Tributylstannyl Precursor Research Articles

Synthesis and Fundamental Evaluation of Radioiodinated Rociletinib (CO-1686) as a Probe to Lung Cancer with L858R/T790M Mutations of Epidermal Growth Factor Receptor (EGFR).

Rociletinib (CO-1686), a 2,4-diaminopyrimidine derivative, is a highly potent tyrosine kinase inhibitor (TKI) that acts on epidermal growth factor receptor (EGFR) with L858R/T790M mutations. We supposed radioiodinated CO-1686 would function as a useful tool for monitoring EGFR L858R/T790M mutations. To aid in patient selection before therapy with EGFR-TKIs, this study aimed to develop a 125I-labeled derivative of CO-1686, N-{3-[(2-{[4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl]amino}-5-(trifluoromethyl)pyrimidine-4-yl] amino}-5-([125I]iodophenyl)acrylamide ([125I]ICO1686) and evaluate its selectivity toward EGFR L858R/T790M. Radiosynthesis was performed by iododestannylation of the corresponding tributylstannyl precursor with [125I]NaI and N-chlorosuccinimide. The selectivity of the tracer for detecting EGFR L858R/T790M was evaluated using three relevant non-small cell lung cancer (NSCLC) cell lines—H1975, H3255 and H441 overexpressing the dual mutation EGFR L858R/T790M, active mutant EGFR L858R and wild-type EGFR, respectively. The nonradioactive ICO1686 and the precursor compound were successfully synthesized. A novel radiolabeled probe, [125I]ICO1686, was prepared with high radiochemical yield (77%) and purity (>99%). ICO1686 exhibited high cytotoxicity toward H1975 (IC50 0.20 ± 0.05 μM) and H3255 (IC50 0.50 ± 0.21 μM), which is comparable to that of CO-1686. In contrast, the cytotoxicity of ICO1686 toward H441 was 10-fold lower than that toward H1975. In the cell uptake study, the radioactivity uptake of [125I]ICO1686 in H1975 was 101.52% dose/mg, whereas the uptakes in H3255 and H441 were 33.52 and 8.95% dose/mg, respectively. The uptake of [125I]ICO1686 in H1975 was greatly reduced to 45.61% dose/mg protein by treatment with excess CO-1686. In vivo biodistribution study of the radiotracer found that its accumulation in H1975 tumor (1.77 ± 0.43% ID/g) was comparable to that in H3255 tumor (1.63 ± 0.23% ID/g) and the accumulation in H1975 tumor was not reduced by pretreatment with an excess dose of CO-1686. Although this radiotracer exhibited highly specific in vitro uptake in target cancer cells, structural modification is required to improve in vivo biodistribution.

Synthesis and characterization of novel radiofluorinated probes for positron emission tomography imaging of monoamine oxidase B.

Monoamine oxidase B (MAO-B), predominantly expressed in glial cells, plays an important role in neurotransmitter regulation, and MAO-B activity relates to several neuronal diseases. Here, we aimed to develop a radiofluorinated MAO-B imaging probe based on the structure of a selective MAO-B inhibitor, MD-230254. We synthesized and evaluated a series of compounds in vitro and in vivo. A series of fluorinated analogs of MD-230254 were synthesized and evaluated for inhibitory potency and selectivity toward MAO-B. 5-[4-(2-[18 F]Fluorobenzyloxy)phenyl]-3-(2-cyanoethyl)-1,3,4-oxadiazol-2(3H)-one (2-[18 F]FBPO) was synthesized from a corresponding tributylstannyl precursor and [18 F]CH3 COOF. Biodistribution after intravenous injection of 2-[18 F]FBPO was evaluated in male ddY mice with or without pretreatment by inhibitors. Among the compounds synthesized and evaluated, 2-FBPO showed high inhibitory potency and selectivity toward MAO-B comparable with MD-230254. 2-[18 F]FBPO was successfully synthesized by an electrophilic reaction with a high radiochemical purity of more than 99%. 2-[18 F]FBPO was efficiently taken up by the brain and showed rapid blood clearance, which provided a brain/blood radioactivity ratio of 3.7 at 90 minutes postinjection. The brain radioactivity was significantly decreased by pretreatment with an MAO-B selective inhibitor. The great potential of 2-[18 F]FBPO as an MAO-B imaging probe, applicable to a variety of diseases, is indicated.

Synthesis and cellular uptake ofp-[123I]-phenyl-amino-thiazole (123I-PAT) as a potential agent for targeting tubulin polymerization in tumors

The phenyl-amino-thiazole (PAT) templates of methoxylbenzoyl-aryl-thiazole are potent agents against cancer by inhibiting tubulin polymerization in the nanomolar range. Herein, a radioiodinated PAT, [(123)I]-PAT 1, was prepared via a tributylstannyl precursor and [(123)I]iodide through electrophilic aromatic radioiodination. Radiolabelling of [(123)I]-PAT 1 was achieved in less than 15 min, with a radiochemical purity of over 99%. The accumulated radioactivity in tumor cellular uptake experiments suggested that [(123) I]-PAT could serve as a potential radioprobe for targeting tumor cells.

Radioiodinated sunitinib as a potential radiotracer for imaging angiogenesis—radiosynthesis and first radiopharmacological evaluation of 5-[125I]Iodo-sunitinib

Sunitinib® (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of RTKs might be useful tools for monitoring RTKs levels in tumour tissue giving valuable information for anti-angiogenic therapy. We report here the synthesis of a 125I-labeled derivative of sunitinib® and its first radiopharmaceutical characterization.The non-radioactive reference compound 5-iodo-sunitinib 4 was prepared by Knoevenagel condensation of 5-iodo-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. In a competition binding assay against VEGFR-2 a binding constant (Kd) of 16nM for 4 was found. The ability of 4 to inhibit tyrosine kinase activity was demonstrated on RTK expressing cells suggesting this radiotracer as a useful tool for monitoring VEGFR expression. 5-[125I]lodo-sunitinib, [125I]-4 was obtained via destannylation of the corresponding tributylstannyl precursor with [125I]NaI in the presence of H2O2 in high radiochemical yield (>95%) and radiochemical purity (<98%) after HPLC purification. Determination of human plasma protein binding at time intervals of 0; 1; 2; 4 and 24h suggested a low non-specific binding of 5-10%. Preliminary biodistribution studies of [125I]-4 in healthy CD-1 mice showed a relatively high uptake in VEGFR-2 rich tissues like kidney and lung followed by rapid washout (9.6 and 9.7; 4.5 and 3.8% ID/g of kidney and lung at 1 and 4h, respectively).

Synthesis and evaluation of radioiodinated phenoxyquinazoline and benzylaminoquinazoline derivatives as new EGF receptor tyrosine kinase imaging ligands for tumor diagnosis using SPECT

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) represents an attractive target for tumor diagnosis agents. Previously, the radioiodinated 4-(3-iodoanilino)-6,7-diethoxyquinazoline ([(125)I]m-IPQ) has been reported to possess good characteristics as a tumor imaging agent; however, it was also found to have low in vivo stability. To improve the in vivo stability, m-IPQ derivatives, 4-(3-iodophenoxy)-6,7-diethoxyquinazoline (PHY) and 4-(3-iodobenzylamino)-6,7-diethoxyquinazoline (BAY) were designed and synthesized, and the biological studies of [(125)I]PHY and [(125)I]BAY were performed to evaluate these new ligands as in vivo tumor diagnosis agents. PHY and BAY were synthesized according to previous reports. The EGFR-TK inhibitory potency of these new compounds was measured and compared to other EGFR-TK inhibitors. Radiolabeled [(125)I]PHY and [(125)I]BAY were synthesized by an iododestannylation reaction. Biodistribution studies of these radioligands were conducted in normal mice and tumor-bearing mice. Furthermore, selectivity and binding characteristics of [(125)I]PHY were analyzed by in vitro blocking studies and a binding assay. The new derivatives were found to have high inhibitory potency against EGFR-TK (PHY: IC50 = 12.7 ± 7.2 nM, BAY: IC50 = 51.0 ± 8.9 nM). [(125)I]PHY and [(125)I]BAY were conveniently synthesized from tributylstannyl precursors. In in vivo biodistribution studies, [(125)I]PHY and [(125)I]BAY were observed to have lower uptake in the stomach, an indication of deiodination, than [(125)I]m-IPQ. Moreover, [(125)I]PHY showed high uptake and prolonged retention in tumors and low accumulation in blood and muscle tissue resulting in a good tumor-to-blood ratio (0.94-1.50) and tumor-to-muscle ratio (1.02-1.95). The EGFR-TK selectivity of [(125)I]PHY was confirmed by pretreatment experiments with specific EGFR-TK inhibitors. New radioiodinated quinazoline derivatives were synthesized, which were found to have improved in vivo stability. In particular, [(125)I]PHY showed higher tumor accumulation than the other ligands which was indicative of selective binding to EGFR-TK. These desirable characteristics for [(125)I]PHY suggest that the (123)I-labeled counterpart, [(123)I]PHY, could be a possible candidate for cancer diagnosis radiopharmaceutical.

Efficient synthesis of [11C]H-1152, a PET probe specific for Rho-kinases, highly potential targets in diagnostic medicine and drug development

A novel PET probe, [11C]H-1152, specific for Rho-kinases, was synthesized efficiently for the first time based on rapid Pd0-mediated C-[11C]methylation using [11C]methyl iodide and a tributylstannyl precursor, giving the isolated radioactivity of 3.8±1.2 GBq (n=3), specific radioactivity of 97±10 GBq μmol−1 (n=3), and high chemical and radiochemical purities (>99%). The decay-corrected radiochemical yield based on 11CH3I was 63±14%. New potentials of a trifluoroacetyl (TFA) group as a tolerant protecting and facile deprotecting group of a secondary amine were demonstrated by highly selective stannylation (via lithiation of isoquinoline moiety using t-C4H9Li) and ready removal by hydroxide ion leaving the arylstannyl moiety intact, respectively. The role of excess hexamethylphosphoric triamide (HMPA) to realize an efficient stannylation of the isoquinoline lithium species is also discussed briefly.

5-O-(4-[125I]Iodobenzyl)-L-ascorbic Acid: Electrophilic Radioiodination and Biodistribution in Mice

As a part of our efforts to develop potential imaging agents for ascorbate bioactivity, 5-O-(4-[(125)I]iodobenzyl)-L-ascorbic acid ([(125)I]1) was prepared through a two-step sequence which involved radioiodo-destannylation of a protected tributylstannyl precursor 6, followed by hydrolysis in acidic methanol of the protecting groups in 61% overall radiochemical yield, with a radiochemical purity of over 98% and a specific activity of more than 15.4 GBq/μmol. Tissue distribution of [(125)I]1 in tumor-bearing mice showed signs of distribution profiles similar to the reported results for 6-deoxy-6-[(18)F]fluoro-L-ascorbic (6-(18)FAsA) acid and 6-deoxy-6-[(131)I]iodo-L-ascorbic acid (6-(131)IAsA) but with notable differences in the adrenal glands, in which considerably lower uptake of radioactivity and rapid clearance with time were observed. Pretreatment of mice with a known inhibitor of ascorbate transport, sulfinpyrazone, did not produce any significant change in the adrenal uptake of radioactivity after injection of [(125)I]1 compared to the control, suggesting that uptake in the adrenal glands is independent of the sodium-dependent vitamin C transporter 2 transport mechanism. Introduction of a bulky substituent at C-5 on AsA, such as an iodobenzyloxy group, may not be suitable for the design of analogs that may still be able to maintain characteristic distribution properties in vivo seen with AsA itself.

Radiohalogenated 4-anilinoquinazoline-based EGFR-TK inhibitors as potential cancer imaging agents

The overexpression of epidermal growth factor receptor (EGFR) in tumors underlines the recent interest in EGFR as attractive target for the development of new cancer imaging agents. EGFR-tyrosine kinase inhibitors (EGFR-TKIs) based on the anilinoquinazoline scaffold have been explored as potential probes for EGFR imaging. However, up to now, no optimal radiotracer is available. Herein, we report the synthesis and biological evaluation of three novel halogenated 6-substituted 4-anilinoquinazoline based EGFR-TKIs. Radiosynthesis ((125)I and (18)F) of the corresponding analogues was also performed. 6a, 6b and 8 were obtained by reaction of 6-amino-4-anilinoquinazoline (5) with 3-/4-iodobenzoyl and 4-fluorobenzoyl chlorides. Inhibition of EGFR autophosphorylation and A431 cellular proliferation were assessed by Western blot and MTT assays. (125)I-anilinoquinazolines [(125)I]6a/b were prepared via destannylation of the corresponding tributylstannyl precursors with [(125)I]NaI. Cellular uptake studies were conducted in A431 cells. Optimization of the radiosynthesis of the (18)F-anilinoquinazoline [(18)F]8 was attempted by nucleophilic substitution of the trimethylammonium- and nitro-6-substituted 4-anilinoquinazoline precursors. 6a, 6b and 8 were synthesized in high chemical yield. All of them are inhibitors of EGFR autophosphorylation (0.1<IC(50)<1 μM) and A431 cell proliferation (IC(50)<3.5 μM). [(125)I]6a/b, obtained in high radiochemical purity and specific activity, were highly taken up by A431 cells. Biodistribution profile in mice indicated fast blood clearance and hepatobiliary excretion. Despite all attempts, [(18)F]8 was only formed in 4% yield, hampering further biological evaluation. This study suggests that these quinazoline derivatives can act as EGFR-TKI, warranting further modifications in the chemical structure in order to be explored as potential molecular imaging agents for single photon emission computerized tomography and positron emission tomography.

PET study using [11C]FTIMD with ultra-high specific activity to evaluate I2-imidazoline receptors binding in rat brains

We recently developed a selective (11)C-labeled I(2)-imidazoline receptor (I(2)R) ligand, 2-(3-fluoro-4-[(11)C]tolyl)-4,5-dihydro-1H-imidazole ([(11)C]FTIMD). [(11)C]FTIMD showed specific binding to I(2)Rs in rat brains having a high density of I(2)R, as well as to I(2)Rs those in monkey brains, as illustrated by positron emission tomography (PET) and autoradiography. However, [(11)C]FTIMD also showed moderate non-specific binding in rat brains. In order to increase the specificity for I(2)R in rat brains, we synthesized [(11)C]FTIMD with ultra-high specific activity and evaluated its binding. [(11)C]FTIMD with ultra-high specific activity was prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [(11)C]methyl iodide, which was produced by iodination of [(11)C]methane using the single-pass method. Dynamic PET scans were conducted in rats, and the kinetic parameters were estimated. [(11)C]FTIMD with ultra-high specific activity was successfully synthesized with an appropriate level of radioactivity and ultra-high specific activity (4470 ± 1660 GBq/μmol at end of synthesis, n = 11) for injection. In the PET study, distribution volume (V(T)) values in all the brain regions investigated whether I(2)R expression was greatly reduced in BU224-pretreatead rats compared with control rats (29-45% decrease). Differences in V(T) values between control and BU224-pretreated rats using [(11)C]FTIMD with ultra-high specific activity were greater than those using [(11)C]FTIMD with normal specific activity (17-34% decrease) in all brain regions investigated. Quantitative PET using [(11)C]FTIMD with ultra-high specific activity can contribute to the detection of small changes in I(2)R expression in the brain.

Synthesis and evaluation of PET probes for the imaging of I2 imidazoline receptors in peripheral tissues

To explore the possible use of positron emission tomography (PET) probes for imaging of I(2)-imidazoline receptors (I(2)Rs) in peripheral tissues, we labeled two new I(2)R ligands, 2-[2-(o-tolyl)vinyl]-4,5-dihydro-1H-imidazole (K(i) for I(2)Rs, 3.7 nM) and 2-[2-(o-tolyl)ethyl]-4,5-dihydro-1H-imidazole (K(i) for I(2)Rs, 1.7 nM) with (11)C ([(11)C]metrazoline and [(11)C]TEIMD), respectively, and evaluated these ligands and the recently developed I(2)R ligand 2-[3-fluoro-[4-(11)C]tolyl]-4,5-dihydro-1H-imidazole ([(11)C]FTIMD) by in vivo studies. [(11)C]Metrazoline and [(11)C]TEIMD were prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [(11)C]methyl iodide. Their biodistribution in mice was investigated by tissue dissection. In addition, PET scans and metabolite analysis were performed. [(11)C]Metrazoline and [(11)C]TEIMD were successfully synthesized with a suitable radioactivity for injection. In the liver and pancreas expressing I(2)Rs, coinjection with the high-affinity I(2)R ligand, BU224, induced a reduction in the radioactivity level at 30 min after injection of [(11)C]metrazoline and [(11)C]FTIMD. However, the radioactivity level after injection of [(11)C]TEIMD was unchanged. In the PET study, coinjection with BU224 induced a decrease in the radioactivity level in the liver and pancreas after more than 15 min of injection of [(11)C]metrazoline and [(11)C]FTIMD as compared with the results obtained for controls. In metabolite analysis, coinjection with BU224 induced a significant reduction in the percentage of unchanged [(11)C]metrazoline at 30 min after injection as compared with that in the control, although no significant difference was observed in the percentage of unchanged [(11)C]FTIMD. [(11)C]Metrazoline may be a more useful PET probe than [(11)C]FTIMD for imaging of I(2)Rs in peripheral tissues.

Radiosynthesis of 123I‐labelled benzimidazoles as novel single‐photon emission computed tomography tracers for the histamine H3 receptor

The histamine H3 receptor is implicated in many neurological and psychological disorders and is therefore of interest as a target for pharmacological intervention. The drug development process can be facilitated by using a radiotracer for this receptor in conjunction with single‐photon emission computed tomography or positron emission tomography imaging studies, such as drug occupancy studies. This study investigates methods for the radiosynthesis of three compounds to be evaluated as novel radiotracers for the histamine H3 receptor. The radiosyntheses of the three target compounds, 123I‐WYE230949, 123I‐WYE126734 and 123I‐WYE127044 were evaluated using electrophilic iododesilylation and iododestannylation of the corresponding trimethylsilyl and tributylstannyl precursors. All three compounds could be produced in high radiochemical yield by electrophilic iododestannylation. By contrast, only two of the three (WYE230949 and WYE126734) could be produced by electrophilic iododesilylation. This is because of the fact that the trimethylsilyl precursors are less reactive than the stannyl precursors. Electrophilic iododestannylation of the tributylstannyl precursors is therefore the method of choice for radiosynthesis of these compounds because all three target compounds could be produced in high radiochemical yield.

Synthesis and preliminary biodistribution studies of [131I]SIB-PEG4-CHC in tumor-bearing mice

This work reports the synthesis and preliminary biodistribution results of [131I]SIB-PEG4-CHC in tumor-bearing mice. The tributylstannyl precursor ATE-PEG4-CHC was synthesized by conjugation of ATE to amino pegylated colchicine NH2-PEG4-CHC. [131I]SIB-PEG4-CHC was radiosynthesized by electrophilic destannylation of the precursor with a yield of ~44%. The radiochemical purity (RCP) appeared to be >95% by a Sep-Pak cartridge purification. [131I]SIB-PEG4-CHC was lipophilic and was stable at room temperature. Biodistribution studies in tumor-bearing mice showed that [131I]SIB-PEG4-CHC cleared from background rapidly, and didn’t deiodinate in vivo. However, the poor tumor localization excluded it from further investigations as a tumor-targeted radiopharmaceuticals.

Imaging of I2-imidazoline receptors by small-animal PET using 2-(3-fluoro-[4-11C]tolyl)-4,5-dihydro-1H-imidazole ([11C]FTIMD)

Imidazoline receptors (IRs) have been established as distinct receptors, and have been categorized into at least two subtypes (I(1)R and I(2)R). I(2)Rs are associated with depression, Alzheimer's disease, Huntington's disease and Parkinson's disease. A few positron emission tomography (PET) probes for I(2)Rs have been synthesized, but a selective PET probe has not been evaluated for the imaging of I(2)Rs by PET. We labeled a selective I(2)R ligand 2-(3-fluoro-4-tolyl)-4,5-dihydro-1H-imidazole (FTIMD) with (11)C and performed the first imaging of I(2)Rs by PET using 2-(3-fluoro-[4-(11)C]tolyl)-4,5-dihydro-1H-imidazole ([(11)C]FTIMD). [(11)C]FTIMD was prepared by a palladium-promoted cross-coupling reaction of the tributylstannyl precursor and [(11)C]methyl iodide in the presence of tris(dibenzylideneacetone)dipalladium(0) and tri(o-tol)phosphine. Biodistribution was investigated in rats by tissue dissection. [(11)C]FTIMD metabolites were measured in brain tissues and plasma. Dynamic PET scans were acquired in rats, and the kinetic parameters estimated. [(11)C]FTIMD was successfully synthesized with a suitable radioactivity for the injection. Co-injection with 0.1 mg/kg of cold FTIMD and BU224 induced a significant reduction in the brain-to-blood ratio 15 and 30 min after the injection. In metabolite analysis, unchanged [(11)C]FTIMD in the brain was high (98%) 30 min after the injection. In PET studies, high radioactivity levels were observed in regions with a high density of I(2)R. The radioactivity levels and V(T) values in the brain regions were prominently reduced by 1.0 mg/kg of BU224 pretreatment as compared with control. [(11)C]FTIMD showed specific binding to I(2)Rs in rat brains with a high density of I(2)R.

Synthesis and Characterization of Iodinated Tetrahydroquinolines Targeting the G Protein-Coupled Estrogen Receptor GPR30

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ER alpha/beta and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC(50) values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with (125)I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics.

Synthesis, radiosynthesis and in vivo evaluation of [123I]‐4‐(2‐(bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine as a selective tracer for imaging the dopamine transporter

Dopamine transporter (DAT) neuroimaging is a useful tool in Parkinson's disease diagnosis, staging and follow-up providing information on the integrity of the dopaminergic neurotransmitter system in vivo. 4-(2-(Bis(4-fluorophenyl)-methoxy)ethyl)-1-(4-iodobenzyl)piperidine (7) has nanomolar affinity for DAT and better selectivity over the other monoamine transporters compared with the existing SPECT radioligands for DAT. The aim of this study was to synthesize and evaluate [(123)I]-7 as an in vivo tracer for DAT.The tributylstannyl precursor was synthesized with an overall yield of 25%. [(123)I]-7 was synthesized by electrophilic destannylation with a yield of 40±10%. Radiochemical purity appeared to be >98%, whereas specific activity was at least 667 GBq/μmol. Biodistribution studies in mice showed brain uptake of 0.96±0.53%ID/g at 30 s post injection (p.i.) and 0.26±0.02%ID/g at 3 h p.i. High blood activity was observed at all time points. Pretreatment with Cyclosporin A raised brain uptake indicating that [(123)I]-7 is transported by P-glycoprotein (P-gp) pumps. In rats, regional brain distribution of [(123)I]-7 was not in agreement with DAT distribution. These results indicate that [(123)I]-7 is not suitable for mapping DAT in vivo but could be a useful tracer for the P-gp transporter.

Preclinical and the first clinical studies on [11C]CHIBA-1001 for mapping α7 nicotinic receptors by positron emission tomography

4-[(11)C]methylphenyl 2,5-diazabicyclo[3.2.2]nonane-2-carboxylate ([(11)C]CHIBA-1001), a 4-methyl-substituted derivative of the selective alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) partial agonist 4-bromophenyl 1,4 diazabicyclo[3.2.2]nonane-4-carboxylate (SSR180711), is a potential radioligand for mapping alpha7 nAChRs in the brain by positron emission tomography (PET). In this study, we performed preclinical and first clinical PET studies using [(11)C]CHIBA-1001 for imaging alpha7 nAChRs in the human brain. [(11)C]CHIBA-1001 was synthesized by methylation of the tributylstannyl precursor with [(11)C]CH(3)I in a palladium-promoted Stille cross-coupling reaction. The radiation absorbed-dose of [(11)C]CHIBA-1001 in humans was calculated from distribution data in mice. The acute toxicity of CHIBA-1001 at a dose of 3.20 mg/kg body weight, which is more than 41,000-fold the clinical equivalent dose of [(11)C]CHIBA-1001, was evaluated. The mutagenicity of CHIBA-1001 was studied by a reverse mutation test in Salmonella typhimurium (Ames test). Metabolite analysis in the mouse brain was carried out by high-performance liquid chromatography. The first clinical PET imaging of alpha7 nAChRs with [(11)C]CHIBA-1001 in a normal volunteer was also performed. A suitable preparation method for [(11)C]CHIBA-1001 injection was established. The radiation absorbed-dose by [(11)C]CHIBA-1001 in humans was low enough for clinical use, and no acute toxicity or mutagenicity of CHIBA-1001 was found. Most radioactivity in the mouse brain was detected as an unchanged form, although peripherally [(11)C]CHIBA-1001 was degraded. We successfully performed brain imaging by PET with [(11)C]CHIBA-1001 in a normal volunteer. A 90-min dynamic scan showed a rapid accumulation and gradual washout of radioactivity in the brain. The highest distribution volume of [(11)C]CHIBA-1001 was found in the thalamus; however, regional differences in brain radioactivity were small. Peripherally, [(11)C]CHIBA-1001 was stable in humans: >80% of the radioactivity in plasma was detected as the unchanged form for 60 min. These results demonstrate that [(11)C]CHIBA-1001 is a suitable radioligand to use in clinical trials for imaging alpha7 nAChRs in the human brain, providing acceptable dosimetry and pharmacological safety at the dose required for adequate PET imaging.

Radioiodinated Agents for Imaging Multidrug Resistant Tumors

Diagnostic agents enabling characterization of multidrug resistance (MDR) in tumors can aid in the selection of chemotherapy regimens. We report here synthesis and evaluation of radiopharmaceuticals based on the second-generation MDR-reversing drug MS-209. 5-[3-{4-(2-Phenyl-2-(4'-[(125)I]iodo-phenyl)acetyl)piperazin-1-yl}-2-hydroxypropoxy]quino-line (17) was prepared from the 4'-tributylstannyl precursor (16) in >95% radiochemical yield. (16) was synthesized in a six-step process with the overall yield of 25%. In vitro studies were conducted in MES-SA (drug-sensitive) and MES-SA/Dx5 (MDR) human uterine sarcoma cell lines. In vivo studies were performed in athymic mice bearing MES-SA and MES-SA/Dx5 xenografts. The uptake of (17) is higher in MES-SA than MES-SA/Dx5 cells. The uptake and efflux of (17) depend on temperature and concentration, and indicate active transport mechanism(s). Incubation of drug sensitive MES-SA cells with verapamil or (15), a nonradioactive analog of (17), alters the cellular retention of radioactivity only marginally. However, MES-SA/Dx5 cells retain approximately 12% more of (17) when incubated with 10 muM verapamil. The addition of (15) or high concentrations of (17) also increase the uptake of (17) in MES-SA/Dx5 up to 200%, depending on the concentration and temperature. The dependence of (17) uptake on the MDR status is also evident in the ex vivo binding studies. In vivo tests in mice xenografted simultaneously with both tumor cell lines indicate distinct pharmacokinetics for each tumor. The absorption half-life in MES-SA/Dx5 xenograft is approximately 10x shorter and the mean residence time approximately 50% shorter compared to MES-SA xenograft in the same mouse. Radioiodinated derivatives of MS-209 appear to be good indicators of multidrug resistance.

Synthesis of 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles and their in vitro and in vivo evaluation as imaging agents for amyloid plaque

A series of novel 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles was synthesized and their potential as imaging probes for Alzheimer’s Disease (AD)-related amyloid plaque was evaluated in vitro and in vivo. In vitro binding affinities for Aβ1–40 peptide of several of these compounds were in the low-nanomolar range . The lowest K i of 9.3 nM was found for N-(2-(4-(dimethylamino)phenyl)-1,3-benzoxazol-5-yl)-4-iodobenzamide ( 1e). Its 123I-radiolabeled form ([ 123I] 1e) was subsequently prepared by iododestannylation of the corresponding tributylstannyl precursor and evaluated in vivo in a baboon model using SPECT imaging. Contrary to our expectations, 1e did not cross the blood–brain barrier (BBB) to any significant extent.

A change of in vivo characteristics depending on specific activity of radioiodinated (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-pIV] as a ligand for sigma receptor imaging

The radioiodinated (+)-p-iodovesamicol [(+)-pIV], which shows a high binding affinity for sigma-1 (sigma-1) receptors, is prepared by an exchange reaction. The specific activity (SA) is fairly low and therefore is insufficient for clinical use. In this study, we prepared (+)-[(125)I]pIV with a high SA from tributylstannyl precursor and compared the in vivo characteristics between high and low SA by imaging sigma-1 receptors in the central nervous system. In the biodistribution study, a difference in brain accumulation was observed between the two methods. At 30 min postinjection, the brain accumulation (1.58%ID/g) of low SA [0.6-1.1 TBq/mmol (16-30 Ci/mmol)] (+)-[(125)I]pIV was higher than that (1.34%ID/g) of high SA [>88.8 TBq/mmol (>2400 Ci/mmol)] (+)-[(125)I]pIV. In the blocking study, the brain uptake of high SA (+)-[(125)I]pIV was reduced more significantly by the coadministration of sigma ligands such as pentazocine, haloperidol or SA4503 than that of low SA (+)-[(125)I]pIV. These results showed that nonspecific binding of high SA (+)-[(125)I]pIV in the brain was lower than that of low SA (+)-[(125)I]pIV, and high SA (+)-[(125)I]pIV bound more specifically to sigma-1 receptors in the brain than low SA (+)-[(125)I]pIV. In contrast, in the blood-binding study, high SA (+)-[(125)I]pIV (58.4%) bound to blood cells with higher affinity than low SA (+)-[(125)I]pIV (46.0%). In metabolite studies, blood metabolites of high SA (+)-[(125)I]pIV (57.3+/-3.5%) were higher than those of low SA (+)-[(125)I]pIV (45.5+/-4.1%) at 30 min postinjection. Higher SA may be apt to bind to blood cells with higher affinity and to be metabolized faster.

Mitochondrial avid radioprobes. Preparation and evaluation of 7′(Z)-[125I]iodorotenone and 7′(Z)-[125I]iodorotenol

The loss of mitochondrial function has been implicated in a number of maladies such as Huntington's disease, Parkinson's disease (PD), cancer and cardiovascular disease. The objective of this research was to develop a radiolabeled mitochondrial probe. Two tracers, 7'-Z-iodorotenol and 7'-Z-iodorotenone, analogs of rotenone a natural product that inhibits Complex I of the mitochondrial electron transport chain, have been labeled with iodine-125 in 45-85% yield in a single step from the corresponding tributylstannyl precursor. In vivo distribution in adult male Sprague-Dawley rats for both compounds showed high accumulation in the heart (1.7-3.7 %ID/g at 1 h), a tissue with high mitochondrial content. Z-Iodorotenol did not washout of most tissues between 1 and 2 h postinjection, whereas Z-iodorotenone showed moderate washout (7-26%) over the same period. By 24 h, there was significant loss of both compounds from most tissues including the heart. Heart-to-blood, -lung and -liver ratios for Z-iodorotenone of 28.9, 10.7 and 2.4, respectively, were two- to fourfold higher than the Z-iodorotenol ratios. Compared to the current clinical perfusion tracers, 99mTc-sestamibi and 99mTc-tetrofosmin, Z-iodorotenone demonstrates similar 1 h heart accumulation and significantly higher heart-to-lung ratio (P<.001). Z-Iodorotenone heart-to-liver ratio is equivalent to 99mTc-sestamibi. 7'-Z-Iodorotenone possesses distribution characteristics of an improved tracer for SPECT perfusion studies.