Design, Synthesis and Evaluation of Diphenylamines As MEK5 Inhibitors Suravi Chakrabarty

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Fall 2014 Design, Synthesis and Evaluation of Diphenylamines as MEK5 Inhibitors Suravi Chakrabarty

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DESIGN, SYNTHESIS AND EVALUATION OF DIPHENYLAMINES AS MEK5

INHIBITORS

A Thesis

Submitted to the Graduate School of Pharmaceutical Sciences

Duquesne University

In partial fulfillment of the requirements for

the degree of Master of Science

(Medicinal Chemistry)

Suravi Chakrabarty

December 2014

Suravi Chakrabarty

2014

DESIGN, SYNTHESIS AND EVALUATION OF DIPHENYLAMINES AS MEK5

INHIBITORS

Suravi Chakrabarty

Approved August 7, 2014

______Patrick Flaherty, Ph.D. Aleem Gangjee, Ph.D., Chair, Thesis Committee Professor of Medicinal Chemistry Associate Professor of Medicinal Mylan School of Pharmacy Chemistry Distinguished Professor Graduate School Pharmaceutical Sciences Graduate School Pharmaceutical Sciences Duquesne University Duquesne University Pittsburgh, PA Pittsburgh, PA

______David J. Lapinsky, Ph.D. Jane E. Cavanaugh, Ph.D. Associate Professor of Medicinal Assistant Professor of Pharmacology Chemistry Graduate School Pharmaceutical Sciences Graduate School Pharmaceutical Sciences Duquesne University Duquesne University Pittsburgh, PA Pittsburgh, PA

______David A. Johnson, Ph.D. Director of Graduate Studies Associate Professor of Pharmacology- Toxicology Graduate School Pharmaceutical Sciences Duquesne University Pittsburgh, PA

iii ABSTRACT

DESIGN, SYNTHESIS AND EVALUATION OF DIPHENYLAMINES AS MEK5

INHIBITORS

Suravi Chakrabarty

December 2014

Dissertation supervised by Dr. Patrick T. Flaherty

The mitogen-activated protein kinases (MAPK) are a family of interrelated signal transduction kinases mediating intracellular responses to extracellular events. They are involved in mediating complex cellular events including, cell differentiation, cell proliferation, and cell death. Extracellular mitogens or the binding of other ligands to cell-surface receptors begin a signaling cascade that activates MEK resulting in phosphorylation of its corresponding and specific and parallel ERK (Extracellular signal-

Regulated Kinase) substrate. The MEK5/ERK5 pathway is involved in cell survival, anti-apoptotic signaling, angiogenesis, and cell motility. It is significantly up-regulated in specific tumor types including breast and prostate cancers. A novel series of compounds utilizing the diphenylamine scaffold was designed and synthesized based on a homology model of MEK5 using the X-ray crystal structure of MEK1 (PDB ID: 3EQC)

iv as a template. The compounds were tested for their MEK12, and MEK5 inhibition in a cell based assay.

v DEDICATION

To my grandparents,

Shri Dwijapada Chakrabarty and Smt. Shyamala Chakrabarty

Shri Nripendra Mohan Chaudhuri and Smt. Arati Chaudhuri

vi ACKNOWLEDGEMENT

First and foremost, I would like to sincerely thank my advisor, Dr. Patrick T.

Flaherty. I am so thankful to him for giving me the opportunity to work in his research group and his guidance throughout this process, which has helped me to become an inquisitive learner. His optimism, encouragement and support helped me tremendously in my journey as a researcher.

Additionally, I would also like to thank Drs. Aleem Gangjee, David J. Lapinsky and Jane E. Cavanaugh for serving on my committee. I have learned a great deal, not only from the courses they taught, but more importantly, their valuable suggestions and encouragement in our many research meetings. Likewise, I thank Drs. Mark W. Harrold and Kevin J. Tidgewell for the knowledge they shared with me throughout my course work. I am especially grateful to Dr. Fraser F. Fleming for continually trying to bring out the best in me in my learning, and research.

A great deal of appreciation is shared with Dr. James K. Drennen and the

Graduate School of Pharmaceutical Sciences for their financial support. Mrs. Mary

Caruso, Mrs. Jackie Farrer, Mrs. Nancy Hosni and Mrs. Deb Wilson have helped me personally and emotionally throughout my several years at Duquesne. Tom and Miss T. deserve special thanks for working so diligently to keep our lab safe, clean, and healthy.

Their daily hard work, often unrecognized, is very much appreciated.

I am very grateful and give many thanks to Darlene Monlish and Sneha Potdar, from Dr. Cavanaugh’s lab and Dr. Matthew Burow from Tulane University, New

Orleans, for the fruitful collaboration and for performing the biological evaluation of my

vii compounds. Likewise, the company of my lab mates Prashi Jain, Dhruv Shah, Mohit

Gupta and Si Qin provided not only serious reflection on the work we were conducting, but countless hours of joy and laughter. Similarly, the support of Sudhir Raghavan, Ravi

Devambatla and Shaili Aggarwal throughout this process has been extremely helpful, and

I thank them for assisting me through many scientific and personal concerns. Again, I thank all of them for their support and encouragement; it was a pleasure working with you all.

I would also like to acknowledge and thank Diane Rhodes for her help as an instructor during my many TA sessions. Furthermore, I am thankful to Erin Rentschler and Michael McGravey for encouraging me to become a better teacher.

I truly believe that life is incomplete without friends, and I am so lucky to have such great life companions. Many thanks are shared with Isha Makan, Upasana Banerjee,

Shruti Choudhary, Manasa Ravindra, Khushbu Shah, Rishabh Mohan and Arpit Doshi.

Each of you have unconditionally, and in your own ways, added to my life three most important ingredients of happiness: love, laughter and friendship. I will forever cherish the time spent with you all throughout my life.

I would like to thank Abhishek Singh for his affection, patience and endurance in every step I have taken in my academic pursuits.

Finally, and most importantly, I would like to express my gratitude for my mother and father for their never-ending love, patience and understanding. I am so fortunate to have parents who have always let me live my dream. Last, but not least, I would like to thank my younger brother, Debopam, for being my biggest strength, always and forever.

viii TABLE OF CONTENTS

Page

Abstract ...... iv

Dedication ...... vi

Acknowledgement ...... vii

List of Tables ...... x

List of Figures ...... xi

List of Schemes ...... xiv

List of Abbreviations ...... xvi

Biological Literature Review ...... 1

Chemical Literature Review ...... 45

Statement of the Problem ...... 56

Chemical Discussion ...... 70

Biological Discussion ...... 91

Experimental ...... 99

Summary ...... 114

Bibliography ...... 122

Appendix ...... 134

ix LIST OF TABLES

Page

Table 1. Factors associated with increased risk of breast cancer ...... 3

Table 2. Characteristics of the basic molecular/intrinsic breast cancer subtypes ...... 10

Table 3. Kinase inhibitors approved by FDA in 2013 ...... 15

Table 4. Optimization of the Carboxamide Side Chain ...... 39

Table 5. Substitution on the Pyridone Nitrogen ...... 40

Table 6. Replacement of the 4′-Iodide ...... 40

Table 7. Commonly used chlorinating agents ...... 53

Table 8. Carbodiimide coupling reagents ...... 55

Table 9. Ocular toxicities of MEK inhibitors ...... 59

Table 10. Ocular toxicity of BRAF inhibitor Dabrafenib ...... 60

Table 11. Different reaction conditions with different precursor amines ...... 75

Table 12. MEK1/2 and MEK5 inhibition data ...... 97

Table 13. MEK1/2 and MEK5 inhibition data ...... 137

x LIST OF FIGURES

Page

Figure 1. Incidence Rates of Female Breast Cancer, by Age, US, 1975-2010 In Situ (a) and Invasive (b) ...... 2

Figure 2. DCIS: Non-invasive abnormal cells found in the epithelial lining of the milk ducts ...... 5

Figure 3. LCIS: Non-invasive abnormal cells found in the lobules of the breast ...... 6

Figure 4. Extensive overlap of expressed genes between the basal and TN subtypes ...... 8

Figure 5. External contributions to EMT ...... 12

Figure 6. EMT/MET ...... 13

Figure 7. Schematic diagram of protein kinase catalytic mechanism in case of PKA

(Protein kinase A) ...... 16

Figure 8. Active catalytic site of PKA with ATP and substrate. PDB id: 1ATP.1 The structure represents a ‘DFG in’ conformation ...... 17

Figure 9. Design strategy of Type I inhibitors mimicking select ATP interactions ...... 18

Figure 10. Examples of Type I inhibitor ...... 19

Figure 11. Type II inhibitors design with DFG “out” conformation ...... 20

Figure 12. Designing of a Type I ½ inhibitor ...... 21

Figure 13. Type III and Type IV binding pockets with a type III inhibitor (PD-325089) bound in the allosteric pocket ...... 22

Figure 14. The MAPK signaling pathway ...... 24

Figure 15. Crystal structure of MEK1 with Mg++ATP in the active site...... 25

Figure 16. ERK1/2, ERK5, p38 MAPKs (α,β,γ,δ) and JNK 1/2/3 are arranged into a hierarchical three-tier system known as a MAPK module ...... 26

xi Figure 17. MEK5 splice variants and the pertinent functional domains ...... 27

Figure 18. Structure and functional domains of ERK5 ...... 28

Figure 19. Mechanism of nucleocytoplasmatic transport of ERK5; TAD: transcriptional- activation domain ...... 30

Figure 20. Crystal structure of ERK5 with inhibitor 3. PDB ID 3H9F ...... 31

Figure 21. MEK5/ERK5 pathway ...... 32

Figure 22. Compound 4 and analogues ...... 35

Figure 23. MEK1 and MEK2 homodimer structures respectively ...... 36

Figure 24. MEK1/2 interactions with inhibitor 5 (PD325901) ...... 37

Figure 25. Schematic of halogen bond...... 37

Figure 26. Halogen bond from 5 to acyl lone pair of backbone Val127 in 3EQC ...... 38

Figure 27. SAR studies on 17 and development of 18...... 42

Figure 28. Substitution on the aniline and heteroaromatic core transformation ...... 42

Figure 29. Polar group substitution SAR let to the development of GSK1120212...... 43

Figure 30. MEK1/2 inhibitors U0126 and PD98059 ...... 44

Figure 31. Li+ ion directs the attack of the aniline anion ...... 50

Figure 32. The MEK5 signaling cascade ...... 57

Figure 33. MEK1/2 inhibitors and the lead compounds ...... 61

Figure 34. BRAF inhibitor Dabrafenib (Tafinlar) ...... 61

Figure 35. ATP and Type III binding site in MEK1 crystal structure and superimposed homology model of MEK5 ...... 62

Figure 36. Type III inhibitor docked in 3EQC ...... 62

Figure 37. Most selective MEK5 inhibitor from our initial studies ...... 63

xii Figure 38. Ligand specific interactions plot of 56 in MEK5 homology model ...... 64

Figure 39. The presence of the N-methyl on conformation ...... 69

Figure 40. MEK1/2 interaction with inhibitor 5 (PD325901) ...... 91

Figure 41. MEK1/2 and MEK5 inhibition graphs ...... 138

Figure 42. Dose-response effect of 57 (SC-1-151) at different concentrations ...... 139

Figure 43. MDA-MB-231 – SC-1-151 treatment at day 3 ...... 140

Figure 44. MDA-MB-231 – SC-1-151 treatment at day 7 ...... 140

Figure 45. Fold change of E-Cadherin upon administration of 57 (SC-1-151) ...... 141

Figure 46. Sequence Alignment of Human MEK5 β DD with Human MEK1 ...... 142

xiii LIST OF SCHEMES

Scheme 1. Synthesis of diarylamines by Ullmann Coupling ...... 46

Scheme 2. Goldberg synthesis of diarylamines in 1906...... 47

Scheme 3. Synthesis of diphenylamines using Buchwald Coupling ...... 47

Scheme 4. Synthetic route for diphenylamines utilizing LiN(iPr)2...... 49

Scheme 5. Synthesis of diphenylamine 39 utilizing LiNH2 ...... 49

Scheme 6. Synthetic scheme of amides via acid chloride ...... 52

Scheme 7. Mechanism of chlorination by oxalyl chloride or phosgene ...... 52

Scheme 8. Mechanism of carbodiimide coupling ...... 54

Scheme 9. DIC coupling reaction...... 54

Scheme 10. Lithium amide displacement approach ...... 71

Scheme 11. Acid chloride formation...... 73

Scheme 12. Mechanism of acid chloride formation ...... 73

Scheme 13. Synthesis of simple amides using the acid chloride method ...... 74

Scheme 14. DCC coupling reaction ...... 76

Scheme 15. DIC coupling reaction ...... 76

Scheme 16. Pd/C reductive de-iodination reaction ...... 77

Scheme 17. Lithium amide displacement method ...... 77

Scheme 18. T3P coupling reaction ...... 78

Scheme 19. Synthesis of 69 Fumarate by EDCI coupling...... 79

Scheme 20. Synthesis of 71 using lithium amide displacement method ...... 80

Scheme 21. Eschweiler-Clarke reaction approach towards the synthesis of 72 ...... 81

Scheme 22. EDCI coupling for the synthesis of 72...... 82

xiv Scheme 23. Synthesis of the primary amides 57, 73 and 70 ...... 82

Scheme 24. Sodium hydride SNAr reactions for the synthesis of 25...... 83

Scheme 25. Lithium amide SNAr reaction for the synthesis of 25 ...... 83

Scheme 26. Ullmann coupling strategy to synthesize 25...... 84

Scheme 27. The acid chloride method for the synthesis of amide 75 ...... 85

Scheme 28. Synthesis of 75 via Eschweiler-Clarke reaction ...... 86

Scheme 29. DIC coupling method to synthesize 75...... 86

Scheme 30. Synthesis of amides 75 and 74 ...... 87

Scheme 31. Eschweiler-Clarke strategy to synthesize 76 ...... 88

Scheme 32. Mechanism of Eschweiler-Clarke reaction ...... 88

Scheme 33. Synthesis of 76 by sodium hydride and methyl iodide reaction ...... 89

Scheme 34. Monomethylation reaction of 38 ...... 89

Scheme 35. Synthesis of acid 76 by lithium amide displacement method ...... 90

xv LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

BrCA Breast cancer

CRE cAMP response element

DCIS Ductal carcinoma in situ

DCM Dichloromethane

DIAD Diisopropyl azodicarboxylate

DMAP N,N-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO Dimethyl sulfoxide

DIEA Diisopropylethylamine

DCC N,N-dicyclohexylcarbodiimide

EA Ethyl acetate

E-cadherin Epithelial cadherin

EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

EGF Endothelial growth factor

EMT Epithelial to mesenchymal transition

ER Estrogen receptor

ERK Extracellular signal-regulated kinase

EtOH Ethanol

FGF Fibroblast growth factor

HIF1α Hypoxia-inducible factor 1α

xvi Hsp90 Heat shock protein 90

HTS High-throughput screen

LCIS Lobular carcinoma in situ

Ile Isoleucine

MAPK Mitogen activated protein kinase

MDR Multidrug resistance

Mef2 Myocyte enhance factor 2

MEKK Mitogen-activated kinase kinase kinase

MEK Mitogen-activated kinase kinase mp Melting point

N-cadherin Neural cadherin

NGF Nerve growth factor

NLS Nuclear localization signal

PR Progesterone receptor

Pro Proline

SAR Structure-activity relationship sat Saturated

SERMs Selective estrogen receptor modulators

TEA Triethylamine

TGFβ Transforming growth factor beta

THF Tetrahydrofuran

TNBC Triple negative breast cancer

Zeb Zinc finger E-box binding homeobox

xvii Chapter 1

Biological Literature Review

1.1 Breast Cancer

Cancer accounted for 8.2 million deaths worldwide in 2012 (the last complete year of

WHO record) establishing it as one of the leading causes of death.1 Breast cancer ranks fifth for all cancer deaths and is the most lethal cancer for women from less developed countries. In more developed societies breast cancer is the second highest cause of cancer death (198,000 deaths, 15.4%) after lung cancer.1

Incidence rates of in situ as well as invasive breast cancer (vide infra) rose rapidly during

1980s as shown in the demographic (Figure 1).2 Possible explanations for this rise may include increased mammography screenings, better diagnosis and an increase in the total number of individuals in this demographic group.

Figure 1: Incidence Rates of Female Breast Cancer, by Age, US, 1975-2010 In Situ (a) and Invasive (b).2

Breast cancer (BrCA) is a heterogeneous collection of cancers targeting the breast which vary by anatomical origin, protein expression, and cell-cycle phase. Although these variations make the disease complex, understanding the details of this variation provides multiple opportunities for therapeutic intervention. While it is reported that there are over 2 million breast cancer survivors in the United States, in reality 5-10 % of these individuals will experience a relapse within the next 10 years.

Breast cancer is multifactorial and results from a complex -yet poorly understood- interplay between environment and genetics. This is consistent with the “two-hit” or soil and seed hypothesis for cancer originally proposed by Knudon in 1971.3 Subsequent models have been proposed, but most retain this complex inter-play of both environmental and genetic contributions.4 Environmental factors increasing the development of breast cancer include alcohol use, obesity, and physical inactivity. These factors contribute to 21% of all breast cancer deaths worldwide.5 Prolonged exposure to

2 endogenous estrogens, arising from an early menarche, late menopause, and a late age for first childbirth are also significant risk factors for breast cancer. Though controversial, the use of exogenous hormones for oral contraceptive or hormone replacement therapy may also present an increased risk for developing breast cancer.6

Several genetic risk factors for breast cancer have previously been identified, and many are well-documented (Table 1). These include BRCA1 (chromosome 17q), BRCA2

(chromosome 13q12), BRCATA (chromosome 11q), BRCA3 (chromosome 13q21),

BWSCR1A (chromosome 11p15.5), TP53 (chromosome 17p), and RB1CC1

(chromosome 8q11).7 BRCA1, BRCA2 and p53 mutations can result in a very high risk for breast cancer and are the most extensively studied.

Race White

Age >50

Genetics BRCA1 or BRCA2 mutation

Previous medical history Endometrial cancer

Proliferative forms of fibrocystic disease

Cancer in other breast

Menstrual history Early menarche (under age 12)

Late menopause (after age 50)

Reproductive history Nulliparous or late first pregnancy

Table 1: Factors associated with increased risk of breast cancer.6b

The recognition that a significant amount of the genetic risk is understood, yet not quantitatively predictive has focused an increased attention to early adaptive changes in

3 pre-cancerous and cancerous lesions. Presumably this would represent an adaptive change to the initial event and the genetic predisposition would represent the ability for this event to precipitate cancer. Also, this refocusing on early events is the result of earlier identification of breast cancers due to increased screenings. The challenge now becomes to treat breast cancer in its earlier stages. These early stages are phenotypically different than the later stages of breast cancer. Of particular note is that the proto- oncogenes, the phorbol esters, activate the MAPK cascade, specifically the MEK5/ERK5 cascade. Members of this cascade are dramatically up-regulated in early and aggressive breast cancer.8 Thus there is a need for the development of new therapeutic approaches that can treat breast cancer identified in its early stages.

There are currently 53 drugs approved by the US Food and Drug Administration for the treatment of breast cancer. Amongst these, chemotherapeutic drugs such as DNA alkylating agents (e.g. Cyclophosphamide in combination with Doxorubicin), antimetabolites (e.g. Methotrexate), microtubule targeted antimitotic agents (e.g.

Paclitaxel), antineoplastic antibiotics (e.g. Doxorubicin) and SERMs (selective estrogen receptor modulators) (e.g. Tamoxifen and Toremifene) are most commonly employed.

Other newer, but less commonly used agents target the EGFR/ERRB2 or ESR1 pathways

(e.g. Erlotinib). The current recommendation9 of profiling of cancer phenotype and genotype provides the opportunity to better characterize the specific nature of the cancer from each individual. The development of new drugs that specifically target the adaptive change10 unique to each cancer phenotype/genotype is a current and active goal of drug development.

4 1.1.1 Categories of Breast Cancer

Breast cancer is categorized on the basis of tissue origin and phenotype of the cancer.

Overtime, this phenotypic expression of proteins may change; noninvasive

(nonmetastatic or cancer that has not spread outside of its tissue of origin) BrCAs may develop into malignant or invasive cancers through a series of changes that increase the rate of cancer growth, replication, and invasiveness. Ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS or Lobular Neoplasia) are presented diagrammatically in

Figure. 2 and 3. DCIS begins in the epithelial lining of the breast ducts. LCIS begins in the milk-producing lobules of the breast tissue. Both of these cancers are classified as

“in-situ” because they are -at the time of characterization- localized and not invading past the basement membranes into other breast tissue structure.

Figure 2: DCIS: Non-invasive abnormal cells found in the epithelial lining of the milk ducts.11

Figure 3: LCIS: Non-invasive abnormal cells found in the lobules of the breast.11

Either of these cancers can become more aggressive and invasive due to changes in protein expression resulting in metastatic breast cancer. Due to the changing character of these cancers -regardless of tissue origin- it has been found useful to further categorize breast cancer types based on hormone receptor expression. The receptors most commonly altered in breast cancer are ER (Estrogen Receptor), PR (Progesterone

Receptor) and HER2 (Human Epidermal Growth factor receptor-2).

1.1.1.1 Estrogen Receptor+

75% of BrCAs are Estrogen Receptor + (ER+) and express ER on the cell surface. These receptors are normally inactive and can be activated by the presence of endogenous steroids or synthetic steroid agonists. Conversely, they can be inactivated by the presence of steroid antagonists. These receptors are internalized following agonist

6 binding, relocate to the nucleus, and initiate gene transcription. These receptors can also be internalized without steroid binding, in which case they normally do not result in gene activation. Amongst these ER+ cells, 65% are also Progesterone Receptor+ (PR+).

Estrogen-receptor positive breast cancers are further categorized into luminal A and luminal B subtypes. Luminal A breast cancers have the highest levels of estrogen receptor expression and tend to be low-grade and less invasive cancer. Luminal B breast cancers are ER+ (and either PR+ or PR-) and HER2+. The designation of a negative receptor profile indicates that the cell does not express the receptor on the cell surface and consequently the ligand cannot bind and subsequently induce gene expression.

These cancer cells, however, may activate the expression of agonist-dependent genes regardless of receptor occupancy. In such a case, these receptor negative cell types are designated constitutively active and receptor negative cells.

1.1.1.2 Her2 type12

The second class of relevant breast cancer receptors are designated as Her2 for Human

Epidermal Growth factor receptor-2 type. These breast cancer cells express Her2 receptors which upon activation express Her2-related genes. Breast cancer cells can be either ER+ or ER– and independently can also be PR+ or PR-. Approximately 20-30% of all breast cancers over-express Her2; as a result Her2 designations have been clinically determined routinely since 2007 and serve as a prognostic indicator of the disease. Her2 does not become activated upon ligand binding and is therefore classified as an orphan receptor. Activation of Her2 requires either ligand-dependent heterodimerization with

EGFR (Epidermal Growth Factor Receptor) or a different membrane receptor.

7 Alternatively, ligand-independent homodimerization can also generate an active complex.12 Activation of Her2 receptors is an early event in breast tumorogenesis and its unique expression in human breast tumors has established this receptor as a clinically validated therapeutic target.

1.1.1.3 TNBCs

The third major breast cancer subtype defined by receptor expression is designated

“basal-like” and shares significant overlap with the triple-negative class of breast tumors

(TNBC) (Figure 4).13 Both basal-like and TNCB cancers are characterized by a lack of all three characteristic breast-cancer receptors: ER, PR, and HER2. These triple-negative/

TNBC cancers display aggressive growth, invasiveness, and are associated with characteristic histologic features. These histological features include: solid-pushing borders, geographic areas of necrosis, and regions with dense lymphocytic infiltration.

TNBCs constitute 10-25% of the breast cancers commonly presented in patient populations, and are the most aggressive histological subtype with respect to both morbidity and mortality.14

Figure 4 A, B: Extensive overlap of expressed genes between the basal and TN subtypes. A set of 50 genes (Pam50 or Prediction Analysis of Microarray 50) was developed for classifying subtypes of breast cancer.13-14

8 TNBCs are a diverse and heterogeneous group of tumors that -by definition- lack estrogen receptors, progesterone receptors and do not show over-expression of HER2 receptors. The majority of the tumors classified as TNBCs are highly malignant and only a subgroup of TNBCs respond to conventional chemotherapy resulting in a less than favorable prognosis in most cases. Results from decades of research have identified important molecular characteristics that can subdivide this group of breast cancers further. High-throughput cellular profiling techniques including sequencing, pathway analyses, and integrated analyses of alterations at the genomic level the gene transcription level have improved understanding of how bimolecular alteration results in tumor development and progression. The current challenge is to exploit this knowledge to develop new therapeutic agents.15

9 Table 2 below delineates the subtypes of breast cancer and their histology.

ER+ Her2 Basal Molecular Luminal A Luminal B Subtype Genetic profile Amplified ER related genes Amplified Her2 ER-/ PR-/ (A>B) related gene on Her2 - chromosome Increased Basal 17q CKs Histologic correlates

Higher-grade Lower-grade ER+ High-grade, High-grade, ER+ +/– apocrine sheet-like, features necrosis, inflammation

Prognosis Good Worse than Poor Worse luminal A Less Response to responsive; Intermediate Higher Higher Chemotherapy most likely to respond to endocrine therapy HER2-targeted Currently Targeted Hormone therapies therapies investigational therapies

Table 2: The characteristics of the basic molecular/intrinsic breast cancer subtypes.16

The luminal subtypes A & B share expression of estrogen receptor (ER)–related genes and have better overall survival than the HER2-related and the basal-like subtypes, which are typically ER negative. To establish the prognosis of the disease, tumor grade and factors such as, the stage of cancer, patient’s age and general health is determined. A lower grade cancer is well differentiated and indicates a better prognosis, whereas, a higher grade cancer is poorly differentiated and may grow and be invasive more quickly.

10 Thus, a more aggressive treatment may be required for higher grade breast cancer subtypes. Targeted therapies are available for patients with ER-positive and HER2- positive tumors, while those with TNBC are left without the option for the above therapies.

1.2 Epithelial to Mesenchymal transition/ Mesenchymal to epithelial transition

This invasive property of breast cancers was recognized over 120 years ago by Ram´on y

Cajal who described the process of how breast ductal epithelial cells developed a different cellular morphology and also developed the ability to migrate into the stroma to become an invasive cell type. EMT has been described as a process of cell phenotype alteration where epithelial cells are altered to a cell phenotype more representative of a mesenchymal type. Epithelial cells are attached to the basement membrane and are polarized. They are tethered together as a flat 2-dimensional matrix through E-cadherin and other protein strands. Mesenchymal cells, on the other hand, have decreased E- cadherin and are less strongly tethered to adjacent cells. Other cadherins such as N- cadherin and cadherin 11 are significantly up-regulated. It has been shown that breast cancers that lack expression of E-cadherin show increased tumorigenesis and metastatic potential.17 Therefore, it has been proposed that EMT is a key transitional program invoked during cancer invasion, metastasis, and development to chemotherapeutic resistance.18 It was subsequently recognized that pro-oncogenic transcription factors could produce this EMT change of phenotype.19 The most commonly associated pro- oncogenic transcription factors are Snail, Twist and Zeb. Snail is an oncogene that is

11 activated by FGF, Wnt, and TGF Activation of Snail directly suppresses the epithelial phenotype by repression of E-cadherin expression. Additionally Snail promotes cellular motility. Both Snail1 and Snail2 are potent inducers of EMT20 and are highly expressed in breast, GI tract, ovarian, and pancreatic tissue.21 The amount of Twist1 activation, the loss of E-cadherin, and the increase of vimentin are all correlated with increased tumor cell motility, metastasis, tumor cell presence in the blood, and development of new remote lung tumors.22 ZEB proteins are zinc finger E-box binding homeobox1 transcription factors. Elevation of ZEB1 levels inhibit expression of the T-lymphocyte- specific IL2 gene. ZEB1 also represses the E-cadherin promoter indirectly and induces

EMT in the presence of SMARCA4/BRG1.23 It has been found that activation of MAPK cascades cause an up regulation of Snail, Twist, and Zeb genes24 and thus activation of these pathways may represent early pro-oncogenic events.

Figure 5: External contributions to EMT.24

Figure 6: EMT/MET.24

1.3 Protein kinases as drug targets:

Protein kinases catalyze many of the most vital physiological and biochemical processes in the human body.25 Protein kinases can directly phosphorylate proteins, typically at

Ser, Thr, or Tyr residues. This biochemical event alters the local and net charge of the protein. This alteration can result in the acquisition of new chemical, biological, and physical properties for the substrate protein. These kinase-mediated phosphorylation events can change the shape of proteins, modify biochemical activity, alter protein- protein interactions, and alter sub-cellular localization. There are in excess of 510 known

13 human kinases. Of these, about 90 % of the known kinases, or kinome, phosphorylate

Ser, Thr, or Tyr and are known as “Typical” kinases. The biochemical mechanism of these typical kinases are increasingly well understood.25 As a group of related structures, these typical kinases often display the ability to exist in either an active or inactive form

(often based on their own post-transnationally modified state) and bind ATP and Mg++ ions in well-conserved manner. This allows them to have significant homology in the

ATP binding domain, whereas significant differences in the substrate-binding domains, and to have a carefully conserved enzymatic mechanism for both substrate binding and product release. The remaining 10 % of the kinases in the human kinome are structurally very different than the typical kinases and display less homology, and significant differences in their activation strategy and proposed enzymatic mechanism. These atypical, or “pseudo-kinases” are often compartmentalized into sub-cellular regions with other typical kinases and are proposed to mediate cell-signal transduction, scaffold proteins, or modify gene transcription.25

Due to the large number of different kinases and their important biological roles, many kinases have been utilized as therapeutic targets for drug action. A list of kinase inhibitors approved in 2013 are listed in Table 3. The involvement of kinases in the regulation of basic cellular functions including proliferation, growth, differentiation, and apoptosis has resulted in several kinases as being important therapeutic targets for the treatment of cancer. Although at the current time there are more than 510 known human kinases, only 50 kinases have been the focus of significant medicinal chemistry and drug- design efforts.26 The therapeutic category for most, but not all, of these kinases is cancer.

14 Drug Structure Inhibitor Known Indication

type target

Afatinib27 Irreversible EGFR NSCLC

(Gilotrif) Type I

Dabrafenib28 Reversible B-RAF Melanoma

(Tafinlar) Type I

Ibrutinib29 Irreversible Bruton’s Mantle cell

(Imbruvica) Michael kinase lymphoma

acceptor and CLL

Trametinib30 Type III MEK 1/2 Melanoma

(Mekinist)

Table 3: Kinase inhibitors approved by FDA in 2013.

15 The biomechanical events for typical kinases are increasingly well understood. Ser/Thr- as well as Tyr- protein kinases share a catalytic domain of ̴ 290 residues in which the active site is sandwiched between an N-terminal lobe composed of a β-sheet, a single α- helix (the “C helix”), and a larger C-terminal lobe, connected by a linker or “hinge” region. The C-terminal lobe is comprised predominantly of α-helices and includes an activation segment, a region of 20–35 residues located near a highly conserved DFG motif (this abbreviation is the single letter amino acid codes for Asp, Phe, and Gly), and an APE motif, which is less conserved.31 In its active conformation, the C helix packs against the N-terminal lobe, and the Asp of the DFG motif chelates a Mg++ ion that coordinates with the phosphoryl oxygen atoms of ATP and helps coordinate the nucleotide’s geometry and electronics to facilitate transfer of the terminal -phosphate.

Kinases then phosphorylate the hydroxyl group of Ser, Thr, or the Tyr residues on their protein substrates using the γ-phosphate group of ATP carefully positioned in the ATP binding site of the kinase (Figure 7).

Figure 7: Schematic diagram of protein kinase catalytic mechanism in case of PKA (Protein kinase A).25

In the inactive conformation of typical kinases, the DFG motif exists in a different conformation where the Phe of the DFG motif is turned in toward the ATP site25 and the

16 Asp is directed away from the ATP site. This is referred to as the inactive ‘DFG out’ conformation indicating the relative location of the Asp residue of the DFG motif.

Conversely, the active form of the kinase where the Asp is directed toward the ATP site

(to assist with the binding of ATP and Mg++ ions) is referred to as the “DFG” in conformation (Figure 8).

Figure 8: Active catalytic site of PKA with ATP and substrate. PDB ID: 1ATP.25 The structure represents a ‘DFG in’ conformation.25

1.3.1 Kinase catalysis and inhibitors

As knowledge of kinases has increased, largely from X-ray crystal structures (there are in excess of 170 protein kinase structures deposited in the PDB) and enzyme kinetic studies, it has been recognized that there are multiple ways to inhibit a kinase. These different

17 ways to inhibit a kinase have been categorized by the geometry of the inhibitor binding on the enzyme surface.

Type I inhibitors: The initial kinase inhibitors were designed as ATP-site inhibitors. The small molecule competed with the endogenous ligands, ATP and Mg++, for the ATP binding site. These reversible competitive inhibitors bound the catalytic domain with the

DFG motif oriented with a “DFG-in” conformation. These type I inhibitors competed with ATP for the active form of the protein kinase. The chemical structure of these inhibitor molecules mimic ATP and form hydrogen bonds with the hinge region at the catalytic domain (Figure 9).

Figure 9 a, b: Design strategy of Type I inhibitors mimicking select ATP interactions

In recent past, two Type I small molecule inhibitors of the MEK5/ERK5 pathway, 1

(BIX02188) and 2 (BIX02189) were identified with IC50 values of 4.3 and 1.5 nM, respectively.32

Figure 10: Examples of Type I inhibitor.

Achieving kinase isoform selectivity, however, has still remained as a major concern because most kinases share a highly conserved ATP-binding site with similar glycine- rich loop.33 Recently, inhibitor binding sites other than the catalytic -or the ATP site- have been exploited for inhibitor design with the goal of improving target selectivity based on the observation that regions adjacent to the ATP binding domain vary among the different kinases.

Type II kinase inhibitors34 : Type II kinase inhibitors bind to the ATP site after the phosphorylation event. The ATP site is then converted to the ADP release site as the

DFG motif converts to the “DFG-out” conformation, releasing the side-product molecule of ADP and generating the inactive form of the kinase. This DFG-out conformation exposes a new unoccupied hydrophobic pocket (previously occupied by the phenyl side chain of the Phe in the DFG motif) permitting new interactions to be explored and exploited by small-molecule inhibitors. This practically results in more potent compounds (by a factor of a 20-fold increase in potency) with better selectivity than is achievable by targeting the ATP site with the DFG-in conformation. However, mutations can occur at non-essential sites that prevents drug binding (binding the DFG-out

19 conformation), but not the endogenous substrate ATP from binding. ATP binds the

DFG-in conformation. Overall, type II kinase inhibitors may be more prone to the development of resistance than type I kinase inhibitors.

Figure 11: Type II inhibitors design with DFG “out” conformation.

Type I ½ kinase inhibitors34: The type I½ inhibitors bind to the ATP site with the DFG motif in the ‘DFG in’ conformation. These inhibitors utilize the ATP binding pocket, extend past the “gatekeeper” reside at the back cavity of the catalytic domain, and then fill a smaller hydrophobic pocket proximal to the “Phe” binding site. These kinase inhibitors have all the features of type I inhibitors and some of the features of type II inhibitors. They are likely to be more potent than just type I inhibitors (typically 5-times more potent than structurally similar inhibitors that do not utilize the smaller hydrophobic pocket) and selective than type I inhibitors. However, type I½ inhibitors may still be prone to the development of resistance as are type II inhibitors, but perhaps to a lesser extent.

Figure 12: Designing of a Type I ½ inhibitor.

Type III or allosteric site inhibitors:35 An allosteric site, also known as the Type III site, lies adjacent to the ATP-binding domain in many kinases. This feature, however may not be present in all kinases. Type III kinase inhibitors are small -mostly hydrophobic- molecules that do not make any interaction with the hinge region of the

ATP binding pocket. These compounds bind in a “side pocket” of the kinase adjacent to where the -phsophate of ATP is located. Binding of these type III inhibitors lead to a conformational change in the α-C-helix region and results in a global conformational change in the overall enzyme shape. The conformational change prevents activation of the t-loop via phosphorylation. Theoretically type III kinase inhibitors could be stoichiometric (with respect to the enzyme) inhibitors of kinase activity without competition for occupancy by endogenous substrates such as ATP or ADP. The reality of this type of kinase inhibition was confirmed by X-ray crystallographic analysis approximately 7 years ago and has been confirmed for MEK1,2 for several small molecules since. Despite this, it has only recently been designated as a specific inhibitor site and infrequently exploited for medicinal chemistry design strategies.35

Figure 13: Type III and Type IV binding pockets with a type III inhibitor (PD-325089) bound in the allosteric pocket, adjacent to the ATP pocket; PDB id: 3EQG.35

The practical utility of a type III inhibitor was in question, but the recent FDA approval of the MEK1/2 inhibitor Mekinist as an anti-cancer agent confirms the success in using this design strategy for drug design. There is the possibility of development of resistance to a type III inhibitor.36 As a result targeting synergistic kinases or appropriate biological targets may be necessary to develop a type III inhibitor into successful drug.

Therapeutically kinases play a very important role in interpreting the external/environmental stimuli into the internal cellular responses/signaling and thus can be exploited to study the underlying causes of various diseases. Mitogen-activated protein kinases cascade are a family of such kinases which have been found to be upregulated in breast and prostate cancers and thus holds the key to the better understanding and treatment of the disease.

22 1.4 The Mitogen-activated Protein Kinase (MAPK) Cascades

The mitogen activated protein kinase pathway (MAPK) is a ubiquitous family of interrelated, serine/threonine and tyrosine protein kinases which participate in signal- transduction pathways that mediate important cellular functions. These cascades were originally discovered in yeast37, but were subsequently found to be distributed across multiple species including humans. The activation of these pathways can have multiple and complex initiation events. In many cases these pathways are activated by G protein- linked cell surface receptors including receptor tyrosine kinases and thus are activated by signaling molecules such as growth factors, cytokines, neurotransmitters, hormones or various cell stressors. The eventual outcome can be equally diverse including protein phosphorylation, activation of transcription factors, post-translational modification of cytoskeletal proteins and enzymes. These outcomes may dramatically modify embryogenesis, cell survival, differentiation, proliferation, and cell death.

These MAPK pathways all involve a sequence of ordered phosphorylation events, in which a mitogen-activated kinase kinase kinase (MAPKKK or MEKK) phosphorylates the next downstream mitogen-activated kinase kinase (MAPKK or MEK), which subsequently phosphorylates the third mitogen-activated kinase (MAPK or ERK). The

ERKs activate the various downstream transcriptional factors which regulate the important cellular processes such as proliferation, gene expression and cell death. This three-tiered kinase cascade is highly conserved across the species. In humans the most extensively studied is the Raf/MEK/ERK pathway due to its relevance to multiple types of cancer.38 In Figure 14, a simplified outline of the human three-tiered kinase cascade is presented.

Figure 14: The MAPK signaling pathway.8

MAPK cascades may utilize a specific kinase for several different signaling cascades.

This is true for both yeast and human MAPK cascades. Although these pathways have been extensively studied and are increasingly well characterized in yeast37, there is a substantial deficiency in the current understanding of human MAPK cascades. In yeast several strategies have been identified that produce many well-defined cascades with seemingly redundant use of specific MAPK enzymes. These strategies result in very little erroneous cross-talk between the pathways. Some of the yeast MAPK pathway strategies identified to date include the use of scaffolding proteins that carefully recognize and order the MAPK members correctly, dephosphorylation by phosphatases, and conformational changes resulting from kinase oligomerization. In humans these

MAPK strategies have also been identified, along with sub-cellular compartmentalization, association with structural biomolecules, and possibly other

24 strategies. In a manner similar to yeast MAPK cascades it has been proposed that these strategies when employed in human MAPK cascades may result in well-defined specific

MAPK pathways with –at first glance- seemingly redundant phosphorylation events. To date it appears that the MEK members of the MAPK kinases are specific for a single

ERK substrate. Thus understanding the physiological role of each MEK may assist in understanding the pathways that utilize that specific MEK. The development of small- molecule inhibitors for each isoform of MEK may assist in understanding the normal and pathological roles for each MEK isoform specific pathway.

1.5 MAPK kinase or MEK

The MEK/ERK1/2 pathway (namely, the Ras–ERK pathway) is the best characterized of the MAPK pathways (Figure 15).39

Figure 15: Crystal structure of MEK1 with Mg++ATP in the active site. PDB ID: 3EQC.

25 1.6 MAP kinase

Of the eight different MAP kinases known to exist within the mammalian cells, extracellular signal-regulated kinases (ERK) 1/2, c-Jun NH2-terminal kinases (JNK)

1/2/3, p38 and ERK5 have been well recognized and studied. The other ERKs and their associated pathways: ERK 3, 4, 7 and 8, still require significant studies. Activation of the

ERKs by its respective MEK requires dual-phosphorylation of specific Thr and Tyr residues on the substrate ERKs activation loop near the kinase domain within a highly conserved T–X–Y motif. Residue X corresponds to a glutamic acid (Glu; E) for ERKs, proline (Pro; P) for JNK and glycine (Gly; G) for p38 subfamilies substrates. This is shown below in Figure 16. As a result MEKs can be categorized as TY dual-specificity kinases. Note that the sequence presented to both MEK1/2 and MEK5 are identical. The substrate sequence presented to MEK3/6 or MEK 4/7 is TGY and TPY respectively.

Figure 16: ERK1/2, ERK5, p38 MAPKs (α,β,γ,δ) and JNK 1/2/3 are arranged into a hierarchical three-tier system known as a MAPK module.40

26 1.7 MEK5

MEK5 is the MAPK kinase upstream of ERK5. It is activated by either MEKK2 or

MEKK3. MEKK2 has higher binding affinity for MEK5 than MEKK3 although both bind the N-terminal of MEK5.8, 41 MEKK2 and MEKK3 phosphorylate MEK5 at both

Ser311 and Thr315 residues, resulting in the dually phosphorylated pSer-pThr-MEK5, the enzymatically active form of MEK5.42 Two splice variants of MEK5 have been identified: MEK5-α and MEK5-β (Figure 17). The difference between the splice variants is the length of the variants; MEK5- is 89 amino acids longer than MEK5- in

N-terminal domain. Although, specific functional differences between the two splice variants has yet to be identified, it has been shown that the ratio of MEK5- to MEK5- expression was elevated in cancer cell lines.43 It has been postulated that this structural difference may result in a functional difference between the isoforms.

Figure 17: MEK5 splice variants and the pertinent functional domains.43

MEK5 shares a 48% overall sequence homology with MEK1.8 ERK5 is the only substrate of MEK5. This makes MEK5 a unique target for the selective treatment of disease states such as cancer.

27 1.8 ERK5

ERK5, also known as MAPK7 and BMK1 (big map kinase), was first recognized in

1995. MEK5 is the only known upstream MAPKK that can directly activate ERK5.

Similar to ERK1/2, ERK5 also has a dual phosphorylation site characterized by a TEY sequence (Figure 18). The N-terminus catalytic domain shares 51% identity to ERK1/2.

ERK5 and is characterized by the presence of a larger C-terminus extension than any other ERK enzyme.44 This large C-terminus domain has been proposed as being responsible for activation, autophosphorylation, subcellular localization, and nuclear shuttling. ERK5 is an approximately 100 kDa protein and at 816 residues long, hERK5 is one of the largest of the MAPK family members.44-45 It has been proposed that the large size of ERK5 is a functional replacement for the scaffold proteins commonly associated with the smaller ERK isoforms.37 The functional domains of ERK5 are shown below in Figure 7. Although platform proteins do not associate with ERK5, other proteins do associate with MEK5 and modify its biological function.

Figure 18: Structure and functional domains of ERK5.39

28 1.8.1 ERK5 localization

The unique C-terminal tail of ERK5 contains a transcriptional activation domain, which can either phosphorylate the various transcription factors, or can directly act as a transcriptional co-activator. In the resting state, non-phosphorylated ERK5 folds into a

“U” shape and the N-terminal domain interacts with the C-terminal domain, to form an

NES region that has been shown to interact with Hsp90. In this instance, Hsp90 behaves as a cytoplasmic anchor protein (Figure 19). This interaction is responsible for keeping resting-state (non-phosphorylated and Hsp90-associated) ERK5 in the cytosol.

MEK5-mediated phosphorylation of ERK5’s TEY motif results in ERK5 activation, and permits ERK5’s additional auto-phosphorylation at its C-terminal which leads to the disruption of the intra-molecular interactions. This induces a conformational change in pERK5 that results in dissociation of Hsp90unfolding of ERK5, exposition of the NLS region, and subsequent nuclear translocation of pERK5.

Figure 19: Mechanism of nucleocytoplasmatic transport of ERK5; TAD: transcriptional- activation domain.46

1.8.2 ERK5 crystal structure

The X-ray crystal structure of human ERK5 kinase domain in complex with inhibitor 3 was recently determined by Knapp et al (Figure 20).47 The structure revealed differences in residues in the ATP-binding site, compared to the other ERKs, p38s and JNKs and has lead the way for development of selective ERK5 inhibitors.

Figure 20: Crystal structure of ERK5 with inhibitor 3. PDB ID: 3H9F.48

1.8.3 Downstream activation of ERK5

ERK5 regulates a number of downstream transcription factors including the myocyte enhancer factor (MEF) family, MEF2A, C, and D.49 ERK5 phosphorylates MEF2C on

Ser387, which increases MEF2C transcriptional activity resulting in an increase in gene expression of c-Jun. ERK5 is also able to directly enhance the transcription of c-Myc,

CREB and Sap1a and c-Fos.49b, 50

1.9 MEK5/ERK5 Pathway and Cancer

The MEK5/ERK5 pathway is a unique signaling pathway triggered by external stimuli such as cytokines (LIF, CT-1, IL6), mitogens (EGF, NGF, FGF-2, BDNF), and stress including radiation51, palyotoxin52, and phorbol ester treatment52. The MEK-5 pathway allows cells to survive extreme metabolic demands and is significantly up-regulated in response to stress.

During signal transmission, the N-terminus of the activated form of pMEK5 binds to the functional domain of its unique substrate ERK5 to convey the activation signal via

31 phosphorylation events described above. Active pERK5 has two possible cellular pathways for signaling; either by activating downstream molecules, such as, Sap1, cFOS, c-Myc and MEF2 in the cytosol or by nuclear translocation (described above) and transcription activation.53

Figure 21: MEK5/ERK5 pathway.8

1.9.1 Role in tumor development

Significant effort has been directed toward the development of MEK1/2 inhibitors as anticancer agents. In July 2013, Mekinist, a type III MEK1/2 inhbitor, has been approved for cancer therapy. Due to the development of full inhibition of MEK1 and the partial inhibition of MEK2, it is currently recommended that Mekinist be given with a BRAF inhibitor.36a This is the first in class type III MEK inhibitor and it is recommended that other MEKs may deserve additional scrutiny. Over-expression of the MEK5/ERK5

32 pathway has been demonstrated in many cancers. Most importantly the ERK5/MEK5 pathways has been shown to be upregulated in breast cancers.10, 54 This upregulation is most important in the potential treatment of triple negative breast cancers, which at the current time are aggressive and refractory to the majority of present therapeutic strategies.14 The up-regulation in breast cancer occurs early and is correlated with the expression of ER  and  gene products in a receptor independent manner.10 This is consistent with MEK5/ERK5 as an early mediator of ER(-) breast cancers.10

Additionally it was found that immunohistochemical staining of excised human breast cancer tissue for ERK5 (phospho-form not specified) indicated decreased survival time for patients who over-expressed ERK5.54a Analysis of gene products from these same tissues identified that cells overexpressing ERK5 were consistent with excessive constitutively active HER2 activity. Thus MEK5 inhibitors have been10 suggested as potentially useful for the treatment of the most dangerous and least successfully treated variety of breast cancer.

The MEK5/ERK5 pathway is also significantly up-regulated in squamous cell carcinoma and prostate cancer. Sticht et al. discovered high levels of ERK5 association, but not

ERK1 expression, in advanced stage tumors and lymph node metastases from oral squamous cell carcinoma (OSCC) patients.55 Mehta et al. not only documented moderate expression of MEK5 in 44% of prostate tumors overall, but also identified that in 37% of tissues excised from patients, MEK5 was highly expressed.56 In malignant mesotheliomas, HGF (Hepatocyte growth factor) has been shown to be highly expressed and stimulates proliferation through the PIK3/ERK5/fos-related antigen 1 (fra-1) pathway. Furthermore, ERK5, but not ERK1, was shown to contribute to the Src-

33 mediated structural disruption of the actin cytoskeleton. This disruption may be the first phase in development of metastatic disease.48, 55 This observation is consistent with

MEK5/ERK5 representing an early conversion to an invasive cancerous phenotype.

Taken together, these observations suggest that MEK5/ERK5, as a downstream target of

EGFR, may be a novel molecular target and potential interference point in future therapeutic approaches given its association with advanced tumor stage particularly if mutations have occurred upstream at the level of the tyrosine receptor kinases.

Two commonly used human breast cancer cell lines, BT474 and SKBR3, have been shown to express a constitutively active form of ERK5 in activated ErbB2, ErbB3 and

ErbB4 cells. When these cells were treated to ErbB receptor activity inhibitor, ERK5 was found to be inactivated thus indicating the involvement of ERK5 in proliferation of these breast cancer cells.57 Thus it is reasonable to speculate that the MEK5/ERK5 cascade may be a pro-cancerous phenotypic adaptation.

After considering the above recent findings regarding MEK5, it is apparent that this MEK isoform requires additional study. The development of new small molecule inhibitors of the MEK5/ERK5 signaling pathway could contribute to these studies that examine the pathophysiological role of MEK5, particularly in the previously mentioned cancers.

It may well be that multiple activity MEK inhibitors may be desirable and perhaps useful, but at the current time it is unclear how the various MAPK pathways interact. It is known that one MAPK pathway can modify another MAPK pathway.58 Additionally, there are more complex biological mechanisms that that can apparently upregulate the activity of other MAPK pathways.59

34 1.10 Development of Mekinist as MEK1/2 inhibitor60

Figure 22: Compound 4 and analogues.61

Compound 4 (CI-1040/ PD184352) is a well characterized, highly potent and selective

Type III inhibitor of MEK1 and MEK2 initially identified in 1999. It displays significant antitumor activity in vivo54b, 62 and was the subject of a multicenter Phase II clinical trial in patients with advanced non small-cell lung cancer, breast cancer, colon cancer or pancreatic cancer.63 However, the advancement of 4 into clinical trials revealed a number of pharmacological problems, including metabolic instability and low overall bioavailability.64 Second generation analogs, for example compounds 5, 6 and 7, displayed greater potency and improved oral bioavailability. The crystal structures of

MEK1 and MEK2 with compounds 6 and 7 respectively (Figure 23), identified that these

35 small molecule inhibitors bind to the novel allosteric (or type III) binding pocket adjacent to -but not overlapping with- the Mg++/ATP binding site.65

Figure 23 a, b: MEK1 and MEK2 homodimer structures respectively. The nucleotide- binding loops are blue, the catalytic residues are gold, the activation loops are red, ATP is pink and the ligand is orange.66

The key interactions observed between the inhibitors and the adjacent amino acid residues of this binding site were (Figure 24):61

1. Numerous van der Waals interactions of the B ring of the inhibitor with the amino

acid side chains of the hydrophobic pocket formed by Met143, Ile141, Leu118,

and Phe209. Phe209 also formed a critical edge-to-face aromatic interaction with

the B ring.

2. A hydrogen bond interaction was shown to exist between the 4-fluoro atom of the

inhibitor A ring and the backbone amide of Ser212.

3. The backbone carbonyl oxygen of Val127 formed an electrostatic interaction with

the 4′-iodide at the back of the pocket. This interaction is now known as the

halogen bond.67

Figure 24: MEK1/2 interactions with inhibitor 5 (PD325901).

A halogen bond is a newly identified, short-range electrostatic interaction of a halogen atom with a lone electron pair from heteroatoms including O, N and S. The electron density from the lone pair of the heteroatom is loaned to the antibonding orbital of the halogen (Figure 25).

Figure 25: Schematic of halogen bond.

Figure 26: Halogen bond from compound 5 to acyl lone pair of backbone Val127 in 3EQC. Bond angle: 127.8 o, Bond distance: 3.11Å

This led to the structure-guided design of a new class of potent inhibitors of MEK1 and

MEK2, represented by 4-(phenylamino)-5-carboxamido-2-pyridone 8, where, the difluorophenyl group from the diphenylamine class was replaced with the pyridine in order to ascertain the improvement of the hydrogen bond interaction with Ser212. SAR studies were conducted to optimize the R1, R2, and Ar on a variety of relevant parameters.

1. Optimization of the Carboxamide Side Chain61

From the second generation diphenylamines (5 and 6), it was expected that

introduction of a carboxamide would both increase the potency and improve the

pharmacokinetic properties relative to the acid. The simple carboxamide 9 (table 4)

on the series 4-(phenylamino)-5-carboxamido-2-pyridone was highly potent against

the Raf-MEK-ERK cascade and was significantly active in the colon26 cellular assay.

38 The colon26 cellular assay utilizes murine colon adenocarcinomas originally isolated from mouse lung that are stable yet metastatic after 26 passages. It is used as a model for aggressive metastatic cancer.68 This cell line is convenient to maintain sensitive to MEK1 inhibitors and is a standard in vitro test for cellular efficacy of MEK inhibitors against malignant tumors.69 The mono- or dimethylation of the amide nitrogen greatly diminished this potency.

Compound R1 Cascade Cellular

IC50(µM) IC50(µM)

9 NH2 0.023 0.004

10 CH3NH 0.195 --

11 (CH3)2NH 0.627 --

12 HOCH2CH2ONH 0.018 0.005

Table 4: Optimization of the Carboxamide Side Chain.

2. Substitution on the Pyridone Nitrogen61

39 The primary pyridone, 13, was the most potent among variations examined at R2.

Other compounds displayed a loss of potency in both isolated enzyme and in cellular

assays when compared to compound 9 (table 5).

Compound R2 Cascade Cellular

IC50(µM) IC50(µM)

9 NH2 0.023 0.004

13 H 0.100 0.63

Table 5: Substitution on the Pyridone Nitrogen.

3. Replacement of the 4′-Iodide61

The 4′-iodide was replaced with various halogens and/or alkyl groups to examine the

contribution of the 4′-iodide to MEK1 activity when the molecule also contained the

pyridone acyl as an H-bond acceptor in the central ring (table 6).

Compd R1 R3 Cascade Cellular

IC50(µM) IC50(µM)

9 NH2 2-F-4-I 0.023 0.004

14 NH2 2-F-4-CH3 2.97 --

15 NH2 2-F-4-Br 1.91 0.093

16 NH2 3,4-dichloro 5.72 3.9

Table 6: Replacement of the 4′-Iodide.

40 From the results obtained, it was clear with hindsight that the interaction between the 4′- iodide and the lone pair of the backbone amide acyl of Val127 (evidenced in both the

MEK1 and MEK2 X-ray crystal structures) was critical to the activity of this series of

MEK1/2 inhibitors.

1.10.1 Development of Mekinist from the 4-(phenylamino)-5-carboxamido-2- pyridones60

Compound 17 was identified by high-throughput screening based on the previous studies which showed 4-(phenylamino)-5-carboxamido-2-pyridones to be potent MEK inhibitors.

It was shown to have an anti-proliferative activity against human cancer cell lines ACHN and HT-29 with IC50 values of 4800 and 990 nM respectively. However, due to the high hydrophobicity of the molecule (ClogP 6.3)60, SAR studies were conducted and led to 18 which displayed improved potency 4-fold and a bit reduced hydrophobicity (ClogP

5.0).60

Figure 27: SAR studies on 17 and development of 18.60

To further increase the potency, substitution on the aniline ring was examined and various alkyl groups and halogens were prepared. Variations on the heteroaromatic core were also conducted to generate the highly potent 19. Unfortunately compound 19 also displayed very low aqueous solubility and high hydrophibicity (ClogP 6.0).

42 Figure 28: Substitution on the aniline and heteroaromatic core transformation.60

Substitutions were tried on the indicated phenyl ring on the heterocyclic core of 19 with more polar groups was examined to optimize the aqueous solubility and oral bioavailability while improving potency if possible (Figure. 29). GSK1120212,

Mekinist, shown as compound 20 was developed as the DMSO mono solvate. It showed good primary activity, desirable solid state properties, oral bioavailability and acceptable although not ideal aqueous solubility.

Figure 29: Polar group (R) substitution SAR let to the development of GSK1120212.60

Compound-immobilized affinity chromatography with compound 20, identified MEK1/2 as the target kinase.70 Starting from an initial lead 17, SAR studies led to the discovery of 20, GSK1120212 (JTP-74057 DMSO Solvate), a highly potent, selective, and orally active MEK inhibitor. It also displayed a remarkable potency in cellular assays for

ERK1/2 phosphorylation and growth inhibition. Significant dose-dependent tumor growth inhibition was observed for a span of 21 days of daily oral administration.60

43 Mekinist (GSK1120212) (trametinib) was approved in May of 2013 for use in treatment in melanoma patients with BRAF V600E or V600K mutation either alone or with co- administration with Dabrafenib (tafinlar).30a Dabrafenib is a BRAF inhibitor with

IC50 values of 0.65, 0.5, and 1.84 nM for BRAF V600E, BRAF V600K, and BRAF

V600D mutants, respectively.

8.3 Other MEK inhibitors

PD98059 and U0126 were early compounds developed to be selective MEK1/2 pathway inhibitors (Figure 30). Full characterization of their specific biological interactions has not been published to date. These compounds do not compete with ATP for binding in isolated enzyme preparations. Both compounds, U0126 and PD 980595, display competitive binding with each other and were assumed to be interacting at single or adjacent site.6a, 71

44b, 71a U0126 inhibited MEK1 in a dose dependent fashion with an IC50 of 0.072 µM.

Both compounds were later shown to poorly inhibit the MEK5/ERK5 kinase cascade in a cellular assay.72

Figure 30: MEK1/2 inhibitors U0126 and PD98059.44b

44 Chapter 2

Chemical Literature Review

The chemistry required for this project can be divided into several discreet categories: aryl bond formation to generate the diphenyl amine portion, amide bond formation, and supporting chemistry.

2.1 Aryl bond formation to generate Diphenylamines

The formation of aryl-nitrogen bonds has been recently reviewed.73 The strategies for forming an aryl-nitrogen bond has its origins back in 1903 with one of the oldest methods to synthesize diarylamines reported by Fritz Ullmann (Scheme 1).74 These Ullmann coupling reactions utilized an aryl halide and an aryl amine along with copper and base to form an aryl-nitrogen bond. Recent variations of the Ullmann reaction have been published75 and modern developments include the use of solvating and activating ligands for the metal, the use of less-reactive electrophilic aryl partners, and the development of methodologies that require less forcing conditions: lower temperature, weaker bases,

45 shorter reaction times, and the use of microwave heating to both increase yield and reduce side reactions. The rationale for these strategies are to permit greater aryl ring variation. The reaction is proposed to proceed through oxidative addition into the aryl- halide bond, followed by ligand displacement by the aryl amid, to yield the final product by reductive elimination of the metal atom. The specific pathway, or more correctly pathways, is (are) still undergoing analysis to confirm the mechanism(s). In certain conditions near quantitative yields have been obtained, although reactions with much lower yields are more typical.

Specifically, Ullmann reported the reaction of aniline with 2-chlorobenzoic acid in the presence of stoichiometric amount of copper to give 2-phenylaminobenzoic acid.74b

Scheme 1: Synthesis of diarylamines by Ullmann Coupling.

Irma Goldberg in 1906, showed that the reaction occurred in the presence of catalytic amounts of Cu (Scheme 2).76 Of particular note for this conversion is that the relative electron flow for the Goldberg variation of the Ullmann coupling has an electron flow in the reverse direction. This raises the possibility that there may be multiple reaction mechanisms for the “Ullmann” reaction, an observation that is still providing ample opportunity for inquiry. Practically, this gives the experimenter wide latitude for variation of reaction precursors to yield the desired product.

Scheme 2: Goldberg synthesis of diarylamines in 1906.76

Additional metal-mediated couplings have been discovered including palladium, nickel, iron and other metal couplings. Of particular note is the development of the Buchawld-

Hartwig palladium couplings.77 This conversion was originally conceived by Hartwig but brought to practical utility by Stephen Buchwald.78 In 2002, Buchwald developed a more efficient palladium catalyst systems for the amidation79 shown below. (Scheme 3).

The adequate solvation of all oxidation forms of the palladium atom, the development of metal/ligand complexes that were more stable to the presence of trace amounts of molecular oxygen, and optimization of the base employed were all advances that brought this reaction from an interesting synthetic observation to a widely-used synthetic methodology.

Scheme 3. Synthesis of diphenylamines using Buchwald Coupling.79

This methodology continues to evolve with an emphasis on new nitrogen sources, improved catalytic turnover achieved by well-solvated pre-catalysts, and the design of more efficient ligands. These reactions require an electron-rich aryl nitrogen and a

47 coupling aryl partner that can readily undergo oxidative palladium insertion. In general, the Buchwald-Hartwig couplings are recommended for aryl groups with substitutions that would be incompatible with the more rigorous Ullman coupling conditions.

In addition to these metal-mediated couplings, ionic SNAr reactions with an appropriately matched nitrogen nuclophile and haloarene behaving as an electrophile have been successfully developed.80 These conversions continue to be explored as useful strategies to form aryl-nitrogen bonds80-81 and find additional utility in forming di-aryl ethers80, 82,

80, 81c-f, 83 and di-aryl thioethers. These SNAr conversions require a very nucleophilic aryl nitrogen atom, typically prepared by deprotonation using an Na+, Li+, or K+ containing strong base. The pKa for these anilines are typically in the 15-25 range so NaH,

NaHMDS, LiNH3, LiHMDS, KH, KOt-Bu, or KHMDS are commonly employed.

The electrophilic partner must be electron deficient and be able to stabilize the proposed

3 sp transition state of the SNAr mechanism. Ortho-halo heterocycles are commonly encountered in this reaction class. Arenes with ortho-substitution by strongly electron- withdrawing groups are also frequently encountered. Specifically, ortho nitro haloarenes,81a, 81f, 84 and multiply-halogenated arenes are commonly used successfully for this reaction.

85 A specific recent example is the conversion by Davis et al. that uses the SNAr reaction to develop a novel route of synthesis of diphenylamines utilizing LiNH2 (Scheme 5).

This route utilizes a less-common, but commercially available base LiNH2. They then

48 surveyed both activated or unactivated aryl halides.85

Scheme 4: Synthetic route for diphenylamines utilizing LiN(iPr)2.

A specific goal and significant finding of this work is that the lithium cation directs the ultimate regioselectivity of this conversion by preferential attack of the aniline anion to the ortho- position of the carboxylic acid. Coordination of the lithium cation with the carboxyl group and the leaving group were postulated as coordinating both the attack and elimination, resulting in a kinetic preference for the ortho substitution product.85

85 Scheme 5. Synthesis of diphenylamine 39 utilizing LiNH2.

Figure 31: Li+ ion directs the attack of the aniline anion to the ortho position of the carboxylic acid.

Attack at the 3-fluoro atom was less likely arising from the absence of a reasonable resonance form with the carboxylate. Attack at the 4-fluoro atom would be lacking any coordination possible, but in the absence of the Li+ coordination effects, could conceivably be as electrophilic as the 2-fluoro atom. Davis confirmed this by using

NaNH2 which did give a mixture of the 2 and 4 substituted produces. Additionally Davis found that only 2-iodo benzoic acid and not 4-iodo benzoic acid was reactive under the experimental conditions examined using LiNH2.

2.2 Amide bond formation to generate Acyl variations

The final relevant chemical conversion is the synthesis of a series of amide molecules.

Many peptide coupling reactions have been reported for the formation of the amide bonds.86 A recent critical review that examines the utility and limitation of many common methods of amide bond-forming strategies provides a convenient basis for surveying strategies employed in this work.86 In general, the carboxylate is activated to convert the relatively weak “OH” group into a better leaving group. Common general strategies include replacement of the OH of a free carboxylic acid with its ester,

50 conversion of the “OH” to a halide anion (acid halide formation), conversion of the “OH” into a carboxylic acid (anhydride formation), conversion of the “OH” into a urea (use of a carbodiimide or uronium reagents), and conversion of the “OH” into a phosphate

(phosphoric acid anhydride and BOP-type reagents). These strategies are listed in order of increasing cost and increasing complexity. The development of multiple different amide bond forming strategies has been driven by the need to have reagents that are reactive, suppress side reactions (specifically loss of stereochemical integrity at the - carbon of -amino carboxylic acids), cost-effective, and have an easy isolation of the desired product from the side products. Practically there is always a compromise the must be reckoned with during the selection of an amide bond formation reaction strategy.

Many times the selection of a specific synthetic strategy or reagent is determined by the specific substrates utilized.

One efficient approach is conversion of an acid to its corresponding acid chloride, and then reacting this now activated acyl with the desired amine to form the required amide.

This reaction usually proceeds at room temperature and employs relatively mild reaction conditions. Potential disadvantages of using an acid chloride to form an amide include: a) generation of very reactive species which is highly moisture sensitive, b) the establishment of acidic reaction environment which makes it unsuitable for acid labile functional groups, c) the possibility of ketene formation if an -proton is present and a sufficiently strong base (pKa 9 or greater) is used, and d) the likely loss of stereochemical integrity if the -carbon has a proton and is stereogenic.

Scheme 6: Synthetic scheme of amides via acid chloride.

Scheme 7: Mechanism of chlorination by oxalyl chloride or phosgene.

Many chlorinating reagents have been reported and the most commonly used are given in table 7.

For some of the compounds required, the formation and use of an acid chloride coupling strategy was found to be a useful strategy. Factors considered for choosing this approach included: the need to significantly activate the carboxylic acid due to its low capacity for nucleophilic attack on the peptide coupling reagent, a decreased likelihood for attached nitrogen atoms to attack the acyl chloride during the course of the reaction, cost of the reagents, precedent, reproducibility, the absence of either a stereogenic -carbon or an extractable -proton, and ease of isolation.

52 Entry Reagent Structure

87 1 Thionyl chloride SOCl2

2 Oxalyl chloride88

Oxalyl chloride with Cat. DMF

89 3 Phosgene COCl2

4 Triphosgene90

5 Pivaloyl chloride91

6 Phthaloyl chloride92

93 7 TPP PPh3/CCl4

Table 7: Commonly used chlorinating agents.

For some of our compounds, the use of a carbodiimide reagent such as DIC and urea- forming coupling strategy was required.

Scheme 8: Mechanism of carbodiimide coupling.

Scheme 9: DIC coupling reaction.

Several of the most commonly used carbodiimide coupling reagents are given below. Of the many carbodiimdide reagents commercially available, DIC and EDCI were found to have an appropriate combination of reactivity, cost, and ease of isolation that recommend their use. The use of a carbodimide coupling strategy was most commonly necessary when the nitrogen atom of the diphenyl amine was sufficiently electron rich to begin to attack the acid chloride during the reaction if an acid chloride coupling strategy was attempted. Thus the carbodiimide strategy was employed when the acid chloride coupling strategy gave a complicated mixture of non-desired products.

54 Entry Abb. Structure

1. DCC94

N,N’-dicyclohexylcarbodiimide

2. DIC95

N,N’-diisopropylcarbodiimide

3. EDCI95

N-ethyl-N’(3-dimethylaminopropyl)carbodiimide

4. CIC96

N-cyclohexyl, N’-isopropylcarbodiimide

5. PIC97

N-phenyl, N’-isopropylcarbodiimide

Table 8: Carbodiimide coupling reagents.

55 Chapter 3

Statement of the Problem

3.1 MEK5

Goal of the research

The goal of this research is to develop potent and selective inhibitors of MEK5 to establish better characterization of the functional role of MEK5 both in vitro and in vivo.

The goals for designing and synthesizing the target compounds are:

 To increase H2O solubility

 To simplify the structure of the compounds and improve ligand efficiency98

 To establish structural requirements for MEK5 versus MEK1 selectivity

 To validate the homology model

Hypothesis:

MEK5 is one component in a signaling pathway that permits cells to survive oxidative stress and is activated by various mitogens (EGF and G-CSF), cytokines (LIF and CT-1), or stress (H2O2 and sorbitol). MEK5 is significantly up-regulated in response to stress including ionizing radiation, palyotoxin, and phorbol ester treatment. These stressors, both external and internal typically activate a cascade of intracellular signaling events.

56 Usually, multiple pathways are activated concurrently. The phosphorylation of various

ERKs by their associated MEKs represents a uniquely parallel event in the signaling cascade. Our hypothesis is that these MEKs represent a non-redundant array of gates for mitogen-activated physiological responses. Development of MEK knockout or knockdown models have not been successful due to either lethal or adaptive phenotypes.

Thus the development of small molecule inhibitors could permit analysis of the role of each MEK across multiple animal models. MEK1 has been the focus of extensive study with Type III inhibitors. MEK5, by contrast has received little attention despite its significant up-regulation in cancers and its up-regulation to oncogenic stimuli. (Figure

32).

Figure 32: The MEK5 signaling cascade.99

57 The need for going towards Type III inhibitors:35

A high degree of sequence homology is displayed amongst the catalytic domain or the

ATP binding site of kinases. The activation loop above the ATP binding site is highly conserved and thus it is a significant challenge to achieve selectivity by targeting the

ATP-site. The Type III inhibitor site is largely a hydrophobic binding pocket outside of the ATP binding site that has been recognized as having the potential for inhibitory kinase activity. Exploration of the type III binding site is a new pharmacological strategy to inhibit kinases that has only appeared within the last 3 years, and has only been applied to non-MEK1 kinases until very recently. The rational design and synthesis of type III site kinase inhibitors provides a recently emergent opportunity for achieving kinase selectivity of protein kinases requiring phosphorylation for T-loop activation. The synthesis of type III inhibitors for the various MEK isoforms, initially MEK5, will assist in discerning the biological role and relevance of these signaling cascades that are indicated in significant human pathological states.

Lead compounds:

CI-1040 (4) and PD325901 (5) were developed by Pfizer in the early 1990’s as MEK1/2 inhibitors with the goal of developing orally-active anti-cancer agents.61-62 These were subsequently found by the Cavanaugh lab to inhibit MEK5 as well. Both CI-1040 and

PD325901 have been presented as Type III or allosteric site inhibitors on the basis of X- ray crystallographic analysis with MEK1 and these compounds have progressed into the clinical trials. CI-1040, however suffered from poor aqueous solubility and rapid clearance.100 SAR studies led to the development of PD325901 wherein the 2-

58 orthochloro was replaced by 2-orthofluoro and the hydroxamate side chain was modified for better pharmacokinetic properties such as solubility and bioavailability.66 PD325901 entered clinical trials for the treatment of advanced non-small cell lung cancer; however clinical trials were terminated due to the occurrence of ocular and neurological toxicity

(ClinicalTrials.gov Identifier: NCT00174369). It has been found that these toxicities are currently believed to reside in MEK2 inhibition in the outflow of the retinal pigment epithelium and results in local increase of luminal pressure in retinal veins.101 The activity and side effects of PD325901 underscores our hypothesis that understanding the

MEK isoform selectivity of specific inhibitors and understanding the in vivo pharmacology are essential goals both for discerning direct activity as well as indirect toxicity.

Name Stage of Frequency of Description of ocular IC50 development ocular toxicities toxicities for MEK CI-1040 Discontinued 6 (9%) out of 67 Blurred vision, altered 17 (Phase II) light perception.63 nM62a PD-0325901 Discontinued 9 (14%) out of 66 Visual disturbances, 1nM62c (Phase I) including 2 cases of 5 (38%) out of 13 RVO.102 (Phase II Blurred vision.103 continuous) 1 (5%) out of 21 (Phase II intermittent) Trametinib FDA Unknown (Phase 2 dose limiting central 3.4 (Mekinist) approved in I) serous retinopathies nM104 (GSK1120212) 2013 Unknown (Phase 2 cases of dry eye, 2 I melanoma) cases of blurred vision Unknown (Phase 2 cases of central II) serous retinopathy

Table 9: Ocular toxicities of MEK inhibitors.

59 Name Stage of Frequency Description IC50 development BRAF mutant Dabrafenib Phase III None reported NA105 0.16 (GSK2118436) (Phase I) nM

Table 10: Ocular toxicity of BRAF inhibitor Dabrafenib.

Recently, CI-1040 finished a multicenter phase 2 Study of CI-1040 in patients with advanced nonsmall-cell lung cancer, breast cancer, colon cancer or pancreatic cancer

(ClinicalTrials.gov Identifier: NCT00033384) (Figure 33). Also, the first MEK1/2 inhibitor ‘Mekinist’ (trametinib) was approved in May of 2013 for use in treatment of patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutations either alone (assuming the absence of prior therapy with a BRAF inhibitor) or with co-administration with Dabrafenib (Tafinlar) (Figure 34). It has recently been suggested that the synergistic effects with dabrafenib may be the result of BRAF mutations resulting from partial inhibition of MEK2 which permit the development of therapeutic resistance through BRAF mutations.

Figure 33: MEK1/2 inhibitors and the lead compounds.

Figure 34: BRAF inhibitor Dabrafenib (Tafinlar).

3.2 Construction of the homology model

The primary protein sequence of MEK5-β-DD (S311D and T315D) (Q13163)106 was aligned with the structure of MEK1 (PDB: 3EQC: 1.8 A resolution) using Acelrys

Discovery studio 2.5.1. The two structures indicated good overlap of the DFG and HRD sequences, DOPE score and Ramachandran plot analysis.

Figure 35: ATP and Type III binding site in MEK1 crystal structure and superimposed homology model of MEK5.107

Figure 36: Type III inhibitor docked in 3EQC.107

PD325901 (5) was docked into the homology model of MEK5DD and made reasonable interactions with adjacent residues (Figure 35). An analysis of the amino acid residues that were proposed to converge to the Type III site by the homology model indicated that the portion of MEK5DD with the right aniline ring could be a source of possible

62 difference between MEK1, 2, and 5. Subsequent CLUSTAL analysis of all 7 canonical

MEK isoforms is consistent with this initial analysis (see Appendix). The glycerol sidechain of PD325901 was originally developed by Pfizer as a surrogate for the triphosphate of ATP with the simultaneous goals of increasing water solubility and potency.

However, upon analysis of the MEK1 structure of 3EQC, it was observed that the binding domains of ATP’s triphosphate and the binding domain of the glycerol portion represented different and non-overlapping portions on the surface of MEK1. The glycerol portion of PD325901 was found to extend into the solvent and although it did increase water solubility, both the more complex synthesis of the hydroxamic acid ester and the toxicity of this group recommended truncation back to the amide. Further analysis and ligand design from the amide portion of the molecule attempted to combine both increased interaction with the MEK5DD surface with better water solubility into the same functional group.

Evaluation of initial compounds during development of the homology model suggested the compound (Figure 37) as one of the most selective MEK5 inhibitors. This compound was bound into the MEK5βDD homology model using both MOE dock and by structural variation from PD325901.

Figure 37: Most selective MEK5 inhibitor from our initial studies (synthesized by Ishveen Chopra).

Figure 38: Ligand specific interactions plot of compound 56 in MEK5 homology model.

Analysis of interaction of the ammonium nitrogen of 56 suggested potential interaction with Asp283 (3.71 Å) and adjacent polar residues through a complex web of interactions

(Figure 38). Thus the N-methyl piperidine moiety was retained with the premise that it contributed to both increased interaction with the MEK5DD surface (on the basis of post-testing rationalization using the MEK5DD homology model) and better water solubility due to probable ammonium ion formation at physiological pH (~ 7).

The designed compounds were proposed as MEK5 type III (allosteric) inhibitors derived from the known MEK1/2 inhibitor PD325901, biological testing, and our MEK5DD homology model.

64 3.3 Specific goals:

The diphenylamine moiety was selected as the core because it exists in the PD325901 structure, provides rapid synthesis, and allows structural variation in locations what were proposed as being both interesting and potentially useful for modification. The major modifications tried were:

I: Side chain variations • Increase aqueous solubility • Examine MEK5 predicted interactions

II: Amide variations

III: Central arene • Establish minimal necessary substitution • Predict MEK5 selective interactions

IV: Terminal arene • Establish minimal necessary substitution • Predict MEK5 selective interactions

1. Side chain variation and amide variation:

65 This is the last synthetic step and represents the most useful point of structural diversification. Goals were to increase interaction with the MEK5DD surface (using the assistance of the MEK5DD homology model) and increase water solubility. Proposed variations included basic amines with various alkyl length linkers (both cyclic and acyclic); these were designed to interact with acidic and polar resides unique to MEK5 versus MEK1. Sidechain variations examined also included synthetic precursors

(amides, acids, tert-butyl carbamates) and characterized side-products.

2. Target compounds with de-iodo diphenylamine:

3. Target compounds with no halogens on the terminal arene ring:

66 4. Central arene variation:

Prior literature61 indicated that the 3 and 4 fluorine atoms, although present on the lead compound PD325901, functioned as more than just electron-withdrawing groups. It has been proposed that the 4-fluorine atom, forms an electrostatic or hydrogen bond interactions with the Ser212 of the C-helix. With an eye toward the eventual design and synthesis of more novel central arene variations, an initial survey of the need and utility of fluorine atom substitution for MEK5 inhibition was conducted.

5. Terminal arene variation:

In a manner analogous to the narrative above, terminal arene variations were prepared to survey of the need and utility of fluorine and iodine atom substitution for MEK5 inhibition. Of particular note, the 4’-iodide atom presented multiple sub-ideal chemical features: high molecular weight (126 Da), large hydrophobic area, and potential photo- instability. However, the 4’-iodidophenyl moiety is scrupulously retained in all compounds that were advanced in the design of MEK1inhibitors; ultimately this 4’- iodidophenyl moiety is present in the structure of the FDA approved MEK1 inhibitor

Mekinist.

67 6. Variations on the nitrogen atom of the diphenylamine:

Analysis of the deposited X-ray crystal structures of MEK1/2 type-III inhibitors indicated the unvarying presence of secondary amines at this position. Additional analysis indicated a geometry of the heavy atoms identified in the deposited X-ray crystal structures consistent with internal hydrogen bonding into a 6-membered ring. Given the rigorous retention of the 4’-iodidophenyl moiety in all MEK1 inhibitors, it was unclear if the highly plausible internal bond from the aniline NH to the acyl lone pair was necessary or useful either for MEK1 or MEK5. The initial compound slated for synthesis to analyze the contribution from internal hydrogen bonding was the acid: the N-methyl diphenyl aniline acid analog, compound 76. This compound is unable to participate in internal H-bonding and although introduction of the N-methyl group would certainly result in additional structural variation, this was viewed as both the most accessible and the most useful first step towards analyzing the contribution of internal hydrogen bonding toward MEK1 or MEK5 activity.

Figure 39: The presence of the N-methyl changes the conformation of the molecule which otherwise had an internal hydrogen bonding leading to a more conformationally restricted analog.

3.4 Results:

For the design and synthesis of MEK-5 inhibitors, medicinal chemistry strategies included: an initial lead and analog design, modification of known Type III MEK-1/2 inhibitors, and computer-assisted design based on a MEK5DD homology model.

Compound 72 was identified as a selective inhibitor of MEK5 mediated phosphorylation of ERK5 in MDA-MB-231 triple negative breast cancer cells, and will be examined in mouse tumor xenograft models.

69 Chapter 4

Chemical discussion

As presented in the statement of the problem, variations were desired at multiple locations on the core structure of the diphenylamine. Although it was initially envisioned to use a single synthetic strategy to prepare all compounds, in practice it was found necessary to use the most appropriate synthesis strategy for each compound required.

Although these required variations in synthetic strategy were determined experimentally, in hindsight these required variations can be readily understood on the basis of understanding the reaction mechanism.

Synthesis of the tetrahalo core: 3,4-difluoro-2-((2-fluoro-4- iodophenyl)amino)benzoic acid (39).

Synthesis of the tetrahalo diphenylamine core, 39, was achieved with the lithium amide displacement approach (Scheme 10) of Davis et al.85 This procedure was scaled up to a

4 gram quantity which was necessary for the desired animal experiments with compound

57 (SC-1-151).

Scheme 10: Lithium amide displacement approach.

Preferential substitution occurs at the ortho-position as indicated above and as identified in the literature.85 The regioselectivity of the addition can be rationalized along the following lines. First, ionic interaction of the carboxylate-oxygen anion and the lithium cation is very likely to occur. This ionic chemistry can be observed experimentally as the initial exotherm noticed as the lithium amide abstracts the carboxylate proton to form the ionic lithium carboxylate. This species is well solvated in the reaction solvent, THF.

Davis et al.85 have postulated that the lithium cation associated with the carboxylate coordinates with, and directs, the aniline anion towards the ortho-position of the carboxylate to facilitate initial nucleophilic attack at the fluorine bearing number 2 carbon. The 2-carbon is significantly electron deficient due to the attached fluorine atom, the adjacent fluorine atom, but most significantly due to the ortho carboxylate. The ortho carboxylate can withdraw electron density from the number 2 carbon through both inductive and resonance effects. Inductive effects are decreased by the inverse square of distance suggesting that the 2-carbon would be the most electrophilic carbon of lithium

2,4,5-trifluorobenzoate In support of this postulate, Davis indicates that similar conditions were successful for nucleophilic attack of lithium (2-fluoro-4- iodophenyl)amide on 2-fluroro-benzoic acid, while nucleophilic attack of lithium (2- fluoro-4-iodophenyl)amide on 2-fluroro-benzoic acid was unsuccessful. Additionally

71 this method was used to prepare compounds that were co-crystallized in the structure of

MEK1. The chemical (1H NMR, elemental analysis) and physical properties were identical with the literature values.

However, additional confirmation for the regioselectivity was sought. Acquisition of the

19F NMR spectra of 2,3,4-trifluorobenzoic acid, 2-fluoro-4-iodoaniline, and 3,4-difluoro-

2-((2-fluoro-4-iodophenyl)amino)benzoic acid was conducted using the hetero-nuclear broad-band probe on the 500 MHz instrument.

Synthesis of the tetrahalo amides.

The simple amides were envisioned as being readily prepared by the addition of the appropriate amine into the acid chloride that could be prepared from the carboxylic acid

39. This acid, 39, was found to be readily converted into the corresponding acid chloride

(Scheme 11). The reagent of choice was oxalyl chloride and catalytic DMF. This system utilized the addition of neat oxalyl chloride to the reaction mixture containing the acid, DMC, and catalytic DMF. The functional role of the DMF is to generate the chloridinium intermediate as shown below. This chloridinium species (84) then activates the carboxylic acid to produce the acid chloride. This reaction proceeded at diffusion- limited rates at 0 oC indicated by the rapid formation and loss of the yellow chloridinium species (Scheme 12). Overall the reaction was completed within 2 hours at or below room temperature to consistently generate acid chloride 79.

Scheme 11: Acid chloride formation.

Scheme 12: Mechanism of acid chloride formation.

Reaction of the acid chloride, 79, with the desired amines to yield the desired amides was not always straight-forward as originally planned.

Scheme 13: Synthesis of simple amides using the acid chloride method.

In case of amides bearing a basic amine, this method was not successful. Although the starting acid chloride was consumed, many TLC spots were obtained and the product could not be isolated using routine silica gel column chromatography. The addition of a base -such as TEA- along with the precursor amine in the reaction mixture was examined and was only modestly successful in a few cases, albeit with poor yields. Table 11 below summarizes the different conditions (reagents and equivalents) examined in an attempt to optimize the yield for this synthetic strategy.

74 Precursor Equivalents Reagents Solvent Reaction Result Conditions amine of amine 2.54 mmol/ 2 eq. TEA, DMAP DCM 25 oC, 4 h 44%*

6 mmol/ 2 eq. DMAP DCM 25 oC, 12 h, Could not isolate the product

2.64 mmol/ 3 eq. TEA, DMAP DCM 25 oC, 12 h 40%*

o 5 mmol/ 10 eq NaHCO3, DCM 25 C, 12 h Could not DMAP isolate the product

3.55 mmol/ 1.18 DIEA, DMAP DCM 25 oC, 12 h Could not eq isolate the product 3.46 mmol/ 2 eq. DMAP DCM 25 oC, 6 h 6 %*

8.04 mmol/ 3 eq. TEA, DMAP DCM 25 oC, 12 h Could not isolate the product

* = Chemical yield based on mass of isolated product.

Table 11: Different reaction conditions with different precursor amines.

Several possibilities could exist to explain the low yields. In many cases isolation of the compound from the column was difficult due to the basicity of the product amine.

An intractable complex mixture resulted if excess starting amine was used. Presence of moisture may also have complicated both identification and isolation of the product from the crude.

Finally it was decided to use DCC108 (Scheme 14) and DIC95 (Scheme 15) coupling to form the aide bond more efficiently. In all cases the use of DCC presented a complex

75 mixture which did not permit isolation of clean product. In most cases reactions with

DIC proceeded well with a comparatively better cleanup than with DCC.

Scheme 14: DCC coupling reaction.

Scheme 15: DIC coupling reaction.

Synthesis of the trihalo core: 3,4-difluoro-2-((2-fluorophenyl)amino)benzoic acid

(68).

Synthesis of 3,4-difluoro-2-((2-fluorophenyl)amino)benzoic acid , or the de-iodo variant of diphenylamine acid 68, was initially attempted directly from 39 using the standard Pd/C reductive de-iodination109. However, only starting material was recovered, with no evidence of conversion by either TLC or 1H NMR. Although this conversion could have been pursued, the reliable yield for addition of 2-fluoro-4-iodioaniline into

76 2,3,4-trifluorobenzoic acid recommended a direct synthesis for compound 68 using the procedure of Davis85.

Scheme 16: Pd/C reductive de-iodination reaction.

The desired carboxylic acid 68 was then prepared successfully using the lithium amide of

2-fluoroaniline in an SNAr displacement on 2,3,4-trifluorobenzoic acid. This conversion proceeded in 59% isolated yield.

Scheme 17: Lithium amide displacement method.

For the formation of the methyl piperazine amide 69, and in an attempt to streamline the isolation, particularly for basic amine variants, a T3P coupling was tried (Scheme 18).

The crude TLC indicated a number of spots which could not be separated using column chromatography. This reagent was not pursued further.

Scheme 18: T3P coupling reaction.

An EDCI coupling was subsequently used successfully and the fumarate salt of the product 69 was isolated (Scheme 19). The side product urea from EDCI was removed more effectively than the side product from the DIC coupling permitting a clean isolation of the basic amine containing product. Formation of the fumarate salt was required because the free base form of the compound was difficult to solidify and recrystallize, as a result direct elemental analysis on the product was unsuccessful. Formation of the HCl salt of 69 produced a very hygroscopic compound that could not be adequately dried to a free-flowing solid. The formation of the fumarate salt addressed isolation and characterization of the desired product in a simple efficient manner.

Scheme 19: Synthesis of 69 Fumarate by EDCI coupling.

The primary amide derivatives of various terminal ring variations were also synthesized and are described below.

Synthesis of the dihalo core: 3,4-difluoro-2-(phenylamino)benzoic acid (71).

The dihalo core, 3,4-difluoro-2-(phenylamino)benzoic acid, retained the central 3,4- difluoro phenyl ring but varied by the lack of halogens on the terminal arene ring. This compound was prepared by the lithium amide displacement method previously described in 43 % isolated yield.85

Scheme 20: Synthesis of 71 using lithium amide displacement method.

Synthesis of the dihalo amides.

Again although the general synthetic strategy proceeding through the acid chloride of carboxylic acid 71 did work, there were complications. The chemical route beginning with the dihaloacid 71 was lower yielding than with the previously described tetrahalo acid 39. A possible explanation is that the attached diphenyl amine was less electron deficient for 71 than for 39. Although the carboxylic acid 71 was anticipated to react in a manner analogous to the tetrahalo acid 39 described above, the less electron deficient diphenyl amine may have participated in side reactions including intra- or inter-molecular condensation of the diphenyl amine with the acyl chloride.

Additional synthetic difficulties were encountered in this series during the preparation of the piperazine derivatives. The desired Boc-piperazine (91) amide was made successfully albeit with 20% yield, however deprotection with 4N HCl and subsequent

Eschweiler-Clarke alkylation, as shown below did not work. A complex crude mixture was obtained with many spots on silica TLC possibly due to the formation of the dimethylated compound along with the unreacted starting acid. Also the presence of the basic amine presented extreme isolation challenges. Deprotection of the Boc group and isolation of the resultant TFA salt was also tried, but the product could not be isolated

80 (Scheme 21). Hence, an EDCI coupling reaction was tried between the carboxylic acid

71 and N-methyl piperazine, compound 72; the free base of the desired product was isolated in 61% yield (Scheme 22). A possible explanation for the success of this strategy is that the carbodiimide coupling circumvented the self-condensations described above. The direct use of N-methyl piperazine was particularly useful. At the beginning of this project it was unclear if the secondary amine or tertiary amine variations of acyl piperazine would be more active. The SAR emerged in such a way that the tertiary N- methyl piperazine containing amide, compound 72, was actually more active. As a result, direct synthesis of the N-methyl piperazine containing amides was established as the preferred synthetic strategy, regardless of amide formation used.

Scheme 21: Eschweiler-Clarke reaction approach towards the synthesis of 72.

Scheme 22: EDCI coupling for the synthesis of 72.

Additionally, the primary amides of these derivatives were also prepared as shown in scheme 23. The synthesis of compound 73 was unsuccessful and will the focus of additional work by another student.

Scheme 23: Synthesis of the primary amides 57, 73 and 70.

82 Synthesis of the unhalogenated core: 2-(phenylamino)benzoic acid.

Initial attempts for synthesis of unhalogenated diphenylamine acid (25) scaffold:

Both lithium amide and sodium hydride SNAr reactions were attempted and did not work.

One possible explanation is that in case of the synthesis of diphenylamine analogs containing no halogens on either of the arene rings that the carbon halogen bond was not adequately polarized to facilitate an SNAr attack from the amid salt.

Scheme 24: Sodium hydride SNAr reactions for the synthesis of 25.

Scheme 25: Lithium amide SNAr reaction for the synthesis of 25.

Due to the different substrates required for the desired analogs, a different synthetic strategy was necessary. A survey of different aryl to nitrogen coupling strategies was conducted (see prior description). Ultimately an Ulllmann coupling strategy using the addition of an aniline into the carbon-halogen bond of 2-iodobenzoid acid was selected.

This approach used an inexpensive commercially available starting material and used a reasonable electron flow from the electron rich aniline into the electron deficient 2-iodo benzoic acid. An Ullmann coupling strategy worked to give the desired acid in a 44%

83 isolated yield. A survey recent variations110 recommended the conditions that were ultimately selected and shown in scheme 26. The specific method employed used copper iodide, potassium carbonate, and a mixed solvent system of 9 to 1 DMF to water.

Efficient heating was provided by microwave irradiation.

Scheme 26: Ullmann coupling strategy to synthesize 25.

In case of the unhalogenated diphenylamine, compound 25, a different amide-forming strategy was required. The acid chloride method described previously did not work.

Probable reasons for the failure of this strategy include the increased nucleophilicity of the amine of compound 25 resulting from the absence of electron-withdrawing groups compared to compounds previously synthesized in the series described above. This greater electron density in compound 95 may have led to self-condensations in either inter- or an intramolecular manner. This explanation is consistent with the observed consumption of starting material with no isolable product.

Scheme 27: The acid chloride method for the synthesis of amide 75.

The next approach attempted for the synthesis of compound 75 was synthesis of the Boc- piperazine derivative 96 followed conversion to the piperazine analog. Synthesis to the tertiary amine using an Eschweiler-Clarke reaction was planned. However, the

Eschweiler-Clarke product could not be isolated (Scheme 28). Learning from prior reactions, this strategy was abandoned for the far more efficient route (DIC coupling) presented in Scheme 29.

Scheme 28: Synthesis of 75 via Eschweiler-Clarke reaction.

This DIC coupling was successful and the product was isolated in a modest yet welcome

17% yield.111

Scheme 29: DIC coupling method to synthesize 75.

86 An attempted SNAr nucleophilic of the lithium amid of 2-fluoro-4-iodoanaline into 2- iodobenzoic acid with excess lithium amide in THF using the now standard procedure of

Davis85 was unsuccessful. This was probably due to a more electropositive carbon at the

2 position when compared to the prior substrate, 2,3,4-trifluorobenzoic acid. Again, based on prior observations, alteration of the synthetic strategy to use an Ullmann coupling was shown to be successful and proceeded reliably in 40 – 60 % isolated then recrystallized yield.

Scheme 30: Synthesis of amides 75 and 74.

87 Synthesis of the N-methyl diphenylamine core:

An Eschweiler-Clarke reaction112 was examined as an initial strategy in an attempt to prepare 3,4-difluoro-2-((2-fluoro-4-iodophenyl)(methyl)amino)benzoic acid, compound

76. The use of standard microwave reaction conditions were examined, but the desired product could not be isolated.

Scheme 31: Eschweiler-Clarke strategy to synthesize 76.

Scheme 32: Mechanism of Eschweiler-Clarke reaction.

An explanation for the failure of the Eschweiler-Clarke reaction likely arises from difficulty when forming the “iminium ion” (104). For the initial step -shown above-

88 nucleophilic attack by the significantly electron deficient nitrogen atom into the molecule of formaldehyde would be difficult and slow and could hinder or halt the reaction.

Another strategy was attempted using sodium hydride abstraction of the proton from the diphenyl amine nitrogen atom followed by alkylation with methyl iodide (Scheme 33).113

A crude TLC analysis revealed many spots, attempted separation by column chromatography was unsuccessful.

Scheme 33: Synthesis of 76 by sodium hydride and methyl iodide reaction.

After the two unsuccessful synthetic approaches a very different strategy was pursued.

Alkylation was conducted on the simple aniline precursor, 2-fluoro-4-iodoaniline 38, to successfully prepare the mono N-methylated product, 2-fluoro-4-iodo-N-methylaniline

108, shown in scheme 34.114 This secondary aniline was then coupled to 2,3,4- trifluorobenzoic acid (37) using the standard SNAr lithium amide displacement approach.85

Scheme 34: Monomethylation reaction of 38.

Scheme 35: Synthesis of acid 76 by lithium amide displacement method.

90 Chapter 5

Biological discussion

There has been extensive prior SAR conducted on what makes a potent Type III MEK1 inhibitor.61, 115 A summary of known SAR for MEK1 overlaid on PD 325901, compound

5, is shown below in figure 39.

Figure 40: MEK1/2 interaction with inhibitor PD325901.115

Terminal B ring forms Van der Waals interactions with the hydrophobic pocket formed by Met143, Ile141, Leu118, and Phe209. Phe209 has been proposed to form an important edge-to-face aromatic interaction with the B ring.

A significant determinant MEK1 activity, both in the literature and also in our testing, is the essential presence of an electrostatic interaction, or halogen interaction between the

91 lone pair of electrons on the oxygen atom of the backbone carbonyl of Val127 and the 4′- iodide on the B ring.67

A second significant determinant MEK1 activity is the hydrogen bond interaction, between the 4-fluoro atom of the central A ring and the backbone amide of Ser212.

Our aim was to rationally design toward MEK5 selectivity and analyze the observed differences in activity on the basis of MEK5 interactions proposed on the basis of the homology model. A rigorous SAR in the absence of a direct enzymatic assay is inappropriate, but the cellular model is likely to be more descriptive of an intact biological cascade.

1. Side chain variation and amide variation:

All amide variations were conducted on the tetra-halogenated ring system. The

rationale for this approach was based in part on the synthetic approach that

permitted these point variations from a common synthetic intermediate. However

this approach permitted better comparison as a single component was altered to

provide a better basis for rational interpretation. These compounds showed potent

92 MEK1/2 inhibition. This is not surprising as these compounds retained the

major/critical MEK1/2 interactions. It was hoped on the basis of the homology

model that differential activity would be observed but this was not seen. The

primary amide (57) was found to be a non-selective MEK1 and MEK5 inhibitor.

2. Terminal arene variation:

Compounds without fluorine on the central A ring yet retaining the 2-F-4-I atoms

on the B ring showed improved MEK5 inhibition plus showed reduced MEK1/2

inhibition. The decrease in MEK1 inhibition was most likely due to the loss of

the hydrogen bond interaction of the 4-fluoro from the A ring to the amide

backbone of Ser212 (Figure 39). This is not well predicted by the homology

model, but as indicated in analysis in the appendix, this is a region of greater

variability between MEK1 and MEK5. Additionally this alpha helix is known to

move relative to the anti-parallel beta sheets comprising the N-terminal domain of

the catalytic (ATP binding) domain of the enzyme and the alpha helix is likely to

rotate or roll to expose a different surface to the type III binding site. An X-ray

crystal structure could partially address these variations.

93 3. Central arene variation:

The compound 72 with 2 fluorine atoms in the central A ring and the complete

absence of halogens on the B ring showed the maximum MEK5 inhibition

amongst all the other variations examined. Also, this compound did not show any

MEK1 inhibition in the cellular assay. The full absence of observed activity for

MEK1 is unsurprising. From a structural perspective the electrostatic (halogen)

interaction is consistently present in all MEK1 or MEK2 X-ray crystallographic

structures deposited in the PDB. From a medicinal chemistry perspective all

potent MEK1 inhibitors in the literature, including the approved MEK1 inhibiting

drug Mekinist, rigorously retain this interaction. The homology model does

predict that this region is smaller in MEK5. It was anticipated that a modest

decrease in MEK1 activity would be observed and any selectivity would result

from less than ideal water displacement from the B ring binding site.

4. Target compounds with de-iodo diphenylamine:

94 The de-iodo acid 68 did not display any MEK5 inhibition and showed only 30%

MEK1 inhibition in the cellular assay. This observation was consistent with the

proposed importance of the 4′-I halogen bond as being essential to MEK1

inhibition activity. However, its N-methyl piperazine 69 analog displayed an

increased MEK5 inhibition with up to 56% inhibition being observed while

retaining the lower 30% MEK1 inhibition of its de-iodo structural variation,

compound 68. This suggests that additional variation of the amide portion

following aromatic ring variation may result in compounds with equally increased

MEK5 activity as well.

5. Target compounds with no halogens on rings A and B:

The compound with no halogens on either of the rings was expected to lose at

least two major MEK1 interactions, however it still displayed 30% MEK1

inhibition. It also inhibited MEK5 up to 71%. Possible explanations include that

halogen and hydrogen bonding interactions are additive to other portions of the

molecule if the minimum structural requirements of an amide and two

hydrophobic regions are present. The homology model also suggests that

variation on the B ring may be useful due to proposed structural variation of the

homology model of MEK5DD from the structure of MEK1 presented by 3EQC.

95 Compound 75 anticipates that central A ring substitution may not be necessary.

In the case of MEK1 inhibitors, as discussed previously, this was a design

strategy for A-ring variations which was successfully used to ultimately design

the drug Mekinist.

6. Variations on the nitrogen atom of the diphenylamine:

The N-methyl compound 76 was a very important compound in our series to

establish the relevance of the internal hydrogen bond. The N-methyl compound

displayed only 18% MEK5 inhibition, but still showed 67% MEK1 inhibition. It

was anticipated that a parallel decrease in both MEK1 and MEK2 activity would

be observed because the majority of MEK1 inhibitors in the PDB display internal

hydrogen bonding. Because the homology model was based on MEK1 it was

anticipated that this would apply to MEK5 as well. Apparently this is not the

case. Conducting the assays concurrently in the same cell population provides an

important internal control and retention of MEK1 inhibition suggests that the

observed loss of MEK5 activity is unlikely to arise from inadequate transport into

the cell. Thus we are examining the geometry imposed by the internal hydrogen

bonding as a design strategy to differentiate structural requirements for type III

96 MEK5 inhibitors versus type III MEK1 inhibitors and synthesizing ring-

constrained diphenylamine analogs to explore this unanticipated finding.

Compounds pERK5 pERK5 pERK1/2 pERK1/2

activation (%) inhibition (%) activation (%) inhibition (%)

DMSO 100 0 100 0

64 (SC-1-75) -- 8.4 -- 70.9

59 (SC-1-65) 7.4 -- -- 5.5

74 (SC-1-24) -- 30.9 -- 33.5

65 (SC-1-122) -- 9.4 -- 27.9

58 (SC-1-69) -- 0.2 -- 87.3

60 (SC-1-72 a) -- 20.4 -- 99.6

62 (SC-1-72 e) -- 13.0 -- 98.9

63 (SC-1-79) 6.2 -- -- 99.0

61 (SC-1-80) -- 8.5 -- 98.6

75 (SC-1-177) -- 71 -- 29.3

72 (SC-1-181) -- 82.4 8.5 --

57 (SC-1-151) -- 59 -- 96.8

39 (SC-1-180) -- 20.1 -- 98.5

71 (SC-1-175) -- 10.4 -- 7.1

68 (SC-2-25) -- 11.1 7.1 --

76 (SC-2-32) 5.5 -- -- 33.5

70 (SC-2-37) -- 56.1 -- 52.8

97 69 (SC-2-45) -- 40.5 -- 16.5

U0126 -- 43 -- 99.6

XMD8-92 -- 95.2 -- 50.1

PD 0325901 -- 95.9 -- 99.7

Table 12: MEK1/2 and MEK5 inhibition data.

Our collaborator Dr. Matthew Burow in Tulane University, New Orleans further tested the compounds in proliferation and cell viability assays in MDA-MB-231 cells. The normal duration of this study was 24 hrs to 3 days, however, a temporary closure of the lab for a week, led to the discovery of a morphological change in the shape of the cells.

The treatment of MDA-MB-231cells presented an MET (mesenchymal to epithelial transition) when exposed to a concentration of 10 M of compound for a 7-day interval.

This unexpected finding suggests the potential for MEK inhibitors to facilitate an MET in vitro breast cancer cells grown in charcoal-stripped media.

98 Chapter 6

Experimental

All solvents and reagents were used as received unless noted otherwise. All reactions were conducted in dry glassware and under an atmosphere of argon unless otherwise noted. Microwave reactions were conducted in sealed tube and utilized a multimode

Milestone Start apparatus for irradiation with power and control parameters as noted.

Melting points were determined on a MelTemp apparatus and are uncorrected. All proton NMR spectra were obtained with a 500 MHz or a 400 MHz Oxford spectrospin cryostat, controlled by a Bruker Avance system, and were acquired using Bruker

TOPSPIN 2.0 acquisition software. Acquired FIDs were analyzed using MestReC 3.2.

Elemental analyses were conducted by Atlantic Microlabs and are ± 0.4 of theoretical.

1 All H NMR spectra were taken in CDCl3 unless otherwise noted and are reported as ppm relative to TMS as an internal standard. Coupling values are reported in Hertz. All TLCs were obtained on Sorbent Technologies polyester backed Silica G TLC Plates of thickness 200 µm.

99 General Method A:

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (SC-1-180) (39). A 250 mL round bottom flask was charged with 2-fluoro-4-iodoaniline, (38; 2.38 g, 10.0 mmol),

2,3,4-trifluorobenzoic acid, (37; 1.80 g, 10.2 mmol), and 30 mL of anhydrous THF. The

o reaction mixture was cooled with an ice-bath to 0 C and LiNH2 (561.2 mg, 24.45 mmol) was added in 3 portions over 10 min. The reaction was then warmed to an internal temperature of 58 oC and stirred for 12 h. The mixture was cooled to 0 oC and 1 N HCl was added maintaining the reaction mixture at 0 oC to yield a final pH of 1.0 (red to pHydrion paper). The reaction mixture was then extracted three times with 10 mL portions of Et2O, washed three times with 5 mL portions of 1 N HCl, washed with NaCl

(aq, sat), and dried over Na2SO4. The extract was decanted and the solvent was removed under reduced pressure. The crude product was isolated on SiO2 using 2:1 hexane/EA to provide 2.11 g (53%) of a white solid. MP = 199.0 - 200.1 oC (lit. MP = 200 - 201 oC).85

1 SiO2 TLC Rf 0.51 (2:1 hexane/EA). H NMR (400 MHz, MeOD-d4): δ 6.74 (m, 1 H,

Ar), 6.91 (m, 1 H, Ar), 7.38-7.45 (d, 1 H, J = 8.5 Hz, Ar), 7.47 (dd, 1H, J1 = 1.8 Hz and

J2 = 10.5 Hz, Ar), 7.89 (br, 1 H, Ar). Anal Calcd for C13H7F3INO2: C, 39.72; H, 1.79; N,

3.56. Found: C, 39.41; H, 1.91; N, 3.52.

General procedure B: Acid chloride approach to synthesize amide:

A dry 100 mL round bottomed flask was charged with 3,4-difluoro-2-((2-fluoro-4- iodophenyl)amino)benzoic acid, (39) and 5 mL of DCM. The reaction mixture was cooled with an ice-bath to 0 oC. 100 μL of anhydrous DMF was added followed by dropwise addition of neat oxalyl chloride (2 equiv.) over 5 min. The reaction was stirred

100 at 23 oC for 4 h. The solvent was then removed under reduced pressure. Excess oxalyl chloride was azeotropically removed with 2 X 5 mL portions of DCM under reduced pressure. The crude product was dissolved into 5 mL of DCM and the appropriate amine was added neat at 0 oC. The ice bath was removed after 10 min and the reaction was permitted to warm to room temperature. The reaction was then stirred at 23 oC for 6 h; completion of reaction was determined by TLC. A mixture of 10 mL of H2O and 10 mL of Et2O was added and the resultant mixture was extracted with Et2O, washed with NaCl

(aq, sat), and dried over Na2SO4. The extract was decanted and then the solvent was removed under reduced pressure. The crude product was isolated on SiO2 using hexane/EA.

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (SC-1-151) (57): was synthesized using procedure B from 3,4-difluoro-2-((2-fluoro-4- iodophenyl)amino)benzoic acid, (39; 1.2 g, 3.0 mmol) and 7 N NH3 in methanol (2 mL,

15.73 mmol). The crude product was isolated on SiO2 using 2:1 hexane/EA to give 900

o mg (73%) of a pink-white powder. MP = 160.9 – 162.0 C. SiO2 TLC Rf 0.29 (2:1

1 hexane/EA). H NMR (400 MHz, CDCl3): δ 5.72-6.22 (br. d, 2 H, NH2), 6.60-6.64 (m, 1

H, NH), 6.85-6.90 (m, 1 H, Ar), 7.34 (d, 1 H, J = 8.5 Hz, Ar), 7.39-7.43 (m, 2 H, Ar),

8.71 (s, 1 H, Ar). Anal Calcd for C13H8F3IN2O: C, 39.82; H, 2.06; N, 7.14. Found: C,

39.86; H, 2.18; N, 7.24.

N,N-diethyl-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (SC-1-65) (59):

Compound 59 was synthesized using procedure B (acid chloride approach) from 3,4-

101 difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, (39; 200 mg, 0.51 mmol) and diethyl amine (0.16 mL, 1.53 mmol). The crude product was isolated on SiO2 using 1:1 hexane/EA and recrystallised from hexanes to give 100.2 mg (45%) of a white solid. MP

o 1 = 78.9 – 80.1 C. SiO2 TLC Rf 0.7 (1:1 hexane/EA). H NMR (400 MHz, CDCl3): δ 1.07

(br, 6 H), 3.22-3.46 (br. d, 4 H, 2N-CH2), 6.50 (s, 1 H, NH) 6.51-6.55 (m, 1 H, Ar), 6.9-

7.04 (m, 2 H, Ar), 7.27-7.29 (d, 1 H, J = 8.5 Hz, Ar), 7.34-7.37 (dd, 1 H, J1 = 1.9 Hz and

J2 = 10.5 Hz, Ar). Anal Calcd for C17H16F3IN2O: C, 45.5; H, 3.60; N, 6.25; F, 12.72; I,

28.31. Found: C, 45.25; H, 3.57; N, 6.25; F, 12.86; I, 28.22.

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N,N-dimethylbenzamide (SC-1-69)

(58): Compound 58 was synthesized using procedure B from 3,4-difluoro-2-((2-fluoro-

4-iodophenyl)amino)benzoic acid, (39; 200.0 mg, 0.51 mmol) and dimethyl amine HCl

(408 mg, 5.0 mmol). A solution of dimethyl amine HCl in 5 mL H2O was added with dropwise addition to a suspension of Na2CO3 (7 mmol), H2O (5 mL), DCM (25 mL), and

DMAP (5.0 mg, 0.04 mmol) at 0 oC. The solution of acid chloride in DCM was added

o over 5 min and the reaction was stirred at 23 C for 2 h. A mixture of 10 mL of H2O and

50 mL of DCM was added and the resultant mixture was washed with H2O (2 X 10 mL), washed with NaCl (aq, sat), and then dried over Na2SO4. The crude product was isolated on SiO2 using 2:1 hexane/EA to give 47 mg (22%) of a white solid. MP = 115.4 – 117.7 o 1 C. SiO2 TLC Rf 0.61 (2:1 hexane/EA). H NMR (400 MHz, CDCl3): δ 2.96 (br, 3 H,

CH3), 2.91 (br, 3 H, CH3), 6.53-6.59 (m, 1 H, Ar), 6.82 (s, 1 H, NH), 6.91-6.95 (m, 1H,

Ar), 7.02-7.06 (m, 1 H, Ar), 7.29-7.31 (d, 1 H, J = 8.5 Hz, Ar), 7.36-7.39 (dd, 1 H, J1 =

102 1.9 Hz and J2 = 10.4 Hz, Ar). Anal Calcd for C15H12F3IN2O: C, 42.8; H, 2.8; N, 6.67; F,

13.5; I, 30.2. Found: C, 42.69; H, 2.89; N, 6.53; F, 13.45; I, 29.99.

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-methylbenzamide (SC-1-72 amide)

(60): was synthesized using procedure B from 3,4-difluoro-2-((2-fluoro-4- iodophenyl)amino)benzoic acid, (39; 315 mg, 0.8 mmol) and methylamine in methanol

(0.5 mL, 4 mmol, 8 M solution). The crude product was isolated on SiO2 using 5:1 hexane/EA and recrystallized from hot EtOH to give 203 mg (63%) of a white solid. MP

o 1 = 159.0 – 160.2 C. SiO2 TLC Rf 0.51 (1:1 hexane/EA). H NMR (500 MHz, CDCl3): δ

2.95 (d, 3 H, J = 4.8 Hz), 6.23 (br, 1 H, NH), 6.54-6.59 (m, 1 H, Ar), 6.82-6.88 (m, 1 H,

Ar), 7.28-7.32 (m, 2 H, Ar), 7.40 (dd, 1 H, J = 1.9 Hz and J = 10.3 Hz, Ar), 8.61 (s, 1 H,

NH). Anal Calcd for C14H10F3IN2O: C, 41.40; H, 2.48; N, 6.90; F, 14.03; I, 31.25. Found:

C, 41.67; H, 2.51; N, 6.79; F, 13.79; I, 31.35.

Methyl 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoate (SC-1-72 ester) (62): was obtained as a side-product using procedure B during the synthesis of 60 from 3,4- difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, (39; 315 mg, 0.8 mmol) and methylamine in methanol (0.5 mL, 4 mmol, 8 M solution). The product was obtained as

o 60 mg (17%) of a white solid. MP = 118.5 – 111.4 C. SiO2 TLC Rf 0.82 (2:1

1 hexane/EA). H NMR (400 MHz, CDCl3): δ 3.91 (s, 3 H), 6.65-6.71 (m, 1 H, Ar), 6.74-

6.80 (m, 1 H, Ar), 7.35 (d, 1 H, J = 8.6 Hz), 7.42 (dd, 1 H, J = 1.9 Hz and J = 10.2 Hz,

Ar), 7.78-7.82 (m, 1 H, Ar), 9.04 (s, 1 H, NH). Anal Calcd for C14H9F3INO2: C, 41.30; H,

2.23; N, 3.44; F, 14.00; I, 31.17. Found: C, 41.43; H, 2.08; N, 3.52; F, 14.18; I, 31.31.

103 Tert-butyl 4-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoyl)piperazine-1- carboxylate (SC-1-75) (64): Compound 64 was synthesized using procedure B from

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, (39; 1.00 g, 2.54 mmol) and

N-Boc-piperazine (2.85 g, 5.08 mmol). A solution of acid chloride in 6 mL DCM was added with dropwise addition to a solution of N-Boc-piperazine, TEA (0.70 mL, 5.08 mmol), DCM (12 mL) and DMAP (5.0 mg, 0.04 mmol) at 0 oC and the reaction was

o stirred at 23 C for 2 h. The crude product was isolated on SiO2 using 2:1 hexane/EA to

o give 630 mg (44%) of a white solid. MP = 188.4 C. SiO2 TLC Rf 0.5 (1:1 hexane/EA).

1 H NMR (400 MHz, CDCl3): δ 1.45 (s, 9 H), 3.34-3.55 (m, 8 H), 6.53-6.58 (m, 1 H, Ar),

6.62 (s, 1 H, NH), 6.92-6.96 (m, 1 H, Ar), 7.03 (m, 1 H, Ar), 7.30 (d, 1 H, J = 8.9 Hz,

Ar), 7.39 (dd, 1 H, J = 1.9 Hz and J = 10.4 Hz, Ar). Anal Calcd for C22H23F3IN3O3: C,

47.07; H, 4.13; N, 7.49; F, 10.15; I, 22.61. Found: C, 47.22; H, 4.18; N, 7.40; F, 9.94; I,

22.64.

(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(piperazin-1-yl)methanone hydrochloride (SC-1-79) (63): was synthesized from 64 (530 mg, 0.94 mmol) and 1:1

15 mL (v/v) HCl/Dioxane. A solution of 64 was taken in a 250 mL RBF and 15 mL of

1:1 conc. HCl in Dioxane was added with constant stirring at 23 oC for 3.5 hrs. The crude product was recrystallized from hot EtOH to give 340 mg (79%) of a white solid; MP =

o 1 201.2 – 203.6 C. H NMR (400 MHz, MeOD-d4): δ 3.08 (br, 4 H), 3.60 (m, 4 H), 6.58-

6.62 (t, 1 H, Ar), 7.12-7.22 (m, 2 H, Ar), 7.32 (d, 1 H, J = 8.5 Hz, Ar) 7.46 (dd, 1 H, J =

1.9 Hz and J = 10.8 Hz, Ar). Anal Calcd for C17H15F3IN3O: C, 41.03; H, 3.24; N, 8.44; F,

11.45; I, 25.50. Found: C, 40.77; H, 3.38; N, 8.34; F, 11.20; I, 25.24.

104 N-ethyl-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (SC-1-80) (61): was synthesized using procedure B from 3,4-difluoro-2-((2-fluoro-4- iodophenyl)amino)benzoic acid, (39; 315 mg, 0.8 mmol) and ethyl amine 2M solution in

THF (2.25 mL, 4.5 mmol). The crude product was isolated on SiO2 using 2:1 hexane/EA and recrystallized from hot EtOH to give 155 mg (41%) of a white solid. MP = 172.5 –

o 1 173.6 C. SiO2 TLC Rf 0.7 (2:1 hexane/EA). H NMR (400 MHz, CDCl3): δ 1.19 (t, 3 H,

J = 7.3 Hz), 3.38-3.45 (m, 2 H), 6.22 (br, 1 H, NH), 6.54-6.59 (m, 1 H, Ar), 6.82-6.89 (m,

1 H, Ar), 7.31 (m, 2 H, Ar), 7.40 (dd, 1 H, J = 2.0 Hz, J = 10.3 Hz, Ar), 8.52 (s, 1 H,

NH). Anal Calcd for C15H12F3IN2O: C, 42.88; H, 2.88; N, 6.67; F, 13.56; I, 30.20. Found:

C, 42.89; H, 2.89; N, 6.60; F, 13.57; I, 30.47.

N-(2-(dimethylamino)ethyl)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N- methylbenzamide hydrochloride (SC-1-122) (65): was synthesized using procedure B from 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid, (39; 360 mg, 0.88 mmol) and N,N,N’-trimethylethane-1,2-diamine (0.34 ml, 2.64 mmol). A solution of

N,N,N’-trimethylethane-1,2-diamine (0.34 ml, 2.64 mmol), TEA (0.37 mL, 2.64 mmol) and DMAP (6 mg, 0.05 mmol) in 3 mL DCM was added dropwise to a solution of the acid chloride in DCM over 5 min and the reaction was stirred at 23 oC for 12 h. The crude product was isolated on SiO2 using CHCl3 and 5% MeOH to give 290 mg. HCl salt was made from ethereal HCl and recrystallized from hot ethanol to afford 168.2 mg (40%) of

o a white solid. MP = 211 – 213 C. SiO2 TLC Rf 0.7 (DCM/ 5% MeOH/ 0.1% NH4OH).

1 H NMR (500 MHz, DMSO-d6) : δ 2.78 (s, 6 H, 2CH3), 2.84 (s, 3 H, CH3), 3.13 (m, 2 H,

CH2), 3.61 (t, 2 H, CH2), 6.59-6.63 (t, 1 H, Ar), 7.27-7.33 (m, 2 H, Ar), 7.52-7.53 (dd, 1

105 H, J = 1.6 Hz, J = 11.3 Hz, Ar), 8.05 (s, 1 H, Ar), 10.06 (s, 1 H, NH). Anal Calcd for

C18H20ClF3IN3O: C, 42.08; H, 3.92; N, 8.18; F, 11.09; I, 24.70. Found: C, 42.21; H, 3.98;

N, 8.09; F, 11.22; I, 24.52.

Procedure C (Ullmann coupling):

2-((2-fluoro-4-iodophenyl)amino)benzoic acid (SC-1-14) (98): A microwave reactor tube was charged with ortho-iodo benzoic acid (496 mg, 2 mmol), 2-fluoro-4-iodo aniline

(237 mg, 1 mmol), K2CO3 (416 mg, 3 mmol), CuI (200 mg, 1.04 mmol) and 5 mL

DMF/H2O (9:1). The reaction was subjected to 300 Watt microwave irradiation with the internal temperature maintained at 100 oC for 2 h. After completion of the reaction was analyzed by TLC, 1 N HCl (~ 4 mL) was added to the reaction mixture to obtain a final solution pH of 6.0. The solvent was then removed under reduced pressure. The crude compound was isolated on SiO2 using 1:1 hexane/EA to give 217 mg (61%) of white

o 1 solid; MP = 186.2 – 186.5 C. SiO2 TLC Rf 0.70 (1:1 hexane/EA). H NMR (400 MHz,

CDCl3): δ 6.85 (t, 1 H, J = 7.1 Hz, Ar), 7.11 (d, 1 H, J = 8.6 Hz, Ar), 7.20 (t, 1 H, J = 8.4

Hz, Ar), 7.42 (m, 2 H, Ar), 7.50 (dd, 1 H, J = 2.0 Hz and J = 9.8 Hz, Ar), 8.06 (dd, 1 H, J

= 1.6 Hz and J = 8.1 Hz, Ar), 9.25 (s, 1 H, CO2H). Anal Calcd for C13H9FINO2: C, 43.72;

H, 2.54; N, 3.92. Found: C, 43.81; H, 2.65; N, 3.80.

(2-((2-fluoro-4-iodophenyl)amino)phenyl)(4-methylpiperazin-1-yl)methanone hydrochloride (SC-1-24) (74): A dry 100 mL round bottom flask was charged with 98,

(140 mg, 0.39 mmol) and 5 mL of DCM. The reaction mixture was cooled on ice-bath to

0 oC. 100 μL of anhydrous DMF was added followed by dropwise addition of oxalyl

106 chloride (70 μL, 0.8 mmol) over 2 min at 0 oC. The reaction was stirred at 23 oC for 2 h.

The solvent was then removed under reduced pressure. The crude product was dissolved into 5 mL of DCM and N-methyl piperazine (0.5 mL, 4.5 mmol) was added neat at 23 oC.

The reaction was stirred at 23 oC for 2 h; completion of reaction was determined by TLC.

A mixture of 10 mL of DCM and 5 mL of 5% Na2CO3 was added and the resultant mixture was extracted with DCM, washed with NaCl (aq, sat), and dried over Na2SO4.

The extract was decanted and then the solvent was removed under reduced pressure and water chased with toluene. The crude product was isolated on SiO2 using EA/0.5%

TEA/10% ethanol and recrystallised from HCl salt (ethereal HCl) to give 20 mg (12%) of

o 1 off-white powder. MP = 217.2 – 217.5 C. SiO2 TLC Rf 0.24 (20% EA/EtOH). H NMR

(400 MHz, MeOD-d4): δ 1.18 (t, 4 H, J = 7.0 Hz), 2.89 (s, 3 H), 3.49 (q, 2 H, J = 7.0 Hz),

3.60 (q, 2 H, J = 7.1 Hz), 6.92 (t, 1 H, J = 8.7 Hz), 7.09 (t, 1 H, J = 7.5 Hz), 7.15 (d, 1 H,

J = 8.2 Hz), 7.34-7.42 (m, 3 H), 7.49 (dd, 1 H, J = 2.0 Hz and J = 10.7 Hz). Anal Calcd for C18H20ClFIN3O. 0.38 % EtOH: C, 45.68; H, 4.55; N, 8.51; F, 3.85; I, 25.71; Cl, 7.18.

Found: C, 45.9; H, 4.49; N, 8.54; F, 3.66; I, 25.68; Cl, 7.49.

2-(phenylamino)benzoic acid (SC-1-39) (25): A microwave reactor tube was charged with ortho-iodo benzoic acid (496 mg, 2 mmol), aniline (24) (0.45 mL, 4 mmol), K2CO3

(832 mg, 6 mmol), CuI (400 mg, 2.08 mmol) and 10 mL DMF/H2O (9:1). The reaction was subjected to 300 Watt microwave irradiation with the internal temperature maintained at 100 oC for 1 h. After completion of the reaction was observed by TLC, 1 N

HCl (~ 9 mL) was added to the reaction mixture to obtain a final pH of 6.0. The solvent was then removed under reduced pressure and water was azeotropically removed with 3

107 X 10 mL of toluene. The crude compound was isolated on SiO2 using hexane/EA and recrystallised from toluene to give 267 mg (63%) of white solid; MP = 176.6 – 177.0 oC.

1 H NMR (400 MHz, CDCl3): δ 6.76 (t, 1 H, J = 7.5 Hz, Ar), 7.13 (t, 1 H, J = 7.3 Hz, Ar),

7.23 (d, 1 H, J = 8.7 Hz, Ar), 7.26-7.28 (m, 2 H, Ar), 7.33-7.39 (m, 3 H, Ar), 8.04 (dd, 1

H, J = 1.6 Hz and J = 8.1 Hz, Ar), 9.33 (s, 1 H, CO2H).

Procedure D (DIC coupling)

(4-methylpiperazin-1-yl)(2-(phenylamino)phenyl)methanone (SC-1-177 amide) (75):

A dry 100 mL round bottom flask was charged with 25, (1.00 g, 4.69 mmol) and 12 mL of DCM. N-methyl piperazine (2.59 mL, 23.45 mmol) and DMAP (9 mg, 0.07 mmol) was added followed by DIC (1.08 mL, 7 mmol) and the reaction mixture was stirred at 23 oC for 22 h. The solvent was removed under reduced pressure. A mixture of 10 mL of ether and HCl was added and the resultant mixture was extracted into HCl (3 X 5 mL) and washed with ether (2 X 5 mL). The aqueous layer was basified with 5% Na2CO3 and the crude material was extracted into DCM (3 X 8 mL). The crude product was loaded onto SiO2 and eluted with chloroform: methanol (95:5). Collection of appropriate fractions, removal of solvent, and recrystallization from hot ethanol gave 232 mg (17%)

o of transparent colorless needles. MP = 105.9 – 108.0 C. SiO2 TLC Rf 0.35 (chloroform/

1 1% methanol). H NMR (400 MHz, MeOD-d4): δ 2.23 (s, 3 H, CH3), 2.35 (br, 4 H,

2CH2), 3.73-3.82 (m, 4 H, 2CH2), 6.88 (t, 1 H, J = 7.3 Hz), 6.97-7.01 (m, 3 H, Ar), 7.19-

7.26 (m, 4 H, Ar), 7.30-7.34 (m, 1 H, Ar). Anal Calcd for C18H21N3O: C, 73.19; H, 7.17;

N, 14.23. Found: C, 73.14; H, 7.22; N, 14.23.

108 3,4-difluoro-2-(phenylamino)benzoic acid (SC-1-175 acid) (71): A 250 mL round bottom flask was charged with aniline (24) (0.57 mL, 5.7 mmol), 2,3,4-trifluorobenzoic acid (37), (1 g, 5.7 mmol), and 15 mL of anhydrous THF. The reaction mixture was

o cooled with an ice-bath to 0 C and LiNH2 (327 mg, 14.25 mmol) was added in portions

2 portions over 10 min. The reaction was then warmed to 58 oC (external temperature) and stirred for 7 h. 1 N HCl was then added to the reaction mixture at 0 oC to obtain a final pH of 1.0 (red to pHydrion paper). The reaction mixture was extracted three times with 5 mL portions of Et2O, washed three times with 5 mL portions of 1 N HCl, washed with NaCl (aq, sat) and dried over Na2SO4. The extract was decanted and the solvent was removed under reduced pressure. The crude product was isolated on SiO2 using

o hexane/EA and provided 606 mg (44 %) yellow crystals. MP = 162.1 – 162.6 C. SiO2

1 TLC Rf 0.61 (2:1 hexane/EA). H NMR (500 MHz, CDCl3): δ 6.73-6.78 (m, 1 H, Ar),

7.05 (d, 2 H, J = 7.5 Hz, Ar), 7.10 (t, 1 H, J = 7.4 Hz), 7.32 (t, 2 H, J = 7.6 Hz), 7.87-7.90

(m, 1 H, Ar), 8.99 (s, 1 H, OH).

Procedure E (EDCI coupling)

3,4-difluoro-2-(phenylamino)phenyl)(4-methylpiperazin-1-yl)methanone (SC-1-181)

(72): A solution of 71 (249 mg, 1 mmol), N-methyl piperazine (0.25 mL, 2 mmol) and

DMAP (6 mg, 0.05 mmol) was prepared in 10 mL anhydrous THF, then EDCI (382 mg,

2 mmol) was added in one portion. The reaction mixture was stirred at 23 oC for 12 hrs.

The solvent was removed under reduced pressure and a mixture of 50 mL of ether and 1 mL H2O was added. The resultant mixture was washed three times with 1mL portions of

H2O, 5 mL of saturated NaCl and then dried over anhydrous Na2SO4. The crude product

109 was isolated on SiO2 using CHCl3, 1% MeOH, 1% TEA to give 204 mg (62%) of a white

o 1 solid. MP = 153.0 – 155.3 C. H NMR (400 MHz, CDCl3): δ 2.22 (s, 4 H, 2CH2), 2.27

(br, 3 H, CH3), 3.47 (br, 4 H, 2CH2), 6.60 (s, 1 H, NH), 6.82-6.95 (m, 4 H, Ar), 6.99-7.03

(m, 1 H, Ar), 7.22-7.24 (m, 2 H, Ar). Anal Calcd for C18H19F2N3O: C, 65.24; H, 5.78; N,

12.68; F, 11.47. Found: C, 65.38; H, 5.89; N, 12.72; F, 11.46.

3,4-difluoro-2-((2-fluorophenyl)amino)benzoic acid (SC-2-25 acid) (68): A 100 mL dry round bottom flask was charged with 2-fluoroaniline (0.27 mL, 2.97 mmol), 2,3,4- trifluorobenzoic acid, (528 mg, 3 mmol), and 7 mL of anhydrous THF. The reaction

o mixture was cooled with an ice-bath to 0 C and LiNH2 (165.2 mg, 7.2 mmol) was added in 2 portions over a 10 min interval. The reaction was then warmed to 58 oC (external temperature) and stirred for 4 h. 1 N HCl was then added to the reaction mixture at 0 oC to obtain a final pH of 1.0 (red to pHydrion paper). The reaction mixture was extracted three times with 5 mL portions of Et2O, washed three times with 5 mL portions of 1 N

HCl, washed with NaCl (aq, sat), and dried over Na2SO4. The extract was decanted and the solvent was removed under reduced pressure. The crude product was isolated on SiO2

o using hexane/EA to provide 471 mg (59 %) of white crystals. MP = 170 – 172 C. SiO2

1 TLC Rf 0.55 (2:1 hexane/EA). H NMR (400 MHz, CDCl3): δ 6.72-6.78 (dt, 1 H, J = 6.8

Hz and J = 9.1 Hz, Ar), 7.00-7.13 (m, 4 H, Ar), 7.87-7.91 (ddd, 1 H, J = 2.1 Hz, J = 5.8

Hz and J = 9.1 Hz, Ar), 8.92 (s, 1 H, CO2H). Anal Calcd for C13H8F3NO2: C, 58.44; H,

3.02; N, 5.24. Found: C, 58.41; H, 3.02; N, 5.23.

110 (3,4-difluoro-2-((2-fluorophenyl)amino)phenyl)(4-methylpiperazin-1-yl)methanone mono-fumarate (SC-2-45) (69): was synthesized using procedure E from 68, N-methyl piperazine and EDCI. A solution of 68 (430 mg, 1.61 mmol), N-Methyl piperazine (0.35 mL, 3.22 mmol) and DMAP (6 mg, 0.05 mmol) was prepared in 12 mL anhydrous THF and then EDCI (615 mg, 3.22 mmol) was added. The reaction mixture was stirred at 23 oC for 6 hrs; completion of reaction was followed by TLC. The crude product was isolated on SiO2 using CHCl3, 1% MeOH, 1% TEA. The fumarate salt of the compound was prepared from fumaric acid (186 mg, 1.61 mmol) followed by recrystallization from

o hot EtOH to give 81 mg (14%) of a white solid. MP = 155.0 – 160 C. SiO2 TLC Rf 0.2

1 (CHCl3 + 2% MeOH). H NMR (400 MHz, CDCl3): δ 2.68 (s, 3 H, CH3), 2.83-2.94 (br,

4 H, 2CH2), 3.57 (br, 4 H, 2CH2), 6.72 (s, 2 H, CH=CH), 6.82-6.86 (m, 1 H, Ar), 6.89-

6.94 (m, 1 H, Ar), 6.97-7.01 (m, 1 H, Ar), 7.04-7.12 (m, 2 H, Ar), 7.14-7.18 (m, 1 H, Ar).

Anal Calcd for C22H22F3N3O5. 0.55 % Fumaric acid. 0.78 % EA: C, 54.87; H, 5.13; N,

7.02. Found: C, 54.98; H, 4.88; N, 6.86.

3,4-difluoro-2-((2-fluorophenyl)amino)benzamide (SC-2-37) (70): was synthesized using procedure B from 68, (267 mg, 1 mmol) and 7 N NH3 in methanol (0.65 mL, 5.03 mmol). The crude product was isolated on SiO2 using hexane/EA to give 96 mg (36%) of

o 1 a white powder. MP = 157.6 – 161.2 C. H NMR (400 MHz, CDCl3): δ 6.82-6.88 (m, 2

H, NH2), 6.91-6.97 (m, 1 H, Ar), 6.99-7.10 (m, 3 H, Ar), 7.44 (ddd, 1 H, J = 2.1 Hz, J =

5.5 Hz and J = 8.8 Hz, Ar), 8.45 (s, 1 H, Ar). Anal Calcd for C13H9F3N2O: C, 58.65; H,

3.41; N, 10.52; F, 21.41. Found: C, 58.22; H, 3.27; N, 10.20; F, 21.71.

111 2-fluoro-4-iodo-N-methylaniline (SC-2-20 amine) (108): 2-fluoro-4-iodoaniline (474 mg, 2 mmol) was added to a dry 100 mL round bottom flask containing a suspension of

NaOMe (540 mg, 10 mmol) in MeOH (5 mL). This mixture was poured into a suspension of paraformaldehyde (84 mg, 2.8 mmol) in anhydrous MeOH (4 mL) and the reaction

o mixture was stirred at 25 C for 5 h. After 5 h, NaBH4 (75 mg, 2 mmol) was added and the reaction mixture was refluxed at 90 oC for 2.5 h. The solvent was evaporated and the reaction mixture was treated with 5 mL 1 M KOH. The product was extracted into diethyl ether (2 X 8 mL) and dried over Na2SO4. The extract was decanted and the solvent was removed under reduced pressure. The crude product was isolated on SiO2 using 20% EA/

o hexane to provide 270 mg (54%) of white needles. MP = 44 C. SiO2 TLC Rf 0.75 (2:1

1 hexane/EA). H NMR (400 MHz, CDCl3): δ 2.85 (d, 3 H, J = 4.6 Hz), 3.97 (s, 1 H, NH),

6.43 (t, 1 H, J = 8.8 Hz), 7.22-7.26 (m, 1 H, Ar), 7.30 (d, 1 H, J = 9.3 Hz, Ar). Anal

Calcd for C7H7FIN: C, 33.39; H, 2.81; N, 5.58. Found: C, 33.69; H, 2.67; N, 5.64.

3,4-difluoro-2-((2-fluoro-4-iodophenyl)(methyl)amino)benzoic acid (SC-2-32) (76): A

100 mL dry round bottom flask was charged with 2-fluoro-4-iodo-N-methylaniline (270 mg, 1.07 mmol), 2,3,4-trifluorobenzoic acid, (192 mg, 1.09 mmol), and 10 mL of

o anhydrous THF. The reaction mixture was cooled with an ice-bath to 0 C and LiNH2 (60 mg, 2.6 mmol) was added in portions 2 portions over 5 min. The reaction was then warmed to 58 oC (external temperature) and stirred for 48 h. 1 N HCl was then added to the reaction mixture at 0 oC to obtain a final pH of 1.0 (red to pHydrion paper). The reaction mixture was extracted three times with 5 mL portions of Et2O, washed three times with 5 mL portions of 1 N HCl, washed with NaCl (aq, sat) and dried over Na2SO4.

112 The extract was decanted and the solvent was removed under reduced pressure. The crude product was isolated on SiO2 using 3:1 hexane/EA and recrystallized from toluene

o and hexanes to provide 286 mg (66 %) of brown crystals. MP = 86.2 – 89.1 C. SiO2

1 TLC Rf 0.45 (2:1 hexane/EA). H NMR (500 MHz, CDCl3): δ 3.34 (s, 3 H), 6.97 (t, 1 H,

J = 8.8 Hz, Ar), 7.24-7.27 (m, 1 H, Ar), 7.35-7.37 (dd, 1 H, J = 2.0 Hz and J = 11.4 Hz,

Ar), 7.50 (d, 1 H, J = 8.6 Hz, Ar), 8.07-8.10 (m, 1 H, Ar). Anal Calcd for C14H9F3INO2.

0.0436 % C6H5CH3: C, 41.78; H, 2.29; N, 3.40. Found: C, 41.77; H, 2.42; N, 3.35.

113 Chapter 7

Summary

List of compounds prepared

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoic acid (SC-1-180).

391,2,3

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (SC-1-151 primary amide)

571,2,3

114

N,N-diethyl-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (SC-1-65)

591,2

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N,N-dimethylbenzamide (SC-1-69)

581,2

3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-methylbenzamide (SC-1-72 amide)

601,2

115 Methyl 3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoate (SC-1-72 ester)

621,2

Tert-butyl 4-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzoyl)piperazine-1- carboxylate (SC-1-75)

641,2

(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(piperazin-1-yl)methanone hydrochloride (SC-1-79)

631,2

116 N-ethyl-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)benzamide (SC-1-80)

611,2

N-(2-(dimethylamino)ethyl)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)-N- methylbenzamide hydrochloride (SC-1-122)

651,2

2-((2-fluoro-4-iodophenyl)amino)benzoic acid (SC-1-14 acid)

117 (2-((2-fluoro-4-iodophenyl)amino)phenyl)(4-methylpiperazin-1-yl)methanone hydrochloride (SC-1-24 amide)

741,2

2-(phenylamino)benzoic acid (SC-1-39 acid)

(4-methylpiperazin-1-yl)(2-(phenylamino)phenyl)methanone (SC-1-177 amide)

751,2,3

118 3,4-difluoro-2-(phenylamino)benzoic acid (SC-1-175 acid)

711,2

3,4-difluoro-2-(phenylamino)phenyl)(4-methylpiperazin-1-yl)methanone (SC-1-181)

721,2,3

3,4-difluoro-2-((2-fluorophenyl)amino)benzoic acid (SC-2-25 acid)

681,2

119 (3,4-difluoro-2-((2-fluorophenyl)amino)phenyl)(4-methylpiperazin-1-yl)methanone mono-fumarate (SC-2-45)

691,2

3,4-difluoro-2-((2-fluorophenyl)amino)benzamide (SC-2-37)

701,2,3

2-fluoro-4-iodo-N-methylaniline (SC-2-20 amine)

108

120 3,4-difluoro-2-((2-fluoro-4-iodophenyl)(methyl)amino)benzoic acid (SC-2-32 acid)

761,2

1: Biological testing: Cellular assay for inhibition for pERK isoforms (MEK inhibition) 2: Biological testing: MDA-MB-231 proliferation studies and survey for EMT activity 3: Mouse hollow fiber assay

Acheivments:

Synthesis of analogs of diphenylamines and identificaion of compounds with the best

MEK5 selectivity and potency (compound 72).

Idenitification of small molecule compouds that can effect the phenomenon of reversal of epithilial to mesenchymal transition in MDA-MB-231 triple negative breast cancer cell line (compound 57).

Our collaborators are doing experiments to determine the IC50 values of the compounds which will give us a clear path to go forward with the animal model testing.

121 Chapter 8

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133 Appendix

Biological evaluation

MEK5

Biological assay

The biological testing of these compounds was conducted in the laboratory of Dr. Jane

Cavanaugh (Duquesne University, Pittsburgh).

Cell Culture and treatment

MDA-MB-231 cells were grown on 10cm cell culture plates [Sarstedt] in Dulbecco’s

Modified Eagle’s Medium (DMEM; Gibco) with Ham's F12 Nutrient Mixture (1:1)

(Invitrogen), 10% heat-inactivated FBS [Atlanta Biological and 0.5% penicillin/ streptomycin [Gibco]. Cells were maintained at 37°C with 5% CO2. Plating of the cells was done 36 hours before treatment in 35mm culture plates [Sarstedt] and allowed to reach confluence. To test MEK-5 inhibitors, the cells were treated with epidermal growth factor (EGF; Sigma-Aldrich) 30 min after treatment with the compounds. 15 min after the addition of EGF, the cells were washed with 1XPBS [Sigma-Aldrich] and then lysed in

1% Triton X-100 buffer containing 20 mM Tris (pH 6.8), 137 mM NaCl, 25 mM beta glycerophosphate, 2 mM NaPPi, 2 mM EDTA, 1 mM Na3VO4, 10% glycerol, 5 μg/mL

134 leupeptin, 5 μg/mL aprotinin, 2 mM benzamidine, 0.5 mM DTT, and 1 mM PMSF. The lysates were then centrifuged at 10,000 rpm for 10 min at 4°C.

Western Blot Analysis

Total protein content was assessed by Bradford Bio-Rad protein assay (Cat. No. 500-

0006, Bio-Rad, Hercules, CA) and 30 μg of protein was loaded on a 8% SDS-PAGE gel for phosphorylated and total ERK1/2 and ERK5 proteins. After running the samples, gels were transferred to a nitrocellulose membrane (Cat. No. 926-31092, Licor Biosciences,

Lincoln, NE). After transfer, membranes were washed for 5 min with 1X PBS and blocked for 1 h in a Casein Blocking Buffer (Cat. No. 927-40200, Licor Biosciences) at room temperature. Membranes were then incubated overnight at 4 ºC in primary antibody in CBB with 0.2% Tween-20. Antibodies included rabbit anti-phospho-ERK1/2 (Dilution

– 1:1000, Cat. No. 9101, Cell Signaling, Beverly, MA), mouse anti-total ERK1/2

(Dilution – 1:1000, Cat. No. 9107, Cell Signaling), and rabbit anti-total ERK5 (Dilution –

1:1,000, Cat. No. 3372, Cell Signaling). Mouse anti-α-Tubulin (Dilution – 1:10,000, Cat.

No. T5168, Sigma–Aldrich) was used as a loading control. After incubation with primary antibody, blots were washed in 1X PBS solution with 0.2% Tween-20 (1X PBS-T) and incubated with goat anti-rabbit (Dilution – 1: 10,000, Cat. No. 926-68021, LICOR

Biosciences) and goat anti-mouse (Dilution – 1: 10,000, Cat. No. 926-32210, LICOR

Biosciences) secondary antibodies for 1 h at room temperature. After washing the membranes with 1X PBS-T, the protein bands were visualized on an Odyssey Infrared

Imager and quantified with Odyssey software (LICOR Biosciences).

135 pERK-5 pERK-1/2 pERK-5 decrease pERK-1/2 decrease Compounds relative activity relative activity (%) (%) (%) (%) DMSO 100 0 100 0

SC-1-75 -- 8.4 -- 70.9

SC-1-65 7.4 -- -- 5.5

SC-1-24 -- 30.9 -- 33.5

SC-1-122 -- 9.4 -- 27.9

SC-1-69 -- 0.2 -- 87.3

SC-1-72 amide -- 20.4 -- 99.6

SC-1-72 ester -- 13.0 -- 98.9

SC-1-79 6.2 -- -- 99.0

SC-1-80 -- 8.5 -- 98.6

SC-1-177 -- 71 -- 29.3

SC-1-181 -- 82.4 8.5 --

SC-1-151 -- 59 -- 96.8

SC-1-180 -- 20.1 -- 98.5

SC-1-175 -- 10.4 -- 7.1

SC-2-25 -- 11.1 7.1 --

SC-2-32 5.5 -- -- 33.5

SC-2-37 -- 56.1 -- 52.8

SC-2-45 -- 40.5 -- 16.5

U0126 -- 43 -- 99.6

136 XMD8-92 -- 95.2 -- 50.1

PD 0325901 -- 95.9 -- 99.7

Table 13: MEK1/2 and MEK inhibition data

)

e v

2 0 0

g 1 5 0

/ o

5 1 0 0

;

n e

t 5 0

t a

r 0 g

r F 5 5 2 7 5 2 1 e G 7 2 3 3 4 -9 0 t 1 - - - - 9 E - 2 2 2 2 -8 5 n + 1 - - - - 2 I e - C C C C D ( l 3 C S S S S M 0 ic S h X D e P V C o m p o u n d s (1 0  M )

137

)

e v

1 5 0

1 a

K 1 0 0

;

i R

s 5 0

t a

r 0 g

r F 5 5 2 7 5 6 2 1 e G 7 2 3 3 4 2 -9 0 t 1 - - - - 1 9 E - 2 2 2 2 0 8 5 n + 1 - - - - D 2 I e - C C C C U ( l 3 C S S S S M 0 ic S X h D e P V C o m p o u n d s (1 0  M )

Figure 41: MEK1/2 and MEK5 inhibition graphs.

138 Biological data from Dr. Matthew Burow

The biological testing of the compounds was conducted in the laboratory of Dr. Matthew

Burow (Tulane University, New Orleans, LA).

Crystal violet proliferation assay Protocol:

MDA-MB 231 cells were used as they express the triple negative cancer phenotype.

1) MDA-MB-231 were seeded in 96-well plates at a density of 2,000 cells per well

in 5% charcoal-stripped phenol free DMEM, allowed to attach overnight, and

subsequently treated with DMSO and MEK inhibitor compounds.

2) Plates were harvested on days 3, 5 and 7, fixed with glutaraldehyde and stained

with Crystal Violet. Cells were observed for morphological changes under an

inverted microscope.

3) The cells were washed, lysed, and the absorbance of Crystal Violet sequestered in

living cells was determined at 630 nM in a Biotek Synergy plate reader.

4) Wells were conducted in duplicate. Experiments were run in triplicate.

5) Cells were normalized to initial cell count.

Biological data

Figure 42: Dose-response effect of compound 57 (SC-1-151) at different concentrations.

139

Figure 43: MDA-MB-231 – SC-1-151 treatment (Dual MEK1/2 and MEK5 inhibitor) – Morphology at day 3.

DMSO 0.001 µM

0.01 µM 0.1 µM

1.0 µM 10 µM

Figure 44: MDA-MB-231 – SC-1-151 treatment (Dual MEK1/2 and MEK5 inhibitor) – Morphology at day 14.

140

Figure 45: Fold change of E-Cadherin upon administration of 57 (SC-1-151).

141 Construction of the homology model:

• Of the three human isoforms; A, B and C, a model of the B isoform (b) (Q13163)

was built following CLUSTAL W225-26 sequence alignment.

• 3EQC (MEK1)27 was used as the 3-D structural template and the low resolution

residues in the template were modeled prior to model building.

• The constitutively active mutant of human MEK5 β [MEK5 β DD] was modeled

by mutating Ser 311 and Thr 315 to Asp.

• Care was taken to ensure that the DFG and HRD motifs were aligned properly

with the template.

Figure 46: Sequence Alignment of Human MEK5 β DD with Human MEK1. Conserved residues are highlighted in blue, similar residues are highlighted in magenta. Helices represented by coils, beta-strand by blue arrow and loops by black lines. Domains, HRD and DFG aligned were highlighted by vertical boxes. (The homology model analysis was conducted by Dr. Sankar Manepalli in Madura lab and is currently being prepared as a manuscript for submission in September 2014).

142 Gene Entry Entry name Protein names names Length Dual specificity mitogen-activated protein kinase kinase 1 (MAP kinase kinase 1) (MAPKK 1) (MKK1) (EC MAP2K1 2.7.12.2) (ERK activator kinase 1) MEK1 Q02750 MP2K1_HUMAN (MAPK/ERK kinase 1) (MEK 1) PRKMK1 393 Dual specificity mitogen-activated protein kinase kinase 2 (MAP kinase MAP2K2 kinase 2) (MAPKK 2) (EC 2.7.12.2) MEK2 (ERK activator kinase 2) (MAPK/ERK MKK2 P36507 MP2K2_HUMAN kinase 2) (MEK 2) PRKMK2 400 Dual specificity mitogen-activated protein kinase kinase 3 (MAP kinase kinase 3) (MAPKK 3) (EC 2.7.12.2) MAP2K3 (MAPK/ERK kinase 3) (MEK 3) MEK3 (Stress-activated protein kinase MKK3 kinase 2) (SAPK kinase 2) (SAPKK-2) PRKMK3 P46734 MP2K3_HUMAN (SAPKK2) SKK2 347 Dual specificity mitogen-activated protein kinase kinase 4 (MAP kinase kinase 4) (MAPKK 4) (EC 2.7.12.2) MAP2K4 (JNK-activating kinase 1) JNKK1 (MAPK/ERK kinase 4) (MEK 4) MEK4 (SAPK/ERK kinase 1) (SEK1) (Stress- MKK4 activated protein kinase kinase 1) PRKMK4 (SAPK kinase 1) (SAPKK-1) (SAPKK1) SEK1 (c-Jun N-terminal kinase kinase 1) SERK1 P45985 MP2K4_HUMAN (JNKK) SKK1 399 Dual specificity mitogen-activated MAP2K5 protein kinase kinase 5 (MAP kinase MEK5 kinase 5) (MAPKK 5) (EC 2.7.12.2) MKK5 Q13163 MP2K5_HUMAN (MAPK/ERK kinase 5) (MEK 5) PRKMK5 448 Dual specificity mitogen-activated protein kinase kinase 6 (MAP kinase kinase 6) (MAPKK 6) (EC 2.7.12.2) MAP2K6 (MAPK/ERK kinase 6) (MEK 6) MEK6 (Stress-activated protein kinase MKK6 kinase 3) (SAPK kinase 3) (SAPKK-3) PRKMK6 P52564 MP2K6_HUMAN (SAPKK3) SKK3 334 Dual specificity mitogen-activated protein kinase kinase 7 (MAP kinase MAP2K7 kinase 7) (MAPKK 7) (EC 2.7.12.2) JNKK2 (JNK-activating kinase 2) MEK7 (MAPK/ERK kinase 7) (MEK 7) MKK7 (Stress-activated protein kinase PRKMK7 O14733 MP2K7_HUMAN kinase 4) (SAPK kinase 4) (SAPKK-4) SKK4 419

143 (SAPKK4) (c-Jun N-terminal kinase kinase 2) (JNK kinase 2) (JNKK 2)

FASTA SEQUENCES: >sp|Q02750|MP2K1_HUMAN Dual specificity mitogen-activated protein kinase kinase 1 OS=Homo sapiens GN=MAP2K1 PE=1 SV=2 MPKKKPTPIQLNPAPDGSAVNGTSSAETNLEALQKKLEELELDEQQRKRLEAFLTQKQKV GELKDDDFEKISELGAGNGGVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHE CNSPYIVGFYGAFYSDGEISICMEHMDGGSLDQVLKKAGRIPEQILGKVSIAVIKGLTYL REKHKIMHRDVKPSNILVNSRGEIKLCDFGVSGQLIDSMANSFVGTRSYMSPERLQGTHY SVQSDIWSMGLSLVEMAVGRYPIPPPDAKELELMFGCQVEGDAAETPPRPRTPGRPLSSY GMDSRPPMAIFELLDYIVNEPPPKLPSGVFSLEFQDFVNKCLIKNPAERADLKQLMVHAF IKRSDAEEVDFAGWLCSTIGLNQPSTPTHAAGV

>sp|P36507|MP2K2_HUMAN Dual specificity mitogen-activated protein kinase kinase 2 OS=Homo sapiens GN=MAP2K2 PE=1 SV=1 MLARRKPVLPALTINPTIAEGPSPTSEGASEANLVDLQKKLEELELDEQQKKRLEAFLTQ KAKVGELKDDDFERISELGAGNGGVVTKVQHRPSGLIMARKLIHLEIKPAIRNQIIRELQ VLHECNSPYIVGFYGAFYSDGEISICMEHMDGGSLDQVLKEAKRIPEEILGKVSIAVLRG LAYLREKHQIMHRDVKPSNILVNSRGEIKLCDFGVSGQLIDSMANSFVGTRSYMAPERLQ GTHYSVQSDIWSMGLSLVELAVGRYPIPPPDAKELEAIFGRPVVDGEEGEPHSISPRPRP PGRPVSGHGMDSRPAMAIFELLDYIVNEPPPKLPNGVFTPDFQEFVNKCLIKNPAERADL KMLTNHTFIKRSEVEEVDFAGWLCKTLRLNQPGTPTRTAV

>sp|P46734|MP2K3_HUMAN Dual specificity mitogen-activated protein kinase kinase 3 OS=Homo sapiens GN=MAP2K3 PE=1 SV=2 MESPASSQPASMPQSKGKSKRKKDLRISCMSKPPAPNPTPPRNLDSRTFITIGDRNFEVE ADDLVTISELGRGAYGVVEKVRHAQSGTIMAVKRIRATVNSQEQKRLLMDLDINMRTVDC FYTVTFYGALFREGDVWICMELMDTSLDKFYRKVLDKNMTIPEDILGEIAVSIVRALEHL HSKLSVIHRDVKPSNVLINKEGHVKMCDFGISGYLVDSVAKTMDAGCKPYMAPERINPEL NQKGYNVKSDVWSLGITMIEMAILRFPYESWGTPFQQLKQVVEEPSPQLPADRFSPEFVD FTAQCLRKNPAERMSYLELMEHPFFTLHKTKKTDIAAFVKEILGEDS

>sp|P45985|MP2K4_HUMAN Dual specificity mitogen-activated protein kinase kinase 4 OS=Homo sapiens GN=MAP2K4 PE=1 SV=1 MAAPSPSGGGGSGGGSGSGTPGPVGSPAPGHPAVSSMQGKRKALKLNFANPPFKSTARFT LNPNPTGVQNPHIERLRTHSIESSGKLKISPEQHWDFTAEDLKDLGEIGRGAYGSVNKMV HKPSGQIMAVKRIRSTVDEKEQKQLLMDLDVVMRSSDCPYIVQFYGALFREGDCWICMEL MSTSFDKFYKYVYSVLDDVIPEEILGKITLATVKALNHLKENLKIIHRDIKPSNILLDRS GNIKLCDFGISGQLVDSIAKTRDAGCRPYMAPERIDPSASRQGYDVRSDVWSLGITLYEL ATGRFPYPKWNSVFDQLTQVVKGDPPQLSNSEEREFSPSFINFVNLCLTKDESKRPKYKE LLKHPFILMYEERAVEVACYVCKILDQMPATPSSPMYVD

>sp|Q13163|MP2K5_HUMAN Dual specificity mitogen-activated protein kinase kinase 5 OS=Homo sapiens GN=MAP2K5 PE=1 SV=2 MLWLALGPFPAMENQVLVIRIKIPNSGAVDWTVHSGPQLLFRDVLDVIGQVLPEATTTAF EYEDEDGDRITVRSDEEMKAMLSYYYSTVMEQQVNGQLIEPLQIFPRACKPPGERNIHGL KVNTRAGPSQHSSPAVSDSLPSNSLKKSSAELKKILANGQMNEQDIRYRDTLGHGNGGTV YKAYHVPSGKILAVKVILLDITLELQKQIMSELEILYKCDSSYIIGFYGAFFVENRISIC TEFMDGGSLDVYRKMPEHVLGRIAVAVVKGLTYLWSLKILHRDVKPSNMLVNTRGQVKLC DFGVSTQLVNSIAKTYVGTNAYMAPERISGEQYGIHSDVWSLGISFMELALGRFPYPQIQ KNQGSLMPLQLLQCIVDEDSPVLPVGEFSEPFVHFITQCMRKQPKERPAPEELMGHPFIV QFNDGNAAVVSMWVCRALEERRSQQGPP

>sp|P52564|MP2K6_HUMAN Dual specificity mitogen-activated protein kinase kinase 6 OS=Homo sapiens GN=MAP2K6 PE=1 SV=1

144 MSQSKGKKRNPGLKIPKEAFEQPQTSSTPPRDLDSKACISIGNQNFEVKADDLEPIMELG RGAYGVVEKMRHVPSGQIMAVKRIRATVNSQEQKRLLMDLDISMRTVDCPFTVTFYGALF REGDVWICMELMDTSLDKFYKQVIDKGQTIPEDILGKIAVSIVKALEHLHSKLSVIHRDV KPSNVLINALGQVKMCDFGISGYLVDSVAKTIDAGCKPYMAPERINPELNQKGYSVKSDI WSLGITMIELAILRFPYDSWGTPFQQLKQVVEEPSPQLPADKFSAEFVDFTSQCLKKNSK ERPTYPELMQHPFFTLHESKGTDVASFVKLILGD

>sp|O14733|MP2K7_HUMAN Dual specificity mitogen-activated protein kinase kinase 7 OS=Homo sapiens GN=MAP2K7 PE=1 SV=2 MAASSLEQKLSRLEAKLKQENREARRRIDLNLDISPQRPRPTLQLPLANDGGSRSPSSES SPQHPTPPARPRHMLGLPSTLFTPRSMESIEIDQKLQEIMKQTGYLTIGGQRYQAEINDL ENLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRILMDLDVVLKSHDCPYIV QCFGTFITNTDVFIAMELMGTCAEKLKKRMQGPIPERILGKMTVAIVKALYYLKEKHGVI HRDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYMAPERIDPPDPTKPDYD IRADVWSLGISLVELATGQFPYKNCKTDFEVLTKVLQEEPPLLPGHMGFSGDFQSFVKDC LTKDHRKRPKYNKLLEHSFIKRYETLEVDVASWFKDVMAKTESPRTSGVLSQPHLPFFR

145

MEK Isozymes: (Last update 7/9/2014)

MEK1: Common MEK1 abbreviation MEK1, MAPKK1, MKK1, PRKMK1, Mek1, ERK activator kinase Previous and 1, MAP kinase kinase 1, MAP kinase/Erk kinase 1, MAPK/ERK unofficial kinase 1, MAPKK 1, dual specificity mitogen-activated protein kinase names kinase 1, Prkmk1

Genes MAP2K1 (Hs), Map2k1 (Mm), Map2k1 (Rn)

ENSG00000169032 (Hs), ENSMUSG00000004936 (Mm), Ensembl ID ENSRNOG00000010176 (Rn) UniProtKB Q02750 (Hs), P31938 (Mm), Q01986 (Rn) EC number 2.7.12.2

Post-translational modifications: Residue Modification 1 N-acetylmethionine 222 O-acetylserine 222 Phosphoserine 226 O-acetylserine 226 Phosphoserine 293 Phosphoserine 295 Phosphoserine 394 Phosphothreonine 396 Phosphothreonine

Nucleotide binding site: 74-82

MEK2: Common MEK2 abbreviation PRKMK2, MEK2, MKK2, Prkmk2, ERK activator kinase 2, MAP Previous and kinase kinase 2, MAPK/ERK kinase 2, MAPKK 2, MEK 2, dual unofficial names specificity mitogen-activated protein kinase kinase 2, MAP kinase/Erk kinase

146 Genes MAP2K2 (Hs), Map2k2 (Mm), Map2k2 (Rn)

ENSG00000126934 (Hs), ENSMUSG00000035027 (Mm), Ensembl ID ENSRNOG00000020005 (Rn) UniProtKB P36507 (Hs), Q63932 (Mm), P36506 (Rn)

EC number 2.7.12.2

Post-translational modifications: Residue Modification: 1 N-acetylmethionine 222 O-acetylserine 222 Phosphoserine 226 O-acetylserine; 226 Phosphoserine 293 Phosphoserine 295 Phosphoserine 394 Phosphothreonine 396 Phosphothreonine

Nucleotide binding site: 78-86

MEK3: Common MKK3 abbreviation PRKMK3, MAPK/ERK kinase 3, MAP kinase kinase 3, dual Previous and specificity mitogen activated protein kinase kinase 3, MEK3, MKK3, unofficial names MAPKK3, LOC303200, Map2k3_predicted, Prkmk3

Genes MAP2K3 (Hs), Map2k3 (Mm), Map2k3 (Rn)

ENSG00000034152 (Hs), ENSMUSG00000018932 (Mm), Ensembl ID ENSRNOG00000006612 (Rn) UniProtKB P46734 (Hs), O09110 (Mm)

EC number 2.7.12.2

Post-translational modifications: Residue Modification: 1 N-acetylmethionine 3 Phosphoserine 15 Phosphoserine 218 Phosphoserine [S → E: Constitutive activation] 222 Phosphothreonine [T → E: Constitutive activation]

147

Splice variants: Isoform 1: [VSP_004878] Residues 1-29 missing: MESPASSQPA SMPQSKGKSK RKKDLRISC Isoform 2: [VSP_004877] Residues 1-16 altered: MGVQGTLMSR DSQTPHLLSI LGKS

Isoform 3: (canonical form)

Nucleotide binding site: 70-78

MEK4: Common MKK4 abbreviation Previous and SERK1, MEK4, JNKK1, PRKMK4, MKK4, LOC287398, Map2k4_predicted, Sek1, unofficial SAPK/ERK kinase 1, dual specificity mitogen-activated protein kinase kinase 4, names mitogen activated protein kinase kinase 4, Serk1

Genes MAP2K4 (Hs), Map2k4 (Mm), Map2k4 (Rn)

Ensembl ID ENSG00000065559 (Hs), ENSMUSG00000033352 (Mm), ENSRNOG00000003834 (Rn) UniProtKB P45985 (Hs), P47809 (Mm)

EC number 2.7.12.2

Post-translational modifications: Residue Modification: 2 N-acetylalanine 90 Phosphoserine 257 Phosphoserine [by MAP3K] 261 Phosphothreonine [by MAP3K]

Splice variants: Isoform 1: (canonical form)

Isoform 2: [VSP_038838] Residue 39 altered: G → GFQINFCEKAQS

Nucleotide binding site: 108-116

148 MEK5: Common MEK5 abbreviation PRKMK5, MEK5, MAPKK5, HsT17454, Mek5, MAP kinase Previous and kinase 5, MAPK/ERK kinase 5, MAPKK 5, MEK 5, dual specificity unofficial names mitogen-activated protein kinase kinase 5

Genes MAP2K5 (Hs), Map2k5 (Mm), Map2k5 (Rn)

ENSG00000137764 (Hs), ENSMUSG00000058444 (Mm), Ensembl ID ENSRNOG00000007926 (Rn) UniProtKB Q13163 (Hs), Q9WVS7 (Mm), Q62862 (Rn)

EC number 2.7.12.2

Post-translational modifications: Residue Modification: 311 Phosphoserine 315 Phosphothreonine

Splice variants: Isoform A: 349-358: Missing.

Isoform B: (canonical form)

Isoform C: (partial sequence) 349-358: Missing. 444-448: QQGPP → LASLPSPSPSV

Isoform 4: [VSP_043333] (inferred from mRNA, protein not isoated) Alternate sequence 1-45: MLWLALGPFP AMENQVLVIR IKIPNSGAVD WTVHSGPQLL FRDVL → MMEGHFPQS

Nucleotide binding site: 172-180

MEK6: Common MKK6 abbreviation PRKMK6, MAPKK 6, MEK6, protein kinase mitogen-activated kinase Previous and 6, MAP kinase kinase 6, MGC93287, Mkk6, dual specificity mitogen- unofficial activated protein kinase kinase 6, MKK6, SAPKK3, Prkmk6, Stress- names activated protein kinase kinase 3, SAPK kinase 3, SKK3

149

Genes MAP2K6 (Hs), Map2k6 (Mm), Map2k6 (Rn)

ENSG00000108984 (Hs), ENSMUSG00000020623 (Mm), Ensembl ID ENSRNOG00000004437 (Rn) UniProtKB P52564 (Hs), P70236 (Mm), Q925D6 (Rn)

EC number 2.7.12.2

Post-translational modifications: Residue Modification: 311 Phosphoserine 315 Phosphothreonine

Splice variants: Isoform 1: (canonical form) [MKK6b]

Isoform 2: [MKK6] Missing residues 1-56: [VSP_004882]

Nucleotide binding site: 59-67

MEK7: Common MKK7 abbreviation PRKMK7, MKK7, Jnkk2, JNKK2, JNK kinase 2, Stress-activated Previous and protein kinase kinase 4, SAPKK4, SKK4, JNK-activating kinase 2, unofficial MAP kinase kinase 7, MAPK/ERK kinase 7, MAPKK 7, MEK 7, c- names Jun N-terminal kinase kinase 2, dual specificity mitogen-activated protein kinase kinase 7, sek2, Prkmk7

Genes MAP2K7 (Hs), Map2k7 (Mm), Map2k7 (Rn)

ENSG00000076984 (Hs), ENSMUSG00000002948 (Mm), Ensembl ID ENSRNOG00000001047 (Rn) UniProtKB O14733 (Hs), Q8CE90 (Mm), Q4KSH7 (Rn)

EC number 2.7.12.2

Post-translational modifications: Residue Modification: 311 Phosphoserine 315 Phosphothreonine

150

Splice variants: Isoform 1: (canonical form) [Isoform A,]

Isoform 2: [Isoform B] [VSP_004883] Insertion at 111: T → IIVITLSPAPAPSQRAA

Isoform 3: (1) [VSP_022309] Insertion at 42: T → IIVITLSPAPAPSQRAA [VSP_022309]

Isoform 4: [VSP_022310] (extrapolation from cDNA library) Insertion at 312-312: L → LPCPSPSQ

Nucleotide binding site: 122-134

Analysis of isoform homology at the ATP site:

MEK 1: LGAGNGGVV MEK 2: LGAGNGGVV MEK 3: LGRGAYGVV MEK 4: IGRGAYGSV MEK 5: LGHGNGGTV MEK 6: LGRGAYGVV MEK 7: LGEMMGSGTCGQV

151 Domain analysis: Lower platform near triphosphate: 140- 147: HRDVKPSNM (MEK5DD) MEK5DD with amide docked. The HRDVKPSNM sequence is displayed.

152

CLUSTAL analysis of the HRDVKPSNM sequence; for all 7 MEK’s: 78-88 % homology: SP|sp|Q13163|MP2K5_HUMAN|MP2K5 HRDVKPSNM ||||||||. SP|sp|Q02750|MP2K1_HUMAN|MP2K1 HRDVKPSNI

Percentage ID = 88.89

153

C-helix: 163-175: VSTQLVNSIAKTY

CLUSTAL analysis of the VSTQLVNSIAKTY sequence; for all 7 MEK’s: 46-77 % homology: SP|sp|Q13163|MP2K5_HUMAN|MP2K5 VSTQLVNSIAKTY || ||..|.|... SP|sp|Q02750|MP2K1_HUMAN|MP2K1 VSGQLIDSMANSF

Percentage ID = 46.15

154

Back to C-helix: 159-162: CDFG

155

CLUSTAL analysis of the CDFG sequence; for all 7 MEK’s: 100 % homology:

Hinge and -sheet: 35-37: NGG 54-56: KVI 98-100: ICT

156 CLUSTAL analysis of the Hinge: 35-37: NGG; for all 7 MEK’s: 25-100 % homology: CLUSTAL analysis of the -sheet: 54-56: KVI; for all 7 MEK’s: 46-77 % homology: CLUSTAL analysis of the -sheet: 98-100: ICT; for all 7 MEK’s: 50-67 % homology:

Hinge and -sheet: 35-37: NGG 54-56: KVI 98-100: ICT

157 Homology tree for MEK isoforms:

Overall Homology: MEK5 with MEK1: 38 % MEK5 with MEK2: 37 % MEK5 with MEK3: 39 % MEK5 with MEK4: 35 % MEK5 with MEK6: 40 % MEK5 with MEK7: 30 %

158 Swiss-Prot references: MEK1 Entry name MP2K1_HUMAN Accession Primary (citable) accession number: Q02750 Entry history Integrated into UniProtKB/Swiss-Prot: July 1, 1993 Last sequence update: January 23, 2007 Last modified: July 9, 2014

MEK2 Entry name MP2K2_HUMAN Accession Primary (citable) accession number: P36507 Entry history Integrated into UniProtKB/Swiss-Prot: June 1, 1994 Last sequence update: June 1, 1994 Last modified: July 9, 2014

MEK3 Entry name MP2K3_HUMAN Accession Primary (citable) accession number: P46734 Secondary accession number(s): B3KSK7 expand/collapse secondary AC list Q9UE72 Entry history Integrated into UniProtKB/Swiss-Prot: November 1, 1995 Last sequence update: November 1, 2002 Last modified: July 9, 2014

MEK4 Entry name MP2K4_HUMAN Accession Primary (citable) accession number: P45985 Secondary accession number(s): B2R7N7 expand/collapse secondary AC list Q6PIE6 Entry history Integrated into UniProtKB/Swiss-Prot: November 1, 1995 Last sequence update: November 1, 1995 Last modified: July 9, 2014

MEK5 Entry name MP2K5_HUMAN Accession Primary (citable) accession number: Q13163 Secondary accession number(s): B4DE43, Q92961, Q92962 Entry history Integrated into UniProtKB/Swiss-Prot: November 1, 1997 Last sequence update: November 28, 2006 Last modified: July 9, 2014

159 MEK6 Entry name MP2K6_HUMAN Accession Primary (citable) accession number: P52564 Entry history Integrated into UniProtKB/Swiss-Prot: October 1, 1996 Last sequence update: October 1, 1996 Last modified: July 9, 2014

MEK7 Entry name MP2K7_HUMAN Accession Primary (citable) accession number: O14733 Secondary accession number(s): B2R9S5 expand/collapse secondary AC list Q8IY10 Entry history Integrated into UniProtKB/Swiss-Prot: May 30, 2000 Last sequence update: May 30, 2000 Last modified: July 9, 2014

Concise Guide to PHARMACOLOGY Database page citation:

STE7 family. Accessed on 09/07/2014. IUPHAR/BPS Guide to PHARMACOLOGY, http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=623.

Concise Guide to PHARMACOLOGY citation:

Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, Peters

JA and Harmar AJ, CGTP Collaborators. (2013) The Concise Guide to

PHARMACOLOGY 2013/14: Enzymes. Br J Pharmacol. 170: 1797–1867.

160