University of South FloridaScholar Commons

Graduate Theses and Dissertations Graduate School

7-2-2014

Synthesis of [1,2,4]-Triazines as Kinase Inhibitorsand of Novel Fluorine Capture Reagents for PETprobesFenger ZhouUniversity of South Florida, fzhou2@mail.usf.edu

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Scholar Commons CitationZhou, Fenger, "Synthesis of [1,2,4]-Triazines as Kinase Inhibitors and of Novel Fluorine Capture Reagents for PET probes" (2014).Graduate Theses and Dissertations.https://scholarcommons.usf.edu/etd/5339

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Synthesis of [1, 2, 4]-Triazines as Kinase Inhibitors and of Novel Fluorine

Capture Reagents for PET Probes

by

Fenger Zhou���

A dissertation submitted in partial fulfillmentof the requirements for the degree of

Doctor of PhilosophyDepartment of Chemistry

College of Arts and SciencesUniversity of South Florida

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Major Professor: Mark L. McLaughlin, Ph.D.Jon Antilla, Ph.D.

Jianfeng Cai, Ph.D.David L. Morse, Ph.D.

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Date of Approval: July 2, 2014

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Keywords: Triazines, Kinase Inhibitor, PET imaging, Fluorine Capture, PET Probe �

Copyright© 2014, Fenger Zhou

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DEDICATION

To my wife Dan Qin, my daughter Elaine and my newborn

baby Ethan

To my parents, brother and sister

To my mentor Mark and all my teachers

To my friends

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ACKNOWLEDGMENTS

�I would like to thank a number of people who offered invaluable assistance to the

completion of this dissertation. First of all, I would like to express my heartfelt gratitude

to my major professor, Dr. Mark L. Mclaughlin, for his tremendous encouragement,

unreserved support, understanding and invaluable ideas during my graduate study and,

for his devotion to my professional growth on this learning journey. My sincere thanks

also go to all my committee members, Dr. Jon Antilla, Dr. Jianfeng Cai, Dr. David L.

Morse for their guidance towards the completion of my degree. I am also very thankful

to Dr. Hongdao Meng for agreeing to chair my Defense Committee.

Secondly, I would like to thank Dr. Courtney DuBoulay who helped me with

molecular modeling studies. I would like to acknowledge Dr. Edwin Rivera and his

NMR TAs for helping me in obtaining NMR data and NMR training at USF. I would like

to extend my thanks to Dr. Mohanraja Kumar and my lab mate Michael Doligalski, for

helping me in peptide synthesis and mass spectrum analysis. I would also like to thank

my friends Dr. Jingran Tao, Yi Liang, Dr. Guilong Li and Dr. Zuhui Zhang for their

valuable suggestions and unselfish help in my life in the past six years.

In addition, I also thank my previous and current group members: Dr. Priyesh

Jain, Dr. Sridhar Kaulagari, Dr. Mingzhou Zhou, Dr. David Badger, Dr. Philip Murray,

Dr. Missy Topper, Dr . Hyun Joo Kil, Josanne-Dee Woodroffe for their constant

support and sharing time with me during my stay at University of South Florida.

Finally, I would like to express my special thanks to my beloved wife, Dan Qin

for her sacrifice. I would not have been able to complete the Ph.D. program and the

�

dissertation without her unselfish love and unconditional support. Thank you very much

for accompanying with me all the hardships and sharing with me all the happiness over

the years. Thank you very much for bringing me two lovely child, those are the precious

gifts towards my PhD study in the past six years. I would also like to thank my family

members, my parents, my brother and my sister who inspired me to pursue the Doctorate

degree and for your infinite love.

i  

TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………. iii

LIST OF FIGURES……………………………………………………………………… iv

LIST OF SCHEMES………………………………………………………………………v

LIST OF ABBREVIATIONS……………………………………………………………vii

ABSTRACT………………………………………………………………………………xi

CHAPTER ONE: SYNTHESIS OF [1, 2, 4]-DIHYDROTRIAZINE NUCLEUS AS A NEW KINASE INHIBITOR………………….. 1

1.1 Introduction………………………………………………………………. 1 1.2 Results & Discussions………………………………………………… 4

1.2.1 Design of the Potential Kinase Inhibitor………………………... . 4 1.2.2 Synthesis of [1, 2, 4]-dihydrotriazine Dimer Library…………....7

1.3 Conclusion and Future Plan…………………………………………………. 11 1.4 Experimental Section……………………………………………………….. 11

1.4.1 Materials and Methods…………………………………………….. 11 1.4.2 Experimental Procedures……………………………………… 12

1.5 References………………………………………………………………..31

CHAPTER TWO: NOVEL BUILDING BLOCKS FOR 18F-RADIOLABELING OF MOLECULAR PROBE FOR PET IMAGING………………... 32

2. 1 Introduction…………………………………………………………….. 32 2.1.1 PET Imaging, Molecular Probe and Radiotracers……………... 32

2. 2 Current Strategies for Fluorine-18 Radiolabeling……………………… 35 2.2.1 Carbon-Fluorine Bond Formation……………………………... 35

2.2.1.1 Electrophilic Fluorine-18 Labeling Reactions……….. 35 2.2.1.2 Nucleophilic 18F-Substitution Reactions…………….. 36

2.2.2 Boron-Fluorine Bond Formation………………………………. 37 2.2.3 Silicon-Fluorine Bond Formation……………………………... 38 2.2.4 Aluminium-Fluorine Bond Formation………………………… 39 2.2.5 Common 18F Reagents for Labelling Peptides, Proteins

and Oligonucleotides…………………………………………... 41 2.3 Results & Discussions……………………………………………….. . 43

2.3.1 Current Challenges in 18F Radiolabelling for Molecular Probe in PET Imaging............................................................... 43

ii  

2.3.2 Design of the Potential 18F-Radiolabelling Molecular Probe………………………………………………. 43

2.3.3 Fluorine Introduction Strategy: Building Block………………. 45 2.3.4 Fluorine Introduction Strategy: RCOOH Series Substrates…… 46 2.3.5 Fluorine Introduction Strategy: RNH2 Series Substrates……… 47 2.3.6 New Approach for Boron-Fluorine Bond Formation 1………... 49 2.3.7 New Approach for Boron-Fluorine Bond Formation 2………... 50 2.3.8 Fluorine Capture Application in PET Probe…………………... 51

2.3.8.1 Folic Acid as a Targeting Ligand................................. 51 2.3.8.2 Synthesis of New Folate-Targeted

Molecular Probe……………………………………... 53 2.4 Conclusion…………………………………………………………………... 54 2.5 Experimental Section……………………………………………………….. 55

2.5.1 Materials and Methods……………………………………………. .55 2.5.2 Experimental Procedures……………………………………… 56

2.6 References………………………………………………………………. 74 APPENDICES……………………………………………………………………… 78

Appendix A: Chapter One - Selected 1H and 13C NMR Spectra…………... 79 Appendix B: Chapter Two - Selected 1H, 13C, 19F,

11B NMR Spectra & HRMS…………………………………. 107

About the Author……………………………………………………………...End Page

iii  

LIST OF TABLES

Table 1.1 XP Ligand Dockings to 2XB7 6

Table 1.2 Preparation of thio-ester 1.3 9

Table 1.3 Preparation of [1, 2, 4]-dihydrotriazine precursor 1.4 10

Table 1.4 Library of the synthesized [1, 2, 4]-dihydrotriazine dimer 1.5 10

Table 2.1 Commonly Used Positron-Emitting Radionuclides 34

Table 2.2 Bond Dissociation Energy 44

Table 2.3 Preparation of RCOOH series compound 2.5 46

Table 2.4 Preparation of boron-fluoride complex adduct 2.6 for RCOOH series substrates 47

Table 2.5 Preparation of RNH2 series compound 2.10 48

Table 2.6 Preparation of boron-fluoride complex adduct 2.11 for RNH2 series substrates 48

iv  

LIST OF FIGURES

Figure 1.1 Anaplastic lymphoma kinase (ALK)-positive cancers and its fusion Proteins 2

Figure 1.2 ALK fusion oncogene and major downstream signaling pathways 2

Figure 1.3 Crystal structure of human ALK with NVP-TAE684 3

Figure 1.4 A model of ALK in complex with NVP-TAE684 4

Figure 1.5 [1,2,4]-dihydrotriazine scaffold as small molecule inhibitors targeting ALK-driven cancers 5

Figure 1.6 Ligand interaction at ALK binding site based on [1, 2, 4]-dihydrotriazine scaffold 5

Figure 1.7 Comparison of ligand 1.5L and NVP-TAE684 at the ALK binding site 7

Figure 1.8 Ligand 1.5L binding pose at the ALK binding site 8

Figure 2.1 Principle of PET imaging. 18F atom on the sugar molecule decays by emitting a positron 33

Figure 2.2 Complexation of a potassium ion (blue) by the azacryptand kryptofix-222 (K222); green: fluoride ion 36

Figure 2.3 Biomolecule labelling using an aluminium chelate as a binding site for [18F]-fluoride 40

Figure 2.4 Currently used bifunctional chelators 41

Figure 2.5 Boron’s electron configuration and its sp2 hybridization model 44

Figure 2.6 Flow chart of boron ester used as a fluorine capture reagent 45

Figure 2.7 Structure of folate conjugate 52

Figure 2.8 Receptor-mediated endocytosis of folate conjugate 52

v  

LIST OF SCHEMES

Scheme 1.1 Synthesis of [1, 2, 4]-dihydrotriazine dimer 9

Scheme 2.1 Electrophilic 18F fluorination using [18F] acetyl hypofluorite for the preparation of [18F]FDG 35

Scheme 2.2 Preparation of the hypoxia biomarker [18F]EF5 by direct electrophilic substitution using [18F]F2 35

Scheme 2.3 Synthesis of [18F]FDG precursor: protected [18F] sugar 36

Scheme 2.4 Synthesis and reaction of simple [18F] fluoroaliphatic derivatives 37

Scheme 2.5 18F-radiolabelled boronic ester conjugates by reaction with nucleophilic 18F (most often KHF2). X= a linker group, e.g. amide 37

Scheme 2.6 Ting and co -worker boron-fluorine bond formation strategy for 18F-

radiolabelling 38 Scheme 2.7 Rosenthal and coworkers silicon-fluorine bond formation strategy

for 18F-radiolabelling 38 Scheme 2.8 Ting and co -worker silicon-fluorine bond formation approach

for 18F-radiolabelling 39 Scheme 2.9 Synthesis of [18F]-fluorodi-tert-butylphenylsilyl by 19F/18F isotopic

Exchange 39 Scheme 2.10 Prosthetic reagents for the 18F radiolabelling of peptides, proteins 42

Scheme 2.11 “Click chemistry” strategy for the 18F radiolabelling of peptides, Proteins 43

Scheme 2.12 Newly discovered strategy for 19F introduction 45

Scheme 2.13 19F introduction into RCOOH series substrates 46

Scheme 2.14 19F introduction into RNH2 series substrates 47

vi  

Scheme 2.15 New Approach for 19F introduction 49

Scheme 2.16 New Approach for 19F introduction based on ion-exchange resin 49

Scheme 2.17 New Approach for 19F introduction 50

Scheme 2.18 New Approach for 19F introduction based on ion-exchange resin 50

Scheme 2.19 New Approach for 19F introduction into folic acid 53

Scheme 2.20 New Approach for the synthesis of folic acid molecular probe 54

vii  

LIST OF ABBREVIATIONS

AA = Amino acid

ALCL = Anaplastic large cell lymphomas

ALK = Anaplastic lymphoma kinase

ATP = Adenosine

Bn = Benzyl

Boc = tert-Butyloxy carbonyl

BOP = Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate

BOP-Cl = Bis(2-oxo-3-oxazolidinyl)phosphonic chloride

Calcd = calculated

Cbz = Benzyloxycarbonyl

CD = Circular Dichroism

DBU = 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCC = N,N'-dicyclohexylcarbodiimide

DCM = Dichloromethane

DIC = N,N'-diisopropylcarbodiimide

DIEA = N,N-Diisopropylethylamine

DMF = N,N-Dimethyl Formamide

DMSO = Dimethylsulfoxide

DPPA = Azidodiphenoxyoxophosphorane

viii  

DNA = Deoxyribonucleic acid

DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

DTPA = Diethylenetriamine pentaacetic acid

ECM = Extracellular matrix

EDC = 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide

ELISA = Enzyme-linked immunosorbent assay

ESI MS= Electrospray ionization mass spectrometry

EtOAc = Ethyl acetate

EtOH = Ethanol

Et3SiH = Triethylsilane

Fmoc = 9-Fluorenylmethoxycarbonyl

Fmoc-Cl = 9-Fluorenylmethoxycarbonyl chloride

Fmoc-OSu = 9-Fluorenylmethyl N-succinimidyl carbonate

FR = Folate receptors

FTIR = Fourier Transform InfraRed

HATU = N,N,N′,N′-Tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate

HBTU = 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HCTU = N,N,N′,N′-Tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uronium hexafluorophosphate

HOBT = 1-hydroxybenzotriazole

HPLC = High-performance liquid chromatography

HRMS = High resolution mass spectrum

Hz = Hertz

ix  

J = coupling constants

KF = Potassium fluoride

KOH = Potassium hydroxide

KHF2 = Potassium hydrogen fluoride

LCMS = Liquid chromatography – mass spectrometry

MRI = Magnetic resonance imaging

NMM = N-Methylmorpholine

NMP = N-Methylpyrrolidone

NMR = Nuclear magnetic resonance

NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid

NPM = Nucleophosmin

NOE = Nuclear Overhauser effect

PDB = Protein Data Bank

PET = Positron emission tomography

Ph = Phenyl

PTSCl = p-Toluenesulfonyl chloride

PyAOP = 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

RT = Room temperature

SN2 = Nucleophilic Substitution bimolecular

SPPS = Solid Phase Peptide Synthesis

T3P = Propylphosphonic anhydride

TBAI = Tetrabutylammonium iodide

TATU = 2-(7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

x  

TBTU = 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

TEA = Triethylamine

TEMPO = 2,2,6,6-tetramethylpiperidinoxyl TFA = Trifluoroacetic acid

TFMSA = Trifluoromethanesulfonic acid THF = Tetrahydrofuran

TLC = Thin Layer Chromatography TMS = Tetramethylsilane

TMSCl = Chlorotrimethylsilane

Tris = Tris(hydroxymethyl)aminomethane

Tryp = Tryptophan

US = Ultrasound

xi  

ABSTRACT

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor, which plays a

pivotal part in the development of the central nervous system. Aberrant expression of

full-length ALK occurs in neuroblastoma and chromosomal translocation or inversion of

the ALK gene can generate novel fusion-ALK proteins that possess constitutive kinase

activity and contribute to oncogenic processes. One of the well-studied fusion proteins is

nucleophosmin (NPM-ALK), which draws a lot of attention for medicinal chemists to

design small molecules as kinase inhibitors for this target. In this dissertation, [1, 2, 4]-

Dihydrotriazine dimers as competitors of the lead compound NVP-TAE684 targeting

NPM-ALK have been designed and synthesized. Molecular modelling studies show that

those dihydrotriazine dimers have a great potential to be better kinase inhibitors.

Chapter two describes imaging in the drug discovery and development arena. One

of important imaging techniques is positron emission tomography (PET). PET is a

radionuclide based molecular imaging technique, which can be used for early detection,

characterization, “real time” monitoring of diseases, and investigation of the efficacy of

drugs. Fluorine-18 (18F) based molecular probes for PET imaging still remain big

challenging to prepare but have gained increased interest by radiochemists in the past two

decades. In this study, a novel approach to introduce fluorine into a molecular probe has

been discovered based on boron chemistry. A few novel fluorine capture reagents have

been synthesized and described in this Chapter.

1 �

CHAPTER ONE:

SYNTHESIS OF [1, 2, 4]-DIHYDROTRIAZINE NUCLEUS AS A NEW KINASE

INHIBITOR

1.1 Introduction

Anaplastic lymphoma kinase (ALK)[1,2] is a receptor tyrosine kinase of the insulin

receptor superfamily, as shown in Figure 1.1. It is believed that it plays a pivotal part in

the development of the central nervous system. Aberrant expression of full-length ALK

occurs in neuroblastoma and chromosomal translocation or inversion of the ALK gene

can generate novel fusion-ALK proteins that possess constitutive kinase activity and

contribute to oncogenic processes[3, 4]. One of the well-studied fusion proteins is�

nucleophosmin (NPM-ALK), which has constitutive tyrosine kinase activity and a strong

oncogenic potential and is responsible for neoplastic transformation5. It is believed that

overexpression and activation of NPM-ALK fusion protein can drive the survival and

proliferation of anaplastic large cell lymphomas (ALCLs)6. The signal transduction

pathway activated by NPM-ALK is shown in Figure7 1.2.

There are a lot of ALK inhibitors that have been reported, the most important

ligand is NVP-TAE684, which is a highly potent and selective small molecule inhibitor

targeting NPM-ALK protein with an IC50 value at 2~10 nM in vitro activity[5, 6, 8].

�

Figu

Figu

ure 1.1 Ana

ure 1.2 ALK

aplastic lymp

K fusion onc

phoma kinas

cogene and m2

se (ALK)-po

major downs

ositive cance

stream signa

ers and its fu

aling pathwa

sion protein

ays.

s.

�

TAE

Figu

to fu

The cry

E684 bound

ure 1.3 Crys

The mo

urther invest

ystal structur

d is shown in

stal structure

lecular mod

tigate the se

re of human

n Figure 1.3

e of human A

delling of N

electivity of

3

n anaplastic

3 (PDB ID: 2

ALK with N

NVP-TAE68

f this ligand,

c lymphoma

2XB7)5.

NVP-TAE684

84 at ALK b

, as shown i

a kinase (A

4.

binding site

in Figure 1.4

ALK) with N

�

e was carrie

49.

NVP-

ed out

�

Figu

with

grou

L25

L25

oppo

site.

1.2 R

resid

dihy

ALK

ure 1.4 A m

TAE684

h residue L2

up on the 2-

8 and M259

8 is one of

ortunity to d

Results & D

1.2.1 De

The mo

due L258 a

ydro-1,2,4-tr

K-driven can

model of ALK

4 binds to t

258 at the ki

-aniline sub

9, it leads to

the major k

design bette

Discussions

esign of the

olecular mod

at the ALK

riazin-6(1H

ncers, as sho

K in comple

the ATP bin

inase “hinge

bstituted (rin

o a steric cl

kinase select

er ligands to

e Potential K

delling show

K binding s

H)-one was

own in Figu

4

x with NVP

nding site th

e” region of

ng A) points

lash for this

tivity determ

o improve t

Kinase Inhi

ws that liga

site, so a n

designed a

ure 1.5.

P-TAE684.

hrough the

f ALK[6, 10, 1

s into a sma

s binding po

minants for

the binding

ibitor

and TAE684

novel scaff

as a small-m

bidentate h

11]. Second,

all cleft betw

ocket. It sug

TAE684, w

affinity wit

4 has a ster

fold of 3-(1

molecule in

hydrogen bo

the orthome

ween the re

ggests that r

which gives

thin ALK b

ric clash wi

1H-imidazo

nhibitor tar

onding

ethoxy

esidues

esidue

us the

inding

ith the

l)-4,5-

rgeting

�

Figudrive

avoi

main

1.6.

hydr

Figu

ure 1.5 [1, en cancers.

The me

id steric hin

ntain the cru

On the ot

rophobic int

ure 1.6 ligan

Docking

2, 4]-dihydr

thoxy group

ndrance with

ucial hydro

ther hand,

teractions w

nd interaction

g studies w

rotriazine sc

p on the le

h residue L2

gen bondin

two hydrop

with relevant

n at ALK bi

ere carried

5

NN

NHN

NH

OR

R1A

B

caffold as sm

ft hand in r

258, but sti

ng with Glu1

phobic grou

t residues an

nding site ba

out to supp

R2

1

mall molecu

ring A was

ll keep thre

1197 and M

ups R1 and

nd to increa

ased on [1, 2

port this de

ule inhibitor

s removed a

ee nitrogen

Met 1199, as

d R2 were u

ase permeab

2, 4]-dihydro

sign based

rs targeting

at this scaff

atom in ring

s shown in F

used to ma

bility.

otriazine sca

on this [1,

ALK-

fold to

g B to

Figure

aintain

affold.

2, 4]-

6 �

dihydrotriazine scaffold. X-ray crystal structures of ALK enzymes, PDB ID: 2XB7, were

prepared, optimized, and refined using Schrodinger’s Protein Preparation Wizard at a

neutral pH. The ligands to be docked were prepared using Schrodinger’s Ligprep in

which the ionization states were generated within a pH range of 5-9 using Epik. Grids

were prepared using the Glide Grid Generation. Ligands were docked with SP (standard

precision) and XP (extra precision) using default settings.

The docking scores of the XP docking of the ligands with the greatest affinity

ranged from -6.48kcal/mol to -9.484 kcal/mol. (As shown in Table 1.1)

Table 1.1 XP Ligand Dockings to 2XB7

Protein: 2XB7

Water in binding site: Yes

Ligand R1 R2 Docking Score

Known ligand NVP-TAE684 -8.575

Experimental Ligands

1.5a Methyl Methyl -8.12 1.5b Methyl Isobutyl -8.125 1.5c Methyl Benzyl -8.941 1.5d Methyl 3-Methylindole -7.746 1.5e Benzyl Methyl -9.029 1.5f Benzyl Isobutyl -9.094 1.5g Benzyl Benzyl -8.234 1.5h Benzyl 3-Methylindole -8.664 1.5i Isobutyl Methyl -6.48 1.5j Isobutyl Isobutyl -8.495 1.5k Isobutyl Benzyl -8.774 1.5l Isobutyl 3-Methylindole -9.484

�

scaf

supe

corr

inter

Figu

avai

1.3 b

Based o

ffold for th

erimposed,

responding l

ractions of l

ure 1.7 Com

1.2.2 Sy

The 1, 2

ilable imida

by treating

on the dock

his target (N

this mime

ligand resid

ligand 1.5L

mparison of l

ynthesis of [

2, 4-dihydr

azole as sho

with n-buty

king results

NPM-ALK)

etic had s

dues as show

L at the ALK

ligand 1.5L

[1, 2, 4]-dih

rotriazine di

wn in Sche

ylithium and

7

, ligand 1.5

) and it m

side chain

wn in Figure

K binding sit

and NVP-TA

hydrotriazin

imer analog

eme 1.1. The

d carbon dis

5L seems t

may be a b

residues p

e 1.7. Figur

te.

AE684 at th

ne Dimer L

gs were syn

en imidazol

sulfide, follo

to be the be

better ALK

perfectly a

re 1.8 show

he ALK bin

Library

nthesized fro

le was conv

owed by co

est ligand i

inhibitor.

aligned wit

s the dockin

nding site.

om commer

verted to thio

oupled with

in this

When

th the

ng and

rcially

o-ester

amino

�

acid

the

treat

dihy

Figu

d methyl este

last step fo

ted with hy

ydrotriazine

ure 1.8 Liga

er hydrochlo

r formation

ydrazine und

dimer analo

and 1.5L bin

oride to affo

n of 1, 2, 4

der reflux co

ogs 1.5 in u

N

NHN

Li

nding pose at

8

ord compoun

4-dihydrotria

ondition in

up to 78% is

N

NH

O

igand 1.5L

t the ALK b

nd 1.4. The

azine ring.

1,4-dioxane

olated yield

NH

inding site.

e key step in

Ideally, co

e to give th

ds.

n this schem

ompound 1.

he desired 1

�

me was

.4 was

, 2, 4-

9 �

(a) KOH, DMF, Isobutyl bromide, 0-25 oC, 53% (b) n-BuLi, LiBr, CuBr, -45 oC, then CS2,followed by CH3I, 64-70% (c) Amino acid methyl ester hydrochloride, TEA, DCM, 20-25 oC, 63-95%(d) H2NNH2.H2O, 1,4-dioxane, ref lux, 47-78%

b c

d

N

N

R1

N

N

R1

S

S

NN

NHN

NH

OR2

N

N

R1

HN

OS

O

R2

R1

1.2 a-c 1.3 a-c 1.4 a-l

1.5 a-l

aNH

N

1.1

Scheme 1.1 Synthesis of 1, 2, 4-dihydrotriazine dimer.

The preparation of thio-ester 1.3 is listed in Table 1.2, and the following [1, 2, 4]-

dihydrotriazine precursor 1.4 is listed in Table 1.3 and final library compound of [1, 2,

4]-dihydrotriazine dimer was shown in Table 1.5.

Table 1.2 Preparation of thio-ester 1.3

R1 Percent yield

1.3a Methyl 70%

1.3b Benzyl 69%

1.3c Isobutyl 64%

10 �

Table 1.3 Preparation of [1, 2, 4]-dihydrotriazine precursor 1.4

R1 R2 Percent yield

1.4a Methyl Methyl 84%

1.4b Methyl Isobutyl 71%

1.4c Methyl Benzyl 85%

1.4d Methyl 3-Methylindole 69%

1.4e Benzyl Methyl 94%

1.4f Benzyl Isobutyl 63%

1.4g Benzyl Benzyl 88%

1.4h Benzyl 3-Methylindole 72%

1.4i Isobutyl Methyl 85%

1.4j Isobutyl Isobutyl 81%

1.4k Isobutyl Benzyl 95%

1.4l Isobutyl 3-Methylindole 70%

Table 1.4 Library of the synthesized [1, 2, 4]-dihydrotriazine dimer 1.5

R1 R2 Percent yield

1.5a Methyl Methyl 49%

1.5b Methyl Isobutyl 79%

1.5c Methyl Benzyl 57%

1.5d Methyl 3-Methylindole 76%

1.5e Benzyl Methyl 58%

1.5f Benzyl Isobutyl 47%

1.5g Benzyl Benzyl 52%

1.5h Benzyl 3-Methylindole 68%

1.5i Isobutyl Methyl 67%

1.5j Isobutyl Isobutyl 68%

1.5k Isobutyl Benzyl 78%

1.5l Isobutyl 3-Methylindole 72%

11 �

1.3 Conclusion and Future Plan

From the docking studies, a few experimental ligands are better than the known

ligand (NVP-TAE684), especially ligand 1.5L has highest docking score, indicating it

may be the potential candidate as inhibitor of anaplastic lymphoma kinase (ALK)-driven

cancers.

In summary, [1, 2, 4]-dihydrotriazine scaffold has been successfully designed and

library compound has been built up against NPM-ALK. Totally twenty-eight compounds

have been made and twelve [1, 2, 4]-dihydrotriazine dimers have been successfully

synthesized, the overall yield is up to 51% in three step synthesize. All the [1, 2, 4]-

dihydrotriazine dimers will be screened and tested against NPM-ALK enzyme as well as

other ALK-driven enzyme assay for potential anticancer activity in the future study.

1.4 Experimental Section

1.4.1 Materials and Methods

Organic and inorganic reagents (ACS grade) and solvents were obtained from

commercial sources and used without further purification, unless otherwise noted.

Moisture and air-sensitive reactions were carried out under an inert atmosphere of

nitrogen. Thin layer chromatography (TLC) was performed on glass plates pre-coated

with 0.25mm thickness of silica gel (60F-254) with fluorescent indicator. Column

chromatographic purification was performed using silica gel 60 Å, (# 70-230 mesh). All

1H NMR and 13C NMR spectra were recorded on Varian INOVA 400 MHz or Bruker

250 MHz spectrometer at 25 oC in chloroform-d (CDCl3) or dimethyl sulfoxide-d6

(DMSO-d6), unless otherwise specified. Chemical shifts are reported in parts per million

12 �

(ppm) relative to internal standard tetramethylsilane (TMS). Multiplicity is expressed as

(s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, or m = multiplet) and

the values of coupling constants ( J ) are given in Hertz (Hz). High Resolution Mass

Spectrometry (HRMS) spectra were carried out on an Agilent 1100 Series in the ESI-

TOF mode.��

�

1.4.2 Experimental Procedures

1-Isobutyl-1H-imidazole (1.2c)12

To a solution of imidazole 1.1 (3.404 g, 50.0 mmol) in dimethylformamide

(DMF) (100 mL) was added potassium hydroxide (KOH) (4.20 g, 75.0 mmol) at room

temperature under nitrogen. The reaction mixture was stirred at room temperature for 5

hrs, then cooled down to 0 oC with ice bath. Isobutyl bromide (6.85 g, 50.0 mmol) was

then added dropwise, the resulting mixture was allowed to warm up to room temperature

and stirred overnight. The reaction mixture was then concentrated in vacuum and the

residue was partitioned between dichloromethane (DCM) (100 mL) and water (50 mL).

The aqueous layer was extracted with DCM (2* 50 mL), and organic layer was

combined, dried over anhydrous sodium sulfate (Na2SO4), filtered and concentrated to

give a light yellow oil as crude compound. The crude oil was purified by vacuum

distillation to afford compound 1.2c as a colorless oil (3.32 g, 53%). 1HNMR (400MHz,

CDCl3) � ppm = 7.32 (s, 1 H), 6.92 (s, 1 H), 6.77 (s, 1 H), 3.61 (d, J=7.0 Hz, 2 H), 1.74 -

13 �

2.07 (m, 1 H), 0.79 (d, J=6.6 Hz, 6 H). 13CNMR (101MHz, CDCl3) � ppm = 137.3,

129.0, 119.1, 54.4, 30.0, 19.7. HRMS-ESI (m/z): [M+H]+ calcd. for C7H13N2: 125.1073,

found, 125.1078.

Methyl 1-methyl-1H-imidazole-2-carbodithioate (1.3a)13

General Procedure A. To a solution of 10.0 mmol of n-butyllithium in hexanes

was added 20 mL of THF, cooled down to below -40 oC under nitrogen. 1-

Methylimidazole 1.2a (821.0 mg, 10.0 mmol) in 5 mL of tetrahydrofuran (THF) was then

added dropwise during 5 mins at -55 oC. After an additional 10 mins (at the above

temperature mentioned) stirring, a solution of copper (I) bromide (261.08 mg, 1.82 mmol)

and anhydrous lithium bromide (316.1 mg, 3.64 mmol) in 30 mL of THF was added

dropwise during 10mins, followed by carbon disulfide (761.4 mg, 10.0 mmol) in 5 mL of

THF at the same temperature. Methyl iodide (1.562 g, 11.0 mmol) was then added in one

portion. The reaction temperature was then allowed to warm to +15 oC. Once the reaction

was completed, a solution of 1.8 g of potassium cyanide in 40 mL of water was added to

quench this reaction. The reaction solution was then separated, the aqueous layer was

extracted with ethyl acetate, and organic layer was combined, dried over anhydrous

magnesium sulfate (MgSO4), filtered and concentrated in vacuum to obtain a red-brown

oil as crude compound. The resulting crude was purified by flash column

chromatography (silica gel, EtOAc: Hexanes = 1:4 as eluent) to afford compound 1.3a as

14 �

a bright-red solid (1.21 g, 70%). 1HNMR (400MHz, CDCl3) � ppm = 7.05 (s, 1 H), 7.03

(s, 1 H), 3.96 (s, 3 H), 2.57 (s, 3 H). 13CNMR (101MHz, CDCl3) � ppm = 211.8, 147.5,

128.4, 128.2, 37.9, 18.9. HRMS-ESI (m/z): [M+H]+ calcd. for C6H9N2S2: 173.0202,

found, 173.0209.

Methyl 1-benzyl-1H-imidazole-2-carbodithioate (1.3b)

Compound 1.3b was prepared from 1-benzylimidazole 1.2b according to general

procedure A to afford 1.3b as a bright-red oil (69% yield).� 1HNMR (400MHz, CDCl3) �

ppm = 7.21 - 7.34 (m, 3 H), 7.17 (s, 1 H), 7.01 - 7.11 (m, 3 H), 5.81 (s, 2 H), 2.62 (s, 3

H). 13CNMR (101MHz, CDCl3) � ppm = 212.3, 147.1, 136.0, 128.8, 128.5, 127.9, 127.2,

52.0, 19.0. HRMS-ESI (m/z): [M+H]+ calcd. for C12H13N2S2: 249.0515, found,

249.0527.

�

Methyl 1-isobutyl-1H-imidazole-2-carbodithioate (1.3c)

Compound 1.3c was prepared from 1-isobutylimidazole 1.2c according to general

15 �

procedure A to afford 1.3c as a bright-red oil (64% yield). 1HNMR (400MHz, CDCl3) �

ppm = 7.10 (d, 2 H), 4.33 (d, J=7.4 Hz, 2 H), 2.63 (s, 3 H), 2.01 - 2.18 (m, 1 H), 0.86 (d,

J=6.6 Hz, 6 H). 13CNMR (101MHz, CDCl3) � ppm = 212.7, 147.0, 127.9, 127.8, 55.9,

29.5, 19.6, 19.0. HRMS-ESI (m/z): [M+H]+ calcd. for C9H15N2S2: 215.0671, found,

215.0678.

Methyl 2-(1-methyl-1H-imidazole-2-carbothioamido)propanoate (1.4a)14

General Procedure B. To a solution of alanine methyl ester hydrochloride (844.5

mg, 6.05 mmol) in 40 mL of anhydrous CH2Cl2 at room temperature (RT) under nitrogen

was added triethylamine (TEA) (0.914 mL, 6.6 mmol), followed by compound 1.3a

(947.5 mg, 5.5 mmol).

The reaction mixture was then stirred for 15 hrs at room temperature, and the

solvent was evaporated. The resulting residue was purified by flash column

chromatography (silica gel, EtOAc: Hexanes = 1:4 as eluent) to afford compound 1.4a as

a light yellow solid (1.05 g, 84%). 1HNMR (400MHz, CDCl3) � ppm = 9.49 - 9.87 (m, 1

H), 7.00 (d, J=7.8 Hz, 2 H), 5.15 (t, J=7.2 Hz, 1 H), 4.15 (s, 3 H), 3.76 (s, 3 H), 1.58 (d, 3

H). 13CNMR (101MHz, CDCl3) � ppm = 182.0, 172.1, 142.2, 127.5, 126.3, 52.5, 52.4,

38.0, 17.1. HRMS-ESI (m/z): [M+H]+ calcd. for C9H14N3O2S: 228.0801, found,

228.081.

16 �

Methyl 4-methyl-2-(1-methyl-1H-imidazole-2-carbothioamido)pentanoate

(1.4b)

Compound 1.4b was prepared from compound 1.3a and leucine methyl ester

hydrochloride according to general procedure B to afford 1.4b as a light yellow solid (71%

yield).� 1HNMR (400MHz, CDCl3) � ppm = 9.59 (d, J=5.9 Hz, 1 H), 7.01 (d, J=8.6 Hz, 2

H), 5.13 - 5.25 (m, 1 H), 4.17 (s, 3 H), 3.74 (s, 3 H), 1.81 - 1.89 (m, 2 H), 1.69 - 1.81 (m,

1 H), 0.96 (dd, 6 H). 13CNMR (101MHz, CDCl3) � ppm = 182.4, 171.9, 142.0, 127.5,

126.2, 55.3, 52.3, 40.6, 38.1, 25.0, 22.7, 22.1. HRMS-ESI (m/z): [M+H]+ calcd. for

C12H20N3O2S: 270.1271, found, 270.1282.�

Methyl 2-(1-methyl-1H-imidazole-2-carbothioamido)-3-phenylpropanoate

(1.4c)

Compound 1.4c was prepared from compound 1.3a and phenylalanine methyl

ester hydrochloride according to general procedure B to afford 1.4c as a light yellow oil

(85% yield).� 1HNMR (400MHz, CDCl3) � ppm = 9.78 (d, J=5.9 Hz, 1 H), 7.21 (m, 5 H),

7.00 (d, J=10.9 Hz, 2 H), 5.39 - 5.51 (m, 1 H), 4.15 (s, 3 H), 3.70 (s, 3 H), 3.32 (t, J=6.8

17 �

Hz, 2 H). 13CNMR (101MHz, CDCl3) � ppm = 181.9, 170.7, 142.0, 135.7, 129.2, 128.6,

127.5, 127.1, 126.2, 57.8, 52.3, 38.1, 37.1. HRMS-ESI (m/z): [M+H]+ calcd. for

C15H18N3O2S: 304.1114, found, 304.1119.�

Methyl 3-(1H-indol-3-yl)-2-(1-methyl-1H-imidazole-2-

carbothioamido)propanoate (1.4d)

N

NHN

O

O

SNH

Compound 1.4d was prepared from compound 1.3a and tryptophan methyl ester

hydrochloride according to general procedure B to afford 1.4d as yellow foamy powder

(69% yield).� 1HNMR (400MHz, CDCl3) � ppm = 9.75 (d, 1 H), 8.31 (br. s., 1 H), 7.57 (d,

J=7.8 Hz, 1 H), 7.23 - 7.31 (m, 1 H), 7.11 - 7.18 (m, 1 H), 7.04 - 7.11 (m, 2 H), 6.94 (d,

J=3.9 Hz, 2 H), 5.49 (d, J=7.4 Hz, 1 H), 4.13 (s, 3 H), 3.65 (s, 3 H), 3.48 - 3.53 (m, 2 H).

13CNMR (101MHz, CDCl3) � ppm = 182.0, 171.1, 142.2, 136.1, 127.4, 127.3, 126.2,

123.1, 122.1, 119.5, 118.6, 111.2, 109.6, 57.2, 52.5, 38.0, 27.0. HRMS-ESI (m/z):

[M+H]+ calcd. for C17H19N4O2S: 343.1223, found, 343.1238.�

Methyl 2-(1-benzyl-1H-imidazole-2-carbothioamido)propanoate (1.4e)

18 �

Compound 1.4e was prepared from compound 1.3b and alanine methyl ester

hydrochloride according to general procedure B to afford 1.4e as yellow oil (94% yield).�

1HNMR (400MHz, CDCl3) � ppm = 9.85 (d, J=7.0 Hz, 1 H), 7.23 - 7.38 (m, 3 H), 7.17

(d, J=7.0 Hz, 2 H), 7.03 (s, 1 H), 6.96 (s, 1 H), 6.05 (s, 2 H), 5.01 - 5.27 (m, 1 H), 3.76 (s,

3 H), 1.58 (d, J=7.4 Hz, 3 H). 13CNMR (101MHz, CDCl3) � ppm = 182.1, 172.1, 142.1,

136.6, 128.7, 127.8, 127.6, 126.9, 126.1, 52.5, 52.4, 52.1, 17.1. HRMS-ESI (m/z):

[M+H]+ calcd. for C15H18N3O2S: 304.1114, found, 304.1107.��

Methyl 2-(1-benzyl-1H-imidazole-2-carbothioamido)-4-methylpentanoate

(1.4f)

Compound 1.4f was prepared from compound 1.3b and leucine methyl ester

hydrochloride according to general procedure B to afford 1.4f as yellow oil (63% yield).�

1HNMR (400MHz, CDCl3) � ppm = 9.69 (d, J=7.0 Hz, 1 H), 7.22 - 7.39 (m, 3 H), 7.18

(d, J=7.0 Hz, 2 H), 7.04 (s, 1 H), 6.96 (s, 1 H), 6.06 (d, J=6.2 Hz, 2 H), 5.21 (d, J=7.0 Hz,

1 H), 3.74 (s, 3 H), 1.81 - 1.89 (m, 2 H), 1.68 - 1.81 (m, 1 H), 0.97 (dd, J=10.5, 6.6 Hz, 6

H). 13CNMR (101MHz, CDCl3) � ppm = 182.5, 171.8, 142.1, 136.5, 128.7, 127.8, 127.6,

126.8, 126.1, 55.3, 52.4, 52.1, 40.6, 25.0, 22.7 22.2. HRMS-ESI (m/z): [M+H]+ calcd. for

C18H24N3O2S: 346.1584, found, 346.1601.��

19 �

Methyl 2-(1-benzyl-1H-imidazole-2-carbothioamido)-3-phenylpropanoate

(1.4g)

Compound 1.4g was prepared from compound 1.3b and phenylalaine methyl ester

hydrochloride according to general procedure B to afford 1.4g as yellow oil (88% yield).�

1HNMR (400MHz, CDCl3) � ppm = 9.79 (d, 1 H), 7.21 - 7.41 (m, 6 H), 7.17 (d, J=7.0 Hz,

4 H), 6.94 - 7.07 (m, 2 H), 5.97 - 6.17 (m, 2 H), 5.48 (d, J=7.0 Hz, 1 H), 3.71 (s, 3 H),

3.23 - 3.42 (m, 2 H). 13CNMR (101MHz, CDCl3) � ppm = 182.1, 170.7, 142.1, 136.6,

135.7, 129.2, 128.7, 128.6, 127.8, 127.5, 127.1, 127.0, 126.2, 57.7, 52.4, 52.1, 37.0.

HRMS-ESI (m/z): [M+H]+ calcd. for C21H22N3O2S: 380.1427, found, 380.1411.��

Methyl 2-(1-benzyl-1H-imidazole-2-carbothioamido)-3-(1H-indol-3-

yl)propanoate (1.4h)

Compound 1.4h was prepared from compound 1.3b and tryptophan methyl ester

hydrochloride according to general procedure B to afford 1.4h as yellow foamy powder

(72% yield). 1HNMR (400MHz, CDCl3) � ppm = 9.75 (d, 1 H), 8.11 (br. s., 1 H), 7.56 (d,

20 �

J=7.8 Hz, 1 H), 7.26 - 7.37 (m, 4 H), 7.12 - 7.21 (m, 3 H), 7.03 - 7.10 (m, 2 H), 6.97 (d,

J=14.8 Hz, 2 H), 5.97 - 6.16 (m, 2 H), 5.50 (d, J=7.4 Hz, 1 H), 3.65 (s, 3 H), 3.51 (t,

J=6.4 Hz, 2 H). 13CNMR (101MHz, CDCl3) � ppm = 182.1, 171.1, 142.3, 136.7, 136.0,

128.7, 127.8, 127.6, 127.3, 127.0, 126.1, 123.0, 122.1, 119.5, 118.7, 111.1, 109.8, 57.1,

52.4, 52.1, 26.8. HRMS-ESI (m/z): [M+H]+ calcd. for C23H23N4O2S: 419.1536, found,

419.1553.��

Methyl 2-(1-isobutyl-1H-imidazole-2-carbothioamido)propanoate (1.4i)

Compound 1.4i was prepared from compound 1.3c and alanine methyl ester

hydrochloride according to general procedure B to afford 1.4i as yellow oil (85% yield).�

1HNMR (400MHz, CDCl3) � ppm = 9.79 (d, J=4.4 Hz, 1 H), 7.00 (d, J=5.9 Hz, 2 H),

5.15 (t, J=7.0 Hz, 1 H), 4.50 (t, J=7.8 Hz, 2 H), 3.76 (s, 3 H), 2.13 - 2.32 (m, 1 H), 1.58

(d, J=7.0 Hz, 3 H), 0.89 (d, J=7.0 Hz, 6 H). 13CNMR (101MHz, CDCl3) � ppm = 182.1,

172.1, 141.9, 126.9, 125.9, 56.1, 52.5, 52.3, 29.7, 19.6, 17.1. HRMS-ESI (m/z): [M+H]+

calcd. for C12H20N3O2S: 270.1271, found, 270.1277.��

� �

21 �

Methyl 2-(1-isobutyl-1H-imidazole-2-carbothioamido)-4-methylpentanoate

(1.4j)

Compound 1.4j was prepared from compound 1.3c and leucine methyl ester

hydrochloride according to general procedure B to afford 1.4j as yellow oil (81% yield).�

1HNMR (400MHz, CDCl3) � ppm = 9.66 (d, J=6.0 Hz, 1 H), 6.99 (d, J=4.7 Hz, 2 H),

5.19 (d, J=7.0 Hz, 1 H), 4.50 (dd, J=9.8, 7.4 Hz, 2 H), 3.73 (s, 3 H), 2.15 - 2.31 (m, 1 H),

1.80 - 1.87 (m, 2 H), 1.70 - 1.79 (m, 1 H), 0.95 (dd, J=10.2, 6.2 Hz, 6 H), 0.88 (dd, J=6.8,

2.5 Hz, 6 H). 13CNMR (101MHz, CDCl3) � ppm = 182.6, 171.9, 141.9, 126.9, 126.0,

56.1, 55.3, 52.3, 40.6, 29.7, 25.0, 22.6, 22.1, 19.7, 19.6. HRMS-ESI (m/z): [M+H]+ calcd.

for C15H26N3O2S: 312.174, found, 312.1757.��

Methyl 2-(1-isobutyl-1H-imidazole-2-carbothioamido)-3-phenylpropanoate

(1.4k)

Compound 1.4k was prepared from compound 1.3c and phenylalanine methyl

ester hydrochloride according to general procedure B to afford 1.4k as yellow oil (95%

yield).� 1HNMR (400MHz, CDCl3) � ppm = 9.78 (d, J=7.2 Hz, 1 H), 7.13 - 7.33 (m, 5 H),

22 �

6.98 (d, J=6.2 Hz, 2 H), 5.45 (d, J=7.4 Hz, 1 H), 4.50 (d, J=7.4 Hz, 2 H), 3.69 (s, 3 H),

3.31 (dd, J=9.4, 6.2 Hz, 2 H), 2.13 - 2.28 (m, 1 H), 0.88 (dd, J=6.6, 3.1 Hz, 6 H).

13CNMR (101MHz, CDCl3) � ppm = 182.1, 170.7, 141.8, 135.7, 129.2, 128.5, 127.1,

126.9, 126.0, 57.7, 56.1, 52.3, 37.1, 29.7, 19.6. HRMS-ESI (m/z): [M+H]+ calcd. for

C18H24N3O2S: 346.1584, found, 346.1569.��

Methyl 3-(1H-indol-3-yl)-2-(1-isobutyl-1H-imidazole-2-

carbothioamido)propanoate (1.4l)

Compound 1.4l was prepared from compound 1.3c and tryptophan methyl ester

hydrochloride according to general procedure B to afford 1.4l as yellow foamy solid (70%

yield). 1HNMR (400MHz, CDCl3) � ppm = 9.87 (d, J=7.6 Hz, 1 H), 8.26 (br. s., 1 H),

7.56 (d, J=7.8 Hz, 1 H), 7.21 - 7.32 (m, 1 H), 7.02 - 7.17 (m, 3 H), 6.94 (s, 2 H), 5.49 (d,

J=7.4 Hz, 1 H), 4.48 (d, J=7.0 Hz, 2 H), 3.64 (s, 3 H), 3.51 (d, J=5.5 Hz, 2 H), 2.14 - 2.30

(m, 1 H), 0.88 (d, J=6.6 Hz, 6 H). 13CNMR (101MHz, CDCl3) � ppm = 181.9, 171.1,

141.8, 136.1, 127.3, 126.7, 125.7, 123.1, 122.0, 119.4, 118.6, 111.1, 109.7, 57.3, 56.1,

52.4, 29.7, 26.9, 19.6. HRMS-ESI (m/z): [M+H]+ calcd. for C20H25N4O2S: 385.1693,

found, 385.1699.

23 �

5-Methyl-3-(1-methyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5a)15

General Procedure C. To a solution of compound 1.4a (754 mg, 3.32 mmol) in

25 mL of 1,4-dioxane at room temperature (RT) under nitrogen was added hydrazine

hydrate (830 mg, 16.6 mmol), then the reaction solution was heated up to reflux for

24hrs. The solvent was then evaporated in vacuum, and the resulting residue was purified

by flash column chromatography to afford compound 1.5a as an off-white solid (315 mg,

49%). 1HNMR (400MHz, DMSO-d6) � ppm = 10.46 (s, 1 H), 7.28 (s, 1 H), 7.21 (s, 1 H),

6.97 (s, 1 H), 3.90 (q, J=6.6 Hz, 1 H), 3.82 (s, 3 H), 1.26 (d, J=6.6 Hz, 3 H). 13CNMR

(101MHz, DMSO-d6) � ppm = 164.7, 139.8, 138.5, 127.2, 125.8, 48.7, 36.0, 19.0.

HRMS-ESI (m/z): [M+H]+ calcd. for C8H12N5O: 194.1036, found, 194.1046.

5-Isobutyl-3-(1-methyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5b)

Compound 1.5b was prepared from compound 1.4b according to general

24 �

procedure C to afford 1.5b as an off-white solid (79% yield).� 1HNMR (400MHz, DMSO-

d6) � ppm = 10.49 (s, 1 H), 7.28 (s, 1 H), 7.14 (s, 1 H), 6.96 (s, 1 H), 3.78 - 3.86 (m, 4 H),

1.69 - 1.83 (m, 1 H), 1.38 - 1.47 (m, 2 H), 0.87 (d, J=6.6 Hz, 6 H). 13CNMR (101MHz,

DMSO-d6) � ppm = 164.3, 139.7, 138.6, 127.5, 125.9, 51.4, 41.5, 35.9, 23.9, 23.2, 22.6.

HRMS-ESI (m/z): [M+H]+ calcd. for C11H18N5O: 236.1506, found, 236.152.

5-Benzyl-3-(1-methyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5c)

Compound 1.5c was prepared from compound 1.4c according to general

procedure C to afford 1.5c as an off-white solid (57% yield). 1HNMR (400MHz, DMSO-

d6) � ppm = 10.41 (s, 1 H), 7.14 - 7.26 (m, 6 H), 7.05 (s, 1 H), 6.95 (s, 1 H), 4.21 (t, J=4.7

Hz, 1 H), 3.67 (s, 3 H), 2.97 (dd, J=14.4, 5.1 Hz, 2 H). 13CNMR (101MHz, DMSO-d6) �

ppm = 162.9, 139.4, 138.5, 137.0, 130.3, 128.4, 127.4, 126.8, 125.6, 54.4, 38.7, 35.8.

HRMS-ESI (m/z): [M+H]+ calcd. for C14H16N5O: 270.1349, found, 270.1362.

25 �

5-((1H-indol-3-yl)methyl)-3-(1-methyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-

triazin-6(1H)-one (1.5d)

Compound 1.5d was prepared from compound 1.4d according to general

procedure C to afford 1.5d as an off-white solid (76% yield). 1HNMR (400MHz, DMSO-

d6) � ppm = 10.84 (br. s., 1 H), 10.36 (s, 1 H), 7.50 (d, J=8.2 Hz, 1 H), 7.29 (d, J=8.2 Hz,

1 H), 7.20 (s, 1 H), 7.11 (d, J=1.6 Hz, 1 H), 6.81 - 7.05 (m, 4 H), 4.19 (t, J=4.9 Hz, 1 H),

3.60 (s, 3 H), 3.11 - 3.21 (m, 1 H), 2.98 - 3.10 (m, 1 H). 13CNMR (101MHz, DMSO-d6) �

ppm = 163.5, 139.4, 138.6, 136.4, 127.9, 127.3, 125.6, 124.6, 121.2, 118.9, 118.5, 111.6,

109.2, 53.8, 35.7, 28.9. HRMS-ESI (m/z): [M+H]+ calcd. for C16H17N6O: 309.1458,

found, 309.1463.

3-(1-Benzyl-1H-imidazol-2-yl)-5-methyl-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5e)

Compound 1.5e was prepared from compound 1.4e according to general procedure C

26 �

to afford 1.5e as a light yellow solid (58% yield). 1HNMR (400MHz, DMSO-d6) � ppm =

10.45 (s, 1 H), 7.38 (s, 1 H), 7.16 - 7.35 (m, 6 H), 7.02 (s, 1 H), 5.54 - 5.67 (m, 2 H), 3.89 (q,

J=6.5 Hz, 1 H), 1.24 (d, J=6.6 Hz, 3 H). 13CNMR (101MHz, DMSO-d6) � ppm = 164.6,

139.8, 138.3, 128.9, 128.0, 127.9, 127.8, 124.9, 50.5, 48.8, 19.0. HRMS-ESI (m/z): [M+H]+

calcd. for C14H16N5O: 270.1349, found, 270.1351.

3-(1-Benzyl-1H-imidazol-2-yl)-5-isobutyl-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5f)

Compound 1.5f was prepared from compound 1.4f according to general procedure C

to afford 1.5f as a light yellow solid (47% yield). 1HNMR (400MHz, CDCl3) � ppm = 8.58

(s, 1 H), 7.23 - 7.38 (m, 3 H), 7.15 (d, J=7.0 Hz, 2 H), 7.04 (s, 1 H), 6.92 (s, 1 H), 6.47 (br. s.,

1 H), 5.52 - 5.69 (m, 2 H), 4.08 (dd, J=7.4, 4.7 Hz, 1 H), 1.81 - 1.94 (m, 1 H), 1.69 - 1.78 (m,

1 H), 1.59 - 1.68 (m, 1 H), 0.96 (dd, J=9.0, 6.6 Hz, 6 H). 13CNMR (101MHz, CDCl3) � ppm

= 164.6, 139.4, 137.9, 136.6, 128.8, 128.0, 127.8, 127.5, 123.9, 51.7, 51.6, 41.5, 23.9, 23.1,

21.5. HRMS-ESI (m/z): [M+H]+ calcd. for C17H22N5O: 312.1819, found, 312.1827.

�

27 �

5-Benzyl-3-(1-benzyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5g)

Compound 1.5g was prepared from compound 1.4g according to general

procedure C to afford 1.5g as a white foamy powder (52% yield). 1HNMR (400MHz,

CDCl3) � ppm = 8.50 (s, 1 H), 7.17 - 7.42 (m, 8 H), 7.10 (d, J=7.4 Hz, 2 H), 7.02 (s, 1 H),

6.89 (s, 1 H), 6.52 (br. s., 1 H), 5.45 - 5.60 (m, 2 H), 4.28 - 4.38 (m, 1 H), 3.24 (dd,

J=13.7, 3.5 Hz, 1 H), 3.01 (dd, J=13.7, 8.2 Hz, 1 H). 13CNMR (101MHz, CDCl3) � ppm

= 163.4, 139.0, 137.6, 136.5, 135.7, 129.7, 128.8, 128.7, 127.9, 127.5, 127.4, 127.0,

123.8, 55.0, 51.6, 39.3. HRMS-ESI (m/z): [M+H]+ calcd. for C20H20N5O: 346.1662,

found, 346.1673.

5-((1H-indol-3-yl)methyl)-3-(1-benzyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-

triazin-6(1H)-one (1.5h)

Compound 1.5h was prepared from compound 1.4h according to general

28 �

procedure C to afford 1.5h as an off-white solid (68% yield). 1HNMR (400MHz, CDCl3)

� ppm = 8.48 (s, 1 H), 8.25 (br. s., 1 H), 7.64 (d, J=7.8 Hz, 1 H), 7.22 - 7.38 (m, 4 H),

6.96 - 7.21 (m, 6 H), 6.84 (s, 1 H), 6.64 (br. s., 1 H), 5.35 - 5.53 (m, 2 H), 4.36 (d, J=5.1

Hz, 1 H), 3.44 (dd, J=14.6, 2.9 Hz, 1 H), 3.13 (dd, J=14.4, 8.2 Hz, 1 H). 13CNMR

(101MHz, CDCl3) � ppm = 163.9, 138.9, 137.6, 136.4, 136.3, 128.8, 128.0, 127.5, 127.1,

127.0, 123.8, 123.6, 122.1, 119.4, 118.7, 111.1, 109.6, 53.8, 51.6, 29.2. HRMS-ESI

(m/z): [M+H]+ calcd. for C22H21N6O: 385.1771, found, 385.1781.

3-(1-Isobutyl-1H-imidazol-2-yl)-5-methyl-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5i)

Compound 1.5i was prepared from compound 1.4i according to general procedure

C to afford 1.5i as an off-white solid (67% yield). 1HNMR (400MHz, CDCl3) � ppm =

9.14 (br. s., 1 H), 7.04 (s, 1 H), 6.96 (s, 1 H), 6.57 (br. s., 1 H), 4.16 (d, J=7.0 Hz, 3 H),

2.04 - 2.22 (m, 1 H), 1.48 (d, J=6.6 Hz, 3 H), 0.89 (d, J=6.6 Hz, 6 H). 13CNMR

(101MHz, CDCl3) � ppm = 165.2, 139.8, 137.8, 127.0, 124.6, 55.4, 49.0, 29.5, 19.7, 18.7.

HRMS-ESI (m/z): [M+H]+ calcd. for C11H18N5O: 236.1506, found, 236.1517.

�

29 �

5-Isobutyl-3-(1-isobutyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-

one (1.5j)

Compound 1.5j was prepared from compound 1.4j according to general procedure

C to afford 1.5j as an off-white solid (68% yield). 1HNMR (400MHz, CDCl3) � ppm =

9.00 (s, 1 H), 7.01 (s, 1 H), 6.93 (s, 1 H), 6.53 (br. s., 1 H), 4.10 - 4.22 (m, 2 H), 4.02 -

4.10 (m, 1 H), 2.04 - 2.17 (m, 1 H), 1.80 - 1.92 (m, 1 H), 1.67 - 1.77 (m, 1 H), 1.56 - 1.66

(m, 1 H), 0.94 (dd, J=9.8, 6.6 Hz, 6 H), 0.83 - 0.90 (m, 6 H). 13CNMR (101MHz, CDCl3)

� ppm = 164.9, 139.5, 137.9, 127.2, 124.6, 55.4, 51.6, 41.5, 29.5, 23.9, 23.0, 21.6, 19.8,

19.7. HRMS-ESI (m/z): [M+H]+ calcd. for C14H24N5O: 278.1975, found, 278.1991.

5-Benzyl-3-(1-isobutyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-one

(1.5k)

Compound 1.5k was prepared from compound 1.4k according to general

procedure C to afford 1.5k as a pink foamy solid (78% yield). 1HNMR (400MHz, CDCl3)

30 �

� ppm = 8.57 (s, 1 H), 7.14 - 7.34 (m, 5 H), 6.99 (s, 1 H), 6.89 (s, 1 H), 6.48 (br. s., 1 H),

4.31 (dd, J=7.2, 3.3 Hz, 1 H), 3.97 - 4.14 (m, 2 H), 3.21 (dd, J=13.7, 3.5 Hz, 1 H), 3.01

(dd, J=13.7, 7.8 Hz, 1 H), 1.91 - 2.05 (m, 1 H), 0.79 - 0.85 (m, 6 H). 13CNMR (101MHz,

CDCl3) � ppm = 163.4, 139.1, 137.5, 135.8, 129.7, 128.6, 127.0, 126.9, 124.5, 55.4, 55.0,

39.3, 29.3, 19.7. HRMS-ESI (m/z): [M+Na]+ calcd. for C17H21N5ONa: 334.1638,

found, 334.1639.

5-((1H-indol-3-yl)methyl)-3-(1-isobutyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4-

triazin-6(1H)-one (1.5l)

Compound 1.5l was prepared from compound 1.4l according to general procedure

C to afford 1.5l as a white foamy solid (72% yield). 1HNMR (400MHz, CDCl3)� �� ppm�=

8.55 (s, 1 H), 8.36 (br. s., 1 H), 7.63 (d, J=8.2 Hz, 1 H), 7.22 - 7.32 (m, 1 H), 7.07 - 7.17

(m, 2 H), 7.00 - 7.07 (m, 1 H), 6.96 (s, 1 H), 6.86 (s, 1 H), 6.53 (s, 1 H), 4.33 (dd, J=8.6,

2.3 Hz, 1 H), 4.01 (d, J=7.4 Hz, 2 H), 3.47 (d, J=3.5 Hz, 1 H), 3.43 (d, J=3.1 Hz, 1 H),

1.88 - 2.04 (m, 1 H), 0.81 (t, J=6.2 Hz, 6 H). 13CNMR (101MHz, CDCl3) �� ppm = 164.1,

139.2, 137.6, 136.3, 127.1, 126.7, 124.4, 123.8, 122.0, 119.4, 118.7, 111.1, 109.6, 55.4,

53.7, 29.3, 29.1, 19.7, 19.6. HRMS-ESI (m/z): [M+H]+ calcd. for C19H23N6O:

351.1928, found, 351.1926.�

31 �

1.5 References

1. Iwahara, T.; Fujimoto, J.; et al. Oncogene, 1997, 14 (4), 439-449.

2. Hallberg, B.; Palmer, R. H. Nature Reviews Cancer, 2013, 13, 685-700.

3. George, R. E.; Sand, T.; et al. Nature, 2008, 455, 975-978.

4. Morris, S. W.; Kirstein, M. N.; et al. Science, 1994, 263, 1281-4.

5. Bossi, R. T.; Saccardo, M. B.; et al. Biochemistry, 2010, 49(32), 6813-6825.

6. Galkin, A. V.; Melnick, J. S.; et al. Proc. Natl. Acad. Sci. U.S. A. , 2007, 104, 270-275.

7. Shaw, A. T.; Solomon, B. Clin Cancer Res, 2011, 17 (8), 2081-2086.

8. Lewis, R. T.; Bode, C. M.; et al. J. Med. Chem., 2012, 55 (14), 6523–6540.

9. Schindler, T.; Bornmann, W.; et al. Science, 2000, 289, 1938-1942.

10. Mol, C. D.; Dougan, D. R.; et al. J. Biol. Chem., 2004, 279, 31655-31663.

11. Hubbard, S. R.; Wei, L.; et al. Nature, 1994, 372, 746-754.

12. Starikova, O. V.; Dolgushin, G. V.; Larina, L. I.; et al. ARKIVOC (Gainesville, FL,

United States) 2003, (13), 119-124.

13. Verkruijsse, H. D.; Brandsma, L. Journal of Organometallic Chemistry 1987, 332,

95-8.

14. Pfund, E.; Masson, S.; Vazeux, M.; Lequeux, T. Journal of organic

Chemistry 2004, 69(14), 4670-6.

15. Saniere, L.; Schmitt, M.; Pellegrini, N.; Bourguignon, J.J.

Heterocycles 2001, 55(4), 671-688.

�

32 �

CHAPTER TWO:

NOVEL BUILDING BLOCKS FOR 18F-RADIOLABELING OF MOLECULAR

PROBE FOR PET IMAGING

2.1 Introduction

2.1.1 PET Imaging, Molecular Probe and Radiotracers

Current imaging techniques including X-rays, ultrasound (US) and magnetic

resonance imaging (MRI) have many anatomical applications but give very limited

information on metabolic or molecular events. Accordingly, the development of novel

approaches to image and monitor real-time molecular events in vivo has seen increasing

demand[1-4]. One such technique that has been developed is positron emission

tomography (PET). PET is a radionuclide based molecular imaging technique, which can

be used for early detection, characterization, “real time” monitoring of diseases, and

investigating the efficacy of drugs[5-8]. More recently, it has become an important clinical

diagnostic and research method as well as a valuable tool in the drug discovery and

development arena.

PET imaging techniques rely on the use of exogenous radioactive probe

(molecular probe) to provide a detectable signal. The probes can be designed as tissue-

based or receptor-based molecules and give a detailed picture of the targeted structure or

biological processes[9, 10]. A few biologically interesting molecular probes for PET

imaging have been reported in the last few years and used for diagnostic clinical studies,

�

how

for s

radio

posi

then

and

Figuposi

far,

wever, the de

synthetic ch

Molecul

onuclides, w

itron emitte

n is taken up

recorded, a

ure 2.1 Printron.

Several

such as 11C

evelopment

hemists.

lar probes

which emit

d from the

p by an elect

s shown in F

ciple of PET

positron-em

, 13N, 15O, 1

t of highly e

for PET i

a positively

nucleus tra

tron, which

Figure 2.1.

T imaging5.

mitting radio

8F, 68Ga, 64C

33

efficient mo

imaging ar

y charged p

avels a short

h generates e

18F atom on

onuclides fo

Cu, as show

olecular pro

re radiolabe

particle, pos

t distance in

energy (as a

n the sugar m

or PET imag

wn in Table 2

obes still rem

eled with p

sitron during

n the surrou

a photon) tha

molecule dec

ging have b

2.1.

mains a cha

positron em

g decay[5, 11

unding tissu

at can be de

cays by emi

been develop

allenge

mitting

1]. The

ue and

etected

itting a

ped so

�

Tab

the m

prop

posi

avai

minu

com

cons

ble 2.1 Comm

Among

most ideal

perties and

itron range

ilable positr

ute half-lif

mmercially

sidered an e

monly Used

all the com

radionuclid

nuclear ch

in tissue)

ron emitters

fe allows

available d

excellent pos

Positron-Em

mmon radion

de as 18F-lab

haracteristics

that gives

, as shown i

for comp

distribution

sitron emitti

34

mitting Radio

nuclides used

beled PET a

s[12, 13]. Fir

18F the hig

in Table 2.1

lex radiosy

to clinica

ing radionu

onuclides5

d in PET, fl

agents have

st, 18F has

ghest resolu

1. Secondly

ynthesis, l

al PET cen

clide for PE

luorine-18 (

the most f

low positr

ution PET i

, a short but

longer in

nters. In s

ET in clinica

(18F) is cons

favorable ph

ron energy

images of a

t manageab

vivo study

summary,

al practice.

�

sidered

hysical

(short

all the

ble 110

y and

18F is

�

2.2 C

18F

deve

cow

[18F

Scheof [1

biom

show

Schesubs

Current Str

2.2.1 Ca

2.2.1.1 E

Currentl

radiolabelin

elopment o

workers repo

F]FDG, as s

eme 2.1 Ele8F]FDG.

Another

marker [18F]

wn in Schem

eme 2.2 Pstitution usin

rategies for

arbon-Fluo

Electrophil

ly, electroph

ng. Howev

f 18F-labele

orted electr

shown in Sch

ectrophilic 18

r example w

]EF5 was p

me 2.2.

Preparation ng [18F]F2.

r Fluorine-1

orine Bond

lic Fluorine

hilic 18F fluo

ver, historic

ed PET ima

rophilic flu

heme 2.1.�

8F fluorinati

was reporte

prepared by

of the hyp

35

18 Radiolab

Formation

e-18 Labelin

orinations a

cally they h

aging agent

uorination u

ion using [18

ed by Dolb

y direct elec

poxia biom

beling

n

ng Reaction

are not comm

have played

ts. For exam

using [18F]a

8F] acetyl hy

bier15 in 20

ctrophilic s

marker [18F]

ns

mon synthet

d an impo

mple, Ehren

acetyl hypo

ypofluorite f

001, a new

substitution

EF5 by di

tic approach

rtant role

nkaufer14 an

ofluorite to

for the prepa

type of hy

using [18F]

irect electro

hes for

in the

nd his

make

aration

ypoxia

]F2, as

ophilic

�

impo

com

in F

Figugree

whic

disso

the p

Sche

Sche

2.2.1.2 N

Nucleop

ortant 18F P

mmon strateg

igure 2.2.

ure 2.2 Comen: fluoride i

The aza

ch leaves th

olved in pol

A comm

precursor o

eme 2.3)

eme 2.3 Syn

Nucleophili

philic 18F-f

PET radiotr

gies is to ad

mplexation oion.

acryptand K2

he fluoride a

lar aprotic s

mon exampl

f [18F]FDG

nthesis of [18

ic 18F-Subs

fluorination

tracers and

dd the phas

f a potassium

222 (Figure

anion expos

solvents suc

le of direct

, which was

F]FDG prec

36

titution Re

reactions

many exam

e-transfer re

m ion (blue)

e 2.2) has a

sed, inducin

h as DMF, D

ed nucleoph

s first repor

cursor: prote

eactions

are comm

mples have

eagent kryp

) by the azac

strong attra

ng its strong

DMSO or a

hilic 18F sub

rted by Ham

cted [18F] su

monly used

been repo

ptofix-222 (

cryptand kry

action to the

nucleophili

acetonitrile.

bstitution is

macher17 in

ugar.

to make

orted. One

K222)16, as s

yptofix-222

e potassium

ic character

s the synthe

1986. (Sho

some

of the

shown

(K222);

cation

r when

esis of

own in

�

nucl

Sche

Sche

form

carb

For

therm

work

aryl

Sche(mos

2005

fluor

Another

leophilic [18

eme[18-21] 2.

eme 2.4 Syn

2.2.2 Bo

The stra

mation was

bon based bo

example,

modynamic

k with the b

boronic est

eme 2.5 18Fst often KHF

The first

5, the [18F]-

ride with the

r strategy to

8F] fluoride

4.

nthesis and r

oron-Fluori

ategy of ra

a major co

ond formati

boron-flu

cally stable

boron-fluori

ters with flu

F-radiolabellF2). X= a lin

t example to

organotriflu

e carrier pota

o introduce

with dihalo

eaction of si

ine Bond F

adiolabelling

ontribution t

ion has also

uorine bond

covalent b

ine bond fo

uoride conta

led boronic nker group, e

o adopt this

uoroborates w

assium hydr37

18F by a nu

o or disulfon

imple [18F] f

Formation

g target m

to the PET

attracted at

ds are we

bonds (� 73

or biomolecu

ining syntho

ester conjue.g. amide.

strategy was

was prepare

rogen fluorid

ucleophilic s

nate alkyl st

fluoroaliphat

molecules th

T imaging fi

ttention and

ell known

30KJ mol-1)

ule labelling

ons, as show

ugates by re

s reported by

d by boroni

de (KHF2), a

substitution

tarting mate

tic derivative

hrough carb

field, recent

d demonstrat

to be som

)22. Currentl

g is based o

wn in Schem

eaction with

y Ting and c

ic esters and

as shown in S

n was prepar

erials, as sho

es

bon-fluorine

ly however

ted some su

me of the

ly, most rep

on the react

me 2.5.

h nucleophil

co-workers[2

d nucleophil

Scheme 2.6.

red by

own in

bond

r, non-

uccess.

most

ported

tion of

lic 18F

23, 24] in

lic 18F-

38 �

Scheme 2.6 Ting and co -worker boron-fluorine bond formation strategy for 18F-radiolabelling.

2.2.3 Silicon-Fluorine Bond Formation

The silicon-fluorine bond energy (� 570KJ mol-1)22 is much higher than that of

carbon-fluorine bond (~ 480KJ mol-1), and therefore has drawn the attention of

radiochemists for its potential in 18F-radiolabelling. The first example was described by

Rosenthal and coworkers in 1985, Scheme 2.7.

Scheme 2.7 Rosenthal and coworkers silicon-fluorine bond formation strategy for 18F-radiolabelling.

Another example of this approach was reported by Ting and co-workers23 in 2005,

a triethoxysilane precursor (ex. biotin derivatives) are reacted with nucleophilic 18F-

fluoride using the carrier potassium hydrogen fluoride (KHF2) to give the anionic

alkyltetrafluorosilicate, as shown in Scheme 2.8.

39 �

Scheme 2.8 Ting and co-worker silicon-fluorine bond formation approach for 18F-radiolabelling.

In 2007, Schirrmacher26 and coworkers reported another approach based on the

trialkylsilane binding sites through 19F/18F isotopic exchange instead of nucleophilic

substitution of hydroxyl or alkoxy groups, as shown in Scheme 2.9.

Si Cl18F

CH3CN, rt15min80-95% RCY

Si 18F

Si 19F18F

CH3CN, rt15minisotopic exchange

Scheme 2.9 Synthesis of [18F]-fluorodi-tert-butylphenylsilyl by 19F/18F isotopic exchange

2.2.4 Aluminium-Fluorine Bond Formation

The aluminium-fluorine bond also has high bond energy (� 670KJ mol-1)22,

offering the opportunity for fluoride to act as coordination ligand to aluminium (Al3+).

The general strategy of this coordinate bond is based on the selective binding of [18F]-

�

fluo

show

Figufluor

(DT

tetra

ride to alum

wn in Figure

ure 2.3 Biomride.

The co

TPA), 1,4,

aazacyclodo

minum whi

e 2.3.

molecule la

ommonly u

7-triazacycl

odecane-1,4,

ich attached

abelling usin

used chelato

lononane-1,

,7,10-tetraac

40

d to biomo

ng an alumin

ors[28-30] ar

,4,7-triacetic

cetic acid (D

olecules via

nium chelat

re diethylen

c acid (

DOTA), as s

a bifunction

te as a bind

netriamine

(NOTA)

shown in Fi

nal chelators

ding site for

pentaacetic

and 1,4

igure 2.4.

s27, as

r [18F]-

c acid

4,7,10-

41 �

NN NOH

O

OHN

OHO

OHO

HO

O

DTPA: diethylenetriamine pentaacetic acid

N N

NOH

O

HO

OHN

O

NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid

NN

NN

HN

O

OH

O

HO

O

HO

O

DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

Figure 2.4 Currently used bifunctional chelators .

2.2.5 Common 18F Reagents for Labelling Peptides, Proteins and

Oligonucleotides

Owing to the potentials of biomolecules in the treatment of diseases and

diagnosis, the application of 18F radiolabelling biomolecules including peptides,

oligonucleotides and proteins in PET is becoming more important and has drawn the

attention of radiochemists. The traditional approach using direct radiolabelling of most

peptides and proteins through nucleophilic [18F] fluoride is not appropriate due to harsh

reaction conditions such as high temperatures, strong acidic/basic conditions, and longer

reaction times. The alternative approach of indirect introduction of 18F radioisotope into

peptides and proteins has been developed in PET imaging. The general strategy is that

peptides and proteins are reacted with suitable prosthetic 18F groups under mild reaction

42 �

conditions such as room temperature, aqueous solution and short reaction time. In

addition, the prosthetic groups in this reaction should be chemo-selective and have no

adverse effects on the biological properties of peptides and proteins.

A few prosthetic18F-radiolabelled groups have been reported for labelling

peptides[31-34], and they are shown in Scheme 2.10. Each method has its own strengths

and weakness based on the synthesis and reactivity of the target peptide. No general

protocol is available for the synthesis of radiolabelled peptides. For any particular ligand,

the best approach is to review several radiolabelling procedures and optimized one to suit

the particular needs.

18F labelled peptide or proteins

18FON

O

O

O

[18F]SFBCHO

18F[18F]FBA

HO

O

18F[18F]FBzA

HO18F

O

[18F]FPA

NO2

N3

18F

[18F]ANBAFN3

18F

O

[18F]APF

labelling at

N terminus or

lysine residue

solid phasesynthesisphotochemical

conjugation

oxime orhydrazonebond formation

�

Scheme 2.10 Prosthetic reagents for the 18F radiolabelling of peptides, proteins.

Another Approach for the preparation of [18F]-radiolabelled peptides has been

reported through “Click” chemistry[35, 36], is shown in Scheme 2.11. This technique uses

Huisgen 1,3-dipolar cycloaddition of terminal alkynes and azides to form 1,2,3-triazoles.

43 �

TsO18F-, K222-K+

CH3CNn 18F n

N3 peptide

O

CuI, Na ascorbateDIEA

N peptide

ON

N

18F nn = 1-3 RCY = 54-99%

TsON3

18F-, K222-K+

CH3CN18F

N3peptide

O

CuSO4, Na ascorbateSodium phosphate bufferpH = 6.0

NNN

18F

RCY = 92%

peptide

O

Scheme 2.11 “Click chemistry” strategy for the 18F radiolabelling of peptides, proteins.

2.3 Results & Discussions

2.3.1 Current Challenges in 18F Radiolabelling for Molecular Probe in PET

Imaging

One of the main challenges in 18F radiolabelling for PET imaging is the

development of efficient synthetic methods to introduce 18F into biomolecules, with the

shortest reaction time possible. Another challenge is to avoid harsh reaction conditions

(high temperature, high pressure, strong acidic or basic, longer reaction time), especially

for biomolecules (peptides, proteins). Only a few peptide based 18F-radiopharmaceuticals

for diagnostic application with PET have entered into clinical trials so far.

2.3.2 Design of the Potential 18F-Radiolabelling Molecular Probe

In order to explore potential molecular probes for peptides/proteins, the boron-

fluorine bond formation approach attracted our attention due to the bond energy already

discussed22 (Table 2.2).

�

Tab

orbi

with

mole

with

2.5.

Figu

B

ble 2.2 Bond

Boron’s

tals and in f

h other atom

ecular orbit

h other ligan

ure 2.5 Boro

To take

B (1s2,

Dissociation

Bond

C-F

Si-F

Al-F

B-F

s atomic orb

fifth electro

ms through

tal makes it

nd. Its electr

on’s electron

advantage

2s22p

1)

n Energy�

bitals are fill

on in a 2p or

h sp2 hybrid

t a strong L

ron configur

n configurati

of this attr

)

44

Bond Disso

led by five e

rbital. Boro

dization to

Lewis acid,

ration and h

on and its sp

ribute of bor

ociation Ene/�Hf298,

513.8 ±

576.4 ±

67

732

electrons, fo

on arranges i

form a co

which allow

hybridizatio

p2 hybridiza

ron, Tris(hy

ergy (enthalpkJ/mol

± 10.0

± 17.0

5

2

our electron

its three out

ovalent bond

ws for poten

on model are

ation model.

ydroxymeth

py chage)

ns in the 1s a

ter-shell ele

d. The em

ntial coordi

e shown in F

hyl)aminome

and 2s

ectrons

mpty p

ination

Figure

ethane

45 �

(Tris) was proposed as building block to introduce 19F/18F into a biomolecule. Tris can be

converted to the borate ester, and then the empty p orbital on boron will allow for

additional coordination from a fluoride ion, making the boron ester a fluorine capture

reagent. The proposed scheme is shown in Figure 2.6.

Figure 2.6 Flow chart of boron ester used as a fluorine capture reagent.

2.3.3 Fluorine Introduction Strategy: Building Block

Scheme 2.12 Newly discovered strategy for 19F introduction.

Tris(hydroxymethyl)aminomethane (Tris) (2.1) was used as building block to

introduce 19F/18F into biomolecule in our new discovery. The strategy first treated

commercially available Tris 2.1 with trimethyl borate to give the Tris borate ester 2.2,

followed by treatment with potassium fluoride in ethanol to afford the potassium salt of

the boron-fluoride complex adduct 2.3 in quantitative yield, as shown in Scheme 2.12.

46 �

2.3.4 Fluorine Introduction Strategy: RCOOH Series Substrates

Scheme 2.13 19F introduction into RCOOH series substrates

The strategy to introduce 19F/18F into RCOOH series substrates was initiated with

coupling RCOOH 2.4 with Tris 2.1 to afford the Tris analogs 2.5. Generally, carboxylic

acid 2.4 (or amino acid) was coupled with Tris 2.1 through a conventional peptide

coupling method with HCTU as the coupling reagent. The resulting Tris-OH intermediate

2.5, was treated with trimethyl borate to give a Tris borate ester. Finally, treatment with

potassium fluoride in ethanol to afforded the potassium salt of boron-fluoride complex

adduct 2.6 in quantitative yield, as shown in Scheme 2.13. The preparation of Tris

analogs compound 2.5 (RCOOH series substrates) is listed in Table 2.3, and the

following potassium salt of boron-fluoride complex adduct 2.6 is listed in Table 2.4.

Table 2.3 Preparation of RCOOH series compound 2.5�

Compound 2.4 Compound 2.1 Percent yield

2.5a 3-chlorobenzoic acid Tris 72%

2.5b Z-Ala-OH Tris 52%

2.5c Z-Val-OH Tris 66%

2.5d Z-Phe-OH Tris 81%

2.5e Z-Typ-OH Tris 42%

2.5f Boc-Lys-Z-OH Tris 51%

47 �

Table 2.4 Preparation of boron-fluoride complex adduct 2.6 for�RCOOH series substrates �

Compound 2.5 Percent yield

2.6a 2.5a 100%

2.6b 2.5b 100%

2.6c 2.5c 100%

2.6d 2.5d 100%

2.6e 2.5e 100%

2.6f 2.5f 100%

2.3.5 Fluorine Introduction Strategy: RNH2 Series Substrates

Scheme 2.14 19F introduction into RNH2 series substrates

A parallel strategy to introduce 19F/18F into a RNH2 series substrate was also

devised following a functional group conversion. Firstly, primary amine 2.7 was reacted

with succinic anhydride 2.8 to give the carboxylic acid 2.9. Then compound 2.9 was

coupled with Tris 2.1 through the conventional peptide coupling method with HCTU as

48 �

the coupling reagent. The resulting Tris-OH intermediate 2.10, was treated with trimethyl

borate to give the Tris borate ester which was finally, treated with potassium fluoride in

ethanol to afford the potassium salt of boron-fluoride complex adduct 2.11 in quantitative

yield, as shown in Scheme 2.14. The preparation of Tris analogs compound 2.10 (RNH2

series substrates) is listed in Table 2.5, and the following potassium salt of boron-fluoride

complex adduct 2.11 is listed in Table 2.6.

Table 2.5 Preparation of RNH2 series compounds 2.10.

Compound 2.7 Compound 2.8 Compound 2.1 Percent yield

2.10a 2,4-Dimethoxybenzylamine Succinic anhydride Tris 41%

2.10b 4-Methoxybenzylamine Succinic anhydride Tris 35%

2.10c 2-(3,4-dimethoxyphenyl)ethanamine Succinic anhydride Tris 56%

2.10d Benzylamine Succinic anhydride Tris 68%

2.10e H-Trp-OMe Succinic anhydride Tris 36%

Table 2.6 Preparation of boron-fluoride complex adducts 2.11 for�RNH2 series substrates.

Compound 2.10 Percent yield

2.11a 2.10a 100%

2.11b 2.10b 100%

2.11c 2.10c 100%

2.11d 2.10d 100%

2.11e 2.10e 100%

49 �

2.3.6 New Approach for Boron-Fluorine Bond Formation 1

Scheme 2.15 New Approach for 19F introduction.

Scheme 2.16 New Approach for 19F introduction based on ion-exchange resin.

1,1,1-Tris(hydroxymethyl)ethane (2.12) was also used as a substrate to introduce

19F/18F into a biomolecule. The approach first treated commercially available reagent 2.12

with trimethyl borate to give the Tris borate ester 2.13. Next, the fluoride ion was added

through an ion-exchange resin to form a fluoride adduct on the resin, and then the Tris

borate ester 2.13 was added onto resin. Following elution with ammonium bicarbonate,

the ammonium salt of the desired boron-fluoride complex adduct 2.14 was isolated in

quantitative yield, Schemes 2.15 & 2.16.

50 �

2.3.7 New Approach for Boron-Fluorine Bond Formation 2

HO NHO

O OH

OHOH

HO NHO

O O

OO

B

OHOH

OHH2N + O OO

HO NHO

O O

OO B

2.17

FNH4

2.82.1 2.15

a

2.16

(a) DMF, RT, 3hrs (b) B(OMe)3, MeOH, Ion-exchange resin(c) Ion-exchange resin, KF, MeOH, then NH4HCO3

b

c

Scheme 2.17 New Approach for 19F introduction.

Scheme 2.18 New Approach for 19F introduction based on ion-exchange resin.

Another novel approach for the introduction of the fluoride ion to boron ester

involved ion-exchange resin, Tris 2.1 was reacted with succinic anhydride 2.8 to give

51 �

compound 2.15. Then compound 2.15 was attached to ion-exchange resin. The resulting

intermediate on resin was treated with trimethyl borate to yield the Tris borate ester 2.16.

Finally the fluoride ion was added into ion-exchange resin to form the boron-fluoride

adduct. Elution with ammonium bicarbonate afforded the ammonium salt of boron-

fluoride complex adduct 2.17 in quantitative yield, Scheme 2.17 & 2.18.

2.3.8 Fluorine Capture Application in PET Probe

2.3.8.1 Folic Acid as a Targeting Ligand

Folate receptors (FR) have very limited expression on healthy cells, but are

commonly expressed in cancer cells. For example, folate receptors are found in epithelial

cancers among ovary, mammary gland, colon, lung, prostate, nose, throat and brain,

which make them to be good drug targets[37-41]. As the development of targeted

pharmaceuticals and drug delivery, folate-drug conjugate (as shown in Figure 2.7)

become well studied example in receptor targeted therapeutics, because folic acid can

selectively binds to the pathologic cell and delivers attached drugs into cell while normal

tissues lacking FR will not get involved42.

�

Figu

thro

drug

Figu

imag

ure 2.7 Struc

Literatu

ugh recepto

g incorporat

ure 2.8 Rece

In the

ge FR-expre

cture of folat

ure has descr

or-mediated

tion is show

eptor-mediat

last two de

essing cells

te conjugate

ribed how a

endocytosi

wn in Figure

ted endocyto

ecades, folat

through flu

52

e.

a folate-drug

s after bindi

2.8.

osis of folate

te-targeted

uorescent dy

g conjugate

ing to cell s

e conjugate.

imaging ag

yes[46, 47], M

can be trans

urface FR[4

gents have b

MRI contrast

sported into

3-45]. This ro

been develop

t agents[48, 4

o a cell

oute of

ped to

9], and

53 �

PET imaging agents50. Among all the other radionuclides, technetium-99m (99mTc) has

been shown to be the best diagnostic radionuclide, and folate targeted 99mTc-

radiopharmaceuticals have been successfully used to image human cancers[51-54].

However, the current (first generation) folate conjugates of MRI and PET imaging probes

have enjoyed very limited success. Opportunities for the design of novel folate-targeted

imaging probe have arisen from the demand for development of higher resolution and

more sensitive imaging technologies.

2.3.8.2 Synthesis of New Folate-Targeted Molecular Probe

Scheme 2.19 New Approach for 19F introduction into folic acid.

54 �

Scheme 2.20 New Approach for the synthesis of folic acid molecular probe.

This strategy introduced 19F/18F into folic acid. First, folic acid was coupled with

Tris to afford a Tris-OH intermediate through traditional peptide coupling method with

HCTU as coupling reagent. Next, the Tris-OH intermediate was treated with trimethyl

borate to afford Tris borate ester. The fluoride ion was added to an ion-exchange resin to

form fluoride adduct, and then the Tris borate ester is added onto resin. Finally, elution

with ammonium bicarbonate afforded the desired ammonium salt of the boron-fluoride

complex adduct, a novel folic acid molecular probe. This strategy is shown in Scheme

2.19 & 2.20.

2.4 Conclusion

In this chapter, we have successfully developed the general protocol to introduce

fluorine into peptide-based molecules using the novel building block

Tris(hydroxymethyl)aminomethane (Tris). This approach allows us to introduce fluorine

into biomolecules within 30 minutes based on the resin technology. The novel building

block offer us several advantages. First, short reaction time for 18F introduction into

55 �

molecular probe; second, no harsh reaction conditions applied for this series of reactions,

such as room temperature, no strong acidic or strong basic condition; third, 18F can be

introduced in the last step of the synthetic sequence, which give the radiochemists

enough time to prepare the precursor of molecular probe; fourth, Tris could be a very

versatile building block for peptides, non-peptides, proteins and amines, which can be

easily introduced fluorine-18 as molecular probe for PET imaging. Finally, Tris could

have a lot of variants as the next generation of building blocks for PET imaging.

2.5 Experimental Section

2.5.1 Materials and Methods

Organic and inorganic reagents (ACS grade) and solvents were obtained from

commercial sources and used without further purification, unless otherwise noted.

Moisture and air-sensitive reactions were carried out under an inert atmosphere of

nitrogen. Thin layer chromatography (TLC) was performed on glass plates pre-coated

with 0.25mm thickness of silica gel (60F-254) with fluorescent indicator. Column

chromatographic purification was performed using silica gel 60 Å, (# 70-230 mesh). All

1H NMR , 13C NMR, 19F-NMR, 11B-NMR spectra were recorded on Varian INOVA 400,

500 MHz or Bruker 250 MHz spectrometer at 25 oC in chloroform-d (CDCl3) or dimethyl

sulfoxide-d6 (DMSO-d6), unless otherwise specified. Chemical shifts are reported in parts

per million (ppm) relative to internal standard tetramethylsilane (TMS). Multiplicity is

expressed as (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, or m =

multiplet) and the values of coupling constants ( J ) are given in Hertz (Hz). High

Resolution Mass Spectrometry (HRMS) spetra were carried out on an Agilent 6540

56 �

QToF in the ESI-TOF mode.��

2.5.2 Experimental Procedures

Potassium salt of boron-fluoride complex adduct (2.3)55

To a solution of tris(hydroxymethyl)aminomethane (Tris) (2.1) (1.09g, 9.0mmol)

in dichloromethane (DCM) (30 mL) was added trimethyl borate (B(OMe)3) (0.935 g, 9.0

mmol) at room temperature under nitrogen. The reaction mixture was then heated to

reflux for 5 hrs. The reaction mixture was then concentrated in vacuo to remove all the

solvent. The resulting crude compound 2.2 (Tris borate ester) was dried overnight under

high vacuum, and directly used for the next step without further purification. Then Tris

borate ester 2.2 was dissolved in anhydrous ethanol (EtOH), and potassium fluoride (KF)

(1.0 eqv.) was added, the resulting suspension was stirred at room temperature (RT)

overnight. The reaction mixture was then concentrated in vacuo to remove all the solvent

and, the crude was then dried overnight under high vacuum to afford compound 2.3

(potassium salt of boron-fluoride complex adduct 2.3) as a white solid in quantitative

yield. 1HNMR (400MHz, METHANOL-d4) ��ppm = 3.65 (s, 6 H). 13CNMR (125.6MHz,

METHANOL-d4) ��ppm = 61.3, 59.6. 19FNMR (376MHz, METHANOL-d4) ��ppm = -

154.9. 11BNMR (80.2MHz, METHANOL-d4) ��ppm = 1.01. HRMS-ESI (m/z): calcd. for

C4H8[11B]FNO3: 148.0587, found, 148.0586.

57 �

3-Chloro-N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)benzamide (2.5a)

General Procedure A. To a solution of 3-chlorobenzoic acid (1.01g, 6.45mmol)

in DMF (30mL) was added T3P (2.70g, 8.49mmol) or HCTU (1.1 eqv.) at RT under N2,

followed by triethylamine (TEA) (1.2mL, 1.3eqv.). The mixture was stirred for 5mins,

then Tris (2.1) (3.50g, 28.89mmol) was added in several portions at RT and, after that,

the mixture was stirred at RT overnight. Once the reaction was completed, the reaction

solution was concentrated to remove all the solvents. The resulting residue was extracted

with hot ethyl acetate (EtOAc), sequentially washed with sat. sodium bicarbonate

solution, brine. Then the organic layer was dried over sodium sulfate (Na2SO4) and,

concentrated to yield crude product. The resulting crude was then purified by flash

column chromatography (silica gel, 100% EtOAc as eluent) to afford compound 2.5a as a

white powder (1.21 g, 72%). 1HNMR (400MHz, DMSO-d6) ��ppm = 3.65 (d, J=5.86 Hz,

6 H), 4.64 (t, J=6.05 Hz, 3 H), 7.36 (s, 1 H), 7.42 - 7.48 (m, 1 H), 7.55 (dd, J=8.20, 1.17

Hz, 1 H), 7.71 (d, J=7.81 Hz, 1 H), 7.81 (t, J=1.76 Hz, 1 H). 13CNMR (101MHz, DMSO-

d6) ��ppm = 166.2, 137.8, 133.3, 131.2, 130.5, 127.7, 126.6, 63.4, 60.5. HRMS-ESI

(m/z): [M+H]+ calcd. for C11H15ClNO4: 260.0684, found, 260.0693.

58 �

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-1-

oxopropan-2-yl)carbamate (2.5b)

Compound 2.5b was prepared from Z-Ala-OH and Tris (2.1) according to general

procedure A to afford 2.5b as a white solid (52% yield).� 1HNMR (400MHz, DMSO-d6) �

ppm = 1.13 - 1.22 (m, 3 H), 3.5(s, 6H), 4.05 (quin, J=7.22 Hz, 1 H), 4.68 (br. s., 3 H),

4.97 - 5.05 (m, 2 H), 7.14 (s, 1 H), 7.25 - 7.40 (m, 5 H), 7.47 (d, J=7.03 Hz, 1 H).

13CNMR (101MHz, DMSO-d6) � ppm = 173.8, 156.1, 137.4, 128.8, 128.2, 128.1, 65.9,

62.3, 60.7, 51.0, 18.7. HRMS-ESI (m/z): [M+H]+ calcd. for C15H23N2O6: 327.1551,

found, 327.1544.

�

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-

methyl-1-oxobutan-2-yl)carbamate (2.5c)

Compound 2.5c was prepared from Z-Val-OH and Tris (2.1) according to general

procedure A to afford 2.5c as a white solid (66% yield).� 1HNMR (400MHz, D2O) � ppm

= 0.67 - 0.96 (m, 6 H), 1.79 - 2.06 (m, 1 H), 3.49 (br. s., 1 H), 3.62 (br. s., 6 H), 3.79 (d,

J=6.64 Hz, 1 H), 5.0(s., 2H), 7.29 (br. s., 5 H). 13CNMR (101MHz, D2O) � ppm = 174.4,

158.1, 136.4, 128.7, 128.3, 127.5, 67.0, 61.9, 61.2, 60.4, 29.9, 18.3. HRMS-ESI (m/z):

59 �

[M+H]+ calcd. for C17H27N2O6: 355.1864, found, 355.1866.

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-1-oxo-3-

phenylpropan-2-yl)carbamate (2.5d)

Compound 2.5d was prepared from Z-Phe-OH and Tris (2.1) according to general

procedure A to afford 2.5d as a white solid (81% yield).� 1HNMR (400MHz, DMSO-d6) �

ppm = 2.66 - 2.79 (m, 1 H), 3.00 (dd, J=13.67, 3.91 Hz, 1 H), 3.51(br. s., 9H), 4.23 - 4.32

(m, 1 H), 4.93 (s, 2 H), 7.17 - 7.35 (m, 10 H), 7.51 (d, J=8.20 Hz, 1 H), 8.04 (br. s., 1 H).

13CNMR (101MHz, DMSO-d6) � ppm = 172.8, 156.2, 138.5, 137.4, 129.6, 128.7, 128.4,

128.0, 127.8, 126.6, 65.6, 62.5, 60.8, 56.9, 38.0. HRMS-ESI (m/z): [M+H]+ calcd. for

C21H27N2O6: 403.1864, found, 403.1870.

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-(1H-

indol-3-yl)-1-oxopropan-2-yl)carbamate (2.5e)

HNCbz N

H

O OH

OHOH

HN

Compound 2.5e was prepared from Z-Tryp-OH and Tris (2.1) according to

60 �

general procedure A to afford 2.5e as a white solid (42% yield).� 1HNMR (400MHz,

DMSO-d6) � ppm = 2.83 - 2.97 (m, 1 H), 3.07 - 3.14 (m, 1 H), 3.52 (br. s., 9H), 4.24 -

4.34 (m, 1 H), 4.89 - 4.98 (m, 2 H), 6.90 - 7.01 (m, 1 H), 7.01 - 7.10 (m, 1 H), 7.14 (s, 1

H), 7.19 - 7.38 (m, 6 H), 7.44 (d, J=7.81 Hz, 1 H), 7.55 - 7.62 (m, 1 H), 8.06 (br. s., 1 H),

10.69 - 10.94 (m, 1 H). 13CNMR (101MHz, DMSO-d6) � ppm = 173.1, 156.3, 137.4,

136.5, 128.8, 128.7, 128.0, 127.8, 124.1, 121.2, 118.8, 118.6, 111.7, 110.6, 65.7, 62.5,

60.8, 56.5, 28.2. HRMS-ESI (m/z): [M+H]+ calcd. for C23H28N3O6: 442.1973, found,

442.1973.

(S)-benzyl tert-butyl (6-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-

yl)amino)-6-oxohexane-1,5-diyl)dicarbamate (2.5f)

HNBoc N

H

O OH

OHOH

NHCbz

Compound 2.5f was prepared from Boc-Lys-Z-OH and Tris (2.1) according to

general procedure A to afford 2.5f as a white solid (51% yield). 1HNMR (400MHz,

DMSO-d6) ��ppm = 1.25 (d, J=7.03 Hz, 2 H), 1.35 (s, 11 H), 1.42 - 1.62 (m, 2 H), 2.94

(d, J=6.25 Hz, 2 H), 3.51 (br. s., 6 H), 3.81 (d, J=3.51 Hz, 3 H), 4.09 (d, J=6.25 Hz, 1 H),

4.98 (s, 2 H), 6.93 - 7.13 (m, 1 H), 7.15 - 7.25 (m, 1 H), 7.26 - 7.39 (m, 5 H), 7.98 (br. s.,

1 H). 13CNMR (101MHz, DMSO-d6) ��ppm = 173.6, 156.5, 155.9, 137.7, 128.8, 128.1,

78.7, 65.5, 62.3, 60.6, 55.4, 31.7, 29.5, 28.6, 23.2, 10.7. HRMS-ESI (m/z): [M+H]+ calcd.

for C23H38N3O8: 484.2653, found, 484.2652.

61 �

3-Chloro-N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)benzamide boron-

fluoride complex adduct (2.6a)

General Procedure B. To a solution of 3-Chloro-N-(1,3-dihydroxy-2-

(hydroxymethyl)propan-2-yl)benzamide (2.5a) (1.0mmol) in dichloromethane (DCM) (5

mL) was added trimethyl borate (B(OMe)3) (1.0mmol) at room temperature under

nitrogen. The reaction mixture was then heated to reflux for 5 hrs. The reaction mixture

was then concentrated in vacuo to remove all the solvent. The resulting crude compound

(Tris borate ester) was dried overnight under high vacuum, and directly used for the next

step without further purification. Then Tris borate ester was dissolved in anhydrous

ethanol (EtOH), and potassium fluoride (KF) (1.0 eqv.) was added, the resulting

suspension was stirred at room temperature (RT) overnight. The reaction mixture was

then concentrated in vacuo to remove all the solvent, the crude was then dried overnight

under high vacuum to afford compound 2.6a (potassium salt of boron-fluoride complex

adduct 2.6a) as a white solid in quantitative yield. 1HNMR (400MHz, D2O) ��ppm = 3.73

(s, 6 H) 7.25 - 7.33 (m, 1 H) 7.42 (d, J=7.81 Hz, 1 H) 7.48 (d, J=7.42 Hz, 1 H) 7.58 (br.

s., 1 H). 13CNMR (101MHz, D2O) ��ppm = 169.9, 135.9, 133.8, 131.7, 130.0, 127.1,

125.5, 62.4, 60.2. 19FNMR (376MHz, D2O) ��ppm = -123.0. 11BNMR (160MHz, D2O)

��ppm = 16.3. HRMS-ESI (m/z): calcd. for C11H11[11B]FClNO4: 286.0459, found,

286.0468.

62 �

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-1-

oxopropan-2-yl)carbamate boron-fluoride complex adduct (2.6b)

Compound 2.6b was prepared from compound 2.5b, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.6b as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 1.28 - 1.37 (m, 3 H), 3.57 -

3.84 (m, 6 H), 4.14 (dq, J=14.21, 6.98 Hz, 1 H), 5.03 - 5.14 (m, 2 H), 7.22 - 7.42 (m, 5

H). 13CNMR (101MHz, CD3OD) � ppm = 156.9, 136.6, 128.0, 127.6, 127.4, 127.3, 66.3,

61.8, 60.7, 57.1, 32.7. 19FNMR (376MHz, CD3OD) ��ppm = -152.5. 11BNMR (160MHz,

CD3OD) ��ppm = 2.4. HRMS-ESI (m/z): calcd. for C15H19[11B]FN2O6: 353.1326,

found, 353.1329.

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-

methyl-1-oxobutan-2-yl)carbamate boron-fluoride complex adduct (2.6c)

HNCbz N

H

O O

OO B F

K

Compound 2.6c was prepared from compound 2.5c, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.6c as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 0.94 (dd, J=11.72, 7.03 Hz, 6

H), 2.05 (dt, J=12.69, 6.54 Hz, 1 H), 3.61 - 3.81 (m, 6 H), 3.88 - 3.99 (m, 1 H), 5.03 -

63 �

5.12 (m, 2 H), 7.24 - 7.37 (m, 5 H). 13CNMR (101MHz, CD3OD) � ppm = 173.5, 157.3,

136.7, 128.0, 127.6, 127.4, 66.3, 62.1, 60.9, 30.4, 16.9. 19FNMR (376MHz, CD3OD)

��ppm = -152.7. 11BNMR (160MHz, CD3OD) ��ppm = 2.7. HRMS-ESI (m/z): calcd. for

C17H23[11B]FN2O6: 381.1639, found, 381.1643.

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-1-oxo-3-

phenylpropan-2-yl)carbamate boron-fluoride complex adduct (2.6d)

Compound 2.6d was prepared from compound 2.5d, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.6d as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 2.86 (dd, J=13.47, 9.57 Hz, 1

H), 3.05 - 3.19 (m, 1 H), 3.53 - 3.79 (m, 6 H), 4.29 - 4.44 (m, 1 H), 4.96 - 5.05 (m, 2 H),

7.11 - 7.36 (m, 12 H). 13CNMR (101MHz, CD3OD) � ppm = 173.3, 156.9, 137.1, 136.7,

136.6, 129.0, 128.9, 128.1, 128.0, 127.5, 127.2, 127.1, 66.1, 62.0, 60.7, 57.0, 37.5.

19FNMR (376MHz, CD3OD) ��ppm = -152.4. 11BNMR (160MHz, CD3OD) ��ppm = 2.3.

HRMS-ESI (m/z): calcd. for C21H23[11B]FN2O6: 429.1639, found, 429.1641.

64 �

(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-3-(1H-

indol-3-yl)-1-oxopropan-2-yl)carbamate boron-fluoride complex adduct (2.6e)

Compound 2.6e was prepared from compound 2.5e, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.6e as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 3.01 - 3.15 (m, 1 H), 3.20 -

3.26 (m, 1 H), 3.51 - 3.71 (m, 6 H), 4.36 - 4.49 (m, 1 H), 5.01 (br. s., 2 H), 6.94 - 7.01 (m,

1 H), 7.03 - 7.12 (m, 3 H), 7.18 - 7.36 (m, 7 H), 7.51 - 7.63 (m, 1 H). 13CNMR (101MHz,

CD3OD) � ppm = 163.7, 155.3, 136.8, 136.6, 128.1, 128.0, 127.7, 127.5, 127.3, 127.2,

121.0, 120.9, 118.3, 110.8, 61.9, 60.7, 59.9, 56.6, 32.8. 19FNMR (376MHz, CD3OD)

��ppm = -152.3. 11BNMR (160MHz, CD3OD) ��ppm = 2.3. HRMS-ESI (m/z): calcd. for

C23H24[11B]FN3O6: 468.1748, found, 468.1766.

(S)-benzyl tert-butyl (6-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-

yl)amino)-6-oxohexane-1,5-diyl)dicarbamate boron-fluoride complex adduct (2.6f)

65 �

Compound 2.6f was prepared from compound 2.5f, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.6f as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 1.42 (s, 11 H) 1.49 (d, J=6.64

Hz, 2 H) 1.54 - 1.82 (m, 2 H) 3.10 (t, J=6.64 Hz, 2 H), 3.52 (d, J=5.08 Hz, 1 H), 3.68-

3.74 (m, 6 H), 3.92 (d, J=3.91 Hz, 1 H), 5.04 (s, 2 H), 7.22 - 7.38 (m, 5 H). 13CNMR

(101MHz, CD3OD) � ppm = 171.3, 156.9, 137.0, 128.0, 127.5, 127.4, 127.3, 79.4, 65.8,

61.8, 60.7, 55.4, 39.9, 30.2, 29.0, 27.2, 22.7. 19FNMR (376MHz, CD3OD) ��ppm = -

152.7. 11BNMR (160MHz, CD3OD) ��ppm = 2.9. HRMS-ESI (m/z): calcd. for

C23H34[11B]FN3O8: 510.2428, found, 510.2446.

N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(2,4-

dimethoxybenzyl)succinamide (2.10a)

General Procedure C. To a solution of 2,4-dimethoxybenzylamine (2.7a) (1.0

mmol) in DMF (1mL) was added succinic anhydride (2.8) (1.0 mmol) at RT, the mixture

was stirred at RT for 3hrs. Check TLC or LCMS to make sure the reaction was complete.

Then another 1mL of DMF was added, followed by HCTU (1.3 mmol) and N,N-

Diisopropylethylamine (DIEA) (1.5 mmol) at RT under N2, the mixture was stirred for

5mins. Then Tris (2.1) (1.5 mmol) was added in several portions, and the mixture was

stirred at RT overnight. Once the reaction was completed, the reaction solution was

concentrated to remove all the solvents, and resulting residue was extracted with hot ethyl

66 �

acetate (EtOAc), sequentially washed with sat. sodium bicarbonate solution, brine. Then

the organic layer was dried over sodium sulfate (Na2SO4), concentrate to get crude

product. The resulting crude was then purified by flash column chromatography (silica

gel, 100% EtOAc as eluent) to afford compound 2.10a as a white powder (41% yield).

1HNMR (400MHz, D2O) ��ppm = 2.41 (dd, J=15.62, 5.86 Hz, 4 H), 3.53 - 3.62 (m, 6 H),

3.70 (d, J=7.03 Hz, 6 H), 4.13 (s, 2 H) 6.41 - 6.47 (m, 1 H), 6.50 (d, J=2.34 Hz, 1 H),

7.06 (d, J=8.20 Hz, 1 H). 13CNMR (101MHz, D2O) ��ppm = 175.1, 174.3, 159.8, 158.1,

129.9, 118.5, 104.8, 98.7, 61.9, 60.4, 55.5, 55.4, 38.3, 31.5, 31.0. HRMS-ESI (m/z):

[M+H]+ calcd. for C17H27N2O7: 371.1813, found, 371.1823.

N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(4-

methoxybenzyl)succinamide (2.10b)

Compound 2.10b was prepared from 4-methoxybenzylamine, succinic anhydride

(2.8) and Tris (2.1) according to general procedure C to afford 2.10b as a white solid (35%

yield). 1HNMR (400MHz, D2O) ��ppm = 2.36 - 2.57 (m, 4 H), 3.20 - 3.24 (m, 1 H), 3.59

(s, 6 H), 3.70 (s, 3 H), 4.19 (s, 2 H), 6.83 - 6.90 (m, 2 H), 7.12 - 7.19 (m, 2 H). 13CNMR

(101MHz, D2O) ��ppm = 175.1, 174.6, 158.0, 130.6, 128.7, 114.0, 61.9, 60.4, 55.3, 42.3,

31.4, 31.0. HRMS-ESI (m/z): [M+H]+ calcd. for C16H25N2O6: 341.1707, found,

341.1712.

67 �

N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(3,4-

dimethoxyphenethyl)succinamide (2.10c)

Compound 2.10c was prepared from 2-(3,4-dimethoxyphenyl)ethanamine,

succinic anhydride (2.8) and Tris (2.1) according to general procedure C to afford 2.10c

as a white solid (56% yield).� 1HNMR (400MHz, DMSO-d6) � ppm = 2.22 - 2.40 (m, 4

H), 2.60 (t, J=7.22 Hz, 2 H), 3.21 (quin, J=6.54 Hz, 2 H), 3.39 (d, J=5.86 Hz, 3 H),

3.50(s, 6H), 3.69 (s, 3 H), 3.72 (s, 3 H), 6.68 (d, J=7.81 Hz, 1 H), 6.77 (s, 1 H), 6.83 (d,

J=8.20 Hz, 1 H), 7.89 (t, J=5.47 Hz, 1 H), 7.97 (br. s., 1 H). 13CNMR (101MHz, DMSO-

d6) � ppm = 173.4, 171.8, 149.0, 147.6, 132.4, 120.8, 112.9, 112.3, 62.7, 60.9, 55.9, 55.8,

40.9, 35.1, 31.8, 31.3. HRMS-ESI (m/z): [M+H]+ calcd. for C18H29N2O7: 385.1969,

found, 385.1972.

N1-benzyl-N4-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)succinamide

(2.10d)

Compound 2.10d was prepared from benzylamine, succinic anhydride (2.8) and

Tris (2.1) according to general procedure C to afford 2.10d as a white solid (68% yield).�

1HNMR (400MHz, DMSO-d6) � ppm = 2.30 - 2.43 (m, 4 H), 3.50(s, 6H), 4.20 - 4.28 (m,

68 �

2 H), 4.64 (br. s., 3 H), 7.14 - 7.25 (m, 4 H), 7.25 - 7.33 (m, 2 H), 8.35 (t, J=5.86 Hz, 1

H). 13CNMR (101MHz, DMSO-d6) � ppm = 173.3, 171.9, 139.9, 128.6, 127.5, 127.0,

62.7, 60.9, 42.4, 31.8, 31.3. HRMS-ESI (m/z): [M+H]+ calcd. for C15H23N2O5:

311.1601, found, 311.1585.

Methyl 2-(4-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-4-

oxobutanamido)-3-(1H-indol-3-yl)propanoate (2.10e)

HN N

HO

O OH

OHOH

MeO

O

HN

Compound 2.10e was prepared from H-Trp-OMe, succinic anhydride (2.8) and

Tris (2.1) according to general procedure C to afford 2.10e as a white solid (36% yield).�

1HNMR (400MHz, DMSO-d6) � ppm = 2.23 - 2.45 (m, 4 H), 2.95 - 3.17 (m, 2 H),

3.38(br. s., 3H), 3.49 (s, 6 H), 3.54 (s, 3 H), 4.40 - 4.52 (m, 1 H), 6.93 - 7.00 (m, 1 H),

7.05 (t, J=7.62 Hz, 1 H), 7.12 (s, 1 H), 7.32 (d, J=7.81 Hz, 1 H), 7.46 (d, J=7.81 Hz, 1 H),

7.94 (br. s., 1 H), 8.28 - 8.40 (m, 1 H), 10.85 (d, J=6.25 Hz, 1 H). 13CNMR (101MHz,

DMSO-d6) � ppm = 173.1, 172.3, 171.3, 136.5, 127.5, 124.1, 121.4, 118.8, 118.4, 111.9,

109.9, 109.8, 62.7, 60.9, 60.0, 59.8, 53.7, 52.2, 31.8, 31.6, 29.8. HRMS-ESI (m/z):

[M+H]+ calcd. for C20H28N3O7: 422.1922, found, 422.1928.

69 �

N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(2,4-

dimethoxybenzyl)succinamide boron-fluoride complex adduct (2.11a)

Compound 2.11a was prepared from compound 2.10a, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.11a as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 2.47 - 2.58 (m, 4 H), 3.66 -

3.73 (m, 6 H), 3.77 (d, J=18.75 Hz, 6 H), 4.25 (s, 2 H), 6.44 (d, J=8.59 Hz, 1 H), 6.49 (d,

J=1.95 Hz, 1 H), 7.10 (d, J=8.20 Hz, 1 H). 13CNMR (101MHz, CD3OD) � ppm = 174.4,

173.0, 160.5, 158.3, 129.1, 118.3, 103.8, 97.7, 62.2, 61.0, 54.4, 54.3, 37.7, 31.3, 31.2.

19FNMR (376MHz, CD3OD) ��ppm = -153.0. 11BNMR (160MHz, CD3OD) ��ppm = 4.4.

HRMS-ESI (m/z): calcd. for C17H23[11B]FN2O7: 397.1588, found, 397.1610.

N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(4-

methoxybenzyl)succinamide boron-fluoride complex adduct (2.11b)

Compound 2.11b was prepared from compound 2.10b, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.11b as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 2.46 - 2.59 (m, 4 H), 3.69 (s,

70 �

6 H), 3.74 (s, 3 H), 4.27 (s, 2 H), 6.84 (d, J=8.59 Hz, 2 H), 7.18 (d, J=8.59 Hz, 2 H).

13CNMR (101MHz, CD3OD) � ppm = 174.1, 172.8, 158.5, 131.9, 128.9, 128.8, 128.7,

114.0, 61.1, 58.0, 55.4, 41.9, 31.8, 26.4. 19FNMR (376MHz, CD3OD) ��ppm = -151.9.

11BNMR (160MHz, CD3OD) ��ppm = 3.2. HRMS-ESI (m/z): calcd. for

C16H21[11B]FN2O6: 367.1482, found, 367.1499.

N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(3,4-

dimethoxyphenethyl)succinamide boron-fluoride complex adduct (2.11c)

Compound 2.11c was prepared from compound 2.10c, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.11c as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 2.43 - 2.52 (m, 4 H), 2.71 (t,

J=7.42 Hz, 2 H), 3.35 (t, J=7.42 Hz, 2 H), 3.52 (d, J=1.17 Hz, 1 H), 3.66 - 3.73 (m, 6 H),

3.78 (s, 3 H), 3.81 (s, 3 H), 6.74 (dd, J=8.01, 1.37 Hz, 1 H), 6.81 - 6.88 (m, 2 H).

13CNMR (101MHz, CD3OD) � ppm = 174.3, 173.1, 148.8, 147.4, 132.1, 120.7, 112.3,

111.6, 62.2, 60.9, 55.1, 55.0, 40.7, 34.6, 31.3, 30.8. 19FNMR (376MHz, CD3OD) ��ppm =

-151.2. 11BNMR (160MHz, CD3OD) ��ppm = 2.7. HRMS-ESI (m/z): calcd. for

C18H25[11B]FN2O7: 411.1744, found, 411.1767.

71 �

N1-benzyl-N4-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)succinamide

boron-fluoride complex adduct (2.11d)

HN N

HO

O O

OO B F

K

Compound 2.11d was prepared from compound 2.10d, trimethyl borate and

potassium fluoride (KF) according to general procedure B to afford 2.11d as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 2.53 - 2.58 (m, 4 H), 3.52 (d,

J=1.17 Hz, 1 H), 3.69 - 3.73 (m, 6 H) 4.34 (s, 2 H), 7.20 - 7.30 (m, 5 H). 13CNMR

(101MHz, CD3OD) � ppm = 173.1, 171.3, 131.5, 128.0, 127.0, 126.6, 61.0, 57.9, 42.6,

30.7, 30.3. 19FNMR (376MHz, CD3OD) ��ppm = -151.6. 11BNMR (160MHz, CD3OD)

��ppm = 2.8. HRMS-ESI (m/z): calcd. for C15H19[11B]FN2O5: 337.1377, found,

337.1386.

Methyl 2-(4-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-4-

oxobutanamido)-3-(1H-indol-3-yl)propanoate boron-fluoride complex adduct

(2.11e)

Compound 2.11e was prepared from compound 2.10e, trimethyl borate and

72 �

potassium fluoride (KF) according to general procedure B to afford 2.11e as a white solid

in quantitative yield.� 1HNMR (400MHz, CD3OD) � ppm = 2.44 - 2.51 (m, 4 H), 3.11 -

3.25 (m, 2 H), 3.48 (s, 3 H), 3.63 - 3.68 (m, 6 H), 4.17 (s, 1 H), 4.69 (t, J=6.64 Hz, 1 H),

6.95 - 7.02 (m, 1 H), 7.03 - 7.10 (m, 2 H), 7.32 (d, J=8.20 Hz, 1 H), 7.49 (d, J=7.81 Hz, 1

H). 13CNMR (101MHz, CD3OD) � ppm = 174.2, 172.7, 169.1, 136.6, 127.2, 123.1,

120.9, 118.3, 117.6, 110.9, 109.1, 75.6, 71.1, 64.0, 60.9, 53.6, 51.2, 30.8, 30.4, 27.0.

19FNMR (376MHz, CD3OD) ��ppm = -151.5. 11BNMR (160MHz, CD3OD) ��ppm =

2.83. HRMS-ESI (m/z): calcd. for C20H24[11B]FN3O7: 448.1697, found, 448.1674.

Ammonium salt of boron-fluoride complex adduct (2.14)

General Procedure D. Preparation of Tris borate ester (2.13): to a solution of

1,1,1-Tris(hydroxymethyl)ethane (2.12) (961.2 mg, 8.0mmol) in dichloromethane (DCM)

(30 mL) was added trimethyl borate (B(OMe)3) (832 mg, 8.0 mmol) at room temperature

under nitrogen. The reaction mixture was then heated to reflux for 3 hrs. The reaction

mixture was then concentrated in vacuo to remove all the solvent. The resulting crude

compound 2.13 (Tris borate ester) was dried overnight under high vacuum, and directly

used for the next step without further purification.

Preparation of Ammonium salt of boron-fluoride complex adduct(2.14): 1.0g of

ion-exchange resin (Amberlite_IRA-67, free base) was loaded into a syringe column,

then rinsed with 10mL of 2M HCl solution, then rinsed with 10mL of 2M potassium

73 �

fluoride (KF) solution for 3mins. Deionized water was added to remove excess KF

solution, followed by rinses of 15mL of methanol, 10mL of anhydrous ethanol. After

that, the solution of Tris borate ester (2.13) (50mg) in ethanol (2mL) was added to the

resin and kept for 3mins. Once the reaction was complete, 10mL of 1N ammonium

bicarbonate solution was added to elute the product as an ammonium salt solution. The

collected ammonium solution was freeze-dried to afford the final product: Ammonium

salt of boron-fluoride complex adduct as a white solid in 80% yield. 1HNMR (400MHz,

D2O) ��ppm = 0.758 (3, 3H), 3.39 (s, 6 H). 13CNMR (101MHz, D2O) ��ppm = 64.4, 41.0,

15.5. 19FNMR (376MHz, D2O) ��ppm = -143.5. 11BNMR (80.2MHz, D2O) ��ppm = 0.34.

HRMS-ESI (m/z): calcd. for C5H9[11B]FO3: 147.0634, found, 147.0632.

Ammonium salt of boron-fluoride complex adduct (2.17)

General Procedure E. Preparation of Tris analog (2.15): To a solution of Tris 2.1

(1.0 mmol) in DMF (1mL) was added succinic anhydride (2.8) (1.0 mmol) at RT, the

mixture was stirred at RT for 3hrs. The reaction was monitored by TLC or LCMS to

insure completion. The reaction was concentrated to remove all the solvent, and the

resulting crude compound was directly used for the next step without further purification.

Preparation of Ammonium salt of boron-fluoride complex adduct(2.17): 1.0g of ion-

exchange resin (Amberlite_IRA-67, free base) was loaded into a syringe column, then a

74 �

solution of Tris analog (2.15) (1.0mmol) in methanol (3mL) was added into resin for

3mins and, rinsed with methanol (15mL), anhydrous ethanol (10mL). Next, trimethyl

borate (3mL) was added and kept for 3mins. Once the reaction was complete, 10mL of

anhydrous ethanol was added into resin to remove excess trimethyl borate. After that, a

solution of potassium fluoride (KF) / 18-crown-6 in methanol was added and kept for

3mins. Once the reaction was complete, 10mL of 1N ammonium bicarbonate solution

was added to elute the product as an ammonium salt solution. The collected ammonium

solution was freeze-dried to afford the final product: Ammonium salt of boron-fluoride

complex adduct as a white solid in 30% yield. 1HRMS-ESI (m/z): calcd. for

C8H12[11B]FNO6: 248.0747, found, 248.0751.

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United States patent Application Publication, US20110171112A1.

� �

78 �

APPENDICES

�

APP

1-Iso

PENDIX A

obutyl-1H-i

: Chapter O

imidazole (1

One - Selecte

1.2c)

79�

ed 1H and 113C NMR Spectra

�

Met

thyl 1-methyyl-1H-imidaazole-2-carb

80�

bodithioate (1.3a)

�

Metthyl 1-benzyyl-1H-imidaazole-2-carb

81�

bodithioate ((1.3b)

�

Metthyl 1-isobutyl-1H-imiddazole-2-car

82�

rbodithioatee (1.3c)

�

Metthyl 2-(1-meethyl-1H-immidazole-2-c

83�

arbothioammido)propannoate (1.4a)

�

Metthyl 4-methyyl-2-(1-methhyl-1H-imid

84�

dazole-2-carrbothioamiddo)pentanoate (1.4b)

�

Metthyl 2-(1-meethyl-1H-immidazole-2-c

85�

arbothioammido)-3-phennylpropanooate (1.4c)

�

Met

(1.4d

thyl 3-

d)

-(1H-indol-33-yl)-2-(1-m

86�

methyl-1H-immidazole-2--carbothioaamido)propa

anoate

�

Metthyl 2-(1-bennzyl-1H-imiidazole-2-ca

87�

arbothioammido)propannoate (1.4e)

�

Metthyl 2-(1-bennzyl-1H-imiidazole-2-ca

88�

arbothioammido)-4-methhylpentanoaate (1.4f)

�

Metthyl 2-(1-bennzyl-1H-imiidazole-2-ca

89�

arbothioammido)-3-phennylpropanoaate (1.4g)

�

Met

(1.4h

thyl 2

h)

2-(1-benzyl-1H-imidazo

90�

ole-2-carbotthioamido)--3-(1H-indoll-3-yl)propaanoate

�

Metthyl 2-(1-isoobutyl-1H-immidazole-2-

91�

carbothioammido)propaanoate (1.4i))

�

Metthyl 2-(1-isoobutyl-1H-immidazole-2-

92�

carbothioammido)-4-meethylpentanooate (1.4j)

�

Metthyl 2-(1-isoobutyl-1H-immidazole-2-

93�

carbothioammido)-3-pheenylpropannoate (1.4k)

�

Met

(1.4l

thyl 3-(

l)

(1H-indol-3-yl)-2-(1-iso

94�

obutyl-1H-immidazole-2--carbothioaamido)propa

anoate

�

5-MMethyl-3-(1-mmethyl-1H-iimidazol-2-

95�

-yl)-4,5-dihyydro-1,2,4-trriazin-6(1HH)-one (1.5a))

�

5-Isoobutyl-3-(1--methyl-1HH-imidazol-2

96�

2-yl)-4,5-dihhydro-1,2,4--triazin-6(1HH)-one (1.5bb)

�

5-Beenzyl-3-(1-mmethyl-1H-iimidazol-2-y

97�

yl)-4,5-dihyydro-1,2,4-trriazin-6(1H))-one (1.5c)

�

5-((1

6(1H

1H-indol-3-

H)-one (1.5d

-yl)methyl)-

d)

3-(1-methyl

98�

l-1H-imidazzol-2-yl)-4,55-dihydro-1,2,4-triazin-

-

�

3-(1-Benzyl-1HH-imidazol-22-yl)-5-meth

99�

hyl-4,5-dihyydro-1,2,4-trriazin-6(1H))-one (1.5e)

�

3-(1-Benzyl-1HH-imidazol-22-yl)-5-isobu

100�

utyl-4,5-dihyydro-1,2,4-ttriazin-6(1HH)-one (1.5ff)

�

5-Beenzyl-3-(1-bbenzyl-1H-immidazol-2-y

101�

yl)-4,5-dihyddro-1,2,4-trriazin-6(1H))-one (1.5g)

�

5-((1

6(1H

1H-indol-3-

H)-one (1.5h

-yl)methyl)-

h)

3-(1-benzyl

102�

l-1H-imidazzol-2-yl)-4,5-dihydro-1,,2,4-triazin-

-

�

3-(1-Isobutyl-1HH-imidazol--2-yl)-5-met

103�

thyl-4,5-dihhydro-1,2,4--triazin-6(1HH)-one (1.5ii)

�

5-Isoobutyl-3-(1--isobutyl-1HH-imidazol-

104�

-2-yl)-4,5-diihydro-1,2,44-triazin-6(11H)-one (1.55j)

�

5-Beenzyl-3-(1-issobutyl-1H--imidazol-2

105�

-yl)-4,5-dihyydro-1,2,4-ttriazin-6(1HH)-one (1.5kk)

�

5-((1

6(1H

1H-indol-3-

H)-one (1.5l)

-yl)methyl)-

)

3-(1-isobuty

106�

yl-1H-imidaazol-2-yl)-4,,5-dihydro-11,2,4-triazin

n-

�

APP

Com

PENDIX B:

mpound (2.3

: Chapter T

3) -1H and 13

Two - Select

3C NMR Sp

107�

ed 1H, 13C,

ectra

19F, 11B NMMR Spectra & HRMS

�

Commpound (2.33) -19F and 111B NMR Sp

108�

pectra

�

Com

�

mpound (2.33) –High Resolution Ma

109�

ass Spectra

�

Com

�

mpound (2.55a) -1H and 113C NMR Sp

110�

pectra

�

Com

mpound (2.55b) -1H and 13C NMR Sp

111�

pectra

�

Com

�

mpound (2.55c) -1H and 113C NMR Sp

112�

pectra

�

Com

�

mpound (2.55d) -1H and 13C NMR Sp

113�

pectra

�

Com

mpound (2.55e) -1H and 113C NMR Sp

114�

pectra

�

Com

mpound (2.55f) -1H and 113C NMR Sp

115�

pectra

�

Com

�

mpound (2.66a) -1H and 113C NMR Sp

116�

pectra

�

Com

mpound (2.66a) -19F and 11B NMR S

117�

pectra

�

Com

mpound (2.66a) –High RResolution M

118�

Mass Spectra

a

�

Com

�

mpound (2.66b) -1H and 13C NMR Sp

119�

pectra

�

Commpound (2.66b) -19F and 11B NMR S

120�

Spectra

�

Com

mpound (2.66b) –High RResolution M

121�

Mass Spectra

a

�

Com

�

mpound (2.66c) -1H and 113C NMR Sp

122�

pectra

�

Com

mpound (2.66c) -19F and 11B NMR Sp

123�

pectra

�

Com

mpound (2.66c) –High Resolution M

124�

Mass Spectra

a

�

Com

mpound (2.66d) -1H and 13C NMR Sp

125�

pectra

�

Com

�

mpound (2.66d) -19F and 11B NMR S

126�

Spectra

�

Com

mpound (2.66d) –High RResolution M

127�

Mass Spectra

a

�

Com

�

mpound (2.66e) -1H and 113C NMR Sp

128�

pectra

�

Com

mpound (2.66e) -19F and 11B NMR Sp

129�

pectra

�

Com

mpound (2.66e) –High Resolution M

130�

Mass Spectra

a

�

Com

�

mpound (2.66f) -1H and 113C NMR Sp

131�

pectra

�

Com

�

mpound (2.66f) -19F and 111B NMR Sp

132�

pectra

�

Com

�

mpound (2.66f) –High Reesolution M

133�

Mass Spectra

a

�

Commpound (2.110a) -1H andd 13C NMR S

134�

Spectra

�

Com

�

mpound (2.110b) -1H andd 13C NMR S

135�

Spectra

�

Com

�

mpound (2.110c) -1H andd 13C NMR S

136�

Spectra

�

Com

mpound (2.110d) -1H andd 13C NMR S

137�

Spectra

�

Com

�

mpound (2.110e) -1H andd 13C NMR S

138�

Spectra

�

Com

mpound (2.111a) -1H andd 13C NMR S

139�

Spectra

�

Com

mpound (2.111a) -19F andd 11B NMR S

140�

Spectra

�

Com

�

mpound (2.111a) –High RResolution M

141�

Mass Spectr

ra

�

Com

�

mpound (2.111b) -1H andd 13C NMR S

142�

Spectra

�

Com

mpound (2.111b) -19F andd 11B NMR

143�

Spectra

�

Com

�

mpound (2.111b) –High RResolution M

144�

Mass Spectr

ra

�

Com

�

mpound (2.111c) -1H andd 13C NMR S

145�

Spectra

�

Com

mpound (2.111c) -19F andd 11B NMR S

146�

Spectra

�

Com

�

mpound (2.111c) –High RResolution M

147�

Mass Spectr

ra

�

Com

mpound (2.111d) -1H andd 13C NMR S

148�

Spectra

�

Com

�

mpound (2.111d) -19F andd 11B NMR

149�

Spectra

�

Com

�

mpound (2.111d) –High RResolution M

150�

Mass Spectr

ra

�

Com

mpound (2.111e) -1H andd 13C NMR S

151�

Spectra

�

Com

mpound (2.111e) -19F andd 11B NMR S

152�

Spectra

�

Com

�

mpound (2.111e) –High RResolution M

153�

Mass Spectr

ra

�

Com

mpound (2.114) -1H and 113C NMR Sp

154�

pectra

�

Com

�

mpound (2.114) -19F and 11B NMR S

155�

pectra

�

Com

�

mpound (2.114) –High RResolution M

156�

Mass Spectraa

�

�

Com

mpound (2.117) –High RResolution M

157�

Mass Spectra

a

   

ABOUT THE AUTHOR

Mr. Fenger Zhou is currently working as medicinal chemistry

intern at GlaxoSmithKline at Research Triangle Park (RTP), NC. He

received his Bachelor’s degree in Pharmaceutical Engineering from Nanjing University

of Science & Technology (Nanjing, China) in 2002, and then he continued his Master’s

study in Applied Chemistry at the same school and got Master degree in July 2004.

After that, he directly jumped into industry and worked for two different chemical &

pharmaceuticals companies in China from 2004 to 2008. He started his career at USF in

Fall 2008 as a graduate student in Dr. Mark McLaughlin’s group. He continued his

research in synthetic organic chemistry and medicinal chemistry and will receive h i s

doctoral degree in Summer 2014.