PET RADIOTRACERS TECHNICAL FIELD The present invention generally relates to compounds capable of binding to α7 nicotinic acetylcholine receptor ( α7-nAChR), and in particular such compounds that can be used as PET radiotracers. BACKGROUND Nicotinic acetylcholine receptors (nAChRs) are receptor polypeptides that respond to the neurotransmitter acetylcholine. Based on the compositions of the subunits that form the ion channel, nAChRs can be classified in two different types; muscle nAChR types and neuronal nAChR types. The neuronal nAChR types vary in homomeric or heteromeric combinations of twelve different nicotinic receptor subunits: α2− α10 and β2− β4. Homomeric α7 nAChRs ( α7-nAChR) mainly expressed in the central nervous system (CNS) and spinal cord are distinguished from neuronal heteromeric nAChRs by their high-affinity binding to α-bungarotoxin ( α- BTX). For decades it was believed that neuronal nAChRs are exclusively expressed only on neurons. However, it has recently been clear that functional nAChR responses can be found in non-excitable cells, including microglia and astrocytes. Thus, α7-nAChR is involved in several cognitive and physiologic processes. Its expression levels and patterns change in neurodegenerative and psychiatric diseases, such as Parkinson´s disease, Alzheimer’s disease or schizophrenia, which makes it a relevant drug target. Positron emission tomography (PET), a high-resolution, sensitive and non-invasive molecular imaging technique, has been successfully utilized in visualizing the localization of α7-nAChR. ¹¹ C-CHIBA-1001 was the first PET radiotracers to image α7-nAChRs in humans but displayed poor specificity for α7-nAChR and high nonspecific uptake. [ ¹⁸ F]ASEM and [ ¹⁸ F]DBT-10 are structural isomers (Fig. 8) based on the dibenzothiophene scaffold, differing only in the position of the fluoro substituent. More recent studies using [ ¹⁸ F]ASEM and [ ¹⁸ F]DBT-10 supported the suitability of the PET radiotracers, showing high and reversible brain uptake with a regional binding pattern consistent with the distribution of α7-nAChRs in the non-human primate brain. Several human PET studies have suggested that novel α7-nAChR radiotracers might be complicated by the fact that α7 subunits can form heteromeric receptors together with other subunits, such as β2, remaining unclear how this affects the selectivity of the radiotracer binding. Furthermore, the development of PET radiotracers for α7-nAChR is challenging because α7-nAChR exhibits very low density in the CNS. Accordingly, successful radiotracers for α7-nAChR should exhibit a combination of high binding affinity and high specificity. There is still a need for compounds that could be used as PET radiotracers or radioligands for α7-nAChR. SUMMARY It is a general objective to provide compounds capable of binding to α7-nAChR. It is a particular objective to provide compounds that could be used as PET radiotracers or radioligands for α7-nAChR. These and other objectives are met by embodiments as disclosed herein. The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims. An aspect of the invention relates to a compound as defined by formula I (I) R ₁ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, with the proviso that R ₁ and R ₂ are not both hydrogen. R ₃ and R ₄ are independently selected from the group consisting of hydrogen, C _1- C ₄ alkyl and C ₁ -C ₄ haloalkyl with the proviso that R ₃ and R ₄ are not both hydrogen. R ₅ is selected from the group consisting of C ₁ -C ₄ alkyl, aryl, C ₁ -C ₄ alkyl phenyl, pyridyl, and C ₁ -C ₄ alkyl pyridine. Another aspect of the invention relates to a PET radiotracer as defined by formula I (I) R ₁ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, with the proviso that R ₁ and R ₂ are not both hydrogen. At least one of R ₁ and R ₂ other than hydrogen comprises a ³ H radioactive isotope and/or a ¹¹ C radioactive isotope. R ₃ and R ₄ are independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl and C ₁ -C ₄ haloalkyl with the proviso that R ₃ and R ₄ are not both hydrogen. R ₅ is selected from the group consisting of C ₁ -C ₄ alkyl, aryl, C ₁ -C ₄ alkyl phenyl, pyridyl, and C ₁ -C ₄ alkyl pyridine. A further aspect of the invention relates to an in vitro competition binding assay method. The method comprises contacting a sample comprising α7-nAChRs with a PET radiotracer according to above and a test agent. The method also comprises filtering the sample through a filter and measuring radiation on the filter. The method further comprises determining binding of the test agent to α7-nAChRs based on the measured radiation. The invention also relates to use of a PET radiotracer according to above to visualize localization and/or distribution of homomeric α7-nAChRs in vitro in a tissue or in a subject by means of PET imaging. Yet another aspect of the invention relates to a method of diagnosing a neurodegenerative or psychiatric disease, or monitoring progression of the neurodegenerative or psychiatric disease. The method comprises administering a PET radiotracer according to above to a subject and taking PET images of the subject to detect location of α7-nAChRs in the subject. The compounds are ASEM analogs with different ortho- and para-substitutions that can be labelled with ³ H and/or ¹¹ C to be used as PET radiotracers capable of binding to α7-nAChR both in vitro and in vivo in a subject body. The PET radiotracers thereby enable visualization and quantification of α7-nAChR in various target tissues, including monitoring the distribution of α7-nAChRs in such a target tissue. The compounds exhibit of high binding affinity and specificity towards α7-nAChR and are able to pass the blood-brain barrier (BBB). BRIEF DESCRIPTION OF THE DRAWINGS The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which: Fig.1 (a) Structures of α7-AChBP and its binding site, (b) the binding mode of epibatidine with α7-AChBP, and (c) the binding mode of ASEM with α7-AChBP. Epibatidine and ASEM are shown in thick stick mode while other residues in thin stick mode. Non-polar hydrogens are not shown for clarity. Fig.2 The predicted binding mode of ASEM (a) and DBT-10 (b). Fig.3 Binding mode of ASEM with α7-AChBP and the structure of ASEM analogues. Fig.4 Histogram for inhibition of ASEM analogues with substitutions at the R ₁ and R ₂ positions. Fig.5 Comparison of the in silico binding free energy difference calculated by FEP+ and in vitro inhibition. Fig. 6 The binding mode of R ₁ - and R ₂ -analogues. (a) Representative structures of R ₁ -analogues. (b) Representative structures of R ₂ -analogues. Fig.7 Time evolutions of ASEM and loop C from t = 0 (full arrow) to unbound (hatched arrow) during the unbinding process. Fig.8 Structures of radiotracers ¹⁸ F-ASEM and ¹⁸ F-DBT-10. Fig. 9 Radiosynthesis of [ ¹¹ C]KIn74, [ ¹¹ C]KIn75, [ ¹¹ C]KIn77, [ ¹¹ C]KIn83, [ ¹¹ C]KIn84 and [ ¹¹ C]KIn85. Conditions: a: [ ¹¹ C]CH ₃ I/DMF/CsCO ₃ , 80°C/4 min; b: [ ¹¹ C]CH ₃ I/DMF/KOH, 90°C/5 min; c: [ ¹¹ C]CH ₃ I/DMF/KOH, 90°C/ 5 min; d: [ ¹¹ C]CH ₃ I/DMF/CsCO ₃ , 80°C/4 min; e: [ ¹¹ C]CH ₃ I/DMF/KOH, 90°C/3 min; f: [ ¹¹ C]CH ₃ I/DMF/KOH, 90°C/5 min and Radiosynthesis of [ ³ H]KIn74, [ ³ H]KIn83 and [ ³ H]KIn84. Conditions: g: [ ³ H]CH ₃ I/DMSO/KOH, 90°C/30 min; h: [ ³ H]CH ₃ I/DMSO/KOH, 90°C/30 min; i: [ ¹¹ H]CH ₃ I/DMF/CsCO ₃ , 90°C/30 min. Fig.10 (A) Autoradiogram obtained showing total binding obtained with [ ¹¹ C]KIn83 (0.01 MBq/ml) and non- specific binding using different blockers at 10 µM (Nicotine, ASEM, KIn83, KIn84 and KIn85) in rat at hippocampus level section. (B) Quantification of [ ¹¹ C]KIn83 (0.01 MBq/ml) total and non-specific binding (expressed in PSL/mm ² ). Fig. 11 (A) Autoradiograms showing the total and non-specific binding (blocked with homologous cold compound and ASEM at 10 µM) obtained in rat when using [ ³ H]KIn83 at 1 nM concentration. (B) Autoradiograms showing the total and non-specific binding (blocked with homologous cold compound (10 µM), ASEM (10 µM), KIn77 (10 µM) and nicotine (100 µM) obtained in rat when using [ ³ H]KIn83 at 0.8 nM concentration. (C) Quantification of total and nonspecific binding for [ ³ H]KIn83 expressed as percentage over total binding (100%). Fig.12 (A) Autoradiogram showing the total binding obtained with using [ ³ H]KIn83 (1 nM) and non-specific binding (blocked with KIn83 and ASEM at 10 µM) obtained in temporal cortex of human tissue from a healthy control (CT) and an Alzheimer´s disease patient (AD). (B) Quantification of total and non-specific binding for [ ³ H]KIn83 in control (white bars) and PD tissue (black bars) obtained when blocking with KIn83 or ASEM. (C) Specific binding obtained blocking with KIn83 and ASEM. Data is expressed in fmol/mg. Fig.13 (a, c) Autoradiographical localization of ¹²⁵ I-BTX (1.4 nM) binding and [ ³ H]KIn83 (1 nM) binding is shown in a 14 pm coronal section of the telencephalon. Approximate level: bregma -0.3 mm, bar = 1 mm. Abbreviations: 1, anterior cingulate cortex; 2, frontoparietal cortex, motor arca; 3, frontoparietal cortex, somatosensory area; 4, piriform cortex; 5, rhinal fissure; 6, claustrum, endopiriform nucleus; 7, corpus callosum; 8, septofimbrial nucleus; 9, anterior commissure; 10, lateral septal nucleus intermediate part; 11, caudate putamen; 12, bed nucleus stria terminalis, lateral part; 13, bed nucleus stria terminalis, medial part; 14, septohypothalamic nucleus; 15, median preoptic nucleus; 16, medial preoptic area; 17, olfactory tuhercle; 18, nucleus of the horizontal limb diagonal band; 19, ventral pallidum; 20, optic tract; 21, preoptic suprachiasmatic nucleus. (b, d) Autoradiographic localization of ¹²⁵ I-BTX (1.4 nM) and [ ³ H]KIn83 (1 nM) binding is shown in a 14 µm coronal section of the diencephalon. Approximate level: bregma - 1.5 mm, bar = 1 mm. Abbreviations: 1, anterior cingulate cortex; 2, frontoparietal cortex, motor area; 3, frontoparietal cortex, somatosensory area; 4, piriform cortex; 5, rhinal fissure; 6, claustrum, endopiriform nucleus; 7, corpus callosum; 8, optic tract; 9, hippocampus; 10, ventro-lateral thalamic nucleus; 11, anteroventral thalamic nucleus; 12, reuniens thalamic nucleus; 13, paraventricular thalamic nucleus; 14, paraventricular hypothalamic nucleus; 15, fornix; 16, third ventricle; 17, anterior hypothalamic area; 18, central amygdaloid nucleus; 19, lateral hypothalamic area; 20, medial amygdaloid nucleus; 21, ciudate putamcn; 22, supraoptic hypothalamic nucleus. Fig.14. PET images of [ ¹¹ C]KIn83 co-registered with MRI at baseline, after pre-treatment with ASEM and after pre-treatment with tariquidar. Fig.15. Average time activity curves for [ ¹¹ C]KIn83 in different brain regions at baseline condition. Fig.16 (A) Radiochromatogram of plasma taken 15 min after injection of [ ¹¹ C]KIn83 at baseline condition, (B) Radiochromatogram of plasma taken 15 min after injection of [ ¹¹ C]KIn83 after pre-treatment with ASEM, (C) The in vivo metabolism of [ ¹¹ C]KIn83 is shown as the relative plasma composition of parent compound (PET1: baseline, PET2: after pre-treatment with ASEM). Fig.17 Saturation binding assay performed in GH ₃ -ha7 cells with ³ H-Epibatidine from 0-10 nM. Fig.18 Competition binding studies in GH ₃ -ha7 cells using ³ H-Epibatidine (A), ³ H-ASEM (B) and ³ H-KIn83 (C) with increasing concentration of unlabeled epibatidine, ASEM, KIn83, KIn84, Kln77, KIn74, Kln90, or KIn60. Fig. 19 Competition binding studies in α4β2 cells using ³ H-Epibatidine with increasing concentration of unlabeled epibatidine, ASEM, KIn83, KIn84, Kln77 or KIn74. Fig.20 Saturation binding assay performed in P2 fraction from human control tissue with ³ H-ASEM from 0- 1.5 nM with 1 µM nicotine as non-specific. Fig.21 Competition binding studies in P2 fraction from human control tissue with ³ H-ASEM (A) and [ ³ H]KIn83 (B) with increasing concentration of unlabeled epibatidine, ASEM, KIn83, KIn84, KIn74. Fig.22 Comparison of binding studies in P2 fraction from human control tissue and AD brain using [ ³ H]KIn83. Fig.23 [ ³ H]KIn83 Autoradiography on large frozen brain section from one control, one AD and one arctic case. Fig.24 The synthesis of KIn83 is outlined. Fig.25 The synthesis of PRE-1 is outlined. Fig.26 The synthesis of PRE-3 is outlined. Fig.27 The synthesis of PRE-2 is outlined. Fig.28 The synthesis of PRE-4 is outlined. DETAILED DESCRIPTION The present invention generally relates to compounds capable of binding to α7 nicotinic acetylcholine receptor ( α7-nAChR), and in particular compounds that can be used as positron emission tomography (PET) radiotracers. The compounds of the present invention are ASEM (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6- dibenzothiophene 5,5-dioxide) analogs with different ortho- and para-substitutions. These compounds are capable of binding to α7-nAChR and can be labelled with ³ H and/or ¹¹ C, for example, by means of various alkylation and/or carbonylation reactions. Hence, the compounds can be used as PET radiotracers capable of binding to α7-nAChR both in vitro and in vivo in a subject body. The PET radiotracers thereby enable visualization and quantification of α7-nAChR in various target tissues, including monitoring the distribution of α7-nAChRs in such a target tissue. The PET radiotracers can be used to diagnose various neurodegenerative and neuropsychiatric disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and schizophrenia, characterized by altered and abnormal density and/or distribution of α7-nAChRs. In addition, or alternatively, the PET radiotracers can be used to measure receptor occupancies of drugs targeting α7-nAChR and dose-response relationships. Hence, the PET tracers of the embodiments find uses in drug development and drug characterization and verification. The compounds of the invention have several properties making them suitable for PET radiotracers in addition to binding to α7-nAChR. Firstly, the compounds exhibit a high binding affinity and specificity towards α7-nAChR. Such a property is important since α7-nAChRs are generally present in very low density in central nervous system (CNS) (0.13 – 15 fmol/mg protein in humans). Furthermore, the compounds are predicted to pass the blood-brain barrier (BBB), thereby enabling transport of the compounds and PET radiotracers to target tissue in the CNS and the brain when administered to a subject. An aspect of the invention relates to a compound as defined by formula I (I) According to the invention, R ₁ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, with the proviso that R ₁ and R ₂ are not both hydrogen. R ₃ and R ₄ are independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl and C ₁ -C ₄ haloalkyl with the proviso that R ₃ and R ₄ are not both hydrogen. R ₅ is selected from the group consisting of C ₁ -C ₄ alkyl, aryl, C ₁ -C ₄ alkyl phenyl, pyridyl, and C ₁ -C ₄ alkyl pyridine. ASEM (R ₁ and R ₂ are both hydrogen) is available as a PET radiotracer in the form of [ ¹⁸ F]ASEM, i.e., having R ₁ as ¹⁸ F and R ₂ as hydrogen, or as [ ¹⁸ F]DBT-10, i.e., having R ₁ as hydrogen and R ₂ as ¹⁸ F, see Fig.8. In clear contrast to ASEM, the compounds of the present invention comprise an ortho-substitution (R ₁ ) and/or a para-substitution (R ₂ ) in terms of an amine, an amide, or an alkoxy group. In an embodiment, both of R ₁ and R ₂ are different than hydrogen, i.e., are independently selected from the group consisting of -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy. In a preferred embodiment, one but not both of R ₁ and R ₂ is hydrogen. In an embodiment, R ₁ is selected from the group consisting of -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy and R ₂ is hydrogen. In another embodiment, R ₁ is hydrogen and R ₂ is selected from the group consisting of -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy. The compounds of the present invention are, as shown herein, capable of binding to the same binding site in α7-nAChR (see Fig.3) as ASEM (Figs.1c, 2a), DBT-10 (Fig.2b) and epibatidine (Figs.1a, 1b), which is an agonist of α7-nAChR. In this binding site, the ortho-substitution (R ₁ ) of the compounds points towards the solvent, whereas the para-substitution (R ₂ ) points towards a hydrophilic region between serine 32 (Ser32) and Ser34 as indicated in Fig.3c. Hence, the size of the substitutions at the ortho-position (R ₁ ) of the compounds in formula I can generally be larger as compared to the size of the substitutions at the para-position (R ₂ ) without negatively affecting the binding of the compounds to α7-nAChR. In more detail, substitutions at the ortho-position had a negative relative free energy (ΔΔG) as compared to ASEM, which indicated that substitutions at this position improved the binding of the compounds to α7- nAChR as compared to ASEM, i.e., the compounds had higher binding affinities than ASEM (Table 1). Hence, in a currently, preferred embodiment, R ₁ is selected from the group consisting of -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy and R ₂ is hydrogen. In an embodiment, the C ₁ -C ₄ alkoxy is a C ₁ -C ₂ alkoxy, i.e., is selected from the group consisting of methoxy and ethoxy. In a particular embodiment, C ₁ -C ₄ alkoxy is a C ₁ alkoxy, i.e., methoxy. In an embodiment, R ₃ and R ₄ are independently selected from the group consisting of hydrogen, C ₁ -C ₃ alkyl and C ₁ -C ₃ haloalkyl with the proviso that R ₃ and R ₄ are not both hydrogen. In a particular embodiment, R ₃ and R ₄ are independently selected from the group consisting of methyl, ethyl, propyl and halopropyl with the proviso that R ₃ and R ₄ are not both hydrogen, more preferably R ₃ and R ₄ are independently selected from the group consisting of hydrogen, methyl, propyl and halopropyl with the proviso that R ₃ and R ₄ are not both hydrogen. In an embodiment, the halopropyl is fluoropropyl. Other examples of halopropyl groups that could be used according to the invention include chloropropyl and iodopropyl. In a preferred embodiment, R ₃ and R ₄ are independently selected from the group consisting of hydrogen and methyl with the proviso that R ₃ and R ₄ are not both hydrogen. Hence, preferred -NR ₃ R ₄ groups include methylamine (–NHCH ₃ ) and dimethylamine (–NH(CH ₃ ) ₂ ). In an embodiment, R ₅ is selected from the group consisting of C ₁ -C ₂ alkyl, C ₁ -C ₂ alkyl phenyl, and C ₁ -C ₂ alkyl pyridine. In a particular embodiment, R ₅ is selected from the group consisting of methyl, ethyl, benzyl and pyridinylmethyl. In a preferred particular embodiment, R ₅ is selected from the group consisting of methyl, ethyl, benzyl and 4-pydinylmethyl. In an embodiment, the compound is selected from the group consisting of: N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)acetamide (KIn60); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-methoxydibenzo[b,d]t hiophene 5,5-dioxide (KIn74); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-methylaminodibenzo[b ,d]thiophene 5,5-dioxide) (KIn75); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((4-fluoroethyl)(met hyl)amino)dibenzo[b,d]thiophene 5,5- dioxide (KIn76); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-N,N-dimethylaminodib enzo[b,d]thiophene 5,5-dioxide (KIn77); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methoxydibenzo[b,d]t hiophene 5,5-dioxide (KIn83); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methylaminodibenzo[b ,d]thiophene 5,5-dioxide (KIn84); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-N,N-dimethylaminodib enzo[b,d]thiophene 5,5-dioxide (KIn85); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((2-fluoroethyl)(met hyl)amino)dibenzo[b,d]thiophene 5,5- dioxide (KIn86); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-(methyl(propyl)amino )dibenzo[b,d]thiophene 5,5-dioxide (KIn87); N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenz [b,d]thiophene-4-yl)propionamide (KIn88); N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-phenylacetamide (KIn89); N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)acetamide (KIn91); N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)phenylacetamide (KIn93); N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)-2-(pyridine-4- yl)acetamide (KIn94); and N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-(pyridine-4- yl)acetamide (KInXX). In a preferred embodiment, the compound is selected from the group consisting of: 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-methoxydibenzo[b,d]t hiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methoxydibenzo[b,d]t hiophene 5,5-dioxide; and 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methylaminodibenzo[b ,d]thiophene 5,5-dioxide. A currently preferred compound is 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methoxydibenzo[b,d]t hiophene 5,5-dioxide. The compounds of the present invention can be manufactured as disclosed in Example 1 and 2 and as disclosed in Example 3 but using CH ₃ I rather than [ ¹¹ C]CH ₃ I as alkylating agent. The PET radiotracers of the present invention are defined by formula I

(I) According to the invention, R ₁ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, with the proviso that R ₁ and R ₂ are not both hydrogen. At least one of R ₁ and R ₂ other than hydrogen comprises a ³ H radioactive isotope and/or a ¹¹ C radioactive isotope. R ₃ and R ₄ are independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl and C ₁ -C ₄ haloalkyl with the proviso that R ₃ and R ₄ are not both hydrogen. R ₅ is selected from the group consisting of C ₁ -C ₄ alkyl, aryl, C ₁ -C ₄ alkyl phenyl, pyridyl, and C ₁ -C ₄ alkyl pyridine. The PET radiotracers of the present invention are thereby ³ H PET radiotracers, ¹¹ C PET radiotracers or ³ H and ¹¹ C PET radiotracers since at least one of the R ₁ and R ₂ groups that is other than hydrogen, i.e., -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, comprises a ³ H radioactive isotope and/or a ¹¹ C radioactive isotope. In an embodiment, one of R ₁ and R ₂ is selected from the group consisting of –NR ₆ R ₇ , -NHC(O)R ₈ , [ ³ H]C ₁ -C ₄ alkoxy and [ ¹¹ C]C ₁ -C ₄ alkoxy. One of R ₆ and R ₇ is selected from the group consisting of [ ³ H]C ₁ -C ₄ alkyl, [ ¹¹ C]C ₁ -C ₄ alkyl, [ ³ H]C ₁ -C ₄ haloalkyl and [ ¹¹ C]C ₁ -C ₄ haloalkyl and the other of R ₆ and R ₇ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, [ ³ H]C ₁ -C ₄ alkyl, [ ¹¹ C]C ₁ -C ₄ alkyl, C ₁ -C ₄ haloalkyl, [ ³ H]C ₁ -C ₄ haloalkyl and [ ¹¹ C]C ₁ -C ₄ haloalkyl. R ₈ is selected from the group consisting of [ ³ H]C ₁ -C ₄ alkyl, [ ¹¹ C]C ₁ -C ₄ alkyl, [ ³ H]aryl, [ ¹¹ C]aryl, C ₁ -C ₄ alkyl [ ³ H]phenyl, C ₁ -C ₄ alkyl [ ¹¹ C]phenyl, [ ³ H]pyridyl, [ ¹¹ C]pyridyl, C ₁ -C ₄ alkyl [ ³ H]pyridine and C ₁ -C ₄ alkyl [ ¹¹ C]pyridine. The other of R ₁ and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, –NR ₆ R ₇ , -NHC(O)R ₈ , [ ³ H]C ₁ -C ₄ alkoxy and [ ¹¹ C]C ₁ -C ₄ alkoxy. In an embodiment, the PET radiotracers are ³ H PET radiotracers, in which at least one of the R ₁ and R ₂ groups that is other than hydrogen comprises a ³ H radioactive isotope. In such an embodiment, one of R ₁ and R ₂ is selected from the group consisting of –NR ₉ R ₁₀ , -NHC(O)R ₁₁ , and [ ³ H]C ₁ -C ₄ alkoxy. One of R ₉ and R ₁₀ is selected from the group consisting of [ ³ H]C ₁ -C ₄ alkyl and [ ³ H]C ₁ -C ₄ haloalkyl and the other of R ₉ and R ₁₀ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, [ ³ H]C ₁ -C ₄ alkyl, C ₁ -C ₄ haloalkyl, and [ ³ H]C ₁ -C ₄ haloalkyl. R ₁₁ is selected from the group consisting of [ ³ H]C ₁ -C ₄ alkyl, [ ³ H]aryl, C ₁ -C ₄ alkyl [ ³ H]phenyl, [ ³ H]pyridyl and C ₁ -C ₄ alkyl [ ³ H]pyridine. The other of R ₁ and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, –NR ₉ R ₁₀ , -NHC(O)R ₁₁ , and [ ³ H]C ₁ -C ₄ alkoxy. In another embodiment, the PET radiotracers are ¹¹ C PET radiotracers, in which at least one of the R ₁ and R ₂ groups that is other than hydrogen comprises a ¹¹ C radioactive isotope. In such an embodiment, one of R ₁ and R ₂ is selected from the group consisting of –NR ₁₂ R ₁₃ , -NHC(O)R ₁₄ , and [ ¹¹ C]C ₁ -C ₄ alkoxy. One of R ₁₂ and R ₁₃ is selected from the group consisting of [ ¹¹ C]C ₁ -C ₄ alkyl and [ ¹¹ C]C ₁ -C ₄ haloalkyl and the other of R ₁₂ and R ₁₃ is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, [ ¹¹ C]C ₁ -C ₄ alkyl, C ₁ -C ₄ haloalkyl, and [ ¹¹ C]C ₁ -C ₄ haloalkyl. R ₁₄ is selected from the group consisting of [ ¹¹ C]C ₁ -C ₄ alkyl, [ ¹¹ C]aryl, C ₁ -C ₄ alkyl [ ¹¹ C]phenyl, [ ¹¹ C]pyridyl and C ₁ -C ₄ alkyl [ ¹¹ C]pyridine. The other of R ₁ and R ₂ is selected from the group consisting of hydrogen, -NR ₃ R ₄ , -NHC(O)R ₅ and C ₁ -C ₄ alkoxy, –NR ₁₂ R ₁₃ , -NHC(O)R ₁₄ , and [ ¹¹ C]C ₁ -C ₄ alkoxy. The PET radiotracers of the invention use ¹¹ C and/or ³ H, preferably ¹¹ C or ³ H, as radiolabel. The PET radiotracers can then be manufactured using ¹¹ C or ³ H alkylation, preferably methylation, or carbonylation reactions as described in Example 3 herein. ¹¹ C radioactive isotopes have the advantage of enabling synthesis of PET radiotracers using methylation and carbon monoxide chemistry. Synthesis of PET radiotracers with ¹⁸ F is more limited. An advantage of the compounds of the invention is that they can be used as either ¹¹ C PET radiotracers or ³ H PET radiotracers. ³ H PET radiotracers are in particular useful in experimental studies due to their longer half-life (T1/2 of about 12.32 years). ¹¹ C and ¹⁸ F PET radiotracers generally have too short half-life to be used in such experiments. Hence, the PET radiotracers of the invention can be synthesized using the same methylation reaction for both ¹¹ C radioactive isotopes ([ ¹¹ C]CH ₄ ) and ³ H radioactive isotopes ([ ³ H]CH ₄ ). The ³ H PET radiotracers can then be used in experimental studies to validate the effects of the corresponding ¹¹ C PET radiotracers. In an embodiment, both of R ₁ and R ₂ are different than hydrogen, i.e., are independently selected from the group consisting of -NR ₃ R ₄ , -NHC(O)R ₅ , C ₁ -C ₄ alkoxy and C ₁ -C ₄ alkoxy. At least one of R ₁ and R ₂ comprises a ³ H radioactive isotope and/or a ¹¹ C radioactive isotope. In a preferred embodiment, one but not both of R ₁ and R ₂ is hydrogen. In an embodiment, R ₂ is hydrogen and R ₁ is other than hydrogen. In a particular embodiment, R ₁ is selected from the group consisting of– NR ₆ R ₇ , -NHC(O)R ₈ , [ ³ H]C ₁ -C ₄ alkoxy and [ ¹¹ C]C ₁ -C ₄ alkoxy and R ₂ is hydrogen. In another particular embodiment, R ₁ is selected from the group consisting of –NR ₉ R ₁₀ , -NHC(O)R ₁₁ , and [ ³ H]C ₁ -C ₄ alkoxy and R ₂ is hydrogen. In a further particular embodiment, R ₁ is selected from the group consisting of –NR ₁₂ R ₁₃ , - NHC(O)R ₁₄ , and [ ¹¹ C]C ₁ -C ₄ alkoxy and R ₂ is hydrogen. In another embodiment, R ₁ is hydrogen and R ₂ is other than hydrogen. In a particular embodiment, R ₁ is hydrogen and R ₂ is selected from the group consisting of NR ₆ R ₇ , -NHC(O)R ₈ , [ ³ H]C ₁ -C ₄ alkoxy and [ ¹¹ C]C ₁ - C ₄ alkoxy. In another particular embodiment, R ₁ is hydrogen and R ₂ is selected from the group consisting of –NR ₉ R ₁₀ , -NHC(O)R ₁₁ , and [ ³ H]C ₁ -C ₄ alkoxy. In a further particular embodiment, R ₁ is hydrogen and R ₂ is selected from the group consisting of –NR ₁₂ R ₁₃ , -NHC(O)R ₁₄ , and [ ¹¹ C]C ₁ -C ₄ alkoxy. In an embodiment, the [ ³ H]C ₁ -C ₄ alkoxy is a [ ³ H]C ₁ -C ₂ alkoxy, i.e., is selected from the group consisting of [ ³ H]methoxy and [ ³ H]ethoxy. In a particular embodiment, [ ³ H]C ₁ -C ₄ alkoxy is a [ ³ H]C ₁ alkoxy, i.e., [ ³ H]methoxy. Correspondingly, in an embodiment, the [ ¹¹ C]C ₁ -C ₄ alkoxy is a [ ¹¹ C]C ₁ -C ₂ alkoxy, i.e., is selected from the group consisting of [ ¹¹ C]methoxy and [ ¹¹ C]ethoxy. In a particular embodiment, [ ¹¹ C]C ₁ -C ₄ alkoxy is a [ ¹¹ C]C ₁ alkoxy, i.e., [ ¹¹ C]methoxy. In an embodiment, one of R ₆ and R ₇ is selected from the group consisting of [ ³ H]C ₁ -C ₃ alkyl, [ ¹¹ C]C ₁ -C ₃ alkyl, [ ³ H]C ₁ -C ₃ haloalkyl and [ ¹¹ C]C ₁ -C ₃ haloalkyl and the other of R ₆ and R ₇ is selected from the group consisting of hydrogen, C ₁ -C ₃ alkyl, [ ³ H]C ₁ -C ₃ alkyl, [ ¹¹ C]C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, [ ³ H]C ₁ -C ₃ haloalkyl and [ ¹¹ C]C ₁ -C ₃ haloalkyl. In a particular embodiment, one of R ₆ and R ₇ is selected from the group consisting of [ ³ H]methyl, [ ¹¹ C]methyl, [ ³ H]ethyl, [ ¹¹ C]ethyl, [ ³ H]propyl, [ ¹¹ C]propyl, [ ³ H]halopropyl and [ ¹¹ C]halopropyl and the other of R ₆ and R ₇ is selected from the group consisting of hydrogen, methyl, [ ³ H]methyl, [ ¹¹ C]methyl, ethyl, [ ³ H]ethyl, [ ¹¹ C]ethyl, propyl, [ ³ H]propyl, [ ¹¹ C]propyl, halopropyl, [ ³ H]halopropyl and [ ¹¹ C]halopropyl. In an embodiment, one of R ₉ and R ₁₀ is selected from the group consisting of hydrogen, [ ³ H]C ₁ -C ₃ alkyl and [ ³ H]C ₁ -C ₃ haloalkyl and the other of R ₉ and R ₁₀ is selected from the group consisting of hydrogen, C ₁ -C ₃ alkyl, [ ³ H]C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, and [ ³ H]C ₁ -C ₃ haloalkyl. In a particular embodiment, one of R ₉ and R ₁₀ is selected from the group consisting of [ ³ H]methyl, [ ³ H]ethyl, [ ³ H]propyl and [ ³ H]halopropyl and the other of R ₆ and R ₇ is selected from the group consisting of hydrogen, methyl, [ ³ H]methyl, ethyl, [ ³ H]ethyl, propyl, [ ³ H]propyl, halopropyl and [ ³ H]halopropyl. In an embodiment, one of R ₁₂ and R ₁₃ is selected from the group consisting of [ ¹¹ C]C ₁ -C ₃ alkyl and [ ¹¹ C]C ₁ - C ₃ haloalkyl and the other of R ₁₂ and R ₁₃ is selected from the group consisting of hydrogen, C ₁ -C ₃ alkyl, [ ¹¹ C]C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, and [ ¹¹ C]C ₁ -C ₃ haloalkyl. In a particular embodiment, one of R ₁₂ and R ₁₃ is selected from the group consisting of [ ¹¹ C]methyl, [ ¹¹ C]ethyl, [ ¹¹ C]propyl, and [ ¹¹ C]halopropyl and the other of R ₁₂ and R ₁₃ is selected from the group consisting of hydrogen, methyl, [ ¹¹ C]methyl, ethyl, [ ¹¹ C]ethyl, propyl, [ ¹¹ C]propyl, halopropyl, and [ ¹¹ C]halopropyl In an embodiment, the [ ³ H]halopropyl is [ ³ H]fluoropropyl and [ ¹¹ C]halopropyl is [ ¹¹ C]fluoropropyl. Other examples of halopropyl groups that could be used according to the invention include [ ³ H]chloropropyl, [ ¹¹ C]chloropropyl, [ ³ H]iodopropyl and [ ¹¹ C]iodopropyl In an embodiment, R ₈ is selected from the group consisting of [ ³ H]C ₁ -C ₂ alkyl, [ ¹¹ C]C ₁ -C ₂ alkyl, [ ³ H]C ₁ -C ₂ alkyl phenyl, [ ¹¹ C]C ₁ -C ₂ alkyl phenyl, [ ³ H]C ₁ -C ₂ alkyl pyridine and [ ¹¹ C]C ₁ -C ₂ alkyl pyridine. In a particular embodiment, R ₈ is selected from the group consisting of [ ³ H]methyl, [ ¹¹ C]methyl, [ ³ H]ethyl, [ ¹¹ C]ethyl, [ ³ H]benzyl, [ ¹¹ C]benzyl, [ ³ H]pyridinylmethyl and [ ¹¹ C]pyridinylmethyl. In a preferred particular embodiment, R ₈ is selected from the group consisting of [ ³ H]methyl, [ ¹¹ C]methyl, [ ³ H]ethyl, [ ¹¹ C]ethyl, [ ³ H]benzyl, [ ¹¹ C]benzyl, [ ³ H]4-pyridinylmethyl and [ ¹¹ C]4-pyridinylmethyl. In an embodiment, R ₁₁ is selected from the group consisting of [ ³ H]C ₁ -C ₂ alkyl, [ ³ H]C ₁ -C ₂ alkyl phenyl, and [ ³ H]C ₁ -C ₂ alkyl pyridine. In a particular embodiment, R ₁₁ is selected from the group consisting of [ ³ H]methyl, [ ³ H]ethyl, [ ³ H]benzyl, and [ ³ H]pyridinylmethyl. In a preferred particular embodiment, R ₁₁ is selected from the group consisting of [ ³ H]methyl, [ ³ H]ethyl, [ ³ H]benzyl, and [ ³ H]4-pyridinylmethyl. In an embodiment, R ₁₄ is selected from the group consisting of [ ¹¹ C]C ₁ -C ₂ alkyl, [ ¹¹ C]C ₁ -C ₂ alkyl phenyl, and [ ¹¹ C]C ₁ -C ₂ alkyl pyridine. In a particular embodiment, R ₁₄ is selected from the group consisting of [ ¹¹ C]methyl, [ ¹¹ C]ethyl, [ ¹¹ C]benzyl, and [ ¹¹ C]pyridinylmethyl. In a preferred particular embodiment, R ₁₄ is selected from the group consisting of [ ¹¹ C]methyl, [ ¹¹ C]ethyl, [ ¹¹ C]benzyl, and [ ¹¹ C]4-pyridinylmethyl. In an embodiment, the PET radiotracer is selected from the group consisting of: N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)[ ³ H]CH ₃ -acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)[ ¹¹ C]CO-acetamide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ³ H]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ³ H]methylaminodibenzo[b,d]thiophene 5,5-dioxide); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methylaminodibenzo[b,d]thiophene 5,5-dioxide); 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((4-fluoroethyl)([ ³ H]methyl)amino)dibenzo[b,d]thiophene 5,5- dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((4-fluoroethyl)([ ¹¹ C]methyl)amino)dibenzo[b,d]thiophene 5,5- dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ³ H]N,N-dimethylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]N,N-dimethylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]methylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]N,N-dimethylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]N,N-dimethylaminodibenzo[b,d]thiophene-5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((2-fluoroethyl)([ ³ H]methyl)amino)dibenzo[b,d]thiophene 5,5- dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((2-fluoroethyl)([ ¹¹ C]methyl)amino)dibenzo[b,d]thiophene 5,5- dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-([ ³ H]methyl(propyl)amino)dibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-([ ¹¹ C]methyl(propyl)amino)dibenzo[b,d]thiophene 5,5-dioxide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenz[b ,d]thiophene-4-yl)[ ³ H]propionamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenz[b ,d]thiophene-4-yl)[ ¹¹ C]CO-propionamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2- phenyl[ ³ H]acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-[ ¹¹ C]CO- phenylacetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)[ ³ H]acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)[ ¹¹ C]CO-acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)phenyl- [ ³ H]acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)[ ¹¹ C]CO- phenylacetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)-2-(pyridine-4-yl)[ ³ H]- acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)-2-(pyridine-4- yl)[ ¹¹ C]CO-acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-(pyridine-4-yl)[ ³ H]- acetamide; and N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-(pyridine-4- yl)[ ¹¹ C]CO-acetamide. In another embodiment, the PET radiotracer is selected from the group consisting of: N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)[ ¹¹ C]CO-acetamide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methylaminodibenzo[b,d]thiophene-5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((4-fluoroethyl)([ ¹¹ C]methyl)amino)dibenzo[b,d]thiophene 5,5- dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]N,N-dimethylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]N,N-dimethylaminodibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-((2-fluoroethyl)([ ¹¹ C]methyl)amino)dibenzo[b,d]thiophene 5,5- dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-([ ¹¹ C]methyl(propyl)amino)dibenzo[b,d]thiophene 5,5-dioxide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenz[b ,d]thiophene-4-yl)[ ³ H]propionamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-[ ¹¹ C]CO- phenylacetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)[ ¹¹ C]CO-acetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)[ ¹¹ C]CO- phenylacetamide; N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-2-yl)-2-(pyridine-4- yl)[ ¹¹ C]CO-acetamide; and N-(7-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5,5-dioxidodibenzo[ b,d]thiophene-4-yl)-2-(pyridine-4- yl)[ ¹¹ C]CO-acetamide. In a particular embodiment, the PET radiotracer is selected from the group consisting of: 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ³ H]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6- ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]methylaminodibenzo[b,d]thiophene 5,5-dioxide; and 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methylaminodibenzo[b,d]thiophene 5,5-dioxide. In another particular embodiment, the PET radiotracer is selected from the group consisting of: 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide; and 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methylaminodibenzo[b,d]thiophene 5,5-dioxide. In a preferred embodiment, the PET radiotracer is selected from the group consisting of: 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]methoxydibenzo[b,d]thiophene 5,5-dioxide; and 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide. A currently preferred PET radiotracer is 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6- [ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide. The PET radiotracers of the invention can be manufactured as disclosed in Examples 1 and 3. The PET radiotracers of the invention can be used to visualize localization and/or distribution of α7-nAChRs in vitro in a target tissue or in vivo in a subject by means or using PET imaging. The PET radiotracers of the invention have high affinity for α7-nAChR and, in addition, have high specificity for α7-nAChR. The PET radiotracers are thereby useful for visualizing, by PET imaging, nAChRs in a target tissue or in a subject’s body. The PET radiotracers can thereby be used to detect the locations and distributions of nAChRs in the target tissue or the subject’s body, and optionally quantify nAChRs in the target tissue of the subject’s body. The PET radiotracers can also be used in vitro in binding studies of isolated nAChRs, cell membrane preparations or homogenates comprising nAChRs and/or tissue preparations or homogenates comprising nAChRs. In such in vitro binding studies, the binding of drugs, drug candidates or other compounds to nAChRs could be detected or even quantified in competition binding assays using PET radiotracers and measuring radiation using, for instance, a scintillation counter. Hence, the invention relates to an in vitro competition binding assay method. The method comprises contacting, in vitro, a sample comprising α7-nAChRs with a PET radiotracer according to the invention and a test agent. The method also comprises filtering the sample through a filter and measuring radiation on the filter. The binding of the test agent to α7-nAChRs can then be determined based on the measured radiation. The sample could be any sample comprising α7-nAChRs, including isolated and purified α7-nAChRs, a cell preparation or homogenate comprising α7-nAChRs and a tissue preparation or homogenate comprising α7- nAChRs. For instance, a defined concentration of the PET radiotracer and a defined concentration of the test agent are added to the sample. In another embodiment, a defined concentration of the PET radiotracer is added to different aliquots of the sample together with different concentrations of the test agent, or a defined concentration of the test agent is added to different aliquots of the sample together with different concentrations of the PET radiotracer. In either case, following a period of time for incubation, such as from 15 min up to 6 hours, preferably from 30 min up to 4 hours, or about 1 to 2 hours, preferably in room temperature (20-25 °C), the sample or sample aliquots is or are filtered through a filter or filters, such as a glass fiber filter or glass fiber filters, optionally presoaked with polyethylenimine (PEI) to minimize binding to the filter by neutralizing negative charges of the glass fiber filter. In an optional embodiment, the filter or filters is or are rinsed and filtered once or multiple times with a binding buffer (such as 50 mM Tris HCl 120 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , at pH 7.4). The radiation on the filter or filters can then be measured, for instance, with a scintillation counter. The measured radiation on the filter or filters can then be used to determine or at least estimate the binding of the test agent to α7-nAChRs. This method is therefore useful in drug discovery applications when investigating whether a test agent, such as a drug or drug candidate, is capable of binding to α7-nAChR and preferably also to quantify this binding. The invention also relates to a method of diagnosing a neurodegenerative or psychiatric disease, or monitoring progression of the neurodegenerative or psychiatric disease. The method comprises administering a PET radiotracer according to the invention to a subject and taking PET images of the subject to detect location and/or distribution of α7-nAChRs in the subject. In an embodiment, the neurodegenerative or psychiatric disease is selected from the group consisting of Parkinson’s disease, Alzheimer’s disease and schizophrenia. Hence, in an embodiment, the disease is Alzheimer’s disease. In another embodiment, the disease is Parkinson’s disease. In a further embodiment, the disease is schizophrenia. In a particular embodiment, the neurodegenerative or psychiatric disease is selected from the group consisting of Parkinson’s disease and Alzheimer’s disease. The PET radiotracers according to the invention are preferably administered to the subject by injection, preferably intravenous injection or subcutaneous injection, preferably intravenous injection. The PET radiotracers are then preferably dissolved or dispersed in an injection solution, preferably an aqueous injection solution. Non-limiting, but illustrative, examples of such aqueous injection solutions include saline, and buffer solutions, such as phosphate-buffered saline (PBS). In a particular embodiment, the PET radiotracers are injected intravenously in PBS, such as in a volume of from about 5 to about 10 ml. In an embodiment, the solution comprises a P-glycoprotein inhibitor. An illustrative, but non-limiting, example of such a P-glycoprotein inhibitor is tariquidar (N-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2- yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-c arboxamide). Other examples of P- glycoprotein inhibitors that could be used include amiodarone, clarithromycin, ciclosporin, colchicine, diltiazem, erythromycin, felodipine, ketoconazole, lansoprazole, omeprazole and other proton-pump inhibitors, nifedipine, paroxetine, reserpine, saquinavir, sertraline, quinidine, tamoxifen, verapamil, duloxetine, elacridar, CP 100356 zosuquidar, valspodar and reversan. Co-administration or sequential administration of a PET radiotracer of the invention and a P-glycoprotein inhibitor increases the brain uptake of the PET radiotracer. The subject is preferably a human subject, or a non-human mammal, such as selected among mouse, rat, Guinea pig, rabbit, cat, dog, sheep, goat, cattle, horse and non-human primate. EXAMPLES Example 1 - In silico studies of ASEM analogues targeting α7-nAChR and experimental verification The α7 nicotinic acetylcholine receptor ( α7-nAChR) is implicated in a variety of neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease (AD) and schizophrenia. The progress of these disorders can be studied using positron emission tomography (PET) with radiotracers for α7-nAChR. [ ¹⁸ F]ASEM and [ ¹⁸ F] para-ASEM (also referred to as [ ¹⁸ F]DBT-10) are novel and potent α7-nAChR PET radiotracers, which have successfully been used in human subjects and non-human primates, though further improvement of them is still a pressing task in the community of neurodegeneration research. In this Example, we demonstrate the use of modern in silico techniques to predict the binding modes, binding strengths, and residence times for molecular PET tracers binding to proteins, using ASEM and DBT-10 as a showcase of the predictive and interpretational power of such techniques, in particular free energy perturbation theory. The corresponding compounds were synthesized and further tested by in vitro binding experiment for validation. Encouragingly, our in silico modeling can correctly predict the binding affinities of the ASEM analogues. The structure–activity relationships for the ortho- and para-substitutions are well explained at the atomistic level and provide structure-based guiding for the future development of PET tracers for α7-nAChR. Results Binding mode of ASEM with α7-AChBP Like the crystallized ligand epibatidine (see Fig.1), ASEM has a diazobicyclic head group and is protonated under physiological conditions. However, the diazobicyclic head group of ASEM is bulkier than the counterpart of epibatidine, which should have an impact on its binding with the receptor. Besides, the dibenzothiophene ring of ASEM is also much bigger than the pyridine ring of epibatidine. These two structural differences make epibatidine and ASEM have different potency profiles, namely, epibatidine is an agonist whereas ASEM is an antagonist. This is consistent with the general knowledge that α7-nAChR antagonists, such as methyllycaconitine (MLA) and abungarotoxin, tend to be much bulkier than the agonists, such as nicotine and acetylcholine. In a standard docking procedure where the receptor was held rigid, ASEM could not be docked properly, with a less favorable docking score (4.53 kcal mol ^-1 ), which is most probably due to the small size of the binding pocket occupied by epibatidine (docking score: 8.84 kcal mol ^-1 ). However, with the induced fit docking (IFD) procedure, ASEM could be docked to the binding site with a much more favorable docking score (10.8 kcal mol ^-1 ). This is reasonable because ASEM is bulkier and would need more space for binding. The residues relaxed most significantly are Trp53, Tyr91, Trp145, Tyr184, Cys186, Cys187, and Tyr191. The tip nitrogen (N1, pKa ~9.6) of ASEM is protonated under physiological conditions and has cation– π interactions with the aromatic rings of Tyr91, Trp145, Tyr184, and Try191. These cation– π interactions are believed to be important for the affinity of α7-nAChR ligands. The protonated nitrogen also forms a hydrogen bond with the backbone oxygen of Trp145 (Fig.1c). Besides, the diazobicyclic group has extensive van der Waals interactions with the side chains of Tyr91, Trp145, Tyr184, and Try191. Glide docking score decomposition of residues around the binding site shows that van der Waals interactions from Tyr91, Trp145, and Tyr191 has a major contribution to the docking score, which helps to stabilize ASEM in the binding site. The most significant difference between the binding modes of epibatidine and ASEM was seen in the aromatic tail part (Figs.1b and 1c). For epibatidine, the chloro-pyridine ring lies in the cavity formed by Leu106, Gln114, and Leu116 and has van der Waals or hydrophobic interactions with these residues. Besides, the chlorine atom is thought to have halogen-bond interaction with the backbone oxygen atom of Gln114, which also supports the binding of epibatidine. However, for ASEM, the dibenzothiophene ring is too big to fit into the site originally occupied by the pyridine ring of epibatidine. As a result, it adopts a different orientation and lies in the cavity on the other side which is formed by Ser34, Leu36, Trp53, Asp160, Gly163, Tyr184, Glu185, Cys186, and Cys187 (Fig.1c). The dibenzothiophene ring is clenched by van der Waals interactions with Glu185, Cys186, and Cys187 from loop C (residues 180–193) on one side and π-π stacking interaction with Trp53 from the complementary subunit on the other side (Fig.1c). Ser34, Leu116, and Asp160 also have some contact with the dibenzothiophene ring. The fluorine and oxygen atoms of ASEM point towards the solvent and do not have much interaction with surrounding residues. With induced-fit docking, we managed to produce a reasonable docking mode of ASEM with α7-AChBP, which will be used as the starting point for subsequent analysis. Comparison of the binding modes of ASEM and DBT-10 We compared the difference in the binding mode between ASEM and DBT-10 (Fig.2). The binding mode of ASEM suggests that the fluorine atom of ASEM points towards the solvent and does not have much interaction with the surrounding residues, while the fluorine atom of DBT-10 is predicted to point toward the inside of the pocket. The fluorine atom of DBT-10 occupies the hydrophilic region near Ser32 and Ser34. The formation of extra interactions between the fluorine atom of DBT-10 and protein residues increases the binding affinity. Free energy perturbation calculations by FEP+ shows that DBT-10 has a lower relative binding free energy than ASEM (ΔΔG = -0.24 ± 0.02 kcal mol ^-1 ), indicating that DBT-10 has a slightly higher binding affinity than ASEM. The higher binding affinity of DBT10 is in agreement with the in vitro experiment using the human α7-nAChR and is in contradiction to the result from rat cortical membranes. This may be due to that the chimera structure of AChBP we use in the FEP calculations has a higher identity to the human α7-nAChR. Ortho- and para-substitutions of ASEM and binding affinities prediction The binding modes of ASEM and DBT-10 indicate that the ortho-substitution points towards the solvent (R ₁ in Fig.3), while the para-substitution points towards the hydrophilic region between Ser32 and Ser34 (R ₂ in Fig.3). To explore the effect of ortho- and para-substitutions on binding, we designed a series of ASEM analogues (Fig. 3, right). Chemical groups with different sizes and properties, such as N-methyl, N,N- dimethyl, N-methyl-N-propyl, acetyl, propionyl, and phenylpropionyl, were designed to substitute the ortho- fluorine on ASEM and para-fluorine on DBT-10. Free energy perturbation calculations were carried out for the designed ASEM analogues. The relative binding free energies of the analogues relative to ASEM are shown in Table 1. Compared with ASEM, the substitutions on R ₁ position all have a negative ΔΔG, which means that the modifications at this position can remain or improve the activity of the compounds. The substitutions on R ₂ position result in a positive ΔΔG relative to ASEM, which means that these compounds could have lower binding affinities than ASEM. For the R ₂ substitution, even a small substituent such as N-methyl can cause a positive ΔΔG. The ΔΔG is higher when substitutions are large groups such as propionyl and phenylpropionyl. Table 1 - Relative free energies and physiochemical properties of ASEM analogues. ^a The relative free energy is calculated with ASEM as the reference. The standard errors are in the range of 0.1 to 0.5 kcal mol ^-1 . ^b Alog P is calculated using Schrodinger. ^c BBB and P-gp were predicted by our in-house machine learning tools based on cheminformatics using Python, sklearn and rdkit. ^d The residence time was calculated with potential scaled MD simulations. The standard errors are in the range of 1 to 5 ns. In addition to changing the binding ability of the compound, using substituents to adjust the physical and chemical properties of the compound is also an important part on the development of PET radioligands for α7-nAChR. The physiochemical properties were calculated and are shown in Table 1. All designed compounds can pass the blood–brain barrier and are P-gp substrate in the prediction. The log P increases when the substituents are lipophilic groups. The same substitution at R ₁ or R ₂ has little effect on the log P, plasma protein binding, blood–brain barrier permeability, or whether it is a P-gp substrate. In vitro validation and explanations for the activity from a structure of view The ASEM analogues with substitutions at the R ₁ - and R ₂ -positions were synthesized and tested by in vitro experiment using recombinant human α7-nAChR expressed in SH-SY5Y cells with [ ¹²⁵ I]- α-bungarotoxin (see Methods). The inhibition at a test concentration of 10 mM is shown in Fig.4. Substitutions at R ₁ position can retain the binding affinity with inhibition above 90%. At R ₁ , even the bulky substituents such as propionyl and phenylpropionyl can remain the inhibition as high as 90%. At the R ₁ position, only the pyridine ring substituent has inhibition of 55%. Substitutions at R ₂ position almost abolished the activity of the analogues. The inhibition of substituents at R ₂ position is less than 20%. The small groups such as N-methyl and N,N- dimethyl are slightly better with the inhibition of 15% and 12%, respectively. The experimental results are in good agreement with the previous ΔΔG predictions, which demonstrates that the calculated binding affinities for the R ₁ -analogues are more favorable than those for the R ₂ -analogues. Note that a completely linear relationship is not expected since there is an underlying dose concentration dependence. As shown in Fig. 5, with a cutoff of 50% inhibition, the true positive rate (TPR or sensitivity) and false-positive rate (FPR or fall- out) are 100% and 0%, respectively. The binding affinity differences of the R ₁ -and R ₂ -analogues can be explained by their binding modes in the protein. As shown in Fig.6a, the R ₁ -position is close to loop C of the binding pocket. Loop C is flexible and showed an open-closed mechanism. For substitutions at R ₁ position, loop C can open up and offer extra spaces for the chemical groups. Therefore, R ₁ -analogues can maintain the binding affinity even when the substituents are as large as propionyl and phenylpropionyl. In contrast, the R ₂ -position is deeply buried and has close interactions with nearby residues Ser32 and Ser34. Substituents at R ₂ position can lead to steric conflict between the protein and ligand. As shown in Fig.6b, the N-methyl substituent at the R ₂ position leads a steric conflict to Ser32 and Ser34. When the substituent is large, such as phenylpropionyl group, there is no more space for such a group, and the dibenzothiophene ring flips to let the phenylpropionyl group point towards the solvent via the egress portal between Glu185 and Asp160. The π-π stacking interaction between the dibenzothiophene ring and Trp53 is therefore lost. The steric conflict also changes the position of the diazobicyclic ring. The hydrogen bond between the protonated nitrogen of the diazobicyclic ring and the backbone oxygen of Trp145 is weakened. The cation–π interactions between the diazobicyclic ring and the aromatic rings of Tyr91, Trp145, Tyr184, and Try191 are also weakened because of the conformational changes. As a result, R ₂ -substitutions are generally less favorable than the R ₁ -substitutions. R ₁ -position substitutions are thus quite tolerant of bulky chemical groups. Prediction of the unbinding kinetics The measurements of kinetic parameters for ligands cannot disclose the way the tracer leaves the protein receptor nor give the information on the molecular determinants that dictate the dissociation process. That leaves out the key interactions and conformational changes essential for structural optimization of tracers concerning the kinetic properties. Luckily, enhanced sampling techniques have emerged recently as effective tools for studying unbinding kinetics of protein–ligand systems at the atomistic level. However, for practical computational studies, it is necessary to consider the fact that the ligand unbinding processes are strongly coupled to protein conformational changes and that there may be hidden degrees of freedom to disclose. This poses still a great challenge for sampling. In [1] it was shown how potential scaled molecular dynamics (sMD) and infrequent metadynamics (InMetaD) simulation techniques could be combined to successfully reveal the unbinding mechanism of ASEM from the chimera structure of the a7-AChBP receptor. It was possible to utilize these simulation techniques to pinpoint the important role of certain structural units in the unbinding process, and in particular, to identify that the motion of “loop C” (see Fig.7) could be critical for the ASEM unbinding process. Here one could follow the progression of opening and closing of this loop as the most relevant slow degree of freedom for the unbinding. One could furthermore see that there is more than one metastable state involved in the unbinding process and that the rate-limiting steps, and associated transition state structures, are associated with the interactions between the residues of the binding pocket and the sulfone group of the ASEM tracer. With the applied simulation techniques, it is possible to sample the slow conformational rearrangement of a fibrillar-tracer system occurring at the timescale beyond seconds, which thus makes it possible to consider these slow conformational changes, which are critical to the ligand unbinding process. Thus, with modern molecular dynamics techniques, the detailed mechanisms of the unbinding process can be revealed which paves the way for studying the unbinding kinetics of protein–ligand systems in general and for the optimization of new tracers towards certain receptors with maximum properties. In addition to the binding affinities and binding modes, the unbinding kinetics of the tracer-receptor systems is of great importance for the design of tracers with the desired specificity. To explore the effect of R ₁ -and R ₂ -substitutions on the kinetics property of the tracers, we recall here sMD simulations of the residence time(s) of the ASEM analogues as described in method section. The results are recapitulated in Table 1. We would like to point out that the estimated residence times are not real residence times. However, they can be used to rank the experimental residence times. In sMD simulations, the reference residence time of ASEM is predicted to be 53.4 ns, while compound 1 (KIn75) with N,N-dimethyl substituent ( τ = 75.6 ns) and compound 4 (KIn87) with N-methyl-N-propyl at the R ₁ -position ( τ = 87.3 ns) have longer residence times although they have comparable binding affinity than ASEM. Compound 6 (KIn86) and 8 (KIn60) with N- fluoroethyl-N-methyl ( τ = 51.2 ns) and acetyl substituent ( τ = 58.3 ns) at R ₁ -position share comparable residence time to ASEM. Substitutions at the R ₂ -position are found to decrease the binding affinity and residence time, such as N-methyl (compound 2 (KIn75), τ = 30.0 ns) and phenylpropionyl (compound 10 (KIn83), τ = 14.4 ns). Large groups at R ₁ -position, such as phenylpropionyl (compound 11, τ = 14.2 ns) and pyridylpropionyl (compound 13 (KInXX), τ = 40.8 ns) have shorter residence time than ASEM. Considering that compound 11 (KIn89) has a comparable binding affinity to that of ASEM, and that a compound with a shorter residence time is desired in the structural modification of ASEM analogues, compound 11 (KIn89) may be a better choice as PET tracer for α7-nAChR. Discussion Owing to the importance of developing potent PET radioligands, which can be used to study the roles of the α7 nicotinic acetylcholine receptor ( α7-nAChR), to facilitate drug discovery and to monitor the progress of diseases related to α7-nAChR, molecular modeling methods have been used in this Example to investigate the binding profile of [ ¹⁸ F]ASEM and α7-AChBP (a structural homologue of the extracellular domain of α7- nAChR). We studied the binding features of [ ¹⁸ F]ASEM at the orthosteric site of α7-AChR. Several structural details of this binding are found to be important. The diazabicyclo[3.2.2]nonane ring has cation–π and extensive van der Waals interactions with Tyr91, Trp145, Tyr184, and Try191, which fixes [ ¹⁸ F]ASEM tightly in the binding site. The dibenzothiophene ring turns to the other side of the pyridine ring of epibatidine (the crystallized agonist) and has van der Waals interactions with residues from loop C on one side and π-π stacking interaction with Trp53 of the complementary subunit on the other side. A series of ASEM analogues were calculated by FEP+ in silico and tested in vitro. A second purpose of the present Example was to demonstrate the general power of modern in silico approaches based on rational principles to predict the binding mode and binding energies of PET tracers to various protein structures, using Free Energy Perturbation Theory (FEP+) as the basic theoretical approach. Indeed, the consistency between in silico and, a posteriori, in vitro results indicates that FEP+ can accurately predict the binding free energy difference of ASEM analogues. This Example, thus, indicates that the FEP+ utility as implemented in the Schrödinger suite of programs can greatly facilitate the development of α7- nAChR PET tracers using rational drug design strategies. In addition to the focus on binding modes and energies, we also reviewed some results on the kinetics of the [ ¹⁸ F]ASEM unbinding from a chimera structure α7-nAChR as this is just as an important factor for the evaluation of tracer performance. This information is required to understand how long time it stays there (the residence time). We furthermore note that the techniques for binding modes and kinetics also can be used not only to explore the competitive binding of tracers for a given protein target and the competitive binding sites for a given tracer – protein pair but also to distinguish binding for a given tracer and different protein targets, which is a crucial aspect to design a tracer with sufficient selectivity on top of its efficiency. As a final note, we emphasize that the validation between in silico and results from measurements have been conducted using in vitro binding assays. Going to in vivo a whole new situation appears for the modeling, as now factors like blood–brain-barrier (BBB) penetration, membrane protein binding (PgP) and lipophilicity, enter into the evaluation of the potency of tracers. Methods Molecular docking In a standard molecular docking study, the receptor is held rigid, and the ligand can change its position and conformation freely. However, this procedure is problematic when the ligand to be docked is rather different from the crystallized one in shape or size. In reality, the receptor structure will undergo sidechain or backbone movements upon ligand binding to conform to the shape of the ligand, a process known as induced fit. The induced-fit docking (IFD) workflow of Schrödinger implements this idea through a combination of Glide and Prime jobs, which account for the conformational changes of the ligand and receptor, respectively. In this Example, the crystal structure of the α7-AChBP chimera (PDB code 3SQ6) [2] was used as the protein target. Before docking, the crystal structure was prepared with the protein preparation workflow of Schrödinger, where the hydrogen atoms were added and optimized, and the bond order was fixed. For the glide docking procedure, the centroid of the crystalized ligand epibatidine was chosen as the grid center and the residues within 20 Å of it are treated as binding pocket. A van der Waals scaling factor of 0.5 was used for both receptor and ligand. In the induce fit docking process, protein residues within 5 Å of the ligand were optimized by prime. The glide standard precision (SP) scoring function was adopted to rank the optimized docking poses. The α7-AChBP/ASEM complex with the most favorable binding energy was chosen for subsequent analysis. The radionuclide fluorine-18 of ASEM, which is used in PET studies, is not indicated hereafter unless otherwise specified, as radiation is supposed not to affect the binding with α7-AChBP. Free energy calculation using FEP+ The FEP+ utility of Schrödinger was used to calculate the free energy differences of ASEM analogues [3]. The OPLS3 force field was used to describe the protein and ligands [4]. Ligand atomic partial charges are computed via the CM1A-BCC [5]. The REST (replica exchange with solute tempering) algorithm [6] has been incorporated using Desmond as the MD engine.2 Tesla K80 GPUs are used for FEP calculations. LOMAP mapping algorithm [7] was used to set up the calculations and perturbation pathways. The maximum common substructure (MCS) between any pair of compounds is generated and their similarity is measured. Then ligand pairs with high similarity scores are connected by edges, where each edge represents one FEP calculation that will be performed between the two ligands. The systems of α7-AChBP with ASEM analogues were first relaxed and equilibrated using the default Desmond relaxation protocol. The whole system with the solute molecules restrained to their initial positions was first minimized using the Brownie integrator and then simulated at 10 K using an NVT ensemble followed by an NPT ensemble. After that the system was simulated at room temperature using the NPT ensemble with the restraints retained. Then the whole system without any restraint was simulated at room temperature using the NPT ensemble for 240 ps followed by the production simulation. A total of 12 λ windows were used for all the FEP/REST calculations. The production stage lasted 5 ns for both the complex and the solvent simulations using NPT ensemble conditions. Replica exchanges between neighboring l windows were attempted every 1.2 ps. The Bennett acceptance ratio method (BAR) was used to calculate the free energy [8]. Errors were estimated for each free energy calculation using both bootstrapping and the BAR analytical error prediction. Potential scaled MD simulations The sMD simulations were carried out in line with the previous study [1]. In brief, we employed ff99SB-ildn and GAFF force field for the protein and the ligands, respectively. The restrained electrostatic potential- derived charges were used for ASEM with the electrostatic potential calculated at the Hartree–Fock level with the 6-31G* basis set using Gaussian 09.28 The TIP3P [9] water model was used to solvate the complex, and 140 Na ⁺ and 138 Cl- ions were used to neutralize the system. The systems were equilibrated in the NVT ensemble (T = 300 K) for 200 ps, followed by a 500 ps simulation carried out in the NPT ensemble (T = 300 K, P = 1 atm). The GROMACS [10] program was employed for the MD simulations. In the sMD simulations, the force field was scaled by a factor of 0.4. Heavy atoms of the protein backbone were restrained with a weak harmonic potential (with the force constant k = 50 kJ mol ^-1 nm ^-2 ) except for residues within 6 Å of ASEM and its analogues. The unrestrained residues are Ser32–Ser34, Leu36, Phe52–Gln55, Ala89, Tyr91, Thr101–Pro102, Leu106, Leu116–S118, Gln143–His148, Glu158–Asp160, Ser162–Gly163, Arg182– Asp193, and Phe196. For each system, twenty simulation runs were performed with the scaled force field. Each simulation was stopped once the ligand was fully unbound from the pocket. The residence times thus obtained as the characteristic parameter of the unbinding times of each simulation run. The mass of the ¹⁸ F isotope of [ ¹⁸ F]ASEM is not considered in the simulations. In vitro binding assay The 14 compounds calculated and tested in this work were commercially synthesized by Piramal Pharma solution, Ahmedabad India. Purity was checked by HPLC and ¹ H NMR. All compounds had a purity of 95%. The compounds were diluted in DMSO to a concentration of 10 mM. Proportions of 50 ml aliquots of the 10 mM solution were dispensed into plastic vials which were kept frozen at -20 °C until sent to Cerep, Eurofins, France for measurement of α7 nicotinic receptor activity. The molecular weight of each compound was calculated and provided to Cerep. Before analysis each tube with compound was thawed and a solution of 100 nM was prepared for each compound. The test system consisted of an in vitro binding study was performed using human neuronal α7 transfected neuroblastoma cells SH-SY5Y cells (human recombinant) incubated with 0.05 nM ¹²⁵ I-α-bungarotoxin and 100 nM test compound at 37 °C for 120 minutes. The value of bound radioactivity was calculated with a scintillation counter. The inhibition was calculated as the percentage of displacement of ¹²⁵ I-α-bungarotoxin by each compound. Epibatidine (IC50 = 94 nM, Ki = 82 nM) was used as a reference in this Example. Example 2 – Synthesis of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methoxydibenzo[b,d]t hiophene 5,5- dioxide (KIn83) Materials and Methods The purity of compounds was determined by HPLC and all final target compounds had purities of >95% unless otherwise stated. Proton nuclear magnetic resonance ( ¹ H NMR) spectra were recorded in the deuterated solvents specified on a Varian 400 spectrometer operating at 400 MHz Mass spectra were determined by using Shimadzu LCMS 2020 with N-Series DUIS (ESI) system using positive-negative switching. HPLC spectra were determined by using Agilent 1200 series. The synthesis of KIn83 is outlined in Fig.24. Preparation of (5-bromo-2-nitrophenyl)(2-methoxyphenyl)sulfane (3) Cesium carbonate (17.8 g, 54.5 mmol) was added to a solution of 4-bromo- 2-fluoronitrobenzene (2) (10 g, 45.4 mmol) and 2-methoxybenzene thiol (1) (6.37 g, 45.4 mmol) in DMF (240 mL), and the mixture was stirred for 16 h at room temperature. The reaction mixture was poured into the ice-cold water (1000 mL), the solid was precipitated out and was filtered through Buchner funnel washed with water and dried in vacuum to yield a yellow solid without further purification taken for next step (3) (14 g, 91%). Preparation of 4-bromo-2-((2-methoxyphenyl)thio)aniline (4) A mixture of (5-bromo-2-nitrophenyl)(2-methoxyphenyl)sulfane (3) (14 g, 41.1 mmol), iron powder (9.2 g ,164 mmol), ammonium chloride (2.64 g, 49.32 mmol) in methanol (200 mL), THF (200 mL), and water (65 mL) was heated to reflux (80 °C) for 48 h. The resulting mixture was filtered through celite and washed with methanol, the solvent was concentrated, and dried under vacuum to give the corresponding aniline derivative, The crude was taken for next step (11 g, 86%) without further purification. Preparation of 3-bromo-6-methoxydibenzo[b,d]thiophene (5) 4-bromo-2-((2-methoxyphenyl)thio)aniline (4) (11 g, 35.4 mmol) was dissolved in 37% HCl (110 mL), and the solution was cooled below 5 °C. To this reaction mixture, sodium nitrite (3.67 g, 53.2mmol) was added slowly at a temperature below 5 °C. After addition, the mixture was stirred for 30 min below 5 °C. Then sodium tetrafluoroborate (7.8 g, 70.9 mmol) was added, and the reaction mixture was stirred for another 30 min at a temperature below 5 °C. This reaction solution was then added to the stirred solution of copper(I) oxide (10.1 g, 70.9 mmol) in 0.1 N sulfuric acid (3.5 L) at 35−40 °C. The reaction mixture was stirred for 15−30 min. Ethyl acetate was added to the reaction mixture, and the mixture wasfiltered to remove inorganic compound. The filtrate was then extracted with ethyl acetate (3 x 1000 mL). The organic extract was washed with water followed by brine and then concentrated under vacuum. The residue was purified by silica gel chromatography (hexanes) to give 5 (4.8 g, 47%). Preparation of 3-bromo-6-methoxydibenzo[b,d]thiophene 5,5-dioxide (6) 3-bromo-6-methoxydibenzo[b,d]thiophene (5) (4.8 g,16.3 mmol) was dissolved in glacial acetic acid (50 mL) at room temperature. Aqueous hydrogen peroxide (30%, 25 mL) was added in small portions to the stirred solution. The addition of H ₂ O ₂ resulted initially in some precipitation. The mixture was stirred at 60 °C for 24 h, then cooled to room temperature. The solid wasfiltered off, sequentially washed with 70% aqueous acetic acid, then 30% aqueous acetic acid, then water, and dried to afford the title compound as white solid 6 (2.8g, 45%). Preparation of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methoxydibenzo[b,d]t hiophene 5,5-dioxide (Kln83) A catalyst solution was prepared by mixing tris(dibenzylideneacetone)dipalladium (Pd ₂ (dba) ₃ , 0.27 g, 0.29 mmol) and racemic BINAP (0.36 g, 0.59 mmol) in toluene (30 mL) and heating the mixture to 90 °C for 15 min. The solution was cooled and then added to a mixture of 1,4-diazabicyclo [3.2.2]nonane (0.93 g, 7.3 mmol) and 3-bromo-6-methoxydibenzo[b,d]thiophene 5,5-dioxide (6) (2.4, 7.3 mmol), in toluene (60 mL). Cs ₂ CO ₃ (3.63 g, 11.0 mmol) was added, and the reaction mixture wasflushed with nitrogen and heated overnight at 80−85 °C. After cooling to room temperature, the mixture was concentrated and purified by silica gelflash chromatography (CH ₂ Cl ₂ /MeOH/Et ₃ N 10:1:0.2). The title compound Kln83 (1.3 g, 48% yield) was obtained as a pale-yellow solid. Example 3 - Development of novel ¹¹ C-labeled ASEM analogs for detection of α7-nAChR The homo-pentameric alpha 7 receptor is one of the major types of neuronal nicotinic acetylcholine receptor ( α7-nAChR) related to cognition, memory formation, and attention processing. The mapping of α7-nAChR by PET draws a lot of attention to understand mechanism and the progress of CNS diseases such as AD, PD, schizophrenia. Several PET ligands have been explored for imaging of the α7-nAChR, but [ ¹⁸ F]ASEM is so far the most interesting for in vivo quantification of α7-nAChR in human. The aims of this Example were to label derivatives of ASEM with ¹¹ C ^/3 H and to evaluate binding characteristics in vitro as well as in vivo. Six analogs of ASEM (KIn74, KIn75, KIn77, KIn83, KIn84 and KIn85) were labeled with ¹¹ C and KIn83, KIn84 and KIn74 were additionally labeled with ³ H. Binding properties were evaluated using autoradiography (ARG) and PET measurements in non-human primates (NHPs). Radiometabolites were measured in NHPs plasma using gradient radio HPLC. All six ASEM analogs were successfully radiolabeled with high purity >99%. Evaluation with ARG showed that [ ¹¹ C]KIn83 binds to α7-nAChR. Competition studies showed that 80% of the total binding was displaced by adding 10 µM of unlabeled KIn83 and ASEM. Further, ARG was performed with [ ³ H]KIn83, replicating the results. In vivo [ ¹¹ C]KIn83 brain uptake was 1.6 SUV at peak. Regional distribution of [ ¹¹ C]KIn83 was similar to [ ¹⁸ F]ASEM, with relatively high uptake in thalamus, cortex and basal ganglia. Low uptake was observed in cerebellum and white matter. Evaluation of KIn83 by ARG with both ¹¹ C and ³ H as well as in vivo evaluation in NHP showed favorable properties for imaging α7-nAChR. Materials and Methods Radiochemistry All the precursors (PRE-1, PRE-2, PRE-3 and PRE-4, see Example 4) and all the non-radioactive reference standards (KIn74, KIn75, KIn77, KIn83, KIn84 and KIn85) were synthesized by Syngene International, India. All other chemicals and reagents were obtained from commercial sources and used without any further purification. Solid-phase extraction (SPE) cartridges SepPak C18 Plus were purchased from Waters (Milford, Mass U.S.A). C-18 Plus cartridge was activated using EtOH (10 mL) and followed by sterile water (10 mL). Liquid chromatographic analysis (LC) was performed with a Merck-Hitachi gradient pump and a Merck- Hitachi, L-4000 variable wavelength UV-detector. [ ³ H]Methyl Iodide ([ ³ H]CH ₃ I) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Synthesis of ¹¹ C-methyl iodide ([ ¹¹ C]CH ₃ I) ¹¹ C-Methane ([ ¹¹ C]CH ₄ ) was produced in-target via the ¹⁴ N(p, α) ¹¹ C reaction on nitrogen mixed with 10% of hydrogen, with 16.4 MeV protons using a GEMS PET trace cyclotron (GE, Uppsala, Sweden). Typically the target gas was irradiated for 15–20 min with a beam current of 35 µA. ¹¹ C-Labeled methyl iodide, [ ¹¹ C]CH ₃ I, was produced following the previously published method [11]. In short, the produced [ ¹¹ C]CH ₄ was released from the target and collected in a Porapak Q trap cooled in liquid nitrogen. After collection, the [ ¹¹ C]CH ₄ was released from the trap by heating with pressurized air and subsequently [ ¹¹ C]CH ₄ was mixed with vapors from of iodine crystals at 60°C followed by a radical reaction at 720°C in a closed circulation system. The formed [ ¹¹ C]CH ₃ I was collected in a porapak Q trap at room temperature and the unreacted [ ¹¹ C]CH ₄ was recirculated for 3 min. [ ¹¹ C]CH ₃ I was released from the Porapak Q trap by heating the trap using a custom- made oven at 180°C with the flow of helium. Radiosynthesis of [ ¹¹ C]KIn74 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide) Carbon-11 labelled KIn74 ([ ¹¹ C]KIn74) was obtained by trapping [ ¹¹ C]CH ₃ I at room temperature in a reaction vessel containing the mixture of precursor PRE-4 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-hydroxyl- dibenzo[b,d]thiophene 5,5-dioxide) (1.0 mg, 2.8 µmol) and cesium carbonate (5,0 mg) in dimethylformamide (DMF) (500 µL) (Fig.9). After end of trapping, the reaction mixture was heated at 80°C for 4 minutes. The reaction mixture was diluted with sterile water (500 µL) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound. The HPLC system consisted of a semi-preparative reverse phase (RP) ACE column ( C18, 10 × 250 mm, 5 µm particle size) and a Merck Hitachi UV detector ( λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection. The product was eluted with mobile phase of 40% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 5 mL/min which gave a radioactive fraction corresponding to pure [ ¹¹ C]KIn74 with a retention time (tR) 13-14 min. Radiosynthesis of [ ¹¹ C]KIn75 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]methylaminodibenzo[b,d] thiophene 5,5-dioxide) Carbon-11 labelled KIn75 ([ ¹¹ C]KIn75) was obtained by trapping [ ¹¹ C]CH ₃ I at room temperature in a reaction vessel containing the mixture of precursor PRE-2 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-amino- dibenzo[b,d]thiophene 5,5-dioxide) (0.5 mg, 1.4 µmol) and KOH (5,0 mg) in dimethylsulphoxide (DMSO) (500 µL) (Fig.9). After end of trapping, the reaction mixture was heated at 90°C for 5 minutes. The reaction mixture was diluted with sterile water (500 µL) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound. The HPLC system consisted of a semi-preparative reverse phase (RP) ACE column (C18, 10 × 250 mm, 5 µm particle size) and a Merck Hitachi UV detector ( λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection. The product was eluted with mobile phase of 30% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 6 mL/min which gave a radioactive fraction corresponding to pure [ ¹¹ C]KIn75 with a retention time (tR) 9-10 min. Radiosynthesis of [ ¹¹ C]KIn77 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ¹¹ C]N,N-dimethylaminodibenzo[b,d] thiophene 5,5-dioxide) Carbon-11 labelled KIn77 ([ ¹¹ C]KIn77) was obtained by trapping [ ¹¹ C]CH ₃ I at room temperature in a reaction vessel containing the mixture of precursor KIn75 (1.0 mg, 2.7 µmol) and NaOH (5,0 mg) in dimethylsulphoxide (DMSO) (500 µL) (Fig.9). After end of trapping, the reaction mixture was heated at 90°C for 5 minutes. The reaction mixture was diluted with sterile water (500 µL) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound. The HPLC system consisted of a semi-preparative reverse phase (RP) ACE column (C18, 10 × 250 mm, 5 µm particle size) and a Merck Hitachi UV detector ( λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection. The product was eluted with mobile phase of 35% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 5 mL/min which gave a radioactive fraction corresponding to pure [ ¹¹ C]KIn77 with a retention time (tR) 12-14 min. Radiosynthesis of [ ¹¹ C]KIn83 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methoxydibenzo[b,d]thiophene 5,5-dioxide) Carbon-11 labelled KIn83 ([ ¹¹ C-KIn83]) was obtained by trapping [ ¹¹ C]CH ₃ I at room temperature in a reaction vessel containing the mixture of precursor PRE-3 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-hydroxyl- dibenzo[b,d]thiophene 5,5-dioxide) (0.50 mg, 1.2 µmol) and cesium carbonate (5,0 mg) in dimethylformamide (DMF) (500 µL) (Fig.9). After end of trapping, the reaction mixture was heated at 80°C for 4 minutes. The reaction mixture was diluted with sterile water (500 µL) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound. The HPLC system consisted of a semi-preparative reverse phase (RP) ACE column (C18, 10 × 250 mm, 5 µm particle size) and a Merck Hitachi UV detector ( λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection. The product was eluted with mobile phase of 40% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 5 mL/min which gave a radioactive fraction corresponding to pure [ ¹¹ C]KIn83 with a retention time (tR) 13-14 min. Radiosynthesis of [ ¹¹ C]KIn84 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]methylaminodibenzo[b,d] thiophene 5,5-dioxide) Carbon-11 labelled ([ ¹¹ C]KIn84) was obtained by trapping [ ¹¹ C]CH ₃ I at room temperature in a reaction vessel containing the mixture of precursor PRE-1 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-amino- dibenzo[b,d]thiophene 5,5-dioxide) (1.5-2.0 mg, 4.2-5.6 µmol) and KOH (7.0-8.0 mg) in dimethylsulphoxide (DMSO) (500 µL) (Fig.9). After end of trapping, the reaction mixture was heated at 80°C for 3 minutes. The reaction mixture was diluted with sterile water (500 µL) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound. The HPLC system consisted of a semi-preparative reverse phase (RP) ACE column (C18, 10 × 250 mm, 5 µm particle size) and a Merck Hitachi UV detector ( λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection. The product was eluted with mobile phase of 40% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 5 mL/min which gave a radioactive fraction corresponding to pure [ ¹¹ C]KIn84 with a retention time (tR) 13-14 min. Radiosynthesis of [ ¹¹ C]KIn85 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ¹¹ C]N,N-dimethylaminodibenzo[b,d] thiophene 5,5-dioxide) Carbon-11 labelled KIn85 ([ ¹¹ C]KIn85) was obtained by trapping [ ¹¹ C]CH ₃ I at room temperature in a reaction vessel containing the mixture of precursor KIn84 (1.0 mg, 2.8 µmol) and KOH (5,0 mg) in dimethylsulphoxide (DMSO) (500 µL) (Fig.9). After end of trapping, the reaction mixture was heated at 90°C for 5 minutes. The reaction mixture was diluted with sterile water (500 µL) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound. The HPLC system consisted of a semi-preparative reverse phase (RP) ACE column (C18, 10 × 250 mm, 5 µm particle size) and a Merck Hitachi UV detector ( λ = 254 nm) (VWR, International, Stockholm, Sweden) in series with a GM-tube (Carroll-Ramsey, Berkley, CA, USA) used for radioactivity detection. The product was eluted with mobile phase of 40% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 6 mL/min which gave a radioactive fraction corresponding to pure ¹¹ C-KIn85 with a retention time (tR) 12-14 min. Radiosynthesis of [ ³ H]KIn74 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-[ ³ H]methoxydibenzo[b,d]thiophene 5,5- dioxide) and [ ³ H]KIn83 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]methoxydibenzo[b,d]thiophene 5,5- dioxide) The radiosynthesis was performed following the similar procedure described for ¹¹ C-labeling of KIn74 and KIn83. [ ³ H]CH ₃ I was added in the reaction vessel containing the corresponding precursors PRE-4 or PRE-3 (1.0 mg-2.0 mg, 2.7 µmol-5.4 µmol) potassium hydroxide powder (4-7 mg) in DMSO (300 µL) and the mixture was sonicated for 15 minutes. A solution of ³ H-methyl iodide in toluene (~1 mCi) was added and then heated at 90ºC for 30 minutes.300 µL of water was adjoined. Analysis and purification were performed by LaChrom HPLC on an ACE 5 C18 HL column (250 x100mm). The product was eluted with mobile phase of 40% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 5 mL/min monitored with UV (254 nm) and radioactivity detectors. After repeats of synthesis and combination of collected fractions, solvents in fraction were removed by solid phase extraction, the product was formulated in ethanol/water. The products [ ³ H]Kin74 and [ ³ H]Kin83 were analyzed and identified by HPLC. Retest of radiochemical purity was performed before it was used for ARG experiment. Radiosynthesis of [ ³ H]KIn84 (3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[ ³ H]N,N-dimethylaminodibenzo[b,d] thiophene 5,5-dioxide) The radiosynthesis was performed following the similar procedure described for ¹¹ C-labeling of KIn84. [ ³ H]CH ₃ I was added in the reaction vessel containing the corresponding precursors PRE-1 (1.0 mg-2.0 mg, 2.7 µmol-5.4 µmol) cesium carbonate (4-5 mg) in dry DMF (300 µL) and the mixture was vortexed for 5 minutes. A solution of ³ H-methyl iodide in toluene (~1 mCi) was added and then heated at 90ºC for 30 minutes.300 µL of water was adjoined. Analysis and purification were performed by LaChrom HPLC on an ACE 5 C18 HL column (250 x100mm). The product was eluted with mobile phase of 40% acetonitrile in ammonium formate (AF, 0.1 M) with a flow rate of 5 mL/min monitored with UV (254 nm) and radioactivity detectors. After repeats of synthesis and combination of collected fractions, solvents in fraction were removed by solid phase extraction, the product was formulated in ethanol/water. The product [ ³ H]KIn84 was analyzed and identified by HPLC. Retest of radiochemical purity was performed before it was used for ARG experiment. Isolation of ¹¹ C/ ³ H labelled KIn74, KIn75, KIn77, KIn83, KIn84 and KIn85 The corresponding radioactive fraction collected from HPLC was diluted with sterile water (50 mL). The resulting mixture was loaded on to a pre-conditioned (10 mL ethanol followed by 10 mL sterile water) SepPak tC18 plus cartridge. The cartridge was washed with sterile water (10 mL) and the corresponding isolated ¹¹ C/ ³ H-product was eluted with 1 mL of ethanol into a sterile vial containing phosphate buffered saline solution (PBS, 9 mL). Quality control and Molar activity (MA) determination The radiochemical purity, identity and stability of [ ¹¹ C]KIn74, [ ¹¹ C]KIn75, [ ¹¹ C]KIn77, [ ¹¹ C]KIn83, [ ¹¹ C]KIn84 and [ ¹¹ C]KIn85 were determined by analytical HPLC system which included a ACE RP column (C18, 3.9 Ø × 250 mm, 5 µm particle size), Merck-Hitatchi L-7100 Pump, L-7400 UV detector and GM-tube for radioactivity detection (VWR International). The mobile phase CH ₃ CN/0.1% TFA with a gradient HPLC method (15-90% in 10 min) and flow rate of 2 mL/min was used to elute the product. The effluent was monitored with an UV absorbance detector ( λ = 254 nm) coupled to a radioactive detector (b-flow, Beckman, Fullerton, CA). The identity of fluorine-18 labelled compounds was confirmed by using HPLC with the co- injection of the corresponding authentic non-radioactive reference standard. The MA of the final product was measured by analytical HPLC which included a ACE RP column (C18, 3.9 Ø × 250 mm, 5 µm particle size) using mobile phase CH ₃ CN/0.1 M ammonium formate with a gradient HPLC method (10-90% in 10 min) and flow rate of 2 mL/min. MA was calibrated for UV absorbance ( λ = 254 nm) response per mass of ligand and calculated as the radioactivity of the radioligand (GBq) divided by the amount of the associated carrier substance (µmol). Each sample was analyzed three times and compared to a reference standard also analyzed three times. In vitro autoradiography The temporal cortex from an AD patient and a matched normal healthy individuals were obtained from the Netherlands Brain Bank, Amsterdam, the Netherlands. Autopsies were performed on donors from whom written informed consent had been obtained either from the donor or direct next of kin and were handled in a manner similar to that described previously [12-14]. Cases were neuropathologically confirmed using conventional histopathological stains in fresh frozen tissue. Details of the control subject and AD patient are listed below. Both human and rat brains, fresh frozen postmortem tissue was sectioned on a cryomicrotome (Leica CM 1860 Leica, Nussloch, Germany), thaw mounted to poly-L-lysine-treated glass plates, dried at room temperature and stored at -20°C until use. The thickness differed from human tissue (20 µm) and rat tissue (10 µm). In vitro autoradiography (ARG) using ¹¹ C-labelled compounds For the preliminary screening of compounds, labelling with ¹¹ C was carried out in rat brain tissue using autoradiography for testing the binding to the target. Slides were thawed at room temperature and pre- incubated in phosphate-buffered saline (PBS) for 10 min following incubation with the labeled compound at 0.01 MBq/mL for 30 min at 37 °C. Non-specific binding was determined in the presence of excess of unlabeled reference compounds and/or other ASEM analogues at 10 µM. After incubation, the slides were washed twice for 3 × 3 min each in ice-cold PBS buffer followed by a brief wash in distilled water. The slides were then dried and exposed to phosphor imaging plates (Fujifilm Plate BAS-TR2025, Fujifilm, Tokyo, Japan) before scanning in a Fujifilm BAS-5000 phosphor imager (Fujifilm, Tokyo, Japan) at a resolution of 25 µm/pixel. For calibration, 20 µL aliquots of the incubation solution were dropped onto a filter paper and scanned together with the sections. The sections were analyzed by Multi Gauge 3.2 phosphor imager software (Fujifilm, Tokyo, Japan). The specific binding was defined as subtracting the non-specific binding from the total binding, expressed as percentage of total binding (100%). If the compound did not show specific binding to the brain regions of interest, it was discarded for further analysis. In vitro Autoradiography (ARG) using [ ³ H]KIn83 ARG experimental procedures using tritiated compounds were previously described elsewhere [15] and in brief carried out as follows; slides were thawed at room temperature and incubated with radioligand in binding buffer (50 mM Tris HCl) at the desired concentration (0.8 or 1 nM) for 1 hour. The binding was displaced on adjacent sections with the cold compound (unlabeled compound), other ASEM analogues and ASEM at 10 µM. After incubation, the slides were washed three times in washing buffer (50 mM Tris HCl 120 mM NaCl, 5mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , at pH 7.4) followed by a brief wash in distilled water. The slides were dried and exposed to new phosphor imaging plates (Fujifilm Plate BAS-TR2025, Fujifilm, Tokyo, Japan). Tritium micro scales standards (American Radiolabeled Chemicals Inc.) were placed in cassettes together with the sections for calibration and quantification of the binding density. For image analysis, the phosphor imaging plates were exposed for approximately ninety hours. Then, the films were scanned, and the resulting images were processed in a Fujifilm BAS-5000 phosphor imager (Fujifilm, Tokyo, Japan). Analysis was performed using Multi Gauge 3.2 phosphor imager software (Fujifilm, Tokyo, Japan). Manual delineation of each region of interest (ROI) was performed visually on each digital image using three- to fourfold magnification. Mean pixel values of the ROIs from each section were transformed into radioactivity values using the droplets of incubation solution used as calibrating standard and recalculated as binding density (fmol/mg protein). Based on these measurements, specific binding values were calculated in the absence or presence of the inhibitor (total binding – non-specific binding), expressed as percentage of total binding (100%) or fmol/mg. Study Design in non-human primates, PET experimental procedure and Quantification The study was approved by the Animal Ethics Committee of the Swedish Animal Welfare Agency (N185/14) and was performed according to “Guidelines for planning, conducting and documenting experimental research” (Dnr 4820/06-600) of Karolinska Institutet. The NHPs were housed in the Astrid Fagraeus Laboratory of the Swedish Institute for Infectious Disease Control, Solna, Sweden. Four cynomolgus monkeys (two females and two males, body weight 4500 – 8410 g) were used. For three NHPs, brain PET was performed at a baseline condition. For one of these NHPs, measurement after tariquidar (2.2 mg/kg) administration was also performed. For these three experiments, only venous blood sampling was done. Another NHP was measured before and after ASEM (1.24 mg/kg) administration. Arterial blood sampling was performed in this experiment for measurement of plasma input function. Anesthesia was induced by intramuscular injection of ketamine hydrochloride (10 mg/kg) at Astrid Fagraeus Laboratory and maintained by the administration of a mixture of sevoflurane, oxygen, and medical air through endotracheal intubation. The head was immobilized with a fixation device. Body temperature was maintained by a Bair Hugger model 505 warming unit (Arizant Healthcare, MN) and monitored by an esophageal thermometer. Heart rate, blood pressure, respiratory rate and oxygen saturation were continuously monitored throughout the experiments. Fluid balance was maintained by continuous infusion of saline. PET measurements were conducted using a High Resolution Research Tomograph (HRRT) (Siemens Molecular Imaging). A transmission scan of 6 min using a single ¹³⁷ Cs source was performed before the [ ¹¹ C]KIn83 injection. List mode data were acquired continuously for 123 min (three NHPs) or 93 min (NHP for ASEM administration) immediately after intravenous injection of the radioligand. Images were reconstructed by the ordinary Poisson-3D-ordered subset expectation maximization (OP-3D-OSEM) algorithm with 10 iterations and 16 subsets including modeling of the point spread function (PSF). The regions of interest (ROIs) were delineated manually on MRI images of each NHP for the whole brain, cerebellum, caudate, putamen, thalamus, frontal cortex, temporal cortex, and hippocampus. The summed PET images of the whole duration were co-registered to the MRI image of the individual NHP. After applying the co-registration parameters to the dynamic PET data, the time-activity curves of brain regions were generated for each PET measurement. Average standardized uptake value (SUV) was calculated for each brain regions. For the experiment of ASEM administration, the target occupancy was estimated by the Lassen occupancy plot using VT calculated by two tissue compartment (2TC) using metabolite corrected plasma radioactivity. Radiometabolite analysis Radiometabolite analysis was performed following a previously published method [16]. In short, a reverse- phase HPLC method was used for the determination of the percentages of radioactivity corresponding to unchanged radioligand [ ¹¹ C]KIn83 and its radioactive metabolites during the course of a PET measurement. Arterial blood samples (2 mL) were obtained from the monkey at different time point such as 4, 15, 30, 60 and 90 min after injection of [ ¹¹ C]KIn83. Collected blood (2 mL) was centrifuged at 2000 g for 2 min to obtain the plasma (0.5 mL). The plasma obtained after centrifugation of blood at 2000 g for 2 min was mixed with 1.4 times volume of acetonitrile. The mixture was then centrifuged at 2000 g for 4 min and the extract was separated from the pellet and was diluted with water before injecting into the HPLC system coupled to an on- line radioactivity detector. An Agilent binary pump (Agilent 1200 series) coupled to amanual injection valve (7725i, Rheodyne), 1-3.0 mL loop and a radiation detector (Oyokoken, S-2493Z) housed in a shield of 50 mm thick lead was used for metabolite measurements. Data collection and control of the LC system was performed using chromatographic software (ChemStation Rev. B.04.03; Agilent). The accumulation time of radiation detector was 10 sec. Chromatographic separation was achieved on an ACE C18 column, (250 mm × 10 mm I.D) by gradient elution. Acetonitrile (A) and 10 mM ammonium format (B) were used as the mobile phase at 5.0 mL/min, according to the following program: 0−8.5 min, (A/B) 50:50 → 95:5 v/v; 8.5−11.0 min, (A/B) 95:5 v/v. Peaks for radioactive compounds eluting from the column were integrated and their areas were expressed as a percentage of the sum of the areas of all detected radioactive compounds (decay- corrected to the time of injection on the HPLC). To calculate the recovery of radioactivity from the system, an aliquot (2 mL) of the eluate from the HPLC column was measured and divided with the amount of total injected radioanalyte. Results and Discussions Radiochemistry Target produced [ ¹¹ C]CH ₄ was utilized for production of [ ¹¹ C]CH ₃ I or ¹¹ C-CH ₃ OTf in all preparations of radioligands. The total radiosynthesis time including purification and formulation of all six radioligands were 30-32 minutes after end of bombardment (EOB). The one step radiosynthesis for all was highly reproducible and it was possible to produce 550 MBq – 1600 MBq of the pure product for specific radioligand following irradiation of the target with a beam current of 35 µA for 15-20 minutes. Molar activities of all six radioligands were > 165 GBq/µmol. The radiochemical purity was >99% at EOS and the identity of the radioligand was confirmed by co-injection of the radioligand with an authentic standard by radio-HPLC. The formulated solution of respective radioligand was found to be radiochemically stable for up to 1h. A rapid and effective one step radiosynthesis of six novel radioligands [ ¹¹ C]KIn74, [ ¹¹ C]KIn75, [ ¹¹ C]KIn77, [ ¹¹ C]KIn83, [ ¹¹ C]KIn84 and [ ¹¹ C]KIn85 was developed with high yield purity and MA. Selective N- methylation/O-methylation of the corresponding precursor was achieved using [ ¹¹ C]CH ₃ I as alkylating agent. Two different alkylating agents [ ¹¹ C]CH ₃ I or [ ¹¹ C]CH ₃ OTf were used to do the radiolabeling. Several different bases such as NaOH, KOH, NaH, Na ₂ CO ₃ , CsCO ₃ , and different reaction solvents such as acetone, DMSO, DMF and MeOH were explored to develop the optimal radiosynthesis conditions. For all the radiosynthesis it was found that the combination of [ ¹¹ C]CH ₃ I as alkylating agent, DMF/DMSO with specific base at ambient temperatures were suitable for optimal radiochemical yield. A method to isolate the product on a SPE cartridge after HPLC purification was developed allowing removal of the mobile phase. The final product was eluted using ethanol and formulated into saline which yielded >98% radiochemical pure compound containing less than 10% ethanol. ³ H-Methyl iodide ([ ³ H]CH ₃ I) used to synthesize [ ³ H]KIn74, [ ³ H]KIn83 and [ ³ H]KIn84 via one step N- methylation/O-methylation of the corresponding precursor. The obtained molar activity of all three compounds were >1 GBq/µmol and the radiochemical purity was >96% up to several months after radiosynthesis when stored at -20°C. Autoradiography Binding selectivity of all six compounds for α7 nicotinic receptors ( α7-nAChR) was evaluated by autoradiography, as a preliminary screening (data not shown). Taking the library concept to a radiochemical environment is a promising approach towards experimental tracer development for PET studies. Evaluation with ARG showed that only [ ¹¹ C]KIn83 (0.01 MBq/ml) binds to α7-nAChR in rat brain, showing the best signal to the brain regions containing the highest density of α7 nicotinic receptors; hippocampus, hypothalamus and the cerebral cortex (Fig.10A). ARG competition studies showed that 80% of the total binding exerted by [ ¹¹ C]KIn83 in rat bran tissue was displaced by adding 10 µM of ASEM and unlabeled KIn83 (Fig.10B). KIn84 and KIn85 (other ASEM analogues sharing the same binding sites for α7-nAChR) were also able to displace this binding at the same extent (Fig.10). KIn83 was then tritiated, in order to get a higher image resolution and the possibility of quantifying the specific binding to each brain region, separately. Thus, further ARG studies were performed with the tritiated version of KIn83 ([ ³ H]KIn83), replicating the results obtained with [ ¹¹ C]KIn83 using a low concentration of tracer (0.8 nM – 1 nM). As it is observed in Figs.11A and 11C, autoradiogram showed a high specific binding to the brain regions of interest, which was completely blocked by both unlabeled KIn83 and ASEM (10 µM), suggesting that both compounds share the same binding sites for α7-nAChR. Fig.11B shows how KIn77 was also able to block [ ¹¹ C]KIn83 at the same extent as both unlabeled KIn83 and ASEM, in principle suggesting that other binding sites (apart of the one shared with ASEM) could be also targeted by KIn83 for α7-nAChR. [ ¹¹ C]KIn83 (1 nM) was also tested with ARG using human brain from a single Alzheimer’s disease case (AD) and a healthy control (CT) as is depicted in Fig.12A. Fig.12B shows the total binding obtained in control tissue (around 40 fmol/mg) and the AD case (around 75 fmol/mg). However, the non-specific binding levels were also high for both AD and control. A higher specific binding was observed in the grey matter of the AD case (around 25-30 fmol/mg) compared to control (around 15-20 fmol/mg) regardless of the blocker used (ASEM or unlabeled KIn83, both at 10 µM (Fig.12C). ¹²⁵ I- α-bungarotoxin has been suggested as the in vitro gold-standard radioligand for α7-nAChR. The 7α- nAChRs are widely distributed in the mammalian brain, with highest receptor density in hippocampus, hypothalamus, amygdala and the cerebral cortex, and lowest receptor density in cerebellum. [ ³ H]KIn83 binding matched the pattern of ¹²⁵ I- α-bungarotoxin, showing high specific binding to hippocampus, hypothalamus, amygdala and the cerebral cortex using rat tissue (Fig. 13). This signal was completely abolished by ASEM, unlabeled KIn83 and other ASEM derivatives included for the autoradiographic blocking study. In a previous recent study from Donat and collaborators, it was described that the specific binding of ¹²⁵ I- iodo-ASEM was lower in the rat and mouse brain when comparing to ¹²⁵ I- α-bungarotoxin [17]. However, in the present study [ ³ H]KIn83 showed similar binding signal as ¹²⁵ I- α-bungarotoxin using a lower concentration of tracer (0.8 nM vs 1.4 nM, respectively). Although ¹²⁵ I-iodo-ASEM allows sensitive and selective imaging of α7-nAChR in vitro, with better signal-to-noise ratio than previous described tracers [17], our data suggests that [ ³ H]KIn83 binds to the brain regions of interest at a higher extent, showing a high affinity and becoming a promising more selective target for α7-nAChR. When [ ³ H]KIn83 was tested with a preliminary ARG using human brain from a single Alzheimer’s disease case (AD) and a healthy control, a higher specific binding was observed in the grey matter of the AD case. NHP brain PET At the time of injection, the injected radioactivity of [ ¹¹ C]KIn83 was 146 ± 10 MBq and the injected mass was 6.6 ± 2.6 µg. Fusion images of MRI and summated PET are shown in Fig. 14. Whole brain uptake of [ ¹¹ C]KIn83 was 1.6 SUV on average at peak for the baseline condition. Distribution of [ ¹¹ C]KIn83 showed high in thalamus (1.5 SUV on average), middle in cortex (1.07-1.17 SUV), and low in basal ganglia and cerebellum (0.99-1.07 SUV) (Fig.15). Metabolism of [ ¹¹ C]KIn83 was relatively fast, showing less than 50% at 30 min. Several radiometabolite peaks were close to parent, but only polar ones. Clear increasing of brain uptake was observed after administration of tariquidar as 98% increase of average SUV. VTs decreased in all regions after administration of ASEM with estimated occupancy as 43%. The distribution pattern of [ ¹¹ C]KIn83 was similar as other α7-nAChR PET ligand such as ¹⁸ F-ASEM. Additionally, administration of ASEM decreased VTs of [ ¹¹ C]KIn83, showing similar occupancy values as previous study using ¹⁸ F-ASEM [18]. These data indicate that [ ¹¹ C]KIn83 is a promising PET ligand for α7- nAChR. After tariquidar administration, the brain uptake increased almost in double. This indicates that [ ¹¹ C]KIn83 is a substrate of P-gp. Radiometabolite analysis The recovery of radioactivity from plasma into acetonitrile after deproteinization was higher than 95%. HPLC analysis of plasma following injection of [ ¹¹ C]KIn83, which eluted at 5.3 minutes (Fig. 16A). The parent compound was more abundant at 4 min representing approximately 96% and it decreased to <10 at 90 min for PET at baseline condition (Fig. 16C). Whereas the abundance of the parent compound for PET after pretreatment with ASEM or tariquidar decreased to about 20% (Fig.16C). Two more radiometabolite peaks were observed which were which eluted at 3.9 and 4.6 minutes (Figs.16A and 16B). The identity of the radio metabolite [ ¹¹ C]KIn83 was confirmed by co-injection with the authentic non-radioactive KIn83. In the present Example an efficient synthesis strategy for six novel ¹¹ C-labeled ASEM analogues were established yielding the target compounds. Specific binding in the autoradiography studies was further studied by the tritium labeled compound KIn83, [ ³ H]KIn83, which showed the most promising features by the initial autoradiography screening with six ¹¹ C-labelled compounds. In vivo evaluation in NHP showed favorable properties for imaging α7-nAChR. These results together suggest that [ ¹¹ C]KIn83 may be an improved PET radioligand for for the detection of neuronal nicotinic acetylcholine receptors ( α7-nAChR). Example 4 – Synthesis of precursors This Example describes the synthesis of the precursors PRE-1 to PRE-4 mentioned in Example 3. Materials and Methods PRE-1 and PRE-3 The precursors PRE-1 and PRE-3 and the synthesis schemes are shown in Figs.25 and 26. Step-1 AgNO ₃ (8.15 g, 48.25 mmol) and trimethylsilyl chloride (TMSCl; 6.5 ml, 48.25 mmol) were added to a solution of dibenzo[b,d]thiophen-4-ylboronic acid 1 (5.0 g, 21.92 mmol) in dichloromethane (DCM; 50 ml) and stirred at room temperature for 12 h. After completion, the reaction mixture was filtered out and the filtrate was washed with water and brine. The organic layer was dried over sodium sulphate, concentrated to obtained 1,20 g crude compound 2. Step-2 H ₂ O ₂ (60 ml) was added dropwise to a solution of 4-nitrodibenzo[b,d]thiophene 2 (5.0 g, 21.83 mmol) in acetic acid (50 mL) at room temperature followed by heating to 60 °C for 14 h. After completion, the reaction mixture was cooled and quenched with ice cold water. The reaction mixture was filtered and the solid was washed with water to obtained white solid compound 3 (1.6 g) with around 28 % yield. ¹ H NMR (400 MHz, DMSO) δ 8.684 (d, J=8 Hz, 1H), 8.411 (d, J= 8.0Hz, 1H), 8.320 (d, J=8Hz, 1H), 8.063- 8.115 (m, 2H), 7.868-7.906 (m, 1H), 7.755 – 7.793 (m, 1H). Step-3 N-Bromosuccinimide (NBS; 3.51 g, 20.11 mmol) was added to a solution of 4-nitrodibenzo[b,d]thiophene 5,5- dioxide 3 (3.5 g, 13.40 mmol) in H ₂ SO ₄ (52 mL) at room temperature and allowed to stir at room temperature for 24 h. After completion, the reaction mixture was quenched with ice cold water and stirred for 1 h and the solid compound was filtered to obtain a crude compound, which was purified by column chromatography in MeOH in DCM. The pure compound was eluted at 3-6 % MeOH in DCM as compound 4 (2.80 g) after drying over vacuum. ¹ H NMR (400 MHz, DMSO) δ 8.698 (d,1H), 8.438-8.509 (m, 2H), 8.273 (m, 1H), 8.096-8.141 (m, 2H). Step-4 A solution of Pd ₂ dba ₃ (0.96 g, 1.05 mmol), 2,2’-bis(diphenylphosphino)-1,1’-binaphthyl (BINAP; 1.10 g, 1.76 mmol) in toluene 60 ml was purged with N2 for 30 minute and then heated to 90 °C for 15 minute. The reaction mixture was cooled to room temperature and 3-bromo-6-nitrodibenzo[b,d]thiophene 5,5-dioxide 4 (5.0 g, 14.70 mmol), nonane (2.24 g, 17.46 mmol), and Cs ₂ CO ₃ ( 8.59g, 26.0 mmol) were added and the reaction mixture was heated to 100 °C for 48 h. After completion, the reaction mixture was filtered and the filtrate was concentrated to obtain a crude product, which was purified by column chromatography in MeOH in DCM. The desired compound was eluted at 8-10 % MeOH in DCM, the pure fraction was concentrated to obtain compound 5 (1.20 g) confirmed on LCMS. Solubility of compound was very poor. Step-5 Fe (0.42 g, 15.50 mmol) and acetic acid (8 ml) were added to a solution of 3-(1,4-diazabicyclo[3.2.2]nonan- 4-yl)-6-nitrodibenzo[b,d]thiophene 5,5-dioxide 5 (1.2 g, 3.116 mmol) in THF (8 ml) and water (8 ml) and stirred at 60 °C for 2 h. After completion, the reaction mixture was quenched with water and filtered through celite pad and the aqueous layer washed with DCM and was neutralized with saturated NaHCO ₃ . The compound was extracted in 10 % MeOH in DCM and the organic layer dried over sodium sulphate and concentrated to obtain crude precursor PRE-1 as 0.40g.400 mg crude compound was purified by column chromatography using MeOH in DCM, and the desired compound was eluted in 8-12% MeOH in DCM to obtained the compound at 40 mg. ¹ H NMR (400 MHz, DMSO) δ 7.747 (d, J= 8.8Hz, 1H), 7.247-7.286 (m, 1H), 6.999-7.112 (m, 3H), 6.625 (d, J= 8 Hz,1H), 5.878 (s, 2H), 4.166 (s, 1H), 3.651-3.673 (m, 2H), 2.880-3.041 (m, 6H), 2.008 (s, 2H), 1.681- 1.714 (m, 2H). Step-6 Fe (0.635 g, 23.52 mmol) and acetic acid (8 ml) were added to a solution of 3-bromo-6- nitrodibenzo[b,d]thiophene 5,5-dioxide 4 (2.0 g, 5.88 mmol) in THF (8 ml) and water (8 ml), stirred at 60 °C for 2 h. After completion, the reaction mixture was cooled and saturated NaHCO ₃ was added and the compound was extracted in ethyl acetate and the organic layer was dried over sodium sulphate, concentrated to obtain the crude compound 6 as 1.0 g. Step-7 H ₂ SO ₄ (1.4 ml) was added to a solution of 6-amino-3-bromodibenzo[b,d]thiophene 5,5-dioxide 6 (1.50 g, 5.10 mmol) in H ₂ O (25 ml) at 0 °C and then NaNO ₂ (0.352 g, 5.10 mmol) was added and stirred at 100 °C for 2 h. After completion, the reaction mixture was cooled and saturated NaHCO ₃ was added. The compound was extracted in ethyl acetate and the organic layer was dried over sodium sulphate, and concentrated to obtain the crude compound 7 as 1.0 g. Step-8 K ₂ CO ₃ (0.893 g, 6.80 mmol) was added to a solution of 3-bromo-6-hydroxydibenzo[b,d]thiophene 5,5-dioxide 7 (1.0 g, 3.20 mmol) in DMF (30 ml) and stirred under nitrogen atmosphere and benzyl bromide (0.620 g, 4.86 mmol) was added and stirred at 70 °C for 4 h. After completion, the reaction mixture was cooled and water was added. The compound was extracted in ethyl acetate and the organic layer was dried over sodium sulphate and concentrated to obtain crude compound 8 as 920 mg. Step-9 A solution of Pd ₂ dba ₃ (0.069 g, 0.076 mmol), BINAP (0.079 g, 0.126 mmol) in toluene 10 ml was purged in N ₂ for 30 minute ad then heated at 90 °C for 20 minute. The reaction mixture was cooled to room temperature and added to 6-(benzyloxy)-3-bromodibenzo[b,d]thiophene 5,5-dioxide 8 (0.50 g, 1.2 mmol), nonane (0.174 g, 1.37 mmol), K ₂ CO ₃ (0.400g, 1.25 mmol) and t-BuOH (0.140 g, 1.25 mmol). The reaction mixture was heated at 100 °C for 24 h. After completion, the reaction mixture was filtered and the filtrate was concentrated to obtain the crude compound, which was purified by column chromatography in MeOH in DCM. The desired compound was eluted at 6-8 % MeOH in DCM, the pure fraction was concentrated to obtain compound 9 (0.280 g). Step-10 Pd/C ( 0.208 g, 0.869 mmol) was added to a solution of 6-(benzyloxy)-3-(1,4-diazabicyclo[3.2.2]nonan-4- yl)dibenzo[b,d]thiophene 5,5-dioxide 9 (0.20 g, 0.434 mmol) in MeOH (20 ml) and stirred under hydrogen atmosphere for 2 h. After completion, the reaction mixture was filtered through celite and the filtrate was concentrated to obtain 110 mg of the crude product, which was purified by prep-HPLC to obtain the precursor PRE-3 as 45 mg. ¹ H NMR (400 MHz, DMSO) δ 8.382 (m, 2H), 7.752 (d, J= 8.8Hz, 1H), 7.380-7.419 (m, 1H), 7.234 (m, 1H), 7.049 – 7.113 (m, 1H), 6.818 (d, J= 8 Hz,1H), 4.164 (s, 2H), 3.596-3.677 (m, 2H),3.183 (s, 2H), 2.915-3.016 (m, 5H), 2.006 (s, 2H), 1.721 (s, 2H). PRE-2 and PRE-4 The precursors PRE-2 and PRE-4 and the synthesis schemes are shown in Figs.27 and 28. Step-1 H ₂ O ₂ (7.4g, 217 mmol) was added dropwise to a solution of nitrodibenzo[b,d]thiophene 1 (10.0 g, 43.60 mmol) in acetic acid (80 mL), stirred at room temperature, heated to 60 °C for 2 h and then cooled to room temperature. H ₂ O ₂ (2.9 g, 87 mmol) was added and heated to 60 °C for 12 h. After completion, the reaction mixture was cooled. The reaction mixture was filtered and the solid was washed with water to obtain a white solid compound 2 (10.30 g) with 93% yield. ¹ H NMR (400 MHz, DMSO) δ 9.034 (s, 1H), 8.421-8.474 (m, 2H), 8.271 (m, 1H), 8.051 (d, J=8 Hz, 1H), 7.839-7.876 (m, 1H), 7.710 – 7.743 (m, 1H). Step-2 NBS (9.81g, 55.14 mmol) was added to a solution of 2-nitrobenzo[b,d]thiophene 5,5-dioxide 2 (12.0 g, 45.97 mmol) dissolved in H ₂ SO ₄ (120 mL), stirred at room temperature and stirring continued for 24 h. After completion, the reaction mixture was quenched with ice cold water and stirred for 1 h and the solid compound was filtered to obtain compound 3 (9.10 g) after drying over vacuum. ¹ H NMR (400 MHz, DMSO) δ 9.538 (s, 1H), 8.564-8.647 (m, 3H), 8.307-8.327 (m, 1H), 8.069-8.075 (m, 1H). Step-3 A solution of Pd ₂ dba ₃ (0.84 g, 0.9 mmol), BINAP (1.0 g, 1.60 mmol) in toluene 50 ml was purged in N2 for 30 minute and then heated at 90 °C for 15 minute. The reaction mixture was cooled to room temperature and added to 7-bromo-2-nitrodibenzo[b,d]thiophene 5,5-dioxide 3 (5.0 g, 14.70 mmol), nonane (1.88 g, 14.90 mmol), Cs ₂ CO ₃ (7.16g, 22.0 mmol) and the reaction mixture was further heated to 100 °C for 48 h. After completion, the reaction mixture was filtered and the filtrate was concentrated to obtain the crude product, which was purified by column chromatography in MeOH in DCM. The desired compound was eluted at 8- 10 % MeOH in DCM, the pure fraction was concentrated to obtain compound 4 (1.40 g). Step-4 Fe (1.26 g, 46.60 mmol) and acetic acid (36 ml) were added to a solution of 7-(1,4-diazabicyclo[3.2.2]nonan- 4-yl)-2-nitrodibenzo[b,d]thiophene 5,5-dioxide 4 (3.6 g, 9.330 mmol) in THF (18 ml) and water (18 ml) and stirred at 60 °C for 2 h. After completion, the reaction mixture was cooled and saturated NaHCO ₃ was added. The compound was extracted in 10 % MeOH in DCM, the organic layer was dried over sodium sulphate and concentrated to obtain crude precursor PRE-2 as 1.30 g.300 mg of the crude compound was purified by prep-HPLC to obtain the compound as 40 mg. ¹ H NMR (400 MHz, DMSO) δ 7.632-7.654 (d, J= 8.0Hz, 1H), 7.412-7.433 (m, 1H), 7.083 – 7.177 (m, 2H), 6.888 (m, 1H), 6.515 (m, 1H), 6.143(m, 1H), 4.250 (s, 1H), 3.728-3.754 (m, 2H), 3.085-3.174 (m, 4H), 1.924- 1.989 (m, 2H), 1.819 (m, 2H). Step-5 Fe (0.397 g, 14.70 mmol) and acetic acid (10 ml) were added to a solution of 7-bromo-2- nitrodibenzo[b,d]thiophene 5,5-dioxide 4 (1.0 g, 2.941 mmol) in THF (5 ml) and water (5 ml) and stirred at 60 °C for 2 h. After completion, the reaction mixture was cooled and saturated NaHCO ₃ was added. The compound was extracted in ethyl acetate and the organic layer was dried over sodium sulphate and concentrated to obtain the crude compound 5 as 0.70 g. Step-6 H ₂ SO ₄ (0.2 ml) was added to a solution of 2-amino-7-bromodibenzo[b,d]thiophene 5,5-dioxide 5 (0.10 g, 0.340 mmol) in water (5 ml) at 0 °C and then NaNO ₂ ( 0.024 g, 0.340 mmol) was added and the reaction mixture was stirred at 100 °C for 2 h. After completion, the reaction mixture was cooled and saturated NaHCO ₃ was added. The compound was extracted in ethyl acetate and the organic layer was dried over sodium sulphate and concentrated to obtain the crude compound 6 as 70 mg. Step-7 K ₂ CO ₃ (0.447 g, 3.40 mmol) was added to a solution of 7-bromo-2-hydroxydibenzo[b,d]thiophene 5,5-dioxide 6 (0.50 g, 1.62 mmol) in DMF (10 ml) and stirred under nitrogen atmosphere. Benzyl bromide (0.306 g, 2.43 mmol) was added and stirred at 70 °C for 4 h. After completion, the reaction mixture was cooled and water was added and the compound was extracted in ethyl acetate. The organic layer was dried over sodium sulphate, concentrated to obtain the crude product, which was purified by Combiflash in ethyl acetate in hexane. The desired compound was eluted at 25-30% ethyl acetate in hexane, the pure fraction was concentrated to obtain compound 7 as 400 mg. Step-8 A solution of Pd ₂ dba ₃ (0.161 g, 0.176 mmol), BINAP (0.73 g, 0.116 mmol) in toluene (5 ml) was purged in N2 for 30 minute and then heated at 90 °C for 20 minute. The reaction mixture was cooled to room temperature and added to 2-(benzyloxy)-7-bromodibenzo[b,d]thiophene 5,5-dioxide 7 (0.40 g, 1.0 mmol), nonane (0.126 g, 1.0 mmol), K ₂ CO ₃ (0.208g, 1.50 mmol) and t-BuOH (0.112 g, 1.0 mml). The reaction mixture was heated to 100 °C for 24 h. After completion, the reaction mixture was filtered and concentrated to obtain the crude product, which was purified by column chromatography in MeOH in DCM. The desired compound was eluted at 8-10 % MeOH in DCM, the pure fraction was concentrated to obtained compound 8 (0.040 g). Step-9 Pd/C ( 0.208 g, 0.869 mmol) was added to a solution of 2-(benzyloxy)-7-(1,4-diazabicyclo[3.2.2] nonan-4- yl)dibenzo[b,d]thiophene 5,5-dioxide 8 (0.20 g, 0.434 mmol) in MeOH (20 ml) and stirred under hydrogen atmosphere for 2 h. After completion, the reaction mixture was filtered through celite and the filtrate was concentrated to obtain the crude product, which was purified by Combiflash by MeOH in DCM mobile phase. The desired compound was eluted at 6-8% MeOH in DCM, the pure fraction was concentrated to obtain precursor PRE-4 as 85 mg. ¹ H NMR (400 MHz, DMSO) δ 10.597 (bs, 1H), 7.775 (d, J= 8.8Hz, 1H), 7.622 (d, J=8.4Hz, 1H) 7.196 – 7.232 (m, 2H), 7.091 (m, 1H), 6.782 (m, 1H), 4.208 (s, 1H), 3.701-3.711 (m, 2H), 2.915-3.016 (m, 5H), 2.031 (m, 2H), 1.925 (m, 2H). Example 5 - In vitro characterization ASEM analogue targeting alpha 7 nicotinic acid receptor in transfected cell line and human brain tissue The aim of this Example is to characterize α7-nAChR PET tracers and to compare them in vitro with already characterized α7-nAChR PET tracers such as ASEM, epibatidine and NS14492 using binding assay in GH3- ha7-22 transfected cells (rat pituitary epithelial like cells transfected with human neuronal α7-nACh receptors) (hα7) and HEK293- α4β2 transfected cells (human embryonic kidney cells transfected with human α4β2 receptors) as well as in post-mortem human brain tissues from AD and control. Additional experiment using ³ H-PIB (amyloid PET tracers) was performed to determine the interaction of the newly developed PET tracers and amyloid depositions. Material and Methods Chemicals ³ H-ASEM and unlabeled ASEM were synthesized by Novandi chemistry AB (Södertälje, Sweden; specific activity (SA) =  21 Ci/mmol). ³ H-NS144992 was purchased Novandi chemistry AB (Södertälje, Sweden; specific activity (SA)  =  81Ci/mmol). ³ H-Epibatidine was purchased from Perkin Elmer (SA: 55Ci/mmol). [ ³ H]KIn83 and [ ³ H]KIn84 (respectively SA = 24.8Ci/mmol and 27Ci/mmol) were synthesized and labeled at the Centre for Psychiatric Research in the Department of Clinical Neuroscience (Karolinska Institutet, Solna, Sweden). Unlabeled compound were synthesized by Syngene International Limited, India. Preparation of cell membrane homogenate Cell lines transfected with different subtypes of nicotinic receptor derived from human were used to identify the selectivity and specificity of the tracers. The cells were cultured and grown until confluency in Dulbecco’s Modified Eagle Medium (DMEM)/F12 medium supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml of Geneticin (G418). The preparation of cell membrane homogenate was performed as previously described [19], the cells were collected by scraping with 1xphosphate buffered saline (PBS), and then homogenized in buffer containing 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 1.3 mM MgCl ₂ , 33 mM Tris (pH 7.4). Then the homogenate was centrifuged at 20,000 g for 20 minutes. The resulting membrane pellets were re-suspended in the buffer containing 50 mM KH ₂ PO ₄ , 1 mM EDTA, 0.005% Triton X 100, protease inhibitor and used for further experiments. In vitro binding assay on cells All binding assay were performed using membrane preparation. The cells were collected by scraping with 1xPBS, and then homogenized using PBS (pH 7.4). Then the cell homogenate is centrifuged at 20,000 g for 20 minutes. The resulting membrane pellets were suspended in 50 mM KH ₂ PO ₄ , 1 mM EDTA containing protease inhibitor. Saturation binding experiment was carried out using increasing concentration (0.001 nM to 10 nM) of ³ H- epibatidine in GH ₃ -ha7 cells to determine the dissociation constant (Kd). Non-specific (NSP) binding was determined by using 1 µM unlabeled epibatidine. After 1 hour incubation, the binding assay was terminated by filtration through glass fiber filters presoaked for at least 3 hours in 0.3% polyethylenimine. To do so, the filters were rinsed and filtered three times using cold binding buffer, and then the radiation on the filter was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA). Competition binding assay was performed on GH ₃ -ha7 cells using ³ H-ASEM (0.2 nM), ³ H-NS14492 (0.2 nM), [ ³ H]KIn83 (0.2 nM) or ³ H-Epibatidine (1 nM) with increasing concentration of unlabeled nicotinic ligands (10- ¹⁴ to 10 ^-6 M) such as epibatidine, SSR180711, ASEM, and KIn compounds (KIn83, KIn74, KIn60, KIn77, KIn90, KIn84) to determine the inhibitory constant (Ki). After 1 hour the binding assay was terminated by filtration through glass fiber filters presoaked for at least 3 hours in 0.3% polyethylenimine. To do so, the filters were rinsed and filtered three times using cold binding buffer, and then the radiation on the filter was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA). Competition binding assay were performed on α4β2 cells using ³ H-Epibatidine (1 nM) with increasing concentration of unlabeled nicotinic ligands (10 ^-14 to 10 ^-6 ), such as epibatidine, ASEM, KIn83, KIn74, KIn77, KIn84, to determine the inhibitory constant (Ki). After 1-hour incubation the binding assay was terminated by filtration through glass fiber filters presoaked for at least 3 hours in 0.3% polyethylenimine. To do so, the filters were rinsed and filtered three times using cold binding buffer, and then the radiation on the filter was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA). In vitro binding assay on human brain homogenates All binding assay were performed using membrane preparation. The brain samples were homogenized using PBS containing protease inhibitor. The homogenate were centrifuged at 2800 rpm for 10 minutes. The resulting (P1) pellet was discarded and the supernatant was again centrifuged at 11,000 rpm for 20 minutes. The membrane fractions were collected and resuspended in 20 volumes of 1xPBS and used for receptor binding studies. Saturation binding experiments were carried out using increasing concentration (0.001 nM to 2 nM) of ³ H- ASEM in the P2 fractions of human brain to determine the dissociation constant (Kd). Non-specific (NSP) binding was determined by using 1 µM unlabeled nicotine or 1 µM of unlabeled ASEM. After 2 hours of incubation at room temperature for brain membrane homogenate the binding assay was terminated by filtration through glass fiber filters presoaked for at least 3 hours in 0.3% polyethylenimine. To do so, the filters were rinsed and filtered three times using cold binding buffer, and then the radiation on the filter was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA). Competition binding assay were performed using ³ H-ASEM (0.2 nM), ³ H-NS14492 (0.2 nM), [ ³ H]KIn83 (0.2 nM) or ³ H-epibatidine (1 nM) with increasing concentration of unlabeled nicotinic ligands (10 ^-14 to 10 ^-6 ), such as epibatidine, SSR ₁ 80711, ASEM, and the KI compounds (KIn83, KIn74, KIn60, KIn77, KIn90, KIn84) to determine the inhibitory constant (Ki). After 2 hours of incubation at room temperature the binding assay was terminated by filtration through glass fiber filters presoaked for at least 3 hours in 0.3% polyethylenimine. To do so, the filters were rinsed and filtered three times using cold binding buffer, and then the radiation on the filter was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA). Interaction between ³ H-PIB and unlabeled potential alpha 7 were studied as follow. P2 fractions of AD were pre-incubated with unlabeled KIn83, KIn84, NS14492 (10 ^-6 -10 ^-8 M) 30 min prior adding ³ H-PIB (1 nM), non- specific binding determined by adding 1 µM of unlabeled BTA-1. After 1 hour of incubation at room temperature the binding assay was terminated by filtration through glass fiber filters presoaked for at least 3 hours in 0.3% polyethylenimine. To do so, the filters were rinsed and filtered three times using cold binding buffer, and then the radiation on the filter was quantified using a scintillation counter (Beckman Coulter, Brea, CA, USA). Autoradiography on human and rat brain sections Post mortem frozen right hemispheres of 1 AD, 1 control and 1 APParc mutation were allowed to reach room temperature, pre-incubated for 10 minutes with 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 1.3 mM MgCl ₂ , 33 mM Tris (pH 7.4) and then incubated for 1 h at room temperature with [ ³ H]KIn83 (1 nM). The sections were rinsed three times in ice-cold buffer for 5 minutes, followed by a quick dip in cold distilled water. Non-specific binding was determined using 1 µM unlabeled KIn83. After waiting 24 h for the sections to dry, a phosphor imaging plate was placed on the sections together with a tritium standard on a phosphor plate for 7 days and then scanned using a BAS-2500 phosphor imager (Fujifilm, Tokyo, Japan). For the autoradiography studies, the regions of interest were drawn manually on the autoradiogram using multigauge software and were used for the semi quantitative analysis. Photo stimulated luminescence per square millimeter (PSL/mm ² ) was transformed to fmol/mm ² using the tritium standard. Results In vitro binding studies in GH3-ha7 cells Saturation binding studies with ³ H-epibatidine (0.001-10 nM) in α7 transfected GE3-ha cells showed a single site binding with a Bmax of 247 fmol/g and a Kd of 2.3 nM (Fig.17). Competition binding assay in α7 cell line are presented in Fig.18. The competition using ³ H-epibatidine with 7 unlabeled compounds is presented in Fig.18A. All 7 unlabeled compounds showed one high affinity site ranked from the more potent IC50 to less potent IC50 as follow: ASEM (9.0 x10 ^-12 M) < KIn83 (2.2x10 ^-11 ) < KIn84 (9.8 x10 ^-11 ) < KIn77 (5.0 x10 ^-11 ) < epibatidine (2.4 x10 ^-9 ). In clear contrast, compound KIn74 and KIn90 both showed two binding sites at 8.8x10 ^-13 and 3.9x10 ^-9 with 50 % of high affinity site and 1.5x10 ^-12 and 1.5x10 ^-9 with 59 % of high affinity site, respectively (Fig.18A). When similar competitions studies were performed with ³ H-ASEM (0.2 nM), four unlabeled compounds showed one binding site with IC50 ranked in the following order with KIn84 (2.4x10 ^-11 ) < ASEM (0.2 10 ^-9 ) ≈ KIn83 (0.3 x10 ^-9 ) < epibatidine (1.7 x10 ^-8 ). KIn60 demonstrated two binding sites (0.1 x10 ^-9 and 4.6 x10 ^-9 with 38 % of high affinity site) (Fig.18B). Finally, similar competition studies with [ ³ H]KIn83 (0.2 nM) were performed with four compounds. Only unlabeled epibatidine demonstrated one binding site with an IC50 value of 3.2 x10 ^-8 . The other three compounds tested all showed two binding sites with different proportion of high affinity sites in the order KIn83 (1.2 x10 ^-12 and 0.2 x10 ^-9 with 45 % of high affinity site) < KIn74 (4.5 x10 ^-12 and 6.6 x10 ^-8 with 18 % of high affinity site) < ASEM (6.1 x10 ^-12 and 0.7 x10 ^-9 with 61 % of high affinity site) (Fig.18C). In vitro binding studies in HEK293-alpha α4β2 cells In the Fig. 19, unlabeled ASEM, KIn83, KIn84, Kin77, Kin74 (10 ^-11 to 10 ^-6 M) were tested against ³ H- epibatidine (1 nM) in transfected HEK293- α4β2 cells no competition was obtained. Unlabeled epibatidine showed an IC50 value of 2.12 x10 ^-9 , which was comparable to the IC50 demonstrated by epibatidine towards ³ H-epibatidine in GH ₃ -ha7 cells (Fig.18A). Binding studies with ³ H-ASEM and [ ³ H]KIn83 in control human brain Saturation binding studies with ³ H-ASEM in P2 fractions prepared from post-mortem human frontal cortical brain tissue showed one binding site with a Bmax of 44.48 fmol/g and Kd value of 0,38 nM) (Fig.20). Competition studies with five unlabeled compounds against ³ H-ASEM (0.2 nM) showed two binding sites for all tested compounds with two IC50 comparable for KIn83, ASEM, KIn84 one at 10 ^-13 and one at 10 ^-9 , with 65 %, 49 %, and 63 % for the high affinity sites respectively. KIn74 showed one binding site (4.5 x10 ^-13 ). Epibatidine showed two binding sites (4.2 x10-13 and 1.2 x10 ^-7 with 37% high affinity sites) (Fig.21A). When [ ³ H]KIn83 was used as labelled compounds in the competitions studies KIn83 and ASEM showed comparable competition data of two binding sites 1.8 x10 ^-13 and 3.0 x10 ^-9 (60 % high affinity sites) and 2.3 x10- ¹³ and 0.6 x10 ^-9 (56 % high affinity sites), respectively. KIn74 showed somewhat lower IC50 values of 4.4 x10- ¹² and 8.4 x10 ^-8 (64 % high affinity sites). Epibatidine showed one binding site with IC501.4 x10 ^-11 (Fig.21B). Binding studies with [ ³ H]KIn83 in Alzheimer brain tissue Fig. 22 illustrates a competition experiment with unlabeled KIn83 against [ ³ H]KIn83 performed in frontal cortical tissue (P2 fractions) from an Alzheimer brain compared to control brain. The competition studies showed two binding sites with IC50 values of 0.4 x10 ^-12 and 0.2 x10 ^-9 (50 % high affinity sites) in the Alzheimer brain tissue. The IC50 value for the low affinity site for the Alzheimer brain was 10 times lower (IC500.2 x10- ⁹ ) compared with control brain (3.4 x10 ^-9 ) (Fig.22). Quantitative in vitro autoradiography with [ ³ H]KIn83 human brain sections Autoradiography on large frozen section from AD and control brain were performed and illustrated in Fig.23. We can observe total binding both in control and AD with less unspecific binding in the AD case. The binding in APParc, mutation known to not have typical amyloid cored plaques showed less binding than sporadic AD. Development of PET tracers for α7-nAChR is challenge because of the moderate concentration of the receptor in the CNS. In this perspective, the present Example focus on identifying and characterizing suitable PET tracer for α7-nAChR in vitro using the ASEM chemical structure as pharmacophore. The ¹⁸ F-ASEM, is one of the only PET tracers for α7-nAChR that have been used and injected in healthy volunteers. In a first step, in silico screening of a large library of potential α7-nAChR compound was performed using ASEM chemical structure as lead compound. After in silico screening, the most potent ones were synthesized and then sent to CEREP for affinity screening in α7 cells. The compounds showing the best affinity towards α7 in vitro have been tritiated labeled to performed in vitro binding studies both in transfected α7, α4β2 as well as in cell membrane preparation from control and Alzheimer disease cases. To have an idea of the regional distribution of the α7 receptors, large autoradiography in AD, control and Arctic mutation cases were performed as well. Several ligands specific to nicotinic receptor are already available and well characterized in vitro, such as epibatidine and ASEM. In this Example, those two compound were tritiated to compare the data with the new ligands in house synthesized. The binding properties of the transfected cells have been investigated first, by doing a saturation binding studies using ³ H-epibatidine. We could observe a Kd of 2 nM with a Bmax of 247 fmol/mg, data in the same range of previously describe data in literature. It is important to keep in mind the complexity of binding to α7 receptor with compound having different properties towards the receptor being full agonist, partial agonist or even antagonist. In this Example we looked at epibatidine, which is an agonist, as well as the ASEM, which is an antagonist. When we made competition study in the α7 cells we could observe that unlabeled epibatidine was having a different IC50 than unlabeled ASEM and unlabeled KIn83. Unlabeled epibatidine seems to behave similarly towards ³ H-epibatidine and [ ³ H]KIn83 with a IC50 of 2.4 nM when it was about 17 nM using ³ H-ASEM. In competition binding assay with ³ H-epibatidine both unlabeled ASEM and unlabeled KIn83 showed a super high IC50 value with one binding site in pmolar range. When studies were performed with ³ H-ASEM and unlabeled ASEM and KIn83 both compounds showed somewhat lower IC50 values around 0,2 nM. [ ³ H]KIn83 was the only tracer where we obtained two binding sites for both unlabeled ASEM and unlabeled KIn83 both with IC50 in the picomolar range and the nanomolar range. We also tested other KIn compounds than KIn83 that were part of the in silico screening and even though the in silico screening started from the same structure, we could observe different binding properties for different compounds. For example, the KIn74 is showing two binding sites both in ³ H-epibatidine and in [ ³ H]KIn83. KIn84 also showed similar binding IC50 value in ³ H-epibatdine and ³ H-ASEM in the picomolar range but with a totally different distribution. To ensure that the compounds were specific to α7 we also performed competition with ³ H-epibatidine in ^4 β2 cells. Indeed, epibatidine was not specific to α7 and we could observe similar IC50 of 2 nM for unlabeled epibatidine when none of the KIn compounds, nor ASEM where competing in α7 cells. The compounds were then tested in control and AD human P2 fractions. In the competition binding experiment more unspecific binding was observed for both ³ H-ASEM and [ ³ H]KIn83 in the human P2 fraction homogenates compared to in transfected alpha7 transfected neuroblastoma cells. However, all unlabeled compounds, except KIn74, showed two binding sites in the competition studies with ³ H-ASEM. KIn74, which showed one binding in the picomolar range in human brain tissue, showed in contrast two binding sites (picomolar and nanomolar range) in competitions experiments with ³ H-ASEM in transfected neuroblastoma cells, KIn74 also showed two binding sites in human P2 cell membranes with [ ³ H]KIn83 with similar values as for the two binding sites observed for the cells. However, the proportion (%) of high affinity binding site was greater in competition studies in human P2 cell membrane homogenate than in the transfected neuroblastoma cells. Unlabeled ASEM showed higher IC50 in competition with [ ³ H]KIn83 than ³ H-ASEM in the nanomolar range when the IC50 in the picomolar range was similar. Unlabeled KIn83 behaved in the same way when in competition with ³ H-ASEM or [ ³ H]KIn83. We looked at the behavior of the [ ³ H]KIn83 in AD cases in comparison to control. We could observe two binding sites one in picomolar range and one in nanomolar range. Interestingly, in the AD, we could see a shift of the proportion of high affinity binding sites and also higher binding affinity in nanomolar range in AD than in control. To have an idea of the regional distribution, we looked at large autoradiography. We could see different binding pattern in AD compared to control. The NSP binding in AD seems to be less important than in control cases. Interestingly, when we looked at the APParc cases, which do not have typical amyloid plaques with a core, we could observe less binding than in sporadic AD. It has already been demonstrated that an interaction with α7 compound and amyloid plaques influences the binding of ³ H-PIB. In this Example, we could characterize newly developed compounds for α7 receptor, which showed good specificity in human transfected cell as well as good selectivity with no binding in ^4 β2 cells. The experiments using P2 fraction in human brain tissue (control and AD) showed that the compounds can bind and have good affinity towards human α7 receptor in postmortem tissue. Altogether, the finding indicates that KIn83 is a potent α7 PET tracer that could be used in human studies. The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. REFERENCES [1] Zhou et al., Enhanced Sampling Simulations of Ligand Unbinding Kinetics Controlled by Protein Conformational Changes, J. Chem. Inf. Model., 2019, 59(9), 3910–3918 [2] Brejc et al., Crystal structure of an AChbinding protein reveals the ligand-binding domain of nicotinic receptors, Nature, 2001, 411, 269–276 [3] Li et al., Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist, Nat. Neurosci., 2011, 14, 1253–1259 [4] Schrödinger Release 2016-4: Glide, Schr¨ odinger, LLC, New York, NY, 2016 [5] Harder et al., OPLS3: A Force Field Providing Broad Coverage of Drug-like Small Molecules and Proteins, J. Chem. Theory Comput., 2016, 12, 281–296 [6] Shivakumar et al., Improving the Prediction of Absolute Solvation Free Energies Using the Next Generation OPLS Force Field, J. Chem. Theory Comput., 2012, 8(8), 2553–2558 [7] Liu et al., Replica exchange with solute tempering: a method for sampling biological systems in explicit water, Proc. Natl. Acad. Sci., 2005, 102, 13749–13754 [8] Liu et al., Lead optimization mapper: automating free energy calculations for lead optimization, J. Comput. Aided Mol. Des., 2013, 27, 755–770 [9] Price and Brooks, A Modified TIP3P Water Potential for Simulation with Ewald Summation, J. Chem. Phys., 2004, 121, 10096–10103 [10] Abraham et al., Gromacs: High Performance Molecular Simulations through Multi-Level Parallelism from Laptops to Supercomputers, SoftwareX, 2015, 1–2, 19–25 [11] Andersson et al., In-target produced [ ¹¹ C]methane: Increased specific radioactivity. Appl Radiat Isot 2009, 67(1), 106-10 [12] Hall et al., Whole hemisphere autoradiography of the postmortem human brain. Nucl Med Biol 1998, 25(8), 715-9 [13] Schou et al., Post-mortem human brain autoradiography of the norepinephrine transporter using (S,S)- [ ¹⁸ F]FMeNER-D2. Eur Neuropsychopharmacol 2005, 15(5), 517-20 [14] Gillberg et al., Large section cryomicrotomy for invitro receptor autography. Journal of Pharmacological Methods 1986, 15(2), 169-180 [15] Jahan et al., The development of a GPR44 targeting radioligand [ ¹¹ C]AZ12204657 for in vivo assessment of beta cell mass. Ejnmmi Res 2018, 8, 113 [16] Moein et alSample preparation techniques for radiometabolite analysis of positron emission tomography radioligands; trends, progress, limitations and future prospects. Trac-Trend Anal Chem 2019, 110, 1-7 [17] Donat et al., In Vitro and In Vivo Characterization of Dibenzothiophene Derivatives [ ¹²⁵ I]Iodo-ASEM and [ ¹⁸ F]ASEM as Radiotracers of Homo- and Heteromeric alpha 7 Nicotinic Acetylcholine Receptors. Molecules 2020, 25(6), 1425 [18] Hillmer et al., PET imaging of alpha(7) nicotinic acetylcholine receptors: a comparative study of [ ¹⁸ F]ASEM and [ ¹⁸ F]DBT-10 in nonhuman primates, and further evaluation of [ ¹⁸ F]ASEM in humans. Eur J Nucl Med Mol I 2017, 44(6), 1042-1050 [19] Xiao et al., Rat neuronal nicotinic acetylcholine receptors containing α7 subunit: pharmacological properties of ligand binding and function. Acta Pharmacologica Sinica 2009, 30(6), 842