Karin A. Stephenson

The medicinal chemistry of carboranes

The medicinal chemistry of dicarba-closo-dodecaboranes (otherwise referred to as carboranes) has traditionally centered on their use in boron neutron capture therapy (BNCT). More recently, work has begun to exploit the unique chemical and physical properties of carboranes for the preparation of novel inorganic pharmaceuticals and biological probes. This review is designed to highlight some of the recent work concerning medicinal carborane chemistry including the synthesis and testing of new BNCT agents. Following this review, as an appendix, is an illustrated summary of reactions involving carboranes reported in literature since 1992.

Radiosynthesis and initial evaluation of [18F]-FEPPA for PET imaging of peripheral benzodiazepine receptors

INTRODUCTION A novel [18F]-radiolabelled phenoxyanilide, [18F]-FEPPA, has been synthesized and evaluated, in vitro and ex vivo, as a potential positron emission tomography imaging agent for the peripheral benzodiazepine receptor (PBR). METHODS [18F]-FEPPA and two other radiotracers for imaging PBR, namely [11C]-PBR28 and [11C]-PBR28-d3, were synthesised and evaluated in vitro and ex vivo as potential PBR imaging agents. RESULTS [18F]-FEPPA is efficiently prepared in one step from its tosylate precursor and [18F]-fluoride in high radiochemical yields and at high specific activity. FEPPA displayed a Ki of 0.07 nM for PBR in rat mitochondrial membrane preparations and a suitable lipophilicity for brain penetration (log P of 2.99 at pH 7.4). Upon intravenous injection into rats, [18F]-FEPPA showed moderate brain uptake [standard uptake value (SUV) of 0.6 at 5 min] and a slow washout (SUV of 0.35 after 60 min). Highest uptake of radioactivity was seen in the hypothalamus and olfactory bulb, regions previously reported to be enriched in PBR in rat brain. Analysis of plasma and brain extracts demonstrated that [18F]-FEPPA was rapidly metabolized, but no lipophilic metabolites were observed in either preparation and only 5% radioactive metabolites were present in brain tissue extracts. Blocking studies to determine the extent of specific binding of [18F]-FEPPA in rat brain were problematic due to large perturbations in circulating radiotracer and the lack of a reference region. CONCLUSIONS Further evaluation of the potential of [18F]-FEPPA will require the employment of rigorous kinetic models and/or appropriate animal models.

Synthesis and Screening of a Library of Re/Tc-Based Amyloid Probes Derived from β-Breaker Peptides

Through the development and application of a unique approach for producing Re-metallopeptides, a new class of peptide-derived probes that are designed to target beta-amyloid plaques was developed. Derivatives of a class of beta-breaker peptides having the core sequence lvffa or affvl (lower case letters represent D-amino acids) and the single amino acid chelate quinoline (SAACQ) ligand which can bind Re and (99m)Tc were prepared on an automated peptide synthesizer. Both monomeric and dimeric peptides were synthesized in modest to good yields where in select examples a biotin-containing amino acid derivative was included to act as a linker point for further conjugation to carrier proteins. The Re complexes for all reported peptides were prepared similarly and screened for their ability to inhibit fibrillogenesis. Two of the reported compounds showed excellent inhibitory properties (8a: 40 +/- 5% amyloid formation versus control; 16: 40 +/- 4%) and warrant further investigation. For one of these leads, the (99m)Tc analogue was synthesized and the product showed high stability toward histidine and cysteine challenges, making it a viable candidate for in vivo biodistribution studies.

An improved radiosynthesis of the muscarinic M2 radiopharmaceutical, [18F]FP-TZTP.

The radioligand 3-(4-(3-[(18)F]fluoropropylthio)-1,2,5-thiadiazol-3-yl)-1-methyl-1,2,5,6-tetrahydropyridine ([(18)F]FP-TZTP) is an agonist with specificity towards subtype 2 of muscarinic acetylcholine (M2) receptors. It is currently the only radiotracer available for imaging M2 receptors in human subjects with positron emission tomography. The present study reports on an improved method for the synthesis of [(18)F]FP-TZTP, automated using a GE TRACERlab FX(FN) radiosynthesis module. A key facet was the use of a new precursor, 3-(4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazol-3-ylthio)propyl 4-methylbenzenesulfonate. The precursor was fluorinated via nucleophilic displacement of the tosyloxy group by potassium cryptand [(18)F]fluoride (K[(18)F]/K(222)) in CH(3)CN at 80 degrees C for 5 min, and purified by HPLC. Formulated [(18)F]FP-TZTP was prepared in an uncorrected radiochemical yield of 29+/-4%, with a specific activity of 138+/-41 GBq/micromol (3732+/-1109 mCi/micromol) at the end of synthesis (35 min; n=3). This methodology offers higher yields, faster synthesis times, an optimized precursor, and simpler automation than previously reported.

Synthesis and in vitro evaluation of derivatives of the β1-adrenergic receptor antagonist HX-CH 44

Isopropyl- and fluoroisopropyl-amino derivatives of the β(1)-adrenergic receptor antagonist 2-[4-[3-(tert-butyl-amino)-2-hydroxypropoxy]phenyl]-3-methyl-6-methoxy-4(3H)-quinazolinone ((±)HX-CH 44) were synthesized, including a concise and efficient preparation of the core, 2-(4-hydroxyphenyl)-6-methoxy-3-methylquinazolin-4(3H)-one. In vitro binding assays showed that the fluorinated analog was selective towards β(1)-adrenergic receptors over β(2)-adrenergic and 5-HT(1A) receptors. An X-ray crystallographic characterization of the fluorinated analog is also reported.

Synthesis and preliminary evaluation of [18F]-fluoro-(2S)-Exaprolol for imaging cerebral β-adrenergic receptors with PET

Cerebral beta-adrenergic receptors (beta-ARs) are of interest in several disorders including Parkinsons disease, Alzheimers disease and in particular major depressive disorder. Development of a positron emission tomography (PET) ligand for imaging beta-ARs would allow the quantification of these receptors in the living human brain so as to better understand both the pathophysiology of depression and how to improve treatment. Currently there are no radioligands suitable for this purpose. In an attempt to achieve this goal, we prepared [(18)F]-labeled (2S)-1-(1-fluoropropan-2-ylamino)-3-(2-cyclohexylphenoxy)propan-2-ol (fluoro-Exaprolol; (2S)-1). Radiolabeling with fluorine-18 was accomplished via preparation of a precursor containing a tosyl leaving group (10), and utilizes the 2-oxazolidinone group to simultaneously protect both the amine and hydroxy groups. The oxazolidinone was readily removed with lithium aluminum hydride following a nucleophilic [(18)F]-fluoride for tosyl displacement to prepare [(18)F]-(2S)-1 in 31% radiochemical yield (uncorrected for decay), with >98% radiochemical purity in <1h. The specific activity of the formulated product was 927 mCi/micromol and the log P (pH 7.4) was 2.97. Preliminary biological evaluations in conscious rats indicated that [(18)F]-(2S)-1 had good brain uptake for imaging (0.8-1.3% injected dose/gram (% ID/g) of wet tissue, 5 min post-injection of the radiotracer) with a slow washout (>0.5% ID/g at 60 min post-injection) in all brain regions. Pharmacological challenges indicate that the binding is largely non-specific, as administration of Propranolol, authentic (2S)-1, or WAY 100635 prior to injection of [(18)F]-(2S)-1 did not block uptake of the radiotracer. These results indicate that [(18)F]-(2S)-1 is not a suitable candidate for PET imaging of cerebral beta-ARs.

Facile Radiosynthesis of Fluorine-18 Labeled β-Blockers. Synthesis, Radiolabeling, and ex Vivo Biodistribution of [18F]-(2S and 2R)-1-(1-Fluoropropan-2-ylamino)-3-(m-tolyloxy)propan-2-ol

An efficient and general method has been developed for fluorine-18 labeling of beta-blockers that possess the propanolamine moiety. A new synthetically versatile intermediate, 3-(1-(benzyloxy)propan-2-yl)-2-oxooxazolidin-5-yl)methyl 4-methylbenzenesulfonate (13), was prepared and can be conjugated to any phenoxy core. To demonstrate the synthetic methodology, fluorinated derivatives of toliprolol were prepared, namely, [(18)F]-(2S and 2R)-1-(1-fluoropropan-2-ylamino)-3-(m-tolyloxy)propan-2-ol ((2S and 2R)-[(18)F]1). The radiosyntheses were accomplished in <1 h, with 20-24% (uncorrected for decay, n = 7) radiochemical yields, >96% radiochemical and >99% enantiomeric purities, with specific activities of 0.9-1.1 Ci/micromol (EOS). Ex vivo biodistribution studies with the radiotracers demonstrated excessively rapid washout that may limit their use for cerebral PET imaging.

Emulsion Reactors: A New Technique for the Preparation of Molecular Imaging Probes

There is an ongoing global effort to prepare targeted molecular imaging (MI) agents for positron emission tomography (PET) and single photon emission computed tomography (SPECT) by labeling small molecules, peptides, and antibodies with radionuclides. More recently, the library of available vectors has expanded to include naturally occurring and genetically engineered proteins, such as affibodies, antibody fragments, hormones, and growth factors. Although modern MI agents offer the opportunity to image and interrogate specific biochemical pathways, they also present unique challenges with respect to labeling the parent targeting vectors with medical isotopes. In many cases, conventional labeling methods fail to produce the desired products in high yield because of the complex structures of derivatized vectors and (in the case of biological agents) because of sensitivities to elevated reaction temperatures and non-physiological pH. Furthermore, production of the parent ligands is often non-trivial, resulting in their limited availability and high cost. These challenges have created the need for a new generation of high yielding and chemoselective labeling methods for both traditional and emerging classes of vectors. In response to these challenges, new radiolabeling strategies have been developed that include solid-phase and fluorous-phase radiolabeling, more reactive prosthetic groups and radiometal chelating His-tag analogues. Microfluidics has also been used increasingly for radiochemical applications because it allows for reactions to be run by using small quantities of precursor in which the unique reaction environment can improve yields and reduce labeling times. Here we report a new strategy that utilizes an emulsion-based labeling platform that requires no specialized equipment, provides a biocompatible reaction environment and that affords a significant increase in radiochemical yields. Aqueous dispersed systems, heterogeneous interfaces, and micelles have been found to accelerate organic reactions and catalyze molecular transformations, therefore, it was postulated that these reaction environments could similarly enhance radiolabeling reactions while providing a biocompatible reaction media. The utility of the emulsion platform was initially explored by using a chelate-derivatized peptide 1 (Scheme 1) that binds to the urokinase plasminogen activator receptor (uPAR)—a protein that is associated with aggressive forms of cancer. The peptide was modified with a tridentate amino acid-derived chelate (SAACII) that binds the technetium tricarbonyl ([Tc(CO)3] ) core. This is an attractive system for evaluating the emulsion platform since technetium is the most widely used radionuclide in diagnostic medicine and the tricarbonyl core typically requires a large excess of the ligand to achieve reasonable conversion yields in less than one half-life. A series of different emulsions were prepared in which the amount and type of surfactant and oil, and the volume ratio between the aqueous and oil phase, were varied. The labeling yields were evaluated as a function of ligand concentration and reaction conditions, and results were compared directly to conventional solution phase labeling reactions run in parallel. The initial emulsions investigated were prepared by varying the amount of the non-ionic surfactant sorbitan mono-oleate (Span-80) in isooctane from 0–5 wt% (0–8.02 10 2 molL ). The reaction mixture contained 1 (10 nmol), 37–55 MBq of [Tc(CO)3 ACHTUNGTRENNUNG(OH2)3]+ (an amount that enabled imaging and biodistribution studies to be completed), the aqueous phase (0.1 mL), and isooctane (1.0 mL). The reactions were conducted at ambient temperature and monitored for 60 min. Emulsification was performed by using a 35 kHz sonication bath with the conventional aqueous reaction placed in the sonicator beside the emulsion reactor so that the process for the two methods was identical (except for the addition of the isooctane) to account for any effects from sonication. All of the labeling reactions that were conducted in the emulsion system outperformed the corresponding conventional labeling, which had a radiochemical yield of less than 5%. The highest radiochemical yield (98%) was achieved when no surfactant was added to the system. [a] Dr. R. W. Simms, D. M. Weaver, M. Blacker, Dr. K. A. Stephenson, Prof. Dr. J. F. Valliant Centre for Probe Development and Commercialization Burke Science Building, Room B231, McMaster University 1280 Main Street West, Hamilton, Ontario L8S 4K1 (Canada) Fax: (+1)905-522-2509 E-mail : valliant@mcmaster.ca [b] Dr. D. H. Kim, Dr. C. Sundararajan, Prof. Dr. J. F. Valliant Department of Chemistry and Chemical Biology McMaster University, 1280 Main Street West Hamilton, Ontario L8S 4L8 (Canada) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201200049.

125I-Labelled 2-Iodoestrone-3-sulfate: synthesis, characterization and OATP mediated transport studies in hormone dependent and independent breast cancer cells

INTRODUCTION Organic Anion Transporting Polypeptides (OATP) are a family of membrane associated transporters that facilitate estrone-3-sulphate (E3S) uptake by hormone dependent, post-menopausal breast cancers. We have established E3S as a potential ligand for targeting hormone dependent breast cancer cells, and in this study sought to prepare and investigate radioiodinated E3S as a tool to study the OATP system. METHODS 2- and 4-Iodoestrone-3-sulfates were prepared from estrone via aromatic iodination followed by a rapid and high yielding sulfation procedure. The resulting isomers were separated by preparative HPLC and verified by (1)H NMR and analytical HPLC. Transport studies of 2- and 4-[(125)I]-E3S were conducted in hormone dependent (i.e. MCF-7) and hormone independent (i.e. MDA-MB-231) breast cancer cells in the presence or absence of the specific transport inhibitor, bromosulfophthalein (BSP). Cellular localization of OATP1A2, OATP2B1, OATP3A1 and OATP4A1 were determined by immunofluorescence analysis using anti-Na(+)/K(+) ATPase-α (1:100 dilution) and DAPI as plasma membrane and nuclear markers, respectively. RESULTS Significantly (p<0.01) higher total accumulation of 2-[(125)I]-E3S was observed in hormone dependent MCF-7 as compared to hormone independent MDA-MB-231 breast cancer cells. In contrast 4-[(125)I]-E3S did not show cellular accumulation in either case. The efficiency of 2-[(125)I]-E3S transport (expressed as a ratio of Vmax/Km) was 2.4 times greater in the MCF-7 as compared to the MDA-MB-231 breast cancer cells. OATP1A2, OATP3A1 and OATP4A1 expression was localized in plasma membranes of MCF-7 and MDA-MB-231 cells confirming the functional role of these transporters in radioiodinated E3S cellular uptake. CONCLUSION An efficient method for the preparation of 2- and 4-[(125)I]-E3S was developed and where the former demonstrated potential as an in vitro probe for the OATP system. The new E3S probe can be used to study the OATP system and as a platform to create radiopharmaceuticals for imaging breast cancer.

Recent advances towards imaging cerebral β-adrenergic receptors with PET

Introduction: Imaging cerebral β-Adrenergic receptors (βARs) with PET remains elusive in humans due to the lack of a suitable radiotracer. Our goal was to develop general synthetic and radiosynthetic methods amenable to the preparation of small libraries of novel Fand F-labelled β-blockers. We report our preliminary cerebral ex vivo biodistribution studies of new [F]-labelled β-blockers, prepared via efficient synthetic methodologies, in rodents.