Constantin Mamat

Recent Applications of Click Chemistry for the Synthesis of Radiotracers for Molecular Imaging

Click chemistry has received considerable attention as powerful modular synthesis approach, which has found numerous ap- plications in many areas of modern organic chemistry, drug discovery and material science. Recently, click chemistry, and in particular the copper-mediated 1,3-dipolar (3+2) cycloaddition between azides and alkynes, has also entered the field of radiopharmaceutical sci- ences. This review addresses the recent developments of click chemistry for the synthesis of various radiotracers for molecular imaging purposes. Click chemistry-based radiotracers that will be covered include peptides and small organic molecules containing the short-lived positron emitter fluorine-18, and the gamma-emitters technetium-99m, indium-111, and iodine-125. Current biomedical research is revealing the fundamental mo- lecular processes of life and diseases. The understanding of how molecular components of living cells are organized, how they inter- act, how they move and how they are formed and eliminated within the life cycle of an organism represents an integrative approach, which requires the direct observation of biochemical and physio- logical processes at the molecular level in vivo. The molecular processes of life can be studied and visualized at various levels of resolution by means of in vivo imaging techniques (1-3). Molecular imaging techniques span the electromagnetic spectrum from ultra- sonic to gamma-ray frequencies. In this line, especially magnetic resonance imaging (MRI), optical imaging and nuclear imaging are emerging as key molecular imaging techniques. Nuclear imaging techniques are based on the application and detection of decaying radioisotopes. In most cases, the radioiso- topes are combined with biologically active compounds to form radiolabeled probes capable of imaging specific biochemical and physiological events in vivo. The radiolabed probe is administered to follow a metabolic pathway connected with the pathophysiology of disease processes. The detection of the emitted radiation can either be performed by positron emission tomography (PET) or single photon computered tomography (SPECT) (2-4). PET and SPECT have extensively been used for noninvasive diagnosis, stag- ing and therapy control of diseases at the molecular level. Both techniques have found numerous applications in the field of clinical oncology, cardiology and neurology. Moreover, especially PET has also been recognized and applied as a valuable research tool in the process of drug development and evaluation. The success of radiotracer-based molecular imaging techniques like PET and SPECT stems mainly from the availability of suitable radiolabed probes, also referred to as radiotracers. The design and synthesis of radiotracers as molecular probes is subject of radio- pharmaceutical chemistry. Todays radiopharmaceutical chemistry has evolved into a complex chemical science, combining recent advances in synthetic organic and inorganic chemistry with devel- opments and achievements in the field of molecular biology. The development of radiotracers for molecular imaging purposes has to address important questions on target selection and target validation while considering the special requirements encountered in ra- diotracer synthesis such as choice of the appropriate radionuclide and suitable labeling position.

Recent Trends in Bioorthogonal Click-Radiolabeling Reactions Using Fluorine-18

The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of novel and versatile bioorthogonal conjugation techniques especially for the radiolabeling of biologically active high molecular weight compounds like peptides, proteins or antibodies. Taking into consideration that the introduction of fluorine-18 (t1/2 = 109.8 min) proceeds under harsh conditions, radiolabeling of these biologically active molecules represents an outstanding challenge and is of enormous interest. Special attention has to be paid to the method of 18F-introduction. It should proceed in a regioselective manner under mild physiological conditions, in an acceptable time span, with high yields and high specific activities. For these reasons and due to the high number of functional groups found in these compounds, a specific labeling procedure has to be developed for every bioactive macromolecule. Bioorthogonal strategies including the Cu-assisted Huisgen cycloaddition and its copper-free click variant, both Staudinger Ligations or the tetrazine-click reaction have been successfully applied and represent valuable alternatives for the selective introduction of fluorine-18 to overcome the afore mentioned obstacles. This comprehensive review deals with the progress and illustrates the latest developments in the field of bioorthogonal labeling with the focus on the preparation of radiofluorinated building blocks and tracers for molecular imaging.

Cross-coupling reactions as valuable tool for the preparation of PET radiotracers.

The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of new radiotracers and novel radiolabeling procedures with the most prominent short-lived positron emitters carbon-11 and fluorine-18. Radiolabeling with these radionuclides represents a remarkable challenge. Special attention has to be paid to synthesis time and specific labeling techniques due to the short physical half life of the respective radionuclides 11C (t1/2 = 20.4 min) and 18F (t1/2 = 109.8 min). In the past, numerous transition metal-catalyzed reactions were employed in organic chemistry, even though only a handful of these coupling reactions were adopted in radiochemical practice. Thus, the implementation of modern synthesis methods like cross-coupling reactions offers the possibility to develop a wide variety of novel radiotracers. The introduction of catalysts based on transition metal complexes bears a high potential for rapid, efficient, highly selective and functional group-tolerating incorporation of carbon-11 and fluorine-18 into target molecules. This review deals with design, application and improvement of transition metal-mediated carbon-carbon as well as carbon-heteroatom cross-coupling reactions as a labeling feature with the focus on the preparation of radiolabeled compounds for molecular imaging.

Fluorine-18 Radiolabeling and Radiopharmacological Characterization of a Benzodioxolylpyrimidine-based Radiotracer Targeting the Receptor Tyrosine Kinase EphB4

Members of the Eph receptor tyrosine kinase family play essential roles in the pathogenesis of cancer and are therefore promising candidates for molecular imaging by positron emission tomography (PET), for example. In this regard, radiochemical access to novel PET radiotracers derived from potent inhibitors that target the EphB4 kinase domain and which bear a benzodioxolylpyrimidine structural motif was developed. A synthetic route was established for a new fluorine‐18‐containing radiotracer and for the desired precursor based on a high‐affinity benzodioxolylpyrimidine receptor tyrosine kinase inhibitor lead structure. The radiotracer [18F]15 was obtained in 16 % radiochemical yield with a specific activity of ∼7 GBq μmol−1 and >95 % radiochemical purity. Due to the implication of EphB4, particularly in the progression, angiogenesis, and metastasis of melanoma, EphB4‐overexpressing human melanoma cells were generated and used as a novel in vitro model for radiopharmacological evaluation of the radiotracer. We demonstrate that the corresponding non‐radioactive reference compound regained its functionality as an inhibitor for both EphB4 receptor tyrosine kinase and Src kinase. EphB4 was significantly inhibited at compound concentrations >1 μM. Cellular uptake studies with [18F]15 revealed substantial uptake in both EphB4‐overexpressing and control cells. Moreover, NMRI nu/nu mice bearing both EphB4‐overexpressing tumors and control tumors were used for radiopharmacological characterization by biodistribution studies ex vivo and by dynamic small‐animal PET experiments in vivo. Despite the high metabolic stability of the novel radiotracer observed in vivo, no substantial binding or accumulation in EphB4‐overexpressing and control tumors was observed. Nevertheless, we point out that the approach presented herein gives convenient access to novel 18F‐labeled benzodioxolylpyrimidines and is a promising strategy for the further development of novel radiotracers for imaging Eph receptor tyrosine kinases in cancer.

Methods to Increase the Metabolic Stability of 18 F-Radiotracers

The majority of pharmaceuticals and other organic compounds incorporating radiotracers that are considered foreign to the body undergo metabolic changes in vivo. Metabolic degradation of these drugs is commonly caused by a system of enzymes of low substrate specificity requirement, which is present mainly in the liver, but drug metabolism may also take place in the kidneys or other organs. Thus, radiotracers and all other pharmaceuticals are faced with enormous challenges to maintain their stability in vivo highlighting the importance of their structure. Often in practice, such biologically active molecules exhibit these properties in vitro, but fail during in vivo studies due to obtaining an increased metabolism within minutes. Many pharmacologically and biologically interesting compounds never see application due to their lack of stability. One of the most important issues of radiotracers development based on fluorine-18 is the stability in vitro and in vivo. Sometimes, the metabolism of 18F-radiotracers goes along with the cleavage of the C-F bond and with the rejection of [18F]fluoride mostly combined with high background and accumulation in the skeleton. This review deals with the impact of radiodefluorination and with approaches to stabilize the C-F bond to avoid the cleavage between fluorine and carbon.

An Efficient Bioorthogonal Strategy Using CuAAC Click Chemistry for Radiofluorinations of SNEW Peptides and the Role of Copper Depletion

The EphB2 receptor is known to be overexpressed in various types of cancer and is therefore a promising target for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of EphB2‐overexpressing tumors, monitoring responses to therapy directed toward EphB2, and thus improvement in patient outcomes. We report the synthesis and evaluation of several fluorine‐18‐labeled peptides containing the SNEW amino acid motif, with high affinity for the EphB2 receptor, for their potential as radiotracers in the non‐invasive imaging of cancer using PET. For the purposes of radiofluorination, EphB2‐antagonistic SNEW peptides were varied at the C terminus by the introduction of L‐cysteine, and further by alkyne‐ or azide‐modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [18F]AFP and [18F]BFP were applied, and their capacity to introduce fluorine‐18 was compared with that of the established building block [18F]FBAM. Copper‐assisted Huisgen 1,3‐dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide‐ or alkyne‐modified SNEW peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.

18F-Labeled 1,4-Dioxa-8-azaspiro[4.5]decane Derivative: Synthesis and Biological Evaluation of a σ1 Receptor Radioligand with Low Lipophilicity as Potent Tumor Imaging Agent

We report the syntheses and evaluation of series of novel piperidine compounds with low lipophilicity as σ1 receptor ligands. 8-(4-(2-Fluoroethoxy)benzyl)-1,4-dioxa-8-azaspiro[4.5]decane (5a) possessed high affinity (K(i) = 5.4 ± 0.4 nM) for σ1 receptors and selectivity for σ2 receptors (30-fold) and the vesicular acetylcholine transporter (1404-fold). [(18)F]5a was prepared using a one-pot, two-step labeling procedure in an automated synthesis module, with a radiochemical purity of >95%, and a specific activity of 25-45 GBq/μmol. Cellular association, biodistribution, and autoradiography with blocking experiments indicated specific binding of [(18)F]5a to σ1 receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging using mouse tumor xenograft models demonstrated a high accumulation in human carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity and appropriate affinity for σ1 receptors could be used for tumor imaging.

Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers

Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [(11)C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired (18)F-radiotracer under the chosen conditions.

Novel indole -based sigma-2 receptor ligands: synthesis , structure–affinity relationship and antiproliferative activity

We report the synthesis and biological evaluation of a series of indole-based σ2 receptor ligands derived from siramesine. In vitro competition binding assays showed that these analogues possessed high to moderate affinity and selectivity for σ2 receptors. Structure–affinity relationship analyses of these indole-based σ2 receptor ligands were performed. In the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 1a and 1b displayed significant and comparable antiproliferative activity in DU145, MCF7 and C6 cells to siramesine. In cell cycle analyses, compounds 1a, 1b and siramesine were found to induce a G1 phase cell cycle arrest in DU145 cells using flow cytometry. The combination of 5,6-dimethoxyisoindoline scaffold and N-(4-fluorophenyl)indole moiety was identified as a new σ2 receptor ligand deserving further investigation as an antitumor agent.

Synthesis and Radiofluorination of Iodophenyl Esters as Tool for the Traceless Staudinger Ligation

A new synthetic pathway for the preparation of ω-functionalized 2-iodophenyl esters as starting materials for the synthesis of substituted phosphanes is described. A radiolabeling of these esters with fluorine-18 has led to building blocks which were reacted with HPPh2 in a Pd-catalyzed P-C cross coupling to establish new phosphanes. These compounds can be applied as mild and bioorthogonal radiolabeling agents by means of the traceless Staudinger ligation. A route to access this class of compounds has been established. Graphical Abstract Synthesis and Radiofluorination of Iodophenyl Esters as Tool for the Traceless Staudinger Ligation