Design and Synthesis of Potential Anticancer Agents a Dissertation

Home , Benzoyl chloride

Design and Synthesis of Potential Anticancer Agents

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Weihe Zhang

November 2010

This dissertation titled

Design and Synthesis of Potential Anticancer Agents

WEIHE ZHANG

has been approved for

the Department of Chemistry and Biochemistry

and the College of Arts and Sciences by

Stephen C. Bergmeier

Professor of Chemistry and Biochemistry

Benjamin M. Ogles

Dean, College of Arts and Sciences 3

ABSTRACT

ZHANG WEIHE, Ph.D., November 2010, Chemistry

Design and Synthesis of Potential Anticancer Agents (223 pp.)

Director of Dissertation: Stephen C. Bergmeier

Most cancers mainly rely on glucose as a source of biosynthesis material and energy for their fast growth and proliferation. We proposed inhibiting glucose uptake might kill cancer cells by restricting their energy supply. Based on this hypothesis, we designed and synthesized small molecules which can inhibit the glucose uptake and cell growth in lung cancer line H1299. These small molecules acted as potential novel anticancer agents. First, we designed and synthesized phenolic benzoate esters, from which most compounds displayed mild to good inhibitory activity in glucose uptake and cell growth. The Structure-Activity-Relationship (SAR) was studied by changing different substituents on the core aromatic ring and the pendant aromatic rings, as well as changing the number of hydroxyl groups on the pendant aromatic rings. SAR showed the meta-hydroxyl group played an important role in both inhibitory activity. Due to the instability of the benzoate esters, we designed and synthesized hydrolytically more stable analogs-phenolic ethers, N-linked and S-linked phenolic derivatives. The phenolic ethers showed better inhibitory activity than the N-linked and S-linked phenolic analogs.

Finally, the nitro phenolic ether WZB-173 was selected as the lead compound.

Approved: ______

Stephen C. Bergmeier

Professor of Chemistry and Biochemistry 4

Dedicated to my wife Junling Yang and my two wonderful daughters Jasmine Hongshun Zhang and Angela Hongli Zhang

ACKNOWLEDGMENTS

First, I would like to express my deepest gratitude to my academic advisor Professor

Stephen C. Bergmeier for his mentorship, guidance, insightful discussions, continuous support, patience and encouragements during the five years of my study at Ohio

University.

Then I would like to thank Professor Xiaozhuo Chen, one of my dissertation committee members and research collaborators, for his valuable discussions and suggestions during my research. I would also like to thank my dissertation committee members: Professor

Mark C. McMills and Professor Shiyong Wu for their valuable discussions and suggestions during my study.

Special thanks go to the members of Prof. Bergmeier’s research group both former members (Dr. Junfeng Huang, Dr. Pulipaka B. Aravinda, Dr. Ahbigit Nayek, Ms. Nova

Emerald, and Dr. Iwona Maciagiewicz) and present members (Ms. Fang Fang, Dr.

George Acquaah-Harrison, Dr. Crina M. Orac, Ms. Susann Krake, Mr. Gregg Wells and

Mr. Dennis Roberts) for their useful discussions and building a productive laboratory culture. I would also like to express special thanks to Ms. Yi Liu in Prof. Xiaozhuo

Chen’s laboratory for her hard work on testing all the compounds and her valuable discussions on the bioactivity data.

I am thankful to the faculty of the Department of Chemistry and Biochemistry, especially

Dr. Klaus Himmeldirk, Professor Hao Chen and Dr. Kumar Pichumani as well as the administrative staff of the Department of Chemistry and Biochemistry for their 6 suggestions and support during my study. I also want to thank my friends Dr. Yao Lu, Dr.

Dan Wang, Ms. Jingxing Li, Mr. Xiaoxi Ling, Ms. Zhixin Miao, Dr. Yuwu Zhong, Dr.

Kun Zhang, Mr. Xiaoyong Lu, Mr. Huaibo Ma, Mr. Haoshuang Wang, Ms. Yan An, Mr.

Huamin Li, Ms Yu Cai, Mr. Zhiguo Wang, Mr. Yueran Yan, Dr. Jing He and Ms. Yun

Zhang for their friendship.

Finally I want to thank my parents, my younger sister and my parents-in-law for their support and encouragement throughout my study and my daily life. Specialty is for my wife, Junling Yang, and two wonderful daughters, Jasmine Hongshun Zhang and Angela

Hongli Zhang, for your love, encouragement, patience and sharing life with me.

TABLE OF CONTENTS

Page

ABSTRACT ...... 3 DEDICATION ...... 4 ACKNOWLEDGMENTS ...... 5 LIST OF TABLES ...... 9 LIST OF FIGURES ...... 10 LIST OF SCHEMES...... 13 ABBREVIATIONS ...... 14 CHAPTER 1: INTRODUCTION ...... 15 CHAPTER 2: BACKGROUND ...... 17 2.1 Warburg Effect ...... 17 2.2 Glucose Metabolism ...... 17 2.3 Anticancer Agents ...... 21 2.3.1 Known anticancer drugs ...... 21 2.3.1.1 Alkylating agents ...... 21 2.3.1.2 Antibiotics ...... 22 2.3.1.3 Antimetabolites ...... 24 2.3.1.4 Anti-mitotic agents ...... 25 2.3.1.5 Topoisomerase inhibitors ...... 27 2.3.2 Phenols and polyphenols ...... 29 2.3.2.1 Antioxidants ...... 29 2.3.2.2 Glucose transport (GLUT) inhibitors ...... 31 2.4 Research Design and Significance ...... 33 CHAPTER 3: DESIGN AND SYNTHESIS OF PHENOLIC BENZOATE ESTERS .... 36 3.1 Design and Synthesis of Phenolic benzoate Esters ...... 36 3.1.1 Design and synthesis of 3,4,5-tri-hydroxyl benzoate esters ...... 37 3.1.2 Design and synthesis of di-hydroxyl benzoate esters ...... 48 3.1.2.1 Synthesis of 3, 5-dihydroxyl benzoate esters ...... 50 3.1.2.2 Synthesis of 3, 4-dihydroxyl benzoate esters ...... 51 8

3.1.3 Design and synthesis of mono-hydroxyl benzoate esters ...... 58 3.1.3.1 Synthesis of ortho-hydroxyl benzoate esters ...... 59 3.1.3.2 Synthesis of para-hydroxyl benzoate esters ...... 63 3.1.3.3 Synthesis of meta-hydroxyl benzoate esters ...... 68 3.2 Design and Synthesis of Analogs of Phenolic Benzoate Esters ...... 75 3.2.1 Synthesis of the fluoro-benzoate ester derivatives ...... 75 3.2.2 Synthesis of the methoxyl-benzoate ester derivatives ...... 80 3.2.3 Other derivatives of phenolic benzoate ester ...... 84 3.3 Stability Study on Selected Benzoate Esters ...... 88 3.4 Structure-Activity-Relationship (SAR) ...... 90 CHAPTER 4: DESIGN AND SYNTHESIS OF MORE STABLE ANALOGS ...... 93 4.1 Design and Synthesis of Phenolic Ether Derivatives ...... 94 4.2 Design and Synthesis of N-linked Phenolic Derivatives ...... 102 4.2.1 Design and synthesis of phenolic amide derivatives ...... 103 4.2.2 Design and synthesis of phenolic amine derivatives ...... 108 4.2.3 Design and synthesis of phenolic amide/ester derivatives ...... 113 4.3 Design and Synthesis of S-linked Derivatives ...... 117 4.4 Modification of the Phenolic Ether Derivatives ...... 122 4.5 Structure-Activity-Relationship (SAR) ...... 129 CHAPTER 5: EXPERIMENTAL SECTION ...... 133 REFERENCES ...... 202

LIST OF TABLES

Page Table 3.1 Glucose uptake inhibition and cell growth inhibition for trihydroxyl benzoate esters ...... 42 Table 3.2 Glucose uptake inhibition and cell growth inhibition for dihydroxyl benzoate esters ...... 54 Table 3.3 Glucose uptake inhibition and cell growth inhibition for ortho-hydroxyl benzoate esters ...... 61 Table 3.4 Glucose uptake inhibition and cell growth inhibition for para-hydroxyl benzoate esters ...... 66 Table 3.5 Glucose uptake inhibition and cell growth inhibition for meta-hydroxyl benzoate esters ...... 72 Table 3.6 Glucose uptake inhibition and cell growth inhibition for fluoro-benzoate esters ...... 79 Table 3.7 Glucose uptake inhibition and cell growth inhibition for methoxybenzoate esters ...... 83 Table 3.8 Glucose uptake inhibition and cell growth inhibition of WZB-158 ...... 86 Table 3.9 Glucose uptake inhibition and cell growth inhibition of WZB-159 and WZB- 162...... 87 Table 3.10 Stability study of WZB-115 and WZB-117 in human serum ...... 89 Table 4.1 Synthesis of phenolic ether derivatives ...... 96 Table 4.2 Glucose uptake inhibition and cell growth inhibition for phenolic ether derivatives ...... 97 Table 4.3 Glucose uptake inhibition and cell growth inhibition for phenolic amide derivatives ...... 105 Table 4.4 Glucose uptake inhibition and cell growth inhibition for phenolic amine derivatives ...... 110 Table 4.5 Glucose uptake inhibition and cell growth inhibition for phenolic amide/ester derivatives ...... 115 Table 4.6 Glucose uptake inhibition and cell growth inhibition of S-linked derivatives120 Table 4.7 Glucose uptake inhibition and cell growth inhibition of selected the best compounds from each group ...... 122 Table 4.8 Synthesis of substituted phenolic ether derivatives ...... 125 Table 4.9 Glucose uptake inhibition and cell growth inhibition for substituted phenolic ether analogs ...... 126

LIST OF FIGURES Page Figure 1.1 Structure of WZB-115 ...... 16 Figure 1.2 Structure of WZB-173 ...... 16 Figure 2.1 Glycolysis under aerobic conditions ...... 18 Figure 2.2 Glucose metabolism in mammalian cells ...... 20 Figure 2.3 Known alkylating anticancer drugs ...... 22 Figure 2.4 Known antibiotic anticancer drugs ...... 24 Figure 2.5 Known antimetabolites anticancer drugs ...... 25 Figure 2.6 Structure of paclitaxel (Taxol®) and docetaxel ...... 26 Figure 2.7 Structure of vincristine and vinblastine ...... 27 Figure 2.8 Structure of colchicine ...... 27 Figure 2.9 Well-known anticancer topoisomerase inhibitors ...... 28 Figure 2.10 Flavonoids scavenge free radicals ...... 30 Figure 2.11 Important phenolic and polyphenolic antioxidants ...... 31 Figure 2.12 Structure of GLUT-1 molecule with the predicted 12 transmembrane helixes ...... 32 Figure 2.13 Structure of fasentin and apigenin ...... 32 Figure 2.14 Structure of α-PGG, WZB-26 and WZB-27 ...... 34 Figure 3.1 Design the analogs of WZB-27 ...... 36 Figure 3.2 Reduction of MTT to formazan ...... 41 Figure 3.3 Tube view of energy-minimized structure of WZB-26, 89, 90 and 110 ...... 44 Figure 3.4 Tube view of energy-minimized structure of WZB-27 with WZB-26, 89, 90 and 110 ...... 45 Figure 3.5 View of energy-minimized structure of WZB-27 (in tube view) and WZB-75 (in wire view) ...... 46 Figure 3.6 Representative tube view of energy-minimized structure of tri-hydroxyl benzoate esters ...... 47 Figure 3.7 Calculated pKa values of 3,4,5-trihydroxy benzoate esters ...... 48 Figure 3.8 Design and retro-synthetic analysis of di-hydroxyl benzoate esters ...... 49 Figure 3.9 Calculated pKa values of 3, 5-di-hydroxyl benzoate esters ...... 55 Figure 3.10 Calculated pKa values of 3, 4-di-hydroxyl benzoate esters ...... 55 Figure 3.11 Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-111 and 119 (in tube view) ...... 56 Figure 3.12 Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-121 and 122 (in tube view) based on core aromatic ring ...... 57 Figure 3.13 Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-121 and 122 (in tube view) based on one pendant ring ...... 57 Figure 3.14 Design the mono-hydroxyl benzoate esters ...... 59 Figure 3.15 Calculated pKa values of ortho-hydroxyl benzoate esters ...... 62 Figure 3.16 Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-127 and 128 (in tube view) based on core ring ...... 63 Figure 3.17 Calculated pKa values of para-hydroxyl benzoate esters ...... 67 11

Figure 3.18 Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-91 and 92 (in tube view) based core ring ...... 68 Figure 3.19 Calculated pKa values of meta-hydroxyl benzoate esters ...... 73 Figure 3.20 Tube views of energy-minimized structure of WZB-115, WZB-117 ...... 74 Figure 3.21 Tube view of angles between two planar rings in energy-minimized structure of WZB-27 and WZB-115 ...... 74 Figure 3.22 Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-115 and 117 (in tube view) based core ring ...... 75 Figure 3.23 View of energy-minimized structure of WZB-27 (in wire view) and WZB-78 (in tube view) based core ring overlap ...... 80 Figure 3.24 View of energy-minimized structure of WZB-27 (in wire view), WZB-76 and WZB-98 (in tube view) based on core ring overlap ...... 84 Figure 3.25 Structure-Activity-Relationship of benzoate ester derivatives ...... 91 Figure 4.1 Design hydrolytically more stable analogs ...... 93 Figure 4.2 Design of the phenolic ether derivatives ...... 94 Figure 4.3 Retro-synthetic analysis of synthesizing phenolic ether derivatives ...... 95 Figure 4.4 Calculated pKa values for the phenolic ether derivatives ...... 99 Figure 4.5 Overlap view of energy-minimized structure of WZB-134 (in tube model) and WZB-117 (in wire model) ...... 100 Figure 4.6 Overlap view of energy-minimized structure of WZB-131 (in tube model) and WZB-115 (in wire model) ...... 101 Figure 4.7 Overlap view of energy-minimized structure of WZB-141 (in tube model) and WZB-117 (in wire model, left side) and WZB-115 (in wire model, right side) ...... 101 Figure 4.8 Design the N-linked phenolic derivatives...... 103 Figure 4.9 Design the phenolic amide derivatives ...... 103 Figure 4.10 Calculated pKa values for the phenolic amide derivatives ...... 106 Figure 4.11 Overlap view of energy-minimized structure of WZB-124 (in tube model) and WZB-117 (in wire model) ...... 107 Figure 4.12 Overlap view of energy-minimized structure of WZB-143 (in tube model) and WZB-117 (in wire model) ...... 107 Figure 4.13 Retro-synthetic analysis of phenolic amine derivatives ...... 108 Figure 4.14 Calculated pKa values for the phenolic amide derivatives ...... 111 Figure 4.15 Overlap view of energy minimized structure of WZB-138 (in tube model) and WZB-139 (in wire model) ...... 112 Figure 4.16 Overlap view of energy minimized structure of WZB-145 (in tube model) and WZB-144 (in wire model) ...... 113 Figure 4.17 Retrosynthetic analysis of phenolic amide/ester derivatives ...... 113 Figure 4.18 Calculated pKa values for the phenolic amide and amide/ester derivatives116 Figure 4.19 Overlap view of WZB-153 (in tube model) and WZB-157 (in wire model) ...... 117 Figure 4.20 Design the S-linked derivatives of WZB-117 ...... 118 Figure 4.21 Calculated pKa values for the S-linked derivatives ...... 120 Figure 4.22 Overlap view of WZB-165 (in tube model) and WZB-117 (in wire model) ...... 121 12

Figure 4.23 Modification of the phenolic ether derivatives ...... 123 Figure 4.24 Calculated pKa values for the modified phenolic ethers ...... 127 Figure 4.25 Energy minimized structure of WZB-173 ...... 128 Figure 4.26 Energy minimized structure of WZB-172 ...... 129 Figure 4.27 Structure-Activity-Relationship of hydrolytically more stable analogs ....131 Figure 4.28 Structures of small compounds in future plans...... 132

LIST OF SCHEMES Page Scheme 3.1 Synthesis of 3,4,5-tribenzyloxybenzoyl chloride ...... 37 Scheme 3.2 Synthesis of WZB-27 and WZB-75 ...... 38 Scheme 3.3 Synthesis of WZB-26, WZB-89, WZB-90 and WZB-110 ...... 39 Scheme 3.4 Synthesis of dibenzyloxybenzoyl chlorides 11a and 11b ...... 50 Scheme 3.5 Synthesis of 3, 5-dihydroxyl benzoate ester WZB-111 ...... 50 Scheme 3.6 Synthesis of 3, 5-dihydroxyl benzoate esters WZB-112, 113 and 114 ...... 51 Scheme 3.7 Synthesis of 3, 4-dihydroxyl benzoate ester WZB-119 ...... 52 Scheme 3.8 Synthesis of 3, 4-dihydroxyl benzoate esters WZB-120, 121 and 122 ...... 52 Scheme 3.9 Synthesis of ortho-benzyloxybenzoyl chloride 19 ...... 59 Scheme 3.10 Synthesis of ortho-hydroxyl benzoate esters WZB-127~130 ...... 60 Scheme 3.11 Synthesis of p-benzyloxybenzoyl chloride 25 ...... 64 Scheme 3.12 Synthesis of 4-hydroxyl benzoate esters WZB-91~94 and WZB-103 ...... 65 Scheme 3.13 Synthesis of 3-benzyloxybenzoyl chloride 31 ...... 69 Scheme 3.14 Synthesis of meta-hydroxyl benzoate ester WZB-115 ...... 69 Scheme 3.15 Synthesis of meta-hydroxyl benzoate esters WZB-116~118 ...... 70 Scheme 3.16 Synthesis of meta-hydroxyl benzoate esters WZB-136, WZB-140 ...... 70 Scheme 3.17 Synthesis of 3,4,5-trifluorobenzoate esters WZB-78~80 ...... 76 Scheme 3.18 Synthesis of 3, 4-di-fluorobenzoate esters WZB-82~84 ...... 77 Scheme 3.19 Synthesis of 4-fluoro-3-(trifluoromethyl) benzoate esters WZB-85~88 ...78 Scheme 3.20 Synthesis of 3,4,5-trimethoxybenzoate esters ...... 81 Scheme 3.21 Synthesis of di-methoxybenzoate esters WZB-95 and WZB-98 ...... 82 Scheme 3.22 Synthesis of 3-methoxybenzoate esters WZB-96 and WZB-97 ...... 82 Scheme 3.23 Synthesis of WZB-158 ...... 85 Scheme 3.24 Synthesis of WZB-159 ...... 86 Scheme 3.25 Synthesis of WZB-162 ...... 87 Scheme 4.1 Synthesis of the benzyl chloride 51 ...... 95 Scheme 4.2 Synthesis of N-H phenolic amides and N-methyl phenolic amides ...... 104 Scheme 4.3 Synthesis of N-H and N-methyl phenolic amine derivatives ...... 108 Scheme 4.4 Synthesis of WZB-152~154 and WZB-157 ...... 114 Scheme 4.5 Synthesis of the S-linked derivatives ...... 119 Scheme 4.6 Synthesis of benzyl chlorides 61 and 64 ...... 123 Scheme 4.7 Synthesis of benzyl bromide 68 ...... 123 Scheme 4.8 Deprotection of the MOM group ...... 124 14

ABBREVIATIONS

ATP Adenosine triphosphate

DCC N, N’-dicyclohexylcarbodiimide

DCM Dichloromethane

DMAP 4-dimethylaminopyridine

DMF Dimethylformamide

EDG Electron donating group

EWG Electron withdrawing group

GLUT Glucose transporter

HPLC High performance liquid chromatography

LAH Lithium aluminum hydride m-CPBA meta-Chloroperbenzoic acid

MOMCl Chloromethyl methyl ether

MsCl Methanesulfonyl chloride

MWT Molecular weight

NMR Nuclear magnetic resonance

NSCLC Non-small cell lung cancer

PGG Penta-O-Galloyl-D-glucopyranose

SAR Structure activity relationship

TLC Thin layer chromatography

WZB Initials of Weihe Zhang & Bergmeier CHAPTER 1: INTRODUCTION

Cancer is a worldwide public health concern. It has been estimated that 1,529,560 new cancer cases are expected in the United States in 2010.1 The mortality in the United

States of the year 2009 was 562,340.2 More and more research groups have devoted into the cancer area in order to slow down its death rate. Based on the mechanism of glucose metabolism, we proposed that inhibiting the glucose uptake might kill cancer cells.

In order to prove our hypothesis, we designed and synthesized several series of small molecules which can inhibit the glucose transport and act as novel anticancer agents.3-5

We synthesized a series of benzoate esters based on the structure of α-PGG. Most compounds from this small library showed moderate to good activity in both glucose uptake inhibition and cell growth inhibition.3, 5 The Structure-Activity-Relationship

(SAR) was studied by: 1) introducing different substituents to the core aromatic ring as well as the pendant aromatic rings; 2) changing the number of hydroxyl groups on the pendant aromatic rings. The SAR showed the introduction of electron donating group on the core aromatic ring decreased both inhibitory activity in glucose uptake and cell growth, whereas the electron withdrawing groups kept or increased both inhibitory activity; the meta-hydroxyl group played a very important role in both inhibitory activity in glucose uptake and cell growth. WZB-115 (Figure 1.1) was selected as the lead compound from the phenolic benzoate esters.

Figure 1.1 Structure of WZB-115

Since the esters can be easily hydrolyzed, the stability of WZB-115 and WZB-117

(which both showed good inhibitory activity in glucose uptake and cell growth) were studied in human serum. The result showed that both compounds were quickly degraded in human serum from this stability study. Several series of hydrolytically stable analogs

(phenolic ethers, N-linked and S-linked phenolic analogs) were designed and synthesized based on the structure of WZB-115. The N-linked and S-linked phenolic analogs showed very poor inhibitory activity in both glucose uptake and cell growth, while most phenolic ethers showed better activity. Some phenolic ether analogs showed similar activity to

WZB-115. WZB-173, which showed the best activity in cell growth inhibition, was selected as our lead compound. (Figure 1.2)

NO2 NO2 O O

OH OH WZB-173

Figure 1.2 Structure of WZB-173 17

CHAPTER 2: BACKGROUND

Cancer is now a major global public health problem. In the United States of America,

562,340 deaths from cancer were reported in 2009 and an estimation of 1,529,560 new cancer cases were expected in 2010.1, 2 Although the overall cancer incident rate has decreased in recent years, lung cancer is still the leading cause of cancer mortality in men and women.6 Novel approaches are urgently required for further improvements to current cancer therapies, especially the lung cancer, in order to slow down the cancer death rate, and therefore, to lower the overall cancer mortality.

2.1 Warburg Effect

In 1924, Warburg and Negelein observed that cancer cells exhibited a higher glycolysis rate than normal cells, even in the presence of oxygen.7 This feature is well known as the

Warburg effect, and is one of the most important characteristics of cancer cells.8, 9 Since then, the Warburg effect has been well investigated and revisited.10-26 It has also been an important biochemical basis for the development anticancer therapeutic strategies and new potential anticancer agents.11, 21, 25

2.2 Glucose Metabolism

Adenosine triphosphate (ATP) is the energy fuel for all cells.27 There are two main metabolic pathways to generate ATP in normal cells: oxidative phosphorylation in mitochondria and anaerobic glycolysis in the cytoplasm.28-30 All cells use both pathways to generate ATP but most healthy cells rely more on the oxidative phosphorylation pathway, and switch to glycolysis during oxygen deprivation (hypoxia). Most cancer cells exist in a hypoxic state, and mainly rely on the latter method to generate ATP.31 18

Glucose metabolism starts after blood delivers glucose and oxygen (on hemoglobin) to tissues. First, glucose is transported into cells by specific glucose transporters and converted to glucose-6-phosphate after glucose phosphorylation catalyzed by hexokinase.

Then glucose-6-phosphate is finally converted to pyruvate and generates two ATPs under aerobic conditions. Therefore, one molecule of glucose generated two molecules of ATPs in this process. (Figure 2.1)

Figure 2.1. Glycolysis under aerobic conditions.

The generated pyruvate is reduced to lactate under anaerobic conditions, which is taken out of cells by the monocarboxylate transporter. Malate-aspartate shuttle carries two pairs 19 of electrons generated from glycolysis under anaerobic conditions into mitochondria.

Then the two electrons enter into the electron transport chain in the presence of oxygen.

Two molecules of NADH are oxidized to two NAD+ and generate six ATPs.29 (Equation

+ + 2 NADH + 2H + 6 Pi + 6 ADP + O2 2 NAD + 6 ATP + 8 H2O (1)

Pyruvate is dehydrogenated to generate acetyl-CoA, NADH and CO2 by the pyruvate dehydrogenase complex, an enzyme located in the mitochondria of eukaryotic cells and in the cytoplasm of prokaryotes. The newly formed two NADH are oxidized and lead to six ATPs. (Equation 2)

2 Pyruvate + 2 CoA + 6 Pi + 6 ADP + O2

2 acetyl-CoA + 2 CO2 + 6 ATP + 8 H2O (2)

Then the two molecules of acetyl-CoA are oxidized via the citric acid cycle to CO2, water and three ATPs. Oxidative phosphorylations of isocitrate, α-ketoglutarate, and malate in the presence of oxygen, each of which leads to three ATPs; oxidation of succinate yields two ATPs; and two ATPs are produced from succinyl-CoA via GTP. By this means, a total of twenty-four ATPs are generated. (Equation 3)

2 Acetyl-CoA + 24 Pi +24 ADP + 4 O2 2 CoA + 4 CO2 + 24 ATP +26 H2O (3) 20

Overall, thirty-six additional ATPs are generated after complete oxidative phosphorylation of glucose in the mitochondria under aerobic conditions. (Figure 2.2)

Blood vessel HbO2 Glucose

O2 Glucose - HCO3 Anion H+ exchanger Lactate Glucose transporter Mitochondrion 36 ATP

Lactate

Glucose He Pyruvate H+ xo ki 2ATP na se Animal cell Glucose-6- H+ phosphate

Sodium-Hydrogen exchanger

Figure 2.2. Glucose metabolism in mammalian cells.32

With respect to the ATP generation, the pathway of oxidative phosphorylation in the presence of oxygen is much more efficient. However, cancer cells mainly rely on anaerobic glycolysis to produce ATPs as their predominate glucose metabolism pathway.32 Such a glycolytic switch not only gives cancer higher potentials for metastasis and invasiveness,33 but also increases cancer’s vulnerability to external interference in glycolysis since cancer cells are “addicted” to glucose as their predominate energy supply.34, 35, 32 However, the healthy cells could not be fatally affected by shrinking their 21 glucose supply because they could also use amino acids, lipids and other metabolic intermediate compounds as their energy sources.

2.3 Anticancer Agents

Anticancer agents have played an important role in the treatment of cancer. More than half of anticancer agents are of natural origin, which includes natural products, synthetic or naturally existing derivatives of natural products.36 So far, several anticancer agents have been in clinical use worldwide, such as vinblastine, vincristine, taxol, topotecan, irinotecan, etoposide, teniposide, and so on. A number of important anticancer agents are in clinical or preclinical development. 37-39

2.3.1 Known anticancer drugs

Anticancer drugs have been classified into several different classes according to various categorizations.40-44 Classical classification of anticancer drugs includes chemotherapy, hormonal therapy and immunotherapy.41 The chemotherapeutic drugs are divided into several groups according to chemical structures and biological mechanisms, such as alkylating agents, antibiotics, antimetabolites, antimitotic agents and topoisomerase inhibitors.41, 45, 42 Hormonal therapy includes steroids, anti-estrogens, anti-androgens, luteinizing hormone-releasing hormone (LH-RH) analogs and anti-aromatase agents.41, 42,

44 Immunotherapeutic drugs are grouped into interferon, interleukin 2 and vaccines.40, 41,

43, 44

2.3.1.1 Alkylating agents

Alkylating agents have been used in cancer treatment by forming a variety of inter-strand cross-linking adducts to alter DNA structure and function. The most common alkylating 22 site is the N-7 position of guanine, but different drugs could have various alkylating sites.41 Several groups of alkylating agents are well-known: nitrogen mustards, platinum compounds, ethylenimes, alkylsulfonates, triazenes, piperazines, and nitrosoureas.

Procarbazine is an antineoplastic chemotherapy drug for the treatment of Hodgkin's lymphoma and certain brain cancer. The common side effect is that this drug can cause a temporary drop in the number of blood cells generated by the bone marrow in many patients. The well-known alkylating drugs are cyclophosphamide, procarbazine, chlorambucil, melphalan, mechlorethamine, dacarbazine, cisplatin. (Figure 2.3) The most important toxic effects are bone marrow suppression, leucopenia and thrombocytopenia.46-48

Figure 2.3. Known alkylating anticancer drugs.

2.3.1.2 Antibiotics

Many clinically useful anticancer antibiotics have been derived from Streptomyces.49 The antibiotics are used in the treatment with anticancer drugs by intercalating DNA to block 23 synthesis of DNA and RNA. The antibiotics used clinically include doxorubicin, daunorubicin, mitomycin C, plicamycin, idarubicin and bleomycin.50-53 (Figure 2.4)

Figure 2.4. Known antibiotic anticancer drugs.

Doxorubicin is mainly used in the treatment of breast carcinoma, ovarian carcinoma, thyroid carcinoma, and lung carcinoma.54-58 Daunorubicin is used to treat breast cancer, acute lymphocytic-leukemia (ALL) and lung cancer.59-61 Mitomycin C is used in the treatment of lung cancer, colon cancer and stomach cancer.62-64 Plicamycin is very useful in managing severe hypercalcemia associated with cancer.65 Idarubicin in combination with cytarabine is more active than daunorubicin in the treatment of acute myelogenous leukemia.66, 67 Bleomycin is mainly useful in testicular cancer, head, neck and cervix cancers.68-70 The common adverse effects from antibiotics are vomiting and anorexia.71-73

2.3.1.3 Antimetabolites

Antimetabolites have been used in cancer treatment because they can interfere with DNA production, cell division and tumor growth.41, 48 Antimetabolites can stop normal cell development and division by replacing a purine or a pyrimidine to become the building blocks of DNA. Most antimetabolites interfere with nucleic acid synthesis or nucleotide synthesis. Therefore, antimetabolites inhibit cell division, which harms cancer cells more than normal cells because tumor cells have a faster proliferation rate than normal cells.

Well-known anti-metabolites include azathioprine, mercaptopurine, fluorouracil, methotrexate, thioguanine, pentostatin, and cytarabine.74-77, 67, 78 (Figure 2.5) The toxicity of antimetabolites is due to the halting of cell growth and cell division.79

Figure 2.5.Known antimetabolites anticancer drugs.

2.3.1.4 Anti-mitotic agents

The mechanism for anti-mitotic agents in the treatment of cancer is to inhibit the function of microtubules through binding their subunits or direct cessation of their growth.80-82 So far, three distinct classes of anti-mitotic agents (taxanes, vinca alkaloids and colchicines) have been identified by the targeting sites of tubulin.81

Two important anti-mitotic agents from taxanes are paclitaxel (Taxol®) and docetaxel.

(Figure 2.6) Taxol was isolated from the bark of Taxus brevifolia.83 The generic name was changed to paclitaxel after it was commercially developed. Paclitaxel is used in the treatment of ovarian cancer, breast cancer, non-small cell lung cancer (NSCLC) as well as some other cancers by overexpression of Class III beta-tubulin.84-87 The common side effects include nausea, vomiting, neutropenia, thrombocytopenia, and loss of appetite.88-90

Docetaxel is a synthetic derivative of taxol,91 which is used mainly for the treatment of 26 breast, ovarian and non-small cell lung cancer (NSCLC).92-95 Docetaxel kills cancer cells in the same way as paclitaxel.

Figure 2.6. Structure of paclitaxel (Taxol®) and docetaxel.

The representative anti-mitotic agents from vinca alkaloids are vincristine and vinblastine, both of which were isolated from Catharanthus roseus (L.) G. Don. (Figure

2.7) Vinblastine and vincristine are the first anticancer agents advanced into clinical use from plant alkaloids.37 Vinblastine is used in the treatment with several kinds of cancers, such as lung cancer, breast cancer, head and neck cancer, and so on.96-99 Vincristine, combined with prednisone, is used in induction of remission in children with acute leukemia.100 Both compounds act through mitotic arrest at metaphase and disrupting the chromosome segregation.101, 102 Nausea, vomiting and alopecia are the common adverse effects caused by vinblastine.103 Significant neurotoxic reactions, cortical blindness and bone marrow depression have been frequently observed after using vincristine.104-106

Figure 2.7. Structure of vincristine and vinblastine.

Colchicine, the third group of anti-mitotic agents, was isolated from Colchicum autumnale.107 Colchicine displays the ability to kill cancer cells by binding with a high affinity site on the heterodimer of tubulin; however, the cytotoxicity prevented it to be further investigated in the treatment of cancers.108 Currently, colchicine is used in the treatment of gout.109 (Figure 2.8)

Figure 2.8. Structure of colchicines.

2.3.1.5 Topoisomerase inhibitors

Several important topoisomerase inhibitors have been identified and used in the treatment of cancers, which include etoposide, teniposide, topotecan and irinotecan.81 (Figure 2.9)

Figure 2.9. Well-known anticancer topoisomerase inhibitors.

Etoposide treats cancer by inhibiting the enzyme topoisomerase II (which unwinds DNA) to break DNA strands.110 It is often given in combination with other drugs for several cancers, such as Ewing's sarcoma, non-small cell lung cancer, testicular cancer and lymphoma.111-118 The common side effects caused by using etoposide are low blood pressure, hair loss and bone marrow suppression.119, 120 Teniposide is mainly used in the treatment of childhood acute lymphocytic leukemia (ALL) via the same mechanism as etoposide.121, 122 Teniposide could lead to the same side effects as etoposide, and can cause severe myelosuppression when used with other chemotherapeutic agents for the treatment of ALL. Topotecan is used to treat ovarian cancer and lung cancer, as well as cervical cancer by inhibiting the topoisomerase I.123-127 The most significant toxic effect from topotecan is myelosuppressive, which leads to low blood count.128 Irinotecan is an 29 active topoisomerase I inhibitor, mainly used in the treatment of colon cancer.129-133 The most common adverse effects of irinotecan is diarrhea.134

2.3.2 Phenols and polyphenols

Phenolic and polyphenolic compounds, which widely exist in plants, include flavonoids, gallocatechins, tannins, stilbenes, curcuminoids, and others. Phenols and polyphenols possess a wide range of pharmacological properties, and they were recognized as antioxidants and anticancer agents.135-150

2.3.2.1 Antioxidants

A well-known and studied member from the phenolic and polyphenolic compounds is flavonoids, which is widely distributed in food and diets.151-154 Most flavonoids show strong antioxidant activity in vitro and in vivo.135, 136, 138, 155, 147, 156, 148, 157 Flavonoids act as antioxidants by scavenging a free radical and forming less reactive radical intermediate to inhibit the oxidative process.136, 158-162, 156 Flavonoids (Fl-OH) can thermodynamically reduce highly oxidizing free radicals (R·, such as superoxide, peroxyl, alkoxyl and hydroxyl radicals) with redox potentials in the range of 2.13-1.0 V by donating hydrogen atom.162 (Equation 4)

R· + Fl-OH RH + Fl-O· (4)

Where R· represents superoxide, peroxyl, alkoxyl and hydroxyl radicals. The aroxyl radical (Fl-O·) is less reactive intermediate and may further react with another R· to give a stable quinone structure.163 (Figure 2.10)

Figure 2.10 Flavonoids scavenge free radicals

Some important antioxidants from the phenolic and polyphenolic compounds are quercetin, (-)-epicatechin, (+)-catechin, chlorogenic acid, phloretin and (-)-Epicatechin gallate.164, 165 (Figure 2.11) Besides the anti-oxidative activity, phloretin also showed good glucose transporter (GLUT) inhibitory activity,166-168 which induced apoptosis of hepatoma cells both in culture and in tumor-bearing SCID mice.168

Figure 2.11. Important phenolic and polyphenolic antioxidants.

2.3.2.2 Glucose transport (GLUT) inhibitors

Glucose transporters (GLUT) are a family of membrane proteins found in most mammalian cells, which contain twelve membrane spanning helices with both N-terminal and C-terminal exposed on the cytoplasmic side of the plasma membrane.169 So far, 13 members of GLUT have been identified.170, 171 Among the glucose transporters, GLUT 1 is responsible for the low-level of basal glucose uptake required to maintain the respiration for mammalian cells.172-174 (Figure 2.12) Inhibition of the GLUT1 transport of glucose could cause energy deficiency for the cancer cells. 175, 176 However, not much attention has been paid on discovery and investigation of the GLUT1 inhibitors until recently.177, 175, 178-181, 4, 5

Figure 2.12. Structure of GLUT-1 molecule with the predicted 12 transmembrane helixes.174

Fasentin and apigenin have recently been identified as novel GLUT1 inhibitors.182, 175, 4

Fasentin induces cancer cells death by altering expression of genes associated with nutrient and glucose deprivation. Moreover, fasentin was found to interact with a unique site in the intracellular channel of GLUT1 protein in a docking study.4 Fasentin acted as

GLUT1 inhibitor to inhibibit glucose uptake and led to a new mechanism on killing cancer cells.4 Apigenin inhibits the GLUT1, the potential anticancer mechanism for apigenin is suggested to be down regulation of GLUT1.182, 175 (Figure 2.13)

Figure 2.13. Structure of fasentin and apigenin.

2.4 Research Design and Significance

All cancers share some common features, one of which is that they require more energy and nutrients for their faster growth and proliferation rates than normal cells.34, 183, 17, 184

One of the common weaknesses of most cancers is the increased glucose uptake and increased dependence on glucose as a source for making building blocks for their fast growth and proliferation.34, 185, 183 Normal cells can utilize different chemicals, such as amino acids, fatty acids, carbohydrates and glucose as their energy supply sources.

However, the cancer cells rely mainly on glucose as their biosynthesis material and energy source,33 and many cancer cells showed increased glycolysis.186, 33, 176 Positron emission tomography (PET) scans on various cancer types, including both primary and secondary metastatic cancers, showed that almost all cancer cells in vivo (solid malignant tumors) have increased glucose supply and metabolism than normal cells. Therefore, strong evidences showed that cancer cells relied mainly on glucose to meet their energy and growth demands.187 Based on this, we proposed that inhibiting glucose uptake might kill the cancer cells. In order to prove the hypothesis, we have designed and synthesized a library of small molecules which inhibited the glucose transport and cell growth.

Initially, we designed and synthesized WZB-26 and WZB-27 based on the structure of α-

PGG, naturally existing polyphenolic compound with strong anti-diabetic activity.188

(Figure 2.14) In order to be effective anti-cancer agents, these compounds should inhibit glucose uptake; meanwhile, they should be able to kill more cancer cells than normal cells. Initial results showed WZB-27 preferentially kill cancer cells (NSCLC H1299 and breast carcinoma MCF-7) than their normal cell counterparts (NL or MCF-12A cells). 34

The results suggest these compounds have excellent potential to be effective anti-cancer agents. Based on the structure of WZB-27, we have designed and synthesized a library of novel compounds to identify a novel target for inhibiting of glucose uptake and inducing cancer cell death.

Figure 2.14. Structure of α-PGG, WZB-26 and WZB-27.

Although several targets along glycolysis pathway have been explored, GLUT1 was only recently targeted. Basal glucose transport has not yet been applied as an anticancer approach, not because it is not a good target, but because there is not enough inhibitors were identified. We have identified such inhibitors from our recently built compounds 35 library. The new anticancer compounds and the new anticancer target will generate novel methods and strategies in fighting with cancers.

CHAPTER 3: DESIGN AND SYNTHESIS OF PHENOLIC BENZOATE ESTERS

From preliminary results, WZB-27 exhibited strong glucose uptake inhibitory activity in the lung cancer cell line H1299, and killed cancer cells without affecting normal cells.

Based on this interesting result, we planned to synthesize more derivatives of WZB-27.

In order to further study the Structure-Activity-Relationship (SAR), we decided to introduce various substituents with different electron affinities-neutral, donating and withdrawing-into the core benzene ring.

3.1 Design and Synthesis of Phenolic Benzoate Esters

Based on the structure of WZB-27, we planned to synthesize several series of analogs of

WZB-27. In order to lower the molecular weight, one galloyl group on the core aromatic ring could be replaced by different substituents, such as methoxyl group, fluorine, chlorine and hydrogen. Different substituents could also be introduced into the pendant aromatic rings and the number of hydroxyl groups on the pendant aromatic rings could also be changed. (Figure 3.1)

Figure 3.1. Design the analogs of WZB-27.

3.1.1 Design and synthesis of 3,4,5-trihydroxy benzoate esters

The 3,4,5-trihydroxy benzoate esters could be prepared from 3,4,5-tribenzyloxybenzoyl chloride and phenols. In order to synthesize 3,4,5-tribenzyloxybenzoyl chloride (4), we started from commercially available methyl 3,4,5-trihydroxybenzoate (1), which reacted with benzyl bromide in the presence of potassium carbonate to give the 3,4,5- tribenzyloxybenzoate (2) in 86% yield. Hydrolysis of 3,4,5-tribenzyloxybenzoate (2) with sodium hydroxide in refluxing methanol, followed by acidic work-up provided 3,4,5- tribenzyloxybenzoic acid (3) in 98% yield, which was treated with oxalyl chloride in the presence of catalytic amount of DMF in toluene to give the 3,4,5-tribenzyloxybenzoyl chloride (4) in 89% yield.189, 190, 188 (Scheme 3.1)

Scheme 3.1. Synthesis of 3,4,5-tribenzyloxybenzoyl chloride.

For the esterification, we tried Steglich esterification191 (DCC and catalytic amount of

DMAP in DCM) and modified alcoholysis of benzoyl chlorides with stoichiometric amount of DMAP.188 In comparison, the latter method gave a clean reaction and easy purification. We used the modified alcoholysis of benzoyl chlorides throughout the synthesis of phenolic benzoate ester derivatives. The benzoyl chloride 4 reacted with pyrogallol (5a) and 3,4,5-trihydroxybenzoate 5b in the presence of stoichiometric amount of DMAP in fresh distilled acetonitrile led to the corresponding O-acylation products 6a 38 and 6b in 86% and 78% yield, respectively.188 Then hydrogenolysis192 of 6a and 6b in the presence of palladium on activated carbon provided WZB-27 and WZB-75, respectively.

(Scheme 3.2)

Scheme 3.2. Synthesis of WZB-27 and WZB-75.

In 1997, Christopher A. Lipinski proposed “Lipinski’s Rule of Five” to evaluate druglikeness, or determine if a compound has a certain properties of pharmacological or biological activity that would make it a potential orally active drug.193, 194 Generally, a potential orally active drug has no more than one violation of the following criteria: 1) not more than 5 H-bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms); 2) not more than 10 H-bond acceptors (nitrogen or oxygen atoms); 3) molecular 39 weight (MWT) under 500; 4) an octanol-water partition coefficient log P of less than

5.193, 194 Two or more violations of “Lipinski’s Rule of Five” could result in poorer absorption or permeability. Compounds WZB-27 and WZB-75 have three violations with respect to the “Lipinski’s Rule of Five”. In order to fit both compounds into

“Lipinski’s Rule of Five”, we decided to cut off one galloyl group from the core ring to decrease the molecular weight, numbers of hydrogen bond donors and acceptors.

Meanwhile, we introduced several different substituents into the core ring so that we can get information on the influence of electron density on selected bioactivity. In this manner, we can figure out the role of the electron withdrawing groups (F, Cl), electron donating group (OMe) as well as neutral group (H) in the glucose transport inhibition.

WZB-26, which has an electron donating group on the core ring, was obtained from 3- methoxycatechol after acylation with benzoyl chloride 4 followed by hydrogenolysis.

WZB-89 and WZB-90, with electron-withdrawing groups on their core aromatic rings, were obtained in the same way from 3-fluorocatechol and 4-chlorocatechol, respectively.

(Scheme 3.3)

Scheme 3.3. Synthesis of WZB-26, WZB-89, WZB-90 and WZB-110. 40

All the compounds were submitted to test their glucose uptake inhibition and cell growth inhibition at the concentration of 30 µM by using H1299 lung cancer cell line in Prof.

Xiaozhuo Chen’s laboratory. The glucose uptake assay was carried out in incubated cells by adding our compounds. After incubating cells in serum-free DMEM media for 2 h and

Krebs-Ringer-Hepes (KRP) buffer for 30 min, glucose uptake was initiated by adding 1.0 mCi tritiated glucose and 1 mM regular glucose as final concentrations. Then glucose uptake was terminated by washing the cells with cold phosphate buffered saline after 20 min incubation with glucose. Finally, the cells were lysed with 0.2 mM NaOH and transferred to scintillation vials. The inhibitory activity of glucose uptake was analyzed by measuring the cell uptake of tritiated glucose in scintillation counter. The cell growth assay was carried out with a MTT proliferation assay kit (Cayman, Ann Arbor, MI). The mechanism of this assay is using the reduction of yellow MTT (3-(4, 5-Dimethylthiazol-

2-yl)-2, 5-diphenyltetrazolium bromide) to purple formazan in the mitochondria of living cells. (Figure 3.2) This reduction reaction only takes place when mitochondrial reductase enzymes are active, thus conversion can be directly correlated to the number of viable cells. The absorbance of this purple solution can be quantified by measuring at 570 nm by a spectrophotometer. The effectiveness of our compound in killing cancer cells can be obtained by measure the amount of purple formazan produced by cells treated with our test compound and compare with the amount from untreated cells. Generally, 5000 seeded cells in each well of a 96-well plate were treated with or without test compounds.

After 48 h treatment, 10 µL of MTT reagent was added to each well and incubated for 3 41 h. Then the culture medium was removed, 100 µL Crystal Dissolving Solution was added to each well, and the absorbance of viable cells was measured at 570 nm.3

Figure 3.2. Reduction of MTT to formazan.

WZB-27 exhibited the best activity in both glucose transport inhibition and cell growth inhibition among the 3,4,5-trihydroxy benzoate esters. When the electron donating group

(Ome) was introduced into the core ring, we prepared WZB-26, the activity of which in glucose transport inhibition was decreased from 86.1% to 58.4% compared with WZB-

27; however, the activity in cell growth inhibition was in the same level as WZB-27.

When strong electron withdrawing groups (F, Cl) were introduced into the core ring, we obtained WZB-89 and WZB-90, respectively. WZB-89 and WZB-90 displayed similar activity as WZB-27 in glucose uptake inhibition and cell growth inhibition. When the mild electron-withdrawing group (CO2Me) was introduced into the core ring, we made

WZB-75. The inhibitory activity in glucose uptake and cell growth of WZB-75 was dropped to a lower level compared with WZB-27. When there were only neutral substituents (H) on the core ring, we synthesized WZB-110, which showed only 32.1% inhibition in glucose uptake, and 27.8% inhibition in cell growth. The ratio of cell growth 42 inhibition to glucose uptake inhibition could inform us of the relationship between cell growth inhibition and glucose uptake inhibition. Ideally, the ratio would be 1.0 if the compound acted the same strength in both glucose uptake and cell growth, in other words, the compound showed inhibitory activity in cell growth by inhibiting glucose uptake. With respect to the ratio of cell growth inhibition to glucose uptake inhibition,

WZB-110 had the highest ratio and WZB-26 had the second highest ratio. Since WZB-

110 showed the poorest inhibitory activity in cell growth and WZB-26 showed the second best inhibitory activity in cell growth, which indicated that the cell growth inhibition was not firmly correlated with the glucose transport inhibition for the 3,4,5- trihydroxy benzoate esters. (Table 3.1)

Table 3.1Glucose uptake inhibition and cell growth inhibition for trihydroxyl benzoate esters

Y Y

O O O

Ar O O Ar X O Ar O O O O Ar Ar

In order to help find out what caused the differences in both inhibition activity, we did a molecular modeling study in Spartan’08 (Version 1.2.0, Wavefunction, Inc). The energy- minimized structure of WZB-26, 89, 90 and 110 shared the same conformation. (Figure

3.3) The pendant aromatic rings are almost stacked in WZB-26, 89, 90 and 110, but perpendicular in WZB-27. Thus the energy-minimized structure of WZB-27 could not overlap with WZB-26, 89, 90 and 110 very well. (Figure 3.4) The methyl ester on the core ring of WZB-75 might have correlation to one of the three pendant aromatic rings, which made the energy-minimized structure of WZB-75 was not well overlapped with the energy-minimized structure of WZB-27. (Figure 3.5) The angle (a) between the two planar rings in energy-minimized structure of WZB-27 is bigger than the angle (b) from the general scaffold of WZB compounds with two galloyl groups. (Figure 3.6) The bigger angle allows WZB-27 to reach more spaces when interacting with potential target, which might be the reason for WZB-27 showing better inhibitory activity.

Figure 3.3. Tube view of energy-minimized structure of WZB-26, 89, 90 and 110

Figure 3.4. Tube view of energy-minimized structure of WZB-27 (in wire view) with WZB-26, 89, 90 and 110 (in tube view).

Figure 3.5. View of energy-minimized structure of WZB-27 (in tube view) and

WZB-75 (in wire view).

Figure 3.6. Representative tube view of energy-minimized structure of tri-hydroxyl benzoate esters.

The pKa values of phenolic compounds have been correlated with their related bioactivity.195-198 The pKa values of the 3,4,5-trihydroxy benzoate esters were calculated in Epik (version 2.1107, Schrodinger, LLC.)199 (Figure 3.7) WZB-27 and WZB-75 had lower pKa values than the other 3,4,5-trihydroxy benzoate esters. Since WZB-27 had better inhibitory activity than the other 3,4,5-trihydroxy benzoate esters, it seemed no tight correlation between the pKa values and their inhibitory activity.

X Y

O O O a a a HO OH OH O O X O b O O b O O b HO OH OH c c OH OHc OH

d f f d HO OH HO OH e e OH OH WZB-27: X=H WZB-26: X=OMe,Y=H;WZB-89: X=F,Y=H

WZB-75: X=CO2CH3 WZB-90: X=H,Y=Cl;WZB-110: X=H,Y=H

HpKa* HpKa*

Ha 8.70 ± 1.0 Ha 9.00 ± 1.0 Hb 8.84 ± 1.0 Hb 9.14 ± 1.0 Hc 8.70 ± 1.0 Hc 9.00 ± 1.0 Hd 9.00 ± 1.0 Hd 9.00 ± 1.0 He 9.14 ± 1.0 He 9.14 ± 1.0 Hf 9.00 ± 1.0 Hf 9.00 ± 1.0 * pKa was calculated in Epik version 2.1107 * pKa was calculated in Epik version 2.1107

Figure 3.7. Calculated pKa values of 3,4,5-trihydroxy benzoate esters.

In conclusion, WZB-27 was the best compound from the 3,4,5-trihydroxy benzoate ester derivatives, which displayed the best inhibitory activity in both glucose transport and cell growth. The larger pocket was formed by two pendant aromatic rings and the core aromatic ring in WZB-27, which showed in Figure 3.1.5, might be the unique reason for

WZB-27 having better inhibitory activity. Further investigation the role of 3,4,5- trihydroxy groups in both inhibitory activity led us changing the number of hydroxyl groups on the pendant aromatic rings.

3.1.2 Design and synthesis of di-hydroxyl benzoate esters

The strategy to further lower the molecular weight, decrease the number of hydrogen bond donors and acceptors is changing the number of hydroxyl groups on the pendant aromatic rings. Based on this idea, 3, 4- di-hydroxyl benzoate esters and 3, 5-di-hydroxyl benzoate esters were designed and synthesized. Retro-synthetically, the di-hydroxyl 49 benzoate esters can be obtained via the modified alcoholysis of di-benzyloxybenzoyl chloride and phenols in the presence of stoichiometric amount of DMAP. (Figure 3.8)

Figure 3.8 Design and retro-synthetic analysis of di-hydroxyl benzoate esters

In order to make the di-hydroxyl benzoate esters, we first synthesized the 3, 4- dibenzyloxybenzoyl chloride (11b) and 3, 5-dibenzyloxybenzoyl chloride (11a).

Commercially available 3, 5-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid were perbenzylated and the resulting esters 10a and 10b were hydrolyzed, followed by converting the acid into the corresponding 3, 5-dibenzyloxybenzoyl chloride (11a)5 and

3, 4-dibenzyloxybenzoyl chloride (11b)200, 201, 5 in good yields. (Scheme 3. 4)

Scheme 3.4. Synthesis of dibenzyloxybenzoyl chlorides 11a and 11b.

3.1.2.1 Synthesis of 3, 5-dihydroxyl benzoate esters

Pyrogallol 5a reacted with 3, 5-dibenzyloxybenzoyl chloride 11a in the presence of stoichiometric amount of DMAP provided benzyl protected ester 12 in 89% yield, followed by hydrogenolysis in the presence of catalytic amount of palladium on activated carbon produced WZB-111 in 91% yield. (Scheme 3.5)

Scheme 3.5. Synthesis of 3, 5-dihydroxyl benzoate ester WZB-111.

WZB-112~114 were synthesized with the same procedure as preparing WZB-111.

WZB-112, with an electron donating group on the core ring, was prepared in 78% yield 51 after two steps from 3-methoxycatechol; WZB-113 and WZB-114, with electron withdrawing groups on their core rings, were obtained in 81% and 87% yield after two steps from 3-fluorocatechol and 4-chlorocatechol, respectively.5 (Scheme 3.6)

Scheme 3.6. Synthesis of 3, 5-dihydroxyl benzoate esters WZB-112, 113 and 114.

3.1.2.2 Synthesis of 3, 4-dihydroxyl benzoate esters

The 3, 4-dihydroxyl benzoate esters WZB-119~122 were prepared via the same procedure as making 3, 5-dihydroxyl benzoate esters. The phenols 5a, 7a, 7b and 7c reacted with the 3, 4-dibenzyloxybenzoyl chloride (11b) led to esters 14, 15a, 15b and

15c, respectively. The final compounds WZB-119~122 were obtained after removing the benzyl groups.5 (Scheme 3.7 and Scheme 3.8)

Scheme 3.7. Synthesis of 3, 4-dihydroxyl benzoate ester WZB-119.

Scheme 3.8. Synthesis of 3, 4-dihydroxyl benzoate esters WZB-120, 121 and 122.

The inhibitory activity in glucose transport and cell growth of the di-hydroxyl benzoate esters were listed in Table 3.2. One hydroxyl group was taken off from 3,4,5-trihydroxy benzoate ester WZB-27 provided the corresponding 3, 5-dihydroxyl benzoate ester

WZB-111 and 3, 4-dihydroxyl benzoate ester WZB-119. The activity for WZB-111 and 53

WZB-119 in glucose transport inhibition were increased to a higher level compared with the 3,4,5-trihydroxy benzoate ester WZB-27, but the cell growth inhibition remained the same level as WZB-27. Interestingly, the WZB-112 and WZB-120, with electron donating group (OMe) in their core aromatic rings, showed 69.4% and zero inhibitory activity in glucose transport, respectively. It is worthwhile to note that the cell growth inhibition of WZB-120 was in the same level as WZB-27, but the cell growth inhibition of WZB-112 was decreased into a lower level. WZB-112, 113, 121, and 122, with electron withdrawn groups on their core aromatic rings, showed increased activity in glucose transport inhibition compared with WZB-27, whereas the inhibitory activity in cell growth were kept in the same level as WZB-27. With respect to the ratio of cell growth inhibition to glucose uptake inhibition, apparently, WZB-122 had the highest ratio; meanwhile, WZB-122 displayed the best inhibitory activity in cell growth among the di-hydroxyl benzoate esters. WZB-121 and WZB-119 had slight lower ratio compared with WZB-122, accordingly, WZB-121 and WZB-119 showed slightly decreased inhibitory activity in cell growth compared with WZB-122. Due to experimental error, we didn’t get reasonable inhibitory activity in glucose uptake for

WZB-120. To some extent, the inhibitory activity of the di-hydroxyl benzoate esters in cell growth related to their corresponding activity in glucose uptake.

Table 3.2. Glucose uptake inhibition and cell growth inhibition for dihydroxyl benzoate esters.

The pKa values of the 3, 5-di-hydroxyl benzoate esters and 3, 4-di-hydroxyl benzoate esters were calculated in Epik (version 2.1107, Schrodinger, LLC.)199 (Figure 3.9 and

Figure 3.10) The 3, 5-di-hydroxyl benzoate esters WZB-112~114 have the same pKa values, but these compounds showed various inhibitory activity in glucose uptake and cell growth. Meanwhile, the 3, 4-di-hydroxyl benzoate esters WZB-120~122 have the same pKa value, but these compounds also showed various inhibitory activity in glucose uptake and cell growth. It seemed no correlation between the pKa values and their inhibitory activity.

Figure 3.9. Calculated pKa values of 3, 5-di-hydroxyl benzoate esters.

Figure 3.10. Calculated pKa values of 3, 4-di-hydroxyl benzoate esters.

As di-hydroxyl benzoate esters WZB-111, 119 and 122 showed similar or better inhibitory activity than WZB-27 in glucose uptake and cell growth, it is important to compare their structure with WZB-27. The energy-minimized structures of WZB-111,

119 and 122 indicated intramolecular hydrogen bond was formed between the two meta-

OH groups on pendant aromatic rings. To some extent, the energy-minimized structures of WZB-111, 119 and 122 can overlap with WZB-27, which might be demanded for binding potential target. The hydrogen bond formed in the di-hydroxyl benzoate esters might be the critical reason for their better inhibitory activity. (Figure 3.11, 3.12 and

3.13)

Figure 3.11. Overlap view of energy-minimized structure of WZB-27 (in wire

view), WZB-111 (in tube view).

Figure 3.12. Overlap view of energy-minimized structure of WZB-27 (in wire view), WZB-119 (in tube view).

Figure 3.13. Overlap view of energy-minimized structure of WZB-27 (in wire view) and 122 (in tube view). 58

In summary, both of the 3, 5-dihydroxyl benzoate esters and 3, 4-dihydroxyl benzoate esters showed better activity in glucose transport inhibition than their corresponding

3,4,5-trihydroxy benzoate esters, but the activity in cell growth inhibition were not enhanced. From the information provided in Table 3.1.2, it showed that the 3, 4-dihydroxyl and 3, 5-dihydroxyl groups were essential for increasing the inhibition activity in glucose transport. The hydrogen bond might play an important role in keeping or enhancing both inhibitory activities in glucose uptake and cell growth.

3.1.3 Design and synthesis of mono-hydroxyl benzoate esters

Comparing the inhibitory activity of the di-hydroxyl benzoate esters with tri-hydroxyl benzoate esters, we found decreasing the number of hydroxyl groups on the pendant aromatic rings helped to increase the inhibitory activity in glucose uptake and keep the inhibitory activity in cell growth. Based on this, we decided to further lower the number of hydroxyl groups on the pendant aromatic rings in order to get more information on the role of hydroxyl groups. Meanwhile, the compounds would be strictly fit into the

“Lipinski's Rule of Five”. Based on this idea, we planned to synthesize ortho-hydroxyl benzoate esters, meta-hydroxyl benzoate esters and para-hydroxyl benzoate esters.

(Figure 3.14)

Figure 3.14.Design the mono-hydroxyl benzoate esters.

3.1.3.1 Synthesis of ortho-hydroxyl benzoate esters

Theoretically, the ortho-hydroxyl benzoate esters can be synthesized from the ortho- benzyloxybenzoyl chloride and phenols via our established method. The ortho- benzyloxybenzoyl chloride 19202 was prepared from salicylic acid (16), which reacted with benzyl bromide to afford benzyl protected ortho-hydroxyl benzoate ester 17.

Followed by hydrolysis of the ester with lithium hydroxide to give the benzoic acid 18, which was treated with oxalyl chloride and catalytic amount of DMF to produce the acid chloride 19 in 77% yield after two steps. (Scheme 3. 9)

Scheme 3.9. Synthesis of ortho-benzyloxybenzoyl chloride 19.

The ortho-hydroxyl benzoate esters WZB-127~130 were prepared from ortho- benzyloxybenzoyl chloride 19 and pyrogallol, 3-methoxycatatechol, 3-fluorocatechol and

4-chlorocatechol, respectively, in the presence of DMAP in dry acetonitrile, followed by 60 deprotection of the benzyl group to liberate the free phenols. All the ortho-hydroxyl benzoate esters were obtained with satisfied yields. (Scheme 3.10)

Scheme 3.10. Synthesis of ortho-hydroxyl benzoate esters WZB-127~130.

The ortho-hydroxyl benzoate esters showed decreased inhibitory activity in both glucose uptake and cell growth compared with WZB-27. Although the ortho-hydroxyl benzoate esters didn’t show good activity, they followed the same trend in the Structure-Activity-

Relationship (SAR): introducing electron-donating group to core aromatic ring decreased the inhibitory activity in glucose uptake, such as WZB-128. Somehow, introducing electron-withdrawing groups to core aromatic ring decreased the inhibitory activity in glucose transport but enhanced the inhibitory activity in cell growth, such as WZB-129 and WZB-130. The ratio of cell growth inhibition to the glucose uptake inhibition showed WZB-129 was the best compound with respect to the inhibitory activity in cell 61 growth. To some extent, the inhibitory activity of the ortho-hydroxyl benzoate esters in cell growth might be caused by their inhibitory activity in glucose uptake. (Table 3. 3)

Table 3.3. Glucose uptake inhibition and cell growth inhibition for ortho-hydroxyl benzoate esters.

The pKa values of the ortho-hydroxyl benzoate esters were calculated in Epik (version

2.1107, Schrodinger, LLC.)199 (Figure 3.15) WZB-92~94 have the same pKa value, but these compounds showed various inhibitory activities in glucose uptake and cell growth.

It seems no correlation between the pKa values and their inhibitory activity. 62

Figure 3.15 Calculated pKa values of ortho-hydroxyl benzoate esters

The energy-minimized structures of WZB-127 and 128 indicate intra-molecular hydrogen-bond was formed between oxygen on the linkage carbonyl (C=O) and the ortho-OH groups on pendant aromatic rings. To some extent, the energy-minimized structures of ortho-hydroxyl benzoate esters overlapped with WZB-27. The intramolecular H-bond formed between ortho-hydroxyl group and carbonyl group on the linkage might be the possible reason for losing inhibitory activity. (Figure 3.16)

Figure 3.16. Overlap view of energy-minimized structure of WZB-27 (in wire

view), WZB-127 and 128 (in tube view) based on core ring.

3.1.3.2 Synthesis of para-hydroxyl benzoate esters

The ortho-hydroxyl benzoate esters showed decreased inhibitions in both glucose transportation and cell growth compared with WZB-27. In order to examine if the location of the hydroxyl groups on the pendant aromatic rings would affect the inhibitory activity, we designed the para-hydroxyl benzoate esters. Based on this idea and the results from the 3, 4-dihydroxyl benzoate esters, we decided to cut off the meta-hydroxyl group and synthesize para-hydroxyl benzoate esters. In order to prepare the para- hydroxyl benzoate esters, we would have to synthesize the para-benzyloxybenzoyl chloride 25. The synthesis started from para-hydroxyl benzoic acid (22), which reacted with benzyl bromide produced the ester 23, followed by hydrolysis led to acid 24, which 64 was transferred into benzoyl chloride 25 via the same methodology as previous.203, 5

(Scheme 3.11)

Scheme 3.1.11. Synthesis of para-benzyloxybenzoyl chloride 25.

With the para-benzyloxybenzoyl chloride 25 in hand, we started making the 4-hydroxyl benzoate esters. The para-hydroxyl benzoate esters WZB-91~94 and WZB-103 were successfully synthesized via alcoholysis of the para-benzyloxybenzoyl chloride 25 with the corresponding phenols (5a, 7a, 7b, 7c and 7d) followed by hydrogenolysis deprotection of the benzyl groups.5 (Scheme 3.12)

Scheme 3.12. Synthesis of para-hydroxyl benzoate esters WZB-91~94 and WZB-103.

The inhibitory activity in glucose uptake and cell growth of the para-hydroxyl benzoate esters WZB-91~94 were increased when comparing with the o-hydroxyl benzoate esters.

The para-hydroxyl benzoate esters WZB-91~94 also showed same level of inhibitory activity in both glucose transportation and cell growth compared with the 3,4,5- trihydroxy benzoate ester WZB-27. (Table 3.1.4) WZB-93, with an electron donating group (OMe) on the core ring, showed the similar inhibitory activity in glucose uptake as

WZB-91 and 3,4,5-trihydroxy benzoate ester WZB-27. Interestingly, the inhibitory activity in glucose transportation led to two directions when different electron withdrawing groups were introduced into the core aromatic ring. WZB-94, which has chlorine on the core ring, showed decreased inhibitory activity in glucose transportation when comparing with WZB-91 or WZB-27. Comparatively, WZB-92, which has 66 fluorine on the core ring, showed increased inhibitory activity in glucose transportation compared with WZB-91 or WZB-27. WZB-103, with only protons on the core ring, displayed decreased inhibitory activity in glucose uptake and cell growth compared with other members in the para-hydroxyl benzoate esters. WZB-103 has the same ratio of cell growth inhibition to glucose uptake inhibition as WZB-27, however, WZB-103 showed poorer inhibitory activity than WZB-27. (Table 3.4)

Table 3.4. Glucose uptake inhibition and cell growth inhibition for para-hydroxyl benzoate esters.

The pKa values of the para-hydroxyl benzoate esters were calculated in Epik (version

2.1107, Schrodinger, LLC.)199 (Figure 3.17) WZB-91 and WZB-103 had lower pKa values than the other para-hydroxyl benzoate esters, but WZB-91 had better inhibitory 67 activity than WZB-103. Meanwhile, WZB-92~94 had the same pKa values, but these compounds showed various inhibitory activity in glucose uptake and cell growth. It seemed no tight correlation between the pKa values and their inhibitory activity.

Figure 3.17. Calculated pKa values of para-hydroxyl benzoate esters.

The para-hydroxyl benzoate esters showed the similar activity in both glucose uptake inhibition and cell growth inhibition. The energy minimized structures of para-hydroxyl benzoate ester WZB-91 and WZB-92 overlapped with WZB-27 very well, which might be the reason for them showing the similar inhibitory activity in glucose uptake and cell growth. (Figure 3.18)

Figure 3.18 Overlap view of energy-minimized structure of WZB-27 (in wire

view), WZB-91 and 92 (in tube view) based core ring

3.1.3.3 Synthesis of meta-hydroxyl benzoate esters

Increased inhibitory activity was observed when the hydroxyl group moved from ortho position to para position. Then we should investigate if it can enhance the activity when the position of hydroxyl group is switched to the meta position on the pendant aromatic rings. Thus, synthesizing the meta-hydroxyl benzoate esters is very essential.

Synthetically, the meta-hydroxyl benzoate esters could be prepared from alcoholysis of meta-benzyloxybenzoyl chloride (31) with different phenols. In order to synthesize the meta-hydroxyl benzoate esters, we started from the commercially available meta- hydroxyl benzoic acid 28. It was reacted with benzyl bromide led to 29, followed by 69 hydrolysis provided the benzoic acid 30, which was converted into the acid chloride 31 in the presence of oxalyl chloride and catalytic amount of DMF.204, 5 (Scheme 3.13)

Scheme 3.13. Synthesis of meta-benzyloxybenzoyl chloride 31.

With the meta-benzyloxybenzoyl chloride 31 in hand, we synthesized four different meta-hydroxyl benzoate esters WZB-115, 116, 117 and 118. WZB-115 was synthesized from pyrogallol, which reacted with meta-benzyloxybenzoyl chloride 31 gave the benzyl protected benzoate ester 32, followed by hydrogenolysis deprotection of the benzyl group. (Scheme 3.14)

Scheme 3.14. Synthesis of meta-hydroxyl benzoate ester WZB-115.

WZB-116, 117 and 118 were prepared with same procedure from 3-methoxycatechol, 3- fluorocatechol and 4-chlorocatechol, respectively. (Scheme 3.15) WZB-136 and WZB- 70

140 were prepared from 4-chlororesorcinol and 3-chlororesorcinol in the same manner.

(Scheme 3.16)

Scheme 3.15. Synthesis of meta-hydroxyl benzoate esters WZB-116~118.

X X Y Y O O O O X 31 H2 DMAP Pd/C Y O O O O

CH3CN CH3OH HO OH OBn OBn OBn OBn

7e:X=H,Y=Cl 33d:X=H,Y=Cl,67% WZB-136:X=H,Y=Cl,100% 7f:X=Cl,Y=H 33e: X = Cl, Y = H, 79% WZB-140:X=Cl,Y=H,90%

Scheme 3.16. Synthesis of meta-hydroxyl benzoate esters WZB-136 and WZB-140.

After successfully synthesized the meta-hydroxyl benzoate esters, we investigated their inhibitory activity in both glucose uptake and cell growth in H1299 lung cancer cell lines.

WZB-115 showed better activity in both glucose transport inhibition and cell growth inhibition than 3,4,5-tri-hydroxyl benzoate esters, 3, 4-dihydroxyl benzoate esters, 3, 5- dihydroxyl benzoate esters, and previous prepared mono-hydroxyl benzoate esters.

WZB-116, with an electron donating group (OMe) on the core aromatic ring, displayed decreased inhibitory activity in glucose transportation and cell growth compared with 71

WZB-27. In the same trends as mentioned above, WZB-117 and WZB-118, with electron withdrawing groups on their core aromatic rings, showed increased inhibitory activity in glucose uptake compared with WZB-27. Both WZB-136 and WZB-140 showed better inhibitory activity in glucose uptake than WZB-27. However, WZB-140 showed decreased inhibitory activity in cell growth and WZB-136 showed no activity on the cell growth inhibition, on the contrary, it promoted the cancer cell growth. WZB-115 had the highest ratio and WZB-117 had the second highest ratio in cell growth inhibition to glucose uptake inhibition; both compounds showed very strong inhibitory activity in glucose uptake and cell growth. Considering the inhibitory activity of all the meta- hydroxyl benzoate esters, the compound with the higher ratio of inhibitory activity in cell growth to glucose uptake showed better inhibitory activity in glucose uptake and cell growth. To some extent, the meta-hydroxyl benzoate esters induced cell death by inhibiting glucose uptake. (Table 3. 5)

Table 3.5. Glucose uptake inhibition and cell growth inhibition for meta-hydroxyl benzoate esters.

The pKa values of the meta-hydroxyl benzoate esters were calculated in Epik (version

2.1107, Schrodinger, LLC.)199 (Figure 3.1.3.6) WZB-115 and WZB-140 had lower pKa values than the other meta-hydroxyl benzoate esters, but WZB-115 had better inhibitory activity than WZB-140. Meanwhile, WZB-116~118 and WZB-136 had the same pKa values, but these compounds showed various inhibitory activity in glucose uptake and cell growth. It indicated that no tight correlation between the pKa values and their inhibitory activity.

Figure 3.19. Calculated pKa values of meta-hydroxyl benzoate esters.

The internal H-bonding and two pockets in the energy-minimized structure of WZB-115 might make it specific and having the best inhibitory activity in both glucose uptake and cell growth. When considering the conformation of WZB-117, the pocket with H-bond might be unique for the inhibitory activity in glucose uptake and cell growth. (Figure

3.20) WZB-115 has a larger pocket than WZB-27 (the angle b between two planar pendant rings is bigger than the angle a), which might be the reason for WZB-115 showing better activity than WZB-27. (Figure 3.21) The energy-minimized structure of

WZB-115 can overlap with WZB-27 very well to some point. (Figure 3.22) The internal

H-bond formed in the meta-hydroxyl benzoate esters might be another contribution to their better inhibitory activity.

Figure 3.20 Tube views of energy-minimized structure of WZB-115, WZB-117

Figure 3.21. Tube view of angles between two planar rings in energy-minimized

structure of WZB-27 and WZB-115.

Figure 3.22. Overlap view of energy-minimized structure of WZB-27 (in wire

view) and WZB-115 (in tube view) based core ring.

3.2 Design and Synthesis of Analogs of Phenolic Benzoate Esters

In order to further investigate the importance of the hydroxyl groups on the pendant rings, we replaced the hydroxyl groups with different substituents. We chose electron- donating group (OMe) and electron-withdrawing group (F) to replace the hydroxyl groups and designed two different analogs of phenolic benzoate esters.

3.2.1 Synthesis of the fluoro-benzoate ester derivatives

Fluoro groups played a very important role in medicinal chemistry. It was reported that fluorine could improve activity and metabolic properties for the potential anticancer agents as well as in the market drugs.205-209 Based on this, we designed and synthesized 76 the fluoro-benzoate esters to investigate the role of fluoro groups in the inhibitory activity. The fluoro-benzoate ester derivatives were prepared in one step from the commercially available fluorobenzoyl chlorides (34~36) and different phenols. WZB-78,

WZB-79 and WZB-80, the relative analogs of the 3,4,5-trihydroxy benzoate esters

WZB-27, WZB-75 and WZB-26 were prepared in good yields after a single step.

(Scheme 3.17)

Scheme 3.17. Synthesis of 3,4,5-trifluorobenzoate esters WZB-78~80.

Then the fluoro analogs of 3, 4-dihydroxyl benzoate esters were designed and synthesized. The 3, 4-difluorobenzoate esters WZB-82, WZB-83 and WZB-84 were synthesized in satisfactory yields from coupling the 3, 4-difluorobenzoyl chloride 35 with the phenols 5a, 5b and 7a. (Scheme 3.18) 77

Scheme 3.18. Synthesis of 3, 4-di-fluorobenzoate esters WZB-82~84.

Finally, we introduced trifluoromethyl on the pendant aromatic rings and synthesized 4- fluoro-3-(trifluoromethyl) benzoate esters WZB-85~88. (Scheme 3.19)

Scheme 3.19. Synthesis of 4-fluoro-3-(trifluoromethyl) benzoate esters WZB-85~88.

3,4,5-trifluorobenzoate ester WZB-78 showed almost no inhibition in glucose uptake compared with the relative 3,4,5-trihydroxy benzoate ester WZB-27. WZB-79 showed decreased inhibitory activity in glucose uptake compared with its parent compound

WZB-75. The 3, 4-difluoro benzoate esters WZB-82 and WZB-83 showed very poor inhibitory activity in glucose uptake. All the other fluorobenzoate esters showed much less inhibitory activity in glucose uptake than their parent compounds from hydroxyl benzoate esters. WZB-85 showed decreased inhibition in cell growth compared with

WZB-121. WZB-88 showed better inhibitory activity in cell growth than WZB-85, but still lower than WZB-119. The rest of fluoro benzoate esters were not tested in cell growth inhibition. WZB-88 showed high ratio in cell growth inhibition to glucose uptake inhibition, which indicated it might act in a different way in the cell growth inhibition with the hydroxyl benzoate esters. (Table 3.6)

Table 3.6. Glucose uptake inhibition and cell growth inhibition for fluoro-benzoate esters

Glucose transport Cell growth Compd # Ar X Y inhibitiona,b(%) inhibitiona,b(%) ratiod

c WZB-27 3,4,5-(OH)3-C6H2 - H 86.1 ± 1.0 39.9 ± 5.0 0.46

WZB-78 3,4,5-(F)3-C6H2 - H 10.8 ± 1.2 - -

WZB-79 3,4,5-(F)3-C6H2 - CO2Me 57.8 ± 3.0 - -

WZB-80 3,4,5-(F)3-C6H2 OMe H 15.7 ± 1.4 - -

WZB-82 3,4-(F)2-C6H3 - H 17.3 ± 2.2 - -

WZB-83 3,4-(F)2-C6H3 OMe H 28.7 ± 4.1 - -

WZB-84 3,4-(F)2-C6H3 - CO2Me 30.8 ± 3.7 - -

WZB-85 3-CF3-4-F-C6H3 F H 27.6 ± 5.1 15.5 ± 5.9 0.56

WZB-86 3-CF3-4-F-C6H3 - H 12.8 ± 5.4 - -

WZB-87 3-CF3-4-F-C6H3 - CO2Me 75.3 ± 9.2 - -

WZB-88 3-CF3-4-F-C6H3 OMe H 10.2 ± 2.2 30.5 ± 5.5 2.99

a Data were provided by Yi Liu in Prof. Xiaozhuo Chen's lab. Compounds were tested at 30 M b Untreated cells served as negative controls (0% inhibition). c Data were presented as mean ± standard deviation. d The ratio is the inhibition of cell growth to the inhibition of glucose transportation.

Comparison of the energy-minimized conformation of WZB-27 and WZB-78 showed the two pendant aromatic rings from WZB-78 were perpendicular to the rings in WZB-

27, which might be the reason for the loss of activity in glucose uptake inhibitory activity. (Figure 3.23) 80

Figure 3.23. View of energy-minimized structure of WZB-27 (in wire view) and

WZB-78 (in tube view) based on core ring overlap.

3.2.2 Synthesis of the methoxyl-benzoate ester derivatives

After our unsuccessful approaches on introducing electron withdrawing groups on the pendant aromatic rings to improve the inhibitory activity in either glucose uptake or cell growth, we found the oxygen (hydrogen acceptor) from the hydroxyl group on pendant aromatic rings might be critical and necessary for both inhibitory activities. Based on this, we introduced electron donating groups (OMe) to seek the possible improvement.

We synthesized the 3,4,5-trimethoxyl benzoate ester analogs WZB-76, WZB-77, WZB-

81, WZB-101 and WZB-102 from 3,4,5-trimethoxybenzoyl chloride 37 and the corresponding phenols 5a, 5b, 7a, 7b and 7c. (Scheme 3.20) 81

Scheme 3.20. Synthesis of 3,4,5-trimethoxybenzoate esters.

Then we synthesized 2, 6-di-methoxybenzoate esters WZB-95 and WZB-98 from pyrogallol together with 2, 6-dimethoxybenzoyl chloride 39 and 3, 4-dimethoxybenzoyl chloride 38 in the presence of DMAP. (Scheme 3.21)

Scheme 3.21. Synthesis of di-methoxybenzoate esters WZB-95 and WZB-98.

We have discovered that the hydroxyl group on the meta position of pendant aromatic rings played an important role in enhancing the inhibitory activity in glucose uptake and cell growth. Thus we decided to synthesize meta-methoxy benzoate ester to see if the meta-methoxyl group has any influence on both inhibitory activities. 3-methoxybenzoate ester WZB-96 and WZB-97 were synthesized in the same manner as mentioned above.

(Scheme 3.22)

Scheme 3.22. Synthesis of meta-methoxybenzoate esters WZB-96 and WZB-97.

All the methoxyl benzoate esters were tested in basal glucose uptake and cell growth to examine their inhibitory activity. All the methoxyl benzoate esters showed decreased and even no inhibition activity in glucose uptake and cell growth. (Table 3.7) WZB-102 has the highest ratio in cell growth inhibition to glucose uptake inhibition, which means

WZB-102 showed better cell growth inhibition than glucose uptake inhibition compared with the other methoxyl benzoate esters, however, WZB-102 showed almost no inhibition in glucose uptake, such compound might act in a different way in the cell growth inhibition. WZB-98 might act in the same manner as WZB-102 as it also has a high ratio in the cell growth inhibition to glucose uptake inhibition. But overall, both compounds still showed lower inhibitory activity in cell growth than the hydroxyl benzoate esters.

Table 3.7. Glucose uptake inhibition and cell growth inhibition for methoxybenzoate esters.

Glucose transport Cell growth Compd # Ar X Y inhibitiona,b(%) inhibitiona,b(%) ratiod

c WZB-27 3,4,5-(OH)3-C6H2 - - 86.1 ± 1.0 39.9 ± 5.0 0.46

WZB-76 3,4,5-(OMe)3-C6H2 - - 41.1 ± 1.5 - -

WZB-81 3,4,5-(OMe)3-C6H2 OMe H 33.3 ± 3.6 22.2 ± 3.4 0.66

WZB-102 3,4,5-(OMe)3-C6H2 H Cl 1.7 ± 1.1 20.6 ± 2.1 12.5

WZB-101 3,4,5-(OMe)3-C6H2 F H 7.9 ± 2.6 19.6 ± 3.6 0.46

WZB-95 2,6-(OMe)2-C6H3 - - 75.8 ± 4.2 22.0 ± 3.8 0.29

WZB-98 3,4-(OMe)2-C6H3 - - 0±3.9 10.0 ± 1.1 10.0

WZB-96 3-OMe-C6H4 - - 66.2 ± 1.8 21.9 ± 5.4 0.29

WZB-97 3-OMe-C6H4 OMe H 32.1 ± 0.2 18.3 ± 4.3 0.57 a Data were provided by Yi Liu in Prof. Xiaozhuo Chen's lab. Compounds were tested at 30 M b Untreated cells served as negative controls (0% inhibition). c Data were presented as mean ± standard deviation. d The ratio is the inhibition of cell growth to the inhibition of glucose transportation. 84

Viewing the energy-minimized conformations of WZB-76 and WZB-98, the pendant aromatic rings are almost perpendicular to the rings of WZB-27, which might be the reason for losing the activity in glucose uptake inhibition. (Figure 3.24)

Figure 3.24. View of energy-minimized structure of WZB-27 (in wire view),

WZB-76 and WZB-98 (in tube view) based on core ring overlap

3.2.3 Other derivatives of phenolic benzoate ester

We have obtained detailed information on the Structure-Activity-Relationship (SAR) of analogs of WZB-27 after introducing different substituents into the core aromatic ring 85 and pendant aromatic rings. Besides these modifications on the aromatic rings, the linkage groups between the aromatic rings are another structure interest for the further understanding of SAR. Thus, we designed and synthesized several analogs of WZB-117 by changing the ester linkage into structurally similar groups so that we could compare the inhibitory activity of these analogs in glucose uptake and cell growth with WZB-117, which would help us get more information on the SAR.

First, we synthesized WZB-158, which is the reverse ester of WZB-117. The synthesis started from mono protection of the hydroxyl groups on resorcinol (41) to 3-

(benzyloxy)phenol (42). The reversed ester WZB-158 was synthesized after Steglich esterification of 42 and 3-fluorophthalic acid (43), followed by deprotection of the benzyl group. (Scheme 3.23)

Scheme 3.23. Synthesis of WZB-158.

WZB-158 showed decreased inhibition activity in glucose transport and cell growth when compared with WZB-117. (Table 3.8) The positions of the carbonyl group and the aryl oxygen seem to play an important role in both inhibition activity, especially in the cell growth inhibition. 86

Table 3.8. Glucose uptake inhibition and cell growth inhibition of WZB-158.

Then we wanted to examine if the length of the linkage played any role in the glucose uptake inhibition and cell growth inhibition. WZB-159 and WZB-162, with one carbon extension from WZB-158 and WZB-117, were prepared. (Scheme 3.2.8 and scheme

3.2.9) The synthesis of WZB-159 started from ethyl 3-hydroxybenzoate (44), after appropriate protection and reduction reaction led to 3-(methoxymethoxy)phenyl- methanol (46), which esterified with 3-fluorophthalic acid 43 followed by deprotection of the MOM group provided WZB-159. (Scheme 3.24)

CO Et CO Et 2 MOMCl 2 LAH OH K2CO3 92% OH 81% OMOM OMOM 44 45 46 O

CO2H 1) DCC, OH OH DMAP O + O CO2H 2) HCl/EtOAc F OMOM 51% for 2 steps F O

43 46 OH WZB-159

Scheme 3.24. Synthesis of WZB-159.

WZB-162 was synthesized from lithium 3-(methoxymethoxy)benzoate (47) and (3- fluoro-1,2-phenylene)dimethanol (48) in 15% yield after two steps. (Scheme 3.25) 87

Scheme 3.25. Synthesis of WZB-162.

Both WZB-159 and WZB-162 showed decreased inhibition activity in glucose transport when compared with WZB-117. Moreover WZB-159 showed stimulatory activity on the lung cancer cell growth instead of inhibitory activity, WZB-162 showed very poor inhibition activity in the cell growth. (Table 3.9) WZB-159 also showed decreased activity in both glucose transport inhibition and cell growth inhibition when compared with WZB-158, which stated the two atoms linkage is better than the extended linkage.

Table 3.9. Glucose uptake inhibition and cell growth inhibition of WZB-159 and WZB-

162.

3.3 Stability Study on Selected Benzoate Esters

The phenolic benzoate esters showed mild to strong activity in glucose transport inhibition and cell growth inhibition in H1299 lung cancer cells. However, the benzoate esters can be easily hydrolyzed under basic condition210-213 or acidic condition,214 even at near-critical water without any presence of acid or base catalyst.215 In order to investigate their activity in vivo, the stability of the phenolic benzoate esters were examined. We chose WZB-115 and WZB-117 for the stability study. The study was carried out in human serums by tracking with HPLC.

The general procedure of testing WZB-115 was as follows. 8.0 mL human blood was donated by Prof. Xiaozhuo Chen. The blood was ready after standing on bench for 15.0 min till blood clots, then centrifuged at 10,000 RPM for 10 min at 4 °C. The serum (up layer, slightly yellow) was separated and stored at -80 °C. The WZB-115 solution (6.8 mM in serum) was prepared with 2.5 µL of WZB-115 (68 mM in CH3CN) and 22.5 µL of serum. The solution of WZB-115 (6.8 mM in serum) was incubated at 37.0 °C and terminated at different time point (60 min, 2 h, 4 h, 8 h, 24 h and 48 h). 200.0 µL of

CH3CN was added to the solution of WZB-115 (6.8 mM in serum), and the solution was centrifuged at 10,000 RPM for 10 min at 4 °C. Then 200.0 µL of solution was taken from the clear up layer to analyze in HPLC (Discovery® C8 HPLC Column, 5 μm particle size,

L × I.D. 15 cm × 4.6 mm; Detector, SPD-10Avp, 214 nm and 254 nm) with water and

CH3CN. Standard curve of WZB-115 (area to compound concentration) was obtained at different concentrations (0 mM, 0.10 mM, 0.25 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 89

4.0 mM, 8.0 mM). The remaining WZB-115 was calculated based on the standard curve of WZB-115. WZB-117 was tested in the same manner as WZB-115.

The stability study showed that both WZB-115 and WZB-117 were degraded very fast.

There was only 41.6% of WZB-115 remaining after incubated at 37 °C for 1 hour, the degradation continued to drop in sixty additional minutes, and no obvious degradation was observed after incubated at 37 °C for another two hours. WZB-115 was completely degraded after being incubation at 37 °C for another four hours. WZB-117 degraded very quickly in the first hour and only 25.1% of WZB-117 was retained. The degradation of

WZB-117 was slowed down in the next hour and no obvious degradation was observed after 44 additional hours. The various numbers shown in Table 3.3.1 during the period of final 44 hours might come from experimental errors. (Table 3.10)

Table 3.10. Stability study of WZB-115 and WZB-117 in human serum.

Due to the instability of the phenolic benzoate esters in vivo, design and synthesis more hydrolytically stable analogs became an important issue.

3.4 Structure-Activity-Relationship (SAR)

Based on these results, we can draw conclusions regarding the the Structure-Activity-

Relationship of the benzoate esters. First of all, one of the pendant rings was not necessary for the activity in both glucose uptake inhibition and cell growth inhibition.

The electron withdrawing group on the core aromatic ring helped to keep or increase inhibitory activity, whereas the electron donating group decreased both inhibitory activities. More importantly, meta-hydroxyl group on the pendant aromatic rings played an important role on both inhibitory activities in glucose uptake and cell growth.

Inhibitory activity was decreased when electron donating or withdrawing groups were introduced to the pendant aromatic rings. So far, mono-meta-hydroxyl benzoate ester

WZB-115 showed the best inhibitory activity in both glucose uptake and cell growth. But considering the molecular weight and inhibition activity, WZB-117 was selected as the lead compound from the library of benzoate esters. (Figure 3.25)

Figure 3.25. Structure-Activity-Relationship of benzoate ester derivatives.

In summary, 3, 5-dihydroxyl and 3, 4-dihydroxyl benzoate esters showed relative stronger activity in both glucose transport inhibition and cell growth inhibition than their corresponding 3,4,5-trihydroxy benzoate esters. The para-hydroxyl and meta-hydroxyl benzoate esters also showed better activity in both glucose transport inhibition and cell growth inhibition than the corresponding 3,4,5-trihydroxy benzoate esters. Overall, the meta-hydroxyl group on the pendant aromatic rings is essential in providing good activity in both glucose uptake inhibition and cell growth inhibition. Electron withdrawing groups on the core aromatic ring increased the inhibitory activity in glucose uptake; meanwhile, electron donating group decreased the inhibitory activity in glucose uptake. The ester linkage played an important role in both inhibitory activity, and two atoms length is also essential in keeping or increasing both inhibition activity. With respect to the structural conformation, the size of the pocket, which formed by the core and pendant aromatic rings, might play some role in both inhibitory activity. On the other hand, the H-bond 92 might also contribute to better inhibitory activity in glucose uptake and cell growth. But the pKa values of the phenols seemed no correlation with both inhibitory activities. 93

CHAPTER 4: DESIGN AND SYNTHESIS OF MORE STABLE ANALOGS

Based on the information from chapter 3, the meta-hydroxyl group is essential for both inhibitory activities in glucose uptake and cell growth. Since the instability of the phenolic benzoate esters, it is necessary for us to design and synthesize hydrolytically more stable analogs of the benzoate esters. We designed a library of hydrolytically more stable compounds by locking the hydroxyl group at meta position on the pendant aromatic rings, and changing the linkage between the core aromatic ring and pendant aromatic rings. The library includes phenolic ethers, phenolic amides, phenolic amines and some other more hydrolytically stable analogs. (Figure 4.1)

Figure 4.1 Design of hydrolytically more stable analogs

The mechanism of hydrolysis of the esters and amides has been studied in detail.216-218

The amides, generally, have a slower hydrolysis rate compared with their corresponding esters. The hydrolysis rate of the amides was also slower than the esters in rat serum and liver homogenate.219 Thus, preparing the phenolic amide derivatives became important in order to understand the role of the stability in the inhibitory activity. Besides the phenolic amids, we also planned to make the phenolic ether derivatives and the phenolic amine 94 derivatives, which are not hydrolyzable in normal conditions. In this way, we built a library of hydrolytically more stable analogs of phenolic benzoate esters. We could answer two questions by preparing this library of hydrolytically stable analogs: 1) Is the ester bond just a linkage between the core aromatic ring and pendant aromatic rings? 2)

Does the carbonyl group or the aryl oxygen play any role in the inhibitory activity?

4.1 Design and Synthesis of Phenolic Ether Derivatives

In order to design hydrolytically more stable analogs and determine the importance of the carbonyl group in the ester linkage, we decided to synthesize carbonyl free phenolic ether derivatives. (Figure 4.2) The phenolic ethers are likely to be more hydrolytically stable than phenolic benzoate esters. The pendant aromatic rings on the phenolic ethers could freely rotate with ether linkage between core aromatic ring and pendant aromatic rings, which might result in different conformations from phenolic benzoate esters. On the other hand, the pKa values on the phenolic ethers might be affected. Therefore, we could get more information on the SAR of the phenolic ethers by investigating their inhibitory activity.

Figure 4.2. Design of phenolic ether derivatives.

Retrosynthetically, the phenolic ether derivatives could be obtained after coupling different phenols with benzyl chlorides. (Figure 4.3)

Figure 4.3. Retrosynthetic analysis of synthesizing phenolic ether derivatives.

First, we prepared the benzyl chlorides for the synthesis of the phenolic ethers. We started from commercially available 3-hydroxyl benzoic acid (49). Fischer esterification220 and MOM protection of the hydroxyl group in the presence of potassium carbonate at 0 °C furnished the methyl 3-(methoxymethoxy)benzoate (50)221 in good yield after two steps. Then the methyl 3-(methoxymethoxy)benzoate was treated with lithium aluminum hydride to generate the 3-(methoxymethoxy)benzyl alcohols,222, 221 which was then reacted with methanesulfonyl chloride (MsCl) in the presence of triethylamine to provide the benzyl chloride 51.223, 224 (Scheme 4. 1)

Scheme 4.1. Synthesis of the benzyl chloride 51. 96

With the benzyl chloride 51 in hand, we started to prepare the phenolic ether derivatives by coupling the benzyl chloride with different phenols. (Table 4.1) Six phenols were used in the coupling reaction with benzyl chloride 51 in a thoroughly degassed suspension of potassium carbonate in DMF at 70-80 oC.225, 226 The MOM protected phenolic ethers were subjected to deprotection with the solution of hydrochloric acid in ethyl acetate (1.2

M). Finally, six meta-hydroxybenzyl ethers (WZB-131~134, 137 and 141) were prepared in reasonable yields after two steps. (Table 4.1)

Table 4.1. Synthesis of phenolic ether derivatives.

a Compd # X Y Z (OR)n Yield

WZB-131 OR H H 1,2-(3-OH-C6H4CH2O)2 54

WZB-132 OMe H H 1,2-(3-OH-C6H4CH2O)2 70

WZB-133 H Cl H 3,4-(3-OH-C6H4CH2O)2 65

WZB-134 F H H 1,2-(3-OH-C6H4CH2O)2 46

WZB-137 H Cl H 1,3-(3-OH-C6H4CH2O)2 57

WZB-141 H H Cl 3,5-(3-OH-C6H4CH2O)2 72 a yield is based on two steps

Most phenolic ether derivatives showed similar inhibitory activity in glucose uptake as

WZB-115. WZB-131, with similar structure as WZB-115, showed the same inhibitory activity in glucose uptake as WZB-115, but lower inhibitory activity in cell growth.

WZB-132, with an electron donating group (OMe) on the core aromatic ring, showed decreased inhibitory activity in glucose uptake compared with WZB-131. The inhibitory 97 activity in glucose uptake was enhanced when electron withdrawing groups were introduced to the core aromatic ring. For example, WZB-133, 134, 137 and WZB-141 showed much better inhibitory activity in glucose uptake than WZB-132. With respect to the inhibitory activity in cell growth, WZB-141 showed the same activity as WZB-27 and was the best compound among the phenolic ethers. Unfortunately, WZB-137 displayed strong stimulatory activity in cell growth. With respect to the ratio of cell growth inhibition to glucose uptake inhibition, WZB-141, the best compound from the phenolic ethers, showed the highest ratio, which indicated the inhibitory activity of

WZB-141 in glucose uptake correlated with cell growth. (Table 4.2)

Table 4.2. Glucose uptake inhibition and cell growth inhibition for phenolic ether derivatives.

The pKa values of the phenolic ethers were calculated in Epik (version 2.1107,

Schrodinger, LLC.)199 There were almost no differences on the calculated pKa values among the phenolic ethers. The phenolic ether derivatives had higher pKa values than

WZB-27 and WZB-115. Most phenolic ether derivatives showed similar inhibitory activity in glucose uptake as WZB-115, and better activity than WZB-27. With respect to the inhibitory activity in cell growth, WZB-141 had the same activity as WZB-27, however, the other phenolic ethers showed poorer activity than WZB-27. Considering the inhibitory activity and the calculated pKa values, it indicated that the pKa values were not correlated with the inhibitory activity. (Figure 4.4)

Figure 4.4. Calculated pKa values for the phenolic ether derivatives.

Both WZB-134 and WZB-117 showed internal H-bonding, but the energy-minimized structures could not overlap well, which might be the reason for the difference in the cell growth inhibitory activity. (Figure 4.5) In the same manner, WZB-131 also could not overlap well with WZB-115, the pendant aromatic rings with no H-bond forming were perpendicular to each other, which might be the reason for losing cell growth inhibitory activity for WZB-131. Moreover, another possibility for the phenolic ether derivatives 100 showing less inhibitory activity might be the lack of a carbonyl group (which could be important for binding to the target). (Figure 4.6) WZB-141, the compound with best inhibitory activity in cell growth from the current phenolic ethers, showed the pendant aromatic rings could reach as much space as WZB-115. (Figure 4.7) WZB-141 could reach a larger space compared with WZB-131and WZB-134, which might be the reason for WZB-141 showing better inhibitory activity than the other members from the phenolic ethers.

Figure 4.5. Overlap view of energy-minimized structure of WZB-134 (in tube

model) and WZB-117 (in wire model).

101

Figure 4.6. Overlap view of energy-minimized structure of WZB-131 (in tube model) and WZB-115 (in wire model).

Figure 4.7. Overlap view of energy-minimized structure of WZB-141 (in tube model) and WZB-115 (in wire model). 102

In conclusion, WZB-141 showed the best inhibitory activity in glucose uptake and cell growth than the other phenolic ether derivatives. Most phenolic ethers showed decreased inhibitory activity in cell growth compared with WZB-27 and WZB-117. The lack of carbonyl groups might be the potential reason for the phenolic ethers losing their inhibitory activity in cell growth.

4.2 Design and Synthesis of N-linked Phenolic Derivatives

In order to find the importance of the carbonyl group in the inhibitory activity in glucose uptake and cell growth, we designed the N-linked phenolic derivatives, which included phenolic amide derivatives, phenolic amide/ester derivatives and phenolic amine derivatives. The phenolic amides, with the N atom replaced the O atom on the linkage, might have a big different conformations with phenolic esters. The phenolic amines had the similar structures as phenolic ethers, but could have different pKa values with phenolic ethers. (Figure 4.8)

103

Figure 4.8. Design the N-linked phenolic derivatives.

4.2.1 Design and synthesis of phenolic amide derivatives

Retro-synthetically, the phenolic amide derivatives could be obtained from acylating phenylenediamine with benzyloxybenzoyl chloride. (Figure 4.9)

Figure 4.9.Design the phenolic amide derivatives.

The synthesis of phenolic amides WZB-142 and WZB-143 started from the easily accessible phenylenediamine 52 and 53, reacted with 3-benzyloxybenzoyl chloride (31) 104 to give rise to amides 54 and 55, respectively. Then hydrogenolysis of amides 54 and 55 with catalytic amount of palladium on activated carbon to furnish the corresponding phenolic amides WZB-125 and WZB-124 in good yields after two steps. Alternatively, alkylation of amides 54 and 55 with iodomethane in the presence of sodium hydride followed by hydrogenolysis to generate N-methyl phenolic amides WZB-142 and WZB-

143 in reasonable yields. (Scheme 4.2)

Scheme 4.2. Synthesis of N-H phenolic amides and N-methyl phenolic amides.

Both N-H phenolic amides (WZB-124 and WZB-125) and the N-methyl phenolic amides

(WZB-142 and WZB-143) showed similar inhibitory activity in glucose uptake compared with the phenolic benzoate esters (WZB-27 or WZB-115). Regarding the cell growth inhibition, the N-H phenolic amides WZB-124 and WZB-125 showed almost no 105 inhibitory activity, however, the N-methyl phenolic amide WZB-143 showed the same level as phenolic benzoate ester WZB-27. Interesting, the WZB-142, which bears a chloride substituent on the core aromatic ring showed half of the inhibitory activity of

WZB-143 in cell growth. From this point, the fluoro-phenolic amides showed better inhibitory activity in cell growth than the chloro-phenolic amides, however, all the phenolic amides showed the similar inhibitory activity in glucose uptake. (Table 4.3)

Table 4.3. Glucose uptake inhibition and cell growth inhibition for phenolic amides.

The pKa values of the phenolic amides were calculated in Epik (version 2.1107,

Schrodinger, LLC.)199 The N-H phenolic amides showed similar calculated pKa values with the N-methyl phenolic amides. With respect to the inhibitory activity in cell growth,

N-methyl phenolic amides showed better activity than the N-H phenolic amides. 106

Therefore, the calculated pKa values seemed no correlation with their inhibitory activity.

(Figure 4.10)

Figure 4.10. Calculated pKa values for the phenolic amide derivatives.

The energy minimized structure of WZB-124 did not overlap with WZB-117, one pendant aromatic ring of WZB-124 was perpendicular to one pendant ring of WZB-117, which might be the reason for WZB-124 losing inhibitory activity in cell growth. (Figure

4.11) In a similar manner, one pendant aromatic ring of WZB-143 was perpendicular to the pendant ring at the same position on WZB-117, and the other aromatic ring overlapped nicely. With respect to the overlap of the energy minimized structures, no direct correlation was found. One possible reason for WZB-143 showing better inhibitory activity in cell growth than the other phenolic amides is still not clear. (Figure 4.12)

107

Figure 4.11. Overlap view of energy-minimized structure of WZB-124 (in tube model)

and WZB-117 (in wire model).

Figure 4.12. Overlap view of energy-minimized structure of WZB-143 (in tube model)

and WZB-117 (in wire model). 108

4.2.2 Design and synthesis of phenolic amine derivatives

Retrosynthetically, the phenolic amine derivatives could be obtained from reduction of the phenolic amide derivatives with lithium aluminum hydride. (Figure 4.13)

Figure 4.13. Retrosynthetic analysis of phenolic amine derivatives.

With the phenolic amides in hands, we proceeded to make the phenolic amine derivatives by reducing their corresponding amides to amines with lithium aluminum hydride. The

N-H phenolic amines (WZB-138 and WZB-139) and the N-methyl phenolic amines

(WZB-144 and WZB-145) were obtained in good yields after this single step. (Scheme

4.3)

X X O

N LiAlH4 N O N R N R R 0 oC, THF R OH OH

OH OH

WZB-124:X=F,R=H WZB-139:X=F,R=H,91% WZB-125:X=Cl,R=H WZB-138: X = Cl, R = H, 90%

WZB-142: X = Cl, R = CH3 WZB-145: X = Cl, R = CH3,93% WZB-143:X=F,R=CH 3 WZB-144:X=F,R=CH3,92%

Scheme 4.3. Synthesis of N-H and N-methyl phenolic amine derivatives. 109

The N-H phenolic amine and N-methyl phenolic amine derivatives showed decreased inhibitory activity in glucose uptake compared with their corresponding phenolic amide derivatives. Interestingly, the fluoro-N-H phenolic amine WZB-139 and chloro-N-methyl phenolic amine WZB-145 showed better inhibitory activity in cell growth than the other amines. WZB-138 showed very strong stimulatory activity in cell growth. With respect to the ratio of cell growth inhibition to glucose uptake inhibition, WZB-139 showed the highest ratio (0.96) of cell growth inhibition to glucose inhibition, which indicated that

WZB-139 inhibited cell growth by inhibiting glucose uptake. WZB-145 showed the same inhibitory activity in cell growth as WZB-139, but the ratio of cell growth inhibition to glucose uptake inhibition was different from WZB-139 due to WZB-139 showed different inhibitory activity in glucose uptake. Overall, the glucose uptake inhibition was not correlated with the cell growth inhibition. (Table 4.4)

110

Table 4.4. Glucose uptake inhibition and cell growth inhibition for phenolic amine derivatives.

The pKa values of the phenolic ethers were calculated in Epik (version 2.1107,

Schrodinger, LLC.)199 Both N-H phenolic amines WZB-138 and WZB-139 showed same calculated pKa values on pendant phenols. But both compounds acted differently in the cell growth, WZB-138 showed strong stimulatory activity in cell growth, whereas WZB-

139 showed inhibitory activity. WZB-145 showed better inhibitory activity in cell growth than WZB-144, but both compounds showed similar calculated pKa values. It seemed pKa values had no tight relationship with inhibitory activity. (Figure 4.14)

111

X X

N N N H N H a a OH OH

b HO HO

WZB-138: X=Cl WZB-144: X=F WZB-139: X=F WZB-145: X=Cl

pKa* value

Comp # Ha Hb WZB-138 9.95 ± 1.0 9.94 ± 1.0 WZB-139 9.94 ± 1.0 9.95 ± 1.0 WZB-144 9.96 ± 1.0 9.96 ± 1.0 WZB-145 9.96 ± 1.0 9.95 ± 1.0 * pKa was calculated in Epik version 2.1107

Figure 4.14 Calculated pKa values for the phenolic amide derivatives

WZB-139 showed good inhibitory activity in cell growth, while WZB-138 showed strong stimulatory activity in cell growth. But WZB-138 and WZB-139 overlapped very well in their energy minimized structures. (Figure 4.15) WZB-145 showed better inhibitory activity in cell growth than WZB-144, however, the energy minimized structures of WZB-144 and WZB-145 overlapped well. (Figure 4.16) One possible reason for the phenolic amines different acting in the cell growth is not clear.

112

Figure 4.15. Overlap view of energy minimized structure of WZB-138 (in tube model) and WZB-139 (in wire model).

113

Figure 4.16. Overlap view of energy minimized structure of WZB-145 (in tube model) and WZB-144 (in wire model).

4.2.3 Design and synthesis of phenolic amide/ester derivatives

Retro-synthetically, the phenolic amide/ester derivatives could be prepared from amino chlorophenol and benzoyl chloride. (Figure 4.17)

Figure 4.17. Retrosynthetic analysis of phenolic amide/ester derivatives. 114

Amino chlorophenol 56 and 57 reacted with benzoyl chloride 31 to provide different acylation products under different reaction conditions: 1) the mono-N-acylated products were obtained in the presence of triethylamine followed by debenzylation to produce

WZB-152 and WZB-154; 2) the O, N-acylated products were obtained in the presence of diisopropyl ethyl amine (DIEA) followed by debenzylation to form WZB-153 and WZB-

157. (Scheme 4.4)

Scheme 4.4. Synthesis of WZB-152~154 and WZB-157.

The inhibitory activity of the phenolic amides (WZB-152 and WZB-154) and the phenolic amide/ester derivatives (WZB-153, 157) in glucose uptake and cell growth were summarized in Table 4.2.3. The phenolic amides WZB-152 and WZB-154 showed extremely low inhibitory activity in glucose uptake, and stimulated the cancer cell growth. The lack of one pendant aromatic ring might be a critical reason for losing their inhibitory activity for WZB-152 and WZB-154. WZB-153 showed low inhibitory activity in glucose uptake and mild inhibitory activity in cell growth. Interesting, WZB-

157 exhibited similar inhibition activity in glucose uptake as WZB-27, but promoted the 115 cancer cell growth. With respect to the inhibitory activity in cell growth, WZB-153 is the best compound, however, WZB-153 showed lower inhibitory activity in cell growth than the previous hydrolytically more stable analogs. (Table 4.5)

Table 4.5. Glucose uptake inhibition and cell growth inhibition for phenolic amide/ester derivatives.

WZB-153 showed the same calculated pKa values as WZB-157, however, WZB-153 and WZB-157 acted differently in cell growth, which indicated the pKa values were not correlated with the inhibitory activity for the amide/ester derivatives. (Figure 4.18)

116

Figure 4.18. Calculated pKa values for the phenolic amide and amide/ester derivatives.

The difference between the energy minimized structures of WZB-153 and WZB-157 was the pendant aromatic ring linked by the amide bond with core aromatic ring. Both pendant aromatic rings were located in different spheres, and almost perpendicular to each other. This might be a reason for WZB-153 and WZB-157 showing different activity in cell growth. (Figure 4.19)

117

Figure 4.19. Overlap view of WZB-153 (in tube model) and WZB-157 (in wire model).

Overall, the N-linked phenolic derivatives showed decreased inhibitory activity compared with phenolic ether derivatives and phenolic benzoate esters. The N-methyl phenolic amides showed better inhibitory activity in cell growth than N-H phenolic amides. The N- methyl phenolic amine WZB-145 showed better inhibitory activity than the other phenolic amines. The phenolic amide/ester analogs showed very poor inhibitory activity in glucose uptake and cell growth.

4.3 Design and Synthesis of S-linked Derivatives

Finally, we designed several S-linked analogs of WZB-117, which include thioether, sulfoxide and sulfone. Sulfoxide and sulfone derivatives were designed by replacing the 118

CO with SO and SO2, both derivatives were hydrolytically more stable than benzoate ester WZB-117.227 The thioether was structurally similar as the phenolic ether derivatives of WZB-117, which was also hydrolytically stable. (Figure 4.20)

Figure 4.20. Design the S-linked derivatives of WZB-117.

Reduction of the 3-fluorophthalic acid (43) with lithium aluminum hydride produced the diol 48 in 87% yield. Then dibromide compound 58 was obtained from diol 48 via bromination in 96% yield. WZB-161 was prepared from 58 and 3-mercaptophenol through nucleophilic substitution in the presence of triethylamine. Several methods were applied to oxidize the thioether to sulfoxide and sulfone, the method with sodium periodate228 or hydroxyl peroxide229-231 gave rise to complex reaction mixtures. Finally,

WZB-163 and WZB-165 were obtained by using m-CPBA232, 233 to oxidize the thioether 119

WZB-161. Sulfone WZB-164 was obtained by further oxidation of WZB-163 and

WZB-165. (Scheme 4.5)

Scheme 4.5. Synthesis of the S-linked derivatives.

WZB-161 showed better inhibitory activity than WZB-117 in glucose uptake, however,

WZB-161 did not show any inhibitory activity in cell growth. Sulfoxide WZB-163 showed the weakest inhibitory activity among the S-linked analogs. Although WZB-165 had a higher ratio in cell growth inhibition to glucose uptake inhibition than the other S- linked derivatives, WZB-165 still showed very poor inhibitory activity in cell growth compared with WZB-117. Overall, the S-linked analogs of WZB-117 showed very poor activity in cell growth compared with WZB-117. (Table 4.6) 120

Table 4.6. Glucose uptake inhibition and cell growth inhibition of S-linked analogs .

The calculated pKa values for the S-linked analogs were listed in Figure 4.5.1. WZB-

161, with the highest pKa values among the S-linked analogs, showed stimulatory activity in cell growth instead of inhibitory activity. With respect to the calculated pKa values, there were no differences among the other S-linked derivatives despite the differences of inhibitory activity in cell growth. It indicated the calculated pKa values were not correlated with the inhibitory activity. (Figure 4.21)

Figure 4.21. Calculated pKa values for the S-linked derivatives.

121

The energy minimized structure of WZB-165 could not overlap with WZB-117 very well; one of the pendant aromatic rings on WZB-165 was perpendicular to the corresponding ring on WZB-117, which might be the potential reason for losing inhibitory activity in cell growth. (Figure 4.22) Overall, the S-linked phenolic derivatives showed decreased inhibitory activity in glucose uptake and cell growth compared with

WZB-117.

Figure 4.22. Overlap view of WZB-165 (in tube model) and WZB-117 (in wire model).

The best compound from each small group (phenolic ethers, amides, amines, amide/esters and S-linked analogs) were selected and listed in Table 4.7. The phenolic ether WZB-141 122 displayed better inhibitory activity in both glucose uptake and cell growth than the other hydrolytically more stable analogs. WZB-145 showed similar inhibitory activity in cell growth as WZB-141; however, it had a lower inhibitory activity in glucose uptake than

WZB-141. The other N-linked phenolic derivatives showed poorer inhibitory activity than WZB-145. The S-linked analog displayed the weakest inhibitory activity in cell growth. Based on these results, it was necessary to further modify the structure of phenolic ethers in order to get more information on the structure-activity-relationship.

Table 4.7. Glucose uptake inhibition and cell growth inhibition of selected the best compounds from each group.

4.4 Modification of the Phenolic Ether Derivatives

The phenolic ether analogs showed the best inhibitory activity among the hydrolytically more stable derivatives. Based on this result, we designed more phenolic ether analogs by introducing different substituents to the pendant aromatic rings in order to get more information on the SAR of the phenolic ethers. Electron donating group (OMe) and electron withdrawing groups (F and NO2) were investigated. (Figure 4.23) 123

Figure 4.23. Modification of the phenolic ether derivatives.

The benzyl chlorides 61 and 64 were prepared from benzoic acids 59 and 62 after 4 steps

(esterification, MOM protection, reduction and SN2 displacement), respectively. (Scheme

4.6) The benzyl bromide 68 was synthesized from aldehyde 65 after 3 steps, which involved MOM protection of the hydroxyl group, boron hydride reduction of aldehyde to alcohol followed by bromination. (Scheme 4.7)

Scheme 4.6. Synthesis of benzyl chlorides 61 and 64.

NO 2 NO2 NO2 NO2 MOMCl CHO CHO NBS NaBH4 OH Br

K2CO3 THF, RT PPh3 DMF, 0oC 97% 83% OH OMOM 86% OMOM OMOM 65 66 67 68

Scheme 4.7. Synthesis of benzyl bromide 68. 124

Six phenols (5a, 7a, 7b, 7c, 7e and 7f) were used to couple with benzyl chlorides 61, 64 or benzyl bromide 68 followed by deprotection with hydrochloric acid solution in ethyl acetate to furnish eighteen phenolic ethers. (Table 4.8) Unfortunately, deprotection of the

MOM protected para-methoxyl phenolic ethers was unsuccessful with hydrochloric acid.

Several different methods were attempted to investigate the deprotection of the phenolic

234 methoxymethyl ethers, such as boron trifluoride diethyl etherate (BF3·OEt2) , p- toluenesulfonic acid (p-TSA)235, and silica-supported sodium hydrogen sulfate

236 (NaHSO4·SiO2), however, none of these methods worked after several trials. (Scheme

4.8) Finally, twelve phenolic ethers were successfully prepared with moderate to good yield after two steps.

Scheme 4.8. Deprotection of the MOM group.

125

Table 4.8 Synthesis of substituted phenolic ether derivatives

The fluorophenolic ether analogs showed relative better activity in glucose uptake than the previous prepared phenolic ether derivatives. WZB-146 showed the best inhibitory activity in glucose uptake and cell growth among the fluorophenolic ethers. Apparently, the electron withdrawing groups on the pendant aromatic rings enhanced the inhibitory activity. Therefore, a stronger electron withdrawing group, nitro group (NO2), was then introduced to the pendant aromatic rings. The nitro phenolic ethers showed slightly decreased inhibitory activity in glucose uptake compared with the fluoro phenolic ethers.

As predicted, most nitro phenolic ether analogs showed better inhibitory activity in cell 126 growth than the fluorophenolic ethers, such as WZB-168, WZB-172 and WZB-173.

WZB-173 showed 66.2% ± 4.7 inhibitory activity in cell growth and was selected as the current lead compound from the library of hydrolytically more stable analogs. (Table 4.9)

Table 4.9. Glucose uptake inhibition and cell growth inhibition for substituted phenolic ether analogs.

The calculated pKa values for the fluoro and nitro phenolic ethers were listed in Figure

4.24. The nitro phenolic ethers showed relative lower pKa values than their corresponding fluoro phenolic ethers. With respect to the inhibitory activity in cell growth, the nitro phenolic ethers showed better activity than the fluoro phenolic ethers, which might indicate the lower pKa values contribute to the better inhibitor activity for the nitro phenolic ethers. WZB-173 and WZB-168 showed better inhibitory activity in 127 cell growth than the other nitro phenolic ethers, their relative lower pKa values might be the reason for this difference. Moreover, WZB-173, with lower pKa values than WZB-

168, displayed better inhibitory activity in cell growth than WZB-168. It indicated that lower pKa values might lead to better inhibitory activity in cell growth. (Figure 4.24)

Figure 4.24. Calculated pKa values for the modified phenolic ethers.

Both pendant aromatic rings from WZB-173 were almost perpendicular to the core aromatic ring, along with the internal H-bond to form a specific pocket in the energy minimized structure of WZB-173. (Figure 4.25) WZB-172, with the similar structure to

WZB-173, showed twisted structure and formed a smaller pocket than WZB-173, which 128 might be the reason for WZB-172 losing inhibitory activity in cell growth. (Figure 4.26)

Overall, the specific pocket formed in WZB-173 might be critical for better inhibitory activity in cell growth.

Figure 4.25. Energy minimized structure of WZB-173.

129

Figure 4.26. Energy minimized structure of WZB-172.

In summary, the phenolic ether derivatives showed better inhibitory activity than the other hydrolytically more stable analogs (phenolic amide, phenolic amine and S-linked analogs). To some extent, the electron withdrawing groups on the pendant aromatic rings of phenolic ether derivatives enhanced both inhibitory activities in glucose uptake and cell growth. Finally, WZB-173 was selected as the current lead compound from the whole library.

4.5 Structure-Activity-Relationship (SAR)

We further investigated the structure activity relationship by preparing hydrolytically more stable analogs of phenolic benzoate esters. A library included phenolic ethers, phenolic amides, phenolic amines, phenolic amide/ester analogs and S-linked phenolic 130 analogs. The phenolic amide/ester analogs and S-linked phenolic derivatives showed very poor inhibitory activity in glucose uptake and cell growth. N-methyl phenolic amines showed better inhibitory activity in glucose uptake and cell growth than the phenolic amide derivatives. With respect to the substituents on the core aromatic ring, chloro phenolic amines showed better activity than the fluoro phenolic amines. Moreover, phenolic ethers showed much better inhibitory activity than the phenolic amines. The electron donating group (OMe) on core aromatic ring decreased inhibitory activity in glucose uptake and cell growth; however, the electron withdrawing groups (F, Cl) enhanced inhibitory activity. The stronger electron withdrawing groups on the pendant aromatic rings showed better inhibitory activity. For example, the nitro phenolic ethers showed relatively better inhibitory activity than the fluoro phenolic ethers. (Figure 4.27)

The pKa values for all the phenolic ethers were calculated, but no obvious relationships with inhibitory activity were found except for the nitro phenolic ether derivatives. WZB-

173, with a lower pKa value than WZB-168, showed better activity in cell growth than

WZB-168, which might indicate the lower pKa values could lead to better inhibitory activity. The energy minimized structures for these more stable analogs were also investigated. The lead compound WZB-173 formed a specific pocket in the energy minimized structure, which might contribute to the better inhibitory activity in cell growth shown by WZB-173. Unfortunately, no obvious evidence was found showing close relationship between the energy minimized structures and inhibitory activity.

Finally, WZB-173 was selected as the current lead compound for the whole library.

131

Figure 4.27 Structure-Activity-Relationship of hydrolytically more stable derivatives

In conclusion, we showed that small compounds, with a scaffold of rigid benzene ring lingked to two pendant aromatic rings with various hydroxyl groups, exhibited inhibitory activity in both basal glucose uptake and cell growth. The varieties of benzoate esters with different numbers of hydroxyl group on the pendant aromatic rings were investigated and we found meta-hydroxyl derivatives displayed the best inhibitory activity. The stability study showed the benzoate ester were not stable, which lead us designed and prepared hydrolytically more stabe analogs. The phenolic esters showed the best inhibitory activity among the hydrolytically stable analogs. Finally, WZB-173, with a very strong withdrawing group (NO2) on the pendant aromatic rings, was slelcted as the lead compound. Based on our rational design and hit to lead modeification, we have identified that a meta-hydroxyphenyl group is an optimal pendant aromatic group.

Usaually, phenols are known to have poor bioavailability and short half time (t1/2) of activity in vivo, which will lead us to develop more metabolically stable analogs in the future. Different substitutents (such as Me, CF3, Cl and OAc) at various positions on the 132 pendant aromatic rings will be explored. A series of bioisosteres will be applied to replace the phenol group. Furthermore, the linkage between the core aromatic ring and pendant aromatic rings will also be investigated. (Figure 4.28) This set of small compounds will provide us more information on discoverying more potent anticancer drug candidiates.

R R O O R O O O NH N O N N HN HN N NH NH HN N N

R R O O R O O O NH N O O N HN N HN O N NH O HN N N HN O

R X R N X R R1 N O 1 N R1 R X N N O R1 X HN N N HN N X

NH X N N NH N N 1 R =H,Me R1 =H,Me X=CH2OH X=CO,SO,SO2 X = NHCONH2 X=CO,SO,SO2 X = NHC(O)CH3 X=NHS(O)2CH3

Figure 4.28. Structures of small compounds in future plans.

133

CHAPTER 5: EXPERIMENTAL SECTION

All reagents and starting materials were purchased from commercial suppliers. All reactions were carried out under an atmosphere of Argon. Analytical thin-layer chromatography (TLC) was performed on a 250 µm layer silica gel (UV254) aluminum plate. Visualization of all compounds with a chromophore was carried out with a UV light. Non-UV active compounds were stained using phosphomolybdic acid in ethanol or ninhydrin in ethanol solution. Purification of desired products was carried out using flash chromatography on silica gel (230-400 mesh) purchased from Silicycle Inc. (Quebec

City, QC). Toluene was fractionally distilled after dried over calcium hydride. DMF was dried over BaO and distilled under reduced pressure and stored over 4Å molecular sieves under argon atmosphere. DCM and THF were dried over a column of packed alumina.

1H-NMR spectra were obtained on a Bruker 300 MHz spectrometer and referenced to tetramethylsilane. 13C-NMR spectra were obtained on a Bruker 300 (75.5 MHz)

1 13 spectrometer and referenced to CDCl3. Both H NMR and C NMR spectra were taken in deuterated chloroform or acetone solution. HPLC data were obtained on Shimadzu using Supelco Discovery C8 column (15cm x 4.6 mm, 5 µm), eluting at 1.0 mL/min with a gradient elution starting at 30% of CH3CN-H2O going to 75% over 26 minutes as the mobile phase eluent unless otherwise stated. Retention times are reported in minutes.

Mass verifications were carried out on a Shimadzu 2010A LC/MS using APCI probe. IR data were acquired on a Shimadzu Advantage FTIR-8400. Melting points were obtained on a Mel-temp П, from Laboratory Devices, USA. Melting points were uncorrected. The 134 energy-minimized structure conformations were obtained in Sparton’08 with the minimize function.

O BnO Cl

BnO OBn 4

3,4,5-Tris(benzyloxy)benzoyl chloride 4: Anhydrous potassium carbonate (16.8 g,

121.6 mmol) and potassium iodide (2.8 g, 16.7 mmol) were added to a solution of methyl

3,4,5-trihydroxybenzoate (7.0 g, 38 mmol) in dried acetone (400 mL) at room temperature. After stirred for 30 min, benzyl bromide (14.4 mL, 121.6 mmol) was added dropwise via a syringe and stirred for another 30 min at room temperature. The mixture of the reaction was then heated to reflux for 12 h. The solid was filtered through Celite after the completion of the reaction as indicated by TLC, the solvent was evaporated under vacuum to give methyl 3,4,5-tribenzyloxybenzoate as white solid which was used to the next step without further purification.

Lithium hydroxide monohydrate (7.9 g, 190 mmol) was added to a solution of 3,4,5- tribenzyloxybenzoate in THF/H2O (300 mL, 4:1) at room temperature. After stirred at room temperature for 8 h, the organic solvent was removed. The residue was diluted with water (150 mL), washed with diethyl ether (3 X 60 mL) and the combined organic phases were disposed. The aqueous phase was acidified to pH 3 with hydrochloric acid (1 M), and a thick, voluminous suspension was formed. The hot mixture was stirred for 15 min before the precipitate was collected by filtration and washed successively with distilled water (60 mL), methanol/H2O (60 mL, 1:1), methanol (2 X 30 mL), and diethyl ether (2 135

X 30 mL). The resulting solid was dried for 5 h and can be used directly into the next step.

DMF (0.2 mL) was added to a solution of 3,4,5-tribenzyloxybenzoic acid (13.5 g, 30.7 mmol) in dry toluene (150 mL) at room temperature and stirred for 10 min. Oxalyl chloride (5.8 g, 46.1 mmol) in toluene (15 mL) was added dropwise to the stirring reaction mixture afterwards. The mixture was stirred for additional 20 min at room temperature before heated to 50 °C. The yellow solution was evaporated after the ceasing of gas evolution. The residue was dissolved in hot toluene (50 mL, 70 °C) and the solution was decanted to another flask and the yellow, undissolved sticky side product was disposed. The chloride was crystallized by adding cold hexane to the hot toluene solution. The solid was filtered and washed with hexane to give the title compound as white solid in 68% yield. Mp 116-117 °C (lit. 237 116-117 °C).

3-Methoxy-1, 2-phenylene bis(3,4,5-tris(benzyloxy)benzoate), 8a: 3,4,5-tris(benzyloxy)-benzoyl chloride (941 mg, 2.05 mmol) was added to a solution of 3- methoxycatechol (140 mg, 1.00 mmol) in anhydrous acetonitrile (10 mL) at room temperature. DMAP (268 mg, 2.20 mmol) was added to the stirring reaction mixture after

30 min at room temperature. The mixture was stirred for 12 h and the solvent was removed, the crude was purified by column chromatography on silica gel 136

(EtOAc/Hexane, 1: 4) to afford 708 mg of the title compound in 72% yield. 1H NMR

(300MHz, CDCl3) δ 7.64 (d, 4H, J = 16.7 Hz), 7.51-7.33 (m, 31H), 7.16 (d, 1H, J = 8.2

Hz), 7.06 (d, 1H, J = 8.4 Hz), 5.18 (d, 4H, J = 7.0 Hz), 5.06 (d, 8H, J = 11 Hz), 3.96 (s,

13 3H); C NMR (75 MHz, CDCl3) δ 164.0, 163.6, 153.1, 152.8, 152.3, 144.1, 143.2,

137.6, 137.5, 136.5, 136.4, 132.3, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.7,

126.6, 123.9, 123.8, 115.4, 110.2, 109.7, 109.6, 75.3, 71.2, 56.4.

3-Methoxy-1, 2-phenylene bis(3,4,5-trihydroxybenzoate), WZB-26: 10% palladium on activated carbon (43 mg, 0.04 mmol) was added to a solution of 3-methoxy-1, 2- phenylene bis(3,4,5-tris(benzyloxy)benzoate) (500 mg, 0.51 mmol) in methanol/DCM

(10.0 mL, 4: 1). The reaction mixture was stirred under hydrogen gas atmosphere for 12 h at room temperature. Then the mixture was filtered through a small pad of silica gel. The filtrate was concentrated and purified by column chromatography on silica gel to provide

1 125 mg of title compound in 89% yield. H NMR (300MHz, CD3COCD3) δ 8.30 (br s,

6H), 7.29 (t, 5H, J = 8.3 Hz), 7.17 (d, 4H, J = 4.8 Hz), 7.06 (dd, 1H, J = 1.2, 8.5 Hz),

6.94 (dd, 1H, J = 1.3, 8.2 Hz), 3.84 (s, 3H); HPLC (CH3CN: H2O) RT 4.39 (99%); IR

(KBr) 3354, 1720, 1610, 1537, 1494, 1447, 1319, 1208, 1094, 1033, 950, 872, 756 cm-1. 137

Benzene-1, 2, 3-triyl tris(3,4,5-tris(benzyloxy)benzoate), 6a: 3,4,5-tris(benzyloxy)- benzoyl chloride (1.40 g, 3.05 mmol) was added to a solution of pyrogallol (126 mg, 1.00 mmol) in anhydrous acetonitrile (15 mL) at room temperature. DMAP (391 mg, 3.20 mmol) was added to the stirring reaction mixture after 30 min at room temperature. The mixture was stirred for 12 h and the solvent was removed, the crude was purified by column chromatography on silica gel (EtOAc/Hexane, 1: 4) to afford 1.0 g of 6a in 72%

1 yield. H NMR (300MHz, CDCl3) δ 7.53 (s, 4H), 7.49-7.20 (m, 50H), 5.10 (s, 4H), 5.01

13 (s, 8H), 4.94 (s, 2H), 4.85 (s, 4H); C NMR (75 MHz, CD3COCD3) δ 163.6, 163.0,

152.7, 144.2, 143.6, 143.3, 137.4, 137.3, 136.3, 136.1, 135.2, 128.6, 128.5, 128.4, 128.2,

128.1, 128.0, 127.9, 127.8, 126.3, 123.6, 123.1, 120.9, 109.6, 75.2, 75.1, 71.2.

Benzene-1, 2, 3-triyl tris(3,4,5-trihydroxybenzoate), WZB-27: 10% palladium on activated carbon (16 mg, 0.024 mmol) was added to a solution of benzene-1, 2, 3-triyl tris(3,4,5-tris(benzyloxy)benzoate) (260 mg, 0.29 mmol) in methanol/DCM (10.0 mL,

4:1), the mixture was stirred under hydrogen gas atmosphere for overnight at room 138 temperature. Then the mixture was filtered through a small pad of silica gel. The filtrate was concentrated and purified by column chromatography on silica gel (EtOAc/Hexane,

1 1: 3) to give 113.2 mg of title compound. H NMR (300 MHz, CD3COCD3) δ 8.24 (br s,

4H), 7.47-7.41 (m, 1H), 7.34-7.32 (m, 2H), 7.17 (s, 4H), 7.09 (s, 2H), 2.92 (br s, 5H);

HPLC (CH3CN: H2O) RT 3.48 (99%); IR (KBr) 3331, 1720, 1617, 1540, 1450, 1350,

1315, 1185, 1085, 1031, 755 cm-1.

5-(Methoxycarbonyl)benzene-1, 2, 3-triyl tris(3,4,5-tris(benzyloxy)benzoate):

Following the procedure described for the preparation of 6a, methyl 3,4,5- trihydroxybenzoate was acylated with 3,4,5-tris(benzyloxy)-benzoyl chloride in the

1 presence of DMAP to 6b in 74% yield. H NMR (300 MHz, CDCl3) δ 8.17 (d, 2H, J =

4.3 Hz), 7.55 (s, 4H), 7.52-7.21 (m, 47 H), 5.14 (s, 4H), 5.01 (d, 10H, J = 20.1 Hz), 4.88

(s, 4H), 4.01 (s, 3H). c

5-(Methoxycarbonyl)benzene-1, 2, 3-triyl tris(3,4,5-trihydroxybenzoate), WZB-75:

Following the procedure described for the preparation of WZB-27, debenylation of 6b with palladium on activated carbon as catalyst to afford WZB-75 in 85% yield, 85%; 1H

NMR (300 MHz, CD3COCD3) δ 8.30 (br s, 5H), 7.96 (s, 2H), 7.22-7.09 (m, 6H), 3.97 (s, 139

13 3H), 2.97 (br s, 4H); C NMR (75 MHz, CD3COCD3) δ 165.7, 164.3, 163.3, 146.2,

146.1, 145.5, 140.9, 139.9, 128.9, 122.8, 119.6, 119.0, 110.6, 52.9.

Benzene-1, 2, 3-triyl tris(3,4,5-trimethoxybenzoate), WZB-76: 3,4,5- trimethoxybenzoyl chloride (696.6 mg, 3.0 mmol) was added into a solution of pyrogallol

(126 mg, 1.0 mmol) in anhydrous acetonitrile (15 mL) at room temperature. DMAP (391 mg, 3.2 mmol) was added to the stirring mixture after 30 min at room temperature. The reaction mixture was stirred for 12 h and the solvent was removed under vacuum, the crude was purified by column chromatography on silica gel (EtOAc/Hexane, 1: 4) to

1 afford 609 mg of WZB-76 in 86% yield. H NMR (300 MHz, CDCl3) δ 7.34-7.31 (m,

13 2H), 7.24 (s, 3H), 7.17-6.68 (m, 3H), 3.84-3.61 (m, 27H); C NMR (75 MHz, CDCl3) δ

171.2, 164.2, 163.9, 163.8, 163.5, 162.8, 152.9, 149.6, 144.0, 143.4, 142.9, 140.6, 140.4,

135.0, 131.3, 126.5, 126.2, 123.6, 123.4, 123.0, 122.9, 120.8, 120.4, 119.9, 115.3, 114.7,

107.4, 60.9, 60.4, 56.2, 56.0.

CO2CH3

O O MeO OMe O O O O MeO OMe OMe OMe

MeO OMe OMe WZB-77 140

Following the procedure described for the preparation of WZB-76, WZB-77 was

1 obtained in 89% yield. H NMR (300 MHz, CDCl3) δ 7.98 (s, 1H), 7.71 (s, 1H), 7.27 (s,

2H), 7.22 (s, 12H), 7.13 (d, 2H, J = 8.7 Hz), 3.86-3.75 (m, 18 H), 3.68 (s, 6H), 3.63 (s,

3H), 3.61 (s, 3H).

General procedure for preparation fluoro benzoic ester derivatives: Fluoro benzoyl chloride (2.2 eq) was added into a solution of substituted catechol (1.0 eq) in dry acetonitrile at room temperature. Then DMAP (2.2 eq) was added into the stirring reaction mixture after 10 min. The reaction mixture was stirred for 8 h at room temperature. The solvent was removed after the completion of the reaction as indicated by TLC. The residue was purified by column chromatography on silica gel to provide the fluoro benzoic ester derivatives.

Benzene-1, 2, 3-triyl tris(3,4,5-trifluorobenzoate), WZB-78: 1H NMR (300 MHz,

13 CDCl3) δ 7.69-7.56 (m, 6H), 7.42-7.18 (m, 3H); C NMR (75 MHz, CDCl3) δ 160.9,

152.9, 152.7, 143.3, 134.4, 127.0, 124.2, 1221.2, 115.0, 114.7.

CO2CH3 O O F F O O O O F F F F

F F F WZB-79 141

5-(Methoxycarbonyl)benzene-1, 2, 3-triyl tris(3,4,5-trifluorobenzoate), WZB-79:

1 Yield, 93%; H NMR (300 MHz, CDCl3) δ 8.06 (s, 2H), 7.76-7.65 (m, 6H), 3.98 (s, 3H);

13 C NMR (75 MHz, CDCl3) δ 164.4, 160.8, 159.8, 152.9, 152.8, 149.6, 149.5, 145.7,

143.2, 142.2, 138.2, 129.4, 123.8, 123.1, 122.6, 115.0, 114.9, 114.8, 114.7, 52.8.

3-Methoxy-1, 2-phenylene bis(3,4,5-trifluorobenzoate), WZB-80: Yield, 93%; 1H

NMR (300 MHz, CDCl3) δ 7.81-7.73 (m, 4H), 7.37-7.28 (m, 1H), 7.01-6.95 (m, 2H),

3.88 (s, 3H).

3-Methoxy-1, 2-phenylene bis(3,4,5-trimethoxybenzoate), WZB-81: Yield, 90%; 1H

NMR (300 MHz, CDCl3) δ 7.37 (s, 2H), 7.35-7.28 (m, 3H), 7.04-6.96 (m, 2H), 3.90-3.89

13 (d, 9H, J = 2.7 Hz), 3.82 (s, 6H), 3.78 (s, 6H); C NMR (75 MHz, CDCl3) δ 163.8,

163.4, 152.9, 143.9, 142.8, 142.7, 132.1, 126.3, 123.7, 123.6, 115.1, 109.9, 107.5, 107.4,

60.9, 56.3, 56.2, 56.1. HPLC (CH3CN: H2O) RT 16.10 (100%). 142

Benzene-1, 2, 3-triyl tris(3, 4-difluorobenzoate), WZB-82: Yield, 78%; 1H NMR (300

MHz, CDCl3) δ 7.93-7.79 (m, 6H), 7.50-7.42 (m, 1H), 7.39-7.35 (m, 2H), 7.28-7.13 (m,

13 3H); C NMR (75 MHz, CDCl3) δ 159.7, 153.8, 149.9, 149.7, 146.4, 141.6, 132.7,

125.3, 125.2, 124.6, 123.3, 119.0, 117.6, 117.3, 115.9, 115.6.

3-Methoxy-1, 2-phenylene bis(3, 4-difluorobenzoate), WZB-83: Yield: 85%; 1H NMR

(300 MHz, CDCl3) δ 7.98-7.87 (m, 4H), 7.35-7.17 (m, 3H), 7.00-6.96 (m, 2H), 3.87 (s,

13 3H); C NMR (75 MHz, CDCl3) δ 162.0, 156.1, 152.5, 151.7, 148.4, 143.7, 131.9,

127.4, 126.7, 125.7, 119.8, 119.4, 117.9, 117.7, 114.9, 110.2, 56.3.

5-(Methoxycarbonyl)benzene-1, 2, 3-triyl tris(3, 4-difluorobenzoate), WZB-84:

1 Yield, 79%; H NMR (300 MHz, CDCl3) δ 8.08-8.05 (m, 2H), 7.93-7.78 (m, 6H), 7.28- 143

13 7.15 (m, 3H), 3.98 (s, 3H); C NMR (75 MHz, CDCl3) δ 164.7, 161.7, 160.7, 156.2,

156.0, 152.8, 152.7, 152.6, 151.9, 151.8, 143.5, 138.7, 129.0, 127.4, 127.3, 125.0, 124.4,

122.6, 119.7, 119.4, 118.1, 118.0, 117.9, 117.8, 52.8.

3-Fluoro-1, 2-phenylene bis(4-fluoro-3-(trifluoromethyl)benzoate), WZB-85: Yield,

1 85%; H NMR (300 MHz, CDCl3) δ 8.29-8.19 (m, 4H), 7.34-7.11 (m, 5H).

Benzene-1, 2, 3-triyl tris(4-fluoro-3-(trifluoromethyl)benzoate), WZB-86: Yield,

1 84%; H NMR (300 MHz, CDCl3) δ 8.24-8.19 (m, 4H), 8.15-8.09 (m, 2H), 7.44-7.38 (m,

1H), 7.34-7.31 (m, 2H), 7.23-7.11 (m, 3H).

144

5-(Methoxycarbonyl)benzene-1, 2, 3-triyl tris(4-fluoro-3-(trifluoromethyl)benzoate),

1 WZB-87: Yield, 83%; H NMR (300 MHz, CDCl3) δ 8.42-8.39 (m, 2H), 8.36-8.30 (m,

2H), 8.26-8.16 (m, 2H), 7.77 (s, 2H), 7.32-7.17 (m, 3H), 3.82 (s, 3H).

O O O O F

CF3

CF3 F WZB-88

3-Methoxy-1, 2-phenylene bis(4-fluoro-3-(trifluoromethyl)benzoate), WZB-88:

1 Yield, 84%; H NMR (300 MHz, CDCl3) δ 8.29-8.18 (m, 4H), 7.27-7.14 (m, 3H), 6.92-

6.89 (d, 2H, J = 8.4 Hz), 3.79 (s, 3H).

3-Fluoro-1, 2-phenylene bis(3, 4 ,5-tris(benzyloxy)benzoate), 8b: Following the procedure described for preparation of 8a, 8b was obtained in 89% yield; 1H NMR (300

13 MHz, CDCl3) δ 7.46-7.20 (m, 37H), 5.20-4.96 (m, 12H); C NMR (75 MHz, CDCl3) δ

163.6, 162.9, 162.2, 157.1, 153.8, 152.9, 152.8, 144.4, 143.5, 143.4, 137.6, 136.3, 131.5,

131.3, 128.7, 128.6, 128.3, 128.2, 128.1, 127.9, 127.6, 126.5, 126.4, 123.6, 123.7, 122.9,

118.9, 114.4, 114.1, 110.2, 109.7, 109.6, 75.2, 71.5, 71.2. 145

4-Chloro-1, 2-phenylene bis(3,4,5-tris(benzyloxy)benzoate), 8c: Following the procedure described for preparation of 8a, 8c was obtained in 91% yield; 1H NMR (300

13 MHz, CDCl3) δ 7.48-7.29 (m, 37H), 5.07-4.97 (m, 12H); C NMR (75 MHz, CDCl3) δ

163.7, 163.5, 152.7, 143.5, 143.4, 143.0, 141.4, 137.4, 136.2, 131.6, 128.6, 128.3, 128.2,

128.1, 127.7, 126.8, 124.5, 124.2, 123.4, 123.3, 109.5, 75.2, 71.2.

4-Benzyloxybenzoyl chloride, 25: Following the procedure described for preparation of compound 4, benzoyl chloride 25 was prepared in 85% yield; 1H NMR (300 MHz,

CD3COCD3) δ 8.13-8.08 (m, 2H), 7.47-7.35 (m, 5H), 7.09-7.04 (m, 2H), 5.18 (s, 2H).

Benzene-1, 2, 3-triyl tris(4-(benzyloxy)benzoate), 26: Following the procedure described for preparation of 6a, compound 26 was obtained in 98% yield; 1H NMR (300 146

MHz, CDCl3) δ 8.08-7.98 (m, 6H), 7.43-7.27 (m, 18H), 6.97-6.87 (m, 6H), 5.09 (d, 6H, J

= 11.7 Hz).

Benzene-1, 2, 3-triyl tris(4-hydroxybenzoate), WZB-91: Yield, 95%; 1H NMR (300

MHz, CD3COCD3) δ 9.27 (br s, 3H), 7.96-7.91 (m, 4H), 7.87-7.82 (m, 2H), 7.50-7.39 (m,

13 3H), 6.91-6.86 (m, 4H), 6.84-6.79 (m, 2H); C NMR (75 MHz, CD3COCD3) δ 161.7,

161.1, 161.0, 143.0, 134.4, 130.7, 124.2, 119.3, 118.4, 117.9, 113.9.

3-Methoxy-1, 2-phenylene bis(4-(benzyloxy)benzoate), 27a: 3-Methoxycatechol (35 mg, 0.25 mmol) and 4-(benzyloxy)benzoyl chloride (126 mg, 0.51 mmol) and DMAP (66 mg, 0.54 mmol) were dissolved in acetonitrile (8 mL), the reaction was stirred for overnight at room temperature. The mixture was concentrated after the completion of the reaction as indicated by TLC. The residue was purified by column chromatography on silica gel (EtOAc/Hexane, 1:4) to provide the title compound in 96% yield. 1H NMR (300

MHz, CDCl3) δ 7.96-7.88 (m, 4H), 7.29-7.18 (m, 10H), 7.15-7.09 (m, 1H), 6.86-6.77 (m,

13 6H), 4.92 (d, 4H, J = 3.9 Hz), 3.68 (s, 3H); C NMR (75 MHz, CDCl3) δ 164.1, 163.7, 147

163.0, 152.9, 136.2, 132.4, 128.7, 128.3, 127.6, 126.2, 121.4, 115.4, 114.6, 109.9, 70.1,

56.2.

3-Fluoro-1, 2-phenylene bis(4-(benzyloxy)benzoate), 27b: Following the procedure described for preparation of 27a, 3-fluorocatechol (32 mg, 0.25 mmol) was acylated with

4-(benzyloxy)benzoyl chloride (126 mg, 0.51 mmol) in the presence of DMAP (64 mg,

0.52 mmol) to give, after purification by column chromatography on silica gel

1 (EtOAc/Hexane, 1: 4), the ester 27b in 96% yield. H NMR (300 MHz, CDCl3) δ 7.97-

7.89 (m, 4H), 7.31-7.19 (m, 10H), 7.17-7.11 (m, 1H), 7.08-6.98 (m, 2H), 6.85-6.81 (m,

13 4H), 4.96 (d, 4H, J = 2.0 Hz); C NMR (75 MHz, CDCl3) δ 163.8, 163.3, 163.1, 157.1,

153.8, 144.6, 136.1, 132.7,131.7, 128.8,128.3, 127.5, 126.2, 121.0, 120.6, 118.9, 114.7,

114.2, 113.9, 70.2.

4-Chloro-1, 2-phenylene bis(4-(benzyloxy)benzoate), 27c: Following the procedure described for preparation of 27a, 4-Chlorocatechol (35 mg, 0.24 mmol) was acylated with 4-(benzyloxy)benzoyl chloride (122 mg, 0.50 mmol) in the presence of DMAP (64 148 mg, 0.52 mmol) to provide, after purification by column chromatography on silica gel

1 (EtOAc/Hexane, 1: 4), title compound 27c in 92% yield; H NMR (300 MHz, CDCl3) δ

8.07-8.02 (m, 4H), 7.46-7.28 (m, 13H), 6.99-6.93 (m, 4H), 5.12 (s, 4H); 13C NMR (75

MHz, CDCl3) δ 163.4, 163.1, 162.8, 142.7, 141.1, 135.6, 132.0, 130.9, 128.4, 128.0,

127.2, 126.2, 124.0, 123.7, 120.6, 120.4, 114.2, 69.7.

3-Fluoro-1,2-phenylene bis(4-hydroxybenzoate), WZB-92: 1H NMR (300 MHz,

CD3COCD3) δ 9.34 (br s, 3H), 7.99-7.91 (m, 4H), 7.47-7.40 (m, 1H), 7.33-7.26 (m, 2H),

13 6.94-6.88 (m, 4H); C NMR (75 MHz, CD3COCD3) δ 164.1, 163.7, 163.5, 163.4, 158.0,

154.7, 145.8, 133.4, 133.3, 132.7, 132.5, 127.3, 127.2, 120.5, 120.3, 120.2, 120.0, 116.5,

116.4, 114.6, 114.4.

MeO O O O OH

OH WZB-93

3-Methoxy-1,2-phenylene bis(4-hydroxybenzoate), WZB-93: 1H NMR (300 MHz,

CD3COCD3) δ 9.24 (br s, 2H), 7.96-7.90 (m, 4H), 7.32 (t, 1H, J = 8.3 Hz), 7.09 (dd, 1H,

J = 1.3, 9.8 Hz), 7.01 (dd, 1H, J = 1.3, 8.2 Hz), 6.92-6.85 (m, 4H), 3.87 (s, 3H); 13C NMR 149

(75 MHz, CD3COCD3) δ 164.3, 163.9, 163.3, 163.2, 154.1, 145.4, 133.6, 133.1, 126.8,

121.0, 116.3, 116.2, 110.7, 56.6.

4-Chloro-1,2-phenylene bis(4-hydroxybenzoate), WZB-94: 1H NMR (300 MHz,

CDCl3) δ 9.30 (br s, 1H), 7.96-7.88 (m, 4H), 7.58-7.37 (m, 3H), 7.92-7.86 (m, 4H), 2.86

13 (br s, 1H); C NMR (75 MHz, CDCl3) δ 164.1, 164.0, 163.5, 163.4, 163.3, 144.7, 143.1,

133.3, 133.2, 131.3, 127.2, 125.9, 124.9, 124.6, 120.6, 120.5, 116.3.

1 Benzene-1, 2, 3-triyl tris(3-methoxybenzoate), WZB-96: H NMR (300 MHz, CDCl3)

δ 7.71-7.68 (m, 2H), 7.64-7.61 (m, 1H), 7.58-7.54 (m, 2H), 7.49-7.47 (m, 1H), 7.45-7.37

(m, 2H), 7.45-7.37 (m, 3H), 7.35-7.21 (m, 3H), 7.13-7.02 (m, 3H), 3.74 (s, 6H), 3.69 (s,

3H).

150

Benzene-1, 2, 3-triyl tris(3, 4-dimethoxybenzoate), WZB-98: 1H NMR (300 MHz,

CDCl3) δ 7.80 (dd, 3H, J = 2.0, 8.4 Hz), 7.62 (d, 3H, J = 2.0 Hz), 7.28 (s, 3H), 6.95 (d,

3H, J = 8.4 Hz), 6.69 (q, 1H, J = 7.6, 8.7 Hz), 6.52-6.49 (m, 2H), 3.97 (d, 18H, J = 3.3

Hz).

3-Fluoro-1, 2-phenylene bis(3,4,5-trimethoxybenzoate), WZB-101: 1H NMR (300

MHz, CDCl3,) δ 7.36-7.14(m, 7H), 3.89 (d, 6H, J = 1.8 Hz), 3.78 (d, 12H, J = 8.6 Hz);

13 C NMR (75 MHz, CDCl3) δ 165.1, 146.1, 144.2, 139.6, 127.4, 127.2, 124.7, 120.3,

117.9, 110.6, 110.5.

4-Chloro-1, 2-phenylene bis(3,4,5-trimethoxybenzoate), WZB-102: 1H NMR (300

MHz, CDCl3) δ 7.48-7.28 (m, 7H), 3.91 (s, 6H), 3.78 (s, 12H). 151

O O O OBn

OBn 27d

1, 2-Phenylene bis(4-(benzyloxy)benzoate), 27d: Following the procedure described for

1 preparation of 27a, ester 27d was obtained in 85% yield; H NMR (300 MHz, CDCl3) δ

8.07-8.02 (m, 4H), 7.44-7.28 (m, 14H), 6.97-6.93(m, 4H), 5.11 (s, 4H).

1 1, 2-Phenylene bis(4-hydroxybenzoate), WZB-103: H NMR (300 MHz, CD3COCD3)

δ 9.11 (br s, 2H), 7.78 (d, 4H, J = 8.2 Hz), 7.32-7.18 (m, 4H), 6.73 (d, 4H, J = 7.9 Hz);

13 C NMR (75 MHz, CD3COCD3) δ 164.4, 163.3, 162.9, 144.1, 133.5, 133.3, 133.2,

132.7, 127.2, 124.6, 121.0, 116.6, 116.3, 115.9.

3-Benzyloxybenzoyl chloride, 3d: Following the procedure described for preparation of compound 4, benzoyl chloride 31 was prepared in 81% yield; 1H NMR (300 MHz,

CDCl3) δ 7.79-7.72 (m, 2H), 7.49-7.36 (m, 5H), 7.33-7.26 (m, 2H), 7.22-7.16 (m, 1H),

5.55 (s, 2H). 152

Benzene-1, 2, 3-triyl tris(3-(benzyloxy)benzoate), 32: Following the procedure described for preparation of 6a, ester 32 was obtained in 86% yield; 1H NMR (300 MHz,

CDCl3) δ 7.79-7.66 (m, 6H), 7.47-7.31 (m, 20H), 7.27-7.19 (m, 3H), 7.12 (dd, 1H, J =

13 2.5, 2.4 Hz), 4.97 (s, 4H), 4.85 (s, 2H); C NMR (75 MHz, CDCl3) δ 163.8, 163.2,

158.8, 144.1, 136.3, 135.2, 130.0, 129.7, 129.5, 128.7, 128.6, 128.2, 127.7, 126.2, 123.1,

121.4, 120.9, 115.3, 115.2, 70.1.

Benzene-1,2,3-triyl tris(3-hydroxybenzoate), WZB-115: 1H NMR (300 MHz,

CD3COCD3) δ 8.58 (br s, 2H), 7.40-7.35 (m, 5H), 7.32-7.26 (m, 4H), 7.17-7.04 (m, 3H),

6.96 (dd, 1H, J = 1.0, 2.5 Hz), 6.93 (q, 1H, J = 1.4, 2.2 Hz), 6.87 (dd, 1H, J = 1.0, 2.5

Hz), 2.85 (br s, 1H); HPLC (CH3CN: H2O) RT 20.37 (100%); IR (KBr) 3455, 3079, 1747,

1719, 1599, 1485, 1456, 1285, 1239, 1207, 1109, 1002, 743 cm-1.

153

3-Methoxy-1,2-phenylene bis(3-(benzyloxy)benzoate), 33a: 1H NMR (300 MHz,

CDCl3) δ 7.88-7.80 (m, 4H), 7.49-7.33 (m, 13H), 7.23 (dd, 2H, J = 2.2, 7.5 Hz), 7.11 (d,

1H, J = 8.2 Hz), 7.01 (d, 1H, J = 8.0 Hz), 5.01 (d, 4H, J = 10.7 Hz), 3.89 (s, 3H); 13C

NMR (75 MHz, CDCl3) δ 164.2, 163.8, 158.9, 153.0, 144.1, 136.6, 136.5, 132.3, 130.3,

130.2, 129.8, 128.7, 128.2, 127.7, 126.5, 123.1, 123.0, 121.2,120.9, 115.9, 115.4, 115.3,

110.1, 70.2, 56.4.

O O O O

HO WZB-116

3-Methoxy-1,2-phenylene bis(3-hydroxybenzoate), WZB-116: 1H NMR (300 MHz,

CD3COCD3) δ 8.74 (br s, 2H), 7.57-7.52 (m, 4H), 7.39-7.28 (m, 3H), 7.15-7.08 (m, 3H),

13 7.04 (dd, 1H, J = 1.3, 8.3 Hz), 3.88 (s, 3H); C NMR (75 MHz, CD3COCD3) δ 163.6,

163.1, 161.1, 157.3, 152.7, 143.9, 132.1, 129.9, 129.8, 129.7, 126.2, 121.1, 120.8, 116.1,

115.2, 109.9, 55.6.

1 3-fluoro-1,2-phenylene bis(3-(benzyloxy)benzoate), 33b: H NMR (300 MHz, CDCl3)

δ 7.84-7.76 (m, 4H), 7.47-7.33 (m, 13H), 7.30-7.20 (m, 4H), 5.00 (d, 4H, J = 5.9 Hz); 13C

NMR (75 MHz, CDCl3) δ 163.9, 163.2, 158.9, 157.1, 153.8, 144.4, 144.3, 136.3, 131.5, 154

131.3, 129.8, 129.5, 128.7, 128.3, 127.7, 126.5, 126.4, 123.2, 123.1, 121.5, 121.3, 118.9,

115.9, 115.6, 114.4, 114.2, 70.2.

3-Fluoro-1,2-phenylene bis(3-hydroxybenzoate), WZB-117: Mp: 173.6- 174.9 °C. 1H

NMR (300 MHz, CD3COCD3) δ 8.79 (br s, 1H), 7.39-7.52 (m, 4H), 7.50-7.45 (m, 1H),

13 7.37-7.30 (m, 4H), 7.16-7.11 (m, 2H), 2.88 (br s, 1H); C NMR (75 MHz, CD3COCD3)

δ 164.4, 163.6, 158.6, 157.9, 154.6, 145.5, 132.5, 132.3, 130.9, 130.8, 130.7, 130.1,

127.7, 127.6, 122.3, 122.1, 122.0, 121.7, 120.3, 117.3, 115.0, 114.7; HPLC (CH3CN:

H2O) RT 17.23 (100%).

1 4-Chloro-1,2-phenylene bis(3-(benzyloxy)benzoate), 33c: H NMR (300 MHz, CDCl3)

δ 7.58-7.52 (m, 4H), 7.32-7.10 (m, 15H), 7.04-7.00 (m, 2H), 4.77 (s, 4H); 13C NMR (75

MHz, CDCl3) δ 164.6, 163.9, 163.7, 158.9, 142.9, 141.3, 136.4, 131.9, 129.9, 129.8,

129.7, 128.7, 128.2, 127.7, 126.8, 124.4, 124.1, 123.0, 121.5, 121.3, 115.3, 70.3. 155

4-Chloro-1,2-phenylene bis(3-hydroxybenzoate), WZB-118: 1H NMR (300 MHz,

CD3COCD3) δ 7.62-7.39 (m, 7H), 7.32-7.21 (m, 2H), 7.12-702 (m, 2H), 2.79 (br s, 2H);

IR (KBr) 3487, 3446, 3411, 1722, 1590, 1493, 1454, 1307, 1247, 1203, 1179, 1120,

1088, 1068, 930, 887, 746, 675, 579 cm-1.

2-Benzyloxy-benzoyl chloride, 19: Following the procedure described for preparation of

1 4, benzoyl chloride 19 was obtained in 81% yield; H NMR (300 MHz, CDCl3) δ 8.13

(dd, 1H, J = 1.7, 8.0 Hz), 7.60-7.50 (m, 3H), 7.45-7.32 (m, 3H), 7.11-7.06 (m, 2H), 5.23

(s, 2H).

3-Methoxy-1,2-phenylene bis(2-(benzyloxy)benzoate), 21a: 1H NMR (300 MHz,

CDCl3) δ 7.91 (dd, 1H, J = 1.8, 7.8 Hz), 7.83 (dd, 1H, J = 1.7, 7.8 Hz), 7.34-7.28 (m,

6H), 7.24-7.13 (m, 7H), 6.89-6.70 (m, 6H), 4.99 (s, 4H), 3.75 (s, 3H); 13C NMR (75

MHz, CDCl3) δ 163.0, 162.4, 159.3, 144.8, 136.9, 136.0, 134.6, 132.9, 132.8, 128.9, 156

128.8, 128.7, 127.7, 127.6, 127.0, 126.7, 126.2, 121.3, 120.7, 120.5, 119.1, 118.7, 114.1,

114.0, 70.6, 70.3.

1 3-Fluoro-1,2-phenylene bis(2-(benzyloxy)benzoate), 21b: H NMR (300 MHz, CDCl3)

δ 7.87 (dd, 1H, J = 1.7, 7.8 Hz), 7.78 (dd, 1H, J = 1.7, 7.8 Hz), 7.29-7.09 (m, 13H), 7.03-

13 6.69 (m, 2H), 6.80-6.67 (m, 4H), 4.93 (s, 4H). C NMR (75 MHz, CDCl3) δ 163.0,

162.0, 159.2, 159.0, 144.7, 136.6, 134.6, 134.5, 132.8, 132.5, 131.9, 128.5, 127.8, 127.6,

126.8, 126.7, 126.2, 126.1, 120.5, 119.2, 119.1, 118.7, 118.2, 114.2, 113.9, 113.8, 70.3.

1 4-Chloro-1,2-phenylene bis(2-(benzyloxy)benzoate), 21c: H NMR (300 MHz, CDCl3)

δ 7.77-7.72 (m, 2H), 7.27-7.05 (m, 15H), 6.75 (d, 2H, J = 8.4 Hz), 6.66 (m, 2H), 4.88 (s,

13 4H); C NMR (75 MHz, CDCl3) δ 163.0, 162.8, 159.1, 143.4, 141.8, 136.6, 136.5,

134.7, 134.6, 132.5, 132.4, 128.6, 127.8, 127.7, 126.9, 126.7, 124.7, 124.4, 120.5, 118.7,

118.5, 113.8, 70.4. 157

Benzene-1,2,3-triyl tris(2-hydroxybenzoate), WZB-127: 1H NMR (300 MHz,

CD3COCD3) δ 10.04 (br s, 2H), 7.94 (dd, 2H, J = 1.7, 8.0 Hz), 7.85 (dd, 1H, J = 1.9, 8.0

Hz), 7.64 (s, 3H), 7.58-7.46 (m, 3H), 7.00-6.78 (m, 6H); 13C NMR (75 MHz,

CD3COCD3) δ 166.3, 165.5, 160.9, 160.8, 142.4, 136.2, 136.0, 133.7, 129.8, 129.2,

129.1, 126.0, 120.8, 118.8, 118.7, 116.6, 110.2, 109.6.

3-Methoxy-1,2-phenylene bis(2-hydroxybenzoate), WZB-128: 1H NMR (300 MHz,

CD3COCD3) δ 10.17 (d, 2H, J = 3.6 Hz), 7.84 (dd, 1H, J = 1.6, 8.0 Hz), 7.78 (dd, 1H, J =

1.6, 8.0 Hz), 7.37-7.30 (m, 2H), 7.22 (t, 1H, J = 8.4 Hz), 6.89-6.84 (m, 4H), 6.76-6.67

13 (m, 2H), 3.75 (s, 3H); C NMR (75 MHz, CD3COCD3) δ 168.0, 167.4, 162.2, 162.0,

152.8, 143.1, 136.7, 136.6, 131.5, 130.5, 130.3, 126.9, 119.6, 117.8, 117.7, 115.1, 111.2,

111.1, 110.4, 56.4; HPLC (CH3CN: H2O) RT 19.75 (98%); IR (KBr) 3231, 2945, 2846,

1705, 1615, 1583, 1480, 1379, 1297, 1248, 1156, 1126, 1092, 858, 805, 761, 728, 698,

662 cm-1.

158

3-Fluoro-1,2-phenylene bis(2-hydroxybenzoate), WZB-129: 1H NMR (300 MHz,

CD3COCD3) δ 10.14 (s, 1H), 10.07 (s, 1H), 7.92-7.85 (m, 2H), 7.49-7.42 (m, 2H), 7.38-

7.31 (m, 1H), 7.23-7.16 (m, 2H), 6.97 (dd, 2H, J = 3.4, 8.4 Hz), 6.86-6.79 (m, 2H); 13C

NMR (75 MHz, CD3COCD3) δ 167.6, 166.7, 162.2, 156.9, 153.6, 143.5, 143.4, 136.9,

130.7, 130.4, 126.9, 119.7, 118.9, 117.9, 114.8, 114.6, 110.8, 110.6.

4-Chloro-1,2-phenylene bis(2-hydroxybenzoate), WZB-130: 1H NMR (300 MHz,

CD3COCD3) δ 9.90 (br s, 2H), 7.79-7.74 (m, 2H), 7.59 (d, 1H, J = 2.4 Hz), 7.52-7.34 (m,

13 4H), 6.85-6.72 (m, 4H); C NMR (75 MHz, CD3COCD3) δ 168.5, 168.2, 168.0, 162.8,

143.4, 142.8, 141.8, 137.9, 137.7, 132.3, 131.0, 128.2, 126.0, 125.1, 124.8, 120.6, 118.5,

112.1, 120.0, 111.9.

methyl 3-(methoxymethoxy)benzoate, 50: Concentrated sulfuric acid (2 mL) was added to a solution of 3-hydroxybenzoic acid (13.8 g, 100 mmol) in methanol (200 mL) at room 159 temperature. The mixture was stirred for 5 h. Then the reaction mixture was poured into water (200 mL) and extracted with diethyl ether (3 X 150 mL), The combined organic phase was washed with saturated sodium bicarbonate aqueous solution, then washed with brine, dried over MgSO4, concentrated to give a white solid, which recrystallized from benzene/diethyl ether to give methyl 3-hydroxybenzoate in 91% yield. Mp: 69 °C. 1H

NMR (300 MHz, CDCl3) δ 7.62-7.61 (m, 1H), 7.59-7.56 (m, 1H), 7.28 (t, 1H, J = 7.9,

13 15.8 Hz), 7.11-7.06 (m, 2H), 3.90 (s, 3H); C NMR (75 MHz, CDCl3) δ 167.9, 156.2,

131.2, 129.8, 122.7, 120.6, 116.5, 52.5.

Then chloromethyl methyl ether (3.94 mL, 52.19 mmol) was slowly added into the solution of methyl 3-hydroxybenzoate (6.90 g, 45.38 mmol) and potassium carbonate

(25.1 g, 181.52 mmol) in DMF (100 mL) at 0 °C. The reaction was stirred for 4 h at 0 °C.

Cold water was added to quench the reaction upon completion as indicated by TLC. The mixture was diluted with EtOAc (150 mL), washed with water (3 X 50 mL), dried over

MgSO4, filtered and condensed; the crude was purified by column chromatography to

1 give the title compound 50 in 85 % yield; H NMR (300 MHz, CDCl3) δ 7.70-7.67 (m,

2H), 7.37-7.32 (m, 1H), 7.26-7.20 (m, 1H), 5.20 (s, 2H), 3.91 (s, 3H), 3.48 (s, 3H).

1-(chloromethyl)-3-(methoxymethoxy)benzene, 51: LiAlH4 (2.78g, 73.10 mmol) was added into a solution of ester 50 (6.52 g, 33.23 mmol) in THF (100 mL) at 0 °C in five portions at an interval of 10 min. The reaction was stirred at 0 °C for 1 h and was diluted with diethyl ether (200 mL) and quenched by carefully adding cold water (4.4 mL), 15% 160 sodium hydroxide (4.4 mL) and cold water (7.2 mL). MgSO4 (5.5 g) was subsequently added to the mixture and the solution was stirred for another 1 h at room temperature.

The precipitate was filtered off, the solvents were removed and the crude was purified by column chromatography to provide (3-(methoxymethoxy)phenyl)methanol in 99% yield;

1 H NMR (300 MHz, CDCl3) δ 7.23 (t, 1H, J = 8.2, 15.7 Hz), 7.00 (s, 1H), 6.93 (t, 2H, J =

7.3, 15.7 Hz), 5.12 (d, 2H, J = 1.3 Hz), 4.56 (d, 2H, J = 1.8 Hz), 3.42 (s, 3H); 13C NMR

(75 MHz, CDCl3) δ 157.3, 142.8, 129.7, 120.5, 115.4, 114.7, 94.4, 64.9, 55.9.

Then triethylamine (3.85 mL, 27.41 mmol) was added into a solution of (3-

(methoxymethoxy)phenyl)methanol (3.84 g, 22.84 mmol) in DCM (45 mL) at 0 °C under an atmosphere of argon. Then methanesulfonyl chloride (2.13 mL, 27.41 mmol) was added dropwise into the reaction mixture. The reaction mixture was allowed to warm to room temperature after the addition was completed and stirred for 18 h. Cold water (20 mL) was poured into the reaction mixture which was subsequently extracted with DCM

(3 X 50 mL). The combined organic phases were washed with brine (30 mL) and dried over MgSO4, filtered and concentrated. The crude was purified by column chromatography on silica gel with the eluent (EtOAc/Hexane, 1: 8) to give the title

1 compound 51 in 84 % yield. H NMR (300 MHz, CDCl3) δ 7.25 (t, 1H, J = 7.9, 15.7 Hz),

7.02-6.96 (m, 3H), 5.16 (s, 2H), 4.53 (s, 2H), 3.46 (s, 3H).

161

3,3',3''-(Benzene-1,2,3-triyltris(oxy))tris(methylene)tris((methoxymethoxy)benzene),

75: Pyrogallol (63 mg, 0.5 mmol) was added into a degassed solution of potassium carbonate (414.6 mg, 3.0 mmol) in DMF (5 mL) at room temperature. Then 51 (297.7 mg, 1.6 mmol) was added into the mixture and the stirring reaction mixture was heated at

70 °C for 12 h under argon atmosphere. After the completion of the reaction as indicated by TLC, cold water (15 mL) was added into the reaction mixture and diluted with EtOAc

(35 mL). The organic phase was separated and washed with water (3 X 20 mL). The organic phases were combined and dried over MgSO4, filtered and concentrated. The crude was purified by column chromatography on silica gel with the eluent

(EtOAc/Hexane, 1: 8) to give the title compound in 67 % yield. 1H NMR (300 MHz,

CDCl3) δ 7.29-7.24 (m, 2H), 7.19-7.05 (m, 7H), 6.99-6.88 (m, 4H), 6.62 (d, 2H, J = 8.3

Hz), 5.14 (s, 4H), 5.08 (s, 6H), 5.06 (s, 2H), 3.45 (s, 6H), 3.42 (s, 3H).

3, 3', 3''-(Benzene-1, 2, 3-triyltris(oxy))tris(methylene)triphenol, WZB-131: HCl (2.4

M, 0.2 mL) in ethyl acetate was added into a solution of 75 (0.12 mmol) in methanol (5 mL) at room temperature. The mixture was heated at 50 °C for 90 min followed by removal of solvent. The crude was purified by column chromatography on silica gel with the eluent (EtOAc/Hexane, 1: 5) to give WZB-131 in 81 % yield. 1H NMR (300 MHz,

CDCl3) δ 8.27 (br s, 3H), 7.22-7.10 (m, 3H), 7.09-6.90 (m, 6H), 6.80-6.65 (m, 6H), 5.10-

13 5.00 (m, 6H); C NMR (75 MHz, CDCl3) δ 157.2, 152.8, 147.1, 139.0, 136.8, 129.2, 162

129.1, 118.4, 118.2, 118.1, 114.3, 114.1, 113.9, 107.4, 70.4, 70.1; MS (APCI): M+H expected 444.16, obtained 445.10.

3, 3'-(3-Methoxy-1, 2-phenylene)bis(oxy)bis(methylene)bis ((methoxymethoxy)- benzene), 76: Following the procedure described for the preparation of 75, compound 7a

(140.0 mg, 1.0 mmol) reacted with 51 (409.3 mg, 2.2 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 7), the title compound 76 in 79% yield. 1H

NMR (300 MHz, CDCl3) δ 7.25-7.17 (m, 3H), 7.12-7.09 (m, 2H), 7.02 (d, 1H, J = 7.6

Hz), 6.96-6.87 (m, 3H), 5.08 (s, 4H), 5.02 (s, 4H), 3.76 (s, 3H), 3.39 (s, 3H), 3.38 (s, 3H);

13 C NMR (75 MHz, CDCl3) δ 157.6, 157.3, 154.1, 152.9, 139.7, 139.0, 137.9, 129.6,

129.2, 123.8, 121.9, 120.8, 116.1, 115.8, 115.7, 115.1, 107.6, 105.9, 94.4, 74.9, 70.9,

56.2, 55.9.

3,3'-(3-Fluoro-1,2-phenylene)bis(oxy)bis(methylene)bis((methoxymethoxy)benzene),

77: Following the procedure described for the preparation of 75, compound 7b (72.0 mg,

0.5 mmol) reacted with 51 (204.6 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 9), the title compound 77 in 78% yield. 1H NMR (300 163

MHz, CDCl3) δ 7.28-6.84 (m, 9H), 6.72-6.66 (m, 2H), 5.12 (s, 2H), 5.09 (s, 2H), 5.08 (s,

13 2H), 5.04 (s, 2H), 3.42 (s, 3H), 3.41 (s, 3H); C NMR (75 MHz, CDCl3) δ 158.3, 157.6,

157.3, 155.0, 153.4, 153.3, 139.0, 138.4, 136.7, 136.6, 129.7, 129.4, 123.6, 123.4, 121.7,

120.7, 116.0, 115.9, 115.1, 109.9, 109.5, 109.3, 94.4, 75.3, 74.0, 55.9.

3,3'-(4-Chloro-1,2-phenylene)bis(oxy)bis(methylene)bis((methoxymethoxy)benzene),

78: Following the procedure described for the preparation of 75, compound 7c (72.0 mg,

0.5 mmol) reacted with 51 (204.6 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 7), the title compound 78 in 80% yield. 1H NMR (300

MHz, CDCl3) δ 7.28-7.20 (m, 2H), 7.11 (d, 2H, J = 7.0 Hz), 7.05 (t, 2H, J = 6.5, 13.4

Hz), 6.98-6.94 (m, 2H), 6.90 (d, 1H, J = 1.6 Hz), 6.81-6.80 (m, 2H), 5.12 (d, 4H, J = 2.94

13 Hz), 5.05 (s, 4H), 3.43 (s, 3H), 3.42 (s, 3H); C NMR (75 MHz, CDCl3) δ 157.5, 149.6,

147.6, 138.7, 138.3, 129.7, 129.6, 126.3, 121.1, 120.7, 115.9, 115.8, 115.3, 115.2, 115.1,

94.4, 71.3, 71.0, 56.0.

3,3'-(3-Methoxy-1,2-phenylene)bis(oxy)bis(methylene)diphenol, WZB-132: With the procedure described for the preparation of WZB-131, compound 76 (348.8 mg, 0.8 164 mmol) was deprotected with the solution of HCl (1.2 M, in EtOAc) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 4), the title compound WZB-132

1 in 89% yield. H NMR (300 MHz, CD3COCD3) δ 7.16-7.05 (m, 3H), 6.97-6.81 (m, 4H),

6.76-6.70 (m, 2H), 6.58-6.53 (m, 2H), 4.95 (s, 2H), 4.93 (s, 2H), 3.81 (s, 3H); 13C NMR

(75 MHz, CD3COCD3) δ 158.5, 158.2, 155.0, 153.8, 149.2, 147.9, 140.9, 140.2, 138.8,

137.5, 130.4, 130.0, 124.6, 120.2, 119.4, 119.3, 115.9, 115.4, 115.1, 108.4, 106.7, 75.2,

71.5, 71.3, 60.7, 56.6; MS (APCI): M+H expected 352.13, obtained 353.05.

3,3'-(4-Chloro-1,2-phenylene)bis(oxy)bis(methylene)diphenol, WZB-133: Following the procedure described for the preparation of WZB-131, compound 78 (178.1 mg, 0.4 mmol) was deprotected with the solution of HCl (1.2 M, in EtOAc) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 4), WZB-133 in 92% yield. 1H

NMR (300 MHz, CDCl3) δ 8.26 (br s, 2H), 7.23-7.16 (m, 2H), 7.06-6.94 (m, 5H), 6.90-

6.76 (m, 4H), 5.14 (s, 2H), 5.10 (s, 2H).

3,3'-(3-Fluoro-1,2-phenylene)bis(oxy)bis(methylene)diphenol, WZB-134: Following the procedure described for the preparation of WZB-131, compound 77 was deprotected 165 with the solution of HCl (1.2 M, in EtOAc) to provide, after purification on silica gel the

1 eluent (EtOAc/Hexane, 1: 4), WZB-134 in 79% yield. H NMR (300 MHz, CDCl3) δ

7.25-7.12 (m, 3H), 7.02-6.87 (m, 4H), 6.81-6.70 (m, 4H), 5.05 (s, 2H), 5.01 (s, 2H); IR

(KBr) 3374, 2949, 2880, 2738, 1594, 1496, 1457, 1379, 1281, 1246, 1211, 1158, 1066,

999, 960, 868, 778, 691 cm-1.

N,N'-(4-fluoro-1,2-phenylene)bis(3-(benzyloxy)benzamide), 55: The acid chloride

(528.9 mg, 2.15 mmol) and DIEA (383 µL, 2.20 mmol) were added into a solution of 4- fluoro-1, 2-phenylenediamine (126 mg, 1.00 mmol) in DCM at room temperature. The mixture was stirred at room temperature for 2 h. Cold water (15 mL) was added into the reaction mixture to quench the reaction upon completion as indicated by TLC. The mixture then was extracted with dichloromethane (4 X 30 mL). The combined organic phases were washed with brine (30 mL), dried over MgSO4, filtered and condensed. The crude was purified by column chromatography on silica gel with the eluent

(EtOAc/Hexane, 1: 10 to 1: 3) to provide the product as a solid in 73% yield. Mp: 151.2-

1 152.2 °C. H NMR (300 MHz, CDCl3) δ 9.45 (s, 1H), 9.16 (s, 1H), 7.63-7.53 (m, 4H),

7.43-7.29 (m, 12H), 7.23-7.14 (m, 4H), 5.08 (d, 4H, J = 3.3 Hz); 13C NMR (75 MHz,

CDCl3) δ 166.5, 166.3, 159.1, 136.5, 134.6, 134.5, 132.6, 132.5, 129.9, 128.6, 127.6, 166

127.1, 126.9, 126.3, 126.2, 119.9, 119.8, 119.6, 113.5, 113.4, 113.1, 112.8, 112.7, 112.4,

70.2.

N, N'-(4-chloro-1,2-phenylene)bis(3-(benzyloxy)benzamide), 54: Following the procedure described for the preparation of 55, compound 52 (213.0 mg, 1.5 mmol) was acylated with 31 (812.1 mg, 3.3 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), the title compound 54 in 73% yield. Mp: 173.2-174.3 °C;

1 H NMR (300 MHz, CDCl3) δ 9.27 (d, 1H, J = 2.0 Hz), 7.63-7.55 (m, 4H), 7.43-7.31 (m,

13 13H), 7.23-7.15 (m, 3H), 6.81-6.72 (m, 1H), 5.09 (s, 4H); C NMR (75 MHz, CDCl3) δ

164.9, 157.6, 134.9, 133.0, 130.3, 130.0, 128.5, 127.6, 127.1, 126.6, 126.1, 125.2, 124.7,

124.0, 118.4, 118.3, 118.2, 111.9, 68.6.

Methyl 4-fluoro-3-(methoxymethoxy)benzoate, 60: Chloromethyl methyl ether (2.55 mL, 33.79 mmol) was slowly added into a solution of methyl 4-fluoro-3- hydroxybenzoate (5.0 g, 29.38 mmol) and potassium carbonate (16.2 g, 117.52 mmol) in

DMF (50 mL) at 0 °C. The reaction was stirred for 4h at 0 °C. Cold water was added to quench the reaction upon completion as indicated by TLC. The mixture was diluted with

EtOAc (150 mL), washed with water (3 X 50 mL), dried over MgSO4, filtered and 167 condensed. The crude was purified by column chromatography on silica gel to give the

1 title compound as oil in 81% yield. H NMR (300 MHz, CDCl3) δ 7.87 (dd, 2H, J = 2.0,

8.0 Hz), 7.73-7.68 (m, 1H), 7.14 (dd, 1H, J = 8.6, 10.5 Hz), 5.27 (s, 2H), 3.91 (s, 3H),

13 3.54 (s, 3H); C NMR (75 MHz, CDCl3) δ 167.3, 159.2, 155.8, 146.1, 128.0, 125.9,

120.4, 117.7, 96.8, 57.8, 53.6.

4-(Chloromethyl)-1-fluoro-2-(methoxymethoxy)benzene, 61: LiAlH4 (1.99g, 52.49 mmol) was added into a solution of ester 60 (5.11 g, 23.86 mmol) in THF (100 mL) at 0

°C in five portions at an interval of 10 min. The reaction was stirred at 0 °C for 1 h and was diluted with diethyl ether (200 mL) and quenched by carefully adding cold water

(3.1 mL), 15% sodium hydroxide (3.1 mL) and cold water (5.2 mL). MgSO4 (5.5 g) was added to the mixture and the solution was stirred for another 1 h at room temperature.

The precipitate was filtered off, the solvents were removed and the crude was purified by column chromatography on silica get to provide (4-fluoro-3-(methoxymethoxy)phenyl)-

1 methanol in 88% yield; H NMR (300 MHz, CDCl3) δ 7.20 (dd, 1H, J = 2.0, 8.0 Hz),

7.06 (dd, 1H, J = 8.3, 10.8 Hz), 6.97-6.92 (m, 1H), 5.21 (s, 2H), 4.61 (d, 2H, J = 5.7 Hz),

13 3.52 (s, 3H), 2.08 (t, 1H, J = 5.8 Hz); C NMR (75 MHz, CDCl3) δ 152.6, 149.4, 143.3,

135.8, 119.5, 115.0, 114.7, 94.0, 63.1, 54.8.

Then triethylamine (1.8 mL, 12.9 mmol) was added into a solution of (4-fluoro-3-

(methoxymethoxy)phenyl)-methanol (2.0 g, 10.7 mmol) in DCM (45 mL) at 0 °C under an atmosphere of argon. Then methanesulfonyl chloride (1.0 mL, 12.9 mmol) was added 168 dropwise into the reaction mixture. The reaction mixture was allowed to warm to room temperature after the addition was completed and continued stirring for 18 h. Cold water

(20 mL) was poured into the reaction mixture which was subsequently extracted with

DCM (3 X 50 mL). The combined organic phases were washed with brine (30 mL) and dried over MgSO4, filtered and concentrated. The crude was purified by column chromatography on silica gel with the eluent (EtOAc/Hexane, 1: 8) to give the title

1 compound 61 in 92 % yield. H NMR (300 MHz, CDCl3) δ 7.23 (dd, 1H, J = 2.1, 7.8

Hz), 7.02 (dd, 1H, J = 8.3, 10.6 Hz), 7.01-6.96 (m, 1H), 5.22 (s, 2H), 4.53 (s, 2H), 3.53

(s, 3H).

O O O F F OMOM OMOM

OMOM F 79

4, 4', 4'' - (Benzene-1, 2, 3-triyltris(oxy))tris(methylene)tris(1-fluoro-2-(methoxy - methoxy)benzene), 79: 4-Fluoro-benzyl chloride 61 (674.7 mg, 3.2 mmol) was added into a solution of pyrogallol (126.1 mg, 1.0 mmol) and potassium carbonate (829.2 mg,

6.0 mmol) in degassed anhydrous DMF (5.0 mL) at room temperature. The reaction mixture was heated to 80 °C for 12 h and diluted with EtOAc (35 mL), then washed with water (5 X 5 mL) and brine (5 mL). Dried over MgSO4, filtered and concentrated. The residue was purified by column chromatography (EtOAc/Hexane, 1: 6) to provide the

1 title compound 79 in 65% yield. H NMR (300 MHz, CDCl3) δ 7.29-7.25 (m, 3H), 7.10-

6.90 (m, 7H), 6.63 (s, 1H), 6.60 (s, 1H), 5.17 (s, 4H), 5.07 (s, 2H), 5.01 (s, 4H), 4.99 (s, 169

13 2H), 3.47 (s, 6H), 3.44 (s, 3H); C NMR (75 MHz, CDCl3) δ 154.4, 123.8, 151.1, 145.1,

144.9, 144.7, 144.5, 138.3, 134.4, 133.6, 123.8, 122.6, 121.5, 118.2, 117.1, 116.4, 116.1,

116.0, 115.7, 108.1, 95.7, 95.6, 74.4, 70.5, 56.3.

4,4'-(3-Fluoro-1,2-phenylene)bis(oxy)bis(methylene)bis(1-fluoro-2-(methoxymethoxy)benzene), 80: Following the procedure described for the preparation of 79, 7b (64.1 mg, 0.5 mmol) was reacted with 61 (230.3 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), 215.5 mg of the title compound 80 in 93%

1 yield. H NMR (300 MHz, CDCl3) δ 7.28 (d, 2H, J = 8.1 Hz), 7.10-6.88 (m, 5H), 6.74-

6.68 (m, 2H), 5.17 (s, 2H), 5.13 (s, 2H), 5.03 (s, 2H), 5.01 (s, 2H), 3.46 (s, 6H); 13C NMR

(75 MHz, CDCl3) δ 158.2, 154.9, 154.6, 153.1, 151.3, 145.2, 136.4, 133.8, 133.1, 123.5,

122.8, 121.5, 118.1, 117.2, 116.5, 116.2, 116.1, 115.9, 109.8, 109.7, 109.4, 95.6, 74.9,

70.6, 56.3.

4,4'-(3-Methoxy-1,2-phenylene)bis(oxy)bis(methylene)bis(1-fluoro-2-(methoxymethoxy)benzene), 81: Following the procedure described for the preparation of 79, 7a 170

(70.1 mg, 0.5 mmol) was reacted with 61 (230.3 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), 189.7 mg of the title

1 compound 81 in 80% yield. H NMR (300 MHz, CDCl3) δ 7.34 (dd, 1H, J = 9.8, 1.7 Hz),

7.27 (dd, 1H, J = 9.7, 1.9 Hz), 7.09-6.91 (m, 5H), 6.59 (s, 1H), 6.56 (s, 1H), 5.15 (s, 2H),

5.13 (s, 2H), 4.99 (s, 2H), 4.97 (s, 2H), 3.82 (s, 3H), 3.46 (s, 3H), 3.45 (s, 3H); 13C NMR

(75 MHz, CDCl3) δ 154.4, 153.9, 152.6, 151.1, 145.0, 137.6, 134.5, 133.7, 123.9, 122.5,

121.5, 118.2, 117.2, 116.4, 115.9, 115.7, 107.5, 105.9, 74.3, 70.6, 56.3.

4,4'-(4-Chloro-1,2-phenylene)bis(oxy)bis(methylene)bis(1-fluoro-2-(methoxymethoxy)benzene), 82: Following the procedure described for the preparation of 79, 7c (72.1 mg, 0.5 mmol) was reacted with 61 (230.3 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), the title compound 82 in 94% yield. 1H

NMR (300 MHz, CDCl3) δ 7.29-7.19 (m, 2H), 7.09-6.96 (m, 4H), 6.91-6.79 (m, 3H),

5.16 (d, 4H, J = 3.2 Hz), 5.00 (s, 4H), 3.47 (s, 3H), 3.46 (s, 3H); 13C NMR (75 MHz,

CDCl3) δ 160.6, 154.4, 151.2, 149.4, 147.4, 145.0, 133.3, 132.9, 126.5, 122.7, 121.5,

118.2, 117.2, 116.5, 116.2, 115.4, 95.6, 70.9, 70.7, 56.4, 56.3. 171

F Cl O OMOM

OMOM F 83

4,4'-(4-Chloro-1,3-phenylene)bis(oxy)bis(methylene)bis(1-fluoro-2-(methoxymethoxy)benzene), 83: Following the procedure described for the preparation of 79, 7e (72.1 mg, 0.5 mmol) was reacted with 61 (230.3 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), the title compound 83 in 70% yield. 1H

NMR (300 MHz, CDCl3) δ 7.30 (d, 1H, J = 1.1 Hz), 7.23 (d, 2H, J = 8.3 Hz), 7.11-6.96

(m, 4H), 6.58 (d, 1H, J = 2.5 Hz), 6.48 (dd, 1H, J = 8.7, 2.2 Hz), 5.20 (s, 4H), 4.99 (s,

13 2H), 4.90 (s, 2H), 3.50 (s, 6H); C NMR (75 MHz, CDCl3) δ 158.2, 154.6, 154.4, 151.2,

145.0, 132.9, 130.3, 121.7, 121.6, 121.3, 121.2, 117.3, 117.0, 116.5, 116.3, 115.3, 106.9,

102.6, 95.7, 70.1, 69.7, 56.4.

4,4'-(5-Chloro-1,3-phenylene)bis(oxy)bis(methylene)bis(1-fluoro-2-(methoxymethoxy)benzene), 84: Following the procedure described for the preparation of 79, 7f (72.1 mg, 0.5 mmol) was reacted with 61 (230.3 mg, 1.1 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), the title compound 84 in 71% yield. 1H

NMR (300 MHz, CDCl3) δ 7.25 (d, 1H, J = 1.6 Hz), 7.23 (d, 1H, J = 1.6 Hz), 7.11-7.04 172

(m, 2H), 7.00-6.95 (m, 2H), 6.58 (d, 2H, J = 1.8 Hz), 6.45 (t, 1H, J = 1.8 Hz), 5.21 (s,

13 4H), 4.89 (s, 4H), 3.50 (s, 6H); C NMR (75 MHz, CDCl3) δ 160.0, 154.7, 151.3, 145.1,

135.3, 132.3, 121.7, 117.3, 116.6, 116.3, 108.2, 100.9, 95.5, 69.7, 56.5.

5,5',5''-(Benzene-1,2,3-triyltris(oxy))tris(methylene)tris(2-fluorophenol), WZB-146:

Hydrochloride in EtOAc (1.2 M, 0.5 mL) was added into a solution of 79 (0.16 mmol) in methanol. The mixture was heated to 50 °C and stirred for 2 h. The mixture was condensed and purified by flash column chromatography (EtOAc/Hexane, 1: 4) to provide WZB-146 as a white solid in 89% yield. Mp, 163.1-163.9 °C; 1H NMR (300

MHz, CD3OCD3) δ 7.18-7.07 (m, 5H), 7.00-6.92 (m, 5H), 6.75 (s, 1H), 6.72 (s, 1H), 5.07

13 (s, 4H), 4.95 (s, 2H), 2.80 (br s, 3H); C NMR (75 MHz, CD3OCD3) δ 153.8, 153.5,

150.3, 147.9, 145.6, 145.5, 145.3, 145.1, 139.1, 135.9, 135.3, 135.1, 124.5, 120.9, 120.7,

120.1, 120.0, 119.9, 119.8, 119.2, 118.7, 118.0, 117.9, 117.8, 116.8, 116.4, 116.1, 108.8,

108.6, 74.8, 71.0, 70.8; HPLC (CH3CN: H2O) RT 15.77 (92%); IR (KBr) 3392, 3240,

2949, 2877, 1608, 1531, 1513, 1458, 1436, 1311, 1294, 1261, 1239, 1203, 1111, 966,

808, 770, 628 cm-1; MS (APCI): M+H expected 498.45, obtained 499.10. 173

5,5'-(3-Fluoro-1,2-phenylene)bis(oxy)bis(methylene)bis(2-fluorophenol), WZB-147:

Following the procedure described for the preparation of WZB-146, compound 80 was deprotected with HCl solution in EtOAc (1.2 M) to give WZB-147 in 91% yield. Mp,

1 99.8-101.5 °C; H NMR (300 MHz, CD3OCD3) δ 7.20-7.03 (m, 4H), 7.01-6.96 (m, 2H),

6.94-6.87 (m, 2H), 6.79-6.73 (m, 1H), 5.12 (s, 2H), 5.02 (s, 2H); 13C NMR (75 MHz,

CD3OCD3) δ 158.4, 155.4, 153.7, 153.1, 150.0, 145.2, 145.0, 144.9, 144.7, 136.6, 136.4,

134.6, 124.1, 123.9, 120.2, 120.1, 119.5, 119.4, 118.1, 117.4, 116.3, 116.0, 115.9, 115.7,

110.3, 109.3, 109.0, 74.7, 70.5; HPLC (CH3CN: H2O) RT 15.73 (94%); IR (KBr) 3347,

2954, 2866, 1611, 1514, 1476, 1434, 1379, 1338, 1283, 1247, 1204, 1150, 1113, 1081,

959, 873, 805, 770 cm-1.

5,5'-(4-Chloro-1,2-phenylene)bis(oxy)bis(methylene)bis(2-fluorophenol), WZB-149:

Following the procedure described for the preparation of WZB-146, compound 82 was deprotected with HCl solution in EtOAc (1.2 M) to give WZB-149 in 85% yield. Mp,

1 132.6-133.2 °C; H NMR (300 MHz, CD3OCD3) δ 7.18-7.04 (m, 5H), 7.01-6.87 (m, 4H), 174

13 5.11 (s, 2H), 5.08 (s, 2H), 2.95 (br s, 2H); C NMR (75 MHz, CD3OCD3) δ 153.5, 150.5,

150.3, 148.7, 145.7, 145.4, 135.0, 134.7, 126.5, 121.8, 120.0, 119.9, 117.9, 116.9, 116.8,

116.6, 116.5, 115.9, 71.2, 71.1; HPLC (CH3CN: H2O) RT 16.43 (98%); IR (KBr) 3575,

3413, 3075, 3045, 2936, 2874, 1612, 1588, 1515, 1500, 1439, 1381, 1304, 1280, 1246,

1205, 1160, 1131, 995, 868, 811, 776 cm-1.

5,5'-(4-Chloro-1,3-phenylene)bis(oxy)bis(methylene)bis(2-fluorophenol), WZB-150:

Following the procedure described for the preparation of WZB-146, compound 83 was deprotected with HCl solution in EtOAc (1.2 M) to give WZB-150 in 86% yield. 1H

NMR (300 MHz, CD3OCD3) δ 8.73 (br s, 2H), 7.14-7.07 (m, 4H), 6.95-6.90 (m, 2H),

6.64 (d, 1H, J = 2.2 Hz), 6.59 (t, 1H, J = 2.2 Hz), 5.03 (s, 4H); 13C NMR (75 MHz,

CD3OCD3) δ 161.2, 153.6, 150.4, 145.6, 135.6, 134.4, 120.0, 117.9, 116.8, 116.5, 108.7,

101.7, 70.1. HPLC (CH3CN: H2O) RT 16.53 (93%); IR (KBr) 3561, 3407, 3084, 2935,

2874, 1607, 1585, 1512, 1458, 1432, 1381, 1311, 1287, 1242, 1188, 1148, 1106, 1075,

1029, 816, 797, 763 cm-1.

175

5, 5'-(5-Chloro-1, 3-phenylene)bis(oxy)bis(methylene)bis(2-fluorophenol), WZB-151:

Following the procedure described for the preparation of WZB-146, compound 84 was deprotected with HCl solution in EtOAc (1.2 M) to give WZB-151 in 83% yield. 1H

NMR (300 MHz, CD3OCD3) δ 8.77 (br s, 2H), 7.28 (d, 1H, J = 8.7 Hz), 7.20-7.07 (m,

4H), 6.98-6.88 (m, 2H), 6.81 (d, 1H, J = 2.7 Hz), 6.59 (dd, 1H, J = 8.7, 2.7 Hz), 5.11 (s,

13 2H), 5.00 (s, 2H); C NMR (75 MHz, CD3OCD3) δ 159.5, 155.4, 153.5, 150.3, 145.7,

145.5, 134.5, 134.3, 130.8, 120.0, 119.9, 119.8, 119.7, 119.6, 118.0, 117.9, 117.6, 116.8,

116.7, 116.5, 115.0, 108.0, 103.1, 70.4, 70.1; HPLC (CH3CN: H2O) RT 17.11 (94%); IR

(KBr) 3371, 3092, 2901, 2866, 1611, 1575, 1517, 1440, 1384, 1279, 1172, 1109, 1063,

889, 867, 803, 772 cm-1.

Methyl 4-methoxy-3-(methoxymethoxy)benzoate, 63: Concentrated sulfuric acid (1.46 g, 14.86 mmol) was added dropwise into a stirring solution of 3-hydroxy-4- methoxybenzoic acid (5.00 g, 29.74 mmol) in methanol (25 mL) at room temperature.

The reaction mixture was heated to reflux for 12 h. The solvent was removed in vacuo and the residue was dissolved in EtOAc (100 mL), the solution was washed with saturated sodium bicarbonate (3 X 20 mL), water (20 mL) and brine (20 mL), then dried over MgSO4, filtered and condensed to give the crude product which was purified by column chromatography on silica gel with the eluent (EtOAc/Hexane, 1: 4) to give

1 methyl 4-methoxy-3-hydroxybenzoate in 98% yield. H NMR (300 MHz, CDCl3) δ 7.63- 176

7.59 (m, 2H), 6.86 (d, 1H, J = 8.2 Hz), 5.75 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H); 13C NMR

(75 MHz, CDCl3) δ 164.7, 148.3, 143.1, 121.2, 120.7, 113.5, 107.7, 53.9, 49.9.

Then, chloromethyl methyl ether (2.6 mL, 34.6 mmol) was slowly added into a solution of methyl 4-methoxy-3-hydroxybenzoate (5.7 g, 31.4 mmol) and potassium carbonate

(15.2 g, 109.9 mmol) in DMF (30 mL) at 0 °C. The reaction was stirred for 4 h at 0 °C.

Cold water was added to quench the reaction upon completion as indicated by TLC. The mixture was diluted with EtOAc (150 mL), washed with water (3 X 50 mL), dried over

MgSO4, filtered and condensed; the crude was purified by column chromatography on silica gel to give the product 12 as clear oil in 91 % yield. Mp: 49-49.5 °C. 1H NMR (300

MHz, CDCl3) δ 7.79 (d, 1H, J = 2.0 Hz), 7.71 (dd, 1H, J = 8.5, 2.0 Hz), 6.90 (d, 1H, J =

13 8.5 Hz), 5.26 (s, 2H), 3.91 (s, 3H), 3.87 (s, 3H), 3.52 (s, 3H); C NMR (75 MHz, CDCl3)

δ 165.6, 152.6, 144.8, 123.9, 121.7, 116.0, 109.7, 94.3, 55.2, 54.9, 50.8.

4-(Chloromethyl)-1-methoxy-2-(methoxymethoxy)benzene, 64: LiAlH4 (1.4g, 35.9 mmol) was added into a solution of ester 12 (3.7 g, 16.3 mmol) in THF (100 mL) at 0 °C in five portions at an interval of 10 min. The reaction was stirred at 0 °C for 1 h and was diluted with diethyl ether (200 mL) and quenched by carefully adding cold water (2.2 mL), 15% sodium hydroxide (2.2 mL) and cold water (3.5 mL). MgSO4 (5.5 g) was added to the mixture and the solution was stirred for another 1 h at room temperature.

The precipitate was filtered off, the solvents were removed and the crude was purified by column chromatography on silica gel to provide (4-methoxy-3-(methoxymethoxy)- 177

1 phenyl)methanolin 83% yield. H NMR (300 MHz, CDCl3) δ 7.11 (d, 1H, J = 1.8 Hz),

6.92 (dd, 1H, J = 8.3, 1.8 Hz), 6.83 (d, 1H, J = 8.3 Hz), 5.17 (s, 2H), 4.51 (d, 2H, J = 5.3

13 Hz), 3.83 (s, 3H), 3.47 (s, 3H), 3.20 (br s, 1H); C NMR (75 MHz, CDCl3) δ 149.0,

146.2, 134.3, 121.1,115.4, 111.6, 95.3, 64.9, 55.8.

Then, methanesulfonyl chloride (295.0 µL, 3.8 mmol) was added into the solution of (4- methoxy-3-(methoxymethoxy)phenyl)methanol (716.2 mg, 3.6 mmol) and triethylamine

(558.3 µL, 3.9 mmol) in DCM (50 mL) at 0 °C. The reaction mixture was stirred for 1 h and allowed to warm to room temperature slowly and stirred for overnight at room temperature. The reaction was quenched with addition of cold water (2.5 mL) upon completion of the reaction as indicated by TLC. The mixture was extracted with DCM (3

X 30 mL), combined organic phases were washed with brine (50 mL), then dried over

MgSO4, filtered and condensed. The residue was purified by flash chromatography on silica gel in a short column to give the title compound 64 as clear oil in 95% yield. 1H

NMR (300 MHz, CDCl3) δ 7.19 (d, 1H, J = 2.1 Hz), 7.01 (dd, 1H, J = 8.3, 2.1 Hz), 6.85

(d, 1H, J = 8.3 Hz), 5.24 (s, 2H), 4.54 (s, 2H), 3.88 (s, 3H), 3.52 (s, 3H); 13C NMR (75

MHz, CDCl3) δ 149.8, 146.5, 130.1, 122.8, 120.5, 111.6, 95.5, 56.3, 55.9, 46.4.

4,4',4''-(benzene-1,2,3-triyltris(oxy))tris(methylene)tris(1-methoxy-2-(methoxymethoxy)benzene), 69: Following the procedure described for the preparation of 79, 178 compound 5 (40.1 mg, 0.3 mmol) was reacted with 64 (209.6 mg, 1.0 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 8), the title compound 69 in

1 54% yield. H NMR (300 MHz, CDCl3) δ7.26-7.24 (m, 3H), 7.08-7.02 (m, 3H), 6.90-

6.86 (m, 3H), 6.78 (d, 1H, J = 8.3 Hz), 6.62 (d, 2H, J = 8.3 Hz), 5.20 (s, 4H), 5.09 (s,

2H), 5.02 (s, 4H), 4.99 (s, 2H), 3.88 (s, 6H), 3.85 (s, 3H), 3.48 (s, 6H), 3.44 (s, 3H); 13C

NMR (75 MHz, CDCl3) δ 152.1, 148.4, 145.5, 145.1, 137.6, 129.8, 128.9, 122.5, 121.9,

120.8, 115.9, 114.9, 110.6, 110.3, 107.2, 99.5, 99.4, 73.8, 70.0, 55.2, 54.9.

4,4'-(3-methoxy-1,2-phenylene)bis(oxy)bis(methylene)bis(1-methoxy-2-(methoxymethoxy)benzene), 70: Following the procedure described for the preparation of 79, compound 7a (50.0 mg, 0.3 mmol) was reacted with 64 (160.8 mg, 0.7mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 7), the title compound 70 in

1 69% yield. H NMR (300 MHz, CDCl3) δ 7.29 (d, 1H, J = 1.8 Hz), 7.25 (d, 1H, J = 1.8

Hz), 7.09-7.02 (m, 2H), 6.91-6.74 (m, 3H), 6.59 (t, 2H, J = 8.8 Hz), 5.20 (s, 2H), 5.17 (s,

2H), 5.03 (s, 2H), 4.97 (s, 2H), 3.88 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.48 (s, 6H); 13C

NMR (75 MHz, CDCl3) δ 153.9, 152.9, 149.4, 146.4, 146.1, 137.8, 130.7, 129.9, 123.5,

122.8, 121.8, 116.9, 115.9, 111.5, 111.2, 107.7, 105.7, 95.5, 95.4, 74.7, 70.9, 56.2, 56.1,

56.0. 179

O O OMe OMOM

OMOM OMe 71

4, 4'-(4-Chloro-1, 2-phenylene)bis(oxy)bis(methylene)bis(1-methoxy-2-(methoxymethoxy)benzene): Following the procedure described for the preparation of 79, compound 7c (50.7 mg, 0.35 mmol) was reacted with 64 (158.3 mg, 0.7 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 5), the title

1 compound 71 in 82% yield. Mp: 103.8-104.2 °C; H NMR (300 MHz, CDCl3) δ 7.22 (dd,

2H, J = 9.2, 2.0 Hz), 7.06-7.00 (m, 2H), 6.93-6.82 (m, 5H), 5.19 (d, 4H, J = 3.9 Hz), 5.02

(d, 4H, J = 3.1 Hz), 3.88 (s, 3H), 3.87 (s, 3H), 3.49 (s, 3H), 3.48 (s, 3H); 13C NMR (75

MHz, CDCl3) δ 148.2, 148.0, 146.1, 144.9, 128.1, 127.7, 124.8, 120.3, 119.6, 114.9,

114.5, 114.1, 110.1, 94.0, 70.0, 69.7, 54.7, 54.4.

F O O OMe OMOM

OMOM OMe 72

4, 4'-(3-Fluoro-1,2-phenylene)bis(oxy)bis(methylene)bis(1-methoxy-2-(methoxy methoxy)benzene), 72: Following the procedure described for the preparation of 79, compound 7b (45.0 mg, 0.35 mmol) was reacted with 64 (158.3 mg, 0.7 mmol) to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 5), 112.2 mg of the

1 title compound 72. H NMR (300 MHz, CDCl3) δ 7.25 (q, 2H, J = 6.0, 4.0, 2.0 Hz), 7.04 180

(d, 2H, J = 8.3 Hz), 6.92-6.86 (m, 2H), 6.81 (d, 1H, J = 8.3 Hz), 6.73-6.66 (m, 2H), 5.20

(s, 2H), 5.16 (s, 2H), 5.03 (s, 2H), 5.02 (s, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.47 (s, 6H);

13 C NMR (75 MHz, CDCl3) δ 157.0, 153.7, 152.1, 148.4, 145.2, 144.9, 135.3, 135.1,

128.7, 128.1, 122.1, 122.0, 121.6, 120.6, 115.6, 114.7, 110.4, 110.1, 108.7, 108.1, 107.9,

94.3, 74.0, 69.8, 54.9, 54.7.

4,4'-(4-Chloro-1,3-phenylene)bis(oxy)bis(methylene)bis(1-methoxy-2-(methoxymethoxy)benzene), 73: 72: Following the procedure described for the preparation of 79,

1 compound 73 was obtained in 79% yield; H NMR (300 MHz, CDCl3) δ 7.26-7.20 (m,

3H), 7.08-7.00 (m, 2H), 6.89 (d, 2H, J = 8.3 Hz), 6.62 (d, 1H, J = 2.6 Hz), 6.50 (dd, 1H, J

= 2.6, 8.7 Hz), 5.23 (s, 4H), 5.01 (s, 2H), 4.91 (s, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 3.51

13 (s,3H), 3.50 (s, 3H); C NMR (75 MHz, CDCl3) δ 158.4, 154.8, 149.8, 149.7, 146.6,

146.5, 130.2, 129.1, 129.0, 122.1, 121.6, 116.2, 115.9, 115.2, 111.8, 107.1, 102.7, 95.6,

70.7, 70.3, 56.3, 55.9.

O O

MeO OMe OMOM OMOM 74 181

4,4'-(5-Chloro-1,3-phenylene)bis(oxy)bis(methylene)bis(1-methoxy-2-(methoxymethoxy)benzene), 74: Following the procedure described for the preparation of 79,

1 compound 74 was obtained in 84% yield; H NMR (300 MHz, CDCl3) δ 7.20 (d, 2H, J =

1.9 Hz), 7.02 (dd, 2H, J = 1.9, 8.3 Hz), 6.89 (d, 2H, J = 8.3 Hz), 6.59 (d, 2H, J = 2.2 Hz),

6.48 (t, 1H, J = 2.2 Hz), 5.23 (s, 4H), 4.90 (s, 4H), 3.87 (s, 6H), 3.51 (s, 6H); 13C NMR

(75 MHz, CDCl3) δ 160.2, 149.8, 146.6, 135.2, 128.9, 122.1, 116.3, 111.8, 108.2, 100.9,

95.6, 70.2, 56.2, 55.9.

N-(5-Chloro-2-hydroxyphenyl)-3-hydroxybenzamide, WZB-152: 3-(benzyloxy) - benzoyl chloride (775.1 mg, 3.2 mmol) was added dropwise into a solution of 2-amino-4- chlorophenol (430.7 mg, 3.0 mmol) and triethylamine (632.0 µL, 4.5 mmol) in anhydrous toluene at room temperature. The reaction mixture was heated to reflux and stirred for overnight. The solvent was removed after the completion of the reaction as indicated by

TLC. The residue was purified by column chromatography (EtOAc/Hexane, 1: 4) to give

3-(benzyloxy)-N-(5-chloro-2-hydroxyphenyl)benzamide as a red solid in 82% yield. Mp,

1 180.6-181.3 °C; H NMR (300 MHz, CDCl3) δ 10.20 (br s, 1H), 9.49 (s, 1H), 7.80 (d,

1H, J = 2.6 Hz), 7.60-7.34 (m, 8H) 7.25 (dd, 1H, J = 8.0, 1.7 Hz), 7.08 (dd, 1H, J = 8.6,

2.6 Hz), 6.92 (d, 1H, J = 8.6 Hz), 5.20 (s, 2H).

Then hydrogenolysis of 3-(benzyloxy)-N-(5-chloro-2-hydroxyphenyl)benzamide with palladium on activated carbon as catalyst to give WZB-152 in 84% yield; 1H NMR (300 182

MHz, CDCl3) δ 9.22 (br s, 1H), 9.07 (br s, 1H), 8.05 (d, 1H, J = 2.4 Hz), 7.51-7.47 (m,

2H), 7.41-7.36 (m, 1H), 7.11-7.05 (m, 2H), 6.97 (d, 1H, J = 8.6 Hz); 13C NMR (75 MHz,

CDCl3) δ 166.7, 158.6, 147.6, 136.4, 130.8, 128.9, 125.6, 124.8, 122.2, 120.0, 119.3,

118.6, 115.3.

4-Chloro-2-(3-hydroxybenzamido)phenyl 3-hydroxybenzoate, WZB-153: 3-

(benzyloxy) -benzoyl chloride (1.1 g, 4.5 mmol) was added dropwise into a solution of 2- amino-4-chlorophenol (287.1 mg, 2.0 mmol) and DIEA (801.2 µL, 4.6 mmol) in anhydrous DCM (40 mL) at room temperature. The reaction mixture was stirred for 2 h.

Then cold water (10 mL) was added to quench the reaction upon completion as indicated by TLC. Extracted with DCM (3 X 30 mL), the combined organic phases were washed with water (20 mL) and brine (20 mL), dried over MgSO4, filtered and concentrated, the crude was purified by column chromatography (EtOAc/Hexane, 1 :6) to afford 2-(3-

(benzyloxy)benzamido)-4-chlorophenyl 3-(benzyloxy)benzoate in 80% yield. 1H NMR

(300 MHz, CDCl3) δ 8.35 (d, 1H, J = 2.3 Hz), 8.22 (s, 1H), 7.76-7.71 (m, 2H), 7.34-7.24

(m, 14H), 7.21-7.14 (m, 3H), 7.08-7.00 (m, 2H), 4.95 (s, 2H), 4.86 (s, 2H).

The debenzylation of 2-(3-(benzyloxy)benzamido)-4-chlorophenyl 3-

1 (benzyloxy)benzoate to give WZB-153 in 81% yield; H NMR (300 MHz, CDCl3) δ 9.11

(s, 1H), 8.73 (br s, 2H), 8.08 (d, 1H, J = 1.4, 8.0 Hz), 7.69 (t, 2H, J = 7.7 Hz), 7.41-7.16 183

13 (m, 7H), 6.98 (dd, 1H, J = 1.4, 8.0 Hz); C NMR (75 MHz, CDCl3) δ 165.5, 164.2,

157.8, 157.7, 143.4, 136.6, 131.0, 130.0, 129.7, 126.0, 125.3, 124.8, 123.2, 121.4, 121.0,

118.8, 118.4, 116.7, 114.7.

3-(Benzyloxy)-N-(4-chloro-2-hydroxyphenyl)benzamide: Yield: 84%. Mp, 166.5-

1 167.4 °C; H NMR (300 MHz, CDCl3) δ 9.64 (br s, 1H), 9.43 (s, 1H), 7.79 (d, 1H, J = 8.6

Hz), 7.67-7.59 (m, 2H), 7.53-7.25 (m, 7H), 7.00 (d, 1H, J = 2.3 Hz), 6.93 (dd, 1H, J =

13 8.6, 2.3 Hz), 5.22 (s, 2H); C NMR (75 MHz, CDCl3) δ 164.9, 157.9, 148.4, 135.9,

134.1, 128.9, 128.7, 127.3, 126.7, 126.5, 124.6, 122.5, 118.7, 117.6, 116.0, 112.8, 68.7.

N-(4-Chloro-2-hydroxyphenyl)-3-hydroxybenzamide, WZB-154: Yield, 76%; 1H

NMR (300 MHz, CDCl3) δ9.40 (s, 1H), 9.00 (br s, 1H), 7.76 (dd, 1H, J = 1.5, 8.0 Hz),

7.51-7.48 (m, 2H), 7.40-7.35 (m, 1H), 7.10-7.05 (m, 2H), 6.98-6.95 (m, 1H), 6.91-6.86

13 (m,1H) ; C NMR (75 MHz, CDCl3) δ 165.5, 156.6, 147.5, 134.6, 128.8, 125.7, 124.7,

121.3, 118.8, 118.1, 117.4, 116.4, 113.4. 184

O O NH

OBn

2-(3-(Benzyloxy)benzamido)-5-chlorophenyl 3-(benzyloxy)benzoate: Yield: 82%; 1H

NMR (300 MHz, CDCl3) δ 8.22 (s, 1H), 8.07 (d, 1H, J = 8.8 Hz), 7.73-7.70 (m, 2H),

7.33-7.23 (m, 15H), 7.17-7.10 (m, 3H), 6.99 (dd, 1H, J = 8.1, 2.3 Hz), 4.92 (s, 2H), 4.83

13 (s, 2H); C NMR (75 MHz, CDCl3) δ 165.0, 163.8, 158.9, 141.7, 136.3, 136.1, 135.5,

129.9, 129.7, 129.6, 129.4, 128.6, 128.5, 128.4, 128.1, 127.5, 126.5,124.1, 122.6, 121.4,

119.1, 119.0, 115.7, 113.0, 70.1, 69.8.

5-Chloro-2-(3-hydroxybenzamido)phenyl 3-hydroxybenzoate, WZB-157: 1H NMR

(300 MHz, CD3OCD3) δ 9.20 (d, 1H, J = 32.2 Hz), 8.74 (br s, 2H), 8.12-8.06 (m, 1H),

7.72-7.63 (m, 2H), 7.41-7.14 (m, 7H), 7.01-6.96 (m, 1H); 13C NMR (75 MHz,

CD3OCD3) δ 165.0, 163.6, 157.1, 143.1, 142.7, 135.9, 135.7, 130.3, 130.0, 129.4, 129.0,

128.6, 125.4, 125.2, 124.7, 124.3, 122.9, 122.6, 120.9, 120.8, 120.6, 120.4, 118.3, 118.1,

117.9, 117.8, 116.1,116.0, 114.1. IR (KBr) 3443, 3309, 3183, 2955, 2925, 1750, 1663,

1589, 1533, 1493, 1455, 1325, 1282, 1250, 1215, 1189, 1072, 923, 880, 744, 678 cm-1;

MS (APCI): M+H expected 383.78, obtained 383.95. 185

3-(Benzyloxy)phenol, 42: Benzyl bromide (9.7 g, 56.75 mmol) was added dropwise into a solution of resorcinol (25 g, 227 mmol) and potassium carbonate (15.66 g, 113.5 mmol) in dry acetone (150 mL) at room temperature for 1.5 h. The mixture was then heated to reflux and stirred for overnight. The precipitate was filtered off after the completion of benzyl bromide as indicated by TLC. The solvent was removed and the residue was purified by column chromatography on silica gel to provide the title compound in 57%

1 yield. H NMR (300 MHz, CDCl3) δ 7.38-7.25 (m, 5H), 7.07 (t, 1H, J = 8.2 Hz), 6.53

(ddd, 1H, J = 0.57, 2.3, 8.2 Hz), 6.44 (t, 1H, J = 2.3 Hz), 6.38 (ddd, 1H, J = 0.69, 2.3, 8.1

13 Hz), 5.63 (s, 1H), 4.94 (s, 2H); C NMR (75 MHz, CDCl3) δ 158.3, 154.8, 135.0, 128.6,

126.9, 126.3, 125.9, 106.6, 105.8, 100.9, 68.4.

Bis(3-(benzyloxy)phenyl) 3-fluorophthalate: DCC (177.0 mg, 0.86 mmol) was added into the solution of 3-fluorophthalic acid (72.2 mg, 0.39 mmol) in DCM (15 mL) at room temperature and stirred for 10 min. Then DMAP (5.1 mg, 0.04 mmol) was added into the reaction mixture and stirred for additional 30 min. Then 42 (196.0 mg, 0.98 mmol) was added, and the reaction mixture was stirred for 12 h. Cold water was added to quench the reaction upon completion as indicated by TLC. DCM (30 mL) was added to dilute the reaction, then washed with water, the aqueous solution was extracted with DCM (3 X 30 mL). The organic layers were combined and washed with brine (30mL) and dried over 186

MgSO4, filtered and condensed, the residue was purified by column chromatography on silica gel (EtOAc/Hexane, 1: 8) to provide the title compound in 59% yield. 1H NMR

(300 MHz, CDCl3) δ 7.99 (d, 1H, J = 7.8 Hz), 7.58-7.51 (m, 1H), 7.43-7.23 (m, 13H),

13 6.89-6.83 (m, 6H), 4.95 (s, 2H), 4.84 (s, 2H); C NMR (75 MHz, CDCl3) δ 162.9, 162.8,

162.7, 160.7, 159.5, 159.4, 157.4, 151.1, 136.2, 136.1, 131.4, 131.3, 129.8, 129.7, 129.3,

129.2, 128.3, 128.2, 127.8, 127.4, 127.3, 126.2, 126.1, 123.3, 123.0, 120.8, 120.5, 113.8,

113.7, 113.0, 112.9, 108.2, 107.9, 69.9, 69.8.

Bis(3-hydroxyphenyl) 3-fluorophthalate, WZB-158: Debenzylation of the compound bis(3-(benzyloxy)phenyl) 3-fluorophthalate to give WZB-158 in 87% yield; 1H NMR

(300 MHz, CD3OCD3) δ 8.72 (br s, 2H), 8.14-8.11 (m, 1H), 7.85-7.77 (m, 1H), 7.72-7.64

13 (m, 1H), 7.33-7.22 (m, 2H), 6.86-6.73 (m, 6H); C NMR (75 MHz, CD3OCD3) δ 161.7,

161.2, 159.5, 157.2, 157.1, 156.2, 150.4, 131.1, 128.8, 128.7, 128.3, 125.2, 122.1, 121.9,

119.8, 119.5, 112.2, 112.1, 111.2, 111.1, 107.7, 107.6; IR (KBr) 3452, 3088, 3070, 2926,

2550, 1729, 1613, 1462, 1258, 1185, 1160, 1129, 1095, 1054, 998, 969, 942, 908, 877,

771, 744, 681 cm-1.

187

1 Ethyl 3-(methoxymethoxy)benzoate, 45: Yield, 81%; H NMR (300 MHz, CDCl3) δ

7.0-7.68 (m, 2H), 7.35 (t, 1H, J = 16.3 Hz), 7.26-7.20 (m, 1H), 5.21 (s, 2H), 4.37 (q, 2H,

13 J = 7.1, 14.3 Hz), 3.48 (s, 3H), 1.39 (t, 3H, J = 7.1 Hz); C NMR (75 MHz, CDCl3) δ

164.1, 154.9, 129.2, 130.8, 118.6, 114.8, 92.2, 58.8, 53.9, 12.1.

Bis(3-(methoxymethoxy)benzyl) 3-fluorophthalate: DCC (454.0 mg, 2.2 mmol) was added into the solution of 3-fluorophthalic acid (184.12 mg, 1.0 mmol) in DCM at room temperature and stirred for 10 min. Then DMAP (13 mg, 0.1 mmol) was added into the reaction mixture and stirred for additional 30 min. Then 46 (420.0 mg, 2.5 mmol) was added afterwards, and the reaction mixture was stirred for 12 h. Cold water was added to quench the reaction upon completion as indicated by TLC. DCM (50 mL) was added to dilute the reaction, then washed with water, the aqueous solution was extracted with

DCM (3 X 50 mL). The organic layers were combined and washed with brine (30 mL) and dried over MgSO4, filtered and condensed. The residue was purified by column chromatography on silica gel to provide the title compound in 51% yield. 1H NMR (300

MHz, CDCl3) δ 7.79 (d, 1H, J = 7.3 Hz), 7.45-7.38 (m, 1H), 7.30-7.21 (m, 2H), 7.08-6.96

(m, 6H), 5.24 (s, 2H), 5.18 (s, 2H), 5.16 (s, 2H), 5.14 (s, 2H), 3.44 (s, 6H); 13C NMR (75

MHz, CDCl3) δ 164.6, 164.4, 160.9, 157.6, 157.4, 136.8, 136.6, 131.1, 131.0, 129.8,

129.6, 125.96, 123.6, 123.4, 121.9, 121.7, 120.3, 120.0, 116.3, 94.4, 67.5, 55.9. 188

O O

F O OH

OH WZB-159

Bis(3-hydroxybenzyl) 3-fluorophthalate, WZB-159: Hydrochloride (396.0µL, 1.2 M in

EtOAc) was added into a solution of bis(3-(methoxymethoxy)benzyl) 3-fluorophthalate

(92.3 mg, 0.19 mmol) in methanol/DCM (3.0 mL, 3:1) at room temperature. The reaction was then heated to 48 °C for overnight. The solvent was removed after TLC showed the reaction was completed. The residue obtained was purified by column chromatography on silica gel (EtOAc/Hexane, 1: 5) to provide WZB-159 in 84% yield. 1H NMR (300

MHz, CD3OCD3) δ 7.78 (d, 1H, J = 7.7 Hz), 7.46-7.39 (m, 1H), 7.29-7.14 (m, 3H), 6.91-

13 6.78 (m, 6H), 6.16 (br s, 2H), 5.16 (s, 2H), 5.11 (s, 2H); C NMR (75 MHz, CD3OCD3)

δ 164.5, 163.9, 159.9, 156.7, 154.9, 154.8, 135.8, 135.7, 130.5, 130.4, 129.2, 129.0,

125.2, 122.5, 122.2, 120.0, 119.9, 119.7, 119.4, 115.0, 114.8, 114.7, 67.1, 66.7; HPLC

(CH3CN: H2O) RT 15.01 (98%).

1, 2-Bis(bromomethyl)-3-fluorobenzene, 58: Phosphorus tribromide (109 µL, 1.16 mmol) was added into a solution of 3-fluorophthalic acid (129.0 mg, 0.58 mmol) in dry

DCM (15 mL) at 0 °C. The reaction was allowed to warm to room temperature slowly and stirred for overnight. Then cold water was added into the reaction and extracted with

DCM (3 X 30 mL). The organic layers were combined and dried over MgSO4. The 189 solvent was removed and the crude was purified by column chromatography on silica gel

1 to afford the title compound in 96% yield. H NMR (300 MHz, CDCl3) δ 7.32-7.25 (m,

1H), 7.17 (d, 1H, J = 7.5 Hz), 7.05 (t, 1H, J = 18.6 Hz), 4.69 (d, 2H, J = 1.4 Hz), 4.62 (s,

13 2H); C NMR (75 MHz, CDCl3) δ 162.7, 159.3, 138.7, 130.5, 126.5, 124.5, 124.3,

116.4, 116.1, 28.9, 21.0.

3, 3'-(3-Fluoro-1, 2-phenylene)bis(methylene)bis(sulfanediyl)diphenol (WZB-161):

3-Hydroxythiophenol (27.7 mg, 0.22 mmol) was added into a solution of 1,2- bis(bromomethyl)-3-fluorobenzene (28.0 mg, 0.1 mmol) and triethylamine (35.0 µL, 0.25 mmol) in DCM (4.0 mL) at 0 °C. The reaction was allowed to warm to room temperature and stirred for overnight. Then the solvent was removed and the residue was purified by column chromatography on silica gel (EtOAc/Hexane, 1: 8 to 1: 5) to provide the title

1 compound (37.0 mg) in 99% yield. H NMR (300 MHz, CD3OCD3) δ 7.08-6.95 (m, 4H),

6.91-6.79 (m, 4H), 6.74-6.63 (m, 3H), 6.31 (br s, 2H), 4.13 (s, 2H), 3.99 (s, 2H); 13C

NMR (75 MHz, CD3OCD3) δ 162.1, 158.8, 155.1, 154.8, 154.7, 137.6, 137.1, 136.7,

136.4, 129.5, 128.2, 128.1, 125.2, 123.1, 122.7, 122.4, 121.9, 119.0, 117.1, 115.8, 141.1,

113.9, 113.4, 35.1, 28.4. 190

m-CPBA (77.0 mg, 0.313 mmol, 77% mixture) was added into a solution of WZB-161

(58.2 mg, 0.16 mmol) in DCM (3 mL) at 0 °C. The mixture was stirred for 30 min and quenched with cold saturated sodium bicarbonate (10 mL). Extracted with DCM (3 X 20 mL), combined organic layers were dried over MgSO4, filtered and concentrated in vacuo, the residue was separated by flash chromatography on silica gel to provide WZB-

163 (20.3 mg) and WZB-165 (13.5 mg).

1 WZB-163: H NMR (300 MHz, CD3OCD3) δ 8.87 (br s, 1H), 7.34 (t, 2H, J = 7.6 Hz),

7.27-7.21 (m, 1H), 7.06-6.92 (m, 8H), 4.48-4.08 (m, 4H), 2.92 (br s, 1H); 13C NMR (75

MHz, CD3OCD3) δ 159.0, 146.3, 146.2, 134.8, 131.1, 130.2, 130.0, 128.6, 119.9, 119.2,

118.9, 115.9, 115.8, 115.5, 111.6, 111.3, 60.5, 54.0; IR (KBr) 3163, 1694, 1586, 1449,

1342, 1306, 1257, 1225, 1024, 991, 783, 684 cm-1; MS (APCI): M+H expected 404.47, obtained 404.80.

1 WZB-165: H NMR (300 MHz, CD3OCD3) δ 8.99 (br s, 1H), 7.45-7.09 (m, 7H), 7.06-

6.97 (m, 4H), 4.97-4.61 (m, 2H), 4.52-4.23 (m, 2H), 2.92 (m, 1H); 13C NMR (75 MHz,

CD3OCD3) δ 159.0, 158.8, 146.2, 141.0, 132.8, 131.4, 131.1, 130.4, 130.3, 129.7, 128.9,

121.9, 120.4, 120.1, 119.3, 119.0, 116.5, 116.2, 115.7, 111.5, 111.2, 59.8, 53.5; IR (KBr)

3385, 2923, 1587, 1450, 1302, 1260, 1137, 1021, 987, 742 cm-1; MS (APCI): M+H expected 420.47, obtained 420.75. 191

(3-Fluoro-1,2-phenylene)bis(methylene) bis(3-hydroxybenzoate), WZB-162: 1H NMR

(300 MHz, CD3OCD3) δ 7.56-7.44 (m, 6H), 7.28-7.20 (m, 3H), 7.07-7.03 (m, 2H), 5.60

13 (s, 4H); C NMR (75 MHz, CD3OCD3) δ 166.4, 166.3, 158.4, 139.5, 132.1, 131.8,

131.7, 130.5, 126.4, 121.5, 121.1, 116.8, 116.6, 116.3, 64.4, 57.8.

WZB-164: m-CPBA (2 eq) was added into a solution of the mixture of WZB-163 and

WZB-165 in DCM/EtOAc (4: 1) at 0 °C. The mixture was stirred for 45 min, saturated sodium bicarbonate was then added upon the completion of the reaction as indicated by

TLC. The mixture was extracted with EtOAc. The organic layers were combined and dried over MgSO4, filtered and condensed. The residue was purified by column chromatography on silica gel to provide WZB-164 in 71% yield. 1H NMR (300 MHz,

CD3OCD3) δ 9.15 (br s, 1H), 7.45 (dt, 2H, J = 2.6, 7.9 Hz), 7.37-7.30 (m, 1H), 7.28-7.17

(m, 6H), 7.08 (q, 2H, J = 9.0, 8.5 Hz), 4.78 (s, 2H), 4.72 (s, 2H), 2.96 (br s, 1H); 13C

NMR (75 MHz, CD3OCD3) δ 164.4, 161.1, 158.8, 140.9, 140.7, 132.6, 131.5, 131.2,

130.1, 130.0, 122.1, 122.0, 120.1, 120.00, 118.3, 118.1, 116.8, 116.5, 115.6, 115.5, 59.6,

53.2; MS (APCI): M+H expected 436.47, obtained 436.80. 192

5-(Methoxymethoxy)-2-nitrobenzaldehyde, 66: Chloromethyl methyl ether (1.7 mL,

22.4 mmol) was added into a solution of 5-hydroxy-2-nitrobenzaldehyde (2.5 g, 14.9 mmol) and potassium carbonate (8.3 g, 59.8 mmol) in dry DMF (50.0 mL) at 0 °C. The reaction mixture was stirred for 4 h at 0 °C. Cold water then was added upon the completion of the reaction as indicated by TLC. The mixture was diluted with

EtOAc(120 mL) and washed with water (5 X 10 mL), the organic phase was dried over

MgSO4, filtered and condensed. The residue was purified by column chromatography to afford the title compound (2.84 g) as a slight green solid in 86% yield. Mp, 61.5 °C; 1H

NMR (300 MHz, CDCl3) δ 10.45 (s, 1H), 8.15 (d, 1H, J = 9.0 Hz), 7.47 (d, 1H, J = 2.8

13 Hz), 7.33-7.28 (m, 1H), 5.31 (s, 2H), 3.50 (s, 3H); C NMR (75 MHz, CDCl3) δ 188.3,

161.7, 134.3, 127.2, 119.9, 116.1, 94.5, 93.6, 56.7.

(5-(Methoxymethoxy)-2-nitrophenyl)methanol, 67: NaBH4 (462.0 mg, 12.2 mmol) was added into a solution of 66 (2.7g, 12.2 mmol) in anhydrous THF (50 mL) at room temperature. The reaction mixture was quenched with NH4Cl (sat. 5 mL) after stirred for

90 min. Then the mixture was diluted with diethyl ether (100 mL) and extracted with diethyl ether (3 X 40 mL), the combined organic phases were dried over MgSO4. The solvent was removed and the residue was purified by chromatography on silica gel to 193

1 give 67 in 97% yield; H NMR (300 MHz, CDCl3) δ 8.07 (d, 1H, J = 9.1 Hz), 7.38 (d,

1H, J = 1.8 Hz), 6.98 (dd, 1H, J = 2.2, 9.0 Hz), 5.26 (s, 2H), 4.96 (s, 2H), 3.86 (br s, 1H),

13 3.48 (s, 3H); C NMR (75 MHz, CDCl3) δ 161.7, 140.7, 140.4, 127.5, 115.4, 114.9,

94.1, 62.1, 56.4.

2-(Bromomethyl)-4-(methoxymethoxy)-1-nitrobenzene, 68: NBS (847.3 mg, 4.8 mmol) was added into a solution of 67 (707.7 mg, 3.2 mmol) and triphenylphosphine

(1.25 g, 4.8 mmol) at 0°C. The reaction mixture was allowed to warm to room temperature slowly stirred for 30 min. Then the reaction was quenched with cold water

(6.0 mL) and extracted with diethyl ether (3 X 30 mL). The combined organic phases were dried over MgSO4, filtered and condensed in vacuo. The residue was purified by

1 chromatography on silica gel to provide 68 in 83% yield; H NMR (300 MHz, CDCl3) δ

8.09 (dd, 1H, J = 2.6, 9.1 Hz), 7.17 (d, 1H, J = 2.6 Hz), 7.08 (dd, 1H, J = 2.6, 9.1 Hz),

13 5.27 (s, 2H), 4.84 (s, 2H), 3.50 (s, 3H); C NMR (75 MHz, CDCl3) δ 159.3, 139.6,

133.7, 126.3, 117.7, 114.3, 92.6, 54.7, 27.8.

2, 2', 2''-(Benzene-1, 2, 3-triyltris(oxy))tris(methylene)tris(4-(methoxymethoxy)-1- nitrobenzene), 85: Following the procedure described for the preparation of 79, 5a 194

(20.0mg, 0.16 mmol) was reacted with 68 (137.9 mg, 0.50 mmol) in dry acetone at 55 °C to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 6), 50.1 mg of the

1 title compound 85 in 44% yield. H NMR (300 MHz, CDCl3) δ 8.22 (q, 3H, J = 6.0, 9.1

Hz), 7.76 (d, 1H, J = 2.4 Hz), 7.47 (d, 1H, J = 2.4 Hz), 7.33-7.20 (m, 2H), 7.08 (dd, 3H, J

= 2.5, 9.1 Hz), 6.77 (d, 1H, J = 2.5 Hz), 6.57 (dd, 1H, J = 2.6, 8.7 Hz), 5.49 (d, 6H, J =

13 18.9 Hz), 5.27 (d, 6H, J = 10.1 Hz), 3.49 (d, 9H, J = 5.3 Hz); C NMR (75 MHz, CDCl3)

δ 161.8, 161.7, 161.6, 153.9, 152.7, 152.1, 149.8, 141.0, 140.4, 140.2, 138.7, 137.4,

137.2, 136.7, 134.0, 127.8, 127.6, 124.7, 124.4, 116.6, 115.5, 115.2, 114.9, 114.8, 114.4,

108.5, 107.3, 106.2, 104.1, 94.3, 94.2, 94.1, 71.7, 71.3, 68.1, 56.5, 56.4, 56.1, 55.9.

2, 2'-(3-Methoxy-1, 2-phenylene)bis(oxy)bis(methylene)bis(4-(methoxymethoxy)-1- nitrobenzene), 86: Following the procedure described for the preparation of 79, 7a

(24.0mg, 0.17 mmol) was reacted with 68 (99.4 mg, 0.36 mmol) in dry acetone at 55 °C to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 6), 79.1 mg of the

1 title compound 86 in 87% yield. H NMR (300 MHz, CDCl3) δ 8.25-8.20 (m, 2H), 7.76

(s, 1H), 7.47 (s, 1H), 7.30 (t, 1H, J = 8.7 Hz), 7.10-7.06 (m, 2H), 6.76 (s, 1H), 6.58 (dd,

1H, J = 2.3, 8.6 Hz), 5.52 (s, 2H), 5.46 (s, 2H), 5.29 (s, 2H), 5.26 (s, 2H), 3.50 (s, 3H),

13 3.48 (s, 3H); C NMR (75 MHz, CDCl3) δ 162.6, 162.4, 158.5, 154.9, 141.0, 140.7,

131.1, 128.4, 128.3, 116.5, 116.2, 115.8, 115.6, 115.2, 107.6, 103.5, 94.9, 68.3, 68.1,

57.1. 195

2, 2'-(3-Fluoro-1,2-phenylene)bis(oxy)bis(methylene)bis(4-(methoxymethoxy)-1- nitrobenzene), 87: Following the procedure described for the preparation of 79, 7b

(20.0mg, 0.16 mmol) was reacted with 68 (90.5 mg, 0.31 mmol) in dry acetone at 55 °C to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 6), 74.0 mg of the

1 title compound 87 in 91% yield. H NMR (300 MHz, CDCl3) δ 8.18 (dd, 2H, J = 3.8, 9.1

Hz), 7.83 (s, 1H), 7.48 (d, 1H, J = 2.1 Hz), 7.12-6.99 (m, 3H), 6.84-6.74 (m, 2H), 5.59 (s,

2H), 5.54 (s, 2H), 5.27 (s, 2H), 5.10 (s, 2H), 3.47 (s, 3H), 3.34 (s, 3H); 13C NMR (75

MHz, CDCl3) δ 162.4, 158.6, 155.4, 163.1, 140.8, 140.7, 138.5, 137.2, 137.1, 137.0,

128.4, 128.3, 127.9, 124.8, 124.7, 117.1, 116.0, 115.7, 115.6, 115.4, 115.2, 112.1, 110.7,

110.5, 110.4, 94.9, 94.7, 73.1, 73.0, 68.8, 57.1, 57.0.

2, 2'-(4-Chloro-1,2-phenylene)bis(oxy)bis(methylene)bis(4-(methoxymethoxy)-1- nitrobenzene), 88: Following the procedure described for the preparation of 79, 7c

(30.0mg, 0.2 mmol) was reacted with 68 (120.3 mg, 0.4 mmol) in dry acetone at 55 °C to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 6), 59.2 mg of the 196

1 title compound 88 in 53% yield. H NMR (300 MHz, CDCl3) δ 8.20 (dd, 2H, J = 3.3, 9.1

Hz), 7.56 (d, 2H, J = 2.4 Hz), 7.08-7.00 (m, 3H), 6.93 (d, 2H, J = 1.1 Hz), 5.55 (s, 4H),

13 5.19 (s, 4H), 3.40 (s, 6H); C NMR (75 MHz, CDCl3) δ 161.8, 149.0, 147.2, 145.6,

144.7, 140.4, 136.9, 136.7, 135.5, 128.1, 127.8, 127.7, 127.1, 124.8, 122.3, 121.9, 116.1,

115.7, 115.5, 115.2, 115.0, 114.9, 111.5, 94.4, 94.3, 68.8, 68.7, 56.6, 56.5.

2,2'-(4-chloro-1,3-phenylene)bis(oxy)bis(methylene)bis(4-(methoxymethoxy)-1- nitrobenzene), 89: Following the procedure described for the preparation of 79, 7e

(20.0mg, 0.14 mmol) was reacted with 68 (80.2 mg, 0.29 mmol) in dry acetone at 55 °C to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 5), 65.0 mg of the

1 title compound 89 in 88% yield. H NMR (300 MHz, CDCl3) δ 8.17 (dd, 2H, J = 1.8, 9.0

Hz), 7.42 (d, 2H, J = 2.4 Hz), 7.17-7.11 (m, 2H), 6.99-6.96 (m, 3H), 5.62 (s, 2H), 5.58 (s,

13 2H); C NMR (75 MHz, CDCl3) δ 161.4, 147.8, 146.0, 137.9, 135.8, 135.5, 126.7,

126.6, 124.7, 120.1, 114.5, 113.7, 113.3, 113.2, 113.1, 67.0, 66.9.

197

2, 2'-(5-Chloro-1, 3-phenylene)bis(oxy)bis(methylene)bis(4-(methoxymethoxy)-1- nitrobenzene), 90: Following the procedure described for the preparation of 79, 7f

(20.0mg, 0.14 mmol) was reacted with 68 (80.2 mg, 0.29 mmol) in dry acetone at 55 °C to provide, after purification on silica gel the eluent (EtOAc/Hexane, 1: 5), 72.5 mg of the

1 title compound 90 in 98% yield. H NMR (300 MHz, CDCl3) δ 8.22 (d, 2H, J = 9.1 Hz),

7.46 (d, 2H, J = 2.6 Hz), 7.08 (dd, 2H, J = 2.7, 9.1 Hz), 6.67 (d, 2H, J = 2.2 Hz), 6.59 (t,

13 1H, 2.2 Hz), 5.54 (s, 4H), 5.26 (s, 4H), 3.48 (s, 6H); C NMR (75 MHz, CDCl3) δ 160.6,

158.4, 139.1, 135.0, 134.9, 126.6, 114.3, 114.1, 113.4, 107.5, 100.0, 93.1, 66.1, 55.3.

3, 3', 3''-(Benzene-1, 2, 3-triyltris(oxy))tris(methylene)tris(4-nitrophenol), WZB-168:

Following the procedure described for the preparation of WZB-146, compound 85 was deprotected with HCl solution in EtOAc (1.2 M) to give 36.5 mg of WZB-168 in 89%

1 yield. H NMR (300 MHz, CD3OCD3) δ 9.79 (br s, 1H), 8.17 (t, 3H, J = 9.1 Hz), 7.44 (d,

1H, J = 2.7 Hz), 7.38 (d, 2H, J = 8.8 Hz), 7.26 (d, 1H, J = 2.6 Hz), 7.00-6.92 (m, 4H),

13 6.72 (dd, 1H, J = 2.7, 8.8 Hz), 5.56 (d, 6H, J = 27.5 Hz); C NMR (75 MHz, CD3OCD3)

δ 163.0, 162.8, 154.6, 139.5, 139.4, 136.9, 136.8, 130.6, 128.3, 114.9, 114.8, 114.7,

114.5, 107.6, 102.7, 100.2, 67.8, 67.6; HPLC (CH3CN: H2O) RT 16.18 (99%); IR (KBr)

3375, 2920, 1693, 1596, 1516, 1489, 1450, 1378, 1305, 1259, 1194, 1086, 1035, 880,

129, 756 cm-1; MS (APCI): M-H expected 579.47, obtained 579.40. 198

3, 3'-(3-Fluoro-1, 2-phenylene)bis(oxy)bis(methylene)bis(4-nitrophenol), WZB-169:

Following the procedure described for the preparation of WZB-146, compound 87 was deprotected with HCl solution in EtOAc (1.2 M) to give 45.9 mg of WZB-169 in 75%

1 yield. H NMR (300 MHz, CD3OCD3) δ 9.69 (br s, 1H), 8.14 (dd, 2H, J = 7.1, 9.0 Hz),

7.59 (d, 1H, J = 2.6 Hz), 7.27 (d, 1H, J = 2.6 Hz), 7.14-7.07 (m, 1H), 7.00-6.85 (m, 4H),

13 5.59 (s, 2H), 5.56 (s, 2H), 2.97 (br s, 1H); C NMR (75 MHz, CD3OCD3) δ 162.2, 157.4,

154.2, 152.3, 138.8, 138.6, 137.1, 136.2, 136.0, 135.9, 127.6, 127.3, 123.8, 123.7, 114.3,

114.2, 114.1, 114.0, 110.0, 109.9, 109.1, 108.8, 71.9, 67.9; HPLC (CH3CN: H2O) RT

15.34 (99%); IR (KBr) 3334, 3120, 3080, 2933, 2809, 1693, 1601, 1581, 1513, 1475,

1449, 1384, 1331, 1251, 1221, 1095, 1065, 1007, 970, 858, 757 cm-1; MS (APCI): M-H expected 430.34, obtained 429.75.

3, 3'-(3-Methoxy-1, 2-phenylene)bis(oxy)bis(methylene)bis(4-nitrophenol), WZB-

170: Following the procedure described for the preparation of WZB-146, compound 86 was deprotected with HCl solution in EtOAc (1.2 M) to give 61.0 mg of WZB-170 in

1 92% yield. H NMR (300 MHz, CD3OCD3) δ 8.12 (q, 2H, J = 6.8 Hz), 7.68 (d, 1H, J = 199

2.5 Hz), 7.29 (d, 1H, J = 2.5 Hz), 7.05 (t, 1H, J = 8.4 Hz), 6.92 (dd, 2H, J = 1.4, 9.0 Hz),

6.76-6.71 (m, 2H), 5.54 (s, 2H), 5.44 (s, 2H), 3.85 (s, 3H); 13C NMR (75 MHz,

CD3OCD3) δ 161.9, 151.3, 138.3, 137.7, 136.5, 127.3, 126.8, 123.5, 114.3, 113.7, 113.4,

106.7, 105.5, 70.7, 67.4; HPLC (CH3CN: H2O) RT 15.20 (96%); IR (KBr) 3295, 2934,

2841, 1690, 1594, 1520, 1477, 1442, 1380, 1336, 1303, 1254, 1222, 1162, 1117, 1080,

1010, 970, 866, 826, 753 cm-1; MS (APCI): M+H expected 442.38, obtained 442.85.

3, 3'-(4-Chloro-1, 2-phenylene)bis(oxy)bis(methylene)bis(4-nitrophenol), WZB-171:

Following the procedure described for the preparation of WZB-146, compound 88 was deprotected with HCl solution in EtOAc (1.2 M) to give 24.3 mg of WZB-171 in 83%

1 yield. H NMR (300 MHz, CD3OCD3) δ 8.17 (dd, 2H, J = 1.8, 9.0 Hz), 7.42 (d, 2H, J =

2.4 Hz), 7.17- 7.11 (m, 2H), 6.99-6.96 (m, 3H), 5.62 (s, 2H), 5.58 (s, 2H); 13C NMR (75

MHz, CD3OCD3) δ 161.4, 147.8, 146.0, 137.9, 135.8, 135.5, 126.7, 126.6, 124.7, 120.1,

114.5, 113.7, 113.3, 113.2, 113.1, 67.0, 66.9; HPLC (CH3CN: H2O) RT 15.80 (99%); IR

(KBr) 3380, 3079, 2923, 1689, 1594, 1510, 1446, 1411, 1379, 1332, 1256, 1219, 1161,

1131, 1080, 1034, 973, 906, 870, 795, 756 cm-1; MS (APCI): M-H expected 446.05, obtained 445.10. 200

3, 3'-(4-Chloro-1, 3-phenylene)bis(oxy)bis(methylene)bis(4-nitrophenol), WZB-172:

Following the procedure described for the preparation of WZB-146, compound 89 was deprotected with HCl solution in EtOAc (1.2 M) to give 53.2 mg of WZB-172 in 99%

1 yield. H NMR (300 MHz, CD3OCD3) δ 8.15-8.06 (m, 2H), 7.68 (d, 1H, J = 2.7 Hz),

2.30 (d, 1H, J = 2.7 Hz), 7.06 (t, 1H, J = 8.5 Hz), 6.95-6.80 (m, 4H), 5.56 (s, 2H), 5.54 (s,

13 2H); C NMR (75 MHz, CD3OCD3) δ 162.1, 151.9, 138.5, 138.2, 137.7, 137.5, 136.5,

127.3, 126.9, 123.7, 114.4, 113.9, 113.7, 107.6, 71.3, 67.7; HPLC (CH3CN: H2O) RT

15.43 (97%); IR (KBr) 3353, 2926, 1599, 1516, 1474, 1447, 1382, 1335, 1299, 1252,

1219, 1165, 1107, 1082, 1015, 155, 830, 754 cm-1; MS (APCI): M-H expected 446.05, obtained 445.10.

3, 3'-(5-Chloro-1, 3-phenylene)bis(oxy)bis(methylene)bis(4-nitrophenol), WZB-173:

Following the procedure described for the preparation of WZB-146, compound 90 was deprotected with HCl solution in EtOAc (1.2 M) to give 41.8 mg of WZB-168 in 70%

1 yield. H NMR (300 MHz, CD3OCD3) δ 9.78 (br s, 1H), 8.17 (d, 2H, J = 9.0 Hz), 7.25 (d,

2H, J = 2.6 Hz), 6.98 (dd, 2H, J = 2.7, 9.0 Hz), 6.75 (d, 2H, J = 2.3 Hz), 6.72 (t, 1H, J =

13 2.1 Hz), 5.54 (s, 4H), 2.94 (br s, 1H); C NMR (75 MHz, CD3OCD3) δ 163.4, 160.9, 201

140.1, 137.3, 135.9, 128.9, 115.5, 115.2, 109.1, 68.1; HPLC (CH3CN: H2O) RT 16.77 (98

%); IR (KBr) 3468, 3334, 3107, 2921, 1696, 1587, 1510, 1447, 1377, 1332, 1297, 1253,

1212, 1180, 1083, 1059, 1043, 967, 881, 827, 755 cm-1; MS (APCI): M-H expected

446.05, obtained 445.05.

202

REFERENCES

1 American Cancer Society, Cancer Facts & Figures, Atlanta: American Cancer Society 2010.

2 A. Jemal, R. Siegel, E. Ward, Y. P. Hao, J. Q. Xu, M. J. Thun, Cancer Statistics, 2009, CA Cancer J.Clin. 2009, 59, 225-249.

3 Y. Liu, W. Zhang, Y. Cao, Y. Liu, S. Bergmeier, X. Chen, Small compound inhibitors of basal glucose transport inhibit cell proliferation and induce apoptosis in cancer cells via glucose-deprivation-like mechanisms, Cancer Lett. 2010, 298, 176-185.

4 T. E. Wood, S. Dalili, C. D. Simpson, R. Hurren, X. L. Mao, F. S. Saiz, M. Gronda, Y. Eberhard, M. D. Minden, P. J. Bilan, A. Klip, R. A. Batey, A. D. Schimmer, A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death, Mol. Cancer Ther. 2008, 7, 3546-3555.

5 W. H. Zhang, Y. Liu, X. Z. Chen, S. C. Bergmeier, Novel inhibitors of basal glucose transport as potential anticancer agents, Bioorg. Med. Chem. Lett. 2010, 20, 2191-2194.

6 N. C. I. Cancer Trends Progress Report – 2009/2010 Update, NIH, DHHS, Bethesda, MD, April 2010, http://progressreport.cancer.gov.

7 N. E. Warburg O, On the metabolism of cancer cells, Biochem. Z. 1924, 319-344.

8 H. Kondoh, M. E. Lleonart, D. Bernard, J. Gil, Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization, Histol. Histopathol. 2007, 22, 85-90.

9 O. H. Warburg, On the origin of cancer cells, Science 1956, 123, 309-314.

10 G. Bonuccelli, D. Whitaker-Menezes, R. Castello-Cros, S. Pavlides, R. G. Pestell, A. Fatatis, A. K. Witkiewicz, M. G. Vander Heiden, G. Migneco, B. Chiavarina, P. G. Frank, F. Capozza, N. Flomenberg, U. E. Martinez-Outschoorn, F. Sotgia, M. P. Lisanti, The reverse Warburg effect Glycolysis inhibitors prevent the tumor promoting effects of caveolin-1 deficient cancer associated fibroblasts, Cell Cycle 2010, 9, 1960-1971.

11 Z. Chen, W. Q. Lu, C. Garcia-Prieto, P. Huang, The Warburg effect and its cancer therapeutic implications, J. Bioenerg. Biomembr. 2007, 39, 267-274.

12 C. V. Dang, p32 (C1QBP) and cancer cell metabolism: Is the warburg effect a lot of hot air?, Mol. Cell. Biol. 2009, 30, 1300-1302. 203

13 D. T. Dang, S. A. Knock, S. W. Chen, F. Chen, L. H. Dang, HIF-1 alpha mediates aerobic glycolysis (the Warburg effect) and normoxic growth in colorectal cancer cells, Gastroenterology 2005, 128, A479-A479.

14 K. Garber, Energy boost: The Warburg effect returns in a new theory of cancer, J. Natl. Cancer Inst. 2004, 96, 1805-1806.

15 V. Gogvadze, B. Zhivotovsky, S. Orrenius, The Warburg effect and mitochondrial stability in cancer cells, Mol. Aspects Med. 2009, 31, 60-74.

16 R. Kannagi, Molecular mechanism for cancer-associated induction of sialyl Lewis X and sialyl Lewis A expression - The Warburg effect revisited, Glycoconjugate J. 2003, 20, 353-364.

17 J. W. Kim, C. V. Dang, Cancer's molecular sweet tooth and the Warburg effect, Cancer Res. 2006, 66, 8927-8930.

18 T. Koike, N. Kimura, K. Miyazaki, T. Yabuta, K. Kumamoto, S. Takenoshita, J. Chen, M. Kobayashi, M. Hosokawa, A. Taniguchi, T. Kojima, N. Ishida, M. Kawakita, H. Yamamoto, H. Takematsu, A. Suzuki, Y. Kozutsumi, R. Kanangi, Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates, Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 8132-8137.

19 S. Langbein, M. Zerilli, A. zur Hausen, W. Staiger, K. Rensch-Boschert, N. Lukan, J. Popa, M. P. Ternullo, A. Steidler, C. Weiss, R. Grobholz, F. Willeke, P. Alken, G. Stassi, P. Schubert, J. F. Coy, Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted, Br. J. Cancer 2006, 94, 578-585.

20 M. Lopez-Lazaro, The Warburg effect: Why and how do cancer cells activate glycolysis in the presence of oxygen?, Anti. Canc. Agents Med. Chem. 2008, 8, 305-312.

21 S. P. Mathupala, Y. H. Ko, P. L. Pedersen, Hexokinase-2 bound to mitochondria: Cancer's stygian link to the "Warburg effect" and a pivotal target for effective therapy, Semin. Cancer Biol. 2009, 19, 17-24.

22 P. L. Pedersen, Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment's roles together with hexokinase-2 in the "Warburg effect" in cancer, J. Bioenerg. Biomembr. 2008, 40, 123-126.

23 I. F. Robey, R. M. Stephen, K. S. Brown, B. Baggett, R. A. Gatenby, R. J. Gillies, Regulation of the Warburg effect in early-passage breast cancer cells, Neoplasia 2008, 10, 745-756. 204

24 C. Ruckenstuhl, S. Buttner, D. Carmona-Gutierrez, T. Eisenberg, G. Kroemer, S. J. Sigrist, K. U. Frohlich, F. Madeo, The Warburg Effect Suppresses Oxidative Stress Induced Apoptosis in a Yeast Model for Cancer, Plos One 2009, 4, 4592- 4597.

25 A. Shakya, D. Tantin, Ablation of transcription factor Oct-1 antagonizes the Warburg effect and offers a potential new target for anticancer therapy, Mol. Cancer Ther. 2007, 6, 3364S-3364S.

26 B. Zhivotovsky, S. Orrenius, The Warburg Effect returns to the cancer stage, Semin. Cancer Biol. 2009, 19, 1-3.

27 B. J. Collins, ATP, not glucose, is energy currency, Behav. Brain Sci. 1981, 4, 579-580.

28 A. Atlante, S. Giannattasio, A. Bobba, S. Gagliardi, V. Petragallo, P. Calissano, E. Marra, S. Passarella, An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis, Biochim. Biophys. Act. 2005, 1708, 50- 62.

29 A. L. Lehninger, in Principles of Biochemistry (Ed.: J. F. Sally Anderson), Worth Pubilishers, Inc., New York, 1982, pp. 467-510.

30 P. Mitchell, J. Moyle, Chemiosmotic hypothesis of oxidative phosphorylation, Nature 1967, 213, 137-139.

31 G. L. Semenza, Intratumoral hypoxia, radiation resistance, and HIF-1, Cancer Cell 2004, 5, 405-406.

32 R. A. Gatenby, R. J. Gillies, Why do cancers have high aerobic glycolysis?, Nat. Rev. Cancer 2004, 4, 891-899.

33 K. Garber, Energy deregulation: Licensing tumors to grow, Science 2006, 312, 1158-1159.

34 T. Bui, C. B. Thompson, Cancer's sweet tooth, Cancer Cell 2006, 9, 419-420.

35 V. R. Fantin, J. St-Pierre, P. Leder, Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance (vol 9, pg 425, 2006), Cancer Cell 2006, 10, 172-172.

36 G. M. Cragg, D. J. Newman, K. M. Snader, Natural products in drug discovery and development, J. Nat. Prod. 1997, 60, 52-60. 205

37 G. M. Cragg, D. J. Newman, Plants as a source of anti-cancer agents, J. Ethnopharmacol. 2005, 100, 72-79.

38 G. M. Cragg, D. J. Newman, Plants as a source of anti-cancer and anti-HIV agents, Ann. Appl. Biol. 2003, 143, 127-133.

39 M. Shoeb, Anticancer agents from medicinal plants, Bangladesh J. Pharmacol. 2006, 1, 35-41.

40 S. Benefraim, S. Shoval, T. Gal, R. Ophir, Classification of anticancer chemotherapeutic drugs according to their immunopotentiating activity, Anticancer Res. 1986, 6, 801-806.

41 E. Espinosa, P. Zamora, J. Feliu, M. G. Baron, Classification of anticancer drugs - a new system based on therapeutic targets, Cancer Treat. Rev. 2003, 29, 515-523.

42 A. H. Kyle, A. I. Minchinton, Classification of anticancer drugs based on tissue penetration using a novel in vitro screening assay, Mol.Cancer Thera. 2007, 6, 3405-3406.

43 T. S. Matney, T. V. Nguyen, T. H. Connor, W. J. Dana, J. C. Theiss, Genotoxic classification of anticancer drugs, Teratog. Carcinog. and Mutag. 1985, 5, 319- 328.

44 X. Z. Wu, A new classification system of anticancer drugs - Based on cell biological mechanisms, Med. Hypotheses 2006, 66, 883-887.

45 M. S. Furness, T. P. Robinson, T. Ehlers, R. B. Hubbard, J. L. Arbiser, D. J. Goldsmith, J. P. Bowen, Antiangiogenic agents: Studies on fumagillin and curcumin analogs, Curr. Pharm. Des. 2005, 11, 357-373.

46 S. Cruet-Hennequart, M. T. Glynn, L. S. Murillo, S. Coyne, M. P. Carty, Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin, DNA Repair 2008, 7, 582-596.

47 S. McClean, C. Costelloe, W. A. Denny, M. Searcey, L. P. G. Wakelin, Sequence selectivity, cross-linking efficiency and cytotoxicity of DNA-targeted 4- anilinoquinoline aniline mustards, Anti-Cancer Drug Des. 1999, 14, 187-204.

48 R. Pazdur, Cancer Management: A Multidisciplinary Approach, Medical, Surgical & Radiation Oncology, 11th ed., And the publishers of the journal ONCOLOGY, 2008.

49 M. G. Watve, R. Tickoo, M. M. Jog, B. D. Bhole, How many antibiotics are produced by the genus Streptomyces?, Arch. Microbiol. 2001, 176, 386-390. 206

50 G. Aubelsadron, D. Londosgagliardi, Daunorubicin and doxorubicin, anthracycline antibiotics, a physicochemical and biological review, Biochimie 1984, 66, 333-352.

51 J. R. Carignan, Anticancer antibiotics - bleomycin, mitomycin, and plicamycin, Hosp. Formul. 1986, 21, 1025-1033.

52 E. Ferrazzi, J. M. Woynarowski, A. Arakali, D. E. Brenner, T. A. Beerman, Dna damage and cytotoxicity induced by metabolites of anthracycline antibiotics, doxorubicin and idarubicin, Cancer Commun. 1991, 3, 173-180.

53 J. W. Lown, A. Begleiter, D. Johnson, A. R. Morgan, Studies related to antitumor antibiotics .V. Reactions of mitomycin-c with dna examined by ethidium fluorescence assay, Can. J. Biochem. 1976, 54, 110-119.

54 V. Gebbia, L. Blasi, N. Borsellino, M. Caruso, V. Leonardi, B. Agostara, R. Valenza, Paclitaxel and epidoxorubicin or doxorubicin versus cyclophosphamide and epidoxorubicin as first-line chemotherapy for metastatic breast carcinoma: A randomised phase II study, Anticancer Res. 2003, 23, 765-771.

55 A. N. Gordon, J. T. Fleagle, D. Guthrie, D. E. Parkin, M. E. Gore, A. J. Lacave, Recurrent epithelial ovarian carcinoma: A randomized phase III study of pegylated liposomal doxorubicin versus topotecan, J. Clin. Oncol. 2001, 19, 3312-3322.

56 W. E. Hardman, M. P. Moyer, I. L. Cameron, Efficacy of treatment of colon, lung and breast human carcinoma xenografts with: Doxorubicin, cisplatin, irinotecan or topotecan, Anticancer Res. 1999, 19, 2269-2274.

57 B. D. Nelkin, D. W. Ball, Combretastatin A-4 and doxorubicin combination treatment is effective in a preclinical model of human medullary thyroid carcinoma, Oncol. Rep. 2001, 8, 157-160.

58 A. K. Tyagi, C. Agarwal, D. C. F. Chan, R. Agarwal, Synergistic anti-cancer effects of silibinin with conventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breast carcinoma MCF-7 and MDA-MB468 cells, Oncol. Rep. 2004, 11, 493-499.

59 R. R. Ellison, R. Mick, J. Cuttner, C. A. Schiffer, R. T. Silver, E. S. Henderson, T. Woliver, I. Royston, F. R. Davey, A. S. Glicksman, C. D. Bloomfield, J. F. Holland, The effects of postinduction intensification treatment with cytarabine and daunorubicin in adult acute lymphocytic-leukemia - a prospective randomized clinical-trial by cancer and leukemia group, J. Clin. Oncol. 1991, 9, 2002-2015.

60 P. B. Jensen, P. S. Jensen, E. J. F. Demant, E. Friche, B. S. Sorensen, M. Sehested, K. Wassermann, L. Vindelov, O. Westergaard, H. H. Hansen, 207

Antagonistic effect of aclarubicin on daunorubicin-induced cytotoxicity in human small-cell lung-cancer cells - relationship to dna integrity and topoisomerase-ii, Cancer Research 1991, 51, 5093-5099.

61 K. J. O'Byrne, A. L. Thomas, R. A. Sharma, M. DeCatris, F. Shields, S. Beare, W. P. Steward, A phase I dose-escalating study of DaunoXome, liposomal daunorubicin, in metastatic breast cancer, Br. J. Cancer 2002, 87, 15-20.

62 S. Fujimoto, K. Igarashi, R. D. Shrestha, M. Miyazaki, K. Okui, Antitumor effects of 2 polyamine antimetabolites combined with mitomycin-c on human stomach- cancer cells xenotransplanted into nude-mice, Int. J. Cancer 1985, 35, 821-825.

63 A. Paccagnella, A. Favaretto, F. Oniga, G. Festi, S. Lauro, A. Morabito, L. Ossana, F. Sartore, F. DePoli, M. V. Fiorentino, Mitomycin C, vinblastine, and carboplatin regimen in patients with nonsmall cell lung cancer - A phase II trial, Cancer 1996, 78, 1701-1707.

64 R. R. Perry, B. R. Greaves, Y. Kang, Development and initial characterization of a mitomycin-c-resistant colon-cancer cell-line variant, Cancer Chemother. Pharmacol. 1993, 32, 326-328.

65 B. Thurlimann, R. Waldburger, H. J. Senn, D. Thiebaud, Plicamycin and pamidronate in symptomatic tumor-related hypercalcemia - a prospective randomized crossover trial, Ann. Oncol. 1992, 3, 619-623.

66 E. C. Attar, D. J. De Angelo, J. G. Supko, F. D'Amato, D. Zahrieh, A. Sirulnik, M. Wadleigh, K. K. Ballen, S. McAfee, K. B. Miller, J. Levine, I. Galinsky, E. G. Trehu, D. Schenkein, D. Neuberg, R. M. Stone, P. C. Amrein, Phase I and pharmacokinetic study of bortezomib in combination with idarubicin and cytarabine in patients with acute myelogenous leukemia, Clin. Cancer. Res. 2008, 14, 1446-1454.

67 W. R. Vogler, E. Velezgarcia, R. S. Weiner, M. A. Flaum, A. A. Bartolucci, G. A. Omura, M. C. Gerber, P. L. C. Banks, A phase-III trial comparing idarubicin and daunorubicin in combination with cytarabine in acute myelogenous leukemia - a southeastern cancer study-group study, J. Clin. Oncol. 1992, 10, 1103-1111.

68 R. deWit, G. Stoter, S. B. Kaye, D. T. Sleijfer, W. G. Jones, W. W. T. Huinink, L. A. Rea, L. Collette, R. Sylvester, Importance of bleomycin in combination chemotherapy for good-prognosis testicular nonseminoma: A randomized study of the European Organization for Research and Treatment of Cancer Genitourinary Tract Cancer Cooperative Group, J. Clin. Oncol. 1997, 15, 1837- 1843. 208

69 B. H. Kaplan, S. E. Vogl, Methotrexate (m), bleomycin(b) and diammine dichloro-platinum(d) in squamous cancers (sq ca) of head and neck, cervix and other sites, Proc. Am. Asso. Cancer Res. 1978, 19, 323-323.

70 G. Stoter, A. Koopman, C. P. J. Vendrik, A. Struyvenberg, D. T. Sleyfer, P. H. B. Willemse, H. S. Koops, A. T. Vanoosterom, W. W. T. Huinink, H. M. Pinedo, 10- year survival and late sequelae in testicular cancer-patients treated with cisplatin, vinblastine, and bleomycin, J. Clin. Oncol. 1989, 7, 1099-1104.

71 R. E. Polk, Anaphylactoid reactions to glycopeptide antibiotics, J. Antimicrob. Chemother. 1991, 27, 17-29.

72 T. G. Slama, A. Amin, S. A. Brunton, T. M. File, G. Milkovich, K. A. Rodvold, D. F. Sahm, J. Varon, D. Weiland, A clinician's guide to the appropriate and accurate use of antibiotics: the Council for Appropriate and Rational Antibiotic Therapy (CARAT) criteria, Am. J. Med. 2005, 118, 1-6.

73 J. Verwey, J. Devries, H. M. Pinedo, Mitomycin-c-induced renal toxicity, a dose- dependent side-effect, Eur. J. Cancer Clin. Oncol. 1987, 23, 195-199.

74 M. Das, S. E. Livingstone, Metal-chelates as anti-cancer agents .2. Cytotoxic action of palladium and platinum complexes of 6-mercaptopurine and thioguanine, Br. J. Cancer 1978, 38, 325-328.

75 R. O. Dillman, R. Mick, O. R. McIntyre, Pentostatin in chronic lymphocytic- leukemia - a phase-ii trial of cancer and leukemia group, J. Clin. Oncol. 1989, 7, 433-438.

76 B. L. Hillcoat, P. B. McCulloch, A. T. Figueredo, M. H. Ehsan, J. M. Rosenfeld, Clinical response and plasma-levels of 5-fluorouracil in patients with colonic cancer treated by drug infusion, Br. J. Cancer 1978, 38, 719-724.

77 A. E. M. Taylor, S. Shuster, Skin-cancer after renal-transplantation - the causal role of azathioprine, Acta Derm. Venereol. 1992, 72, 115-119.

78 R. J. Wells, M. T. Adams, T. A. Alonzo, R. J. Arceci, J. Buckley, A. B. Buxton, K. Dusenbery, A. Gamis, M. Masterson, T. Vik, P. Warkentin, J. A. Whitlock, Mitoxantrone and cytarabine induction, high-dose cytarabine, and etoposide intensification for pediatric patients with relapsed or refractory acute myeloid leukemia: Children's Cancer Group study 2951, J. Clin. Oncol. 2003, 21, 2940- 2947.

79 A. L. Smith, Oxford dictionary of biochemistry and molecular biology, Oxford [Oxfordshire]: Oxford University Press, 1997. 209

80 T. H. Bestor, G. Schatten, Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea-urchin fertilization, Dev. Biol. 1981, 88, 80-91.

81 John A. Hadfield, Sylvie Ducki, Nicholas Hirst, A. T. McGown, in Progress in Cell Cycle Research (Ed.: L. Meijer, Jézéquel, A., and Roberge, M.,), 2003, pp. 309-325.

82 C. W. Lloyd, A. R. Slabas, A. J. Powell, G. Macdonald, R. A. Badley, Cytoplasmic microtubules of higher-plant cells visualized with anti-tubulin antibodies, Nature 1979, 279, 239-241.

83 M. C. Wani, H. L. Taylor, M. E. Wall, P. Coggon, A. T. McPhail, Plant antitumor agents .VI. Isolation and structure of taxol, a novel antileukemic and antitumor agent from taxus-brevifolia, J. Am. Chem. Soc. 1971, 93, 2325-2327.

84 R. B. Cohen, C. J. Langer, G. R. Simon, P. D. Eisenberg, J. D. Hainsworth, S. Madajewicz, T. M. Cosgriff, K. Pierce, H. Xu, K. Liau, D. Healey, A phase I/randomized phase II, non-comparative, multicenter, open label trial of CP- 547,632 in combination with paclitaxel and carboplatin or paclitaxel and carboplatin alone as first-line treatment for advanced non-small cell lung cancer (NSCLC), Cancer Chemother. Pharmacol. 2007, 60, 81-89.

85 S. Mozzetti, C. Ferlini, P. Concolino, F. Filippetti, G. Raspaglio, S. Prislei, D. Gallo, E. Martinelli, F. O. Ranelletti, G. Ferrandina, G. Scambia, Class III beta- tubulin overexpression is a prominent mechanism of paclitaxel resistance in ovarian cancer patients, Clin. Cancer. Res. 2005, 11, 298-305.

86 L. L. Nielsen, P. Lipari, J. Dell, M. Gurnani, G. Hajian, Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer, Clin. Cancer. Res. 1998, 4, 835- 846.

87 L. R. Wiseman, C. M. Spencer, Paclitaxel - An update of its use in the treatment of metastatic breast cancer and ovarian and other gynaecological cancers, Drugs Aging 1998, 12, 305-334.

88 A. D. Guminski, P. R. Harnett, A. deFazio, Carboplatin and paclitaxel interact antagonistically in a megakaryoblast cell line - a potential mechanism for paclitaxel-mediated sparing of carboplatin-induced thrombocytopenia, Cancer Chemother. Pharmacol. 2001, 48, 229-234.

89 B. Ozcelik, C. Turkyilmaz, M. T. Ozgun, I. S. Serin, C. Batukan, S. Ozdamar, A. Ozturk, Prevention of paclitaxel and cisplatin induced ovarian damage in rats by a gonadotropin-releasing hormone agonist, Fertil. Steril. 2010, 93, 1609-1614. 210

90 J. Schwartz, S. M. Domchek, W. T. Hwang, K. Fox, Evaluation of anemia, neutropenia and skin toxicities in standard or dose-dense doxorubicin/cyclophosphamide (AC)-paclitaxel or docetaxel adjuvant chemotherapy in breast cancer, Ann. Oncol. 2005, 16, 247-252.

91 I. Ojima, O. Duclos, M. Zucco, M. C. Bissery, C. Combeau, P. Vrignaud, J. F. Riou, F. Lavelle, Synthesis and structure-activity-relationships of new antitumor taxoids - effects of cyclohexyl substitution at the C-3' and/or C-2 of taxotere (docetaxel), J. Med. Chem. 1994, 37, 2602-2608.

92 G. Frasci, P. Comella, R. Thomas, M. Di Bonito, L. Lapenta, I. Capasso, G. Botti, P. Vallone, V. De Rosa, G. D'Aiuto, G. Comella, Biweekly docetaxel-irinotecan with filgrastim support in pretreated breast and non-small-cell lung cancer patients. A phase I study, Cancer Chemother. Pharmacol. 2004, 53, 25-32.

93 C. Kouroussis, S. Agelaki, D. Mavroudis, S. Kakolyris, N. Androulakis, K. Kalbakis, J. Souglakos, K. Mallas, V. Bozionelou, A. Pallis, H. Adamtziki, V. Georgoulias, A dose escalation study of docetaxel and oxaliplatin combination in patients with metastatic breast and non-small cell lung cancer, Anticancer Res. 2003, 23, 785-791.

94 G. G. Laport, G. F. Fleming, S. Waggoner, T. M. Zimmerman, D. L. Grinblatt, S. F. Williams, A phase II trial of docetaxel for peripheral blood stem cell mobilization for patients with breast cancer and ovarian cancer, Bone Marrow Transplant. 2001, 27, 677-681.

95 S. Zeng, Y. Z. Chen, L. W. Fu, K. R. Johnson, W. M. Fan, In vitro evaluation of schedule-dependent interactions between docetaxel and doxorubicin against human breast and ovarian cancer cells, Clin. Cancer. Res. 2000, 6, 3766-3773.

96 P. A. Ellis, I. E. Smith, J. R. Hardy, M. C. Nicolson, D. C. Talbot, S. E. Ashley, K. Priest, Symptom relief with MVP (mitomycin-C, vinblastine and cisplatin) chemotherapy in advanced non-small-cell lung-cancer, Br. J. Cancer 1995, 71, 366-370.

97 S. P. Leitner, G. J. Bosl, E. W. Strong, F. P. Gerold, R. H. Spiro, J. P. Shah, R. B. Sessions, B. Vikram, A pilot-study of cisplatin-vinblastine as the initial treatment of advanced head and neck-cancer, Cancer 1986, 58, 1014-1017.

98 J. M. Nabholtz, H. J. Senn, W. R. Bezwoda, D. Melnychuk, L. Deschenes, J. Douma, T. A. Vandenberg, B. Rapoport, R. Rosso, V. Trillet-Lenoir, J. Drbal, A. Molino, J. W. R. Nortier, D. J. Richel, T. Nagykalnai, P. Siedlecki, N. Wilking, J. Y. Genot, P. Hupperets, F. Pannuti, D. Skarlos, E. M. Tomiak, M. Murawsky, M. Alakl, A. Riva, M. Aapro, Prospective randomized trial of docetaxel versus mitomycin plus vinblastine in patients with metastatic breast cancer progressing 211

despite previous anthracycline-containing chemotherapy, J. Clin. Oncol. 1999, 17, 1413-1424.

99 A. Paccagnella, A. Favaretto, F. Oniga, F. Barbieri, G. Ceresoli, W. Torri, E. Villa, C. Verusio, G. L. Cetto, A. Santo, M. G. Ghi, A. Morabito, R. Biason, M. Giusto, P. Mosconi, V. C. Sileni, Cisplatin versus carboplatin in combination with mitomycin and vinblastine in advanced non small cell lung cancer. A multicenter, randomized phase III trial, Lung Cancer 2004, 43, 83-91.

100 W. W. Sutow, T. J. Vietti, D. J. Fernbach, D. M. Lane, Donaldso.Mh, D. H. Berry, Combination of vincristine and prednisone in therapy of acute leukemia in children, J. Pediatr. 1968, 73, 426-&.

101 M. A. Jordan, L. Wilson, Microtubules as a target for anticancer drugs, Nat. Rev. Cancer 2004, 4, 253-265.

102 H. Tanaka, H. Matsushima, N. Mizumoto, A. Takashima, Induction of protective immunity against melanoma by intra-tumor administration of a dendritic cell- stimulatory anticancer drug, vinblastine, J. Invest. Dermatol. 2006, 126, 225.

103 C. Teutsch, A. Lipton, H. A. Harvey, Raynauds-phenomenon as a side-effect of chemotherapy with vinblastine and bleomycin for testicular carcinoma, Cancer Treat. Rep. 1977, 61, 925-926.

104 M. Boranic, F. Raci, Parkinson-like syndrome as side-effect of chemotherapy with vincristine and adriamycin in a child with acute-leukemia, Biomedicine 1979, 31, 124-125.

105 O. Merimsky, A. Loewenstein, S. Chaitchik, Cortical blindness - a catastrophic side-effect of vincristine, Anti-Cancer Drugs 1992, 3, 371-373.

106 M. Riga, M. Varvutsi, D. Doganis, M. Baka, I. Psarommatis, H. Kosmidis, Abnormal olivocochlear reflex: A neurotoxic side-effect of vincristine, Pediatr. Blood Cancer 2007, 49, 571-571.

107 C. J. Pelletier PS, Ann. Chim. Phys. 1820, 14, 69.

108 P. Verdier-Pinard, J. Y. Lai, H. D. Yoo, J. R. Yu, B. Marquez, D. G. Nagle, M. Nambu, J. D. White, J. R. Falck, W. H. Gerwick, B. W. Day, E. Hamel, Structure- activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells, Mol. Pharmacol. 1998, 53, 62-76.

109 M. J. Ahern, C. Reid, T. P. Gordon, M. McCredie, P. M. Brooks, M. Jones, Does colchicine work - the results of the 1st controlled-study in acute gout, Aust. N. Z. J. Med. 1987, 17, 301-304. 212

110 A. J. Wozniak, W. E. Ross, Dna damage as a basis for 4'- demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyranoside ) (etoposide) cyto-toxicity, Cancer Research 1983, 43, 120-124.

111 C. Bokemeyer, H. J. Schmoll, M. A. Kuczyk, J. Beyer, W. Siegert, Risk of secondary leukemia following high cumulative doses of etoposide during chemotherapy for testicular cancer, J. Natl. Cancer Inst. 1995, 87, 58-60.

112 C. C. Chiu, C. H. Li, M. W. Ung, T. S. Fuh, W. L. Chen, K. Fang, Etoposide (VP- 16) elicits apoptosis following prolonged G(2)-M cell arrest in p53-mutated human non-small cell lung cancer cells, Cancer Lett. 2005, 223, 249-258.

113 R. Fernandez, I. Rubio, J. M. Mane, J. R. Barcelo, A. Munoz, G. Abon, G. L. Vivanco, Treatment of small cell lung cancer (SCLC) with 3-day out-patient administration of cisplatin (CDDP) and etoposide (VP-16): Efficacy, toxicity and survival analysis, Ann. Oncol. 1998, 9, 510.

114 F. A. Greco, D. H. Johnson, K. R. Hande, L. L. Porter, J. D. Hainsworth, S. N. Wolff, High-dose etoposide (VP-16) in small-cell lung-cancer, Semin. Oncol. 1985, 12, 42-44.

115 H. E. Grier, M. D. Krailo, N. J. Tarbell, M. P. Link, C. J. H. Fryer, D. J. Pritchard, M. C. Gebhardt, P. S. Dickman, E. J. Perlman, P. A. Meyers, S. S. Donaldson, S. Moore, A. R. Rausen, T. J. Vietti, J. S. Miser, Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone, New Engl. J. Med. 2003, 348, 694-701.

116 I. R. Judson, E. Wiltshaw, Cis-dichlorodiammineplatinum (cisplatinum) and etoposide (VP16) - an effective salvage regimen in malignant-lymphoma, Br. J. Cancer 1984, 50, 248-248.

117 P. J. Loehrer, Etoposide therapy for testicular cancer, Cancer 1991, 67, 220-224.

118 A. G. Pedersen, H. H. Hansen, Etoposide (VP-16) in the treatment of lung-cancer, Cancer Treat. Rev. 1983, 10, 245-264.

119 K. Marigny, F. Aubin, G. Burgot, E. Le Gall, V. Gandemer, Particular cutaneous side effects with etoposide-containing courses: is VP16 or etoposide phosphate responsible?, Cancer Chemother. Pharmacol. 2005, 55, 244-250.

120 M. Muller, R. Heicappell, U. Steiner, C. Goessl, K. Miller, Side effects of chemotherapy for advanced urothelial carcinoma with etoposide and ifosfamide, Urol. Int. 1997, 59, 248-251.

121 Hanny Haaxma-Reiche, Henk H. Berendsen, P. E. Postmus, Podophyliotoxins for brain metastases of small cell lung cancer, J. Neuro. Oncol. 1988, 231-232. 213

122 M. G. Saulnier, K. L. Leboulluec, B. H. Long, D. M. Vyas, A. R. Crosswell, T. W. Doyle, Synthesis and biological evaluation of 4'-deshydroxy-4'-methyl etoposide and teniposide analogs, Bioorg. Med. Chem. Lett. 1992, 2, 1213-1218.

123 D. K. Armstrong, D. Spriggs, J. Levin, R. Poulin, S. Lane, Hematologic safety and tolerability of topotecan in recurrent ovarian cancer and small cell lung cancer: An integrated analysis, Oncologist 2005, 10, 686-694.

124 J. V. Fiorica, The role of topotecan in the treatment of advanced cervical cancer, Gynecol. Oncol. 2003, 90, S16-S21.

125 G. Frasci, N. Panza, P. Comella, G. Carteni, T. Guida, G. P. Nicolella, M. Natale, R. Lombardi, A. Apicella, C. Pacilio, A. Gravina, L. Lapenta, G. Comella, Cisplatin-topotecan-paclitaxel weekly administration with G-CSF support for ovarian and small-cell lung cancer patients: A dose-finding study, Ann. Oncol. 1999, 10, 355-358.

126 J. Garst, Safety of topotecan in the treatment of recurrent small-cell lung cancer and ovarian cancer, Expert Opin. Drug Saf. 2007, 6, 53-62.

127 C. Kollmannsberger, K. Mross, A. Jakob, L. Kanz, C. Bokemeyer, Topotecan - A novel topoisomerase I inhibitor: Pharmacology and clinical experience, Oncology 1999, 56, 1-12.

128 S. B. Hizel, C. Sanli, N. B. Muluk, M. Albayrak, A. Ozyazici, A. Apan, Topotecan treatment and its toxic effects on hematologic parameters and trace elements, Biol. Trace Elem. Res. 2008, 124, 129-134.

129 M. Ando, K. Eguchi, T. Shinkai, T. Tamura, Y. Ohe, N. Yamamoto, T. Kurata, T. Kasai, H. Ohmatsu, K. Kubota, I. Sekine, N. Hojo, T. Matsumoto, T. Kodama, R. Kakinuma, Y. Nishiwaki, N. Saijo, Phase I study of sequentially administered topoisomerase I inhibitor (irinotecan) and topoisomerase II inhibitor (etoposide) for metastatic non-small-cell lung cancer, Br. J. Cancer 1997, 76, 1494-1499.

130 F. Kanzawa, F. Koizumi, Y. Koh, T. Nakamura, Y. Tatsumi, H. Fukumoto, N. Saijo, T. Yoshioka, K. Nishio, In vitro synergistic interactions between the cisplatin analogue nedaplatin and the DNA topoisomerase I inhibitor irinotecan and the mechanism of this interaction, Clin. Cancer. Res. 2001, 7, 202-209.

131 L. B. Saltz, D. Niedzwiecki, D. Hollis, R. M. Goldberg, A. Hantel, J. P. Thomas, A. L. A. Fields, R. J. Mayer, Irinotecan fluorouracil plus leucovorin is not superior to fluorouracil plus leucovorin alone as adjuvant treatment for stage III colon cancer: Results of CALGB 89803, J. Clin. Oncol. 2007, 25, 3456-3461.

132 Y. Shimada, M. Rothenberg, S. G. Hilsenbeck, H. A. Burris, D. Degen, D. D. Vonhoff, Activity of CPT-11 (irinotecan hydrochloride), a topoisomerase-i 214

inhibitor, against human tumor colony-forming-units, Anti-Cancer Drugs 1994, 5, 202-206.

133 E. Van Cutsem, R. Labianca, G. Bodoky, C. Barone, E. Aranda, B. Nordlinger, C. Topham, J. Tabernero, T. Andre, A. F. Sobrero, E. Mini, R. Greil, F. Di Costanzo, L. Collette, L. Cisar, X. X. Zhang, D. Khayat, C. Bokemeyer, A. D. Roth, D. Cunningham, Randomized phase III trial comparing biweekly infusional fluorouracil/leucovorin alone or with irinotecan in the adjuvant treatment of stage III colon cancer: PETACC-3 J. Clin. Oncol. 2009, 27, 3117-3125.

134 H. Y. Xue, M. B. Sawyer, C. J. Field, L. A. Dieleman, V. E. Baracos, Nutritional modulation of antitumor efficacy and diarrhea toxicity related to irinotecan chemotherapy in rats bearing the ward colon tumor, Clin. Cancer. Res. 2007, 13, 7146-7154.

135 D. Amic, D. Davidovic-Amic, D. Beslo, V. Rastija, B. Lucic, N. Trinajstic, SAR and QSAR of the antioxidant activity of flavonoids, Curr. Med. Chem. 2007, 14, 827-845.

136 A. Arora, M. G. Nair, G. M. Strasburg, Structure-activity relationships for antioxidant activities of a series of flavonoids in a liposomal system, Free Radical Biol. Med. 1998, 24, 1355-1363.

137 S. Azam, N. Hadi, N. U. Khan, S. M. Hadi, Prooxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: implications for anticancer properties, Toxicol. In Vitro 2004, 18, 555-561.

138 G. H. Cao, E. Sofic, R. L. Prior, Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships, Free Radical Biol. Med. 1997, 22, 749-760.

139 H. Chang, M. T. Mi, W. H. Ling, J. D. Zhu, Q. Y. Zhang, N. Wei, Y. Zhou, Y. Tang, X. P. Yu, T. Zhang, J. Wang, J. L. Yuan, Structurally Related Anticancer Activity Of Flavonoids: Involvement Of Reactive Oxygen Species Generation, J. Food Biochem. 2010, 34, 1-14.

140 P. Fresco, F. Borges, C. Diniz, M. P. M. Marques, New insights on the anticancer properties of dietary polyphenols, Med. Res. Rev. 2006, 26, 747-766.

141 E. B. Golden, P. Y. Lam, A. Kardosh, K. J. Gaffney, E. Cadenas, S. G. Louie, N. A. Petasis, T. C. Chen, A. H. Schonthal, Green tea polyphenols block the anticancer effects of bortezomib and other boronic acid-based proteasome inhibitors, Blood 2009, 113, 5927-5937. 215

142 S. M. Hadi, S. H. Bhat, A. S. Azmi, S. Hanif, U. Shamim, M. F. Ullah, Oxidative breakage of cellular DNA by plant polyphenols: A putative mechanism for anticancer properties, Semin. Cancer Biol. 2007, 17, 370-376.

143 D. Lamoral-Theys, L. Pottier, F. Dufrasne, J. Neve, J. Dubois, A. Kornienko, R. Kiss, L. Ingrassia, Natural Polyphenols that Display Anticancer Properties through Inhibition of Kinase Activity, Curr. Med. Chem. 2010, 17, 812-825.

144 K. R. Landis-Piwowar, C. D. Huo, D. Chen, V. Milacic, G. Q. Shi, T. H. Chan, Q. P. Dou, A novel prodrug of the green tea polyphenol (-)-epigallocatechin-3- gallate as a potential anticancer agent, Cancer Res. 2007, 67, 4303-4310.

145 S. B. Lotito, B. Frei, Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in humans: Cause, consequence, or epiphenomenon?, Free Radical Biol. Med. 2006, 41, 1727-1746.

146 J. Lu, L. V. Papp, J. G. Fang, S. Rodriguez-Nieto, B. Zhivotovsky, A. Holmgren, Inhibition of mammalian thioredoxin reductase by some flavonoids: Implications for myricetin and quercetin anticancer activity, Cancer Res. 2006, 66, 4410-4418.

147 L. Mira, M. T. Fernandez, M. Santos, R. Rocha, M. H. Florencio, K. R. Jennings, Interactions of flavonoids with iron and copper ions: A mechanism for their antioxidant activity, Free Radical Res. 2002, 36, 1199-1208.

148 S. Teixeira, C. Siquet, C. Alves, I. Boal, M. P. Marques, F. Borges, J. Lima, S. Reis, Structure-property studies on the antioxidant activity of flavonoids present in diet, Free Radical Biol. Med. 2005, 39, 1099-1108.

149 K. L. Wolfe, R. H. Liu, Structure-activity relationships of flavonoids in the cellular antioxidant activity assay, J. Agric. Food Chem. 2008, 56, 8404-8411.

150 H. Z. Ying, Y. Z. Hu, Q. J. He, R. P. Li, B. Yang, Synthesis and anticancer activity of a novel class of flavonoids: 2,4-diarylchromane[4,3-d]-Delta(1,9b)- 1,2,3-thiadiazolines, Eur. J. Med. Chem. 2007, 42, 226-234.

151 J. A. Kennedy, M. A. Matthews, A. L. Waterhouse, Effect of maturity and vine water status on grape skin and wine flavonoids, Am. J. Enol. Vitic. 2002, 53, 268- 274.

152 Y. Miyagi, K. Miwa, H. Inoue, Inhibition of human low-density lipoprotein oxidation by flavonoids in red wine and grape juice, Am. J. Cardiol. 1997, 80, 1627-1631.

153 C. A. RiceEvans, N. J. Miller, Antioxidant activities of flavonoids as bioactive components of food, Biochem. Soc. Trans. 1996, 24, 790-795. 216

154 T. Ridgway, J. Oreilly, G. West, G. Tucker, H. Wiseman, Potent antioxidant properties of novel apple-derived flavonoids with commercial potential as food additives, Biochem. Soc. Trans. 1996, 24, S391-S391.

155 B. Fuhrman, A. Lavy, M. Aviram, Consumption of red wine with meals reduces the susceptibility of human plasma and low-density lipoprotein to lipid peroxidation, Am. J. Clin. Nutr. 1995, 61, 549-554.

156 C. A. RiceEvans, N. J. Miller, G. Paganga, Structure-antioxidant activity relationships of flavonoids and phenolic acids, Free Radical Biol. Med. 1996, 20, 933-956.

157 S. vanAcker, D. J. vandenBerg, M. Tromp, D. H. Griffioen, W. P. VanBennekom, W. J. F. VanderVijgh, A. Bast, Structural aspects of antioxidant activity of flavonoids, Free Radical Biol. Med. 1996, 20, 331-342.

158 N. Cotelle, J. L. Bernier, J. P. Henichart, J. P. Catteau, E. Gaydou, J. C. Wallet, Scavenger and antioxidant properties of 10 synthetic flavones, Free Radical Biol. Med. 1992, 13, 211-219.

159 B. Fauconneau, P. WaffoTeguo, F. Huguet, L. Barrier, A. Decendit, J. M. Merillon, Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests, Life Sci. 1997, 61, 2103-2110.

160 C. F. F. Grael, A. Kanashiro, L. M. Kabeya, C. O. Jordao, R. Takeara, L. Gobbo- Neto, A. C. M. Polizello, Y. M. Lucisano-Valim, N. P. Lopes, J. L. C. Lopes, In vitro study of antioxidant and scavenger properties of phenolic compounds from lychnophora species, Quim. Nova 2010, 33, 867-870.

161 F. N. Ko, Z. J. Cheng, C. N. Lin, C. M. Teng, Scavenger and antioxidant properties of prenylflavones isolated from Artocarpus heterophyllus, Free Radical Biol. Med. 1998, 25, 160-168.

162 P. G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 2000, 63, 1035-1042.

163 Y. Porat, A. Abramowitz, E. Gazit, Inhibition of amyloid fibril formation by polyphenols: Structural similarity and aromatic interactions as a common inhibition mechanism, Chemical Biology & Drug Design 2006, 67, 27-37.

164 S. B. Lotito, B. Frei, Relevance of apple polyphenols as antioxidants in human plasma: Contrasting in vitro and in vivo effects, Free Radical Biol. Med. 2004, 36, 201-211. 217

165 J. A. Vinson, Y. A. Dabbagh, M. M. Serry, J. H. Jang, Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in-vitro oxidation model for heart-disease, J. Agric. Food Chem. 1995, 43, 2800-2802.

166 M. Kobori, H. Shinmoto, T. Tsushida, K. Shinohara, Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport, Cancer Lett. 1997, 119, 207-212.

167 J. A. S. Nelson, R. E. Falk, Phlorhizin and phloretin inhibition of 2-deoxy-d- glucose uptake by tumor-cells in-vitro and in-vivo, Anticancer Res. 1993, 13, 2293-2299.

168 C. H. Wu, Y. S. Ho, C. Y. Tsai, Y. J. Wang, H. Tseng, P. L. Wei, C. H. Lee, R. S. Liu, S. Y. Lin, In vitro and in vivo study of phloretin-induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter, International Journal of Cancer 2009, 124, 2210-2219.

169 Y. Oka, T. Asano, Y. Shibasaki, J. L. Lin, K. Tsukuda, H. Katagiri, Y. Akanuma, F. Takaku, C-terminal truncated glucose transporter is locked into an inward- facing form without transport activity, Nature 1990, 345, 550-553.

170 H. G. Joost, B. Thorens, The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members, Mol. Membr. Biol. 2001, 18, 247-256.

171 M. Mueckler, C. Caruso, S. A. Baldwin, M. Panico, I. Blench, H. R. Morris, W. J. Allard, G. E. Lienhard, H. F. Lodish, Sequence and structure of a human glucose transporter, Science 1985, 229, 941-945.

172 D. N. Hebert, A. Carruthers, Glucose transporter oligomeric structure determines transporter function - reversible redox-dependent interconversions of tetrameric and dimeric GLUT1, J. Biol. Chem. 1992, 267, 23829-23838.

173 A. L. Olson, J. E. Pessin, Structure, function, and regulation of the mammalian facilitative glucose transporter gene family, Annu. Rev. Nutr. 1996, 16, 235-256.

174 B. Thorens, Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes, Am.J.Physiol.-Gastrointestinal and Liver Physiology 1996, 270, G541-G553.

175 L. G. Melstrom, M. R. Salabat, X. Z. Ding, B. M. Milam, M. Strouch, J. C. Pelling, D. J. Bentrem, Apigenin inhibits the GLUT-1 glucose transporter and the phosphoinositide 3-kinase/akt pathway in human pancreatic cancer cells, Pancreas 2008, 37, 426-431. 218

176 H. Pelicano, D. S. Martin, R. H. Xu, P. Huang, Glycolysis inhibition for anticancer treatment, Oncogene 2006, 25, 4633-4646.

177 R. Airley, A. Evans, A. Mobasheri, S. M. Hewitt, Glucose transporter Glut-1 is detectable in peri-necrotic regions in many human tumor types but not normal tissues: Study using tissue microarrays, Ann. Anatomy 2010, 192, 133-138.

178 J. B. Park, Flavonoids are potential inhibitors of glucose uptake in U937 cells, Biochem. Biophys. Res. Commun. 1999, 260, 568-574.

179 K. N. Stephenson, R. L. Croxen, A. El-Barbary, J. D. Fenstermacher, H. C. Haspel, Inhibition of glucose transport and direct interactions with type 1 facilitative glucose transporter (GLUT-1) by etomidate, ketamine, and propofol - A comparison with barbiturates, Biochem. Pharmacol. 2000, 60, 651-659.

180 S. O. Wei, S. K. Kulp, C. S. Chen, Energy restriction as an antitumor target of thiazolidinediones, J. Biol. Chem. 2010, 285, 9780-9791.

181 H. J. Won, T. K. Ha, S. J. Kwon, H. Y. Cho, S. J. Hur, H. H. Baik, S. I. Suh, E. Ha, Y. H. Kim, Differential effects of 5-fluorouracil on glucose transport and expressions of glucose transporter proteins in gastric cancer cells, Anti-Cancer Drugs 2010, 21, 270-276.

182 L. Golkar, M. R. Salabat, X. Z. Ding, M. B. Ujiki, S. Rao, J. J. McGill, C. Chen, D. M. Heiferman, J. Pelling, R. H. Bell, M. S. Talamonti, D. J. Bentrem, Apigenin inhibits pancreatic cancer cell proliferation via down-regulation of the GLUT-1 glucose transporter through the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, Pancreas 2006, 33, 464-464.

183 E. Gottlieb, I. P. M. Tomlinson, Mitochondrial tumour suppressors: A genetic and biochemical update, Nat. Rev. Cancer 2005, 5, 857-866.

184 P. L. Pedersen, Bioenergetics of cancer cells - A brief orientation to this minireview series, J. Bioenerg. Biomembr. 1997, 29, 301-302.

185 E. Gottlieb, What Does Bioenergetics Have to Do with Cancer?, Am. Assoc. Cancer Res. Educ. Book 2005, 341-344.

186 X. H. Cao, L. Y. Fang, S. Gibbs, Y. Huang, Z. Y. Dai, P. Wen, X. C. Zheng, W. Sadee, D. X. Sun, Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia, Cancer Chemother. Pharmacol. 2007, 59, 495-505.

187 J. M. Brown, W. R. William, Exploiting tumour hypoxia in cancer treatment, Nat. Rev. Cancer 2004, 4, 437-447. 219

188 Y. L. Ren, K. Himmeldirk, X. Z. Chen, Synthesis and structure-activity relationship study of antidiabetic penta-O-galloyl-D-glucopyranose and its analogues, J. Med. Chem. 2006, 49, 2829-2837.

189 P. Arapitsas, S. Menichetti, F. F. Vincieri, A. Romani, Hydrolyzable tannins with the hexahydroxydiphenoyl unit and the m-depsidic link: HPLC-DAD-MS identification and model synthesis, J. Agric. Food Chem. 2007, 55, 48-55.

190 K. Dodo, T. Minato, T. Noguchi-Yachide, M. Suganuma, Y. Hashimoto, Antiproliferative and apoptosis-inducing activities of alkyl gallate and gallamide derivatives related to (-)-epigallocatechin gallate, Bioorg. Med. Chem. 2008, 16, 7975-7982.

191 B. Neises, W. Steglich, 4-dialkylaminopyridines as acylation catalysts .5. Simple method for esterification of carboxylic-acids, Angew. Chem. Int. Ed. 1978, 17, 522-524.

192 R. Connor, H. Adkins, Hydrogenolysis of oxygenated organic compounds., J. Am. Chem. Soc. 1932, 54, 4678-4690.

193 C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Del. Rev. 1997, 23, 3-25.

194 C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Del. Rev. 2001, 46, 3-26.

195 R. M. Czerwinski, T. K. Harris, W. H. Johnson, P. M. Legler, J. T. Stivers, A. S. Mildvan, C. P. Whitman, Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pK(a) values of active site residues and on the pH dependence of catalysis, Biochem. 1999, 38, 12358-12366.

196 L. Konishiimamura, D. H. Kim, K. Kobashi, Relationship between substrate activity and pka value of phenols on sulfotransferase from eubacterium a-44, Biochem. Int. 1992, 28, 725-734.

197 W. L. Mock, J. T. Tsay, pK values for active-site residues of carboxypeptidase-A, J. Biol. Chem. 1988, 263, 8635-8641.

198 J. T. Stivers, C. Abeygunawardana, A. S. Mildvan, G. Hajipour, C. P. Whitman, 4-Oxalocrotonate tautomerase: pH dependence of catalysis and pK(a) values of active site residues, Biochem. 1996, 35, 814-823.

199 Epik, 2.1107 ed., Schrodinger, LLC, New York, 2010. 220

200 R. A. Gardner, R. Kinkade, C. J. Wang, O. Phanstiel, Total synthesis of petrobactin and its homologues as potential growth stimuli for Marinobacter hydrocarbonoclasticus, an oil-degrading bacteria, J. Org. Chem. 2004, 69, 3530- 3537.

201 W. L. Wang, S. C. Chai, Q. Z. Ye, Synthesis and structure-function analysis of Fe(II)-form-selective antibacterial inhibitors of Escherichia coli methionine aminopeptidase, Bioorg. Med. Chem. Lett. 2009, 19, 1080-1083.

202 S. T. Huang, I. J. Hsei, C. P. Chen, Synthesis and anticancer evaluation of bis(benzimidazoles), bis(benzoxazoles), and benzothiazoles, Bioorg. Med. Chem. 2006, 14, 6106-6119.

203 M. Cowart, R. Faghih, M. P. Curtis, G. A. Gfesser, Y. L. Bennani, L. A. Black, L. P. Pan, K. C. Marsh, J. P. Sullivan, T. A. Esbenshade, G. B. Fox, A. A. Hancock, 4-(2-[2-(2(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl)benzonitrile and related 2-aminoethylbenzofuran H-3 receptor antagonists potently enhance cognition and attention, J. Med. Chem. 2005, 48, 38-55.

204 G. Tamagnan, Y. G. Gao, V. Bakthavachalam, W. L. White, J. L. Neumeyer, An efficient synthesis of m-hydroxycocaine and m-hydroxybenzoylecgonine, two metabolites of cocaine, Tetrahedron Lett. 1995, 36, 5861-5864.

205 H. J. Bohm, D. Banner, S. Bendels, M. Kansy, B. Kuhn, K. Muller, U. Obst- Sander, M. Stahl, Fluorine in medicinal chemistry, ChemBioChem 2004, 5, 637- 643.

206 W. K. Hagmann, The many roles for fluorine in medicinal chemistry, J. Med. Chem. 2008, 51, 4359-4369.

207 C. Isanbor, D. O'Hagan, Fluorine in medicinal chemistry: A review of anti-cancer agents, J. Fluorine Chem. 2006, 127, 303-319.

208 K. L. Kirk, Fluorine in medicinal chemistry: Recent therapeutic applications of fluorinated small molecules, J. Fluorine Chem. 2006, 127, 1013-1029.

209 S. Purser, P. R. Moore, S. Swallow, V. Gouverneur, Fluorine in medicinal chemistry, Chem. Soc. Rev. 2008, 37, 320-330.

210 I. Bauerova, M. Ludwig, Substituent effects on the base-catalysed hydrolysis of phenyl esters of ortho-substituted benzoic acids, Collect. Czech. Chem. Commun. 2001, 66, 770-784.

211 I. Bauerova, M. Ludwig, Substituent effects on the base-catalysed hydrolysis of phenyl esters of para-substituted benzoic acids, Collect. Czech. Chem. Commun. 2000, 65, 1777-1790. 221

212 V. Nummert, M. Piirsalu, V. Maemets, I. Koppel, Kinetic study of hydrolysis of benzoates. Part XXV. Ortho substituent effect in alkaline hydrolysis of phenyl esters of substituted benzoic acids in water, Collect. Czech. Chem. Commun. 2006, 71, 107-128.

213 V. Nummert, M. Piirsalu, S. Vahur, O. Travnikova, I. A. Koppel, Kinetic Study of Hydrolysis of Benzoates. Part xxvii. Ortho Substituent Effect in Alkaline Hydrolysis of Phenyl Esters of Substituted Benzoic Acids in Aqueous Bu4NBr, Collect. Czech. Chem. Commun. 2009, 74, 29-42.

214 Chester T. Chmiel, F. A. Long, The hydrolysis of esters of some substituted benzoic acids in strongly acid aqueous solutions, J. Am. Chem. Soc. 1956, 78, 3326-3320.

215 H. P. Lesutis, R. Glaser, C. L. Liotta, C. A. Eckert, Acid/base-catalyzed ester hydrolysis in near-critical water, Chem. Commun. 1999, 2063-2064.

216 T. J. Broxton, N. W. Duddy, Hydrolysis of esters and amides in strongly basic solution - evidence for the intermediacy of carbanions, J. Org. Chem. 1981, 46, 1186-1191.

217 R. A. Cox, A comparison of the mechanisms of hydrolysis of benzimidates, esters, and amides in sulfuric acid media, Can. J. Chem. Rev. Can. Chim. 2005, 83, 1391-1399.

218 K. Hori, Y. Ikenaga, K. Arata, T. Takahashi, K. Kasai, Y. Noguchi, M. Sumimoto, H. Yamamoto, Theoretical study on the reaction mechanism for the hydrolysis of esters and amides under acidic conditions, Tetrahedron 2007, 63, 1264-1269.

219 Kue Jeong Yeon, Si Myung Byun, Henry J. Lee, S. H. Lee, a. H. P. Kim, Hydrolysis of esters and amides of 20R- and 20S-dihydroprednisolonic acid in rat serum and liver homogenate, Arch. Pharm. Res. 12(2), 68-72 (1989) 1989, 12, 68- 72.

220 E. Fischer, A. Speier, Darstellung der Ester, Chem. Ber. 1895, 28, 3252-3258.

221 M. R. Winkle, R. C. Ronald, Regioselective metalation reactions of some substituted (methoxymethoxy)arenes, J. Org. Chem. 1982, 47, 2101-2108.

222 Robert C. Ronald, M. C. Winkle, Regioselective metallations of (methoxymethoxy)arenes, Tetrahedron 1983, 39, 2031-2042.

223 M. G. Banwell, S. Chand, G. P. Savage, An enantioselective total synthesis of the stilbenolignan (-)-aiphanol and the determination of its absolute stereochemistry, Tetrahedron-Asymmetry 2005, 16, 1645-1654. 222

224 R. V. Connors, Z. L. Wang, M. Harrison, A. Zhang, M. Wanska, S. Hiscock, B. Fox, M. Dore, M. Labelle, A. Sudom, S. Johnstone, J. S. Liu, N. P. C. Walker, A. Chai, K. Siegler, Y. Li, P. Coward, Identification of a PPAR delta agonist with partial agonistic activity on PPAR gamma, Bioorg. Med. Chem. Lett. 2009, 19, 3550-3554.

225 V. Percec, M. Peterca, A. E. Dulcey, M. R. Imam, S. D. Hudson, S. Nummelin, P. Adelman, P. A. Heiney, Hollow spherical supramolecular dendrimers, J. Am. Chem. Soc. 2008, 130, 13079-13094.

226 V. Percec, M. Peterca, Y. Tsuda, B. M. Rosen, S. Uchida, M. R. Imam, G. Ungar, P. A. Heiney, Elucidating the structure of the pm(3)over-barn cubic phase of supramolecular dendrimers through the modification of their aliphatic to aromatic volume ratio, Chem. Eur. J. 2009, 15, 8994-9004.

227 H. R. Pfaendler, R. Weishaupt, C. Medicus, Synthesis and biological activity of novel penem sulfoxides and sulfones, Bioorg. Med. Chem. Lett. 1997, 7, 2217- 2222.

228 A. Carotti, C. Altomare, M. Catto, C. Gnerre, L. Summo, A. De Marco, S. Rose, P. Jenner, B. Testa, Lipophilicity plays a major role in modulating the inhibition of monoamine oxidase B by 7-substituted coumarins, Chem. Biodivers. 2006, 3, 134-149.

229 M. M. Khodaei, K. Bahrami, Y. Tirandaz, POCl3 as a catalytic activator for H2O2 activation in selective sulfide oxidation, Journal of Sulfur Chemistry 2009, 30, 581-584.

230 J. P. Scott, S. F. Oliver, K. M. J. Brands, S. E. Brewer, A. J. Davies, A. D. Gibb, D. Hands, S. P. Keen, F. J. Sheen, R. A. Reamer, R. D. Wilson, U. H. Dolling, Practical asymmetric synthesis of a gamma-secretase inhibitor exploiting substrate-controlled intramolecular nitrile oxide-olefin cycloaddition, J. Org. Chem. 2006, 71, 3086-3092.

231 E. A. Serebryakov, P. G. Kislitsin, V. V. Semenov, S. G. Zlotin, Selective synthesis of 1,2-benzisothiazol-3-one-1-oxide nitro derivatives, Synthesis 2001, 1659-1664.

232 G. Jeges, T. Nagy, T. Meszaros, J. Kovacs, G. Dorman, A. Kowalczyk, R. A. Goodnow, Practical Synthesis of 5-Aryl-3-alkylsulfonyl-phenol and 5-Aryl-3- arylsulfonyl-phenol Libraries, J. Comb. Chem. 2009, 11, 327-334.

233 S. G. Zlotin, P. G. Kislitsin, F. A. Kucherov, A. A. Gakh, Synthetic utilization of polynitroaromatic compounds. 4. Synthesis of nitro-free 4,6-disubstituted 3- aminobenzothiophene derivatives based on 2,4,6-trinitrobenzamide, Heterocycles 2006, 68, 1109-1119. 223

234 A. K. Ghosh, G. Gong, Total synthesis and structural revision of (+)- amphidinolide W, J. Am. Chem. Soc. 2004, 126, 3704-3705.

235 T. Katoh, O. Ohmori, Studies toward the total synthesis of Sch 202596, an antagonist of the galanin receptor subtype GalR1: synthesis of geodin, the spirocoumaranone subunit of Sch 202596, Tetrahedron Lett. 2000, 41, 465-469.

236 C. Ramesh, N. Ravindranath, B. Das, Simple, efficient, and selective deprotection of phenolic methoxymethyl ethers using silica-supported sodium hydrogen sulfate as a heterogeneous catalyst, J. Org. Chem. 2003, 68, 7101-7103.

237 Y. L. Ren, K. Himmeldirk, X. Z. Chen, Synthesis and structure-activity relationship study of antidiabetic penta-O-galloyl-D-glucopyranose and its analogues, Journal of Medicinal Chemistry 2006, 49, 2829-2837.