Tricarbonyl Complexes for the Design of Specific ... · UNIVERSIDADE DE LISBOA FACULDADE DE...

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA

Tricarbonyl Complexes for the Design

of Specific Radiopharmaceuticals for

Endogenous Gene Expression and

Membrane Receptor Imaging

Catarina Alexandra Henriques Xavier Doutoramento em Química Especialidade de Química Inorgânica Tese orientada pela Doutora Isabel Rego dos Santos

2009

i

The work described in this thesis was performed in Grupo de Ciências

Radiofarmacêuticas, Unidade de Ciências Químicas e Radiofarmacêuticas,

Instituto Tecnológico e Nuclear, Sacavém, Portugal, under supervision of Dr.

Isabel Rego dos Santos.

The research project was a collaboration between the Grupo de

Ciências Radiofarmacêuticas and the Medicinal Inorganic Chemistry Group,

Institute of Inorganic Chemistry, University of Zurich, Switzerland. The work

performed in Zurich had the supervision of Prof. Dr. Roger Alberto. The

synthesis of Peptide Nucleic Acids and melting temperature studies were

performed in the group of Prof. Dr. Stefano Maiorana, in the Dipartimento

di Chimica Organica e Industriale, Università degli Studi di Milano, Italy,

under supervision of Dr. Clelia Giannini.

The work was financially supported by Fundação para a Ciência e a

Tecnologia through the PhD grant SFRH/BD/16680/2004.

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Acknowledgements

À Doutora Isabel Rego dos Santos agradeço a forma empenhada e interessada com

que me orientou e todo o apoio científico, essenciais à realização do trabalho. Agradeço

ainda o apoio humano e amizade demonstrada.

I would like to thank Prof. Dr. Roger Alberto for the supervision and the opportunity

to work in his group that was very important for me and for my scientific career.

I would like to thank Prof. Dr. Stefano Maiorana for the opportunity to do part of my

work in his lab. I am grateful to Dr. Clelia Giannini for the supervision and all the support

during my time in Milan.

À Prof. Doutora Maria Helena Garcia agradeço ter aceite ser responsável por esta

tese na Faculdade de Ciências da Universidade de Lisboa.

À Doutora Lurdes Gano e à Doutora Paula Raposinho agradeço o empenho na

realização dos estudos de biodistribuição e ensaios biológicos in vitro, assim como todo o

apoio prestado.

À Doutora Fernanda Marques agradeço os estudos de microscopia de fluorescência

assim como alguns dos ensaios biológicos in vitro.

À Doutora Célia Fernandes agradeço a sua disponibilidade para resolver todos os

problemas relacionados com o HPLC e não só. Agradeço também a sua amizade, boa

disposição, a sua capacidade de transmitir conhecimentos e de nunca se recusar a nada e a

ninguém.

À Elisabete Correia agradeço a dedicação com que executou os ensaios de

biodistribuição e ao Amadeu Rodrigues o apoio técnico.

Ao Doutor Joaquim Marçalo agradeço a disponibilidade na realização de alguns dos

espectros de massa apresentados neste trabalho.

Ao Sr. António Soares agradeço a realização das análises elementares de C, H, N e S

assim como a sua boa disposição e simpatia.

Ao Doutor José Rino (Instituto de Medicina Molecular) agradeço a realização dos

estudos de microscopia de fluorescência.

Aos meus colegas do Grupo de Ciências Radiofarmacêuticas agradeço a colaboração,

o incentivo e a forma carinhosa como sempre me trataram. Agradeço em especial às

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Doutoras Raquel, Susana e Rute pela amizade, paciência e todo o tipo de ajuda. À Teresa

obrigada pelo apoio no trabalho na parte da “Orange”.

Aos restantes elementos da Unidade de Ciências Químicas e Radiofarmacêuticas

agradeço a simpatia com que me receberam e, em particular, agradeço a todos aqueles que,

directa ou indirectamente, colaboraram na realização deste trabalho.

To Dr. Sergio Dall’Angelo I would like to thank the help in the work with the peptide

synthesizer as well as all the help in the lab; thanks for all the good times, good mood and

friendship. To other people in the lab, Dr. Paulo, Dr. Giusepe, Lubna, Dr. Marco, Dr. Alberto

and others that I do not mention, thanks for the good times, friendship and help.

To Dr. Nikos, Dr. Lukas, Dr. Yu, Dr. Paul Schmutz, Dr. Susana, Dr. Fabio, Tooyama and

Dr. Selvi thanks for all the help in the lab, friendship and all the good times that we spent

together.

To all my Vinzenz friends, especially Utkum, Estelle, Marta, Nerijus, Vaibhav,

Tooyama, Yu, Rohit, Ahmad, Tahmineh, Virgil, Irina and Nico, thanks for the good times in

the Vinzenz House, for the parties, trips and especially for the company and friendship.

Às minhas amigas Orquídea, Raquel, Margarida e Maria João, um especial obrigada

pela amizade, pelas conversas, pelos bons momentos passados e apoio.

Ao Nuno agradeço o design da capa da tese.

Agradeço ao Instituto Tecnológico e Nuclear por me ter acolhido e à Fundação para a

Ciência e a Tecnologia pela bolsa de doutoramento (SFRH/BD/16680/2004).

À minha família.

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Resumo

A imagiologia molecular é a visualização in vivo de biomoléculas ou processos

biológicos associados a certas patologias, por métodos não invasivos e quantitativos, e

mediante interacção da molécula alvo com uma sonda molecular. A imagiologia molecular

pode ser utilizada para detecção, caracterização e monitorização em tempo real de certas

patologias, assim como para seguir o efeito das terapias.

As técnicas nucleares de imagem, tomografia por emissão de fotão único (SPECT) e

tomografia por emissão de positrões (PET), são das mais relevantes em imagiologia

molecular devido ao seu carácter não invasivo e à sua elevada sensibilidade, características

que se devem essencialmente ao facto de utilizarem sondas radioactivas (compostos que

têm na sua composição um elemento radioactivo) com elevada actividade específica e

especificidade.

A descodificação do genoma humano e consequente desenvolvimento na área da

proteómica tem permitido identificar alvos moleculares (receptores, enzimas, genes, etc.)

associados a diferentes patologias, com um impacto significativo nas áreas da oncologia e

neurologia. A concepção de sondas radioactivas para visualização desses alvos moleculares é

uma área de investigação multidisciplinar que necessita da contribuição de disciplinas várias,

tais como a medicina, a bioquímica, a biologia molecular, a química e a radioquímica, entre

outras.

O principal objectivo do trabalho apresentado nesta tese foi contribuir para o

aumento do conhecimento ao nível da concepção de sondas radioactivas para imagiologia

molecular por SPECT. Pretendia‐se conceber compostos contendo a unidade fac‐

[99mTc(CO)3]+ estabilizada por ligandos bifuncionais potencialmente tridentados úteis para

visualizar a expressão génica endógena (mRNA N‐MYC) e receptores de membrana (MC1R).

Utilizando a mesma unidade metálica e o mesmo tipo de ligandos, pretendia‐se também

isolar e avaliar compostos de Re e 99mTc contendo na sua composição um derivado do

alaranjado de acridina. Este grupo é fluorescente e intercala no DNA, pelo que estes

resultados abririam caminho ao estudo de sondas multimodais e/ou ao estudo do interesse

teurapêutico do 99mTc como emissor de electrões Auger. Os ligandos bifuncionais

vi

seleccionados são derivados de pirazolo‐diaminas e da cisteína, sendo a sua fórmula geral

apresentada no esquema seguinte:

Ligandos bifuncionais contendo unidades pirazolo e cisteína para coordenação ao metal e

conjugação a biomoléculas (BM) e moléculas fluorescentes (MF).

Para visualizar o mRNA N‐MYC (sobre‐expresso em certos tumores do sistema

nervoso central e periférico) foi escolhida uma sequência de ácidos peptídico‐nucleicos

(PNA), uma vez que estes ácidos mimetizam o ácido desoxirribonucleico (DNA) e apresentam

propriedades notáveis de estabilidade e hibridação com o RNA complementar.

O trabalho iniciou‐se com a síntese e caracterização química e biológica de

complexos modelo de Re e 99mTc estabilizados pelos ligandos indicados no esquema acima e

contendo unidades de PNA (monómero e dímero). Estes estudos preliminares mostraram

que os complexos de 99mTc se formavam com elevado rendimento e pureza radioquímica,

apresentavam uma elevada estabilidade in vitro e in vivo e um bom perfil biológico (rápida

depuração sanguínea e eliminação renal relativamente rápida). Assim, concluímos que

qualquer dos ligandos acima referidos eram adequados para conjugação a uma sequência de

PNA clinicamente relevante e posterior ligação à unidade fac‐[99mTc(CO)3]+.

A sequência de PNA escolhida foi N‐A GAT CAT GCC CGG CAT‐C, pois é complementar

de uma região do mRNA N‐MYC que pretendíamos visualizar. A preparação de HA GAT CAT

GCC CGG CAT‐LysNH2 foi realizada utilizando técnicas de síntese em fase sólida e a sua

conjugação aos dois ligandos bifuncionais apresentados no esquema acima foi realizada com

sucesso. A conjugação à cisteína, embora possível, processou‐se com um rendimento muito

baixo. Embora este processo de síntese pudesse ser optimizado, limitações de tempo

levaram a não prosseguir os estudos com a cisteína. O ligando contendo a unidade pirazolo

SNH2

O

HO

O

OH

NN

NNH2

O

OH

MF

M

BM

BM

M

vii

foi conjugado à sequência HA GAT CAT GCC CGG CAT‐LysNH2, originando o composto Pz‐A

GAT CAT GCC CGG CAT‐Lys‐NH2 com um rendimento dentro do que seria expectável. O

complexo fac‐[Re(CO)3(3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ foi também conjugado à

sequência HA GAT CAT GCC CGG CAT‐LysNH2, originando fac‐[Re(CO)3(κ3‐Pz‐A GAT CAT GCC

CGG CAT‐Lys‐NH2)]+ (Pz = 3,5‐Me2pz(CH2)2N((CH2)3CO)(CH2)2NH2). Este complexo e a

sequência HA GAT CAT GCC CGG CAT‐LysNH2 foram utilizados em estudos de temperatura

de fusão (Tm) e os resultados obtidos mostraram que a metalação da sequência não afectava

o reconhecimento da sequência complementar, nem a estabilidade do duplex formado com

o DNA. O elevado valor de Tm, 83,5 ± 0,1 °C, deixa também prever uma elevada estabilidade

in vivo do duplex formado pelo complexo análogo de 99mTc e o mRNA alvo. O complexo

análogo de 99mTc, fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+, foi preparado

quantitativamente fazendo reagir o conjugado Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 com fac‐

[99mTc(CO)3(OH2)3]+. Estudos preliminares com células SH‐SY5Y do neuroblastoma

(expressando o N‐MYC), MCF7 do cancro da mama e PC3 do cancro da próstata mostraram

uma elevada internalização do complexo fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+, assim como uma retenção elevada pelas células SH‐SY5Y. Para que possamos

afirmar que esta retenção resulta da interacção do complexo radioactivo com o mRNA N‐

MYC, são necessários estudos com células IMR32 que sobre‐expressam o mRNA N‐MYC,

assim como experiências controlo utilizando compostos com uma sequência de PNA análoga

à que se quer visualizar e/ou com bases diferentes da complementar. O perfil biológico de

fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ em ratinhos CD1 mostrou existir

uma elevada retenção do complexo no rim (84,64 ± 6,07 % ID/g 4 h p.i.) e no fígado (24,06 ±

4,99 % ID/g 4 h p.i.). A co‐injeccão do complexo de 99mTc e L‐lisina mostrou que era possível

reduzir em 63% a retenção renal. No entanto, os valores finais obtidos eram ainda elevados.

O ligando pirazolo foi também ligado com sucesso a um derivado do alaranjado de

acridina (unidade fluorescente e com capacidade de intercalar com o DNA). O conjugado

resultante reagiu com fac‐[M(CO)3(OH2)3]+ (M = Re, 99mTc), conduzindo à formação de

complexos tricarbonilo de Re e 99mTc contendo a unidade alaranjado de acridina. O

radioconjugado apresentou níveis de internalização celular moderados em células B16F1 do

melanoma murino (7,79% após 5 h incubação), sendo que uma percentagem significativa de

composto se deslocava rapidamente para o núcleo. Através da microscopia de fluorescência

e usando o complexo de Re, foi possível visualizar a sua localização no citoplasma e no

viii

núcleo das células. Utilizando o complexo de 99mTc foi possível determinar quanto composto

permanecia no citoplasma das células e quanto migrava para o núcleo das mesmas. Tendo

em conta que estamos na presença de complexos isoestruturais, a combinação desta

informação indica que o que estamos a ver e a quantificar são os complexos de Re e 99mTc na

sua forma intacta.

Tendo em conta que o radioconjugado tem na sua composição um emissor de

electrões Auger (99mTc) e uma unidade com capacidade para interactuar com o DNA

(alaranjado de acridina), e que se consegue chegar ao núcleo das células, foi considerado

interessante estudar a radiotoxicidade do complexo de 99mTc para avaliar o potencial

interesse deste tipo de compostos na concepção de agentes radioterapêuticos. Os estudos

foram realizados com células B16F1 mas não foi observado qualquer efeito radiotóxico,

atribuível aos electrões Auger. O perfil biológico do radioconjugado também foi avaliado,

apresentando uma elevada retenção no fígado (36.12 ± 12.76 % ID/g 4 h p.i.) e intestino

(17.01 ± 5.50 % ID/g 4 h p.i.) e uma excreção total reduzida. Um melhor perfil biológico

poderá ser alcançado desde que se aumente o carácter hidrofílico do complexo, o que

poderá acontecer quando se conjugar ao complexo um péptido biologicamente activo.

Para visualizar os receptores MC1, sobre‐expressos nas células do melanoma, um

análogo cíclico da α‐MSH (hormona estimulante do crescimento dos melanócitos) foi

preparado e conjugado ao ligando pirazolo‐diamina. O composto obtido, Pz‐βAla‐Nle‐

cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2 reagiu com fac‐[99mTc(CO)3(OH2)3]+, conduzindo à

formação de fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ com

elevado rendimento e pureza radioquímica. O radioconjugado apresentou uma elevada

estabilidade in vitro e in vivo. A ciclização levou à obtenção de uma estrutura mais compacta

que aumentou a internalização celular (41,8% após 4 h de incubação) e retenção (75% após

4 h de incubação) relativamente ao análogo linear previamente estudado (1,6% e 35,3%,

internalização e retenção celular, respectivamente). O complexo fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐

Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ foi injectado em ratinhos com melanoma tendo‐se

verificado uma fixação elevada no tumor (11,31% ID/g 4 h p.i.), relativamente ao complexo

análogo linear (0,99% ID/g 4 h p.i.). Apesar dos bons resultados de retenção tumoral, o perfil

biológico deste complexo necessita ser melhorado no que se refere à retenção no rim e à

excreção total. Tal melhoria poderá ser alcançada usando agentes bifuncionais mais

ix

hidrofílicos ou introduzindo outros aminoácidos na sequência peptídica, desde que seja

preservada a parte biologicamente activa.

xi

Abstract The work described in this thesis is focused on the design of 99mTc specific probes for

imaging endogenous gene expression (N‐MYC mRNA) or membrane receptors (MC1R). The

fac‐[99mTc(CO)3]+ was the selected core and its conjugation to the biomolecules was

performed using pyrazolyl‐ and cysteine‐containing bifunctional chelators.

We have chosen a PNA sequence for imaging the N‐MYC mRNA, overexpressed in

certain tumors. Once there was no experience on the conjugation of the chelators to PNA

sequences, model tricarbonyl complexes anchored on cysteine‐ and pyrazolyl‐containing

chelators bearing PNA units (monomer and dimer) were synthesized and characterized. The

preparation of the 99mTc model complexes in high yield and the favourable tissue distribution

profile confirmed the possibility of attaching these chelators to the PNA sequence, H‐A GAT

CAT GCC CGG CAT‐LysNH2, complementary to the N‐MYC mRNA. Conjugation of the

cysteine‐containing chelator to the PNA sequence was possible but the resulting conjugate

was obtained in very low yield. Optimization of the reaction conditions could certainly be

possible, but it was not tried due to time limitations. The pyrazolyl‐containing chelator (Pz‐

Boc) and fac‐[Re(CO)3(3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ (RePz) were conjugated to

the PNA sequence yielding Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 and the complex fac‐

[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]+. The radioactive analogous fac‐

[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ was prepared, characterized and

biologically evaluated.

The pyrazolyl‐containing chelator was also conjugated to the fluorescent moiety

acridine orange, and isostructural Re and 99mTc tricarbonyl complexes were prepared and

evaluated in vitro and in vivo. These preliminary studies were important to evaluate the

possibility of designing multimodal probes and/or to explore the Auger emitter 99mTc for

therapy.

For imaging the MC1 receptors overexpressed in melanoma cells, a cyclic α‐MSH

analogue was conjugated to the pyrazolyl‐containing chelator yielding Pz‐βAla‐Nle‐

cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2. The fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐

Arg‐Trp‐Lys]‐NH2)]2+ was prepared and studied with B16F1 murine melanoma cells and with

melanoma bearing mice.

xiii

Palavras‐chave

Complexos específicos

Ácidos peptídico‐nucleicos

Alaranjado de acridina

Visualização da expressão génica endógena

Visualização de receptores de membrana

Keywords

Specific complexes

Peptide nucleic acids

Acridine orange

Endogenous gene expression imaging

Membrane receptors imaging

xv

Contents Acknowledgements ............................................................................................................... iii

Resumo ..................................................................................................................................... v

Abstract.................................................................................................................................... xi

Palavras‐chave .......................................................................................................................xiii

Keywords ................................................................................................................................xiii

Contents...................................................................................................................................xv

Figures .....................................................................................................................................xxi

Tables .................................................................................................................................... xxvi

Schemes .............................................................................................................................. xxviii

Abbreviations........................................................................................................................xxx

Scope and Aim ......................................................................................................................... 1

1. Introduction ..................................................................................................................... 5

1.1. Radiopharmaceuticals ............................................................................................... 5

1.1.1. Diagnosis vs Therapy .............................................................................................. 5

1.1.2. Perfusion and Specific Radiopharmaceuticals ..................................................... 10

1.2. Technetium and Rhenium Coordination Chemistry Relevant for Nuclear Medicine

................................................................................................................................ 16

1.2.1. The Technetium‐ and Rhenium‐Tricarbonyl Core................................................ 19

1.3. Specific Probes for Endogenous Gene Expression and Membrane Receptors

Imaging................................................................................................................... 22

1.3.1. Antisense Imaging of Endogenous Gene Expression ........................................... 22

1.3.1.1. Peptide Nucleic Acids and Diagnosis............................................................. 27

1.3.1.1.1. PNA Labelling with Different Radionuclides for Imaging Endogenous Gene

Expression ..................................................................................................... 28

1.3.2. Imaging Membrane Receptors with Small Radiopeptides................................... 34

1.3.2.1. α‐Melanocyte Stimulating Hormone‐Based Radiopharmaceuticals ............ 35

xvi

1.3.2.1.1. α‐MSH Analogues.................................................................................... 36

2. Evaluation of Chelators for Labelling Biologically Relevant Molecules ............... 41

2.1. Introduction ............................................................................................................. 41

2.2. Evaluation of Two Chelators for Labelling PNA Units ............................................ 42

2.2.1. Synthesis and Characterization of a PNA Monomer and Dimer .......................... 42

2.2.2. Synthesis and Characterization of Bifunctional Chelators Bearing a PNA

Monomer............................................................................................................. 47

2.2.3. Synthesis and Characterization of a Bifunctional Chelator Bearing a PNA

Dimer................................................................................................................... 52

2.2.4. Synthesis and Characterization of Rhenium Tricarbonyl Complexes .................. 54

2.2.5. Synthesis, Characterization and Biological Behavior of 99mTc Tricarbonyl

Complexes ........................................................................................................... 61

2.2.5.1. Stability in the Presence of Cysteine and Histidine....................................... 63

2.2.5.2. Biodistribution and In Vivo Stability.............................................................. 65

2.3. Synthesis of Tricarbonyl Complexes Bearing Acridine Orange .............................. 69

2.3.1. Synthesis and Characterization of a Pyrazolyl‐Diamine Acridine Orange

Conjugate ............................................................................................................ 69

2.3.2. Synthesis and Characterization of Rhenium and Technetium Tricarbonyl

Complexes Bearing the Acridine Orange Moiety................................................ 74

2.3.3. Studies with B16F1 Cells ...................................................................................... 77

2.3.3.1. Cytotoxicity Studies....................................................................................... 78

2.3.3.2. Cellular Localization by Fluorescence Microscopy Studies........................... 79

2.3.3.3. Internalization, Retention and Radiotoxicity Studies ................................... 80

2.3.4. Biodistribution and In Vivo Stability..................................................................... 86

3. Peptide Nucleic Acids ‐ Synthesis, Conjugation to Bifunctional Chelators,

Labelling and Biological Evaluation........................................................................... 93

3.1. Introduction ............................................................................................................. 93

3.2. Solid Phase Synthesis .............................................................................................. 94

3.2.1. Resin ..................................................................................................................... 95

3.3. PNA Synthesis .......................................................................................................... 96

3.4. Automated Solid Phase Synthesis........................................................................... 98

xvii

3.5. Synthesis of the PNA Sequence ............................................................................ 100

3.5.1. Synthesis of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin...................................... 100

3.5.2. Synthesis and Characterization of H‐A GAT CAT GCC CGG CAT‐Lys‐NH2........... 108

3.6. Synthesis and Characterization of Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 and fac‐

[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]+.......................................... 111

3.7. Coupling of the Cysteine‐Containing Chelator to the PNA Sequence ................. 113

3.8. UV‐Melting Temperature ...................................................................................... 114

3.9. Synthesis and Characterization of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐

Lys‐NH2)]2+............................................................................................................ 116

3.10. In Vitro Stability Studies ........................................................................................ 118

3.11. In Vitro Studies in Cells .......................................................................................... 119

3.12. Biodistribution and In Vivo Stability ..................................................................... 123

3.12.1. Biodistribution.................................................................................................... 123

3.12.1.1. Inhibition of Kidney Uptake ........................................................................ 125

3.12.2. In Vivo Stability................................................................................................... 126

4. Melanoma Targeting with an α‐Melanocyte Stimulating Hormone Analogue

Labelled with fac‐[99mTc(CO)3]+ ................................................................................ 133

4.1. Introduction ........................................................................................................... 133

4.2. Synthesis and Characterization of Pz‐βAla‐Nle‐cyclo[Asp‐His‐DPhe‐Arg‐Trp‐Lys]‐

NH2 ....................................................................................................................... 134

4.3. Synthesis of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys] ‐

NH2)]2+ .................................................................................................................. 135


4.5. In Vitro Studies in B16F1 Murine Melanoma Cells – Internalization and Cellular

Retention ............................................................................................................. 138

4.6. Biodistribution and In Vivo Stability of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐Asp‐DPhe‐

Arg‐Trp‐Lys‐NH2)]2+ .............................................................................................. 141

5. Concluding Remarks and Outlook ............................................................................ 149

6. Experimental Part ....................................................................................................... 159

6.1. Materials ................................................................................................................ 159

6.2. Characterization and Purification Techniques ..................................................... 159

xviii

6.3. Synthesis of Compounds 1 – 17 ............................................................................ 166

6.3.1. Tert‐butyl 2‐aminoethylcarbamate (1) .............................................................. 166

6.3.2. Methyl 2‐(2‐(tert‐butoxycarbonylamino)ethylamino)acetate (2) ..................... 166

6.3.3. 2‐(5‐methyl‐2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐yl) acetic acid (3) .............. 167

6.3.4. Methyl‐2‐(N‐(2‐(tert‐butoxycarbonylamino)ethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐

dihydropyrimidin‐1(2H)‐yl)acetamido)acetate (4)............................................ 168

6.3.5. 2‐(N‐(2‐methoxy‐2‐oxoethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐

yl)acetamido)ethanaminium 2,2,2‐trifluoroacetate (5).................................... 169

6.3.6. 2‐(N‐(2‐(tert‐butoxycarbonylamino)ethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐

dihydropyrimidin‐1(2H)‐yl)acetamido)acetic acid (6)....................................... 169

6.3.7. Methyl N‐[2‐[N’‐[2‐(Boc‐amino)ethyl]‐N’‐(thymin‐1‐

ylacethyl)glycyl]amino]ethyl]‐N‐[(4‐thymini‐1‐yl)acetyl]glycinate (7).............. 170

6.3.8. 5,11‐bis(2‐(5‐methyl‐2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐yl)acetyl)‐3,9‐dioxo‐

2‐oxa‐5,8,11‐triazatridecan‐13‐aminium 2,2,2‐trifluoroacetate (8)................. 171

6.3.9. (R)‐methyl 2,2,11,11‐tetramethyl‐4‐oxo‐3,10‐dioxa‐8‐thia‐5‐azadodecane‐6‐

carboxylate (9)................................................................................................... 172

6.3.10. (R)‐2‐(2‐amino‐3‐methoxy‐3‐oxopropylthio)acetic acid (10) ............................ 172

6.3.11. Methyl 3‐(2‐oxo‐2‐(perfluorophenoxy)ethylthio)‐2‐(2,2,2‐

trifluoroacetamido)propanoate (11)................................................................. 173

6.3.12. Protected Cysteine‐PNA Monomer Conjugate (12)........................................... 173

6.3.13. 2‐amino‐3‐(2‐(2‐(N‐(carboxymethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐

dihydropyrimidin‐1(2H)‐yl)acetamido)ethylamino)‐2‐oxoethylthio)propanoic

acid (13)............................................................................................................. 174

6.3.14. Protected Pz‐PNA Monomer Conjugate (14) ..................................................... 175

6.3.15. 2‐(N‐(2‐(4‐((2‐aminoethyl)(2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐

yl)ethyl)amino)butanamido)ethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐dihydropyrimidin‐

1(2H)‐yl)acetamido)acetic acid (15).................................................................. 176

6.3.16. Protected Pz‐PNA Dimer Conjugate (16) ........................................................... 177

6.3.17. Deprotected Pz‐Dimer (17) ................................................................................ 178

6.4. Synthesis of the Rhenium Complexes 18 – 21...................................................... 179

6.4.1. Synthesis of fac‐[Re(CO)3(κ3‐ 13)] (18)............................................................... 179

6.4.2. Synthesis of fac‐[Re(CO)3(κ3‐15)]CF3COO (19)................................................... 180

xix

6.4.3. Synthesis of fac‐[Re(CO)3(κ3 – 17)]CF3COO (20) ................................................ 181

6.5. Synthesis of 99mTc(I) Complexes 21 – 23 ............................................................... 182

6.5.1. General Method for Preparing the 99mTc Complexes ........................................ 183

6.5.1.1. Synthesis of fac‐[99mTc(CO)3(κ3‐13)] (21)..................................................... 183

6.5.1.2. Synthesis of fac‐[99mTc(CO)3(κ3‐15)]+ (22) ................................................... 183

6.5.1.3. Synthesis of fac‐[99mTc(CO)3(κ3‐17)]+ (23) ................................................... 184

6.6. Synthesis of compounds 24 – 33........................................................................... 184

6.6.1. 2‐(4‐bromobutyl) isoindoline‐1,3‐dione (24) ..................................................... 184

6.6.2. 3,6‐bis(dimethylamino)‐10‐(4‐(1,3‐dioxoisoindolin‐2‐yl)butyl)acridinium (25) 185

6.6.3. 10‐(4‐Amino‐butyl)‐3,6‐bis‐dimethylamino‐acridinium (26) ............................. 185

6.6.4. Ethyl 3‐acetyl‐4‐oxopentanoate (27) ................................................................. 186

6.6.5. Tert‐butyl 2‐(2,4‐dinitrophenylsulfonamido)ethylcarbamate (28).................... 186

6.6.6. Ethyl‐2‐(1‐(2‐hydroxyethyl)‐3,5‐dimethyl‐1H‐pyrazol‐4‐yl)acetate (29) ........... 187

6.6.7. Ethyl 2‐(1‐(2‐(N‐(2‐(tert‐butoxycarbonylamino)ethyl)‐2,4‐

dinitrophenylsulfonamido)ethyl)‐3,5‐dimethyl‐1H‐pyrazol‐4‐yl)acetate (30) . 188

6.6.8. 2‐(1‐(2‐(2‐(tert‐butoxycarbonylamino)ethylamino)ethyl)‐3,5‐dimethyl‐1H‐

pyrazol‐4‐yl)acetic acid (31) .............................................................................. 188

6.6.9. 10‐(4‐(2‐(1‐(2‐(2‐(tert‐butoxycarbonylamino)ethylamino)ethyl)‐3,5‐dimethyl‐1H‐

pyrazol‐4‐yl)acetamido)butyl)‐3,6‐bis(dimethylamino)acridinium (32) ........... 189

6.6.10. 10‐(4‐(2‐(1‐(2‐(2‐aminoethylamino)ethyl)‐3,5‐dimethyl‐1H‐pyrazol‐4‐

yl)acetamido)butyl)‐3,6‐bis(dimethylamino)acridinium (33) ........................... 190

6.6.11. Synthesis of fac‐[Re(CO)3(κ3‐42)]2Br (34) .......................................................... 191

6.6.12. Synthesis of fac‐[99mTc(CO)3(κ3‐33)]2+ (35)......................................................... 192

6.7. Synthesis of Peptide Nucleic Acids ....................................................................... 192

6.7.1. Synthesis of N‐α‐Fmoc‐L‐Lys(Boc)‐resin Novasyn TGR‐PEG‐PS (36).................. 192

6.7.1.1. Resin Loading .............................................................................................. 193

6.7.2. Automated Solid Phase Synthesis of Peptide Nucleic Acids on the ABI 433A

Synthesizer ........................................................................................................ 195

6.7.2.1. Automated Synthesis of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin (37).... 195

6.7.2.1.1. General Cleavage Procedure................................................................. 196

6.7.3. Manual Solid Phase Synthesis of Peptide Nucleic Acids .................................... 197

xx

6.7.3.1. Manual Synthesis of Fmoc‐ A GAT CAT GCC CGG CAT‐Lys‐resin (37) ......... 197

6.7.3.2. H‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (38) ................................................... 198

6.7.3.3. Synthesis of Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) and fac‐[Re(CO)3(κ3‐

Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]+ (40)............................................... 199

6.7.3.4. Synthesis of Cyst‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (41)........................... 201

6.7.4. Synthesis of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ (42) . 202

6.8. Synthesis of compounds 43 – 44........................................................................... 202

6.8.1. Synthesis of Pz‐βAla‐Nle‐cyclo[Asp‐His‐DPhe‐Arg‐Trp‐Lys]‐NH2 (43) ................ 202

6.8.2. Synthesis of fac‐[99mTc(CO)3(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐Dphe‐Arg‐Trp‐Lys]‐NH2]2+

(44) .................................................................................................................... 203

6.9. Partition Coefficient .............................................................................................. 204


6.10.1. Stability in the Presence of Cysteine and Histidine............................................ 204

6.10.2. Stability in Fresh Human Serum......................................................................... 205

6.10.3. Stability in PBS with 0.2% BSA............................................................................ 205

6.10.4. Stability in Cell Medium ..................................................................................... 205

6.11. Cell Studies............................................................................................................. 205

6.11.1. Cell Cultures ....................................................................................................... 206

6.11.2. Cellular Internalization and Retention Studies .................................................. 207

6.11.3. Nuclear Internalization....................................................................................... 207

6.11.4. Cell Viability........................................................................................................ 208

6.11.5. Cytotoxicity of Compounds 33 and 34 ............................................................... 208

6.11.6. Radiotoxicity of fac‐[99mTc(CO)3(κ3‐33)]2+ (35)................................................... 209

6.11.7. Evaluation of Cell Uptake by Fluorescence Microscopy .................................... 209

6.12. Biodistribution and In Vivo Stability ..................................................................... 210

6.12.1. Biodistribution Studies ....................................................................................... 210

6.12.1.1. Biodistribution Studies in CD‐1 Female Mice.............................................. 210

6.12.1.2. Biodistribution Studies in C57BL/6 female mice......................................... 213

6.12.2. In Vivo Stability/Metabolization......................................................................... 215

References ................................................................................................................. 219

xxi

Figures

Figure 1.1 – Schematic representation of Auger electrons, α and β‐ particles path lengths

in a cellular and subcellular environment (arbitrary scaling). 8

Figure 1.2 – Structures of two specific radiopharmaceuticals in clinical use. 13

Figure 1.3 – Representation of three approaches used in the design of specific metal‐

based radiopharmaceuticals. 14

Figure 1.4 – Rhenium complexes which mimic the structure of dihydrotestosterone,

progesterone and estradiol. 14

Figure 1.5 – Structural model of 99mTc‐CCMSH for melanoma imaging. 15

Figure 1.6 – Most studied technetium and rhenium cores for the development of

radiopharmaceuticals (M = Tc, Re). 18

Figure 1.7 – IsoLink® kit and synthesis of fac‐[99mTc(CO)3(OH2)3]+. 19

Figure 1.8 – Examples of bidentate and tridentate bifunctional chelating agents for the

tricarbonyl core. 21

Figure 1.9 – Schematic illustration of the reporter gene approach. 23

Figure 1.10 – Schematic image showing the radiolabelled antisense oligonucleotide

upon hybridization to the complementary target mRNA. 24

Figure 1.11 – Illustration of antisense hybridization imaging approach. 24

Figure 1.12 – General structure of some DNA and RNA analogues and artificial analogues

used in antisense imaging. 27

Figure 1.13 – DNA and aminoethylglycine‐PNA backbones. 28

Figure 1.14 – Proposed structure for 99mTc‐N‐GlyD(Ala)GlyGly‐Aba‐GCATCGTCGCGG. 29

Figure 1.15 – Illustration of 99mTc‐AcGlyD(Ala)GlyGlyAba‐PNA‐AEEA‐D(CysSerLysCys)

conjugates designed to bind to IGF1 receptor, internalize and hybridize

with the CCND1 or c‐MYC mRNA. 30

Figure 1.16 – Illustration of M‐N2S2‐K‐RAS PNA‐IGF1 peptide probes (M = 99mTc or 64Cu). 31

Figure 1.17 – Illustration of 64Cu‐DOTA‐KRAS PNA‐IGF1 peptide radiohybridization probe

designed to bind to the IGF1 receptor, internalize and hybridize with

KRAS mRNA. 32

Figure 1.18 – Illustration of 64Cu‐DOTA‐PNA–(Lys)4 probes for UNR mRNA imaging. 32

xxii

Figure 1.19 – Illustration of 111In‐DOTA‐anti‐BCL‐2 PNA‐Tyr3‐octreotate for targeting

BCL‐2 mRNA. 33

Figure 1.20 – Structures of DOTA‐ReCCMSH(Arg11) for labelling with 111In and 64Cu, and

CBTE2A‐ReCCMSH(Arg11) for labelling with 64Cu. 38

Figure 2.1 – 1H NMR spectrum of PNA monomer 4 in CDCl3. 46

Figure 2.2 – 1H NMR spectrum of PNA dimer 7 in CD3OD. 46

Figure 2.3 – Mass spectrum of compound 7 in the negative mode obtained by

ESI/QITMS. 47

Figure 2.4 – 1H NMR spectrum of compound 12 in dmso‐d6. 49

Figure 2.5 – 13C NMR spectrum of compound 12 in dmso‐d6. 50

Figure 2.6 – 1H NMR spectrum of compound 15 in CD3OD. 51


Figure 2.8 – Mass spectrum of compound 17 in the positive mode obtained by

ESI/QITMS. 53

Figure 2.9 – Mass spectrum of compound 18 in the negative mode obtained by

ESI/QITMS. 55

Figure 2.10 – IR spectra of compounds 18, 19 and 20 (KBr). 56

Figure 2.11 – 1H‐1H g‐COSY spectrum of complex 19 in CD3OD and respective

attributions. 57

Figure 2.12 – Expansion of the 1H‐1H g‐COSY spectrum of complex 19 in the range 5 – 1.2

ppm. 58


Figure 2.14 – Analytical RP‐HPLC chromatograms of Re complexes 18 (a), 19 (c) and 20

(e) (254 nm), and corresponding 99mTc complexes 21 (b), 22 (d) and 23 (f)

(γ trace). 62

Figure 2.15 – Stability of 21 and 23 in the presence of excess of histidine and cysteine

(37 °C , PBS pH 7.4). 64

Figure 2.16 – Biological data for complexes 21 and 23 66

Figure 2.17 – Analytical RP‐HPLC chromatograms of complex 23, urine and blood serum

samples collected 1 h after injection (γ trace). 67

Figure 2.18 – 1H NMR spectrum of compound 31 in D2O. 72

xxiii

Figure 2.19 – Mass spectrum of compound 33 in the positive mode obtained by

ESI/QITMS. 73

Figure 2.20 – 1H NMR spectrum of compound 33 in D2O. 74

Figure 2.21 – 1H NMR spectrum of complex 43 in CD3OD. 76

Figure 2.22 – Analytical RP‐HPLC chromatograms of the Re complex 34 (254 nm) and the

purified 99mTc complex 35 (γ trace). 77

Figure 2.23 – Confocal fluorescence microscopy images of B16F1 murine melanoma cells

after 3 h of exposure to 60 μM of compound 33 and complex 34 (green

colour) followed by fixation and DNA staining with DAPI (blue colour). 80

Figure 2.24 – Internalization at 37 °C of the radioconjugate 35 in B16F1 cells at different

time‐points. 82

Figure 2.25 – Internalization at 37 °C expressed as a percentage of total activity for the

non‐purified and purified radioconjugate 35 (mean ± standard deviation,

n = 3). 82

Figure 2.26 – Nuclear internalization at 37 °C in B16F1 murine melanoma cells of

purified and non‐purified radiocomplex 35 (mean ± standard deviation, n

= 3). 83

Figure 2.27 – Activity internalized in the nucleus (white and black) and activity outside

the nucleus (black) in B16F1 murine melanoma cells, after incubation

with purified or non‐purified radiocomplex 35 (mean ± standard

deviation, n = 3). 84

Figure 2.28 – Cellular retention of the internalized radioconjugate 44 (purified and non‐

purified) in B16F1 cells over time at 37 °C (mean ± standard deviation, n =

3). 85

Figure 2.29 – Cytotoxicity studies of purified radioconjugate 35 (0.9 – 60 μCi), TcO4‐ =

[99mTcO4]‐ (40 μCi), Carb. = [99mTc(CO)3(OH2)3]

+ (40 μCi ) and Pz‐Ao =

compound 33 (2 x 10‐9 M) in B16F1 cells at 37 °C (mean ± standard


Figure 2.30 – Analytical RP‐HPLC chromatograms of compound 35, blood serum and

urine samples collected 1 h after injection (γ trace). 88

xxiv

Figure 3.1 – General structure of a PNA monomer. 96

Figure 3.2 – Peptide synthesizer Applied Biosystems ABI 433A. 99

Figure 3.3 – Calibration curve for DBU/DMF deprotection of Fmoc‐Lys and quantification

by gas chromatography. 101

Figure 3.4 – Automated synthesis (ABI 433A): RP‐HPLC chromatogram of the crude

product Fmoc‐A GAT CAT GCC CGG CAT‐Lys (Absorbance at 260 nm). 109

Figure 3.5 – Manual synthesis: RP‐HPLC chromatogram of the crude product Fmoc‐ A

GAT CAT GCC CGG CAT‐Lys (Absorbance at 260 nm). 109

Figure 3.6 – Analytical RP‐HPLC chromatograms of 39 (left) and 40 (right), after

purification (Absorbance at 260 nm). 112

Figure 3.7 – LC‐ESI/QITMS of the crude product 41. A) HPLC chromatogram at 260 nm.

B) ESI/QITMS of the peak at 17.70 min. 114

Figure 3.8 – Melting profiles of 40:DNA (gray curve) and 38:DNA (black curve). 115

Figure 3.9 – Analytical RP‐HPLC chromatograms of 40 (absorbance at 260 nm) and 42 ( γ

trace). 117

Figure 3.10 – Analytical RP‐HPLC chromatogram of the reaction of 38 with [99mTc(CO)3]+. 117

Figure 3.11 – Analytical RP‐HPLC chromatograms of fac‐[99mTc(CO)3(κ3‐Pz‐ A GAT CAT

GCC CGG CAT‐Lys‐NH2)]+2 (42) in human serum at 37 °C, at different time

points (γ trace). 118

Figure 3.12 – Analytical RP‐HPLC chromatogram of 42 after 4 h incubation at 37 °C in

culture medium (γ trace) 119

Figure 3.13 – ITLC‐SG chromatogram of compound 42 after 4 h incubation at 37 °C in

culture medium (γ trace). 119

Figure 3.14 – Cell‐associated radioactivity and cellular internalization of 42 at different

time points (37 °C) in SH‐SY5Y cells (A), MCF7 cells (B) and PC3 cells (C). 120

Figure 3.15 – Cellular internalization in SH‐SH5Y, MCF7 and PC3 cells at different time

points (37 °C). 121

Figure 3.16 – Cellular retention of internalized radioconjugate 42 in SH‐SY5Y cells over

time at 37 °C (mean ± standard deviation, four replicates) 121

Figure 3.17 – Uptake of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]

2+ (42)

in some more relevant organs (CD‐1 Charles River mice at 1 and 4 h after

intravenous injection). 124

xxv

Figure 3.18 – Biological data for complexes fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG

CAT‐Lys‐NH2)]2+ (42) and fac‐[99mTc(CO)3(κ3‐17)]+ (23) in CD‐1 Charles

River female mice. 124

Figure 3.19 – Reversed‐phase HPLC chromatograms of the injected preparation of

complex 42, blood serum, urine and liver homogenate samples collected

1 h after injection and treated before analysis (γ trace). 127

Figure 4.1 – Structure of Pz‐βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2 (43). 135

Figure 4.2 – Analytical RP‐HPLC chromatogram of purified of Pz‐βAla‐Nle‐cyclo[Asp‐D‐

Phe‐Arg‐Trp‐Lys]‐NH2 (43) 135

Figure 4.3 – RP‐HPLC chromatogram of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐c[Asp‐D‐Phe‐Arg‐

Trp‐Lys]‐NH2)]2+ (44) (γ trace). 136

Figure 4.4 – TLC chromatogram of 44, after 24 h incubation at 37 °C in culture medium

(γ trace). 137

Figure 4.5 – Analytical RP‐HPLC chromatogram of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐

cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ after incubation in human serum at

37 °C at different time points (γ trace). 138

Figure 4.6 – Cell studies of the radioconjugate 44 in B16F1 cells at different time points

and temperatures. 139

Figure 4.7 – Cellular retention of internalized cyclic radioconjugate 44 in B16F1 cells

over time at 37 °C (mean ± standard deviation, n = 3). 140

Figure 4.8‐ Biodistribution results (% ID/organ) and total excretion (% ID) of the fac‐

[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐c[Asp‐DPhe‐Arg‐Trp‐Lys]‐NH2)]

2+ (44) in

B16F1 murine melanoma‐bearing C57BL/6 mice at 1, 4 and 24 h after

intravenous injection. 142

Figure 4.9 – RP‐HPLC chromatograms of complex 44, blood serum and urine samples

collected 1 h after injection (γ trace). 145

xxvi

Tables

Table 1.1 ‐ γ emitters used in SPECT. 6

Table 1.2 – Positron emitters used in PET. 7

Table 1.3 – Range of Auger electrons, β‐and α particles in tissues. 8

Table 1.4 – Some radionuclides in clinical use or potentially interesting for therapy. 9

Table 1.5 – Perfusion radiopharmaceuticals for diagnosis in clinical use. 11

Table 1.6 – Specific radiopharmaceuticals for diagnosis in clinical use. 12

Table 1.7 – Regulatory peptides, their function, target disease, cells expressing

receptors, and receptor subtypes. 35

Table 1.8 – Structure of α‐melanocyte stimulating hormone (α‐MSH) and some α‐MSH

analogues. 37

Table 2.1 – Experimental conditions used to synthesize complexes 21 – 23 and

respective retention times. 63

Table 2.2 – Biodistribution results of the 99mTc compounds 21 and 23 at 1 and 4 h after

intravenous injection (mean ± standard deviation, n = 3). 66

Table 2.3 – Biodistribution of the radioconjugate 35 in CD1 female mice at 1 and 4 h

after intravenous injection (mean ± standard deviation, n = 4). 87

Table 3.1 – Commonly used protecting groups for PNA synthesis. 97

Table 3.2 – Biodistribution data of the fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐

Lys‐NH2)]2+ (42) in CD‐1 Charles River mice, at 1 and 4 h after intravenous

injection (mean ± standard deviation, n = 4). 123

Table 3.3 – Biodistribution results of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+ (42) with coinjected or not L‐Lys (15 mg) in CD‐1 Charles River

mice, at 4 h after intravenous injection (mean ± standard deviation, n =

4). 126

Table 4.1 – Biodistribution of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐

Lys]‐NH2)]2+ (44) in B16F1 murine melanoma‐bearing C57BL/6 mice at 1, 4

xxvii

and 24 h, after intravenous injection (mean ± standard deviation, n = 4 –

5). 141

Table 4.2 – Biodistribution of 44 co‐injected or not with 15 mg of L‐lysine in healthy

C57BL/6 mice, at 1h after intravenous injection (mean ± standard


Table 6.1 – Calculations for the calibration curve by gas chromatography. 194

Table 6.2 – Data for calculating the Fmoc substitution (FS). 194

Table 6.3 – Experimental conditions for the automated synthesis. 195

Table 6.4 – Experimental conditions for the manual synthesis. 197

Table 6.5 – Experimental conditions for the synthesis of compound 39. 199



Table 6.8 – Biodistribution results of the 99mTc compounds 21 and 23 at 1 and 4 h in CD‐

1 Charles River mice, after intravenous injection (mean ± standard


Table 6.9 – Biodistribution results of the 99mTc compound 35 at 1 and 4 h in CD‐1 Charles

River mice, after intravenous injection (mean ± standard deviation, n = 4). 212

Table 6.10 – Biodistribution results of the 99mTc compound 42 at 1 and 4 h in CD‐1

Charles River mice, after intravenous injection (mean ± standard


Table 6.11 – Biodistribution results of the 99mTc compound 42 co‐injected with lysine at

4 h in CD‐1 Charles River mice, after intravenous injection (mean ±

standard deviation, n = 5). 213

Table 6.12 – Biodistribution results of the 99mTc compound 44 in B16F1 murine

melanoma‐bearing C57BL/6 mice at 1, 4 and 24 h after intravenous

injection (mean ± standard deviation, n = 4 ‐ 5) 214

Table 6.13 – Biodistribution of the 99mTc compound 44 co‐injected with 15 mg of L‐lysine

in healthy C57BL/6 mice, at 1 h after intravenous injection (mean ±

standard deviation, n = 4). 215

xxviii

Schemes Scheme 2.1 – Pyrazolyl‐ and cysteine‐containing bifunctional chelators for coordination

to the metal and conjugation to biomolecules (BM) and fluorescent

molecule (FM). 41

Scheme 2.2 – Synthesis of the protected PNA backbone (2). 42

Scheme 2.3 – Synthesis of the PNA monomer (4). 43

Scheme 2.4 – Synthesis of a PNA Dimer (7). 44

Scheme 2.5 – cis and trans PNA rotamers. 45

Scheme 2.6 – Synthesis of compounds 9 – 13. 48

Scheme 2.7 – Synthesis of the conjugate 15. 49

Scheme 2.8 – Synthesis of 17. 52

Scheme 2.9 – Synthesis of the rhenium complexes 18, 19 and 20. 54

Scheme 2.10 – Synthesis of 99mTc complexes 21, 22 and 23. 61

Scheme 2.11 – Synthesis of compound 26. 70



Scheme 2.14 – Synthesis of rhenium (34) and technetium (35) complexes. 75

Scheme 3.1 – Download of the resin. 100

Scheme 3.2 – Piperidine vs. DBU cleavage of the Fmoc group. 101

Scheme 3.3 – Synthesis cycle of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin (37). 103

Scheme 3.4 – Removal of the Fmoc protecting group with piperidine. 104

Scheme 3.5 – Base catalyzed reactions of the amine terminus. 105

Scheme 3.6 – PNA monomer activation with HATU. 106

Scheme 3.7 – Coupling of the activated monomer to the growing PNA chain. 106

Scheme 3.8 – Potential guanidine by‐product when using HATU. 107

Scheme 3.9 – Acetylation of the oligomer with acetic anhydride. 107

Scheme 3.10 – Synthesis of the PNA sequences 38 and conjugates 39 and 40. 111

Scheme 3.11 – Synthesis of conjugate 41. 113

xxix

Scheme 3.12 – Synthesis of the radioconjugate fac‐[99mTc(CO)3(κ3‐Pz‐ A GAT CAT GCC

CGG CAT‐Lys‐NH2)]2+ (42). 116

Scheme 4.1 – Synthesis of the radioconjugate fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐

His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ (44). 136

Scheme 5.1 ‐ Pyrazolyl‐ and cysteine‐containing bifunctional chelators. 149

Scheme 5.2 ‐ Illustration of the main compounds described in this thesis – tricarbonyl

metal complexes and PNA conjugates. 155

xxx

Abbreviations A

A Adenine

aa Amino acid

Aba 4‐Aminobutyric acid

Ac Acetyl

Ac2O Acetic anhydride

AEEA Aminoethoxyethoxyacetic acid

aeg Aminoethylglycine

Ao Acridine orange

Arg Arginine

ASON Antisense oligonucleotide

Asp Aspartate

B

BFCA Bifunctional chelating agent

Bhoc Benzylhydryloxycarbonyl

Boc Tert‐butoxycarbonyl

Bq Becquerel

br Broad

C

C Cytosine

CBTE2A 4,11‐Bis(carboxymethyl)‐1,4,8,11‐tetraazabicyclo[6.6.2]hexadecane

Cbz Benzyloxycarbonyl

Ci Curie (1 Ci = 3.7 x 107 Bq)

Cys Cysteine

D

d Doublet

DBF Dibenzofulvene

DBU 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene

DCA Dichloroacetic acid

xxxi

DCM Dichloromethane

DIPEA N,N‐Diisopropylethylamine

DMF N,N‐Dimethylformamide

DNA Deoxyribonucleic acid

DOTA 1,4,7,10‐tetraazacyclododecane‐N,N’,N’’,N’’’‐tetracetic acid

DTPA Diethylenetriaminepentaacetic acid

E

ESI Electrospray ionization

EtOH Ethanol

F

FDA Food and Drug Administration

FDG Fluorodeoxyglucose

Fmoc 9‐Fluorenylmethoxycarbonyl

FTICR Fourier Transform Ion Cyclotron Resonance

G

G Guanine

GC Gas chromatography

Glu Glutamic acid

Gly Glycine

H

h Hour

HATU 2‐(1H‐7‐Azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium

hexafluorophosphate

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

His Histidine

HPLC High‐performance liquid chromatography

HYNIC 6‐Hydrazinopyridine‐3‐carboxylic acid

I

IR Infrared

ITLC Instant thin‐layer chromatography

ITN Instituto Tecnológico e Nuclear

xxxii

L

LC Liquid chromatography

Lys Lysine

M

m Multiplet

MAG3 Mercaptoacetyl‐triglycine

Met Methionine

min Minute

Mmt Monomethoxytrityl or 4‐methoxyphenyldiphenylmethyl

mRNA Messenger ribonucleic acid

MS Mass spectrometry

MSH Melanocyte‐stimulating hormone

N

Nle Norleucine

NMP N‐methylpyrrolidone

NMR Nuclear Magnetic Resonance

P

PBS Phosphate buffer saline

PE Polyethylene

PEG Polyethylene glycol

PET Positron emission tomography

Phe Phenylalanine

p.i. Post injection

PNA Peptide nucleic acid

ppm Part per million

Pro Proline

PS Polystyrene

Pz 3,5‐Me2pz(CH2)2N((CH2)3CO)(CH2)2NH2

Pz‐Boc 3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc

Q

q Quartet

xxxiii

QIT Quadrupole Ion Trap

quint Quintuplet

R

RePz fac‐[Re(CO)3(3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+

Rf Retention factor

RGD Arginine‐glycine‐aspartic acid

RNA Ribonucleic acid

RNase H Ribonuclease H

RP Reversed phase

rpm Rotation per minute

S

s Singlet

Ser Serine

SPECT Single Photon Emission Computed Tomography

SPPS Solid Phase Peptide Synthesis

T

t Triplet

T Thymine

t1/2 Semi‐disintegration period or Half‐life

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

TFA Trifluoroacetic acid

TFMSA Trifluormethanesulfonic acid

TGR TentaGel Rink Amide

THF Tetrahydrofuran

TIS Triisopropylsilane

Tm Melting temperature

tR Retention time

Trp Triptophan

Tyr Tyrosine

V

Val Valine

xxxiv

κ Denticity

δ Chemical shift

ν Frequency

1

Scope and Aim

Recent advances in non‐invasive imaging modalities have opened endless

opportunities for molecular diagnostic and therapeutic procedures.

Molecular imaging is the visualization of in vivo biological or biochemical processes

associated with certain pathologies, at the cellular or molecular level. The target molecule is

visualized in vivo by virtue of its interaction with a molecular imaging probe. Molecular

imaging may be used for early detection, characterization and real‐time monitoring of

diseases as well as for the follow‐up of therapies.

In recent years, the field of molecular imaging has broadened and includes different

imaging modalities. The most explored are computed tomography (CT), magnetic resonance

imaging (MRI), ultrasound (US), nuclear imaging (single photon emission computed

tomography (SPECT) and positron emission tomography (PET)), fluorescence and

bioluminescence imaging (BLI). From all these only fluorescence and bioluminescence

imaging are not in clinical use. From the clinical point of view CT, MRI and US provide

anatomical and morphological information and are in widespread clinical use. On the other

hand, PET and SPECT are the ones in clinical use that can provide physiological, functional

and molecular information.1

Nuclear molecular imaging with PET and SPECT represent the prototype for non‐

invasive quantitative tracing of biochemical processes in vivo, because radioactive probes

can be synthesized at sufficiently high specific activity, enabling the use of tracer

concentrations to detect small‐capacity molecular systems in vivo without interfering with

the processes being studied. These two techniques have some advantages such as

sensitivity, the availability and specificity of radioactive probes, good temporal resolution

(seconds to minutes) and fast examination time (minutes to hours).2

The decoding of the human genome and subsequent developments in proteomics

enabled the identification of a new spectrum of targets (receptors, enzymes, genes, etc.)

associated with certain pathologies, with a significant impact in areas such as oncology and

neurology. However, the development of specific probes for imaging such targets is a great

challenge and a demanding task. To achieve such task the contribution of disciplines such as

2

medicine, biochemistry, chemistry, radiochemistry, molecular biology, among others, is

highly desired.

The main goal of the work described in this thesis was to contribute for the design of

radioactive probes for molecular imaging. Our work was based on the radionuclide 99mTc,

which still motivates an intense research, due to its ideal properties for SPECT imaging,

interesting chemistry, availability and cost. We have explored the chemistry and

radiochemistry of the recently introduced fac‐[M(CO)3]+ (M = Re, 99mTc) core using

bifunctional chelating agents bearing vectors with interest for imaging endogenous gene

expression as well as membrane receptors. Isostructural Re/99mTc tricarbonyl compounds

bearing an acridine‐orange moiety were also studied aiming to explore in a latter stage

multimodal probes and/or the utility of 99mTc as a therapeutic agent.

This thesis is organized in six chapters. In the first chapter a brief and general

introduction is presented, giving particular attention to the work published about

endogenous gene expression imaging and membrane receptors imaging, namely the MC1R.

The second chapter is divided in two parts. In the first part are described the synthesis and

characterization of the model tricarbonyl complexes anchored on pyrazolyl‐ and cysteine‐

containing chelators bearing PNA units (monomer and dimer) and the biological evaluation

of the 99mTc complexes. In the second part of the chapter, the conjugation of the pyrazolyl‐

containing chelator to an acridine orange moiety is described as well as the synthesis and

characterization of the isostructural Re and 99mTc tricarbonyl complexes. In vitro and in vivo

studies will be also presented and discussed. In the third chapter are described the synthesis

and characterization of a PNA sequence with clinical relevance, its conjugation to the

bifunctional chelators evaluated in the second chapter, the preparation of Re and 99mTc

complexes and the biological evaluation of the 99mTc complex. In the fourth chapter are

presented the conjugation of the pyrazolyl‐containing chelator to a cyclic α‐MSH analogue,

the labelling of the resulting conjugate with the fac‐[99mTc(CO)3]+, and the in vitro and in vivo

studies with the 99mTc complex. The conclusions as well as some suggestions for future work

are presented in the fifth chapter. Finally, in the sixth chapter, experimental details about

the work presented in chapters 2 ‐ 4 are described.

1. Introduction

1. Introduction

5

1. Introduction

1.1. Radiopharmaceuticals

Radiopharmaceuticals are drugs containing a radionuclide in its composition, used

routinely in nuclear medicine for the diagnosis or therapy of various diseases. These drugs

are used in tracer quantities and therefore have no pharmacological effects. A

radiopharmaceutical can be a small organic molecule or an inorganic or organometallic

complex which can contain or not in its composition a biologically active molecule. These

biologically active molecules can be macromolecules, such as monoclonal antibodies or

antibody fragments, small peptides, inhibitors or substrates of enzymes, among others.

A radiopharmaceutical approved for clinical use should be sterile, apyrogenic and of

safe administration. Almost all radiopharmaceuticals are administered via intravenous

injection but they can also be administered orally (gastric emptying) or by inhalation

(pulmonary ventilation).3

The radionuclide, essential in the composition of a radiopharmaceutical, is an unstable

nuclide which undergoes a radioactive decay emitting gamma rays (γ) and/or subatomic

particles (α, β‐, β+, Auger electrons).

1.1.1. Diagnosis vs Therapy

Depending on the medical application, diagnosis or therapy, different physical

properties are required for the radionuclide. The important physical properties of the

radionuclide are the type and energy of the emitted radiation and/or particles and half‐life

(t1/2).

Diagnostic radiopharmaceuticals have in their composition a gamma‐emitting (γ)

radionuclide for SPECT, or a positron‐emitting (β+) radionuclide for PET. For diagnosis the

radionuclides should not emit α or β‐ particles and should have a relatively short half‐life.

The radiation emitted by the radionuclide or resulting from annihilation crosses the body

tissues and is detected by instrumentation that is external to the patient. The half‐life must

be short enough to minimize the radiation dose but sufficiently long for preparation,

1. Introduction

6

administration, distribution and accumulation of the radiopharmaceutical in the target organ

or tissue, as well as for image acquisition.

For SPECT, gamma‐ray energies between 80 and 300 keV are the most indicated, the

optimum being between 100 and 200 keV for the instrumentation currently in use in nuclear

medicine departments.4 Examples of the most used gamma emitters in SPECT are shown in

table 1.1.

Table 1.1 ‐ γ emitters used in SPECT.5,6

Radionuclide Half‐life γ‐Energy (keV) Abundance (%) Decay mode* Production method

67Ga 3.26 d 93

184

300

393

40

20

17

5

EC Cyclotron: 68Zn(p,2n)67Ga

99mTc 6.02 h 140 90 IT 99Mo/99mTc Generator

111In 2.83 d 171

245

90

94

EC Cyclotron: 111Cd(p,n)111In

123I 13.2 h 159 83 EC Cyclotron: 124Te(p,2n)123I 121Sb(α, 2n)123I

201Tl 3.04 d 135

167

3

9.4

EC Cyclotron: 203Tl(p,3n)201Pb

* EC ‐ electron capture, IT ‐ isomeric transition

Due to its physical properties and availability (generator), 99mTc is the most used

radionuclide in SPECT. Today, over 90% of all diagnostic nuclear medicine imaging studies

carried out worldwide use this isotope.

PET is based on the physical principle of annihilation of a positron/electron (β+/β‐)

pair, from which two 511 keV photons result. These arise from the mass‐energy conversion

of the two massive particles and are emitted with an angle of 180° at opposite sides from

the annihilation center.5 The double coincidence in energy and emission of the annihilation

photons is advantageous for imaging purposes because it allows a simple and comparatively

precise localization of the annihilation site. Most of the radionuclides used in PET are non‐

99mTc β‐ 66 h

99Mo

201Pb 201Tl EC

9.3 h

1. Introduction

7

metallic isotopes (table 1.2), such as 11C, 15O or 18F. These radionuclides can replace the

natural elements in biologically active molecules, without disturbing significantly their

biological activity. However, some positron‐emitting radiometals, such as 64Cu and 68Ga, are

of great interest for developing new PET radiopharmaceuticals, due to their physical and

chemical properties, and availability.

Most of the PET radionuclides have the disadvantage of requiring costly technology,

and automated and sophisticated methods of synthesis, since they are produced in

cyclotrons and have short half‐lives. 18F‐FDG is the most widely used positron‐emitting

radiopharmaceutical for PET imaging, making 18F the most used radionuclide in this nuclear

imaging technique. 18F‐FDG has shown clinical usefulness in cardiology and neurology but is

used mainly in oncology, in the diagnosis, staging and post‐therapy evaluation of oncologic

patients.7

Table 1.2 ‐ Positron emitters used in PET.5,8,9

Radionuclide Half‐life Decay mode Abundance (%) Eβ+(max) (MeV) Production method

11C 20.4 min β+ 99.8 0.96 Cyclotron: 14N(p,α)11C; 10B(d,n)11C;

13N 10.0 min β+ 100 1.19 Cyclotron: 12C(d,n)13N; 16O(p,α)13N; 13C(p,n)13N

15O 2.0 min β+ 99.9 1.72 Cyclotron: 14N(d,n)15O; 15N(p,n)15O

18F 1.83 h β+

EC

97

3

0.64 Cyclotron: 18O(p,n)18F

64Cu 12.7 h β+

EC

β‐

19

41

40

0.66 Cyclotron: 64Ni(p,n)64Cu

68Ga 1.1 h β+

EC

90

10

1.88: 68Ge/68Ga Generator

Therapeutic radiopharmaceuticals have in their composition a radionuclide that

emits ionizing radiation with a high linear energy transfer (LET), to destroy selectively cells or

1. Introduction

8

tissues.5 For therapy the most used radionuclides are β‐emitters, but some research

involving α and Auger electron emitters is also underway. These particles have different

ranges in the tissues: distance travelled until all their kinetic energy is transferred (table 1.3).

In figure 1.1 the range of Auger electrons, α and β‐ particles in a cellular and subcellular

environment is represented.

Table 1.3 ‐ Range of Auger electrons, β‐and α particles in tissues.10

Particle Mean tissue range Best suited for treatment of:

β‐ (high energy) 1 ‐ 10 mm Tumor masses

β‐ (low and moderate energy) 0.1 ‐ 1 mm Tumor masses

α (5.3 MeV) 30 ‐ 80 μm (some cells) Clusters and individual cells

Auger electrons < 1 μm (cell nucleus) Individual cells

Figure 1.1 ‐ Schematic representation of Auger electrons, α and β‐ particles path lengths in a cellular

and subcellular environment (arbitrary scaling). Note that the major energy deposition of an Auger decay

occurs in the close vicinity of a few nm, while that of α and β‐ occurs on tracks of 40 ‐ 80 μm and 0.1 ‐ 10 mm,

respectively.10

Table 1.4 shows some of the radionuclides in clinical use or with potential interest for

therapy.

1. Introduction

9

Table 1.4 ‐ Some radionuclides in clinical use or potentially interesting for therapy.11,12

Radionuclide Half‐life Emax (MeV) 111Ag 7.5 d 1.05 67Cu 2.6 d 0.57 131I 8.0 d 0.81 90Y 2.7 d 2.27 186Re 3.8 d 1.07

β‐ emitters

188Re 17.0 h 2.11

Emed.a (keV)

99mTc 6.0 h 0.96 111In 2.8 d 4.00 123I 13.2 h 7.33

Auger electrons

125I 60.0 h 11.9

aValues obtained taking into account just the energy released by the Auger electrons. The values of total energy released by the radionuclides by decay are higher.

The choice of a radionuclide for therapy depends on the type, energy, half‐life and

range of the emitted particles, and should take into account the size of the tumor or tissue

to irradiate. The β‐ particles have a long range tissue penetration (mm) (table 1.3 and figure

1.1), and can be used for large solid tumor, taking advantage of the crossfire effect of β‐

particles.10 However, this crossfire might be a disadvantage when targeting small tumor cells

because it irradiates healthy tissues, leading to undesirable side effects. The α particles have

higher LET compared to β‐ particles and a path length in tissue in the range of 30 ‐ 80 μm

(table 1.3 and figure 1.1), being very cytotoxic. For this reason, the α‐emitting

radiopharmaceuticals are optimal for the treatment of small tumors. The Auger electrons

are less energetic particles and have a smaller range (< 1 μm), having a LET similar to that of

the α particles, which makes them potentially interesting for therapy. In contrast to α and β‐

particles, treatment based on Auger electron emitters requires the targeting of individual

cells, specifically the DNA in the nucleus. Despite the multiple obstacles that have to be

overpassed, Auger electron therapy approaches remain very appealing.

1. Introduction

10

1.1.2. Perfusion and Specific Radiopharmaceuticals

The radiopharmaceuticals can also be classified according to their biodistribution

characteristics as perfusion or first generation radiopharmaceuticals and specific or second

generation radiopharmaceuticals. Most of the radiopharmaceuticals in clinical use are

perfusion agents but presently most of the research efforts are directed to specific

radiopharmaceuticals for diagnostic and/or therapy.

First generation radiopharmaceuticals are those whose biodistribution and targeting

capability depend mainly on their lipophilicity, size and charge. These perfusion agents are

transported in the blood and delivered to the target organ in the proportion of the blood

flow. A wide variety of compounds were successfully developed for imaging organs, such as

liver, kidney, bone, heart or brain, giving information on a pathologic condition. Table 1.5

lists selected examples of perfusion radiopharmaceuticals in clinical use.

1. Introduction

11

Table 1.5 ‐ Perfusion radiopharmaceuticals for diagnosis in clinical use.3,4,13,14

Radiopharmaceutical Chemical structure Trade name Applications

Sodium iodide‐123I Na123I Thyroid imaging

18F‐FDG

Neck, head and lung cancer and lymphoma imaging, brain and heart metabolism study

67Ga(III)‐ citrate GaH2O

H2O O

O

OH2

O

O

O O

OH

(proposed structure)

Neoscan® Hodgkin disease, lymphoma, lung cancer

111In(III)‐oxyquinoline N

O

N

ON

O In

111InOxine® Labelling leukocytes and platelets

111In(III)‐DTPA In

N N

NOOO

OO

OO

O

O O

2‐

111InDTPA® Imaging of cerebrospinal fluid

99mTc(V)‐D,L‐HMPAO N N

N NTc

OH

O

O

Ceretec® Cerebral perfusion imaging

99mTc(V)‐L,L‐ECD N N

S STcOH3CH2COOC COOCH2CH3

Neurolite® Cerebral perfusion imaging

99mTc(I)‐Sestamibi TcC

N

C

N

CN

CNC

N

CN

+OMe

OMe

OMe

OMe

MeO

MeO

Cardiollite®

Miraluma®

Myocardial perfusion imaging Breast cancer imaging

99mTc(V)‐tetrafosmin P P

P PTc

O

O

+

OEt

OEtEtO

EtO

EtO OEt OEtOEt

Myoview® Myocardial perfusion imaging

99mTc(V)‐MAG3 N N

S NTc

O

O

COOH

O

O

‐

Technescan®MAG3 Renal Imaging

99mTc(IV)*‐MDP

(possible structure♦)

TechneScan MDP® Bone imaging

99mTc‐HDP Not known Osteoscan® HDP Bone imaging

* Dominant oxidation state; other oxidation states may be present. ♦ The structure presented is one possibility, but it is

believed that a mixture of 99mTc‐oligomers is formed.

1. Introduction

12

In the last decade, due to the advances in molecular biology and the consequent

identification and comprehension of molecular mechanisms that are in the base of several

diseases, the most recent radiopharmaceuticals in the market are specific

radiopharmaceuticals. The targeting capability of these complexes depends on a biologically

active molecule, which can be, for example, a small organic molecule, a small peptide, an

antibody, sugars, inhibitors or substrates of enzymes, nucleotides or oligonucleotides. The

capacity of the biomolecule to recognize a certain molecular target (receptor, antigene,

enzyme, DNA, mRNA, etc.) will determine the radiopharmaceutical uptake in the target

organ or tissue.

Table 1.6 shows some of the specific radiopharmaceuticals approved by FDA (Food and Drug

Administration) for clinical use. In figure 1.2 the structures of two of these compounds are

shown.

Table 1.6 ‐ Specific radiopharmaceuticals for diagnosis in clinical use.3, 13

Radiopharmaceutical Biomolecule Trade name Applications 111In(III)‐capromab pendetide Peptide ProstaScint® Imaging of prostate cancer

111In(III)‐octreotide Peptide OctreoScan® Imaging of neuroendocrine tumors

111In(III)‐satumomab

pendetide

Peptide OncoScint® Imaging of metastatic disease

associated with colorectal and

ovarian cancers

99mTc(V)‐Apcitide Peptide AcuTect® Imaging of deep vein thrombosis

99mTc‐Arcitumomab Monoclonal

antibody fragment

CEA‐Scan® Colorectal cancer imaging

99mTc(V)‐Depreotide Peptide NeoTect® Imaging of somatostatin receptor‐

bearing pulmonary masses

1. Introduction

13

111In‐Octreotide (OctreoScan®)

99mTc‐Depreotide (NeoTect®)

Figure 1.2 ‐ Structures of two specific radiopharmaceuticals in clinical use.

The overwhelming majority of diagnostic metal‐based radiopharmaceuticals,

specific or not, currently in clinical use have in their composition a radiometal, such as 111In

or 99mTc (table 1.5 and table 1.6).

Since the end of the 1980s, an intensive research for developing specific

radiopharmaceuticals with 99mTc is being carried out, but only some cases were successful in

getting to clinical use (table 1.6). This is a demanding task, since it is necessary to conjugate

the radionuclide to a biomolecule without interfering with its biological properties. For the

design of specific 99mTc radiopharmaceuticals there are three general strategies: the

integrated, the hybrid and the bifunctional approach (figure 1.3).4,13,15,16,17,18

1. Introduction

14

Figure 1.3 ‐ Representation of three approaches used in the design of specific metal‐based

radiopharmaceuticals. BFCA ‐ BiFunctional Chelator Agent.

The integrated approach involves the substitution of part of a biologically active

molecule by a metal unit. This replacement must minimize changes in the structure,

molecular size and conformation. In figure 1.4 some examples of this approach are

presented.18,19

Figure 1.4 ‐ Rhenium complexes which mimic the structure of dihydrotestosterone, progesterone

and estradiol.18

So far, the integrated approach did not provide good results, as the resulting

molecules presented low specificity and biological affinity for the steroid receptors.

M

M

Integrated

Hybrid

Bifunctional

M BFCA Spacer Biomolecule

1. Introduction

15

In the hybrid approach, the 99mTc is stabilized by functional groups naturally

occurring or synthetically introduced in the biomolecule. These groups are usually

tripeptides, such as Gly‐Gly‐Gly, Cys‐Gly‐Gly or Cys‐Gly‐Cys. The small peptide sequence can

be part of a linear or cyclic polypeptide. In some cases the coordination of the radiometal to

such amino acids can promote the cyclization of the polypeptide, improving the affinity for

the receptors and increasing the in vivo stability. An example of such result is shown in

figure 1.5.20,21,22

Figure 1.5 ‐ Structural model of 99mTc‐CCMSH for melanoma imaging.22 The linear peptide Acetyl‐Cys‐

Cys‐Glu‐His‐DPhe‐Arg‐Trp‐Cys‐Lys‐Pro‐Val‐NH2, an α‐MSH analogue, was cyclized with 99mTc through three

cysteine.

The bifunctional approach has been the most exploited for the development of

specific radiopharmaceuticals. This approach uses a bifunctional chelating agent (BFCA) that

strongly coordinates the metal ion, through appropriate coordinating groups, and is

covalently attached to the biomolecule. The nature of the BFCA depends on the metal and

its oxidation state. A spacer/linker between the metal and the biomolecule may exist, to

modulate either the pharmacokinetics of the compound or its biological activity. This

approach has been successfully used for the development of some of the

radiopharmaceuticals shown in table 1.6 and figure 1.2.

1. Introduction

16

1.2. Technetium and Rhenium Coordination Chemistry Relevant for

Nuclear Medicine

The first clinical use of 99mTc was in 1961 and involved the utilization of [99mTcO4]‐ for

thyroid imaging. Since then, 99mTc assumed a great importance in the development of

radiopharmaceuticals, being widely used in SPECT imaging.14,18 The continuing study of the

coordination chemistry of technetium is mainly related with its importance in nuclear

medicine.

The characterization of 99mTc complexes (obtained at very low concentrations, 10‐10 ‐

10‐7) is usually supported by the synthesis of analogous rhenium complexes. This is one of

the reasons why rhenium chemistry has acquired great importance in the development of 99mTc radiopharmaceuticals.

Technetium and rhenium are transition metals of group 7 belonging respectively to

the 2nd and 3rd transition series, with atomic numbers 43 and 75 and electronic

configurations [Kr]4d55s2 and [Xe]4f145d56s2, respectively.

Rhenium, first detected by Noddack, Tacke and Berg in 1925 in the X‐ray spectra of

certain mineral concentrates, was the last of the stable elements to be discovered, and

occurs naturally as a mixture of two isotopes: 185Re (37.4%) and 187Re (62.6%). This element

has two radioactive isotopes with interest in nuclear medicine: 186Re and 188Re, two β‐

emitters that can be used in therapy (table 1.4).23,24 Due to its ready availability from a

generator (188W/188Re) and favourable nuclear properties, 188Re is being intensively studied

for the design of radiotherapeutic pharmaceuticals.

Technetium was first predicted by Mendeleev and first isolated by Segré and Perrier

in 1937.25 It was separated from a molybdenum target plate that had been bombarded with

deuterons in the Berkeley cyclotron. Currently, there are 21 known isotopes (90Tc ‐ 110Tc),

none of them stable, with half‐lives which go from some seconds to millions of years.14 From

all the isotopes 99mTc and 99Tc are the most important. The long‐lived isotope 99Tc (t1/2 = 2.12

x 105 years) is a β‐ emitter (Emax = 0.3 MeV) and is available in milligram amounts. However,

special safety measures are necessary when working with this isotope, such as the use of

1. Introduction

17

glove boxes, safety glasses and specially the ingestion or inhalation should be avoided since

this isotope can cause serious damages to biological tissues. The use of the rhenium

complexes to identify the 99mTc congeners is usually preferable because it avoids the use of

the radioactive 99Tc.

The importance of 99mTc in nuclear medicine is related to its ideal characteristics:3, 4,

13,14, 18, 26

‐ The half‐life of 6 h is optimal for diagnostic. It is long enough to allow the

radiopharmaceutical preparation, quality control, administration to the

patient and image acquisition, and is sufficiently short to induce low doses of

radiation to the patient;

‐ The emission of γ radiation, monoenergetic of 140 keV (90%), sufficiently low

to prevent a high dose burden to the patient, but sufficiently high to

penetrate biological tissues and emerge from internal organs;

‐ The availability at reduced prices in commercial 99Mo/99mTc generators is one

of the greatest advantages of this radionuclide. The 99Mo/99mTc generator was

developed in the early 1960s in the Brookhaven National Laboratory, and its

introduction revolutionized the nuclear medicine research and clinical nuclear

medicine;

‐ The diverse coordination chemistry, which enables the preparation of a wide

variety of complexes with different physico‐chemical and biological

properties.

Technetium and rhenium present several oxidation states being the chemistry of

these elements dominated by the formation of coordination complexes. Technetium

complexes can have the metal in the oxidation sates ‐I (d8) to +VII (d0), while for rhenium

complexes are known where the metal presents oxidation states ‐III (d10) to +VII (d0).27,28

These elements have very similar atomic radii (Tc, 1.36 Å; Re, 1.37 Å) so they form

structurally analogous complexes.29 Despite these similarities, there are differences that

should be taken into consideration in the preparation of Tc and Re complexes, the more

relevant being the higher kinetic inertness and the easier oxidation of rhenium

complexes.14,30

1. Introduction

18

For both metals, the most studied oxidation states for medical applications are +V,

+IV, +III and +I (figure 1.6). The chemistry of Tc(V) and Re(V) is dominated by the formation

of oxo‐complexes with the [M=O]3+ or trans‐[O=M=O]+ moieties. The number of oxo ligands

is influenced by the nature of the ligands coordinated to the metal. Generally, π donor

coligands (ex. alcoxides or thiolates) favour the formation of complexes with the [M=O]3+

core, while the complexes with the trans‐[O=M=O]+ unit are stabilized by neutral ligands, like

primary amines. In lower oxidation states (M(I) ‐ M(III)), the stabilization of the metal center

needs π acceptor ligands, like isonitriles, phosphines or carbonyls.3,13,17,31

In the development of 99mTc radiopharmaceuticals the most studied complexes have

the [Tc=O]3+, [O=Tc=O]+ and [Tc≡N]2+ moieties (figure 1.6), being the [Tc=O]3+ moiety present

in most of the first generation and specific 99mTc radiopharmaceuticals (table 1.5 and figure

1.2).

XX

YL

YM

X

Y

M

Y

X X

Y

N

M

Y

X X

Y

O

ML

L L

LL

MOC

X X

CO

X

CO

M

Y

X X

Y

O

O

N

HNO R

NN

M(III)/M(IV) [MO]3+/M(V) [MO2]+/ M(V)

[MN]2+/ M(V) fac‐[M(CO)3]+/ M(I) [M]HYNIC/ M(V)

Figure 1.6 ‐ Most studied technetium and rhenium cores for the development of

radiopharmaceuticals (M = Tc, Re).

Generally, complexes with the [Tc=O]3+ core are stabilized by tetradentate chelators

with nitrogen and sulfur donor atoms.3,4,13,17 To overcome some limitations of the Tc(V)

oxocomplexes, new cores have been introduced in radiopharmaceutical chemistry such as

1. Introduction

19

[Tc]‐HYNIC, [Tc≡N]2+ and fac‐[Tc(CO)3]+ (figure 1.6). A wide range of bifunctional chelating

agents have been studied and explored to stabilize these moieties.3,13,17,18

1.2.1. The Technetium‐ and Rhenium‐Tricarbonyl Core

The fac‐[M(CO)3]+ (M = Tc, Re) unit is a promising organometallic core for labelling

biomolecules with 99mTc(I). This fragment is very compact, with an almost spherical shape

and the metal center is in the oxidation state +I with electronic configuration d6.

Alberto et al. succeeded in developing a normal pressure and a fully aqueous‐based

preparation of the precursor fac‐[M(OH2)3(CO)3]+ (M = 99mTc, 99Tc, Re) in high yield and with

excellent (radio)chemical purity.32,33,34 Initially, it was prepared by reduction and

carbonylation of pertechnetate, using borohydrides and carbon monoxide. This synthetic

procedure, although being suitable for research, was not adequate for the nuclear medicine

centres due to the use of carbon monoxide. Then, Alberto et al. introduced a remarkable

innovative procedure which consisted in using potassium boranocarbonate, K2[H3BCO2],

which simultaneously reduces the Tc(VII) to Tc(I) and is a source of CO.35 Currently, the

synthesis of fac‐[99mTc(OH2)3(CO)3]+ can be achieved using a kit formulation, IsoLink®

(Mallinckrodt Med. BV). As shown in figure 1.7, by adding the [99mTcO4]‐ to IsoLink®, the

complex fac‐[99mTc(CO)3(OH2)3]+ can be obtained in quantitative yield.

Figure 1.7 ‐ IsoLink® kit and synthesis of fac‐[99mTc(CO)3(OH2)3]

+.

The availability of this precursor represents a significant progress in

radiopharmaceutical chemistry.18,36 The major attractions of the fac‐[99mTc(CO)3]+ approach

include:36,37,38

‐ Its applicability to a wide range of biologically relevant molecules;

+ TcOC

H2O OH2

CO

OH2

CO

[99mTcO4]‐

100 °C, 20 min, pH = 11

Kit composition:

K2[H3BCO2]

Na2(tartrate)

Na2B4O7.10H2O

Na2CO3.

1. Introduction

20

‐ The high thermodynamic and kinetic stability of the resultant 99mTc

complexes;

‐ The high specific activity which can be achieved, often without needing

purification;

‐ The relatively low molecular weight of the moiety.

In the precursor fac‐[99mTc(CO)3(OH2)3]+, the three water molecules and the three

carbonyls are facially arranged around the metal center, in a octahedral coordination

geometry. The three water molecules are labile and can be easily replaced by mono‐, bi‐ or

tridentate ligand systems with different donor atom sets, electronic and stereochemical

properties. This versatility enabled the preparation of a wide variety of complexes, which

can be functionalized with different biomolecules. In figure 1.8 are presented some of the

more explored bi‐ and tridentate chelators suitable for the fac‐[99mTc(CO)3]+ moiety. 18, 26, 36,

1. Introduction

21

Bidentate Bifunctional Chelating Agents

N NTc

COOC CO

OH2

NH2

O

NTc

OC CO

N

OH2

ROC OTc

OC CO

N

OH2

OC

R

+ +

O

R

NN

R

NN

Tc

OC COOH2

OC

+

1 2 43

H

Tridentate Bifunctional Chelating Agents

RSNH2

O

O

Tc

OC CO CO

NTc

OC CO

N NTc

COOC CO

ONH2

O

RN

O O

R

OC

Tc

COCO

OC

NN

N NH2

R +

Tc

OC CO

H

CO

NN

R S

NN R

S

B

H

Tc

OC CO CO

R

Tc

OC CO CO

X XX

R+

Tc

COCO

OC

NN

N NH2

+

R

X=N,P,S5 6 7 8

9 10 11 12

Figure 1.8 ‐ Examples of bidentate and tridentate bifunctional chelating agents for the tricarbonyl

core; R = biomolecule. 1 ‐ Functionalized histidine;39,40,41 2 ‐ Functionalized Schiff base; 42,43 3‐ Picolinic

acid and derivatives;44,45 4 ‐ Bis‐pyrazolyl methane;36 5 ‐ Functionalized cysteine;43,46,47,48,49,50,51,52 6 ‐ N‐

acetic picolinic acid (functionalized);33,40,53 7 ‐ Nε functionalized histidine;40,54 8 –

(triaza/tritia)cyclononane;55 9 and 10 ‐ Functionalized pyrazolyl diamine ligand;56,57,58 11 ‐

Cyclopentadienyl; 59 12 ‐ Bis(mercaptoimidazolyl)borates.60

Pharmacokinetic studies of different complexes with the fac‐[99mTc(CO)3]+ moiety, in

animal models, have shown that tridentate chelators are those presenting, in general, more

favourable biological profiles.36,40 In part, this behaviour reflects the higher stability of the

complexes with tridentate chelators compared with bidentate chelators.13,36 Based on these

studies, tridentate chelators containing N‐heterocycles such as imidazoles,40,54

mercaptoimidazoles,60 pyridines53,61 and pyrazoles56‐57 have been explored to develop

specific radiopharmaceuticals, being conjugated to different biomolecules, in particular

1. Introduction

22

small peptides, sugars, amino acids, nucleotides, agonists or antagonists of central nervous

system receptors.36,60,62

1.3. Specific Probes for Endogenous Gene Expression and

Membrane Receptors Imaging

Molecular imaging owes its existence to the revolution in molecular medicine and

drug discovery. The decoding of the human genome and the subsequent developments in

proteomics provided a completely new spectrum of targets for molecular imaging, being

proteins and gene expression some of these targets. Proteins, such as intracellular,

transmembrane and surface receptors, and cell surface or secreted antigens are often

present in copy numbers ranging from thousands to millions per cell, being attractive for

SPECT or PET in vivo imaging.63

Gene expression imaging can be directed to mRNA providing valuable information at

the cellular level. However, imaging of mRNA based on one‐to‐one antisense‐target

interactions still faces considerable challenges, as mRNA is typically present at levels of a few

hundred to a few thousand molecules per cell. The ability to image the degree, distribution

and persistence of gene expression will unquestionably be useful in connection with gene

therapy.63,64

In this section we will present some of the concepts underlying the targeting of

mRNA and membrane receptors as well as the state of the art relative to these two targets,

directly related with the experimental work described in this thesis.

1.3.1. Antisense Imaging of Endogenous Gene Expression

Molecular imaging of gene expression is directed at cellular processes closer to

transcription rather than translation.64 Basically, there are two main approaches to observe

gene expression non‐invasively, the indirect and the direct. Indirect imaging involves the

expression of a reporter gene that is monitored in order to check the expression of a co‐

transcribed gene of interest. Both genes are translated and the protein product of the

1. Introduction

23

reporter gene can be visualized using reporter probes that trap them inside the cells (figure

1.9).65 In this approach exogenous protein function is being visualized.

Figure 1.9 ‐ Schematic illustration of the reporter gene approach. a, 65

The direct approach involves the in vivo direct imaging of mRNA using the antisense

approach. This approach consists in developing single‐stranded oligonucleotides (13 ‐ 20

bases) with a complementary base sequence (i.e. “antisense”) to effectively locate and

hybridize to its target mRNA (“sense” sequence), through Watson‐Crick base‐pairing

(hydrogen bonds between A and U/T and G with C). The complementary sequence when

radiolabelled (RASON, Radiolabelled Antisense OligoNucleotides) may be used as a probe to

visualize the target mRNA (i.e. antisense imaging) (figure 1.10 and figure 1.11).63,66

a An imaging reporter gene has to be introduced into the cell by a promoter of choice. Transcription of the

imaging reporter gene with subsequent translation of the mRNA leads to an enzyme. This enzyme can

selectively trap an imaging reporter probe. The imaging reporter probe will not be trapped in those cells in

which there is no expression of the imaging reporter gene. Note that it is also possible for the imaging reporter

gene to encode for an intracellular and/or cell surface receptor. This receptor would then bind the imaging

reporter probe. Levels of the trapped probe can be related to levels of imaging reporter gene expression in

either approach.

1. Introduction

24

Figure 1.10 ‐ Schematic image showing the radiolabelled antisense oligonucleotide upon

hybridization to the complementary target mRNA. RASON ‐ Radiolabelled Antisense OligoNucleotides.

Figure 1.11 ‐ Illustration of antisense hybridization imaging approach.b,65

It is expected that an oligonucleotide with more than 12 nucleobases (12‐mer)

represents a unique sequence in the whole genome,67 so a radiolabelled oligonucleotide

with 13 ‐ 20 bases is likely to be specific for its target. Since these short oligonucleotides can

in principle be easily produced, antisense imaging using radiolabelled oligonucleotides may

offer a wide range of new specific probes.

b Small radiolabelled antisense oligonucleotides (RASONs) are used to target a small portion of a chosen mRNA. If sufficient levels of mRNA are present, it may be possible to retain the RASONs only in those cells expressing the mRNA. Efflux would occur in other cells if non‐specific interactions can be minimized. Because radiolabelling chemistry is made independently of the RASON sequence, different types of mRNA can potentially be targeted.

RASONmRNA

1. Introduction

25

In order to be successfully used as probes, antisense oligonucleotides have to meet

the following criteria:64‐66,68,69,70

1. Easy synthesis of the radiolabelled oligonucleotide;

2. High specific activity of the probe;

3. Stability of the radiolabelled antisense molecules ‐ resistance to nucleases and

proteases;

4. Non‐specific interaction with other macromolecules (e.g. proteins) should be

minimized;

5. High cellular uptake rates ‐ the probe should have high accumulation in the

target expressing cells and little in non‐target cells;

6. High binding affinity and specificity to the target mRNA ‐ the antisense

oligonucleotide‐mRNA duplex should have a melting temperature high

enough to prevent dissociation of the base pairs at 37 °C;

7. Prevention of degradation of mRNA after hybridization by RNase H attack.c

Concerning the target mRNA, the number of copies and the concentration of cells

expressing the specific mRNA needs to exceed a certain level for antisense imaging, and the

target region of mRNA needs to be carefully chosen. The mRNA structure is very complex,

composed of secondary and tertiary structures.69,71 The secondary structures are composed

of many regions of intrachain base pairing, stem‐loops, hairpins, etc. These secondary

structures are not good targets for antisense strategies because antisense oligonucleotides

usually show lower affinities for duplex regions.69,72 Therefore, antisense strategies usually

seek to target only single‐strand regions of mRNA. Due to the limited knowledge of the

molecular structures,69 one common approach is to target either the initiation codon (AUG)

and adjacent sequences or the untranslated sequences on either 5’ or 3’ end (5’‐UTR or 3’‐

UTR), hopping the accessibility of these regions.73 Also, in order to identify favourable local

c The enzyme RNase H is a ribonuclease that cleaves the 3'‐O‐P‐bond of RNA in a DNA/RNA duplex to produce 3'‐hydroxyl and 5'‐phosphate terminated products. RNase H is a non‐specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism, aided by an enzyme‐bound divalent metal ion.

1. Introduction

26

target sequences and to predict the secondary structure of RNA, different computer‐based

methods have been established.74

Potential targets for antisense imaging can be the majority of targets for antisense

chemotherapy in preclinical and clinical trials, for example proto‐oncogenes.69,75 Proto‐

oncogenes that have been the object of study for antisense imaging are the c‐MYC that is

overexpressed in a variety of cancers, including breast, bladder, kidney, and lung cancers,

and lymphoma; CCND1 (cyclin D1) and ERBB2 overexpressed in breast and pancreatic

cancers; members of the RAS gene family, H‐RAS, K‐RAS and N‐RAS that are among the most

frequently mutated oncogenes detected in human tumor, such as colon, lung and pancreatic

tumors; B‐cell lymphoma/leukemia‐2 gene (BCL‐2)76 overexpressed in non‐Hodgkin’s

lymphoma, lung, colon, prostate, and neuroendocrine cancers, and malignant melanomas.

Naturally occurring oligonucleotides, DNA or RNA, are not good candidates for being

directly used as probes as they are rapidly degradated in vivo by endo‐ and exonucleases,

and they can promote degradation of the target mRNA by RNaseH.68,77,7879,80 To increase the

in vivo stability of oligonucleotides, without significant alteration of their pharmacokinetics

and targeting properties, many chemical modifications have been made to the sugar‐

phosphate backbone of DNA or RNA (figure 1.12). Some of the analogues include

phosphorothioate,81 methylphosphonate,82 2’‐O‐methyl RNA79,80,83,8485 and complete

replacement of the backbone including morpholino86 and peptide nucleic acids.87,88,89,90

Among these, peptide nucleic acids (PNAs) present remarkable properties and are

considered good candidates for developing radiolabelled antisense probes.

1. Introduction

27

N

HNN

HNN

HN

O

OB

OO

B

OHN

B

O

O

O

B

OO

O

O

B

PO

SO

O

O

B

PS

O

O

O

B

OO

O

O

B

PO

O

O

O

B

PO

O

O

B

OO

O

O

B

PO

O O

O

O

B

PO O

O O O

PN

OPNO

ON

B

O

ON

B

O

ON

B

O PON

O

Phosphorothioate DNA

Methylphosphonate DNA

2'‐O‐methyl RNA

Morpholino

Peptide Nucleic Acids

Figure 1.12 ‐ General structure of some DNA and RNA analogues used in antisense imaging. B = base =

thymine, adenine, cytosine, guanine.

1.3.1.1. Peptide Nucleic Acids and Diagnosis

Peptide nucleic acids have been introduced in 1991, based on the research efforts of

Ole Buchardt and Peter Nielsen. 91,92 They tried to develop PNAs as specific reagents for gene

targeting and gene therapy. Nowadays, applications of PNAs can be arbitrarily split into four

main categories: antigene and antisense therapy, as tools for molecular biology and

functional genomics, as probes for diagnosis and detection, and as biosensors.87,93,94,95,96

A PNA is a DNA mimic in which a 2‐aminoethylglycine (aeg) linkage generally replaces

the normal phosphodiester backbone and a methylcarbonyl linker connects the nucleotide

bases to this backbone at the amino nitrogen (figure 1.13).91,92,94 If we compare the

structure of a nucleotide with that of a PNA monomer, we can see that they are identical ‐

the skeleton of the monomer is formed in both cases by six atoms and the side arm that

connects the base (B) to the backbone is two atoms long (figure 1.13).

1. Introduction

28

NH

N

OB

O2

4

51'

3

2'

61

O O

B

PO

2

45

1'

3

2'

6

1 O

O

Figure 1.13 ‐ DNA and aminoethylglycine‐PNA backbones.

Unmodified PNAs are not charged at neutral pH, are non‐chiral and can be

synthesized without need of any stereoselective pathway, following standard solid‐phase

synthetic protocols for peptides.93‐96,97,98 The amide (or peptide) bonds in PNAs are

sufficiently different from the α‐amino acid bonds present in proteins, and therefore PNAs

are biologically stable and not degradable by cellular proteases and peptidases, and are also

resistant to nucleases.98,99 Despite all the modifications, PNAs are still able of binding

specifically to DNA as well as to RNA, obeying the Watson‐Crick hydrogen bonding rules.

PNAs form very stable duplex structures with DNA and RNA (PNA/DNA and PNA/RNA), with

affinity and specificity substantially exceeding that of DNA/DNA and DNA/RNA

structures.93,100 Such strong binding is attributed to the lack of charge repulsion between the

neutral PNA and the DNA or RNA strand. Another consequence of the neutral backbone is

that the melting temperature (Tm) values of PNA/DNA duplexes are practically independent

of salt concentration. For in vivo applications, very important PNA properties are the fact

that the uncharged PNA backbone is unlikely to interact with cellular proteins that normally

bind to negatively charged macromolecules, PNA hybridization does not induce RNase H

degradation of bound mRNA and they present no sign of any general toxicity.92‐100

1.3.1.1.1. PNA Labelling with Different Radionuclides for Imaging

Endogenous Gene Expression

Using the bifunctional chelator approach mentioned in section 1.1.2, different PNAs

have been labelled with SPECT (99mTc, 111In)75,76,88,90,101,102,103,104,105,106,107,108,109 and PET (18F, 64Cu)78,110,111,112 radionuclides. The labelling of PNAs with 125I for autoradiography studies has

also been described.113,114

1. Introduction

29

The labelling of PNAs with 99mTc was first reported in 1997 by Hnatowich and co‐

workers.90 They used the chelator mercaptoacetyl‐triglycine (MAG3) to stabilize the metal

and to conjugate the PNA and tried to prove that in vivo antisense hybridization is possible.

The authors used mice implanted intra‐muscularly in the left and in the contralateral thigh

with “sense” PNA coupled to polystyrene beads and beads without PNA, respectively. The

antisense PNA labelled with 99mTc was then injected intraperitonially and, 23 h after

injection, a 6‐fold enhancement of binding to “sense” PNA coupled to beads versus

uncoupled beads was found.90

Later, in 2003, Wickstrom and co‐workers reported the labelling of a tetrapeptide‐

PNA dodecamer with 99mTc for antisense imaging of c‐MYC mRNA, overexpressed in different

tumors, namely in breast cancer (figure 1.14).102 The tetrapeptide GlyD(Ala)GlyGly was used

as BFCA, providing an N4 coordination to 99mTc.

N N

O

NN

O O

HN

OAba

HH

Tc O

GCATCGTCGCGG

Figure 1.14 ‐ Proposed structure for 99mTc‐N‐GlyD(Ala)GlyGly‐Aba‐GCATCGTCGCGG.

Tissue distribution studies in mice bearing human breast tumor showed that the

uptake of c‐MYC antisense 99mTc‐GlyD(Ala)GlyGly‐c‐MYC PNA was two‐fold greater than the

corresponding mismatch PNA probe, and such result was considered encouraging. The

sequences also showed a good biological profile in normal tissues and organs at 4 and 24 h

post‐injection. The results were promising, but it was necessary to increase the tumor

uptake and specificity of the c‐MYC antisense 99mTc‐PNA conjugate.102

In order to increase the cellular uptake and specificity of PNA sequences to different

target mRNAs, Wickstrom and co‐workers have conjugated a D‐peptide (D(CysSerLysCys))

analogue of insulin‐like growth factor 1 (IGF1) specific for the IGF1R (insulin‐like growth

factor 1 receptor), overexpressed in malignant cells, to PNA sequences complementary to

CCND1, c‐MYC and KRAS mRNAs.103‐106,111,112 The CCND1 PNA‐IGF1 was labelled with 99mTc

and 64Cu using the chelators GlyD(Ala)GlyGly and 1,4,7,10‐tetraazacyclododecane‐

N,N’,N’’,N’’’‐tetraacetic acid (DOTA), respectively.103,111 The c‐MYC PNA‐IGF1 was labelled

1. Introduction

30

with 99mTc using the GlyD(Ala)GlyGly chelator.104 The conjugate KRAS PNA‐IGF1 was labelled

with 99mTc and 64Cu using as BFCA the bis(S‐benzoylthioglycolyl)diaminoproponoate chelator

(N2S2) for both radionuclides, and also DOTA in the case of 64Cu.105,112

99mTc‐AcGlyD(Ala)GlyGly‐CCND1 PNA‐IGF1 peptide and 99mTc‐GlyD(Ala)GlyGly‐c‐MYC

PNA‐IGF1 peptide antisense probes (figure 1.15) enabled the in vivo visualization of human

MCF‐7 breast cancer xenografts in immunocompromised mice scintigraphically at 4, 12 and

24 h after intravenous administration.103,104 In all experiments, control sequences with no

PNA, mismatched PNA or mismatched peptide yielded no tumor signals, demonstrating that

non‐invasive mRNA tumors imaging was both receptor‐ and sequence‐specific.

Figure 1.15 ‐ Illustration of 99mTc‐AcGlyD(Ala)GlyGlyAba‐PNA‐AEEA‐D(CysSerLysCys) conjugates

designed to bind to IGF1 receptor, internalize and hybridize with the CCND1 or c‐MYC mRNA.d,104

Human MCF‐7 breast cancer xenografts in immunocompromised mice were also

imaged by microPET/CT at 4 and 24 h after intravenous injection of 64Cu‐DOTA‐CCND1 PNA‐

IGF1 peptide antisense probe, but not after administration of control probes (64CuCl2, PNA

mismatch and peptide mismatch). Preblocking the IGF1 receptors with recombinant human

IGF1 peptide 30 min before administration of the antisense probe 64Cu‐CCND1 PNA‐IGF1,

reduced the PET image intensity to the level of the controls, demonstrating the specificity of

the probe. The 64Cu‐DOTA‐CCND1 PNA‐IGF1 antisense probe showed a relatively high tumor

uptake, 2.01 ± 0.43 % ID/g, and high tumor‐to‐muscle ratio, 2.72 ± 0.67, at 4 h after

injection. The results confirmed that a receptor‐specific targeting peptide facilitates the

d Sintigraphic imaging of γ radiation emitted upon decay of 99mTc might identify sites of high c‐MYC or CCND1

mRNA expression. CCND1 PNA = CTGGTGTTCCAT; c‐MYC PNA = GCATCGTCGCGG; Aba ‐ 4‐aminobutyric acid;

AEEA – aminoethoxyethoxyacetate.

PNA

1. Introduction

31

cellular uptake of a PNA probe into the cytoplasm of cancer cells and enables external

imaging of oncogene mRNA expression in vivo.111 The microPET studies were compared with

the scintigraphic studies performed to visualize the same CCND1 mRNA.103 The microPET

studies indicated that the 64Cu‐DOTA‐CCND1 PNA‐IGF1 probe exhibited faster and higher

tumor uptake than the corresponding 99mTc‐Ac‐GlyD(Ala)GlyGlyAba‐CCND1 PNA‐IGF1

analogue.

The K‐RAS radioprobes, 99mTc‐N2S2‐K‐RAS PNA‐IGF1 and 64Cu‐N2S2‐K‐RAS PNA‐IGF1,

enabled scintigraphic or PET imaging of immunocompromised mice bearing human pancreas

cancer (figure 1.16). However, in these studies no control studies were performed that

would be necessary to determine the specificity of the probes, and the labelling yields with 64Cu were very low (10%).105

Figure 1.16 ‐ Illustration of M‐N2S2‐K‐RAS PNA‐IGF1 peptide probes (M = 99mTc or 64Cu).105

The antisense 64Cu‐DOTA‐K‐RAS PNA‐IGF1 probe (figure 1.17), radiolabelled in high

yield using the DOTA chelator, was administered intravenously to mice bearing human

pancreas cancer and yielded strong tumor contrast in PET images, 8.60 ± 1.39 and 8.99 ±

4.17 ‐fold more intense in the center of the tumor than in contralateral muscle at 4 h and 24

h, respectively. Control experiments with single base K‐RAS PNA mismatches, an IGF1

peptide mismatch, and studies in breast cancer xenograft lacking K‐RAS mRNA activation

yielded weak tumor contrast images. The PET imaging results showed that K‐RAS mRNA

overexpression could be detected by radiohybridization with the complementary probe.112

R

R =

1. Introduction

32

Figure 1.17 ‐ Illustration of 64Cu‐DOTA‐KRAS PNA‐IGF1 peptide radiohybridization probe designed to

bind to the IGF1 receptor, internalize and hybridize with KRAS mRNA.112

PNA conjugates for microPET imaging of breast cancer via upstream of N‐RAS mRNA

(UNR mRNA) were reported by Sun et al.78 The UNR mRNA is an oncogene highly abundant

and uniquely overexpressed in MCF‐7 breast cancer cell lines. Three antisense UNR PNA (to

different sites on mRNA) and one sense UNR PNA conjugates, containing four‐lysines for cell

permeation and DOTA for chelating 64Cu were studied (figure 1.18).

Figure 1.18 – Illustration of 64Cu‐DOTA‐PNA–(Lys)4 probes for UNR mRNA imaging.

The MCF‐7 tumor‐bearing mice were imaged with all four 64Cu‐DOTA‐UNR PNA‐(Lys)4

conjugates using a microPET camera, but one antisense conjugate showed better tumor

image quality at all time points, with a tumor/muscle ratio of 7.9 ± 3.3 and 6.6 ± 1.1 at 4 and

24 h post‐injection, respectively. These ratios are among the highest reported for

radiolabelled oligonucleotides. However, these values are less than 4‐fold better than the

corresponding sense sequence, indicating that factors other than the sequence

complementary to the UNR mRNA played a significant role in tumor localization. The

-

-

antisense KRAS PNA

1. Introduction

33

biodistribution date in the tumor‐bearing mice also showed very high kidney uptake for all

sequences studied, presenting values higher than 100% ID/g.78

Taking into account that 84% of non‐Hodgkin’s lymphomas (NHLs) overexpress

somatostatin receptors and BCL‐2 mRNA, Lewis and co‐workers prepared 111In‐DOTA‐ BCL‐2

PNA‐Tyr3‐octreotate complementary to the translational star site of BCL‐2 mRNA (figure

1.19).76 The antisense probe was able to detect Mec‐1 human small lymphocytic tumor

bearing mice by microSPECT/CT, but only after 48 h after injection. The two negative

controls, 111In‐DOTA‐non‐sense PNA‐Tyr3‐octreotate and 111In‐DOTA‐anti bcl‐2 PNA‐

Ala[3,4,5,6]‐substituted congener of octreotate, did not detect the tumors by

microSPECT/CT.76

Figure 1.19 ‐ Illustration of 111In‐DOTA‐anti‐BCL‐2 PNA‐Tyr3‐octreotate for targeting BCL‐2 mRNA.

Pardridge and colleagues have employed ex‐vivo autoradiography for antisense

detection of brain diseases. In one report, stably transfected, ectopic C6‐790 rat gliomas

expressing luciferase were visualized using a 125I‐Tyr‐antisense PNA‐OX26 antibody

conjugate but not with control sequences.113 In a second publication, an antibody against

murine transferrin receptor was conjugated to 125I‐Tyr‐antisense PNA targeting huntingtin

mRNA in a transgenic mouse model of Huntington’s disease. Quantitative autoradiography

showed a 3‐fold selective sequestration of the antisense compound in the brains of HD

(Huntington’s) transgenic mice, compared to controls.114

NH‐CCAGCGTGCGCCAT‐DPhe‐Cys‐Tyr‐D‐Trp‐Lys‐Thr‐Cys‐Thr(OH)

S S

1. Introduction

34

1.3.2. Imaging Membrane Receptors with Small Radiopeptides

The discovery that certain tumor types (over)‐express receptors for certain regulatory

peptide hormones dates back to the mid 1980s and early 1990s.115, 116, Since then, there has

been an exponential growth in the development of radiolabelled peptides for diagnosis

and/or therapy, and currently peptides are the radioactivity vehicles of choice for receptor

targeting.117,118,119 The best examples illustrating this strategy are radiolabelled somatostatin

analogues, which are now routinely used in clinics to image tumors expressing somatostatin

receptors (OctreoScan®, figure 1.2) and are promising for internal radiotherapy in

patients.119,120,121,122,123

Small peptides offer many distinct advantages over other bioactive molecules like

proteins and monoclonal antibodies.115,124,125,126 Small peptides can be easily and

inexpensively synthesized by automated solid phase techniques, and manipulated

molecularly to optimize their affinity for a particular receptor and to display a more specific

biodistribution pattern. Peptides have the ability to tolerate the harsh conditions (pH,

temperature, etc.) of chemical modification and/or radiolabelling and the ability to attach a

bifunctional chelating agent at the C‐ or N‐ terminus of the peptide. They present low

toxicity and low immunogenicity.116,124,126 Because of their small size, peptides usually

display favourable pharmacokinetics characterized by high concentration in the target

tissues and rapid clearance from the blood and non‐target tissues. Small peptides have the

ability to reach the receptors on tumor cells more efficiently, and penetrate into tumors

faster than monoclonal antibodies.17,124,126,127

In table 1.7 examples are given of endogenous peptides whose receptors can be used

as targets for imaging.

1. Introduction

35

Table 1.7 ‐ Regulatory peptides, their function, target disease, cells expressing receptors, and

receptor subtypes.116,117,126,127

Peptide Function Target diseases Receptor

Bombesin CNS and GI tract activity Glioblastomas, SCLC, prostate , breast, gastric, colon and pancreatic cancer

GRP‐bombesin

CCK/gastrin Gallbladder contraction/acid secretion,

SCLC, GI tumors, pancreatic cancer, adenomas and medullary thyroid cancer

CCK 1 and 2

Epidermal growth factor

Growth promoter Breast cancer Epidermal growth factor

Gastrin releasing pepide

Promotes gastrin secretion

Glioblastomas, SCLC, prostate, breast, gastric, colon and pancreatic cancer

GRP‐bombesin

α‐MSH Regulation of skin pigment/CNS

Melanoma Melanocortin 1‐5

Neurotensin GI and cardiac activity, neuromodulator

Prostate and pancreatic cancer Neurotensin 1‐3

Somatostatin and analogues

Growth hormone release inhibiting factor

Neuroendocrine tumors, SCLC, breast cancer, monocytes and lymphocytes

Somatostatina 1‐5

Substance P Vasoactive Glial tumors, astrocytomas, medullary thyroid and breast cancer

NK1, NK2 and NK3

VIP Vasodilator, growth promoter, immunomodulator

NSCLC, breast, colon, pancreatic, prostate, bladder and ovarian cancer

VP 1 and 2

RGD analogues Endothelial adhesion molecule

Tumor– induced angiogenesis αvβ3 integrin

α‐MSH = α‐melanocyte‐stimulating hormone; CCK = cholecystokinin; GI = gastrointestinal; GRP = gastrin‐releasing peptide; NK = neurokinin; NSCLC = non‐SCLC; RGD = single‐letter designation for amino acids, Arg‐Gly‐Asp; SCLC = small cell lung cancer; VIP = vasoactive intestinal peptide

1.3.2.1. α‐Melanocyte Stimulating Hormone‐Based

Radiopharmaceuticals

Malignant melanoma is a serious form of cancer with high mortality rates. Early

detection of primary melanoma is essential because there is no effective treatment for

metastatic melanoma. The melanocortin type 1 receptors (MC1R) have been used as targets

for melanoma imaging due to their overexpression on human and mouse melanoma cells.20,

128,129,130,131 Radiolabelled α‐melanocyte stimulating hormone (α‐MSH) peptide analogues

1. Introduction

36

have been considered very promising candidates for melanoma imaging and therapy due to

their nanomolar MC1 receptor binding affinities and high receptor‐mediated tumor uptake

in murine melanoma‐bearing mice and human melanoma xenografts132,133,134,135,136

α‐Melanocyte stimulating hormone (α‐MSH) is a linear 13‐amino acid peptide (Ac‐

Ser‐Tyr‐Ser‐Met‐Glu‐His–Phe–Arg‐Trp‐Gly‐Lys‐Pro‐Val‐NH2) derived from the proteolytic

cleavage of pro‐opiomelanocortin (POMC) and is the most potent naturally occurring

melanotropic peptide that is produced in the brain and pituitary gland.128,137,138,139

So far, the melanocortin receptor system consists of five isoform receptors (MC1R‐

MC5R) and belongs to the heterodimeric guanine nucleotide‐binding protein (G‐protein)‐

coupled family of receptors, characterized by the presence of seven transmembrane repeat

spanning domains.128,138,140,141,142 The melanocortin‐1 receptor (MC1R) is expressed in skin

cells (keratinocytes, melanocytes and fibroblasts) including melanoma cells, immune cells

and endothelial cells.128 MC1R regulates the amount and type of pigment production and is a

major determinant of skin phototype and sensitivity to ultraviolet‐light induced damage.139.

Taking into account that MC1 receptors are expressed in melanocytes and

overexpressed in melanoma cells,128,143 and that α‐MSH has high affinity for this receptor,

the development of radiolabelled α‐MSH analogues may be useful for both detection and

treatment of melanoma.

1.3.2.1.1. α‐MSH Analogues

The endogenous α‐MSH as such is not a good peptide to be labelled directly with

radionuclides, as it is rapidly degradated by proteolytic enzymes.138,139 As a result, several α‐

MSH analogues that possess increased receptor affinity and stability have been synthesized

(table 1.8).

Structure‐activity relationship studies have shown that the minimal sequence in α‐

MSH required for biological activity is the amino acid sequence His6‐Phe7‐Arg8‐Trp9. The

replacement of Met4 and Phe7 by Nle4 and DPhe7, respectively, led to the more potent [Nle4,

DPhe7]‐α‐MSH (NDP‐MSH) analogue (table 1.8), which has a longer half‐life, and is one of

the most cited α‐MSH analogue, due to its subnanomolar receptor affinity and resistance to

enzymatic degradation.138,139,144

1. Introduction

37

Table 1.8 ‐ Structure of α‐melanocyte stimulating hormone (α‐MSH) and some α‐MSH analogues.

Bold: amino acid residues replaced in different analogues relatively to the endogenous peptide (α‐MSH);

underline: minimal sequence for biological activity.

Peptide Name aa

Ac‐Ser1‐Tyr2‐Ser3‐ Met4‐Glu5‐His6–Phe7–Arg8‐Trp9‐Gly10‐Lys11‐Pro12‐Val13‐NH2 α‐MSH 13

Ac‐Ser‐Tyr‐Ser‐Nle4‐Glu‐His‐D‐Phe7‐Arg‐Trp‐Gly‐Lys‐Pro‐Val‐NH2 NDP‐MSH 13

Ac‐Cys3‐Cys4‐Glu‐His‐D‐Phe7‐Arg‐Trp‐Cys10‐Lys‐Pro‐Val‐NH2 CCMSH 11

Ac‐Cys3‐Cys4‐Glu5‐His6‐D‐Phe7‐Arg8‐Trp9‐Cys10‐Arg11‐Pro‐Val‐NH2 CCMSH(Arg11) 11

Ac‐ Nle4‐Asp5‐His‐D‐Phe7‐Arg‐Trp‐Gly‐Lys10‐NH2 NAPamide 8

Ac‐ Nle4‐cyclo[Asp5‐His‐D‐Phe7‐Arg‐Trp‐Lys10]‐NH2 MT‐II 7

βAla3‐Nle4‐Asp5‐His‐D‐Phe7‐Arg‐Trp‐Lys10‐NH2 MSHoct 8

For potential diagnostic application, some linear α‐MSH analogues such as

NAPamide, MSHoct, have been labelled with different PET (68Ga, 64Cu, 18F)135,145,146 and SPECT

(111In, 67Ga, 99mTc)22,58,134,147,148 radionuclides, using DOTA or a pyrazolyl‐diamine as

bifunctional chelators, and in the case of 18F the N‐succinimidyl‐4‐18F‐fluorobenzoate.

Introduction of a cyclic constraint may restrict the flexibility of a lead peptide and

may generate peptides with enhanced potency, receptor selectivity and enzymatic

stability.138 The concept of side chain to side chain cyclization of melanocortin peptides has

been successfully applied, namely in the case of Ac‐[Cys4,Cys10]‐α‐MSH, which contains a

disulfide bridge between Cys4 and Cys10 amino acids.138,149 Another very potent peptide

agonist, also used in the characterization of melanocortin receptors, is the cyclic analogue

melanotan II (MT‐II; table 1.8).138,150

To take advantage of the superior biological properties presented by the cyclic

peptides, a new family of Re‐ and Tc‐based cyclic α‐MSH analogues, MO[Cys3,4,10,D‐Phe7]‐α‐

MSH3‐13 [M‐CCMSH] (M = Re, 99mTc) has been developed for melanoma targeting.22,129,151 The

biological evaluation of the cyclic radioanalogues, 99mTc‐CCMSH (figure 1.5, section 1.1.2.)

and 111In‐DOTA‐ReCCMSH, has shown a significant tumor uptake but also a high kidney

accumulation. Substitution of Lys11 by Arg11 led to the compounds 99mTc‐CCMSH(Arg11) and 111In‐DOTA‐ReCCMSH(Arg11) (figure 1.20), which present higher tumor uptake and lower

1. Introduction

38

kidney accumulation. These compounds were considered as potential imaging probes for

primary and pulmonary metastatic melanoma detection.152,153 These analogues were also

labelled with therapeutic radionuclides (188Re and 212Pb) and their tumor uptake and

therapeutic efficacy were evaluated in melanoma models.21,132,154,155 For PET imaging of

melanoma, the cyclic ReCCMSH(Arg11) peptide was labelled with 64Cu, using DOTA and a

cross‐bridged cyclam (CBTE2A = 4,11‐bis(carboxymethyl)‐1,4,8,11‐

tetraazabicyclo[6.6.2]hexadecane) as bifunctional chelators (figure 1.20).156,157

Figure 1.20 ‐ Structures of DOTA‐ReCCMSH(Arg11) for labelling with 111In and 64Cu, and CBTE2A‐

ReCCMSH(Arg11) for labelling with 64Cu.

Chelators =

ReCCMSH(Arg11)

chelator

2.

Evaluation of Chelators for Labelling

Biologically Relevant Molecules

2. Evaluation of Chelators for Labelling Biologically Relevant Molecules

41

2. Evaluation of Chelators for Labelling Biologically

Relevant Molecules

2.1. Introduction

The main goal of this work was to contribute for the design of radioactive probes

potentially useful for imaging the N‐MYC mRNA and MC1 membrane receptors. To achieve

such goal we explored the labelling of a peptide nucleic acid sequence and of a cyclic α‐MSH

analogue with the fac‐[99mTc(CO)3]+ moiety. We have also conjugated an acridine orange

derivative to a bifunctional chelator to prepare isostructural rhenium and technetium

tricarbonyl compounds, to evaluate their localization in the cell and also their cytotoxicity

and radiocytotoxicity. In a later stage, and depending on the results, such complexes could

be interesting to explore multimodal probes as well as the therapeutic usefulness of 99mTc

complexes.

The 99mTc, more specifically the fac‐[99mTc(CO)3]+ moiety, was chosen due to its

versatility and excellent properties, in terms of stability in vitro and in vivo, being an

important core for application in radiopharmaceutical chemistry. The bifunctional chelators

selected were the pyrazolyl‐ and cysteine‐containing chelators which are N3 and N, O, S

donors, respectively (scheme 2.1).46‐52,56‐58 Both chelators contain a carboxylic group

available to conjugate a biomolecule and in the case of the pyrazolyl‐containing chelator the

4‐position of the pyrazolyl ring can also be derivatized with a fluorescent molecule (scheme

2.1).

Scheme 2.1 – Pyrazolyl‐ and cysteine‐containing bifunctional chelators for coordination to the metal

and conjugation to biomolecules (BM) and fluorescent molecule (FM).

SNH2

O

HO

O

OH

NN

NNH2

O

OH

FM

M

BM

BM

M


42

Once there was no experience on conjugating the cysteine‐ and pyrazolyl‐containing

chelators to PNAs, we decided to evaluate the possibility of conjugating PNA units

(monomer and dimer) to these chelators and to explore the coordination capability of the

conjugates towards the fac‐[M(CO)3]+ (M = Re, 99mTc) moiety. The evaluation of these model

complexes would be helpful for choosing the most suitable chelator to label the clinical

relevant PNA sequence with the fac‐[99mTc(CO)3]+ moiety.

In the first part of this chapter we describe the synthesis and characterization of a PNA

monomer and dimer, their conjugation to the bifunctional chelators, pyrazolyl‐ and cysteine‐

containing chelators, and reactions of these conjugates with the fac‐[M(OH2)3(CO)3]+ (M =

Re, 99mTc) precursors. The in vitro stability and the biological behaviour of the 99mTc

complexes formed will be also presented.

In the second part of this chapter we describe the synthesis of a pyrazolyl‐diamine

chelator bearing an acridine orange moiety as well as the synthesis of rhenium and

technetium tricarbonyl complexes. The cytotoxicity, radiotoxicity and localization of these

complexes in B16F1 murine melanoma cells will also be reported.

2.2. Evaluation of Two Chelators for Labelling PNA Units

2.2.1. Synthesis and Characterization of a PNA Monomer and Dimer

The synthesis of the classical aminoethylglycine PNA (aeg‐PNA) backbone protected with

Boc in the N‐terminus and with a methyl ester in the carboxylic function is illustrated in

scheme 2.2.

H2N NH2O

HN

ONH

O

O

OHN

ONH2

1 2

i ii

Scheme 2.2 ‐ Synthesis of the protected PNA backbone (2). i) Di‐terbuthyldicarbonate ((Boc)2), 1,4‐

dioxane; ii) methylbromoacetate, CH2Cl2.


43

One of the amino groups of ethylendiamine is selectively protected with tert‐

butoxycarbonyl (Boc), originating compound 1,158 which is alkylated with bromoacetic acid

ester yielding the aeg‐PNA backbone monomer 2.159 Then, the thymin‐1‐ylacetic acid is

attached to 2 via an amide bond affording the aeg‐PNA monomer 4 (scheme 2.3).160,161

NH

N

O

O

OHN

ON

O

Oi

OHN

ONH

O

O2

3

4

OH

O

NH

N

O

O

O

Scheme 2.3 ‐ Synthesis of the PNA monomer (4). i) DMF, DIPEA, HBTU.

The synthesis of the PNA dimer starts with the PNA monomers having the amine and

the acid groups in the free form. To achieve such goal, the PNA monomer 4 was

deprotected, using TFA or NaOH, yielding quantitatively compounds 5 (amine form) and 6

(acid form), respectively (scheme 2.4).161 By reacting compounds 5 and 6 in DMF, in the

presence of NEt3 and HBTU, the PNA dimer 7 was obtained (scheme 2.4) with 62% yield,

after purification by column chromatography (silica gel, MeOH 0 ‐ 10%)/CH2Cl2).


44

N

O

NH

O

NH

N

O

O

N

O

NH

O

O

NH

N

O

O

O

O

N

O

NH

O

NH

N

O

O

O

O

O

H3N

O

O

NH

N

O

O

OH

N

O

NH

O

NH

N

O

O

O

O O

CF3COO‐

4

5 6

7

i ii

iii

Scheme 2.4 ‐ Synthesis of a PNA Dimer (7). i) CH2Cl2, TFA; ii) NaOH 2N, H3O+; iii) NEt3, HBTU, DMF.

The compounds were characterized by the usual analytical techniques. As shown in

figures 2.1 and 2.2, 1H NMR spectra of the PNA monomer and of the dimer are relatively

complex, due to the presence of rotamers.

In solution, the PNA monomer exists as both the cis and trans rotamers, the trans

conformation being slightly favored, as revealed by nuclear magnetic resonance studies

(scheme 2.5).96,162,163,164,165 In the trans form, the methylene protons next to the nucleobase

(8’ in scheme 2.5) are toward the 2‐aminoethyl unit, while in the cis isomer those protons

are on the side of the glycyl unit. These rotamers appear in the NMR spectra due to the high

barriers of rotation and low rate of exchange around the tertiary amide bond (χ1 = C3’‐N4’‐C7’‐

C8’, torsion angle) of the PNA monomer (scheme 2.5). The two rotamers interconvert on the

∼1s time scale at room temperature. For longer sequences, the 1H‐NMR reveals the

presence of a multitude of structural species. If there are N residues, 2N possible structural

species can exist.96


45

NH

N

O O

B

NH

N

O

O

B

trans cis

1'

2'3'

4'

5'

7'8'

χ1

6'

Scheme 2.5 ‐ cis and trans PNA rotamers.

In figure 2.1 and figure 2.2, the 1H NMR spectra obtained for the protected PNA

monomer (4) and for the dimer (7) are presented. In the 1H NMR spectrum of the PNA

monomer, two resonances are observed with different intensities for the H6’ of thymine (T)

nucleobase, for the methylenic protons H1, H2, H6 and for the NHa (figure 2.1). The relative

intensity of the resonances confirmed the presence of a major (ma) species, which was

assigned to the trans isomer, according to what is described in the literature.162 As can be

seen in figure 2.2, the 1H NMR spectrum of the PNA dimer (7) is much more complex, due to

the presence of different rotamers in solution. In this case four different species are

expected because there are two monomers that can adopt two different conformations

each (2N = 22). For the H6’ and H6’’ of thymine, eight different resonances are observed,

confirming the presence of four different species in solution (figure 2.2). The methylenic

protons H1, H2, H5, H9, H9’ and the CH3 of the thymine base also present several resonances

being difficult to make their assignment (figure 2.2).


46

Figure 2.1 ‐ 1H NMR spectrum of PNA monomer 4 in CDCl3. • CH2Cl2, T ‐ thymine, S = solvent.

Figure 2.2 ‐ 1H NMR spectrum of PNA dimer 7 in CD3OD. ∗ Residual water, T‐thymine, S = solvent.

2.03.04.05.06.07.08.09.0 ppm

NH

N

O

NH

N

O

O

O

OO

O

123

45

5a

b

5

6'

6

NHb NHa

NHa6’

6’

S

•

2

2 6

6

1

1

3

4

CH3‐T 5

2.03.04.05.06.07.0

6’, 6’’

CH3‐T

8

1

2, 5, 9, 9’

3, 4, 6, 7

∗

S

N

O

NH

O

NH

N

O

O

N

O

NH

O

O

NH

N

O

O

O

O12356

478

88

9' 9

6'6''

7.207.307.407.50

ppm


47

Compound 7 was also characterized by ESI/QITMS that confirmed its formulation. In

the spectrum of 7, one group of peaks at m/z 663.2 (most abundant mass) is observed,

corresponding to the calculated value for [M‐H]‐ (figure 2.3).

Figure 2.3 ‐ Mass spectrum of compound 7 in the negative mode obtained by ESI/QITMS.

2.2.2. Synthesis and Characterization of Bifunctional Chelators Bearing

a PNA Monomer

The protected S‐(carboxymethyl‐pentafluorphenyl)‐N‐[(trifluor)carbonyl]‐L‐cysteine

methyl ester (cysteine‐containing ligand) (11) and 3,5‐Me2‐

pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (pyrazolyl diamine containing ligand) (Pz‐Boc)

bifunctional chelators were prepared to further conjugate the PNA monomer.

Compound 11 was prepared by a multistep synthetic procedure as illustrated in

scheme 2.6. Briefly, N‐(tert‐Butoxycarbonyl)‐L‐cysteine methyl ester was alkylated with tert‐

buthyl bromoacetate originating compound 9 that was deprotected with trifluoracetic acid

in CH2Cl2 yielding quantitatively compound 10. Compound 10 was protected in the amine

620 630 640 650 660 670 680 690 700m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

663.2

664.2

665.1

631.3

699.1649.3

666.1 701.1633.3 702.0651.3 662.4 667.1 686.1634.3 677.0621.4 648.6 698.4630.7 670.7 703.6637.1 694.7684.1

[M‐H]‐


48

function and activated in the acid position in a one step synthesis, using excess of

pentafluorphenol trifluoracetic acid in DMF and reacting overnight at room temperature.

After removing the solvent and washing with hexane, compound 11 was obtained as a white

solid.

HNS O

O

O

OO

SF3COCHN

O

OO

O

F

F

FF

F

NH2

S O

O

NH2

HS O

Oi iiiii

91110

OHO

CF3COO‐

5

H3NN

O

NH

N

O

O

O

O

13

HN N

O

NH

N

O

O

O

OHS

NH2

O

OHO

iv

v

12

HN N

O

NH

N

O

O

O

OS

NHCOCF3

O

OO

Scheme 2.6 ‐ Synthesis of compounds 9 ‐ 13. i) tert‐buthyl bromoacetate, NEt3, CH2Cl2; ii) TFA/CH2Cl2; iii)

pentafluorphenol trifluoracetic acid, DMF, pyridine; iv) CH3CN, NEt3; v) MeOH, K2CO3.

Conjugation of 11 to the PNA monomer 5 was performed in CH3CN in the presence of

NEt3. Compound 12 was isolated in 25% yield after purification by column chromatography

(silica gel, (MeOH (3 ‐ 7%)/CHCl3). The cysteine‐conjugate 13 was then obtained in 74% yield

after removing the protecting groups with potassium carbonate (scheme 2.6).

The 3,5‐Me2‐pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (Pz‐Boc) was synthesized as

previously described56 and conjugated to compound 5 in CH3CN, in the presence of NEt3 and

HBTU. The conjugate 14 was obtained in 70% yield after purification by column

chromatography (MeOH (5 ‐ 13%)/CHCl3). Compound 15 was obtained in an overall yield of

50% after removing the Boc and the ester protecting groups with TFA and K2CO3,

respectively (scheme 2.7).


49

i

NH

N

O

NH

N

O

O

O

OH

NN NNH2

O

NH

N

O

NH

N

O

O

O

O

NN NNHBoc

O

1514

NN N NHBoc

O

OH

CF3COO‐

5

pz‐Boc

H3NN

O

NH

N

O

O

O

Oii

iii

Scheme 2.7 ‐ Synthesis of the conjugate 15. i) CH3CN, NEt3, HBTU; ii) CH2Cl2, TFA; iii) MeOH, K2CO3.

Compounds 12 ‐ 15 are air stable, soluble in polar organic solvents such as

dichloromethane, chloroform and methanol, being compounds 13 and 15 also soluble in

water. These compounds were characterized by multinuclear NMR spectroscopy (1H, 13C, 19F), RP‐HPLC and elemental analysis in the case of 15. To assign the 1H and 13C NMR

resonances, 2D experiments, 1H‐1H g‐COSY and 1H‐13C g‐HSQC, were performed.

As an example, in figure 2.4 and figure 2.5 are presented the 1H and 13C NMR spectra

of the cysteine conjugate 12 in dmso‐d6.

Figure 2.4 ‐ 1H NMR spectrum of compound 12 in dmso‐d6. S = solvent, * residual water.

2.03.04.05.06.07.08.09.010.011.0

3b

3a

S

CH3‐T

10.00

NHb

NHc

6’’

7

7

9

2

9

1,8

5,6

4

NHa

*

NHc 6’’

ppm

HN N

O

NH

N

O

O

O

OS

NH

O

OO

1 23 4 5

6''

76

9

8

a

c

b

O

FFF


50

Figure 2.5 ‐ 13C NMR spectrum of compound 12 in dmso‐d6. S = solvent

In the 1H NMR spectrum (figure 2.4) we can identify three different NH resonances,

one singlet at 11.29 ppm attributed to the NHa of the thymine nucleobase, one doublet at

10.03 ppm corresponding to the NHb of the cysteine and two triplets at 8.20 (ma) and 8.06

ppm (mi) corresponding to the NHc of the amide bond between the cysteine and the PNA

monomer. The presence of these two triplets at 8.20 and 8.06 ppm confirms the formation

of the conjugate and were attributed to the trans and cis rotamers, respectively. These two

resonances appear as triplets due to the coupling of NHc with the H5 protons of the PNA

monomer. The H2 of the cysteine unit was identified based on 1H‐1H g‐COSY. In fact, this

proton is coupled with NHb and with the diastereotopic protons H3a and H3b, which appear as

separated multiplets at 3.13 and 2.91 ppm. The H4 protons are easily identified because they

appear as a multiplet at 3.17 ppm at higher field than the H3 protons and are not coupled

with any other protons. Another characteristic of this spectrum is the presence of two

resonances for the H6’’, H7, H8 and H9 due to the presence of two rotamers in solution.

The attribution of the different resonances in the 13C NMR spectrum of compound 12

(figure 2.5) was based on 1H‐13C g‐HSQC. The diastereotopic character of the H3a and H3b was

confirmed in the g‐HSQC spectrum by the correlation between these protons and one

resonance at 32.2 ppm corresponding to C3.

102030405060708090100110120130140150160170

1’, 4’, 7’

9’ 4’’

2’

5’’

3’

2’’ 5

6’’ 1’,8,2

7, 9, 6

3

CH3‐T

4

ppm

S

HN N

O

NH

N

O

O

O

OS

NH

O

OO

1 23 4 5

2''

4''5''

6''

76

9

81'

3' 2'

4'9'

a

c7'

b

O

FF

F


51

The 1H and 13C‐NMR spectra obtained for the pyrazolyl‐conjugate 15 in CD3OD

present the expected resonances, which have also been assigned based on g‐COSY and g‐

HSQC experiences.

In figure 2.6 is shown the 1H NMR spectrum of compound 15 with the corresponding

assignments. The spectrum presents one singlet at 5.92 ppm and two resonances at 2.29

and 2.19 ppm attributed to the H4’ and the methyl groups of the pyrazolyl ring, respectively.

The aliphatic protons of the pyrazolyl‐diamine chelator H1, H6 and H7 appear as

diastereotopic protons. As can be observed in the spectrum, the H7 protons appear as two

triplets at 2.51 and 2.35 ppm, the H6 protons that should appear as a quintuplet appear as a

multiplet (mixture of two quintuplets) at 1.97 ppm, and the H1 as a multiplet (two close

triplets) at 4.40 ppm. It should also be referred that the methyl groups do not appear as

singlets but appear as doublets, maybe due to some intra‐ or intermolecular interactions in

solution. The presence of two rotamers in solution can also be clearly assigned based on the

H6’’, H10 and H11 protons of the PNA monomer unit.

Figure 2.6 ‐ 1H NMR spectrum of compound 15 in CD3OD. S = solvent, * residual water.

2.03.04.05.06.07.0

6’’

4’ 10

1

2,3,4,8,9

1110

11

5

3’, 5’ (CH3‐pz)

CH3‐T

6 7b

*

S

ppm

NH

N

O

NH

N

O

O

O

OH

NN NNH2

O

1

2 3

45 6

78

910

11

3' 5'4'

6''

7a


52

2.2.3. Synthesis and Characterization of a Bifunctional Chelator Bearing

a PNA Dimer

After coupling the pyrazolyl‐diamine chelator (Pz‐Boc) to the PNA monomer (5), in a

high yield, we decided to evaluate the possibility of conjugating also a PNA dimer to the

same chelator. For that, the Boc protecting group of the PNA dimer 7 was removed with TFA

and the compound obtained (8) was conjugated to 3,5‐Me2‐

pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (Pz‐Boc) in DMF, in the presence of NEt3 and HBTU

(scheme 2.8). Compound 16 was obtained in 75% yield after purification by RP‐HPLC. After

removing the Boc and ester protecting groups of 16 and purification by RP‐HPLC, conjugate

17 was obtained. Compounds 16 and 17 were characterized by NMR (1H, 13C, g‐COSY, g‐

HSQC), RP‐HPLC, elemental analysis and, in the case of 17, also by ESI/QITMS.

NO

H3NO

NH

N

O

O

NO

NH

O

O

NH

N

O

O NO

NH

O

HN

N

O

O

NO

NH

OO

HN

N

O

O

NN N NH

O

O

ONO

NH

O

HN

N

O

O

NO

NH

OOH

HN

N

O

O

NN N NH2

O

CF3COO‐ ii

8 16 17i

NN N NHBoc

O

OH

pz‐Boc

iii

Scheme 2.8 ‐ Synthesis of 17. i) NEt3, HBTU, DMF; ii) CH2Cl2, TFA; iii) K2CO3, MeOH.

As expected, the 1H and 13C NMR spectra of 16 and 17 in CD3OD are relatively

complex due to the presence of different rotamers.

From all the resonances found in the 1H NMR spectra of these compounds, the one

which gives more information about the number and intensity ratio of rotamers in solution

are the H6’’ and H6’’’ of the thymine nucleobases. As can be seen in figure 2.7, eight

resonances for the H6’’ and H6’’’ are observed, indicating the presence of four different

rotamers in solution. The presence of different rotamers are also identified by the different

resonances found for the CH3 of the thymine and the methylenic protons H10, H13, H14 and

H14’ of the PNA dimer unit (figure 2.7). This spectrum is quite complex and the assignment

shown in figure 2.7 was based on the 1H‐1H g‐COSY.


53

Figure 2.7 ‐ 1H NMR spectrum of compound 17 in CD3OD. * Residual water, S = solvent.

The formulation of compound 17 was also based on ESI/QITMS. In the spectrum, two

main groups of peaks were found at m/z 801.7 (most abundant m/z) and m/z 823.6 (most

abundant m/z), corresponding to [M+H]+ and [M+Na]+, respectively (figure 2.8).

Figure 2.8 ‐ Mass spectrum of compound 17 in the positive mode obtained by ESI/QITMS.

801.71+

802.61+

803.61+

804.61+

823.61+

824.61+

825.71+

800 805 810 815 820 825 m/z0.0

0.5

1.0

1.5

7x10Intens.

[M+H]+

[M+Na]+

2.03.04.05.06.07.0

6’’, 6’’’

4’

10,13,14,14’

2,3,4,8,9, 11,12

*

5 6 7

S

CH3‐T 3’, 5’ (CH3‐pz)

1 7.107.207.307.407.5060

ppm

N

O

NH

O

NH

N

O

O

N

O

NH

O

OH

NH

N

O

O

NN N

NH2

O

1

2 3

45 6

78

9 10

11

3' 5'4'

6'''

12 13

1414'

6''


54

2.2.4. Synthesis and Characterization of Rhenium Tricarbonyl

Complexes

The coordination capability of the BFCA‐PNA conjugates (13, 15 and 17) towards the

fac‐[Re(CO)3]+ moiety was evaluated. Compounds 13, 15 and 17 reacted with equimolar

amounts of the precursor fac‐[Re(CO)3(OH2)3]Br,166 in refluxing methanol for 20 h (15 and

17) or in water at 75 °C for 5 h (13) (scheme 2.9). After purification, by preparative RP‐HPLC

(TFA 0.1% in H2O/MeOH), complexes fac‐[Re(CO)3(κ3‐13)] (18) and fac‐[Re(CO)3(κ3‐L)]+ (L =

15 (19); L = 17 (20)) were obtained as white microcrystalline solids with high purity (> 95%).

ReOC

H2O OH2

CO

OH2

CO

NN N

NH2

Re

OC CO CO

+

OSNH2

O

O

Re

OC CO CO

O

i

NH

N

O

NH

N

O

O

O

OH

NH

N

O

NH

N

O

O

O

OH

15 13

17

NN N

NH2

Re

OC CO CO

+

O

NH

N

O

NH

N

O

O

ONH

N

O

NH

N

O

O

O

OH

1819

20

i

ii

Scheme 2.9 ‐ Synthesis of the rhenium complexes 18, 19 and 20. i) CH3OH, reflux, 20 h; ii) H2O, 75 °C, 5 h.

After purification, compounds 18, 19 and 20 were obtained in 70%, 20% and 43%

yield, respectively.

Compounds 19 and 20 were obtained in relatively low yield due to the formation of

side products, as indicated by the HPLC of the crude. Such side products appear in the HPLC


55

chromatograms with retention times higher than the retention times of 19 and 20. The

formation of these products may be explained by hydrolysis of the amide function between

the pyrazolyl‐diamine chelator and the PNA unit. However, a complete characterization of

such species has not been performed.

Compounds 18, 19 and 20 are soluble in water and in most common polar organic

solvents. These complexes are also stable towards air oxidation. The characterization of 18,

19 and 20 involved the usual spectroscopic techniques (IR, 1H, 13C NMR and 2D NMR

experiences), ESI/QITMS, elemental analysis and RP‐HPLC.

The ESI/QITMS spectra of 18, 19 and 20 present prominent peaks at m/z values which

are in agreement with the expected for those complexes. The isotopic patterns of the peaks

were also in agreement with the presence of rhenium. As an example, in figure 2.9 is

presented the mass spectrum of 18, where a group of peaks at m/z 713.9 (most abundant

m/z) is shown, with the expected isotopic distribution for [M‐H]‐ (C19H21N5O11SRe).

Figure 2.9 ‐ Mass spectrum of compound 18 in the negative mode obtained by ESI/QITMS.

In the IR spectra of 18, 19 and 20, two strong bands appear in the range 1912 ‐ 2039

cm‐1 which can be assigned to the ν(CO) stretching bands of the CO ligands of the fac‐

[M‐H]‐


56

[Re(CO)3]+ moiety.46‐52, 56‐58 The IR spectra of these compounds also show medium to strong

bands in the 1678 ‐ 1673 cm‐1 range. These bands were assigned to the υ(CO) stretching

mode associated to the carbonyl groups of compounds 18, 19 and 20.

Figure 2.10 ‐ IR spectra of compounds 18, 19 and 20 (KBr).

No X‐ray structure was obtained for 19 and 20 and the 1H, 13C and 2D (g‐COSY, g‐

HSQC) NMR studies were essential for the characterization of the complexes. Based on the

splitting of some resonances, the tridentate coordination mode of the pyrazolyl‐diamine

19

20

18

υ(C≡O) υ(C=O)


57

conjugates was clear. As an example, it is shown in figure 2.11 and figure 2.12 how the 1H‐1H

g‐COSY for 19 has been used for the characterization of the complex.

Figure 2.11 ‐ 1H‐1H g‐COSY spectrum of complex 19 in CD3OD and respective attributions. * Residual

water, S = solvent. Protons X = 1 ‐2 and 4 ‐7 are diastereotopic and are assigned as Hxa and Hxb.

H(4)‐pz

H(6’’)‐T

NH

NH

CH3‐T

3

10

10

11

11

*

CH3‐pz

5a

2b

1a

8,9,2a

1b5b

4a

4b

7a

7b

6b

S

H(4)‐pz CH3‐pz

H(6’’)‐TCH3‐T

NH 4b

NH 4a

NH NH

6a

NN N

NH2

Re

CO CO CO

O

NHN

O

NH

N

O

O

O

11

12 3 4

5 6

78

910

6''

OH

+

H(4)‐pz

Expansion in figure 2.12

H(6’’)‐ CH3‐T

H(4)‐pzCH3‐pz

NH 4b

NH NHNH 4a


58

Figure 2.12 ‐ Expansion of the 1H‐1H g‐COSY spectrum of complex 19 in the range 5 ‐ 1.2 ppm.

In the 1H NMR spectra of 19 (figure 2.11) and 20, the pattern obtained for the H6’’,

methyl groups of the thymine nucleobase and the methylenic protons of the PNA

monomer/dimer compares well with the pattern found for the free conjugates (15, 17),

being consistent with the presence of rotamers. The assignment of the H(4) proton and the

methyl groups of the pyrazolyl ring was also very clear. The H(4) and the methyl protons

NH

CH3‐T

3

10

10

11

11

* CH3‐pz

5a

2b

1a

8,9,2a

1b5b

4a

4b

7a

7b

6b

S

6a

1b 2b

1a 2a

1a 1b

5a 6b

5b 6a

2a 2b

4a 3

4b 3

4a 4b 6b 7b

7a 7b

7a 6a

NN N

NH2

Re

CO CO CO

O

NHN

O

NH

N

O

O

O

11

12 3 4

5 6

78

910

6''

OH

+

H(4)‐pz


59

(Me) of the pyrazolyl ring are significantly shifted downfield (H(4), Δ= 0.28 ppm; Me, Δ =

0.15 ppm) relatively to the same resonances in the corresponding free conjugates (15 (figure

2.6) or 17 (figure 2.7)), indicating coordination to the metal center. The tridentate

coordination mode of the pyrazolyl‐diamine conjugate was confirmed by the splitting of the

NH2 protons of the primary amine, as well as by the splitting of the H1, H2 and H4 protons.

For each of these protons, two resonances assigned as Hxa and Hxb (x = 1, 2 and 4),

integrating for one proton each, were found, due to the diastereotopic character of the

resonances after coordination to the metal center. The assignment of all the other

resonances of 19 and 20 was also mainly based on 2D NMR studies.

An important characteristic of the 13C spectra of compounds 19 and 20 is the

presence of three resonances for the CO ligands (C≡O, 195.2, 194.9 and 193.8 ppm for 19;

194.9, 194.6 and 193.5 ppm for 20) coordinated facially to rhenium. The assignment of the 13C resonances was based on 1H‐13C g‐HSQC. The most important characteristic of the

spectra was the correlation between the geminal diastereotopic protons and the respective

carbons.

The formulation of complex 18 was mainly based on ESI/QITMS and elemental

analyses of the HPLC purified product. In fact, the 1H (figure 2.13) and 13C NMR spectra of 18

were very complex and the assignment was very difficult, mainly in the region where the

protons of the cysteine fragment may appear.

As shown in scheme 2.9, we have considered that conjugate 13 coordinates to the

metal through the terminal carboxylic acid, the primary amine and the sulfur atom.

However, in this conjugate there are other potential coordination sites on the cysteine

fragment. We do not have data in the solid state or in solution to support completely such

proposal. However, the affinity of the fac‐[M(CO)3]+ moiety for cysteine and cysteine‐

containing ligands are well documented and recognized.43,46‐52 Any other possibility would

require coordination of the amide groups to the metal fragment and this would certainly

lead to a less stable complex.

The complexity of the NMR spectrum was certainly due to the presence of different

species in solution, as indicated by the number of signals due to the H6’’ proton of the

thymine base. As can be seen in figure 2.13, at least three resonances with different


60

intensities appear at 7.31, 7.26 and 7.22 ppm. Also, the presence of different resonances for

the NH protons of the cysteine chelator indicates the presence of different species in

solution (figure 2.13). These species can most probably be rotamers and diastereoisomers.

The formation of diastereoisomers upon coordination of the cysteine chelator is clearly

possible because the sulfur atom has two lone pairs of electrons and the cysteine chelator

has a chiral center. Two isomers can equilibrate by inversion of the sulfur configuration. The

presence of diastereoisomers in rhenium tricarbonyl complexes with cysteine derivatives has

been observed and studied by others.46,51,52

The presence of two peaks in the HPLC chromatogram of 18, with retention times of

19.90 and 19.70 min, may be related to the presence of two diastereoisomers which do not

interconvert in solution at room temperature (figure 2.14).

Figure 2.13 ‐ 1H NMR spectrum of compound 18 in CD3OD. S = solvent, *residual water.

The tertiary amine of the pyrazolyl‐diamine chelator is also a pro‐chiral center, but in

complexes 19 and 20 we did not observe isomers, probably because there is a fast

interchange between them. There is a long experience with this chelator conjugated or not

2.03.04.05.06.07.0

6’’

NH NH NH

NH

67

4, 5

CH3‐T

S*

OSNH2

O

O

Re

OC CO CO

NH

N

O

NH

N

O

O

O

OH12 3

5

6''

7

6

4

ppm


61

to different biomolecules and only recently, in a complex bearing guanidine derivatives, the

presence of isomers was observed.167

2.2.5. Synthesis, Characterization and Biological Behavior of 99mTc

Tricarbonyl Complexes

The coordination capability of 13, 15 and 17 with 99mTc(I) was evaluated by reacting

the conjugates with the aquo complex fac‐[99mTc(CO)3(OH2)3]+ (scheme 2.10).

Tc

OC

H2O OH2

CO

OH2

CO

NN N

NH2

Tc

OC CO CO

OSNH2

O

O

Tc

OC CO CO

O

NH

N

O

NH

N

O

O

O

OH

NH

N

O

NH

N

O

O

O

OH

15 13

17

NN N

NH2

Tc

OC CO CO

O

NH

N

O

NH

N

O

O

ONH

N

O

NH

N

O

O

O

OH

2122

23

99m

99m99m

99m

+

+

Scheme 2.10 ‐ Synthesis of 99mTc complexes 21, 22 and 23.

The precursor fac‐[99mTc(CO)3(H2O)3]+ was obtained quantitatively by adding a

solution of Na[99mTcO4] to an IsoLink® Kit and heating at 100 °C for 30 minutes. After cooling,

the pH was adjusted (∼7.4) with HCl 1N, to destroy the remaining boranocarbonate. The

radiochemical purity of the precursor was controlled by RP‐HPLC.


62

The precursor fac‐[99mTc(OH2)3(CO)3]+ reacted then with a solution of 13, 15 or 17

(10‐4 M) in water, in a total volume of 1 mL. The experimental conditions were optimized in

terms of reaction time and temperature.

The concentration of 99mTc in these reactions is very low (10‐9 M ‐ 10‐7 M), so the

characterization of the 99mTc compounds are made by comparing their HPLC profiles with

those of the corresponding Re(I) tricarbonyl complexes, which were synthesized and

characterized at the macroscopic level. As can be observed in figure 2.14, based on HPLC we

confirmed that the 99mTc complexes corresponded to the expected compounds, which

means they had the same structure as the rhenium congeners, being formulated as fac‐

[99mTc(CO)3(κ3‐13)] (21) and fac‐[99mTc(CO)3(κ3‐L)]+ (L = 15 (22), 17 (23)).

The small differences found in the retention times of the Re and the corresponding 99mTc complexes are due to the detection mode of those complexes. For Re complexes a UV‐

vis detector is used and for 99mTc complexes a γ detector. These two detectors are connected

in line, which justifies the small differences found in the retention times.

Figure 2.14 ‐ Analytical RP‐HPLC chromatograms of Re complexes 18 (a), 19 (c) and 20 (e) (254 nm),

and corresponding 99mTc complexes 21 (b), 22 (d) and 23 (f) (γ trace).

In table 2.1 are shown the experimental conditions used for the preparation of 21 ‐

23, the yield of the reactions and the retention time of the 99mTc and Re complexes.

10 15 20 25 10 15 20 25 10 15 20 25

a

b

c

df

e

Time (min) Time (min) Time (min)


63

Table 2.1 ‐ Experimental conditions used to synthesize complexes 21 ‐ 23 and respective retention

times.

Complex

(conjugate)

Yield

(%)

[conjugate]

(M)

Time

(min)

Temperature

(°C)

Retention time

(min)a

21 (13) > 97 10‐4 45 75 19.9, 20.1 (19.7, 19.9)

22 (15) > 99 10‐4 45 100 23 (22.4)

23 (17) >99 10‐4 45 100 23 (22.5) a The values in parentheses refer to the Re complexes.

Using the experimental conditions indicated in table 2.1, all complexes were

obtained in almost quantitative yield, using relatively low final concentrations of the

conjugates (13, 15, 17), which means that 21 ‐ 23 were obtained with high specific activity.

2.2.5.1. Stability in the Presence of Cysteine and Histidine

The in vitro stability studies can predict the stability of the 99mTc complexes in vivo,

towards reoxidation and transquelation of the conjugates (BFCA‐PNA units) by biological

substrates with affinity to the metal. These biological substrates can be proteins, amino

acids or other molecules present in vivo.

For complexes 21 and 23, the in vitro stability was evaluated in the presence of

cysteine and histidine, two amino acids present in vivo, with known affinity for the fac‐

[99mTc(CO)3]+.39‐41,46‐52

The organometallic 99mTc complexes 21 and 23 were incubated with a large excess of

the two amino acids (amino acid:conjugates 13 or 17 = 100:1) under physiological conditions

(37 °C, phosphate buffer saline pH 7.4). After 1, 2, 4 and 6 h of incubation, aliquots of the

solutions were collected and analysed by RP‐HPLC.

As can be seen in figure 2.15, no significant ligand exchange and/or decomposition

were observed 6 h after incubation. For 23, 6 h after incubation with histidine or cysteine,

the percentage of intact complex is higher than 93%. For 21, the transquelation with

histidine and cysteine is slightly higher but not significant, as 90% of the complex remains

intact after 6 h of incubation. Also, no reoxidation of the complexes to [99mTcO4]‐ was


64

observed. These findings indicate that 21 and 23 have a high kinetic inertness and were

promising to be studied in vivo.

0

20

40

60

80

100

0 2 4 6

incubation time/h

% com

plex 21

cysteine

histidine

0

20

40

60

80

100

0 2 4 6

Incubation time/h

% com

plex 23

cysteinehistidine

Figure 2.15 ‐ Stability of 21 and 23 in the presence of excess of histidine and cysteine (37 °C, PBS pH

7.4).

These studies were relatively important for compound 21, as the coordination of the

cysteine conjugate (13) to the fac‐[M(CO)3]+ (M=Re, 99mTc) moiety was ambiguous. If the

coordination of 13 was not through the cysteine chelator (ONS) but through other

coordinating groups existing in the molecule (e.g. amide groups), a much lower stability for

complex 21 would be expected in the presence of histidine and cysteine. Since the Re (18)

and 99mTc (21) complexes have the same chemical structure (figure 2.14), these studies were

helpful to characterize both, reinforcing the fact that conjugate 13 is most probably

coordinated by the cysteine chelator to the Re and 99mTc metals.


65

2.2.5.2. Biodistribution and In Vivo Stability

Biodistribution

The biological behavior of 21 and 23 was studied in mice, to evaluate their

pharmacokinetics and in vivo stability. In particular, we were interested in the evaluation of

blood clearance, route of elimination and preferential retention in some organs.

In general, the blood clearance must be fast enough to avoid long exposure to

radiation, and slow enough to allow the radioactive compound to reach the target site. The

best route of elimination would be renal, as it is normally the fastest.

The pharmacokinetic studies of 21 and 23 were performed in CD‐1 Charles River

female mice. The mice were intravenously injected with 100 μl of each preparation via the

tail vein. At 1 and 2 h post‐injection (p.i.), mice were sacrificed by cervical dislocation and

tissues and organs were removed, weighted and their activity measured. Then, the

percentage of injected dose per gram (% ID/g) (table 2.2) or percentage of injected dose per

organ (% ID/organ) was calculated. The difference between the radioactivity injected and

that in the sacrificed animals is assumed to be the excretion. For blood, bone and muscle,

the activity is estimated assuming that it is 6, 10 and 40% of the total body weight,

respectively. The biological profile for both complexes was comparable. A relatively fast

blood clearance was found (1.65 ± 0.26 and 0.50 ± 0.03 % ID/g at 1 h p.i., for 21 and 23,

respectively), and 1 h after injection a fast distribution has taken place (table 2.2). There was

no significant uptake in the stomach (table 2.2), indicating a high stability of the complexes

to reoxidation. No preferential uptake in the main organs or tissues was observed, except in

the liver and intestine.


66

Table 2.2 ‐ Biodistribution results of the 99mTc compounds 21 and 23 at 1 and 4 h after intravenous

injection (mean ± standard deviation, n = 3).

% ID/g ± standard deviation

fac‐[99mTc(CO)3(κ3‐13)] (21) fac‐[99mTc(CO)3(κ

3‐17)]+ (23) Tissue/organ

1h 4h 1h 4h

Blood 1.65 ± 0.26 1.24 ± 0.50 0.50 ± 0.03 0.27 ± 0.01

Liver 4.46 ± 0.71 4.87 ± 0.66 5.85 ± 1.82 3.57 ± 0.89

Intestine 9.30 ± 0.83 14.16 ± 2.61 13.41 ± 0.30 17.49 ± 1.44

Spleen 0.90 ± 0.50 1.39 ± 1.30 1.53 ± 0.53 1.74 ± 0.52

Heart 0.37 ± 0.09 0.24 ± 0.04 0.24 ± 0.11 0.16 ± 0.01

Lung 0.90 ± 0.29 0.62 ± 0.22 0.55 ± 0.14 0.46 ± 0.13

Kidney 3.72 ± 0.74 3.29 ± 0.04 1.34 ± 0.01 0.94 ± 0.02

Muscle 0.10 ± 0.02 0.09 ± 0.02 0.06 ± 0.01 0.03 ± 0.01

Bone 0.20 ± 0.02 0.19 ± 0.06 0.11 ± 0.02 0.08 ± 0.01

Stomach 0.85 ± 0.26 0.52 ± 0.11 1.53 ± 0.19 0.44 ± 0.21

Pancreas 0.32 ± 0.07 0.22 ± 0.05 0.20 ± 0.05 0.08 ± 0.01

Excretion (% ID) 69.5 ± 1.4 67.6 ± 3.5 61.2 ± 4.8 64.4 ± 2.8

In order to compare the excretion of 21 and 23, figure 2.16 shows the activity

present in the liver, intestine and kidney, as well as the total excretion 4 h post‐injection. The

results are expressed as % of injected dose per organ (% ID/organ).

0

20

40

60

80

100

% ID

/organ

1 4 1 4

KidneyLiver

IntestineExcretion

time/h

21 23

Figure 2.16 ‐ Biological data for complexes 21 and 23

(% ID)


67

As can be seen in figure 2.16, the overall radioactivity elimination occurred mainly via

the renal‐urinary pathway for both complexes, as indicated by the percentage of total

excretion for compound 21 (69.5 ± 1.4 %) and for compound 23 (61.2 ± 4.8 %), at 1 h after

injection. Nevertheless, some part of the remaining radioactivity was retained in the liver

(4.81 ± 1.04 and 5.57 ± 1.51 % ID/organ 1 h p.i. for 21 and 23, respectively) and intestine

(16.03 ± 0.77 and 24.05 ± 5.28 % ID/organ 1 h p.i for 21 and 23, respectively), indicating that

these complexes are also eliminated via the hepatobiliary pathway.

In vivo Stability Studies

To verify the in vivo stability of 21 and 23, serum and urine were also analyzed by

radiometric HPLC. The murine serum, isolated from blood collected 1h after administration

of the complexes, was treated with ethanol to precipitate the proteins. Analysis of the

supernatant by RP‐HPLC showed no pertechnetate or other signal of decomposition

products, being complexes 21 or 23 the only species present. The urine collected at 1 h post‐

injection also did not show any metabolites, demonstrating the in vivo stability of the

complexes. As an example, the HPLC chromatograms of biological samples collected from

mice injected with 23 are shown in figure 2.17.

Figure 2.17 ‐ Analytical RP‐HPLC chromatograms of complex 23, urine and blood serum samples

collected 1 h after injection (γ trace).

0 5 10 15 20 25

Urine

Serum

Initial preparation

Time (min)


68

Summarizing, to evaluate the possibility of labeling with fac‐[99mTc(CO)3]+ a clinically

relevant PNA sequence, we have isolated and characterized two potentially tridentate

chelators bearing PNA units (13, 15 and 17). Compounds 13, 15 and 17 react with fac‐

[M(CO)3(OH2)3]+ yielding the complexes fac‐[M(CO)3(κ3‐13)] (M = Re (18) and 99mTc (21)) and

fac‐[M(CO)3(κ3‐L)]+ (L = 15 (M = Re (19), 99mTc (22)); L = 17 (M = Re (20), 99mTc (23)). For the

Re complexes 19 and 20 NMR studies have been very informative and a complete

assignment of the resonances was possible using different techniques. The tridentate

coordination mode of the pyrazolyl‐containing ligand was clear and also the presence of

rotamers in solution. For 18 the NMR data was more complex and not very informative,

possibly due to the presence of diastereoisomers and rotamers in solution, which do not

interconvert at room temperature. In fact, in the HPLC chromatograms of 18 and 21 two

peaks were observed corresponding, most probably, to the diastereoisomers that do not

interconvert at room temperature. The 99mTc tricarbonyl model complexes 21, 22 and 23

were prepared in high yield and high radiochemical purity. Complexes 21 and 23 are stable

against cysteine and histidine exchange. The biological profiles of 21 and 23 indicate that

they were rapidly cleared from blood and other main organs into urine, being the major

excretory route the renal‐urinary pathway.

The formation of stable model complexes in high yield, together with the favorable

pharmacokinetic profile of complexes 21 and 23, encourage the use of cysteine‐ and

pyrazolyl‐containing chelators for labeling with fac‐[99mTc(CO)3]+ a PNA sequence

complementary to the N‐MYC mRNA, to be evaluated as an antisense nuclear imaging probe.


69

2.3. Synthesis of Tricarbonyl Complexes Bearing Acridine Orange

2.3.1. Synthesis and Characterization of a Pyrazolyl‐Diamine Acridine

Orange Conjugate

As mentioned before, the bifunctional chelators pyrazolyl‐diamine are powerful

tridentate chelators towards the fac‐[M(CO)3]+ (M = Re, 99mTc) moiety, forming tricarbonyl

complexes with excellent in vitro and in vivo stability.56‐58 Due to their versatile nature, these

chelators can be easily conjugated to biologically active molecules and/or to fluorescent

moieties through different positions of their framework. In this work we have used the 4‐

position of the pyrazolyl ring to conjugate an acridine orange moiety, while keeping the

central amine for later coupling of a biologically relevant molecule.

For the incorporation of the acridine orange fragment at the 4‐position of the

pyrazolyl ring it was necessary to prepare the compound 4‐(HOOCCH2)‐3,5‐

Me2pz(CH2)NH(CH2)2NHBoc, functionalized at the 4‐position of the pyrazolyl ring with an

acid group, and also the amine derivative of acridine orange.

Synthesis of the amine derivative of acridine orange (26)

The synthetic procedure used to prepare the acridine orange amine derivative 26 is

shown in scheme 2.11. The first step involved the alkylation of the commercial acridine

orange with N‐(4‐bromobutyl)‐phthalimide (2‐(4‐bromobutyl) isoindoline‐1,3‐dione) (24), in

p‐xylol and reflux for 24 h. The mixture was then filtered and the collected solid washed

with acetone yielding a red compound which was formulated as 25. Then, compound 25

reacted with excess of hydrazine in ethanol to cleave the phthalimide group. After adequate

work‐up, compound 26 was quantitatively obtained as a red solid.


70

H2N NH2NN N NN N

H2N

NN Ni

N

O

OBr

N OO

ii

24

25 26

Scheme 2.11 ‐ Synthesis of compound 26. i) p‐xylol, reflux 24 h; ii) methanol, room temperature.

Synthesis of compound 4‐(HOOCCH2)‐3,5‐Me2pz(CH2)NH(CH2)2NHBoc (31)

The bifunctional chelator 4‐(HOOCCH2)‐3,5‐Me2pz(CH2)NH(CH2)2NHBoc (31) was

prepared by a multi‐step synthetic procedure which is depicted in scheme 2.12. This

procedure followed one previously described, although with some modifications.168 A key

step in this synthesis is the preparation of the 4‐(EtOOCCH2)Me2pz(CH2)2OH (29) (scheme

2.12). Compound 29 was obtained by a cyclization reaction between a diketone compound

(27) and 2‐hydroxyethylhydrazine, following a previously described method.169 Compound

31 was then obtained by a Mitsunobu reaction,170 i.e., by reacting compound 29, in excess,

with NH(DNS)(CH2)2NHBoc (28)171 in THF, in the presence of diethylazodicarboxylate (DEAD)

and triphenilphosphine (PPh3), at room temperature and overnight. The solvent was

removed under vacuum and the obtained solid was purified by column chromatography in

silica gel (ethyl acetate (30 ‐ 100%)/hexane). Then, the protecting groups 2,4‐

dinitrobenzenosulfonilo (DNS) and ester were removed from 30 with K2CO3 in the presence

of H2O/MeOH, by overnight reaction at room temperature. After evaporation of the solvent,

the residue was purified by column chromatography (MeOH) yielding compound 31 as oil.


71

NN NNHBoc

O O

+

NN OH

O O

NO2

SO

O HN

NHBoc

NO2

SO O

NNHN

NHBoc

O OH

OO

OO

27

iHN

H2N OH

NHBocH2N

O2NS OOCl

1

O2N

NO2

NO2ii

iii

iv

29 28

30

31

Scheme 2.12 ‐ Synthesis of compound 31. i) Ethanol, 0 °C, overnight; ii ) CH2Cl2, pyridine; iii) DEAD, PPh3,

THF; iv) H2O/MeOH, K2CO3.

All the intermediates of these reactions were characterized by NMR spectroscopy. As

an example, in figure 2.18 is shown the 1H NMR spectrum of compound 31. The spectrum

presents one singlet at 3.08 ppm attributed to the H6, two resonances at 2.00 and 1.93 ppm

assigned to the methyl groups of the pyrazol ring, four triplets (3.97 ‐ 2.55 ppm) due to the

aliphatic chain and one singlet at 1.24 ppm due to the protons of the Boc protecting group.


72

Figure 2.18 ‐ 1H NMR spectrum of compound 31 in D2O. * Solvent, • impurity.

Coupling of the acridine orange derivative 26 to the pyrazolyl‐diamine 31

The conjugation of the acridine orange derivative 26 to compound 4‐(HOOCCH2)‐3,5‐

Me2pz(CH2)NH(CH2)2NHBoc (31) was performed in DMF, in the presence of NEt3 and using

HBTU as activating agent (scheme 2.13). The mixture was stirred for 24 h at room

temperature. Then, the solvent was evaporated and the red solid obtained was extracted

with chloroform. The extracts were vacuum dried and the solid obtained was finally purified

by column chromatography (15% MeOH/2% NH4+/CHCl3), yielding a red solid in a very low

yield. The Boc group was then removed from this compound using TFA and CH2Cl2 as

solvent. Compound 33 was obtained as a red solid.

NNHN

NHBoc

O OH

NN N

H2N

NNHN

NH2

O

N

N

N

NH

+

31 26 33

i

ii

Scheme 2.13 ‐ Synthesis of compound 33. i) DMF, NEt3, HBTU. ii) TFA, CH2Cl2.

1.502.002.503.003.504.004.50

6

1 2 3 4

5

3’, 5’ (CH3‐pz)

NNHN

NH

O OH

1

2 3

45

3' 5'4'

O

O

5

5

6*

ppm

•


73

This compound is air stable, soluble in polar organic solvents such as methanol and

also soluble in water. Compound 33 was characterized by ESI/QITMS and multinuclear NMR

spectroscopy (1H, 13C, g‐COSY, g‐HSQC).

The mass spectrum of compound 33 presented two main groups of peaks at m/z

280.1 (most abundant mass) and m/z 559.2 (most abundant m/z), corresponding to

[M+H]2+and [M]+, respectively, confirming the expected formulation for 33 (figure 2.19).

Figure 2.19 ‐ Mass spectrum of compound 33 in the positive mode obtained by ESI/QITMS.

The 1H NMR spectrum of 33 (figure 2.20) presented two resonances at high field,

attributed to the methyl protons of the pyrazolyl ring (1.90 and 1.75 ppm). For the acridine

orange, the aromatic protons are observed between 7.15 and 5.68 ppm and the methyl

protons at 2.90 ppm. The methylenic protons of the aliphatic chain between the pyrazolyl

ring and the acridine appear at 1.42 ppm (H7 and H8), 3.00 ppm (H6) and 3.85 ppm (H9). The

singlet at 3.10 ppm corresponds to the CH2 protons (H5) linked to the C4’ of the pyrazolyl

ring. The aliphatic protons of the pyrazolyl‐diamine chelator appear at 4.12 ppm (H1) and

between 3.34 and 3.27 ppm (H2, H3 and H4).

[M + H]2+

280.1

[M]+

559.2

280.1

280.5

281.0

560.2

561.2

559.2


74

Figure 2.20 ‐ 1H NMR spectrum of compound 33 in D2O. S = solvent.

In the 13C NMR spectrum of 33 all the expected resonances could be observed, being

the presence of the carbon atoms C5 and C=O easily assigned at 32.1 ppm and 175.8 ppm,

respectively.

In conclusion, the 1H and 13C NMR spectra obtained for 33 in D2O presented the

expected resonances for the acridine orange fragment, the pyrazolyl ring and its

substituents, as well as for the methylenic protons of the respective aliphatic chains, being

in agreement with the proposed structure.

2.3.2. Synthesis and Characterization of Rhenium and Technetium

Tricarbonyl Complexes Bearing the Acridine Orange Moiety

The reaction of compound 33 with the precursor fac‐[Re(CO)3(OH2)3]Br was studied

using equimolar amounts of the reagents, methanol as solvent and overnight reflux (scheme

2.14). After evaporation of the solvent, compound fac‐[Re(CO)3(κ3‐33)]2+ (34) was obtained

as a red microcrystalline solid with high purity (> 95%) and with 91% of yield.

2.03.04.05.06.07.08.0

13 12 1110

1

5

3, 4

29

6

CH3‐Ao

3’, 5’ CH3‐pz

7, 8

S

NNHN

NH2

O

N

N

N

NH

1

2 3

4

5

6 7

89 10

1112

13

10

11

12

3'

4'5'

ppm


75

Compound 34 is air and water stable and soluble in methanol and water. The

characterization of 34 involved the usual spectroscopic techniques (IR, 1H, 13C NMR and 2D

NMR experiences) and RP‐HPLC.

O

NN

N

NH

NN NH

NH2

Re

OC CO CO

2+

MOC

H2O OH2

CO

OH2

CO

MeOH/ reflux

33

34

( 40', 100 °C)

O

NN

N

NH

NN NH

NH2

Tc

OC CO CO

35

99m33

2+

M = Re, 99mTc

Scheme 2.14 ‐ Synthesis of rhenium (34) and technetium (35) complexes.

The IR spectrum of 34 shows the typical fac‐[Re(CO)3]+ carbonyl vibrations at around

2023 and 1889 cm‐1, together with the bands assigned to the acridine orange and pyrazolyl‐

diamine chelator.

Analysing the 1H NMR of complex 34 (figure 2.21), the pattern observed for the

pyrazolyl‐diamine chelator is very similar to that obtained for the congener complex

described in the literature56 and that of complexes 19 (figure 2.11) and 20 described

previously. In particular, this similarity is reflected in the multiplicity and chemical shift of

the diastereotopic protons CH2 (H1, H2, H3 and H4) and NHa (figure 2.11) of the aliphatic

chain, confirming that chelator 33 is coordinated to the metal center in a tridentate fashion,

through the nitrogen of the pyrazolyl ring and through the two nitrogen atoms of the

primary and secondary amines. The resonances of the methyl protons of the pyrazolyl ring

are significantly shifted downfield (Δ = 0.20 ppm) relatively to the same resonances in the

free chelator 33, confirming also the coordination of the nitrogen of the pyrazolyl ring to the

metal center. The resonances corresponding to the acridine orange and to the aliphatic

chain appear with a pattern and chemical shifts similar to what was observed for the free

chelator 33.


76

Figure 2.21 ‐ 1H NMR spectrum of complex 34 in CD3OD. S = solvent, * residual water, • impurities.

Protons X = 1, 2, 3 and 4 are diastereotopic and are assigned as Hxa and Hxb; NH2a are also diastereotopic and

are assigned as NHa and NHa’.

The 13C NMR spectrum of 34 presents the expected resonances for the acridine

orange group, pyrazolyl ring and aliphatic protons, and the presence of the amide C=O

group was clearly seen at 172.9 ppm. In the 13C NMR spectrum are also observed three

resonances at 194.7, 194.5 and 194.1 ppm due to the carbon atoms of the CO ligands. The

presence of three resonaces for the CO ligands is due to the asymmetric character of the Re

complex.

The fac‐[99mTc(CO)3(κ3‐33)]2+ (35) complex was prepared by reacting the precursor

fac‐[99mTc(OH2)3(CO)3]+ with an aqueous solution of 33 ([33] = 8 x 10‐5 M), at 100 °C for 40

min (scheme 2.15). Complex 35 was obtained in high yield (90%) and was characterized by

comparing its HPLC profile with that of the corresponding Re(I) tricarbonyl complex (34)

(figure 2.22).

2.03.04.05.06.07.08.0

NHa’

13

12

11

10 3a, 4a

9

2a

1a

6

CH3‐Ao 3’, 5’ CH3‐pz

7

*

NHb NHa

8 3b, 4b

1b 2b

5

S

•

•

•

ppm

O

NN

N

NH

NN NH

NH2

Re

OC CO CO

1 2 34

5

6

78

910

11

12

13

10

11

12

3'

5'

2+

a

b


77

Figure 2.22 ‐ Analytical RP‐HPLC chromatograms of the Re complex 34 (254 nm) and the purified

99mTc complex 35 (γ trace).

Although there are many factors that can affect the in vitro or in vivo biological

behaviour of the 99mTc complex, the lipophilicity is a physico‐chemical characteristic that can

affect the biodistribution profile, including the route of elimination, as well as the capacity to

cross the cell membrane.172,173 The lipophilicity (log Po/w) was determined by the partition

coefficient (Po/w) in the biphasic system octanol/PBS (0.1 M, pH 7.4). The log Po/w was

determined to be 0.56 ± 0.02, indicating that complex 35 is moderately lipophilic.

2.3.3. Studies with B16F1 Cells

Acridine‐based compounds are fluorescent and represent a class of compounds with

relevance in the development of chemotherapeutic agents, due to their ability to intercalate

DNA inhibiting topoisomerase enzymes.45,174,,175,176,177 On the other hand, conjugation of

ligands containing intercalators to transition metals has been one of the explored strategies

to modulate the cytotoxicity of metal‐based drugs.178 Due to this, it was considered to

evaluate the cytotoxicity of the conjugate 33 and of the corresponding rhenium complex 34.

These results would also be informative for an evaluation of the radiotoxic effect of the

Auger electrons emitted by the 99mTc complex 35.

The approach of designing new potential therapeutic agents based on the Auger

electron‐emitter 99mTc has been explored previously.45,57,179,180 Due to the short range of

Auger electrons, there is a need for preferential accumulation of the complexes into the

nucleus of neoplasic cells in order to obtain significant DNA damage and therefore a

therapeutic effect. The strategy used to achive this goal is the design of multifunctional

10 20

Re 99mTc

Time (min)


78

complexes that include a DNA‐binding moiety, a carrier to transport the complex into the

nucleus, and/or a tumor‐seeking vector.45,57,179,180,181 Encouraging in vitro results have been

reported that highlighted the relevance of 99mTc for the development of therapeutic

radiopharmaceuticals.45,57,179,180

2.3.3.1. Cytotoxicity Studies

The cytotoxicity of the Re tricarbonyl complex 34 and of the compound 33 was

determined by the MTT test. This test was created in 1983 by Tim Mosmann182 and has been

used by others with some modifications. The test indirectly evaluates the cellular viability,

measuring the capacity of the mitochondrial enzyme succinato dehydrogenase to reduce the

3‐(4,5‐dimethylthiazol‐2‐yl‐)‐2,5‐diphenyltetrazolium bromide (MTT) to formazan (insoluble

crystals). In the viable cells (metabolically active), the formazan crystals are retained, being

necessary its solubilisation with DMSO. The number of viable cells is directly proportional to

the quantity of formazan formed, being this value estimated by the intensity of the blue

colour of the formazan solution.

The studies were performed using B16F1 murine melanoma cell lines. The cells were

incubated for 24 h at 37 °C with different concentrations (1.2 x 10‐9 ‐ 6 x 10‐5 M) of

compounds 33 and 34. After incubation, the cells were washed with PBS and incubated with

MTT for 3 h at 37 °C. After dissolution of the formazan crystals with DMSO, the solution

absorbance was measured at 595 nm.

The IC50 values (concentration at which there is 50% of cell death) were found to be

6.0 ± 1.4 x 10‐6 M and 6.6 ± 1.5 x 10‐6 M for compounds 33 and 34, respectively. Comparing

these results with previous results obtained in the Radiopharmacuetical Sciences Group of

ITN (RSG/ITN), it can be said that the cytotoxic effects of 33 and 34 are mainly due to the

acridine orange fragment.183 In fact, the corresponding compounds without the acridine

orange moiety, previously studied, did not present any toxicity when incubated with B16F1

cells at concentrations as high as 1 mM (conjugate) and 100 μM (rhenium complex).183


79

2.3.3.2. Cellular Localization by Fluorescence Microscopy Studies

When designing a radiopharmaceutical, it is highly desirable to follow the trafficking

and fate of the compounds at the cellular and subcellular level in real time. However, this is

not achievable with the radioactive probes (99mTc), which are more suitable to obtain

scintigraphic images from tissues and organs inside the body, and for quantification.

Profiting from the similarities of Re and Tc chemistry, fluorescent probes that are based on

organometallic Re(I) complexes emerged recently as an attractive alternative to bridge this

gap between in vitro fluorescence‐imaging studies and in vivo radioimaging.45,57,172,179,180

As stated above, the objective is to prove that the intrinsic fluorescence of the

acridine orange chromophore, which was introduced in the 4‐position of the pyrazolyl ring of

the pyrazolyl‐diamine chelator, enables the visualization of the intracellular localisation of

the Re(I)‐tricarbonyl complex. To confirm this possibility, the uptake of complex 34 by B16F1

murine melanoma cells was studied by fluorescence microscopy. The cells were incubated

for 3 h at 37 °C with 6 x 10‐5 M of compounds 33 and 34. After appropriate treatment, the

images were acquired by confocal fluorescence microscopy. As depicted in figure 2.24,

complex 34 and compound 33 could be both detected (green fluorescence) inside the cells,

in the cytoplasm and in the nucleus. The cells DNA was stained with DAPI (4’,6‐diamino‐2‐

phenylindol) to localize the uptake in the cell, in particular in the nucleus. DAPI is a

fluorescent probe commercially available that emits in the blue/cyan and enables the

localization of the cell nucleus because it binds strongly to DNA. Because DAPI emits in

wavelengths different from acridine orange, it is possible to visualize the co‐localization of

DAPI and of compounds 33 or 34.


80

33

34

Figure 2.23 – Confocal fluorescence microscopy images of B16F1 murine melanoma cells after 3 h of

exposure to 60 μM of compound 33 and complex 34 (green colour) followed by fixation and DNA

staining with DAPI (blue colour). The detection of green fluorescence in the cells reveals that the

compounds entered the cell and localized in the cytoplasm and nucleus. The white arrows indicate the

presence of green fluorescence in the nucleus.

The fluorescence microscopy studies revealed that compound 33 and complex 34

entered the cells and localized in the cytoplasm and in the nucleus.

2.3.3.3. Internalization, Retention and Radiotoxicity Studies

Before performing internalization/retention and radiotoxicity studies, we have

evaluated the stability of the 99mTc tricarbonyl complex 35 in cell culture medium (0.2% BSA

solution in MEM). For that we have incubated complex 35 with cell culture medium and


81

analysed the mixture by TLC at different time points. The radiochromatogram of the TLC

indicated only the presence of complex 35 (Rf = 0.34). No hydrolysed 99mTc species (Rf = 0),

labelled BSA (Rf = 0), [99mTcO4]

‐ (Rf = 0.85) or fac‐[99mTc(CO)3(OH2)3]

+ precursor (Rf = 0.8) were

detected. These results indicate that the radioconjugate is stable in the presence of BSA and

MEM, and confirmed the high ability of the pyrazolyl‐diamine chelator to stabilize the fac‐

[99mTc(CO)3]+ moiety.

The in vitro biological evaluation of the radioconjugate 35 involved studies of cellular

internalization and retention, nuclear internalization and evaluation of the radiotoxicity in

B16F1 murine melanoma cells, the same cell line used for the cytotoxicity and fluorescence

studies of 33 and 34. These studies were performed with the HPLC purified and non‐purified

radioactive complex 35. Purification of compound 35 has been performed to increase the

specific activity and radiochemical purity. This purification was performed by semi‐

preparative RP‐HPLC. The evaporated product was checked by RP‐HPLC and by TLC to

confirm its radiochemical purity, which was higher than 95%. The stability of the purified

complex in cells medium was also evaluated, and it was found that the complex was as

stable as the non‐purified radiocomplex.

Cellular internalization and retention

Cellular internalization

The cellular internalization studies were performed at 37 °C for the non‐purified and

for the purified 99mTc tricarbonyl conjugate 35 and revealed to be time dependent (figure

2.25). Moderate levels of internalization were reached with no significant difference

between the non‐purified and purified radioconjugates. At 5 h post‐incubation, 32% of the

total cell‐associated activity for the non‐purified radioconjugate 35 and approximately 34%

for the purified radioconjugate were taken up and internalized by the cells (figure 2.24A and

figure 2.24B). When this parameter is expressed as a percentage of total activity, for the

same time of incubation, a significant percentage, 6.3% and 7.8% for the non‐purified and

purified radioconjugates, respectively, were internalized by the cells (figure 2.25).


82

Figure 2.24 ‐ Internalization at 37 °C of the radioconjugate 35 in B16F1 cells at different time‐points.

Internalized and surface bound activity expressed as a fraction of bound activity (activity on the membrane

surface and inside the cell) for the non‐purified radioconjugate (A) and purified radioconjugate (B) (mean ±

standard deviation, n = 3 ).

0

2

4

6

8

10

12

0 2 4 6 8 10 12 14 16 18 20 22 24

Incubation time (h)

% Internalization/ to

tal activity

non‐purifiedpurified

Figure 2.25 ‐ Internalization at 37 °C expressed as a percentage of total activity for the non‐purified

and purified radioconjugate 35 (mean ± standard deviation, n = 3).

Nuclear internalization

Cell studies to calculate the percentage of activity internalized in the nucleus of

B16F1 cells were also performed. The purified and non‐purified compounds were incubated

with the cells at 37 °C. At different time points the internalization was stopped and the

cellular membrane was disrupted with Nonidet P‐40. The resulting cells suspension was

centrifuged. The precipitate contained the activity retained in the nucleus and the

0.1 0.5 1 2 3 4 5 24

Incubation time (h)

interna l i zedsurface bound

0

20

40

60

80

100

0.1 0.5 1 2 3 4 5 24

incubation time (h)

% uptake /bou

nd activity

internal ized surface bound

A B


83

supernatant the activity dispersed in other cellular components. The percentage of activity

internalized in the nucleus per total activity is presented in figure 2.26. The nuclear

internalization presented slightly higher values for the purified compound 35 and this

difference increased with time. At 6 h post‐incubation, 3.8% of the administered purified

compound and 1.5% of the non‐purified compound were internalized in the cells nucleus.

One possible reason for the higher nuclear and cellular internalization of the purified

compound is that with the purification we have removed the inactive compound 33, which

certainly competes with the 99mTc complex 35 in the diffusion process.

0

1

2

3

4

5

0 1 2 3 4 5 6

Incubation time (h)

% activity

in th

e nu

cleu

s/ to

tal activity

non‐purified

purified

Figure 2.26 ‐ Nuclear internalization at 37 °C in B16F1 murine melanoma cells of purified and non‐

purified radiocomplex 35 (mean ± standard deviation, n = 3 ).

The nuclear internalization was also determined by the ratio between the activity

internalized in the nucleus and that associated to the cells. Such calculation gives a more

accurate measure of the ability of the complex to pass from the cytoplasm to the nucleus. In

figure 2.27 it can be seen that, the nuclear internalization is very fast and the percentage of

compound inside the nucleus is higher than that outside (cytoplasm and other cellular

structures) for both purified and non‐purified compounds. The radioactive compound is

internalized by the cell and rapidly diffuses to the cell nucleus. After 30 min, 67.2% of

purified complex and 55.5% of non‐purified complex associated to the cells were in the

nucleus.


84

Purified compound

01020304050607080

0.5 1 2 3 4 5 6

Incubation time (h)

% activity

Non‐Purified compound

0

1020

3040

50

6070

80

0.5 1 2 3 4 5 6

Incubation time (h)

% activity

Figure 2.27 ‐ Activity internalized in the nucleus (white and black) and activity outside the nucleus

(black) in B16F1 murine melanoma cells, after incubation with purified or non‐purified radiocomplex

35 (mean ± standard deviation, n = 3).

Cellular retention

The cellular retention of the radioconjugate was evaluated at different time points,

after 3 h of internalization (figure 2.28). A good degree of cellular retention was observed.

The radioconjugates were slowly released from the cells into the culture medium, with

about 50% and 60% of the initially internalized non‐purified and purified radioconjugates,

respectively, still remaining inside the cells after 3 h of incubation. In this case, the


85

difference between the non‐purified and purified radioconjugates seems to be more

significant, being the cellular retention slightly higher for the purified radioconjugate.

40

50

60

70

80

90

100

0 0.5 1 1.5 2 2.5 3 3.5 4

Incubation time (h)

% cellular reten

tion

purified compound non‐purified compound

Figure 2.28 ‐ Cellular retention of the internalized radioconjugate 44 (purified and non‐purified) in

B16F1 cells over time at 37 °C (mean ± standard deviation, n = 3).

Radiotoxicity studies of complex 35 in B16F1 murine melanoma cells

The radiotoxicity studies of 35 were performed using the purified radioconjugate 35

and [99mTcO4]‐ (40 μCi), [99mTc(CO)3(OH2)3]

+ (40 μCi) and the conjugate 33 ([33] = 7.5 x 10‐9

M) as controls (figure 2.29). These studies involved the determination of the cellular

viability by the MTT test, after incubating the cells for a period of 36 ‐ 40 h, in the presence

of different radioactive concentrations of complex 35. In these studies no cell death was

observed, indicating that the radiocompound 35 is not radiotoxic even at high radiochemical

concentrations (30 ‐ 60 μCi). These results also showed that the radiocomplex 35 has no

cytotoxic effect in the concentration range 10‐9 ‐ 10‐12 M, as expected from the cytotoxic

studies with the rhenium complexes.


86

0

20

40

60

80

100

0.9 1.9 3.8 7.5 15 20 30 60 TcO4‐ Carb. Pz‐Ao

activity (μCi)

survival fraction (%

)

40 μCi

Figure 2.29 ‐ Cytotoxicity studies of purified radioconjugate 35 (0.9 ‐ 60 μCi), TcO4‐ = [99mTcO4]

‐ (40

μCi), Carb. = [99mTc(CO)3(OH2)3]+ (40 μCi ) and Pz‐Ao = compound 33 (2 x 10‐9 M) in B16F1 cells at 37

°C (mean ± standard deviation, n = 4).

These studies indicate no effect of the Auger electrons, as no radiotoxic effect was

observed, even with compound 35 entering in the nucleus of the cells. Further work related

with this subject is being performed in the RSG/ITN.

2.3.4. Biodistribution and In Vivo Stability

The biodistribution of the purified fac‐[99mTc(CO)3(κ3‐33)]2+(35) was examined in CD1

female mice at 1 and 4 h after intravenous injection, and expressed as percentage of

injected dose per gram (% ID/g) (table 2.3) and percentage of injected dose per organ

(%ID/organ).

Biodistribution data indicate a relatively fast clearance from the bloodstream, as 1 h

after injection only 0.65 ± 0.53 % of the complex was in circulation (table 2.3). No

preferential uptake in the main organs or tissues was observed, except in the liver, intestine

and kidney. The radioconjugate was excreted very slowly via both the renal and

hepatobiliary pathways and the total excretion was very low, only 3% of the activity was


87

eliminated at 1 h after injection, reaching 10.2% at 4 h p.i.. The high retention of the

radioconjugate in the liver can be attributed to the lipophilicity of the complex.

Table 2.3 ‐ Biodistribution of the radioconjugate 35 in CD1 female mice at 1 and 4 h after intravenous

injection (mean ± standard deviation, n = 4).

% ID/g ± standard deviation Tissue/organ

1h 4h

Blood 0.65 ± 0.53 0.12 ± 0.04

Liver 48.32 ± 7.81 36.12 ± 12.76

Intestine 11.61 ± 3.2 17.01 ± 5.50

Spleen 0.62 ± 0.12 0.35 ± 0.08

Heart 0.28 ± 0.06 0.28 ± 0.09

Lung 5.98 ± 2.89 0.63 ± 0.21

Kidney 26.92 ±3.56 6.66 ± 2.47

Muscle 0.07 ± 0.03 0.04 ± 0.01

Bone 0.15 ± 0.04 0.14 ± 0.02

Stomach 0.88 ± 0.23 1.28 ± 0.40

Pancreas 0.12 ± 0.05 0.10 ± 0.02

Total Excretion (% ID) 3.5 ± 1.1 10.2 ± 6.5

In vivo stability studies revealed that the radioconjugate was stable in blood (1 h

after injection) as no metabolites could be detected (figure 2.30). In contrast, metabolites

from the radioconjugate were found in urine. The metabolites formed have lower retention

time, appearing the major metabolite at ca. 12 min.


88

Figure 2.30 ‐ Analytical RP‐HPLC chromatograms of compound 35, blood serum and urine samples

collected 1 h after injection (γ trace).

In conclusion, we have prepared a new pyrazolyl‐acridine orange conjugate (33) that

reacted with the fac‐[M(CO)3(OH2)3]+ (M = Re, 99mTc) moiety originating the fac‐[M(CO)3(κ3‐

33)]2+ (M = Re (34), M = 99mTc (35)) complexes in high yield and radiochemical purity (99mTc).

The radioconjugate presented moderate cellular internalization levels in B16F1 cells, with a

significant percentage of the internalized compound moving rapidly to the nucleus of the

cells. The radioconjugate displayed good cellular retention with levels slightly different

between the non‐purified and purified conjugate, ca. 50% and ca. 60% respectively, after 3h

of incubation.

The combination of in vitro fluorescence microscopy studies and the cellular and

nuclear internalization studies for the isostructural Re (34) and 99mTc (35) complexes,

provided complementary information for a better understanding of the in vitro behaviour of

these complexes. In fact, by fluorescence microscopy it was directly observed the presence

of the fluorophore acridine orange in the cytoplasm and nucleus of the B16F1 cells. In the

internalization studies with the 99mTc complex what was detected was the presence of

radiometal in the cytoplasm and nucleus of the same cells. Taking into account that Re and

0 10 20

Serum

Urine

Time (min)

Initial preparation


89

99mTc complexes are isostructural, the combination of this information shows that the

Re/99mTc complexes anchored by pyrazolyl‐diamine‐acridine orange are located in the

cytoplasm and nucleus of the cells. These results suggest that the Re and 99mTc complexes

reach the cytoplasm and specially the nucleus of the cells in an intact form.

Cytotoxicity studies using the purified radioconjugate 35 indicated no radiotoxic

effect, meaning that despite entering the nucleus of the cells the Auger electrons do not

have any effect. The pharmacokinetic profile of the radioconjugate is not a good one and

needs to be improved. Lower kidney, liver and intestine uptake and faster excretion need to

be reached. Such improvement may be achieved by increasing the hydrophilic character of

the complex, which can be done by removing the methyl groups from the pyrazolyl ring, by

incorporating carboxylic groups in the chelate backbone or by coupling a peptide to the

pyrazolyl‐acridine orange conjugate.

3. Peptide Nucleic Acids ‐

Synthesis, Conjugation to

Bifunctional Chelators, Labelling and

Biological Evaluation

3. Peptide Nucleic Acids ‐ Synthesis, Conjugation to Bifunctional Chelators, Labelling and Biological Evaluation

93

3. Peptide Nucleic Acids ‐ Synthesis, Conjugation to

Bifunctional Chelators, Labelling and Biological Evaluation

3.1. Introduction

As referred in chapter 1, imaging gene expression can be directed to mRNA using

radiolabelled antisense oligonucleotides. This strategy combines the superior sensitivity of

radionuclide detection and the high specificity of antisense‐sense interactions. This approach

may detect alterations in overexpressed oncogene mRNA or identify sites of neoplasic

transformation non‐invasively at relatively early stages, providing opportunities for early

diagnostic and therapeutic interventions.66,66,106 As discussed before, PNAs are good

candidates for developing radiolabelled antisense probes for mRNA imaging and it was our

choice to start exploring the field of antisense probes.

The results described in chapter 2 confirmed that it was possible to couple the

pyrazolyl‐ and cysteine‐containing chelators to PNA units (monomer and dimer), and

prepare the rhenium and 99mTc tricarbonyl complexes. The formation of stable model 99mTc

complexes in high yield and with good pharmacokinetics, led us to attach these bifunctional

chelators to a PNA sequence clinically relevant to prepare 99mTc complexes.

The N‐MYC mRNA was selected as the target mRNA. N‐MYC is amplified in certain

peripheral nervous system tumors such as neuroblastomas,184,185,186,187,188,189

retinoblastomas185,190 and small cell lung carcinoma,185,191 and in central nervous system

tumors such as medulloblastoma185,192 and glioblastoma.185,193

Antisense and antigene oligonucleotides have been studied as selective inhibitors of

N‐MYC for the development of more effective and less toxic specific therapeutic agents for

tumors with N‐MYC overexpression. Inhibition of N‐MYC expression in vitro has been studied

with antisense oligodeoxynucleotides194 and antisense PNA188 targeted against the N‐MYC

mRNA, antigene PNA targeted against the N‐MYC DNA187 and also with antisense

phosphorothioate radiolabelled with the Auger emitter 111In targeted against the N‐MYC

mRNA.186 Each of these molecules has inhibited the translation or transcription of N‐MYC,


94

encouraging research on the development of therapeutic agents for neuroblastoma or other

neoplasms with N‐MYC expression.

The 16‐mer PNA sequence N‐A GAT CAT GCC CGG CAT‐C, complementary to the

region of the N‐MYC mRNA beginning with the ATG start codon at position 1650,184 was

chosen based on some studies mentioned above, namely on 111In‐Phosphorothioate186 and

on antigene studies with the same PNA sequence and the complementary one to target the

DNA.187

In this chapter we will present:

1. A brief introduction to solid phase synthesis;

2. The synthesis and characterization of the 16‐mer PNA sequence H‐A GAT CAT

GCC CGG CAT Lys‐NH2;

3. The conjugation of bifunctional chelators to the 16‐mer PNA and their

characterization;

4. The conjugation of the Re complex fac‐[Re(CO)3(3,5‐

Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ to the 16‐mer PNA sequence, using

solid phase techniques, and characterization of the final complex;

5. UV melting experiments of H‐A GAT CAT GCC CGG CAT Lys‐NH2 and fac‐

[Re(CO)3(κ3‐Pz‐ A GAT CAT GCC CGG CAT‐Lys‐NH2)]+ with the complementary

ssDNA sequence;

6. The synthesis and characterization of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC

CGG CAT‐Lys‐NH2)]2+;

7. The biological evaluation of the 99mTc complex, which includes in vitro cell

studies, and in vivo studies in normal mice.

3.2. Solid Phase Synthesis

The principle of solid phase chemistry was developed by Bruce Merrifield (Nobel

Prize in Chemistry 1984) in the 1950s and 1960s, and the synthesis of oligomeric molecules

on the surface of polymeric supports was first reported in 1963.195


95

Construction of a peptide chain on a solid support has several benefits: separation of

the intermediate peptides from soluble reagents and solvents, simply by filtration and

washing, saving time and labour over the corresponding operations in solution; many of the

operations are amenable to automation; excess reagents can be employed to force reactions

to completion; and losses can be minimized as the peptide remains attached to the support

throughout the synthesis. However, this technique does have limitations: by‐products arising

either from incomplete reactions, side reactions, or impure reagents will accumulate on the

resin during chain assembly and contaminate the final product.

3.2.1. Resin

Suitable polymers for solid phase synthesis must be insoluble in all solvents used in

the synthesis and must have a stable physical form for a ready filtration. These polymers

must also contain a functional group to which the first protected monomer could be firmly

linked by a covalent bond.

The most used resin in solid phase synthesis is 1% divinylbenzene cross‐linked

polystyrene (PS). It is relatively inexpensive to produce, swells in the most commonly used

solvents in peptide synthesis, namely dichloromethane (DCM), dimethylformamide (DMF)

and N‐methylpyrrolidone (NMP) and can be functionalized using chloromethyl, aminomethyl

and benzhydrylamino groups.

An important aspect of solid phase organic synthesis is the diffusion of reagents to

the reactive sites on bound molecules (swelling). For lightly cross‐linked polystyrene and

polyethylene glycol‐PS resins it is generally required that the beads swell in the reaction

solvent, establishing a phase that is approximately 10 ‐ 20% polymer and 80 ‐ 90% solvent.

The mobility of polymer‐bound molecules and reactants within the swollen gel is directly

related to the level of swelling.196,197


96

Linkers

The purpose of the linker is to provide a reversible linkage between the synthetic

peptide chain and the solid support, and to protect the C‐terminus during the process of

chain extension.

The choice of linker determines the C‐terminal functional group in the final product.

Most linkers are designed to release peptide acids or amides upon treatment with TFA.

3.3. PNA Synthesis

A requirement in the field of peptide nucleic acids research is the preparation of

monomers for subsequent oligomerization.198 A PNA monomer consists of N‐protected (2‐

aminoethyl)glycine (Pg1) to which a protected nucleobase (Pg2) is attached (figure 3.1).

These two protecting groups have to be orthogonal, i.e. must be removed in different

conditions.199

NH

N

O

Base

O

ORPg1

(Pg2)

Figure 3.1 ‐ General structure of a PNA monomer. Pg1 ‐ protecting group 1, Pg2 ‐ protecting group 2.

There are several combinations of protecting groups reported for the PNA synthesis,

being the most commonly used summarised in table 3.1.


97

Table 3.1 ‐ Commonly used protecting groups for PNA synthesis.199

Protecting group 1 (Pg1) Protecting group 2 (Pg2) Removal of Pg1 Removal of Pg2

Boc

COO

Cbz

COO

50% TFA HF or TFMSA

Fmoc

COO

Bhoc

COO

20% piperidine 95% TFA

Mmt

C

OMe

Acyla

O for A and C O

for G

2% DCAb NH3c

Fmoc Mmt 20% piperidine 2% DCAb

a Benzoyl for Adenine and Cytosine, isobutyryl for Gua; b can be replaced by 1% TFA; c saturated aqueous or methanolic

Usually, PNA synthesis uses solid‐phase peptide synthesis protocols.94,198 There are

three synthetic strategies used for the PNA synthesis. The strategies are classified according

to the nature of the protective groups on the aliphatic amino group (Pg1) in the PNA

monomers: Boc, Fmoc and Mmt strategies.200

After choosing the appropriate resin and strategy of synthesis, including the PNA

monomers with appropriate protecting groups, a PNA synthesis cycle can be summarized in

the following way:

‐ First it is necessary to download the resin, i.e., attach the first

monomer by the C‐terminus (carboxyl group) to the resin. After

coupling it is necessary to perform the capping of the resin, to

deactivate its active sites;

‐ The aliphatic amino protective group is then removed in the

deprotection step. An excess of the second monomer is introduced,

with the carboxyl group activated for amide bond formation.


98

‐ The capping is then performed to avoid further reactions of the non‐

reacted amine groups. This process is repeated until the desired PNA

sequence is assembled.

In a final step, the PNA is released from the support and the base protective groups

removed. Generally, base protecting groups and resin linkage are chosen such that

protecting groups are removed and the assembled PNA released under the same conditions.

3.4. Automated Solid Phase Synthesis

There are several peptide synthesizers; the one we have used was the ABI 433A

synthesizer.201 In this section a general idea of this apparatus will be given.

The synthesis unit is composed by one computer and a peptide synthesizer ABI 433A.

The software used for the synthesis is SynthAssist 2.0. The software allows to program and

analyse the synthesis.

The peptide synthesizer ABI 433A is shown in figure 3.2. It is composed of a display, a

keyboard, a sliding track on which the cartridges are placed (figure 3.2a), a barcode reader, a

system of two needles placed on a mobile arm driven by pressure, an activator vessel (figure

3.2c) and a reaction vessel (figure 3.2b) where oligomerization occurs. In particular for the

synthesis of PNAs, a 3 mL reaction vessel (figure 3.2b) that enables the PNA synthesis at

different scales, 5, 10 and 20 μmol, is used, reducing the cost of the synthesis. There are also

a number of reagents and solvents placed in numbered bottles (figure 3.2).


99

ccba ba

Figure 3.2 ‐ Peptide synthesizer Applied Biosystems ABI 433A. a) Sliding track, b) reaction vessel, c)

activator vessel.

All moving parts, except the part that vortexes the reaction vessel, are based on

pneumatic systems due to the incoming pressure into the instrument (5 bar). The agitation

of the reaction vessel is very important in PNA synthesis for efficient coupling and washing,

preventing the formation of agglomerates.

The strategies frequently used in the automated synthesis are the Fmoc or Boc with

the advantage of existing commercial available Boc/Cbz and Fmoc/Bhoc monomers.

The solvents used in the synthesizer are N‐methylpyrrolidone (NMP) and

dichlorometane. For automated PNA synthesis, each cartridge contains a solution of 5.3 eq

of monomer in NMP for 1 eq of resin loaded in the reactor. The activating agents used can

be HBTU or HATU, the base is N,N‐diisopropylethylamine (DIPEA) and, depending on the

strategy used, TFA or piperidine is used for deprotection of the aliphatic amine. The capping

solution to be used can be acetic anhydride/DIPEA/NMP 5:6:89 or acetic

anhydride/pyridine/NMP 1:25:25.


100

The synthesis in an automatic synthesizer needs some manual steps, which are the

download of the resin and the cleavage of the oligomers from the resin at the end of the

synthesis. In the manual solid phase synthesis a shaker is used to shake the reactor.

3.5. Synthesis of the PNA Sequence

3.5.1. Synthesis of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin

To synthesize the PNA sequence, the Fmoc/Bhoc chemistry was chosen and the PNA

monomers used were Fmoc‐A(Bhoc)‐OH, Fmoc‐C(Bhoc)‐OH, Fmoc‐G(Bhoc)‐OH and Fmoc‐T‐

OH.

The Novasyn TentaGel Rink Amide polystyrene (TGR PS) resin was used as solid

support and was downloaded with N‐α‐Fmoc‐L‐Lys(Boc)‐OH to get a final loading of ∼0.2

mmol/g (scheme 3.1). After the coupling of the N‐α‐Fmoc‐L‐Lys(Boc)‐OH to the amine group

of the resin, a capping solution (Ac2O/NMP/Pyridine 2:4:4) was added to acetylate the resin

unreacted amine groups.

MeO

MeO

HN

O NHO

i

NHBoc

O

FmocHN

PEG

MeO

MeO

NH2

O NHO

PEG

36Novasyn TGR PEG‐PS resin

ii

Scheme 3.1 ‐ Download of the resin. i) N‐α‐Fmoc‐L‐Lys(Boc)‐OH, HBTU, DIPEA, NMP; ii) Ac2O/NMP/Pyridine

(2:4:4).

From the different techniques used to evaluate the resin loading,202,203,204 we have

chosen gas chromatography.204 Such method consists on quantifying the Fmoc loading

utilizing the non‐nucleophilic amidine 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) as a Fmoc

cleavage reagent. In this method dibenzofulvene (DBF) is formed as single cleavage product

(scheme 3.3) which can be analyzed quantitatively by GC, using anthracene as an internal

standard. DBU is used instead of piperidine because deprotection with piperidine originates


101

the dibenzofulvene and the dibenzofulvene‐piperidine adduct, affecting the reproducibility

of the GC method (scheme 3.2).

CO O

RHNN

NH

DMF DMF

NN

piperidineDBU DBFDBF‐piperidine adduct

NH +

DBF

Scheme 3.2 ‐ Piperidine vs. DBU cleavage of the Fmoc group.

To start, a calibration curve was built using solutions of Lysine‐Fmoc (N‐α‐Fmoc‐L‐

Lys(Boc)‐OH) with different concentrations in DMF, containing 2% DBU/DMF. After

formation of DBF (scheme 3.3), anthracene was added as an internal standard and the

resulting mixture analyzed by GC. The calibration curve was obtained by plotting GC peak

area ratio of dibenzofulvene:anthracene vs. the molar ratio of Lys‐Fmoc:anthracene (figure

3.3).

y = 0.9945x + 0.0075

R2 = 0.9999

0.000.200.400.600.801.001.20

0.00 0.20 0.40 0.60 0.80 1.00 1.20

Lys‐Fmoc/Anthracene (mmol/mmol)

Area DBF/Area Anthracen

e

Figure 3.3 ‐ Calibration curve for DBU/DMF deprotection of Fmoc‐Lys and quantification by gas

chromatography.

Afterwards, accurately weighted samples of the vacuum‐dried resin 36 were placed

in tubes and a 2% DBU/DMF solution was added. After stirring the reaction mixture for 1h,

anthracene was added and the samples were analyzed by GC. The area of the DBF and

anthracene were then determined. The Fmoc substitution (FS, mmol/g) for each sample was

calculated using the following equation:


102

FS= (C)(V)(1/178.23)(Afmoc/Aanth + B)(M) (1/W)

C = exact concentration of anthracene solution in mg/mL; V = volume of anthracene

solution added; 178.23 = molar mass of anthracene; Afmoc and Aanthr = GC peak areas

of dibenzofulvene and anthracene, respectively; B = y‐intercept of the calibration

curve; M = slope of the calibration curve; W = mass of resin in grams.

The resin loading was calculated to be 0.137 ± 0.001 mmol/g. During the work

described in this thesis, resins with loadings in the range 0.14 – 0.18 mmol/g were used.

The Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin (37) was synthesized on an ABI 433A

peptide synthesizer or manually using solid phase Fmoc sythesis. The resin 36 was used on a

20 μmol scale and PNA assembly comprised repetitive cycles of deprotection, activation ‐

coupling and capping (scheme 3.3).

Before starting the synthesis, the dry resin was swelled with CH2Cl2 and DMF or

NMP. In the manual synthesis, CH2Cl2 was added to the resin and agitated for 1h, the CH2Cl2

was drained and DMF was added and agitated for 15 min. In the automated synthesis, the

swelling was performed by setting several steps of CH2Cl2 wash and then NMP wash before

starting the synthesis. When the resin beads swell and reach approximately 10 times their

dry volume, the resin appears as an insoluble solid support. However, at the molecular level

the resin is “in solution” or fully solvated, which enhances the coupling of the protected

monomers to the PNA‐resin.196,197,201


103

Deprotection

NFmocHN

O

O

B

OH

MeO

OMe

HN

O

NH

O

NHBoc

OHNN

FmocHN

O

O

B

Capping

Acetic anhydrideDIEANMP

piperidineDMF

HATUDIPEA/LutidineNMP

36

Fmoc‐A GAT CAT GCC CGG CAT

MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

37

Coupling

MeO

MeO

HN

O NHO

NHBoc

O

FmocHN

PEG

MeO

MeO

HN

O NHO

NHBoc

O

H2N

PEG

PEG

MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

O

repeat cycles

Scheme 3.3 ‐ Synthesis cycle of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin (37).


104

Deprotection

The first step in chain assembly is deprotection, or removal of the Fmoc protecting

group. The Fmoc group, which protects the aliphatic amine, is removed by the addition of

20 ‐ 25% piperidine in DMF or NMP to the synthesis support (scheme 3.4).

NH

NNH

OO

Base

ON

NH

OBase

OH

NH

NH

NNH

OO

Base

ON

NH

OBase

OO

O

NH

NNH

OO

Base

ON

NH

OBase

OO

NH2

NH

+

N

+ NH

NNH

OO

Base

ON

H2N

OBase

CO2

HN

NHBoc

O

HN

NHBoc

O

HN

NHBoc

O

HN

NHBoc

O

+ NH2

H

N

proton transfer

Scheme 3.4 ‐ Removal of the Fmoc protecting group with piperidine.


105

Care must be taken in this stage since the primary amine group at the N‐terminus of

a PNA oligomer can attack either the carbonyl group of the nucleobase or the carbonyl

group of the inter‐unit amide bond (scheme 3.5).205

HN

NH2N

O

O

Base

NO

O

Base

NH

LINKER NH

HN

O

O

Base

NH

LINKER

H2NN

NH

OBase

ON

NH

OBase

O

LINKER H2NN

NH

OBase

O

LINKER+

Scheme 3.5 ‐ Base catalyzed reactions of the amine terminus.

The increased basicity of the 2‐ethyl amine in the PNA and the fact that it is less

sterically hindered makes this amine more reactive than the α‐amine in amino acids.

The first reaction in scheme 3.5 consists on the acyl migration of the nucleobase

acetyl moiety to the N‐terminal position leaving the central secondary amine available for

subsequent coupling, which result in a PNA oligomer with strongly altered hybridization

properties. In the second reaction, the N‐terminal unit is lost and cleaved off in the form of

a ketopiperizine derivative (scheme 3.5).

Coupling

Before coupling, the carboxylic group of the PNA monomer must be “activated”.

HATU was selected as the activator for the coupling reaction (scheme 3.6). The activation of

the PNA monomer with HATU requires a base to proceed and we have used DIPEA or DIPEA

and 2,6‐lutidine in the automated and manual synthesis, respectively.


106

Activation

ON

NH

OO

Base

Fmoc

N NN

N

O

N NO

NNH

OO

Base

FmocN

N

N

NNN

O

NNH

OO

Base

FmocN

NN

N

O

O

N N+

PNA monomer activated tetramethyl urea

PF6

Scheme 3.6 ‐ PNA monomer activation with HATU.

In the coupling step, the activated monomer reacts with the deprotected amino acid

terminal of the growing PNA, as shown in scheme 3.7.

Coupling

N

NH

O

O

Base

Fmoc

N

NN

NO NH

NNH

OO

Base

ON

H2N

OBase

HN

NHBoc

O+

NH

NNH

OO

Base

ON

NH

OBase

HN

NHBoc

O

ON

FmocHN

OBase

+NN

NN

OH

Scheme 3.7 ‐ Coupling of the activated monomer to the growing PNA chain.


107

Generally, the PNA synthesis compared with peptide synthesis requires a slight

excess of monomers relative to the activation agent. These conditions prevent the

undesired tetramethyl guanidine capping, stemming from the reaction of HATU directly with

the primary amine on the growing chain (scheme 3.8).205,206 This side reaction is easily

identified by MALDI‐TOF mass spectrometry by the presence of M + 100 m/z.

N NN

N

O

N N

PF6

+

NH

NNH

OO

Base

ON

H2N

OBase

HN

NHBoc

ONH

NNH

OO

Base

ON

NH

OBase

HN

NHBoc

O

N

N

N NN

N

OH

+

Scheme 3.8 ‐ Potential guanidine by‐product when using HATU.

Capping

The acetylation of the oligomers that failed to extend post coupling was performed

during each cycle of the stepwise synthesis using Ac2O/DIPEA/NMP = 5/6/89 as capping

solution (scheme 3.9). This capping simplifies the purification of the full length PNA and also

prevents the side reactions illustrated in scheme 3.6 to occur.

NH

NNH

OO

Base

ON

NH2

OBase

NH

BocHN

O

OO

O

OO

O

OO

O+ NH

NNH

OO

Base

ON

NH2

OBase

NH

BocHN

O

O

O O

NH

NNH

OO

Base

ON

NH2

OBase

NH

BocHN

O

O+

O

O NH

NNH

OO

Base

ON

NH

OBase

NH

BocHN

O

O O

HO+

resonances structures of acetic anhydride

Scheme 3.9 ‐ Acetylation of the oligomer with acetic anhydride.


108

In the automated synthesis, the ratio monomer/resin is 5.3:1 eq and a single

coupling is performed for each monomer. In the manual synthesis, the ratio monomer/resin

is 3:1 eq but two cycles of coupling are performed. The time for each coupling is

approximately the same in the two methodologies (30 ‐ 35 min).

The synthesis of the 16‐mer PNA was performed in the synthesizer in two steps.

First, we synthesized the 8‐mer Fmoc‐CC CGG CAT–resin, removed 5 mg of resin, cleaved,

and analysed the product by ESI/QITMS or LC‐ESI/QITMS. After confirming the formation of

the 8‐mer sequence, we went on with the reaction until the 16‐mer PNA sequence (Fmoc‐ A

GAT CAT GCC CGG CAT‐Lys‐resin) was obtained. When we synthesized manually the 16‐mer

PNA sequence, we have followed a similar procedure but we checked the synthesis by

ESI/QITMS after 7 or 9 and 16 cycles.

3.5.2. Synthesis and Characterization of H‐A GAT CAT GCC CGG CAT‐Lys‐

NH2

Compound 38 was obtained by treating 37 with TFA/H2O/TISe = 90/5/5 for 1.5 h,

followed by precipitation with cold diethyl ether, after concentrating the final solution. The

precipitates were washed with cold diethyl ether 3 times, centrifuged at each time,

collected and analysed by LC‐ESI/QITMS.

In figure 3.4 is shown a RP‐HPLC chromatogram of the Fmoc‐A GAT CAT GCC CGG

CAT‐Lys sequence synthesized in the ABI 433A peptide synthesizer and in figure 3.5 the

chromatogram of the same sequence synthesized manually.

e Triisopropylsilane (TIS) was used as a scavenger for the benzhydryl cations generated by the trifluoracetic acid treatment. The use of a scavenger is required because the electron‐rich aromatic rings of the nucleobases can be alkylated by these cations


109

Figure 3.4 – Automated synthesis (ABI 433A): RP‐HPLC chromatogram of the crude product Fmoc‐A

GAT CAT GCC CGG CAT‐Lys (Absorbance at 260 nm).

Figure 3.5 – Manual synthesis: RP‐HPLC chromatogram of the crude product Fmoc‐ A GAT CAT GCC

CGG CAT‐Lys (Absorbance at 260 nm).

The peak corresponding to the Fmoc‐A GAT CAT GCC CGG CAT‐Lys sequence was

identified as the one with retention time ∼20 min by analysing the LC‐ESI/QITMS spectra.

This sequence has the Fmoc group attached to the last base (scheme 3.3), which is

responsible for the high retention time of the 16‐mer sequence and allows its discrimination

from the shorter acetylated sequences, which were not extended.

16‐mer sequence

Shorter sequences

Time (min)

Shorter sequences 16‐mer sequence

Time (min)


110

The crude product was then purified by semi‐preparative RP‐HPLC and the peak with

retention time ∼20 min was recovered. The white solid obtained was deprotected at the N‐

terminus with 25% piperidine in DMF, followed by RP‐HPLC purification (scheme 3.10). The

H‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (38) sequence was then characterized by ESI/FTICRMS

and RP‐HPLC. In the ESI/FTICRMS spectrum two main prominent peaks could be found at

m/z 1489.6196 and m/z 1117.4661, corresponding to [M+3H]3+ and [M+4H]4+, respectively.

HPLC chromatographic analysis has also shown only one peak with retention time 15.9 min.

Sequence 38 was only used for melting temperature experiments and its yield was not

calculated. However, taking into account the HPLC chromatogram of the crude product

(figures 3.4 and 3.5) the yield does not seem that high. This is not unexpected considering

the yields described in the literature for the same type of methodologies (Fmoc/HATU, 26%

to 38%).200

The overall yield of PNA sequences are affected by their lengths and base

sequence.200 In the case of purine‐rich PNAs, if more than two consecutive purine residues

are present the attachment efficiency of the subsequent purine monomer decreases.

Another problem of the purine‐rich PNAs is the aggregation of the chains. Our sequence is

relatively long (16‐mer) and has seven consecutive purine bases, so these two parameters

may justify the formation of shorter sequences in the synthesis of our PNA, as shown by LC‐

ESI/QITMS (figures 3.4 and 3.5).


111

ii

iii iii

NN NNHBoc

OOH

NN N

NH2Re

OC CO CO

O

OH+

NN N

NH2Re

OC CO CO

O

ii

NN NNH2

O

A GAT CAT GCC CGG CAT A GAT CAT GCC CGG CAT

NN N

NH2Re

OC CO CO

O

NN NNHBoc

O

39 40

H‐A GAT CAT GCC CGG CAT Lys NH2 i

+

Pz‐Boc

RePz

ii3738

MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG


MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

A GAT CAT GCC CGG CAT

MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG


H2N

NH2

OHN

H2N

NH2

OHN+

Scheme 3.10 ‐ Synthesis of the PNA sequences 38 and conjugates 39 and 40. i) Cleavage from resin:

TFA/TIS/H2O=90:5:5 (1.5 h), ii) Fmoc‐deprotection: 25% piperidine/DMF, iii) coupling: HATU/DIPEA/2,6‐

Lutidine.

3.6. Synthesis and Characterization of Pz‐A GAT CAT GCC CGG CAT‐

Lys‐NH2 and fac‐[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]+

The bifunctional chelator 3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (Pz‐Boc) and

the complex fac‐[Re(CO)3(3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ (RePz) were coupled to

the 16‐mer PNA sequence 37 using manual solid phase techniques (scheme 3.10). Taking

advantage of the free carboxylic acid group of fac‐[Re(CO)3(3,5‐

Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2)]+ (RePz), this complex has been coupled to the PNA


112

sequence, an approach which had been previously used with success by Hamzavi et al., for

the conjugation of [Re(bip‐O)(CO)3(H2O)] to different PNA sequences.207

Before coupling the RePz to the PNA sequence using solid phase techniques, the

stability of the complex was tested in the cleavage conditions (TFA/TIS/H2O = 90:5:5). The

complex was mixed with the cleavage solution for 1.5 h and after this time was analysed by

RP‐HPLC, ESI/QITMS and 1H NMR. All these techniques have shown that the complex was

stable.

Briefly, Pz‐Boc and RePz were coupled to the N‐terminus of 37 after Fmoc

deprotection and activation with piperidine/DMF (25%) and HATU/DIPEA/2,6‐lutidine,

respectively (scheme 3.10).

In these reactions the activation and coupling were performed during 20 and 40

minutes, respectively. The molar ratios Pz‐Boc and RePz:HATU were also changed and

instead of 3:2.7 we have used 3.5:2.7 and 4:2.7, respectively. The success of the coupling

was evaluated using the Kaiser test.208 Cleavage from the resin was carried out by treatment

with TFA/H2O/TIS (90:5:5) and the products isolated by precipitation with cold diethyl ether.

The crude was analysed by LC‐ESI/QITMS and purified by semi‐preparative reversed phase

HPLC (figure 3.6). The characterization of the conjugate and/or complex was based on

ESI/FTICRMS. In the spectrum of Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) two strong peaks

were observed at m/z 1180.0126 and 944.2102, corresponding to the calculated values for

[M+4H]4+ and [M+5H]5+, respectively. For fac‐[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]+ (40) three strong peaks appeared at m/z 1247.4959, 998.1970 and 831.9986,

corresponding to the calculated values for [M+3H]4+, [M+4H]5+ and [M+5H]6+, respectively.

Figure 3.6 ‐ Analytical RP‐HPLC chromatograms of 39 (left) and 40 (right), after purification

(Absorbance at 260 nm).

Time (min)

0

200

400

600

0 5 10 15 20 25

100

200

300

0 5 10 15 20 25

Time (min)


113

3.7. Coupling of the Cysteine‐Containing Chelator to the PNA

Sequence

The cysteine containing chelator 11 was coupled to the N‐terminus of the

deprotected PNA sequence according to scheme 3.11. No activating agents were necessary

because compound 11 was activated with pentafluorphenol. After double coupling, a Kaiser

test was performed.

iii

SF3COCHN

O

OO

O

F

F

FF

F

SF3COCHN

O

OO

iii

SF3COCHN

O

OO



11

MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

37


MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

H2N

NH2

ONH

41

Scheme 3.11 ‐ Synthesis of conjugate 41. i) Fmoc‐deprotection: 25% piperidine/DMF; ii) coupling in NMP;

iii) cleave from resin: TFA/H2O/TIS=90:5:5.

Then, a small portion of the reaction mixture was taken and reacted with

TFA/H2O/TIS (90:5:5) to cleave our compound from the resin. The resulting product was

analysed by LC‐ESI/QITMS (figure 3.7). Despite the complex HPLC chromatogram obtained

(figure 3.7A), we have assigned the peak at 17.70 min to compound 41. The ESI/QITMS of


114

this species presents two main peaks at m/z values 1580.4 and 1185.7, which agree with the

values calculated for [M+3H]3+ (1580.3) and [M+4H]4+ (1185.5), respectively (figure 3.7 B).

Figure 3.7 ‐ LC‐ESI/QITMS of the crude product 41. A) HPLC chromatogram at 260 nm. B) ESI/QITMS

of the peak at 17.70 min.

As can be observed in the HPLC chromatogram (figure 3.7A), compound 41 was

obtained in a very low yield. For further studies, the amine and the carboxylic groups in 41

had still to be deprotected. These results, together with time limitations, did not encourage

further studies with this conjugate.

3.8. UV‐Melting Temperature

The interaction of fac‐[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]+ (40) with

the complementary single strand DNA was studied by temperature‐dependent UV

spectroscopy. These studies were performed in phosphate buffered aqueous solution (pH

7.2) and in a temperature range of 20 ‐ 95 °C. For comparison, the behaviour of the PNA

sequence H‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (38) was also studied using the same

8 10 12 14 16 18Time (min)

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

uAU

15,07

13,10

11,66

10,51

17,70

16,40

9,10

BA

900 1000 1100 1200 1300 1400 1500 1600 170m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1580,2

1185,5

1186,5

1581,1

1190,91593,3

963,0 1098,8 1595,11195,4 1281,8 1491,2 11424,7


115

experimental conditions. For 38 and 40 well‐defined sigmoidal curves were obtained for

melting and annealing. The melting profiles for 38:DNA and 40:DNA are shown in figure 3.8.

The melting temperatures (UV‐Tm), determined as the maximum of the first

derivative of the melting curves, were found to be 83.5 ± 0.1 °C for both sequences. These

results indicate that the introduction of the Re tricarbonyl fragment in the PNA sequence did

not affect the recognition of its complementary, as well as the duplex stability. So far, it is

difficult to compare our results with others in the literature, as there is only one Re

tricarbonyl‐PNA conjugate described.207 However, this complex was anchored on a bidentate

bis(imidazolyl)propionic acid and the effect of the metal fragment on the Tm of the PNA

sequence was not evaluated.

Other studies with ferrocene209 and tris(bipyridine)ruthenium(II)210 have shown,

respectively, an increase (ΔTm = +4.3 °C) and a decrease (ΔTm = ‐2.3 °C) of the duplex stability

after conjugation. The decrease found for the ruthenium complex has been attributed to the

disruption of hydrogen bonding between the N‐terminal A:T base pair, most probably due to

the bulkiness of the metal fragment. For complex 40, the position of the PNA sequence

relatively to the metal fragment [Re(CO)3]+ and/or the existence of a spacer may be

responsible for the high stability found for 40:DNA which can be compared to 38:DNA.

0.6

0.7

0.8

20 30 40 50 60 70 80 90

T/ºC

Abs at 260 nm

Figure 3.8 ‐ Melting profiles of 40:DNA (blue curve) and 38:DNA (black curve).


116

3.9. Synthesis and Characterization of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT

CAT GCC CGG CAT‐Lys‐NH2)]2+

The reactivity of Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) towards the fac‐

[99mTc(CO)3(OH2)3]+ was studied according to the same procedure described in chapter 2 and

detailed in the experimental part (scheme 3.14).

42

NH

N

O

N

NN

NNH2

O

NH

N

O O

NH

NN

NO

NH2

NH

N

O

N

NN

NNH2

O

NH

N

O O

NH

N O

O

NH

N

O O

N

N O

NH2

NH

N

O

N

NN

NH2N

O

NH

N

O O

NH

N O

O

NH

N

O O

NH

NN

NO

NH2

NH

N

O O

N

N O

NH2

NH

N

O O

N

N O

NH2

NH

N

O O

N

N O

NH2

NH

N

O O

NH

NN

NO

NH2

NH

N

O O

NH

NN

NO

NH2

NH

N

O O

N

N O

NH2

NH

N

O

N

NN

NH2N

O

NH

N

O O

NH

N O

O

H2N

NH3

ONH

NN NNH2

O

NH

N

O

N

NN

NNH2

O

NH

N

O O

NH

NN

NO

NH2

NH

N

O

N

NN

NNH2

O

NH

N

O O

NH

N O

O

NH

N

O O

N

N O

NH2

NH

N

O

N

NN

NH2N

O

NH

N

O O

NH

N O

O

NH

NO O

NH

NN

NO

NH2

NH

N

O O

N

N O

NH2

NH

N

O O

N

N O

NH2

NH

N

O O

N

N O

NH2

NH

N

O O

NH

NN

NO

NH2

NH

N

O O

NH

NN

NO

NH2

NH

N

O O

N

N O

NH2

NH

N

O

N

NN

NH2N

O

NH

N

O O

NH

N O

O

H2N

NH3

ONH

NN N

NH2Tc

OC CO CO

O

99m

2+

TcOC

H2O OH2

CO

OH2

CO

99m 1h at 100 °CpH = 7

39

+

Scheme 3.12 – Synthesis of the radioconjugate fac‐[99mTc(CO)3(κ3‐Pz‐ A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+ (42).

Using a final concentration ∼5 x 10‐5 M of 39, complex fac‐[99mTc(CO)3(κ3‐Pz‐A GAT

CAT GCC CGG CAT‐Lys‐NH2)]2+ (42) was obtained in high yield (more than 90%), with high

radiochemical purity and high specific activity. Complex 42 was characterized by comparing

its HPLC profile with the profile of the corresponding rhenium tricarbonyl complex fac‐

[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]+ (40) (figure 3.9). No formation of colloids

(Rf = 0) was detected in the preparation, as indicated by ITLC‐SG analysis, developed with

pyridine/HOAc/H2O (3:5:1.5).


117

Figure 3.9 ‐ Analytical RP‐HPLC chromatograms of 40 (absorbance at 260 nm) and 42 ( γ trace).

As can be observed in figure 3.9, the compounds present well‐defined peaks with a

difference of 0.3 min in retention time, confirming that the 99mTc compound 42 has the same

chemical structure as the rhenium congener 40. In order to confirm that the 99mTc

coordination was through the pyrazolyl‐diamine chelator, an aqueous solution of H‐A GAT

CAT GCC CGG CAT‐Lys‐NH2 (38) was reacted with the precursor fac‐[99mTc(CO)3(OH2)3]

+, at

100 °C for 1 h, pH 7. As can be observed in figure 3.10, the main peak detected was the

tricarbonyl precursor and no peak was found with a retention time identical to 42.

Figure 3.10 ‐ Analytical RP‐HPLC chromatogram of the reaction of 38 with [99mTc(CO)3(OH2)3]+ (γ

trace).

0

100

200

0 20 40

[99mTc(CO)3(H2O)3]+

Time (min)

Re 99mTc

Time (min)


118

3.10. In Vitro Stability Studies

To assess the resistance of the radiocomplex 42 to proteolytic degradation caused by

endogenous peptidases and to predict its in vivo stability, stability assays in fresh human

serum at 37 °C were performed. Analysis by RP‐HPLC at different time points (0 min, 5 min,

45 min, 2 h and 4 h) indicated high serum stability, with negligible degradation of 42 (figure

3.11). These results confirmed the high ability of the pyrazolyl‐diamine chelator to stabilize

fac‐[99mTc(CO)3]+, avoiding transmetallation to serum‐based proteins and/or reoxidation of

the 99mTc(I), and confirmed also the resistance of the PNA to enzymatic degradation.99

Figure 3.11 ‐ Analytical RP‐HPLC chromatograms of fac‐[99mTc(CO)3(κ3‐Pz‐ A GAT CAT GCC CGG CAT‐

Lys‐NH2)]+2 (42) after incubation with human serum at 37 °C, at different time points (γ trace).

In vitro internalization/retention in cells is also an important test which can predict

the possibility of a radioactive compound to be retained in vivo in a target organ. However,

before such studies, it was important to evaluate the stability of the 42 in the cell medium.

Compound 42 was incubated with culture medium and the misture was analysed by RP‐HPLC

and ITLC‐SG. As can be seen in figure 3.12, after 4 h incubation at 37 °C, the RP‐HPLC

5 10 15 20 25

Initial preparation

2 h

4 h

24 h

Time (min)


119

chromatogram displayed only one peak due to 42 (γ detection). By ITLC‐SG

radiochromatography no hydrolyzed 99mTc species (Rf = 0) or labelled BSA (Rf = 0) were

detected, being 42 the only product present (Rf = 1) (figure 3.13). These results confirmed

that 42 was stable, as no interaction with BSA and/or transmetallation, oxidation or

formation of colloids took place.

Figure 3.12 ‐ Analytical RP‐HPLC chromatogram of 42 after 4 h incubation at 37 °C in culture medium

(γ trace)

Figure 3.13 ‐ ITLC‐SG chromatogram of compound 42 after 4 h incubation at 37 °C in culture medium

(γ trace).

3.11. In Vitro Studies in Cells

SH‐SY5Y human neuroblastoma cells, where N‐MYC is expressed but not amplified,211

were selected for a first screening of the internalization/externalization of complex 42. For

comparison, the same studies were performed using MCF7 breast212 and the PC3 prostate

cancer cell lines, which do not express the N‐MYC gene.

Uptake and internalization studies performed for 42 at 37 °C, revealed a time‐

dependent behaviour for all cell lines (figure 3.14). As shown in figure 3.14A, high levels of

0

200

400

0 5 10 15 20 25Time (min)

0


120

cellular uptake and internalization were achieved for 42 in SH‐SY5Y cells. For instance, 4 h

after incubation, while approximately 15% of the administered radioactivity was associated

with the cells (activity on the membrane surface and inside the cell), 7.5% of the

radioactivity was internalized. The percentage of internalized radioconjugate increased with

time and after 24 h almost all cell‐associated 42 was inside the cell.

0

5

10

15

20

25

0.1 0.5 1 2 4 6 24Incubation time (h)

% internalized activity% cell associated activity

0

5

10

15

20

25

0.1 0.5 2 4 6Incubation time (h)


0

5

10

15

20

25

0.1 0.5 2 4 6

Incubation time (h)


Figure 3.14 ‐ Cell‐associated radioactivity and cellular internalization of 42 at different time points

(37 °C) in SH‐SY5Y cells (A), MCF7 cells (B) and PC3 cells (C). Internalized and cell‐associated activity

expressed as a percentage of the total activity (mean ± standard deviation, n = 4).

A

B

C


121

For the MCF7 (figure 3.14B) and PC3 cells (figure 3.14C) high levels of cellular uptake

and internalization were also observed. After 4 h of incubation, the cell‐associated activity in

PC3 cells was ∼17%, while in MCF7 and SH‐SY5Y was ∼15%. At all time points internalized

activity was higher for the PC3 than for the other cell lines (figure 3.15).

0246810121416

0.1 0.5 2 4 6

Incubation time (h)

% internalizaed activ

ity MCF7SH‐SY5YPC3

Figure 3.15 ‐ Cellular internalization in SH‐SH5Y, MCF7 and PC3 cells at different time points (37 °C).

Internalized activity expressed as a percentage of total activity (mean ± standard deviation, n = 4).

The cellular retention of 42 was also evaluated at different time points, but only for

SH‐SY5Y cells (figure 3.16). A high cellular retention was observed for the compound, as

almost 60% of the initially internalized compound still remained inside the cells, after 5 h of

incubation.

0

20

40

60

80

100

120

0 0.5 1 2 3 5

Incubation time (h)

% cellular retention

Figure 3.16 ‐ Cellular retention of internalized radioconjugate 42 in SH‐SY5Y cells over time at 37 °C

(mean ± standard deviation, n = 4)


122

The internalization of 42 in SH‐SY‐5Y cells is significant but a correlation between our

cell‐associated activity and other values described in the literature is difficult, as such activity

depends on the cell type and PNA sequence. Moreover, the values reported for unassisted

PNA were evaluated either by fluorescence microscopy or using a 14C‐labelled compound

with a much lower specific activity than 42.108,188,213,214,215

In general, most of the studies are with PNA assisted with specific or non‐specific

biological vectors, to facilitate the internalization and/or selectivity.76,78,103,104,105,111,187,215,216

There are several examples of radiolabelled antisense PNA sequences conjugated to

specific or non‐specific peptides, such as analogues of insulin growth factor 1,103‐106,111,112 of

somatostatin (Tyr3‐octreotate),76 or cell membrane‐permeating peptides.78,108 In all these

studies, the cellular internalization of the complexes increased as well as the specificity,

when they are receptor‐mediated. However, just in one case (somatostatin analogue) the in

vitro cellular uptake was quantified.76 In this work the 111In‐DOTA‐ BCL‐2 PNA‐Tyr3‐

octreotate (antisense probe) showed, after 4 h after incubation, a cellular internalization of

5.2% and a cellular retention of 60%. Comparing these results with ours (7.5%

internalization, after 4 h incubation and 60% cellular retention after 5 h), we can say that our

results were relatively high and encouraging, as our PNA sequence is unassisted.

Considering the half‐life (t1/2) of 99mTc, we can say that the internalization/retention

of 42 is encouraging, as the values found are relatively high within a period of time well

adjusted to t1/2 of 99mTc. Nevertheless, it must be noted that the cell‐associated activity as a

function of time, although being an important factor to predict the potential interest of a

radiocompound for in vivo imaging, must be considered only as an indication. Clinically

useful probes for in vivo imaging must reach the target organ but the target/non‐target ratio

has to be high, and that can only be analysed by in vivo studies.


123

3.12. Biodistribution and In Vivo Stability

3.12.1. Biodistribution

The biological behaviour of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+ (42) was examined in CD‐1 Charles River female mice to evaluate the

pharmacokinetics and in vivo stability. Animals were intravenously injected with 100 μl of a

preparation of 42 via the tail vein, using the same procedure as for the radiocomplexes

described in chapter 2. Biodistribution data were expressed as percentage of injected dose

per gram (%ID/g) (table 3.2) and percentage of injected dose per organ (%ID/organ) (figure

3.17).

Table 3.2 ‐ Biodistribution data of the fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ (42)

in CD‐1 Charles River mice, at 1 and 4 h after intravenous injection (mean ± standard deviation, n =

4).


1h 4h

Blood 0.9 ± 0.17 0.54 ± 0.13

Liver 17.09 ± 1.2 24.06 ± 4.99

Intestine 0.47 ± 0.06 0.53 ± 0.08

Spleen 2.61 ± 1.06 2.58 ± 0.45

Heart 0.52 ± 0.05 0.41 ± 0.07

Lung 2.94 ± 2.29 4.51 ± 3.11

Kidney 82.23 ± 1.26 84.64 ± 6.07

Muscle 0.23 ± 0.05 0.17 ± 0.05

Bone 0.85 ± 0.26 0.89 ± 0.52

Stomach 1.21 ± 0.29 0.54 ± 0.10

Excretion (% ID) 15.5 ± 1.4 18.9 ± 2.1


124

0

5

10

15

20

25

30

35

40

Kidney Liver Intestine Muscle Bone Stomach Spleen

% ID

/ organ

1 h4 h

Figure 3.17 ‐ Uptake of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ (42) in some more

relevant organs (CD‐1 Charles River mice at 1 and 4 h after intravenous injection).

Biodistribution data indicate a fast clearance from the bloodstream (0.9 ± 0.17 %

ID/g, 1 h after injection) and no preferential uptake in the main organs or tissues, except in

the kidney and liver (table 3.2). Also, there was no significant fixation or retention in the

stomach (table 3.2 and figure 3.17), indicating that in vivo the reoxidation of Tc(I) to

[99mTcO4]‐ does not take place.

Figure 3.18 shows some biological data for complexes 42 and 23. Comparing such

data, it is possible to get an idea of the effect of the 16‐mer PNA sequence.

0

20

40

60

80

% ID

/ organ

1 h 4 h 1h 4 h

excretionIntestine

LiverKidney

time (h)

23

42

Figure 3.18 – Biological data for complexes fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+

(42) and fac‐[99mTc(CO)3(κ3‐17)]+ (23) in CD‐1 Charles River female mice.

(% ID)


125

The total excretion found for 42 (15.5 ± 1.4 %, 1 h p.i.) is significantly lower than the

value found for 23 (1.2 ± 4.8 %, 1 h p.i.). Such decrease results from the high liver and kidney

uptake found for 42. These results clearly show that the incorporation of the 16‐mer PNA

sequence affected significantly the biological profile of the radioconjugate.

Previous results have shown that other PNA sequences labelled with 99mTc, 64Cu and 111In also present high kidney uptake. For the 99mTc and 64Cu‐PNA radiocompounds the

kidney uptake had values between 30 ‐ 90% (ID/g)78,103,104,112 and for the 111In‐PNA

radiocompound values higher than 100% (ID/g) were found.76

In terms of liver uptake, our values are higher than those reported for other PNA

radioconjugates.76,78,103,104,112 Only a 125I‐PNA conjugated to a monoclonal antibody

presented a high liver uptake (29% ID/g at 1 h after intravenous injection).144

3.12.1.1. Inhibition of Kidney Uptake

Since it is well established that administration of cationic amino acids (Lys or Arg) can

inhibit renal accumulation of radiolabelled proteins and peptides,217,218,219 a second set of

biodistribution experiments in mice were performed. In these studies the Lys and 42 were

co‐injected intravenously (table 3.3). Interestingly, coadministration of this basic amino acid

reduced the kidney accumulation of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+ (42) in about 63% (table 3.3). Most probably the positively charged lysine interacts

with the cationic transporters on the renal tubules of the kidney, decreasing the interaction

of the negatively charged proximal tubule cells with the positively charged compound 42.

Such incubation blocks the mechanism of tubular reabsorption of 42. Such effect has already

been observed for other radiolabelled peptides and antibody fragments.22,217,218,219 For

example, co‐injection of L‐lysine with 111In‐octreotide reduced the kidney uptake in 35%, 24

h after injection.219


126

Table 3.3 ‐ Biodistribution of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ (42) when co‐

injected or not with L‐Lys (15 mg), at 4 h after intravenous injection (mean ± standard deviation, n =

4).


With L‐Lys Without L‐Lys

Blood 0.52 ± 0.04 0.54 ± 0.13

Liver 29.87 ± 2.08 24.06 ± 4.99

Intestine 1.29 ± 0.28 0.53 ± 0.08

Spleen 37.12 ± 10.75 2.58 ± 0.45

Heart 0.53 ± 0.14 0.41 ± 0.07

Lung 3.72 ± 2.75 4.51 ± 3.11

Kidney 31.11 ± 2.75 84.64 ± 6.07

Muscle 0.23 ± 0.03 0.17 ± 0.05

Bone 1.39 ± 0.50 0.89 ± 0.52

Stomach 0.46 ± 0.26 0.54 ± 0.10

Excretion (% ID) 31.0 ± 3.0 18.9 ± 2.1

3.12.2. In Vivo Stability

To study the in vivo stability of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+ (42), urine, blood and liver samples were collected from the sacrificed CD‐1 mice, 1 h

after injection of the radioactive preparation. After appropriate treatment, the biological

samples were analyzed by RP‐HPLC (figure 3.19).


127

Figure 3.19 ‐ Reversed‐phase HPLC chromatograms of the injected preparation of complex 42, blood

serum, urine and liver homogenate samples collected 1 h after injection and treated before analysis

(γ trace).

In the mice serum, isolated from the collected blood, no [99mTcO4]‐ or other products

resulting from the complex degradation were detected, being all the radioactivity due to

complex 42. Analysis of the urine collected at the sacrifice time presented only a small

metabolite at approximately 18 min, demonstrating the high stability of the complex in vivo

and the resistance of the PNA sequence to metabolization. The liver homogenate analysis

also did not reveal the presence of any metabolite.

The resistance to oxidation and to metabolic degradation in blood, liver, and kidney

demonstrated the high in vivo robustness of complex 42.

Initial Preparation

Serum

Urine

0 10 20 30

Liver homogenate

Time (min)


128

Summarizing, it was possible to synthesize the 16‐mer PNA sequence H‐A GAT CAT

GCC CGG CAT‐Lys‐NH2 (38) and to incorporate the pyrazolyl‐diamine bifunctional chelator

into this sequence, originating the conjugate Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39).

Conjugation of the cysteine containing chelator 11 to the PNA sequence was also possible.

However, the very low yield of the preparation, together with time limitations, did not

encourage further studies with such conjugate.

The fac‐[Re(CO)3(3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ was also successfully

coupled to the PNA sequence originating the fac‐[Re(CO)3(κ3‐Pz‐ A GAT CAT GCC CGG CAT‐

Lys‐NH2)]2+ (40) complex that was used for melting temperature experiments and to

characterize the congener 99mTc complex.

The melting temperature (Tm) of 40:DNA was determined and compared with the Tm

found for the same sequence without the metal fragment (38:DNA). The Tm for both

sequences was 83.5 ± 0.1 °C, indicating that the introduction of the Re tricarbonyl complex

in the PNA sequence did not affect the recognition of the complementary, as well as the

duplex stability.

The conjugate, Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) reacted quantitatively with

fac‐[99mTc(CO)3(OH2)3]+ yielding the radioactive complex fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT

GCC CGG CAT‐Lys‐NH2)]2+ (42) in high yield and with high radiochemical purity and specific

activity. Compound 42 is stable in human serum and in cell medium and its characterization

was done by comparing its HPLC chromatogram with the one obtained for the rhenium

surrogate fac‐[Re(CO)3(κ3‐Pz‐ A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ (40).

Preliminary studies with SH‐SY5Y neuroblastoma, MCF7 breast cancer and PC3

prostate cancer cells have shown a relatively high internalization of complex 42 in all the cell

lines and a high retention in SH‐SY5Y. So far, we cannot say if the cellular retention is due to

the interaction of 42 with the complementary N‐MYC mRNA. Such confirmation can only be

obtained after studying the internalization/retention of 42 in IMR32 cells, which overexpress

N‐MYC mRNA. Control experiments with the same cell line and using mismatch probes are

still necessary. The conjugation of a specific vector to 42 could also be part of future work, to

confer specificity and to increase the cellular uptake of the conjugate.

The pharmacokinetic profile of 42 has to be improved, mainly the kidney and liver

excretion has to be increased. The kidney retention was reduced by 63% by co‐injection of

42 and L‐lysine but the values are still very high and unacceptable.


129

Conjugation of a biologically active peptide to 42, as mentioned before, can introduce

more specificity, but can also modify the pharmacological profile of the radioconjugate.

4.

Melanoma Targeting with an α‐

Melanocyte Stimulating Hormone

Analogue Labelled with fac‐

[99mTc(CO)3]+

4. Melanoma Targeting with an α‐Melanocyte Stimulating Hormone Analogue Labelled with fac‐[99mTc(CO)3]+

133

4. Melanoma Targeting with an α‐Melanocyte Stimulating

Hormone Analogue Labelled with fac‐[99mTc(CO)3]+

4.1. Introduction

Malignant melanoma is the most serious form of skin cancer and its incidence and

mortality rate are still increasing in most western countries.220 Early diagnosis and adequate

surgical removal of primary melanoma lesions provide the best opportunities for cures or

prolonged survival to melanoma patients.221 Melanoma metastases are very aggressive and

the survival time for patients with metastatic melanoma is short (3 ‐ 15 months).221 This has

motivated several investigations aiming melanoma‐specific targeting for imaging and staging

but also for internal radiotherapy. One approach is the use of radiolabelled monoclonal

antibodies or antibody fragments directed against specific melanoma cell epitopes,222 which

accumulates preferentially in melanin‐producing melanoma by virtue of its affinity for the

pigment. So far, their clinical impact has been low because of the lack of specificity due, in

part, to the presence of individual tumor variants and the high occurrence of amelanotic

melanoma.223

Another approach was based on the finding that most murine and human melanoma

cells and melanoma tissues overexpress α‐melanocyte stimulating hormone (α‐MSH)

receptors, namely the melanocortin type 1 receptor (MC1R), making possible the use of

radiolabelled α‐MSH analogues as imaging tools.128‐135 These imaging tools would have the

advantage of being melanoma‐specific, must probably resulting in higher selectivity and

fewer false positives than imaging agents, such as 18F‐FDG and 99mTc‐MIBI that are non‐

specific for melanoma.221

Compared with the use of radiolabelled antibodies or antibody fragments, the use of

small peptides offers several advantages, already referred in section 1.3.2.115,124,125,126

The encouraging data on the use of cyclic α‐MSH analogues (section 1.3.2.) and

following previous work developed at the RSG/ITN with a linear α‐MSH analogue,224 it was


134

decided to prepare a new cyclic radioconjugate and to evaluate the effect of lactam

cyclization on its pharmacokinetic profile and tumor‐targeting properties.

The cyclic peptide βAla3‐Nle4‐cyclo[Asp5‐His‐D‐Phe7‐Arg‐Trp‐Lys10]‐NH2 formed from

lactam cyclization through the Lys and Asp side chains is an analogue of MT‐II peptide (Ac‐

Nle4‐cyclo[Asp5‐His‐D‐Phe7‐Arg‐Trp‐Lys10]‐NH2). Studies on the bioactive conformation of

MT‐II indicated that the type II β‐turn occurs in the His‐D‐Phe‐Arg‐Trp region, as compared

with a type III β‐turn of the linear analogue. The studies also indicated that the His, D‐Phe

and Trp side chains (affinity for the hydrophobic receptor pocket) are located on one surface

of the peptide, whereas the Arg side chain (affinity for the hydrophilic receptor pocket) was

on the opposite face.138 As a consequence, MT‐II has a high affinity for the melanocortin

receptors.

Thus, in this chapter, we report on the synthesis and characterization of the cyclic

peptide conjugate Pz‐βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2, as well as on its

radiolabelling with the fac‐[99mTc(CO)3]+ moiety. The in vitro/in vivo stability studies,

internalization/externalization in B16F1 cells, and biodistribution in murine melanoma‐

bearing mice of the radiolabelled conjugate will be also described. The advantages of using

the 99mTc(CO)3‐labelled cyclic analogue over the corresponding linear peptide for melanoma

targeting will also be discussed.

4.2. Synthesis and Characterization of Pz‐βAla‐Nle‐cyclo[Asp‐His‐

DPhe‐Arg‐Trp‐Lys]‐NH2

The Pz‐βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2 (43) (figure 4.1) was prepared

by standard solid‐phase synthetic methods,225 purified by preparative RP‐HPLC (purity ≥

97%), and characterized by ESI/QITMS and RP‐HPLC. In the ESI/QITMS spectra two main

peaks could be found at m/z 1303.9 and m/z 653.2 corresponding to [M+H]+ and [M+2H]2+,

respectively. RP‐HPLC chromatographic analysis showed only one peak with a retention time

of 9.9 min (figure 4.2).


135

NH

HN NH

OH2N

NH

O

NH

HN

O

NH

NH2

HN

NH

O

NH

N

HN O

HNO

HNO

O

O

NN NNH2

O

Figure 4.1 ‐ Structure of Pz‐βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2 (43).

Figure 4.2 ‐ Analytical RP‐HPLC chromatogram of the purified Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐

Lys]‐NH2 (43) (absorbance at 280 nm).

4.3. Synthesis of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐

Arg‐Trp‐Lys]‐NH2)]2+

The fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]+2 (44) was

prepared by reaction of the precursor fac‐[99mTc(CO)3(OH2)3]+ with the Pz‐βAla‐Nle‐

cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2 (43) as indicated in scheme 4.1.

50

100

0 2 4 6 8 10 12 14Time (min)


136

TcOC

H2O OH2

CO

OH2

CO

+99m

90 °C50 minpH = 7

43

NH

HN NH

OH2N

NH

O

NH

HN

O

NH

NH2

HN

NH

O

NH

NHN O

HNO

HNO

O

O

NN NNH2

O

99mNN N

NH2

TcOC

COCO

O HN

HN NH

OH2N

NH

O

NH

HN

O

NH

NH2

H2N

NH

O

NH

NHN O

HNO

HNO

O

O

44

2+

Scheme 4.1‐ Synthesis of the radioconjugate fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐

Arg‐Trp‐Lys]‐NH2)]2+ (44).

After optimization of the labelling conditions, namely temperature and reaction time,

radiochemical yields higher than 90% were obtained, using 6 x 10‐5 M of 43. The RP‐HPLC

analytical retention time was 11.7 min for fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐c[Asp‐D‐Phe‐Arg‐

Trp‐Lys]‐NH2)]2+ (44) (figure 4.3).

Figure 4.3 ‐ RP‐HPLC chromatogram of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐c[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐

NH2)]2+ (44) (γ trace).

50

100

0 5 10 15 20 25

Time (min)


137

The lipophilicity of the radiopeptide was evaluated by determination of the partition

coefficient in physiological conditions. The radioconjugate 44 revealed a moderately

hydrophobic character (log Po/w = 0.20 ± 0.03).


In order to increase specific activity and to maximize cellular and tumor uptake, the

fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ was separated from

the corresponding non‐metallated conjugate (Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐

NH2) by semi‐preparative RP‐HPLC. The fraction corresponding to the radioconjugate was

collected in a 50 mL Falcon vial, containing PBS or MEM solutions with 0.2% BSA (for

biodistribution or cell studies, respectively), to minimize the adsorption phenomena during

the evaporation of the elution solvents.

The stability of the purified radioconjugate in PBS and MEM solutions was evaluated

by RP‐HPLC and TLC, being found that no interaction with BSA and/or transmetallation took

place under those conditions. Indeed, the HPLC chromatogram (Supelguard LC 3 DP 2 cm x

4.6 mm, ID column) of the radioconjugate (44), after 4 h incubation at 37 °C in a 0.2% BSA

solution in MEM, displayed only the peak assigned to 44 (γ‐detection, retention time 6.0

min) and another peak due to BSA (UV‐detection, retention time 9.8 min). Furthermore, on

analyzing the TLC radiochromatogram of 44 after 24 h incubation at 37 °C in a 0.2% BSA

solution in MEM, no hydrolysed 99mTc species (Rf = 0), labelled BSA (Rf = 0), [99mTcO4]

‐ (Rf =

0.85) or fac‐[99mTc(CO)3(OH2)3]+ (Rf = 0.80) were detected, being the radiolabelled cyclic

conjugate the only radiochemical species present (Rf = 0.44) (figure 4.4). The results

obtained with HPLC and TLC indicate that 44 was stable in the presence of BSA and MEM.

Figure 4.4 ‐ TLC chromatogram of 44, after 24 h incubation at 37 °C in culture medium (γ trace).

0


138

To assess the resistance of the purified radioconjugate 44 to proteolytic degradation

caused by endogenous peptidases and to predict its in vivo stability, compound 44 was

incubated with fresh human plasma at 37 °C. Analysis by RP‐HPLC at different time points (0

min, 5 min, 45 min, and 4 h) indicated high plasma stability (purity ≥ 98%) with negligible

degradation of the radioconjugate (figure 4.5).

Figure 4.5 ‐ Analytical RP‐HPLC chromatogram of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐

Arg‐Trp‐Lys]‐NH2)]2+ after incubation in human serum at 37 °C at different time points (γ trace).

4.5. In Vitro Studies in B16F1 Murine Melanoma Cells ‐

Internalization and Cellular Retention

The internalization of cell receptors, after binding to the radiolabelled peptide,

promotes and extends the retention of the radioactivity inside the target cells, improving the

5 10 15

Initial preparation

2 h

4 h

24 h

Time (min)


139

quality of the scintigraphic image in diagnostic applications.125 The conjugate must enter the

cells by a specific process but must also be retained enough time to allow the acquisition of

an image. For this reason, the cellular retention of the 99mTc complex, after internalization,

must also be studied.

Internalization studies performed for the radioconjugate 44 at room temperature

and at 37 °C revealed that the cellular uptake was temperature and time‐dependent (figure

4.6).

0

10

20

30

40

50

60

0.1 0.5 1 2 3 4 5 6

Incubation time (h)

% Uptake/total activity Internalized

Surface bound

0

20

40

60

80

100

0.1 0.5 1 2 3 4 5 6

Incubation time (h)

% Uptake/bo

und activity

Surface boundinterna l i zed

0

20

40

60

80

100

0.1 0.5 1 2 3 4 5 6

Incubation time (h)

% Uptake/

bou

nd activity Surface bound

Internal ized

0

10

20

30

40

50

60

0.1 0.5 1 2 3 4 5 6

Incubation time (h)

% Uptake/total activity

InternalizedSurface bound

A B

C D

Figure 4.6 ‐ Cell studies of the radioconjugate 44 in B16F1 cells at different time points and

temperatures. Internalized and surface bound activity expressed as a fraction of bound activity (activity on

the membrane surface and inside the cell) at room temperature (A) and 37 °C (B); Internalized and surface

bound activity expressed as a percentage of total activity at room temperature (C) and at 37 °C (D) (mean ±

standard deviation, n = 3).

As shown in figure 4.6, high levels of internalization were reached for the fac‐

[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+(44). For instance, at 4 h

post‐incubation (37 °C), 79% of the total cell‐associated activity was taken up and

internalized by the cells (figure 4.6B). When the internalization is expressed as a percentage

of total activity, 41.8% of 44 was internalized by the cells 4 h after incubation (figure 4.6D).


140

These values are particularly high when compared with data previously reported for other

radiolabelled α‐MSH analogues. Indeed, for the linear analogue fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐

Nle‐Asp‐D‐Phe‐Arg‐Trp‐Lys‐NH2)]+2 only 1.6% of the total administered activity was

internalized by the cells, after 4 h incubation (37 °C).224 For the 64Cu‐DOTA‐NAPamide and 99mTc(CO)3‐Pz‐NAPamide the maximum internalization observed was ca. 4.5% of applied

activity 3 h after incubation.58,145 In the case of the cyclic radiopeptide 99mTc‐CCMSH, using

the same experimental conditions as those used for the radioconjugate 44 (0.2 x 106

melanoma B16F1 cells per well and 200 000 cpm of radioconjugate per well), the maximum

internalization achieved was less than 4%.22

The cellular retention of the radioconjugate was evaluated at different time points in

the same type of cells at 37 °C, after 3 h of incubation. A remarkably high degree of cellular

retention was observed for the conjugate 44 (figure 4.7). Indeed, 44 was slowly released

from the cells into the medium, with about 75% of the initially internalized compound still

remaining inside the cells after 4 h of incubation. This value was only 35.3% for the linear

radioconjugate fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐Asp‐DPhe‐Arg‐Trp‐Lys‐NH2)]+2 at the same

time point.224 The enhanced internalization and intracellular retention of the cyclic

radioconjugate 44, compared with that of its linear counterpart (fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐

Nle‐Asp‐DPhe‐Arg‐Trp‐Lys‐NH2)]2+), is probably related with the compact structure of the

cyclic radioconjugate. In fact, this structure seems to confer a more potent agonistic binding

behaviour to the peptide and an increased resistance to proteolysis.

70

75

80

85

90

95

100

0 1 2 3 4 5

Incubation time (h)

% cellular retention

Figure 4.7 ‐ Cellular retention of internalized cyclic radioconjugate 44 in B16F1 cells over time at 37

°C (mean ± standard deviation, n = 3).


141

4.6. Biodistribution and In Vivo Stability of fac‐[99mTc(CO)3‐(κ3‐Pz‐

βAla‐Nle‐Asp‐DPhe‐Arg‐Trp‐Lys‐NH2)]2+

The biodistribution of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐

NH2)]2+ (44) was examined in B16F1 murine melanoma‐bearing C57BL/6 female mice. The

primary skin melanoma was generated by implanting subcutaneously 1 x 106 B16F1 cells.

Ten to twelve days after the inoculation, tumors reached a weight of 0.2 ‐ 1 g. Animals were

intravenously injected into the retroorbital sinus with the radioconjugate 44 diluted in 100 μl

of PBS, pH 7.2. The biodistribution was evaluated at 1, 4, and 24 h post‐injection (p.i.).

Biodistribution data was expressed as percentage of injected dose per gram (%ID/g) (table

4.1) and percentage of injected dose per organ (% ID/organ) (figure 4.8).

Table 4.1 ‐ Biodistribution of fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+

(44) in B16F1 murine melanoma‐bearing C57BL/6 mice at 1, 4 and 24 h, after intravenous injection

(mean ± standard deviation, n = 4 ‐ 5).


1 h 4 h 4 h with NDPa 24 h

Tumor 9.26 ± 0.83 11.31 ± 1.83 2.97 ± 0.62 3.48 ± 0.40

Blood 2.71 ± 0.64 1.67 ± 0.24 1.46 ± 0.13 0.20 ± 0.05

Liver 42.19 ± 5.05 22.86 ± 1.17 24.69 ± 3.16 1.72 ± 0.12

Intestine 5.17 ± 0.91 8.45 ± 0.76 11.00 ± 1.04 1.15 ± 0.37

Spleen 2.54 ± 0.28 2.24 ± 0.37 1.86 ± 0.37 0.46 ± 0.09

Heart 1.04 ± 0.19 0.48 ± 0.07 0.44 ± 0.05 0.15 ± 0.01

Lung 3.85 ± 0.46 1.54 ± 0.16 1.72 ± 0.32 0.90 ± 0.52

Kidney 71.06 ± 6.44 32.12 ± 1.57 42.37 ± 3.07 1.48 ± 0.24

Muscle 0.35 ± 0.07 0.19 ± 0.08 0.18 ± 0.06 0.03 ± 0.00

Bone 1.14 ± 0.19 0.70 ± 0.13 0.48 ± 0.04 0.09 ± 0.01

Stomach 1.97 ± 0.75 0.88 ± 0.46 0.93 ± 0.27 0.25 ± 0.30

Pancreas 0.73 ± 0.32 0.39 ± 0.10 0.26 ± 0.04 0.07 ± 0.01

Skin 0.84 ± 0.12 0.53 ± 0.10 0.57 ± 0.10 0.13 ± 0.08

Uptake ratio of tumor to normal tissue:

Tumor: blood 3.4 6.8 2.0 17.4

Tumor: muscle 26.5 61.4 16.5 116

Excretion (% ID) 14.2 ± 2.3 37.4 ± 2.4 33.0 ± 1.5 87.6 ± 3.2 aNDP (Nle4, DPhe7)‐αMSH (see table 1.8); aCoinjection of the radioconjugate with NDP


142

0

10

20

30

40

50

60

70

80

90

Bloo

d

skin

muscule

bone

tumor

Liver

Intestine

kidn

eys

excretion

% ID

/organ

24 h

4 h

1 h

Figure 4.8‐ Biodistribution results (% ID/organ) and total excretion (% ID) of the fac‐[99mTc(CO)3‐(κ3‐

Pz‐βAla‐Nle‐c[Asp‐DPhe‐Arg‐Trp‐Lys]‐NH2)]2+ (44) in B16F1 murine melanoma‐bearing C57BL/6 mice

at 1, 4 and 24 h after intravenous injection.

Biodistribution data indicate a relatively fast clearance from the bloodstream (2.71 ±

0.24 % ID/g, 4 h p.i), as 1 h after injection most of the complex was not in circulation. No

preferential uptake in the main organs or tissues was observed, including skeletal and soft

tissues like muscle, except for the liver and kidney. As a consequence of the high retention

and slow clearance from the liver and kidney, the overall excretion from the whole animal

body was significantly low. Indeed, 1 h and 4 h after injection only 14.2% and 37.4% of the

total activity was eliminated. After 24 h, the kidney and liver uptake decreased to values

lower than 2% ID/g and the excretion was almost total (87.6%).

The high liver uptake must be probably related with the lipophilic character of 44 (log

Po/w = 0.20 ± 0.03). A shift from the hepatobiliary towards the renal excretion pathway could

be achieved by reducing the lipophilicity of the radiopeptide, which means to introduce Asp

or Glu residues (polar, negatively charged, and very hydrophilic amino acids) in the peptide

sequence.

The most remarkable result observed in the biodistribution data was the fast, high

and specific activity accumulation in the tumor. Indeed, 1 h after injection a tumor uptake of


143

9.26 ± 0.83 % ID/g was found, with this value increasing slowly to 11.31 ± 1.83 % ID/g at 4 h

after injection. The activity was then slowly washed out from the tumor, and the uptake

dropped to a still relevant value of 3.48 ± 0.40 % ID/g over 24 h. The high tumor uptake and

retention are in agreement with the in vitro studies, which show high internalization and

slow washout.

The in vivo specificity of the radioconjugate 44 for the MC1 receptor was evaluated

by its co‐administration with NDP‐α‐MSH peptide (linear α‐MSH peptide analogue with

picomolar affinity for the MC1 receptors expressed on murine melanoma cells) in the same

animal model. The receptor‐blocking studies at 4 h after injection revealed that the tumor

uptake was significantly reduced by 73.7% in the presence of NDP, without significant

changes in the biodistribution profile of the radioconjugate in healthy tissues (table 4.2).

These results confirmed that the tumor uptake of the radioconjugate 44 is MC1 receptor‐

mediated, and that the peptide kept its biological activity upon conjugation to the pyrazolyl‐

diamine chelator and coordination to the moiety fac‐[99mTc(CO)3]+. Tumor‐to‐blood and

tumor‐to‐muscle ratios (table 4.1) were high despite the slow clearance from non‐target

organs (liver and kidney). Furthermore, the tumor‐to‐blood and tumor‐to‐muscle ratios

increased from 6.8 and 61.4 (4 h after injection) to 17.4 and 116 (24 h after injection), which

also indicates a receptor‐mediated transport and subsequent intracellular trapping of this

radiocomplex in the MC1 receptor‐expressing tumor.

The high tumor uptake and retention found for the radioconjugate 44 (9.26 ± 0.83

and 11.31 ± 1.83 % ID/g at 1 h and 4 h after injection, respectively), compared to its linear

counterpart (1.96 ± 0.17 and 0.99 ± 0.08 % ID/g at 1 h and 4 h after injection,

respectively),224 are in full agreement with the data obtained for the cyclic 99mTc‐CCMSH

analogue (11.64 ± 1.54 and 9.51 ± 1.97 % ID/g at 1 h and 4 h after injection, respectively),

using the same animal model.22,151 Also, the linear 111In‐DOTA‐CCMSH exhibited a much

lower tumor uptake than the cyclic homolog 111In‐DOTA‐ReCCMSH (4.32 ± 0.59 vs. 9.49 ±

0.90 % ID/g, 4 h after injection). All those data show clearly the benefit of peptide

cyclization.151

However, cyclization of the linear peptide through lactam formation led to an

impressive increase in kidney uptake (for the linear peptide 1.64 ± 0.19 % ID/g224 and for the

cyclic peptide 32.12 ± 0.19 % ID/g, at 4 h after injection). It is believed that one hypothesis of


144

non‐specific renal activity accumulation is due to the electrostatic interaction between

positively charged peptides and the negatively charged surface of the renal tubules. The co‐

administration of cationic amino acids, such as lysine or arginine, has been a strategy to

decrease the renal uptake of radiolabelled peptides and proteins.217‐219,226,227

Taking this into account, a second set of biodistribution experiments in healthy mice

were performed. In these experiments lysine was co‐injected with the radioconjugate 44

(table 4.2). Interestingly, co‐administration of this basic amino acid did not reduce

significantly kidney accumulation of 44, which seems to indicate that the electrostatic

interaction between the positively charged complex and the negatively charged surface of

the proximal tubules is not the major factor for tubular reabsorption. This behaviour has

already been observed for similar α‐MSH analogues labelled with other radiometals (M‐

DOTA‐Re(Arg11)CCMSH; M = 90Y, 177Lu).226 In these studies, the megalin was proposed to be

involved in the renal uptake of the complexes, because megalin is expressed in the proximal

tubules and acts as a scavenger receptor for endocytosis of multiple ligands in the kidney.228

Table 4.2 ‐ Biodistribution of 44 co‐injected or not with 15 mg of L‐lysine in healthy C57BL/6 mice, at

1h after intravenous injection (mean ± standard deviation, n = 5).


With lysine Without lysine

Blood 3.03 ± 1.75 2.83 ± 1.04

Liver 28.15 ± 4.05 24.71 ± 1.94

Intestine 2.79 ± 0.72 2.70 ± 0.68

Spleen 1.87 ± 0.36 2.39 ± 0.66

Heart 0.96 ± 0.30 0.86 ± 0.14

Lung 2.67 ± 0.95 2.07 ± 1.03

Kidney 49.44 ± 5.87 59.38 ± 11.79

Muscle 0.30 ± 0.08 0.31 ± 0.05

Bone 0.75 ± 0.30 0.79 ± 0.21

Stomach 3.36 ± 1.57 4.62 ± 2.94

Adrenals 11.31 ± 8.61 4.98 ± 2.29

Pancreas 0.44 ± 0.15 0.66 ± 0.48

Skin 1.08 ± 0.15 1.15 ± 0.18

Total Excretion (%) 17.0 ± 2.2 12.7 ± 2.2


145

In vivo stability studies

To study the in vivo stability of complex fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐

Phe‐Arg‐Trp‐Lys]‐NH2)]2+ (44), urine and blood samples were collected from the sacrificed

C57BL/6 mice 1 h after injection of 44. After appropriate treatment, the biological samples

were analysed by RP‐HPLC (figure 4.9).

Figure 4.9 ‐ RP‐HPLC chromatograms of complex 44, blood serum and urine samples collected 1 h

after injection (γ trace).

In vivo stability studies revealed that the radioconjugate was stable in blood serum (1

h after injection), as no metabolites could be clearly detected (figure 4.9). These results

indicate again that metal complexation via the tridentate pyrazolyl‐diamine backbone

overcomes potential binding/transmetallation to coordinating residues in circulating

proteins, such as histidine, cysteine or methionine. In contrast, several metabolites were

found in urine. These metabolites present lower retention time than the injected compound,

but were not identified.

5 10 15Time (min)

Initial preparation

serum

urine


146

In conclusion, a MT‐II‐based cyclic radiopeptide, fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐

cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ (44), was prepared in high yield and radiochemical

purity. The radioconjugate 44 displayed good stability in vitro and in vivo. The cyclization

through a lactam bridge resulted in a compact structure, which enhanced remarkably the

cellular internalization and retention, as well as the MC1R‐mediated tumor uptake in B16F1

melanoma‐bearing mice. Despite the promising tumor‐targeting properties exhibited by the

cyclic radiopeptide 44, its pharmacokinetic profile still has to be improved. Higher excretion

and lower kidney uptake must be reached, either by using bifunctional chelating agents with

different physicochemical properties (e.g. charge and hydrophilicity) or by adding negatively

charged amino acids such as glutamate or aspartate to the peptide sequence studied in this

work.

5.

Concluding Remarks and Outlook

5. Conclusions

149

5. Concluding Remarks and Outlook

The aim of this thesis was to increase the knowledge on the design of 99mTc specific

radiopharmaceuticals for imaging endogenous gene expression or membrane receptors. The

chosen targets were N‐MYC mRNA, overexpressed in peripheral and central nervous system

tumors, and MC1 receptors overexpressed in malignant melanoma.

The fac‐[99mTc(CO)3]+ was the selected core and its conjugation to the biomolecules

was performed using the bifunctional approach. Knowing that tridentate chelators form

stable complexes with the fac‐[99mTc(CO)3]+ core, we have selected pyrazolyl‐ and cysteine‐

containing chelators, which are N3 and N, O, S donors, respectively (scheme 5.1).46‐52,56‐58

Scheme 5.1 ‐ Pyrazolyl‐ and cysteine‐containing bifunctional chelators.

We have chosen a PNA sequence for imaging the N‐MYC mRNA, knowing that PNA is

a DNA mimic that presents remarkable properties. Once there was no experience on the

conjugation of the cysteine‐ and the pyrazolyl‐containing chelators to PNA sequences, the

possibility of conjugating PNA units (monomer and dimer) to these chelators was first

explored, as well as the coordination capability of the resulting conjugates towards the fac‐

[M(CO)3]+ (M = Re, 99mTc) moiety. We have isolated and characterized cysteine‐ and

pyrazolyl‐containing chelators bearing PNA units (13, 15 and 17). Compounds 13, 15 and 17

react with fac‐[M(CO)3(OH2)3]+ yielding the complexes fac‐[M(CO)3(κ3‐13)] (M = Re (18),

99mTc (21)), fac‐[M(CO)3(κ3‐15)]+ (M = Re (19), 99mTc (22)) and fac‐[M(CO)3(κ3‐17)]+ (M = Re

(20), 99mTc (23)) (scheme 5.2). For the Re complexes, 19 and 20, NMR studies have been

very informative: the tridentate coordination mode of the pyrazolyl‐containing ligand was

SNH2

O

HO

O

OH

NN

NNH2

O

OH

FM

M

BM

BM

M

5. Conclusions

150

clear as well as the presence of rotamers in solution, due to the nature of the PNA units. For

18 the NMR data were more complex and not very informative, possibly due to the

presence of diastereoisomers and rotamers in solution. The 99mTc tricarbonyl model

complexes 21, 22 and 23 were prepared in high yield. Complexes 21 and 23 have high in

vitro and in vivo stability and a good biological profile. The excretory route was mainly the

renal‐urinary pathway, with a small contribution of the hepatobiliary tract.

The formation of these model complexes in high yield, their high stability and the

favorable tissue distribution profile, confirmed the possibility of attaching the pyrazolyl‐ and

cysteine‐containing chelators to a PNA sequence complementary to the N‐MYC mRNA.

The 16‐mer PNA sequence, N‐A GAT CAT GCC CGG CAT‐C, complementary to the

region of the N‐MYC mRNA beginning with the ATG start codon, was chosen based on

published data.184,186,187 The sequence H‐A GAT CAT GCC CGG CAT‐LysNH2 (38) was

synthesized using solid phase techniques, on a resin downloaded with lysine and using Fmoc

chemistry. Conjugation of the cysteine‐containing chelator (11) to the PNA sequence was

possible but the conjugate 41 was obtained in very low yield (scheme 5.2). Optimization of

the reaction conditions could certainly be achieved, but it was not tried due to time

limitations. The pyrazolyl‐containing chelator (Pz‐Boc) and fac‐[Re(CO)3(3,5‐

Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ (RePz) were conjugated to the PNA sequence

yielding Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) and the complex fac‐[Re(CO)3(κ3‐Pz‐ A GAT

CAT GCC CGG CAT‐Lys‐NH2)]+ (40) (scheme 5.2). Complex 40 and sequence 38 were used for

melting temperature (Tm) experiments. Melting temperature studies indicated that the

introduction of the Re tricarbonyl complex in the PNA sequence did not affect the

recognition of the complementary sequence, as well as the duplex stability. The high Tm

value of 83.5 ± 0.1 °C also predicts a high duplex stability in vivo between the 99mTc complex

congener and the target mRNA.

The Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) reacted quantitatively with fac‐

[99mTc(CO)3(OH2)3]+ yielding the radioactive complex fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC

CGG CAT‐Lys‐NH2)]2+ (42) with high specific activity (scheme 5.2). The characterization of 42

was made by comparing its HPLC profile with the profile of complex 40.

Preliminary cell internalization studies of 42 with SH‐SY5Y neuroblastoma (ca. 7.5%),

MCF7 breast cancer (ca. 7.5%) and PC3 prostate cancer cells (ca. 10%) have shown relatively

high internalization levels of the complex. With the cells SH‐SY5Y a relatively high cellular

5. Conclusions

151

retention was also found (ca. 60%) 5 h after incubation. A correlation between our values

and other reported in the literature is difficult, because most of the values reported for

unassisted PNA sequences were evaluated either by fluorescence microscopy or using a 14C‐

labelled compound. There is only one example of radiolabelled PNA assisted with a

somatostatin analogue (111In‐DOTA‐ BCL‐2 PNA‐Tyr3‐octreotate).76 In this work, recently

reported, the in vitro cellular uptake was quantified and the values found for the cellular

internalization and cellular retention, after 4 h incubation, were 5.2% and 60%, respectively.

Comparing these values with ours and taking into account that our PNA conjugate is not

assisted by any vector, we can say that our results are relatively high and encouraging for

further work.

With the studies performed we cannot say if the cellular retention is due to the

interaction of 42 with the complementary N‐MYC mRNA. Further studies with IMR32 cells,

overexpressing N‐MYC mRNA, as well as control experiments using mismatch and/or sense

probes, are still necessary. The conjugation of a specific vector to 42 would also be

interesting to do, to confer specificity and also to increase the cellular uptake and retention

of the labelled PNA.

The biological profile of 42 in normal mice has not been very satisfactory. We have

found a high kidney (84.64 ± 6.07 % ID/g, 4 h p.i.) and liver uptake (24.06 ± 4.99 % ID/g, 4 h

p.i.). In a second set of biodistribution studies, 42 and L‐lysine were co‐injected to see

whether the kidney uptake would decrease. After this co‐injection the kidney retention was

reduced by 63%, but the values are still unaceptable. So, the pharmacokinetics of 42 has to

be improved either by modifying the PNA length and/or by adding a specific vector.

We have synthesized and characterized a pyrazolyl‐containing chelator bearing an

acridine orange moiety at the 4‐position of the pyrazolyl ring, while keeping the central

amine for latter coupling of a biologically active molecule. The main goal of these

preliminary studies was to evaluate the possibility of designing multimodal probes and/or to

explore the Auger emitter 99mTc for therapy. The resulting conjugate 33 reacts with fac‐

[M(CO)3(OH2)3]+ (M = Re, 99mTc) originating fac‐[M(CO)3(κ3‐33)]2+ (M = Re (34), M = 99mTc

(35)) in high yield (scheme 5.2). The radioactive complex 35 presents moderate cellular

internalization in B16F1 cells (7.8%, 5 h after incubation, purified compound), with a high

amount of the internalized compound entering the cell nucleus. Complex 35 also presents a

high cellular retention (ca. 60%).

5. Conclusions

152

Using isostructural Re (34) and 99mTc (35) complexes, it was possible to visualize the

localization of the complex in the cells and at the same time to quantify the amount of

complex entering the cell. The localization was achieved by fluorescence microscopy studies

using complex 34 and the quantification was possible by measuring the activity of 35 in the

cytoplasm and in the nucleus. Taking into account that the Re and 99mTc complexes are

isostructural, the combination of this information shows that the acridine orange derivative

and the metals (Re/99mTc) are located in the cytoplasm and the nucleus of the cells.

Therefore, these results put together indicate that there is a strong probability that the Re

and 99mTc complexes reach the cytoplasm and especially the nucleus of the cells in an intact

form.

The radiocomplex 35 has in its composition an Auger emitter (99mTc) and a planar

molecule, the acridine orange moiety, which is known to intercalate DNA. Moreover, this

complex has the ability to reach the nucleus of the tumoral B16F1 murine melanoma cells.

Taking these aspects into account, the radiotoxic effect of 35 was studied in order to

evaluate its potential usefulness for designing radiotherapeutic compounds. The

radiotoxicity studies were performed in B16F1 cells using the MTT test. Our results have

shown no radiotoxic effect, despite the high amount of compound in the cell nucleus.

Further studies are being performed in the RSG/ITN to better understand such result.

The biodistribution of 35 was also studied. The profile was not encouraging as the

complex presented a high liver (36.12 ± 12.76 % ID/g, 4 h p.i.) and intestine retention (17.01

± 5.50 % ID/g, 4 h p.i.) and a low overall total excretion. Lower liver and intestine uptake and

consequently higher excretion could be reached by increasing the hydrophilic character of

the complex. This could be achieved by incorporating for example carboxylic groups in the

chelator backbone or after coupling peptide sequences to the pyrazolyl‐acridine orange

conjugate.

For imaging the MC1 receptors overexpressed in melanoma cells, radiolabelled α‐

MSH analogues were considered potentially interesting imaging tools. Following previous

work at the RSG/ITN,224 a new cyclic α‐MSH analogue, βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐Arg‐

Trp‐Lys]‐NH2, was synthesized and conjugated to the bifunctional pyrazolyl‐containing

ligand, yielding Pz‐βAla‐Nle‐cyclo[Asp‐His‐D‐Phe‐Arg‐Trp‐Lys]‐NH2 (43). Compound 43 was

prepared using solid phase synthetic methods225 and reacts with fac‐[99mTc(CO)3(OH2)3]+

originating fac‐[99mTc(CO)3‐(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐D‐Phe‐Arg‐Trp‐Lys]‐NH2)]2+ (44), in high

5. Conclusions

153

yield (scheme 5.2). The radioconjugate 44 displayed good stability in vitro and in vivo.

Compound 44 has shown a remarkable cellular internalization (41.8% after 4 h incubation)

and retention (75% after 4 h incubation) in B16F1 murine melanoma cells compared to the

linear analogue (1.6% and 35.3%, respectively). Such result may be due to the compact

structure of the peptide due to the cyclization through a lactam bridge. In vivo studies, using

melanoma‐bearing mice, have also shown a significant tumor uptake that has been

confirmed to be specific, i.e. MC1R‐mediated. Despite the promising tumor‐targeting

properties exhibited by the cyclic radiopeptide 44 (11.31% ID/g, 4 h p.i.), its pharmacokinetic

profile still has to be improved. In fact, the overall excretion has to be increased. One

possibility would be to use bifunctional chelating agents with different physicochemical

properties (e.g. charge and hydrophilicity) or to introduce other amino acids (glutamate or

aspartate) in the sequence of the peptide, preserving the biologically active part.

5. Conclusions

155

Scheme 5.2 ‐ Illustration of the main compounds described in this thesis – tricarbonyl metal complexes and PNA conjugates.

M

OC

H2O OH2

CO

OH2

CO

OSNH2

O

O

M

OC CO CO

NH

N

O

NH

N

O

O

O

OHNN N

NH2

M

OC CO CO

O

NH

N

O

NH

N

O

O

O

OH

+

99mNN N

NH2

TcOC

COCO

O HN

HN NH

OH2N

NH

O

NH

HN

O

NH

NH2

H2N

NH

O

NH

NHN O

HNO

HNO

O

O

2+

O

NN

N

NH

NN NH

NH2

M

OC CO CO

2+

NN N

NH2

M

OC CO CO

O

NH

N

O

NH

N

O

O

ONH

N

O

NH

N

O

O

O

OH

+

13 15

17

33

43


NN N

NH2

Tc

OC CO CO

O H2N

NH3

OHN2+

99m

M = Re (18), 99mTc (21)

M = Re (19), 99mTc (22) M = Re (20), 99mTc (23)

4244

M = Re (34), 99mTc (35)

MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG


NN N

NH2

O


H2N

NH2

OHN

38

41

H‐A GAT CAT GCC CGG CAT Lys NH2

SF3COCHN

O

OO


H2N

NH2

ONH

40


NN N

NH2

Re

OC CO CO

O H2N

NH2

ONH+

39

37

SF3COCHN

O

OO

O

F

F

FF

F

11

M = Re, 99mTc

NN NNHBoc

OOH

Pz‐Boc

NN N

NH2Re

OC CO CO

O

OH+

RePz

6.

Experimental Part

6. Experimental Part

159


6.1. Materials

All chemicals and solvents were of reagent grade and were used without purification,

unless stated otherwise. The organometallic precursor fac‐[Re(CO)3(H2O)3]Br was prepared

according to a published method;229 compounds 3,5‐

Me2pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (Pz‐Boc) and fac‐[Re(CO)3(3,5‐

Me2pz(CH2)2N((CH2)3COOH)(CH2)2NH2]+ (RePz) were prepared according to the literature.56

6.2. Characterization and Purification Techniques

Elemental Analysis C, H, N, S

C, H, N and S analyses were performed in a EA110 CE Instruments automatic

analyzer.

Infrared Spectroscopy (IR)

Infrared spectra were recorded as KBr pellets in a Bruker Tensor 27 spectrometer.

Nuclear Magnetic Resonance Spectrometry (NMR)

1H, 13C and 19F NMR spectra were recorded in a Varian Unity 300 MHz spectrometer;

1H and 13C chemical shifts were referenced to the residual solvent resonances relative to

tetramethylsilane (SiMe4) and 19F chemical shifts were referenced relatively to trifluorotoluol

or trifluoracetic acid.

In the NMR spectra of some compounds rotamers were observed and were assigned

as major (ma) or minor (mi) species.


160

Multiplicities are reported using the following abbreviations: s (singlet), d (doublet), t

(triplet), q (quartet), quint (quintuplet), m (multiplet), br (broad) or a suitable combination of

them.

Column Chromatography

Some compounds were purified by column chromatography, using silica gel 60 with

70 ‐ 230 mesh granulometry (ASTM Merck) and glass columns with dimensions appropriate

to the amount of compound to purify.

Thin Layer Chromatography (TLC)

Some chemical reactions were monitored by TLC, using silica‐gel plates MERCK 60‐

F254 with 0.25 mm of thickness, in an aluminium support; the plates were analysed with UV

radiation or I2.

High Performance Liquid Chromatography (HPLC)

Reverse phase HPLC analyses were performed with a Perkin Elmer LC pump 200

coupled to an UV/VIS detector (Shimadzu SPD‐10 AV or Perkin Helmer LC 290) or to a γ

detector (Berthold‐LB 507A or LB 509) (ITN, Portugal). Some HPLC analyses of peptide

nucleic acids were performed in an Agilent 1100 series coupled to an UV‐VIS diode‐array

detector (UNIMI, Milan, Italy).

The solvents were of HPLC grade and the water bidistilled from a quartz distillation

unit. The solvents were filtered by Millipore 0.22 μm filters and purged with helium.

Analytical Control of Compounds 5 ‐ 8, 12, 13, 15 ‐ 23:

Column: Analytical, EC250/3 Nucleosil 100‐5 C18, Macherey Nagel; Pre‐column: EP 30/8

Nucleosil 100‐7 C18, Macherey Nagel; Flow: 0.5 mL/min; γ detection; UV detection: λ = 254

nm; Eluents: A ‐ TFA 0.1 % in H2O; B ‐ CH3OH.


161

Analytical Control of compounds 33 ‐ 35:

Column: Analytical, EC 250/4 Nucleosil 100‐5 C18, Macherey Nagel; Pre‐column: EP 30/8

Nucleosil 100‐7 C18, Macherey Nagel; Flow: 0.9 mL/min; γ detection; UV detection: λ = 254

nm; Eluents: A ‐ TFA 0.1 % in H2O; B ‐ CH3OH or CH3CN.

Purification of Compounds 13, 15 ‐ 17:

Column: Preparative, Waters Bondapak C18 (150/19); Pre‐column: Hypersil C18 (ODS), 4.6 ×

25 mm, 10 μm; Flow: 5.0 mL/min; UV detection: λ = 254 nm; Eluents: A ‐ TFA 0.1 % in H2O;

B ‐ CH3OH or CH3CN.

Purification of Compounds 18 ‐ 20:

Column: Semi‐Preparative, EP 250/8 Nucleosil 100‐7 C18, Macherey Nagel; Pre‐column: EP

30/8 Nucleosil 100‐7 C18, Macherey Nagel; Flow: 2.0 mL/min; γ detection, UV detection: λ=

254 nm; Eluents: A ‐ TFA 0.1 % in H2O; B ‐ CH3OH or CH3CN.

HPLC Method 1:

B ‐ CH3OH

Step Time (min) % B

0 10 0

1 0 ‐ 3 0

2 3 ‐ 3.1 0 → 25

3 3.1 ‐ 9 25

4 9 ‐ 9.1 25 → 34

5 9.1 ‐ 18 34 → 100

6 18 ‐ 25 100

7 25 ‐ 25.1 0

8 25.1 ‐ 30 0


162

HPLC Method 2:

B ‐ CH3OH

Step Time (min) % B

0 10 10

1 0 ‐ 5 10

2 5.1 ‐ 30 10 → 100

3 30 ‐ 34 100

4 34 ‐ 35 100 → 10

5 35 ‐ 40 10

HPLC Method 3:

As method 1 with B = CH3CN.

Chromatographic Conditions for PNA Compounds 38 ‐ 42:

Analytical Control

Column: Analytical, 5 μm (250 X 4.6 mm) C18, Discovery® BioWide Pore analytic; Flow: 1.0

mL/min; γ detection, UV detection: λ = 220 nm; 260 nm; 280 nm; diode‐array; Eluents: A ‐

0.1% TFA in H2O; B ‐ 0.1% TFA in CH3CN.

Purification

Column: Semi‐Preparative, 5 μm (250x10 mm) C18, Discovery® BioWide Pore; Flow: 4.0

mL/min; γ detection, UV detection: λ= 220 nm; 260 nm; 280 nm; Eluents: A ‐ 0.1 % TFA in

H2O; B ‐ 0.1% TFA in CH3CN.

HPLC Method 4:

Step Time (min) % B

0 5 0

1 0 ‐ 3 0

2 3 ‐ 50 0 → 100

3 50 ‐ 60 100 → 0


163

Chromatographic Conditions for Compounds 43 ‐ 44:

Analytical Control:

Column: Hypersil ODS column (250/4 mm, 10 μm): Flow: 1 mL/min; γ detection, UV

Detection: λ = 280 nm; Eluents: A ‐ 0.5 % TFA in H2O; B ‐ 0.5% TFA in CH3CN

HPLC Method 5:

Step Time (min) % B

0 5 15

1 0 ‐ 18 15 → 100

2 18 ‐ 20 100 → 15

3 20 ‐ 25 15

Purification:

Column: Semi‐Preparative, Hypersil ODS column (250/8 mm, 10 μm); Flow: 3 mL/min; γ

detection, UV detection: λ= 280 nm; Eluents: A ‐ 0.5 % TFA in H2O; B ‐ 0.5% TFA in CH3CN.

Method 6:

Step Time (min) % B

0 5 15

1 0 ‐ 15 15 → 30

2 15 ‐ 30 30

3 30 ‐ 45 30 → 40

4 45 ‐ 60 40

Mass Spectrometry

ESI/QITMS analyses of compounds 7, 17, 19 and 20 were performed with a Bruker

Esquire 3000 plus (Mass Spectrometry Laboratory, ITQB/IBET, Oeiras, Portugal).


164

LC‐ESI/QITMS and ESI/QITMS analyses of PNA compounds were performed with a

Thermo Finnigan LCQ Advantage (UNIMI, Milan, Italy). A Discovery® BioWide Pore C18

reversed‐phase column (5 μm, 250X4.6 mm) was used for the LC‐MS analyses.

High resolution ESI/FTICR mass spectra were recorded using a Bruker Daltonics APEX

II (UNIMI, Milan, Italy).

MALDI‐TOF mass spectra were obtained with a Bruker Daltonics Microflex LT, using

sinapinic acid (3,5‐dimetoxy‐4‐hydroxycinamic acid 98%) as matrix (UNIMI, Milan, Italy).

ESI/QITMS analyses of compounds 18, 33 and some PNA compounds were performed

with a Bruker HCT (ITN, Sacavém, Portugal; the instrument was acquired with the support of

the Programa Nacional de Reequipamento Científico of FCT and is part of Rede Nacional de

Espectrometria de Massa ‐ RNEM).

Gas Chromatography (GC)

Gas chromatography analyses were performed in an Agilent HP 1100 6890 with

autosampler, split/splitless injector, thermal conductivity detector, and a capillary column.

The temperature profile for the run was as follows: initial temperature 190 °C, hold 2 min,

ramp at 15 °C/min to 260 °C, ramp at 20 °C/min to 280 °C; injections were 5 or 10 μl.

Solid Phase Synthesis

An ABI 433A peptide synthesizer (Applied Biosystems) was used for automated

Peptide Nucleic Acids solid phase synthesis.

UV‐Melting Temperature Experiments

The 16‐mer single strand DNA sequence 5’‐ATG CCG GGC ATG ATC T‐3’ was

purchased from Primm srl, Italy. The concentration of the ssDNA was determined by UV

absorption measurements at 260 nm, using an extinction coefficient of 148 280 M‐1 cm‐1.

PNA concentrations were also determined by UV absorption measurements at 260 nm using

the following molar extinction coefficients:230 ε260 adenine = 13 700 M‐1 cm‐1, ε260 guanine =


165

11 700 M‐1 cm‐1, ε260 thymine = 8 600 M‐1 cm‐1, ε260 cytosine = 6 600 M‐1 cm‐1 and ε260

RePzCOOH = 2 939 M‐1 cm‐1 (calculated).

Main stock solutions of PNA and ssDNA were prepared by dissolution in deionised

distilled water. Equimolar mixtures (1:1 stoichiometry in single strands) of the PNA and its

complementary ssDNA were dissolved in 10 mM phosphate buffer (pH = 7.2) containing 100

mM NaCl and 0.1 mM EDTA. The duplexes were prepared by heating the samples up to 90 °C

and then cooling slowly to room temperature to allow proper annealing.

The thermal melting temperatures were measured with a Perkin‐Elmer Lambda2S

spectrophotometer attached to a Peltier temperature controller PTP‐6. Cuvettes of 1.0 cm

path‐length and 1.0 mL volume were used for all experiments.

Thermal melting and annealing profiles were obtained using heating‐cooling cycles

between 0 and 95 °C. The absorption was registered at 260 nm against temperature.

Temperature profile: the mixtures were kept at 20 °C for 20 min and heated to 95 °C

at a rate of 0.5 °C per minute, then stayed at 95 °C for 10 min and were then cooled to 20 °C

at a rate of 0.5 °C per minute.

The melting temperature (Tm) was determined as the maximum of the first‐derivative

of the absorption vs. temperature curves of the melting.

Activity Measurements of Radioactive Solutions

The activity of the 99mTc solutions was measured in an ionization chamber (Aloka,

Curiemeter IGC‐3). The samples with activity less than 2 μCi were measured in a Gamma

Counter (Berthold LB2111).

Instant Thin‐Layer Chromatography

Instant thin‐layer chromatography (ITLC) was performed using ITLC silica gel strips

(PALL) developed with a mobile phase of pyridine/acetic acid/H2O (3:5:1.5).

Radioactive distribution on the ITLC strips was detected using a Berthold LB 505

detector, equipped with a NaI(Tl) scintillation crystal, coupled to a radiochromatogram

scanner.


166

Thin Layer Chromatography of Radioactive Compounds

Thin‐layer chromatography (TLC) was performed using silica gel TLC strips (Polygram

Sil G, Macherey–Nagel) developed with a mobile phase of 0.5% or 5% HCl 6 N in methanol.

Radioactive distribution on the TLC strips was detected using a Berthold LB 505 detector,

equipped with a NaI(Tl) scintillation crystal, coupled to a radiochromatogram scanner.

6.3. Synthesis of Compounds 1 ‐ 17

6.3.1. Tert‐butyl 2‐aminoethylcarbamate (1)158

H2N NH

O

O

1

2 3

3

3

A solution of di‐tert‐butyldicarbonate (8.86 g, 39.6 mmol) in dioxane (100 mL) was

added to a solution of ethylendiamine (19.2 g, 318.2 mmol) in dioxane in an ice bath for 2.5

h. After stirring overnight at room temperature the solvent was removed. To the residue

were added 150 mL of H2O. The bis alkylated by‐product precipitated and was removed by

filtration. The aqueous solution was extracted with chloroform (3 x 100 mL) and the organic

phases were dried with MgSO4, filtered and evaporated under vacuum.

Yield: 5.33 g, 84%, oil. 1H‐NMR (300 MHz, CDCl3): δ (ppm) 4.99 (s br, 1H, NH); 3.12 (q, 2H, CH2

2); 2.76 (t, 2H,

CH21); 1.38 (s, 9H, CH3

3); 1.21 (s, 2H, NH2).

6.3.2. Methyl 2‐(2‐(tert‐butoxycarbonylamino)ethylamino)acetate (2)159

NH

HN

O

O 123O

O 45

a

b5

5

To a solution of tert‐butyl 2‐aminoethylcarbamate (1) (2.4 g, 15 mmol) and

triethylamine (1.57 mL, 17 mmol) in CH2Cl2 (25 mL), methyl bromoacetate (1.43 mL, 15


167

mmol) was added dropwise. The solution was stirred overnight at room temperature, then

diluted with CH2Cl2 (25 mL) and washed with brine. The organic phase was dried over

Na2SO4, filtered and evaporated. The residue was purified by column chromatography on

silica gel using a gradient of ethyl acetate (25 ‐ 100%) in hexane.

Yield: 1.7 g, 50%, oil. 1H NMR (300 MHz, CDCl3): δ ppm 4.99 (s br, 1H, NHa), 3.74 (s, 3H, CH3

1), 3.42 (s, 2H,

CH22), 3.20 (q, 2H, CH2

4), 2.75 (t, 2H, CH23), 1.45 (s, 9H, CH3

5), 1.75 (br s, 1H, NHb).

6.3.3. 2‐(5‐methyl‐2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐yl) acetic acid

(3)160

NH

N

O

O

O

OH

5

6

1'

2

3

1

4

To a suspension of thymine (5.0 g, 39 mmol) and K2CO3 (5.5 g, 39 mmol) in dry DMF

(40 mL) was added methyl bromoacetate (3.8 mL, 39 mmol) and the mixture was stirred

vigorously overnight at room temperature. The mixture was evaporated under vacuum and

the obtained white solid was washed with water. The solid was cooled to 0 °C, treated with

water (40 mL) and 4 M HCl (aqueous, 2 mL), and stirred for 30 min. The precipitate was

collected by filtration and washed with water (3 x 20 mL). The collected solid was treated

with water (40 mL) and 2 M NaOH (aqueous, 20 mL), and refluxed for 10 min. The mixture

was cooled to 0 °C, treated with 4 M HCl (aqueous, 13.5 mL), and stirred for 30 min. The

precipitate was collected by filtration, washed with water (3 x 20 mL) and dried.

Yield: 4.2 g, 60%, white solid. 1H NMR (300 MHz, dmso‐d6): δ (ppm) 11.29 (s, 1H. NH), 7.47 (s, 1H, CH6), 4.34 (s, 2H,

CH21’), 1.74 (s, 3H, CH3).


168

6.3.4. Methyl‐2‐(N‐(2‐(tert‐butoxycarbonylamino)ethyl)‐2‐(5‐methyl‐

2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐yl)acetamido)acetate (4)161

NH

N

O

NH

N

O

O

O

OO

O

123

45

5a

b

5

5'

6'

6

4'

2'

5'' 6''

5'''

2''

To a solution of 2 (1.4 g, 6 mmol) in dry DMF (10 mL) were added N,N‐

diisopropylethylamine (DIPEA) (2 mL, 12 mmol), HBTU (2.26 g, 5.9 mmol) and compound 3

(1.2 g, 5.9 mmol). The mixture was stirred at room temperature for 19 h and the solvent

evaporated under vacuum. The residue was dissolved in ethyl acetate (20 mL) and the

resulting solution was washed with NaHCO3, H2O, HCl 0.01 N and H2O. The organic phase

was dried over MgSO4, filtered and the solvent evaporated under vacuum. The obtained oil

was purified by column chromatography, using a gradient of MeOH (0 ‐ 5%) in CH2Cl2.

Yield: 1.8 g, 77%, white foam. 1H NMR (300 MHz, CDCl3): δ (ppm) 8.86 (br, 1H, NHb); 7.01 (mi) and 6.94 (ma) (s, 1H,

H6’), 5.57 (ma) and 4.96 (mi) (br t, 1H, NHa); 4.55 (ma) and 4.40 (mi) (s, 2H, CH22); 4.19 (mi)

and 4.03 (ma) (s, 2H, CH26), 3.79 (mi) and 3.73 (ma) (s, 3H, CH3

1); 3.51 (t, 2H, CH23); 3.31 (q,

2H, CH24); 1.89 (s, 3H, 5’‐CH3‐T); 1.42 (s, 9H, CH3

5). 13C NMR (75.373 MHz, CDCl3): 170.2 (ma) and 169.9 (mi) (C2’’=O); 167.8 (mi) and

167.4 (ma) (C6’’=O); 164.4 (C4’=O); 156.1 (C5’’’=O); 151.2 (C2’=O); 141.1 (C6’); 110.7 (C5’); 79.9

(C5’’); 55.2 (mi), 52.9 (mi), 52.5 (ma), 50.2 (mi), 48.9 (ma), 48.6 (ma) and 47.9 (ma) (C1 + C2 +

C3 + C6); 38.6 (C4); 28.3 (C5); 12.3 (5’‐CH3‐T).


169

6.3.5. 2‐(N‐(2‐methoxy‐2‐oxoethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐

dihydropyrimidin‐1(2H)‐yl)acetamido)ethanaminium 2,2,2‐

trifluoroacetate (5)

H3NN

O

NH

N

O

O

O

O12

3

4

b5'

6'

5

4'

2'

CF3COO‐

Compound 4 (220 mg, 0.5 mmol) was dissolved in a mixture of CH2Cl2/TFA (5:2 mL).

The reaction mixture was stirred at room temperature for 4 h and the solvent evaporated

under vacuum. The obtained crude oil was washed with diethylether and dried under

vacuum, yielding quantitatively a white‐yellow solid.

RP‐HPLC (method 1): tR = 14.2 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.36 (mi) and 7.28 (ma) (s, 1H, H6’); 4.71 (mi) and

4.58 (ma) (s, 2H, CH22); 4.38 (ma) and 4.17 (mi) (s, 2H, CH2

5); 3.82 (ma) and 3.75 (mi) (s, 3H,

CH31); 3.70 (t, 2H, CH2

3); 3.15 (t, 2H, CH24); 1.87 (s, 3H, 5’‐CH3).

6.3.6. 2‐(N‐(2‐(tert‐butoxycarbonylamino)ethyl)‐2‐(5‐methyl‐2,4‐dioxo‐

3,4‐dihydropyrimidin‐1(2H)‐yl)acetamido)acetic acid (6)

NH

N

O

NH

N

O

O

O

OHO

O

12

34

4

4a

b

5

5'

6'

A suspension of 4 in 2 N aqueous NaOH was stirred for 45 min at room temperature.

The aqueous solution was cooled to 0 °C and the pH was adjusted to 2 with HCl 2 N. The

aqueous phase was extracted with ethyl acetate several times and the joined organic phases


170

were dried over sodium sulphate, filtered and evaporated in vacuum. The compound was

obtained quantitatively as a white solid.

RP‐HPLC (method 1): tR = 21.6 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.30 (ma) and 7.26 (mi) (s, 1H, H6’); 4.74 (ma)

and 4.56 (mi) (s, 2H, CH21); 4.27 (mi) and 4.10 (ma) (s, 2H, CH2

5); 3.51 (t, 2H, CH22); 3.14 (t,

2H, CH23); 1.86 (s, 3H, 5’‐CH3‐T); 1.42 (s, 9H, CH3

5).

6.3.7. Methyl N‐[2‐[N’‐[2‐(Boc‐amino)ethyl]‐N’‐(thymin‐1‐

ylacethyl)glycyl]amino]ethyl]‐N‐[(4‐thymini‐1‐yl)acetyl]glycinate

(7)

N

O

NH

O

NH

N

O

O

N

O

NH

O

O

NH

N

O

O

O

O12356

478

2'

88

9' 9

4'5'

6'2''

4''

5''

6''

8'

NEt3 was added to a solution of compound 5 (374 mg, 1.25 mmol) in dry DMF (5 mL)

(6.24 mmol, 0.9 mL) and the mixture stirred for 45 min at room temperature.

Compound 6 (420 mg, 1.09 mmol) was dissolved in DMF (5mL) and NEt3 (6.24 mmol,

0.9 mL) and HBTU (414 mg, 1.09 mmol) were added. After stirring for 45 min, this solution

was added to the solution of compound 5 and the resulting one was stirred for 19 h. The

solvent was removed under vacuum and the obtained residue was purified by column

chromatography, using a gradient of MeOH (0 ‐ 10%) in CH2Cl2.

Yield: 450 mg, 62%.

RP‐HPLC (method 1): tR = 22.2 min.

ESI/QITMS (most abundant m/z): Molecular Formula ‐ C28H40N8O11; [M‐H]‐ ‐

calculated m/z 663.3, found m/z 663.2. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.47 (ma), 7.38 (mi), 7.37 (mi), 7.34 (mi), 7.30

(mi), 7.26 (mi), 7.25 (mi) and 7.22 (ma) (s, 2H, CH6’ + CH6’’); 4.75 – 3.97 (s (several singlets),

8H, CH22 + CH2

5 + CH29 + CH2

9’)); 3.81 (mi), 3.80 (mi), 3.78 (mi), 3.77 (mi), 3.74 (ma), 3.71 (ma)


171

(s, 3H, CH31); 3.56 – 3.37 (m, 6H, CH2

3 + CH24 + CH2

6); 3.32 (t, 2H, CH27), 1.85 (mi), 1.83 (mi),

1.79 (ma), 1.76 (ma) (s, 6H, 5’‐CH3‐T + 5’’‐CH3‐T); 1.43 (br s, 9H, CH38).

13C NMR (75.373 MHz, CD3OD) (several CH2 were obscured under the solvent peak):

δ (ppm) 172.5 (C=O); 171.5 (C=O); 169.8 (2C=O); 166.9 (C=O); 166.8 (C=O); 158.4 (C=O);

153.0 (2C=O); 144.2 (mi), 144.0 (mi), 143.7 (ma) (C6’ + C6’’); 111.2 (mi), 111.0 (ma), 110.6(mi)

(C5’+ C5’’); 80.6 (C8’); 53.2 (mi), 53.0 (ma), 52.8 (mi) (C1), 51.2 (CH2); 39.5 (C7), 37.8 (C4); 28.7

(C8); 12.2 (5’‐ CH3‐T).

6.3.8. 5,11‐bis(2‐(5‐methyl‐2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐

yl)acetyl)‐3,9‐dioxo‐2‐oxa‐5,8,11‐triazatridecan‐13‐aminium 2,2,2‐

trifluoroacetate (8)

N

O

H3N

O

NH

N

O

O

N

O

NH

O

O

NH

N

O

O

12356

47

2'

8' 8

4'5'

6'2''

4''

5''

6''

CF3COO‐

Compound 7 was dissolved in a mixture of CH2Cl2/TFA (8:2 mL). The reaction mixture

was stirred at room temperature for 4 h and the solvent evaporated under vacuum. The

obtained oil was washed with diethyl ether and dried under vacuum, yielding quantitatively

a white solid.

RP‐HPLC (method 1): tR = 17.3 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.40 ‐ 7.22 (s (several singulet’s), 2H, CH6’ +

CH6’’); 4.89 ‐ 4.13 (s (several singlets), 8H, CH22 + CH2

5 + CH28 + CH2

8’)); 3.81 (mi), 3.80 (mi),

3.72 (ma)(s, 3H, CH31); 3.60 – 3.4 (m, 6H, CH2

3 + CH24 + CH2

6); 3.01 (t, 2H, CH27), 1.85 (ma),

1.83 (mi), 1.81 (ma), (s, 6H, 5’‐CH3‐T + 5’’‐CH3‐T).


172

6.3.9. (R)‐methyl 2,2,11,11‐tetramethyl‐4‐oxo‐3,10‐dioxa‐8‐thia‐5‐

azadodecane‐6‐carboxylate (9)

1

23

46

HNS O

O

O

OO

55

56

6

To a solution of N‐(tert‐butoxycarbonyl)‐L‐cysteine methyl ester (1.2 g, 4.9 mmol) in

CH2Cl2 (50 mL) NEt3 (1.0 mL, 7.3 mmol) was added, followed by dropwise addition of tert‐

buthyl bromoacetate (1.1 mL, 7.3 mmol). The reaction mixture was stirred for 20 h at room

temperature. After this time, the mixture was diluted with 170 mL of CH2Cl2, washed with

H2O, 1 M HCl and H2O. The organic phase was separated and dried over Na2SO4, filtered and

the solvent evaporated under vacuum. The residue was purified by column chromatography,

using hexane/ethyl acetate (7:1) as eluent.

Yield: 1.2 g, 70%. 1H NMR (300 MHz, CDCl3): δ (ppm) 5.46 (d br, 1H, NH); 4.56 (m, 1H, CH2); 3.77 (s, 3H,

CH31); 3.23‐3.13 (m, 2H, CH2

4); 3.11‐3.00 (m, 2H, CH23); 1.48 (s, 9H, CH3

5); 1.46 (s, 9H, OtBu).

6.3.10. (R)‐2‐(2‐amino‐3‐methoxy‐3‐oxopropylthio)acetic acid (10)

1

234

NH2

S

O

OO

HO

Compound 9 (1.2 g, 3.4 mmol) was dissolved in 7 mL of CH2Cl2. After addition of 7 mL

of TFA, the resulting mixture was stirred at room temperature. The deprotection was

complete after 4 h of reaction, as indicated by HPLC. The solvent was evaporated under

vacuum and the expected product was recovered almost quantitatively. 1H NMR (300 MHz, D2O): δ (ppm) 4.30 (m, 1H, CH2); 3.73 (s, 3H, CH3

1); 3.41‐3.30 (m,

2H, CH24); 3.20 ‐ 3.13 (m, 1H, HC‐H3); 3.03 ‐ 2.98 (m, 1H, HC‐H3).


173

6.3.11. Methyl 3‐(2‐oxo‐2‐(perfluorophenoxy)ethylthio)‐2‐(2,2,2‐

trifluoroacetamido)propanoate (11)

SF3COCHN

O

OO

O

F

F

FF

F

1 2 3 46'

1' 5'4'

3' 2'6'

7'

7'

8'

To a solution of compound 10 (400 mg; 1.3 mmol) in DMF (2 mL) was added pyridine

(1 mL, 13 mmol) and pentafluorphenol trifluoracetic acid (1.5 mL; 9.1 mmol). The resulting

yellow reaction mixture was stirred overnight at room temperature. The solvent was

removed under vacuum and the obtained residue was dissolved in 60 mL of ethyl acetate.

After washing the resulting solution with 0.01 N HCl (3 x 10 mL) and 5% NaHCO3 aq. (3 x 10

mL), the organic phase was dried over MgSO4, filtered and the solvent evaporated under

vacuum. The residue was washed several times with hexane and dried under vacuum,

yielding a white powder.

Yield: 580 mg, 98%. 1H NMR (300 MHz, CDCl3): δ 7.2 (br, 1H, NH); 4.88 (m, 1H, CH2); 3.82 (s, 3H, CH3

1);

3.64‐3.49 (m, 2H, CH24); 3.32‐3.26 (m, 1H, HC‐H3); 3.22‐3.15 (m, 1H, HC‐H3).

13C NMR (75.373 MHz, CDCl3): 169.4 (C1’); 166.2 (C4’); 157.4 (C2’); 156.9 (C6’); 142.7

(C5’); 139.4 (C8’); 136.2 (C7’); 117.4 (C3’); 53.4 (C2); 51.8 (C1); 33.8 (C4); 32.7 (C3). 19F NMR (CDCl3): ‐77.5 (s, 3F), ‐154.2 (d, 2F), ‐158.5 (t, 2F), ‐163.3 (t, 1F).

Anal. Calc. for C14H9F8NO5S: C, 36.93; H, 1.99; N, 3.08; S, 7.04. Found: C, 36.99; H,

2.40; N, 3.25; S, 7.40.

6.3.12. Protected Cysteine‐PNA Monomer Conjugate (12)

HN N

O

NH

N

O

O

O

OS

F3COCHN

O

OO

1 23 4 5

2''

4''5''

6''

76

9

81'

3' 2'

4'9'

a

b

c7'


174

NEt3 (0.2 mL, 1.5 mmol) was added to a solution of compound 5 (78 mg, 0.26 mmol)

in dry CH3CN (10 mL). After stirring for 2 h at room temperature, the solution was cooled to

0 °C and compound 11 (148 mg, 0.26 mmol) was added. The mixture was stirred for 12 h at

room temperature, the solvent removed under vacuum and the resulting residue purified by

column chromatography (MeOH (3 ‐ 7%)/CHCl3).

RP‐HPLC (method 1, analytic): tR = 21.2 min.

Yield: 37 mg, 25%, white solid. 1H NMR (300 MHz, dmso‐d6): δ (ppm) 11.30 (s, 1H, NHa); 10.03 (d, 1H, NHb); 8.21 (mi)

and 8.08 (ma) (t, 1H, NHc); 7.32 (ma) and 7.25 (mi) (s, 2H, H6’); 4.67 (1H, 1H, CH2); 4.63 (ma)

and 4.45 (mi) (s, 2H, CH27); 4.30 (mi) and 4.05 (ma) ((s, 2H, CH2

9); 3.71 (mi), 3.66 (ma) and

3.62 (mi) (s, 6H, CH31 + CH3

8); 3.43 (2H, CH26); 3.28 (2H, CH2

5); 3.22 (m, 2H, CH24); 3.15‐3.10

(m, 1H, HC‐H3); 2.95‐2.87 (m, 1H, HC‐H3); 1.73 (s, 3H, 5’’‐CH3‐T). 13C NMR (75.373 MHz, dmso‐d6): δ (ppm) 169.2 (C1’=O + C4’=O + C7’=O); 167.4 (C9’=O);

164.4 (C4’’=O); 156.8 (C2’=O); 151.0 (C2’’=O); 142.2 (C6’’); 117.7 (C3’); 108.1 (C5’’); 52.6 (ma),

52.3 (mi), 52.0 (ma) and 51.8 (ma) (C1 + C8 + C2); 48.7 (mi), 47.8 (ma), 47.4 (ma) and 46.3 (mi)

(C7 + C9); 46.5 (C6); 37.2 (C5); 34.2 (C4); 32.2 (C3); 11.9 (5’’‐CH3‐T). 19F NMR (CD3OD): δ (ppm) ‐75.9.

6.3.13. 2‐amino‐3‐(2‐(2‐(N‐(carboxymethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐

dihydropyrimidin‐1(2H)‐yl)acetamido)ethylamino)‐2‐

oxoethylthio)propanoic acid (13)

HN N

O

NH

N

O

O

O

OHS

H2N

O

OHO

12 3

45

2''

4''

5''

6''

7

61'

3'6'7'

Compound 12 was dissolved in MeOH and 10 eq of K2CO3 were added. After stirring

overnight the solvent was removed. The obtained solid was dissolved in H2O and the

solution was neutralized and purified by preparative RP‐HPLC (method 1).

Yield: 21 mg, 74 %, white solid.


175

RP‐HPLC (method 1, analytic): tR = 13.8 min. 1H NMR (300 MHz, D2O): δ (ppm) 7.20 (ma) and 7.18 (mi) (s, 1H, H6’’); 4.61 (ma) and

4.47 (mi) (s, 2H, CH26); 4.18 (mi) and 4.01 (ma) (s, 2H, CH2

7); 4.03‐3.99 (m, 1H, CH1); 3.45 (q,

2H, CH24); 3.39 (t, 2H, CH2

5); 3.29‐3.17 (m, 2H, CH23), 3.17 ‐ 3.07 (m, 1H, HC‐H2); 3.00 ‐ 2.87

(m, 1H, HC‐H2); 1.71 (s, 3H, 5’’‐CH3‐T). 13C NMR (75.373 MHz, D2O): δ (ppm) 181.0 (C1’=O); 178.1 (C6’=O); 174.9 (C3’=O);

172.3 (C7’=O); 165.7 (C4’’=O); 160.6 (C2’’=O); 144.6 (C6’’); 112.9 (C5’’); 58.4 (CH2); 53.6 (CH2);

52.5 (CH2); 51.7 (CH2); 49.1 (CH2); 39.5 (CH2); 37.7 (CH2); 14.6 (5’’‐CH3‐T).

6.3.14. Protected Pz‐PNA Monomer Conjugate (14)

NH

N

O

NH

N

O

O

O

O

NN NHN

O

1

2 3

45 6

78

910 11

3' 5'4'

2''

4''

5''

6''

O

O

a

c

b

12

4'''

4'''4'''

4''''4'''''

7'

12' 10'

To a solution of compound 5 (200 mg, 0.5 mmol) in dry CH3CN (10 mL) NEt3 (0.2 mL,

1.5 mmol), HBTU (189 mg, 0.5 mmol) and 3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (Pz‐

Boc) (184 mg, 0.5 mmol) were successively added and the mixture was stirred for 19 h. After

evaporation of the solvent, the residue obtained was purified by column chromatography

(MeOH (5 ‐ 13%)/CHCl3).

Yield: 230 mg, 70%. 1H NMR (300 MHz, CDCl3): δ (ppm) 9.32 (s br, 1H, NHa); 7.48 (s br, 1H, NHb); 6.96 (mi)

and 6.91 (ma) (s, 1H, H6’); 5.78 (s, 1H, H4’); 5.18 (s br, 1H, NHc); 4.57 (ma) and 4.37 (mi) (s, 2H,

CH210); 4.20 (mi) and 4.04 (ma) (s, 2H, CH2

12); 4.01 (t, 2H, CH21); 3.79 (mi) and 3.72 (ma) (s,

3H, CH311); 3.53 (t, 2H, CH2

8); 3.38 (t, 2H, CH29); 3.02 (t, 2H, CH2

4); 2.72 (t, 2H, CH22); 2.44 (m,

2H, CH23 + CH2

5); 2.21 (s br, 8H, 2CH3‐pz, CH27); 1.87 (s, 3H, 5’’‐ CH3‐T), 1.72 (quint, 2H, CH2

6);

1.42 (s, 9H, CH34’’’).

13C NMR (75.373 MHz, CDCl3): δ (ppm) 174.4 (C7’’=O); 169.7 (C10’=O); 167.4 (C12’=O);

164.4 (C4’’=O); 156.1 (C4’’’’’=O); 151.2 (C2’’=O); 147.2 (C3’); 140.9 (C5’); 138.8 (C6’’); 110.5 (C5’’);


176

105.1 (C4’); 78.9 (C4’’’’); 53.6, 53.0 and 52.3 (C2 + C3+ C5 + C11); 48.1, 47.7 and 46.7 (C9 + C10 + C1

+ C12); 38.3 (C3); 37.3 (C8); 33.1 (C7); 28.3 (C4’’’); 22.5 (C6); 13.2 (3’‐CH3‐pz); 12.2 (6’’‐CH3‐T);

(5’‐CH3‐pz).

6.3.15. 2‐(N‐(2‐(4‐((2‐aminoethyl)(2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐

yl)ethyl)amino)butanamido)ethyl)‐2‐(5‐methyl‐2,4‐dioxo‐3,4‐

dihydropyrimidin‐1(2H)‐yl)acetamido)acetic acid (15)

NH

N

O

NH

N

O

O

O

OH

NN NNH2

O

1

2 3

45 6

78

910

11

3' 5'4'

2''

4''

5''

6''

Compound 14 (200 mg, 0.3 mmol) was dissolved in a mixture of CH2Cl2/TFA (4:2).

After 1 h at room temperature, the solvent was evaporated, the residue dissolved in MeOH

and 5 eq K2CO3 added. After stirring overnight, the solvent was removed. The obtained solid

was dissolved in H2O neutralized and purified by preparative RP‐HPLC (method 2).

Yield: 115 mg, 72%.

RP‐HPLC (method 1, analytic): tR = 19 min 1H NMR (300 MHz, CD3OD): δ (ppm) 7.30 (ma) and 7.27 (mi) (s, 1H, H6’); 5.92 (s, 1H,

H4’); 4.69 (ma) and 4.55 (mi) (s, 2H, CH210); 4.38 (t, 2H, CH2

1); 4.28 (mi) and 4.11 (ma) (s, 2H,

CH211); 3.62‐3.41 (m, 10H, CH2

2 + CH23 + CH2

4 + CH28 + CH2

9); 3.29 (t, 2H, CH25); 2.51 (t, 1H,

HC‐H7); 2.35 (t, 1H, HC‐H7); 2.27 and 2.26 (s, 3H, CH3‐pz); 2.19 and 2.18 (s, 3H, CH3‐pz); 1.97

(m, 2H, CH26); 1.85 (s, 3H, 5’’‐CH3‐T).

13C NMR (75.373 MHz, CD3OD): δ (ppm) (three CH2 are obscured under the solvent)

176.1 (HOC=O); 175.8 (C=O); 170.4 (C=O); 166.9 (C4’’=O); 153.2 (C2’’=O); 149.8 (C3’); 143.7

(C6’’); 142.3 (C5’); 111.1 (C4’); 107.0 (C5’’); 55.5 (C5); 54.2 (CH2); 51.4 (CH2), 50.0 (CH2); 49.7

(CH2); 48.0 (CH2); 44.0 (C1); 38.2 (C2); 36.1 (C8); 33.8 (C7); 21.0 (C6); 13.2 (5’‐CH3‐pz); 12.2 (5’’‐

CH3‐T); 10.7 (3’‐CH3‐pz).


177

Anal. Calc. for C24H38N8O6.3CF3COOH: C, 41.08; H, 4.67; N, 12.78. Found: C, 42.14; H,

3.53; N, 12.94.

6.3.16. Protected Pz‐PNA Dimer Conjugate (16)

N

O

NH

O

NH

N

O

O

N

O

NH

O

O

NH

N

O

O

NN N

NH

O

1

2 3

45 6

78

9 10

11

3' 5'4'

2'''

4'''

5'''

6'''

12 1314

1515'

O

O

2''

4''

5''

6''

16

16'

16

16

16''

To a solution of compound 8 (145 mg, 0.26 mmol) in dry DMF (5 mL), NEt3 (0.3 mL, 2

mmol), HBTU (97 mg, 0.26 mmol) and 3,5‐Me2pz(CH2)2N((CH2)3COOH)(CH2)2NHBoc (94 mg,

0.26 mmol) were successively added and the mixture was stirred for 19 h. After evaporation

of the solvent, the obtained residue was purified by preparative RP‐HPLC (method 1).

Yield: 176 mg, 75%, white solid.

RP‐HPLC (method 1, analytic): tR = 23.2 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.45 (ma), 7. 36 (mi), 7.33 (mi), 7.30 (mi), 7.25

(mi) and 7.19 (ma) (s, 2H, CH6’ + CH6’’); 5.89 (s, 1H, CH4’); 4.75–4..5 and 4.30‐4.00 (s (several

singlets), 8H, CH210 + CH2

13 + CH215 + CH2

15’)); 4.38 (t, 2H, CH21); 3.81 (mi), 3.78 (mi), 3.74 (ma)

and 3.71 (mi) (s, 3H, CH31); 3.67 (t, 2H, CH2

2); 3.60 – 3.45 (m, 12H, CH23 + CH2

4 + CH28 + CH2

9 +

CH211 + CH2

12); 3.36 (2H, CH25); 2.49 (t, 1H, HC‐H7); 2.32 (t, 1H, HC‐H7); 2.27 (s, 3H, CH3‐pz);

2.18 (s, 3H, CH3‐pz); 1.99 (m, 2H, CH26); 1.84 (mi), 1.82 (mi), 1.81 (mi), 1.80 (mi), 1.77 (ma),

1.75 (ma) (s, 6H, 5’‐CH3‐T + 5’’‐CH3‐T); 1.38 (br s, 9H, CH316).

13C NMR (75.373 MHz, CD3OD) (several CH2 were obscured under the solvent peak):δ

(ppm) 175.3 (C=O); 174.9 (C=O); 172.6 (C=O); 171.6 (C=O); 171.4 (C=O); 170.6 (C=O); 170.4

(C=O); 169.9 (C=O); 169.8 (C=O); 166.9 (C=O); 166.7 (C=O); 158.3 (C16’’=O); 153.0 (C2’’=O and

(C2’’’=O); 149.9 (C3’); 144.1 and 143.8 (C6’’ and C6’’’); 142.2 (C5’); 106.8 (C4’); 111.0 and 110.5

(C5’’ and C5’’’); 81.1 (C16’); 54.4 and 54.2 (C2 and 3CH2); 53.3 (mi), 53.1 (ma) and 52.8 (mi) (C14);

48.1 (CH2); 47.7 (CH2); 42.7 (C1); 38.2 (CH2); 37 8 (CH2); 33.5 and 33.3 (C

7); 28.6 (C16); 20.3

(C6); 13.5 (3’‐CH3‐pz); 12.3 and 12.1 (5’’‐ CH3‐T and 5’’’‐CH3‐pz), 10.7 (5’‐CH3‐pz).


178

Anal. Calc. for C41H64N12O12. 2CF3COOH: C, 47.28; H, 5.64; N, 14.70. Found: C, 47.70; H,

5.64; N, 14.56.

6.3.17. Deprotected Pz‐Dimer (17)

N

O

NH

O

NH

N

O

O

N

O

NH

O

OH

NH

N

O

O

NN N

NH2

O

1

2 3

45 6

78

9 10

11

3' 5'4'

2'''

4'''

5'''

6'''

12 13

1414'

2''

4''

5''

6''

Compound 16 (75 mg, 0.082 mmol) was dissolved in a mixture of CH2Cl2/TFA (2:2).

After 1h at room temperature, the solvent was evaporated and the obtained residue was

dissolved in MeOH and 5 eq of K2CO3 added. After stirring overnight the solvent was

removed. The obtained solid was dissolved in H2O neutralized and purified by preparative

RP‐HPLC (method 1).

Yield: 49 mg, 75%, white solid.

RP‐HPLC (method 1, analytic): tR = 19.1 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.48 (ma), 7.35 (mi), 7.32 (mi), 7.30 (mi), 7.29

(mi), 7.26 (mi), 7.25 (mi), 7.16 (ma) (s, 2H, CH6’’ + CH6’’’); 5.93 (s, 1H, CH4’); 4.75–3.9 (s

(several singulet’s), 8H, CH210 + CH2

13 + CH214 + CH2

14’); 4.40 (t, 2H, CH21); 3.6); 3.65 – 3.35 (m,

14H, CH22 + CH2

3 + CH24 + CH2

8 + CH29 + CH2

11 + CH212); 3.26 (2H, CH2

5); 2.51 (t, 1H, HC‐H7);

2.34 (t, 1H, HC‐H7); 2.28 (s, 3H, CH3‐pz); 2.18 (s, 3H, CH3‐pz); 1.99 (m, 2H, CH26); 1.84 (mi),

1.82 (mi), 1.80 (mi), 1.80 (mi), 1.77 (ma), 1.73 (ma) (s, 6H, 5’‐CH3‐T + 5’’‐CH3‐T). 13C NMR (75.373 MHz, CD3OD) (several CH2 were obscured under the solvent peak): δ

(ppm) 177.4 (C=O); 176.1 (C=O); 174.2 (C=O); 172.8 (C=O); 171.2 (C=O); 170.7 (C=O); 170.5

(C=O); 170.1 (C=O); 169.6 (C=O); 166.9 (C=O); 166.7 (C=O); 153.0 (C2’’=O and C2’’’=O); 149.7

(C3’); 144.2, 144.0 and 143.8 (C6’’ and C6’’’); 142.4 (C5’); 111.1 and 110.5 (C5’’ + C5’’’); 107.1 (C4’);

55.7 (CH2); 54.2 (CH2); 51.3 (CH2); 43.9 (CH2); 44.0 (CH2); 38.2 (CH2); 37.6 (CH2); 36.1 (CH2);

33.6 (C7); 20.9 (C6); 13.2 (5’‐CH3‐pz); 12.3 (5’’‐CH3‐T and 5’’’‐CH3‐T); 10.7 (3’‐CH3‐pz).


179

ESI/QITMS (most abundant m/z): Molecular formula ‐ [C35H52N12O10]; [M+H]+ ‐

calculated m/z 801.4, found m/z 801.7; [M+Na]+ ‐ calculated m/z 823.4, found m/z 823.6.

Anal. Calc. for C35H52N12O10.3CF3COOH: C, 43.09; H, 4.85; N, 14.71. Found: C, 43.66;

H, 4.34; N, 15.08.

6.4. Synthesis of the Rhenium Complexes 18 ‐ 20

6.4.1. Synthesis of fac‐[Re(CO)3(κ3‐ 13)] (18)

OSNH2

O

O

Re

OC CO CO

NH

N

O

NH

N

O

O

O

OH12 3

5

2''

4''

5''

6''

7

61'3' 6'

7'

[Re(CO)3(OH2)3]Br (12 mg, 0.03 mmol) was reacted with equimolar amounts of 13

(0.03 mmol) in water/75 °C/5h. After removing the solvent under vacuum, the residue was

purified by preparative RP‐HPLC (method 1).

Yield: 15 mg, 70%.

RP‐HPLC (method 1): Rt = 19.7 min, 19.9 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.32 (mi), 7.31 (ma), 7.26 (mi) and 7.22 (mi) (s,

1H, H6’’); 6.31, 6.06, 5.53, 5.38, 5.20 (m, HN‐H); 4.75‐4.3 (m, CH or CH2); 4.30 (s, CH26); 4.14

(s, CH27); 4.1‐3.2 (m, CH2); 3.7‐ 3.5 (m, 4H, CH2

4 + CH25); 1.86 (s, 3H, 5’’‐CH3‐T).

13C NMR (75.373 MHz, CD3OD): δ (ppm) 195.4 (ReCO); 193.7(ReCO); 193.4 (ReCO);

183.4, 182.3, 181.9, 181.4 (C1’=O); 172.7, 170.5, 169.6 (C6’=O + C3’=O + C7’=O); 167.0 (C4’’=O);

153.0 (C2’’=O); 143.8 (C6’’); 111.2 (C5’’); 62.6, 61.6, 59.5, 57.7, 57.0, 47.6, 46.5, 46.2, 41.1,

40.1, 39.1, 38.7, 38.0, (CH + 6CH2); 12.2 (5’’‐CH3‐T).

IR (KBr) (cm‐1): υ(C≡O), 2039, 1913; υ(C=O) 1673.

ESI/QITMS (most abundant m/z): Molecular formula: [C19H22N5O11SRe]; [M‐H]‐ ‐

calculated m/z 714.1, found m/z 713.9.


180

Anal. Calc. for C19H22N5O11ReS.2CF3COOH: C, 29.30; H, 2.57; N, 7.43; S, 3.40. Found:

C, 29.88; H, 3.50; N, 7.62; S, 3.33.

6.4.2. Synthesis of fac‐[Re(CO)3(κ3‐15)]CF3COO (19)

NN N

NH2

Re

OC CO CO

O

NH

N

O

NH

N

O

O

O

3'

5'

4'

11

12 3 4

5 6

7 8

910

2''

4''5''

6''

OH10'

7'

11'

+


(0.075 mmol) in methanol/reflux for 20 h. After removing the solvent under vacuum the

residue was purified by preparative RP‐HPLC (method 2).

Yield: 11 mg, 20%.

RP‐HPLC (method 1): tR = 22.4 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.31 (ma) and 7.30 (mi) (s, 1H, H6’’); 6.2 (s, 1H,

H4’); 5.63 (m, 1H, HN‐H); 4.73 (ma) and 4.57 (mi) (s, 2H, CH210); 4.47 (m, 1H, HC‐H1); 4.21 (m,

1H HC‐H1); 4.30 (mi) and 4.13 (ma) (s, 2H, CH211); 4.05 (m, 1H, HN‐H); 3.65 (m, 1H, HC‐H5);

3.60‐3.40 (m, 6H, CH28 + CH2

9 + HC‐H2); 3.36 (m, 1H, HC‐H5); 3.15 (m, 1H, HC‐H4); 2.85 (m,

2H, CH23); 2.65 (m, 1H, HC‐H2); 2.51 (m, 1H, HC‐H4); 2.43 (s + m, 3H + 1H, CH3‐pz + HC‐H

7);

2.36 (s, 3H, CH3‐pz); 2.26 (m, 1H, HC‐H7); 2.17 (m, 1H, HC‐H6); 2.05 (m, 1H, HC‐H6); 1.86 (s,

3H, 5’’‐CH3‐T). 13C NMR (75.373 MHz, CD3OD): δ (ppm) (four CH2 are under the solvent peak) 195.2

(ReCO); 194.9 (ReCO); 193.8 (ReCO); 175.2 (C10’=O); 172.5 (C7’=O); 169.8 (C11’=O); 167.0

(C4’’=O); 155.1 (C3’); 153.0 (C2’’=O); 145.4 (C5’); 143.8 (C6’’); 111.0 (C5’’); 109.2 (C4’); 67.3 (C5);

62.5 (C3); 53.8 (C2); 43.7 (C4); 37.9 (C8); 33.2 (C7); 21.0 (C6); 16.1 (3’‐CH3‐pz); 12.2 (6’’‐CH3‐T);

11.6 (5’‐ CH3‐pz). 18F NMR (CD3OD): δ (ppm) ‐75.905.

IR (KBr) (cm‐1): υ(C≡O) 2029, 1912; υ(C=O) 1678.


181

ESI/QITMS (most abundant m/z): Molecular formula ‐ [C27H38N8O9Re]+; [M]+ ‐

calculated m/z 805.2, found m/z 805.5; [M+H]+ ‐ calculated m/z 806.2, found m/z 806.5; [(M‐

H)+Na]+ ‐ calculated m/z 827.2, found m/z 827.5; [M+Na]+ ‐ calculated m/z 828.2, found m/z

828.5.

Anal. Calc. for [C27H38N8O9Re]+(CF3COO)

‐.2CF3COOH: C, 34.59; H, 3.52; N, 9.78. Found

C, 35.42; H, 3.95; N, 9.16.

6.4.3. Synthesis of fac‐[Re(CO)3(κ3 ‐ 17)]CF3COO (20)

NN N

NH2

Re

OC CO CO

+

O

NH

N

O

NH

N

O

O

ONH

N

O

NH

N

O

O

O

OH

3'

5'

4'

1213

1414'

12 3 4

5 6

7 8

910

11

2''

4''5''

6''

2'''

4'''5'''

6'''


(0.055 mmol) in methanol/reflux for 20 h. After removing the solvent under vacuum the

residue was purified by preparative RP‐HPLC (method 2).

Yield: 30 mg, 43%

RP‐HPLC (method 1, analytic): tR = 22.5 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 7.52 (ma), 7.36 (mi), 7.34 (mi), 7.0 (mi), 7.25 (mi)

and 7.17 (ma) (s, 2H, CH6’’ + CH6’’’); 6.2 (s, 1H, H4’); 5.53 (m, 1H, HN‐H); 4.75 – 4.12 (s (several

singulet’s), 8H, CH210 + CH2

13 + CH214 + CH2

14’); 4.54 (m, 1H, HC‐H1); 4.16 (m, 1H, HC‐H1); 4.07

(br, 1H, HN‐H); 3.70 (m, 1H, HC‐H5); 3.61‐ 3.52 (m, 9H, HC‐H2 + CH28 + CH2

9 + CH211 + CH2

12);

3.42 (m, 1H, HC‐H5); 3.18 (m, 1H, HC‐H4); 2.85 (m, 2H, CH23); 2.65 (m, 1H, HC‐H2); 2.51 (m,

1H, HC‐H4); 2.43 (s + m, 3H + 1H, CH3‐pz + HC‐H7); 2.36 (s, 3H, CH3‐pz); 2.26 (m, 1H, HC‐H7);

2.16 (m, 1H, HC‐H6); 2.04 (m, 1H, HC‐H6); 1.85, 1.83, 1.80, 1.78 and1.74 (s, 3H, 5’’‐CH3‐T and

5’’’‐CH3‐T ). 13C NMR (75.373 MHz, CD3OD): δ (ppm) (several CH2 were obscured under the

solvent peak) 194.9 (ReCO); 194.6 (ReCO); 193.5 (ReCO); 175.0(C=O); 173.9 (C=O); 171


182

(C=O); 170.9 (C=O); 170.3 (C=O); 169.8 (C=O); 169.5 (C=O); 169.4 (C=O); 166.9 (C=O); 154.8

(C2’’=O and C2’’’=O); 152.8 (C3’); 145.1 (C5’); 144.0, 143.7 and 143.5 (C6’’ and C6’’’); 110.7 and

110.2 (C5’’ + C5’’’); 108.9 (C4’); 67.0 (C5); 62.1 (C3); 53.4 (C2); 43.4 (C4); 37.8 (CH2), 37.3 (CH2);

33.3 and 32.9 (C7); 20.9 and 20.5 (C6); 15.8 (3’‐CH3‐pz); 12.0 and 11.8 (5’’‐CH3‐T and 5’’’‐CH3‐

T); 11.3 (5’‐CH3‐pz).

IR (KBr) (cm‐1): υ(C≡O), 2029, 1915; υ(C=O) 1673.

ESI/QITMS (most abundant m/z): Molecular formula ‐ [C38H52N12O13Re]+: [M]+ ‐

calculated m/z 1071.3, found m/z 1071.7; [M+H]+ ‐ calculated m/z 1072.3, found m/z 1072.8;

[(M‐H)+Na]+ ‐ calculated m/z 1093.3, found m/z 1093.7; [M+Na]+ ‐ calculated m/z 1094.3,

found m/z 1094.7.

Anal. Calc. for [C38H52N12O13Re]+CF3COO

‐.CF3COOH: C, 38.86; H, 4.12; N, 12.95.

Found: C, 38.17; H, 3.98; N, 12.81.

6.5. Synthesis of 99mTc(I) Complexes 21 ‐ 23

Manipulations of radioactive substances were performed under radiation protection

conditions, with protecting gloves and lead shielding in a ventilated hood.

The vials containing the radioactive products were kept inside lead containers with

appropriate thickness.

The exposure to radiation during manipulation was monitored by reading the

individual and the finger dosimeters (radiation detector).

Preparation of the fac‐[99mTc(OH2)3(CO)3]+ precursor

Na[99mTcO4] in NaCl 0.9% was eluted from a 99Mo/99mTc generator Ultra‐TechneKow®

from Mallinckrodt.

The radioactive synthon fac‐[99mTc(CO)3(H2O)3]+ was prepared by adding 1 ‐ 2 mL of

freshly eluted Na[99mTcO4] to the Mallinckrodt IsoLink® kit, and heating for 30 min at 100 °C.

The reaction vial was cooled and the solution neutralized with HCl 1 M, to destroy the

remaining boranocarbonate. The product was controlled by RP‐HPLC; the yield was

quantitative and did not depend on the total amount of activity.


183

6.5.1. General Method for Preparing the 99mTc Complexes

In a glass vial under nitrogen, 100 μl of 10‐3 M aqueous solutions of compounds 13,

15 and 17 were added to 900 μl of fac‐[99mTc(OH2)3(CO)3]+ in NaCl 0.9%. The reaction was

incubated at 100 °C for 45 min in the case of conjugates 15 and 17, and at 75 °C for 45 min in

the case of 13. The resulting complexes were analyzed by RP‐HPLC and TLC.

6.5.1.1. Synthesis of fac‐[99mTc(CO)3(κ3‐13)] (21)

OSNH2

O

O

Tc

OC CO CO

NH

N

O

NH

N

O

O

O

OH

99m

RP‐HPLC (method 1): tR = 19.9, 20.1 min.

TLC (0.5% HCl 6 N/MeOH): Rf = 0.67.

6.5.1.2. Synthesis of fac‐[99mTc(CO)3(κ3‐15)]+ (22)

NN N

NH2

Tc

OC CO CO

O

NH

N

O

NH

N

O

O

O

OH

99m

+



184

6.5.1.3. Synthesis of fac‐[99mTc(CO)3(κ3‐17)]+ (23)

NN N

NH2

Tc

OC CO CO

O

NH

N

O

NH

N

O

O

ONH

N

O

NH

N

O

O

O

OH

99m

+

RP‐HPLC (method 1): tR= 23.0 min.

TLC (0.5% HCl 6 N/MeOH): Rf = 0.57.

6.6. Synthesis of compounds 24 ‐ 33

6.6.1. 2‐(4‐bromobutyl) isoindoline‐1,3‐dione (24)231

N

O

OBr

1

1

2

2

3

4

5

6

10 g (54 mmol) of potassium phthalimide and 35 g (162 mmol) of dibromobutane

were refluxed in 200 mL of acetonitrile for 6 h. After separation of the precipitated KBr, the

solvent was removed in vacuum. The remaining yellow oil was dissolved in 50 mL of

methanol. The desired compound crystallized as a white powder at ‐20 °C overnight.

Yield: 6 g, 40% 1H NMR (300 MHz, CDCl3): δ (ppm) 7.88 ‐ 7.80 (m, 2H, H1); 7.73 ‐ 7.68 (m, 2H, H2);

3.70 (t, 2H, CH23); 3.42 (t, 2H, CH2

6); 1.84 (2 quint, 4H, CH24+5).


185

6.6.2. 3,6‐bis(dimethylamino)‐10‐(4‐(1,3‐dioxoisoindolin‐2‐

yl)butyl)acridinium (25)

N

N

N

N

O

O

1

12

23

4

5

67 9

8

8

7

10

9

Acridine orange (N3,N3,N6,N6‐tetramethylacridine‐3,6‐diamine) (1.54 g, 5.8 mmol)

and 2‐(4‐bromobutyl) isoindoline‐1,3‐dione (24) (4.9 g, 17.4 mmol) in p‐xylol (50 mL) were

refluxed for 24 h. After cooling, the mixture was filtered and the collected solid was dried

under vacuum. The solid was washed with acetone to remove compound 24, present as an

impurity.

Yield: 1.7 g, 54 %. 1H NMR (300 MHz, CDCl3): δ (ppm) 8.67 (s, 1H, CH10); 7.89 ‐ 7.86 (d, J = 9.3 Hz, 2H,

2CH9); 7.74 (m, 2H, 2CH2); 7.68 (m, 2H, 2CH1); 7.00 ‐ 6.97 (dd, J = 9.3 Hz, 2H, 2CH8); 6.61 (s,

2H, 2CH7); 4.97 (t, 2H, CH26); 3.80 (t, 2H, CH2

3); 3.30 (s, 12H, CH3); 2.09 (quint, 2H, CH25); 1.67

(quint, 2H, CH24).

6.6.3. 10‐(4‐Amino‐butyl)‐3,6‐bis‐dimethylamino‐acridinium (26)

NN N

H2N1

23

455

6 687 7

To a suspension of compound 25 (1.23 g, 2.25 mmol) in methanol (20 mL) was added

hydrazine hydrate (5 mL) and the mixture was stirred overnight. After addition of 6 mL of

conc. HCl (37%), a solid of phthalic acid hydrazide precipitated. After its separation the pH

was adjusted to about 9 with NaOH 40%. The reaction mixture was extracted three times

with chloroform (30 mL for each extraction) and the combined organic phases were dried


186

over MgSO4. The solvent was removed under vacuum originating 26 quantitatively as a red

solid that was used without further purification.

RP‐HPLC (method 1, analytic): tR = 24.6 min. 1H NMR (300 MHz, CD3OD): δ (ppm) 8.61 (s, 1H, CH8); 7.88 ‐ 7.85 (d, J = 9.3 Hz, 2H,

2CH7); 7.24 ‐ 7.21 (dd, J = 9.3 Hz, 2H, 2CH6); 6.63 (s, 2H, 2CH5); 4.74 (t, 2H, CH24); 3.32 (s, 12H,

CH3); 3.00 (t, 2H, CH21); 2.04 (quint, 2H, CH2

3); 1.90 (quint, 2H, CH22).

6.6.4. Ethyl 3‐acetyl‐4‐oxopentanoate (27)232

OO

OO

OHO

OO

A B

2,4 ‐pentanedione (7.66 g, 76 mmol) was added slowly at 0 °C to a suspension of NaH

(3.3 g, 84 mmol) in 120 mL of THF. After stirring 1 h, ethyl bromoacetate (8.43 mL, 76 mmol))

was added. Stirring was continued for 3 h at 0 °C and overnight at room temperature.

Addition of 1 N HCl (∼ 500 μl) was followed by extraction with diethyl ether. The ether

extracts were dried over MgSO4 and evaporated. The residue was used without further

purification.

Yield: 10.8 g, 76%. 1H NMR (300 MHz, CDCl3): δ (ppm) 16.77 (s, 1H, OH (B)); 4.13‐4.06 (q + t, 2H+1H, CH2

(A and B) + CH (A)); 3.21 (s, 2H, CH2 (B)); 2.85, 2.83 (d, J = 7.5 Hz, 2H, CH2, (A)); 2.23 (s,

6H,2CH3 (A)); 2.14, 2.13 (s, 6H, 2CH3); 1.21 (t, 3H, CH3).

6.6.5. Tert‐butyl 2‐(2,4‐dinitrophenylsulfonamido)ethylcarbamate

(28)171

NO2

SO

O HN

NH

1

2

3

4

5

O

O 1

1

5a

b

O2N


187

Tert‐butyl‐2‐aminoethylcarbamate (1) (5.33 g, 33.3 mmol), 2,4‐

dinitrobenzenesulfonyl chloride (8.9 g, 33.3 mmol) and pyridine (3.3 mL, 40.2 mmol) were

dissolved in dry CH2Cl2. The solution was stirred at room temperature for 4 h. The resulting

suspension was filtered and the CH2Cl2 phase was washed with water (3 times). The solvent

was evaporated and the obtained solid was purified by column chromatography (eluent:

ethyl acetate (30 ‐ 100%)/hexane).

Yield: 7 g, 54 %. 1H‐NMR (300 MHz, CDCl3): δ (ppm) 8.65 ‐ 8.31 (3H, m, CH5+ CH4); 6.08 (s br, 1H, NHb);

4.79 (s, br, NHa); 3.26 (s br, 4H, 2CH2); 1.38 (s, 9H, CH3).

6.6.6. Ethyl‐2‐(1‐(2‐hydroxyethyl)‐3,5‐dimethyl‐1H‐pyrazol‐4‐yl)acetate

(29)168,169

NN OH

O O

2

3

4

1

5

A solution of 2‐hydroxyethylhydrazine (3.5 mL, 56 mmol) in EtOH (100 mL) was added

dropwise to a solution of ethyl 3‐acetyl‐4‐oxopentanoate (27) (56 mmol) in EtOH at 0 °C.

After overnight reaction at room temperature, the solvent was removed by vacuum and

compound 29 obtained as a yellow oil.

Yield: 12 g, 95%. 1H‐NMR (300 MHz, CDCl3): δ (ppm) 4.04 (q, 2H, CH2

4); 4.00 (t, 2H, CH21); 3.89 (t, 2H,

CH22); 3.28 (s, 2H, CH2

3); 2.15 (s, 3H, CH3‐pz); 2.13 (s, 3H, CH3‐pz); 1.20 (t, 3H, CH35).


188

6.6.7. Ethyl 2‐(1‐(2‐(N‐(2‐(tert‐butoxycarbonylamino)ethyl)‐2,4‐

dinitrophenylsulfonamido)ethyl)‐3,5‐dimethyl‐1H‐pyrazol‐4‐

yl)acetate (30)168,170

NN NNH

OO

NO2SO O

7

2 3

41

5

1'O

O 1'1'

6

9

8

8

NO2

Compound 28 (4.95 g, 12.7 mmol), compound 29 (5.72 g, 25.3 mmol) and

diethylazodicarboxylate (DEAD) (3.28 g, 19 mmol) were dissolved in dry THF, and PPh3 (4.98

g, 19 mmol) was added to the resulting solution. The mixture was allowed to react overnight

at room temperature. After this time, the solvent was removed under vacuum and the

obtained solid purified by column chromatography (ethyl acetate (30 ‐ 100%)/hexane). The

obtained solid was contaminated with OPPh3 and was washed with diethyl ether; the wash

did not remove all the contamination but the product was used in the next deprotection

step.

Yield: 51% (based on NMR spectra), yellow solid. 1H‐NMR (300 MHz, CDCl3): δ (ppm) 8.44 (m, 2H, CH8); 8.15 (d, 1H, CH9); 5.00 (t, 1H,

NH); 4.18 (t, 2H, CH21); 4.11 (q, 2H, CH2

6); 3.78 (t, 2H, CH22); 3.29 (t, 2H, CH2

3); 3.24 (s, 2H,

CH25); 3.14 (q, 2H, CH2

4); 2.18 (s, 3H, CH3‐pz); 2.06 (s, 3H, CH3‐pz); 1.37 (s, 9H, CH31’); 1.24 (t,

3H, CH37).

6.6.8. 2‐(1‐(2‐(2‐(tert‐butoxycarbonylamino)ethylamino)ethyl)‐3,5‐

dimethyl‐1H‐pyrazol‐4‐yl)acetic acid (31)

NNHN

NH

O OH

1

2 3

45

3' 5'4'

O

O

5

5

6

5''4''

6''


189

Compound 30 was dissolved in a mixture of H2O/MeOH (1:2), 5 equivalents of K2CO3

were added and the mixture was stirred overnight at room temperature. The solvent was

evaporated and the obtained residue was purified by column chromatography (MeOH).

Yield: 1.4 g, 70%. 1H‐NMR (300 MHz, D2O): 3.97 (t, 2H, CH2

1); 3.08 (s, 2H, CH26); 3.00 (t, 2H, CH2

4); 2.85

(t, 2H, CH22); 2.55 (t, 2H, CH2

3); 2.00 (s, 3H, CH3‐pz); 1.93 (s, 3H, CH3‐pz); 1.24 (s, 9H, CH35).

13C‐NMR (75.373 MHz, CDCl3): 178.6 (C6’’=O); 156.4 (C4’’=O); 147.0 (C3’); 137.3 (C5’);

112.7 (C4’); 79.2 (C5’’); 50.4 (C1); 48.3 (C3); 47.7 (C2); 38.8 (C4); 32.1 (C6); 28.4 (C5); 11.7 (3’‐

CH3‐pz); 9.4 (5’‐CH3‐pz).

6.6.9. 10‐(4‐(2‐(1‐(2‐(2‐(tert‐butoxycarbonylamino)ethylamino)ethyl)‐

3,5‐dimethyl‐1H‐pyrazol‐4‐yl)acetamido)butyl)‐3,6‐

bis(dimethylamino)acridinium (32)

NNHN

NH

O

N

N

N

NH

1

2 3

4

5

6 7

89 10

1112

13

10

11

12

O

O

4''

4''

4''

4'''

4''''

5'

a

b

c4'

3'

5''

11'

11'

10'

10'

12'

12'

Compound 26 (532 mg, 1.27 mmol) was suspended in DMF (50 mL) and triethylamine

(1mL, 6.9 mmol) added. After 30 min stirring, HBTU (260 mg, 0.69 mmol) and compound 31

(234 mg, 0.69 mmol) were added and the mixture stirred for 24 h. The reaction mixture was

filtered and the collected solid (285 mg) was identified as unreacted compound 26. The

supernatant was evaporated originating a crude solid. Extractions with chloroform removed

a red solid (47 mg) identified as the desired compound that was purified by column

chromatography (15% MeOH/2% NH4+/CHCl3).

Yield: 20 mg, 4%.


190

1H‐NMR (300 MHz, CDCl3): δ (ppm) 8.32 (s, 1H, CH13); 7.69 ‐ 7.66 (d, J = 9.3 Hz, 2H,

2CH12); 6.98‐6.95 (dd, J = 9.3 Hz, J = 2.1 Hz, 2H, 2CH11); 6.47 (t, 1H, NHa); 6.45 (s, 2H, 2CH10);

5.35 (t , 1H, NHc); 4.49 (t, 2H, CH29); 4.08 (t, 2H, CH2

1); 3.31 (t, 2H, CH26); 3.23 (12H + 2 H,

4CH3 (Ao) + CH24); 3.07 (t, 2H, CH2

2); 2.78 (t, 2H, CH23); 2.56 (s br, 1H, NHb); 2.16 (s, 3H, 5’‐

CH3‐pz); 2.09 (s, 3H, 3’‐CH3‐pz); 1.83 (br, 4H, CH27 + CH2

8), 1.39 (s, 9H, CH34’’).

13C‐NMR (75.373 MHz, CDCl3): δ (ppm) 171.6 (C5’’=O); 155.6 (C4’’’’=O); 146.9 (C3’);

142.5, 142.0 (C13 + C11’ + C10’); 138.1 (C5’); 132.9 (C12); 132.0 (C12’); 116.9 (C4’); 114.0 (C11);

92.4 (C10); 79.2 (C4’’’); 48.2, 48.3, 47.3 and 47.8 (C2 + C3 + C1 + C9); 40.7 (4CH3 (Ao)); 39.7 and

38.9 (C4 + C6); 31.5 (C5); 28.4 (C4’’); 26.8 (C7); 23.5 (C8); 11.8 (3’‐CH3‐pz); 9.5 (5’‐CH3‐pz).

6.6.10. 10‐(4‐(2‐(1‐(2‐(2‐aminoethylamino)ethyl)‐3,5‐dimethyl‐1H‐

pyrazol‐4‐yl)acetamido)butyl)‐3,6‐bis(dimethylamino)acridinium

(33)

NNHN

NH2

O

N

N

N

NH

1

2 3

4

5

6 7

89 10

1112

13

10

11

12

10'

10'

11'

11'

12'

12'

3'

4'5'

Compound 32 was dissolved in 2 mL of CHCl3 and 1 mL of TFA was added. After 1 h at

room temperature, the solvent was evaporated originating compound 33 quantitatively.

RP‐HPLC (method 3, analytic): tR = 15.1 min.

ESI/QITMS (most abundant m/z): Molecular Formula ‐ C32H47N8O+. [M]+ ‐ calculated

m/z 559.4, found m/z 559.2; [M+H]2+ ‐ calculated m/z 280.2, found m/z 280.1. 1H‐NMR (300 MHz, D2O): δ (ppm) 7.75 (s, 1H, CH13); 7.21‐7.18 (d, J = 9.3 Hz, 2H,

2CH12); 6.67‐6.64 (dd, J = 9.3 Hz, J = 2.1 Hz, 2H, 2CH11); 5.68 (s, 2H, 2CH10); 4.12 (t, 2H, CH21);

3.85 (t, 2H, CH29); 3.34 (t, 2H, CH2

2); 3.27 (t +t, 4H, CH24 + CH2

3); 3.1 (s, 2H, CH25); 3.00 (t, 2H,

CH26); 2.90 (s, 12H, 4CH3 (Ao)); 1.90 (s, 3H, CH3‐pz); 1.75 (s, 3H, CH3‐pz); 1.42 (br, 4H, CH2

7 +

CH28).


191

13C‐NMR (75.373 MHz, D2O): δ (ppm) 175.8 (C=O); 157.1 (2C(Ar)); 150.5 (C3’); 143.8,

143.4, 142.5 (3C(Ar) + C5’‐pz); 134.6 (4C(Ar); 118.1 (2C(Ar)); 115.7 (2C(Ar)); 112.9 (C4’); 93.3

(C10); 49.3 (C9); 48.4 (C1); 46.6 (C3); 46.2 (C2); 41.9 (4CH3 (Ao)); 41.1 (C4); 37.4 (C6); 32.1 (C5);

28.2 (C7); 25.0 (C8); 12.5 (3’‐CH3‐pz); 10.6 (5’‐CH3‐pz).

6.6.11. Synthesis of fac‐[Re(CO)3(κ3‐42)]2Br (34)

O

NN

N

NH

NN NH

NH2

Re

OC CO CO

2+

[Re(CO)3(OH2)3]Br (6 mg, 0.015 mmol) was reacted with compound 33 (11 mg, 0.015

mmol) in methanol (5 mL) and the mixture was refluxed overnight under N2. After removing

the solvent compound 34 was obtained.

Yield: 13.3 mg, 91%.

RP‐HPLC (method 3): tR = 17.8 min. 1H‐NMR (300 MHz, CD3OD): δ (ppm) 8.64 (s, 1H, CH13); 7.88 (d, J = 9.3 Hz, 2H, 2CH12);

7.24 (dd, J = 9.3 Hz, 2H, 2CH11); 6.92 (br, 1H, NHb); 6.65 (s, 2H, 2CH10); 5.42 (m, 1H, HN‐Ha);

4.75 (t, 2H, CH29); 4.51 (m, 1H, HC‐H1); 4.1 (m, 1H, HC‐H1); 3.84 (m, 1H, HN‐Ha); 3.54 ‐ 3.45

(m, 1H, HC‐H2); 3.39 (s, 2H, CH25); 3.34 (s, 12H, 4CH3 (Ao)); 3.30 (2H, CH2

6); 2.97 ‐ 2.82 (m, 2H,

HC‐H3 + HC‐H4); 2.64 (m, 1H, HC‐H2); 2. 55 ‐ 2.52 (m, 2H, HC‐H3 + HC‐H4); 2.32 (s, 3H, 3’‐CH3‐

pz); 2.25 (s, 3H, 5’‐CH3‐pz); 1.98 (quint, 2H, CH28); 1.84 (quint, 2H, CH2

7). 13C‐NMR (75.373 MHz, CD3OD): δ (ppm) (several signals were obscured under the

solvent) 194.7 (ReCO); 194.5 (ReCO); 194.1 (ReCO); 172.9 (C=O); 157.3 (C11’); 153.4 (C3’);

144.3 (C13); 143.7, 143.7 (C5’+ C10’); 134.4 (C12); 118.5 (C12’); 115.5 (C11); 113.9 (C4’); 93.7 (C10);

55.9 (C3); 43.2 (C4); 40.9 (4CH3 (Ao)); 40.3 (C6); 31.3 (C5); 28.3 (C7); 24.9 (C8); 14.6 (3’‐CH3‐pz);

10.4 (5’‐CH3‐pz).

IR (KBr) (cm‐1): υ(C≡O), 2023, 1889; υ(C=O) 1596.


192

6.6.12. Synthesis of fac‐[99mTc(CO)3(κ3‐33)]2+ (35)

O

NN

N

NH

NN NH

NH2

Tc

OC CO CO

99m

2+

In a glass vial under nitrogen, 100 μl of 8 x 10‐4 M aqueous solution of compound 33

was added to 900 μl of [99mTc(OH2)3(CO)3]+ in NaCl 0.9%. The reaction was incubated at 100

°C for 45 min and the resulting complex was analyzed by RP‐HPLC and TLC.

HPLC (method 1): tR = 17.9 min.

TLC (5% HCl 6N/MeOH): Rf = 0.30.

6.7. Synthesis of Peptide Nucleic Acids

6.7.1. Synthesis of N‐α‐Fmoc‐L‐Lys(Boc)‐resin Novasyn TGR‐PEG‐PS (36)

MeO

MeO

HN

O NHO

i

NHBoc

O

FmocHN

PEG

MeO

MeO

NH2

O NHO

PEG

36 i) N‐α‐Fmoc‐L‐Lys(Boc)‐OH, HBTU, DIPEA, NMP.

Procedure

1. Preparation of the resin

The resin Novasyn TGR‐PEG‐PS (1 g, 0.2 mmol) in a reactor for solid phase synthesis

was dried for 2h. Then, CH2Cl2 was added (until the resin was covered) and agitated for 1


193

h. The CH2Cl2 was removed and the resin was washed with CH2Cl2 (2 x 4mL), 5% DIPEA in

CH2Cl2 (6 mL, 1min) and finally CH2Cl2 (2 x 4 mL).

2. Coupling N‐α‐Fmoc‐L‐Lys(Boc)‐OH to the resin

N‐α‐Fmoc‐L‐Lys(Boc)‐OH (94 mg, 0.20 mmol) was dissolved in NMP (1 mL) and DIPEA

(69 μl, 0.40 mmol) was added. A solution of HBTU (76 mg, 0.20 mmol) in 1 mL of NMP

was added to the lysine solution, the mixture was stirred for 1 min and added to the

resin and shaked for 1 h. The resin was filtrated and washed with DMF (2 x 4mL, 1 min

each wash), CH2Cl2 (4 x 4mL, 1 min each), 5% DIPEA in CH2Cl2 (1 x 4 mL, 30 sec each) and

CH2Cl2 (4 x 2mL, 1 min each).

3. Capping of the resin

A solution of Ac2O/NMP/Pyridine 2:4:4 (10 mL) was added to the resin and shaked for

1.5 h. The capping solution was removed and the resin was washed with CH2Cl2 (3 x 3

mL). A Kaiser test was performed and the yellow colour indicated that there was no free

amine groups. Then, the resin was washed with CH2Cl2 (3 x 2mL), DMF (3 x 2mL), CH2Cl2

(3 x 2mL) and dried under vacuum.

6.7.1.1. Resin Loading204

1. Calibration Curve for Gas Chromatography (GC) Analysis of Fmoc

Deprotection

A 10 mg/mL solution of N‐α‐Fmoc‐L‐Lys(Boc)‐OH in DMF was prepared and different

amounts of this solution were placed in vials. DBU (2% in DMF, 4 mL) was added to each vial

and the vials were shaken to mix. After 1 h, anthracene (10 mg/mL in DMF, 0.6 mL) was

added to each vial. After shaking, an aliquot was drawn from each vial and transferred to a

GC autosampler vial for analysis. A standard curve was prepared by plotting the ratio of the

GC areas of dibenzofulvene and anthracene vs. the molar ratio of Fmoc‐Lys:anthracene (the

calculated weight of Fmoc‐Lys was corrected taking into account the purity of the compound

(98%)). The slope and intercept of the resulting curve were determined and used in the

calculations.


194

Table 6.1 ‐ Calculations for the calibration curve by gas chromatography.

Solution mmol of

Lys‐Fmoc

mmol of

Anthracene

Lys‐Fmoc/Anthracene

(mmol/mmol)

Area of

DBF

Area of

Anthracene

Area DBF/Area

Anthracene

A 4.27E‐03 3.37E‐02 1.27E‐01 1.242 9.493 1.31E‐01

B 8.54E‐03 3.37E‐02 2.53E‐01 2.403 9.307 2.58E‐01

C 1.71E‐02 3.37E‐02 5.07E‐01 4.737 9.192 5.15E‐01

D 2.56E‐02 3.37E‐02 7.60E‐01 6.857 8.919 7.69E‐01

E 3.63E‐02 3.37E‐02 1.08E+00 9.377 8.733 1.07E+00

2. DBU Cleavage of 36

Accurately weighted samples of the vacuum‐dried resin (12.7 mg, 12.4 mg, 13.1 mg,

14.9 mg, 14.8 mg) were placed in tubes and 2 mL of DBU/DMF (2% v/v) was added to each

tube. After 1h stirring, anthracene (10 mg/mL in DMF, 0.2 mL) was added to each tube. After

shaking, an aliquot was drawn from each tube and transferred to a GC autosampler vial. GC

analyses were performed and the peak areas were determined. The Fmoc substitution (FS,

mmol/g) for each sample was calculated as shown in pages 101 and 102

The values found are indicated in table 6.2.

Table 6.2 ‐ Data for calculating the Fmoc substitution (FS).

Solution Area DBF Area

Anthracene

Area DBF/Area

Anthracene

Resin

Weight (g)

[Anthracene]

(mg/mL)

V

(mL)

FS

(mmol/g)

1 12.792 87.208 0.147 1.27E‐02 10 0.2 0.137

2 12.557 87.443 0.144 1.24E‐02 10 0.2 0.138

3 13.265 86.735 0.153 1.31E‐02 10 0.2 0.138

4 14.733 85.267 0.173 1.49E‐02 10 0.2 0.137

5 14.482 85.518 0.169 1.48E‐02 10 0.2 0.135

The average loading for the rection was 0.137 ± 0.001 mmol/g.


195

6.7.2. Automated Solid Phase Synthesis of Peptide Nucleic Acids on the

ABI 433A Synthesizer

6.7.2.1. Automated Synthesis of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐

resin (37)


MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

The calculations for the automated synthesis are presented in table 6.3.

Table 6.3 – Experimental conditions for the automated synthesis.

Reagent M.W (g/mol)

eq mmol Loading of the resin (mmol/g)

mass (mg)

μl of NMP

mg of monomer in 500 μl of

NMP Resin novasyn

TGR‐Lys‐Fmoc (36) 1 0.02 0.18 ‐ 0.14 111 ‐ 143

Fmoc‐T‐Aeg‐OH 506.51 5.3 0.106 53.7 448 60.3

Fmoc‐C(Bhoc)‐OH 701.72 5.3 0.106 74.4 460 81.5

Fmoc‐A(Bhoc)‐OH 725.75 5.3 0.106 76.9 464 83

Fmoc‐G(Bhoc)‐OH 741.75 5.3 0.106 78.6 464 85 1Base Solution 2Capping Solution 3HATU 4Piperidine

1‐ Base solution ‐ DIPEA (1.6 M in NMP) ‐ bottle 5

2‐ Capping solution ‐ Ac2O/ DIPEA/NMP 5:6:89 (v/v/v) ‐ bottle 4

3‐ 3.6 g HATU (2‐(1H‐7‐Azabenzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate) in 25 mL

of NMP, [] = 0.38 M ‐ bottle 7

4‐ Piperidine ‐ bottle 1


196

In the reaction vessel was added the Novasyn TGR‐PEG‐PS resin downloaded with N‐

α‐Fmoc‐L‐Lys(Boc)‐OH (36) (0.14 ‐ 0.18 mmol/g; 0.02 mmol; 111 ‐ 143 mg). The Fmoc‐PNA

sequence (Fmoc‐ A GAT CAT GCC CGG CAT‐Lys‐resin) was synthesized on a 20 μmol scale, by

using Fmoc solid phase peptide synthesis procedures. For each monomer, the following

steps were performed: Fmoc deprotection by piperidine (20% piperidine in NMP), activation

and coupling with HATU as activating agent and DIPEA as base, and capping with acetic

anhydride/DIPEA/NMP (5:6:89).

The 16‐mer sequence was synthesized in two parts. First was synthesized the 8‐mer

sequence (Fmoc‐CC CGG CAT–resin. Then, 5 mg of resin were taken and cleaved with TFA:

H2O: TIS (90:5:5) for 1.5 h. ESI/QITMS analysis was performed to confirm if the observed m/z

were consistent with the calculated for the 8‐mer sequence. When the 8‐mer PNA was

identified, the synthesis restarted in the same way, until the 16‐mer sequence was

completed. At the end of the synthesis the resin was transferred to a manual reaction vessel,

the increase of weight was checked and again 5 mg of resin were cleaved to confirm the

formation of the 16‐mer PNA sequence.

6.7.2.1.1. General Cleavage Procedure

1. A solution of TFA/TIS/H2O 90:5:5 was added to the resin until it was covered;

2. Agitation for 1.5 h in a shaker;

3. The solution was filtered using a N2 flow and the solution was collected in a

tube;

4. The resin was washed 3 times with TFA and the filtrate collected and joined to

the previous one;

5. The solution was concentrated using a N2 flow;

6. After the volume was reduced to 0.5 mL, diethyl ether 4 ‐ 5 mL was added

and the oligomers precipitated;

7. More diethyl ether was added and the solution was frozen to ‐20 °C for 1h to

complete the precipitation;

8. The precipitate was centrifuged and the supernatant removed;


197

9. The solid was washed 3 or more times with diethyl ether and removed at

each time;

10. The solid was dried in vacuum.

The solid was analysed by LC‐ESI/QITMS or ESI/QITMS. If necessary the product was

purified by RP‐HPLC.

6.7.3. Manual Solid Phase Synthesis of Peptide Nucleic Acids

6.7.3.1. Manual Synthesis of Fmoc‐ A GAT CAT GCC CGG CAT‐Lys‐resin

(37)


MeO

MeO

HN

O NHO

NHBoc

O

NH

PEG

The manual synthesis of 37 was performed using experimental conditions indicated

in table 6.43.

Table 6.4 – Experimental conditions for the manual synthesis

Reagent M.W

(g/mol)

eq mmol Loading of

the resin

(mmol/g)

mass

(mg)

V (μl)

Resin novasyn TGR‐

Lys‐fmoc (36) 1 0.02 0.18 ‐ 0.14 111 ‐ 143

Fmoc‐T‐Aeg‐OH 506.51 3 0.06 30.4

Fmoc‐C(Bhoc)‐OH 701.72 3 0.06 42.1

Fmoc‐A(Bhoc)‐OH 725.75 3 0.06 43.5

Fmoc‐G(Bhoc)‐OH 741.75 3 0.06 44.5

DIPEA

N

129.25 3 0.06 11

2,6 – lutidine

N

107.16 4.5 0.09 11

HATU 380.2 2.7 0.054 20.5


198

A polyethylene syringe with a frit at the bottom was used as reactor for manual solid

phase synthesis and the reactions were carried out on a Shaker. Fmoc/Bhoc chemistry was

used with the PNA monomers shown in table 6.4. Fmoc‐Lys(Boc)‐Novasyn TGR‐PEG‐PS resin

(36) was used as solid support at a substitution level of 0.18 – 0.14 mmol/g. Before starting

the synthesis, the resin was swelled for 1 h in CH2Cl2 and 15 min in DMF. Each cycle of

elongation consisted of:

1. Fmoc deprotection with 25% piperidine in DMF for 2 cycles of 1 min at room

temperature;

2. Washing with DMF, CH2Cl2, twice with NMP for 1 min each at room

temperature;

3. Coupling using a molar ratio of resin: monomer: HATU: DIPEA: 2,6‐lutidine=

1:3:2.7:3:4.5, 5 min of activation followed by a 30 min coupling at room

temperature, after which the process was repeated (double coupling)‐ repeat

steps 2 and 3;

4. Capping with 2 mL of a solution of Ac2O/DIPEA/NMP= 5/6/89 for 5 min at

room temperature;

5. Washing with DMF, CH2Cl2, NMP, CH2Cl2.

The Fmoc deprotection was followed by UV on a TLC plate. After 7/9 and 16 cycles,

aliquots of the resin‐PNA were cleaved and Bhoc‐deprotected with TFA:H2O:TIS (90:5:5) for

1.5 h. ESI/QITMS or LC‐ESI/QITMS analyses were performed to confirm that the observed

m/z were consistent with the calculated m/z of the Fmoc‐protected intermediates.

6.7.3.2. H‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (38)

After cleavage of Fmoc‐A GAT CAT GCC CGG CAT‐Lys‐resin (58 mg) from the resin

with TFA/H2O/TIS (90:5:5) for 1.5 h, the Fmoc‐16‐mer PNA was isolated by precipitation with

cold diethylether. The precipitate was then washed three times with the same solvent,

centrifuged and finally dried under vacuum. The crude product was purified by semi‐

preparative RP‐HPLC. The purified Fmoc‐PNA sequence was then deprotected at the N‐

terminus with 25% piperidine in DMF, followed by purification by RP‐HPLC. Compound 38

was then analysed by HPLC and ESI/FTICRMS.


199

RP‐HPLC: tR = 15.9 min.

High resolution ESI/FTICRMS (most abundant m/z): Molecular formula ‐

C177H226N96O48. [M+3H]3+ ‐ calculated m/z 1489.6176, found m/z 1489.6196; [M+4H]4+ ‐

calculated m/z 1117.4657, found m/z 1117.4661.

6.7.3.3. Synthesis of Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) and fac‐

[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]

+ (40)

NN N

NH2

O


39

H2N

NH2

OHN


NN N

NH2

Re

OC CO CO

O

40

H2N

NH2

ONH

+

The syntheses of 39 and 40 were performed using the experimental conditions

indicated in tables 6.5 and 6.6, respectively.

Table 6.5 – Experimental conditions for the synthesis of compound 39.

Reagent M.W. (g/mol) Density eq mmol mg V (μl)

Fmoc‐ A GAT CAT GCC

CGG CAT‐Lys‐resin (36)

0.02

Pz‐Boc

NN NNHBoc

O

OH

368.47 3.5 0.07 25

DIPEA 129.25 0.755 3 0.06 11

2,6‐ lutidine 107.16 0.923 4.5 0.09 11

HATU 380.2 2.7 0.054 20.5


200

Table 6.6 ‐ Experimental conditions for the synthesis of compound 40.

Reagent Molecular weight Eq mmol mg V (μl)

Fmoc‐ A GAT CAT GCC

CGG CAT‐Lys‐resin

0.01

RePz

NN N

NH2

Re

OC CO CO

O

OH

+ Br‐

618.05 4 0.04 23

DIPEA 129.25 3 0.03 5

2,6 LUTIDINA 107.16 4.5 0.045 5

HATU 380.2 2.7 0.027 10

The procedure described in section 6.7.3.1 was used. In the coupling step was used a

molar ratio of resin:Pz‐Boc: HATU: DIPEA: 2,6‐lutidine of 1.0:3.5:2.7:3.0:4.5 or resin:RePz:

HATU: DIPEA: 2,6‐lutidine of 1.0:4:2.7:3.0:4.5; 20 min of pre‐activation were followed by 40

min coupling at room temperature.

The crude products 39 and 40 were obtained in 75 and 50% yield, respectively. These

products were then purified by semipreparative RP‐HPLC and recovered in 25% (39) and 10%

(40) overall yield. Their identity was confirmed by ESI/FTICRMS.

• Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39): molecular formula: C190H248N100O49. High

resolution ESI/FTICRMS (most abundant m/z): [M+4H]4+ ‐ calculated m/z 1180.0082,

found m/z 1180.0126; [M+5H]5+ ‐ calculated m/z 944.2080, found m/z 944.2102. RP‐

HPLC: tR = 17.1 min.

• fac‐[Re(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]+ (40): molecular formula:

C193H248N100O52Re+. High resolution ESI/FTICRMS (most abundant m/z): [M+3H]4+ ‐

calculated m/z 1247.4916, found m/z 1247.4959; [M+4H]5+ ‐ calculated m/z

998.1947, found m/z 998.1970; [M+5H]6+ ‐ calculated m/z 831.9968, found m/z

831.9986. RP‐HPLC: tR = 19.3 min.


201

6.7.3.4. Synthesis of Cyst‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (41)

SF3COCHN

O

OO

A GAT CAT GCC CGG CAT‐ Lys‐ NH2

41

Table 6.7 – Experimental conditions for the synthesis of compound 41

Reagent Molecular Weight eq mmol mg

Fmoc‐ A GAT CAT GCC CGG

CAT‐Lys‐resin 0.02

SF3COCHN

O

OO

O

F F

F

FF

11

455.28 4.4 0.088 40

The procedure described in section 6.7.3.1 was used. In the coupling step was used a

molar ratio of resin:11 = 1.0:4.4, and 40 min coupling at room temperature.

5 mg of the resin were cleaved with TFA/H2O/TIS (90:5:5) for 1.5 h. The product was

isolated by precipitation with cold diethylether. The precipitate was washed with cold

diethylether, centrifuged at each time and collected. The crude product was analysed by LC‐

ESI/QITMS. The peak at retention time 17.7 min was attributed to compound:

• Cys‐A GAT CAT GCC CGG CAT‐Lys: CH3OCOC(NHCOCF3)CH2SCH2CO‐ A GAT CAT

GCC CGG CAT‐Lys‐NH2. Molecular formula: C185H234F3N97O52S. ESI/QITMS

(most abundant m/z): [M+3H]3+ ‐ calculated m/z 1580.3, found m/z 1580.4;

[M+4H]4+ ‐ calculated m/z 1185.5, found m/z 1185.7.


202

6.7.4. Synthesis of fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐

NH2)]2+ (42)


NN N

NH2

Tc

OC CO CO

O H2N

NH3

ONH

2+

99m

In a nitrogen purged glass vial, 20 μl of ∼5x10‐4 M aqueous solution of the purified

conjugate Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2 (39) was added to 180 μl of fac‐

[99mTc(CO)3(H2O)3]+ (37–74 MBq) in NaCl 0.9%. The reaction mixture was incubated at 100 °C

for 1h at pH ∼ 7.4. The resulting complex fac‐[99mTc(CO)3(κ3‐Pz‐ A GAT CAT GCC CGG CAT‐

Lys‐NH2)]2+ was analyzed by RP‐HPLC. The presence of colloids was checked by instant thin

layer chromatography.

RP‐HPLC (analytic, method 4): tR = 19.6 min.

ITLC (Pyridine/acetic acid/H2O (3:5:1.5)): Rf = 1.

6.8. Synthesis of compounds 43 ‐ 44

6.8.1. Synthesis of Pz‐βAla‐Nle‐cyclo[Asp‐His‐DPhe‐Arg‐Trp‐Lys]‐NH2

(43)

NH

HN NH

OH2N

NH

O

NH

HN

O

NH

NH2

HN

NH

O

NH

NHN O

HNO

HNO

O

O

NN NNH2

O


203

The Pz‐βAla‐Nle‐cyclo[Asp‐His‐DPhe‐Arg‐Trp‐Lys]‐NH2 was synthesized by Soraia

Falcão and Dr. Paula Gomes at the Universidade do Porto followed the methodology

described.225

Full deprotection and cleavage of the final peptide from the solid support was

performed by treatment of the peptidyl resin with TFA containing 2.5% H2O and 2.5%

triisopropylsilane. The crude product was purified by semipreparative reversed‐phase HPLC

(better than 97%) and successfully characterized as the target peptide by amino acid analysis

and ESI/QITMS.


ESI/QITMS (most abundant m/z): Molecular formula ‐ C64H94N20O10. [M+H]+ ‐

calculated m/z 1303.7, found m/z 1303.9; [M+2H]2+ ‐ calculated m/z 652.4, found m/z 653.2.

6.8.2. Synthesis of fac‐[99mTc(CO)3(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐DPhe‐Arg‐

Trp‐Lys]‐NH2]2+ (44)

99mNN N

NH2

TcOC

COCO

O HN

HN NH

OH2N

NH

O

NH

HN

O

NH

NH2

H2N

NH

O

NH

NHN O

HNO

HNO

O

O

2+

In a nitrogen purged glass vial, 100 μl of 6x10‐4 M aqueous solution of Pz‐βAla‐Nle‐

cyclo[Asp‐His‐DPhe‐Arg‐Trp‐Lys]‐NH2 (43) was added to 900 μl of fac‐[99mTc(CO)3(OH2)3]+ (37

‐ 74 MBq) in NaCl 0.9%. The reaction mixture was incubated at 90 °C for 50 min and then

analyzed by RP‐HPLC and TLC.

RP‐HPLC (analytic, method 5): tR = 11.8 min.

TLC (5% HCl 6N/MeOH): Rf = 0.45.


204

The radiolabelled compound was purified by semi‐preparative RP‐HPLC. The activity

corresponding to the radioconjugate 44 was collected in a 50 mL Falcon flask containing 200

μL of PBS with 0.2% BSA or modified Eagle’s medium (MEM) with 0.2% BSA, for

biodistribution or cell studies, respectively. The solutions were concentrated to a final

volume of 100 μL under a nitrogen stream, and the product was checked by thin‐layer

chromatography and HPLC (see in vitro stability) to confirm its purity and stability after

purification and evaporation.

6.9. Partition Coefficient

The lipophilicity of the radioconjugates was evaluated by the ‘‘shakeflask’’ method233

which consists on determining the partition coefficient (P) in a biphasic system n‐

octanol/PBS 0.1 M, pH 7.4.

The radioconjugates were added to a mixture of octanol (1 mL) and 0.1 M PBS, pH =

7.4 (1 mL), previously saturated in each other by stirring the mixture (1 min). This mixture

was vortexed and centrifuged (3000 rpm, 10 min) to allow phase separation. 500 μl of each

phase were counted in an ionization chamber and the phase with higher activity was used

for a new partition by adding 500 μl of the opposite phase. The mixture was again vortexed

and centrifuged (3000 rpm, 10 min) to allow phase separation. 50 μl aliquots of both octanol

and PBS were counted using a γ‐counter. The partition coefficient (Po/w) was calculated by

dividing the counts in the octanol phase by those in the buffer, and the results were

expressed as log Po/w.


6.10.1. Stability in the Presence of Cysteine and Histidine

100 μl of 99mTc complexes solutions (21 and 23) were added to 900 μl of a 1.1 x 10‐3

M solution of cysteine and to 900 μl of a 1.1 x 10‐3 M solution of histidine, in PBS, pH = 7.4 (it

was added a 100‐fold excess of cysteine or histidine relative to compounds 15 and 17). The


205

solutions were incubated at 37 °C and aliquots were analyzed by RP‐HPLC at different time

points (1, 2, 4 and 6 h).

6.10.2. Stability in Fresh Human Serum

The radiocomplexes fac‐[99mTc(CO)3(κ3‐Pz‐A GAT CAT GCC CGG CAT‐Lys‐NH2)]2+ (42)

and fac‐[99mTc(CO)3(κ3‐Pz‐βAla‐Nle‐cyclo[Asp‐DPhe‐Arg‐Trp‐Lys]‐NH2]2+ (44) (100 μl) were

added to fresh human serum (700 μl ‐ 1 mL), and the mixtures were incubated at 37 °C. At

appropriate time points (5 min, 45 min, 1 h, 2 h, 4 h and 24 h), 100 μl aliquots were sampled

and treated with 200 μl of ethanol to precipitate the proteins. Samples were centrifuged at

3000 rpm for 15 min at 4 °C. The supernatant was analyzed by RP‐HPLC.

6.10.3. Stability in PBS with 0.2% BSA

The stability of the purified compound 44 in PBS containing 0.2% BSA was checked by

TLC (5% HCl 6 N /MeOH) at different time points, after incubation at 37 °C.

6.10.4. Stability in Cell Medium

The radioactive compounds (purified or non‐purified) used for cell tests were added

to cell medium and incubated at 37 °C. At appropriate periods of time (0 min, 1 h, 2 h, 4 h

and 24 h), aliquots were analysed by ITLC (pyridine/HOAC/H2O (3:5:1.5) for 42, TLC (5% HCl 6

N /MeOH) for 35 and 44, and RP‐HPLC for 42 (Discovery® BioWide Pore analytic) and 44

(column: Supelguard LC 3 DP 2 cm x 4.6 mm ID; eluents: A – 10% iso‐propanol and 90% TFA

0.1% in H2O, B ‐ 90% iso‐propanol and 10% TFA 0.1% in H2O; flow: 1 mL/min).

6.11. Cell Studies

Cell experiments were performed by the RSG/ITN cell experimentation group. The

fluorescence microscopy studies were performed at Instituto de Medicina Molecular by Dr.

Fernanda Marques and Dr. José Rino.


206

The cell studies of compounds 35 and 44 were performed with the purified

radiocompounds. The fraction corresponding to each radioconjugate was collected in a 50

mL Falcon flask containing 200 μl of modified Eagle’s medium (MEM) with 0.2% BSA. The

solutions were concentrated to dryness and after that cell medium was added to the

purified product.

6.11.1. Cell Cultures

SH‐SY5Y human neuroblastoma cells, MCF7 human breast cancer cells and PC3

human prostate cancer cells

SH‐SY5Y human neuroblastoma cells, kindly provided by Dr. R. Perfeito from Centro

de Neurociências, Coimbra, Portugal, and MCF7 human breast cancer cells (ATCC, Manassas,

VA USA) were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing GlutaMax I

supplemented with 10% heat‐inactivated fetal bovine serum and 1% penicillin/streptomycin

antibitiotic solution (all from Gibco, Invitrogen, UK). PC3 human prostate cancer cells (ATCC,

Manassas, VA USA) were grown in RPMI 1640 supplemented with 10% heat‐inactivated fetal

bovine serum and 1% penicillin/streptomycin antibitiotic solution (Gibco, Invitrogen, UK).

Cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C, with

the medium changed every two days. The cells were adherent in monolayers and, when

confluent, were harvested from the cell culture flasks with trypsin–EDTA (Gibco, Invitrogen,

UK) and seeded farther apart.

B16F1 murine melanoma cells

B16F1 murine melanoma cells (ECACC, England) were grown in Dulbecco’s Modified

Eagle Medium (DMEM) containing GlutaMax I supplemented with 10% heat‐inactivated fetal

bovine serum and 1% penicillin/streptomycin antibitiotic solution (all from Gibco, Alfagene,

Lisbon). Cells were cultured in a humidified atmosphere of 95 % air and 5 % CO2 at 37 °C

(Heraeus, Germany), with the medium changed every other day. The cells were adherent in


207

monolayers and, when confluent, were harvested from the cell culture flasks with trypsin

EDTA (Gibco, Alfagene, Lisbon) and seeded farther apart.

6.11.2. Cellular Internalization and Retention Studies

Internalization assays of the radioconjugates were performed in the appropriate cell

line. The cells were seeded at a density of 0.2 million/well into 24‐well tissue culture plates

and allowed to attach overnight. Cells were incubated in humidified 5 % CO2/95 % air, at

room temperature or 37 °C for a period of 5 min to 24 h with about 200 000 cpm of

radioconjugates in 0.5 mL of assay medium (Modified Eagle’s Medium with 25 mM HEPES

(N‐(2‐hydroxyethyl)piperazine‐N’‐ethanesulfonic acid and 0.2% BSA or RPMI). After

incubation, cells were washed with ice‐cold assay medium. Cell‐surface bound

radiocompound was removed by two steps of acid wash (50 mM glycine, HCl/100 mM NaCl,

pH 2.8) at room temperature for 5 min. The pH was neutralized with cold PBS with 0.2% BSA,

and subsequently the cells were lysed by 10 min incubation with 1N NaOH at 37 °C and the

radioactivity measured to determine the percentage of internalized complex.

The cellular retention of the internalized radioconjugate was determined by pre‐

incubation of cells with the radiolabelled compound for 3 or 4 h in humidified 5 % CO2/95 %

air, at 37 °C. Afterwards, cells were washed with cold assay medium, the membrane‐bound

radioactivity removed with acid buffer. The cells were then neutralized with cold PBS with

0.2% BSA and incubated with culture medium (0.5 mL) at 37 °C. Radioactivity release into

the culture media and that in the cell lysates were monitored in a gamma camera at

different time points over a 5 ‐ 6 h incubation period.

6.11.3. Nuclear Internalization

The cells were seeded at a density of 0.2 million/well into 24‐well tissue culture

plates and allowed to attach overnight. Cells were incubated in humidified 5% CO2/ 95% air,

37 °C for a period of 30 min, 1, 2, 3, 4, 5 and 6 h with about 200 000 cpm of radioconjugate

in 0.5 mL of assay medium. After incubation cells were washed with PBS with 0.2% BSA (250

μl/well) and removed from the plate with trypsin (100 μl/well). The inactivation of trypsin


208

was performed with 250 μl of culture medium. The cells in suspension were centrifuged (800

rpm, 2 min) and washed twice with cold PBS with 0.2% BSA. To disrupt the cell membrane 1

μl of Nonidet P‐40 10% (Roche) in 500 μl of lyse buffer (Tris 10 mM, MgCl2 1.5 mM, NaCl 140

mM, pH 8.0 ‐ 8.3) were added. After 15 min of incubation in ice the cells suspension was

centrifuged at 1300 g at 4 °C for 1 min.

The activities of supernatant (activity outside the nucleus) and of the precipitate

(activity retained in the nucleus) were measured (4 replicates) for different incubation times.

The cellular viability of the cells subjected to the radioactive product for 4 h was

tested. Three wells of each plate were selected and the cells were harvested with trypsin

and trypan blue was added. The viable cells do not get the blue colour because they are not

permeable to trypan blue.

6.11.4. Cell Viability

The cytotoxicity was analysed by MTT test that evaluates the cellular viability after

incubation with the compound to test. The cells that survive to the test reduced the MTT182

(3‐(4,5‐dimethylthiazol‐2‐yl‐)‐2,5‐diphenyltetrazolium bromide) to formazan, during the

cellular respiration in the mitochondria. The formazan detection is an indication of the

mitochondria integrity and, indirectly, of cellular viability.

6.11.5. Cytotoxicity of Compounds 33 and 34

The cells were seeded in 96‐well culture plates at 9000 cells/well and allowed to

attach for 6 h at 37 °C. Then the cells were exposed to compounds 33 or 34 (6.0 x 10‐5 M, 1.2

x 10‐5 M, 6.0 x 10‐6 M, 1.2 x 10‐6 M, 6.0 x 10‐7 M, 1.2 x 10‐7 M, 6.0 x 10‐8 M, 1.2 x 10‐8 M, 6.0 x

10‐9 M, 1.2 x 10‐9 M) for 24 h at 37 °C. After removing the compound solutions (pump

aspiration) and after PBS washing (200 μl), MTT (0.5 mg/mL, 200 μl, 0.1 mg/well) in MEM

without phenol red with 10% FBS or PBS was incubated with the cells for 3 h at 37 °C. After

removing the MTT solution and PBS washing (200 μl), the insoluble formazan crystals formed

were dissolved (with micropipettes) and homogenized with dimethylsulfoxide (200 μl/well).


209

6.11.6. Radiotoxicity of fac‐[99mTc(CO)3(κ3‐33)]2+ (35)

The cells were seeded in 96‐well culture plates at 7000 cells/well (100μL, culture

medium (DMEM complete medium)) and allowed to attach for 6 h at 37 °C. Then the cells

were exposed to the radioactive complex (40; 20; 10; 4; 2; 1; 0.4 μCi in culture medium, by

dilutions from the most concentrated) for 36 ‐ 40 h at 37 °C. After removing the radioactive

solutions (pump aspiration) and after PBS washing (200 μL), MTT (0.5mg/mL, 200μl,

0.1mg/well) in MEM without phenol red with 10% FBS or PBS was incubated with the cells

for 3 h at 37 °C. After removing the MTT solution and PBS washing (200 μL), the insoluble

formazan crystals formed were dissolved (with micropipettes) and homogenized with

dimethylsulfoxide (200 μL/well).

The absorbance was measured at 595 nm using a microplate spectrophotomer (Power

Wave Xs, Bio‐Tek). The blank solution was prepared with 200 μl of DMSO without cells and

the procedure with MTT (first column of the microplate). The negative control was

performed with the cells without radioactive complex (only DMEM complete medium and all

procedure with MTT). The IC50 values were calculated with the GraphPad Prism 5 Demo

program.

6.11.7. Evaluation of Cell Uptake by Fluorescence Microscopy

B16F1 murine melanoma cells were grown at 37 °C in DMEM medium (GIBCO) that

was supplemented with 10% fetal bovine serum, under a humidified 5% CO2 atmosphere.

For microscopy, cells were cultured overnight on coverslips that had been sterilised in

ethanol, and then placed in sterile six‐well plates in which ∼5x104 cells per well were plated.

The next day the medium was discarded and replaced by fresh medium that contained

ligand or complex (60 μM). The cells were exposed to compounds 33 or 34 for 3 h. After the

loading, the cells were washed with PBS and fixed for 20 min at room temperature with 3%

paraformaldehyde in PBS. After three washings with PBS, the cells on the coverslips were

incubated with 60 μM DAPI (4’, 6‐diamidino‐2‐phenylindole) for nuclear staining for 20 min

at room temperature, and then washed three times with PBS. After washing, the coverslips

were mounted on standard microscope slides with 3% N‐propyl gallate in glycerol to


210

improve the optical conditions and to prevent photobleaching. The samples were then

imaged on a Leica DMRA2 upright microscope by using a 100 x 1.4NA objective and a Leica

L5 filter cube for evaluating acridine orange fluorescence (λex = 440‐480 nm, λem max = 530

nm (green)) and a Chroma +A4 UV filter for DAPI (λex max = 340 nm, λem max = 449 nm

(blue)). Images were acquired and colour‐combined by using a CoolSNAP HQ 1.3

Mpixelcooled CCD camera and the MetaMorph software.

6.12. Biodistribution and In Vivo Stability

All animal experiments were performed in compliance with Portuguese Law and

European Directives (Portaria 1131/97, Decretos‐Lei nº 129/92 de 6 de Julho e 197/96 de 16

de Outubro and 86/609/CEE) regarding ethics, care and protection of animals used for

experimental and other scientific proposes. The animals were housed in a temperature (22‐

23 °C) and humidity (45 ‐ 65 %) controlled room with a 12 h light/12 h dark schedule.

Animal studies were performed by the RSG/ITN animal experimentation group.

6.12.1. Biodistribution Studies

6.12.1.1. Biodistribution Studies in CD‐1 Female Mice

Biodistribution of the 99mTc complexes 21, 23, 35 and 42 was evaluated in groups of 3

‐ 4 CD‐1 female mice (Charles River outbred strain, from IFFA CREDO, Barcelona, Spain).

Animals were 30 ‐ 39 days old and 22 ‐ 29 g weight. Mice were intravenously injected with

100 μl of each radiolabelled complex (in PBS) (3 ‐ 10 MBq) via the tail vein and the animals

were kept in normal diet ad libitum. The effect of L‐lysine on non‐specific kidney uptake of

radiocomplex 42 was checked up by co‐injection with L‐Lys (15 mg/50 μl saline) in order to

block the cationic transporters on renal tubules.

Animals were sacrificed by cervical dislocation at 1 and 4 h after injection. The

administered dose and the radioactivity in the sacrificed animals were measured with a dose

calibrator (Curiemeter IGC‐3, Aloka, Tokyo, Japan or Carpintec CRC‐15W, Ramsey, USA). The


211

difference in radioactivity between the injected animal and the sacrificed animal was

assumed to be due to excretion. Tissues and organs of interest were dissected, rinsed to

remove excess blood, weighted, and their radioactivity was measured using the dose

calibrator or a γ‐counter. The uptake in the tissues or organs was calculated and expressed

as percent injected dose per gram of tissue or organ (%ID/g) or percent of injected dose per

total organ or tissue (%ID/organ). For blood, bone, muscle, and skin, total activity was

estimated assuming that they represent 6, 10, 40, and 15% of the total body weight,

respectively. Blood samples were taken by cardiac puncture at sacrifice. The blood was then

centrifuged and the serum separated and treated for HPLC analysis. Urine was also collected

and pooled together at sacrifice time.

Table 6.8 ‐ Biodistribution results of the 99mTc compounds 21 and 23 at 1 and 4 h in CD‐1 Charles

River mice, after intravenous injection (mean ± standard deviation, n = 3).

% ID/organ ± standard deviation

fac‐[99mTc(CO)3(κ3‐13)] (21) fac‐[99mTc(CO)3(κ

3‐17)]+ (23) Tissue/organ

1h 4h 1h 4h

Blood 1.89 ± 0.23 1.39 ± 0.57 0.69 ± 0.12 0.31 ± 0.01

Liver 4.81 ± 1.04 4.16 ± 0.54 5.57 ±1.51 3.24 ± 0.94

Intestine 16.03 ± 0.77 20.38 ± 2.95 24.05 ±5.28 29.15 ± 1.72

Spleen 0.06 ± 0.04 0.09 ± 0.08 0.12 ± 0.03 0.12 ± 0.05

Heart 0.04 ± 0.01 0.02 ± 0.00 0.03 ± 0.01 0.01 ± 0.00

Lung 0.13 ± 0.03 0.09 ± 0.03 0.10 ± 0.02 0.07 ± 0.03

Kidney 0.98 ± 0.15 0.80 ± 0.01 0.33 ± 0.09 0.23 ± 0.02

Muscle 0.74 ± 0.11 0.67 ± 0.14 0.46 ± 0.06 0.26 ± 0.04

Bone 0.38 ±0.03 0.36 ± 0.11 0.23 ± 0.02 0.14 ± 0.02

Stomach 0.28 ± 0.13 0.07 ± 0.04 0.39 ± 0.10 0.09 ± 0.03

Pancreas 0.05 ± 0.01 0.03 ± 0.01 0.03 ±0.02 0.01 ± 0.00

Excretion (% ID) 69.5 ± 1.4 67.6 ± 3.5 61.2 ± 4.8 64.4 ± 2.8


212

Table 6.9 ‐ Biodistribution results of the 99mTc compound 35 at 1 and 4 h in CD‐1 Charles River mice,

after intravenous injection (mean ± standard deviation, n = 4).

% ID/organ ± standard deviation Tissue/organ

1h 4h

Blood 0.90 ± 0.68 0.17 ± 0.06

Liver 54.89 ± 8.48 43.81 ± 15.71

Intestine 26.41 ± 5.95 38.83 ± 11.98

Spleen 0.06 ± 0.01 0.34 ± 0.01

Heart 0.03 ± 0.01 0.03 ± 0.01

Lung 0.91 ± 0.37 0.12 ± 0.04

Kidney 8.80 ± 1.39 6.66 ± 2.47

Muscle 0.64 ± 0.27 0.38 ± 0.09

Bone 0.35 ± 0.07 0.34 ± 0.06

Stomach 0.32 ± 0.11 0.40 ± 0.10

Excretion (% ID) 3.5 ± 1.1 10.2 ± 6.5

Table 6.10 ‐ Biodistribution results of the 99mTc compound 42 at 1 and 4 h in CD‐1 Charles River mice,

after intravenous injection (mean %ID/organ ± standard deviation, n = 4).


1h 4h

Blood 1.40 ± 0.14 0.80 ± 0.05

Liver 26.24 ± 3.31 32.39 ± 1.34

Intestine 1.22 ± 0.23 1.29 ± 0.12

Spleen 0.34 ± 0.14 0.38 ± 0.12

Heart 0.08 ± 0.02 0.07 ± 0.01

Lung 0.80 ± 0.52 1.21 ± 0.74

Kidney 36.66 ± 1.03 34.71 ± 0.95

Muscle 2.38 ± 0.21 1.71 ± 0.15

Bone 2.19 ± 0.42 2.09 ± 0.87

Stomach 0.65 ± 0.19 0.30 ± 0.04

Excretion (% ID) 15.5 ± 1.4 18.9 ± 2.1


213

Table 6.11 ‐ Biodistribution results of the 99mTc compound 42 co‐injected with lysine at 4 h in CD‐1

Charles River mice, after intravenous injection (mean %ID/organ ± standard deviation, n = 4).

Tissue/organ % ID/organ ± standard deviation

Blood 0.73 ± 0.06

Liver 38.60 ± 2.37

Intestine 3.86 ± 0.73

Spleen 3.59 ± 0.73

Heart 0.10 ± 0.02

Lung 1.14 ± 1.00

Kidney 10.87 ± 1.47

Muscle 2.15 ± 0.28

Bone 3.26 ± 1.20

Stomach 0.57 ± 0.34

Excretion (% ID) 31.0 ± 3.0

6.12.1.2. Biodistribution Studies in C57BL/6 female mice

Biodistribution of the purified fac‐[99mTc(CO)3‐Pz‐βAla‐Nle‐cyclo[Asp‐DPhe‐Arg‐Trp‐

Lys]‐NH2] (44) was evaluated in healthy and melanoma‐bearing C57BL/6 female mice (8–10

weeks old). Mice were previously implanted subcutaneously with 1 X 106 B16F1 cells to

generate a primary skin melanoma. 10 to 12 days after the inoculation, tumors reached a

weight of 0.2‐1 g. C57BL/6 female mice were intravenously injected into the retroorbital

sinus with the radiolabelled complex (3 ‐ 10 MBq) diluted in 100 μl of PBS pH 7.2. For

confirming the specific tumor uptake, 10 μg of NDP‐MSH was co‐injected with the

radioactive complex. The effect of L‐Lys coi‐njection on non‐specific kidney uptake of the

radiopeptide (44) was examined in healthy C57BL/6 female mice. The animals were injected

with a mixture of 3 MBq of the radiocomplex 44 and 15 mg of L‐Lys in 100 μl.

The animals were sacrificed by cervical dislocation at 1, 4 and 24 h after injection. The

administered dose and the radioactivity in the sacrificed animals were measured with a dose

calibrator (Curiemeter IGC‐3, Aloka, Tokyo, Japan or Carpintec CRC‐15W, Ramsey, USA). The

difference in radioactivity between the injected animal and the sacrificed animal was


214

assumed to be due to excretion. Tumor, normal tissues and normal tissues and organs of

interest were dissected, rinsed to remove excess blood, weighted, and their radioactivity

was measured using a γ‐counter (LB2111, Berthold, Germany). The uptake in the tumor,

tissues and organs of interest was calculated and expressed as percent injected dose per

gram of tissue or organ (%ID/g) or percent of injected dose per organ or tissue (%ID/organ).

For blood, bone, muscle, and skin, total activity was estimated assuming that they represent

6, 10, 40, and 15% of the total body weight, respectively. Urine was also collected and

pooled together at the sacrificed time.

Table 6.12 ‐ Biodistribution results of the 99mTc compound 44 in B16F1 murine melanoma‐bearing

C57BL/6 mice at 1, 4 and 24 h after intravenous injection (mean ± standard deviation, n = 4 ‐ 5)


1 h 4 h 4 h with NDPa 24 h

Tumor 2.9 ± 1.01 4.65 ± 1.17 0.75 ± 0.46 1.43 ± 0.46

Blood 3.33 ± 0.93 2.18 ± 0.25 1.83 ± 0.17 0.25 ± 0.07

Liver 39.33 ± 1.85 23.51 ± 1.28 24.93 ± 0.60 1.87 ± 0.04

Intestine 8.59 ± 0.39 16.68 ± 2.00 18.59 ± 0.65 2.39 ± 0.76

Spleen 0.22 ± 0.02 0.18 ± 0.02 0.16 ± 0.01 0.04 ± 0.00

Heart 0.11 ± 0.02 0.05 ± 0.01 0.47 ± 0.01 0.02 ± 0.00

Lung 0.56 ± 0.10 0.23 ± 0.02 0.25 ± 0.07 0.12 ± 0.06

Kidney 19.22 ± 1.64 9.24 ± 0.67 11.77 ± 0.48 0.43 ± 0.07

Muscle 2.86 ± 0.77 1.71 ± 0.84 1.52 ± 0.48 0.24 ± 0.03

Bone 2.30 ± 0.25 1.53 ± 0.35 1.00 ± 0.13 0.20 ± 0.03

Stomach 0.40 ± 0.15 0.20 ± 0.06 0.27 ± 0.09 0.05 ± 0.06

Pancreas 0.11 ± 0.04 0.06 ± 0.02 0.04 ± 0.00 0.01 ± 0.00

Skin 2.55 ± 0.30 1.74 ± 0.31 1.77 ± 0.30 0.43 ± 0.24

Excretion (% ID) 14.2 ± 2.3 37.4 ± 2.4 33.0 ± 1.5 87.6 ± 3.2 aNDP (Nle4, DPhe7)‐αMSH (see table 1.8); aCoinjection of the radioconjugate with NDP


215

Table 6.13 ‐ Biodistribution of the 99mTc compound 44 co‐injected with 15 mg of L‐lysine in healthy

C57BL/6 mice, at 1 h after intravenous injection (mean ± standard deviation, n = 4).

Tissue/organ % ID/organ ± standard deviation

Blood 3.56 ± 1.77

Liver 29.81 ± 3.16

Intestine 4.32 ± 0.90

Spleen 0.16 ± 0.02

Heart 0.12 ± 0.02

Lung 0.67 ± 0.29

Kidney 14.70 ± 1.51

Muscle 2.39 ± 0.49

Bone 1.49 ± 0.55

Stomach 0.84 ± 0.40

Adrenals 0.06 ± 0.04

Pancreas 0.07 ± 0.02

Skin 3.30 ± 0.43

Total Excretion (%) 17.0 ± 2.2

6.12.2. In Vivo Stability/Metabolization

The in vivo stability of the radiocomplexes was evaluated by RP‐HPLC analysis of

urine, blood and liver homogenate.

Urine: The urine was collected at the sacrificed time, centrifuged at 3000 rpm for 15

min and analysed by RP‐HPLC.

Blood: The blood was collected at the sacrificed time, centrifuged at 3000 rpm for 15

min at 4 °C, and the serum was separated. 100 μl aliquots of serum were sampled and

treated with 200 μl of ethanol to precipitate the proteins. Samples were then centrifuged at

3000 rpm for 15 min at 4 °C. The supernatant was analyzed by RP‐HPLC.


216

Liver homogenate: After injection of the radiocompound, the animals were kept with

ad libitum diet. Immediately after sacrificed, the liver was excised, washed and added to 50

mM TRIS/ 0.2 M sacarose, pH 7.4 at 4 °C, and homogenised. Liver homogenate aliquots (in

duplicate) were treated with ethanol in a proportion EtOH/aliquot 2:1 v/v. The samples were

centrifuged at 25 000 rpm, 15 min, 4 °C, and the supernatant analysed by RP‐HPLC.

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