PART I: OVERVIEW OF THE COORDINATED RESEARCH PROJECT
CHAPTER 1. OVERVIEW OF THE COORDINATED RESEARCH PROJECT ........ 3
1.1. Introduction ........................................... 3
1.1.1. Background ..................................... 3
1.1.2. Objectives of the coordinated research
project ........................................ 4
1.1.3. Work plan of the coordinated research
project ........................................ 4
1.2. Scientific background on technetium-99m cores .......... 6
1.2.1. [99mTc(CO)3]+-carbonyl core ..................... 6
1.2.1.1. Tridentate chelators ................. 6
1.2.1.2. Combination of bidentate and
monodentate chelators (2 + 1
approach) ............................ 7
1.2.1.3. Higher technetium-carbonyls .......... 8
1.2.2. [99mTc≡N]2+-nitrido core ........................ 8
1.2.3. Technetium-99m-HYNIC core ..................... 10
1.2.4. 99mTc(III)/Re(III)-(4+1) core .................. 12
1.3. Summary of scientific work ............................ 14
1.3.1. Chart 1: Technetium-99m labelling of RGD
peptides targeting αvβ3 integrin receptors .... 14
1.3.1.1. Radiolabelling ...................... 14
1.3.1.2. Biological characterization ......... 17
1.3.1.3. Conclusion (chart 1) ................ 19
1.3.2. Chart 2: Labelling of annexin V fragments ..... 20
1.3.2.1. Radiolabelling ...................... 21
1.3.2.2. Biological characterization ......... 23
1.3.2.3. Conclusion (chart 2) ................ 24
1.3.3. Chart 3: Labelling of fatty acids using
the 99mTc-tricarbonyl core .................... 24
1.3.4. Chart 4: Technetium-99m labelling of
quinazoline derivatives ....................... 26
1.3.5. Chart 5: Development of technetium-99m
glucose analogues ............................. 27
1.4. Achievements of the coordinated research project ...... 30
1.5. Intergroup collaborations ............................. 31
1.5.1. Exchanges of reagents and precursors .......... 31
1.5.2. Exchange visits ............................... 32
1.6. Conclusion ............................................ 33
PART II: REPORTS BY THE PARTICIPANTS IN THE COORDINATED
RESEARCH PROJECT
CHAPTER 2. TECHNETIUM-99m LABELLING OF RGD PEPTIDES
TARGETING INTEGRIN RECEPTORS: COMPARISON OF
DIFFERENT CONJUGATES OF cRGDyK UTILIZING
DIFFERENT TECHNETIUM-99m CORES ..................... 37
C. Decristoforo, R. Haubner, M. Rupprich, E.
von Guggenberg
2.1. Introduction .......................................... 38
2.2. Peptide synthesis and distribution .................... 39
2.3. Experimental methods .................................. 40
2.3.1. Analytical methods ............................ 40
2.3.1.1. HPLC ................................ 40
2.3.1.2. Purification by solid phase
extraction .......................... 41
2.3.2. Technetium-99m labelling ...................... 41
2.3.2.1. HYNIC-cRGDyK ........................ 41
2.3.2.2. Labelling with
[99mTc(OH2)3(CO)3]+ ................... 43
2.3.2.3. 4 + 1 approach ...................... 43
2.3.2.4. Labelling of Cys-RGD with the
99mTc-nitrido fragment ............... 44
2.3.3. In vitro evaluation of radiolabelled
peptides ...................................... 44
2.3.3.1. Stability ........................... 44
2.3.3.2. Protein binding ..................... 44
2.3.3.3. Log P values ........................ 45
2.3.4. Internalization and binding studies in αvβ3
positive and αvβ3 negative cells .............. 45
2.3.5. In vivo evaluation of radiolabelled
peptides ...................................... 45
2.4. Results ............................................... 46
2.4.1. Characterization of cold peptides ............. 46
2.4.2. Labelling experiments ......................... 46
2.4.3. Stability experiments, log P and protein
binding ....................................... 48
2.4.4. Internalization studies ....................... 48
2.4.5. In vivo tumour model .......................... 50
2.5. Discussion and outlook ................................ 52
References ............................................ 52
CHAPTER 3. RADIOLABELLING ANGIOGENIC AND APOPTOTIC AGENT
WITH DIFFERENT TECHNETIUM-99m TECHNIQUES ........... 55
B. Linkowski Faintuch, R.L.S. Ribeiro Santos,
R. Teodoro, E. Muramoto, L. Morganti, I.V.
Da Silva Nunes, M.R.Y. Okamoto
3.1. Introduction .......................................... 56
3.1.1. Angiogenesis markers .......................... 56
3.1.2. Investigation of apoptosis .................... 56
3.1.3. Labelling procedures .......................... 57
3.2. Materials ............................................. 58
3.3. Methods ............................................... 59
3.3.1. Labelling of HYNIC-RGD and HYNIC-annexin
with technetium-99m using EDDA/tricine
as exchange products .......................... 59
3.3.2. Preparation of 99mTc-nitrido precursor ......... 59
3.3.3. Labelling of Cys-RGD and Cys-annexin using
the 99mTc-nitrido precursor .................... 59
3.3.4. Radiochemical stability, transchelation
towards cysteine and partition coefficient .... 60
3.3.5. Synthesis of 99mTc-carbonyl .................... 60
3.3.6. Labelling of Ter-Cys-RGD, PZ1-RGD, His-RGD
and His-annexin with the precursor
99mTc(CO)3 ..................................... 60
3.3.7. Quality control ............................... 60
3.3.7.1. 99mTc-HYNIC peptide complexes ........ 60
3.3.7.2. 99mTc(CO)3 peptide complexes ......... 61
3.3.7.3. 99mTcN(PNP6) peptide complexes ....... 61
3.3.8. Purification .................................. 61
3.3.9. Biodistribution studies in healthy animals .... 61
3.3.10. Animal model with tumour cells ................ 61
3.3.11. Animal model for annexin studies .............. 62
3.3.12. Imaging of tumour ............................. 62
3.4. Results and discussion ................................ 62
3.4.1. Labelling of HYNIC-RGD and HYNIC-annexin
with technetium-99m using EDDA/tricine
as exchange products .......................... 62
3.4.2. Preparation of 99mTc-nitrido and labelling
of Cys-RGD and Cys-annexin .................... 63
3.4.3. Radiochemical stability, transchelation
towards cysteine and partition coefficient .... 64
3.4.4. Synthesis of 99mTc-carbonyl and labelling
of Ter-Cys-RGD, PZ1-RGD, His-RGD and
His-annexin ................................... 65
3.4.5. Biodistribution studies in normal and
tumour bearing animals ........................ 66
3.4.6. Animal model with tumour cells ................ 66
3.4.7. Animal model for annexin studies .............. 69
3.4.8. Imaging of tumour uptake ...................... 69
3.5. Conclusions ........................................... 70
Acknowledgements ...................................... 72
References ............................................ 72
CHAPTER 4. RADIOLABELLING OF RGD PEPTIDES USING
NOVEL TECHNETIUM-99m CORES ......................... 75
Ji Нu, Baojun Chen, Jixin Liang, Lianzhe Luo,
Hongyu Li, Yang Chen, Langtao Shen, Zhifu Luo
4.1. Introduction .......................................... 75
4.2. Materials and methods ................................. 76
4.2.1. Radiolabelling ................................ 78
4.2.1.1. Radiolabelling of Cys-RGD with the
[99mTc(N)]2+ core .................... 78
4.2.1.2. Radiolabelling of HYNIC-RGD
with 99mTc .......................... 78
4.2.1.3. Radiolabelling of His-RGD and
Cys(X)-RGD with
[99mТс(Н20)3(СО)3]+ .................. 78
4.2.1.4. Purification of radiolabeled RGD
peptides ............................ 79
4.2.1.5. In vitro stability .................. 79
4.2.2. In vivo animal experiment ..................... 79
4.2.2.1. Tumour xenograft .................... 79
4.2.2.2. Biodistribution studies ............. 80
4.2.2.3. Blocking studies .................... 80
4.2.2.4. Gamma camera imaging ................ 80
4.3. Results and discussion ................................ 80
4.3.1. Radiolabelling of RGD peptides ................ 80
4.3.1.1. In vitro stability ................... 82
4.3.2. Biodistribution studies ....................... 83
4.4. Conclusion ............................................ 85
References ............................................ 86
CHAPTER 5. 99mTc-HYNIC-Anxl3: PREPARATION, STABILITY,
BIODISTRIBUTION AND IMAGING OF APOPTOSIS ........... 89
Hongyu Li, Ji Нu, Jixin Liang, Baojun Chen, Ja
Lu, Lianzhe Luo, Yang Chen, Zhifu Luo
5.1. Introduction .......................................... 89
5.2. Materials and methods ................................. 90
5.2.1. Radiolabelling of technetium-99m .............. 90
5.2.1.1. Tricine as a coligand ............... 90
5.2.1.2. EDDA as a coligand .................. 90
5.2.1.3. Tricine/EDDA as coligands ........... 90
5.2.2. Quality control ............................... 91
5.2.2.1. Thin layer chomatography ............ 91
5.2.2.2. HPLC ................................ 91
5.2.2.3. Sep-Pak purification procedure ...... 91
5.2.3. Stability of radiolabeled peptides ............ 92
5.2.4. Biodistribution in normal mice ................ 92
5.2.5. Imaging and biodistribution in apoptotic
model animals ................................. 92
5.3. Results and discussion ................................ 93
5.3.1. Preparation of 99mTc-tricine-HYNIC-Anxl3
using tricine as a coligand ................... 93
5.3.2. Preparation of 99mTc-EDDA-HYNIC-Anxl3 using
EDDA as a coligand ............................ 93
5.3.3. Technetium-99m labelling of HYNIC-Anxl3
using EDDA/tricine as coligands ............... 94
5.3.4. Stability in vivo and in vitro ................ 96
5.3.5. Bioditribution in normal mice ................. 97
5.3.6. Biodistribution and radionuclide imaging in
model animals ................................. 98
5.4. Conclusion ........................................... 101
References ........................................... 101
CHAPTER 6. PREPARATION AND COMPARATIVE EVALUATION OF
TECHNETIUM-99m LABELLED FATTY ACIDS ............... 103
Ji Нu, Jixin Liang, Baojun Chen, Lianzhe Luo,
Hongyu Li, Langtao Shen, Zhifu Luo
6.1. Introduction ......................................... 103
6.2. Materials ............................................ 104
6.3. Methods .............................................. 104
6.3.1. Preparation of [99mTc(CO)3(H20)3]+
intermediate ................................. 104
6.3.2. Labelling of CYST FAC11 and IDA FAC1 ......... 105
6.3.3. Purification of radiolabelled products by
the SPE method ............................... 105
6.3.4. In vitro stability ........................... 105
6.3.5. Biodistribution studies in normal mice ....... 106
6.4. Results and discussion ............................... 106
6.4.1. Radiolabelling ............................... 106
6.4.2. In vitro stability ........................... 107
6.4.3. Biodistribution .............................. 108
6.5. Conclusions .......................................... 110
References ........................................... 112
CHAPTER 7. TECHNETIUM-99m LABELLING OF AN ANNEXIN FRAGMENT
USING THE 4+1 MIXED LIGAND CHELATE SYSTEM ......... 113
J.-U. Künstler, В. Pawelke, A. Duatti, J.
Környei, H.-J. Pietzsch
7.1. Introduction ......................................... 113
7.2. Materials and methods ................................ 114
7.2.1. General ...................................... 114
7.2.2. Ll-AF, L2-AF, Re(NS3)(Ll-AF) and
Re(NS3)(L2-AF) ............................... 114
7.2.3. 99mTc(NS3)(Ll-AF) and 99mTc(NS3)(L2-AF) ........ 115
7.2.4. In vivo stability ............................ 115
7.2.5. Cell binding assay ........................... 116
7.2.5.1. Binding studies .................... 116
7.3. Results and discussion ............................... 116
7.4. Conclusions .......................................... 119
References ........................................... 120
CHAPTER 8. TECHNETIUM-99m LABELLING OF A QUINAZOLINE
DERIVATIVE USING THE 4+1 MIXED LIGAND
CHELATE SYSTEM .................................... 121
J.-U. Künstler, I. Santos, I. Pirmettis,
H.-J. Pietzsch
8.1. Introduction ......................................... 121
8.2. Results and discussion ............................... 122
8.2.1. In vitro evaluation .......................... 124
8.2.1.1. Inhibition of cell proliferation ... 124
8.2.1.2. Inhibition of EGFR-TK .............. 126
8.3. Conclusions .......................................... 126
References ........................................... 127
CHAPTER 9. TECHNETIUM-99m LABELLING OF THE RGD PEPTIDE
c(RGDyK) USING THE 4+1 MIXED LIGAND APPROACH ...... 129
J.-U. Künstler, P. Ansorge, R. Bergmann, E.
Gniazdowska, С Decristoforo, A. Rey, H. Stephan,
H.-J. Pietzsch
9.1. Introduction ......................................... 130
9.2. Materials and methods ................................ 130
9.2.1. General ...................................... 130
9.2.2. Ll-c(RGDyK), L2-c(RGDyK), Re(NS3)
(Ll-c(RGDyK)) and Re(NS3)(L2-c(RGDyK)) ....... 131
9.2.3. Re(NS3(COOH)3)(L2-c(RGDyK)) .................. 131
9.2.4. Technetium-99m labelling ..................... 132
9.2.5. Analytical data .............................. 132
9.2.6. Distribution ratio (log D, octanol/PBS,
pH7.4) ....................................... 132
9.2.7. Biodistribution studies ...................... 133
9.3. Results and discussion ............................... 133
9.3.1. Biodistribution studies in mice .............. 136
9.4. Conclusions .......................................... 136
References ........................................... 140
CHAPTER 10. DEVELOPMENT OF TECHNETIUM-99m LABELLED ANNEXIN
V FRAGMENTS USING THE Tc-TRICARBONYL CORE ......... 141
D. Psimadas, С Zikos, M. Fani, E. Livaniou, M.
Papadopoulos, I. Pirmettis
10.1. Introduction ......................................... 141
10.2. Materials and methods ................................ 141
10.2.1. General ...................................... 141
10.2.2. Derivatization of CAQVLRGTVTDFPGx2TFA with
vinyl pyridine ............................... 142
10.2.3. Preparation of the precursor
[99mTc(H20)3(CO)]+ ............................ 142
10.2.4. Radiolabelling of peptides ................... 142
10.2.5. Cysteine and histidine challenge of
technetium-99m labelled peptides ............. 143
10.2.6. Biodistribution studies in mice .............. 143
10.3. Results and discussion ............................... 143
10.4. Conclusion ........................................... 145
CHAPTER 11. DEVELOPMENT OF TECHNETIUM-99m LABELLED
BIOMARKERS FOR HEART METABOLISM USING
THE Tc-TRICARBONYL CORE ........................... 147
A. Papadopoulos, C. Tsoukalas, A.
Panagiotopoulou, M. Pelecanou, M.
Papadopoulos, I. Pirmettis
11.1. Introduction ......................................... 147
11.2. Materials and methods ................................ 148
11.2.1. General ...................................... 148
11.2.2. Synthesis of ligands ......................... 148
11.2.2.1. Synthesis of ll-[bis
(carboxymethyl)amino] undecanoic
acid (IDA-FA11) .................... 148
11.2.2.2. Synthesis of ll-(S-cysteine
undecanoic acid Cyst-FA11 .......... 149
11.2.2.3. Synthesis of 16-(S-cysteine)
hexadecanoic acid Cyst-FA16 ........ 149
11.2.2.4. Synthesis of 16-(S-cysteine)
hexadecanoic acid dimethylester,
Cyst-FA16 dimethylester ............ 150
11.2.2.5. Synthesis of N-undecanoicdithio-
carbamate sodium salt CS2-FA11 ..... 150
11.2.2.6. Synthesis of 16-(N-(pyridine-2-
yl-methyl)-N-aminoethyl acetate)
hexadecanoic ethylester PAM-FA16 ... 150
11.2.2.7. Synthesis of NSC-FA11 .............. 150
11.2.3. Synthesis of rhenium complexes ............... 151
11.2.3.1. Synthesis of Re(CO)3(IDA-FAll) ..... 151
11.2.3.2. Synthesis of the complex Re(CO)3
(Cyst-FA11) ........................ 151
11.2.3.3. Synthesis of the complex Re(CO)3
(NSC-FA11) ......................... 152
11.2.3.4. Synthesis of the complex Re(CO)3
PAM-FA16 ........................... 152
11.2.4. Labelling with technetium-99m ................ 152
11.2.4.1. Preparation of the precursor
[99mTc(H20)3(CO)]+ .................. 152
11.2.4.2. General method for the preparation
of the technetium-99m labelled
fatty acids with [99mTc(CO)3]+ ...... 153
11.2.4.3. Synthesis of the technetium-99m
complex with 11-[bis(carboxy-
methyl)amino]undecanoic acid ....... 153
11.2.4.4. Synthesis of the complex
99mTc(CO)3 (Cyst-FA11) .............. 153
11.2.4.5. Synthesis of the complex
99mTc(CO)3 (NSC-FA11) ............... 153
11.3. Results and discussion ............................... 153
11.4. Conclusions .......................................... 156
References ........................................... 156
CHAPTER 12. DEVELOPMENT OF TECHNETlUM-99m LABELLED
BIOMARKERS FOR EGFR-TK USING THE Tc-
TRICARBONYL CORE .................................. 157
N. Margaritis, N. Bourkoula, M. Paravatou, E.
Livaniou, A. Papadopoulos, A. Panagiotopoulou,
C. Tsoukalas, M. Pelecanou, M. Papadopoulos,
I. Pirmettis
12.1. Introduction ......................................... 157
12.2. Materials and methods ................................ 158
12.2.1. General ...................................... 158
12.2.2. Synthesis .................................... 158
12.2.2.1. 6-nitroquinazoline (1) ............. 158
12.2.2.2. 4-chloro-6-nitroquinazoline (2) .... 159
12.2.2.3. 4-[(3-bromophenyl)amino]-6-nitro-
quinazoline (3) .................... 159
12.2.2.4. 6-amino-4-[(3-bromophenyl)amino]-
quinazoline (4) .................... 159
12.2.2.5. 6-(piridine-2-methylimin)-4-
[(3-bromophenyl) amino]-
quinazoline (5) .................... 160
12.2.2.6. N-{4-[(3-bromophenyl)amino]-
quinazoline-6-yl}-2-
chloroacetamide (6) ................ 160
12.2.3. Synthesis of rhenium complexes, general
method ....................................... 160
12.2.4. Labelling with technetium-99m ................ 161
12.2.4.1. Preparation of the precursor
[99mTc(H20)3(CO)]+ .................. 161
12.2.4.2. General method for the
preparation of the 99mTc
labelled quinazolines
[99mTc(CO)3]+ ...................... 161
12.2.4.3. In vitro evaluation of
quinazoline analogues .............. 161
12.3. Results and discussion ............................... 162
12.4. Conclusions .......................................... 164
References ........................................... 164
CHAPTER 13. DEVELOPMENT OF ANNEXIN V FRAGMENTS FOR
LABELLING WITH TECHNETIUM-99m ..................... 167
J. Környei, F. Tóth, E. Szemenyei, A. Duatti
13.1. Introduction ......................................... 167
13.2. Materials and methods ................................ 168
13.2.1. Synthesis of annexin V fragments ............. 168
13.2.2. Stability studies ............................ 168
13.2.3. 99mTc-nitrido labelling ...................... 169
13.2.4. Determination of the radiochemical purity .... 169
13.3. Results and discussion ............................... 169
13.3.1. Peptide synthesis and stability .............. 169
13.3.2. 99mTc-nitrido labelling ...................... 172
13.3.3. Other studies with derivatized Anxl3
fragments .................................... 174
13.4. Conclusions .......................................... 175
References ........................................... 175
CHAPTER 14. DEVELOPMENT OF TECHNETIUM-99m BASED SMALL
BIOMOLECLES USING NOVEL TECHNETIUM-99m CORES:
99mTc-TRICARBONYL, 99mTc-HYNIC AND 99mTc-NITRIDO
CHEMICAL APPROACHES ............................... 177
K. Kothari, A. Mukherjee, D. Satpati, A. Korde,
S. Joshi, H.D. Sarma, A. Mathur, M. Mallia, S.
Banerjee, M. Venkatesh
14.1. Introduction ......................................... 178
14.2. Materials ............................................ 180
14.3. Methods .............................................. 181
14.3.1. Synthesis and characterization of
99mTc(CO)3-RGD, 99mTc(N)-RGD and
99mTc-HYNIC-RGD complexes for tumour
targeting .................................... 181
14.3.1.1. Synthesis of 99mTc-tricarbonyl
complexes of RGD ................... 181
14.3.1.2. Preparation of 99mTc-nitrido
complexes of RGD ................... 182
14.3.1.3. Synthesis of 99mTc-HYNIC-RGD ........ 182
14.3.1.4. Quality control .................... 183
14.3.1.5. In vitro studies ................... 183
14.3.1.6. In vivo studies .................... 184
14.3.2. Synthesis and evaluation of 99mTc(N)-Anxl3
and 99mTc(CO)3-Anxl3 as apoptosis marker ..... 184
14.3.2.1. Preparation and characterization
of technetium-99m Anxl3 ............ 185
14.3.2.2. In vitro studies ................... 185
14.3.2.3. In vivo studies .................... 186
14.3.3. Synthesis and characterization of
technetium-99m fatty acid analogues for
myocardial imaging ........................... 187
14.3.3.1. Radiolabelling studies ............. 187
14.3.3.2. Quality control .................... 188
14.3.3.3. In vitro studies ................... 188
14.3.3.4. In vivo studies .................... 188
14.4. Results .............................................. 189
14.4.1. Synthesis and characterization of
99mTc(CO)3-RGD and 99mTc(N)-RGD for
tumour targeting ............................. 189
14.4.1.1. 99mTc-tricarbonyl complexes of
RGD ................................ 189
14.4.1.2. 99mTc(N)Cys-RGD (symmetric
complexes) ......................... 189
14.4.1.3. 99mTc(N)(PNP)-Cys-RGD
(asymmetric complex) ............... 190
14.4.1.4. Preparation of 99mTc-HYNIC-RGD ..... 190
14.4.1.5. In vitro studies ................... 190
14.4.1.6. In vivo studies .................... 191
14.4.2. Synthesis and evaluation of 99mTc(N)-Anxl3
and 99mTc(CO)3-Anxl3 as an apoptosis
marker ....................................... 191
14.4.2.1. Radiolabelling of Anxl3
analogues .......................... 191
14.4.2.2. In vitro studies ................... 192
14.4.2.3. In vivo studies .................... 195
14.4.3. Synthesis and characterization of
99mTc(CO)3 undeconic acid for myocardial
imaging ...................................... 197
14.4.3.1. 99mTc(CO)3-CYSFA11 .................. 197
14.4.3.2. 99mTc(CO)3-CYSFA16 .................. 198
14.4.3.3. 99mTc(N)-CS2FA11 .................... 198
14.5. Conclusion ........................................... 200
Acknowledgements ..................................... 200
References ........................................... 201
CHAPTER 15. LABELLING OF SMALL MOLECULES WITH THE
99mTc-NITRIDO CORE ................................ 203
M. Pasquali, L. Uccelli, A. Boschi, A. Duatti
15.1. Introduction ......................................... 203
15.2. Labelling methods .................................... 204
15.2.1. Symmetrical complexes ........................ 204
15.2.2. Asymmetrical complexes ....................... 204
15.3. Results and discussion ............................... 206
15.3.1. Development of 99mTc glucose analogues ....... 206
15.3.2. Labelling of bioactive peptides .............. 207
15.3.2.1. RGD peptides ....................... 208
15.3.2.2. Annexin V .......................... 210
15.3.2.3. Quinazoline ligands ................ 211
15.4. Experimental procedures .............................. 215
15.4.1. Symmetrical complexes ........................ 215
15.4.1.1. Bis(dithiocarbamate) nitrido
99mTc complexes .................... 215
15.4.2. Asymmetrical complexes ....................... 215
15.4.2.1. Mixed [99mTc(N)(PNP)(XY)]0/+
(PNP = amino-bis-phosphino lignad;
XY - monoanionic or dianionic
bidentate ligand) complexes ........ 215
15.4.2.2. Mixed [99mTc(N)(PS)(DTC)]
(PS = phosphino-thiol lignad; DTC
= monoanionic dithiocarbamate
ligand) complexes .................. 217
References ........................................... 218
CHAPTER 16. LABELLING OF QUINAZOLINE AND RGD PEPTIDES
WITH THE [99mTc(CO)3]+ CORE: SYNTHESIS,
CHARACTERIZATION AND BIOLOGICAL EVALUATION ........ 219
I. Santos, С. Fernandes, S. Alves, J. Galamba,
L. Gano, I. Pirmettis, С Decristoforo, C.J.
Smith, R. Alberto
16.1. Introduction ......................................... 220
16.2. Results .............................................. 221
16.2.1. Synthesis and characterization of
quinazoline derivatives to be coupled
to different chelators and synthesis of
a novel chelator for tricarbonyl ............. 221
16.2.2. Evaluation of L1 as a new ligand for the
[99mTc(CO)3]+ core ........................... 222
16.2.3. Conjugation of a quinazoline fragment to L1
and labelling of the conjugate with the
[99mTc(CO)3]+ core ............................ 223
16.2.4. Cell growth and EGFR-TK inhibition ........... 225
16.2.5. Labelling of an RGD peptide with the fac-
[99mTc(CO)3]+ moiety .......................... 227
16.2.6. Internalization and binding studies in
αvβ3 positive and αvβ3 negative cells ......... 227
16.2.7. Biodistribution in healthy and in nu/nu
mice ......................................... 228
16.3. Summary and conclusions .............................. 229
References ........................................... 230
CHAPTER 17. LIGANDS OF LOW DENTICITY AS COORDINATION UNITS
FOR TETHERING THE 99mTc-CARBONYL CORE TO
BIOMOLECULES ...................................... 233
N.I. Gorshkov, E.M. Levitskaya, A.A. Lumpov,
A.E. Miroslavov, G.V. Sidorenko, D.N. Suglobov
17.1. Introduction ......................................... 233
17.2. Experimental 234
17.3. 2+1 ligand systems for binding Tc-tricarbonyl core ... 235
17.3.1. Complexation with bidentate ligands .......... 235
17.3.2. 2+1 systems with dithiocarbamates ............ 236
17.3.3. 2+1 systems with bidentate nitrogen bases .... 237
17.3.4. Biodistribution studies of 2+1 systems
with bipyridine and related compounds ........ 238
17.4. Derivatives of higher technetium-carbonyls ........... 240
17.4.1. Preparation of higher technetium-carbonyls
at the non-carrier added level ............... 241
17.4.2. Tetracarbonyltechnetium
diethyldithiocarbamate ....................... 243
17.4.3. Pentacarbonyltechnetium phosphine and
isonitrile complexes ......................... 243
17.4.4. Hexacarbonyltechnetium(I) cation ............. 244
17.5. Conclusion ........................................... 247
Acknowledgements ..................................... 247
References ........................................... 247
CHAPTER 18. SMALL MOLECULE LABELLING WITH [99mTc(OH2)3
(CO)3]+: LIGANDS, AMINO ACIDS AND INTERCALATORS ... 249
R. Alberto, Y. Liu, N. Agorastos, I. Santos,
R. Raposinho, H. Knight, J. Mertens, M. Bauwens
18.1. Introduction ......................................... 250
18.2. Results .............................................. 251
18.2.1. New ligands for the [Tc(CO)3]+ core .......... 251
18.2.2. Labelling of amino acids ..................... 252
18.2.3. Targeting the nucleus: intercalators ......... 254
18.2.4. Kit preparation of [188/186Re(OH2)3(CO)3]+ ...... 256
18.3. Summary and conclusions .............................. 257
18.4. Experimental ......................................... 258
18.4.1. Triethyl 1,6-diacetamido-6-cyanohexane-
1,1,6-tricarboxylate (II) .................... 258
18.4.2. Triethyl l,6-diacetamido-7-(tert-
butoxycarbonylamino) heptane-l,l,6-
tricarboxylate (III) ......................... 259
18.4.3. Preparation of V ............................. 259
Acknowledgements ..................................... 260
References ........................................... 260
CHAPTER 19. DESIGN AND EVALUATION OF POTENTIAL 99mTc
RADIOPHARMACEUTICALS BASED ON THE Tc-CARBONYL,
4+1 MIXED LIGAND CHELATE SYSTEM AND Tc-NITRIDO
APPROACHES ........................................ 261
J. Giglio, A. Muslera, M. Incerti, R. Fernández,
E. León, A. Paolino, A. Brugnini, E. Manta, A.
Chabalgoity, A. León, A. Rey
19.1. Introduction ......................................... 262
19.2. Materials ............................................ 263
19.3. Methods .............................................. 263
19.3.1. Synthesis of glucose derivatives ............. 263
19.3.1.1. N-(3,4,6-tri-0-acetyl-β-D-
glucopyranosyl) piperidine (1) ..... 263
19.3.1.2. N-[3,4,6-tri-0-acetyl-2-0-(6-
bromohexyl)β-D-glucopyranosyl)
piperidine (2) ..................... 264
19.3.1.3. N-[3,4,6-tri-0-acetyl-2-0-(6-
bromobutyl)β-D-glucopyranosyl)
piperidine (2') .................... 264
19.3.1.4. (2R,3S,4R,5R)-2-(acetoxymethyl)-
5-(4-azidobutyl)-6-(piperidin-
l-yl)-tetrahydro-2H-pyran-3,4-
diyl diacetate (3) ................. 265
19.3.1.5. (2R,3S,4R,5R)-2-(acetoxymethyl)-
5-(4-azidobutyl)-6-(piperidin-l-
yl)-tetrahydro-2H-pyran-3,4-diyl
diacetate (4) ...................... 265
19.3.1.6. (2-aminoethylamino)acetic
acid (5) ........................... 265
19.3.2. Preparation and evaluation of technetium-
99m labelled RGD peptides .................... 266
19.3.2.1. Technitium-99m labelling ........... 266
19.3.2.2. In vitro evaluation ................ 267
19.3.2.3. In vivo evaluation ................. 268
19.3.3. Preparation and evaluation of technetium-
99m labelled annexin 13 peptides ............. 268
19.3.3.1. Biodistribution in aminals
treated with doxorubicin ........... 269
19.4. Results and discussion ............................... 269
19.4.1. Glucose derivatives .......................... 269
19.4.2. RGD peptides ................................. 270
19.4.2.1. Labelling of RGD peptides with
HYNIC .............................. 271
19.4.2.2. Labelling of RGD peptides with
tricarbonyl ........................ 274
19.4.2.3. Labelling of RGD peptides with
4+1 chemistry ...................... 277
19.4.2.4. Labelling of RGD peptides with
nitrido chemistry .................. 279
19.4.3. Annexin ...................................... 280
19.4.3.1. Labelling of annexin peptides
with HYNIC and carbonyl ............ 281
19.5. Conclusions .......................................... 283
Acknowledgements ..................................... 284
References ........................................... 284
CHAPTER 20. IN VITRO AND IN VIVO EVALUATION OF
[99mTc(CO)3CYCLO[Arg-Gly-Asp-D-Tyr-Lys(PZl)]] ...... 287
S. Alves, J.D.G. Correia, I. Santos, С.
Decristoforo, R. Alberto, C.J. Smith
20.1. Introduction ......................................... 287
20.2. Materials ............................................ 290
20.3. Methods .............................................. 290
20.3.1. Synthesis of PZ1(3,5Me2-PZ(CH2)2N((CH2)3
COOH)(CH2)2NH-BOC) ........................... 290
20.3.2. Peptide synthesis ............................ 291
20.3.3. Radiolabelling of PZ1-RGD conjugate
cyclo[Arg-Gly-Asp-D-Tyr-Lys(PZl)] ............ 292
20.3.4. In vitro serum stability ..................... 292
20.3.5. In vitro internalization analysis ............ 292
20.3.6. In vivo evaluation of [99mTc(CO)3-cyclo
[Arg-Gly-Asp-D-Tyr-Lys(PZl)]] in normal
mouse models ................................. 293
20.4. Results .............................................. 294
20.5. Conclusion ........................................... 300
References ........................................... 301
PAPERS PUBLISHED BY THE PARTICIPANTS RELATED TO
THE COORDINATED RESEARCH PROJECT .............................. 305
PARTICIPANTS IN THE COORDINATED RESEARCH PROJECT .............. 311
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