Synthesis and preliminary PET study of the 5‐HT7 receptor antagonist [11C]DR4446

DR4446 (1‐methyl‐2a‐[4‐(4,5,6,7‐tetrahydrothieno[3,2‐c]pyridin‐5‐yl)butyl]‐2a,3,4,5‐tetrahydro‐1H‐benz[cd]indole‐2‐one) is a potent 5‐HT₇ receptor antagonist (K_i=9.7 nM) with a high selectivity over other 5‐HT family receptors (K_i for 5‐HT_1A: 770 nM; for other 5‐HT receptors: >1000 nM). As a positron emission tomography (PET) tracer for the 5‐HT₇ receptor, [¹¹C]DR4446 was synthesized at high radiochemical purity ( >98%) with specific activity of 73–120 GBq/μmol at the end of synthesis by the alkylation of the desmethyl precursor (1) with [¹¹C]CH₃I in the presence of NaH. A PET study in monkey demonstrated that [¹¹C]DR4446 had good permeability into the brain, and had a specific binding component in the brain regions including the thalamus, possibly an area in the 5‐HT₇ receptors. Metabolite analysis showed that [¹¹C]DR4446 was relatively stable and low percentages of two radio‐labeled metabolites were detected in the plasma of monkey using HPLC. Copyright © 2002 John Wiley & Sons, Ltd.     

Optimization and synthesis of an 18F‐labeled dopamine D3 receptor ligand using [18F]fluorophenylazocarboxylic tert‐butylester

There is still no efficient fluorine‐18‐labeled dopamine D₃ subtype selective receptor ligand for studies with positron emission tomography. We aim at improving the D₃ selectivity and hydrophilicity of a candidate ligand by changing the substitution pattern to a 2,3‐dichlorophenylpiperazine and hydroxylation of the butyl chain. The compound [¹⁸F]3 exhibited D₃ affinity of K_i = 3.6 nM, increased subtype selectivity (K_i(D₂/D₃) = 60), and low affinity to 5‐HT_1A and α₁ receptors (K_i (5‐HT_1A/D₃) = 34; K_i (α₁/D₃) = 100). The two‐step radiosynthesis was optimized for analog [¹⁸F]4 by reducing the necessary concentration of the precursor amine (57 mM), which reacted with [¹⁸F]fluorophenylazocarboxylic tert‐butylester under basic conditions. The optimization of the base (Cs ₂CO₃, 23 mM) and the adjustment of reaction temperature led to the radiochemical yield of 63% after 5 min at 35°C. The optimized reaction conditions were transferred on to the synthesis of [¹⁸F]3 with an overall non‐decay corrected yield of 8‐12% in a specific activity of 32‐102 GBq/µmol after a total synthesis time of 30‐35 min. This provides a D ₃ radioligand candidate with improved attributes concerning selectivity and radiosynthesis for further preclinical studies.     

Synthesis and evaluation of a 18F‐labeled 4‐phenylpiperidine‐4‐carbonitrile radioligand for σ1 receptor imaging

We report the design and synthesis of several 4‐phenylpiperidine‐4‐carbonitrile derivatives as σ₁ receptor ligands. In vitro radioligand competition binding assays showed that all the ligands exhibited low nanomolar affinity for σ₁ receptors (K_i(σ₁) = 1.22–2.14 nM) and extremely high subtype selectivity (K_i(σ₂) = 830–1710 nM; K_i(σ₂)/K_i(σ₁) = 680–887). [¹⁸F]9 was prepared in 42–46% isolated radiochemical yield, with a radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic ¹⁸F^‐ substitution of the corresponding tosylate precursor. Biodistribution studies in mice demonstrated high initial brain uptakes and high brain‐to‐blood ratios. Administration of SA4503 or haloperidol 5 min prior to injection of [¹⁸F]9 significantly reduced the accumulation of radiotracers in organs known to contain σ₁ receptors. Two radioactive metabolites were observed in the brain at 30 min after radiotracer injection. [¹⁸F]9 may serve as a lead compound to develop suitable radiotracers for σ₁ receptor imaging with positron emission tomography.     

Synthesis,radiofluorination and first evaluation of (±)‐[18F]MDL 100907 as serotonin 5‐HT2A receptor antagonist for PET

In some psychiatric disorders 5‐HT_2A receptors play an important role. In order to investigate those in vivo there is an increasing interest in obtaining a metabolically stable, subtype selective and high affinity radioligand for receptor binding studies using positron emission tomography (PET). Combining the excellent in vivo properties of [¹¹C]MDL 100907 for PET imaging of 5‐HT_2A receptors and the more suitable half‐life of fluorine‐18, MDL 100907 was radiofluorinated in four steps using 1‐(2‐bromoethyl)‐4‐[¹⁸F]fluorobenzene as a secondary labelling precursor. The complex reaction required an overall reaction time of 140 min and (±)‐[¹⁸F]MDL 100907 was obtained with a specific activity of at least 30 GBq/µmol (EOS) and an overall radiochemical yield of 1–2%. In order to verify its binding to 5‐HT_2A receptors, in vitro rat brain autoradiography was conducted showing the typical distribution of 5‐HT_2A receptors and a very low non‐specific binding of about 6% in frontal cortex, using ketanserin or spiperone for blocking. Thus, [¹⁸F]MDL 100907 appears to be a promising new 5‐HT_2A PET ligand. Copyright © 2008 John Wiley & Sons, Ltd.     

The synthetic cathinone psychostimulant α‐PPP antagonizes serotonin 5‐HT2A receptors: In vitro and in vivo evidence

Synthetic cathinones (SCs) are β‐keto analogs of amphetamines. Like amphetamines, SCs target monoamine transporters; however, unusual neuropsychiatric symptoms have been associated with abuse of some SCs, suggesting SCs might possess additional pharmacological properties. We performed radioligand competition binding assays to assess the affinities of nine SCs at human 5‐HT_2A receptors (5‐HT_2AR) and muscarinic M₁ receptors (M₁R) transiently expressed in HEK293 cells. None of the SCs exhibited affinity at M₁R (minimal displacement of [~K_d] [³H]scopolamine up to 10 μM). However, two SCs, α‐pyrrolidinopropiophenone (α‐PPP) and 4‐methyl‐α‐PPP, had low μM K_i values at 5‐HT_2AR. In 5‐HT_2AR–phosphoinositide hydrolysis assays, α‐PPP and 4‐methyl‐α‐PPP displayed inverse agonist activity. We further assessed the 5‐HT_2AR functional activity of α‐PPP, and observed it competitively antagonized 5‐HT_2AR signaling stimulated by the 5‐HT₂R agonist (±)‐2,5‐dimethoxy‐4‐iodoamphetamine (DOI; K_b = 851 nM). To assess in vivo 5‐HT_2AR activity, we examined the effects of α‐PPP on the DOI‐elicited head‐twitch response (HTR) in mice. α‐PPP dose‐dependently blocked the HTR with maximal suppression at 10 mg/kg (P < 0.0001), which is a moderate dose used in studies investigating psychostimulant properties of α‐PPP. To corroborate a 5‐HT_2AR mechanism, we also tested 3,4‐methylenedioxy‐α‐PPP (MDPPP) and 3‐bromomethcathinone (3‐BMC), SCs that we observed had 5‐HT_2AR K_is > 10 μM. Neither MDPPP nor 3‐BMC, at 10 mg/kg doses, attenuated the DOI HTR. Our results suggest α‐PPP has antagonist interactions at 5‐HT_2AR in vitro that may translate at physiologically‐relevant doses in vivo. Considering 5‐HT_2AR antagonism has been shown to mitigate effects of psychostimulants, this property may contribute to α‐PPPs unpopularity compared to other monoamine transporter inhibitors.     

Synthesis,labelling and first evaluation of [18F]R91150 as a serotonin 5‐HT2A receptor antagonist for PET

In psychiatric disorders such as anxiety, depression and schizophrenia, 5‐HT_2A receptors play an important role. In order to investigate them in vivo there is an increasing interest in selective and high‐affinity radioligands for receptor binding studies using positron emission tomography (PET). Since available radioligands have disadvantages, R91150, which is a selective and high‐affinity ligand for 5‐HT_2A receptors, was labelled with fluorine‐18. This was accomplished in six steps via 4‐[¹⁸F]fluorophenol and 1‐(3‐bromopropoxy)‐4‐[¹⁸F]fluorobenzene within 190 min starting from no‐carrier‐added [¹⁸F]fluoride. The overall radiochemical yield was 3.8±2% and the specific activity was at least 335 GBq/µmol at the end of the synthesis. First ex vivo studies in mice proved the uptake of [¹⁸F]R91150 in the brain. Radiometabolite studies revealed no radiometabolites in the brain, whereas in the plasma at least two could be detected 30 min p.i. Further preclinical studies are encouraged to evaluate the potential of this new 5‐HT_2A ligand as a radiotracer for PET. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis,pharmacological evaluations,and molecular docking studies on a new 1,3,4,11b‐tetrahydro‐1H‐fluoreno[9,1‐cd]azepine framework: Rigidification of D1 receptor selective 1‐phenylbenzazepines and discovery of a new 5‐HT6 receptor scaffold

The novel 1,3,4,11b‐tetrahydro‐1H‐fluoreno[9,1‐cd]azepine framework, a structurally rigidified variant of the 1‐phenylbenzazepine template, was synthesized via direct arylation as a key reaction. Evaluation of the binding affinities of the rigidified compounds across a battery of serotonin, dopamine, and adrenergic receptors indicates that this scaffold unexpectedly has minimal affinity for D₁ and other dopamine receptors and is selective for the 5‐HT₆ receptor. The affinity of these systems at the 5‐HT₆ receptor is significantly influenced by electronic and hydrophobic interactions as well as the enhanced rigidity of the ligands. Molecular docking studies indicate that the reduced D₁ receptor affinity of the rigidified compounds may be due in part to weaker H‐bonding interactions between the oxygenated moieties on the compounds and specific receptor residues. Key receptor–ligand H‐bonding interactions, salt bridges, and π–π interactions appear to be responsible for the 5‐HT₆ receptor affinity of the compounds. Compounds 10 (6,7‐dimethoxy‐2,3,4,11b‐tetrahydro‐1H‐fluoreno[9,1‐cd]azepine) and 12 (6,7‐dimethoxy‐2‐methyl‐2,3,4,11b‐tetrahydro‐1H‐fluoreno[9,1‐cd]azepine) have been identified as structurally novel, high affinity (K_i = 5 nM), selective 5‐HT₆ receptor ligands.     

Synthesis and biological evaluation of S‐[11C]methylated mercaptoimidazole piperazinyl derivatives as potential radioligands for imaging 5‐HT1A receptors by positron emission tomography (PET)

The novel 2‐mercaptoimidazole derivatives, 1‐[4‐((2‐methoxyphenyl)‐1‐piperazinyl)butyl]‐2‐mercaptoimidazole ( 3 ) and methyl[4‐((2‐methoxyphenyl)‐1‐piperazinyl))butyl] (2‐mercapto‐1‐methylimidazol‐5‐yl)methanamide ( 8 ), were efficiently labelled with ¹¹C through methylation of the thioketone function with [¹¹C]methyl iodide. The resulting radioligands 1‐[4‐((2‐methoxyphenyl)‐1‐piperazinyl))butyl]‐2‐thio[¹¹C]methylimidazole ([¹¹C] 9 ) and methyl[4‐((2‐methoxyphenyl)‐1‐piperazinyl))butyl] (2‐thio[¹¹C]methyl‐1‐methylimidazol‐5‐yl)‐methanamide ([¹¹C] 10 ) were synthesized in radiochemical yields of 20–30% (decay‐corrected, related to [¹¹C]CO₂) at a specific radioactivity of 0.2–0.4 Ci/µmol within 40–45 min including HPLC‐purification. The radiochemical purity exceeded 99%. The reference compounds 9 and 10 were tested in a competitive receptor binding assay to determine their affinity toward the 5‐HT_1A receptor. Both compounds exhibit excellent sub‐nanomolar affinities (IC₅₀=0.576±0.008 nM ( 9 ); IC₅₀=0.86±0.02 nM ( 10 )) for the 5‐HT_1A receptor while displaying a high selectivity towards the 5‐HT_2A subtype of receptors (IC₅₀>480 nM). By contrast, compound 9 also shows substantial binding for the alpha1‐adrenergic receptor (IC₅₀=3.00±0.02 nM) when compared with compound 10 (IC₅₀=54.5±0.6 nM). Preliminary biodistribution studies in rats showed an initial brain uptake of 1.14±0.11 and 0.37±0.04% ID/g after 5 min, which decreased to 0.18±0.04 and 0.16±0.01% ID/g after 60 min for compounds [¹¹C] 9 and [¹¹C] 10 , respectively. For both compounds, the cerebellum and rest of the brain uptake are very similar at the different time points. Unlike [¹¹C] 9 , the radioligand [¹¹C] 10 has significant uptake and retention in the adrenal glands. Due to their washout from the brain compounds [¹¹C] 9 and [¹¹C] 10 seem not to be good candidates as radioligands for imaging 5‐HT_1A receptors by PET. Copyright © 2005 John Wiley & Sons, Ltd.     

18F‐Labeled benzylpiperazine derivatives as highly selective ligands for imaging σ1 receptor with positron emission tomography

We report the design, synthesis, and evaluation of a new series of benzylpiperazine derivatives as selective σ₁ receptor ligands. All seven ligands possessed low nanomolar affinity for σ₁ receptors (K_i(σ₁) = 0.31‐4.19 nM) and high subtype selectivity (K_i(σ₂)/K_i(σ₁) = 50‐2448). The fluoroethoxy analogues also exhibited high selectivity toward the vesicular acetylcholine transporter (K_i(VAChT)/K_i(σ₁) = 99‐18252). The corresponding radiotracers [¹⁸F] 13 , [¹⁸F] 14 , and [¹⁸F] 16 with high selectivity (K_i(σ₂)/K_i(σ₁) > 100, K_i(VAChT)/K_i(σ₁) > 1000) were prepared in 42% to 55% radiochemical yields (corrected for decay), greater than 99% radiochemical purity (RCP), and molar activity of about 120 GBq/μmol at the end of synthesis (EOS). All three radiotracers showed high initial brain uptake in mouse (8.37‐11.48% ID/g at 2 min), which was not affected by pretreatment with cyclosporine A, suggesting that they are not substrates for permeability‐glycoprotein (P‐gp). Pretreatment with SA4503 or haloperidol resulted in significantly reduced brain uptake (35%‐62% decrease at 30 min). In particular, [¹⁸F] 16 displayed high brain‐to‐blood ratios and high in vivo metabolic stability. Although it may not be an optimal neuroimaging agent because of its slow kinetics in the mouse brain, [¹⁸F] 16 can serve as a lead compound for further structural modifications to explore new potential radiotracers for σ₁ receptors.     

Antinociceptive Synergism of MD‐354 and Clonidine. Part II. The α2‐Adrenoceptor Component

Abstract: Previously, we reported that antinociceptive synergism of a 5‐HT₃/α₂‐adrenoceptor ligand MD‐354 (m‐chlorophenylguanidine) and clonidine combination occurs, in part, through a 5‐HT₃ receptor antagonist mechanism. In the present investigation, a possible role for α₂‐adrenoceptors was examined. Mechanistic studies using yohimbine (a subtype non‐selective α₂‐adrenoceptor antagonist), BRL 44408 (a preferential α_2A‐adrenoceptor antagonist) and imiloxan (a preferential α_2B/C‐adrenoceptor antagonist) on the antinociceptive actions of a MD‐354/clonidine combination were conducted. Subcutaneous pre‐treatment with all three antagonists inhibited the antinociceptive synergism of MD‐354 and clonidine in the mouse tail‐flick assay in a dose‐dependent manner (AD₅₀ = 0.33, 2.1, and 0.17 mg/kg, respectively). Enhancement of clonidine antinociception by MD‐354 did not potentiate clonidine’s locomotor suppressant activity in a mouse locomotor assay. When [ethyl‐³H]RS‐79948‐197 was used as radioligand, MD‐354 displayed almost equal affinity to α_2A‐ and α_2B‐adrenoceptors (K_i = 110 and 220 nM) and showed lower affinity at α_2C‐adrenoceptors (K_i = 4,700 nM). MD‐354 had no subtype‐selectivity for the α₂‐adrenoceptor subtypes as an antagonist in functional [³⁵S]GTPγS binding assays. MD‐354 was a weak partial agonist at α_2A‐adrenoceptors. Overall, in addition to the 5‐HT₃ receptor component, the present investigation found MD‐354 to be a weak partial α_2A‐adrenoceptor agonist that enhances clonidine’s thermal antinociceptive actions through an α₂‐adrenoceptor‐mediated mechanism without augmenting sedation.     

Potent antagonists for the human adenosine A2B receptor. Derivatives of the triazolotriazine adenosine receptor antagonist ZM241385 with high affinity

A series of novel and known 5‐substituted 7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[1,5‐a][1,3,5]triazine derivatives were synthesized and tested for adenosine receptor antagonism in radioligand binding assays at all four adenosine receptor subtypes and for inhibition of the agonist‐induced cyclic AMP response at human A_2B receptors. The known potent adenosine A_2A receptor antagonist, 7‐amino‐2‐(2‐furyl)‐5‐[2‐(4‐hydroxyphenyl)ethyl]amino[1,2,4]triazolo[1,5‐a][1,3,5]triazine (ZM241385, K_iA_2A = 1.78 nM) had a K_i value of 16.5 nM at A_2B receptors in radioligand binding studies on Chinese hamster ovary cells expressing A_2B receptors. A pA₂ value of 7.9 was measured for the inhibition of the cyclic AMP response by A_2B receptors induced by 5′‐N‐ethylcarboxamidoadenosine (NECA). In a series of 5‐phenyl(alkyl)amino analogs the 5‐(2‐phenylethyl)amino analog LUF5452 and the 5‐benzylamino analog LUF5451 were both more potent than ZM241385 in the cyclic AMP assay at A_2B receptors. Moreover, K_i values of 9.9 and 7.6 nM were found in binding studies at this receptor subtype, indicating that LUF5451 and LUF5452 are more potent A_2B receptor antagonists than ZM241385. The affinity of LUF5451 for the A_2A receptor (K_i value = 13 nM) showed that the selectivity for this receptor subtype was lost and that a modest A_2B receptor selectivity was achieved. The 5‐(2‐phenylhydrazino) derivative LUF5475 showed a high A_2B receptor affinity (K_i = 7.6 nM), while it was equally active at A_2A receptors, being A_2B receptor‐selective with respect to A₁ and A₃ receptors. Drug Dev. Res. 48:95–103, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Synthesis,docking studies,and pharmacological evaluation of 5HT2C ligands containing the N′‐cyanoisonicotinamidine or N′‐cyanopicolinamidine nucleus

N′‐Cyanoisonicotinamidine and N′‐cyanopicolinamidine derivatives, linked to an arylpiperazine moiety, were prepared and their affinities to the 5‐HT_1A, 5‐HT_2A, and 5‐HT_2C receptors were evaluated. Several of the newly synthesized compounds, tested by binding studies, showed nanomolar affinity at the 5‐HT_1A and 5‐HT_2C receptors and moderate or no affinity for other relevant receptors (D₁, D₂, α₁, and α₂). Compound 8e (K_i = 21.4 nM) was the most affine for the 5‐HT_2C receptor, showing, at the same time, a high selectivity with respect to the other receptors analyzed. Compounds 4a and 4c , instead, showed an interesting mixed 5‐HT_1A/5‐HT_2C activity with K_i values of 21.3/11.5 and 23.2/6.48 nM, respectively. The compounds with better affinity and selectivity binding profiles toward 5‐HT_2C ( 4a , 4c , 8b , and 8e ) were selected for further in vivo assays to determine their functional activity. Finally, to rationalize the obtained results, molecular docking studies were performed. The results of the pharmacological studies showed that compounds 4a , 8b , and 8e exerted antidepressant‐like effects and 4a and 8e revealed also significant anxiolytic properties. Among the developed derivatives, the most promising compound seems to be 4a , which displayed antipsychotic‐, antidepressant‐ and anxiolytic‐like properties. No side effects, like catalepsy, motor‐impairment or ethanol‐potentiating effects, were observed after the injection of the tested compounds.     

Synthesis and biological evaluation of a radioiodinated spiropiperidine ligand as a potential σ1 receptor imaging agent

We report the synthesis and evaluation of 1′‐(4‐[¹²⁵I]iodobenzyl)‐3H‐spiro[isobenzofuran‐1,4′‐piperidine] ([¹²⁵I]Spiro‐I) as a potential SPECT tracer for imaging of σ₁ receptors. [¹²⁵I]Spiro‐I was prepared in 55–65% isolated radiochemical yield, with radiochemical purity of >99%, via iododestannylation of the corresponding tributyltin precursor. In receptor binding studies, Spiro‐I displayed low nanomolar affinity for σ₁ receptors (σ₁: K_i=2.75±0.12 nM; σ₂: K_i=340 nM) and high subtype selectivity (σ₂/σ₁=124). Biodistribution in mice demonstrated relatively high concentration of radioactivity in organs known to contain σ₁ receptors, including the lung, kidney, heart, spleen, and brain. Administration of haloperidol 5 min prior to injection of [¹²⁵I]Spiro‐I significantly reduced the concentration of radioactivity in the above‐mentioned organs. These findings suggest that the binding of [¹²⁵I]Spiro‐I to σ₁ receptors in vivo is specific. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and preliminary in vivo evaluation of 4‐[18F]fluoro‐N‐{2‐[4‐(6‐trifluoromethylpyridin‐2‐yl)piperazin‐1‐yl]ethyl}benzamide,a potential PET radioligand for the 5‐HT1A receptor

4‐Fluoro‐N‐{2‐[4‐(6‐trifluoromethylpyridin‐2‐yl)piperazin‐1‐yl]ethyl}benzamide is a full 5‐HT_1A agonist with high affinity (pK_i=9.3), selectivity and a c log P of 3.045. The corresponding PET radioligand 4‐[¹⁸F]fluoro‐N‐{2‐[4‐(6‐trifluoromethylpyridin‐2‐yl)piperazin‐1‐yl]ethyl}benzamide was synthesized by nucleophilic aromatic substitution on the nitro precursor. The fluorinating agent K[¹⁸F]F/Kryptofix 2.2.2 was both dried (9 min, 700 W) and incorporated in the precursor (5 min, 700 W) using a commercially available microwave oven. In a total synthesis time of 60 min, an overall radiochemical yield of 18% (SD=5, n=7, EOS) was obtained. Radiochemical purity was always higher than 99% and specific activity always higher than 81.4 GBq/µmol (2.2 Ci/µmol). Initial brain uptake in mice was 2.19% ID (5.47% ID/g, 2 min) but decreased rapidly (0.17% ID, 0.45% ID/g (60 min)). During the first 20 min p.i., radioactivity concentration of the brain was significantly higher than that of blood demonstrating good brain entry of the tracer. Copyright © 2004 John Wiley & Sons, Ltd.     

Comparison of CGS 15943, ZM 241385 and SCH 58261 as antagonists at human adenosine receptors

Three structurally related non-xanthine compounds, CGS 15943, ZM 241385 and SCH 58261, are potent A_2A adenosine receptor antagonists and have been used as tools in many pharmacological studies. We have now characterized their affinity and selectivity profile on human adenosine receptors stably transfected into either CHO cells (A₁ and A_2B receptors) or HEK-293 cells (A_2A and A₃ receptors). In binding studies using [³H]SCH 58261 as a radioligand, the three compounds were equally potent at A_2A receptors, their K _i value being less than 1 nM. Affinity for A₁ and A₃ receptors was measured using [³H]DPCPX and [¹²⁵I]AB-MECA as radioligands. Given the lack of selective ligands, interaction with A_2B receptors was assessed using the cAMP accumulation assay following stimulation by the adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA). CGS 15943 was almost as potent at A₁ receptors (K _i 3.5 nM) as at A_2A receptors, showed moderate affinity for A₃ receptors (K _i 95 nM) and also interacted with A_2B receptors (K _i 44 nM; pA₂ 7.5). ZM 241385 showed little affinity for A₁ receptors (K _i 255 nM), and did not interact with A₃ receptors (K _i>10 μM); however, it displayed moderate affinity for A_2B receptors (K _i 50 nM; pA₂ 7.3). SCH 58261 had weak affinity for A₁ receptors (K _i 287 nM), no interaction with A₃ receptors (K _i>10 μM), and showed negligible interaction with A_2B receptors (K _i 5 μM; pA₂ 6.0). These data indicate that SCH 58261 is the most selective A_2A antagonist currently available. Moreover, the different receptor selectivity of these three chemically related compounds provides useful information to progress with structure-activity relationship studies. Received: 2 July 1998 / Accepted: 6 October 1998     

Synthesis,radiofluorination, and preliminary evaluation of the potential 5‐HT2A receptor agonists [18F]Cimbi‐92 and [18F]Cimbi‐150

An agonist PET tracer is of key interest for the imaging of the 5‐HT_2A receptor, as exemplified by the previously reported success of [¹¹C]Cimbi‐36. Fluorine‐18 holds several advantages over carbon‐11, making it the radionuclide of choice for clinical purposes. In this respect, an ¹⁸F‐labelled agonist 5‐HT_2A receptor (5‐HT_2AR) tracer is highly sought after. Herein, we report a 2‐step, 1‐pot labelling methodology of 2 tracer candidates. Both ligands display high in vitro affinities for the 5‐HT_2AR. The compounds were synthesised from easily accessible labelling precursors, and radiolabelled in acceptable radiochemical yields, sufficient for in vivo studies in domestic pigs. PET images partially conformed to the expected brain distribution of the 5‐HT_2AR; a notable exception however being significant uptake in the striatum and thalamus. Additionally, a within‐scan displacement challenge with a 5‐HT_2AR antagonist was unsuccessful, indicating that the tracers cannot be considered optimal for neuroimaging of the 5‐HT_2AR.     

Synthesis of [18F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine ([18F]NicFP): a potential α4β2 nicotinic acetylcholine receptor radioligand for PET

Nicotinic acetylcholine receptors are widely distributed throughout the human brain and are believed to play a role in several neurological and psychiatric disorders. In order to identify an effective PET radioligand for in vivo assessment of the α4β2 subtype of nicotinic receptor, we synthesized [¹⁸F]3‐[1‐(3‐fluoropropyl)‐(S)‐pyrrolidin‐2‐ylmethoxy]pyridine (NicFP). The in vitro K_D of NicFP was determined to be 1.1 nM, and the log P value obtained by HPLC analysis of the unlabelled standard was found to be 2.2. The radiosynthesis of [¹⁸F]NicFP was carried out by a nucleophilic substitution reaction of anhydrous [¹⁸F]fluoride and the corresponding mesylate precursor. After purification by HPLC, the radiochemical yield was determined to be 11.3±2.1% and the specific activity was 0.47±0.18 Ci/μmol (EOS, n = 3). The time of synthesis and purification was 99±2 min. The final product was prepared as a sterile saline solution suitable for in vivo use. Copyright © 2003 John Wiley & Sons, Ltd.     

G Protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors

The effect of guanine nucleotide-binding protein (G protein) modifiers on the binding of the adenosine A_2A receptor agonist 2-[4-(2-p-carboxyethyl[³H])phenyl-amino]-5’-N-ethylcarboxamidoadenosine ([³H]CGS 21680) and of the adenosine A₁ receptor agonist [³H]R-phenylisopropyladenosine ([³H]R-PIA) to rat cortical and striatal membranes was studied. Guanosine 5’-(β,γ-imido)triphosphate (1–300 μM), which uncouples all G proteins, more effectively inhibited [³H]CGS 21680 (30 nM) binding in the cortex than [³H]R-PIA (2 nM) binding to cortical or striatal membranes or [³H]CGS 21680 (30 nM) binding in the striatum. N-Ethylmaleimide (1–300 μM) or pertussis toxin (1–100 μg/ml), which uncouple G_i/G_o protein-coupled receptors, more effectively inhibited [³H]R-PIA binding to cortical or striatal membranes and [³H]CGS 21680 binding in the cortex than [³H]CGS 21680 binding in the striatum. Cholera toxin (2.5–250 μg/ml), which uncouples G_s protein-coupled receptors, more effectively inhibited [³H]CGS 21680 binding in the striatum than [³H]CGS 21680 binding in the cortex and less effectively inhibited [³H]R-PIA binding to cortical or striatal membranes. Treatment of solubilised cortical membranes with pertussis toxin (50 μg/ml) decreased [³H]CGS 21680 (30–100 nM) binding which almost fully recovered after reconstitution with G_i/G_o proteins. The K _i for displacement of [2-³H]-(4-{2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl}phenol) ([³H]ZM 241385, 1 nM) by CGS 21680 was 110 nM (95%CI: 98–122 nM) in non-treated, 230 (167–292) nM in pertussis toxin (25 μg/ml)-treated and 222 (150–295) nM in cholera toxin (50 μg/ml)-treated cortical membranes; in contrast, the K _i for displacement of [³H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine ([³H]SCH 58261, 1 nM) by CGS 21680 was 74 (57–91) nM in non-treated, 71 (44–100) nM in pertussis toxin-treated and 147 (100–193) nM in cholera toxin-treated cortical membranes. Finally, CGS 21680 displaced monophasically the binding of the A₁ antagonist, [³H]8-cyclopentyl-1,3-dipropylxanthine (2 nM), and the A₁ agonist, [³H]R-PIA (2 nM), in 2 or 10 mM Mg²⁺-medium, either at 25°C or 37°C, in cortical or striatal membranes. These results indicate that CGS 21680 does not bind to A₁ receptors and that limbic CGS 21680 binding sites differ from striatal-like A_2A receptors since they couple to G_i/G_o proteins, as well as to G_s proteins. Received: 22 July 1998 / Accepted: 18 January 1999     

2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors

Summary 2-Chloro-N⁶-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for A₁ adenosine receptors. Binding of [³H]PIA to A₁ receptors of rat brain membranes was inhibited by CCPA with a K _i-value of 0.4 nM, compared to a K _i-value of 0.8 nM for the parent compound N⁶-cyclopentyladenosine (CPA). Binding of [³H]NECA to A₂ receptors of rat striatal membranes was inhibited with a K _i-value of 3900 nM, demonstrating an almost 10,000-fold A₁-selectivity of CCPA.CCPA inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A₁ receptor, with an IC₅₀-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC₅₀-value of 3500 nM. The more than 100-fold A₁-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A₁ adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard A₁ receptor agonist R-N⁶-phenylisopropyladenosine (R-PIA). CCPA represents the agonist with the highest selectivity for A₁ receptors reported so far.Abbreviations CCPA 2-choro-N⁶-cyclopentyladenosine - CPA N⁶-cyclopentyladenosine - NECA 5-N-ethylcarboxamidoadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to M. J. Lohse at the above address