University of South Florida
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Graduate Theses and Dissertations
Graduate School
January 2014
Synthesis of [1,2,4]-Triazines as Kinase Inhibitors
and of Novel Fluorine Capture Reagents for PET
probes
Fenger Zhou
University of South Florida, [email protected]
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Zhou, Fenger, "Synthesis of [1,2,4]-Triazines as Kinase Inhibitors and of Novel Fluorine Capture Reagents for PET probes" (2014).
Graduate Theses and Dissertations.
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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 fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida
Major Professor: Mark L. McLaughlin, Ph.D.
Jon Antilla, Ph.D.
Jianfeng Cai, Ph.D.
David L. Morse, Ph.D.
Date of Approval:
July 2, 2014
Keywords: Triazines, Kinase Inhibitor, PET imaging, Fluorine Capture, PET Probe
Copyright© 2014, Fenger Zhou
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
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, D r . Hyun Joo Kil, J o s a n n e - D e e W o o d r o f f e 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.
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
i
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 1 H and 13 C NMR Spectra…………... 79
Appendix B: Chapter Two - Selected 1 H, 13C, 19F,
11
B NMR Spectra & HRMS…………………………………. 107
About the Author……………………………………………………………...End Page
ii
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
iii
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
iv
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
18
F-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 18Fradiolabelling
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
Scheme 2.14
19
F introduction into RCOOH series substrates
46
19
F introduction into RNH2 series substrates
47
v
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
vi
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
vii
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
viii
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
ix
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
x
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.
xi
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].
1
Figu
ure 1.1 Anaaplastic lympphoma kinasse (ALK)-poositive canceers and its fusion proteins.
Figu
ure 1.2 ALK
K fusion onccogene and m
major downsstream signaaling pathwaays.
2
The cryystal structurre of humann anaplasticc lymphomaa kinase (A
ALK) with N
NVP5
TAE
E684 boundd is shown inn Figure 1.33 (PDB ID: 2XB7)
2
.
Figu
ure 1.3 Crysstal structuree of human A
ALK with N
NVP-TAE6844.
NVP-TAE6884 at ALK binding
b
sitee was carrieed out
The molecular moddelling of N
to fuurther investtigate the seelectivity off this ligand,, as shown iin Figure 1.449.
3
Figu
ure 1.4 A m
model of ALK
K in complex with NVP
P-TAE684.
TAE6844 binds to tthe ATP binnding site thhrough the bidentate hhydrogen boonding
1
withh residue L2258 at the kiinase “hingee” region off ALK[6, 10, 11]
. Second, the orthomeethoxy
grouup on the 2--aniline subbstituted (rinng A) pointss into a smaall cleft betw
ween the reesidues
L258 and M2599, it leads too a steric cllash for thiss binding poocket. It sugggests that residue
minants for TAE684, which
w
gives us the
L258 is one of the major kkinase selecttivity determ
t binding affinity witthin ALK binding
oppoortunity to ddesign betteer ligands too improve the
site.
1.2 R
Results & D
Discussions
1.2.1 Deesign of thee Potential K
Kinase Inhiibitor
The moolecular moddelling show
ws that ligaand TAE6844 has a sterric clash wiith the
residdue L258 at
a the ALK
K binding site,
s
so a novel
n
scafffold of 3-(11H-imidazol)-4,5dihyydro-1,2,4-trriazin-6(1H
H)-one was designed aas a small-m
molecule innhibitor tarrgeting
ALK
K-driven canncers, as shoown in Figuure 1.5.
4
N A N R1
N NH
HN B
R2
O
Figu
ure 1.5 [1, 2, 4]-dihydrrotriazine sccaffold as sm
mall molecuule inhibitorrs targeting ALKdriveen cancers.
The methoxy groupp on the left hand in rring A wass removed aat this scafffold to
avoiid steric hinndrance withh residue L2258, but still keep threee nitrogen atom in ringg B to
mainntain the cruucial hydrogen bondinng with Glu11197 and Met
M 1199, ass shown in F
Figure
1.6. On the otther hand, two hydropphobic grouups R1 andd R2 were uused to maaintain
with relevantt residues annd to increaase permeabbility.
hydrrophobic intteractions w
Figu
ure 1.6 ligannd interactionn at ALK binding site baased on [1, 22, 4]-dihydrootriazine scaaffold.
Dockingg studies were carried out to suppport this design based on this [1, 2, 4]-
5
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
Known ligand
R1
NVP-TAE684
Docking Score
-8.575
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
Experimental Ligands
R2
6
Based on
o the dockking results, ligand 1.55L seems to
t be the beest ligand in
i this
NPM-ALK)) and it may
m be a bbetter ALK inhibitor. When
scafffold for thhis target (N
supeerimposed, this mimeetic had sside chain residues pperfectly aaligned witth the
corrresponding lligand residdues as show
wn in Figuree 1.7. Figurre 1.8 shows the dockinng and
interractions of lligand 1.5L
L at the ALK
K binding sitte.
Figu
ure 1.7 Com
mparison of ligand
l
1.5L and NVP-TA
AE684 at thhe ALK binnding site.
hydrotriazin
ne Dimer Library
L
1.2.2 Syynthesis of [[1, 2, 4]-dih
The 1, 22, 4-dihydrrotriazine diimer analoggs were synnthesized froom commerrcially
avaiilable imidaazole as shown in Scheeme 1.1. Theen imidazolle was convverted to thioo-ester
1.3 bby treating with n-butyylithium andd carbon dissulfide, folloowed by cooupled with amino
7
acidd methyl esteer hydrochlooride to affoord compounnd 1.4. Thee key step inn this schem
me was
the last step for formationn of 1, 2, 44-dihydrotriaazine ring. Ideally, coompound 1..4 was
treatted with hyydrazine undder reflux coondition in 1,4-dioxanee to give thhe desired 1, 2, 4dihyydrotriazine dimer analoogs 1.5 in uup to 78% isolated yieldds.
N
N
N
HN
NH
NH
O
Liigand 1.5L
Figu
ure 1.8 Ligaand 1.5L binnding pose att the ALK binding site.
8
N
N
a
b
N
R1
1.2 a-c
N
H
1.1
N
d
N
HN
N
S
c
S
N
N
R1 S
N
R1
1.3 a-c
O
H
N
O
R2
1.4 a-l
N R1
NH
R2
O
1.5 a-l
(a) KOH, DMF, Isobutyl bromide, 0-25 oC, 53% (b) n-BuLi, LiBr, CuBr, -45 oC, then CS2,
f ollowed 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%
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%
9
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%
10
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
1
H NMR and
13
C 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
11
(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 ESITOF 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) G 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 12
2.07 (m, 1 H), 0.79 (d, J=6.6 Hz, 6 H).
13
CNMR (101MHz, CDCl3) G 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. 1Methylimidazole 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
13
a bright-red solid (1.21 g, 70%). 1HNMR (400MHz, CDCl3) G ppm = 7.05 (s, 1 H), 7.03
(s, 1 H), 3.96 (s, 3 H), 2.57 (s, 3 H). 13CNMR (101MHz, CDCl3) G 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) G
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) G 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
14
procedure A to afford 1.3c as a bright-red oil (64% yield). 1HNMR (400MHz, CDCl3) G
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).
13
CNMR (101MHz, CDCl3) G 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) G 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).
13
CNMR (101MHz, CDCl3) G 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.
15
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) G 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).
CNMR (101MHz, CDCl3) G ppm = 182.4, 171.9, 142.0, 127.5,
13
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) G 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
16
Hz, 2 H). 13CNMR (101MHz, CDCl3) G 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
H
N
N
O
O
S
NH
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) G 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).
13
CNMR (101MHz, CDCl3) G 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)
17
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).
1
HNMR (400MHz, CDCl3) G 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) G 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).
1
HNMR (400MHz, CDCl3) G 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) G 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.
18
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).
1
HNMR (400MHz, CDCl3) G 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).
CNMR (101MHz, CDCl3) G ppm = 182.1, 170.7, 142.1, 136.6,
13
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) G ppm = 9.75 (d, 1 H), 8.11 (br. s., 1 H), 7.56 (d,
19
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) G 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).
1
HNMR (400MHz, CDCl3) G 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) G 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.
20
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).
1
HNMR (400MHz, CDCl3) G 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).
CNMR (101MHz, CDCl3) G ppm = 182.6, 171.9, 141.9, 126.9, 126.0,
13
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) G ppm = 9.78 (d, J=7.2 Hz, 1 H), 7.13 - 7.33 (m, 5 H),
21
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).
13
CNMR (101MHz, CDCl3) G 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) G 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).
13
CNMR (101MHz, CDCl3) G 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.
22
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) G 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).
13
CNMR
(101MHz, DMSO-d6) G 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
23
procedure C to afford 1.5b as an off-white solid (79% yield). 1HNMR (400MHz, DMSOd6) G 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) G 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, DMSOd6) G 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) G
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.
24
5-((1H-indol-3-yl)methyl)-3-(1-methyl-1H-imidazol-2-yl)-4,5-dihydro-1,2,4triazin-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, DMSOd6) G 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) G
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
25
to afford 1.5e as a light yellow solid (58% yield). 1HNMR (400MHz, DMSO-d6) G 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).
CNMR (101MHz, DMSO-d6) G ppm = 164.6,
13
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) G 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) G 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.
26
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) G 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) G 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-1,2,4triazin-6(1H)-one (1.5h)
Compound 1.5h was prepared from compound 1.4h according to general
27
procedure C to afford 1.5h as an off-white solid (68% yield). 1HNMR (400MHz, CDCl3)
G 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).
13
CNMR
(101MHz, CDCl3) G 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) G 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).
13
CNMR
(101MHz, CDCl3) G 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.
28
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) G 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)
G 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)
29
G 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) G 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-1,2,4triazin-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) G 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) G 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
351.1928, found, 351.1926.
30
C19H23N6O:
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,
Heterocycles 2001, 55(4), 671-688.
31
N.;
Bourguignon,
J.J.
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 tissuebased 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,
32
how
wever, the deevelopmentt of highly eefficient moolecular proobes still rem
mains a chaallenge
for ssynthetic chhemists.
Molecullar probes for PET imaging
i
arre radiolabeeled with ppositron em
mitting
radioonuclides, w
which emit a positivelyy charged pparticle, possitron duringg decay[5, 111]. The
posiitron emitted from the nucleus traavels a shortt distance inn the surrouunding tissuue and
thenn is taken upp by an electtron, whichh generates energy
e
(as a photon) thaat can be deetected
and recorded, as shown in F
Figure 2.1.
Figu
ure 2.1 Principle of PET
T imaging5. 18F atom onn the sugar m
molecule deccays by emiitting a
positron.
Several positron-em
mitting radioonuclides foor PET imagging have bbeen developped so
far, such as 11C, 13N, 15O, 18F, 68Ga, 64C
Cu, as show
wn in Table 2.1.
2
33
Tab
ble 2.1 Comm
monly Used Positron-Em
mitting Radioonuclides5
mmon radionnuclides usedd in PET, flluorine-18 (18F) is conssidered
Among all the com
the m
most ideal radionuclidde as
18
F-labbeled PET aagents have the most ffavorable phhysical
propperties and nuclear chharacteristicss[12,
posiitron range in tissue) that gives
18
13]
. First,
18
F has low positrron energy (short
F the higghest resoluution PET iimages of aall the
avaiilable positrron emitters, as shown iin Table 2.11. Secondly, a short butt manageabble 110
minuute half-liffe allows for complex radiosyynthesis, llonger in vivo studyy and
com
mmercially available ddistribution to clinicaal PET cennters. In ssummary,
conssidered an eexcellent possitron emittiing radionuclide for PE
ET in clinicaal practice.
34
18
F is
2.2 C
Current Strrategies forr Fluorine-118 Radiolab
beling
2.2.1 Caarbon-Fluoorine Bond Formation
n
2.2.1.1 E
Electrophillic Fluorinee-18 Labelin
ng Reaction
ns
Currentlly, electrophhilic 18F fluoorinations aare not comm
mon synthettic approachhes for
18
F radiolabelinng. Howevver, historiccally they have
h
playedd an important role in the
deveelopment of
18
F-labeleed PET imaaging agentts. For exam
mple, Ehrennkaufer14 annd his
cow
workers repoorted electrrophilic fluuorination uusing [18F]aacetyl hypoofluorite to make
[18F
F]FDG, as sshown in Schheme 2.1.
Scheeme 2.1 Eleectrophilic 188F fluorinatiion using [188F] acetyl hyypofluorite ffor the prepaaration
of [18F]FDG.
Anotherr example w
was reporteed by Dolbbier15 in 20001, a new type of hyypoxia
biom
marker [18F]]EF5 was pprepared byy direct elecctrophilic ssubstitution using [18F]]F2, as
show
wn in Schem
me 2.2.
Scheeme 2.2 P
Preparation of the hyppoxia biom
marker [18F]EF5 by diirect electroophilic
18
subsstitution usinng [ F]F2.
35
2.2.1.2 N
Nucleophiliic 18F-Substitution Reeactions
Nucleopphilic
impoortant
18
F-ffluorination reactions are comm
monly used to make some
18
F P
PET radiotr
tracers and many exam
mples have been repoorted. One of the
com
mmon strateggies is to addd the phase-transfer reeagent krypptofix-222 (K222)16, as sshown
in Figure 2.2.
Figu
ure 2.2 Com
mplexation of a potassium
m ion (blue)) by the azaccryptand kryyptofix-222 (K222);
greeen: fluoride iion.
The azaacryptand K2222 (Figuree 2.2) has a strong attraaction to thee potassium cation
whicch leaves thhe fluoride aanion expossed, inducinng its strong nucleophiliic characterr when
dissoolved in pollar aprotic ssolvents such as DMF, D
DMSO or aacetonitrile.
A comm
mon examplle of directed nucleophhilic
18
F subbstitution iss the syntheesis of
the pprecursor of [18F]FDG, which wass first reporrted by Ham
macher17 in 1986. (Shoown in
Scheeme 2.3)
Scheeme 2.3 Synnthesis of [18F]FDG preccursor: protected [18F] suugar.
36
Anotherr strategy too introduce
18
F by a nuucleophilic substitution
s
n was preparred by
nuclleophilic [188F] fluoride with dihaloo or disulfonnate alkyl sttarting mateerials, as shoown in
Scheeme[18-21] 2.4.
Scheeme 2.4 Synnthesis and reaction of siimple [18F] fluoroaliphat
f
tic derivativees
2.2.2 Booron-Fluoriine Bond F
Formation
The straategy of raadiolabellingg target m
molecules thhrough carbbon-fluorine bond
form
mation was a major coontribution tto the PET
T imaging fi
field, recently howeverr, noncarbbon based boond formatiion has also attracted atttention andd demonstratted some suuccess.
For example,
boron-fluuorine bondds are weell known to be som
me of the most
modynamiccally stable covalent bonds
b
( 7330KJ mol-1)22. Currentlly, most repported
therm
workk with the boron-fluori
b
ine bond foor biomolecuule labellingg is based oon the reacttion of
aryl boronic estters with fluuoride containing synthoons, as show
wn in Schem
me 2.5.
Scheeme 2.5 18F
F-radiolabellled boronic ester conjuugates by reeaction withh nucleophillic
(mosst often KHF
F2). X= a linnker group, e.g.
e amide.
18
F
[223, 24]
The firstt example too adopt this strategy wass reported byy Ting and co-workers
c
in
20055, the [18F]-organotrifluuoroborates w
was prepared by boroniic esters andd nucleophillic
fluorride with thee carrier potaassium hydrrogen fluoridde (KHF2), aas shown in S
Scheme 2.6.
37
18
F-
Scheme 2.6 Ting and co -worker boron-fluorine bond formation strategy for
radiolabelling.
18
F-
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
18
F-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
radiolabelling.
18
F-
Another example of this approach was reported by Ting and co-workers23 in 2005,
a triethoxysilane precursor (ex. biotin derivatives) are reacted with nucleophilic
18
F-
fluoride using the carrier potassium hydrogen fluoride (KHF2) to give the anionic
alkyltetrafluorosilicate, as shown in Scheme 2.8.
38
Scheme 2.8 Ting and co-worker silicon-fluorine bond formation approach for
radiolabelling.
18
F-
In 2007, Schirrmacher26 and coworkers reported another approach based on the
trialkylsilane binding sites through
19
F/18F isotopic exchange instead of nucleophilic
substitution of hydroxyl or alkoxy groups, as shown in Scheme 2.9.
18F
Si Cl
Si 18F
CH3CN, rt
15min
80-95% RCY
18
F
19
Si F
CH3CN, rt
15min
isotopic 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]-
39
fluoride to alum
minum whiich attachedd to biomoolecules viaa bifunctionnal chelatorss27, as
wn in Figuree 2.3.
show
Figu
ure 2.3 Biom
molecule laabelling usinng an aluminnium chelatte as a bindding site forr [18F]fluorride.
The coommonly uused chelatoors[28-30] arre diethylennetriamine pentaaceticc acid
(DT
TPA),
1,4,7-triazacycllononane-1,,4,7-triaceticc
acid
((NOTA)
and
1,44,7,10-
tetraaazacyclodoodecane-1,4,,7,10-tetraaccetic acid (D
DOTA), as shown
s
in Fiigure 2.4.
40
OH
O
HN
O
O
N
N
HO
O
N
O
N
OH
OH
O
O
N
HO
N
OH
H
N
O
DTPA: diethylenetriamine pentaacetic acid
NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid
HN
HO
O
N
N
O
O
N
N
O
HO
OH
DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
Figure 2.4 Currently used bifunctional chelators .
2.2.5 Common
18
F Reagents for Labelling Peptides, Proteins and
Oligonucleotides
Owing to the potentials of biomolecules in the treatment of diseases and
diagnosis, the application of
18
F 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
41
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.
O
O
18F
18F
[18F]FBA
O
N O
CHO
oxim
e
hydr or
azon
e
bond
f orm
ation
O
[18F]SFB
t
ga r
lin nus o
l
e
lab ermi sidue
N t ine re
lys
HO
18
[18F]FBzA
F
18
F labelled peptide or proteins
NO2
l
ica
m
e
ch n
oto gatio
h
p nju
co
sol
syn id ph
the ase
sis
18
F
N3
[18F]ANBAF
18F
N3
O
HO
18F
[18F]FPA
O
[18F]APF
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.
42
O
O
N3
18 -
F , K222-K+
TsO
n
18
F
CH3CN
n
peptide
CuI, Na ascorbate
DIEA
N
N
n
n = 1-3
18
F
peptide
N
RCY = 54-99%
O
TsO
F , K222-K+
peptide
18 -
N3
CH3CN
18
F
O
N3
18
F
CuSO4, Na ascorbate
Sodium phosphate buffer
pH = 6.0
peptide
N
N
N
RCY = 92%
Scheme 2.11 “Click chemistry” strategy for the 18F radiolabelling of peptides, proteins.
2.3 Results & Discussions
2.3.1 Current Challenges in
18
F Radiolabelling for Molecular Probe in PET
Imaging
One of the main challenges in
18
F 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 boronfluorine bond formation approach attracted our attention due to the bond energy already
discussed22 (Table 2.2).
43
Tab
ble 2.2 Bond Dissociationn Energy
Bond
py chage)
Bond Dissoociation Eneergy (enthalp
/Hf298, kJ/mol
C-F
513.8 ± 10.0
Si-F
576.4 ± 17.0
Al-F
675
B-F
7322
Boron’ss atomic orbbitals are fillled by five eelectrons, foour electronns in the 1s aand 2s
orbitals and in ffifth electroon in a 2p orrbital. Boroon arranges iits three outter-shell eleectrons
ms throughh sp2 hybriddization to form a coovalent bondd. The em
mpty p
withh other atom
moleecular orbittal makes itt a strong L
Lewis acid, which allow
ws for potenntial coordiination
withh other ligannd. Its electrron configurration and hhybridizatioon model aree shown in F
Figure
2.5.
2
2
1
B (1s , 2s 2p )
Figu
ure 2.5 Boroon’s electronn configuration and its spp2 hybridizaation model.
To take advantage of this attrribute of borron, Tris(hyydroxymethhyl)aminomeethane
44
(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
19
F/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.
45
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%
46
Table 2.4 Preparation of boron-fluoride complex adduct 2.6 forRCOOH 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
19
F/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
47
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
Succinic anhydride
Tris
35%
2.10c
4-Methoxybenzylamine
2-(3,4dimethoxyphenyl)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 forRNH2 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%
48
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
19
F/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.
49
2.3.7 New Approach for Boron-Fluorine Bond Formation 2
OH
OH
OH
O
OH
H2N
2.1
OH
OH
O
O
HO
N
H
O
+ O
O
O
O
2.8
HO
a
O
N
H
O
b
2.15
B
O
O
c
HO
N
H
O
2.16
O
O
B F
NH4
2.17
(a) DMF, RT, 3hrs (b) B(OMe)3, MeOH, Ion-exchange resin
(c) Ion-exchange resin, KF, MeOH, then NH4HCO3
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
50
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 boronfluoride 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.
51
Figu
ure 2.7 Struccture of folatte conjugatee.
Literatuure has descrribed how a folate-drugg conjugate can be transsported intoo a cell
through receptoor-mediated endocytosis after bindiing to cell surface FR[43-45]. This rooute of
wn in Figure 2.8.
drugg incorporattion is show
Figu
ure 2.8 Receeptor-mediatted endocytoosis of folatee conjugate.
In the last two deecades, folatte-targeted imaging aggents have bbeen developped to
MRI contrastt agents[48, 49], and
imagge FR-expreessing cells through fluuorescent dyyes[46, 47], M
52
PET imaging agents50. Among all the other radionuclides, technetium-99m (99mTc) has
been shown to be the best diagnostic radionuclide, and folate targeted
99m
Tc-
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.
53
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
54
18
F introduction into
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,
18
F 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
1
H 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
55
QToF in the ESI-TOF mode.
2.5.2 Experimental Procedures
55
Potassium salt of boron-fluoride complex adduct (2.3)
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) Gppm = 3.65 (s, 6 H). 13CNMR (125.6MHz,
METHANOL-d4) Gppm = 61.3, 59.6.
19
FNMR (376MHz, METHANOL-d4) Gppm = -
154.9. 11BNMR (80.2MHz, METHANOL-d4) Gppm = 1.01. HRMS-ESI (m/z): calcd. for
C4H8[11B]FNO3: 148.0587, found, 148.0586.
56
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) Gppm = 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, DMSOd6) Gppm = 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.
57
(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) G
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).
13
CNMR (101MHz, DMSO-d6) G 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) G 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) G 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):
58
[M+H]+ calcd. for C17H27N2O6: 355.1864, found, 355.1866.
(S)-benzyl (1-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)-1-oxo-3phenylpropan-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) G
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).
13
CNMR (101MHz, DMSO-d6) G 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)
H
Cbz N
O
N
H
OH
OH
OH
HN
Compound 2.5e was prepared from Z-Tryp-OH and Tris (2.1) according to
59
general procedure A to afford 2.5e as a white solid (42% yield). 1HNMR (400MHz,
DMSO-d6) G 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).
13
CNMR (101MHz, DMSO-d6) G 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)
H
Boc N
O
N
H
OH
OH
OH
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) Gppm = 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).
13
CNMR (101MHz, DMSO-d6) Gppm = 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.
60
3-Chloro-N-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)benzamide boronfluoride 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) Gppm = 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).
CNMR (101MHz, D2O) Gppm = 169.9, 135.9, 133.8, 131.7, 130.0, 127.1,
13
125.5, 62.4, 60.2.
19
FNMR (376MHz, D2O) Gppm = -123.0.
11
BNMR (160MHz, D2O)
Gppm = 16.3. HRMS-ESI (m/z): calcd. for C11H11[11B]FClNO4: 286.0459, found,
286.0468.
61
(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) G 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) G 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) Gppm = -152.5. 11BNMR (160MHz,
CD3OD) Gppm = 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)
H
Cbz N
O
O
N
H
F
O B
O
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) G 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 62
5.12 (m, 2 H), 7.24 - 7.37 (m, 5 H). 13CNMR (101MHz, CD3OD) G ppm = 173.5, 157.3,
136.7, 128.0, 127.6, 127.4, 66.3, 62.1, 60.9, 30.4, 16.9.
19
FNMR (376MHz, CD3OD)
Gppm = -152.7. 11BNMR (160MHz, CD3OD) Gppm = 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-3phenylpropan-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) G 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) G 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.
19
FNMR (376MHz, CD3OD) Gppm = -152.4. 11BNMR (160MHz, CD3OD) Gppm = 2.3.
HRMS-ESI (m/z): calcd. for C21H23[11B]FN2O6: 429.1639, found, 429.1641.
63
(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) G 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) G 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.
19
FNMR (376MHz, CD3OD)
Gppm = -152.3. 11BNMR (160MHz, CD3OD) Gppm = 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)
64
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) G 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), 3.683.74 (m, 6 H), 3.92 (d, J=3.91 Hz, 1 H), 5.04 (s, 2 H), 7.22 - 7.38 (m, 5 H).
13
CNMR
(101MHz, CD3OD) G 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.
152.7.
19
FNMR (376MHz, CD3OD) Gppm = -
BNMR (160MHz, CD3OD) Gppm = 2.9. HRMS-ESI (m/z): calcd. for
11
C23H34[11B]FN3O8: 510.2428, found, 510.2446.
N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(2,4dimethoxybenzyl)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,NDiisopropylethylamine (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
65
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).
1
HNMR (400MHz, D2O) Gppm = 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) Gppm = 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-(4methoxybenzyl)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) Gppm = 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) Gppm = 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.
66
N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(3,4dimethoxyphenethyl)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) G 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, DMSOd6) G 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).
1
HNMR (400MHz, DMSO-d6) G ppm = 2.30 - 2.43 (m, 4 H), 3.50(s, 6H), 4.20 - 4.28 (m,
67
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).
CNMR (101MHz, DMSO-d6) G ppm = 173.3, 171.9, 139.9, 128.6, 127.5, 127.0,
13
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)
O
MeO
O
H
N
O
N
H
OH
OH
OH
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).
1
HNMR (400MHz, DMSO-d6) G 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).
13
CNMR (101MHz,
DMSO-d6) G 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.
68
N1-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)-N4-(2,4dimethoxybenzyl)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) G 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) G 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.
19
FNMR (376MHz, CD3OD) Gppm = -153.0. 11BNMR (160MHz, CD3OD) Gppm = 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-(4methoxybenzyl)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) G ppm = 2.46 - 2.59 (m, 4 H), 3.69 (s,
69
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).
13
CNMR (101MHz, CD3OD) G 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.
11
BNMR
(160MHz,
CD3OD)
Gppm
19
FNMR (376MHz, CD3OD) Gppm = -151.9.
=
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-(3,4dimethoxyphenethyl)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) G 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).
13
CNMR (101MHz, CD3OD) G 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) Gppm =
-151.2.
BNMR (160MHz, CD3OD) Gppm = 2.7. HRMS-ESI (m/z): calcd. for
11
C18H25[11B]FN2O7: 411.1744, found, 411.1767.
70
N1-benzyl-N4-(1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)succinamide
boron-fluoride complex adduct (2.11d)
O
O
H
N
N
H
O
F
O B
O
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) G 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).
13
CNMR
(101MHz, CD3OD) G 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) Gppm =
-151.6. 11BNMR (160MHz, CD3OD)
Gppm = 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
71
potassium fluoride (KF) according to general procedure B to afford 2.11e as a white solid
in quantitative yield. 1HNMR (400MHz, CD3OD) G 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).
CNMR (101MHz, CD3OD) G ppm = 174.2, 172.7, 169.1, 136.6, 127.2, 123.1,
13
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.
19
FNMR (376MHz, CD3OD) Gppm = -151.5.
11
BNMR (160MHz, CD3OD) Gppm =
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
72
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) Gppm = 0.758 (3, 3H), 3.39 (s, 6 H). 13CNMR (101MHz, D2O) Gppm = 64.4, 41.0,
15.5. 19FNMR (376MHz, D2O) Gppm = -143.5. 11BNMR (80.2MHz, D2O) Gppm = 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 ionexchange resin (Amberlite_IRA-67, free base) was loaded into a syringe column, then a
73
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.
1
HRMS-ESI (m/z): calcd. for
C8H12[11B]FNO6: 248.0747, found, 248.0751.
2.6 References
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11
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77
APPENDICES
78
APP
PENDIX A: Chapter One
O - Selecteed 1H and 13C NMR Spectra
1-Isoobutyl-1H-iimidazole (11.2c)
79
Metthyl 1-methyyl-1H-imidaazole-2-carb
bodithioate (1.3a)
80
Metthyl 1-benzyyl-1H-imidaazole-2-carb
bodithioate (1.3b)
81
Metthyl 1-isobutyl-1H-imid
dazole-2-carrbodithioatee (1.3c)
82
Metthyl 2-(1-meethyl-1H-im
midazole-2-carbothioam
mido)propan
noate (1.4a)
83
Metthyl 4-methyyl-2-(1-meth
hyl-1H-imid
dazole-2-carrbothioamid
do)pentanoate (1.4b)
84
Metthyl 2-(1-meethyl-1H-im
midazole-2-carbothioam
mido)-3-phen
nylpropanooate (1.4c)
85
Metthyl
3--(1H-indol-33-yl)-2-(1-m
methyl-1H-im
midazole-2--carbothioaamido)propaanoate
(1.4d
d)
86
Metthyl 2-(1-ben
nzyl-1H-imiidazole-2-caarbothioam
mido)propan
noate (1.4e)
87
Metthyl 2-(1-ben
nzyl-1H-imiidazole-2-caarbothioam
mido)-4-meth
hylpentanoaate (1.4f)
88
Metthyl 2-(1-ben
nzyl-1H-imiidazole-2-caarbothioam
mido)-3-phen
nylpropanoaate (1.4g)
89
Metthyl
22-(1-benzyl-1H-imidazoole-2-carbotthioamido)--3-(1H-indoll-3-yl)propaanoate
(1.4h
h)
90
Metthyl 2-(1-isoobutyl-1H-im
midazole-2-carbothioam
mido)propaanoate (1.4i))
91
Metthyl 2-(1-isoobutyl-1H-im
midazole-2-carbothioam
mido)-4-meethylpentanooate (1.4j)
92
Metthyl 2-(1-isoobutyl-1H-im
midazole-2-carbothioam
mido)-3-pheenylpropan
noate (1.4k)
93
Metthyl
3-((1H-indol-3-yl)-2-(1-isoobutyl-1H-im
midazole-2--carbothioaamido)propaanoate
(1.4ll)
94
5-M
Methyl-3-(1-m
methyl-1H-iimidazol-2--yl)-4,5-dihyydro-1,2,4-trriazin-6(1H
H)-one (1.5a))
95
5-Isoobutyl-3-(1--methyl-1H
H-imidazol-22-yl)-4,5-dih
hydro-1,2,4--triazin-6(1H
H)-one (1.5b
b)
96
5-Beenzyl-3-(1-m
methyl-1H-iimidazol-2-yyl)-4,5-dihyydro-1,2,4-trriazin-6(1H))-one (1.5c)
97
5-((11H-indol-3--yl)methyl)-3-(1-methyll-1H-imidazzol-2-yl)-4,55-dihydro-1,2,4-triazin-6(1H
H)-one (1.5d
d)
98
3-(1-Benzyl-1H
H-imidazol-22-yl)-5-meth
hyl-4,5-dihyydro-1,2,4-trriazin-6(1H))-one (1.5e)
99
3-(1-Benzyl-1H
H-imidazol-22-yl)-5-isobu
utyl-4,5-dihyydro-1,2,4-ttriazin-6(1H
H)-one (1.5ff)
100
5-Beenzyl-3-(1-b
benzyl-1H-im
midazol-2-yyl)-4,5-dihyd
dro-1,2,4-trriazin-6(1H))-one (1.5g)
101
5-((11H-indol-3--yl)methyl)-3-(1-benzyll-1H-imidazzol-2-yl)-4,5-dihydro-1,,2,4-triazin-6(1H
H)-one (1.5h
h)
102
3-(1-Isobutyl-1H
H-imidazol--2-yl)-5-metthyl-4,5-dih
hydro-1,2,4--triazin-6(1H
H)-one (1.5ii)
103
5-Isoobutyl-3-(1--isobutyl-1H
H-imidazol--2-yl)-4,5-diihydro-1,2,44-triazin-6(11H)-one (1.55j)
104
5-Beenzyl-3-(1-issobutyl-1H--imidazol-2-yl)-4,5-dihyydro-1,2,4-ttriazin-6(1H
H)-one (1.5k
k)
105
5-((11H-indol-3--yl)methyl)-3-(1-isobutyyl-1H-imidaazol-2-yl)-4,,5-dihydro-11,2,4-triazin
n6(1H
H)-one (1.5l))
106
APP
PENDIX B:: Chapter T
Two - Selected 1H, 13C, 19F, 11B NM
MR Spectra & HRMS
Com
mpound (2.33) -1H and 133C NMR Spectra
107
Com
mpound (2.33) -19F and 111B NMR Sp
pectra
108
Com
mpound (2.33) –High Resolution Maass Spectra
109
Com
mpound (2.55a) -1H and 13C NMR Sp
pectra
110
Com
mpound (2.55b) -1H and 13C NMR Sp
pectra
111
Com
mpound (2.55c) -1H and 13C NMR Sp
pectra
112
Com
mpound (2.55d) -1H and 13C NMR Sp
pectra
113
Com
mpound (2.55e) -1H and 13C NMR Sp
pectra
114
Com
mpound (2.55f) -1H and 13C NMR Sp
pectra
115
Com
mpound (2.66a) -1H and 13C NMR Sp
pectra
116
Com
mpound (2.66a) -19F and 11B NMR Spectra
117
Com
mpound (2.66a) –High Resolution Mass
M
Spectraa
118
Com
mpound (2.66b) -1H and 13C NMR Sp
pectra
119
Com
mpound (2.66b) -19F and 11B NMR S
Spectra
120
Com
mpound (2.66b) –High Resolution M
Mass Spectraa
121
Com
mpound (2.66c) -1H and 13C NMR Sp
pectra
122
Com
mpound (2.66c) -19F and 11B NMR Sp
pectra
123
Com
mpound (2.66c) –High Resolution M
Mass Spectraa
124
Com
mpound (2.66d) -1H and 13C NMR Sp
pectra
125
Com
mpound (2.66d) -19F and 11B NMR S
Spectra
126
Com
mpound (2.66d) –High Resolution M
Mass Spectraa
127
Com
mpound (2.66e) -1H and 13C NMR Sp
pectra
128
Com
mpound (2.66e) -19F and 11B NMR Sp
pectra
129
Com
mpound (2.66e) –High Resolution M
Mass Spectraa
130
Com
mpound (2.66f) -1H and 13C NMR Sp
pectra
131
Com
mpound (2.66f) -19F and 11B NMR Sp
pectra
132
Com
mpound (2.66f) –High Reesolution M
Mass Spectraa
133
Com
mpound (2.110a) -1H and
d 13C NMR S
Spectra
134
Com
mpound (2.110b) -1H and
d 13C NMR Spectra
S
135
Com
mpound (2.110c) -1H and
d 13C NMR S
Spectra
136
Com
mpound (2.110d) -1H and
d 13C NMR Spectra
S
137
Com
mpound (2.110e) -1H and
d 13C NMR S
Spectra
138
Com
mpound (2.111a) -1H and
d 13C NMR S
Spectra
139
Com
mpound (2.111a) -19F and
d 11B NMR S
Spectra
140
Com
mpound (2.111a) –High Resolution M
Mass Spectrra
141
Com
mpound (2.111b) -1H and
d 13C NMR Spectra
S
142
Com
mpound (2.111b) -19F and
d 11B NMR Spectra
143
Com
mpound (2.111b) –High Resolution Mass
M
Spectrra
144
Com
mpound (2.111c) -1H and
d 13C NMR S
Spectra
145
Com
mpound (2.111c) -19F and
d 11B NMR Spectra
S
146
Com
mpound (2.111c) –High Resolution M
Mass Spectrra
147
Com
mpound (2.111d) -1H and
d 13C NMR Spectra
S
148
Com
mpound (2.111d) -19F and
d 11B NMR Spectra
149
Com
mpound (2.111d) –High Resolution Mass
M
Spectrra
150
Com
mpound (2.111e) -1H and
d 13C NMR S
Spectra
151
Com
mpound (2.111e) -19F and
d 11B NMR Spectra
S
152
Com
mpound (2.111e) –High Resolution M
Mass Spectrra
153
Com
mpound (2.114) -1H and 13C NMR Sp
pectra
154
Com
mpound (2.114) -19F and 11B NMR Spectra
155
Com
mpound (2.114) –High Resolution Mass
M
Spectraa
156
Com
mpound (2.117) –High Resolution Mass
M
Spectraa
157
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.