WO2023109745A1 - 用于α-突触核蛋白聚集体成像的小分子探针 - Google Patents

用于α-突触核蛋白聚集体成像的小分子探针 Download PDF

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WO2023109745A1
WO2023109745A1 PCT/CN2022/138356 CN2022138356W WO2023109745A1 WO 2023109745 A1 WO2023109745 A1 WO 2023109745A1 CN 2022138356 W CN2022138356 W CN 2022138356W WO 2023109745 A1 WO2023109745 A1 WO 2023109745A1
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compound
synuclein
imaging
pharmaceutically acceptable
solvate
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French (fr)
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楚勇
王坚
边江
刘逸奇
林欣
邱辰旸
何洁
叶德泳
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复旦大学
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Definitions

  • the invention belongs to the technical field of medicine, and relates to a small molecular probe for imaging alpha-synuclein aggregates and its application.
  • ⁇ -synuclein lesion is an important pathogenesis of neurodegenerative diseases (Vekrellis, 2010).
  • An important pathological feature of Parkinson's disease (PD), Parkinsonian dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA) and other neurodegenerative disorders is the expression of ⁇ -synuclein. Abnormal aggregation, and then the formation of Lewy bodies and Lewy neurites, which are the main components, lead to pathogenesis.
  • ⁇ -synuclein deposition The process from the formation of ⁇ -synuclein deposition to the appearance of clinical symptoms is relatively long, usually lasting several years or even more than ten years, and it is too late to intervene when the patient has already developed clinical symptoms.
  • Early clinical intervention is extremely important to delay the progression of the disease and improve the quality of life and prognosis of patients. Therefore, the development of reliable early detection methods is very important for the early diagnosis, prevention and treatment of neurodegenerative diseases.
  • regulating the aggregation process of ⁇ -synuclein is also an important strategy for the treatment of these neurological diseases.
  • ⁇ -synuclein Based on its important role in the pathogenesis and progression of the above-mentioned various neurodegenerative diseases, ⁇ -synuclein has become an important biomarker for early diagnosis of these diseases and an important target for drug treatment.
  • the current detection of ⁇ -synuclein aggregates can only be based on histological analysis of autopsy materials, and non-invasive detection of living bodies cannot be performed.
  • the use of molecular imaging is the best way to solve this problem.
  • Molecular imaging is based on the specific binding of molecular tracer probes (such as radioactive tracer probes, fluorescent tracer probes, etc.) to biomarkers (e.g., receptors, enzymes, ion channels, misfolded proteins), Then it is visualized and imaged by PET, SPECT, nuclear magnetic resonance, near-infrared or other methods to provide diagnostic information of the living body.
  • biomarkers e.g., receptors, enzymes, ion channels, misfolded proteins
  • the imaging probe of a specific protein not only needs to have a strong enough affinity for the target protein aggregates, It must also be sufficiently selective for abnormal accumulations of other proteins to enable selective imaging.
  • few small molecule tracer probes capable of imaging ⁇ synuclein deposition in the brain of patients have been reported.
  • the purpose of the present invention is to provide a class of small molecule tracer probes capable of imaging ⁇ -synuclein aggregates, and small radionuclide-labeled probes for the imaging diagnosis of ⁇ -synuclein accumulation diseases.
  • Molecular Tracer Probes capable of imaging ⁇ -synuclein aggregates, and small radionuclide-labeled probes for the imaging diagnosis of ⁇ -synuclein accumulation diseases.
  • the small molecule tracer probe of the present invention can combine staining and imaging of ⁇ -synuclein lesions in the patient's brain, and its radiolabeled tracer probe can be used as an imaging tracer for PET and SPECT, thereby realizing In vivo non-invasive visual detection of ⁇ -synuclein lesions provides early diagnosis, disease monitoring and drug efficacy evaluation for patients with Parkinson's disease, Lewy body dementia, multiple system atrophy and other neurodegenerative diseases.
  • the present invention provides a compound represented by the following general formula I, a salt or a solvate thereof.
  • the compound has a strong affinity for ⁇ -synuclein aggregates, and has good blood-brain barrier permeability, and can specifically bind/stain Lewy bodies and Lewy neurite in the patient's brain tissue, and has A ⁇ and Tau lesions showed good selectivity. Therefore, the compound of the present invention can be used as a fluorescent imaging tracer of ⁇ -synuclein aggregates, or as a radiographic tracer required by imaging techniques such as PET and SPECT after radiolabeling, for in vivo (such as non-invasive visualization of ⁇ -synuclein pathology in the brain.
  • Ring A is selected from phenyl, pyridyl, pyrimidyl;
  • R 1 is selected from nitrogen-containing cycloalkane group containing 4-6 atoms, N, N-two C 1-3 alkyl substituted amino, C 1-3 alkoxy, nitric
  • Ring B is selected from pyridyl, piperazinyl, piperazinone;
  • R 2 is selected from H, halo, hydroxyl, C 1-3 alkyl, C 1-4 alkoxy, halogenated C 1-4 alkoxy;
  • the halogen atom is derived from fluorine, chlorine, bromine or iodine.
  • one or more atoms of the compound of formula I are radioactive isotopes of the atoms, and the radioactive isotopes are preferably
  • the present invention also provides a precursor compound for preparing the labeled compound of formula I, the structure of which is as follows:
  • R 3 is independently selected from hydroxyl, fluorine, bromine, iodine, nitro, borate, Ts O-(CH 2 ) m -, Ms O-(CH 2 ) m -, m is 0-4 Integer;
  • R 4 is a hydrogen atom, a C 1-3 alkyl group, or NR 4 R 4 is connected to form a nitrogen-containing cycloalkane containing 4-6 atoms.
  • One or more atoms in the compound of formula I can be labeled as a radionuclide of that atom by means of the above-mentioned precursor compounds. Therefore, the present invention also provides labeled compounds of formula I, preferably taken from the following structure:
  • the present invention also provides the use of the compound represented by formula I that can specifically bind to ⁇ -synuclein aggregates.
  • the compound has autofluorescence and can be used as a fluorescent imaging tracer of ⁇ -synuclein aggregates; when one or more atoms in the compound are replaced by radioactive atoms, it can be used as a variety of imaging techniques.
  • Radioactive imaging tracer probes for example, after one or more fluorine atoms or carbon atoms in the compound are replaced by radionuclide 18 F or 11 C, they can be used as imaging tracer probes for PET imaging techniques, or It is used for preparing the imaging tracking probe and the composition including the imaging tracking probe.
  • imaging tracers can be used to detect neurological diseases associated with ⁇ -synuclein misfolding and aggregation, or to screen for therapeutic or preventive drugs for diseases associated with ⁇ -synuclein aggregates in the brain, or It is used to quantify or determine the accumulation of ⁇ -synuclein aggregates in the brain.
  • PET Positron emission computed tomography
  • SPECT single photon emission computed tomography
  • the use of PET and SPECT radioactive tracer probes that specifically bind to a given molecular target can provide real-time diagnostic information that is closest to pathology in the living body, and prove and quantify the pathophysiological changes caused by the disease. It is an early clinical diagnosis and disease progression monitoring. and the most powerful tool for therapeutic drug development.
  • the radionuclides used in PET generally include 11 C, 13 N, 15 O, and 18 F, whose radioactive half-lives are 20 minutes, 10 minutes, 2 minutes, and 110 minutes, respectively.
  • 18 F has relatively the longest half-life and is the most convenient to use, 18 F is usually the best choice as the radionuclide for PET.
  • 99m Tc, 123 I, 131 I, 111 In are the most commonly used radionuclides for SPECT. In principle, these nuclides could be used to replace any corresponding non-radioactive isotopic atom in the target ligand molecule to render it radioactive.
  • the specific binding ligands of ⁇ synuclein aggregates can be labeled and used as tracking probes for in vitro autoradiography and in vivo PET or SPECT imaging, realizing Pathological imaging of ⁇ -synuclein in vitro and in vivo has greatly promoted the diagnosis, management, mechanism research and development of therapeutic drugs for neurological disorders related to ⁇ -synuclein misfolding and aggregation.
  • the key to imaging is to find small ligand molecules with high affinity and high selectivity for ⁇ -synuclein, and further, to label them with radionuclides as imaging probes for PET and SPECT.
  • the present invention provides a class of compounds with strong affinity and high specificity for ⁇ -synuclein aggregates and can penetrate the blood-brain barrier. These compounds are autofluorescent, can bind highly specifically and clearly stain Lewy bodies and Lewy neurites (the main component of which is ⁇ -synuclein aggregates) in patient brain tissue, and can be used as ⁇ - A fluorescent imaging imaging agent for synuclein.
  • radionuclide 18 F or 11 C When one or more fluorine atoms, or one or more carbon atoms in the compound of the present invention are replaced by radionuclide 18 F or 11 C, it can be used as an imaging tracer probe for autoradiography or PET Visualization of alpha-synuclein aggregates in vitro and in vivo, especially in the brain.
  • the halogen atoms in the compounds of the present invention are replaced by radioactive isotopes or other nuclides, they can be used as tracking probes for SPECT to visualize ⁇ -synuclein aggregates.
  • the present invention also provides the preparation method of the compound of formula I and its radiolabeled compound, as well as the precursor compound used for preparing the radiolabeled compound and its preparation method. Further, an imaging diagnosis method of the compound of formula I or its composition, a drug screening method for preventing or treating ⁇ -synuclein accumulation diseases, and the accumulation of ⁇ -synuclein in the brain are also provided. A method of quantification or determination.
  • Fig. 1 is a fluorescence microscope photograph of the staining result of the tracer probe of the present invention on the brain slice of a patient with dementia with Lewy bodies (DLB).
  • white arrows indicate Lewy bodies (left image) or Lewy neurites (right image). This result shows that the tracer probe of the present invention can strongly bind to the ⁇ -synuclein aggregate lesion in the patient's brain.
  • Fig. 2 is a fluorescent microscope photograph of the staining result of the tracer probe of the present invention on brain slices of Alzheimer's disease (AD) patients.
  • white arrows indicate A ⁇ plaques. This result shows that the tracer probe of the present invention has weak or even no binding to A ⁇ and Tau lesions in the patient's brain, and has good tissue target selectivity.
  • ⁇ -synuclein accumulation disease refers to diseases in which ⁇ -synuclein is abnormally folded and accumulated in the brain, including but not limited to Parkinson's disease (PD), Parkinson's disease mental disorder (PDD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), etc.
  • PD Parkinson's disease
  • PPD Parkinson's disease mental disorder
  • MSA multiple system atrophy
  • DLB dementia with Lewy bodies
  • the present invention uses the compound of general formula I, its salt or its solvate as an imaging tracer probe to image ⁇ -synuclein in patients with ⁇ -synuclein accumulation disease in vivo and in vitro, so as to provide Diagnosis and evaluation information of these diseases.
  • the tracer probe of the present invention that can be used for imaging diagnosis of ⁇ -synuclein accumulation disease is the compound represented by general formula I, or its salt, or its solvate.
  • Preferred compounds are I-1 to I-11.
  • I-4, I-5, and I-6 can well label the ⁇ -synuclein lesion Lewy bodies and Lewy neurites in the brain tissue of patients with dementia with Lewy bodies (DLB), and are effective for Alzheimer's disease (AD).
  • DLB dementia with Lewy bodies
  • AD Alzheimer's disease
  • the A ⁇ lesion in the patient's brain tissue only weakly binds, does not bind to the Tau lesion, and shows good binding specificity.
  • the present invention also includes the salts of the compounds of general formula I.
  • Nitrogen atoms or other functional groups in the compounds of general formula I can be used to form pharmaceutically acceptable salts.
  • any formula given herein is also intended to represent isotopically labeled forms of the compound. Its isotope-labeled compound has the structure shown in the chemical formula of formula I provided by the present invention, the difference is only that one or more atoms are replaced by its radioactive isotope.
  • Isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, chlorine and iodine such as 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, respectively , 35 S, 18 F, 36 Cl, 123 I and 125 I and 131 I.
  • Substitution with heavier isotopes may afford certain advantages resulting from greater metabolic stability (eg increased in vivo half-life or reduced dosage requirements).
  • Substitution with 2 H can be used in particular to prevent the formation of undesired radiometabolites or to block radiodefluorination.
  • 11 C, 13 N, 15 O, and 18 F are preferred for PET imaging among positron radioactive nuclides, 18 F is the most preferred, and 11 C is the second preferred labeling; Among the radionuclides, 123 I is preferred for SPECT imaging.
  • the present invention also encompasses radiolabeled compounds of general formula I.
  • any position of the compound of general formula I can be replaced by a radionuclide, but it is preferred to replace the halogen group, nitro group shown in the examples, or to label the alkyl group.
  • any position of the compound can be labeled with 18 F, preferably replacing the nitro group or fluorine atom in the compound with 18 F.
  • Radiolabeled compounds of the present invention and their required precursor compounds can generally be prepared by conventional schemes, or by schemes disclosed in the Examples, or by the following preparations by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. preparation.
  • Many methods have been reported to label 11 C, 15 N, 18 O, 18 F or other isotopes into compounds (Angew. Chem. Int. Ed. Miller, Philip W, 2008, 47, 8998-9033; Peter JHScott, 2009, 48, 6001-6004; Chem. Rev., Sean Preshlock, 2016, 116, 719-766; Frederic Dollé, Fluorine-18 chemistry for molecular imaging with positron emission tomography.
  • the radionuclide-labeled compound of formula I can be used as a PET or SPECT tracer probe for imaging ⁇ -synuclein accumulation in vivo.
  • the present invention also provides precursor compounds for the synthesis of compounds of formula I labeled with radionuclides.
  • Those skilled in the art can design and synthesize the precursor compound according to the structure shown in the present invention. That is, the precursor compound can be obtained by structurally modifying a commercially available compound or the compound of the present invention.
  • the radiolabeled compounds of the present invention can be synthesized from different precursor compounds.
  • the labeling position of the precursor compound contains hydroxyl or nitro, bromine, iodine, borate or other easy-leaving groups (such as MsO-, TsO-, etc.), which can be labeled by 11 C or 18 F, respectively. .
  • the methoxy group contained in the compound of formula I of the present invention can be demethylated to obtain a precursor compound containing a hydroxyl group, which can then be labeled with 11 C; or a brominated alkane that has been labeled with 18 F, such as 18 F-CH 2 CH 2 -Br undergoes an oxyalkylation reaction to generate 18 F-CH 2 CH 2 -O-substitution products, thereby realizing radiolabeling.
  • the precursor compound may also contain nitro, bromine, iodine, borate or TsO-, MsO-, and these groups can be replaced by 18 F according to known conventional methods.
  • synthesizing compounds such as I-5, I-6, I-10, etc., it is usually preferred to convert the position to be labeled in the precursor compound into a group containing TsO-, MsO-, etc. that are easy to leave.
  • the nuclide used for labeling is produced by a cyclotron, and those skilled in the art can select corresponding methods and instruments according to the nuclide to be produced. Methods for labeling compounds using these radionuclides are known in the art, mainly including chemical synthesis, isotope exchange and biosynthesis.
  • the radiolabeled compound of the present invention can be administered locally or systemically to the patient, and after a sufficient time of binding and dissociation with ⁇ -synuclein, the detection site can be visualized and imaged by PET or SPECT.
  • the route of administration can be subcutaneous, intraperitoneal, intravenous, arterial or intraspinal fluid injection or infusion, or oral, with due attention to the patient's exposure dose, and the specific use depends on the type of disease, the nuclide used, and the compound used , patient condition, detection site and other factors.
  • the present invention also provides a composition for imaging diagnosis of ⁇ -synuclein accumulation disease, which comprises the compound of the present invention, its pharmaceutically acceptable salt, or its solvate, and a pharmaceutically acceptable carrier.
  • a composition for imaging diagnosis of ⁇ -synuclein accumulation disease which comprises the compound of the present invention, its pharmaceutically acceptable salt, or its solvate, and a pharmaceutically acceptable carrier.
  • Compounds of the invention in preferred compositions are labeled, wherein labeling with radionuclides (especially positron-radiating nuclides 11 C, 13 N, 15 O, 18 F, etc.) is preferred for in vivo imaging diagnostics.
  • the compound of the present invention or a composition thereof is preferably in a form that allows injection.
  • pharmaceutically acceptable carriers are preferably liquids, including, but not limited to, aqueous solvents (such as potassium phosphate buffer, saline, Ringer's solution, and distilled water) or anhydrous solvents (such as polyethylene glycol, vegetable oils, ethanol , glycerin, dimethyl sulfoxide and propylene glycol).
  • aqueous solvents such as potassium phosphate buffer, saline, Ringer's solution, and distilled water
  • anhydrous solvents such as polyethylene glycol, vegetable oils, ethanol , glycerin, dimethyl sulfoxide and propylene glycol.
  • the formulation ratio of the carrier and the compound of the present invention can be appropriately selected, depending on the site of action, detection means, and the like.
  • composition of the present invention may contain commonly used antimicrobial agents (such as antibiotics, etc.), local anesthetics (such as procaine hydrochloride, tibucaine hydrochloride, etc.), buffers (such as trihydrochloride buffer, HEPES buffer, etc.) ), osmotic pressure regulators (such as glucose, sorbitol, sodium chloride, etc.), etc.
  • antimicrobial agents such as antibiotics, etc.
  • local anesthetics such as procaine hydrochloride, tibucaine hydrochloride, etc.
  • buffers such as trihydrochloride buffer, HEPES buffer, etc.
  • osmotic pressure regulators such as glucose, sorbitol, sodium chloride, etc.
  • Compounds of the invention may be labeled or unlabeled. When not labeled, the compound of the present invention can be labeled by the usual methods described above before use.
  • the compound of the present invention has the ability of highly specific binding to ⁇ -synuclein, so the labeled or unlabeled compound of the present invention can be used for staining and quantifying ⁇ -synuclein in vitro.
  • the compounds of the present invention have autofluorescent properties, they can be directly used to stain ⁇ -synuclein in specimens and observe through laser confocal or fluorescence microscopy, or to compare ⁇ -synuclein in samples. Quantification of ⁇ -synuclein using a scintillation counter after radiolabeling.
  • the early pathological basis of synuclein diseases such as Parkinson's disease, Lewy body dementia, multiple system atrophy, etc.
  • Lewy bodies is the formation of Lewy bodies, and its main component is abnormal accumulation of ⁇ -synuclein.
  • the detection of Lewy bodies can provide Early onset information for these diseases. Since the compound of the present invention can clearly stain Lewy bodies and Lewy neurites, it can be used for research on relevant pathological mechanisms and diagnosis before and after death of patients. Staining of brain sections with the compound of the present invention can be carried out by common methods.
  • the compound of the present invention that is, the compound represented by the general formula I or its salt or its solvate can be used as an imaging probe for ⁇ -synuclein accumulation, preferably using a radionuclide-labeled imaging probe. Needle.
  • the present invention provides:
  • Optical and radioactive tracer probes for imaging diagnosis of ⁇ -synuclein accumulation diseases especially imaging tracer probes labeled with positron radionuclides;
  • a composition for imaging and diagnosing ⁇ -synuclein accumulation diseases which comprises a compound of general formula I, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier;
  • the present invention also provides:
  • a method for the detection/staining of ⁇ -synuclein aggregates, ⁇ -synuclein lesion-depositing Lewy bodies and Lewy neurites in the patient's brain which can be used to provide evidence of ⁇ -synuclein accumulation disorders Early diagnosis and progression assessment information;
  • a method for screening drugs for the prevention and/or treatment of ⁇ -synuclein accumulation diseases
  • the above-mentioned methods all include using the compound of general formula I, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.
  • compound of formula I refers to any compound selected from the class of compounds defined by formula I, including stereoisomers, cis Transisomers, tautomers, solvates and salts (eg pharmaceutically acceptable salts).
  • the term "one or more" means from one substituent to the largest chemically possible number of substitution, ie replacement of one hydrogen to replacement of all hydrogens by a substituent.
  • substituted refers to an atom or group of atoms that replaces a hydrogen atom on a parent molecule.
  • halogen refers to fluorine (-F), chlorine (-Cl), bromine (-Br), and iodine (-I).
  • TsO- means "MsO-" means
  • C 1-4 alkoxy refers to a group of formula -O-R', wherein R' refers to a linear or branched saturated alkyl group containing 1 to 4 carbon atoms. Examples thereof include methoxy.
  • halogenated C 1-4 alkoxy denotes an alkoxy group in which one or more hydrogen atoms of the alkoxy group have been replaced by the same or different halogen atoms (especially fluorine atoms). Examples thereof include 1-fluoroethoxy.
  • C 1-3 alkyl means a linear or branched saturated hydrocarbon group containing 1 to 3 carbon atoms. Examples thereof include methyl.
  • aromatic denotes the general concept of aromaticity as defined eg in the literature (in particular IUPAC - Catalog of Chemical Terms 2nd Edition, A.D. McNaught & A. Wilkinson. Blackwell Scientific Publications, Oxford (1997)).
  • pharmaceutically acceptable salt refers to a salt that is not harmful to mammals, especially humans.
  • Pharmaceutically acceptable salts may be formed using non-toxic acids or bases comprising inorganic acids or bases, or organic acids or bases.
  • examples of pharmaceutically acceptable salts include: metal salts formed with aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc; or lysine, N,N'-dibenzylethylenediamine , chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and organic salts formed by procaine, etc.
  • the pharmaceutically acceptable salts include acid addition salts and base addition salts.
  • pharmaceutically acceptable carrier refers to physiological saline solution; liquid or solid fillers, diluents, solvents, or packaging materials that are pharmaceutically acceptable materials, compositions, or excipients.
  • pharmaceutically acceptable carriers include water, saline, normal saline or phosphate-buffered saline (PBS), sodium chloride injection, Ringer's injection, dextrose injection, sterile water injection, dextrose, And Lactated Ringer's Injection, etc.
  • solvate refers to a solvent-containing compound formed by the association of one or more solvent molecules with a compound.
  • monosolvates, disolvates, trisolvates, and tetrasolvates may be included.
  • solvates also include hydrates.
  • hydrate refers to a compound or a salt thereof containing water bound by non-covalent intermolecular forces, and the amount of water contained may be stoichiometric or non-stoichiometric. For example, monohydrate, dihydrate, trihydrate, and tetrahydrate etc. are contained.
  • the tracer probe (hereinafter, also referred to as tracer probe) of ⁇ -synuclein aggregate provided by the present invention contains the compound shown in the following formula I, its pharmaceutically acceptable salt, or its solvate .
  • the compound represented by the following formula I has autofluorescence.
  • one or more atoms of the compound may be the radioactive isotope of the atom. Therefore, the compound of the present invention can be used as a small molecule tracking probe for optical imaging of ⁇ -synuclein aggregates, or PET and SPECT imaging after radiolabeling.
  • Ring A is selected from phenyl, pyridyl, pyrimidyl;
  • R 1 is selected from nitrogen-containing cycloalkane group containing 4-6 atoms, N, N-two C 1-3 alkyl substituted amino, C 1-3 alkoxy, nitric
  • Ring B is selected from pyridyl, piperazinyl, piperazinone;
  • R 2 is selected from H, halo, hydroxyl, C 1-3 alkyl, C 1-4 alkoxy, halogenated C 1-4 alkoxy;
  • the halogen atom is taken from fluorine, chlorine, bromine or iodine;
  • one or more atoms of the compound of formula I are radioactive isotopes of the atoms, and the radioactive isotopes are preferably taken from 11 C, 13 N, 15 O, 18 F, 76 Br, 123 I, 125 I, 131 I.
  • the atoms marked with * in the above specific compound structural formula may be the radioactive isotope of the atom, such as 11 C or 18 F.
  • F in the specific compounds above is the radioisotope18F ; preferably, the carbon atom of the methoxy or dimethylamino group linked to the aryl group is the radioisotope11C .
  • composition for optical imaging of ⁇ -synuclein aggregates of the present invention comprises the compound of formula I above, its pharmaceutically acceptable salt, or its solvate.
  • the optical imaging includes in vitro imaging, in vitro imaging, and in vivo imaging.
  • the optical imaging methods include, but are not limited to, fluorescence microscopy, multiphoton imaging, two-photon imaging, and near-infrared fluorescence imaging.
  • composition for radiographic imaging of ⁇ -synuclein aggregates of the present invention comprises a radiolabeled compound of formula I, a pharmaceutically acceptable salt thereof, or a solvent thereof compounds.
  • the radiographic imaging includes in vitro imaging, in vitro imaging, and in vivo imaging.
  • the radiographic methods include, but are not limited to, PET, SPECT, autoradiography.
  • compositions for optical imaging and the composition for radiographic imaging can be contained in the aforementioned pharmaceutically acceptable carrier.
  • the content of the compound of formula I contained therein, its pharmaceutically acceptable salt, or its solvate, and the pharmaceutically acceptable carrier are not particularly limited, and can be determined according to: the compound used; the mammal to be administered The age, weight, health status, gender and meal content of the patient; the frequency and route of administration; the treatment period; other drugs used at the same time should be adjusted.
  • the diagnostic drug of the ⁇ -synuclein aggregate-related disease of the present invention, or the companion diagnostic drug for the treatment or prevention of the disease includes the compound of the present invention.
  • the therapeutic accompanying diagnostic drug refers to a diagnostic drug used to judge whether or not treatment is expected when the above-mentioned disease is identified.
  • the prophylactic companion diagnostic drug refers to a diagnostic drug for predicting future onset or for judging whether preventive onset suppression is expected when the precursor symptoms of the above-mentioned disease are known.
  • the relevant data on the amount and/or distribution of ⁇ -synuclein aggregates in the subject obtained by using the above-mentioned diagnostic drugs or companion diagnostic drugs, and the previously known relationship between the above-mentioned diseases and ⁇ -synuclein
  • the subject can be diagnosed with the above-mentioned diseases (specifically, such as whether he suffers from the above-mentioned diseases, severity, possibility of attack, etc.) ); or understand the above-mentioned disease state of the subject, and based on this, formulate a prevention/treatment plan for the above-mentioned disease (types of preventive administration/treatment drugs and their combinations, dosage, usage, etc.).
  • the optical imaging method of the present invention includes the following steps.
  • the method for detecting ⁇ -synuclein aggregates in the brain is used as an example for illustration, and the method for detecting other parts is similar.
  • the tracer probe of the present invention When an effective amount of the tracer probe of the present invention is administered to a test organism, the tracer probe that reaches the brain of the organism will bind to ⁇ -synuclein aggregates in the brain. Then, the light of the first wavelength for exciting the tracer probe is irradiated from outside the brain, and the light of the second wavelength (such as fluorescence) emitted from the tracer probe in the brain is detected, thereby achieving detection of ⁇ -synuclein aggregates Optical visualization imaging (picture).
  • the tracer probe comprises a compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof.
  • the radiation imaging method of the present invention includes the following steps, which are illustrated below by using the method for detecting ⁇ -synuclein aggregates in the brain, and the method for detecting other parts is similar.
  • the tracking probe When an effective amount of the radiolabeled tracking probe of the present invention is administered to a test organism, the tracking probe that reaches the brain of the organism will bind to ⁇ -synuclein aggregates in the brain. Then, radiation emitted from the tracer probe in the brain is detected by PET or SPECT, thereby realizing radiographic imaging (imaging) of ⁇ -synuclein aggregates.
  • the tracer probe comprises a compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein one or more atoms of the compound of formula I are radioactive isotopes of the atom.
  • optical imaging and radioimaging test organisms include mammals, such as human, rat, mouse, rabbit, guinea pig, hamster, monkey, dog, mink, or miniature pig.
  • the mammal is a human.
  • the administration method of the tracer probe is not particularly limited, and it can be administered orally, intravenously or intraperitoneally. Intravenous or intraperitoneal injection is preferred, and intravenous injection is most preferred.
  • the accumulation of synuclein is quantified and the presence or absence of accumulation of alpha-synuclein aggregates in vivo (eg, in the brain) is determined.
  • the screening drug Based on the imaging method described in [Optical Imaging Method] or [Radiographic Imaging Method] above, detect the light or radiation emitted by the test organism before and after administration of the screening drug, and judge according to the difference in intensity and/or distribution Changes in alpha-synuclein accumulation to screen for therapeutic or preventive drugs. For example, after administration of a screening drug, if the amount (intensity) of light (such as fluorescence) or radiation from the tracer probe is reduced compared to before administration of the screening drug, the screening drug may be used as a drug for the treatment or prevention of the disease or condition .
  • the The screened drug may be used as a treatment or preventive drug for the disease or condition.
  • test organism The species of the test organism and the method of administration are the same as described above in [Optical Imaging Method] and [Radiographic Imaging Method].
  • the compound of the present invention can be synthesized from known materials (for example, commercially available materials) by known methods. Those skilled in the art can appropriately select starting materials and synthesis methods according to the desired compound of the present invention. The present invention will be further described below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, conventional conditions or the conditions suggested by the manufacturer are usually adopted.
  • the known starting materials of the present invention can be used or prepared according to methods known in the art, or purchased from commercial products. Compound structures were determined by nuclear magnetic resonance spectroscopy (NMR) and/or mass spectroscopy.
  • Embodiment 1 the preparation of compound 1-1, as follows:
  • Embodiment 2 the preparation of compound 1-2, its structure is as follows:
  • step a 2-amino-5-trifluoromethylpyridine is replaced by 2-amino-5-trifluoromethylbenzene to obtain a yellow solid with a yield of 45%.
  • Embodiment 3 the preparation of compound 1-3, its structure is as follows:
  • Embodiment 4 the preparation of compound 1-4, as follows:
  • Embodiment 5 the preparation of compound 1-5, as follows:
  • Embodiment 6 the preparation of compound 1-6, as follows:
  • Embodiment 7 be the preparation of compound 1-7, as follows:
  • Embodiment 8 the preparation of compound 1-8, as follows:
  • Embodiment 9 the preparation of compound 1-9, its structure is as follows:
  • Embodiment 10 the preparation of compound 1-10, as follows:
  • Labeling of various radionuclides can be performed by conventionally known methods.
  • the following uses the preparation of ( 18 F)I-5, ( 18 F)I-10 and ( 11 C)I-4 as an example to illustrate the methods of labeling 18 F and 11 C respectively.
  • Other radioactive tracer probes can be prepared in the same way. Method preparation.
  • Labeling of the radionuclide18F can be performed by a number of different precursor compounds as shown below.
  • the synthesis methods of four precursor compounds are exemplified below, but not limited thereto.
  • Example 5 replace 2-fluoro-5-pyridineboronic acid with 2-nitro-5-pyridineboronic acid, 2-bromo-5-pyridineboronic acid or 2-hydroxyl-5-pyridineboronic acid, respectively Preparation of nitro-containing precursor compound I-5N, bromine-containing precursor compound I-5B, and hydroxyl-containing precursor compound I-5H.
  • the bromine-containing precursor I-5B can be coupled with pinacol borate under palladium catalysis to prepare more active borate-containing precursor compound I-5O, hydroxyl-containing precursor compound I-5H and p-Toluenesulfonyl chloride (TsCl) reacts under basic conditions to generate TsO-group-containing precursor compound I-5T.
  • TsCl p-Toluenesulfonyl chloride
  • Method 1 Synthesis from borate-containing precursor I-5O. 18 F- is produced by a cyclotron, then adsorbed by QMA, and the K 222 /K 2 CO 3 eluent is extruded from the No. 1 bottle to elute 18 F ions into the reaction tube, and evaporated to dryness at 116°C under nitrogen flow.
  • the No. 2 bottle solution (2 mL of acetonitrile) was injected into the reaction tube, and the water was removed by azeotropic evaporation at 116° C. under nitrogen flow. The reaction tube was cooled for 60 s.
  • the No. 1 Synthesis from borate-containing precursor I-5O. 18 F- is produced by a cyclotron, then adsorbed by QMA, and the K 222 /K 2 CO 3 eluent is extruded from the No. 1 bottle to elute 18 F ions into the reaction tube, and evaporated to dryness at 116°C under nitrogen flow.
  • Method 2 Synthesis from nitro-containing precursor I-5N. After dissolving ( 18 F) fluoride ions into a 50% acetonitrile solution (0.4 mL) containing K 222 (Kryptofix 222) (7.5 mg) and potassium carbonate (2.77 mg), and introducing the solution into the reaction vessel, Heating under nitrogen flow allowed the solvent to dry and solidify. Anhydrous acetonitrile (0.1 mL) was added for azeotropic distillation to fully dry the inside of the reaction vessel. A solution of nitro precursor compound I-5N (1 mg) in DMSO (300 ⁇ L) was added to the reaction vessel, and heated at 110° C. for 10 minutes. After cooling, it was separated and purified by HPLC to obtain the pure product of ( 18 F)I-5.
  • bromine-containing precursor I-5B and the TsO-group-containing precursor I-5T can also be labeled with 18 F under the similar conditions of the above method 2 to synthesize ( 18 F)I-5.
  • the radioactive tracer probe ( 18 F)I-10 can be prepared by direct nitrogen alkylation of I-9 with 18 F-labeled bromoalkane 18 F-CH 2 CH 2 -Br. Or react I-9 with 1,2-bis(toluenesulfonyloxy)ethane to generate the precursor compound I-10T containing leaving group TsO-; also react I-9 with ethylene oxide to generate The precursor compound I-10O containing a terminal hydroxyl group is reacted with p-toluenesulfonyl chloride (TsCl) or methanesulfonyl chloride (MsCl) under basic conditions to generate a labeling site as an easily leaving group TsO-, or Precursor compounds of MsO- (such as I-10T). Finally, I-10T was reacted with radioactive K 18 F to generate radioactive tracer probe ( 18 F)I-10.
  • TsCl p-toluenesulfonyl chloride
  • the binding activity of the compounds of the present invention to human ⁇ -synuclein aggregates was determined by the fluorescence method described below.
  • Bacteria were collected by centrifugation, ultrasonically crushed and then centrifuged at high speed for 30 minutes, the supernatant was collected, DNA and foreign proteins were removed by Ni-NTA affinity column chromatography, and then purified by molecular exclusion chromatography to obtain ⁇ -synuclein monomer. Purity was verified by SDS-PAGE discontinuous electrophoresis.
  • ⁇ -synuclein monomer into a Buffer solution containing 1 ⁇ PBS, in which the final protein concentration is 100 ⁇ M (about 5 mg/mL), and incubate at 37°C in a 1000 rpm shaker for 7 days to obtain ⁇ -synuclein Aggregates. Both initial protein monomer concentration and final concentration were accurately determined by BCA method.
  • Example compound Kd ( ⁇ M) Example compound Kd ( ⁇ M) Example compound Kd ( ⁇ M) Example compound Kd ( ⁇ M) I-1 * I-2 * I-4 *** I-5 ** I-6 ** I-10 **
  • the brain slices of a patient with dementia with Lewy bodies were obtained from the amygdala tissue of a 75-year-old male deceased who suffered from stage 2 dementia with Lewy bodies. Cryosections of amygdala tissues enriched in ⁇ -synuclein lesions were performed at a thickness of 20 ⁇ m.
  • the compound to be tested was diluted to 30 ⁇ M with 50% EtOH-containing PBS solution, incubated with the obtained fresh frozen brain slices at room temperature for 30 minutes, then washed with 50% ethanol solution for 5 minutes, and then washed twice with ultrapure water, each 3 minutes each time. After embedding the slices with embedding agent (VECTASHIELD H-1000, Vector Laboratories), photographs were taken through a fluorescence microscope to obtain images of the lesion accumulation area on the slices. Binding selectivity was assessed using analysis software (Image J) to quantify the fluorescence intensity of the lesion area and the lesion non-formation area (background).
  • image J analysis software
  • Fluorescent image results show that compounds I-4, I-5, and I-6 can clearly stain Lewy bodies and Lewy neurites in the brain slices of patients with dementia with Lewy bodies (accompanying drawing 1), indicating that they can all be combined with Lewy bodies in the patient's brain. Strong binding occurs for ⁇ -synuclein lesions.
  • Alzheimer's patient brain slices were obtained from the postmortem superior temporal gyrus of a stage 3 patient.
  • the dewaxed brain tissue was fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned with a thickness of 6 ⁇ m.
  • the detection method is the same as the above method for the brain slices of patients with dementia with Lewy bodies (DLB).
  • the results of fluorescence images are shown in Figure 2.
  • Compounds I-4, I-5, and I-6 can also detect A ⁇ lesions in brain slices of AD patients without binding to Tau neurofilaments.
  • the staining signals of these compounds were far weaker than those in the brain slices of DLB patients, indicating that their binding to A ⁇ and Tau pathological tissues was very weak, or even not binding.
  • the compounds of the present invention were injected into the tail vein of rats to determine their blood-brain barrier permeability in vivo.
  • the blood taken out was centrifuged at 9000rpm for 5min, 200 ⁇ L of supernatant was taken, 800 ⁇ L of methanol was added, centrifuged at 14000rpm for 10min, the supernatant was passed through a 0.22 ⁇ m filter membrane, and stored at -80°C for later use.
  • LC-MS/MS was used to detect the concentration of the compounds in the blood sample and the brain homogenate supernatant sample respectively.
  • the brain/blood ratio is ⁇ 0.1, 0.1-0.3 or >0.3, it means that the degree of compound penetration through the blood-brain barrier is difficult, moderate or good, respectively.
  • the test results show that the brain/blood ratio of compounds I-4, I-5 and I-6 of the present invention is close to 1.0 or greater than 1.0, which proves that they all have good blood-brain barrier penetration ability. Since the compounds of the present invention have similar structures and the clogP values are basically between 1.0 and 3.0, it can be predicted that other compounds of the present invention should also have acceptable blood-brain barrier penetration.
  • the probe compound capable of binding/staining ⁇ -synuclein accumulation of the present invention and its composition are extremely important for the early detection, treatment and prevention of difficult diseases such as Parkinson's disease, one of the current very important medical difficulties, and It has a high possibility of being used in the medical field.
  • the compound of the present invention can be used as an imaging tracer for imaging the accumulation of ⁇ -synuclein, thereby being able to treat various neurodegenerative diseases, such as Parkinson's disease (PD), Parkinson's disease dementia (PDD) ), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), etc. provide early diagnosis and disease progression information.
  • PD Parkinson's disease
  • PDD Parkinson's disease dementia
  • DLB dementia with Lewy bodies
  • MSA multiple system atrophy

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Abstract

提供一类如通式I所示的能特异性结合α-突触核蛋白聚集体的化合物、其放射性标记的化合物、制备方法及用途。所述化合物可作为α-突触核蛋白聚集体的小分子探针，用于生物样本或生物体内（如脑中）的α-突触核蛋白聚集体的可视化成像检测，其放射性标记的化合物可用作临床疾病诊断的PET、SPECT等影响检查技术所需的显像示踪探针，实现对生物体内（如脑中）的α-突触核蛋白病变的非侵入性的可视化成像检测。

Description

用于α-突触核蛋白聚集体成像的小分子探针 技术领域
本发明属于医药技术领域，涉及用于α-突触核蛋白聚集体成像的小分子探针及其用途。
背景技术
α-突触核蛋白(α-synuclein,α-Syn)病变是神经变性疾病的重要发病机制(Vekrellis，2010)。帕金森病(PD)、帕金森病性失智症(PDD)、路易体痴呆症(DLB)、多***萎缩(MSA)及其它神经变性病症的重要病理特征即为α-突触核蛋白的异常聚集，进而形成以其为主要成分的路易小体和路易神经突并导致发病。从α-突触核蛋白沉积形成到出现临床症状的过程较为漫长，通常持续数年甚至十年以上，而当病人已经出现临床症状时再行干预则为时已晚。越早的临床干预对延缓病程进展、改善患者的生活质量和预后都极为重要。因此，发展可靠的早期检测方法对神经变性疾病的早期诊断、预防和治疗十分重要。同时，调节α-突触核蛋白的聚集进程也是治疗这些神经疾病的重要策略。
基于在上述多种神经退行性疾病发病及病程进展中的重要作用，α-突触核蛋白已成为针对这些疾病进行早期诊断的重要生物标志物和药物治疗的重要靶点。但目前对α-突触核蛋白聚集体的检测只能基于尸检材料的组织学分析，还不能针对活体进行非侵入性的检测，运用分子影像学是解决这一难题的最佳方法。
分子成像学是基于分子示踪探针(例如放射性示踪探针、荧光示踪探针等)对生物标志物(例如，受体、酶、离子通道、错误折叠的蛋白质)的特异性结合，再通过PET、SPECT、核磁共振、近红外或其他方法对其进行可视化成像，从而提供活体的诊断信息。要实现分子影像其关键是必须具有能对给定分子靶标特异结合的小分子化合物作为显像示踪探针。由于神经退行性病人脑中常常共沉积α‐突触核蛋白、Aβ和Tau蛋白的病理改变，因此，某一特定蛋白的显像探针不仅需要对该靶蛋白聚集体有足够强的亲和力，还必须对其他蛋白的异常积聚体具有足够高的选择性，才能实现选择性成像。迄今为止，鲜见报道能显像病人脑内α‐突触核蛋白沉积的小分子示踪探针。
发明的公开
本发明的目的是提供一类能够显像α‐突触核蛋白聚集体的小分子示踪探针、以及用于α‐突触核蛋白积聚性疾病的成像诊断的被放射核素标记的小分子示踪探针。本发明的小分子示踪探针能够对病人脑内的α-突触核蛋白病变结合染色成像，其放射标记的示踪探针可用作PET、SPECT的显像示踪剂，从而实现对α-突触核蛋白病变的活体非侵入性的可视化检测，对帕金森病、路易体痴呆症、多***萎缩等神经退行性疾病患者提供早期诊断、疾病监测和药物疗效评估。
为达到上述目的，本发明提供了如下通式I所示的化合物、其盐或其溶剂化物。该化合物对α-突触核蛋白聚集体具有强亲和力，并具有良好的血脑屏障透过性，尤其能特异性地对病人脑组织中的路易体和路易神经突进行结合/染色，且对Aβ和Tau蛋白病变显示出良好的选择性。因此，本发明化合物可用作α-突触核蛋白聚集体的荧光成像示踪剂，或经放射标记后用作PET、SPECT等影像技术所需的放射显像示踪剂，对体内(如脑中)α‐突触核蛋白的病理进行非侵入性的可视化成像。
Figure PCTCN2022138356-appb-000001
其中，
环A选自苯基、吡啶基、嘧啶基；
R 1选自含4-6个原子的含氮环烷烃基、N，N-双C 1-3烷基取代的氨基、C 1-3烷氧基、硝
基、卤素基；
环B选自吡啶基、哌嗪基、哌嗪酮基；
R 2选自H、卤素基、羟基、C 1-3烷基、C 1-4烷氧基、卤代的C 1-4烷氧基；
其中，所述卤素原子取自氟、氯、溴或碘。
其中，式I化合物的1个或1个以上原子是该原子的放射性同位素，该放射性同位素优
选取自 11C、 13N、 15O、 18F、 76Br、 123I、 125I、 131I。
本发明还提供了用于制备被标记的式I化合物的前体化合物，其结构如下所示：
Figure PCTCN2022138356-appb-000002
其中，R 3独立地取自羟基、氟、溴、碘、硝基、硼酸酯基、Ts O-(CH 2) m-、Ms O-(CH 2) m-，m为0～4的整数；R 4为氢原子、C 1-3烷基，或NR 4R 4连接成含4-6个原子的含氮环烷烃。通过上述前体化合物，可将式I化合物中的1个或多个原子标记为该原子的放射性核素。因此，本发明还提供了被标记的式I化合物，优选地取自如下结构:
Figure PCTCN2022138356-appb-000003
其中，具有*的原子中至少有一个是该原子的放射性同位素。
本发明还提供了式I所示能特异性结合α-突触核蛋白聚集体的化合物的用途。该化合物具有自发荧光，可用作α-突触核蛋白聚集体的荧光显像示踪剂；当该化合物中1个或多个原子被放射性原子取代后可用作多种影像技术所需的放射性显像示踪探针，例如该化合物中1个或多个氟原子或碳原子被取代为放射性核素 18F或 11C后，可用作PET影像技术的显像示踪探针，或用于制备该显像示踪探针，以及制备包括该显像示踪探针的组合物。这些显像示踪剂可用于检测与α-突触核蛋白错误折叠和聚集相关的神经疾病、或用于筛选与脑内α-突触核蛋白聚集体相关的疾病的治疗或预防药物，或用于对脑内α-突触核蛋白聚集体的积聚进行定量或判定。
本发明的有益效果：
正电子发射计算机断层显像(PET)和单光子发射计算机断层显像(SPECT)技术是当前最先进的非侵入性的三维显像技术。运用对给定分子靶标特异结合的PET、SPECT放射性示踪探针，能够实时提供活体最接近于病理学的诊断信息，证明和量化由于疾病产生的病理生理变化，是临床早期诊断，疾病进展监测及治疗药物开发的最有力工具。用于PET的放射性核素通常包括 11C、 13N、 15O和 18F，其放射性半衰期分别为20分钟、10分钟、2分钟和110分钟。由于 18F具有相对最长的半衰期，使用最为方便，故通常最优选择 18F作为PET的放射核素。此外， 99mTc, 123I, 131I, 111In是最常用于SPECT的放射核素。原则上，可以使用这些核素替换靶标配体分子中的任一对应的非放射性同位素原子，以使其具有放射性。
因此，当标记上放射性核素后，α‐突触核蛋白聚集体的特异结合配体就可以对其进行标记，用作体外放射自显影和体内PET或SPECT显像的示踪探针，实现体内外α‐突触核蛋白的病理学成像，极大促进对α‐突触核蛋白错误折叠和聚集相关的神经病症的诊断、管理、机制研究和治疗药物的发展。显然，实现显像的关键在于发现对α‐突触核蛋白具有高亲和力和高选择性的配体小分子，进一步地，将其标记上放射核素作为PET、SPECT的显像探针。
本发明提供了一类对α-突触核蛋白聚集体具有强亲和力和高度特异性、并能透过血脑屏障的化合物。这些化合物具有自发荧光，能对病人脑组织中的路易体和路易神经突(其主要成分为α-突触核蛋白聚集体)发生高度特异性的结合并显示出清晰的染色，可用作α-突触核蛋白的荧光成像显像剂。当将本发明化合物中的1个或多个氟原子、或1个或多个碳原子替换为放射性核素 18F或 11C，可用作放射自显影或PET的显像示踪探针进行体外、体内(尤其是脑中)α-突触核蛋白聚集体的可视化成像。当将本发明化合物中的卤素原子替换为放射性的同位素或其他核素时，可用作SPECT的示踪探针以显像α-突触核蛋白聚集体。
本发明还提供了式I化合物及其放射标记化合物的制备方法，以及用于制备放射标记化合物的前体化合物及其制备方法。进一步地，还提供了式I化合物或其组合物的成像诊断方法、用于预防或治疗α‐突触核蛋白积聚性疾病的药物的筛选方法，及对脑内α‐突触核蛋白的积聚进行定量或判定的方法。
附图的简要说明
图1为本发明示踪探针对路易体痴呆症(DLB)病人脑片的染色结果的荧光显微镜拍照。图中，白色箭头表示路易体(左图)或路易神经突(右图)。该结果表明本发明示踪探针能与病人脑中的α-突触核蛋白聚集体病变发生强结合。
图2为本发明示踪探针对阿尔茨海默症(AD)病人脑片的染色结果的荧光显微镜拍照。图中，白色箭头表示Aβ斑块。该结果表明本发明示踪探针对病人脑中的Aβ和Tau病变结合很弱甚至不结合，具有良好的组织靶标选择性。
实现本发明的最佳方式
在本说明书中，“α‐突触核蛋白积聚性疾病”是指α‐突触核蛋白在大脑内异常折叠和积聚的疾病，包括但不限于帕金森病(PD)、帕金森病性失智症(PDD)、多***萎缩(MSA)、路易体痴呆症(DLB)等。本发明通过在α‐突触核蛋白积聚性疾病患者体内外使用通式I化合物、其盐或其溶剂化物，作为显像示踪探针对α‐突触核蛋白进行显像，以提供对这些疾病的诊断、评估信息。
本发明的可被用作成像诊断α-突触核蛋白积聚性疾病的示踪探针是通式I表示的化合物，或其盐，或其溶剂化物。优选的化合物为I-1～I-11。其中，I-4、I-5、I-6能良好标记路 易体痴呆症病人(DLB)脑组织中的α-突触核蛋白病变路易体和路易神经突，对阿尔茨海默症(AD)病人脑组织中的Aβ病变仅有弱结合，不结合其中的Tau病变，表现出了良好的结合特异性。
本发明还包括通式I化合物的盐。通式I化合物中的氮原子或其他官能团可以用于形成医药上可接受的盐。
本发明给出的任何化学式还旨在表示该化合物的同位素标记的形式。其同位素标记化合物具有由本发明给出的式I化学式所示结构，不同之处仅为其中的一个或多个原子被其放射性同位素替代。可以掺入本发明化合物的同位素包括氢、碳、氮、氧、氟、氯和碘的同位素，分别诸如 2H、 3H、 11C、 13C、 14C、 15N、 18O、 17O、 35S、 18F、 36Cl、 123I和 125I和 131I。用较重的同位素(诸如氘， 2H))取代可以提供某些由更大的代谢稳定性(例如增加的体内半衰期或降低的剂量需求)产生的优势。用 2H取代可以特别地用于防止形成不希望的放射代谢物或者阻断放射脱氟。用于标记本发明化合物时，在正电子放射性核素中优选 11C、 13N、 15O、 18F用于PET显像，最优选使用 18F，次优选使用 11C进行标记；在γ射线放射性核素中优选 123I用于SPECT显像。
本发明还包含通式I的放射标记的化合物。理论上，通式I化合物的任何位置都可被放射性核素替换，但优选对实施例中所示的卤素基、硝基进行取代，或对烷基进行标记。例如，当用 18F标记本发明的化合物时，可以用 18F标记化合物的任何位置，优选用 18F替换化合物中的硝基或氟原子。
本发明的放射标记的化合物及其所需的前体化合物通常可以通过常规方案，或实施例中公开的方案，或下述制备方法(通过用易得的同位素标记试剂替代非同位素标记试剂)而制备。已报道了诸多方法可将 11C、 15N、 18O、 18F或其他同位素标记到化合物中(Angew.Chem.Int.Ed.Miller,Philip W,2008,47,8998-9033；Peter J.H.Scott，2009,48,6001-6004；Chem.Rev.,Sean Preshlock,2016,116,719-766；Frederic Dollé,Fluorine-18 chemistry for molecular imaging with positron emission tomography.Fluorine and Health：Molecular Imaging，Biomedical Materials and Pharmaceuticals(Tressaud，A.Haufe，G.),2008,pp.3-66，Elsevier)。经过放射性核素标记后的式I化合物可用作PET或SPECT的示踪探针进行活体中α‐突触核蛋白积聚体的显像。
本发明还提供了前体化合物以用于合成被放射核素标记的式I化合物。本领域技术人员可以根据本发明所示结构设计并合成该前体化合物。即，该前体化合物可以通过对市售化合物或对本发明化合物进行结构修饰而得到。
本发明的放射标记化合物可以通过不同的前体化合物合成。通常地，前体化合物的标记位置含有羟基或硝基、溴、碘、硼酸酯或其他易离去基团(如MsO-、TsO-等)，从而可以分别被 11C或 18F所标记。特别地，本发明式I化合物中所含的甲氧基可通过脱除甲基获得含有羟基的前体化合物，然后可用 11C进行标记；或用已被 18F标记的溴代烷烃，如 18F-CH 2CH 2-Br进行氧烷基化反应，生成 18F-CH 2CH 2-O-取代产物，从而实现放射标记。同样地，前体化合物中也可以含有硝基、溴、碘、硼酸酯或TsO-、MsO-，这些基团都能按已知的常规方法被 18F替换。在合成如I-5、I-6、I-10等化合物时，通常优先将前体化合物中需标记的位置转变成含易离去的TsO-、MsO-等基团。
通常，用于标记的核素由回旋加速器产生，本领域熟练的技术人员可以根据所要制造的核素来选择相应方法和仪器。使用这些放射性核素进行化合物标记的方法在本领域是已知的，主要包括化学合成法、同位素交换法和生物合成法。放射标记的本发明化合物可以局部地或者全身性地给予患者，经过与α‐突触核蛋白结合和解离的充分时间后，通过PET、SPECT即可对检测部位进行可视化成像。给药途径可采用皮下、腹腔、静脉、动脉或脊髓液内的注 射或输液或经口服，并充分关注患者的暴露剂量，具体使用方式取决于疾病类型、所使用的核素、所使用的化合物、患者状况、检测部位等因素。
本发明还提供用于成像诊断α-突触核蛋白积聚性疾病的组合物，其包含本发明的化合物、其在医药上可接受的盐、或其溶剂化物及医药上可接受的载体。优选组合物中的本发明化合物已被标记，其中使用放射性核素(尤其是正电子放射性核素 11C、 13N、 15O、 18F等)进行标记对于体内成像诊断是优选的。根据其用途，优选本发明化合物或其组合物的形态是一种允许用于注射的形态。因此，医药上可接受的载体优选为液体，包括(但不限于)含水溶剂(如磷酸钾缓冲剂、盐水、林格氏溶液和蒸馏水)或无水溶剂(例如聚乙二醇、植物油、乙醇、甘油、二甲基亚砜和丙二醇)。可以适当选择载体和本发明化合物的配制比例，其取决于作用的部位、检测手段等。此外，本发明组合物可以包含常用的抗微生物剂(如抗菌素等)、局部麻醉剂(如盐酸普鲁卡因、盐酸待布卡因等)、缓冲液(如三盐酸缓冲液、HEPES缓冲液等)、渗透压调节剂(如葡萄糖、山梨糖醇、氯化钠等)等。
本发明化合物可以是标记的或未被标记的。当未被标记时，可以在使用前通过以上描述的常用方法对本发明化合物进行标记。
本发明化合物具有与α-突触核蛋白高度特异性结合的能力，因此可通过标记或未标记的本发明化合物，用于在体外对α-突触核蛋白的染色和定量。例如，由于本发明化合物具有自发荧光特性，因此可被直接用于染色标本中的α-突触核蛋白并通过激光共聚焦或荧光显微镜观察，或用于样本中α-突触核蛋白的比色定量，或经放射标记后使用闪烁计数器用于α-突触核蛋白的定量。共核蛋白病如帕金森病、路易体痴呆症、多***萎缩等的早期病理基础都是形成路易体，其主要成分是异常积聚的α‐突触核蛋白，通过对路易体的检测可以提供这些疾病的早期发病信息。由于本发明化合物能清晰染色路易体和路易神经突，因此可用于研究相关病理机制及患者死亡前后的诊断。使用本发明化合物对大脑切片进行染色可以通过常用方法来进行。
如上所述，本发明化合物，即通式I所示的化合物或盐或其溶剂化物可以用作α-突触核蛋白积聚体的显像探针，优选使用经放射性核素标记的显像探针。
因此，本发明提供了:
用作显像α-突触核蛋白积聚体的示踪探针的通式I化合物、或其医药上可接受的盐或其溶剂化物；
用作成像诊断α‐突触核蛋白积聚性疾病的光学及放射性示踪探针，特别是使用正电子放射性核素标记的显像示踪探针；
用作制备放射性同位素标记的式I化合物的前体化合物；
用作成像诊断α-突触核蛋白积聚性疾病的组合物，其包含通式I的化合物、或其医药上可接受的盐或其溶剂化物，及医药上可接受的载体；
通式I的化合物、其医药上可接受的盐或其溶剂化物在用于诊断α-突触核蛋白积聚性疾病中的用途；
使用通式I的化合物、其医药上可接受的盐或其溶剂化物在生产用于诊断α-突触核蛋白积聚性疾病的组合物中的用途。
此外，本发明还提供了：
用于α‐突触核蛋白聚集体、患者脑中的α‐突触核蛋白病变沉积路易体和路易神经突的检测/染色方法，该方法可用于提供α-突触核蛋白积聚性疾病的早期诊断和进展评估信息；
用于对脑内α-突触核蛋白的积聚进行定量或判定的方法；
用于α-突触核蛋白积聚性疾病的预防和/或治疗药物的筛选方法；
以上所述方法均包含使用通式I的化合物、或其医药上可接受的盐或其溶剂化物，及医药上可接受的载体。
以下，针对式I化合物的取代基进行解释，进而对通式I化合物的盐、溶剂化物和衍生物、以及标记方法进行说明。
【定义】
除非另有指明，本发明的各种术语的含义和范围如下定义进行说明和限定。
术语“式I的化合物”、“式I化合物”、“本发明的化合物”或“本发明化合物”是指选自式I所限定的一类化合物的任意化合物，包括其立体异构体、顺反异构体、互变异构体、溶剂化物和盐(例如药用盐)。
除非另有指明，“或”或“和”的使用意指“和/或”。
当指示取代基的数目时，术语“一个或多个”意指从一个取代基到最大的化学上可能的取代数，即由取代基替代一个氢至替代所有氢。
术语“取代基”是指替代母体分子上的氢原子的原子或原子团。
术语“卤素”或“卤”是指氟(-F)、氯(-Cl)、溴(-Br)、及碘(-I)。
术语“TsO-”是指
Figure PCTCN2022138356-appb-000004
“MsO-”是指
Figure PCTCN2022138356-appb-000005
术语“C 1-4烷氧基”表示式-O-R’的基团，其中R’是指包含1至4个碳原子的直链或支链的饱和烷基。其实例包括甲氧基。
术语“卤代的C 1-4烷氧基”表示这样的烷氧基，其中所述烷氧基的一个或多个氢原子已被相同或不同的卤素原子(特别是氟原子)替代。其实例包括1-氟代乙氧基。
术语“C 1-3烷基”表示包含1至3个碳原子的直链或支链饱和烃基。其实例包括甲基。
术语“芳香族”表示例如在文献(特别是IUPAC-化学术语目录第2版，A.D.McNaught&A.Wilkinson.Blackwell Scientific Publications，Oxford(1997))中限定的芳香性的常规概念。
术语“在医药上可接受的盐”是指对哺乳动物、尤其是对人类无害的盐。可以使用包含无机酸或无机碱，或者包含有机酸或有机碱的、无毒性的酸或碱来形成在医药上可接受的盐。在医药上可接受的盐的例子可举出:用铝、钙、锂、镁、钾、钠及锌等形成的金属盐；或用赖氨酸、N,N’-二苄基乙二胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、甲葡胺(即N-甲基葡糖胺)及普鲁卡因等形成的有机盐等。另外，在医药上可接受的盐包含酸加成盐及碱加成盐。
术语“在医药上可接受的载体”是指生理盐水溶液；液态或固态的填充剂、稀释剂、溶剂、或封包材料等在医药上可接受的材料、组合物、或赋形剂。在医药上可接受的载体的例子包括水、食盐水、生理盐水或磷酸缓冲食盐水(PBS)、氯化钠注射液、林格氏注射液、葡萄糖注射液、无菌水注射液、葡萄糖、及乳酸林格氏注射液等。
术语“溶剂化物”是指，1个或多个溶剂分子与化合物缔合而形成的含溶剂化合物。例如可包含一溶剂化物、二溶剂化物、三溶剂化物、及四溶剂化物。另外，溶剂化物也包括水合物。
术语“水合物”是指，含有被非共价性分子间力所束缚的水的化合物或其盐，所含水的量可以是化学计量或非化学计量的。例如包含一水合物、二水合物、三水合物以及四水合物等。
【α‐突触核蛋白聚集体的示踪探针】
本发明提供的α-突触核蛋白聚集体的示踪探针(以下，也记作示踪探针，含有下式I所示的化合物、其在医药上可接受的盐、或其溶剂化物。
另外，下式I所示的化合物具有自发荧光。其中，化合物的1个或多个原子可为该原子的 放射性同位素。因此，本发明的化合物可作为小分子示踪探针用于α-突触核蛋白聚集体的光学成像，或经放射标记后的PET、SPECT成像。
Figure PCTCN2022138356-appb-000006
其中，
环A选自苯基、吡啶基、嘧啶基；
R 1选自含4-6个原子的含氮环烷烃基、N，N-双C 1-3烷基取代的氨基、C 1-3烷氧基、硝
基、卤素基；
环B选自吡啶基、哌嗪基、哌嗪酮基；
R 2选自H、卤素基、羟基、C 1-3烷基、C 1-4烷氧基、卤代的C 1-4烷氧基；
其中，所述卤素原子取自氟、氯、溴或碘；
其中，式I化合物的1个或1个以上原子是该原子的放射性同位素，该放射性同位素优选取自 11C、 13N、 15O、 18F、 76Br、 123I、 125I、 131I。
作为式I所示的化合物的具体例子，例如可举出以下的化合物:
Figure PCTCN2022138356-appb-000007
上述具体的化合物结构式中所示的*标记的原子(结构式中若有2个*标记，则指其中任意1个或2个)可以是该原子的放射性同位素，例如 11C或 18F。优选地，上述具体化合物中的F是放射性同位素 18F；优选地，与芳基连接的甲氧基或二甲氨基的碳原子是放射性同位素 11C。
本说明书中，( 18F)I-5等名称的含义是指，编号为I-5等的结构式中的具有*标记的原子是 18F；同理，( 11C)I-4等名称的含义是指，编号为I-4等的结构式中的具有*标记的原子是 11C。
【α-突触核蛋白聚集体的光学成像用组合物】
本发明的α-突触核蛋白聚集体的光学成像用组合物(以下也记作光学成像用组合物)包含上述式I化合物，其在药学上可接受的盐、或其溶剂化物。该光学成像包括试管内成像、生物体外成像、及生物体内成像。所述光学成像方法包括但不限于荧光显微镜测定法、多光子成像法、双光子成像法、近红外荧光成像法。
【α-突触核蛋白聚集体的放射成像用组合物】
本发明的α-突触核蛋白聚集体的放射成像用组合物(以下也记作放射成像用组合物)包含经放射标记后的式I化合物，其在药学上可接受的盐、或其溶剂化物。该放射成像包括试管内成像、生物体外成像、及生物体内成像。所述放射成像方法包括但不限于PET、SPECT、放射自显影法(autoradiography)。
上述光学成像用组合物和放射成像用组合物均可包含于前述医药上可接受的载体中。 其中所含的式I化合物、其在医药上可接受的盐、或其溶剂化物、以及在医药上可接受的载体的含量并无特别限定，可根据：所使用的化合物；被给予的哺乳动物的年龄、体重、健康状态、性別及餐食内容；给予的次数和途径；治疗期；同时使用的其他药剂等因素进行调整。
【α-突触核蛋白聚集体相关疾病的诊断药、或所述疾病的治疗或预防上的伴随诊断药】
本发明所述α-突触核蛋白聚集体相关疾病的诊断药、或所述疾病的治疗或预防上的伴随诊断药(以下也称为伴随诊断药)包含本发明的化合物。治疗上的伴随诊断药是指：在判明了所述疾病的情况下，用于判断是否有望实施治疗的诊断药。另外，预防上的伴随诊断药是指：在判明了所述疾病的前兆症状的情况下，用于预测今后的发病或者用于判断是否有望实施预防性发病抑制的诊断药。
将使用上述诊断药或伴随诊断药获得的受试者体内(如脑)α-突触核蛋白聚集体的量及/或分布量的相关数据，与预先已获知的上述疾病与α-突触核蛋白聚集体的量及/或分布量之间的相关性进行对照，则能够对受试者进行上述疾病的相关诊断(具体而言，如是否罹患有上述疾病、危重度、发作可能性等)；或了解受试者的上述疾病状态，基于此来制定上述疾病的预防/治疗计划(预防性给予/治疗药的种类及其组合、用量、用法等)。
【光学成像方法】
本发明所述的光学成像方法包含以下步骤，以下用检测脑中α-突触核蛋白聚集体的方法进行示例说明，检测其他部位时方法类似。
向受试生物体给予有效量的本发明示踪探针，到达生物体脑的示踪探针会与脑内的α-突触核蛋白聚集体结合。然后从脑外照射用于激发示踪探针的第1波长的光，并检测从脑内示踪探针发出的第2波长的光(例如荧光)，从而实现对α-突触核蛋白聚集体的光学可视化成像(图像化)。其中，所述示踪探针包含式I所示的化合物、或其在医药上可接受的盐、或其溶剂化物。
【放射成像方法】
本发明所述的放射成像方法包含以下步骤，以下用检测脑中α-突触核蛋白聚集体的方法进行示例说明，检测其他部位时方法类似。
向受试生物体给予有效量的经放射标记的本发明示踪探针，到达生物体脑的示踪探针会与脑内的α-突触核蛋白聚集体结合。然后通过PET或SPECT检测从该脑内的示踪探针发出的放射线，从而实现对α-突触核蛋白聚集体的放射成像(图像化)。其中，所述示踪探针包含式I所示的化合物、或其在医药上可接受的盐、或其溶剂化物，其中式I化合物的1个或1个以上原子是该原子的放射性同位素。
上述光学成像和放射成像受试生物体包括哺乳动物，例如人类、大鼠、小鼠、兔、豚鼠、仓鼠、猴、狗、貂、或小型猪等。优选地，哺乳动物是人类。所述示踪探针的给予方法无特别限定，可经口、静脉或腹腔给予。优选静脉或腹腔注射，最优选静脉注射。
【对脑内α-突触核蛋白的积聚进行定量或判定的方法】
通过上述成像方法，计算对受试生物体内(如脑内)所检测出的光或放射线的量及/或分布相比于其它正常哺乳动物的差值，则可对体内(如脑内)α-突触核蛋白的积聚进行定量，并判断体内(如脑内)是否存在α-突触核蛋白聚集体的积聚。
【用于与脑内α-突触核蛋白积聚相关疾病的治疗或预防药物的筛选方法】
基于使用以上【光学成像方法】或【放射成像方法】中所述成像方法，检测受试生物体在给予了筛选药物前、后所发出的光或放射线，根据其强度及/或分布的差异判断α-突触 核蛋白积聚的变化，以此筛选治疗或预防药物。例如，给予筛选药物后，如果来自示踪探针的光(如荧光)或放射线的量(强度)比给予筛选药物前减少，则该筛选药物可能用作该疾病或症状的治疗或预防用药物。进一步地，将来自所检测的受试生物体的光或放射线的量及/或分布与其它正常的哺乳动物进行比较，如果给予筛选药物后的结果比给予前更接近正常的哺乳动物，则该筛选药物可能用作该疾病或症状的治疗或预防用药物。
受试生物体种类及给予方法与以上【光学成像方法】及【放射成像方法】中所述相同。
本发明的化合物可以由已知的材料(例如市售的材料)通过公知的方法合成。本领域熟练的技术人员可以根据所需要的本发明的化合物适当选择原材料和合成方法。以下结合实施例进一步描述本发明，应理解，这些实施例仅用于说明本发明而不用于限制本发明的保护范围。下列实施例中未注明具体条件的实验方法，通常采用常规条件或按照制造厂商所建议的条件。本发明的已知的起始原料可以采用或按照本领域已知的方法制备，或购自于市售品。化合物的结构是通过核磁共振谱(NMR)和/或质谱来确定的。
实施例1:化合物I-1的制备，如下所示:
Figure PCTCN2022138356-appb-000008
步骤a:制备中间体b-1
1.0mmol化合物2-氨基-4-溴-苯酚(a-1)溶解于5ml甲醇中,加入2.0mmol NaOH和1.0mmol 2-氨基-5-三氟甲基吡啶,氮气保护下,加热回流10小时，蒸干溶剂后加入乙酸乙酯，硅胶柱层析得到b-1，产物为白色固体，产率40％。ESI-MS(positive):290.1(M+1) +。
步骤b:制备中间体b-2
将1.0mmol产物b-1溶解于4ml N,N-二甲基甲酰胺(DMF),加入2mmol K 2CO 3,4mmol CH 3I,室温反应8小时，加水后用乙酸乙酯萃取，有机相经无水硫酸钠干燥后，硅胶柱层析得到产物b-2为黄色固体，产率57％。ESI-MS(positive):318.1(M+1) +。
步骤c:制备化合物I-1
将1mmol产物b-2溶解于3ml N,N-二甲基甲酰胺，加入2mmol K 2CO 3,1％Pd(PPh 3) 4,1mmol的3-吡啶硼酸(c-1)，氮气保护下加热回流10小时，加水后用乙酸乙酯萃取，有机相经无水硫酸钠干燥后，硅胶柱层析得淡黄色固体，产率30％。 1H NMR(600MHz,DMSO-d 6)δ8.93(d,J＝2.2Hz,1H),8.54(d,J＝4.6Hz,1H),8.22-8.02(m,4H),7.83(d,J＝8.2Hz,1H),7.72(d,J＝8.4,1H),7.55-7.43(m,1H),6.82(d,J＝2.2Hz,1H),3.06(s,6H).ESI-MS(positive):317.1(M+1) +。
实施例2:化合物I-2的制备，其结构如下所示:
Figure PCTCN2022138356-appb-000009
其制备方法同化合物I-1，只是在步骤a中将2-氨基-5-三氟甲基吡啶替换为2-氨基-5-三氟甲基苯，得黄色固体，产率45％。 1H NMR(600MHz,DMSO-d 6)δ8.96(d,J＝2.3Hz,1H),8.59(dd,J＝4.8,1.5Hz,1H),8.18-8.10(m,1H),8.06-8.00(m,3H),7.81(d,J＝8.3Hz,1H),7.67(dd,J＝8.4,1.9Hz,1H),7.57-7.47(m,1H),6.92-6.84(m,2H),3.05(s,6H).ESI-MS(positive):316.1(M+1) +。
实施例3:化合物I-3的制备，其结构如下所示:
Figure PCTCN2022138356-appb-000010
其制备方法同化合物I-1，只是在步骤a中将2-氨基-5-三氟甲基吡啶替换为2-甲氧基-5-三氟甲基吡啶。获得黄色固体，产率26％。 1H NMR(600MHz,DMSO-d 6)δ8.89(d,J＝2.2Hz,1H),8.62(d,J＝4.6Hz,1H),8.21-8.00(m,4H),7.85(d,J＝8.3Hz,1H),7.71(d,J＝8.4,1H),7.52-7.46(m,1H),6.80(d,J＝2.2Hz,1H),3.75(s,3H).ESI-MS(positive):304.2(M+1) +。
实施例4:化合物I-4的制备，如下所示:
Figure PCTCN2022138356-appb-000011
其制备方法同化合物I-1，只是在步骤c中将3-吡啶硼酸(c-1)替换为N-甲基哌嗪酮(c-4)。得黄色固体，产率34％。 1H NMR(600MHz,DMSO-d 6)δ7.04-7.89(m,6H),3.69(s,2H),2.68-3.42(m,4H),2.28(s,3H),3.08(s,6H).ESI-MS(positive):352.4(M+1) +。
实施例5:化合物I-5的制备，如下所示:
Figure PCTCN2022138356-appb-000012
步骤d:制备中间体b-3
将2.0mmol的1,4-二碘丁烷和1.0mmol化合物b-1溶解于5ml N,N-二甲基甲酰胺,加入2mmol K 2CO 3,氮气保护下加热回流12小时，加水后用乙酸乙酯萃取，有机相经无水硫酸钠干燥后，硅胶柱层析得黄色固体，产率8％。ESI-MS(positive):344.1(M+1) +。
步骤e:制备化合物I-5
其制备方法同化合物I-1，只是将中间体b-2替换为反应物b-3，将3-吡啶硼酸(c-1)替换为2-氟-5-吡啶硼酸(c-5)。得淡黄色固体，产率42％。 1H NMR(600MHz,DMSO-d 6)δ8.56(s,1H),8.34(dd,J＝4.6,1.4Hz,1H),8.55(s,1H),8.05(s,1H),7.92-8.06(m,3H),7.57(dd,J＝4.6,1.4,1H),6.96-6.88(m,1H),3.45-3.52(m,4H),1.82-1.98(m,4H).ESI-MS(positive):361.2(M+1) +。
实施例6:化合物I-6的制备，如下所示:
Figure PCTCN2022138356-appb-000013
其制备方法同化合物I-5，其中b-5的制备同b-3，只是将起始原料2-氨基-4-溴-苯酚(a-1)替换成2-氨基-5-溴-苯酚(a-2)。得淡黄色固体，产率38％。 1H NMR(600MHz,DMSO-d 6)δ8.51(s,1H),8.28-8.8.42(m,1H),8.25(s,1H),7.96-8.12(m,3H),7.85(s,1H),7.42-7.49(m,1H),6.61-6.88(m,1H),3.46-3.53(m,4H),1.80-1.95(m,4H).ESI-MS(positive):361.2(M+1) +。
实施例7:即化合物I-7的制备，如下所示:
Figure PCTCN2022138356-appb-000014
其制备方法同化合物I-1，只是在步骤a中将起始原料2-氨基-5-三氟甲基吡啶替换成4-氟-1-三氟甲基苯。获得淡黄色固体，产率38％。 1H NMR(600MHz,DMSO-d 6)δ8.93(s,1H),8.52-7.46(m,10H).ESI-MS(positive):291.1(M+1) +。
实施例8:化合物I-8的制备，如下所示:
Figure PCTCN2022138356-appb-000015
其制备方法同化合物I-4，只是将N-甲基哌嗪酮(c-4)替换为N-(Boc)-哌嗪酮(c-8)。得淡黄色固体，产率48％。 1H NMR(600MHz,DMSO-d 6)δ8.00(d,J＝8.5Hz,2H),7.71(d,J＝8.7Hz,1H),7.28(d,J＝8.6Hz,1H),6.87(d,J＝8.6Hz,2H),4.10(s,2H),3.82-3.68(m,4H),3.05(s,6H),1.46(s,9H).ESI-MS(positive):438.2(M+1) +。
实施例9:化合物I-9的制备，其结构如下所示:
Figure PCTCN2022138356-appb-000016
将I-8溶于乙酸乙酯后加入三氟乙酸搅拌12小时，加水后用乙酸乙酯萃取，有机相经无水硫酸钠干燥后，硅胶柱层析得黄色固体，产率26％。 1H NMR(600MHz,DMSO-d 6)δ8.25(s,1H),7.78-6.85(m,5H),3.81(s,2H),3.61-3.68(m,2H),2.53-2.61(m,2H),3.08(s,6H).ESI-MS(positive):338.2(M+1) +。
实施例10:化合物I-10的制备，如下所示:
Figure PCTCN2022138356-appb-000017
将0.8g化合物I-9溶于3mL N,N-二甲基甲酰胺(DMF)，加入0.08g碳酸钾和0.3g 1-溴-2-氟乙烷，室温反应8小时。反应结束后加入20mL水，用乙酸乙酯萃取并用无水硫酸镁干燥，硅胶柱层析(乙酸乙酯:石油醚＝1:1)纯化，得淡黄色固体，产率19％。 1H NMR(600MHz,DMSO-d 6)δ8.22(s,1H),7.80-6.88(m,5H),4.26-4.32(m,2H),3.58-3.61(m,2H),3.85(s,2H),3.10(s,6H),2.51-2.43(m,4H).ESI-MS(positive):384.2(M+1) +。
【放射性核素的标记】
可采用常规已知的方法进行各种放射性核素的标记。以下用( 18F)I-5，( 18F)I-10和( 11C)I-4的制备为例，分别说明标记 18F和 11C的方法，其他放射性示踪探针可按相同方法制备。
实施例11:放射性示踪探针( 18F)I-5的合成
如下所示，可通过多种不同的前体化合物进行放射性核素 18F的标记。以下通过四种前体化合物(含硝基的前体、含溴的前体、含硼酸酯的前体、含TsO-基的前体)的合成方法进行示例说明，但不限于此。
Figure PCTCN2022138356-appb-000018
按实施例5的方法，将2-氟-5-吡啶硼酸分别替换为2-硝基-5-吡啶硼酸、2-溴-5-吡啶硼酸或2-羟基-5-吡啶硼酸，即可分别制备含硝基前体化合物I-5N、含溴前体化合物I-5B和含羟基前体化合物I-5H。进一步地，含溴前体I-5B在钯催化下与频哪醇硼酸酯偶联可制备活性更高的含有硼酸酯的前体化合物I-5O,含羟基前体化合物I-5H和对甲基苯磺酰氯(TsCl)在碱性条件下反应生成含TsO-基的前体化合物I-5T。这四种前体化合物I-5B、I-5O、I-5N、I-5T都能够与放射性K 18F反应生成放射性示踪探针( 18F)I-5。
放射性示踪探针( 18F)I-5的合成:
方法1:由含硼酸酯的前体I-5O合成。 18F-由回旋加速器生产，然后经过QMA吸附，由1号瓶压出K 222/K 2CO 3洗脱液洗脱 18F离子至反应管中，在116℃及氮气流下蒸干。2号瓶溶液(2mL乙腈)注入反应管，在116℃及氮气流下共沸蒸干除水。反应管冷却60s。3号瓶溶液(8mg前体化合物I-5O溶于1mL N,N-二甲基甲酰胺)注入反应管，反应温度115℃，反应时间30min。冷却100s(≤40℃)。4号瓶溶液注入反应管(10mL注射用水)稀释，传至C-18柱富集，注射用水20mL，将C-18柱用2.5mL无水乙醇洗脱备用。用生理盐水稀释产物的乙醇溶液至乙醇含量低于10％。并用0.22μm滤膜过滤获得可用于注射的( 18F)I-5溶液。所得产物与非放射性I-5比对HPLC图谱，两者保留时间一致，证明放射标记探针制备成功。
方法2:由含硝基的前体I-5N合成。使( 18F)氟化物离子溶入到包含K 222(Kryptofix 222)(7.5mg)及碳酸钾(2.77mg)的50％乙腈溶液(0.4mL)中，将该溶液引入到反应容器中之后，在氮气流下加热，使溶剂干燥固化。添加无水乙腈(0.1mL)共沸蒸馏，使反应容器内充分干燥。将溶有硝基前体化合物I-5N(1mg)的DMSO(300μL)溶液添加到反应容器中，在110℃下加热10分钟。冷却后，经HPLC分离纯化,获得( 18F)I-5纯品。
同理，含溴的前体I-5B和含TsO-基的前体I-5T也可用上述方法2的类似条件进行 18F标记，合成( 18F)I-5。
实施例12:放射性示踪探针( 18F)I-10的合成
如下图所示，放射性示踪探针( 18F)I-10可用I-9与已被 18F标记的溴代烷烃 18F-CH 2CH 2-Br直接进行氮烷基化制备。或将I-9与1,2-双(甲苯磺酰氧基)乙烷反应生成含离 去基TsO-的前体化合物I-10T；也可将I-9与环氧乙烷反应先生成含末端羟基的前体化合物I-10O，再和对甲基苯磺酰氯(TsCl)或甲烷磺酰氯(MsCl)等在碱性条件下反应生成标记位点为易离去基团TsO-、或MsO-的前体化合物(如I-10T)。最后将I-10T与放射性K 18F反应生成放射性示踪探针( 18F)I-10。
Figure PCTCN2022138356-appb-000019
前体化合物I-10T的制备示例如下：
0.16g化合物I-9溶于5mL N,N-二甲基甲酰胺，加入0.11g碳酸钾(2eq)和0.3g化合物1,2-双(甲苯磺酰氧基)乙烷(2eq)，室温搅拌过夜。反应结束后加入20mL水，乙酸乙酯萃取，无水硫酸镁干燥，粗品用硅胶柱层析分离得目标产物。
由含TsO-基的前体化合物I-10T制备( 18F)I-10的示例如下：
将( 18F)氟化物离子溶入到包含K 222(Kryptofix 222)(7.5mg)及碳酸钾(2.77mg)的50％乙腈溶液(0.4mL)中，将该溶液引入到反应容器中之后，在氮气流下加热，使溶剂干燥固化。然后添加无水乙腈(0.1mL)共沸蒸馏，使反应容器内充分干燥。将溶有前体I-10T(1.0mg)的二甲基亚砜(DMSO，300μL)溶液添加到反应容器中，在110℃下加热10分钟。冷却后，经HPLC(C18柱)分离纯化,获得( 18F)I-10。
实施例13:放射性示踪探针( 11C)I-4的合成，如下所示:
Figure PCTCN2022138356-appb-000020
中间体b-1的氨基用Boc保护后得b-7，再按实施例4类似方法合成生成I-4B，然后按实施例9类似方法脱除Boc保护基后得到标记前体化合物I-4N。
以下合成需避光进行。室温下将( 11C)碘甲烷加到溶有I-4N(2mg)的二甲基亚砜(DMSO，300μL)溶液中。反应混合液在120℃下加热5分钟，冷却后用HPLC纯化分离。将( 11C)I-4组分回收至含有乙醇(300μL)、25％抗坏血酸(100μL)及Tween80(75μL)的烧瓶中，减压蒸馏除去溶剂。将残余物溶解在生理盐水(3mL，pH 7.4)中，获得可用于注射的( 11C)I-4溶液。
【对α-突触核蛋白聚集体的结合力测试】
通过以下所述的荧光法测定本发明化合物对人源α-突触核蛋白聚集体的结合活性。
(1)α-突触核蛋白单体制备
取1μL测序正确的载有α-突触核蛋白表达序列的氨苄青霉素抗性质粒与100μL  BL21(DE3)感受态细胞混合均匀，冰浴冷却，加入600μL LB培养液，置于37℃220rpm摇床培养90min。将培养好的菌液150μL加至有氨苄培养基的灭菌培养皿涂布均匀，挑取阳性克隆菌落加入配置好的氨苄培养基中，37℃培养箱中培养。将培养好的阳性克隆菌液倾入1L的2×YT培养基中，在220rpm摇床中以37℃培养至OD 600为0.6时降温至18℃，每瓶培养基加入500mM IPTG 1ml诱导培养16小时。
离心收集菌体，超声破碎后高速离心30min，收集上清液，经Ni-NTA亲和柱层析去除DNA和杂蛋白，再通过分子排阻层析纯化得到α-突触核蛋白单体，以SDS-PAGE不连续电泳验证纯度。
(2)α-突触核蛋白聚集体制备
将α-突触核蛋白单体配置成含1×PBS的Buffer溶液，其中蛋白终浓度100μM(约5mg/mL)，置于37℃下1000rpm摇床中孵育7天获得α-突触核蛋白聚集体。初始蛋白单体浓度和终浓度均以BCA法精确测定。
(3)化合物结合活性测试
称取约1mg化合物，用二甲基亚砜(DMSO)配置成10mM母液，随后用PBS稀释成20μM，并进行7次梯度稀释(每次稀释三倍)；将30μL待测化合物加入384孔板，实验组加入30μLα-突触核蛋白聚集体(3μM)，对照组加入等量PBS，将384孔板于室温中震荡(50rpm)孵育1小时；随后将孔板取出，用酶标仪检测化合物的最大吸收、发射波长，并在该波长下检测荧光值。用实验组扣减对照组计算出分子不同浓度的荧光变化值，用GraphPad Prism的Saturation binding模块计算化合物对蛋白的结合力。
本发明式I化合物对α-突触核蛋白聚集体的结合活性通过以上方法测定，测得的部分化合物的解离平衡常数(K d)结果如表1所示。
表1本发明部分式I化合物对人源α-突触核蛋白聚集体的结合活性(K d)
实施例化合物 K d(μM) 实施例化合物 K d(μM) 实施例化合物 K d(μM)
I-1 * I-2 * I-4 ***
I-5 ** I-6 ** I-10 **
***:0.1～0.2μM；**:0.2～0.5μM；*:0.5～1.0μM
【病人脑的光学成像】
路易体痴呆症(DLB)患者脑片的染色及成像
路易体痴呆病人脑片取自一位75岁男性逝者的脑杏仁核组织，生前患有2期路易体痴呆症。对富含α-突触核蛋白病变的杏仁核组织进行冰冻切片，切片厚度为20μm。
将待测化合物用含50％EtOH的PBS溶液稀释成30μM，与获得的新鲜冷冻脑切片在室温下孵育30分钟，随后用50％乙醇溶液洗涤5分钟，再用超纯水洗涤2次，每次3分钟。使用封埋剂(VECTASHIELD H-1000，Vector Laboratories)封埋切片后，通过荧光显微镜拍照，获得切片上的病变积聚区域的图像。使用分析软件(Image J)对病变区域及病变非形成区域(背景)的荧光辉度进行定量，以评估结合选择性。
荧光图像结果显示化合物I-4、I-5、I-6都能清晰地染色路易体痴呆病人脑片中的路易体和路易神经突(附图1)，表明它们都能与病人脑中的α-突触核蛋白病变发生强结合。
阿尔茨海默症(AD)患者脑片的染色及成像
阿尔茨海默病人脑片取自一位3期患者逝后的脑颞上回组织。将脱蜡后的脑组织在10％中性缓冲***液中固定，石蜡包埋后切片，切片厚度为6μm。检测方法同上述对路易体痴呆患者(DLB)脑片的方法一样。荧光图像结果如附图2所示，化合物I-4、I-5、I-6也能探测到AD病人脑片中的Aβ病变，且不结合Tau神经纤维丝。很明显地，在AD病人脑片中，这些化合物的染色信号都远远弱于对DLB病人脑片的染色信号，表明它们对Aβ和Tau病理组织的结合都非常弱，甚至不结合。
由附图1、附图2结果可知，化合物I-4、I-5、I-6对α-突触核蛋白病理组织的结合明显强于对Aβ、Tau病理组织的结合，表明这些化合物对α-突触核蛋白聚集体都具有很好的选择性。
【血脑屏障渗透能力】
根据下列方法，给大鼠尾静脉注射本发明化合物以测定它们在体内的血脑屏障渗透性。
将待测化合物溶解于DMSO中，加入蓖麻油和PBS进行稀释(DMSO:蓖麻油:PBS＝1:1:8)；对SD大鼠进行称重，以5mg/kg进行尾静脉给药；用异氟烷麻醉，给药20min后取血500μL。随后用200mL PBS进行心脏灌流，待器官褪色后停止灌流，取出脑组织，用PBS冲洗表面。
取出的血液用9000rpm离心5min，取200μL上清液，加入800μL甲醇，14000rpm离心10min，取上清液过0.22μm滤膜，保存于-80℃备用。
取约0.5g脑组织，加入2mL PBS和2mL甲醇进行组织匀浆，取出1mL匀浆液加入2mL甲醇，14000rpm离心10min，取1mL上清液过0.22μm滤膜并保存于-80℃备用。对上述血液样本和脑匀浆上清液样本分别用LC-MS/MS检测其中的化合物浓度。
当脑/血比值<0.1，0.1～0.3或>0.3分别表示化合物透过血脑屏障程度为难、适中或良好。测试结果表明本发明化合物I-4、I-5、I-6的脑/血比接近1.0或大于1.0，证明它们都具有很好的血脑屏障渗透能力。由于本发明化合物结构相似，且clogP值基本都位于1.0～3.0之间，因此可以预测本发明其它化合物也应具有可接受的血脑屏障渗透能力。
工业实用性
本发明的能够结合/染色α-突触核蛋白积聚的探针化合物及其组合物对于当前十分重要的医疗难点之一的帕金森病等疑难病症的早期发现、医疗和预防都极为重要，并且具有用于医疗领域的极高的可能性。本发明化合物可以作为显像示踪剂用于显像α-突触核蛋白的积聚，从而能对多种神经退行性疾病，例如帕金森病(PD)、帕金森病性失智症(PDD)、路易体痴呆症(DLB)、多***萎缩(MSA)等提供早期诊断和疾病进展信息。

Claims (8)

  1. 通式I所示的化合物、其在医药上可接受的盐、或其溶剂化物，其用作诊断α-突触核蛋白积聚性疾病的示踪探针，
    Figure PCTCN2022138356-appb-100001
    通式I中，
    环A选自苯基、吡啶基、嘧啶基；
    R 1选自含4-6个原子的含氮环烷烃、N，N-双C 1-3烷基取代的氨基、C 1-3烷氧基、硝基、卤素基；
    环B选自吡啶基、哌嗪基、哌嗪酮基；
    R 2选自H、卤素基、羟基、C 1-3烷基、C 1-4烷氧基、卤代的C 1-4烷氧基；
    其中，所述卤素原子取自氟、氯、溴或碘。
  2. 根据权利要求1所述的通式I所示的化合物、其在医药上可接受的盐、或其溶剂化物，其特征在于，所述R 1为四氢吡咯基、N，N-二甲基氨基、甲氧基中的一种。
  3. 根据权利要求1或2所述的通式I所示的化合物、其在医药上可接受的盐、或其溶剂化物，其特征在于，所述化合物的1个或1个以上原子是该原子的放射性同位素，其中，所述的放射性同位素取自 11C、 13N、 15O、 18F、 76Br、 123I、 125I、 131I。
  4. 根据权利要求3所述的通式I所示的化合物、其在医药上可接受的盐、或其溶剂化物，其特征在于，所述化合物选自以下结构:
    Figure PCTCN2022138356-appb-100002
    其中，具有*的原子中至少有一个是该原子的放射性同位素。
  5. 下式所示的前体化合物，其特征在于，所述前体化合物用于制备权利要求4所述的化合物，
    Figure PCTCN2022138356-appb-100003
    其中，R 3独立地取自羟基、氟、溴、碘、硝基、硼酸酯基、TsO-(CH 2) m-、MsO-(CH 2) m-，m为0～4的整数；R 4为氢原子、C 1-3烷基，或NR 4R 4连接成含4-6个原子的含氮环烷烃。
  6. α-突触核蛋白聚集体的光学显像用组合物，其特征在于，其包含权利要求1或2所述的化合物、其在医药上可接受的盐、或其溶剂化物，及医药上可接受的载体。
  7. α-突触核蛋白聚集体的放射显像用组合物，其特征在于，包含权利要求3或4所述的化合物、其在医药上可接受的盐、或其溶剂化物，及医药上可接受的载体。
  8. 权利要求1所述的通式I所示的化合物、其在医药上可接受的盐、或其溶剂化物的用途，其特征在于，所述化合物、其在医药上可接受的盐、或其溶剂化物能与α-突触核蛋白聚集体结合，用作诊断α‐突触核蛋白积聚性疾病的显像示踪探针。
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