WO2022255915A1 - Radiotraceurs pour tep - Google Patents

Radiotraceurs pour tep Download PDF

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WO2022255915A1
WO2022255915A1 PCT/SE2022/050413 SE2022050413W WO2022255915A1 WO 2022255915 A1 WO2022255915 A1 WO 2022255915A1 SE 2022050413 W SE2022050413 W SE 2022050413W WO 2022255915 A1 WO2022255915 A1 WO 2022255915A1
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thiophene
diazabicyclo
nonan
group
alkyl
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PCT/SE2022/050413
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English (en)
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Agneta Nordberg
Bengt LÅNGSTRÖM
Hans ÅGREN
Christer Halldin
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Agneta Nordberg
Laangstroem Bengt
Aagren Hans
Christer Halldin
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Publication of WO2022255915A1 publication Critical patent/WO2022255915A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/08—Bridged systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/041—Heterocyclic compounds
    • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
    • A61K51/0468—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/002—Heterocyclic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention generally relates to compounds capable of binding to ⁇ 7 nicotinic acetylcholine receptor ( ⁇ 7-nAChR), and in particular such compounds that can be used as PET radiotracers.
  • ⁇ 7-nAChR nicotinic acetylcholine receptor
  • BACKGROUND Nicotinic acetylcholine receptors 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: ⁇ 2 ⁇ ⁇ 10 and ⁇ 2 ⁇ ⁇ 4.
  • Homomeric ⁇ 7 nAChRs mainly expressed in the central nervous system (CNS) and spinal cord are distinguished from neuronal heteromeric nAChRs by their high-affinity binding to ⁇ -bungarotoxin ( ⁇ - BTX).
  • ⁇ - BTX ⁇ -bungarotoxin
  • ⁇ 7-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 ⁇ 7-nAChR.
  • 11 C-CHIBA-1001 was the first PET radiotracers to image ⁇ 7-nAChRs in humans but displayed poor specificity for ⁇ 7-nAChR and high nonspecific uptake.
  • [ 18 F]ASEM and [ 18 F]DBT-10 are structural isomers (Fig.
  • 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.
  • 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 ⁇ 7-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 ⁇ 7-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 ⁇ 7-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 ⁇ 7-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 ⁇ 7-nAChR both in vitro and in vivo in a subject body.
  • the PET radiotracers thereby enable visualization and quantification of ⁇ 7-nAChR in various target tissues, including monitoring the distribution of ⁇ 7-nAChRs in such a target tissue.
  • the compounds exhibit of high binding affinity and specificity towards ⁇ 7-nAChR and are able to pass the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • Fig.1 (a) Structures of ⁇ 7-AChBP and its binding site, (b) the binding mode of epibatidine with ⁇ 7-AChBP, and (c) the binding mode of ASEM with ⁇ 7-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 ⁇ 7-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.
  • 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 ⁇ M (Nicotine, ASEM, KIn83, KIn84 and KIn85) in rat at hippocampus level section.
  • FIG. 11 (A) Autoradiograms showing the total and non-specific binding (blocked with homologous cold compound and ASEM at 10 ⁇ M) 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 ⁇ M), ASEM (10 ⁇ M), KIn77 (10 ⁇ M) and nicotine (100 ⁇ M) obtained in rat when using [ 3 H]KIn83 at 0.8 nM concentration.
  • FIG.12 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 ⁇ M) obtained in temporal cortex of human tissue from a healthy control (CT) and an Alzheimer ⁇ s disease patient (AD).
  • CT healthy control
  • AD Alzheimer ⁇ s disease patient
  • 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.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 ⁇ 4 ⁇ 2 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 ⁇ M 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.
  • the present invention generally relates to compounds capable of binding to ⁇ 7 nicotinic acetylcholine receptor ( ⁇ 7-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 ⁇ 7-nAChR and can be labelled with 3 H and/or 11 C, for example, by means of various alkylation and/or carbonylation reactions.
  • the compounds can be used as PET radiotracers capable of binding to ⁇ 7-nAChR both in vitro and in vivo in a subject body.
  • the PET radiotracers thereby enable visualization and quantification of ⁇ 7-nAChR in various target tissues, including monitoring the distribution of ⁇ 7-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 ⁇ 7-nAChRs.
  • the PET radiotracers can be used to measure receptor occupancies of drugs targeting ⁇ 7-nAChR and dose-response relationships.
  • 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 ⁇ 7-nAChR. Firstly, the compounds exhibit a high binding affinity and specificity towards ⁇ 7-nAChR.
  • ⁇ 7-nAChRs are generally present in very low density in central nervous system (CNS) (0.13 – 15 fmol/mg protein in humans).
  • 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.
  • BBB blood-brain barrier
  • 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
  • R 1 and R 2 are both hydrogen
  • [ 18 F]ASEM i.e., having R 1 as 18 F and R 2 as hydrogen
  • [ 18 F]DBT-10 i.e., having R 1 as hydrogen and R 2 as 18 F, see Fig.8.
  • 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.
  • 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.
  • one but not both of R 1 and R 2 is hydrogen.
  • 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.
  • 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 ⁇ 7-nAChR (see Fig.3) as ASEM (Figs.1c, 2a), DBT-10 (Fig.2b) and epibatidine (Figs.1a, 1b), which is an agonist of ⁇ 7-nAChR.
  • 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.
  • 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 ⁇ 7-nAChR.
  • 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.
  • 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.
  • C 1 -C 4 alkoxy is a C 1 alkoxy, i.e., methoxy.
  • 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.
  • 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.
  • the halopropyl is fluoropropyl.
  • Other examples of halopropyl groups that could be used according to the invention include chloropropyl and iodopropyl.
  • 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.
  • preferred -NR 3 R 4 groups include methylamine (–NHCH 3 ) and dimethylamine (–NH(CH 3 ) 2 ).
  • 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.
  • R 5 is selected from the group consisting of methyl, ethyl, benzyl and pyridinylmethyl.
  • R 5 is selected from the group consisting of methyl, ethyl, benzyl and 4-pydinylmethyl.
  • 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]thiophene 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-fluoro
  • the compound is selected from the group consisting of: 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-8-methoxydibenzo[b,d]thiophene 5,5-dioxide; 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-methoxydibenzo[b,d]thiophene 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]thiophene 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • R 1 and R 2 comprises a 3 H radioactive isotope and/or a 11 C radioactive isotope.
  • one but not both of R 1 and R 2 is hydrogen.
  • R 2 is hydrogen and R 1 is other than hydrogen.
  • 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.
  • 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.
  • 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.
  • R 1 is hydrogen and R 2 is other than hydrogen.
  • 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.
  • 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.
  • 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.
  • 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.
  • [ 3 H]C 1 -C 4 alkoxy is a [ 3 H]C 1 alkoxy, i.e., [ 3 H]methoxy.
  • 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.
  • [ 11 C]C 1 -C 4 alkoxy is a [ 11 C]C 1 alkoxy, i.e., [ 11 C]methoxy.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the [ 3 H]halopropyl is [ 3 H]fluoropropyl and [ 11 C]halopropyl is [ 11 C]fluoropropyl.
  • 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.
  • 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.
  • 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.
  • 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.
  • R 11 is selected from the group consisting of [ 3 H]methyl, [ 3 H]ethyl, [ 3 H]benzyl, and [ 3 H]pyridinylmethyl.
  • R 11 is selected from the group consisting of [ 3 H]methyl, [ 3 H]ethyl, [ 3 H]benzyl, and [ 3 H]4-pyridinylmethyl.
  • 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.
  • R 14 is selected from the group consisting of [ 11 C]methyl, [ 11 C]ethyl, [ 11 C]benzyl, and [ 11 C]pyridinylmethyl.
  • R 14 is selected from the group consisting of [ 11 C]methyl, [ 11 C]ethyl, [ 11 C]benzyl, and [ 11 C]4-pyridinylmethyl.
  • 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-d
  • 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;
  • 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- 11 C]me
  • 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.
  • 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 ⁇ 7-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 ⁇ 7-nAChR and, in addition, have high specificity for ⁇ 7-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.
  • the invention relates to an in vitro competition binding assay method.
  • the method comprises contacting, in vitro, a sample comprising ⁇ 7-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 ⁇ 7-nAChRs can then be determined based on the measured radiation.
  • the sample could be any sample comprising ⁇ 7-nAChRs, including isolated and purified ⁇ 7-nAChRs, a cell preparation or homogenate comprising ⁇ 7-nAChRs and a tissue preparation or homogenate comprising ⁇ 7- nAChRs.
  • a defined concentration of the PET radiotracer and a defined concentration of the test agent are added to the sample.
  • 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.
  • 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.
  • 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.
  • PEI polyethylenimine
  • 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).
  • 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 ⁇ 7-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 ⁇ 7-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 ⁇ 7-nAChRs in the subject.
  • the neurodegenerative or psychiatric disease is selected from the group consisting of Parkinson’s disease, Alzheimer’s disease and schizophrenia.
  • the disease is Alzheimer’s disease.
  • the disease is Parkinson’s disease.
  • the disease is schizophrenia.
  • 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.
  • aqueous injection solutions include saline, and buffer solutions, such as phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the PET radiotracers are injected intravenously in PBS, such as in a volume of from about 5 to about 10 ml.
  • the solution comprises a P-glycoprotein inhibitor.
  • 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-carboxamide).
  • 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 ⁇ 7-nAChR and experimental verification The ⁇ 7 nicotinic acetylcholine receptor ( ⁇ 7-nAChR) is implicated in a variety of neurodegenerative and neuropsychiatric disorders, such as Alzheimer's disease (AD) and schizophrenia.
  • AD Alzheimer's disease
  • ASEM has a diazobicyclic head group and is protonated under physiological conditions.
  • the diazobicyclic head group of ASEM is bulkier than the counterpart of epibatidine, which should have an impact on its binding with the receptor.
  • epibatidine is an agonist whereas ASEM is an antagonist.
  • ⁇ 7-nAChR antagonists such as methyllycaconitine (MLA) and abungarotoxin, tend to be much bulkier than the agonists, such as nicotine and acetylcholine.
  • the tip nitrogen (N1, pKa ⁇ 9.6) of ASEM is protonated under physiological conditions and has cation– ⁇ interactions with the aromatic rings of Tyr91, Trp145, Tyr184, and Try191. These cation– ⁇ interactions are believed to be important for the affinity of ⁇ 7-nAChR ligands.
  • the protonated nitrogen also forms a hydrogen bond with the backbone oxygen of Trp145 (Fig.1c).
  • 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).
  • 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.
  • the chlorine atom is thought to have halogen-bond interaction with the backbone oxygen atom of Gln114, which also supports the binding of epibatidine.
  • 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 ⁇ - ⁇ 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.
  • induced-fit docking we managed to produce a reasonable docking mode of ASEM with ⁇ 7-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.
  • the higher binding affinity of DBT10 is in agreement with the in vitro experiment using the human ⁇ 7-nAChR and is in contradiction to the result from rat cortical membranes.
  • 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.
  • the N-methyl substituent at the R 2 position leads a steric conflict to Ser32 and Ser34.
  • 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 ⁇ - ⁇ 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.
  • 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 ⁇ 7-nAChR.
  • the diazabicyclo[3.2.2]nonane ring has cation– ⁇ 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 ⁇ - ⁇ 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.
  • FEP+ Free Energy Perturbation Theory
  • 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.
  • IFD induced-fit docking
  • the crystal structure of the ⁇ 7-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.
  • the centroid of the crystalized ligand epibatidine was chosen as the grid center and the residues within 20 ⁇ of it are treated as binding pocket.
  • a van der Waals scaling factor of 0.5 was used for both receptor and ligand.
  • protein residues within 5 ⁇ of the ligand were optimized by prime.
  • the glide standard precision (SP) scoring function was adopted to rank the optimized docking poses.
  • the ⁇ 7-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 ⁇ 7-AChBP.
  • 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 ⁇ 7-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 ⁇ 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 GROMACS [10] program was employed for the MD simulations. In the sMD simulations, the force field was scaled by a factor of 0.4.
  • 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 ⁇ 7 transfected neuroblastoma cells SH-SY5Y cells (human recombinant) incubated with 0.05 nM 125 I- ⁇ -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- ⁇ -bungarotoxin by each compound.
  • 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%).
  • Example 3 Development of novel 11 C-labeled ASEM analogs for detection of ⁇ 7-nAChR
  • the homo-pentameric alpha 7 receptor is one of the major types of neuronal nicotinic acetylcholine receptor ( ⁇ 7-nAChR) related to cognition, memory formation, and attention processing.
  • ⁇ 7-nAChR neuronal nicotinic acetylcholine receptor
  • 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.
  • the reaction mixture was heated at 80°C for 4 minutes.
  • the reaction mixture was diluted with sterile water (500 ⁇ L) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound.
  • HPLC high performance liquid chromatography
  • the reaction mixture was heated at 90°C for 5 minutes.
  • the reaction mixture was diluted with sterile water (500 ⁇ L) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound.
  • HPLC high performance liquid chromatography
  • the reaction mixture was diluted with sterile water (500 ⁇ L) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound.
  • HPLC high performance liquid chromatography
  • the reaction mixture was heated at 80°C for 4 minutes.
  • the reaction mixture was diluted with sterile water (500 ⁇ L) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound.
  • HPLC high performance liquid chromatography
  • the reaction mixture was heated at 80°C for 3 minutes.
  • the reaction mixture was diluted with sterile water (500 ⁇ L) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound.
  • HPLC high performance liquid chromatography
  • the reaction mixture was diluted with sterile water (500 ⁇ L) before injecting to the built-in high performance liquid chromatography (HPLC) system for the purification of the radiolabeled compound.
  • HPLC high performance liquid chromatography
  • 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.
  • AF ammonium formate
  • [ 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 ⁇ mol-5.4 ⁇ mol) potassium hydroxide powder (4-7 mg) in DMSO (300 ⁇ L) 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 ⁇ L 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.
  • AF ammonium formate
  • 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 ⁇ mol-5.4 ⁇ mol) cesium carbonate (4-5 mg) in dry DMF (300 ⁇ L) and the mixture was vortexed for 5 minutes.
  • 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 ⁇ ⁇ 250 mm, 5 ⁇ m 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.
  • 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 ⁇ m/pixel.
  • phosphor imaging plates Flujifilm Plate BAS-TR2025, Fujifilm, Tokyo, Japan
  • a Fujifilm BAS-5000 phosphor imager Flujifilm, Tokyo, Japan
  • 20 ⁇ L 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.
  • 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.
  • the regions of interest 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.
  • the time-activity curves of brain regions were generated for each PET measurement.
  • Average standardized uptake value (SUV) was calculated for each brain regions.
  • 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 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).
  • 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).
  • EOB end of bombardment
  • 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/ ⁇ mol and the radiochemical purity was >96% up to several months after radiosynthesis when stored at -20°C.
  • 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 ⁇ 7-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.
  • 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.
  • DCM dichloromethane
  • 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.
  • 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.
  • BINAP 2,2’-bis(diphenylphosphino)-1,1’-binaphthyl
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 7-nAChR PET tracers and to compare them in vitro with already characterized ⁇ 7-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 ⁇ 7-nACh receptors) (h ⁇ 7) and HEK293- ⁇ 4 ⁇ 2 transfected cells (human embryonic kidney cells transfected with human ⁇ 4 ⁇ 2 receptors) as well as in post-mortem human brain tissues from AD and control.
  • GH3- ha7-22 transfected cells rat pituitary epithelial like cells transfected with human neuronal ⁇ 7-nACh receptors
  • HEK293- ⁇ 4 ⁇ 2 transfected cells human
  • [ 3 H]KIn83 and [ 3 H]KIn84 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 ⁇ g/ml of Geneticin (G418).
  • DMEM Modified Eagle Medium
  • FBS fetal bovine serum
  • G4108 50 ⁇ g/ml of Geneticin
  • 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.
  • PBS 1xphosphate buffered saline
  • 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.
  • Non-specific (NSP) binding was determined by using 1 ⁇ M 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.
  • 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 ⁇ 4 ⁇ 2 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.
  • 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).
  • 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.
  • Non-specific (NSP) binding was determined by using 1 ⁇ M unlabeled nicotine or 1 ⁇ M 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.
  • 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 ⁇ M 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.
  • 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).
  • 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 ⁇ M 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.
  • PSL/mm 2 Photo stimulated luminescence per square millimeter
  • 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).
  • 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.
  • 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.
  • a first step in silico screening of a large library of potential ⁇ 7-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 ⁇ 7 cells.
  • the compounds showing the best affinity towards ⁇ 7 in vitro have been tritiated labeled to performed in vitro binding studies both in transfected ⁇ 7, ⁇ 4 ⁇ 2 as well as in cell membrane preparation from control and Alzheimer disease cases.
  • To have an idea of the regional distribution of the ⁇ 7 receptors large autoradiography in AD, control and Arctic mutation cases were performed as well.
  • 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.
  • 3 H-epibatidine was not specific to ⁇ 7 and we could observe similar IC50 of 2 nM for unlabeled epibatidine when none of the KIn compounds, nor ASEM where competing in ⁇ 7 cells. The compounds were then tested in control and AD human P2 fractions.

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Abstract

Les composés de l'invention sont des analogues d'ASEM présentant différentes substitutions en position ortho et para qui peuvent être marquées par 3H et/ou 11C, destinés à être utilisés en tant que radiotraceurs pour TEP pouvant se lier à α7-nAChR à la fois in vitro et in vivo dans un corps d'un sujet. Les radiotraceurs pour TEP permettent ainsi la visualisation et la quantification d'α7-nAChR dans divers tissus cibles, comprenant la surveillance de la distribution d'α7-nAChR dans un tel tissu cible. Les composés présentent une affinité de liaison élevée et une spécificité élevée envers α7-nAChR et peuvent de traverser la barrière hémato-encéphalique (BHE).
PCT/SE2022/050413 2021-06-01 2022-04-28 Radiotraceurs pour tep WO2022255915A1 (fr)

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