Disposition of a New Alkylating Agent [14C]Mitoclomine (N,N-Bis(2′-Chloroethyl)4-Amino-2-Methyl-1-Methoxy-naphthalene)

1. The absorption, distribution and elimination of a new alkylating agent, N,N-bis(2′-chloroethyl)4-amino-2-methyl-1-methoxynaphthalene (mito-clomine), labelled with ¹⁴C either in the dichloroethyl group or in the methoxy group, have been studied in mice and rats.

2. Expired ¹⁴CO₂ of rats given [chloroethyl-¹⁴C]- or [methoxy-¹⁴C]mitoclomine amounted to 1·0 and 35% dose respectively, indicating that O-dealkylation occurs.

3. The part of the molecule carrying the cytotoxic group displayed a high affinity for lymphoid organs (thymus, spleen). This could explain some biological properties of this drug, especially the selective effects of mitoclomine on circulating lymphocytes.     

The Fate of [14C]Saccharin in Man,Rat and Rabbit and of 2-Sulphamoyl[14C]benzoic Acid in the Rat

1. [¹⁴C]Saccharin administered orally was excreted entirely unchanged by rats on a normal diet and by rats on a 1% and 5% saccharin diet for up to 12 months. Some 90% dose was excreted in 24?h, about 70–80% in urine and 10–20% in faeces. No metabolite was detected in the excreta by chromatography or reverse isotope dilution. No ¹⁴CO₂ was found in the expired air and no ¹⁴CO₃^2- or 2-sulphamoylbenzoic acid in the urine.

2. When [¹⁴C]saccharin was injected into bile-duct cannulated rats kept on a normal diet or on a 1% saccharin diet for 19 and 23 months, 0.1–0.3% dose appeared in the bile in 3?h and no more at 24?h after dosing. Most of the saccharin was excreted in the urine, 0.6% appearing in the faeces.

3. [¹⁴C]Saccharin given orally to rabbits kept on untreated water and on water containing 1% saccharin for 6 months was excreted unchanged, 60–80% in 24?h, with 70% in urine and 3–11% in faeces.

4. [3-¹⁴C]Saccharin taken orally was excreted unchanged mainly in urine (85–92% in 24?h) by 3 adult humans both before and after taking 1 g of saccharin daily for 21 days. No metabolite of saccharin was found.

5. When [¹⁴C]saccharin was administered orally to pregnant rats on the 21st day of gestation only at most 0.6% of dose entered the foetuses. The ¹⁴C cleared more slowly from the urinary bladder than from other maternal or foetal tissues.

6. Saccharin was not metabolized in vitro by liver microsomal preparations or faecal homogenates from rats kept on a normal diet or on a 1% saccharin diet for two years.

7. 2-Sulphamoyl[¹⁴C]benzoic acid given orally to rats was excreted unchanged more slowly than saccharin. It was not cyclized to saccharin in vivo.     

Synthesis of 2-(3-Substituted-1,2,4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]octanes and 2α-(3-Substituted-1,2,4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-enes as Potential Muscarinic Agonists

Radioligand binding affinities of seven muscarinic receptor ligands which possess an oxadiazole ring side chain have been determined in rat heart, rat brain, and m₁- or m₃-transfected CHO cell membrane preparations to determine the selectivity for subtypes of muscarinic receptor. The ratios of binding constants in brain membranes were measured as an indicator of potential agonist activity against [³H]QNB and [³H]Oxo-M. These muscarinic ligands did not discriminate the subtypes of muscarinic receptors. Six muscarinic ligands which have a 3-amino- or 3-methyl-1,2,4-oxadiazol-5-yl groups attached to the 8-methyl-8-azabicyclo[3.2.1]oct-2-ene or 8-methyl-8-azabicyclo[3.2.1]octane head group show binding constants between 2.04 x 10^–6 and 1.79 x 10^–5 M in rat heart, rat brain, and m₁- or m₃-transfected CHO cell membrane preparations. 1-Methyl-2-[3-amino-1,2,4-oxadiazol-5-yl]piperidine shows low binding constants of approximately 10^–4 M in rat heart and rat brain. (1R,5S)-2-[3-Amino-1,2,4-oxadiazol-5-yl]-8-methyl-8-azabicyclo-[3.2.1]oct-2-ene [(1R,5S)-17] was the most active compound.     

Radiosynthesis of 2-[6-chloro-2-(4-iodophenyl)imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-[11C]methyl-acetamide, [11C]CLINME,a novel radioligand for imaging the peripheral benzodiazepine receptors with PET

Recently, a new 2-(iodophenyl)imidazo[1,2-a]pyridineacetamide series has been developed as iodine-123-labelled radioligands for imaging the peripheral benzodiazepine receptors using single photon emission tomography. Within this series, 2-[6-chloro-2-(4-iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl-acetamide (CLINME) was considered as an appropriate candidate for positron emission tomography imaging and was isotopically labelled with carbon-11 (T_1/2: 20.38 min) at the methylacetamide side chain from the corresponding nor-analogue using [¹¹C]methyl iodide and the following experimental conditions: (1) trapping at −10°C of [¹¹C]methyl iodide in a 1/2 (v:v) mixture of DMSO/DMF (300 µl) containing 0.7–1.0 mg of the precursor for labelling and 3–5 mg of powdered potassium hydroxide (excess); (2) heating the reaction mixture at 110°C for 3 min under a nitrogen stream; (3) diluting the residue with 0.6 ml of the HPLC mobile phase; and (4) purification using semi-preparative HPLC (Zorbax^® SB18, Hewlett Packard, 250 × 9.4 mm). Typically, starting from a 1.5 Ci (55.5 GBq) [¹¹C]CO₂ production batch, 120−150 mCi (4.44–5.55 GBq) of [¹¹C]CLINME were obtained (16–23% decay-corrected radiochemical yield, n=12) within a total synthesis time of 24–27 min (Sep-pak^®Plus-based formulation included). Specific radioactivities ranged from 0.9 to 2.7 Ci/µmol (33.3–99.9 GBq/µmol) at the end of radiosynthesis. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis of isotopically labelled [3-14C]- and [3,3-2H2]-5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), a new antifungal agent for the potential treatment of onychomycosis

5-Fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690) is a new antifungal agent for the potential treatment of onychomycosis. During the preclinical development phase, it was necessary to synthesize the radioisotope [3-¹⁴C]-5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole and the deuterium isotope [3,3-²H₂]-5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole for in vitro studies. We report the synthesis of these two isotopically labelled derivatives. Copyright © 2007 John Wiley & Sons, Ltd.     

Synthesis and anti-HIV evaluation of the novel 2-(m-chlorobenzyl)-4-substituted-7-methyl-1, 1, 3-trioxo-pyrazolo[4, 5-e] [1, 2, 4]thiadiazines

A novel series of 2-(m-Chlorobenzyl)-4-substituted-1, 1, 3-trioxo-2H,4H-pyrazolo[4, 5-e][1, 2, 4] thiadiazines (7a-k) were synthesized, and evaluated for their anti-HIV replication in MT-4 cell cultures. Compound (7a) showed activity against HIV-1-induced cytopathicity, with an EC₅₀ value of 45.6 μM, but none of the compounds exhibited inhibitory activity against HIV-2.     

Species differences in the metabolism of a tricyclic psychotropic agent,SQ 11,290-C

The metabolism of SQ 11,290-¹⁴C (4-[3-(7-chloro-5,11-dihydrodibenz[b,e]-[1,4]-oxazepin-5-yl)propyl]-α,β-¹⁴C₂-1-piperazineethanol, dihydrochloride) was studied in mice, rats, guinea pigs, hamsters, New Zealand White or Dutch rabbits, monkeys and man after po administration. The excretion of SQ 11,290-¹⁴C, its metabolites, or both, was chiefly in the feces (with the exception of hamsters and man). Rats and rabbits of either strain excreted 2–5% of the dose—mice and hamsters excreted 20–42%—as ¹⁴CO₂. Hamsters appeared to excrete radioactivity in a quantitative manner most similar to that observed in man, but the metabolites found in the urine and feces of these 2 species were not similar. The disposition of SQ 11,290-¹⁴C in albino and pigmented rabbits cannot be distinguished on the basis of the excretion of radioactivity, but different metabolites appear to be excreted in the urine. No unchanged SQ 11,290-¹⁴C was detected in the excreta of humans. One percent of the dose or less was present as unchanged SQ 11,290-¹⁴C in the urine of any animal species. In the feces, an average of 2–6% of the dose was excreted by animal species as unchanged SQ 11,290-¹⁴C. Whereas albino rabbits excreted in the feces only 3.6% of the dose as unchanged drug, Dutch rabbits excreted about 16.7% of the dose as unchanged drug. In those human subjects excreting large amounts of radioactivity as ¹⁴CO₂, cleavage or degradation of the side chain, or both, rather than hydroxylation of the ring system as had been found previously in dogs, appeared to be a major metabolic pathway.     

Syntheses of [2-14C]penem antibacterials; (FCE 22101 and FCE 22891)

The synthesis of FCE 22101 (sodium (5R,6S)-6-[(1R)-hydroxyethyl]-2-carbamoyloxymethylpenem-3-carboxylate) labelled with carbon-14 in the 2-position of the penem system ring was performed in eight steps, using sodium salt of [1-¹⁴C]glycollic acid 1 as the labelled starting material. The final product, penem [2-¹⁴C]FCE 22101 11, was obtained in an overall radiochemical yield of 21%, 98% radiochemically pure and with a specific activity of 641 MBq/mmol (17.3 mCi/mmol). The acetoxymethyl ester FCE 22891 12 was prepared by condensation of 11 with bromomethyl acetate, with a yield of 41%.     

Studies on Metabolism of Trazodone,I. Metabolic Fate of [14C]Trazodone Hydrochloride in Rats

Abstract

1. One of the main metabolites of [¹⁴C]trazodone hydrochloride by rat liver in vitro is hydroxylated trazodone.

2. [¹⁴C]Trazodone HCI is absorbed very rapidly and the blood level of radioactivity attains a maximum within 15 min after oral administration of 4 mg/kg to rats and thereafter decreases rapidly.

3. Urinary and faecal excretions of radioactivity are 49.0 and 46.1% of the dose respectively, during the first 7 days after ingestion, and biliary excretion is 80.0% in 8 h.

4. After oral administration of [¹⁴C]trazodone HCI to rats the main metabolites in urine and bile are hydroxylated trazodone, β-{3-oxo-s-triazolo[4,3a]-pyridin-2-yl}-propionic acid and their glucuronides.

5. Unchanged and hydroxylated trazodone alone are present in brain of rats after oral administration (20 mg/kg); both compounds in brain decrease with similar half-lives to those in plasma.     

Metabolism of N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide (S-23121) in the rat. II. Absorption,disposition, excretion and biotransformation

1. To examine the metabolic fate of N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide (S-23121), rats were given a single oral dose of [phenyl-¹⁴C]S-23121 at 1 or 250mg/kg.

2. The radiocarbon was almost completely eliminated from the rat within 7 days after administration for both dose groups. Faecal ¹⁴C-excretion was major (71-86% of the dose) and urinary ¹⁴C-excretion was minor (18-30%).

3. ¹⁴C-tissue residues on the seventh day after administration were generally very low. Peak ¹⁴C-concentrations in the kidney and liver occurred 4h after administration and decreased rapidly thereafter. Amounts (percentage of dose) of the parent compound in faeces were 13-26% for low dose, and 22-35% for high dose.

4. The major metabolites in faeces were sulphonic acid conjugates (13-20% of the administered dose), formed by incorporation of a sulphonic acid group into the double bond of the tetrahydrophthalimide. The major metabolites in urine were sulphates and glucuronides of 4-chloro-2-fluoro-5-hydroxyaniline, amounting to 5-7 and 2-3% of the administered dose, respectively. Sulphonic acid conjugates were not detected in urine, blood, kidney or liver.     

Synthesis of carboxy-polyethylene glycol-amine (CA (PEG)n) and [1-14C]-CA (PEG)n via oxa-Michael addition of amino-polyethylene glycols to propiolates vs to acrylates

Synthesis of carboxy-polyethylene glycol-amine (CA (PEG)_n) via oxa-Michael addition of amino-polyethylene glycols to either acrylates or propiolates was investigated. Compared with the oxa-Michael addition to acrylates, the corresponding addition to propiolates was found to proceed under mild reaction conditions and afford the adducts in high yields from a broad scope of substrates. A two-step efficient and convenient synthesis of benzyl [1-¹⁴C]-propiolate from ¹⁴CO₂ was therefore developed and utilized as a common synthon to afford practical and high yielding access to [1-¹⁴C]-CA (PEG)_n.     

Mixed Dopaminergic/Serotonergic Properties of Several 2-Substituted 4-[2-(5-Benzimidazole)ethyl]-1-arylpiperazines

A series of substituted 4-[2-(5-benzimidazole)ethyl]-arylpiperazines was synthesized by introducing different substituents into position 2 of benzimidazole ring of 4-[2-(N,N-di-n-propyl-amino)ethyl]-1,2-diaminobenzenes. They were evaluated for in vitro binding affinity at the D₁ and D₂ dopamine and 5-HT_1A serotonin receptors using synaptosomal membranes of the bovine caudate nuclei and hippocampi, respectively. Tritiated SCH 23390 (D₁ receptor-selective), spiperone (D₂ receptor selective) and 8-OH-DPAT (5-HAT_1A receptor selective) were employed as the radioligands. Only compound 6 expressed a moderate binding affinity at the dopamine D₁ receptor, while the remaining ligands were inefficient or weak competitors of [³H]SCH 23390. Compound 12 was an absolutely inactive competitor of all three radioligands. Also, compound 7 was an inefficient displacer of [³H]-8-OH-DPAT. Compound 19 with a K_i value of 3.5 nM was the most potent competitor of [³H]spiperone and compound 13 (K_i = 3.3 nM) was the most efficient in displacing [³H]-8-OH-DPAT from the 5-HAT_1A serotonin receptor. Ligands 5, 6, 8–11 , and 13–20 expressed mixed dopaminergic/serotonergic activity in nanomolar range of concentrations with varying affinity ratios which strongly depended on the properties of the substituents introduced into position 2 of benzimidazole ring of the parent compounds.     

The metabolism and disposition of [2-14C]diazepam in the streptozotocin-diabetic rat

1. The oral administration of [2-¹⁴C]diazepam to streptozotocin (STZ)-diabetic rats resulted in decreased faecal and biliary levels of metabolites with a concomitant rise in urinary radioactivity when compared to control values. This situation was reversible upon insulin treatment.

2. The increased urinary metabolite excretion could not be ascribed to the diuresis observed in STZ-diabetic rats.

3. No alteration in the phase I or II routes of [¹⁴C]diazepam metabolism in diabetic animals was observed either in vivo or in vitro.

4. Following i.v. administration of [2-¹⁴C]diazepam, blood ¹⁴C levels in diabetic rats were elevated above those observed in normal animals.     

Anellierte Thiopyrone, 4. Mitt.1): Indeno[1,2-b]-, Indolo[3,2,-b]-, [1]Benzofuro[3,2-b]- und [1]Benzothieno[3,2-b]thiopyrone

Fused Thiopyrones, IV: Indeno[1,2-b]-, Indolo[3,2-b]-, [1]Benzofuro[3,2-b]-, and [1]Benzothieno[3,2-b]thiopyrones The thiopyrones 2 are obtained from the corresponding thiopyranones 1 by dehydration with tritylperchlorate or SeO₂. Treatment of the benzofurane 2d with m-chloroperbenzoic acid (mCPBA) yields the thiopyrone-S, S-dioxide 3d. Starting from the benzothienothiopyrone 2e only one product, the thiophene-sulfone 3e , can be isolated.     

Synthesis characterization and biological evaluation of some alkoxyphthalimide derivatives of 3-(4-substituted phenyl)-6,6-diphenyl-3,3a-dihydro-2H-imidazo[2,1-b]pyrazolo[3,4-d][1,3]thiazol-7(6H)-one

A series of 2-N-ethoxyphthalimido 3-(4-substitutedphenyl)-6,6-diphenyl-3,3a-dihydro-2H-imidazo[2,1-b]pyrazolo[3,4-d][1,3]thiazol-7(6H)-one(7a–e) and 4-(4-substituted phenyl)-2-(N-ethoxyphthalimido amino)-7,7-diphenylimidazo[2′,1′:2,3][1,3]thiazolo[4,5-d] pyrimidin-8(7H)-one (9a–e) have been designed and synthesized starting from thiohydentoin (1). The structure of all synthesized compounds has been established by IR, ¹H NMR, ¹³C NMR and mass studies. These compounds have been screened for antimicrobial activities in order to evaluate the possibility of the derivatives to be used as potential chemotherapeutic agents.     

Synthesis and antiviral activity of new 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids derivatives

The synthesis of new 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (3a-l) derivatives and the new 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (5a–c) derivatives was achieved with an efficient synthetic route. Ethyl 4-chloro-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (1) on fusion with appropriate substituted anilines or aminopicolines gave the required new ethyl 4-(phenylamino)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (2a–l) (52–82%) or new ethyl 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylates (4a–c) (50–60%), respectively. Subsequent hydrolysis of the esters afforded the corresponding carboxylic acids (3a–l) (86–93%) and (5a–c) in high yield (80–93%). Inhibitory effects of 4-(phenylamino)/4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acids. Derivatives on Herpes simplex virus type 1 (HSV-1), Mayaro virus (MAY) and vesicular stomatitis virus (VSV) were investigated. Compounds 2d, 3f, 3a, and 3c exhibited antiviral activity against HSV-1, MAY, and VSV virus with EC₅₀ values of 6.8, 2.2, 4.8, 0.52, 2.5, and 1.0. None of these compounds showed toxicity for Vero cells.     

Comparative Metabolic Studies of Phenacetin and Structurally-related Compounds in the Rat

1. A comparative study of the metabolism of [acetyl-¹⁴C]phenacetin, [acetyl-¹⁴C]methacetin, [acetyl-¹⁴C]paracetamol and [acetyl-¹⁴C]acetanilide in the rat is reported.

2. The extent of N-deacetylation, evidenced by the measurement of respired ¹⁴CO₂ varied, being greatest with acetanilide (25—31%) and least with paracetamol (6%).

3. The major urinary metabolites in each case were N-acetyl-p-aminophenyl sulphate and N-acetyl-p-aminophenyl glucuronide; the relative proportions varied with the sex of the animals and as a result of extended dosage.

4. The metabolism of [ethyl-¹⁴C]phenacetin and [ethyl-¹⁴C]phenetidine was investigated and the extent of O-dealkylation determined by measurement of respired ¹⁴CO₂

5. The metabolic pathways of some related glycolanilides and oxanilic acids included N-deacylation, and in the glycolanilides, oxidation of the glycollic group.     

Metabolic fate of [1-14C]isobutyric acid in the rat.

Isobutyric acid (IBA) has potential use as a fungistat for the storage of moist grain. Since a complete accounting of the fate of IBA has not been reported, the metabolic fate of [1-¹⁴C]IBA was investigated. [1-¹⁴C]IBA was administered by gavage to male Charles River CD rats at doses of 4, 40, and 400 mg/kg body weight and to female rats at 400 mg/kg. All rats showed a similar excretion pattern. [1-¹⁴C]IBA was eliminated rapidly in the breath as expired ¹⁴CO₂. At 4 hr, 75.4, 83.3, and 66.7% of the dose was eliminated in the breath by male rats dosed with 4, 40, or 400 mg/kg, respectively. At 48 hr, 85–90% of the dose was eliminated in the breath. Urinary radioactivity averaged 3.5% of the dose, with about $\text{2}\text{3}$ of the radioactivity present as urea. Fecal radioactivity was less than 1% of the dose. The excretion of radioactivity by female rats was similar to that of male rats dosed with [1-¹⁴C]IBA. Isobutyric acid disappeared rapidly from the plasma of rats dosed by gavage with 400 mg/kg. These studies show that IBA is rapidly metabolized to CO₂ and its use as a feed fungistat is unlikely to contribute to the endogenous levels of IBA in the flesh, eggs, or milk or grain-consuming animals.     

STUDIES OF THE INTERACTION OF BOVINE NEUROPHYSIN-II WITH [1-HEMI-d-(3-13 C)CYSTINE] OXYTOCIN AND [1-HEMI-(3-13 C)CYSTINE] OXYTOCIN

The interactions of [1-hemi-(3-¹³C)cystine]oxytocin and [1-hemi-D-(3-¹³C) cystine]oxytocin with bovine neurophysin-II have been studied in aqueous solution under various conditions of pH using carbon-13 (¹³C) nuclear magnetic resonance spectroscopy (n.m.r.). We have monitored changes in ¹³C chemical shifts (δ), spin-lattice relaxation times (T₁) and linewidths (πT₂*)^-1 of the [1-hemi-(3-¹³C)cystine] oxytocin as a function of the concentration of added neurophysin. [1-Hemi-d -(3-¹³C)cystine]oxytocin does not interact with neurophysin and shows no changes in the above spectral parameters as a function of increasing protein concentrations. The ¹³C chemical shifts have been monitored as a function of pH for both ¹³C-enriched, diastereoisomeric oxytocin analogs in the presence and absence of neurophysin. The midpoints of the pH-titration curves for [1-hemi-(3-¹³C)cystine]oxytocin and [1-hemi-d -(3-¹³C) cystine]oxytocin in the absence or presence of neurophysin are 6.2 and 5.5, respectively. T₁ values are independent of pH for both peptides in free solution, and the rotational correlation times (t_c) obtained for the peptides are of the order of magnitude expected from the Stokes-Einstein relation (5.0 times 10^-10 sec rad^-1). T₁ values decrease and linewidths increase for [1-hemi-(3-¹³C)cystine]oxytocin in the presence of neurophysin. Fast-exchange on the n.m.r. time scale occurs at low pH. Only broad lines can be detected at neutral pH rendering ¹³C T₁ measurements difficult. It appears that increasing the concentration of neurophysin in the presence of [1-hemi-(3-¹³C)cystine]oxytocin leads to aggregation of the system as judged by the ¹³C T₁ results and corresponding rotational correlation times. In light of the significant changes in T₁ values and small changes in δ values, it would seem that T₁ measurements are more accurate monitors of peptide-protein interactions than are ¹³C chemical shifts when the interaction of the peptide with the protein does not involve any changes in steric constraints of the peptide being monitored.     

Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.