design, synthesis and analysis of small molecule

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DESIGN, SYNTHESIS AND ANALYSIS OF SMALL MOLECULE DESIGN, SYNTHESIS AND ANALYSIS OF SMALL MOLECULE

HETEROCYCLIC AROMATIC-BASED CXCR4 MODULATORS HETEROCYCLIC AROMATIC-BASED CXCR4 MODULATORS

Theresa D. Gaines Georgia State University

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DESIGN, SYNTHESIS AND ANALYSIS OF SMALL MOLECULE HETEROCYCLIC

AROMATIC-BASED CXCR4 MODULATORS

by

THERESA DENISE GAINES

Under the Direction of Suazette Mooring, PhD

ABSTRACT

CXCR4 is a chemokine receptor that has been linked to several disease related pathways

including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer

metastasis. The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C

chemokine ligand 12 (CXCL12) plays a key role in triggering these disease related pathways.

Various antagonists for these receptors have been synthesized and tested, but many are not useful

clinically either because of toxicity or poor pharmacokinetics. Some of the most extensive CXCR4

antagonist libraries stem from a class of compounds, p-xylyl-enediamines, which all feature a

benzene ring as the core of the compound. This work focuses on the design and synthesis of a new

class of compounds that show potential as CXCR4 antagonists by using heterocyclic aromatic

rings (2,6-pyridine, 2,5-furan, 2,5-pyrazine and 3,4-thiophene) as the core of the scaffold. After

synthesis, these analogues were probed through a variety of assays and techniques by our

collaborators in the Shim lab at Emory University including: preliminary binding assays, Matrigel

invasion assays, carrageenan mouse paw edema tests, and in silico analysis. In silico analysis also

probed 2,5-thiophene-based analogues previously synthesized. This work has produced the

beginnings of a new library of CXCR4 antagonists and has identified fifteen hit compounds that

are promising leads for further testing and modification.

INDEX WORDS: CXCR4, CXCL12, Chemokine, Chemokine receptor, Inflammation, Irritable

Bowel Disease, Rheumatoid arthritis, Cancer, Cancer metastasis, SAR, Structure activity

relationship, Heterocycle, Small molecule, Synthesis, Drug design, Antagonist,

Modulator, Pyridine, Pyrazine, Furan, Thiophene, Reductive amination, Effective

concentration, Matrigel invasion assay, Carrageenan mouse paw edema, Molecular

modeling, Docking, It1t, 3ODU.



by


A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Chemistry

in the College of Arts and Sciences

Georgia State University

2017

Copyright by

Theresa Denise Gaines

2017



by


Committee Chair: Suazette Mooring

Committee: Markus Germann

Maged Henary

Electronic Version Approved:

Office of Graduate Studies

College of Arts and Sciences

Georgia State University

July 2017

v

ACKNOWLEDGEMENTS

I want to give many thanks to everyone who has encouraged me and inspired me to achieve

all I can. Biggest thanks go to my mom, dad and sister, for believing in me and pushing me. I’m

deeply grateful for all the teachers and professors I’ve had through my schooling who invested in

me and guided me, especially Dr. Marvin Miller. I’m thankful to Brett Perkins, the Hendrickson

family, and my ISI, Teen Advisor and NAPC families for providing accountability,

companionship, advice and a loving community.

Thank you to my community of friends from Engi. You’ve encouraged me and helped me

become who I am today since I was thirteen (literally half of my life) and every step of the way

until now. Seriously, thanks for teaching me how to write and I want to give a special thanks to:

Eyad, Merdle, Kana, Kaen, Jami, Buko, Oakapea, WoF, Sushyi, Hitoko, Juushichi and Gransy.

You guys are amazing and I can’t thank you all enough.

I’m thankful to Georgia State University’s Bio-Bus program and to Dr. and Dr. Baumstark

for their mentorship and guidance. I’m grateful to the U.S. Department of Education for funding

this degree through the Graduate Assistance in Areas of National Need Program. I am grateful and

thankful for our collaborators in the Shim laboratory at Emory University for their work and

advice—especially Dr. Renren Bai. Thank you so much for all of the advice you’ve given me.

I’m thankful to my research group—especially Nikita Burrows. Thank you for a wonderful

experience over these five years and for making me a better person and colleague. And finally,

thank you, Dr. Mooring for your guidance, support and confidence over the past five years.

vi

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................. v

LIST OF FIGURES ......................................................................................................... ix

LIST OF SCHEMES ........................................................................................................ 1

LIST OF TABLES ........................................................................................................... 2

1 INTRODUCTION .................................................................................................. 4

1.1 Cancer .................................................................................................................. 4

1.1.1 Hallmarks of Cancer ....................................................................................... 6

1.2 Inflammation ....................................................................................................... 7

2 DESIGN AND SYNTHESIS OF SMALL MOLECULE HETEROCYCLIC-

BASED CXCR4 MODULATORS............................................................................................. 10

2.1 Background ........................................................................................................ 10

2.1.1 Chemokines and CXCR4 .............................................................................. 10

2.1.2 CXCR4 Mechanism of Action ...................................................................... 12

2.1.3 Drug and Disease Related Pathways of CXCR4 .......................................... 13

2.1.4 CXCR4 Antagonists and Modulators ........................................................... 18

2.2 Rationale for Design .......................................................................................... 20

2.3 Results and Discussion ...................................................................................... 26

2.3.1 Chemistry ....................................................................................................... 26

2.3.2 Biology ........................................................................................................... 29

vii

2.4 Summary of Analogues Designed and Synthesized ........................................ 43

3 DOCKING ANALYSIS ........................................................................................... 47

4 CONCLUSIONS AND FUTURE OUTLOOK ...................................................... 59

5 EXPERIMENTAL.................................................................................................... 61

5.1 Biology ................................................................................................................ 61

5.1.1 Primary Binding Affinity Screening ............................................................ 61

5.1.2 Matrigel Invasion Assay ............................................................................... 61

5.1.3 Paw Inflammation Suppression Test ........................................................... 62

5.2 Docking Studies ................................................................................................. 63

5.3 Chemistry ........................................................................................................... 63

5.3.1 General Procedure for the Synthesis of the 2,6-Pyradine Analogues (2). .. 65

5.3.2 General Procedure for the Synthesis of the 2,5-Furan Analogues (4) ....... 72

5.3.3 General Procedure for the Synthesis of the 3,4-Thiophene Analogues (8) 78

5.3.4 General Procedure for the Synthesis of the 2,5-Pyrazine Analogues (14) . 82

REFERENCES ................................................................................................................ 86

APPENDICES ................................................................................................................ 97

NMR Spectra ............................................................................................................... 97

1H NMR Spectra ....................................................................................................... 97

13C NMR Spectra .................................................................................................... 156

Mass Spectra .............................................................................................................. 215

ix

LIST OF FIGURES

Figure 1: This picture shows the six original hallmarks of cancer5. Reprinted from Cell, Volume

144, Issue 5, Hanahan, D., Weinberg, R. A. Hallmarks of cancer: the next generation, page 647,

2011, with permission from Elsevier. ............................................................................................. 7

Figure 2: Structure of CXCR4. Reprinted with permission from Wu, B.; Chien, E. Y. T.; Mol, C.

D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D.

F.; Kuhn, P.; Handel, T. M., Cherezov, V.; Stevens, R. C. Structures of the CXCR4 chemokine

GPCR with small-molecule and cyclic peptide antagonists. Science. 2010. 330, 1066-1071.

Copyright 2010 American Association for the Advancement of Science. ................................... 11

Figure 3: Structure of CXCL12. Reprinted with permission from Murphy, J. W.; Yuan, H.;

Kong, Y.; Lolis, E. J. Heterologous quaternary structure of CXCL12 and its relationship to the

CC chemokine family. Proteins: Structure, Function, and Bioinformatics. 2009. 78 (5), 1331-

1337. Copyright 2009 Wiley-Liss, Inc. ........................................................................................ 12

Figure 4: Reprinted with permission from Choi, W. T.; Duggineni, S.; Xu, Y.; Huang, Z. W.; An,

J., Drug Discovery Research Targeting the CXC Chemokine Receptor 4 (CXCR4). J Med Chem.

2012, 55 (3), 977-994. Copyright 2017 American Chemical Society. ......................................... 14

Figure 5: General structure for tetrahydroquinoline-based CXCR4 antagonists.92 ...................... 18

Figure 6: Examples of indole-based,115 cyclic pentapeptide-based,116 quinoline-based,117 and

pyrimidine-based CXCR4 antagonists.118 ..................................................................................... 19

Figure 7: General Structure for p-xylyl-enediamine-based CXCR4 antagonists. ........................ 19

Figure 8: Progression of AMD3100 from JM1498...................................................................... 21

Figure 9: Progression from AMD3100 to MSX122 ..................................................................... 22

x

Figure 10: Progression from AMD3100 to the compounds synthesized and analyzed in this

work. The 2,5-thiophene series was synthesized by Francisco Garcia. ........................................ 25

Figure 11: Reduction of inflammation observed for selected derivatives. 2i had an EC of 10 nM.

2c had an EC of 100 nM and 2r had an EC of 1000 nM............................................................... 31

Figure 12: Histological assay of compound 2i. Paw tissue sections were stained with H&E. The

whole tissue slices were scanned/digitized by NanoZoomer 2.0 HT. Software NDP.view 2 was

used to zoom in. ............................................................................................................................ 42

Figure 13: Compound 2i is shown here with the central pyridine protonated. ............................. 49

Figure 14: Protonated 1-methyl piperazine substituent in compound 4u. Pyrrolidine, morpholine

and thiomorpholine substituents also had lower docking scores when protonated. ..................... 50

Figure 15: Compound 2o is shown with pi-pi interactions with TRP94. ..................................... 51

Figure 16: Compound 2h had a low docking score and key residue interactions with TRP 94,

ASP97, and HID113 in silico; however, in vivo failed to show activity. ..................................... 51

Figure 17: Compound 15u received a high docking score of -3.782 kcal/mol, contrary to

promising scores the in vitro results in the binding and Matrigel invasion assays. ...................... 52

Figure 18: The docked pyridine compounds in AutoDock Vina. All compounds are located away

from the key residues. The CXCR4 monomer is shown here in ribbon form; however, the key

residues are highlighted as stick representations. ......................................................................... 54

Figure 19: The docked pyridine compounds shown in AutoDock in the docking gridbox. All of

the docked compounds are centered away from the key residues towards the top of the gridbox.

The CXCR4 monomer is shown here in ribbon form; however, the key residues are highlighted

as stick representations. ................................................................................................................ 55

xi

Figure 20: The docked pyridine compounds in AutoDock Vina using a biased gridbox. All

compounds are located away from the key residues. The CXCR4 monomer is shown here in

ribbon form; however, the key residues are highlighted as stick representations. ....................... 57

Figure 21: The docked pyridine compounds shown in AutoDock in the biased gridbox. All of the

docked analogues are centered away from the key residues towards the top of the gridbox. The

CXCR4 monomer is shown here in ribbon form; however, the key residues are highlighted as

stick representations. ..................................................................................................................... 57

1

LIST OF SCHEMES

Scheme 1: Synthesis of 2,6-pyridine analogues. .......................................................................... 27

Scheme 2: Synthesis of 2,5-furan analogues. ............................................................................... 27

Scheme 3: Synthesis of 3,4-thiophenedicarbaldehyde. ................................................................. 28

Scheme 4: Synthesis of 3,4-thiophene analogues. ........................................................................ 28

Scheme 5: Synthesis of 2,5-pyrazinedicarbaldehyde. ................................................................... 29

Scheme 6: Synthesis of 2,5-pyrazine analogues. .......................................................................... 30

2

LIST OF TABLES

Table 1: Ring structures and their corresponding log P values. aValues from Exploring QSAR.138

bValues from Human Metabolome Database.139-141 ...................................................................... 24

Table 2: Binding and invasion assay results for pyridine analogues synthesized. aThe invasion

assay concentration used for all compounds tested was 100nM. .................................................. 33

Table 3: Binding and invasion assay results for furan analogues synthesized. aThe invasion assay

concentration used for all compounds tested was 100nM. ........................................................... 35

Table 4: Binding and invasion assay results for the 3,4-Thiophene analogues synthesized. aThe

invasion assay concentration used for all compounds tested was 100nM. ................................... 36

Table 5: Binding and invasion assay results for pyrazine analogues synthesized. aThe invasion

assay concentration used for all compounds tested was 100nM. .................................................. 37

Table 6: 2,5-Thiophene analogues synthesized by Francisco Garcia148 which will be analyzed in

the discussion section of this work as well as in silico. ................................................................ 38

Table 7: Assay and test results for hit compounds. aThe concentration used in the invasion assay

was 100 nM. bMice were dosed used 10 mg of compound for every kg the mouse weighed in this

assay. cCompounds with a dash in the carrageenan studies have not yet been submitted. ........... 41


was 100nM. bMice were dosed used 10mg of compound for every kg the mouse weighed.

cCompounds with a dash in the carrageenan studies have not yet been tested. dCompounds

synthesized by Francisco Garcia148 which will be analyzed in the discussion section of this work

as well as in silico. ........................................................................................................................ 45

Table 9: In silico results for hit compounds suing the Schrӧdinger Small Molecule Suite. aThe

compound has two or more interactions with this residue. ........................................................... 48

3

Table 10: In silico results for select compounds using AutoDock Vina....................................... 54

Table 11: In silico results for selected compounds using a biased gridbox in AutoDock Vina. .. 56

4

1 INTRODUCTION

This work is centered around synthesizing and analyzing small molecules to disrupt the

interaction between CXCR4, a protein receptor, and CXCL12, a ligand. This interaction triggers

many pathways in the body—including cancer cell metastasis and inflammation. The precedence

in mitigating these diseases will be explained in two parts below.

1.1 Cancer

A formidable foe and wily enemy, cancer has been a troubling adversary to a many people

from all socio-economic and racial backgrounds. Researchers, doctors, grocers, artists, students,

entrepreneurs, sons and daughters—none are immune to cancer. For the year 2014, cancer was

estimated to have claimed the lives of 585,720 American citizens, which is 1.3 times the population

of Atlanta, Georgia. Furthermore, an estimated 1.6 million people were diagnosed with cancer in

2014 in the United States. According to the CDC, 7.6 million people—just over the population of

Hong Kong—worldwide die from cancer.1 As intimidating as cancer is in the world today—as the

second leading cause of death,2 cancer has been a known disease for centuries, with evidence of

tumors in the bones of mummies from 1600 BC and descriptions of tumor removals in ancient

Egyptian inscriptions.3

Cancer is a broad and multi-faceted disease stemming from numerous mutations from the

host’s DNA genome. Each mutation has the possibility of giving the cancer cell a method of

escaping the body’s immune system or an advantage in order to gain more resources from the

surrounding area to thrive. Because of this, cancer is always different from person to person, and

possibly even from tumor to tumor in a single host’s body. This makes the treatment of cancer

5

difficult—not only are there multiple mutations that can cause a tumor to thrive in a person’s body,

but breast cancer cells from two individuals might not respond to the same medicines or treatments.

Cancer occurs when healthy cells fall out of the healthy cell cycle—which includes cell

death after a certain number of divisions. Cancerous cells continue to grow and divide and do not

undergo programmed cell death.3 Mutated DNA causes these deviations from the healthy cell

cycle. In ordinary cell replication, mutations are not extremely rare; however, organisms contain

enzymes and proteins that check DNA for mutations as well as special enzymes capable of fixing

any mutated strands by replacing the mutated parts before the cell division is completed and normal

daughter cells are still formed. DNA mutation can become the devastating disease we know as

cancer when mutations slip by the body’s processes that aim to prevent the mutations during cell

division. Mutations can be triggered and caused by a number of factors. Mutated DNA can be

given from parents to a child, or environmental factors can cause mutations in a person’s DNA.3

Fortunately, cancer survival rates have increased for the past thirty years. In 1975, newly

diagnosed cancer patients had a 48.9% survival rate over a five-year. In 2010, the five-year survival

rate rose to 68.3%.4 This increased survival rate can be attributed to advances in medicine and

novel strategies to combat cancer.

X-rays were discovered in 1896 and quickly became routine in cancer treatment and

diagnosis in only three years. New technologies such as ultrasound, computed tomography,

magnetic resonance imaging and positron emission tomography have all played an important role

in cancer diagnosis and treatment—providing lower risk and non-invasive methods of analyzing

tumors. Better diagnostic tools as well as new methodologies to destroy or remove them via lasers

have improved surgery procedures. Chemotherapy agents have been synthesized and research over

the years has been effective in reducing side effects. Hormonal and immunotherapy are also paving

6

the way for potential anti-cancer vaccines and drugs that try to reprogram a cancer cells to stop

growing and undergo cell death.3 These new technologies and innovations have changed the fight

against cancer for the better. For most cancer patients, multiple treatment strategies are used to

target the cancer. This is because cancer is able to proliferate through multiple pathways.

1.1.1 Hallmarks of Cancer

Robert Weinberg and Douglas Hanahan proposed the ‘Hallmarks of Cancer,’ which are the

fundamental, distinguishing characteristics that transform normal cells into cancerous cells.5

Cancer is different for every person and classification by treatment would prove to be difficult.

Cancers can grow in different parts of the body and sometimes can metastasize to travel to other

parts of the body. The hallmarks are defined as ‘acquired functional capabilities that allow cancer

cells to survive, proliferate and disseminate.’ Cancerous cells can acquire these hallmarks in

different ways at different times and in different orders. The original six hallmarks of cancer are:

the ability to sustain proliferative signaling, the ability to evade growth suppressors, the ability to

activate invasion and metastasis, the ability to enable replicative immortality, the ability to induce

angiogenesis or new blood vessel generation and the ability to resist cell death. Four new

characteristics have recently been added to the list. There are two abilities—the ability to

deregulate cellular energetics and the ability to avoid immune destruction. The final two have been

called ‘enabling characteristics’ which are tumor-promoting inflammation and genome instability

and mutation.5 The original six hallmarks of cancer are below in Figure 1.

7

Figure 1: This picture shows the six original hallmarks of cancer5. Reprinted from Cell, Volume

144, Issue 5, Hanahan, D., Weinberg, R. A. Hallmarks of cancer: the next generation, page 647,

2011, with permission from Elsevier.

Various cancers can possess various hallmarks, or combination of hallmarks. As hallmarks

are mutated abilities to give cancer extra functionality, a common strategy to treat cancer is to

address the various hallmarks. When undergoing chemotherapy, cancer patients are prescribed

several drugs—each often with a different function—to attack specific aspects or hallmarks of

cancer. Chemotherapy can be combined with other cancer treatment strategies such as radiation or

surgery to achieve a higher success rate of eliminating the cancer.

1.2 Inflammation

Irritable Bowel Disease (IBD) is quickly becoming a global concern; however, the

incidences of IBD vary by location. An estimated 1.2 million Americans6 have been diagnosed

8

with IBD, and between 2.5 and 3 million people in Europe. Another concerning feature is that as

nations become industrialized, incidences of IBD increase.7 Even though 0.5% of the western

world has been diagnosed with IBD,7 incidences are expected to rise exponentially over the next

ten years.8 IBD is a low mortality disease; therefore, even with a steady incidence rate, the amount

of people affected is compounded.

Higher rates of IBD lead to higher costs to treat this disease. Immunosuppressive treatment

plans can cost upwards of $25,000 annually.8 Innovation is needed to prepare for a future where

increasing members of our population need to be treated for this disease and lower the cost of said

treatments.

IBD is comprised of several different diseases—including Crohn’s disease (CD) and

ulcerative colitis (UC)—which are environmentally triggered and appear in genetically susceptible

individuals. The trigger causes an unusual immune response to the individual’s intestinal flora

resulting in gastrointestinal inflammation.9-10 Individuals with IBD have exhibited altered

chemokines and receptors in their epithelial cells, which could disrupt the body’s recognition of

their gut bacteria. Not only are individuals with IBD, unable to recognize enteric microbiota, but

it is also possible that they are unable to regulate the amount of pro-inflammatory chemokines that

are sent as the immune response.11-12

Rheumatoid Arthritis (RA) is an auto-inflammatory disease that affects 1.5 million people

in the United States with about 5-500 new diagnoses per 100,000 people each year.13-14 RA

symptoms usually appear in women between the ages of 40 and 50 and a bit later for men.15 Men

and women from diverse ethnicities and races can all suffer from RA.16 Even though men and

women can both be diagnosed with RA, three times as many women are living with RA than

men.17

9

There is no one test to diagnose RA as the disease seems to both have a genetic component

and environmental triggers.14 Because of this, RA is diagnosed by the symptoms displayed.18 RA

is characterized as inflammation in the joints caused by the immune system attacking the joint

tissue. The inflammatory response causes the synovium to become thick, and leads to swelling of

the joints. As this continues, the inflamed synovium degrades the cartilage and bone and weakens

the surrounding muscles, tendons and ligaments.18 This inflammatory response occurs in the joints

primarily; however, organs can be inflamed as a symptom of RA.19 There is no cure for RA,

however, management and treatment are more successful when it is treated early and

aggressively.20

10

2 DESIGN AND SYNTHESIS OF SMALL MOLECULE HETEROCYCLIC


2.1 Background

CXCR4 is a chemokine receptor that has been linked to various disease pathways

including: HIV-1 proliferation, autoimmune disorders, inflammatory disease and cancer

metastasis.21-22 The interaction of the C-X-C chemokine receptor type 4 (CXCR4) with C-X-C

chemokine ligand 12 (CXCL12) plays an important role in cancer metastasis and inflammation.23

Various antagonists for these receptors have been synthesized and tested, but many are not useful

clinically either because of toxicity or poor pharmacokinetics.22, 24 This work focuses on the design

and synthesis of a new class of compounds that show potential as CXCR4 antagonists by using

aromatic heterocyclic rings as the core of the compound.

2.1.1 Chemokines and CXCR4

Cytokines are small proteins (5-20 kDa) involved cell signaling. A large variety of cells

(endothelial cells, macrophages, T cells, B cells) produce these proteins for different functional

purposes. Chemokines are cytokines that chemoattract cells to areas of inflammation by binding

to G protein coupled receptors (GPCRs), chemokine receptors, and creating a chemical gradient.25-

27 Chemokines can belong to four different groups, C, CC, CXC, and CX3C. They are named

based on the location of the first two cystine residues on the N-terminus.28

11

Figure 2: Structure of CXCR4. Reprinted with permission from Wu, B.; Chien, E. Y. T.; Mol, C.

D.; Fenalti, G.; Liu, W.; Katritch, V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. F.;

Kuhn, P.; Handel, T. M., Cherezov, V.; Stevens, R. C. Structures of the CXCR4 chemokine GPCR

with small-molecule and cyclic peptide antagonists. Science. 2010. 330, 1066-1071. Copyright

2010 American Association for the Advancement of Science.

Most chemokines can bind to more than one chemokine receptor and receptors to more

than one ligand. These chemokines are referred to as promiscuous; however, six of the eighteen

known chemokine receptors bind to a single chemokine ligand.29 CXCR4 (shown in Figure 2) is

one of these non-promiscuous receptors. CXCR4 is a seven transmembrane GCPR and has been

known by a few other names—CD184, LESTR30 and fusin.31 CXCR4 binds exclusively to the

chemokine ligand, CXCL12 (SDF-1).32-33 CXCL12 has several isoforms in the body and even

though the alpha isoform is the most prevalent,34 CXCR4 seems to bind to each isoform with

similar affinities.35

12

Figure 3: Structure of CXCL12. Reprinted with permission from Murphy, J. W.; Yuan, H.; Kong,

Y.; Lolis, E. J. Heterologous quaternary structure of CXCL12 and its relationship to the CC

chemokine family. Proteins: Structure, Function, and Bioinformatics. 2009. 78 (5), 1331-1337.

Copyright 2009 Wiley-Liss, Inc.

Because CXCR4, is involved in many drug and disease pathways like HIV-131 entry and

cancer metastasis,36 it is one of the most studied chemokine receptors.37 Through knock out mice

studies, it has been shown that CXCR4 is necessary for neural development and hematopoiesis,

organogenesis and vascularization in embryos.38-41 CXCR4 has different functions, in line with

other chemokine receptors, in adults.42 In healthy cells, the interaction between CXCR4 and

CXCL12 triggers several downstream pathways: mobilization of stem cells,43 inflammatory cells

and immune cells,44 cell survival,45 gene transcription,46 intracellular calcium modulation,47 and

cell adhesion.48

2.1.2 CXCR4 Mechanism of Action

The crystal structure of CXCR4 was first reported in 2007 with the protein bound to a small

molecule antagonist, IT1t.49 The crystal structure was incomplete, in that some amino acids in the

N terminus were missing.50 In 2008, an NMR structure of CXCR4’s N terminus in complex with

CXCL12 was reported and it was able fill in some of the missing in pieces and identify some of

13

the interactions between CXCL12 and CXCR4.51 Unfortunately, there is no crystal structure of

CXCR4 and CXCL12 bound together.

CXCL12 binds to CXCR4 in a two-step process. In the first step, the beta sheets, the 50s

loop (the series of amino acids connecting the last beta sheet to the alpha helix), and N loop (the

series of amino acids connecting the N terminus to the first beta sheet) of CXCL12 interact with

the extracellular region on the top of CXCR4.52 The N terminus of CXCR4 then interacts with the

RFFESH loop (residues 12-17) of CXCL12.51, 53 It has been suggested that this first step causes a

conformational change in CXCR4 which exposes the binding pocket in the helices.54-55 This allows

the N terminus of CXCL12 to interact with the residues in the binding pocket near the top of the

helices of CXCR4.53 The crystal structure of CXCR4 reveals that the binding pocket is larger and

closer to the extracellular surface compared to other GCPRs.50

Because of this two-step process, small molecule antagonists may not completely block the

interactions between CXCR4 and CXCL12. Since the CXCR4/CXCL12 interaction is vital for

other necessary physiological processes, and ideal antagonist would block some functionality but

not all function. Particularly, small molecules that block the N terminus of CXCL12 from binding

in the second step would not block interactions in the first step.56 Key residues involved in the

second binding site influence signaling and have been identified as ASP97 (ECL2), ASP187

(TMII), and GLU288 (TMVII).57-58 ASP171, ASP 87 and ASP262 have also been suggested to be

important binding sites for signaling.59

2.1.3 Drug and Disease Related Pathways of CXCR4

CXCR4 is a seven transmembrane GPCR, which is embedded in the cell membrane and

interacts with other proteins from the cell surface.60-62 This receptor was first discovered as a co-

14

receptor for HIV-1 entry and after more studies were conducted, the other functional

responsibilities of CXCR4 were uncovered.61-64 CXCR4 selectively binds to the chemokine ligand,

CXCL1265 and this interaction plays a very important role in the body as knock out mice for both

of these genes do not survive due to a number of defects in the embryo.61-62 The interaction between

CXCR4 and CXCL12 triggers downstream pathways that induce inflammation; therefore, over

expression of CXCR4 has also been linked to inflammatory diseases such as allergic asthma,66

IBD,67 systemic lupus erythematosus (SLE), atherosclerosis68 and rheumatoid arthritis.69 Lastly,

the CXCR4/CXCL12 promotes cancer cell metastasis.70

Figure 4: Reprinted with permission from Choi, W. T.; Duggineni, S.; Xu, Y.; Huang, Z. W.; An,

J., Drug Discovery Research Targeting the CXC Chemokine Receptor 4 (CXCR4). J Med Chem.

2012, 55 (3), 977-994. Copyright 2017 American Chemical Society.

15

Since the CXCR4-CXCL12 axis plays such an important role in these disease related

pathways, blocking this interaction has become a leading strategy in an attempt to reduce the

progression of cancer and other inflammatory conditions.71 Several small molecule antagonists

have had success in blocking this interaction and have been shown to inhibit cancer metastasis,

HIV-1 entry and have anti-inflammatory activity.72-75

2.1.3.1 CXCR4 and Metastasis

Migration of cells is normal in the human body.76 The immune system is comprised of cells

that migrate through the body in order to facilitate healing in infected areas and routine repair and

upkeep inside the body.76-78 This migration inside the body is made possible by two groups of

proteins. The first group—chemokines—is comprised of small proteins, which interact with the

second group of proteins called chemokine receptors.22 In particular, the interaction between the

chemokine ligand, CXCL12, and the chemokine receptor, CXCR4, plays an important role in

triggering pathways to facilitate physiological functions.22, 62, 79 The interaction between CXCR4

and CXCL12 orients hematopoietic stem cells (HSCs), nonhematopoietic tissue-committed stem

cells (TCSCs), pre-B lymphocytes, t lymphocytes and embryonic stem cells (ESCs) to their

appropriate sites in the body.76-77, 80-81

Metastatic cells break from the primary tumor and travel through the body and create

tumors in other parts of the body.76, 82 Metastatic cancer cells take advantage of the

CXCR4/CXCL12 signaling interaction in order to migrate through the body in the same way that

stem cells in the body migrate.70 Many tumors develop hypoxic conditions as it grows due to

changes in cell vasculature.83 HIF-1 is then activated because of the hypoxia and can promote the

16

expression of CXCR4.84-86 It’s also possible that other growth factors contribute to upregulation

of CXCR4 because of the increased surface area of the tumor.37

Over expression of CXCR4 in cancer cells can lead to production of metalloproteases,87-90

which can degrade membranes, enabling the cells to enter into the blood stream.91 Once

circulation, these cells migrate to CXCL12 rich sites in the body—i.e. lymph nodes, lung, liver

and bone marrow.22, 77, 79-80, 92 These cells migrate through chemotaxis, which is a system that pilots

cells through the body.34 CXCL12, that is secreted by cells diffuses away from their source and

this creates a gradient. This gradient extends from the CXCL12 rich sites and becomes more dilute

as distance from the source increases.34 The CXCR4-rich cancer cells will then be guided through

the gradient to the sites that are CXCL12-rich.93 CXCR4 overexpression triggers integrin activity,

enabling the cancerous cells to adhere to the surrounding tissue once they migrate.94-96

Stage IV cancers—metastatic cancers97—usually yield a poor prognosis compared to other

stages98 and CXCR4 expression on a tumor is used to determine the prognosis for many types of

cancers including leukemia, breast cancer, prostate cancer, lung cancer and ovarian cancer.60, 62, 76,

82, 92 As seventy five percent of all cancers over express CXCR4, it is strategic to use CXCR4

expression as marker in cancer diagnosis and prognosis.60

Tissue invasion and metastasis is one of the hallmarks of cancer outlined by Weinberg and

Hanahan.5 Slowing down metastatic cancer could help boost cancer survival rates and keep tumors

localized for other treatments medicines to work more effectively. Targeting the CXCR4/CXCL12

axis to stop metastasis is an alluring tactic because the CXCR4 proteins that are expressed are less

likely to mutate. The CXCR4/CXCL12 interaction is specific; therefore, if the CXCR4 was

changed as response to a drug preventing it from migrating, it would likely no longer bind to

17

CXCL12, and still would not be able to migrate. And since the CXCL12 is produced by healthy

cells, the risk of mutation of the receptor while retaining activity is low.

2.1.3.2 CXCR4 and Inflammation

CXCR4 and CXCL12 are expressed in healthy intestinal epithelial cells (IEC).99-101 Both

CXCR4 and CXCL12 are needed for necessary physiological functions including electrolyte

secretion,102 migration, and upkeep of the epithelial mucosal membrane.103 CXCR4 and CXCL12

expression, however, are both upregulated in the IEC’s of patients with IBD.67, 104 The presence of

extra CXCL12 chemoattracts CD45RO+ T cells, which are rich with CXCR4. This interaction

triggers inflammation in the intestinal membrane and disrupts the intestinal membrane’s

homeostasis.104 It is possible that blocking the CXCR4, CXCL12 interaction can help mitigate

intestinal inflammation for patients with IBD.

In patients with Rheumatoid arthritis, CD+ T cells, neutrophils and macrophages gather

into the synovial tissue in joints and damage surrounding tissue.105-106 Several studies have

suggested that the overexpression of CXCR4 in the synovial tissue, recruits the T-cells to gather

into those areas.72 Furthermore, treatment of RA in mice with CXCR4 antagonists like

AMD3100107 and TN14003,108 a peptide antagonist, yield a reduction in inflammation.

Furthermore, treatment of RA with low-level laser irradiation (LLLI)109 reduces inflammation by

decreasing the expression of CXCR4 in the affected area. Overexpression of CXCR4 has also been

documented in the monocytes, neutrophils and B cells of patients with lupus.110 Additionally,

treatment in mouse models with CXCR4 antagonists have shown reduction of symptoms.111 As

CXCR4 is a key factor in the inflammatory response and as existing small molecule antagonists

18

have shown to be effective in mitigating symptoms of inflammatory diseases, this sets a

precedence for the creation of new antagonists.

2.1.4 CXCR4 Antagonists and Modulators

Since the CXCR4-CXCL12 interaction plays such an important role in these disease related

pathways, blocking this interaction has become a leading strategy in an attempt to mitigate these

pathways or even selectively accelerate normal physiological pathways.22 Several small molecule

antagonists have had success in blocking this interaction and have been shown to inhibit cancer

growth, inhibit cancer metastasis and have anti-RA activity.23, 112-114

Many compounds that have shown potential as CXCR4 antagonists and share the common

central benzene ring. This class of compounds is called p-xylyl-enediamines. There are several

other categories of CXCR4 antagonists, shown in Figures 5, 6 and 7, including: cyclic

pentapeptide-based antagonists, indole based antagonists, quinoline-based antagonists,

pyrimidine-based antagonists and tetrahydroquinoline-based antagonists;92 however, this work

focuses only on the p-xylyl-enediamines.

Figure 5: General structure for tetrahydroquinoline-based CXCR4 antagonists.92

19

Figure 6: Examples of indole-based,115 cyclic pentapeptide-based,116 quinoline-based,117 and

pyrimidine-based CXCR4 antagonists.118

Figure 7: General Structure for p-xylyl-enediamine-based CXCR4 antagonists.

2.1.4.1 AMD3100 and p-xylyl-enediamines

One of the first small molecule inhibitors of CXCR4 to show promise was AMD3100.64

The precursor to this compound was discovered as an impurity that showed anti-HIV activity by

blocking CXCR4, which is one of the receptors HIV-1 uses to enter the cell. This impurity was

composed of two cyclam rings connected by an aliphatic chain. Replacing the aliphatic linker with

a benzene ring increased the potency of the compound—resulting in AMD3100 (Figure 8).119

Other non-aromatic ring derivatives of AMD3100 were synthesized and tested for activity, but

only the compounds featuring benzene rings were active.120

20

AMD3100 was the first FDA approved CXCR4 antagonist; however, even though the

initial purpose for this compound was to block HIV-1 entry into the cells, this drug was not

approved for this purpose.121 In addition to poor oral bioavailability,122-123 AMD3100 is

cardiotoxic.122, 124 The FDA approved AMD3100 for limited use in patients with multiple myeloma

in order to mobilize and harvest stem cells.119, 125-126

It was speculated that the toxicity and poor oral bioavailability of AMD3100 stemmed from

the bicyclam rings. To verify this, new analogues were designed and synthesized in which the

bicyclams rings were replaced. These studies led to the synthesis of a potent p-xylyl-enediamines

derivate, WZ811 (Figure 9).127 There are several other categories of CXCR4 antagonists

including: cyclic pentapeptide-based antagonists, indole based antagonists, and

tetrahydroquinoline-based antagonists.92 Of these classes, several modulators are in clinical

trials,92 where a majority of them are peptide based.128 Many of the p-xylyl-enediamine compounds

have shown CXCR4 activity but very few have gone on to clinical trials primarily due to toxicity

or bioavailability issues.92 Various substituents and side chains have been analyzed in the p-xylyl-

enediamine class of compounds and have been screened for CXCR4 activity. The cyclams and

bicyclams are among the most studied in this group;127 however, there has not been robust

structure-activity relationship (SAR) studies in which the central phenyl ring has been altered to

another aromatic ring.

2.2 Rationale for Design

AMD3100 and other p-xylyl-enediamines are structurally specific drugs, drugs that

interact at a specific site. Changes to the structure of a lead compound can affect the biological

activity of such a type of drug.129 Using this information, it is possible to map out the relation

21

between structure of a compound and activity of a compound by making small changes to a lead

compound—called Structure Activity Relationship (SAR)—and draw conclusions about the active

site where the compound is interacting.

Figure 8: Progression of AMD3100 from JM1498

It was this process that lead to the development of AMD3100 from JM1498 (Figure 8).

JM1657 was discovered as an HIV inhibitor through screening several commercially available

cyclam rings.119 JM1657 was found to be an impurity from synthesizing JM1498 and was then

characterized.119 A series of small modifications were made to the linker between the two cyclam

rings and the lead out of the compound was a benzene ring, connected to the cyclam rings at the 1

and 4 position by a methylene, JM2987 (also known as AMD3100).119

AMD3100 was the first CXCR4 antagonist to become FDA approved;121 however, it was

for one time use to treat multiple myeloma—not HIV. The compound was cardio-toxic.122, 124

AMD3100 was still a good lead to branch off for more SAR studies to mitigate these negative

effects. This gave birth to the class of CXCR4 antagonists called p-xylyl-enediamines. The central

benzene ring was preserved, and at the 1 and 4 positions, different substituents were used to replace

the cyclam rings.92, 127 The active compounds in this class often have benzene-based, pyridine-

based or pyrimidine-based side chains replacing one or more of the cyclam rings.92 Two

forerunners out of this class of compound (Figure 9), were WZ811 (where the cyclam rings have

22

been replaced with a 2-aminopyridinyl group)130 and MSX122 (where the cyclam rings have been

replaced with a 2-aminopyrimidinyl group).131

Figure 9: Progression from AMD3100 to MSX122

The p-xylyl-enediamine compounds which removed the cyclam rings have eliminated the

toxicity issue due to metal chelation;130 however, only MSX-122 has shown promising oral

bioavailability and has made it to clinical trials but it suspended for undisclosed reasons in 2008.92

Many of the other analogues, including WZ811 are not orally bioavailable and have a short half-

life in the body.120

In designing a new class of compounds using WZ811 and MSX122 as leads to make a

more orally bioavailable analogue, the qualities that make a compound orally bioavailable must

be examined. Lipinski’s rule of five132-134 states that compounds with two or more of the following

traits are likely to be poorly orally bioavailable: molecular weights over 500 g/mol, more than five

hydrogen bond donors, more than ten hydrogen bond acceptors and a log P value greater than 5.

Naturally, there are exceptions to this rule and more detailed parameters have been outlined to

account for the exceptions by Ghose et al. Ghose et al. suggests that a log P value between -0.4

and 5.6 and a molecular weight between 180 g/mol and 500 g/mol increase drug-likeness.135 When

examining WZ811 and MSX122 by each of these parameters, they fall well within the parameters

of rules of five. Both leads have molecular weights above 180 g/mol and below 500 g/mol,

23

Hydrogen bond donors are defined as the sum of hydroxyl and amino groups.129 Both leads

have two each and are again, well within Lipinski’s range. Hydrogen bond acceptors are defined

as the sum nitrogen and oxygen atoms.129 WZ811 and MSX122 have four and six, respectively.

The last parameter is a log P value between -0.4 and 5.6 as modified by Ghose et al.135 Log P is

the partition coefficient which is a measure how lipophilic a molecule is and is calculated by

talking the log of the solubility of the molecule in 1-octanol over the solubility of the compound

in water.129 For WZ811 and MSX122, the calculated log P values, using Molinspiration, were 2.65

and 1.65 respectively.136

Since both compounds fall within the range of these rules, it is possible that the lower log

P for MSX122 contributes to its oral bioavailability. In the structure of the p-xylyl-enediamines,

the common motif is the central benzene ring. In previous studies, benzene ring has been altered

to aliphatic rings130 and aliphatic chains137 and both resulted in a loss of activity; however, a 2,6-

pyridine analogue and a 3,5-pyridine analogue based on AMD3100 were synthesized. They were

not as active as AMD3100, but they were still active.137 Pyridine and other heterocyclic aromatic

rings such as pyrazine, furan, and thiophene have lower log P values than benzene as shown in the

Table 1 below.138-141

Ring Structure Log P

Benzene

2.13a

Pyridine

0.65a

Pyrazine

-0.26b

24

Furan

1.34a

Thiophene

1.81a

Table 1: Ring structures and their corresponding log P values. aValues from Exploring QSAR.138 bValues from Human Metabolome Database.139-141

Analogues that change the linker from benzene to other heterocyclic aromatic rings

following the p-xylyl-enediamine motif may retain activity while lowering the log P value. It is

these such analogues that were synthesized in this work (Figure 10). The potential of lowering the

log P value combined with the frequent use of pyridines,142 thiophenes,143 and furans144 and

pyrazines145 in medicine, set a precedence for this work. This work will focus particularly on anti-

metastatic activity and anti-inflammatory activity of these analogues since HIV-1 binds differently

than CXCL12,146 and the analogues synthesized are generally too small to block HIV-1 as a co-

receptor.

25

Figure 10: Progression from AMD3100 to the compounds synthesized and analyzed in this

work. The 2,5-thiophene series was synthesized by Francisco Garcia.

26

2.3 Results and Discussion

2.3.1 Chemistry

2.3.1.1 2,6-Pyridine analogues

The 2,6-pyridine analogues (2a-x) were synthesized via a reductive amination reaction

between 2,6-pyridinedicarbaldehyde (1) and a substituted amine.

27

Scheme 1: Synthesis of 2,6-pyridine analogues.

2.3.1.2 2,5-Furan analogues

The 2,5-furan analogues (4a-u) were synthesized via a reductive amination reaction

between 2,5-furandicarbaldehyde (3) and a substituted amine.

Scheme 2: Synthesis of 2,5-furan analogues.

28

2.3.1.3 3,4-Thiophene analogues

Synthesized by reducing the 3,4-thiophenedicarboxyllic acid (5) to an alcohol using

diisobutylaluminum hydride. The resulting dialcohol (6) was quenched and isolated before it was

oxidized over manganese dioxide to give the dicarbaldehyde product (7).

Scheme 3: Synthesis of 3,4-thiophenedicarbaldehyde.

The 3,4-thiophene analogues (8a-j) were synthesized via a reductive amination reaction

between 3,4-thiophenedicarbaldehyde (7) and a substituted amine (Scheme 4).

Scheme 4: Synthesis of 3,4-thiophene analogues.

29

In synthesizing the 3,4-thiophene analogues, the bis(amino) product is not the major

product. In this reductive amination, when one of the aldehydes reacts with the amine, the same

amine will undergo a second, intramolecular, reductive amination leading to compound 8” shown

above in Scheme 4. This compound is favorable because of the formation of the five-membered

ring. This percent yield of the desired analogue, compound 8, was increased by breaking up the

reaction into a two-step process where the imine is isolated, and then reduced; however, the

cyclized compound is still the major product.

.

2.3.1.4 2,5-Pyrazine analogues

The 2,5-pyrazinedicarbaldehyde starting material was synthesized through a series of

reactions starting from 2,5-dimethylpyrazine. These reactions are shown below in Scheme 5. The

nitrogens on the pyrazine are oxidized under mCPBA. The resulting oxidized compound is

refluxed in acetic anhydride to encourage a substitution of the anhydride followed by a

rearrangement to the methyl group. The resulting compound (11) is then stirred in sodium

hydroxide to yield the dialcohol product (12). This compound was then oxidized using manganese

dioxide to yield the starting aldehyde.

Scheme 5: Synthesis of 2,5-pyrazinedicarbaldehyde.

30

The 2,5-pyrazine analogues (14a-d) were synthesized via a reductive amination reaction

between 2,5-prazinedicarbaldehyde (13) and a substituted amine (Scheme 6).

Scheme 6: Synthesis of 2,5-pyrazine analogues.

2.3.2 Biology

All biological assays were completed by our collaborators at Emory University in Dr.

Hyunsuk Shim’s laboratory at the Winship Cancer Institute.

2.3.2.1 Binding Assay

The derivatives were tested in a semi-quantitative binding affinity assay. It is important to

emphasize that this assay is used as a preliminary screen for potential CXCR4 antagonists that will

warrant further testing. The MDA-MB-231 breast cancer cells are incubated with the compounds

at 1 nM, 10 nM, 100 nM and 1000 nM concentrations. Next, the cells are incubated with a

biotinylated peptide, TN14003 (a known CXCR4 inhibitor), followed by streptavidin-rhodamine.

The fluorescence of the cells is then measured to obtain effective concentration (EC) is obtained.

31

EC is the lowest concentration of the compound where there is a significant reduction in

fluorescence observed compared to the positive control. All compounds synthesized were screened

using this binding assay. Compounds that scored at 100 nM and below were then subjected to the

Matrigel invasion assay. Figure 11 below shows examples of what the fluorescence looks like at

different effective concentrations.

Figure 11: Reduction of inflammation observed for selected derivatives. 2i had an EC of 10 nM.

2c had an EC of 100 nM and 2r had an EC of 1000 nM.

2.3.2.2 Matrigel Invasion Assay

The Matrigel invasion assay is used as a functional assay to probe if the synthesized

analogues can block CXCR4/CXCR12 mediated chemotaxis and invasion. This assay uses a

special two chambered apparatus. MDA-MB-231 cells that have been incubated in 100 nM

concentrations of the analogues are placed in the top chamber and CXCL12 is placed in the bottom

32

chamber as a chemoattractant. The partition between the two chambers is a Matrigel matrix that

the MDA-MB-231 cells can pass through. The measurement gained in this assay is a percentage

of inhibition of chemotaxis. When the assay is complete, the number of cells that migrated through

the matrix is counted. The percent inhibition is the percentage of cells that were prevented from

migrating compared to the negative control. The stronger the inhibitor, the fewer cells that pass

through the membrane.

Only the compounds that showed promise in the binding assay (EC ≤ 100 nm) were tested

in the Matrigel invasion assay. The two compounds used as benchmarks were AMD3100 and

WZ811. An invasion inhibition above 35% was favorable. The results for this assay have been

consolidated with the results from the binding assay for each class of compound in Tables 2

through 6, where AMD3100 and WZ811 were used as benchmarks for comparison.

Compd R Group EC (nM) Invasiona Compd R Group EC

(nM) Invasiona

2a aniline 100 <1% 2m 4-Cl aniline 100 21%

2b 2-Me aniline >1000 -- 2n 2-OMe aniline 1000 <1%

2c 3-Me aniline 100 60% 2o 3-OMe aniline 100 37%

2d 4-Me aniline 1000 -- 2p 4-OMe aniline 100 5%

2e 2-Et aniline 1 59% 2q 2-CF3 aniline >1000 --

2f 3-Et aniline 1000 -- 2r 3-CF3 aniline 1000 15%

2g 4-Et aniline 1 64% 2s 4-CF3 aniline 1 52%

33

2h 2-F aniline 1000 -- 2t 4-SMe aniline >1000 --

2i 3-F aniline 10 50% 2u 3-NO2 aniline >1000 --

2j 4-F aniline

100

5% 2v 4-NO2 aniline >1000 --

2k 2-Cl aniline >1000 -- 2w Morpholine 10 63%

2l 3-Cl aniline 100 34% 2x Pyrrolidine 1 58%

AMD3100131,

147 --- 1000 62%

WZ811127 --- 10 90%

Table 2: Binding and invasion assay results for pyridine analogues synthesized. aThe invasion

assay concentration used for all compounds tested was 100nM.

In the pyridine analogues (Table 2), eight performed well in both assays. Six of these eight

analogues contained aniline-based side chains and the other two were the morpholino analogue

(2w), and the pyrrolidinyl analougue (2x) which had binding assay resutls of 10 nM and 1 nM

respectively, and invasion assay inhibition of 63% and 58% respectively. Out of these compounds,

it seems that analogues with electron withdrawing groups as side chains are not favoured; the nitro

comounds did not have activity and the chloro compounds did not have favourable activity. The

exception is in the trifluoromethyl family, where none of the analogues were active except for the

4-CF3 anilino analogue (2s); where the binding assay concentration was 1 nM and the invasion

inhibition was 52%. Perhaps the CF3 groups in the para position has a favourable interaction in the

pocket that wasn’t favourable for the 4-Me aniline (2d)—as they would take up nearly the same

amount of space. In silico analyses would be required to gain insight into this.

The 3-Fluoro aniline analogue (2i) showed favourable activity with a binding of 10 nM

and an invasion inhhibition of 50%. Halogens are weakly electron withdrawing and have lone pairs

that may be used for donation. This could be another reason why the chloro anilino analogues had

34

some activity but none favourable. This could also be why this fluoro analogue had favourable

activity. Of the methyl analogues, only the 3-Methyl aniline (2c) had faourable activity with an

effective concentration of 100 nM and an invasion inhibition of 60%. The 2-Ethyl (2e) and 4-Ethyl

(2g) aniline analogues showed promising sctivity with effective concentrations of 1 nM for both

and invasion assay inhibitions of 59% and 64% respectively. Lastly, the 3-Methoxy aniline (2o)

showed favourable activity with an effective concentration of 100 nM and an invasion inhibition

of 37%. It is interesting that this analogue had some activity were the 3-Ethyl (2f) did not. It could

be possible that the lone pairs on the oxygen are able to interact with residues or solvent better in

the active site compared to the 3-Ethyl aniline analogue. Where the ortho and para position ethyl

groups have hydrophobic interactions at those positions.

Compd R Group EC

(nM) Invasiona Compd R Group

EC

(nM) Invasiona

4a Aniline >1000 -- 4l 3-Cl aniline >1000 --

4b 2-Me aniline >1000 -- 4m 4-Cl aniline 10 48%

4c 3-Me aniline >1000 -- 4n 2-OMe aniline 100 5%

4d 4-Me aniline 100 75% 4o 3-OMe aniline >1000 --

4e 2-Et aniline 1000 -- 4p 4-OMe aniline >1000 --

4f 3-Et aniline >1000 -- 4q 2-CF3 aniline 100 24%

4g 4-Et aniline >1000 -- 4r 3-CF3 aniline >1000 --

4h 2-F aniline 10 71% 4s 4-CF3 aniline >1000 --

4i 3-F aniline >1000 -- 4t 4-SMe aniline >1000 --

35

4j 4-F aniline 100 53% 4u 1-methylpiperazine 10 82%

4k 2-Cl aniline >1000 --

AMD3100131, 147 --- 1000 62%

WZ811127 --- 10 90%

Table 3: Binding and invasion assay results for furan analogues synthesized. aThe invasion assay

concentration used for all compounds tested was 100nM.

Of the 2,5-furan analogues synthesized (Table 3), five analogues performed well. First was

the 4-Methyl aniline (4d), which had an effective concentration of 100 nM and an invasion

inhibition of 75%. The 2-Fluoro (4h) and 4-Fluro (4j) aniline derivatives had effective

concentrations of 10 and 100 nM respectively and invasion inhibition of 71% and 53%

respectively. The 4-Chloro aniline analogue (4m) had an effective concentration of 10 nM and

invasion inhibition of 48%. The 1-methylpiperazine analogue (4u) had an effective concentration

of 10 nM and inhibited 82% of metastatic cells from invading—the highest percentage of any of

the other compounds synthesized in this work.

It is interesting to note for the 2,5-furanoaniline-based compounds the only compounds

that showed activity were compounds with ortho and para substitutions. Overall, it seems that

weakly electron donating groups like methyl and weakly electron withdrawing groups like the

fluorine and chlorine had the most activity. The 2-CF3 analogue (4q) had some activity with an

effective concentration of 10 nM and an invasion inhibition of 24%; however, it is not enough for

it to pass on to the paw edema test. All other 2,5-furan based compounds had no significant activity.

36


(nM) Invasiona

8a Aniline 10 72% 8f 4-F aniline >1000 --

8b 3-Me aniline >1000 -- 8g 3-Cl aniline >1000 --

8c 4-Me aniline >1000 9% 8h 4-Cl aniline >1000 --

8d 4-Et aniline >1000 -- 8i 3-CF3 aniline >1000 --

8e 3-F aniline >1000 -- 8j 4-CF3 aniline >1000 --

AMD3100131,

147 --- 1000 62%

WZ811127 --- 10 90%

Table 4: Binding and invasion assay results for the 3,4-Thiophene analogues synthesized. aThe

invasion assay concentration used for all compounds tested was 100nM.

Of the 3,4-thiophene analogues (Table 4), only the anilino analogue (8a) showed

favourable activity with an effective concentration of 10 nM and an invasion inhibition of 72%.

All other compounds had an effective concentration over 1000 nM and did not show significant

activity.


(nM) Invasiona

14a aniline >1000 -- 14c 4-Et aniline >1000 --

14b 3-Me aniline >1000 -- 14d 4-F aniline 10 76%

AMD3100131,

147 --- 1000 62%

WZ811127 --- 10 90%

37

Table 5: Binding and invasion assay results for pyrazine analogues synthesized. aThe invasion

assay concentration used for all compounds tested was 100nM.

Of the 2,5-pyrazine analogues (Table 5), only the 4-Fluoro anilino analogue (14d)

exhibited favorable activity with an effective concentration of 10 nM and an invasion inhibition of

76%. Since only four analogues of this series have been synthesized, there is not enough data to

speculate trends.


(nM) Invasiona

15a aniline 1 68% 15n 2-OMe aniline 10 52%

15b 2-Me aniline 10 61% 15o 3-OMe aniline 1000 --

15c 3-Me aniline >1000 -- 15p 4-OMe aniline 10 84%

15d 4-Me aniline 100 49% 15q 2-CF3 aniline >1000 --

15e 2-Et aniline 1000 -- 15r 3-CF3 aniline 100 77%

15f 3-Et aniline >1000 -- 15s 4-CF3 aniline >1000 --

15g 4-Et aniline >1000 -- 15t Morpholine >1000 --

15h 2-F aniline 1000 -- 15u Thiomorpholine 1 88%

15i 3-F aniline >1000 -- 15v Piperidine 1000 --

15j 4-F aniline >1000 -- 15w 4-CF3 Benzylamine 1 97%

15k 2-Cl aniline 10 93% 15x 3-F Benzylamine >1000 --

15l 3-Cl aniline 100 51% 15y 4-Cl Benzylamine >1000 --

38

15m 4-Cl aniline 1 22%

AMD3100131,

147 --- 1000 62%

WZ811127 --- 10 90%

Table 6: 2,5-Thiophene analogues synthesized by Francisco Garcia148 which will be analyzed in

the discussion section of this work as well as in silico.

A detailed summary of the 2,5-thiophene compound’s synthesis and trends can be found

in Francisco Garcia’s thesis;148 however, the most direct and straightforward comparison to be

made is between the 2,5-thiophene analogues and the 2-5-furan analogues. Unlike the furan

aniline-based compounds which only displayed activity for weakly electron donating and weakly

electron withdrawing groups, the thiophene derivatives seem to show more activity across the

board. None of the thiophene fluoroaniline analogues had activity, where the furan derivatives had

activity with the ortho and para fluoroaniline sidechains. The thiophene chloroaniline derivatives

also had activity where only one of the furan-based chlorine substituents had activity. It is possible

that the electronics of the 2,5-thiophene are such that those derivatives are able to interact better

with the residues in the active site compared to the 2,5-furan. It’s also possible that the size

difference between the 2,5-thiophene and the 2,5-furan contribute to the difference between the

two.

Between the 3,4-thiophene and the 2,5-thiophene, it is clear that the 3,4 positioning leads

to loss of activity. It’s possible that the 2,5-thiophene compounds are able to fit better into the

active site because of the geometry of the analogues. This would need to be explored further in

silico.

Overall, there are no strong motifs for substituents across the different core molecules.

Both the pyridine and 2,5-thiophene analogues had a variety of substituents that had activity in the

39

binding and Matrigel invasion assays with no clear pattern. It is worth noting that even though the

2,6-pyridine morpholino analogue (2w) had activity, the 2,5-thiophene morpholino analogue (15t)

did not. Instead, the 2,5-thiophene thiomorpholino derivative (15u) had activity. Compound 4u,

which also had a heterocyclic substituent, 1-methylpiperazine, follows in this trend and had

significant activity. Further probing would need to be done in synthesizing more derivatives as

well as in silico analysis to paint a bigger picture and complete this library.

2.3.2.3 In vivo carrageenan suppression

The mouse paw edema model is used as a proof-of-concept test.127, 131 If these analogues

are able to disrupt the CXCR4-CXCL12 interaction, then it also has an effect on inflammation. If

inflammation were to be induced in the presence of these compounds, a reduction in said

inflammation would be observed for potent CXCR4 antagonists. All compounds that performed

well in both the binding assay and Matrigel invasion assay were submitted for the paw edema test.

This test is also a way to gain insight into the toxicity of our compounds. None of the mice that

were given the synthesized analogues died during this test.

In this test, the hind paws of mice are inflamed using carrageenan, and one paw is treated

with a solution of the analogue (10 mg of analogue per kg), and the other is treated with a saline

solution. Both the saline solution and analogue solution are delivered via injection into the paw.

The paws are then measured at the end of the test using calipers to determine the percent reduction

in swelling. Compounds that score 100 nM or below in the binding assay and above a 35% in the

invasion assay were considered hit compounds and were submitted for further analysis in the paw

edema test. Of the compounds synthesized, fifteen qualified for the mouse paw edema test (shown

in Table 7).

40

Compd R Group EC (nM) Invasiona Carageenanb,c

2c 3-Me aniline 100 60% 20%

2e 2-Et aniline 1 59% 18%

2g 4-Et aniline 1 64% 42%

2i 3-F aniline 10 50% 20%

2o 3-OMe aniline 100 37% 20%

2s 4-CF3 aniline 1 52% 20%

2w Morpholine 10 63% 39%

2x Pyrrolidine 1 58% --

4d 4-Me aniline 100 75% 15%

4h 2-F aniline 10 71% --

4j 4-F aniline 100 53% 31%

4m 4-Cl aniline 10 48% 17%

4u 1-Methylpiperazine 10 82% --

8a Aniline 10 72% --

14d 4-F aniline 10 76% 32%

AMD3100131, 147 1000 62% --

WZ811127 10 90% 40%

41


was 100 nM. bMice were dosed used 10 mg of compound for every kg the mouse weighed in this

assay. cCompounds with a dash in the carrageenan studies have not yet been submitted.

WZ811 is used as the benchmark in this assay, where paw edema test, WZ811 reduced

inflammation by 40% compared to the control. Two compounds, the 4-Ethyl pyridine (2g) and the

morpholino pyridinee (2w) reduced inflammation on par with WZ811 at 42% and 39%

respectively. The para fluoro furan (4j) ands pyrazine derivative (14d) both follow close behind

with inflammation reduction of 31% and 32% respectively.

The tissue slice shown in Figure 12 was taken from a mouse paw that was treated with the

3-fluoro pyridine-based analogue 2i. Even though 2i did not perform the best out of the compounds

selected for the carrageenan study (20%), these tissue slices show that even though the reduction

in swelling was smaller, these compounds are still mitigating the inflammatory response in the

inflamed tissue—where the inflamed dermal papilla is returning to its normal curved and spiked

shape compared to the inflamed shape where it becomes flat and smooth.

In light of this, it is worth noting that several compounds reduced swelling in the range of

2i. The 3-methyl pyridine derivative (2c), reduced swelling by 20%. The 3-methoxy pyridine (2o)

reduced inflammation by 20%. The para trifluoromethyl pyridine analogue (2s) reduced swelling

by 20%. Since tissue slices were not taken for each of these compounds, it could be a stretch to

assume that all of them are showing this activity in the tissue; however, it is promising to know

that some compounds that don’t reduce swelling as dramatically are restoring normal tissue shape.

42

Figure 12: Histological assay of compound 2i. Paw tissue sections were stained with H&E. The

whole tissue slices were scanned/digitized by NanoZoomer 2.0 HT. Software NDP.view 2 was

used to zoom in.

The other remaining compounds, did not have a reduction in swelling above 20% compared

to the control, but some swelling reduction was observed. There is no clear relation between low

effective concentration, high invasion inhibition and inflammation reduction. Several compounds

that scored well in the preliminary assays showed lower edema reduction. 2e and 4d are prime

examples of this, it could be possible that these compounds do not completely block CXCR4 and

CXCL12 is still able to interact with the receptor to trigger inflammation. Bioavailability may be

an issue as these analogues might not stay in circulation long enough to mitigate inflammation

before they are flushed out by the body. Further studies would be needed to determine the

43

pharmacokinetic properties and discern if this is an issue. There are remaining compounds that

need to be submitted for paw edema analysis; however, the data so far lays a good foundation for

analysis and future plans for this work.

2.4 Summary of Analogues Designed and Synthesized

The compounds shown in Table 8 below show the hit compounds from this work and

Francisco Garcia’s work. Very few of the 2,5-thiophene compounds synthesized have been

submitted for the paw edema test, so it is difficult to make a complete analysis or comparison in

that regard; however out of all the five core molecules that have been synthesized, the 2,5-

thiophenes have had the most active compounds with the best invasion assay inhibitions, where

compounds 15k, 15p, 15u and 15w all inhibited upwards of 80% of had invasion assay results that

high. In the pyridine analogues, the 3-methyl (2c), 4-ethyl (2g), and the morpholino (2w) analogues

all had invasion assay results above 60% inhibition. The 2,5-furan (4d, 4h and 4u) and the 3,4-

thiophene series (8a) both had derivatives with invasion assay inhibition over 70%.

Complete inhibition of invasion—and by extension, the CXCR4/CXCL12 interaction is

undesirable as activity is necessary for normal cell function; however, in probing what about these

2,5-thiophene analogues enables it to block this interaction and prevent metastasis, future

analogues could be used as a lead to further tweak to be a suitable antagonist.

44

Compd R Group EC (nM) Invasiona Carageenanb,c

2c 3-Me aniline 100 60% 20%

2e 2-Et aniline 1 59% 18%

2g 4-Et aniline 1 64% 42%

2i 3-F aniline 10 50% 20%

2o 3-OMe aniline 100 37% 20%

2s 4-CF3 aniline 1 52% 20%

2w Morpholine 10 63% 39%

2x Pyrrolidine 1 58% --

4d 4-Me aniline 100 75% 15%

4h 2-F aniline 10 71% --

4j 4-F aniline 100 53% 31%

4m 4-Cl aniline 10 48% 17%

4u 1-Methylpiperazine 10 82% --

8a Aniline 10 72% --

14d 4-F aniline 10 76% 32%

15ad Aniline 1 68% 30%

15bd 2-Me aniline 10 61% --

15dd 4-Me aniline 100 49% --

15kd 2-Cl aniline 10 93% >5%

15ld 3-Cl aniline 100 51% --

15nd 2-OMe aniline 10 52% --

45

15pd 4-OMe aniline 10 84% --

15rd 3-CF3 aniline 100 77% --

15ud Thiomorpholine 1 88% --

15wd 4-CF3 Benzylamine 1 97% 23%

AMD3100131, 147 1000 62% --

WZ811127 10 90% 40%


was 100nM. bMice were dosed used 10mg of compound for every kg the mouse weighed. cCompounds with a dash in the carrageenan studies have not yet been tested. dCompounds

synthesized by Francisco Garcia148 which will be analyzed in the discussion section of this work

as well as in silico.

Even though the 2,5-thiophene series seem to perform well in the binding and invasion

assay, the preliminary data from the paw edema test shows that they are not reducing inflammation

in vivo as well as they prevented metastasis in vitro compared to the pyridine or furan-based

analogues. Where 15w and 15k, had invasion scores of 93% and 97% respectively, they only

reduced inflammation by 30% and 23% respectively. Compared to 2g which had invasion assay

inhibition of 64% and an inflammation reduction of 42%, it seems that performance in the invasion

assay is not a good predictor for the success of the paw edema test.

As mentioned in the rationale for design, desirable pharmacokinetic properties tend to

increase as the partition coefficient of a potential drug is lowered. Both the pyridine and furan

rings have a lower log P than thiophene. If the 2,5-thiophene analogue’s log P is not low enough,

it may be flushed out of the body too quickly to effectively reduce swelling in this assay. If this

trend continues as more and more 2,5-thiophene analogues are submitted for the paw edema test,

using substituents that lower the overall partition coefficient may yield better results.

46

Similarly, perhaps the pyridine compounds were able to do relatively well in the paw

edema test because of the lower partition coefficient. Even though they did not inhibit invasion of

more than 60% of the metastatic cells compared to the control, perhaps their results in the paw

edema assay are consistently active because of their drug-likeness due to the lowered log P. This

would need to be further evaluated in pharmacokinetic tests.

The 3,4-thiophene analogues overall were not active, with only the aniline derivative (8a)

showing activity. This seems to suggest, that the geometry of the 2,5 positioned five membered

rings may be favorable. This could be analyzed further by synthesizing 3,4-furan-based analogues

for comparison. Since the log P of pyrazine is lower than all of the other heterocyclic aromatic

rings, the pyrazine, further analogue synthesis should be conducted in order to complete this

section of the library; however, the activity of the 4-fluoro pyrazine (14d) is a promising first look.

Overall, analogues 2g, 2w, 4j,14d, and 15a are the five best compounds synthesized and

analyzed so far with favorable effective concentrations, invasion inhibition properties and with

inflammation reduction above 30% compared to the control.

47

3 DOCKING ANALYSIS

3.1 Schrӧdinger Small Molecule Suite

To gain insight as to how these analogues are interacting with the active site, in silico

methods used. In addition to the analogues synthesized in this work, analysis for 2,5-thiophene

(15a-y) derivatives synthesized by Francisco Garcia148 are also provided in this work. Preliminary

docking analysis of several of the 2,6-pyridine compounds were conducted in a previous work by

Dr. Davita Camp. Previous work has identified several key residues that can block the CXCR4-

CXCL12 interaction. Some of these residues include ASP97, GLU288, ASP187, PHE87, ASP171,

and PHE292, where the first three are required for signaling pathways triggered by CXCL12.57-58

Docking scores and key residue interactions for hit compounds are shown in Table 9.

Compd R Group EC (nM) Docking Score

(kcal/mol)

Key Residue

Interactions

2c 3-Me aniline 100 -7.314 GLU288, TRP94,

CYS186

2e 2-Et aniline 1 -5.944 HID113

2g 4-Et aniline 1 -5.716 TRP94, GLU288

2i 3-F aniline 10 -6.822 ASP97a

2o 3-OMe aniline 100 -6.187 TRP94, ASP97a

2s 4-CF3 aniline 1 -6.026 TRP94,a ASP97,

HID113, CYS186

2w Morpholine 10 -7.173 ASP97,a HID113,

TYR116

2x Pyrrolidine 1 -6.400 ASP97, TYR116,

CYS186

48

4d 4-Me aniline 100 -4.809 HID113, ARG188,

GLN200

4h 2-F aniline 10 -4.869 TRP94

4j 4-F aniline 100 -5.524 TRP94, CYS186,

ARG188, GLU288

4m 4-Cl aniline 10 -5.300 HID113, CYS186

4u 1-Methylpiperazine 10 -6.373 TRP94, TYR116,

GLU288

8a Aniline 10 -3.437 ASP97, HID113,

ARG188

14d 4-F aniline 10 -4.600 ASP97, HID113,

ARG188

15ab Aniline 1 -4.773 ASP97

15bb 2-Me aniline 10 -4.354 TRP94, HID113

15db 4-Me aniline 100 -4.506 TRP94, CYS186

15kb 2-Cl aniline 10 -4.840 TRP94, ASP97

15lb 3-Cl aniline 100 -5.975 ASP97

15nb 2-OMe aniline 10 -5.409 ASP97

15pb 4-OMe aniline 10 -4.479 GLU32, ASP97,

ARG183

15rb 3-CF3 aniline 100 -5.944 TRP94, ASP97

15ub Thiomorpholine 1 -3.782 TRP94, GLU288a

Table 9: In silico results for hit compounds suing the Schrӧdinger Small Molecule Suite. aThe

compound has two or more interactions with this residue.

Out of all the compounds docked, the hit pyridine compounds had the lowest docking

scores and out of the hit compounds the pyridine-based compounds had the best scores. The Ligand

Preparation wizard in the Schrödinger program created two different ionized states for each of the

pyridine analogues. The first, where the central ring was deprotonated, and a second where the

nitrogen in the central pyridine was protonated (Figure 13). The protonated pyridine compounds

had lower docking scores compared to the deprotonated version.

49

Figure 13: Compound 2i is shown here with the central pyridine protonated.

Previous studies suggest that small molecules with a positively charged center can interact

with better the acid residues in the active site.59 None of the other heterocyclic cores had an

ionization where the center was protonated; however, certain substituents lead to protonation of

the connecting amine. These substituents—morpholine, thiomorpholine and pyrrolidine have a

generated ionization (Figure 14) where both amines are protonated creating a positive charge in

the active site. These compounds also had a low docking score.

50

Figure 14: Protonated 1-methyl piperazine substituent in compound 4u. Pyrrolidine, morpholine

and thiomorpholine substituents also had lower docking scores when protonated.

Most of the active compounds docked have key residue interactions with ASP97, or

GLU288—two of the three residues mentioned earlier that are necessary for signaling. CYS186,

TRP94, and TYR116, when docked are programmed to freely rotate to allow the ligands to interact

via hydrogen bonding, pi-pi interactions or pi-cation interactions. Compound 2o, shown in Figure

15, displays pi-pi interactions with aromatic residues like TRP94, TYR116 and HID113.

It is worth noting that several ligands which were not active in the binding and Matrigel

assays were docked with low scores. One such example is 2h, shown in Figure 16. This compound

has a docking score of -6.237 kcal/mol and has key residue interactions with TRP94, ASP97, and

HID113. 2h was not active in any of the assays, with an effective concentration of 1000 nM. This

51

was the same, for other inactive compounds with a charged ionization state, including charged

substituents like morpholine.

Figure 15: Compound 2o is shown with pi-pi interactions with TRP94.

Figure 16: Compound 2h had a low docking score and key residue interactions with TRP 94,

ASP97, and HID113 in silico; however, in vivo failed to show activity.

52

Similarly, there were several compounds that had low effective concentrations and high

invasion inhibition in vitro, but obtained a low docking score in silico. Compound 15u (shown in

Figure 17) had an effective concentration of 1 nM and inhibited invasion of metastatic cells by

88%. In silico results yielded a docking score of -3.782 kcal/mol even though it had a key residue

interactions with GLU288. Overall, compared to the activity displayed in the assays, the 2,5-

thiophene analogues received higher docking scores compared to their in vitro activity.

Figure 17: Compound 15u received a high docking score of -3.782 kcal/mol, contrary to

promising scores the in vitro results in the binding and Matrigel invasion assays.

These discrepancies show that the docking model is not perfect; however, there are a few

themes that can be taken away and used to direct future compounds synthesized. Positive charged

ligands are favoured in the active site. All pyridine-based and most furan-based analogues with a

positive charge scored below -5.000 kcal/mol. Synthesis of these analogues with non-aromatic

cyclical amines and heterocyclic as substituents that can be protonated as secondary amines could

yield active compounds.

53

Additionally, as some analogues with aromaticity are able to have pi-pi interactions with

nearby residues, the use of substituted benzylamines, which have an extra sp3 hybridized carbon

linking the substituent to the core molecule, instead of substituted anilines may give more

flexibility to the analogue to fit better inside the pocket to make those pi-pi interactions.

3.2 AutoDock Vina

Due to the ambiguity of the results of the docking analysis using the Schrӧdinger software,

the docking analysis was partially repeated using AutoDock Vina149 on a select subset of the 2,6-

pyridine analogues to determine if the resulting data in these programs yield trends. Ten pyridine

compounds were selected; six had activity in the binding and invasion assays, the other four did

not. The docking scores are displayed below in Table 10. The key residue interactions are not

listed because the AutoDock Vina program does not have an interaction wizard.

Compd R Group EC (nM) Invasion Assay Docking Score

(kcal/mol)

2a Aniline 100 <1% -6.8

2c 3-Me aniline 100 60% -7.3

2e 2-Et aniline 1 59% -6.7

2f 3-Et aniline 1000 -- -6.6

2g 4-Et aniline 1 64% -7.1

2h 2-F aniline 1000 -- -7.2

2i 3-F aniline 100 50% -7.0

2q 2-CF3 aniline >1000 -- -6.3

54

2w Morpholine 1 63% -5.5

2x Pyrrolidine 1 58% -5.3

Table 10: In silico results for select compounds using AutoDock Vina.

Figure 18: The docked pyridine compounds in AutoDock Vina. All compounds are located away

from the key residues. The CXCR4 monomer is shown here in ribbon form; however, the key

residues are highlighted as stick representations.

The docking in this program has no correlation between the assay results compared to in

silico results. The morpholine and pyrrolidine analogues scored the highest; however, all the

other analogues consistently scored between -6.3 and -7.3. The analogues were then visualized in

AutoDock. Figure 18 shows the compounds in relation to the key residues and active site. The

docked analogues are neither interacting with the key residues nor located within the active site.

Figure 19 shows the analogues in relation to the docking gridbox.

55

Figure 19: The docked pyridine compounds shown in AutoDock in the docking gridbox. All of

the docked compounds are centered away from the key residues towards the top of the gridbox.


as stick representations.

In response to the lack of trends in the first set of in silico results, the same selected

pyridine analogues were docked using a biased gridbox. The gridbox was reduced in size and

was re-centered on the active site of the CXCR4 monomer. Docking with a biased gridbox is

investigative in nature as it may help force compounds into a favorable position in the active site

that was not seen in the first gridbox was larger at the same runtime; however, the results from

this set of docking analyses cannot replace the original results from the non-biased docking

analysis. The results of the biased docking analysis can be found in Table 11, below.

56

Compd R Group EC (nM) Invasion Assay Docking Score

(kcal/mol)

2a Aniline 100 <1% -6.8

2c 3-Me aniline 100 60% -3.6

2e 2-Et aniline 1 59% -1.7

2f 3-Et aniline 1000 -- -3.1

2g 4-Et aniline 1 64% -1.8

2h 2-F aniline 1000 -- -5.1

2i 3-F aniline 100 50% -3.8

2q 2-CF3 aniline >1000 -- -1.9

2w Morpholine 1 63% -4.3

2x Pyrrolidine 1 58% -4.4

Table 11: In silico results for selected compounds using a biased gridbox in AutoDock Vina.

The docking scores in the biased gridbox are markedly higher than in the non-biased

gridbox with docking scores ranging from -1.7 to -6.8. Unfortunately the broadness in the range

has not lead to correlation between active compounds and their assay scores. Hit compounds such

as 2e, 2g, and 2w scored -1.7, -1.8 and -4.3 respectively, where inactive compounds such as 2f,

2h, and 2q scored -3.1, -5.1 and -1.9 respectively. The visualization shown in Figure 20 and

Figure 21 show the biased docked compounds and the biased gridbox. Like the unbiased gridbox

first used, the analogues are as far away from the active site as they can be and are clustered in the

top edge of the gridbox.

57

Figure 20: The docked pyridine compounds in AutoDock Vina using a biased gridbox. All

compounds are located away from the key residues. The CXCR4 monomer is shown here in

ribbon form; however, the key residues are highlighted as stick representations.

Figure 21: The docked pyridine compounds shown in AutoDock in the biased gridbox. All of

the docked analogues are centered away from the key residues towards the top of the gridbox.


as stick representations.

58

The docking completed using AutoDock Vina has not yielded any additional information

highlighting how these heterocyclic analogues are interacting with the active site of CXCR4 using

the parameters outlined in this work Further analysis using another program or perhaps more

exhaustiveness may give more information and insight.

59

4 CONCLUSIONS AND FUTURE OUTLOOK

In this work, a small library of heterocyclic-based modulators for CXCR4 mimicking p-

xylyl-enediamines have been synthesized and analyzed in binding assays, Matrigrel invasion

assays and an in vivo paw edema test. Overall, the 2,6-pyridine-based compounds and the 2,5-

thiophene based compounds had the best results in the assays overall; however, more 2,5-thiophen

compounds need to be submitted for the paw edema test. The 3,4-thiophene analogues were largely

inactive, possibly due to the geometry. The 2,5-furan analogues had several with activity; however,

compared to the 2,5-thiophene compounds, they exhibit less activity.

There are several ways to extend this project going into the future. The 2,5-pyraizne library

needs to be completed and analyzed. For the 2,6-pyridine and 2,5-furan compounds, extending the

sidechains by using benzylamines instead of anilines may boost activity as well, potentially

enabling analogues to have pi-pi interactions with nearby residues and granting them more

flexibility in the binding pocket. In addition to benzylamine substituents, more cyclic non-aromatic

analogues need to be synthesized. In silico analysis suggests that these substituents are likely to be

positively charged in physiological conditions and will interact with acidic key residues. The

success of the pyrrolidine, morpholine, thiomorpholine and 1-methylpiperazine compounds set a

precedence to further investigate this trend.

In order to verify if the 3,4-thiophene was largely inactive because of the 3,4 positioning

of the side chains, synthesis of 3,4-furan based derivatives can provide insight into if this is true.

There are a few other core molecules that would contribute to this library as the 2,6-pyridine and

2,5-pyrazine analogues have shown activity. Modeled after the 2,6-pyridine analogues, synthesis

60

of 2,6-pyrazine analogues could possibly increase activity as pyrazine has a lower log P than

pyridine. It would also provide more information as to if the analogues are able to bind better in

the pocket with another nitrogen in the heterocyclic ring can increase activity as in silico analysis

suggests that the pyridine analogues are protonated in the active site and are able to bind differently

be because of the positioning of the cation on the ring instead of on the amine. In silico results did

not suggest protonation for the 2,5-pyrazine cores; however, 2,6-pyrazine should still be explored.

In the same vein, synthesizing 2,5-pyrdine analogues that match the 2,5-pyrazine would be a useful

addition to the library.

For existing core molecules like the 2,5-thiophene and the 2,6-pyridine, using other amines

that have shown to have exception activity in the p-xylyl-enediamine series such as the 2-

aminopyridine and 2-aminopyrimidine and halogen substituted 2-aminopyridine and 2-

aminopyrimidines. Design and synthesis of asymmetrical analogues of these core molecules could

help increase activity by designing a small molecule that can fit better into the active site.

61

5 EXPERIMENTAL

5.1 Biology

All biological assays were completed by our collaborators at Emory University in Dr.

Hyunsuk Shim’s laboratory at the Winship Cancer Institute.

5.1.1 Primary Binding Affinity Screening

The binding affinity assay is a competitive assay where approximately twenty thousand

MDA-MB-231 breast cancer cells are incubated an 8-well slide chamber for two days in 300 μL

of medium. The compounds were also incubated in separate wells at several concentrations (1, 10,

100, 1000 nM) for ten minutes at room temperature. The cells were then fixed in a chilled solution

of 4% paraformaldehyde. After the cells were rehydrated in phosphate-buffered saline (PBS), the

slides were prepared by incubating them with 0.05 μg/mL biotinylated TN14003 for thirty minutes

at room temperature. These slides were washed three times with the PBS solution and were then

incubated for thirty minutes at room temperature in streptavidin-rhodamine (1:150 dilution;

Jackson ImmunoResearch Laboratories, West Grove, PA). The slides were washed again with the

PBS solution and were mounted in an antifade mounting solution (Molecular Probes, Eugene, OR).

A Nikon Eclipse E800 microscope was used to analyze the samples.127, 150

5.1.2 Matrigel Invasion Assay

This assay was performed using a Matrigel invasion chamber (Corning Biocoat; Bedford,

MA). In the bottom chamber, a solution of CXCL12 (200ng/mL; R&D Systems, Minneapolis,

MN) was added to the apparatus. 100nm of the selected compounds (or AMD3100 as a control)

62

were added to the MDA-MB-231. The cells were then placed in the top chamber. The apparatus

was then incubated in a humidified incubator for 22 hours. The remaining cells in the top chamber

were removed using a cotton swab and the invading cells in the bottom chamber were stained

hematoxylin and eosin (H&E) and fixed with methanol. The rate of invasion was calculated by

counting the invading (stained) cells.127, 150

5.1.3 Paw Inflammation Suppression Test

In this test, C57BL/6J does (Jackson Laboratories) are subcutaneously injected with λ-

carrageenan (50 μL in 1% w/v in saline) in the right hind paw to trigger inflammation; the other

hind paw is used as the non-inflammation control. The selected analogues were prepared in 10%

DMSO and 90% of 45% (2-hydroxypropyl)-β-cyclodextrin (CD) in PBS. Doses of the analogues

were set at 10mg/kg and the dose for TN14003 was set at 300 μg/kg. The TN14003 dose was set

lower for this experiment because it was found that 300 μg/kg gave the maximum efficacy at

minimum concentration in breast cancer metastasis in an animal model. The mice were dosed 30

minutes after the carrageenan injection and then once a day following the initial dose. The mice

were sacrificed 74 hours after inflammation was induced and two hours after the last injection of

the selected analogues. The hind paws of the mice were photographed and calipers were used to

measure the thickness of the paw from front to back. To quantify the edema, the measurement

from the untreated paw was subtracted from the volume of the treated paw. The inflammation

suppression percentage was determined by comparing the analogue treated groups to the control

group. Each analogue was tested in quintuplicate using the above procedure.127, 131 Paw tissue slices

were also collected and stained with H&E. Tissue slices were scanned and digitized by

NanoZoomer 2.0 HT. The software NDP.view 2 was used to view the slices in detail.

63

5.2 Docking Studies

5.2.1 Schrodinger Small Molecule Suite

Schrodinger Small Molecule Suite

The structure of the target receptor CXCR4, PDB-ID 3ODU, was retrieved from the RCSB

Protein Data Bank.50 Docking calculations were performed by the Schrodinger suite with default

settings unless otherwise indicated. The Protein Preparation Wizard was used to prepare the

CXCR4 protein for docking. CXCR4 along with its co-crystallized ligand IT1t went through a

preprocess procedure with the additional options to fill in missing side chains and loops using

Prime and removing waters beyond 5Å from heteroatom groups. The heterostate for the co-

crystallized ligand was generated using Epik. The PROPKA feature was utilized to optimize the

hydrogen bond network at a physiological pH. After hydrogen bond optimization, water molecules

were removed with less than 3 H-bonds to non-waters. The protein was then energetically

minimized using a default constraint of 0.3Å RMSD and OPLS 2005 force field. Ligands were

prepared by the LigPrep function of the Schrodinger Suite with default parameters in the gas phase.

Epik generated possible ionization states of the ligands at physiological pH. Receptor grid

generation was performed by inputting the prepared receptor directly from the protein preparation

wizard. Docking was performed using the Extra Precision (XP) feature of the GLIDE program.

Ligands were docked flexibly in the rigid protein devoid of the IT1t ligand and were ranked based

on their GLIDE docking score.

64

5.2.2 Autodock Vina

The structure of the target receptor CXCR4, PDB-ID 3ODU, was retrieved from the RCSB

Protein Data Bank.50 Docking calculations were performed AutoDock Vina and default settings

were used unless otherwise specified. The protein in PDB form was uploaded into the program.

The B dimer of the protein was then selected and removed to create a monomer. The monomer

was then exported as a macromolecule, converting it to a PDBQT file. The gridbox was then

selected; the initial gridbox had the following parameters: center_x = 16.807, center_y = -6.232,

center_z = 69.119, size_x = 28, size_y = 28, size_z = 28, spacing = 1 angstrom. The biased gridbox

had the following parameters: center_x = 19.717, center_y = -6.232, center_z = 69.53, size_x =

22, size_y = 20, size_z = 14, spacing = 1 angstrom. Ligands were uploaded into Autodock as PDB

files. Once inputted, charges were assigned automatically. Ligand was then exported as a PDBQT

file. The PDBQT files were then coded in the configuration files labeled as ligand and receptor

with an exhaustiveness of 16. The compounds were then docked in AutoDock Vina and the

resulting log files were retained.

65

5.3 Chemistry

The 1H NMR (400 MHz) and 13C NMR (100MHz) spectra were recorded on a Bruker Ac

400 FT NMR spectrometer in deuterated chloroform (CDCl3). All chemical shifts were reported

using parts per million (ppm). Mass spectra were recorded on a JEOL spectrometer at Georgia

State University Mass Spectrometry Center. Detailed syntheses for the 2,5-Thiophene analogues

can be found in the thesis of Francisco Garcia.148

5.3.1 General Procedure for the Synthesis of the 2,6-Pyradine Analogues (2).

To a solution of pyridine-2,6-dicarbaldehyde (50 mg, 0.37 mmol) in methanol (2 mL) was

added the aniline derivative of choice (0.81 mmol) and ZnCl2 (100 mg, 0.74 mmol). The solution

was stirred for two hours at room temperature. NaBH3CN (46.5mg, 0.81 mmol) was then added

and the solution as stirred overnight. The crude product was purified by column chromatography.

2,6-Bis(anilinomethyl)pyridine, (2a) Product purified using a 1:1 Hexane/Ethyl Acetate solvent

system and was obtained in 37% yield as an off-white semi-solid. 1H NMR (400 MHz, CDCl3): δ

ppm 4.38 (s, 4H), 6.59 (d, J = 7.8 Hz, 4H), 6.65 (t, J = 7.2 Hz, 2H), 7.06 - 7.15 (m, 6 H), 7.49 (t,

J = 7.7 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 158.1, 148.0, 137.3, 129.3, 119.9, 117.7,

113.1, 49.3; HRMS: m/z [M + H]+ calcd for C19H19N3: 290.1657, found: 290.1657.

2,6-Bis(2-methylanilinomethyl)pyridine, (2b) The product was obtained in a 7:1 Hexanes/Ethyl

Acetate solvent system and was obtained as an orange oil in 21% yield. 1H NMR (400 MHz,

CDCl3): δ ppm 2.25 (s, 6H), 4.52 (s, 4H), 6.60 (d, J = 7.83 Hz, 2H), 6.68 (t, J = 7.33 Hz, 2H), 7.05

- 7.14 (m, 4H), 7.22 (d, J = 7.83 Hz, 2H), 7.60 (t, J = 7.71 Hz, 1H); 13C NMR (100 MHz, CDCl3):

δ ppm 158.02, 145.84, 137.26, 130.08, 127.13, 122.27, 119.99, 117.22, 110.14, 49.22, 17.59;

HRMS: m/z [M + H]+ calcd for C21H23N3: 318.1965, found: 318.1958.

66

2,6-Bis(3-methylanilinomethyl)pyridine, (2c) The product was purified via flash

chromatography using DCM as a solvent and was obtained as an orange oil in 50% yield. 1H NMR

(400 MHz, CDCl3): δ ppm 2.26 (s, 6 H) 4.42 (s, 4 H) 6.42 - 6.52 (m, 4 H) 6.54 (d, J=7.33 Hz, 2

H) 7.06 (t, J=7.58 Hz, 2 H) 7.17 (d, J=7.58 Hz, 2 H) 7.54 (t, J=7.58 Hz, 1 H); 13C NMR (100 MHz,

CDCl3): δ ppm 158.23, 148.06, 139.06, 137.26, 129.20, 119.86, 118.61, 113.95, 110.23, 49.30,

21.70. HRMS: m/z [M+ Z]+ calcd for C21H23N3 : 318.1965, found: 318.1965.

2,6-Bis(4-methylanilinomethyl)pyridine, (2d) The product was purified via flash

chromatography using DCM as a solvent and was obtained was a yellow-orange solid in 34%

yield. 1H NMR (400 MHz, CDCl3): δ ppm 2.24 (s, 6 H) 4.43 (s, 4 H) 6.59 (d, J=8.34 Hz, 4 H) 6.99

(d, J=8.08 Hz, 4 H) 7.19 (d, J=7.83 Hz, 2 H) 7.56 (t, J=7.71 Hz, 1 H); 13C NMR (100 MHz, CDCl3):

δ ppm 158.36, 145.70, 137.22, 129.78, 126.84, 119.85, 113.27, 49.64, 20.42. HRMS: m/z [M+ Z]+

calcd for C21H23N3 : 318.1965, found: 318.1962.

2,6-Bis(2-ethylanilinomethyl)pyridine, (2e) The product was purified in a 1:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 53% yield as a brown solid. 1H NMR (400 MHz,

CDCl3): δ ppm 1.31 (t, J = 7.45 Hz, 6H), 2.60 (q, J = 7.58 Hz, 4H), 4.52 (s, 4H), 6.61 (d, J = 7.83

Hz, 2H), 6.73 (t, J = 7.33 Hz, 2H), 7.06 - 7.14 (m, 4H), 7.18 - 7.25 (m, 2H), 7.58 (t, J = 7.71 Hz,

1H); 13C NMR (100 MHz, CDCl3): δ ppm 158.17, 145.32, 137.29, 127.89, 127.04, 119.98, 117.43,

110.50, 49.36, 23.98, 12.92. HRMS: m/z [M + H]+ calcd for C23H27N3: 346.2278, found: 346.2276.

2,6-Bis(3-ethylanilinomethyl)pyridine, (2f) The product was purified in a 3:1 Hexanes/Ethyl


CDCl3): δ ppm 1.18 - 1.24 (m, 6H), 2.56 (d, J = 6.32 Hz, 4H), 4.46 (br. s., 4H), 6.52 (d, J = 14.65

Hz, 4H), 6.59 (br. s., 2H), 7.02 - 7.15 (m, 2H), 7.21 (d, J = 4.04 Hz, 2H), 7.51 - 7.62 (m, 1H); 13C

67

NMR (100 MHz, CDCl3): δ ppm 158.15, 148.10, 145.45, 137.20, 129.19, 119.83, 117.36, 112.80,

110.34, 49.31, 29.02, 15.57; HRMS: m/z [M + H]+ calcd for C23H27N3: 346.2278, found: 346.2285.

2,6-Bis(4-ethylanilinomethyl)pyridine, (2g) The product was purified in a 7:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 11% yield as an orange oil; 1H NMR (400 MHz,

CDCl3): δ ppm 1.18 (t, J = 7.6 Hz, 6H), 2.50 - 2.57 (m, 4H), 4.41 (s, 4H), 6.59 (d, J = 8.3 Hz, 4H),

6.97 (d, J= 8.1 Hz, 2H), 7.01 (d, J = 8.3 Hz, 4H), 7.53 (t, J = 7.7 Hz, 1H); 13C NMR (100 MHz,

CDCl3): δ ppm 158.4, 146.0, 137.3, 133.6, 128.7, 119.9, 115.3, 113.3, 49.7, 28.0, 16.02. HRMS:

m/z [M + H]+ calcd for C23H27N3: 346.2283, found: 346.2291.

2,6-Bis(2-fluoroanilinomethyl)pyridine, (2h) The product was purified in a 3:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 7% yield as an orange oil. 1H NMR (400 MHz,

CDCl3): δ ppm 4.51 (br. s., 4H), 6.59 - 6.74 (m, 4H), 6.88 - 7.07 (m, 4H), 7.24 (d, J = 5.05 Hz,

2H), 7.61 (d, J = 6.06 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 157.81, 152.93, 137.39,

136.33, 124.56, 119.85, 116.96, 114.57, 112.50, 48.90; HRMS: m/z [M + H]+ calcd for

C19H17N3F2: 326.1463, found: 326.1451.

2,6-Bis(3-fluoroanilinomethyl)pyridine, (2i) Product was purified in a 3:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 62% yield as a brown semi-solid. 1H NMR (400 MHz,

CDCl3): δ ppm 4.34 (s, 4H), 6.21 - 6.40 (m, 6H), 6.95 - 7.06 (m, 2H), 7.10 (d, J = 7.8 Hz, 2H),

7.51 (t, J = 7.71 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 165.4, 162.9, 157.5, 149.8, 149.7,

137.4, 130.3,120.1, 109.0, 104.2, 104.0, 99.9, 99.6, 48.9; HRMS: m/z [M + H]+ calcd for

C19H17N3F2: 326.1469, found: 326.1462.

2,6-Bis(4-fluoroanilinomethyl)pyridine, (2j) Product purified using a 3:1 Hexanes/Ethyl Acetate

solvent system and was obtained in 34% yield as a brown semi-solid; 1H NMR (400 MHz, CDCl3):

δ ppm 4.33 (s, 4H), 6.51 (dd, J = 8.72, 4.17 Hz, 4H), 6.81 (t, J = 8.59 Hz, 4H), 7.12 (d, J = 7.58

68

Hz, 2 H), 7.52 (t, J = 7.71 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 158.0, 157.2, 154.8,

137.4, 137.2, 120.0,115.9, 115.7, 115.6, 115.5, 114.3, 113.5, 113.4, 49.9; HRMS: m/z [M + H]+

calcd for C19H17N3F2: 326.1469, found: 326.1462.

2,6-Bis(2-chloroanilinomethyl)pyridine, (2k) The product was purified in a 3:1 Hexane/Ethyl

Acetate solvent system and was obtained in 39% yield as a yellow solid. 1H NMR (400 MHz,

CDCl3): δ ppm 4.54 (d, J = 5.05 Hz, 4H), 6.61 - 6.69 (m, 4H), 7.10 (t, J = 7.45 Hz, 2H), 7.21 (d,

J = 7.58 Hz, 2H), 7.28 (d, J = 7.58 Hz, 2H), 7.61 (t, J = 7.71 Hz, 1H); 13C NMR (100 MHz,

CDCl3): δ ppm 157.64, 143.70, 137.42, 139.18, 137.80, 119.83, 119.49, 117.45, 111.63, 48.94.

HRMS: m/z [M + H]+ calcd for C19H17N3Cl2: 358.0872, found: 358.0877.

2,6-Bis(3-chloroanilinomethyl)pyridine, (2l) Product was purified in a 4:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 14% yield as a yellow semi-solid; 1H NMR (400 MHz,

CDCl3): δ ppm 4.36 (s, 4 H), 6.46 (dd, J = 8.1, 1.0 Hz, 2 H), 6.58 (s, 2 H) 6.61 (d, J = 7.8 Hz, 2

H), 7.01 (t, J = 8.0 Hz, 2 H), 7.12 (d, J = 7.6 Hz, 2 H), 7.54 (t, J = 7.71 Hz, 1 H); 13C NMR (100

MHz, CDCl3): δ ppm 157.4, 149.0, 137.5, 135.1, 130.3, 120.1, 117.5, 112.7, 111.4, 48.9. HRMS:

m/z [M + H]+ calcd for C19H17N3Cl2: 358.0854, found: 358.0864.

2,6-Bis(4-chloroanilinomethyl)pyridine, (2m) Product was purified in a 4:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 15% yield as a yellow solid; mp 116-118°C. 1H NMR

(400 MHz, CDCl3): δ ppm 4.36 (br, s, 4H), 6.51 (d, J = 8.34 Hz, 4H), 7.06 (d, J = 8.34 Hz, 4H),

7.12 (d, J = 7.6 Hz, 2H), 7.54 (t, J = 7.7 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 157.6,

146.4, 137.4, 129.1, 122.3, 120.0, 114.2, 49.2. HRMS: m/z [M + H]+ calcd for C19H17N3Cl2:

358.0872, found: 358.0864

2,6-Bis(2-methoxyanilinomethyl)pyridine, (2n) The product was purified in a 7:1 Hexane/Ethyl

Acetate solvent system and was obtained in 51% yield as a brown solid; mp 112-114°C. 1H NMR

69

(400 MHz, CDCl3): δ ppm 3.81 (s, 6 H), 4.43 (s, 4 H), 6.49 (d, J = 7.58 Hz, 2H), 6.58 - 6.64 (m,

2H), 6.70 - 6.79 (m, 4H), 7.14 (d, J = 7.8 Hz, 2H), 7.48 (t, J = 7.7 Hz, 1H); 13C NMR (100 MHz,

CDCl3): δ ppm 158.7, 147.0, 137.9, 137.3, 121.3, 119.6, 116.7, 110.3, 109.5, 55.5, 49.4. HRMS:

m/z [M + H]+ calcd for C21H23N3O2: 350.1869, found: 350.1862.

2,6-Bis(3-methoxyanilinomethyl)pyridine, (2o) The product was purified in a 3:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 16% yield as an off white semisolid. 1H NMR (400

MHz, CDCl3): δ ppm 3.76 (br. s., 6H), 4.45 (br. s., 4H), 6.23 (br. s., 2H), 6.30 (m, 4H), 7.09 (d, J

= 3.28 Hz, 2H), 7.20 (m, 2H), 7.59 (m, 1H); 13C NMR (100 MHz, CDCl3): δ ppm 160.79, 157.91,

149.30, 137.28, 130.00, 119.89, 106.18, 102.75, 99.03, 55.08, 49.19. HRMS: m/z [M + H]+ calcd

for C21H23N3O2: 350.1863, found: 350.1854.

2,6-Bis(4-methoxyanilinomethyl)pyridine, (2p) Product was purified in two silica columns. The

fist was purified in a solution of 9:1 DCM/Methanol. The compound was then further purified in

4:5 solution of Hexanes/Ethyl Acetate. Product was obtained in 30% yield as a brown solid; mp

63-65°C. 1H NMR (400 MHz, CDCl3): δ ppm 3.66 (s, 6 H), 4.33 (s, 4 H), 6.55 (d, J = 8.84 Hz, 4

H), 6.70 (d, J = 8.84 Hz, 4 H), 7.12 (d, J = 7.58 Hz, 2 H), 7.49 (t, J = 7.7 Hz, 1 H); 13C NMR (100

MHz, CDCl3): δ ppm 158.39, 152.25, 142.22, 137.20, 119.94, 114.90, 114.36, 55.79, 50.24;

HRMS: m/z [M + H]+ calcd for C21H23N3O2 : 350.1869, found: 350.1865.

2,6-Bis(2-trifluoromethylanilinomethyl)pyridine, (2q) The product was purified in a 7:1

Hexane/Ethyl Acetate solvent system and was obtained in 19% yield as an off white solid. 1H

NMR (400 MHz, CDCl3): δ ppm 4.55 (d, J = 5.05 Hz, 4H), 6.63 - 6.79 (m, 4H), 7.20 (d, J = 7.58

Hz, 2H), 7.32 (t, J = 7.71 Hz, 2H), 7.47 (d, J = 7.58 Hz, 2H), 7.61 (t, J = 7.71 Hz, 1H); 13C NMR

(100 MHz, CDCl3): δ ppm 157.32, 145.14, 137.58, 133.14, 126.72, 126.67, 119.77, 116.27,

113.66, 112.26, 48.69. HRMS: m/z [M + H]+ calcd for C21H17F6N3: 426.1401, found: 426.1399.

70

2,6-Bis(3-trifluoromethylanilinomethyl)pyridine, (2r) The product was purified in a 3:2

Hexanes/Ethyl Acetate solvent system and was obtained in 50% yield as an off white solid. 1H

NMR (400 MHz, CDCl3): δ ppm 4.46 (br. s., 4 H) 6.78 (d, J=8.08 Hz, 2 H) 6.87 (br. s., 2 H) 6.95

(d, J=7.58 Hz, 2 H) 7.19 (d, J=7.83 Hz, 2 H) 7.25 (t, J=1.00 Hz, 2 H) 7.61 (t, J=7.71 Hz, 1 H); 13C

NMR (100 MHz, CDCl3): δ ppm 157.24, 148.06, 137.51, 131.75, 129.71, 125.74, 123.03, 120.20,

116.02, 114.05, 109.23, 48.80. HRMS: m/z [M + H]+ calcd for C21H17F6N3: 426.1399, found:

426.1384.

2,6-Bis(4-trifluoromethylanilinomethyl)pyridine, (2s) The product was purified in a 2:1

Hexanes/Ethyl Acetate solvent system and was obtained in 61% yield as a light orange semi-solid.

1H NMR (400 MHz, CDCl3): δ ppm 4.49 (s, 4 H) 6.65 (d, J=8.34 Hz, 4 H) 7.20 (d, J=7.83 Hz, 2

H) 7.41 (d, J=8.34 Hz, 4 H) 7.63 (t, J=1.00 Hz, 1 H); 13C NMR (100 MHz, CDCl3): δ ppm 157.15,

150.28, 137.59, 126.68, 126.32, 123.64, 120.15, 112.21, 48.46. HRMS: m/z [M + H]+ calcd for

C21H17F6N3: 426.1399, found: 426.1381.

2,6-Bis(4-thiomethylanilinomethyl)pyridine (2t) The product was purified in a 3:1

Hexanes/Ethyl Acetate solvent system and was obtained in 14% yield as a white solid; 1H NMR

(400 MHz, CDCl3): δ ppm 2.41 (s, 6 H) 4.44 (br. s., 4 H) 6.61 (d, J=8.34 Hz, 4 H) 7.16 - 7.31 (m,

6 H) 7.59 (t, J=7.71 Hz, 1 H); 13C NMR (100 MHz, CDCl3): δ ppm 19.02, 49.12, 113.70, 119.94,

124.61, 131.34, 137.30, 146.70, 157.80. HRMS: m/z [M + H]+ calcd for C21H24N3S2: 382.1206,

found: 382.1404.

2,6-Bis(3-nitroanilinomethyl)pyridine, (2u) The product was purified using a 30:1

DCM/Methanol solvent system and was obtained in 34% yield as a yellow-orange solid; mp 147-

149°C. 1H NMR (400 MHz, CDCl3): δ ppm 4.54 (d, J = 5.05 Hz, 4H), 6.96 (d, J = 8.08 Hz, 2H),

7.24 (br s, 2H), 7.31 (t, J = 8.1 Hz, 2H), 7.49 (br s, 2 H), 7.56 (d, J = 8.08 Hz, 2H), 7.65 - 7.71 (m,

71

1H); 13C NMR (100 MHz, CDCl3): δ ppm 156.8, 148.6, 137.7, 129.8, 120.4, 119.2, 112.4, 106.6,

48.7. HRMS: m/z [M + H]+ calcd for C19H17N5O4: 380.1359, found: 380.1358.

2,6-Bis(4-nitroanilinomethyl)pyridine, (2v) The product was purified using a 10:1

DCM/Methanol soluvent system and was obtained in 10% yield as a yellow semi-solid; 1H NMR

(400 MHz, CDCl3): δ ppm 4.51 (d, J = 5.1 Hz, 4H), 6.56 (d, J = 9.1 Hz, 4H), 7.17 (d, J = 7.8 Hz,

2H), 7.64 (t, J=7.71 Hz, 1H), 8.05 (d, J=9.09 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ ppm 156.1,

152.7, 137.9, 126.4, 120.5, 111.6, 48.9. HRMS: m/z [M + H]+ calcd for C19H18N5O4: 380.1359,

found: 380.1359.

2,6-Bis(morpholinylmethyl)pyridine (2w) The product purified in a 25:1 DCM/Methanol

solvent system and was obtained was a white semi-solid in 1% yield. 1H NMR (400 MHz, CDCl3):

δ ppm 7.73 (1H t), 7.47 (2H d), 5.30 (4H s), 2.60 (8H m), 1.65 (8H m), 1.50 (4H, m); 13C NMR

(100 MHz, CDCl3): δ ppm 152.75, 137.55, 123.65, 120.96, 60.43, 63.80, 25.68, 25.11, 23.78.

HRMS: m/z [M + H]+ calcd for C15H23N3O2: 278.1863, found: 278.1852.

2,6-Bis(pyrrolidinylmethyl)pyridine (2x) The product was purified in a 5:1 DCM:Methanol

solvent system and was obtained was a orangeish oil in 9% yield. 1H NMR (400 MHz, CDCl3): δ

ppm 7.59 (t, J=7.7 Hz, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 3.60 (s, 4H), 2.42-2.44 (m, 8H), 1.55-1.61

(m, 8H), 1.40-1.46 (m, 4H); 13C NMR (100 MHz, CDCl3): δ ppm 158.71, 136.67, 121.20, 65.57,

54.99, 26.20, 24.52. HRMS: m/z [M+H]+ calcd for C15H24N3: 246.1965, found: 246.1956.

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5.3.2 General Procedure for the Synthesis of the 2,5-Furan Analogues (4)

General Procedure for the Synthesis of the 2,5-furan-based analogues (4). To a solution of

methanol, 50 mg (0.4029 mmol) of furan-2.5-dicarbaldehyde was combined in a dry vial with the

aniline derivative of choice (0.8865 mmol) and 75.963 mg (1.2088 mmol) of sodium

cyanoborohydride (NaBH3CN). The solution was stirred for five minutes at room temperature

before 164.578 mg (1.2088 mmol) of zinc chloride (ZnCl2) was added. The solution was then

stirred for two hours and purified by flash chromatography.

2,5-Bis(anilinomethyl)furan, (4a) Product was purified in a 7:1 Hexanes/Ethyl Acetate solvent

system and was obtained in 38% yield as a yellow oil. 1H NMR (400 MHz, CDCl3): δ ppm ppm

4.27 (s, 4 H) 6.15 (s, 2 H) 6.66 (d, J=7.58 Hz, 4 H) 6.74 (t, J=7.33 Hz, 2 H) 7.18 (t, J=7.96 Hz, 4

H); 13C NMR (100 MHz, CDCl3): δ ppm 152.11, 147.58, 129.22, 118.04, 113.18, 107.80, 41.49.

HRMS: m/z [M + H]+ calcd for C18H19ON2: 279.1492, found: 279.1487.

2,5-Bis(2-methylanilinomethyl)furan, (4b) The product was purified in a 13:1 solvent system

and was obtained as a yellow solid in 35% yield. 1H NMR (400 MHz, CDCl3): δ ppm 2.14 (s, 6

H) 3.84 (br s, 2 H) 4.33 (s, 4 H) 6.17 (s, 2 H) 6.63 - 6.75 (m, 4 H) 7.00 - 7.17 (m, 4 H); 13C NMR

(100 MHz, CDCl3): δ ppm 152.22, 145.56, 130.14, 127.07, 122.34, 117.61, 110.13, 107.83, 41.50,

17.49. HRMS: m/z [M + H]+ calcd for C20H23ON2 : 307.1805, found: 307.1792.

2,5-Bis(3-methylanilinomethyl)furan, (4c) The product was purified in a 3:1 Hexanes/Ethyl

Acetate solvent system and was obtained as a light orange solid in 49% yield. 1H NMR (400 MHz,

CDCl3): δ ppm2.27 (s, 6 H) 4.25 (s, 4 H) 6.13 (s, 2 H) 6.42 - 6.50 (m, 4 H) 6.56 (d, J=7.58 Hz, 2

H) 7.06 (t, J=1.00 Hz, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 152.17, 147.64, 138.98, 129.9,

73

118.97, 114.01, 110.27, 107.73, 41.53, 21.61. HRMS: m/z [M+ Z]+ calcd for C20H23ON2 :

307.1817, found: 307.1810.

2,5-Bis(4-methylanilinomethyl)furan, (4d) The product was purified in a 3:1 Hexanes/Ethyl

Acetate solvent system and was obtained as an orange solid in 49% yield. 1H NMR (400 MHz,

CDCl3): δ ppm 2.22 (s, 6 H) 4.21 (s, 4 H) 6.10 (s, 2 H) 6.56 (d, J=8.08 Hz, 4 H) 6.97 (d, J=7.83

Hz, 4 H); 13C NMR (100 MHz, CDCl3): δ ppm 152.25, 145.33, 129.68, 127.21, 113.38, 107.67,

41.82, 20.40. HRMS: m/z [M+ Z]+ calcd for C20H22ON2Na: 329.1617, found: 329.1624.

2,5-Bis(2-ethylanilinomethyl)furan, (4e) The product was purified in a 9:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 44% yield as a yellow oil. 1H NMR (400 MHz,

CDCl3): δ ppm 1.24 (br d, J=5.56 Hz, 6 H) 2.44 - 2.56 (m, 4 H) 3.94 (br s, 2 H) 4.33 (br s, 4 H)

6.16 (br s, 2 H) 6.66 - 6.78 (m, 4 H) 7.05 - 7.16 (m, 4 H); 13C NMR (100 MHz, CDCl3): δ ppm

152.28, 144.98, 128.02, 127.92, 126.93, 117.81, 110.52, 107.79, 41.59, 23.79, 12.90. HRMS: m/z

[M + H]+ calcd for C22H27ON2: 335.2118, found: 335.2103.

2,5-Bis(3-ethylanilinomethyl)furan, (4f) The product was purified in a 9:1 Hexanes/Ethyl


CDCl3): δ ppm 1.21 (br d, J=5.31 Hz, 6 H) 2.51 - 2.63 (m, 4 H) 3.93 (br s, 2 H) 4.26 (br s, 4 H)

6.14 (br s, 2 H) 6.45 - 6.54 (m, 4 H) 6.54 - 6.65 (m, 2 H) 7.05 - 7.14 (m, 2 H); 13C NMR (100

MHz, CDCl3): δ ppm 152.20, 147.70, 145.40, 129.15, 117.78, 112.93, 110.47, 107.75, 41.57,

29.00, 15.51. HRMS: m/z [M + H]+ calcd for C22H27ON2: 335.2118, found: 335.2103.

2,5-Bis(4-ethylanilinomethyl)furan, (4g) The product was purified in a 7:1 Hexanes/Ethyl

Acetate solvent system and wass obtained in 27% yield as an orange oil; 1H NMR (400 MHz,

CDCl3): δ ppm 1.18 (t, J=7.58 Hz, 6 H) 2.54 (q, J=7.41 Hz, 4 H) 4.24 (s, 4 H) 6.12 (s, 2 H) 6.60

(d, J=8.34 Hz, 4 H) 7.01 (d, J=8.08 Hz, 4 H); 13C NMR (100 MHz, CDCl3): δ ppm 152.28, 145.58,

74

133.88, 128.52, 113.35, 107.69, 41.83, 27.93, 15.91. HRMS: m/z [M + H]+ calcd for C22H27ON2:

335.2118, found: 335.2111.

2,5-Bis(2-fluoroanilinomethyl)furan, (4h) The product was purified in a 20:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 40% yield as an yellow oil. 1H NMR (400 MHz,

CDCl3): δ ppm 4.28 (br s, 4 H) 6.14 (s, 2 H) 6.59 - 6.78 (m, 4 H) 6.90 - 7.05 (m, 4 H); 13C NMR

(100 MHz, CDCl3): δ ppm 152.84, 151.84, 150.47, 136.04, 135.93, 130.41, 126.93, 124.53,

124.50, 118.58, 117.38, 117.31, 114.90, 114.40, 112.51, 107.94, 41.02. HRMS: m/z [M + H]+

calcd for C18H17ON2F2: 315.13, found: 315.1299.

2,5-Bis(3-fluoroanilinomethyl)furan, (4i) Product was purified in 9:1 Hexanes/Ethyl Acetate

solvent system and was obtained in 35% yield as an orange oil. 1H NMR (400 MHz, CDCl3): δ

ppm 4.23 (s, 4 H) 6.15 (s, 2 H) 6.34 (dd, J=11.49, 1.64 Hz, 2 H) 6.37 - 6.45 (m, 4 H) 7.05 - 7.13

(m, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 165.22, 162.80, 151.69, 149.35, 149.25, 130.35,

130.25, 109.00, 108.98, 108.07, 104.53, 104.31, 99.95, 99.69, 41.27. HRMS: m/z [M + H]+ calcd

for C18H17ON2F2: 315.1303, found: 315.1292.

2,5-Bis(4-fluoroanilinomethyl)furan, (4j) Product was purified in 3:1 Hexanes/Ethyl Acetate


4.22 (s, 4 H) 6.13 (s, 2 H) 6.53 - 6.64 (m, 4 H) 6.88 (m, J=8.70, 8.70 Hz, 4 H); 13C NMR (100

MHz, CDCl3): δ ppm 157.35, 155.00, 152.04, 143.88, 115.77, 115.55, 114.18, 114.11, 107.90,

42.10. HRMS: m/z [M + H]+ calcd for C18H17ON2F2: 315.1294, found: 315.1303.

2,5-Bis(2-chloroanilinomethyl)furan, (4k) The product was purified in a 12:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 6% yield. 1H NMR (400 MHz, CDCl3): δ ppm 4.35

(br s, 4 H) 4.67 (br s, 2 H) 6.17 (br s, 2 H) 6.63 - 6.69 (m, 2 H) 6.73 (br s, 2 H) 7.12 (br s, 2 H)

7.26 (br s, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 151.72, 143.42, 129.19, 127.76, 119.46,

75

117.89, 111.59, 107.98, 41.16. HRMS: m/z [M + H]+ calcd for C18H17ON2Cl2: 347.0712, found:

347.0509.

2,5-Bis(3-chloroanilinomethyl)furan, (4l) Product was purified in a 7:1 Hexanes/Ethyl Acetate


ppm 4.21 (4 H, s) 6.13 (2 H, s) 6.49 (2 H, d, J=8.34 Hz) 6.61 (2 H, br. s.) 6.69 (2 H, d, J=6.82 Hz);

13C NMR (100 MHz, CDCl3): δ ppm 41.18, 108.10, 111.43, 112.79, 114.89, 117.83, 130.21,

148.69, 151.67. HRMS: m/z [M + H]+ calcd for C18H17ON2Cl2: 347.0712, found: 347.0685.

2,5-Bis(4-chloroanilinomethyl)furan, (4m) Product as purified in a 7:1 Hexanes/Ethyl Acetate


ppm 4.21 (4 H, s) 6.12 (2 H, s) 6.54 (4 H, d, J=8.59 Hz) 7.09 (4 H, d, J=8.59 Hz). 13C NMR (100

MHz, CDCl3): δ ppm 41.26, 107.80, 114.06, 122.32, 28.85, 146.01, 151.69. HRMS: m/z [M + H]+

calcd for C18H17ON2Cl2: 347.0712, found: 347.0696.

2,5-Bis(2-methoxyanilinomethyl)furan, (4n) The product was purified in a 9:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 29% yield as a white solid; 1H NMR (400 MHz,

CDCl3): δ ppm 7.12 (t, J = 7.71 Hz, 2H), 7.06 (d, J = 7.07 Hz, 2H), 6.64 - 6.72 (m, 4H), 6.17 (s,

2H), 4.33 (s, 4H), 2.14 (s, 6H); 13C NMR (100 MHz, CDCl3): δ ppm 152.22, 145.56, 130.14,

127.07, 122.34, 117.61, 110.13, 107.83, 41.5, 17.49. HRMS: m/z [M + H]+ calcd for

C20H22N2O3Na: 361.1528, found: 361.1537.

2,5-Bis(3-methoxyanilinomethyl)furan, (4o) The product was purified in a 2:1 Hexane/Ethyl


CDCl3): δ ppm 7.00 - 7.13 (m, 2H), 6.19 - 6.35 (m, 6H), 6.15 (s, 2H), 4.25 (s, 4H), 3.65 - 3.79 (m,


99.20, 55.07, 41.45. HRMS: m/z [M + H]+ calcd for C20H23N2O3: 339.1676, found: 339.1687.

76

2,5-Bis(4-methoxyanilinomethyl)furan, (4p) Product was purified in a 7:1 Hexanes/Ethyl


CDCl3): δ ppm 3.74 (6 H, s) 4.22 (4 H, s) 6.13 (2 H, s) 6.63 (4 H, d, J=8.84 Hz) 6.78 (4 H, d,

J=8.84 Hz); 13C NMR (100 MHz, CDCl3): δ ppm 152.58, 152.34, 141.77, 116.42, 114.81, 107.72,

55.73, 42.52. HRMS: m/z [M + H]+ calcd for C20H23N2O3 : 339.1701, found: 339.1703.

2,5-Bis(2-trifluoromethylanilinomethyl)furan, (4q) The product was purified in a 13:1

Hexanes/Ethyl Acetate solvent system and was obtained in 9% yield as a yellow oil. 1H NMR (400

MHz, CDCl3): δ ppm 4.37 (br d, J=4.55 Hz, 4 H) 4.72 (br s, 2 H) 6.17 (s, 2 H) 6.70 - 6.88 (m, 4

H) 7.31 - 7.41 (m, 2 H) 7.45 (br d, J=7.58 Hz, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 151.48,


C20H17F6N2O: 415.1240, found: 415.1246.

2,5-Bis(3-trifluoromethylanilinomethyl)furan, (4r) The product was purified in a 3:1

Hexanes/Ethyl Acetate solvent system and was obtained in 60% yield as an orange oil. 1H NMR

(400 MHz, CDCl3): δ ppm 7.20 - 7.24 (m, 2H), 6.95 - 6.98 (m, 2H), 6.86 (br. s., 2H), 6.76 - 6.80

(m, 2H), 6.17 (s, 2H), 4.29 (s, 4H); 13C NMR (100 MHz, CDCl3): δ ppm 151.62, 147.67, 129.73,

129.65, 117.97, 116.11, 114.46, 109.34, 108.23, 41.14. MS: m/z [M]+ calcd for C20H16F6N2O:

413.1080.

2,5-Bis(4-trifluoromethylanilinomethyl)furan, (4s) The product was purified in a 3:1

Hexanes/Ethyl Acetate solvent system and was obtained in 33% yield as an orange oil. 1H NMR

(400 MHz, CDCl3): δ ppm 7.41 (d, J=8.08 Hz, 4H), 6.66 (d, J=8.34 Hz, 4H), 6.18 (s, 2H), 4.32

(s, 4H); 13C NM0R (100 MHz, CDCl3): δ ppm 151.54, 149.93, 126.64, 123.53, 119.80, 112.23,

108.23, 40.94. HRMS: m/z [M+ Z]+ calcd for C20H16F6N2ONa: 437.1066, found: 437.1059.

77

2,5-Bis(4-thiomethylanilinomethyl)furan (4t) The product was purified in a 7:1 Hexanes/Ethyl


CDCl3): δ ppm 2.40 (s, 6 H) 4.25 (s, 4 H) 6.14 (s, 2 H) 6.60 (d, J=8.59 Hz, 4 H) 7.20 (d, J=8.59

Hz, 4 H). 13C NMR (100 MHz, CDCl3): δ ppm 151.94, 146.31, 131.12, 125.17, 113.81, 107.92,

41.48, 18.87. HRMS: m/z [M + H]+ calcd for C20H23N2OS2: 371.1238, found: 371.1246.

2,5-Bis(4-methypiperazin-1-ylmethyl)furan, (4u) The product was purified in a 30:1

DCM/Methanol solvent system and was obtained as a yellow oil in 40% yield. 1H NMR (400 MHz,

CDCl3): δ ppm 6.13 (s, 2H), 3.54 (s, 4H), 2.53 (m, J=16.17 Hz, 16H), 2.30 (s, 6H); 13C NMR (100

MHz, CDCl3): δ ppm 151.09, 109.48, 54.89, 54.76, 52.48, 45.84. HRMS: m/z [M + H]+ calcd for

C16H29N4O: 293.2336, found: 293.2323.

78

5.3.3 General Procedure for the Synthesis of the 3,4-Thiophene Analogues (8)

Procedure for the Synthesis of Thiophene-3,4-dimethanol (6).

The dimethanol compound was prepared according to a literature procedure151 where 500

mg of thiophene-3,4-dicarboxylic acid (2.9mmol) was added to 10mL of dry THF in a dry round

bottom flask and was chilled to 0°C. 17.375mL (17.375mmol) of diisobutylaluminimum hydride

(139mL in 1M hexanes) was added to the flask and the solution was stirred at room temperature

for 16 hours. The reaction was quenched with methanol and water and then 25mL of HCl was

added to break up the solid chunks in the reaction. The solution as then extracted with ethyl acetate,

washed with brine and dried with MgSO4. The solution was then evaporated under reduced

pressure to give an orange oil in 77% yield.

Procedure for the Synthesis of Thiophene-3,4-dialdehyde (7). The thiophene aldehyde

was prepared using an adapted literature procedure152 where 2.0 g of Compound 6 (13.9 mmol) was

added to a 100 mL solution of dry 1,4-dioxane. 6.0 g of dry activated MnO2 (69.00 mmol) was added and

the mixture was refluxed under N2 for forty-five minutes at 96oC. The solution was filtered through a fritted

filter funnel and was concentrated under reduced pressure to yield a light yellow solid in 90% yield. 1H

NMR was used to confirm the structure with the literature characterization

General Procedure for the Synthesis of the 3,4-bis(anilino)thiophene analogues (8).

To a solution of methanol, 50mg (0.3568 mmol) of thiophene-3.4-dicarbaldehyde was combined

in a dry vial with the aniline derivative of choice (0.7849 mmol), 145.68 mg (1.070 mmol) of zinc

chloride (ZnCl2). The solution was stirred at room temperature overnight. The solution was

reduced to dryness and was then dissolved in ethanol, where 67.24 mg (1.070 mmol) of sodium

79

cyanoborohydride (NaBH3CN) was added. The solution was then stirred overnight and purified

by flash chromatography.

3,4-Bis(anilinomethyl)thiophene, (8a) The product was purified in a 7:1 Hexanes/Ethyl Acetate

solvent system and was obtained in this reaction was an brown semisolid in 4% yield. 1H NMR

(400 MHz, CDCl3): δ ppm 7.24 (2H s), 7.18 (4H t), 6.65 (4H d), 6.44 (2H d), 4.31 (4H s); 13C

NMR (100 MHz, CDCl3): δ ppm 147.94, 138.38, 129.30, 124.23, 117.98, 113.21, 42.89. HRMS:

m/z [M + H]+ calcd for C18H19N2S: 291.0956, found: 291.0963.

3,4-Bis(3-methylanilinomethyl)thiophene, (8b) The product was purified in a 20:1

Hexanes/Ethyl Acetate solvent system and was obtained as an orange solid in 26% yield. 1H NMR

(400 MHz, CDCl3): δ ppm 2.34 (6 H, s) 4.77 (4 H, s) 6.81 (2 H, d, J=7.83 Hz) 6.84 (2 H, s) 7.00

(2 H, s) 7.18 - 7.28 (4 H, m); 13C NMR (100 MHz, CDCl3): δ ppm 21.83, 49.59, 114.71, 116.91,

118.75, 120.29, 128.74, 138.62, 140.60, 151.66. HRMS: m/z [M + H]+ calcd for C20H23N2S:

323.1576, found: 323.1561.

3,4-Bis(4-methylanilinomethyl)thiophene, (8c) The product was purified in a 20:1

Hexanes/Ethyl Acetate solvent system and was obtained as an orange solid in 19% yield. 1H NMR

(400 MHz, CDCl3): δ ppm 2.33 (6 H, s) 4.77 (4 H, s) 6.90 (4 H, d, J=8.08 Hz) 7.01 (2 H, s) 7.80

(4 H, d, J=8.34 Hz); 13C NMR (100 MHz, CDCl3): δ ppm 20.82, 49.67, 114.69, 120.05, 121.71,

123.21, 129.47. HRMS: m/z [M + H]+ calcd for C20H23N2S: 323.1576, found: 323.1565.

3,4-Bis(4-ethylanilinomethyl)thiophene, (8d) The product was purified in a 15:1 Hexanes/Ethyl

Acetate solvent system and as obtained as an orange oil in 3% yield. 1H NMR (400 MHz, CDCl3):

δ ppm 1.19 (t, J = 7.45 Hz, 6 H) 2.54 (q, J = 7.58 Hz, 4 H) 4.28 (s, 4 H) 6.60 (d, J = 8.08 Hz, 4 H)

7.02 (d, J = 8.08 Hz, 4 H) 7.23 (s, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 147.94, 138.38,

80

129.30, 124.23, 117.98, 113.21, 42.89. HRMS: m/z [M + H]+ calcd for C22H27N2S: 351.1889,

found: 351.1888.

3,4-Bis(3-fluoroanilinomethyl)thiophene, (8e) The product was purified in a 7:1 Hexanes/Ethyl

Acetate solvent system and was obtained as an off-white semi-solid in 12% yield. 1H NMR (400

MHz, CDCl3): δ ppm 4.43 (br. s., 4 H) 6.21 - 6.66 (m, 8 H) 7.14 (d, J = 7.07 Hz, 2 H); 13C NMR

(100 MHz, CDCl3): δ ppm 141.68, 137.70, 130.35, 124.53, 114.69, 107.13, 104.13, 99.97, 49.13.

HRMS: m/z [M + H]+ calcd for C18H17N2SF2: 331.1061, found: 331.1075.

3,4-Bis(4-fluoroanilinomethyl)thiophene, (8f) The product was purified in a 6:1 Hexane/Ethyl

Acetate solvent system and was obtained as a yellowish semisolid in 15% yield. 1H NMR (400

MHz, CDCl3): δ ppm 4.25 (s, 4 H) 6.49 - 6.61 (m, 4 H) 6.88 (t, J = 8.72 Hz, 4 H) 7.23 (s, 2 H);

13C NMR (100 MHz, CDCl3): δ ppm 157.32, 144.26, 138.22, 124.40, 115.86, 115.64, 112.16,

43.58. HRMS: m/z [M + H]+ calcd for C18H17N2SF2: 331.1072, found: 331.1075.

3,4-Bis(3-chloroanilinomethyl)thiophene, (8g) Product was purified in a 15:1 Hexanes/Ethyl

Acetate solvent system and was obtained in 31% yield as an off white oil. 1H NMR (400 MHz,

CDCl3): δ ppm 4.26 (4 H, s) 6.48 (2 H, dd, J=8.21, 1.39 Hz) 6.60 (2 H, s) 6.69 (2 H, dd, J=0.80

Hz) 7.06 (2 H, t, J=7.96 Hz); 13C NMR (100 MHz, CDCl3): δ ppm 42.60, 111.47, 112.76, 117.85,

124.53, 130.27, 135.05, 137.64, 148.95. HRMS: m/z [M + H]+ calcd for C18H17N2Cl2S: 363.0484,

found: 363.0466.

3,4-Bis(4-chloroanilinomethyl)thiophene, (8h) Product was purified in a 15:1 Hexanes/Ethyl


CDCl3): δ ppm 4.26 (s, 4 H) 6.54 (d, J=8.84 Hz, 4 H) 7.11 (d, J=8.59 Hz, 4 H) 7.22 (s, 2 H); 13C

NMR (100 MHz, CDCl3): δ ppm 146.39, 137.82, 129.15, 124.50, 122.67, 114.26, 42.97. HRMS:

m/z [M + H]+ calcd for C18H17N2Cl2S: 363.0408, found: 363.0466.

81

3,4-Bis(3-trifluoromethylanilinomethyl)thiophene, (8i) The product was purified in a 10:1

solvent system of Hexanes/Ethyl Acetate and was obtained in 20% yield as an orange oil. 1H NMR

(400 MHz, CDCl3): δ ppm 4.32 (s, 4 H) 6.74 (d, J=8.08 Hz, 2 H) 6.82 (br. s., 2 H) 6.89 - 7.04 (m,

2 H) 7.23 - 7.33 (m, 4 H); 13C NMR (100 MHz, CDCl3): δ ppm 147.95, 137.47, 131.48, 129.76,

125.63, 125.77, 116.09, 114.50, 109.28, 42.61 HRMS: m/z [M + H]+ calcd for C20H17F6N2S:

431.1011, found: 431.0994.

3,4-Bis(4-trifluoromethylanilinomethyl)thiophene, (8j) The product was purified in a 10:1

Hexanes/Ethyl Acetate solvent system and was obtained in 6% yield as a white semi-solid. 1H

NMR (400 MHz, CDCl3): δ ppm 4.33 (br. s., 4 H) 6.62 (d, J=8.08 Hz, 4 H) 7.25 (s, 2 H) 7.40

(d, J=8.34 Hz, 4 H); 13C NMR (100 MHz, CDCl3): δ ppm 150.17, 137.26, 126.73, 126.69, 126.66,

124.77, 112.21, 42.36. HRMS: m/z [M + H]+ calcd for C20H17F6N2S: 431.1011, found: 431.0995.

82

5.3.4 General Procedure for the Synthesis of the 2,5-Pyrazine Analogues (14)

Procedure for the Synthesis of 2,5-Dimethylpyrazine-1,4-dioxide (10)

The dioxide compound was prepared from a literature procedure152 where 20.20mL

(184.94 mmol) of 2,5-dimethyl pyrazine (9) was added to 50 mL of ethyl acetate. This solution

was combined to a solution of m-CPBA (70% purity, 405.63 mmol) dissolved in 150 mL of ethyl

acetate that had been washed with 150 mL of brine and dried over MgSO4. The mixture was stirred

at room temperature for 24 hours and a white precipitate formed. The precipitate was then filtered

and washed with 100 mL of ethyl acetate in triplicate. This compound was yielded in 90% percent

as a white solid. 1H was used to confirm structure with the literature characterization.

Procedure for the Synthesis of 2,5-Di(acetoxymethyl)pyrazine (11)

The di(acetoxymethyl) compound was prepared from a literature procedure152 where 20 g

of compound 10 (142.72 mmol) was added to 100 mL of acetic anhydride and was refluxed at

158oC for seven hours. The solution was then stirred at room temperature for twelve hours. The

resulting product was a black residue and was acquired by removing the acetic anhydride under

reduced pressure. 500 mL of diethyl ether was added to the black residue and was stirred at room

temperature for two hours. The solution was then filtered and the remaining residue was washed

with 100 mL of diethyl ether. The filtrate was condensed under reduced pressure to yield a dark

yellow residue. The residue was purified via flash column chromatography using a solution of

40% ethyl acetate and 60% hexanes. This yellow product was then recrystallized from ethyl acetate

and hexanes to yield a white solid in 16% yield. 1H NMR was used to confirm the structure with

the literature characterization.

83

Procedure for the Synthesis of 2,5-Di(hydroxymethyl)pyrazine (12)

The di(hydroxymethyl) compound was prepared from a literature procedure152 where 4.0

g of compound 11 (17.84 mmol) was suspended in 100 mL of dry methanol. 0.8 g of NaOMe

(14.80 mmol) was added. The solution was stirred at room temperature for three hours under N2.

105 g of NH4Cl was added to quench the reaction and the methanol was removed under reduced

pressure. The remaining residue was purified by flash chromatography using a solution of 10%

methanol and 90% chloroform to yield a white solid in 91% yield. 1H NMR was used to confirm

the structure with the literature characterization.

Procedure for the Synthesis of 2,5-pyrazinedicarbaldehyde (13)

The di(hydroxymethyl) compound was prepared from a literature procedure152 where 2.0

g of compound 12 (14.27 mmol) was added to a 100 mL solution of dry 1,4-dioxane. 6.0 g of dry

activated MnO2 (69.00 mmol) was added and the mixture was refluxed under N2 for forty-five

minutes at 96oC. The solution was filtered through a fritted filter funnel and was concentrated

under reduced pressure to yield a light yellow solid in 80% yield. 1H NMR was used to confirm

the structure with the literature characterization.

General Procedure for the Synthesis of the 2,5-pyrazinedicarbaldehyde analogues

(14)

To a solution of methanol, 32 mg of compound 13 (0.2351 mmol) was combined in a dry

vial with the aniline derivative of choice (0.5172 mmol) and 96.02 mg (0.7053 mmol) of ZnCl2.

The solution was stirred for two hours at room temperature before 44.32 mg (0.7053 mmol) of

84

NaBH3CN was added. The solution was then stirred overnight and purified by flash

chromatography.

3,4-Bis(anilinomethyl)pyrazine, (14a) The product was purified in a 1:1 Hexane/Ethyl Acetate

solvent system and was obtained as an orange solid in 10% yield. 1H NMR (400 MHz, CDCl3): δ

ppm 4.50 (s, 4 H) 6.68 (d, J=7.83 Hz, 4 H) 6.75 (t, J=7.33 Hz, 2 H) 7.19 (t, J=7.83 Hz, 4 H) 8.60

(s, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 152.42, 147.42, 142.71, 129.37, 118.17, 113.18,

47.08. HRMS: m/z [M + H]+ calcd for C18H19N4: 291.1604, found: 291.1599.

3,4-Bis(3-methylanilinomethyl)pyrazine, (14b) The product was purified in a 1:1 Hexanes/Ethyl

Acetate solvent system and was obtained as a brown solid in 4% yield. 1H NMR (400 MHz,

CDCl3): δ ppm 8.59 (s, 2H), 7.07 (t, J=7.71 Hz, 2H), 6.23 - 6.67 (m, 6H), 4.49 (s, 4H), 2.27 (s,


112.25, 47.13, 21.44. HRMS: m/z [M + H]+ calcd for C20H23N4: 319.1917, found: 319.1909.

3,4-Bis(4-ethylanilinomethyl)pyrazine, (14b) The product was purified in a 1:1 Hexanes/Ethyl

Acetate solvent system and was obtained as a brown solid in 5% yield. 1H NMR (400 MHz,

CDCl3): δ ppm 1.18 (t, J=7.58 Hz, 6 H), 2.54 (d, J=7.58 Hz, 4 H), 4.48 (s, 4 H), 6.62 (d, J=8.08

Hz, 4 H), 7.03 (d, J=8.08 Hz, 4 H), 8.9 (s, 2 H); 13C NMR (100 MHz, CDCl3): δ ppm 152.60,


C22H27N4: 347.2230, found: 347.2225.

3,4-Bis(4-fluoroanilinomethyl)pyrazine, (14d) The product was purified in a 1:1 Hexanes/Ethyl

Acetate solvent system and was obtained as a light orange solid in 5% yield. 1H NMR: δ ppm 4.46

(br. s., 4 H) 6.55 - 6.67 (m, 4 H) 6.90 (m, J=8.20, 8.20 Hz, 4 H) 8.58 (s, 2 H); 13C NMR: δ ppm

85

157.39, 155.05, 152.31, 143.71, 142.73, 115.94, 114.09, 114.02, 47.63. HRMS: m/z [M + H]+

calcd for C18H17N4F2: 327.1416, found: 327.1409.

86

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97

APPENDICES

NMR Spectra

1H NMR Spectra

TG12-1 FINAL PROTON COPY.ESP

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

4.004.002.086.021.00

M04(d)

M05(s)

M03(t)

M01(t)

M02(m)

7.5

17

.49

7.4

77

.13

7.1

17

.09

6.6

76

.65

6.6

06

.58

4.3

8

1H NMR Spectrum for Compound 2a

98

TG99 proton.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

6.204.192.002.054.001.911.05

M02(d)

M07(m)

M03(t)

M04(d)

M05(s)

M06(s)

M01(t)

7.6

2 7.6

07

.58

7.2

3

7.1

07

.08

6.7

06

.68

6.6

16

.59

4.5

2

2.2

5

1H NMR Spectrum for Compound 2b

99

TG34 last spot proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

6.003.506.052.001.711.06

M03(t)

M02(d)

M07(s)

M04(d)

M06(m)

M05(s)

M01(t)

7.5

6 7.5

47

.52

7.1

8

7.0

67

.04

6.5

5

6.4

96

.47

6.4

5

4.4

2

2.2

6

1H NMR Spectrum for Compound 2c

100

TG32-1 Third spot orange oiL PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

No

rma

lize

d I

nte

nsity

6.003.153.263.601.640.92

M02(d)

M03(d)

M05(s)

M04(d)

M06(s)

M01(t)

7.5

8 7.5

67

.54

7.2

07

.00

6.9

8

6.6

06

.58

4.4

3

2.2

4

1H NMR Spectrum for Compound 2d

101

TG82PROTON.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.073.734.071.821.853.692.041.00

M06(m)

M05(d)

M07(m)

M04(t)

M03(t)

M08(t)

M02(q)

M01(s)7

.66

7.6

4

7.2

87

.27

7.1

87

.16

7.1

4

6.8

16

.79

6.6

86

.66

4.5

8

2.6

92

.67

2.6

62

.64

1.3

91

.37

1.3

5

1H NMR Spectrum for Compound 2e

102

TG100.001.001.1r.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

No

rma

lize

d I

nte

nsity

6.004.073.375.871.460.68

M06(m)

M03(d)

M02(m)

M05(d)M04(m)

M01(br. s.)

7.5

87

.57

7.5

6

7.2

1 7.2

0 6.5

96

.53

6.5

0

4.4

6

2.5

72

.55

1.2

11

.21

1.2

0

1H NMR Spectrum for Compound 2f

103

TG22-4 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.004.053.033.833.680.82

M03(d)

M04(s)M06(m)

M05(m)M02(m)

TMS

M01(t)

7.5

57

.53

7.5

1

7.0

06

.98

6.6

06

.58

4.4

1

2.5

42

.52

2.5

12

.51

1.1

81

.16

1H NMR Spectrum for Compound 2g

104

TG98-2 dirty PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

3.733.603.672.221.00

M04(d)

M05(d) M03(m)

M01(br. s.)M02(br. s.)

7.6

2 7.2

47

.23

6.9

96

.97

6.9

5 6.6

6

4.5

1

1H NMR Spectrum for Compound 2h

105

TG13-1 Final proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

4.005.931.971.970.99

M03(d)

M05(s)

M04(m)

M01(t)

M02(m)

7.5

37

.51

7.4

9

7.1

17

.09

7.0

27

.00

6.9

8

6.3

56

.35

6.3

36

.32

6.3

16

.27

6.2

4

4.3

4

1H NMR Spectrum for Compound 2i

106

TG7-1 Final Proton Copy.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

3.923.933.931.881.00

M02(d)

M04(dd)

M05(s)

M01(t)

M03(t)

7.5

2

7.1

3

6.8

1

6.5

2

4.3

3

1H NMR Spectrum for Compound 2j

107

TG83 PROTON.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

4.003.901.991.972.001.00

M03(d)

M02(d)

M05(m)

M06(d)

M01(t)

M04(t)

7.6

37

.61

7.5

97

.29

7.2

77

.22

7.2

07

.10

7.0

8 6.6

66

.65

6.6

2

4.5

54

.54

1H NMR Spectrum for Compound 2k

108

TG16-1 final proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

4.052.001.951.961.991.960.98

M06(s)

M05(d)

M03(d)

M02(t)

M04(t)

M07(dd)

M01(s)

7.5

67

.54

7.5

2

7.1

3 7.0

16

.99

6.6

2

6.5

86

.47

6.4

56

.45

4.3

6

1H NMR Spectrum for Compound 2l

109

TG17-1 final proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

4.003.973.842.021.00

M02(d)

M03(d)

M05(br. s.)

M01(t)

M04(d)7

.56

7.5

47

.52

7.1

37

.07 7.0

5

6.5

26

.50

4.3

6

1H NMR Spectrum for Compound 2m

110

TG24 proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.193.662.001.973.992.051.03

M02(s)

M06(m)M04(m)

M01(s)

M05(m)M03(t)

M07(d)

7.4

8

7.1

67

.15

7.1

3

6.7

7

6.7

5 6.7

36

.72

6.6

16

.50

6.4

8

4.4

3

3.8

1

1H NMR Spectrum for Compound 2n

111

TG93 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

5.653.632.003.652.041.680.90

M02(br. s.)

M07(br. s.)

M05(m)

M06(br. s.)

M04(m)

M03(m)

M01(br. s.)

7.5

9

7.2

27

.09

6.3

16

.24

4.4

5

3.7

6

1H NMR Spectrum for Compound 2o

112

TG14-2 Final proton.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

6.023.483.543.931.750.93

M05(s)

M06(s)

M04(d)

M02(d)

M03(d)

M01(t)

7.5

1 7.4

97

.47

7.1

37

.11

6.7

26

.69

6.5

66

.54

4.3

3

3.6

6

1H NMR Spectrum for Compound 2p

113

TG90 PROTON.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

4.094.001.952.022.011.03

M06(d)

M03(t)

M04(d)

M05(t)

M07(m)

M01(d)

7.6

37

.61

7.4

87

.46

7.3

07

.21

7.1

9

6.7

56

.73 6

.70

6.6

8

4.5

54

.54

1H NMR Spectrum for Compound 2q

114

TG29 proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9N

orm

aliz

ed

In

tensity

4.002.041.991.981.961.970.97

M05(d)

M06(t)

M04(br. s.)

M02(d)

M03(d)

M01(t)

M07(br. s.)

7.6

37

.61

7.5

9 7.2

77

.25 7

.20

7.1

86

.94

6.8

76

.79

6.7

7

4.4

6

1H NMR Spectrum for Compound 2r

115

TG28 2nd Spot PROTON.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8N

orm

aliz

ed

In

tensity

4.004.121.934.071.11

M03(d)

M01(t)

M02(d)

M05(s)

M04(d)

7.6

57

.63

7.4

27

.40

7.2

17

.19

6.6

66

.64

4.4

9

1H NMR Spectrum for Compound 2s

116

TG37 dark spot 2.001.001.1r.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

No

rma

lize

d I

nte

nsity

6.034.084.076.001.03

M05(m)

M01(t)

M03(br. s.)M02(d)

M04(s)

7.6

1 7.5

9

7.2

57

.23

7.2

17

.18

6.6

26

.60

4.4

4

2.4

1

1H NMR Spectrum for Compound 2t

117

TG19-2 PROTON Final.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

No

rma

lize

d I

nte

nsity

4.012.081.702.252.022.091.11

M03(br. s.)

M05(br. s.)M07(d)

M02(d)

M06(d)

M01(m)

M04(t)

TMS

7.6

87

.57

7.5

57

.49

7.3

3 7.3

17

.24

6.9

76

.95

4.5

44

.53

1H NMR Spectrum for Compound 2u

118

TG18-1 proton final.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70N

orm

aliz

ed

In

tensity

4.004.711.811.244.60

M04(d)

M05(d)

M01(t)

M03(d)

M02(d)

8.0

68

.04

7.6

57

.64

7.6

2

7.1

87

.16

6.5

76

.54

4.5

14

.50

1H NMR Spectrum for Compound 2v

119

TG33 proton.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75N

orm

aliz

ed

In

tensity

7.954.408.001.831.05

M05(m)

M04(t)

M02(d)

M01(t)

M03(s)

7.6

5 7.6

37

.61

7.3

37

.31

3.7

53

.73

3.6

7

2.5

32

.52

1H NMR Spectrum for Compound 2w

120

TG39 PROTON.esp

16 14 12 10 8 6 4 2 0 -2 -4


0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11N

orm

aliz

ed

In

tensity

8.048.944.272.221.12

M05(t)

M02(m)

M03(s)

M01(t)

M04(t)

7.6

3 7.6

1

7.2

6

3.7

8

2.5

8

1.8

0

1H NMR Spectrum for Compound 2x

121

TG50 PROTON.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

4.171.994.072.014.00

M05(t) M03(d)

M04(t)M02(s)

M01(s)7

.20

7.1

87

.16

6.7

66

.74

6.6

76

.65

6.1

5

4.2

7


122

TG103 PROTON.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

6.104.041.963.941.992.00

M06(t)

M04(m)

M05(d)

M01(s)

M02(s)

M03(s)

7.1

47

.12

7.0

77

.05

6.6

96

.67

6.1

7

4.3

3

2.1

4


123

TG54 proton.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

6.823.891.824.002.022.22

M06(m)

M05(d)

M03(s)

M04(t)

M01(s)

M02(s)

7.0

87

.06

7.0

4 6.5

7

6.4

86

.46

6.1

3

4.2

5

2.2

7


124

TG55 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.594.091.924.204.00

M05(d)M03(s)

M04(d)

M02(s)

M01(s)

6.9

8 6.9

6

6.5

76

.55 6.1

0

4.2

1

2.2

2


125

TG107 proton.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

6.003.903.781.843.773.76

M04(br. s.)

M02(m)

M01(s)

M06(q)

M03(m)

M05(t)

7.1

27

.10

7.0

96

.75

6.7

46

.72

6.1

6

4.3

3

2.5

02

.48

1.2

5 1.2

4


126

TG108 proton.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.114.024.031.925.932.00

M01(br. s.)

M04(t)

M02(br. s.)

M03(q)

M06(m)

M05(m)

7.1

07

.09 6

.60

6.5

0

6.1

4

4.2

6

2.5

72

.55

1.2

2 1.2

0


127

TG51 proton.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

9.504.623.761.803.624.00

M06(d)

M05(d)

M01(t)

M04(s)

M03(s)

M02(q)

7.0

27

.00

6.6

16

.59

6.1

2

4.2

4

2.5

62

.55

2.5

32

.52

2.5

1

1.2

0 1.1

91

.18

1.1

71

.16


128

TG101 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

4.631.522.001.883.17

M03(m)

M02(m)

M04(m)

M05(s) M01(br. s.)

7.0

06

.99

6.9

76

.95

6.9

56

.76

6.7

46

.63

6.6

16

.61

6.1

4

4.2

8


129

TG52 PROTON.esp

8 7 6 5 4 3 2 1 0 -1


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

3.841.806.212.00

M02(s)

M03(dd)

M04(m)

M05(m)

M01(s)

7.1

17

.09

7.0

87

.05 6.4

36

.41

6.4

16

.38

6.3

56

.15

4.2

3


130

TG53 proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

4.061.944.024.00

M03(m)

M01(s)

M02(s)M04(m)

6.9

06

.88

6.8

66

.59

6.5

86

.57

6.1

3

4.2

2


131

TG102 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

3.891.844.001.912.29

M04(m)

M03(m)

M01(br. s.)

M05(m)

M02(br. s.)

7.2

67

.12

6.7

36

.66

6.1

7

4.3

5

1H NMR Spectrum for Compound 4k

132

TG66 proton.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

4.001.882.622.622.66

J(M05)=6.82 Hz

J(M04)=8.34 Hz

M04(d)

M03(br. s.)

M05(d)

M01(s)

M02(s)

7.0

76

.70

6.7

0

6.6

96

.68

6.6

26

.61

6.6

16

.50

6.4

96

.47

6.1

3

4.2

1

1H NMR Spectrum for Compound 4l

133

TG67 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8N

orm

aliz

ed

In

tensity

4.442.034.814.00

M04(d)

M03(d)

M01(s)

M02(s)

7.1

0 7.0

8 6.5

56

.53 6

.12

4.2

1

1H NMR Spectrum for Compound 4m

134

TG60.001.001.1r.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

6.453.891.884.002.221.92

M05(m)

M04(m)

M06(m)

M01(s)

M03(s)

M02(m)

6.8

66

.86

6.7

9 6.7

76

.77

6.7

06

.69

6.6

76

.16

4.3

0

3.8

43

.84

1H NMR Spectrum for Compound 4n

135

TG105 PROTON.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.404.022.106.002.05

M03(s)

M01(s)

M06(m)

M02(m)

M04(m)

7.1

07

.08

7.0

6

6.3

16

.29

6.2

26

.15

4.2

5

3.7

73

.75

1H NMR Spectrum for Compound 4o

136

TG74.PROTON.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

6.883.711.764.434.00

M03(s)

M04(s)

M02(d)

M05(s)

M01(d)

6.7

96

.77

6.6

46

.62

6.1

3

4.2

2

3.7

4

1H NMR Spectrum for Compound 4p

137

TG104 PROTON.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

No

rma

lize

d I

nte

nsity

4.632.144.312.002.33

M05(t)

M04(d)

M03(m)

M01(s)

M02(d)7

.46

7.4

47

.35

7.3

3

6.8

0 6.7

86

.75

6.7

4

6.1

7 4.3

84

.37

1H NMR Spectrum for Compound 4q

138

TG58 PROTON.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

3.621.681.931.472.031.99

M03(br. s.)

M04(m)

M01(m)

M06(m)

M05(s)

M02(s)7

.24

7.2

27

.20

6.9

86

.97

6.8

66

.78

6.7

6

6.1

7

4.2

9

1H NMR Spectrum for Compound 4r

139

TG59 proton.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

4.002.134.754.77

M04(s)

M01(s)

M03(d)

M02(d)7

.42 7

.40

6.6

76

.65

6.1

8

4.3

2

1H NMR Spectrum for Compound 4s

140

TG75 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

6.294.271.964.244.00

M05(d) M03(d)

M04(s)

M01(s)

M02(s)

7.2

17

.19

6.6

16

.59 6

.14

4.2

5

2.4

0

1H NMR Spectrum for Compound 4t

141

TG111 april10 PROTON.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5 -1.0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

5.9415.984.251.86

M03(s)

M01(s)

M02(s)

M04(d)

6.1

3

3.5

4

2.5

5 2.5

1

2.3

0

1H NMR Spectrum for Compound 4u

142

TG9-1 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

4.202.101.102.002.08

7.3

57

.33

7.3

1

7.0

26

.80 6

.78

6.6

86

.66

4.4

5

1.6

11

.47

1.2

91

.14

0.9

20

.90

0.8

7

0.7

90

.77

0.7

5

0.1

1


143

TG64-1 proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9N

orm

aliz

ed

In

tensity

M05(s)

M04(d)

M03(s)

M02(s)

M06(m) M01(s)

7.2

7

7.2

17

.19 7

.00

6.8

46

.82

6.8

0

4.7

7

2.3

4


144

TG65-1 proton.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

No

rma

lize

d I

nte

nsity

6.484.104.262.004.03

M02(s)

M05(s)

M04(d)

M03(d)

M01(s)

7.8

17

.79

7.0

1 6.9

16

.89

4.7

7

2.3

3


145

TG36 proton.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060N

orm

aliz

ed

In

tensity

6.003.602.903.283.231.42

M04(s)

M01(s)

M03(d)

M02(d)

M06(t)

M05(q)

7.0

37

.01

6.6

16

.59

4.2

8

2.5

72

.55

2.5

32

.52

1.2

11

.19

1.1

7


146

TG10-1 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

4.003.271.951.04

7.2

87

.24

7.0

3

6.4

86

.46

6.4

4 6.4

26

.39

6.3

56

.32 5

.32

4.4

3

1.5

61

.43

1.2

81

.13

0.9

80

.91

0.8

90

.86

0.7

90

.76

0.7

4

0.1

0


147

tg8 SPOT 2 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50N

orm

aliz

ed

In

tensity

4.194.204.102.00

M04(s)

M01(s)

M02(m)

M03(t)

7.2

3

6.9

06

.88

6.8

66

.58

6.5

76

.56

6.5

56

.54

4.2

5


148

TG68 proton.esp

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

No

rma

lize

d I

nte

nsity

4.342.142.032.002.20

M05(dd)

M04(s)

M02(dd)

M03(t)

M01(s)

7.0

87

.06

7.0

4

6.7

0

6.6

06

.49

6.4

96

.47

6.4

7

4.2

6


149

TG69 PROTON.esp

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

No

rma

lize

d I

nte

nsity

4.214.234.082.00

M04(s)

M03(s)

M02(d)

M01(d)

7.2

2

7.1

0

6.5

56

.53

4.2

6


150

TG62-1 PROTON.esp

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

No

rma

lize

d I

nte

nsity

4.001.752.032.263.36

M01(br. s.)

M05(m)

M03(d)

M04(s)

M02(m)

7.2

67

.25

7.2

47

.01

6.9

7

6.8

26

.75

6.7

3

4.3

2


151

TG63-1 PROTON.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75N

orm

aliz

ed

In

tensity

3.943.632.003.54

M04(s)M03(br. s.)

M01(d)

M02(d)

7.4

17

.39

7.2

5

6.6

36

.61

4.3

3


152

TG42 proton.esp

9 8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70N

orm

aliz

ed

In

tensity

4.364.542.494.592.00

M05(s) M02(d)

M01(s)M04(t)

M03(t)

8.6

0

7.2

1 7.1

97

.17

6.7

5 6.6

96

.67

4.5

0


153

TG46 PROTON.esp

16 14 12 10 8 6 4 2 0 -2 -4


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

6.613.436.472.002.02

M02(s)

M05(t)

M03(s)

M04(m)M01(s)

8.5

9

7.0

97

.07

7.0

56

.50

6.4

8

4.4

9

2.2

7


154

TG43 PROTON.esp

9 8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75N

orm

aliz

ed

In

tensity

6.003.914.093.803.791.89

M04(s)

M03(d)

M02(d)M06(t)

M01(s) M05(d)

8.5

9

7.0

47

.02

6.6

36

.61 4

.48

2.5

52

.53

1.2

0 1.1

81

.16


155

TG45 Proton.esp

10 9 8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

No

rma

lize

d I

nte

nsity

3.394.003.821.55

M03(m)

M02(br. s.)

M01(s)

M04(m)8

.58

6.9

26

.90

6.8

86

.61

4.4

6


156

13C NMR Spectra

13C NMR Spectrum for Compound 2a

TG12-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.1

3

14

7.9

6

13

7.2

8

12

9.3

1

11

9.9

1

11

3.1

3

77

.41

77

.09

76

.77

49

.29

29

.75

157

13C NMR Spectrum for Compound 2b

TG99 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.0

2

14

5.8

4

13

7.2

6

13

0.0

81

27.1

31

22.2

71

19.9

91

17.2

2

11

0.1

4

77

.33

77

.02

76

.70

49

.22 1

7.5

9

158

TG34 last spot carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.2

3

14

8.0

6

13

9.0

61

37.2

6

12

9.2

0

11

9.8

61

18.6

11

13.9

51

10.2

3

49

.30

21

.70

13C NMR Spectrum for Compound 2c

159

TG32-1 Third spot orange oil Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS1

58.3

6

14

5.7

0

13

7.2

2

12

9.7

81

26.8

4

11

9.8

5

11

3.2

7

49

.64

20

.42

13C NMR Spectrum for Compound 2d

160

TG82CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.1

7

14

5.3

2

13

7.2

9

12

7.8

91

27.0

41

19.9

81

17.4

3

11

0.5

0

77

.41 77

.09

76

.77

49

.36 23

.98

12

.92

13C NMR Spectrum for Compound 2e

161

TG100.002.001.1r.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.1

5

14

8.0

11

45.4

5

13

7.2

0

12

9.1

9

11

9.8

31

17.3

61

12.8

01

10.3

4

77

.35

77

.04

76

.72

49

.31

29

.71

29

.02

28

.83

15

.57

0.0

0

13C NMR Spectrum for Compound 2f

162

TG22-4 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.4

1

14

6.0

0

13

7.2

81

33.5

51

28.6

5

11

9.9

1

11

3.3

0

77

.48

77

.17

76

.85

49

.71

28

.05

28

.01

16

.02

13C NMR Spectrum for Compound 2g

163

TG98-2 dirty CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS

15

7.8

1

15

2.9

3

13

7.3

91

36.3

3

12

4.5

6 11

9.8

51

14.5

7 11

2.5

0

77

.34

77

.02

76

.70

48

.90

29

.71

13C NMR Spectrum for Compound 2h

164

TG13-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

16

5.3

51

62.9

41

57.5

11

49.7

61

49.6

6

13

7.4

4

13

0.3

2

12

0.0

6

10

9.0

01

04.1

71

03.9

69

9.8

99

9.6

4

77

.41

77

.09

76

.77

48

.99

13C NMR Spectrum for Compound 2i

165

TG7-1 - YELLOW - CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.05

0.10

0.15

0.20

0.25

0.30

No

rma

lize

d I

nte

nsity

15

7.9

51

57.1

51

54.8

1

14

4.2

7

13

7.3

81

37.2

0

12

0.0

31

15.8

61

15.6

41

15.5

41

14.3

21

13.4

4

77

.35

77

.03

76

.72

49

.85

49

.26

13C NMR Spectrum for Compound 2j

166

TG83 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.6

4

14

3.7

0

13

7.4

2

12

9.1

81

27.8

01

19.8

31

17.5

41

11.6

3

77

.34

77

.02

76

.71

48

.94

13C NMR Spectrum for Compound 2k

167

TG16-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.3

9

14

9.0

2

13

7.4

5

13

0.2

6

12

0.0

91

17.5

31

12.7

41

11.4

2

77

.36

77

.05

76

.73

48

.86

13C NMR Spectrum for Compound 2l

168

TG17-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.6

4

14

6.4

3

13

7.3

8

12

9.1

21

22.2

9 12

0.0

2

11

4.1

6

77

.34

77

.03

76

.71

49

.21

13C NMR Spectrum for Compound 2m

169

TG24 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.6

0

14

6.9

7

13

7.8

5

12

1.2

2

11

6.7

21

10.2

31

09.4

3

77

.31

77

.00

76

.68

55

.44

49

.28

13C NMR Spectrum for Compound 2n

170

TG93 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

No

rma

lize

d I

nte

nsity

16

0.8

51

57.9

7

14

9.3

4

13

7.2

9

13

0.0

2

11

9.9

4

10

6.2

21

02.8

29

9.1

1

77

.35

77

.03

76

.71

55

.10

53

.43

49

.24

0.0

0

13C NMR Spectrum for Compound 2o

171

TG14-2 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

8.3

9

15

2.2

5

14

2.2

2

13

7.2

0

11

9.9

41

14.9

01

14.3

6

77

.39

77

.07

76

.76

55

.79

55

.50

50

.24

29

.73

14

.23

13C NMR Spectrum for Compound 2p

172

TG90 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.3

2

14

5.1

6

13

7.5

8 13

3.1

4

12

6.7

21

19.7

71

16.2

71

12.2

6

77

.35 7

7.0

37

6.7

1

48

.69

0.0

0

13C NMR Spectrum for Compound 2q

173

TG29 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.2

4

14

8.0

6

13

7.5

1

13

1.7

51

29.7

1

12

3.0

31

20.2

01

16.0

21

14.0

51

09.2

3

48

.80

13C NMR Spectrum for Compound 2r

174

TG28 2nd Spot carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.1

5

15

0.2

8

13

7.5

9

12

6.6

81

26.3

21

20.1

5

11

2.2

1

48

.46

13C NMR Spectrum for Compound 2s

175

TG37 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.8

0

14

6.7

0

13

7.3

0

13

1.3

4

12

4.6

11

19.9

4

11

3.7

0

77

.35

77

.04

76

.72

49

.16

19

.02

0.0

0

13C NMR Spectrum for Compound 2t

176

TG19-2 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

No

rma

lize

d I

nte

nsity

TMS

15

6.7

9

14

8.5

7

13

7.6

9

12

9.8

1

12

0.4

41

19.1

5

11

2.3

5

10

6.6

3

77

.33

77

.02

76

.70

48

.72

13C NMR Spectrum for Compound 2u

177

TG18-1 Second FRaction attempt 2.002.001.1r.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

No

rma

lize

d I

nte

nsity

15

6.1

01

52.6

9

13

7.8

8

12

6.4

1

12

0.4

3

11

1.5

3

77

.32

77

.00

76

.68

48

.07

13C NMR Spectrum for Compound 2v

178

TG33 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS1

57.6

9

13

6.7

4

12

1.4

7

77

.37

77

.06

76

.74

66

.95

64

.82

53

.73

13C NMR Spectrum for Compound 2w

179

TG39 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS1

58.8

0

13

6.7

0

12

0.9

3

77

.33 7

7.0

17

6.6

9

62

.35

54

.32

23

.57

13C NMR Spectrum for Compound 2x

180

TG50 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.1

11

47.5

8

12

9.2

2

11

8.0

41

13.1

8

10

7.8

0

77

.34

77

.02

76

.71

41

.49

0.0

0


181

TG103 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.2

2

14

5.5

6

13

0.1

41

27.0

71

22.3

41

17.6

1

11

0.1

31

07.8

3

77

.34

77

.02

76

.71

41

.50

17

.49


182

TG54 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.1

71

47.6

4

13

8.9

8

12

9.0

9

11

8.9

7 11

4.0

11

10.2

71

07.7

3

77

.36

77

.04

76

.72

41

.53

21

.61

0.0

0


183

TG55 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.2

5

14

5.3

3

12

9.6

81

27.2

1

11

3.3

8

10

7.6

7

77

.39

77

.07

76

.75

41

.82

20

.40


184

TG107 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.2

8

14

4.9

8

12

8.0

21

27.9

21

26.9

3

11

7.8

1

11

0.5

21

07.7

9

77

.34

77

.03

76

.71

41

.59

23

.79

12

.90


185

TG108 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.2

01

47.7

01

45.4

0

12

9.1

5

11

7.7

81

12.9

31

10.4

71

07.7

5 77

.36

77

.04

76

.72

41

.57

29

.00 15

.51


186

TG51 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.2

8

14

5.5

6

13

3.8

81

28.5

51

28.5

2

11

3.4

11

13.3

5

10

7.6

9

77

.36

77

.04

76

.73

41

.83

27

.93

23

.09

15

.91

0.0

0


187

TG101 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.8

41

51.8

41

50.4

71

44.5

3

13

6.0

41

35.9

31

30.4

11

24.5

01

22.2

91

18.5

81

17.3

8

11

2.5

11

07.9

4

77

.37

77

.05

76

.73

41

.02

17

.31

0.0

0


188

TG52 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

16

5.2

21

62.8

0

15

1.6

91

49.3

51

49.2

5

13

0.3

51

30.2

5 10

9.0

01

08.9

81

08.0

71

04.5

3

99

.95

99

.69

77

.36

77

.04

76

.72

41

.27

0.0

0


189

TG53 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

7.3

51

55.0

01

52.0

4

14

3.8

8

11

5.7

71

15.5

51

14.1

8

10

7.9

0

77

.36 77

.04

76

.72

42

.10

0.0

0


190

TG102 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.7

2

14

3.4

2

12

9.1

91

27.7

61

19.4

61

17.8

9

11

1.5

91

07.9

8

77

.34

77

.02

76

.70

41

.16

13C NMR Spectrum for Compound 4k

191

TG66 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.6

71

48.6

9

13

4.9

5

13

0.2

1

11

8.3

61

17.8

31

12.7

91

11.4

31

08.1

0

77

.43

77

.11

76

.80

60

.44

41

.18

31

.62

22

.69

21

.02

14

.14

13C NMR Spectrum for Compound 4l

192

TG67 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.6

9

14

6.0

1

12

8.8

5

12

2.3

2

11

4.0

6

10

7.8

0

41

.26

13C NMR Spectrum for Compound 4m

193

TG60.002.001.1r.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

2.2

1

14

7.0

0

13

7.6

0

12

1.1

71

17.1

11

10.2

81

09.4

71

07.7

0

77

.35

77

.03

76

.72

55

.40

41

.26

0.0

0

13C NMR Spectrum for Compound 4n

194

TG105 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

16

0.7

4 15

1.9

91

48.9

8

12

9.9

6

10

7.8

7

10

3.0

99

9.2

0

77

.36

77

.04

76

.72

55

.07

41

.45

13C NMR Spectrum for Compound 4o

195

TG75 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.9

4

14

6.3

1

13

1.1

2

12

5.1

7

11

3.8

1

10

7.9

2

77

.36

77

.04

76

.72

41

.48

18

.87

13C NMR Spectrum for Compound 4p

196

TG104 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.4

9

14

4.9

8

13

3.0

7

12

6.6

91

26.4

5

11

6.6

71

12.1

81

08.0

3

77

.34

77

.03

76

.71

41

.07

0.0

0

13C NMR Spectrum for Compound 4q

197

TG58 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.6

21

47.6

7

12

9.7

31

29.6

5

11

7.9

71

16.1

11

14.4

6

10

8.2

3

77

.35

77

.03

76

.71

41

.15

13C NMR Spectrum for Compound 4r

198

TG59.002.001.1r.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

No

rma

lize

d I

nte

nsity

17

0.1

6

15

8.7

41

56.6

7

15

1.5

21

49.9

1

12

6.6

21

21.8

1

11

2.2

11

08.2

0

81

.08

77

.32

77

.00

76

.68

40

.91

13C NMR Spectrum for Compound 4s

199

TG75 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.9

4

14

6.3

1

13

1.1

2

12

5.1

7

11

3.8

1

10

7.9

2

77

.36

77

.04

76

.72

41

.48

18

.87

13C NMR Spectrum for Compound 4t

200

TG111 april10 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

1.0

9

10

9.4

8

77

.32

77

.00

76

.69

54

.89

52

.48

45

.84

13C NMR Spectrum for Compound 4u

201

TG9-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

14

7.6

6

14

2.2

2

12

9.3

8

11

6.3

41

14.4

31

11.4

1

77

.33

77

.01

76

.69

48

.99

31

.94

29

.71

29

.37

22

.70

14

.12


202

TG64-1 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0N

orm

aliz

ed

In

tensity

TMS

15

1.6

6

14

0.6

01

38.6

2

12

8.7

4

12

0.2

91

18.7

51

16.9

11

14.7

1

49

.59

21

.83


203

TG65-1 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0N

orm

aliz

ed

In

tensity

TMS

12

9.4

7

12

3.2

11

21.7

11

20.0

51

14.6

9

49

.67

20

.82


204

TG63-1 PROTON.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75N

orm

aliz

ed

In

tensity

3.943.632.003.54

M04(s)M03(br. s.)

M01(d)

M02(d)

7.4

17

.39

7.2

5

6.6

36

.61

4.3

3


205

TG10-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.05

0.10

0.15

0.20

0.25

No

rma

lize

d I

nte

nsity

16

5.4

3

14

9.1

7

14

1.6

6

13

0.4

51

30.3

5

11

4.6

8

10

7.1

31

02.9

41

02.7

39

8.5

99

8.3

3

77

.34

77

.02

76

.70

49

.13

31

.95

29

.72

29

.38

22

.71

14

.14


206

tg8 SPOT 2 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0N

orm

aliz

ed

In

tensity

TMS

15

7.3

2

14

4.2

6 13

8.2

2

12

4.4

0

11

5.8

61

15.6

41

14.1

6

43

.58


207

TG68 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0N

orm

aliz

ed

In

tensity

14

8.9

5

13

7.6

41

35.0

51

30.2

7

12

4.5

3

11

7.8

5 11

2.7

61

11.4

7

42

.60


208

TG69 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS14

6.3

9

13

7.8

2

12

9.1

51

24.5

01

22.6

7

11

4.2

6

77

.34

77

.03

76

.71

42

.97

31

.60

22

.67

14

.13


209

TG62-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

14

7.9

5

13

7.4

7

12

9.7

6

12

4.7

7

11

6.0

91

14.5

01

09.3

2

77

.36

77

.04

76

.72

42

.61


210

TG63-1 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

15

0.1

7

13

7.2

6

12

6.7

3 12

4.7

7

11

2.2

1

77

.34

77

.02

76

.71

42

.36

0.0

0


211

TG42 carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS

15

2.4

21

47.4

21

42.7

1

12

9.3

7

11

8.1

7

11

3.1

8

77

.35

77

.03

76

.71

47

.08


212

TG46 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS

15

2.5

0

14

6.3

4 14

2.6

91

39.1

8 12

9.2

4 12

9.1

7

11

9.4

71

15.9

21

12.2

51

10.3

3

77

.36

77

.04

76

.72

47

.13

21

.63

21

.44


213

TG43 Carbon.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS

15

2.6

0

14

5.3

71

42.7

3

13

4.0

5

12

8.6

7

11

3.3

3

77

.34 77

.02

76

.70

47

.45

27

.93

15

.91


214

TG45 CARBON.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

No

rma

lize

d I

nte

nsity

TMS

15

7.3

91

55.0

51

52.3

11

43.7

1 14

2.7

3

11

5.9

41

15.7

11

14.0

91

14.0

2

77

.34

77

.02

76

.71

47

.63


215

Mass Spectra

Mass Spectra for compound 2a

in MeOH 100x dilution 01-Jul-201312:44:58

m/z100 150 200 250 300 350 400 450 500 550 600

%

0

100

THERESA_TG12-1_MOORING-ACCU_07012013_ESI-POS02 64 (1.193) AM (Top,2, Ar,5000.0,0.00,1.00); Sm (SG, 2x3.00); Cm (54:64)1.15e4290.1657

197.112593.1245183.1013

286.1455

291.1728

292.1725379.2231

216

Mass Spectra for compound 2b

TG_99_ESIPOS_MOORING_05202015_150520142143 #213-221 RT: 2.98-3.09 AV: 9 NL: 1.68E9T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

318.1958z=1

217

Mass Spectra for compound 2c

TG34_ESIPOS_MOORING_041317 #217-226 RT: 3.00-3.13 AV: 10 NL: 1.38E8T: FTMS + p ESI Full ms [50.00-1000.00]

305 310 315 320 325 330 335 340 345

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

318.1952z=1

319.1980z=1

314.1650z=1

218

Mass Spectra for compound 2d

Therasa_TG32_ESI_pos_Mooring_04032014_2 #3 RT: 0.01 AV: 1 NL: 3.36E7T: FTMS + c ESI Full ms [100.00-1000.00]

314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

318.1952

319.1984

219

Mass Spectra for compound 2e

Theresa_TG78_ESIPOS_Mooring_04292015_150429152657 #186-198 RT: 2.58-2.75 AV: 13 NL: 2.70E9T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

346.2276z=1

220

Mass Spectra for compound 2f

TG_100_MOORING_ESIPOS_021016_160210142315 #150-160 RT: 2.27-2.40 AV: 11 NL: 2.34E8T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

346.2285z=1

122.0966z=1

221

Mass Spectra for compound 2g

QtofMicro 14-Jan-201416:07:33

m/z100 200 300 400 500 600 700 800 900 1000

%

0

100

THERESA_TG22_MOORING-ACCU_01142014_ESI-POS01 19 (0.354) AM (Cen,2, 80.00, Ar,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (13:19)1.49e5346.2291

225.1465

122.1134

342.2165

226.1571

227.1681

347.2480

368.2348

463.3156 687.4909532.3705

711.4645804.5576

222

Mass Spectra for compound 2h


100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

326.1457z=1

223

Mass Spectra for compound 2i


m/z100 150 200 250 300 350 400 450 500 550 600

%

0

100

THERESA_TG13-1_MOORING-ACCU_07012013_ESI-POS01 54 (1.006) AM (Cen,2, 80.00, Ar,5000.0,0.00,1.00); Sm (SG, 2x3.00); Cm (51:60)6.93e3326.1462

215.0945195.0959152.1014 238.0572

327.1527

328.1463 376.1253

224

Mass Spectra for compound 2j

in MeOH+0.1%HCOOH 556.2771 10:26:20 15-Mar-2013

m/z300 320 340 360 380 400 420 440 460 480 500 520 540 560 580

%

0

100

THERESA_GG7_HR_ESI_POS_MOORING_03152013_1 181 (3.610) AM (Cen,2, 80.00, Ar,5000.0,556.28,0.70); Cm (177:201)1.70e4417.2165

322.1155

391.2851 556.2771482.1673

225

Mass Spectra for compound 2k

Theresa_TG83_ESIPOS_Mooring_04292015 #189-195 RT: 2.57-2.65 AV: 7 NL: 1.18E9T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

358.0877z=1

360.0842z=1

194.0965z=1 238.0865

z=1

226

Mass Spectra for compound 2l

Theresa_TG16-1_Mooring-ACCU_08272013_ESI-POS #165-169 RT: 2.25-2.30 AV: 5 SB: 8T: FTMS + p ESI Full ms [100.00-1000.00]

200 400 600 800 1000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

358.0864

282.2782

563.5491 815.3322686.0469191.0365 921.4147392.1545

227

Mass Spectra for compound 2m

Theresa_TG17-1_Mooring-ACCU_08272013_ESI-POS_130827185100 #168-173 RT: 2.29-2.36T: FTMS + p ESI Full ms [100.00-1000.00]

200 400 600 800 1000

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

358.0862

282.2781247.0623 686.0467 815.3318571.1619 921.4132

377.0623

455.1868

228

Mass Spectra for compound 2n


m/z338 340 342 344 346 348 350 352 354 356 358 360 362 364 366 368

%

0

100


346.1608

342.1349337.1736347.1673

351.1931

364.1717360.1421352.1934

357.1512 365.1776

229

Mass Spectra for compound 2o

TG93_FLUSH_ESIPOS_MOORING_06172015 #183-189 RT: 2.50-2.58 AV: 7 NL: 8.45E7T: FTMS + p ESI Full ms [100.00-1000.00]

200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

350.1854z=1

230

Mass Spectra for compound 2p


m/z100 150 200 250 300 350 400 450 500 550 600

%

0

100

THERESA_TG14_MOORING-ACCU_07012013_ESI-POS01 86 (1.590) AM (Cen,2, 80.00, Ar,5000.0,0.00,1.00); Cm (75:87)1.69e4350.1865

123.0933

227.1080213.0961

124.0976212.0974

346.1485229.1191

351.1888

469.2160351.2566 470.2405

231

Mass Spectra for compound 2q


100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

426.1401z=1

232

Mass Spectra for compound 2r


100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

426.1384

233

Mass Spectra for compound 2s


100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

426.1381

234

Mass Spectra for compound 2t

Theresa_TG37_ESIPOS_Mooring_04232014_3 #120 RT: 1.64 AV: 1 NL: 3.40E8T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

382.1404z=1

235

Mass Spectra for compound 2u


m/z376 377 378 379 380 381 382 383 384 385 386 387 388 389 390

%

0

100


378.1206376.1051

379.1213

381.1391

384.1781382.1494 385.1989387.2310 390.1345

388.1492

236

Mass Spectra for compound 2v


m/z378 379 380 381 382 383

%

0

100


378.1191 379.0827 379.7990

381.1438

382.1359383.2566

237

Mass Spectra for compound 2w

TG33__ESIPOS_MOORING_072716 #109-184 RT: 1.72-2.82 AV: 76 NL: 5.56E6T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

278.1853z=1

251.1383z=1

209.1277z=1

139.5964z=2

238

Mass Spectra for compound 2x

Theresa_TG39_ESIPOS_Mooring_05142014_2 #71 RT: 0.96 AV: 1 NL: 1.50E7T: FTMS + c ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

246.1956

239


Theresa_TG50_ESIPOS_Mooring_061114 #169 RT: 2.39 AV: 1 NL: 2.49E7T: FTMS + p ESI Full ms [200.00-2000.00]

278.2 278.4 278.6 278.8 279.0 279.2 279.4 279.6 279.8 280.0 280.2 280.4 280.6

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

279.1487

280.1519

280.2628

279.2313278.2322279.4247278.8739 280.0693279.0532 280.4678278.7065

240


GAINES_103_ESIPOS_MOORING_062415 #158-234 RT: 2.15-3.20 AV: 77 NL: 2.63E5T: FTMS + p ESI Full ms [100.00-1000.00]

285 290 295 300 305 310 315 320 325 330

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

307.1792z=1

305.1638z=1

308.1826z=1

321.1585z=1303.1479

z=1312.3247

z=1

241


QtofMicroin ACN+0.1%HCOOH 556.2771 as ITSD 23-Jun-201416:18:29

m/z300 350 400 450 500 550 600 650 700 750

%

0

100

THERESA_TG54_HR_ESIPOS_MOORING_062314 300 (5.578) AM (Cen,4, 80.00, Ar,6000.0,556.28,0.80)2.29e3307.1817

319.1493

321.1647

426.2225

329.1757

*556.2771

578.2488

242


TG_55_ESIPOS_MOORING_070516 #136-143 RT: 1.93-2.02 AV: 8 NL: 4.20E5T: FTMS + p ESI Full ms [50.00-1000.00]

310 315 320 325 330 335 340 345 350 355 360

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

329.1617z=1

307.1798z=1 346.2081

z=1

319.1258z=1

330.1654z=1324.1226

z=1 337.1045z=1

243



322 324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

335.2103z=1

336.2136z=1

333.1947z=1

244



320 322 324 326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

335.2103z=1

336.2136z=1

333.1948z=1

245


Theresa_BG51_ESIPOS_Mooring_061114 #154 RT: 2.19 AV: 1 NL: 5.38E7T: FTMS + p ESI Full ms [200.00-2000.00]

334.4 334.6 334.8 335.0 335.2 335.4 335.6 335.8 336.0 336.2 336.4 336.6

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

335.2111

336.2143

246



307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

313.1142z=1

315.1299z=1

314.1382z=1

312.1226z=1

316.1332z=1

311.0986z=1

247


Theresa_TG52_ESIPOS_POS_Mooring_061214 #207 RT: 2.91 AV: 1 NL: 9.58E6T: FTMS + p ESI Full ms [100.00-1000.00]

250 300 350 400 450 500 550 600 650 700

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

315.1292

313.1136

629.2512

248



250 300 350 400 450 500 550 600 650 700 750 800 850

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

315.1294

629.2521

313.1138

337.1112

249

Mass Spectra for compound 4k


330 335 340 345 350 355 360 365 370

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

345.0539z=1

347.0509z=1

354.2832z=1

371.0992z=1

361.0488z=1

355.0680z=1

343.1191z=1

348.0543z=1339.1685

z=1363.0458

z=1369.0511

z=1355.2866

z=1329.0849

z=1359.2386

z=1350.0513

z=1335.1060

z=1364.0490

z=1337.1030

z=1

353.1478z=1

250

Mass Spectra for compound 4l

TGraines_TG66_ESIPOS_Mooring_100182014_1 #171 RT: 2.42 AV: 1 NL: 4.04E6T: FTMS + p ESI Full ms [100.00-1000.00]

300 320 340 360 380 400 420 440 460 480

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

363.0457

347.0685

319.1238

367.0393

377.0788329.0850311.0921 486.0495470.0809445.1154395.3106

251

Mass Spectra for compound 4m

TGraines_TG67_ESIPOS_Mooring_100182014_1 #168 RT: 2.38 AV: 1 NL: 1.65E6T: FTMS + p ESI Full ms [100.00-1000.00]

325 330 335 340 345 350 355 360 365

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

347.0696

349.0667

345.0541

353.2647363.0467

365.0438

252

Mass Spectra for compound 4n

m/z326 328 330 332 334 336 338 340 342 344 346 348 350 352 354 356 358 360 362 364 366 368 370 372 374 376 378 380

%

0

100

TG60_ESIPOS_MOORING_062016_2 420 (7.813) Cm (420:429) TOF MS ES+ 1.78e4362.9202

361.1523

339.1754

340.1850 363.9440

253

Mass Spectra for compound 4o


100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

216.1007z=1

339.1686z=1

124.0749z=1

188.1060z=1

254

Mass Spectra for compound 4p


338.6 338.8 339.0 339.2 339.4 339.6 339.8 340.0 340.2 340.4 340.6 340.8 341.0 341.2 341.4 341.6

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

339.1701z=1

340.1737z=1

255

Mass Spectra for compound 4q


410 411 412 413 414 415 416 417 418 419 420 421 422 423

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

415.1246z=1

417.1404z=1

416.1279z=1413.1093

z=1419.1565

z=1

256

Mass Spectra for compound 4r


390 395 400 405 410 415 420 425 430 435

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

413.1080z=1

411.0925z=1

414.1117z=1

437.1063z=1

393.2976z=1

397.1134z=1

257

Mass Spectra for compound 4s


436.0 436.5 437.0 437.5 438.0 438.5 439.0 439.5

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

437.1066z=?

437.1651z=1

436.3936z=? 438.1096

z=?437.3236z=?

438.4096z=?

436.1713z=1

439.4133z=?

258

Mass Spectra for compound 4t


330 340 350 360 370 380 390 400 410 420 430 440

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

403.1139z=1

371.1238z=1

387.1190z=1

369.1084z=1332.9636

z=1

404.1174z=1

372.1275z=1 425.0963

z=1388.1227

z=1385.1036z=1

259

Mass Spectra for compound 4u

TG111_ESIPOS_MOORING_041217 #114 RT: 1.61 AV: 1 NL: 2.04E7T: FTMS + p ESI Full ms [50.00-1000.00]

280 285 290 295 300 305 310 315 320

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

293.2323z=1

294.2367z=1

260


261

Mass spectra for compound 8b


305 310 315 320 325 330 335 340 345 350 355

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

319.1241z=1

323.1561z=1

320.1284z=1

262

Mass spectra for compound 8c

TG_65_ESIPOS_dil_MOORING_071217_1_170713094936 #84-107 RT: 1.19-1.52 AV: 24 NL: 5.18E3T: FTMS + p ESI Full ms [50.00-1000.00]

321.5 322.0 322.5 323.0 323.5 324.0 324.5 325.0 325.5 326.0

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

323.1565z=1

324.1598z=1

325.1722z=1

263


Theresa_TG36_ESIPOS_Mooring_04232014_2 #6-7 RT: 0.08-0.09 AV: 2 NL: 9.56E7T: FTMS + p ESI Full ms [100.00-1000.00]

344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

351.1888z=1

352.1919z=1

264



318 320 322 324 326 328 330 332 334 336 338 340 342 344 346 348

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

331.1061z=1

332.1106z=1

265


Theresa_TG8_ESIPOS_Mooring_04232014_2 #75 RT: 1.01 AV: 1 NL: 2.82E8T: FTMS + p ESI Full ms [150.00-2000.00]

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

331.1077z=1

220.0592z=1

266


TGraines_TG68_ESIPOS_Mooring_100182014 #175 RT: 2.48 AV: 1 NL: 1.01E7T: FTMS + p ESI Full ms [100.00-1000.00]

361 362 363 364 365 366 367

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

363.0466

365.0432

364.0496

366.0462 367.0397

267


TGraines_TG69_ESIPOS_Mooring_100182014 #162 RT: 2.29 AV: 1 NL: 1.24E7T: FTMS + p ESI Full ms [100.00-1000.00]

356 358 360 362 364 366 368 370 372

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

363.0466

365.0433

366.0465

268


Theresa_TG62_ESIPos_Mooring_09102014 #183 RT: 2.60 AV: 1 NL: 2.23E7T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

431.0994z=1

270.0548z=1

286.0497z=1 427.0693

z=1133.0522z=1

166.0456z=1

269


Theresa_TG63_ESIPos_Mooring_09102014 #195 RT: 2.77 AV: 1 NL: 3.06E7T: FTMS + p ESI Full ms [100.00-1000.00]

100 200 300 400 500 600 700 800 900 1000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

431.0995z=1

270.0548z=1

133.0522z=1 166.0456

z=1 286.0497z=1

270


Theresa_TG42_ESIPOS_Nooring_06111401 #104-234 RT: 1.48-3.27 AV: 131 NL: 2.05E8T: FTMS + p ESI Full ms [200.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

291.1599

271



200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

319.1909

422.2328

272



200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

347.2225

291.1601

273



200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lative

Ab

un

da

nce

327.1409

262.0983

design, synthesis and analysis of small molecule

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