Apr 17, 2009 - Magnus Janard, Robert Steen, Dr. Olle Rengby, Stefan Röstlund, Fredrik. Nordborg, Henrik Friberg and Calle Carlsson. My parents Inez ...
!" # $ !" %#&' &'%( !! " # )&*+ (!
,-%. +,/01/--.0.- 2 2 22 3 /1-//
! " # $% &&' $!($)* + * * ,+ +-.+ / 0 + 1 2- &&'- 3 + *$$4 4 * + 5 *06" 706" #.$ #. 8 - -# - ! ! "# ! # # # 9$9-9)- -'%:'$)) % )$).+/ ; / + + / + ,0. / + + $$./ + * + + *$$ + -" &? $$ * / + &? - # + + / * ; + / + + + ; -3* * A *+ / * + * + .+ 55 #. / $$ + >&? $9!9D -2 * + / + ,0.-.+ / A * - + / * + + * * %& '+
(EE -;-E I B ( ((( ':)''?
Att doktorera innebär att man lär sig mer och mer om allt mindre och mindre, för att till slut veta nästan allting om nästan ingenting.
List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals I-VII. I
Synthesis of [11C]-/(13C)-Acrylamides by Palladium-Mediated Carbonylation. Jonas Eriksson, Ola Åberg, Bengt Långström. Eur J Org Chem, 2007, 3, 455-461
II
Synthesis of a Library of 11C-Carbonyl-labelled Irreversibly Binding EGFr Inhibitors as Potential Biomarkers for Tumours. Ola Åberg, Bengt Långström, Manuscript
III Rhodium-mediated [11C]Carbonylation: a library of N-phenyl-N’-{4-(4quinolyloxy)-phenyl}-[11C]-urea derivatives as potential PET angiogenic probes. Ohad Ilovich, Ola Åberg, Bengt Långström and Eyal Mishani, J Label Compd Radiopharm, 2009, available online IV Synthesis of Asymmetric [11C]Ureas and [11C]Sulphonylureas by Rh(I)Mediated Carbonylation. Ola Åberg, Bengt Långström, Manuscript V
Synthesis and Biological Evaluation of [Carboxyl-11C]Eprosartan. Ola Åberg, Örjan Lindhe, Håkan Hall, Per Hellman, Tor Kihlberg, Bengt Långström, J Label Compd Radiopharm, 2009, accepted
VI Synthesis and Biological Evaluation of a 11C-Labelled Angiotensin II AT2 Receptor Ligand by Molecular Imaging using PET. Ola Åberg, Mark Stevens, Jonas Lindh, Charlotta Wallinder, Mats Larhed, Bengt Långström, Manuscript VII Appendix I: Synthesis of the Aryl Iodide Precursor of [Carboxyl11 C]Candesartan Ola Åberg, Tor Kihlberg, Bengt Långström Reprints were made with permission from the publishers.
Contribution Report
The work presented in this thesis belongs to a multidisciplinary field of research and was accomplished in collaboration with many people. The author would therefore like to clarify his contribution. I
Contributed significantly to the planning and labelling experiments, to interpreting the results and to writing the paper. Synthesized and characterized the reference compounds.
II Contributed the original idea, synthesized and characterized the precursors, references, and labelled compounds, interpreted the results and wrote the paper. III Contributed significantly to the planning, synthesis and characterization of the azide precursors, labelling experiments, to interpreting the results and to writing the paper. IV Contributed the original idea, synthesized and characterized the precursors, references, and labelled compounds, interpreted the results and wrote the paper. V Contributed significantly to the planning and labelling experiments and to interpreting the results. Wrote the paper. VI Contributed the original idea, contributed significantly to the planning, performed the labelling experiments and wrote the paper. Planned and synthesized the precursor and wrote the paper. VII Planned and synthesized the precursor and wrote the paper.
Contents
Introduction...................................................................................................11 PET Technology.......................................................................................11 PET-Tracer Development ........................................................................13 Affinity ................................................................................................13 Specific Activity ..................................................................................14 Lipophilicity and pKa...........................................................................14 Membranes and Active Efflux Systems...............................................15 Metabolism and Labelling Position .....................................................15 Selectivity, Specificity, and Sensitivity ...............................................15 Perspectives on 11C and [11C]Carbon Monoxide ......................................16 Production of 11C as [11C]O2 ................................................................16 Online Production of [11C]Carbon Monoxide......................................16 The [11C]Carbon Monoxide Synthesis System ....................................17 Specific Activity and Isotopic Dilution ...............................................18 [11C]Carbon Monoxide and the Library Concept ................................19 Purification, Analysis and Characterization .............................................20 Aim...........................................................................................................22 Tyrosine Kinases as Targets for Molecular Imaging ....................................23 The ErbB Recptor Family ........................................................................23 Active and Inactive Conformations of EGFr.......................................24 Reversible Versus Irreversible Binding PET Tracers for EGFr ..........24 Docking Studies on the EGFr ..............................................................25 Carbonyl-11C-Acrylamide Labelling (Paper I) .............................................27 Benzyl[carbonyl-11C]acrylamide from Acetylene....................................27 [Carbonyl-11C]Acrylamides from Vinyl Iodide .......................................27 Configuration of the Carbon-Carbon Double Bond .................................30 Carbonyl-11C-Acrylamide Labelling of EGFr Inhibitors (Paper II) .............32 Synthesis of a Library of Potential Angiogenic Probes (Paper III) ..............34 Synthesis of Asymmetric [11C]Ureas and [11C]Sulphonylureas by Rh(I)Mediated (Paper IV) .....................................................................................37 Targeting the Angiotensin II Receptors ........................................................39
The Renin-Angiotensin System................................................................39 The AT1 receptor (AT1R) ....................................................................40 The AT2 receptor (AT2R) ....................................................................40 The Ang II Receptors as Imaging Targets................................................40 Strategy in Choice of Ligands ..................................................................41 Synthesis of the AT1R Antagonist [Carboxyl-11C]Eprosartan (Paper V).42 Precursor synthesis ..............................................................................42 Labelling reaction ................................................................................44 Synthesis of the Aryl Iodide Precursor of [Carboxyl-11C]Candesartan (Appendix I) .............................................................................................46 11 C-Labelling of a Series of Angiotensin II AT2 Agonists (Paper VII)....48 Biological Evaluation ...............................................................................50 Conclusions...................................................................................................52 Populärvetenskaplig sammanfattning på svenska.........................................53 Acknowledgements.......................................................................................55 References.....................................................................................................57
Definitions
The following definitions are used throughout this thesis. Analytical radiochemical yield: The conversion of [11C]O multiplied by the radiochemical purity. The analytical radiochemical yields are decaycorrected. Conversion of [11C]O: The decay-corrected fraction of [11C]carbon monoxide converted to non-volatile products (also known as trapping efficiency, TE). Decay-corrected: The yield of a product obtained at a specific time At can be decay-corrected to the time point, A0, using the following formulas:
At = A0 × e
λ=
− λ ×t
where
ln 2 t1 / 2
Labelled compound or product: A mixture of an isotopically unmodified compound with the analogous singly and specifically isotopically modified compound. Radiochemical purity: The proportion of the total radioactivity that is in the chemical form stated (assessed by decay-corrected analytical radio-HPLC). Radiochemical yield: The yield based on the initial amount of radioactivity as [11C]carbon monoxide at the start of synthesis and the radioactivity of the isolated compound. All the radiochemical yields are decay-corrected. Specific activity: The radioactivity of the labelled compound divided by the molar amount of the compound, here expressed in GBq·mol-1 and at a specific time point from EOB.
Abbreviations
3D-QSAR ACE AIBN AMF ATP BBB CT dc DMF EGFr EOB EOS ESI [18F]DG HPLC GBq GC GPCR LC-MS LC-MS/MS MBq MRI NMR PET RAS RCY or rcy SPE SUV TE TEA THF UV VEGFr-2 *
3-Dimensional quantitative structure activity relationship Angiotensin converting enzyme ,’-Azoisobutyronitrile Ammonium formate Adenosine 5’-triphosphate Blood-brain barrier Computed tomography Decay-corrected Dimethylformamide Epidermal growth factor receptor End of bombardment End of synthesis Electron spray ionization [18F]fluorodeoxyglucose High performance liquid chromatography Giga Becquerel Gas chromatography G-protein coupled receptor Liquid chromatography-mass spectrometry Liquid chromatography-mass spectrometry-mass spectrometry Mega Becquerel Magnetic resonance imaging Nuclear magnetic resonance Positron emission tomography Renin-Angiotensin System Radiochemical yield Solid-phase extraction Standardized uptake value Trapping efficiency Triethylamine Tetrahydrofuran Ultraviolet Vascular endothelial growth factor receptor 2 = [11C]
Introduction
Radiochemistry, radio·chem·is·try, /reǹ di oșɎkǪm stri/ “That part of chemistry which deals with radioactive materials. It includes the production of radionuclides and their compounds by processing irradiated materials or naturally occurring radioactive materials, the application of chemical techniques to nuclear studies, and the application of radioactivity to the investigation of chemical, biochemical or biomedical problems.” (IUPAC Gold Book, 2009)
Modern technologies have made it possible to track and quantify the spatial distribution patterns and dynamics of radioactivity in a living organism over time. One of the most powerful technologies for this purpose is positron emission tomography (PET), which utilizes positron-emitting nuclides as the radioactive source. The applications of PET are very diverse. For example, it can be used to follow changes in the biochemistry of a system, such as altered metabolism in cancer or the progress of neurodegenerative diseases such as Alzheimer’s. It is also widely used in drug development to study the distribution and receptor occupancy of a candidate drug. Many endogenous compounds have been radiolabelled to study their functions in the human body. Today, 30 years after PET’s first clinical implementation, [18F]DG is still the tracer used in the vast majority of all PET scans performed. This is due partly to the difficulties associated with the development and clinical validation of new radiotracers. This thesis deals with radiochemistry and new approaches to developing novel PET tracers, in particular using [11C]carbon monoxide.
PET Technology PET has been widely used to visualize and quantify different biochemical processes, such as metabolic processes, and receptor densities in vivo. The tracer molecule, labelled with a short-lived positron-emitting radionuclide such as 11C (t1/2 = 20.4 min), 15O (t1/2 = 2.04 min), 13N (t1/2 = 9.97 min), 18F (t1/2 = 110 min), or 68Ga (t1/2 = 67.8 min), can be synthesized with high spe11
cific activity. As a consequence, very small amounts can be administered, which allows biochemical processes to be studied in vivo over time without perturbing the system. The short half-life of 11C enables a sufficient amount of statistical data to be collected during the PET scan while keeping the radiation dose to a minimum. Another advantage is that the use of 11C permits repeated studies in the same subject on the same day. As a rule of thumb, labelling and purification should be accomplished within three half-lives from EOB, i.e. around 60 min. Therefore, the radionuclide is incorporated as late as possible in the synthetic route. Positron-emitting radionuclides are neutron-deficient and decay by the conversion of a proton into a neutron with concomitant emission of a positron ( +) and a neutrino (ve). 11C has a short half-life and positron emission accounts for 99% of its decay. e
Neutrino
Radioactive decay
11
Positron
C 6 protons 5 neutrons
11
+
B 5 protons 5 neutrons
+
Photon 511 keV
e-
Photon 511 keV
Annihilation 180°±0.25
e-
Electron
Figure 1. Schematic picture of the radioactive decay of 11C to 11B upon emission of a neutrino ( e) and a positron ( +).
The positron ( +) travels up to a few millimetres in wet tissue before encountering its antiparticle, an electron (e-), causing an annihilation that generates two high-energy (511 keV) gamma ray photons, which are emitted in essentially opposite directions (Figure 1). The photons are detected in coincidence as they reach the scintillators, creating a burst of light that in turn is amplified by photomultipliers, giving rise to an electrical signal that can be further processed. Only signals that are detected in pairs within a few nanoseconds are considered to originate from the same radioactive decay; the singlephoton detections that result from scattering are ignored. The detectors in the PET camera are arranged in a ring around the object under study, allowing 12
the location of the decay to be determined. The data points collected are used to reconstruct a series of 3-D images with a resolution as low as 3-4 mm in a whole body scanner and even better in a small-animal PET scanner. The lower resolution limit is ultimately set by the distance the positron travels in the tissue before encountering an electron, which is dependent on its kinetic energy. In modern facilities, PET is used together with CT to produce both biochemical and anatomical images.1 The most fashionable technique is the PET-MRI, which enables simultaneous PET and MRI.2 In contrast to CT, MRI requires no additional radiation dose and can be used with a range of endogenous as well as exogenous contrasts.3
PET-Tracer Development The development of a new radiotracer is complex and multidisciplinary. A successful radiotracer gives a sufficient signal-to-noise ratio for a validated and pathology-specific target and allows radioactivity to be quantified within the timeframe of the radionuclide used. There are many similarities between drug development and PET-tracer development, but there are also important differences. In drug development, candidate drugs fail for many reasons; poor oral bioavailability, high toxicity, lack of efficacy and inappropriate therapeutic window are the most common. These parameters rarely play a role in PET-tracer development because PET tracers are injected intravenously and in very low amounts, making toxicity and unwanted pharmacological effects rare. Thus failed candidate drugs may well serve as lead compounds for PET-tracer development. A PET tracer with appropriate imaging characteristics must find its way to the target in very low concentration and in a relatively short time, so increasing the availability of the tracer by increasing its concentration is not an option, nor is time a parameter that can be compromised.4 There are many parameters determining the likeliness of success of a candidate tracer and all parameters are directly related to its chemical structure. The most important factors are briefly discussed below.
Affinity Affinity can be expressed as the inverse equilibrium dissociation constant Kd = koff/kon. The affinity required for a tracer to be successful depends on the concentration of binding sites in the region of interest. Typically, the most successful tracers have subnanomolar or low nanomolar affinity to the target of interest in vitro. Less is known about the influence of the absolute values of kon and koff in PET applications, but they have recently gained interest in drug discovery.5,6 A low concentration of binding sites means that the affinity must be high, and because these are easier to saturate the specific activity 13
also needs to be sufficiently high (see next paragraph). For a reversibly binding tracer, increased affinity is associated with a slower approach to equilibrium following a single bolus injection; equilibrium is the time point when quantification is most easy performed. Quantification of a reversibly-binding ligand is generally preferable over an irreversible one. 7-9 A well-known exception is [18F]DG, which is trapped irreversibly inside the cell.10
Specific Activity High specific activity allows a very small amount of the tracer to be administration and thus decreases the risk of perturbing or saturating the biological system under study. It has been suggested that the tracer should occupy 90% in under 5 min.
Specific Activity and Isotopic Dilution The theoretical maximum specific activity of 11C is 3.4×105 GBq·μmol-1, i.e. when all carbon atoms are 11C. However, current methods of producing 11Clabelled molecules also give isotopic dilution with stable carbon isotopes. The radioactive 11CO2 obtained from the cyclotron target is written as [11C]O2 to indicate that the chemical entity that is specifically and singly isotopically labelled makes up only a fraction of a percent of a sample.29 In practice, isotopic dilution of the final labelled product may be affected by all of the materials used in the experimental setup including the foil separating the target gas from the vacuum in the cyclotron target, the target wall material, the gas in the target, tubing, reactor materials, reagents, reagent handling equipment, solvents and purification equipment. When dealing with [11C]O2, atmospheric carbon dioxide is a source of isotopic dilution. Tropospheric air contains about 380 ppm carbon dioxide. Carbon monoxide, on the other hand, is much less abundant with an overall tropospheric concentration of 0.05-0.2 ppm,30 and around 10 ppm in urban areas.31 Thus the strategy of using [11C]carbon monoxide as the labelled precursor allows a higher specific activity to be achieved than does the traditional use of [11C]carbon dioxide and Grignard reagents. When [11C]carbon monoxide is used, the specific activity obtained is typically in the range of 100 to 1000 GBq·μmol-1 at EOS (normally within 40 min from EOB). In other words, 0.03 to 0.3% of the molecules in the purified labelled product 18
actually contain a 11C atom. Whereas the radioactivity is dependent on the radioactivity produced and the time, the amount of [11C]carbon monoxide remains rather stable at 20 nmol when a well-conditioned synthesis system is used. This gas volume can be represented by a 1.0-mm diameter bubble under normal pressure at r.t., thus the scale in radiochemistry is very small.
[11C]Carbon Monoxide and the Library Concept Compound libraries and combinatorial chemistry have since the early nineties been extensively used for acceleration of SAR analyses and lead optimization in drug discovery.32 Though a drug may serve as a PET-tracer lead, the criteria discussed above mean that often several analogues need to be synthesized and screened. Three-component carbonylations (i.e. substrate, CO and a nucleophile) have been used in large-scale syntheses of carbonyl compounds and transition-metal catalysts have proven very useful for these reactions.20 The micro-autoclave system described above has been used in the small-scale synthesis of an array of functional groups containing a 11Ccarbonyl group via transition-metal mediated carbonylation to form esters,33 carbamates,34 carbonates,35 imides,36 amides,37-40 hydrazides,38 ureas,34,35,41 ketones,42 aldehydes43 and carboxylic acids40,44 (Scheme 1). Substrates such as alkyl halides that contain -hydrogens are prone to undergo -hydride elimination to form a hydridometal-alkene complex in the presence of transition metals.45 -Elimination can be circumvented using photo-initiated radical carbonylation in a photoreactor.46 The broad range of labelling chemistry available using [11C]carbon monoxide makes it an ideal labelled precursor for synthesizing libraries of compounds in a combinatorial fashion (Figure 3). In a first selection, the candidates are tested in vitro. The most promising candidates are then further investigated in vivo. Examination of analogues of a lead compound make it possible in principle to fine-tune the imaging characteristics of a potential PET tracer and also to gain knowledge about which physiochemical properties are important for a tracer to fit its purpose.
19
Esters
Caroxylic acids O Aldehydes
R * OH
O
O R'
R * O
O R
R * H
Carbamates
N * O R'
O Ketones
O R
[11C]O
R * R' O R
Ureas
R''
O * O
R' Carbonates
O
O R''
N * N R' R'''
O
R'
R * N R'
O
Imides
R'' R * N R' Amides
R * N NH2 H Hydrazides
* = [11C]
11
Scheme 1. The use of [ C]carbon monoxide in labelling chemistry.
O
O R' X
Nu'
R'
R'' X
Nu''
R'
Nu'
R''
Nu''
R''
O
O Nu'
R'''
Nu''
R'''
O
O Nu'
R''''
O
Nu' O
Nu'' R''''
Nu''
transition-metal + [11C]O +
O
R''' X
Nu'''
R'''' X
Nu''''
R'
Nucleophiles
Nu'''
R''
Nu''''
R''
O R'
Organohalides
O
O Nu'''
R'''
O
O Nu''' R''''
O Nu'''' R'''
Nu''' O
Nu'''' R''''
Nu''''
Library of 11C-labelled compounds
Figure 3. [11C]Carbon monoxide can be used in a three-component combinatorial approach.
Purification, Analysis and Characterization Purification of products labelled with short-lived radionuclides is usually performed by semi-preparative UV-radio-HPLC and/or solid-phase extraction (SPE). The fraction containing the purified labelled product is often concentrated via SPE during a formulation process in order to change the solvent, e.g. from acetonitrile to the less toxic ethanol and aqueous buffer solution before biological delivery.
20
A radiochemical purity of >95% for biological examinations is desirable, and is obtainable in most cases. The acceptable amounts of a non-radioactive impurity depend on its properties. Such impurities may be solvent residues, starting materials, reagents, decomposition products, etc. For primate examinations, the product solution must also be sterile and free of pyrogens‡. An important factor sometimes overlooked is the chemical stability of the product over time, and here decomposition induced by radiolysis can be of significance.47 The small amount of product used in PET precludes NMR analysis. Instead, an aliquot of the purified or crude product is generally characterized by co-eluting with an isotopically unmodified sample using UV-radioHPLC. The concentration of the purified product is usually in the range 1-10 μM, and in most cases a UV-flow cell is sufficiently sensitive to determine the concentration, which can be used to determine the specific activity. In samples with very low UV absorbance, LC-MS/MS can be used. This technique is also used to identify labelled substances that lack an appropriate reference, and is especially useful for analogues when synthesizing libraries. The labelling position can be verified in a scale-up experiment by the co-labelling with both [11C]carbon monoxide and e.g. (13C)carbon monoxide. After purification, radio-decay and work-up, the (13C)-compound may be analyzed by NMR spectroscopy and compared to an isotopically unmodified sample as shown in Figure 4. The intensity of the carbonyl peak is enhanced in the 13Clabelled sample, indicating the labelling position. The amount of product is sometimes sufficient for 1H NMR as shown in Figure 5. The incorporated 13 C carbonyl causes 2JC,H and 3JC,H couplings as well as 2JC,C couplings. O C
13
(13C)carbonyl peak
N H
O N H
180
13
160
140
120
100
13
80
60
40
20
ppm
13
Figure 4. C NMR spectra of: Upper: C-labelled N-benzyl(carbonyl- C)acrylamide indicates the labelling position. Lower: Isotopically unmodified reference compound.
‡
Substances that induce fever, often endotoxins excreted from bacterial cell walls.
21
O C
13
N H
O N H
6.3
6.2
6.1
6.0
5.9
5.8
5.7
ppm
Figure 5. 1H NMR spectra of: Upper: 13C-labelled N-benzyl(carbonyl-13C)acrylamide with additional JC,H couplings. Lower: Isotopically unmodified reference compound.
Aim The work presented in this thesis aims to introduce a strategy that takes advantage of [11C]carbon monoxide as the labelled precursor in combinatorial radiochemistry for PET applications. The biological targets in this work are the tyrosine kinases EGFr and VEGFr-2, involved in cancer, and the angiotensin II receptors AT1 and AT2, involved in the renin angiotensin system. Specific aims: • To develop a labelling method for acrylamides using [11C]carbon monoxide and use it in a combinatorial approach to synthesize a library of EGFr tyrosine kinase inhibitors targeting either the active or inactive form of the kinase based upon a computational strategy • To perform the 11C-labelling of a library of dual VEGFr-2/PDGFr- tyrosine kinase inhibitors • To investigate the tolerance of different functional groups in the Rh(I)mediated 11C-labelling of unsymmetrical ureas and sulphonylureas • To perform the 11C-labelling of angiotensin II AT1 and AT2 ligands
22
Tyrosine Kinases as Targets for Molecular Imaging
A tyrosine kinase (or phosphokinase) is a protein enzyme that transfers a phosphate group from ATP and covalently attaches it to a tyrosine residue in the kinase itself (in autophosphorylation), or in another protein.48 This process activates the target protein for further downstream processes. Cells control many important processes like cell growth, movement and apoptosis in this way. Bioinformatic studies of the human genome have revealed over 500 expressed kinase genes,49 and finding a selective ligand is a big challenge. Disregulated kinase activity is a frequent cause of disease, particularly cancer, and the kinases were recognized as drug targets in the 1980s. Since the publication of the human genome,50 the term “personalized medicine” has frequently been used. The current classification system used in oncology is largely based on anatomical criteria (e.g. tumour shrinkage), but as new specific kinase inhibitors are becoming more available, it will be broadened to include individual expression patterns and the activity of certain biochemical processes, and in this development PET can be a valuable tool.
The ErbB Recptor Family The ErbB family of receptor kinases consists of four members; EGFr (ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). The epidermal growth factor receptor (EGFr) is a single transmembrane receptor that is intrinsically autoinhibited and is believed to exist as homo or heterodimers in the cell membrane. Signalling is triggered by binding of growth factors to the extracellular domain, which induces conformational change and phosphorylation.51,52 Heterodimerization occurs with the other members of the Her family, such as Her-2, Her-3 and Her4.53 Overexpression of the EGFr is associated with aggressive tumours and poor survival. In 2003 the EGFr inhibitor gefitinib (Iressa, ZD1839) was approved for the treatment of non-small cell lung cancer, but only a subpopulation (~10%) of the lung cancer patients treated responded to therapy. Lots of resources have been allocated to find a predictive method for selecting responsive patients. PET technology with a selective EGFr tracer could 23
be used to determine the receptor expression in tumour tissue in vivo as well as the accessibility of a drug and thereby identify potential responders to specific therapies.
Active and Inactive Conformations of EGFr The X-ray structure of erlotinib-EGFr complex (1M17.pdb) displays the protein in its active conformation,54 whereas lapatinib-EGFr complex (1XKK.pdb) shows the inactive conformation (Figure 6).55 The inactive conformations of kinases in general display significant diversity in and around the ATP-binding pocket. The inactive conformations are not subject to the chemical constraints that the active conformations must satisfy to bind ATP, so different kinases have evolved distinct off-states.56,57 This could be an advantage to achieve high selectivity.
HN O
O O
O
N N
Erlotinib–active EGFR complex in white O O S O
H N
HN O
F
Cl N
N
Lapatinib-inactive EGFR complex in grey
Figure 6. A shift in the C helix opens up a back pocket making room for an extra benzyl group.
A recent study where a number of kinase inhibitors were screened for 317 kinases showed that the irreversible EKB-569 and CI-1033, having reactive acrylamide functionalities had lower selectivity than the reversibly binding erlotinib and gefitinib, all targeting the active form. Lapatinib, targeting the inactive form of EGFr, had a remarkable selectivity. The extra benzyl group in lapatinib reaches deep into a hydrophobic pocket in the protein and allows it to bind to the inactive conformation, and thereby may gain higher selectivity over other kinases.58,59
Reversible Versus Irreversible Binding PET Tracers for EGFr The intracellular concentration of ATP can exceed 5 mM, particularly in tumour cells, whereas Km for ATP in most kinase active sites is in the mi24
cromolar range, thus ensuring full-time saturation by ATP. An ATPcompetitive inhibitor would need a Ki in the nanomolar range to compete and function as a drug in this circumstance.60,61 Several studies conclude that reversible EGFr PET tracers are rapidly washed out of tumour tissue without giving adequate target-to-noise ratios.62,63 Next-generation tracers were based on an irreversibly binding acrylamide functionality, and were more successful. However, their pharmacokinetic properties are not yet optimal.6466 A not-yet-reported alternative used for PET is to target the inactive conformation with an irreversibly binding ligand, a design used in the compound HKI-272 now in clinical trials.67
Docking Studies on the EGFr Computational efforts in predicting the activity of kinase inhibitors of the EGFr in its active conformation, like 3D-QSAR, docking, molecular dynamics etc has gained a lot of interest lately. In our study, both of the known conformations of the EGFr kinase domain were used; the active conformation complexed with erlotinib (1M17.pdb) and the inactive conformation complexed with lapatinib (1XKK.pdb) (Figure 6). The dataset used to explore the docking protocol in the software Glide consisted of 54 analogues to lapatinib with known IC50 values.68-70 The poses obtained when docking to the inactive EGFr structure resembled that of the co-crystallized ligand and there was no need to force the ligands into place by constraints. The hydrophobic back pocket is opened by a shift of the C helix when going from active to inactive conformation and, as expected, the software was unable to find appropriate poses for the lapatinib analogues in the active EGFr structure, suggesting that ligands with an extra hydrophobic group are specific for the inactive conformation, or possibly induce a conformational change. O
Solvent accessible area X
3
O
N
7
H
O
N Cys797
1
H N
F
H
N
N
H S O Asp800
Cl
HN
H N 6
O H
O H
Thr790 "Gatekeeper"
H O
N Thr854
Cys775
Met793
Figure 7. Schematic representation of a ligand docked into the active site of the inactive form of the EGFr kinase domain.
25
Many irreversible inhibitors that target the active form of the EGFr possess a linear acrylamide functionality.71,72 At the time of the investigation (2005) there were only three crystal structures available of the kinase domain of EGFr and no co-crystallized irreversible binding had yet been published.54,55 Therefore, we wanted to investigate (for the inactive conformation) the spatial distance between the -carbon in a number of hypothetical acrylamidederivatives and Cys797 to predict whether a covalent bonding was possible. The extra hydrophobic group that is required to target the inactive conformation will also increase lipophilicity, which may lead to non-specific binding. To compensate for the increased lipophilicity, hydrophilic and solubilizing functional groups were added at the -position of the acrylamide that aims at the solvent interface of the active site. The poses found in the docking had binding modes similar to lapatinib. The N1 hydrogen bound to the backbone of Met793 and N3 was bridged to Thr854 via a water molecule held in the active site. The substituent of the acrylamide faced the solvent interface and may therefore serve as a solubilizing group without affecting the binding mode. The distance from the -acrylamide carbon to the nucleophilic sulphur atom in Cys797 was found to be in the range 3.5-4.5 Å. Assuming a certain degree of rotational freedom in the molecule and the protein, this distance is close enough to promote C-S bond formation (~1.8 Å), possibly via the 5membered transition state depicted in Figure 7. Today, there are over thirty crystal structures available of EGFr kinase domain. Recently, a close analogue, HKI-272, which features a nitrile moiety in the 3 position instead of the bridging water molecule, was cocrystallized with the kinase domain of EGFr, confirming our docking results that revealed covalent binding to the inactive conformation.73
26
Carbonyl-11C-Acrylamide Labelling (Paper I)
Benzyl[carbonyl-11C]acrylamide from Acetylene The results from the computational docking studies gave us an incentive to develop methods for labelling acrylamides with high specific activity using [11C]carbon monoxide. The feasibility of using acetylene in palladiummediated aminocarbonylation was investigated first. Mixing Pd2(dba)3, PPh3, p-TsOH·H2O and a nucleophile such as benzylamine resulted in precipitation of the amine salt, which was a practical hindrance to continuing this one-pot reaction path. In the absence of the amine nucleophile, we noticed that [111 C]acrylic acid 1 was formed in approximately 60% analytical radiochemical yield (Scheme 2). The acid 1 could subsequently be converted to benzyl[carbonyl-11C]acrylamide 2 via the acyl chloride in 51 ± 4% (n = 2) analytical radiochemical yield based on [11C]carbon monoxide (Scheme 2). Unfortunately, a high concentration (400 mM) of the amine was needed to give satisfactory yields. Moreover, with time and automation in mind, a one-step synthesis it is always desirable.
+ [11C]O + H2O
PPh3 p-TsOH Pd2(dba)3 THF
O * OH 1
i) SOCl2 ii) Bn-NH2
O N * H
THF
* = [11C] 2
Scheme 2. N-Benzyl[carbonyl-11C]acrylamide via hydroxycarbonylation of acetylene.
There is no consensus on the mechanism of hydroxycarbonylation of acetylenes and different paths have been suggested: the “metalhydroxycarbonyl”,74,75 the “metal-hydride”76,77 and the “metallo-cyclic”78 mechanism.
[Carbonyl-11C]Acrylamides from Vinyl Iodide As the carbonylation of higher acetylenes often results in a mixture of linear and branched products,79,80 we explored the feasibility of using vinyl halides 27
as substrates for aminocarbonylation as outlined in Scheme 4.81 The configuration of the double bond formed from E and Z vinyl halides was also investigated. I or R Br + [11C]O + HN R'
PPh3 Pd2(dba)3
O R''
N R'
THF
R
or Br
2-14
Scheme 3. One-step synthesis of [carbonyl-11C]acrylamides from [11C]carbon monoxide.
The general catalytic cycle of the palladium-catalyzed carbonylation of organohalides is described in Scheme 4. Before oxidatively adding an organohalide, a coordinatively saturated palladium complex a [e.g. 18-electron Pd(PPh3)4] must be converted to an unsaturated complex by dissociation of ligands to form b [16-electron Pd(PPh3)3 and even 14-electron Pd(PPh3)2]. Irrespective of the exact mechanism, the oxidative addition of the organohalide to form RPdII(PPh3)2X c requires two of the four ligands to dissociate so that the palladium complex effectively acts as a 14-electron species.82 The next step is the coordination of carbon monoxide. It occurs via displacement of a ligand (L) by CO (d). Commonly the anionic organo ligand then migrates and attacks the electrophilic CO to form the acyl-Pd(II)LnX complex e. A concerted mechanism has been proposed for this step.20 The acyl ligand formed is not as good a donor, but overall the metal has replaced an electronwithdrawing CO ligand with a better donating phosphine, so the equilibrium usually lies towards the acyl-Pd(II)LnX-complex. The next step is the nucleophilic displacement in which the product f is formed. The mechanistic path is reaction-system dependent and several paths have been suggested. In one, reductive elimination forms the free acyl halide, which then reacts with the nucleophile. In another path the nucleophile may displace one of the ligands to provide the acyl-Pd-NuLn complex before reductive elimination occurs. In some reaction systems the nucleophile attacks the carbonyl of the coordinated acyl group directly.20 It should be noted that, in the labelling reactions, about 1-2 μmol of Pd complex was used, whereas the amount of [11C]carbon monoxide was about 25 nmol. As a consequence of this stoichiometry, the palladium complex is believed to participate in only one catalytic cycle; thus the labelling reaction can be described as metal-mediated rather than metal-catalyzed. Tetrakis(triphenylphosphane)palladium(0) [Pd(PPh3)4] was generated in situ from Pd2(dba)3 and triphenylphosphine in THF.83 When a vinyl halide
28
was added to the Pd(PPh3)4 solution, the solution changed colour from yellow to pale yellow. This suggests that a reaction, likely the oxidative addition, occurred. When using vinyl iodide the colour change occurred instantly at r.t. whereas the use of the bromopropenes required heating. Amine was subsequently added and the resulting solution was transferred to a microautoclave that had been pre-charged with [11C]carbon monoxide. The reagents were kept in the micro-autoclave for 4 min at 110 °C. For the model reaction using vinyl iodide and benzylamine (Figure 8, Product 2), temperatures 110 °C gave comparable yields (data not shown). This method allowed the amine concentration to be decreased from 400 mM (used in the acetylene method) to 20 mM. After purification using semipreparative HPLC, the products were characterized by co-eluting with an isotopically unmodified reference sample. PdL4
O R
f
Nu
a
+ R3NHX
R X
PdL4-n
b
R3N NuH
R
O L R Pd X L
L Pd X L
c
e
CO L
CO R Pd X L
L
Nu = RO-, OH- or R2N-
d Scheme 4. Generalized catalytic cycle of the palladium-catalyzed carbonylation of organohalides.
A range of different amine nucleophiles were used in the aminocarbonylation of vinyl iodide to form the acrylamides shown in Figure 8. The concentration of amine was 20 mM for all reactions except with the weakly deactivated and sterically hindered o-chloroaniline, which gave 6a in moderate yield using 95 mM amine. The outcome of the reactions correlated well with the nucleophilicity of the amines. The highest yield was achieved with benzylamine, whereas sterically hindered anilines such as 2,6-dimethylaniline gave lower yields. With sterically non-hindered nucleophiles the reaction proceeded even at r.t. (e.g. p-chloroaniline), albeit in lower yields. In the case of sterically hindered and strongly deactivated o-nitroaniline, the de29
sired product 7 was not formed even when the temperature and the concentration of the nucleophile were increased. O N * H x 2: 81 ± 3% (4)
H N *
H N *
H N * O 3: 74 ± 6% (2)
NO2
H N *
O
O
H N *
O
O Me 4a: ortho 71 ± 4% (2) 5a: ortho 65 ± 4% (2) 4b: para 72 ± 2% (2) 5b: para 72 ± 2% (2) 5c: orthodimethyl 52 ± 2% (2) O O * N N * OMe
Cl 6a: ortho 51 ± 1% (2)a 6b: meta 63 ± 1% (3) 6c: para 73 ± 1% (2)b O N *
10: 76 ± 1% (2)
7: 0%
8: 66 ± 4% (2)
9: 75 ± 1% (2)
O N N * H 11a: 2-amino 46 ± 2% (2) 11b: 3-amino 70 ± 1% (2)
* = [11C] x = (13C)
Figure 8. Isolated radiochemical yields of products obtained from aminocarbonylation of vinyl iodide. Numbers in parentheses are the number of experiments. Standard conditions were helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave pressurized to 35 MPa with a solution of amine (20 mM), vinyl iodide (10 mM), Pd2(dba)3 (2 mM) and PPh3 (17 mM) in THF, 4 min, 110 °C. a 95 mM amine and 145 °C were used. b 25 °C gave 49% rcy.
Configuration of the Carbon-Carbon Double Bond Many of the EGFr inhibitors proposed in the computational docking studies were substituted at the -carbon of the acrylamide moiety. We therefore wanted to determine whether reaction proceeded with retention of the C=C double bond configuration. Using the same reaction conditions as above, the aminocarbonylations of E- and Z-bromopropene were performed to give the products 12-14 (Figure 9). The configuration was preserved during the reaction. Benzylamine was used to give both E-12 and Z-12 in good yields. When a weak nucleophile such as m-chloroaniline was used, leading to Eand Z-14, the yields were significantly lower. To confirm the labelling position as well as the configuration around the double bond, larger-scale syntheses of 2, E-12 and Z-12 were performed using (13C)-carrier-addded [11C]carbon monoxide. Z-14 was synthesized using 12CO-carrier-added [11C]carbon monoxide. The products were subse30
quently analysed by NMR spectroscopy. The same amounts of palladium and triphenylphosphane used for the labelling experiments were used for the larger-scale syntheses, but the amounts of vinylhalide, amine and carbon monoxide were increased, thus the palladium complex is acting as an actual catalyst here because the amount product of formed exceeds the amount of catalyst. H x N *
H N *
H N *
O
O
O E-12 72%
E-13 67%
Cl E-14 41% H N *
H x N * O Z-12 67%
O Cl Z-14 16%
* = [11C] x = (13C)
Figure 9 Isolated radiochemical yields of products obtained from aminocarbonylation of Z- and E-bromopropene.
The specific activity of N-benzyl[carbonyl-11C]acrylamide 2 was assessed from 10-μAh cyclotron bombardments, resulting in 10 ± 0.5 GBq of [11C]carbon monoxide (n = 2). After 20 min, the product was isolated with a radioactivity of 4.2 ± 0.3 GBq. The total amount of product was 22 ± 1 nmol as determined by LC-MS/MS standard addition analysis; this corresponds to a specific activity of 190 ± 4 GBq·μmol-1.
31
Carbonyl-11C-Acrylamide Labelling of EGFr Inhibitors (Paper II)
A combinatorial approach has been utilized to synthesize a small library of potential tracers for the EGFr. The structures based on the 6-amino-4anilino-quinazoline scaffold and were designed to target either the active (20 and 21) or the inactive (22) form of EGFr. With an acrylamide moiety at the 6-position, these compounds are known to have inhibitory effects in isolated enzyme as well as in cell-based assays (Scheme 6).71 Previously, related structures labelled with either with 11C or 18F have shown uptake in tumour tissue. However, the imaging characteristics and kinetic properties of these compounds are not yet optimal. (See refs 84 and 85 for reviews). Using combinatorial labelling chemistry, a large number of labelled compounds may be synthesized using similar labelling conditions. The 6-amino-quinazolines 19a-c were synthesized starting from antralinic acid according to literature procedures. The vinyl iodide substrates 18a-c were synthesized starting from propargyl alcohol (Scheme 5). Treating propargyl alcohol with tri-n-butyltin hydride gave a mixture of the three possible addition products. The desired syn-addition product 15 was the major product when tri-n-butyltin hydride was used in excess.86 Iodination with molecular iodine was performed to form the vinyl iodide 16. The hydroxyl group was converted to a bromide using carbon tetrabromide and triphenylphosphane to give 17. a
HO
Sn
b
16 (86%)
15 (44%) d
c Br
I 17 (75%)
I
HO
HO
R'
I
18 (37-58 %)
Scheme 5. a) Bu3SnH, AIBN, 80 °C, 2 h. b) I2, dichloromethane, r.t., 2 h. c) CBr4, Ph3P, dichloromethane, 0 °C, 10 min. d) Secondary amine.
32
With access to the intermediate 17, it was possible to synthesize the vinyl iodide building blocks 18a-c in a parallel fashion by the reaction with different amine nucleophiles. These attached groups serve as solubilizers and point towards the solvent interface of the kinase ATP-binding site (Figure 7). As neighbouring groups, they may also affect the reactivity of the acrylamide Michael acceptor. This may play an important role for the metabolism of the tracer as well as its rate of covalent binding to Cys797. The pKa of the amines may affect their passage through the cell membrane to reach the binding site. R''' R''
HN R'
I + H2N
R'''
N
a
R'
H * N
R''
HN N
O
N
N * = [11C] 20 a-d R'' = Br R'''= H 21 a-d R'' = Cl R''' = F 22 a-d R'' = Cl R''' = OCH2(3-F-Ph)
19a-c
Scheme 6. a) [11C]O, Pd2(dba)3, PPh3 in THF, 110 °C, 5 min. Table 1. 11C-Labelled acrylamide derivatives targeting the EGFr. cLogD7.4a
Calc.a Isolated [11C]O conv. (%) pKa of R’ RCY (%) -H 4.2 84 ± 11 (4) 47 ± 15 20a -H 4.2 89 ± 7 (6) 48 ± 9 21a -H 5.8 95 ± 5 (4) 61 ± 13 22a -CH2piperidine 3.7 8.6 92 ± 1(3) 37 ± 2 20b 3.6 8.6 86 ± 4 (3) 31 ± 6 -CH2piperidine 21b -CH2piperidine 5.2 8.6 93 ± 3 (2) 25 ± 9 22b 3.8 6.3 89 ± 9 (2) 39 ± 0 -CH2morpholine 20c -CH2morpholine 3.8 6.3 94 ± 2 (3) 35 ± 0 21c 5.3 6.3 92 ± 0 (2) 36 ± 2 -CH2morpholine 22c -CH2thiomorpholine 4.0 7.6 90 31 20d 4.0 7.6 90 7 -CH2thiomorpholine 21d 5.6 7.6 90 32 -CH2thiomorpholine 22d Standard conditions were: helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave pressurized to 35 MPa with a solution of Pd2(dba)3 (2 mM), PPh3 (17 mM), vinyl iodide (10 mM) and amine (21 mM) in THF, 5 min, 110 °C. a Calculations were made using Chemaxon Marwin online software. Product
R’
Twelve inhibitors were 11C-labelled in the carbonyl position using the earlier-developed method using [11C]carbon monoxide. The products were isolated in 7 to 61% decay-corrected radiochemical yield calculated from [11C]carbon monoxide, usually within 30 min of EOB. Product 21a was obtained in 48% rcy with a specific activity of 60 GBq·mol-1 (EOS) as compared to 12% and 10 GBq·mol-1 (EOS) by a published method (Scheme 6).87 33
Synthesis of a Library of Potential Angiogenic Probes (Paper III)
Angiogenesis is the process by which new capillaries are formed by outgrowth from existing blood vessels. This is an essential part of many physiological processes, such as wound repair. On the other hand, angiogenesis is also associated with the progression of tumour growth and metastasis, as tumours cannot grow larger than a few millimetres without new blood vessels being formed to supply them with nutrients. A non-invasive method for monitoring angiogenesis-related events would be a valuable tool in the clinic as well as in research. The vascular endothelial growth factor receptor 2 (VEGFr-2/KDR/flk-1), plays a key role in angiogenic processes and overexpression of both the VEGFr-2 protein and mRNA has been found in tumour-associated endothelial cells, but not in the vasculature that surrounds normal tissues. This makes VEGFr-2 a possible biomarker for angiogenesis that could be targeted with PET.88,89 In a parallel study the 18F-labelling of the dual VEGFr-2/PDGFr inhibitor 25 was reported.90 Its urea moiety is suitable for 11C-labelling via the rhodium-mediated carbonylative coupling of the corresponding azide and aniline, which also opens up the possibility to synthesize a library of analogues.34 Both synthetic paths (A and B) to [11C]25 were explored (Scheme 7). Initially path A was followed using the conditions earlier reported for the labelling of N,N’-diphenyl[11C]urea, but the solubility of the amine nucleophile 24 in THF was limited to around 50 mM, as compared to the 350 mM used in the published method.34 This resulted in only 3% rcy. Varying the concentrations of the other reagents did not significantly affect the yield (0 to 7%). Instead, path B was tried and increased the yield from 7% to 27% using otherwise identical conditions. This effect is associated with the higher nucleophilicity and smaller steric bulk of the amine. The metal-coordination of azide 23 was insensitive to the greater steric hindrance of that component. The concentrations of rhodium complex and azide could both be decreased by a factor of approx. 20 as compared to the initial conditions tried. The concentration of nucleophile was found to be the most important parameter, and rather high concentrations (>180 mM) were needed in order to obtain high yields. The readily soluble 4-fluoroaniline could be used at a concentration of 371 mM, giving an analytical rcy over 90% in a very clean reaction. 34
F
NH2
O O O
N3
Path A
N
24 F
[11C]O, Rh complex, solvent, heat F O
H H N * N O
F
O F
N3
O
H2N
N [11C]25
O F
O
* = [11C] O
N 23
[11C]O,
Rh complex, solvent, heat
Path B
Scheme 7. Paths A and B to [11C]4. Table 2. Optimization of reaction conditions in the synthesis of [11C]25. Azide Anal. Ligand T [11C]O conv. 4-FRh(I)23 (%) RCY (mM) (°C) aniline complex (mM) (mM) (%) (mM) 1 37 371 0.39 dppe, 0.39 140 71 70 2 37 371 0.39 PPh3, 0.78 140 83± 0(2) 81±3 3 37 371 0.39 dppe, 0.78 110 79 67 84 71 4 37 371 0.39 PPh3, 0.78 110 5 19 186 0.19 dppe, 0.39 110 81 69 74±5(2) 61±8 6 37 74 0.39 PPh3, 0.78 110 46±5(2) 41±9 7 48 37 0.39 PPh3, 0.78 110 8 37 371 0.39 dppe, 0.78 80 84 77 80 95±1(2) 91±1 9 37 371 0.39 PPh3, 0.78 80 95±4(2) 90±6 10 19 371 0.19 PPh3, 0.39 80 92 87 11 19 186 0.19 PPh3, 0.39 12 9.4 371 0.10 PPh3, 0.20 80 90±1(2) 86±3 80 77±10(2) 71±10 13 4.7 371 0.05 PPh3, 0.10 9.4 74 0.19 PPh3, 0.39 80 96 32 14a Standard conditions were helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave pressurized to 35 MPa with a solution of [Rh(cod)Cl]2, phosphane ligand, and azide 23 in THF. a n-BuLi (0.9 eq relative to aniline) was added. Entry
35
Table 3. Potential angiogenic probes synthesized via Path B in Scheme 7. Product
Aniline substituent(s)
cLogD7.4a
IC50 (nM)b
PDGFr
VEGFr-2
Isolated RCY (%)
[11C]25 4-F 4.7 2-5 8-20 78 ± 1 (2) 11 [ C]26 2-F 4.1 2-5 4-10 38 2,4-diF 4.2 2-5 n.d. 48 [11C]27 4-Cl 5.2 n.d. n.d. 70 [11C]28 [11C]29 2-OMe 4.4 n.d. n.d. 77 11 4-OMe 4.4 n.d. n.d. 50 [ C]30 2-Me 4.4 n.d. n.d. 74 [11C]31 [11C]32 4-Me 5.1 n.d. n.d. 76 Standard conditions were helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave and a solution of [Rh(cod)Cl]2 (0.19 mM), PPh3 (0.39 mM), azide 23 (19 mM) and an aniline (371 mM) in THF, pressurized to 35 MPa. a Caclulated with Chemaxon Marwin online software. b Values from ref 91. VEGFr-2 phosphorylation assay, PAE/KDRcells incubated with inhibitor for 1 h. PDGFr phosphorylation assay, NIH/PDGFr cells, incubated with inhibitor for 1 h.
The conditions used in Table 2, Entry 10 were used to label a library of 7 analogues of [11C]25. The identities of the radiolabelled compounds were established by co-eluting (Table 3, Products 25 to 27) or LC-MS/MS (Table 3, Products 28 to 32). During the optimization of the labelling reaction, the amount of 11 [ C]carbon monoxide was estimated after each synthesis. It was observed that [11C]carbon dioxide is trapped on the zinc surface at r.t. and can be readily released at temperatures >330 °C. This correlates well with the observation of high isotopic dilution directly after system service, when the old zinc granules were replaced with fresh ones (stored in air at r.t.). Up to 150 nmol [11C]carbon monoxide was observed in the first experiment directly after a service, an amount that was gradually decreasing in the following experiments. Conditioning the zinc for 3 h at 400 °C under a flow of helium (20 mL·min-1) removed the absorbed carbon dioxide and gave normal levels of [11C]carbon monoxide around 20 nmol. In a well-conditioned system, starting with 10.7 GBq of [11C]carbon monoxide, [11C]25 was isolated with a specific activity of 92 ± 4 GBq·mol-1, 45 min from EOB.
36
Synthesis of Asymmetric [11C]Ureas and [11C]Sulphonylureas by Rh(I)-Mediated (Paper IV)
The urea is a common functional group in biologically active compounds, and labelling is commonly performed by the use of [11C]phosgene.92,93 The drawbacks of these methods include low reproducibility and low specific activity. To demonstrate the versatility of the Rh(I)-mediated carbonylative cross-coupling of an azide and an amine to form asymmetric [11C]ureas, we have explored its tolerance of a number of functional groups present in the nucleophile (Scheme 8). Path A 33a
Path B
N3 a
or
+ amine
R
H H N * N
I
a R'
amine + N3
O O S O N3
34-43
33b
33c
Scheme 8. a) [11C]O, [Rh(cod)Cl]2, PPh3, in THF, 80 °C, 5 min.
In general, when azide 33a was used as the substrate, high yields were obtained in very clean reactions using strong nucleophiles (Figure 10, Path A, Products 34, 39-41). Aromatic iodides are known to be good substrates for oxidative addition to transitions metal complexes. When 33b was used as the substrate (Scheme 8, Path B), two major products accounted for over 95% of the converted [11C]carbon monoxide, and the desired urea 36 was the minor product of the two. When instead substrate 33a and (Path A) was used leading to urea 36, only one major product was obtained. Thus, an aromatic iodide in the substrate is more problematic than an iodide in the nucleophile, despite that there is five times as much nucleophile present. A plausible explanation is that the azide 33a reacts with the metal-complex, possibly via formation of a nitrene or by coordiation,94 and thereby occupies the available sites and hinders oxidative addition of the 2-iodo aniline. Electron withdrawing groups and steric hindrance in the nucleophile results in lower yields (Figure 10, Products 36-38). The sulphonylazide 33c was used as a substrate 37
in the synthesis of the cytotoxic compound LY-181984 43, which was isolated in 66% yield (calculated from [11C]carbon monoxide).
O
HO
O
HOOC
34
O
O 38
[11C]O-conv. (%): 71 ± 7 (4) Anal. dc. rcy. (%): 24 ± 7 (4)
27 ± 1 (2) 14 ± 1 (2) NH
H H N * N
H N * N
H N * N
O
O
O
40 96 ± 1 (2) 96 ± 1 (2)
41 84 ± 0 (2) 62 ± 2 (2)
39 [11C]O-conv. (%): 84 ± 0 (2) Anal. dc. rcy. (%): 62 ± 2 (2)
O
Path A Path B 52 ± 3 (2) 76 ± 9 (2) 36 ± 0 (2) 26 ± 13 (2)
H H N * N
37
S
O 36
59 ±3 (2) 56 ±4 (2)
H H N * N O
I
35
[11C]O-conv. (%): 89 ± 1 (2) Anal. dc. rcy. (%): 88 ± 1 (2) NC
H H N * N
H H N * N
H H N * N
H H N * N O O
42 (Tolbutamide) [11C]O-conv. (%): 90 ± 3 (2) Anal. dc. rcy. (%): 83 ± 1 (2)
S O
H H N * N O O
* = [11C] Cl
43 (LY-181984) 93a 92b 79 78
Figure 10. Urea derivatives labelled with 11C at the carbonyl position. Standard conditions were helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave pressurized to 35 MPa with a solution of [Rh(cod)Cl]2 (0.40 mM), triphenylphosphane (0.80 mM), phenyl azide (20 mM) or p-toluene sulphonyl azide and an amine (100 mM) in THF at 80 °C for 5 min. aAzide (16 mM), amine (370 mM). bAzide (16 mM), amine (185 mM), [Rh(cod)Cl]2 (0.20 mM), triphenylphosphane (0.40 mM).
The readily available starting materials,95 generality and mild conditions of this method make it an interesting alternative to [11C]phosgene and other methods, especially for the synthesis of asymmetric [11C]ureas and [11C]sulphonylureas.
38
Targeting the Angiotensin II Receptors
The Renin-Angiotensin System The most active component of the renin-angiotensin system (RAS) is the octapeptide angiotensin II (Ang II). In the endocrine RAS, angiotensinogen is released from the liver and cleaved in the circulatory system by renin (released from the kidney) to form the decapeptide angiotensin (Ang I). Angiotensin-converting enzyme (ACE) then cleaves off two residues to form Ang II. Ang II acts mainly on AT1 and AT2 receptors (Scheme 9). Several biologically active truncated peptides, such as Ang III, Ang (1-7) and Ang IV, derived from angiotensin have been characterized separately from Ang II.96 Angiotensinogen 452 amino acids long
Renin Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
ACE Angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
AT1R Expressed in healthy adult human: Heart Kidney Adrenals Liver Brain Lung Endometrium Vasculature Skin Colon Prostate
Actions: Vasocontriction Salt/water retention Aldosterone release Proliferation Renal functions
Expressed in healthy adult human: Adrenals Uterus Ovary Vasculature Brain Heart Kidney Lung Myometrium Pancreas Retina Skin
AT2R Actions: Vasodilatation Antiproliferation Apoptosis Differentiation Regeneration
Scheme 9. Synthesis and actions of angiotensin II.
39
In addition to the circulating RAS, local RAS have been documented in a number of human tissues such as brain, adrenals, etc. These local systems are not isolated entities but can interact with the circulating RAS.97
The AT1 receptor (AT1R) The AT1R is a seven-transmembrane receptor with 395 amino acids and belongs to the GPCR superfamily. Upon binding angiotensin II, the receptor undergoes a conformational change that promotes its interaction with the Gprotein(s). The AT1 receptor is expressed in the heart, kidneys, adrenals, liver, brain, lung, endometrium, vasculature, skin, colon and prostate. The classical actions of AT1R are vasoconstriction, salt/water retention, aldosterone release, proliferation and renal functions.98 Overactivity of the RAS has been implicated in the development of cardiovascular diseases such as hypertension and renal insufficiency. Factors such as angiotensin, all-trans retinoic acid and oestrogen have been reported to down-regulate the AT1R, whereas up-regulation is mediated by low-density protein cholesterol, insulin, insulin-like growth factor, nitric oxide and EGF.99
The AT2 receptor (AT2R) The AT2R is about 30% homologous to AT1R. Its physiological role is not yet fully understood. Many of the effects it mediates oppose those of the AT1R, such as vasodilatation and anti-proliferation. The AT2R is widely expressed in foetal tissue and is rapidly down-regulated after birth. In healthy adults the AT2R is localized to the heart, kidney, adrenal, brain, lung, uterus, pancreas, ovary, myometrium, retina, and both the endothelial and vascular smooth muscle cells of the vasculature. AT2R down-regulation is induced by oestrogen, aldosterone, and basic fibroblast growth factor, while stimulating factors include insulin, insulin-like growth factor, interferon regulatory factors 1 and 2, interleukin-1 , PDGF, and glucocorticoids.99
The Ang II Receptors as Imaging Targets The regulation of the human AT1R and AT2R expression is not understood in great detail, but it has been shown that the expression pattern is altered in a range of settings, e.g. cancer and inflammation.100 Since the AT1R and AT2R often exert opposing biological actions, it has been hypothesized that both the receptor densities and the ratio between the two receptors is important. This makes both the Ang II receptors interesting as imaging targets in a multitracer concept.101 Up-regulation of AT1R has been reported in infarcted hearts and in a number of cancers such as breast, pancreas, kidney, squamous-cell carci40
noma, keratoacanthanoma, larynx, and lung.100 Benign prostatic hyperplasia has been associated with down-regulation of AT1R.102 Altered expression is reported in aldosterone-producing adenomas.103 The AT1 receptor has previously been examined in rats, dogs and baboons using PET.104 The AT2R is up-regulated in heart failure, cardiac fibrosis, stroke, renal disease, diabetes, atherosclerosis, and cutaneous wounds as well as in certain brain areas during Alzheimer’s disease.105-107
Strategy in Choice of Ligands Selective antagonists of the AT1R, i.e. the sartan compounds, are used clinically for lowering blood pressure. The labelling of a marketed drug has the advantage that the substance has already been subjected to extensive toxicology testing, and the tracer can therefore be brought faster into clinical trials. O O
HO
N O
O N
N OH
N
O
S
O S
S Eprosartan IC50 (nM) AT1 1.4-9.2 (antagonist) AT2 >10,000
N
N H
N O
HO
O
N O
L-159,686 IC50 (nM) AT1 >10,000 AT2 1.5 (antagonist)
44 Ki (nM) AT1 >10,000 AT2 3.0 (agonist) N
N O
N
O
O
N
OH H N N N N NH Candesartan Ki (nM) AT1 0.1 (antagonist) AT2 >10,000
O OH
O 45 Ki (nM) AT1 >10,000 AT2 17
O HO
N
N N
O
N
PD123319 IC50 (nM) AT1 >10,000 AT2 34 (antagonist)
Figure 11. Small-molecule antagonists and agonists of the AT1R and AT2R. The arrows indicate potential labelling positions with 11C.
41
Two approved selective AT1R antagonists from the sartan family, eprosartan and candesartan, were chosen for labelling on the basis of their different binding modes, metabolic stabilities, and synthetic routes (Figure 11). Eprosartan is classed as a competitive antagonist and competes individually with angiotensin II for receptor binding; it is also very metabolically stable.108 Candesartan, on the other hand, has a higher relative affinity than eprosartan for the receptor and displays insurmountable§ binding caused by a very slow dissociation rate.109-112 Such differences could be important for the ability of the molecules to function as PET tracers. Eprosartan could potentially be labelled in either of the two carboxyl positions. Candesartan could potentially be labelled in the tetrazoyl, ethyl or carboxyl position. Only a few classes of potent and selective small-molecule AT2R antagonists are known in literature; these include L-159,686 and PD123319 (Figure 11).113-115 L-159,686 could potentially be labelled in the carbonyl position, but this would require the availability of [11C]phosgene. PD123319 has slightly lower affinity but could potentially be labelled using readily available [11C]methyl iodide. Recently several potent, selective and drug-like AT2 receptor agonists (44) possessing two possible labelling sites, the amide and the sulphoncarbamate, have been reported (Figure 11).116-118 Three potent AT2 agonists were chosen for labelling in the amide position using [11C]carbon monoxide.
Synthesis of the AT1R Antagonist [Carboxyl11 C]Eprosartan (Paper V) Precursor synthesis The synthesis of the iodide precursor of the AT1R antagonist eprosartan was performed as outlined in Scheme 10 and 11.119 The sequence of a Knoevenagel reaction, reduction and ester hydrolysis gave the ester/carboxylic acid 47 in 69% overall yield starting from 2-thiophenecarboxaldehyde. The aldehyde 50 was prepared from 2-butyl-4(5)-(hydroxymethyl)-imidazole in 4 steps in 57% yield. The position of the 4-iodobenzyl group in alcohol 49 was confirmed by NOE NMR spectroscopy by selective irradiation of the CH2 groups. A Knoevenagel-like condensation between the acid 47 and the aldehyde 50 and subsequent elimination of CO2 formed the intermediate ester 51.120 The acid 47 was added in excess due to a competing decarboxylation of the acid (Scheme 12). Using refluxing toluene as a solvent resulted in a tarry mixture and only 3% yield. When the reaction was instead performed
§
Insurmountable antagonism means that an agonist cannot reach its maximal effect in the presence of the antagonist, irrespective of the agonist concentration.
42
using a solvent mixture with a lower boiling point (cyclohexane with a small amount of toluene), the product 51 was obtained in 73% yield. O O
O O
O S
a
S
O
O
b
O O
O S
OH
O S
c
H 45 (87%)
O
46 (89%)
47 (89%)
Scheme 10. a) Diethyl malonate, piperidine, benzoic acid in cyclohexane, reflux 20 h. b) NaBH4 in EtOH, 40 min. c) KOH 1.0 eq in EtOH, r.t., 48 h.
I H N
OH
N
a
N
b, c
O
N
N
O
O 48 (90%) I
OH
N 49 (68%) I
O
d
N
e H
N
O N O N
50 (93%)
S 51 (73%)
I f
O N OH N
S
52 (81%)
Scheme 11. a) Acetic anhydride. b) (4-Iodo-phenyl)methanol, triflic anhydride, DIPEA in DCM. c) K2CO3 in MeOH. d) Activated MnO2 in DCM. e) Piperidine and 47 in cyclohexane and toluene. f) NaOH, EtOH.
The E geometry of the double bond was confirmed by measuring the 3JH-C coupling constants of the vinyl proton by means of an HMBC experiment. The cis coupling to the carbonyl carbon was 7.4 Hz and the trans coupling to the CH2 carbon was 11.2 Hz. The precursor 52 was finally obtained by basic hydrolysis of the ester.
43
O
H
O
O
Δ S
O O
O
O
O
H
- CO2
S
S
53
29
Scheme 12. Decarboxylation side-reaction.
Labelling reaction The 11C-carboxylation of 52 was performed according to a reported method, using a palladium-mediated carboxylation of the aryl iodide with [11C]carbon monoxide in the presence of a quaternary ammonium hydroxide in a micro-autoclave (Scheme 13).44 O I
HO * O a
N OH N
S
O N OH N
S * = [11C]
52
[Carboxyl-11C]Eprosartan 54
Scheme 13. a) [11C]Carbon monoxide, Pd(PPh3)4, tetra-n-butylammonium hydroxide 30 hydrate, THF, 150 ºC, 5 min. Additives were H2O and DMSO.
Different temperatures were tested using constant reagent concentrations. Temperatures below 120 °C gave high conversion of [11C]carbon monoxide, but this was 61-77% in [11C]carbonate and only 11-31% radiochemical yield of [carboxyl-11C]eprosartan 54 (Figure 12). The side-reaction that trapped the radioactivity as [11C]carbonate probably occurs via the water-gas shift reaction (Scheme 14) and subsequent reaction of [11C]carbon dioxide with OH-. [11C]O + H2O
[11C]O2 + H2
Scheme 14. The water-gas shift reaction.
44
(%) of initial [11C]carbon monoxide
100
80
60
40
20
0 65
90
110
120 130 140 Tem perature ( oC)
150
160
180
Figure 12. = [11C]O Conversion. = [11C]Carbonate. = [Carboxyl11 C]Eprosartan 54. = 11C-labelled lipophilic compounds. Reaction conditions were 11 [ C]carbon monoxide (0.05 to 0.5 mM), and 200 L solution of 52 (17 mM), Pd(0) (5 mM), n-butyl ammonium hydroxide (25 mM), H2O (2.5 M), DMSO (0.7 M) in THF.
When the amounts of base, water and DMSO were decreased and the temperature was raised from r.t. to around 140 °C over 5 min, the reaction was cleaner and formed less [11C]carbonate (Table 4, Entry 1). However, 51% conversion of [11C]carbon monoxide was not satisfactory, so the amount of Pd complex was increased in order to favour the insertion that forms the Pd[11C]acyl complex. This gave 90% [11C]O conversion in a very clean reaction that produced radiochemically pure product (Table 4, Entry 2). The presence of DMSO as a co-solvent had no effect on the radiochemical yields but helped to prevent precipitation while diluting the nitrogen-purged crude product for HPLC injection (Table 4, Entries 3 and 4). When base, water and DMSO were omitted, the reaction resulted in an unidentified compound (47%) along with the product (33%) (Table 4, Entry 5). [Carboxyl-11C]Eprosartan was purified using semipreparative HPLC and formulated in a phosphate buffer solution with pH = 7.2. The decaycorrected radiochemical yield was 37-54% (n = 5), within 35 min from end of bombardment. The specific activities up to 360 GBq·mol-1 were achieved. The labelled product was characterized with LC-MS and by coeluting with a sample of isotopically unmodified eprosartan. Eprosartan has been reported to undergo photolytic cis/trans isomerization to yield the Z isomer.108 Radiation can initiate decomposition via direct or indirect radiolysis that gives rise to reactive radical and ionic species. In general, the risk of radiolysis increases with specific activity and concentra45
tion.47 [Carboxyl-11C]Eprosartan 54 was analysed up to 90 min after formulation and showed no signs of radiolabelled decomposition products. Table 4. Synthesis of [carboxyl-11C]eprosartan 54. [11C]O Anal. conv. RCY (%) (%) 1 5 25 0.75 25 to 140 51±6(2) 41±2(2) 2 14 6.8 0.90 0.35 25 to 140 91 91 3 14 6.8 0.20 0.35 25 to 140 86 82 4 14 6.8 0.20 25 to 140 89 79 5 14 25 to 140 81 33 Standard conditions were helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave pressurized to 35 MPa with a solution of Pd(PPh3)4, tetra-nbutylammonium hydroxide (Q-OH), H2O and DMSO. Entry
Pd(0) (mM)
Q-OH (mM)
H2O (M)
DMSO (M)
T (°C)
Synthesis of the Aryl Iodide Precursor of [Carboxyl11 C]Candesartan (Appendix I) Synthesis of the aryl iodide precursor 61a of [carboxyl-11C]candesartan was carried out as outlined in Scheme 15. A synthetic-strategy question was where in the sequence to introduce the biphenyl moiety in order to give sufficient yield of the desired regioisomer. Rasmussen et al have shown that both the substituents at the benzoimidazole and the properties of the leaving group on the electrophile influence the ratio.121-123 N-Alkylation of the benzoimidazole was attempted at different stages in the synthetic route; Nalkylation of the 7-nitro compound 57 gave a 2:1 ratio (1H NMR) of the 4nitro:7-nitro products, but the following diazotization became problematic. When instead the diazotization was performed on the aniline 58, the resulting tetrafluoroborate salt was simply filtered off and was stable for several months at 4 °C. The ethoxy group was unstable under aqueous acidic conditions, so diazotization was performed using nitrosonium tetrafluoroborate in DCM. To favour iodination over elimination in the next step, a solvent mixture of methyl iodide and DMF was used.124 Alkylation of the 7-iodo compound 59 gave a less favourable 5:1 mixture of the regioisomers. Sodium hydride and potassium carbonate were tried as bases, and both gave the same product ratio. Tetrazole formation using trimethyltin azide was not compatible with the aromatic iodide and resulted in iodide elimination, so the biphenyl tetrazole had to be synthesized separately and subsequently protected and brominated and combined with 59. Finally, deprotection using dilute HCl produced the desired precursor 61b. However, the overall yield was less than 0.25% over 8 steps and insufficient to optimize 11C-labelling and subsequent biological 46
evaluation. To continue with radiolabelling of this precursor, a more efficient synthetic route is desirable.
NO2
O
NH2
a
N
b
O
NH2
NH2
NH2
NO2
NO2
NO2
55 (65%) N
c
d
O N H
NO2
56 (97%) N
NH2
O
N H I
58 (89%)
57 (80%)
7
1
N
5
2
N
5 6
6
3 O
N 2 7 1 I
+ N N N CH(Ph)3 N
O
N I
4
N H 59 (9%)
4
N CH(Ph)3 N N N
g
N
e, f
O
3
60a 4-Iodo (53%)
60b 7-Iodo (16%)
h
h N NH N N
N O N I
N O N I
61a 4-Iodo (72%)
N N NH N
61b 7-Iodo (41%)
Scheme 15. a) SnCl2·2H2O, HCl/EtOH. b) (EtO)4C, AcOH in THF. c) AcOH, M.W. heating at 140 °C. d) H2 (1 atm), Pd/C in THF. e) NOBF4 in DCM f) Me3SiI in MeI and DMF. g) NaH, 5-(4'-bromomethyl-biphenyl-2-yl)-1-trityl-1H-tetrazole in DMF. h) HCl in MeOH, 0 °C.
47
11
C-Labelling of a Series of Angiotensin II AT2 Agonists (Paper VII)
The aryl iodide precursor 63a, Pd(PPh3)4 and diethylamine were used in the optimization of reaction conditions. The parameters that were varied were the ratios between aryl iodide and palladium complex, the amount of amine and the reaction temperature (Table 5, Entries 1 to 10). The best conditions were a 1:1 ratio of aryl halide:palladium complex, temperatures between 90 and 120 °C and large amount of amine; these gave comparable yields from 38 to 44% (Table 5, Entries 6-8). I
Nu
O *
O S
O S N R H
62a R = H 63a R = COOnBu
O
a
S
b
O S
N R H
* = [11C]
62b NuH = Diethylamine, R = H 63b NuH = Diethylamine, R = COOnBu 63c NuH = Isopropylamine, R = COOnBu 63d NuH = Benzylamine, R = COOnBu
Scheme 16. a) [11C]O, Pd(PPh3)4, NuH, THF, , 5 min. b) Base, n-butyl chloroformate.
In an attempt to avoid the formation of an unidentified, more polar byproduct, we briefly explored the possibility of a two-step route to 63b starting from the aryl iodide 62a (Scheme 16). In contrast to reactions using 63a as substrate, no significant increase in radiochemical yield was observed when 62a was used with a large amount of amine (Table 5, Entries 11 and 12). In both cases, most of the radioactivity was bound to very lipophilic compounds, possibly products arising from the sulphonamide itself acting as the nucleophile. All conditions tested gave poor yields (2 to 10%, Table 5 , Entries 11, 12 and 13) of the intermediate 62b, so the two-step route was abandoned. The reaction conditions used in Table 5, Entry 6 were used to synthesize the isopropylamide and benzylamide analogues labelled with 11C (Table 6).
48
Table 5. Synthesis of [11C]63b and [11C]62b labelled at the carbonyl position via Pd(0)-mediated aminocarbonylation. Entry
Product
1 2 3 4 5 6 7
[11C]63b [11C]63b [11C]63b [11C]63b [11C]63b [11C]63b [11C]63b
8 9 10
[11C]63b [11C]63b [11C]63b
Ar-I (mM)
Pd(0) (mM)
Amine (mM)
63a 14 14 9 9 6.9 9 9
12 5 9 9 9 9 9
100 100 1000 500 500 500 500
T (°C)
[11C]Oconv. (%)
Anal. RCY (%)
25-140 130 120 120 110 110 100
91 87 93 93 89 92±1(2) 93±1(2)
3 9 34 34 16 44±1(2) 38±1(2)
9 9 500 90 95 44 9 9 500 80 91 35 18 9 43 70 93 10 62a 18 9 43 90 89 6 11 [11C]62b 9 9 500 110 91 10 12 [11C]62b 4.5 9 500 110 78 2 13 [11C]62b Standard conditions were helium-carrier-added [11C]carbon monoxide (0.05 to 0.5 mM) in a 200-L autoclave pressurized to 35 MPa with a solution of aryl halide, Pd(PPh3)4, and diethylamine in THF for 5 min.
Table 6. Synthesis of a small library of [11C-carbonyl]-AT2R ligands via Pdmediated aminocarbonylation. Product
Amine (mM)
[11C]63b
cLogP a
cLogD7.4a
AT2
AT1
Anal. RCY (%)
Ki b (nM)
Isol. RCY (%)
5.94
5.00
3.0 ± 0.3
>10 000
44 ± 1 (2)
16
5.77
4.84
2.6 ± 0.2
>10 000
49
29
6.73
5.79
1.0 ± 0.08
>10 000
58
36
N
[11C]63c
[11C]63d
H N
H N
Reaction conditions were as for Table 5, entry 6. a Calculated using Marwin online software by Chemaxon. b Data from reference 117. Binding assay relying on displacement of [125I]Ang II from pig uterus myometrium (AT2) and rat liver membranes (AT1).
49
Biological Evaluation In vitro autoradiography of frozen organ sections from rat, [carboxyl11 C]eprosartan 54 showed specific binding in lung, adrenal and kidney and to some degree also in spleen and liver (left panel Figure 13). In vivo organ distribution of [carboxyl-11C]eprosartan in rat showed high accumulation in kidney, liver and intestinal wall. Quantitative analysis of binding in pig adrenal followed by saturation analysis revealed that the binding was saturable, with a Kd-value of approx. 10 nM and the binding was most concentrated to the outer layer of the cortex. Autoradiography of a limited number of human adrenals showed an increased uptake in aldosterone producing adenomas compared to controls, which encourages further investigation of this tracer (Paper V). [Carboxyl-11C]Eprosartan 54 (AT1-selective) Total binding Rat Heart
[11C]63c (AT2-selective) Total binding
Nonspecific binding
Nonspecific binding
Rat heart
Rat Lung Rat pancreas Rat Liver Rat liver Rat Spleen
Pig Adrenal
Pig adrenal
Rat Kidney
Rat kidney
Rat Striatum Rat brain Rat Cerebellum
Figure 13. Autoradiography of selected tissue sections (25 m). The non-specific binding was affected by simultaneous incubation with unlabelled eprosartan (10 M) and 63c (3.5 M), respectively.
Autoradiography of the AT2 selective ligand [11C]63c revealed specific binding in pancreas and kidney cortex of rat, and to some degree in pig adrenals and rat brain (Figure 13, right panel). In vivo organ distribution in rat at 40 min post injection showed very high accumulation of radioactivity in the liver. Substantial radioactivity was also found in the intestine, kidney and adrenal. All other organs measured had SUVs lower than blood.
50
Head
Liver
Kidneys
Tail
Urinary Bladder
Figure 14. Small animal PET scan of a female rat injected with 12.2 MBq of [11C]63c. The whole body view shows accumulation of radioactivity in liver, urinary bladder and kidneys.
A series of dynamic animal PET scans showed rapid elimination of radioactivity from the kidneys and high accumulation and retention in the liver and urinary bladder (Figure 14). The rapid urinary excretion and the high accumulation in the liver limit the information that can be gained about AT2receptors in vivo with this tracer. Therefore, more metabolically stable compounds are desirable for future development.
51
Conclusions
Based upon this work, we concluded that: • It is likely that lapatinib analogues possessing a -substituted acrylamide functionality in the 6 position can bind irreversibly to the inactive form of the EGFr kinase domain. This was concluded by computational docking studies. • A range of acrylamides that are 11C-labelled at the carbonyl position can be synthesized from acetylene as well as from vinyl halides using a palladium(0)-mediated carbonylation. The vinyl halide method is superior and the conformation of the carbon-carbon double bond is preserved in the reaction. • Inhibitors designed to bind irreversibly to the EGFr kinase domain can be labelled at the 11C-carbonyl position in useful yields using the acrylamide method. This can be performed in a facile manner using a combinatorial approach to synthesize libraries of labelled analogues. • A library of dual VEGFr-2/PDGFr inhibitors as potential PET tracers for angiogenesis could be 11C-labelled at the urea position in useful yields using a rhodium(I)-mediated carbonylation. The more nucleophilic of the two nitrogens forming the urea should be used as nucleophile in the reaction to obtain high yields. • The rhodium(I)-mediated 11C-urea labelling method is compatible with a range of common functional groups and is therefore a useful alternative to the [11C]phosgene method. • The AT1 tracer [carboxyl-11C]eprosartan could be synthesized in useful yields with high specific activity in a one-step reaction from [11C]carbon monoxide. The tracer shows low but saturable binding to pig adrenals and higher uptake in human adrenocortical adenomas than in controls in autoradiography. This makes this tracer interesting for further evaluation. • A small series of AT2-ligands could be 11C-labelled at the amide position. One ligand showed specific binding in some rat organs using autoradiography, and very fast metabolism was shown by small animal PET. For future development, ligands that are more metabolically stable are desired.
52
Populärvetenskaplig sammanfattning på svenska
Denna avhandling handlar om ämnet radiokemi och dess tillämpningar inom molekylär avbildning. Den kortlivade radioaktiva isotopen 11C kan framställas i relativt små men dock praktiskt tillräckliga mängder i en partikelaccelerator, en sk cyklotron, där en protonstråle bombarderar en behållare med kvävgas. De bildade 11C-atomerna sönderfaller till 11B och samtidigt bildas en positron. Positronen är elektronens antipartikel och när dessa två kolliderar bildas det två fotoner som rör sig i motsatt riktning. Dessa fotoner kan fångas upp av en ring av detektorer och denna princip används i positronemissions-tomografi (PET) och är en mycket användbar metod för att avbilda och studera biologiska förlopp i levande vävnad. PET används inom bl a cancerdiagnostik, forskning inom Alzheimer, samt inom testning och utveckling av nya läkemedel. För att kunna göra detta behöver 11C-atomen inkorporeras med kemiska metoder i en för ändamålet intressant molekyl, dvs kemisk syntes av en 11C-märkt molekyl. Denna avhandling fokuserar speciellt på användandet av det radioaktiva startmaterialet [11C]kolmonoxid för att syntetisera bibliotek av närbesläktade 11C-märkta substanser för att senare kunna jämföra vilken påverkan skillnaderna i molekylerna har för hur de kan fungera som spårmolekyler. I synteserna används olika typer av övergångsmetall-medierad karbonylering med [11C]kolmonoxid. Användandet av [11C]kolmonoxid har fördelar mot tidigare kända metoder som istället använder [11C]koldioxid. Då halten kolmonoxid är mycket lägre än koldioxid i vår omgivning kan man undvika isotoputspädning och därmed uppnå högre specifik aktivitet som är viktig för att inte oavsiktligt störa det biologiska system man vill studera. Att blockera utvalda enzymer kallade receptorbundna tyrosinkinaser är ett relativt nytt sätt att angripa cancer. Här beskrivs designen, metodutvecklingen och syntesen av ett bibliotek av spårmolekyler som är tänkta att binda irreversibelt till kinasdelen av receptorn EGFr. Den irreversibla bindningen är tänkt att bildas mellan en reaktiv funktionell grupp i spårmolekylen kallad akrylamid och aminosyran cystein i bindningsfickan i kinaset som annars binder ATP. EGFr överuttrycks in vissa cancerformer och är associerad med aggressiva tumörer och dålig prognos, och att kunna identifiera och mängdbestämma uttrycket av denna receptor med hjälp av PET i ett tidigt stadium skulle vara av stort diagnostiskt värde. På liknande vis har molekylbibliotek 53
syntetiserats som har för avsikt att avbilda receptorn VEGFr-2 som är inblandad i blodförsörjningen av tumörer, sk angiogenes. Nästa del av avhandlingen handlar om syntes och biologisk utvärdering av 11C-märkta molekyler som binder till angiotensin II receptorer. Angiotensin II är en peptid som är involverad i regleringen av blodtrycket och regleringen av uttrycket av dess receptorer AT1 och AT2 tros vara störd i många sjukdomstillstånd inom det kardiovaskulära området. För AT1 receptorn märktes läkemedlet eprosartan med 11C i karboxylposition i en palladium-medierad hydroxykarbonylering med [11C]kolmonoxid. Dess förmåga att avbilda AT1 receptorer i frysta organsnitt studerades med autoradiografi och dess distribution i olika organ studerades i råtta. [Carboxyl-11C]Eprosartan visade ett relativt högt upptag i binjurebarken från patologiska prover från adrenokortikala adenom jämfört med frisk binjurebark, men även ett högt upptag i tarmar, njure och lever. Då dessa organ ligger relativt nära binjurarna är det inte optimalt ur avbildningssynpunkt. För AT2 receptorn 11C-märktes ett litet bibliotek av nyligen publicerade molekyler som binder effektivt till AT2-receptorn. Autoradiografi på en av molekylerna visade specifik bindning till bukspottkörteln och njurbarken. PET på råtta visade att spårmolekylen väldigt snabbt eliminerades via njurarna till urinen medan en viss del fastnade i levern. Ett högt upptag i levern bekräftades även genom organdistribution. För att bättre kunna avbilda AT2receptorer med hjälp av PET i framtiden är det önskvärt att mer metaboliskt stabila molekyler används.
54
Acknowledgements
First of all I would like to thank my supervisor Prof. Bengt Långström for accepting me as a Ph.D-student and for your never-ending enthusiasm for research and radiochemistry and for keeping up the good spirit. Assoc. Prof. Tor Kihlberg, my co-supervisor, for introducing me to the [11C]O-system in the HL, the CO2-system in our environment and strategic discussions regarding both chemistry and technical gizmos as well as commenting of this thesis. Dr. Torben Rasmussen, my co-supervisor, for the many hours of computational discussions and helping me with the docking studies. Dr. Jonas Eriksson, for extraordinarily good collaboration with the acrylamide project, and for sharing with me the inner secrets of the [11C]Osystem, as well as other gizmos in the hotlab. Prof. Håkan Hall, for all your efforts in the biological evaluation of my tracers. Ohad Ilovich and Prof. Eyal Mishani, for good collaboration in the angiogenesis project. Past and present members of the BLå-group, especially Elisabeth Blom and Maria Erlandsson for all good moments in the lab, Dr. Oleksiy Itsenko for sharing your deep philosophical thoughts and commenting on this thesis, Dr. Julien Barletta for collaboration with 11C-methylation, Dr. Obaidur Rahman for taking care of me the first time when I was a new Ph. D. student, Dr. Farhad Karimi for chemistry related discussions, Sofia Karlsson for being a nice fumehood neighbour during your exam work. I would like to thank all people at Uppsala Imanet, GE Healthcare, and Uppsala ASL, for always being friendly and creating a nice atmosphere in the lab as well as in the lunchroom. Especially Dr. Azita Monazzam and My Quach for collaboration in the AT2-project, Dr. Örjan Lindhe and Elisabeth Bergström-Pettermann for collaboration with the AT1-project, Madeleine Svennebäck for ordering of chemicals, Eva-Lotta Nyberg for taking care of HL booking, Dr. Pernilla Johansen and Dr. Niklas Tyrefors for LC-MS/MS related issues, Associate Prof. Alf Thibblin for HPLC related issues, Joachim Schultz for keeping the cyclotron running, Johan Ulin and Sven-Åke Gustavsson for a little bit of everything. All people at the department of Biochemistry and Organic chemistry at Uppsala University. Especially Assoc. Prof. Per Arvidsson for discussions regarding tetrazole chemistry, Prof. Adolf Gogoll for NMR-related ques55
tions, Dr. Tamara Church for linguistic corrections and comments on manuscripts and this thesis, the Midnattsloppet gang for nice after-work exercise, Claes-Henrik Andersson for giving me insight to the nanotube world, Julius Tibbelin for late evening Si discussions and Henrik Johansson for Se chemistry and all bad and sometimes good jokes ;) Eva Pylvänen, Gunnar Svensson, and Tomas Kronberg for keeping the department running. Dr. Stina Syvänen and Dr. Karl Andersson for your efforts in the biological evaluation of EGFR tracers. Prof. Mats Larhed, Dr. Charlotta Wallinder, Jonas Lindh and Marc Stevens at the Department of Medicinal Chemistry Organic Pharmaceutical Chemistry at Uppsala University for good collaboration in the AT2-project. The members of FBAB for all good musical moments during the years. I miss playing and performing a lot. All my friends from the time when we studied chemistry at Mälardalen University in Eskilstuna, Kemi-98, and -99, especially Göran Wennerberg, Magnus Janard, Robert Steen, Dr. Olle Rengby, Stefan Röstlund, Fredrik Nordborg, Henrik Friberg and Calle Carlsson. My parents Inez Karlsson and Bengt Åberg for all your endless support over the years and my sister Åsa Åberg for forcing me to think about other things than chemistry sometimes. Last but not least the love of my life Emelie Wistrand for all your support, patience and understanding. Life is more fun when you are around! Also our cat Tigris for being furry and keeping me company when writing this thesis. And of course, all of you that I unfortunately have forgot to mention above.
56
References
1. 2. 3. 4. 5.
6. 7.
8.
9. 10.
11. 12. 13.
Valk, P.E., et al., Positron Emmision Tomography, Basic Science and Clinical Practice. 2003: Springer-Verlag. Judenhofer, M.S., et al., Simultaneous PET-MRI: a new approach for functional and morphological imaging. Nat. Med., 2008. 14(4): 459-465. Pichler, B.J., H.F. Wehrl, and M.S. Judenhofer, Latest advances in molecular imaging instrumentation. J. Nucl. Med., 2008. 49: 5S-23S. Krogsgaard-Larsen, P., T. Liljefors, and U. Madsen, Textbook of Drug Design and Discovery. 3rd ed. 2002: Crc Press Llc. Huber, W., A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions. J. Mol. Recognit., 2005. 18(4): 273281. Regan, J., et al., The kinetics of binding to p38 MAP kinase by analogues of BIRB 796. Bioorg. Med. Chem. Lett., 2003. 13(18): 31013104. Laruelle, M., M. Slifstein, and Y. Huang, Relationships between radiotracer properties and image quality in molecular imaging of the brain with positron emission tomography. Molecular Imaging and Biology, 2003. 5(6): 363-375. Wong, D.F. and M.G. Pomper, Predicting the success of a radiopharmaceutical for in vivo imaging of central nervous system neuroreceptor systems. Molecular Imaging and Biology, 2003. 5(6): 350-362. Patel, S. and R. Gibson, In vivo site-directed radiotracers: a minireview. Nucl. Med. Biol., 2008. 35(8): 805-815. Pauwels, E.K.J., et al., Positron-emission tomography with [18F]fluorodeoxyglucose Part I. Biochemical uptake mechanism and its implication for clinical studies. J. Cancer Res. Clin. Oncol., 2000. 126(10): 549-559. Waterhouse, R.N., Determination of lipophilicity and its use as a predictor of blood-brain barrier penetration of molecular imaging agents. Molecular Imaging and Biology, 2003. 5(6): 376-389. Casey, D.R., et al., Formulation affects the rate of membrane degradation catalyzed by cationic amphiphilic drugs. Industrial & Engineering Chemistry Research, 2008. 47(3): 650-655. Syvänen, S., Blood-Brain Barrier Transport : Investigation of Active Efflux using Positron Emission Tomography and Modelling Studies,
57
14. 15. 16. 17.
18.
19. 20. 21.
22. 23. 24. 25. 26.
27. 28.
58
in Faculty of Pharmacy, Department of Pharmaceutical Biosciences. 2008, Uppsala University, Sweden: Uppsala. Bergström, M., et al., In vivo demonstration of enzyme activity in endocrine pancreatic tumors: Decarboxylation of carbon-11-DOPA to carbon-11-dopamine. J. Nucl. Med., 1996. 37(1): 32-37. Gambhir, S.S., et al., A tabulated summary of the FDG PET literature. J. Nucl. Med., 2001. 42(5): 1S-93S. Ache, H.J. and A.P. Wolf, Effect of Radiation on Reactions of Recoil Carbon-11 in Nitrogen-Oxygen System. J. Phys. Chem., 1968. 72(6): 1988-&. Zeisler, S.K., et al., Conversion of no-carrier-added [11C]carbon dioxide to [11C]carbon monoxide on molybdenum for the synthesis of 11C-labelled aromatic ketones. Appl. Radiat. Isot., 1997. 48(8): 1091-1095. Al-Qahtani, M.H. and V.W. Pike, Rapid mild syntheses of [11C]benzophenones by Pd(0)-catalysed 11C-carbonylative coupling of iodoarenes with phenyltributylstannane in DME-water. J. Labelled Compd. Radiopharm., 2000. 43(8): 825-835. Tobias, C.A., et al., The Elimination of Carbon Monoxide from the Human Body with Reference to the Possible Conversion of Co to Co2. Am. J. Physiol., 1945. 145(2): 253-263. Colquhoun, H.M., D.J. Thompson, and M.V. Twigg, Carbonylation Direct Synthesis of Carbonyl Compounds. 1991: Plenum Press. Andersson, Y. and B. Långström, Synthesis of 11C Labeled Ketones Via Carbonylative Coupling Reactions Using [11C] CarbonMonoxide. Journal of the Chemical Society-Perkin Transactions 1, 1995(4): 287-289. Kihlberg, T. and B. Långström, Method and apparatus for production and use of [11C]carbon monoxide in labelling synthesis., WO/2002/102711, 2002 Kihlberg, T., et al., Methods and apparatus for production and use of [11C]carbon monoxide in labeling synthesis, WO/2006/008603, 2006 Hostetler, E.D. and H.D. Burns, A remote-controlled high pressure reactor for radiotracer synthesis with [11C]carbon monoxide. Nucl. Med. Biol., 2002. 29(8): 845-848. Audrain, H., et al., Utilisation of [11C]-labelled boron carbonyl complexes in palladium carbonylation reaction. Chemical Communications, 2004(5): 558-559. Miller, P.W., et al., Rapid multiphase carbonylation reactions by using a microtube reactor: Applications in positron emission tomography 11C-radiolabeling. Angewandte Chemie-International Edition, 2007. 46(16): 2875-2878. Audrain, H., Positron emission tomography (PET) and microfluidic devices: A breakthrough on the microscale? Angewandte ChemieInternational Edition, 2007. 46(11): 1772-1775. Langstrom, B., et al., Compounds labelled with short-lived beta(+)emitting radionuclides and some applications in life sciences. The
29. 30. 31. 32.
33. 34.
35. 36. 37. 38. 39.
40. 41.
42. 43.
importance of time as a parameter. Acta Chem. Scand., 1999. 53(9): 651-669. IUPAC. R-8.3.1 Specifically labelled compounds 2008 [cited 2008]; Available from: http://www.acdlabs.com/iupac/nomenclature/93/r93_656.htm. Von Burg, R., Carbon monoxide. J. Appl. Toxicol., 1999. 19(5): 379-386. Russell, R. Windows to the Universe - Carbon Monoxide - CO. 2006 [cited 2009 -03-08]; Available from: http://www.windows.ucar.edu/. Gordon, E.M., et al., Applications of Combinatorial Technologies to Drug Discovery .2. Combinatorial Organic-Synthesis, Library Screening Strategies, and Future-Directions. J. Med. Chem., 1994. 37(10): 1385-1401. Kihlberg, T. and B. Långström, J. Labelled Compd. Radiopharm., 2001. 44(Suppl. 1): S990–S992. Doi, H., et al., Synthesis of 11C-labelled N,N '-diphenylurea and ethyl phenylcarbamate by a rhodium-promoted carbonylation via [11C]isocyanatobenzene using phenyl azide and [11C]carbon monoxide. Org. Biomol. Chem., 2004. 2(21): 3063-3066. Kihlberg, T., F. Karimi, and B. Långström, [11C]carbon monoxide in selenium-mediated synthesis of 11C-carbamoyl compounds. J. Org. Chem., 2002. 67(11): 3687-3692. Karimi, F., T. Kihlberg, and B. Långström, [11C]/(13C)Carbon monoxide in palladium-mediated synthesis of imides. J. Chem. Soc. [Perkin. 1]. 2001(13): 1528-1531. Kihlberg, T. and B. Långstrom, Biologically active 11C-labeled amides using palladium-mediated reactions with aryl halides and [11C]carbon monoxide. J. Org. Chem., 1999. 64(25): 9201-9205. Karimi, F. and B. Långström, Palladium-mediated synthesis of [carbonyl-11C]amides and hydrazides using [11C]carbon monoxide. J. Chem. Soc. [Perkin. 1]. 2002(18): 2111-2115. Karimi, F. and B. Långström, Synthesis of 11C-labelled amides by palladium-mediated carboxamination using [11C]Carbon monoxide, in situ activated amines and 1,2,2,6,6-pentamethylpiperidine. Eur. J. Org. Chem., 2003(11): 2132-2137. Rahman, O., T. Kihlberg, and B. Långström, Aryl triflates and [11C]/(13C)carbon monoxide in the synthesis of 11C-/13C-amides. J. Org. Chem., 2003. 68(9): 3558-3562. Barletta, J., F. Karimi, and B. Långström, Synthesis of [11Ccarbonyl]hydroxyureas by a rhodium-mediated carbonylation reaction using [11C]carbon monoxide. J. Labelled Compd. Radiopharm., 2006. 49(5): 429-436. Rahman, O., T. Kihlberg, and B. Långström, Synthesis of 11C-/13Cketones by Suzuki coupling. Eur. J. Org. Chem., 2004(3): 474-478. Björkman, M., Palladium-Promoted Synthesis of Compounds Labelled with 11C. 2000, Uppsala University: Uppsala. 59
44.
45. 46.
47. 48. 49. 50. 51. 52. 53. 54.
55.
56. 57. 58. 59. 60.
60
Karimi, F. and B. Långström, Palladium-mediated carboxylation of aryl halides (triflates) or benzyl halides using [13C]/[11C]carbon monoxide with tetrabutylammonium hydroxide or trimethylphenylammonium hydroxide. J. Chem. Soc. [Perkin. 1]. 2002(20): 22562259. Crabtree, R.H., The Organometallic Chemistry of the Transition Metals. 4th ed. 2005: Wiley-Interscience. Itsenko, O., Photoinitiated Radical Carbonylation Using [11C]Carbon Monoxide: Labelling of Aliphatic Carboxylic Acids, Esters, and Amides, in Department of Biochemistry and Organic Chemistry. 2005, Uppsala University: Uppsala. Fukumura, T., et al., Stability of 11C-labeled PET radiopharmaceuticals. Appl. Radiat. Isot., 2004. 61(6): 1279-1287. Smith, J.A., S.H. Francis, and J.D. Corbin, Autophosphorylation - a Salient Feature of Protein-Kinases. Mol. Cell. Biochem., 1993. 128: 51-70. Manning, G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): 1912-+. Lander, E.S., et al., Initial sequencing and analysis of the human genome. Nature, 2001. 409(6822): 860-921. Yarden, Y. and M.X. Sliwkowski, Untangling the ErbB signalling network. Nature Reviews Molecular Cell Biology, 2001. 2(2): 127137. Sako, Y., S. Minoghchi, and T. Yanagida, Single-molecule imaging of EGFR signalling on the surface of living cells. Nature Cell Biology, 2000. 2(3): 168-172. Linggi, B. and G. Carpenter, ErbB receptors: new insights on mechanisms and biology. Trends Cell Biol., 2006. 16(12): 649-656. Stamos, J., M.X. Sliwkowski, and C. Eigenbrot, Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol. Chem., 2002. 277(48): 46265-46272. Wood, E.R., et al., A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): Relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res., 2004. 64(18): 6652-6659. Huse, M. and J. Kuriyan, The conformational plasticity of protein kinases. Cell, 2002. 109(3): 275-282. Cowan-Jacob, S.W., Structural biology of protein tyrosine kinases. Cell. Mol. Life Sci., 2006. 63(22): 2608-2625. Karaman, M.W., et al., A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol., 2008. 26(1): 127-132. Fabian, M.A., et al., A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol., 2005. 23(3): 329-336. Morin, M.J., From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents. Oncogene, 2000. 19(56): 6574-6583.
61. 62. 63. 64.
65.
66. 67. 68. 69. 70. 71.
72.
73. 74.
Lawrence, D.S. and J.K. Niu, Protein kinase inhibitors: The tyrosine-specific protein kinases. Pharmacol. Ther., 1998. 77(2): 81114. Fredriksson, A., et al., In vivo evaluation of the biodistribution of 11 C-labeled PD153035 in rats without and with neuroblastoma implants. Life Sci., 1999. 65(2): 165-174. Bonasera, T.A., et al., Potential 18F-labeled biomarkers for epidermal growth factor receptor tyrosine kinase. Nucl. Med. Biol., 2001. 28(4): 359-374. Mishani, E., et al., Novel carbon-11 labeled 4-dimethylamino-but-2enoic acid [4-(phenylamino)-quinazoline-6-yl]-amides: potential PET bioprobes for molecular imaging of EGFR-positive tumors. Nucl. Med. Biol., 2004. 31(4): 469-476. Ortu, G., et al., Labeled EGFr-TK irreversible inhibitor (ML03): In vitro and in vivo properties, potential as PET biomarker for cancer and feasibility as anticancer drug. Int. J. Cancer, 2002. 101(4): 360370. Pal, A., et al., Molecular imaging of EGFR kinase activity in tumors with 124I-labeled small molecular tracer and positron emission tomography. Molecular Imaging and Biology, 2006. 8(5): 262-277. Wissner, A. and T.S. Mansour, The development of HKI-272 and related compounds for the treatment of cancer. Arch. Pharm. (Weinheim). 2008. 341(8): 465-477. Cockerill, G. and M. Carter, Bicyclic heteroaromatic compounds as protein tyrosine kinase inhibitors, WO 98/02438, 1998 Cockerill, G. and M. Carter, Fused heterocyclic compounds as protein tyrosine kinase inhibitors, WO 98/02434, 1998 Cockerill, G. and M. Carter, Bicyclic heteroaromatic compounds as protein tyrosine-kinase inhibitors WO 98/02437, 1998 Smaill, J.B., et al., Tyrosine kinase inhibitors. 15. 4(phenylamino)quinazoline and 4-(phenylamino)pyrido[d] pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor. J. Med. Chem., 1999. 42(10): 1803-1815. Smaill, J.B., et al., Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2d]pyrimidine-6-acrylamides bearing additional solubilizing functions. J. Med. Chem., 2000. 43(7): 1380-1397. Yun, C.H., et al., The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl. Acad. Sci. U. S. A., 2008. 105(6): 2070-2075. Murray, T.F. and J.R. Norton, Design and Mechanism of Palladium Catalysts for Synthesis of Methylene Lactones by Cyclocarbonylation of Acetylenic Alcohols. J. Am. Chem. Soc., 1979. 101(15): 4107-4119.
61
75. 76. 77. 78.
79.
80. 81. 82. 83. 84. 85.
86. 87.
88. 89.
62
Samsel, E.G. and J.R. Norton, Mechanism of Acetylene and Olefin Insertion into Palladium Carbon-Omicron-Bonds. J. Am. Chem. Soc., 1984. 106(19): 5505-5512. Tsuji, J., Carbon-Carbon Bond Formation Via Palladium Complexes. Acc. Chem. Res., 1969. 2(5): 144-&. Zargarian, D. and H. Alper, Palladium-Catalyzed Hydrocarboxylation of Alkynes with Formic-Acid. Organometallics, 1993. 12(3): 712-724. Zeigarnik, A.V., et al., Computer-aided mechanism elucidation of acetylene hydrocarboxylation to acrylic acid based on a novel union of empirical and formal methods. Organometallics, 1997. 16(14): 3114-3127. El Ali, B., et al., Palladium(II)-catalyzed regioselective carbonylative coupling of aniline derivatives with terminal aryl acetylenes to give acrylamides under syngas conditions. Tetrahedron Lett., 2000. 41(30): 5761-5764. El Ali, B., et al., Regioselective control of the thiocarbonylation of terminal acetylenes with arylthiols catalyzed by Pd(II) and diphosphine ligands. Tetrahedron Lett., 2001. 42(8): 1567-1570. Schoenberg, A. and R.F. Heck, Palladium-Catalyzed Amidation of Aryl, Heterocyclic, and Vinylic Halides. J. Org. Chem., 1974. 39(23): 3327-3331. Negishi, A.-i. and A.d. Meijere, Handbook of Organopalladium Chemistry for Organic Synthesis. 2 ed. 2003: Wiley-IEEE. Townsend, J.M., et al., Dehydrobromination of Alpha-Bromo Ketones with Palladium Tetrakis(Triphenylphosphine). J. Org. Chem., 1975. 40(20): 2976-2978. Mishani, E., et al., Imaging of EGFR and EGFR Tyrosine Kinase Overexpression in Tumors by Nuclear Medicine Modalities. Curr. Pharm. Des., 2008. 14(28): 2983-2998. Gelovani, J.G., Molecular imaging of epidermal growth factor receptor expression-activity at the kinase level in tumors with positron emission tomography. Cancer Metastasis Rev., 2008. 27(4): 645653. Jung, M.E. and L.A. Light, Preparation of Iodoallylic Alcohols Via Hydrostannylation - Spectroscopic Proof of Structures. Tetrahedron Lett., 1982. 23(38): 3851-3854. Ben-David, I., et al., Radiosynthesis of ML03, a novel positron emission tomography biomarker for targeting epidermal growth factor receptor via the labeling synthon: [C-11]acryloyl chloride. Appl. Radiat. Isot., 2003. 58(2): 209-217. Duff, S.E., et al., Vascular endothelial growth factors and receptors in colorectal cancer: Implications for anti-angiogenic therapy. Eur. J. Cancer, 2006. 42(1): 112-117. Kolibaba, K.S. and B.J. Druker, Protein tyrosine kinases and cancer. Biochimica Et Biophysica Acta-Reviews on Cancer, 1997. 1333(3): F217-F248.
90.
91.
92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106.
Ilovich, O., et al., Rhodium-mediated [11C]carbonylation: a library of N-phenyl-N'-{4-(4-quinolyloxy)-phenyl}-[11C]-urea derivatives as potential PET angiogenic probes J. Labelled Compd. Radiopharm., 2009. 52: in press. Ilovich, O., et al., Formation of fluorine-18 labeled diaryl ureas labeled VEGFR-2/PDGFR dual inhibitors as molecular imaging agents for angiogenesis. Bioorg. Med. Chem., 2008. 16(8): 42424251. Crouzel, C., et al., 11C-Labelled Phosgene - an Improved Procedure and Synthesis Device. Int. J. Appl. Radiat. Isot., 1983. 34(11): 15581559. Roeda, D. and G. Westera, A Uv-Induced Online Synthesis of 11CPhosgene and the Preparation of Some of Its Derivatives. Int. J. Appl. Radiat. Isot., 1981. 32(12): 931-932. Cenini, Coordination chemistry of organic azides and amination reactions catalyzed by transition metal complexes. Coord. Chem. Rev., 2006. 250: 1234-1253. Bräse, S., et al., Organic Azides: An exploding diversity of a Unique Class of Compounds. Angew. Chem., 2005. 44: 5188-5240. Dinh, D.T., et al., Angiotensin receptors: distribution, signalling and function. Clin. Sci., 2001. 100(5): 481-492. Paul, M., A.P. Mehr, and R. Kreutz, Physiology of local reninangiotensin systems. Physiol. Rev., 2006. 86(3): 747-803. de Gasparo, M., et al., International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol. Rev., 2000. 52(3): 415472. Kaschina, E. and T. Unger, Angiotensin AT1/AT2 receptors: Regulation, signalling and function. Blood Press., 2003. 12(2): 70-88. Smith, G.R. and S. Missailidis, Cancer, inflammation and the AT1 and AT2 receptors. J. Inflamm., 2004. 1(3). Engler, H., et al., Multitracer study with positron emission tomography in Creutzfeldt-Jakob disease. European Journal of Nuclear Medicine and Molecular Imaging, 2003. 30(1): 85-95. Dinh, D.T., et al., Identification, distribution, and expression of angiotensin II receptors in the normal human prostate and benign prostatic hyperplasia. Endocrinology, 2001. 142(3): 1349-1356. Ohtani, S., et al., Up-regulated expression of angiotensin II type 1 receptor gene in human pathologic hearts. J. Card. Fail., 1997. 3(4): 303-310. Zober, T.G., et al., PET Imaging of the AT(1) receptor with [11C]KR31173. Nucl. Med. Biol., 2006. 33(1): 5-13. Jones, E.S., et al., AT(2) receptors: Functional relevance in cardiovascular disease. Pharmacol. Ther., 2008. 120(3): 292-316. Wharton, J., et al., Differential distribution of angiotensin AT(2) receptors in the normal and failing human heart. J. Pharmacol. Exp. Ther., 1998. 284(1): 323-336.
63
107.
108. 109. 110. 111. 112.
113. 114. 115.
116. 117. 118. 119. 120. 121.
64
Ge, J.A. and N.M. Barnes, Alterations in angiotensin AT(1) and AT(2) receptor subtype levels in brain regions from patients with neurodegenerative disorders. Eur. J. Pharmacol., 1996. 297(3): 299306. SmithKlineBeecham. New Drug Application, Tevetan/Eprosartan mesylate. 1997 ; Available from: http://www.fda.gov/cder/foi/nda/index97.htm. Timmermans, P.B., Pharmacological properties of angiotensin II receptor antagonists. Can. J. Cardiol., 1999. 15: Suppl F:26F-8F. Ojima, M., et al., Candesartan (CV-11974) dissociates slowly from the angiotensin AT(1) receptor. Eur. J. Pharmacol., 1997. 319(1): 137-146. Fierens, F.L.P., et al., Insurmountable angiotensin AT(1) receptor antagonists: the role of tight antagonist binding. Eur. J. Pharmacol., 1999. 372(2): 199-206. Pinheiro, H., et al., A comparison of AT(1) angiotensin II antagonists at pre- and postjunctional angiotensin II receptors of the rat tall artery. Naunyn. Schmiedebergs Arch. Pharmacol., 2002. 366(6): 537-542. Wu, M.T., et al., Synthesis and Structure-Activity-Relationships of a Novel Series of Nonpeptide at(2)-Selective Angiotensin-Ii Receptor Antagonists. Bioorg. Med. Chem. Lett., 1993. 3(10): 2023-2028. Brechler, V., et al., Agonistic and Antagonistic Properties of Angiotensin Analogs at the At2-Receptor in Pc12w Cells. Regul. Pept., 1993. 44(2): 207-213. Blankley, C.J., et al., Synthesis and Structure-Activity-Relationships of a Novel Series of Nonpeptide Angiotensin-II Receptor-Binding Inhibitors Specific for the AT2 Subtype. J. Med. Chem., 1991. 34(11): 3248-3260. Wan, Y.Q., et al., Design, synthesis, and biological evaluation of the first selective nonpeptide AT(2) receptor agonist. J. Med. Chem., 2004. 47(24): 5995-6008. Wallinder, C., et al., Selective angiotensin II AT(2) receptor agonists: Benzamide structure-activity relationships. Bioorg. Med. Chem., 2008. 16(14): 6841-6849. Georgsson, J., et al., Synthesis of a new class of druglike angiotensin II C-terminal mimics with affinity for the AT(2) receptor. J. Med. Chem., 2007. 50(7): 1711-1715. Keenan, R.M., et al., Potent Nonpeptide Angiotensin-Ii Receptor Antagonists .2. 1-(Carboxybenzyl)Imidazole-5-Acrylic Acids. J. Med. Chem., 1993. 36(13): 1880-1892. Dell'Orco, P., et al., Monitoring process-scale reactions using API mass spectrometry. Anal. Chem., 1999. 71(22): 5165-5170. Haque, M.R. and M. Rasmussen, Ambident Heterocyclic Reactivity - Alkylation of 4-Substituted and 2,4-Disubstituted Benzimidazoles. Aust. J. Chem., 1994. 47(8): 1523-1535.
122. 123. 124.
Haque, M.R. and M. Rasmussen, Ambident heterocyclic reactivity: Intramolecular alkylations of 2,4-disubstituted benzimidazoles. Tetrahedron, 1997. 53(20): 6937-6958. Haque, M.R. and M. Rasmussen, Ambident Heterocyclic Reactivity - Alkylation of 2-Substituted-4-Methylbenzimidazoles. Tetrahedron, 1994. 50(18): 5535-5554. Keumi, T., et al., The Decomposition of Arenediazonium Tetrafluoroborates with Halotrimethylsilane and Azidotrimethylsilane in Nonaqueous Solvents. Bull. Chem. Soc. Jpn., 1989. 62(1): 89-95.
65
& 4 3 4
2' " ' & " ' #4 4 3 # 5&6
7 8 6 #6 3 4 " ' 5 9: ; #%
