A simple and fast method for the preparation of n.c.a. 2-[18F]F-A85380 for human use.

2-[18F]F-A85380 is the first subtype selective PET-radiotracer to visualize the distribution of alpha4beta2 nicotinic acetylcholine receptors in human brain in vivo. We investigated a fast and safe automated production of 2-[18F]F-A85380 by purification of the BOC-protected intermediate product with a combination of solid phase extraction cartridges. After deprotection, adjustment of the pH and sterile filtration n.c.a. 2-[18F]F-A85380 was applicable for the use in human studies with a high specific activity and an overall radiochemical yield of 55% in 35 minutes.

Nicotinic acetylcholine receptors and imaging.

In vivo imaging techniques like positron emission tomography (PET) and single photon emission computed tomography (SPECT) offer the possibility to monitor human central nicotinic acetylcholine receptors (nAChRs) in a variety of central nervous system disorders. In the past, the only available PET radiotracer for imaging nAChRs in the human brain, [11C]-(-)-nicotine, suffered from a spectrum of not suitable properties for in vivo imaging. Current efforts are focused on the development of new, highly specific and highly selective radioligands based on different structural classes (e.g. nicotine, epibatidine, 3-pyridyl ether analogues) for central nAChRs. The most promising compounds are halogenated 3-pyridyl ether compounds for imaging alpha 4 beta 2 nAChRs. But there is still a lack for radiotracers for other subtypes of nicotinic acetylcholine receptors being a promising area of interest.

2-, 5-, and 6-Halo-3-(2(S)-azetidinylmethoxy)pyridines: synthesis, affinity for nicotinic acetylcholine receptors, and molecular modeling.

3-(2(S)-Azetidinylmethoxy)pyridine (A-85380) has been identified recently as a ligand with high affinity for nicotinic acetylcholine receptors (nAChRs). Here we report the synthesis and in vitro nAChR binding of a series of 10 pyridine-modified analogues of A-85380. The novel compounds feature a halogen substituent at position 2, 5, or 6 of the 3-pyridyl fragment. Those with the substituents at position 5 or 6, as well as the 2-fluoro analogue, possess subnanomolar affinity for nAChRs in membranes from rat brain. For these ligands, Ki values range from 11 to 210 pM, as measured by competition with (+/-)-[3H]epibatidine. In contrast, 2-chloro, 2-bromo, and 2-iodo analogues exhibit substantially lower affinity. AM1 quantum chemical calculations demonstrate that the bulky substituents at position 2 cause notable changes in the molecular geometry. The high-affinity members of the series and (+)-epibatidine display a tight fit superposition of low-energy stable conformers. The new ligands with high affinity for nAChRs may be of interest as pharmacological probes, potential medications, and candidates for developing radiohalogenated tracers to study nAChRs.

Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates.

A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.

2-[18F]F-A-85380: a PET radioligand for alpha4beta2 nicotinic acetylcholine receptors.

2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]F-A-85380), a ligand for nicotinic acetylcholine receptors (nAChRs) was evaluated in an in vitro binding assay with membranes of rat brain and in vivo by PET in Rhesus monkey brain. The ligand has high affinity for alpha4beta2 nAChRs (K(D)=50 pM), crosses the blood-brain barrier, and distributes in the monkey brain in a pattern consistent with that of alpha4beta2 nAChRs. The specific/non-specific binding ratio increased steadily, reaching a value of 3.3 in the thalamus at 4 h. The specific binding of 2-[18F]F-A-85380 was reversed by cytisine. These results, in combination with the data demonstrating low toxicity of 2-[18F]F-A-85380, indicate that this ligand shows promise for use with PET in human subjects.

6-[18F]fluoro-A-85380, a novel radioligand for in vivo imaging of central nicotinic acetylcholine receptors.

A novel positron emission tomography (PET) radiotracer, 6-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[18F]fluoro-A-85380, 6-[18F]FA) was synthesized by a no-carrier-added fluorination. In vitro 6-[18F]FA bound to nicotinic acetylcholine receptors (nAChRs), with very high affinity (Kd 28 pM). In PET studies, 6-[18F]FA specifically labeled central nAChRs in the brain of the Rhesus monkey and demonstrated highest levels of accumulation of radioactivity in brain regions enriched with the alpha4beta2 subtype of nAChR. 6-[18F]FA exhibited a target-to-non-target ratio (estimated as radioactivity in the thalamus to that in the cerebellum) of binding in primate brain similar to that previously determined for a labeled analog of epibatidine, [18F]FPH. In contrast to [18F]FPH, the novel tracer is expected to exhibit substantially less toxicity. Thus, the novel radioligand, 6-[18F]FA, appears to be a suitable candidate for imaging nAChRs in human brain.

Syntheses and evaluation of halogenated cytisine derivatives and of bioisosteric thiocytisine as potent and selective nAChR ligands.

We have developed one-step syntheses of halogenated derivatives of (-)-cytisine featuring a halogen substituent at positions 3, 5 or 3 and 5 of the 2-pyridone fragment, and prepared the novel bioisosteric thiocytisine by oxygen-sulphur exchange. The affinities of these pyridone-modified analogs of (-)-cytisine for (alpha 4)(2)(beta 2)(3) and alpha 7* nAChRs in rat forebrain membranes were determined by competition with (+/-)-[(3)H]epibatidine and [(3)H]MLA, respectively. The 3-halocytisines 7 possess subnanomolar affinities for (alpha 4)(2)(beta 2)(3) nAChRs, higher than those found for (-)-cytisine as well as for the 5-halocytisines 8 and 3,5-dihalocytisines 6. In contrast to the parent alkaloid the 3-halogenated species display much a higher affinity for the alpha 7* nAChR subtype. The most potent molecule was 3-bromocytisine (7b) with preferential selectivity (200-fold) for the (alpha 4)(2)(beta 2)(3) subtype [K(i)=10 pM (alpha 4 beta 2) and 2.0 nM (alpha 7*)]. Replacement of the lactam with a thiolactam pharmacophore to thiocytisine (12) resulted in a subnanomolar affinity for the (alpha 4)(2)(beta 2)(3) nAChR subtype (K(i)=0.832 nM), but in a drastic decrease of affinity for the alpha 7* subtype; thiocytisine (12) has a K(i) value of 4000 nM (alpha 7*), giving a selectivity of 4800-fold for the neuronal (alpha 4)(2)(beta 2)(3)-nAChR and thus displaying the best affinity-selectivity profile in the series under consideration.

Imaging of central nAChReceptors with 2-[18F]F-A85380: optimized synthesis and in vitro evaluation in Alzheimer's disease.

In vivo labeling of the nicotinic acetylcholine receptors (nAChR) could be a useful tool for early diagnosis of neurodegenerative disorders. 2-[18F]F-A85380 (2-[18F]Fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine), a ligand with high affinity to the beta2 subunit of the nAChRs, has been shown to label neurons in the nAChR-rich thalamus, cortex and striatum in baboons. We report an optimized synthesis resulting in an uncorrected yield of 58% in 45 min (precursor 2), enabling efficient production intended for clinical use. Incubation of normal rat brain sections with 2-[18F]F-A85380 with subsequent autoradiographic analyses showed the expected distribution in nAChR areas. In human brain sections of Alzheimer's disease (AD) a decrease of 2-[18F]F-A85380 uptake to 36% of the control group was measured in the thalamus and also in the occipital cortex. These findings suggest that 2-[18F]F-A85380 is a promising PET-ligand in the diagnosis of AD.

Synthesis and nicotinic binding studies on enantiopure pinnamine variants with an 8-azabicyclo[3.2.1]octane moiety.

Bioisosteric replacement of the 9-azabicyclo[4.2.1]nonane pharmacophoric element of the novel alkaloidal marine toxine pinnamine (5) by the 8-azabicyclo[3.2.1]octane moiety resulted in conformationally restricted analogues 6a and 6c of (-)-ferruginine (4). Key step in the diastereoselective synthesis of these pyranotropanes was the condensation of enantiopure ecgonine methyl ester (9) from the "chiral pool" with the lithium anion of N-tert-butylbutyraldimin and subsequent cyclisation with TFA. The potential nAChR ligands were tested for their in vitro affinity for the (alpha4)2(beta2)3 and the alpha7* nAChR subtypes. Despite obvious structural similarities with the potent alkaloids pinnamine (5) and (-)-ferruginine (4) the pyranotropanes 6a and 6c exhibited distinctly lower nAChR affinities.

Novel enantiopure ferrugininoids active as nicotinic agents: synthesis and radioligand binding studies.

A series of hitherto unknown enantiopure (-)-ferruginine analogues of type 8 and 9 was prepared and tested for the affinity toward the nicotinic acetylcholine receptor (nAChR) subtypes (alpha4)2(beta2)3, alpha7*, alpha3beta4*, and (alpha1)2beta1gamma delta. The stereoconservative asymmetric syntheses started with (-)-cocainhydrochloride (10) from the chiral pool which was transformed into the chiral building blocks (+)-2-tropanone (11) and to (-)-anhydroecgonine (18). Key steps of the syntheses are novel extensions to existing methodology e.g. a Suzuki Pd(0)-mediated cross-coupling of vinyl triflate (12) with the heteroaryl organoboranes 13-15 and an inverse type [4+2]-cycloaddition with 1,2,4,5-tetrazine (21). The bioisosteric replacement of the 3-acetyl pharmacophoric element of the lead 6 by a 3-pyridyl, 5-chloropyridyl, 5-pyrimidinyl, 2-pyrazinyl, or 4-pyridazinyl moiety resulted in nAChR ligands with Ki-values ranging from 1.1-713 nM toward the (alpha4)2(beta2)3 subtype combined with significant differentiation among the nAChR subtypes when tested in vitro by radioligand binding studies. Generally the ferrugininoids are less potent than the corresponding norferrugininoids. Similar to results of the norferrugininoid series the novel azine substituted ferrugininoids 8 proved to be more potent than the diazine analogues 9; both exhibited higher affinities compared to the lead 6. The 5-chloropyridyl-containing variant 8b [1R, 5S)-enantiomer] turned out to be the most active nAChR ligand with a 12-fold higher affinity toward the (alpha4)2(beta2)3 subtype than the corresponding (1S, 5R)-form ent-8b.

Bisquaternary caracurine V and iso-caracurine V salts as ligands for the muscle type of nicotinic acetylcholine receptors: SAR and QSAR studies.

The binding constants (K(i) values) of 24 caracurine V and 6 iso-caracurine V analogues for the muscle type of nicotinic ACh receptors (nAChR) from Torpedo californica were determined in a binding assay using (+/-)-[(3)H]epibatidine as a radioligand. The allyl alcohol group present in the iso-caracurine V ring system was found to be essential for high binding affinity. The most potent compounds are the dimethyl and di-(4-nitrobenzyl)-iso-caracurinium V salts 29 (18 nM), and 31 (79 nM), respectively. Compound 29 and the corresponding diallyl analogue 30 (350 nM) exhibited similar binding affinities as the equally substituted neuromuscular-blocking agents toxiferine I (14 nM) and alcuronium (234 nM), respectively. The SAR results were confirmed by QSAR studies, which additionally revealed that the presence of hydrogen-bond acceptor groups close to the quaternary nitrogen, is detrimental for the nicotinic binding affinity. The diallyl- and dimethylcaracurinium V salts 13 and 27, respectively, which are known to be among the most potent allosteric modulators of M(2) receptors (EC(50)=10 and 8nM, respectively), exhibited rather low nicotinic binding affinities for muscle type nAChR (K(i)=1.5 and 5.2 microM, respectively). Such a large difference in affinity suggests that it is possible to develop compounds with high muscarinic allosteric potency and low or negligible affinities for (alpha1)(2)beta1gammadelta nAChR. Additionally, the iso-caracurine V analogues with binding affinities comparable to those of (+)-tubocurarine and alcuronium could become a new class of neuromuscular-blocking agents.

Synthesis and evaluation of diazine containing bioisosteres of (-)-ferruginine as ligands for nicotinic acetylcholine receptors.

In this structure-affinity relationship (SAFIR) study, the bioisosteric potential of diazines in the field of ferruginine-type nAChR ligands was investigated. Novel enantiopure analogues of (-)-Ferruginine (3) such as 6-8 were synthesized utilizing enantiomerically pure N-protected (+)-2-tropanone 9 from the 'chiral pool' as versatile chiral building block and a palladium-catalyzed Stille cross-coupling of the tributylstannyl diazines 12, 14 and 16 with the vinyl triflate 11 of (+)-2-tropanone 9. The structures of the novel diazine analogues 6-8 of (-)-ferruginine (3) were assigned on the basis of spectral data, that of ligand 7 being additionally verified by X-ray crystallography. The bioisosteric replacement of the acetyl moiety as structural part of the lead compound 3 with the pyridazine, pyrimidine and pyrazine nucleus resulted in ligands with high to moderate affinity for the central alpha4beta2 and remarkably low affinity for the alpha7* nAChR subtypes. Among the compounds synthesized and tested, 7 was the most active one with K(i)=3.7 nM (alpha4beta2). Compared with the lead 3, this value represents a 30-fold improvement in the affinity for the alpha4beta2 subtype combined with a substantially improved selectivity ratio between the alpha4beta2 and alpha7* subtypes.

5-Iodo-A-85380, an alpha4beta2 subtype-selective ligand for nicotinic acetylcholine receptors.

In an effort to develop selective radioligands for in vivo imaging of neuronal nicotinic acetylcholine receptors (nAChRs), we synthesized 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380) and labeled it with (125)I and (123)I. Here we present the results of experiments characterizing this radioiodinated ligand in vitro. The affinity of 5-[(125)I]iodo-A-85380 for alpha4beta2 nAChRs in rat and human brain is defined by K(d) values of 10 and 12 pM, respectively, similar to that of epibatidine (8 pM). In contrast to epibatidine, however, 5-iodo-A-85380 is more selective in binding to the alpha4beta2 subtype than to other nAChR subtypes. In rat adrenal glands, 5-iodo-A-85380 binds to nAChRs containing alpha3 and beta4 subunits with 1/1000th the affinity of epibatidine, and exhibits 1/60th and 1/190th the affinity of epibatidine at alpha7 and muscle-type nAChRs, respectively. Moreover, unlike epibatidine and cytisine, 5-[(125)I]iodo-A-85380 shows no binding in any brain regions in mice homozygous for a mutation in the beta2 subunit of nAChRs. Binding of 5-[(125)I]iodo-A-85380 in rat brain is reversible, and is characterized by high specificity and a slow rate of dissociation of the receptor-ligand complex (t(1/2) for dissociation approximately 2 h). These properties, along with other features observed previously in in vivo experiments (low toxicity, rapid penetration of the blood-brain barrier, and a high ratio of specific to nonspecific binding), suggest that this compound, labeled with (125)I or (123)I, is superior to other radioligands available for in vitro and in vivo studies of alpha4beta2 nAChRs, respectively.