Development of a Tc-99m labeled sigma-2 receptor-specific ligand as a potential breast tumor imaging agent.

A novel in vivo imaging agent, 99mTc labeled [(N-[2-((3'-N'-propyl-[3,3,1]aza-bicyclononan-3alpha-yl)(2"-methoxy-5-methyl-phenylcarbamate)(2-mercaptoethyl)amino)acetyl]-2-aminoethanethiolato] technetium(V) oxide), [99mTc]2, displaying specific binding towards sigma-2 receptors was prepared and characterized. In vitro binding assays showed that the rhenium surrogate of [99mTc]2, Re-2, displayed excellent binding affinity and selectivity towards sigma-2 receptors (K(i) = 2,723 and 22 nM for sigma-1 and sigma-2 receptor, respectively). Preparation of [99mTc]2 was achieved by heating the S-protected starting material, 1, in the presence of acid, reducing agent (stannous glucoheptonate) and sodium [99mTc]pertechnetate. The lipophilic racemic mixture was successfully prepared in 10 to 50% yield and the radiochemical purity was >98%. Separation of the isomers, peak A and peak B, was successfully achieved by using a chiralpak AD column eluted with an isocratic solvent (n-hexane/isopropanol; 3:1; v/v). The peak A and peak B appear to co-elute with the isomers of the surrogate, Re-2, under the same HPLC condition. Biodistribution studies in tumor bearing mice (mouse mammary adenocarcinoma, cell line 66, which is known to over-express sigma-2 receptors) showed that the racemic [99mTc]2 localized in the tumor. Uptake in the tumor was 2.11, 1.30 and 1.11 %dose/gram at 1, 4 and 8 hr post iv injection, respectively, suggesting good uptake and retention in the tumor cells. The tumor uptake was significantly, but incompletely, blocked (about 25-30% blockage) by co-injection of "cold" (+)pentazocine or haloperidol (1 mg/Kg). A majority of the radioactivity localized in the tumor tissue was extractable (>60%), and the HPLC analysis showed that it is the original compound, racemic [99mTc]2 (>98% pure). The distribution of the purified peak A and peak B was determined in the same tumor bearing mice at 4 hr post iv injection. The tumor uptake was similar for both isomers, but the blood and peripheral tissue content for the isomer in peak B was higher than that for the isomer in peak A. It is evident that the isomer in peak A displayed significantly better tumor/blood and tumor/muscle ratios. The higher rate of in vivo metabolism was also confirmed by the higher thyroid uptake values for the isomer in peak B as compared to peak A. In summary, a 99mTc-labeled sigma receptor imaging agent, [99mTc]2, has demonstrated the feasibility of using a 99mTc-labeled agent for imaging sigma receptor expression in tumor cells. This is the first time a subtype-selective 99mTc-labeled agent for imaging sigma receptor sites is reported.

Characterization of [125I]S(-)5-OH-PIPAT binding to dopamine D2-like receptors expressed in cell lines.

Recently, [125I]S(-)5-OH-PIPAT [5-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)aminotetralin], a derivative of S(-)5-OH-DPAT (5-hydroxy-N,N-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine D2-like receptor ligand than the previously reported iodinated ligand, [125I]R(+)7-OH-PIPAT. Therefore, in the present study, the binding profile of [125I]S(-)5-OH-PIPAT to D2-like receptors expressed in cell lines was established. High binding affinity (Kd = 0.3-0.4 nM) and NaCl sensitivity were displayed with this ligand in membranes of human embryonic kidney (HEK293) cells expressing either human D2 or rat D3 receptors and in Chinese hamster ovary (CHO) cells expressing human dopamine D4 receptors. Specific binding to D2 and D4 receptors was significantly increased in the presence of 2 mM MgCl2 and decreased in the presence of 100 microM 5'-guanylyl-imidodiphosphate (GMP-PNP). This finding is consistent with reports that 2-aminotetralin compounds display agonist properties. The specific binding to D3 receptors however, was not affected by either MgCl2 or GMP-PNP. This lack of GMP-PNP sensitivity for D3 receptors may result from inadequate G protein-receptor coupling in this cell line. The rank order of potency for inhibition of [125I]S(-)5-OH-PIPAT binding with various dopamine agents was consistent with reported values for D2, D3 and D4 receptors. In membranes prepared from Spodoptera frugiperda (Sf9) cells infected with baculovirus that contains DNA encoding D3 receptors, [125I]S(-)5-OH-PIPAT recognized only 70% of the receptor population labeled by [125I]NCQ298. This new ligand offers several unique advantages, including high specific activity, high binding affinity and selectivity for D2-like receptors, that make it an excellent probe for the investigation and the characterization of dopamine D2-like receptors.

Synthesis and characterization of technetium-99m-labeled tropanes as dopamine transporter-imaging agents.

In the development of novel Tc-99m-labeled tropane derivatives as dopamine transporter (reuptake site)-imaging agents, a series of neutral and lipophilic complexes containing bis-(aminoethanethiol) as a neutral complexing moiety for a [99mTc]TcO3+ center core was successfully prepared. Biological evaluation of the Tc-99m-labeled complexes 13-16 as central nervous system (CNS) dopamine transporter-imaging agents was reported. Synthesis of the tropane derivatives was achieved by stepwise reactions adding two aminoethanethiol units. The final free thiol ligands were obtained by deblocking the 4-methoxybenzyl protecting group with Hg(OAc)2 to obtain trifluoroacetate salts. All of the Tc-99m complexes, with the exception of 16, displayed good initial brain uptake and selective uptake in the striatal area, where dopamine transporters are concentrated. One of the compounds, [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo [3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethy] amino]ethanethiolato-(3-)-N2,N2',S2,S2'] oxo-[1R-(exo-exo)]- [99mTc]technetium,[99mTc]TRODAT-1 (13), displayed the highest initial uptake in rat brain (0.4% at 2 min post iv injection); the striatal/cerebellar (ST/ CB) ratio reached 2.8 at 60 min after an iv injection. The specific uptake in rat brain can be blocked by pretreating rats with a competing dopamine transporter binding agent, beta-CIT (RTI-55, 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane; iv, 1 mg/kg), which reduced the regional brain uptake ratio (ST/CB) to 1.2. In contrast, the specific striatal uptake was not affected by pretreating rats with a noncompeting ligand, haldol (iv, 1 mg/kg). After an iv injection of 9 mCi of [99mTc]TRODAT-1 (13), in vivo images of baboon brain using single-photon emission-computed tomography exhibited excellent localization in striatum (basal ganglia), where dopamine neurons are known to be concentrated. This series of compounds may provide a convenient source of short-lived imaging agents for routine diagnosis of CNS diseases (i.e., Parkinson's disease) in which changes in the dopamine transporter concentration are implicated.

Characterization of a novel iodinated ligand, IPMPP, for human dopamine D4 receptors expressed in CHO cells.

A novel radioiodinated ligand with a high specific activity (2,200 Ci/mmol), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine ([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 +/- 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(-)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [125I]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contrast to [125I]S(-)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPgammaS binding further demonstrated the antagonist property of IPMPP.

Quantitative autoradiographic studies of dopamine D3 receptors in rat cerebellum using [125I]S(-)5-OH-PIPAT.

Recently, [125I]S(-)5-OH-PIPAT (5-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)amino-tetralin) was reported to be a selective radioiodinated ligand for dopamine D2-like receptors. This ligand displayed a high binding affinity (Kd = 0.3-0.4 nM) and an agonist binding profile to dopamine D2 and D3 receptors expressed in HEK293 cells and dopamine D4 receptors expressed in CHO cells. Herein, a series of studies to characterize D3 receptors in native tissues is presented. Based on studies of the distribution of receptor mRNA, D3, but not D2, receptors are present in the rat cerebellum. Quantitative autoradiographic experiments using [125I]S(-)5-OH-PIPAT to label molecular layers 9 and 10 of rat cerebellum were conducted. Saturation experiments demonstrated that [125I]S(-)5-OH-PIPAT bound with high affinity (Kd = 0.1 nM) to a low density (approximately 3 fmol/mg protein) of sites in molecular layers 9 and 10 of rat cerebellum. Increasing concentrations of Gpp(NH)p, but not ATP, decreased the specific binding of [125I]S(-)5-OH-PIPAT in rat cerebellum slices. In comparison studies, binding of [125I]NCQ298, a dopamine D2/D3 receptor antagonist, with a similar affinity (Kd = 0.2 nM) for D3 receptors as [125]S(-)5-OH-PIPAT, was not sensitive to Gpp(NH)p. Analysis of inhibition by S(-)5-OH-PIPAT of [125I]NCQ298 binding to rat cerebellum resulted in two-site binding with IC50 values of 0.07 nM and 6.0 nM. In the presence of GTP (300 microM), the data best fit a one-site model with an IC50 value of 1.6 nM. Agonists and antagonists inhibited the binding of [125I]S(-)5-OH-PIPAT in the cerebellum with a rank order of potency consistent with an interaction at D3 receptors. These results indicate that [125I]S(-)5-OH-PIPAT binds to D3 receptors in rat cerebellum. Furthermore, [125I]S(-)5-OH-PIPAT binds to GTP sensitive and GTP insensitive states of D3 receptors with distinctive high and low affinity states, respectively.

Tc-99m-labeled tropanes as dopamine transporter imaging agents.

The development of novel Tc-9m-labeled tropane derivatives as dopamine transporter imaging agents is reported. A series of neutral and lipophilic conjugated complexes, containing N-(alkylthiolato)-tropane, aminobis(ethylthiolato), and a [99mTc]TcO3+ center core, were prepared and evaluated as central nervous system (CNS) dopamine transporter imaging agents in rats. One of the compounds, [99mTC]technetium, [methyl 3-(4-chlorophenyl)-8-(2-mercaptoethyl-8-azabicyclo [3.2.1]octane-2-carboxylato-S][[2,2'-(methylimino)bis[eth anethiolato]] (2-)-N,S,S']oxo (25), displayed low initial uptake in rat brain (0.1% at 2 min post i.v. injection); the striatal/cerebellar (ST/CB) ratio reached 3.50 at 60 min after an i.v. injection. The specific uptake can be blocked by pretreating rats with a competing dopamine transporter binding agent, beta-CIT (RTI-55, N-methyl-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane; i.v. 1 mg/kg), which reduced the regional brain uptake ratio (ST/CB) to 1.0. In contrast, the specific uptake in striatum was not affected by pretreating rats with a noncompeting ligand, haldol (i.v., 1 mg/kg). In vitro autoradiography of rat brain sections exhibited elevated labeling in striatum, major islands of Calleja, and olfactory tubercle regions, where dopamine neurons are known to be concentrated. This series of compounds is the first example of technetium-99m labeled CNS receptor-specific imaging agents and may provide a convenient source of short-lived imaging agents for routine diagnosis of CNS abnormality in conjunction with single photon emission computed tomography.

In vitro and in vivo characterization of R(+)-FIDA2: a dopamine D2-like imaging agent.

UNLABELLED: R(+)-FIDA2, (R)-(+)-2,3-dimethoxy-5-iodo-N-[(1-(4'-fluorobenzyl)-2-pyrrolid iny l)- methyl]benzamide, is a new dopamine D2-like receptor imaging agent that can be labeled with either 123I or 18F for SPECT or PET imaging. The purpose of this study was to characterize its in vitro and in vivo binding properties. METHODS: In vitro binding studies using [125I]R(+)-FIDA2 were performed in Sf9 cells expressing dopamine D2 or D3 receptors and in rat basal forebrain homogenates, which contain a high density of dopamine D2-like receptors. A series of in vivo SPECT imaging studies in nonhuman primates (cynomologous monkeys) were performed by intravenously injecting 7.1 +/- 1.0 mCi of [123I]R(+)-FIDA2. At least one control study and one displacement experiment, in which a cold compound was injected intravenously 90 min after tracer injection, was performed in each monkey. Data were acquired in 10-min frames for 180 min, and the activity in regions of interest (basal ganglia and cerebellum) were plotted versus time. RESULTS: Iodine-125-R(+)-FIDA2 displayed Kd values for D2 and D3 receptor subtypes expressed in Sf9 cells of 0.11 and 0.04 nM, respectively. As expected, SPECT images of monkey brain (transaxial sections, 2 mm) showed that the radioactivity was localized in the area of the basal ganglia and reached peak concentrations in 11.5 +/- 5.8 min postinjection. An injection of R(+)7-OH-PIPAT, a new ligand that is selective for dopamine D3 receptors and the high affinity state of dopamine D2 receptors, did not show significant displacement of [123I]R(+)-FIDA2 binding in the basal ganglia. CONCLUSION: These studies indicate that R(+)-FIDA2 may be a useful ligand for in vitro pharmacological characterization and in vivo imaging of CNS dopamine D2-like receptors.

Dosimetry of a D2/D3 dopamine receptor antagonist that can be used with PET or SPECT.

UNLABELLED: FIDA-2 (R-(+)-2,3-dimethoxy-5-iodo-N-[(1-4'-fluorobenzyl)-2-pyrrolidinyl) methyl] benzamide) is a simultaneously fluorinated and iodinated D2/D3 dopamine receptor antagonist. The purpose of this study was to measure its biodistribution and radiation dosimetry in humans. METHODS: Whole-body emission scans were sequentially acquired in eight healthy volunteers 24-43 hr after the intravenous administration of 101-150 MBq 123I FIDA-2. Regions of interest (ROIs) were placed on the initial set of conjugate emission images and transposed as a single set onto all the other scans without manipulating any of the regions for solid organs independently. The counts in each ROI were corrected for attenuation with transmission scans and compared to the net counts in images of the injection syringe containing the administered dose. The radiation doses were estimated with the MIRD formalism from the residence times for both the 18F- and 123I-labeled ligands. RESULTS: There were no subjective or objective pharmacological effects of the tracer on any of the subjects. The findings showed that the dose-limiting organ for the 123I-labeled product was the thyroid gland in this sample. If the 18F-labeled product had been used, then the urinary bladder would have received 0.086 mGy/MBq (0.32 rads/mCi) and become the dose-limiting organ. The effective dose equivalents were 0.025 mSv/MBq (0.092 rem/mCi) for both the 123I- and the 18F-labeled versions of the tracer. CONCLUSION: The data suggest that FIDA-2 can be used to produce relatively high contrast images of the D2/D3 dopaminergic system with substantially less than the maximum allowable radiation dose for research volunteers.

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain.

Binding characteristics of a novel radioiodinated ligand, [125I]R(+)trans-7-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl) aminotetralin ([125I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [125I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (Kd = 0.42 nM). A majority (70%) of the sites labeled by [125I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [125I]R(+)trans-7-OH-PIPAT to 5-HT1A receptors, with a Kd value of 1.4 nM and a Bmax value of 210 fmol/mg protein, was observed. Binding of [125I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D2 and 5-HT1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [125I]R(+)trans-7-OH-PIPAT. [125I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D3 receptors.

Derivatives of 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl-p-iodobenzamido)ethyl]pipera zine (p-MPPI) as 5-HT1A ligands.

A series of new p-alkylbenzamido derivatives of 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p- iodobenzamido)ethyl]piperazines (p-MPPI) were prepared. In vitro binding studies suggest that p-methyl and p-ethyl substituents on the benzamido group display the same high binding affinity to 5-HT1A receptors (Ki = 2.2 and 9.3 nM, rat hippocampal homogenates). However, when the substitution groups were larger than a C5 pentyl group, the affinity to 5-HT1A receptors dropped below a useful level (Ki > 50 nM). Several irreversible binding agents (CH2Cl, NHCOCH2Cl) and a photoaffinity labeling compound (m-iodo p-azido) which showed good binding affinity to 5-HT1A receptors were successfully prepared.

Iodinated 2-aminotetralins and 3-amino-1-benzopyrans: ligands for dopamine D2 and D3 receptors.

In developing selective ligands for dopamine D2 and D3 receptors, several iodinated 2-aminotetralins and 3-amino-1-benzopyrans, trans-7-hydroxy-2-[N-(3'-iodo-2'- propenyl)amino]tetralin (1), trans-monohydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin (7-, 5-, and 6-OH-PIPAT) (2, 3, and 4), and trans-monohydroxy-3,4-dihydro-3-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]-2H-1-benzopyran (6- and 8-OH-benzopyrans) (5 and 6), were prepared. These compounds were evaluated for their binding profiles in several membrane preparations: Spodoptera frugiperda (Sf9) cells expressing dopamine D2 (non-GTP coupled, low-affinity states) and D3 receptors, HEK293 cells expressing dopamine D2 receptors in high-affinity states (D2H), rat hippocampal homogenates for 5-HT1A receptors, and cerebellar homogenates for sigma receptors. The mono-N-alkylated 2-aminotetralin, 1, displayed high sigma binding (Ki = 1.68 nM) with a moderate D3 binding (Ki = 30.2 nM). Derivatives with one N-propyl and one N-(3'-iodo-2'-propeny) group generally displayed high to moderate affinity to D3 receptors (Ki = 2.90, 1.85, 0.99, 2.20, 31.4, and 6.69 nM for 7-OH-DPAT [7-hydroxy-2-(N,N-di-n-propylamino)tetralin], 2, 3, 4, 5, and 6, respectively). It is interesting to note that all of the active D3 ligands also displayed comparable binding to the high affinity states of D2 receptors in HEK293 cells (Ki = 6.6, 3.6, 9.7, and 10.8 nM for 2, 3, 4, and 6, respectively). Among all of the tetralin derivatives tested, 5-OH-PIPAT, 3, showed the highest binding affinity to D3 receptors (Ki = 0.99 nM) and better selectivity (KiD2H/KiD3, KiD2/KiD3, Ki5-HT1A/KiD3 and Ki sigma/KiD3 = 3.64, 327, 48.4, and 1250 nM, respectively), making it the best ligand for studying dopamine D2H and D3 receptors.

Characterization of [125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin binding to dopamine D3 receptors in rat olfactory tubercle.

[125I](R,S)-trans-7-Hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)- amino]tetralin ([125](R,S)-trans-7-OH-PIPAT) has been shown to bind with high affinity to dopamine D3 receptors expressed in Spodoptera frugiperda cells. No specific binding was seen in Spodoptera frugiperda cells expressing a high density of D2 receptors. It was therefore, suggested that [125I] (R,S)-trans-7-OH-PIPAT selectively labels D3 receptors. In the present study, saturation binding of [125I](R)-trans-7-OH-PIPAT to membranes from rat olfactory tubercle resulted in markedly curvilinear Scatchard plots, suggesting that the radioligand was binding to more than one receptor class or affinity state. [125I] (R)-trans-7-OH-PIPAT bound with high affinity to membranes from human embryonic kidney-293 cells expressing transfected D2 or D3 receptors and to membranes from Chinese hamster ovary cells expressing serotonin1A receptors. Binding of [125I](R)-trans-7-OH-PIPAT to serotonin1A and D2 receptors was decreased or eliminated in the presence of NaCl and/or guanylyl-imidodiphosphate [Gpp(NH)p]. In the presence of Gpp(NH)p and NaCl, a linear Scatchard plot with a Kd value of 0.4 nM and a density of 100 fmol/mg of protein was obtained in membranes from rat olfactory tubercle. Agonists and antagonists inhibited binding of [125I](R)-trans-7-OH-PIPAT with a rank order of potency consistent with an interaction at D3 receptors. These results suggest that, in the presence of Gpp(NH)p and NaCl, [125I](R)-trans-7-OH-PIPAT specifically labels D3 receptors.

[125I](S)-trans-7-OH-PIPAT: a potential spect imaging agent for sigma binding sites.

[125I](S)-trans-7-hydroxy-2-[(N-n-propyl-N-(3'-iodo-2'-propenyl)] aminotetralin ([125I](S)-trans-7-OH-PIPAT) has been prepared as an iodinated radioligand for studying the sigma binding site. [125I](S)-trans-7-OH-PIPAT binds to rat cerebellar membranes with a Kd = 1.67 +/- 0.07 nM and Bmax = 240 +/- 72 fmol/mg of protein (determined in the presence of 15 nM spiperone). This new ligand appears to bind to only one site with Hill coefficients close to unity. Inhibition constants for competing ligands determined in the cerebellar tissue homogenates (in the presence of 15 nM spiperone) are closely comparable to inhibition constants determined in the whole brain tissue homogenates (in the absence of spiperone). Furthermore, these inhibition constants are consistent with the values reported for typical sigma ligands. In vivo uptake of [125I](S)-trans-7-OH-PIPAT in the rat brain is initially high (2.52% dose/organ at 2 min post i.v. injection) and displays a rapid washout from the brain (0.8% dose/organ at 30 min post i.v. injection). Uptake of [125I](S)-trans-7-OH-PIPAT shows moderate target to non-target ratios at 30 minutes (1.54, 1.66 and 1.92 for cerebellar, hypothalamic and hindbrain uptake over striatal uptake, respectively). Pre-injection with haloperidol reduced these ratios to unity suggesting that the ligand binds specifically to haloperidol-sensitive sites in vivo. The selectivity and affinity of [125I](S)-trans-7-OH-PIPAT suggest that this new iodinated ligand may be useful for in vitro studies of the sigma sites and can be used in vivo as a potential SPECT imaging agent.

Synthesis and optical resolution of (R)- and (S)-trans-7-Hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin: a new D3 dopamine receptor ligand.

An improved method for synthesis and resolution of (R,S)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino]tetralin (trans-7-OH-PlPAT,5), a new D3 dopamine receptor ligand, was reported. Both isomers, (R)-(+)- and (S)-(-)-5, were prepared and characterized. HPLC retention times obtained on a chiral column for these isomers were consistent with those observed for [125I]-(R)-(+)- and (S)-(-)-5. Direct radioiodination of an optically resolved tin precursor, (R)-(+)-7, yielded the desired [125I](R)-(+)-5, which is a simpler method for synthesis of this ligand. Binding studies with membrane preparations containing D3 dopamine receptors expressed in Spodoptera frugiperda (Sf9) cells also suggested that the [125I](R)-(+)-5 is the active isomer (Kd = 0.05 nM). The schemes described may provide an efficient way for synthesizing a large quantity of this new D3 dopamine receptor ligand for in vivo behavior studies.

In vitro binding of a novel dopamine D3 receptor ligand: [125I]trans-7-OH-PIPAT-A.

An iodinated dopamine D3 receptor ligand, [125I]trans-7-OH-PIPAT-A-trans-7-hydroxy-2-(N-n-propyl-N-3'-iodo-2 '- propenyl)aminotetralin, was developed. This ligand demonstrated unique high affinity and selectivity toward the dopamine D3 receptor; Kd = 0.48 nM and Bmax = 240 fmol/mg of protein in rat striatal membrane homogenates.

Fluorinated and iodinated dopamine agents: D2 imaging agents for PET and SPECT.

A novel series of dual-labeling D2 dopamine agents (labeled with either 18F or 123I for PET or SPECT imaging, respectively) was investigated. Two desired fluorinated and iodinated dopamine agents, FIDA1, (S)-(-)-2-(2-fluoroethoxy)-5-iodo-3-methoxy-N-[(1-ethyl-2- pyrrolidinyl)methyl]-benzamide, and FIDA2, (R)-(+)-2,3-dimethoxy-5-iodo-N-[(1-(4'-fluorobenzyl)-2- pyrrolidinyl)-methyl]benzamide, were synthesized. Both compounds displayed high affinity to the D2 receptor of rat striatal membrane preparations (Kd = 0.13 and 0.02 nM for FIDA1 and FIDA2, respectively). The biodistribution study in rats exhibited high localization in the striata of the brain with the striatum/cerebellum ratio reaching 29.3 and 13.1 at 1 h post iv injection for FIDA1 and FIDA2, respectively. Imaging studies with [18F]- and [123I]FIDA2 in monkeys, with PET and SPECT, respectively, showed comparable high selective striatal uptake. These results suggest that they are potentially useful D2 dopamine receptor imaging agents for PET and SPECT.

Iodinated tomoxetine derivatives as selective ligands for serotonin and norepinephrine uptake sites.

In order to develop selective radioactive ligands for the study of presynaptic monoamine uptake sites, iodinated derivatives of tomoxetine were synthesized and evaluated in radioligand binding assays. Iodotomoxetine derivatives showed high affinity for serotonin (5-HT) uptake sites using a rat cortical membrane preparation. Compound 1R,(R)-(-)-N-methyl-3-(4-iodo-2-methylphenoxy)-3-phenylpropanamine , was the most potent and showed high stereoselectivity for 5-HT uptake sites (Ki, R isomer = 0.65 nM, S isomer = 13.9 nM). Changing the position of the methyl group or eliminating the methyl group at the phenoxy ring resulted in a loss of stereoselectivity. Substitution of the methyl group of tomoxetine with iodine gave the R and S isomers of N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine 4R and 4S. These compounds displayed stereoselectivity for the norepinephrine (NE) (Ki values = 0.24 and 9.35 nM for R and S isomers, respectively). The in vitro binding data suggest that 1R and 4R are potential radioiodinated ligands for pharmacological studies of 5-HT and NE uptake sites, respectively.

4-Iodotomoxetine: a novel ligand for serotonin uptake sites.

The tomoxetine analog, R-4-iodotomoxetine, binds in vitro to a single site of rat cortical membranes with high affinity (Kd = 0.03 +/- 0.01 nM, n = 4) and can be blocked by a selective serotonin reuptake site inhibitor, paroxetine. The [125I]R-4-iodotomoxetine binding at equilibrium is saturable and is temperature- and Na(+)-dependent. The number of specific [125I]R-4-iodotomoxetine binding sites (Bmax = 356 +/- 20 fmol/mg protein) is similar to that of [3H]citalopram (329 +/- 30 fmol/mg protein), a known serotonin uptake inhibitor. The binding of [125I]R-4-iodotomoxetine is selectively inhibited by several serotonin uptake blockers, and a good correlation is demonstrated between the potency of various drugs to inhibit in vitro binding of [125I]R-4-iodotomoxetine and [3H]citalopram. In addition, lesions performed with the neurotoxin p-chloroamphetamine, which destroys monoamine neurons, including serotonergic neuronal system, result in a 90% reduction of [125I]R-4-iodotomoxetine binding when compared to sham controls. These results indicate that the binding sites labeled by [125I]R-4-iodotomoxetine are associated with the neuronal serotonin uptake sites. However, the in vivo and ex vivo results do not show regional localization corresponding to the distribution of serotonin uptake sites. The nonspecific uptake may be related to this compound's high lipophilicity (octanol-buffer partition coefficient = 1100 - 1400 at pH 7). Although the in vivo properties of [125I]R-4-iodotomoxetine make it an unlikely candidate for mapping serotonin uptake sites with SPECT, the high affinity and selectivity should make it a useful tool for in vitro studies of the serotonin uptake sites.

Characterization of radioiodinated TISCH: a high-affinity and selective ligand for mapping CNS D1 dopamine receptor.

In developing CNS D1 dopamine receptor-imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine], an analog of SCH 23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)-[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(-) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD = 0.21 +/- 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 greater than (+/-)-TISCH greater than (+)-butaclamol = (+/-)-FISCH [7-chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3-benzazepine] much greater than WB4101 = spiperone greater than dopamine, serotonin, (+/-)-propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)-[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)-TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine-123, it may be suitable for noninvasive imaging in humans.

Synthesis and resolution of (+-)-7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro- 1H-3- benzazepine (TISCH): a high affinity and selective iodinated ligand for CNS D1 dopamine receptor.

The synthesis and resolution of (+-)-7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3- benzazepine, (+/-)-TISCH (8) has been achieved by resolution of intermediate 4, the O-methoxyl, 3'-bromo derivative, as the diastereomeric camphor sulfonate salt. The final products, R-(+)-8 and S-(-)-8, were prepared by treatment of R-(+)- or S-(-)-7, the 3'-tributyltin intermediates, with iodine in chloroform, followed by O-demethylation. By using HPLC with a chiral column, the optical purity (greater than 99%) of the intermediates and the final compounds was determined. Radioiodination was achieved by an iodo-destannylation reaction with sodium [125I]iodide and hydrogen peroxide. As expected, the R-(+)-[125I]-8 (the active isomer) displayed high affinity and selectivity to the CNS D-1 receptor in rat striatum tissue preparation (Kd = 0.205 nM). The rank order of potency was as follows: SCH-23390 (1a) greater than (+/-)-8 greater than (+)-butaclamol greater than spiperone, WB4101 greater than dopamine, 5-HT. After an iv injection, the R-(+)-[125I]-8 penetrated the blood-brain barrier with ease and displayed specific regional distribution corresponding to the D-1 receptor density, while the S-(-)-[125I]-8 showed no specific uptake. The data suggest that the ligand may be useful as a pharmacological tool for characterizing the D-1 dopamine receptor. When labeled with I-123, this ligand is a potential agent for in vivo imaging of CNS D-1 dopamine receptor.