Developing Small Molecule Inhibitors of ALK2: a Serine/Threonine Kinase Implicated in Diffuse Intrinsic Pontine Glioma

Deeba Ensan

A thesis submitted in conformity with the requirements for the degree of Master of Science Pharmacology and Toxicology University of Toronto

Deeba Ensan

Master of Science

Pharmacology and Toxicology University of Toronto

2020 Abstract

Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we developed two separate series of potent type I inhibitors of ALK2 based on the previously reported inhibitor LDN-214117. The first structure-activity relationship (SAR) study focuses on improving the selectivity, permeability and pharmacokinetic profile of M4K2149, a benzamide analogue with reduced off-target affinity for the hERG potassium channel. The second part of this thesis highlights the efforts made to develop a conformationally constrained inhibitor that was rigidified into the biologically active configuration of M4K2009, the lead compound of this project. Future studies will assess the permeability of select compounds in a Caco-2 permeability assay and their PK profiles in vivo.

Acknowledgments

Completing my Master’s degree at the Ontario Institute for Cancer Research (OICR) was truly an eye-opening experience. The beginning of my training was most certainly taxing. However, the support I received from the Drug Discovery team facilitated my adaptation to a new environment. As time went on, I discovered that I possessed competencies and capabilities of which I was previously unaware. There were many individuals at the institute who helped me to realize my potential. To them, I am much obliged.

I would like to primarily thank Dr. Carlos A. Zepeda-Velázquez for being my mentor for the past two years. Carlos was instrumental in bringing my research project to fruition. Drug discovery and design is quite a niche field, incorporating elements of biochemistry, physiology, pharmacology and organic chemistry. It is a subject that is not taught in university. Therefore, Carlos ensuring that I was able to fully grasp its core principles was no small feat. From setting aside time to answer my inquiries to helping me troubleshoot reactions and develop synthetic methods, Carlos was able to instill in me the importance of scientific reasoning, critical thinking and independence.

I am grateful to Dr. David Smil for his guidance throughout the ALK2 project and for actively promoting the exchange of ideas related to drug discovery and research. I also acknowledge Babu Joseph, Dr. Iain D. G. Watson, Brian J. Wilson, Dr. Methvin B. Isaac, Dr. Taira Kiyota, Dr. Shiva Kalhor-Monfared and Julie Owen for their invaluable assistance. Lastly, I wish to express my deepest gratitude to Dr. Rima Al-awar for giving me the opportunity to complete my graduate studies in the Drug Discovery Program at the OICR. Rima created an environment conducive to student learning and always took the time to ensure that I was making progress throughout my degree.

The work presented herein could not have been done without the continuous support of my parents, sister and friends. They were always there when I needed them most.

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Table of Contents

Acknowledgments...... iii

Table of Contents ...... iv

List of Tables ...... vi

List of Figures ...... vii

List of Abbreviations ...... viii

Chapter 1 Introduction ...... 1

Introduction ...... 1

1.1 Diffuse Intrinsic Pontine Glioma ...... 1

1.1.1 Pathology and Prognosis ...... 1

1.1.2 Current Standard of Care and Prospective Therapies ...... 1

1.1.3 Epigenetic Drug Targets ...... 3

1.1.4 Activin receptor-like kinase 2 (ALK2) ...... 5

1.2 Kinase inhibitors ...... 7

1.2.1 Targeting Kinases: Strengths and Limitations ...... 7

1.2.2 Brain Penetrant Kinase Inhibitors ...... 9

1.3 Study Objective ...... 11

Chapter 2 Structure Activity Relationship of M4K2149 ...... 15

Structure Activity Relationship of M4K2149 ...... 15

2.1 Chemical Syntheses ...... 15

2.2 Results and Discussion ...... 22

2.2.1 Binding Mode of M4K2149 with ALK2 ...... 22

2.2.2 Optimizing Potency and Selectivity...... 23

2.2.3 Caco-2 Studies ...... 29

2.2.4 Pharmacokinetic Studies and Assessment of Off-target Activity ...... 32

2.2.5 Exploring Alternative Ortho Substituents ...... 34

Chapter 3 Structure Activity Relationship of Conformationally Constrained Derivatives of M4K2009 ...... 38

Structure Activity Relationship of Conformationally Constrained Derivatives of M4K2009 ...... 38

3.1 Chemical Syntheses ...... 38

3.2 Results and Discussion ...... 41

3.2.1 Binding Mode of M4K2009 with ALK2 ...... 41

3.2.2 Optimizing Potency and Selectivity...... 43

Chapter 4 Conclusions and Future Studies ...... 48

Conclusions and Future Studies ...... 48

Chapter 5 Synthetic Procedures and Compound Characterizations ...... 50

Synthetic Procedures and Compound Characterizations ...... 50

5.1 Chemical Syntheses ...... 50

5.2 NMR Spectra ...... 85

References ...... 119

Appendices ...... 131

List of Tables

Table 1: Target values of lead ALK2 inhibitor 13

Table 2: Inhibitory and off-target activities of 14a-b, 7a-b and 8a 25

Table 3: Inhibitory and off-target activities of 8b-c, 18a-b, 20a-e and 31b 26

Table 4: Inhibitory and off-target activities of 2-fluoro-6-methoxybenzamide and 2,6-

dimethoxybenzamide analogues, 26a-f 30

Table 5: In vitro permeability and oral in vivo PK studies of 2-fluoro-6-methoxybenzamide

analogues 33

Table 6: Inhibitory activity of benzamide analogues against WT and DIPG-linked mutant forms

of ALK2 34

Table 7: Inhibitory and off-target activities of 31a, 31c-f and 36 36

Table 8: Caco-2 permeability of 31a and 31e-f 37

Table 9: Inhibitory and off-target activities of M4K2009 and 49 45

Table 10: Inhibitory and off-target activities of 56, 64a and 64b 46

List of Figures

Figure 1. BMP signaling cascade. 6

Figure 2. ALK2 inhibitors currently in clinical trials. 7

Figure 3. Co-crystal of the kinase domain of ALK2 with inhibitor M4K2149 (PDB: 6T6D). 8

Figure 4. Development of lorlatinib from the first-generation ALK inhibitor crizotinib. 10

Figure 5. Development of the clinical candidate AZD3759 from the first-generation EGFR

inhibitor gefitinib. 11

Figure 6: Inhibitory and off-target activities of previously reported ALK2 inhibitors and 12

OICR analogues. 13

Figure 7. Determining the optimal 3D configuration of M4K2009 for binding to ALK2. 14

Figure 8. (A) Co-crystal structure of M4K2149 (light yellow) with ALK2 (PDB code 6T6D).

(B) Co-crystal structure of LDN-213844 (light yellow) with ALK2 (PDB code 4BGG). 23

Figure 9. Conformation of EZH2 inhibitor. 27

Figure 10. Docking of 39, an isoquinoline analogue of M4K2121, with ALK2. 31

Figure 11. Altering the 3D configuration of the 3,5-diarylpyridine inhibitors of ALK2. 42

Figure 12. (A) Co-crystal structure of M4K2009 (dark blue) with ALK2 (PDB code 6SZM). 43

vii

List of Abbreviations

ACN: acetonitrile

ADME: absorption, distribution, metabolism & excretion

ALK: anaplastic lymphoma kinase

ALK2: activin receptor-like kinase 2

ALK5: activin receptor-like kinase 5

Amphos: (4-(N,N-dimethylamino)phenyl)di-tert-butyl phosphine

ATP: adenosine triphosphate

AUCinf: area under the curve extrapolated to infinity

BBB: blood-brain barrier

BCR-Abl: breakpoint cluster region protein – Abl tyrosine kinase fusion protein

BET: bromodomain and extra-terminal domain

BMP: bone morphogenetic protein

B/P: total brain-to-plasma ratio

B2pin2: bis(pinacolato)diboron

BRD2: bromodomain-containing protein 2

BRD4: bromodomain-containing protein 4

Caco-2: colorectal adenocarcinoma cell line

CAR T cell therapy: chimeric antigen receptor T cell therapy

Cb,u: unbound brain concentration

CED: convection-enhanced delivery

CFTR: cystic fibrosis transmembrane conductance regulator

COD: 1,5-cyclooctadiene cLogP: calculated lipophilicity

Cmax: maximum serum concentration reached in vivo

CML: chronic myeloid leukemia viii

CNS: central nervous system

CuSO4·5H2O: copper (II) sulfate pentahydrate

CYP: cytochrome P450 enzyme

DCM: dichloromethane

DFG: aspartate-phenylalanine-glycine motif

DIPEA: N,N-diisopropylethylamine

DIPG: diffuse intrinsic pontine glioma

DLA: dual-luciferase reporter assay

DLT: dose-limiting toxicities

DMF: dimethylformamide

DMSO: dimethyl sulfoxide dppf: 1,1'-bis(diphenylphosphino)ferrocene

DTBPY: 4,4′-di-tert-butyl-2,2′-dipyridyl

EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EGFR: epidermal growth factor receptor

ESI: electrospray ionization

Et2O: diethyl ether

EtOAc: ethyl acetate

EtOH: ethanol

EZH2: enhancer of zeste homolog 2

FDA: food and drug administration

FOP: fibrodysplasia ossificans progressiva

GS: glycine-serine rich domain

HATU: hexafluorophosphate azabenzotriazole tetramethyl uranium

HEK293: human embryonic kidney cell line

H3K27ac: acetylated lysine residue 27 of histone 3 ix

H3K27me3: trimethylated lysine residue 27 of histone 3

HBD: hydrogen bond donor

HCl: hydrochloric acid

HDAC: histone deacetylase hERG: human ether-a-go-go related gene

HLM assay: human liver microsomal stability assay

H2O2: hydrogen peroxide

HOBt: hydroxybenzotriazole

HRMS: high-resolution mass spectrometry

IC50: half maximal inhibitory concentration

IV: intravenous

K2CO3: potassium carbonate

KCN: potassium cyanide

KOAc: potassium acetate

KOH: potassium hydroxide

K3PO4: potassium phosphate tribasic

Kp,uu: unbound brain concentration to unbound plasma concentration ratio

ME-FUS: microbubble-enhanced focused ultrasound

MeOH: methanol

MgSO4: magnesium sulfate

MLM assay: mouse liver microsomal stability assay

MOMCl: methoxymethyl chloride

MS: mass spectrometry

MTD: maximum tolerated dose mTOR: mammalian target of rapamycin

MRI: magnetic resonance imaging x

NaBH4: sodium borohydride

Na2CO3: sodium carbonate

NaH: sodium hydride

NaHCO3: sodium bicarbonate

NaHMDS: hexamethyldisilazane sodium salt

NanoBRET: Nanoluciferase bioluminescence resonance energy transfer

NaOH: sodium hydroxide

NaOtBu: sodium tert-butoxide

NaSO4: sodium sulfate

NH3: ammonia

NH4Cl: ammonium chloride

NMP: N-methyl-2-pyrrolidone

NMR: nuclear magnetic resonance

NOBF4: nitrosonium tetrafluoroborate

NSCLC: non-small-cell lung carcinoma

OAc: acetate

PDGFB: platelet-derived growth factor subunit B

P-gp: P-glycoprotein

PK: pharmacokinetic pKa: negative logarithm (base 10) of the acid dissociation constant

PTM: post-translational modification

PRC2: polycomb repressive complex 2 rac-BINAP: (±)-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene

RuPhos: 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl

SAR: structure-activity relationship shRNA: small hairpin RNA xi

SNAr: nucleophilic aromatic substitution

SPE: solid-phase extraction t1/2: half-life of drug in vivo

TFA: trifluoroacetic acid

TGF-βR1: transforming growth factor beta receptor I

THF: tetrahydrofuran tPSA: topological polar surface area

WT: wild type

XPhos: 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

ºC: degrees Celsius cm: centimeter d: day h: hour kg: kilogram mg: milligram min: minute ng: nanogram nM: nanomolar

μM: micromolar rt: room temperature s: second

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Chapter 1 Introduction Introduction 1.1 Diffuse Intrinsic Pontine Glioma

1.1.1 Pathology and Prognosis

Diffuse intrinsic pontine glioma (DIPG) represents a subset of pediatric brainstem cancers that affects around one in a million people.1,2 It is the leading cause of brain tumor-related death in the pediatric setting.3 The age of onset varies from 3 to 12 years old.4 Children afflicted with the disease may exhibit symptoms, some of which are indicative of cranial neuropathy, however, they are usually transient.1 These symptoms include diplopia (double vision), facial asymmetry, abnormal gait, the occurrence of the Babinski sign and the most diagnostic symptom, abducens palsy, which manifests as restricted eye movement.1,5 Prognosis for the disease is ominous. The median overall survival is below a year, while the 5-year relative survival rate is less than a percent.1-5 Poor survival outcomes are attributed in part to the infiltrative nature of the tumor. Pathologic features of DIPG confirm that the disease is a type of fibrillary astrocytoma, distinct from pilocytic astrocytomas, as the tumor is diffuse in nature, originates in the pons and has an associated poor prognosis.1,6-7 Brainstem pilocytic astrocytomas tend to be discrete, are not restricted to the pons and the overall survival is greater for those afflicted.6 Given that DIPG lesions are poorly demarcated by MRI,4 total surgical resection of the tumor is not possible.

1.1.2 Current Standard of Care and Prospective Therapies

Focal radiation therapy is currently the only effective treatment for DIPG, however, it merely delays tumor progression by approximately 2-4 months before the patients succumb to the disease.1,3 Radiosensitizing agents are unable to improve survival outcomes for those afflicted.1,5,8 Several clinical studies evaluating the efficacy of carbogen and motexafin- gadolinium in DIPG patients were to no avail.9-10 The addition of alkylating agents, such as carboplatin and temozolomide, to radiation therapy proved futile in this cohort as well.1,5,11-12 The failure of conventional chemotherapy drugs at improving patient outcomes has brought into question their ability to reach therapeutically relevant concentrations in the pons, which ostensibly has a tighter BBB than other areas of the CNS.13 This has prompted the emergence of

1 2 drug delivery methods which either impair the integrity of the BBB or avoid it altogether.13 Several of these techniques are described below:

Cytokine-mediated enhancement in BBB permeability

Enhancing drug delivery to the CNS via the administration of cytokines that can modulate the integrity of the BBB has been explored.13 One such study investigated the effects that lobradimil, an analogue of bradykinin, had on enhancing the efficacy of carboplatin in patients with primary brain tumors.14 The cohort included children with brainstem gliomas. Lobradimil (RMP-7) is a 14 bradykinin B2 receptor agonist which increases BBB permeability. Although lobradimil has been shown to augment carboplatin delivery to the brain parenchyma in animal models, this combinatorial approach did not provide a survival benefit to the brainstem glioma and high- grade glioma patients enrolled in this study.14 It is conceivable that carboplatin is ineffective against some of the types of brain cancers that were included in this phase II clinical trial.13 Future studies should look into the effectiveness of lobradimil use in conjunction with drugs of demonstrated efficacy against DIPG.

Convection-enhanced delivery (CED)

CED is a procedure that has gained traction in recent years. The technique utilizes a microcatheter to infuse target tissues with drugs.5,15 CED is distinct from intrathecal delivery as it relies on pressure exerted by a pump instead of passive diffusion for adequate drug distribution within the brainstem.5,15 It has been shown that gadolinium-bound albumin can be delivered successfully to the pons of Macaca mulatta monkeys using this method.15 Several clinical trials are currently evaluating the efficacy of drugs administered to the pons via CED in DIPG patients.16-17 Although CED is able to sidestep the limitations associated with traditional routes of drug administration, such as systemic toxicity and the BBB,15 it is a highly invasive procedure and its applicability in DIPG treatment warrants further investigation.

Microbubble-enhanced focused ultrasound (ME-FUS)

ME-FUS utilizes mechanical force to purportedly increase both paracellular and transcellular transport across the BBB. The procedure entails intravenous (IV) injection of microbubbles and subsequent exposure to short pulse-wave ultrasound to temporarily enhance drug delivery to the

3 brain parenchyma.18 Marquet and co-workers were able to visualize BBB opening by MRI in the visual cortex of Macaca mulatta monkeys after IV administration of gadodiamide (BBB impenetrable) and ME-FUS exposure.18 While the integrity of the BBB is restored after treatment, some adverse effects of ME-FUS include edema and hemorrhaging. The long-term effects of repeated BBB disruption should be investigated further.

Beyond the development of drug delivery methods which modify or bypass the BBB, interest in exploring other treatment options for patients has surged. Immunotherapy is a rapidly evolving treatment modality with broad applications in cancer therapy. One such strategy, known as chimeric antigen receptor (CAR) T cell therapy, utilizes autologous T cells that have been engineered to express receptors that recognize antigens on the surface of cancer cells.19-20 A phase I study investigating the safety and efficacy of B7-H3-specific CAR T cell therapy in children with DIPG is currently being conducted.21 B7-H3 (CD276) is a protein that has been shown to be expressed by pediatric tumor cells, including DIPG.20 Manipulating the immune system to activate anti-tumor adaptive immune responses is most certainly an emerging area of interest with potential applications in DIPG therapy.

Although the results of several of the aforementioned pre-clinical studies are encouraging, their limitations have been an impetus for the development of targeted molecular therapies. Very little was known about the biology of DIPG for decades. This was in part due to the limited number of biopsies that had been performed.1,5,8 This has changed drastically, however, with the advent of stereotactic biopsy procedures.5,8 The recent identification of potential targets for therapeutic intervention has generated hope that the standard of care will evolve to include small molecule inhibitors.

1.1.3 Epigenetic Drug Targets

Whole-genome sequencing of biopsy samples has revealed a plethora of dysregulated pathways that may play critical roles in driving tumorigenesis. Approximately 80% of DIPG samples harbor missense mutations in H3F3A and HIST1H3B genes,2 which encode the histone variants H3.3 and H3.1, respectively. The mutation, which substitutes lysine for a methionine at residue 27 (K27M), alters the spatial distribution of post-translational modifications (PTMs) across the genome, which is believed to change the expression of certain genes, including those involved in

4 tumor suppression. Targeting the following proteins has the potential to restore normal epigenetic signatures in DIPG cells:

Enhancer of zeste homolog 2 (EZH2)

EZH2 is a subunit of the polycomb repressive complex 2 (PRC2), which catalyzes the methylation of H3K27 residues, a mark of transcriptional silencing. A global reduction in H3K27me3 is a hallmark of H3K27M mutant DIPG. Paradoxically, increased levels of H3K27me3 have been shown to localize near specific genes.22 To investigate the potential involvement of PRC2 in DIPG pathogenesis, Mohammad and co-workers treated murine neuronal stem cells co-expressing platelet-derived growth factor subunit B (PDGFB) and mutant H3.3K27M with the EZH2 inhibitors, GSK343 and EPZ6438.22 They found that the inhibitors induced senescence in these cells and correspondingly, hindered cell proliferation. Cognizant of the fact that H3K27me3 levels were elevated at the Ink4a promoter, Mohammad and co-workers attributed the anti-proliferative effects of the EZH2 inhibitors to an increase in the expression of the cell-cycle regulator and tumor-suppressor protein p16INK4A. Their work suggests that targeting EZH2 may offer therapeutic benefit in children afflicted with H3K27M mutant DIPG.

Bromodomain and extraterminal domain (BET)

BET proteins are readers of histone PTMs.23 They specifically recognize acetylated lysine residues, which are marks of transcriptional activation. Piunti and co-workers showed that H3K27M mutations can increase H3K27ac levels and localize at regions containing these PTMs.24 This explained the accumulation of bromodomain-containing proteins 2 and 4 (BRD2 and BRD4) at H3K27M-K27ac sites in SF8628 human DIPG cells. Treatment of this cell line with JQ1, a small molecule inhibitor of BET, reduced proliferation. Its efficacy in a murine xenograft model of DIPG was quite encouraging, as well.

Histone deacetylase (HDAC)

In a drug screen against patient-derived DIPG cells, panobinostat was identified as a potential therapeutic agent.25 The drug, which has been approved by the FDA, is a potent inhibitor of HDAC. Grasso and co-workers demonstrated that panobinostat partially restored normal epigenetic patterns in H3K27M mutant DIPG cell lines, SU-DIPG-VI and SU-DIPG-XIII,

5 particularly with respect to H3K27me3 levels.25 Recent studies evaluating the efficacy of HDAC inhibitors as a therapy for DIPG have proposed that other proteins involved in epigenetic regulation also be targeted in conjunction with HDAC, as monotherapies alone show little promise.26 A handful of clinical trials are currently investigating the effects of HDAC inhibition in children afflicted with DIPG.27-28 One such trial is testing the use of vorinostat in conjunction with the mTOR inhibitor, temsirolimus.

1.1.4 Activin receptor-like kinase 2 (ALK2)

The bone morphogenetic protein (BMP) signaling pathway is another biochemical cascade that is dysregulated in DIPG. BMPs are a group of cytokines that modulate a plethora of physiological processes, including musculoskeletal growth and CNS development.2 The signal elicited by BMP binding to type II BMP receptors is transduced by type I BMP receptors, which promote the translocation of downstream effector proteins (SMADs) to the nucleus where they can regulate the transcription of target genes via chromatin remodeling.29-30 Aberrant BMP signaling is implicated in several diseases, the most notable being fibrodysplasia ossificans progressiva (FOP). Germline mutations (c.617G>A; p.R206H) in the juxtamembrane glycine-serine (GS) rich domain of activin receptor-like kinase 2 (ALK2) confer gain-of-function activity to the type I BMP receptor and contribute to the abnormal skeletal phenotype observed in individuals affected by FOP.2,31

Somatic gain-of-function mutations in the ACVR1 gene encoding ALK2 have been reported in approximately 24% of DIPG patients,32 with a higher prevalence of mutation occurring in the serine/threonine kinase domain.32-33 The mechanism by which ALK2 contributes to DIPG pathogenesis has not yet been elucidated.2,33-34 However, a recent study by Carvalho and co- workers demonstrated that shRNA knockdown of ACVR1 elicits apoptosis in HSJD-DIPG-007 cells harboring both ACVR1 R206H and histone H3.3 K27M mutations.32 Their work suggests that these DIPG cells are dependent on enhanced ALK2 signaling.

BMP BMPRII ALK2

Cytoplasm P FKBP12

Small Molecule ALk2 Inhibitors SMAD 1/5/8

SMAD P 1/5/8

SMAD P 1/5/8 SMAD 4

Nucleus

SMAD P 1/5/8 SMAD 4

Gene Transcription Figure 1. BMP signaling cascade.31 BMP ligand binding to BMPRII triggers the phosphorylation of ALK2 at its GS domain. ALK2 phosphorylates SMAD1/5/8, which then interact with the co- mediator SMAD4 to create a complex that can translocate to the nucleus.29 FOP and DIPG-linked ACVR1 mutations weaken interactions between ALK2 and FKBP12, a regulatory protein which keeps ALK2 in its inactive configuration.31 Small molecule inhibitors of ALK2, such as dorsomorphin and LDN193189, decrease phosphoSMAD levels. Mutations in ACVR1 are often observed in conjunction with H3.1K27M mutations.32

Clinical trials evaluating the efficacy of ALK2 inhibitors in different disease areas are underway. A phase I study is currently determining the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of TP-0184 (Figure 2), developed by Tolero Pharmaceuticals, in adults with advanced solid tumors.35-36 Anti-tumor activity will be measured as a secondary outcome. BLU- 782 is another orally bioavailable ALK2 inhibitor that was created by Blueprint Medicines Corporation as a therapy for FOP patients (Figure 2).37 A phase I study evaluating the pharmacokinetic profile and adverse effects of the drug was recently completed in 2019 and the small molecule inhibitor has also received orphan drug status by the FDA.38-39 BioCryst

Pharmaceuticals has also developed a potent and orally bioavailable ALK2 inhibitor known as BCX9250. Its structure has not yet been disclosed. The molecule has shown promise in preclinical animal models of FOP and a phase I clinical trial evaluating dosing regimens was initiated by BioCryst in 2019.40

Figure 2. ALK2 inhibitors currently in clinical trials.

Given the interest in ALK2 as a target for therapeutic intervention and that mutations in ACVR1 are highly specific to DIPG, targeting ALK2 represents a new approach that may provide therapeutic benefit in patients harboring these genetic alterations.

1.2 Kinase inhibitors

1.2.1 Targeting Kinases: Strengths and Limitations

Although targeting kinases constitutes a relatively new therapeutic modality,41 there have been significant advances in the development of kinase inhibitors of clinical efficacy. Considering the implications that aberrant kinase activity has in disease, it is unsurprising that more than 50 kinase inhibitors have been approved by the FDA since the turn of the century.42 Imatinib (marketed as Gleevec) was the first small molecule kinase inhibitor approved for medical use in 2001.41-42 Since then, much progress has been made in the understanding of kinase structural biology in addition to the development of cellular target engagement assays and kinome-wide selectivity profiling.43

Kinases are structurally very similar, particularly with respect to their kinase domains.41 The domain consists of primarily two lobes: the N-terminal lobe (N-lobe), which is comprised of a five-stranded antiparallel β-sheet, and the C-terminal lobe (C-lobe), which is made up of primarily α-helices.41-42 The hinge region joins the two lobes and can hydrogen bond with ATP.41 One notable conserved sequence resides in the activation loop of the kinase and is referred to as the DFG motif. The aspartic residue of the DFG motif coordinates to magnesium, which in turn interacts with the β and γ phosphates of ATP.44 The aspartate can adopt multiple orientations, either positioned towards the active site (DFG-in) or away from the active site (DFG-out).41-42 Depending on the configuration of the DFG motif and the binding location, small molecule kinase inhibitors can be categorized into several groups. Type I inhibitors compete with ATP to bind to the active conformation (DFG-in) of kinases, whereas Type II inhibitors bind to the inactive conformation (DFG-out).41 Type III inhibitors on the other hand do not bind to the active site of the enzyme, rather to an area adjoining it, while Type IV inhibitors interact with allosteric pockets that are distant from the site of catalysis.41

N-lobe

Hinge

C-lobe

Figure 3. Co-crystal of the kinase domain of ALK2 with inhibitor M4K2149 (PDB code: 6T6D). The kinase is colored from its N-terminus to its C-terminus (purple to red). The N-lobe consists of a five-stranded antiparallel β-sheet, while α-helices make up the C-lobe. The ligand

9 binds to the hinge region, which connects the two lobes together. The ligand can be observed in cyan.

Despite the various ways that kinases can be targeted, there are limitations associated with the use of kinase inhibitors as a therapy, the most notable being antineoplastic resistance.43 A classic example of the phenomenon can be observed in chronic myeloid leukemia (CML) patients that become resistant to imatinib.44 Imatinib targets the fusion protein BCR-Abl, a dysregulated tyrosine kinase, which drives the development of the leukemia.41-42 Cells expressing the chimeric protein can evade inhibition by mutating key residues in the ATP-binding pocket of the kinase. One such residue is the gatekeeper threonine of Abl. Substitution of T315 with an isoleucine drastically reduces the potency of imatinib against BCR-Abl.44 Consequently, a third- generation BCR-Abl inhibitor ponatinib had to be developed which bypassed the gatekeeper residue.41

Despite these drawbacks, the pivotal roles that kinases play in the regulation of a vast array of cellular processes make them very alluring molecular targets. The clinical success of kinase inhibitors explains why the focus of approximately 20-30% of drug discovery programs is on kinases.42 Furthermore, the administration of potent and selective kinase inhibitors that are orally bioavailable represents a non-invasive therapeutic modality, which offers the additional benefit of increasing patient compliance.43

1.2.2 Brain Penetrant Kinase Inhibitors

Developing brain penetrant kinase inhibitors entails so many challenges that not a single drug has been approved for the treatment of malignant primary CNS tumors.45 There are numerous obstacles that a drug must overcome in order to enter the brain parenchyma from the systemic circulation, the most notable being the BBB. CNS drug exposure is restricted by the endothelial tight junctions of the BBB, which impede paracellular transport. Consequently, many approved CNS drugs reach the brain tissue via transcellular passive diffusion.46 The expression of efflux transporters, such as the ATP-binding cassette transporter, P-glycoprotein (P-gp), on the luminal surface of these endothelial cells further limits drug entry into target tissues.46

For CNS indications, several structural properties must be considered when designing drugs that are meant to reach therapeutically relevant concentrations at their intended site of action. These

10 parameters include lipophilicity (cLogP), topological polar surface area (tPSA), hydrogen bonding potential, molecular rigidity and pKa.45-46 Several drug discovery programs have been successful in developing brain penetrant kinase inhibitors with optimized ADME profiles.47

The pioneering work done by Pfizer in their rational design of a macrocyclic analogue of the first-generation inhibitor crizotinib is a noteworthy example.48 The rigidification of small molecule ligands into their protein-bound configurations is an energetically favorable modification from an entropic standpoint. In addition to possibly enhancing potency, reducing the molecular flexibility of drugs also has the potential to increase their permeability across the BBB.46,48 Johnson and co-workers suggested that the maintenance of a low molecular weight and the minimization of the hydrogen bond donating potential via the introduction of an intramolecular hydrogen bond may have contributed to the reduction in P-gp-mediated efflux of their lead candidate.48 Their work resulted in the identification of lorlatinib, which received accelerated approval by the FDA in 2018 for anaplastic lymphoma kinase (ALK)-positive metastatic non-small-cell lung carcinoma (NSCLC).49

Figure 4. Development of lorlatinib from the first-generation ALK inhibitor crizotinib.

AztraZeneca also took a multi-pronged approach to develop a brain penetrant epidermal growth factor receptor (EGFR) inhibitor for patients with metastatic NSCLC harboring EGFR activating mutations.50 They were able to optimize the clinically approved EGFR inhibitor gefitinib by incorporating intramolecular electrostatic interactions to mask hydrogen bond donors (HBD), reducing the number of rotatable bonds and introducing aliphatic substituents to limit metabolic oxidation. The resulting clinical candidate AZD3759 demonstrated anticancer activity in a murine brain metastasis model.

Figure 5. Development of the clinical candidate AZD3759 from the first-generation EGFR inhibitor gefitinib.

Although the application of kinase inhibitors as a therapy for the treatment of CNS tumors is still in its infancy, these examples demonstrate that the development of brain penetrant kinase inhibitors is attainable.

1.3 Study Objective

Given that a significant portion of DIPG patients harbor gain-of-function mutations in the ACVR1 gene, ALK2 may be involved in driving its pathogenesis. Therefore, targeting ALK2 has the potential to provide therapeutic benefit in children afflicted with the disease. Small molecule kinase inhibitors of clinical efficacy that can be taken orally represent a minimally invasive therapeutic strategy that could impose little hardship on children with already poor prognostic outcomes. The main purpose of this study was to optimize an existing type I inhibitor of ALK2 to make it more potent, selective, orally bioavailable and brain penetrant. Such an inhibitor could also be used to further our understanding of disease biology.

Several inhibitors of ALK2 have emerged in the past decade.51 The very first ALK2 inhibitor dorsomorphin was discovered in an in vivo screening of small molecules that could elicit a dorsalization phenotype in zebrafish embryos, a hallmark of BMP signaling inhibition.52 Modifications to the scaffold of dorsomorphin resulted in the identification of a more potent analogue LDN-193189, which showed improved metabolic stability in liver microsomal stability assays.53 K02288 was identified as a hit in a screen against human kinases utilizing a differential scanning fluorimetry assay.54 At the time of discovery, K02288 represented a new chemotype that differed substantially from the typical pyrazolo[1,5-ɑ]pyrimidine core of the previously reported ALK2 inhibitors. The 2-aminopyridine inhibitor eventually evolved into a new class of

3,5-diarylpyridine compounds, which included LDN-214117 and LDN-213844. LDN-214117 has been shown to have low cytotoxic activity and excellent kinome-wide selectivity, making it a promising compound on which to expand SAR.55

Prior to my joining the OICR, the drug discovery team had previously explored whether modifications to the hinge-binding pyridyl core of LDN-214117 could improve ALK2 potency and selectivity over the closely related TGF-βRI receptor ALK5. Small molecule inhibitors of ALK5 have been shown to elicit cardiotoxic effects in animals.56 Correspondingly, a major focus of the drug discovery team’s initial medicinal chemistry efforts was to synthesize analogues with reduced off-target affinity for this receptor. Shifting the methyl substituent from the C-2 position of the pyridyl core of LDN-214117 to the C-4 position generated M4K2009, the project’s lead compound. This transposition maintained potency and selectivity as determined by the in vitro and cell-based assays employed throughout this study (Figure 6).

ALK2 Inhibitor Dorsomorphin LDN-193189 K02288 LDN-213844 LDN-214117 M4K2009 M4K2149

Reported vs measured biochemical 9.8, - 0.7, 17 35, - 15, 18 24, 115 -, 13 -, 17 ALK2 IC50 (nM) Reported vs measured biochemical 7829, - 117, 468 280, - 240, 747 3000, >2000 -, 1830 -, 576

ALK5 IC50 (nM) Reported vs measured fold 799, - 167, 28 8, - 16, 42 125, >17 -, 141 -, 34 selectivity (ALK5/ALK2)

Figure 6: Inhibitory and off-target activities of previously reported ALK2 inhibitors and

OICR analogues. Reported values were obtained from the corresponding references.55,57-58 Measured values were conducted by Reaction Biology Corporation utilizing a radioactive biochemical kinase assay.

Table 1: Target values of lead ALK2 inhibitor

Assay M4K2009 Target value

ALK2 IC50 13 nM <20 nM

ALK5 IC50 1830 nM >1000 nM

NanoBRET ALK2 IC50 49 nM <100 nM

Dual-Luciferase Reporter Cell-based ALK5/ALK2 = Cell-based ALK5/ALK2 =

Assay ALK5 IC50 44 >30

MLM t1/2 67 min >60 min

HLM t1/2 70 min >60 min

-6 -6 Caco-2 Papp (A→B) 7.0 x 10 cm/s (>3 x 10 cm/s)

Caco-2 1.3 <2 Papp(B→A)/Papp(A→B)

hERG IC50 8.25 µM >10 µM

The work presented herein has two distinct objectives, both of which expand on the SAR of M4K2009. Although this compound met most of the target values for potency, selectivity (over ALK5), permeability and metabolic stability (Table 1), there was concern regarding its risk for

14 eliciting torsades de pointes arrythmia in vivo due to the moderate affinity it has for the protein 59 product encoded by the human ether-a-go-go related gene (hERG) (IC50 = 8.25 μM). This protein product forms part of a potassium channel in cardiac muscle cells. Its blockage can interfere with cardiac repolarization, which can be fatal.59 The drug discovery team, prior to my start at the OICR, made additional modifications to the trimethoxyphenyl moiety of M4K2009, which resulted in the identification of M4K2149. The benzamide analogue has a hERG IC50 of >50 μM and comparable inhibitory activity against ALK2 (Figure 6). Chapter 2 of this thesis focuses on the efforts made to preserve the favourable hERG profile of this analogue while making further structural modifications to enhance its oral bioavailability. Improving the Caco-2 permeability and oral PK of this series was a major challenge and is discussed in detail within the chapter.

The second topic of discussion relates to the preferred binding orientation of M4K2009. Co- crystal images of the molecule with ALK2 reveal its non-planar configuration. Specifically, the dihedral angle between the pyridyl and 4-piperazinylphenyl motifs (C-1 to C-4) deviates substantially from 0º. Taking advantage of the potential enhancements that intramolecular conformational constraints can bestow in terms of potency and permeability resulted in the development of rigidified analogues of M4K2009 featuring tricyclic cores of varying size. This part of the project is discussed in Chapter 3.

Figure 7. Determining the optimal 3D configuration of M4K2009 for binding to ALK2. Co- crystal images of the ligand bound to ALK2 reveal that M4K2009 adopts a non-planar configuration. The dihedral angle between carbons C-1 to C-4 deviates from 0º. Reducing the rotation about the C-2 to C-3 bond has the potential to enhance both potency and permeability.

Chapter 2 Structure Activity Relationship of M4K2149 Structure Activity Relationship of M4K2149 2.1 Chemical Syntheses

The synthetic route employed to prepare M4K2149 and related analogues ultimately depended on the commercial availability of starting materials, ease of synthesis and reaction efficiency. The compounds were initially accessed as depicted in Scheme 1. Suzuki-Miyaura coupling of 1.0 equivalent of 3,5-dibromo-4-methylpyridine (2) with 1.05 equivalents of 1 generated intermediate 3 in 47% yield. Formation of di-tert-butyl 4,4'-((4-methylpyridine-3,5-diyl)bis(4,1- phenylene))bis(piperazine-1-carboxylate) contributed to the lower yield obtained for 3, however, given that the starting materials were relatively cheap and that the reaction was easily scalable, high quantities of the desired product could be easily isolated. 3 subsequently underwent Miyaura borylation to yield the boronate ester 4a. Aromatic methyl esters 5a-b were coupled with 4a to afford intermediates 6a-b, which were transformed to the corresponding primary amides 7a-b by refluxing in methanolic ammonia, which was then followed by the removal of the carbamate protecting groups with trifluoroacetic acid (TFA). The preparation of analogues 8a-c followed a similar synthetic route in which 4a was coupled with the aromatic amides 5c-e then deprotected with TFA. Almost 50% of intermediate 3 was converted to the undesired protodehalogenated side-product 4b in step b (as determined by UV absorbance at 254 nm during tandem liquid chromatography/mass spectrometry (LCMS)), which not only contributed to lower yields obtained for the final products, but 4b was at times difficult to remove from reaction mixtures during chromatographic purification, especially after the intermediate had been deprotected by TFA.

To overcome these limitations, a second synthetic scheme was devised in which a wide variety of boronate esters were coupled with the pyridyl derivatives 3, 10 or 15 (Scheme 2). The synthesis of the carboxylic acid intermediates 13a-b was accomplished via a two-step, one-pot Suzuki-Miyaura coupling sequence. HATU-mediated coupling with ammonium chloride followed by deprotection, furnished the final amide regioisomers 14a-b.

Scheme 1. Synthesis of Compounds 7a-b and 8a-cɑ

ɑ Reagents and conditions: (a) Pd(dppf)Cl2·CH2Cl2, Na2CO3·H2O, dioxane/H2O, 85 ºC, overnight (3 (47%)); (b) B2pin2, Pd(dppf)Cl2·CH2Cl2, KOAc, dioxane, 110 ºC, 4 h (4a (57%));

6b (52%)); (d) 7 N NH3 in MeOH, 90 ºC, 3 d; (e) TFA, DCM, rt, overnight (7a (47% over 2 steps), 7b (53% over 2 steps), 8a (15% over 2 steps), 8b (29% over 2 steps), 8c (32% over 3 steps)); (f) aryl halide (5c-e), XPhos Pd G2, K3PO4, dioxane/H2O, 100 ºC, 3 h.

Suzuki-Miyaura coupling of 15 with the boronate ester 16 afforded the methyl ester intermediate 17, which was converted to the corresponding primary, secondary or tertiary amide via aminolysis or base-catalyzed hydrolysis followed by EDC-mediated coupling with the desired amine. Deprotection using TFA or HCl afforded the compounds M4K2149 and 18a-b. These three compounds were synthesized by Dr. David Smil and Dr. Dimitrios Panagopoulos at the OICR. A similar synthetic route was used to access analogues 20a-e, although additional transformations beyond the standard Suzuki-Miyaura coupling and deprotection were not required, as the boronate esters 19a-e already had the desired amide substituents installed. The final compound 20d was synthesized by Dr. David Smil.

Scheme 2. Synthesis of Compounds 14a-b, M4K2149, 18a-b and 20a-eɑ

ɑ Reagents and conditions: (a) Pd(dppf)Cl2·CH2Cl2, Na2CO3·H2O, DMF/H2O, 100 ºC, 4 h (11a

(87%), 11b (90%)); (b) XPhos Pd G2, K3PO4, dioxane/H2O, 100 ºC, overnight (17 (99%)); (c)

NH4Cl, HATU, DIPEA, DCM, rt, 3 h; (d) TFA, DCM, rt, 1 h (14a (7% over 4 steps), 14b (6% yield over 4 steps), 18a (20% over 2 steps), 18b (23% over 3 steps), 20a (44% over 2 steps), 20b (45% yield over 2 steps), 20c (29% yield over 2 steps), 20e (36% yield over 2 steps)); (e) 7 N

NH3 in MeOH, 75 ºC, 3 d; (f) methylamine, EtOH/MeOH, 85 ºC, 5 h; (g) KOH, THF/H2O, rt, 2

18 h; (h) dimethylamine, HOBt, EDC, DIPEA, DCM/DMF, 50 ºC, overnight; (i) 4 M HCl in dioxane, MeOH, rt, 30 min (M4K2149 (60% over 3 steps), 20d (50% yield over 2 steps)).

The synthesis of analogues 26a-f (Scheme 3) was initiated by the nucleophilic aromatic substitution of 4-bromo-2,6-difluorobenzonitrile (21) with sodium methoxide to yield both 4- bromo-2-fluoro-6-methoxybenzonitrile (22a) and 2,6-dimethoxybenzonitrile (22b). Both intermediates were hydrolyzed to the corresponding amides 5d-e using hydrogen peroxide and an aqueous solution of sodium hydroxide. Miyaura borylation of 5d-e followed by Suzuki- Miyaura coupling with 3-bromo-5-chloro-4-methylpyridine (10) afforded 24a-b, which were subjected to a second coupling reaction with a variety of (4-(piperazin-1-yl)phenyl)boronate ester derivatives (25a-c) to furnish the final compounds 26a-f in excellent yields.

Scheme 3: Synthesis of Compounds 26a-fɑ

ɑReagents and conditions: (a) NaH, MeOH, dioxane, rt, overnight (22a (46%), 22b (34%)); (b)

H2O2, NaOH, EtOH/H2O, overnight (5d (80%), 5e (87%)); (c) B2pin2, Pd(dppf)Cl2·CH2Cl2,

KOAc, dioxane, 110 ºC, 4 h (23a (84%), 23b (70%)); (d) Pd(dppf)Cl2·CH2Cl2, Na2CO3·H2O, dioxane/H2O, 100 ºC, overnight (24a (56%), 24b (79%)); (e) XPhos Pd G3, K3PO4, dioxane/H2O, 100 ºC, overnight (26a (83%), 26b (60%), 26c (43%), 26d (87%), 26e (75%), 26f (26%)).

The generation of analogues 31a-f (Scheme 4) was more synthetically challenging. The borylation of 2-methoxybenzonitrile or 2-methoxybenzamide derivatives (27a-d) via C-H ʹ activation was accomplished using a catalytic system comprising of [Ir(COD)(OMe)]2 and 4,4 - di-tert-butylbipyridine.60 Functionalization of the C-H bond para to the nitrile/benzamide moiety was quite high. The boronate esters 28a-d were subsequently coupled with the pyridyl compounds 3 and 29 to yield the precursors 30a-f. Hydrolysis of the nitrile functional groups under basic aqueous conditions followed by the deprotection of carbamate protecting groups, as well as the N-(2,4,4-trimethylpentan-2-yl) motif of 30b using TFA, afforded the final compounds 31a-f in low to moderate yields.

The synthesis of the chlorine analogue 36 was mainly linear (Scheme 5), consisting of a single convergent step (c). The transformation of the aniline starting material 32 to the corresponding benzonitrile derivative 33 was achieved under Sandmeyer reaction conditions in quite low yield.

SNAr of the fluorine substituent for a methoxy group (34) preceded the hydrolysis of the nitrile to form the amide intermediate 35. Miyaura borylation yielded 19e, which was coupled with 29 at low temperature to afford the final compound. The isoquinoline analogue of 26d was prepared in 2 steps. Suzuki-Miyaura coupling of 37 with 23b, followed by Buchwald-Hartwig amination utilizing RuPhos Pd G3 as the catalyst furnished 39 in relatively low yield. Once again, a significant amount of the protodehalogenated side-product 40 was formed during step g as determined by UV absorbance at 254 nm during tandem liquid chromatography/mass spectrometry (LCMS), which contributed to the low yield obtained for 39.

Scheme 4: Synthesis of Compounds 31a-fɑ

ɑ Reagents and conditions: (a) B2pin2, [Ir(OMe)(1,5-cod)]2, DTBPY, hexane, 60 ºC, overnight

(28a (41%), 28b (75%), 28c (51%), 28d (36%)); (b) Pd(dppf)Cl2·CH2Cl2, Na2CO3·H2O, dioxane/H2O, 100 ºC, 4 h (30c (32%)); (c) XPhos Pd G2, K3PO4, dioxane/H2O, 100 ºC, overnight; (d) NaOH, EtOH/H2O, 100 ºC, overnight (31a (15% over 2 steps), 31d (7% over 2 steps), 31f (27% over 2 steps)); (e) TFA, DCM, 25-60 ºC, overnight (31b (7% over 2 steps), 31c (59% over 2 steps), 31e (49% over 3 steps)).

Scheme 5: Synthesis of Compounds 36 and 39ɑ

ɑ Reagents and conditions: (a) NOBF4, KCN, CuSO4·5H2O, CH2Cl2/H2O, rt, 3h (33 (10%)); (b)

NaH, MeOH, dioxane, rt, 2 h (34 (49%)); (c) H2O2, NaOH, EtOH/H2O, 90 ºC, 6 h (35 (77%));

(d) B2pin2, Pd(dppf)Cl2·CH2Cl2, KOAc, dioxane, 110 ºC, 4 h (19e (36%)); (e) XPhos Pd G2,

K3PO4, dioxane/H2O, 80 ºC, overnight (36 (14%)); (f) Pd(dppf)Cl2, Na2CO3·H2O, dioxane/H2O, 95 ºC, overnight (38 (37%)); 1-methylpiperazine, RuPhos Pd G3, NaOtBu, dioxane, 95 ºC, overnight (39, 18%)).

2.2 Results and Discussion

2.2.1 Binding Mode of M4K2149 with ALK2

It has been disclosed by Mohedas and co-workers that the pyridyl nitrogen of LDN-213844 participates in a key hydrogen bond interaction with the backbone amide of H286 in the hinge region of ALK2 (Figure 8B).55 Our own crystallographic efforts led to the generation of a co- crystal structure of M4K2149 with the kinase in high resolution, which revealed that the same interaction had been preserved (PDB code 6T6D). Furthermore, the trimethoxyphenyl motif of LDN-213844 was reported to occupy a hydrophobic pocket of ALK2, where the meta-methoxy group participates in a water-mediated hydrogen bond with K235 (PDB code 4BGG).55 Substitution of the para-methoxy group of LDN-213844 with a primary amide results in the + establishment of a direct hydrogen bond between the carbonyl O of the amide and the NH3 group of K235 (Figure 8A). Additionally, the phenyl ring of M4K2149 stacks between G289 and 61 + V214, while the protonated piperazine NH2 is in close proximity to D293. This is suggestive of an electrostatic interaction. An intramolecular hydrogen bond between the amide NH2 and O of the ortho-methoxy substituent can also be observed in the co-crystal structure.

A M4K2149 B LDN-213844

Figure 8. (A) Co-crystal structure of M4K2149 (light yellow) with ALK2 (PDB code 6T6D). Hydrogen bonds are established with H286 and K235. The benzamide moiety of M4K2149 occupies a hydrophobic pocket (green) of ALK2 and is flanked by several hydrogen bond donating (blue; K235) and hydrogen bond accepting residues (red; D354 and E248). The protonated piperazine motif is in close proximity to D293, indicative of an electrostatic interaction. (B) Co- crystal structure of LDN-213844 (light yellow) with ALK2 (PDB code 4BGG). M4K2149 and LDN-213844 have similar modes of binding.

2.2.2 Optimizing Potency and Selectivity

The potency and selectivity of our analogues was assessed using a radioactive in vitro kinase assay, employing LDN-193189 as a control. To test the activity of the compounds in cells, a HEK293 cell-based NanoBRET assay from Promega was used. In this assay, the competitive displacement of a fluorescent tracer (PBI-6908) from the binding pocket of ALK2 by test compounds elicits reductions in BRET ratios, which are used to generate IC50 values. Cell-based potency against ALK5 was subsequently determined using a dual luciferase assay (DLA) in HEK293 cells.

Our initial SAR studies focused on varying the substitution pattern of the amide in order to determine if the group could interact with other residues in the pocket, such as D354 of the DLG motif or E248.61 Inverting the amide and methoxy substituents (14a) resulted in a complete loss of activity against ALK2 in the biochemical kinase assay. This correlated well with the results obtained by NanoBRET and DLA (Table 2). The biochemical potency of the regioisomer 14b was superior to 14a, however this did not translate into a significant improvement in cell-based potency. Replacement of the phenyl ring with 5-membered heterocycles gave rise to the thiophene analogues 7a-b and 8a, which were profiled to further investigate the effect that amide geometry had on potency and selectivity. A greater than 40-fold decrease in inhibitory activity against ALK2 was measured for 7a and 7b relative to M4K2149, while 8a was found to be completely inactive against the kinase in both assays. It became evident that positioning the primary amide on a six-membered aromatic ring para to the hinge-binding pyridyl core was critical for maintaining key binding interactions with ALK2.

The consideration of several structural parameters, such as cLogP, tPSA and number of HBD is pertinent in the design of small molecule inhibitors that must penetrate the BBB to exert their pharmacological effects. The ideal values that brain penetrant drugs should have vary between reviews.46 In the case of lipophilicity, the consensus is that substantial increases in cLogP should be avoided. Although increasing the lipophilicity of a drug typically enhances potency and permeability, concomitant increases in nonspecific tissue binding also occur, which would ultimately decrease the concentration of free drug at its intended site of action within the brain.46 The number of HBD that a molecule possesses, in addition to its tPSA, can also influence its ability to permeate the BBB. Increasing the value of either parameter also risks recognition by efflux transporters, such as P-gp.45

With these guidelines in mind, we sought to determine if mono- and di-N-methylation of the primary amide, as well as its cyclization to form the corresponding isoindolinone analogue, could be tolerated. The effects of these modifications were two-fold. In addition to decreasing the number of HBD, the tPSA was reduced to below 70 Å2 for 18a-b and 20a,62 which is in an optimal range for CNS penetrant drugs.46 These structural changes had only moderate effects on the calculated lipophilicity of the three analogues. Unfortunately, these compounds were discovered to be poorly active against ALK2 (Table 3). Consequently, we decided to incorporate only the primary amide motif in the rest of our analogues.

To determine if the methoxy group of M4K2149 was critical for maintaining potency, compound 20b was profiled. Removal of the methoxy substituent reduced inhibitory activity against ALK2 by 28-fold. This result lead us to suspect that the methoxy group oriented the amide into a conformation that was ideal for ALK2 binding. The incorporation of intramolecular hydrogen bonds and electrostatic interactions to mask HBD is a technique commonly employed to enhance brain penetration.45-46,48,63-64 In an attempt to exploit these interactions and probe for additional ones in the vicinity of the benzamide ring, we profiled compounds 20c and 20d, which featured a one-carbon homologation of the methoxy group and a bioisosteric replacement of the methoxy for a fluorine atom, respectively. Both modifications, however, failed to improve biochemical potency against ALK2.

Table 2: Inhibitory and off-target activities of 14a-b, 7a-b and 8a

NanoBRET DLA Cell-based ALK2 IC ALK5 IC Fold Compound R 50 50 Fold 1 (nM) (nM) Selectivity ALK2 IC50 ALK5 IC50 (nM) (nM) Selectivity

M4K2149 17 576 34 55a 704b 13

14a >1000 >5000 - 694 >5000 >7

14b 102 >5000 >49 588 >5000 >8

7a 751 >5000 >6 3371 >5000 >1

7b 712 >5000 >7 2811 >5000 >1

8a >1000 >5000 - >5000 >5000 -

aAverage of duplicate measurements. bAverage of triplicate measurements.

Table 3: Inhibitory and off-target activities of 8b-c, 18a-b, 20a-e and 31b

NanoBRET DLA Cell-based ALK2 IC ALK5 IC Fold Compound R 50 50 Fold 1 (nM) (nM) Selectivity ALK2 IC50 ALK5 IC50 (nM) (nM) Selectivity

M4K2149 17 576 34 55a 704b 13

18a >1000 >5000 - >5000 >5000 -

18b >1000 >5000 - >5000 >5000 -

20a 652 >5000 >7 2336 4810 2

20b 477 >5000 >10 2771 >5000 >1

20c 687 >5000 >7 3845 >5000 >1

20d 263 >5000 >19 1013 >5000 >4

8b 24 1920 80 81 3554 44

20e 9 2080 231 93 >5000a >54

31b 14 2210 158 38 4188 110

8c 5 46 9 178 216 1

aAverage of duplicate measurements. bAverage of triplicate measurements.

Incorporation of both a fluorine and methoxy substituent ortho to the amide gave rise to compound 8b, which not only had a biochemical ALK2 potency comparable to that of

M4K2149 (ALK2 IC50 = 24 nM), but also an improved selectivity profile over ALK5. These results correlated well with those obtained in the NanoBRET and DLA assays (Table 3). Exactly how the fluorine substituent contributes to this enhancement in selectivity has yet to be elucidated, although two possible explanations exist. We surmised that the electron-withdrawing nature of the fluorine atom decreases the ability of the carbonyl O to act as a hydrogen bond acceptor,64 thereby reducing the strength of intermolecular interactions that may be more critical for ligand binding to ALK5 than ALK2. The second possible explanation focuses on how the halogen substituent affects the conformation of the amide with respect to the phenyl ring. Planar topologies can be adopted by aromatic motifs with substituents capable of electron donation or withdrawal via resonance. This includes benzamide groups. However, ortho substituents, especially methyl and chloro substituents, can cause the dihedral angle between the amide carbonyl and phenyl ring of benzamide moieties to deviate from 0º.65-66 This is a direct consequence of the increased allylic strain ortho substituents incur in these motifs. Several reports in the literature have attributed potency differences to this type of conformational change. Prominent examples include EZH2 inhibitors comprised of a pyridone group connected to an aromatic ring via an amide linker (Figure 9).66-67 We postulated that a similar phenomenon could be occurring in the case of 8b and that this change in conformation induced by the fluorine substituent is better tolerated by ALK2 than ALK5.

Figure 9. Conformation of EZH2 inhibitor.66 Kung et al. reported that variation of the substituent ortho to the amide linker of their EZH2 inhibitors had drastic effects on potency. They

28 suspected that a dihedral angle of 50º between the phenyl ring and amide carbonyl was optimal for ligand binding to EZH2. Chlorine and methyl substituents provided the best potencies.

We decided to introduce a larger substituent ortho to the amide in order to confirm whether the latter hypothesis was true. Replacing the fluorine atom of 8b with a chlorine atom (20e) increased the biochemical selectivity over ALK5 to greater than 200-fold. This translated to a slight improvement in selectivity in the cell-based assay as well, however, NanoBRET ALK2

IC50 values were similar for both 8b and 20e, indicating that the modification has little impact on ALK2 potency. Incorporating an even larger bromine substituent (31b) did not appear to enhance selectivity any further than chlorine did in the biochemical assay, suggesting that there is a limit to which increasing the size of the halogen improves selectivity. Although it is enticing to ascribe these selectivity differences to the size of the ortho substituent, it is important to be cognizant of the fact that because the inhibitory activity of these compounds are in the nM range, small changes in ALK2 potency can cause drastic changes in fold-selectivity. It is possible that the true selectivity profiles of 8b, 20e and 31b are comparable.

To determine if electron-donating groups could be tolerated at this position as well, 8c was prepared. This analogue had the greatest structural similarity to our lead compound M4K2009. Although 8c was the most potent analogue in our series, it suffered from poor selectivity (biochemical selectivity of 9-fold over ALK5). Additionally, there was a substantial difference in its biochemical and cell-based potencies (biochemical ALK2 IC50 = 5 nM vs NanoBRET

ALK2 IC50 = 178 nM).

2.2.3 Caco-2 Studies

Having identified several potent analogues, we decided to focus our efforts on improving the pharmacokinetic (PK) profiles of two; 8b and 8c. In order to assess the permeability of these compounds, they were tested in a Caco-2 assay, which revealed that both analogues were poorly permeable and being recognized by efflux transporters (efflux ratios for both 8b and 8c were >30) (see Table 5 and Table A1). In an attempt to reduce efflux, the terminal piperazine nitrogens of 8b and 8c were capped with various alkyl groups to generate 1-methyl-, 1-isopropyl- and 1,2,6-trimethylpiperazine analogues (26a-f).68 In addition to reducing the number of HBD, methylation of the terminal piperazine nitrogen offered the additional advantage of attenuating pKa,62,69 which is often associated with a decrease in P-gp-mediated efflux.46 The rationale behind incorporating methyl groups at positions 1, 2 and 6 of the piperazine groups was to increase the molecular rigidity of analogues 26c and 26f. This is a strategy that is commonly employed to enhance brain penetration and oral bioavailability.46 As it has been reported that the piperazine motif of LDN-193189 is a metabolic liability,70 increasing the steric bulk around this group was also done to improve the inhibitors’ ADME profile in vivo.

As anticipated, the permeability (Papp_AB) of the 2-fluoro-6-methoxybenzamide analogues was increased from 0.3 x 10-6 cm/s (8b) to around 5.0 x 10-6 cm/s for 26a-c. This was accompanied by a concomitant reduction in the efflux ratio (from >30 for 8b to less than 3.0 for 26a and 26b) (Table 5). An enhancement in selectivity over ALK5 was also observed for these compounds. This was more pronounced in the biochemical kinase assay (Table 4). Unfortunately, similar results were not obtained for the 2,6-dimethoxybenzamide analogues (26d-f). Piperazine alkylation appeared to have little effect on reducing efflux (see Table A1). However, 26d-f demonstrated excellent inhibitory activity against ALK2 in the NanoBRET assay. Although M4K2149 and 8c differ by only one methoxy group, the latter analogue had a significantly higher efflux ratio (8.1 vs >30). We suspected that the extra electron-donating group was increasing the hydrogen bond acceptor potential of the amide carbonyl, which was potentially being recognized by one of the efflux transporters expressed by the Caco-2 cells.

Table 4: Inhibitory and off-target activities of 2-fluoro-6-methoxybenzamide and 2,6-

dimethoxybenzamide analogues, 26a-f

NanoBRET DLA Cell-based ALK2 IC ALK5 IC Fold Compound R R 50 50 Fold 1 2 (nM) (nM) Selectivity ALK2 IC50 ALK5 IC50 (nM) (nM) Selectivity

8b 24 1920 80 81 3554 44

26a 3 1050 350 93 4297a 46

26b 5a 2144a 429 52 3900a 75

26c 10 2910 291 100 >5000a >50

8c 5 46 9 178 216 1

26d <3b 75b - 19c 249d 13

26e 5 66 13 18 276a 15

26f 3 52 17 21 283a 13

aAverage of duplicate measurements. bAverage of triplicate measurements. cAverage of quadruplicate measurements. dAverage of quintuplicate measurement.

Because the affinity of the 2,6-dimethoxybenzamide warhead for ALK2 was one of the highest in the series, we decided to design another analogue, which had this motif installed onto a different chemical scaffold. Previous SAR studies at the OICR have shown that varying the length and size of the inhibitors could significantly alter their off-target activities. Docking studies of M4K2121 (which had been synthesized prior to my start date at the OICR) with ALK2 revealed that substitution of the 5-phenylpyridyl core for isoquinoline could potentially maintain critical interactions between the inhibitor and kinase (Figure 10). By fusing the 5-phenyl and pyridyl rings, the molecular weight of the analogue was reduced and the molecular flexibility decreased. Both of these modifications had the potential to enhance not only permeability in the Caco-2 assay, but we hypothesized that the efflux ratio would be affected (preferably attenuated) as well. The corresponding analogue 39 was quite potent, having a biochemical and cell-based

ALK2 IC50 of 26 nM. However, the selectivity over ALK5 was once again quite poor (biochemical ALK5/ALK2 fold selectivity = 13 and cell-based ALK5/ALK2 fold selectivity = 14). Given that the 2-fluoro-6-methoxy benzamide analogues had better structural and physicochemical properties, the 2,6-dimethoxybenzamide analogues were excluded from further profiling.

Figure 10. Docking of 39, an isoquinoline analogue of M4K2121, with ALK2. The isoquinoline analogue of 26d (39) was quite potent against ALK2, suggesting that critical

32 interactions between the ligand and kinase were maintained. However, the analogue suffered from poor selectivity over ALK5.

2.2.4 Pharmacokinetic Studies and Assessment of Off-target Activity

Prior to assessing the PK profiles of the 2-fluoro-6-methoxybenzamide analogues in vivo, the metabolic stabilities of 8b and 26a-c were evaluated in mouse and human liver microsomal (MLM and HLM) stability assays. All four analogues exhibited moderate to high stability in both in vitro assays, with compounds 8b and 26b demonstrating the highest degree of stability after a 60-minute incubation period at 37 ºC (>85% remaining) (see Table A2). Oral administration of a 10 mg/kg dose of 8b in female CB17 SCID mice (n = 3) gave rise to suboptimal values for Cmax (97 ng/mL), t1/2 (1.31 h) and AUCinf (296 ng·h/mL). Given the poor intrinsic permeability of 8b, these results were not surprising. A significant improvement in PK properties was observed for analogues 26a and 26b, which both yielded a greater than 17-fold increase in Cmax, 13-fold increase in AUCinf and a doubling of t1/2 (Table 5). The two analogues were also assessed for their ability to penetrate the BBB in the same strain of mice. Oral administration of these compounds at a 100 mg/kg dose gave rise to average total brain concentrations of 777 and 1595 ng/g and total brain-to-plasma ratios (B/P) of 0.178 and 0.132 for 26a and 26b, respectively. Although these values are moderate, the use of B/P ratios to assess brain permeability is generally not encouraged. The extent of brain penetration is typically evaluated based on the ratio of the unbound brain concentration to the unbound plasma 71 concentration (Kp,uu). Whether these analogues require additional modifications to enhance BBB permeability will ultimately depend on the value of this parameter.

Table 5: In vitro permeability and oral in vivo PK studies of 2-fluoro-6-methoxybenzamide analogues

PK in Female SCID Mice (10 mg/kg PO Caco-2 Permeability Assay dose) (n = 3) Compound R1 R2 P (10-6 P (10-6 AUC app_AB app_BA Efflux ratio C (ng/mL) inf t (h) cm/s cm/s) max (ng·h/mL) 1/2

8b 0.3 10.7 >30 97 296 1.31

26a 5.5 16.2 2.9 2140 4056 2.56

26b 5.7 15.1 2.6 1650 4630 2.61

26c 4.3 14.5 3.4 - - -

To ensure that a favourable hERG profile had been maintained for the 2-fluoro-6- methoxybenzamide analogues, their potencies against the hERG potassium channel were assessed using a HEK293 cell-based patch-clamp assay. 26a and 26b had optimal hERG IC50 values of >30 μM, while 26c was slightly more potent against the ion channel (IC50 = 19 μM) (see Table A3). To further investigate the off-target activity of these analogues, we profiled them in a 375-member kinase panel. At a concentration of 1 μM for each of the three compounds, fewer than 5% of the kinases showed a greater than 50% reduction in enzymatic activity. Excluding ALK1, 2, 3 and 6, the kinases ARAF, MAP4K4, MINK and TNK1 were the most sensitive to inhibition by 26a-c (see Table A4). We were also encouraged by the results obtained in an in vitro CYP inhibition assay, which showed that these analogues had negligible inhibitory activity (IC50 > 50 μM) against 7 CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) (see Table A3). Altogether, these results demonstrate that we were able to meet our

34 objective of designing selective and orally bioavailable inhibitors of ALK2. Furthermore, profiling 7 analogues against 3 ALK2 mutants (R206H, G328V and R258G) in a radioactive in vitro kinase assay revealed that the analogues had comparable potencies against both WT and mutant ALK2 (Table 6). These findings confirm that the benzamide inhibitors developed in this series have the potential to regulate aberrant BMP signaling in patients harboring these mutations.

Table 6: Inhibitory activity of benzamide analogues against WT and DIPG-linked mutant forms of ALK2

Compound

ALK2 (WT) 17 24 652 9 3 5 <3 IC50 (nM)

ALK2 (G328V) 5 4 171 <2 16 3 <2 IC50 (nM)

ALK2 (R206H) 9 8 226 5 19 6 4 IC50 (nM)

ALK2 (R258G) 9 7 363 4 17 6 <2 IC50 (nM)

2.2.5 Exploring Alternative Ortho Substituents

Having investigated how minor modifications to the M4K2149 scaffold can have significant effects on biological activity, we decided to return to one of our previously proposed hypotheses: that the electronic properties of the ortho substituent affect the hydrogen bond acceptor potential of the amide carbonyl oxygen and ultimately, influence both selectivity over ALK5 and P-gp

35 recognition. To test the validity of this argument, we prepared the methyl analogue 31a, which was to serve as a direct comparator to the chlorine analogue 36.

The bioisosteric exchange of methyl and chloro substituents in aromatic ring systems is commonly done in medicinal chemistry. Both motifs have comparable van der Waals radii and thus, impose similar steric constraints in chemical scaffolds.72 However, they differ substantially in terms of their electronic properties. Chlorine is inductively electron-withdrawing (Hammett constant σp = 0.23, F = 0.42, R = -0.19), while methyl groups are inductively electron-donating 64,73 (Hammett constant σp = -0.17, F = 0.01, R = -0.18). That the selectivity of 36 would exceed that of 31a was a plausible conjecture. However, this proved to not be the case. Both analogues had comparable potencies and selectivities as determined by the biochemical kinase assay, although the NanoBRET ALK5 IC50 of 36 was slightly greater than that of 31a (Table 7). If the chloro substituent imparts greater selectivity, it is not discernible. The permeability (Papp_AB) of 31a was also superior to that of the capped 2-fluoro-6-methoxybenzamide analogues. Its efflux ratio was even less than 2 (Table 8), suggesting that inductively electron-withdrawing substituents do not pose any significant advantage from a selectivity or permeability standpoint.

Expanding on how the electronic properties of the ortho substituent affect potency and selectivity, the trifluoromethyl (CF3) and difluoromethyl (CF2H) analogues 31c-f were prepared. Each functional group imparts a unique set of properties to their corresponding molecule. Unlike 64 fluorine, CF3 and CF2H groups cannot stabilize aromatic systems via π-resonance. As such, the

Hammett substituent constants (σp) for these two motifs are significantly larger than that of 73 fluorine. The CF3 motif was the largest substituent tested in this series. It has been reported to have a similar van der Waals volume as an ethyl group.74 We had originally hypothesized that due to its size and inductive electron-withdrawing nature, it would give rise to the most selective analogues. Unfortunately, 31c and 31d were not as active against ALK2 in the biochemical or cell-based assays in comparison to other lead amides.

CF2H groups represent an atypical class of hydrogen bond donors and are reputed to be lipophilic bioisosteres of OH groups.75 A study by Sessler and co-workers revealed the group’s ability to engage in intramolecular hydrogen bond interactions with ortho-substituted hydrogen bond acceptor moieties, specifically nitro groups, in aromatic ring systems.76 We speculated

36 whether an intramolecular shielding of the carbonyl oxygen of the primary amide of M4K2149 could be established via the introduction of a CF2H substituent on the benzamide ring. Such an interaction had the potential to make the compound more cell-permeable. The installation of the

CF2H group instead of a OH group provided the additional benefit of maintaining the tPSA within the recommended range for brain penetrant compounds.62 Both 31e and f were moderately active against ALK2 in the biochemical kinase assay. Their cell-based potencies were consistent with those of the halogenated compounds, although the capped piperazine analogue 31f was slightly less potent (Table 7). 31e and f were also quite selective. Once again, it can be observed that capping the terminal piperazine nitrogen significantly reduces efflux (compare 31e to 31f in

Table 8). 31f demonstrated excellent permeability (Papp_AB) in the Caco-2 assay as well.

Table 7: Inhibitory and off-target activities of 31a, 31c-f and 36

NanoBRET DLA Cell-based ALK2 IC ALK5 IC Fold Compound R R 50 50 Fold 1 2 (nM) (nM) Selectivity ALK2 IC50 ALK5 IC50 (nM) (nM) Selectivity

36 Me 10 2010 201 84 >10000 >119

31a Me 18 3280 182 59 6018 102

31c H 54 4920 91 305 >10000 >33

31d Me 45 >5000 >111 409 >10000 >24

31e H 22 4990 227 84 >10000 >119

31f Me 22 >5000 >227 127 >10000 >78

Table 8: Caco-2 permeability of 31a and 31e-f

Caco-2 Permeability Assay

Compound R1 R2 P (10-6 P (10-6 app_AB app_BA Efflux ratio cm/s cm/s)

31a Me 11.1 19.3 1.7

31e H 0.8 13.7 >10

31f Me 12.3 18.5 1.5

Chapter 3 Structure Activity Relationship of Conformationally Constrained Derivatives of M4K2009 Structure Activity Relationship of Conformationally Constrained Derivatives of M4K2009 3.1 Chemical Syntheses

The cyclic ether series was instigated by primarily generating the acyclic analogue 45. Coupling 3,5-dibromoisonicotinaldehyde (41) with (3,4,5-trimethoxyphenyl)boronic acid (42) gave rise to 43. Formation of 3,5-bis(3,4,5-trimethoxyphenyl)isonicotinaldehyde contributed to the lower yield obtained for 43, however, given that the starting materials were relatively cheap and that the reaction was easily scalable, high quantities of the desired product could be easily isolated. 43 was subjected to a second cross-coupling reaction with the commercially available boronic acid 25a after it was reduced to the common intermediate 44 using NaBH4. This cross-coupling step was completed and the final product (45) purified by PhD student, Hector Gonzalez- Alvarez. The coupling of 44 with 46 using a co-solvent mixture of dioxane and water hydrolyzed the benzyl bromide moiety of 46 in situ to afford the benzyl alcohol motif found in compound 47. The isolated yield of 47 was particularly low in step d due to a significant amount of 44 undergoing protodehalogenation (as determined by UV absorbance at 254 nm during tandem liquid chromatography/mass spectrometry (LCMS)). At a later point in the project, it was determined that protection of the benzylic alcohol of 44 by transforming it into the corresponding methoxy methyl ether failed to improve the yield of the cross-coupled product, in this case 48. As a result, 48 and 50 were never isolated and an alternative route to synthesize 50 will be devised by the medicinal chemists continuing the project at the OICR. An acid-mediated intramolecular condensation reaction generated the dihydrobenzo[5,6]oxepino[4,3-c]pyridine core of 49 from 47. Buchwald-Hartwig amination and deprotection of the Boc-protected piperazine, furnished 49 as the first cyclic analogue of the series.

Scheme 6: Synthesis of Compounds 45 and 49-50ɑ

ɑ Reagents and conditions: (a) Pd(dppf)Cl2, Na2CO3·H2O, dioxane/H2O, 100 ºC, 4 h (43 (56%));

(b) NaBH4, THF/MeOH, rt, 15 min (44 (71%)); (c) NaH, MOMCl, THF, rt, overnight (52%); (d)

Pd(amphos)Cl2, K2CO3, dioxane/H2O, 90 ºC, 3 h (47 (21%)); (e) HCl, THF/H2O, 90 ºC, 1 ½ d;

(f) 1-Boc-piperazine, Pd(OAc)2, rac-BINAP, CsCO3, dioxane, 130 ºC, 5 ½ h; (g) TFA, DCM, rt, 1 h (49 (9% over 3 steps)).

To determine the optimal ring size of the cyclic ether, analogues 56 and 64a-b were prepared. The 5-membered ring system was fully synthesized by the PhD student Hector Gonzalez- Alvarez. 3,5-dibromopyridin-4-ol (51) was coupled with the boronic acid 42 to afford 52, which underwent another cross-coupling reaction with (4-chloro-2-fluorophenyl)boronic acid (53) to yield the acyclic precursor 54. The fluoride substituent of 54 was a critical synthetic handle, which was rapidly displaced by the adjoining phenolic hydroxyl group via an intramolecular nucleophilic aromatic substitution (SNAr) reaction to generate the benzofuro[3,2-c]pyridine core. Buchwald-Hartwig amination of 55 afforded the final compound 56.

Scheme 7: Synthesis of Compounds 56ɑ

ɑ Reagents and conditions: (a) Pd(dppf)Cl2, Na2CO3·H2O, dioxane/H2O, 100 ºC, overnight; (b)

K2CO3, NMP, 100 ºC, overnight; (c) 1-methylpiperazine, RuPhos Pd G3, NaOtBu, dioxane, 90 ºC, 3 h.

The preparation of 64a and b followed similar synthetic routes. The common intermediate 57 was synthesized from 3,5-dibromoisonicotinaldehyde (41) and (4-chloro-2-fluorophenyl)boronic acid (53) in one step. Once again, formation of 3,5-bis(4-chloro-2- fluorophenyl)isonicotinaldehyde contributed to the lower yield obtained for 57. For the 7- membered ring system, homologation of the aldehyde substituent of 57 was accomplished via Wittig olefination using (methoxymethyl)triphenylphosphonium chloride, which preceded the hydrolysis of the enol ether under acidic conditions (route A, Scheme 8). The retention times of both the E and Z alkenes were identical by LCMS but the diastereomers could be detected by 1H

NMR. Cyclization was promoted once again via an intramolecular SNAr reaction after the aldehyde moieties of 57 and 60 were reduced and the corresponding alkoxides created. 61a was formed almost immediately upon the addition of sodium hydride to a solution of the alcohol precursor. This was not the case for the 7-membered cyclic analogue 61b. The same reaction took several days to go to completion at elevated temperatures. A significant portion of the starting material underwent β-elimination to form the olefin side-product 62,77 contributing to the very low yield obtained for 61b. Suzuki-Miyaura coupling of 61a-b with 42 furnished the penultimate intermediates 63a-b. Yields for the subsequent Buchwald-Hartwig amination were quite low as a substantial amount of 63a and b was transformed to the protodehalogenated side- products 65a-b.

Scheme 8: Synthesis of Compounds 64a-bɑ

ɑ Reagents and conditions: (a) Pd(dppf)Cl2, Na2CO3·H2O, dioxane/H2O, 90 ºC, overnight (57 (36%), 63a (47%), 63b (61%)); (b) (methoxymethyl)triphenylphosphonium chloride, NaHMDS,

THF, rt, 4 h (58 & 59 (66%)); (c) HCl, DCM/H2O, 80 ºC, 2 h (60 (88%)); (d) NaBH4, THF/MeOH, rt, 30 min; (e) NaH, THF, rt - 70 ºC, 10 min - 3 d (61a (107%), 61b (8%)); (f) 1- methylpiperazine, RuPhos Pd G3, NaOtBu, dioxane, 90 ºC, overnight (64a (37%), 64b (16%)).

3.2 Results and Discussion

3.2.1 Binding Mode of M4K2009 with ALK2

The discovery of M4K2009 was not serendipitous. Rather, the translocation of the methyl substituent from the C-2 position of the pyridyl core of LDN-214117 to the C-4 position was a mark of rational design. Replacing the C-4 hydrogen with a bulkier aliphatic group introduces steric strain between the methyl substituent and the aromatic hydrogens of the adjoining phenyl rings. We anticipated that the dihedral angle between both the pyridyl and 4-piperazinylphenyl rings and the pyridyl and trimethoxyphenyl rings would shift from 0º in order to minimize steric clash and that M4K2009 would potentially adopt a configuration reminiscent of the biologically active conformer (Figure 11).

Figure 11. Altering the 3D configuration of the 3,5-diarylpyridine inhibitors of ALK2. The introduction of a methyl substituent at C-4 of the pyridyl ring shifts the dihedral angle of the adjoining aromatic rings from 0º.

Co-crystal images of M4K2009 with ALK2 confirmed the plausibility of our approach. It can be seen in Figure 12 that the modes of binding of M4K2009 and LDN-213844 are almost identical. As observed with other 3,5-diarylpyridine inhibitors of ALK2, M4K2009 forms the quintessential hydrogen bond with the backbone amide of H286 in the hinge region of the kinase. The methoxy groups also participate in a water-mediated bond with K235. On closer inspection, it can be observed that the analogue adopts a non-planar, almost U-shaped, configuration. A dihedral angle of approximately 43º was calculated between the pyridyl and 4-piperazinylphenyl rings.

A B

Figure 12. (A) Co-crystal structure of M4K2009 (dark blue) with ALK2 (PDB code 6SZM). A direct hydrogen bond is established with H286 and a water-mediated hydrogen bond is established with K235 (not shown). The trimethoxy moiety of M4K2009 occupies a hydrophobic pocket (green) of ALK2 and is flanked by several hydrogen bond donating (blue; K235) and hydrogen bond accepting residues (red; D354 and E248). (B) The dihedral angle between the pyridyl and 4-piperazinylphenyl rings deviates from 0º suggesting that M4K2009 adopts a non-planar configuration when bound to the hinge region of ALK2.

Although X-ray crystallographic images are merely snapshots of ligand binding and fail to provide a complete comprehensive view of protein dynamics as well as the complex intermolecular interactions that occur between a ligand and its target, we were fairly confident that the conformational change induced by the methyl group was responsible for the improvement in the biological activity of M4K2009. We decided to rigidify the molecule into its biologically active configuration by reducing the rotational freedom about the pyridyl and 4- piperazinylphenyl bond.

Developing conformationally constrained analogues is a strategy that is commonplace in medicinal chemistry. Numerous drug discovery programs have adopted such an approach. Inhibitors of bacterial type II DNA topoisomerase, EZH2, viral RNA polymerase as well as small molecule correctors of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein have been developed with internal covalent restraints incorporated.66, 78-80 Rigidifying small molecule inhibitors can offer several advantages. Such a technique has the potential to enhance potency by mitigating the entropic penalty associated with ligand binding to its protein target.81 Additionally, reducing the number of rotatable bonds, at the cost of increasing molecular weight, could conceivably augment permeability, which is crucial for the development of brain penetrant inhibitors.46

3.2.2 Optimizing Potency and Selectivity

The analogues in this series were similarly tested in the radioactive in vitro kinase assay and NanoBRET and DLA assays used for assessing the potency and selectivity of the amides presented in Chapter 2. Scaffold exploration was initiated with the development of the 7- membered cyclic analogue 49, which featured a dihydrobenzo[5,6]oxepino[4,3-c]pyridine core.

The incorporation of this three-atom linker had discernible effects on the biological activity of M4K2009. 49 was more potent against ALK2 in the biochemical kinase assay, however, there was a marked reduction in selectivity over ALK5. These trends corresponded well with those observed in the NanoBRET assay (Table 9). The near 4-fold enhancement in the cell-based potency of 49 suggests that reducing the molecular flexibility of M4K2009 may augment permeability, although this needs to be verified through Caco-2 permeability testing.

In light of the fact that the bridging of the pyridyl and 4-piperazinylphenyl moieties can offer a significant advantage from the standpoint of binding affinity, we were prompted to further investigate the effects that ring size and heteroatom substitution had on potency, as well as selectivity over ALK5.

To determine the optimal dihedral angle between the two rings, the length of the tether conjoining them was varied to generate the 5-membered (56), 6-membered (64a) and 7- membered (64b) ring systems. All three analogues contained an oxygen atom positioned at C-2 of the 4-piperazinylphenyl ring. It was synthetically challenging to generate analogues which did not incorporate heteroatoms at this position.

Table 9: Inhibitory and off-target activities of M4K2009 and 49

ALK2 Inhibitor

M4K2009 49

Biochemical ALK2 IC 50 13 3 (nM)

Biochemical ALK5 IC 50 1830 89 (nM)

Fold selectivity 141 30 (ALK5/ALK2)

NanoBRET 49 15 ALK2 IC50 (nM)

Cell-based Fold Selectivity 44 20 (ALK5/ALK2)

Table 10: Inhibitory and off-target activities of 56, 64a and 64b

Dihedral ALK2 IC ALK5 IC Fold Compound n 50 50 angle θ (o)a (nM) (nM) Selectivity

2009 - - 13 1830 141

56 0 0.7 >1000 >5000 -

64a 1 14.4 24 1490 62

64b 2 42.8 5 618 124

aDihedral angles were calculated by Dr. Lisa Rooney from the Institute of Cancer Research. Geometry optimisation was done using Schrodinger Jaguar, M06-2X-D3/6-31G** density functional theory.

Given that the dihedral angle between the pyridyl and 4-piperazinylphenyl rings was suspected to be around 0º for the 5-membered ring system,78-79 we hypothesized that 56 would be weakly active against ALK2. Although the ALK2 IC50 of 56 was >1000 nM in the biochemical kinase assay, it should be noted that the compound was found to be poorly soluble in DMSO. Taking into account the planarity of the molecule, it was to be expected that there would be issues with respect to the solubility of the compound in both organic and aqueous solvent mixtures. This makes it difficult to interpret the biochemical data. Therefore, the results for this compound should not be taken at face value.

One-carbon homologation of the cyclic ether significantly altered both the structural and physicochemical properties of the molecule, as 64a was determined to be completely soluble in DMSO. The change in conformation conferred by the expansion of the ring system by a single

CH2 unit increased the inhibitory activity of the compound considerably (Table 10). The selectivity of 64a over ALK5 was also moderate. The 7-membered analogue 64b, however, was one of the most potent compounds in the series with an ALK2 IC50 of 5 nM. Intriguingly, the fold selectivity of 64b was appreciably higher than that of 49 for reasons unbeknownst to us. It is possible that the oxygen atom of the dihydrobenzo[5,6]oxepino[4,3-c]pyridine core of 49 engages in additional interactions with ALK5, making the analogue more potent against the kinase and correspondingly less selective for its primary target. Although 50 would serve as a better comparator to 64b, it is evident that the selectivity of the 7-membered analogues can be modulated by altering the location of the oxygen atom within the linker.

In this miniseries, we not only identified the optimal size of the tether joining the pyridyl and 4- piperazinylphenyl rings of M4K2009, but we also determined that the selectivity of the analogue over ALK5 could be enhanced by changing the position of the oxygen heteroatom within the bridging tether. The weaker inhibitory activity of the acyclic analogue 45 (biochemical ALK2

IC50 = 73 nM) indicates that rigidifying M4K2009 into its biologically active configuration offers several advantages.

47 48

Chapter 4 Conclusions and Future Studies Conclusions and Future Studies

Advances in the development of effective chemotherapeutic agents for the treatment of DIPG have been limited. This is in part due to the convoluted genomic signatures of DIPG, which has made our understanding of its pathogenesis difficult. Recent identification of ALK2 as a target for therapeutic intervention has prompted the emergence of several classes of type I kinase inhibitors. The work presented herein featured two distinct SAR studies based on the 3,5- diarylpyridine inhibitor LDN-214117. Enhancing the physiochemical and biochemical properties of M4K2149, a potent benzamide analogue with an attenuated affinity for the hERG potassium channel, was a major focus of this project. We determined that we could tailor the selectivity of our analogues over ALK5 by altering substituents at a position ortho to the amide group of M4K2149. We were also able to address issues of permeability by capping the NH of the solvent-exposed piperazine group. The 2-fluoro-6-methoxybenzamide derivatives 26a-c are an excellent demonstration of how small structural modifications can drastically influence PK properties. The co-crystal structure of M4K2149 with ALK2 helped us rationalize potency differences between analogues in the series and highlighted structural motifs that were crucial for maintaining key interactions with the protein. Despite these optimizations, total brain-to-plasma ratios are inadequate for accurately assessing the pharmacological activity of 26a-b in the brain.

Measuring the unbound brain concentrations (Cb,u) of these analogues in vivo is therefore warranted. Caco-2 permeability testing of the methyl and difluoromethyl analogues 31a and 31f showed favorable permeability and efflux ratios. In fact, 31a met the target values from the standpoint of potency, selectivity over ALK5 and Caco-2 permeability. Further investigation regarding how this compound performs in vivo and whether it poses the risk of hERG blockade should be conducted in the future.

Developing conformationally constrained analogues of the lead compound M4K2009 was the second focus of this project. We were able to determine that a 7-membered ring system, which incorporated a 3-atom tether between the pyridyl and 4-piperazinylphenyl rings was optimal for potency and that the selectivity of the analogue could be modulated by altering the location of the oxygen heteroatom within the linker. Once again, understanding the binding geometry of

M4K2009 with ALK2 via the analysis of co-crystal images enabled the rational design of rigidified analogues with greater binding affinities for the kinase than their acyclic counterparts. Future studies will assess whether reducing the molecular flexibility of our lead compound increases its ability to penetrate both intestinal and CNS barriers. We plan on assessing the permeability of 64b in our Caco-2 assay in the next few months.

In summary, compounds presented herein represent new chemotypes possessing high inhibitory activity against ALK2. They have the potential to deepen our understanding of the biology of DIPG and will hopefully pave the way for future chemotherapies.

Chapter 5 Synthetic Procedures and Compound Characterizations Synthetic Procedures and Compound Characterizations 5.1 Chemical Syntheses

All reagents were purchased from commercial vendors and used without further purification. Volatiles were removed under reduced pressure by rotary evaporation or by using the V-10 solvent evaporator system by Biotage®. Very high boiling point (6000 rpm, 0 mbar, 56 oC), mixed volatile (7000 rpm, 30 mbar, 36 ºC) and volatile (6000 rpm, 30 mbar, 36 oC) methods were used to evaporate solvents. The yields given refer to chromatographically purified and spectroscopically pure compounds unless indicated otherwise. Compounds were purified using a Biotage Isolera One system by normal phase chromatography using Biotage® SNAP KP-Sil or Sfär Silica D columns (Part No.: FSKO-1107/FSRD-0445) or by reverse-phase chromatography using Biotage® SNAP KP-C18-HS or Sfär C18 D columns (Part No.: FSLO-1118/FSUD-040). If additional purification was required, compounds were purified by solid phase extraction (SPE) using Biotage Isolute Flash SCX-2 cation exchange cartridges (Part No.: 532-0050-C and 456- 0200-D). Products were washed with 2 cartridge volumes of MeOH and eluted with a solution of

MeOH and NH4OH (9:1 v/v). Preparative chromatography was carried out using a Waters 2767 injector with the collector attached to PDA UV/Vis and SQD mass detectors. An XSelect CSH Prep C18 5µm OBD 19 mm x 100 mm (Part No.: 186005421) or Xselect CSH Prep C18 5µm 10 mm x 100 mm (Part No.: 186005415) column was used for purification. Final compounds were TM TM 1 dried using the Labconco Benchtop FreeZone Freeze-Dry System (4.5 L Model). H and proton-decoupled 19F NMRs were recorded on a Bruker Avance-III 500 MHz spectrometer at ambient temperature. 13C NMRs were recorded at Lash Miller Chemical Laboratories. Residual protons of CDCl3, DMSO-d6 and CD3OD solvents were used as internal references. Spectral data are reported as follows: chemical shift (δ in ppm), multiplicity (br = broad, s = singlet, d = doublet, dd = doublet of doublets, m = multiplet), coupling constants (J in Hz) and proton integration. Compound purity was determined by UV absorbance at 254 nm during tandem liquid chromatography/mass spectrometry (LCMS) using a Waters Acquity separations module. All final compounds had a purity of ≥95% as determined using this method. Low resolution mass spectrometry (LRMS) was conducted in positive ion mode using a Waters

Acquity SQD mass spectrometer (electrospray ionization source) fitted with a PDA detector. Mobile phase A consisted of 0.1% formic acid in water, while mobile phase B consisted of 0.1% formic acid in acetonitrile. One of three types of columns were used: Column 1: Acquity UPLC CSH C18 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186005296), Column 2: Acquity UPLC BEH C8 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186002877) or Column 3: Acquity UPLC HSS T3 (2.1 x 50 mm, 100 Å, 1.8 µm. Part No. 186003538). For columns 1 and 2, the gradient went from 90% to 5% mobile phase A over 1.8 min, maintained at 5% for 0.5 min, then increased to 90% over 0.2 min for a total run time of 3 min. For column 3, the gradient went from 98% to 5% mobile phase A over 1.8 min, maintained at 5% for 0.5 min, then increased to 98% over 0.2 min for a total run time of 3 min, as well. The flow rate was 0.4 mL/min throughout both runs. All columns were used with the temperature maintained at 25 °C. High resolution mass spectrometry was conducted using a Waters Synapt G2-S quadrupole-time-of-flight (QTOF) hybrid mass spectrometer system coupled with an Acquity ultra-performance liquid chromatography (UPLC) system. Chromatographic separations were carried out on an Acquity UPLC CSH C18 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186005296), Acquity UPLC BEH C8 (2.1 x 50 mm, 130 Å, 1.7 µm. Part No. 186002877) or Acquity UPLC HSS T3 (2.1 x 50 mm, 100 Å, 1.8 µm. Part No. 186003538). The mobile phases were 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Leucine Enkephalin was used as lock mass. MassLynx 4.1 was used for data analysis.

Tert-butyl 4-(4-(5-bromo-4-methylpyridin-3-yl)phenyl)piperazine-1-carboxylate (3) A solution of tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine-1- carboxylate (1) (1.549 g, 3.99 mmol), 3,5-dibromo-4-methylpyridine (2) (0.953 g, 3.80 mmol), [1,12-bis(diphenylphosphino)ferrocene]dichloropalladium(II)·DCM complex (0.310 g, 0.38 mmol) and sodium carbonate monohydrate (1.414 g, 11.40 mmol) in 1,4-dioxane (16.3 mL) and water (2.7 mL) was heated to 85 °C and stirred overnight. The reaction mixture was concentrated under reduced pressure prior to dilution with water (30 mL) and extraction with

EtOAc (3 x 30 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated under reduced pressure to yield a brown oil. The crude material was purified by silica gel chromatography (0-50% EtOAc in hexanes) to afford a white solid (0.770 g, 47% 1 yield). H NMR (500 MHz, CDCl3) δ 8.61 (s, 1H), 8.31 (s, 1H), 7.20 (d, J = 8.6 Hz, 2H), 6.99 (d,

J = 8.8 Hz, 2H), 3.63 – 3.58 (m, 4H), 3.25 – 3.18 (m, 4H), 2.36 (s, 3H), 1.49 (s, 9H). MS (ESI): + + m/z = 432.31 [M + H] , 434.38 [M + H] + 2. (tR = 2.40 min).

(5-(4-(4-(Tert-butoxycarbonyl)piperazin-1-yl)phenyl)-4-methylpyridin-3-yl)boronic acid (4a) A solution of 3 (171 mg, 0.396 mmol), bis(pinacolato)diboron (201 mg, 0.792 mmol), [1,12- bis(diphenylphosphino)ferrocene]dichloropalladium(II)·DCM complex (32 mg, 0.040 mmol) and potassium acetate (78 mg, 0.792 mmol) in 1,4-dioxane (4 mL) was microwaved at 110 °C for 4 h. The mixture was transferred to a 15 mL Falcon tube and centrifuged for 1 min at 4000 rpm. The dark brown supernatant was used without further purification in subsequent reactions (190 mg, 57% yield as determined by LCMS). MS (ESI): m/z = 397.90 [M + H]+.

5-Chloro-4-methoxythiophene-3-carboxamide (5c) The title compound was prepared according to a modified literature procedure.82 To a solution of 5-chloro-4-methoxythiophene-3-carboxylic acid (100 mg, 0.519 mmol) in DCM (1.5 mL) was added ammonium chloride (33 mg, 0.623 mmol), HATU (237 mg, 0.623 mmol) and DIPEA (0.27 mL, 1.558 mmol). The reaction mixture was stirred at room temperature for 3 h. Volatiles were removed under reduced pressure and the crude product was loaded onto Celite and purified by silica gel chromatography (0-100% EtOAc in hexanes) to afford the final compound (71 mg, 1 72% yield). H NMR (500 MHz, CDCl3) δ 7.91 (s, 1H), 7.21 (br s, 1H), 5.72 (br s, 1H), 4.06 (s, 3H). MS (ESI): m/z = 192.27 [M + H]+, 194.28 [M + H]+ + 2.

4-Bromo-2-fluoro-6-methoxybenzamide (5d) The title compound was prepared according to modified literature procedures.83-84 A solution of 4-bromo-2-fluoro-6-methoxybenzonitrile (22a) (5.00 g, 21.7 mmol) in EtOH (100 mL) was cooled in an ice bath prior to the addition of an aqueous solution of sodium hydroxide (0.43 M, 65 mL). This was followed by the addition of hydrogen peroxide (30 wt. % solution in water) (27 mL). The solution was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure prior to dilution with water (250 mL) and extraction with

EtOAc (3 x 250 mL). The combined organic extracts were dried over Na2SO4, filtered and

53 concentrated under reduced pressure to give the final product (4.63 g, 80% yield). 1H NMR (500 MHz, DMSO) δ 7.84 (br s, 1H), 7.58 (br s, 1H), 7.17 (d, J = 8.5 Hz, 1H), 7.13 (s, 1H), 3.82 (s, 3H). 19F NMR (471 MHz, DMSO) δ -115.03. MS (ESI): m/z = 248.20 [M + H]+, 250.27 [M + + H] + 2. (tR = 1.40 min).

4-Bromo-2,6-dimethoxybenzamide (5e) The title compound was synthesized according to the procedure described for 5c from 4-bromo- 2,6-dimethoxybenzoic acid (157 mg, 0.600 mmol) using ammonium chloride (38 mg, 0.708 mmol), HATU (269 mg, 0.708 mmol) and DIPEA (0.31 mL, 1.782 mmol). The reaction was stirred at room temperature overnight. The crude was diluted with water and the aqueous layer extracted three times with EtOAc. The combined organic extracts were dried over MgSO4, filtered and concentrated in vacuo prior to loading onto Celite and purifying by silica gel chromatography (10-100% EtOAc in hexanes). The final product was a white solid (100 mg, 64% yield). 1H NMR (500 MHz, DMSO) δ 7.51 (br s, 1H), 7.23 (br s, 1H), 6.87 (s, 2H), 3.75 (s, + + 6H). MS (ESI): m/z = 260.35 [M + H] , 262.29 [M + H] + 2. (tr = 1.48 min).

The title compound was alternatively synthesized according to the procedure described for 5d from 4-bromo-2,6-dimethoxybenzonitrile (22b) (968 mg, 4.00 mmol), hydrogen peroxide (30 wt. % solution in water) (9.8 mL) and an aqueous solution of sodium hydroxide (2 M, 25.0 mL). The reaction mixture was heated to 110 oC for 8 h. The solvents were evaporated and the crude material was suspended in water, filtered and dried under high vacuum to afford a white crystalline solid (903 mg, 87% yield).

Tert-butyl 4-(4-(5-(4-methoxy-5-(methoxycarbonyl)thiophen-2-yl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (6a) A solution of 4a (80 mg, 0.167 mmol), methyl 5-bromo-3-methoxythiophene-2-carboxylate (42 mg, 0.167 mmol) (5a), [1,12-bis(diphenylphosphino)ferrocene]dichloropalladium(II)·DCM complex (14 mg, 0.017 mmol) and sodium carbonate monohydrate (62 mg, 0.501 mmol) in 1,4- dioxane (2.9 mL) and water (0.48 mL) was heated to 100 oC and stirred for 2 h. The reaction mixture was adsorbed onto Celite and the volatiles were removed under reduced pressure. The

54 crude product was purified by silica gel chromatography (0-100% EtOAc in hexanes) to afford the final compound (45 mg, 50% yield). MS (ESI): m/z = 524.70 [M + H]+.

Tert-butyl 4-(4-(5-(4-methoxy-5-(methoxycarbonyl)thiophen-3-yl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (6b) The title compound was synthesized according to the procedure described for 6a from 4a (80 mg, 0.167 mmol) and methyl 4-bromo-3-methoxythiophene-2-carboxylate (5b) (42 mg, 0.167 mmol). The final product was a light yellow powder (47 mg, 52% yield). MS (ESI): m/z = 524.70 [M + H]+.

3-Methoxy-5-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)thiophene-2-carboxamide (7a) A 5 mL MW vial was charged with 6a (20 mg, 0.038 mmol). The material was dissolved in a solution of ammonia in methanol (7N, 4.0 mL). The vial was sealed and the solution was stirred at 90 oC for 3 days. Volatiles were removed under reduced pressure and the crude material was loaded onto Celite and purified by silica gel chromatography (0-100% EtOAc in hexanes). The purified product was dissolved in DCM (1 mL) and treated with trifluoroacetic acid (0.1 mL, 1.146 mmol). The solution was stirred overnight. The product was purified by SPE. Drying under high vacuum overnight afforded an off-white powder (7.8 mg, 47% yield). 1H NMR (500 MHz, DMSO) δ 8.37 (s, 1H), 8.36 (s, 1H), 7.76 (s, 1H), 7.66 (br s, 1H), 7.31 – 7.25 (m, 3H), 7.05 (d, J = 8.7 Hz, 2H), 3.51 (s, 3H), 3.22 – 3.20 (m, 4H), 2.98 – 2.94 (m, 4H), 2.13 (s, 3H). + HRMS (ESI) for C22H24N4O2S [M + H] : m/z = calcd, 409.1693; found, 409.1691. (tR = 0.99 min).

3-Methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)thiophene-2-carboxamide (7b) The title compound was synthesized according to the procedure described for 7a from 6b (20 mg, 0.038 mmol). The final product was an off-white powder (8.9 mg, 53% yield). 1H NMR (500 MHz, DMSO) δ 8.38 (s, 1H), 8.36 (s, 1H), 7.76 (s, 1H), 7.66 (br s, 1H), 7.30 – 7.26 (m, 3H), 7.04 (d, J = 8.7 Hz, 2H), 3.52 (s, 3H), 3.19 – 3.17 (m, 4H), 2.95 – 2.91 (m, 4H), 2.13 (s,

+ 3H). HRMS (ESI) for C22H24N4O2S [M + H] : m/z = calcd, 409.1693; found, 409.1689. (tR = 0.99 min).

4-Methoxy-5-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)thiophene-3-carboxamide (8a) A solution of 4a (60 mg, 0.125 mmol), 5c (20 mg, 0.104 mmol), XPhos Pd G2 (8 mg, 0.010 mmol) and potassium phosphate tribasic (44 mg, 0.209 mmol) in 1,4-dioxane (1.8 mL) and water (0.30 mL) was heated to 100 oC and stirred for 3 h. The reaction mixture was adsorbed onto Celite and volatiles were removed under reduced pressure. The crude product was purified by silica gel chromatography (0-100% EtOAc in hexanes). Further purification was carried out by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The product was dissolved in DCM (1 mL) and treated with trifluoroacetic acid (0.48 mL, 6.26 mmol). The solution was stirred for 1 h. The product was purified by SPE. Freeze drying for 3 days afforded an off-white powder (6.5 mg, 15% yield). 1H NMR (500 MHz, MeOD) δ 8.46 (s, 1H), 8.36 (s, 1H), 8.18 (s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.7 Hz, 2H), 3.63 (s, 3H), + 3.44 – 3.40 (m, 4H), 3.29 – 3.27 (m, 4H), 2.25 (s, 3H). HRMS (ESI) for C22H24N4O2S [M + H] : m/z = calcd, 409.1693; found, 409.1694. (tR = 1.06 min).

2-Fluoro-6-methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (8b) The title compound was synthesized according to the procedure described for 8a from 4a (77 mg, 0.161 mmol) and 5d (40 mg, 0.161 mmol). The final product was an off-white powder (20 mg, 29% yield). 1H NMR (500 MHz, DMSO) δ 8.38 (s, 1H), 8.34 (s, 1H), 7.89 (br s, 1H), 7.58 (br s, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.98 – 6.95 (m, 2H), 3.85 (s, 3H), 3.19 – 3.15 (m, 4H), 2.94 – 2.89 (m, 4H), 2.19 (s, 3H). 19F NMR (471 MHz, DMSO) δ -116.73. 13 C NMR (126 MHz, CD3OD) δ 168.10, 160.82 (d, J = 247.6 Hz), 159.01 (d, J = 8.4 Hz), 152.56, 149.64, 147.95, 144.90, 142.82 (d, J = 10.2 Hz), 140.08, 138.82 (d, J = 2.2 Hz), 131.35, 129.98, 117.22, 115.57 (d, J = 20.8 Hz), 110.24 (d, J = 23.0 Hz), 109.74 (d, J = 2.7 Hz), 57.02, + 49.98, 46.12, 18.35. HRMS (ESI) for C24H25FN4O2 [M + H] : m/z = calcd, 421.2034; found,

421.2040. (tR = 1.05 min).

2,6-Dimethoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (8c)

The title compound was synthesized according to the procedure described for 8a from 4a (190 mg, 0.396 mmol) and 5e (82 mg, 0.317 mmol). The final product was an off-white powder (45 mg, 32% yield over 3 steps). 1H NMR (500 MHz, DMSO) δ 8.36 (s, 1H), 8.35 (s, 1H), 7.57 (br s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.23 (br s, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.72 (s, 2H), 3.78 (s, 6H), 3.19 – 3.15 (m, 4H), 2.93 – 2.89 (m, 4H), 2.20 (s, 3H). 13C NMR (126 MHz, DMSO) δ 166.32, 156.24, 150.51, 148.48, 147.49, 141.63, 139.43, 137.59, 137.36, 130.13, 127.79, 116.37, + 115.11, 105.65, 55.89, 47.87, 44.76, 18.09. HRMS (ESI) for C25H28N4O3 [M + H] : m/z = calcd,

433.2234; found, 433.2228. (tR = 0.99 min).

5-(5-Chloro-4-methylpyridin-3-yl)-2-methoxybenzoic acid (11a) The title compound was synthesized according to the procedure described for 6a from 3-bromo- 5-chloro-4-methylpyridine (10) (41 mg, 0.200 mmol) and 5-borono-2-methoxybenzoic acid (9a) (39 mg, 0.200 mmol). DMF (1.6 mL) and water (0.43 mL) were used as the solvents. The crude material was used without purification in the subsequent cross-coupling reaction (56 mg, 87% yield by LCMS). MS (ESI): m/z = 278.30 [M + H]+, 280.30 [M + H]+ + 2.

3-(5-Chloro-4-methylpyridin-3-yl)-5-methoxybenzoic acid (11b) The title compound was synthesized according to the procedure described for 6a from 3-bromo- 5-chloro-4-methylpyridine (10) (41 mg, 0.200 mmol) and 3-carboxy-5-methoxyphenylboronic acid (9b) (39 mg, 0.200 mmol). DMF (1.6 mL) and water (0.43 mL) were used as the solvents. The crude material was used without purification in the subsequent cross-coupling reaction (56 mg, 90% yield by LCMS). MS (ESI): m/z = 278.23 [M + H]+, 280.30 [M + H]+ + 2.

5-(5-(4-(4-(Tert-butoxycarbonyl)piperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-2- methoxybenzoic acid (13a) The title compound was synthesized according to the procedure described for 8a from 11a (56 mg, 0.200 mmol) and 1 (140 mg, 0.360 mmol). The crude product was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The purified intermediate was used immediately in the subsequent reaction.

3-(5-(4-(4-(Tert-butoxycarbonyl)piperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-5- methoxybenzoic acid (13b) The title compound was synthesized according to the procedure described for 8a from 11b (56 mg, 0.200 mmol) and 1 (140 mg, 0.360 mmol). The crude product was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The purified intermediate was used immediately in the subsequent reaction.

2-Methoxy-5-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (14a) The title compound was synthesized according to the procedure described for 5c from 13a. Deprotection with trifluoroacetic acid (0.46 mL, 6.00 mmol), purification by SPE and freeze drying for 2 days afforded the final product as a yellow powder (6 mg, 7% yield over 4 steps). 1H NMR (500 MHz, DMSO) δ 8.32 (s, 1H), 8.29 (s, 1H), 7.81 (d, J = 2.1 Hz, 1H), 7.71 (br s, 1H), 7.59 (br s, 1H), 7.56 (dd, J = 8.5, 2.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H), 3.95 (s, 3H), 3.21 – 3.18 (m, 4H), 2.97 – 2.93 (m, 4H), 2.14 (s, + 3H). HRMS (ESI) for C24H26N4O2 [M + H] : m/z = calcd, 403.2129; found, 403.2126. (tR = 1.05 min).

3-Methoxy-5-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (14b) The title compound was synthesized according to the procedure described for 5c from 13b. Deprotection with TFA (0.46 mL, 6.00 mmol), purification by reverse-phase chromatography and SPE and freeze drying for 2 days afforded the final product as an off-white powder (4 mg, 6% yield over 4 steps). 1H NMR (500 MHz, DMSO) δ 8.36 (s, 1H), 8.35 (s, 1H), 8.02 (br s, 1H), 7.52 – 7.49 (m, 1H), 7.49 – 7.47 (m, 1H), 7.44 (br s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.17 – 7.15 (m, 1H), 7.05 (d, J = 8.7 Hz, 2H), 3.85 (s, 3H), 3.23 – 3.20 (m, 4H), 2.99 – 2.95 (m, 4H), + 2.15 (s, 3H). HRMS (ESI) for C24H26N4O2 [M + H] : m/z = calcd, 403.2129; found, 403.2137.

(tR = 1.07 min).

Tert-butyl 4-(4-(5-chloro-4-methylpyridin-3-yl)phenyl)piperazine-1-carboxylate (15) The title compound was synthesized according to the procedure described for 6a from 10 (320 mg, 1.550 mmol) and 1 (722 mg, 1.860 mmol). The final product was an off-white crystalline 1 solid (510 mg, 85% yield). H NMR (500 MHz, CDCl3) δ 8.48 (s, 1H), 8.30 (s, 1H), 7.21 (d, J =

8.7 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), 3.63 – 3.59 (m, 4H), 3.24 – 3.19 (m, 4H), 2.33 (s, 3H), + + 1.49 (s, 9H). MS (ESI): m/z = 388.56 [M + H] , 390.57 [M + H] + 2. (tR = 2.38 min).

Tert-butyl 4-(4-(5-(3-methoxy-4-(methoxycarbonyl)phenyl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (17) The title compound was fully synthesized by Dr. David Smil at the OICR. The title compound was synthesized according to the procedure described for 8a from 15 (120 mg, 0.309 mmol) and 3-methoxy-4-methoxycarbonylphenylboronic acid, pinacol ester (16) (90 mg, 0.309 mmol). The solvents used were butan-1-ol (2.0 mL) and water (0.48 mL). The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic fractions were dried over Na2SO4, filtered and concentrated under reduced pressure to afford a light beige solid (160 mg, 99% yield), which was used without further purification in the subsequent reaction. MS (ESI): m/z = 518.57 [M + H]+.

Tert-butyl 4-(4-(5-(4-carbamoyl-3-methoxyphenyl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (M4K2149) The title compound was fully synthesized by Dr. David Smil at the OICR. To a solution of 17 (0.160 g, 0.309 mmol) in MeOH (3.0 mL) at room temperature was added a solution of ammonia in MeOH (7N) (4.4 mL). The resulting mixture was heated to 75 °C for 3 days prior to cooling back down to room temperature, removing all solvents under reduced pressure, and triturating the residue from EtOAc with hexanes. The beige precipitate was collected by filtration and washed with hexanes. The product was subsequently dissolved in MeOH (5.0 mL) and treated with HCl (4.0 M in dioxane, 1.0 mL). The solution was stirred for 30 minutes prior to the removal of solvents under reduced pressure. The product was purified by SPE. The final compound was dried under vacuum overnight to give an off-white solid (75 mg, 60% yield). 1H NMR (500 MHz, MeOD) δ 8.34 – 8.31 (m, 2H), 8.08 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.18 (s, 1H), 7.12 – 7.08 (m, 3H), 4.02 (s, 3H), 3.29 – 3.26 (m, 4H), 3.10 – + 3.06 (m, 4H), 2.22 (s, 3H). HRMS (ESI) for C24H26N4O2 [M + H] : m/z = calcd, 403.2129; found, 403.2128. (tR = 1.09 min).

Tert-butyl 4-(4-(5-(3-methoxy-4-(methylcarbamoyl)phenyl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (18a)

The title compound was fully synthesized by Dr. Dimitrios Panagopoulos at the OICR. To a solution of 17 (42 mg, 0.081 mmol) in MeOH (0.81 mL) was added methylamine, 33 wt. % in EtOH (1.0 mL). The solution was stirred at 85 °C for 5h. The solvents were removed under reduced pressure prior to the crude material being triturated from a minimum amount of EtOAc and hexanes. The product was filtered and dried under air, then dissolved in DCM (0.21 mL), treated with trifluoroacetic acid (0.10 mL, 1.310 mmol) and stirred for 1 h. The solution was concentrated under reduced pressure prior to purification by reverse-phase (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The product was purified by SPE. Freeze-drying for 2 days afforded a white powder (8 mg, 20% yield). 1H NMR (500 MHz, MeOD) δ 8.32 (s, 1H), 8.30 (s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.15 (s, 1H), 7.12 – 7.06 (m, 3H), 4.00 (s, 3H), 3.30 – 3.27 (m, 4H), 3.12 – 3.08 (m, 4H), 2.98 (s, 3H), 2.21 (s, 3H). HRMS (ESI+) + for C25H28N4O2 [M + H] : m/z = calcd, 417.2285; found, 417.2288. (tR = 0.94 min).

4-(5-(4-(4-(Tert-butoxycarbonyl)piperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-2- methoxybenzoic acid The title compound was synthesized by Dr. Dimitrios Panagopoulos at the OICR. To a suspension of 17 (54 mg, 0.104 mmol) in THF (0.70 mL) and water (0.70 mL) was added potassium hydroxide pellets (12 mg, 0.209 mmol). The suspension was stirred at room temperature for 2 h. The reaction mixture was diluted with water (35 mL) and extracted with

Et2O (1 x 20 mL). The aqueous layer was carefully acidified to a pH of 5 and extracted with

DCM (3 x 20 mL). The Et2O and DCM layers were combined, dried over MgSO4, filtered and concentrated under reduced pressure to afford an off-white solid (50 mg, 93% yield). MS (ESI): m/z = 504.60 [M + H]+.

2-Methoxy-N,N-dimethyl-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (18b) The title compound was fully synthesized by Dr. Dimitrios Panagopoulos at the OICR. To a solution of 4-(5-(4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-2- methoxybenzoic acid (50 mg, 0.099 mmol), HOBt (16 mg, 0.119 mmol) and EDC (18 mg, 0.119 mmol) in DCM (0.89 mL) and DMF (0.10 mL) was added DIPEA (43 µL, 0.248 mmol) and dimethylamine, 2.0 M in THF (50 µL, 0.099 mmol). The solution was stirred at 50 oC overnight. The reaction mixture was diluted with water (5 mL) and DCM (5 mL). The organic layer was

separated, dried over MgSO4, filtered and concentrated under reduced pressure to afford a sticky yellow solid. The solid was dissolved in DCM (0.89 mL) and treated with trifluoroacetic acid (0.34 mL, 4.430 mmol). The solution was stirred for 45 minutes prior to purification by reverse- phase (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The product was purified by SPE. Freeze-drying for 2 days afforded a white powder (13 mg, 23% yield). 1H NMR (500 MHz, MeOD) δ 8.33 – 8.30 (m, 2H), 7.35 – 7.28 (m, 3H), 7.12 – 7.04 (m, 4H), 3.90 (s, 3H), 3.28 – 3.24 (m, 4H), 3.12 (s, 3H), 3.08 – 3.04 (m, 4H), 2.94 (s, 3H), 2.22 (s, 3H). HRMS (ESI) for + C25H28N4O2 [M + H] : m/z = calcd, 431.2442; found, 431.2439. (tR = 1.08 min).

4-Bromo-2-(hydroxymethyl)benzonitrile To a solution of 4-bromo-2-formylbenzonitrile (630 mg, 3.00 mmol) in MeOH (7.5 mL) cooled in an ice bath was added sodium borohydride (125 mg, 3.30 mmol). The reaction mixture was stirred for an hour at 0 °C prior to quenching with water (20 mL). Volatiles were removed under reduced pressure and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic fractions were washed with brine, dried over NaSO4, filtered and concentrated under reduced pressure to afford a yellow-brown solid, which was used without further purification in the subsequent reaction (637 mg, 85% yield). MS (ESI): m/z = 212.28 [M + H]+, 214.22 [M + H]+ + 2.

4-Bromo-2-(methoxymethyl)benzonitrile To a solution of 4-bromo-2-(hydroxymethyl)benzonitrile (400 mg, 1.89 mmol) in THF (6.3 mL) cooled in an ice bath was added sodium hydride, 60% in mineral oil (181 mg, 4.52 mmol). The solution was stirred for 30 min prior to the addition of iodomethane (1.4 mL, 22.63 mmol). The reaction mixture was stirred for an additional 2 h, then quenched with water (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic fractions were dried over Na2SO4, filtered and concentrated under reduced pressure prior to purification by silica gel chromatography (0-80% EtOAc in hexanes) to afford the final product (89 mg, 20% yield). 1H

NMR (500 MHz, CDCl3) δ 7.76 (d, J = 1.1 Hz, 1H), 7.55 – 7.48 (m, 2H), 4.62 (s, 2H), 3.49 (s, 3H).

4-Bromo-2-(methoxymethyl)benzamide

The title compound was synthesized according to the procedure described for 5d from 4-bromo- 2-(methoxymethyl)benzonitrile (80 mg, 0.354 mmol). The reaction mixture was stirred at 90 oC for 2 h then at room temperature overnight. The crude mixture was diluted with water (10 mL) and extracted with EtOAc (3 x 10 mL). The organic layers were combined and dried over 1 Mg2SO4 to afford an off-white solid (71 mg, 81% yield). H NMR (500 MHz, DMSO) δ 7.84 (br s, 1H), 7.64 (d, J = 1.9 Hz, 1H), 7.55 (dd, J = 8.2, 2.0 Hz, 1H), 7.46 (br s, 1H), 7.42 (d, J = 8.2 Hz, 1H), 4.58 (s, 2H).

2-(Methoxymethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (19c) The title compound was synthesized according to the procedure described for 4a from 4-bromo- 2-(methoxymethyl)benzamide (50 mg, 0.205 mmol). The dark brown supernatant was used without purification in the subsequent reaction (60 mg, 78% yield by LCMS). MS (ESI): m/z = 292.53 [M + H]+.

2-Chloro-6-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (19e) The title compound was synthesized according to the procedure described for 4a from 4-bromo- 2-chloro-6-methoxybenzamide (100 mg, 0.378 mmol). The dark supernatant was used without purification in the subsequent reaction (113 mg, 36% yield by LCMS). MS (ESI): m/z = 312.46 [M + H]+, 314.41 [M + H]+ + 2.

5-(4-Methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)isoindolin-1-one (20a) The title compound was synthesized according to the procedure described for 8a from 15 (75 mg, 0.193 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-1-one (19a) (50 mg, 0.193 mmol). The final product was a white powder (33 mg, 44% yield). 1H NMR (500 MHz, DMSO) δ 8.62 (br s, 1H), 8.37 (s, 1H), 8.34 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.65 (s, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 4.44 (s, 2H), 3.19 – + 3.16 (m, 4H), 2.94 – 2.90 (m, 4H), 2.15 (s, 3H). HRMS (ESI) for C24H24N4O [M + H] : m/z = calcd, 385.2023; found, 385.2018. (tR = 0.96 min).

4-(4-Methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (20b) The title compound was synthesized according to the procedure described for 8a from 15 (19 mg, 0.048 mmol) and 4-aminocarbonylphenylboronic acid (19b) (8 mg, 0.048 mmol). The final

62 product was a light pink-orange powder (8 mg, 45% yield). 1H NMR (500 MHz, DMSO) δ 8.36 (s, 1H), 8.33 (s, 1H), 8.06 (br s, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.42 (br s, 1H), 7.31 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.7 Hz, 2H), 3.23 – 3.19 (m, 4H), 3.02 – 2.92 (m, + 4H), 2.15 (s, 3H). HRMS (ESI) for C23H24N4O [M + H] : m/z = calcd, 373.2023; found,

373.2014. (tR = 0.93 min).

2-(Methoxymethyl)-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (20c) The title compound was synthesized according to the procedure described for 8a from 15 (73 mg, 0.188 mmol) and 19c (60 mg, 0.205 mmol). The final compound was an off-white powder 1 (23 mg, 29% yield). H NMR (500 MHz, CDCl3) δ 8.45 (s, 1H), 8.37 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.44 (dd, J = 7.9, 1.7 Hz, 1H), 7.40 (d, J = 1.4 Hz, 1H), 7.28 – 7.26 (m, 2H), 7.01 (d, J = 8.7 Hz, 2H), 5.74 (br s, 1H), 4.65 (s, 2H), 3.46 (s, 3H), 3.26 – 3.22 (m, 4H), 3.10 – 3.06 (m, + 4H), 2.17 (s, 3H). HRMS (ESI) for C25H28N4O2 [M + H] : m/z = calcd, 417.2285; found,

417.2283. (tR = 0.98 min).

2-Fluoro-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (20d) The title compound was fully synthesized by Dr. David Smil at the OICR. The title compound was synthesized according to the procedure described for 8a from 15 (100 mg, 0.258 mmol) and 4-carbamoyl-3-fluorophenylboronic acid (19d) (47 mg, 0.258 mmol). The final compound was a white powder (50 mg, 50% yield over 2 steps). 1H NMR (500 MHz, MeOD) δ 8.35 (s, 1H), 8.30 (s, 1H), 7.97 – 7.92 (m, 1H), 7.37 – 7.28 (m, 4H), 7.09 (d, J = 8.7 Hz, 2H), 3.26 – 3.22 (m, 4H), 3.05 – 3.01 (m, 4H), 2.22 (s, 3H). 19F NMR (471 MHz, MeOD) δ + -114.60. HRMS (ESI) for C23H23FN4O [M + H] : m/z = calcd, 391.1929; found, 391.1926. (tR = 1.13 min).

2-Chloro-6-methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (20e) The title compound was synthesized according to the procedure described for 6a from 3 (66 mg, 0.153 mmol) and 19e (59 mg, 0.189 mmol). The material was deprotected with TFA acid (0.35 mL, 4.579 mmol) and purified by SPE. Freeze-drying for a day and a half afforded an off-white 1 powder (24 mg, 36% yield). H NMR (500 MHz, CDCl3) δ 8.45 (s, 1H), 8.31 (s, 1H), 7.26 – 7.24 (m, 2H), 7.03 (d, J = 1.1 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 1.0 Hz, 1H), 5.94 –

5.87 (br m, 2H), 3.89 (s, 3H), 3.26 – 3.22 (m, 4H), 3.10 – 3.06 (m, 4H), 2.18 (s, 3H). HRMS + (ESI) for C24H25ClN4O2 [M + H] : m/z = calcd, 437.1739; found, 437.1740. (tR = 1.01 min).

4-Bromo-2-fluoro-6-methoxybenzonitrile (22a) The title compound was prepared according to a modified literature procedure.85 Sodium hydride, 60% in mineral oil (0.733 g, 18.34 mmol) was added gradually to a solution of 4- bromo-2,6-difluorobenzonitrile (21) (2.00 g, 9.17 mmol) and MeOH (0.75 mL, 18.34 mmol) in 1,4-dioxane (25 mL). The solution was allowed to stir overnight. The solvents were evaporated and the solution was diluted with water and filtered through a Buchner funnel. The filter cake was loaded onto Celite and purified by silica gel chromatography (0-80% DCM in hexanes) to afford the final compound as a white crystalline solid (965 mg, 46% yield). 1H NMR (500 MHz, 19 CDCl3) δ 7.00 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 3.95 (s, 3H). F NMR (471 MHz, CDCl3) δ - + + 103.78. MS (ESI): m/z = 230.21 [M + H] , 232.21 [M + H] + 2. (tR = 1.90 min).

4-bromo-2,6-dimethoxybenzonitrile (22b) The title compound was prepared according to the procedure described for 22a from 21 (2.00 g, 9.17 mmol). Both 22a and 22b were synthesized in the same reaction. 4-bromo-2,6- dimethoxybenzonitrile was isolated as a white crystalline solid (754 mg, 34% yield). 1H NMR + (500 MHz, CDCl3) δ 6.73 (s, 2H), 3.91 (s, 6H). MS (ESI): m/z = 242.25 [M + H] , 244.19 [M + + H] + 2. (tR = 1.85 min).

(4-Carbamoyl-3-fluoro-5-methoxyphenyl)boronic acid (23a) The title compound was synthesized according to the procedure described for 4a from 5d (2.48 g, 10.0 mmol). The dark brown supernatant was used without further purification in subsequent reactions (2.46 g, 84% yield by LCMS). MS (ESI): m/z = 214.34 [M + H]+.

2,6-Dimethoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (23b) The title compound was synthesized according to the procedure described for 4a from 5e (800 mg, 3.08 mmol). The dark brown supernatant was used without further purification in subsequent reactions (945 mg, 70% yield by LCMS). MS (ESI): m/z = 308.26 [M + H]+.

4-(5-Chloro-4-methylpyridin-3-yl)-2-fluoro-6-methoxybenzamide (24a)

The title compound was synthesized according to the procedure described for 6a from 10 (2.06 g, 10.0 mmol) and 23a (2.95g, 10.0 mmol). The final compound was an off-white crystalline solid (1.67 g, 56% yield). 1H NMR (500 MHz, DMSO) δ 8.63 (s, 1H), 8.38 (s, 1H), 7.90 (br s, 1H), 7.60 (br s, 1H), 6.97 – 6.94 (m, 2H), 3.83 (s, 3H), 2.33 (s, 3H). 19F NMR (471 MHz, + + DMSO) δ -116.46. MS (ESI): m/z = 294.97 [M + H] , 297.10 [M + H] + 2. (tR = 1.51 min).

4-(5-Chloro-4-methylpyridin-3-yl)-2,6-dimethoxybenzamide (24b) The title compound was synthesized according to the procedure described for 6a from 10 (954 mg, 4.62 mmol) and 23b (946 mg, 3.08 mmol). The final compound was an off-white crystalline solid (747 mg, 79% yield). 1H NMR (500 MHz, DMSO) δ 8.61 (s, 1H), 8.38 (s, 1H), 7.58 (br s, 1H), 7.26 (br s, 1H), 6.70 (s, 2H), 3.77 (s, 6H), 2.34 (s, 3H). MS (ESI): m/z = 307.26 [M + H]+.

(tR = 1.47 min).

(2R,6S)-4-(4-bromophenyl)-1,2,6-trimethylpiperazine The title compound was prepared according to modified literature procedures.86-87 A solution of (2R,6S)-1,2,6-trimethylpiperazine (100 mg, 0.780 mmol), 1-bromo-4-iodobenzene (221 mg, 0.780 mmol), bis(dibenzylideneacetone)palladium(0) (22 mg, 0.039 mmol), Xantphos (68 mg, 0.117 mmol) and lithium tert-butoxide 1.0 M in THF (2.34 mL, 2.340 mmol) in 1,4-dioxane (3.12 mL) was heated to 110 oC and stirred overnight. Volatiles were removed under reduced pressure and the crude material was purified by silica gel chromatography (0-10% MeOH in EtOAc). 1H NMR (500 MHz, MeOD) δ 7.32 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 3.55 – 3.49 (m, 2H), 2.52 – 2.46 (m, 2H), 2.44 – 2.38 (m, 2H), 2.33 (s, 3H), 1.19 (d, J = 6.1 Hz, 6H). MS (ESI): m/z = 283.37 [M + H]+, 285.32 [M + H]+ + 2.

(2R,6S)-1,2,6-trimethyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine (25c) The title compound was prepared according to the procedure described for 4a from (2R,6S)-4-(4- bromophenyl)-1,2,6-trimethylpiperazine (100 mg, 0.353 mmol). The dark brown reaction mixture was used without purification in subsequent reactions (117 mg, 91% yield by LCMS). MS (ESI): m/z = 331.46 [M + H]+.

2-Fluoro-6-methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3- yl)benzamide (26a) The title compound was synthesized according to the procedure described for 8a from 24a (150 mg, 0.509 mmol) and 4-(4-methylpiperazin-1-yl)phenylboronic acid (25a) (134 mg, 0.611 mmol). XPhos Pd G3 (22 mg, 0.025 mmol) was used as the catalyst. The reaction mixture was adsorbed onto Celite and the volatiles were removed under reduced pressure. The crude material was purified by silica gel chromatography (0-15% MeOH in EtOAc). Freeze drying for 1 day afforded a white powder (183 mg, 83% yield). 1H NMR (500 MHz, DMSO) δ 8.37 (s, 1H), 8.34 (s, 1H), 7.89 (br s, 1H), 7.58 (br s, 1H), 7.30 (d, J = 8.7 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 6.98 – 6.94 (m, 2H), 3.85 (s, 3H), 3.23 – 3.19 (m, 4H), 2.48 – 2.45 (m, 4H), 2.23 (s, 3H), 2.19 (s, 3H). 19F NMR (471 MHz, DMSO) δ -116.74. 13C NMR (126 MHz, DMSO) δ 163.77 (d, J = 0.9 Hz), 158.51 (d, J = 244.1 Hz), 156.80 (d, J = 9.4 Hz), 150.37, 148.86, 147.39, 141.65, 140.29 (d, J = 10.3 Hz), 137.43, 136.39 (d, J = 2.2 Hz), 130.10, 127.33, 115.45 (d, J = 22.2 Hz), 114.96, 109.01 (d, J = 2.4 Hz), 108.79 (d, J = 22.9 Hz), 56.33, 54.56, 47.66, 45.72, 18.02. HRMS (ESI) for + C25H27FN4O2 [M + H] : m/z = calcd, 435.2191; found, 435.2191. (tR = 1.02 min).

2-Fluoro-4-(5-(4-(4-isopropylpiperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-6- methoxybenzamide (26b) The title compound was synthesized according to the procedure described for 26a from 24a (150 mg, 0.509 mmol) and 4-(4-isopropylpiperazinyl)phenylboronic acid, pinacol ester (25b) (202 mg, 0.611 mmol). The final compound was a white powder (142 mg, 60% yield). 1H NMR (500 MHz, DMSO) δ 8.38 (s, 1H), 8.34 (s, 1H), 7.89 (br s, 1H), 7.58 (br s, 1H), 7.30 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.98 – 6.95 (m, 2H), 3.85 (s, 3H), 3.23 – 3.17 (m, 4H), 2.72 – 2.66 (m, 1H), 2.63 – 2.57 (m, 4H), 2.19 (s, 3H), 1.02 (d, J = 6.5 Hz, 6H). 19F NMR (471 MHz, DMSO) δ -116.74. 13C NMR (126 MHz, DMSO) δ 163.76 (d, J = 0.9 Hz), 158.50 (d, J = 244.2 Hz), 156.80 (d, J = 9.4 Hz), 150.47, 148.87, 147.38, 141.64, 140.29 (d, J = 10.3 Hz), 137.44, 136.38 (d, J = 2.2 Hz), 130.09, 127.27, 115.44 (d, J = 22.2 Hz), 114.90, 109.01 (d, J = 2.4 Hz),

108.79 (d, J = 22.9 Hz), 56.33, 53.74, 48.18, 48.11, 18.25, 18.02. HRMS (ESI) for C27H31FN4O2 + [M + H] : m/z = calcd, 463.2504; found, 463.2506. (tR = 1.08 min)

2-Fluoro-6-methoxy-4-(4-methyl-5-(4-((3R,5S)-3,4,5-trimethylpiperazin-1- yl)phenyl)pyridin-3-yl)benzamide (26c)

The title compound was synthesized according to the procedure described for 26a from 24a (156 mg, 0.531 mmol) and (2R,6S)-1,2,6-trimethyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)phenyl)piperazine (25c) (58 mg, 0.177 mmol). The final compound was a light yellow 1 powder (36 mg, 43% yield). H NMR (500 MHz, CDCl3) δ 8.43 (s, 1H), 8.30 (s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H), 6.77 (dd, J = 9.7, 0.5 Hz, 1H), 6.71 (s, 1H), 6.32 (br s, 1H), 6.09 (br s, 1H), 3.91 (s, 3H), 3.57 (d, J = 11.5 Hz, 2H), 2.86 – 2.67 (m, 2H), 2.60 – 2.45 (m, 2H), 2.40 (s, 3H), 2.16 (s, 3H), 1.26 (d, J = 5.8 Hz, 6H). 19F NMR (471 MHz, MeOD) δ -117.13. 13C NMR (126 MHz, DMSO) δ 163.74 (d, J = 0.9 Hz), 158.48 (d, J = 244.2 Hz), 156.79 (d, J = 9.5 Hz), 149.54, 148.85, 147.39, 141.63, 140.26 (d, J = 10.5 Hz), 137.38, 136.37 (d, J = 2.1 Hz), 130.13, 127.31, 115.44 (d, J = 22.2 Hz), 114.84, 108.99 (d, J = 2.5 Hz), 108.79 (d, J = 22.9 Hz), + 57.38, 56.32, 54.45, 17.99, 17.29. HRMS (ESI) for C27H31FN4O2 [M + H] : m/z = calcd,

463.2504; found, 463.2510. (tR = 1.12 min).

2,6-Dimethoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3-yl)benzamide (26d) The title compound was synthesized according to the procedure described for 26a from 24b (150 mg, 0.489 mmol) and 25a (129 mg, 0.587 mmol). The final compound was a white powder (189 mg, 87% yield). 1H NMR (500 MHz, DMSO) δ 8.36 (s, 1H), 8.35 (s, 1H), 7.57 (br s, 1H), 7.31 (d, J = 8.7 Hz, 2H), 7.24 (br s, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.72 (s, 2H), 3.78 (s, 6H), 3.28 – 3.18 (m, 4H), 2.61 – 2.52 (m, 4H), 2.30 (s, 3H), 2.20 (s, 3H). 13C NMR (126 MHz, DMSO) δ 166.34, 156.25, 150.16, 148.49, 147.47, 141.63, 139.45, 137.60, 137.38, 130.12, 127.69, 116.37, + 115.05, 105.65, 55.90, 54.23, 47.36, 45.23, 18.09. HRMS (ESI) for C26H30N4O3 [M + H] : m/z = calcd, 477.2391; found, 447.2394. (tR = 1.01 min).

4-(5-(4-(4-Isopropylpiperazin-1-yl)phenyl)-4-methylpyridin-3-yl)-2,6-dimethoxybenzamide (26e) The title compound was synthesized according to the procedure described for 26a from 24b (150 mg, 0.489 mmol) and 25b (194 mg, 0.587 mmol). The final compound was an off-white powder (179 mg, 75% yield). 1H NMR (500 MHz, MeOD) δ 8.31 (s, 1H), 8.30 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.7 Hz, 2H), 6.69 (s, 2H), 3.86 (s, 6H), 3.47 – 3.36 (m, 4H), 3.15 – 3.01 (m, 5H), 2.22 (s, 3H), 1.27 (d, J = 6.6 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 166.33, 156.24, 150.10, 148.49, 147.49, 141.64, 139.44, 137.59, 137.36, 130.14, 127.84, 116.37, 115.09, 105.65,

+ 55.90, 54.56, 47.87, 47.46, 18.09, 17.76. HRMS (ESI) for C28H34N4O3 [M + H] : m/z = calcd,

475.2704; found, 475.2705. (tR = 1.07 min).

2,6-Dimethoxy-4-(4-methyl-5-(4-((3R,5S)-3,4,5-trimethylpiperazin-1-yl)phenyl)pyridin-3- yl)benzamide (26f) The title compound was synthesized according to the procedure described for 26a from 24b (109 mg, 0.354 mmol) and 25c (58 mg, 0.177 mmol). The final compound was a white powder (22 mg, 26% yield). 1H NMR (500 MHz, MeOD) δ 8.31 (s, 1H), 8.29 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.69 (s, 2H), 3.86 (s, 6H), 3.65 (d, J = 11.8 Hz, 2H), 2.62 – 2.55 (m, 2H), 2.51 – 2.44 (m, 2H), 2.37 (s, 3H), 2.23 (s, 3H), 2.16 (s, 2H), 1.23 (d, J = 6.2 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 166.29, 156.22, 149.71, 148.49, 147.43, 141.58, 139.44, 137.57, 137.37, 130.10, 127.32, 116.37, 114.76, 105.64, 57.15, 55.88, 54.78, 37.39, 18.05, 17.79. + HRMS (ESI) for C28H34N4O3 [M + H] : m/z = calcd, 475.2704; found, 475.2699. (tR = 1.10 min).

2-Bromo-6-methoxy-N-(2,4,4-trimethylpentan-2-yl)benzamide (27b) The title compound was synthesized according to the procedure described for 5c from 2-bromo- 6-methoxybenzoic acid (500 mg, 2.16 mmol) and tert-octylamine (0.42 mL, 2.60 mmol). The reaction mixture was diluted with saturated sodium bicarbonate and extracted with DCM (2 x

100 mL). The organic fractions were dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-60% EtOAc in hexanes) to afford the final 1 product (649 mg, 88% yield). H NMR (500 MHz, CDCl3) δ 7.17 – 7.11 (m, 2H), 6.83 (dd, J = 7.2, 1.9 Hz, 1H), 5.46 (br s, 1H), 3.80 (s, 3H), 1.79 (s, 2H), 1.56 (s, 6H), 1.05 (s, 9H). MS (ESI): m/z = 342.32 [M + H]+, 344.33 [M + H]+ + 2.

2-(Dibromomethyl)-6-methoxybenzonitrile The title compound was prepared according to a modified literature procedure.88 Under an argon atmosphere, a mixture of 2-methoxy-6-methylbenzonitrile (0.786 g, 5.34 mmol), N- bromosuccinimide (2.85 g, 16.0 mmol) and benzoyl peroxide (0.129 g, 0.534 mmol) was dissolved in carbon tetrachloride (7.6 mL). The solution was stirred at 85 °C for 4 h. The reaction mixture was filtered through a fritted funnel and the filter cake washed with EtOAc. The crude material was purified by silica gel chromatography (0-20% EtOAc in hexanes) to 1 afford the final product (1.40 g, 77% yield). H NMR (500 MHz, CDCl3) δ 7.64 – 7.56 (m, 2H),

6.96 – 6.92 (m, 2H), 3.96 (s, 3H). MS (ESI): m/z = 304.02 [M + H]+, 306.21 [M + H]+ + 2, 308.16 [M + H]+ + 4.

2-Formyl-6-methoxybenzonitrile The title compound was prepared according to a modified literature procedure.88 To a solution of 2-(dibromomethyl)-6-methoxybenzonitrile (1.20 g, 3.93 mmol) in ACN (9.8 mL) was added a solution of silver nitrate (2.00 g, 11.80 mmol) in 2.0 mL of water. The reaction mixture was stirred in an oil bath at 85 °C for 30 min. The mixture was filtered through a fritted funnel and the filter cake washed with EtOAc. The organic layer was diluted with water and extracted with

EtOAc (3 x). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo to afford the final compound (668 mg, 101% yield). 1H NMR (500 MHz, DMSO) δ 10.08 (s, 1H), 7.92 – 7.88 (m, 1H), 7.66 (dd, J = 7.6, 0.7 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 3.99 (s, 3H). MS (ESI): m/z = 162.19 [M + H]+.

2-(Difluoromethyl)-6-methoxybenzonitrile (27d) To a solution of 2-formyl-6-methoxybenzonitrile (250 mg, 1.55 mmol) in anhydrous DCM (6.0 ml), cooled in an ice bath, was added (diethylamino)sulfur trifluoride (0.81 mL, 6.21 mmol). The solution was warmed to room temperature and stirred overnight. The reaction was cooled in an ice bath, quenched with saturated sodium bicarbonate and extracted with EtOAc (3x). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-70% EtOAc in hexanes) to afford the final 1 product (210 mg, 72% yield). H NMR (500 MHz, CDCl3) δ 7.68 – 7.62 (m, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H), 7.00 – 6.73 (m, 1H), 3.98 (s, 3H). 19F NMR (471 MHz, + CDCl3) δ -112.44. MS (ESI): m/z = 184.27 [M + H] .

2-Methoxy-6-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (28a) To a solution of bis(pinacolato)diboron (397 mg, 1.56 mmol) and 4,4'-di-tert-butyl-2,2'-dipyridyl (11 mg, 0.041 mmol) in dry hexane (3.6 mL) was added di-mu-methoxobis(1,5- cyclooctadiene)diiridium (I) (14 mg, 0.020 mmol). After purging with argon, the solution was stirred for 5 min at room temperature prior to the addition of 2-methoxy-6-methylbenzonitrile (27a) (85 mg, 0.58 mmol). The reaction mixture was stirred at 60 °C for 3 h. The solvents were

69 evaporated under air and the crude material was purified by silica gel chromatography (0%-25% EtOAc in hexanes). The product was collected in two batches (Batch 1 = 32 mg, 20% yield; 1 Batch 2 = 69 mg, 21% yield). H NMR (500 MHz, CDCl3) δ 7.30 (s, 1H), 7.16 (s, 1H), 3.95 (s, 3H), 2.50 (s, 3H), 1.35 (s, 12H). MS (ESI): m/z = 274.43 [M + H]+.

2-Bromo-6-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (28b) The title compound was synthesized according to the procedure described for 28a from 27b (300 mg, 0.876 mmol) and bis(pinacolato)diboron (334 mg, 1.315 mmol) using 4,4'-di-tert-butyl-2,2'- dipyridyl (14 mg, 0.053 mmol) and di-mu-methoxobis(1,5-cyclooctadiene)diiridium (I) (17 mg, 0.026 mmol). The final product was isolated in 75% yield (334 mg). 1H NMR (500 MHz,

CDCl3) δ 7.58 (s, 1H), 7.21 (s, 1H), 5.44 (s, 1H), 3.84 (s, 3H), 1.77 (s, 2H), 1.55 (s, 6H), 1.34 (s, 12H), 1.04 (s, 9H). MS (ESI): m/z = 468.43 [M + H]+, 470.38 [M + H]+ + 2.

2-Methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6-(trifluoromethyl)benzonitrile (28c) The title compound was synthesized according to the procedure described for 28a from 27c (200 mg, 0.994 mmol) and bis(pinacolato)diboron (278 mg, 1.094 mmol) using di-mu- methoxobis(1,5-cyclooctadiene)diiridium (I) (20 mg, 0.030 mmol) and 4,4'-di-tert-butyl-2,2'- dipyridyl (16 mg, 0.060 mmol). The final product was isolated in 51% yield (325 mg). 1H NMR 19 (500 MHz, CDCl3) δ 7.73 (s, 1H), 7.57 (s, 1H), 4.03 (s, 3H), 1.36 (s, 12H). F NMR (471 MHz, + CDCl3) δ -61.95. MS (ESI): m/z = 245.94 [M + H] .

2-(Difluoromethyl)-6-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile (28d) The title compound was synthesized according to the procedure described for 28a from 27d (200 mg, 1.092 mmol) and bis(pinacolato)diboron (416 mg, 1.638 mmol) using 4,4'-di-tert-butyl-2,2'- dipyridyl (35 mg, 0.130 mmol) and di-mu-methoxobis(1,5-cyclooctadiene)diiridium (I) (22 mg, 0.033 mmol). The final product was isolated in 36% yield (278 mg). 1H NMR (500 MHz, 19 CDCl3) δ 7.71 (s, 1H), 7.48 (s, 1H), 6.98 – 6.73 (m, 1H), 4.01 (s, 3H), 1.36 (s, 12H). F NMR + (471 MHz, CDCl3) δ -112.03. MS (ESI): m/z = 310.35 [M + H] .

1-(4-(5-Chloro-4-methylpyridin-3-yl)phenyl)-4-methylpiperazine (29)

The title compound was synthesized according to the procedure described for 3 from 3-bromo-5- chloro-4-methylpyridine (10) (0.270 g, 1.308 mmol) and 1-methyl-4-(4-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)phenyl)piperazine (25a) (0.474 g, 1.569 mmol) using sodium carbonate monohydrate (0.486 g, 3.919 mmol) and [1,12- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.096 g, 0.131 mmol). The resulting mixture was heated at 100 °C overnight. The final product was a dark brown solid (368 mg, 1 89% yield). H NMR (500 MHz, CDCl3) δ 8.47 (s, 1H), 8.30 (s, 1H), 7.19 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H), 3.33 – 3.29 (m, 4H), 2.65 – 2.61 (m, 4H), 2.39 (s, 3H), 2.34 (s, 3H). MS + + (ESI): m/z = 302.43 [M + H] , 304.50 [M + H] + 2. (tR = 1.33 min).

2-Methoxy-6-methyl-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3- yl)benzonitrile (30a) The title compound was synthesized according to the procedure described for 8a from 29 (42 mg, 0.139 mmol) and 28a (38 mg, 0.139 mmol) using potassium phosphate tribasic (59 mg, 0.278 mmol) and XPhos Pd G2 (11 mg, 0.014 mmol). The reaction mixture was stirred at 100 °C overnight. The crude mixture was diluted with water and extracted with EtOAc (3x). The organic layers were dried over MgSO4, filtered and concentrated in vacuo prior to purification by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The product was used immediately in the subsequent reaction. MS (ESI): m/z = 413.40 [M + H]+.

Tert-butyl 4-(4-(5-(3-bromo-5-methoxy-4-((2,4,4-trimethylpentan-2-yl)carbamoyl)phenyl)- 4-methylpyridin-3-yl)phenyl)piperazine-1-carboxylate (30b) The title compound was synthesized according to the procedure described for 3 from 28b (117 mg, 0.250 mmol) and 3 (108 mg, 0.250 mmol) using [1,12- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (18 mg, 0.025 mmol) and sodium carbonate monohydrate (93 mg, 0.749 mmol). The reaction mixture was stirred at 90 °C overnight. Additional amounts of 28b were added to increase conversion (total of 105 mg, 0.224 mmol). The reaction mixture was diluted with water and extracted with EtOAc (3x). The organic fractions were combined, dried over NaSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-85% EtOAc in hexanes). The product was used immediately in the subsequent reaction. MS (ESI): m/z = 693.47 [M + H]+, 695.48 [M + H]+ + 2.

Tert-butyl 4-(4-(5-(4-cyano-3-methoxy-5-(trifluoromethyl)phenyl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (30c) The title compound was synthesized according to the procedure described for 3 from 28c (75 mg, 0.229 mmol) and 3 (99 mg, 0.229 mmol) using sodium carbonate monohydrate (85 mg, 0.688 mmol) and [1,12-bis(diphenylphosphino)ferrocene]dichloropalladium(II)·DCM complex (19 mg, 0.023 mmol). The resulting solution was heated in an oil bath at 100 °C for 4 h. The reaction mixture was diluted with water (40 mL) and extracted with EtOAc (4 x 40 mL). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo. The compound was purified by silica gel chromatography (0%-75% EtOAc in hexanes). The final product was isolated in 32% yield (40 mg). 1H NMR (500 MHz, DMSO) δ 8.46 – 8.42 (m, 2H), 7.73 (s, 1H), 7.65 (s, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.7 Hz, 2H), 4.07 (s, 3H), 3.51 – 3.45 (m, 4H), 3.22 – 3.17 (m, 4H), 2.18 (s, 3H), 1.43 (s, 9H). 19F NMR (471 MHz, DMSO) δ -60.72. MS (ESI): m/z = 553.54 [M + H]+.

2-Methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3-yl)-6- (trifluoromethyl)benzonitrile (30d) The title compound was synthesized according to the procedure described for 8a from 28c (75 mg, 0.229 mmol) and 29 (69 mg, 0.229 mmol) using sodium carbonate monohydrate (85 mg, 0.688 mmol) and XPhos Pd G2 (18 mg, 0.023 mmol). The resulting mixture was heated at 100 °C for 4 h. The reaction mixture was diluted with water (40 mL) and extracted with EtOAc (4 x

40 mL). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo. The compound was purified by silica gel chromatography (0%-100% EtOAc in hexanes, then 0%-15% MeOH in EtOAc). The final product was isolated in 23% yield (35 mg). 1H NMR (500 MHz, DMSO) δ 8.45 – 8.43 (m, 2H), 7.73 (s, 1H), 7.65 (s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 4.07 (s, 3H), 3.23 – 3.21 (m, 4H), 2.47 – 2.46 (m, 4H), 2.23 (s, 3H), 2.18 (s, 3H). 19F NMR (471 MHz, DMSO) δ -60.72. MS (ESI): m/z = 467.49 [M + H]+.

Tert-butyl 4-(4-(5-(4-cyano-3-(difluoromethyl)-5-methoxyphenyl)-4-methylpyridin-3- yl)phenyl)piperazine-1-carboxylate (30e) The title compound was synthesized according to the procedure described for 3 from 28d (139 mg, 0.450 mmol) and 3 (43 mg, 0.099 mmol) using XPhos Pd G2 (8 mg, 0.010 mmol) as the catalyst and sodium carbonate monohydrate (37 mg, 0.298 mmol) as the base. The resulting

72 mixture was stirred at 100 °C for 3 h. The reaction was diluted with water and extracted with

EtOAc (3x). The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-100% EtOAc in hexanes). The product was used immediately in the subsequent reaction. MS (ESI): m/z = 535.43 [M + H]+.

2-(Difluoromethyl)-6-methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3- yl)benzonitrile (30f) The title compound was synthesized according to the procedure described for 8a from 28d (139 mg, 0.450 mmol) and 29 (55 mg, 0.182 mmol) using XPhos Pd G2 (14 mg, 0.018 mmol) and sodium carbonate monohydrate (68 mg, 0.547 mmol). The resulting mixture was stirred at 100 °C for 3 and a half hours. The reaction was diluted with water and extracted with EtOAc (3x).

The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-100% EtOAc in hexanes, then 0-20% MeOH in EtOAc). The product was used immediately in the subsequent reaction. MS (ESI): m/z = 449.44 [M + H]+.

2-Methoxy-6-methyl-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3- yl)benzamide (31a) To a solution of 30a in ethanol (0.50 mL) was added an aqueous solution of sodium hydroxide (0.14 mL, 5.0 M). The reaction mixture was stirred at 100 °C overnight. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The product was purified by SPE. Freeze drying for 2 days afforded a white powder (9 mg, 15% yield over 2 steps). 1H NMR (500 MHz, DMSO) δ 8.34 (s, 1H), 8.30 (s, 1H), 7.68 (br s, 1H), 7.39 (br s, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 6.93 (s, 1H), 6.87 (s, 1H), 3.79 (s, 3H), 3.24 – 3.19 (m, 4H), 2.28 (s, 3H), 2.26 (s, 3H), 2.18 (s, 3H). + HRMS (ESI) for C26H31N4O2 [M + H] : m/z = calcd, 431.2447; found, 431.2457. (tR = 1.11 min).

2-Bromo-6-methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)benzamide (31b) To a solution of 30b dissolved in DCM (0.8 mL) was added TFA (0.18 mL, 2.38 mmol). The reaction mixture was stirred at 60 °C for 1 h and left to stir with the heat turned off overnight. An additional 0.50 mL (6.53 mmol) of TFA was added and the solution was stirred at 60 °C

73 overnight. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). Further purification was required. The compound was purified by prep-HPLC followed by SPE. Freeze drying for 2 days afforded a white powder (9 mg, 7% yield over 2 steps). 1H NMR (500 MHz, DMSO) δ 8.38 (s, 1H), 8.33 (s, 1H), 7.85 (br s, 1H), 7.53 (br s, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.27 (s, 1H), 7.15 (s, 1H), 7.04 (d, J = 8.7 Hz, 2H), 3.83 (s, 3H), 3.19 – 3.12 (m, 4H), 2.93 – 2.85 (m, 4H), 2.18 (s, 3H). HRMS + (ESI) for C24H26BrN4O2 [M + H] : m/z = calcd, 481.1239; found, 481.1235. (tR = 1.23 min).

2-Methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3-yl)-6- (trifluoromethyl)benzamide (31c) The title compound was synthesized according to the procedure described for 5d from 30c (40 mg, 0.072 mmol) using an aqueous solution of sodium hydroxide (0.43 mL, 2.0 M) and hydrogen peroxide (30 wt. % solution in water) (0.20 mL). The reaction mixture was heated at 110 °C for 3 and a half hours. The solution was diluted with water (30 mL) and extracted with

EtOAc (3 x). The organic layers were combined and washed with brine, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-100% EtOAc in hexanes). The product was dissolved in DCM and treated with TFA (0.17 mL, 2.172 mmol). The product was purified by SPE. Freeze drying for 3 days afforded a white powder (20 mg, 59% yield over 2 steps). 1H NMR (500 MHz, DMSO) δ 8.40 (s, 1H), 8.37 (s, 1H), 7.90 (br s, 1H), 7.61 (br s, 1H), 7.46 (s, 1H), 7.36 (s, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.7 Hz, 2H), 3.89 (s, 3H), 3.16 – 3.12 (m, 4H), 2.90 – 2.86 (m, 4H), 2.18 (s, 3H). 19F NMR (471 MHz, + DMSO) δ -57.86. HRMS (ESI) for C25H26F3N4O2 [M + H] : m/z = calcd, 471.2008; found,

471.2002. (tR = 1.12 min).

2-Methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3-yl)-6- (trifluoromethyl)benzamide (31d) The title compound was synthesized according to the procedure described for 31c from 30d using an aqueous solution of sodium hydroxide (0.45 mL, 2.0 M) and hydrogen peroxide (30 wt. % solution in water) (0.20 mL). The reaction mixture was heated at 110 °C for 3 and a half hours. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)) and SPE. Freeze drying for 3 days afforded a white 1 powder (8 mg, 7% yield over 2 steps). H NMR (500 MHz, CDCl3) δ 8.47 (s, 1H), 8.32 (s, 1H),

7.28 (s, 1H), 7.27 – 7.24 (m, 2H), 7.11 (s, 1H), 7.01 (d, J = 8.7 Hz, 2H), 5.94 – 5.86 (m, 2H), 3.93 (s, 3H), 3.33 – 3.30 (m, 4H), 2.66 – 2.62 (m, 4H), 2.40 (s, 3H), 2.17 (s, 3H). 19F NMR (471 + MHz, CDCl3) δ -59.21. HRMS (ESI) for C26H28F3N4O2 [M + H] : m/z = calcd, 485.2164; found,

485.2160. (tR = 1.08 min).

2-(Difluoromethyl)-6-methoxy-4-(4-methyl-5-(4-(piperazin-1-yl)phenyl)pyridin-3- yl)benzamide (31e) The title compound was synthesized according to the procedure described for 31a from 30e using an aqueous solution of sodium hydroxide (0.43 mL, 5.0 M). The reaction mixture was stirred at 100 °C for 2 h. The solution was diluted with water and extracted with EtOAc (5x).

The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-100% EtOAc in hexanes). The product was dissolved in DCM and treated with TFA (0.33 mL, 4.26 mmol) prior to purification by SPE. Freeze drying for 1 day afforded a white powder (22 mg, 49% yield over 3 steps). 1H NMR (500 MHz, DMSO) δ 8.39 (s, 1H), 8.36 (s, 1H), 7.93 (br s, 1H), 7.70 (br s, 1H), 7.35 – 7.30 (m, 3H), 7.25 (s, 1H), 7.12 – 6.86 (m, 3H), 3.87 (s, 3H), 3.16 – 3.11 (m, 4H), 2.91 – 2.85 (m, 4H), 2.18 (s, 19 + 3H). F NMR (471 MHz, DMSO) δ -109.60. HRMS (ESI) for C25H27F2N4O2 [M + H] : m/z = calcd, 453.2102; found, 453.2089. (tR = 0.91 min).

2-(Difluoromethyl)-6-methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3- yl)benzamide (31f) The title compound was synthesized according to the procedure described for 31a from 30f using an aqueous solution of sodium hydroxide (0.29 mL, 5.0 M). The reaction mixture was stirred at 100 °C for 2 h. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)) and SPE. Freeze drying for 1 day afforded a white powder (23 mg, 27% yield over 2 steps). 1H NMR (500 MHz, DMSO) δ 8.39 (s, 1H), 8.36 (s, 1H), 7.93 (br s, 1H), 7.70 (br s, 1H), 7.35 – 7.31 (m, 3H), 7.25 (s, 1H), 7.11 – 6.86 (m, 3H), 3.87 (s, 3H), 3.26 – 3.21 (m, 4H), 2.56 – 2.52 (m, 4H), 2.28 (s, 3H), 2.18 (s, 3H). 19F NMR (471 + MHz, DMSO) δ -109.60. HRMS (ESI) for C26H29F2N4O2 [M + H] : m/z = calcd, 467.2259; found, 467.2248. (tR = 0.92 min).

4-Bromo-2-chloro-6-fluorobenzonitrile (33) The title compound was prepared according to a modified literature procedure.89 To a solution of 4-bromo-2-chloro-6-fluoroaniline (2.00 g, 8.91 mmol) in DCM (17.8 mL) was added nitrosonium tetrafluoroborate (1.14 g, 9.80 mmol). The solution was stirred for 1 h at room temperature, then cooled in an ice bath. Potassium cyanide (1.16 g, 17.82 mmol) was added. A solution of copper (II) sulfate pentahydrate (4.45 g, 17.82 mmol) in water (35.0 mL) was then added gradually. The suspension was stirred for 1 h on ice, then at room temperature for an additional hour. The reaction mixture was diluted with DCM and a saturated sodium bicarbonate solution, then filtered through Celite. The organic layer was washed with brine, separated, dried over Na2SO4, filtered and concentrated under reduced pressure prior to purification by silica gel chromatography (0-50% EtOAc in hexanes) to give the final product (0.398 g, 10% yield).

4-Bromo-2-chloro-6-methoxybenzonitrile (34) The title compound was prepared using a modified literature procedure.85 To a solution of 4- bromo-2-chloro-6-fluorobenzonitrile (33) (0.398 g, 1.697 mmol) in 1,4-dioxane (4.6 mL) was added MeOH (0.18 mL, 4.412 mmol). Sodium hydride, 60% in mineral oil (106 mg, 2.647 mmol) was added gradually over 1 h. The reaction mixture was stirred for 1 h at room temperature. The solvents were removed under reduced pressure and the crude material was suspended in water and filtered. The filter cake was dissolved in DCM, concentrated and purified by silica gel chromatography (0-100% DCM in hexanes) to give the final product (223 1 mg, 49% yield). H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 1.5 Hz, 1H), 7.04 (d, J = 1.4 Hz, 1H), 3.95 (s, 3H). MS (ESI): m/z = 246.26 [M + H]+, 248.26 [M + H]+ + 2, 250.21 [M + H]+ + 4.

4-Bromo-2-chloro-6-methoxybenzamide (35) 4-bromo-2-chloro-6-methoxybenzamide was synthesized according to the procedure described for 5d from 4-bromo-2-chloro-6-methoxybenzonitrile (34) (219 mg, 0.889 mmol). The solution was stirred at 90 oC for 6 h. The crude was diluted with water (50 mL) and extracted with

EtOAc (3 x 50 mL). The organic layers were combined, dried over Na2SO4, filtered and concentrated under reduced pressure to give the final product (219 mg, 77% yield). MS (ESI): + + + 1 m/z = 264.25 [M + H] , 266.26 [M + H] + 2, 268.20 [M + H] + 4. H NMR (500 MHz, CDCl3) δ 7.20 (d, J = 1.4 Hz, 1H), 6.99 (d, J = 1.3 Hz, 1H), 5.93 (br s, 1H), 5.72 (br s, 1H), 3.85 (s, 3H).

2-Chloro-6-methoxy-4-(4-methyl-5-(4-(4-methylpiperazin-1-yl)phenyl)pyridin-3- yl)benzamide (36) The title compound was synthesized according to the procedure described for 8a from 29 (57 mg, 0.189 mmol) and 19e (59 mg, 0.189 mmol) using potassium phosphate tribasic (80 mg, 0.378 mmol) and XPhos Pd G2 (7 mg, 0.010 mmol). The reaction mixture was stirred at 80 °C overnight. The crude material was loaded onto Celite and purified by silica gel chromatography (0%-15% MeOH in EtOAc). Further purification was required. The product was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)) and SPE. Freeze drying for 1 day and a half afforded a white powder (12 mg, 14% yield). 1H

NMR (500 MHz, CDCl3) δ 8.45 (s, 1H), 8.32 (s, 1H), 7.27 – 7.23 (m, 2H), 7.03 (d, J = 1.2 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 1.1 Hz, 1H), 5.89 – 5.83 (m, 2H), 3.89 (s, 3H), 3.33 –

3.28 (m, 4H), 2.65 – 2.59 (m, 4H), 2.38 (s, 3H), 2.18 (s, 3H). HRMS (ESI) for C25H28ClN4O2 [M + + H] : m/z = calcd, 451.1901; found, 451.1894. (tR = 1.04 min).

4-(7-Chloroisoquinolin-4-yl)-2,6-dimethoxybenzamide (38) The title compound was synthesized according to the procedure described for 3 from 23b (35 mg, 0.114 mmol) and 4-bromo-7-chloroisoquinoline (28 mg, 0.114 mmol) using [1,12- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (8 mg, 0.011 mmol) and sodium carbonate monohydrate (42 mg, 0.342 mmol). The reaction mixture was stirred at 95 °C overnight. The crude material was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)) to afford the final product (15 mg, 37% yield). MS (ESI): m/z = 343.33 [M + H]+, 345.33 [M + H]+ + 2.

2,6-Dimethoxy-4-(7-(4-methylpiperazin-1-yl)isoquinolin-4-yl)benzamide (39) To a solution of 38 (15 mg, 0.044 mmol) in 1,4-dioxane (0.90 mL) was added RuPhos Pd G3 (7 mg, 9 µmol), sodium tert-butoxide (17 mg, 0.175 mmol) and 1-methylpiperazine (20 µL, 0.175 mmol). The reaction mixture was heated in an oil bath at 95 °C overnight. The crude material was passed through a Celite plug prior to purification by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)) and SPE. Freeze drying for 2 days afforded an off-white compound (3 mg, 18% yield). 1H NMR (500 MHz, MeOD) δ 9.08 (s, 1H), 8.19 (s, 1H), 7.85 (d, J = 9.3 Hz, 1H), 7.65 (dd, J = 9.3, 2.3 Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H),

6.78 (s, 2H), 3.86 (s, 6H), 3.53 – 3.46 (m, 4H), 2.95 – 2.88 (m, 4H), 2.56 (s, 3H). HRMS (ESI) + for C23H27N4O3 [M + H] : m/z = calcd, 407.2083; found, 407.2079. (tR = 0.95 min).

3-Bromo-5-(3,4,5-trimethoxyphenyl)isonicotinaldehyde (43) The title compound was synthesized according to the procedure described for 3 from 3,4,5- trimethoxyphenylboronic acid (42) (4.03 g, 19.0 mmol) and 3,5-dibromoisonicotinaldehyde (41) (5.03 g, 19.0 mmol) using sodium carbonate monohydrate (7.07 g, 57.0 mmol) and [1,12- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.417 g, 0.57 mmol). The resulting mixture was heated at 100 °C for 4 h, then stirred with the heat turned off over the long weekend. The mixture was diluted with water (200 mL) and extracted with EtOAc (3 x 200 mL). The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-35% EtOAc in hexanes). The final compound 1 was a yellow solid (3.89 g, 56% yield). H NMR (500 MHz, CDCl3) δ 9.93 (s, 1H), 8.87 (s, 1H), 8.67 (s, 1H), 6.51 (s, 2H), 3.91 (s, 3H), 3.87 (s, 6H). MS (ESI): m/z = 352.29 [M + H]+, 354.23 + [M + H] + 2. (tR = 1.87 min).

3-Bromo-5-(3,4,5-trimethoxyphenyl)pyridin-4-yl)methanol (44) A solution of 43 (2.50 g, 7.10 mmol) in THF (10.0 mL) and MeOH (10.0 mL) was cooled to 0 °C and treated with sodium borohydride (0.269 g, 7.10 mmol) in one portion. The reaction mixture was stirred at room temperature for 15 min. The mixture was quenched with water and extracted with EtOAc (3x). The organic layers were dried over NaSO4, filtered and concentrated in vacuo. The material was used without further purification (2.40 g, 71% yield). MS (ESI): m/z + + = 354.30 [M + H] , 356.30 [M + H] + 2. (tR = 1.60 min).

3-bromo-4-((methoxymethoxy)methyl)-5-(3,4,5-trimethoxyphenyl)pyridine To a solution of 44 (150 mg, 0.423 mmol) in THF (1.0 ml) was added sodium hydride, 60% in mineral oil (25 mg, 0.635 mmol) on ice. The solution was removed from the ice bath and chloromethyl methyl ether (0.05 ml, 0.635 mmol) was added in one portion. The reaction was allowed to stir at room temperature overnight. The reaction had not gone to completion. As such, sodium hydride, 60% in mineral oil (25 mg, 0.635 mmol) and chloromethyl methyl ether (0.05 ml, 0.635 mmol) were added again. The reaction mixture was quenched with saturated

78 sodium bicarbonate, diluted with water and extracted with EtOAc (3x). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification on a Biotage SNAP KP-Sil 10 g column (0%-50% EtOAc in Hexanes) to afford the final compound 1 (88 mg, 52% yield). H NMR (500 MHz, CDCl3) δ 8.75 (s, 1H), 8.48 (s, 1H), 6.63 (s, 2H), 4.72 (s, 2H), 4.53 (s, 2H), 3.91 (s, 3H), 3.87 (s, 6H), 3.39 (s, 3H). MS (ESI): m/z = 398.36 [M + H]+, 400.30 [M + H]+ + 2.

(5-Chloro-2-(4-(hydroxymethyl)-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)methanol (47) The title compound was synthesized according to a modified literature procedure.90 To a solution of 44 (485 mg, 1.109 mmol), 2-bromomethyl-4-chlorophenylboronic acid, pinacol ester (46) (368 mg, 1.109 mmol) and potassium carbonate (463 mg, 3.352 mmol) in 1,4-dioxane (5.5 mL) and water (5.5 mL) was added Pd(amphos)Cl2 (79 mg, 0.111 mmol). The reaction mixture was microwaved at 90 °C for 3 h. The reaction mixture was diluted with water (40 mL) and the aqueous layer extracted with EtOAc (3 x 40 mL). The organic layers were combined, dried over

MgSO4, and concentrated in vacuo prior to purification by silica gel chromatography (10-100% EtOAc in hexanes). The final compound was a green solid (102 mg, 21% yield). 1H NMR (500 MHz, DMSO) δ 8.59 (s, 1H), 8.31 (s, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.1, 2.0 Hz, 1H), 7.29 (d, J = 8.1 Hz, 1H), 6.93 (s, 2H), 5.31 – 5.24 (m, 1H), 5.04 – 4.99 (m, 1H), 4.28 – 4.19 (m, 2H), 4.18 – 4.10 (m, 1H), 4.06 – 4.00 (m, 1H), 3.82 (s, 6H), 3.72 (s, 3H). MS (ESI): m/z = + + 416.20 [M + H] , 418.40 [M + H] + 2. (tR = 1.57 min).

9-Chloro-4-(3,4,5-trimethoxyphenyl)-5,7-dihydrobenzo[5,6]oxepino[4,3-c]pyridine The title compound was synthesized according to a modified literature procedure.91 To a solution of 47 (90 mg, 0.216 mmol) in THF (0.85 mL) was added 0.85 mL of 2 M HCl and 0.43 mL of concentrated HCl. The reaction mixture was heated at 90 °C for approximately 37 h. The solution was diluted with water (5 mL) and the pH adjusted to between 7-9 using a saturated

NaHCO3 solution (5 mL). The aqueous layer was extracted with EtOAc (3 x 10 mL) and the organic layers were combined and passed through a MgSO4 plug. The solvents were evaporated under reduced pressure and the material was used immediately in the subsequent reaction. MS + + (ESI): m/z = 398.40 [M + H] , 400.41 [M + H] + 2. (tR = 2.14 min).

9-(Piperazin-1-yl)-4-(3,4,5-trimethoxyphenyl)-5,7-dihydrobenzo[5,6]oxepino[4,3-c]pyridine (49) Multiple reaction conditions had been tested on a small scale prior to scaling up. This includes having used different catalysts such as Pd(II) acetate and BINAP, Pd-PEPPSI-IPent and RuPhos Pd G3. For the larger scale reaction, RuPhos Pd G3 (13 mg, 0.017 mmol), RuPhos (8 mg, 0.017 mmol) and 1-boc-piperazine (38 mg, 0.202 mmol) were added to a solution of 9-chloro-4-(3,4,5- trimethoxyphenyl)-5,7-dihydrobenzo[5,6]oxepino[4,3-c]pyridine in THF (3.5 mL). Lithium tert- butoxide 1.0 M in THF (0.20 mL, 0.202 mmol) was added in sequentially. The solution was microwaved for 3 h at 100 °C. Doubling the amount of the catalyst, ligand, amine and base and heating at 90 °C overnight failed to improve the yield. A different catalyst system was used. Cesium carbonate (110 mg, 0.337 mmol) was added to the reaction mixture. In a separate vial, 2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (21 mg, 0.034 mmol) and Pd(II) acetate (4 mg, 0.017 mmol) were dissolved in 1,4-dioxane (1.7 mL). The mixture was heated until the solution turned red/orange in colour and was added to the main reaction vessel, as well as an additional 88 mg (0.472 mmol) of 1-boc-piperazine. The vial was heated at 130 °C for approximately 5 h and 30 min. The crude material was purified by silica gel chromatography (0-80% EtOAc in hexanes). Further purification was required. The product was purified by reverse-phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)). The fractions were combined with those isolated from the small-scale reaction. The product was dissolved in DCM and treated with TFA (0.06 mL, 0.784 mmol). This was followed by purification by SPE. Freeze drying for a day and a half afforded an off-white powder (8 mg, 9% yield over 3 steps). 1H NMR (500 MHz, DMSO) δ 8.74 (s, 1H), 8.65 (s, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.13 (dd, J = 8.5, 2.6 Hz, 1H), 6.84 (s, 2H), 4.41 (s, 2H), 4.17 (s, 2H), 3.84 (s, 6H), + 3.73 (s, 3H), 3.26 – 3.23 (m, 4H), 2.97 – 2.92 (m, 4H). HRMS (ESI) for C26H30N3O4 [M + H] : m/z = calcd, 448.2236; found, 448.2242. (tR = 1.29 min).

3-Bromo-5-(4-chloro-2-fluorophenyl)isonicotinaldehyde (57) The title compound was synthesized according to the procedure described for 3 from 41 (1.85 g, 7.00 mmol) and 4-chloro-2-fluorophenylboronic acid (53) (1.22 g, 7.00 mmol) using sodium carbonate monohydrate (2.60 g, 21.00 mmol) and [1,12- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.180 g, 0.246 mmol). The resulting

80 mixture was heated at 90 °C overnight. The solution was diluted with water (70 mL) and extracted with EtOAc (3 x 70 mL). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-30% EtOAc in hexanes). The final compound was isolated in 36% yield (849 mg). 1H NMR (500

MHz, CDCl3) δ 10.24 (d, J = 1.3 Hz, 1H), 8.91 (s, 1H), 8.54 (s, 1H), 7.33 – 7.22 (m, 2H), 7.19 19 (dd, J = 9.6, 1.8 Hz, 1H). F NMR (471 MHz, CDCl3) δ -112.30. MS (ESI): m/z = 314.18 [M + + + + H] , 316.24 [M + H] + 2, 318.,19 [M + H] + 4. (tR = 2.07 min).

(3-Bromo-5-(4-chloro-2-fluorophenyl)pyridin-4-yl)methanol The title compound was synthesized according to the procedure described for 44 from 57 (600 mg, 1.908 mmol) and sodium borohydride (72 mg, 1.145 mmol). The crude material was diluted with water and extracted with EtOAc (3x). The fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-50% 1 EtOAc in hexanes) to afford the final product (408 mg, 62% yield). H NMR (500 MHz, CDCl3) δ 8.78 (s, 1H), 8.42 (s, 1H), 7.34 – 7.23 (m, 3H), 4.60 (br s, 2H), 2.22 (t, J = 6.6 Hz, 1H). 19F + + NMR (471 MHz, CDCl3) δ -112.12. MS (ESI): m/z = 316.09 [M + H] , 318.03 [M + H] + 2, 320.04 [M + H]+ + 4.

(E)-3-bromo-5-(4-chloro-2-fluorophenyl)-4-(2-methoxyvinyl)pyridine and (Z)-3-bromo-5- (4-chloro-2-fluorophenyl)-4-(2-methoxyvinyl)pyridine (58-59) The title compound was synthesized according to a modified literature procedure.92 (Methoxymethyl)triphenylphosphonium chloride (3.27 g, 9.54 mmol) was dissolved in THF (20 mL). This was followed by the addition of sodium bis(trimethylsilyl)amide solution, 1.0 M in THF (9.5 mL, 9.54 mmol) at 0 °C. The solution became dark red. After 30 minutes of stirring at 0 °C, 57 (1.50 g, 4.77 mmol) dissolved in 20 mL of THF was added in gradually and the solution was stirred for 30 min at this temperature. The solution was then stirred for 4 h at room temperature. The reaction mixture was quenched with water and extracted with EtOAc (3 x 100 mL). The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-30% EtOAc in hexanes) to afford the final 1 product (1.12 g, 66% yield). H NMR (E diastereomer) (500 MHz, CDCl3) δ 8.71 (s, 1H), 8.29 (s, 1H), 7.29 – 7.18 (m, 3H), 6.55 (d, J = 13.1 Hz, 1H), 5.69 (d, J = 13.1 Hz, 1H), 3.61 (s, 3H).

19 + F NMR (471 MHz, CDCl3) δ -110.76, -112.21. MS (ESI): m/z = 342.13 [M + H] , 344.20 [M + + + H] + 2, 346.21 [M + H] + 4. (tR = 2.19 min)

2-(3-Bromo-5-(4-chloro-2-fluorophenyl)pyridin-4-yl)acetaldehyde (60) The title compound was synthesized according to a modified literature procedure.93 To a solution of 58 and 59 (800 mg, 2.335 mmol) in DCM (13 mL) was added HCl (22 mL, 3 M). The reaction mixture was stirred at 80 °C for 2 h. The mixture was diluted with water and extracted with DCM. The organic layers were combined, dried over MgSO4, filtered and concentrated in vacuo. The material was used without further purification in the subsequent reaction (826 mg, 88% yield). MS (ESI): m/z = 328.22 [M + H]+, 330.10 [M + H]+ + 2, 332.17 [M + H]+ + 4.

2-(3-Bromo-5-(4-chloro-2-fluorophenyl)pyridin-4-yl)ethan-1-ol The title compound was synthesized according to the procedure described for 44 from 60 (700 mg, 2.130 mmol) and sodium borohydride (91 mg, 1.443 mmol). The crude material was diluted with water and extracted with EtOAc (3x). The fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-60% EtOAc in hexanes) to afford the final compound (417 mg, 58% yield). 1H NMR (500 MHz,

CDCl3) δ 8.72 (s, 1H), 8.28 (s, 1H), 7.29 – 7.18 (m, 3H), 3.75 – 3.68 (m, 2H), 3.09 – 2.84 (m, 19 2H), 1.41 – 1.34 (m, 1H). F NMR (471 MHz, CDCl3) δ -111.04. MS (ESI): m/z = 330.22 [M + H]+, 332.11 [M + H]+ + 2, 334.17 [M + H]+ + 4.

4-Bromo-8-chloro-5H-chromeno[4,3-c]pyridine (61a) The title compound was synthesized according to a modified literature procedure.94 (3-bromo-5-(4-chloro-2-fluorophenyl)pyridin-4-yl)methanol (250 mg, 0.790 mmol) was dissolved in THF (7.9 mL) and the solution cooled in an ice bath. To this was added sodium hydride, 60% in mineral oil (151 mg, 3.78 mmol) in one portion. The solution turned a clear yellow upon addition of the sodium hydride. Gas formation was noted. The solution was allowed to stir in the ice bath for 10 min prior to quenching with water (50 mL). The aqueous fraction was extracted with EtOAc (50 mL x 3). The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo. The material was used without further 1 purification in the subsequent reaction (265 mg, 107% yield). H NMR (500 MHz, CDCl3) δ 8.80 (s, 1H), 8.61 (s, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.07 (dd, J = 8.3, 2.0 Hz, 1H), 7.03 (d, J =

2.0 Hz, 1H), 5.22 (s, 2H). MS (ESI): m/z = 296.10 [M + H]+, 298.04 [M + H]+ + 2, 300.11 [M + H]+ + 4.

4-Bromo-9-chloro-5,6-dihydrobenzo[2,3]oxepino[4,5-c]pyridine (61b) 2-(3-bromo-5-(4-chloro-2-fluorophenyl)pyridin-4-yl)ethan-1-ol (400 mg, 1.210 mmol) was dissolved in THF (20 mL) and the solution cooled in an ice bath. To this was added sodium hydride, 60% in mineral oil (80 mg, 2.000 mmol) in one portion. After 30 min, product was beginning to form. An additional 90 mg (2.250 mmol) of sodium hydride was added and the solution was stirred at room temperature overnight. The reaction mixture was capped and stirred at 60 °C for 8 h. The solution was then stirred at 70 °C for 6 h and left to stir at room temperature overnight. The reaction mixture was quenched with water and the aqueous layer was extracted with EtOAc (3x). The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo. The material was purified by silica gel chromatography (0- 40% EtOAc in hexanes). A significant amount of 2-(5-bromo-4-vinylpyridin-3-yl)-5- chlorophenol formed, which contributed to the low yield obtained for the desired product (33 1 mg, 8% yield). H NMR (500 MHz, CDCl3) δ 8.71 (s, 1H), 8.48 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.28 (dd, J = 8.2, 2.1 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 4.59 (t, J = 6.4 Hz, 2H), 3.08 (t, J = 6.4 Hz, 2H). MS (ESI): m/z = 310.16 [M + H]+, 312.11 [M + H]+ + 2, 314.11 [M + H]+ + 4.

2-(5-bromo-4-vinylpyridin-3-yl)-5-chlorophenol (62) 1 This side product was isolated in the reaction for 61b. H NMR (500 MHz, CDCl3) δ 8.63 (s, 1H), 8.29 (s, 1H), 7.04 (d, J = 8.1 Hz, 1H), 6.99 – 6.94 (m, 2H), 6.65 – 6.56 (m, 2H), 5.46 (dd, J = 11.8, 0.5 Hz, 1H), 5.35 (d, J = 17.8 Hz, 1H). The side product has the same mass as 61b.

8-Chloro-4-(3,4,5-trimethoxyphenyl)-5H-chromeno[4,3-c]pyridine (63a) The title compound was synthesized according to the procedure described for 3 from 61a (75 mg, 0.253 mmol) and 42 (59 mg, 0.278 mmol) using sodium carbonate monohydrate (94 mg, 0.759 mmol) and [1,12-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (9 mg, 0.013 mmol. The resulting mixture was microwaved at 90 °C for 4 h. The reaction mixture was diluted with water (30 mL) and the aqueous layer extracted with EtOAc (3 x 30 mL). The combined organic extracts were dried with MgSO4, filtered and concentrated under reduced pressure prior to purification by silica gel chromatography (0%-50% EtOAc in hexanes) to afford the final

1 product (50 mg, 47% yield). H NMR (500 MHz, CDCl3) δ 8.91 (s, 1H), 8.53 (s, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.11 (dd, J = 8.3, 2.0 Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H), 6.47 (s, 2H), 5.13 (s, 2H), + + 3.92 (s, 3H), 3.89 (s, 6H). MS (ESI): m/z = 384.27 [M + H] , 386.28 [M + H] + 2. (tR = 2.11 min).

9-Chloro-4-(3,4,5-trimethoxyphenyl)-5,6-dihydrobenzo[2,3]oxepino[4,5-c]pyridine (63b) The title compound was synthesized according to the procedure described for 3 from 61b (30 mg, 0.097 mmol) and 42 (25 mg, 0.116 mmol) using sodium carbonate monohydrate (36 mg, 0.290 mmol) and [1,12-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (7 mg, 10 µmol). The resulting mixture was stirred at 100 °C for 2 h. The mixture was diluted with water and extracted with EtOAc (3x). The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-45% EtOAc in hexanes) to afford the final product (28 mg, 61% yield). MS (ESI): m/z = 398.36 [M + H]+, + 400.37 [M + H] + 2. (tR = 2.01 min).

8-(4-Methylpiperazin-1-yl)-4-(3,4,5-trimethoxyphenyl)-5H-chromeno[4,3-c]pyridine (64a) The title compound was synthesized according to the procedure described for 39 from 63a (49 mg, 0.128 mmol) and 1-methylpiperazine (29 μL, 0.261 mmol) using RuPhos Pd G3 (19 mg, 0.026 mmol) and sodium tert-butoxide (24 mg, 0.255 mmol). The resulting mixture was stirred at 90 °C overnight. The crude material was passed through a Celite plug and purified by reverse- phase chromatography (2-95% ACN (0.1% formic acid) in water (0.1% formic acid)) and SPE. Freeze drying for 2 days afforded a light yellow powder (21 mg, 37% yield). 1H NMR (500 MHz, DMSO) δ 8.93 (s, 1H), 8.44 (s, 1H), 7.85 (d, J = 8.8 Hz, 1H), 6.77 – 6.72 (m, 1H), 6.68 (s, 2H), 6.53 (d, J = 1.5 Hz, 1H), 5.14 (s, 2H), 3.82 (s, 6H), 3.72 (s, 3H), 3.28 – 3.22 (m, 4H), 2.60 – 2.52 (m, 4H), 2.31 (s, 3H). 13C NMR (126 MHz, DMSO) δ 155.54, 152.96, 147.33, 141.36, 137.32, 134.98, 133.26, 130.95, 125.53, 124.34, 110.50, 109.63, 106.62, 102.52, 64.70, 60.06, + 56.08, 53.93, 46.69. HRMS (ESI) for C26H30N3O4 [M + H] : m/z = calcd, 448.2236; found,

448.2226. (tR = 1.26 min).

9-(4-Methylpiperazin-1-yl)-4-(3,4,5-trimethoxyphenyl)-5,6-dihydrobenzo[2,3]oxepino[4,5- c]pyridine (64b)

The title compound was synthesized according to the procedure described for 39 from 63b (28 mg, 0.070 mmol) and 1-methylpiperazine (66 μL, 0.591 mmol) using RuPhos Pd G3 (21 mg, 0.028 mmol) and sodium tert-butoxide (27 mg, 0.282 mmol). The resulting mixture was stirred at 100 °C for 6 h. The crude material was diluted with water and extracted with EtOAc (3x).

The organic fractions were combined, dried over MgSO4, filtered and concentrated in vacuo prior to purification by silica gel chromatography (0%-100% EtOAc in hexanes then 0-20% MeOH in EtOAc). Further purification was required and the product was purified by Prep- HPLC and SPE. Freeze drying for 2 days afforded a white powder (5 mg, 16% yield). 1H NMR (500 MHz, DMSO) δ 8.53 (s, 1H), 8.43 (s, 1H), 7.39 (d, J = 8.6 Hz, 1H), 6.88 (dd, J = 8.6, 2.4 Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 6.70 (s, 2H), 4.56 (t, J = 6.3 Hz, 2H), 3.82 (s, 6H), 3.71 (s, 3H), 3.25 – 3.19 (m, 4H), 2.70 (t, J = 6.2 Hz, 2H), 2.49 – 2.43 (m, 4H), 2.24 (s, 3H). 13C NMR (126 MHz, DMSO) δ 155.39, 152.86, 152.49, 147.59, 146.22, 142.67, 137.00, 136.26, 134.72, 132.76, 129.38, 120.84, 111.30, 108.34, 106.86, 77.69, 60.03, 56.02, 54.34, 47.24, 29.33. HRMS + (ESI) for C27H32N3O4 [M + H] : m/z = calcd, 462.2393; found, 462.2401. (tR = 1.26 min).

5.2 NMR Spectra

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, DMSO)

19F NMR (471 MHz, DMSO)

1H NMR (500 MHz, DMSO)

1H NMR (500 MHz, MeOD)

1H NMR (500 MHz, DMSO)

19F NMR (471 MHz, DMSO)

13 C NMR (126 MHz, CD3OD)

1H NMR (500 MHz, DMSO)

13C NMR (126 MHz, DMSO)

1H NMR (500 MHz, DMSO)

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, MeOD)

1H NMR (500 MHz, DMSO)

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, MeOD)

1 H NMR (500 MHz, CDCl3)

19 F NMR (471 MHz, CDCl3)

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, DMSO)

19F NMR (471 MHz, DMSO)

100

13C NMR (126 MHz, DMSO)

1H NMR (500 MHz, DMSO)

101

19F NMR (471 MHz, DMSO)

13C NMR (126 MHz, DMSO)

102

1 H NMR (500 MHz, CDCl3)

19F NMR (471 MHz, MeOD)

103

13C NMR (126 MHz, DMSO)

1H NMR (500 MHz, DMSO)

104

13C NMR (126 MHz, DMSO)

1H NMR (500 MHz, MeOD)

105

13C NMR (126 MHz, DMSO)

1H NMR (500 MHz, MeOD)

106

13C NMR (126 MHz, DMSO)

1 H NMR (500 MHz, CDCl3)

107

1H NMR (500 MHz, DMSO)

108

1H NMR (500 MHz, DMSO)

1 H NMR (500 MHz, CDCl3)

109

1H NMR (500 MHz, DMSO)

110

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, MeOD)

111

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, DMSO)

112

1H NMR (500 MHz, DMSO)

1 H NMR (500 MHz, CDCl3)

113

19 F NMR (471 MHz, CDCl3)

1 H NMR (500 MHz, CDCl3)

114

19 F NMR (471 MHz, CDCl3) δ -110.76, - 112.21.

1 H NMR (500 MHz, CDCl3)

115

1 H NMR (500 MHz, CDCl3)

116

1 H NMR (500 MHz, CDCl3)

1H NMR (500 MHz, DMSO)

117

13C NMR (126 MHz, DMSO)

1H NMR (500 MHz, DMSO)

118

13C NMR (126 MHz, DMSO)

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Appendices

Table A1: Caco-2 permeability of 2,6-dimethoxybenzamide analogs

Compound

P (10-6 cm/s) app_AB 0.1 0.7 0.9 0.7

P (10-6 cm/s) app_BA 6.1 13.9 14.4 12.6

Efflux Ratio >10 >10 >10 >10

Table A2: HLM and MLM stability of lead amides

131 132

Table A3: Off-target activity of 2-fluoro-6-methoxybenzamide analogs (26a-c); CYP and hERG inhibition

Compound

CYP1A2 >50 >50 >50

CYP2B6 >50 >50 >50

CYP2C8 >50 >50 >50

CYP2C9 >50 >50 >50

CYP2C19 >50 >50 >50

CYP2D6 >50 >50 >50

CYP3A4-Midazolam >50 >50 >50

CYP3A4-Testosterone >50 >50 >50

hERG >30 >30 18.8

Table A4: 375-member kinase selectivity panel: % enzyme activity at 1 μM

26b 26a 26c Kinase Run 1 Run 2 Run 1 Run 2 Run 1 Run 2

ABL1 84.48 80.78 75.19 75.12 83.92 82.07

ABL2/ARG 98.76 98.48 97.35 95.77 97.10 96.59

133

ACK1 66.77 61.59 65.49 65.46 79.52 76.07

AKT1 101.90 99.01 105.74 102.39 105.40 104.50

AKT2 94.01 93.58 91.93 91.70 93.01 92.08

AKT3 98.54 97.15 94.96 94.73 98.68 98.56

ALK 103.35 102.24 99.92 99.31 100.77 96.51

ALK1/ACVRL1 2.90 2.55 2.93 2.65 4.67 4.38

ALK2/ACVR1 7.69 5.67 9.92 8.63 13.70 11.70

ALK3/BMPR1A 39.92 39.34 40.21 38.35 50.46 49.95

ALK4/ACVR1B 81.34 79.81 68.83 68.79 82.37 81.71

ALK5/TGFBR1 84.09 83.42 82.46 82.28 97.24 96.60

ALK6/BMPR1B 27.92 27.02 18.19 17.03 30.16 30.06

ARAF 52.62 51.73 29.31 28.83 48.15 43.32

ARK5/NUAK1 92.50 92.31 97.59 90.26 96.00 93.75

ASK1/MAP3K5 101.03 99.17 95.53 94.20 97.79 96.09

Aurora A 93.85 91.68 100.62 97.24 102.45 101.37

AURORA B 112.26 110.21 102.08 100.58 105.03 104.62

Aurora C 100.58 98.40 105.38 103.00 100.02 99.34

AXL 81.11 81.04 81.85 81.59 91.27 86.95

BLK 101.20 100.60 97.21 95.94 103.82 102.50

BMPR2 94.33 94.05 89.10 88.40 103.72 100.11

BMX/ETK 102.78 102.35 103.19 102.04 101.61 100.72

BRAF 44.45 44.03 28.84 28.77 41.09 40.52

134

BRK 65.32 64.33 58.34 58.28 73.93 73.52

BRSK1 87.89 84.48 84.66 83.89 83.95 78.18

BRSK2 73.81 71.41 61.34 60.95 77.89 76.84

BTK 97.71 97.62 95.31 94.35 96.55 94.07

c-Kit 80.81 76.90 74.10 74.03 75.75 74.89

c-MER 96.98 95.70 100.00 99.93 101.13 96.34

c-MET 98.44 98.41 100.40 98.28 107.02 106.76

c-Src 98.33 98.08 96.63 96.22 100.08 97.10

CAMK1a 94.69 91.83 94.86 92.25 97.52 96.82

CAMK1b 100.78 99.74 97.89 95.63 99.36 99.02

CAMK1d 100.93 98.72 98.48 98.20 99.93 97.72

CAMK1g 98.99 97.85 102.44 99.10 107.90 107.66

CAMK2a 89.93 89.17 85.47 83.69 96.65 93.72

CAMK2b 89.04 86.76 83.94 83.40 92.83 91.32

CAMK2d 93.56 93.45 95.08 94.73 96.21 95.15

CAMK2g 96.32 96.27 102.51 100.65 109.89 107.66

CAMK4 103.80 101.77 100.25 98.68 103.35 100.90

CAMKK1 92.70 88.83 94.66 92.05 94.05 93.52

CAMKK2 79.40 79.29 71.74 70.52 76.42 75.78

CDC7/DBF4 98.14 94.08 92.20 91.84 94.17 93.55

CDK1/cyclin A 84.48 83.71 94.73 92.50 90.77 89.52

CDK1/cyclin B 90.35 89.90 95.67 92.35 94.04 91.98

135

CDK1/cyclin E 100.40 99.30 98.29 97.19 92.40 91.92

CDK14/cyclin Y (PFTK1) 97.79 97.41 98.41 98.24 99.84 99.61

CDK16/cyclin Y (PCTAIRE) 93.46 91.35 93.35 92.85 92.26 91.60

CDK17/cyclin Y (PCTK2) 94.88 93.99 96.13 95.45 100.57 99.91

CDK18/cyclin Y (PCTK3) 101.52 100.66 102.15 100.43 99.05 97.60

CDK19/cyclin C 82.42 82.07 79.08 77.81 87.08 85.09

CDK2/cyclin A 92.33 92.29 99.96 97.95 99.98 98.65

CDK2/Cyclin A1 111.93 111.68 99.92 99.67 98.22 97.14

CDK2/cyclin E 106.40 104.69 104.56 104.52 102.22 102.10

CDK2/cyclin E2 104.97 104.46 108.37 104.96 95.00 93.14

CDK2/cyclin O 92.35 92.30 95.09 93.78 92.72 88.53

CDK3/cyclin E 85.83 85.35 82.66 81.63 78.70 78.38

CDK3/cyclin E2 85.07 84.43 89.55 88.05 86.16 83.94

CDK4/cyclin D1 100.09 99.89 99.36 98.46 100.47 100.29

CDK4/cyclin D3 93.74 93.67 100.73 96.96 96.05 95.54

CDK5/P25 93.58 93.23 94.60 91.77 94.85 94.09

CDK5/p35 95.63 95.08 96.22 95.26 97.61 96.90

CDK6/cyclin D1 101.19 100.82 100.38 97.80 95.90 92.80

CDK6/cyclin D3 89.55 88.85 86.29 86.25 103.38 102.19

136

CDK7/cyclin H 103.42 101.47 97.01 93.27 96.12 95.50

CDK8/cyclin C 90.11 89.57 84.60 83.64 93.06 92.05

CDK9/cyclin K 105.53 104.69 97.53 96.95 105.74 102.30

CDK9/cyclin T1 93.52 92.35 90.84 90.82 97.07 96.85

CDK9/cyclin T2 102.05 102.02 100.00 99.00 101.72 100.35

CHK1 106.02 105.34 98.22 94.26 116.91 100.95

CHK2 95.42 95.41 99.69 99.41 102.33 101.19

CK1a1 99.28 99.26 96.07 91.31 100.33 99.44

CK1a1L 103.89 102.82 98.38 97.55 99.41 99.15

CK1d 100.50 99.03 98.06 94.27 108.24 108.05

CK1epsilon 95.99 91.70 93.82 93.07 94.74 90.08

CK1g1 61.14 60.03 57.68 55.41 71.74 69.61

CK1g2 73.25 70.17 77.96 75.72 86.97 85.61

CK1G3 71.45 69.81 65.92 64.87 78.82 77.64

CK2a 88.72 85.73 92.50 91.32 87.19 86.32

CK2a2 98.13 91.24 95.26 92.27 101.45 98.98

CLK1 93.61 92.82 97.84 94.42 88.03 86.91

CLK2 88.31 87.08 91.72 91.71 90.33 85.73

CLK3 92.75 91.99 103.06 99.59 88.26 86.89

CLK4 94.89 93.34 88.92 85.58 92.56 91.14

COT1/MAP3K8 94.82 93.40 90.60 88.67 93.89 92.86

CSK 94.12 92.13 99.55 99.36 103.67 102.04

137

CTK/MATK 95.25 94.38 95.23 94.85 99.24 98.62

DAPK1 98.50 98.30 95.66 94.56 98.78 96.25

DAPK2 104.25 103.70 103.30 101.48 100.40 95.98

DCAMKL1 84.74 83.59 81.84 81.46 89.11 85.85

DCAMKL2 70.71 69.99 64.44 62.07 84.54 82.24

DDR1 40.20 39.34 32.12 31.23 57.22 55.50

DDR2 92.60 91.94 100.02 96.68 92.70 92.27

DLK/MAP3K12 94.50 94.38 93.71 91.76 99.18 97.93

DMPK 111.79 110.69 108.78 108.75 105.34 99.12

DMPK2 87.02 85.28 83.84 82.52 87.55 86.97

DRAK1/STK17A 97.03 96.54 96.37 95.60 96.60 95.69

DYRK1/DYRK1A 57.20 56.39 43.26 43.07 62.42 60.05

DYRK1B 53.64 52.79 40.65 39.80 59.24 58.52

DYRK2 97.52 95.78 96.34 95.06 100.45 98.44

DYRK3 93.39 92.56 90.42 88.30 96.03 96.01

DYRK4 97.15 96.54 102.84 102.49 101.22 100.24

EGFR 101.33 101.05 96.02 95.66 95.30 93.91

EPHA1 97.99 97.40 97.49 96.36 93.32 89.93

EPHA2 102.33 98.97 102.55 97.38 108.18 104.56

EPHA3 101.11 100.47 95.66 94.70 101.16 98.11

EPHA4 101.47 99.89 95.31 91.81 99.30 98.76

EPHA5 94.67 94.61 92.21 90.37 92.65 90.66

138

EPHA6 103.23 101.59 104.89 103.54 105.77 103.73

EPHA7 102.78 102.43 100.53 98.85 98.39 98.36

EPHA8 102.03 95.79 95.26 94.54 97.48 97.01

EPHB1 91.48 91.06 95.49 91.99 92.59 89.37

EPHB2 96.21 95.97 93.91 92.89 98.85 97.40

EPHB3 94.79 92.25 95.50 95.16 98.92 96.08

EPHB4 91.05 85.87 94.68 93.42 91.55 91.07

ERBB2/HER2 87.21 85.10 89.74 89.07 96.83 95.40

ERBB4/HER4 100.83 100.25 99.50 99.40 94.94 93.88

ERK1 98.69 97.39 97.34 94.73 95.26 94.56

ERK2/MAPK1 89.35 88.25 92.54 91.58 89.78 87.49

ERK5/MAPK7 99.60 98.66 96.51 92.77 100.55 98.01

ERK7/MAPK15 90.48 89.98 86.79 85.69 98.32 96.45

ERN1/IRE1 98.26 96.56 92.80 92.47 90.99 90.44

ERN2/IRE2 104.16 103.94 97.51 97.32 98.87 96.29

FAK/PTK2 96.05 87.69 94.56 93.97 97.12 95.42

FER 83.86 83.48 89.18 86.16 86.01 84.91

FES/FPS 97.02 96.09 99.93 99.20 98.72 98.66

FGFR1 101.69 101.63 97.43 96.52 101.12 99.91

FGFR2 97.84 97.43 95.51 95.46 91.89 90.89

FGFR3 104.85 104.10 101.07 100.98 102.79 102.08

FGFR4 95.18 94.51 98.18 97.61 99.61 98.99

139

FGR 93.41 91.32 83.07 74.69 89.43 86.35

FLT1/VEGFR1 98.17 95.86 107.26 105.29 107.93 104.04

FLT3 90.68 85.34 77.06 76.15 84.31 81.48

FLT4/VEGFR3 96.83 96.06 98.40 97.81 97.06 96.61

FMS 49.28 48.82 82.24 75.66 79.55 78.83

FRK/PTK5 100.39 99.17 100.82 95.48 98.20 95.52

FYN 121.72 119.07 99.86 97.69 97.01 96.30

GCK/MAP4K2 68.25 67.48 61.77 59.95 75.44 74.91

GLK/MAP4K3 83.93 83.55 88.42 88.16 90.29 88.71

GRK1 95.38 93.08 94.01 93.15 95.27 94.01

GRK2 106.03 105.76 105.04 103.58 100.58 99.17

GRK3 97.93 96.85 94.39 94.07 96.12 95.20

GRK4 91.85 90.10 80.43 79.15 97.14 92.15

GRK5 99.91 97.81 103.50 99.06 93.13 91.62

GRK6 104.08 103.22 108.53 107.43 97.07 94.12

GRK7 100.81 100.06 95.12 93.81 91.79 91.35

GSK3a 102.65 100.51 101.64 100.80 102.24 101.44

GSK3b 99.45 96.28 88.53 84.98 88.84 88.83

Haspin 84.75 83.53 83.33 82.77 84.79 82.31

HCK 95.09 93.44 93.59 91.96 91.82 88.06

HGK/MAP4K4 29.70 29.61 21.96 20.97 30.63 28.35

HIPK1 100.16 99.62 106.43 102.36 99.91 96.64

140

HIPK2 101.68 96.91 99.85 97.88 103.25 101.85

HIPK3 87.90 85.10 97.83 97.73 90.44 89.08

HIPK4 104.07 103.79 103.63 102.90 100.58 97.53

HPK1/MAP4K1 56.39 55.64 58.71 57.53 74.71 74.31

IGF1R 100.72 99.60 99.22 98.31 96.08 94.80

IKKa/CHUK 85.62 84.25 83.26 82.74 87.42 85.96

IKKb/IKBKB 108.96 108.24 104.06 101.39 101.07 100.59

IKKe/IKBKE 92.20 82.11 91.06 87.51 96.73 94.75

IR 101.24 100.67 102.37 100.44 100.50 97.15

IRAK1 100.81 100.16 102.79 101.36 98.92 97.90

IRAK4 88.83 88.31 82.71 78.47 88.61 84.94

IRR/INSRR 123.57 121.01 108.49 99.85 121.06 113.76

ITK 99.45 95.85 105.57 103.48 102.11 100.51

JAK1 96.67 96.26 102.44 99.53 101.38 99.07

JAK2 95.80 92.56 93.48 92.09 88.59 88.17

JAK3 102.64 101.59 106.09 102.92 100.59 99.31

JNK1 86.44 83.52 90.64 88.65 86.86 85.74

JNK2 91.34 90.94 93.42 91.99 94.72 94.14

JNK3 98.83 95.11 96.38 93.66 104.05 100.92

KDR/VEGFR2 97.27 95.43 95.86 92.99 88.36 85.59

KHS/MAP4K5 42.81 39.41 39.69 39.28 41.74 40.60

KSR1 93.57 92.67 94.48 93.83 96.28 95.91

141

KSR2 97.23 96.38 96.92 93.25 98.52 97.71

LATS1 101.28 99.48 97.36 97.31 102.26 100.53

LATS2 110.37 109.35 105.71 104.67 101.91 100.36

LCK 96.99 96.67 93.16 92.53 100.83 98.93

LCK2/ICK 89.44 86.13 107.87 104.57 97.53 95.60

LIMK1 77.94 76.31 65.09 64.18 74.16 72.63

LIMK2 105.28 102.22 102.34 99.63 97.82 95.75

LKB1 92.49 92.06 80.69 79.50 96.08 90.86

LOK/STK10 61.59 61.54 51.83 51.51 68.66 68.53

LRRK2 74.06 73.65 60.10 59.73 78.27 77.27

LYN 84.03 82.25 79.87 79.23 91.04 88.67

LYN B 95.08 91.23 99.25 96.56 98.25 97.80

MAK 111.75 110.09 99.16 98.28 118.59 100.81

MAPKAPK2 99.92 98.48 103.16 101.22 95.40 93.08

MAPKAPK3 99.55 97.50 99.51 99.20 97.25 96.39

MAPKAPK5/PRAK 96.08 95.81 93.24 90.60 94.65 94.46

MARK1 92.51 91.67 88.02 87.63 94.12 89.90

MARK2/PAR-1Ba 93.74 92.98 92.76 91.66 104.07 103.03

MARK3 93.00 91.46 83.46 82.91 94.97 93.66

MARK4 83.45 83.34 83.71 82.62 96.82 95.76

MAST3 97.45 97.29 94.66 93.52 95.55 95.17

MASTL 94.94 93.05 99.27 98.59 102.39 100.30

142

MEK1 103.50 102.97 97.45 97.44 95.10 93.40

MEK2 91.30 89.42 87.11 85.87 93.98 91.58

MEK3 104.87 103.04 102.24 100.94 101.41 101.33

MEK5 96.31 93.44 86.86 85.95 93.15 92.97

MEKK1 89.43 87.93 94.26 92.92 97.67 97.31

MEKK2 82.92 81.13 95.51 95.02 100.62 98.82

MEKK3 99.81 92.94 97.97 93.57 108.60 100.00

MEKK6 95.17 89.86 98.28 96.75 95.93 95.16

MELK 99.63 97.92 99.24 98.82 99.44 99.30

MINK/MINK1 37.11 36.56 24.06 22.65 30.16 29.65

MKK4 94.33 94.30 101.22 98.66 94.64 93.94

MKK6 68.55 64.71 84.36 80.71 79.73 79.33

MKK7 100.41 98.52 93.05 92.71 98.71 97.52

MLCK/MYLK 106.35 106.15 106.34 106.15 103.44 101.49

MLCK2/MYLK2 82.26 81.15 66.82 66.59 84.82 82.74

MLK1/MAP3K9 103.62 102.91 111.75 110.44 99.78 98.95

MLK2/MAP3K10 115.37 106.44 86.36 86.29 98.53 96.07

MLK3/MAP3K11 106.15 101.95 99.36 99.32 96.73 96.66

MLK4 91.19 89.41 95.27 95.21 96.15 93.38

MNK1 60.95 59.43 51.11 50.34 71.38 70.81

MNK2 68.96 63.87 48.23 46.54 70.76 68.28

MRCKa/CDC42BPA 104.45 102.98 102.59 102.07 105.33 104.72

143

MRCKb/CDC42BPB 97.02 95.88 94.59 94.42 99.74 99.47

MSK1/RPS6KA5 99.70 96.47 87.13 82.15 90.51 89.44

MSK2/RPS6KA4 96.00 95.45 97.14 96.56 95.42 92.01

MSSK1/STK23 98.18 91.96 99.31 98.71 99.84 99.23

MST1/STK4 100.28 99.22 101.34 100.26 103.09 101.65

MST2/STK3 98.35 93.52 95.63 93.91 101.75 100.46

MST3/STK24 113.21 107.63 90.20 87.91 96.29 94.57

MST4 90.69 90.53 86.90 85.32 94.97 90.97

MUSK 90.30 88.04 92.79 91.48 92.16 90.22

MYLK3 102.98 101.18 99.83 98.90 100.08 99.34

MYLK4 95.87 95.46 97.68 95.79 103.63 102.67

MYO3A 99.37 95.48 84.03 81.86 94.74 94.68

MYO3b 84.96 84.87 81.60 81.31 86.55 85.13

NEK1 100.04 93.20 87.56 84.54 88.09 84.46

NEK11 92.05 89.23 90.59 85.69 91.90 87.79

NEK2 93.02 91.46 92.28 90.39 98.45 96.23

NEK3 96.09 95.23 88.26 86.84 96.16 94.54

NEK4 100.85 99.62 98.49 97.67 98.05 97.25

NEK5 105.78 96.39 98.71 97.51 93.22 93.10

NEK6 100.84 100.11 99.26 98.81 103.64 103.36

NEK7 96.75 93.87 97.56 96.68 97.42 97.32

NEK8 101.75 100.17 98.40 98.06 109.21 106.54

144

NEK9 87.78 82.66 91.72 91.25 93.51 91.59

NIM1 94.49 93.25 96.64 95.18 98.15 97.81

NLK 44.71 44.67 21.99 20.81 50.44 47.74

OSR1/OXSR1 96.08 95.05 93.41 93.34 94.97 94.75

P38a/MAPK14 99.75 99.05 92.73 91.67 97.01 96.02

P38b/MAPK11 101.90 99.99 92.29 92.20 95.54 95.51

P38d/MAPK13 103.50 101.10 100.64 98.26 97.65 97.52

P38g 94.38 90.04 109.56 107.36 107.64 105.83

p70S6K/RPS6KB1 97.82 94.87 97.80 96.69 100.22 99.76 p70S6Kb/RPS6KB2 102.82 98.03 97.27 97.21 101.90 99.09

PAK1 103.65 103.63 104.85 102.29 103.23 100.98

PAK2 106.75 104.17 100.73 100.35 104.48 103.95

PAK3 90.17 89.47 89.45 87.14 124.51 124.21

PAK4 103.71 103.30 98.47 98.21 99.91 98.82

PAK5 96.74 96.36 94.56 92.74 97.29 94.99

PAK6 98.78 98.70 102.00 97.22 96.32 95.67

PASK 90.71 89.01 93.95 93.23 99.15 98.21

PBK/TOPK 95.05 94.31 91.79 89.81 102.69 102.56

PDGFRa 80.05 79.03 82.87 81.22 81.89 81.08

PDGFRb 90.14 88.87 77.30 76.01 92.12 89.56

PDK1/PDPK1 96.26 93.89 94.32 91.49 95.91 94.36

PEAK1 112.02 110.20 99.07 97.59 96.57 95.56

145

PHKg1 96.68 92.81 85.63 82.79 96.97 95.24

PHKg2 95.85 95.06 94.94 93.32 96.68 96.43

PIM1 105.63 103.63 100.02 97.92 95.25 92.25

PIM2 98.05 96.36 99.17 95.51 99.70 98.82

PIM3 107.18 106.04 100.03 99.10 107.12 106.19

PKA 97.99 97.30 97.90 96.94 96.87 95.62

PKAcb 99.42 98.30 96.17 95.19 97.92 96.20

PKAcg 91.97 88.31 94.08 93.99 100.89 98.97

PKCa 88.30 85.60 89.76 89.62 90.26 89.76

PKCb1 87.68 86.84 85.45 82.63 85.35 83.58

PKCb2 104.64 103.20 106.80 106.51 99.11 97.56

PKCd 93.40 92.22 98.99 97.72 101.05 100.12

PKCepsilon 97.83 97.53 99.77 96.64 106.14 105.76

PKCeta 96.80 96.60 94.91 93.90 98.29 98.10

PKCg 98.34 97.90 97.01 96.27 97.16 96.41

PKCiota 100.39 95.18 107.22 100.36 99.14 98.91

PKCmu/PRKD1 93.33 90.68 90.08 88.26 97.61 95.09

PKCnu/PRKD3 144.24 142.24 83.26 82.18 89.59 89.04

PKCtheta 102.63 95.99 85.06 81.31 87.00 84.96

PKCzeta 92.41 91.33 91.17 90.40 92.51 91.43

PKD2/PRKD2 105.17 102.86 96.07 95.72 99.15 98.89

PKG1a 103.94 103.73 97.90 97.81 101.80 101.45

146

PKG1b 99.52 98.95 101.57 98.83 98.81 96.31

PKG2/PRKG2 95.65 91.81 98.88 97.48 98.49 97.08

PKMYT1 124.81 121.94 101.28 100.09 94.46 92.38

PKN1/PRK1 94.64 94.01 98.54 96.76 98.68 96.39

PKN2/PRK2 102.96 100.36 94.53 91.84 95.93 95.77

PKN3/PRK3 100.61 98.77 95.02 93.55 98.38 96.44

PLK1 98.82 97.79 92.38 91.24 92.72 92.70

PLK2 93.59 92.87 98.76 97.24 102.70 102.38

PLK3 96.05 93.88 94.99 93.70 97.67 94.47

PLK4/SAK 94.00 93.89 94.12 93.32 98.82 98.79

PRKX 96.97 96.87 99.94 99.24 91.29 90.55

PYK2 83.76 81.78 85.92 85.65 88.59 85.73

RAF1 85.03 84.08 78.66 78.57 85.46 83.85

RET 92.55 89.18 94.44 94.34 96.01 93.64

RIPK2 80.92 79.92 71.12 66.31 89.66 89.10

RIPK3 112.17 111.71 106.23 101.40 103.87 102.83

RIPK4 79.68 77.02 75.50 70.28 86.09 82.04

RIPK5 84.75 83.86 83.14 82.21 86.85 86.47

ROCK1 96.97 96.20 94.95 91.98 94.71 92.61

ROCK2 121.91 119.86 115.65 115.13 100.51 93.98

RON/MST1R 113.67 108.77 98.74 97.00 105.33 104.29

ROS/ROS1 95.56 95.52 87.73 86.47 97.39 95.54

147

RSK1 97.01 95.84 98.93 97.99 101.97 101.00

RSK2 97.43 97.24 100.90 99.92 102.57 101.06

RSK3 101.40 99.21 92.16 87.93 101.35 99.74

RSK4 97.30 94.31 95.95 95.64 95.46 94.61

SBK1 96.31 96.20 94.60 93.74 103.20 101.08

SGK1 91.09 90.95 94.93 93.48 98.75 95.22

SGK2 95.78 94.93 102.03 100.43 102.54 100.91

SGK3/SGKL 86.00 83.45 102.87 99.35 94.81 93.33

SIK1 88.51 87.47 82.87 82.71 93.76 91.60

SIK2 36.68 35.99 31.97 30.68 43.90 43.50

SIK3 72.61 71.66 59.47 58.19 88.98 82.93

SLK/STK2 105.63 104.60 100.24 96.16 100.70 100.64

SNARK/NUAK2 93.54 92.65 93.71 91.64 94.38 93.96

SNRK 113.18 107.97 105.10 104.94 100.30 97.75

SRMS 98.82 97.08 96.58 95.09 96.08 95.91

SRPK1 104.82 102.86 111.76 105.40 89.32 81.70

SRPK2 97.17 95.75 97.75 94.13 121.46 115.95

SSTK/TSSK6 110.28 107.83 110.04 107.86 98.18 98.04

STK16 102.11 99.20 106.91 103.69 100.72 99.45

STK21/CIT 102.54 99.05 88.76 84.42 89.07 84.97

STK22D/TSSK1 101.05 99.56 97.48 97.01 100.64 99.16

STK25/YSK1 92.82 91.63 95.70 94.98 96.14 94.06

148

STK32B/YANK2 86.51 82.92 87.61 87.20 98.22 94.20

STK32C/YANK3 92.80 90.70 86.77 85.01 93.31 91.96

STK33 97.29 96.42 91.03 88.77 97.37 96.94

STK38/NDR1 91.48 88.77 91.87 90.38 96.68 93.15

STK38L/NDR2 95.32 93.58 99.37 98.69 103.76 102.97

STK39/STLK3 97.61 94.07 89.10 88.76 95.70 93.62

SYK 104.83 99.79 101.32 100.12 101.79 101.24

TAK1 98.69 97.41 98.52 97.44 99.08 98.46

TAOK1 38.45 36.37 60.28 60.19 62.19 60.59

TAOK2/TAO1 82.27 81.16 88.57 87.58 100.90 99.37

TAOK3/JIK 68.90 65.47 69.70 69.26 84.52 81.51

TBK1 87.00 83.46 93.61 92.56 87.18 85.62

TEC 127.66 124.33 107.92 107.42 106.34 106.19

TESK1 108.31 108.28 113.76 108.88 105.36 105.23

TESK2 93.29 93.23 93.94 93.89 92.15 92.03

TGFBR2 111.40 110.01 102.91 97.44 105.82 105.33

TIE2/TEK 93.07 91.97 93.84 92.85 93.36 92.15

TLK1 111.42 109.03 108.27 107.91 103.74 100.84

TLK2 95.15 94.99 96.63 95.61 93.27 89.84

TNIK 5.27 4.57 -0.77 -2.62 4.85 3.92

TNK1 108.03 102.51 105.47 99.53 98.56 98.22

TRKA 91.94 89.07 91.84 90.11 95.24 94.00

149

TRKB 109.41 105.99 100.49 99.94 98.97 98.44

TRKC 118.59 117.99 102.66 101.44 99.02 97.50

TSSK2 107.44 102.75 100.20 98.77 100.08 99.71

TSSK3/STK22C 95.08 94.89 96.57 96.02 96.56 95.64

TTBK1 96.89 93.79 97.29 96.78 99.41 98.52

TTBK2 97.99 94.77 97.12 94.62 97.80 96.48

TXK 103.85 100.24 101.32 100.80 98.23 95.85

TYK1/LTK 100.49 99.87 99.76 99.01 99.99 99.29

TYK2 103.25 100.78 92.64 91.37 102.60 99.47

TYRO3/SKY 97.39 95.17 84.13 83.48 94.52 93.66

ULK1 86.28 82.29 84.69 83.53 89.77 89.24

ULK2 81.06 80.11 73.70 73.24 85.24 84.46

ULK3 94.55 93.28 91.50 91.23 98.85 97.24

VRK1 108.53 108.29 104.27 100.69 101.56 98.74

VRK2 88.02 85.61 89.06 87.47 92.46 89.63

WEE1 128.65 120.95 104.70 104.15 104.62 99.73

WNK1 101.95 100.13 107.57 107.33 104.49 102.29

WNK2 98.86 98.34 95.45 95.09 96.93 93.64

WNK3 92.11 90.95 89.56 88.15 93.78 92.59

YES/YES1 100.50 98.27 104.81 104.56 109.01 108.87

YSK4/MAP3K19 105.08 101.54 98.16 97.39 97.00 96.61

ZAK/MLTK 38.53 36.39 31.26 30.86 46.64 46.59

150

ZAP70 99.62 98.79 96.23 94.79 98.70 98.47

ZIPK/DAPK3 108.04 105.01 103.90 103.31 103.72 102.34

Chapters of this thesis are reproduced with permission from Ensan, D.; Smil, D.; Zepeda- Velázquez, C. A.; Panagopoulos, D.; Wong, J. F.; Williams, E. P.; Adamson, R.; Bullock, A. N.; Kiyota, T.; Aman, A.; Roberts, O. G.; Edwards, A. M.; O’Meara, J. A.; Isaac, M. B.; Al-awar, R. Targeting ALK2: An Open Science Approach to Developing Therapeutics for the Treatment of Diffuse Intrinsic Pontine Glioma. J. Med. Chem. 2020, 63, 4978-4996. https://doi.org/10.1021/acs.jmedchem.0c00395. Copyright 2020 American Chemical Society. Further permission related to the material should be directed to the ACS.

M4K2009, M4K2149 and compound 20d were designed and synthesized by David Smil. Compounds 18a and 18b were synthesized by Dimitrios Panagopolous. Compounds 45 and 56 was synthesized by PhD student Hector Gonzalez-Alvarez. The compounds were designed and their syntheses devised by David Smil, Methvin B. Isaac, Carlos A. Zepeda-Velázquez and Deeba Ensan. All NanoBRET data was generated by Jong Fu Wong under the supervision of Alex N. Bullock from the Structural Genomics Consortium (Oxford). Biochemical kinase data was generated by Reaction Biology Corporation. Crystallography experiments were performed by Eleanor P. Williams and Roslin Adamson from the Structural Genomics Consortium (Oxford). Caco-2 and microsomal stability studies were conducted by Taira Kiyota at the OICR. HRMS were also generated by Taira Kiyota. Dihedral angle optimizations were done by Lisa Rooney at the Institute of Cancer Research using Schrodinger Jaguar, M06-2X-D3/6-31G** density functional theory. CYP inhibition assay and in vivo PK experiments were performed by Pharmaron Inc. hERG inhibition assay was conducted by Charles River Laboratories. For more information regarding detailed experimentals, we refer you to the publication in which chapters of this thesis were reproduced.

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