Je Remercie Également Monsieur

Ludovic Jullien

Wadih Ghattas

Charles Skarbek

Yoan Chevallier

Kariyawasam Kalani

Guillermo- Allexandro Bowithantri

Antoine D ' Olivera-Udry

Antoine Alemand

Ivanna Schlichter

Benjamin Amarsy

ces côtés et je lui adresse tous mes remerciements pour

Résumé : L'inactivation pharmacologique des médicaments antitumoraux vis-à-vis des cellules saines est un facteur critique dans le développement de prodrogues. Généralement, les chimistes médicinaux greffent des motifs temporaires aux principes actifs antitumoraux existants afin de réduire autant que possible leur activité pharmacologique. Nous avons développé une plateforme où la structure de la prodrogue n'inclut pas le principe actif préexistant. Celle-ci est basée sur un précurseur synthétique inactif capable de générer l'agent cytotoxique par cyclisation bioorthogonale dans un environnement tumoral. En utilisant les phénanthridines comme composés modèles cytotoxiques, nous avons conçu des précurseurs biaryles à cycle ouvert qui génèrent des phénanthridines par imination bioorthogonale irréversible. Cette réaction a été déclenchée par des espèces réactives de l'oxygène, communément surproduites dans les cellules cancéreuses et ainsi capables de convertir une fonction ester de vinylboronate en une fonction cétone qui réagit avec une fonction aniline en attente. Nous avons préparé un précurseur inactif qui engendre une phénanthridine cytotoxique sur la lignée cancéreuse KB. De plus, la cinétique de cyclisation de cette prodrogue est extrêmement rapide (˂ 10 ms) dans des sphéroïdes de cellules vivante KB. Cette formation rapide permet de circonscrire l'action du médicament à la tumeur.

Title:

In-cell synthesis of cytotoxic phenanthridine through bioorthogonal cyclization: a paradigm in antitumor prodrug development Keywords: Spontaneous reaction, organic chemistry, cancer, prodrug, bioorthogonal reaction, ROS.

Abstract: Pharmacological inactivation of antitumor drugs toward healthy cells is a critical factor in prodrug development. Typically, pharmaceutical chemists graft temporary moieties to existing antitumor drugs to reduce their pharmacological activity as much as possible. Here, we report a platform where the structure of the prodrug excludes the preexisting antitumor drug motif and is based on an inactive synthetic precursor able to generate the cytotoxic agent by bioorthogonal cyclization within a tumor environment. Using phenanthridines as cytotoxic model compounds, we designed ringopened biaryl precursors that generated the phenanthridines through bioorthogonal irreversible imination. This reaction was triggered by reactive oxygen species, commonly overproduced in cancer cells, able to convert a vinyl boronate ester function into a ketone that subsequently reacted with a pendant aniline. An inactive precursor was shown to engender a cytotoxic phenanthridine against KB cancer cells. Moreover, the kinetic of cyclization of this prodrug was extremely rapid (˂ 10 ms) inside living cells of KB cancer spheroids so as to circumvent drug action.

Ce projet de développement de prodrogues antitumorales est à l'interface de la chimie, de la biologie et de la biophysique.

La conception, la synthèse et l'étude biologique des différents dérivés ont été réalisées dans le groupe du Dr Raphaël LABRUERE du laboratoire de Chimie Bioorganique et Bioinorganique de l'Institut de Chimie Moléculaire et des Matériaux d'Orsay. En particulier, les essais biologiques ont été réalisés par le Dr Charles SKARBEK qui était chercheur associé post-doctoral dans le groupe. D'autre part, l'équipe du Pr Ludovic JULLIEN ainsi que le Dr Thomas LE SAUX du laboratoire PASTEUR de l'Ecole Normale Supérieure de Paris ont été impliqués dans l'analyse cinétique et les études de microscopie de fluorescence.

Un nombre important de thérapies utilisées contre le cancer ont une toxicité sur les cellules saines proche de celle observée sur les cellules cancéreuses. Il y a donc un besoin urgent de développer de nouvelles chimiothérapies. Pour répondre à cette problématique, il serait souhaitable de cibler spécifiquement les cellules cancéreuses afin de réduire la toxicité générale des molécules utilisées en clinique en exploitant les différences entre cellules saines et cellules cancéreuses. Une réponse rapide et adaptée réside dans le développement de prodrogues, des molécules à activité masquée, conçues pour être activées après une réaction enzymatique ou chimique. La stratégie des prodrogues peut non seulement améliorer l'efficacité du médicament mais aussi diminuer sa toxicité sur les organes ou tissus non ciblés. En général, il est nécessaire que la prodrogue soit activée par un stimulus spécifiquement produit par les cellules cancéreuses afin d'obtenir une libération du médicament dans l'environnement tumoral. Dans la majorité des cas, l'activation de ces prodrogues est réalisée par des enzymes fortement exprimées dans la tumeur.

Parmi les différentes voies métaboliques, l'activation des prodrogues antitumorales par les espèces réactives de l'oxygène (ROS) semble être particulièrement prometteuse. En effet, des études biologiques sur les tumeurs ont révélé que les cellules cancéreuses présentent un niveau de stress oxydatif plus élevé que les cellules saines. Ce stress oxydatif se traduit par la surproduction d'espèces réactives de l'oxygène dont le peroxyde d'hydrogène (H₂O₂), le radical hydroxyle (HO•), l'anion superoxyde (O₂ •⁻ ) ou le peroxynitrite (ONO₂⁻).

Les acides boroniques et leurs esters ont été développés comme groupes de masquage temporaire activés par les ROS. En particulier, il a été démontré que le peroxyde d'hydrogène et le peroxynitrite surproduits dans l'environnement tumoral réagissent sélectivement avec les acides et les esters boroniques par oxydation de la liaison carbone-bore pour donner un groupe alcool.

Le principal inconvénient de la stratégie typique des prodrogues antitumorales est la faible capacité de la partie temporaire à masquer l'activité pharmacologique du médicament lié de manière covalente. Cette situation nous a conduits à pousser à mener des recherches via cette thèse visant à développer une plateforme où le médicament antitumoral préexistant n'est pas inséré dans la structure de la prodrogue. La conception de la prodrogue repose plutôt sur un précurseur inactif à cycle ouvert capable de générer l'agent cytotoxique par cyclisation bioorthogonale dans le microenvironnement tumoral.

Ce manuscrit de thèse a été organisé en cinq chapitres en fonction des publications des résultats.

Le premier chapitre de ce manuscrit présente des généralités sur le cancer et les traitements actuels. La majeure partie de ce chapitre a été publiée dans European Journal of Medicinal Chemistry, 2020, 112670. Dans ce chapitre, nous avons rassemblé les prodrogues anticancéreuses contenant du bore et répondant au stress oxydatif du microenvironnement tumoral qui ont été rapportées dans la littérature.

Le deuxième chapitre de cette thèse annonce le projet de cette thèse et les principaux objectifs sont présentés ici.

Le troisième chapitre de ce manuscrit présente la preuve de concept de la nouvelle stratégie de prodrogue anticancéreuse. Ce chapitre est basé sur la première partie de notre publication dans Angewandte Chemie International Edition, 2021, 133, 45, 24245-24249. En utilisant la phénanthridine comme modèle de médicament, nous avons préparé un précurseur biaryle à cycle ouvert qui a généré la 6-méthylphénanthridine correspondante par imination bioorthogonale intramoléculaire. Cette réaction a été déclenchée par des espèces réactives de l'oxygène capables de convertir une fonction ester de vinylboronate en une cétone qui a ensuite réagi avec une aniline. Les cinétiques d'oxydation et de cyclisation ont été déterminées avec précision par une méthode de stopped flow combinée à la spectrofluorométrie.

Dans le quatrième chapitre de ce manuscrit, a été développée une voie originale de synthèse one pot de diverses 6-méthylphénanthridines. L'identification d'un dérivé cytotoxique de la 6méthylphénanthridine a été réalisée via la détermination de la viabilité des cellules cancéreuses KB 3.1. Le contenu de ce chapitre sera soumis pour publication prochainement.

Le dernier chapitre de ce manuscrit est basé sur la deuxième partie de notre publication dans Angewandte Chemie International Edition, 2021, 133, 45, 24245-24249. Dans ce chapitre, le précurseur biarly ouvert de la 6-méthylphénanthridine cytotoxique sélectionnée a été synthétisé et trouvé inactif contre les sphéroïdes de KB 3.1. La stabilité de la prodrogue a été évaluée dans le plasma de rat et la cinétique d'oxydation et de cyclisation dans les sphéroïdes a été mesurée par épifluorescence. La cinétique de cyclisation de cette prodrogue a été trouvée extrêmement rapide (<10 ms) à l'intérieur de cellules vivantes de sphéroïdes de cancer KB.

Cette thèse se termine par une conclusion générale et une brève mise en perspective de ces résultats.

This project of antitumor prodrug development is at the interface of chemistry, biology and biophysics.

The design, synthesis and biological study of the different derivatives were carried out in the group of Dr Raphaël LABRUERE from the laboratoire de Chimie Bioorganique et Bioinorganique of the Institut de Chimie Moléculaire et des Matériaux d'Orsay. In particular, the biological assays were performed by Dr Charles SKARBEK who was postdoctoral research associate in the group. On the other hand, the team of Pr Ludovic JULLIEN together with Dr Thomas LE SAUX from the laboratoire PASTEUR at Ecole Normale Supérieure in Paris were involved in the kinetic analysis and the fluorescence microscopy studies.

A significant number of therapies used against cancer have a toxicity against healthy cells close to that observed on cancer cells. There is therefore an urgent need to develop new chemotherapies. To address this issue, it would be desirable to specifically target cancer cells in order to reduce the general toxicity of molecules in clinical use by exploiting the differences between healthy and cancer cells. A rapid and adapted answer lies in the development of prodrugs, molecules with masked activity, designed to be activated after an enzymatic or a chemical reaction. The prodrug strategy can not only improve the efficacy of the drug but also decrease its toxicity on non-target organs or tissues. In general, it is necessary for the prodrug to be activated by a stimulus specifically made by the cancer cells in order to obtain a release of the drug within the tumor environment. In the majority of cases, the activation of these prodrugs is performed by enzymes highly expressed in the tumor.

Among the different metabolic pathways, the activation of antitumor prodrugs by reactive oxygen species (ROS) seems to be particularly promising. Indeed, biological studies of tumors have revealed that cancer cells exhibit a higher level of oxidative stress compared to healthy cells. This oxidative stress results in the overproduction of reactive oxygen species including hydrogen peroxide (H₂O₂), hydroxyl radical (HO•), superoxide anion (O₂ •⁻ ) or peroxynitrite (ONO₂⁻).

Boronic acids and their esters were developed as temporary masking groups triggered by ROS. In particular, hydrogen peroxide and peroxynitrite overproduced within tumor environment were shown to react selectively with boronic acids and esters by oxidation of the carbon-boron bond to engender an alcohol group.

The main disadvantage of the typical antitumor prodrug strategy is the often weak ability of the temporary promoiety to extinguish the pharmacological activity of the covalently linked drug. This situation led us to motivate this PhD study aiming to develop a platform where the preexisting antitumor drug is not inserted in the structure of the prodrug. The design of the prodrug rather relies on an inactive ring-opened precursor able to generate the cytotoxic agent by internal bioorthogonal cyclization within a tumor microenvironment.

This PhD manuscript has been organized into five chapters driven by the publications of the results.

The first chapter of this manuscript introduces generalities on cancer and the current treatments. The major part of this chapter was published in European Journal of Medicinal Chemistry, 2020, 112670. In this chapter, we gathered the anticancer boron-containing prodrugs responsive to the oxidative stress from the tumor microenvironment which have been reported in the literature.

The second chapter of this dissertation announces the PhD project from the origin and the main goals are presented herein.

The third chapter of this manuscript introduces the proof of concept of the novel anticancer prodrug strategy. This chapter is based on the first part of our publication in Angewandte Chemie International Edition, 2021, 133, 45, 24245-24249. Using phenanthridine as drug model, we prepared a ring-opened biaryl precursor that generated the corresponding 6-methylphenanthridine through intramolecular bioorthogonal imination. This reaction was triggered by reactive oxygen species able to convert a vinyl boronate ester function into a ketone that subsequently reacted with a pendant aniline. The kinetics of oxidation and cyclization were accurately determined by a method of stopped flow combined to spectrofluorometry.

In the fourth chapter of this manuscript, an original one pot synthetic pathway to various 6methylphenanthridines was developed. Identification of a cytotoxic 6-methylphenanthridine derivate was achieved by determination of the KB 3.1 cancer cells viability. The content of this chapter will be submitted for publication in due course.

The last chapter of this manuscript is based on the second part of our publication in Angewandte Chemie International Edition, 2021, 133, 45, 24245-24249. In this chapter, the precursor of the selected cytotoxic 6-methylphenanthridine was then synthesized and found inactive against spheroids of KB 3.1. The stability of the prodrug have been assessed in rat plasma and the kinetic of oxidation and cyclization in spheroids have been measured by epifluorescence. The kinetics of cyclization of this prodrug was found extremely rapid (<10 ms) inside living cells of KB cancer spheroids This dissertation ends with a general conclusion and a brief outlook of these results. A significant number of therapies used against cancer have a toxicity against healthy cells close to that observed on cancer cells. There is therefore an urgent need to develop new chemotherapies. To address this issue, it would be desirable to specifically target cancer cells in order to reduce the general toxicity of molecules in clinical use by exploiting the differences between healthy and cancer cells. A rapid and adapted answer lies in the development of prodrugs, molecules with masked activity, designed to be activated after an enzymatic or a chemical reaction. The prodrug strategy can not only improve the efficacy of the drug but also decrease its toxicity on non-target organs or tissues. In general, it is necessary for the prodrug to be activated by a stimulus specifically made by the cancer cells in order to obtain a release of the drug within the tumor environment. In the majority of cases, the activation of these prodrugs is performed by enzymes highly expressed in the tumor.

Among the different metabolic pathways, the activation of antitumor prodrugs by reactive oxygen species (ROS) seems to be particularly promising. Indeed, biological studies of tumors have revealed that cancer cells exhibit a higher level of oxidative stress compared to healthy cells. This oxidative stress results in the overproduction of reactive oxygen species including hydrogen peroxide (H2O2), hydroxyl radical (HO . ), superoxide anion (O2 .-) or peroxynitrite (NO3 -).

Boronic acids and their esters were developed as temporary masking groups triggered by ROS. In particular, hydrogen peroxide and peroxynitrite overproduced within tumor environment were shown to react selectively with boronic acids and esters by oxidation of the carbon-boron bond to engender an alcohol group.

The main disadvantage of the typical antitumor prodrug strategy is the often weak ability of the temporary promoiety to extinguish the pharmacological activity of the covalently linked drug.

This situation led us to motivate this PhD study aiming to develop a platform where the preexisting antitumor drug is not inserted in the structure of the prodrug. The design of the prodrug rather relies on an inactive ring-opened precursor able to generate the cytotoxic agent by internal bioorthogonal cyclization within a tumor microenvironment.

This PhD manuscript has been organized into five chapters driven by the publications of the results.

The first chapter of this manuscript introduces generalities on cancer and the current treatments. The major part of this chapter was published in European Journal of Medicinal Chemistry, 2020, 112670. In this chapter, we gathered the anticancer boron-containing prodrugs responsive to the oxidative stress from the tumor microenvironment which have been reported in the literature.

The second chapter of this dissertation announces the PhD project from the origin and the main goals are presented herein.

The third chapter of this manuscript introduces the proof of concept of the novel anticancer prodrug strategy. This chapter is based on the first part of our publication in Angewandte Chemie International Edition, 2021, 133, 45, 24245-24249. Using phenanthridine as drug model, we prepared a ring-opened biaryl precursor that generated the corresponding 6methylphenanthridine through intramolecular bioorthogonal imination. This reaction was triggered by reactive oxygen species able to convert a vinyl boronate ester function into a ketone that subsequently reacted with a pendant aniline. The kinetics of oxidation and cyclization were accurately determined by a method of stopped flow combined to spectrofluorometry.

In the fourth chapter of this manuscript, an original one pot synthetic pathway to various 6methylphenanthridines was developed. Identification of a cytotoxic 6-methylphenanthridine derivate was achieved by determination of the KB 3.1 cancer cells viability. The content of this chapter will be submitted for publication in due course.

The last chapter of this manuscript is based on the second part of our publication in Angewandte Chemie International Edition, 2021, 133, 45, 24245-24249. In this chapter, the precursor of the selected cytotoxic 6-methylphenanthridine was then synthesized and found inactive against spheroids of KB 3.1. The stability of the prodrug have been assessed in rat plasma and the kinetic of oxidation and cyclization in spheroids have been measured by epifluorescence. The kinetics of cyclization of this prodrug was found extremely rapid (˂ 10 ms) inside living cells of KB cancer spheroids This dissertation ends with a general conclusion and a brief outlook of these results.

Chapter 1: Cancer treatments and the place of ROS-activated prodrugs

Generality

Cancer is a generic term for a large group of diseases that can affect any part of the body. 1

Other terms used are malignant tumors and neoplasms. One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries. Indeed, healthy cells develop, divide, and die according to a process written in their genetic material. Cancer cells, on the other hand, are the result of this cycle, thus escaping the mechanisms of regulation and control of cell proliferation by multiplying in an anarchic way, these cancerous cells will give birth to a tumor that will develop by invading and then destroy the organ from which it originated. Throughout the evolution of the pathology, cancer cells may detach from the tumor and may break away from the primary tumor, migrate through the bloodstream or lymphatic bloodstream and attach themself and new organs to give rise to new tumors called metastases 2 .

Cancer is a major public health problem today due to the number of people affected (19 million per years) and the number of deaths (10 million per years). According to estimations from the World Health Organization (WHO) in 2020, cancer is the second leading cause of death before the age of 70. In 2018, the International Agency for Research on Cancer (IARC) estimated that: 1 in 5 men will develop cancer in their lifetime and 1 in 8 men will die from this disease; 1 in 6 women will develop cancer in their lifetime and 1 in 11 women will die from this disease (figure 1). 3 In men, the four most frequently encountered cancers are lung, prostate, colorectal and stomach cancers and the deadliest are lung, liver colorectal and stomach cancers. These four types of cancer represent more than 50% of worldwide men deaths (figure 2). 3 There are many different cancers with different causes for each cancer:

-Colorectal cancer is increasing in men and women between the ages of 40 and 50 due to poor diet, sedentary lifestyle, and obesity.

-Lung cancer in women has been increasing at a rate of 5.3% per year for the last 30 years. This is mainly due to women who started smoking in the 70's.

-Liver cancer is increasing in men and women due to obesity, alcohol consumption, tobacco and industrial food.

-Breast cancer increases in women under 40 years old, the main causes are alcohol, hormonal treatments (contraceptives or in case of menopause, delay of the age of the first pregnancy [START_REF] Macmahon | Age at First Birth and Breast Cancer Risk[END_REF] , early puberty or decrease of breastfeeding duration).

According to the INCa, in 30 years there has been an increase in the number of cancers in the French population: +65% in men and + 93% in women. 5 A part of this increase comes from the increase of the population. We are 8 million more in 2018 compared to 1990. Also, the population is older and therefore more at risk of cancer. If we remove this demographic effect, there remains an increase of 6% in men and 45% in women. This may be because life expectancy has increased and that diagnoses via screening campaigns and imaging techniques are more accurate and done earlier (figure 4). In 2011, the French health insurance system (sécurité sociale) spent 14.5 billion € on treating cancer patients. This equates to average expenditure of 10,000 € per patient per year.

Treatments to cure cancer

Four different approaches coexist to cure cancer, these therapies can be used in combination depending on the type of cancer, its aggressiveness, size, location, and the patient's general condition.

Surgery

The goal of curative surgical treatment of cancer is to remove the malignant tumor and local lymph nodes at a stage where the tumor has not yet metastasized. Surgical resection is said to be complete when all tumor tissue has been resected and the surgical margins are healthy.

In some very specific cancers, surgical treatment is indicated to reduce the tumor mass (debulking) in order to facilitate subsequent eradication of the tumor by chemotherapy and/or radiotherapy. It may also be indicated for the removal of a single metastasis in the liver, lung or brain.

In 2015, 447,391 hospitalizations for cancer surgery (including biopsies and removals of malignant, in situ or invasive tumors) were performed, up by 8.5% from 2010. This represents 8.3% of total hospital activity in oncology (figure 5). In France, in 2016, 408,276 people underwent surgery for cancer and 26.6% of stays were performed on an outpatient basis.

Radiotherapy

Radiotherapy is the use of radiation to destroy cancer cells by blocking their ability to multiply. [START_REF] Baskar | Biological Response of Cancer Cells to Radiation Treatment[END_REF] This is a local treatment for cancer. Different types of radiation (x-rays, gamma rays and particles) damage and destroy tumors and, can be combined with surgery or chemotherapy.

The radiation can be external (teletherapy) or internal (brachytherapy). The goal of radiation is to destroy cancer cells while preserving healthy tissue and nearby organs as much as possible.

More than half of all cancer patients are treated with radiation therapy at some point in their treatment. Radiotherapy may sometimes be combined with chemotherapy (called radiochemotherapy), which makes cancer cells more sensitive to radiation. [START_REF] Ozsaran | Radiochemotherapy for Patients with Locally Advanced Cervical Cancer: Early Results[END_REF] The goal is to reduce the risk of tumor recurrence and the size of the tumor before surgery. The length of the treatment, as well as the number of sessions, varies according to the area to be treated and the technique used.

Immunotherapy

Immunotherapy involves the body's own immune defenses to attack and destroy cancer cells. [START_REF]How Immunotherapy Is Used to Treat Cancer[END_REF] Different approaches have been used for decades, but recently there has been a shift to a new and very promising strategy: targeting immune cells with drugs to break their tolerance to cancer cells and stimulate their destruction by the body. Immunotherapy is an effective treatment in 20 to 40% of patients. However, they are often accompanied by severe side effects related to the overactivation of the immune system, including the development of autoimmune diseases. These effects are explained by the fact that these therapies make our white blood cells very aggressive: they start to kill the patient's normal cells in addition to the tumor.

Chemotherapy

Chemotherapy is a treatment based on the administration of chemical substances and used in different types of cancer to destroy cancer cells. 10 There are many chemotherapy drugs with different mechanisms of action; in the treatment of cancer, these chemotherapy drugs can be used alone or in combination for better efficiency. Each drug differs in the way it works in the cell cycle. Most agents destroy cancer cells by affecting the synthesis or function of DNA during the cell's reproductive cycle or by interacting with RNA and proteins. Herein, only the cytotoxic agents, a major family of chemical drugs used in clinic will be discussed. There are 4 classes of cytotoxic agents.

Antimetabolite drugs

The antimetabolites are divided into several subfamilies ( Antimetabolites can be used in the treatment of cancer because they interfere with DNA production and therefore with cell division and tumor growth because cancer cells divide more often than other cells, therefore inhibiting cell division will affect tumor cells more than other cells.

Alkylating agents

The idea of using alkylating agents to treat cancer arose out of Edward Bell Krumbhaar, Indeed, Canadian soldiers who were victims of the combat gas (mustard gas) used in 1917 presented respiratory problems associated to disappearance of lymphocytes in the blood (lymphoid aplasia). 12 Alkylating agents are able to link an alkyl group to the nitrogen atom (position 7) of guanine in DNA strands. 13 Cancer cells are more susceptible to DNA deterioration than normal cells and they proliferate faster and are therefore less able to correct mistakes made during the DNA replication. Alkylating agents are used to treat several cancers but present serious side effects on normal cells. This pharmacological class includes a very large number of substances and the most used are presented in figure 7.

Antitumor antibiotics 14

Topoisomerases are enzymes that cut and re-ligate a single strand of the DNA molecule. Their levels are much higher in tumor cells than in normal cells. They inhibit the re-ligation step of the two ends of the DNA strand, held by the enzyme, forming a complex at the DNA replication fork. They induce an arrest of cell division. These drugs act in synergy with platinum salts, alkylating drugs, ionizing radiation. The current products used in clinic are derivatives of camptothecin found in the ornamental plant Camptotheca acuminata found in China or doxorubicin which is a topoisomerase inhibitor and an intercalating agent produced by Streptomyces bacteria (figure 8).

Mitotic inhibitors 15

The common feature of spindle poisons is their biding to tubulin, which is part of the mitotic spindle and plays an important role in maintaining the cytoskeleton. 15 They disorganize the segregation of chromosomes at the time of mitosis and lead to the death of the cell. Several types of mitotic inhibitors can be distinguished according to their structure, their origin (often natural) and their binding site on tubulin, resulting in various consequences on the microtubules.

-Periwinkle alkaloids or vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine, vinflunine) prevent microtubule polymerization and destabilize them.

-Taxanes (paclitaxel, derived from the Pacific yew Taxus brevifolia, docetaxel and cabazitaxel), on the other hand, are spindle stabilizers because they prevent spindle depolymerization and stimulate spindle polymerization.

-Dolastatin 10 is one of numerous potent cytotoxic natural products that have been isolated from the marine sea hare Dolabella auricularia. This drug has strong antiproliferative activity against murine PS leukemia cells by activating tubulin binding leading to lengthening cellular retention and is cytotoxic in vitro against several human cancer cell lines, such as melanoma, sarcoma, colorectal, and ovarian cancer.

These structures are represented in figure 9. Overall, most clinically approved anticancer drugs are characterized by a narrow therapeutic window that results mainly from a high systemic toxicity of the drugs in combination with an evident lack of tumor selectivity.

Tumor targeted chemotherapy

The main problem of the cytotoxic agents is often their lack of cancer cells selectivity. When the drug is injected in the body, it diffuses within the body and operates in the tumor area to kill cancer cells, but normal cells are also damaged which generates many side effects like swelling, vomiting, nausea, hair loss etc. Thus, there is an urgent need for the development of new and specific chemotherapies, capable of exploiting the differences between normal and tumor cells to increase the potency and decrease the dose and side effects (figure 10). The prodrug strategy was initially introduced by Albert in 1958. [START_REF] Albert | Chemical Aspects of Selective Toxicity[END_REF] Prodrugs are activity-masked molecules designed to be activated in vivo by an enzymatic or a chemical reaction. In most cases, these molecules are covalent assemblies made of the active drug linked to a temporary moiety (figure 10a). The latter chemical unit confers new physicochemical properties to the entire entity optimizing its absorption, distribution, metabolism, excretion and toxicity (ADMET). [START_REF] Rautio | Prodrugs: Design and Clinical Applications[END_REF][START_REF] Huttunen | Prodrugs--from Serendipity to Rational Design[END_REF][START_REF] Rautio | The Expanding Role of Prodrugs in Contemporary Drug Design and Development[END_REF] The prodrug strategy used in anticancer chemotherapy intends to deliver the active drug only at the tumor site in order to avoid deleterious side effects on normal tissues.

This approach ideally ensures a high selectivity against targets and low toxicity, since drugs are liberated specifically at the tumor area, generally by enzymes strongly overexpressed at the tumor site, by reactive oxygen species (ROS) or by hypoxia. Furthermore, the prodrug tactic remains faster and less expensive than the development of novel therapeutic agents with appropriate drug-like characteristics. [START_REF] Denny | Prodrug Strategies in Cancer Therapy[END_REF][START_REF] Zhang | Prodrug Strategy for Cancer Cell-Specific Targeting: A Recent Overview[END_REF][START_REF] Taresco | Stimuli-Responsive Prodrug Chemistries for Drug Delivery[END_REF] The development of an antitumor prodrug mainly depends on 3 factors:

-Tumor targeting: It is important to distinguish the tumor specificity of cancerous tissue from healthy tissue in order to accumulate anticancer drug selectively and efficiently at the tumor site. The exploited targets used are found either in cancer cells or in the tumor microenvironment.

-Controlled release: The release of the active ingredient should take place only when in contact with the tumor area to avoid uncontrolled release of the drug throughout the body. Therefore, it is essential that the prodrug would be completely stable in the body and only breaks down under the stimulus associated to cancer cells via chemical or enzymatic release processes. In addition, the kinetics of activation of the prodrug and release of the active ingredient must be rapid to act specifically on cancer cells and not on healthy cells through diffusion.

-Difference in activity between the prodrug and the drug: The chemical grafting of an organic temporary group to a given drug is sometimes not sufficient to mask its pharmacological activity. It is therefore important to control that the biological inactivation of the prodrug is achieved.

Active targeting of cancer cells

Tumor cell targeting

Drug delivery in oncology is of particular interest owing to the narrow therapeutic window of anticancer agents. Cancer cells expressed on their surface particular receptors and antigens as shown in table Targeting the membrane receptors on the different type of cancers cells is a promising approach. This strategy is quite selective and may be tailored to a wide range of tumors by modulating the ligand or antibody in addition to the nature of the cytotoxic agent. In 2009, only gemtuzumab ozogamicin, Mylotarg® (figure 12) was used clinically (approved by the US FDA in 2000, withdrawn in 2010 and approved again in 2017). [START_REF] Research | FDA Approves Gemtuzumab Ozogamicin for CD33-Positive AML in Pediatric Patients[END_REF][START_REF]Pfizer Receives FDA Approval for MYLOTARG TM (gemtuzumab ozogamicin)[END_REF] More than 80 others are currently in clinical trials. Mylotarg® is an anti-CD33 humanized monoclonal antibody conjugated to calicheamicin. It represents an interesting option for patients over 60 years old with acute myelogenous leukemia. [START_REF]Pfizer Receives FDA Approval for MYLOTARG TM (gemtuzumab ozogamicin)[END_REF] The potent DNA damaging agent calicheamicin was conjugated to a humanized monoclonal antibody that specifically binds to the CD33 cell surface antigen on acute myeloid leukemia cells. After internalization of the conjugate into the cancer cell by endocytosis, the drug is released by hydrolysis of the hydrazone bond in the acidic endosomes/lysosomes, the enediyne is then activated by reduction of the trisulfide bond (scheme 1).

Scheme 1. DNA-cleaving mechanism of calicheamicin γ1.

After DNA binding, the trisulfide part is reduced by a thiol (e.g. glutathione) followed by intramolecular Michael addition of the thiolate to the adjacent α,β-unsaturated ketone leading to the loss of the bridgehead double bond. The enediyne moiety thus cyclize by Bergman cyclization reaction to generate a diradical, which abstracts a hydrogen from DNA resulting in DNA cleavage. [START_REF] Elmroth | Cleavage of Cellular DNA by Calicheamicin Γ1[END_REF]

Small Molecule Drug Conjugates (SMDCs)

In lieu of an antibody or other macromolecules, the ligand-based prodrugs are made of the covalent assembly of a small molecule and a cytotoxic agent. [START_REF] Cazzamalli | Chemically Defined Antibody-and Small Molecule-Drug Conjugates for in Vivo Tumor Targeting Applications: A Comparative Analysis[END_REF] As observed for ADCs, the SMDC binds to the targeted receptor and the ligand-receptor is subsequently taken up by receptor-mediated endocytosis. Typical ligands are vitamins, peptides, or sugars. Folic acid is one of the mostly employed ligand for active tumor targeting. Interestingly, it was shown that folic acid conjugated to various drugs still present high affinity [START_REF] Lu | Folate Receptor-Targeted Immunotherapy of Cancer: Mechanism and Therapeutic Potential[END_REF][START_REF] Pohl | D-19575--a Sugar-Linked Isophosphoramide Mustard Derivative Exploiting Transmembrane Glucose Transport[END_REF][START_REF] Mchugh | Demonstration of a High Affinity Folate Binder in Human Cell Membranes and Its Characterization in Cultured Human KB Cells[END_REF][START_REF] Antony | The Biological Chemistry of Folate Receptors[END_REF][START_REF] Antony | Folate Receptors[END_REF] for the overexpressed folate receptor of cancer cells. [START_REF] Dixon | Effects of Folate Receptor Expression Following Stable Transfection into Wild Type and Methotrexate Transport-Deficient ZR-75-1 Human Breast Cancer Cells[END_REF][START_REF] Ross | Differential Regulation of Folate Receptor Isoforms in Normal and Malignant Tissues in Vivo and in Established Cell Lines. Physiologic and Clinical Implications[END_REF][START_REF] Holm | Folate Receptors in Malignant and Benign Tissues of Human Female Genital Tract[END_REF][START_REF] Franklin | New Anti-Lung-Cancer Antibody Cluster 12 Reacts with Human Folate Receptors Present on Adenocarcinoma[END_REF][START_REF] Campbell | Folate-Binding Protein Is a Marker for Ovarian Cancer[END_REF] Thus folate conjugated to camptothecin, doxorubicin, alkylating agents, platinum complexes, paclitaxel and 5-FU were studied. [START_REF] Campbell | Folate-Binding Protein Is a Marker for Ovarian Cancer[END_REF][START_REF] Henne | Synthesis and Activity of a Folate Peptide Camptothecin Prodrug[END_REF][START_REF] Liu | Targeted Drug Delivery to Chemoresistant Cells: Folic Acid Derivatization of FdUMP[10] Enhances Cytotoxicity toward 5-FU-Resistant Human Colorectal Tumor Cells[END_REF][START_REF] Lee | Synthesis and Evaluation of Taxol-Folic Acid Conjugates as Targeted Antineoplastics[END_REF][START_REF] Bae | Multifunctional Polymeric Micelles with Folate-Mediated Cancer Cell Targeting and PH-Triggered Drug Releasing Properties for Active Intracellular Drug Delivery[END_REF][START_REF] Steinberg | Synthesis and Evaluation of Pteroic Acid-Conjugated Nitroheterocyclic Phosphoramidates as Folate Receptor-Targeted Alkylating Agents[END_REF] EC145, a conjugate of the Vinca alkaloid desacetylvinblastine monohydrazide coupled to folic acid was introduced by Leamon et al. [START_REF] Reddy | Preclinical Evaluation of EC145, a Folate-Vinca Alkaloid Conjugate[END_REF][START_REF] Vlahov | Design and Regioselective Synthesis of a New Generation of Targeted Chemotherapeutics. Part 1: EC145, a Folic Acid Conjugate of Desacetylvinblastine Monohydrazide[END_REF] and evaluated in clinical phase I. The peptidic linker improved the water solubility of the conjugate whereas the disulfide bond was inserted in order to allow the drug release by reaction with glutathione in the endosomes. In vivo preclinical studies have shown that EC145 is better tolerated than the free drug in mice and moreover antitumor efficacy was stronger when using the conjugate (figure 13).

Metabolic particularities of cancer cells and tumor microenvironment for specific activation of prodrugs

Cancer cells are markedly different to cells from healthy tissues. In addition, the tumor microenvironment includes blood vessels, immune cells, fibroblasts, signaling molecules, such as chemokines, and the extracellular matrix displaying biochemical particularities. The biologic singularities of cancer cells and tumor microenvironment are therefore exploited in anticancer prodrug strategy in order to localize the delivery of the active drug at the tumor site.

Abnormalities of the tumor environment has led to distinct strategies in cancer-responsive prodrug research. First, certain enzymes such as cathepsins are overexpressed in cancer cells and specific prodrugs were developed to target this characteristic. [START_REF] Bildstein | Prodrug-Based Intracellular Delivery of Anticancer Agents[END_REF][START_REF] Walther | Prodrugs in Medicinal Chemistry and Enzyme Prodrug Therapies[END_REF][START_REF] Hamada | Recent Progress in Prodrug Design Strategies Based on Generally Applicable Modifications[END_REF] Prodrugs capitalizing on the hypoxia of cancer cells inside solid tumors are also the object of intensive research. 50- 52 Antitumor targeted-prodrugs responsive to the pronounced oxidative stress in cancer cells have been recently developed. 53,54

Drug release by enzymatic activation

The enzyme allowing activation of the prodrug must be naturally expressed or overexpressed in tumor cells. Activation of a prodrug can be carried out by tumor-associated enzymes such as proteases, glucuronidase, or carboxylesterases, which are expressed either intra-or extracellularly by cancer cells. Therefore, the use of a substrate recognized by the enzymatic site is an approach for tumor targeting. In the past, a great number of carrier-linked prodrugs have been developed with enzyme-specific substrates such as peptides or sugars. An overview of enzymes overexpressed by tumors and examples for respective substrates is given in table 3. Lysosomal degradation of proteins Arg-Arg, Leu, Ala-Leu, Gly-Leu-Phe-Gly, Gly-Phe-Leu-Gly,Ala-Leu-Ala-Leu Cathepsin D (EC 3.

Degradation of extracellular matrix and collagens

Ac-Pro-Leu-Gly-Leu, Ac-γE-Pro-Cit-Gly-Hof-Tyr-Leu, Gly-Pro-Leu-Gly-lle-Ala-Gly-GIn β-Gluconuronidase (EC3.

Drug release by chemical activation

The various chemical activation processes exploited over the last few decades are based on the reactivity of certain functional groups in acidic pH (≈ 6.5-6.8) [START_REF] Neri | Interfering with PH Regulation in Tumours as a Therapeutic Strategy[END_REF] , reducing environment (hypoxia) or oxidizing environment (ROS) characterizing cancer cells/tumors. Chemically activatable prodrugs must remain stable and inert under the physiological conditions encountered in healthy tissues.

pH sensitive

Acidic extracellular pH is a major feature of tumor tissue; extracellular acidification is considered to occur in the presence of lactic acid, which accumulates due to anaerobic glycolysis in hypoxic cancer cells; pentose phosphate pathway produces CO2, which is then oxidized by the enzyme carbonic anhydrase (CA) leading to the increase of the proton A pH-sensitive prodrug of doxorubicin was prepared by Chen et al. [START_REF] Chen | PolyMPC-Doxorubicin Prodrugs[END_REF] The ability of the prodrug to release the cytotoxic in a weakly acidic medium was confirmed. Using in vitro cytotoxicity studies, it was shown that the pH-sensitive doxorubicin prodrug was able to accumulate in cancer tissues, therefore reducing nonspecific cytotoxicity associated with systemic administration of the free doxorubicin (scheme 3). [START_REF] Chen | PolyMPC-Doxorubicin Prodrugs[END_REF] Scheme 3. pH-Sensitive prodrug of doxorubicin.

Reducing environment (hypoxia sensitive prodrug)

Hypoxia corresponds to the situation of low concentration of oxygen in tissue in comparison with appropriate oxygen supply. [START_REF] Al Tameemi | Hypoxia-Modified Cancer Cell Metabolism[END_REF] In solid tumors, hypoxia is essentially observed in the center of a tumor mass (100 -150 μm) and gradually decrease with the distance to the oxygen supplying blood vessels (figure 14). This lack of oxygen resulting from reduced blood supply to the surrounding tissue is a common characteristic of solid tumors because of rapid cell growth. Hypoxia in tumors is a predictor of both resistance to chemotherapy and of a metastatic/aggressive form of cancer, and as a result, development of cancer therapies which target hypoxia is of great importance. The strategy of hypoxia-activated prodrugs (HAPs) was then developed for the release of chemotherapeutic agents within hypoxic tumor regions. This targeting strategy is accomplished by attaching a hypoxia activated trigger (quinones, Noxides, nitro-substituted aromatics and derivatives with a disulfide bridge) to a chemotherapeutic agent. Jin et al. [START_REF] Jin | Synthesis and Biological Evaluation of Hypoxia-Activated Prodrugs of SN-38[END_REF] have prepared a hypoxia-activated prodrug of SN-38 by conjugating a 2-nitroimidazol nucleus with SN-38 (scheme 4). This prodrug was designed to be activated by nitro reduction under hypoxia condition to release the drug in the tumor.

Scheme 4.

Hypoxia-activated prodrug (HAP) of SN-38.

Oxidizing environment (ROS sensitive prodrug)

Biological studies of tumors have revealed that cancer cells have a higher degree of oxidative stress compared to healthy cells. [START_REF] Liou | Reactive Oxygen Species in Cancer[END_REF] This stress is produced by deregulated and enhanced mitochondrial functions and cellular metabolism. Oxidative stress stimulates the tumor progression processes such as cell proliferation and angiogenesis. Cancer cells migration and neovascularization will ultimately lead to the dissemination and growth of cancer cells in metastatic sites. Macrophages have been described to promote the generation of oxidative stress of cancer cells through the secretion of molecular mediators. [START_REF] Tan | The Reactive Oxygen Species in Macrophage Polarization: Reflecting Its Dual Role in Progression and Treatment of Human Diseases[END_REF] These immune cells are primarily present around the tumor cells in order to eradicate them by both phagocytosis and production of oxygen-containing reactive molecules. Nonetheless, they will indirectly contribute to cancer progression. [START_REF] Qian | Macrophage Diversity Enhances Tumor Progression and Metastasis[END_REF] These cellular events provide an increase of the oxygenated reactive molecules level in the tumor microenvironment.

These highly reactive molecules are composed of reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS). [START_REF] Liou | Reactive Oxygen Species in Cancer[END_REF] Among ROS, superoxide anion (O2• -) is engendered from O2 via NADPH oxidase action and mitochondrial metabolism. O2• -is subsequently converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) catalysis. H2O2 further evolves into hypochlorous acid (HOCl) and hydroxyl radical (HO•). Among RNOS, nitric oxide (NO•) is produced by reaction of molecular oxygen and L-arginine catalyzed by nitric oxide synthases (NOSs). NO• spontaneously reacts with O2• -to form peroxynitrite/peroxinitrous acid (ONOO - /ONOOH). The latter species is unstable and generate hydroxyl radical and nitrogen dioxide radical (NO2•).

Boronic acid (and ester) prodrugs targeting the overexpressed level of reactive oxygen species within tumor microenvironment represent a promising area for the discovery of new selective anticancer chemotherapy. This strategy that emerged only ten years ago is exponentially growing and could demonstrate its clinical usefulness in the near future. Herein, the previously described small-molecule and macromolecular anticancer prodrugs activated by carbon-boron oxidation are gathered. This chapter reports on the most interesting derivatives mentioned in the literature based on the in vitro and in vivo activity when available. Eventually, the pharmacological applicability of this strategy is discussed, in particular, the kinetic aspect of the prodrug oxidation and the selectivity of this reaction towards certain ROS from the tumor microenvironment are specified.

Boronic acid /boronate prodrugs

Boronic acids and their corresponding esters have been developed as temporary masking groups for drugs or probes specifically triggered by oxygen-containing reactive molecules. [START_REF] Peng | ROS-Activated Anticancer Prodrugs: A New Strategy for Tumor-Specific Damage[END_REF][START_REF] Peiró Cadahía | Prodrug Strategies for Targeted Therapy Triggered by Reactive Oxygen Species[END_REF][START_REF] Ye | Recent Advances on Reactive Oxygen Species-Responsive Delivery and Diagnosis System[END_REF][START_REF] Antonio | Boronic Acids as Building Blocks for the Construction of Therapeutically Useful Bioconjugates[END_REF][START_REF] Andina | Ratiometric Fluorescent Probes for the Detection of Reactive Oxygen Species[END_REF] In biological aqueous media, boronate esters are rapidly hydrolyzed into boronic acid. [START_REF] Cao | Substituent Effects on Oxidation-Induced Formation of Quinone Methides from Arylboronic Ester Precursors[END_REF] Among the different reactive species, hydrogen peroxide, hypochlorite anion (ClO•) and peroxynitrite were shown to react with boronic acids and esters to further oxidize the carbon-boron bond to form an alcohol group. [START_REF] Zielonka | Boronate Probes as Diagnostic Tools for Real Time Monitoring of Peroxynitrite and Hydroperoxides[END_REF] In biological conditions, hypochlorite anion will almost exclusively react with cellular amines and thiols since the latter reactions will be faster than the oxidation of carbon-boron bonds. Nucleophilic oxygen of the reactive species will be able to attack the empty p orbital of the boron atom (scheme 5). [START_REF] Sikora | Direct Oxidation of Boronates by Peroxynitrite: Mechanism and Implications in Fluorescence Imaging of Peroxynitrite[END_REF][START_REF] Sikora | Reaction between Peroxynitrite and Boronates: EPR Spin-Trapping, HPLC Analyses, and Quantum Mechanical Study of the Free Radical Pathway[END_REF] Carbon migration to oxygen further gives a borate that will be hydrolyzed to afford the corresponding alcohol and non-toxic boric acid. [START_REF] Baker | Therapeutic Potential of Boron-Containing Compounds[END_REF] At physiological pH, conversion by peroxinitrite is 10 6 times faster than the hydrogen peroxidemediated oxidation likely due to the anionic form of peroxinitrite in comparison to neutral H2O2. [START_REF] Zielonka | Boronate Probes as Diagnostic Tools for Real Time Monitoring of Peroxynitrite and Hydroperoxides[END_REF] Scheme 5. Mechanism of boronic acid oxidation into alcohol by either hydrogen peroxyde or peroxynitrite.

The boronic/boronates functionalities were used as temporary moieties to either mask aliphatic alcohols, 70,74 enols [START_REF] Mosey | Versatile Approach to α-Alkoxy Carbamate Synthesis and Stimulus-Responsive Alcohol Release[END_REF][START_REF] Brooks | Synthesis, and Characterization of Small-Molecule Reagents That Cooperatively Provide Dual Readouts for Triaging and, When Necessary, Quantifying Point-of-Need Enzyme Assays[END_REF] or aryl-alcohols. [START_REF] Peng | ROS-Activated Anticancer Prodrugs: A New Strategy for Tumor-Specific Damage[END_REF][START_REF] Andina | Ratiometric Fluorescent Probes for the Detection of Reactive Oxygen Species[END_REF] The active effector can be connected to the boronic/boronates groups directly or via self-immolative spacers. [START_REF] Alouane | Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications[END_REF][START_REF] Gisbert-Garzarán | Self-Immolative Chemistry in Nanomedicine[END_REF] The use of these spacers allows the bounding of different effectors through either their alcohol or amine functions generating ether, carbonate or carbamate bonds. The volume added by the spacer to the final entity contributes to a better masking of the biological activity. Oxidation of such spacer lead to the formation of an aryl-alcohol motif that will undergo spontaneous elimination leading to the release of an effector (scheme 6). Scheme 6. Release of the active drug from boron-containing prodrug by either direct C-B bond oxidation or by oxidation and subsequent self-immolative process.

The strategy of boronic acid/boronate oxidation has been widely used for the emergence of many probes for tumor imaging and/or prodrugs with tumor targeting properties leading to the release of the parent drug. Herein, we comprehensively review the ROS responsive boronate prodrugs that were developed and evaluated as anticancer agents. In the next section, we gathered the different platforms used to construct the prodrug of interest: a majority of small molecules were found but a non-negligible amount of drug delivery-based polymeric matrix and nanoparticles were also described.

Small-molecule and macromolecular prodrugs

Precursors of selective estrogen receptor modulators (SERM)

The selective estrogen receptor modulators have been developed to treat breast cancer tumors presenting the estrogen receptor. [START_REF] Patel | Selective Estrogen Receptor Modulators (SERMs) and Selective Estrogen Receptor Degraders (SERDs) in Cancer Treatment[END_REF] Antiestrogens, such as tamoxifen 1, bind to the hormone receptor and block the proliferation of the cancer cells. Moreover, tamoxifen 1 is a prodrug converted to both 4-hydroxytamoxifen 2 and endoxifen 4 by cytochrome P450

enzymes. The Wang group has therefore synthesized and evaluated boron-based prodrugs of 4-hydroxytamoxifen 2 and endoxifen 4 as potential anticancer agents (scheme 7). [START_REF] Jiang | Boron-Based 4-Hydroxytamoxifen Bioisosteres for Treatment of de Novo Tamoxifen Resistant Breast Cancer[END_REF][START_REF] Zhang | Boronic Prodrug of Endoxifen as an Effective Hormone Therapy for Breast Cancer[END_REF][START_REF] Zhong | Boronic Prodrug of 4-Hydroxytamoxifen Is More Efficacious than Tamoxifen with Enhanced Bioavailability Independent of CYP2D6 Status[END_REF] Scheme 7. Structures of 4-hydroxytamoxifen and endoxifen (oxidative metabolites of tamoxifen) and theirs prodrugs.

This strategy is indeed meaningful since these two active metabolites would be similarly obtained by oxidative cleavage of the boronic acid function of 3 and 5 in the tumor environment.

In vitro cytotoxic activity of 3 and 5 was determined against MCF-7 and T47D, two breast cancer cell lines expressing the estrogen receptor. The prodrug 3 and its active metabolite 4hydroxytamoxifen 2 were as active against a given cell line. 80% conversion of prodrug 3 to 4hydroxitamoxifen 2 was observed in cell culture media and the cellular uptake of 3 was 4 times higher than 4-hydroxytamoxifen 2 in MCF-7 culture. Comparable in vitro observations were made for the prodrug/drug couple: 5/endoxifen 4. Growth inhibition of MCF-7 cancer xenografts in mice was similar for the prodrug 5 and the active metabolite endoxifen 4 at the optimal dose of 1 mg/kg. Metabolism and pharmacokinetic studies in mice showed that the plasma concentration of boronic prodrugs 3 and 5 were 40-fold higher than their respective active ingredients or the other parent prodrug tamoxifen 1 administrated at the same dose.

Noteworthy, the conversion of both prodrugs was due to the oxidation of the boronic acid functions by cytochrome P450 enzyme rather than the ROS/RNOS overexpressed by cancer cells. The enhanced bioavailability from the prodrugs 3 and 5 is therefore due to the prolonged release of the phenol counterparts. Hence, rapid excretion of 4-hydroxytamoxifen 2 and endoxifen 4 is observed when these molecules are directly injected and this was attributed to the glucuronidation of the hydroxy groups during phase II metabolism.

Precursor of epigenetic regulators

Histone deacetylase (HDAC) inhibitors

Histone deacetylases (HDACs) remove acetyl groups from histone and other nonhistone targets. HDACs control the transcription of oncogenes leading to the production of proteins involved in cancer induction and proliferation and HDAC inhibitors were developed to prevent the progression of cancer cells. [START_REF] Manal | Inhibitors of Histone Deacetylase as Antitumor Agents: A Critical Review[END_REF] Interestingly, two hydroxamate molecules: vorinostat 6 and belinostat 8, FDA-approved drugs for the treatment of cutaneous T-cell lymphoma, were the basis for the preparation of prodrugs (scheme 8). [START_REF] Liao | H(2)O(2)/Peroxynitrite-Activated Hydroxamic Acid HDAC Inhibitor Prodrugs Show Antileukemic Activities against AML Cells[END_REF][START_REF] Zheng | Biocompatible Boron-Containing Prodrugs of Belinostat for the Potential Treatment of Solid Tumors[END_REF] Indeed, these pharmaceuticals are good candidates for prodrug development since they present important drawbacks such as toxicity and weak bioavailability due to both the lack of hydroxamic acid stability and to rapid excretion by sulfation and glucuronidation. Liao et al. [START_REF] Liao | H(2)O(2)/Peroxynitrite-Activated Hydroxamic Acid HDAC Inhibitor Prodrugs Show Antileukemic Activities against AML Cells[END_REF] proposed the prodrug 7 of vorinostat 6 targeting the acute myeloid leukemia owing to the high level of ROS produced by these tumor cells. [START_REF] Hole | Overproduction of NOX-Derived ROS in AML Promotes Proliferation and Is Associated with Defective Oxidative Stress Signaling[END_REF] Scheme 8. Prodrugs of vorinostat and belinostat.

Here, the drug was linked to a self-immolative benzene boronic acid moiety to afford the prodrug 7. The viability of two leukemia cell lines, U937 and MV4-11, treated by 7 and vorinostat 6 was determined in vitro and showed a partial recovery (~ 20%) of the drug activity after incubation.

Furthermore, Wang and colleagues prepared a prodrug 9 made of a benzene boronate nucleus branched to the hydroxamic acid moiety of belinostat 8 for the chemotherapy of solid tumors. [START_REF] Zheng | Biocompatible Boron-Containing Prodrugs of Belinostat for the Potential Treatment of Solid Tumors[END_REF] Belinostat 8 and 9 were tested in vitro against four cancer cell lines: A549 (lung carcinoma), HeLa (cervical cancer) and, MDA-MB-231 and MCF-7 (breast adenocarcinoma). Again, around 25% of the parent drug activity was recovered after incubation except for the MCF-7 cells where prodrug 9 displayed only 5% of the corresponding drug activity. Conversion of the prodrug 9 into belinostat 8 in MDA-MB-231 culture cell media was ~ 10% after 24h of incubation. The HDAC inhibitory activity of the prodrug 9 was then measured and was ten times lower than that of belinostat 8. Altogether, these observations account for the noticeable difference of cancer cells viability between the drug and the prodrug. In vivo activity of prodrug 9 was compared to belinostat 8 in a MCF-7 xenograft model in mice at the dose of 10 mg/kg/day, a slight increase of efficacy was observed for 9 toward the free drug. The belinostat 8 concentration was twice higher in tumor tissue after prodrug 9 subcutaneous injection in contrast with the same injected dose of the parent drug. The higher efficacy of the prodrug is explained by the increased bioavailability of the benzene boronate precursor 9. The plasmatic concentration in mice was quantified for both belinostat 8 and its prodrug 9: 10 mg/kg were independently injected and after 3h, the concentration of belinostat 8 was 7-fold higher when using the prodrug 9. [START_REF] Zhang | Metabolism and Pharmacokinetic Study of the Boron-Containing Prodrug of Belinostat (ZL277), a Pan HDAC Inhibitor with Enhanced Bioavailability[END_REF] Moreover, study of 9 metabolism in liver S9 fraction clearly brought to light the role of the enzymatic metabolism (via cytochrome P450) in the conversion of the boronic acid function into the corresponding phenol leading to belinostat 8 through selfimmolation.

Lysine-specific histone demethylase inhibitor

Among histone post-translational modifications, lysine methylation of histone governs the structure of chromatin and the methylation degree of histones is involved in cancer formation. [START_REF] Varier | Histone Lysine Methylation and Demethylation Pathways in Cancer[END_REF] Lysine-specific histone demethylase inhibitor are not currently used in clinic but these molecules are studied for the development of anticancer epigenetic drugs. Engel et al. [START_REF] Engel | Novel Dual-Action Prodrug Triggers Apoptosis in Glioblastoma Cells by Releasing a Glutathione Quencher and Lysine-Specific Histone Demethylase 1A Inhibitor[END_REF] have prepared a benzene boronate prodrug 11 of the promising histone demethylase inhibitor trans-2-phenylcyclopropylamine 10 (scheme 9).

hydroxybenzyl adduct of crizotinib was as active as the free drug against all the tested cell lines pointing out that benzyl moiety is not sufficient to mask the drug activity in this case.

Precursors of antimetabolite drugs

Methotrexate (MTX) 15, a potent inhibitor of the dihydrofolate reductase implied in the purine and pyrimidine synthesis, is used in clinic to treat cancer and autoimmune diseases. The Clausen group has described several benzene boronate prodrugs of methotrexate 15 originally developed for the treatment of rheumatoid arthritis (Scheme 11). [START_REF] Peiró Cadahía | Synthesis and Evaluation of Hydrogen Peroxide Sensitive Prodrugs of Methotrexate and Aminopterin for the Treatment of Rheumatoid[END_REF][START_REF] Previtali | Auxiliary in Vitro and in Vivo Biological Evaluation of Hydrogen Peroxide Sensitive Prodrugs of Methotrexate and Aminopterin for the Treatment of Rheumatoid Arthritis[END_REF] Scheme 11. Prodrugs of MTX, 5-FU and gemcitabin.

Recently, the in vitro activity of the prodrug 16 (scheme 11) was reported on HL-60 (human leukemia cancer cell line), HeLa cancer cell line and HEK293 (human embryonic kidney cell line). [START_REF] Bildstein | Prodrug-Based Intracellular Delivery of Anticancer Agents[END_REF] The HL-60 cells were shown to produce around ten times more ROS in vitro than of the HEK293 cells. However, prodrug 16 displayed close in vitro activity against the two latter cell lines (IC50 ~ 0.05 µM), a growth inhibition decreased by a factor 2-4 in comparison with the free methotrexate 15.

The antimetabolite 5-fluorouracil (5-FU) 17, a widely used antitumor drug, presents several side effects such as myelosuppression and neurotoxicity. In order to reduce these drawbacks, the Xue group has developed the benzene boronate prodrug of 5-fluorouracil (scheme 11). [START_REF] Ai | Enhanced Tumor Selectivity of 5-Fluorouracil Using a Reactive Oxygen Species-Activated Prodrug Approach[END_REF] Boronate prodrug 18 and its corresponding boronic acid were tested in vitro against the 60 cancer cell lines from the National Cancer Institute. Prodrug 18 was more active against all cell lines than its boronic acid counterpart which is quite surprising since boronate esters are hydrolyzed into boronic acid in aqueous media (vide supra). Then, the authors particularly compared breast cancer cells MCF-7 to immortalized mouse embryonic epicardial cells MEC1.

5-Fluorouracil 17 was cytotoxic against two cell lines with an IC50 around 1 µM whereas 18 had no effect against MEC1 up to a concentration of 100 µM and was slightly active on the MCF-7 cancer cells (IC50 = 10-50 µM). Aside from that, prodrug 18 showed a better drug safety profile than 5-fluorouracil 17 on wild-type C57BL/6 male mice.

Another nucleotide analog used in clinic, gemcitabin 19, was linked to a benzene boronate moiety to build the prodrug 20 (scheme 11). [START_REF] Matsushita | A Hydrogen Peroxide Prodrug for the Selective Treatment of Pancreatic Ductal Adenocarcinoma[END_REF] Gemcitabin 19 and prodrug 20 were evaluated in vitro against pancreatic cell lines: cancerous (PSN1 and BxPC3) and normal (NPEC). The H2O2 level produced by each cell line was measured using the ROS-Glo™ assay which demonstrated a weakly higher production of ROS by the cancer cells. Gemcitabin 19 displayed an IC50 of 0.005-0.015 µM against the three cell lines and prodrug 20 was around 20 times less active in each case. Nonetheless, prodrug 20 was able to retrieve the activity of gemcitabin 19 when ROS were artificially produced by the cancer cell lines via preliminary addition of 2deoxy-D-glucose. In vivo activity was determined using PSN1 xenograft model in mice and showed a similar activity of both gemcitabin 19 and prodrug 20. However, myelosuppression was lowered in mice treated with the prodrug 20 and the authors showed that concentration of the resulting gemcitabin 19 in bone marrow was reduced in comparison with the gemcitabintreated group.

Precursors of cytotoxic drugs

Campthotecin 21

Campthotecin 21 is a strongly cytotoxic natural product as a result of its topoisomerase I inhibitory activity. The 10-hydroxycampthotecin known as SN-38 22, is the toxic hydrolytic metabolite of the commercialized prodrug irinotecan 23. [START_REF] Rothenberg | Irinotecan (CPT-11): Recent Developments and Future Directions--Colorectal Cancer and Beyond[END_REF] Thus, Lei et al. [START_REF] Wang | 10-Boronic Acid Substituted Camptothecin as Prodrug of SN-38[END_REF] The fluorescence appeared only one minute after prodrug injection and the cyanine dye was still observable 6 hours after administration to the mice.

Doxorubicin 27

Doxorubicin 27 is a cytotoxic natural product acting as DNA intercalating agent and topoisomerase II inhibitor (scheme 13). Although used in clinic as antitumor drug, important side effects such as cardiotoxicity were described. [START_REF] Octavia | Doxorubicin-Induced Cardiomyopathy: From Molecular Mechanisms to Therapeutic Strategies[END_REF] Our group therefore prepared arylboronate-based prodrugs of doxorubicin by branching the free amine function of this drug. 100

Scheme 13. Prodrugs of doxorubicin. A theranostic prodrug 29 of doxorubicin was built based on a central benzenic self-immolative spacer able to release simultaneously the free drug and a fluorophore, a tetraphenylethene with aggregation-induced emission properties (scheme 13). 102 The construct 29 was evaluated in vitro on Hela cells that were first treated with 1-phorbol-12-myristate-13-acetate (PMA), an agent inducing the ROS production in cells. After incubation, fluorescence and cytotoxicity (IC50 ~ 2 µM) were observed in PMA-treated cells whereas no effect was detected on the PMAfree cells.

Organo-silica nanoparticles 30 displaying phenyl boronic moiety at their surface were prepared by the Shi group. 103 Doxorubicin was then covalently linked via its cis diol part to generate a boronate ester bond leading to nanoparticles covered with the drug (Scheme 19). These nanoparticles were designed to specifically release the doxorubicin within a tumor by ROSmediated oxidation or by acidic cleavage of the boronate ester bond (due to particularly acidic pH in cancer cells). Hyaluronic acid was also coated at the surface of the nanoparticles in order to specifically target the CD44 receptor which is overexpressed at the surface of certain cancer cells. Efficient in vitro release of doxorubicin 27 was first demonstrated in presence of H2O2 and then the particles were evaluated against Hep G2 cancer cells (CD44+++) and NIH/3T3 fibroblasts (CD44+). Nanoparticles 30 bearing hyaluronic acid and doxorubicin were: (i) more toxic against Hep G2 (IC50 ~ 0.5 µM) than their hyaluronic acid free counterparts (IC50 = 2 µM) and (ii) more active against Hep G2 than NIH/3T3 (IC50 ~ 2 µM).

Paclitaxel 31

Paclitaxel is an antitumor drug inhibiting microtubules depolymerization therefore blocking the mechanism of mitosis. 104 This form of the drug presently used suffers from several side effects and a micellar prodrug 32 built from conjugates "polyethylene glycol-benzene boronatepaclitaxel (scheme 14) was developed by Dong et al. 105 Scheme 14. Prodrug of paclitaxel.

Prodrug 32 was evaluated in vitro against breast cancer MCF-7 and U251 human glioma cells, the latter cell being less sensible to paclitaxel. Interestingly, prodrug 32 was three times more active than paclitaxel 31 against U251 cells when pre-incubation with PMA, the ROS inductor, was applied. A non-activatable analog of micellar prodrug 32 devoid of the boron trigger was also tested and showed significant cytotoxicity decreased by only a factor 5 in comparison with free paclitaxel 31. This highlights the uncertain ability of the temporary promoiety, although very bulky, to clearly impair the paclitaxel activity. In vivo, a similar trend was observed on a MCF-7 xenograft tumor model in mice. Micelles 32 displayed the best activity with almost 90% of tumor inhibition attributable to its advantageous pharmacokinetics compared to paclitaxel 31 (~ 70% inhibition). Noticeably, the non-activatable prodrug was almost as active as paclitaxel 31 (~ 60% inhibition).

Precursor of nitric oxide

In cells, nitric oxide is an endogenous signaling molecule involved in several metabolic pathways. High concentration of nitric oxide usually lead to elevated formation of cytostatic peroxinitrite (vide infra). The Chakrapani group has prepared a theranostic precursor 33 of nitric oxide based on a self-immolative coumarin core linked to a nitrosohydroxylamine (scheme 15). 106 The release of the latter moiety will spontaneously generate two molecules of nitric oxide.

Scheme 15. Theranostic prodrug of nitric oxide.

Theranostic prodrug 33 was evaluated in vitro against MRC5 fibroblasts and three cancer cell lines: A549, HeLa, MDA-MB-231. Incubation with the cancer cells in particular with A549, showed greater conversion of 33 into turned-on coumarin and nitric oxide using fluorescence microscopy experiment. A similar activity was observed against all three cell lines (IC50 ~ 10 µM) and a lower effect was noted on the MRC5 cells (IC50 ~ 25 µM).

Precursors of DNA alkylating agents

Nitrogen mustards

Nitrogen mustards are cytotoxic drugs reacting with DNA to form interstrand cross-links (ICLs).

The Peng group designed and synthesized benzene boronate prodrugs of chlormethine 34 since this drug is weakly selective of cancer cells (scheme 16). 107

Scheme 16. Prodrugs of chlormethine.

When linked to benzene boronate, chlormethine is not able to form the electrophilic aziridinium cycle strongly reacting with DNA. In vitro, prodrug 35 showed efficient ability to alkylate DNA in presence of H2O2 and 35 was further evaluated against four cancer cells: leukemia cells SR, lung cancer cells NCI-H460, renal cancer cells SN12C and CAKI-1, and healthy lymphocytes.

Prodrug 35 (with no added H2O2) was especially active against SR and NCI-H460 cells with 85-90% of growth inhibition at 10 µM while no activity was detected at this concentration on the normal lymphocytes. Rationally, the same activity for 35 and its corresponding free boronic acid was found in vitro against all cell lines. This benzene boronate prodrug of chlormethine

Quinone methides as alkylating agents

The Peng group also envisioned the generation of quinone methides as Michael acceptor for DNA strand (scheme 18). 112-114 Among several benzene boronate precursors, prodrug 41 was the most interesting of their series.

Scheme 18. Prodrug of quinone methide as Michael acceptor.

Prodrug 41 was able to form ICLs in presence of H2O2 and its cytotoxicity was evaluated against the NCI cancer cells panel. IC50s of 41 against several cancer cells was about 10-20 µM which was significantly more active than chlorambucil and melphalan.

Precursors of ROS/RNOS amplifier/accumulator for oxidation therapy

Aminoferrocene

Over the last decade, the group of Mokhir has developed benzene boronate precursors of aminoferrocene. [115][116][117][118][119][120][121][122][123][124] These precursor were conceived to increase drastically ROS concentrations inside cancer cells leading ultimately to cell death, i.e oxidation therapy. 125 The released aminoferrocene 42 reacts with hydrogen peroxide or oxygen to afford both cytotoxic hydroxyl radical and ferrocenium ion 43 (scheme 19). These ROS are usually detoxified by antioxidants such as glutathione but in this strategy, electrophilic quinone methide is also formed and acts as glutathione scavenger blocking this protective mechanism.

Scheme 19.

Cycle for ROS amplification within tumor environment initiated by the presence of free aminoferrocene.

From a first series of aminoferrocene prodrugs, 115 the authors found that the prodrug 44 (Scheme 20) was the most efficient at both penetrating and amplifiying ROS within HL-60 leukemia cells. This is attributable to the greater stability of the liberated Nbenzylaminoferrocene and 44 was indeed the most cytotoxic prodrug against HL-60 (IC50 = 9 μM) whereas no activity was found against healthy fibroblasts (IC50 > 100 μM).

Scheme 20. Aminoferrocene prodrugs.

The authors further showed that the cytotoxicity was decreased when the ferrocene part was partly constituted of a cyclopentadiene carboxylic acid moiety. 116 Modification of the N-benzyl group by either introducing ortho-or para-substitutent or replacement of the benzene ring by a pyridine nucleus was also counterproductive to the cytotoxicity on HL-60 cells. The corresponding free boronic acid of 46 was injected in mice bearing two tumors: PC3

(human prostate cancer) and AR42J (rat pancreatic cancer). PET analysis showed a moderate localization of the tracer in both tumors but the measured radioactivity remain relatively stable in the tumors for 60 minutes in comparison with the healthy organs of the mice.

An organic moiety able to target the lysosome was alternatively added on the benzene boronate precursors of aminoferrocene. 121 The piperidine prodrug 47 (scheme 20) was prepared in order to obtain an ammonium form at physiological pH leading to lysosome trapping and accumulation. Biophysical properties of 47 were compared to the reference prodrug 44 and were shown significantly improved. In particular, the hydroxyl radical formation was more efficient from prodrug 47 and this was transcribed with the higher in vitro cytotoxicity observed for 47 than for 44 against several cancer cell lines (6 to 8 folds more active). The Scheme 21. Bis "aminoferrocene-platinum" prodrugs.

The bis "aminoferrocene-cisplatin" prodrug 51 was shown to effectively release cisplatin 49 after addition of H2O2, indeed, the free aminoferrocenes generated are able to transfer one electron that both reduces the metal complex into cisplatin and forms the activated ferrocenium. 51 was evaluated against normal fibroblasts HDFa, ovarian carcinoma cell line A2780 and the cisplatin resistant counterpart A2780cis cell line. Prodrug 51 was not toxic against the healthy fibroblasts while activity of free cisplatin 49 was fully recovered when A2780 cells were treated with 51 (IC50 ~ 2 µM). Interestingly, the resistant A2780cis cells behaved differently: activity of prodrug 51 (IC50 = 6 µM) was twice greater than cisplatin 49 (IC50 = 13 µM). The authors revealed that prodrug 51 was not generating an aminoferrocene which strongly produces ROS but the activity of prodrug 51 was mostly due to the release of cisplatin 50 rather than the generally modest activity provided by ferrocenium. The oxaliplatin prodrug 52 was built with the same scaffold used for cisplatin prodrug 51 (Scheme 21). 123 In construct 52, the spacer between the ferrocene and the platinum-based drug was truncated (C4 in lieu of C5) in order to facilitate the intramolecular electron transfer within these two moieties.

Prodrug 52 was indeed more active than 51 against both ovarian cancer cell lines in vitro : IC50 (A2780) = 0.4 µM and IC50 (A2780cis) = 0.7 µM. Analog 52 followed the trend described for prodrug 51 in view of the stronger activity (over free cisplatin 49) displayed by the free drug oxaliplatin 50 : IC50 (A2780) = 0.9 µM and IC50 (A2780cis) = 3 µM. The authors further synthesized a mono-"carboplatin-aminoferrocene" prodrug 48 (scheme 20). 124 Accumulation of 48 in the mitochondria of A2780 cancer cells was demonstrated using the mitochondrial tracker rhodamine R123 and it was assumed that this uptake was due to the release of a delocalized lipophilic cation: the generated ferrocenium. In vitro activity of 48 was then determined against the three aforementioned cell lines and adopted the tendency observed for cisplatin prodrug 51 but to a weaker extent.

Diethyldithiocarbamate 54

Diethyldithiocarbamate 54 is a metabolite of disulfiram 56, a drug used in clinic to treat chronic alcoholism and recently, copper complex of diethyldithiocarbamate 53 was shown to have anticancer activity. 126 The formation of this complex 53 generates ROS in the cancer cells and the Pu group prepared a prodrug 55 capitalizing on the ROS amplification due to both formation of the latter complex and a quinone methide (Scheme 22). 127

Scheme 22. Structures of disulfiram and the prodrug of diethyldithiocarbamate.

Prodrug 55 was tested against 4T1 breast cancer cells in vitro : In the presence of the nontoxic concentration of copper (1 µM), activity was six times more potent on cancer cells (IC50 = 1 µM) than on normal fibroblasts NIH3T3 (IC50 = 6 µM). This difference of activity could be explained by the high ROS production of 4T1 which was essentially due to the diethyldithiocarbamate copper complex formation and minimally as a result of quinone methide generation. In vivo testing was made on a 4T1 xenograft mice and the most potent result was obtained using a co-injection of copper gluconate at 1.5 mg/kg with 55 at 37 mg/kg. Applying this protocol, a decrease of 80% of the tumors volume was observed in comparison to the inactive vehicle or copper gluconate alone.

Cinnamaldehyde 57

Cinnamaldehyde 57 is a natural product found in cinnamon essential oil and used as food flavoring. This aldehyde was shown to amplify ROS in cancer cells but this action is very limited on healthy cells. Poor bioavailability of cinnamaldehyde 57 has prevented its application in clinic and therefore this compound was a good candidate for prodrug development. Thus, the Lee group has prepared a benzene boronate prodrug of cinnamaldehyde 58 (scheme 23). 128

Glucose oxidase

The glucose oxidase enzyme catalyze the reaction of glucose and oxygen into gluconolactone and hydrogen peroxide. This enzyme can therefore generate ROS from two compounds naturally abundant in cells. To capitalize on this, Li et al. 129 have prepared nanocapsules 59 able to entrap glucose oxidase (scheme 24).

Scheme 24. Nanocapsules loaded with glucose oxidase for the amplification of ROS within the tumor microenvironment.

The shell of the capsule was made of a diblock copolymer of PEG and polymethacrylate grafted with benzene boronate and piperidine moieties. Protonation of piperidine in the acidic tumor environment increases the permeability of the nanocapsule and allows the entry of glucose which lead to the strong production of H2O2. This enhanced level of hydrogen peroxide further reinforces the oxidation of the benzene boronate part into glutathione scavenger quinone methide. The authors verified that this dual acting nanoconstruct was operational in vitro by measuring H2O2 production and quinone methide formation. Mice bearing lung carcinoma A549 were treated with nanocapsule 59 and showed complete inhibition of tumor growth in comparison with either glucose oxidase or empty nanocapsule alone.

Pharmacological relevance

In this section, the strategy of boron-containing prodrugs responsive to oxidative stress is discussed in regards to its potential to reach the market as effective cancer treatment. Indeed, boronic acid/boronate prodrug have been largely evaluated in animal models but no clinical trials have been started with any of these molecules so far.

Nonetheless, the potential of these constructs is strong considering that several drugs containing a boronic acid function, are already on the market. [START_REF] Baker | Therapeutic Potential of Boron-Containing Compounds[END_REF]130 Moreover, the development of boron-containing classical drug remains a dynamic area of research. 131 In terms of drug delivery, the boronic acid function is particularly relevant since it allows facilitated internalization in cancer cells. 132,133 The reversible formation of ester bonds between boronic acid and the diol of saccharides ensure this membrane crossing. Precisely, high affinity between boronic acids and the glycocalix of cancer cells lead to the selective uptake of the molecules bearing this moiety.

Furthermore, to improve the strategy of the small-molecule prodrugs, the kinetic of prodrug conversion must be considered. At the tumor site, activation of boron-containing prodrugs by ROS can either occur extra-or intracellularly. As the system is not closed, the liberated drug will then freely diffuse around the tumor (~10 -3 mm 3 ; a mass of around 1,000 cancer cells) and that will influence the concentration profile of the drug. Scission of the prodrugs should arise locally to preserve an efficient dose of the liberated drugs within the tumor. In view of the diffusion coefficient of small molecules (10 -10 m 2 .s -1 ) and considering the aforementioned tumor volume, oxidation of precursors into drugs should last approximately 2 minutes in order to maintain the concentration of the free drugs equal to that of the corresponding prodrugs. When the prodrug conversion implies an additional self-immolative step, the complete process must not exceed the latter duration.

The reaction of boron-containing prodrug with the ROS species is bimolecular and the rate will therefore depend on the concentration of both reactants:

v = k[prodrug][ROS].
The concentration of the prodrug is mostly guided by the dose injected to the biologic system but levels of peroxinitrite and hydrogen peroxide is dictated by the living organism. Specific activation in the tumor microenvironment can occur since the concentrations of peroxinitrite and hydrogen peroxide are strongly increased in comparison with healthy tissues. Cellular concentration of peroxinitrite can reach 50-100 µM in pathological conditions whereas a basal concentration of 0.1-1 µM was estimated in healthy cells. 134,135 Physiological hydrogen peroxide is produced at a steady-state concentration of 0.1-1 µM which rises to 10-100 µM in malignant organs. 136,137 In this activation strategy, two parameters must be taken into account: the half-life of each ROS in vivo and the rate constant associated to a ROS and a particular prodrug structure.

As mentioned earlier, at physiological pH, oxidation of the carbon-boron bond by peroxinitrite is 10 6 faster than that obtained with hydrogen peroxide: e.g. conversion of phenylboronic acid to phenol occurred with rate constant of 1 x 10 6 M -1 .s -1 with peroxinitrite and approximatively 1-2 M -1 .s -1 with hydrogen peroxide. [START_REF] Research | FDA Approves Gemtuzumab Ozogamicin for CD33-Positive AML in Pediatric Patients[END_REF] In vivo, peroxinitrite has a half-life of ~ 10 ms and reacts preponderantly but reversibly with carbon dioxide (1-2 mM) to form nitrosoperoxocarbonate anion (ONOOCO2 -) at a rate constant of 0.5 x 10 5 M -1 .s -1 . As opposed to hypochlorite anion which react faster with biomolecules than boronate/boronic acid in vivo, peroxinitrite is able to oxidize the carbon-boron bond due to the rapid kinetic of this reaction over the addition of carbon dioxide.

In biological systems, hydrogen peroxide also reacts with biomolecules such as amino acids and metals (free or inside metallo-enzymes). Consequently, its half-life in cells is ~ 1 ms. The reaction with the thiol group of cysteine residues was described as the fastest physiological transformation of hydrogen peroxide with a rate constant up to 10 6 M -1 .s -1 . 138,139 Given the differences of reactivity of both involved oxidative species, peroxynitrite is likely to be the main actor for the conversion of these prodrugs into the corresponding free drugs. In view of the short half-life of oxidative species, increasing the rate constant of this oxidation reaction should result to a greater number of available peroxynitrite molecules that can react with the boronic acid prodrug. This would lead to a better yield of prodrug transformation at the tumor site and then improve the chance of treatment efficacy. Notably, the structure of the prodrug will also greatly affect the kinetic of oxidation in regards of the ROS involved in the reaction. The possibility of modulating the prodrug scaffold in favor of a faster reactivity toward ROS is crucial and can be operated by medicinal chemists.

In term of structure-kinetic studies of boronic acids conversion into alcohols, we noted that aliphatic boronic acids are 10 times slower than benzene boronic acids (scheme 25). 70 The oxidation kinetic of benzene boronic acids is dependent of the substitutions of the aromatic nucleus. [START_REF] Cao | Substituent Effects on Oxidation-Induced Formation of Quinone Methides from Arylboronic Ester Precursors[END_REF][START_REF] Mosey | Versatile Approach to α-Alkoxy Carbamate Synthesis and Stimulus-Responsive Alcohol Release[END_REF] Adding a strongly electron-withdrawing substituent such as a nitro group at the para position of the ring increased the oxidation rate by 20-fold whereas an electron-donating methoxy group at the same position divided this rate by a factor of three. Although electronwithdrawing group substitution of the benzenic ring lead to a faster formation of the quinone methide intermediate, this latter modification also cause a dramatic reduction of the kinetic of the following self-immolation step. [START_REF] Alouane | Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications[END_REF] Therefore, no substitution of benzene boronate nucleus as self-immolative promoiety can be envisioned to increase the global rate of the oxidation/disassembly steps of such constructs.

The vinyl boronic acid group was also shown to release an effector through oxidation and subsquent self-immolation process. [START_REF] Mosey | Versatile Approach to α-Alkoxy Carbamate Synthesis and Stimulus-Responsive Alcohol Release[END_REF][START_REF] Brooks | Synthesis, and Characterization of Small-Molecule Reagents That Cooperatively Provide Dual Readouts for Triaging and, When Necessary, Quantifying Point-of-Need Enzyme Assays[END_REF]140 The oxidation of the vinyl boronic acid motif was 20 times more rapid than that of the equivalent unsubstituted benzene boronic acid group. [START_REF] Mosey | Versatile Approach to α-Alkoxy Carbamate Synthesis and Stimulus-Responsive Alcohol Release[END_REF] This difference of reactivity could be attributed to a faster self-immolation step since the ϖ electrons of the double bond are not delocalized within a benzene nucleus and therefore available for the elimination reaction. Moreover, the empty p-orbital of the boron in vinyl boronic acid motif is less hindered than in the benzenic equivalent leading to an easiest access of the nucleophilic oxidative species. Eventually, this motif could be further engineered to potentially increase the global rate of effector release.

With the condition that an average drug concentration of 10 µM is required within the tumor of around 10 -3 mm 3 , the rate of the reaction (v = k[prodrug][ROS]) should not fall belowc 10 µM per 2 minutes which is about 0.1 µM.s -1 (total conversion inside the tumor). Given the concentration of peroxinitrite (~ 10 µM; the most reactive ROS) in cancer cells, only prodrugs oxidized at rate constant (k) higher than 10 3 M -1 .s -1 would be effective. Nonetheless, due to the short half-life of peroxinitrite (~10 ms), we estimate that the reaction of the latter oxidant with a particular boronic acid prodrug should react at a rate constant superior to 10 7 M -1 .s -1 in order to afford 10 µM of drug localized at the tumor site. In light of this short physiological half-life, targeting mitochondria is a relevant strategy in order to efficiently reach the cellular ROS. Such vectorized prodrug containing a delocalized lipophilic cation was already developed by the Mokhir group. 124 In regards of the aforementioned boronic acid promoieties, we noticed that the quinone methide species generated from benzene boronates were not strongly cytotoxic but very efficient at scavenging antioxidants. [START_REF] Engel | Novel Dual-Action Prodrug Triggers Apoptosis in Glioblastoma Cells by Releasing a Glutathione Quencher and Lysine-Specific Histone Demethylase 1A Inhibitor[END_REF]115,128,129 Remarkably, it was occasionally reported that the boronic promoiety was not completely masking the pharmacological activity of the branched drug. [START_REF] Reddy | Preclinical Evaluation of EC145, a Folate-Vinca Alkaloid Conjugate[END_REF][START_REF] Jin | Synthesis and Biological Evaluation of Hypoxia-Activated Prodrugs of SN-38[END_REF] This suggests that the attachment of a benzene boronic acid promoiety to a drug do not provoke the automatic discontinuation of the bioactivity. Furthermore, the nominal boronic acid function is often used in this reviewed prodrug strategy and it was recently proposed that the latter function could be used as a bioisoster of a hydroxyl group. Some successful drug analogs were indeed developed by the Wang group 141-143 that formerly developed boronic acid-containing prodrugs (vide supra).

The same group showed that boronic acid prodrug were also activated by the cytochromes P450. [START_REF] Zhang | Metabolism and Pharmacokinetic Study of the Boron-Containing Prodrug of Belinostat (ZL277), a Pan HDAC Inhibitor with Enhanced Bioavailability[END_REF] Thus, the benzene boronic promoiety could even be employed for the development of several types of prodrug allowing the prolonged release of any drug for the treatment of any disease. 144 Consequently, this activation could compete with the expected oxidation by the ROS of cancer-targeting boronic acid prodrugs within the tumor microenvironment. It is important to note, however, that unlike a direct chemical reaction between an oxidant and a boronic acid molecule, the reaction of the latter function with the cytochromes P450 is an enzymatic reaction. This implies the entrance of the prodrug into the active site before oxidation of the C-B bond. In order to develop boronic acid prodrugs specifically targeting tumors, their oxidation by cytochromes P450 should be evaluated. In case of unwanted interaction, modulation of the structural core of the boronic acid promoiety should be therefore considered to impede the prodrug access to the catalytic sites of the cytochromes P450.

Conclusion

This chapter reports on the different small-molecule, polymeric and nanoparticle-based prodrugs activated through oxidation of intrinsic carbon-boron bonds by ROS from tumor environment. Since we published this chapter as a review, two articles reported the development of boronic acid based anticancer prodrugs of evodiamine 145 and melatonine. 143 The literature reveals several constructs with interesting activity and until now, none of these prodrugs have entered into clinical trial. In view of the in vivo data, this prodrug strategy is promising for the treatment of several tumors such as leukemia, breast and pancreatic cancers which produce high concentrations of ROS. Chemical system leading to both ROS amplification and anticancer agent delivery were particularly effective. In order to obtain a ROS-activated boronic acid prodrug with a great therapeutic potential, it would be beneficial to develop a structure which could both intensify the ROS level within cancer cells and ensure rapid release of a drug by virtue of a rapid C-B oxidation.

In cancer chemotherapy, the prodrug strategy has risen as the cytotoxic drugs available in clinic often present similar toxicity towards both cancer and normal cells. Many research groups have made significant efforts in the development of innovative antitumor prodrug but the typical prodrug approach remains the branching of an organic promoiety to antitumor drugs in order to mask their original activity (scheme 26). [START_REF] Denny | Prodrug Strategies in Cancer Therapy[END_REF]142,[146][147][148][149] Subsequently, masked drugs are therefore liberated at the tumor site as a result of modified cancer cell metabolism. One major drawback in this prodrug strategy is often the poor ability of the promoiety (regardless Frequently, the assembly can be almost as pharmacologically active as the free drug owing to the preserved binding to the biological target.

As significant example, the natural product camptothecin is not used in clinic due to its strong general toxicity, lack of selectivity and low solubility. These problems have led researchers to develop camptothecin prodrugs based on conjugates of promoieties associated to hydroxycamptothecin. 154,155 However, pharmacological activity was reduced by a factor 10 to 50 which is not sufficient to prevent the cytotoxic effect on healthy cells (Scheme 27).

Scheme 27. General structure of camptothecin prodrugs.

In the literature, the enediynes are natural products that cyclize spontaneously into cytotoxic antibiotics (scheme 1). [156][157][158][159] Moreover, this scaffold is the sole described as able to afford cytotoxic agents after internal cyclization. Conjugation of these molecules to antibodies lead to the development of the antibody-drug conjugates Mylotarg® and Besponsa® used in clinic to treat cancer. 160 Furthermore, precursors of coumarins relying on an intramolecular cyclization to eventually generate the fluorophore were also studied. [161][162][163] In this thesis, we intend to adapt this feature by establishing chemist-made precursors of antitumor agents operating analogously: (i) no branched organic promoiety is used but masked reactive functions are rather inserted; (ii) the proposed prodrug is not a drug linked to a hindered moiety but a synthetic precursor of the drug that will need an in-cell cyclization step to produce the final drug (scheme 28).

Scheme 28. Generation of anticancer agents from ring-opened precursors through bioorthogonal cyclization.

The prodrug is made of a relatively flexible precursor of the final drug, in order to strongly reduce the possibility of prodrug potency in respect to the free drug. In this approach, the prodrug displays lower molecular mass in comparison to classic prodrugs, a favorable characteristic for both crossing biological membranes and complying with the Lipinski's rule of 5.

The phenanthridines present large and planar structures which are frequently encountered features of intercalating agents and topo isomerase I inhibitors. Moreover, phenanthridines are not used in clinic due to their general toxicity, [164][165][166] and are therefore candidates of choice to explore this strategy. In this context, we first envisioned to prepare ring-opened precursors of phenanthridine (scheme 29). Starting from a biaryl precursor, we proposed a cyclization by imination that would generate the nitrogen-containing central ring of the aromatic phenanthridine Phen. We then designed a biaryl derivative containing a nitro and a ketone functions to test the ability of this precursor to spontaneously cyclize. The final step of formation of the phenanthridine would be then possible via spontaneous intramolecular cyclization due to entropic favorability as the driving force. Using the commercially available nitrophenylboronic acid 60 and the iodoacetophenone 61, we were able to efficiently obtain the needed precursor 62 in one step by palladium catalyzed coupling. In order to initiate the expected cyclization reaction, we reduced the nitro by hydrogenation at 37°C in presence of Raney nickel. This reaction was carried out in organic solvent and then in cell medium.

Scheme 29. A nitro-biaryl derivative was chemically reduced to afford its corresponding aniline that spontaneously generated the aromatic phenanthridine.

By following the reaction by TLC, we monitored the rapid formation of the cyclic compound whereas no observation of the aniline intermediate was possible by this method. Interestingly, the formed phenanthridine was fluorescent and it would be therefore straightforward to develop a strategy to measure the kinetic of phenanthridine assembly. Two steps are involved in this process: first the nitroreduction and the subsequent imination of the ketone function by the newly formed aniline. The kinetics of the whole process must be fast in order to rapidly form the final compound once the reaction is triggered. This is of particular importance when developing prodrugs. Indeed, in an open system, molecular motion affects the concentration profiles. Hence, co-localizing product liberation with precursor activation at the concentration of the non-activated precursor (a much sought after goal in the prodrug design) is kinetically constrained: the fastest activation and self-assembly chemistry must be therefore found.

As discussed in chapter 1, the reactive oxygen species (hydrogen peroxide or peroxynitrite) overproduction in cancer cells is a common way of anticancer prodrug activation. Boronic acid precursors are oxidized into corresponding alcohols and these precursors were largely designed to generate phenols (scheme 30). However, vinyl boronic precursors were less studied, in particular, the goal of generating a ketone function starting from such motif was not previously described. Indeed, in the presence of ROS, the vinyl boronic acid would be transformed into an enol moiety tautomerizing into its keto equivalent.

Scheme 30.

Strategy to generate either an electrophile or a nucleophile from boronic acids. In the presence of ROS, delocalization of π electrons can generate a nucleophile starting from arylboronic acid or an electrophile can be generated from a vinylboronic acid by transformation into enol tautomerizing into ketone.

Due to the strong interest of Raphaël Labruere's group in boronic acid-containing prodrugs, we decided to focus our strategy toward insertion of a vinyl boronic acid motif into our precursor scaffold. The designed prodrugs of phenanthridine would thus contain one masked ketone (vinyl boronic acid motif). Once the ketone is selectively unmasked in the tumor microenvironment, this latter group would react with a pendant amine function to build up an imine bond finally shaping the non-hydrolysable phenanthridine scaffold (scheme 31).

1.Synthesis of biaryl precursor ProPhen

In our strategy for the preparation of the biaryl precursor ProPhen (able to generate phenanthridine Phen after activation), we envisioned the synthesis of each aryl moiety bearing the appropriate substituents and subsequent bonding of these two units by a palladium catalyzed coupling reaction (Scheme 32).

Scheme 32. Disconnection envisionned for the preparation of ProPhen.

We first investigated the possible synthetic pathways to generate 1-phenylvinylboronic motif from phenylacetylene 65 (synthetic pathway A) or from acetophenone (synthetic pathway B and C) (Scheme 33).

-The synthetic pathway A allowed to obtain the vinyl boronate in two steps from phenylacetylene 65: the first step consisted to the α-bromation of phenylacetylene using the protocol of Dong et al. 167 with HBr in acetic acid as solvent to generate a mixture of α-bromo styrene 66 in 92% yield. The α-bromo styrene was engaged in boron-bromide exchange using the method of Miyaura et al. 168 to afford the vinyl boronate motif 71 in a global yield of 32%.

-The synthetic pathway B also provided the vinyl boronate 71 in two steps starting from acetophenone 67: the first step consisted in the enol formation which was trapped using At this stage, we decided to exploit the biaryl 62 prepared as the model precursor of this project as it displays the appropriate ketone function and a nitrobenzene moiety that could be further reduced into aniline (scheme 29 and 34). We first attempted to prepare the corresponding αboryl alcohol 72 using the protocol developed by Hanna et al. [START_REF] Hanna | Alcohol, Aldehyde, and Ketone Liberation and Intracellular Cargo Release through Peroxide-Mediated α-Boryl Ether Fragmentation[END_REF] but we surprisingly observed the direct formation of the phenanthridine compound Phen. The second synthetic pathway consisted in the generation of the vinyl triflate derivate 73 using Tf2O but this route was also unsuccessful.

Scheme 34. Synthetic pathway to generate biaryl 74.

A B C

We then returned to our initial strategy of preparing separately both aryl moieties in order to couple these molecules in the end of the reaction. Vinyl boronate 76 was then prepared in a two-step procedure: (i) starting from iodoacetophenone 61, α-boryl alcohol 75 was obtained in excellent yield using the protocol developed by Hanna et al. [START_REF] Hanna | Alcohol, Aldehyde, and Ketone Liberation and Intracellular Cargo Release through Peroxide-Mediated α-Boryl Ether Fragmentation[END_REF] The last step to produce ProPhen would be then the nitro-reduction under the conditions described in Table 5. Unfortunately, none of the employed conditions for the reduction of the nitro group afforded ProPhen. Again, we only obtained Phen as observed by TLC.

Kinetic analysis of phenanthridine cyclization from open biaryl precursor

Then, we launched the accurate determination of the kinetic constants of both oxidation and cyclization reactions and, advantageously, phenanthridine Phen is fluorescent as opposed to its precursor ProPhen.

The absorption spectra of ProPhen and Phen were measured in a solvent mixture:

CH3CN:PBS buffer:DMSO 48.5:48.5:1 v/v/v. at T = 298 K in figure 16. The set of differential equations governing the evolution of the concentration in the species shown in scheme 37 is given in Eqs.(1-3):

𝑑𝑅 𝑑𝑡 = -𝜅 1 𝑅 (1) 𝑑𝐵 𝑑𝑡 = 𝜅 1 𝑅 -𝑘 2 𝐵 (2) 𝑑𝑃 𝑑𝑡 = 𝑘 2 𝐵 (3)
with the additional conservation law

𝑅 0 = 𝑅 + 𝐵 + 𝑃 (4)
since the system is closed. In Eqs.

(1-4), R and P respectively designate ProPhen and Phen, and k1 = κ1[H2O2] where the concentration [H2O2] is assumed to be constant since H2O2 is in excess of the R, B, and P species.

In the most general case, the rate constants κ1 and k2 are different and the solutions of Eqs.(1-3) are given in Eqs.

(5-7):

𝑅(𝑡) 𝑅 0 = 𝑒 -𝜅 1 𝑡 (5) 𝐵(𝑡) 𝑅 0 = 𝜅 1 𝑘 2 -𝜅 1 (𝑒 -𝜅 1 𝑡 -𝑒 -𝑘 2 𝑡 ) (6) 𝑃(𝑡) 𝑅 0 = 1 - 𝑘 2 𝑒 -𝜅 1 𝑡 -𝜅 1 𝑒 -𝑘 2 𝑡 𝑘 2 -𝜅 1 (7)
In the case k2 ≫ k1, this set of equations transforms into Eqs.(8-10) at t≫1/k2.

𝑅(𝑡) 𝑅 0 = 𝑒 -𝜅 1 𝑡 (8) 𝐵(𝑡) 𝑅 0 ≈ 0 (9)
𝑃(𝑡) 𝑅 0 = 1 -𝑒 -𝜅 1 𝑡 (10)

Experimental result

For this study, we devised a stopped-flow fluorescence spectrometry experiment (scheme 39)

to analyze the kinetics of Phen formation initiated by hydrogen peroxide addition to precursor

ProPhen.

The For a total absorbance lower than 0.15 (as in the present series of experiments), fluorescence emission is proportional to the sum of the contributions of the fluorescent components, which are individually proportional to their respective concentrations. By neglecting the contributions of non-absorbing and non-emissive species, we derived:

𝐼 𝐹 𝑃 (𝑡) = 𝛼𝑄 𝑃 𝑃(𝑡) (11)
The temporal evolution of the fluorescence signal was satisfactorly adjusted with Eqs. (10,11).

In particular, the relevance of Eq.(10) suggests to be in a kinetic regime such that acquisition is performed with t ≫1/k2. Since we used a 100 Hz acquisition frequency, we deduced k2 ≫ 10 2 s -1 . As we monitor the steps occurring beyond the millisecond, we reduced the preceding reactive scheme from three to two steps considering that tautomerization reactions typically occur at the sub-millisecond time scale. 169 In the corresponding reduced scheme, the rate constant k1 was associated to oxidation of the C-B bond whereas k2 refers to the cyclization step.

Satisfactory fits (figures 18 and 19) were obtained and we analyzed the dependence of κ1 on the H2O2 concentration, which led us to retrieve k1 ~ 130 M -1 .s -1 . This value indicates a rate of oxidation by hydrogen peroxide of the vinyl boronate ester motif enhanced by a factor 10 2 in comparison with the intensively studied benzene boronate esters (1-2 M -1 .s -1 ). 170 Then, we extracted k2 > 10 2 s -1 which represent a time of assembly (𝜏2 = 1/k2) below 10 ms.

Introduction

Phenanthridines are an important class of heterocyclic compounds that have attracted much attention due to their medicinal importance (figure 20). 171-173 Indeed, molecules containing the phenanthridine motif were the subject of numerous studies as antitumor agents, 165,174,175 antiparasitic agents, and antibacterial agents. 176 The mode of action of these alkaloids is characterized by their potency as topoisomerase I inhibitors and DNA intercalating agents.

Furthermore, ethidium bromide (figure 20) is a widely used fluorescent markers of both DNA and RNA and was also studied for its potential pharmacological activity but its mutagenic property interrupted further study. As of today, no phenanthridines are currently used in clinic due to the general toxicity observed for these structures. As seen in chapter 2 and 3, in our strategy, only 6-methylphenanthridine derivatives can be produced as potential drug based on the internal imination reaction between an aniline and an acetophenone. In order to identify a toxic 6-methylphenanthridine, we decided to prepare several derivatives bearing various substituents and evaluate their cytotoxic activity against KB 3.1 cells, a cancer cell line frequently used to ascertain the activity of phenanthridines. 175,177,178

Synthesis

Due to their versatile applications, the development of new methodologies for the preparation of substituted phenanthridines was the subject of vigorous research (scheme 40). The Pictet-Hubert reaction is carried out at high temperatures which has limited the application of this method for the preparation of variously substituted phenanthridines 179,180 . Other approaches to synthetize phenanthridines have been developed using transition metal, [181][182][183][184] cycloaddition, 185-187 and intramolecular cyclization. [188][189][190][191][192] Recently, Ge et al. have developed a one pot synthesis of phenanthridine via a Suzuki palladium coupling followed by a N-Boc deprotection and intramolecular cyclization by imination (scheme 40). 193 Scheme 40. Methods to generate phenanthridine derivatives.

We then became interested in that straithforward method for the generation of 6methylphenanthridines. In our strategy, the prodrug is bearing a vinyl boronate moiety and the preparation of our phenanthridine prodrugs cannot rely on a Suzuki reaction. We therefore decided to adapt this method using a Stille coupling for the synthesis of the biaryl bond (scheme 41). The choice of the 6-methylphenanthridines substituents in this study was mainly guided by the abundant reported cytotoxicity results from the LaVoie group. 165,174,175,177,178 In particular, benzannulated phenanthridines and/or the presence of alkoxy substituents were found as efficient features for anti-proliferative activity. Moreover, the di-ortho-substitution of the aryl building blocks of the Stille reaction was ruled out in order to enable the biaryl bond formation.

Scheme 41. Retrosynthetic analysis for the synthesis of 6-methylphenanthridines.

First, we synthetized the phenyl stannane building blocks 80, 85 and 88 in two steps starting from the commercial anilines 78, 83 and 86 (scheme 42). N-Boc anilines 79, 84 and 87 were prepared in very good yield using Boc2O in THF at room temperature overnight. An ortholithiation was then carried out with t-BuLi followed by the addition of SnMe3Cl in freshly distillated diethyl ether to afford the protected stannanes 80, 85 and 88 in good yield.

Scheme 42. Reaction pathway to phenyl stannane building blocks.

In regards to the acetophenone building blocks, the iodoacetophenone 61 and bromo-(ethylenedioxy)acetophenone 99 are commercially available except 96 which was prepared according to a convergent synthesis starting from 3,4-dimethoxyphenethyl alcohol 89 (scheme 43). The primary alcohol of 89 was oxidized with Dess-Martin periodinane reagent in dry dichlomethane to give the aldehyde 90 in excellent yield. 92 was obtained from 3-butyn-2-ol 91 which was protected with a tert-butyldimethylsilyl (TBS) group in 81% yield. Terminal alkyne 92 was deprotonated with n-BuLi in dry THF and nucleophilic addition was then performed onto compound 90 to provide alcohol 93 in 50% yield.

According to a described procedure, 194 the next step consisted in the iodocyclization of 93 in the presence of ICl during 5 min resulting in the formation of two products: compound 95 as unprotected iodo-naphthalene and compound 94 as TBS-protected iodo-naphthalene in 80%

and 15% yield respectively. The unprotected iodo-naphthalene 95 was directly formed as HCl and H2O were generated during the reaction. The TBS group of the iodo-naphthalene 94 was finally cleaved using TFAB in THF in 95% yield. In this latter sequence, deprotected iodonaphthalene 95 was thus obtained in 95% yield. Oxidation of the alcohol group of 95 with PCC in dry DCM at room temperature during 24h gave compound 96 in almost quantitative yield.

The synthesis of ketone 96 was then achieved in 5 steps with 32% overall yield.

Scheme 43. Synthetic pathway to generate building block 96.

The 6-methyl phenanthridines were then prepared via a three-step sequence of reaction in one pot (scheme 44): (i) biaryl coupling using Pd(PPh3)4, CuCN in freshly distilled toluene at 110°C during 24h under argon; (ii) Thermolytic deprotection of the N-Boc group due to the high temperature of the reaction; (iii) spontaneous cyclization by imination. In the first few hours of the reaction, we observed the formation of the biaryl with the protected amine. In order to allow the reaction to go to completion and therefore increase the yield of phenanthridine formation, the reaction must be maintained for 24 hours at 110°C. Scheme 44. 6-methyl phenanthridines synthetized by one pot reaction.

In scheme 45, we detailed the different steps of this synthesis. First, a phenyl stannane undergoes a tin-copper exchange reaction. 195 The resulting intermediate and a 2halogenoacetophenone will then react following a classical catalytic cycle of the Stille reaction.

Then, the Boc group will be removed by thermolysis 196 and subsequent intramolecular cyclization between the aniline and the ketone will finally afford the 6-methylphenanthridine. Table 6. Biological activity of 6-methylphenanthridines against KB 3.1 cancer cells.

Name

We first observed that the unsubstituted 6-methylphenanthridine Phen was completely inactive up to 50 µM which was the maximum concentration not exceeding Phen solubility limit in the assay conditions. Addition of one supplementary cycle to the latter scaffold was achieved employing either a methylenedioxy bridge 100, an ethylenedioxy motif (101 and 104) or a dimethoxybenzene nucleus (100) but resulted in no improvement of the antiproliferative activity.

We then evaluated the two phenanthridines 107 and 108 previously reported as moderately cytotoxic but we were not able to retrieve the same level of activity whether it be on the same cell line with the methylenedioxy 107: compare IC50 (KB 3.1) = 12 µM 175 with IC50 (KB 3.1) > 50 µM or a different cell line with 108: compare IC50 (CPT-K5) = 11 µM 174 with IC50 (KB 3.1) > 50 µM. In our optimized protocol for the determination of cytotoxicity on KB 3.1 cell, we incubated the different compounds with the cells for three days instead of the four days mentioned in the described procedure 174,175 and we assume that this shorter duration of exposure to the tested compounds decreases the observed activity. In regards to 107, isomer 103 where both the methylenedioxy motif and the dimethoxy substituents are placed on the opposite side of the phenanthridine scaffold is also inactive. However, phenanthridines ToxPhen, 104 and 106 substituted on both sides with at least one ethylenedioxy moiety were all cytotoxic.

Interestingly, replacement of the methylenedioxy motif in 103 by an ethylenedioxy group in 105 lead to an increase of the activity. Nonetheless, it is the reverse modification from 106 (IC50 ≈ 6 µM) that produced the most cytotoxic phenanthridine ToxPhen of this series with an activity enhanced by a factor 10 (IC50 ≈ 0.7 µM). This phenanthridine has been therefore selected for the preparation of the corresponding ring-opened biaryl prodrug.

Chapter 5: Preparation of the biaryl precursor of ToxPhen and in-cell studies of cyclization

Having identified a fast-cyclizing scaffold for spontaneous phenanthridine generation, we next examined the feasibility of using such reaction to generate a cytotoxic phenanthridine within tumor tissue. Beforehand, several 6-methylphenanthridines have been tested against KB cancer cells. We chose the most cytotoxic phenanthridine ToxPhen of our series displaying two alkoxy groups: a methylene dioxy and an ethylene dioxy.

The biaryl precursor of ToxPhen which we named ProToxPhen was thus designed based on the ProPhen scaffold.

Synthesis of ProToxPhen

The prodrug ProToxPhen of phenanthridine ToxPhen was prepared (scheme 46) following the same synthetic pathway devised for unsubstituted precursor ProPhen.

Cytotoxicity experiments on KB 3.1 human cancer cells

In vitro cell-proliferation assays

The antitumoral activity of ProToxPhen was evaluated on KB cells using the same procedure devised for the cytotoxicity determination of the phenanthridines in chapter 4.

The cytotoxic activity of ProToxPhen was then compared to the one obtained with ToxPhen (figure 21). We observed that biaryl prodrug ProToxPhen activity was significantly decreased by a factor superior to 700 (IC50 > 500 µM): considered as completely inactive in comparison to the corresponding drug ToxPhen (IC50 ≈ 0.7 µM).

Furthermore, the cytotoxic activity of ProToxPhen and ToxPhen was then investigated using a KB 3.1 spheroid, a 3D model of in vivo tumors 197 . In a round-bottom 96-well plate, 1,000 cells (100 µL) were seeded with 100 L of each compound at different concentrations (from 50 M to 0.05 nM for the ProToxPhen compound and from 500 M to 100 nM for the ToxPhen compound) and incubated at 37 °C in presence of 5% CO2 and under 95% hygrometry. After 72h, the formed KB spheroids were observed using an optical microscope (Olympus CK 2)

and their morphology were imaged with a digital camera (Moticam 1000 1.3MP Live resolution) mounted on the optical microscope. The pictures were then normalized using Image J software (figure 22). We observed that biaryl prodrug ProToxPhen have no activity at 500 µM in comparison to the corresponding drug ToxPhen active at 5 µM.

Conformational analysis

Such difference of pharmacological activity was expected as ProToxPhen displays a nonplanar structure over the completely planar phenanthridine ToxPhen. The dihedral angle of ProToxPhen was then calculated from MM2 energy minimization which a dihedral angle of 40° (47.18 kcal.mol -1 ) whereas ToxPhen was completely planar as expected. These data are in agreement with the reported optimal dihedral angle (ca. 43°) obtained on the elementary biphenyl from MP2 calculations. 198 As depicted in figure 24, cell shrinkage, loss of cell-cell contact, and membrane blebbing is observed after treatment with ToxPhen (150 μM). An enhanced effect was detected after 45, 60 and 180 min incubation (white arrows). Nuclear fragmentation was also observed after 180 min of incubation (grey arrows). These whole-cell and nuclear deformations are induced by ToxPhen and have been pointed out to be characteristic features of cell apoptosis 199,200 .

For this experience, we used a home-built epifluorescence microscopy set-up. We then achieved the accurate determination of the prodrug conversion kinetics within KB spheroids. KB cells were incubated for three days in a 150 µM solution of ProToxPhen in DMEM:DMSO 99:1 v:v in order to form observable spheroids. The spheroids were washed with PBS and deposited on a 35 mm glass circular round microscope slide. The prodrug oxidation was then triggered by peroxynitrite, a powerful oxidant of the C-B bond having a physiological halflife ten times higher than that of hydrogen peroxide. 153 The peroxynitrite was generated from the decomposition of 3-morpholinosydnonimine (SIN-1, scheme 47). 201

Kinetic analysis of ProToxPhen conversion into

Scheme 47. Decomposition of 3-morpholinosydnonimine into peroxinitrite. 201 To initiate the reaction, a 2 mM solution of peroxynitrite generator 3-morpholinosydnonimine (SIN-1) 202 leading to peroxynitrite production rate of ca. 30 µM.min -1 in PBS was added onto the ProToxPhen containing cells. This continual flux mimics the tumor microenvironment where peroxynitrite production is estimated at ca. 50 µM.min -1 essentially supplied by tumor cells and tumor-associated macrophages 135,203 . Considering the rapid decomposition of peroxinitrite in cells (ca. 1.5 s -1 ), 202 we calculated a steady-state peroxynitrite concentration of ca. 0.3 µM in our experiment. Moreover, peroxynitrite was shown to rapidly diffuse through cell membranes (< 10 ms). 204 The in-cell kinetic of cyclisation of ProToxPhen in ToxPhen was studied unsing homemade epi-fluorescence microscopy using KB 3.1 spheroid models. In a round-bottom 96-well plate, 500 cells were seeded in a 150 µM ProToxPhen solution (200 µL, DMEM) and incubated at 37 °C in presence of 5 % CO2 and under 95 % hygrometry. After 3 days, the formed KB spheroids were observed using an optical microscope. 100 µL of media were aspirated and the spheroids were washed twice with PBS (100 µL). The spheroid was then aspirated (20 µL) The kinetics of ToxPhen formation within KB cells was analyzed as mentioned in chapter 3.

The temporal evolution of the fluorescence signal (figure 29) was satisfactorly adjusted with Eq. (10,11) and yielded k2≫10 2 .s -1 in agreement with the value found in vitro. Next, we derived the rate constant of oxidation k1 = 1.8 10 4 M -1 .s -1 which is 10 2 times faster than the reaction with hydrogen peroxide. A sluggish decrease in fluorescence was subsequently observed as expected from unbalancing production of ToxPhen with its diffusion out of the cells (figures 29 and 30).

The decrease in fluorescence after generation of ToxPhen was observed at similar rate in both an isolated cell (green) and the whole spheroid (red). We assume that this drop originates from diffusion of ToxPhen out of the cells. times slower than the flux found in the pathological condition.

Plasma stability of ProToxPhen

Rat plasma (Sigma-Aldrich) was reconstituted with deionized water to a final volume of 1 mL.

1 mL of PBS buffer at pH 7.5 was then added to the freshly prepared plasma. ProToxPhen solutions at 10 µM were prepared (100 µL) in the mixture described above and were incubated at 37°C at different time interval (t0, 2h, 6h, 24h and 72h). Each sample were then extracted under vigorous mixing with 300 µL of LCMS grade acetonitrile. After centrifugation at 4,000 rpm for 2 min, the supernatant was filtered before being analyzed by LCMS. This compound was present at t0 and remained stable up to 72h of incubation in the plasma (area under curve ≈ 60% in all cases) associated to no appearance of metabolites. This compound with m/z = 342 was analyzed as the corresponding free boronic acid of the parent ProToxPhen structure (figure 32). Samples at t0 and 72h were further subjected to HRMS analysis (ESI + , m/z calculated for C17H17BNO6, [M+H] + : 342.1146, found: 342.1139) confirming the presence of the hydrolyzed counterpart of ProToxPhen even when no incubation with plasma was performed. As expected, hydrolysis of boronate ester into corresponding boronic acid was very fast [START_REF] Cao | Substituent Effects on Oxidation-Induced Formation of Quinone Methides from Arylboronic Ester Precursors[END_REF] in aqueous media and interestingly the obtained boronic acid was highly stable in plasma.

Conclusion and perspectives

The objective of this dissertation in chemical biology was the development of a novel approach in the design of antitumor prodrugs. Inspired by the bioorthogonal chemistry field and previous data observed in our group, we have described a methodology for the preparation of inactive antitumor prodrugs. These prodrugs were built as ring-opened precursors of the drugs that will cyclize within tumors upon cancer cell-specific stimuli in contrast to the conventional grafting of a bulky temporary moiety hardly inactivating the drug.

After a general introduction about cancer and the different treatments available in clinic, a bibliographic work on anticancer boronic acid/boronate-containing prodrugs responsive to oxidative stress from the tumor microenvironment was carried out in chapter 1. We selected the phenanthridines as drug model for the implementation of our strategy. We then developed a biaryl precursor of phenanthridine bearing both an aniline and a vinyl boronate ester motif able to get oxidized by hydrogen peroxide or peroxynitrite to afford a ketone function. We first measured the kinetics of oxidation and observed that the rate of oxidation of our vinyl boronate ester motif was oxidized 10 2 times faster than the benzene boronate ester moiety. In spite of the presence of a poorly nucleophilic aniline, a spontaneous and fast cyclization of our precursors was measured by virtue of both the geometry of their biaryl scaffolds and the final formation of a six-membered aromatic nucleus. Common imines are rapidly hydrolyzed under physiological conditions and, oxime and hydrazone bonds were introduced to prohibit the reversibility of the former link but these reactions display sluggish kinetics. In this work, the generated imine is inserted in an aromatic ring conferring high stability toward hydrolysis of the newly formed bond. We then devised a new method for the one pot preparation of 6methylphenanthridines and then synthesized several derivatives. The most cytotoxic was selected, the corresponding prodrug was then prepared and we observed that this prodrug was completely devoid of cytotoxicity. Indeed, the radically different structures geometry of the prodrug toward that of the drug induced a vast change of cytotoxic activity. Using this prodrug, we recorded a very fast cyclization (3.5 s) into the phenanthridine counterpart within tumor spheroids under tumor microenvironment conditions.

New bioorthogonal reactions are constantly investigated in order to either improve existing reactions (i.e. rate constant modifications) or solely access to more complex applications. Thus, this system based on fast cyclization by biorthogonal imination could be expanded to other platforms integrating other types of (i) chemical switch to trigger the reaction and (ii) in vivo-compatible synthetic steps. The design of such programed "cycl'in-cell" precursors may find applications in the development of new tools for imaging aside from prodrugs involved in several pathologies.

Cytotoxicity experiments on KB 3.1 human cancer cells 1.2.1 In vitro cell-proliferation assay (2D)

The cytotoxic activity phenanthridines was investigated using the MTS (3-( % hygrometry. After 72 h, the media was aspirated and 100 µL of a MTS solution (10% in media) were added in each well. After a 3 h incubation period at 37 °C in presence 5% CO2 and 95% hygrometry, the optical density was measured at 490 nm wavelength using a microplate reader (Infinite M200 Pro, Tecan trading AG, Switzerland). Untreated cells were used as control. Each concentration was tested in six replicates and the experiment was fulfilled in triplicates. The concentration inhibiting 50% of the cell proliferation (IC50) was determined using GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA).

In vitro 3D tumor spheroid viability assay

The cytotoxic activity of ProToxPhen and ToxPhen was investigated using a KB 3.1 spheroid model. In a round-bottom 96-well plate, 1 000 cells (100 µL) were seeded with 100 L of each compound at different concentrations (from 50 M to 0.05 nM for the ProToxPhen compound and from 500 M to 100 nM for the ProToxPhen compound) and incubated at 37 °C in presence of 5% CO2 and under 95% hygrometry.

After 72h, the formed KB spheroids were observed using an optical microscope (Olympus CK 2) and their morphology were imaged with a digital camera (Moticam 1000 1.3MP Live resolution) mounted on the optical microscope. The pictures were then normalized using Image J software.

Photophysical analyses

UV-visible absorption

UV-Vis absorption spectra were recorded in 1 cm × 1 cm quartz cuvettes (Hellma) on a diode array UV-Vis spectrophotometer (Evolution array, Thermo Scientific) at 293 K. The molar absorption coefficients were extracted while checking the validity of the Beer-Lambert law.

Epifluorescence spectroscopy

Photon Technology International QuantaMaster QM-1 spectrofluorimeter (PTI, Monmouth Junction, NJ) equipped with a Peltier cell holder (TLC50, Quantum Northwest, Shoreline, WA) was used.

Epifluorescence microscopy

We used a home-built epifluorescence microscopy set-up. The samples were illuminated using a NCSU033B LED (Nichia Corp., Tokushima, Japan) filtered at 365 ± 40 nm (Chroma

123

Compound 80. N-Boc-aniline (79) (100 mg, 0.52 mmol, 1.0 eq) was placed in an oven-dried round-bottom flask and dissolved in 1 mL of dry Et2O. The solution was cooled to -40 °C under an Ar atmosphere. t-BuLi (1.7 M in pentane, 647 µL, 1.1 mmol, 2.1 eq) was added via a syringe pump over 30 min, during which the mixture became homogenous, and the resulting solution was stirred at 0 °C for 3 h. The reaction mixture was further cooled to -78 °C, treated with a solution of Me3SnCl (104 mg, 0.52 mmol, 1.0 eq) in 1 mL of Et2O and stirred at 0 °C for 6 h.

The reaction mixture was quenched with saturated aqueous NaHCO3 solution (5 mL) at 0 °C.

The organic phase was extracted with Et2O (2 x 30 mL) and then washed with brine (50 mL).

The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. Flash chromatography using neutral alumina pre-washed with a 40:4:1 mixture of cyclohexane:Et3N:EtOAc and eluted with a 20:1 mixture of cyclohexane:EtOAc gave the expected product (94 mg, 51%) as a colorless oil. Compound 87. To a solution of 1,4-benzodioxan-6-amine 86 (2,3 g, 20 mmol, 1 eq) in 200 mL of THF was added triethylamine (5.57 mL, 40 mmol, 2 eq) and Boc2O (4.4 g, 20 mmol, 1 eq).

The mixture was then stirred at RT overnight. When the reaction was complete (checked by TLC), the THF was removed by evaporation under reduced pressure and the crude product was purified by flash chromatography (cyclohexane:EtOAc = 7:3) to afford the expected product (5.0 g, 100%) as a yellow powder. .

6-methylphenanthridines "

Phen/ToxPhen/100-106". To a flame-dried round-bottom flask containing a stir bar was added the acetophenone derivatives (1.1 eq), the phenyl stannane derivatives (1 eq), Pd(PPh3)4 (0.02 eq) and CuCN (0.2 eq). 4 mL of dry toluene was then added and the solution was refluxed during 24 h. The mixture was filtered through a pad of celite and the filtrate was concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 9:1 to 6:4) to give the expected product.

Yield = 65%

Compound 110. To a solution of 109 (470 mg, 1.22 mmol, 1 eq) in toluene (40 mL) was added p-toluenesulfonic acid (630 mg, 3.66 mmol, 3 eq). The mixture was heated at 60°C for 3 h. 5 mL of a Na2CO3 saturated aqueous solution was added and the aqueous layer was extracted with EtOAc (3x20 mL). The organic phases were combined, washed with brine, dried with MgSO4 and concentrated under reduced pressure to give the expected product (447 mg, 100%) as a white solid.

Titre:

  Développement de prodrogues antitumorales générant des phénanthridines cytotoxiques par cyclisation bioorthogonale in cellulo Mots clés : Réaction spontanée, Chimie organique, cancer, prodrogue, réaction bioorthogonale, ROS.

1 2 «Introduction

 2 La réussite est la combinaison de la passion, l'effort et le sacrifice » This project of antitumor prodrug development is at the interface of chemistry, biology and biophysics. The design, synthesis and biological study of the different derivatives were carried out in the group of Dr Raphaël LABRUERE from the laboratoire de Chimie Bioorganique et Bioinorganique at the Institut de Chimie Moléculaire et des Matériaux d'Orsay. In particular, the biological assays were performed by Dr Charles SKARBEK who was postdoctoral research associate in the group. On the other hand, the team of Pr Ludovic JULLIEN together with Dr Thomas LE SAUX from the laboratoire PASTEUR at Ecole Normale Supérieure in Paris were involved in the kinetic analysis and the fluorescence microscopy studies.

Figure 1 .

 1 Figure 1. National ranking of cancer as a cause of death at ages below 70 in 2019.3

Figure 2 .

 2 Figure 2. Estimated new cases and number of deaths (all age) in males. 3

Figure 3 .

 3 Figure 3. Estimated new cases and number of deaths (all age) in females.3

Figure 4 .

 4 Figure 4. Evolution of the number of new cases and deaths due to cancer in France between 1980 and 2018. 6

Figure 5 .

 5 Figure 5. Evolution of the number of people operated for cancer since 2010. 5

Figure 6 .

 6 Figure 6. Structures of several antimetabolites drugs.

Figure 7 .

 7 Figure 7. Structures of alkylating agent.

Figure 8 .

 8 Figure 8. Structures of several antitumor antibiotics.

Figure 9 .

 9 Figure 9. Structures of mitotic inhibitor drugs.

Figure 10 .

 10 Figure 10. (a) General structure of a prodrug; (b) activation of the prodrug in the tumor area.

Figure 11 .

 11 Figure 11. List of linker-payloads used in approved antibody-drug conjugates (as of September 2021).24

Figure 12 .

 12 Figure 12. Structure of Mylotarg®.

Figure 13 .

 13 Figure 13. Structure of the folate-vinblastine prodrug EC145, containing desacetylvinblastine monohydrazide DAVLBH.

  4.23.5) Degradation of extracellular matrix Phe-Ala-Ala-Phe(NO,)-Phe-Val-Leu-OM4P, Bz-Arg-Gly-Phe-Phe-Pro-4MβNA Plasmin (EC 3.4.21.7) Fibrinolysis, degradation of blood plasma proteins D-Val-Leu-Lys,D-Ala-Phe-Lys,D-Ala-Trp-Lys uPA (EC 3.4.21.73) tPA (EC 3.4.21.68) Activation of plasmin formation Gly-Gly-Gly-Arg-Arg Arg-Val Prostate-specific antigen [PSA (EC 3.4.21.77)] Liquefecation of semen Mu-His-Ser-Ser-Lys-Leu-GIn-Leu, L-377,202 Matrix metalloproteases [MMP-2 (EC 3.4.24.24), MMP-9 (EC 3.4.24.35)]

Figure 14 .

 14 Figure 14. Hypoxia in a solid tumor.

First, using an

  arylboronate profluorescent probe of coumarin, we determined the most efficient cell line at converting the probe into free coumarin among a panel of six cancers from different organs (breast cancer MCF-7 and the resistant counterpart MCF-7 MDR, hepatocellular carcinoma Hep G2, lung adenocarcinoma A549, glioblastoma U87, pancreatic cancer cell line MiaPaCa-2). Hep G2 and MiaPaCa-2 were the most ROS-producing cell lines but the cytotoxicity of the prodrugs was nonetheless evaluated on the different cell lines. Prodrug 28 was the most interesting since it displayed an IC50 of 0.3 µM corresponding to 67% of the total effect obtained with the same concentration of doxorubicin 27 (IC50 = 0.2 µM). Using MiaPaCa-2 pancreatic tumor in ovo xenograft via the HET-CAM (Hen's Egg Test-Chorioallantoïc Membrane) assay, 28 induced the same tumor regression as doxorubicin 27 intratumorally injected at the same dose. Ye et al.101 have efficiently entrapped the prodrug 28 in polymeric nanoparticles of polyethylene glycol conjugated to niacin (PEG-NAM, scheme 13). The ROS generator β-lapachone was co-encapsulated within the micelles in order to enhance the release of free doxorubicin. The authors observed that β-lapachone was first liberated and was then able to amplify the conversion of 28 into doxorubicin 27. The loaded nanoparticles together with doxorubicin 27, β-lapachone and 28 were evaluated in vitro against three breast cancer cell lines (4T1, MCF-7 and MCF-7 ADR, i.e resistant to doxorubicin) and against NIH/3T3 fibroblasts. Prodrug 28 was less active than doxorubicin 27 showing weak in vitro conversion of 28 into free drug against the four cell lines. Furthermore, the IC50s of doxorubicine 27, β-lapachone and the loaded nanoparticles obtained were equals in every cases except with the resistant MCF-7 cells where the micelles and β-lapachone were 4-5 times more active. This last observation highlight the particular efficacy of the latter molecules against doxorubicin resistant cancer cells. In vivo pharmacokinetics assays showed that both bioavailability and tumor concentration of doxorubicin 27 is improved when the doxorubicincontaining polymeric micelles are injected to mice in comparison with the administration of free drug at the same dose. The different molecules were tested on a MCF-7 ADR tumor xenograft model by intravenous injections at 5 mg/kg. An important tumor regression was observed (~ 90%) when treated with doxorubicin 27 loaded nanoparticles which was higher than the free doxorubicin 27 (~ 50%) and prodrug 28 (~ 30%).

34Scheme 17 .

 17 Scheme 17. Structures of chlorambucil, melphalan and the prodrugs of aromatic nitrogen mustard.

  therefore designed in order to reduce this property. A N-propargyl prodrug 45 displaying lower hydrophobicity was indeed less susceptible to aggregation and was also shown to display in vitro cytotoxicity similar to that of 44. Growth inhibiton of Guerin's carcinoma in rat was observed when 45 was administrated intraperitoneally at a single dose of 30 mg/kg seven days after tumor transplantation.119 Aminoferrocene theranostic prodrugs (scheme 20) were then developed by branching benzene boronate precursors of aminoferrocene to a [ 18 F]-fluoroglucose for positron emission tomography (PET).120 Theranostic prodrug 46 had an in vitro cytotoxicity close to that of the reference prodrug 44 (e.g. against HL-60, IC50(46) = 26 µM compared to IC50(44) ~ 10 µM).

  ability of prodrug 47 to disrupt lysosomal function was confirmed by a fluorescence imaging experiment. Prodrug 47 was evaluated in Nemeth-Kellner lymphoma xenograft mice model by intraperitoneal injection of eight doses of 40 mg/kg every two days for 15 days. The tumor weight of treated animals was reduced in comparison with the untreated cohort but the results were very contrasted.These aminoferrocene precursors were further complexified by the increment of a platinumbased antineoplastic prodrug moiety (48, 51 and 52) (scheme 20 and 21).[122][123][124]

Scheme 23 .

 23 Scheme 23. Prodrug of cinnamaldehyde.

≤<Scheme 25 .

 25 Scheme 25.Relative rate of C-B bonds oxidation of alkyl, benzyl and vinyl boronic acid derivatives.

Scheme 26 .

 26 Scheme 26. Antitumor drug and cancer-targeting prodrug strategies. (a) Typical antitumor drug binds to its biological target inducing a pharmacological effect. (b) Typical produg for cancer-targeted therapy is constituted of an existing drug linked to a bioresponsive promoiety.

Figure 15 .

 15 Figure 15. Oxidation of ProPhen into Phen and its deuterated isotope with 6.25 equivalents of H2O2. a: Chemical reaction and structures; b: Alignment of 1 H NMR spectra recorded every two minutes; c: Extraction of initial, intermediate (#50), final spectra and Phen; d: Mass spectrum of the mixture at the end of the reaction.

Figure 16 .

 16 Figure 16. Absorption spectra of ProPhen (black) and Phen (grey).

Figure 17 .

 17 Figure 17. Emission spectra of ProPhen (grey) and Phen (black) upon illuminating at λexc = 300 nm.

Scheme 39 .

 39 Scheme 39. Stopped flow method.

Figure 18 .

 18 Figure 18. Temporal evolution of the fluorescence emission at λem = 365 nm of a 0.5 µM ProPhen solution upon addition of H2O2 at various concentrations at 298 K: a: 50 µM; b: 100 µM; c: 200 µM. Grey dots: experimental data; black solid line: fit with Eq. 11.From the fits, we retrieved κ1 values equal to 6.5×10 -3 ± 2.1×10 -5 , 1.26×10 -2 ± 9×10 -5 , and 2.6×10 -2 ± 1.0×10 -4 s - 1 at 50, 100 and 200 µM H2O2 at 298 K. Solvent: PBS buffer:DMSO 99:1 v/v (pH 7.4). T = 298 K.

Figure 18 displays

 18 Figure 18 displays the temporal evolution of the fluorescence emission originating from adding various concentrations of H2O2 to a continuously illuminated 0.5 µM solution of ProPhen at 298K using the stopped-flow apparatus. As anticipated, an increase of fluorescence emission was observed, which is associated to the formation of the fluorescent phenanthridine Phen.

Figure 19 .

 19 Figure 19. Dependence of k1 on H2O2 concentration. Solvent: PBS buffer:DMSO 99:1 v/v (pH 7.4). T = 298 K.

Figure 20 .

 20 Figure 20. Bioactive phenanthridines.

Scheme 46 .

 46 Scheme 46. Synthetic pathway to precursors ProToxPhen.

Figure 21 .

 21 Figure 21. Cytotoxicity of ProToxPhen and ToxPhen on KB 3.1 cancer cells.

Figure 22 .

 22 Figure 22. Cytotoxicity on KB spheroids in function of the tested compounds at several increasing concentrations (scale bar: 250 µm).

  Such twist engenders a significant variation in the 3D structure of the biaryl in comparison to the phenanthridine scaffold. The molecular geometry of the open precursor is therefore drastically different from the cytotoxic phenanthridine ToxPhen (figure 23).

Figure 23 .Figure 24 .

 2324 Figure 23. Optimized 3D representations of ProToxPhen and ToxPhen and relative conformation energy diagrams as a function of dihedral angle of ProToxPhen.

  ToxPhen within KB 3.1 spheroids by video microscopy The absorption spectra of ProToxPhen and ToxPhen were measured in a solvent mixture: CH3CN:PBS buffer:DMSO 48.5:48.5:1 v/v/v. at T = 298 K in figure 25.

Figure 25 . 26 )Figure 26 .Figure 27 .

 25262627 Figure 25. Absorption spectra of ProToxPhen (grey) and ToxPhen (black). CH3CN:PBS buffer:DMSO 48.5:48.5:1 v/v/v. T = 298 K.

  and deposited on a 35 mm glass circular round microscope slide. 20 µL of SIN-1 (4 mM) in PBS was dropped on the spheroid to initiate the cyclization kinetic. Using a homemade epi-fluorescence microscope, we observed ToxPhen fluorescence emission in real time at 440 nm upon illumination at 365 nm (figure 28 and 29). This experiment was carried out in triplicates. We observed the same cyclization behavior among several cell populations and different spheroids. No cyclization was observed outside the living cells showing the absence of prodrug leakage over the time scale of our experiments.

Figure 28 .

 28 Figure 28. In-spheroid kinetic of cyclisation of ProToxPhen into ToxPhen studied by epifluorescence microscopy. (a) Image of the spheroid at time t = 5 s after SIN-1 (2 mM) was added to initiate the generation of ToxPhen from ProToxPhen; (b) Time evolution of the average fluorescence intensity following the addition of SIN-1. Signal extracted from regions of interest inside (red) and outside (green) the spheroid. Solvent: 100 mM PBS pH 7.4 buffer/DMSO 99:1 (v:v). T = 37°C.

Figure 29 .

 29 Figure 29. Temporal evolution of the fluorescence emission at λem = 440 nm of a 150 µM ProToxPhen solution upon addition of SIN-1 (2 mM). Black dots: experimental data; red solid line: fit with Eq. 11. From the fits, we retrieved τ = 3.5 s. Solvent: PBS buffer (pH 7.4). T = 298K.

Figure 30 .

 30 Figure 30. In-spheroid kinetics of cyclisation of 150 µM of ProToxPhen into ToxPhen studied by epifluorescence microscopy. (a): Image of the spheroid at t = 0 s (corresponding to t = 20 s after SIN-1 (2 mM) addition. (b): Time evolution of the average fluorescence intensity following the addition of SIN-1. Signal extracted from regions of interest at the edge of the spheroid (red) and in an isolated cell (green). Red and green dots: experimental data; red and green solid lines: fit with simple mono-exponential equation. From the fits, we retrieved 𝜏 = 11.8 s (red, edge of the spheroid) and 𝜏 = 13.3 s (green, isolated cell). Solvent: 100 mM PBS pH 7.4 buffer/DMSO 99:1 (v:v). T = 298 K.

Figure 31 .

 31 Figure 31. Plasma stability of ProToxPhen.

Figure 32 .

 32 Figure 32. Conversion of ProToxPhen into its Boronic acid counterpart via ester hydrolysis.

  ,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method (Promega). 7.10 3 KB 3.1 cells (100 µL) were seeded in 96-well plates with 100 µL of each compound at different concentrations (from 50 µM to 0.05 nM for ToxPhen compound and from 500 µM to 100 nM for the ProToxPhen compound) and incubated at 37 °C in presence of 5 % CO2 and under 95

table 1

 1

	and figure 6): 11

Table 1 .

 1 Subfamilies of antimetabolites drugs.

Table 2 .

 2 Examples of membrane-associated targets for prodrug therapy.23

	2. Medicinal chemists have therefore developed prodrugs composed of an
	antibody or a ligand coupled to an anticancer agent allowing targeting of cancer cells via ligand-
	receptor or antibody-antigen interactions. Cell surface targets can be classified as internalizing
	and non-internalizing systems. In non-internalizing systems, the drug conjugate has to be
	cleaved extracellularly, whereas in internalizing systems cleavage should occur intracellularly
	after endocytosis. 23

Table 3 .

 3 Overview of enzymes that are overexpressed in tumors.

	CA4-βGal

As shown in scheme 2, the Suzuki group developed a β-galactosidase-responsive prodrug composed of a galactose linked to the cytotoxic and antivascular Combretastatin A4 (CA4).

[START_REF] Doura | Combretastatin A4-β-Galactosyl Conjugates for Ovarian Cancer Prodrug Monotherapy[END_REF]

Table 4 .

 4 Overview of pH-sensitive linkers used in anticancer prodrug development.[START_REF] Patel | Activation Approaches on Delivery of Imaging and Therapeutic Agents[END_REF]

	Linkers	Chemical structures	Therapeutics agents
			Diethylstilboestrol
	Acetal		Doxorubicin
	Ester		Bortezomib Doxorubicin
	Hydrazone		Cisplatin Doxorubicin
			Geldanamycin
	Imine		Gemcitabine Chromone
			Doxorubicin
	Othoester		Paclitaxel
			Daunorubicin
	Oxime		Doxorubicin
			Oligonucleotide
	Phosphoramidate		Oligonucleotide
	Vinyl ether		Oligonucleotide
	β-Thiopropionate		Oligonucleotide

concentration. An overview of pH sensitive linkers used in anticancer prodrug development is given in table

4

.

[START_REF] Patel | Activation Approaches on Delivery of Imaging and Therapeutic Agents[END_REF]

  Theranostic boronate-based prodrugs of campthotecin were also prepared. In cancer chemotherapy, this approach combining therapy and diagnosis allows the visualization of the drug by imaging its localization within the organism. The Kim group prepared a prodrug 25[START_REF] Kim | An Activatable Prodrug for the Treatment of Metastatic Tumors[END_REF] able to release a fluorophore (a coumarin) and SN-38 22 whereas the Shabat group developed a precursor 26 liberating a near-infrared cyanine fluorophore and campthotecin 21 (scheme 12).[START_REF] Redy-Keisar | NIR Fluorogenic Dye as a Modular Platform for Prodrug Assembly: Real-Time in Vivo Monitoring of Drug Release[END_REF] Prodrug 25 and SN-38 22 were evaluated in vitro against melanoma cells B16F10 and cervical cancer cells Hela. The prodrug 25 was able to restore around 60% of the SN-38 22 activity on both cancer cells and a complete recovery was obtained when adding 100 µM of H2O2. This latter observation was made at 100 µM of tested compounds, a concentration way beyond the IC50 of SN-38 22. The authors treated a B16F10 melanoma xenograft in mice with 25 at 0.25 mg/kg or with a saline solution. Again, a relatively limited efficacy was observed with a slightly prolonged survival time (1-6 days) of the treated mice over the non-treated group.

	prepared the
	oxidative prodrug 24 of SN-38 22 by replacing the original phenol group into a boronic acid
	function (scheme 12).

Scheme 12. Prodrugs of camptothecin.

Prodrug 24 and the corresponding active metabolite SN-38 22 were tested in vitro against six cancer cell lines: colon cancer cells HCT-15 and HT-29, breast cancer cells MCF-7 and MDA-MB-231 and, glioblastoma cells U87MG and U251. The activity of SN-38 22 was fully restored upon incubation of 24 with the different cell lines (IC50 ranging from 0.01 to 5 µM). The authors observed that the conversion of 24 into SN-38 22 in MCF-7 culture cell media was around 40% after 48h of incubation. Moreover, the topoisomerase I inhibitory evaluation of 24 showed that the boronic acid derivative was as potent as SN-38 22. These two latter observations therefore account for the good in vitro activity of 24 against this panel of cancer cells. Prodrug 24 was evaluated in vivo using U87MG cancer xenograft in mice and displayed a modest efficacy at 2 mg/kg in comparison with the reference irinotecan 23 (antitumor effect of 8% T/C with irinotecan vs. 28% T/C with 24).

Regrettably, irinotecan 23 or the active ingredient SN-38 22 were not evaluated in this in vivo study. In regards to the cyanine prodrug 26, the activity against U87MG cells was first determined in vitro: the authors observed an IC50 of 0.25 µM for the prodrug 26 whereas campthotecin 21 was ten times more active (IC50 = 0.02 µM). Nonetheless, the pre-incubation of H2O2 with 26 lead to the full release of campthotecin 21 and therefore the original IC50 of this free drug was re-established. In vivo imaging of the prodrug 26 using U87MG cancer xenograft in mice showed a precisely localized activation within the tumor after intratumoral administration.

  Tf2O, Et3N in DCM during 24h at RT to give the triflate 68 in 28% yield. The triflate group was exchanged into boronate pinacol ester group with the method of Miyaura et al. 168 to give vinyl boronate motif 71 in a global yield of 13%.

	according	to	the	procedure	involving	bis(pinacolato)diboron,	1,3-
	bis(cyclohexyl)imidazolium tetrafluoroborate (ICyBF4), t-BuONa and CuCl by Hanna et
	al. 74 Subsequent dehydration reaction with p-toluenesulfonic acid (PTSA) gave vinyl
	boronate 71 in a global yield of 57%.

-In the synthetic pathway C, α-boryl alcohol 70 was prepared from acetophenone 69 Scheme 33. Synthetic pathway to generate vinyl boronate 71.

Table 5 .

 5 Conditions used for the reduction of the nitro group.We therefore decided to replace the nitrobenzene used in the previous synthetic scheme by a protected aniline (Scheme 36). N-Boc aniline 78 was treated with t-BuLi leading to an ortholithiated intermediate which was directly reacted with trimethyltin chloride to afford stannane 80 in 51% yield. Formation of biaryl 82 was achieved via a Stille coupling reaction with 76.Again, we observed a total selectivity for the Stille reaction. Eventually, removal of the Boc protecting group afforded the precursor ProPhen in 32% yield over two steps.

	Scheme 36. Synthetic pathway to precursors ProPhen.
	In order to study the cyclization of precursor ProPhen, the corresponding product 6-
	methylphenanthridine Phen previously prepared (scheme 29) of this reaction was used as a
	reference product.

  All chemical reagents were of analytical grade, obtained from Acros, Alfa Aesar, or Aldrich, and used without further purification. Solvents were obtained from SDS or VWR-Prolabo. MHz), AV 300 ( 1 H at 300 MHz and 13 C at 75 MHz and 11 B at 96 MHz) or AV 360 ( 1 H at 360 MHz and 13 C at 90 MHz) spectrometers and analyzed using MestReNova software.

	3. Chemistry
	Dichloromethane (DCM) was freshly distilled under CaH2 and used immediately.
	Tetrahydrofurane (THF) was freshly distilled under sodium and used immediately. Toluene Compound 79. To a solution of aniline 78 (300 mg, 3.2 mmol, 1.0 eq) in 65 mL of THF was
	was freshly distilled under CaH2 and used immediately. Flash chromatography was performed added triethylamine (850 µL, 6.4 mmol, 2.0 eq) and Boc2O (700 mg, 3.2 mmol, 1.0 eq). The
	using silica gel (35-70 μm, Merck). Evaporation of solutions was performed under reduced pressure at temperature below 40 °C using rotary evaporator. Analytical TLC was performed using Silica Gel 60 F254 pre-coated aluminum plates (Merck). Spots were visualized by treatment with phosphomolybdic acid (PMA) revelator (for molecules containing boron atom) followed by heating and/or by absorbance/emission under UV light at 254 or 360 nm, or diode or KMnO4 on silica. NMR spectra were collected on Bruker DPX 250 ( 1 H at 250 MHz and 13 C bis(pinacolato)diboron (3.16 g, 12.5 mmol, 1.5 eq), 1,3-bis(cyclohexyl)imidazolium tetrafluoroborate (266 mg, 0.83 mmol, 0.1 eq), t-BuONa (160 mg, 1.6 mmol, 0.2 eq), and CuCl (164 mg, 1.6 mmol, 0.2 eq). 60 mL of toluene were added, the flask was sealed, and the reaction was stirred for 30 min. Benzophenone 69 (1 g, 8.3 mmol, 1 eq) was then added via a syringe followed immediately by MeOH (672 µL, 16.6 mmol, 2 eq) addition. After 24 h, the mixture was then stirred at r.t. overnight. When the reaction was complete (checked by TLC), the THF was removed by evaporation and the mixture was poured into water (10 mL). The residual water phase was extracted with EtOAc (3 x 30 mL) and dried over Na2SO4. Removal of solvent under reduced pressure gave a crude product that was purified by flash chromatography (cyclohexane: EtOAc = 3:1) to afford the expected product (490 mg, 80%) as a white powder. at 62.5 Compound 70. To a flame-dried 100 mL round-bottom flask containing a stir bar was added mixture was loaded directly onto a short pad of silica gel pretreated with DCM. The pad was 1 H NMR (360 MHz, CDCl3) δ 7.40 -7.26 (m, 3H), 7.07 -7.00 (m, 2H), 6.36 (s, 1H), 1.52 (s,
	thoroughly washed with Et2O and the filtrate was evaporated under reduced pressure. The 9H).
	resulting residue was purified by flash chromatography on silica gel (cyclohexane:EtOAc =
	95:5 to 70:30) to give the expected product (1.16 g, 57%) as a light yellow oil.
	13 C NMR (75 MHz, CDCl3) δ 140.92, 129.30, 127.12, 126.45, 97.63, 84.54, 83.48, 26.89,
	25.06, 17.16.
	Technology, Bellows Falls, VT) and a LXZ1-PL02 LED (Lumileds, Schipol, Netherlands)
	filtered at 585 ± 10 nm as light sources. The LEDs were supplied by a LED driver (LEDD1B,
	Thorlabs Inc, Newton, NJ. A lens (ACL2520U; Thorlabs, f = 20 mm) and were placed just after
	each diode to collimate the light source. A dichroic mirror (T425LPXR, Chroma, Bellows Falls,
	VT; or a FF505-606-Di01, Semrock, Rochester, NY for excitation at 585 nm) and a second pair
	of lenses were used to focus the light at the back focal plane of the objective. Fluorescence
	images at 440 ± 20 nm (HQ 440/40x; Chroma Technology) or at 628 ± 16 (FF628-32, Semrock)
	were acquired with a 50× MPLFLN (NA 0.8, Olympus Corporation, Tokyo, Japan) with a Luca-
	R CCD camera (Andor Technology, Belfast, UK).
	122

Chemical shifts are reported in ppm (δ) and coupling constants in Hz (J). NMR spectra were performed in CDCl3, (CD3)2SO, CD3CN or D2O. High-resolution mass spectrometry (HRMS) analyses were performed by electrospray with positive or negative (ESI + or ESI -). 1 H NMR (300 MHz, CDCl3) δ 7.48-7.22 (m, 5H), 1.65 (s, 3H), 1.25 (s, 12H).

The spectral data are similar to those reported in: J. Org. Chem. 1988, 53, 6138-6139.

  °C for 6 h. The reaction mixture was quenched with saturated NaHCO3 aqueous solution (5 mL) at 0 °C. The organic phase were extracted with Et2O (2 x 30 mL) and washed with brine (50 mL). The combined organic phases were dried over Na2SO4 and concentrated under reduced pressure. Flash chromatography using neutral alumina prewashed with a 40:4:1 mixture of hexane:Et3N:EtOAc and eluted with 20:1 mixture of hexane:EtOAc gave the expected product (77 mg, 46%) as an yellow oil. CDCl3) δ 154.42, 153.38, 143.20, 136.44, 108.53, 100.23, 89.64, 80.13, 28.33, -7.85. HRMS (ESI + ): m/z Calcd for C15H23NO4Sn + Na, [M+Na] + : 424.0541, found: 424.0544.

	Compound 85. N-Boc-3,4-methylenedioxyaniline 84 (100 mg, 0.42 mmol, 1.0 eq) was placed
	Compound 81. To a flame-dried 50 mL round-bottom flask containing a stir bar was added Compound 76. To a solution of 81 (1.5 g, 4 mmol, 1 eq) in 80 mL of toluene was added p-in an oven-dried round-bottom flask and dissolved in 1 mL of dry Et2O. The solution was cooled
	bis(pinacolato)diboron (1.52 g, 6.0 mmol, 1.5 eq), 1,3-bis(cyclohexyl)imidazolium toluenesulfonic acid (2.1 g, 12 mmol, 3 eq). The mixture was heated at 60°C during 2 h. 50 mL to -40 °C under an Ar atmosphere. t-BuLi (1.7 M in pentane, 524 µL, 0.89 mmol, 2.1 eq) was
	tetrafluoroborate (128 mg, 0.4 mmol, 0.1 eq), t-BuONa (77 mg, 0.8 mmol, 0.2 eq), and CuCl of a Na2CO3 saturated aqueous solution was added and the aqueous layer was extracted with ProPhen. To a solution of Boc derivative 82 (50 mg, 0.12 mmol, 1.0 eq) in 1 mL of dry added via a syringe pump over 30 min, during which the mixture became homogenous, and
	(79 mg, 0.8 mmol, 0.2 eq). 30 mL of Toluene was added, the flask was sealed, and the reaction EtOAc (3x50 mL). The organic phases were combined, washed with brine, dried with MgSO4 dichloromethane was added 2,6-lutidine (162 µL, 1.4 mmol, 12 eq) and TMSOTf (128 µL, 0.71 the resulting solution was stirred at 0 °C for 3 h. The reaction mixture was further cooled to -
	was stirred for 30 min. Iodobenzophenone 61 (1 g, 4.0 mmol, 1 eq) was added via a syringe followed immediately by MeOH (342 µL, 8 mmol, 2 eq) addition. After 24 h, the mixture was loaded directly onto a short pad of silica gel pretreated with DCM. The pad was thoroughly 1 H NMR (300 MHz, CDCl3) δ 7.32 (s, 4H), 7.14 -6.98 (m, 2H), 6.81 -6.66 (m, 2H), 5.98 (d, J (cyclohexane:EtOAc = 80:20); Rf = 0.71; revelation by PMA. purification. expected product (1.12 g, 75%) as a light yellow oil. TLC conditions: eluent (cyclohexane:EtOAc = 70:30) to give the expected product (12 mg, 32%) as a brown power. reduced pressure to give a crude product that was engaged in the next step without further residue was purified by flash chromatography (cyclohexane:EtOAc = 95:5 to 70:30) to give the 1 H NMR (360 MHz, CDCl3) δ 7.80 (dd, J = 7.6, 1.3 Hz, 1H), 7.31 (td, J = 7.6, 1.3 Hz, 1H), 7.18 chromatography (cyclohexane:EtOAc = 70:30) followed by purification by PTLC celite which was thoroughly washed with EtOAc. The filtrate was then concentrated under washed with Et2O and the filtrate was evaporated under reduced pressure. The resulting and concentrated under reduced pressure to give the expected product (1.41 g, 100%) as a colorless oil. toluene under argon for 1h. The mixture was quenched with EtOAc, filtered through a pad of over MgSO4 and concentrated on a rotary evaporator. The crude product was purified by flash TLC conditions: eluent (cyclohexane:EtOAc = 70:30); Rf = 0.78. mmol, 6 eq) at r.t. and under Ar atmosphere. The solution was stirred for 1h30 before water 78 °C and Me3SnCl (84 mg, 0.42 mmol, 1.0 eq) in 1 mL of Et2O solution was added and the (222 mg, 0.66 mmol, 1.1 eq), 76 (210 mg, 0.6 mmol, 1 eq), Pd(PPh3)4 (14 mg, 0.012 mmol, vigorously for 15 min. The organic phase was separated, washed with water and brine, dried 0.02 eq) and CuCN (10 mg, 0.12 mmol, 0.2 eq) and the mixture was refluxed in 6.5 mL of (10 mL) was added. The resulting mixture was diluted with CH2Cl2 (50 mL) and stirred reaction was stirred at 0
	13 C NMR (91 MHz, CDCl3) δ 147.77, 138.38, 132.74, 128.78, 128.34, 128.25, 98.62, 83.94, TLC conditions: eluent (cyclohexane:EtOAc = 80:20); Rf = 0.75. = 3.0 Hz, 1H), 5.86 (d, J = 3.0 Hz, 1H), 1.04 (s, 6H), 1.02 (s, 6H).
	1 H NMR (250 MHz, CDCl3) δ 7.52 (d, J = 8.2 Hz, 1H), 7.40 (dd, J = 7.2, 1.5 Hz, 1H), 7.32 (td, J = 7.7, 1.7 Hz, 1H), 7.12 (td, J = 7.3, 1.1 Hz, 1H), 6.28 (s, 1H), 1.51 (s, 9H), 0.34 (s, 9H). (td, J = 7.6, 1.3 Hz, 1H), 6.93 (td, J = 7.6, 1.3 Hz,1H), 2.26 (s, 1H), 1.67 (s, 3H), 1.33 (s, 12H). 13 C NMR (75 MHz, CDCl3) δ 148.62, 140.42, 128.56, 128.34, 126.45, 97.63, 84.54, 83.48, 26.89, 25.06. 11 B NMR (96 MHz, CDCl3) δ 31.47. HRMS (ESI + ): m/z Calcd for C14H20BIO3 + Na, [M+Na] + : 397.0445, found [M+Na] + : 397.0421. 24.90. 11 B NMR (96 MHz, CDCl3) δ 29.67. HRMS (ESI + ): m/z Calcd for C14H18BIO2 + H, [M+H] + : 357.0522, found: 357.0519. HRMS (ESI + ): m/z Calcd for = C25H32BNO4 + H, [M+H] + : 422.2502, found: 422.2501. HRMS (ESI + ): m/z Calcd for C25H32BNO4 + Na, [M+Na] + : 444.2322, found: 444.2321. 13 C NMR (75 MHz, CDCl3) δ 144.10, 142.30, 137.08, 132.18, 131.87, 130.39, 129.28, 128.25, 127.59, 127.20, 118.07, 115.68, 83.45, 24.77, 24.45. 11 B NMR (96 MHz, CDCl3) δ 29.99. that was purified by flash chromatography (cyclohexane:EtOAc = 3:1) to give the expected product (507 mg, 98%) as an white powder 1 H NMR (360 MHz, CDCl3) δ 7.03 (bs, 1H), 6.74 -6.67 (m, 1H), 6.67 -6.57 (m, 1H), 6.37 (bs, HRMS (ESI and dried over Na2SO4. Removal of solvents under reduced pressure gave a crude product 1H), 5.91 (s, 2H), 1.50 (s, 9H).

The spectral data are similar to those reported in: J. Org. Chem. 1988, 53, 6138-6139. 1 H NMR (300 MHz, CDCl3) δ 7.82 (dd, J = 7.8, 1.3 Hz, 1H), 7.57 (dd, J = 7.9, 1.7 Hz, 1H), 7.36 (dd, J = 7.6, 1.8 Hz, 1H), 6.93 (td, J = 7.6, 1.3 Hz, 1H), 6.12 (d, J = 3.3 Hz, 1H), 5.73 (d, J = 3.3 Hz, 1H), 1.32 (s, 12H). + ): m/z Calcd for = C20H24BNO2 + H, [M+H] + : 322.1976, found: 322.1966. HRMS (ESI + ): m/z Calcd for C20H24BNO2 + Na, [M+Na] + : 344.1797, found: 344.1781. 128 13 C NMR spectra of ProPhen 129 Compound 84.

To a solution of 3,4-methylenedioxyaniline 83 (300 mg, 2.19 mmol, 1 eq) in 45 mL of THF was added triethylamine (581 µL, 4.4 mmol, 2 eq) and Boc2O (478 mg, 2.19 mmol, 1 eq). Then the mixture was stirred at r.t overnight. When the reaction was complete (checked by TLC), the THF was removed under reduced pressure and the resulting mixture was poured onto water (10 mL). The aqueous mixture was extracted with EtOAc (3 x 30 mL)

The spectral data are similar to those reported in: Eur. J. Org. Chem. 2010, 3704-3710. 1 H NMR (250 MHz, CDCl3) δ 7.52 (d, J = 8.2 Hz, 1H), 7.40 (dd, J = 7.2, 1.5 Hz, 1H), 7.32 (td, J = 7.7, 1.7 Hz, 1H), 7.17 -7.08 (m, 1H), 6.29 (s, 1H), 1.51 (s, 9H), 0.34 (s, 9H). 13 C NMR (91 MHz,

  NMR (300 MHz, CDCl3) δ 6.95 (s, 1H), 6.76-6.74 (m, 2H), 6.49 (s, 1H), 4.20 (s, 4H), 1.48 (s, 9H). CDCl3) δ 152.93, 143.41, 139.60, 132.05, 117.02, 112.29, 108.43, 80.14, 64.34, 64.13, 28.24, 26.79. HRMS (ESI + ): m/z Calcd for C13H17NO4 + Na, [M+Na] + : 274.1049, found: 274.1044Compound 92. To a solution of but-3-yn-2-ol 91 (10 g, 143 mmol, 1 eq) in anhydrous DCM (0.5 M) were added TBDMSCl (28.0 g, 186 mmol, 1.3 eq) and imidazole (12.3 g, 186 mmol, 1.3 eq) at 0°C under argon. The resulting mixture was warmed to room temperature and stirred at the same temperature overnight. Then, the reaction mixture was treated with saturated aqueous NH4Cl and extracted with EtOAc, washed with water and brine, dried over MgSO4, and concentrated under reduced pressure to give 21.3 g (81%) of silylated ether. 250 MHz, CDCl3): δ = 4.51 (qd, J = 6.6, 2.1 Hz, 1H), 2.37 (d, J = 2.0 Hz, 1H), 1.42 (d, J = 6.6 Hz, 3H), 0.90 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H).The spectral data are similar to those reported in: J. Am. Chem. Soc. 2004, 126, 16553-16558. To a solution of 2-(3,4-dimethoxyphenyl)ethanol 89 (4.0 g, 11.0 mmol, 1eq) in 100 mL dichloromethane was added Dess-Martin reagent (11.2 g, 13.2 mmol, 1.2 eq). The mixture was stirred at r.t. overnight. The suspension was filtered and the solid was rinsed with dichloromethane. The filtrate was concentrated under reduced pressure to give the expected product that was engaged in the next step without further purification. NMR (360 MHz, CDCl3) δ 9.71 (t, J = 2.4 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H), 6.76 (dt, J = 8.1, 2.0 Hz, 1H), 6.69 (d, J = 2.0 Hz, 1H), 3.86 (s, 6H), 3.61 (d, J = 2.4 Hz, 2H). To a solution of 92 (6.8 g, 37 mmol, 2 eq) in 50 mL of anhydrous THF was added n-BuLi 2.5 M (14.8 mL, 37 mmol, 2 eq) at 0°C under Ar atmosphere. The resulting solution was stirred at the same temperature for 1 h. Then 90 (3.31 g, 18 mmol, 1 eq) in 11 mL of THF was added dropwise by syringe. The reaction mixture was kept under the inert atmosphere and stirred for 16 h while it warmed up to ambient temperature. The mixture was then quenched by adding saturated aqueous NH4Cl and extracted twice with EtOAc. The combined organic fractions were dried over MgSO4 and concentrated under vacuum to yield the crude product, which was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 95:5 to 60:40) to give 3.30 g (50%) of expected product. CDCl3) δ 6.80-6.82 (m, 3H), 4.65 -4.40 (m, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.00 -2.85 (m, 2H), 1.38 (d, J = 6.5 Hz, 3H), 0.92 -0.86 (m, 9H), 0.12 -0.06 (m, 6H). CDCl3) δ 148.82, 148.03, 129.30, 121.79, 112.95, 111.18, 88.23, 83.44, 63.26, 63.13, 58.94, 55.83, 43.57, 25.78, 25.38, -4.60, -4.95. HRMS (ESI + ): m/z Calcd for C20H32O4Si + Na, [M+Na] + : 387.1962, found: 387.1949.Compounds 94 and 95. To a solution of 93 (3.30 g, 9.1 mmol 1 eq) in 70 mL of dry MeCN was added NaHCO3 (1.53 g, 18.2 mmol, 2 eq). The solution was stirred for 1 min at room temperature then ICl (2.95 g 18.2 mmol, 2 eq) in 10 mL of dry CH3CN was added dropwise.The reaction mixture was stirred at room temperature during 5 min. The reaction mixture was then diluted with 100 mL of EtOAc and washed with 50 mL of saturated aqueous Na2S2O3. The organic layer was separated, and the aqueous layer was extracted three time with 30 mL of EtOAc. The combined organic layers were dried over MgSO4 and filtered. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 95:5 to 60:40) to give 641 mg of 94 (15%) and 2.76 g (85%) of 95. CDCl3) δ 149.24, 148.21, 140.63, 134.58, 130.69, 127.69, 127.46, 106.58, 105.96, 94.38, 79.09, 55.70, 26.90, 25.86, 23.29, -4.76, -4.97. HRMS (ESI + ): m/z Calcd for C20H29IO3Si + Na, [M+Na] + : 495.0822, found: 495.0805. NMR (360 MHz, CDCl3) δ 8.22 (s, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.18 (dd, J = 11.3, 5.6 Hz, 1H), 6.98 (s, 1H), 5.67 (q, J = 6.7 Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 1.63 (d, J = 6.8 Hz, 3H). 13 C NMR (91 MHz, CDCl3) δ 149.39, 148.63, 139.59, 134.77, 130.72, 128.07, 127.28, 106.90, 104.93, 78.30, 55.89, 55.75, 21.63. Compound 96. To a solution of compound 95 (1.50 g, 4.2 mmol, 1 eq) in 60 mL of dry DCM was added PCC (4.5 g, 21 mmol, 5 eq). The dark solution was stirred at r.t. during 24h. 50 mL of Et2O was added, and the suspension was filtered under celite. The pad of celite was washed with DCM and the was concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 95:5 to 60:40) to give 1.46 g (98%) of expected product as an orange powder. 300 MHz, CDCl3) δ 7.64 (d, J = 8.6 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.08 (s, J = 5.6 Hz, 1H), 6.80 (s, 1H), 3.98 (s, 3H), 3.93 (s, 3H), 2.70 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ 206.52, 150.59, 150.07, 143.10, 133.37, 128.66, 128.49, 125.63, 106.67, 102.71, 56.4, 28.05. HRMS (ESI + ): m/z Calcd for C14H13IO3 + H, [M+H] + : 356.9987, found: 356.9975. HRMS (ESI + ): m/z Calcd for C14H13IO3 + Na, [M+Na] + : 378.9801, found: 378.9801.

	Compound 93. Compound 94:
	1 H NMR (300 MHz, CDCl 3 ) δ 8.53 (s, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.47 -7.39 (m, 1H), 7.21
	(s, 1H), 5.80 (q, J = 6.6 Hz, 1H), 4.17 (s, 3H), 4.16 (s, 3H), 1.80 (d, J = 6.6 Hz, 3H), 1.04 (s,
	9H), 0.28 (s, 3H), 0.10 (s, 3H).
	C NMR (91 MHz, CDCl3) δ 199.40, 149.22, 148.30, 124.11, 121.74, 112.50, 111.50, 55.84, Compound 90. 13 Compound 95:
	55.77, 49.98.
	The spectral data are similar to those reported in: Monatshefte fur Chemie, 2004, 135, 1289-
	1295.

1 H 13 C NMR (75 MHz, 1 H NMR (1 H 1 H NMR (300 MHz, 13 C NMR (75 MHz, 13 C NMR (75 MHz, 1 H HRMS (ESI+): m/z Calcd for C14H15IO3 + Na, [M+Na] + : 380.9963, found: 380.9956. 1 H NMR (

  NMR (75 MHz, CDCl3) δ 155.36, 148.54, 147.26, 143.43, 128.60, 120.94, 118.99, 112.87, 108.51, 107.05, 101.52, 100.76, 99.21, 97.72, 64.72, 64.40, 26.79. HRMS (ESI + ): m/z Calcd for C14H11N + H, [M+H] + : 194.0929, found: 194.0964. CDCl3) δ 156.66, 149.34, 148.97, 143.30, 132.66, 130.77, 129.14, 128.72, 128.46, 126.21, 125.78, 123.71, 122.61, 122.32, 118.62, 108.24, 108.11, 56.02, 55.92, 31.22. HRMS (ESI + ): m/z Calcd for C20H17NO2 + H, [M+H] + : 296.0917, found: 296.0913. CDCl3) δ 8.32 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.1 Hz, 1H), 7.99 (s, 1H), 7.63 (s, 1H), 7.53 (dd, J = 19.4, 12.2 Hz, 1H), 7.26 (dt, J = 15.1, 7.2 Hz, 1H), 4.53 -4.35 (m, 4H), 2.93 (s, 3H). 13 C NMR (91 MHz, CDCl3) δ 157.65, 147.10, 143.95, 142.98, 129.13, 128.36, 127.88, 126.04, 123.47, 121.74, 121.69, 113.27, 109.08, 64.69, 64.40, 23.35. HRMS (ESI + ): m/z Calcd for C16H13NO2 + H, [M+H] + : 252.1019, found: 252.1018. CDCl3) δ 8.43 (d, J = 8.3 Hz, 1H), 8.20 (d, J = 8.2 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.28 (s, 1H), 6.15 (s, 2H), 3.03 (s, 3H). CDCl3) δ 156.57, 149.16, 147.58, 140.87, 132.62, 130.16, 126.52, 126.40, 125.06, 122.08, 119.46, 107.18, 101.67, 99.35, 22.99. HRMS (ESI + ): m/z Calcd for C15H11NO2 + H, [M+H] + : 238.0863, found: 238.0859. NMR (91 MHz, CDCl3) δ 154.83, 149.14, 149.02, 144.60, 141.82, 139.93, 130.71, 130.13, 129.35, 125.36, 122.78, 122.67, 122.23, 111.49, 110.93, 107.95, 107.87, 101.66, 55.98, 55.90, 31.08. HRMS (ESI + ): m/z Calcd for C21H17NO4 + H, [M+H] + : 348.1235, found: 348.1219. CDCl3) δ 155.36, 148.54, 147.26, 143.43, 128.60, 120.94, 118.99, 112.87, 108.51, 107.05, 101.52, 100.76, 99.21, 97.72, 64.72, 64.40, 26.79. HRMS (ESI + ): m/z Calcd for C17H13NO4 + H, [M+H] + : 296.0917, found: 296.0913. CDCl3) δ 9.40 (dd, J = 8.6, 0.6 Hz, 1H), 8.13 (dd, J = 8.2, 1.0 Hz, 1H), 7.79 -7.54 (m, 3H), 7.26 -7.15 (m, 1H), 4.53 -4.32 (m, 4H), 2.92 (s, 3H). 13 C NMR (91 MHz, CDCl3) δ 156.81, 141.06, 140.44, 139.94, 132.13, 129.87, 128.14, 126.84, 126.30, 125.96, 122.37, 119.01, 114.93, 64.30, 63.91, 23.26. HRMS (ESI + ): m/z Calcd for C16H13NO2 + H, [M+H] + : 252.1019, found: 252.1018. CDCl3) δ 150.60, 150.09, 143.52, 143.12, 139.51, 133.41, 132.04, 128.68, 128.52, 125.65, 117.13, 112.34, 108.47, 106.68, 102.72, 84.79, 64.43, 64.22, 55.97, 55.92, 31.43. HRMS (ESI + ): m/z Calcd for C22H19NO4 + H, [M+H] + : 362.1386, found: 362.1373. NMR (75 MHz, CDCl3) δ 155.58, 153.31, 143.31, 143.09, 139.79, 133.62, 129.68, 124.30, 120.33, 118.73, 116.81, 115.26, 112.86, 65.08, 64.29, 63.92, 63.22, 28.29. HRMS (ESI + ): m/z Calcd for C18H15NO4 + H, [M+H] + : 310.1073, found: 310.1063.

	Phen 103 104
	1 H NMR (300 MHz, CDCl3) δ 7.79 (s, 1H), 7.65 (s, 1H), 7.59 (s, 1H), 7.43 (bs, 1H), 6.10 (s, 2H), 4.41 (m, 4H), 2.90 (s, 3H). Yield = 41% 101 1 Yield = 63% H NMR (400 MHz, Yield = 47% 1 H NMR (360 MHz, CDCl3) δ 8.90 (d, J = 8.9 Hz, 1H), 8.30 (s, 1H), 7.99 (d, J = 8.9 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.42 -7.30 (m, 2H), 6.29 (s, 2H), 4.12 (s, 3H), 4.08 (s, 3H), 3.39 (s, 3H). 1 106 1 H NMR (360 MHz, CDCl3) δ 7.57 (s, 1H), 7.47 -7.29 (m, 1H), 7.27 -7.08 (m, 1H), 6.86 -H NMR (360 MHz, Yield = 51% 6.48 (m, 1H), 4.55 -3.95 (m, 8H), 1.36 (s, 3H).
	100
	1 H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 8.2 Hz, 1H), 8.51 (d, J = 8.9 Hz, 1H), 8.32 (s, 1H),
	8.16 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.76 (t, J = 7.4 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.34 (s, 1H), 4.14 (s, 3H), 4.10 (s, 3H), 3.47 (s, 3H). ToxPhen 105
	102 H NMR (300 MHz, CDCl3) δ 7.79 (s, 1H), 7.65 (s, 1H), 7.59 (s, 1H), 7.43 (s, 1H), 6.10 (s, 2H), 1 1 H NMR (360 MHz, CDCl3) δ 7.59 (d, J = 8.6 Hz, 1H), 7.33 (t, J = 9.2 Hz, 1H), 7.19 (s, 1H),
	Yield = 55% 1 7.02 (s, 1H), 6.74 (s, 1H), 6.70 (s, 1H), 4.20 -4.08 (m, 4H), 3.93 (s, 3H), 3.87 (s, 3H), 2.65 (s, 4.41 (m, 4H), 2.90 (s, 3H). Yield = 91% 3H). H NMR (300 MHz, Yield = 76% Yield = 42%

13 C 13 C NMR (101 MHz, 13 C NMR (75 MHz, 13 C 13 C NMR (75 MHz, 13 C NMR (91 MHz, 13 C

  NMR (400 MHz, CDCl3) δ 7.04 (s, 1H), 6.75 (s, 1H),6.03 (d, J = 3.3 Hz, 1H), 5.75 (d, J = 3.3 Hz, 1H), 4.22 (s, 4H), 1.30 (s, 12H). CDCl3) δ 128.88, 128.64, 123.74, 120.11, 111.04, 109.06, 105.34, 82.37, 67.03, 66.98, 36.22, 36.04. Calcd for C16H20BBrO4 + H, [M+H] + : 367.0716, found: 367.0698. HRMS (ESI + ): m/z Calcd for C16H20BBrO4 + NH4, [M+NH4] + : 384.0979, found: 384.0964.(67 mg, 0.17 mmol, 1.1 eq), 110 (56 mg, 0.15 mmol, 1 eq), Pd(PPh3)4 (3.5 mg, 0.0036 mmol, 0.02 eq) and CuCN (2.7 mg, 0.036 mmol, 0.2 eq) and the mixture was refluxed in 4 mL of toluene under argon for 14h. The mixture was quenched with EtOAc, filtered through a pad of celite which was thoroughly washed with EtOAc. The filtrate was then concentrated under reduced pressure to give a crude product that was engaged in the next step without further purification.HRMS (ESI+ ): m/z Calcd for = C28H34BNO8 + H, [M+H] + : 524.2455, found: 524.2441. HRMS (ESI + ): m/z Calcd for C28H34BNO8 + Na, [M+Na] + : 546.2257, found: 546.2274.ProToxPhen. To a solution of Boc derivative 111 (13 mg, 0.025 mmol, 1.0 eq) in 1 mL of dry dichloromethane was added 2,6-lutidine (35 µL, 0.30 mmol, 12 eq) and TMSOTf (27 µL, 0.15 mmol, 6.0 eq) at r.t. and under Ar atmosphere. The solution was stirred for 1h30 before water (10 mL) was added. The resulting mixture was diluted with CH2Cl2 (50 mL) and stirred vigorously for 15 min. The organic phase was separated, washed with water and brine, dried over MgSO4 and concentrated on a rotary evaporator. The crude product was purified by flash chromatography (cyclohexane:EtOAc = 70:30) followed by purification by PTLC (cyclohexane:EtOAc = 70:30) to give the expected product (4.1 mg, 39%) as a brown power. 91 MHz, CD3CN) δ 149.21, 146.40, 144.47, 144.19, 137.49, 132.43, 120.86, 118.58, 113.53, 111.66, 103.27, 103.17, 84.77, 65.29, 65.23, 25.05.

1 H 13 C NMR (96 MHz, 11 B NMR (96 MHz, CDCl3) δ 29.60. HRMS (ESI + ): m/z 1 H NMR (360 MHz, CD3CN) δ 7.08 (s, 1H), 6.90 (s, 1H), 6.84 (s, 1H), 6.76 (s, 1H), 6.10 -5.98 (m, 2H), 5.98 (d, J = 3.2 Hz, 1H), 5.76 (d, J = 2.9 Hz, 1H), 4.27 (s, 4H), 1.29 (s, 12H). 13 C NMR (11 B NMR (96 MHz, CD3CN) δ 32.78. HRMS (ESI + ): m/z Calcd for = C23H26BNO6 + H, [M+H] + : 424.1930, found: 424.1912. 150 Bibliography

Remerciements

Je souhaite remercier en premier lieu Monsieur Laurent Salmon, Professeur de l'Université Paris Saclay, pour m'avoir accueilli au sein de l'équipe « Chimie Bioorganique et Bioinorganique » de l'Institut de Chimie des Milieux Moléculaire et des Matériaux d'Orsay.

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Antibody Drug Conjugates (ADCs)

Many ADCs have been developed in the last 10 years (figure 11). Scheme 31. Generation of anticancer agents from ring-opened precursors through bioorthogonal cyclization. In lieu of the chemical modification of the existing drug, the ringopened antitumor prodrug displaying a spatial structure markedly distinct from the drug is designed to cyclize in cancer cells to finally synthesize the antitumor agent. Interaction of this prodrug with the biological target is extremely unlikely.

Abbreviation

°C

Degrees Celsius 4T1 Scheme 9. Prodrug of trans-2-phenylcyclopropylamine.

Prodrug 11 was tested in vitro against glioblastoma U87 cells and primary glioblastoma cells, and healthy astrocytes. No activity was observed up to 300 µM on healthy astrocytes while primary glioblastoma and U87 cells were impacted at 10 µM and 1 µM respectively. The authors also showed the impact of the quinone methide (formed from the benzene boronate promoiety) which reacts covalently with gluthatione in cells reducing the intracellular concentration of this antioxidant (vide infra).

Precursor of tyrosine kinase inhibitors

Crizotinib 12 is a tyrosine kinase inhibitor developed to treat non-small cell lung cancers. [START_REF] Bielec | Reactive Oxygen Species (ROS)-Sensitive Prodrugs of the Tyrosine Kinase Inhibitor Crizotinib[END_REF] This drug however presents important side effects such as hepatoxicity and neutropenia. These drawbacks prompted the Kowol group to prepare a benzene boronate prodrug 13 of this molecule in an attempt to selectively reach the cancer cells (scheme 10). [START_REF] Bielec | Reactive Oxygen Species (ROS)-Sensitive Prodrugs of the Tyrosine Kinase Inhibitor Crizotinib[END_REF] Scheme 10. Benzyloxycarbonyl prodrug of crizotinib and non-immolated metabolite 14.

Three non-small cell lung cancer cell lines (H1993, RUMH and H2228) known to respond to crizotinib 12 were evaluated toward their production of ROS by flow cytometry using 2′,7′dichlorofluorescein diacetate. The in vitro ROS production of the H1993 cells was found to be twice as much as the two other cell lines. In vitro cytotoxicity assays showed that approximately 50% of free crizotinib 12 (IC50 ~ 3-15 µM) activity was found with the prodrug 13. Addition of H2O2 prior to incubation with the different cell lines completely restored the activity of the original drug, except when in contact with H1993 cells where the activity remains surprisingly lower. The authors also observed that a stable non-immolated metabolite 14, a p- H2O2-mediated oxidation of the vinyl boronate moiety of ProPhen was followed by 1 H NMR performed with a Bruker avance II ( 1 H at 600 MHz). The NMR spectrometer was programmed to acquire a spectrum of 16 scans (1 minute, 44 seconds) with a relaxation time of 2 seconds to ensure accurate integration values. Between two acquisitions, we have a timeout of two minutes (total between two acquisition = 3 minutes, 44 seconds).

We followed the protocol described by Hanna et al. [START_REF] Hanna | Alcohol, Aldehyde, and Ketone Liberation and Intracellular Cargo Release through Peroxide-Mediated α-Boryl Ether Fragmentation[END_REF] to carry out this experiment: ProPhen

Phen T initial T intermediate T final (c)

ProPhen oxidation and subsequent cyclization into Phen

The kinetic analysis of the phenanthridine assembly following oxidation of the C-B bond, tautomerization and subsequent imination has been performed and validated stepwise. We analyzed the most general kinetic scheme relevant for the open precursors. The kinetic analysis of oxidation-cyclization was performed using ProPhen conversion into Phen in presence of hydrogen peroxide (scheme 37).

Scheme 37. ProPhen conversion into Phen with H2O2 as oxidizing agent.

The oxidative cleavage of the boronate ester moiety initiates the cascade of reactions. The enol tautomerizes into its keto counterpart that subsequently undergoes an intramolecular imine formation by reaction with the pending aniline group. Note that the intermediate C leads to a stable aromatized phenanthridine.

Theoretical model

The tautomerization reaction is expected to occur below the millisecond time scale. [6] A fast equilibrium is also assumed for the attack of aniline on the keto group of the acetophenone. Nine different 6-methylphenanthridines were synthetized according to this method and two reportedly cytotoxic 6-methylphenanthridines 107 and 108 were also prepared using the procedure described. 174

Cytotoxicity

The in vitro cytotoxicity (IC50) of the eleven 6-methylphenanthridines were evaluated on human papillomavirus-related endocervical adenocarcinoma (KB 3.1) cells, a well responsive cell line toward the activity of phenanthridines. The results are depicted in table 6.

Experimental part

Compound 66. To a flask containing a magnetic stir bar was added phenylacetylene 65 (1.02 g, 10.0 mmol, 1.0 eq). Then HBr (1.8 mL, 11.0 mmol, 1.1 eq, 33% in acetic acid) was added dropwise at 0 °C and the solution was stirred for 1 h at room temperature. Then, the reaction mixture was cooled to 0 °C and 20 mL of saturated aqueous solution of NaHCO3 was added.

This solution was extracted with DCM (3 x 20 mL) and the combined organic phases were dried with Na2SO4. The solvent was evaporated to afford the crude product which was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 95:5 to 70:30) to give the expected product (1.68 g, 92%) as a dark brown oil. The spectral data are similar to those reported in: Journal of Heterocyclic Chemistry 1988, 25

(4), 1237-1241.

Synthetic pathway A & B.

To a solution of PdCl2(PPh3)2 (0.03 eq), PPh3 (0.06 eq), B2(Pin)2

(1.1 eq), PhOK (1.5 eq) in 2.3 mL of toluene was added 66 or 68 (1 eq). The solution was stirred under Ar at 50°C during 4h and at 100°C during 3h. Water was added, the aqueous layer was extracted with EtOAC, washed with brine, dried with MgSO4, and concentrated under reduced pressure. The crude was purified by flash chromatography on silica gel (cylcohexane-EtOAC = 100:0 to 90:10) to give the expected product as a colorless oil.

Reaction followed by TLC (revelation: I2 and PMA)

Yield from synthetic pathway A = 35%

Yield from synthetic pathway B = 46%

Synthetic pathway C

To a solution of 70 (248 g, 1 mmol, 1 eq) in 20 mL of toluene was added p-toluenesulfonic acid (525 mg, 3 mmol, 3 eq). The mixture was heated at 60°C during 2 h. 50 mL of a saturated aqueous solution of Na2CO3 was added and the aqueous layer was extracted with EtOAc (3 x 50 mL). The organic phases were combined, washed with brine, dried with MgSO4 and concentrated under reduced pressure to give the expected product (230 mg, 100%) as a colorless oil. (No further purification was needed)

Yield from synthetic pathway C = 100%

Compound 95 (from 94).

To a solution of 94 (500 mg, 1.0 mmol, 1 eq) in dry THF was added TBAF (1.1 mmol, 1.1 mL, 1.0 M in THF, 1.1 eq) and the reaction was stirred at r.t. during 5 h. Water (20 mL) was added and the mixture was extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 95:5 to 60:40) to give 340 mg of 95 (95%).

Compound 109. To a flame-dried 50 mL round-bottom flask containing a stir bar was added bis(pinacolato)diboron (1.47 g, 5.8 mmol, 1.5 eq), 1,3-bis(cyclohexyl)imidazolium tetrafluoroborate (125 mg, 0.39 mmol, 0.1 eq), t-BuONa (75 mg, 0.78 mmol, 0.2 eq), and CuCl (77 mg, 0.78 mmol, 0.2 eq). Then 10 mL of toluene was added, the flask was sealed and the reaction was stirred for 30 min. Compound 99 (1 g, 3.9 mmol, 1 eq) was added via a syringe followed immediately by MeOH (342 µL, 8 mmol, 2 eq) addition. After 3 h, the mixture was loaded directly onto a short pad of silica gel pretreated with DCM. The pad was thoroughly washed with Et2O and the filtrate was evaporated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel (cyclohexane:EtOAc = 95:5 to 60:40) to give the expected product (470 mg, 31%) of expected product as a colorless oil.

TLC condition: eluent (cyclohexane:EtOAc = 8:2); Rf = 0.