0 Hz, 2H, CH2 aromatic), 6.74 (s, 1H, CH pyrazole), 4.97 (s, 2H, NH2), 2.39 (s, 3H, CH3) ppm. Deuterium incorporation notified by the coloured dots. Determined against integral at δ 2.39 (blue arrow). Conditions: 9.1 mg (24.0 µmol) 13, 5 mol% cat., PhCl (3 mL), 4h. a) 1.9 mg (1.2 µmol) catalyst E, 100°C. b) 1.0 mg (1.2 µmol) catalyst A, 0°C. c) 1.8 mg (1.2 µmol) catalyst F, 0°C. LC-MS (positive ESI): a) m/z 330.4 [M(2D)+H] + (5%), 331.3 [M(3D)+H] + (25%), 332.3 [M(4D)+H] + (60%), 333.1 [M(5D)+H] + (10%). b) m/z 329.4 [M(1D)+H] + (40%), 330.

Conditions : 11 1 H

 111 .8 mg (24.0 µmol) 14, 5 mol% cat., PhCl (3 mL), 4h. a) 1.9 mg (1.2 µmol) catalyst G, 100°C. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yield: a) 10.6 mg, 21.0 µmol, 90%, 99% D for δ 8, MeOD): δ 7.51 (d, 3 J = 9.0 Hz, 2H, CH2 aromatic meta-methoxy), 7.42 (d, 3 J = 8.5 Hz, 2H, CH2 aromatic ortho-amide), 7.32 (d, 3 J = 8.5 Hz, 2H, CH2 aromatic orthocyclohxylamide), 7.00 (d, 3 J = 9.0 Hz, 2H, CH2 aromatic ortho-methoxy), 4.14 (m, 2H, CH2 ortho-Nitrogen amide), 3.85 (s, 3H, CH3 methoxy), 3.68 (m, 2H, CH2 ortho-Nitrogen amide cyclohexyl), 3.34 (m, 2H, CH2 ortho-pyrazole cycle), 2.52 (m, 2H, CH2 cyclohexyl), 1.97 (m, 4H, 2CH2 cyclohexyl) ppm. Deuterium incorporation notified by the coloured dots. Determined against integral at δ 3.85 (blue arrow). Conditions: 11.00 mg (24.0 µmol) 15, 5 mol% cat., PhCl (3 mL), 4h. a) 1.9 mg (1.2 µmol) catalyst E, 100°C. b) 1.9 mg (1.2 µmol) catalyst G, 100°C. LC-MS (positive ESI): a) m/z 462.4 [M(2D)+H] + (5%), 463.3 [M(3D)+H] + (10%), 464.3 [M(4D)+H] + (55%), 465.3 [M(5D)+H] + (30%). b) m/z 460.5 [M+H] + (5%), 461.4 [M(1D)+H] + (80%), 462.4 [M(2D)+H] , 20 µmol, 83%, 60% D for δ 7.51, 55% D for δ 7.42, 83% D for δ 7.32. b) 11.5 mg, 25 µmol, 100%, 50% D for δ 7.32.

Figure III. 2 . 1 :

 21 Figure III.2. 1: Analysis of 68 aromatic drugs and their different aromatic directing groups(analysed from 200 drugs rated by number of prescriptions, 2016, united states)10

3 : 1 :

 31 Scheme III.2. 3: Experimentally found order of different aryl-DGs in competition HIE experiments

Figure III. 2 . 2 :

 22 Figure III.2. 2: Calculated potential energy surfaces (PES) for the HIE reaction of substrates 2-10 with catalyst Ba a

Scheme III. 2 . 4 :

 24 Scheme III.2. 4: HIE experiment of disubstituted aromatic substrates 12-22a,b,c

2 : 1 H 6 :

 216 , DMSO-d6): δ 7.45-7.39 (m, 3H, CHC3H3CH), 7.35-.7.29 (m, 2H, CHC3H3CH), 3.51-3.02 (br d, 3 J= 75 Hz, 4H, N(CH2CH3)2), 1.21-0.96 (br d, 3 J= 21.5 Hz, 29 mg, 200 µmol 2, 36 mg, 200 µmol 8, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 7.92 ppm (red arrow). Determined against reference-integral at δ 12.50 ppm (blue arrow). Incorporation expected at δ 7.45-7.39 ppm (red arrow). Determined against referenceintegral at δ 7.35-7.29 ppm (blue arrow).

2 : 1 H 8 :

 218 29 mg, 200 µmol 2, 27 mg, 200 µmol 8, 10.1 mg, 10 µmol catalyst Ba Incorporation expected at δ 7.92 ppm (red arrow). Determined against reference-integral at δ 12.50 ppm (blue arrow). Incorporation expected at δ 7.96 ppm (red arrow). Determined against reference-integral at δ 3.82 ppm (blue arrow).

4 : 1 H 4 O 10 :

 41410 ESI): Reference: m/z = 136.2 (M+H + ) (129), 137.2 (M+H + +1 Natural isotope).(12). 9% natural isotope M+1. ESI): Reference: m/z = 136.2 (M+H + ) (129), 137.2 (M+H + +1 Natural isotope). (12). 9% natural isotope M+1. 39 mg, 200 µmol 4, 34 mg, 200 µmol 10, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 8.05 ppm (red arrow). Determined against reference-integral at δ 2.63 ppm (blue arrow). Incorporation expected at δ 7.72 ppm (red arrow). Determined against referenceintegral at δ 2.36 ppm (blue arrow).

3 :

 3 27 mg, 200 µmol 3, 25 mg, 200 µmol 5, 10.1 mg, 10 µmol catalyst Ba. N.B.: These reactions were not separated before NMR analysis, since the respective spectra of the compounds do not overlap. Incorporation expected at δ 7.56 ppm (red arrow). Determined against reference-integral at δ 2.03 ppm (blue arrow).

.

1 H 5 : 1 H 3 :

 1513 Incorporation expected at δ 8.21 ppm (red arrow). Determined against reference-integral at δ 7.66 ppm (blue arrow).Result:1) 72% D for compound 3, 28% D for compound 5.2) 76% D for compound 3, 13% D for compound 5 ESI): Reference: m/z = 178.2 (M+H + ) (129), 179.2 (M+H + +1 Natural isotope). (15). 10% natural isotope M+1. 27 mg, 200 µmol 3, 27 mg, 200 µmol 9, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 7.56 ppm (red arrow). Determined against reference-integral at δ 2.03 ppm (blue arrow).

1 H 9 :

 19 Incorporation expected at δ 7.12 ppm (red arrow). Determined against reference-integral at δ 2.26 ppm (blue arrow).

3 : 1 H 10 :

 3110 27 mg, 200 µmol 3,34 mg, 200 µmol 10, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 7.56 ppm (red arrow). Determined against reference-integral at δ 2.03 ppm (blue arrow). Incorporation expected at δ 7.72 ppm (red arrow). Determined against referenceintegral at δ 2.36 ppm (blue arrow).

5 : 1 H 7 :

 517 25 mg, 200 µmol 5, 21 mg, 200 µmol 7, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 8.21. ppm (red arrow). Determined against reference-integral at δ 7.66 ppm (blue arrow). Incorporation expected at δ 7.91 ppm (red arrow). Determined against reference-integral at δ 7.60 ppm (blue arrow).

4 : 1 H 5 :

 415 39 mg, 200 µmol 4, 25 mg, 200 µmol 5, 10.1 mg, 10 µmol catalyst Ba. N.B.: Two separate reactions were done but were pooled before purification. Incorporation expected at δ 8.05 ppm (red arrow). Determined against reference-integral at δ 2.63 ppm (blue arrow). Incorporation expected at δ 8.05 ppm (red arrow). Determined against reference-integral at δ 2.63 ppm (blue arrow).

6 : 1 H 8 :

 618 36 mg, 200 µmol 6, 27 mg, 200 µmol 8, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 7.35-7.29 ppm (red arrow). Determined against referenceintegral at δ 7.45-7.39 ppm (blue arrow). Incorporation expected at δ 7.96 ppm (red arrow). Determined against reference-integral at δ 3.82 ppm (blue arrow).

6 : 1 H 9 :

 619 36 mg, 200 µmol 6, 27 mg, 200 µmol 9, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 7.35-7.29 ppm (red arrow). Determined against referenceintegral at δ 7.45-7.39 ppm (blue arrow). Incorporation expected at δ 7.12 ppm (red arrow). Determined against reference-integral at δ 2.26 ppm (blue arrow).

6 : 1 H

 61 mg, 200 µmol 6, 34 mg, 200 µmol 10, 10.1 mg, 10 µmol catalyst Ba. Incorporation expected at δ 7.35-7.29 ppm (red arrow). Determined against referenceintegral at δ 7.45-7.39 ppm (blue arrow). D for compound 6, 2% D for compound 10.2) 82% D for compound 6, 2% D for compound 10.

7 :

 7 21 mg, 200 µmol 7, 27 mg, 200 µmol 8, 10.1 mg, 10 µmol catalyst Ba. N.B.: Two separate reactions were done but were pooled before purification. Incorporation expected at δ 7.60 ppm (red arrow). Determined against reference-integral at δ 7.91 ppm (blue arrow).

1 H 8 :

 18 Incorporation expected at δ 7.96 ppm (red arrow). Determined against reference-integral at δ 3.82 ppm (blue arrow).

1 H

 1

  ): m/z 271.3 [M+H] + (53%), 272.2 [M(1D)+H] + (38%), 273.1 [M(2D)+H] + (9%).

1 H

 1

  17

Method B: 10

 10 mg, 61 µmol 17, 3.1 mg, 3.1 µmol catalyst Ba. LC-MS (positive ESI): m/z 165.1 [M(1D)+H] + (7%), 166.1 [M(2D)+H] + (15%), 167.1 [M(3D)+H] + (75%), 168.1 [M(4D)+H] + (3%).

1 H

 1

  , 3.15 (s, 3H, NMe), 2.97 (s, 3H, NMe) ppm. Incorporation expected at δ 8.29 and/or δ 7.59 (red arrow). Determined against reference-integral at δ 3.15 ppm (blue arrow). Method B: 7.7 mg, 40 µmol 20, 2.0 mg, 2.0 µmol catalyst Ba. LC-MS (positive ESI): m/z 196.1 [M(1D)+H] + (5%), 197.1 [M(2D)+H] + (46%), 198.1 [M(3D)+H] + (49%).Yield: 1) 7.5 mg, 39 µmol, 97%; 27% D for δ 8.29, 93% D for 7.59.2) 7.1 mg, 36 µmol, 92%; 31% D for δ 8.29, 94% D for 7.59.

  ): 10 mg, 42 µmol 22, 2.1 mg, 2.1 µmol catalyst Ba. Yield: 1) 9.5 mg, µmol, 95%; 37% D for δ 7.64, 0% D for δ 7.64, 60% D for δ 7.16.2) 8.9 mg, µmol, 89%; 37% D for δ 7.64, 0% D for δ 7.64, 56% D for δ 7.16.Average: y= 92%, 58% D at position a, 37% D at position b, 0% D at position c.

Yield: 1 1 H

 11 ) 9.9 mg, 26 µmol, 99%; 13% D for δ 7.86, 86% D for δ 7.53. 2) 9.6 mg, 25 µmol, 96%; 10% D for δ 7.86, 91% D for δ 7.53. , MeOD): δ 8.03 (m, 3H, CH(a) and 2 CH(b)), 6.92 (d, 3 J= 8.7 Hz, 2H, 2 CH(c)), 4.04 (s, 3H, CH3), 2.67 (s, 3H, CH3) ppm. Incorporation expected at δ 8.03 ppm (red arrow). Determined against reference-integral at δ 4.04 ppm (blue arrow). Method B (80°C): 5.7 mg, 20 µmol 25, 1.0 mg, 1.0 µmol catalyst Ba. LC-MS (positive ESI): m/z, 270.2 [M+H] + (9%), 271.2 [M(1D)+H] + (79%), 272.1 [M(2D)+H] + (12%).Yield: 1) 5.2 mg, 18 µmol, 91%; 50%D for δ 8.03.

  Method B (80°C): 5.3 mg, 20 µmol 26, 1.0 mg, 1.0 µmol catalyst Ba. LC-MS (positive ESI): m/z, 264.2 [M+H] + (5%), 265.1 [M(1D)+H] + (85%), 266.1 [M(2D)+H] + (10%). Yield: 1) 4.7 mg, 18 µmol, 89%; 54%D for δ 8, DMSO-d6: δ 10.30 (s, 1H), 8.26 (t, 3 J= 5.6 Hz, 1H), 7.83 (d, 3 J= 8.3 Hz, 2H, Hb), 7.63 (d, 4 J= 2.9 Hz, 1H, Ha), 7.53-7.44 (m, 3H), 7.14 (d, 3 J= 9.0 Hz, 1H), 6.32 (d, 3 J= 7.8 Hz, 1H), 3.79 (s, 3H), 3.54 (q, 3 J= 6.7 Hz, 2H), 2.92 (t, 3 J= 7.0 Hz, 2H), 1.70-1.41 (m, 5H), 1.14 (sep, 3 J= 9.7 Hz, 6H) ppm. Incorporation expected at δ 7.83 and/or δ 7.63 ppm (red arrow). Determined against reference-integral at δ 3.79 ppm (blue arrow). Method B: 10 mg, 20 µmol 25, 1.0 mg, 1.0 µmol catalyst Ba. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yield: 1) 10.0 mg, 20 µmol, 100%; 82% for δ 7.63. 2) 9.5 mg, 19 µmol, 95%; 83% for δ 7.63. , DMSO-d6: δ 12.42 (s, 1H, NH), 7.81 (d, 3 J= 2.5 Hz, 1H, CHc), 7.77 (d, 3 J= 8.8 Hz, 2H, 2 CHa), 7.68 (s, 1H, CHf), 7.56 (dd, 3 J= 8.8 Hz and 4 J= 2.5 Hz, 1H, CHd), 7.34 (d, 3 J= 8.8 Hz, 1H, CHe), 7.11 (d, 3 J= 8.8 Hz, 2H, 2 CHa), 1.88 (m, 2H, CH2), 1.62 (m, 3H, CH3), 0.97 (m, 3H, CH3) ppm. Incorporation expected at δ 7.81 and/or δ 7.56 ppm (red arrow). Determined against reference-integral at δ 1.62 ppm (blue arrow). Method B: 5.0 mg, 12.3 µmol 28, 1.2 mg, 1.2 µmol catalyst Ba. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl.

1 H 1 H

 11 , DMSO-d6: δ 9.76 (s, 1H, CHO), 8.08 (dd, 3 J= 7.7 Hz and 4 J= 1.5 Hz, 1H, CH ring f), 8.02 (d, 3 J= 1.89 Hz, 1H, CHc), 7.67 (m, 3H, CHe, CH ring f), 7.58 (m, 3H, 3 CHa), 7.52 (m, 2H, 2 CHb), 7.39 (m, 1H, CH ring f), 6.93 (m, 1H, CH ring f), 5.93 (s, 2H, CH2), 3.34 (s, 2H, CH2), 0.97 (s, 9H, tBu) ppm. Incorporation expected at δ 7.58 and/or δ [ring f] ppm (red arrow). Determined against reference-integral at δ 0.97 ppm (blue arrow). Method B: 5.0 mg, 8.5 µmol 29, 0.9 mg, 0.9 µmol catalyst Ba. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yield: 1) 5.2 mg, 8.8 µmol 29, quant.; 67%D for δ 7.58 ppm. δ 9.56 (s, 1H, NH), 8.42 (m, 1H, CH pyridine), 8.04 (dd, 3 J= 8.6 Hz and 2.6 Hz, 1H, CHe), 7.89 (d, 4 J= 1.6 Hz, 1H, CHa), 7.72 (m, 4H, 1 CH pyridine, CHb, CHc, CHd), 7.60 (d, 3 J= 8.0 Hz, 1H, CH pyridine), 7.50 (d, 3 J= 8.6 Hz, 1H, CHf), 7.21 (m, 1H, CH pyridine), 3.02 (s, 3H, CH3) ppm. Incorporation expected at δ 7.72 ppm (red arrow). Determined against reference-integral at δ 3.02 ppm (blue arrow). Method B: 10.5 mg, 25 µmol 30, 1.3 mg, 1.3 µmol catalyst Ba. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yield: 1) 8.6 mg, 20 µmol, 82%; 62%D for δ 7.72. 2) 9.9 mg, 23 µmol, 94%; 72%D for δ 7.72. , DMSO-d6: δ 11.52 (s, 1H, CHO), 9.98 (s, 1H, OH), 8.35 (d, 3 J= 2.2 Hz, 1H, ), 8.29 (s, 1H, ), 7.87 (dd, 3 J= 8.2 Hz, 4 J= 2.0 Hz, 1H, ), 7.77 (d, 3 J= 8.4 Hz, 1H, ), 7.73 (s, 1H, ), 3.20 (s, 3H, CH3), 2.93 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.21 (s, 3H, CH3) ppm. Incorporation expected at δ and δ ppm (red arrow). Determined against reference-integral at δ 2.21 ppm (blue arrow). Method B: 5.0 mg, 12.3 µmol 31, 1.2 mg, 1.2 µmol catalyst Ba. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl. Yield: 1) 5.3 mg, 12.6 µmol, quant.; 95% D for δ 7.87 and 89% D for δ 7.73 ppm.

Figure IV. 2 :

 2 Figure IV. 2: Structure of an ADC (left) and comparison of unselective and selective toxic agent (right).

Besides DM1 2

 2 utilized for example in Trastuzumab emtansine (T-DM1), DM4 1a is another frequently applied synthetic maytansinoid payload. In DM4 1a, next to the C-S bond, two germinal dimethyl groups have been installed to increase sterical hindrance for improved chemical stability (figure IV.1).8 Examples of DM4-conjugated antibodies in clinical development include oltuximab ravtansine (SAR3419)9 targeting CD19 to treat acute lymphoblastic leukemia (ALL), mirvetuximab soravtansine (IMGN853),10 a FRα-targeting ADC for the treatment of FRαpositive, platinum-resistant ovarian cancer and lorvotuzumab mertansine (IMGN901), a CD56-targeting ADC for treatment of small-cell lung cancer (SCLC) (figure IV.4).11

Figure IV. 4 :

 4 Figure IV. 4: Maytansinoid-conjugated antibodies examples9,10,13

Figure IV. 5 :

 5 Figure IV. 5: 3 H-NMR of 3 H-DM4-SMe 1c a

Figure IV. 6 :

 6 Figure IV. 6: HIE reaction with catalyst Bb on 1a-c

IV. 2 :

 2 Scheme IV. 2: HIE reaction ofDM4 side chain precursor 10-12 with catalyst Bb a

  Scheme IV. 3: HIE reaction of DM4 side chain precursors 13-17 with catalyst Bb a

Figure IV. 7 :

 7 Figure IV. 7: Enantioselective HPLC-Separation of L-Ac-Ala-OMe 19a and D-Ac-Ala-OMe 19b

IV. 4 :

 4 Scheme IV. 4: HIE reaction with amino acids 20-22 a

IV. 5 :

 5 Scheme IV. 5: HIE reaction on dipeptides with N-terminal glycine 23-29a

Scheme IV. 6 :

 6 Scheme IV. 6: HIE reaction on dipeptides with C-terminal glycine 30-34a

  Scheme IV. 7: HIE reaction on peptides 35-40 a

Figure IV. 8 :

 8 Figure IV. 8: Calculated free energy profile for selective deuteration of Ac-Gly-OMe 18a with catalyst Bb (ligands not drawn)

Figure IV. 9 :

 9 Figure IV. 9: 5-membered transition state of catalyst Bb and Ac-Gly-OMe 18a (left) and of Boc-Gly-OMe 18d (right)

  ): m/z 808.3 [M-H2O+H] + , 810.3 [M(T)-H2O+H] + ; 848.3 [M+Na] + ; 826.3 [M+H] + MS (DM4-SMe 1c, unlabelled) MS (DM4-SMe 1c, 3 H-labelled) H-NMR: DM4-SMe 1c unlabeled comparison (upper spectra); 3 H-DM4-SMe 1c (below) H-NMR (533 MHz): 3 H-DM4-SMe 1c H NMR 1c (533 MHz, DMSO-d6): δ 2.52-2.43, 2.25-2.16 (m, 2H, 6%); 1.98-1.66 (m, 2H, 94%) ppm.

  All spectra about the HIE experiments are available from the following page.4-[(4-methyl)dithio]-4-methyl-N-(benzyl)-pentanamide 10 δ 7. 5H), 5.90 (br s, 1H, NH), 4.36 (d, 3 J=6.9 Hz, 2H, CH2Ph),2.32 (s, 3H, SCH3), 2.22-2.16 (m, 2H), 1.90-1.85 (m, 2H), 1.22 (s, 6H, CH3) ppm. Incorporation expected at δ 1.90-1.85 (red arrow). Determined against integral at δ 4.36 (blue arrow). HRMS (positive ESI): m/z calculated for C14H21NOS2 [M+H] + : 284.1137; found: 284.1151. Conditions: 3.5 mg (12.0 µmol) 10; 2.1 mg (1.2 µmol) catalyst Bb, 3h. Yield: 3.2 mg, 11.0 µmol, 91%; 99%D for δ 1.90-1.85.4-[(4-methyl)dithio]-4-methyl-N-(propyl)-pentanamide 11 δ 5.56 (br s, 1H, NH), 3.17 (dd, 3 J= 6.9 Hz, 3 J= 6.9 Hz, 2H), 2.35 (s, 3H, SCH3), 2.23-2.18 (m, 2H), 1.91-1.86 (m, 2H), 1.51 (dt, 3 J= 6.9 Hz,3 J= 7.5 Hz, 2H), 1.23 (s, 6H, CH3), 0.88 (t, 3 J= 7.5 Hz, 3H) ppm. Incorporation expected at δ 1.91-1.86 (red arrow). Determined against integral at δ 2.35 (blue arrow). HRMS (positive ESI): m/z calculated for C10H21NOS2 [M +H] + : 236.1137; found: 236.1139. Conditions: 3.0 mg (13.0 µmol) 11; 2.3 mg (1.3 µmol) catalyst Bb, 3h. Yield: 2.6 mg, 11.0 µmol, 88%; 99%D for δ 1.91-1.86. 3.33-3.22 (m, 4H), 2.34 (s, 3H, SCH3), 2.27-2.24 (m, 2H), 1.87-1.80 (m, 2H) 1.25 (s, 6H), 1.15 (t, 3 J = 7.2 Hz, 3H), 1.06 (t, 3 J = 7.2 Hz, 3H) ppm. Incorporation expected at δ 1.87-1.80 (red arrow). Determined against integral at δ 3.33-3.22 (blue arrow). HRMS (positive ESI): m/z calculated for C11H23NOS2 [M+H] + : 250.1293; found: 250.1300. Conditions: 3.0 mg (12.0 µmol) 12; 2.1 mg (1.2 µmol) catalyst Bb, 3h. Yield: 2.8 mg, 11.0 µmol, 93%; 99%D for δ 1.87-1.80

  HRMS (positive ESI): m/z calculated for C11H19NO3S2 [M+H] + : 280.1035; found: 280.1045. Conditions: 5.0 mg (19.0 µmol) 13; 3.3 mg (1.9 µmol) catalyst Bb, 3h. Yield: a) 4.4 mg, 15.0 µmol, 87%; 99%D for δ 1.91-1.85. b) 4.5 mg, 16.0 µmol, 91%; 99% D for δ 91-1.85.

  ppm. Incorporation expected at δ 1.96-1.90 (red arrow). Determined against integral at δ 4.25 (blue arrow). HRMS (positive ESI): m/z calculated for C12H23NO4S2 + [M+H] + : 310.1141; found: 310.1144. Conditions: 5.0 mg (16.0 µmol) 14; 2.8 mg (1.6 µmol) catalyst Bb, 3h. Yield: a) 4.5 mg, 15.0 µmol, 90%; 98% D for δ 1.96-1.90. b) 4.9 mg, 16.0 µmol, 98%; 91% D for δ 1.96-1.90.

1 H

 1 , CDCl3): δ 6.11 (br s, 1H, NH), 3.99(d, 3 J=5.4 Hz, 2H, CH2NH), 3.70 (s, 3H, COOCH3), 2.35 (s, 3H, SCH3), 2.29-2.23 (m, 2H), 1.89-1.84 (m, 2H), 1.24 (s, 6H, CH3) ppm. Incorporation expected at δ 1.89-1.84 and 3.99 (red arrows). Determined against integral at δ 3.70 (blue arrow). HRMS (positive ESI): m/z calculated for C10H19NO3S2 + [M+H] + : 266.0879; found: 266.0888. Conditions: 5.0 mg (19.0 µmol) 15; 3.3 mg (1.9 µmol) catalyst Bb, 3h. Yield: a) 4.3 mg, 16.0 µmol, 85%; 42% D for δ 1.89-1.84 and 88% D for δ 3.99. b) 4.9 mg, 18.0 µmol, 98%; 30% D for δ 1.89-1.84 and 80% D for δ 3.99.

  , 2.31 (t, 3 J=7.5 Hz, 2H), 1.81-1.65 (m, 2H), 1.09 (d, 3 J=6.6 Hz, 3H) ppm. Incorporation expected at δ 3.99 (red arrow). Determined against integral at δ 3.69 (blue arrow). HRMS (positive ESI): m/z calculated for C9H17NO4 + [M+H] + : 204.1230; found: 204.1234. Conditions: 7.0 mg (35.0 µmol) 16; 6.1 mg (3.5 µmol) catalyst Bb, 3h. Yield: 5.3 mg, 26.0 µmol, 76%; 93%D for δ 3.99.

Yields 18a: 9 D

 9 .0 mg, 75.0 µmol, 99%; 93%D for δ 3.89. 18b: 9.8 mg, 43.0 µmol, 98%; 50%D for δ 3.99. 18c: 7.20 mg, 23.0 µmol, 72%; 30%D for δ 3.95. 18d: 0%D for δ 3.65. 18e: 0%D for δ 3.82. δ 6.36 (br s, 1H, NH), 4.51-4.42 (m, 1H), 3.61 (s, 3H, COOCH3), 1.89 (s, 3H, COCH3), 1.28 (d, 3J= 9.0 Hz, 3H) ppm. Incorporation expected at δ 4.51-4.42 (red arrow). Determined against integral at δ 3.61 (blue arrow). Conditions: 10.0 mg (69.0 µmol) 19a; 12 mg (6.9 µmol) catalyst Bb, 8h. Yield: 6.5 mg, 45.0 µmol, 65%; 50%D for δ 4.51-4.42 δ 6.75 (br s, 1H, NH), 4.55-4.45 (m, 1H), 3.67 (s, 3H, COOCH3), 1.94 (s, 3H, COCH3), 1.33 (d, 3J= 9.0 Hz, 3H) ppm. Incorporation expected at δ 4.51-4.42 (red arrow). Determined against integral at δ 3.61 (blue arrow). Conditions: 10.0 mg (69.0 µmol) 19b; 12 mg (6.9 µmol) catalyst Bb, 8h. Yield: 7.8 mg, 54.0 µmol, 78%; 50%D for 4.51-4HIE reaction 19b Enantioselective HPLC-Separation of L-Ac-Ala-OMe 19a and D-Ac-Ala-OMe 19b In all HIE reactions of 19a and 19b no isomerization of the stereocenter was observed. Ac-beta-Ala-ethyl ester 22

Conditions: 10 1 H 1 H 1 H

 10111 .0 mg (63.0 µmol) 22; 11 mg (6.3 µmol) catalyst Bb, 8h. Yield: 9.5 mg, 60.0 µmol, 95%; 91%D for δ 3.46. δ 7.32-7.05 (m, 5H, arom. CH),6.51 (br s, 1H, NH), 6.28 (br s, 1H, NH),4.88-4.83 (m, 1H), 3.95-3.88 (m, 2H),3.74 (s, 3H, COOCH3), 3.22-3.05 (m, 2H), 2.01 (s, 3H, COCH3) ppm. Incorporation expected at δ 3.95-3.88 (red arrow). Determined against integral at δ 3.74 (blue arrow). Conditions: 10.0 mg (36.0 µmol) 23; 6.2 mg (3.6 µmol) catalyst Bb, 8h. Yield: a) 9.0 mg, 32.0 µmol, 90%; 50%D for δ 3.95-3.88. b) 8.7 mg, 31.0 µmol, 87%; 60% D for δ 3.95-3.88. δ 7.39 (m, 5H, 5 CH Phe), 7.28 (m, 3H, 2 CH meta and 1 CH para Cbz), 7.10 (m, 2H, 2 CH ortho Cbz), 6.45 (br s, 1H, NH), 5.38 (br s, 1H, NH), 5.15 (s, 2H, CH2 benzylic), 4.45 (m, 1H, CH Phe), 3.90 (m, 2H, CH2 Gly), 3.75 (s, 3H, OCH3), 3.15 (m, 2H, CH2 Phe) ppm. Incorporation expected at δ 3.90 (red arrow). Determined against integral at δ 5.15 (blue arrow). Conditions: 10.0 mg (27.0 µmol) 24; 4.7 mg (2.7 µmol) catalyst Bb, 8h. Yield: a) 8.4 mg, 23.0 µmol, 84%; 14%D for δ 3.90. L-Ac-Gly-Tyr(OtBu)-tert-butyl ester 2527 , CDCl3): δ 7.06 (d, 3 J = 8.7 Hz, 2H), 6.93 (d, 3 J = 8.7 Hz, 2H), 6.26 (br s, 1H, NH), 6.12 (br s, 1H, NH), 4.75-4.68 (m, 1H), 3.94-3.91 (m, 2H), 3.06 (d, 3 J = 7.2 Hz, 2H), 2.03 (s, 3H, COCH3), 1.52-1.35 (m, 18H, OtBu) ppm. Incorporation expected at δ 3.94-3.91 (red arrow). Determined against integral at δ 3.06 (blue arrow). Conditions: 10.0 mg (26.0 µmol) 27; 4.5 mg (2.6 µmol) catalyst Bb, 8h. Yield: a) 7.4 mg, 19.0 µmol, 74%; 93% D for δ 3.94-3.91. b) 6.6 mg, 17.0 µmol, 66%; 93% D for δ 3.94-3.91. δ 6.45 (br s, 1H, NH), 6.17 (br s, 1H, NH), 4.00 (d, 3 J = 5.4 Hz, 2H), 3.93 (d, 3 J = 5.4 Hz, 2H), 3.70 (s, 3H, COOCH3), 1.99 (s, 3H, COCH3) ppm. Incorporation expected at δ 3.93 and 4.00 (red arrow). Determined against integral at δ 3.70 (blue arrow). Conditions: 10.0 mg (53.0 µmol) 28; 9.2 mg (5.3 µmol) catalyst Bb, 8h. Yield: a) 9.7 mg, 52.0 µmol, 97%; 37% D for δ 4.00 and 55% D for δ 3.93. b) 9.2 mg, 49.0 µmol, 92%; 37% D for δ 4.00 and 57% D for δ 3.93. δ 6.55 (br s, 1H, NH), 5.06 (br s, 1H, NH), 4.01 (d, 3 J = 5.7 Hz, 2H), 3.80 (d, 3 J = 5.7 Hz, 2H), 3.70 (s, 3H, COOCH3), 1.39 (s, 9H) ppm. Incorporation expected at δ 4.01 (red arrow). Determined against integral at δ 3.70 (blue arrow). Conditions: 10.0 mg (41.0 µmol) 29; 7.1 mg (4.1 µmol) catalyst Bb, 8h. Yield: a) 7.8 mg, 32.0 µmol, 78%; 50% D for δ 4.01. b) 8.9 mg, 36.0 µmol, 89%; 54% D for δ 4.01. δ 6.56 (br s, 1H, NH), 4.87 (br s, 1H, NH), 4.21-4.09 (m, 1H), 3.98 (br s, 2H), 3.70 (s, 3H, COOCH3), 1.39 (s, 9H) ppm. Incorporation expected at δ 3.98 (red arrow). Determined against integral at δ 3.70 (blue arrow). Conditions: 10.0 mg (38.0 µmol) 30; 6.6 mg (3.8 µmol) catalyst Bb, 8h. Yields: a) 9.7 mg, 37.0 µmol, 97%; 59% D for δ 3.98. b) 9.5 mg, 36.0 µmol, 95%; 60% D for δ 3.98. , CDCl3): δ 6.63 (br s, 1H, NH), 4.85 (br s, 1H, NH), 4.21-4.11 (m, 1H), 4.06 (d, 3 J = 6.3 Hz, 2H), 3.77 (s, 3H, COOCH3), 1.67-1.55 (m, 3H), 1.46 (s, 9H), 0.99-0.94 (m, 6H) ppm. Incorporation expected at δ 4.06 (red arrow). Determined against integral at δ 3.77 (blue arrow). Conditions: 10.0 mg (33.0 µmol) 31; 5.7 mg (3.3 µmol) catalyst Bb, 8h. Yield: a) 7.1 mg, 23.0 µmol, 71%; 72% D for δ 4.06. b) 7.3 mg, 24.0 µmol, 73%; 76% D for δ 4.06. , CDCl3): δ 7.36-7.21 (m, 5H, arom. CH), 6.35 (br s, 1H, NH), 4.95 (br s, 1H, NH), 4.45-4.37 (m, 1H), 4.09 (dd, 2 J = 15.6 Hz, 3 J = 5.7 Hz, 2H), 3.75 (s, 3H, COOCH3), 3.11-3.05 (m, 2H), 1.42 (s, 9H) ppm. Incorporation expected at δ 4.09 (red arrow). Determined against integral at δ 3.75 (blue arrow). Conditions: 12.0 mg (35.0 µmol) 32; 6.0 mg (3.5 µmol) catalyst Bb, 8h. Yield: a) 11.1 mg, 33.0 µmol, 93%; 95% D for δ 4.09. b) 10.8 mg, 32.0 µmol, 90%; 89% D for δ 4.09. , CDCl3): δ 7.69 (br s, 1H, NH), 6.61 (br s, 1H, NH), 5.91 (br s, 1H, NH), 4.28-4.16 (m, 1H), 4.15-3.88 (m, 2H), 3.72 (s, 3H, COOCH3), 3.30-3.19 (m, 2H, NCH2CH3), 2.39-2.28 (m, 2H), 2.15-1.95 (m, 2H), 1.42 (s, 9H), 1.12 (t, 3 J = 7.2 Hz, NCH2CH3) ppm. Incorporation expected at δ 4.15-3.88 and 2.15-1.95 (red arrow). Determined against integral at δ 3.72 (blue arrow). Conditions: 10.4 mg (30.0 µmol) 33; 5.2 mg (3.0 µmol) catalyst Bb, 8h. Yield: a) 8.7 mg, 25.0 µmol, 84%; 49% D for δ 4.15-3.88 and 69% D for δ 2.15-1.95. b) 8.2 mg, 24.0 µmol, 79%; 54% D for δ 4.15-3.88 and 58% D for δ 2.15-1.95.

  δ 4.32 (br s, 1H, CH alpha Pro), 4.08 (br s, 2H, CH2 Gly),3.75 (s, 3H, OCH3), 3.48 (m, 3H, CH2),1.83 (m, 3H, CH2),1.40 (s, 9H, Boc) ppm. Incorporation expected at δ 4.32 and/or 4.08 (red arrow). Determined against integral at δ 3.75 (blue arrow). Conditions: 10.0 mg (35.0 µmol) 34; 6.0 mg (3.5 µmol) catalyst Bb, 8h. LCMS (positive ESI): m/z 188.2 [M(1D)+H-Boc] (7%), 189.2 [M(2D)+H-Boc] (46%), 190.2 [M(3D)+H-Boc] (37%), 191.2 [M(4D)+H-Boc] (10%). Yield: a) 10.3 mg, 36.0 µmol, quant.; 36% D for δ 4.32 and 93% D for δ 4.08. L-Cbz-Tyr(OtBu)-Gly-Leu-tert-butyl ester 35 δ 7.39-7.24 (m, 5H), 7.04 (d, 3 J = 8.7 Hz, 2H), 6.89 (d, 3 J = 8.7 Hz, 2H), 6.64 (br s, 2H, NH), 5.45-5.32 (m, 1H), 5.11-4.95 (m, 2H), 4.52-4.42 (m, 1H), 4.39-4.28 (m, 1H), 4.11-3.96 (m, 1H), 3.79-3.68 (m, 1H), 3.13-2.91 (m, 2H), 1.71-1.50 (m, 3H), 1.44 (s, 9H), 1.31 (s, 9H), 0.96-0.88 (m, 6H) ppm. Incorporation expected at δ 4.11-3.96 (red arrow). Determined against integral at δ 5.11-4.95 (blue arrow). Conditions: 12.0 mg (20.0 µmol) 35; 3.5 mg (2.0 µmol) catalyst Bb, 8h. Yield: a) 11.9 mg, 19.0 µmol, 99%; 98% D for δ 4.11-3.96 and 97% D for δ 3.79-3.68. b) 11.7 mg, 19.0 µmol, 98%; 97% D for δ 4.11-3.96 and 82% D for δ 3.79-3.68. (L)-Boc-Tyr-Gly-Ala-Phe-methyl ester 36 , CDCl3): δ 8.50 (br s, 1H, NH), 7.62 (d, 3 J= 8.4 Hz, 1H), 7.36-7.08 (m, 8H), 7.00 (s, 1H), 6.84 (br s, 1H, NH), 6.63 (br s, 1H, NH), 6.52 (br s, 1H, NH), 5.22 (d, 3 J= 7.2 Hz, 1H, NH), 4.83-4.76 (m, 1H), 4.49-4.37 (m, 2H), 3.85-3.73 (m, 2H), 3.70 (s, 3H, COOCH3), 3.33-3.17 (m, 2H), 3.15-3.02 (m, 2H), 1.41 (s, 9H), 1.23 (d, 3 J = 7.2 Hz, 3H) ppm. Incorporation expected at δ 3.85-3.73 and 3.33-3.17 (red arrow). Determined against integral at δ 3.70. Conditions: 10.0 mg (17.0 µmol) 36; 2.9 mg (1.7 µmol) catalyst Bb, 8h. Yield: a) 9.6 mg, 16.0 µmol, 96%; 96% D for δ 3.85-3.73 and 50% D for δ 3.33-3.17. b) 9.2 mg, 15.0 µmol, 92%; 94% D for δ 3.85-3.73 and 50% D for δ 3.33-3.17

  .

1 H 2 )

 12 , CDCl3): δ 7.80 (m, 2H, 2 CH aromatic), 7.60 (m, 2H, 2 CH aromatic), 7.30 (m, 8H, 8 CH aromatic), 7.10 (m, 2H, 2 CH aromatic), 6.45 (br s, 1H, NH), 5.45 (br s, 1H, NH), 4.90 (m, 2H, OCH2), 4.25 (m, 1H, CH Fmoc), 3.90 (m, 2H, CH2 Gly), 3.75 (s, 3H, OCH3), 3.15 (m, 2H, CH2 benzylic) ppm. Incorporation expected at δ 3.90. Determined against integral at δ 3.75. Conditions: 10.0 mg (22.0 µmol) 37; 2.2 mg (3.8 µmol) catalyst Bb, 8h. LCMS (positive ESI): m/z 459.2 [M+H] (47%), 460.2 [M(1D)+H] (38%), 461.3 [M(2D)+H] (13%), 462.23 [M(3D)+H] (2%). Yield: a) 8.6 mg, 19.0 µmol, 86%; 23% D for δ 3.90. L-Boc-Lys(boc)-Met-methyl ester 38 δ 6.79 (br s, 1H, NH), 5.16 (br s, 1H, NH), 4.76-4.68 (m, 2H, NH,CH), 4.11-4.02 (m, 1H), 3.76 (s, 3H, COOCH3), 3.18-3.05 (m, 2H), 2.53 (t, 3J=7.4 Hz, 2H), 2.24-2.11 (m, 1H), 2.09 (s, 3H, SCH3), 2.05-1.93 (m, 2H), 1.90-1.55 (m, 2H), 1.54-1.35 (m, 20H) ppm. Incorporation expected at δ 4.76-4.68. Determined against integral at δ 3.76. Conditions: 10.3 mg (21.0 µmol) 38; 3.6 mg (2.1 µmol) catalyst Bb, 8h. Yield: a) 10.0 mg, 21.0 µmol, 100%; 95% D for δ 4.76-4.68. b) 9.8 mg, 20.0 µmol, 98%; 95% D for δ 4.76-4.68. Due to the pro-chiral environment of the Gly-alpha carbon and catalyst Bb, four reaction pathways are conceivable: Two enter into the reaction by engaging the Ir-center with the Pro-R hydrogen while the Pro-S hydrogen is either facing towards the triphenylphosphine ligand (up, u) or towards the NHC ligand (down, d). All four pathways have been calculated up to the stage of the insertion product and the results are summarized in Figure S1.

  Figure S1

Figure S2 V. 1 .

 S21 Figure S2

Figure V. 1 :

 1 Figure V. 1: Size comparison between homogeneous and heterogeneous catalysts

Scheme V. 1 :Figure V. 2 :V. 1 . 3 . 9 ,

 12139 Scheme V. 1: Synthesis of N-heterocyclic carbene stabilized iridium nanoparticles IrICy 3a-c

  1). These nanoparticles have been evaluated in the HIE reaction of our model substrate 4butylaniline 17. A study of the deuterium incorporation versus time has been done for each of them (figure V.3). The IrICy 3b provided the best result with incorporation of 1.85 D/molecule, in comparison with IrICy 3a (1.79 D/molecule) and IrICy 3c (1.72 D/molecule). Consequently, the IrICy 3b was used for continuing the project.

Figure V. 3 :

 3 Figure V. 3: Comparison of the IrICy nanoparticles 3a-c in the HIE reaction of aniline 17 a,b

Figure V. 4 :

 4 Figure V. 4: Evaluation of the catalyst loading of IrICy 3b in the HIE reaction of aniline 17 a,b

Figure V. 5 :

 5 Figure V. 5: Evaluation of the homogeneity between the different batches of IrICy 3b. a,b

  Scheme V. 5: HIE reaction with various anilines 17-30a,b

  : substrate 21 (1 eq.), IrICy 3b (4.0 mol%), solvent (3 mL), D2 (1 bar), 3h, Temp. (°C). b) Method A (blue): 55°C, THF. Method B (green): 80°C, THF/D2O (1:1). Method C (pink): 80°C, THF/H2O (1:1). c) Position and percentage of deuterium incorporation determined by 1 H NMR or LC-MS.

  : substrate (1 eq.), IrICy 3b (4.0 mol%), THF/D2O (1:1) (3 mL), D2 (1 bar), 3h, 80°C. b) Position and percentage of deuterium incorporation determined by 1 H NMR and LC-MS.

Scheme V. 9 :

 9 Scheme V. 9: Deuteration and tritiation of Volixibat precursor 43a,b,c

  , MeOD): δ 6.59 (m, 2H, CH ortho-NH2 and CH ortho-OH), 6.42 (dd, 3 J = 8.1 Hz and 1.7 Hz, 1H, CH ortho-methyl), 2.18 (s, 3H, CH3) ppm. Incorporation expected at δ 6.59 (red arrow). Determined against integral at δ 2.18 (blue arrow). General method A: 18.4 mg (150.0 µmol) 19; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 124.1 [M+H] + (18%), 125.1 [M(1D)+H] + (76%), 126.1 [M(2D)+H] + (6%).

1 H

 1 , MeOD): δ 7.25 (t, 3 J = 7.6 Hz, 1H, CH meta-NH2), 7.02 (d, 3 J = 7.6 Hz, 1H, CH ortho-NH2), 6.92 (s, 1H, CH ortho-NH2), 6.82 (dd, 3 J = 7.6 Hz and 2.3 Hz, 1H, CH ortho-CF3) ppm. Incorporation expected at δ 7.02 (red arrow). Determined against integral at δ 7.25 (blue arrow). General method A: 24.1 mg (150.0 µmol) 20; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 162.1 [M+H] + (1%), 163.1 [M(1D)+H] + (94%), 164.1 [M(2D)+H] + (5%). Yield: 22.2 mg, 137.7 µmol, 92%; 99% D for δ 7.02. General method B: 28.6 mg (150.0 µmol) 21; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80 °C, 3h. LC-MS (positive ESI): m/z 192.1 [M+H] + (1%), 193.1 [M(1D)+H] + (95%), 194.1 [M(2D)+H] + (4%). Yield: 22.3 mg, 116.8 µmol, 78%; 99% D for δ 6.35.

1 H 1 H 1 H

 111 , MeOD): δ 7.84 (m, 2H, CH(9) and CH(7)),7.48 (m, 2H, CH(8) and CH(6)),7.33 (m, 2H, CH(3) and CH(4)), 6.80 (m, 1H, CH(2)), 4.09 (br s, 2H, NH2) ppm. Incorporation expected at δ 7.84 and 6.80 (red arrow). Determined against integral at δ 7.48 (blue arrow). General method A: 21.4 mg (150.0 µmol) 22; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 144.1 [M+H] + (4%), 145.1 [M(1D)+H] + (30%), 146.1 [M(2D)+H] + (55%), 147.1 [M(3D)+H] + (9%), 148.1 [M(4D)+H] + (2%).Yield: 18.8 mg, 131.4 µmol, 88%; 56% D for δ 7.84 and 81% D for δ 6.80. , MeOD): δ 7.82 (m, 2H, CH(9) and CH(7)),7.46 (m, 2H, CH(8) and CH(6)),7.29 (m, 2H, CH(3) and CH(4)), 4.11 (br s, 2H, NH2), 2.40 (s, 3H, CH3) ppm. Incorporation expected at δ 7.82 (red arrow). Determined against integral at δ 7.29 (blue arrow). General method A: 23.5 mg (150.0 µmol) 23; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 158.2 [M+H] + (10%), 159.2 [M(1D)+H] + (85%), 160.2 [M(2D)+H] + (5%). Yield: 23.1 mg, 146.7 µmol, 98%; 91% D for δ 7.82.

2 1 H

 1 NMR (300 MHz, MeOD): δ 6.98 (m, 1H, CH(

1 H

 1 General method A: 22.0 mg (150.0 µmol) 24; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h.LC-MS (positive ESI): m/z 148.2 [M+H] + (2%), 149.2 [M(1D)+H] + (94%), 150.2 [M(2D)+H] + (4%). Yield: 20.5 mg, 139.1 µmol, 93%; 99% D for δ 6.56. , MeOD): δ 7.69 (m, 2H, CH(4) and CH phenyl), 7.61 (m, 1H, CH phenyl), 7.38 (m, 1H, CH phenyl), 7.22 (m, 1H, CH phenyl), 7.00 (s, 1H, CH(1)), 6.96 (m, 1H, CH(3)), 3.80 (br s, 2H, NH2) ppm. Incorporation expected at δ 7.00 and 6.96 (red arrow). Determined against integral at δ 7.38 (blue arrow). General method A: 21.4 mg (150.0 µmol) 25; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 144.1 [M+H] + (3%), 145.1 [M(1D)+H] + (38%), 146.1 [M(2D)+H] + (48%), 147.1 [M(3D)+H] + (9%), 148.1 [M(4D)+H] + (2%).Yield: 20.3 mg, 142.2 µmol, 81%; 58% D for δ 7.00 and 87% D for δ 6.96.

  General method A: 19.8 mg (150.0 µmol) 29; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 134.1 [M(1D)+H] + (24%), 135.1 [M(2D)+H] + (41%) 136.1 [M(3D)+H] + (29%), 137.1 [M(4)+H] + (6%). Yield: 17.6 mg, 133.5 µmol, 89%; 61% D for δ 7.14, 77% D for δ 6.96 and 75% D for δ 6, MeOD): δ 7.85(d, 3 J = 8.7 Hz, 2H, 6.64 (d, 3 J = 8.7 Hz, 2H, CH2 ortho-NH2), 4.34 (t, 3 J = 6.4 Hz, 2H, OCH2), 4.04 (br s, 2H, NH2), 2.83 (t, 3 J = 6.4 Hz, 2H, CH2-N), 2.63 (q, 3 J = 7.1 Hz, 4H, 2xCH2 Ethyl), 1.08 (t, 3 J = 7.1 Hz, 6H, 2xCH3 Ethyl) ppm. Incorporation expected at δ 6.64 (red arrow). Determined against integral at δ 1.08 (blue arrow). General method A: 35.4 mg (150.0 µmol) 30; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. LC-MS (positive ESI): m/z 237.2 [M+H] + (50%), 238.2 [M(1D)+H] + (33%), 239.1 [M(2D)+H] + (17%). Yield: 34.3 mg, 145.5 µmol, 97%; 25% D for δ 635.4 mg (150.0 µmol) 30; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80 °C, 3h. LC-MS (positive ESI): m/z 239.1 [M(2D)+H] + (61%), 240.3 [M(3D)+H] + (26%), 241.2 [M(4D)+H] + (13%). Yield: 32.5 mg, 138.0 µmol, 92%; 97% D for δ 6, MeOD): δ 7.80 (s, 1H, CH alkene), 7.34 (d, 3 J = 8.7 Hz, 1H, CH meta-NH2), 7.03 (d, 3 J = 2.3 Hz, 1H, CH ortho-NH2), 6.98 (dd, 3 J = 8.7 Hz and 2.3 Hz, 1H, CH ortho-NH2) ppm. Incorporation expected at δ 7.80, 7.03 and 6.98 (red arrow). Determined against integral at δ 7.34 (blue arrow). General method B: 20.0 mg (150.0 µmol) 31; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80 °C, 3h. LC-MS (positive ESI): m/z 134.1 [M+H] + (45%), 135.1 [M(1D)+H] + (37%), 136.1 [M(2D)+H] + (15%), 137.1 [M(3D)+H] + (3%). Yield: 17.8 mg, 133.8 µmol, 89%; 25% D for δ 7.80, 18% D for δ 7.03 and 28% D for δ 6.98. General method A: no deuterium incorporation. , MeOD): δ 8.83 (m, 1H, CH ortho-N), 8.55 (d, 3 J = 2.0 Hz, 1H, CH ortho-NH2), 8.25 (dd, 3 J = 6.5 Hz and 1.8 Hz, 1H, CH para-N), 7.46 (d, 3 J = 2.0 Hz, 1H, CH ortho-NH2), 7.38 (m, 1H, CH meta-NH2), 4.16 (br s, 2H, NH2) ppm. Incorporation expected at δ 8.83, 8.55 and 8.25 (red arrow). Determined against integral at δ 7.46 (blue arrow). General method B: 21.8 mg (150.0 µmol) 32, 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80 °C, 3h. LC-MS (positive ESI): m/z 146.1 [M+H] + (34%), 147.1 [M(1D)+H] + (39%), 148.1 [M(2D)+H] + (20%), 149.1 [M(3D)+H] + (7%). Yield: 17.5 mg, 120.2 µmol, 80%; 30% D for δ 8.83, 15% D for δ 8.55 and 10% D for δ 8.25. General method A: no deuterium incorporation. , MeOD): δ 7.18 (d, 3 J = 8.1 Hz, 2H, 2 CH ortho-Cl), 7.09 (m, 2H, CH meta-NH2 and para-NH2), 6.80 (m, 2H, CH ortho-NH2 and meta-NH2), 6.67 (d, 3 J = 8.1 Hz, 2H, 2 CH ortho-NH), 5.16 (br s, 1H, NH), 3.76 (br s, 2H, NH2) ppm. Incorporation expected at δ 6.80 (red arrow). Determined against integral at δ 5.16 (blue arrow). General method B: 32.8 mg (150.0 µmol) 33; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80 °C, 3h. LC-MS (positive ESI): no interpretation because of the natural isotopes of the Br. Yield: 25.6 mg, 117.4 µmol, 78%; 53% D for δ 6.80. , MeOD): δ 7.60 (d, 3 J = 8.7 Hz, 2H, 2 CH ortho-SO2NH2), 6.70 (d, 3 J = 8.7 Hz, 2H, 2 CH ortho-NH2) ppm. Incorporation expected at δ 6.70 (red arrow). Determined against integral at δ 7.60 (blue arrow). General method B: 25.8 mg (150.0 µmol) 34; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80 °C, 3h. LC-MS (positive ESI): m/z 174.1 [M(1D)+H] + (12%), 175.0 [M(2D)+H] + (82%), 176.1 [M(3D)+H] + (6%).

1 H

 1 General method B: 34.0 mg (150.0 µmol) 37; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h.LC-MS (positive ESI): m/z 230.2 [M+H] + (7%), 231.1 [M(1D)+H] + (57%), 232.1 [M(2D)+H] + (32%), 233.1 [M(3D)+H] + (4%). Yield: 28.6 mg, 124.7 µmol, 84%; 64% D for δ 6.26 and 23% D for δ 6, MeOD): δ 7.58 (d, 3 J = 3.1 Hz, 1H, CH ortho-COOH), 7.12 (dd, 3 J = 8.6 Hz and 3.1 Hz, 1H, CH ortho-NH2), 6.84 (d, 3 J = 8.6 Hz, 1H, CH meta-NH2) ppm. Incorporation expected at δ 7.12 (red arrow). Determined against integral at δ 6.84 (blue arrow). General method B: 23.0 mg (150.0 µmol) 38; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 154.1 [M+H] + (5%), 155.1 [M(1D)+H] + (91%), 156.1 [M(2D)+H] + (4%).Yield: 17.7 mg, 116.0 µmol, 77%; 90% D for δ 7.12. , MeOD): δ 6.76 (m, 2H, 2 CH ortho-COOMe), 6.33 (m, 1H, CH ortho-NH2), 3.84 (s, 3H, CH3) ppm. Incorporation expected at δ 6.33 (red arrow). Determined against integral at δ 3.84 (blue arrow).General method A: 24.9 mg (150.0 µmol) 39; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55°C, 3h.LC-MS (positive ESI): m/z 167.1 [M+H] + (58%), 168.1 [M(1D)+H] + (32%), 169.1 [M(2D)+H] + (10%). Yield: 21.4 mg, 129.0 µmol, 86%; 32% D for δ 6.33. 24.9 mg (150.0 µmol) 39; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 168.1 [M(1D)+H] + (73%), 169.1 [M(2D)+H] + (27%).

1 H 1 H 1 H 1 H 1 H

 11111 , MeOD): δ 6.97(d, 3 J = 8.2 Hz, 2H, 2 CH meta-NH2), 6.64(d, 3 J = 8.2 Hz, 2H, 2 CH ortho-NH2), 4.93 (br s, 1H, OH or NH), 4.50 (br s, 1H, CH), 3.66 (br s, 3H, NH2 and OH or NH), 3.04 (m, 2H, CH2),1.43 (s, 9H, Boc) ppm. Incorporation expected at δ 6.64 (red arrow). Determined against integral at δ 1.43 (blue arrow).General method B:42.1 mg (150.0 µmol) 40; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 182.1 [M(1D)-Boc+H] + (5%), 183.1 [M(2D)-Boc+H] + (90%), 184.1 [M(3D)-Boc+H] + (5%). Yield: 35.4 mg, 125.9 µmol, 84%; 99% D for δ 6.64. , MeOD): δ 7.64 (d, 3 J = 8.8 Hz, 2H, 2 CH meta-NH2), 6.69 (d, 3 J = 8.8 Hz, 2H, 2 CH ortho-NH2) ppm. Incorporation expected at δ 6.69 (red arrow). Determined against integral at δ 7.64 (blue arrow). General method B: 32.0 mg (150.0 µmol) 41; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 216.1 [M+H] + (40%), 217.1 [M(1D)+H] + (55%), 218.0 [M(2D)+H] + (5%). Yield: 25.6 mg, 119.0 µmol, 80%; 55% D for δ 6.69. General method A: no deuterium incorporation. General method B: 22.3 mg (150.0 µmol) 42; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 150.1 [M+H] + (25%), 151.1 [M(1D)+H] + (46%), 152.0 [M(2D)+H] + (29%). Yield: 18.3 mg, 122.7 µmol, 82%; 15% D for δ 7.30 and 85% D for δ 7.10. , MeOD): δ 7.64 (d, 3 J = 8.2 Hz, 2H, 2 CH meta-NH2), 6.67 (d, 3 J = 8.2 Hz, 2H, 2 CH ortho-NH2), 6.05 (s, 1H, CH ortho-OMe), 3.87 (s, 6H, 2 OCH3) ppm. Incorporation expected at δ 6.67 (red arrow). Determined against integral at δ 3.87 (blue arrow). General method B: 46.5 mg (150.0 µmol) 5; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 312.1 [M(1D)+H] + (11%), 313.0 [M(2D)+H] + (86%), 314.1 [M(3D)+H] + (3%). Yield: 44.2 mg, 142.5 µmol, 95%; 79% D for δ 6, MeOD): δ 6.95 (d, 3 J = 8.0 Hz, 2H, 2 CH meta-NH2), 6.70 (d, 3 J = 8.0 Hz, 2H, 2 CH ortho-NH2), 4.69 (m, 1H, CH), 3.90 (m, 1H, CH), 3.77 (m, 3H, 3 CH), 3.59 (m, 2H, 2 CH) ppm. Incorporation expected at δ 6.70 (red arrow). Determined against integral at δ 6.95 (blue arrow). General method B: 40.6mg (150.0 µmol) 7; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 274.1 [M(2D)+H] + (95%), 275.1 [M(3D)+H] + (5%). Yield: 36,4 mg, 134.3 µmol, 91%; 20% D for δ 6.95 and 96% D for δ 6.70. , MeOD): δ 7.05 (d, 3 J = 8.6 Hz, 2H, 2 CH meta-NH2), 6.72 (d, 3 J = 8.6 Hz, 2H, 2 CH ortho-NH2), 2.05 (m, 1H, CH aliphatic), 2.34 (m, 2H, CH2 aliphatic), 2.19 (m, 1H, CH aliphatic), 1.90 (m, 2H, CH2 aliphatic), 0.85 (m, 3H, CH3 aliphatic) ppm. Incorporation expected at δ 7.10 (red arrow). Determined against integral at δ 0.85 (blue arrow). General method B: 34.8 mg (150.0 µmol) 8; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 234.2 [M(1D)+H] + (5%), 235.2[M(2D)+H] + (90%), 236.2[M(3D)+H] + (5%). Yield: 28.5 mg, 12 3.0 µmol, 82%; 99% D for δ 6.72. , MeOD): δ 7.62 (d, 3 J = 8.5 Hz, 2H, 2 CH meta-NH2), 6.66 (d, 3 J = 8.5 Hz, 2H, 2 CH ortho-NH2), 2.08 (s, 3H, CH3), 1.99 (s, 3H, CH3) ppm. Incorporation expected at δ 6.66 (red arrow). Determined against integral at δ 2.08 (blue arrow). General method B: 39.9 mg (150.0 µmol) 10; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF/D2O (1:1) (3 mL), 80°C, 3h. LC-MS (positive ESI): m/z 267.3 [M+H] + (48%), 268.3 [M(1D)+H] + (47%), 269.3 [M(2D)+H] + (5%).Yield: 30.3 mg, 114.0 µmol, 76%; 50% D for δ 6.66.

Figure 2 :

 2 Figure 2 : XRD diffractograms for Ir@ICy NPs (0.125 eq) 3b, showing the peaks corresponding to fcc-Ir(0).

Figure 3 :Figure 4 :

 34 Figure 3 : X-ray photoelectron spectroscopy (XPS) of the N 1s signals of Ir@ICy (0.125 eq) 3b.

Figure 5 :Figure 6 : 2 H

 562 Figure 5 : ATR FT-IR spectra of Ir@ICy 3b before and after CO adsorption.

2 ). 1 Scheme VI. 2 :

 212 Scheme VI. 2: Hydrogen Isotope Exchange reactions of Sulphonamides, Phoshonamides and N-oxides with iridium P,N-ligated catalyst F (Part II -Chapter 1)

2

 2

Scheme VI. 3 :

 3 Scheme VI. 3: Hydrogen Isotope Exchange reactions of Phenylacetic acid derivatives withiridium P,N-ligated catalyst F (Part II -Chapter 2)

3 Scheme VI. 4 :

 34 Scheme VI. 4: Temperature-mediated Hydrogen Isotope Exchange reactions with different iridium catalysts (Part III -Chapter 1)

6 Scheme VI. 7 :

 67 Scheme VI. 7: Hydrogen Isotope Exchange reactions of anilines 17-21 with NHC-ligated iridium nanoparticles (Part V)

Figure 3 :

 3 Figure 3: The deuterium manifold

Figure 4 :

 4 Figure 4: The tritium manifold

  Fonctionnalisation de liaisons C-H par catalyse à l'iridium: Développement de nouvelles méthodes pour l'échange d'isotopes d'hydrogène sur des petites molécules et des composés biothérapeutiques Mots clés : fonctionnalisation-CH, échange d'isotopes d'hydrogène, catalyse, Drug Discovery Résumé : Le développement de nouvelles méthodes, efficaces, rapides et facile à mettre en oeuvre pour le marquage avec du deutérium ou du tritium de composés organiques tels que des médicaments est indispensable dans le secteur industriel comme académique. Ces composés isotopiquement marqués sont essentiels lors des études précliniques. En effet, ces méthodes de marquages permettent d'obtenir plus rapidement les données nécessaires concernant la métabolisation et le profil de sécurité d'un candidat médicament, ce qui permet de réduire le temps investit dans cette recherche. Les méthodes classiques de marquages sont basées sur la synthèse d'un précurseur qui est ensuite marqué isotopiquement puis modifié jusqu'à l'obtention de la structure finale du candidat médicament. Ces anciennes méthodes, coûteuses en temps et en argent et génératrices dstructure finale du composé d'intérêt, en particulier par échange d'isotope d'hydrogène. Au cours de ces travaux de thèse, nous avons développé la première méthode de marquage catalytique à l'iridium dans des conditions douces (température ambiante) sur les motifs dérivés d'acides phenylacetiques tels que esters et amides, motifs très présents dans les structures pharmacologiques. Cette méthode permet d'introduire du deutérium ou du tritium en position ortho du groupement directeur de façon sélective. Lors d'un autre projet, nous avons mis en évidence que la position de marquage au sein d'une molécule avec plusieurs groupements directeurs peut être modulée en fonction du catalyseur d'iridium utilisé ainsi que de la température réactionnelle (de -60°C à +130°C). L'utilisation de calculs de DFT nous a permis de postuler un ordre classifiant différents groupements directeurs par rapport à leur réactivité vis-à-vis de la réaction d'échange d'isotopes d'hydrogène en situation de compétition (deux substrats et un catalyseur d'iridium). Ces données théoriques ont été confirmées par les données expérimentales. Lors d'un autre projet, le marquage sélectif au tritium d'une structure complexe tel qu'un médicament cytotoxique (maytansine DM4) a débouché sur le marquage de différentes chaînes latérales employées dans des méthodes de conjugaison d'anticorps. Ces données ont permis d'étendre cette méthodologie au marquage au deutérium d'acides aminés et de di-tri et tétra-peptide, sélectivement sur le carbone alpha d'un résidu glycine. Finalement, en collaboration avec le CEASaclay et le CNRS, nous avons développé les premières nanoparticules d'iridium stable à l'air et actives vis-à-vis de l'échange d'isotopes d'hydrogène sur des anilines. L'ensemble des travaux réalisés pendant cette thèse a permis le marquage au tritium de composés médicamenteux tels que le DM4, le Benalaxyl, ou encore le pharmacophore du Volixibat. Title : Iridium-Catalyzed CH-Functionalization: Development and Applications of Innovative Strategies for Hydrogen Isotope Exchange on Small Molecules and Biotherapeutic Drugs for Drug Discovery Keywords : CH-functionalization, hydrogen isotope exchange, catalysis, Drug Discovery

ketones 74, esters 75, amides 76 or 77, acetanilide 78, nitro 79 and heterocycles 80-83 (

	catalyzed by chloro-Kerr catalyst C, 87 and only one example of secondary sulfonamide 85
	catalyzed by Pfaltz catalyst D. 88
	Scheme I.22: Scope of directing groups described in C(sp 2 )-H Iridium catalysed
				ortho-directed HIE
	O				O	O
						O		NH 2
	O	NEt 2		N H		O N +	O
						O
	N					N	N
	N					O	S
	N N H	N N	R	O	S NH 2 O	O	H S N O
	Cl

Kerr catalyst Ba and providing a broad range of functionalities employable for the exchange, like scheme I.22).

86

More challenging, there were some examples of primary sulfonamides 84

[93] [91] [93] [92] [94] [98] [97] [52] [98] [90] [90] [95] [8] 74 75 76 77 78 79 80 81 82 83 84 85 Some applications on carbon-sp 3 centers are also described even with tritium labelling on more complex structures like 86, 87 and 88 (scheme I.23). 33

	N D [95]	D	N	N	T [38]	T [41] O	T N	[21] O	OH	F	T [60]	T N H	O H H T N	[40]	N	S	O O
	Mirtazapine 86		87						88
				Scheme I.23: C(sp 3 )-H Iridium catalysed HIE on complex structures
								30

Part II Homogeneous iridium catalysis on C(sp 2 )-H centers Chapter 1: Performance and analysis of iridium(I)-catalyzed hydrogen isotope exchange reactions of aryl-and heteroaryl compounds

	In 2014 catalyst F was synthesized by the working group of Prof. Tamm for the first time and
	in collaboration with Sanofi tested in HIE reactions. Based on this initial work which showed
	HIE reactivity for N-Boc indoles (1), N-Boc anilines (2) and anisoles (3, scheme II.1.1), 1 we
	became interested in the broader scope and limitations of this catalyst.
	Scheme II.1. 1: Previous work with catalyst F in the HIE reactions of N-Boc anilines and
		anisoles 1
		R'
		N
		N R'	N	Ir	PR 2
	H				D
	DG				DG
	H	D (1 bar)		D
	NBoc	O H	NHBoc	O
					a ITN European
	Scholarship network, parts of this work should be done in collaboration with other groups
	and universities. It was planned to collaborate especially with the group of Prof Bruno
	Chaudret (INSA, Toulouse, France) and Dr. Gregory Pieters (CEA Saclay, Paris, France) on
	nanoparticle development. Furthermore, a collaboration with the University of
	Braunschweig and Prof. Matthias Tamm was envisioned to elaborate homogeneous iridium
	catalysis.

2

[

92] [93] [96] [96] [98] [10]

		+
	BArF 24	-
	F (5 mol%)
	DCM, RT, 1 h
	Selected examples:

1 2 3 II.1.1. Sulphonamides, N-oxides and phosphonamides -unexplored directing groups in HIE reactions

	Sildenafil (Viagra) 6, 5 sulphonyl ureas for treatment of diabetes mellitus like Glibenclamide
	7 6 (scheme II.1.2). 7
	Scheme II.1. 2: Secondary and tertiary sulphonamide (urea) drugs 7
	N H 2
	Cl
		N H
	O	O	S	N H	N H
			O O	O
	As mentioned in the introduction, Kerr's catalysts B and C are widely applied for mild and
	selective ortho-labelling employing a broad range of directing groups. 2 Despite recent
	progresses, a number of interesting functionalities still present significant challenges in HIE
	protocols, such as secondary and tertiary sulphonamides (also sulphonyl ureas), which are
	important structural moieties in several groups of drugs called sulpha drugs. These groups
	include sulphonamide antibiotics like sulfamethoxazole 4, 3 protease inhibitors for HIV
	treatment like Darunavir 5, 4 PDE5 inhibitors for treatment of erectile dysfunction like

[H]- 14 in different solvents at room temperature. Sulphonamide [H]-14 was chosen as

	model
	compound due to its tertiary sulphonamide directing group (DG),. Moreover, it is a currently
	available product, iprompt to ionization and UV-detectable to be easily seen in LC-MS.
	Results are summarized in table II.1).
	The catalytic HIE reaction with our model substrate [H]-14 was strongly influenced by the
	solvent. In most solvents, none or only poor deuterium exchange was observed; however,
	high deuterium incorporation was found in chlorobenzene (entry 9). The strong influence of
	chlorobenzene in HIE reactions has been observed earlier, 8,7 however the effect is still not
	understood.

Table II .1. 1: Solvent screening of catalyst F in the HIE reaction with tertiary sulphonamide [H]-14. a

 II

	H	O			D	O
		S O H	N	N	D 2 , rt, 2 h catalyst F	S D O	N	N
		[H]

-14 14 Entry Solvent %D 14 b 1 dichloromethane 22 2 chloroform 12 3 MTBE 11 4 ethanol 0 5 cyclohexane 15 6 MeTHF 0 7 isopropylacetate 0 8 1-butanol 0 9 chlorobenzene 82 10 fluorobenzene 16 a Conditions: substrate [H]-14 (10 µmol

	, 2.5 mg), catalyst F (1.0 µmol, 1.5 mg, 10 mol%), solvent (2
	mL), D2 (1 atm), rt, 2 h .

b

Percentage of deuterium incorporation determined by LC-MS.

Table II .1. 2: Optimization

 II of catalyst content and reaction time in the HIE reaction of tertiary sulphonamide [

H]-14. a Entry Catalyst F (mol%) Time (min) %D 14 b

	1	3	300	0
	2	5	300	0
	3	7,5	120	32
	4	10	5	15
	5	10	10	44
	6	10	15	69
	7	10	30	80
	8	10	60	82
	9	10	120	85
	10	10	300	89
	a Conditions: substrate [H]-14 (2.5 mg, 10 µmol), catalyst F (0.5-1.5 mg, 0.3-1 μmol, 3-10 mol%, 0.03-
	0.1 eq.), chlorobenzene (2 mL), D2 (1 atm), rt. b Percentage of deuterium incorporation determined
	by LC-MS.

General deuteration procedure: see Annex 1. Solvent screening of catalyst F in the HIE reaction with tertiary sulfonamide [H]-14 (table II.1.1)

	For the screening of different solvents, 1-methyl-4-[(4-methylphenyl)sulfonyl]-piperazine
	[H]-14 in solvent (10 μmol, 500 μL, 1 eq.), the catalyst F (1.5 mg, 1 μmol, 10 mol%, 0.1 eq.)
	and the different solvents were used. All reactions were performed according to the general
	deuteration procedure at rt for 2 h. Deuterium incorporation was determined by LC-MS and
	labelling position was by 1 H-NMR.

Optimization of catalyst content and reaction time in the HIE reaction of tertiary sulfonamide [H]-14 (table II.1.2)

	1-methyl-4-[(4-methylphenyl)sulfonyl]-piperazine [H]-14 in chlorobenzene (2.5 mg, 10 μmol,
	500 μL, 1 eq.) and catalyst F indicated in table 2 (0.5-1.5 mg, 0.3-1 μmol, 3-10 mol%, 0.03-0.1
	eq.) were used for the optimization of the HIE reaction. The reaction was stirred for the
	declared time and at the stated temperature under D2 atmosphere.

12, 15-17 Ref 6 Up to 97%D Ref 1 Up to 69%D Ref 1 Up to 7%D Not describedTable II . 2

 II2

		D
	O	Ir-cat (10 mol%)		O
	O	D , PhCl, rt, 2 h	D	O
	[H]-12		12

.2.1)

. The results clearly demonstrated the high activity of catalyst F (entry 8) with incorporation of 1.8 deuterium atoms into the molecule. Especially compared to the other tested catalysts A-E, 18-20 (entry 1-7) which could incorporate 0.13 deuterium atoms at the maximum under the described reaction conditions. 1

HIE substrates [H]-. 1: Comparison of different commercially available iridium(I) catalysts and catalyst F in HIE reactions of methyl phenylacetate [H]-12

2 a Literature data; b Conditions: substrate [H]-12 (1.5 mg, 10 µmol, 1 eq.), D2 (1 atm), chlorobenzene (1 mL), rt, 2h. c determined by LC-MS II.

2.2.2. Time screening

	Entry	Catalyst	Loading (mol%) D/molecule (12) c
	1 a	[Ir COD(PPh3)2]BF4 (18)	100	<0.02 a
	2 a	[Ir COD(dppe)]BF4 (19)	5	0.13 a

A reaction advancement (deuterium incorporation) was studied versus time on model compound [H]-12 with catalyst F in chlorobenzene at room temperature. Samples of the ongoing reaction were taken off at different time points, from 15 min to 24 h, and analysed by LC-MS. A maximum plateau corresponding to 1.8 deuterium/molecule was reached after 105 min (figure II.2.2). Consequently, an optimal reaction time of two hours was kept for the entire project.

Table II . 2

 II2

	Entry	Solvent	Temp.	D/molecule
	1	chloroform	RT	0.9
	2	MTBE	RT	0.77
	3	ethanol	RT	0.65
	4	isopropylacetate	RT	0.9
	5	dichloromethane	RT	1.06
	6	chlorobenzene	RT	1.8
	7	cyclohexane	RT	1.05
	8	methylcyclohexane	RT	0.9
	9	1-butanol	RT	0.72
		toluene	RT	1.02
		dioxane	RT	0.4
		chloroform	55°C	1.06
		MTBE	50°C	0.68
		ethanol	70°C	0.41
		isopropylacetate	85°C	0.93
		dichloromethane	35°C	1.03
		chlorobenzene	110°C	1.71
		cyclohexane	75°C	0.99
		methylcyclohexane	95°C	0.99
		1-butanol	110C	0.83
		toluene	105°C	0.94
		dioxane	95°C	0.52

. 2: Solvent and temperature screening of catalyst F in the HIE reaction of methylphenylacetate ester [H]-12. a,b a Conditions: substrate [H]-12 (1.5 mg, 10 µmol, 1 eq.), catalyst F (1.5 mg, 1.0 μmol, 10 mol%), solvent (1 mL), D2 (1atm), 2 h. b Position determined by 1 H NMR and percentage of deuterium incorporation analysed by LC-MS.

Table II .

 II

	Entry	Substrate	P1 D2 (mbar)	P2 D2 (mbar)	Eq D2	D/molecule
	1	Camylofine 5	1013	1013	n.d.	1.5
	2	Camylofine 5	248	166	10	1.1
	3	Camylofine 5	422	270	20	1.2
	4	Camylofine 5	642	407	30	1.3
	5	Benalaxyl 10	1013	1013	n.d.	1.9
	6	Benalaxyl 10	125	107	2.5	1.2
	7	Benalaxyl 10	155	119	5	1.7
	8	Benalaxyl 10	180	107	10	1.7

2. 4:

Optimization of the deuterium pressure on the deuterium manifold a Conditions: substrate (1 eq.), catalyst F (10 mol%), chlorobenzene (1 mL), rt, 2 h. b Percentage of deuterium incorporation determined by LC-MS.

  Incorporation expected at δ 7.05 and 7.01 (red arrow). Determined against integral at δ 3.72 (blue arrow).

	F Cl	O O O O
	Cl Br	O O O O	Molecular Weight =184.6239 Molecular Formula =C9H9ClO2 Molecular Weight =168.1693 Molecular Weight =184.6239 Molecular Weight =229.0749 Molecular Formula =C9H9BrO2 Molecular Formula =C9H9FO2 Molecular Formula =C9H9ClO2
	28b 28c 29a 29b
	1 H NMR (300 MHz, CDCl3): δ 7.31 (d, 3 J=8.3 Hz, 2H, CH2 meta-phenyl), 7.22 (d, 3 J=8.3 Hz, 2H, CH2 ortho-phenyl), 3,71 (s, 3H, CH3), 3.61 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.22 and δ 7.31 (red arrow). Determined against integral at δ 3.71 (blue arrow). 1 H NMR (300 MHz, CDCl3): δ 7.37 (d, 3 J=8.0 Hz, 2H, CH2 meta-phenyl), 7.08 (d, 3 J=8.0 Hz, 2H, CH2 ortho-phenyl), 3.62 (s, 3H, OCH3), 3.50 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ 7.08 (red arrow). Determined against integral at δ 3.62 (blue arrow). 1 H NMR (300 MHz, CDCl3): δ 7.29 (s, 1H, CH ortho), 7.26 (m, 2H, CH meta and para), 7.17 (m, 1 H NMR (300 MHz, CDCl3): δ 7.29 (m, 1H, arom. CH), 7.05 (m, 1H, arom. CH), 7.01 (m, 1H, 1H, CH ortho), 3.71 (s, 3H, CH3), 3.61 (s, 2H, CH2 benzylic) ppm. Incorporation expected at δ arom. CH), 6.98 (m, 1H, arom. CH), 3.72 (s, 3H, CH3), 3.63 (s, 2H, CH2 benzylic) ppm. 7.29 and 7.17
	General method: 11.1 mg (60.0 µmol) 28b; 9.0 mg (6.0 µmol) catalyst F, 2h. General method: 13.70 mg (60.0 µmol) 28c; 9.20 mg (6.0 µmol) catalyst F, 2h.
	General method: 10.2 mg (61.0 µmol) 29a; 9.3 mg (6.1 µmol) catalyst F, 2h.
			Average: y=88%
			Average: y=90% 85% D δ 7.08 77% D for δ 7.22 16% D for δ 7.31 Average: y=75% 15% D δ 7.37 86% D δ 7.01
			53% D δ 7.05

Yield: a) 9.7 mg, 53.0 µmol, 87%; 77% D for δ 7.22 and 16% for δ 7.31. b) 10.2 mg, 55.0 µmol, 92%; 77% D for δ 7.22 and 16% for δ 7.31. 1 H-NMR Reference 28b 1 H-NMR HIE reaction 28b Yield: a) 12.5 mg, 55.0 µmol, 91%; 14% D for δ 7.37 and 84% D for δ 7.08. c) 11.7 mg, 51.0 µmol, 85%; 15% D for δ 7.37 and 86% D for δ 7.08. 1 H-NMR Reference 28c 1 H-NMR HIE reaction 28c Yield: a) 8.0 mg, 48.0 µmol, 78%; 88% D for δ 7.01 and 53% D for δ 7.05. d) 7.3 mg, 43.0 µmol, 72%; 83% D for δ 7.01 and 53% D for δ 7.05. 1 H-NMR Reference 29a 1 H-NMR HIE reaction 29a

1 H NMR (500 MHz, CD2Cl2):

δ 7.71 (m, 16H, ortho CH BAr F 24)

,

7.63 (m, 2H, CH aromatic), 7.55 (m, 8H, para CH BAr F 24)

, 7.23 (m, 2H, CH aromatic), 6.88 (m, 2H, CH aromatic), 6.07 (m, 2H, CH aromatic), 3.28 (s, 12H, N-CH3), 2.18 (s, 12H, C-CH3), 1.28 (d 3 JHP = 14.9 Hz, 36H, tertbutyl), -28.14 (m, 4H, Ir-H).

Table 5 .

 5 Crystallographic data

	Compound	61
	CCDC	1886537
	Empirical formula	C107H96B2Cl2F48Ir2N6P2
	Formula weight	2916.75
	Temperature	100(2) K
	Wavelength	0.71073 Å
	Instrument (scan mode)	XtaLAB Synergy S, HyPix (ω scan)
	Crystal system	Triclinic
	Space group	P1
	Unit cell dimensions	a = 12.0377(2) Å	ɑ= 100.0440(10)°
		b = 14.8035(2) Å.	β= 101.6020(10)°
		c = 16.8587(3) Å.	γ = 96.9800(10)°
	Volume	2859.29(8) Å 3
	Z	1
	Density (calculated)	1.694 Mg/m 3
	Absorption coefficient	2.528 mm -1
	F(000)	1440
	Crystal habitus	plate (violet)
	Crystal size	0.311 x 0.177 x 0.147 mm 3
	θ range for data collection	2.475 to 38.602°
	Index ranges	-21 ≤ h ≤ 20, -25 ≤ k ≤ 25, -29 ≤ l ≤ 28
	Reflections collected	327737
	Independent reflections	30311 [R(int) = 0.0652]
	Completeness to θ = 25.242°	99.9 %
	Absorption correction	Gaussian
	Max. and min. transmission	1.000 and 0.317
	Refinement method	Full-matrix least-squares on F 2
	Data / restraints / parameters 30311 / 52 / 802
	Goodness-of-fit on F 2	1.092
	Final R indices [I>2σ(I)]	R1 = 0.0476, wR2 = 0.1329
	R indices (all data)	R1 = 0.0608, wR2 = 0.1383
	Largest diff. peak and hole	5.580 and -2.623 e•Å -3

12

Scheme 2: Forward reaction (deuteration).

Table 6 .

 6 Energies for all optimized structures

Compound E0K a / [Ha] E298K b / [Ha] H298K b / [Ha] G298K b / [Ha]

  standard conditions T = 298.15 K and p = 1 atm.

				IN1	-1890.517163	-1890.477118	-1890.476173	-1890.585022
	HIE of phenyl	acetone		starting point (ed.) TS1 IN1	-1854.572270 -1854.554900 -1854.560326	-1854.531451 -1854.513893 -1854.519657	-1854.530507 -1854.512948 -1854.518712	-1854.641918 -1854.624576 -1854.628742
				starting point	-	-	-	-
				(ed.)	1929.776523	1929.735103	1929.734159	1929.846725
	catalytic H/D exchange of	(2) methyl phenylacetate		TS1 IN1 TS2 IN2 TS3	-1929.762204 -1929.767073 -1929.766763 -1929.767039 -1929.761817	-1929.721008 -1929.725775 -1929.725852 -1929.725754 -1929.720726	-1929.720064 -1929.724831 -1929.724908 -1929.724809 -1929.719781	-1929.831777 -1929.836191 -1929.835384 -1929.836144 -1929.831344
			terminal point	-	-	-	-
				(prod.)	1929.777271	1929.735940	1929.734996	1929.847414
	Kinetic Isotope	(2) (KIE) of Effect		starting point (ed.) TS1 IN1	-1929.777961 -1929.762458 -1929.767656	-1929.736691 -1929.721415 -1929.726419	-1929.735747 -1929.720471 -1929.725475	-1929.848108 -1929.831985 -1929.836756
				starting point	-	-	-	-
	HIE of N-	phenyl	acetanilide	(ed.) TS1 IN1	1870.642419 -1870.636885 -1870.643128	1870.602203 -1870.596656 -1870.603023	1870.601258 -1870.595712 -1870.602079	1870.711315 -1870.704967 -1870.710680
	HIE of	phenyl	acetate	starting point (ed.) TS1	-1890.522761 -1890.511071	-1890.481916 -1890.470753	-1890.480972 -1890.469809	-1890.592887 -1890.580556

a DFT energy incl. ZPE. b

.2.2. Comparison study at high temperatures

  Kerr et al. reported the different free energy profiles for HIE reactions of aromatic compounds in the gas phase with catalyst Ba. A ΔH activation energy in the transition state (9) of 18.13 kcal/mol (298 K) was calculated for acetophenone whereas a value of 23.04 kcal/mol(10) was reported for acetanilide (scheme III.1.3). 2c This difference of 4.91 kcal/mol makes acetanilide DG more energetically demanding to proceed the HIE reaction, which is in line with the observed selectivities of all used catalysts in table III.1.1 and III.1.2. Indeed, the deuterium incorporation in ortho-position of the ketone is higher or at least equal to the one related to the acetanilide. This might suggest that all catalysts A-G could either go through a five-or six-membered-ring transition-state as both positions were exchanged by deuterium in the products.Next, the HIE reaction of the model compound 8 has been studied at elevated temperatures up to 130°C (table III.1.2). Therefore, the solvent had to be changed to chlorobenzene (bp:

	ketone coordination	anilide coordination
	5-membered transition state	6-membered transition state
				N H
				O H
	H	Ir	O	Ir
		9		10
	H =18.13 kcal/mol	H =23.04 kcal/mol
	III.1

Scheme III.1. 3: Simplified transition states in the HIE reaction of acetophenone and

acetanilide with a iridium catalyst. DFT calculation values of catalyst Ba. 2c 131°C) due to the low boiling point of dichloromethane (bp: 40°C). Interestingly, catalysts A, Ba, E, G showed their highest deuterium incorporation at 75-100°C with nearly full deuteration of the aromatic positions

(entry 3, 8, 19, 24)

.

Table III .1. 3:

 III

Evaluations of iridium(I) catalysts A-G in a HIE reaction of 4-acetamidoacetophenone 8 at temperatures from 25 to 130°C in chlorobenzene

  .2.1, have enabled us to determine experimentally the general relative directing group strength in the following order: imidazole 2 (heterocycle 14 ) > acetylanilide 3 ~ ketone 4 > nitro 5 > tertiary amide 6 > aldehyde 7 > ester 8 > carbamate 9 > primary sulfonamide 10 (scheme III.2.3). In an earlier contribution, Derdau and Atzrodt et al. have studied the relative directing group strength of different heterocyclic directing groups such as imidazole 2 in HIE reactions using similar competition experiments.14 These results add to the proposed reactivity order in the HIE reactions.

Table III .2. 2:

 III Calculated values of different DGs to form coordination complex 11a(sorted by Grel) and the activation energy H to generate 11c via transition state 11b

		DG	Grel complex 11a	H (11a=>11b)
		-N-imidazole 2	-27.5	23.9
		-N-CO-Me 3	-23.7	27.4
		-CO-Me 4	-23.0	22.7
		-CO-NEt2 6	-23.0	27.6
	Ar	-COOMe 8	-20.9	24.4
		-CO-H 7	-20.3	20.6
		-NO2 5	-16.6	18.5
		-OCOMe 9	-14.3	23.0
		-SO2NH2 10	-14.0	23.8

  Yield: 1) 8.7 mg, 36 µmol, 87%; 0% D for δ 8.33, 40% D for δ 8.09.2) 8.2 mg, 34 µmol, 82%; 4% D for δ 8.33, 55% D for δ 8.09.

	4-Acetyl-4'-nitrobiphenyl 15 Methyl 4-acetoxy-4'-biphenylcarboxylate 16
	a Method B (-15°C): 10 mg, 41 µmol 15, 2.1 mg, 2.1 µmol catalyst Ba. b
	O LC-MS (positive ESI): m/z 242.2 [M+H] + (52%), 243.2 [M(1D)+H] + (40%), 244.1 [M(2D)+H] + NO 2 Molecular Weight =241,25 Molecular Formula =C14H11NO3 O (8%). O O Molecular Weight =270.2876 O Molecular Formula =C16H14O4
	15	16
	1 H NMR (300 MHz, DMSO-d6): δ 8.33 (d, 3 J= 9.0 Hz, 2H, Hb), 8.09 (d, 3 J= 8.5 Hz, 2H, Ha), 8.04 Average: y=85%, (d, 3 J= 9.0 Hz, 2H), 7.94 (d, 3 J= 8.5 Hz, 2H), 2.63 (s, 3H) ppm. Incorporation expected at δ 8.33 and/or δ 8.09 ppm (red arrow). Determined against reference-integral at δ 2.63 ppm (blue 47% D at position a,
	arrow).	2% D at position b.
	Method B: 10 mg, 41 µmol 15, 2.1 mg, 2.1 µmol catalyst Ba.
		Average: y=91%,
		98% D at position a,
		96% D at position b.

LC-MS (positive ESI): m/z 244.2 [M(2D)+H] + (4%), 245.2 [M(3D)+H] + (13%), 246.1 [M(4D)+H] + (77%), 247.1 [M(5D)+H] + (6%).

Yield: 1) 9.3 mg, 38 µmol, 93%; 96% D for δ 8.33, 98% D for δ 8.09.

2) 8.9 mg, 37 µmol, 89%; 96% D for δ 8.33, 98% D for δ 8.09.

  Incorporation expected at δ 7.64 and/or δ 7.46 and/or δ 7.16 ppm (red arrow). Determined against reference-integral at δ 1.33 ppm.

	Methyl-4-acetamido-2-ethoxybenzoate 22
	H N	a	b	O	Molecular Weight =237,26
	O	c	O	O	Molecular Formula =C12H15NO4
		22
	1 H NMR (300 MHz, DMSO-d6): δ 10.16 (s, 1H), 7.64 (d, 3 J= 8.5 Hz, 1H, Hb), 7.46 (s, 1H, Hc), 1 H NMR (300 MHz, DMSO-d6): δ 9.56 (s, 1H), 7.81 (s, 1H, Ha), 7.73 (s, 1H, Hb), 3.78 (s, 3H), 7.16 (d, 3 J= 8.5 Hz, 1H, Ha), 4.02 (q, 3 J= 7 Hz, 2H), 3.73 (s, 3H), 2.06 (s, 3H), 1.33 (t, 3 J= 7 Hz,
	3.76 (s, 3H), 2.16 (s, 3H) ppm. Incorporation expected at δ 7.81 and/or δ 7.73 ppm (red arrow). Determined against reference-integral at δ 2.16 ppm (blue arrow). 3H) ppm. Method B: 10 mg, 42 µmol 22, 2.1 mg, 2.1 µmol catalyst Ba.
					Average: y=92%,
					99% D at position b.

Method B: 10 mg, 39 µmol 21, 2.0 mg, 1.9 µmol catalyst Ba.

LC-MS (positive ESI): no interpretation because of the natural isotopes of the Cl.

Yield: 1) 9.1 mg, 35 µmol, 91%; 99% D for δ 7.73.

2) 9.2 mg, 36 µmol, 92%; 99% D for δ 7.73. 1 H-NMR Reference 21 LC-MS (positive ESI): m/z 239.2 [M(1D)+H] + (10%), 240.1 [M(2D)+H] + (80%), 241.1 [M(3D)+H] + (10%). Yield: 1) 9.5 mg, 40 µmol, 95%; 75% D for δ 7.64, 6% D for δ 7.64, 89% D for δ 7.16. 2) 8.5 mg, 36 µmol, 85%; 81% D for δ 7.64, 9% D for δ 7.64, 90% D for δ 7.16.

  Incorporation expected at δ 8.02 and δ 7.53 and/or 7.73 ppm (red arrow). Determined against reference-integral at δ 3.19 ppm (blue arrow).

	F	F
	F	N	N	23 a b	S O O
					NH 2	Molecular Weight =381,38 Molecular Formula =C17H14F3N3O2S
				24
			b
	1 H NMR (300 MHz, DMSO-d6: δ 10.13 (s, 1H), 8.02 (dd, 3 J= 9.0 Hz, 4 J= 2.6 Hz, 1H, Hb), 7.73 (d, 1 H NMR (300 MHz, DMSO-d6: δ 7.86 (d, 3 J= 8.7 Hz, 2H, Hb), 7.53 (d, 3 J= 8.7 Hz, 2H, Ha), 7.51 3 J= 9.0 Hz, 1H, Ha), 7.53 (d, 4 J= 2.6 Hz, 1H, Hb), 7.48 (t, 3 J= 8.1 Hz, 2H), 7.27 (t, 3 J= 7.4 Hz, 1H), (s, 2H), 7.25-7.15 (m, 5H), 2.31 (s, 3H) ppm. Incorporation expected at δ 7.86 and/or δ 7.53 7.16 (d, 3 J= 7.6 Hz, 2H), 3.19 (s, 3H) ppm. Average: y=95%, ppm (red arrow). Determined against reference-integral at δ 2.31 ppm (blue arrow).
						94% D at position b
						72% D at position c

Method B: 10 mg, 32 µmol 23, 1.6 mg, 1.6 µmol catalyst Ba. LC-MS (positive ESI): m/z, 309.2 [M+H] + (3%), 310.2 [M(1D)+H] + (20%), 311.2 [M(2D)+H] + (63%), 312.2 [M(3D)+H] + (14%).

Yield: 1) 9.6 mg, 31 µmol, 96%; 92%D for δ 8.02 and 64%D for δ 7.53 ppm.

2) 9.3 mg, 30 µmol, 93%; 95%D for δ 8.02 and 80%D for δ 7.53 ppm. 1 H-NMR Reference 23 1 H-NMR HIE reaction 23 Method B: 10 mg, 26 µmol 24, 1.3 mg, 1.3 µmol catalyst Ba. LC-MS (positive ESI): m/z, 382.3 [M+H] + (3%), 383.2 [M(1D)+H] + (10%), 384.2 [M(2D)+H] + (63%), 312.2 [M(3D)+H] + (24%).

Table IV . 2 :

 IV2 Catalyst screening of [H]-10 in HIE a

	O					O	D	D
	N H	S	S	Ir-catalyst	N H			S	S
				D (1 atm), 3 h 2
	[H]-10		isopropyl acetate		10
	Entry	Catalyst		Loading (mol%)	Temp.	D/molecule 10
	1	Crabtree A		10	50		0.5
	2	Kerr PF6 Ba		10	50		0.6
	3	Kerr Barf Bb		10	50		1.9
	4	Burgess E		10	50		0.8
	5	Tamm F		10	50			0
	6	Ding G		10	50			0
	7	Kerr Barf Bb		10	80			2
	8	Kerr Barf Bb		5	80		0.6

a)Conditions: substrate [H]-10 (1 eq.), isopropylacetate (1 mL), D2 (1 atm), 3 h. b) Position determined by 1 H NMR and percentage of deuterium incorporation analysed by LC-MS.

Table IV . 3: Solvent screening in HIE reaction of [H]-10 with catalyst Bb

 IV

	Entry	Solvent	D/molecule
	1	chloroform	1.1
	2	MTBE	0.5
	3	ethanol	1.2
	4	MeTHF	0.5
	5	Isopropyl acetate	1.9
	6	chlorobenzene	1.8

a Conditions: substrate [H]-10 (1 eq.), catalyst Bb (10 mol%), solvent (1 mL), D2 (1atm), 50°C,

3 h. b

Position determined by 1 H NMR and percentage of deuterium incorporation analysed by LC-MS.

  -methyl ester 16

			O
	O	O	H N	O	Molecular Weight =203,2401 Molecular Formula =C9H17NO4
			16

reaction 17 Protected glycine methyl ester 18 a-e

	Glycine, N-[4-methoxy-1-oxopentyl]-methyl ester 17
	Conditions: 10.0 mg (76.0 µmol) 18a; 13.0 mg (7.6 µmol) catalyst Bb, 8h. O O O 10.0 mg (45.0 µmol) 18b; 7.8 mg (4.5 µmol) catalyst Bb, 8h. Molecular Weight =189,2131 Molecular Formula =C8H15NO4 10.0 mg (32.0 µmol) 18c; 5.5 mg (3.2 µmol) catalyst Bb, 8h. N 10.0 mg (53.0 µmol) 18d; 9.2 mg (5.3 µmol) catalyst Bb, 8h. H O 17 10.0 mg (56.0 µmol) 18e; 9.7 mg (5.6 µmol) catalyst Bb, 8h.
	1 H NMR (300 MHz, CDCl3): δ 6.43 (br s, 1H, NH), 3.98 (d, 3 J=8.1 Hz, 2H, CH2NH), 3.69 (s, 3H,
	COOCH3), 3.38 (t, 3 J=12.0 Hz, 2H), 3.27 (s, 3H, OCH3), 2.31 (t, 3 J=7.2 Hz, 2H), 1.89-1.80 (m, 2H)
	ppm. Incorporation expected at δ 3.99 (red arrow). Determined against integral at δ 3.69
	(blue arrow).
	HRMS (positive ESI): m/z calculated for C8H15NO4 + [M+H] + : 190.1074; found: 190.1074.

Conditions: 9.5 mg (50.0 µmol) 17; 8.7 mg (5.0 µmol) catalyst Bb, 3h. Yield: 6.8 mg, 36.0 µmol, 72%; 92%D for δ 3.99.

1 H-NMR Reference 17 1 H-NMR HIE

  CH ester ring), 7.19 (m, 2H, CH2 ester ring), 6.26 (dd, 3 J = 9.1 Hz and 2.2 Hz, 1H, CH ortho-NH2), 6.14 (d, 3 J = 2.2 Hz, 1H, CH ortho-OH) ppm. Incorporation expected at δ 6.26 and 6.14 (red arrow). Determined against integral at δ 7.76 (blue arrow).

	Aniline 35 General method B: 22.6 mg (150.0 µmol) 35; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, Aniline 36 General method B: 33.7 mg (150.0 µmol) 36; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, Aniline 37
	O THF/D2O (1:1) (3 mL), 80 °C, 3h. O O THF/D2O (1:1) (3 mL), 80°C, 3h. N H 2
	N H 2 N H 2	S	O F O	F F	Molecular Weight =151.1664 Molecular Formula =C8H9NO2 Molecular Weight =225.1906 Molecular Weight =229.2375 Molecular Formula =C7H6F3NO2S Molecular Formula =C13H11NO3
		OH O
		36 35 37
	1 H NMR (300 MHz, MeOD): δ 7.36 (m, 1H, CH ortho-NH2), 7.31 (m, 1H,CH ortho-CO2Me), 7.18 (m, 1H, CH meta-NH2), 6.92 (m, 1H, CH ortho-NH2), 3.88 (s, 3H, OCH3) ppm. Incorporation expected at δ 6.92 (red arrow). Determined against integral at δ 3.88 (blue arrow). 1 H NMR (300 MHz, MeOD): δ 7.41 (m, 1H, CH meta-NH2), 7.27 (s, 1H, CH ortho-NH2), 7.22 (m, 1 H NMR (300 MHz, MeOD): δ 7.76 (d, 3 J = 9.1 Hz, 1H, CH meta-NH2), 7.43 (m, 2H, CH2 ester 1H, CH para-NH2), 7.13 (m, 1H, CH ortho-NH2) ppm. Incorporation expected at δ 7.13 (red arrow). Determined against integral at δ 7.27 (blue arrow). ring), 7.28 (m, 1H,
	General method A: 33.7 mg (150.0 µmol) 36; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, General method A: 22.6 mg (150.0 µmol) 35; 1.5 mg (6.0 µmol, 4 mol%) IrICy(0.125NHC) 3b, THF (3 mL), 55 °C, 3h. THF (3 mL), 55°C, 3h.
	1 H-NMR HIE reaction 35b 1 H-NMR HIE reaction 36b
	1 H-NMR HIE reaction 35
					331

LC-MS (positive ESI): m/z 152.1 [M+H] + (60%), 153.1 [M(1D)+H] + (36%), 154.1 [M(2D)+H] + (4%). Yield: 21.0 mg, 139.1 µmol, 93%; 39% D for δ 6.92. 1 H-NMR Reference 35 LC-MS (positive ESI): m/z 153.1 [M(1D)+H] + (61%), 154.1 [M(2D)+H] + (34%), 155.1 [M(3D)+H] + (5%). Yield: 19.9 mg, 131.7 µmol, 88%; 23% D for δ δ 7.36, 33% D for δ 7.18 and 99% D for δ 6.92. 1 H-NMR Reference 35 LC-MS (positive ESI): m/z 226.0 [M+H] + (61%), 227.1 [M(1D)+H] + (33%), 228.0 [M(2D)+H] + (6%). Yield: 32.0 mg, 142.2 µmol, 95%; 33% D for δ 7.13. 1 H-NMR Reference 36 1 H-NMR HIE reaction 36 LC-MS (positive ESI): m/z 227.1 [M(1D)+H] + (67%), 228.0 [M(2D)+H] + (33%). Yield: 29.3 mg, 130.3 µmol, 87%; 99% D for δ δ 7.13 and 23% D for δ 7.41. 1 H-NMR Reference 36

  Pieters et al. reported the selective labelling of sulfurethers in the neighboring position of the sulfur-atom with Ru/C.7 Furthermore, they reported the HIE with oligonucleotides 8 -a compound class never reported before. Audisio et al. developed a concept for carbon isotope exchange with [ 14 C]CO2,9 and a very fast approach to benzimidazolines and cyclic ureas with 14 C and 11 C carbonylation.10 The PET chemistry was further improved by Genicot et al. reporting new activation methods of C-H bonds in order to introduce [ 18 F] into F, 11 CHF2 or CF3 moieties12 to cite only a few.

a) V.Derdau, J. Atzrodt, W. Holla, J. Label. Compd. Radiopharm. 2007, 50, 295-298;b) V. Derdau, J.Atzrodt, J. Zimmermann, C. Kroll, F. Brückner, Chem. Eur. J. 2009,

15, 10397-10404; c) W. J. S.Lockley, J. Label. Compd. Radiopharm.2010, 53, 668-673. 15 E. Alexakis, J. R. Jones, W.J.S.Lockley, Tetrahedron Lett. 2006, 47, 5025-5028.

H NMR (300 MHz, DMSO-d6): δ 7.71 (d, 3 J = 8.2 Hz,

2H, 2 CH ortho-SO2NH2), 7.35 (d, 3 J = 8.2 Hz, 2H, 2 CH ortho-CH3), 2.38 (s, 3H, CH3) ppm. Incorporation expected at δ 7.71 (red arrow). Determined against integral at δ 2.38 (blue arrow). Yield: 83%. 1 H-NMR Reference 15 1 H-NMR HIE reaction 15

H NMR (300 MHz, DMSO-d6): δ 8.50 (m, 1H, CH ortho-SO2NH2), 8.30 (m, 1H, CH), 8.08 (m, 1H, CH ortho-SO2NH2), 7.63 (m,

2H, 2 CH), 7.25 (m, 1H, CH), 2.85 (s, 6H, 2 CH3), 2.75 (s, 6H, 2 CH3) ppm. Incorporation expected at δ 8.50 and/or 8.08 (red arrow). Determined against integral at δ 2.75 (blue arrow). Yield: 76%. 1 H-NMR Reference 19 1 H-NMR HIE reaction 19

H-NMR Reference

1 H-NMR HIE reaction 22

H-NMR Reference

1 H-NMR Reference 28 1 H-NMR HIE reaction 28 1 H-NMR HIE reaction 29

H-NMR HIE reaction 30 1 H-NMR Reference 31 1 H-NMR HIE reaction 31

H-NMR Reference 33 1 H-NMR Reference 32 1 H-NMR HIE reaction 32 1 H-NMR HIE reaction 33

H-NMR Reference12 1 H-NMR HIE reaction 12 Average: y=83%, 87% D

H-NMR Reference 11 1 H-NMR HIE reaction 11 Average: y=78%, 68% D

H-NMR Reference

1 H-NMR HIE reaction 21Average: y=81%, 49% D

H-NMR Reference

1 H-NMR HIE reaction 22Average: y=87%, 85% D

H-NMR HIE reaction

1 H-NMR Reference 24Average: y=82%, 94% D

H-NMR Reference

1 H-NMR HIE reaction 25Average: y=89%, 82% D

H-NMR Reference

28a 1 H-NMR HIE reaction 28a Average: y=32% 87% D δ 7.25 9% D δ 7.02

H-NMR Reference30 1 H-NMR HIE reaction 30 Average: y=82%, 76% D δ 7.16

H-NMR Reference40 1 H-NMR HIE reaction 40 Average: y=97%, 99% D δ 7.20

H-NMR Reference42 1 H-NMR HIE reaction 42 Average: y=95%, 79% D δ 7.10

H-NMR Reference43 1 H-NMR HIE reaction 43 Average: y=96%, 96% D δ 7.09

H-NMR Reference 50a1 H-NMR HIE reaction 50a

H-NMR Reference 50b1 H-NMR HIE reaction 50b Average: y=98%, 81% D δ 7.19

H-NMR Reference 50c1 H-NMR HIE reaction 50cAverage: y=100%, 96% D δ 7.16

H-NMR Reference50d 1 H-NMR HIE reaction 50d Average: y=95%, 88% D δ 7.18

H-NMR Reference 51 1 H-NMR HIE reaction 51 Average: y=87%, 88% D δ 7.71

H-NMR Reference 52a1 H-NMR HIE reaction 52aAverage: y=79%, 75% D δ 7.40

H-NMR HIE reaction 5 1 H-NMR Reference 5

H-NMR Reference 10 1 H-NMR HIE reaction 10Average: y=90%, 96% D δ 6.95

H-NMR Reference14 1 H-NMR: reaction with catalyst G

H NMR (300 MHz, DMSO-d6): δ 8.05(d, 3 J= 8.5 Hz,

2H, CHC(COCH3)CH), 7.83 (d, 3 J= 8.5 Hz, 2H, CHC(C6H5)CH), 7.75 (d, 3 J= 8.5 Hz, 2H, CHC3H3CH), 7,48 (sex, 3 J= 7.5 Hz, 3H, CHC3H3CH), 2.63 (s, 1H, CH3) ppm.

H NMR (300 MHz, DMSO-d6): δ 7.96(d, 3 J= 8.0 Hz,

2H, CHC(C2O2H3)CH), 7.66 (t, 3 J= 7.5 Hz, 1H, C2H2CHC2H2), 7.52 (t, 3 J= 7.5 Hz, 2H, CHCHCHCHCH), 3.82 (s, 3H, CH3) ppm.

H NMR (300 MHz, DMSO-d6: δ 7.41 (t, 3 J= 8.0 Hz,

2H, CHCHCHCHCH), 7.25 (t, 3 J= 7.5 Hz, 1H, C2H2CHC2H2), 7.12 (d, 3 J= 8.5 Hz, 2H, CHC(C2O2H3)CH), 2.26 (s, 3H, CH3) ppm.

H NMR (300 MHz, DMSO-d6): δ 7.72(d, 3 J= 8.2 Hz,

2H),7.36 (d, 3 J= 8.2 Hz, 2H), 7.28 (s, 2H), 2.36 (s, 3H) ppm.

H-NMR Reference12 1 H-NMR HIE reaction 12

H-NMR Reference13 1 H-NMR HIE reaction 13

H-NMR Reference14 1 H-NMR HIE reaction 14

H-NMR Reference15 1 H-NMR HIE reaction 15

H-NMR Reference 15 1 H-NMR HIE reaction 15b

H-NMR Reference17 1 H-NMR HIE reaction 17

H-NMR Reference18 1 H-NMR HIE reaction 18

H-NMR Reference

1 H-NMR HIE reaction 20

H-NMR HIE reaction

H-NMR Reference1 H-NMR HIE reaction

H-NMR Reference

1 H-NMR HIE reaction 24

H-NMR HIE reaction

H-NMR Reference

1 H-NMR HIE reaction 25

H-NMR Reference30 1 H-NMR HIE reaction 30

H-NMR Reference31 1 H-NMR HIE reaction 31

H-NMR Reference 10 1 H-NMR HIE reaction 10

H-NMR Reference 11 1 H-NMR HIE reaction 11

H-NMR Reference 161 H-NMR HIE reaction 16

H-NMR Reference 18a1 H-NMR HIE reaction 18a

H-NMR Reference18b 1 H-NMR HIE reaction 18b 1 H-NMR Reference 18c 1 H-NMR HIE reaction 18c

H-NMR Reference19a 1 H-NMR HIE reaction 19a

H-NMR Reference

1 H-NMR HIE reaction 23Average: y=89%, 55% D

H-NMR Reference

1 H-NMR HIE reaction 27

H-NMR Reference34 1 H-NMR HIE reaction 34

H-NMR Reference35 1 H-NMR HIE reaction 35 Average: y=99%, 94% D

H-NMR Reference37 1 H-NMR HIE reaction 37

H-NMR Reference

1 H-NMR HIE reaction 26

H-NMR Reference

1 H-NMR HIE reaction 14

H-NMR Reference

1 H-NMR HIE reaction 28

H-NMR Reference

1 H-NMR HIE reaction 29

H-NMR Reference32 1 H-NMR HIE reaction 32

H-NMR Reference33 1 H-NMR HIE reaction 33

H-NMR Reference38 1 H-NMR HIE reaction 38

H-NMR Reference 41 1 H-NMR HIE reaction 41

H-NMR HIE reaction 101 H-NMR Reference 10

H-NMR HIE reaction 111 H-NMR Reference 11

H-NMR HIE reaction 431 H-NMR Reference 43

Acknowledgements

Acknowledgments Abbreviations

DFT DMAP DMSO EMEA Equiv./ Eq. EW FDA Fmoc Lys MeV mL MPa MS MRI MW n-Bu NDA NHC Ni NMP NMR NP

II.1.2. Explanation of the HIE reaction process at Sanofi laboratories

The general reaction optimization process to establish a tritium method is structured into three main parts: the reactions in a parallel synthesizer with atmospheric deuterium gas (D2), the optimization in the deuterium manifold with low D2 pressure and finally the application on the tritium manifold (scheme II. 1.6 and see Annexes 1 &2 for details We studied the HIE reaction of apixaban 15 (scheme III. 1.8), a reversible direct inhibitor of factor Xa to prevent stroke, with two different iridium catalysts at the same temperature (100°C). Interestingly, with catalyst G only deuteration of position C was observed, with a total of one deuterium introduced into the molecule. In comparison the HIE reaction with catalyst E generated a highly deuterated product with overall four deuterium atoms, however all three possible positions A, B and C were deuterated.

General deuteration procedure (Annex 1):

-Method A (table III.2.1): two competing substrates (0.2 mmol each, 1 eq.), catalyst Ba (10.0 µmol, 5 mol%, 0.05 eq.), DCM (6 mL), D2 (1 atm), rt, 2 h. After 2 h the reaction was stopped, and the DCM was evaporated in vacuo. For substrates with overlapping 1 H-NMR spectra, the products were separated on a silica flash column with a gradient of pentane and MTBE. The products were analyzed by 1 H-NMR or LC/MS. Each competition reactions were run twice and are noted as 1) and 2). -Method B (scheme III.2.4): substrate (10.0 mg, 1 eq.), catalyst Ba (5 mol%, 0.05 eq), DCM (3 mL), D2 (1 atm), rt unless otherwise specified, 2 h. After 2 h the reaction was stopped, and the DCM was evaporated in vacuo. The products were analyzed by 1 H-NMR.

Computational Studies

Density functional theory calculations were done by Dr. Stefan Güssregen from the Computational Drug Design group in Sanofi.

Details of Computational Methods

Density functional theory (DFT) [1,2] has been employed to calculate gas-phase electronic structures and energies for all species in this paper. We followed the approach validated by Kerr et al. [3] and used the hybrid meta-GGA exchange correlation functional M06 [4] in combination with the 6-31G(d) [5] basis set for non-metal atoms and the Stuttgart RSC [6] effective core potential for Iridium to optimize all structures. Transition states were located at the same level of theory. Harmonic vibrational frequencies have been calculated at the same level of theory to characterize the respective minima (i.e.) and first order saddle points (TS). Thermal energies have been calculated at 298.15K corresponding to experimental conditions. Atomic coordinates for Iridium, Deuterium and the Triphenylphosphine and NHC ligands were taken from Kerr et al. [3] as starting point of the calculations. All calculations have been performed using the Gaussian 09 [7] quantum chemistry program package. All coordinates provided are listed in Cartesian format, with charge and multiplicity of each system given at the top of the coordinate list (i.e. 0 1 = neutral singlet; 1 1 = 1+ charged singlet).

Calculation of stationary points

Following the approach published by Kerr et al. [3] a catalytic complex was calculated assuming that two molecules of dichloromethane (DCM) are coordinated to the iridium center. The relative free energies (Grel) of the stationary points (SP) on the potential energy surface (PES) for coordination complexes 11a, transition states 11b and insertion products 11c were calculated based on the sums of the free and electronic energies for the respective reaction partners using the following calculation scheme:

If directing groups bear more than one atom that is suitable for coordination, all possible reactions routes were investigated, in order to identify the one with the lowest energy profile. All results are summarized in Table S1.

Table S1. Relative free energy values (Grel) for different directing groups to form the coordination complex (CC), insertion product (IP) and transition state (TS) in the HIE reaction.

Deuteration of DM4-H 1a

Conditions: 2.0 mg (2.5 µmol) 1a; 0. Calculated D-content/molecule: 1.9.

Yield: 1.6 mg (2.0 µmol) 1c, 80%.

No determination of labelling position with 1 H-NMR possible.

Tritiation of DM4-SMe 1c (reaction 2; 12 eq tritium)

The tritiation of DM4-SMe 8c was carried out using a standard Tritec ® tritium manifold. DM4-SMe (3.0 mg, 3.3 µmol) 1c, and catalyst Ba (3.0 mg, 1.7 µmol, 51 mol%) were dissolved in isopropyl acetate (0.7 mL) in a 1.0 mL reaction flask before being connected to the manifold.

The solution was frozen in liquid nitrogen and the flask was evacuated, then charged with tritium (2.5 Ci, 74 mbar, 12eq.). The reaction mixture was then allowed to warm to room temperature over 15 min before stirring at r.t. for a further 30 min (186 mbar pressure). The reaction mixture was then heated to 50 °C (270 mbar pressure) and stirred for 180 min before cooling again to r.t. The solvents was evaporated; methanol added and the solvent evaporated into a waste ampulle. This procedure was repeated three times in total. The residue was dissolved in methanol and filtered. Purification and isolation via HPLC gave the tritiated product (1.7 mg, 1.9 µmol 1c, 96.7% radiochemical purity, 2787 MBq; 1.15 TBq/mmol = 31 Ci/mmol).

MS (positive ESI

Computational Studies

Details of Computational Methods

DFT calculations (M06 functional) have been done by Dr. Stefan Güssregen from the Computational Drug Design group in Sanofi. DFT [1,2] have been employed to calculate gasphase electronic structures and energies for all species in this paper. The hybrid meta-GGA exchange correlation functional M06 [3] in combination with the 6-31G(d) [4] basis set for nonmetal atoms and Stuttgart RSC [5] effective core potential for Iridium was used to optimize all structures, as this approach with validated by Kerr et al. Transition states were located at the same level of theory. Harmonic vibrational frequencies have been calculated at the same level of theory to characterize the respective minima (i.e.) and first order saddle points (TS). Thermal energies have been calculated at 353.15K corresponding to experimental conditions.

All calculations have been performed using the Gaussian 09 quantum chemistry program package. All coordinates provided are listed in Cartesian format, with charge and multiplicity of each system given at the top of the coordinate list (i.e. 0 1 = neutral singlet; 1 1 = 1+ charged singlet).

Calculation of the catalytic complex of catalyst 1

Following the approach published by Kerr et al. a catalytic complex was calculated assuming that two molecules isopropyl acetate are coordinated to the iridium center.

Conformational analysis of Ac-Gly-OMe 18a

The conformations were taken from the respective coordination products and optimized to the next local minimum. The conformation with the lowest energy was chosen for calculation of the reference point.

Calculation of the Potential Energy Surface (PES) for deuteration of Boc-Gly-OMe 18b with catalyst Bb.

The PES for (D/L) Boc-Gly-OMe was calculated in the analogous to Ac-Gly-OMe. The results are summarized in Figure S3. The energies of the transition states increase even more and the overall reaction is endergonic.

Figure S3

The details about the global DFT calculations are available in the supporting information of the online publication. [6] References (1) Kohn, W.; Sham, L., J., Phys. Rev. 1965, 140, 1133 -1138

Part V

Heterogeneous iridium catalysis on C(sp 2 )-H centers

Supporting information Part V Materials: see Annex 1

General deuteration procedure (Annex 1):

-Method A: THF (3 mL), 55 °C. Deuterium incorporation showed in blue brackets.

-Method B: THF/D2O (1:1) (3 mL), 80 °C. Deuterium incorporation showed in green brackets.

Hydrogen isotope exchange reactions data

Tritium labelling of 43

This work was done in the LMT laboratory at CEA-Saclay (France), within our collaboration with Dr. Pieters, Dr. Feuillastre and Dr. Dugave.

A 5 mL Fischer-Porter bottle was equipped with a magnetic stir bar and charged with IrICy 3b (8.8 µmol, 80 mol%) under air. Aniline 43 (5.0 mg, 11.0 µmol) was dissolved in THF (0.5 mL) and added to the Fischer-Porter bottle and the latter was then connected to the tritium manifold. The Fischer-Porter glassware was then frozen using a liquid nitrogen bath, left under vacuum for 5 min and then pressurized with T2 gas (0.8 bar at room temperature, 10 Ci). The reaction mixture was magnetically stirred and heated at 55°C (sand bath) for 3 hours. The solution was then cooled down to room temperature, further cooled using a liquid nitrogen bath, the extra T2 gas was then removed. Then the solution was warmed to room temperature and 4 mL of methanol was added to precipitate the IrNp. The resulted suspension was filtred using Nylon filter membranes (0.45 µm) and the filtrate was evaporated under vacuum. The described procedure gave a crude total activity of 10.6 GBq (287 mCi). 25% of the crude was purified by preparative HPLC leading to a total activity of 1.4 GBq (38 mCi) with a molar activity of 962 GBq/mmol (26 Ci/mmol).

LC-MS (positive ESI

Part VI Conclusion and future perspectives

Part VI: Conclusion and future perspectives

In this PhD project, it was aimed to develop new efficient, fast and easy-to-handle methods to label drug compounds with deuterium and tritium in one single step. These labelled compounds are an essential part in drug discovery and help to speed up the generation of the safety profile of a drug candidate. The development of new and efficient radioactive labelling methods can therefore lead to an improved attrition rate and a faster approval of necessary medications (scheme VI.1).

Scheme VI. 1: Acceleration of the Drug Discovery Pathway by Isotope Chemistry

If working with radioactive material, the most convenient way to introduce the radioactive label is as late as possible to reduce waste, to increase the overall yield and to reduce the handling time with a potentially dangerous compound. In this regards, Isotope Chemistry can be considered as an ancestor of Late-Stage Functionalization. For deuterated compounds applied as internal MS standard, the selective introduction in very high percentages is essential to generate a suitable MS pattern. Therefore, the developed synthetic HIE methods need to be highly selective, highly efficient (>95%D) and should generally introduce 3-6 deuterium atoms in total. activation energy H. Only if the energies of two competing coordination complexes are very similar, the formation of the CH activation transition-state became the most demanding parameter. These results are not only allowing predictions in regioselective HIE reactions of complex molecules as was shown for compounds 10-12, they also give an outreach to develop the concept of directing group-induced late-stage functionalization in general. Understanding the influence of the directing groups could allow a much better planning and successful prediction of late-stage functionalization pathways. Such a prediction rule could save time and resources in drug discovery research or could even broaden the applications of tritiated molecules to new fields of the life sciences. 4

Scheme VI. 5: Prediction of Iridium catalysed Hydrogen Isotope Exchange reactions in competition situation (Part III -Chapter 2)

Meanwhile the Part II-III were focused on HIE on C(sp 2 )-centers, the labelling of C(sp 3 )centers were still a challenge. Consequently, the Part IV focused on developing a new method for highly selective HIE of unactivated C(sp 3 )-centers. Starting from a complex tritium labelling of the maytansine DM4 drug 13, the HIE reaction on a series of common linker side-chains of antibody-drug-conjugates proceeded with high yields, high regioselectivity and with deuterium incorporations up to 99% (scheme VI.6). The scope of the method was further extended to amino acids, di-and tripeptides (14-16), with

Annex 1 Materials and General deuteration procedure

Materials:

-All substrates, catalysts and solvents were obtained from commercial suppliers and used without further purification, unless otherwise stated. Deuterium gas (99.9%D) was purchased in from Sigma Aldrich in 12 L bottles (CAS 7782-39-0).

-The degree and regioselectivity of deuterium incorporation in the substrates were determined by 1 H-NMR. 1 H (300, 500 MHz) and 13 C (75, 125 MHz) NMR spectra were obtained on Bruker spectrometers in the indicated solvents. Chemical shifts (δ) are reported in ppm. Coupling constants are reported in Hz and refer to 3 J H-H couplings, unless otherwise stated. NMR assignments were made using additional 2D NMR experiments. The listed 1 H-NMR spectra show sequential the substrate before reaction and the spectrum of the substrate after HIE reaction with the respective catalyst. Due to the automatic calibration from the Bruker instrument there is sometimes a small shift between the 2 compared spectra. The red arrow shows the position of the deuterium incorporation and the blue arrow shows the peak of calibration.

-The deuterium incorporation result of 1 H-NMR is calculated according to the following approach: [initial number of protons at carbon in reference] -[amount remaining, same position in HIE reaction] = X. Then: (X*100)/[initial number of proton] = % of D.

-Silica-gel column chromatography was carried out with SiO2 (Merck, 0.063-0.200 mesh) with the indicated eluant.

-The distribution of hydrogen isotopes in the products was determined by a liquid chromatography-mass spectrometry (LC-MS) system with a Symmetry Shield RP18 column, 3.9 x 150 mm, with a gradient program. LC column conditions were as follows: mobile phase A: water (900 mL), acetonitrile (100 mL), TFA (1 mL); mobile phase B: water (100 mL), acetonitrile (900 mL), TFA (0.75 mL), Flow rate: 0.6 mL/min. Detection: UV 254 nm and UV 210 nm.

-The deuterium incorporation result of LC-MS is calculated according to the following approach: the area values of MS peaks M0, M0+1D, M0+2D, M0+3D, M0+4D, etc. are added to a total sum.

[D/molecule] = M1 area/total sum + M2 area/total sum*2 + M3 area/total sum*3 etc…

Annex 2 Detailed description of the HIE reaction process

The general reaction optimization process to establish a tritium method is structured into three main parts: the reactions in a parallel synthesizer with atmospheric deuterium gas (D2), the optimization in the deuterium manifold with low D2 pressure and finally the application on the tritium manifold (figure 1).