Svetlana V. Selivanova

Development of 3,4-dihydroisoquinolin-1(2H)-one derivatives for the Positron Emission Tomography (PET) imaging of σ2 receptors

σ₂ Receptors are promising biomarkers for cancer diagnosis given the relationship between the proliferative status of tumors and their density. With the aim of contributing to the research of σ₂ receptor Positron Emission Tomography (PET) probes, we developed 2-[3-[6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-3,4-dihydroisoquinolin-1(2H)-one (3), with optimal σ₂ pharmacological properties and appropriate lipophilicity. Hence, 3 served as the lead compound for the development of a series of dihydroisoquinolinones amenable to radiolabeling. Radiosynthesis for compound 26, which displayed the most appropriate σ₂ profile, was developed and σ₂ specific binding for the corresponding [(18)F]-26 was confirmed by in vitro autoradiography on rat brain slices. Despite the excellent in vitro properties, [(18)F]-26 could not successfully image σ₂ receptors in the rat brain in vivo, maybe because of its interaction with P-gp. Nevertheless, [(18)F]-26 may still be worthy of further investigation for the imaging of σ₂ receptors in peripheral tumors devoid of P-gp overexpression.

Studies toward the Development of New Silicon-Containing Building Blocks for the Direct 18F-Labeling of Peptides

Silicon-containing prosthetic groups have been conjugated to peptides to allow for a single-step labeling with (18)F radioisotope. The fairly lipophilic di-tert-butylphenylsilane building block contributes unfavorably to the pharmacokinetic profile of bombesin conjugates. In this article, theoretical and experimental studies toward the development of more hydrophilic silicon-based building blocks are presented. Density functional theory calculations were used to predict the hydrolytic stability of di-tert-butylfluorosilanes 2-23 with the aim to improve the in vivo properties of (18)F-labeled silicon-containing biomolecules. As a further step toward improving the pharmacokinetic profile, hydrophilic linkers were introduced between the lipophilic di-tert-butylphenylsilane building block and the bombesin congeners. Increased tumor uptake was shown with two of these peptides in xenograft-bearing mice using positron emission tomography and biodistribution studies. The introduction of a hydrophilic linker is thus a viable approach to improve the tumor uptake of (18)F-labeled silicon-bombesin conjugates.

Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability

INTRODUCTION Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. METHODS Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. RESULTS Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. CONCLUSIONS MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively.

Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9.

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no carrier added (18)F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO4(-) > Br(-) > TFA(-) > tosylate). (18)F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/μmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Synthesis and biological evaluation of 18F-labeled fluoropropyl tryptophan analogs as potential PET probes for tumor imaging

In the search for an efficient, fluorine-18 labeled amino acid based radiotracer for tumor imaging with positron emission tomography (PET), two new tryptophan analogs were synthesized and characterized in vitro and in vivo. Both are tryptophan alkyl-derivatives, namely 2-(3-[(18)F]fluoropropyl)-DL-tryptophan ([(18)F]2-FPTRP) and 5-(3-[(18)F]fluoro-propyl)-DL-tryptophan ([(18)F]5-FPTRP). Standard reference compounds and precursors were prepared by multi step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 29-34% decay corrected yields with radiochemical purity over 99%. In vitro cell uptake assays showed that both compounds are substrates for amino acid transport and enter small cell lung cancer cells (NCI-H69) most probably almost exclusively via large neutral amino acids transporter(s) (LAT). Small animal PET imaging with xenograft bearing mice revealed high tumor/background ratios for [(18)F]2-FPTRP comparable to the well established tyrosine analog O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET). Radiometabolite studies showed no evidence of involvement of a biotransformation step in tumor accumulation.

Single-step radiofluorination of peptides using continuous flow microreactor

18F radiolabelling of peptides bearing two different prosthetic groups was successfully conducted in a continuous flow microfluidic device for the first time. Radiochemical yields were dependent on precursor concentration, reaction temperature and flow rate. The choice of leaving group had a dramatic influence on the reaction outcome. Rapid reaction optimization was possible.

Radiofluorinated histamine H3 receptor antagonist as a potential probe for in vivo PET imaging: Radiosynthesis and pharmacological evaluation

The histamine H(3) receptor (H(3)R) plays a role in cognition and memory processes and is implicated in different neurological disorders, including Alzheimers disease, schizophrenia, and narcolepsy. In vivo studies of the H(3)R occupancy using a radiolabeled PET tracer would be very useful for CNS drug discovery and development. We report here the radiosynthesis, in vitro and in vivo evaluation of a novel (18)F-labeled high-affinity H(3)R antagonist (18)F-ST889. The radiosynthesis was accomplished via nucleophilic substitution of the mesylate leaving group with a radiochemical yield of 8-20%, radiochemical purity >99%, and specific radioactivity > 65 GBq/μmol. (18)F-ST889 exhibited high in vivo stability and rather low lipophilicity (logD(7.4)=0.35 ± 0.09). In vitro autoradiography showed specific binding in H(3)R-rich brain regions such as striatum and cortex. However, in vivo PET imaging of the rat brain with (18)F-ST889 was not successful. Possible reasons are discussed.

Synthesis and pharmacological evaluation of 11C-labeled piperazine derivative as a PET probe for sigma-2 receptor imaging

INTRODUCTION Both subtypes of sigma (σ) receptors, σ₁ and σ₂, are over-expressed in many cancers with σ₂ proposed as a biomarker of tumor proliferation. We are interested in developing a high affinity selective σ₂ radioligand for in vivo monitoring of proliferative status of solid tumors and response to anti-cancer therapies. 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) represents one of the lead candidates in the development of σ receptor ligands for therapeutic and diagnostic applications. However, the utility of PB28 is limited due to its relatively high lipophilicity. METHODS A more hydrophilic analogue (-)-(S)-1 was radiolabeled with (11)C via standard O-alkylation. In vitro autoradiography with [(11)C](-)-(S)-1 was done using rat brain slices. PET imaging was performed in mice bearing EMT6, C6 or PC-3 tumors after i.v. injection of [(11)C](-)-(S)-1. RESULTS [(11)C](-)-(S)-1 was produced in 53%±7% isolated decay-corrected yield with radiochemical and chemical purity over 99% and specific activity greater than 100 GBq/μmol. In vitro autoradiography with [(11)C(-)-(S)-1 resulted in a heterogeneous binding of the tracer in the rat brain with the highest radioactivity signals in the cortex region followed by cerebellum. This binding was successfully blocked by 10 μM of either haloperidol, (+)-(R)-1 or PB28. For C6 xenografts low target-to-nontarget ratio and high non-specific binding did not allow clear tumor visualization. No accumulation was visible in EMT6 tumor or in PC-3 tumor. Rat and mouse brain uptake was low and homogeneous while stronger signal was detected in the spinal cord. High accumulation of radioactivity was observed in liver and intestine suggesting hepatobiliary clearance. CONCLUSIONS Despite excellent in vitro properties, [(11)C](-)-(S)-1 did not provide high enough specific binding in vivo and is, therefore, not a useful PET tracer for imaging σ₂ expression in tumors.

In Vitro and In Vivo Evaluation of N-{2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[11C]methoxybenz­amide, a Positron Emission Tomography (PET) Radioligand for Dopamine D4 Receptors, in Rodents

The D4 dopamine receptor belongs to the D2‐like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D4 receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D4 receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high‐affinity D4 receptor‐selective ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[11C]methoxybenzamide ([11C]2) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D4 radioligand should possess in order to have improved in vivo utility in rodents.

Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein Inhibitors

Kinesin spindle protein (KSP), an ATP‐dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide)) assays, combined with fluorescence‐assisted cell sorting (FACS) and Western blot studies analyzing cell‐cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7, exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.