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Synthesis of small molecule inhibitors targeting signal transduction pathways

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Synthesis of small molecule inhibitors targeting signal transduction pathways
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Ramamoorthy, Divya
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Bcl-xL
Shp
Ras pathway
EGFR pathway
DiFMUP
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bibliography   ( marcgt )
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ABSTRACT: The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transduction pathways, thereby treating cancer. We attempted to synthesize compounds based on the hits obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, history of chemotherapeutic drugs and an introduction to signal transduction pathways. The following two chapters briefly introduce the biological targets in the authors study. Chapter Two describes the role of B-cell lymphoma type xL (Bcl-xL), in apoptosis and the development of drugs targeting Bcl-xL. Examples of Bcl-xL drugs relevant to this study have been provided.Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathways and the recent developments in drug development towards the inhibition of this oncogene. Chapter Four gives a general introduction to microwave-assisted organic synthesis and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author's own findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibitors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL. Our strategy focused on identifying an isoquinolinol lead with increased potency.Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency.
Thesis:
Thesis (M.S.)--University of South Florida, 2009.
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by Divya Ramamoorthy.
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Synthesis of Small Molecule Inhibitors Targeting Signal Transduction Pathways by Divya Ramamoorthy A dissertation submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Nicholas J Lawrence, Ph.D. Wayne C. Guida, Ph.D. David Merkler, Ph.D. Jerry Wu, Ph.D. Date of Approval: June 10, 2009 Keywords: Bcl-xL, Shp, Ras pathway, EGFR pathway, DiFMUP Copyright 2009, Divya Ramamoorthy

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Dedication Dedicated to my parents

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Acknowledgments I would like to thank my research advisor, Prof. Nicholas J Lawrence for his guidance. For the use of HTS instruments, I would like to thank Dr Harshani Lawrence and Yunting Luo. For the biological analysis and computational studies, I thank all the members of the Drug Discovery Program, Prof. Jerry Wu (Shp-2 phosphatase project), Dr LeiWei Chen (Shp-2 phosphatase project), Dr Shen-Shu Sung (modeling for Shp2 phosphatase project), and Dr George Sun (Bcl-Xl). I would like to thank my committee members, Prof. Wayne C. Guida, Prof. David Merkler, Prof. Roman Manetsch and Prof. Jerry Wu for their support and suggestions. I would like to thank the crystallographer, Gregory McManus, and Prof. Mike Zaworotkos lab at the University of South Florida, who helped me in obtaining all the crystal data. I want to thank present and past members of Lawrence lab: Jayan Narayanan, Danielle Pernazza, Roberta Pireddu, Jing He, Jon Underwood and Xin Wu for their patience, support and help throughout. I would like to thank my current advisor, Dr Wayne Guida for his encouragement, support and help. I would also like to thank Guida lab members: Daniel Santiago, Courtney Duboulay and Sai Lakshman Vankayala for their help and constant support. Last but not the least; I want to thank my husband, Arun who has been very understanding and supportive.

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i Table of Contents List of Abbreviations.............................. ................................................... .....................iv List of Tables..................................... ................................................... ............................v List of Figures.................................... ................................................... ...........................vi Abstract........................................... ................................................... ...........................viii Chapter 1 Signal Transduction and Cancer .......... ................................................... ....1 1.1 Introduction................................... ................................................... .....................1 1.2 Homeostasis.................................... ................................................... ...................1 1.3 Apoptosis...................................... ................................................... .....................2 1.4 Chemotherapy .................................. ................................................... .................3 1.5 Signal Transduction............................ ................................................... ...............4 1.6 Protein tyrosine kinase inhibitor: Imatinib.... ................................................... ....6 1.7 Conclusion..................................... ................................................... ....................9 Chapter 2 Design of Bcl-xL inhibitors as therapeuti c agents for cancer therapy ........................................ ................................................... ............10 2.1 Introduction............................... ................................................... .........................10 2.2 General characteristics of Bcl family prote ins................................................ ......12 2.3 Bcl-xL..................................... ................................................... ...........................12 2.4 Bcl-xL – Bad Complex ...................... ................................................... ...............14 2.5 Known inhibitors of Bcl-xL ................ ................................................... ..............15 2.6 Conclusion ................................ ................................................... ........................17

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ii Chapter 3 Shp2 Phosphatase as a Target for Cancer T herapy ...................................18 3.1 Introduction .............................. ................................................... .........................18 3.2 Protein Tyrosine Phosphatases (PTPs)....... ................................................... .......19 3.3 SHPs ...................................... ................................................... ............................20 3.3.1 Shp1 PTPase ............................... ................................................... ...........21 3.3.2 Shp2 PTPase ................................ ................................................... ..........21 3.4 Shp2 targets .............................. ................................................... .........................22 3.5 Ras/MAPK pathway .......................... ................................................... ...............23 3.6 Epidermal growth factor signaling pathway ................................................... ....24 3.7 Known inhibitors of Shp2 .................. ................................................... ...............25 3.8 Conclusion ................................ ................................................... ........................27 Chapter 4 Microwaves and Flow reactors in organic s ynthesis ................................28 4.1 Introduction: Microwave-assisted synthesis ................................................... .....28 4.2 Flow reactors: Hydrogenator ............... ................................................... .............31 4.3 Conclusion ................................ ................................................... ........................32 Chapter 5 Synthesis and evaluation of small molecul es as Bcl-xL Inhibitors......................................... ................................................... .....33 5.1 Introduction .............................. ................................................... .........................33 5.2 Biological evaluation of compounds, 12a-z .................................................. ......39 5.3 Conclusion ................................ ................................................... ........................39 Chapter 6 Synthesis and evaluation of small molecul es as Shp2 inhibitors ........................................ ................................................... .....40 6.1 Hit-to-Lead approach based on HTS screening .................................................. .40 6.1.1 Synthesis of HLM000661 .................................................. ......................41 6.1.2 Synthesis of HLM019544 .................................................. ......................42 6.1.3 Synthesis of HLM002903 ................................................... ......................48 6.1.4 Synthesis of HLM001038 and HLM 001426 ..........................................49 6.2 Hit-to-Lead approach based on hits of relat ed phosphatases ...............................51

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iii 6.2.1 HePTP hit evaluated for Shp2 ............... ................................................... .51 6.3 Hit-to-Lead approach based on previous hits .................................................. ....59 6.3.1 Synthesis of urea derivatives .............. ................................................... ...59 6.3.2 Synthesis of Isatin derivatives ............ ................................................... ...60 6.4 Conclusion ................................ ................................................... ........................63 Chapter 7 Experimental ............................ ................................................... ..............64 7.1 General Methods and Instrumentation ....... ................................................... .......64 7.2 General procedure for the synthesis of 2,2-Di ethoxy-N-(2,3,4substituted benzyl) ethanamines ............ ................................................... ..........64 7.3 General procedure for the synthesis of 4-(4 -aryl)-6,7,8 substituted isoquinolines .................................... ................................................... .................67 7.4 General procedure for the synthesis of tetr ahydro-cyclopentane quinoline-4-carboxylic acids ..................... ................................................... ........96 7.5 General procedure for the synthesis of subs tituted cyclohexane carboxylic acids ................................. ................................................... .............115 7.6 General procedure for the synthesis of subs tituted oxobutanoic acids ..............119 7.7 General procedure for the synthesis of urea derivatives ....................................12 3 References ........................................ ................................................... .........................151 Appendices......................................... ................................................... ........................161 Appendix 1......................................... ................................................... ........................162

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iv List of Abbreviations DiFMUP 6,8-Difluoro-4-methylumbelliferyl phosphate EYA Eyes absent EGF Epidermal growth factor EGFR Epidermal growth factor receptor ERK Extra-cellular signal-regulated kinase FRS Fibroblast growth factor Receptor Substrate IRS Insulin receptor substrate KDa Kilo dalton PDGFR Platelet derived growth factor receptor PI3K Phosphodyl inositol 3-kinase PK Protein kinase pNPP para-nitrophenyl phosphate PP Protein phosphatase PTP Protein tyrosine phosphatase Ser Serine SH Src homology Shc Src homology 2/ -collagen-related TK Tyrosine kinase Tyr Tyrosine Thr Threonine

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v List of Tables Table 1 Synthesis of substituted isoquinolinols, 20a-z from 19a-e 37 Table 2 Summary of the reaction conditions towards the synthesis of 39a 47 Table 3 Synthesis of quinolones 39a-h 48 Table 4 Synthesis of isooxindole derivatives 54 Table 5 Synthesis of substituted cyclohexane carbox ylic acids 63a-h 56 Table 6 Synthesis of substituted oxobutanoic acids 52a-g 56 Table 7 Synthesis of urea derivatives 78a-e 60 Table 8 Synthesis of 82a-x 61

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vi List of Figures Figure 1 Apoptosis by intrinsic and extrinsic pathw ays 3 Figure 2 Structures of early chemotherapeutic agent s 4 Figure 3 Structures of Actinomycin and folate antag onists 4 Figure 4 Representation of signaling cascade at the cell membrane 5 Figure 5 Structure of Imatinib (Glivec) 7 Figure 6 Schematic representation of the BCR-Abl kinase pathway 7 Figure 7 Mechanism of Action of BCR-ABL and of Its Inhibition by Imatinib 8 Figure 8 Crystal structure of Imatinib with BCR-ABL protein 9 Figure 9 Bcl-2 family members 11 Figure 10 Structural features of anti-apoptotic and pro-apoptotic Bcl-2 proteins 12 Figure 11 Stucture of Human Bcl-xL in complex with the BH3 binding domain of Bak 13 Figure 12 Crystal structure of Bcl-XL in complex wi th Bad 15 Figure 13 Known inhibitors of Bcl-2 family 16 Figure 14 Structures of ABT-737 and ABT-263 16 Figure 15 Crystal structure of Bcl-xL in complex wi th ABT-737 17 Figure 16 Protein kinases and protein phosphatases 18 Figure 17 Mechanism of dephosphorylation by PTP 20 Figure 18 SHP proteins domain structures 20 Figure 19 Crystal structure of Shp1 SH2 domain comp lexed with a tyrosine-phosphorylated 21 Figure 20 Crystal structure of the human tyrosine p hosphatase Shp2 22 Figure 21 Schematic representation of basal and act ive states of Shp2 22

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vii Figure 22 Role of Shp2 in Ras-Raf Pathway 24 Figure 23 Schematic representation of the EGFR path way 25 Figure 24 Known Shp2 inhibitors 25 Figure 25 PHPS1 and Isatin derivatives and their ac tivity against Shp2 26 Figure 26 A Active site of Shp2 showing the catalyt ic cleft 26 B Ligand interaction of PHPS1 in Shp2 active site 26 Figure 27 Structure of Isatin ( 6 ) and ( 7 ) docked to Shp2 27 Figure 28 dipole moments align parallel in an elect ric field 29 Figure 29 Biginelli reaction 30 Figure 30 Biotage microwave reactor and H-cube hydr ogenator 31 Figure 31 Structures of HL2-100 and HL2-100-2 33 Figure 32 Pomeranz-Fritsch reaction 34 Figure 33 Mechanism of Pomeranz-Fritsch reaction 35 Figure 34 Modifications to Pomeranz-Fritsch reactio n 35 Figure 35 Synthesis of substituted isoquinolinols, 20a-z 36 Figure 36 Mechanism of synthesis of isoquinolinols 38 Figure 37 FP assay 39 Figure 38 Shp2 hits derived from a high throughput screen at the Moffitt Cancer Center 40 Figure 39 X-ray crystal structure of 41 44 Figure 40 X-ray Crystal structure of 44 49 Figure 41 X-ray Crystal Structure of 46a 50 Figure 42 pNPP Assay: Dephosphorylation by Shp2 51 Figure 43 DiFMUP Assay: Dephosphorylation by Shp2 51 Figure 44 Docking structure of 52a with the Shp2 PTPase domain 53 Figure 45 Interactions of 52a amino acid residues of the Shp2 protein, in the docking structure 53 Figure 46 Isatin hits for Shp2 59

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viii Synthesis of Small Molecule Inhibitors Targeting Si gnal Transduction Pathways Divya Ramamoorthy ABSTRACT The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transducti on pathways, thereby treating cancer. We attempted to synthesize compounds based on the h its obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, h istory of chemotherapeutic drugs and an introduction to signal transduction pa thways. The following two chapters briefly introduce the biological targets in the aut hors study. Chapter Two describes the role of B-cell lymphoma t ype xL (Bcl-xL), in apoptosis and the development of drugs targeting Bc l-xL. Examples of Bcl-xL drugs relevant to this study have been provided. Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathwa ys and the recent developments in drug development towards the inhibition of this oncogene Chapter Four gives a general introduction to microwave-assisted organic synthesi s and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author’s ow n findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibit ors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL Our strategy focused on identifying an isoquinolinol le ad with increased potency. We focused on improving the synt hetic N O OH NSC-131734,R= HL2-100,R= R

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ix procedure using microwave-assisted heating, thereby reducing the reaction time and facilitating a combinatorial approach for the synthesis of these isoquinolinols. Isoquinolinols with different aryl groups (R) were synthesized to study the structure activity relationship. A series of isoquinolinols were synthesized by varying the position of the hydroxyland methoxygroups in the isoquinoline core. Chapter Six focuses on the development of Shp2 inhibitors. Based on the hits obtained from HTS screening, compounds were re-synthesized and were evaluated for biological activity for Shp2. A series of compounds were synthesized, containing the isooxindole scaffold, based on the hematopoetic protein tyrosine phosphatase (HePTP) hits reported earlier in literature. Modifications were performed on the isooxindole core to study the SAR. Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency. N H Cl S O O N H N O RPM744, IC50:0.98 m M N H O OH N CO2H O HePTPhit,IC50=1.17 m M O

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1 Chapter 1 1.0 Signal transduction and cancer 1.1 Introduction Cancer is characterized by uncontrolled cell growt h, invasion, and metastasis (spread to other locations in the body).1 Cancer cells, derived from the normal cells in the body, undergo alterations, which distinguish them from the normal cells. The a lterations in the cells allow them to proliferate. Cancer can be caused by genetic abnorm alities in the genetic material of the transformed cells. The abnormalities may be caused by carcinogens, errors in DNA replication or by inheritance. Genetic abnormalitie s affect oncogenes (genes that contribute to cancer in a gain-of-function manner) and, tumor suppressor genes (genes inactivated in cancer cells), resulting in the loss of normal functions in those cells.1 Apoptosis or programmed cell death plays a very imp ortant role to maintain the balance of cell growth and cell death, essential for homeos tasis. 1.2 Homeostasis Homeostasis is a property that refers to the regul ation of the internal environment to maintain a stable, constant condition in any liv ing organism. It is a property of either the open-system (system interacting with the enviro nment) or the closed-system (system isolated from the environment) and is maintained by a balance of cell-proliferation and cell-death in multicellular organisms.2 Homeostasis is achieved when the rate of cell divi sion by mitosis is balanced by cell death. If the balance is disrupted, it might r esult in the formation of a tumor, by cells dividing much faster than they die, or a disorder o f cell loss, by cells dividing much slower than they die. Thus it is a very important p rocess in living organisms to maintain their internal states within certain limits. Many t ypes of cell signaling are involved in homeostasis making it essential to be a tightly con trolled process: any impairment in this

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2 process might lead to a diseased state.2 1.3 Apoptosis A balance of cell division and cell death is essen tial to maintain homeostasis in multicellular organisms. Apoptosis is a form of pro grammed cell death of unwanted or damaged cells, which is essential for the developme nt and the maintenance of all multicellular organisms. It is critical for the removal of superfluous cells during diverse physiological processes in all organisms.3 As a highly ordered process, apoptosis allows cells to degrade themselves as a way of eliminating unwanted or dysfunctional cells from the body. Cells die in response to a variety of sti muli and during apoptosis they do so in a controlled, regulated fashion.4 Apoptosis is critical for normal development and thermostatics (response to temperature changes) and serves as a defense mechanism against cellular abnormalities. It is tightly regul ated at the transcriptional and posttranscriptional levels. Any dysregulation in the pr ocess, either activation or inhibition, is usually associated with degenerative disorders and cancer.5 Thus apoptotic pathways are of immense interest in drug discovery and developme nt. Apoptosis can be triggered by internal events or a n intrinsic pathway in which the disruption of the mitochondria and cytochrome C lea d to the downstream activation of caspases (Fig. a). Alternatively, extrinsic pathway s also lead to apoptosis: specific ligands bind to the death surface receptors, such as the re ceptors of the tumor necrosis factor (TNF)/ nerve growth factor (NGF) super family (Fig. 1). The immune cells mediate the extrinsic pathway to initiate intracellular signali ng and downstream activation of caspases.6-9

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3 IntrinsicExtrinsic Cytochrome c Death receptors Caspases Apaf-1 APOPTOSIS Fig. 1: Apoptosis by intrinsic and extrinsic pathways 1.4 Chemotherapy Detection of cancer at an early stage is critical, as prevention of metastasis is a major cancer-fighting tool. Cancer can be diagnosed by performing a biopsy on the tissue sample retrieved from a tumor near the body surface Staging refers to the extent or spread of disease at the time of diagnosis. It is b ased on the primary tumor's size and location and is essential to determine the choice o f therapy to treat cancer. Statistics from American Cancer Society reveal th at, in 2004, cancer prevalence in the US was about 10.3 million.10 In 2007 alone, about 7.6 million people died of cancer. Cancer can be treated by surgery, chemother apy, and radiotherapy or by using targeted therapies for specific types of cancer (so urce: www.cancer.org). In the early 20th century, Paul Ehrlich, who had coined the term ‘ch emotherapy’ also developed some aniline drugs to treat cancer. Later, in 1939, Charles Huggins stated that estrogens could be used to treat breast cancer .11 During World War II, the accidental spillage of sul fur mustards on troops, showed a remarkable depletion in the bone marrow and lymph nodes in men exposed to the gas.12, 13 In 1946, researches at Yale University published t he use of nitrogen mustard (Fig. 2) to treat lymphomas; unfortunately the remissions were brief and incomplete.14

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4 S Cl Cl N Cl Cl N Cl Cl N Cl Cl Cl Sulfurmustard Nitrogenmustards 1 2 3 4 Fig. 2 : Structures of early chemotherapeutic agents Farber and coworkers found that folic acid acceler ated leukemia cell growth. Based on the above observation, folate antagonists were developed; these include aminopterin and amethopterin15 (methotrexate) (Fig. 3). These drugs exhibited unquestionable remissions. In 1951, 6-thioquanine a nd 6-mercaptopurine were developed for the treatment of acute leukemia.16, 17 Fluoropyrimidine 5-fluorouracil (5-FU), was developed by Charles Heidelberger and co-workers to treat non-hematologic cancers. This drug was found to have broad-spectrum activity against a range of solid tumors and is currently used in the treatment of colorectal ca ncer. The drug 5-FU targets thymidylate synthase (TS), which is an important chemotherapeut ic target since the inhibition of TS has a positive effect on patients with colorectal a nd breast cancers. It has been used in combination therapy along with other folates to tre at cancer.18 N O O NH2 O HN O NH N N NN NH2 NH2 N NH O HO O HO O NN N N NH2 NH2 Aminopterin Methotrexate Actinomycin HN HN O O F 5-Fluorouracil NH NH O HO O HO O O O N O O N O N O N O O O N O O N O Fig 3 : Structures of Actinomycin and folate antagonists Targeted cancer therapy focuses on treating cancer by attacking only the cancer

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5 cells without damaging the normal cells, thus leadi ng to fewer side effects. Targeted therapy varies with the type of cancer. They all in terfere with the cell proliferation of the cancer cells. Usually targeted therapy involves use of small molecules that enter the cell and disrupt their function leading to cell death. T argeted therapy involves molecules acting on the signaling transduction pathways, disr upting the signaling cascade. 1.5 Signal Transduction Signal transduction refers to the process by which a cell converts one kind of signal to another involving a series of biochemical transformations that are caused by either enzymes19 or protein-protein interactions. Modular protein-pr otein interaction domains, controlled by the phosphorylation status, convey signals from activated receptors using a variety of recognition motifs. In many signal transduction processes, the number of proteins and other molecules participatin g in these events increases as the process emanates from the initial stimulus, resulti ng in “signal cascade” (Fig. 4). Defects in signal transduction pathways often lead to uncon trolled cell growth and tumorigenesis20 indicating the importance of signal transduction i n medicine. Receptor Receptor Membrane Gene expression Growth Survival Differentiation Metabolism PP S S p PPKInput signals Signal-Integrating complex Output USK PTP PTP PP Fig. 4: ( Reproduced from lit21) Representation of signaling cascade at the cell mem brane; signals received at the cell membrane stimulate the activat ion that incorporates both a protein kinase and a protein phosphatase. The agent that signals a cell to respond is a molec ule that binds to a cell surface

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6 receptor or to a cytoplasmic receptor. These signal s can activate protein kinases and phosphatases in the cell. The activation or inactiv ation of the protein kinase or phosphatase, the affinity of the protein target for these enzymes, the concentration and access of the kinase, phosphatase and target protei n affect the phosphorylation or dephosphorylation state of the protein. Phosphoryla tion or dephosphorylation produces specific changes in the function of a target protei n and these changes may increase or decrease its activity. It can also modify the inter actions between the phosphoprotein and other proteins, DNA, phospholipids, or other cellul ar constituents, which can thereby alter the function of the phosphoprotein in a signa ling pathway, ultimately leading to changes in gene expression.22 Signaling pathways are vital for cell growth and wh en some of these become aberrant, it leads to an increase in proliferative potential, sustained angiogenesis, tissue invasion and metastasis, and apoptosis inhibition.23 The proteins involved in the signaling pathways may provide druggable targets for cancer t herapy since inhibition of any one of these proteins stops the signaling cascade. Signal transduction is a cascade process involving a sequence of steps. The receptors in the cell membrane transfer information from the cell exterior to the interior. The receptor (primary messengers) has both extracel lular and intracellular domains: a binding site in the extracellular domain recognizes the chemical signal, forming a receptor-ligand complex, and then passes the signal to the interior of the cell. Secondary messengers, which include cyclic AMP, cyclic GMP, c alcium ion, inositol 1,4,5triphosphate and diacylglycerol, relay information from the receptor-ligand complex to other components of the cell, where gene expression and other processes could be altered. The secondary messengers elicit responses by activa ting protein kinases. After the signaling process is initiated and information tran sduced to other components of the cell, it has to be terminated otherwise cells would lose their responsiveness to new signals or might lead to uncontrolled cell growth. Protein pho sphatases usually terminate the signal transduction processes.24 1.6 Protein tyrosine kinase inhibitor: Imatinib Among the enzymes and the proteins involved in sign al transduction, kinases remain the most established target for cancer thera py. Imatinib (Glivec)25 (Fig. 5),

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7 formerly referred as ST1571, is protein tyrosine ki nase inhibitor, which targets platelet derived growth factor receptor (PDGFR). This is the first example of target-based drug design. The Philadelphia chromosome (Ph) of chronic myelogenous leukemia (CML) and c-kit (CD117) combine to form a product, which is o ver-expressed in gastro-intestinal stromal tumors, which is targeted by Imatinib.26 N N N HN HN O N N Fig. 5 : Structure of Imatinib (Glivec) Protein kinases activate the signal transduction pa thways by phosphorylating proteins (Fig. 6). Several protein kinases are dere gulated and over-expressed in human cancers, thus posing as attractive targets for deve loping selective pharmacologic inhibitors. BCR-Abl kinase of CML is an extensively studied kinase and Imatinib disrupts BCR-Abl mediated transfer of phosphate to its substrates.27-31 BCR-ablgene Chromosome9-22translocation Stat5 Stat1 JAK2 CRKL GRB2 SOS1 BCR/ABLBCR/ABL Nucleus B C R a b l f u s i o n p r o t e i n T3151 Fig. 6: (Reproduced from biocarta pathways) Schematic representation of the BCR-Abl kinase pathway

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8 When ATP is bound to BCR-ABL in the kinase-binding site, the substrate is activated by the phosphorylation of one of its tyrosine residues and can activate other downstream effector molecules. Imatinib bound to the kinase pocket inhibits the action of BCR-ABL, by preventing the phosphorylation on the substrate (Fig. 7). BCR-ABL ATP SubstrateP h o s p h a t eTyrosine SubstrateE f f e c t o rATP P h o s p h a t eTyrosine ChronicMyelogenousLeukemia BCR-ABL Substrate Tyrosine SubstrateE f f e c t o r Tyrosine ChronicMyelogenousLeukemia Imatinib XA B Fig. 7 : (Reproduced from lit32) Mechanism of Action of BCR-ABL and of Its Inhibition by Imatinib A : Activated BCR-ABL oncoprotein with ATP in the kinase pocket; B : Inhibited BCR-ABL oncoprotein with Imatinib in the kinase pocket. Although the precise oncogenic mechanism of BCR-ABL is unknown,30, 33-35 its tyrosine kinase activity leads to the chronic phase of CML.35 BCR-Abl protein kinase C and EGFR were among the first protein kinases to be targeted for selective inhibition because of their unregulated activity in various human cancers.29 Tyrphostins were reported to be specific for epidermal growth factor receptor (EGFR) in 1988. 2Phenylaminopyridine compounds were discovered by chemists at Ciba-Geigy to be specific for tyrosine kinases. Based on SAR studies, imatinib was developed as an inhibitor of PGDF receptor while EGFR, FLT1 and FLT3 remained unaffected.36, 37 Druker et al reported that Imatinib inhibited proliferating myeloid cell lines containing BCR-ABL, but minimally harming normal cells.38 The crystal structure of Imatinib bound to the BCR-ABL kinase is shown in Fig. 8.39

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9 Fig. 8 : Crystal structure of Imatinib with BCR-ABL protei n (PDB ID: 2HYY) 1.7 Conclusion Cancer is recognized to be a major killer, next to heart disease. Development of new drugs to treat cancer is very important, given the fact that the drugs currently used in treatment of cancer have numerous side effects. The discovery of anti-cancer drugs is a challenging endeavor; in spite of the biochemical m echanisms of many signaling pathways being extensively studied. Although immens ely challenging, it is not impossible to develop drug candidates that target s pecific molecular targets or signaling pathways. Some progress to this end is the subject of this dissertation.

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10 Chapter 2 2.0 Design of Bcl-xL inhibitors as therapeutic agen ts for cancer therapy 2.1 Introduction A balance of cell division and cell death is essent ial to maintain homeostasis in multicellular organisms. Apoptosis is a form of pro grammed cell death of unwanted or damaged cells, which is essential for development a nd the maintenance of all multicellular organisms. The Bcl-2 (Basal cell lymphoma) family of proteins plays a very important role in apoptosis. Numerous proteins are involved in the control of ap optosis. The Bcl-2 family of proteins (Fig. 9) are thought to be the key regulat ors functioning at or near the cell death pathway.40 A key component in the process of apoptosis is the activation of the caspase family in a cascade, which is in turn responsible f or the apoptotic-specific changes, and dissembly of the cell. Bcl-2 proteins, present in the outer mitochondrial membrane, play a major role in inhibiting apoptosis by playing a c ritical role in caspase activation. Deactivation of Bcl-2 proteins initiates apoptosis through a well-controlled chain of enzymatic reactions.41

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11 B c l s u p e r f a m i l y Proapoptotic Antiapoptotic BH3-onlyproteins Multidomain(BH) Bcl-2 Bcl-xL Bcl-W Mcl1 Bcl-B +viralhomologs BIK BAD BIM +others BAKBAX BOK +others Fig. 9: (Reproduced from researchapoptosis.com) Bcl-2 family members The Bcl-2 family includes the anti-apoptotic pro teins (proteins that inhibit apoposis), Bcl-xL, Bcl-2, which are the death antag onists, and the proapoptotic proteins (proteins that promote apoptosis), Bak, Bax, Bad,42 which are the death agonists. In normal cells, the anti-apoptotic proteins bind to t he pro-apoptotic proteins and hence block apoptosis, whereas in damaged cells this bala nce is disrupted, triggering apoptosis. The mechanism by which the Bcl-2 proteins regulate apoptosis is not quite clear; however these protein-protein interactions are crit ical for the apoptosis to occur.43 The ratio of the heterodimerization of the death an tagonist Bcl-xL to the agonists Bax, Bak, Bad determines whether a cell will respon d to an apoptotic signal;40 however, the importance of hetero-dimerization is not fully understood. The ability of Bcl-xL to regulate cell survival may involve other dimerizati on partners. Non Bcl-2 family members like Raf-1, calcineurin, CED-4, Apaf-1 and other caspases are also shown to interact with the anti-apoptotic Bcl-2 family membe rs.44, 45 The contribution of these interactions to the regulation of apoptosis is diff icult to address due to the extensive network of protein-protein interactions between the Bcl-2 and non Bcl-2 family members. Cancer cells restrain apoptosis by overproducing an ti-apoptotic proteins such as Bcl-2 and Bcl-xL.46 Over-expression of Bcl-2 or Bcl-xL (or both) has be en found in a majority of human cancers.

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12 2.2 General characteristics of Bcl family proteins Bcl-2 codes for a 25-kDa protein having a hydrophob ic C-terminus located in the outer mitochondrial membrane, the endoplasmic retic ulum and the nuclear membrane, and an N-terminus facing the cytosol. Bcl-2 family members possess conserved motifs known as Bcl-2 homology motifs (BH1 to BH4) (Fig. 1 0). They contain at least one of the four conserved BH domains.5 The anti-apoptotic members, like Bcl-xL and Bcl-2 show sequence conservation in all four domains whil e the pro-apoptotic members, Bax, Bak, and Bok do not show conservation in the first helical sequence, BH4, but only contain BH1, BH2 and BH3, resembling the Bcl-2 fami ly closely. Almost all BH3domain-only members are pro-apoptotic. The Bcl-2 fa mily members form homoor hetero-dimers with their anti-apoptotic counterpart s. The Bcl-x member generates two proteins through alternate splicing mechanism – Bcl -xL and Bcl-xS. Bcl-2 over-expressed cells seem to be deprived of their ATP by glycolysis, which is a reversible process. Bcl-xL blocks cell death b y preventing the release of cytochrome C from mitochondria. Bcl-2 acts as a checkpoint ups tream of caspases and mitochondrial dysfunction. Bcl-2 Proteins Anti-apoptotic Proteins Pro-apoptotic Proteins NN-N-N-C -C-C-CBcl-Xl, Bcl-2, Mcl-1, Al, Bcl-W Bax, Bak, Bok Blk, BNIPBad, Bid, Bim, EGL-1 BH4 BH3 BH1 BH2 TM BH1 BH2 TM BH3 BH3 BH3 TM Fig. 10: (Reproduced from lit47) Structural features of anti-apoptotic and pro-apopt otic Bcl-2 proteins 2.3 Bcl-xL Bcl-xL (Fig. 11) is a transmembrane molecule in mit ochondria that plays a very critical role in cancer development by inhibiting a poptosis. It is a 241 amino acid protein, which shares 43% sequence similarity with Bcl-2; it appears to function in the same apoptotic pathway.48 Bcl-xL has two central hydrophobic a -helices surrounded by

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13 amphipathic helices. A flexible 60-residue loop, no t essential for the anti-apoptotic activity, connects a 1 and a 2. The Bcl-2 homology domains, BH1, BH2 and BH3 are close to each other to form an elongated hydrophobi c cleft. This hydrophobic cleft represents the binding site for Bcl-2 family protei ns. The X-ray crystal structure of human Bcl-xL (PDB ID: 1r2d),49 a close analog of Bcl2, provided the structural ba sis for the design and development of small molecules that bind to Bcl-2.40 The X-ray crystal structure of human Bcl-xL reveals a hydrophobic poc ket essential for the anti-apoptotic activity, formed by the conserved BH1, BH2 and BH3 domains; mutations in this pocket might abolish their biological function.50, 51 Fig. 11: Stucture of Human Bcl-xL in complex with the BH3 bi nding domain of Bak (shown in blue) (in the a -helix binding pocket) (PDB ID: 1bxl) Bcl-xL is an important contributor to the progressi on of vascular diseases, being a critical determinant of intimal lesion formation.52 Expression of Bcl-xL has been found in a range of normal tissues, especially in the centra l nervous system and thymus. In the Bcl-2 family proteins, such as Bcl-xL, Bax, BID and Bcl-2, proteolytic cleavage follows a cell-death stimulus which enhanc es the pro-apoptotic activity in these molecules. In Bcl-xL, a C-terminal fragment exhibit ing Bax-like properties is produced after the endogeneous cleavage by caspases or capli ns.53-55 The NBcl-xL protein which results from the cleavage, lacks the BH4 domain whi ch confers protection against

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14 apoptosis.54, 56 Conformational rearrangements at the N terminus mi ght contribute to its pro-apoptotic activity, especially in the brain, wh ere Bcl-xL is highly expressed.57 A conductance ion channel is produced by NBcl-xL wh ich may induce cell death by increasing the conductance of this membrane and rel easing cytochrome c from mitochondria following a cell-death stimulus.54, 56 Bcl-xL proteolytic cleavage causes a change of fun ction from antito proapoptotic. Both the proand anti-apoptotic forms c ontribute to cell death and some nervous system activities. Therefore long term inhi bition of Bcl-xL could result in the disruption of neuron response to cell death and syn aptic function impairment in the absence of a death stimulus.56, 58 Bcl-xL is over-expressed in most human cancer type s, thus it is a very attractive target for the development of anticancer agents.11 High expression of Bcl-2 or Bcl-xL in human cancer contributes to neoplastic cell expansi on and interferes with the therapeutic effect of the chemotherapeutic agents used in cance r treatment, by blocking apoptosis. Inhibiting the function of Bcl-xL could restore apo ptosis or sensitize the tumors for therapeutic agents. Interaction between the pro-apo ptotic and anti-apoptotic proteins involves the hydrophobic groove, essential for the anti-apoptotic activity, on the surface of the anti-apoptotic members and the BH3 dimerizat ion domain of the pro-apoptotic counterparts.59 Therefore, the BH3 domains of the pro-apoptotic Bc l2 family members, provide natural templates for the development of sm all molecule BH3 mimics that can serve as surrogates for designing inhibitors of ant i-apoptotic Bcl-2 family proteins.43 2.4 Bcl-xL Bad Complex The three dimensional structure of human Bcl-xL in complex with the pro-apoptic partner Bad has been determined and published (PDB ID: 2bzw) (Fig. 12). This provides a structural basis for the design of Bcl-xL inhibit ors. The Bcl-2 family of proteins contains conserved Bcl-2 homology regions which med iate the formation of homoor heterodimers, critical for enhancing or suppressing apoptosis. Bad (Bcl-xL/Bcl-2 associated death promoter) is a new member of the BH3-only fam ily, a subfamily of the Bcl-2 family of proteins.3 However, Bad shares the conserved Bcl-2 homology B H1 and BH2 domains with other Bcl-2 family members. It cou nters the anti-apoptotic effects of Bcl-xL in an interleukin 3-dependent cell line. Bad has no C-terminal trans membrane

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15 domain for targeting to the outer mitochondrial mem brane and nuclear envelope. Hsu et. al. reported that Bad might regulate apoptosis in a di fferent manner from the Bcl-2 family members and interact with cellular proteins outside the Bcl-2 family.3 The pro-apoptotic protein Bad is known to promote apoptosis by dimeri zation with Bcl-xL and displace the apoptosis promoter Bax.3 Fig. 12: Crystal structure of Bcl-XL in complex with Bad (sh own in yellow) (PDB ID: 2bzw) 2.5 Known inhibitors of Bcl-xL Chelerythrine (Fig. 13) was found to be a Bcl-xL in hibitor by high throughput screening based on Fluorescence Polarization. Chele rythrine was found to disrupt the interaction between Bcl-xL and Bax with an IC50 of 1.5 m M.60 Abbott laboratories recently published their discovery of a novel inhib itor of Bcl-xL/Bcl-2, ABT 73761 (IC50 of 35+ 1 nM). HA14-1 was identified to inhibit BAK BH3/Bcl -2 interaction with an IC50 of 9 m M.62 Gossypol and Theaflavanin were found to inhibit Bad BH3/Bcl-xL interactions with an IC50 of 0.5 m M63, 64 and 120-1230 nM respectively.65

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16 N+ O O O O Chelerythine O NH2 O O CN O O Br HA14-1 OH OH O OH H OH HO HO O Gossypol O OH OH OH O OH HO OH Theaflavanin OH O OH HO HO PurpurogallinH H Fig. 13 : Known inhibitors of Bcl-2 family ABT-73766 (Fig. 14) is a Bcl-xL inhibitor currently in clini cal trials. It exhibits an IC50 of 35 nM. However, the lack of oral bioavailabilit y of ABT-737 represents an obstacle for chronic agent therapy and limits the f lexibility to dose in combination regimens.67 Fig. 15 shows the docking x-ray crystal structure of ABT-737 with the protein, Bcl-xL (PDB ID: 2yxj).68 Cl N N O NH S O O NH N S NO2 ABT-737 Cl N N O NH S O O NH N S S ABT-263 O O F3C O Fig. 14 : Structures of ABT-737 and ABT-263 ABT-26369 (Fig. 14) is another drug launched by Abbott labor aties, which is in clinical trials and is a potent, and orally bioavai lable Bad-like BH3 mimetic ( Ki's of <1 nmol/L for Bcl-2, Bcl-xL, and Bcl-w). The oral bioa vailability of ABT-263 in preclinical animal models is 20% to 50%, depending on formulati on. ABT-263 disrupts Bcl-2/BclxL interactions with pro-death proteins leading to the initiation of apoptosis within two hours post-treatment. ABT-263 exhibits modest or no single agent activity and ye t it significantly enhances the efficacy of clinically r elevant therapeutic regimens.70

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17 Fig. 15 : Crystal structure of Bcl-xL in complex with ABT-737 (PDB ID: 2yxj) 2.6 Conclusion Bcl-xL, being an anti-apoptotic protein, plays a v ery important role in cancer. Since the over-expression of Bcl-xL in cancer cells render resistance to chemotherapeutic agents or radiation therapy, there is an urgent need for molecules that specifically target the anti-apoptotic activity of Bcl-xL. Small molecule BH3 surrogates can serve as the starting point to develop new mole cules that could validate the use of Bcl-xL in cancer therapy. Compounds developed by Ab bott laboratories currently in clinical trials could provide an in-depth knowledge of the biological function of Bcl-xL and help in developing new molecules as inhibitors for targeted therapy.

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18 Chapter 3 3.0 Shp2 Phosphatase as a Target for Cancer Therapy 3.1 Introduction Signal transduction refers to the process by which a cell converts one kind of signal to another involving a series of biochemical transformations that are caused by either enzymes19 or protein-protein interactions. In the signaling p athways, the secondary messengers elicit responses by activating protein k inases. Protein phosphatases are the terminators of the signal transduction process. Kinases and phosphatases regulate all aspects of ce llular function. Kinases are enzymes that phosphorylate molecules within the cel l using ATP.71 Phosphatases are enzymes, which dephosphorylate through hydrolysis ( Fig. 16). If the phosphorylated proteins are not dephosphorylated by phosphatases, then the protein would be in a constant state of being activated or inhibited. Protein-OH+ OH N H HO OH H H O N NN NH2 O P O O OP O O OP O OOATP Protein P OO OO + OH N H HO OH H H O N NN NH2 O P O O OP O OOADP PiH2O ProteinKinase ProteinPhosphatase Fig. 16: Protein kinases and protein phosphatases About 518 protein kinases are known in humans.71 Tyrosine phosphorylation is catalyzed by protein tyrosine kinases, which are re presented by 90 genes in the human genome.72 Some of these proteins have a common 100 amino aci d domain, called SH,

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19 (Src homology) and are phosphorylated by the recept or tyrosine kinase. The intracellular receptor kinase, Src, is activated when it binds th rough it’s SH domain(s) to the autophosphorylated receptor Tyr kinase. There are two main kinds of protein phosphatases: p hospho-tyrosine phosphatases and phospho-serine/threonine phosphatases. Most of the active phosphatases consist of a phosphatase catalytic subunit and a regulatory subu nit. Regulatory subunits for tyrosine phosphatases may contain SH2 domain allowing bindin g of the binary complex to autophosphorylated membrane receptor tyrosine kinas es. 3.2 Protein Tyrosine Phosphatases (PTPases) The 107 PTPs can further be divided into Class-I c ysteine-based PTPs, which are 99 in number and contain the 38-well known tyrosine -specific PTPs.73 These cysteinebased PTPs can be further subdivided into trans-mem brane receptor-like enzymes (RPTPs) and intracellular non-receptor PTPs (NRPTPs ). Class-II cysteine-based PTPs family is represented by a single gene, ACP1 which encodes the low Mr phosphatase (LMPTP). Class-III cysteine-based PTPs comprise the three cell cycle regulators, CDC25A, CDC25B, CDC25C, in humans. The CDCs dephosp horylate the Cdks at their dually phosphorylated N-terminal Thr-Tyr motifs, wh ich is critical for the activation of these kinases to drive cell cycle.74 The fourth class of PTP is the Asp-based PTPs, whi ch has only 4 EYA genes that have recently shown Tyr/S er phosphatase activity.75, 76 Protein tyrosine phosphatases dephosphorylate many cell surface receptors such as EGF and PDGF that have been phosphorylated on ty rosine residues.77 They are increasingly viewed as integral components of signa l transduction cascades and have been implicated in regulation of many cellular proc esses, including cell growth, cellular differentiation, mitotic cycles and oncogenic trans formation. Phospho tyrosine kinases (PTKs) and PTPs work together to regulate protein f unction in response to a variety of signals, including hormones, mitogens and oncogenes PTPs utilize a common mechanism for catalysis going through a covalent th iophosphate intermediate (Fig. 17) that involves the nucleophilic Cys residue in the P TPase signature motif (HCXXGXXRS/T).78

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20 PTP-Cys-SH -H+PTP-Cys-SNH O P O OOP-Tyr-Protein O H O Asp PTP-Cys-S P O O-O OH H : (Slow) PTP-Cys-SH + P O -O OH OH O O Fig. 17: Mechanism of dephosphorylation by PTP Most of the PTP family members contain at least one domain outside the PTP catalaytic domain. Although molecular and cellular biology studies ha ve suggested that PTPs are potential therapeutic targets for diabetes, cancer, and immunologic diseases, PTP inhibitor design is a new area in the field of drug development. For the majority of PTPs, chemical genetic studies are not yet possible becau se of the lack of specific inhibitors. 3.3 SHPs The SHP family consists of Shp1, Shp2 and Csw (from Drosophila) that form the subfamily of the intracellular PTPs.79 They contain two SH2 domains, a PTP catalytic domain and an inhibitory C terminus (Fig. 18). Thes e enzymes remain inactive within resting cells which is attributed to the insertion of the D’-E loop of the N-terminal SH2 somain into the susbtrate binding pocket. They tran slocate to the plasma membrane from the cytoplasm after the cell stimulation and become activated by binding to the tyrosinephosphorylated receptors through their SH2 domains.79 Human Human Drosophila(Csw) Xenopus C.elegens(PTP-2) Shp1 Shp2 PTPPTP PTP PTP PTP PTP Insert N-SH2C-SH2N-SH2C-SH2N-SH2C-SH2 N-SH2C-SH2 N-SH2C-SH2 536 Y 564 Y bipartileNLS Proline-richregion 542 Y 580 Y 542 Y 582 Y 666 Y Fig. 18 : (Reproduced from lit80) SHP proteins domain structures

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21 3.3.1 Shp1 PTPase Shp1 (Fig. 19) is a cytostolic PTP, 65 KDa protein, and is highly expressed in hematopoietic cells that is found to cause profound abnormalities in the immune system. It functions as a negative regulator in eukaryotic cellular signaling pathways81 and terminates the signal transduction pathway by depho sphorylation of appropriate substrates. The catalytic site of Shp1 has a core region and t he extended N and C termini. Shp1 shows 38% sequence identity with PTP1B and 60% with Shp2. Shp1 and Shp2 share extremely high similarity in both the seconda ry and tertiary structures, the major difference in the catalytic site being that the Nand C-termini in Shp1 are extended away from the molecule. Shp1 and Shp2 have different sub strate specificities explained by the electrostatic potentials being more positive in Shp 2 than in Shp1.81 Fig. 19 : Crystal structure of Shp1 SH2 domain complexed wi th a tyrosine-phosphorylated (PDB ID: 2yu7)81 3.3.2 Shp2 PTPase Shp2 is a ubiquitously expressed, non-receptor Src homology protein containing tyrosine phosphatase (PTPase), also known as PTP1D, PTP2C, SHPTP2 or Syp. It is ubiquitously expressed in mammalian tissues82 and is essential for embryonic development. It is a signal-enhancing component of growth factor, cytokine, and extracellular matrix receptor signaling, and plays an im portant role in regulating cell proliferation, differentiation and migration.

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22 Fig. 20: Crystal structure of the human tyrosine phosphatase Shp2 (PTPN11) showing N-SH2 (Yellow), C-SH2 (Green) and PTP catalytic domains ( Blue) (PDB ID: 3b7o)83 Shp2 is a 68-kDa protein82 (Fig. 20) and is composed of a single phosphotyros ine phosphatase (PTP) domain and two Src homology 2 (SH 2) domains at its NH2 terminus (N-SH2 and C-SH2, respectively, where N-SH2 is clos est to the N-terminus). The Cterminal region of Shp2 contains sites of tyrosine phosphorylation and a proline-rich region (Fig. 20).84 In the basal state, the PTP domain is inhibited by intramolecular interaction with N-SH2. Phosphotyrosyl peptide bind ing to the N-SH2 domain induces a conformational change that reverses this inhibition and activates Shp2 (Fig. 21).85, 86 PTP PTP BasalActive N-SH2C-SH2 pY pY biphosphotyrosyl peptide C-SH2 N-SH2 Fig. 21: (Reproduced frim lit87) Schematic representation of basal and active states of Shp2 Mutations in PTPN11, the human Shp2 gene, have know n to cause Leopard syndrome, Noonan syndrome,88 juvenile myelomonocytic leukemia89 and other malignancies. Most PTPN11 mutations affect N-SH2 or PTP domain residue s involved in the basal inhibition of Shp2.90 Gain-of-function mutations in Shp291 may lead to an increased activation of the Ras/MAPK pathway and increased cell proliferati on. 3.4 Shp2 targets There has been a great deal of efforts to identify Shp2 substrates. It has been found that Shp2 dephosphorylates Gab1 and partially dephosphorylates EGFR indicating

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23 that Gab1 and EGFR can be the substrates for Shp2. RasGAP has been found to be a downstream target of Shp288 since Shp2 dephosphorylates RasGAP to facilitate Ra s activation.88 Shp2 is an important downstream signaling molecule of Met/Gab1 for the activation of Mitogen activated protein kinase (MAP K) pathway, which may lead to cell transformation, a pre-requisite for cancer. Shp2 ac tivates a signaling step downstream of Gab1 and upstream of Src and Ras in the MAPK pathwa y. Dephosphorylation of a RasGAP binding site on Gab1 by shp2 negatively regu lates the association of Gab1 with RasGAP.91 Shp2 gets phosphorylated at its C-terminal end aft er stimulation, which then binds to Grb2 with the C-terminal end and to other receptors as EGFR, PDGFR and IRS1 via its sh2 domains. Grb2 functioning as an adapt er molecule facilitates MSOS binding to Shp2. This complex activates Ras/MAPK pathway an d thus Shp2 acts as a positive regulator of cell proliferation.92 These suggest that Shp2 is a great target for cance r therapy since, inhibition of Gab1 dephosphorylation by Shp293 may ultimately stop the MAPK/ERK signaling cascade, thereby preventing the alteration of gene expression. Being a positive mediator of GF signaling, especially in the Ras pathway, Shp 2 inhibition by small molecules would be of great therapeutic interest in drug disc overy. Therefore, the search for small molecules, which can restore the basal state of Shp 2, by interacting with the PTP catalytic domain, represents an exciting and novel area for a nti-cancer drug development. 3.5 Ras/MAPK pathway Shp2 plays a critical role in cell development by promoting the Ras/MAPK activation in response to various agonists. Ras/MAP K is a major signaling cascade modulating the cell cycle. Shp2 functions as a posi tive regulator/signal-enhancer in this pathway. Although studies indicate that Shp2 plays a signal-enhancing action in this pathway, the mechanism by which it functions is not quite clear.94

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24 Shp2 Y P542580 Grb2 SOS Ras GTP Raf1 Erk1/2 SHPS-1 Src MVP Shp2 Shp2 RTK Fig. 22 : (Reproduced from lit95) Role of Shp2 in Ras-Raf Pathway Shp2 participates in Ras/MAPK activation under EGF stimulation. Gab1 is one of the substrates of Shp2. Shp2 down-regulates the rec ruitment of RasGAP by dephosphorylating RasGAP binding sites on Gab1 (Fig 22). Thus this Ras inhibitor is excluded out of the signaling complex, promoting su stained Ras/MAPK activation in response to EGF. 3.6 Epidermal growth factor receptor signaling path way Epidermal growth factors (EGFs) are critical for ce ll cycle, cell differentiation and growth. Epidermal growth factor receptors (EGFRs) a re present on the cell surface, and their mutations could lead to uncontrolled cell div ision and thereby other malignancies. In normal cells, EGFR exists in equilibrium between its inactive monomeric and its active homodimeric form. Dimerization stimulates it s intrinsic intracellular proteintyrosine kinase activity.96 The protein tyrosine phosphatase, Shp2, plays a vi tal role in the EGFR transduction pathway leading to src activation ; Gab1 phosphorylation; Grb2 binding to Sos1 (and thereby stimulation of the MAP K/ERK cascade); PI3K cascade; and the JNK cascade (Fig. 23).91 This implies that Gab1 is a substrate for Shp297 since Shp2 dephosphorylation of Gab1 leads to the PI3K cascade

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25 EGFRSHP-1 SHP-2 Sos1 Shc Grb2 ERK1/2 RSK2 CellCycle MAPK Grb2 Sos1 Gab1 Ras Raf ERK1/2 RSK2 Cellcycle MAPK P13K Grb2 P P P P P RasGAP Ras Raf MAPK Stat-3 Gab1 SHP-2 Transcription Stat-1 EGFRStimulation Homodimerization& AutophosphorylationY1173 Y1148 Y1086 Y1068 Y992 Y1068/Y1086 CellMembrane Fig. 23: (Reproduced from biocarta pathways) Schematic representation of the EGFR pathway 3.7 Known inhibitors of Shp2 There are several compounds known to inhibit Shp2 without selectivity. They include CDL 4340-0580 (from ChemDiv Inc.)91 and NAT6-297775 (Fig. 24), discovered from a screen of a natural product-like library.98 O N H N S N S O O O O S O O CDL 4340-0580 IC 50 (Shp2) : 2.2 m m m m M O O H N H H N N N N N NAT6-297775 IC 50 (Shp2) : 2.5 m m m m M Fig. 24: Known Shp2 inhibitors Recently, Birchmeier reported a new class of Shp2 selective inhibitor, PHPS1 (5) (Fig. 25) which exhibited a Ki of 0.73 0.34 m M. This compound was identified via in silico screening of ~2.7 million compounds, out of which 843 compounds showed potency and 235 were specific to Shp2. Out of these compounds, 5 exhibited good predicted ADME properties and the compound synthesized showed an IC50 less than 1 m M. Compound 5 is a potential phosphotyrosine mimetic because of the presence of a sulfonic acid group and is reported to penetrate into the substrate-binding pocket of Shp2.

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26 6,IC50:0.80.22 m M N H N O HO2C NH HO2C N N N O H N N+ O -O PHPS1(5),Ki:0.730.34 m m M O N+ NH N N H -O O 7,IC50:46.810.2 m m M S HO O O S OH O O N H Cl S O O N H N O N H O OH 8,IC50:1.40.6 m M N N S N S OO HO O O HO NSC-87877,IC50:0.318mM Fig. 25 : PHPS1 and Isatin derivatives and their activity against Shp2 According to the high-throughput docking studies,99 the pyrazolone core of 5 and its substituents make contacts to residues at the periphery of this cleft. The amino acid residues Lys-280, Asn 281, Arg-362, and His-426 of Shp2 are the only residues of the catalytic cleft that are not conserved between Shp2 and PTP1B. These residues (Fig. 26) may be of particular importance for inhibitor specificity, because they are all involved in binding to 5 .99 N N N O NH N + O O S HO O O ASN281 Gly464A r g 4 6 5 A r g 3 6 2 Ser460 LYS280 Asn281 His426 Arg362 A B Ile463 Ala461 Fig. 26: (Reproduced from lit 100 ) A Active site of Shp2 showing the catalytic cleft, B Ligand interaction of PHPS1 in Shp2 active site. Our lab has been actively involved in the investigation of oxindole-based small m olecules as potential selective inhibitors of Shp2. The isatin derivative 7 (Fig. 25)

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27 emerged as a hit of modest potency. This was based on compound NSC-87877101 obtained from the HTS screening of the NCI Diversit y set at the Moffitt Cancer Center. Extensive SAR studies around the oxindole scaffold led us to the discovery of novel, potent and selective inhibitors 6 and 8 The carboxylic acid group in the isatin compounds was found to be critical for the phosphat ase activity, as a mimic of the phospho-tyrosine. It is reported that the hydrazone aromatic system is pointing into the active site PTP signature motif (VHCSAGIGRTG).102 The nitro group in compound 7 mimics the phosphate group of the tyrosine phosphat e and the sulfonic acid group is hydrogen-bonded with the basic residues Arg362 and Lys366 (Fig. 27). Compounds with an oxindole core have been studied by other groups as potential therapeutic agents.103, 104 Fig. 27 : (Adapted from lit105) Structure of Isatin ( 6 ) and ( 7 ) docked to Shp2 3.8 Conclusion Shp2 has been identified as a positive regulator of growth factor signaling. Gainof-function mutations in many types of human cancer s indicate that Shp2 is an oncogene. Over-expression of Shp2 in many types of cancers, i ncluding childhood leukemias makes it a potential anti-cancer target, suitable for dru g development. Recently, Shp2 is in the highlight of medicinal chemistry research amongst i ts related PTPs because of its involvement in many signaling pathways. Although, t he biological function of Shp2 is known, its role in the signaling pathways remains u nclear. Inhibiting Shp2 activation using small molecules has gained enormous interest lately, in the area of drug discovery. To validate Shp2 as a therapeutic target and to get a clear understanding of its underlying mechanism in the signaling pathways, there is need for inhibitors that are specific to Shp2.

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28 Chapter 4 4.0 Microwaves and Flow reactors in organic synthes is 4.1 Introduction: Microwave-assisted synthesis High-speed microwave-assisted synthesis has gained substantial interest lately, especially in the area of drug discovery where it i s important to generate collections of compounds rapidly and efficiently. Commercial use o f microwave ovens began in 1947. High water content and consequent efficient convers ion of microwave energy into thermal energy by water molecules at microwave freq uencies was well recognized in the earlier days.106 Microwave chemistry involves the use of microwave radiation to conduct chemical reactions. Microwaves are electromagnetic waves consisting of an oscillating magnetic field with a perpendicular oscillating ele ctric field and lie between the infrared and the radio waves in the electromagnetic spectrum They have wavelengths between 0.01 and 1m and frequencies ranging from 30GHz to 3 00 MHz respectively. The microwave frequency most commonly used is 2.45 GHz, which is ideal for the interaction with water molecules and aqueous soluti ons. Microwaves heat up the sample by utilizing the abil ity of the solid or liquid to convert the electromagnetic radiation into heat. Th e charged particles or dipoles present in the sample tend to align with the incident radia tion, when it interacts with an electric field (Fig. 28). The alternating nature of the radi ation realigns the polarized particles in the opposite direction as the field alternates. The time taken for the dipoles to realign with the microwave field is the same as that of the alte rnating oscillation in the microwave frequency. Thus the particles are in constant motio n, which results in heating.29

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29 E=0 E = = 0 \ Fig. 28 : dipole moments align parallel in an electric field Ideal solvents for use in the microwave reactors depend on the reaction itself and the polarity of the solvent. Usually reactions in the microwaves are conducted using the same solvent as the conventional method. Since microwave heating occurs through dipolar polarization or conduction mechanism, solvents that are dipolar or ionic can only absorb microwave energy. Polar solvents like water, N, N-dimethyl formamide (DMF), N-mehyl pyrrolidine (NMP), Dimethyl sulfoxide (DMSO), acetone, dichloromethane, dichloroethane, methanol, ethanol and acetic acid work well in microwaves because of their high dielectric constants. Usually non-polar solvents like toluene, benzene, diethyl ether, dioxane and THF do not absorb microwave energy well and are considered poor solvents for use in microwaves. Ionic liquids are suitable for use in microwaves due to their dielectric properties. In the microwave reactor, in the presence of an electric field, the dipole moments align parallel to the applied field, they realign if the electrical field is oscillating. The molecules become agitated and the molecular collisions give rise to dipolar heating, approximately 10 degrees per second.107 In conventional heating, an external heat source is employed, which is relatively slow and inefficient to transfer energy into the system, as it is dependent on the thermal conductivity of the materials that must be penetrated. In this case, the temperature of the reaction vessel is much hotter than the reaction mixture itself. In microwave irradiation, direct coupling of microwave energy with the molecules present in the reaction mixture produces an efficient internal heating.108 In the conventional heating, thermal lag, the time required to bring the reactants to the reaction temperature from the room temperature, is quite long, which is a major drawback. In the microwave heating, the desired temperature can be attained in less than three minutes. The ramp time which is the time required to reach the desired temperature can be changed accordingly.

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30 Microwave synthesis can be conducted with a modifie d commercial microwave oven. However, there are microwave reactors specifi cally designed to perform reactions, which has controls for pressure and temperature and the microwave energy source is well shielded. The microwave reactor used in this work i s a bench-top model from Biotage Inc. (Fig. 30). It has an infrared sensor, which me asures the temperature. It is an automated system and has sample containers that can accommodate 4-8 reactions at a time. The vessels used for the reactions are made o f contaminant free microwave-safe glass. They are designed to hold volumes from 0.2 m L to 20 mL and withstand pressures of up to 300 psi (20 bars). The caps are made of Te flon lined septum, which can withstand high pressure and temperature. The septum allows repeated reactions and insitu sampling, since it can be resealed after it has be en penetrated. Magnetic stirring allows even temperature distribution in the reactio n mixture. Advantages of microwave synthesis include higher r eaction temperatures, significantly reduced reaction times, use of low-bo iling solvents under pressure, uniform heating of the reaction components, ease of tempera ture and pressure control, energy efficiency and parallel synthesis.107 Microwave assisted synthesis is ideal for synthesi zing compounds in pharmaceutical drug development. Micro wave-assisted heating enhances the reaction rates compared to the conventional hea ting because of the high temperatures used. We also observed that there were fewer impuri ties when we used microwaveassisted heating. For example, the Biginelli reaction (Fig. 29) invol ves a one-pot condensation of an aldehyde, a ketone and either a substituted thio urea, urea or guanidine in ethanol using a strong acid catalyst like acetic acid and a Lewis acid at reflux temperature of ethanol for 2-12 hrs; and the yield obtained varies from 20-60% ; the same reaction performed in the microwave reactor takes 10-20 minutes at 120 C, with yields of 30-90%109. O H R2 R1 O E HN Z NH2 R3 Ethanol,Aceticacid Lewisacidcatalyst, m W,120oC,10-20min N NH Z R2 R3 R1 E E=ester,amide,nitro;Z=O,S,NR;R1-R3=H,alkyl,aryl Fig. 29 : Biginelli reaction

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31 Reactions performed in the microwave reactors are u sually cleaner and more environment friendly than the conventional heating methods; usage of solvent can be minimized or even eliminated since microwaves heat the compounds directly. Higher temperatures using low boiling solvents could be ac hieved in the microwave by using the autoclave technology. Higher yields and cleaner rea ctions allow rapid reaction optimization and library synthesis. Combinatorial s ynthesis plays a very critical role in the generation of libraries in drug discovery. Comb ining combinatorial synthesis and microwave-assisted heating to generate compounds ha ve recently evolved much interest due to its enormous benefits. However, there are limitations to microwave chemis try; the reaction scale is usually limited to grams; Solvents that absorb micr owave energy can only be used. In spite of the adverse effects of the microwave, it s till remains an efficient source of heating, which results in saving energy and time.108, 110 One of the major drawbacks of the microwave technology is the scalability of reac tions in the process industry. But despite the limitations, microwave chemistry plays a major role in organic synthesis. Many reactions, which were not possible previously using conventional methods of heating have been effectively performed in the micr owave reactors quickly and efficiently in a few minutes.107 Fig. 30 : Biotage microwave reactor and H-cube hydrogenator 4.2 Flow reactors: Hydrogenator The competitive nature of pharmaceutical research requires lead optimization and library synthesis to be performed easier and faster A recent development in the field of drug discovery is the use of continuous flow reacto rs.111 The use of flow reactors in organic synthesis is highly attractive since it can handle a wide range of scale that can be

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32 performed and the ease of use. Reductions of nitro compounds to the corresponding amino compounds were performed on a H-cube flow rea ctor (Fig. 32). It is a bench-top hydrogenator, in which the hydrogen is generated by electrolysis of water. A HPLC pump flows a continuous stream of solvent into reac tor. Hydrogen, generated in-situ, is mixed with the sample, heated and passed through a catalyst cartridge (30-70mm), where the reaction takes place and the hydrogenated produ ct is collected into the product vial. Reductions varying from 10mg-1 g scale can be perfo rmed in the flow reactor. The maximum temperature and pressure that can be attain ed in the H-cube flow reactor are 100 C and 14.5 psi (100 bars) respectively. The flow of reactant can be controlled by the HPLC pump from 1-10 mL. This reactor is relatively safe compared to other hydrogenation devices since no cylinders or externa l hydrogen source is required in the H-cube. There is no risk of handling the metal cata lyst or filtering it. 4.3 Conclusion Flow reactors have drastically changed and improved the way in which organic reactions are performed. Many reactions that were p reviously impossible using the conventional method can now be performed using micr owave technique. Hydrogenations, generally considered dangerous can easily be performed in the benchtop hydrogenator, which not only has cut down the r isks involved with the handling of catalysts but also enables pressurized reactions to be performed at elevated temperatures.

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33 Chapter 5 5.0 Synthesis and evaluation of small molecules as Bcl-xL inhibitors 5.1 Introduction Bcl-xL is over-expressed in most human cancer types making it a very attractive target in the area of anti-cancer drug development.11 High expression of Bcl-2 or Bcl-xL in human cancer contributes to neoplastic cell expa nsion and interferes with the therapeutic effect of the chemotherapeutic agents u sed in cancer treatment, by blocking apoptosis. Inhibiting the function of Bcl-xL could restore apoptosis or sensitize the tumors for therapeutic agents. From a high throughput screening of the NCI Diversi ty Set at the Moffitt Cancer Center, the isoquinolinol NSC-131734 was identified as an inhibitor of p53-HDM2 binding and Bcl-xL-Bax interactions having an IC50 of less than 15 m M in the FP (fluorescent polarization) assay. Isoquinolinols are very important compounds in the field of drug discovery, especially as short-acting cardiovascular agents an d anti-hypertensive agents. The opium alkaloid, Papaverine is an isoquinolinol, which has a relaxant effect on the vascular and vis ceral smooth muscle.112 Based on the screening, two analogs of NSC131734 isoquinolinols HL2-100 and HL2-101 (Fig. 31) were synthesized and identified as a new starting point in our hit-to-lead process in the search of new inhibitor of Bcl-xL-Bax interactions. Molecu les bearing a fused anthracene or an N MeO MeO OMe OMe Papaverine N O OH NSC-131734 N O OH HL2-100 N O OH HL2-100_2 Fig. 31 Structures of HL2-100 and HL2-100-2

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34 anthracycline ring system are known to intercalate the DNA.113 Since NSC-131734 has a fused anthracene ring system, it was suspected that it would act as a DNA intercalating drug. DNA intercalators are drugs that bind to DNA tightly and reversibly by a combination of hydrophobic, electrostatic, hydrogen bonding and dipolar forces causing programmed cell-death. DNA intercalation usually gi ves rise to high toxicity, which is undesirable in drug discovery and medicine. We have therefore designed and prepared focused libraries of 4-methylarylisoquinolines114 (Table 1) related to these structures, which we hoped would retain the Bcl-xL inhibition b ut without acting as DNA intercalators. Initially, we focused on synthesizing a library by varying the substituents on the aromatic ring (Ring A) attached to the isoquinoline ring. A variety of benzaldehydes were chosen to generate th e first library for the initial screening against Bcl-xL. W e then focused on the isoquinoline core (Ring B) and synth esized a few compounds by varying the groups in ring B. All these compounds were tested for Bcl-xL. The isoquinolines, 12a-z (Table 1) were synthesized using the Pomeranz-Frit sch reaction115 (Fig. 32). Amongst the other methods to prepare is oquinolines such as the Pictet-Spengler116 and Bishler-Napieralski cyclization,117 the Pomeranz-Fritsch reaction is widely used to synthesize completely unsaturated isoquinolines.115 The PomeranzFritsch reaction involves the synthesis of isoquino lines via the acid-mediated cyclization of the appropriate aminoacetal intermediate iii (Fi g. 33). Benzaldehydes bearing electrondonating substituents in the 3or 4positions are favorable substrates for the PomeranzFritsch reaction.118 Fig. 32 : Pomeranz-Fritsch reaction N RingA RingB

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35 The conventional mechanism of this reaction involv es the formation of an imine 11 from the amine 10 and the aldehyde 9 which on cyclization and subsequent elimination affords the isoquinoline 14 (Fig. 33)115. Fig. 33 : Mechanism of Pomeranz-Fritsch reaction Modified Pomeranz-Fritsch reactions were performed, employing milder reaction conditions (Fig. 34). The Bobbitt modification form s the 4-hydroxy isoquinolines by employing mild acidic conditions for cyclization. T he Jackson modification involves the cyclization of the N-tosylated aminoacetal intermed iate.119 The Schlittler-Mueller modification120 involves the formation of imine from a benzyl amin e and 2,2diethoxyacetaldehyde, which on cyclization yields t he corresponding isoquinoline.121 Fig. 34 : Modifications to Pomeranz-Fritsch reaction The Pomeranz-Fritsch reaction has also been carrie d out in the presence of Lewis-

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36 acid catalysts like BF3-trifluoroacetic anhydride or polyphosphoric acid.122, 123 In such cases, only the activated imines, bearing electron donating groups in the 3or 4positions, cyclize to give the isoquinolines, where as the inactivated benzaldehydes fail to cyclize. We synthesized the isoquinolinols 20a-b following the literature reported method described by Bobbitt and Dyke.114 The aniline 9a was treated with aminoacetal diethyl amine at 80 C to afford the imines 10a Reduction of 10a with sodium borohydride afforded the amines 19a Compound 19a was then treated with the corresponding benzaldehydes at reflux temperatures to afford the isoquinolinols, 20a-b To facilitate parallel synthesis of compounds for our initial lib rary and reduce the reaction time, we carried out reactions under microwave conditions. H igh-speed microwave-assisted synthesis has gained substantial interest lately, e specially in the area of drug discovery where it is important to generate collection of com pounds rapidly and efficiently. Compounds 20c-z were synthesized using the microwave-assisted Pome ranzFritsch reaction (Fig. 35). Compounds 10c-e were synthesized by treatment of the appropriate benzaldehydes with aminoacetaldehyde di ethylacetal in EtOH at 130 C for 15 min in the Biotage microwave reactor afforded th e imines 10c-e Sodium borohydride was directly added to the reaction mixture to affor d amines 19c-e The amines 19c-e were then reacted with a wide range of commercially avai lable aldehydes in the presence of concentrated hydrochloric acid to provide the desir ed 4-aryl isoquinolinols 20c-z (Table 1) Fig. 35: Synthesis of substituted isoquinolinols, 20a-z

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37 Reagents and conditions: a. EtOH, reflux, 30 min; b. EtOH, m w, 130 C, 15 min; c. NaBH4, MeOH, 30 min, rt; d. ArCHO, HCl, H2O, reflux, 40 min or e. ArCHO, HCl, H2O, m w, 140 C, 15 min Table 1: Synthesis of substituted isoquinolinols, 20a-z from 19a-e Entry R1 R2 R3 Ar Yield (%) (from 19) 20a OH OMe H 2-Cl 70a 20b OH OMe H 4-Br 69a 20c OH OMe H 2,4-Cl2 82b 20d OH OMe H 3,5-(CH3)2 71b 20e OH OMe H 4-Biphenyl 90b 20f OH OMe H 3-Pyridyl 99b 20g OH OMe H 4-Et 61b 20h OH OMe H 3-Cl,4-F 99b 20i OH OMe H 4-CH(CH3)2 23b 20j OH OMe H 2,4-(CH3)2 50b 20k OH OMe H 4-(4-Pyridin-2-yl)benzyl 99b 20l OH OMe H 2-CF3 55b 20m OH OMe H 3,5-Br2 99b 20n OH OMe H 2,3-Cl2 80b 20o OH OMe H 4-Pyridyl 65b 20p OH OMe H 2,6-Cl2 64b 20q OH OMe H 3-F 64b 20r OH OMe H 4-F 95b 20s H OMe OH 4-Br 89b 20t H OMe OH 3-Cl,4-F 82b 20u H OMe OH 4-F quantitativeb 20v H OMe OH 3-F 64b 20w H OMe OH 2,3-Cl2 99b 20x H OMe OH 2,4-Cl2 quantitativeb 20y H OMe OMe 4-NH2 73b 20z H OMe OMe 3-Cl,4-F 94b a reflux, 40 min; b m w, 130 C, 15 min. The reaction is believed to take place by the mecha nism shown (Fig. 36). The formation of intermediate 23 is similar to that shown in Fig. 33. Intermediate 23 being an enamine is highly reactive and hence it reacts furt her with the aryl aldehyde to form the hemi-acetal 21 which on elimination of water, rearranges to give the isoquinolinol 20

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38 Fig. 36: Mechanism of synthesis of isoquinolinols All the compounds synthesized were analyzed by 1H NMR, 13C NMR, IR and HRMS. The structures of these compounds were establ ished from their unique NMR spectra. Compound 12a showed a singlet at d 3.7 ppm in the 1H NMR spectrum, indicative of the methoxy group. The singlet for two protons at d 4.4 ppm indicates the benzylic protons. In the 13C NMR spectrum, the signals for the methoxy and methy lene carbons appeared at 37 ppm and 57 ppm respectively. A series of peaks between 6.84 and 7.44 ppm in the 1H NMR spectrum indicates the protons on the benzyl group containing the chlorine and the protons on the ring containing the methoxy group (Ha and Hb). Two sharp singlets at d 8.22 and d 9.56 ppm indicate the protons on the isoquinoline ring containing the nitrogen, Hc and Hd respectively. Mass spectrometry of the compound (MS (m/z) 300.1 (M+H)+) further confirmed the structure of this compound. The first library of compounds (Table 1) was tested in the fluorescence N O OH Cl Ha Hb H d H c

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39 polarization (FP) assay.124 FP is a technique amply used to analyze protein-pr otein interactions in solution. The measurements are base d on the assessment of the rotational motions of species. The theory of FP was establishe d by Perrin, in 1926.125 Fluorescent molecules when excited with plane-polarized light r otate and tumble during the fluorophore's excitation. When a protein binds to a fluorophore-labeled peptide, it leads to a decrease in peptide rotation and an increase i n polarization upon excitation with polarized light (Fig. 37). The fluorescence of the small molecule bound to the Bcl-xL protein is measured, which is proportional to the p olarization value.124, 126 Interaction Peptide Fluorophore Fasttumbling Depolarizedemission Slowtumbling Polarizedemission ProteinProtein Fig. 37 : FP assay 5.2 Biological evaluation of compounds, 12a-z Initial screening of the NCI diversity set compound s in the FP assay led to the discovery of the hit, NSC-131734 Although initial screening of the commercially available compounds did show substantial activity f or Bcl-xL using the in-house FP assay, later screening of the NCI diversity set hit s and the in-house synthesized compounds in the same assay did not exhibit any inh ibitory activity. This, in part, might be due to some impurities present in the commercial ly purchased compounds, which were undetectable using NMR or high-resolution MS. The impurities could have been responsible for the detection of the compounds as f alse positives. Compounds 12a-z exhibited IC50 greater than 300 m M for Bcl-xL in the FP assay. 5.3 Conclusion In the search for novel small molecules as disrupt ors of Bcl-xL activity, an efficient microwave-assisted synthesis of isoquinol inols has been developed. Isoquinolinols, being one of the most important pha rmacophores in drug discovery, the applicability of this reaction to a broad range of substrates and the ease of synthesis makes it valuable in combinatorial chemistry and dr ug discovery.

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40 Chapter 6 6.0 Synthesis and evaluation of small molecules as Shp2 inhibitors 6.1 Hit-to-Lead approach based on HTS screening From a high throughput screen of the NCI Diversity Set and the Moffitt ChemDiv 20000 compound collection, a diverse set of small molecules (Fig. 37) were identified as hits, capable of inhibiting Shp2 PTP. The original hits were chosen based on their initial Shp1/Shp2 selectivity profile and whether they were amenable to analog synthesis. The initial step in our hit-to-lead approach is re-synthesis of the original hits to confirm the biological activity. The second step involves the design and synthesis of new analogs to improve the potency of the initial hits. The lead optimization is also directed towards the design and synthesis of new inhibitors showing potency and selectivity for Shp2 rather than the related phosphatase Shp1. S H N O HO O NH N H O NH O N S O OH OH N N N H OH O O H H N H OH O OH H H HLM000661, ( 34 ) IC50(Shp2):0.67 m M IC50(Shp1):7.06 m M HLM019544, ( 41 ) IC50(Shp2):3.59 m m M IC50(Shp1):52.35 m M HLM002903, ( 44 ) IC50(Shp2):6.41 m M IC50(Shp1):>133 m M HLM001038, ( 46a ) IC50(Shp2):12.2 m m M IC50(Shp1):14.97 m M HLM001426, ( 46b ) IC50(Shp2):2.45 m M IC50(Shp1):19.7 m M S O O Fig. 38 : Shp2 hits derived from a high throughput screen at the Moffitt Cancer Center

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41 6.1.1 Synthesis of HLM000661 From the screening of the Moffitt ChemDiv library b y HTS for Shp2 inhibitory action, HLM000661 ( 34) was identified as a hit of remarkable potency with an IC50 of 0.67 m M. Hence, re-synthesis of the compound HLM000661 ( 34 ) was undertaken using the synthetic route as depicted scheme 1.127, 128 The oxime 28 was synthesized by reacting 6,7-dihydrobenzo[ b ]thiophen-4( 5H )-one 27 with hydroxylamine hydrochloride under microwave assisted heating at 120 C for 15 min. The tosylated oxime 29 was obtained by reaction of 28 with p -toluenesulfonyl chloride in presence of pyridine. Treatment of 29 with KOH and ethanol under the Beckman conditions g ave the 7-membered lactams 30 and 30a .128 This reaction provided both regio-isomers 30 and 30a in equal yield. The two isomers were separated by column chromatography and analyzed using 1H NMR and 13C NMR. The protons on the thiophene ring displayed signals at 6.51 ppm and 7.06 ppm in the 1H NMR for the desired product compared to the peaks at 7.23 and 7.83 ppm for the regioisomer respectively. The melting points of the samples were taken which further confirmed the desired product. Compound 30a had a melting point of 85-87 C while the desired isomer had a melting point of 133-134 C, which was comparable to the reported data ( 30a : lit. m.p. 92-93 C; 30 : lit. m.p. 134-135 C).129 Acylation of 30 under the Friedel-Crafts conditions (AlCl3, 0 C) proved unsuccessful. The desired isomer 30 was acylated with methyl 4-chloro-4-oxobutyrate in pres ence of SnCl4 at 0 C to give 31 The reduction of the carbonyl group to the correspondin g alcohol was performed using NaBH4. Treatment of 32 with Et3SiH/BF3.Et2O yielded compound 33 Prior attempts to perform direct reduction of 31 under Wolf-Kishner (hydrazine hydrate KOH, Diethyle ne glycol, 150 C, 4hrs) or Clemmenson (potassium acetate, ethanolwater, reflux, 22hrs) conditions, failed to provide the target product 33 Saponification (1M NaOH) of the methyl ester 33 afforded the desired product 34 Compound 34 was analyzed by 1H NMR, 13C NMR and mass spectrometry. A singlet at 6.41 ppm (proton on the thiophene ring), seven –CH2 peaks at 1.59-2.89 ppm in the 1H NMR and the signals for 2 carbonyl groups and four aromatic carbon peaks in the 13C NMR confirmed the structure of 34 The mass spectrometry of compound 34 (MS m/z 268 (M+H)+) further confirmed the structure of 34 The analytical data were comparable to the literatu re reported data129 and the analytical data of the HLM000661 sample purchased from ChemDiv.

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42 Scheme 1 : Synthesis of HLM000661 ( 34 ) Reagents & Conditions: a. NH2OH.HCl, NaHCO3, m w, 120 C, 10 min; b. p -TsCl, Pyridine, 0 C, 1 hr; c. K2CO3, EtOH, H2O, reflux, 22 hrs; d. Methyl 5-chloro-5-oxo-pentano ate, SnCl4, DCM, 0 C rt, 30 min, Ar; e. NaBH4, MeOH, 30 min, rt; f. BF3. Et2O, Et3SiH, DCM, rt, 30 min; g. 1M NaOH, rt, 1 hr. 6.1.2 Synthesis of HLM019544 The quinolone 41 (initially purchased from ChemDiv) was found to i nhibit Shp2 activity with an IC50 of 3.59 m M. Synthesis of HLM019544 ( 41 ) was performed as shown (scheme 4).130-132 Amide coupling, followed by Pd-catalyzed hydrogena tion of the nitro group, performed in a Thales H-cube Flow Reac tor, provided the key intermediates 37a-h The nitro group in 36i bearing chlorine at the ortho-position of the sulfonamide terminus, was reduced to the correspond ing amine 37i by treatment with NiCl2 and NaBH4 (Table 2, note a). In fact, the cleavage of the C (aromatic)-Cl bond under Pd-catalyzed hydroge nation has been amply described and documented in the literature.133 The aminomethylenemalonates 38a-i were prepared by reacting compounds 37a-i with diethyl ethoxymethylenemalonate under solvent free heating (180 C, 1h) (Scheme 2). NH 41 S O O NH O O NH

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43 Scheme 2: Synthesis of quinolone, 41 Reagents and Conditions: a. p -Nitrosulfonyl chloride, DCE, Pyridine, m w, 150 C, 10 min; b. NiCl2 6H2O, NaBH4, 0 C, 30 min; c. H2, 10% Pd/C, Methanol, H-cube; d. Diethylethoxymethy lene malonate, 180 C, 1 hr; e. o -Dichlorobenzne, 250 C, m w, 15 min; f. 10% KOH, EtOH, reflux, 1.5 hrs; g. SOCl2, refux, 1 hr; h. CH3NH2, Pyridine, DCM, rt, 1 hr. Ring closure to form the quinolone was a key step in this synthesis. The synthesis of 39 is discussed later. Base-catalyzed hydrolysis of th e ester 39 afforded acid 40 Reaction of 40 with thionyl chloride afforded the acid chloride, w hich was treated with methylamine in presence of pyridine gave 41 Compound 41 was analyzed using 1H NMR, 13C NMR, MS and single crystal X-ray diffraction anal ysis (Fig. 39, for data, see Appendix). The presence of two peaks (for the two m ethyl groups) in the upfield region of 1H NMR, a multiplet (four protons of the aromatic r ing attached to the sulfonamide unit), a doublet with a coupling constant of 2 Hz ( meta coupling, Ha), a doublet of doublet showing meta and ortho coupling (Hd) and a doublet showing ortho coupling (Hc), a singlet at 8.78 ppm (proton in the quinolone ring, Hb) confirmed the structure of 41 The 1H NMR and MS obtained for compound 41 were comparable with the data obtained for the HLM019544 sample purchased from ChemDiv. NH S O O NH O O NH Hb Ha Hd H c

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44 Fig. 39 : X-ray crystal structure of 41 Quinolones are a family of broad-spectrum antibioti cs representing an area of great interest in medicinal chemistry. In fact, the y are widely used for the treatment of many infectious diseases as antimicrobial as well a s anti-bacterial agents. Discovered in the 1960s, Nalidixic acid was the first quinolone u sed as a clinical agent in the treatment of urinary tract infections caused by gram-negative organisms.134, 135 Due to its pharmacological relevance, medicinal chemistry effo rts have been directed towards the search and the synthesis of Nalidixic acid derivatives of increased potency and improved pharmacokinetic and pharmacodynamic properties. Ciprofloxacin belonging to the fluoroquinolones group was introduced as a syntheti c antibiotic in the 1980s effective against gram-positive and gram-negative bacteria. C iprofloxacin possesses a remarkably improved profile in terms of potency, spectrum and pharmacodynamic properties compared to the first generation quinolones and the parent compound, Nalidixic acid.136, 137 Quinolones have been found to show their antibacte rial properties through the stabilization of the bacterial type II topoisomeras es-DNA complex.134, 135 DNA topoisomerases are essential for DNA replication, transcription, chromosome segregation, and DNA recombination. Quinolone antibacterials display a r emarkable selectivity between the bacterial and mammalian II topoisomerase and, therefore, a good toxicity profi le.134, 135 Quinolones like 42 show both topoisomerase II mediated DNA cleavage a nd anti-tumor activity.138 Many of the quinolones show their cytotoxicity thr ough the stabilization of N N HO O O N OH O O F N HN Nalidixicacid Ciprofloxacin N OH O O F N HN 42 F

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45 the topoisomerase-DNA complex (also referred to as cleavage complex), forming ternary complexes.139 The mechanistic similarities of the antibacterial quinolones and anti-tumor agents have stimulated a new interest in the quinol one class of compounds in an effort to change their biological profile from antibacterial to cytotoxic agent.140 Due to their therapeutical potential, there is alwa ys the need for new and optimized synthetic routes to access novel quinolon es in a combinatorial fashion. In the synthesis of quinolones 41 we investigated the optimization of a microwave a ssisted Gould-Jacob cyclization for the synthesis of ethyl 6-sulfamoyl-4-oxoquinoline-3carboxylates 39 employing o -dichlorobenzene (ODCB) as solvent. The synthesis of a small library of ethyl 6-sulfamo yl-4-oxoquinoline-3carboxylates 39 as precursors for the synthesis of cystic fibrosis transmembrane conductance ‘potentiators’ h as been recently published by Kurth and coworkers.141 Kurth’s protocol involves the microwave irradiation of a diphenylether solution of diethyl 2-[(4-phenylsulfa moyl-phenylamino)-methylene]malonates containing catalytic p -chlorobenzoic acid (250 C, 2h, w). We initially attempted to build our library upon Kurth’s protoco ls (Scheme 3). Literature reported protocols for the synthesis of quinolones involve the condensation between anilines and diethyl ethoxymet hylenemalonate followed by thermal cyclization (Gould-Jacob reaction). The cyl clization is usually performed very high temperatures (180-250 C) under solvent free classical or microwave heatin g, or employing diphenyl ether or biphenyl or Dowtherm (a n eutectic mixture of biphenyl and diphenylether) as solvents. NH EtO2C CO2Et NH O O O 38 39 R R aorborc Scheme 3 : Synthesis of quinolones Reagents and Conditions: a. Diphenyl ether, 250 C, 2hrs; b. Diphenyl ether, pchlorobenzoic acid, 250 C, m w, 2 hrs; c. Dowtherm, 250 C, 2 hrs As a simple model, we initially studied the reacti on of 38a (Table 2). Our initial experiment was carried out in the Biotage microwave reactor under the following 39 NR1 S O O NH O O O R

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46 conditions: Ph2O, cat. p -chlorobenzoic acid, 250 C, 2 h (Table 2, entry 1) However, a maximum temperature of 150 C could be achieved usi ng diphenyl ether as solvent in the microwave reactor. A mixture of starting material a nd aniline 37a was recovered and no product formation was observed. A series of experim ents were performed in the CEM microwave reactor under different conditions (Table 2, entries 2, 3), which proved unsuccessful. Classical heating diphenyl ether as s olvent (cat. p -chlorobenzoic acid, reflux, 15 min), afforded a complex reaction mixtur e (analyzed by 1H NMR spectroscopy). No starting material was recovered. In an effort to optimize the synthesis of ethyl 6-s ulfamoyl-4-oxoquinoline-3carboxilates 39 we addressed the possibility of performing the Go uld-Jacob reaction in different solvents under microwave heating. Employi ng ethanol or dioxane as solvents, allowed us to reach the maximum temperature of 180 C but no reaction was observed and a mixture of starting material and the correspo nding amine 37a was recovered (Table 2, entry 4, 5). o -Dichlorobenzene has a high boiling point (250 C) and also a high dipole moment (2.14 debye units at 20 C),142 a feature of solvents that absorb microwave radiation efficiently. Since high temperatures are required to promote thermal cyclization, the reaction was performed using o -dichlorobenzene as solvent. The quinolones formed could be easily isolated by washing the reaction ma ss with hexane to remove ODCB. Thus, ODCB was found to be the best choice of solve nt to reach high temperature under microwave heating (250 C) and effective to promote the quantitative thermal cyclization of 38a (as determined by analysis of the crude 1H NMR spectrum), shortening significantly the reaction time to 15 min compared to Kurth’s protocol)141 (Table 2, entry 6).

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47 Table 2: Summary of the reaction conditions towards the synt hesis of 39a NH 38a 39a NH S O O CO2Et CO2Et NH S O O NH O O CO2Et Entry Microwave reactor Solvent p -chloro benzoic acid Max. T (C) Power (W) Time (min) Reaction outcomea 1 Biotage Ph2O Catalytic 150 N.A. 15 Starting material & 37a 2 CEM Ph2O Catalytic 250 275 30 Decomposition 3 CEM Ph2O Catalytic 212 250 30 Complex mixture 4 Biotage Ethanol Catalytic 180 N.A 15 Starting material & 37a 5 Biotage Dioxane Catalytic 180 N.A 15 Starting material & 37a 6 Biotage ODCB Catalytic 250 N.A 15 39a quantitative 7 Biotage ODCB None 250 N.A 15 39a 84%b aDetermined by NMR spectroscopy, bIsolated yield The reaction also proved to be successful in absenc e of cat. p -chlorobenzoic acid ( 39a 84%, Table 2, entry 7). Moreover, the pure produc t could be easily precipitated from the reaction mixture by addition of hexane. No further purification was required. Meeting our need of a straightforward and robust pr otocol to synthesize the target molecules, the optimized conditions (Table 2, entry 7) proved to be reproducible, reliable, and applicable to a variety of substrates in good to excellent yields (Table 2, 39b-i )139. The structures of the compounds 39a-i were determined by NMR and Mass spectroscopy.

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48 Table 3 : Synthesis of quinolones ( 39a-h ) NH ODCB MW,250oC,15min 38b-h 39b-h NH S O O CO2Et CO2Et NH S O O NH O O O R R Entry R R1 Yield (%) 39b 2-Cl H 90 39c 4-OMe H 95 39d H Me 70 39e 2-Et H 81 39f 3-Me H 57 39g 2-OMe H 90 39h H H 89 39i 4-Me H 88 6.1.3 Synthesis of HLM002903 HLM002903, 44 (purchased from ChemDiv library) exhibited Shp2 ac tivity with an IC50 of 6.41 m M. Compound 44 ( HLM002903 ) was synthesized as shown in Scheme 4. Pseudothiohydantoin was treated with formaldehyde to give compound 43 .143 Compound 43 was further treated with formaldehyde and ethylamine in the presence of potassium carbonate to give 44 DEPT spectral analysis showed five negative signa ls at 135 pulse (5 -CH2 groups), which had the appropriate s plitting pattern in the 1H NMR, and one positive signal (CH3 group) confirmed the presence of 44 Although, the 2 -CH2 groups (a & b) appear to be equivalent, they have different chemical shifts on the 13C NMR as well as the 1H NMR due to the presence of the ethyl group on the n itrogen atom (in the triazine ring), which makes the carbon (C1), a diastereotopic center. The mass spectrometry a nd single crystal X-ray diffraction analysis further confirme d the structure of compound 44 (Fig. 40, for data see Appendix). The 1H NMR and MS obtained for compound 44 were comparable with the analytical data obtained for th e HLM002903 sample purchased from ChemDiv. N S O OH OH N N 44 a b C1

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49 N S O OH OH N N N S O OH OH H2N N S O H2N a b 43, quantitaive 44, 60% 42 Scheme 4: Synthesis of HLM002903 ( 44 ) Reagents & Conditions: a. HCHO, triethylamine, ammonium carbonate, rt, 1 day; b. HCHO, 0.2M ethylamine in THF, K2CO3, rt, 30 min. Fig. 40: X-ray crystal structure of 44 6.1.4 Synthesis of HLM001038 and HLM001426 HLM001038, 46a and HLM001426, 46b exhibited an IC50 of 12.2 m M and 2.45 m M respectively in the HTS screening. Compounds 46a-b were synthesized by Ytterbium triflate catalysed intermolecular imino Diels-Alder reaction of 45a-b with freshly cracked cyclopentadiene and glyoxylic acid as shown in Sche me 5.144 Compounds 46a and 46b were analyzed by 1H NMR, 13C NMR, and mass spectrometry, which confirmed their structures. DEPT spectral analysis showed one negat ive signal at 135 pulse (-CH2 group) and six positive signals (-CH3 and –CH groups) for compound 46a and one negative signal and five positive signals (-CH grou ps) for Compound 46b respectively. The 1H NMR and MS obtained for Compound 46a and 46b were comparable with the analytical data obtained for the HLM001038 and HLM001426 samples purchased from ChemDiv. The structure of compound 46a was further confirmed by single crystal X-ray diffraction analysis (Fig. 41, for data see Appendi x).

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50 NH OH O O R H H O R NH2 46a, R=OMe,58% 46b, R=OH,33% 45a, R=OMe 45b, R=OH Scheme 5 : Synthesis of HLM001038 and HLM001426 ( 46a and 46b ) Reagents & Conditions: a. cyclopentadiene, glyoxalic acid, ytterbium trif late (10 mol%), magnesium sulfate, DCM, 14 hrs. Fig. 41 : X-ray crystal Structure of 46a All the synthesized compounds were evaluated in th e fluorogenic DiFMUP91 assay for their ability to inhibit Shp2 phosphatase activity. The enzyme activity of Shp2 can be assessed by a number of known methods. They include the pNPP assay,91 in which the activity is measured by the absorption generate d by the dephosphorylation of p nitrophenyl phosphate yielding the fluorescent 4-ni trophenolate (Fig. 42) and the DiFMUP assay, in which the activity is measured by the absorption generated by the dephosphorylation of the fluorogenic 6,8-difluoro-4 -methylumbelliferyl phosphate (DiFMUP) yielding the fluorescent 6,8-difluoro-7-hy droxy-4-methyl-2H-chromen-2-one as the substrate (Fig. 43). For the screening of our compounds, we have used th e DiFMUP assay to test the enzyme activity of Shp2.

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51 Shp2 O NO2 P -O O O -O NO2 pNPP;Notfluorescent pNP;Colorchange Fig. 42: pNPP Assay: Dephosphorylation by Shp2 O O O F F P O Shp2 O O -O F F -O -O DiFMU;Fluorescent DiFMUP;Notfluorescent Fig. 43: DiFMUP Assay: Dephosphorylation by Shp2 The biological results showed that the IC50 of the re-synthesized compounds were greater than 300 m M. From the results obtained, we concluded that not all compounds obtained from commercial sources contain the struct ure that is claimed. Since all the analytical data obtained for the re-synthesized com pounds matched the analytical data of the outsourced compounds, we hypothesized that the initial activity observed in the HTS screen must have resulted from some minor impurity present in the sample. According to Hubbard,145 it is not unusual for 5-10% of compounds purchased from commercial suppliers to either be not what the y claim to be, or to contain major contaminants that can give false positive (or false negative) results.145 It is also known that the phosphatase assay is particularly sensitiv e to oxidants, because of the catalytic cysteine residue Cys459.146 6.2 Hit-to-Lead approach based on hits of related p hosphatases Since the above approach towards the development o f Shp2 inhibitors was not successful, we focused our attention towards an alt ernative approach involving the synthesis of compounds known to inhibit related PTP s. We focused on synthesizing compounds, which were previously reported as hits f or HePTP, hematopoietic protein tyrosine phosphatase. 6.2.1 HePTP hit evaluated for Shp2 HePTP, a protein tyrosine phosphatase found in the hematopoetic cells in the myeloid and lymphatic tis sues. It is a 38 KDa protein having a C-terminus, a short N-terminus and a PTP N CO2H O 52a O

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52 catalytic domain. Homology modeling shows 38% ident ity in the conserved region of the PTP catalytic domains of Shp2 and HePTP.11 The results from San Diego Center for Chemical Genomics compound collection screening for HePTP inhibitors, obtained via Pubchem, showed that 52a is active as a HePTP inhibitor with an IC50 of 1.17 m M in DiFMUP assay. The isooxindole 52a was chosen for hit-to-lead optimization since it is a low molecular weight compound, which may bind in th e PTPase catalytic site of Shp2. The synthesis of the isooxindole 52a was performed modifying a literature procedure (Scheme 6).147, 148 The imine 49a were synthesized from 2-furaldehyde and p anisidine using microwave assisted heating at 130 C for 15 minutes. The imine 49a was reduced to the corresponding amine 50a using NaBH4 in MeOH. The amine 50a was then treated with maleic anhydride to give the Diels-Ald er adduct 51a The compound 51a was aromatized using H3PO4 to give the isooxindole 7-carboxylic acid, 52a .The 1H NMR analysis showed 2 singlets at d 3.78 ppm and d 5.17 ppm respectively which denotes the methoxy protons in the phenyl ring and the methylen e protons present on the isoosindole ring. Two doublets with a coupling constant of 8.8 Hz at d 7.04 and d 7.71 ppm respectively showed the presence of the protons in the phenyl ring (AB pattern). A triplet and 2 doublets above d 7.8 ppm showed the presence of the 3 protons on th e isooxindole ring. The carbons of the carbonyl group in the isoo xindole ring and that of the carboxylic acid showed at d 165.69 and d 168.40 ppm respectively in the 13C NMR spectrum. An m/z of 284 (M+H)+ on the mass spectral analysis further confirmed th e structure. O HN O N N CO2H O O O H H2N O CO2H N O + a bc d 50a51a 52a, 49% 47a 48 49a O O O O O Scheme 6 : Synthesis of isooxindole derivative ( 52a ) Reagents & Conditions : a. EtOH, MW, 130 C, 15 min; b. NaBH4, MeOH, 20 min, rt; c. maleic anhydride, toluene, rt, 3 days; d. H3PO4, reflux, 1 hr.

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53 Compound 52a was found to have IC50 of 3.89 1 m M in the DiFMUP assay for Shp2. The docking structure of compound 52a with the Shp2 PTPase domain (PDB ID: 3b7o) is illustrated in Fig. 44. Fig. 44 : Docking structure of 52a with the Shp2 PTPase domain (PDB ID: 3b7o) Fig. 5 shows that the compound 52a binds relatively well into the PTP binding pocket of the Shp2 protein. The docking structure below (Fig. 45) shows the interactions of the compound 52a with the amino acid residues LYS366, SER460, GLY42 7, GLN510, and CYS459. Fig. 45 : Interactions of 52a amino acid residues of the Shp2 protein, in the do cking structure We hoped to build upon the isooxindole scaffold to achieve selectivity for Shp2 by capturing interactions outside the catalytic sit e. The synthesis of a series of GLN510 LYS366 SER460 CYS459 GLY427

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54 isooxindoles was performed using the procedure outlined (Table 4). Table 4: Synthesis of isooxindole derivatives O H N O N N CO2H O O O H H2N O CO2H N O + a b c d 50a-o 51a-o 52a-o R R R R R 47b-o 48 49a-o Entry R Yield (%) IC50 ( m m M) 52b 4-CH(CH3)2 39 >300 52c 2,4-F2 61 Not tested 52d 4-Cl 44 95 52e 4-Br 41 12.3 52f 4-OEt 47 8.9 52g 2-OMe 19 3.6 52h 3,4-Methylenedioxy 30 0.7 To probe the molecular features of 52a responsible for Shp2 activity, we also synthesized compound 55 (Scheme 7) from phthaldialdehyde 55 and p -anisidine in acetic acid, under microwave assisted heating (130 C for 15 min).149 These compounds lacked the carboxylic acid group at position 7 but retained all rings. These were synthesized to probe the function of the carboxylic acid group for Shp2 activity. H H O O N O O 56 ,9% 55 Scheme 7: Synthesis of Isooxindole derivative 55 Reagents & Conditions : a. p -anisidine, acetic acid, m w, 130 C, 10 min. Compound 59 (Scheme 8), was synthesized by treating p -anisidine 57a with 2chloroethyl isocyanate 56 and closing the ring to form the imidazolidinone using NaH in THF. In compound 59 the imidazolidinone ring was introduced to replace the

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55 pyrrolidinone ring and probe the function of the py rrolidinone ring for Shp2 activity. O NH2 O N NH O 59, 35% O NH HN a b Cl 57a 58O Scheme 8 : Synthesis of imidazolidinone derivative Reagents & Conditions : a. 1-chloro-2-isocyanatoethane, THF, m w, 100 C, 35 min; b. NaH, THF, 0 C rt, 30 min. Compounds 61a-b (Scheme 9), were synthesized to provide analogs th at have the pyrolidonone ring attached to the aryl group, but l ack the phenyl group with the carboxylic acid. R N O R NH2 61a, R = OMe, 47% 61b, R = Me, 47% R HN O Cl ab 57a, R = OMe 57b, R = Me 60 Scheme 9: Synthesis of pyrrolidinone derivatives 60a-b Reagents & Conditions : a. 4-chlorobutanoyl chloride, Na2HPO4, CHCl3, rt, 22 hrs; b. NaH, THF, 0 C rt, 30 min. To probe the function of the isooxindole ring (ring B), compounds 63a-h (Table 5) were synthesized by the ringopening of cyclohexane dicarboxylic acid anhydride by an aniline. These were synthesized based on compounds 52 These were synthesized to introduce more flexibilit y to the molecule by removing ring B while keeping the other rings intact. The aromatic ring in 39 was replaced by the cyclohexyl ring in compounds 63a-h N O OH O RingB RingA RingC

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56 Table 5: Synthesis of substituted cyclohexane carboxylic aci ds ( 63a-h ) NH O O OH R (+ )-63a-h O O O 62 Reagents & Conditions : a. RC5H5NH2, CHCl3, rt, 2 hrs Entry R Yield (% ) 63a 4-OCH3 85 63b 2,4-F2 69 63c 4-Cl 93 63d 4-Br 74 63e 4-CH3 87 63f 4-CH(CH3)2 83 63g 4-CF3 98 63h 4-CO2Et 84 Compounds 65a-g, (Table 6), were synthesized by ring opening of succ inic anhydride with an aniline. In these cases, the indo le carboxylic acid has been replaced in part by a more conformationally flexible succinamid e group while keeping the carbon backbone intact. Table 6 : Synthesis of substituted oxobutanoic acids ( 52a-g ) 65a-g HN O COOH R O O O 64 Reagents & Conditions : a. RC5H5NH2, CHCl3, rt, 2 hrs. Entry R Yield (%) 65a 4-OCH3 81 65b 2,4-F2 99 65c 4-Cl 92 65d 4-Br 97 65e 4-CH3 91 65f 4-CH(CH3)2 98 65g 4-CO2Et 82 Compounds 56-65 exhibited no significant activity for Shp2. The abo ve results

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57 proved that the isooxindole ring and the carboxylic acid group were critical for Shp2 activity. It was found that the compound 52a lost its activity completely when the carboxylic acid is removed. This showed that the ca rboxylic acid was very important for the activity as it possibly mimics the phosphate gr oup of phospho-tyrosine. Having proved that the isooxindole ring with the carboxyli c acid is important for Shp2 activity, other isooxindole compounds (Table 6), were synthes ized using the synthetic route shown in Scheme 5 and were tested using DiFMUP assa y for Shp2 activity. As shown in table 6, compounds 52f, 52g and 52h were found to have IC50’s 8.9 m M, 3.6 m M and 0.7 m M respectively. Compound 52h was found to be the most potent amongst these compounds with an IC50 of 700 nM. The sodium salt 54 of the parent compound, 52a was found to be equally potent as compound 54h with an IC50 of 700 nM. The free carboxylic acid was converted i nto the corresponding methyl ester ( 53a ) and ethyl ester ( 53b ) using thionyl chloride and the corresponding alcohol (Scheme 10). Unfortunately, t he compounds 53a and 53b showed no activity for Shp2. N O O O O R N O O O O H 53a, R=CH3,quantitative 53b, R=CH2CH3,quantitative 52a Scheme 10 : Synthesis of esters of 53a and 54b Reagents & Conditions : a. SOCl2, ROH, m w, 60 C, 15 min. The carboxylic acid group in 53a and 53b were also replaced with the nitro group. This compound was synthesized with the objective of replacing the carboxylic acid with a tetrazole ring, a non-classical bioisostere of ca rboxylic acid. The 7-nitro isooxindole 69 was synthesized as shown in Scheme 11,150 starting from 2-methyl-6-nitrobenzoic acid 66 The carboxylic acid was converted into the corres ponding methyl ester 67 under basic conditions using methyl iodide and potassium carbon ate. Bromination of 67 using NBS in CCl4 afforded methyl 2-(bromomethyl)-6-nitrobenzoate 68 Compound 68 on treatment with p -anisidine in the microwave reactor afforded 7-nitr o isooxindole 69.

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58 Compound 69 was tested for Shp2 inhibition in the DiFMUP assay and was found to exhibit no activity for Shp2. N O O 69, 44%NO2 O OH a O O 67, 86%b O O Br NO2 NO2 NO2 68 ,39% c66 Scheme 11 : Synthesis of 7-nitro isooxindole ( 56 ) Reagents and conditions: a. Methyl iodide, Potassium carbonate, acetone, re flux, 18 hrs; b. NBS, CCl4, reflux, 38 hrs, c. p -anisidine, DMF; 150 C, 15 min; Using the synthetic route shown in scheme 6 for com pounds 52a-h the point of diversity could be introduced only in the initial s tage of the reaction. To facilitate parallel synthesis and vary the substituents at a later stag e, an alternate route was followed in our lab and the isooxindole hit 52a, was synthesized using the new route (Scheme 12). O O O O O O O O O O O O O O O O Br N R O O O N R O OH O a bc d e f70 71 72 73 7475 52Scheme 12: Synthesis of isoindoline hits 52a, 52g and 52h Reagents and conditions: a. maleic anhydride, toluene, rt, 16 hrs; b. tetra methylene sulfone, Con. H2SO4, -55 C – rt, 6 hrs; c. H2SO4, MeOH, reflux, 16 hrs; d. NBS, DCM, rt, 16 hrs; e. substituted aniline, m w, 150 C, 15 min; f. 1M LiOH, MeOH, m w, 150 C, 15 min, 1M HCl. The analytical data (1H NMR, 13C NMR, LCMS) obtained for compound 52 (from Scheme 12) was similar when compared to the a nalytical data of the compound prepared using the route outlined in Scheme 6. Howe ver, compound 52a synthesized through the new synthetic route (scheme 12) exhibit ed no inhibitory activity for Shp2. Compounds 52g and 52h were also synthesized using scheme 12 because of th is abnormal change in activity of 52a and they had similar analytical data to the compounds

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59 prepared using the earlier route. The compounds 52g and 52h synthesized by the new route also turned out to be inactive. This unusual behavior made us re-confirm the activity of the original hit 52a using the approach illustrated in scheme 6. While the compounds re-synthesized by the initial route (scheme 6) showed activity in the same scale as before, the compounds synthesized using the alternate route (Scheme 12) failed to exhibit any inhibitory activity for Shp2 in the DifMUP assay. Hence we concluded that the activity of the compounds would have been due to some minor impurity produced during the course of the synthesis, which was not detectable either by NMR or by LCMS. Since all the above compounds proved unsuccessful as Shp2 inhibitors, we decided to turn our attention towards the synthesis of isatin derivatives (Fig. 46) based on the hits obtained previously. 6.3 Hit-to-Lead approach based on previous hits, NSC117199 and RPM744 Isatin derivatives 6 and 7 were found to exhibit Shp2 activity with an IC50 of a few micromolar. These compounds were based on the compounds NSC-87877 and NSC127199101 (Fig. 46) obtained from the screen of the NCI Diversity set at the Moffitt Cancer Center. O N+ NH N NH -O O S O O HO N N N S S O O OH HO O O NSC-87877,IC50:0.318 m M NSC-117199,IC50:47 m m M 6,IC50:0.80.22 m m M N H N O HO2C NH HO2C O N+ NH N N H -O O 7,IC50:46.810.2 m m M S OH O O Fig. 46: Isatin hits for Shp2 6.3.1 Synthesis of urea derivatives Substituted ureas were synthesized based on the results of isatin compounds previously screened for Shp2 inhibition. Ureas are considered non-classical bioisosteres of amides; the amide group in isatin was replaced with the much flexible urea linker. The urea linker can orientate the two-aryl groups, in a similar manner to the isatin, NSC-87877 Diarylureas 78a-e were synthesized using an efficient microwave synthesis of methyl 2-isocyanatobenzoate and the corresponding aniline by 6,IC50:0.80.22mM N H N O HO2C NH HO2C N H HO2C O N H R 78a-e

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60 heating at at 100 C for 10 min to give 77a-e Then a microwave-assisted saponification of the esters 77a-e gave the desired compounds 78a-e (Table 7). Table 7: Synthesis of urea derivatives ( 78a-e ) NH2 77a-e 78a-ea b N H N O H O O N H N O H O OH R R1 R2 R3 R R1 R2 R3 R R1 R2 R3 76 Reagents & Conditions: a. Methyl 2-isocyanatobenzoate, THF, m w, 100 C, 10 min; b. 1M NaOH, MeOH, m w, 60 C, 10 min. Entry R R1 R2 R3 Yield (%) 78a H COOH H H 73 78b COOH H H H 73 78c H H COOH H 79 78d H OH COOH H 88 78e H H OH COOH 60 6.3.2 Synthesis of isatin derivatives Compound RPM744 was found to have an IC50 of 0.98 m M for Shp2. Although RPM744 exhibited good inhibitory activity, it lacked cell permeability. Therefore we decided to synthesize isatin derivatives introducing hydrophilic groups at the sulfonamide terminus of RPM744 aiming at improving the cell permeability. Compounds 82a-x were synthesized as illustrated (Table 8). Isatin-5-sulf onic acid 79 was treated with POCl3 at 60 C to give the Isatin-5-sulfonyl chloride 80 Compound 80 was then treated with the corresponding amines in a microwave-assisted reacti on using diisopropyl ethylamine as base to afford the sulfonamides 81a-m The sulfonamides 81a-m were treated with the corresponding hydrazine benzoic acids to afford com pounds 82 a-x. NH Cl S O O NH N O RPM744, IC50:0.98+ 2mM N H O OH

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61 Table 8: Synthesis of 82a-x 80 ,56%a b c81a-m 82a-x N H O O S HO O O N H O O S Cl O O N H O O S O O N H N O S O O N H R R R1 79 Reagents and Conditions: a. POCl3, Tetramethylene sulfone, 60 C, 3 hrs; b. DIPEA, 4-fluorophenyl hydrazine, m w, 60 C, 10 min; c. 4-hydrazinobenzoic acid, EtOH, 1M HCl, m w, 120 C, 15 min. Entry R R1 Yield (%) IC50 ( m m M) 82a 4-fluorophenylpiperazine 4-COOH 34 15.1 82b 4-morpholinoaniline 4-COOH 29 3.2 82c 2-morpholinoethanamine 4-COOH 17 >300 82d 1-((1,3-dihydroisobenzofuran-5-yl)methyl)piperazine 4-COOH 46 >300 82e isoxazol-3-amine 4-COOH 22 >300 82f 2H-tetrazol-5-amine 4-COOH 10 >300 82g 1-(3-chlorophenyl)piperazine 4-COOH 25 >300 82h 1-(3-(trifluoromethyl)phenyl)piperazine 4-COOH 65 >300 82i 1-(3,4-dichlorophenyl)piperazine 4-COOH 40 16.7 82j 1-(2,3-dichlorophenyl)piperazine 4-COOH 13 39.8 82k 1-(3-methoxyphenyl)piperazine 4-COOH 60 57.5 82l 1-(3-chlorobenzyl)piperazine 4-COOH 25 39.8 82m 1-cyclohexylpiperazine 4-COOH 62 >300 82n 4-fluorophenylpiperazine 3-COOH Quantitative >300 82o 4-morpholinoaniline 3-COOH 37 >300 82p 2-morpholinoethanamine 3-COOH 43 >300 82q isoxazol-3-amine 3-COOH 47 17.8 82r 1-(3-chlorophenyl)piperazine 3-COOH 22 >300 82s 1-(3-(trifluoromethyl)phenyl)piperazine 3-COOH 39 42.7 82t 1-(3,4-dichlorophenyl)piperazine 3-COOH 35 29.5 82u 1-(2,3-dichlorophenyl)piperazine 3-COOH 11 52.5 82v 1-(3-methoxyphenyl)piperazine 3-COOH 51 >300 82w 1-(3-chlorobenzyl)piperazine 3-COOH 49 22.1 82x 1-cyclohexylpiperazine 3-COOH 49 >300 The piperazine substituents, R were chosen based on other hits for Shp2. The

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62 piperazine ring structure is also found in some antibiotics (piperacillin) and anti-psycotic drugs (clozapine). The morpholino, isoxazol and tetrazol substituents were chosen to introduce more diversity in the sulfonamide terminus of the molecule. The compounds 82a-x were analyzed by 1H NMR, 13C NMR and LCMS. All the compounds except 82e 82f and 82q displayed multiplets in the upfield region of the 1H NMR indicating the piperazine/morpholine protons. Compound 82b showed two triplets at 2.95 and 3.63 ppm in the 1H NMR spectrum indicative of the morpholine protons. The protons in the phenyl ring, attached to the morpholine group showed as two doublets in the 1H NMR spectrum and so do the phenyl group attached to the hydrazine group. The latter is shifted downfield indicative of the electron withdrawing groups present. A singlet at 9.71 ppm indicated the NH group of the isatin ring, while a sharp singlet at 11.44 ppm indicates the NH group of the hydrazine. The sharp peak of the hydrazone proton confirmed the Z configuration of the compound, since the NH of the hydrazone is involved in intramolecular hydrogen bonding with the carbonyl group of the oxindole. Another sharp singlet at 12.74 ppm indicates the proton of the carboxylic acid. The isatin compounds 82a-x synthesized, were tested for Shp2 inhibitory activity using the DifMUP assay (Table 8). Some piperazine substituents also exhibited moderate inhibitory activity for Shp2. Compound 82a showed an IC50 of 15.1 m M, the 3-chloro phenylpiperazine derivative 82w exhibited an IC50 of 22.1 m M, however compound 82g exhibited no inhibitory activity for Shp2. The 1-(3,4 dichlorophenyl)piperazine derivatives, 82i and 82t had IC50s of 16.7 and 29.5 mM respectively The most potent compound in this series, 82b exhibited an IC50 of 3.2 1 m M, but less potent than the previous hit RPM744 (IC50 0.98 2 m M). Although compound 82b displayed less potency than the previous hit RPM744 it opens up further avenues for SAR studies. Morpholine group is chemically very stable and has a strong dipole. The morpholine ring structure is also found in some drugs: anti-depressent drug minaprine (cantor) and the vasodilating drug molsidomine. We anticipate that the hydrophilicity of the morpholine ring might improve the water solubility of the compound, and hence its cell permeability. 63b N H N O S O O N H HN O OH Hydrogen bonding N O

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63 Testing compound 82b in cell-based assay would open-up further avenues f or SAR and cell permeability studies on these compounds. 6.4 Conclusion In the search for novel small molecule inhibitors for Shp2, an efficient and fast microwave-assisted synthesis of quinolones has been developed. Applicability to a broad range of substrate and ease of synthesis makes this reaction valuable for the synthesis of various quinolones in a combinatorial fashion. An e fficient microwave-assisted synthesis of imines has been successfully developed. This rea ction was found to be applicable to a variety of substrates. Imines are one of the most i mportant intermediates in organic synthesis and this makes the microwave-assisted syn thesis valuable in the field of organic chemistry.

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64 Chapter 7 7.0 Experimental Section 7.1 General Methods and Instrumentation Infra red spectra were recorded on a Jasco FT-IR 40 0 spectrometer. NMR spectra were recorded on a 400 MHz Varian Mercury plus instrumen t at 25 C and chemical shifts are referenced to TMS using the residual protio form of the solvent used (e.g. CHCl3, MeOH) as internal standard. Data is expressed in pa rts per million and reported as chemical shift ( H), multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet or m = multiplet) and relative integral. High-resolution mass spectrometry was carried out on an ESI-TOF Agilent Technologies LC/M SD instrument. Automated flash chromatography was conducted using a Flashmaster II system (Argonaut-Biotage), using Biotage silica cartridges. Thin layer chromatograph y was performed using 250 m m silica gel 60 F254 plates (Fisher or Whatman). Compounds w ere visualized by ultraviolet fluorescence or by staining with ceric ammonium mol ybdate in H2SO4. Anhydrous solvents were obtained as follows: dichloromethane (anhydrous, 99.8% contains 50-150 ppm hydrocarbon as stabilizer from Aldrich), dimeth yl formamide (anhydrous, 99.9% from Aldrich), tetrahydrofuran (anhydrous, 99.9%, i nhibitor free, Aldrich), acetonitrile (anhydrous, 99.8%, Aldrich), toluene (anhydrous, 99 .8%, Aldrich), methanol (anhydrous, 99.8%, Aldrich). High performance liquid chromatogr aphy was performed on a Jasco LC-NetII/ADC instrument. Microwave reactions were p erformed in a Biotage Initiator I microwave reactor. Melting points were measured on a Mel-Temp apparatus. 7.2 General procedure for the synthesis of 2,2-Diet hoxyN -(2,3,4-substituted benzyl) ethanamines (19a-e): Aminoacetaldehyde diethyl acetal (1 eq) was added t o a stirred solution of the aldehyde

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65 in EtOH (2 mL/ mmol). The resulting solution was he ated at 130 C for 15 minutes in the microwave reactor. After the reaction, EtOH was eva porated and MeOH (1 mL/ mmol) was added. To the resulting solution, NaBH4 (2.5 eq) was added in portions and stirred at rt for 30 minutes. Methanol was evaporated from the resulting mixture; the aqueous layer was extracted with DCM, dried (Na2SO4) and evaporated to afford the product. 2-((2,2-Diethoxyethylamino)methyl)-6-methoxyphenol (19a)114 HN OEt OEt OH O This was prepared from o -vanillin (5.00 g, 33.0 mmol) and aminoacetaldehyde diethylacetal (4.80 mL, 33.0 mmol) in a similar man ner as described above. The product was obtained as a yellow solid, (7.40 g, 85%); The crude product was taken as such for the next without further purification. 1H NMR (400 MHz, CDCl3) d 1.22 (t, J = 7.2 Hz, 6H), 2.78 (d, J = 5.6 Hz, 2H), 3.49 (q, J = 7.2 Hz, 2H), 3.76 (q, J = 7.2 Hz, 2H), 3.88 (s, 3H), 4.02 (s, 2H), 4.62 (t, J = 5.6 Hz, 1H), 6.63 (dd, J = 1.6, 8.0 Hz, 1H), 6.76 (t, J = 8.0 Hz, 1H), 6.82 (dd, J = 1.6, 8.0 Hz, 1H). 5-((2,2-Diethoxyethylamino)methyl)-2-methoxyphenol (19b) HN OEt OEt OH O This was prepared from 3-hydroxy-4-methoxybenzaldeh yde (0.500 g, 3.30 mmol) and aminoacetaldehyde diethylacetal (0.480 mL, 3.30 mmo l) in a similar manner as described above. The product was obtained as a yellow solid, (0.740 g, 85%); The crude product was taken as such for the next without further puri fication. 1H NMR (400 MHz, CDCl3) d 1.22 (t, J = 7.2 Hz, 6H), 2.74 (t, J = 5.8 Hz, 2H), 3.48 (q, J = 7.2 Hz, 2H), 3.64 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 4.62 (t, J = 5.8 Hz, 1H), 6.79 (s, 2H), 6.89 (s, 1H). 4-((2,2-Diethoxyethylamino)methyl)-2-methoxyphenol (19c)

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66 HN OEt OEt O O H This was prepared from 4-hydroxy-3-methoxybenzaldeh yde (0.500 g, 3.30 mmol) and aminoacetaldehyde diethylacetal (0.480 mL, 3.30 mmo l) in a similar manner as described above. The product was obtained as a yellow solid, (0.730 g, 82%); The crude product was taken as such for the next without further puri fication. 1H NMR (400 MHz, CDCl3) d 1.22 (t, J = 7.2 Hz, 6H), 2.75 (d, J = 5.6 Hz, 2H), 3.50 (q, J = 7.2 Hz, 2H), 3.66 (q, J = 7.2 Hz, 4H), 3.86 (s, 3H), 4.64 (t, J = 5.6 Hz, 1H), 6.88 (s, 1H), 6.78-6.80 (m, 2H). N-(3,4-Dimethoxybenzyl)-2,2-diethoxyethanamine (19d ) HN OEt OEt O O This was prepared from 3,4-dimethoxybenzaldehyde (5 .00 g, 31.0 mmol) and aminoacetaldehyde diethylacetal (4.40 mL, 31.0 mmol ) in a similar manner as described above. The product was obtained as a yellow solid, (8.00 g, 87%); The crude product was taken as such for the next without further purifica tion. 1H NMR (400 MHz, CDCl3) d 1.28 (t, J = 7.2 Hz, 6H), 2.75 (d, J = 5.6 Hz, 2H), 3.49 (q, J = 7.2 Hz, 2H), 3.65 (q, J = 7.2 Hz, 2H), 3.75 (s, 2H), 3.88 (s, 3H), 3.89 (s, 3 H), 4.64 (t, J = 5.6 Hz, 1H), 6.80-6.89 (m, 3H). 2-((2,2-Diethoxyethylamino)methyl)-4-methoxyphenol (19e) HN OEt OEt O HO This was prepared from 2-hydroxy-5-methoxybenzaldeh yde (5.00 g, 32.8 mmol) and aminoacetaldehyde diethylacetal (4.78 mL, 32.8 mmol ) in a similar manner as described above. The product was obtained as a pale yellow so lid, (8.83 g, 88%); The crude product was taken as such for the next without further puri fication. 1H NMR (400 MHz, CDCl3) d

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67 1.28 (t, J = 7.2 Hz, 6H), 2.75 (d, J = 5.6 Hz, 2H), 3.49 (q, J = 7.2 Hz, 2H), 3.65 (q, J = 7.2 Hz, 2H), 3.75 (s, 2H), 3.88 (s, 3H), 3.89 (s, 3H), 4.64 (t, J = 5.6 Hz, 1H), 6.80-6.89 (m, 3H). 7.3 General procedure for the synthesis of 4-(4-aryl)-6,7,8 substituted isoquinolines (20a-z): The corresponding benzaldehyde (1.25 eq) was added to a stirred solution of the amine 710 in HCl (8.0 mL/ mmol) and the resulting mixture was heated at 140 C for 15 minutes in the microwave reactor. After the reaction, ammonium hydroxide was added till pH = 8 and the mixture was filtered. The residue was washed with water and dried to give the product. 4-(2-Chlorobenzyl)-7-methoxyisoquinolin-8-ol (20a) N O OH Cl This was prepared from 2-chlorobenzaldehyde (0.190 g, 1.40 mmol) and amine 19a (0.370 g, 1.40 mmol) in a similar manner as described above. The product was collected as a red solid (0.240 g, 83%); m.p 238-240 C; 1H NMR (400 MHz, CDCl3)d 3.73-4.04 (s, 3H), 4.42 (s, 2H), 6.84 (dd, J = 1.2, 8.0 Hz, 1H), 7.04 (dt, J = 1.2, 7.6 Hz, 1H), 7.13 (dt, J = 1.6, 8.0 Hz, 1H), 7.33 (dd, J = 0.8, 9.2 Hz, 1H), 7.39 (s, 1H), 7.41-7.44 (m, 2H), 8.22 (s, 1H), 9.56 (s, 1H); 13C NMR (101 MHz, CDCl3) d 33.79, 57.19, 115.25, 127.09, 127.97, 129.62, 130.48, 137.83, 142.35; IR (cm-1) n 2992, 2839, 1735, 1569, 1512, 1074, 1052; API-ES ( m/z ) found 300.1 (M+H)+ (100%), calculated for C17H14NO2Cl 300.0785, found 300.0792. 4-(4-Bromobenzyl)-7-methoxyisoquinolin-8-ol (20b) N O OH B r This was prepared from 4-bromobenzaldehyde (0.260 g, 1.40 mmol) and compound 19a (0.370 g, 1.40 mmol) in a similar manner as described above. The product was collected

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68 as a red solid (0.170 g, quantitative); m.p 210-211 C; 1H NMR (400 MHz, CDCl3)d 3.96 (s, 3H), 4.32 9s, 2H), 7.17 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 8.11 (s, 1H), 9.39 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.17, 57.17, 115.06, 117.99, 119.69, 120.36, 127.76, 130.40, 139.03, 142.55, 146.93; IR (cm-1) n 3009, 2963, 2949, 1739, 1541, 1456, 1371, 1074, 1014; API-ES ( m/z ) 344 (M+H)+ (100%), 346 (M+2)+ (100%), calculated for C17H14NO2Br 344.0281, found 344.0285. 4-(2,4-Dichlorobenzyl)-7-methoxyisoquinolin-8-ol (20c) N O OH C l Cl This was prepared from 2,4-dichlorobenzaldehyde (0.240 g, 1.40 mmol) and compound 19a (0.370 g, 1.40 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.170 g, 82%); m.p 208-210 C; 1H NMR (400 MHz, CDCl3)d 4.00 (s, 3H), 4.37 (s, 2H), 6.74 (d, J = 8.4 Hz, 1H), 7.02 (dd, J = 2.0, 8.4 Hz, 1H), 7.29 (br s, 1H), 7.39 (d, J = 9.2 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 8.22 (s, 1H), 9.57 (s, 1H); 13C NMR (101 MHz, CDCl3) d 33.01, 57.62, 114.35, 120.38, 120.74, 127.79, 128.19, 129.42, 130.14, 132.47, 132.55, 134.64, 137.15, 141.67, 142.42, 143.85, 147.07; IR (cm-1) n 3013, 2966, 1739, 1559, 1541, 1467, 1361, 1222, 1081, 1031; API-ES ( m/z ) found 334 (M+H)+ (100%), 336 (M+2)+ (70%), calculated for C17H13NO2Cl2 334.0396, found 334.0400. 4-(3,5-Dimethylbenzyl)-7-methoxyisoquinolin-8-ol (20d) N O OH This was prepared from 3,5-dimethylbenzaldehyde (0.110 g, 0.840 mmol) and compound 19a (0.230 g, 0.840 mmol) in a similar manner as described above. The product was collected as a reddish solid (0.140 g, 71%); m.p 196-197 C; 1H NMR (400 MHz, CDCl3)d 2.20 (s, 6 H), 3.95 (s, 3H), 4.25 (s, 2H), 6.80 (br s, 3H), 7.46 (d, J = 9.2 Hz,

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69 1H), 7.54 (d, J = 9.2 Hz, 1H), 8.06 (s, 1H), 9.37 (s, 1H); 13C NMR (101 MHz, CDCl3) d 21.48, 36.56, 57.51, 115.14, 117.99, 119.97, 126.58, 128.15, 129.51, 130.68, 138.21, 139.97, 141.53, 142.11, 142.49, 146.73; IR (cm-1) n 3009, 2970, 2931, 1735, 1555, 1541, 1456, 1361, 1233, 1219, 1074, 1031; API-ES ( m/z ) found 294.1 (M+H)+ (100%), calculated for C19H19NO2 294.1488 found 294.1491. 4-(Biphenyl-4-ylmethyl)-7-methoxyisoquinolin-8-ol (20e) N O OH This was prepared from 4-biphenylbenzaldehyde (0.153 g, 0.90 mmol) and compound 19a (0.240 g, 0.90 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.220 g, 90%); m.p 252-254 C; 1H NMR (400 MHz, CDCl3)d 3.99 (s, 3H), 4.37 (s, 2H), 7.24 (br s, 1H), 7.31 (td, J = 1.6, 7.6 Hz, 1H), 7.38 (d, J = 3.6 Hz, 1H), 7.40-7.44 (m, 4H), 7.48 (d, J = 8.0 Hz, 2H), 7.53-7.55 (m, 2H), 8.15 (s, 1H), 10.02 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.38, 57.18, 104.98, 115.51, 127.17, 127.36, 127.48, 129.09, 139.12, 141.00; IR (cm-1) n 3181, 3150, 3056, 2987, 2960, 2905, 2833, 1793, 1686, 1590, 1278, 1075; API-ES ( m/z ) 342 (M+H)+ (100%) calculated for C23H19NO2 342.1488, found 342.1492. 7-Methoxy-4-(pyridin-3-ylmethyl)isoquinolin-8-ol (20f) N O OH N This was prepared from pyridine-3-carboxaldehyde (0.080 mL, 0.90 mmol) and compound 19a (0.240 g, 0.90 mmol) in a similar manner as described above. The product was collected as a red solid (0.240, quantitative); m.p 215-217 C; 1H NMR (400 MHz, CDCl3)d 3.98 (s, 3H), 4.33 (s, 2H), 7.14 (dd, J = 4.8, 7.6 Hz, 1H), 7.34-7.41 (m, 3H), 8.30 (s, 1H), 8.45 (d, J = 4.4 Hz, 1H), 8.58 (s, 1H), 9.57 (s, 1H); 13C NMR (101 MHz, CDCl3) d 15.77, 28.61, 36.30, 57.18, 115.39, 117.65, 128.19, 128.21, 128.63, 130.56, 137.24, 141.73, 146.70; IR (cm-1) n 3019, 2970, 1866, 1739, 1622, 1559, 1279, 1078;

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70 API-ES ( m/z ) 267 (M+H)+ (100%), calculated for C16H14N2O2 267.1128, found 267.1130. 4-(4-Ethylbenzyl)-7-methoxyisoquinolin-8-ol (20g) N O OH This was prepared from 4-ethylbenzaldehyde (0.120 mL, 0.90 mmol) and compound 19a (0.240 g, 0.90 mmol) in a similar manner as described above. The product was collected as a red solid (0.160 g, 61%); m.p 179-180 C; 1H NMR (400 MHz, CDCl3)d 1.17 (t, J = 7.6 Hz, 3H), 2.56 (q, J = 7.6 Hz, 2H), 3.97 (s, 3H), 4.29 (s, 2H), 7.09 (s, 4H), 7.36 (d, J = 9.2 Hz, 1H), 7.43 (d, J = 9.2 Hz, 1H), 8.29 (s, 1H), 9.53 (s, 1H); 13C NMR (101 MHz, CDCl3) d 16.28, 28.37, 35.67, 57.63, 115.07, 120.09, 128.42, 128.99, 130.28, 141.75, 141.80, 142.08, 142.22, 143.57, 146.58; IR (cm-1) n 3021, 1740, 1563, 1461, 1349, 1267, 1105; API-ES ( m/z ) 294 (M+H)+ (100%), calculated for C19H19NO2 294.1489, found 294.1493. 4-(3-Chloro-4-fluorobenzyl)-7-methoxyisoquinolin-8-ol (20h) N O OH F Cl This was prepared from 3-fluoro-4-chlorobenzaldehyde (0.140 g, 0.90 mmol) and compound 19a (0.240 g, 0.90 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.290 g, quantitative); m.p 217-219 C; 1H NMR (400 MHz, CDCl3)d 3.99 (s, 3H), 4.27 (s, 2H), 7.01 (br s, 1H), 7.02 (d, J = 1.2 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 8.28 (s, 1H), 9.56 (s, 1H); 13C NMR (101 MHz, CDCl3) d 35.73, 57.18, 115.06, 116.64, 116.84, 117.73, 128.16, 128.22, 130.57, 141.97, 147.31; IR (cm-1) n 2991, 2968, 1736, 1590, 1496, 1366, 1281, 1178, 1115, 1060; API-ES ( m/z ) 318 (M+2)+ (100%) 320 (M+4)+ (30%), calculated for C17H13NO2ClF 318.0692, found 318.0691. 4-(4-Isopropylbenzyl)-7-methoxyisoquinolin-8-ol (20i)

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71 N O OH This was prepared from 4-isopropylbenzaldehyde (0.130 mL, 0.90 mmol) and compound 19a (0.240 g, 0.90 mmol) in a similar manner as described above. The product was collected as a red solid (0.0640 g, 23%); m.p 200-201 C; 1H NMR (400 MHz, CDCl3)d 1.20 (d, J = 6.8 Hz, 6H), 2.83 (heptet, J = 6.8 Hz, 1H), 3.98 (s, 3H), 4.29 (s, 2H), 7.11 (br s, 4H), 7.37 (d, J = 9.0 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 8.29 (s, 1H), 9.52 (s, 1H); 13C NMR (101 MHz, CDCl3) d 24.22, 33.87, 36.26, 57.16, 115.16, 117.89, 126.77, 128.65, 130.63, 137.36, 141.60, 142.09, 142.47, 146.78, 147.03; IR (cm-1) n 3016, 2966, 1735, 1421, 1364, 1219; API-ES ( m/z ) 308 (M+H)+ (100%), calculated for C20H21NO2 308.1645, found 308.1653. 4-(2,4-Dimethylbenzyl)-7-methoxyisoquinolin-8-ol (20j) N O OH This was prepared from 2,4-dimethylbenzaldehyde (0.120 g, 0.900 mmol) and compound 19a (0.240 g, 0.90 mmol) in a similar manner as described above. The product was collected as a red solid (0.130 g, 50%); m.p 222-224 C; 1H NMR (400 MHz, CDCl3)d 2.29 (s, 3H), 2.33 (s, 3H), 3.99 (s, 3H), 4.24 (s, 2H), 6.69 (d, J = 7.8 Hz, 1H), 6.84 (d, J = 7.8 Hz, 1H), 7.04 (br s, 1H), 7.39 (br s, 2H), 8.08 (s, 1H), 9.53 (s, 1H); 13C NMR (101 MHz, CDCl3) d 24.22, 33.87, 36.26, 57.16, 115.16, 117.89, 126.77, 128.65, 130.63, 137.36, 141.60, 142.09, 142.47, 146.78, 147.03; IR (cm-1) n 3019, 2963, 1735, 1537, 1474, 1446, 1364, 1215, 1092, 1060; API-ES ( m/z ) 293 (M+H)+ (100%), calculated for C19H19NO2 294.1489, found 294.1491. 7-Methoxy-4-(4-(pyridin-2-yl)benzyl)isoquinolin-8-ol (20k) N O OH N

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72 This was prepared from 4-(2-pyridyl)benzaldehyde (0.150 g, 0.900 mmol) and compound 19a (0.240 g, 0.900 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.300 g, 99%); m.p 232-234 C; 1H NMR (400 MHz, CDCl3)d 3.96 (s, 3H), 4.38 (s, 2H), 7.18 (ddd, J = 1.2, 4.8, 7.2 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.65 (td, J = 1.0, 8.0 Hz, 1H), 7.69 (dt, J = 2.0, 7.2 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 8.35 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H), 9.56 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.60, 57.16, 115.37, 117.77, 120.58, 122.16, 127.30, 127.69, 128.93, 130.47, 136.92, 137.71, 141.15, 141.94, 142.38, 147.03, 149.80, 157.41; IR (cm-1) n 3019, 2966, 2938, 1739, 1541, 1456, 1364, 1215, 1081; MS API-ES ( m/z ) 343 (M+H)+, calculated for C22H18N2O2 343.1441, found 343.1443. 7-Methoxy-4-(2-(trifluoromethyl)benzyl)isoquinolin-8-ol (20l) N O OH CF3 This was prepared from 2-trifluoromethylbenzaldehyde (0.110 mL, 0.900 mmol) and compound 19a (0.240 g, 0.900 mmol) in a similar manner as described above. The product was collected as a red solid (0.170 g, 55%); m.p 200-202 C; 1H NMR (400 MHz, CDCl3)d 3.98 (s, 3H), 4.38 (s, 2H), 7.30-7.36 (m, 3H), 7.38 (d, J = 9.2 Hz, 1H), 7.44-7.49 (m, 2H), 8.29 (s, 1H), 9.58 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.50, 57.17, 115.07, 117.66, 123.47, 129.21, 130.18, 132.00, 141.05, 147.30; IR (cm-1) n 2960, 1735, 1565, 1357, 1286, 1156, 1071; API-ES ( m/z ) 333 (M+H)+ (100%), calculated for C18H14NO2F3 334.1049, found 334.1056. 4-(3,5-Dibromobenzyl)-7-methoxyisoquinolin-8-ol (20m) N O OH Br Br This was prepared from 3,5-dibromobenzaldehyde (0.110 g, 0.420 mmol) and compound 19a (0.110 g, 0.420 mmol) in a similar manner as described above. The product was

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73 collected as a red solid (0.210 g, quantitative); m.p 244-246 C; 1H NMR (400 MHz, CDCl3)d 4.00 (s, 3H), 4.26 (s, 2H), 7.25 (br s, 2H), 7.28 (d, J = 9.2 Hz, 1H), 7.40 (d, J = 9.2 Hz, 1H), 7.49 (t, J = 1.6 Hz, 1H), 8.28 (s, 1H), 9.57 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.07, 57.19, 114.93, 117.91, 123.32, 127.47, 130.03, 130.44, 131.41, 132.36, 142.02, 142.50, 144.17, 147.50; IR (cm-1) n 3009, 1743, 1364, 1226, 1081; API-ES ( m/z ) 422 (M+H)+ (45%), 424 (M+2)+ (100%), 427 (M+4)+ (45%), calculated for C17H13Br2NO2 343.1441, found 343.1443. 4-(2,3-Dichlorobenzyl)-7-methoxyisoquinolin-8-ol (20n) N O OH Cl Cl This was prepared from 2,3-dichlorobenzaldehyde (0.0740 g, 0.420 mmol) and compound 19a (0.110 g, 0.420 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.100 g, 80%); m.p 228-230 C; 1H NMR (400 MHz, CDCl3)d 4.00 (s, 3H), 4.45 (s, 2H), 6.71 (dd, J = 1.6, 7.8 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 9.0 Hz, 1H), 7.33 (dd, J = 1.6, 7.6 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 8.22 (s, 1H), 9.58 (s, 1H); 13C NMR (101 MHz, CDCl3) d 34.73, 57.18, 115.01, 117.78, 127.36, 127.48, 128.52, 128.79, 130.28, 142.11, 147.28; API-ES ( m/z ) 334 (M+H)+ (100%) 336 (M+2)+ (70%), calculated for C17H13NO2Cl2 334.0396, found 334.0394. 7-Methoxy-4-(pyridin-4-ylmethyl)isoquinolin-8-ol (20o) N O OH N This was prepared form 4-pyridylbenzaldehyde (0.0800 mL, 0.840 mmol) and compound 19a (0.230 g, 0.840 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.150 g, 65%); m.p 221-222 C; 1H NMR (400 MHz, CDCl3)d 3.99 (s, 3H), 4.32 (s, 2H), 7.08 (d, J = 5.6 Hz, 2H), 7.26 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 8.33 (s, 1H), 8.46 (d, J = 5.6 Hz, 2H), 9.58 (s, 1H); IR (cm-1)

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74 n 3026, 2966, 1739, 1555, 1272, 1156, 1084; API-ES ( m/z ) 267 (100%), calculated for C16H14N2O2 267.1128, found 267.1134. 4-(2,6-Dichlorobenzyl)-7-methoxyisoquinolin-8-ol (20p) N O OH Cl Cl This was prepared from 2,6-dichlorobenzaldehyde (0.150 g, 0.840 mmol) and compound 19a (0.230 g, 0.840 mmol) in a similar manner as described above. The product was collected as a red solid (0.180 g, 64%); m.p 219-221 C; 1H NMR (400 MHz, CDCl3)d 4.05 (s, 3H), 4.65 (s, 2H), 7.21 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.8 Hz, 1H), 7.62 (br s, 1H), 7.67 (dd, J = 0.8, 8.8 Hz, 1H), 9.50 (s, 1H); 13C NMR (101 MHz, CDCl3) d 31.68, 57.27, 113.98, 117.70, 119.27, 126.27, 129.03, 134.95, 136.68, 142.50, 146.52; IR (cm-1) n3023,2970,1739,1435,1226,1084; API-ES ( m/z ) 334 (M+H)+ (100%) 336 (M+2)+ (70%), calculated for C17H13NO2Cl2 334.0396, found 334.0404. 4-(3-Fluorobenzyl)-7-methoxyisoquinolin-8-ol (20q) N O F OH This was prepared from 3-fluorobenzaldehyde (0.200 g, 0.675 mmol) and compound 19a (0.104 g, 0.844 mmol) in a similar manner as described above. The product was collected as a red solid (0.224 g, 64%); m.p. 189-191 C; 1H NMR (400 MHz, CDCl3)d 3.96 (s, 3H), 4.15 (s, 2H), 6.87-6.96 (m, 2H), 6.98 (d, J = 8.0 Hz, 1H), 7.19 (m, 2H), 7.34 (m, 1H), 8.30 (s, 1H), 9.57 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.43, 51.18, 104.98, 113.35, 113.56, 115.26, 115.51, 117.67, 124.30, 124.33, 130.12, 130.20, 130.31, 141.95, 142.30, 147.07; MS API-ES ( m/z ) 284 (M+H)+ (100%), calculated for C17H14NO2F 284.1081 found 284.1086. 4-(3-Fluorobenzyl)-7-methoxyisoquinolin-8-ol (20r)

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75 N O OH F This was prepared from 4-fluorobenzaldehyde (0.100 g, 0.337 mmol) and compound 19a (0.0836 g, 0.422 mmol) in a similar manner as described above. The product was collected as a red solid (0.090 g, 95%); m.p. 230-231 C; 1H NMR (400 MHz, CDCl3)d 3.98 (s, 3H), 4.29 (s, 2H), 6.92 (m, 2H), 7.11 (m, 2H), 7.37 (s, 2H), 8.28 (s, 1H), 9.54 (s, 1H); 13C NMR (101 MHz, CDCl3) d 32.91, 35.93, 50.28, 56.23, 57.17, 115.19, 115.64, 116.19, 116.68, 117.66, 117.97, 130.01, 130.36, 132.39, 141.85, 142.32, 147.00, 190.69; MS API-ES ( m/z ) 284 (M+H)+ (100%), calculated for C17H14NO2F 284.1081 found 284.1087. 4-(4-bromobenzyl)-7-methoxyisoquinolin-6-ol (20s) N OH O B r This was prepared form 4-bromobenzaldehyde (0.100 g, 0.337 mmol) and compound 19c (0.0780 g, 0.422 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.0995 g, 89%); m.p 209-210 C; 1H NMR (400 MHz, CDCl3)d 4.06 (s, 3H), 4.22 (s, 2H), 7.01-7.04 (m, 2H), 7.15 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 0.8 Hz, 1H), 7.24 (s, 1H), 8.22 (s, 1H), 8.96 (s, 1H); 13C NMR (101 MHz, CDCl3) d 29.92, 35.65, 56.34, 105.54, 106.06, 116.63, 116.84, 121.10, 121.84, 124.83, 127.81, 128.15, 128.22, 130.52, 132.13, 136.87, 136.91, 142.24, 148.61, 149.71, 150.62, 155.71, 158.17; API-ES ( m/z ) 344 (M+H)+ (100%), 346 (M+2)+ (100%), calculated for C17H14NO2Br 344.0280, found 344.0285. 4-(3-chloro-4-fluorobenzyl)-7-methoxyisoquinolin-6-ol (20t) N OH O F Cl This was prepared form 3-chloro-4-fluorobenzaldehyde (0.140 g, 0.840 mmol) and

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76 compound 19c (0.220 g, 0.840 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.0900 g, 82%); m.p 209-210 C; 1H NMR (400 MHz, CDCl3)d 4.06 (s, 3H), 4.22 (s, 2H), 7.01-7.04 (m, 2H), 7.15 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 0.8 Hz, 1H), 7.24 (s, 1H), 8.22 (s, 1H), 8.96 (s, 1H); 13C NMR (101 MHz, CDCl3) d 29.92, 35.65, 56.34, 105.54, 106.06, 116.63, 116.84, 121.10, 121.84, 124.83, 127.81, 128.15, 128.22, 130.52, 132.13, 136.87, 136.91, 142.24, 148.61, 149.71, 150.62, 155.71, 158.17; API-ES ( m/z ) 318 (M+H)+ (100%) 320 (M+2)+ (30%), calculated for C17H13NO2ClF 318.0692, found 318.0694. 4-(4-fluorobenzyl)-7-methoxyisoquinolin-6-ol (20u) N OH O F This was prepared from 4-fluorobenzaldehyde (0.0500 g, 0.420 mmol) and compound 19c (0.110 g, 0.420 mmol) in a similar manner as described above. The product was collected as a red solid (0.170 g, quantitative); m.p. 125-126 C; 1H NMR (400 MHz, CDCl3) d1H NMR (400 MHz, CDCl3) d 4.06 (s, 3H), 4.25 (s, 2H), 6.92 (d, J = 8.8 Hz, 2H), 7.11 (dd, J = 5.6, 7.4 Hz, 2H), 7.25-7.26 (m, 2H), 8.21 (s, 1H), 8.97 (s, 1H); 13C NMR (101 MHz, CDCl3) d 35.17, 56.37, 106.11, 107.51, 115.69, 115.90, 130.75, 131.97, 136.75, 136.78, 140.92, 148.85, 150.40, 152.41, 160.17, 162.57; MS API-ES ( m/z ) 284 (M+H)+ (100%), calculated for C17H14NO2F 284.1081, found 284.1088. 4-(3-fluorobenzyl)-7-methoxyisoquinolin-6-ol (20v) N OH O F This was prepared from 3-fluorobenzaldehyde (0.150 g, 0.840 mmol) and compound 19c (0.230 g, 0.840 mmol) in a similar manner as described above. The product was collected as a red solid (0.220 g, 64%); m.p. 132-133 C; 1H NMR (400 MHz, CDCl3)d 4.06 (s, 3H), 4.27 (s, 2H), 6.82-6.90 (m, 2H), 6.96 (d, J = 7.6 Hz, 1H), 7.21-7.24 (m, 3H), 8.24 (s,

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77 1H), 8.97 (s, 1H); 13C NMR (101 MHz, CDCl3) d 35.72, 56.34, 106.00, 107.38, 113.46, 113.67, 115.67, 115.88, 124.69, 125.12, 125.14, 128.08, 130.91, 130.99, 131.71, 141.84, 143.77, 149.45, 150.28, 152.09, 161.68, 164.10; MS API-ES ( m/z ) 284 (M+H)+ (100%), calculated for C17H14NO2F 284.1081 found 284.1086. 4-(2,3-dichlorobenzyl)-7-methoxyisoquinolin-6-ol (20w) N OH O Cl Cl This was prepared from 2,3-dichlorobenzaldehyde (0.100 g, 0.337 mmol) and compound 19c (0.0738 g, 0.422 mmol) in a similar manner as described above. The product was collected as a red solid (0.111 g, 99%); m.p. 132-133 C; 1H NMR (400 MHz, CDCl3)d 4.06 (s, 3H), 4.27 (s, 2H), 6.82-6.90 (m, 2H), 6.96 (d, J = 7.6 Hz, 1H), 7.21-7.24 (m, 3H), 8.24 (s, 1H), 8.97 (s, 1H); 13C NMR (101 MHz, CDCl3) d 34.49, 56.32, 105.47, 107.35, 124.57, 126.20, 128.80, 129.31, 129.55, 131.61, 131.81, 132.59, 140.61, 142.29, 149.97, 150.38, 152.25; MS API-ES ( m/z ) 334 (M+H)+ (100%) 336 (M+2)+ (30%), calculated for C17H13NO2Cl2 334.0396 found 334.0396. 4-(2,4-dichlorobenzyl)-7-methoxyisoquinolin-6-ol (20x) N OH O Cl Cl This was prepared from 3-fluorobenzaldehyde (0.100 g, 0.337 mmol) and compound 19c (0.0738 g, 0.422 mmol) in a similar manner as described above. The product was collected as a red solid (0.113 g, quantitative%); m.p. 132-133 C; 1H NMR (400 MHz, CDCl3)d 4.06 (s, 3H), 4.27 (s, 2H), 6.82-6.90 (m, 2H), 6.96 (d, J = 7.6 Hz, 1H), 7.217.24 (m, 3H), 8.24 (s, 1H), 8.97 (s, 1H); 13C NMR (101 MHz, CDCl3) d 33.07, 56.34, 105.52, 107.41, 124.61, 126.32, 128.15, 129.40, 131.59, 132.28, 132.44, 134.73, 137.03, 142.10, 149.85, 150.34, 152.17; MS API-ES ( m/z ) 334 (M+H)+ (100%) 336 (M+2)+ (30%), calculated for C17H13NO2Cl2 334.0396 found 334.0404.

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78 4-((6,7-Dimethoxyisoquinolin-4-yl)methyl)aniline (20y) N O O H2N This was prepared from p -aminobenzaldehyde (0.240 g, 2.00 mmol) and compound 19d (0.500 g, 1.60 mmol) in a similar manner as described above. The product was collected as a yellow solid (0.0950 g, 73%); m.p. 160-161 C (lit151 166-167 C); 1H NMR (400 MHz, CDCl3)d 3.87 (s, 3H), 4.00 (s, 3H), 4.19 (s, 2H), 6.58 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 7.10 (s, 1H), 7.18 (s, 1H), 8.27 (s, 1H), 8.95 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.21, 56.20, 102.49, 106.01, 115.57, 125.01, 129.40, 129.55, 129.94, 131.70, 142.60, 144.88, 149.31, 150.10, 152.86; API-ES ( m/z ) 296 (M+H)+ (100%), calculated for C18H18N2O2 295.1441, found 295.1450. 4-(3-Chloro-4-fluorobenzyl)-6,7-dimethoxyisoquinoline (20z) S H N HO O This was prepared from compound 19d (0.170 g, 0.600 mmol) and 3-chloro-4-fluoro benzaldehyde (0.200 g, 0.600 mmol) in a similar manner as described above. The product was collected as a red solid (0.170 g, 94%); m.p. 126-127 C; 1H NMR (400 MHz, CDCl3) d 3.88 (s, 3H), 4.02 (s, 3H), 4.26 (s, 2H), 6.97 (s, 1H), 7.03-7.05 (m, 2H), 7.237.24 (m, 2H), 8.27 (s, 1H), 9.00 (s, 1H); 13C NMR (101 MHz, CDCl3) d 34.74, 56.33, 56.43, 102.68, 106.98, 117.35, 119.94, 125.15, 129.77, 130.94, 132.65, 138.89, 142.62, 150.45, 153.32, 155.16, 157.59, 191.53; MS API-ES ( m/z ) found 332 (M+H)+ (100%), 334 (30%), calculated for C18H15NO2ClF 332.0396 found 332.0404. N-(4-((6,7-Dimethoxyisoquinolin-4-yl)methyl)phenyl)benzamide (26a) N O O N H O

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79 4-((6,7-Dimethoxyisoquinolin-4-yl)methyl)aniline (20y) (0.200 g, 0.700 mmol) was dissolved in THF (5 mL). To this solution, benzoic acid (0.080 g, 0.700 mmol), HATU (0.390 g, 1.00 mmol) and DIPEA (0.450 mL, 2.77 mmol) were added and heated at 100 C for 10 min. in the microwave reactor. The resulting mixture was extracted with EtOAc (3 5 mL), washed with satd. NaHCO3 (5 mL), dried (Na2SO4) and evaporated. The crude product was column purified using EtOAc-hexane (1:3, v/v) to give 26a (0.030 g, 9.2%) as a yellow solid; m.p. 185-186 C; 1H NMR (400 MHz, CDCl3)d 3.90 (s, 3H), 4.02 (s, 3H), 4.32 (s, 2H), 7.09 (s, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.46-7.49 (m, 2H), 7.537.58 (m, 3H), 7.86 (dd, J = 1.6, 6.8 Hz, 3H), 8.27 (s, 1H), 9.01 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.44, 56.24, 102.28, 106.13, 120.65, 127.24, 128.80, 129.29, 131.67, 132.02, 135.14, 136.12, 136.62, 142.55, 149.45, 150.25, 153.12, 165.98; MS API-ES ( m/z ) found 398 (M+H)+ (100%); calculated for C25H22N2O3 399.1703, found 399.1704. N-(4-((6,7-Dimethoxyisoquinolin-4-yl)methyl)phenyl)-3,4,5-trimethoxybenzamide (26b) N O O N H O O O O 4-((6,7-Dimethoxyisoquinolin-4-yl)methyl)aniline (20y) (0.100 g, 0.340 mmol) was dissolved in DCM (2 mL). To this solution, 3,4,5 trimethoxybenzoyl chloride (0.0800 g, 0.340 mmol) and pyridine (0.0300 mL, 0.400 mmol) were added and heated at 60 C for 10 minutes in the microwave reactor. The resulting solution was evaporated and washed with CHCl3 to give 26b (0.0250 g, 15%) as an off-white solid; m.p. 110-112 C; 1H NMR (400 MHz, CDCl3)d 3.86 (s, 6H), 3.88 (s, 6H), 3.99 (s, 3H), 4.28 (s, 2H), 7.04 (s, 1H), 7.13-7.18 (m, 5H), 7.52 (d, J = 8.4 Hz, 2H), 8.25 (s, 1H), 8.39 (s, 1H), 8.93 (s, 1H); 13C NMR (101 MHz, CDCl3) d 36.39, 56.23, 56.55, 61.15, 102.25, 104.82, 106.09, 115.57, 120.76, 125.05, 128.85, 129.24, 129.54, 130.58, 131.63, 136.04, 136.70, 141.35, 142.60, 149.47, 150.25, 153.10, 153.46, 165.81; MS API-ES ( m/z ) 489 (M+H)+ (100%), calculated for C28H28N2O6 489.2020, found 489.2026;

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80 2,4-Dichloro-N-(4-((6,7-dimethoxyisoquinolin-4-yl)methyl)phenyl)benzamide (26c) N O O N H C l O Cl 4-((6,7-Dimethoxyisoquinolin-4-yl)methyl)aniline (20y) (0.100 g, 0.340 mmol) was dissolved in THf (2 mL). To this solution, 2,4-dichlorobenzoyl chloride (0.070 g, 0.340 mmol), HATU (0.190 g, 0.510 mmol) and DIPEA (0.170 g, 1.360 mmol) were added and heated at 100 C for 10 min. in the microwave reactor. The resulting mixture was purified by flash column chromatography using DCM-MeOH (96-4, v/v) to give 26c (0.0200 g, 12%); m.p. 219-220 C; 1H NMR (400 MHz, CDCl3)d 3.90 (s, 3H), 4.02 (s, 3H), 4.32 (s, 2H), 7.08 (s, 1H), 7.21-7.24 (m, 3H), 7.37 (dd, J = 1.6, 6.4 Hz, 1H), 7.46 (d, J = 1.6 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 1H), 7.90 (s, 1H), 8.27 (s, 1H), 9.00 (s, 1H); MS API-ES ( m/z ) 467 (M+H)+ (100%) 469 (M+2)+ (30%), calculated for 467.0924, found 467.0922. 4-(3-(4-(3-Chloro-4-fluorobenzyl)-7-methoxyisoquinolin-6-yloxy)propyl)morpholine (26d) N O O F Cl N O 4-(3-Chloro-4-fluorobenzyl)-7-methoxyisoquinolin-6-ol (20t) (0.060 g, 0.180 mmol) was dissolved in DMF (2 mL). To this solution, K2CO3 (0.0780 g, 0.560 mmol), TBAI (0.069 g, 0.190 mmol) and 4-(3-chloropropyl)morpholine (0.0380 g, 0.190 mmol) was added and heated at 80 C for 4 hrs. The resulting solution was diluted with water (5 mL), extracted three times with DCM (5 mL), dried (Na2SO4) and evaporated to afford 26d (0.0400 g, 38%) as a yellow solid, m.p. 82-84 C; 1H NMR (400 MHz, CDCl3)d 1.972.04 (quintet, J = 7.0 Hz, 2H), 2.45 (br s, 4H), 2.50 (t, J = 7.0 Hz, 2H), 3.70 (t, J = 4.6

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81 Hz, 4H), 3.99 (s, 3H), 4.04 (t, J = 6.8 Hz, 2H), 4.25 (br s, 2H), 6.96 (s, 1H), 7.02-7.04 (m, 2H), 7.21-7.27 (m, 2H), 8.26 (s, 1H), 8.98 (s, 1H); 19F NMR (376 MHz, CDCl3)d 119.145 -119.198 (q); 13C NMR (101 MHz, CDCl3) d 26.02, 35.96, 53.90, 55.54, 56.22, 67.16, 67.28, 76.73, 76.91, 77.23, 77.43, 77.55, 102.89, 106.38, 116.74, 121.12, 121.30, 128.19, 137.12, 142.78, 149.96, 150.59, 152.58, 155.72, 158.18; IR (cm-1) n 3002, 2956, 1502, 1463, 1247, 1166, 1116; MS API_ES ( m/z ) 445 (M+H)+ (100%) 447 (M+2)+ (30%), calculated for C24H26N2FCl 445.1689 found 445.1700. 6,7-Dihydrobenzo[ b ]thiophen-4( 5H )-one oxime (28)127 S NOH Hydroxylamine hydrochloride (5.10 g, 73.9 mmol) was added to a suspension of 6,7dihydrobenzo[ b ]thiophen-4( 5H )-one (5.00 g, 32.8 mmol) and sodium bicarbonate (5.00 g, 59.1 mmol) in water (75 mL). The mixture was stirred at room temperature until the evolution of CO2 ceased. The reaction mixture was heated at 120 C in the microwave reactor for 10 minutes. After cooling to room temperature, the mixture was acidified to pH 3.0 (1M HCl), filtered and dried, to give the oxime 28 (4.60 g, 86%) as an off-white solid, m.p. 125-127 C (lit.1, m.p. 125-127 C) as colorless crystals; 1H NMR (CDCl3) d 1.97-2.04 (m, 2H), 2.79 (t, J = 6.8 Hz, 2H), 2.88 (t, J = 6.4 Hz, 2H), 7.08 (d, J = 5.4 Hz, 1H), 7.35 (d, J = 5.4 Hz, 1H). 6,7-Dihydrobenzo[ b ]thiophen-4( 5H )-one O -tosyl oxime (29)127 S NOTs 6,7-Dihydrobenzo[ b ]thiophen-4( 5H )-one oxime (28) (2.00 g, 11.9 mmol) was dissolved in dry pyridine (4.20 mL) and cooled to 0 C. Para -toluenesulfonyl chloride (2.50 g, 13.2 mmol) was added and the reaction mixture was stirred at 0 C for 1 hr. The resulting mixture was filtered and the residue washed with pyridine and dried to give the O -tosyl oxime 29 (3.84 g, 93%) as an off-white crystals, m.p. 130-131 C (lit.1, m.p. 130-132 C); 1H NMR (400 MHz, CDCl3) d 1.96-1.98 (m, 2H), 2.87 (t, J = 6.4 Hz, 2H), 2.84 (t, J =

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82 6.4 Hz, 2H)), 7.05 (d, J = 4.2 Hz, 1H), 7.23 (d, J = 4.2 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H). 7,8-Dihydro-4 H -thieno[ 3,2-b ]azepin-5( 6H )-one (30)128 S H N O 6,7-Dihydrobenzo[ b ]thiophen-4( 5H )-one O -tosyl oxime (29) (2.50 g, 7.80 mmol) was dissolved in EtOH-H2O mixture (1:1.6, 160 mL). Potassium carbonate (16.00 g, 161.00 mmol) was added and the reaction mixture was refluxed for 22 hrs. After evaporation of EtOH, the aqueous layer was filtered, washed with water and dried to give the lactam 30 (0.840 g, 42%) as a yellow solid, m.p. 133-134 C (lit.3, m.p. 134-135 C); 1H NMR (400 MHz, CDCl3) d 2.17-2.23 (m, 2H), 2.61-2.66 (m, 2H), 2. 97 (t, J = 6.8 Hz, 2H), 6.426.50 (m, 1H), 6.61 (d, J = 5.4 Hz, 1H), 7.07 (d, J = 5.4 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 23.70, 27.57, 35.89, 122.59, 122.99, 125.00, 133.01, 175.65; MS m/z ( APIES ): 168 (M+H)+ (100%). Methyl 5-oxo-5-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl)pentanoate (31) S H N O O O O Methyl 5-chloro-5-oxopentanoate (0.280 mL, 2.02 mmol) was dissolved in dry DCM (2 mL) and cooled to 0 C. 7,8-Dihydro-4H-thieno[ 3,2-b ]azepin-5( 6H )-one (30) (0.200 g, 1.20 mmol) was added dropwise to the above solution. To this mixture, SnCl4 (0.200 mL, 1.80 mmol) was added in such a rate that the temperature of the reaction mixture was maintained between 0 C and 10 C. The reaction mixture was allowed to come to room temperature over a period of 30 min and was quenched with ice. The aqueous layer was extracted with DCM (3 5 mL), washed with 20% HCl solution (5 mL), brine, dried (Na2SO4) and evaporated to give 31 (0.280 g, 82%) as a brown crystalline solid; m.p. 106-108 C; 1H NMR (400 MHz, CDCl3)d 1.98 (t, J = 6.8 Hz, 2H), 2.14-2.21 (m, 2H), 2.38 (t, J = 6.8 Hz, 2H), 2.56-2.59 (m, 2H), 2.87 (t, J = 7.0 Hz, 2H), 2.97 (t, J = 7.0 Hz,

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83 2H), 3.64 (s, 3H), 7.31 (s, 1H); 13C NMR (101 MHz, CDCl3)d 20.05, 20.12, 20.17, 23.95, 27.22, 32.58, 50.91, 127.37, 134.54, 135.89, 139.29, 174.14, 175.51, 192.70; MS m/z ( API-ES): 296 (M+H)+ (100%), calculated for C14H17NO4S 296.0951, found 296.0959. Methyl 5-hydroxy-5-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl) pentanoate (32) S H N O O O OH Methyl 5-oxo-5-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl)pentanoate (31) (0.150 g, 0.500 mmol) was dissolved in MeOH (1.5 mL). To this mixture, NaBH4 (0.050 g, 1.26 mmol) was added in portions such that the temperature was maintained between 25 C and 30 C. The mixture was stirred at rt for 30 min. Methanol was evaporated from the reaction mixture, and the aqueous layer was extracted with DCM (3 10 mL), dried (Na2SO4) and evaporated. The crude product was purified using column chromatography to afford pure 32 (0.130 g, 54%) as a brown oil; 1H NMR (400 MHz, CDCl3) 1.73-1.76 (m, 4H), 2.05-2.08 (m, 2H), 2.32 (t, J = 6.8 Hz, 2H), 2.53-2.58 (m, 2H), 2.86 (t, J = 6.8 Hz, 2H), 3.63 (s, 3H), 4.71-4.72 (m, 1H), 6.52 (s, 1H), 8.81 (s, 1H); 13C NMR (101 MHz, CDCl3) 19.67, 20.12, 23.95, 27.22, 32.65, 34.98, 37.19, 50.86, 127.37, 134.54, 135.89, 139.29, 174.07, 175.51; MS m/z ( API-ES ): 298 (M+H)+ (100%) 280 (M-H2O)+ (50%), calculated for C14H19NO4S 298.1108, found 298.1116. Methyl 5-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl)pentanoate (33) S H N O O O Methyl 5-hydroxy-5-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl) pentanoate (32) (0.240 g, 0.800 mmol) was dissolved in DCM (2.5 mL) and cooled to 0 C. To this mixture, Et3SiH (0.140 mL, 0.880 mmol) was added. To the resulting mixture, BF3.Et2O (0.110 mL, 0.880 mmol) was added dropwise and the reaction mixture was allowed to come to rt over a period of 1 hr. The reaction mixture was quenched slowly with water,

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84 the aqueous layer was extracted with DCM (3 3 mL) dried (Na2SO4) and evaporated to afford 33 as a yellow crystalline solid (0.170 g, 77%); m.p. 95-96 C; 1H NMR (400 MHz, CDCl3) d 1.56-1.81 (m, 4H), 2.06-2.51 (m, 4H), 2.60-2.65 (m 4H), 2.90 (t, J = 6.8 Hz, 2H), 3.53-3.90 (m, 3H), 6.33 (s, 1H), 8.19 (s, 1H); 13C NMR (101 MHz, CDCl3) 23.39, 24.48, 27.78, 28.15, 29.82, 30.95, 33.93, 51 .76, 120.01, 122.15, 131.65, 141.99, 174.08, 175.39; MS m/z ( API-ES ): 282 (M+H)+ (100%), calculated for C14H19NO3S 282.1158, found 282.1179. 5-(5-Oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl)pentanoic acid (34) S HN O HO O Methyl 5-(5-oxo-5,6,7,8-tetrahydro-4H-thieno[ 3,2-b ]azepin-2-yl)pentanoate (33) (0.050 g, 0.170 mmol) was dissolved in MeOH (1 mL). Sodium hydroxide (1M, 0.200 mL) was added and the reaction mixture was stirred at rt fo r 1 hr. The resulting mixture was acidified to pH 5.0 (1M HCl), filtered, washed with water and dried to afford the final compound 34 (0.040 g, 89%) as an off-white solid, m.p. charred >150 C; 1H NMR (400 MHz, CD3OD) d 1.59-1.71 (m, 4H), 2.10-2.14 (m, 2H), 2.25-2.40 (m 2H), 2.46-2.56 (m, 2H), 2.68-2.74 (m, 2H), 2.89 (t, J = 6.8 Hz, 2H), 6.41 (s, 1H); 13C NMR (101 MHz, CD3OD) 23.72, 25.71, 26.97, 29.47, 31.22, 35.35, 37.21, 1 19.80, 122.49, 131.85, 142.14, 174.51, 176.25; MS m/z ( API-ES ): 268 (M+H)+ (100%), calculated for C13H17NO3S 268.0929, found 268.0918. 4-NitroN o -tolylbenzenesulfonamide (36)130 NH S NO 2 O O oToluedine (0.640 g, 6.00 mmol) was dissolved in DCE (10 mL). To this solution, p nitrosulfonylchloride (1.25 g, 5.40 mmol) in dry py ridine (0.500 mL) was added. The above reaction mixture was heated at 150 C for 10 min in the microwave reactor. The resulting mixture was acidified (1M HCl) to pH = 3. The aqueous layer was extracted with DCM (3 5 mL), dried (Na2SO4) and evaporated to give 36 (1.76 g, quantitative) as

PAGE 97

85 a brown solid, m.p. 157-158 C (lit.i, 157-159 C) 1H NMR (400 MHz, CD3OD) d 2.01 (s, 3H), 7.13-7.14 (m, 3H), 7.14-7.25 (m, 1H), 7.89 (d, J = 8.8 Hz, 2H), 8.26 (d, J = 8.8 Hz, 2H). 4-AminoN o -tolylbenzenesulfonamide (37)131, 132 NH S NH 2 O O 4-NitroN o -tolylbenzenesulfonamide (36) (1.70 g, 6.00 mmol) was dissolved in MeOHTHF (50:50, 20 mL) and cooled to 0 C. To this solu tion, NiCl2.6H2O (5.50 g, 23.0 mmol) was added. To the resulting mixture, NaBH4 (1.80 g, 48.0 mmol) was added in portions such that the temperature is maintained be tween 0-10 C. The resulting slurry was filtered through celite (Sigma-Aldrich, Celite 521). The filtrate was extracted with DCM (3 15 mL) dried (Na2SO4) and evaporated to give 37 (1.29 g, 85%) as a brown solid, m.p. 154-155 C (lit.ii 154-155C) 1H NMR (400 MHz, CD3OD) d 1.23 (d, J = 3.2 Hz, 2H), 2.01 (s, 3H), 6.56 (d, J = 8.8 Hz, 2H), 7.04-7.07 (m, 2H), 7.10-7.11 (m, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.4 (d, J = 8.8 Hz, 2H). Diethyl 2-((4-( N o -tolylsulfamoyl)phenylamino)methylene)malonate (38a ) NH S NH O O O O O O Diethyl ethoxymethylenemalonate (0.900 mL, 4.60 mmo l) was added to 4-aminoN o tolylbenzenesulfonamide (37) (1.25 g, 4.20 mmol) and heated 180 C for 1 hr. To the resulting solution, Et2O (5 mL) was added, filtered, the residue dried and washed with Et2O to give 38a (1.66 g, 80%) as a colorless solid; m.p. 1158-160 C; 1H NMR (400 MHz, CDCl3) d 1.32 (t, J = 7.2 Hz, 3H), 1.37 (t, J = 7.2 Hz, 3H), 2.00 (s, 3H), 4.26 (q, J = 7.2 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 6.31 (d, J = 5.2 Hz, 1H), 7.04-7.36 (m, 5H), 7.70 (d, J = 8.7 Hz, 2H), 8.46 (d, J = 13.0 Hz, 1H), 11.09 (d, J = 13.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.44, 14.60, 17.84, 60.75, 61.05, 116.75, 124.90, 126.78, 127.26, 129.53, 131.12, 131.82, 134.39, 135.10, 143.21, 150 .32, 165.46, 168.85; MS m/z ( API-

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86 ES ): 433 (M+H)+ (100%), calculated for C21H24N2O6S 433.1428, found 433.1428. Diethyl 2-((4-( N -(3-ethylphenyl)sulfamoyl)phenylamino)methylene)malonate (38b) N H S N H O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.120 mL, 0.600 mmol) and 4-aminoN -(3-ethylphenyl)benzenesulfonamide (building block available in the lab, 0.150 g, 0.540 mmol), in a similar manner as described for preparation of 38a ; (0.200 g, 84%); m.p. 119-121 C; 1H NMR (400 MHz, CDCl3) d 1.09 (t, J = 7.6 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.30 (t, J = 7.0 Hz, 3H), 2.50 (q, J = 7.6 Hz, 2H), 4.19 (q, J = 7.0 Hz, 2H), 4.24 (q, J = 7.0 Hz, 2H), 6.35 (s, 1H), 6.77 (dd, J = 1.2, 7.6 Hz, 1H), 6.83 (s, 1H), 6.90 (d, J = 8.0 Hz, 1H), 7.04-7.10 (m, 3H), 7.67 (d, J = 8.8 Hz, 2H), 8.39 (d, J = 13.2 Hz, 1H), 11.01 (d, J = 13.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.43, 14.58, 16.61, 28.87, 60.79, 61.04, 96.44, 116.79, 119.06, 121.38, 125.30, 129.44, 134.62, 136.60, 143.13, 150.46, 165.61, 168.83; nmax (solid)/(cm-1) 3420, 3338, 3210, 1638, 1496, 1307, 1144, 1077; MS m/z ( API-ES ): found 447 (M+H)+ (100%), calculated for C22H26N2O6S 446.1512, found 446.1523. Diethyl 2-((4-( N -methylN -phenylsulfamoyl)phenylamino)methylene)malonate (38c) N S N H O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.130 mL, 0.700 mmol) and 4-aminoN -methylN -phenylbenzenesulfonamide (building block available in the lab, 0.150 g, 0.600 mmol) in a similar manner as described for preparation of 38a ; (0.070 g, 30%); m.p. 103-104 C; 1H NMR (400 MHz, CDCl3) d 1.33 (t, J = 7.2 Hz, 3H), 1.39 (t, J = 7.2 Hz, 3H), 3.18 (s, 3H), 4.28 (q, J = 7.2 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 7.10 (dd, J = 1.2, 6.8 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 7.29-7.31 (m, 3H), 7.54 (d, J = 8.6 Hz, 2H), 8.51 (d, J = 13.2 Hz, 1H), 11.12 (d, J = 13.2 Hz, 1H); 13C NMR (101 MHz, CDCl3)

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87 d 14.41, 15.43, 60.65, 60.95, 72.50, 96.43, 116.56, 126.74, 127.63, 129.12, 130.11, 131.97, 141.54, 143.08, 150.38, 163.83, 165.40, 168.78; nmax (solid)/(cm-1) 2988, 1688, 1640, 1573, 1442, 1347, 1239, 1145; MS m/z ( API-ES ): found 433.1 (M+H)+ (100%), calculated for C21H24N2O6S 433.1428, found 433.1445. Diethyl 2-((4-( N m -tolylsulfamoyl)phenylamino)methylene)malonate (38d) N H S N H O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.130 mL, 0.630 mmol) and 4-aminoN m -tolylbenzenesulfonamide (building block available in the lab, 0.150 g, 0.600 mmol) in a similar manner as described for preparation of 38a ; (0.21 g, 82%); m.p. 128-130 C; 1H NMR (400 MHz, CDCl3) d 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 2.27 (s, 3H), 4.27 (q, J = 7.2 Hz, 2H), 4.32 (d, J = 7.2 Hz, 2H), 6.60 (s, 1H) 6.95 (d, J = 8.2 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H) 7.12 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.8 Hz, 1H), 8.43 (d, J = 13.2 Hz, 1H), 11.05 (d, J = 13.2 Hz, 1H) ; 13C NMR (101 MHz, CDCl3) d 14.42, 14.58, 60.77, 61.04, 96.47, 116.80, 118.79, 122.53, 126.55, 134.63, 139.65, 143.15, 150.42, 165.56, 168.84; nmax (solid)/(cm-1); MS m/z ( API-ES ): 433.1 (M+H)+ (100%), calculated for C21H24N2O6S 433.1428, found 433.1425. Diethyl 2-((4-( N -(3-methoxyphenyl)sulfamoyl)phenylamino)methylene)malonate (38e) N H S N H O O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.0800 mL, 0.700 mmol) and 4-aminoN -(3-methoxyphenyl)benzenesulfonamide (building block available in the lab, 0.100 g, 0.360 mmol) in a similar manner as described for preparation of 38a ; (0.060 g, 40%); m.p. 130-131 C; 1H NMR (400 MHz, CDCl3) d 1.32 (t, J = 7.2 Hz, 3H), 1.35 (t, J

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88 = 7.2 Hz, 3H), 3.75 (s, 3H), 4.18 (q, J = 7.2 Hz, 2H), 4.37 (q, J = 7.2 Hz, 2H), 6.47 (d, J = 6.4 Hz, 1H), 6.52 (d, J = 6.4 Hz, 1H), 6.73 (d, J = 1.6 Hz, 2H), 7.12-7.14 (m, 3H), 7.75 (d, J = 8.8 Hz, 2H), 8.45 (d, J = 13.2 Hz, 1H), 11.09 (d, J = 13.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.42, 14.58, 55.56, 60.75, 61.04, 95.59, 107.64, 111.21, 113.79, 116.80, 129.6, 130.36, 134.46, 137.68, 143.27, 150.33, 160.58, 165.48, 268.83 ;nmax (solid)/(cm1) 3420, 3417, 3342, 3213, 1595, 1492, 1414, 1307, 1180; 1144( st); MS m/z ( API-ES ): 449 (M+H)+ (100%), calculated for C21H24N2O7S 449.1377, found 449.1380. Diethyl 2-((4-( N -phenylsulfamoyl)phenylamino)methylene)malonate (38f) N H S N H O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.130 mL, 0.630 mmol) and 4-aminoN -phenylbenzenesulfonamide (building block available in the lab, 0.150 g, 0.570 mmol) in a similar manner as described for preparation of 38a ; (0.200 g, 78%); m.p. 154-155 C; 1H NMR (400 MHz, CDCl3) d 1.25 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H), 4.19 (q, J = 7.2 Hz, 2H), 4.24 (q, J = 7.2 Hz, 2H), 6.98-7.06 (m, 5H), 7.16-7.19 (m, 2H), 7.68 (d, J = 2.8 Hz, 2H), 8.38 (d, J = 13.2 Hz, 1H), 11.00 (d, J = 13.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.42, 14.58, 60.81, 61.04, 96.44, 116.83, 121.90, 125.68, 129.58, 134.53, 143.15, 150.46, 165.66, 168.80; nmax (solid)/(cm-1) 3420, 3348, 3217, 1635, 1591, 1492, 1418, 1311, 1147; MS m/z ( API-ES ): 419.1 (M+H)+ (100%), calculated for C20H22N2O6S 419.1271, found 419.1269. Diethyl 2-((4-( N p -tolylsulfamoyl)phenylamino)methylene)malonate (38g) N H S N H O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.130 mL, 0.700 mmol) and 4-aminoN p -tolylbenzenesulfonamide (building block available in the lab, 0.150 g,

PAGE 101

89 0.600 mmol) in a similar manner as described for preparation of 38a ; (0.230 g, 88%); m.p. 179-181 C; 1H NMR (400 MHz, CDCl3) d 1.32 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 2.28 (s, 3H), 4.26 (q, J = 7.2 Hz, 2H), 4.31 (q, J = 7.2 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 8.47 (d, J = 13.2 Hz, 1H), 11.09 (d, J = 13.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.42, 14.58, 21.05, 60.78, 61.02, 96.39, 116.79, 122.63, 129.64, 130.12, 133.88, 135.77, 143.06, 150.48, 165.63, 168.82; nmax (solid)/(cm-1) 3427, 3345, 3224, 1642, 1589, 1492, 1414, 1315, 1144; MS m/z ( API-ES ): 433.1 (M+H)+ (100%), calculated for C21H24N2O6S 433.1428, found 433.1424. Diethyl 2-((4-( N -(2-chlorophenyl)sulfamoyl)phenylamino)methylene)malonate (38h) N H S N H O O O O O O Cl This was prepared from diethyl ethoxymethylenemalonate (0.110 mL, 0.540 mmol) and 4-aminoN -(2-chlorophenyl)benzenesulfonamide (building block available in the lab, 0.150 g, 0.500 mmol) in a similar manner as described for preparation of 38a ; (0.190 g, 80%); m.p. 134-136 C; 1H NMR (400 MHz, CD3OD) d 1.31 (t, J = 7.2 Hz, 3H), 1.34 (t, J = 7.2 Hz, 3H), 4.22 (q, J = 7.2 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 7.14 (dt, J = 1.6, 6.0 Hz, 1H), 7.24-7.30 (m, 2H), 7.34 (d, J = 9.2 Hz, 2H), 7.54 (dd, J = 1.4, 6.8 Hz, 1H), 7.73 (d, J = 8.8 Hz, 2H), 8.54 (s, 1H); 13C NMR (101 MHz, CDCl3) d 14.63, 14.72, 60.42, 60.91, 72.34, 96.48, 106.27, 117.97, 128.00, 128.38, 126.67, 134.23, 135.92, 143.72, 150.15, 164.17, 164.28, 164.83, 165.38, 167.50; nmax (solid)/(cm-1) 3168, 2984, 1675, 1590, 1569, 1478, 1438, 1343, 1162; MS m/z ( API-ES ): 453.0 (M+H)+ (100%), calculated for C20H21ClN2O6S 453.0882, found 453.0887. Diethyl 2-((4-( N -methylN o -tolylsulfamoyl)phenylamino)methylene)malonate (38i)

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90 N S N H O O O O O O This was prepared from diethyl ethoxymethylenemalonate (0.110 mL, 0.540 mmol) and 4-aminoN -methylN o -tolylbenzenesulfonamide (building block available in the lab, 0.150 g, 0.600 mmol) in a similar manner as described for preparation of 38a ; (0.150 g, 61%); m.p. 136-137 C; 1H NMR (400 MHz, CD3OD) d 1.27 (t, J = 7.2 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H), 2.37 (s, 3H), 3.13 (s, 3H), 4.18 (q, J = 7.2 Hz, 2H), 4.21 (q, J = 7.2 Hz, 2H), 6.63 (dd, J = 0.8, 6.8 Hz), 7.07 (dt, J = 0.4, 6.8 Hz), 7.21 (dt, J = 1.2, 6.8 Hz), 7.30 (dd, J = 0.4, 7.2 Hz), 7.45 (dd, J = 2, 6.8 Hz), 7.69 (d, J = 6.8 Hz), 7.73 (s, 2H), 8.61 (s, 1H); 13C NMR (101 MHz, CDCl3) d 14.61, 14.82, 14.91, 18.64, 60.47, 60.67, 61.48, 96.63, 118.12, 127.33, 127.50, 128.91, 130.01, 138.77, 144.03, 150.12, 165.43, 167.43; nmax (solid)/(cm-1) 3642, 2992, 1682, 1633, 1569, 1343, 1237, 1148; MS m/z ( API-ES ): 447.1 (M+H)+ (100%), calculated for C22H26N2O6S 446.1584, found 446.1581. Ethyl 4-oxo-7-( N o -tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxylate (39a) N H S N H O O O O O o -Dichlorobenzene (10 mL) was added to diethyl 2-((4-( N o -tolylsulfamoyl) phenylamino)methylene)malonate (38a) (1.500 g, 3.500 mmol) and heated at 250 C for 15 min in the microwave reactor. Hexane (10 mL) was added to the reaction mixture, which was filtered and the precipitate washed with hexane (20 mL) to afford 39a (1.17 g, 84%) as an off-white solid; m.p. 270-271 C; 1H NMR (400 MHz, CD3OD) d 1.34 (t, J = 7.2 Hz, 3H), 2.00 (s, 3H), 4.30 (q, J = 7.2 Hz, 2H), 7.05-7.12 (m, 4H), 7.63 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 2 Hz, 1H), 8.66 (d, J = 2.0 Hz, 1H), 8.69 (s, 1H); 13C NMR (101 MHz, CDCl3) d 14.45, 132.42, 134.46, 143.12, 165.55, 168.80, 14.59, 17.89, 60.75, 61.02, 77.02, 77.34, 77.66, 96.45, 116.77, 125.27, 126.78, 127.10, 129.51, 131.12, 135.24, 150.41; MS m/z ( API-ES ): 387 (M+H)+ (100%), calculated for C19H18N2O5S 387.0970, found 387.0987.

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91 Ethyl 7-( N -(3-ethylphenyl)sulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (39b) N H S N H O O O O O This was prepared from compound 38b (0.100 g, 0.220 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.0700 g, 81%), m.p. 273274 C; 1H NMR (400 MHz, CD3OD) d 1.10 (t, J = 7.6 Hz, 3H), 1.36 (t, J = 7.2 Hz, 3H), 2.51 (q, J = 7.6 Hz, 2H), 4.32 (q, J = 7.2 Hz, 2H), 6.89-6.92 (m, 3H), 7.08 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.97 (dd, J = 2.0, 8.8 Hz, 1H), 8.66 (s, 1H), 8.72 (d, J = 2.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.96, 38.63, 60.56, 111.89, 120.95, 126.78, 127.12, 128.10, 132.42, 141.60, 146.61, 165.01, 173.41; nmax (solid)/(cm-1) 3423, 3345, 3217, 1627, 1591, 1496, 1407, 1315, 1133 (st); MS m/z ( API-ES ): 401.1 (M+H)+ (100%), calculated for C20H20N2O5S 401.1165, found 401.1161. Ethyl 7-( N -methylN -phenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (39c) N S N H O O O O O This was prepared from compound 38c (0.100 g, 0.230 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.060 g, 70%); m.p. 266267 C; 1H NMR (400 MHz, CDCl3)d 1.24 (t, J = 7.2 Hz, 3H), 3.12 (s, 3H), 4.21 (q, J = 7.2 Hz, 2H), 7.07-7.10 (m, 2H), 7.27-7.34 (m, 4H), 7.70 (dd, J = 2.4, 8.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 8.21 (d, J = 2.0 Hz, 1H), 8.63 (d, J = 6.4 Hz, 1H), 11.07 (d, J = 13.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.96, 15.98, 28.68, 60.53, 111.76, 118.13, 124.47, 126.11, 127.20, 129.76, 138.07, 142.07, 145.51, 146.52, 164.94, 173.41; nmax (solid)/(cm-1) (st); MS m/z ( API-ES ): 387.1 (M+H)+ (100%), calculated for C19H18N2O5S 387.1009, found 387.1013. Ethyl 4-oxo-7-( N m -tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxylate (39d)

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92 N H S N H O O O O O This was prepared from compound 38d (0.100 g, 0.220 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.0500 g, 57%); m.p. 265266 C; 1H NMR (400 MHz, CD3OD )d 1.36 (t, J = 7.2 Hz, 3H), 2.21 (s, 3H), 4.32 (d, J = 7.2 Hz, 2H), 6.79-6.98 (m, 3H), 7.04 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.99 (dd, J = 0.8, 8.8 Hz, 1H), 8.66 (s, 1H), 8.72 (d, J = 1.6 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.96, 21.70, 60.53, 111.77, 117.75, 121.04, 125.65, 130.38, 136.05, 139.22, 146.56, 164.96, 173.43; nmax (solid)/(cm-1) 1638 (st), 1591, 1496, 1403, 1307, 1186; 1133; MS m/z ( API-ES ): 387.0 (M+H)+ (100%), calculated for C19H18N2O5S 387.1009, found 387.1009. Ethyl 7-( N -(3-methoxyphenyl)sulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (39e) N H S N H O O O O O O This was prepared form compound 38e (0.0500 g, 0.110 mmol) and o -dichlorobenzene (1 mL) in a similar manner as described for preparation of 39a (0.0400 g, 90%); m.p. 268270 C; 1H NMR (400 MHz, CD3OD) d 1.37 (t, J = 7.2 Hz, 2H), 3.69 (s, 3H), 4.32 (q, J = 7.2 Hz, 2H), 6.57 (dd, J = 2.0, 6.8 Hz, 1H), 6.63 (dd, J = 2.0, 6.0 Hz, 1H), 6.72 (t, J = 2.2 Hz, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 8.01 (dd, J = 2.0, 8.8 Hz, 1H), 8.66 (s, 1H), 8.74 (d, J = 1.6 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.96, 55.67, 60.53, 106.40, 109.93, 111.81, 112.59, 121.08, 126.10, 127.21, 130.37, 130.77, 135.91, 139.34, 146.55, 160.37, 164.93, 173.40; MS m/z ( API-ES ): found 403.0 (M+H)+ (100%), calculated for C19H18N2O6S 403.0958, found 403.0964. Ethyl 4-oxo-7-( N -phenylsulfamoyl)-1,4-dihydroquinoline-3-carboxylate (39f) N H S N H O O O O O

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93 This was prepared form compound 38f (0.100 g, 0.240 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.0800 g, 89%), m.p. 275276 C; 1H NMR (400 MHz, CD3OD )d 1.37 (t, J = 7.2 Hz, 3H), 4.32 (q, J = 7.2 Hz, 2H), 6.98-7.25 (m, 3H), 7.46-7.54 (m, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 8.7 Hz, 1H), 8.62-8.77 (m, 1H); 13C NMR (101 MHz, CDCl3) d 14.95, 60.52, 111.79, 120.90, 121.02, 124.96, 129.50, 135.97, 142.09, 146.53, 165.94, 173.40; nmax (solid)/(cm-1) 1653, 1603, 1441, 1240, 1184, 1140; MS m/z ( API-ES ): 373.0 (M+H)+ (100%), calculated for C18H16N2O5S 373.0852, found 373.0859. Ethyl 4-oxo-7-( N -p-tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxylate (39g) N H S N H O O O O O This was prepared form compound 38g (0.100 g, 0.230 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.0800 g, 88%); m.p. 274276 C; 1H NMR (400 MHz, CD3OD )d 1.36 (t, J = 7.2 Hz, 3H), 2.15 (s, 3H), 4.32 (q, J = 7.2 Hz, 2H), 7.02 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 2H), 7.18 (t, J = 7.6 Hz, 2H), 7.63 (d, J = 8.7 Hz, 1H), 7.98 (dd, J = 2.2, 8.7 Hz, 1H), 8.66 (s, 1H), 8.73 (d, J = 2.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.93, 20.93, 60.51, 111.74, 120.96, 121.45, 126.08, 129.45, 130.39, 135.41, 142.03, 146.54, 164.93, 173.47; nmax (solid)/(cm-1) 1767, 1721, 1664, 1642, 1599, 1481, 1438, 1320, 1244, 1173; MS m/z ( API-ES ): 387.1 (M+H)+ (100%), calculated for C19H18N2O5S 387.1009, found 387.1005. Ethyl 7-( N -(2-chlorophenyl)sulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (39h) N H S N H O O O O O Cl This was prepared form compound 38h (0.100 g, 0.200 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.0500 g, 60%); m.p. 274275 C; 1H NMR (400 MHz, CD3OD )d 1.36 (t, J = 7.1 Hz, 1H), 4.33 (d, J = 7.1 Hz, 1H), 7.15 (t, J = 7.0 Hz, 1H), 7.24-7.29 (m, 2H), 7.57 (dd, J = 1.2, 8 Hz, 1H), 7.62 (d, J

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94 = 8.5 Hz, 1H), 7.97 (dd, J = 2.2, 8.5 Hz, 1H), 8.66 (d, J = 2.4 Hz, 1H), 8.69 (s, 1H); 13C NMR (101 MHz, CDCl3) d 14.97, 60.54, 111.81, 120.94, 126.06, 128.65, 130.06, 131.38, 142.14, 146.57, 164.99, 173.44; nmax (solid)/(cm-1) 1697, 1563, 1361, 1198, 1035; MS m/z ( API-ES ): 407.0 (M+H)+ (100%), calculated for C18H15ClN2O5S 407.0463, found 407.0459. Ethyl 7-( N -methylN o -tolylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (39i) N S N H O O O O O This was prepared form compound 38i (0.100 g, 0.220 mmol) and o -dichlorobenzene (2 mL) in a similar manner as described for preparation of 39a (0.0800 g, 88%); m.p. 298300 C; 1H NMR (400 MHz, DMSO )d 1.26 (t, J = 7.2 Hz, 3H), 2.30 (s, 3H), 3.07 (s, 3H), 4.20 (q, J = 7.2 Hz, 2H), 6.56 (d, J = 7.2 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.88 (dd, J = 2.0, 8.8 Hz, 1H), 8.33 (d, J = 2.0 Hz, 1H), 8.65 (d, J = 6.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 14.97, 18.65, 60.56, 104.99, 111.87, 121.12, 126.79, 127.41, 129.04, 138.73, 142.36, 146.64, 165.04, 173.51; nmax (solid)/(cm-1) 1697, 1650, 1559, 1541, 1506, 1456, 1361, 1194; MS m/z ( API-ES ): found 401.1 (M+H)+ (100%), calculated for C20H20N2O5S 401.1165, found 401.1175. 4-Oxo-7-( N o -tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxylic acid (40) N H S N H O O O OH O Ethyl 4-oxo-7-( N o -tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxylate (39a) (0.200 g, 0.500 mmol) was dissolved in EtOH (10 mL). Potassium hydroxide (10%, 10 mL) was added and heated to reflux for 1.5 hrs. EtOH was evaporated and the resulting mixture was acidified to pH 5.0 (HCl, 1.0 M) and the precipitate was filtered, the residue dried and washed with water to afford 40 (0.180 g, 98%); 1H NMR (400 MHz, CD3OD) d 2.04

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95 (s, 3H), 7.03-7.11 (m, 4H), 7.77 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 8.8 Hz, 1H), 8.69 (d, J = 2 Hz, 1H), 8.86 (s, 1H); 13C NMR (101 MHz, CDCl3) d 16.98, 108.90, 120.51, 124.70, 125.38, 126.40, 126.79, 126.96, 130.86, 131.29, 134.47, 134.68, 138.36, 141.80, 146.34, 167.88, 178.81;MS m/z ( API-ES ): 357 (M-H)+ (100%), calculated for C17H14N2O5S 359.0696, found 359.0694. N-Methyl-4-oxo-7-( N o -tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxamide (41) N H S N H O O O N H O 4-Oxo-7-( N o -tolylsulfamoyl)-1,4-dihydroquinoline-3-carboxylic acid (40) (0.150 g, 0.420 mmol) was refluxed with SOCl2 (2.00 mL) for 10 minutes. Excess SOCl2 was evaporated and 0.0500 g of the resulting mass was dissolved in DCM (1 mL). To the resulting mixture, pyridine (0.012 mL) and CH3NH2 (1.0 M in THF, 0.0700 mL, 0.14 mmol) was added and stirred at rt for 1 hr. Pyridine-DCM mixture was evaporated and the resulting mass was column purified using EtoAc-Hexane (50:50, v/v) to give 41 (0.00900 g, 23%) as an off-white solid, m.p. 280-282 C; 1H NMR (400 MHz, CD3OD) d 2.04 (s, 3H), 2.97 (d, J = 3.5 Hz, 3H), 6.97-7.15 (m, 4H), 7.68 (d, J = 8.7 Hz, 1H), 7.94 (dd, J = 2.0 Hz, 8.7 Hz, 1H), 8.68 (d, J = 2.0 Hz, 1H), 8.78 (s, 1H); 13C NMR (101 MHz, CDCl3) d 16.90, 24.80, 112.24, 119.76, 125.91, 126.01, 126.34, 126.88, 130.45, 130.81, 134.65, 134.71, 137.46, 141.62, 144.56, 166.22, 176.71; MS m/z ( API-ES ): 372.1 (M+H)+ (100%), calculated for C18H17N3O4S 372.1012, found 372.1006; 2-Amino-5,5-bis(hydroxymethyl)thiazol-4( 5H )-one (43)143 N S O OH OH H2N Formaldehyde (37% solution in water, 6.00 mL, 78.0 mmol) was added to pseudothiohydantoin (2.00 g, 17.0 mmol). To the above mixture, triethylamine (0.300 mL) was added in three portions over a period of 30 min and stirred at rt for 1.5 hrs. To this reaction mixture, ammonium carbonate (2.80 g, 29.0 mmol) was added and stirred at rt for 16 hrs. The reaction mass was filtered, and the filtrate was evaporated and dried to

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96 afford 43 (2.76 g, quantitative) as a colorless solid. The crude product was taken to the next step without further purification; m.p 189-191 C (lit.68 191-192 C); 1H NMR (400 MHz, D2O) d 3.61 (d, J = 11.6 Hz, 2H), 3.72 (d, J = 11.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 64.12, 73.19, 76.47, 183.72, 190.49; MS m/z ( API-ES ): 401.1 (M+H)+ (100%). 3-Ethyl-7,7-bis(hydroxymethyl)-3,4-dihydro-2H-thiazolo[ 3,2-a ][ 1,3,5 ]triazin-6( 7H )one (44)143 N S O OH OH N N Formaldehyde (35% solution in water, 2.60 mL, 31.0 mmol) was added to 2-amino-5,5bis(hydroxymethyl)thiazol-4( 5H )-one (43) (2.17 g, 12.3 mmol). To this mixture, ethylamine (70% solution in water, 0.790 mL, 12.3 mmol) and potassium carbonate (0.300 g, 3.00 mmol) was added and stirred at rt for 30 min. Water was evaporated from the reaction mixture and the crude reaction mass was purified by column chromatography using DCM-MeOH (75 : 25, v/v) to afford 44 (1.81 g, 60%) as a white solid; m.p 129-131 C (lit.68 131-133 C); 1H NMR (400 MHz, CD3OD) d 1.09 (t, J = 7.2 Hz, 3H), 2.65 (q, J = 7.2 Hz, 2H), 3.75 (d, J = 11.6 Hz, 2H), 3.88 (d, J = 11.6 Hz, 2H), 4.36 (s, 2H), 4.67 (s, 2H); 13C NMR (101 MHz, CDCl3) d 12.27, 45.19, 60.97, 63.58, 66.53, 73.25, 153.54, 173.48; Anal. Calcd for C9H15N3SO3; C, 44.07; H, 6.16; N, 17.13; S, 13.07; Found: C, 42.59; H, 5.89; N, 18.83; S, 9.52; MS m/z ( API-ES ): 246.0 (M+H)+ (100%). 7.4 General procedure for the synthesis of tetrahydro-cyclopentane quinoline-4carboxylic acids 46a-b: Ytterbium triflate (0.100 mol eq) and magnesium sulfate (6.60 mol eq) were suspended in MeCN (0.800 mL) and cooled to 0 C. To this solution, a mixture of the aniline (1 mol eq), glyoxylic acid monohydrate (1 mol eq) and freshly cracked cyclopentadiene (3 mol eq) in MeCN (0.800 mL) was added. The resulting mixture was stirred at rt for 16 hrs. Acetonitrile was evaporated and the crude mass was extracted with EtOAc (3 10 mL), dried (Na2SO4) and evaporated to give the crude product. Purification was performed as described below.

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97 ( 3aRS,4SR,9bSR )-6-Methoxy-3a,4,5,9b-tetrahydro-3H-cyclopenta[ c ]quinoline-4carboxylic acid (46a)144 N H OH O O H H This was prepared from o -anisidine (0.100 g, 0.800 mmol), glyoxylic acid monohydrate (0.0700 g, 0.800 mmol) and cyclopentadiene (0.150 g, 2.40 mmol) in a similar manner as described above. The reaction mass was extracted three times with DCM, dried (Na2SO4) and evaporated. Purification of the crude reaction mass was done by column chromatography using EtOAc-hexane (Rf = 0.2, 30:70 EtOAc-hexane, v/v) (greenish solid, 0.0700 g, 58%); m.p 186-188 C; 1H NMR (400 MHz, CDCl3) d 2.38-2.46 (m, 1H), 2.54-2.61 (m, 1H), 3.33 (dq, J = 3.6, 8.8 Hz, 1H), 3.83 (s, 3H), 4.11 (d, J = 8.8 Hz, 1H), 4.16 (d, J = 3.2 Hz, 1H), 5.68-5.67 (m, 1H), 5.76-5.79 (m, 1H), 6.60 (dd, J = 1.6, 7.2 Hz, 1H), 6.73-6.74 (m, 2H); 13C NMR (101 MHz, CDCl3) d 32.71, 40.82, 46.48, 55.66, 56.20, 107.46, 118.78, 120.72, 126.22, 130.18, 133.45, 134.16, 147.34, 178.19; Anal Calcd for C13H13NO3; C, 68.56; H, 6.16; N, 5.71; found; C, 68.13; H, 5.96; N, 5.59;MS m/z ( API-ES ): 243.1 (M+H)+ (100%). ( 3aRS,4SR,9bSR )-6-Hydroxy-3a,4,5,9b-tetrahydro-3H-cyclopenta[ c ]quinoline-4carboxylic acid (46b)152 N H OH O OH H H This was prepared from 2-aminophenol (0.360 g, 3.300 mmol), glyoxylic acid monohydrate (0.300 g, 3.30 mmol) and cyclopentadiene (0.800 mL, 3.200 mmol) in a similar manner as described above. The crude product was purified by washing with MeOH, (greenish solid, 0.250 g, 33%), m.p. 180-182 C; 1H NMR (400 MHz, DMSO) d 2.31-2.48 (m, 2H), 3.13 (dq, J = 3.6, 5.6 Hz, 1H), 3.90 (d, J = 3.2 Hz, 1H), 3.98 (dd, J = 0.8, 7.6 Hz, 1H), 4.42 (br s, 1H), 5.56-5.58 (m, 1H), 5.70-5.71 (m, 1H)m 6.42-6.48 (m, 3H), 9.36 (s, 1H); 13C NMR (101 MHz, CDCl3) d 32.39, 40.66, 46.65, 56.47, 111.21, 119.01, 119.43, 127.23, 129.07, 132.48, 144.86, 174.28; MS m/z ( API-ES ): 232.09

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98 (M+H)+ (100%). N -(Furan-2-ylmethyl)-4-methoxyaniline (50a)147, 148 O H N O p -Anisidine (0.280 g, 2.26 mmol) was dissolved in EtOH (5 mL). To this mixture, 2furaldehyde (0.180 mL, 2.26 mmol) was added and heated at 130 C for 15 min in the microwave reactor. To the resulting solution, MeOH (2 mL) was added. To this mixture, NaBH4 (2.5 mol eq) was added in portions till effervescence ceased. After stirring the reaction mixture at rt for 30 minutes, the solvent was evaporated and the aqueous layer was extracted with DCM (3 5 mL), dried (Na2SO4) and evaporated to give 50a, (brown oil, 0.42 g, 97%); The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 3.75 (s, 3H), 4.27 (s, 2H), 6.22 (dd, J = 0.7, 3.2 Hz, 1H), 6.32 (dd, J = 1.8, 3.2 Hz, 1H), 6.65 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 7.36 (dd, J = 0.7, 1.8 Hz, 1H). N -(Furan-2-ylmethyl)-4-isopropylaniline (50b) O H N This was prepared from p -isopropylaniline (0.440 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a, (brown oil, quantitative). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 1.22 (d, J = 6.8 Hz, 6H), 2.81 (heptet, J = 6.8 Hz, 1H), 4.30 (s, 2H), 6.24 (d, J = 3.2 Hz, 1H), 6.32 (dd, J = 1.8, 3.2 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 2H), 7.25 (s, 1H), 7.36 (br s, 1H). 2,4-DifluoroN -(furan-2-ylmethyl)aniline (50c) O H N F F This was prepared from 2,4-difluoroaniline (0.400 g, 3.12 mmol) and 2-furaldehyde

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99 (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.530 g, 82%). The crude product was taken as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 4.32 (s, 2H), 6.23 (d, J = 3.2 Hz, 1H), 6.32-6.33 (m, 1H), 6.68-6.80 (m, 3H), 7.37 (br s, 1H). 4-ChloroN -(furan-2-ylmethyl)aniline (50d) O H N C l This was prepared from p -chloroaniline (0.390 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.620 g, 96%). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 4.28 (s, 2H), 6.22 (d, J = 3.2 Hz, 1H), 6.316.32 (m, 1H), 6.60 (d, J = 8.8 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 7.26 (s, 1H), 7.36 (br s, 1H). 4-BromoN -(furan-2-ylmethyl)aniline (50e) O H N B r This was prepared from p -bromoaniline (0.530 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a, (brown oil, 0.790 g, quantitative). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 4.28 (s, 3H), 6.23 (d, J = 3.2 Hz, 1H), 6.31-6.32 (m, 1H), 6.56 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 7.36 (br s, 1H). N -(Furan-2-ylmethyl)-4-(methylthio)aniline (50f) O H N S This was prepared from 4-(thiomethyl)aniline (0.300 g, 2.15 mmol) and 2-furaldehyde (0.180 mL, 2.15 mmol) in a similar manner as described for the preparation of 50a, (brown oil, 0.470 g, quantitative). The crude product was used as such to the next step

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100 without further purification. 1H NMR (400 MHz, CDCl3)d 2.40 (s, 3H), 4.30 (s, 2H), 6.22 (d, 3.2 Hz, 1H), 6.31 (dd, J = 1.6, 3.2 Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 7.36 (br s, 1H). 4-EthoxyN -(furan-2-ylmethyl)aniline (50g) O H N O This was prepared from 4-ethoxyaniline (0.430 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.870 g, quantitative). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 1.35 (t, J = 6.8 Hz, 3H), 3.73 (br s, 1H), 3.94 (q, J = 6.8 Hz, 2H), 4.29 (d, J = 6.0 Hz, 2H), 6.20 (d, J = 3.2 Hz, 1H), 6.30 (dd, J = 1.8, 3.2 Hz, 1H), 6.62 (d, J = 8.8 Hz, 2H), 6.76 (d, J = 8.8 Hz, 2H), 7.35 (br s, 1H). 4-EthylN -(furan-2-ylmethyl)aniline (50h) O H N This was prepared from 4-ethylaniline (0.300 g, 2.47 mmol) and 2-furaldehyde (0.200 mL, 2.47 mmol) in a similar manner as described for the preparation of 50a, (brown oil, 0.500 g, quantitative). The crude product was taken as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 1.36 (t, J = 7.6 Hz, 3H), 3.94 (q, J = 7.6 Hz, 2H), 4.29 (s, 2H), 6.22 (d, J = 3.2 Hz, 1H), 6.31 (dd, J = 1.6, 3.2 Hz, 1H), 6.61 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 7.35 (br s, 1H). N -(Furan-2-ylmethyl)-2-methoxyaniline (50i) O H N O This was prepared from o -anisidine (0.300 g, 2.40 mmol) and 2-furaldehyde (0.200 mL, 2.40 mmol) in a similar manner as described for the preparation of 50a, (brown oil, quantitative). The crude product was used as such to the next step without further

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101 purification. 1H NMR (400 MHz, CDCl3)d 3.84 (s, 3H), 4.34 (d, J = 6.0 Hz, 2H), 4.61 (br s, 1H), 6.24 (d, J = 3.2 Hz, 1H), 6.29-6.34 (m, 1H), 6.67-6.73 (m, 2H), 6.77 (d, J = 6.8 Hz, 1H), 6.84 (t, J = 7.6 Hz, 1H), 7.36 (br s, 1H). N -(Furan-2-ylmethyl)-3-methoxyaniline (50j) O H N O This was prepared from m -anisidine (0.300 g, 2.400 mmol) and 2-furaldehyde (0.200 mL, 2.40 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.580 g, quantitative). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 3.77 (s, 3H), 4.30 (s, 2H), 6.23 (d, J = 2.4 Hz, 2H), 6.29(m, 4H), 7.07-7.10 (m, 1H), 7.36 (br s, 1H). N -(Furan-2-ylmethyl)benzo[ d ][ 1,3 ]dioxol-5-amine (50k) O H N O O This was prepared from 3,4-methylenedioxyaniline (0.430 g, 3.120 mmol) and 2furaldehyde (0.260 mL, 3.120 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.620 g, 92%). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 3.81 (br s, 1H), 4.24 (s, 2H), 5.85 (s, 2H), 6.09 (dd, J = 2.2, 8.2 Hz, 1H), 6.21 (d, J = 3.2 Hz, 1H), 6.30-6.32 (m, 2H), 6.64 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H). 4-(Furan-2-ylmethylamino)benzamide (50l) O H N NH2 O This was prepared from paminobenzamide (0.420 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.300 g, 44%). The crude product was used as such to the next step without

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102 further purification. 1H NMR (400 MHz, CDCl3)d 4.30 (s, 2H), 6.18-6.28 (m, 2H), 6.57 (d, J = 8.8 Hz, 1H), 7.30-7.33 (m, 1H), 7.59 (d, J = 8.8 Hz, 2H). N -(Furan-2-ylmethyl)-3,4-dimethoxyaniline (50m) O H N O O This was prepared from 4-aminoveratrole (0.500 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.500 g, 76%). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 3.79 (s, 3H), 3.82 (s, 3H), 4.27 (s, 2H), 6.20 (dd, J = 2.6, 8.0 Hz, 2H), 6.29-6.32 (m, 2H), 6.72 (d, J = 8.8 Hz, 1H), 7.35 (br s, 1H). N -(Furan-2-ylmethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (50n) O H N O O This was prepared from 1,4-benzodioxan-6-amine (0.300 g, 1.98 mmol) and 2furaldehyde (0.260 mL, 1.98 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.490 g, quantitative). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 3.75 (br s, 1H), 4.17-4.19 (m, 2H), 4.21-4.24 (m, 4H), 6.19-6.24 (m, 3H), 6.30 (dd, J = 1.8, 3.0 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 7.35 (br s, 1H). N -(Furan-2-ylmethyl)-4-methylaniline (50o) O H N This was prepared from p -toluidine (0.330 g, 3.12 mmol) and 2-furaldehyde (0.260 mL, 3.12 mmol) in a similar manner as described for the preparation of 50a (brown oil, 0.520 g, 89%). The crude product was taken as such to the next step without further purification. 1H NMR (400 MHz, CDCl3)d 2.24 (s, 3H), 4.29 (s, 2H), 6.21 (dd, J = 3.2, 7.2 Hz, 1H), 6.30 (dd, J = 2.0, 3.2 Hz, 1H), 6.59 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.2 Hz,

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103 2H), 7.35 (br s, 1H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4methoxy-phenyl)-1-oxo-, [ 3aR-(3aa,6a,7b,7ab)] (51a) N O OH O O O N -(Furan-2-ylmethyl)-4-methoxyaniline ( 50a) (0.300 g, 1.47 mmol) was dissolved in toluene. To this mixture, maleic anhydride (0.140 g, 1.47 mmol) was added and the reaction mixture was stirred at rt for 3 days. Toluene was evaporated from the mixture to afford the Diels-Alder adduct as a brown solid (0.380 g, 87%). The crude product was used as such to the next step without further purification; 1H NMR (400 MHz, DMSO )d 2.55 (d, J = 9.1 Hz, 1H), 3.02 (d, J = 9.1 Hz, 1H), 3.73 (s, 3H), 3.99 (d, J = 11.5 Hz, 1H), 4.50 (d, J = 11.5 Hz, 1H), 5.02 (d, J = 1.4 Hz, 1H), 6.24 (s, 1H), 6.47 (d, J = 5.6 Hz, 1H), 6.62 (d, J = 5.6 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H); MS m/z ( API-ES ): 324 (M+Na)+ (100%). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4isopropyl-phenyl)-1-oxo-, [ 3aR-(3aa,6 a a, 7b,7ab)] (51b) N O OH O O This was prepared from maleic anhydride (0.300 g, 3.12 mmol) and compound 50b (0.670 g, 3.12 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.270 g, 28%). The crude product was used as such to the next step without further purification; 1H NMR (400 MHz, DMSO )d 1.18 (d, J = 6.8 Hz, 6H), 2.57 (d, J = 9.2 Hz, 1H), 2.87 (heptet, J = 6.8 Hz, 1H), 3.04 (d, J = 9.2 Hz, 1H), 4.03 (d, J = 11.6 Hz, 1H), 4.52 (d, J = 11.6 Hz, 1H), 5.02 (d, J = 1.6 Hz, 1H), 6.46 (dd, J = 1.6, 4.4 Hz, 1H), 6.63 (d, J = 5.6 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H); MS m/z ( APIES ): Calculated for C18H19NO4 314.1386, found 314.1390 (M+H)+ (100%), 336.1213

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104 (M+Na)+. ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(2,4difluorophenyl)-1-oxo-, [ 3aR-(3aa, 6a,7b,7ab)] (51c) N O OH O F O F This was prepared from maleic anhydride (0.180 g, 1.90 mmol) and compound 50c (0.400 g, 1.90 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.460 g, 79%). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, DMSO )d 2.54 (d, J = 9.2 Hz, 1H), 2.98 (d, J = 9.2 Hz, 1H), 3.91 (d, J = 11.4 Hz, 1H), 4.46 (d, J = 11.4 Hz, 1H), 5.03 (d, J = 1.6 Hz, 1H), 6.46 (dd, J = 1.6, 5.6 Hz, 1H), 6.61 (d, 6.0 Hz, 1H), 7.13-7.14 (m, 1H), 7.34-7.47 (m, 2H); MS m/z ( API-ES ): 308 (M+H)+ (100%). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4-chlorophenyl)-1-oxo-, [ 3aR-(3a a a,6a,7 b b, 7ab)] (51d) N O OH O Cl O This was prepared from maleic anhydride (0.180 g, 1.92 mmol) and compound 50d (0.400 g, 1.92 mmol) in a similar manner as described for the preparation of 51a, (brown solid, 0.470 g, 80%). The crude product was used as such to the next step without further purification. 1H NMR (400 MHz, DMSO )d 2.56 (d, J = 9.2 Hz, 1H), 3.06 (d, J = 9.2 Hz, 1H), 3.14 (br s, 1H), 4.05 (d, J = 11.4 Hz, 1H), 4.50 (d, J = 11.4 Hz, 1H), 5.03 (s, 1H), 6.46 (dd, J = 1.6, 5.6 Hz, 1H), 6.61 (d, J = 6.0 Hz, 1H), 7.41 (d, J = 9.0 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H); MS m/z ( API-ES ): Calculated for C15H12ClNO4 305.0528 found 305.0531 (M+H)+. ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4-bromophenyl)-1-oxo-, [ 3aR-(3a a a,6a,7 b b, 7ab) (51e)

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105 N O OH O Br O This was prepared from maleic anhydride (0.140 g, 1.47 mmol) and compound 50e (0.300 g, 1.47 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.380 g, 87%); 1H NMR (400 MHz, DMSO )d 2.57 (d, J = 9.2 Hz, 1H), 3.05 (d, J = 8.8 Hz, 1H), 4.07 (d, J = 11.6 Hz, 1H), 4.52 (d, J = 11.6 Hz, 1H), 5.03 (s, 1H), 6.47 (d, J = 5.6 Hz, 1H), 6.61 (d, J = 5.6 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H); MS m/z ( API-ES ): Calculated for C15H12BrNO4 350.0023 found 350.0011 (M+H)+. ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4thiomethyl-phenyl)-1-oxo-, [ 3aR-(3aa, 6 a a,7b,7ab)] (51f) N O OH O S O This was prepared from maleic anhydride (0.230 g, 1.80 mmol) and compound 50f (0.400 g, 1.80 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.630 g, 88%), 1H NMR (400 MHz, DMSO )d 2.43 (s, 3H), 2.54 (d, J = 9.2 Hz, 1H), 3.03 (d, J = 9.2 Hz, 1H), 4.00 (d, J = 11.6 Hz, 1H), 4.48 (d, J = 11.6 Hz, 1H), 5.01 (d, J = 1.6 Hz, 1H), 6.45 (dd, J = 1.6, 4.4 Hz, 1H), 6.60 (d, J = 5.6 Hz, 1H), 7.24 (d, J = 9.0 Hz, 2H), 7.58 (d, J = 9.0 Hz, 2H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4-ethoxyphenyl)-1-oxo-, [ 3aR-(3a a a,6a,7 b b, 7ab)] (51g) N O OH O O O This was prepared from maleic anhydride (0.160 g, 1.60 mmol) and compound 50g (0.350 g, 1.60 mmol) in a similar manner as described for the preparation of 51a (Brown solid, 0.540 g, quantitative); 1H NMR (400 MHz, DMSO )d 1.27 (t, J = 7.2 Hz, 3H), 2.53 (d, J = 9.2 Hz, 1H), 3.00 (d, J = 9.2 Hz, 1H), 3.96 (q, J = 7.2 Hz, 2H), 4.47 (d, J = 11.6 Hz, 1H), 6.45 (dd, J = 1.6, 5.6 Hz, 1H), 6.60 (d, J = 5.6 Hz, 1H), 6.89 (d, J = 9.0 Hz,

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106 2H), 7.51 (d, J = 9.0 Hz, 2H), 12.22 (s, 1H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4-ethylphenyl)-1-oxo-, [ 3aR-(3a a a,6a,7 b b, 7ab)] (51h) N O OH O O This was prepared from maleic anhydride (0.250 g, 2.50 mmol) and compound 50h (0.500 g, 2.50 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.720 g, 97%); 1H NMR (400 MHz, DMSO )d 1.11 (t, J = 7.6 Hz, 3H), 2.53-2.58 (m, 3H), 3.01 (d, J = 8.8 Hz, 1H), 3.99 (d, J = 11.6 Hz, 1H), 4.48 (d, J = 11.6 Hz, 1H), 5.01 (d, J = 1.6 Hz, 1H), 6.44 (dd, J = 1.2, 4.4 Hz, 1H), 6.60 (5.6 Hz, 1H), 7.17 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.6 Hz, 2H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(2methoxy-phenyl)-1-oxo-, [ 3aR-(3aa,6a,7b,7ab)] (51i) N O OH O O O This was prepared from maleic anhydride (0.240 g, 2.40 mmol) and compound 50i (0.500 g, 2.40 mmol) in a similar manner as described for the preparation of 52a The crude reaction mass was purified by column chromatography using EtOAc-Hexane (50:50, v/v) to give 27i (0.310 g, 44%) as a yellowish solid; 1H NMR (400 MHz, DMSO )d 2.89 (d, J = 9.0 Hz, 1H), 3.07 (d, J = 9.0 Hz, 1H), 3.85 (s, 3H), 3.97 (d, J = 12.0 Hz, 1H), 4.55 (d, J = 12.0 Hz, 1H), 5.35 (d, 1H), 6.50 (dd, J = 1.6, 5.8 Hz, 1H), 6.55 (d, J = 5.8 Hz, 1H), 6.97-6.99 (m, 2H), 7.25-7.31 (m, 2H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(3methoxy-phenyl)-1-oxo-, [ 3aR-(3aa,6a,7b,7ab)] (51j) N O OH O O O

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107 This was prepared from maleic anhydride (0.230 g, 2.40 mmol) and compound 50j (0.580 g, 2.40 mmol) in a similar manner as described for the preparation of 51a, (brown solid, 0.110 g, 20%); 1H NMR (400 MHz, DMSO )d 2.95 (d, J = 9.0 Hz, 1H), 3.06 (d, J = 9.0 Hz, 1H), 3.83 (s, 3H), 4.22 (d, J = 11.6 Hz, 1H), 4.46 (d, J = 11.6 Hz, 1H), 5.41 (s, 1H), 6.36 (d, J = 12.8 Hz, 1H), 6.44 (d, J = 12.8 Hz, 1H), 6.74 (ddd, J = 2.4, 8.0, 16.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 7.27-7.31 (m, 2H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(3,4methylenedioxy phenyl)-1-oxo-, [ 3aR-(3aa,6 a a, 7b, 7ab)] (51k) N O OH O O O O This was prepared from maleic anhydride (0.230 g, 2.30 mmol) and compound 50k (0.500 g, 2.30 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.570 g, 78%); 1H NMR (400 MHz, DMSO )d 2.53 (d, J = 9.2 Hz, 1H), 3.01 (d, J = 9.2 Hz, 1H), 3.97 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 11.2 Hz, 1H), 5.01 (d, J = 1.2 Hz, 1H), 6.00 (s, 2H), 6.45 (dd, J = 1.6, 5.6 Hz, 1H), 6.59 (d, J = 5.6 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.96 (dd, J = 2.0, 8.4 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4benzamido-phenyl)-1-oxo-, [ 3aR-(3aa, 6a,7b,7ab)] (51l) N O OH O O O NH2 This was prepared from maleic anhydride (0.140 g, 1.40 mmol) and compound 50l (0.300 g, 1.40 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.0780 g, 17%); 1H NMR (400 MHz, DMSO )d 2.58 (d, J = 9.2 Hz, 1H), 3.11 (d, J = 9.2 Hz, 1H), 4.11 (d, J = 11.6 Hz, 1H), 4.26 (br s, 2H), 4.56 (d, J = 11.6 Hz, 1H), 5.04 (d, J = 1.6 Hz, 1H), 6.48 (dd, J = 1.6, 5.6 Hz, 1H), 6.62 (d, J = 5.6 Hz, 1H), 7.28 (Br s, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 8.8 Hz, 2H), 7.91 (s, 1H).

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108 ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(3,4dimethoxy-phenyl)-1-oxo-, [ 3aR-(3a a a,6a aa a,7 b b, 7a b b)] (51m) N O OH O O O O This was prepared from maleic anhydride (0.200 g, 2.16 mmol) and compound 50m (0.500 g, 2.16 mmol) in a similar manner as described for the preparation of 51a, (brown solid, 0.710 g, 98%); 1H NMR (400 MHz, DMSO )d 2.55 (d, J = 9.2 Hz, 1H), 3.02 (d, J = 9.2 Hz, 1H), 3.71 (s, 6H), 4.00 (d, J = 11.2 Hz, 1H), 4.47 (d, J = 11.2 Hz, 1H), 5.01 (d, J = 1.8 Hz, 1H), 6.45 (dd, J = 1.8, 5.6 Hz, 1H), 6.59 (d, J = 5.6 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 7.00 (dd, J = 2.4, 8.8 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(2,3dihydrobenzo[b][1,4]dioxin)-1-oxo-, [ 3aR-(3aa,6a, 7b, 7a b b)] (51n) N O OH O O O O This was prepared from maleic anhydride (0.0800 g, 0.870 mmol) and compound 50n (0.200 g, 0.870 mmol) in a similar manner as described for the preparation of 51a (brown solid, 0.27 g, 95%); 1H NMR (400 MHz, DMSO )d 2.55 (d, J = 9.2 Hz, 1H), 3.00 (d, J = 9.2 Hz, 1H), 3.95 (d, J = 11.2 Hz, 1H), 4.19-4.23 (m, 4H), 4.44 (d, J = 11.2 Hz, 1H), 5.01 (d, J = 1.8 Hz, 1H), 6.45 (dd, J = 1.8, 5.6 Hz, 1H), 6.59 (d, J = 5.6 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 7.02 (dd, J = 2.6, 8.8 Hz, 1H), 7.22 (d, 2.6 Hz, 1H). ()3a,6-Epoxy-3aH-isoindole-7-carboxylic acid, 1,2,3,6,7,7a-hexahydro-2-(4-methylphenyl)-1-oxo-, [ 3aR-(3a a a,6a,7 b b, 7ab)] (51o) N O OH O O This was prepared from maleic anhydride (0.210 g, 2.13 mmol) and compound 50o (0.400 g, 2.13 mmol) in a similar manner as described for the preparation of 51a (White

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109 solid, 0.430 g, 70%); 1H NMR (400 MHz, DMSO )d 2.26 (s, 3H), 2.54 (d, J = 9.0 Hz, 1H), 3.03 (d, J = 9.0 Hz, 1H), 3.99 (d, J = 11.2 Hz, 1H), 4.48 (d, J = 11.2 Hz, 1H), 5.01 (d, J = 1.6 Hz, 1H), 6.45 (dd, J = 1.6, 5.6 Hz, 1H), 6.61 (d, J = 5.6 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H); MS API-ES ( m/z ) calculated for C16H15NO4 286.1078, found 286.1078. 2-(4-Methoxyphenyl)-3-oxoisoindoline-4-carboxylic acid (52a) NH O NH O OH HO OH O The acid (51a) (0.300 g, 0.990 mmol) was refluxed with phosphoric acid (85% solution, 4 mL) for 1 hr. The resulting solution was poured into water, filtered and the residue washed with water to give 52a (0.130 g, 49%) as a brown solid; m.p 200-202 C; 1H NMR (400 MHz, DMSO )d 3.78 (s, 3H), 5.17 (s, 2H), 7.04 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 7.82 (t, J = 7.4 Hz, 1H), 7.92 (d, J = 7.4 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 53.23, 56.01, 114.93, 123.89, 127.97, 129.51, 131.18, 132.33, 133.28, 143.29, 158.05, 165.69, 168.40; MS m/z ( API-ES ): 284 (M+H)+ (100%) 306.07 (M+Na)+, calculated for C16H13NO4 282.0772, found 282.0792. 2-(4-Isopropylphenyl)-3-oxoisoindoline-4-carboxylic acid (52b) N CO2H O This was prepared from compound 51b (0.200 g, 0.640 mmol) and H3PO4 (4 mL) in a similar manner as described in the preparation of 52a, (brown solid, 0.0700 g, 39%); m.p. 204-206 C; 1H NMR (400 MHz, DMSO )d 1.21 (d, J = 6.8 Hz, 6H), 2.92 (heptet, J = 6.8 Hz, 1H), 5.19 (s, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H), 7.85 (t, J = 7.6 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 8.11 (d, J = 7.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 24.50, 33.68, 52.88, 122.13, 127.55, 127.95, 129.75, 130.01, 132.15, 133.44, 136.16, 143.38, 147.08, 165.83, 168.52; MS m/z ( API-ES ): 284.09 (M+H)+ (100%), calculated for C18H17NO3, 296.1281, found 296.1266.

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110 2-(2,4-Difluorophenyl)-3-oxoisoindoline-4-carboxylic acid (52c) N CO2H O F F This was prepared from compound 51c (0.200 g, 0.650 mmol) and H3PO4 (2.60 mL) in a similar manner as described in the preparation of 52a (brown solid, 0.110 g, 61%), m.p. 196-198 C; 1H NMR (400 MHz, DMSO )d 5.08 (s, 2H), 7.28 (t, J = 9.0 Hz, 1H), 7.527.53 (m, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H), 7.94 (d, J = 7.3 Hz, 1H), 8.08 (d, J = 7.4 Hz, 1H); 13C NMR (101 MHz, DMSO) d 53.90, 105.85, 112.98, 121.83, 124.25, 128.06, 128.47, 128.88, 130.07, 131.72, 132.89, 133.64, 142.94, 144.45, 156.50, 159.13, 160.87, 163.45, 165.98, 168.90, MS m/z ( API-ES ): 290 (M+H)+ (100%), calculated for C15H9F2NO3 289.0550, found 289.0532. 2-(4-Chlorophenyl)-3-oxoisoindoline-4-carboxylic acid (52d) N CO2H O Cl This was prepared from compound 51d (0.200 g, 0.650 mmol) and H3PO4 (2.60 mL) in a similar manner as described in the preparation of 52a (brown solid, 0.0820 g, 44%); m.p. 227-229 C; 1H NMR (400 MHz, DMSO )d 5.18 (s, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 9.0 Hz, 4H), 87.98-8.09 (m, 1H); 13C NMR (101 MHz, DMSO) d 52.46, 123.25, 127.61, 129.61, 129.73, 130.24, 131.50, 133.60, 137.60, 143.20, 166.21, 168.40; MS m/z ( API-ES ): 287 (M+H)+ (100%), calculated for C15H10NO3Cl 288.0422, found 288.0412. 2-(4-Bromophenyl)-3-oxoisoindoline-4-carboxylic acid (52e) N CO2H O Br This was prepared from compound 51e (0.200 g, 0.650 mmol) and H3PO4 (2.30 mL) in a similar manner as described in the preparation of 52a (brown solid, 0.0780 g, 41%); m.p. 230-232 C; 1H NMR (400 MHz, DMSO )d 5.18 (s, 2H), 7.68 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 8.1 Hz, 3H), 7.90 (d, J = 7.7 Hz, 1H), 7.98-8.15 (m, 1H); 13C NMR (101 MHz,

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111 DMSO) d 52.36, 118.62, 123.44, 127.59, 129.59, 130.22, 131.51, 132.63, 133.59, 138.01, 143.14, 166.18, 168.39; MS m/z ( API-ES ): 330 (M+H)+ (100%), calculated for C15H10NO3Br 331.9917, found 331.9910. 2-(4-Ethoxyphenyl)-3-oxoisoindoline-4-carboxylic acid (52f) N CO2H O O This was prepared from compound 51g (0.300 g, 0.960 mmol) and H3PO4 (2.60 mL) in a similar manner as described in the preparation of 52a (brown solid, 0.134 g, 47%); m.p. 182-184 C; 1H NMR (400 MHz, DMSO )d 1.33 (t, J = 6.9 Hz, 3H), 4.05 (q, J = 6.9 Hz, 2H), 5.17 (s, 2H), 7.04 (d, J = 9.7 Hz, 2H), 7.71 (d, J = 9.7 Hz, 2H), 7.88 (td, J = 15.2, 7.4 Hz, 2H), 8.13 (d, J = 7.6 Hz, 1H); 13C NMR (101 MHz, DMSO) d 15.26, 53.18, 63.96, 115.34, 123.79, 127.96, 129.47, 131.03, 132.35, 133.26, 143.24, 157.31, 165.65, 168.36; MS m/z ( API-ES ): 297 (M+H)+ (100%), calculated for C17H15NO4 296.0928, found 298.0938. 2-(2-Methoxyphenyl)-3-oxoisoindoline-4-carboxylic acid (52g) N CO2H O O This was prepared from compound 51i (0.300 g, 0.996 mmol) and H3PO4 (3.00 mL) in a similar manner as described in the preparation of 52a, (brown solid, 0.0500 g, 19%); m.p. 207-208 C; 1H NMR (400 MHz, DMSO )d 4.23 (s, 3H), 5.44 (s, 2H), 7.49 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.86-7.93 (m, 2H), 8.29 (t, J = 7.6 Hz, 1H), 8.37 (d, J = 7.6 Hz, 1H), 8.59 (d, J = 7.6 Hz); 13C NMR (101 MHz, DMSO) d 54.03, 56.52, 113.30, 121.35, 125.39, 128.45, 128.63, 129.32, 129.55, 130.78, 132.45, 132.58, 133.48, 144.48, 155.29, 165.58, 169.48, MS m/z ( API-ES ): 283 (M+H)+ (100%), calculated for C16H13NO4 282.0772, found 282.0784; 2-(Benzo[ d ][ 1,3 ]dioxol-5-yl)-3-oxoisoindoline-4-carboxylic acid (52h)

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112 N CO2H O O O This was prepared from compound 51k (0.300 g, 0.960 mmol) and H3PO4 (3.00 mL) in a similar manner as described in the preparation of 52a (brown solid, 0.0270 g, 30%); m.p. 266-267 C; 1H NMR (400 MHz, DMSO )d 5.14 (s, 2H), 6.08 (s, 2H), 7.04 (d, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.46 (s, 1H), 7.85 (d, J = 6.6 Hz, 1H), 7.94 (s, 1H), 8.11 (d, J = 6.6 Hz, 1H); 13C NMR (101 MHz, DMSO) d 53.53, 102.31, 104.50, 108.92, 116.24, 127.87, 129.71, 129.88, 132.10, 132.34, 133.41, 143.29, 146.10, 148.24, 165.86, 168.44; MS m/z ( API-ES ): 298 (M-H)(100%), calculated for C16H11NO5 296.0564, found 296.0569. Methyl 2-(4-methoxyphenyl)-3-oxoisoindoline-4-carboxylate (53a) N O O O O Compound 52a (0.0150 g, 0.0500 mmol) was dissolved in MeOH (0.3 mL). To this solution, SOCl2 (0.200 mL) was added drop wise and heated at 140 C for 2 min. Methanol was evaporated at reduced pressure to afford 53a (0.0120 g, quantitative) as a brown solid, m.p. 196-198 C; 1H NMR (400 MHz, CD3OD )d 3.88 (s, 3H), 4.13 (s, 3H), 4.82 (s, 2H), 6.94 (d, J = 9.0 Hz, 2H), 7.56-7.65 (m, 3H), 7.71 (d, J = 9.0 Hz, 2H); 13C NMR (101 MHz, DMSO) d 51.04, 52.05, 55.72, 114.54, 122.00, 124.93, 128.07, 130.61, 131.10, 131.67, 132.46, 141.20, 157.06, 165.20, 168.04; MS m/z ( API-ES ): 297 (M+H)+ (100%), calculated for C17H15NO4 298.1074, found 298.1085. Ethyl 2-(4-methoxyphenyl)-3-oxoisoindoline-4-carboxylate (53b) N O O O O Compound 52a (0.0140 g, 0.0500 mmol) was dissolved in EtOH (0.30 mL). To this solution, SOCl2 (0.200 mL) was added drop wise and heated at 140 C for 2 min. Ethanol was evaporated at reduced pressure to afford 53b (0.0120 g, quantitative) as a brown

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113 solid; m.p >250 C; 1H NMR (400 MHz, CD3OD )d 1.42 (t, J = 7.2 Hz, 3H), 3.72 (s, 3H), 4.49 (q, J = 7.2 Hz, 2H), 4.82 (s, 2H), 6.94 (d, J = 9.0 Hz, 2H), 7.58-7.60 (m, 3H), 7.71 (d, J = 9.0 Hz, 2H); 13C NMR (101 MHz, DMSO) d 14.41, 51.04, 55.72, 62.18, 114.55, 121.98, 124.74, 127.90, 130.53, 131.59, 131.64, 132.54, 141.17, 157.02, 162.44, 167.58; MS m/z ( API-ES ): 297 (M+H)+ (100%), calculated for C18H17NO4 312.1230, found 312.1252. Sodium 2-(4-methoxyphenyl)-3-oxoisoindoline-4-carboxylate (54) N O O -O O Na+ Compound 52a (0.100 g, 0.350 mmol) was dissolved in NaOEt (0.20 M, 1.75 mL). This mixture was stirred at rt for 16 hrs. Ethanol was evaporated and dried to give 54 (0.100 g, quantitative) as a brown solid, m.p. >300 C; 1H NMR (400 MHz, DMSO )d 3.76 (s, 3H), 5.01 (s, 2H), 7.00 (d, J = 9.2 Hz, 2H), 7.65 (br s, 3H), 7.72 (d, J = 9.2 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) d 52.53, 56.00, 114.87, 123.42, 126.03, 129.28, 130.50, 131.88, 132.88, 133.47, 142.82, 157.61, 167.52, 167.95; MS m/z ( API-ES ): 284 (M+H)+ (100%), calculated for C16H13NO4 282.0772, found 228.0790. 2-(4-Methoxyphenyl)isoindolin-1-one (56) N O O Phthaldialdehyde 55 (0.400 g, 3.00 mmol) was dissolved in AcOH (8.60 mL). To this solution, p -anisidine (0.360 g, 3.00 mmol) was added and the reaction mixture was heated at 130 C for 10 min. Water (2 mL) was added and the AcOH-water was evaporated under reduced pressure. The aqueous layer was extracted three times with DCM (5.00 mL), washed with HCl (1.0 M, 5 mL), dried (Na2SO4) and evaporated. The crude product was purified by column chromatography using EtOAc-hexane (1:3, v/v). The product obtained from column chromatography was further recrystallised from EtOAc-hexane (1:5, v/v) to give 56 (0.0660 g, 9.2%) as a colourless solid; m.p. 133-134 C; 1H NMR (400 MHz, CDCl3) d 3.83 (s, 3H), 4.83 (s, 2H), 6.96 (d, J = 8.8 Hz, 2H),

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114 7.48-7.52 (m, 2H), 7.57-7.60 (m, 1H), 7.73 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 7.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 51.37, 55.71, 114.54, 121.68, 124.22, 128.51, 132.01, 132.86, 140.35, 156.82, 167.44; MS m/z ( API-ES ): 239 (M+H)+ (100%), calculated for C15H13NO2 240.1019, found 240.1010; 1-(4-Methoxyphenyl)pyrrolidin-2-one (59)149 O N NH O 2-Chloroethylisocyanate (0.0700 mL, 0.800 mmol) in THF (1 mL) was added to p anisidine (0.100 g, 0.800 mmol) and heated at 100 C for 10 min in the microwave reactor. The crude reaction mass was added dropwise to a solution of NaH (60% dispersion in paraffin oil, 0.0400 g, 0.960 mmol) in THF (0.2 mL). The reaction mixture was stirred at rt for 30 min. The aqueous layer was extracted with DCM (3 5 mL), dried (Na2SO4) and evaporated. The crude product was recrystallised from DCM (3 mL) to give 59 (0.0500 g, 35%) as brown crystals; m.p 214-215 C; 1H NMR (400 MHz, DMSO )d 3.60-3.85 (m, 4H), 6.69-6.94 (m, 2H), 7.41 (d, J = 7.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 37.34, 45.49, 55.85, 114.45, 119.37, 134.76, 154.91, 159.92; MS m/z ( API-ES ) 192 (M+H)+ (100%), calculated for C10H12N2O2 192.0893, found 192.0897. 1-(4-Methoxyphenyl)pyrrolidin-2-one (61a)153 O N O p -Anisidine (0.200 g, 1.60 mmol) was dissolved in CHCl3 (3 mL). To this solution, Na2HPO4 (0.46 g, 3.20 mmol) was added. After the aniline dissolved, 4-chloro butyryl chloride (0.180 mL, 1.60 mmol) was added and stirred at rt for 22 hrs. The crude reaction mass was filtered over cotton and the filtrate was evaporated to afford 4-chloroN -(4methoxyphenyl)butanamide. The resulting crude product was added dropwise to a solution of NaH (0.0840 g, 2.11 mmol) in THF (2 mL). After the addition was over, the

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115 product was extracted with DCM (3 5 mL), dried (Na2SO4) and evaporated to afford 61a (0.600 g, 49%) as a reddish solid, m.p. 111-112 C; 1H NMR (400 MHz, DMSO )d 2.09-2.17 (quintet, J = 7.2 Hz, 2H), 2.60 (t, J = 8.0 Hz, 2H), 3.79 (s, 3H), 3.84 (t, J = 7.2 Hz, 2H), 7.16 (d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 21.64, 23.74, 36.27, 53.36, 124.65, 133.07, 138.64, 140.81, 179.40; MS ( m/z) (API-ES) 191 (M+H)+ (100%), calculated for C11H13NO2 192.1019, found 192.1018. 1p -Tolylpyrrolidin-2-one (61b) N O p -Toluidine (0.200 g, 1.86 mmol) was dissolved in CHCl3 (3 mL). To this solution, Na2HPO4 (0.530 g, 3.73 mmol) was added. After the aniline dissolved, 4-chlorobutyryl chloride (0.210 mL, 1.86 mmol) was added and stirred at rt for 22 hrs. The crude reaction mass was filtered over cotton and the filtrate was evaporated to afford 4-chloroN p tolylbutanamide. The resulting crude product was added drop wise to a solution of NaH (0.0840 g, 2.11 mmol) in THF (2 mL). After the addition was over, the product was extracted three times with DCM (5 mL), dried (Na2SO4) and evaporated to afford 61b (0.600 g, 49%) as a reddish solid, m.p. 82-84 C; 1H NMR (400 MHz, CDCl3)d 2.09 (quintet, J = 7.4 Hz, 2H), 2.56 (t, J = 8.0 Hz, 2H), 3.80 (t, J = 7.4 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 21.68, 36.05, 52.01, 58.66, 117.77, 126.61, 136.29, 161.33, 179.34; MS ( m/z) (API-ES) 175 (M+H)+ (100%), calculated for C11H13NO 176.1070, found 176.1080. 7.5 General procedure for the synthesis of substituted cyclohexanecarboxylic acids 63a-h154 cis -Cyclohexane dicarboxylic anhydride was dissolved in CHCl3 (1 mL). To this solution, the aniline (1 Mol eq) was added and stirred at rt for 30 min. The slurry was filtered and the filtrate washed with CHCl3 (3 mL) and dried to give the pure product. (1S,2R) -2-(4-Methoxyphenylcarbamoyl)cyclohexanecarboxylic acid (63a)

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116 O NH O O OH This was prepared from p -anisidine (0.0800 g, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.150 g, 85%), m.p. 180-182 C; 1H NMR (400 MHz, CD3OD )d 1.44-1.49 (m, 2H), 1.79-1.83 (m, 2H), 2.74-2.76 (m, 2H), 2.92-2.93 (m, 2H), 3.75 (s, 3H), 6.85 (d, J = 9.01 Hz, 2H), 7.36 (d, J = 9.01 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 23.14, 24.64, 25.96, 28.44, 42.70, 43.15, 55.78, 114.31, 121.35, 133.41, 155.56, 172.79, 175.81; MS ( m/z) (API-ES) 277 (M+H)+ (100%), calculated for C15H19NO4 278.1387, found 278.1391. (1S,2R) -2-(2,4-Difluorophenylcarbamoyl)cyclohexanecarboxylic acid (63b) F NH O O OH F This was prepared from 2,4-difluoroaniline (0.0800 g, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.130 g, 69%), m.p. 140-142 C; 1H NMR (400 MHz, CD3OD )d 1.44-1.49 (m, 2H), 1.79-1.83 (m, 2H), 2.74-2.76 (m, 2H), 2.92-2.93 (m, 2H), 2.98 (q, J = 5.0 Hz, 1H), 3.15 (d, J = 5.0 Hz, 1H), 7.01-7.04 (m, 1H), 7.28 (dt, J = 2.8, 8.0 Hz, 1H), 7.61-7.68 (m, 1H), 9.47 (s, 1H); 13C NMR (101 MHz, CD3OD) d 23.07, 24.59, 25.94, 28.29, 42.70, 104.65 (dd, J = 2.0, 24.2 Hz), 111.50 (dd, J = 4.0, 18.18 Hz), 123.60 (dd, J = 4.0, 8.0 Hz), 126.87 (d, J = 9.0), 153.65, 156.11, 157.83, 160.25, 173.60, 175.70; MS ( m/z) (API-ES) 283 (M+H)+ (100%), calculated for C14H15F2NO3 284.1093, found 284.1096. (1S,2R) -2-(4-Chlorophenylcarbamoyl)cyclohexanecarboxylic acid (63c)

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117 Cl NH O O OH This was prepared from 4-chloroaniline (0.0820 g, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.170 g, 93%), m.p. 190-192 C; 1H NMR (400 MHz, CD3OD )d 1.44-1.49 (m, 2H), 1.79-1.83 (m, 2H), 2.74-2.76 (m, 2H), 2.92-2.93 (m, 2H), 2.90 (q, J = 5.0 Hz, 1H), 3.15 (d, J = 5.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 9.83 (s, 1H); 13C NMR (101 MHz, CD3OD) d 23.03, 24.65, 25.89, 28.30, 42.69, 43.25, 121.30, 126.95, 129.05, 139.17, 173.46, 175.73; MS ( m/z) (API-ES) 281 (M+H)+ (100%), calculated for C14H16NO3Cl 282.0891, found 282.0897. (1S,2R) -2-(4-Bromophenylcarbamoyl)cyclohexanecarboxylic acid (63d) Br NH O O OH This was prepared from 4-bromoaniline (0.110 g, 0.650 mmol) in a similar manner as described above, (Colorless solid, 0.160 g, 74%), m.p. 196-198 C; 1H NMR (400 MHz, CD3OD )d 1.44-1.49 (m, 2H), 1.79-1.83 (m, 2H), 2.74-2.76 (m, 2H), 2.92-2.93 (m, 2H), 2.90 (q, J = 5.0 Hz, 1H), 3.15 (d, J = 5.0 Hz, 1H), 7.43 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H), 9.83 (s, 1H); 13C NMR (101 MHz, CD3OD) d 23.03, 24.66, 25.88, 28.29, 31.33, 42.67, 43.26, 114.90, 121.70, 131.98, 139.59, 173.49, 175.73; MS ( m/z) (API-ES) 325 (M+H)+ (100%), calculated for C14H16BrNO3 326.0386, found 326.0387. (1S,2R) -2-( p -Tolylcarbamoyl)cyclohexanecarboxylic acid (63e) NH O O OH This was prepared form p-toluidine (0.070 g, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.150 g, 87%), m.p. 186-188 C; 1H NMR (400 MHz,

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118 CD3OD )d 1.44-1.49 (m, 2H), 1.79-1.82 (m, 2H), 2.21 (s, 3H), 2.73-2.76 (m, 2H), 2.912.92 (m, 2H), 2.90 (q, J = 5.0 Hz, 1H), 3.15 (d, J = 5.0 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 9.58 (s, 1H); 13C NMR (101 MHz, CD3OD) d 21.06, 23.11, 24.68, 25.93, 28.47, 42.71, 43.23, 119.85, 129.53, 132.21, 137.74, 173.06, 175.81; MS ( m/z) (API-ES) 261 (M+H)+ (100%), calculated for C15H19NO3 262.1438, found 262.1439. (1S,2R) -2-(4-Isopropylphenylcarbamoyl)cyclohexanecarboxylic acid (63f) NH O O OH This was prepared from 4-isopropylaniline (0.0870 g, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.160 g, 83%), m.p. 176-178 C; 1H NMR (400 MHz, CD3OD) d 1.15 (d, J = 7.2 Hz, 6H), 1.45-1.46 (m, 2H), 1.78-1.80 (m, 2H), 2.73-2.74 (m, 2H), 2.78-2.80 (m, 1H), 2.91-2.93 (m, 2H), 7.11 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 23.09, 24.66, 25.92, 28.46, 33.53, 42.70, 43.19, 119.88, 126.84, 138.02, 143.43, 173.06, 175.78; MS ( m/z) (API-ES) 289 (M+H)+ (100%), calculated for C17H23NO3 290.1751, found 290.1754. (1S,2R) -2-(4-(Trifluoromethyl)phenylcarbamoyl)cyclohexanecarboxylic acid (63g) CF3 NH O O OH This was prepared from 4-trifluoromethylaniline (0.080 mL, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.200 g, 98%), m.p. 176-182 C; 1H NMR (400 MHz, CD3OD )d 1.43-1.45 (m, 2H), 1.79-1.82 (m, 2H), 2.73-2.75 (m, 2H), 2.912.93 (m, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 22.96, 24.63, 25.86, 28.20, 39.71 (t, J = 21.2 Hz), 40.60 (d, J = 65.6 Hz), 79.81, 119.53, 123.00 (m), 123.95, 129.13, 143.81, 173.98, 175.71; MS ( m/z) (API-ES)

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119 315 (M+H)+ (100%), calculated for C15H16F3NO3 316.1155, found 316.1165. (1S,2R) -2-(4-(Ethoxycarbonyl)phenylcarbamoyl)cyclohexanecarboxylic acid (63h) NH O O OH O O This was prepared form 4-(aminoethyl) benzoate (0.110 g, 0.650 mmol) in a similar manner as described above, (colorless solid, 0.170 g, 84%), m.p. 158-160 C; 1H NMR (400 MHz, CD3OD) d 1.30 (t, J = 7.2 Hz, 3H), 1.44-1.46 (m, 2H), 1.78-1.80 (m, 2H), 2.73-2.75 (m, 2H), 2.91-2.93 (m, 2H), 4.28 (q, J = 7.2 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 14.87, 22.96, 24.67, 25.82, 28.26, 42.66, 43.32, 61.02, 119.01, 124.39, 130.76, 144.62, 166.03, 173.94, 175.71; MS ( m/z) (API-ES) 319 (M+H)+ (100%), calculated for C17H21NO5 320.1493, found 320.1497. 7.6 General procedure for the synthesis of substituted oxobutanoic acids 65a-g155 The aniline was dissolved in CHCl3 (2 mL). To this solution, succinic anhydride (1 eq) was added and stirred at rt for 30 minutes. After 30 min, the reaction mixture was filtered, the residue washed with CHCl3 and dried to give the pure product. 4-(4-Methoxyphenylamino)-4-oxobutanoic acid (65a) HN O OH O O This was prepared from p -anisidine (0.120 g, 1.00 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.180 g, 81%), m.p. 162164 C; 1H NMR (400 MHz, CD3OD )d 2.63 (dd, J = 3.6, 5.6 Hz, 4H), 3.76 (s, 3H), 6.84 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.8 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 28.96,

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120 31.02, 54.66, 113.72, 121.82, 121.93, 131.65, 156.62, 171.41, 175.20; MS ( m/z) (APIES) 223 (M+H)+ (100%), calculated for C11H13NO4 222.0772, found 222.0776. 4-(2,4-Difluorophenylamino)-4-oxobutanoic acid (65b) HN F OH O O F This was prepared from 2,4-difluoroaniline (0.130 g, 1.00 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.240 g, quantitative), m.p. 158-160 C; 1H NMR (400 MHz, CD3OD )d 2.65 (d, J = 5.8 Hz, 2H), 2.68 (d, J = 5.8 Hz, 2H), 6.90-6.94 (m, 1H), 6.98 (dt, J = 2.8, 9.6 Hz, 1H), 7.77-7.83 (m, 1H); 13C NMR (101 MHz, CD3OD) d 28.75, 30.63, 103.57 (dd, J = 2.3, 24.2 Hz, 1C), 110.60 (dd, J = 3.8, 18.4 Hz, 1C); 122.30 (dd, J = 3.8, 8.2 Hz, 1C), 125.97 (dd, J = 2.6, 6.8 Hz, 1C), 153.71 (d, J = 12.1 Hz, 1C), 156.18 (d, J = 12.0 Hz, 1C), 158.662 (d, J = 11.4 Hz, 1C), 161.06 (d, J = 11.4 Hz, 1C); MS ( m/z) (API-ES) 229 (M+H)+ (100%), calculated for C10H9F2NO3 228.0478, found 228.0493. 4-(4-Chlorophenylamino)-4-oxobutanoic acid (65c) HN Cl OH O O This was prepared using 4-chloroaniline (0.127 g, 1.00 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.210 g, 92%), m.p. 168170 C; 1H NMR (400 MHz, CD3OD )d 2.65 (s, 4H), 7.27 (d, J = 8.4 Hz, 2H), 7.53 (d J = 8.4 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 28.72, 31.12, 121.19, 121.29, 128.52, 128.57, 137.59, 171.66, 175.11; MS ( m/z) (API-ES) 227 (M+H)+ (100%), calculated for C10H10ClNO3 226.0276, found 226.0278.

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121 4-(4-Bromophenylamino)-4-oxobutanoic acid (65d) HN Br OH O O This was prepared from 4-bromoaniline (0.170 g, 1.00 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.260 g, 97%), m.p. 194196 C; 1H NMR (400 MHz, CD3OD )d 2.65 (s, 4H), 7.42 (d, J = 9.0 Hz), 7.50 (d, J = 9.0 Hz); 13C NMR (101 MHz, CD3OD) d 28.71, 31.15, 116.05, 121.51, 121.61, 131.54, 138.05, 171.67, 175.13; MS ( m/z) (API-ES) 270 (M+H)+ (100%), calculated for C10H10BrNO3 269.9771, found 269.9771. 4-Oxo-4-( p -tolylamino)butanoic acid (65e) HN OH O O This was prepared form p -toluidine (0.100 g, 1.00 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.190 g, 91%), m.p. 180182 C; 1H NMR (400 MHz, CD3OD )d 2.28 (s, 3H), 2.60 (s, 4H), 7.09 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 19.74, 28.89, 31.12, 120.12, 129.03, 135.58, 136.07, 171.50, 175.18; MS ( m/z) (API-ES) 207 (M+H)+ (100%), calculated for C11H13NO3 206.0823, found 206.0830. 4-(4-Isopropylphenylamino)-4-oxobutanoic acid (65f) HN OH O O This was prepared from 4-isopropylaniline (0.142 g, 1.00 mmol) in a similar manner as

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122 described above. The product was collected as a colorless solid (0.230 g, 98%), m.p. 156158 C; 1H NMR (400 MHz, CD3OD )d 1.22 (d, J = 6.8 Hz, 6H), 2.64 (s, 4H), 2.82 (heptet, J = 6.8 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 23.32, 28.90, 31.12, 33.67, 120.20, 126.40, 136.35, 144.74, 171.52, 175.17; MS ( m/z) (API-ES) 235 (M+H)+ (100%), calculated for C13H17NO3 234.36, found 234.1136. 4-(4-(Ethoxycarbonyl)phenylamino)-4-oxobutanoic acid (65g) HN OH O O O O This was prepared from ethyl 4-aminobenzoate (0.165 g, 1.00 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.210 g, 82%), m.p. 159-161 C; 1H NMR (400 MHz, CD3OD )d 1.37 (t, J = 7.2 Hz, 3H), 2.66 (t, J = 3.6 Hz, 4H), 4.30 (q, J = 7.2 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CD3OD) d 13.47, 28.67, 31.29, 60.79, 118.80, 118.89, 125.20, 130.29, 143.31, 166.55, 171.91, 175.13; MS ( m/z) (API-ES) 265 (M+H)+ (100%), calculated for C13H15NO5 264.0878, found 264.0881. Methyl 2-methyl-6-nitrobenzoate (67) NO2 O O 2-Methyl 6-nitro benzoic acid (0.500 g, 3.00 mmol) was dissolved in acetone (10 mL). To the resulting solution, K2CO3 (2.00 g, 15.4 mmol) and iodomethane (0.900 mL, 15.4 mmol) was added and refluxed for 15 hrs. The reaction mass was filtered after cooling to rt and extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to afford 67 (0.530 g, 90%) as a yellow solid. The crude product was taken to the next step without further purification; m.p 50-51 C; 1H NMR (400 MHz, CDCl3) d 2.41 (s, 2H), 3.96 (s, 3H), 7.50 (t, J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H).

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123 Methyl 2-(bromomethyl)-6-nitrobenzoate (68)156 NO 2 O O B r Methyl 2-methyl-6-nitrobenzoate 67 (0.500 g, 2.56 mmol) was dissolved in CCl4 (5 mL). to this solution, NBS (0.431 g, 2.82 mmol) and catalytic amount of AIBN were added and refluxed for 40 hrs. The crude mass was extracted with DCM (3 5 mL), dried (Na2SO4) and evaporated to afford 68 (0.270 g, 39%) as a yellow solid in 39% yield. The crude product was taken to the next step without further purification; m.p 50-51 C; 1H NMR (400 MHz, CDCl3) d 3.97 (s, 3H), 4.56 (s, 2H), 7.58 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H). 2-(4-Methoxyphenyl)-7-nitroisoindolin-1-one (69) NO2 N O O Methyl 2-(bromomethyl)-6-nitrobenzoate 68 (0.0500 g, 0.180 mmol) was dissolved in DMF (0.1 mL). To this solution, p -anisidine (0.0220 g, 0.180 mmol) was added heated at 150 C for 15 min in the microwave reactor. After the completion of the reaction, DMF was evaporated from the reaction mixture and the crude product was recrystallized from DCM to afford 69 (0.02 g, 44%) as a yellow crystalline solid; m.p 226-228 C; 1H NMR (400 MHz, DMSO )d ppm 3.83 (s, 3H), 4.89 (s, 2H), 6.96 (d, J = 9.2 Hz, 2H), 7.70-7.75 (m, 5H); 13C NMR (101 MHz, DMSO) d 50.67, 55.73, 114.62, 122.00, 123.16, 124.38, 126.48, 130.16, 131.18, 131.89, 132.57, 142.71, 157.37; MS ( m/z) (API-ES) 285 (M+H)+ (100%), calculated for C15H12N2O4 285.0870, found 285.0877. 7.7 General procedure for the synthesis of urea derivatives 78a-e: 2-Isocyanatobenzoic acid (1 mol eq) was dissolved in THF (4 mL). To this solution, the aniline (1 mol eq) was added and heated at 100 C for 10 min in the microwave reactor. The THF in the resulting mixture was evaporated to dryness to get the product.

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124 2-(3-(2-(Methoxycarbonyl)phenyl)ureido)benzoic acid (77a) NH O OH O NH O O This was prepared from 2-aminobenzoic acid (0.300 g, 2.20 mmol) and 2isocyanatobenzoic acid (0.380 g, 2.20 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.600 g, 88%); This was taken to the next step without further purification. 1H NMR (400 MHz, CD3OD) d 490 (s, 3H), 7.06-7.10 (m, 2H), 7.52-7.57 (m, 2H), 8.01 (dd, J = 1.2, 8.0 Hz, 2H), 8.05 (dd, J = 1.2, 8.0 Hz, 2H), 8.37 (dd, J = 0.4, 8.8, 2H), 8.41 (dd, J = 0.6, 8.4 Hz, 2H). 3-(3-(2-(Methoxycarbonyl)phenyl)ureido)benzoic acid (77b) NH O NH O O C O O H This was prepared from 3-aminobenzoic acid (0.300 g, 2.20 mmol) and 2isocyanatobenzoic acid (0.380 g, 2.20 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.670 g, 97%); This was taken to the next step without further purification. 1H NMR (400 MHz, CD3OD) d 3.94 (s, 3H), 7.04 (dt, J = 1.2, 8.0 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.51 (dt, J = 1.2, 8.0 Hz, 1H), 7.68 (td, J = 1.2, 8.0 Hz, 1H), 7.74-7.77 (m, 1H), 8.03 (dd, J = 1.6, 8.0 Hz, 1H), 8.15 (t, J = 1.6 Hz, 1H), 8.36 (dd, J = 0.8, 8.8 Hz, 1H). 4-(3-(2-(Methoxycarbonyl)phenyl)ureido)benzoic acid (77c) NH O NH O O H O O C This was prepared from 4-aminobenzoic acid (0.300 g, 2.20 mmol) and 2isocyanatobenzoic acid (0.380 g, 2.20 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.390 g, 57%); This was taken to the next step without further purification. 1H NMR (400 MHz, CD3OD) d 3.94 (s, 3H), 7.05 (dt, J =

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125 1.2, 7.8 Hz, 1H), 7.52 (dt, J = 1.8, 9.2 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 8.01 (dd, J = 1.6, 8.0 Hz, 1H), 8.36 (dd, J = 1.2, 8.6 Hz). 2-Hydroxy-4-(3-(2-(methoxycarbonyl)phenyl)ureido)benzoic acid (77d) NH O NH O O O H HOOC This was prepared from 4-aminosalicylic acid (0.300 g, 2.20 mmol) and 2isocyanatobenzoic acid (0.350 g, 2.20 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.640 g, 98%); This was taken to the next step without further purification. 1H NMR (400 MHz, CD3OD) d 3.94 (s, 3H), 6.97 (dd, J = 2.0, 4.8 Hz, 1H), 7.05 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.52 (dt, J = 1.6, 8.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 8.01 (dd, J = 1.6, 8.2 Hz, 1H), 8.35 (d, J = 8.2 Hz, 1H). 2-Hydroxy-5-(3-(2-(methoxycarbonyl)phenyl)ureido)benzoic acid (77e) NH O NH O O C O O H HO This was prepared from 3-aminosalicylic acid (0.300 g, 2.20 mmol) and 2isocyanatobenzoic acid (0.350 g, 2.20 mmol) in a similar manner as described above. The product was collected as a colorless solid (0.300 g, 46%); This was taken to the next step without further purification. 1H NMR (400 MHz, CD3OD) d 3.90 (s, 3H), 6.88 (d, J = 9.2 Hz, 1H0, 7.02 (t, J = 7.6 Hz, 1H), 7.50-7.55 (m, 2H), 7.96 (d, J = 2.8 Hz, 1H), 7.99 (dd, J = 1.2, 8.0 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 10.30 (s, 1H). 2,2'-Carbonylbis(azanediyl)dibenzoic acid (78a) NH O OH O NH O OH

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126 2-(3-(2-(Methoxycarbonyl)phenyl)ureido)benzoic acid (77a) (0.100 g, 0.0300 mmol) was dissolved in MeOH (3 mL). To this solution, NaOH (1M, 1 mL) was added and heated at 60 C for 10 min in the microwave reactor. The resulting solution was acidified to pH 3.0 (HCl, 1.0 M), filtered, the residue washed with water and dried to give 78a as a white solid (0.0600 g, 73%), m.p. > 300 C; 1H NMR (400 MHz, CD3OD) d 7.18-7.32 (m, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.53-7.80 (m, 3H), 8.04 (d, J = 7.9 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 114.91, 114.94, 115.85, 115.92, 123.21, 128.24, 131.68, 135.95, 140.40, 140.54, 150.69, 150.78, 162.94, 162.96, 166.46; MS m/z ( APIES ): 283 (M-17) (100%), calculated for C15H12N2O5 300.0980, found 300.0986. 2-(3-(3-Carboxyphenyl)ureido)benzoic acid (78b) NH O NH O OH OH O This was prepared from compound 77b (0.100 g, 0.0300 mmol) in a similar manner in a similar manner as described in the preparation of 78a The product was obtained as an off-white solid, (0.0600 g, 73%), m.p. >300 C; 1H NMR (400 MHz, CD3OD )d 7.247.30 (m, 2H), 7.56 (qd, J = 1.2, 0.4 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.95 (t, J = 1.6Hz, 1H), 8.08 (dd, J = 1.2, 6.4 Hz, 1H), 8.13 (td, J = 1.6, 4.8 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 115.04, 115.95, 123.19, 128.24, 129.68, 129.80, 130.86, 132.30, 132.30, 134.40, 135.90, 136.71, 140.53, 150.82, 162.91, 167.41; MS m/z ( API-ES ): 283 (M-17) (100%), calculated for C15H12N2O4 300.0980, found 300.0982. 2-(3-(4-Carboxyphenyl)ureido)benzoic acid (78c) NH O NH O OH O OH This was prepared from compound 77c (0.100 g, 0.0300 mmol) in a similar manner in a similar manner as described in the preparation of 78a The product was obtained as an off-white solid, (0.0750 g, 79%), m.p. > 300 C; 1H NMR (400 MHz, CD3OD )d 7.19-

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127 7.35 (m, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.65-7.78 (m, 1H), 8.01-8.23 (m, 3H); 13C NMR (101 MHz, CDCl3) d 114.95, 115.97, 123.25, 128.23, 130.17, 130.47, 131.25, 135.97, 140.46, 140.51, 150.61, 162.74, 167.52; MS m/z ( API-ES ): 283 (M-17) (100%), calculated for C15H12N2O4 300.0980, found 300.0983. 4-(3-(2-Carboxyphenyl)ureido)-2-hydroxybenzoic acid (78d) NH O NH O OH HO OH O This was prepared from compound 77d (0.100 g, 0.0300 mmol) in a similar manner in a similar manner as described in the preparation of 78a The product was obtained as a grayish solid, (0.0840 g, 88%), m.p. 298-300 C; 1H NMR (400 MHz, CD3OD )d 6.87 (dd, J = 2, 6.4 Hz, 1H), 6.94 (d, J = 1.6 Hz, 1H), 7.22-7.29 (m, 2H), 7.73 (dd, J = 1.2, 7.2 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 8.07 (dd, J = 0.8, 7.2 Hz); 13C NMR (101 MHz, CDCl3) d 113.54, 114.94, 115.94, 118.83, 121.00, 123.22, 128.22, 131.15, 135.94, 140.49, 142.69, 150.42, 162.00, 162.56, 172.13; MS m/z ( API-ES ): found 299 (M-17) (100%), calculated for C15H12N2O6 316.0945, found 316.0928. 5-(3-(2-Carboxyphenyl)ureido)-2-hydroxybenzoic acid (78e) NH O NH O OH O OH O H This was prepared from compound 77e in a similar manner as described in the preparation for 78a. The product was collected as an orange solid, (0.0570 g, 60%), m.p. 298-300 C; 1H NMR (400 MHz, CD3OD )d 6.94 (d, J = 8.77 Hz, 1H), 7.08-7.33 (m, 3H), 7.54-7.74 (m, 2H), 7.97 (d, J = 8.01 Hz, 1H); 13C NMR (101 MHz, CDCl3) d 113.84, 115.02, 115.88, 118.10, 123.12, 127.42, 128.24, 131.43, 135.80, 137.05, 140.47, 151.01, 161.36, 172.07; MS m/z ( API-ES ): found 299 (M-17) (100%), calculated for C15H12N2O6 316.0945, found 316.0933.

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128 2,3-Dioxoindoline-5-sulfonyl chloride (80)105 N H O O S O O Cl 5-Isatin sulfonic acid sodium salt dihydrate (10.0 g, 35.1 mmol) was dissolved in tetramethylene sulfone (50 mL). To this solution, POCl3 (16.0 ml, 177.0 mmol) was added and the reaction mixture was heated at 60 C for 3 hrs. After 3 hrs, the reaction mixture was cooled to 0 C and water (150 mL) was added. The greenish yellow residue was filtered, re-dissolved in EtOAc (75 mL) and filtered. Hexane (100 mL) was added to the filtrate, the yellow solid was filtered, the residue washed with hexane to give 80 (5.97 g, 56%) as yellow solid; m.p 197-198 C (lit. 200-202 C); 1H NMR (400 MHz, DMSO) d 6.82 (d, J = 8.8 Hz, 1H), 7.55 (s, 1H), 7.76 (d, J = 8.8 Hz, 1H), 11.08 (s, 1H). 5-(4-(4-Fluorophenyl)piperazin-1-ylsulfonyl)indoline-2,3-dione (81a) N H O O S O O N N F 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.330 mmol) was dissolved in THF (3 mL). To this solution, 1-(4-fluorophenyl)piperazine (0.0770 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81a (0.130 g, 88%) as a yellow solid. The crude product was used as such for the formation of 82a without further purification; 1H NMR (400 MHz, DMSO) d 3.00 (br s, 4H), 3.13 (br s, 4H). 6.89 (dd, J = 4.4, 9.0 Hz, 2H), 7.00 (t, J = 9.0 Hz, 2H), 7.10 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.91 (dd, J = 1.6, 8.4 Hz); MS m/z (API-ES) 388 (M+H)+, calculated for C18H16FN3O4S 388.0773, found 388.0793. 3-IminoN -(4-morpholinophenyl)-2-oxoindoline-5-sulfonamide (81b)

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129 NH O O S O O HN N O 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, N -(4-morpholinophenyl)-2,3-dioxoindoline-5-sulfonami de (0.0760 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL ) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81b (0.240 g, 88%) as a yellow solid. The crude produc t was used as such for the formation of 82b without further purification. N -(2-Morpholinoethyl)-2,3-dioxoindoline-5-sulfonamid e (81c) NH O O S O O HN N O 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 2-morpholinoethanamine (0.05 60 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81c (0.107 g, 95%) as a reddish solid. The crude product was used as such for the formation of 82c without further purification. 2,3-DioxoN -(1H-tetrazol-5-yl)indoline-5-sulfonamide (81d) NH O O S O O HN N N N NH 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1H-tetrazol-5-amine (0.040 g 0.430 mmol) was added. To the

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130 resulting mixture, DIPEA (0.100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was add ed to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81d (0.092 g, 98%) as a yellow solid. The crude product was used as such fo r the formation of 82d without further purification. N -(Isoxazol-3-yl)-2,3-dioxoindoline-5-sulfonamide (8 1e) NH O O S O O HN O N 2,3-Dioxoindoline-5-sulfonyl chloride (0.050 g, 0.1 60 mmol) was dissolved in THF (3 mL). To this solution, isoxazol-3-amine (0.0140 g, 0.180 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was add ed to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81e (0.21 g, 22%) as a yellow solid. The crude product was used as such fo r the formation of 82e without further purification. 5-(4-(Benzo [d][1,3] dioxol-5-ylmethyl)piperazin-1-ylsulfonyl)indoline-2 ,3-dione (81f) N H O O S O O N N O O 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-(benzo[ d ][ 1,3 ]dioxol-5-ylmethyl)piperazine (0.0900 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0 .100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL ) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81f (0.141 g, quantitative) as a yellow solid. The cru de product was used as such for the formation of 82f without further purification. 5-(4-(3-Chlorophenyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione (81g)

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131 NH O O S O O N N Cl 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-(3-chlorophenyl)piperazine (0.0760 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mm ol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81g (0.0320 g, 53%) as a yellow solid. The crude product was used as such for the formation of 82g without further purification. 5-(4-(3-(Trifluoromethyl)phenyl)piperazin-1-ylsulfo nyl)indoline-2,3-dione (81h) NH O O S O O N N CF3 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-(3-(trifluoromethyl)phenyl )piperazine (0.114 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 m L, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL ) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81h (0.145 g, quantitative) as a yellow solid. The cru de product was used as such for the formation of 82h without further purification. 5-(4-(3,4-Dichlorophenyl)piperazin-1-ylsulfonyl)ind oline-2,3-dione (81i) NH O O S O O N N Cl Cl 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3

PAGE 144

132 mL). To this solution, 1-(3,4-dichlorophenyl)pipera zine (0.0760 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0 .660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL ) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81i (0.0310 g, 51%) as a yellow solid. The crude produc t was used as such for the formation of 82i without further purification. 5-(4-(2,3-Dichlorophenyl)piperazin-1-ylsulfonyl)ind oline-2,3-dione (81j) NH O O S O O N N Cl Cl 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-(2,3-dichlorophenyl)pipera zine (0.0760 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0 .660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL ) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81j (0.010 g, 30%) as a yellow solid. The crude product was used as such for the formation of 82j without further purification. 5-(4-(3-Methoxyphenyl)piperazin-1-ylsulfonyl)indoli ne-2,3-dione (81k) NH O O S O O N N O 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-(3-methoxyphenyl)piperazin e (0.0760 g, 0.430 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0 .660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL ) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81k

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133 (0.0480 g, 80%) as a yellow solid. The crude produc t was used as such for the formation of 82k without further purification. 5-(4-(3-Chlorobenzyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione (81l) NH O O S O O N N Cl 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-(3-chlorobenzyl)piperazine (0.0900 g, 0.420 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mm ol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was added to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81l (0.138 g, quantitative) as a yellow solid. The crude product was used as such for the formation of 82l without further purification. 5-(4-cyclohexylpiperazin-1-ylsulfonyl)indoline-2,3dione (81m) NH O O S O O N N 2,3-Dioxoindoline-5-sulfonyl chloride (0.100 g, 0.3 30 mmol) was dissolved in THF (3 mL). To this solution, 1-cyclohexylpiperazine (0.07 00 g, 0.415 mmol) was added. To the resulting mixture, DIPEA (0.100 mL, 0.660 mmol) was added and heated at 60 C for 10 min. in the microwave reactor. Water (4 mL) was add ed to the reaction mixture, extracted with EtOAc (3 5 mL), dried (Na2SO4) and evaporated to give 81m (0.123 g, quantitative) as a yellow solid. The crude product was used as such for the formation of 82m without further purification. (Z)-4-(2-(5-(4-(4-Fluorophenyl)piperazin-1-ylsulfon yl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82a)

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134 NH N O S O O N NH O OH N F 5-(4-(4-Fluorophenyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81a ) (0.100 g, 0.220 mmol) was dissolved in EtOH (5 mL). To this solutio n 4-hydrazinobenzoic acid (0.0400 g, 0.250 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82a (0.044 g, 34%) as a yellow solid, m.p. 290 C, deco mposed; 1H NMR (400 MHz, DMSO) d 3.00-3.04 (m, 4H), 3.14-3.16 (m, 4H), 6.92-6.93 (m 2H), 7.01 (t, J = 7.2, 9.2 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.67 (dd, J = 1.6, 8.0 Hz, 1H), 7.87 (d, J = 1.6 Hz, 1H), 7.92 (d, J = 8.8 Hz, 2H), 11.57 (s, 1H), 12.80 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.47, 49.54, 111.70, 114.97, 115.97, 116.19, 118. 77, 122.26, 125.88, 128.40, 128.73, 129.61, 131.72, 144 .56, 146.48, 163.62, 167.55; MS m/z (ESI-ve) 522 (M-H)(100%), calculated for C25H22FN5O5S 522.1253, found 522.1253. (Z)-4-(2-(5-( N -(4-Morpholinophenyl)sulfamoyl)-2-oxoindolin-3-ylid ene)hydrazinyl) benzoic acid (82b) NH N O S O O NH O OH HN N O 3-IminoN -(4-morpholinophenyl)-2-oxoindoline-5-sulfonamide ( 81b ) (0.100 g, 0.220 mmol) was dissolved in EtOH (5 mL). To this solutio n 4-hydrazinobenzoic acid (0.040 g, 0.250 mmol) was added. To the resulting mixture, HC l (1.0 M, 2 drops) was added and

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135 heated at 120 C for 15 min in the microwave reactor. After the reaction, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82b (0.0380 g, 29%) as a yellow solid, m.p. >300 C; 1H NMR (400 MHz, DMSO) d 2.95 (t, J = 4.8 Hz, 4H), 3.63 (t, J = 4.8 Hz, 4H), 6.79 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 1.6 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 9.71 (s, 1H), 11.44 (s, 1H), 12.74 (s, 1H); 13C NMR (101 MHz, DMSO) d 49.84, 66.36, 114.80, 117.11, 118.00, 121.92, 123.42, 123.45, 125.79, 128.86, 131.77, 133.75, 143.93, 146.52, 163.58, 167.54; MS m/z (ESI-ve) 520 (M-H)(100%), calculated for C25H23N5O6S 520.1296, found 520.1301. (Z)-4-(2-(5-( N -(2-Morpholinoethyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl) benzoic acid (82c) N H N O S O O HN NH O OH N O N -(2-Morpholinoethyl)-2,3-dioxoindoline-5-sulfonamide ( 81c ) (0.100 g, 0.220 mmol) was dissolved in EtOH (5 mL). To this solution 4-hydrazinobenzoic acid (0.040 g, 0.250 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the reaction, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82c (0.023 g, 17%) as a yellow solid, m.p. 243-245 C; 1H NMR (400 MHz, DMSO) d 3.11 (m, 4H), 3.17 (br s, 2H), 3.35 (d, J = 4.2 Hz, 2H), 3.64 (t, J = 8.0 Hz, 2H), 3.93 (d, J = 12.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 4.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.97 (s, 1H), 11.52 (s, 1H), 12.85 (s, 1H); 13C NMR (101 MHz, DMSO) d 51.92, 55.09, 55.86, 63.76, 111.52, 114.85, 118.04, 122.19, 125.82, 128.72, 128.87, 131.75, 133.56, 144.09, 146.55, 163.65, 167.54; MS m/z (ESI+ve) 474 (M+H)+ (100%), calculated for C21H23N5O6S 474.1441, found 474.1454.

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136 (Z)-4-(2-(5-( N -1H-Tetrazol-5-ylsulfamoyl)-2-oxoindolin-3-ylidene) hydrazinyl) benzoic acid (82d) NH N O S O O HN NH O OH N N N NH 2,3-DioxoN -(1H-tetrazol-5-yl)indoline-5-sulfonamide ( 81d ) (0.0610 g, 0.160 mmol) was dissolved in EtOH (2 mL). To this solution 4-hydraz inobenzoic acid (0.027 g, 0.180 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 m L) and dried to give 82d (0.00800 g, 10%) as a yellow solid, m.p. 270 C, decomposed; 1H NMR (400 MHz, DMSO) d 7.05 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.81 (dd, J = 1.6, 8.4 Hz, 1H), 8.04 (d, J = 8.8 Hz, 2H), 8.16 (d, J = 1.6 Hz, 1H); 13C NMR (101 MHz, DMSO) d 111.58, 114.88, 118.20, 122.05, 125.83, 128.72, 128.75, 128.86, 131 .79, 144.56, 146.49, 146.51, 156.82, 163.63, 167.54, 167.56, 179.10; MS m/z (API-ES) 427 (M-H)(100%), calculated for C16H12N8O5S 427.0578, found 427.0594. (Z)-4-(2-(5-( N -Isoxazol-3-ylsulfamoyl)-2-oxoindolin-3-ylidene)hyd razinyl)benzoic acid (82e) NH N O S O O HN NH O OH O N N -(Isoxazol-3-yl)-2,3-dioxoindoline-5-sulfonamide ( 81e ) (0.0700 g, 0.190 mmol) was dissolved in EtOH (1 mL). To this solution 4-hydraz inobenzoic acid (0.0330 g, 0.219 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 m L) and dried to give 82e (0.021 g,

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137 22%) as a yellow solid, m.p. 295 C, decomposed; 1H NMR (400 MHz, DMSO) d 6.44 (s, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.73 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.99 (d, J = 1.6 Hz, 1H), 8.71 (s, 1H), 11.53 (s, 1H), 12.76 (s, 1H); 13C NMR (101 MHz, DMSO) d 98.99, 111.46, 114.89, 118.05, 122.16, 125.84, 128.71, 128.84, 131.78, 133.17, 144.53, 146.49, 157.71, 161.62, 161.67, 163.61, 167.56; MS m/z (API-ES) 426 (M-H)(100%), calculated for C18H13N5O6S 426.0513, found 426.0511. (Z)-4-(2-(5-(4-(Benzo[ d ][ 1,3 ]dioxol-5-ylmethyl)piperazin-1-ylsulfonyl)-2-oxoindolin3-ylidene)hydrazinyl)benzoic acid (82f) N H N O S O O N NH O OH N O O 5-(4-(Benzo[ d ][ 1,3 ]dioxol-5-ylmethyl)piperazin-1-ylsulfonyl)indoline-2,3-dione ( 81f ) (0.141 g, 0.330 mmol) was dissolved in EtOH (5 mL). To this solution 4hydrazinobenzoic acid (0.0500 g, 0.410 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the reaction, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82f (0.0930 g, 46%) as a yellow solid, m.p. >300 C; 1H NMR (400 MHz, DMSO) d 2.64 (br s, 2H), 3.12 (be s, 2H), 3.75 (br s, 2H), 4.20 (br s, 2H), 6.01 (be s, 2H), 6.91 (d, J = 12.0 Hz, 2H), 7.03 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.84 (s, 1H), 7.92 (8.8 Hz, 2H), 11.62 (s, 1H), 12.81 (s, 1H); 13C NMR (101 MHz, DMSO) d 101.98, 102.08, 102.20, 108.96, 112.88, 122.28, 125.89, 128.25, 128.50, 131.70, 139.12, 144.71, 146.42, 148.02, 148.75, 163.54, 167.55; MS m/z (ESI+ve) 564 (M+H)+ (100%), calculated for C27H25N5O7S 564.1548, found 564.1547. (Z)-4-(2-(5-(4-(3-Chlorophenyl)piperazin-1-ylsulfonyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82g)

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138 NH N O S O O N NH O OH N C l 5-(4-(3-Chlorophenyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81g ) (0.0700 g, 0.170 mmol) was dissolved in EtOH (2 mL). To this solutio n 4-hydrazinobenzoic acid (0.0250 g, 0.160 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82g (0.044 g, 34%) as a yellow solid, m.p. 285 C, deco mposed; 1H NMR (400 MHz, DMSO) d 2.99 (br s, 4H), 3.25 (br s, 4H), 6.78 (d, J = 8.8 Hz, 1H), 6.82 (d, J = 10.8 Hz, 1H), 6.90 (sm 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.87 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 11.55 (s, 1H), 12.80 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.40, 53.43, 111.65, 111.72, 114.97, 115.92, 119. 47, 125.84, 128.22, 128.29, 128.70, 131.72, 144.50, 144.53, 146 .47, 152.22, 163.63, 167.56; MS m/z (ESI-ve) 538 (M-H)(100%), calculated for C25H22FN5O5S 538.0957, found 538.0960. (Z)-4-(2-(2-Oxo-5-(4-(3-(trifluoromethyl)phenyl)pip erazin-1-ylsulfonyl)indolin-3ylidene)hydrazinyl)benzoic acid (82h) NH N O S O O N NH O OH N CF3 5-(4-(3-(trifluoromethyl)phenyl)piperazin-1-ylsulfo nyl)indoline-2,3-dione ( 81h ) (0.0725 g, 0.165 mmol) was dissolved in EtOH (2 mL). To thi s solution 4-hydrazinobenzoic acid (0.02500 g, 0.118 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was

PAGE 151

139 added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82h (0.072 g, 65%) as a yellow solid, m.p. >300 C; 1H NMR (400 MHz, DMSO) d 3.02 (br s, 4H), 3.28 (br s, 4H), 7.04 (d, J = 8.8 Hz, 1H), 7.14-7.16 (m, 3H), 7.37 (t, J = 8.8 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.65 (dd, J = 4.2 Hz, 8.0 Hz, 1H), 7.87 (d, J = 4.2 Hz, 1H), 7.92 (d, J= 8.8 Hz, 2H), 11.56 (s, 1H), 12 .80 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.47, 49.54, 111.70, 114.97, 115.97, 116.19, 118. 77, 122.26, 125.88, 128.40, 128.73, 129.61, 131.72, 144.56, 146.48, 163.62, 167 .55; MS m/z (ESI-ve) 572 (M-H)(100%), calculated for C26H22F3N5O5S 572.1221, found 572.1239. (Z)-4-(2-(5-(4-(3,4-Dichlorophenyl)piperazin-1-ylsu lfonyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82i) NH N O S O O N N C l C l NH O OH 5-(4-(3,4-Dichlorophenyl)piperazin-1-ylsulfonyl)ind oline-2,3-dione ( 81i ) (0.0700 g, 0.150 mmol) was dissolved in EtOH (2 mL). To this s olution 4-hydrazinobenzoic acid (0.020 g, 0.125 mmol) was added. To the resulting m ixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82i (0.032 g, 36%) as a yellow solid, m.p. 280 C, deco mposed; 1H NMR (400 MHz, DMSO) d 2.99 (br s, 4H), 3.29 (br s, 4H), 6.85 (dd, J = 2.8 Hz, 9.2 Hz, 1H), 7.08 (d, J = 2.8 Hz, 1H), 7.13 (d J = 8.0 Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.86 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, DMSO) d 46.29, 47.81, 111.66, 114.94, 116.58, 117.56, 118. 77, 121.01, 121.23, 122.28, 126.01, 128.27, 128.66, 129.62, 131.20, 131 .72, 132.15, 144.54, 146.41, 150.68, 163.60, 167.60; MS m/z (ESI-ve) 572 (M-H)(100%), calculated for C25H22FN5O5S

PAGE 152

140 572.0567, found 572.0534. (Z)-4-(2-(5-(4-(2,3-Dichlorophenyl)piperazin-1-ylsu lfonyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82j) NH N O S O O N NH O OH N Cl C l 5-(4-(2,3-Dichlorophenyl)piperazin-1-ylsulfonyl)ind oline-2,3-dione ( 81j ) (0.0700 g, 0.150 mmol) was dissolved in EtOH (2 mL). To this s olution 4-hydrazinobenzoic acid (0.020 g, 0.125 mmol) was added. To the resulting m ixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82j (0.00900 g, 11%) as a yellow solid, m.p. 250 C, de composed; 1H NMR (400 MHz, DMSO) d 3.06 (br s, 8H), 7.14-7.18 (m, 2H), 7.29-7.30 (m, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 10.0 Hz, 1H), 7.88 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 11.57 (s, 1H), 12.81 (s, 1H); 13C NMR (101 MHz, DMSO) d 50.86, 62.33, 114.98, 118.83, 120.70, 122.30, 125.83, 128.46, 128.76, 129.29, 131.72, 131 .80, 133.23, 144.56, 146.48, 151.02, 161.54, 163.65, 167.57; MS m/z (ESI-ve) 572 (M-H)(100%), calculated for C25H21Cl2N5O5S 572.0567, found 572.0552. (Z)-4-(2-(5-(4-(3-methoxyphenyl)piperazin-1-ylsulfo nyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82k)

PAGE 153

141 NH N O S O O N NH O OH N O 5-(4-(3-Methoxyphenyl)piperazin-1-ylsulfonyl)indoli ne-2,3-dione ( 81k ) (0.0700 g, 0.170 mmol) was dissolved in EtOH (2 mL). To this solutio n 3-hydrazinobenzoic acid (0.0250 g, 0.160 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82k (0.039 g, 41%) as a yellow solid, m.p. 275 C, deco mposed; 1H NMR (400 MHz, DMSO) d 2.99 (br s, 4H), 3.26 (be s, 4H), 3.64 (s, 3H), 6. 33 (d, 8.4 Hz, 1H), 6.40 (s, 1H), 6.44 (d, J = 8.4 Hz, 1H), 7.03 (t, J = 8.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 4.2 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 11.55 (s, 1H), 12.80 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.51, 48.50, 55.41, 102.97, 105.54, 109.23, 114.94, 118.78, 122.24, 125.83, 128 .28, 128.72, 131.72, 144.52, 152.24, 152.30, 160.79, 167.57, 180.36; MS m/z (ESI-ve) 534 (M-H)(100%), calculated for C26H25N5O6S 534.1452, found 534.1459. (Z)-4-(2-(5-(4-(3-chlorobenzyl)piperazin-1-ylsulfon yl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82l) NH N O S O O N N C l NH O OH 5-(4-(3-Chlorobenzyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81l ) (0.0700 g, 0.170 mmol) was dissolved in EtOH (2 mL). To this solutio n 4-hydrazinobenzoic acid (0.0250

PAGE 154

142 g, 0.160 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82l (0.031 g, 34%) as a yellow solid, m.p. 260 C, deco mposed; 1H NMR (400 MHz, DMSO) d 2.70 (br s, 2H), 3.14 (br s, 4H), 3.76 (br s, 2H), 4.30 (br s, 2H), 7.15 (d, J = 8.0 Hz, 1H), 7.43 (br s, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.63 (dd, J = 2.0 Hz, 8.0 Hz, 2H), 7.85 (s, 1H), 7.91 (d, 8.4 Hz, 2H), 11.62 (s, 1H), 12.81 (s, 1H); 13C NMR (101 MHz, DMSO) d 40.70, 50.46, 58.36, 111.87, 114.98, 118.68, 122.2 7, 125.90, 128.46, 130.16, 131.24, 133.94, 144.70, 146.39, 163.54, 167.56; MS m/z (ESI-ve) 553 (M-H)(100%), calculated for C26H24ClN5O5S 552.1114, found, 552.1134. (Z)-4-(2-(5-(4-cyclohexylpiperazin-1-ylsulfonyl)-2 -oxoindolin-3-ylidene)hydrazinyl) benzoic acid (82m) NH N O S O O N NH O OH N 5-(4-cyclohexylpiperazin-1-ylsulfonyl)indoline-2,3dione ( 81m ) (0.0900 g, 0.209 mmol) was dissolved in EtOH (2 mL). To this solution 4-hy drazinobenzoic acid (0.0400 g, 0.250 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 m L) and dried to give 82m (0.0750 g, 64%) as a yellow solid, m.p. >300 C; 1H NMR (400 MHz, DMSO) d 1.17-1.24 (m, 4H), 1.55 (d, J = 8.0 Hz, 1H), 1.74 (br s, 2H), 1.94 (br s, 2H), 2.6 7 (br s, 2H), 3.14-3.16 (m, 4H), 3.46 (d, J = 12.0 Hz, 2H), 3.76 (d, J = 12.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H), 7.61 (d, 8.4 Hz, 2H), 7.67 (d, J = 8.4, 1H), 7.90 (s, 1H), 7.92 (d, J = 8.4 Hz, 2H), 11.63 (s, 1H), 12.82 (s, 1H); 13C NMR (101 MHz, DMSO) d 25.05, 25.29, 26.63, 40.54, 47.61, 59.18, 111.83, 111.89, 115.00, 118.81, 122.28, 125. 90, 127.98, 128.49, 131.70, 142.18, 144.74, 146.43, 148.21, 163.53, 167.56; MS m/z (ESI-ve) 510 (M-H)-, calculated for

PAGE 155

143 C25H29N5O5S 510.1817, found 510.1827. (Z)-3-(2-(5-(4-(4-Fluorophenyl)piperazin-1-ylsulfon yl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82n) NH N O S O O N NH N F O OH 5-(4-(4-Fluorophenyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81a ) (0.100 g, 0.220 mmol) was dissolved in EtOH (5 mL). To this solutio n 3-hydrazinobenzoic acid (0.0400 g, 0.250 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82n (0.0135 g, quantitative) as a yellow solid, m.p. 27 0 C, decomposed; 1H NMR (400 MHz, DMSO) d 3.01 (br s, 4H), 3.14 (br s, 4H), 6.89-6.91 (m, 2H ), 6.99 (t, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 8.4 Hz, 1H), 7.63-7.65 (m, 2H), 7.77 (d, J = 1H), 7.82 (s, 1H), 8.03 (s, 1H), 11.54 (s, 1H), 12.81 (s 1H); 13C NMR (101 MHz, DMSO) d 45.80, 50.95, 111.66, 115.71, 116.65, 118.37, 120.7 1, 124.94, 128.02, 132.74, 143.09, 143.18, 144.27, 157.70, 157.94, 160.08, 163.63, 167 .62, 167.67; MS m/z (ESI-ve) 522 (M-H)(100%), calculated for C25H22FN5O5S 522.1253, found 522.1256. (Z)-3-(2-(5-( N -(4-Morpholinophenyl)sulfamoyl)-2-oxoindolin-3-ylid ene)hydrazinyl) benzoic acid (82o) NH N O S O O NH HN N O O OH

PAGE 156

144 3-IminoN -(4-morpholinophenyl)-2-oxoindoline-5-sulfonamide ( 81b ) (0.100 g, 0.220 mmol) was dissolved in EtOH (5 mL). To this solutio n 3-hydrazinobenzoic acid (0.040 g, 0.250 mmol) was added. To the resulting mixture, HC l (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82o (0.0500 g, 37%) as a yellow solid, m.p. 230 C, decomposed; 1H NMR (400 MHz, DMSO) d 3.66 (s, 4H), 3.63 (s, 4H), 6.79 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.2 Hz, 2H), 6.97 (d, J = 8.4 Hz, 1H), 7.48-7.51 (m, 2H), 7.62 (d, J = 4.2 Hz, 1H), 7.66 (dd, J = 4.2 Hz, 1H), 7.84 (d, J = 4.2 Hz, 1H), 8.05 (s, 1H), 9.71 (s, 1H), 11.49 (s, 1H), 12.78 (s, 1H); 13C NMR (101 MHz, DMSO) d 47.05, 58.56, 104.98, 111.21, 115.39, 117.63, 119. 84, 122.43, 127.60, 128.36, 131.81, 132.73, 133.32, 143 .25, 143.77, 144.63, 163.55, 167.70; MS m/z (ESI-ve) 522 (M-H)(100%), calculated for C25H22FN5O5S 522.1250, found 522.1223. (Z)-3-(2-(5-(N-(2-Morpholinoethyl)sulfamoyl)-2-oxoi ndolin-3-ylidene)hydrazinyl) benzoic acid (82p) NH N O S O O HN NH N O O OH N -(2-Morpholinoethyl)-2,3-dioxoindoline-5-sulfonamid e ( 81c ) (0.100 g, 0.220 mmol) was dissolved in EtOH (5 mL). To this solution 3-hy drazinobenzoic acid (0.040 g, 0.250 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 m L) and dried to give 82p (0.057 g, 43%) as a yellow solid, m.p. 255 C, decomposed; 1H NMR (400 MHz, DMSO) d 1.24 (t, J = 6.4 Hz, 3H), 3.08 (q, J = 6.4 Hz, 2H), 3.61 (br s, 4H), 3.92 (br s, 4H), 7.1 0 (d, J = 8.4 Hz, 1H), 7.47-7.51 (m, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.94 (s, 1H), 11.51 (s, 1H), 12.79 (s, 1H); 13C NMR (101 MHz, DMSO) d 40.34, 52.06, 55.95,

PAGE 157

145 63.99, 111.34, 115.25, 117.63, 120.88, 124.84, 128. 40, 133.35, 140.74, 143.26, 143.58, 163.69, 167.70; MS m/z (ESI-ve) 472 (M-H)(100%), calculated for C21H23N5O6S 472.1296, found 472.1314. (Z)-3-(2-(5-( N -Isoxazol-3-ylsulfamoyl)-2-oxoindolin-3-ylidene)hyd razinyl)benzoic acid (82q) NH N O S O O HN NH O N O OH 5-(4-(4-Fluorophenyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81e ) (0.0 g, 0.220 mmol) was dissolved in EtOH (1 mL). To this solutio n 3-hydrazinobenzoic acid (0.0370 g, 0.240 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82q (0.043 g, 47%) as a yellow solid, m.p. 280 C, deco mposed; 1H NMR (400 MHz, DMSO) d 6.44 (d, J = 1.6 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.70 (t, J = 8.4 Hz, 2H), 7.96 (s, 1H), 8.06 (s, 1H), 8.70 (d, J = 1.6 Hz, 1H), 11.57 (s, 1H), 12.81 (s, 1H); 13C NMR (101 MHz, DMSO) d 98.99, 104.99, 111.26, 115.47, 117.69, 122.34, 124.88, 127.63, 130 .43, 131.57, 133.02, 143.23, 144.17, 161.60, 163.64, 167.70; MS m/z (ESI-ve) 426 (100%), calculated for C16H12N8O5S 427.0579, found 427.0562. (Z)-3-(2-(5-(4-(3-Chlorophenyl)piperazin-1-ylsulfo nyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82r) NH N O S O O N NH N C l O OH

PAGE 158

146 5-(4-(3-Chlorophenyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81g ) (0.0700 g, 0.170 mmol) was dissolved in EtOH (2 mL). To this solutio n 3-hydrazinobenzoic acid (0.0250 g, 0.160 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82r (0.0200 g, 22%) as a yellow solid, m.p. 280 C, dec omposed; 1H NMR (400 MHz, DMSO) d 2.99 (br s, 4H), 3.25 (br s, 4H), 6.75 (d, J = 7.6 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.89 (s, 1H), 7.13 (d, J = 8.0 Hz, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.8 Hz, 1H), 7.81 (s, 1H), 8.02 (s, 1H), 11.52 (s, 1H), 12.81 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.41, 47.96, 104.99, 115.73, 122.41, 127.52, 128. 13, 131.14, 134.44, 143.22, 144.18, 152.24, 163.65, 167 .69; MS m/z (ESI-ve) 537 (M-H)(100%), calculated for C25H22ClN5O5S 538.0957, found 538.0955. (Z)-3-(2-(2-Oxo-5-(4-(3-(trifluoromethyl)phenyl)pip erazin-1-ylsulfonyl)indolin-3ylidene)hydrazinyl)benzoic acid (82s) NH N O S O O N N CF3 NH O OH 5-(4-(3-(trifluoromethyl)phenyl)piperazin-1-ylsulfo nyl)indoline-2,3-dione ( 81h ) (0.147 g, 0.330 mmol) was dissolved in EtOH (2 mL). To this s olution 3-hydrazinobenzoic acid (0.0500 g, 0.360 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82s (0.0860 g, 39%) as a yellow solid, m.p. 295 C, dec omposed; 1H NMR (400 MHz, DMSO) d 3.02 (br s, 4H), 3.31 (br s, 4H), 7.04 (d, J = 7.6 Hz, 1H), 7.14-7.16 (m, 3H), 7.37 (t, J = 8.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.62 (t, J = 4.2 Hz, 2H), 7.75 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 4.2 Hz, 1H), 8.02 (s, 1H), 11.57 (s, 1H), 12.80 (s 1H); 13C NMR (101 MHz, DMSO) d 46.48, 47.93, 111.57, 112.40, 115.72, 118.37, 120. 03,

PAGE 159

147 123.62, 126.33, 127.83, 132.75, 143.21, 151.29, 163 .66, 167.69; MS m/z (ESI-ve) 572 (M-H)(100%), calculated for C26H22F3N5O5S 572.1221, found 572.1236. (Z)-3-(2-(5-(4-(3,4-Dichlorophenyl)piperazin-1-ylsu lfonyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82t) NH N O S O O N N C l C l NH O OH 5-(4-(3,4-Dichlorophenyl)piperazin-1-ylsulfonyl)ind oline-2,3-dione ( 81i ) (0.0700 g, 0.140 mmol) was dissolved in EtOH (2 mL). To this s olution 3-hydrazinobenzoic acid (0.020 g, 0.150 mmol) was added. To the resulting m ixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82t (0.031 g, 35%) as a yellow solid, m.p. 280 C, deco mposed; 1H NMR (400 MHz, DMSO) d 2.99 (br s, 4H), 3.26 (br s, 4H), 6.85 (dd, J = 2.8 Hz, 8.8 Hz, 1H), 7.08 (d, J = 4.2 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 9.2 Hz, 1H), 7.47 (t, J = 9.2 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 8.02 (s, 1H), 11.52 (s, 1H), 12.80 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.28, 47.80, 111.63, 115.71, 116.54, 118.33, 119.75, 121.00, 122.41, 127 .50, 128.10, 131.11, 131.77, 132.74, 143.19, 144.17, 150.66; MS m/z (ESI-ve) 572 (M-H)(100%), calculated for C25H21Cl2N5O5S 572.0567, found 572.0534. (Z)-3-(2-(5-(4-(2,3-Dichlorophenyl)piperazin-1-ylsu lfonyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82u)

PAGE 160

148 NH N O S O O N NH N Cl C l O OH 5-(4-(2,3-Dichlorophenyl)piperazin-1-ylsulfonyl)ind oline-2,3-dione ( 81j ) (0.0700 g, 0.140 mmol) was dissolved in EtOH (2 mL). To this s olution 3-hydrazinobenzoic acid (0.020 g, 0.150 mmol) was added. To the resulting m ixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 mL) and dried to give 82u (0.0100 g, 11%) as a yellow solid, m.p. 280 C, dec omposed; 1H NMR (400 MHz, DMSO) d 3.05 (br s, 8H), 7.13-7.18 (m, 2H), 7.29 (d, J = 4.2 Hz, 1H), 7.30 (s, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.62-7.67 (m, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 4.2 Hz, 1H), 8.03 (s, 1H), 11.54 (s, 1H), 12.82 (s, 1H); 13C NMR (101 MHz, DMSO) d 46.41, 47.96, 104.99, 115.73, 122.41, 127.52, 128.1 3, 131.14, 134.44, 143.22, 144.18, 152.24, 163.65, 167.69; MS m/z (ESI-ve) 572 (M-H)(100%), calculated for C25H21Cl2N5O5S 572.0567, found 572.0556. (Z)-3-(2-(5-(4-(3-Methoxyphenyl)piperazin-1-ylsulfo nyl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82v) NH N O S O O N N O NH O OH 5-(4-(3-Methoxyphenyl)piperazin-1-ylsulfonyl)indoli ne-2,3-dione ( 81k ) (0.0700 g, 0.170 mmol) was dissolved in EtOH (2 mL). To this solutio n 3-hydrazinobenzoic acid (0.0250 g, 0.160 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow

PAGE 161

149 precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82v (0.048 g, 51%) as a yellow solid, m.p. 253 C, deco mposed; 1H NMR (400 MHz, DMSO) d 3.00 (br s, 4H), 3.20 (br s, 4H), 3.64 (s, 3H), 6. 34 (d, J = 8.0 Hz, 1H), 6.40 (s, 1H), 6.44 (d, 8.8 Hz, 1H), 7.03 (t, J = 8.4 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 8.02 (s, 1H), 11.5 (s, 1H), 12.81 (s, 1H); 13C NMR (101 MHz, DMSO) d 45.85, 50.05, 55.87, 104.41, 108.42, 111.64, 115.71, 117.03, 119.74, 122.39, 124 .90, 132.74, 143.18, 149.92, 160.76, 163.63, 167.83; MS m/z (ESI-ve) 534 (M-H)(100%), calculated for C26H25N5O6S 534.1452, found 534.1460. (Z)-3-(2-(5-(4-(3-Chlorobenzyl)piperazin-1-ylsulfon yl)-2-oxoindolin-3-ylidene) hydrazinyl)benzoic acid (82w) NH N O S O O N NH N C l O OH 5-(4-(3-Chlorobenzyl)piperazin-1-ylsulfonyl)indolin e-2,3-dione ( 81l ) (0.100 g, 0.210 mmol) was dissolved in EtOH (2 mL). To this solutio n 3-hydrazinobenzoic acid (0.0400 g, 0.230 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with M eOH (3 3 mL) and dried to give 82w (0.0640 g, 49%) as a yellow solid, m.p. 260C, deco mposed; 1H NMR (400 MHz, DMSO) d 2.70 (br s, 2H), 3.14 (br s, 4H), 3.76 (br s, 2H), 4.30 (br s, 2H), 7.15 (d, J = 8.4 Hz, 1H), 7.43 (br s, 2H), 7.47 (t, J = 8.0 Hz, 2H), 7.61-7.64 (m, 2H), 7.75 (d, J = 9.2 Hz, 2H), 7.79 (s, 1H), 8.02 (s, 1H), 11.59 (s, 1H), 12. 82 (s, 1H); 13C NMR (101 MHz, DMSO) d 40.35, 50.42, 58.25, 111.75, 115.77, 119.77, 124.9 9, 128.09, 129.12, 130.13, 130.56, 131.24, 132.75, 133.94, 135.29, 143.15, 144 .36, 154.20, 163.58, 167.60; MS m/z (ESI-ve) 553 (M-H)(100%), calculated for C26H24ClN5O5S 552.1114, found 552.1136.

PAGE 162

150 (Z)-3-(2-(5-(4-cyclohexylpiperazin-1-ylsulfonyl)-2oxoindolin-3-ylidene) hydrazinyl) benzoic acid (82x) NH N O S O O N NH N O OH 5-(4-Cyclohexylpiperazin-1-ylsulfonyl)indoline-2,3dione ( 81m ) (0.0900 g, 0.209 mmol) was dissolved in EtOH (2 mL). To this solution 3-hy drazinobenzoic acid (0.0400 g, 0.250 mmol) was added. To the resulting mixture, HCl (1.0 M, 2 drops) was added and heated at 120 C for 15 min in the microwave reactor. After the re action, the yellow precipitate was filtered, the residue washed with MeOH (3 3 m L) and dried to give 82x (0.0600 g, 49%) as a yellow solid, m.p. > 300 C; 1H NMR (400 MHz, DMSO) d 1.17-1.24 (m, 4H), 1.55 (d, J = 8.0 Hz, 1H), 1.74 (br s, 2H), 1.94 (br s, 2H), 2.6 7 (br s, 2H), 3.14-3.16 (m, 4H), 3.46 (d, J = 12.0 Hz, 2H), 3.76 (d, J = 12.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 1H), 7.50 (t, 8.0 Hz Hz, 1H), 7.63 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 8.8 Hz, 1H), 7.82 (br s, 1H), 8.03 (s, 1H); 13C NMR (101 MHz, DMSO) d 25.05, 25.29, 26.63, 40.54, 47.61, 64.77, 111.83, 115.68, 116.19, 118.77, 122.33, 125.88, 128 .40, 128.73, 127.94, 132.94, 143.14, 146.48, 163.65, 167.74; MS m/z (ESI-ve) 510 (M-H)(100%), calculated for C25H29N5O5S 510.1817, found 510.1830.

PAGE 163

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161 Appendices

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162 Appendix 1 Crystal data and structure refinement for compound 41 Empirical formula C18H17N3O5S Formula weight 387.41 Temperature 100(2) K Wavelength 0.71073 Crystal system Orthorhombic Space group Fdd2 Unit cell dimensions a = 65.08(2) a = 90. b = 7.743(2) b = 90. c = 13.990(5) g = 90. Volume 7049(4) 3 Z 16 Density (calculated) 1.460 Mg/m 3 Absorption coefficient 0.220 mm -1 F(000) 3232 Crystal size 0.40 x 0.30 x 0.15 mm 3 Theta range for data collection 1.25 to 24.99. Index ranges -68<=h<=76, -8<=k<=8, -13<=l<=16 Reflections collected 6875 Independent reflections 2575 [R(int) = 0.0338] Completeness to theta = 24.99 98.3 % Absorption correction SADABS Max. and min. transmission 1.000 and 0.412 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 2575 / 1 / 246 Goodness-of-fit on F 2 0.915 Final R indices [I>2sigma(I)] R1 = 0.0539, wR2 = 0. 1519 R indices (all data) R1 = 0.0591, wR2 = 0.1604

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163 Absolute structure parameter -0.04(14) Largest diff. peak and hole 0.439 and -0.844 e. -3 Crystal data and structure refinement for compound 44 Empirical formula C9H15N3O3S Formula weight 245.30 Temperature 100(2) K Wavelength 0.71073 Crystal system Triclinic Space group P-1 Unit cell dimensions a = 6.7622(10) a = 67.425(2). b = 9.2133(14) b = 78.946(3). c = 10.1660(16) g = 74.897(2). Volume 561.64(15) 3 Z 2 Density (calculated) 1.451 Mg/m 3 Absorption coefficient 0.285 mm -1 F(000) 260 Crystal size 0.20 x 0.18 x 0.14 mm 3 Theta range for data collection 2.18 to 25.12. Index ranges -7<=h<=8, -10<=k<=11, -8<=l<=12 Reflections collected 2837 Independent reflections 1951 [R(int) = 0.0117] Completeness to theta = 25.12 98.0 % Absorption correction SADABS Max. and min. transmission 1.000 and 0.765 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 1951 / 0 / 147 Goodness-of-fit on F 2 0.880 Final R indices [I>2sigma(I)] R1 = 0.0339, wR2 = 0. 0894 R indices (all data) R1 = 0.0359, wR2 = 0.0914 Largest diff. peak and hole 0.377 and -0.273 e. -3 Crystal data and structure refinement for compound 46a Empirical formula C14H15NO3

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164 Formula weight 245.27 Temperature 100(2) K Wavelength 0.71073 Crystal system Monoclinic Space group P2(1)/c Unit cell dimensions a = 16.035(3) a = 90. b = 30.243(5) b = 93.420(4). c = 4.9189(9) g = 90. Volume 2381.2(7) 3 Z 8 Density (calculated) 1.368 Mg/m 3 Absorption coefficient 0.097 mm -1 F(000) 1040 Crystal size 0.30 x 0.25 x 0.20 mm 3 Theta range for data collection 1.44 to 25.04. Index ranges -15<=h<=14, -35<=k<=26, -3<=l<=5 Reflections collected 5543 Independent reflections 3588 [R(int) = 0.0228] Completeness to theta = 25.04 85.5 % Absorption correction SADABS Max. and min. transmission 1.000 and 0.642 Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 3588 / 0 / 345 Goodness-of-fit on F 2 1.123 Final R indices [I>2sigma(I)] R1 = 0.0613, wR2 = 0. 1352 R indices (all data) R1 = 0.0770, wR2 = 0.1428 Largest diff. peak and hole 0.470 and -0.421 e. -3


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Synthesis of small molecule inhibitors targeting signal transduction pathways
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ABSTRACT: The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transduction pathways, thereby treating cancer. We attempted to synthesize compounds based on the hits obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, history of chemotherapeutic drugs and an introduction to signal transduction pathways. The following two chapters briefly introduce the biological targets in the authors study. Chapter Two describes the role of B-cell lymphoma type xL (Bcl-xL), in apoptosis and the development of drugs targeting Bcl-xL. Examples of Bcl-xL drugs relevant to this study have been provided.Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathways and the recent developments in drug development towards the inhibition of this oncogene. Chapter Four gives a general introduction to microwave-assisted organic synthesis and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author's own findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibitors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL. Our strategy focused on identifying an isoquinolinol lead with increased potency.Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency.
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