development of water-soluble mn(iii) porphyrin as ... · teaching and interacting with students;...

Development of Water-Soluble Mn(III) Porphyrin as Extracellular MRI Contrast Agents: Optimizing Relaxivity at

High Magnetic Field

by

Yong Le Zhu

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Yong Le Zhu 2015

ii

Development of Water-Soluble Mn(III) porphyrin as Extracellular

MRI Contrast Agents: Optimizing Relaxivity at High Magnetic

Field

Yong Le Zhu

Master of Science

Department of Chemistry

University of Toronto

2015

Abstract

In magnetic resonance imaging (MRI), contrast agent (CA) plays a significant role in

modulating signal intensity and enhancing tissue contrast. Gadolinium-based CAs (GBCAs) are

routinely applied in clinical settings for detecting diseases involving disrupted vascularity, such

as tumors and stroke. Despite their clinical success, the applications are constrained by their

suboptimal contrast enhancement efficiency (relaxivity) at high clinical field and their associated

Gd-toxicity. To overcome these limitations, we focus on the development of Gd-free CAs based

on manganese(III) porphyrin (MnP). MnTCP was developed as the first MnP-based ECF agents

with superior properties.. In the thesis, three different chemical approaches have been explored to

further optimize MnTCP. Approaches of improving relaxivity by dimerization (Chapter 2),

incorporating biocompatible polyhydroxyl amide (Chapter 3) and tuning electronic properties in

porphyrin ligand (Chapter 4) are investigated. Among all novel MnTCP derivatives, MnTrCP2

showed the most promising results. Preliminary studies on relaxivity optimization have yielded

encouraging results.

iii

Acknowledgments

Throughout my journey, firstly, I would like to express my deepest gratitude to my research

supervisor, Dr. Xiao-an Zhang, for his support in my graduate studies. As a Master Student, I am

grateful to have the opportunities to work on various projects with him, supervise research

undergraduate students and participate to laboratory organization. Those are valuable

experiences, which help me to develop essential skills as a mature, independent person.

I would like to thank the senior graduate students in our research group: Weiran Cheng, Inga

Haedicke, and Loïse Perruchoud. Before the starting of my graduate school, I was a research

volunteer, who had closely collaborated with PhD students Weiran Cheng and Inga Haedicke.

During my volunteer, they had demonstrated good qualities to be my mentor and taught me a lot

of practical skills in the organic syntheses. They had acted as a senior student and offered me

help while there were major obstacles. Dr. Yuanyuan Lyu, our postdoctoral researcher who

joined us in 2015, was kind enough to share her research experience and instructed me in various

aspects, which were applicable and useful for my whole life. I am grateful to have the

opportunity to work with her.

There are always challenges that we have to face in life and it also applies on research. In a lot of

down moment, Dr. Zhe She, a postdoctoral from the research group of Professor Heinz-Bernhard

Kraatz, and Dr. Yuanyuan Lyu had helped me in providing me mental supports and suggestions

to improve.

Among the three years in the Zhang Research Group, it was a great pleasure to work with the

past and current undergraduate members, especially Taleen Karnieg, Lily Yang, Kelly Mo, Shili

Dong, Elva Huang, Ying Fu, Camille Zhang, Hanlin Liu and Wenbo Gao. As colleagues and

friends in the research group, they provided a lot of support and I enjoyed the time that we had

spent together in the laboratory and private.

Being a

ching assistant is a part of the graduate studies; it is an important duties as I am responsible for

training senior undergraduate students for technical skills and positive work ethics in preparation

of the society. From the aspect as a teaching assistant, I would like to thank Dr. Scott Ballantyne,

iv

the undergraduate laboratory manger, and Loïse Perruchoud, who had given me the opportunities

to teach their corresponding courses. They had kindly introduced me a lot of positive attitudes in

teaching and interacting with students; those communication skills, leadership skills and thinking

strategies are essential for me to prepare for the workforce.

Research is a group effort and in certain circumstances, there are collaboration studies to share

specialties for optimal experimental outcomes. In my graduate studies, I had the opportunity to

collaborate with Dr. Hai-Ling Margaret Cheng and Tameshwar Ganesh for the in vivo studies. I

appreciate their support throughout the studies.

In addition, thank you Professor Kagan Kerman for being my thesis reader and providing

valuable information to me about planning of graduate school. During my research, he had

reminded us about the recent updates to ensure a health working environment in the laboratory

setting.

In the UTSC, TRACES center is essential for researchers as it is the location for a lot of

important analytical instruments. Taking this opportunity, I would like to express my gratitude to

TRACES Centre Lab Manger Tony Adamo and Dr. Ronald Soong for their effort to maintain

every instrument to ensure their functionalities. Despite the fact the complexities of the

instruments, they were responsible and highly qualified as the TRACES Lab Manager.

Finally, I would like to thank my family for providing me emotional, physical and financial

supports. In a lot of cases, my mother and brother had made adjustments based on my schedule,

even though it had compromised them to their daily lives.

Being a Master Student was not an easy job. Commitment and self-motivation are important

personal criteria to succeed in graduate school. However, the reasons that I can graduate are the

opportunities and supports I received from everyone that I had interacted with. Everyone, thank

you for everything!

v

Table of Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ............................................................................................................................ v

List of Tables ............................................................................................................................... viii

List of Figures ................................................................................................................................ ix

List of Schemes ............................................................................................................................... x

List of Abbreviations ..................................................................................................................... xi

1 Introduction ................................................................................................................................ 1

1.1 Magnetic Resonance Imaging ............................................................................................. 1

1.2 MRI Contrast Agents and Their Classification ................................................................... 1

1.2.1 Extracellular CAs and Clinic Importance ............................................................... 4

1.2.2 Limitations of Gd-Based Contrast Agents .............................................................. 5

1.2.3 Manganese(III) Porphyrin as MRI Contrast Agent ................................................ 7

1.3 Objectives of the Thesis .................................................................................................... 10

2 Development of a Compact Mn-Porphyrin Dimer as Extracellular MRI Contrast Agent:

Combination of high T1 Relaxivity and Fast Renal Clearance ................................................ 11

2.1 Introduction ....................................................................................................................... 11

2.2 Results and Discussion ..................................................................................................... 12

2.2.1 Design of MnTrCP2 .............................................................................................. 12

2.2.2 Synthesis of MnTrCP2 .......................................................................................... 14

2.2.3 Evaluation of MnTrCP2 as Extracellular Agent .................................................. 19

2.3 Materials and Methods ..................................................................................................... 23

2.3.1 Instrumentation ..................................................................................................... 23

2.3.2 Materials ............................................................................................................... 24

2.3.3 Synthesis ............................................................................................................... 26

2.3.4 Field-Dependent Relaxivity Measurement ........................................................... 30

vi

2.3.5 HSA Binding Study .............................................................................................. 31

2.3.6 In vivo Study ......................................................................................................... 31

2.4 Conclusion ........................................................................................................................ 31

2.5 Contents of Appendix to Chapter 2 .................................................................................. 32

3 Development of Low Osmolality MRI Contrast Agent Based on Modification of MnTCP ... 33

3.1 Introduction ....................................................................................................................... 33


3.3 Materials and Method ....................................................................................................... 37


3.3.2 Materials ............................................................................................................... 38

3.3.3 Synthesis ............................................................................................................... 39

3.4 Conclusion ........................................................................................................................ 41


4 Tuning Electronic Properties on Mn(III) Porphyrin: Structural Relaxivity Relationship

Studies ..................................................................................................................................... 43

4.1 Introduction ....................................................................................................................... 43


4.2.1 Synthesis of Monomeric MnTriCP Analogs ......................................................... 45

4.2.2 Field-Dependent Relaxivity of MnTCP Derivatives ............................................ 48

4.3 Materials and Methods ...................................................................................................... 49


4.3.2 Materials ............................................................................................................... 50

4.3.3 Synthesis ............................................................................................................... 52

4.4 Conclusion ........................................................................................................................ 55


5 Conclusion and Future Perspective .......................................................................................... 56

vii

References ..................................................................................................................................... 58

Appendix: NMR, ESI-MS, UV-vis Spectra and HPLC Chromatograms of Synthetic Targets ... 63

viii

List of Tables

Table 1: Structures, names and abbreviations of FDA approved linear GBCAs. ........................... 2

Table 2: Structures, names, and abbreviations of the FDA approved macrocyclic GBCAs. ......... 3

Table 3: Synthetic results for TriCP, (3) by reagent ratio adjustments of Lindsey method. ........ 16

ix

List of Figures

Figure 1: NMRD Profiles of Gd-DTPA, Gd-HP-DO3A and Gd-DTPA-BMA from 0.2 mT to 1 T

in PBS buffer. .................................................................................................................................. 6

Figure 2: NMRD Profiles of MnTCP and Gd-DTPA from 0.2 mT to 1 T in HEPES buffer. ......... 9

Figure 3: T1-weighted spin-echo MRIs at 3 T. A dose of 0.05 mmol Mn/kg of MnTCP and MnP2

were introduced into rats via tail vein injection. The MRI slices highlighted the kidney (yellow

circle) and the bladder (blue circle). ............................................................................................. 10

Figure 4: Schematic structure of the metallostar {Fe[Gd2bpy(DTTA)2(H2O)4]3}4-. .................... 12

Figure 5 : Schematic structure of Gd3L. ....................................................................................... 12

Figure 6: NMRD Profiles of MnTrCP2 and MnTCP from 0.2 mT to 3 T at 25oC in 25 mM

HEPES buffer, pH 7.2. .................................................................................................................. 20

Figure 7: HSA binding assay of MnTrCP2 upon one equivalent of HSA in 25 mM HEPES buffer.

....................................................................................................................................................... 21

Figure 8: In vivo T1-weighted gradient-echo MRI of rats at 3 Tesla. Images were acquired at

different intervals post-injection. A dose of 0.032 mmol Gd or Mn/kg was administered for both

Gd-DTPA and MnTrCP2. The MRI slices highlighted the kidney (yellow circle). ...................... 22

Figure 9: Dynamic contrast-enhanced MRI signal enhancement curves in the kidney (solid line)

and muscle (dashed line) after injection of MnTrCP2 (black) or Gd-DTPA (gray). .................... 23

Figure 10: NMRD Profiles of MnTPPS and MnTPPAS from 0.2 mT to 1 T at pH 7.0 (adopted

from Bradshaw, 1998). .................................................................................................................. 34

Figure 11: NMRD Profiles of MnTPPS and MnTPPSBr6 from 0.2 mT to 1 T (adopted from

Bryant et al, 1999). ........................................................................................................................ 44

Figure 12: NMRD Profiles of MnTriCP, MnTriCP-NO2 and MnTCP from 0.2 mT to 3 T at 25oC

in 25 mM HEPES buffer, pH 7.2. ................................................................................................. 48

x

List of Schemes

Scheme 1: Molecular Designs of MnTCP and MnP2 from the structural modifications of

MnTPPS .......................................................................................................................................... 8

Scheme 2: Molecular Design of MnTrCP2 from the combination structural characteristics of

MnTCP and MnP2 ........................................................................................................................ 12

Scheme 3: Final synthetic procedure for the production of MnTrCP2 ......................................... 14

Scheme 4: Synthetic procedure for TriCP, (3) by the conventional Lindsey Method ................. 15

Scheme 5: Synthetic procedure for TriCP, (3) by the [2+2] dipyrromethane 1,9 dicarbinol

porphyrin condensation method .................................................................................................... 17

Scheme 6: Molecular Design of MnTAP from structural modification of MnTCP ..................... 35

Scheme 7: Proposed synthetic route for the production of MnTAP ............................................. 35

Scheme 8: Proposed amide coupling reaction mechanism medicated by EEDQ ......................... 36

Scheme 9: Molecular Design of MnTriCP-NO2 and MnTriCP-NH2 from structural modification

of MnTriCP ................................................................................................................................... 45

Scheme 10: Proposed synthetic routes for the production of MnTriCP, MnTriCP-NO2 and

MnTriCP-NH2 .............................................................................................................................. 45

xi

List of Abbreviations

BBB blood brain barrier

BiCP 5, 15-bis(ethoxycarbonyl)porphyrin

BPA blood pool agent

CA contrast agent

CT computed tomography

DCE dynamic contrast enhanced

DCM dichloromethane

DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone

DIPEA N,N-Diisopropylethylamine

DMF dimethylformamide

DMSO dimethyl sulfoxide

ECF extracellular fluid

EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

EPR enhanced permeability retention

ESI-MS electrospray ionization mass spectrometry

FAAS flame atomic absorption spectroscopy

FDA Food and Drug Administration

FFC fast field cycling

GBCA gadolinium based contrast agent

Gd-DTPA Magnevist, gadopentetate dimeglumine

Gd-DTPA-BMA Omniscan, gadodiamide

Gd-HP-DO3A ProHance, gadoteridol

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HPLC high-performance liquid chromatography

HRMS high resolution mass spectroscopy

HSA human serum albumin

LD50 lethal dose, 50%

MnP manganese(III) porphyrin

MnP2 4,4-bis(manganese(III) 5,10,15-tris(4-sulfonatophenyl)-

porphin-20-yl)biphenyl

xii

MnTAP manganese(III) 5, 10, 15, 20-tetraakis(N-tris(hydroxymethyl)-methyl-

amide)porphyrin

MnTCP manganese(III) tetracarboxylporphyrin

MnTPPS manganese(III) meso-tetra(4-sulfonatophenyl) porphine

MnTrCP2 5,5’-Bis(manganese(III) 10, 15, 20-tricarboxylporphyrin)

MnTriCP manganese(III) 5, 10, 15-tri(carboxyl)porphyrin

MnTriCP-NH2 manganese(III) 5-amino-10, 15, 20-tri(carbonyl) porphyrin

MnTriCP-NO2 manganese(III) 5, 10, 15-tri(carboxyl)-20-nitro-porphyrin

MRI magnetic resonance imaging

NHS N-hydroxysuccinimide

NMRD nuclear magnetic resonance dispersion

NSF nephrogenic systemic fibrosis

PBS phosphate buffered saline

PIFA (bis(trifluoroacetoxy)iodo)benzene

q water exchange coordination sites

r1 T1 relaxivity

RT room temperature

SA serum albumin

SBM Solomon-Bloembergen-Morgan

E electron spin relaxation time

R molecular rotational correlation time

M water residual correlation time

T1 spin-lattice relaxation time

T2 spin-spin relaxation time

TCP 5, 10, 15, 20-tetrakis(ethoxycarbonyl)porphyrin

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TriCP 5, 10, 15-tri(ethoxycarbonyl)porphyrin

Tris tris(hydroxymethyl)aminomethane

UV-vis ultraviolent – visible spectroscopy

1

1 Introduction

1.1 Magnetic Resonance Imaging

Magnetic Resonance Imaging (MRI) is a non-invasive imaging modality that allows

visualization of the internal system. In comparison with other medical imaging modalities, MRI

has proven to be superior for producing high spatial 3D resolution images with excellent soft

tissue contrast, without exposure to ionizing radiation. Because of these advantages, MRI has

been frequently incorporated in both clinical and research applications such as tumor detection,

visualization of blood flow and monitoring brain activities.1,2 Despite the usefulness, the intrinsic

MRI signal sensitivity is limited by a number of factors, such as proton density and the natural

rate of proton relaxation. As the consequence, the differentiation between unhealthy and normal

tissue is weakened. To improve the signal sensitivity and selectivity of image, contrast agents

(CAs) are employed in MR scans.3

1.2 MRI Contrast Agents and Their Classification

Contrast enhancement in MR scans can be assisted by the administration of paramagnetic

CA, which is able to catalytically shortens the relaxation time of the proton nuclei from the

surrounding water molecules, thus improving the signal contrast and the differentiation based on

the CA distribution.3

The CAs are classified in two groups based on their relaxation mechanism: the CAs that

dominantly shorten longitudinal relaxation time (T1) and transverse relaxation time (T2) are

referred to T1 agents and T2 agents, respectively.4 For T1 agents, gadolinium-based contrast

agents (GBCAs) is the dominating class in the disease diagnosis. As a paramagnetic metal,

gadolinium ions have seven unpaired electrons (S = 7/2) that can efficiently accelerate the T1

relaxation. The quantitative T1 relaxation efficiency of a CA is termed relaxivity (r1).

GBCAs have been routinely applied for clinical MRI scans for the past decades. They are

typically administrated as metal complexes with ligands to prevent gadolinium dissociation,

since free Gd ion is highly toxic. In 1987, Gd-DTPA, an eight-coordinated Gd-complex , was

approved as the first GBCA for clinical applications. Several derivatives were later developed

based on ligand modification.5 The ligands are categorized in two different ways, either by

2

structural property (linear vs macrocyclic) or by ionicity (ionic vs non-ionic). The FDA approved

GBCAs are shown in Table 1 (linear) and Table 2 (macrocyclic).

Table 1: Structures, names and abbreviations of FDA approved linear GBCAs.

Linear, Ionic GBCAs Linear, Non-ionic GBCAs

Magnevist

Gadopentetate dimeglumine

Gd-DTPA

Omniscan

Gadodiamide

Gd-DTPA-BMA

MultiHance

Gadobenate dimeglumine

Gd-BOPTA

Optimark

Gadoversetamide

Gd-DTPA-BMEA

Eovist

Gadoxetate disodium

Gd-EOB-DTPA

3

Ablavar

Gadofosveset trisodium

MS-325

Table 2: Structures, names, and abbreviations of the FDA approved macrocyclic GBCAs.

Macrocyclic, Ionic GBCAs Macrocyclic, Non-ionic GBCAs

Dotarem

Gadoterate meglumine

Gd-DOTA

ProHance

Gadoteridol

Gd-HP-DO3A

Gadovist

Gadobutrol

Gd-BT-DO3A

4

These molecular characteristics influence the stabilities, relaxivity, pharmacokinetics and

applications of GBCAs. The macrocyclic property favors metal chelation in the proper geometry

in the complex and the ionicity strengthens the metal coordination; in general, macrocyclic CAs

are more stable than the linear group, and ionic CAs are more stable than the non-ionic class.

The applications of CAs are affected by their molecular properties. Extracellular fluid (ECF)

agents are one class of CA that is typically low molecular size, hydrophilic compounds. They are

known to have a fast biodistribution, rapid extravasation and efficient elimination through kidney

filtration. They are widely applied in the detection of diseases with vascular lesion, such as brain

tumors and breast cancer. Blood pool agents (BPAs) are another class of CA. They have

elongated retention time in the vasculature due to large hydrodynamic size, which prevent them

from extravasation or renal filtration.2,5

1.2.1 Extracellular CAs and Clinic Importance

ECF agent is the major category of MRI CA that is the most widely applied in clinical

settings. After intravenous administration, these agents are freely distributed in the extracellular

space; equilibrium between the intravascular and interstitial fluid compartment is rapidly

established. These compounds remain intact without metabolism in the physiological system.

They are exclusively eliminated in the original form by glomerular filtration with a half-life

about 1-2 hours. These pharmacokinetic properties are contributed by their structural design in

the molecular level.6, 7

Chemically, these gadolinium-based ECF agents exhibit a couple of common properties:

(1) they are gadolinium complexes with low molecular weight; (2) the multi dentate ligands

consist of water solubilizing functional groups such as poly (amino carboxylates) and poly

hydroxyl groups. These features of ECF agents serve for multiple purposes: they prevent any

specific interaction with the bio-macromolecules such as serum albumin (SA) in the circulatory

system and allow rapid distribution through the extracellular space. In addition, the small,

hydrophilic natures facilitate the fast renal clearance. The multi dentate ligand encapsulates the

Gd(III) tightly, resulting in the inertness to avoid biotransformation. These chemical properties

are essential for safe and efficient in vivo applications, especially for detecting lesion.5-7

Through MR scans in combination of CA, the information of tumor vascularity and blood

vessel leakage can be investigated. In normal tissue, the membrane permeability is highly

5

regulated and the transport of hydrophilic ECF agent is controlled. In pathological tissue, the

elevated vascularity and permeability allows selectively accumulation of the ECF agent in the

damaged region, which produces contrast differences between the pathogenic tissue and

surrounding normal tissue. These properties are essential for the evaluation of tumoral, traumatic

and infectious disease processes. The most common application is for the imaging of the central

nervous system such as brain metastases and demyelinating diseases.7,8 The brain is protected by

the blood brain barrier (BBB), an intact membrane that prevents the diffusion of hydrophilic

compounds. In brain tumors, the permeability of BBB is increased in the vicinity of the brain

tumors, and the leaky vascular properties facilitate a greater quantity of contrast agent transiently

accumulate in the ECF compartment via extravasation. . Therefore, in the MR scans, the

abnormal regions appear hyper intense in the image relative to the surrounding brain. This well

characterized phenomenon is called enhanced permeability retention (EPR) effect, and has been

exploited for contrast enhanced imaging with MRI or CT scans. With the administration of ECF

agent, the characterization of lesion in brain, liver, renal system, breast, and musculoskeletal

system is greatly enhanced in the images, which provides valuable information for the medical

diagnosis.8,9

1.2.2 Limitations of Gd-Based Contrast Agents

Despite the clinical importance of Gd-based ECF agents, there are two major drawbacks

that constraint their applications. In MR scans, increase of magnetic field improves the spatial

resolution of image, and there is an increasing tendency to replace the old low-field scanners or

to install new scanners with high clinical field of 3 T. The relaxivity of conventional GBCA

gradually diminishes at high magnetic field (Figure 1), which constrains its future application.

6

P ro to n L a rm o r F re q u e n c y / M H z

r1

/ m

M-1

s-1

0 .0 1 0 .1 1 1 0 1 0 0

0 .0

2 .0

4 .0

6 .0

8 .0

1 0 .0

G d -H P -D O 3 A

G d -D T P A -B M A

G d -D T P A

M a g n e tic F ie ld S tre n g th / T

0 .0 0 1 0 .0 1 0 .1 1

Figure 1: NMRD Profiles of Gd-DTPA, Gd-HP-DO3A and Gd-DTPA-BMA from 0.2 mT to 1 T in PBS buffer.

To compensate the low efficiency, higher dose is necessary to maintain the level of

contrast enhancement, thus further elevates the toxicity risk associated with GBCAs. Free

gadolinium ions are highly toxic to our body.11, 12 After administration of GBCA, it is essential to

avoid prolonged GBCA retention in the body, which leads to in vivo release and accumulation of

free Gd3+ ions. When the GBCAs are not excreted, there is a potential risk to cause a severe

adverse effect called nephrogenic systemic fibrosis (NSF), which occurred in patients with renal

dysfunction. NSF is a lethal disease that causes fibrosis at skin and internal organs.12- 14 Stability

of Gd-based agent should be taken in consideration. In the in vivo environment, transmetallation

is the major pathway for gadolinium release. Endogenous transition metal ions, such as Zn2+ can

replace Gd3+ under the physiological condition and the released Gd3+ ions accumulate in vivo.13,

15 More recently, in patients with healthy kidney, Gd accumulation in the brain was identified.

To address these challenges, our research group focus is on the development of novel class of

Gd-free CA that has a lower toxicity and higher efficiency for contrast enhanced MRI.

7

1.2.3 Manganese(III) Porphyrin as MRI Contrast Agent

In 1980s, water-soluble manganese(III) porphyrin (MnP) was found to demonstrate

features as an alternative to GBCA. MnIII meso-tetra(4-sulfonatophenyl)porphine (MnTPPS) was

reported to exhibit a tremendously high relaxivity at high field (r1 =10.4 mM-1s-1 at 1 T), which

was one fold higher than Gd-DTPA. In comparison to conventional GBCAs with decreasing r1

beyond 0.2 T, MnTPPS showed an optimal at 1 T and dropped slightly as the magnetic field

increases to 1.5 T.16 Those important findings directed our attention to investigate the MnP

platform for MRI application.

The MnP scaffold offers many criteria to become ideal MRI contrast agents. From the

contrast enhancement perspective, the molecular platform of MnP attributes to the high r1 at high

field. According to the Solomon-Bloembergen-Morgan (SBM) model,17 the r1 is governed by a

number of parameters: electron spin relaxation time (E), molecular rotational correlation time

(R), water residual correlation time (M), and the water exchange coordination sites (q).17 In

typical GBCAs, these parameters are not fully optimized. Due to the flexibility of the single

bonds, small macrocyclic or linear ligands in GBCAs have a higher tumbling rate (shorter R) in

solution. Furthermore, to ensure the stability of chelating, there is only one water binding site to

the gadolinium center. Efforts to increase q comprise the stability of Gd-complex. It is even more

challenging and less predictable to tune other parameters, M and e. Thus, the clinical GBCAs

typically exhibit suboptimal r1 especially at high magnetic fields. For MnPs, the rigidity of the

porphyrin backbone greatly reduces the rotational motion and there are two coordinated water

molecules in the manganese center. These unique features in MnP contribute to the high

relaxivity at high field.

From the safety perspective, manganese displays a better tolerability profile than

gadolinium. Manganese is a micronutrient that can be found in our body. A 70 kg adult human

contains 12 to 20 mg of manganese.17 Manganese is required as a co-factor for the metabolic

activities such as bone production and as an antioxidant against radical oxygen-related damage.

As a result, there is a higher in vivo tolerance of manganese than gadolinium, as the latter is an

external lanthanide element that is uncommonly found in human body. Furthermore, the high

thermodynamic stability and kinetic inertness of MnP prevent metal dissociation, which avoids

8

the release free manganese ions in the biological system.19, 20 In addition, the safety of MnP

derivatives can be further optimized by structural changes on porphyrin ligand.

As a promising class of compounds with high relaxivity and biocompatibility, MnP-based

CAs has become our focus on the development of next generation MRI contrast agents. MnTPPS

has been served as a lead compound for structural modifications and new derivatives were

designed, synthesized and evaluated. Among the novel MnP analogs developed from our

research group, MnTCP and MnP2 represent two distinct types of the CAs with finely tuned

biocompatibilities based on different approaches of rational designs (Scheme 1).21

Scheme 1: Molecular Designs of MnTCP and MnP2 from the structural modifications of MnTPPS.

MnTCP was designed as the first MnP-based ECF agent.21 By eliminating the four

phenyl rings on MnTPPS, the novel low molecular weight, hydrophilic compound was

developed by incorporation of highly polar carboxylate groups directly at the meso positions of

porphyrin. The molecular design served two purposes: (1) the molecular size was reduced by the

replacement. (2) The hydrophobic interactions between MnP and components macromolecules in

the serum can be minimized. As an ECF agent, low molecular size and high hydrophilicity thus

facilitated fast elimination via kidney, which was an important feature for application to avoid

toxicity. The potential of MnTCP towards clinical application was evaluated using Gd-DTPA as

a reference compound. Overall, MnTCP demonstrated a higher relaxivity than Gd-DTPA

9

(Figure 2). Furthermore, there was a one fold improvement in the r1 at 3 T, the magnetic field

used in clinical scanner currently.21, 22 In the pharmacokinetic studies conducted in rats, MnTCP

demonstrated rapid renal elimination comparable to that of Gd-DTPA; MnTCP was extensively

excreted through the renal system within one hour (Figure 3).Hence, MnTCP could be an

alternative extracellular agent and potentially used for the diagnosis of diseases such as cerebral

capillary lesion, cancer, and kidney diseases.22, 23


r1

/ m

M-1

s-1

0 .0 1 0 .1 1 1 0 1 0 0

0 .0

2 .5

5 .0

7 .5

1 0 .0

1 2 .5

1 5 .0

M nT C P

G d -D T P A


0 .0 0 1 0 .0 1 0 .1 1

Figure 2: NMRD Profiles of MnTCP and Gd-DTPA from 0.2 mT to 1 T in HEPES buffer.

MnP2 was designed as a dimeric MnP that could greatly enhance the r1 by decreasing the

molecular tumbling rate. At 3 T, MnP2 exhibited a high molar relaxivity with sevenfold

improvement than Gd-DTPA.20 Unlike MnTCP with rapid renal clearance, MnP2 was a BPA

that has long retention time in the circulatory system (Figure 3), which was achieved by

promoting strong interactions to the serum protein. In MnP2, the hydrophobic biphenyl moiety

which bridge two porphyrin units facilitates the strong binding interactions with serum

albumin.22, 24

10

Figure 3: T1-weighted spin-echo MRIs at 3 T. A dose of 0.05 mmol Mn/kg of MnTCP and MnP2 were introduced

into rats via tail vein injection. The MRI slices highlighted the kidney (yellow circle) and the bladder (blue circle).

MnTCP and MnP2 are the first generation MnP-based CAs developed by our group with

promising properties: MnTCP served as a better alternative of classical ECF agents and MnP2

demonstrated superior properties as new BPA with high relaxivity at high field. These novel

contrast agents suggested the remarkable potentials and flexibility of MnP molecular platform by

offering high relaxivity, good biocompatibilities and tunable pharmacokinetics.. In contrast to

GBCAs, the unique advantages of MnPs have not been explored. Currently, we would like to

further investigate the MnP platform for optimizing the contrast efficiency and application

through structural designs.

1.3 Objectives of the Thesis

This thesis will further explore the potential of MnP scaffold as Gd-free MRI contrast

agent for applications in the high clinical field, with the focus on the modification of MnTCP

derivatives. Three chemical approaches towards the development of second generation MnP-

based extracellular agent, with improved relaxivity and biocompatibility, will be presented.

Based on distinct rational of designs, synthesis and evaluation of novel MnP derivatives will be

discussed and analyzed.

11

2 Development of a Compact Mn-Porphyrin Dimer as Extracellular MRI Contrast Agent: Combination of high T1 Relaxivity and Fast Renal Clearance

This chapter has been reproduced in part with a Manuscript in preparation.

2.1 Introduction

A highly efficient CA with fast clearance rate is beneficial for the clinical applications; as

it can provide strong contrast enhancement in clinical scans while minimize the toxicity

associated to CA by lowering the dose. Hence, CA with high relaxivity is important as it is able

to enhance the contrast level with a low dose. Conventional approaches for relaxivity

improvement are incorporation of multiple paramagnetic centers and the increase of molecular

size to facilitate slow tumbling rate. However, the pharmacokinetic property would be influenced,

particularly slowing down the clearance, as the molecular size increases. Therefore, our goal is to

develop a highly compact oligomers that would encapsulate multiple paramagnetic centers in a

low molecular weight compound, thus generating a novel CA with high relaxivity and rapid

renal filtration. MnTCP would be a building block to create a dimeric porphyrin with small size

and high r1.

In the past, similar strategy had been employed in the design of high relaxivity

oligomeric Gd-ECF agents.25, 26 An elegantly designed self-assembling compound, metallostar

(Figure 4), was an intermediate size GBCA with multiple paramagnetic centers and ligand which

is capable to form a rigid structure.26, 27 In each ligand, it consisted of bipyridine derivative

poly(amino carboxylate) ligand which is able to bind two gadolinium centers; the entire complex

with the Fe2+ tris-(bipyridine) core had six gadolinium centers to achieve high relaxivity. At

4.7 T, relaxivity was measured, and high r1 was found 16.4 mM-1s-1.28 The slow tumbling was

achieved by the formation of supramolecular Fe2+ complex, for which the in vivo stability need

to be concerned. In 2008, it was reported that the development of a Gd3L compound (Figure 5), a

complex bearing a trimethylbenzene core covalently linked to three methylene-

diethylenetriamine-N,N,N”,N”-tetraacetate moieties that chelate three gadolinium ions in total.27

Overall, Gd3L is more stable than metallostar, since it does not rely on relatively week

supramolecular assembly to cluster all Gd-centers. It’s relatively smaller with tumbling rate

better tuned for high-field. , It demonstrated remarkable relaxivity at 4.7 T (r1 of 17.0 mM-1s-1)

12

and pharmacokinetics that resemble to typical ECF agent.28 However, metallostar and Gd3L were

both Gd-based chelates, toxicity raised as a concern.27, 28 In particular, for both compounds, Gd

are seven-coordinated with linear ligand, which increase the possibility of Gd-dissociation. In

that aspect, the MnP is a safer alternative building block to construct a high relaxivity ECF agent.

The major challenge is to connect MnP together without significantly increase the size and

hydrophobicity.

2.2 Results and Discussion

2.2.1 Design of MnTrCP2

Scheme 2: Molecular Design of MnTrCP2 from the combination structural characteristics of MnTCP and MnP2.

Considering the rapid renal clearance of MnTCP and the prominent relaxivity at high

field of MnP2, it is intuitive to develop a second generation of extracellular agent with high

relaxivity. MnTrCP2, a compact dimeric porphyrin that combines the selected properties of both

Figure 5: Schematic structure of Gd3L.

Figure 4: Schematic structure of the metallostar

{Fe[Gd2bpy(DTTA)2(H2O)4]3}4-.

13

MnTCP and MnP2, was designed from the rationale (Scheme 2). In the molecular aspect, the

highly polar carboxyl terminals were preserved and the biphenyl bridge was eliminated and

replaced by direct meso-meso linkage.

As a novel MnP-based CA, MnTrCP2 was predicted to have the following

characteristics. (1) A compact compound, with high relaxivity while small, would be an

important feature of MnTrCP2. As a low molecular weight (molecular weight: 982 g/mol)

dimeric porphyrin, it carries two paramagnetic metal centers that can efficiently increase the

relaxation rate of surrounding water in the tissue. The rigidity of the dimer structure further

improved the relaxivity as it facilitated a slower molecular tumbling frequency in contrast to

MnTCP, which already has significantly higher relaxivity than most clinical GBCAs. As a result,

MnTrCP2 would be a low molecular size compound with high r1. (2) The similar

pharmacokinetics properties and applications as conventional ECF agents. As MnTCP consists

of carboxyl groups at the meso positions, these functional groups increased the hydrophilicity

and minimize the size increase. These molecular parameters contributed to the resemblance of

pharmacokinetics to be similar as Gd-DTPA. In MnTrCP2, the carboxyl groups were preserved,

which maintained the high polarity as MnTCP. With similar structural characteristics, the

pharmacokinetics would be expected to be similar to typical gadolinium extracellular agent. (3)

Minimizing HSA interaction by removal of hydrophobic binding site. In MnP2, it was

hypothesized that the strong interaction to HSA was due to the biphenyl moiety. These

important characteristics would contribute MnTrCP2 to be a compact compound with high

relaxivity and pharmacokinetics of extracellular agent, which is suitable for high clinical field

application.

14

2.2.2 Synthesis of MnTrCP2

MnTrCP2 is a dimeric MnP with direct meso,meso linkage and one of the challenges

MnTrCP2 is the development of a reaction that promotes covalently connecting the two

porphyrin subunits at the meso position; another challenge is to generate a novel asymmetric

monomer building block. The final synthetic route of MnTrCP2 was illustrated in Scheme 3.

Two dipyrromethanes with different substitutes, 1 and 2, were synthesized as precursors for the

porphyrin.29, 30 A porphyrin that resembled to TCP, TriCP, was generated. TriCP (3) is a

monomeric porphyrin with three ethyl ester groups. By bearing a vacant position, dimerization

was facilitated from TriCP as the meso position was readily for oxidative coupling reaction. Prior

to dimerization, zinc insertion was performed to increase the reactivity. PIFA medicated

homolytic coupling was a self-dimerization reaction that employed hypervalent iodine species to

attain oxidative coupling from aromatic proton positions.31, 32, 33 From the PIFA oxidative

dimerization, the zinc inserted dimer, 5, was obtained. The acid assisted demetallation was

performed for removal of zinc. As the hydrolysis generated water-soluble compound, which

Scheme 3: Final synthetic procedure for the production of MnTrCP2.

15

increased the difficulty in the synthetic aspect, hydrolysis was arranged as the last step. The apo-

dimer was treated with manganese to generate the Mn-hexa-ethyl ester- dimer 7. After the base

catalyzed hydrolysis, the desired product, MnTrCP2, was generated. The details for certain key

reactions are discussed below.

Optimization of TriCP Synthesis

Two methods can be possibly applied to prepare the asymmetric TriCP monomeric

porphyrin building block. Our first approach is the conventional Lindsey method – a Lewis acid

catalyzed condensation reaction that generated porphyrin using mixed aldehyde and pyrrole in a

one pot synthesis.34, 35 Alternatively, TriCP can be synthesized through a multiple step, [2+2]

condensation approach.30, 34 These two methods were both tested and their results were discussed

below in details.

Lindsey method

Scheme 4: Synthetic procedure for TriCP, (3) by the conventional Lindsey method.

Lindsey method is a traditional porphyrin synthesis using Lewis acid to catalyze the

condensation between pyrrole and aldehyde. Under inert and dark environment, the intermediate

porphyrinogen was generated, which was readily oxidized to porphyrin by DDQ. By

incorporating specific ratios of mixed aldehydes, Lindsey method is efficient to synthesize

asymmetric porphyrin. The ratios of aldehydes affect the selectivity and the yield to the

porphyrin derivatives.30

16

Synthesis of TriCP was originally performed by Lindsey method using

paraformaldehyde, ethyl glyoxalate, and pyrrole in a 1: 3: 4 ratio respectively. However, the

yield of TriCP was 0.19% as a minor porphyrin product; in comparison to the major porphyrin

product bis-(ethoxycarbonyl)porphyrin (BiCP) with a yield of 1.0%. We hypothesized that the

low yield of TriCP was attributed to the ratio of paraformaldehyde used and manipulation of the

ratio was the important parameter for reaction optimization. We had investigated the reaction by

adjusting the ratios of paraformaldehyde to 0.5 and 0.75. In the reaction with 0.5 ratio

paraformaldehyde, TriCP was generated in 0.19% as the only porphyrin product. As the ratio of

0.5 did not improve the yield, the 0.75 ratio was performed. It generated TriCP as the minor

porphyrin product at 0.85% yield and TCP at 4%. The results were summarized in the Table 3.

Due to the low yielding, non-selective with tedious purification of Lindsey Method, The

traditional Lindsey method was not practically feasible for the synthesis of TriCP and we

decided to investigate alternative synthetic method.

Table 3: Synthetic results for TriCP, (3) by reagent ratio adjustments of Lindsey method.

17

[2+2] porphyrin synthesis

Scheme 5: Synthetic procedure for TriCP, (3) by the [2+2] dipyrromethane 1,9 dicarbinol porphyrin condensation

method.

The [2+2] porphyrin synthesis method consists of using dipyrromethane as the precursor

and aldehydes to produce the desired porphyrin. Although the [2+2] approach required additional

synthetic steps for the dipyrromethanes, attention was directed to the [2+2] method and the

feasibility was investigated on whether it can be a more selective synthesis as oppose to the low-

yield Lindsey method to generate TriCP. A variety of symmetries porphyrin products could be

prepared by controlling the ratio among dipyrromethanes and aldehydes.

The dipyrromethane (1) synthesis was followed as the literature while the purification

was modified.29 The original synthesis of dipyrromethane employed 100:1 ratio of pyrrole and

paraformaldehyde to direct the reaction to completion; unreacted pyrrole was regenerated by

cold acetone trap in vacuum. To improve the efficiency, the excess pyrrole was vacuum

distillated at 75oC, which shortened the required time and minimized the product decomposition.

A yield of 83% was obtained and 1H NMR was used for characterization and purity confirmation.

The 5-ethoxycarbonyl dipyrromethane (2) synthesis was adapted from the literature.30

Compared to the Lindsey method, similar strategy of using excess pyrrole (14:1 ratio of pyrrole:

18

aldehyde) was employed to persuade the reaction to favor dipyrromethane formation. Vacuum

distillation was employed for the removal of pyrrole in the purification. The purified product was

obtained as a clear oil at 70 % yield, with high purity confirmed by 1H NMR.

As reported in the literature, a novel method of 1,9 dipyrromethane-dicarbinol was firstly

prepared by Friedel-Crafts alkylation of dipyrromethanes. Dipyrromethane, 5-ethoxycarbonyl

dipyrromethane and commercially available ethyl glyoxalate were employed as the reactant.

According to the report, the 1,9 dipyrromethane-dicarbinol from non-substituted dipyrromethane

had a shorter reaction time (for dipyrromethane and 5-ethoxycarbonyl dipyrromethane, the

reaction time were 3 hours and 24 hours respectively), thus minimized possible decomposition

during the prolonged reaction. Followed by the subsequent condensation with dipyrromethane

under BF3·OEt2 catalysis, porphyrinogen was generated and readily oxidized by DDQ. The target

porphyrin was synthesized in reasonable yield of 4.0%. The selectivity of synthesis allowed

TriCP as the major porphyrin product, which reduced the difficulty in purification. Overall, [2+2]

porphyrin synthesis was a feasible reaction to produce TriCP. The details for preparing all the

intermediates involved in this method, as well as the following steps to obtain the final product,

are discussed below.

Synthesis of Zinc(II) 5, 10, 15-tri-(ethoxycarbonyl) porphyrin (4)

Zinc Insertion of 3 was performed with 10 equivalence of Zn(OAc)2 at RT for 24 hours.

The optimized reaction condition would be reflux, which could accelerate the insertion progress.

Under reflux condition, the zinc insertion was completed within 3 hours, as suggested by the

TLC (solvent: 2% MeOH in DCM). In addition, isocratic solvent system in silica gel column

purification was found to cause co-elution of reaction side product. Gradient column (starting

0.5% MeOH in DCM, followed by 1% MeOH in DCM) was employed to isolate pure 4, as

suggested by TLC and NMR.

Synthesis of 5,5’-Bis-(zinc(II) 5, 10, 15-tri-(ethoxycarbonyl) porphyrin) (5)

The synthetic protocol of PIFA oxidative homolytic coupling was adapted from the

dimerization of Zinc-5, 10, 15-triphenylporphyrin.31, 32 However, the dimerization of 4 required

longer reaction time. As the reaction is a radical reaction initiated by a hypervalent iodine

reagent, we had optimized the oxidative reaction through performing reaction in Argon

19

atmosphere. The reaction was repeatable with yield up to 82%; compared to the reaction under

air with 64% yield, inert environment was found to be critical to allow high yield and consistent

reaction. In the purification, traces of unreacted 4 was found and gradient silica gel column was

an efficient way to isolate the pure 5, as suggested by TLC and NMR.

Synthesis of 5,5’-Bis-(5, 10, 15-tri-(ethoxycarbonyl) porphyrin) (6)

Demetallation was an acid assisted reaction that protonated the inner (NH) from the

zincated porphyrin. Concentrated HCl were selected and testified its performance to the reaction.

Traces of concentrated HCl was successful to remove the zinc ions to generate apo-dimeric

porphyrin 6.

Synthesis of 5,5’-Bis-(manganese(III) 5, 10, 15-tri-(ethoxycarbonyl) porphyrin) (7)

Manganese insertion was adapted from the literature with 10 equivalence of MnCl2∙4H2O

and 6 equivalence of DIPEA. Manganese inserted porphyrin was subsequently oxidized by air.

Gradient silica gel column was efficient to purify 7; the desired product was characterized by

ESI-MS and UV-vis. In ESI-MS, the signal at m/z = 578.20 confirmed the structure of 7, which

matched the calculated m/z 578.0998 of C58H46Mn2N8O122 +.

Synthesis of 5,5’-Bis-(manganese(III) 5, 10, 15-tri-carboxylporphyrin) (MnTrCP2)

In the last step, the base-catalyzed hydrolysis was adapted from the literature, using the

ethyl ester: hydroxide in a ratio of 1: 200. The final compound, MnTrCP2, was characterized by

ESI-MS for structural confirmation; by Mn-FAAS and UV-vis, the extinction coefficient at

468 nm was determined as 125,000 M-1cm-1.

2.2.3 Evaluation of MnTrCP2 as Extracellular Agent

The potentials of MnTrCP2 were evaluated by NMRD profile, HSA binding assay and in

vivo studies. Based on the NMRD profile, MnTrCP2 was found to exhibit a higher relaxivity

than monomeric MnTCP, particularly beyond 0.03 T, due to increase of paramagnetic centre and

molecular rotational correlation time. At 3 T (current magnetic field for clinical scanner),

MnTrCP2 exhibited a threefold improvement on molar relaxivity than Gd-DTPA, a conventional

ECF agent applied in MR scans. The in vivo performance of MnTrCP2 illustrated that it was an

20

efficient CA. In the health rat injection, MnTrCP2 displayed a significant higher T1 contrast

enhancement at 3 T in clinical scanner.

From the HSA binding studies, the interaction between MnTrCP2 and serum albumin

was not observable, which suggested that there was non-selective interactions with serum

albumin and would not compromise the renal clearance rate. In addition, the in vivo studies

further confirmed that the fast renal clearance was preserved, as strong enhancement was

observed in the kidney, particularly in the renal pelvis. Overall, MnTrCP2 is the second

generation MnP-based ECF agent with high sensitivity, which is ideal for applications at high

clinical field.

2.2.3.1 Field-Dependent Relaxivity of MnTrCP2


r1

/ m

M-1

s-1

0 .0 1 0 .1 1 1 0 1 0 0

6 .0

8 .0

1 0 .0

1 2 .0

1 4 .0

M nT C P

M n T rC P 2


0 .0 0 1 0 .0 1 0 .1 1

Figure 6: NMRD Profiles of MnTrCP2 and MnTCP from 0.2 mT to 3 T at 25oC in 25 mM HEPES buffer, pH 7.2.

The NMRD profiles of MnTrCP2 and MnTCP were shown in Figure 6. At the low field

from 0.2 mT to 0.03 T, MnTCP and MnTrCP2 exhibited similar relaxivity. Beyond 0.03 T, the

relaxivity of MnTrCP2 had increased rapidly. Beyond 0.3 T, the r1 of MnTCP had reached its

21

optimum and started to decrease while the r1 of MnTrCP2 was increased gradually. At 0.5 T, the

maximum r1 of MnTrCP2 was attained. At 3 T, MnTrCP2 demonstrated a sustained r1 at

10.8 mM-1s-1; compared to MnTCP, the r1 dropped significantly to 8.3 mM-1s-1. As the r1 was

measured by the manganese concentration, the molar relaxivity of MnTrCP2 would be doubled

from the r1 values in Figure 6, which indicated that the molar relaxivity was 21.6 mM-1s-1 at 3 T.

At 3 T, a threefold improvement in molar relaxivity of MnTrCP2 than Gd-DTPA was observed.

Compare to Gd-DTPA, a triple contrast enhancement would be expected from MnTrCP2 at the

same concentration.

2.2.3.2 HSA Binding Studies of MnTrCP2

Figure 7: HSA binding assay of MnTrCP2 upon one equivalent of HSA in 25 mM HEPES buffer.

Human serum albumin (HSA) binding studies of MnTrCP2 were conducted by the UV-

vis (25 mM HEPES buffer) to determine if there was any binding affinity towards HSA. Upon

the 1:1 equivalence of MnTrCP2: HSA, there was no observable shifting of the Soret band at

468 nm, which suggested that there was no significant protein binding interaction. As MnTrCP2

did not interact with HSA, the retention time in the circulatory system would be comparable to

that of ECF agent, thus favoring rapid clearance rate.

22

2.2.3.3 In vivo Studies of MnTrCP2

Figure 8: In vivo T1-weighted gradient-echo MRI of rats at 3 Tesla. Images were acquired at different

intervals post-injection. A dose of 0.032 mmol Gd or Mn/kg was administered for both Gd-DTPA and

MnTrCP2. The MRI slices highlighted the kidney (yellow circle).

To investigate the in vivo performance of the novel dimeric MnP, MnTrCP2 was

administered to healthy rats with subsequent MR scans on a 3 T clinical scanner, with the

commercially available Gd-DTPA as reference compound. The recommended dose of typical

Gd-based CA was 0.1 mmol/kg; in our previous studies from our groups, a relative small dose

(0.05 mmol/kg) was injected for MnTCP. In our experiments, a dose of 0.016 mmol/kg of

MnTrCP2 (0.032 mmol/kg in Mn concentration) was applied. As the control, Gd-DTPA was

injected at 0.032 mmol/kg of Gd. Comparing MR images of different time intervals in Figure 8,

MnTrCP2 demonstrated a significant stronger contrast than conventional Gd-DTPA at identical

concentration of paramagnetic ions.

23

Figure 9: Dynamic contrast-enhanced MRI signal enhancement curves in the kidney (solid line) and muscle (dashed

line) after injection of MnTrCP2 (black) or Gd-DTPA (gray).

For the dynamic contrast-enhanced MRI signal enhancement curves in the kidney and

muscle, MnTrCP2 displayed a 100% improvement compared to Gd-DTPA in the kidney. One

hour after the injection, majority of the MnTrCP2 was excreted through renal filtration. These

experimental results suggested that the MnTrCP2 had higher in vivo contrast efficiency than Gd-

DTPA, while maintaining the rapid renal clearance as typical extracellular agent.

2.3 Materials and Methods

2.3.1 Instrumentation

All the spectroscopy data for structural characterizations were obtained using the research

facilities at University of Toronto Scarborough Campus (TRACES Center).

Instrumentation Description Company

Analytical high

performance liquid

chromatography system

Agilent 1100 system equipped with an

Agilent 1100 series diode array UV-vis

detector and an Eclipse C-18 reverse

Agilent Technologies

24

phase column (4.6 mm × 150 mm, 5 μm)

Ultraviolent-visible

spectrophotometer

Agilent 8453 Spectroscopy System Agilent Technologies

Nuclear magnetic

resonance (NMR)

spectrometer

Bruker-500 MHz Bruker

Electrospray ionization

mass spectrometer

Agilent 1100 LC MSD Model G1946D Agilent Technologies

High resolution mass

spectrometer

QStarXL mass spectrometer with an

Ionics HSID interface, ESI source,

MS/MS and accurate mass capabilities

AB Sciex

Flame atomic absorption

spectrometer

Thermo iCE 3500 Flame AA

Spectrometer equipped with Mn lamp

Thermo Scientific

Fast field cycling (FFC)

NMR Relaxometer

SMARtracer™ Bench-top Fast Field

Cycling (FFC) NMR Relaxometer with

high temperature superconductor (HTS)-

110 magnet system

Stelar s. r. l. and

Scott® Technology

Limited

2.3.2 Materials

Chemical Purity Source

Dichloromethane (DCM) ACS reagent grade, > 95% Fisher

Methanol ACS reagent grade, 99.9% Fisher

Chloroform ACS reagent grade, 99.8% Caledon

25

Ethyl acetate Reagent grade, ≥99.5% Caledon

Ethanol Reagent grade, ≥85% Caledon

Tetrahydrofuran (THF) Anhydrous, inhibitor free, 99.7% Caledon

Dimethylformamide (DMF) Anhydrous, septum, 99.8% Caledon

Hexane ACS reagent grade, ≥98.5% Fisher

Pyrrole 99%, extra pure, distillated prior to use Acros Organics

Paraformaldehyde Reagent grade Sigma-Aldrich

Indium(III) chloride 98% Sigma-Aldrich

Sodium hydroxide GR ACS grade EMD Millipore

Ethyl glyoxalate 50% in toluene; ≥48.0%, Alfa Asaer

Sodium bicarbonate Reagent grade Caledon

Boron trifluoro etherate ca. 48% Acros Organics

Sodium sulfate 99.0% Caledon

2,3-dichloro-5,6-dicyano-1,4-

benzoquinone (DDQ)

98.0% AK Scientific

Triethylamine 99.0% Caledon

Basic alumina Standard activity, reagent grade Caledon

Celite Reagent grade Acros Organics

Zinc acetate Anhydrous, 99.98% Alfa Asaer

bis(trifluoroacetoxy)iodo) 97% Alfa Asaer

26

benzene (PIFA)

Sodium chloride Reagent grade Caledon

Hydrochloric acid 36.5% – 38.0% Caledon

Manganese chloride

tetrahydrate

Reagent grade Caledon

N,N-diisopropylethylamine

(DIPEA)

≥99.5% Acros Organics

4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid

(HEPES)

Molecular biology grade, ≥99% Fisher

Thin layer chromatography

(TLC) plate

Silica gel 60 F254 EMD Millipore

Silica gel CC grade silica gel, 230 – 400 mesh Desican

Dialysis Membrane Spectra/Por® 7 dialysis membrane:

pre-treated RC tubing – MWCO 1000

Spectrum Labs

2.3.3 Synthesis

Dipyrromethane (1)

Pyrrole (38.5 mL, 555 mmol) and paraformaldehyde (0.167g, 5.55 mmol)

were degassed with argon for 30 minutes in dark. The mixture was stirred

for 10 minutes at 55 oC. InCl3 (0.123 g, 0.56 mmol) was added and the mixture was stirred for

2.5 h at 55 oC under argon atmosphere. Grounded NaOH(s) (0.666 g, 16.65 mmol) was added and

the mixture was stirred for an hour at room temperature to afford a brown mixture. The mixture

was vacuum filtered and concentrated by vacuum distillation. The mixture was washed by ethyl

acetate (2 x 5 mL) and hexane (2 x 5 mL) and distillated off. The brown crude was vacuum dried

and purified via flash chromatography (stationary phase, silica gel; mobile phase, hexane: DCM:

ethyl acetate 7:2:1) to yield 1 (575 mg, 83%) as a white solid. 1H NMR (500 MHz, CDCl3): δ

27

7.91 (s, 2H, NH), 6.69 – 6.65 (dd, 4H, pyr-α H and pyr-β Hb), 6.15 (dd, J = 2.8 Hz, 2H, pyr-β

Ha), 6.04 (s, 2H, pyr-β), 3.99 (s, 2H, meso H).

5-ethoxycarbonyl dipyrromethane (2)

Sodium bicarbonate (5.0 g, 60 mmol) in DCM (50 mL) was degassed with

Argon for 20 minutes. Pyrrole (10.0 mL, 144 mmol) and ethyl glyoxalate

(50% in toluene, 2.0 mL, 10.1 mmol) were added and stirred for 30 minutes

at room temperature under argon atmosphere. The mixture was filtered and washed with DCM

(50 mL). BF3·OEt2 (8 µL, 0.065 mmol) was added to the combined DCM filtrate. The solution

was stirred for 10 minutes and was washed with 5% NaHCO3 solution (1 × 100 mL). The

organic layer was dried by sodium sulfate and concentrated by rotary evaporator, followed by

vacuum distillation. The resulting viscous oil was vacuum-dried and purified via flash

chromatography (stationary phase, silica gel; mobile phase, DCM) to yield a clear oil of 2

(520 mg, 50%). 1H NMR (500 MHz, CDCl3): δ 8.45 (s, 2H, NH), 6.71 (td, J = 2.7, 1.6 Hz, 2H,

pyr-β Ha), 5.92 – 6.37 (m, 4H, pyr-α H and pyr-β Hb), 5.12 (s, 1H, meso H), 4.26 (q, J = 7.1 Hz,

2H, -OCH2-), 1.33 (t, J = 7.1 Hz, 3H, -OCH2CH3).

5, 10, 15-tri-(ethoxycarbonyl) porphyrin (3, TriCP)

Lindsey method

Paraformaldehyde (0.162 g, 5.40 mmol) and DCM (850 mL) were

degassed with argon for 45 minutes at RT. Pyrrole (2.0 mL,

28.8 mmol) and ethyl glyoxalate (50% in toluene, 4.28 mL,

21.6 mmol) were added and the mixture was stirred for 10 minutes at

RT in dark. BF3·OEt2 (0.36 mL, 2.80 mmol) was added dropwise and the reaction was

performed at RT for one hour. DDQ (4.90 g, 21.6 mmol) was added and the mixture was stirred

for 2 hours at RT. Triethylamine (0.40 mL, 2.80 mmol) was added to quench the reaction. The

dark reaction mixture was concentrated and filtered through triple layer column consisted of

silica gel, basic alumina and celite. The filtrate was concentrated and purified via

chromatography (stationary phase, silica; mobile phase, DCM) to afford 3 (32 mg, 0.8%). 1H

NMR (500 MHz, CDCl3): δ 10.32 (s, 1H, por-meso H), 9.62 (d, J = 4.9 Hz, 2H, por-β Ha), 9.59

(d, J = 4.6 Hz, 2H, por-β Hb), 9.52 (d, J = 4.9 Hz, 2H, por-β Hc), 9.43 (d, J = 4.7 Hz, 2H, por-β

28

Hd),5.13 (qd, J = 7.2, 3.0 Hz, 6H, -OCH2-), 1.79 – 1.87 (td, J = 9.0, 7.2 Hz, 9H, -OCH2CH3), -

3.40 (s, 2H, por-NH).

[2+2] dicarbinol porphyrin synthesis method

Dipyrromethane 1 (876mg, 5.99mmol), ethyl glyoxalate (50% in toluene, 2.37 mL, 11.27 mmol),

and sodium bicarbonate (427.5 mg, 5.09 mmol) were mixed in DCM (8.9 mL) in dark. The

mixture was stirred for 3 hours at RT. Sodium bicarbonate was filtered off and the DCM filtrate

was collected. The filtrate, 2 (1.35 g, 6.0 mmol), and DCM (1.23 L) were added in a 2 L four

necked reaction flask, and the mixture was degassed with Argon at 0oC for 30 minutes in dark.

BF3·OEt2 (148 μL, 1.20 mmol) was added dropwise and the mixture was stirred at RT. The

reaction was monitored by UV-Vis. After 30 minutes, DDQ (4.08 g, 18.0 mmol) was added and

the mixture was stirred for 2 hours at RT. The reaction mixture was concentrated and filtered

using triple layer column consisted of silica gel, basic alumina and celite. The filtrate was

concentrated and purified via chromatography (stationary phase, silica; mobile phase, DCM) to

afford 3 (120 mg, 4.0%).

Zinc(II) 5, 10, 15-tri-(ethoxycarbonyl) porphyrin (4)

Porphyrin 3 (120 mg, 0.228 mmol) and zinc acetate (418 mg,

2.28 mmol) were stirred in 9:1 chloroform: methanol solution (23 mL)

under argon atmosphere. The mixture was refluxed and reaction was

monitored by TLC (solvent: 2% MeOH in DCM). After 3 hours, the

reaction was completed, suggested by TLC. The intense pink solution

was filtered through Celite and rinsed by DCM. The solution was

concentrated and purified via chromatography (stationary phase, silica; mobile phase, 0.5%

MeOH in DCM) to afford 4 (127.9 mg, 95% yield). 1H NMR (500 MHz, d6-DMSO): δ 10.59 (s,

1H, por-meso H), 9.68 (d, J = 4.5 Hz, 2H, por-β Ha), 9.50 – 9.63 (m, 6H, por-β Hb, c, d), 5.09 (q,

J = 7.1 Hz, 6H, -OCH2-), 1.74 (td, J = 7.1, 3.1 Hz, 9H, -OCH2CH3).

29

5,5’-Bis-(zinc(II) 5, 10, 15-tri-(ethoxycarbonyl) porphyrin) (5)

Zinc porphyrin 4 (152.3 mg, 0.258 mmol) was

dissolved in DCM (210 mL) and the pink solution

was degassed with argon for 20 minutes.

(bis(trifluoroacetoxy)iodo) benzene (PIFA, 77.8 mg,

0.181 mmol) was added and the reaction was

stirred at RT under argon atmosphere. The reaction

was monitored by TLC (solvent: 2% MeOH in DCM). After 2 hours, the reaction was completed.

The resulting solution was washed by water (2 × 200 mL) and dried by sodium sulfate. After

concentration, the crude was purified via gradient column chromatography (stationary phase,

silica gel; mobile phase, starting with 0.5% MeOH in DCM and eluted with 1% MeOH in DCM)

to yield 5 (125.9 mg, 82%). 1H NMR (500 MHz, d6-DMSO): δ 9.54 – 9.70 (m, 8H, por-β Hc, d),

9.14 (d, J = 4.7 Hz, 4H, por-β Hb), 7.99 (d, J = 4.7 Hz, 4H, por-β Ha), 5.15 (q, J = 7.1 Hz, 4H, -

OCHb2-), 4.98 (q, J = 7.1 Hz, 8H, -OCHa2-), 1.81 (t, J = 7.2 Hz, 6H, -OCH2CHb3), 1.61 (t, J =

7.1 Hz, 12H, -OCH2CHa3).

5,5’-Bis-(5, 10, 15-tri-(ethoxycarbonyl) porphyrin) (6)

Zinc dimeric porphyrin 5 (125.9 mg, 0.1069 mmol)

was dissolved in DCM (110 mL) and concentrated

HCl (60 drops) was added to the solution.

The reaction mixture was stirred at RT under argon

atmosphere and monitored by TLC (solvent: DCM).

After three hours, reaction was completed and the

mixture was neutralized by saturated NaHCO3 washing (2 × 100 mL). The organic layer was

washed with water (1 x 100 mL) and dried by Na2SO4. The crude product was purified via

column chromatography (stationary phase: silica; mobile phase: 0.5% MeOH in DCM) to afford

6 (110.9 mg, 98.7%). 1H NMR (500 MHz, CDCl3): δ 9.62 (q, J = 4.9 Hz, 8H por-β Hc, d), 9.14

(d, J = 4.9 Hz, 4H, por-β Hb), 8.08 (d, J = 4.9 Hz, 4H, por-β Ha), 5.19 (q, J = 7.2 Hz, 4H, -

OCHb2-), 5.03 (q, J = 7.1 Hz, 8H, -OCHa2-), 1.90 (t, J = 7.2 Hz, 6H, -OCH2CHb3), 1.72 (t, J =

7.1 Hz, 12H, -OCH2CHa3), -2.58 (s, 4H, por-NH).

30

5,5’-Bis-(manganese(III) 5, 10, 15-tri-(ethoxycarbonyl) porphyrin) (7)

Dimeric porphyrin 6 (110.9 mg, 0.106 mmol) was

dissolved in DMF (19 mL). MnCl2∙4H2O

(208.8 mg, 1.055 mmol) and DIPEA (110 μL,

0.635 mmol) were added to the solution. The

mixture was refluxed and the reaction was

monitored by TLC (solvent: 10% MeOH in DCM).

After 4 hours, reaction was completed. The mixture was stirred in open air for 16 hours. The

DMF was distillated and the mixture was dried on vacuum. The crude product was purified via

gradient column chromatography (stationary phase: silica; mobile phase: starting 2% MeOH in

DCM and eluted with 5% MeOH in DCM) to yield 7 (110.2 mg, 90%). ESI-MS found m/z =

578.20 ([M]2+), calcd for C58H46Mn2N8O122+, m/z = 578.0998. UV-vis (MeOH): λabs = 321, 364,

390, 464, 471, 559 nm, λmax = 471 nm.

5,5’-Bis-(manganese(III) 5, 10, 15-tri-carboxylporphyrin) (8, MnTrCP2)

Intermediate 7 (96.7 mg, 0.079 mmol) was dissolved

in an ethanol: THF solution (26.4 mL: 20.3 mL). 2 M

NaOH solution (47.5 mL) was added to the orange

solution. The green mixture was refluxed at 72oC.

After 24 hours, the reaction was quenched by 1 M

HCl until pH was neutral, indicating by pH paper. The

brown mixture was concentrated and extracted by ethyl acetate (10 x 100 mL). The aqueous

layer was concentrated and dialyzed using MWCO 1000 dialysis membrane. The product was

lyophilized to afford 8 (55 mg, 70%). ESI-MS found m/z = 245.4909 ([M]4-), calcd for

C46H16Mn2N8O124-, m/z = 245.4912. UV-vis (HEPES buffer): λabs = 378, 397, 468, 564, 698 nm,

λmax = 468 nm, ε = 125,000 M-1cm-1.

2.3.4 Field-Dependent Relaxivity Measurement

Nuclear magnetic resonance dispersion (NMRD) profile from 0.2 mT to 3 T was

measured using fast field cycling (FFC) NMR relaxometer from Stelar s. r. l. A sample of

31

MnTrCP2 was prepared by dissolving in HEPES buffer (25 mM, pH 7.2) and quantified by Mn-

FAAS and UV-vis. The relaxation rate was corrected by a blank consisted of HEPES buffer.

2.3.5 HSA Binding Study

HSA binding study was performed by the UV-vis Spectroscopy System (Agilent 8453).

The measurement was acquired in HEPES buffer (25mM, pH 7.2) at 25oC

2.3.6 In vivo Study

In vivo experiments were conducted on healthy rats. After administration through tail

vein injection, T1-weighted gradient-echo MRI studies were performed on a 3 T clinical scanner.

A dose of 0.032 mmol Gd or Mn/kg was administrated for administered for all contrast agents.

2.4 Conclusion

MnTrCP2 was designed as a highly compact, high polar MnP dimer with high molar

relaxivity and rapid clearance. To construct this dimer with direct meso-meso porphyrin

conjugation, an asymmetric monomeric porphyrin building block was synthesized by

modification of a novel [2+2] porphyrin synthetic method. The unique oxidative coupling

reaction allow efficient porphyrin dimerization. With eight steps, MnTrCP2 was successfully

synthesized and chemically characterized. The field dependent relaxation efficiency of MnTrCP2

was evaluated by the NMRD profile, demonstrating a significant improved r1 at high clinical

field, which is about threefold higher than that of typical Gd-based ECF agent. UV-vis spectrum

suggested that MnTrCP2 does not bind to HSA, thus favoring rapid renal clearance. The in vivo

MRI experiment with rat revealed a rapid and efficient clearance via renal filtration, a similar

pharmacokinetic property as clinical ECF agent and MnTCP. Overall, MnTrCP2 was a novel

Gd-free extracellular agent based on MnP, with considerably high contrast enhancement at

clinical fields (1 – 3 T) as well as rapid renal clearance, which is compatible for future clinical

applications. To further investigate the potentials of MnTrCP2, pharmacokinetics and tumor

perfusion studies would be performed.

The properties of MnTrCP2 further suggested that the molecular tumbling rate and

incorporation of multiple paramagnetic centers were important parameters for contributing high

32

relaxivity. The increase in molecular size and the meso-meso linkage were postulated to decrease

tumbling rate while maintaining the hydrophilicity of the compound, which was essential to

facilitate rapid renal clearance. Slightly larger oligomeric porphyrin with carboxyl terminals,

which were connected by meso-meso linkage, thus becoming interesting targets for the

development novel MnP-based ECF agent in the future.

2.5 Contents of Appendix to Chapter 2

1H NMR spectra of all synthetic targets are found in the appendix. HPLC chromatogram, HRMS

and UV-vis spectra of the MnTrCP2 are located in the appendix.

33

3 Development of Low Osmolality MRI Contrast Agent Based on Modification of MnTCP

Contribution: Haedicke, I. E. developed the syntheses and characterizations of compounds 9-11.

3.1 Introduction

Water solubilizing compounds are created by the incorporation of charged functional

moieties, such as carboxylate, sulfonate, and ammonium groups. Although the highly

hydrophilic compounds make them preferable for the extracellular applications, the osmolarity

also increase by the charged groups and their counter ions at physiological pH. As a common

limitation for multi-charged CAs, the serum osmolarity increases after intravenous injection of

these exogenous agents, which causes the adverse effects including nausea, and headache. In

computed tomography (CT), administration of ionic iodinated contrast medium was reported to

cause these problems after large quantity contrast medium administrated.36 To reduce the side

effects, non-ionic contrast medium are needed to minimize the serum osmolar changes in

administration.

Similar concern was raised for MRI CAs; through formulation of functional group, Gd-

DTPA-BMA (structure is available in Table 1) was developed to reduce the osmolarity by

changing the carboxylate groups to neutral amide, thus converting it to a non-ionic ligand.36, 37

However, it decreases the stability as the metal coordination was weakened compared to

negatively charged carboxyl groups. In the clinical report, administration of Gd-DTPA-BMA

had the most incidents of NSF. In GBCAs, as the peripheral functional groups were directly

coordinated to the metal, the iconicity of functional group would influence the stability. As a

result, non-ionic GBCAs could reduce osmolarity while potentially increase the risk of

gadolinium dissociation.37, 38

In 1988, MnTPPAS was reported as a MnP-based contrast agent with neutral, water-

soluble ligand.39 The water solubility was partially preserved as a neutral compound, which was

accomplished by the poly-hydroxyl amide substitutes. However, MnTPPAS was not applicable

as a contrast agent for its in vivo tolerance toxicity and contrast efficiency. The relaxivity of

MnTPPAS was compared with MnTPPS in the NMRD profiles (Figure 10). Overall, the r1 of

MnTPPAS was lower than MnTPPS throughout 0.2 mT to 1 T. Moreover, MnTPPAS displayed

34

a lower LD50 than MnTPPS.38 Although MnTPPAS was an unsuccessful candidate for MRI

application, it provided insights to the biocompatibility optimization. We hypothesize that the

four phenyl rings in MnTPPAS compromise the water-solubility and possibly the relaxivity;

furthermore, it may also contribute to the elevated toxicity.39

Figure 10: NMRD Profiles of MnTPPS and MnTPPAS from 0.2 mT to 1 T at pH 7.0 (adopted from Bradshaw,

1998).39

MnTCP was the first generation MnP-based extracellular agent with moderate r1 and fast

renal clearance. However, the multiple carboxyl terminals introduced high osmolarity when

administrated. In the project, our goal is to develop a more biocompatible ECF agent derived

from MnTCP. A neutral water-soluble compound, MnTAP, would be generated by the amide

coupling using tris(hydroxymethyl)aminomethane (Tris), a poly hydroxyl amine, and MnTCP.

As a MnP analog, the substitution of poly hydroxyl amide would not compromise the stability as

the peripheral hydroxyl groups did not participate in the metal coordination. The rational design

resembled to the MnTPPAS, but the hydrophobic phenyl groups in MnTPPAS was eliminated

and direct amide groups were at the meso positions (Scheme 6). MnTAP was predicted to

demonstrate similar relaxivity and pharmacokinetic behaviors as MnTCP with higher tolerance

as a non-ionic, low osmolar ECF agent. The multiple hydroxyl groups were responsible to


r1

/ m

M-1

s-1

0 .0 1 0 .1 1 1 0 1 0 0

2 .0

4 .0

6 .0

8 .0

1 0 .0

1 2 .0

M n T P P S

M n T P P A S


0 .0 0 1 0 .0 1 0 .1 1

35

attribute high hydrophilicity, thus promoted fast renal clearance to avoid prolonged in vivo

interactions.

Scheme 6: Molecular Design of MnTAP from structural modification of MnTCP.


The synthetic route of MnTCP was adapted from the optimized condition of our

group.20, 35 Since Tris has both amino and hydroxyl groups, amide bond formation using EEDQ

had raised our attention. EEDQ was found to be a reagent with selectivity to facilitate amide

bond formation in the presence of hydroxyl groups and it had been reported that water-soluble

Scheme 7: Proposed synthetic route for the production of MnTAP.

36

amide was successfully synthesized using EEDQ and Tris.40, 41 As the method had preference

towards amide instead of esterification, we had interested to perform the reaction using EEDQ.

The reaction mechanism are described in Scheme 8.

Scheme 8: Proposed amide coupling reaction mechanism medicated by EEDQ.

Amide coupling using EEDQ method was first investigated using apo-porphyrin as

reactant; as the structural characterization was possible for 1H NMR. Thus we had performed the

hydrolysis of 9 to generate 12 for the preparation of amide formation.

12 was characterized by 1H NMR; as 12 processed a C4 symmetric, singlets from protons

at the inner NH and the porphyrin β-position were identified. The synthesis of 13 was performed

and the NMR supported the formation of the amide product. With the additional singlet signal at

the 6.94 ppm, it was hypothesized that the signal belonged to proton from amide NH.

The non-metal inserted porphyrin was synthesized, which suggested that the EEDQ

medicated amidation was applicable on the metal free porphyrins. Our next attempt was to

perform the amide coupling using MnTCP. Using the same synthetic protocol of 12, the amide

coupling was performed.

The HPLC monitoring results were inconclusive in supporting the formation of MnTAP.

The purified product and the MnTCP were characterized by HPLC. Similar retention time for the

two samples was observed, which could be explained by the high polarities that minimized the

interactions with the reverse phase solid support. From the UV-vis spectra, both of the MnTCP

and the purified sample suggested that the direct amide coupling was unsuccessful, as the spectra

were identical. Additionally, the precipitation test further confirmed the reaction was

37

unsuccessful. In MnTCP, the carboxyl groups were readily protonated at acidic environment,

thus resulted in precipitation of protonated MnTCP. In the test, the purified sample was

precipitated under acidic environment. A plausible explanation would be the difference in

chemical properties of apo-porphyrin and MnTCP. As EEDQ medicated direct amide coupling

was not applicable on MnTCP, alternative reagent that facilitate peptide bond formation such as

EDC/NHS method could be performed and investigated the feasibility.40, 42

3.3 Materials and Method





Analytical high

performance liquid








spectrophotometer


Nuclear magnetic

resonance (NMR)

spectrometer



mass spectrometer



spectrometer



Thermo Scientific

38

3.3.2 Materials













benzoquinone (DDQ)

98.0% AK Scientific




Sodium chloride Reagent grade Caledon


Manganese chloride

tetrahydrate


39

Tris-(hydroxymethyl)-

aminomethane (TRIS)

Ultrapure, 99.9% US Biological

Potassium hydroxide Reagent grade, ≥85.0% Caledon

2-Ethoxy-1-ethoxycarbonyl-

1,2-dihydroquinoline (EEDQ)

≥99% Sigma-Aldrich



(HEPES)



(TLC) plate



Dialysis Membrane Spectra/Por® 7 dialysis membrane:

pre-treated RC tubing – MWCO 1000

Spectrum Labs

Dialysis Membrane Spectra/Por® dialysis membrane:

Biotech CE Tubing – MWCO: 100 –

500 D

Spectrum Labs

3.3.3 Synthesis

5, 10, 15, 20-tetraakis(ethoxycarbonyl)porphyrin (9)

Pyrrole (1.01 mL, 15 mmol) and ethyl glyoxalate (50% in toluene,

3.00 mL, 15 mmol) were added into DCM (500 mL) and stirred at

RT, in the dark and under argon atmosphere. After 10 minutes,

BF3·OEt2 (0.21 mL, 1.65 mmol) was added dropwise. The reaction

was stirred at RT for 50 minutes, followed by the addition of DDQ

(2.55 g, 11.25 mmol). After a stirring period at RT for 2.25 h,

triethylamine (0.23 mL, 1.65 mmol) was added to quench the reaction. The reaction mixture was

concentrated and filtered over a triple layer column, consisted of silica, basic alumina and celite,

40

using DCM as solvent. The product was purified via column (stationary phase: silica, mobile

phase: DCM) to afford 9 (173.1 mg, 7.6%). 1H NMR (500 MHz, CDCl3): = 9.52 (8 H, s, por-β),

5.11 (8 H, q, J = 7.2 Hz, -OCH2-), 1.81(12 H, t, J = 7.2 Hz, -OCH2CH3), -3.33 (2 H, s, NH); UV-

vis (MeOH) max = 409 nm.

[5, 10, 15, 20-tetraakis(ethoxycarbonyl)porphyrinato]manganese (III) chloride (10)

Intermediate 9 (418.8 mg, 0.70 mmol) was dissolved in DMF

(16 mL). MnCl2·4H2O (693 mg, 3.5 mmol) was added and the

reaction was refluxed open to air for 3 hours. After confirmation

with TLC, the reaction was stirred at RT open to air for extended 16

hours. The mixture was dried by vacuum distillation and purified

via gradient column chromatography (stationary phase: silica;

mobile phase: initial 2% MeOH in DCM and subsequent elution with 7% MeOH in DCM). 10

(394.1 mg, 82%) was isolated as black purple solid. ESI-MS found m/z = 651.1 ([M]+ ), calcd for

C32H28MnN4O8+ , m/z = 651.1; UV-vis (MeOH): abs = 328, 366, 387, 413, 456, 552 nm,

max = 456 nm.

Manganese(III) 5, 10, 15, 20-tetraakis(carboxyl)porphyrin (11)

Intermediate 10 (111.1 mg, 0.171 mmol) was dissolved in THF

(26 mL) and Ethanol (35 mL). 2 M NaOH solution (69 mL) were

added and the reaction was refluxed for 20 hours. The mixture was

neutralized by HCl and concentrated, followed by ethyl acetate

washing (10 × 100 mL). The product was precipitated by acidification

to pH = 2 and washed with cold 0.1M HCl. The brown solid was

dialyzed in MWCO 500 membrane and dried to afford 11 (64.0 mg, 70%). ESI MS found

m/z = 539.0032 ([M] +), calcd for C24H12MnN4O8+, m/z = 539.0030. UV−vis (HEPES buffer):

λabs = 325, 377, 397, 421, 465, 561, 592 nm, λmax = 465 nm, ε = 68, 000M−1cm−1.

41

5, 10, 15, 20-tetraakis(carboxyl)porphyrin (12)

A solution of 2 M KOH (3.02 mL) was added to a solution of 9

(136.1 mg, 0.227 mmol) in THF (6.04 mL). The mixture was

refluxed for 6 days at 61oC and extracted by THF (5 × 10 mL). The

KOH layer was neutralized by 3 M H2SO4 solution. Desired product

was purified with ethyl acetate extractions and precipitated out at pH

= 2. The purple solid was washed with cold 0.01M HCl and cold

acetone to afford 12 (50 mg, 45%). 1H NMR (500 MHz, d6-DMSO): = 9.67 (8 H, s, por-β), -

3.51 (2 H, s, NH).

5, 10, 15, 20-tetraakis(N-tris-(hydroxymethyl)-methyl-amide)porphyrin (13)

Intermediate 12 (12.4 mg, 25.5 μmol) was dissolved

in pyridine (4 mL). Tris (34.2 mg, 255 μmol) and

EEDQ (38.8 mg, 153 μmol) were added and the

mixture was degassed with argon by freeze thaw

cycle (three times). The mixture was refluxed for 24

hours under argon. Pyridine was distillated off from

the mixture. The crude solid was dissolved in water

and washed by ethyl acetate (5 × 25 mL). The

aqueous layer was concentrated and purified via

cation exchange column, followed by dialyzed in

MWCO 1000 membrane. The purified product was lyophilized to afford 13 (12.1 mg, 53%).

1H NMR (500 MHz, d6-DMSO): = 9.28 (8 H, s, por-β), 6.94 (2 H, s, amide NH), -2.94 (2 H, s,

NH).

3.4 Conclusion

We have illustrated the development of a non-ionic extracellular agent by amide coupling

of MnTCP. In current stage, the metal free poly hydroxyl amide porphyrin was synthesized using

EEDQ and characterized by 1H NMR. However, the direct peptide bond formation from MnTCP

was unsuccessful by the EEDQ medicated method. In future, EDC/NHS method could be

investigated for its feasibility on the synthesis of MnTAP.41 Chemical characterization of

42

MnTAP would be necessary to confirm the compound identity. In addition, osmolar

measurements of MnTCP and MnTAP would be performed to compare their osmolarities.


1H NMR spectra of all synthetic targets are located in the appendix.

43

4 Tuning Electronic Properties on Mn(III) Porphyrin: Structural Relaxivity Relationship Studies

4.1 Introduction

Modulation of molecular rotational correlation rate is arguably the most popular strategy

for the relaxivity optimization, which had been widely applied in the development of Gd-based

novel contrast agents in particular. However, it has been demonstrated computationally and

experimentally that the contribution of molecular rotational motion to relaxivity gradually

decrease as the magnetic field strength increases at high fields. For the development of high r1 at

high clinical field, it would be necessary to investigate other parameters that contribute to high

relaxivity. In conventional GBCAs, the ligands were mainly consisted of macrocyclic rings or

linear. The contribution of electronic effect to the relaxivity is considerably much less explored.

For GBCAs, since the large f-orbitals have relatively less overlap with the ligand donor

electrons, it is challenging to control the electron spin via ligand modification. In the MnP

platform, however, Mn d-orbitals have significant interaction with the porphyrin ligand, which

allowed us to explore the structural relaxivity relationship in the electronic properties. By tuning

the functional groups in the porphyrin ring, the effects on relaxivity could be analyzed.

44

Figure 11: NMRD Profiles of MnTPPS and MnTPPSBr6 from 0.2 mT to 1 T (adopted from Bryant et al, 1999).43

There was a study conducted on the effect of relaxivity by tuning the electron density in

the conjugated porphyrin system. MnTPPSBr6 was generated from the bromination of MnTPPS

at the β-pyrrolic positions.43 In Figure 11, the NMRD profile was measured at 0.2 mT to 1 T.

Comparing the r1 of MnTPPS and MnTPPSBr6, there was significant alternation in the NMRD

profile of MnTPPSBr6, particularly in the high field with observable improvement. It was

hypothesized that the electron spin delocalization facilitated by heavy bromine atoms induced the

change in the relaxivity and we would like to further investigate this phenomena

systematically.43

Our curiosity towards the electronic effect was first investigated on MnTriCP platform

(see Chapter 3), in which the electronic effect of different substitution could be achievable as the

meso position in MnTriCP is synthetically accessible for functional group interconversion.

The study would be conducted by the incorporation of prototypical electron withdrawing

function group and electron donating function group. Two functional groups, nitro group and

amino group with extreme difference in their electronic properties, were first substituted at the

meso positions of MnTriCP as the initial step. Determination of electronic effect on relaxivity


r1

/ m

M-1

s-1

0 .0 1 0 .1 1 1 0 1 0 0

4 .0

6 .0

8 .0

1 0 .0

1 2 .0

1 4 .0

M n T P P S B r6

M n T P P S


0 .0 0 1 0 .0 1 0 .1 1

45

would be studied. In the next stage, consideration of functional groups in their biocompatibilities

would be taken in account for the contrast agent development.

Scheme 9: Molecular Design of MnTriCP-NO2 and MnTriCP-NH2 from structural modification of MnTriCP.


4.2.1 Synthesis of Monomeric MnTriCP Analogs

Scheme 10: Proposed synthetic routes for the production of MnTriCP, MnTriCP-NO2 and MnTriCP-NH2.

In the molecular aspect, TriCP has a vacant site at the meso position, which allows

extensive chemistry for the aromatic substitution. To compare the effect on relaxivity, MnTriCP

was synthesized as the reference compound through manganese insertion and hydrolysis. Our

goal is to synthesize MnTriCP-NO2 and MnTriCP-NH2. Nitration of TriCP yielded meso-nitro

46

porphyrin 14. Followed by the manganese insertion and hydrolysis, MnTriCP-NO2 was

generated. Our other interest compound MnTriCP-NH2 was developed by the reduction of 14 to

generate meso-amino porphyrin 15. Finally, metal insertion and hydrolysis was planned to yield

the final product MnTriCP-NH2.

Synthesis of 5, 10, 15-tri-(ethoxycarbonyl)-20-nitro porphyrin (14)

Nitration of 3 was performed using NaNO2 with an organic acid.44 Conventional nitration

was performed in trifluoroacetic acid (TFA) or a mixture of trichloroacetic acid and glacial acetic

acid. However, the rate of nitration was slow with TCA/glacial acetic acid; on the other hand, the

reaction proceeded rapidly under TFA with formation of side product. Acidity was essential to

the reaction. We developed a method to control the reaction rate through addition of traces of

TFA while monitoring by TLC. After three hours, the nitration was completed while minimizing

the generation of nitrite and TFA-related by-products. The purity was confirmed by 1H NMR

with 84% yield.

Synthesis of 5-amino-10, 15, 20-tri-(ethoxycarbonyl) porphyrin (15)

Reduction of 12 was performed using SnCl2·2H2O in 25% HCl at room temperature.45 As

conventional reduction using tin chloride was at 65oC, amino-TriCP might be sensitive and

readily decomposed under acidic, heating environment. Reduction was run with room

temperature with elongated time to avoid degradation. During the reaction and work up, the

process was monitored by TLC and a major polar fluorescent band was observed. During the

silica gel column purification, the multiple bands were observed, which suggested that

decomposition occurred in the silica gel column. It was hypothesized that the amino group at the

meso position caused the instability of 15. As the porphyrin consisted of a highly conjugated

system, the imine group might form due to resonance to the π system, which favored the

nucleophilic attack and led to degradation.

Synthesis of [manganese(III) 5, 10, 15-tri-(ethoxycarbonyl)porphyrinato] chloride(16)

Manganese insertion of 3 was performed using an adopted protocol.35 Using 5

equivalents of manganese chloride in a reflux system, reaction was completed within 4 hours. 16

47

was purified by gradient silica gel column chromatography with 3% MeOH in DCM as eluent

and characterized by UV-vis and ESI-MS.

Synthesis of manganese(III) [5, 10, 15-tri-(ethoxycarbonyl)-20-nitro-porphyrinato] chloride (17)

The reaction was modified from the literature procedure.35 5 equivalent of manganese

chloride was employed for the metal insertion of 14. By monitoring by TLC, the reaction was

completed in 3 hours. 17 was purified by gradient silica gel column chromatography with 3%

MeOH in DCM as eluent and characterized by UV-Vis.

Synthesis of manganese(III) 5, 10, 15-tri-(carboxyl)porphyrin (18, MnTriCP)

The reaction was modified from the base catalyzed hydrolysis of MnTCP. A ratio of

1:200 of 16: hydroxide was employed to hydrolyze the ethyl ester groups. The intermediates

bearing ester groups were observed to have lower polarities compared to the 18. Automated

chromatography system using gradient elution was employed as an efficient purification of

hydrolysis intermediates. The purity was confirmed by HPLC; the characterization by Mn-FAAS

and UV-vis was performed. However, from the UV-vis spectrum in Figure S14, the peak at

419 nm might be an indication of demetallated porphyrin. The extinction coefficient at 462 nm

was determined to be 43,000 M-1cm-1, which was slightly lowered than MnTCP and Mn TriCP-

NO2. Partial demetallation of the MnTriCP sample might be the reason for the abnormal UV-vis

spectrum and lower extinction coefficient values.

Synthesis of manganese(III) 5, 10, 15-tri-(carboxyl)-20-nitro-porphyrin (19, MnTriCP-NO2)

The reaction was modified from the base catalyzed hydrolysis of MnTCP. A ratio of

1:200 of 17: hydroxide was employed for the reaction. The high polarities of MnTriCP and

MnTriCP-NO2 were important for effective purification using the automated chromatography

system. The purity was confirmed by HPLC. By the Mn-FAAS and UV-vis characterization, the

extinction coefficient at 430 nm was determined as 64,000 M-1cm-1.

48

4.2.2 Field-Dependent Relaxivity of MnTCP Derivatives

Figure 12: NMRD Profiles of MnTriCP, MnTriCP-NO2 and MnTCP from 0.2 mT to 3 T at 25oC in 25 mM

HEPES buffer, pH 7.2.

The effects of electronic properties were evaluated by the NMRD profiles. From the

preliminary results, the MnTriCP-NO2 demonstrated a lower r1 than the reference compound

MnTCP from 0.2 mT to 3 T, which was among the full profile. MnTriCP showed similar

relaxivity behavior as MnTriCP-NO2. However, the sample might not be pure due to

demetallation of MnP. However, as the Mn concentration, quantified by Mn-FAAS, was based

on the MnP, it would be safe to assume that apo-porphyrin exhibited insignificant influence on

NMRD. The results suggested the substitutes on porphyrin had significant impact on relaxivity.

Furthermore, strong electron withdrawing group would compromise the relaxivity and decreased

the contrast efficiency. Currently, MnTPPS-NH2, another compound from the ongoing project in

the group, demonstrated a higher relaxivity than MnTPPS and their structural difference was the

functional groups: amino and sulfonate, respectively. As the amino group had electron donating

property, we hypothesized that the electron donating group could improve the r1 through

increasing electron spin density. However, due to the instability of meso-amino group, the

relaxivity characteristics of Mn TriCP-NH2 were yet unexplored. To investigate the r1 behavior


r1

/ m

M-1

s-1

0 .0 1 0 .1 1 1 0 1 0 0

6 .0

8 .0

1 0 .0

1 2 .0

M nT C P

M n T riC P

M n T riC P -N O 2


0 .0 0 1 0 .0 1 0 .1 1

49

of electron donating group, incorporations of aniline, phenyl and nitro phenyl groups at the meso

positions would be an alternative. Although the electronic effect would be affect by the indirect

conjugation to the porphyrin system, it would be intuitive to access the relaxivity changes due to

electronic properties of functional groups. In addition, monitoring the direct reduction of Mn

TriCP-NO2 by relaxometer might be informative. As the r1 of Mn TriCP-NO2 was

compromised, any significant changes in r1 might be explained by the presence of Mn TriCP-

NH2. Thus the relaxivity effect due to amino group could be evaluated.

4.3 Materials and Methods





Analytical high

performance liquid








spectrophotometer


Nuclear magnetic

resonance (NMR)

spectrometer



mass spectrometer



spectrometer



Thermo Scientific

50

Fast field cycling (FFC)

NMR Relaxometer

SMARtracer™ Bench-top Fast Field

Cycling (FFC) NMR Relaxometer with

high temperature superconductor (HTS)-

110 magnet system

Stelar s. r. l. and

Scott® Technology

Limited

Automated liquid


CombiFlash Rf+ Lumen Flash

Chromatography System with integrated

Evaporative Light Scattering Detection

and UV-vis variable wavelength detector

Teledyne Isco

4.3.2 Materials








Hexane ACS reagent grade, ≥98.5% Fisher


Paraformaldehyde Reagent grade Sigma-Aldrich

Indium(III) chloride 98% Sigma-Aldrich


51


Sodium bicarbonate Reagent grade Caledon


Sodium sulfate 99.0% Caledon


benzoquinone (DDQ)

98.0% AK Scientific




Trifluoroacetic acid Reagent grade, ≥99.9% Caledon

Trichloroacetic acid Certified, ≥99.0% Fisher

Glacial acetic acid Reagent grade, ≥99.7% Caledon


Manganese chloride

tetrahydrate


Stannous chloride dihydrate ACS grade, ≥98% Fisher



(HEPES)



(TLC) plate



52

Dialysis Membrane Spectra/Por® dialysis membrane:

Biotech CE Tubing – MWCO: 100 –

500 D

Spectrum Labs

4.3.3 Synthesis

5, 10, 15-tri-(ethoxycarbonyl)-20-nitro porphyrin (3, TriCP)

Porphyrin 3 was synthesized by the same protocol in the Synthesis in

Chapter 2. Dipyrromethane (876mg, 5.99mmol), ethyl glyoxalate

(50% in toluene, 2.37 mL, 11.27 mmol), and sodium bicarbonate

(427.5 mg, 5.09 mmol) were mixed in DCM (8.9 mL) in dark. The

mixture was stirred for 3 hours at RT. Sodium bicarbonate was

filtered off and the DCM filtrate was collected. The filtrate, 5-

ethoxydipyrromethane (1.35 g, 6.0 mmol), and DCM (1.23 L) were mixed and degassed with

Argon at 0oC for 30 minutes in dark. BF3·OEt2 (148 μL, 1.20 mmol) was added dropwise and the

mixture was stirred at RT. The reaction was monitored by UV-Vis. After 30 minutes, DDQ (4.08

g, 18.0 mmol) was added and the mixture was stirred for 2 hours at RT. The reaction mixture

was concentrated and filtered using triple layer column consisted of silica gel, basic alumina and

celite. The filtrate was concentrated and purified via chromatography (stationary phase, silica;

mobile phase, DCM) to afford 3 (120 mg, 4.0%).

5, 10, 15-tri-(ethoxycarbonyl)-20-nitro porphyrin (14)

Porphyrin 3 (42 mg, 0.0798 mmol) was transferred to a 10 mL

reaction flask and a solution of glacial acetic acid (2.63 g, 16.1

mmol) in trichloroacetic acid (1.76 mL, 30.8 mmol) was added.

NaNO2 (23.5 mg, 0.341 mmol) was added and the reaction was

monitored by TLC (solvent: DCM). A few drops of trifluoroacetic

acid were added during the process. After 3 hours, saturated

NaHCO3 was added to quench the reaction and the mixture was extracted by DCM (2 x 30 mL).

The DCM fraction was washed with water (2 x 60 mL), dried by Na2SO4. The organic fraction

was concentrated and purified via chromatography (stationary phase, silica; mobile phase: 7:3

53

DCM: hexane), which afforded 4 (38.5 mg, 84%). 1H NMR (500 MHz, CDCl3): 9.55 (dd, J = 9.4,

4.9 Hz, 4H, por-β Ha, b), 9.50 (d, J = 4.9 Hz, 2H, por-β Hc ), 9.40 (d, J = 5.0 Hz, 2H, por-β Hd),

5.12 (qd, J = 7.2, 1.7 Hz, 6H, -OCH2-), 1.82 (td, J = 7.2, 2.4 Hz, 9H, -OCH2CH3), -3.35 (s, 2H,

por-NH).

5-amino-10, 15, 20-tri-(ethoxycarbonyl) porphyrin (15)

Porphyrin 14 (10 mg, 0.0175 mmol), SnCl2·2H2O (60 mg, 0.266

mmol) and 25% HCl (2 mL) were added in a 5 mL reaction flask

and the mixture was stirred at RT for 48 hours. After TLC

suggested that the starting material 14 was consumed, the reaction

was quenched with 1 M NaOH until the solution was basic. The

mixture was extracted with DCM (2 × 30 mL). The organic layer

was washed with saturated NaCl solution (1 × 75 mL) and dried by Na2SO4. The mixture was

concentrated and purified by gradient column chromatography (stationary phase: silica gel;

mobile phase: starting with 1% MeOH in DCM to 5% MeOH in DCM). During the column

purification, decomposition occurred with multiple bands on the TLC.

[manganese(III) 5, 10, 15-tri-(ethoxycarbonyl)porphyrinato] chloride (16)

Porphyrin 3 (20 mg, 0.038 mmol) was dissolved in DMF (1.6 mL).

MnCl2∙4H2O (34.6 mg, 0.175 mmol) was added and the mixture was

refluxed at 130oC while monitoring by TLC. After 3 hours, the

reaction was completed and the DMF was removed by vacuum

distillation. The mixture was purified via chromatography (stationary

phase, silica; mobile phase: initial: 1% MeOH in DCM, final: 3.2%

MeOH in DCM) to afford 16 (18.7 mg, 85%). ESI-MS found m/z = 579.10 ([M]+), calcd for

C29H24MnN4O6+, m/z = 579.1076. UV-vis (MeOH): λabs = 470 nm.

54

[manganese(III) 5, 10, 15-tri-(ethoxycarbonyl)-20-nitro-porphyrinato] chloride (17)

Intermediate 14 (32.5 mg, 0.0329 mmol) was dissolved in DMF

(1.5 mL). MnCl2∙4H2O (32.5 mg, 0.0164 mmol) was added and the

mixture was refluxed open to air while monitoring by TLC. After 3

hours, the reaction was completed and the DMF was removed by

vacuum distillation. The mixture was purified via chromatography

(stationary phase, silica; mobile phase: initial: 1% MeOH in DCM,

final: 3% MeOH in DCM) to afford 17 (18.7 mg, 91%). ESI-MS found m/z = 624.10 ([M]+),

calcd for C29H23MnN5O8+, m/z = 624.0927. UV-vis (MeOH): λabs = 478 nm.

Manganese(III) 5, 10, 15-tri-(carboxyl)porphyrin (18, MnTriCP)

Intermediate 16 (5.0 mg, 0.0069 mmol) was dissolved in ethanol (1.09

mL) and THF (0.82mL). 2 M NaOH (1.91 mL) was added and the green

mixture was refluxed at 65oC under argon atmosphere. The reaction was

monitored by UV-vis until no further change in the UV-vis spectra.

After 6 hours, the reaction mixture was neutralized by 1 M HCl and

concentrated by rotary evaporation. HPLC was performed on the

mixture and the desired product was separated by the automated liquid chromatography system.

The eluent was dialyzed using MWCO 500 membrane and lyophilized to yield 18 (3.7 mg, 74%).

UV-vis (HEPES): λmax = 462 nm, ε = 43,000 M-1cm-1

Manganese(III) 5, 10, 15-tri-(carboxyl)-20-nitro-porphyrin (19, MnTriCP-NO2)

Intermediate 17 (5.0 mg, 0.0080 mmol) was dissolved in ethanol

(1.09 mL) and THF (0.82 mL). 2 M NaOH (1.91 mL) was added and

the green mixture was refluxed at 65oC under argon atmosphere. The

reaction was monitored by UV-vis until no further change in the UV-

vis spectrum. After 6 hours, the reaction was quenched by addition of

1 M HCl until the pH was neutral. The mixture was concentrated and

purified by the automated liquid chromatography system. The eluent was dialyzed using MWCO

500 membrane and lyophilized to yield 19 (3.5 mg, 70%). UV-vis (HEPES): λmax = 430 nm,

ε = 64,000 M-1cm-1.

55

4.4 Conclusion

In order to investigate the influence of electronic properties on relaxivity, our approach

was to incorporate interchangeable functional group that was directly attached to porphyrin at the

meso position. As nitro and amino represented the extremes of electron withdrawing and electron

donating properties respectively, they were chosen as the substitutes at the first stage. Two novel

monomeric MnTCP derivatives were designed and synthesized. The synthesis of these

asymmetric porphyrins were achieved by using TriCP as the porphyrin precursor. The major

challenge at the first stage was the synthesis of TriCP through a stepwise porphyrin condensation

synthesis. The desired product, MnTriCP-NO2, achieved by nitration, manganese insertion and

hydrolysis. By the employment of automated chromatography system, the efficiency of

purification of water-soluble MnP was improved. Two novel compounds, MnTriCP and

MnTriCP-NO2, provided important starting point in the studies of structural relaxivity

relationship. From the preliminary NMRD profile results, the r1 was compromised by replacing

one of four carboxyl groups in MnTCP with a strong electron withdrawing nitro group or with a

hydrogen atom. These findings suggest that the direct substations on porphyrin ring has clear

impact on relaxivity. Another ongoing project suggested that electron-donating group at meso

positon may lead to improvement on r1. In the past, water solubility was the major consideration

for functional groups in molecular design; for now, there would be additional factor for

optimizing relaxivity.

In the future, further purification and more-detailed structural characterizations of

MnTriCP and MnTriCP-NO2 are necessary. Diverse functional groups will be examined to give

a more comprehensive understanding on structure-relaxivity relationship. In particular, the

modification of high stability electron donating group at meso position would be important to

confirm the hypothesis.


1H NMR of synthetic targets are found in the appendix. HPLC chromatograms and UV-vis

spectra of 18 and 19 are located in the appendix.

56

5 Conclusion and Future Perspective

In this thesis, three distinct approaches were explored to improve MnTCP as MnP-based

extracellular MRI contrast agent. The first approach (Chapter 2) was to develop a compact

compound with low molecular size and high relaxivity, thus facilitating strong contrast

efficiency and rapid renal clearance. MnTrCP2, a dimeric porphyrin that resembles to MnTCP,

was designed, synthesized and spectroscopically characterized. In this project, we had developed

a selective synthesis of a novel asymmetric porphyrin precursor, TriCP. Furthermore, we had

generated meso,meso linked porphyrin dimer by employment of oxidative homolytic coupling

method. The desired final product, MnTrCP2, was generated in an eight step synthesis.

MnTrCP2 was evaluated by the field-dependent relaxivity measurement, HSA binding assay and

in vivo studies. MnTrCP2 exhibited a high molar relaxivity with a threefold improvement to that

of Gd-DTPA, a conventional Gd-ECF agent. In addition, no observable HSA binding affinity

was found in MnTrCP2, thus facilitating fast clearance. The in vivo performance was assessed on

healthy rats at 3 T, a significant stronger contrast enhancement was found in MnTrCP2 to that of

Gd-DTPA. Overall, MnTrCP2 demonstrated as a high relaxivity with fast renal clearance, which

is suitable for high clinical field application.

The second approach (Chapter 3) was to further optimize the biocompatibility of MnTCP.

By the replacement of neutral, water solubilizing polyhydroxyl amide from carboxylates, the

high osmolarity of MnTCP would be reduced, thus improving the in vivo tolerability. MnTAP,

an amide porphyrin that consists of four highly polar polyhydroxyl groups at the peripheral

regions, was designed by the amide coupling of MnTCP and Tris. Currently, we are developing a

suitable peptide coupling reagent to meditate the amide bond formation,

The third approach (Chapter 4) was to investigate the structural relaxivity relationship by

tuning the electronic property. Our initial goal was to examine the influences of electron

withdrawing and electron donating properties on the relaxivity, which was performed by

incorporating nitro and amino groups to the porphyrin ligands. In this project, TriCP was

employed as the porphyrin precursor to facilitate those studies; as TriCP has a vacant site at the

meso position, interconversion of functional group is feasible in comparison to the MnTCP. A

novel MnP, MnTriCP-NO2, was designed and generated in a six step synthesis. From the

57

NMRD profile, the electronic property had influenced the relaxivity performance, particularly

that the electron withdrawing property would compromise the overall relaxivity.

For the future work, these three approaches provided insights for the development of next

generation of MnP-based ECF agents. The pharmacokinetic studies and tumor perfusion of

MnTrCP2 would be investigated for the application in disease diagnosis. Furthermore, as

MnTrCP2 was a compact dimeric porphyrin that combined high relaxivity and low molecular

size, MnTCP oligomer with meso-meso bridging and carboxyl terminals would be interesting to

further investigate, thus generating MnTCP derivative of exhibiting high relaxivity and

preserving fast renal clearance.

To further optimize the biocompatibility of MnTCP, a synthetic method for peptide

synthesis from MnTCP and polyhydroxyl amine would be investigated. Furthermore, the

spectroscopic characterization would be performed; osmolarity and viscosity studies would be

our interests to compare MnTAP and MnTCP.

In the project of structural relaxivity relationship, the experimental results suggested that

the electron withdrawing property compromised relaxivity performance. In addition, from

another ongoing project in our group, we hypothesized that the electron donating property lead to

improvement in relaxivity performance. Currently, we faced a challenge from the instability of

meso-amino porphyrin, thus restricting us from evaluating the relaxivity performance of

MnTriCP-NH2. A strategy to tackle the challenging would be performing reduction on

MnTriCP-NO2 and monitoring the relaxivity changes.

These three approaches would be combined to develop future generation of MnTCP

extracellular agents. A substitute with optimized electronic property would be taken in account

for its biocompatibility under the physiological environment. Furthermore, MnP oligomer would

be generated base on the optimized electronic property and biocompatibility, thus further

promoting high relaxivity and low toxicity in novel compounds.

58

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63

Appendix: NMR, ESI-MS, UV-vis Spectra and HPLC Chromatograms of Synthetic Targets

Figure S1: 1H NMR spectrum of (1) acquired in CDCl3. The residual solvent peaks are water and CHCl3.

64

Figure S2: 1H NMR spectrum of (2) acquired in CDCl3. The residual solvent peaks are water, CH2Cl2 and CHCl3.

Figure S3:1H NMR spectrum of (3) acquired in CDCl3. The residual peaks are grease, water, and CHCl3.

65

Figure S4: 1H NMR spectrum of (4) acquired in d6-DMSO. The residual peaks are grease, water, and DMSO.

Figure S5: 1H NMR spectrum of (5) acquired in d6-DMSO. The residual peaks are grease, water, and DMSO.

66

Figure S6: 1H NMR spectrum of (6) acquired in CDCl3. The residual peaks are grease, water, CH2Cl2 and CHCl3.

67

Figure S7: High resolution ESI-MS of MnTrCP2 acquired in water.

68

W a v e le n g th (n m )

Ab

so

rb

an

ce

3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0 8 0 0

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

Figure S8: UV-vis spectrum of MnTrCP2 in HEPES buffer.

T im e (m in )

Ab

so

rb

an

ce

0 2 4 6 8 1 0 1 2

0

1 0 0

2 0 0

3 0 0 3.8

24

Figure S9: HPLC chromatogram of MnTrCP2 detected at 468 nm. Elution occurred at 3.8 min with 98% purity.

69

Figure S10: 1H NMR spectrum of (9) acquired in CDCl3. The residual peaks are grease, water and CHCl3.

70

Figure S11: 1H NMR spectrum of (12) acquired in d6-DMSO. The residual solvent peaks are water and DMSO.

Figure S12: 1H NMR spectrum of (13) acquired in d6-DMSO. The residual solvent peaks are water and DMSO.

71

Figure S13: 1H NMR spectrum of (14) acquired in CDCl3. The residual peaks are grease, water, CH2Cl2 and CHCl3.

72


Ab

so

rb

an

ce

3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

Figure S14: UV-vis spectrum of MnTriCP in HEPES buffer.

T im e (m in )

Ab

so

rb

an

ce

0 2 4 6 8 1 0 1 2

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

3.7

85

Figure S15: HPLC chromatogram of MnTriCP detected at 462 nm. Elution occurred at 3.8 min with 97% purity.

73


Ab

so

rb

an

ce

3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 7 0 0 7 5 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

Figure S16: UV-vis spectrum of MnTriCP-NO2 in HEPES buffer.

T im e (m in )

Ab

so

rb

an

ce

0 2 4 6 8 1 0 1 2

0

1 0 0

2 0 0

3 0 0

4 0 0

3.5

18

Figure S17: HPLC chromatogram of MnTriCP-NO2 detected at 430 nm. Elution occurred at 3.5 min with 96%

purity.

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