France. Without their help, life here would not be like this. I also want to express my sincere thanks to Colette, who is so kind and gives

First and foremost I would like to pay my sincere thanks to my PhD supervisors, Dr Huixiong CHEN and Prof Mélanie ETHEVE-QUELQUEJEU. I thank Dr Huixiong CHEN for helping me to apply for the scholarship and giving me the chance to work on this project.

He cultivated my working ability independently, which is a useful quality in my future career.

I am grateful to Prof Mélanie ETHEVE-QUELQUEJEU, who welcomed me into her team.

Her rigorous and meticulous spirits in science have deeply influenced me. In addition, her encouragement and support have always kept me to move forward.

Next, I would like to thank Prof Delphine JOSEPH and Dr Sandrine PIGUEL for agreeing to be the reporters of my thesis. I thank Dr Charles TRUILLET for being the jury of my defence and examining the manuscript. I would like to express my profound gratitude to Pr Christiane GARBAY for her help during the preparation of this thesis and truly appreciate her willingness to be the jury.

I also would like to express my gratitude to all the collaborators: Mr Daniel LIETHA, who cultured and resolved the crystal structures of our inhibitors in complex with FAK kinase domain; Mr John DAWSON and Mr Yongliang LI, who contributed a lot of time on the biological evaluation in cells. I also would like to thank Mrs Céline TOMKIEWICZ-RAULAE, who gave me the training of biological experiments and performed many in vitro evaluations of our compounds. In addition, I want to say thanks to Mr Cédric CARADEUC and Mr Gildas BERTHO, who gave a lot of help with NMR spectroscopy, and Mr Patrice GERARDO, who did the analyse of HR-MS for me.

I am grateful to all my current and ex lab members in the team of Pr Melanie ETHEVE-QUELQUEJEU. I am especially grateful to Dr Laura INNAZZO and Dr Emmanuelle BRAUD for the discussion of chemistry, for the help in dealing with 'tous les merdes' in

Table of content

General introduction

Cancer: A burden all of the world

Cancer, also called malignant tumour, is a group of diseases involving uncontrolled malignant cell growth which has the potential to invade or transfer to other parts of the body.

Cancer is the second cause of death across the world behind cardiovascular diseases and the single most important block to increasing life expectancy in the 21 st century. In accordance with the statistics of WHO, approximately 14 million cases were diagnosed in 2012, and 8 million lives were deprived by the cancer-related diseases. 1 This number has significantly climbed during recent years. 2 The latest statistics for the most common cancer (excluding non-melanoma skin cancer) in 2018 all over the world was provided by World Cancer Research Fund. There were around 18 million cancer cases diagnosed in 2018, of 9.5 million cases were in man and 8.5 million in women. This trend will continue and it is predicted that in 2040 the number of cases will reach 29.5 million (Figure1).

Current treatments for cancers

Cancer is historically conceived as a cell-autonomous disease driven by the activation of (proto)oncogenes or the inactivation of onco-suppressor genes. Since cancer severely threats human health, a lot of endeavours have been made to conquer this problem. After the diagnosis, different treatments may be given separately, successively or in association to the patient, which depend on the type of cancer and how advanced it is. These treatments in terms of technical approaches are essential including surgery, radiation therapy, chemotherapy and immunotherapy. Targeted therapy and hormone therapy can be considered as sub-classes of chemotherapy and immunotherapy.

Surgery

Surgery is a procedure used to remove solid tumours from the body. This is an effective and valuable treatment for non-metastatic solid tumours, such as breast cancer, colorectal cancer, pancreatic cancer, etc. However, generally, surgery is not sufficient. In most conditions, surgery is considered as primary treatment and it is also combined with other treatments to control the progress of cancer. Moreover, for most types of cancer, biopsy is the main way for the diagnosis. During a surgical biopsy, the suspicious tissues are removed in order to find the origins of the tumour through anatomopathological analyses, which allow improving treatments.

Radiotherapy

Radiotherapy, also called radiation therapy, uses ionizing radiation, usually X-ray in order to treat cancer cells and shrink tumours. Only the solid and local cancers could be treated with radiotherapy. Ionizing radiation could damage the DNA of cancerous tissues.

Radiotherapy is usually used as adjuvant therapy after surgery to prevent tumour recurrence and is generally accompanied by chemotherapy.

Chemotherapy

Chemotherapy is the way to eradicate cancer cells by use of chemical drugs. Cancer chemotherapy history began in the 1940s after the 2 nd world war with the use of nitrogen mustards and folic acid antagonist drugs. Chemotherapy was developed on the observation that cancer cells grow more rapidly than normal cells. Chemotherapy is the most important component of cancer treatment for patients. This treatment is available to both solid tumours and non-solid cancers, such as leukaemia.

Chemotherapeutic agents display their anticancer activities by various mechanisms and have some specificity. For instance, paclitaxel is widely used for the treatment of several types of cancer. It kills cancer cells by promoting polymerization of tubulin dimers to form microtubules and preventing depolymerisation. 4 5-Fluorouracil (5-FU) is an antimetabolite drug. It takes effect by misincorporation of fluoronucleotides into RNA and DNA and inhibition of the nucleotide synthetic enzyme thymidylate synthase. 5 However, despite their success, conventional chemotherapies were limited due to nonsufficient specificity for tumour cells over normal cells resulting in insufficient drug concentrations in tumours or toxic effects on normal cells. In order to decrease the systemic toxicity and to prevent the appearance of drug-resistant tumour cells, several strategies have been proposed. These include alternative formulations e.g., liposomes, resistance modulation e.g., PSC833, antidotes/toxicity modifiers e.g., ICRF-187. 6 Finally targeted therapies have gained importance due to their specificity towards cancer cells while sparing toxicity to offtarget cells.

Hormone therapy

Hormone therapies are chemotherapeutic agents which allow to slow or stop the growth of cancer that dependent on hormones. Generally, hormone therapy is used to treat prostate and breast cancers along with other treatments. Androgen suppression therapy is used for men with prostate cancer. Estrogen deprivation therapy is used for women with estrogen receptor-positive breast cancer.

Targeted therapy

Targeted therapy includes several types of agents. In a first time, chemotherapeutic drugs were conjugated to agents able to recognize specific markers of certain tumour cells. This was possible with the progressive understanding of cancer biology. Various targeted drugs and monoclonal antibodies have been discovered and marketed. Now there are many treatments that use targeted drugs or antibodies alone which target specific genes or proteins in cancer cells that help them grow, divide and spread in cancer. Thus, the targeted therapy is the most effective treatment for most types of cancers and it's also the basis of precision treatment. Protein kinases involved in cell growth and sometimes mutated or overexpressed in different cancer types constitute important targets. Imatinib was designed for targeting the fusion protein tyrosine kinase bcr-abl, caused by the Philadelphia chromosome abnormality which is specific in chronic myeloid leukaemia (CML). Imatinib is the first small molecule that was used in the targeted therapy of the chronic myeloid leukaemia (CML). It binds to the active site of bcr-abl kinase and locked it in a self-inhibited conformation. 7 Hence, the effect of imatinib on normal cells is less than that on cancer cells. Antibodies are also largely developed since they can be expressed under a humanized form. Thus monoclonal antibodies, (mAbs) working mainly by attaching to and blocking antigens on cancer cells are designed.

For example, trastuzumab (Herceptin ®) is an antibody against the HER2 protein, which inhibits its activity in cellular growth. The approach with mAbs refers to targeted therapy and immunotherapy.

Immunotherapy

Immunotherapy is constituted by the ways to help the body's immune system to fight cancer, either by helping the immune system to attack the cancer cells directly or by stimulating the immune response against cancer cells. The immunotherapy has been well developed with specific monoclonal antibodies and has been approved to treat numerous types of cancer. There are several ways to help the immune system act against cancer development, such as adoptive cell transfer and monoclonal antibody mark, vaccination and immune checkpoint inhibition.

In this field, it was recently shown that immune checkpoint proteins such as Programmed death 1 (PD-1)/ programmed cell death 1 ligand (PDL-1) constitute very good targets. PD-1 protein, which is on the surface of T cells, regulates the immune response of T cell by binding with programmed cell death 1 ligand (PD-L1) or programmed cell death 2 ligand (PD-L2).

Many cancer cells can express PD-L1 which inactivates T cells and protects cancer cells from the attacking of T cells. 8 The anti-PD-1 monoclonal antibody, such as Nivolumab (Opdivo ®), that selectively block the interaction of PD-1 receptor with its ligands PD-L1

and PD-L2, 9 can treat inoperable or metastatic melanoma, squamous non-small cell lung cancer.

Summary

The anti-cancer treatments are always on the rise, particularly with targeted approaches and in the field of immunotherapy. A variety of efforts are also underway to propose new anti-cancer therapies. For instance, in the field of gene therapy, the CAR T-cell therapy is very promising and expected to receive several approvals in the near future. 10 It must be noted that important progress in terms of diagnosis allows treating patients earlier and with more appropriate targeted therapies depending on the type and stage of cancers. However, at this time, people with cancer generally receive several types of treatment, often combined to prevent cancer growth and invasion. Due to the complexity and variety of cancer, and even in spite that there are existing a great number of treatment strategies, it's still hard to cure cancers. Hence, there is still a strong demand to develop new drugs against cancers.

So this thesis will describe an approach for targeting the focal adhesion kinase (FAK), due to its role in oncogenic signalling pathways and immune response when it is over-expressed.

In chapter 1, a review on the protein kinase structure and different types of small molecular inhibitors will be described. The structure of focal adhesion kinase (FAK) and the progress of FAK targeted inhibitors are also presented. Next chapters will describe the design, synthesis and properties of our new small molecular inhibitors of FAK. In chapter 3, a microwave-assisted palladium-catalyzed amination of 5-bromopyrimidine study will be presented. The last chapter is dedicated to the synthesis of fluorescent compounds for the FAK imaging study.

Chapter I. Protein kinase and targeted small molecular inhibitors

Protein kinases

Kinases are enzymes that catalyze the transfer of the terminal phosphate group of ATP in two steps to a substrate giving phospho-substrate and releasing ADP (Scheme 1). The substrate of kinases varies from small molecules to various types of compounds, such as oligonucleotides, lipids, carbohydrates, proteins and receptors. Hence, kinases are classified into broad groups by their substrate: protein kinases, lipid kinases, carbohydrate kinases and others. Protein kinase is the largest group and constitutes 2% of human genes. 11 The phosphorylation process of protein kinase mediates most signal transductions and regulates various cellular activities in eukaryotes. 12 By modification of substrate activity, protein kinases are involved in many cellular processes such as metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation . 11

Scheme 1:Kinase phosphorylation process.

Protein Kinase structure

Thanks to the unfolding of the human genome, 518 protein kinases have been identified, which consist of 1.7% of human genes. These 518 protein kinases are classified into 9 groups, 90 families and 145 subfamilies by sequence comparison. 11,13 Though their primary sequence is various, almost all protein kinases share a highly similar 3D structure, especially in the catalytic active domain.

The 3D structure of a protein kinase consists of an N-terminal and a C-terminal domain connected by a hinge region (Figure 2). At the N-terminus, residues are mostly folded to βsheets and usually only one α-helix structure exists, which is termed C-helix. The C-terminal domain contains mostly α-helixes with a small number of sheets. The ATP molecule binds in a cleft formed between the two lobes of the protein kinase. Residues in the ATP binding pocket are highly conserved. The adenine of ATP was anchored to the hinge region through hydrogen bonds. The residue just before the hinge region is called gatekeeper residue, whose side-chain forms hydrophobic interaction with ATP. Around the ATP binding pocket, all the kinases conserve two flexible loops, a glycine-rich loop (called P-loop) and an activation loop (called A-loop). The glycine-rich loop is also termed as the phosphate-binding loop because phosphates of ATP form magnesium-mediated hydrogen bonds to this region. The activation loop controls the access of the substrate to the active site and can be characterized by a conserved amino acid sequence Asp-Phe-Gly (DFG). 12,13 Protein kinases exist in active and inactive conformations (Scheme 2). 14 When the kinase is active, its activation loop is phosphorylated and adopts an open and extended conformation offering a space for the substrate. The peptide substrate binds into the platform provided by the activation loop in front of the nucleotide binding pocket of ATP. Asp-Phe-Gly residues (DFG motif) are highly conserved in the activation loop. The Asp residue of the DFG motif is required for the recognition of the ATP nucleotide through a divalent Mg 2+ cation and the DFG motif adopts a conserved spatial conformation among the majority of kinases. Therefore, the active conformation is also called "DFG-in conformation", while in an inactive state of kinase, the activation loop changes conformation and destroys the platform for the substrate and ATP binding. For many kinases, the conformational changes involve crankshaft-like motions in the N-lobe of the activation loop resulting in the appropriate orientation of the DFG motif within the active site. 15 Hence, the inactive conformation is also called DFG-out conformation.

Scheme 2: The kinase active and inactive conformations. 14

Protein kinase targeted small molecules

For the last 30 years, kinases have been intensively investigated and emerged as important therapeutic targets for the treatment of numerous diseases such as cancers, inflammatory diseases, 16 central nervous system disorders, 17 cardiovascular diseases, 18 and complications of diabetes. 19 FDA approved the first kinase inhibitor Imatinib (Figure 3) in 2001 for the treatment of chronic myeloid leukemia (CML), which was a milestone for the kinase inhibitor design. Until the year of 2019, 48 kinase inhibitors have been approved by the FDA. 20 Among the 48 approved inhibitors, 45 small molecules interact directly with the kinase domain. 20 These inhibitors usually target the ATP binding site of the kinases, resulting in a decrease of enzyme activity. In light of the binding mode, kinase inhibitors can be divided into two categories: reversible inhibitors and irreversible inhibitors. The former categories can be further classified into 4 types on the basis of binding pocket: 1) type I inhibitor that occupies the ATP binding site; 2) type II inhibitor that occupies the ATP binding site and an adjacent hydrophobic pocket formed by activation loop; 3) type III inhibitor that binds to an allosteric site near the active site; 4) type IV inhibitor that occupies a pocket far away from the active site (Figure 4).

Type I inhibitor Definition

A type I inhibitor is a small molecule that competes with ATP for binding to the active site of kinases. Most kinase inhibitors discovered belong to the type I. The predominance of type I inhibitors in the discovery is a consequence of the fact that the design of inhibitors was made to mimic the structure of ATP and used highly activated recombinant kinase catalytic domain in biochemical screening. 22 Due to the similarity of the ATP pocket, type I inhibitors can often inhibit several kinases at the same time leading to a loss of selectivity.

For example, the natural product Staurosporine (Figure 5) is a type I inhibitor, found by the screening of protein kinase C (PKC) inhibitors. This compound has excellent inhibitory activity against PKC with an IC50 value of 2.7 nM. 23 Unfortunately, Staurosporine doesn't have any selectivity and inhibits nearly over 90% kinase. 24 This promiscuous inhibitory activity prevented the development of kinase inhibitor until selective epidermal growth factor receptor (EGFR) inhibitors were reported.

Search for selectivity

The possibility to selectively inhibit tyrosine kinase activity with synthetic small molecules was demonstrated by the discovery of "tyrphostins" (Figure 6), which were found to effectively block the autophosphorylation of EGF receptor, but not the homologous insulin receptor kinases. 25 In 2003, the FDA approved the first targeted EGFR inhibitor Gefitinib (Figure 7) for the treatment of non-small cell lung cancer. 26 An in vitro kinase inhibition profile showed that Gefitinib inhibits EGFR at least 100-fold more potently than other tyrosine kinases, including erbB2, KDR and Flt-1. Additionally, Gefitinib didn't inhibit the activity of the serine/threonine kinase Raf, MEK-1 and ERK-2. 27 The crystallographic structure of the complex of Gefitinib with wild type EGFR unambiguously elucidated the binding mode of type I inhibitor. As shown in Figure 7, the quinazoline is oriented with the 1-N in the back of the ATP-binding pockets, where it forms hydrogen bonds with the hinge region residue Met793. In addition, the 3-chloro-4-fluoro aniline substituent extends into the hydrophobic pocket behind the ATP-binding cleft. The 6-propylmorpholino group exposures to the solvent. 28 With the deep understanding of kinase structure, selective type I inhibitors could be designed by exploiting the subtle differences of residues in the active site 29,30 and by elaborately tailoring the molecular structure to complement the kinase pocket geometry 31 and form hydrogen bonds with the pocket residues. For instance, during the design of selective p38α mitogen-activated protein kinase (MAPK) inhibitors, two differences of residues between p38α MAPK and other kinases were found to improve the selectivity. 32 The first one is that p38α MAPK has a small gatekeeper residue (Thr106) (Figure 8).

However, among most of the protein kinases, it's a bulky residue serving as the gatekeeper, such as methionine, leucine or phenylalanine. The presence of a small gatekeeper residue creates a hydrophobic "back pocket" in the active site. [START_REF] Zhang | A Second-Site Suppressor Strategy for Chemical Genetic Analysis of Diverse Protein Kinases[END_REF] Therefore, an additional aromatic ring was introduced to the hinge region binding moiety for the exploitation of the small gatekeeper region. [START_REF] Koeberle | Skepinone-L Is a Selective P38 Mitogen-Activated Protein Kinase Inhibitor[END_REF] The second residual difference is that in p38α MAPK a glycine (Gly110) is directly adjacent to the linker residue (Met109), which only exists in 9.2% of all kinases (Figure 8). 32 The glycine can undergo a so-called glycine flip causing a position change between the glycine carbonyl oxygen and the glycine NH group, which resulted in forming two hydrogen bonds with a carbonyl oxygen atom in the inhibitors. 32 By taking advantage of above-mentioned features, researchers successfully discovered very selective type I inhibitors of p38α MAPK, such as Skepinone-L (Figure 8).

Type II inhibitor

Definition and discovery

Type II inhibitors are small molecules that recognize the inactive conformation of the kinase domain and occupy the active site and an adjacent hydrophobic pocket created by a unique conformation of the activation loop (DFG-out). The DFG-out conformation is not universal for kinases and has been observed by crystallography only in 48 kinases among the 518 kinases. [START_REF] Zhao | Exploration of Type II Binding Mode: A Privileged Approach for Kinase Inhibitor Focused Drug Discovery?[END_REF] In addition, as a consequence of the flexibility of the activation loop which constitutes of 20~30 residues in length, the allosteric pocket is not conserved among kinases. This provides a new opportunity for selective inhibition. represented with dash lines. 22 At the beginning, the discovery of type II inhibitors was serendipitous, such as Imatinib and Sorafenib (Figure 10). Imatinib was the first type II inhibitor, and its binding mode to kinase Abl was elucidated by crystallography. [START_REF] Nagar | Crystal Structures of the Kinase Domain of C-Abl in Complex with the Small Molecule Inhibitors PD173955 and Imatinib (STI-571)[END_REF] The crystal structure (PDB: 1IEP) demonstrated that the inhibitor takes an outstretched conformation rather than a U-shape conformation required for the type I binding mode in the complex with protein Abl. As shown in Figure 9, the pyridine ring of Imatinib inserts into the hinge region and forms a hydrogen bond with Met318. A hydrophobic para-substituted benzamide region extends to the allosteric pocket created by the DFG motif.

Rational design of type II inhibitor

The good selectivity of type II inhibitors has attracted researchers to put more efforts into the screening and design of such inhibitors. Until 2018, 7 type II inhibitors have applied in the clinic [START_REF] Carles | A Curated, Annotated and Updated Database of Protein Kinase Inhibitors in Clinical Trials[END_REF] and several dozen are under clinical trials. Since the first generation of type II inhibitors was serendipitously discovered, a pharmacophore-based rational design strategy was proposed. As shown in Figure 10, the pharmacophore of type II inhibitors consists of four parts. 22,[START_REF] Zhao | Exploration of Type II Binding Mode: A Privileged Approach for Kinase Inhibitor Focused Drug Discovery?[END_REF] A hinge region binding moiety. Usually, a heterocyclic structure serves as the hinge region binding moiety. Like type I inhibitors, the heterocycle can be decorated with appropriate substituents to fit the geometry of the active site.

A linker segment. A linker segment is responsible for traverse of the gatekeeper region.

Therefore, the linker part can adopt a flexible aliphatic chain or rigid aromatic ring depending on the structures of gatekeeper and adjacent residues.

A hydrogen bonding moiety. The hydrogen bonding moiety is necessary for type II inhibitors. It's usually an amide or urea structure, which forms several hydrogen bonds with the C-helix and DFG motif. The location of hydrogen bonding moiety plays an important role in tuning the kinase selectivity.

A hydrophobic moiety. The hydrophobic moiety usually is a rigid aromatic ring, which is introduced to occupy the allosteric pocket created by the DFG-out flip. Sometimes, the aromatic ring can be decorated with nitrogen-containing substituents to adjust the water solubility.

A number of type II inhibitors can be mapped by the above-mentioned pharmacophore. [START_REF] Zhao | Exploration of Type II Binding Mode: A Privileged Approach for Kinase Inhibitor Focused Drug Discovery?[END_REF] Thus, the pharmacophore guided approach provided a good strategy to design new type II inhibitors.

Allosteric inhibitors (type III and type IV) Definition

Allosteric inhibitors are defined as molecules that bind to an allosteric pocket with neither occupation of the ATP binding pocket nor interaction with the hinge region.

Allosteric inhibitors include type III inhibitors and type IV inhibitors. Type III inhibitors bind in an adjacent pocket of the active site, whereas this pocket does not overlap with the ATP binding site. Type IV inhibitors bind to an allosteric site that is distant from the ATP binding pocket (Figure 11). [START_REF] Wu | FDA-Approved Small-Molecule Kinase Inhibitors[END_REF] Generally, allosteric inhibitors have a strong preference for their targeted kinase because the allosteric pocket is distinct in each kinase. [START_REF] Gavrin | Approaches to Discover Non-ATP Site Kinase Inhibitors[END_REF] Most of the allosteric kinase inhibitors were discovered by serendipity. There is no real rational strategy to design allosteric inhibitors except for deliberately designed screening.

Owing to the high selectivity, a massive effort has been made to identify allosteric inhibitors, some of which have completed the clinical trials and entered the market (Figure 12). Trametinib was the first type III inhibitor of MEK 1/2 that received approval from FDA in 2013 for the treatment of patients with either B-Raf V600E or V600K mutated metastatic melanoma. [START_REF] Wright | Trametinib: First Global Approval[END_REF] Trametinib has a high selectivity. It was shown on in vitro test to have no obvious inhibition on a panel of 98 kinases at 10 μM, while it strongly inhibited the activity of MEK1/2 with a value of IC50 from 0.92 nM ~ 3.4 nM. [START_REF] Takauki | Antitumor Activities of JTP-74057 (GSK1120212), a Novel MEK1/2 Inhibitor, on Colorectal Cancer Cell Lines in Vitro and in Vivo[END_REF] Though there is no crystal structure of Trametinib with MEK, mutually exclusive binding study with PD0325901, which bound in an adjacent pocket of the active site confirmed by X-ray crystallography (Figure 13), [START_REF] Ohren | Structures of Human MAP Kinase Kinase 1 (MEK1) and MEK2 Describe Novel Noncompetitive Kinase Inhibition[END_REF] showed that Trametinib had the same binding mode as PD0325901. 43 GNF2 (Figure 14), discovered by phenotypic screening, is a well-identified type IV inhibitor of Bcl-Abl. [START_REF] Adrián | Allosteric Inhibitors of Bcr-Abl-Dependent Cell Proliferation[END_REF] In spite of the lack of in vitro inhibition of Abl kinase, GNF2 resulted in the inhibition of Bcr-Abl autophosphorylation, inhibition of cell proliferation, and increase of apoptosis on Ba/F3.p210 cells. Through the molecular docking studies, the binding site of GNF2 was presumed at the myristoyl pocket located in the C lobe of the kinase. Thus, the mutation in the myristoyl pocket of bcr-abl gave the Ba/F3 cells resistance to GNF2 while this mutation had little impact on the ATP competitive inhibitor Imatinib, which supported the allosteric inhibition of GNF2. [START_REF] Adrián | Allosteric Inhibitors of Bcr-Abl-Dependent Cell Proliferation[END_REF] In 2010, the binding mode of GNF2 to Abl was confirmed by X-ray crystallography. [START_REF] Zhang | Targeting Bcr-Abl by Combining Allosteric with ATP-Binding-Site Inhibitors[END_REF] The overall Alb kinase structure of complexed to GNF2 was similar to that of the complex of myristic acid, and binding position of GNF2 was overlapped with that of myristic acid in the complex of Abl/imatinib/myristate (Figure 14). Abl/imatinib/myristate (white), crystal complex Abl/imatinib/GNF-2 (green and yellow) and crystal complex Abl/imatinib (red). GNF-2 is coloured in cyan and myristic acid in magenta. 45

Covalent kinase inhibitors

The interactions between inhibitor and kinase are usually due to hydrogen bonds, salt bridges or Van der Waals interactions. The other trend for the design of kinase inhibitors is covalent inhibitor which binds to enzyme irreversibly through a covalent bond. The covalent binding to the enzymes or receptors was not very well considered in drug design for a long time due to the fear of potential toxicity caused by non-specific modifications of proteins, nucleic acids, and other key biomolecules. [START_REF] Park | Idiosyncratic Drug Reactions: A Mechanistic Evaluation of Risk Factors[END_REF] However, covalent drugs already exist on the market (Scheme 3A) and some of them became blockbusters, such as the esomeprazole (an S isomer of omeprazole). These covalent inhibitors are usually derivatives of natural products and their covalent binding mode was elucidated later. For example, Aspirin exerts its pharmacological effects through irreversible acetylation of a serine residue in the active site of cyclooxygenase 2. Omeprazole, a proton pump inhibitor for the treatment of gastric ulcers, translates into the reactive species under the acidic condition in the stomach and binds to the ATPase by forming a disulfide bridge (Scheme 3B). The success of these predecessors of covalent inhibitors has paved the way for the design of covalent kinase inhibitors. The new generation of covalent inhibitors is designed by addition of an electrophilic functional group with low reactivity to a highly selective reversible motif. The innovative inhibitors are termed as targeted covalent inhibitors (TCI), which obtain the most attention for the treatment of cancers.

Binding mode of covalent inhibitors

In general, covalent inhibitors take effect through a two-step binding mode (Scheme 4).

Firstly, the inhibitors form a reversible complex with the enzyme. This step is driven by the affinity of inhibitors to the enzyme. Then in the second step, the electrophile in the inhibitor attacks the targeted residue in the enzyme forming a covalent liaison. [START_REF] Lonsdale | Structure-Based Design of Targeted Covalent Inhibitors[END_REF] Due to the covalent bond, the inhibitors tightly bind to the targeted proteins and can't be replaced by the natural substrate or cofactors of the targets. Therefore, the covalent inhibitors have an increased target potency.

Scheme 4: Cartoon showing the binding of a covalent inhibitor to a target binding pocket. [START_REF] Lonsdale | Structure-Based Design of Targeted Covalent Inhibitors[END_REF] For reversible inhibitors, the potency is usually described by the inhibitory binding constant (Ki) or IC50. On account of the covalent binding, IC50 values for covalent inhibitors are time-dependent, so that it's difficult to compare IC50 values of covalent inhibitors. Based on the two-step binding mode of covalent inhibitor, it's more suitable to interpret the potency of covalent inhibitors in terms of kinact / Ki besides IC50. Ki corresponds to the affinity of initial non-covalent binding and kinact is the kinetic rate constant for the formation of the covalent bond between protein and inhibitor. 48

The covalently modified residues

Recently a lot of efforts have been put to discover new covalent inhibitors because of their advantages. The properties of targeted residue play a pivotal role in the development of selective covalent inhibitors. The successful covalent modifications of cysteine, lysine, tyrosine, serine, and threonine residues have been reported.

a) Cysteine

Cysteine is the most exploited residue in the targeted covalent inhibitor design in spite of the fact that cysteine exists rarely in proteins with a low prevalence of 1.9%. [START_REF] Jones | Covalent Probe Finds Carboxylic Acid[END_REF] The thiol group of cysteine is a moderate nucleophile with a typical pKa of 8.5. [START_REF] Platzer | PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements[END_REF] Owing to the big size and the high energy of lone pairs in sulfur, the thiol group is readily deprotonated resulting in the formation of highly nucleophilic thiolate form. Therefore, all the six approved covalent kinase inhibitor drugs targeted non-conserved cysteine. 20 The cysteine is not conserved in the kinase and is amenable to covalent modification in at least 200 kinases. The distribution of cysteine in the vicinity of ATP pocket is classified into five groups according to the relative positions (Figure 15). [START_REF] Barf | Irreversible Protein Kinase Inhibitors: Balancing the Benefits and Risks[END_REF][START_REF] Zhang | Targeting Cancer with Small Molecule Kinase Inhibitors[END_REF] Figure 15: Distribution of cysteine in the vicinity of the ATP pocket. The sphere indicates the location of cysteines that are in principle accessible for covalent modification. [START_REF] Barf | Irreversible Protein Kinase Inhibitors: Balancing the Benefits and Risks[END_REF] The group 1 shown in the dark blue sphere is located in the glycine-rich loop, which anchors the phosphate of ATP. There are 16 kinases containing a cysteine at this position. [START_REF] Leproult | Cysteine Mapping in Conformationally Distinct Kinase Nucleotide Binding Sites: Application to the Design of Selective Covalent Inhibitors[END_REF] The pink sphere represents the group 2 which is on the roof of the ATP binding pockets.

There are 11 kinases which dropped into the group 2 region.

The group 3 represented with the yellow sphere is located on the hinge region and front pocket. The third group covers more than 100 kinases. [START_REF] Leproult | Cysteine Mapping in Conformationally Distinct Kinase Nucleotide Binding Sites: Application to the Design of Selective Covalent Inhibitors[END_REF] The cysteine in group 3 receives by far the most attention.

The group 4 highlighted with the light blue sphere is near the DFG-motif. The cysteine on this special position is limited in 48 human kinases. [START_REF] Leproult | Cysteine Mapping in Conformationally Distinct Kinase Nucleotide Binding Sites: Application to the Design of Selective Covalent Inhibitors[END_REF] The group 5 shown in dark red sphere represents the cysteine on the activation loop.

There are 8 kinases containing a cysteine on the activation loop. Nevertheless, it is hard to target the cysteine of group 5 because of the flexibility of A-loop. [START_REF] Barf | Irreversible Protein Kinase Inhibitors: Balancing the Benefits and Risks[END_REF] Afatinib is one of the FDA-approved covalent kinase inhibitors targeting the epidermal growth factor receptor (EGFR). 21 The gatekeeper residue mutation (T790M) confers EGFR resistance against the ATP competitive inhibitors. The irreversible inhibitor afatinib can form a covalent ligation with the residue Cys797 and effectively overcome the drug resistance induced by gatekeeper mutation. The anilinoquinazoline core of afatinib enters into the ATP binding pocket and has a conserved hydrogen bond with the hinge region. The substituted acrylamide induces the formation of a covalent bond with Cys797 (Figure 16). 54

b) Lysine

The covalent modification of lysine has been exploited when the kinase didn't have a suitable cysteine around the ATP pocket. [START_REF] Pettinger | Lysine-Targeting Covalent Inhibitors[END_REF] Lysine exists more abundantly than cysteine in the structure of kinases. The covalent modification of lysine is more challenging in comparison with cysteine. The typical pKa of lysine is 10.4. [START_REF] Platzer | PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements[END_REF] Therefore, under the physiological condition where the pKa is 7.4, most surface lysine residues are protonated and are not nucleophilic. The buried lysine residues are usually in the neutral form because of the less access of solvent and perturbation in pKa. [START_REF] Isom | Large Shifts in PKa Values of Lysine Residues Buried inside a Protein[END_REF] According to the hard-soft-acid-base concept, the ε-amino in lysine is a harder nucleophile [START_REF] Pearson | Hard and Soft Acids and Bases[END_REF] in comparison with the thiol in the cysteine or glutathione (GSH), and thus should react more readily with harder electrophiles, such as aldehyde. [START_REF] Migneault | Behavior in Aqueous Solution, Reaction with Crosslinking[END_REF] It provides the possibility of selective modification of lysine. The intrinsic reactivity of lysine towards various electrophiles was determined by Dahal et al. [START_REF] Dahal | Intrinsic Reactivity Profile of Electrophilic Moieties to Guide Covalent Drug Design: N-α-Acetyl-L -Lysine as an Amine Nucleophile[END_REF] It was reported that lysine is more reactive than GSH towards vinyl sulfones, while the commonly used acrylamide electrophiles are prone to react with cysteine.

CDKs are crucial protein kinases in the regulation of the eukaryotic cell cycle and in transcription. Irreversible inhibition of CDK2 with a small molecule NU6300 (Figure 17)

was reported by Anscombe et al. [START_REF] Anscombe | Identification and Characterization of an Irreversible Inhibitor of CDK2[END_REF] This covalent inhibitor of CDK2 entered ATP pocket and bonded to the hinge region by the purine ring. The vinyl sulfone group formed a covalent bond with Lys89, which was confirmed by the X-Ray crystallography.

c) Tyrosine

The modification of neutral tyrosine is challenging due to its relatively low intrinsic nucleophilicity. The pKa value of tyrosine is 9.7 which is lower than that of protonated lysine. [START_REF] Platzer | PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements[END_REF] However, it is worth to be noticed that the pKa value of tyrosine can be profoundly perturbed in the enzyme active site environment. In the bacterial ketosteroid isomerase, the pKa of Tyr57 was perturbed by around 4 units to 6.3. [START_REF] Schwans | Uncovering the Determinants of a Highly Perturbed Tyrosine PKa in the Active Site of Ketosteroid Isomerase[END_REF] The lowered pKa is favourable for the formation of phenoxy anion, which is a high reactive nucleophile. Sulfonyl fluorides are usually used to label tyrosine in the proteome. Whereas sulfonyl fluoride is reactive enough to modify lysine and tyrosine, the preference to tyrosine is observed when the tyrosine is proximal to basic amino acid residues. [START_REF] Hett | Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes[END_REF] Rational design of tyrosine targeted covalent inhibitor was reported by Gray's group. [START_REF] Hatcher | SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform[END_REF] The lead compound alectinib, which is an anaplastic lymphoma kinase (ALK) inhibitor, exhibited strong inhibitory activity toward the SR-protein kinase SRPK1 with an IC50 value of 11 nM. The X-ray structure of a complex of alectinib with SRPK1 showed that Tyr227 in the front region of ATP binding site might be covalently modified by the installation of an electrophilic warhead to the lead compound (Figure 18).

d) Serine and threonine

Serine and threonine have a side-chain containing hydroxyl group which can be covalently modified. When serine or threonine is located at the catalytic site of proteases and other hydrolase enzymes, the side chain hydroxyl group is extensively activated due to the perturbation of pKa by surrounding residues. [START_REF] Czerwinski | The Structural Basis for the Perturbed p K a of the Catalytic Base in 4-Oxalocrotonate Tautomerase: Kinetic and Structural Effects of Mutations of Phe-50 †[END_REF] However, the non-catalytic serine and threonine are less nucleophilic owing to the intrinsic low activity and acidity (typical pKa 13). In spite of the low reactivity, the mechanism study found that in cyclooxygenase the non-catalytic serine Ser530 was acetylated by aspirin. [START_REF] Lei | Mechanistic Insights into a Classic Wonder Drug-Aspirin[END_REF] Recently, researchers from Pfizer reported a pioneering work, [START_REF] Fadeyi | Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue[END_REF] in which a non-catalytic serine (Ser272) in the mRNA decapping scavenger enzyme (DcpS) was covalently targeted using fluorosulfate electrophilic warhead (FS-P1, Scheme 5). The authors emphasized that the covalent modification of serine might be attributed to the appropriate orientation of ligand and p Ka perturbation of serine induced by the nearby basic residues. Therefore, targeting serine or threonine provides an opportunity to develop next-generation of covalent modulators.

Scheme 5: Covalent binding of serine and fluorosulfate. 66

Electrophilic groups for the covalent inhibitor

The reactivity of electrophile is correlated with the kinetic process of covalent bond formation. On the other hand, it's also responsible for off-target labelling. Therefore, most electrophiles used in the covalent inhibitors have modest reactivity, such as the Michael acceptor acrylamide.

In recent years, in order to reduce the risks concerning promiscuous covalent binding, Taunton's laboratory developed chemically tuned electrophiles that could form a reversible covalent bond with non-catalytic cysteine. [START_REF] Serafimova | Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles[END_REF][START_REF] Bradshaw | Prolonged and Tunable Residence Time Using Reversible Covalent Kinase Inhibitors[END_REF] Installation of a nitrile group to electrondeficient olefins increased the olefins' reactivity, but paradoxically eliminated the formation of irreversible adducts. Shindo and colleagues 69 reported electrophile such as chlorofluoroacetamide which also exhibited a reversible covalent modification of kinase cysteine. They demonstrated that the covalent adduct of chlorofluoroacetamide and cysteine was stable in solvent-sequestered ATP-binding pocket of EGFR, while under aqueous conditions it was hydrolyzed to free thiol and hydrated glyxamide. [START_REF] Shindo | Selective and Reversible Modification of Kinase Cysteines with Chlorofluoroacetamides[END_REF] During the development of covalent inhibitors, the scope of electrophile groups is well studied and expanded, including acrylamide, fumaric acid ester, propiolonitrile, αhalomethyl amide/ester/ketone, α-cyanoacrylamide, aldehyde, isothiocyanate, vinyl sulfone, sulfonyl fluoride, aryl fluorosulfate etc. [START_REF] Gehringer | Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology[END_REF] The common warheads used in the drug design and their binding mode were summarized in the Table 1.

Focal adhesion kinase (FAK)

The focal adhesion kinase (FAK) was initially identified using homology-based cDNA cloning approach in 1992. [START_REF] Hanks | Focal Adhesion Protein -Tyrosine Kinase Phosphorylated in Response to Cell Attachment to Fibronectin[END_REF] It's a cytoplasmic 125-kDa non-receptor protein tyrosine kinase.

FAK and proline-rich tyrosine kinase 2 (PYK2) constitute the FAK family of tyrosine kinase, and they share a 65% similarity in sequence. [START_REF] Avraham | Identification and Characterization of a Novel Related Adhesion Focal Tyrosine Kinase (RAFTK) from Megakaryocytes and Brain[END_REF] FAK is ubiquitously expressed in most tissues and cells throughout the human body, while the expression of PYK2 is conserved in the brain tissues. 72

Structure and function of FAK

Figure 19: Schematic representation of FAK. [START_REF] Lee | FAK Signaling in Human Cancer as a Target for Therapeutics[END_REF] The FAK protein comprises three major domains: a central kinase domain, flanked by an N-terminal domain and a C-terminal non-catalytic domain (Figure 19).

a) N-terminal domain

The large N-terminal domain in FAK is composed of 415 amino acids and contains the FERM (four-point-one, ezrin, radixin, moesin) domain, a nuclear export sequence 1 (NES1)

and nuclear localization sequence (NLS). The FERM domain plays a pivotal role in the regulation of the FAK kinase activity and is involved in protein-protein interactions as a scaffold for the signal transduction. For example, the tyrosine kinase Etk mediates the cell migration in respond of the stimuli of extracellular proteins, while the activation process of Etk is dependent on an interaction between the PH domain of Etk and FERM domain of FAK. [START_REF] Chen | Regulation of the PH-Domain-Containing Tyrosine Kinase Etk by Focal Adhesion Kinase through the FERM Domain[END_REF] The nuclear function of FAK also associates with the FERM domain as it regulates the subcellular localization of FAK. 75

b) C-terminal domain

The C-terminal domain of FAK is composed of 414 amino acids and contains two proline-rich regions (PR2 and PR3) and a focal adhesion targeting region (FAT). [START_REF] Parsons | Focal Adhesion Kinase: The First Ten Years[END_REF] The FAT domain shares a similar four-helix bundle structure among FAK and other adhesion proteins, such as vinculin, Cas, and α-catenin. The FAT sequence is essential for the localization of FAK to the adhesion complexes and required for FAK signalling. [START_REF] Parsons | Focal Adhesion Kinase: The First Ten Years[END_REF] The non-catalytic domain in C-terminal of FAK, termed FRNK (FAK-related-non-kinase) serves as a negative regulator of kinase activity and is expressed independently in certain cells. It has been demonstrated that forced overexpression of FRNK inhibits cell spreading, cell migration and growth-factor-mediated signals to MAP kinase. 76

c) Kinase domain

The central kinase domain in FAK (residue 417-676) is structurally similar to the other receptor and non-receptor tyrosine kinases with a small N-lobe and a big C-lobe connected with the hinge region (Figure 20A). The N-lobe consists of four β-sheets and an αC-helix.

Residues from Gly429 to Gly434 constitute the glycine-rich-loop (GXGXXG) also called a P-loop, which forms Mg 2+ -mediated interaction with the phosphates of ATP. The C-lobe comprises several α-helix and two important loops for the kinase catalytic function. Residues from His544 to Asn551 consist of the catalytic loop (C-loop) and residues from Gly563 to Ser568 are part of the activation loop (A-loop). The FAK kinase is autoinhibited by the interaction with the FERM domain. The activation process requires a displacement of the FERM domain. Superposition of the active kinase domain on the structure of the autoinhibited kinase showed that the αC-helix adopts the same position ("αC-helix in" conformation) in these two states, while the activation loop in the inactive conformation extends to the FERM domain and blocks the entrance of ATP to the catalytic region (Figure 20B). The active site of FAK Figure 21: binding mode of ATP to FAK kinase. [START_REF] Nowakowski | Structures of the Cancer-Related Aurora-A, FAK, and EphA2 Protein Kinases from Nanovolume Crystallography[END_REF] Nowakowski et al [START_REF] Nowakowski | Structures of the Cancer-Related Aurora-A, FAK, and EphA2 Protein Kinases from Nanovolume Crystallography[END_REF]

FAK signalling pathway and biological functions a) The activation process of FAK

FAK is a pivotal protein in the integrin signalling. The stimuli of extracellular matrix to integrins assembles FAK localized to focal adhesion. Then the auto-inhibitory interaction of the kinase domain and FERM domain is disrupted to release FAK into activated conformation. In this conformation autophosphorylation of Tyr397 reveals the binding site for SH2 domain of Src. The Src protein is activated by binding to FAK and then it conversely phosphorylates residues Tyr576 and Tyr577 in the activation loop of FAK. The mutually activated FAK/Src complex triggers a cascade of phosphorylation events and protein-protein interactions to activate several signalling pathways (Scheme 6). [START_REF] Parsons | Focal Adhesion Kinase: The First Ten Years[END_REF][START_REF] Schaller | Autophosphorylation of the Focal Adhesion Kinase, Pp125FAK, Directs SH2 -Dependent Binding of Pp60src[END_REF] Scheme 6: Activation process of FAK. 80

b) Biological functions of FAK

FAK kinase plays an important role in regulating cell motility, invasion, and cell survival. [START_REF] Lee | FAK Signaling in Human Cancer as a Target for Therapeutics[END_REF][START_REF] Parsons | Focal Adhesion Kinase: The First Ten Years[END_REF][START_REF] Sulzmaier | FAK in Cancer: Mechanistic Findings and Clinical Applications[END_REF][START_REF] Golubovskaya | Targeting FAK in Human Cancer: From Finding to First Clinical Trials[END_REF][START_REF] Tai | Emerging Roles of Focal Adhesion Kinase in Cancer[END_REF] Deregulation of FAK also participates in the development of cancers.

Enhanced expression of FAK protein has been observed in a number of cancers, including breast [START_REF] Lark | High Focal Adhesion Kinase Expression in Invasive Breast Carcinomas Is Associated with an Aggressive Phenotype[END_REF] , colon [START_REF] Lark | Overexpression of Focal Adhesion Kinase in Primary Colorectal Carcinomas and Colorectal Liver Metastases: Immunohistochemistry and Real-Time PCR Analyses[END_REF] , thyroid [START_REF] Kim | Increased Expression of Focal Adhesion Kinase in Thyroid Cancer: Immunohistochemical Study[END_REF] , prostate [START_REF] Tremblay | Focal Adhesion Kinase (Pp125FAK) Expression, Activation and Association with Paxillin and P50CSK in Human Metastatic Prostate Carcinoma[END_REF] , lung [START_REF] Carelli | Up-Regulation of Focal Adhesion Kinase in Non-Small Cell Lung Cancer[END_REF] , stomach [START_REF] Su | Expression of Focal Adhesion Kinase and Alpha5 and Beta1 Integrins in Carcinomas and Its Clinical Significance[END_REF] and ovary. [START_REF] Judson | Overexpression of Focal Adhesion Kinase, a Protein Tyrosine Kinase, in Ovarian Carcinoma[END_REF] As shown in Table 2, it was observed that advanced cancer cells exhibited higher-level expression of FAK protein compared with benign tumours and normal tissues. 91 The expression of FAK promotes the survival of cancer cells. The first clue of the relationship between FAK and cell survival was demonstrated in 1996 that expression of active FAK could suppress anoikis of MDCK cells. [START_REF] Frisch | Control of Adhesion-Dependent Cell Survival by Focal Adhesion Kinase[END_REF] It was also demonstrated that the function of tumour suppressor protein p53 which induces apoptosis and cell cycle arrest was disturbed due to interaction of FAK and p53. [START_REF] Golubovskaya | Direct Interaction of the N-Terminal Domain of Focal Adhesion Kinase with the N-Terminal Transactivation Domain of P53[END_REF] The nuclear FAK also promotes p53 ubiquitination and degradation. [START_REF] Lim | Nuclear FAK Promotes Cell Proliferation and Survival through FERM-Enhanced P53 Degradation[END_REF] In addition, inhibition of FAK in tumour cells promotes apoptosis and loss of adhesion. [START_REF] Xu | Attenuation of the Expression of the Focal Adhesion Kinase Induces Apoptosis in Tumor Cells[END_REF][START_REF] Xu | The Focal Adhesion Kinase Suppresses Transformation-Associated, Anchorage-Independent Apoptosis in Human Breast Cancer Cells[END_REF] Studies have demonstrated that FAK is an important regulator of cell migration 100 which was required for the invasion and metastasis of cancer cells. FAK integrates several signalling responses that control focal-adhesion assembly and disassembly. Phosphorylation of FAK recruits a number of signalling and structural proteins promoting the assembly of focal adhesion, such as Src, Cas and paxillin. On the other hand, FAK also recruits extracellular signal-regulated kinase 2 (ERK2) and calpain-2 to focal adhesion and promotes calpain-2-mediated cleavage of focal adhesion. 101,102 FAK functions also related to the angiogenesis and lymphangiogenesis based on the observations that FAK-knock-out mice displayed embryonic lethal phenotype with defects of vasculogenesis. 103 In addition, it was demonstrated that inhibition of FAK expression resulted in reduced VEGF expression through FAK-Grb2-MAPK signalling pathway and caused small avascular tumours in mice. 104 Another interesting point is that recent studies indicated that FAK might be a crucial driver of immune escape and inhibition of FAK might have a synergetic effect with immunotherapy. Regulatory T cells (Tregs) could dull the anti-tumour immune response of immune-base therapy which aimed at activation of CD8+ T cell. A study in squamous cell carcinoma cells showed that nuclear FAK regulates chemokine/cytokine and ligand-receptor networks to recruit Tregs in the tumour microenvironment, which resulted in the inhibition of antigen-primed cytotoxic CD8+ T cell activity, and promoted immunological tolerance and permitted tumour growth. Furthermore, this study also demonstrated inhibition of FAK activity with small molecule could promote immune-mediated tumour regression. 105 Another study performed in human pancreatic ductal adenocarcinoma (PDAC) showed that FAK was highly activated in human PDAC and correlated with the immunosuppressive tumour environment. Treatment with FAK inhibitor increased the response of KPC mouse to T cell immunotherapy and PD-1 antagonists. 106 These findings indicate that FAK signalling may play a significant role in tumour immunity. Therefore, immunotherapy treatment in combination with FAK kinase inhibition may provide promising therapeutic opportunities.

FAK inhibitors

Because of the importance of FAK functions in cancer, many efforts have been made for the development of FAK inhibitors. Here, the inhibitors of FAK both under development and in preclinical studies are summarized according to the classification of kinase inhibitors.

Type I FAK inhibitors

Type I inhibitors of FAK have been extensively studied. Up to now, there are 7 type I inhibitors of FAK under clinical trials. In structure, type I inhibitors of FAK conserve a multisubstituted heterocycle scaffold, such as pyrimidine, pyridine, etc. Herein, the represented type I inhibitors of FAK are introduced according to the heterocyclic scaffold.

a. pyrimidine, pyridine, and triazine based inhibitors.

Pyrimidine-based inhibitors

The 2,4-diaminopyrimidines (Figure 22) constitute the most reported FAK inhibitors, and this kind of scaffold delivered several clinical candidates, such as VS-6062, 107 Defactinib, 108 and CEP-37440. Several groups 109,112,113,114 VS-6062 (Figure 22) is also an ATP-competitive inhibitor of FAK, which selectively inhibited the enzymatic activity of FAK with an IC50 value of 1.5 nM in vitro and inhibited autophosphorylation of FAK with an IC50 value of 5 nM in cells. 107 Kinase selectivity profile showed that VS-6062 had a good kinase selectivity on a panel of 42 kinases, and the concentration for half inhibition of IGF-IR was over 500 nM. 107 The good selectivity results from a helical conformation of "DFG motif" induced by VS-6062. The methane sulfonamide moiety of VS-6062 forms a hydrogen bond with backbone NH of Asp564, and the pyridinyl of VS-6062 has a hydrophobic interaction with the side chain of Leu567 (Figure 23). 107 The results of a phase I clinical trial showed that VS-6062 was a potent CYP3A inhibitor, which may cause toxicity in the situation of co-administration with other drugs. 115 The analogue of VS-6062, Defactinib (Figure 22), is the second generation of FAK inhibitor, which has weak inhibitory activity against CYP3A and a more favourable PK/PD profile. 108 The small molecule GSK-2256098 (Figure 22) developed by GlaxoSmithKline inhibited the FAK activity by blocking the phosphorylation of Tyr397. GSK-2256098

showed strong preferential inhibition of FAK with an IC50 value of 0.4 nM, 121 which is 1000fold less than that of Pyk2. 122 GSK-2256098 inhibited cell growth, decreased cell motility in anchorage-independent conditions. 122 Up to now, three phase I clinical trials of GSK-2256098 have been completed. The Phase I clinical trial (NCT 01138033) in the treatment of patients with advanced solid tumours, showed that the adverse events included light grades of nausea, diarrhea, vomiting and decreased appetite. 123

Triazine-based inhibitors and others

In 2013, our team 124 synthesized a family of diarylamino-1,3,5-triazine derivatives as

b. fused heterocycles-based inhibitors

Choi et al 131,132 elucidated the discovery of a series of 7H-pyrrolo [2,3-d]pyrimidines as FAK inhibitors. Through extensively structural modification study, compound 32 (Figure 25)

was obtained as a potent FAK inhibitor with an IC50 value of 4 nM. 132 It was demonstrated that the integrity of pyrrolo-pyrimidine was important to the inhibition of FAK because the reduction or expansion of the five-member ring caused a loss of activity. 131 In addition, the existence of an extended carboxyl group on the pyridinyl ring significantly improved the anti-FAK enzymatic activity. 132 Molecular docking showed that the carboxyl group formed a salt bridge with K454 (Figure 26) Kinase selectivity profiling on a panel of 53 oncology-related kinases showed that 14b displayed over 90% inhibition against 19 kinases at the concentration of 1 μM and it showed inhibition of insulin receptor with an IC50 value of 380 nM. 133 Herinrich et al 134 reported a fragment-based discovery of highly substituted 7-azaindole as FAK inhibitors. Compound 37 (Figure 25) was the most potent inhibitor of FAK among all the 7-azaindole derivatives. An electron-withdrawing substituent at position 5 of the 7azaindole core could improve the binding affinity, and introduction of a CF3 group improved the inhibitory activity against FAK and selectivity. The substituent at position 4 pointed to the DFG-motif of FAK. The substituents, like benzamide or sulphonamide which provide a hydrogen bond acceptor, were beneficial to improve the activity. The substituents at position 2 played a significant role in kinase selectivity. The best kinase selectivity was obtained when 4-F-phenyl group was introduced. 134 Compound 37 strongly inhibited enzymatic activity of FAK in vitro (IC50 = 37 nM) and Tyr397 phosphorylation of FAK in HT-29 cells (IC50 = 2.015 µM). Kinase selectivity profiling showed that only 1 kinase (IGF-IR) was inhibited with higher potency than FAK among 120 tested kinases. 134 The dihydropyrrolopyrimidine derivative, CT-707 (Figure 25), is a multi-kinase inhibitor targeting FAK, ALK, and Pyk2, with significant inhibitory effect on FAK (IC50 value of 1.6 nM). In vitro, CT-707 displayed an effect on cancer cell growth and cell detachment.

Type II FAK inhibitors

Researchers from the Merck Serono Research group disclosed the structure of a type II inhibitor (compound 13, Figure 27) which was discovered by high throughput screening. 137 Compound 13 displayed submicromolar inhibitory potency against FAK (IC50 = 266 nM).

The crystal structure of FAK in complex with compound 13 revealed that the activation loop adopted a DFG-out conformation and the 5-tert-butyl-2-p-tolyl-pyrazole ring of 13 is buried into a hydrophobic pocket induced by a conformational change of activation loop. In addition, the authors conducted replacement of purine ring of compound 13 with the fragments containing smaller molecular weight and heteroatom content which were identified as a good hinge region-binder in a surface plasmon resonance binding assay, but the new analogues unexpectedly showed significantly decrease in binding affinity to FAK. The researchers from Takeda Pharmaceutical Company reported tricyclic sulfonamides as type III inhibitors of FAK. 139,140 A cellular-active allosteric inhibitor of FAK exemplified compound 30 (Figure 28) was reported in 2013. 139 Compound 30 exhibited time-dependent inhibitory activity against FAK (IC50 = 2.2 μM, pre-incubation 5 min; IC50 = 0.64 μM, preincubation 60 min) with 0.5 µM ATP in the testing system and significantly reduced FAK phosphorylation with an IC50 value of 7.1 μM. 139 Kinase selective profiles suggested that compound 30 was a specific FAK allosteric inhibitor with an IC50 value over 10 μM against a panel of kinases including Pyk2, Scr, HER2, Akt1, Aurora B, MEK1 and p38α. The X-ray crystal structure of compound 30 in complex with FAK kinase (Figure 29) showed that compound 30 bound to an allosteric site. The structure-activity relationship analysis demonstrated that the methyl group at 1-position of pyrazole ring is embedded in a hydrophobic pocket and prevents the pyrazolo[4,3-c][2,1]benzothiazine core from entering the ATP binding site.

Inhibitors targeting FAK-protein interactions

The tyrosine kinase FAK plays its biological roles in cells through both kinasedependent and independent ways. Vascular endothelial growth factor receptor 3 (VEGFR3) is a tyrosine receptor kinase, which is involved in lymphangiogenesis, angiogenesis and tumourigenesis. In tumour cells, VEGFR3 binds to the FAT domain in C-terminal of FAK and promotes cell survival. Small drug-like molecules that interfere in the protein-protein interaction of FAK were reported as well. In 2009, Kurenova et al 141 identified a small molecule chloropyramine hydrochloride (C4, Figure 30) through screening in silico as an inhibitor of FAK-VEGFR3 interaction.

Compound C4 bound to a pocket in FAT domain containing residue His1025, which was a key residue for FAK-VEGFR3 interaction. Subsequent studies confirmed that C4 interrupted the colocalisation of FAK-VEGFR3 complex and disrupted FAK and VEGFR3 binding in breast cancer cells. C4 caused apoptosis and reduced viability of breast cancer cells that expressed VEGFR3. In addition, C4 could inhibit the tumour growth in xenograft mouse models with VEGFR3 high-expressed breast cancer.

In 2014, Gogate 142 reported an analogue of C4, compound 29 (Figure 30) which was a more potent inhibitor of FAK-VEGFR3 interaction. Kinase selectivity screening on a panel of 10 kinases, which are related to FAK or VEGFR3 proteins or in FAK downstream signalling cascades showed that none of the tested kinases was inhibited over 50% at the concentration of 1 μM. The cellular assay showed that compound 29 was specifically cytotoxic to pancreatic cancer lines, Panc-1-luc and MiaPacCA-2-luc which expressed a high level of FAK and VEGFR3 proteins. The initial work of the development of covalent inhibitors was dedicated to exploring suitable linkers and electrophiles. Several potential covalent inhibitors with three types of linkers including aliphatic chain, triazole, and squaramide were synthesized in modest yields.

Previous work in the laboratory

The preliminary biological evaluations showed that the compounds bearing squaramide linker and triazole linker displayed strong inhibitory activity against FAK.

Objectives

As mentioned in chapter 1, FAK is emerging as a promising therapeutic target, because it is over-expressed in many types of solid and non-solid tumours, whereas no inhibitors of FAK is on the market except those in clinical trials. On the other hand, the novel role of FAK in the regulation of cancer stem cell functions and immunology still need to be clarified and explored in the future. For these reasons, it is still necessary to develop selective and potent inhibitors of FAK.

Based on the encouraging results obtained in our team, the primary objective of my thesis focused on the development of covalent inhibitors of FAK. For this work, a) The first step was to improve the synthesis of diaminopyrimidine based covalent inhibitors and prepare the corresponding reversible inhibitor without the electrophilic warhead in order to perform the biochemical characterization of the covalent inhibitor of FAK. b) The second step was to design and synthesize new irreversible and reversible covalent inhibitors in order to improve the selectivity and bioavailability.

The second objective was to optimize the synthetic route of 2,5-disubstituted pyrimidines which are important scaffolds for developing type II inhibitors of FAK. Despite the simple structural features of these scaffolds, the substitution at 5 position of pyrimidine encountered major setbacks due to the low reactivity at the 5 position of the pyrimidine ring.

Therefore, it was necessary to develop a concise approach for the synthesis of these compounds with large functional group compatibility and in good yield.

The last objective of my thesis was the synthesis of fluorescent probes of FAK as tools for the visualization of FAK in cells, which could benefit the study of FAK signalling cascades.

Chapter II. Development of covalent inhibitors of FAK

The objective of this chapter is to develop targeted covalent inhibitors of FAK. As In the FAK kinase domain, there exist Cys427 and Cys502 two cysteines in proximity to ATP pocket, which belong to the group 1 and group 3 respectively according to the cysteine classification mentioned in chapter I. Cys502 is located in the hinge region. Cys427 is on the top of the ATP pocket close to the glycine rich-loop (Figure 31). This region is exposed to the solvent resulting in a decrease of pKa of Cys427, 146 which means Cys427 is more reactive than Cys502. In the first part, we performed a kinome-wide sequence cysteine alignment, optimization of the synthetic route to obtain enough amount of compounds for biological evaluation, characterization of irreversible inhibition by biochemical experiments and crystallographic data.

Sequences alignment

For the covalent inhibitor design, the targeted nucleophilic residue should be nonconserved in human kinases, which could increase the selectivity of the covalent inhibitor.

Hence, a kinome wide kinase sequence alignment was important.

Kinase-Ligand Interaction Fingerprints and Structures database 147 (KLIFS, http://klifs.vu-compmedchem.nl/index.php), developed at the division of medicinal chemistry in VU University Amsterdam, is a database that revolves around the protein structure of catalytic kinase domains and the way that kinase inhibitors can interact with them. This database collected 4616 PDB entries which include 295 kinases of 98 families. A sequence search by binding pocket composition was realized in this database.

Cys427 in FAK is the second residue preceding the glycine-rich loop. In the search parameters, we set this position as cysteine and limited the searching range to the wild type sequence. The results showed that only 5 kinases possess a cysteine at this position including FAK, activin receptor-like kinase 1 (ALK1), activin receptor-like kinase 2 (ALK2), pancreatic eukaryotic initiation factor-2alfa kinase (PEK) and serine/threonine-protein kinase RIO1 (RIOK1) (Figure 33). Subsequently, the protein sequences of these 5 kinases and other 15 kinases such as Pyk2, ABL1, IGFR1, HER2, IR, EGFR, SRC, FGFR1, ALK, VEGFR, PDGFRa, BTK, C-KIT, B-Raf and AKT2 were aligned using EMBL-EBI web service tool Clustal Omega. 148 The multiple sequence alignment results (Figure 34) were in accordance with the searching results in KLIFS. PEK, RIO1, ALK1, and ALK2 were the only kinases that have a cysteine at the same position as FAK. Therefore, Cys427 is non-conserved in human kinases.

Covalently targeting Cys427 of FAK might provide improved selectivity to pyrimidine based

Synthesis of the desired inhibitors

Synthesis of inhibitors with a squaramide linker 1) Retrosynthetic analysis

The retrosynthesis for the desired molecules is shown in Scheme 7. To obtain the intermediate A, there are two strategies to introduce the squaramide moiety. In strategy A, squarate is directly introduced to the diaminopyrimidine C followed by condensation with the aliphatic amine. In strategy B, the intermediate A is synthesized from fragment C and a squaramide monoester which is prepared beforehand from squarate and amine. The diaminopyrimidine C can be prepared from 2,4,5-trichloropyrimidine and substituted arylamines.

In previous work, the desired compound was obtained only in small quantities with a very low yield (6.8%). In addition, it's also difficult to get this compound in sufficient purity for further study. To progress the development of covalent inhibitors of FAK, the two strategies were both attempted, and the conditions of key steps were optimized to ensure that the desired compounds could be obtained in good yield and purity. The chlorine at position 2 can be substituted by nucleophile under harsh conditions, like heating over 100 o C overnight 149 or palladium-catalyzed coupling. 155 In general, the yield of this step is moderate.

3) Synthesis of diarylaminopyrimidine

Arylamine 1 was synthesized in excellent yield from commercial available isatoic anhydride and methylamine. 155 Then compound 1 was refluxed in ethanol with commercial starting material 2,4,5-trichloropyrimidine in the presence of NaHCO3 affording monosubstituted pyrimidine 2 in 93% yield (Scheme 8). 150

Scheme 8: synthesis of compound 2.

Two (4-aminobenzyl) carbamates (4 and 6) were synthesized in the process of optimizing the synthetic conditions for substitution of the chlorine at the position 2 of pyrimidine. Starting from 4-aminobenzylamine and Fmoc chloride, compound 4 was obtained only in 10% yield accompanied by the di-protected product. In order to optimize the yield, this compound was prepared in two steps from 4-nitrobenzylamine. The Fmoc protection of the amine was done in a classical condition giving intermediate 3 in 94% yield, 156 then the nitro group was reduced to an amino group by catalytic hydrogenation using 5% Pd/C in good yield (Scheme 9). Allyl (4-aminobenzyl) carbamate 6 was prepared through two steps as well. The reaction of 4-nitro benzylamine and allyl chloroformate gave compound 5 in 98% yield. 157 Reduction of the nitro group was easily completed after reflux for 2 h in ethanol with SnCl2 as reductant without damage to the double bond of the Allocprotecting group (Scheme 9). 158

Scheme 9: synthesis of (4-aminobenzyl) carbamates

To substitute the 2-chlorine on the pyrimidine ring of compound 2, aniline 4 was first used to optimize the reaction conditions (Scheme 10). With ethanol or isopropanol as the solvent, the reactants were refluxed for 20 h in the presence of HCl, but only traces of the desired product were generated. When n-butanol was used as the solvent, which has a boiling point at 118 o C, we isolated product 7 in 38% yield with 0.1 eq of HCl and in 52% yield with 1.0 eq of HCl, respectively. Camphorsulfonic acid (CSA) was then tested as a replacement for HCl, but there was no improvement in the yield. The poor solubility of compound 7 in organic solvent caused difficulties in purification. After removal of protecting group Fmoc in solution of 20% piperidine in DMF, compound 9 was obtained in 53% yield.

4) Incorporation of squaramide linker

In order to prepare compound 13, strategy A and strategy B described in the retrosynthetic analysis (Scheme 7) were both employed. Excess of ethylenediamine was treated with Boc2O in DCM at room temperature giving the mono-amino group protected product 10. Condensation of dialkoxysquarate and excess aliphatic amine usually generates symmetrical squaramide in high yield under mild basic conditions at room temperature, while the mono-substitution could be controlled by adding only one equivalent amine in the system. 160 Condensation of amine 10 with a slight excess of dimethoxysquarate in methanol gave mono-squaramide 11 in 65% yield. In strategy A, amine 9 reacted with dimethoxysquarate firstly giving compound 12 in 81% followed by condensation with linker

5) Incorporation of electrophilic groups

The Boc group in compound 13 was removed by treatment with TFA at room temperature for 30 min. After removing the solvent, the crude product 14 was directly acylated by acryloyl chloride or propionyl chloride to give the desired covalent inhibitor 15

or the corresponding non-covalent inhibitor 16 in yield of 49% and 51% for two steps, respectively (Scheme 12).

Scheme 12: Synthesis of compounds 15 and 16.

Synthesis of inhibitors with a triazole linker.

1) Retrosynthetic analysis 4-Nitrobenzaldehyde and pinacol with p-toluenesulfonic acid (PTSA) were refluxed in toluene for 4 h giving acetal 17 in 92% yield. 161 Then the reduction of the nitro group by catalytic hydrogenation gave aniline 18 in 99% yield (Scheme 14).

Scheme 15: Synthesis of diarylaminopyrimidine 20.

To substitute the chlorine atom at position 2 of compound 2, we initially attempted the conditions used before. The compound 2 and 18 refluxed in butanol with HCl gave no product, and the aniline 18 was degraded. Without adding acid to the reaction system, deprotected compound 20 was isolated in a low yield (8%). Given the instability of acetal 18 to acid, we tried to add one equivalent base to consume the produced HCl, but the substitution reaction didn't proceed.

Hence, we attempted the palladium-catalyzed C-N coupling reaction to prepare compound 19. This compound was obtained in 57% yield with Pd2dba3 as catalyst and XPhos as ligand. Afterwards, the aldehyde group was recovered with Firstenberge's protocol 162 using 10% of HCl solution in THF affording compound 20 in 86% yield (Scheme 15). The alkyne 21 was obtained through a reductive amination reaction (Scheme 16). 163 Aldehyde 20 reacted with the propargylamine in DMF at room temperature. A drop of acetic acid as catalyst was added to generate an iminium intermediate, which was then reduced by NaBH3CN.

3) Synthesis of azide part A.

Compound 22 was prepared in 96% yield using the method described by In this part, we have optimized the synthetic route to obtain the potential covalent inhibitors of FAK and synthesized two squaramide containing inhibitors (15, 16, Scheme 12)

and a triazole containing inhibitor 24. Next, we evaluated the in vitro enzymatic activity and performed biochemical characterization to confirm the formation of the covalent bond between the kinase and inhibitor.

Characterization of irreversible inhibition

The biological evaluation and characterization of irreversible inhibitors of Lietha in the Spanish National Cancer Research Center in Madrid, Spain.

In vitro evaluation of the enzymatic activity

In order to evaluate the in vitro inhibitory activity of FAK, we used ADP-Glo TM kinase assay. 166 The ADP-Glo kinase assay is a luminescent ADP detection assay that measures kinase activity by quantifying the amount of ADP produced during a kinase reaction. The principle of ADP-Glo kinase assay is a two-step process (Figure 36). In the first step, the kinase reaction is stopped by addition of ADP-Glo reagent, and the remaining ATP is depleted. The second step is the addition of kinase detection reagent to convert the ADP to ATP and allows the new synthesized ATP to be measured using a luciferase/luciferin reaction. The light generated correlates to the amount of ADP produced in the kinase assay, which indicates the kinase activity.

1) Inhibition of FAK enzymatic activity

The newly synthesized compounds 15, 16 and 24, intermediate 9, compound EYP (synthesized before in our laboratory, for the structure of EYP see Table 3), and TAE-226 as control were evaluated for their inhibitory activity against FAK. The results given as IC50

values are shown in 2) The effect of exposure time on the enzymatic activity Owing to the two-step binding mode, the covalent inhibitors should show increased inhibitory potency over exposure time. Therefore, the enzymatic activity was tested at 30

3) Effect of ATP concentration on the inhibition of FAK

The inhibitory effect of covalent inhibitor usually is not affected by the concentration of ATP in the testing system since they are not competitive with ATP. Therefore, the ability of compounds 15 and 16 to inhibit FAK was evaluated in the presence of 1 µM, 10 µM, 50µM

and 100 µM ATP, respectively. The results showed that the FAK activity remains inhibited by compound 15 with the concentrations of ATP rising from 1 µM to 100 µM. In contrast, the inhibition of FAK declined in the presence of a high concentration of ATP for reversible inhibitor 16 (Figure 38). The in vitro enzymatic inhibition results are in accordance with the fact that compound 15 could irreversibly inhibit the activity of FAK.

4) Kinase selectivity of compound 15

The kinase selectivity profiling was performed by Eurofins Scientific. The compound 15 was assayed on a panel of 9 kinases at a concentration of 1 µM. The results (Table 4)

showed that at this concentration compound 15 inhibited several oncogenes related kinases and receptors, such as Scr, c-kit, EGFR, IGF1-R, IRR, PDGF-Rα, and Pyk2, whereas HER2

was not inhibited by compound 15 at all. It is worth noting that compound 15 at a concentration of 1 µM only showed 21% inhibition of insulin receptor (IR), which was strongly inhibited by TAE-226 (IC50 = 260 nM). 111

Inhibition of FAK autophosphorylation in SCC cells

FAK was overexpressed in squamous cell carcinoma (SCC) cells and involved in the immunosuppressive microenvironment. [START_REF] Lim | Nuclear FAK Promotes Cell Proliferation and Survival through FERM-Enhanced P53 Degradation[END_REF] The inhibition of FAK autophosphorylation in SCC cells was evaluated with compound 15 and a reversible inhibitor VS-4718 as control.

As shown in Figure 39a and

X-Ray crystallography

Compounds 15, 16 and 24 were crystallized with FAK kinase domain in the group of D.

Lietha. From the crystal complex (Figure 40), it shows that the diarylaminopyrimidine moiety entered the active site of FAK and formed two hydrogen bonds with Cys502 in the hinge region. In the crystal complex of compound 15 and FAK, there is a consecutive electron density from compound 15 to Cys427 (Figure 40A) and a covalent bond between the acrylamide electrophile and Cys427 is observed (Figure 40D). The carbonyl group in squaramide formed a hydrogen bond with the side chain of Arg426. This hydrogen bond is important to maintain the orientation of the linker part and to well place the electrophilic group toward the vicinity of Cys427 (Figure 40D), and facilitate the formation of the covalent bond. In contrast, compound 24 didn't form covalent interaction with Cys427 due to an unsuitable orientation of the electrophilic group (Figure 40E). Therefore, the covalent interaction was formed only when the electrophile was positioned stably near the targeted cysteine. Compound 16 without the electrophilic warhead bound to the hinge region through pyrimidine scaffold, whereas the linker moiety exposed to the solvent (Figure 40F).

Conclusion

In this part, we have developed a feasible synthetic route for the preparation of diarylaminopyrimidine scaffold for covalent inhibitors in a good yield. Then, we established the biochemical characterization of the first covalent irreversible inhibitor of FAK. 145 Finally, we successfully obtained a crystal complex of FAK kinase domain and compound 15, which revealed the inhibitor's binding mode in the ATP binding site and confirmed the covalent linkage between the targeted Cys427 of FAK and the electrophilic group of the inhibitor. In addition, the cocrystal structures underlined the importance of squaramide linker to satisfy the structural requirements for the formation of the covalent bond.

Part 2. Design, synthesis and biological evaluation of new irreversible and reversible covalent inhibitors

This part is devoted to the synthesis of new pyrimidine-based irreversible and reversible inhibitors for improvement of kinase selectivity and decrease of off-target toxicity. Our compounds were tested for their inhibitory activity against FAK in vitro and anticancer activity in cells. The effect on cell growth, cell cycle, apoptosis, cellular morphology and cell migration were evaluated.

Design of new irreversible covalent inhibitors

The diaminopyrimidine moiety in covalent inhibitors of FAK is responsible for binding into the ATP pocket. Due to the two-step binding process of covalent inhibitors, the binding affinity and selectivity of the first step directly affect the overall biochemical and cellular potency. 167 In order to improve the kinase selectivity, new irreversible inhibitors were designed. The modification was carried out from two aspects. As shown in Figure 41, the substituent at position 4 of pyrimidine (part A) was replaced by N-methoxybenzamide, aryl-N-methylmethanesulfonamides or arylmethylsulfonyl. In addition, to increase the hydrophobic interaction with gatekeeper residue a trifluoromethyl group was introduced to replace the chlorine (part B). To synthesize compounds 27 and 28, N-methylmethanesulfonamide 26 was prepared according to a described method in a patent. 169 Methane sulfonyl chloride was slowly dropped to an ice-cooled solution of methylamine in water. Then the mixture was stirred at room temperature overnight to generate 26 in 77% yield. The reaction of 26 and 2fluorobenzonitrile in DMF in the presence of K2CO3 generated compound 27 in 51% yield. 170 Using the same conditions, compound 28 was obtained in 21% yield from 2chloronicotinonitrile. To introduce a sulfone group at the meta-position of benzonitrile, we used a reported method 171 starting from 3-aminobenzonitrile. The reaction of arylamine with tert-butyl nitrite produced an aromatic radical. Then it attacked the dimethyl disulfide which used as the solvent to give sulfide 31. The oxidation of sulfide 31 with Oxone (2KHSO5• KHSO4• K 2SO4) generated sulfone 32 in 87% yield (Scheme 23).

Synthesis of irreversible inhibitors modified on the part A 1) Retrosynthetic pathway

The nitrile group could be reduced to an amine by a reducing agent or transition metal. 172 To obtain the desired benzylamines, we used 10% Pd/C under 50 Psi hydrogen and ammonia solution (7 M in methanol) as solvent to suppress the formation of by-products. Under these conditions, four nitrile compounds were reduced to the corresponding primary amines (33 -36) in excellent yield (Scheme 24). To obtain diarylaminopyrimidine 43, the SNAr reaction between 37 and arylamine 6

under acidic conditions was attempted, but the benzamide moiety of compound 37 was degraded. Therefore, the palladium-catalyzed C-N coupling reaction was employed. We prepared arylamine 42 from 4-aminobenzylamine and Boc2O, 173

Synthesis of irreversible inhibitor modified on the part B 1). Synthesis of 2,4-diarylamino-5-trifluoromethylpyrimidine

Scheme 32: SNAr substitution of 2,4-dichloro-5-trifluoromethylpyrimidine

To synthesize 2,4-diarylamino-5-trifluoromethylpyrimidine, a base-catalyzed aromatic nucleophilic substitution protocol 174 was used (Scheme 32). Under the basic conditions, the substitution reaction gave two products. The ratio of these two compounds was about 1:1.6

determined by NMR (Figure 42). It was difficult to separate these two compounds by column, even after the use of several mixtures of eluents. With eluent of DCM/THF = 200/1, a small part of pure compound was separated. The NMR of each fraction showed that these two compounds were products substituted in position 2 and position 4, respectively (Figure 42).

From the subtle difference of chemical shift of the NH proton and the proton on pyrimidine, we proposed that fraction 1 was compound 68a and fraction 2 was compound 68b. Richter et al 175 reported the study of selective substitution of 2,4-dichloropyrimidines.

The nucleophilic substitution of 2,4-dichloro-5-trifluoromethylpyrimidine was preferential to position 2 with Lewis acid additive ZnCl2. The authors mentioned that the 2-position substituted product was less polar than the 4-position substituted product, which was consistent with our analysis that compound 68a was fraction 1. With the addition of 1 equivalence of ZnCl2, we obtained 69a and 69b in a ratio about 5:1, and the ratio was increased to 10:1 with addition of two equivalent ZnCl2 (Figure 43).

2). Coupling of squaramide linker and incorporation of electrophilic functional group

The condensation with squaramate 11 and deprotection were going smoothly under the conditions previously used to give intermediate 73. Then the amino group was acylated by Then the authors demonstrated that the fusion of the 2-cyanoacrylamide structure with a known p90 ribosomal protein S6 kinase RSK2 inhibitor provided a reversible covalent inhibitor. Their experiments showed that the reversible inhibitor has a sustained and quasiirreversible occupancy of RSK in cells. More importantly, the reversible inhibitor could be recycled after proteolytic digestion of the protein. [START_REF] Serafimova | Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles[END_REF] Therefore, no matter the irreversible or reversible covalent inhibitors, they both can form a covalent interaction with targeted cysteine in proteins. While the reversible one can reduce the risk of off-target labelling and immunogenic response (Figure 45). 177

Design of reversible covalent inhibitors

Owing to the advantages of reversible covalent inhibition over irreversible covalent inhibition, the reversible covalent inhibitors were designed by the introduction of reversible electrophilic functional groups at the extremity of the linker (Figure 46).

Synthesis of reversible covalent inhibitors

1) Synthesis of cyanoacetamide.

Scheme 36: Synthesis of compound 75.

To synthesize compound 75, two synthetic routes were attempted. An eco-friendly protocol to synthesize cyanoacetamides was reported by Pronença, 178 in which primary or secondary amines were directly mixed with methyl cyanoacetate without solvent and stirred for 2 h to give cyanoacetamide in high yield. We tried this method to prepare compound 75

with DMF as solvent because of poor solubility of compound 14. After 4 hours at room temperature, no product was observed by TLC. In the second synthetic route, cyanoacetic acid was used to prepare compound 75 under peptide coupling conditions (EDCI, HOBT).

The cyanoacetamide 75 was obtained in 41% yield.

2) Aldol condensation

Scheme 37: Synthesis of compounds 76 -78.

The aldol condensation of aliphatic aldehyde was achieved using the method described by Basu. 179 Compound 75 reacted with an appropriate aldehyde in a mixed solution of DMF and distilled acetic acid in the presence of piperidine to give the final products 76 -78 with yields of 34% -63% (Scheme 37).

Biological evaluation

In vitro enzymatic activity

The in vitro enzymatic activity assays were conducted by C. Tomkiewicz-Raulet in the laboratory of UMR S-1124 University Paris Descartes. The inhibitory activity of our compounds against FAK was evaluated as described previously using ADP-Glo kinase kit.

Kinetic analysis

In order to further evaluate the potency of covalent inhibitors, the enzymatic kinetics 180,181 were studied by our collaborator YL Li in the Guangdong University of Technology.

The covalent inhibitors require a reversible interaction first and then undergoes the covalent bond formation (Scheme 38). In contrast to the conventional drugs, due to the resulting covalent bond, the biomolecular targets theoretically can be completely inhibited with sufficient time. As a result, the IC50 values of covalent inhibitors can be time dependent.

In order to better rank the covalent inhibitors, it is important to consider the rate of inactivation of a target (kinact). The ability of non-covalent interaction in the first step was measured by Ki (Ki = kon/koff). The kinact/Ki ratio describes the ability of a covalent irreversible inhibitor to interact with and neutralize a target, which is better than IC50 values to reflect the potency of covalent inhibitors against the target. 48

Scheme 38: Covalent binding of inhibitor with its target. [START_REF] Bauer | Covalent Inhibitors in Drug Discovery: From Accidental Discoveries to Avoided Liabilities and Designed Therapies[END_REF] Time-dependent half-maximal inhibitory concentration (IC50(t)) was determined using ADP-Glo kinase kit and values of Ki and kinact were calculated according to a reported procedure with following equations: 182 It was reported that overexpression of FAK in oesophageal SCC cells was correlated to the tumour invasiveness and lymph node metastasis. 183,184 Patients with overexpression of FAK had a poor prognosis and their 5-year survival rate (38%) was almost half of that of patients without FAK overexpression (69%). 183 The effects of newly synthesized compounds on cell growth and cell cycle of human oesophageal cell lines including JH-eso-AD1 (AD1), Flo1, MFD1, OE33, OAC-P4C (P4C), and SKGT4 were evaluated by J. Dawson in Edinburgh cancer research centre. FAK inhibitor VS-4718 and chemotherapeutic drug Paclitaxel (Pac) were chosen as control.

IC 50 (t) = 𝐾 𝑖 (1 + 𝑆 𝐾 𝑀 ) • ( 2 -2e -𝜂 IC

Inhibition of cell growth

Influence on cell cycle

6.2.2.

Effect of new inhibitors on glioblastoma cells.

Since a lot of studies have shown the association between FAK and grade of malignancy, proliferation and migration of gliomas, FAK is a promising therapeutic target for malignant gliomas. 111,185 In addition, it was reported that TAE-226 treatment inhibited the glioma cell proliferation in vitro and in vivo. 111 Therefore, the anticancer activity of a subset of our compounds was evaluated on glioblastoma cells by our collaborators YL Li and C Tomkiewicz-Raulet, respectively.

Antiproliferation activity evaluation

In order to explore the antitumour activity of compounds 15, 74, 77 and 78, which showed good inhibition of FAK in vitro, the ability of antiproliferation was evaluated using MTT assay on three glioblastoma cancer cell lines, including U87-MG, A172 and U251.

Influence on cell cycle of U87-MG cells

To further investigate the influence of FAK inhibitors on the cell cycle, the distribution of cell cycle was analyzed by flow cytometry. When U87-MG cells were exposed to FAK inhibitors (15, 74, 77 or 78) for 24 h, the number of cells in G2-M greatly increased, which was consistent with the results of TAE-226 (Figure 48). It means that our inhibitors effectively block the G2/M process.

Influence on cell morphology

The morphology of U87-MG cells was studied when the cells were exposed to 1 µM

Influence on cell migration

Kinase selectivity profiling

Next, we investigated the kinase selectivity of 2 newly synthesized compounds with compound 15 as control. The kinase selectivity profiling was conducted by Eurofins on a panel of 9 kinases. The inhibition of each kinase for the corresponding compound at a concentration of 100 nM was shown in Table 9. The evaluation results revealed that compound 77 has improved kinase selectivity. Compared with compound 15, compound 74

and 77 showed decreased inhibition of EGFR and PDGFRα. In addition, compound 77

showed significantly reduced inhibitory potency on PDGFRα, IR and Src.

Conclusion and perspective

We synthesized 9 new covalent inhibitors of FAK. Among them, 6 compounds (group A and B) bear an irreversible electrophilic functional group and 3 compounds (group C) have a reversible electrophile (Figure 53).

In vitro enzymatic activity evaluation showed that these newly synthesized compounds The inhibitors 64 -66 inhibited FAK enzymatic activity in vitro, but they didn't show anti-growth activity in cells. Similar results were also reported for FAK inhibitor VS-4718, which inhibits cell survival selectively in suspension culture, while it doesn't affect c ell survival or proliferation under normally attached conditions. 120 In order to confirm their anticancer activity, it's necessary to evaluate their ability of inhibition of FAK autophosphorylation in cells. Moreover, evaluation of antiproliferation or cell survival in three-dimensional environments is also necessary.

On the other hand, in light of reversible covalent inhibition, the crystallization of our potential reversible covalent inhibitors (76 -78) with FAK kinase domain is required to confirm the covalent bond formation. Otherwise, mass spectra of inhibitors with kinase can permit to observe the formation of covalent adducts.

Furtherly, the in vivo anticancer activity assay of several candidate compounds (15, 77 and 78) is under planning and the pharmacokinetic properties will be studied.

Part 3. Synthesis of imidazopyrimidine precursors for the development of bicyclic covalent inhibitor.

In this part, I will present the synthesis of imidazopyrimidine scaffold and discuss the cocrystal structure obtained with imidazopyrimidine precursor and FAK kinase.

Introduction

In 2015, our team reported an imidazotriazine compound as the FAK inhibitor with an IC50 value of 50 nM. 136 Kinase selectivity profiling showed that this compound only inhibits FAK over 50% at the concentration of 1 µM on a panel of 32 kinases, including Pyk2, ABL1, AKT1, EGFR, IR and so on. 136 Molecular modelling showed that the binding mode of imidazotriazine was overlapped with TAE-226, and the imidazole ring has a hydrophobic interaction with the gatekeeper residue. Therefore, the replacement of pyrimidine ring in our covalent inhibitors by a bicyclic scaffold may provide a new approach to improve the selectivity. In consideration of solubility, we chose imidazopyrimidine scaffold to develop a new family of FAK inhibitors (Scheme 39).

Scheme 39: Design of bicyclic covalent inhibitor. Following the same procedure, from regioisomer 80b, another desired compound 88 was also prepared in good yield (Scheme 44).

Synthesis of imidazopyrimidine-based covalent inhibitor

Scheme 44: Synthesis of compounds 85 and 88.

4) Synthesis of bicyclic covalent inhibitors

The squaramide linker was condensed with compounds 86 and 88 under the same conditions used before to give compounds 89 and 90 in 65% and 81% yield, respectively.

The protecting group was removed by treatment with TFA giving intermediates 91 and 92 as trifluoroacetate salts. In the final step, acryloyl chloride was added to the solution of amine in DMF in the presence of DIPEA. Though this reaction worked well with pyrimidine compounds, unfortunately, we couldn't see any product on TLC (Scheme 45).

Scheme 45: Tentative synthesis of imidazopyrimidine-based covalent inhibitor.

To obtain the desired molecule, a venturesome synthetic strategy was proposed. Owing to the difference in reactivity of mono-squaramide and acrylamide towards amine, we attempted to introduce the electrophile to the linker part before the condensation with compound 88.

Starting from N-Boc-ethylenediamine Compounds 88 and 94 were added to a solution of DMF in the presence of DIPEA and stirred at 86 o C overnight. As expected, the reaction of the squaramate and the amine was favoured in this condition and the desired compound 95 was obtained in 70% yield (Scheme

47).

Scheme 47: Synthesis of compound 95.

X-Ray crystallography

In order to confirm that the binding mode of imidazopyrimidine scaffold in the active

Conclusion and perspective

We have established a feasible synthetic route to obtain 5,7-diaminosubstituted imidazo[1,2-a]pyrimidines and synthesized a potential bicyclic covalent inhibitor of FAK.

X-ray crystallography solved the binding mode of imidazo[1,2-a]pyrimidine in the active site of FAK. Interestingly, the bicyclic binds to the hinge region through the nitrogen of imidazole rather than the nitrogen of pyrimidine. Besides, the 4-aminomethylanilino substituent at position 7 of imidazopyrimidine pointed to the hinge region. As shown in Figure 57, the distance between Cys427 and the 7-aryl in 88 is about 8.9 Å. The metaposition seems better than the para-position for the installation of a linker to deliver the electrophilic functional group to the vicinity of Cys427 (Figure 57). Besides, the introduction of a small hydrophobic substituent on the position 2 or 3 of the imidazole ring which will have a hydrophobic interaction with the gatekeeper residue Met499 may improve the affinity. To synthesize this kind of molecules, the amination on the 5-position of pyrimidine is a key step. In the previous work, we attempted Pd-catalysed amination. For the purpose of the total conversion of starting material, it's inevitable that the reaction needs to be heated at high temperature for a long time. Nevertheless, the yield was not satisfied. In addition, under this condition, some substrates with ester or amide functional groups were degraded. It's necessary to explore a new synthetic method for the preparation of 2,5-disubstituted pyrimidine.

The Pd-catalysed C-N coupling reaction

The palladium catalysed aromatic amination of aryl bromide with N, N-diethylaminotributyltin was first reported as early as 1983. 193 Due to the limitation of substrate scope of the tin-reagent, this method didn't obtain extensive applications. About ten years later,

Stephen L. Buchwald and John F. Hartwig reported directly Pd-catalysed amination of aromatic halide with amines without tin-reagents, respectively (Scheme 48). 194,195 Then the Buchwald-Hartwig amination has been widely studied and became an important method for construction of C-N bond.

Microwave in organic reaction

The microwave heating is more efficient than traditional conductive heating. In the conductive heating such as oil bath or heating jacket, the temperature of the reaction vessel is usually higher than that in the reaction mixture, so that the transferring of energy is slow and inefficient. In contrast, microwave irradiation produces efficient internal heating by molecules' dipolar polarization and ionic conductance in chemical reactions. 197 In accordance with the dipolar polarization mechanism, the dipolar molecules will rotate to align with an oscillating electric field. With a suitable frequency, the re-orientation of dipole can't follow the phase change of applied field resulting in a phase difference between the orientation of the field and that of the dipole. This phase difference causes molecular friction and collision and energy released from the dipole producing dielectric heating. 197 The other energy source of heating in microwave irradiation is from the movement of ions through the solution in consequence of an electric field. The increased collisions of ions convert the kinetic energy to heat. 197 The main advantage of microwave irradiation is the shorter reaction time from days or hours to mere minutes or even seconds. For example, the Heck reaction between aryl triflate and N, N-dimethylallylamine need more than 20 hours to achieve the full conversion of aryl triflate with standard thermal heating, while under microwave irradiation the heck coupling could be finished within 5 min (Scheme 50). 198 Therefore, the reaction parameters such as reaction temperature and time, solvents, catalysts and additives can be investigated quickly to optimize the desired chemical reactions. Microwave-assisted organic synthesis has been widely applied in present synthetic chemistry, including transition-metal catalysed carbon-carbon bond formation 199 or carbon-heteroatom bond formation, 200 heterocycles synthesis. 201 Nevertheless, the microwaveassisted Buchwald-Hartwig amination is relatively under-explored compared with the transition-metal catalysed C-C bond forming reactions.

Hence, we studied the Pd-catalysed amination of 2-substituted 5-bromopyrimidine under microwave irradiation and explored the substrate scope.

Optimization of coupling conditions

In order to study the Pd-catalysed amination for the synthesis of 2,5-disubstituted pyrimidine derivatives, we choose the reaction of 2-methyl-5-bromopyrimidine (M1) and para-aminobenzonitrile (M2) as a model for optimization of coupling conditions (Scheme 51). The microwave conditions were set as maximum temperature: 120 o C, heating as fast as possible and duration for 60 minutes. The initial experiment was conducted with Pd2dba3 and XPhos in the presence of Cs2CO3 in 1,4-dioxane (Entry 1, Table 10). Encouragingly, desired compound 96 was isolated in 65% yield. However, other palladium catalysts did not efficiently catalyze this reaction, such as Pd(OAc)2 or Pd(PPh3)4 (Entry 2 and 3, Table 10). Next, the combination of Pd2dba3 and SPhos was employed resulting in a decrease of yield to 26% (Entry 4, Table 10). With XantPhos as ligand, the product was only separated in 14% yield (Entry 5, Table 10).

Reactions with BINAP or P(t-Bu)3 as ligands did not work well either affording a very small amount of product (Entry 6 ad 7, Table 10). Therefore, Pd2dba3 in combination with XPhos was the most efficient reagents to catalyze the amination of 5-bromo-2-methylpyrimidine. a, Cs2CO3 was used as base and 1,4-dioxane was sued as solvent; b, Isolated yield

2) Influence of base and solvent.

Given the fact that product 96 was delivered in 65% yield with Pd2dba3 and XPhos in the presence of Cs2CO3 in 1,4-dioxane (Entry 1, Table 11), then we investigated the influence of base and solvent. With K3PO4 as base, the yield of this reaction declined to 45% (Entry 2, Table 11). When base NaOtBu was used, the yield rose to 80% (Entry3, Table 11). However, the change of solvents didn't give a better result than 80% yield in 1,4-dioxane (Entry 4 and 5, Table 11). In conclusion, through a screening of catalysts, ligands, bases and solvents, the best conditions to promote this amination reaction appeared as the combination of 2 mol% Pd2dba3, 8 mol% XPhos and 1.1 eq NaOtBu in 1,4-dioxane.

Expanding the scope of substrates

The substrate scope of amines.

Firstly, we investigated the substrate scope of amines. The compound M1 and aniline reacted well under our optimal conditions giving the corresponding product in 82% yield (Entry 1, Table 12). Three different methoxyanilines all coupled to bromide M1 in a yield 81% -86% (Entry 2, 3, 4, Table 12), which means that electron-donating group on aromatic ring slightly favours the C-N coupling reaction. The product of reaction M1 and 3methylaniline was also obtained in 85% yield. However, the influence of electronwithdrawing substituent on arylamine depended on the position and property. The compound with a CF3 group on the meta position of aniline was converted to the product in 82% yield (Entry 6, Table 12), while reaction with para-trifluoromethylaniline gave product in 59% yield (Entry 7, Table 12). In addition, reaction of M1 and 4-aminobenzonitrile (M2) that we studied as model generated product in 80% yield (Entry 3, Table 11), whereas with 3-aminobenzonitrile or 2aminobenzonitrile as substrate the products were collected in a yield of 69% and 37%, respectively (Entry 8 and 9, Table 12). Nevertheless, 2-fluoroaniline was converted to products in 98% yield (Entry 10, Table 12). Then the substrate scope was expanded to heteroaryl amines and the products were generated in low to good yield. When pyridine-2amine or 6-methoxypyridin-3-amine was used, the reaction gave the product in a yield of 23% and 64%, respectively (Entry 11, 12 Table 12), but the yield was very good (84%) for pyrazolyl-amine substrate (Entry 13, Table 12). Ester is an important functional group in the bioactive molecules. To investigate if the ester was tolerated in the microwave radiated condition, we chose the ethyl 4-aminobenzoate as substrate. Microwave irradiation for 60 minutes under the coupling conditions was long enough to hydrolyse the ester bond. The irradiation time was shortened to 30 minutes, the ethyl ester product was collected in 52% yield (Entry 14, Table 12). When 2-aminobenzoic acid was used as the substrate, the product was got in 67% yield (Entry 15, Table 12). At last, two aliphatic secondary amines were reacted with M1 giving corresponding products in yield of 93% and 77% (Entry 16, 17, Table 12). In summary, this microwave-assisted amination method had a broad amine substrate scope including aryl amines, heteroaryl amines and aliphatic amines so that prepare a variety of 2,5-disubstituted pyrimidines.

The substrate scope of 5-bromo-2-substituted pyrimidines

In order to study the influence of substituents at position 2 of pyrimidine on the coupling reaction, we prepared three kinds of 2-substituted-5-bromopyrimidine substrates and these substrates were used to react with 4-aminobenzonitrile (M2) under microwave-assisted palladium catalytic conditions.

Preparation of substrates

We firstly prepared 2-aminosubstituted-5-bromopyrimidines from 5-bromo-2chloropyrimidine and appropriate amines through nucleophilic substitution. The substitution was prior at position 2 of the pyrimidine ring. In an optimized protocol of Li, Then the substrates containing a methoxy or aryloxy at position 2 of 5-bromopyrimidine were prepared. 5-Bromo-2-methoxypyrimidine 121 was synthesized from 5-bromo-2chloropyrimidine and freshly prepared sodium methoxide in 58% yield using a method described in the literature (Scheme 54). 204

C-N coupling reaction of various 5-bromo-2substituted pyrimidine

The C-N coupling of 5-bromo-2-substituted pyrimidine with 4-aminobenzonitrile (M2) was carried out under our optimal conditions, with Pd2dba3 as catalyst, XPhos as ligand, NaOtBu as base and 1,4-dioxane as solvent. Firstly, we tried 5-bromopyrimidine as substrate, this coupling reaction gave the desired product in 84% isolated yield (Entry 1, Table 13). For N-substituted-2-amino-5bromopyrimidines, the amination reaction was influenced by the substituents on the amine.

Substrates with disubstituted amino group at position 2 of 5-bromo-pyrimidine generally gave the coupling product in good to high yields (80% -99%) (Entry 3,4,5,8 Table 13), while the amination of substrates with monosubstituted amino group at position 2 of 5bromopyrimidine was hindered (Entry 6,7 Table 13), only N-methyl-2-amino-5bromopyrimidine gave product in 44% yield (Entry2 Table 13). In the beginning, we thought that the low conversion of 2-arylamino-5-bromopyrimidine was due to the occupancy of palladium catalysts by the amino group. Hence, 10 mol% catalysts were loaded for the reaction of entry 6. Unfortunately, the reaction didn't get better monitored by TLC. For 5bromopyrimidine with 2-methoxy or 2-aryloxy substituents, the yields of C-N coupling reaction under microwave irradiation were around 60% (Entry 9, 10, 11 Table 13), which was consistent with the yields of such reactions carried out in thermal conditions. 207 The reaction of 2-aryl-5-bromo-pyrimidines and 4-aminobenzonitrile worked smoothly and afforded products in high yield (86% -90%) (Entry 12, 13 Table 13).

In order to convert 2-arylamino-5-bromopyrimidine (117) into the aminated product, we protected the amino group with Boc2O 208 giving intermediate 137. Then compound 137 underwent the microwave-assisted amination giving products as a mixture with or without Boc group, then the mixture was treated by TFA to generate compound 138 in 52% yield for 2 steps (Scheme 57).

Scheme 57: Synthesis of compound 140.

In summary, this microwave-assisted C-N coupling method could tolerate different substituents at position 2 of 5-bromopyrimidine as substrates including aliphatic amino, methoxy, aryloxy and phenyl. The reaction of 2-arylamino-5-bromopyrimidine was hindered, which could be circumvented by a Boc protecting strategy to give the desired compounds.

Conclusion

We approach is limited to the systems in which genetic editing is available. Therefore, fluorescent small molecular probes that specifically target a protein can offer complementary information in biological experiments.

In the kinase cascades study, it's an effective strategy to inhibit the kinase activity with small molecules, which provide a dose-dependent response of kinase activity. However, this method sometimes gives ambiguous results because of the off-target effect and non-accurate concentration of inhibitors in cells, which are influenced by cell permeability, drug efflux, and cellular metabolism, etc. 213,214 Blair and colleagues 215 demonstrated proof-of-concept of using fluorescent affinity probe to report the necessary fraction of EGFR kinase for cellular signalling and to quantitate the relationship between EGFR stimulation and its downstream outputs. The targeted fluorescent probe can be used to quantitate the percentage of inhibited kinase and identify the most sensitive node in signalling pathway.

In order to study the enzyme occupancy of our covalent inhibitors in cells and related kinetic properties, a fluorescent covalent binding tool is required. On the other hand, FAK signalling is related to a number of downstream proteins. FAK targeted fluorescent probes are likely to be useful for the FAK cascade study. Moreover, it was reported that nuclear FAK reduces p53-mediated cell cycle arrest and enhances cell survival by promoting p53 ubiquitination and degradation. 216 The fluorescent probe is able to visualize FAK in living cells and to detect the nuclear FAK. Therefore, we are interested in the synthesis of fluorescence-labelled covalent inhibitors of FAK as imaging tools.

To maintain the strong affinity to FAK, the structural similarity between the fluorescent probe and the FAK inhibitor is important. 7-Nitrobenz-2-oxa-1,3-diazole (NBD) was selected as a fluorophore because its volume is similar to that of aniline substituent at position 2 of pyrimidine in the FAK inhibitors. Therefore, we introduced the NBD fluorophore to the pyrimidine scaffold through an amino group or an ethylenediamino linker (Figure 62). After stirring overnight the desired product 145 was collected as hydrochloride. In addition, deprotection of compound 144 using 5% NaOH solution in methanol 224 also led to the degradation of compound 144, which was in consistence with our hypothesis. It was reported that 7-alkylamino-4-nitrobenzo-2-oxa-1,3-diazoles (NBD-amines) were reasonably strong acids with pKa values of around 10. 225 The C-N bond at position 7 of NBD-amino derivatives had a partial double bond property with a bond length of 1.336 Å which was apparently shorter than a pure C-N single bond (1.467 Å). 226 In addition, the negative charge on the nitrogen atom of NBD-amine conjugate base could be stabilized by resonance 225 (Scheme 61) making it a good leaving group from pyrimidine. For condensation with the mono-squaramide linker, the synthesis work encountered the same problem. Usually the second substitution of mono-squaramide need base to promote the formation of the product. We attempted it at room temperature with 2 equivalent DIPEA, the starting materials were stable but no product was formed. When the reaction was heated to 80 o C, the base caused decomposition of 145. For the preparation of the desired compound in a non-basic condition, it might be achieved by activating the squarate. Rostami 227 reported a Lewis-acid catalyzed method for condensation of anilines and squarate ester, which may be an alternative solution. (Scheme 62)

Scheme 62: condensation of the linker.

Synthesis of compounds with a fluorophore connected to pyrimidine through an aliphatic linker

To

In vitro enzymatic activity

The inhibitory activity against FAK of newly synthesized fluorescent compounds was evaluated using the ADP-Glo kinase kit. The results are shown in

Conclusion and perspective

We synthesized 3 compounds with an NBD fluorophore. The small fluorophore NBD was chosen to replace the aniline substituent at position 2 of pyrimidine in covalent FAK inhibitor. The preliminary enzymatic activity evaluation showed that these fluorescent compounds had modest inhibition against FAK, which suggested that in the ATP binding site, the NBD fluorophore is not tolerated. Maybe the fluorescent group can be introduced through a longer linker into the para-position of arylamino substituent at the position 2 of pyrimidine.

General conclusion

Targeted therapy is a promising therapeutic strategy to cure cancer, which is one of the most threats to human health. The non-receptor tyrosine kinase FAK attracted our attention due to its important biological function in cancer cells and its overexpression in many cancers, such as glioblastoma, oesophageal carcinoma. This thesis is dedicated to the design and synthesis of selective FAK inhibitors for the development of targeted anticancer drugs. Three parallel projects were described in the manuscript. In chapter II, I continued the development of covalent inhibitors of FAK (Figure 68).

The acrylamide electrophile was incorporated to the diaminosubstituted pyrimidine scaffold through squaramide linker or triazole linker. The synthetic route was optimized to permit obtaining the desired compounds in good yield. Through the biochemical evaluation and X-Ray crystallography, the first irreversible covalent inhibitor (15) was characterized. Then, in order to improve the kinase selectivity, I conducted the structural modification giving 9 new compounds including irreversible and reversible covalent inhibitors. All the compounds showed excellent inhibitory activity against FAK in vitro. In cells a subset of compounds with 2-amino N-methyl benzamide group at 2-position of pyrimidine showed good anticancer activity. On the other hand, 5,7-disubstituted imidazo[1.2-a]pyrimidines were synthesized and the binding mode of the bicyclic structure in the active site was resolved by X-ray crystallography. The distinct binding mode will be used to design new selective FAK inhibitors.

Scheme 66: microwave-assisted amination of 5-bromo-2-substituted pyrimidine.

In chapter III, the efforts were contributed to the microwave-assisted amination of 5-bromo-2-substituted pyrimidine. The 2,5-disubstituted pyrimidine scaffold is an important fragment in the pharmacophore of type II inhibitor of FAK. The synthesis of 2,5-disubstituted pyrimidine is usually hindered because of the low reactivity of 5-halid pyrimidine. In this chapter, I optimized the conditions of Pd-catalysed Buchwald-Hartwig reaction. Under the optimal conditions, 29 derivatives were prepared in good to high yield (Scheme 66).

Therefore, our microwave-assisted amination of 5-bromo-2-substituted pyrimidine approach will be used to synthesize new type II inhibitors of FAK. In the last chapter, I prepared fluorescent FAK inhibitors, which will be used to visualize FAK in cells and study the FAK kinase cascade. Fluorophore NBD was introduced to the 2position of pyrimidine-based inhibitors through an amino group or ethylenediamino spacer.

Two reversible fluorescent inhibitors (145, 150) and one fluorescent compound bearing an electrophile were successfully prepared (153) (Figure 69). The preliminary in vitro evaluation showed that these compounds had modest inhibition of FAK.

EXPERIMENTAL PART

General experimental methods: Commercially available reagents and solvents were used

General procedure A: reduction of nitriles to primary amines

The compounds 33, 34, 35, and 36 were prepared according to the general procedure A.

A Paar TM reaction bottle (250 mL) was charged with a solution of the corresponding benzonitrile in 7 M ammonia in methanol and 10% palladium on carbon (0.1 equivalent).

The reaction mixture was vacuumed and charged with hydrogen (50 psi). Then the bottle was shaken on the Paar apparatus for 3 h. The mixture was filtered through a pad of celite and evaporated to give the corresponding amine.

General procedure B: Substitution of chlorine at position 4 of 2,4,5-trichloropyrimidine.

The compounds 38, 39, 40, and 41 were prepared according to the general procedure B.

Appropriate amine (1.1 eq) was added to a solution of 2,4,5-trichloropyrimidine (1 eq) in THF at room temperature, followed by the addition of DIPEA (1.5 eq). The mixture was stirred overnight. The solution was diluted with ethyl acetate and washed with water and brine. The organic phase was dried over MgSO4 and concentrated under reduced pressure.

The residue was purified by silica column chromatography to give the desired product.

General procedure C: Substitution of chlorine at position 2 of 4-aminosubstituted-2,5dichloropyrimidine with allyl (4-aminobenzyl)carbamate ( 6)

The compounds 45, 46, 47, and 48 were prepared according to the general procedure C.

To a solution of appropriate 4-aminosubstituted 2,5-dichloropyrimidine (1.0 eq) in n-BuOH, aniline 6 (1.0 eq) and HCl (4M in 1,4-dioxane, 1.0 eq) were added. The mixture was stirred at 100 o C overnight. After cooled to room temperature, the solution was diluted with DCM, and washed with 1M Na2CO3, brine and dried over MgSO4. The organic phase was concentrated under reduced pressure. The residue was purified through silica gel column chromatography to give the desired product.

General procedure D: removing the Alloc protecting group

The compounds 49, 50, 51, 52, and 71 were prepared according to the general procedure D.

To a solution of corresponding Alloc protected compounds (1.0 eq) in anhydrous THF or DMF, morpholine (10 eq) and Pd(PPh3)4 (0.1 eq ~ 0.5 eq) were added under an inert atmosphere. The mixture was stirred at room temperature for 3 h. After completion of the reaction monitored by TLC, the solvent was concentrated under reduced pressure. The residue was purified through silica gel column chromatography to give the desired product.

General procedure E: removing the Boc protecting group

The compounds 14, 44, 58, 60, 61, 62, 63, 72, 91, and 92 were prepared according to the general procedure E.

The Boc protected compound (1 eq) was added to a mixed solution of DCM and TFA (v/v =1:1) at room temperature. The mixture was stirred for 30 minutes before removing the solvent in vacuo. Then diethyl ether was added to the residue and filtered to give the desired compound as the salt of trifluoroacetate. The compound was directly used without further purification.

General procedure F: condensation of amines with squaramate linker 11

The compounds 53, 54, 55, 56, 57, 89, and 90 were prepared according to the general procedure F.

To a solution of appropriate amine (1.0 eq) in dry DMF were added squaramate 11 (1.0 eq) and DIPEA (1.0 eq). The mixture was stirred at 75 o C overnight. After removal of the solvent, the crude product was purified by flash silica gel chromatography to give the desired product as solid.

2-Amino-N-methylbenzamide (1) 155

Methylamine (6 mL, 11.77 mmol, 2 M in THF) was added to a stirred suspension of isatoic anhydride (1.0 g, 5.88 mmol) in water at room temperature and the reaction was left to stir at this temperature for 3 h. The solution was extracted with ethyl acetate 3 times.

Combined organic phases were washed with brine and dried over MgSO4, and the solvent was removed under reduced pressure to give the title compound as off-white solid (0.87 g, 99%). Trichloropyrimidine (282 μL, 2.4 mmol) was added to a stirred solution of 1 (397 mg, 2.64 mmol) and NaHCO3 (242 mg, 2.9 mmol) in anhydrous EtOH (5 mL) at room temperature.

The resulted mixture was heated to reflux and stirred overnight. The reaction mixture was cooled to room temperature and the precipitate was filtered out, washed with water giving the title compound as yellowish solid (660 mg, 93%). To a solution of 3 (490 mg, 1.3 mmol) in MeOH and DCM (250 mL v/v=4:1), Pd/C (5%, 25 mg) was added under H2 atmosphere, the mixture was stirred at room temperature for 3 h with an H2 balloon. Subsequently, the mixture was filtered through celite, and the filtrate was concentrated in vacuo to give the desired compound as off-white solid (460 mg, 98%). The solution ethanol containing 5 (478 mg, 2.02 mmol) and tin (II) chloride dihydrate (3.58 g, 16 mmol) was refluxed for 2 h. After cooling to room temperature, the solution was poured to 100 mL water. Then 2 M NaOH solution was added to adjust pH to 14 to get a milk-like solution. After filtration through celite, the filtrate was extracted with EtOAc 3 times. The organic layer was washed with water, brine and dried over MgSO4.

The solvent was filtered and concentrated under reduced pressure to give the crude product. The crude product was purified through a flash column (eluent: Cy/EA= 4/1 ~ 1/1) to give the title compound as oil (353 mg, 85%). atmosphere. The mixture was stirred at room temperature without light for 3h. After the completion monitored with TLC, the solvent was concentrated under reduced pressure.

The residue was purified through silica gel column chromatography (eluent: DCM/MeOH, 10:1+1% NH4OH) to give the desired product as white solid (580 mg, 85%).

N-Boc-ethylenediamine (10) 228

A solution of di-tertbutyl dicarbonate (1.06 g 4.7 mmol) in DCM was dropped to the flask containing ethylenediamine (2 g, 32.9 mmol) in DCM with stirring at room temperature.

The mixture was stirred for 48 h followed by filtration to remove the white precipitate.

The filtrate was concentrated. Subsequently, 2 mL of water was added to the residue and filtered. The water solution was saturated by NaCl and extracted with DCM (5 times). The combined organic phase was dried over MgSO4. After filtration, the solution was concentrated to give the titled compound as a colourless oil (581 mg, 91%).

2-Amino-N-methoxybenzamide (25) 168

To an ice-cooled solution of O-methyl hydroxylamine hydrochloride (500 mg, 6.0 mmol) in H2O (1.5 mL) was added NaOH (aq) (2 M, 3 mL). Then isatoic anhydride (326 mg, 2.0 mmol) was added in one portion. The mixture was stirred at room temperature overnight.

The solution was diluted by ethyl acetate and washed with water and saturated brine solution. Then the organic layer was dried over MgSO4 and concentrated in vacuo. The residue was purified through silica gel column (eluent: DCM/MeOH = 50/1) to give the title compound as white solid (331 mg, 90%).

1 H NMR (500 MHz, DMSO-d6) δ 12.05 (s, 1H),

N-Methylmethanesulfonamide (26) 169

To a solution of Methylamine (10 mL, 40% in H2O) in an ice bath, methane sulfonyl chloride (3 mL, 38.76 mmol) was dropped slowly and maintained the internal temperature was below 25 o C. Then the mixture was stirred at room temperature overnight. The solution was saturated by adding NaCl (3.6 g) and stirred for 30 min followed by extraction with DCM (3 times). The organic phase was dried over MgSO4 and filtered. The solvent was removed in vacuo to give colourless oil 6.5 g, yield 77%.

N-(2-Cyanophenyl)-N-methylmethanesulfonamide (27)

To a solution of potassium carbonate (3.0 g, 21.8 mmol) in DMF (30 mL) at room temperature was added 26 (1.19 g, 10.9 mmol) and 2-fluorobenzonitrile (777 μL, 7.3 mmol). The mixture was stirred at 100 o C for 20 hours and then was diluted with 100 mL EtOAc and washed by water (50 mL x 3) and brine (50 mL). The organic phase was dried over MgSO4 and evaporated. The resulting residue was purified by flash chromatography on silica gel (Cyclohexane / EtOAc = 2/1) to afford the desired product as white solid (787 mg, 51%).

1 H NMR (500 MHz, Chloroform-d) δ 7.71 (dd, J = 7.8, 1.6 Hz, 1H), 7.66 (td, J = 7.9,

3H). 13 C NMR (126 MHz, Chloroform-d) δ 143.9, 134.

N-(3-Cyanopyridin-2-yl)-N-methylmethanesulfonamide (28)

To a solution of potassium carbonate (1.7 g, 12.6 mmol) in DMF (30 mL) at room temperature was added 26 (830 mg, 7.6 mmol), and 2-chloronicotinonitrile (960 mg, 6.3 mmol). The reaction mixture was stirred at 86 o C for 18 hours. Then the mixture was diluted with 100 mL EtOAc and washed by water (50 mL x 3) and brine (50 mL). The organic phase was dried over MgSO4 and evaporated. The resulting residue was purified by flash chromatography on silica gel (eluent, Cyclohexane / EtOAc = 2/1 ~ 1/1) to afford the desired product as white solid (355 mg, 27%).

1 H NMR (500 MHz, Chloroform-d) δ 8.65 (dd, J = 4.9, 2.0 Hz, 1H), 8.05 (dd, J = 7.8, 1.9

Hz, 1H), 7.39 (dd, J = 7.7, 4.9 Hz, 1H), 3.39 (s, 3H), 3.19 (s, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 156.1, 152.0, 142.6, 122.6, 115.2, 109.4, 37.9, 37.2.

N-(3-Cyanophenyl)methanesulfonamide (29)

To a cold solution of 3-aminobenzonitrile (1.18 g, 10.0 mmol) in pyridine (20 mL) was added methanesulfonyl chloride (770 μL, 9.9 mmol) dropwise and the mixture was stirred at room temperature overnight. The solution was removed and the residue was partitioned between 1 N aqueous HCl and ethyl acetate. The organic layer was washed with brine, dried over MgSO4 and filtered. The filtrate was concentrated to give the desired compound as a pale orange solid (1.6 g, 82%). 1 H NMR (250 MHz, Methanol-d4) δ 7.60 -7.45 (m, 4H), 3.05 (s, 3H).

N-(3-Cyanophenyl)-N-methylmethanesulfonamide (30)

Sodium hydride (220 mg, 60% dispersion in mineral oil, 5.5 mmol)) was carefully added to a solution of 29 (900 mg, 4.59 mmol) in distilled DMF (15 mL). After 20 min, methyl iodide (428 μL, 6.88 mmol) was added, and the reaction mixture was stirred at room temperature for 18 h. Then the reaction mixture was poured into ice water and extracted with ethyl acetate (20 mL x 3). The combined organic layers were dried over MgSO4 and concentrated in vacuo. Purification of the residue by silica gel chromatography (eluent, cyclohexane/ethyl acetate= 3/1~ 2/1) provided the title product as white solid (880 mg, 91%). 3-Aminobenzonitrile (500 mg, 4.23 mmol) was added to dimethyl disulfide (4 mL) and the mixture was stirred magnetically. After heating to about 100 o C, tert-BuONO (744 μL, 6.34 mmol) was added in five portions over 10 min. After a further 20 min stirring, the mixture was cooled to room temperature and the solvent was removed in vacuo (Stench!).

The residue was purified by silica column chromatography (eluent, cyclohexane/ethyl acetate= 20/1) to give the title compound as lemon yellow solid (438 mg, 69%).

1 H NMR (250 MHz, Chloroform-d) δ 7.54 -7.34 (m, 4H), 2.51 (s, 3H).

3-(Methylsulfonyl)benzonitrile (32) 171

To a cooled solution of 31 (420 mg, 2.8 mmol) in acetone and water (10 mL, 1:1), was added Oxone (2KHSO5•KHSO4•K2SO4) (2.6 g, 4.2 mmol) and the mixture was stirred overnight. On completion, the reaction mixture was diluted with water (30 mL) and extracted with DCM (10 mL x 3). The combined organic layers were washed with brine and dried over MgSO4 and concentrated to give the title compound as white solid (443 mg, 87%).

N-(2-(Aminomethyl)phenyl)-N-methylmethanesulfonamide (33)

Following the general procedure A from 27 (740 mg, 3.52 mmol) afforded title compound as a colourless oil (763 mg, 100%).

N-(3-(Aminomethyl)pyridin-2-yl)-N-methylmethanesulfonamide (34)

Following the general procedure A from 27 (311 mg, 1.47 mmol) afforded title compound as a colourless oil (294 mg, 94%).

Hz, 1H), 7.29 (dd, J = 7.6, 4.7 Hz, 1H), 3.99 (s, 2H), 3.21 (s, 3H), 3.04 (s, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 152.6, 147.5, 138.7, 138.1, 124.4, 41.8, 37.8, 35.9.

N-(3-(Aminomethyl)phenyl)-N-methylmethanesulfonamide (35)

Following the general procedure A from 30 (350 mg, 1.66 mmol) afforded title compound as a colourless oil (344 mg, 97%).

(3-(Methylsulfonyl)phenyl)methanamine (36) Following the general procedure A from 32 (443 mg, 2.44 mmol) afforded title compound as a colourless oil (429 mg, 95%).

N-(3-(((2,5-Dichloropyrimidin

-4-yl)amino)methyl)pyridin-2-yl)-N- methylmethanesulfonamide (39)
Following the general procedure B, from amine 34 (280 mg, 1.3 mmol) and 2,4,5trichloropyrimidine (224 mg, 1.2 mmol) the title compound obtained as white solid (401 mg, 92%). In an argon charged sealed tube, to a solution of 37 (337 mg, 1.08 mmol) in 1,4-dioxane (6 mL) was added Pd2(dba)3 (99 mg, 10% mmol), XantPhos (128 mg, 20% mmol) and Cs2CO3 (420 mg, 1.29 mmol), followed by addition of 42 (240 mg, 1.08 mmol). The mixture was stirred at room temperature for 10 minutes, then stirred at 105 o C for 24 h.

Subsequently, the solvent was concentrated and the resulting residue was purified by silica gel chromatography (eluent: DCM/MeOH= 100/1) to give the title compound as white solid (231 mg, 43%). Following the general procedure E, from compound 43 (350 mg, 0.7 mmol) the titled compound was obtained as white solid (464 mg, quantitative). This compound was used in next step without further purification. Allyl (

(methylsulfonyl)benzyl)amino)pyrimidin-2-yl)amino)benzyl) carbamate 4-((5-chloro-4-((3-
Following the general procedure C, from 41 (530 mg, 1.60 mmol), aniline 6 (329 mg, 1.60 mmol) and HCl (400 μL, 1.60 mmol), the title compound was obtained as white solid (443 mg, 55%).

N-(3-(((2-((4-(Aminomethyl)phenyl)amino)-5-chloropyrimidin-4-yl)amino)methyl)

pyridin-2-yl)-N-methylmethanesulfonamide (50) Following the general procedure D, from 46 (471 mg, 0.89 mmol), the title compound was obtained as white solid (377 mg, 94%).

HRMS (ESI) m/z calcd. for C19H23O2N7ClS [M+H] + : 448.13170, found: 448.13054.

Melting point: 124 °C.

N-(3-(((2-((4-(Aminomethyl)phenyl)amino)-5-chloropyrimidin-4-yl)amino)methyl)

phenyl)-N-methylmethanesulfonamide (51) Following the general procedure D, from 47 (200 mg, 0.38 mmol), the title compound was obtained as white solid (138 mg, 81%).

, J = 5.9 Hz, 2H), 1.35 (s, 9H). 13 C NMR (126 MHz, DMSO-d6) δ 182.6, 182.

tert-Butyl (2-((2-((4-((5-chloro-4-((2-(N-methylmethylsulfonamido)benzyl)amino) pyrimidin-2-yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl) carbamate (54)
Following the general procedure F from 49 (230 mg, 0.51 mmol) and 11 (230 mg, 0.51 mmol), the title compound was obtained as white solid (302 mg, 86%).

tert-Butyl(2-((2-((4-((5-chloro-4-(((2-(N-methylmethylsulfonamido)pyridin-3-yl)methyl)

amino)pyrimidin-2-yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl) carbamate (55) Following the general procedure F, from 50 (300 mg, 0.67 mmol) and 11 (181 mg, 0.67 mmol), the title compound was obtained as beige solid (267 mg, 58%).

pyrimidin-2-yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl) carbamate (56)
Following the general procedure F, from 51 (200 mg, 0.45 mmol) and 11 (122 mg, 0.45 mmol), the title compound was obtained as white solid (252 mg, 82%).

tert-Butyl(2-((2-((4-((5-chloro-4-((3-(methylsulfonyl)benzyl)amino)pyrimidin-2-yl)

amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamate (57) Following the general procedure F, from 52 (260 mg, 0.6 mmol) and 11 (162 mg, 0.60 mmol), the title compound was obtained as white solid (269 mg, 68%).

, J = 6.0 Hz, 2H), 1.36 (s, 9H). 13 C NMR (126 MHz, DMSO-d6) δ 182.6, 182.

2-((

-Aminoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)methyl) phenyl)amino)-5-chloropyrimidin-4-yl)amino)-N-methoxybenzamide trifluoroacetate salt 2-((4-(((2-((2
Following the general procedure E, from compound 56 (230 mg, 0.34 mmol) the titled compound was obtained as pale white solid (309 mg, quantitative). The product was directly used for next step without further purification.

pyrimidin-2-yl)amino)benzyl)amino)cyclobut-3-ene-1,2-dione trifluoroacetate salt (63) Following the general procedure E, from compound 57 (50 mg, 0.08 mmol) the titled compound was obtained as pale white solid (70 mg, quantitative). The product was directly used for next step without further purification.

N-(2-((2-((4-((5-Chloro-4-((2-(N-methylmethylsulfonamido)benzyl)amino)pyrimidin-2-

yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)acrylamide ( 64)

To an ice-cooled solution of compound 60 (150 mg, 0.17 mmol) in dry DMF was added Et3N (119 µL, 0.85 mmol) and acryloyl chloride (22 µL, 0.26 mmol). The mixture was stirred at room temperature for 9 h. The solvent was removed in vacuo and the crude product was purified by column chromatography (eluent, DCM/MeOH = 20/1) to give the title compound as white solid (59 mg, 56%). After 1 h, compound 6 (305 mg, 1.48 mmol) was added followed by dropwise addition of triethylamine (207 μL, 1.48 mmol). After stirring for 6 hours, the mixture was diluted with DCM and washed with water and brine. The organic layer was dried over MgSO4. After filtration, the organic solution was concentrated in vacuo, and the residue was purified through silica gel chromatography (eluent: DCM/MeOH = 150/1) to give the titled compound as white solid (477 mg, 83%). to give white solid. Two white solids were combined and dried (185 mg, 89%). To an ice-cold solution of Compound 20 (50 mg, 0.09 mmol), propionaldehyde (9 μL, 0,13 mmol) and piperidine (13 µ L, 0.13 mmol) were added to a mixed solution of DMF and acetic acid (v/v=1/1).

The solution was heated to 100 o C and stirred for 2 hours. After cooling to room temperature, the solvent was removed in vacuo. Subsequently, the crude product was purified through silica gel column chromatography (eluent: DCM/MeOH= 20/1 ~ 15/1) to give the desired product as off white solid (19 mg, 34%). give the desired product as off white solid (69 mg, 63%). (1 mL) and stirred for 30 min at room temperature. The mixture was concentrated under reduced pressure. The residue was triturated with ethyl ether and was filtered to give the title compound as orange solid (140 mg, 95%). This compound was directly used for next step without further purification. ABSTRACT: Focal Adhesion Kinase signaling pathway and its functions have been involved in the development and aggressiveness of tumor malignancy, it then presents a promising cancer therapeutic target. Several reversible FAK inhibitors have been developed and are being conducted in clinical trials. On the other hand, irreversible covalent inhibitors would bring many desirable pharmacological features including high potency and increased duration of action. Herein we report the structure-guided development of the first highly potent and irreversible inhibitor of the FAK kinase. This inhibitor showed a very potent decrease of autophosphorylation of FAK in squamous cell carcinoma. A cocrystal structure of the FAK kinase domain in complex with this compound revealed the inhibitor binding mode within the ATP binding site and confirmed the covalent linkage between the targeted Cys427 of the protein and the inhibitor.

F ocal adhesion kinase (FAK) is a ubiquitously expressed nonreceptor protein tyrosine kinase and a scaffold protein localized to focal adhesions, which plays important roles in many biological processes including survival, proliferation, angiogenesis, adhesion, motility, metastasis, tumor microenvironment, epithelial to mesenchymal transition (EMT), as well as cancer stem cell renewal. 1,2 FAK is also overexpressed in a variety of solid and non-solid tumors, which make it a promising therapeutic target. 3,4 In recent years, numerous clinical trials have been developed with small FAK inhibitor molecules. 5,6 The results of such inhibitors are interesting since the decrease of tumor growth and metastasis development was observed in some preclinical models and in cancer patients, even if some adverse events have been revealed. 7 In order to enhance the efficacy of anti-FAK cancer treatments, the trials are conducted either individually or in combination. Recent advances in the evaluation of FAK inhibitors in preclinical and clinical models are very promising. 7,8 Furthermore, FAK has recently been shown to be localized in the nucleus and could promote survival and cell proliferation by interacting directly with p53. 9 FAK inhibition could also trigger immune-mediated tumor regression through the control of regulatory T-cells (Treg) in the tumor environment. 10 The development of covalent irreversible inhibitors to target kinases has recently garnered significant interest. 11,12 From a therapeutic point of view, the main advantages of irreversible inhibitors are (i) an increase of the target residence time to obtain durable inhibition without maintaining continuous drug exposure and (ii) a better ability to avoid drug resistance by reducing the development of resistance mutations. 13 Although there are only very few irreversible tyrosine kinase inhibitors used as therapeutic agents in the fields of oncology nowadays, the number of these new inhibitors is steadily increasing to enter clinical trials.

■ RESULTS AND DISCUSSION

Design of Irreversible Covalent Inhibitors. In order to assess the feasibility of designing irreversible inhibitors of FAK, our strategy entailed identifying a suitable cysteine residue within or next to the ATP binding pocket. This approach would be amenable by using a kinome-wide sequence analysis in connection with 3D structure of the ATP site. In particular, we noticed that FAK contains a cysteine residue in the glycinerich loop region closer to the ATP site, a feature which is not observed in most of other human kinases (Figure 1A). The distance between Cys427 and a reversible inhibitor, such as TAE226 (PDB ID: 2JKK) 14 or PHM16 (PDB ID: 4BRX) 15 in the ATP binding site is less than 10 Å (Figure 1B).

On the basis of the two-step process for kinase inhibition, we designed our inhibitors, which first bind reversibly to the enzyme, forming a noncovalent complex, and then form a covalent binding with the residue Cys427 of the enzyme to provide an irreversible complex (Figure S1, eq 1). Pyrimidine derivatives, especially 2,4-dianilinopyrimidine derivatives, are one of the important scaffolds found in many compounds described by pharmaceutical companies and academic laboratories. 6,[16][17][18] These pyrimidine-containing inhibitors efficiently target the FAK scaffolding function and suppress tumor growth through inhibition of Akt signaling or activation of p53 signaling and its downstream targets. 10 Our design strategy required the synthesis of 2,4-pyrimidine derivatives as a scaffold to maintain affinity similar to reversible inhibitors (compound 2, Figure 1C) and inclusion of a linker with Michael acceptor which could react with the nucleophilic Cys427 residue. A number of diverse linkers were evaluated; we quickly focused upon two linkers, a squarate (3,4bisaminocyclobutane-1,2-dione; 5 and 6) and a 1,2,3-triazole (7 and 8) motif, which are able to sustain a good orientation to target Cys427 in the glycine-rich loop region of the FAK kinase. Concerning the electrophile motif, an acrylate group was chosen to maintain reactivity (5, 7, and 8 in Figure 1C).

Synthesis of FAK Inhibitors. The synthetic route of new 2,4-pyrimidine compounds is outlined in Scheme 1 (for compounds 5 and 6) and Scheme S1 (for compounds 7 and 8). Starting from commercially available 2,4,5-trichloropyrimidine, the first chlorine atom in position 4 of 2,4,5trichloropyrimidine is displaced by 2-amino-N-methylbenzamide in the presence of NaHCO 3 in EtOH at reflux to afford a monosubstituted intermediate in a yield of 93%. This was further substituted to compound 1 in the presence of the corresponding (9H-fluoren-9-yl)methyl(4-aminobenzyl)carbamate and HCl in n-BuOH in a yield of 53%. The amino protecting Fmoc group of 1 was removed using piperidine to provide the key intermediate 2. Then, the electrophilic 1,4-addition reactions occurred between compound 1 and dimethyl squarate in the presence of DIPEA at RT to afford compound 3 in a yield of 81%. This was further condensed with tert-butyl (2-aminoethyl)carbamate in the presence of DIPEA, followed by the deprotection of the Boc group to give compound 4. Finally, acylation of the primary amine with acryloyl chloride or propionyl chloride gave the expected, targeted covalent inhibitor 5 and the corresponding reversible inhibitor 6, without the electrophilic warhead. Other potential irreversible inhibitors 7 and 8 bearing a 1,3,5-triazole linker were also synthesized in a similar manner (Scheme S1).

Biochemical Characterization of FAK Inhibitors. To establish whether our rationally designed targeted covalent inhibitors 5, 7, and 8 could form irreversible complexes, their inhibitory potency was first assessed using an ADP-Glo kinase assay. One previously reported inhibitor of FAK, TAE226, was used as a control. In this assay, TAE226 showed an IC 50 value of 5.8 nM against the kinase activity of FAK (Table 1), which was similar to previously reported data. 19 As expected, our selected 2,4-dianilinopyrimidine scaffold (compound 2) showed potent inhibitory activity to FAK (IC 50 = 26 nM), as shown in Table 1. Interestingly, the introduction of different groups on the amine function of the benzylamino moiety (ring B) resulted in a very large increase in inhibitory potency on FAK kinase activity (compounds 5, 6, 7, and 8), showing specific contributions of these groups. Compounds bearing a 1,2,3-triazole group (7 and 8) gave an inhibitory potency of 2.3 and 1.2 nM, respectively, which are more potent than TAE226. Interestingly, compound 5 bearing a squarate group demonstrated the best inhibitory activity (IC 50 = 0.6 nM), which was largely better than that of the matched pair 6, without a reactive acryloyl group (IC 50 = 4.7 nM).

As irreversible inhibitors always increase inhibitory potency over time, we then investigated time-dependence inhibition of FAK kinase by the putative irreversible inhibitors 5, 7, and 8,as compared with the reversible matched pair 6. As shown in Figure 2, only compound 5 displayed an increase in its inhibitory potency over 2 h, which was consistent with the formation of a covalent bond. As expected, the reversible matched pair 6 displayed no time dependence. Unfortunately, the putative irreversible inhibitors of FAK 7 and 8 displayed no time dependence in this assay.

Since high intracellular ATP concentrations under physiological conditions influence the cellular potency of ATPcompetitive inhibitors of protein kinases, non-ATP competitive inhibitors provide distinct advantages to conventional ATPcompetitive binders. Thus, developing irreversible inhibitors is an attractive and alternative strategy to achieve the non-ATP competitive inhibition of kinase-mediated signaling. Therefore, we studied noncompetitive character versus ATP of the putative irreversible inhibitor 5, compared with the reversible matched pair 6. Indeed, as shown in Figure 3, inhibitor 5 showed the same inhibition at different concentrations of ATP. On the contrary, the inhibitory potency of the matched pair 6 on FAK kinase activity decreased with increasing concentration of ATP.

Crystal Structure of FAK Bound to Inhibitors. To confirm the formation of a covalent bond and to verify that the binding mode of the bis-anilinopyrimidine part is similar to that of the reversible counterparts, we resolved the cocrystal structures of the FAK kinase domain successively with compounds 5 (PDB ID: 6GCX), 6 (PDB ID: 6GCW), and 7 (PDB ID: 6GCR). As shown in Figure 4, the mode of interaction of these compounds with the FAK kinase domain is very similar to that observed in complex with TAE226 or PHM16. Indeed, our inhibitors stabilize an unusual helical conformation of the DFG motif (D564-F565-G566) in which the phi torsion angle of Asp564 is rotated by 113°, compared to the active kinase domain. Compounds 5, 6, and 7 contain a common 5-chloro-2-anilino-4-(2-methylcarbamoyl)anilinopyrimidine scaffold, which binds well to the hinge region of the FAK kinase. Three hydrogen bonds are observed in the crystal structure of FAK bound to inhibitors. Among them, two are formed between the nitrogen in the pyrimidine and 2-aniline moieties with the backbone nitrogen and the carbonyl group of Cys502 in the kinase hinge (Figure 4A-C). Another one is formed between the CO of the carboxamide group of our inhibitor and the backbone nitrogen of Asp564 of the DFG motif. Some of hydrophobic interactions are observed between the carbon atoms of the 2-aniline ring and Ile428 and Gly505, but also between carbons in the pyrimidine ring and Ala452 and Leu553. As already observed in the crystallographic structure of a complex between the FAK kinase domain and TAE226, the chlorine atom on the pyrimidine ring is located in the ATP binding pocket, near the gatekeeper residue Met499 (Figure S2).

Interestingly, for 5, it is observed that two hydrogen bonds are formed between the side chain nitrogen of Arg426 and the carbonyl of the squarate, and the CO of Ile428 forms a hydrogen bond with the NH of the squaramide attached to the benzylamino moiety (Figure 5). On the contrary, the 1,2,3triazole group cannot make this type of interaction (Figure 5), supporting that the binding of the squarate group to the FAK active site is crucial to guiding the electrophilic group of 5 for reaction with the nucleophilic thiol group of Cys427 (Figure 4A). Furthermore, only the cocrystal structure with 5 revealed a strong and continuous electron density between C427 and the electrophilic carbon in 5 (Figure 4D) and thus clearly confirmed a covalent bond formation. Antiproliferation Activity of Compound 5 in SCC Cells. Having established the potency and irreversibility of inhibitor 5 binding to the FAK active site in vitro,w e investigated the inhibition of the full-length FAK kinase by 5 in squamous cell carcinoma (SCC) cells, which were induced in K14CreER FAK f lox/f lox mice using a two stage chemical skin carcinogenesis assay as previously described. 20 In a previous study, we demonstrated that PF-562,271, a reversible inhibitor of FAK kinase, was able to block the 3D proliferation of the SCC cells, cause squamous cell carcinoma regression, and inhibit tumor growth in vivo. 21 The antiproliferation of the irreversible inhibitor 5 against SCC cells was first assessed using cell nuclei counts, and VS-4718, a highly potent reversible inhibitor of FAK which is under clinical development, 22 was used as a control. Treatment of SCC FAK-WT cells with 5 resulted in a dose-dependent inhibition of proliferation with similar ED 50 (1.73 ± 0.1 μM) to that of VS-4718 (1.49 ± 0.2 μM) (Figure S3).

Covalent Inhibition of FAK by Compound 5 in SCC Cells. We tested the inhibition of FAK autophosphorylation in the SCC cell line with the irreversible inhibitor 5. As shown in Figure 6a, compound 5 blocked Tyr397 phosphorylation in a dose-dependent manner, and FAK autophosphorylation was significantly inhibited at low concentrations in these cancer cell lines, which is comparable to VS-4718. This is also consistent with its inhibitory activity against the FAK kinase, suggesting that this compound is able to effectively inhibit cellular FAK autophosphorylation and phosphorylation of kinase targets at low concentrations. Then, we examined the reversibility of the autophosphorylation of Tyr397 of FAK by performing washout experiments. Upon washout, VS-4718 lost its inhibition capacity against FAK, which is coherent with its reversible property. In contrast, 5 retained potent inhibition of FAK after washing out the inhibitor and demonstrated covalent binding (Figure 6d).

Kinase Selectivity Profile of Covalent Inhibitor 5. In order to investigate the selectivity of the irreversible inhibitor 5, we tested this compound in vitro against a panel of 10 kinases (EGFR, HER2/ERBB2, IGF1R, c-Kit, PDGFR, Pyk2, c-Src, IRR, and IR) determined by Eurofins Pharma Discovery Services, Poitiers, France to investigate its selectivity (Table S1, Supporting Information). A TR-FRET based kinase protocol was used with human recombinant protein in the presence of 1 μM of compound 5. Interestingly, this compound showed only 21% inhibition on the IR (insulin receptor) kinase even at the high concentration of 1 μM. This is in contrast to TAE226, which exhibited high potency toward IR (IC 50 = 26 nM). 19 Even if TAE226 has displayed a potent antitumor effect both in vitro and in vivo in a large variety of cancers such as brain tumors, 23 esophageal cancer, 24 breast cancer, 25 ovarian cancer, 26 and gastrointestinal stroma tumor, 27 TAE226 never entered into clinic studies, because this compound inhibited the insulin receptor with a high potency and showed important side effects in animal studies. 19 ■ CONCLUSIONS In summary, we described the first irreversible subnanomolar inhibitor of FAK containing a pyridimine scaffold and a Michael acceptor as electrophile up to date through structurebased and medicinal chemistry optimization. The crystal structure of the FAK kinase domain in complex with this inhibitor was resolved to display the formation of a covalent bond. Beyond generating and characterizing the covalent FAK inhibitor, our approach can direct the design of new electrophilic inhibitors which target a noncatalytic cysteine in proteins. The irreversible inhibitor 5 showed a highly potent decrease of autophosphorylation of FAK in SCC cells, suggesting that this compound could efficiently block a key event of the intracellular FAK signaling pathway. It is worth noting that 5 exhibited an antiproliferative effect in SCC cells similar to that of VS-4718, which is currently under phase I studies. Moreover, this compound displayed low inhibition against the IR (insulin receptor) kinase, which might provide a new mode of action and overcome the side effects observed with TAE226. Further studies into molecular mechanisms of this type of compound against cancer including antiproliferation of human cancer cells and the regulation of tumorigenesis and metastatic progression is underway in the laboratory to demonstrate the advantages for the development of novel cancer therapies.

■ METHODS

Chemistry. Methods for the synthesis of compounds, characterization, and spectra are described in detail in the Supporting Information.

Enzyme Inhibition Studies. Inhibitory potency in an FAK enzymatic assay was determined using an ADP-Glo kinase assay. Briefly, 4 μL of assay mixture containing 0.5 μg of FAK substrate, 5 ng of FAK, 8 μM ATP, and 1 μL of compounds at desired concentrations in kinase reaction buffer (40 mM Tris, 20 mM MgCl 2 , 0.1 mg mL -1 BSA, 2 mM MnCl 2 ,2μM DTT) was added into a 384-well plate. After incubation at 37 °C for 1 h, the kinase reaction was stopped by the addition of 5 μL of ADP-Glo Reagent to deplete the unconsumed ATP and leave only ADP. The plate was incubated at RT for 40 min and 10 μL of Kinase Detection Reagent added to convert ADP to ATP and introduce luciferase and luciferin to detect ATP. After incubation at RT for 15-20 min, the luminescence signal was detected with an Enspire plate reader. The IC 50 value was determined for each compound, from a sigmoid dose-response curve using Graph-Pad Prism (GraphPad Software, San Diego, CA, USA).

Inhibition of FAK Autophosphorylation in SCC Cells. SCC FAK-WT cells were cultured in Glasgow Minimum Essential Medium (10% fetal calf serum, 2 mM L-glutamine, NEAA, sodium pyruvate, and MEM vitamins). Cells were seeded in six-well plates at 100 000 cells per well. Compounds were made up in DMSO to a 1000× final concentration and 24 h after cell seeding were added to the culture media resulting in a final DMSO concentration of 0.1%. Compounds were incubated on cells for 48 h before cell lysis. For washout experiments, cells were washed with PBS twice and fresh medium was added for 3 h.

For cell lysis, briefly, wells were washed in ice-cold PBS and drained and cells lysed in MD Anderson buffer (1% Triton × 100, 50 mM Hepes at pH 7.4, 150 m M NaCl, 1.5 mM MgCl 2 , and 1 mM EGTA) supplemented with a protease and phosphatase tablet (Sigma-Aldrich). Cell lysates were clarified by centrifugation at 12 000g for 10 min and the protein concentration determined by Bradford assay (Thermo Fisher Scientific). Twenty micrograms of protein of each lysate was resolved using 4-15% polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes, blocked, and probed with primary antibodies overnight. Blots were washed in TBS/0.1% Tween and incubated with antirabbit conjugated horseradish peroxidase. Blots were washed in TBS/0.1% Tween and visualized with BM Chemiluminescence Western Blotting Substrate (Sigma-Aldrich) on a Bio-Rad ChemiDocimager. Blot densitometry was determined using Image Lab software (Bio-Rad).

Crystallization and Structure Determination. The kinase domain of the avian FAK protein (FAK411-686) was expressed and purified following a previously described assay. 28 The inhibitor-FAK complex was formed by incubation of the compound (final concentration 10 mM) with the protein (8 mg mL -1 )a t4°C overnight. Crystals of the complex were grown, mixing an equal volume of the complex with the crystallization condition (100 mM Tris, pH 8.5, 100 mM Li 2 SO 4 ,2 0 -24% PEG4000, 10 mM TCEP). Crystals were cryoprotected by a quick soak in the following cryosolution: 100 mM Tris at pH 8.5, 100 mM Li 2 SO 4 , 24% PEG4000, 10 mM TCEP, 35% ethylene glycol, and then flash-frozen. Diffraction data were collected at synchrotron facilities in beamline ID30-1 at ESRF (Grenoble, France; compounds 5, 7) or the XALOC beamline at ALBA (compound 6) and processed with XDS. 29 To provide an initial set of phases, the molecular replacement protocol in Phaser was used using the FAK kinase model from PDB 2JKK. 30 Refinement was carried out using the program Refmac, and manual rebuilding was performed with Coot. 31,32 The Dundee PRODRG2 Server was used to create the initial model for inhibitors 5, 7, and 10. 33 The structures of FAK domain bound to compounds 5, 6, and 7 have been deposited in the Protein Data Bank with PDB accession codes 6GCX, 6GCW, and 6GCR, respectively.

■ AUTHOR INFORMATION

Corresponding Author *E-mail: huixiong.chen@parisdescartes.fr.

ORCID

Huixiong Chen: 0000-0001-7676-1584

  Abbreviations .......................................................................................................................

3

 3

Figure 1 :

 1 Figure 1: Estimated number of incident cases from 2018 to 2040, all cancers, both sexes, all ages .3

3

 3 Figure 1: Estimated number of incident cases from 2018 to 2040, all cancers, both sexes, all ages .3

Figure 2 :

 2 Figure 2: (A) The 3D structure of protein kinase PDK1 with ATP (PDB: 4RRV); (B) Schematic representation of the ATP binding site.

Figure 3 :

 3 Figure 3: Chemical structure of Imatinib.

Figure 4 :

 4 Figure 4: Four types of reversible binding mode, the activation loop was shown in grey, the DFG-motif in Type I (active conformation) and Type II (Inactive conformation) was shown in grey and red sticks .21

Figure 5 :

 5 Figure 5: Chemical structure of Staurosporine.

Figure 6 :

 6 Figure 6: Chemical structure of tyrphostin.

Figure 7 :

 7 Figure 7: Chemical structure of Gefitinib and crystal complex with EGFR. 21

34

 34

Figure 8 :

 8 Figure 8: (A) Binding mode of selective p38α MAPK inhibitor; (B) Chemical structure of Skepinone-L.[START_REF] Koeberle | Skepinone-L Is a Selective P38 Mitogen-Activated Protein Kinase Inhibitor[END_REF]

Figure 9 :

 9 Figure 9: (A) Crystal structure of Imatinib and Abl kinase complex. The hinge region is highlighted in green, gatekeeper residue in yellow and DFG-motif in purple (PDB: 1IEP), 36 (B) Schematic representation of Imatinib binding site. The hydrogen bonds between the inhibitor and kinase are

22

 22

Figure 10 :

 10 Figure 10: (A) A general pharmacophore model for the rational design of type II inhibitors. A, hydrogen bond acceptor; D, hydrogen bond donor; HRB, hinge region binding; HM, hydrophobic motif; 22 (B) Some examples of known type II kinase inhibitors.

Figure 11 :

 11 Figure 11: Binding site of type III and type IV inhibitor in kinase.[START_REF] Gavrin | Approaches to Discover Non-ATP Site Kinase Inhibitors[END_REF]

Figure 12 :

 12 Figure 12: Examples of allosteric inhibitors approved by FDA

Figure 13 :

 13 Figure 13: Complex of MEK with ATP (pink) and type II ligand (silver) (PDB: 1S9J).[START_REF] Ohren | Structures of Human MAP Kinase Kinase 1 (MEK1) and MEK2 Describe Novel Noncompetitive Kinase Inhibition[END_REF]

Figure 14 :

 14 Figure 14: (A) Chemical structure of GNF2, (B) Superposition of the crystal complex

Scheme 3 :

 3 Scheme 3: (A) Examples of covalent inhibitor and (B) covalent binding mode of omeprazole.

Figure 16 :

 16 Figure 16: Chemical structure of afatinib and co-crystal structure of afatinib and mutant T790M of EGFR (PDB: 4G5P)

Figure 17 :

 17 Figure 17: Structure of NU6300 and binding mode of NU6300 with CDK2.[START_REF] Anscombe | Identification and Characterization of an Irreversible Inhibitor of CDK2[END_REF]

  The 4-morpholinopiperidine moiety was replaced by a meta-substituted phenyl ring bearing a sulfonyl fluoride substituent.This modification in the SRPKIN conferred selectivity towards SRPK1 over ALK with IC50 values of 36 nM and 195 nM respectively. The covalent binding mode of this compound was also supported by washout experiments and further confirmed by MS.[START_REF] Hatcher | SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform[END_REF]

Figure 18 :

 18 Figure 18: Chemical structure of Alectinib and SRPKIN-1 (left), the binding mode of Alectinib in SRPK (purple) and overlap of Alectinib in ALK (green).[START_REF] Hatcher | SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform[END_REF]

77

 77

Figure 20 :

 20 Figure 20: (A) crystal complex of FAK with ATP, (B) superposition of active FAK kinase (green) andautoinhibited kinase (red) by FERM.[START_REF] Lietha | Structural Basis for the Autoinhibition of Focal Adhesion Kinase[END_REF]

21 .

 21 solved the crystal structure of FAK kinase domain in 2002 and revealed the binding mode of ATP to FAK. The ATP binding site of FAK is shown in Figure The N1 nitrogen and the amino group of the adenine ring bind to the main chain of Cys502 and Glu500, respectively. The 2'-, 3'hydroxyl group of the ribose bond to the side chain of residue Glu506 through two hydrogen bonds. The phosphates of ATP are stabilized by interaction with residue Lys454 and Glu471. Moreover, around residue Met499, this region forms a hydrophobic pocket in FAK kinase.

Figure 22 :

 22 Figure 22: Chemical structures of some FAK inhibitors.

Figure 23 :

 23 Figure 23: Binding mode of VS-6062 in the active site of FAK.

FAK inhibitors. The most potent compound 34 (Figure 24 )

 3424 has a similar effect to TAE-226 on inhibition of cell viability of human umbilical vein endothelial cells (HUVEC) with IC50 values of 1.5 μM and 1.0 μM, respectively. However, in vitro enzymatic activity of compound 34 was nearly 45-fold weaker than that of TAE-226. The cocrystal structure of compound 34 and FAK was resolved and revealed that the 1,3,5-triazine derivative 34 bound to the hinge region in a similar conformation of TAE 226. The loss of enzymatic activity of compound 34 might be attributed to the fact that interaction between 5-chlorine of TAE-226 and Met499 was lost in our thiazine derivative. The group of Zhu reported the study of different five-member heterocyclic ring derivatives as cellular-active anticancer agents and FAK inhibitors, such as 1,3,4thiadiazole, 125,126 1,3,4-oxadiazole, 127,128 1,2,4-triazole, 129 and imidazole. 130 A 1,3,4oxadiazol derivative 6i (Figure 22) exhibited the most potent antitumour activity against MCF-7 breast cancer line and A431 epidermoid carcinoma cell line with IC50 values of 140 nM and 10 nM, respectively. Its inhibitory enzymatic activity of FAK kinase gave IC50 value of 20 nM. 127 Unfortunately, no further study of this compound was reported.

Figure 24 :

 24 Figure 24: (A) Structure of compound 34, (B) Superposition of the structures of 34 and TAE-226 bound to FAK.

Figure 25 :

 25 Figure 25: Chemical structures of some fused heterocycles-based FAK inhibitors.

Figure 26 :

 26 Figure 26: Molecular docking result of compound 32 binding in the active site of FAK. 132

  In vivo, CT-707 exhibited inhibition of tumour growth and metastasis in T47D, Karpas299 and 4T1 xenograft models. Combination of CT-707 with the MET inhibitor Cabozantinib showed synergistic anti-tumour effects against HCC in vitro and in vivo. 135 Now a phase I clinical study of CT-707 is initialling. A series of imidazo[1,2-a][1,3,5]triazines were synthesized as FAK inhibitors by our group 136 . The most potent compound 19a (Figure 25) inhibited the FAK enzymatic activity with an IC50 value of 50 nM. Kinase selectivity assay of compound 19a on a panel of 30 kinases showed that compound 19a rather specifically inhibited FAK kinase. The further biological evaluation showed that compound 19a could block the Tyr397 phosphorylation of FAK in the tested cell lines, such as U-87MG, HCT-116, MDA-MB-231 and PC-3, and this compound also inhibited cell proliferation by arresting cell cycle at G2/M phase in U87-MG and HCT-116 cells. 136

Figure 27 : 13 2. 4 . 3 .Figure 28 :

 27134328 Figure 27: Structure of compound 13

Figure 29 :

 29 Figure 29: Co-crystal structure of compound 30 with FAK (PDB: 4I4F).

Figure 30 :

 30 Figure 30: Chemical structure of compounds C4 and 29.

  described in chapter I, FAK kinase is a very promising therapeutic target and a lot of efforts have been made to discover highly selective FAK inhibitors. Though there have been some inhibitors in the clinical research, all these compounds compete with ATP and show a reversible inhibition against FAK. The targeted covalent inhibitors have many advantages over the reversible inhibitors such as increased efficiency, better selectivity, lower risk of drug resistance derived from active site mutation and prolonged duration of inhibition. Up to our work, it was a blank field to covalently inhibit the FAK activity. This chapter of my work dedicates to the discovery of highly selective targeted covalent inhibitors of FAK. This chapter consists of three parts: 1. Improvement of synthesis and characterization of the first covalent inhibitor of FAK; 2. Design, synthesis and biological evaluation of new irreversible and reversible covalent inhibitors; 3. Synthesis of imidazopyrimidine precursors for the development of bicyclic covalent inhibitors.Based on the preliminary results obtained in our laboratory, my first work consisted of improvement of chemistry involved in the synthesis of covalent inhibitors, the chemical and biochemical characterization. The work in this part was published in the journal of ACS Chemical Biology, 2018, 13(8), 2067-2073.145

Figure 31 :

 31 Figure 31: Location of cys427 and cys502.

Figure 32 :

 32 Figure 32: (A) cocrystal structure of TAE-226 and FAK enzymatic site, yellow dash line represents the distance between Cys427 and inhibitor. (B) Chemical structure of TAE-226. (C) The general structure of designed covalent inhibitors, represented linkers and electrophilic group are shown.

Figure 33 :

 33 Figure 33: Distribution of kinases that possess a cysteine at the same position of Cys427 in FAK.

Figure 34 :

 34 Figure 34: Sequences alignment of 20 kinases. Position of Cys427 was highlighted in yellow colo ur, the position of gatekeeper residue was highlighted in cyan.

Scheme 7 :

 7 Scheme 7: Retrosynthetic analysis

Figure 35 :

 35 Figure 35: Meisenheimer intermediates

10 in the presence of 1 Scheme 11 :

 111 Scheme 11: Synthesis of compound 13.

Scheme 13 : 2 )

 132 Scheme 13: retrosynthetic analysis

Scheme 16 :

 16 Scheme 16: Synthesis of Compound 21.

Bandyopadhyay 164 from 2 -Scheme 17 : 4 )Scheme 18 :

 217418 Scheme 17: Synthesis of azide 23.

  FAK were carried out by our collaborators. The enzymatic evaluation was done by C. Tomkiewicz-Raulet in University Paris Descartes, INSERM UMR-S 1124. The FAK-phosphorylation assay in cells and washout experiments were completed by J. Dawson in Edinburgh cancer research UK centre, University of Edinburgh. X-ray crystallography was carried out by D.

Figure 36 :

 36 Figure 36: Principle of the ADP-Glo kinase assay.

min, 1 h

 1 and 2 h for the kinase reaction at 3 concentrations (0.01 nM, 1.0 nM, and 100 nM) of inhibitors 15, 16, 24 and EYP. As shown in Figure37, all these 4 compounds have a dose-dependent inhibitory effect on FAK. The kinase activity declined at a higher tested concentration of inhibitors. In addition, compound 15 showed time-dependent inhibition of FAK (Figure37A). The enzymatic activity was reduced as the incubation time prolonged from 30 min to 2 h. However, this time-dependent inhibition was not observed for other compounds (Figure37B, C, and D). From these results, we could conclude that only compound 15 exhibited covalent inhibition properties.

Figure 37 :

 37 Figure 37: Remaining enzymatic activity after the treatment of inhibitors 15, 16, 24 and EYP at different concentrations and reaction times.

Figure 38 :

 38 Figure 38: Inhibition of the enzymatic activity of FAK by compounds 15 and 16 at different concentrations of ATP.

Figure 39b ,

 39b VS-4718 and compound 15 could effectively block the autophosphorylation of FAK on Tyr397, and the autophosphorylation was significantly inhibited even at the concentration as low as 0.1 nM for compound 15. Next, we studied the reversibility of the FAK autophosphorylation by washout experiments. In this assay, inhibitors were washed out after incubation for 48 h with SCC cells, and fresh medium was added. It was observed that the cancer cells treated with VS-4718 recovered the phosphorylation on Tyr397 (Figure39c), while the phosphorylation was still blocked for the cells treated by compound 15 (Figure39d). It could be concluded that compound 15 produced an irreversible inhibition of FAK phosphorylation in SCC cells.

Figure 39 :

 39 Figure 39: FAK autophosphorylation in SCC cells. a, SCC cells treated by VS-4718; b, SCC cells treated by compound 15; c, After washout of inhibitor VS-4718; d, After washout of inhibitor 15.

Figure 40 :

 40 Figure 40: Crystal structures of the FAK kinase domain in complex with inhibitors 15 (A and D) (PDB ID: 6GCR), 24 (B and E) (PDB ID: 6GCX) and 16 (C and F) (PDB ID: 6GCW). (A-C) 2Fo-Fc electron density countered at 1 σ is shown for the compounds (blue mesh) and Cys427 (cyan mesh), which are shown in stick representation. The disordered region of compounds are omitted; (D-E) The binding mode of compounds 15, 24 and 16 to the active site of FAK are shown. Compounds are shown in grey for ordered regions and in white for disordered regions. Key hydrogen bonds between the compounds and FAK are shown as orange dashed lines.

Figure 41 :

 41 Figure 41: Structures of new potentially irreversible covalent inhibitors.

Scheme 19 : 20 :Scheme 21 :

 192021 Scheme 19: Retrosynthetic pathway

Scheme 24 :Scheme 25 :Scheme 26 : 27 :

 24252627 Scheme 24: Reduction of nitrile compounds 3) Synthesis of 4-amino-2,5-dichloropyrimidines. As shown in Scheme 25, compound 37 was prepared from 2,4,5-trichloropyrimidine and compound 25 in the presence of NaHCO3 in ethanol under the condition of reflux overnight in 77% yield.

Figure 42 :

 42 Figure 42: Superposition of 1 H NMR spectra of 68a and 68b in CDCl3 (500 MHz).

Scheme 33 :

 33 Scheme 33: Substitution of 2,4-dichloro-5-trifluoromethylpyrimidine

Figure 43 :Scheme 34 :

 4334 Figure 43: 1 H NMR spectra of compounds 69a in CDCl3 (500 MHz).

Figure 44 :

 44 Figure 44: 1 H-NMR spectra showing reversible addition of βME to cyanoacrylate 3. (A) Proton spectrum of cyanoacrylate 3. (B) Cyanoacrylate 3 (90 mM) was treated with 100 mM βME in DMSO-d6: PBS-d (3:1), affording a 15:85 mixture that favoured the βME adduct 6. (C) Upon 10-fold dilution,the ratio of 3:6 turned to 45:55.[START_REF] Serafimova | Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles[END_REF]

Figure 45 :

 45 Figure 45: Representation of protein-targeting strategies. (A) Irreversible covalent inhibition by reactive ligands; (B) Reversible covalent inhibition by reactive ligands. 177

Figure 46 :

 46 Figure 46: Structures of reversible covalent inhibitors.

Figure 47 :

 47 Figure 47: Percentage of cell cycle distribution.

Figure 48 :

 48 Figure 48: Cell cycle analysis was done on U87-MG cells treated with DMSO or corresponding inhibitors at 1 µM concentration, and the cell distribution at each phase was drawn in histograms.

Figure 49 :

 49 Figure 49: Analysis of cell apoptosis on U87-MG cells at concentrations of 1 µM and 10 µM.

FAK inhibitors for 48 h. As shown in Figure 50 ,

 50 the cells treated with compounds 15, 74, 77 and 78 exhibited obviously cellular morphologic change, which was similar to that of treated with TAE-226. The cells became bigger than the control group and double nucleus in cells was also observed. However, compounds 64 and 65 showed a limited effect on cellular morphology with a slight swell of cell volume observed.

Figure 50 :

 50 Figure 50: Cell morphologic change observed by confocal microscopy.

Figure 51 :

 51 Figure 51: The ability of cell migration under each inhibitor treatment was determined by the scratch assay. (A) The pictures were taken immediately after making scratch (0 h) and at 24 h with or without 0.2 µM inhibitors. (B) The percentage of cell migrated distance into the scratched areas was displayed in the histogram.

Figure 52 :

 52 Figure 52: Inhibition of FAK phosphorylation on U87-MG cells. (A) Phosphorylation of FAK when U87-MG cells were exposed to FAK inhibitors for 48 h. (B) Phosphorylation of FAK when the inhibitors were washed out and fresh medium was added for 3 h.

  displayed significant inhibitory activity against FAK with IC50 values ranging from 1.0 nM to 16.3 nM. Results of in cell evaluation revealed that the substituent at position 4 of the pyrimidine ring was important to the cellular anticancer activities. Compounds with arylamino substituents (15, 59, 74, 77 -78) showed better inhibition of cell growth on oesophageal cancer cells compared with VS-4718 and blocked the cell cycle at the G2/M stage. In contrast, compounds 64 -66 which have arylmethanamino substituents neither had capacity to arrest cell cycle nor inhibited cell growth on oesophageal cancer cells.

Figure 53 :

 53 Figure 53: The chemical structures of newly synthesized compounds.

1 )Scheme 40 :analysis 2 )

 1402 Scheme 40: Retrosynthetic analysis

Figure 54 :

 54 Figure 54: NMR 2D-NOESY spectrum of compound 80a obtained in CDCl3 (500 MHz).

Figure 55 :Scheme 43 :

 5543 Figure 55: NMR 2D-NOESY spectrum of compound 80b obtained in CDCl3 (500 MHz).

  site of FAK, compounds 85 and 88 were crystallized with the kinase domain of FAK. The crystallography experiments were conducted by D. Lietha at the Spanish National Cancer Research Center (CNIO).

Figure 56 :

 56 Figure 56: (A) Interactions of compound 88 with the FAK kinase domain, (B) Superposition of compound 88 and TAE-226.

Figure 57 :FAK

 57 Figure 57: Crystal structure of compound 88 in FAK active site.

Figure 58 :

 58 Figure 58: Superposition of active (purple) and inactive (yellow) FAK kinase domain structure. The activation loop in the active state was shown in blue. (PDB ID: 2J0L and 2J0J).

Figure 59 :

 59 Figure 59: General structure of type II inhibitor of FAK.

Scheme 48 :

 48 Scheme 48: Buchwald Hartwig amination conditions.

Figure 60 :Scheme 49 :

 6049 Figure 60: Catalytic cycle of Buchwald-Hartwig amination.

Scheme 50 :

 50 Scheme 50: Heck reaction between aryl triflate and N, N-dimethylallylamine.

Scheme 51 :

 51 Scheme 51: Model reaction.

Figure 61 :

 61 Figure 61: Chemical structure of ligands.

Scheme 52 :Scheme 53 :

 5253 Scheme 52: Preparation of 2-aminosubstituted-5-bromopyrimidines.

Scheme 54 :Scheme 55 :Scheme 56 :

 545556 Scheme 54: Synthesis of compound 121.

  studied the microwave-assisted Buchwald-Hartwig amination of 2-substituted 5bromopyrimidine. Under the irradiation of microwave, the amination of 5-bromo-2-methyl pyrimidine could be fully achieved within 1 hour instead of over 20 hours under the conductive heating condition. With catalyst Pd2dba3, ligand XPhos, and base NaOtBu, this method had a broad substrate scope of amines including heteroaryl amines, arylamines, and aliphatic amines. In addition, we investigated the amination of 5-bromopyrimidines which bear various substituents at position 2 of the pyrimidine ring giving corresponding products in good to high yields. The efficient microwave-assisted synthesis of 2-5-disubstituted pyrimidine can be applied to the preparation of type II inhibitors of FAK. This work is under submission to the Tetrahedron Letters. Small molecule-based fluorescent probes are powerful and popular tools to visualize the biological processes because of their high resolution in space and time. Fluorescent chemical probes have been widely used in the tumour diagnosis 209 and in vivo tumour imaging. 210 In addition, these probes are also very useful tools in biological experiments to study the kinase signalling transductions. 211 Visualization of the specific protein in cells is an important strategy to analyze the role of a protein in the signal cascade. The antibody-based immunofluorescence and genetically encoded fluorescent reporters 212 are general methods for monitoring selected proteins in cells, but these methods can't satisfy all the biological experiments. In comparison with antibodybased immunofluorescence, the fluorescent small molecules are able to enter cells without fixation and permeabilization and to provide visualization of dynamic cellular processes. In spite of genetically encoded fluorescent reporter is able to achieve live-cell imaging, this

Figure 62 : 1 . 1 .

 6211 Figure 62: Expected fluorescent covalent inhibitors of FAK.

Figure 63 :

 63 Figure 63: Perrin-Jablonski diagram. IC, internal conversion; ISC, intersystem crossing. 217

Figure 64 :

 64 Figure 64: Chemical structure of TUG-1609 and its dissociation constant.

Figure 65 :

 65 Figure 65: Chemical structures of fluorescent COX-2 inhibitors.

2. Synthesis of Fluorescent compounds 2 . 1 .Scheme 58 :Scheme 59 :

 215859 Scheme 58: Synthesis of NBD-NH2

Figure 66 :Scheme 60 :

 6660 Figure 66: Chemical structures of 4, 6 and 143.

Scheme 61 :

 61 Scheme 61: Resonance of NBD-amines.

Scheme 63 :Scheme 64 :Scheme 65 : 3 . Fluorescence properties and biological evaluation 3 . 1 .

 636465331 Scheme 63: Synthesis of NBD-amine 147.

Figure 67 :

 67 Figure 67: Absorption and fluorescence spectra of compound 153 in PBS.

Figure 68 :

 68 Figure 68: Chemical structures of compound 15 and modified analogues.

Figure 69 :

 69 Figure 69: Chemical structures of fluorescent compounds.

  without further purification if it's not noted. CH2Cl2, DMF, 1,4-dioxane, Et3N and DIPEA were distilled over calcium hydride, THF was distilled from sodium benzophenone. The reaction was monitored by thin layer chromatography carried out on 60F-254 silica gel plates and visualized under UV light at 254 and 365 nm. Column chromatography was performed with silica gel 60 (Merck 40 -63 µM) and eluents were given in v/v. The yield refers to isolated and purified products. Chemical shifts (δ) of 1 H and 13 C NMR are reported in ppm, and the residual proton resonance of the solvents was used as reference. The coupling constants are given in Hz. Multiplets are indicated with following abbreviations, s = singlet, d = doublet, t = triplet, q = quadruplet, bs = broad singlet, m = multiplet. Mass spectra (MS) were recorded with an ion trap mass analyzer under electrospray ionization (ESI) in positive or negative ionization mode detection. High resolution mass spectra (HRMS) were recorded with a TOF mass analyzer under electrospray ionization (ESI) in positive or negative mode detection. Microwave reaction was carried out in Anton Paar monowave 400 reactor.

1 H

 1 NMR (250 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.96 (d, J = 4.7 Hz, 1H), 8.54 (dd, J = 8.5, 1.1 Hz, 1H), 8.50 (s, 1H), 7.84 (dd, J = 7.8, 1.5 Hz, 1H), 7.69 -7.54 (m, 1H), 7.31 -7.16 (m, 1H), 2.82 (d, J = 4.5 Hz, 3H).(9H-Fluoren-9-yl)methyl (4-nitrobenzyl)carbamate (3) 156To a solution of 4-Nitrobenzylamine hydrochloride (1.0 g, 5.1 mmol) in anhydrous DCM (40 mL), DIPEA (4.4 mL, 25 mmol) and Fmoc-Cl (1.5 g, 5.6 mmol) were added at room temperature. The mixture was stirred for 12 h. Subsequently, the reaction was diluted, washed with water and brine, the DCM layer was dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (Cyclyhexane: EA= 6:1~0:1) to give the desired compound as white solid (1.9 g, 99%). 1 H NMR (250 MHz, Chloroform-d) δ 8.21 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.4 Hz, 2H), 7.41 (m, 6H), 5.19 (s, 1H), 4.57 (d, J = 6.4 Hz, 3H), 4.49 (d, J = 6.3 Hz, 2H), 4.25 (t, J = 6.4 Hz, 1H) (9H-Fluoren-9-yl)methyl (4-aminobenzyl)carbamate (4) 156

1 H 13 C

 113 NMR (500 MHz, Chloroform-d) δ 7.07 (d, J = 7.9 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 5.92 (td, J = 10.8, 5.2 Hz, 1H), 5.30 (dd, J = 17.2, 1.6 Hz, 1H), 5.20 (dd, J = 10.5, 1.5 Hz, 1H), 4.94 (s, 1H), 4.58 (d, J = 4.8 Hz, 2H), 4.24 (d, J = 5.8 Hz, 2H), 3.66 (s, 2H). NMR (126 MHz, Chloroform-d) δ 156.3, 146.0, 133.1, 129.0, 128.4, 117.7, 115.3, 65.7, 44.9. (9H-Fluoren-9-yl)methyl (4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin -2-yl)amino)benzyl)carbamate (7)HCl solution (50 μL, 4 M in 1,4-dioxane) was added to a stirred suspension of 4 (345 mg, 1.0 mmol) and 2 (298 mg, 1.0 mmol) in n-BuOH (20 mL). The mixture was heated to 120 o C and stirred at this temperature for 20 h before the mixture was concentrated in vacuo. The residual material was purified by column chromatography (DCM: MeOH, 50:1~10:1) to give the desired product as off-white solid (316 mg, 52%).

  80 o C. Subsequently, NaOH solution (2 M, 15 mL) was added and the mixture aqueous solution was saturated with NaCl and extracted with DCM for 3 times. The combined DCM layers were dried over MgSO4, filtered, and concentrated in vacuo to give the title compound as oil (1.0 g, 96%).1 H NMR (250 MHz, Chloroform-d) δ 3.36 (t, J = 5.7 Hz, 2H), 2.94 -2.81 (m, 2H), 1.34 (s, 2H).N-(2-Azidoethyl)acrylamide (23)To a solution of 22 (340 mg, 3.95 mmol) in anhydrous DCM (10 mL) was added DIPEA (697 μL, 4.0 mmol) and acryloyl chloride (334 μL, 3.95 mmol) dropwise. The mixture was stirred at room temperature for 14 h and quenched with H2O. The mixture was washed with saturated brine. The organic phase was dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography (Cyclohexane: EA= 4:1) to give the title compound as light yellow oil (255 mg, 48%). 10.1 Hz, 1H), 5.93 (s, 1H), 5.72 (dd, J = 10.2, 1.5 Hz, 1H), 3.57 -3.45 (m, 4H). 2-((2-((4-((((1-(2-Acrylamidoethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)phenyl) amino)-5-chloropyrimidin-4-yl)amino)-N-methylbenzamide (24)To a solution of compound 21 (30 mg, 0.07 mmol) and compound 23(19 mg, 0.11 mmol) in DMF and water (1.5 mL, v/v=3:2) was added CuSO4 (3.4 mg, 0.02 mmol) and sodium ascorbate (8.5 mg, 0.04 mmol). The mixture was stirred at room temperature for 24 h. The heterogeneous mixture was concentrated in vacuo and purified by column chromatography (DCM: MeOH = 20:1~10:1) to give the title compound as white solid (13 mg, 33%).

1 H

 1 NMR (250 MHz, Chloroform-d) δ 8.26 (t, J = 1.4 Hz, 1H), 8.19 (dt, J = 7.9, 1.5 Hz, 1H), 7.95 (dt, J = 7.8, 1.4 Hz, 1H), 7.75 (td, J = 7.8, 0.6 Hz, 1H), 3.10 (s, 3H).

1 H

 1 NMR (500 MHz, Chloroform-d) δ 7.52 (dd, J = 7.6, 1.6 Hz, 1H), 7.35 (td, J = 7.5, 1.4 Hz, 1H), 7.28 (td, J = 7.6, 1.7 Hz, 1H), 7.22 (dd, J = 7.9, 1.4 Hz, 1H), 3.97 (s, 2H), 3.23 (s, 3H), 2.95 (s, 3H), 1.99 (s, 2H).13 C NMR (126 MHz, Chloroform-d) δ 143.3, 139.3, 130.0, 129.2, 128.0, 126.6, 42.1, 39.

1 H

 1 NMR (250 MHz, Chloroform-d) δ 7.40 -7.26 (m, 4H),

  HRMS (ESI) m/z calcd. for C24H28O4N6Cl [M+H] + : 499.18551, found: 499.18489. Melting point: 216 °C. 2-((2-((4-(Aminomethyl)phenyl)amino)-5-chloropyrimidin-4-yl)amino)-Nmethoxybenzamide trifluoroacetate(44)

1 H

 1 NMR (500 MHz, Chloroform-d) δ 7.93 (s, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.84 (dt, J = 7.8, 1.5 Hz, 1H), 7.62 (dt, J = 8.0, 1.3 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.44 -7.37 (m, 2H), 7.18 (d, J = 8.1 Hz, 2H), 7.05 (s, 1H), 5.93 (ddt, J = 17.2, 10.4, 5.6 Hz, 1H), 5.80 (t, J = 6.1 Hz, 1H), 5.34 -5.18 (m, 3H), 4.77 (d, J = 6.1 Hz, 2H), 4.59 (d, J = 5.6 Hz, 2H), 4.31 (d, J = 6.0 Hz, 2H), 2.96 (s, 3H).

tert-Butyl ( 2 -

 2 ((2-((4-((5-chloro-4-((2-(methoxycarbamoyl)phenyl)amino)pyrimidin -2yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamate(53) Following the general procedure F, from compound 44 (464 mg, 0.74 mmol) and compound 11 (200 mg, 0.74 mmol), the title compound was obtained as white solid (196 mg, 42%).

1 H

 1 NMR (250 MHz, Methanol-d4) δ 7.91 -7.81 (m, 2H), 7.64 -7.57 (m, 2H), 7.41 -7.33 (m, 3H), 4.84 -4.73 (m, 4H), 3.88 (t, J = 6.0 Hz, 2H), 3.20 (t, J = 6.0 Hz, 2H), 3.02 (s, 3H). HRMS (ESI) m/z calcd. for C25H27O4N7ClS [M+H] + : 556.15283, found: 556.15204.

2-(( 4 -

 4 Chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)-N-methylbenzamide (68a) 2-((2-Chloro-5-(trifluoromethyl)pyrimidin-4-yl)amino)-N-methylbenzamide (68b) To a solution of 2,4-dichloro-5-trifluoropyrimidine (180 µ L, 1.33 mmol) in n-BuOH (3 mL) was added compound 1 (200 mg, 1.33 mmol) and DIPEA (290 µ L, 1.66 mmol) at room temperature. The mixture was stirred at room temperature overnight. The mixture was diluted by DCM, washed with water and brine. The organic layer was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography (eluent, cyclohexane/ethyl acetate = 4/1) to give titled compounds as white solid (365 mg, 83%). 68a: 1 H NMR (250 MHz, Chloroform-d) δ 11.35 (s, 1H), 8.63 -8.54 (m, 2H), 7.57 -7.43 (m, 2H), 7.10 (td, J = 7.6, 1.1 Hz, 1H), 6.23 (s, 1H), 3.03 (d, J = 4.9 Hz, 3H). 68b: 1 H NMR (250 MHz, Chloroform-d) δ 11.61 (s, 1H), 8.56 (dd, J = 8.5, 1.1 Hz, 1H), 8.45 (q, J = 1.0 Hz, 1H), 7.61 -7.44 (m, 2H), 7.21 -7.11 (m, 1H), 6.25 (s, 1H), 3.02 (d, J = 4.9 Hz, 3H). Allyl (4-((4-chloro-5-(trifluoromethyl)pyrimidin-2-yl)amino)benzyl)carbamate (69a) To a solution of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (200 μL, 1.48 mmol) in mixed t-BuOH/DCM (v/v=1/1, 10 mL) was added zinc chloride (403 mg, 2.96 mmol) at 0 o C.

Allyl ( 4 -

 4 ((4-((2-(methylcarbamoyl)phenyl)amino)-5-(trifluoromethyl)pyrimidin-2yl)amino)benzyl)carbamate (70) To a solution 69b (380 mg, 0.98 mmol) in DMF (5 mL) was added compound 1 (147 mg, 0.98 mmol) followed by addition of HCl (250 μL, 4 M in 1,4-dioxane). The mixture was stirred at 90 o C for 7 hours. Then the solvent was removed in vacuo. DCM was added to the residue. Through filtration, the title compound was obtained as white solid (286 mg, 58%). tert-Butyl (2-((2-((4-((4-((2-(methylcarbamoyl)phenyl)amino)-5-(trifluoromethyl) pyrimidin-2-yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl) carbamate (72) To a solution of 71 (132 mg, 0.32 mmol) and 11 (86 mg, 0.32 mmol) in dry DMF (1.5 mL) was added DIPEA (56 µ L, 0.32 mmol) at room temperature. Then the mixture was heated to 80 o C and stirred overnight. The solvent was removed under reduced pressure. MeOH was added to the residue and was filtered to give white solid. Then the filtrate was concentrated and purified by column chromatography (eluent: DCM/MeOH= 30/1 ~ 15/1)

2-(( 5 -

 5 Chloro-2-((4-(((2-((2-(2-cyanoacetamido)ethyl)amino)-3,4-dioxocyclobut-1-en-1yl)amino)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide(75)

  Melting point: 258 o C 2-((5-Chloro-2-((4-(((2-((2-(2-cyanopent-2-enamido)ethyl)amino)-3,4-dioxocyclobut-1en-1-yl)amino)methyl)phenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide(76)

2-(( 5 -

 5 Chloro-2-((4-(((2-((2-(2-cyano-3-cyclopropylacrylamido)ethyl)amino)-3,4dioxocyclobut-1-en-1-yl)amino)methyl)phenyl)amino)pyrimidin-4-yl)amino)-Nmethylbenzamide (78)Compound 20 (100 mg, 0.17 mmol), cyclopropanecarbaldehyde (19 μL, 0.26 mmol) and piperidine (25 µL, 0.26 mmol) were added to a mixed solution of DMF (0.5 mL) and acetic acid (0.5 mL).The solution was heated to 100 o C and stirred for 2 hours. After cooling to room temperature, the solvent was removed in vacuo. Subsequently, the crude product was purified through silica gel column chromatography (eluent: DCM/MeOH= 20/1 ~ 15/1) to

13 C

 13 NMR (126 MHz, Chloroform-d) δ 159.9, 158.0, 105.4, 63.0, 50.4, 44.1, 27.9, 26.5, 24.9. MS ESI + :325.2 [M+H] 5-Bromo-N-methyl-N-phenylpyrimidin-2-amine (120) Compound 116 (250 mg, 1.0 mmol) in anhydrous DMF (2 mL) was added to a suspension of sodium hydride (120 mg, 60 wt% in mineral oil, 3.0 mmol) in anhydrous DMF (2 mL) at 0 °C. After stirring at this temperature for 30 minutes iodomethane (125 µL, 2.0 mmol) was added dropwise and the solution was allowed to warm to room temperature and stir for 4 h. The reaction was quenched by the addition of a saturated NaHCO3 solution (5 mL) and the solution was extracted with ethyl acetate (3 x 5 mL). The combined organic extracts were dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The residue was purified by column chromatography on silica (eluent: cyclohexane/ethyl acetate= 1/0 ~ 20/1) to give the title compound as white solid (225 mg, 85%). 4H), 3.50 (s, 3H).

(d, J = 8 . 1

 81 Hz, 2H), 6.41 (d, J = 8.9 Hz, 1H), 4.30 (d, J = 5.9 Hz, 2H), 3.81 (s, 7H). N 2 -(4-(Aminomethyl)phenyl)-5-chloro-N 4 -(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4yl)amino)ethyl)pyrimidine-2,4-diamine hydrochloride (150) To a suspension of 149 (500 mg, 0.91 mmol) in MeOH (30 mL) was added solution of HCl in 1,4-dioxane (4 M, 30 mL). The suspension was stirred at 65 o C for 24 h in a sealed tube. The solvent was removed under reduced pressure. MeOH (5 mL) was added to the residue and filtered to obtain the title product as orange-brown solid (374 mg, 84%). 3-((2-aminoethyl)amino)-4-((4-((5-chloro-4-((2-((7-nitrobenzo[c][1,2,5]oxadiazol-4yl)amino)ethyl)amino)pyrimidin-2-yl)amino)benzyl)amino)cyclobut-3-ene-1,2-dione trifluoroacetate salt (152) Compound 151 (110 mg, 0.158 mmol) was added to the solution of TFA (1 mL) and DCM

Figure 1 .

 1 Figure 1. Design of a potent and electrophilic inhibitor of FAK guided by structural bioinformatics. (A) Partial sequence alignment of FAK with 15 other protein kinases. (B) Structural assessment of cysteine residue 427 for covalent binding to FAK. The structure shows the FAK kinase (tan ribbon) bound to TAE226 (white sticks; PDB ID: 2JKK). The Cys427 side chain is colored in green and its distance to TAE226 indicated. (C) Reversible and irreversible inhibitors of FAK.

Scheme 1 .

 1 Scheme 1. Synthesis of FAK Targeted Covalent Inhibitor 5 and Reversible Inhibitor Matched-Pair 6 a

Figure 2 .

 2 Figure 2. (A) Targeted covalent inhibitor 5 showing time-dependent inhibition of FAK kinase. (B-D) No time-dependent inhibition of FAK kinase with targeted reversible inhibitors 6, 7, 8.

Figure 3 .

 3 Figure 3. (A) Targeted covalent inhibitor 5 showing non-ATP dependent inhibition of FAK kinase, (B) ATP-dependent inhibition of FAK kinase with targeted reversible inhibitor 6.

Figure 4 .

 4 Figure 4. Crystal structures of the FAK kinase domain in complex with inhibitors 5 (PDB ID: 6GCX), 6 (PDB ID: 6GCW), and 7 (PDB ID: 6GCR). (A-C) The active sites of the FAK kinase domain bound to 5, 6, and 7 are shown. Inhibitors are shown in gray for ordered regions and white for disordered regions. Although the white regions are modeled, they are not supported by electron density as can be seen in panels A-C. Key hydrogen bonds between the compounds and the FAK kinase are shown as orange dashed lines. (D-F) 2F o -F c electron density countered at 1σ is shown for the compounds (blue mesh) and Cys427 (cyan mesh), which are shown in stick representation. The FAK kinase is shown as a ribbon in tan. Note that only compound 5 exhibits continuous density connecting to Cys427.

Figure 5 .

 5 Figure 5. Superposition of the two structures of compounds 5 and 7 bound to FAK. The compounds 5 (green) and 7 (blue) and side chains in FAK interacting with the compound are shown in stick representation. Hydrogen bonds are shown as orange dashed lines.

Figure 6 .

 6 Figure 6. Inhibition of FAK autophosphorylation in SCC cells. (a,b) 5 and VS-4718 inhibited the autophosphorylation of FAK in a dosedependent manner. (c,d) Washout experiments showed that the inhibition of FAK autophosphorylation could be reversed for the reversible inhibitor VS-4718, but not for the irreversible inhibitor 5. The experiments were repeated three times and were consistent.

Table 1 :

 1 covalent warheads for targeted amino acid labelling of kinase and their binding mode

	Chemical	Targeted	Kinetic	Binding mode
	structure	residues	properties
		Cys	Irreversible
			covalent
		Cys	Irreversible
			covalent
	X=Cl, Br
		Cys	Irreversible
			covalent
		Cys	Irreversible
			covalent,
			Metabolically
			limited
			reactivity
		Cys	Reversible
			covalent
		Lys	Irreversible
		Tyr	covalent
		Lys	Irreversible
		Cys	covalent
		His

Table 2 :

 2 FAK expression in tumours

	Tissue	Normal	benign	Pre-invasive	Invasive	metastatic	ref
	Breast	+/-			+++	++++	92
	Colon	+	-	+++	+++	+++	92
	Thyroid	+/-	+		+/+++	++++	93
	Prostate	+/-	+		+	+++	87
	Liver	-			+++		92
	Brain	-	-	+	-	+++++	94

Table 3 .

 3 The

intermediate 9 (IC50 = 26 nM) was less potent than TAE-226 (IC50 = 5.8 nM), whereas the reversible inhibitor 16 showed equivalent inhibition of FAK as TAE-226 with IC50 values of 4.7 nM and 5.8 nM, respectively. However, the compounds bearing an electrophile (15, 24 and EYP) showed improved inhibitory activity against FAK. Indeed, in comparison with TAE-226, compound 15 showed 10-fold improved potency against FAK with an IC50 value of 0.6 nM, while compound 16 and EYP inhibited the activity of FAK with IC50 values of 2.3 nM and 1.2 nM, respectively.

Table 3 :

 3 In vitro evaluation of FAK inhibition

	Compound	IC50 (nM)	Compound	IC50 (nM)
		26±2.2		0.6±0.04
		4.7±0.5		2.3±0.3
		1.2±0.1		5.8±0.6

Table 4 :

 4 Kinase selectivity of compound 15 on a panel of 9 kinases.

	Kinase	Inhibition at 1 μM
	FAK	100%
	Src	87%
	c-kit	70%
	EGFR	88%
	HER2/ErbB2	0%
	IGF1-R	95%
	IRR	96%
	PDGF-Ra	98%
	Pyk2	100%
	IR	21%

  Coupling of the squaramide linker With the same strategy in Part 1, the squaramide linker was directly coupled to the pyrimidine moiety. Squaramate 11 and compound 44 were condensed in DMF in the presence of DIPEA at 85 o C to give intermediate 53 in 42% yield. Compounds 54 -57 were synthesized under the same conditions from 49 -52 in a yield of 58% -86% (Scheme 29).

and then the coupling reaction of compound 37 and 42 catalyzed by Pd2dba3 and XantPhos generated the desired compound 43 in 45% yield. Subsequently, the protecting group was removed quantitatively with TFA giving compound 44 (Scheme 27). Scheme 28: Synthesis of compounds 49 -53.

The nucleophilic substitution of 2-chlorine in compounds 38 -41 by arylamine 6 proceeded well under acidic conditions. The diaminosubstituted pyrimidines 45 -48 were obtained in a yield of 55% -88%. The deprotection was carried out according to a classical protocol

159

with Pd(PPh3)4 to give compounds 49 -53 with yields of 81% -94% (Scheme

28).

5)

Scheme 29: Coupling of squaramide linker 6) Incorporation of the electrophilic functional group. Scheme 30: Synthesis of compound 59.

Compound 53 was deprotected by TFA in DCM giving compound 58. Afterwards, the electrophilic functional group was incorporated to compound 58 through an acylation reaction to provide final product 59 (Scheme 30). As shown in Scheme 31, final products 64 -67 were obtained following the same approach in a yield of 50% -65%. Scheme 31: Synthesis of compounds 64 -67.

Table 5 :

 5 In vitro evaluation of FAK inhibition. The expected reversible covalent inhibitors 76 -78 had improved anti-FAK activity compared with TAE-226. In addition, the compounds with an isopropyl(77) or a cyclopropyl (78) group on the

	Compound	R	X	E	IC50 (nM)
	59		Cl		3.7 ± 0.2
	64		Cl		4.6 ± 0.5
	65		Cl		5.5 ± 0.4
	66		Cl		4.9 ± 0.6
	67		Cl		16.3 ± 1.5

a , IC50 value calculated before.

As shown in Table

5

, the newly synthesized compounds significantly inhibited FAK enzymatic activity with IC50 values ranging from 1.0 to 16.3 nM. In comparison with the irreversible covalent inhibitor 15, introducing a benzohydroxamate group at 4-position of pyrimidine ring (59) is not favourable, giving an IC50 value of 3.7 nM against FAK. The compounds 64 -67 with a sulfonamide function group on the aromatic ring and containing a methylene spacer between the aromatic ring and 4-amino of pyrimidine were less potent than the reference compound 15. Compound 15 had about 10-times higher anti-FAK activity than compounds 64 -67. However, compound 64 -66 exhibited similar inhibitory potency as TAE-226. Furthermore, N-methylmethanesulfonamide substituent at position 3 of the aromatic ring (66, IC50=4.9 nM) had better inhibition activity of FAK than methylsulfonyl substituent (67, IC50=16.3 nM). The replacement of Cl with CF3 group at position 5 of the pyrimidine ring caused a decrease of inhibitory activity on FAK, with an IC50 value of 6.8 nM for compound 74 compared to 0.6 nM for compound 15.

Table 6 ,

 6 compounds 15, 77 and 78 had similar potencies with Ki/kinact values ranging from 8.87 µ M -1 •S -1 ~ 11.6 µ M -1 •S -1 . Compounds 77 and 78 had kinact values about 1.5-fold that of compound 15, which is consistent with the fact that α-cyanoacrylamide electrophile is more reactive than the acrylamide towards thiol. The compound 74 exhibited decreased values of Ki (2.64 nM), which implies that the introduction of the

			•𝐾 𝑖𝑛𝑎𝑐𝑡 •t 𝜂 IC 50 • 𝐾 𝑖𝑛𝑎𝑐𝑡 • t	-1)
	𝜂 IC 50 =	𝐾 𝑖 (1 +	IC 50 (𝑡) 𝑆 𝐾 𝑀 ) + IC 50 (𝑡)
	As shown in

trifluoromethyl group at position 5 of pyrimidine reduced compound's affinity. In addition, the kinact of compound 74 was 3-times lower than that of compound 15, the introduction of trifluoromethyl group maybe is not benefit to the formation of the covalent bond between compound 74 and the Cys427. As a result, compound 74 had poor potency toward FAK with kinact/Ki value of 0.3 µM -1 •S -1 .

Table 6 :

 6 Determination

		Compounds	Ki (nM)	kinact (min -1 )	kinact/Ki (M -1 s -1 )
		15	0.23 ± 0.03	0.16 ± 0.02	11.6 ± 3.4
		74	2.64 ± 0.4	0.047 ± 0.01	0.30 ± 0.12
		77	0.47 ± 0.06	0.25 ± 0.03	8.87 ± 2.51
		78	0.40 ± 0.05	0.24 ± 0.04	10.0 ± 3.33
	6.2. In cell evaluation.
	6.2.1.	Effect of new inhibitors on oesophageal cancer cells.

of kinetic parameters Ki, kinact, and kinact/Ki for 15, 74, 77 and 78.

Table 7 :

 7 Inhibition of cell growth in Oesophageal cancer cells.

	Compound	AD1	Growth inhibition of Oesophageal cells (EC50 nM) Flo1 MFD1 OE33 P4C	SKGT4
	59	375.94	148.27	1844.62	1134.34	482.11	622.1
	64	NA	NA	NA	NA	NA	NA
	65	NA	NA	NA	NA	NA	NA
	66	NA	NA	NA	NA	NA	NA
	67	NA	NA	NA	NA	NA	NA
	74	303.67	79.74	859.59	1411.82	849.18	680.7
	76	613.78	300	1414.52	4069.89	1165.04	1295.85
	77	664.61	160.4	1456.33	1088.88	1061.84	1129.99
	78	226.54	17.39	748.31	166.88	384.42	582.61
	15	367.73	107.12	786.24	1265.89	1094	1158.24
	VS-4718	1316.5	581.4	2133.97	869.86	2160.68	1733.67
	Pac	0.3	0.04	0.81	0.04	0.11	0.3
			NA, no activity; Pac: Paclitaxel.

As shown in Table

7

, most compounds (15, 74, 76 -78) which have aminobenzamide substituent at position 4 of the pyrimidine ring showed good inhibitory effect on cell growth. However, the compounds with arylmethanamino substituent at 4-position of pyrimidine (64 -67) did not inhibit the cell growth. In comparison with the reversible inhibitor VS-4718, compounds 15, 74, and 76 -78 displayed equivalent or stronger inhibitory activity especially against AD1 and Flo1 cancer lines. Additionally, compound 78 was the most potent inhibitor with EC50 values of 17.39 nM ~ 748.31 nM. The Flo1 cells were the most sensitive oesophageal cancer lines to FAK inhibitors. The cell growth of Flo1 was significantly inhibited by compounds 74 and 78 with EC50 values of 79.74 nM and 17.39 nM, respectively.

Table 8 :

 8 Antiproliferative activity against glioblastoma cancer cells

			IC50 (µM)
	Compound
		U87-MG	A172	U251
	15	2.2 ± 0.1 0.65 ± 0.05 0.22 ± 0.01
	74	3.3 ± 0.4 0.13 ± 0.01 0.15 ± 0.02
	77	7.1 ± 0.6 1.3 ± 0.02	9.8 ± 0.8
	78	7.2 ± 0.5 0.89 ± 0.07 1.2 ± 0.01
	TAE-226 2.9 ± 0.4 8.3 ± 0.6	6.3 ± 0.5

As shown in

Table 8, compound 15 (IC50 = 2.2 µ M ) showed a slight improvement on inhibition of proliferation on U87-MG cells than that of TAE-226 (IC50 = 2.9 µ M ), while other compounds (74, 77 and 78) were less potent than TAE-226. Our compounds 15, 74, 77,

Table 9 :

 9 Inhibition of kinase activity (results were indicated as a mean of 2 experiments).

	Kinase		Inhibition (at 100 nM)
		15	74	77
	FAK	98.1%	98.6%	94.1%
	PyK2	97.6%	99.8%	97.5%
	EGFR	47.2%	29.0%	25.1%
	FGFR1	87.1%	88.3%	83.5%
	IGF1R	91.5%	94.4%	71.9%
	PDGFRα	91.0%	78.4%	67.5%
	c-Kit	0.2%	0.1%	-5.9%
	IR	57.6%	82.5%	32.4%
	Src	52.1%	55.6%	31.1%

  10, an acylation step gave compound 93 in 62% yield. The Boc group was removed using Yung's method 192 with 4 M HCl in 1,4-dioxane at room temperature. After concentration, the hydrochloride was neutralized with Et3N and reacted with dimethylsquarate giving key intermediate 94 in 89% yield (Scheme 46).

Scheme 46: Synthesis of compound 94.

Table 11 :

 11 Influence of base and solvent a

	Entry	Base	Solvent	Yield b
	1	Cs2CO3	1,4-Dioxane	65%
	2	K3PO4	1,4-Dioxane	45%
	3	NaOtBu	1,4-Dioxane	80%
	4	NaOtBu	Toluene	57%
	5	NaOtBu	DMF	trace

a, Pd2dba3 and XPhos were used as catalyst and ligand; b, Isolated yield

Table 12 :

 12 Amination of 5-bromo-2-methylpyrimidine.

	Entry	Amine	Product	Yield a
				82%
				86%
				83%
				81%
				85%
				82%
				59%
				69%

a, isolated yield; b, reaction time 30 min.

Table 13 :

 13 C-N coupling of various 5-bromopyrimidines and 4-aminobenzonitrile.

	Entry	5-bromopyrimidine	Product	Yield
	1			84%
	2			44%
	3			81%
	4			99%
	5			89%

Table 14 .

 14 The

	anti-FAK

Table 14 :

 14 In vitro inhibition of FAK.

	compound	Inhibition of FAK (IC50)
		3.3 µM
		2.87 µM
		14.27 µM

  Ethyl ether was added to the resulted syrup and filtered to give the crude product as off-white solid (214 mg, 53%). The crude product was used directly for the next step without further purification.

	Allyl (4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2-yl)amino)benzyl) 2-((2-((4-(Aminomethyl)phenyl)amino)-5-chloropyrimidin-4-yl)amino)-N-
	carbamate (8) methylbenzamide (9)
	To a solution of 2 (600 mg, 2.02 mmol) in n-BuOH, 6 (416 mg, 2.02 mmol) and HCl (505 Synthetic route A: A solution of compound 7 (570 mg, 1.06 mmol) in DMF and piperidine
	μL, 4 M in 1,4-dioxane) were added. The mixtures were heated at 120 o C overnight. After (12 mL, v/v=5/1) was stirred at room temperature for 2 h. The solution was concentrated
	cooled to room temperature, the solution was diluted with DCM, and washed with 1M in vacuo.
	K2CO3, brine and dried over MgSO4. The organic phase was concentrated under reduced
	pressure. The residue was purified through silica gel column chromatography to give the
	desired compound as white solid (419 mg, 44%).
	13 C NMR (126 MHz, DMSO-d6) δ 168.9, 157.7, 156.1, 154.9, 154.6, 139.3, 138.9, 133.8,
	133.1, 131.4, 127.9, 127.2, 121.9, 121.4, 120.7, 119.6, 116.8, 105.0, 64.3, 43.5, 26.3.
	HRMS (ESI) m/z calcd. for C23H24O3N6Cl [M+H] + : 467.15984, found:467.16043.
	1 H NMR (250 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.48 (s, 1H), 8.77 (dd, J = 9.7, 6.7 Hz, Melting point: 229 °C.
	2H), 8.23 (s, 1H), 7.96 -7.67 (m, 6H), 7.60 (d, J = 8.2 Hz, 2H), 7.44 (dq, J = 17.5, 9.8,
	8.7 Hz, 4H), 7.31 (d, J = 7.3 Hz, 2H), 7.12 (dd, J = 16.6, 8.0 Hz, 3H), 4.36 (d, J = 6.8 Hz,
	2H), 4.24 (t, J = 6.8 Hz, 1H), 4.16 (d, J = 6.1 Hz, 1H), 2.82 (d, J = 4.4 Hz, 3H).

HRMS: (ESI) m/z [M+Na] + , C34H29ClN6NaO3, calcul. 627.1887, found, 627.1867. 1 H NMR (500 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.42 (s, 1H), 8.79 -8.68 (m, 2H), 8.21 (s, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H), 7.70 (t, J = 6.2 Hz, 1H), 7.62 -7.57 (m, 2H), 7.48 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.19 -7.10 (m, 3H), 5.93 (ddt, J = 17.3, 10.6, 5.3 Hz, 1H), 5.34 -5.13 (m, 2H), 4.50 (dt, J = 5.4, 1.6 Hz, 2H), 4.15 (d, J = 6.2 Hz, 2H), 2.81 (d, J = 4.5 Hz, 3H). Synthetic route B: To a solution of 8 (836 mg, 1.79 mmol) in anhydrous THF, morpholine (3 mL, 34.8 mmol) and Pd(PPh3)4 (103 mg, 0.09 mmol) were added under an inert

  To a solution of 9 (580 mg, 1.51 mmol) in dry DMF were added compound 11 (409 mg, 1.51 mmol) and DIPEA (256 μM, 1.51 mmol). The mixture was stirred at 75 o C for overnight. After removal of the solvent, the crude product was purified by flash silica gel chromatography to give the desired product as white solid (724 mg, 77%). s, 1H), 7.82 -7.70 (m, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.50 (t, J = 7.9 Hz, 1H), 7.28 -

	2-((5-Chloro-2-((4-(((2-methoxy-3,4-dioxocyclobut-1-en-1-yl)amino)methyl)phenyl) 4,4,5,5-Tetramethyl-2-(4-nitrophenyl)-1,3-dioxolane (17) 161 2-((5-Chloro-2-((4-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)phenyl)amino)pyrimidin-4-2-((5-Chloro-2-((4-formylphenyl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide (20)
	amino) pyrimidin-4-yl)amino)-N-methylbenzamide (12) yl)amino)-N-methylbenzimidamide (19)
	and p-toluenesulfonic acid (57 mg, 0.3 mmol) was refluxed in dry toluene (30 mL) for 4 temperature. Then the solution of NaOH (2 M) was added till pH=14. Then the mixture 7.08 (m, 3H), 3.70 (s, 2H), 3.58 (s, 1H), 3.28 (d, J = 2.4 Hz, 3H), 3.10 (t, J = 2.4 Hz, 1H), 2.82 (d, J = 4.4 Hz, 3H). Route 2. 1 H NMR (250 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.51 (s, 1H), 8.74 (d, J = 8.3 Hz, 2H), A mixture of 4-nitrobenzaldehyde (1.51 g, 10.0 mmol) and pinacol (2.36 g, 20.0 mmol) Compound 19 was treated with 10% HCl solution in THF (20 mL) overnight at room 2-Azidoethan-1-amine (22) 164
	Compound 9 (200 mg, 0.52 mmol) was dissolved in dry DMF (3 mL) and dropped to a 8.23 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.47 (t, J = 7.9 Hz, 1H), h. After cooling to room temperature, a solution of NaOH (80 mg, 2.0 mmol) in ethanol A sealed tube was charged with 2 (272 mg, 1.23 mmol), 18 (36.6 mg, 1.23 mmol), was filtered to give the title compound as a yellow solid (228 mg, 86%).
	solution of dimethyl squarate (91 mg, 0.63 mmol) and DIPEA (0.1 mL, 0.52 mmol) in dry 7.26 (d, J = 8.2 Hz, 2H), 7.14 (t, J = 7.5 Hz, 1H), 6.91 (s, 1H), 4.66 (d, J = 5.9 Hz, 2H), 3.52 (s, 2H), 3.10 (d, J = 6.1 Hz, 2H), 2.82 (d, J = 4.3 Hz, 3H), 1.36 (s, 9H). Pd2(dba)3 (23 mg, 2 mol%), XPhos (47 mg, 8 mol%) and Cs2CO3 (400 mg, 1.23 mmol) (2 mL) was added to the mixture and stirred for 30 min. The suspension was filtered and and degassed 1,4-dioxane (7 mL) under N2 atmosphere. The tube was evacuated and Sodium azide (1.8 g, 27.7 mmol) was added to a solution of 2-bromoethan-1-amine the residue was washed with toluene. The filtrate was washed with brine, dried over MgSO4 and finally concentrated in vacuo to give the desired compound as solid (2.23 g, backfilled 3 times with argon and stirred at room temperature for 10 min. Then the mixture hydrobromide (2.5 g, 12.2 mmol) in water (30 mL). The mixture was stirred for 20 h at
	Melting point: 226 °C. 2-((2-((4-(((2-((2-Aminoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)methyl) phenyl)amino)-5-chloropyrimidin-4-yl)amino)-N-methylbenzamide trifluoroacetate salt (14) 92%). 4-(4,4,5,5-Tetramethyl-1,3-dioxolan-2-yl)aniline (18) 161 was heated at 100 13 C NMR (126 MHz, DMSO-d6) δ 168.9, 157.6, 155.0, 154.5, 140.5, 139.2, 132.8, 131.4, N-methylbenzamide (21)
	127.9, 126.6, 122.0, 121.5, 120.8, 119.0, 105.3, 99.1, 81.9, 26.3, 24.2, 22.0. A solution of compound 20 (110 mg, 0.29 mmol), propargylamine (57 μL, 0.87 mmol) and
	A mixture of 17 (1.0 g, 3.98 mmol) and 10% Pd/C in degassed THF (30 mL) under a a drop of AcOH in distilled DMF (4 mL) was stirred at room temperature under Argon
	1 H NMR (250 MHz, Chloroform-d) δ 4.90 (s, 1H), 4.39 (s, 3H), 3.76 (s, 1H), 3.54(s, 1H), hydrogen atmosphere was stirred for 7 h. The mixture was filtrated through a pad of celite atmosphere for 8 h. Then NaBH3CN (91 mg, 1.45 mmol) was added. The stirring was
	3.35 (q, J = 5.8 Hz, 2H), 1.43 (s, 9H). and the filtrate was concentrated in vacuo to give the desired compound as white solid maintained for 12 h. After quenched with H2O, the mixture was extracted with DCM 3
	Route 1. A solution of compound 12 (91 mg, 0.81 mmol), 10 (58 mg, 0.36 mmol) and Following the general procedure E, from compound 13 (716 mg, 1.15 mmol), the title (870 mg, 99%). times. The combined organic phase was dried over MgSO4, filtered and concentrated in
	compound was obtained as solid (983 mg, quant).

1

H NMR (250 MHz, Chloroform-d) δ 4.86 (br, 1H), 3.16 (q, J = 5.9 Hz, 2H), 2.79 (t, J =5.9 Hz, 2H), 1.44 (s, 9H), 1.39 (s, 2H).

tert-Butyl (2-((2-methoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamate

(11)

To a solution of dimethylsquarate (1278 mg, 9.06 mmol) in MeOH was added the solution of 1 (1320 mg, 8.23 mmol) in MeOH dropwise. After stirring at room temperature overnight, the mixture was concentrated and purified by silica column chromatography (DCM/MeOH= 50/1~30/1) to give the title compound as white solid (1451 mg, 65%). DMF (5 mL) at room temperature. The mixture was left to stir for 24 h and the solution was removed in vacuo. Subsequently, DCM was added to the residue to give the product as a pale solid (207 mg, 81%). The crude product was used directly for the next step without further purification. 1 H NMR (250 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.51 (s, 1H), 8.74 (d, J = 11.2 Hz, 2H), 8.23 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.52 -7.43 (m, 1H), 7.27 -7.07 (m, 3H), 4.68 -4.37 (m, 2H), 4.32 (d, J = 10.1 Hz, 3H), 2.82 (d, J = 4.4 Hz, 3H). tert-Butyl (2-((2-((4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)pyrimidin-2yl)amino) benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)carbamate (

13

) DIPEA (43 μL, 0.2 mmol) in dry DMF (2 mL) was stirred at 70 o C for 8 h. Subsequently, the reaction mixture was concentrated in vacuo to afford the crude product which was purified by column chromatography (DCM: MeOH=20: 1). The desired compound was obtained as a white solid (83 mg, 72%).

13

C NMR (126 MHz, DMSO-d6) δ 182.6, 182.4, 168.8, 162.2, 157.6, 155.7, 154.9, 154.5, 139.5, 139.2, 132.0, 131.3, 127.9, 127.8, 121.9, 121.4, 120.7, 119.8, 105.1, 77.8, 46.5, 43.2, 41.1, 28.1, 26.2. HRMS (ESI) m/z calcd. for C30H33ClN8O5 [M+H] + : 621.2341, found: 621.2308.

1 H NMR (250 MHz, Chloroform-d) δ 8.25 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.6 Hz, 2H), 6.04 (s, 1H), 1.36 (s, 6H), 1.25 (s, 6H). 1 H NMR (250 MHz, Chloroform-d) δ 7.30 (d, J = 8.3 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 5.92 (s, 1H), 3.75 (s, 2H), 1.33 (s, 6H), 1.31 (s, 7H). o C for 20 h. After cooling to room temperature, the reaction mixture was diluted and filtered through a celite pad and concentrated in vacuo. The crude residues were purified by column chromatography (DCM:MeOH=100:1) to give the title compound as yellow solid (337 mg, 57%). 1 H NMR (500 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.55 (s, 1H), 8.81 -8.72 (m, 2H), 8.24 (s, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H), 7.70 -7.64 (m, 2H), 7.49 (td, J = 7.2, 1.6 Hz, 1H), 7.37 -7.30 (m, 2H), 7.16 (td, J = 7.6, 1.2 Hz, 1H), 5.86 (s, 1H), 2.82 (d, J = 4.5 Hz, 3H), 1.24 (d, J = 17.2 Hz, 12H). 1 H NMR (250 MHz, DMSO-d6) δ 11.66 (s, 1H), 10.04 (s, 1H), 9.85 (s, 1H), 8.79 (s, 1H), 8.71 (d, J = 8.3 Hz, 1H), 8.34 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 7.8 Hz, 3H), 7.58 (t, J = 7.9 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 2.82 (d, J = 4.4 Hz, 3H). 2-((5-Chloro-2-((4-((prop-2-yn-1-ylamino)methyl)phenyl)amino)pyrimidin-4-yl)amino)vacuo. The crude residue was purified by column chromatography (DCM: MeOH = 50:1~20:1) to give the title compound as yellowish solid (30 mg, 25%).

(

  8.6, 6.6 Hz, 6H), 7.64 -7.49 (m, 1H), 7.40 (d, J = 8.5 Hz, 4H), 7.32 -7.17 (m, 2H), 4.10 (s, 4H), 3.82 (s, 6H).

	Allyl (4-((5-chloro-4-(((2-(N-methylmethylsulfonamido)pyridin-3-yl)methyl)amino)
	pyrimidin-2-yl)amino)benzyl)carbamate (46)
	HRMS (ESI) m/z calcd for C19H20ClN6O2 399.13308, found: 399.13248.
	Melting point: 240 °C.
	Allyl (4-((5-chloro-4-((2-(N-methylmethylsulfonamido)benzyl)amino)pyrimidin-2-Following the general procedure C, from 39 (389 mg, 1.07mmol), aniline 6 (222 mg,
	yl)amino)benzyl)carbamate (45) 1.08mmol) and HCl (270 μL, 1.08mmol), the title compound was obtained as pale foamy
	solid (493 mg, 86%).
	13 C NMR (126 MHz, Chloroform-d) δ 158.1, 157.9, 156.3, 153.2, 139.9, 139.4, 139.4,
	133.0, 132.1, 130.9, 129.4, 129.0, 128.3, 126.5, 119.5, 117.8, 105.3, 65.7, 44.9, 40.6, 39.5,
	35.7.
	HRMS (ESI) m/z calcd. for C24H28O4N6ClS [M+H] + : 531.15758, found: 531.15652.
	Melting point: 110 °C.

Following the general procedure C, form 38 (400 mg, 1.11 mmol), aniline 6 (229 mg, 1.08 mmol) and HCl (280 μL, 1.11 mmol), the title compound was obtained as pale solid (375 mg, 64%). 1 H NMR (500 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.55 -7.47 (m, 3H), 7.38 -7.27 (m, 3H), 7.22 -7.15 (m, 3H), 6.17 (t, J = 6.1 Hz, 1H), 5.92 (ddt, J = 16.3, 10.8, 5.6 Hz, 1H), 5.34 -5.19 (m, 2H), 5.07 (t, J = 6.2 Hz, 2H), 4.64 (br, 1H), 4.59 (d, J = 5.7 Hz, 2H), 4.30 (d, J = 5.9 Hz, 2H), 3.27 (s, 3H), 2.99 (s, 3H). 1 H NMR (500 MHz, Methanol-d4) δ 8.40 (dd, J = 4.8, 1.8 Hz, 1H), 7.86 (s, 1H), 7.77 (dd, J = 7.7, 1.8 Hz, 1H), 7.35 -7.28 (m, 3H), 7.05 (d, J = 8.2 Hz, 2H), 5.94 (ddd, J =

  .2, 147.3, 139.9, 137.1, 133.9, 131.2, 127.7, 124.0, 118.5, 103.7, 77.8, 46.4, 43.2, 41.1,

	37.1, 36.0, 28.2.
	HRMS (ESI) m/z calcd. for C30H37O6N9ClS [M+H] + : 686.22705 found: 686.22590.
	Melting point: 209 °C.
	tert-Butyl (2-((2-((4-((5-chloro-4-((3-(N-methylmethylsulfonamido)benzyl)amino)
	1 H NMR (500 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.43 (dd, J = 4.7, 1.9 Hz, 1H), 8.01 (s, 1H),
	7.73 -7.68 (m, 1H), 7.66 (t, J = 6.0 Hz, 1H), 7.45 -7.39 (m, 3H), 7.04 (d, J = 8.1 Hz,

2H), 6.87 (d, J = 5.8 Hz, 1H), 4.76 (d, J = 5.9 Hz, 2H), 4.63 -4.52 (m, 2H), 3.51 (s, 2H), 3.15 (s, 6H), 3.09 (q, J = 6.0 Hz, 2H), 1.35 (s, 9H).

152

  , 43.3, 41.1, 28.2.

HRMS (ESI) m/z calcd. for C30H35O6N7ClS [M+H] + : 656.20526, found: 656.20439. Melting point: 186 °C.

  The mixture stirred at room temperature for 3 h. Subsequently, the solvent was removed under reduced pressure. Ethyl ether was added to the remained residue and filtered to give the crude product as beige solid, then purification by silica gel chromatography (DCM/MeOH/NH4OH = 10/1/0.01) to give the title compound as off-After cooling to room temperature, the mixture was diluted by ethyl acetate. The mixture was washed by water, brine. The organic phase was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: ethyl acetate/MeOH = 1/0 ~ 10/1) to give the title compound as white solid (729 mg, 86%).

	Methyl 2-((5-chloroimidazo[1,2-a]pyrimidin-7-yl)amino)benzoate (80a) 5,7-Dichloroimidazo[1,2-a]pyrimidine (82) mixture was refluxed for 20 h with stirring. After cooling to room temperature, the solvent Melting point: 164 o C. Methyl 2-((7-((4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)phenyl)amino) stirred at room temperature for 3 h. The solvent was removed under reduced pressure and 5-Bromo-N-phenylpyrimidin-2-amine (116) gel column chromatography (eluent, cyclohexane/ethyl acetate= 2/1) to give the title
	155.0, 154.6, 139.5, 139.2, 132.1, 131.4, 129.9, 127.8, 121.9, 121.4, 120.7, 119.8, 115.6, Methyl 2-((7-chloroimidazo[1,2-a]pyrimidin-5-yl)amino)benzoate (80b) was removed under reduced pressure, and the residue was purified by silica gel imidazo[1,2-a]pyrimidin-5-yl)amino)benzoate (86) the residue was purified through silica gel column (eluent: DCM/MeOH=30/1) to give the 30.4, 24.8. compound as beige solid (367 mg, 54%).
	107.5, 105.2, 46.5, 42.6, 40.6, 26.3, 15.4, 10.4. chromatography (eluent: DCM/MeOH=100/1~20/1) to give the title compound as white 2-((5-((4-(Aminomethyl)phenyl)amino)imidazo[1,2-a]pyrimidin-7-yl)amino)-N-title compound as white solid (377 mg, 90%). 1 H NMR (500 MHz, Chloroform-d) δ 8.42 (s, 2H), 7.11 (s, 1H), 6.86 (s, 2H), 3.87 (s, 6H),
	1 H NMR (500 MHz, DMSO-d6) δ 11.62 (s, 1H), 9.49 (s, 1H), 8.75 (q, J = 4.0 Hz, 2H), 8.28 (t, J = 5.7 Hz, 1H), 8.23 (s, 1H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.7 Hz, 1H), 7.26 (d, J = 8.3 Hz, 2H), 7.14 (t, J = 7.5 Hz, 1H), 6.90 (d, J = 11.1 Hz, 1H), 4.67 (s, 2H), 3.68 -3.52 (m, 2H), 3.35 (d, J = 6.0 Hz, 2H), 2.82 (d, J = 4.5 Hz, 3H), 1.89 (dtt, J = 11.9, 7.9, 4.2 Hz, 1H), 1.22 (dt, J = 7.7, 3.7 Hz, 2H), 0.97 -0.86 (m, 2H). HRMS (ESI) m/z calcd. for C32H31O4N9Cl [M+H] + : 640.21875, found: 640.21960. Melting point: 248 o C. Methyl 2-((2-amino-6-chloropyrimidin-4-yl)amino)benzoate (79) A suspension of 2-amino-4,6-dichloropyrimidine (1 g, 6.0 mmol) and methyl 2-aminobenzoate (0.78 mL, 6.0 mmol) in i-PrOH (30 mL) was heated and refluxed for 12 h. The solvent was removed under reduced pressure and the residue was triturated with DCM (5 mL). The precipitate was filtered off to give the title compound as yellow solid (1.46 g, 85%). 1 H NMR (250 MHz, DMSO-d6) δ 10.17 (s, 1H), 8.23 (dd, J = 8.4, 1.1 Hz, 1H), 7.91 (dd, J = 7.9, 1.7 Hz, 1H), 7.59 (ddd, J = 8.6, 7.3, 1.7 Hz, 1H), 7.19 (td, J = 7.6, 1.2 Hz, 1H), 6.71 (s, 2H), 6.24 (s, 1H), 3.83 (s, 3H). HRMS (ESI ) calcd. for C12H12ClN4O2 [M+H] + 279.0649; found 279.0644. Melting point: 224 o C. Method A: In a sealed tube chloroacetaldehyde (7 mL, 50% in water, 55 mmol) was added to a solution of 79 (1.43 g, 5.14 mmol) in MeOH (15 mL). The mixture was stirred and refluxed for 14 h. After cooling to room temperature, saturated NaHCO3 solution (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL*3). The combined organic phases were dried over MgSO4 and concentrated. Subsequently, DCM (10 mL) was added to the residue and filtered to give the most part of 80a. The filtrate was concentrated and purified by column chromatography (eluent: DCM/MeOH=80/1 ~50/1) give 80a and 80b. After the combination of two parts of the product, the title compound 80a was obtained as white solid (797 mg, 51%) and 80b was obtained as beige solid (274 mg, 18%). Method B: To a solution of 82 (416 mg, 2.2 mmol) in dry DMF (10 mL) was added methyl 2-aminobenzoate (344 µL, 2.65 mmol). The solution was stirred at 100 o C for 2 h. After cooling to room temperature, NaOH solution (0.5 M, 20 mL) was added, the mixture was extracted with ethyl acetate (20 mL*3). The organic layer was washed with brine and dried over MgSO4. Then the solvent was concentrated and the residue was purified by column chromatography (eluent: DCM/MeOH=100/1 ~ 50/1) to give title compound 80a (42 mg, 6%) and 80b (331 mg, 50%). (80a): 1 H NMR (250 MHz, Chloroform-d) δ 10.91 (s, 1H), 8.17 (dt, J = 8.0, 1.1 Hz, 1H), 7.80 (d, J = 1.6 Hz, 1H), 7.74 -7.62 (m, 4H), 7.33 -7.18 (m, 2H), 6.81 (s, 1H), 5.32 (s, 1H), 4.01 (s, 3H). In a sealed tube 81 (439 mg, 2.39 mmol) was dissolved in a mixed solution of HBr (48% in water, 5.5 mL) and acetic acid (5 mL), The mixture was stirred at 120 o C for 1 day. Subsequently, the solution was removed in vacuo. Then POCl3 (3 mL) was added to the flask containing the residue. The suspension was heated at 80 o C for 2 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in cooled water and pH was adjusted to 8 with 1 M NaOH solution. The solution was extracted with ethyl acetate. The organic phases were combined, washed with water and brine, and dried over MgSO4. After concentration, the residue was purified by column chromatography (eluent: DCM/MeOH= 80/1) to give the title compound as beige solid (170 mg, 38%). 1 H NMR (500 MHz, Chloroform-d) δ 7.74 (d, J = 2.5 Hz, 1H), 7.64 (d, J = 2.6 Hz, 1H), 6.98 (s, 1H). 13 C NMR (126 MHz, Chloroform-d) δ 150.0, 147.1, 137.3, 135.9, 110.2, 109.2. Methyl 2-((5-((4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)phenyl)amino) imidazo[1,2-a]pyrimidin-7-yl)amino)benzoate (83) To a suspension of 80a (200 mg, 0.66 mmol) in i-PrOH (8 mL) was added compound 4 (228 mg, 0.66 mmol) and HCl (20 µ L, 4 M in 1,4-dioxane) at room temperature. The solid (189 mg, 47%). 1 H NMR (250 MHz, Chloroform-d) δ 10.58 (s, 1H), 9.11 (d, J = 8.5 Hz, 1H), 7.93 (dd, J = 8.0, 1.7 Hz, 1H), 7.76 -7.74 (m, 3H), 7.60 (d, J = 7.4 Hz, 2H), 7.41 -7.26 (m, 13H), 6.87 (t, J = 7.6 Hz, 1H), 5.76 (s, 1H), 4.48 (d, J = 6.8 Hz, 2H), 4.38 (d, J = 6.0 Hz, 2H), 4.23 (t, J = 6.6 Hz, 1H), 3.83 (s, 3H). Methyl 2-((5-((4-(aminomethyl)phenyl)amino)imidazo[1,2-a]pyrimidin-7-yl)amino) benzoate (84) Compound 83 (184 mg, 0.3 mmol) was added to the mixed solution of DMF (2 mL) and piperidine (0.5 mL). The mixture stirred at room temperature for 3 h. Subsequently, the solvent was removed under reduced pressure. Ethyl ether was added to the remained residue and filtered to give the crude product as beige solid, then purification by silica gel chromatography (DCM/MeOH/NH4OH = 10/1/0.01) to give the title compound as off-white solid (106 mg, 91%). 1 H NMR (500 MHz, DMSO-d6) δ 10.11 (s, 1H), 8.78 (d, J = 8.5 Hz, 1H), 7.93 -7.84 (m, 2H), 7.64 -7.56 (m, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.37 -7.31 (m, 3H), 7.03 (t, J = 7.6 Hz, 1H), 5.64 (s, 1H), 3.81 (s, 3H), 3.78 (s, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 168.1, 154.4, 149.3, 146.4, 142.5, 141.4, 135.9, 133.9, 131.4, 130.5, 128.3, 124.2, 120.5, 120.1, 115.0, 106.2, 77.3, 52.2, 45.0. methylbenzamide (85) Compound 84 (66 mg, 0.17 mmol) was added to a solution of MeNH2 in ethanol (wt 33%, 5 mL). The mixture was stirred at room temperature overnight. Subsequently, the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (eluent: DCM/MeOH/NH4OH = 10/1/0.01) to give the product as white solid (40mg, 61%). 1 H NMR (500 MHz, DMSO-d6) δ 10.90 (s, 1H), 8.86 (d, J = 8.5 Hz, 1H), 8.70 (q, J = 4.5 Hz, 1H), 7.93 -7.83 (m, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 7.7 Hz, 3H), 7.42 -7.32 (m, 3H), 6.99 (t, J = 7.5 Hz, 1H), 5.50 (s, 1H), 3.79 (s, 2H), 2.75 (d, J = 4.5 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 169.2, 154.5, 149.5, 146.3, 141.4, 141.1, 135.9, 131.6, 131.2, 128.3, 127.9, 124.3, 120.2, 119.8, 119.2, 106.1, 77.1, 45.0, 26.2. HRMS (ESI) calcd. for C21H22N7O [M+H] + : 388.1886; found 388.1884. Melting point: over 260 o C . To a suspension of 80b (150 mg, 0.5 mmol) in i-PrOH (8 mL) was added compound 4 (171 mg, 0.5 mmol) and HCl (15 µ L, 4 M in 1,4-dioxane) at room temperature. The mixture was refluxed for 20 h with stirring. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (eluent: DCM/MeOH=100/1~20/1) to give the title compound as white solid (192 mg, 64%). 1 H NMR (500 MHz, Chloroform-d) δ 10.42 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.67 (dd, J = 7.7, 2.8 Hz, 2H), 7.58 -7.36 (m, 8H), 7.33 -7.24 (m, 3H), 7.25 -7.17 (m, 2H), 7.10 -7.02 (m, 2H), 6.97 (d, J = 8.5 Hz, 1H), 6.38 -6.33 (m, 1H), 4.35 (dd, J = 7.1, 2.9 Hz, 2H), 4.23 (d, J = 4.9 Hz, 2H), 4.14 (q, J = 6.8, 6.0 Hz, 1H), 3.87 (d, J = 3.5 Hz, 3H). Methyl 2-((7-((4-(aminomethyl)phenyl)amino)imidazo[1,2-a]pyrimidin-5-yl)amino) benzoate (87) Compound 86 (192 mg, 0.3 mmol) was added to the mixed solution of DMF (3 mL) and 1 H NMR (500 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.76 -7.67 (m, 13 C NMR (126 MHz, DMSO-d6) δ 166.7, 155.4, 149.7, 144.7, 139.6, 139.3, 136.3, 133.9, 131.3, 130.7, 127.3, 124.9, 124.7, 122.7, 118.5, 105.5, 78.8, 52.3, 45.0. Melting point: 174 o C. 2-((7-((4-(Aminomethyl)phenyl)amino)imidazo[1,2-a]pyrimidin-5-yl)amino)-N-methylbenzamide (88) Compound 87 (25 mg, 0.06 mmol) was added to a solution of MeNH2 in ethanol (wt 33%, 1 mL). The mixture was stirred at room temperature overnight. Subsequently, the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (eluent: DCM/MeOH/NH4OH = 10/1/0.01) to give the product as white solid (20mg, 74%). 1 H NMR (500 MHz, DMSO-d6) δ 8.98 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.71 -7.67 (m, 2H), 7.59 (d, J = 1.8 Hz, 1H), 7.47 (d, J = 4.1 Hz, 2H), 7.29 (d, J = 1.7 Hz, 1H), 7.27 -7.22 (m, 2H), 7.09 (dt, J = 8.1, 4.1 Hz, 1H), 5.87 (s, 1H), 3.74 (s, 2H), 2.77 (d, J = 2.8 Hz, 1 H NMR (500 MHz, Methanol-d4) δ 6.27 -6.14 (m, 2H), 5.72 -5.60 (m, 1H), 4.34 (s, 3H), 3.70 (t, J = 5.9 Hz, 1H), 3.52 (t, J = 5.6 Hz, 1H), 3.48 -3.40 (m, 2H). 2-((7-((4-(((2-((2-Acrylamidoethyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)methyl) phenyl)amino)imidazo[1,2-a]pyrimidin-5-yl)amino)-N-methylbenzamide (95) To a solution of compound 88 (20 mg, 0.05 mmol) in DMF (0.5 mL) was added compound 94 (12 mg, 0.05 mmol) and DIPEA (10 µL, 0.05 mmol). The mixture was stirred at 86 o C overnight. Then the solvent was removed in vacuo. The residue was purified by silica gel column chromatography (eluent: DCM/MeOH/NH4OH = 10/1/0 ~ 10/1/0.01) to give the title compound as off-white solid (24 mg, 70%). 1 H NMR (500 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.27 (t, J = 5.7 Hz, 1H), 7.93 -7.83 (m, 1H), 7.79 -7.62 (m, 3H), 7.56 (s, 2H), 7.41 (d, J = 1.9 Hz, 1H), 7.28 -7.16 (m, 3H), 6.20 (dd, J = 17.1, 10.1 Hz, 1H), 6.09 (dd, J = 17.1, 2.3 Hz, 1H), 5.60 (dd, J = 10.1, 2.3 Hz, 1H), 4.65 (s, 2H), 3.58 (s, 2H), 3.31 (s, 2H), 2.78 (d, J = 4.5 Hz, 3H). 13 C NMR (126 MHz, DMSO-d6) 169.6, 161.4, 150.3, 146.2, 134.2, 133.4, 131.9, 118.4, 113.8, 113.6, 24.9. cooling to room temperature, the solvent was removed in vacuo. The residue was dissolved in ethyl acetate and washed with NaOH (0.5 M), water, brine. The organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent, cyclohexane/ethyl acetate= 16/10) to give the title compound as beige solid (366 mg, 73%). 13 C NMR (126 MHz, CDCl3) δ 158.5, 158.4, 138.9, 129.2, 123.4, 119.7, 108.6. MS ESI + : 250.2 [M+H] 5-Bromo-N-(3,4,5-trimethoxyphenyl)pyrimidin-2-amine (117) To a solution of 5-bromo-2-chloropyrimidine (400 mg, 2.02 mmol) in n-BuOH (10 mL) was added 3,4,5-trimethoxyaniline (732 mg, 4.0 mmol). The mixture was stirred at 120 o C overnight. After cooling to room temperature, the solvent was removed in vacuo. The residue was dissolved in ethyl acetate and washed with NaOH (0.5 M), water, brine. The organic phase was concentrated under reduced pressure. The residue was purified by silica 3.83 (s, 3H). To a solution of 5-bromo-2-chloropyrimidine (200 mg, 1.0 mmol) in n-BuOH (5 mL) was added morpholine (262 µL, 3.0 mmol). The mixture was stirred at 100 MS ESI + : 244.2 [M+H] 1'-(5-Bromopyrimidin-2-yl)-1,4'-bipiperidine (119) 1 H NMR (500 MHz, Chloroform-d) δ 8.25 (s, 2H), 4.72 (dt, J = 13.4, 2.5 Hz, 2H), 2.87 -piperidine (0.6 mL). white solid (90 mg, 74%). 4H), 7.60 (d, J = 8.1 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 1.6 Hz, 1H), 7.23 (d, J = 8.2 Hz, 2H), 5.83 (s, 1H), 3.77 (s, 3H), 3.66 (s, 2H). 13 C NMR (126 MHz, Methanol-d4) δ 188.4, 183.7, 176.7, 173.6, 167.1, 130.5, 125.6, 59.8, 43.7, 39.6. To a solution of 5-bromo-2-chloropyrimidine (400 mg, 2.02 mmol) in n-BuOH (10 mL) was added aniline (370 µL, 4.0 mmol). The mixture was stirred at 120 o C overnight. After 2.79 (m, 2H), 2.51 (d, J = 4.8 Hz, 5H), 1.92 -1.84 (m, 2H), 1.58 (p, J = 5.6 Hz, 4H), 1.52 4-(5-Bromopyrimidin-2-yl)morpholine (118) -1.39 (m, 4H).

HRMS (ESI) m/z calcd. for C13H20N5 [M+H] + : 246.17187, found: 246.17118 Ethyl 4-((2-methylpyrimidin-5-yl)amino)benzoate (110) White solid (42.5 mg, 55%). 1 H NMR (500 MHz, Chloroform-d) 8.55 (s, 2H), 7.96 -7.92 (m, 2H), 6.98 -6.94 (m, 2H), 6.17 (s, 1H), 4.34 (q, J = 7.1 Hz, 2H), 2.70 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3) 166.3, 162.5, 149.0, 146.7, 133.7, 131.8, 123.2, 114.9, 60.8, 25.3, 14.5. HRMS (ESI) m/z calcd. for C14H14N3O2 [M-H] -: 256,10860, found: 256.10909. Melting point: 176 o C. 2-((2-Methylpyrimidin-5-yl)amino)benzoic acid (111) Beige solid (46 mg, 67%).

1 H NMR (500 MHz, DMSO-d6) 8.65 (s, 2H), 7.93 (dd, J = 7.9, 1.7 Hz, 1H), 7.41 (ddd, J = 8.7, 7.1, 1.8 Hz, 1H), 7.14 -7.11 (m, 1H), 6.88 -6.84 (m, 1H), 2.60 (s, 3H). HRMS (ESI) m/z calcd. for C12H10N3O2 [M-H] -: 228.07730, found: 228.07739. 1 H NMR (500 MHz, Chloroform-d) δ 8.42 (s, 2H), 7.59 -7.55 (m, 2H), 7.38 -7.33 (m, 2H), 7.08 (tt, J = 7.3, 1.1 Hz, 1H). o C overnight. After cooling to room temperature, the mixture was diluted by ethyl acetate. The mixture was washed by water, brine. The organic phase was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: cyclohexane/ethyl acetate = 10/1 ~ 5/1) to give the title compound as white solid (229 mg, 93%). 1 H NMR (500 MHz, Chloroform-d) δ 8.30 (s, 2H), 3.75 (d, J = 2.6 Hz, 8H). 13 C NMR (126 MHz, Chloroform-d) δ 160.1, 158.0, 106.3, 66.8, 44.5.

To a solution of 5-bromo-2-chloropyrimidine (500 mg, 2.59 mmol) in ethanol (5 mL) was added 1,4'-bipiperidine (970 mg, 5.18 mmol). The mixture was stirred at 80 o C overnight.

  Then the organic layer was dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: cyclohexane/ethyl acetate= 50/1) to give the title compound as white solid (398 mg, 78%). The residue was triturated in DCM and filtered to give the title compound as brown solid (35 mg, 35%).To an ice-cooled solution of 4-aminobenzylamine (200 μL, 1.76 mmol) in MeOH (2 mL) was added dropwise methyl trifluoroacetate (178 μL, 1.76 mmol). After 15 min, the ice bath was removed and the mixture was stirred at room temperature for a further 1 h. Then HCl (342 μL, 4M in 1,4-dioxane). The mixture was stirred at 105 o C for 18 h. After cooling to room temperature the solvent was removed in vacuo and the resulted residue was washed with DCM and MeOH to give the title product as orange solid (603 mg, 79%). This compound was used directly for next step without further purification.

	5-Bromo-2-phenylpyrimidine (124) HRMS (ESI) m/z calcd. for C21H27N6 [M+H] + : 363.22972, found: 363.22878. concentrated under reduced pressure and the residue diluted by DCM and washed with N-(2,5-Dichloropyrimidin-4-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (140) N 1 -(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)ethane-1,2-diamine (147) N-(4-((5-Chloro-4-((2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethyl)amino)
	13 C NMR (126 MHz, Chloroform-d) δ 160.4, 158.0, 145.3, 129.4, 126.6, 126.4, 107.2, Melting point: 78 o C. 4-((2-Phenylpyrimidin-5-yl)amino)benzonitrile (135) NaOH solution (1M) and brine. The organic phase was dried over MgSO4 and concentrated 1 H NMR (500 MHz, DMSO-d6) δ 9.72 (s, 1H), 9.67 (s, 1H), 8.65 (d, J = 8.4 Hz, 1H), 8.48 pyrimidin-2-yl)amino)benzyl)-2,2,2-trifluoroacetamide (149)
	39.1. in vacuo and purified by flash chromatography (eluent: cyclohexane/ethylacetate = 5/1 ~ (s, 1H), 8.25 (s, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.80 (t, J = 6.1 Hz, 1H), 7.70 (d, J = 7.5 Hz,
	5-Bromo-2-methoxypyrimidine (121) To a solution of NaOMe freshly prepared by dissolving Na metal (350 mg) in dry methanol, was added 2-bromo-5-chloropyrimidine (500 mg, 2.59 mmol), and the solution 5-Bromo-2-phenoxypyrimidine (122) To a solution of 5-bromo-2-chloropyrimidine (400 mg, 2.03 mmol) in DMSO (6 mL) was added phenol (191 mg, 2.03 mmol) and K2CO3 (560 mg, 4.06 mmol). The mixture was J = 6.8 Hz, 1H), 7.20 -7.15 (m, 2H). 1 H NMR (500 MHz, Chloroform-d) δ 8.57 (s, 2H), 6.38 (t, J = 2.2 Hz, 1H), 6.34 (d, J = 2.2 Hz, 2H), 3.78 (s, 6H). Phenylboronic acid (126 mg, 1.03 mmol), 5-bromo-2-iodopyrimidine (300 mg, 1.03 1.03 mmol), K2CO3 (2M in H2O, 1.03 mL), and Pd(PPh3)4 (59 mg, 5 mol%) in argon saturated toluene (4 mL). The mixture was refluxed overnight under argon. The resulting mixture was allowed to cool to room temperature, diluted with chloroform and extracted with brine. The combined organic solution was dried over MgSO4. The concentrated crude product was isolated by column chromatography on silica gel (eluent: cyclohexane/ethyl acetate= 50/1) to give the title compound as white solid (123 mg, 45%). 4-((2-(Methyl(phenyl)amino)pyrimidin-5-yl)amino)benzonitrile (131) White solid (44.2 mg, 65%). 1 H NMR (500 MHz, DMSO-d6) δ 8.81 (s, 1H), 8.58 (s, 2H), 7.63 (d, J = 8.9 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 3.98 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 161.5, 153.6, 149.0, 133.7, 130.2, 119.7, 113.7, 99.3, 54.6. 13 C NMR (126 MHz, DMSO-d6) δ 160.8, 157.1, 148.0, 147.0, 133.8, 133.9, 129.6, 128.6, 119.6, 115.2, 114.0, 100.7, 55.2. HRMS (ESI) m/z calcd. for C18H15N4O [M+H] + : 303.12459, found: 303.12380. Melting point: 207 o C. 0/1) to give the product as white solid (51 mg, 98%). 1 H NMR (500 MHz, Chloroform-d) δ 8.34 (s, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.24 (s, 1H), 1 H NMR (250 MHz, Methanol-d4) δ 8.50 (d, J = 8.7 Hz, 1H), 6.40 (d, J = 8.7 Hz, 1H). To a suspension of 140 (52 mg, 0.16 mmol) in 2-methoxyethanol (2 mL) was added 4 (55 2H), 7.49 -7.29 (m, 6H), 7.12 (d, J = 8.2 Hz, 2H), 4.35 (d, J = 6.9 Hz, 2H), 4.23 (t, J = 7.0 Hz, 1H), 4.13 (d, J = 6.1 Hz, 2H). N-(4-Aminobenzyl)-2,2,2-trifluoroacetamide (143) the solvent was removed under reduced pressure and the residue was purified through silica gel column chromatography (eluent: cyclohexane/ethyl acetate= 4/1~2/1) to obtain 1 H NMR (500 MHz, DMSO-d6) δ 9.93 (t, J = 6.0 Hz, 1H), 9.73 (s, 1H), 8.64 (d, J = 8.4 Hz, 1H), 8.46 (s, 1H), 8.20 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.1 Hz, 2H), 4.33 (d, J = 5.7 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 157.5, 156.6, 156.4, 156.1, 145.9, 143.4, 138.8, 134.9, 131.1, 128.5, 127.7, 119.6, 114.4, 106.4, 42.2. was stirred at room temperature overnight, and then was filtered to give the title compound as orange solid (523 mg, 85%). 1 H NMR (500 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.17 (s, 1H), 8.08 (s, 1H), 6.62 (d, J = 9.0 Hz, 1H), 3.69 (s, 4H). heated at 120 1 H NMR (500 MHz, Chloroform-d) δ 8.55 (s, 2H), 7.42 (dd, J = 8.5, 7.3 Hz, 2H), 7.26 (t, 13 C NMR (126 MHz, Chloroform-d) δ 164.1, 160.1, 152.8, 129.8, 125.9, 121.6, 113.3. HRMS (ESI) m/z calcd. for C10H8BrN2O [M+H] + : 250.9820, found: 250.98157. 5-Bromo-2-(3,5-dimethoxyphenoxy)pyrimidine (123) To a solution of 5-bromo-2-chloropyrimidine (400 mg, 2.03 mmol) in DMSO (6 mL) was added 3,5-dimethoxyphenol (320 mg, 2.03 mmol) and K2CO3 (560 mg, 4.06 mmol). The mixture was heated at 120 o C for 3 h. After cooling to room temperature, the mixture was poured into water and extracted with ethyl acetate. The combined organic phase was washed with water and brine. Then the organic layer was dried over MgSO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: cyclohexane/ethyl acetate= 50/1) to give the title compound as colourless oil (532 mg, 84%). mmol), K2CO3 (2 M in H2O, 1.03 mL), and Pd(PPh3)4 (12 mg, 1 mol%) in argon saturated toluene (2 mL). The mixture was refluxed overnight under argon. The resulting mixture was allowed to cool to room temperature, diluted with chloroform and extracted with brine. The combined organic solution was dried over MgSO4. The concentrated crude product was isolated by column chromatography on silica gel (eluent: cyclohexane/ethyl acetate= 50/1) to give the title compound as white solid (104 mg, 43%). HRMS (ESI) m/z calcd. for C10H8BrN2 [M+H] + : 234.9871, found: 234.98663. 5-Bromo-2-(4-methoxyphenyl)pyrimidine (125) 4-Methoxyphenylboronic acid (247 mg, 1.54 mmol), 5-bromo-2-iodopyrimidine (300 mg, Off-white solid (86.6 mg, 93%). 1 H NMR (500 MHz, Methanol-d4) δ 8.24 (s, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.41 (dd, J = 8.4, 7.3 Hz, 2H), 7.32 (dd, J = 8.5, 1.3 Hz, 2H), 7.25 (td, J = 7.3, 1.3 Hz, 1H), 6.83 (d, J = 8.8 Hz, 2H), 3.52 (s, 3H). Melting point: 119 o C. 4-((2-Methoxypyrimidin-5-yl)amino)benzonitrile (132) White solid (70 mg, 85.7%). 1 H NMR (500 MHz, Chloroform-d) δ 8.71 (s, 2H), 8.42 -8.38 (m, 2H), 7.57 (d, J = 8.8 Hz, 2H), 7.52 -7.47 (m, 3H), 7.05 (d, J = 8.7 Hz, 2H), 6.04 (s, 1H). 13 C NMR (126 MHz, Chloroform-d) δ 160.2, 149.5, 146.4, 137.1, 134.2, 133.5, 130.6, 128.8, 127.9, 119.3, 115.7, 104.0. HRMS (ESI) calcd. for C17H13N4 [M+H] + : 273.11402, found: 273.11386. Melting point: 209 o C. 4-((2-(4-Methoxyphenyl)pyrimidin-5-yl)amino)benzonitrile (136) White solid (81.6 mg, 90%). 1 H NMR (500 MHz, DMSO-d6) δ 9.18 (s, 1H), 8.75 (s, 2H), 8.28 (d, J = 8.9 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 3.84 (s, 3H). 6.91 (s, 2H), 6.75 (d, J = 8.8 Hz, 2H), 5.84 (s, 1H), 3.87 (s, 6H), 3.83 (s, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 158.0, 155.5, 153.5, 149.5, 135.4, 134.1, 126.6, 119.8, 113.8, 101.7, 97.6, 61.1, 56.3. HRMS (ESI) m/z calcd. for C20H19N5O3 [M+H] + : 378.15661, found: 378.15670. Melting point: 178 o C. 7-Nitrobenzo[c][1,2,5]oxadiazol-4-amine (139) 221 5.30 -5.14 (m, 2H), 5.07 (s, 1H), 4.58 (d, J = 5.7 Hz, 2H), 4.36 (d, J = 6.1 Hz, 2H). solid (115 mg, 74%). trichloropyrimidine (191 µL, 1.66 mmol) and DIPEA (580 µL, 3.32 mmol). The mixture 6/1~1/1). The titled compound was obtained as brown solid (530 mg, 59%). pyrimidin-2-yl)amino)benzyl)carbamate (141) 8.2 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.12 (s, 1H), 5.88 (ddd, J = 16.3, 10.8, 5.3 Hz, 1H), chromatography (eluent: DCM/MeOH= 80/1~50/1) to give the title compound as orange To a solution of 147 (561 mg, 1.66 mmol) in MeOH (15 mL) was added 2,4,5-purified through silica gel column chromatography (eluent: cyclohexane/ethyl acetate= (9H-Fluoren-9-yl)methyl (4-((5-chloro-4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino) 1 H NMR (250 MHz, Chloroform-d) δ 8.52 (s, 1H), 8.43 (s, 2H), 8.25 (s, 1H), 7.44 (d, J = o C for 20 h and then was concentrated in vacuo. The residue was purified by column dark environment. The solvent was removed under reduced pressure. The residue was (67 mg, 0.306 mmol) and HCl (77 µL, 4 M in 1,4-dioxane). The mixture was stirred at 110 added ammonia (28% in H2O). The mixture was stirred at room temperature for 24 h in a 117.9, 116.1. To a solution of 140 (100 mg, 0.306 mmol) in 2-methoxyethanol (3 mL) was added 143 To a solution of 4-chloro-7-nitrobenzofurazan (1.0 g, 4.96 mmol) in MeOH (8 mL) was Compound 146 (604 mg, 1.87 mmol) was added to a mixture of DCM (1.5 mL) and the title compound as off-white solid (335 mg, 87%). To an ice-cooled solution of compound 139 (390 mg, 2.16 mmol) in distilled DMF was added NaH (130 mg, 60% in mineral oil, 3.24 mmol) in two portions. After 30 min, 2,4,5-trichloropyrimidine (247 µL, 2.16 mmol) was added. After 1 h, the ice bath was removed N-(4-((5-Chloro-4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pyrimidin-2-yl)amino) benzyl)-2,2,2-trifluoroacetamide (144) diamine (148) and the mixture was heated to 90 13 C NMR (126 MHz, DMSO-d6) δ 157.6, 156.5, 156.4, 146.1, 143.4, 134. 6, 134.1, 131.2, Allyl (4-((5-chloro-4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)pyrimidin-2-yl) amino)benzyl)carbamate (142) trifluoroacetic acid (1.5 mL) and stirred for 30 min. The solvent was removed under 1 H NMR (500 MHz, Chloroform-d) δ 7.07 (d, J = 7.9 Hz, 2H), 6.66 (d, J = 7.9 Hz, 2H), To a suspension of 148 (506 mg, 1.37 mmol) in n-BuOH (17 mL) was added 143 (298 mg, reduced pressure to give the crude product, then diethyl ether was added and filtered to 6.53 (s, 1H), 4.39 (d, J = 5.4 Hz, 2H), 3.72 (s, 2H). 13 C NMR (126 MHz, CDCl3) δ 146.7, 129.6, 125.6, 115.4, 43.8. obtain titled compound as orange solid (578 mg, 92%). 1.37 mmol) and

stirred at room temperature overnight. The reaction was monitored by TLC and quenched after 24 h by addition of water and extraction with ethyl acetate. The combined o rganic extracts were washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: cyclohexane/ethyl acetate= 20/1) to give the title compound as white solid (283 mg, 58%).

1

H NMR (500 MHz, Chloroform-d) δ 8.50 (s, 1H), 3.97 (s, 2H).

13

C NMR (126 MHz, Chloroform-d) δ 164.2, 159.5, 111.8, 55.4. HRMS (ESI) m/z calcd. for C5H6BrN2O [M+H] + : 188.9664, found: 188.96593. o C for 3 h. After cooling to room temperature, the mixture was poured into water and extracted with ethyl acetate. The combined organic phase was washed with water and brine. 1 H NMR (500 MHz, Chloroform-d) δ 8.83 (s, 2H), 8.42 -8.39 (m, 2H), 7.51 -7.47 (m, 3H). 13 C NMR (126 MHz, Chloroform-d) δ 163.0, 158.0, 136.6, 131.3, 128.8, 128.3, 118.4. 13 C NMR (126 MHz, Chloroform-d) δ 159.9, 155.3, 149.9, 145.3, 133.9, 129.3, 126.5, 126.1, 124.9, 119.9, 113.5, 100.8, 39.0. HRMS (ESI) m/z calcd. for C18H14N5 calcd. [M-H] -: 300.12492, found: 300.12548. HRMS (ESI) m/z calcd. for C12H11N4O [M+H] + : 227.09329, found: 227.09316. o C and stirred overnight. Dilute the mixture with ethyl acetate, and washed with water and brine. The ethyl acetate phase was dried over MgSO4 and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: cyclohexane/ethyl acetate= 6/1~ 3/1) to give the title compound as yellow solid (193 mg, 27%).

1 H NMR (500 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.82 (d, J = 8.3, 1H), 8.72 (s, 1H), 7.99 (d, J = 8.4, 1H). mg, 0.16 mmol) and HCl (8 µ L, 4 M in 1,4-dioxane). The mixture was stirred at 110 o C for 20 h and then was concentrated in vacuo. To a solution of 140 (95 mg, 0.29 mmol) in 2-methoxyethanol (3 mL) was added 6 (60 mg, 0.29 mmol) and HCl (15 µL, 4 M in 1,4-dioxane). The mixture was stirred at 110 o C for 20 h and then was concentrated in vacuo. The residue dissolved in ethyl acetate and washed with 1 M NaOH, water and brine. The organic phase was dried over MgSO4 and purified by column chromatography (eluent: DCM/MeOH= 80/1~50/1) to give the title compound as orange solid (58 mg, 40%). 1 H NMR (500 MHz, Methanol-d4) δ 8.51 (d, J = 8.7 Hz, 1H), 6.45 (d, J = 8.7 Hz, 1H), 3.91 (t, J = 6.1 Hz, 2H), 3.38 (t, J = 6.2 Hz, 2H).

13 C NMR (126 MHz, Methanol-d4) δ 146.1, 146.0, 145.3, 138.0, 124.7, 100.6, 42.0, 39.1.

N 1 -(2,5-Dichloropyrimidin-4-yl)-N 2 -(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)ethane-1,2-13 C NMR (126 MHz, DMSO-d6) δ 158.9, 157.1, 153.8, 145.4, 144.5, 144.1, 137.6, 121.0, 113.1, 99.5, 42.1, 38.5.

1 H NMR (500 MHz, DMSO-d6) δ 10.20 (s, 1H), 9.94 (d, J = 6.2 Hz, 1H), 9.47 (s, 1H), 8.62 -8.48 (m, 1H), 8.35 (d, J = 8.9 Hz, 1H), 8.20 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.17

  Then the mixture was stirred at room temperature overnight and then poured into ice water. After filtration, the solid was purified by column chromatography (eluent: DCM/MeOH=30/1~20/1) to give the title compound as orange solid (40 mg, 55%).Structure-Based Design, Synthesis, and Characterization of the First Irreversible Inhibitor of Focal Adhesion KinaseExpedite Yen-Pon, †,⊥ Bo Li, †,⊥ Marta Acebroń-Garcia-de-Eulate, ‡ Ceĺine Tomkiewicz-Raulet, § John Dawson, ∥ Daniel Lietha, ‡ Margaret C. Frame, ∥ Xavier Coumoul, § Christiane Garbay, † Meĺanie Etheve-Quelquejeu, † and Huixiong Chen* , † † Chemistry of RNA, Nucleosides, Peptides and Heterocycles, CNRS UMR8601, UniversitéParis Descartes, PRES Sorbonne Paris Cite, UFR Biomedicale, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France ‡ Cell Signalling and Adhesion Group, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Calle Melchor Fernańdez Almagro 3, Madrid 28029, Spain § Toxicologie, Pharmacologie et Signalisation Cellulaire, INSERM, UMR S 1124, UniversitéParis Descartes, PRES Sorbonne Paris Cite, UFR Biomedicale, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France

	1 H NMR (500 MHz, DMSO-d6) δ 9.49 (s, 1H), 9.17 (s, 1H), 8.41 (d, J = 8.7 Hz, 1H), 8.31
	-8.20 (m, 1H), 7.96 (s, 1H), 7.64 (d, J = 7.9 Hz, 2H), 7.38 (s, 1H), 7.19 (d, J = 7.9 Hz,
	2H), 6.43 (d, J = 9.0 Hz, 1H), 6.27 -6.04 (m, 2H), 5.59 (d, J = 10.2 Hz, 1H), 4.62 (s, 2H),
	3.90 -3.77 (m, 2H), 3.73 (s, 2H), 3.58 (s, 2H).
	13 C NMR (126 MHz, DMSO-d6) δ 182.6, 182.4, 168.0, 167.4, 165.0, 157.8, 157.6, 153.1,
	145.3, 144.4, 144.0, 139.9, 137.7, 131.5, 131.2, 127.8, 125.2, 120.8, 118.7, 103.8, 99.4,
	69.8, 46.5, 42.9, 42.8, 40.0, 39.8, 39.7, 39.5, 39.3, 39.2, 39.0, 38.7.
	7.24 (s, 2H), 6.25 (d, J = 8.9 Hz, 1H), 4.74 (s, 2H), 3.97 (dt, J = 12.0, 5.7 Hz, 4H), 3.81
	(s, 2H), 3.32 (d, J = 6.1 Hz, 2H).
	N-(2-((2-((4-((5-Chloro-4-((2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethyl)
	amino)pyrimidin-2-yl)amino)benzyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)
	acrylamide (153)

1 H NMR (500 MHz, Methanol-d4) δ 8.14 (d, J = 8.8 Hz, 1H), 8.02 (s, 1H), 7.31 (s, 2H), To a solution of 152 (100 mg, 0.107 mmol) in distilled DMF (1 mL), acryloyl chloride (14 μL, 0.16 mmol) and Et3N (76 μL, 0.54 mmol) was added at 0 o C. ∥ Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom * S Supporting Information

Table 1 .

 1 In Vitro Enzymatic Activities of Novel 2,4-Diarylaminopyrimidines Compared with TAE226 Sq(CH 2 ) 2 NHCOCH 2 CH 3 4.7 ± 0.5 7 CH 2 Tr(CH 2 ) 2 NH COCHCH 2 2.3 ± 0.3 8 CH 2 Tr(CH 2 ) 3 NH COCHCH 2 1.2 ± 0.1 TAE226 5.8 ± 0.6

	No.	R a	IC 50 (nM)
	2	H2 6 ± 2.2
	5	Sq(CH 2 ) 2 NHCOCHCH 2	0.6 ± 0.04
	6

a Sq: squaramide group. Tr: 1,2,3-triazole group.

  . Final R-factors are 22.3/26.2 (Rwork/Rfree) for FAK/5, 19.5/ 22.3 for FAK/6, and 21.7/27.4 for FAK/7. S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00250. Additional figures, experimental methods, and analyses (DOC)

	■ ASSOCIATED CONTENT
	Accession Codes

*

H NMR (250 MHz, DMSO-d6) δ 11.59 (s, 1H), 9.43 (s, 1H), 8.81 -8.69 (m,

2H),8.22

H NMR (250 MHz, Chloroform-d) δ 6.34 (dd, J = 17.0, 1.5 Hz, 1H), 6.13 (dd, J = 17.0,

H NMR (250 MHz, Chloroform-d) δ 4.43 (s, 1H),

2.94 (s, 3H), 2.82 (d, J = 3.3 Hz, 3H).

H NMR (500 MHz, Chloroform-d) δ 8.34 (dd, J = 4.7, 1.9 Hz, 1H), 7.88 (dd, J = 7.7, 1.9

H NMR (500 MHz, Chloroform-d) δ 7.97 (s, 1H), 7.60 -7.57 (m, 1H), 7.38 -7.35 (m,

H NMR (250 MHz, Methanol-d4) δ 8.57 (d, J = 8.4 Hz,

2H), 8.17 (s, 2H), 7.66 (dd, J =

C NMR (126 MHz, Chloroform-d) δ 158.0, 157.8, 156.4, 153.7,

141.1, 140.5, 138.9,

H NMR (500 MHz, DMSO-d6) δ 9.11 (s, 1H), 7.97 (s, 1H), 7.81 (t, J = 6.2 Hz, 1H), 7.55

C NMR (126 MHz, DMSO-d6) δ 182.6, 182.4, 168.0, 167.3, 157.9, 157.7, 155.7, 153.3,

H NMR (500 MHz, DMSO-d6) δ 11.35 (s, 1H), 9.81 (s, 1H), 8.79 (q, J = 4.6 Hz, 1H),

H NMR (500 MHz, DMSO-d6) δ 11.61 (s, 1H), 9.49 (s, 1H), 8.74 (t, J = 5.9 Hz,

2H), 8.40

C NMR (126 MHz, DMSO-d6) δ 182.7, 182.5, 168.9, 168.1, 167.5, 163.4, 160.8, 157.7,

C NMR (126 MHz, DMSO-d6) δ 182.7, 182.5, 168.9, 168.1, 167.5, 164.0, 160.8, 157.7,

C NMR (126 MHz, Chloroform-d) δ 169.4, 152.7,

149.0, 142.7, 136.2, 135.0, 133.0,

H NMR (500 MHz, DMSO-d6) δ 8.57 (s,

2H), 7.73 (dd, J = 7.8, 1.5 Hz, 1H), 7.34 (tt, J

H NMR (500 MHz, Chloroform-d) δ 8.86 (s,

2H), 8.23 (dd, J = 5.0, 1.9 Hz, 1H), 7.55

C NMR (126 MHz, Chloroform-d) 161.3, 159.7,

143.5, 139.0, 136.8, 94.7, 34.7, 32.3,

H NMR (500 MHz, Chloroform-d) δ 8.32 (s,

2H), 7.43 -7.39 (m, 2H), 7.30 -7.23 (m,

C NMR (126 MHz, Chloroform-d) δ 164.0, 161.6, 160.2, 154.4, 113.4, 100.2, 98.2, 55.6.

H NMR (500 MHz, Chloroform-d) δ 8.77 (s,

2H), 8.36 (d, J = 8.9 Hz, 2H), 6.99(d, J =

H NMR (500 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.51 (s, 1H), 8.88 (s, 1H), 8.48 (s, 3H),

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to all the people who have supported me professionally and personally in my journey to this PhD.

We thank the ESRF and ALBA synchrotron facilities for providing access to their facilities and the staff for their assistance in the data collection. D.L. was supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) Grant MEIC Project Retos BFU2016-77665-R cofunded by the European Regional Development Fund (ERDF) and the Worldwide Cancer Research (15-1177). B.L. thanks the China Scholarship Council (CSC) for financial support.

express my gratitude to the Chinese Scholarship Council for the financial supporting during my study in France.

Abbreviations

5-FU

Fluorouracil acryloyl chloride to give the final product 74 in 60% yield (Scheme 35).

Scheme 35: Synthesis of compound 74.

Design of reversible covalent inhibitors

Concept of reversible covalent inhibitor

Inspired by the fact that in the reaction between 2-cyanoacrylate and thiol, the addition reaction reached a fast equilibrium, 176 Serafimova and colleagues [START_REF] Serafimova | Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles[END_REF] However, when the solution was diluted 10 times, the reaction moved towards starting materials as the signal of cyanoacrylate 3 becomes more intense. [START_REF] Serafimova | Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles[END_REF] Through this NMR evidence, it's proved that the covalent adduct of thiol with cyanoacrylate could be recovered to Michael receptor and free thiol in biological low concentrations. The cyclization was achieved by refluxing 79 and 2-chloroacetaldehyde at 100 o C overnight in a sealed tube (Scheme 42). 186 As described in the synthesis of imidazo [1,2-a][1,3,5]triazine derivatives, 187 two regioisomers were obtained through cyclization in an isolated ratio of about 3:1 (a:b) because of the steric hindrance. These two regioisomers (80a and 80b) had Rf values of 0.21 and 0.15 on TLC (eluent, DCM/MeOH=100/1), respectively.

The regioisomer 80a was a little less soluble than 80b in DCM so that compound 80a could be precipitated from the mixture. Then the filtrate was purified b y column chromatography to give compound 80b.

Scheme 42: Synthesis of compounds 80a and 80b.

In order to confirm the structure of these two regioisomers, the nuclear magnetic resonance spectroscopy was engaged. The peaks on the 1 H-NMR were fully assigned to corresponding protons in accordance with 2D-COSY, HSQC, and HMBC spectra. In the 2D-NOESY spectrum of compound 80a (Figure 54), we observed cross peaks between H10 and H6. It seems that the benzoate substituent was not spatially close to the protons in the imidazo[1,2-a]pyrimidine scaffold in compound 80a. However, in the 2D-NOESY spectrum of compound 80b (Figure 55), the nuclear overhauser effects of protons H6-H6'and protons H10-H3 were observed, which were consistent with the structure that substitution at position

To an ice-cooled solution of 58 (100 mg, 0.