Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes.

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.

Spiperone: influence of spiro ring substituents on 5-HT2A serotonin receptor binding.

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.

(3-N-[11C]methyl)spiperone, a ligand binding to dopamine receptors: radiochemical synthesis and biodistribution studies in mice.

Carbon-11-labeled 3-N-methylspiperone, a positron-emitting dopamine-receptor antagonist with potential for use in positron emission tomography studies of human neurotransmitter receptors, was synthesized from 11CO2 in 40 min, with a radiochemical yield of approximately 20-40%. The specific activity of the (3-N-[11C]methyl)-spiperone was determined by ultraviolet spectroscopy to be approximately 270 mCi/mumol at the end of synthesis. In in vitro binding experiments, the Ki for 3-N-methylspiperone was found to be approximately 250 pM (against H-3 spiperone). The brain-to-blood ratios in normal ICR mice were 2.8 or greater at the times studied, and the striatum-to-cerebellum ratio at 60 min after injection was 20:1.

No-carrier-added fluorine-18-labeled N-methylspiroperidol: synthesis and biodistribution in mice.

No-carrier-added fluorine-18- (18F) labeled N-methylspiroperidol (4) was synthesized from four different substrates: p-nitrobenzonitrile (1), cyclopropyl p-nitrophenyl ketone (2A), p-cyclopropanoyl-N,N,N-trimethylanilinium iodide (2B) and p-cyclopropanoyl-N,N,N-trimethylanilinium perchlorate (2C) using the nucleophilic aromatic substitution reaction. Radiochemical yield, synthesis time, experimental simplicity, and specific activity were compared. In addition, factors which influence the yield of the nucleophilic aromatic substitution were studied. Based on these studies, the synthesis of 4 from 2A maximizes product specific activity and experimental simplicity and provides 4 in 10-15% radiochemical yield [based on [18F-] with a mass of less than 2 nmol and a specific activity of greater than 10 Ci/mumol (EOB)]. The synthesis of 4 from 8-[4-(4-nitrophenyl)-4-oxobutyl]-3-methyl-1-phenyl-1,3,8-triazaspiro+ ++ [4.5]decan-4-one (5) and Cs[18F] using the nucleophilic aromatic substitution reaction gave unacceptably low and erratic yields. The biodistribution of 4 in mice showed a maximum brain uptake of 1.1% of the administered dose at 5 min and declined to approximately 0.6% at 120 min.

Synthesis and in vivo evaluation of [18F]-N-(p-nitrobenzyl)spiperone ([18F]PNBS) in rats.

[18F]-N-(p-nitrobenzyl)spiperone, [18F]PNBS, was prepared and its in vivo uptake in rat brain was measured. A temporal increase in striatum:cerebellum ratio and frontal cortex:cerebellum (FC:Cb) ratio was observed. Blocking studies suggested the formation of a radiolabeled metabolite, presumably [18F]spiperone, that is capable of crossing the blood-brain barrier and labeling cortical 5-HT2 receptors. This was confirmed using the ISRP method of metabolite analysis indicating [18F]PNBS is not a suitable tracer for studying D2 receptors in vivo with PET.

Synthesis, characterization and biodistribution of neutral and lipid-soluble 99mTc-bisaminoethanethiol spiperone derivatives: possible ligands for receptor imaging with SPECT.

Using parts of the molecular structure of spiperone, two new ligand systems for complexation with [99mTc]technetium were prepared in order to develop potential receptor imaging agents for single photon emission computer tomography (SPECT). The bis-aminoethanethiols (BAT): 1-benzyl-4-(2-mercapto-2-methyl-4-aza-pentyl)-4-(2-mercapto-2-methyl- propylamino)-piperidine (benzylpiperidyl-BAT, BP-BAT) and 1-[3-(4-fluorobenzoyl)-propyl]-4-(2-mercapto-2-methyl-4-aza-pentyl)-4- (2-mercapto-2-methyl-propyl-amino)-piperidine (butyrophenoylpiperidyl-BAT, BUP-BAT) form stable, neutral and lipid soluble complexes with [99mTc]technetium at pH > or = 11 using SnCl2 as reducing agent in nearly quantitative radiochemical yields. Biodistribution of 99mTc-BP-BAT and 99mTc-BUP-BAT in rats showed a moderate clearance from blood and low uptake and retention in the liver, whereas brain uptake was moderate, however with prolonged brain retention. On the other hand, significant accumulations and retentions were observed in heart, kidney and lung with increasing oxygen/blood ratios up to 24 h. Within 24 h p.i. 22 and 29% of the injected dose (i.d.) of 99mTc-BP-BAT and 99mTc-BUP-BAT were eliminated by hepatobiliary excretion whereas 22% i.d. of both 99mTc-BAT complexes were excreted into the urine. Although first biodistribution studies of 99mTc-BP-BAT and 99mTc-BUP-BAT in rats showed relatively low brain uptake, the high uptake in peripheral, receptor rich organs indicates that compounds of this type may be used as a basis for further structural modification to develop agents with optimal properties for cerebral or peripheral receptor imaging with SPECT.

Synthesis and biological evaluation of p-bromospiperone as potential neuroleptic drug.

p-Bromospiperone was prepared from the reaction of spiperone with bromine. It was tested for dopamine receptor binding affinity in vitro and its ability to stimulate prolactin secretion in vivo. The results indicate no significant change of biological activities due to the bromination of spiperone.

Structure-based drug design for dopamine D3 receptor.

D2-like receptors are members of dopamine receptors, including dopamine D2 receptor (D2R), dopamine D3 receptor (D3R), and dopamine D4 receptor (D4R), which modulate behavior, cognition, and emotion. D2-like receptors are critical targets for drug development. Particularly, D3R has been identified as a therapeutic target for antipsychotic and anti-parkinsonian drugs. Recently, the crystal structure of D3R was reported. Here we summarize the available active compounds for D3R and the structure-activity relationships (SAR) studies of them. This provides lead templates for further chemical modification. We describe the structure features of the recent crystal structure of D3R and its difference from other G protein-coupled receptors (GPCRs). We provide the recognition mechanism of the inhibitors of D3R (molecular docking results and molecular dynamics results), which illustrates the interaction between the inhibitors and critical residues of D3R. Finally, we summarize the outlook of drug development for D3R. Our study provides useful information for developing high selective, high potent antagonists and agonists of D3R.

An improved synthesis of (3-N-[11C]methyl)spiperone.

An improved radiochemical synthesis of (3-N-[11C]methyl) spiperone is reported. The radiotracer was prepared by N-alkylation of spiperone in dimethylformamide in the presence of equimolar aqueous tetrabutylammonium hydroxide and was purified by semi-preparative reversed phase high performance liquid chromatography (C-18 HPLC). The average radiochemical yield was approximately 21% end-of-synthesis (EOS) with an average specific activity of over 2750 mCi/mumol EOS. The total time for synthesis and specific activity determination was approximately 21 min.

Synthesis of no-carrier-added N-([18F]fluoroalkyl)spiperone derivatives.

3-N-([18F]fluoroalkyl)spiperone derivatives (2,3) can be prepared by N-alkylation of spiperone (1) with fluoroalkyl halides. The fluoroalkylating species 2-[18F]fluoroethyl bromide (7), 3-[18F]fluoropropyl bromide (8) and 4-[18F]fluorobutyl bromide (9) were prepared by [18F]fluoride ion displacement of the corresponding trifluoromethanesulfonates (triflates 4,5,6). By this method, the 2-[18F]fluoroethyl-, 3-[18F]fluoropropyl-, and 4-[18F]fluorobutyl spiperone derivatives (2a-c) can be prepared and purified rapidly and conveniently, within 40 min, in yields of 30-40% (end of synthesis, EOS). An alternative approach, suitable for the preparation of 2-[18F]fluoroalkyl (ethyl, propyl, butyl, pentyl and hexyl) spiperone derivatives (3a-d), involves iodo[18F]fluorination of terminal olefins, followed by N-alkylation of spiperone. This sequence is less convenient and proceeds in lower overall yields (less than 5%).

Technetium-99m spiperone dithiocarbamate: a potential radiopharmaceutical for dopamine receptor imaging with SPECT.

Spiperone dithiocarbamate (SPDC) was prepared by reacting spiperone with carbon disulfide followed by sodium hydroxide. SPDC was labelled with 99mTc by reduction of pertechnetate with formamidine sulfinic acid or sodium dithionite at alkaline pH, resulting in approximately 40% incorporation of 99mTc. The lipophilic complex was conveniently isolated at high specific activity and high radiochemical purity by extraction into dichloromethane, which was then evaporated and the residue was redissolved in a 1:3 mixture of ethanol and saline containing 0.1 mg/mL gentisic acid. Biodistribution studies following i.p. injection in rats showed low uptake of radioactivity in the brain, but striatum/cortex and striatum/cerebellum ratios were reduced by pretreatment with haloperidol. This agent may allow imaging of dopamine D-2 receptors using single-photon emission computed tomography (SPECT).

3-(2'-[18F]fluoroethyl)spiperone, a potent dopamine antagonist: synthesis, structural analysis and in-vivo utilization in humans.

The synthesis of 3-(2'-[18F]fluoroethyl)spiperone (1c), a radiotracer useful for imaging the brain dopamine receptor system in vivo using positron emission tomography, is described. Precursors of 1c, the functional 3-N-alkyl derivatives of spiperone (4), were prepared by the alkylation of the amide group in spiperone (2a) by 1,2-disubstituted ethanes under phase transfer conditions. A comprehensive evaluation of the reaction of the derivatives 4a-h with no-carrier-added K18F/Kryptofix clearly indicated that the ketalized derivatives 4e-h were the choice of the precursors for 1c. The i.r., MS and NMR spectral data suggested that under phase transfer reaction conditions, the amide nitrogen was preferentially alkylated. To provide a firm basis for comparison with related analogues, an x-ray analysis was performed on a single crystal of 3-(2'-fluoroethyl)spiperone (1d). The tomographic behavior of 1c in human brain tissue was measured for more than 7 h and was consistent with the labeling of dopamine D-2 receptors.

Radiobrominated spiroperidol for the study of dopamine D2 receptors.

Some neuropsychiatric disorders have been suggested to be related to the CNS dopaminergic system. In order to probe this neurotransmitter system, radiobrominated spiroperidol, a potent dopamine (DA) D2-receptor antagonist, was developed. This review deals with the routine synthesis of [75, 76, or 77Br]-p-bromospiroperidol (BrSP), its validation as a radiopharmaceutical directed to DA D2 receptors, and the imaging studies which have been done using this radioligand with either single photon tomography (SPECT) or positron tomography (PET). [*Br]BrSP will be compared to other D2 ligands currently available. The present state and future direction of DA receptor research, in particular, the quantitative aspects, will be discussed.

Affinity chromatography of the anterior pituitary D2-dopamine receptor.

The D2-dopamine receptor from bovine anterior pituitary has been solubilized with digitonin and purified approximately 1000-fold by affinity chromatography on a new affinity support. This support consists of a (carboxymethylene)oximino derivative of the D2-selective antagonist spiperone (CMOS) covalently attached to Sepharose 4B through a long side chain. The interaction of the solubilized receptor activity with the affinity gel was biospecific. Dopaminergic drugs blocked adsorption of solubilized receptor activity to the CMOS-Sepharose with the appropriate D2-dopaminergic potency and stereoselectivity. For agonists, (-)-N-n-propylnorapomorphine greater than 2-amino-6,7-dihydroxytetrahydronaphthalene approximately equal to apomorphine greater than dopamine, whereas for antagonists (+)-butaclamol much greater than (-)-butaclamol. The same D2-dopaminergic specificity was observed for elution of receptor activity from the gel. To observe eluted receptor binding activity, reconstitution of the eluted material into phospholipid vesicles was necessary. Typically, 70-80% of the solubilized receptor was adsorbed by CMOS-Sepharose, and 40-50% of the adsorbed activity could be recovered after reconstitution of the eluted material. The overall recovery of D2-receptor activity from bovine anterior pituitary membranes was 12-15% with specific binding activity of approximately 150 pmol/mg. The reconstituted affinity-purified receptor bound ligands with the expected D2-dopaminergic specificity, stereoselectivity, and rank order of potency.

Syntheses and D2 receptor affinities of derivatives of spiperone containing aliphatic halogens.

The development of a high affinity dopamine receptor ligand labeled with the positron emitting radionuclide, 18F (t 1/2 = 110 min), is of considerable interest for imaging and quantification of dopamine receptors in vivo. Derivatives of spiperone, a dopamine antagonist, labeled with 18F have been prepared, but the syntheses either proceed with inefficient fluoride utilization or involve several synthetic steps subsequent to 18F incorporation. To date, only the short-lived radioisotope of carbon, 11C (t 1/2 = 20.4 min), has been efficiently incorporated in the final synthetic step of 3-N-[11C]methyl-spiperone. 3-N-Fluoroethyl, 3-N-chloroethyl, and 3-N-bromoethyl spiperone derivatives are prepared by alkylation of spiperone with the appropriate 2-tosyloxy ethyl halide. In addition, alpha-fluorospiperone, containing fluorine alpha to the butyrophenone carbonyl, has been prepared. The 3-N-haloethyl spiperones display high affinity for dopamine receptor in vitro. Incorporation of [18F]fluoride during the final synthetic step yields a high affinity, 18F-labeled dopamine receptor-binding ligand.

No-carrier-added 3-(2'-[18F]fluoroethyl)spiperone, a new dopamine receptor-binding tracer for positron emission tomography.

No-carrier-added (NCA)3-(2'-[18F]fluoroethyl)spiperone (5), a new dopamine receptor-binding radiopharmaceutical for positron emission tomography, was synthesized by two different methods. Alkylation of the amide nitrogen in spiperone by NCA [18F]fluorobromoethane in the presence of a strong base gave 5 (Method A). Experimental methods were also developed for the syntheses of functional 3-N-alkylderivatives of spiperone such as 3-(2'-bromoethyl)- or 3-(2'-methylsulfonyloxyethyl)spiperone (4a and 4b, respectively). These derivatives (4) reacted with NCA Ag18F, Cs18F or K18F/Kryptofix 222 in acetonitrile or DMSO to give 5 (Method B). Method B, using K18F/Kryptofix 222 in acetonitrile provided 5 in multimillicure amounts (30-40% isolated radiochemical yield) with a specific activity of 2-10/mumol (EOS) in less than 60 min. This one-step, one-pot synthesis is simple, and the high radiochemical yield of 5, as well as the 110 min half-life of 18F, permit multiple tomographic studies a day with one preparation. Tomographic results in monkey brain with 5 are consistent with the labeling of dopamine-D2 receptor systems.

Preparation of 123I-labeled 2'-iodospiperone and imaging of D2 dopamine receptors in the human brain using SPECT.

[123I]2'-ISP was readily prepared using a radioiodine exchange reaction with a radiochemical yield of approx. 50% after HPLC purification. The radiochemical purity of the product was more than 98% and the specific activity was 5.55-11.1 GBq/mumol. Biodistribution studies performed in mice indicated that injection of [123I]2'-ISP with albumin produced a higher gastric uptake and a lower brain uptake than injection of the radioligand in a weakly acidic solution. In addition, toxicity tests performed in mice demonstrated that acute toxic effects would be very unlikely to be encountered if 2'-ISP was used for diagnostic purposes. A preliminary imaging study with [123I]2'-ISP in a healthy human volunteer showed its specific uptake by the basal ganglia, a region of the brain known to have a high density of D2 dopamine receptors.

Iodoethylspiperone, a new potential agent for exploration of central dopamine D2 receptors: synthesis and preliminary in vivo study.

We synthesized a new spiperone derivative: iodoethylspiperone (IES) to perform dopamine D2 receptor exploration by SPECT. IES was prepared from a precursor: tosylethylspiperone, and characterized by i.r. and 1H-NMR analyses. [125I]IES was obtained with 80% yield. In vivo biodistribution in rats showed a high and specific uptake in the striatum. The uptake ratio between the striatum and the cerebellum reached a maximum value 4 h after injection (10.05 +/- 2.81). IES labeled with 123I should be a promising new agent to investigate D2 receptors in the living human brain.

Multistep solution-phase parallel synthesis of spiperone analogues.

A flexible, multistep parallel synthesis of spiperone analogues is described. A library of 4-substituted piperidines, assembled utilizing reductive amination and acylation protocols, was alkylated either homogeneously or heterogeneously, exploiting a product release only concept, to afford an oxa-series of spiperone analogues. Screening of the products at 5-HT2 and D2 receptors revealed 5-HT2A antagonists with improved selectivity compared to spiperone and AMI-193.