WO2019168170A1 - Chromone derivative and composition for diagnosis of amyloid-related disease - Google Patents

Abstract

The purpose of the present invention is to provide a compound having high binding specificity for amyloid proteins and high blood-brain barrier permeability, and featuring rapid elimination from normal tissues. The present invention relates to: a radionuclide-labeled compound represented by formula (I) (the symbols in the formula are as defined in the description) or a pharmaceutically acceptable salt thereof; and a composition for the diagnosis of amyloid-related disease comprising the same.

Description

Chromone derivative and amyloid-related disease diagnostic composition

The present invention relates to a chromone derivative useful for diagnosis of amyloid-related diseases such as Creutzfeldt-Jakob disease, a composition for diagnosing amyloid-related diseases containing the same, and a method for screening a therapeutic or prophylactic agent for amyloid-related diseases using the same And a method for evaluating a therapeutic or prophylactic agent for amyloid-related diseases using the same.

Prion disease, represented by Creutzfeldt-Jakob disease (CJD), is a fatal neurodegenerative disease classified as sporadic, acquired, or hereditary, and effective diagnosis and treatment methods are still established. Not. The incidence of prion disease in Japan is about 1 in 1 million per year, although the incidence is low, but the incidence rate is such as BSE infection due to the increase in imported beef in the future and hospital infection accidents from surgical instruments due to the increase in the elderly There is concern about the danger of the increase. The prion hypothesis that prion disease is associated with prion, which is a proteinaceous infectious particle, is currently the most promising. In this hypothesis, normal prion protein (PrP ^C ) present in the brain is changed to abnormal prion protein aggregate (PrP ^Sc ) for some reason, and this PrP ^Sc becomes a template, and other PrP ^C It is thought that prion disease develops by changing PrP ^Sc to PrP ^Sc one after another and accumulating PrP ^{Sc as} a result. Such autocatalytic processes are thought to be associated with the pathogenesis of common neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Thus, quinacrine and other compounds that block the conversion of PrP ^C to PrP ^Sc have been clinically tested as a treatment for prion diseases, but early diagnostic methods have not been developed, and most trials only target patients at the end. It is not done, and treatment results are not good. Therefore, the development of an early diagnosis method is indispensable for establishing a future prion disease treatment method. Nuclear medicine diagnosis such as SPECT (single photon emission computed tomography) and PET (positron emission tomography) is a diagnostic imaging method that can visualize target molecules in the brain alive. This is thought to be effective for early diagnosis of disease states and judgment of therapeutic effects.

As imaging drugs for diagnosis of amyloid-related diseases such as Alzheimer's disease, flavone derivatives and styrylchromone (SC) derivatives have been reported (Patent Document 1). Recent studies show that SC derivatives show good binding to prion deposition in PrP ^Sc aggregates and mouse-adapted BSE (mBSE) infected mice, and SPECT / CT also clearly differs between mBSE infected and uninfected mice It has been found that non-patent literature 1 shows the accumulation. However, when aiming at practical application, all of the SC derivatives are prone to photoisomerization (cis-trans photoisomerization), and it is difficult to obtain a single component. There is a problem that it is difficult to obtain, which has been an obstacle to imaging not only prions but also other amyloids.

WO2006 / 057323

The present invention has been made based on the technical background as described above, and aims to develop a molecular probe for nuclear medicine diagnosis that can non-invasively visualize PrP ^Sc for early diagnosis of prion disease. Properties required for such molecular probes include 1) permeability of the blood-brain barrier, 2) selective binding of PrP ^Sc , and 3) rapid disappearance of unbound body from the brain. The molecular probe that achieves the above conditions binds to PrP ^Sc , and the radiation emitted from the target site is detected using PET or SPECT, so that PrP ^Sc in vivo can be tracked over time. Be expected.

As a result of intensive investigations to solve the above problems, the present inventors have found that photoisomerization occurs by converting the styryl group of the SC skeleton, which is considered to be the cause of photoisomerization in the SC skeleton, to a 5-membered ring. A stabilized chromone derivative was synthesized. It was also found that such chromone derivatives show high binding affinity for PrP ^Sc , high blood-brain barrier permeability, and rapid disappearance from normal cells, and show ideal brain behavior as amyloid imaging probes. The present invention has been completed.

That is, the present invention provides the following [1] to [15].

[1] Formula (I):

(Where
X ¹ represents O, S, or NH;
X ² represents CH or N;
R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen atom; halogen atom; hydroxy group; carboxy group; sulfo group; nitro group; amino group; C _1-6 alkyl group; An optionally substituted C _1-6 alkoxy group; a mono (C _1-6 alkyl) amino group; or a di (C _1-6 alkyl) amino group, and R ⁵ , R ⁶ , R ⁷ and R ⁸ are , each independently, a hydrogen atom; a halogen atom; hydroxy group; a carboxy group; a sulfo group; a nitro group; an amino group; C _1-6 alkyl group; a halogen atom or a hydroxy which may be substituted by a group C _1-6 An alkoxy group; a mono (C _1-6 alkyl) amino group; or a di (C _1-6 alkyl) amino group. )
Or a pharmaceutically acceptable salt thereof.
[2] The compound according to [1] or a pharmaceutically acceptable salt thereof, wherein X ¹ in the formula (I) is O and X ² is CH.
[3] The compound or a pharmaceutically acceptable salt thereof according to [1], wherein any of R ¹ , R ² , R ³ and R ⁴ in formula (I) is a halogen atom.
[4] The compound according to [1] or a pharmaceutically acceptable salt thereof, wherein any of R ¹ , R ² , R ³ and R ⁴ in formula (I) is an iodine atom.
[5] X ¹ in the formula (I) is O, X ² is CH, R ¹ , R ² , R ⁴ , R ⁵ and R ⁸ are hydrogen atoms, R ³ is An iodine atom and R ⁶ is a hydrogen atom; a C _1-4 alkyl group; a C _1-4 alkoxy group optionally substituted by a hydroxy group; a mono (C _1-4 alkyl) amino group; or a di (C _1-4 alkyl) amino group and R ⁷ is hydroxy group; C _1-4 alkoxy group optionally substituted by hydroxy group; amino group; mono (C _1-4 alkyl) amino group; or di The compound or a pharmaceutically acceptable salt thereof according to [1], which is a (C _1-4 alkyl) amino group.
[6] The compound or a pharmaceutically acceptable salt thereof according to any one of [1] to [5], which is labeled with a radionuclide.
[7] The compound according to [6] or a pharmaceutically acceptable salt thereof, wherein the radionuclide is a positron emitting nuclide.
[8] The compound according to [6] or a pharmaceutically acceptable salt thereof, wherein the radionuclide is a γ-ray emitting nuclide.
[9] The compound according to [6] or a pharmaceutically acceptable salt thereof, wherein the radionuclide is ¹²⁵ I.
[10] A composition for diagnosing amyloid-related diseases, comprising the compound according to any one of [6] to [9] or a pharmaceutically acceptable salt thereof.
[11] Amyloid-related diseases are Creutzfeldt-Jakob disease, Alzheimer's disease, Lewy body dementia, Mediterranean fever, Maccle-Wells syndrome, idiopathic myeloma, amyloid polyneuropathy, amyloid cardiomyopathy, systemic senile Amyloidosis, hereditary cerebral hemorrhage with amyloidosis, Down's syndrome, scrapie, kuru disease, Gerstman-Stroisler-Scheinker syndrome, medullary thyroid cancer, isolated atrial amyloid, β ₂ -microglobulin amyloid in dialysis patients, inclusion body myositis, The composition according to [10], which is selected from the group consisting of β ₂ -amyloid deposition in muscle wasting disease and Langerhans type II diabetes insulinoma.
[12] An imaging agent for amyloid deposition, comprising the compound according to any one of [6] to [9] or a pharmaceutically acceptable salt thereof.
[13] A mammal into which a detectable amount of the compound according to any one of [6] to [9] or a pharmaceutically acceptable salt thereof is introduced is computed tomography (SPECT) or positron tomography A method for imaging amyloid deposits, comprising a step of photographing by a photographing method (PET).
[14] A step of administering a test substance to a model animal of amyloid-related disease, a step of administering the amyloid-related disease composition described in [10] or [11] to the model animal, and in the brain of the model animal A method for screening a therapeutic or prophylactic agent for amyloid-related diseases, which comprises the step of examining the distribution or amount of the compound represented by formula (I) contained in.
[15] A step of administering a therapeutic or prophylactic agent for amyloid-related disease to a model animal of amyloid-related disease, a step of administering to the model animal the composition for diagnosing amyloid-related disease according to [10] or [11], And a method for evaluating a therapeutic or prophylactic agent for amyloid-related diseases, which comprises the step of examining the distribution or amount of the compound represented by formula (I) contained in the brain of the model animal.

The compound represented by the formula (I) exhibits a high binding affinity for PrP ^Sc, a high permeability of the blood brain barrier, and a property of rapidly disappearing from a normal tissue, so that it can be used as an amyloid imaging probe. Shows ideal brain behavior. It is useful for diagnosis of amyloid-related diseases such as Creutzfeldt-Jakob disease. The cis and trans isomers derived from photoisomerism observed in SC derivatives are not confirmed in the compound represented by the formula (I). As a result, there is an advantage that the structure is stabilized by changing the styryl group of the SC skeleton to a 5-membered ring.

Known aggregates composed of peptides and proteins involved in amyloid-related diseases include abnormal prion protein aggregates (PrP ^Sc ), amyloid-β (Aβ) aggregates, and α-synuclein (α-Syn) aggregates. It has been. Alzheimer's disease (AD) is thought to be closely related to the onset of amyloid-β (Aβ), which is excised from neurons by secretase in the patient's brain and becomes aggregates. Prion hypothesis that prion disease is caused by normal cellular prion protein (PrP ^C ) in brain tissue that changes to abnormal prion protein (PrP ^Sc ) due to some cause, and PrP ^Sc accumulates Is currently the most influential. Parkinson's disease (PD) and Lewy body dementia (DLB) are thought to be caused by the accumulation and aggregation of α-synuclein (α-Syn), a protein localized in neurons. ing.

The compound of the present invention has a high affinity for PrP ^Sc , but does not have a high affinity for other Aβ aggregates or α-Syn aggregates. Thus, the compounds of the invention can be useful as imaging probes selective for PrP ^Sc . In addition, some of the compounds of the present invention (for example, Compound 30 in Examples) have an affinity for α-Syn aggregates and are useful as imaging agents (imaging probes) for α-Syn aggregates. obtain.

The figure which shows the synthesis method (1) of a chromone derivative (the number in a figure shows the number of a compound). The figure which shows the synthesis method (2) of a chromone derivative (the number in a figure shows the number of a compound). The figure which shows the synthesis method (3) of a chromone derivative (the number in a figure shows the number of a compound). The figure which shows the labeling method by the radioactive iodine of a styryl chromone derivative and a vinyl pyridyl chromone derivative. The figure which shows the labeling method by the radioactive iodine of a chromone derivative. ^The figure which shows the HPLC chromatogram of the chromone derivative labeled with ¹²⁵ I. [ ^125I ] 2, [ ^125I ] 10, [ ^125I ] 16, [ ^125I ] 17, [ ^125I ] 30, [ ^125I ] 33, [ ^125I ] 36 and [ ^125I ] 39 recombinant mice The figure which shows the binding rate with respect to a prion protein (rMoPrP) aggregate. ^{[125 I] 30, [125} I] 33, shows the ^[125 I] 36 and ^[125 I] 39 binding parameters for rMoPrP aggregates (K _d and B _max). K _d (nM) and B _max (nmol / mol protein) are shown as mean ± SD (n = 3). Photograph showing fluorescence staining of compounds 30, 33, and 39 in brain sections of uninfected mice (a, d and g) or mBSE infected mice (b, e and h). The amyloid deposition of labeled PrP ^Sc was confirmed by immunostaining of each section using anti-PrP antibody (c, f and i). Scale bar = 50 μm. [ ^125I ] 1, [ ^125I ] 2, [ ^125I ] 10, [ ^125I ] 30, [ ^125I ] 33, [ ^125I ] 36 and [ ^125I ] 39 in normal mice after intravenous administration The figure which shows the radioactive distribution in a brain. Data are shown as mean ± SD (n = 3-6). Data for [ ¹²⁵ I] 1 and [ ¹²⁵ I] 2 are from reference 1. Radioactivity distribution in the brain of normal mice after intravenous administration of [ ^125I ] 1, [ ^125I ] 2, [ ^125I ] 10, [ ^125I ] 30, [ ^125I ] 33 and [ ^125I ] 39 FIG. Data are shown as mean ± SD (n = 3-6). Data for [ ¹²⁵ I] 1 and [ ¹²⁵ I] 2 are from reference 1. Shows the ^[125 I] 1 and ^[125 I] 2 structure was used as a comparative compound. A [beta] _1-42 aggregates shows the fluorescence intensity of the _{(Aβ 1-42 aggregates) (0.554 μM} ) Thioflavin bound to T (ThT). Values are mean ± SE, n = 3. Ex: 450 nm, Em: 485 nm. The figure which shows the fluorescence intensity of ThT couple | bonded with the alpha-Syn aggregate ((alpha) -Syn (aggregate) (1.2 (micro | micron | mu) M). Values are mean ± SE, n = 3. Ex: 440 nm, Em: 485 nm. The figure which shows the content rate (%) of the deposit of the alpha-Syn aggregate (0.5 micromicrometer) after centrifugation. Values are mean ± SE, n = 3. The figure which shows the adsorption rate (%) of the [ ^125I ] BFC derivative with respect to (alpha) -Syn aggregate (250 nM). Values are mean ± SE, n = 3. The figure which shows the saturation curve and Scatchard plot of [ ^125I ] 30 couple | bonded with the alpha-Syn aggregate. K _d and B _max values were determined by saturation experiments with increasing concentrations of [ ¹²⁵ I] 30 (8-200 nM). The photograph which shows the fluorescence image of the compound 30, 33, 36, and 39 in the brain section (a, e, i, m) of an uninfected mouse | mouth, or the brain section (c, g, k, o) of an mBSE infection mouse | mouth. Immunostaining of adjacent sections of uninfected mouse brain sections (b, f, j, n). The amyloid deposition of labeled PrP ^Sc was confirmed by immunostaining of each section using anti-PrP antibody (d, h, l, p). Photographs (ad) showing fluorescence images of compounds 30, 33, 36 and 39 in brain sections of Tg2576 mice. A photograph (eh) showing a fluorescent image of ThT in an adjacent section of a Tg2576 mouse brain section.

Hereinafter, the present invention will be described in detail.

In the present invention, the “halogen atom” is, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

In the present invention, “C _1-6 alkyl group” means, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group Etc. Preferably, it is “C _1-4 alkyl group”, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group.

In the present invention, the “C _1-6 alkoxy group” means, for example, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, a pentyloxy group, Such as a hexyloxy group. The “C _1-6 alkoxy group” is preferably a “C _1-4 alkoxy group”, for example, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, a sec-butoxy group, tert-Butoxy group.

In the present invention, the “mono (C _1-6 alkyl) amino group” is an amino group substituted with one C _1-6 alkyl group, for example, a methylamino group, an ethylamino group, a propylamino group. Isopropylamino group, butylamino group, isobutylamino group, sec-butylamino group, tert-butylamino group, pentylamino group, hexylamino group, and the like. The “mono (C _1-6 alkyl) amino group” is preferably a “mono (C _1-4 alkyl) amino group”, for example, a methylamino group, an ethylamino group, a propylamino group, an isopropylamino group, A butylamino group, an isobutylamino group, a sec-butylamino group, and a tert-butylamino group;

In the present invention, the “di (C _1-6 alkyl) amino group” is an amino group substituted by two C _1-6 alkyl groups, and the two C _1-6 alkyl groups are the same as or May be different. For example, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di-sec-butylamino group, di-tert-butylamino group, dipentylamino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group and the like. The “di (C _1-6 alkyl) amino group” is preferably a “di (C _1-4 alkyl) amino group”, for example, a dimethylamino group, a diethylamino group, a dipropylamino group, a diisopropylamino group, And dibutylamino group, diisobutylamino group, di-sec-butylamino group, di-tert-butylamino group, N-ethyl-N-methylamino group, and N-methyl-N-propylamino group.

In the present invention, “optionally substituted with a halogen atom” means that it may be substituted with at least one (preferably 1 to 5, more preferably 1 to 3) halogen atoms. To do.

In the present invention, “optionally substituted by a hydroxy group” means that it may be substituted by at least one (preferably 1 to 5, more preferably 1 to 3) hydroxy group. To do.

In the present invention, “optionally substituted by a halogen atom or a hydroxy group” means that it is substituted by at least one (preferably 1 to 5, more preferably 1 to 3) halogen atom or hydroxy group. It means you may.

In the present invention, the “C _1-6 alkoxy group optionally substituted by a halogen atom” includes a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, a trifluoromethoxy group, and a 2-fluoroethoxy group. , 3-fluoropropoxy group, 4-fluorobutoxy group, 5-fluoropentyloxy group and the like.

In the present invention, the “C _1-4 alkoxy group optionally substituted by a hydroxy group” includes a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a 2-hydroxyethoxy group, a 3-hydroxypropoxy group, 4- Examples thereof include a hydroxybutoxy group.

In the formula (I), X ¹ is preferably O.
In the formula (I), X ² is preferably CH. In another preferred embodiment, X ² is N.
In formula (I), X ¹ is more preferably O and X ² is CH. In another preferred embodiment, X ¹ is O and X ² is N.

In formula (I), preferably any one of R ¹ , R ² , R ³ and R ⁴ is a halogen atom, and more preferably any one of R ¹ , R ² , R ³ and R ⁴ . Is an iodine atom. More preferably, R ¹ , R ² and R ⁴ are hydrogen atoms and R ³ is an iodine atom.

In the formula (I), R ⁵ and R ⁸ are preferably a hydrogen atom, and R ⁶ is a hydrogen atom; a C _1-4 alkyl group; a C _1-4 alkoxy optionally substituted by a hydroxy group A mono (C _1-4 alkyl) amino group; or a di (C _1-4 alkyl) amino group, and R ⁷ is a hydroxy group; a C _1-4 alkoxy group optionally substituted by a hydroxy group Amino group; mono (C _1-4 alkyl) amino group; or di (C _1-4 alkyl) amino group;

Preferable examples of the compound represented by the formula (I) include the following compounds.

In the formula (I), X ¹ is O, X ² is CH, R ¹ , R ² , R ⁴ , R ⁵ and R ⁸ are hydrogen atoms, R ³ is an iodine atom R ⁶ is a hydrogen atom; a C _1-4 alkyl group; a C _1-4 alkoxy group optionally substituted by a hydroxy group; a mono (C _1-4 alkyl) amino group; or a di (C _1-4) Alkyl) amino group and R ⁷ is hydroxy group; C _1-4 alkoxy group optionally substituted by hydroxy group; amino group; mono (C _1-4 alkyl) amino group; or di (C _{1 -4} alkyl) compound or a pharmaceutically acceptable salt thereof is an amino group.

In the formula (I), X ¹ is O, X ² is CH, R ¹ , R ² , R ⁴ , R ⁵ and R ⁸ are hydrogen atoms, R ³ is an iodine atom R ⁶ is a hydrogen atom, and R ⁷ is a C _1-4 alkoxy group (eg, methoxy group); an amino group; a mono (C _1-4 alkyl) amino group (eg, a methylamino group); Or a compound which is a di (C _1-4 alkyl) amino group (eg, dimethylamino group) or a pharmaceutically acceptable salt thereof.

Compound 27, 28, 30, 31, 33, 34, 36, 37, or 39 or a pharmaceutically acceptable salt thereof described in Examples described later.

More preferable examples include compounds 30, 33, 36, or 39 described in Examples described later, or pharmaceutically acceptable salts thereof.

The compound represented by the formula (I) can be synthesized according to the description in the examples described later.

For example, in formula (I), compound (I) -1 in which X ¹ is O and X ² is CH can be produced by the method shown in the following scheme.

(Wherein R ⁹ represents a C _1-6 alkyl group, Hal represents a halogen atom such as a bromine atom or a chlorine atom, and other symbols are as defined above).
Process 1
Compound (IV) can be produced by subjecting compound (II) and compound (III) to cyclization and dehydration reaction in a solvent in the presence of a base. Examples of the base include potassium carbonate. Examples of the solvent include N, N-dimethylformamide (DMF).
Process 2
Compound (V) can be produced by hydrolyzing compound (IV) in the presence of a base in a solvent. Examples of the base include sodium hydroxide and potassium hydroxide. Examples of the solvent include methanol and ethanol.
Process 3
Compound (VII) can be produced by subjecting compound (V) and compound (VI) to an esterification reaction in the presence of a halogenating agent and a base. Examples of the halogenating agent include chlorinating agents such as phosphoryl chloride and thionyl chloride. Examples of the base include pyridine.
Process 4
Compound (VIII) can be produced by subjecting compound (VII) to a rearrangement reaction in the presence of a base in a solvent. Examples of the base include potassium hydroxide and sodium hydroxide. Examples of the solvent include pyridine.
Process 5
Compound (I) -1 can be produced by subjecting compound (VIII) to a dehydration cyclization reaction in a solvent in the presence of an acid. Examples of the acid include concentrated sulfuric acid. Examples of the solvent include acetic acid.

For example, in formula (I), compound (I) -2 in which X ¹ is O and X ² is N can be produced by the method shown in the following scheme.

(Wherein each symbol is as defined above)
Step 6
Compound (X) can be produced by subjecting compound (IX) to an addition reaction with ethyl glyoxylate in a solvent, followed by an oxidation reaction using ammonium cerium nitrate (CAN) or the like. Examples of the solvent include dioxane.
Step 7
Compound (XI) can be produced by hydrolyzing compound (X) in a solvent in the presence of a base. Examples of the base include sodium hydroxide and potassium hydroxide. Examples of the solvent include methanol and ethanol.
Process 8
Compound (XII) can be produced by subjecting compound (XI) and compound (VI) to an esterification reaction in the presence of a halogenating agent and a base. Examples of the halogenating agent include chlorinating agents such as phosphoryl chloride and thionyl chloride. Examples of the base include pyridine.
Step 9
Compound (XIII) can be produced by subjecting compound (XII) to a rearrangement reaction in the presence of a base in a solvent. Examples of the base include potassium hydroxide and sodium hydroxide. Examples of the solvent include pyridine.
Step 10
Compound (I) -2 can be produced by subjecting compound (XIII) to a dehydration cyclization reaction in a solvent in the presence of an acid. Examples of the acid include concentrated sulfuric acid. Examples of the solvent include acetic acid.

The compound represented by formula (I) is preferably labeled with a labeling substance. As the labeling substance, a fluorescent substance, an affinity substance or the like may be used, but it is preferable to use a radionuclide. The kind of radionuclide used for labeling is not particularly limited, and can be appropriately determined depending on the mode of use. For example, when the compound represented by the formula (I) is used for diagnosis by computed tomography (SPECT), the radionuclide is ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, ²⁰¹ Tl, ¹²³ I, ¹³³ Xe (preferably , ^99m Tc, ¹²³ I) can be used. When used for diagnosis by positron emission tomography (PET), ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶² Cu, ⁶⁸ Ga, ⁷⁶ Br (preferably ¹¹ C, ¹³ N, ¹⁵ Positron emitting nuclides such as O, ¹⁸ F) can be used. In addition, when the compound represented by the formula (I) is administered to an animal other than a human, a radionuclide having a longer half-life, such as ¹²⁵ I, may be used. The radionuclide may be included in the molecule of the compound represented by formula (I) or may be bonded to the compound represented by formula (I).

It is also possible to use a pharmaceutically acceptable salt instead of the compound represented by the formula (I). Examples of pharmaceutically acceptable salts include alkali metal salts (sodium salt, potassium salt, lithium salt), alkaline earth metal salts (calcium salt, magnesium salt), sulfate, hydrochloride, nitrate, phosphate, etc. it can.

The composition of the present invention is used for diagnosis of amyloid-related diseases. Here, “amyloid-related disease” means a disease in which the presence of an amyloid protein aggregate is observed. Since the compound of the present invention binds to a protein having a β sheet structure, the “amyloid-related diseases” in the present invention include amyloid having a β sheet structure such as tau, α-synuclein, prion, etc. in addition to β-amyloid. Diseases in which the presence of protein aggregates are observed are included. Specifically, the “amyloid-related disease” in the present invention includes Creutzfeldt-Jakob disease, Alzheimer's disease, Lewy body dementia, Mediterranean fever, Maccle-Wells syndrome, idiopathic myeloma, amyloid polyneuropathy, Amyloid cardiomyopathy, systemic senile amyloidosis, hereditary cerebral hemorrhage with amyloidosis (including hereditary cerebral hemorrhage with Dutch or Icelandic amyloidosis), Down's syndrome, scrapie, Kourou disease, Gerstmann-Stroisler-Scheinker syndrome, Examples include medullary thyroid cancer, isolated atrial amyloid, β ₂ -microglobulin amyloid in dialysis patients, inclusion body myositis, β ₂ -amyloid deposition in muscle wasting disease, and Langerhans type II diabetes insulinoma. Among these, the diagnostic composition of the present invention is particularly suitable for diagnosis of Creutzfeldt-Jakob disease, Alzheimer's disease, and Lewy body dementia. In addition, precursor symptoms of diseases that are generally not recognized as “diseases” are also included in “amyloid-related diseases” in the present invention. Examples of prodromal symptoms of such diseases include mild cognitive impairment (MCI) seen before the onset of Alzheimer's disease.

Diagnosis of amyloid-related diseases with the composition of the present invention is usually performed by administering the composition of the present invention to a subject to be diagnosed or a laboratory animal, and then taking a brain image, which is represented by the formula (I) in the image. This is based on the state of the compound (amount, distribution, etc.). The administration method of the composition of the present invention is not particularly limited and can be appropriately determined according to the type of compound, the type of labeling substance, etc., but is usually intradermal, intraperitoneal, intravenous, arterial, or spinal fluid. It is administered by injection or infusion. The dose of the composition of the present invention is not particularly limited and can be determined appropriately according to the type of compound, the type of labeling substance, etc. In the case of an adult, the compound represented by formula (I) is 10 per day. ^-10 to 10 ^-3 mg is preferably administered, more preferably 10 ^-8 to 10 ^-5 mg.

As described above, since the composition of the present invention is usually administered by injection or infusion, it may contain components usually contained in an injection solution or an infusion solution. Such components include liquid carriers (for example, potassium phosphate buffer, physiological saline, Ringer's solution, distilled water, polyethylene glycol, vegetable oils, ethanol, glycerin, dimethyl sulfoxide, propylene glycol, etc.), antibacterial agents And local anesthetics (eg, procaine hydrochloride, dibucaine hydrochloride, etc.), buffer solutions (eg, Tris-HCl buffer solution, Hepes buffer solution, etc.), osmotic pressure regulators (eg, glucose, sorbitol, sodium chloride, etc.) .

The present invention also relates to an imaging agent for amyloid deposition, which comprises a compound represented by formula (I) or a pharmaceutically acceptable salt thereof labeled with a radionuclide. “Amyloid deposition” is formed by aggregation of amyloid proteins having a β-sheet structure. Examples of amyloid protein include prion, β-amyloid, tau and α-synuclein. The imaging agent of the present invention is suitable for imaging amyloid deposits formed by prion, β-amyloid and α-synuclein. . The imaging agent of the present invention is particularly suitable for imaging abnormal prion protein aggregates (PrP ^Sc ) (amyloid deposits formed by prions). The imaging agent for amyloid deposition of the present invention can be prepared and used in the same manner as the composition for diagnosing amyloid-related diseases.

The present invention also provides
a. Introducing a detectable amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof labeled with a radionuclide into a mammal;
b. Leaving the compound for a time sufficient to bind to the amyloid deposit; and c. Detecting a compound bound to one or more amyloid deposits;
To a method for imaging amyloid deposits.

The present invention also provides
A mammal introduced with a detectable amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof labeled with a radionuclide is subjected to computed tomography (SPECT) or positron emission tomography ( The present invention relates to a method for imaging amyloid deposits, comprising the step of imaging by PET.

“Mammals” include, but are not limited to, humans, mice, rats, rabbits, dogs, monkeys. Preferably, the “mammal” is a human.

The labeling with the radionuclide and the detection of the labeled compound may be performed as described above. That is, SPECT and PET can be used for detection, and the radionuclide may be selected according to the detection method. Moreover, introduction into mammals may be performed as described for the composition for diagnosing amyloid-related diseases.

“Detectable amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof labeled with a radionuclide” and “a sufficient time for the compound to bind to amyloid deposits” A person skilled in the art can determine appropriately depending on the mammal to be used, the compound used and the detection method. For example, the amount and time of these can be determined by introducing various concentrations of the labeled compound into the mammal of interest, and detecting this labeled compound with the selected detection method at various times after introduction. .

The composition for diagnosing amyloid-related diseases of the present invention can also be used for screening for therapeutic or prophylactic agents for amyloid-related diseases. For example, after administering a test substance to a model animal of “disease” such as Creutzfeldt-Jakob disease or Alzheimer's disease, the model animal is administered with the amyloid-related disease diagnostic composition of the present invention, and then The distribution or amount of the compound represented by formula (I) contained in the brain is examined, and as a result, a significant difference from the control (model animal not administered with the test substance) (for example, reduction of the distribution site) If a decrease in the amount is detected, the test substance can be a candidate for a therapeutic drug for amyloid-related diseases. Further, after administering a test substance to a model animal of “disease symptoms” such as mild cognitive impairment, the composition for amyloid-related disease diagnosis of the present invention is administered to the model animal, and then the brain of the model animal The distribution or amount of the compound represented by the formula (I) contained in is investigated, and as a result, there is a significant difference from the control (for example, the distribution site is reduced or enlarged, the amount is decreased or increased, etc.) ) Is detected, the test substance can be a candidate for a prophylactic agent for amyloid-related diseases.

The composition for diagnosing amyloid-related diseases of the present invention can also be used for evaluation of therapeutic and preventive drugs for amyloid-related diseases whose effects have already been confirmed. That is, after the therapeutic or prophylactic agent for the disease is administered to a model animal for amyloid-related disease, the composition for diagnosing amyloid-related disease of the present invention is administered to the model animal, and then contained in the brain of the model animal. Thus, the distribution or amount of the compound represented by the formula (I) is examined, and thereby the therapeutic agent and the prophylactic agent are evaluated (specifically, effective dose, effective administration method, etc.).

Hereinafter, the present invention will be described in more detail with reference to examples.

〔experimental method〕
As reagents and instrument reagents, commercially available first-class reagents or special-grade reagents were used without special purification unless otherwise specified. Radioiodine-125 is produced by MP Biomedicals, Inc. Iodine-125 (Na ¹²⁵ I, 100 mCi / ml, pH10 NaOH solution) and Muromachi Yakuhin Iodine-125 (Na ¹²⁵ I, 3.7 GBq / mL, 0.01 M NaOH solution) ) Was used.
For medium pressure preparative liquid chromatography, Yamazen PUMP-560, UV-DETECTOR prep-UV254, ULTRA PACK (SiOH, 11 × 300 mm and 26 × 300 mm) and ADVANTEC CHF122SSC FRACTION COLLECTOR were used. .
Shimadzu C-R8A CHROMATOPAC, SPD-10A VP UV-VIS DETECTOR, and LC-10AT VP LIQUID CHROMATOGRAPH were used for high performance liquid chromatography (HPLC). As the column, COSMOSIL Packed column 5C ₁₈ -AR-II (4.6 × 150 mm, 20 × 250 mm) manufactured by Nacalai Tesque was used.
¹ H-NMR measurement was performed using Varian Gemini 300 or JOEL JNM-AL 400 and tetramethylsilane as an internal standard substance. Mass spectrometry used JOEL JMS-T100TD. For measurement of radioactivity, a Wizard Autowell gamma counter manufactured by PerkinElmer Japan was used.
For separation of bound and unbound molecules in binding experiments, Brandel M-24R cell harvester and Whatman GF / B filter (pore size 1 μm) were used. Leica cryostat CM1900 was used for the preparation of BSE-infected mouse brain sections, and Nikon ECLIPSE 80i was used for the fluorescence microscope.

Production Example 1
Synthesis of 2- hydroxy-4-nitrobenzaldehyde (18) 2-Methoxy-4-nitrobenzaldehyde (500 mg, 2.76 mmol) was dissolved in dichloromethane (4 mL). Boron tribromide (4 mL) was added under ice cooling, and the mixture was stirred at room temperature for 4 hours. Thereafter, purified water was added to stop the reaction. The organic layer was separated and extracted with chloroform and purified water, and washed with a saturated aqueous sodium chloride solution. The extract was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; chloroform: hexane = 1: 1) to obtain the title compound as yellow crystals (365 mg, yield 79%). ¹ H NMR (400MHz, CDCl ₃ ) δ 7.84-7.87 (m, 3H), 10.06 (s, 1H), 11.15 (s, 1H). MS (DART) m / z: 167 (M + H ⁺ ).

Production Example 2
Synthesis of ethyl 6-methoxybenzofuran-2-carboxylate (19) 2-hydroxy-4-methoxybenzaldehyde (200 mg, 1.31 mmol) and ethyl bromoacetate (435 μL, 3.93 mmol) were combined with N, N-dimethylformamide ( DMF) (2 mL), K ₂ CO ₃ was added, and the mixture was stirred with heating at 80 ° C. for 6 hr. Thereafter, 1,4-dioxane was added to remove the solvent by azeotropic distillation of DMF. The organic layer was separated and extracted with chloroform and purified water. The extract was washed with a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; chloroform: hexane = 1: 1) to obtain the title compound (43 mg, yield 15%). ¹ H NMR (400MHz, CDCl ₃ ) δ 1.42 (t, J = 14.4 Hz, 3H), 3.86 (s, 3H), 4.42 (q, J = 21.6 Hz, 2H), 6.99 (dd, J = 2.8 Hz, 8.4 Hz, 1H), 7.06 (d, J = 2 Hz, 1H), 7.46 (s, 1H), 7.53 (d, J = 8.8 Hz, 1H). MS (DART) m / z: 221 (M + H ⁺ ).

Production Example 3
Synthesis of ethyl 6-nitrobenzofuran-2-carboxylate (20) Compound 18 (341 mg, 2.04 mmol) and ethyl bromoacetate (639.3 μL, 8.16 mmol) were dissolved in DMF (3 mL) and K ₂ CO ₃ (554 mg , 4.08 mmol), and stirred with heating at 90 ° C. for 18 hours. Thereafter, 1,4-dioxane was added to remove the solvent by azeotropic distillation of DMF. The organic layer was separated and extracted with chloroform and purified water. The extract was washed with a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; chloroform: hexane = 1: 1) to obtain the title compound (75 mg, yield 16%). ¹ H NMR (400MHz, CDCl ₃ ) δ 1.46 (t, J = 14 Hz, 3H), 4.49 (q, J = 21.6 Hz, 2H), 7.60 (d, J = 0.8 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 8.24 (dd, J = 2 Hz, 8.8 Hz, 1H), 8.49 (s, 1H). MS (DART) m / z: 236 (M + H ⁺ ).

Production Example 4
Synthetic compound 19 (166 mg, 0.75 mmol) of 6-methoxybenzofuran-2-carboxylic acid (21) was dissolved in methanol (5 mL), 1M NaOH was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, 1M HCl was added under ice cooling. The organic layer was separated and extracted with chloroform and purified water. The extract was washed with a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain the title compound (100 mg, yield 69.0%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.89 (s, 3H), 6.97 (dd, J = 2 Hz, 8.8 Hz, 1H), 7.08 (s, 1H), 7.57 (d, J = 8.8 Hz, 1H) , 7.63 (s, 1H). MS (DART) m / z: 193 (M + H ⁺ ).

Production Example 5
Synthesis compound 6-nitrobenzofuran-2-carboxylic acid (22) 20 (180 mg, 0.77 mmol) was dissolved in methanol (5 mL), 1M NaOH was added, and the mixture was stirred at room temperature for 2 hours. Thereafter, methanol was distilled off under reduced pressure, and 1M HCl was added under ice cooling. The organic layer was separated and extracted with chloroform and purified water, washed with a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by an open column (elution solvent; ethyl acetate: hexane = 7: 3, 2% acetic acid) to obtain the title compound (71 mg, yield 45%). ¹ H NMR (400MHz, CDCl ₃ ) δ 7.70 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 8.26 (dd, J = 1.6Hz, 8.6 Hz, 1H), 8.52 (s, 1H) MS (DART) m / z: 208 (M + H ⁺ ).

Production Example 6
Synthesis of 2-acetyl-4-bromophenyl 6-methoxybenzofuran-2-carboxylate (23) Phosphoryl chloride (77.8 μL, 1.623 g / mL, 0.825 mmol) was added to pyridine (5 mL) under ice cooling and stirring. did. Compound 21 (53 mg, 0.275 mmol) and 1- (5-bromo-2-hydroxyphenyl) ethanone (59 mg, 0.275 mmol) were added, and the mixture was stirred at room temperature for 2 hours. Thereafter, ice was added under ice cooling, and stirring was continued. The reaction solution was collected by filtration, and the precipitate was collected. At that time, it was washed with a saturated aqueous solution of sodium bicarbonate. Drying at room temperature gave the title compound (76 mg, 71% yield). ¹ H NMR (400MHz, CDCl ₃ ) δ 2.58 (s, 3H), 3.89 (s, 3H), 6.99 (dd, J = 2.4 Hz, 8.6 Hz, 1H), 7.09 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.70 (dd, J = 3.6 Hz, 8.8 Hz, 1H), 7.72 (s, 1H), 7.97 (d, J = 2.4 Hz , 1H).

Production Example 7
Synthesis of 2-acetyl-4-bromophenyl 6-nitrobenzofuran-2-carboxylate (24) Phosphoryl chloride (77.8 μL, 1.623 g / mL, 0.825 mmol) was added to pyridine (5 mL) under ice cooling and stirring. did. Compound 22 (71 mg, 0.343 mmol) and 1- (5-bromo-2-hydroxyphenyl) ethanone (73 mg, 0.343 mmol) were added, and the mixture was stirred at room temperature for 12 hours. Thereafter, ice was added under ice cooling, and stirring was continued. The reaction solution was collected by filtration, and the precipitate was collected. At that time, it was washed with a saturated aqueous solution of sodium bicarbonate. Drying at room temperature gave the title compound (138 mg, 99% yield). ¹ H NMR (400MHz, CDCl ₃ ) δ 2.59 (s, 3H), 7.21 (d, J = 8.8 Hz, 1H), 7.75 (dd, J = 2.4 Hz, 8 Hz, 1H), 7.83 (s, 1H) , 7.90 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 2 Hz, 1H), 8.29 (dd, J = 2 Hz, 8.8 Hz, 1H), 8.54 (s, 1H).

Production Example 8
Synthesis of 1- (5-bromo-2-hydroxyphenyl) -3- (6-methoxybenzofuran-2-yl) propane-1,3-dione (25) Compound 23 (214 mg, 0.55 mmol) was converted to pyridine (7 In addition, powdered KOH (131 mg, 2.37 mmol) was added with stirring under ice cooling. The mixture was stirred at room temperature for 2 hours, and a 10% aqueous acetic acid solution was added under ice cooling to stop the reaction. The reaction solution was collected by filtration, and the precipitate was collected and dried at room temperature. Thereafter, the residue was purified by an open column (elution solvent; chloroform: hexane = 1: 1) to obtain the title compound (36 mg, yield 16.8%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.91 (s, 3H), 6.85 (s, 1H), 6.92 (d, J = 8.8 Hz, 1H), 6.96 (dd, J = 2 Hz, 8.8 Hz, 1H) , 7.11 (d, J = 1.6 Hz, 1H), 7.50 (s, 1H), 7.54 (dd, J = 3.6 Hz, 8.2 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.93 (d , J = 2.4 Hz, 1H) .MS (DART) m / z: 389, 391 (M + H ⁺ ).

Production Example 9
Synthesis of 1- (5-bromo-2-hydroxyphenyl) -3- (6-nitrobenzofuran-2-yl) propane-1,3-dione (26) 24 (138 mg, 0.34 mmol) was converted to pyridine (8 The solution was dissolved in mL) and powdered KOH (38.14 mg, 0.68 mmol) was added with stirring under ice cooling. The mixture was stirred at room temperature for 2 hours, and a 10% aqueous acetic acid solution was added under ice cooling to stop the reaction. The reaction solution was collected by filtration, and the precipitate was collected and dried at room temperature. Thereafter, the residue was purified by an open column (elution solvent; chloroform: hexane = 3: 1) to obtain the title compound (56 mg, yield 41%). ¹ H NMR (400MHz, CDCl ₃ ) δ 7.21 (d, J = 8.8 Hz, 1H), 7.75 (dd, J = 2.4 Hz, 8 Hz, 1H), 7.83 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 8.01 (d, J = 2 Hz, 1H), 8.29 (dd, J = 2 Hz, 8.8 Hz, 1H), 8.54 (s, 1H). MS (DART) m / z: 404, 406 (M + H ⁺ ).

Example 1
Synthesis of 6-bromo-2- (6-methoxybenzofuran-2-yl) -4H-chromen-4-one (27) Compound 25 (29 mg, 0.075 mmol) was suspended in acetic acid (10 mL) at room temperature. Stir. Concentrated sulfuric acid (0.5 mL) was added, and the mixture was stirred with heating at 100 ° C. for 2 hr. Then, it cooled at room temperature and added ice to the reaction liquid, and stirred. The reaction solution was collected by filtration, and the precipitate was collected. Thereafter, the collected precipitate was purified with an open column (elution solvent; chloroform: hexane = 1: 1) to obtain the title compound (20 mg, yield 72%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.93 (s, 3H), 6.87 (s, 1H), 6.95 (dd, J = 2.8 Hz, 8.4 Hz 1H), 7.06 (d, J = 2 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.43 (s, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.77 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H) .MS (DART) m / z: 371, 373 (M + H ⁺ ).

Example 2
Synthesis of 6-bromo-2- (6-nitrobenzofuran-2-yl) -4H-chromen-4-one (28) Compound 26 (580 mg, 1.44 mmol) was suspended in acetic acid (15 mL) at room temperature. Stir. Concentrated sulfuric acid (1 mL) was added, and the mixture was stirred with heating at 100 ° C. for 2 hr. Then, it cooled at room temperature and added ice to the reaction liquid, and stirred. The reaction mixture was collected by filtration, and the precipitate was collected to give the title compound (284 mg, yield 51%). MS (DART) m / z: 386, 388 (M + H ⁺ ).

Production Example 10
2- (6-Methoxybenzofuran-2-yl) -6- (tributylstannyl) -4H-chromen-4-one (29) Compound 27 (46 mg, 0.12 mmol) was converted to 1,4-dioxane: triethylamine = 3: Dissolved in a mixed solvent of 5 (5 mL), bis (tributyltin) (1.15 g / ml) (417.28 μL, 1.00 mmol) and (PPh ₃ ) ₄ Pd (58 mg, 0.05 mmol) were added, and 90 The mixture was stirred at 14 ° C. for 14 hours. Then, it cooled to room temperature and filtered using celite, and the solvent of the solution was depressurizingly distilled. The residue was purified with an open column (elution solvent; ethyl acetate: hexane = 1: 6) to obtain the title compound (11 mg, yield 15%). ¹ H NMR (400MHz, CDCl ₃ ) δ 0.89-1.61 (m, 27H), 3.90 (s, 3H), 6.90 (s, 1H), 6.95 (dd, J = 1.6Hz, 8.6 Hz, 1H), 7.08 ( d, J = 9.2 Hz, 1H), 7.43 (s, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 8.31 (s, 1H) .MS (DART) m / z: 581 (M + H ⁺ ).

Example 3
Synthesis of 6-iodo-2- (6-methoxybenzofuran-2-yl) -4H-chromen-4-one (30) 29 (30 mg, 0.05 mmol) was dissolved in chloroform (4 mL) and 0.2 M Iodine solution (chloroform solution) was added until the reaction solution turned purple, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous sodium hydrogen sulfite solution was added until the color of the reaction solution changed from purple to tan, and the organic layer was separated and extracted with chloroform and purified water, and washed with a saturated aqueous sodium chloride solution. Thereafter, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (19 mg, yield 88%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.90 (s, 3H), 6.88 (s, 1H), 6.95 (dd, J = 2.4 Hz, 8.6 Hz, 1H), 7.07 (d, J = 2 Hz, 1H) , 7.43 (s, 1H), 7.41 (d, J = 2 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 8.22 (dd, J = 2 Hz, 8 Hz, 1H), 8.53 (d , J = 2 Hz, 1H). MS (DART) m / z: 419 (M + H ⁺ ).

Example 4
Synthesis of 2- (6-aminobenzofuran-2-yl) -6-bromo-4H-chromen-4-one (31) Compound 28 (406 mg, 1.05 mmol) was suspended in ethanol (10 mL). Heated to reflux at ° C. While heating under reflux, tin chloride (1319 mg, 5.25 mmol) was added, and the mixture was heated under reflux for 3 hours. Then, it cooled to cooling at room temperature and distilled the solvent off under reduced pressure. 1M NaOH and ethyl acetate were added to the residue, and the suspension was filtered and filtered. The filtrate was partitioned between purified water and ethyl acetate, and the organic layer was extracted. The extract was washed with a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (334 mg, yield 89%). ¹ H NMR (400MHz, CDCl ₃ ) δ 6.69 (dd, J = 1.2 Hz, 8.2 Hz, 1H), 6.84 (s, 1H), 6.87 (s, 1H), 7.40 (d, J = 14.8 Hz, 2H) , 7.42 (d, J = 21.2 Hz, 1H), 7.76 (dd, J = 2.4 Hz, 9.4 Hz, 1H), 8.34 (d, J = 2.8 Hz, 1H). MS (DART) m / z: 356, 358 (M + H ⁺ ).

Production Example 11
Synthesis of 2- (6-aminobenzofuran-2-yl) -6- (tributylstannyl) -4H-chromen-4-one (32) compound 31 (50 mg, 0.14 mmol) in 1,4-dioxane: triethylamine = 3: Dissolved in a mixed solvent of 5 (5 mL), bis (tributyltin) (1.15 g / ml) (417.28 μL, 1.00 mmol) and (PPh ₃ ) ₄ Pd (58 mg, 0.05 mmol) were added, and 90 The mixture was stirred at 14 ° C. for 14 hours. Then, it cooled to room temperature and filtered using celite, and the solvent of the solution was depressurizingly distilled. The residue was purified with an open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (7 mg, yield 9%). ¹ H NMR (400MHz, CDCl ₃ ) δ 0.87-1.613 (m, 27H), 6.66 (dd, J = 2 Hz, 10 Hz, 1H), 6.83 (s, 1H), 6.86 (s, 1H), 7.37 ( s, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.76 (dd, J = 0.8 Hz, 8 Hz, 1H), 8.30 (d, J = 1.2 Hz, 1H). MS (DART) m / z: 566 (M + H ⁺ ).

Example 5
Synthesis of 2- (6-aminobenzofuran-2-yl) -6-iodo-4H-chromen-4-one (33) Compound 32 (8 mg, 0.014 mmol) was dissolved in chloroform (3 mL) and 0.2 M Iodine solution (chloroform solution) was added until the reaction solution turned purple, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous sodium hydrogen sulfite solution was added until the color of the reaction solution changed from purple to tan, and the organic layer was separated and extracted with chloroform and purified water, and washed with a saturated aqueous sodium chloride solution. Thereafter, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (5 mg, yield 88%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.99 (s, 2H), 6.69 (dd, J = 2 Hz, 8 Hz 1H), 6.82 (s, 1H), 6.84 (s, 1H), 7.29 (s, 1H ), 7.38 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.94 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 8.54 (d, J = 2 Hz, 1H). MS ( DART) m / z: 403 (M + H ⁺ ).

Example 6
Synthesis of 6-bromo-2- (6- (methylamino) benzofuran-2-yl) -4H-chromen-4-one (34) Compound 31 (40 mg, 0.112 mmol) was dissolved in methanol (20 mL). Paraformaldehyde (18 mg, 0.599 mmol) and sodium methoxide (30 μL, 27.0-29.7 w / w%, 0.224 mmol) were added, and the mixture was heated to reflux for 1 hour. Thereafter, sodium borohydride (25 mg, 0.66 mmol) was added, and the mixture was heated to reflux for 3 hours. The mixture was cooled to room temperature, and 1M NaOH was added under ice cooling. The organic layer was separated and extracted with chloroform and purified water, and washed with a saturated aqueous sodium chloride solution. Thereafter, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: chloroform = 1: 9) to obtain the title compound (9 mg, yield 22%). ¹ H NMR (400MHz, CDCl ₃ ) δ 2.93 (s, 3H), 6.63 (dd, J = 2 Hz, 8 Hz 1H), 6.69 (s, 1H), 6.83 (s, 1H), 7.38-7.42 (m , 3H), 7.76 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 8.34 (d, J = 2.4 Hz, 1H). MS (DART) m / z: 370 (M + H ⁺ ).

Production Example 12
Synthesis of 2- (6- (methylamino) benzofuran-2-yl) -6- (tributylstannyl) -4H-chromen-4-one (35) 34 (14 mg, 0.038 mmol) was converted to 1,4- Dissolve in a mixed solvent of dioxane: triethylamine = 3: 4 (7 mL), and add bis (tributyltin) (1.15 g / ml) (121.89 μL, 0.015 mmol) and (PPh ₃ ) ₄ Pd (17 mg, 0.015 mmol). In addition, the mixture was heated and stirred at 80 ° C. for 14 hours. Then, it cooled to room temperature and filtered using celite, and the solvent of the solution was depressurizingly distilled. The residue was purified with an open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (9 mg, yield 41%). ¹ H NMR (400MHz, CDCl ₃ ) δ 0.87-1.61 (m, 27H), 2.93 (s, 3H), 6.62 (dd, J = 2 Hz, 8.8 Hz, 1H), 6.71 (s, 1H), 6.85 ( s, 1H), 7.38 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 8.30 ( s, 1H). MS (DART) m / z: 582 (M + H ⁺ ).

Example 7
Synthesis of 6-iodo-2- (6- (methylamino) benzofuran-2-yl) -4H-chromen-4-one (36) 35 (8 mg, 0.014 mmol) was dissolved in chloroform (10 mL). 0.2 M iodine solution (chloroform solution) was added until the reaction solution turned purple, and the mixture was stirred at room temperature for 3 hours. A saturated aqueous sodium hydrogen sulfite solution was added until the color of the reaction solution changed from purple to tan, and the organic layer was separated and extracted with chloroform and purified water, and washed with a saturated aqueous sodium chloride solution. Thereafter, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (5 mg, yield 87%). ¹ H NMR (400MHz, CDCl ₃ ) δ 2.92 (s, 3H), 6.63 (dd, J = 2 Hz, 8.2 Hz, 1H), 6.70 (s, 1H), 6.83 (s, 1H), 7.39-7.42 ( m, 3H), 7.68 (d, J = 1.6 Hz, 1H), 8.22 (dd, J = 2 Hz, 8.2 Hz, 1H). MS (DART) m / z: 418 (M + H ⁺ ).

Example 8
Synthesis of 6-bromo-2- (6- (dimethylamino) benzofuran-2-yl) -4H-chromen-4-one (37) Compound 31 (65 mg, 0.18 mmol) was dissolved in acetic acid (12 mL). Paraformaldehyde (54 mg, 1.80 mmol) and sodium cyanoborohydride (68 mg, 1.08 mmol) were added. Then, it stirred at room temperature for 4 hours. Thereafter, 1M NaOH was added to stop the reaction. The organic layer was separated and extracted with purified water and chloroform, and washed with a saturated aqueous sodium chloride solution. Thereafter, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (50 mg, yield 71%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.04 (s, 6H), 6.77-6.819 (m, 3H), 7.38 (s, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 2.4 Hz, 9.2 Hz, 1H), 8.33 (d, J = 2.4 Hz, 1H) .MS (DART) m / z: 384, 386 (M + H ⁺ ).

Production Example 13
Synthesis of 2- (6- (dimethylamino) benzofuran-2-yl) -6- (tributylstannyl) -4H-chromen-4-one (38) 37 (50 mg, 0.13 mmol) Dissolve in a mixed solvent (5 mL) of dioxane: triethylamine = 3: 2 and add bis (tributyltin) (1.15 g / ml) (417.28 μL, 1.00 mmol) and (PPh ₃ ) ₄ Pd (58 mg, 0.05 mmol). In addition, the mixture was heated and stirred at 90 ° C. for 14 hours. Then, it cooled to room temperature and filtered using celite, and the solvent of the solution was depressurizingly distilled. The residue was purified with an open column (elution solvent; ethyl acetate: hexane = 1: 4) to obtain the title compound (21 mg, yield 27%). ¹ H NMR (400MHz, CDCl ₃ ) δ 0.87-1.613 (m, 27H), 3.06 (s, 6H), 6.78 (d, J = 2.4 Hz, 1H), 6.80 (s, 1H), 6.85 (s, 1H ), 7.38 (s, 1H), 7.44-7.49 (m, 2H), 7.75 (dd, J = 1.6 Hz, 12 Hz, 1H), 8.30 (d, J = 1.6 Hz, 1H). MS (DART) m / z: 594 (M + H ⁺ ).

Example 9
Synthesis of 2- (6- (dimethylamino) benzofuran-2-yl) -6-iodo-4H-chromen-4-one (39) Compound 38 (12 mg, 0.020 mmol) was dissolved in chloroform (3 mL). 0.2 M iodine solution (chloroform solution) was added until the reaction solution turned purple, and the mixture was stirred at room temperature for 2 hours. A saturated aqueous sodium hydrogen sulfite solution was added until the color of the reaction solution changed from purple to tan, and the organic layer was separated and extracted with chloroform and purified water, and washed with a saturated aqueous sodium chloride solution. Thereafter, it was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 3) to obtain the title compound (8 mg, yield 91.9%). ¹ H NMR (400MHz, CDCl ₃ ) δ 3.10 (s, 6H), 6.78 (s, 1H), 6.82 (s, 1H), 7.25 (s, 1H), 7.28 (s, 1H), 7.38 (s, 1H ), 7.48 (d, J = 9.2 Hz, 1H), 7.92 (dd, J = 2 Hz, 8.6 Hz, 1H), 8.53 (d, J = 2.4 Hz, 1H). MS (DART) m / z: 432 (M + H ⁺ ).

Production Example 14
Synthesis of ethyl 6-nitrobenzo [d] oxazole-2-carboxylate (40) 2-Amino-5-nitrophenol (200 mg, 1.30 mmol) was dissolved in dioxane (5.0 mL) and ethyl glyoxylate (664 mg, 3.25 mmol) was added and the mixture was stirred at 50 ° C. for 2 hours. Subsequently, ammonium cerium nitrate (CAN) (1.07 g, 1.95 mmol) was added and heated to reflux for 9 hours. Thereafter, the mixture was filtered through Celite and washed with ethyl acetate, and then the solvent was distilled off under reduced pressure. The residue was purified by open column (elution solvent; ethyl acetate: hexane = 1: 5) to obtain the title compound (20 mg, yield 7%). ¹ H NMR (400MHz, CDCl ₃ ) δ 1.53 (d, J = 3.6 Hz, 3H), 4.60 (d, J = 3.6 Hz, 2H), 8.04 (d, J = 8.8 Hz, 1H), 8.41 (d, J = 9 Hz, 1H), 8.59 (s, 1H). MS (DART) m / z: 237 (M + H ⁺ ).

Production Example 15
Synthesis of 6-nitrobenzo [d] oxazole-2-carboxylic acid (41) Compound 40 (21 mg, 0.091 mmol) was dissolved in methanol (5 mL), 1M NaOH was added, and the mixture was stirred at room temperature for 10 minutes. Thereafter, methanol was distilled off under reduced pressure, and 1M HCl was added under ice cooling. The organic layer was separated and extracted with ethyl acetate and purified water, washed with a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by an open column (elution solvent; chloroform: methanol = 3: 1, 1% acetic acid) to obtain the title compound (6 mg, yield 31.7%). MS (DART) m / z: 207 (M + H -).

Example 10
Using Synthesis Compound 41 of 6-bromo-2- (6-nitrobenzo [d] oxazol-2-yl) -4H-chromen-4-one , the same as in Production Example 7, Production Example 9 and Example 2 By the method, the title compound is obtained.

The following compounds were used as comparative compounds. The structure of the compound is shown in FIGS.
(E) -6- (Iodo-125I) -2- (4-methoxystyryl) -4H-chromen-4-one (1)
(E) -2- (4- (Dimethylamino) styryl) -6- (iodo-125I) -4H-chromen-4-one (2)
(E) -2- (4-((2-hydroxyethyl) amino) styryl) -6-iodo-4H-chromen-4-one (10)
(E) -6-Iodo-2- (2- (6-methoxypyridin-3-yl) vinyl) -4H-chromen-4-one (16)
(E) -2- (2- (6- (Dimethylamino) pyridin-3-yl) vinyl) -6-iodo-4H-chromen-4-one (17)
Compounds 1 and 2 were synthesized according to the methods described in References 1 and 2.

Radioactive iodine-labeled precursor tributyltin (1 mg / mL) in methanol solution (50 μL), Na ¹²⁵ I (0.1-0.2 mCi, specific activity 2200 Ci / mmol), 1.0 M HCl (50 μL), 3 W / v% hydrogen peroxide solution (50 μL) was sequentially added, and the mixture was allowed to stand at room temperature for 10 minutes, or stirred for 10 seconds and then left for 15-20 minutes to react. After completion of the reaction, a saturated sodium hydrogen sulfite aqueous solution (100 μL) was added to stop the reaction, and a saturated sodium hydrogen carbonate aqueous solution (100 μL) was added to adjust the pH of the reaction solution to 9-10. Liquid separation extraction was performed with ethyl acetate, and after dehydration with anhydrous sodium sulfate, the solvent was distilled off with nitrogen gas. The residue was purified by reverse phase HPLC. Absorbance at 254 nm was analyzed by HPLC using a non-radioactive compound as a standard, and it was confirmed that it coincided with the separated radioactive compound. The solvent of the collected solution was distilled off with nitrogen gas, and then stored at 4 ° C. as an ethanol solution until used in various experiments.

Analysis of [ ¹²⁵ I] SC, BFC derivatives by reversed-phase HPLC The cis-trans photoisomerization that occurred due to the styryl group in the SC skeleton was caused by the structural change of the styryl group to a 5-membered ring. In order to confirm that it was lost from the (BFC) derivative, analysis by reverse phase HPLC was performed.
Compound 2 was analyzed by a previously reported method (References 1 and 2). In the analysis of compounds 30, 33 and 39, reverse phase HPLC was set at a flow rate of 1.0 mL / min and acetonitrile / water (7: 3) was used as the mobile phase. The mobile phase was conditioned for about 1 hour before sample injection. The absorbance of the above compound at a wavelength of 254 nm was recorded. The results are shown in FIG.
It was confirmed that the cis and trans isomers derived from the photoisomerization of [ ¹²⁵ I] 2 which is an SC derivative showed peaks at 12 min and 18 min after injection into HPLC.
On the other hand, B ¹²⁵ derivatives [ ¹²⁵ I] 30, [ ¹²⁵ I] 33 and [ ¹²⁵ I] 39 were confirmed to have peaks at 10 min, 5 min and 15 min, respectively, after injection into HPLC. At this time, unlike the SC derivative, cis and trans isomers that were thought to be derived from photoisomerization were not observed. From this, the BFC skeleton formed by structural conversion of the styryl group of the SC skeleton to a 5-membered ring was improved in structure stabilization without photoisomerization.

Evaluation of binding affinity of [ ^125I ] SC, VPC and BFC derivatives to recombinant mouse prion protein (rMoPrP) aggregates to evaluate the binding affinity of SC, vinylpyridylchromone (VPC) and BFC derivatives to PrP ^Sc , RMoPrP aggregates were used as models for PrP ^Sc and [ ¹²⁵ I] SC, VPC and BFC derivatives ([ ¹²⁵ I] 2, [ ¹²⁵ I] 10, [ ¹²⁵ I] 16, [ ¹²⁵ I] 17, [ ¹²⁵ I ], [ ¹²⁵ I] 33, [ ¹²⁵ I] 36, [ ¹²⁵ I] 39) were subjected to in vitro binding experiments.

(1) Expression and purification of rMoPrP Expression and purification of rMoPrP were performed according to a previously reported technique (Reference 3). First, BL21 Escherichia coli into which the 23rd to 23rd DNA sequences of mouse prion protein were introduced was cultured in ampicillin-containing LB medium. Thereafter, it was treated with CalLytic B, lysozyme and benzonase to dissolve E. coli. RMoPrP solubilized with a denaturant (6.0 M Guanidine) was purified by affinity chromatography using Ni-NTA (Nickel-NitriloTriacetic Acid) to refold the protein. The eluted rMoPrP was dialyzed overnight in ultrapure water and stored at −80 ° C. until aggregate formation.

(2) Preparation of rMoPrP aggregate An rMoPrP aggregate was prepared according to a previously reported technique (Reference 4). After rMoPrP (925 mg / L) was diluted to a concentration of 2.8 μM with 50 mM HEPES buffer (containing 300 mM NaCl, pH = 7.5), 200 μL each was dispensed into a 96-well microplate. After placing the plate on Tecan's infinite F200, 9 cycles of shaking for 30 seconds and standing for 30 seconds at 40 ° C were performed, and then rMoPrP aggregates were repeated for 72 hours by standing for 15 seconds. Prepared. Stored at −80 ° C. until used for experiments. When used in the experiment, 2.0 μM, 200 nM rMoPrP aggregates were prepared by thawing at room temperature and diluting with 50 mM HEPES buffer (containing 300 mM NaCl, pH = 7.5).

(3) Adsorption experiment using rMoPrP aggregates ¹²⁵ I-labeled SC, VPC, BFC derivatives Weigh out to 1.3 million cpm, distill off ethanol with nitrogen gas, and then add 50 mM HEPES buffer (1,300 μL, 40 % DMSO, containing 300 mM NaCl, pH = 7.5). For the rMoPrP aggregate solution, prepare 75, 90, 95 100 μL of each mixture diluted with 50 mM HEPES buffer so that the final concentration is 0, 5, 10, 50, 100, 250 nM. After mixing with ¹²⁵ I-labeled SC, VPC, and BFC derivative solution (100 μL) in a silicate glass tube (12 mm × 75 mm), the mixture was shaken at room temperature for 2 hours. After shaking, the reaction solution was permeated through a GF / B filter using an M-24R cell harvester and washed 4 times with 50 mM HEPES buffer (containing 20% DMSO, 300 mM NaCl, pH = 7.5). The radioactivity remaining on the filter was measured with a γ counter, and the adsorptivity of ¹²⁵ I-labeled SC, VPC and BFC derivatives to the rMoPrP aggregate solution was calculated from the obtained values. In recent studies, 6- [ ¹²⁵ I] SC-NMe ₂ [ ¹²⁵ I] 2 showed the highest binding affinity and binding amount of rflavopr aggregates among flavonoid analogues ( Reference 1), used as a positive control.

(4) Results FIG. 7 shows the binding rate of SC, VPC and BFC derivatives to rMoPrP aggregates (250 nM).
As a result, it was confirmed that [ ¹²⁵ I] 2, which showed the highest binding rate to rMoPrP aggregates among the flavonoid analogues, showed high binding properties of about 10%. Furthermore, [ ¹²⁵ I] 10 introduced with an ethanolamino group at the 4′-position of the SC skeleton showed a binding rate equivalent to [ ¹²⁵ I] 2, indicating that it has a high binding property to rMoPrP aggregates. On the other hand, in [ ^125I ] 16 and [ ^125I ] 17, in which a methoxy group and a dimethyl group are introduced at the 4'-position of the VPC skeleton, the bonding rate is 1% or less, which is lower than that of the SC derivative. The rate dropped significantly.

In addition, [ ^125I ] 30, [ ^125I ] 33, in which a methoxy group, an amino group, a monomethylamino group, and a dimethylamino group were introduced at the 4 ′ position of the BFC skeleton obtained by structural conversion of the styryl group of the SC skeleton into a 5-membered ring, The binding rates of [ ¹²⁵ I] 36 and [ ¹²⁵ I] 39 were 14.2%, 7.2%, 10.6%, and 18.0%, respectively. In particular, [ ¹²⁵ I] 30 and [ ¹²⁵ I] 39 were found to have a binding rate to rMoPrPG aggregates equal to or higher than that of the SC derivative. It is suggested that changing the styryl group of the SC skeleton to a 5-membered ring and introducing benzofuran into the skeleton leads to improved binding affinity to rMoPrP aggregates classified as amyloid as well as amyloid β (Aβ) It was done. Compared with benzofuranyl chromone derivatives [ ¹²⁵ I] 33, [ ¹²⁵ I] 36, and [ ¹²⁵ I] 39 with amino groups introduced at the 4 ′ position, tertiary amine derivatives [ ¹²⁵ I] 39> secondary amine derivatives [ ¹²⁵ I] 36> Primary amine derivative [ ¹²⁵ I] 33. It was suggested that introduction of a hydrophobic electron-donating group at the 4 'position of the BFC skeleton leads to improved affinity for rMoPrP aggregates.

Binding saturation experiments for BFC derivatives to rMoPrP aggregates To examine the binding properties of BFC derivatives to rMoPrP aggregates in more detail, [ ¹²⁵ I] 30, [ ¹²⁵ I] 33, [ ¹²⁵ I] 36, and [ ¹²⁵ I ] Was used to perform bond saturation experiments on rMoPrP aggregates.
A mixed solution (200 nM) of [ ¹²⁵ I] 30, [ ¹²⁵ I] 33, [ ¹²⁵ I] 36, and [ ¹²⁵ I] 39 and the corresponding non-radioactive compound was sequentially added to a 50 mM HEPES buffer (20% DMSO, It was diluted with 300 mM NaCl, pH = 7.5) to a final concentration of 3.125-200 nM. 100 μL of various concentrations of the mixture and 100 μL of rMoPrP aggregate solution (100 nM) were mixed in a borosilicate glass tube (12 mm × 75 mm) and shaken for 2 hours at room temperature. Nonspecific binding was calculated by adding 100 μL of HEPES buffer instead of rMoPrP aggregate solution. After shaking, the reaction solution was permeated through a GF / B filter using an M-24R cell harvester. The radioactivity remaining on the filter was measured with an autowell gamma counter, and a saturation curve was prepared from the obtained data. A saturation curve and a Scatchard plot were created from the data obtained using GraphPad Prism (GraphPad Software, San Diego, Calif.), And the dissociation constant _Kd and the maximum binding amount _Bmax were calculated.
The results are shown in FIG. Compounds [ ^125I ] 30, [ ^125I ] 33, [ ^125I ] 36, and [ ^125I ] 39 are fitted with a one binding site model for rMoPrP aggregates with _Kd values of 49.2 ± 7.6 nM, 26.1 ± 2.6 nM, 23.1 ± 1.1 nM, 22.6 ± 2.0 nM, B _max values are 25.7 ± 1.9 mmol / mol protein, 12.6 ± 0.5 mmol / mol protein, 23.0 ± 3.6 mmol / mol protein, 27.6 ± 1.2 mmol / mol protein And calculated.

Evaluation of the binding affinity of BFC derivatives to PrP ^Sc on mBSE-infected mouse brain sections To evaluate the binding affinity to PrP ^Sc deposited on mouse brain sections, mBSE-infected mice of compounds 30, 33 and 39 (mBSE infection Fluorescence staining was performed using brain sections about 20-22 weeks later. As a control group, fluorescent staining was also performed using brain sections prepared from frozen brain blocks of uninfected mice of the same week.

(1) Preparation of mBSE-infected mice Mouse-adapted BSE (mBSE) -infected mice were prepared by inoculating the brain of 20% of 1% brain emulsion of mBSE-infected brain into the right temporal region of 4-week-old ddY male mice. did. Further, 20 μL of PBS solution as non-infected mice was inoculated in the brain on the right temporal region of 4-week-old ddY male mice. Each group was bred for 20-25 weeks and then used to prepare mouse brain slices.

(2) Preparation of mBSE-infected mouse brain sections BSE-infected mice and non-infected mice were fixed by perfusion with physiological saline, and then the brains were collected and immediately immersed in 10% neutral buffered formalin fixative. After fixing with formalin, it was immersed in a 70% ethanol solution, further immersed in an 80, 90, 100% ethanol solution in order, dehydrated, further dealcoholized with xylene, and then embedded in paraffin. The block embedded in paraffin was ice-cooled, sliced to 3.0 μm with a microtome, attached to a slide glass, immersed in a warm 1% acetic acid solution, and then dried overnight in a constant temperature bath at 40 ± 3 ° C.

(3) Staining experiment using BSE-infected mouse brain section
(3-1) Fluorescence staining experiment Brain sections of BSE prion-infected model mouse mice were first deparaffinized. First, it was immersed in xylene for 5 minutes × 3 times, and then immersed in 99.5% ethanol for 5 minutes × 3 times. After deparaffinization, the sections were immersed in each compound (100 μM) dissolved in 50% ethanol for 1 hour. After washing with 50% ethanol for 2 minutes × 2 times, observation was performed using a fluorescence microscope.

(3-2) Immunostaining experiment Using the BSE prion-infected model mouse brain section in which the above fluorescence staining experiment was performed, an immunostaining experiment was subsequently performed. After washing with 50% ethanol for 10 minutes, autoclaving (121 ° C., 12 minutes) in 1.2 mM dilute hydrochloric acid solution for antigen activation was followed by immersion in 90% formic acid for 5 minutes. Subsequently, the membrane was immersed in a 0.3% H ₂ O ₂ / methanol solution for 30 minutes for blocking, and then immersed in normal goat serum diluted 20 times for 30 minutes. React with the primary antibody SAF32 (2.0 μg / mL) that recognizes the N-terminal octarepeat residue of PrP overnight at room temperature. I left it. After adding 3,3′-diaminobenzidine tetrahydrochloride solution to confirm color development, the brain section was placed in a hematoxylin solution and allowed to stand for 1 minute. Thereafter, it was washed with running water, dehydrated, dealcoholized and sealed, and then observed using an optical microscope.

(4) Results In the fluorescence staining experiment of non-infected mouse brain sections, clear fluorescence images of each BFC derivative were not observed, but only background fluorescence was observed (FIGS. 9a, d and g). On the other hand, in the fluorescence staining experiment of brain sections of infected mice, clear fluorescent images of compounds 30, 33 and 39, which are BFC derivatives, were obtained (FIGS. 9b, e and h). As a result, immunostaining was performed on the same section as that used in the fluorescence staining experiments with compounds 30, 33 and 39 to confirm the location of PrP ^Sc. Images were observed (FIGS. 9c, f and i). It was confirmed that the fluorescence image derived from the BFC derivative was coincident with the PrP ^Sc positive site, suggesting that the BFC derivative recognized the PrP ^Sc deposition site in the brain. From this result, it was revealed that BFC derivatives 30, 33 and 39 have binding properties to PrP ^Sc deposited in the mouse brain.

Evaluation of in vivo radioactivity distribution in normal mice of [ ¹²⁵ I] SC and BFC derivatives In vivo radioactivity distribution experiments using normal mice were conducted to examine the brain migration and subsequent clearance of SC and BFC derivatives.
(1) Radioactivity distribution experiments in normal mice Labeled [ ¹²⁵ I] 10, [ ¹²⁵ I] 30, [ ¹²⁵ I] 33, [ ¹²⁵ I] 36 and [ ¹²⁵ I] 39 in 20% DMSO-containing saline Was diluted as above and used as an injection solution. One group of 5 to 7 5-week-old ddY male mice (20-25 g) was labeled with the respective labeled [ ¹²⁵ I] 10, [ ¹²⁵ I] 30, [ ¹²⁵ I] 33, [ ¹²⁵ I] 36 and [ ¹²⁵ I] 39 was administered from the tail vein of 100 μL (6.2-22.8 kBq) per animal. After 2, 30, 60, 120, and 180 minutes after administration, decapitation and blood collection were performed, and then major organs were removed. Next, immediately after measuring the weight of blood and organs, the radioactivity was measured and expressed as% injected dose / g (% ID / g), which is the ratio of the amount of radioactivity accumulated per gram of each organ relative to the dose. .

(2) Results ^{[125 I] 10, [125} I] 30, the body distribution of radioactivity in Tables 1 and 2 in ^{[125 I] 33, [125} I] 36 and ^[125 I] 39 normal mice, ^[125 Figure 10-1 shows the radioactivity distribution in the brain of [I] 1, [ ^125I ] 2, [ ^125I ] 10, [ ^125I ] 30, [ ^125I ] 33, [ ^125I ] 36, and [ ^125I ] 39. Fig. 10-2 shows the radioactivity distribution of [ ¹²⁵ I] 1, [ ¹²⁵ I] 2, [ ¹²⁵ I] 10, [ ¹²⁵ I] 30, [ ¹²⁵ I] 33 and [ ¹²⁵ I] 39 in the brain. In addition, [ ¹²⁵ I] 1, [ ¹²⁵ I] 2, [ ¹²⁵ I] 10, [ ¹²⁵ I] 30, [ ¹²⁵ I] 33, [ ¹²⁵ I] 36 and [ ¹²⁵ I] 39 parameters of radioactivity distribution in the brain The values are shown in Table 3 along with the cLogP values for each derivative.

SC, BFC derivatives [ ^125I ] 10, [ ^125I ] 30, [ ^125I ] 33, [ ^125I ] 36, [ ^125I ] 39 in the early stage of administration (2-30 minutes) The maximum values were 0.88, 4.22, 5.06, 3.08, and 3.16% ID / g, respectively.
To introduce ethanol amino group SC skeleton ^[125 I] 10, the brain resistance and loss of greater than ^[125 I] 1 having the best brain-localizing the fugitive in previously reported SC derivative was observed . The cLogP values tended to be higher in the order of [ ¹²⁵ I] 2, [ ¹²⁵ I] 1, [ ¹²⁵ I] 10, but no relationship was found for brain migration or disappearance.

On the other hand, [ ¹²⁵ I] 30, [ ¹²⁵ I] 33, and [ ¹²⁵ I] 39, in which a methoxy group, an amino group, and a dimethylamino group were introduced into the BFC skeleton, exhibited a brain migration property that exceeded the previously reported SC derivatives. The ratio of radioactivity in the brain 2 minutes after administration of [ ¹²⁵ I] 30, [ ¹²⁵ I] 33 or 180 minutes after administration of [ ¹²⁵ I] 39 was calculated as 6.1, 6.7, and 8.9%, respectively. As a result, these compounds were shown to have very high disappearance from the brain. When [ ¹²⁵ I] 30, [ ¹²⁵ I] 33 and [ ¹²⁵ I] 39 were compared, it was shown that the lower the lipid solubility, the better the brain migration and disappearance. In particular, [ ¹²⁵ I] 33 (cLogP = 4.15) having moderate fat solubility had a ratio of accumulation in the brain of 14.9 and 2.6 after 2 minutes, 180 minutes, 2 minutes and 60 minutes, respectively. 2 and 60 minutes after [ ¹⁸ F] Florbetapir ([ ¹⁸ F] AV-45) and [ ¹⁸ F] Florbetaben ([ ¹⁸ F] BAY94-917218), which are clinically used as PET imaging agents for Aβ The ratio of brain accumulation has been reported to be 3.8 (reference 5) and 4.8 (reference 6), respectively. Therefore, it is considered that the compound [ ¹²⁵ I] 33 of the present invention has a brain behavior useful as a prion imaging agent.

Evaluation of BFC derivatives as PrP ^Sc selective imaging probes
1. Experimental method
1.1. Reagent / Equipment A commercially available first grade reagent or special grade reagent was used.
For analysis and fractionation of the compound using HPLC, a liquid chromatograph LC-10AT equipped with Shimadzu UV-visible fluorescence detector SPD-10A and Chromatopack C-R8A was used. For reverse phase HPLC, a Cosmosil 5C ₁₈ -AR column (4.6 × 150 mm) manufactured by Nacalai Tesque was used, and analysis was performed at a flow rate of 1.0 mL / min using a mixed solution of H ₂ O and acetonitrile as an elution solvent. As radioactive iodine-125, sodium iodide (Na ¹²⁵ I, 3.7 GBq / mL, 0.01 N NaOH solution) manufactured by PerkinElmer Japan was used. M-24 cell harvester (Brandel, Gaithersburg, MD) and GF / B filter (Whatman, Kent, UK) were used for separation of bound and unbound molecules to amyloid aggregates in adsorption and binding saturation experiments. . ¹ H NMR was measured using JEOL JNM-AL 400 using tetramethylsilane (TMS) as an internal standard substance and deuterated chloroform (CDCl ₃ ) or deuterated methanol (CD ₃ OD) as a solvent. DART (Direct Analysis in Real Time) mass spectrometry was measured using JMS-T100TD manufactured by JEOL Ltd. For the radioactivity measurement, a Wizard autowell gamma counter manufactured by PerkinElmer Japan was used. Cytation 3 manufactured by Bio Tek was used for measurement of fluorescence intensity.

Aβ _1-42 manufactured by Peptide Institute was used. α-Syn used was transferred from Nagasaki University School of Medicine. A centrifuge manufactured by Kubota Corporation was used for the centrifugation in the binding saturation experiment for α-Syn aggregates. The fluorescence image of the compound accumulated in the brain section was observed using a fluorescence microscope (BZ-8100) manufactured by Keyence or a fluorescence microscope (ECLIPSE 80i) manufactured by Nikon.

1.2. Preparation of Aβ _1-42 aggregates Aβ _1-42 is dissolved in 10 mM phosphate buffer (1 mM EDTA, pH 7.4), adjusted to a concentration of 0.25 mg / mL (55.4 μM), and adjusted to 60 ° C. And shaken for about 30 minutes. Thereafter, Aβ _1-42 aggregates were prepared by incubating at 37 ° C. for 42 hours, and stored at −80 ° C. until used for experiments. After diluting the Aβ _1-42 aggregate to a concentration of 0.554 μM with 10 mM phosphate buffer (1 mM EDTA, pH 7.4), add thioflavin T (ThT), an amyloid fluorescent dye with a concentration of 20 μM, It was confirmed that a β-sheet structure was formed by measuring fluorescence intensity at an excitation wavelength of 450 nm and a fluorescence wavelength of 485 nm. As a control, the fluorescence intensity of ThT was measured when 10 mM phosphate buffer (1 mM EDTA, pH 7.4) was used instead of Aβ _1-42 aggregates.

1.3.Preparation of α-Syn aggregates α-Syn is dissolved in 30 mM Tris-HCl buffer (200 mM NaCl, pH 6.0, 7.0, 8.0) and adjusted to 1.67 mg / mL (115 μM) at 37 ° C. The mixture was stirred for 72 hours and stored at -80 ° C until used in the experiment. The α-Syn aggregate is diluted to 1.2 μM with PBS, then 20 μM of ThT is added, and the fluorescence intensity at excitation wavelength 440 nm and fluorescence wavelength 485 nm is measured to form a β-sheet structure. It was confirmed. As a control, the fluorescence intensity of ThT when PBS was used instead of the α-Syn aggregate was measured. To confirm the size of the aggregate, dilute the α-Syn aggregate to a concentration of 0.5 μM, then centrifuge at 20,000 g for 15 minutes at 20 ° C. Pre-centrifuge α-Syn, precipitated α-Syn and 20 μM The fluorescence intensity at an excitation wavelength of 440 nm and a fluorescence wavelength of 485 nm was measured. As a control, the fluorescence intensity of ThT when PBS was used instead of the α-Syn aggregate was measured.

1.4. Adsorption experiment for α-Syn aggregates In a borosilicate glass tube (φ12 × 75 mm), 100 μL of [ ¹²⁵ I] -labeled compound (150,000-170,000 cpm) in 20% EtOH and α-Syn aggregates ( (500 nM) 100 μL was mixed (α-Syn final concentration: 250 nM) and shaken at 50 rpm at room temperature for 2 hours. After shaking, the reaction solution was passed through a GF / B filter using an M-24 cell harvester. After washing 4 times with 10% EtOH at room temperature, the radioactivity remaining in the filter was measured with an autowell gamma counter, and the adsorption rate was calculated using the obtained count value (cpm). Nonspecific adsorption was calculated by conducting the same experiment using PBS instead of α-Syn aggregates.

1.5. Binding saturation experiment for α-Syn aggregates [ ^125I ] BFC derivative and corresponding non-radioactive compound mixed solution (2000 nM) were sequentially diluted with 20% EtOH to prepare final concentration of 24.7-2000 nM did. 400 μL of the mixed solution of various concentrations and 400 μL of α-Syn aggregate (200 nM) were mixed in a polypropylene tube and shaken at 50 rpm at room temperature for 2 hours. After shaking, 200 μL was collected before centrifugation. The remaining 600 μL was centrifuged at 20 ° C. and 20,000 g for 15 minutes. The radioactivity of 100 μL of the sample before centrifugation and the supernatant was measured with an autowell gamma counter, and the binding rate was calculated using the obtained count value (cpm). Nonspecific binding was calculated by adding 400 μL of PBS not containing α-Syn aggregates. Subsequently, Scatchard analysis was performed using GraphPad Prism (GraphPad Software, San Diego, Calif.), And the dissociation constant _Kd value and the maximum binding amount _Bmax value were calculated.

1.6. Binding saturation experiment on Aβ _1-42 aggregate [ ¹²⁵ I] BFC derivative and the corresponding non-radioactive compound mixed solution (2000 nM) were sequentially diluted with 20% EtOH to a final concentration of 24.7-2000 nM. . A mixture of 100 μL of various concentrations and 100 μL of Aβ _1-42 aggregate (200 nM) were mixed in a borosilicate glass tube (φ12 × 75 mm) and shaken at 50 rpm for 2 hours at room temperature. Nonspecific binding was calculated by adding 100 μL of a phosphate buffer not containing Aβ _1-42 aggregates. After shaking, the reaction solution was permeated through a GF / B filter using an M-24R cell harvester. After washing 4 times with 10% EtOH at room temperature, the radioactivity remaining on the filter was measured with an autowell gamma counter, and a saturation curve was prepared using the obtained count value (cpm). Subsequently, Scatchard analysis was performed using GraphPad Prism (GraphPad Software, San Diego, Calif.), And the dissociation constant _Kd value and the maximum binding amount _Bmax value were calculated.

1.7. Fluorescence and immunostaining of mouse brain slices
1.7.1. Fluorescent staining of mBSE-infected mouse brain sections Paraffin-embedded mBSE-infected mouse brain sections were soaked in xylene (5 min x 3 times) followed by 99.5% EtOH (5 min x 3 times). Paraffin treatment was performed. Thereafter, it was immersed in each compound (100 μM) dissolved in 50% EtOH for 1 hour. After washing with 50% EtOH for 2 minutes × 2 times, observation was performed using a fluorescence microscope.

1.7.2. Immunostaining of mBSE-infected mouse brain sections Adjacent sections of mBSE-infected mouse brain sections that had undergone the above fluorescence staining were deparaffinized in the same manner as described above, and PrP immunostaining was performed. In order to remove the normal PrP protein and activate the antigen, it was autoclaved (121 ° C., 10 minutes) in 1.2 mM dilute hydrochloric acid solution, and then immersed in 90% formic acid for 5 minutes. Subsequently, it was immersed in a 0.3% H ₂ O ₂ / MeOH solution for 30 minutes for blocking, and then immersed in normal goat serum diluted 20 times for 30 minutes. React with the primary antibody SAF32 (1:20), which recognizes the N-terminal octarepeat residue of PrP, overnight at room temperature. Washed. Thereafter, the secondary antibody was added and left at room temperature for 2 hours, followed by washing in the same manner as in the treatment with the primary antibody. 3,3′-Diaminobenzidine tetrahydrochloride solution was added to confirm color development, and then washed with Tris-HCl buffer and running water. Then, it observed using the optical microscope.

1.7.3. Fluorescence staining of Tg2576 mouse brain sections Frozen brain sections of Tg2576 mice (24 months old) overexpressing amyloid precursor protein (APP) were dissolved in 50% EtOH in each compound (100 μM). Soaked for 1 hour. After washing with 50% EtOH for 2 minutes × 2 times, observation was performed using a fluorescence microscope. Further, the adjacent sections were soaked in ThT (100 μM) dissolved in 50% EtOH for 10 minutes, and washed with 50% EtOH for 1 minute × 2 times to confirm Aβ aggregates in the brain sections.

2. Results
2.1. Check A [beta] _1-42 of A [beta] _1-42 aggregates by fluorescence intensity measurement of ThT with 10 mM phosphate buffer (1 mM EDTA, pH 7.4) using a diluted to 0.25 mg / mL (55.4 μM) Aβ _1-42 aggregates were made by incubating at 60 ° C. for about 30 minutes, followed by incubation at 37 ° C. for 42 hours. In order to confirm that the prepared Aβ _1-42 aggregates formed amyloid, the fluorescence intensity during ThT mixing was measured (FIG. 12). As a result, compared to the fluorescence intensity of ThT when using 10 mM phosphate buffer (1 mM EDTA, pH 7.4) instead of Aβ _1-42 aggregate as a control, the prepared Aβ _1-42 aggregate Fluorescence intensity when mixed with the mixture increased by about 8.5 times, indicating that the prepared Aβ _1-42 aggregate had an amyloid structure.

2.2. Confirmation of α-Syn Aggregate by ThT Fluorescence Intensity Measurement Using α-Syn in three types of 30 mM Tris-HCl buffer (200 mM NaCl, pH6.0, 7.0, 8.0), 1.67 mg / mL ( 115 μM) and then stirred at 37 ° C. for 72 hours. In order to confirm that the produced α-Syn aggregates formed amyloid, the fluorescence intensity during ThT mixing was measured (FIG. 13). As a result, compared with the fluorescence intensity of ThT in the absence of α-Syn, the fluorescence intensity when mixed with the prepared α-Syn aggregates is pH 6.0, 7.0, 8.0 of the buffer used, The increase by 186, 134, and 47-fold showed that the produced α-Syn aggregate had an amyloid structure.

In addition, in the binding affinity evaluation with a compound, it is necessary to separate a compound that is bound to an aggregate and a compound that is not bound by filtration using a filter or centrifugation. For this reason, it is necessary to produce an aggregate that cannot pass through a filter or precipitates by centrifugation. Therefore, the content ratio of the fraction precipitated by centrifugation of the aggregates prepared by the above three methods was examined, and the aggregates more suitable for the binding experiment were selected. First, the α-Syn aggregate was centrifuged at 20 ° C. and 20,000 μg for 15 minutes. Subsequently, the fluorescence intensity during ThT mixing of the α-Syn aggregate before centrifugation and the precipitated α-Syn aggregate after centrifugation was measured to determine the content ratio of the precipitate (FIG. 14). As a result, it was suggested that α-Syn prepared at pH 6.0 had a large proportion of precipitate. Therefore, in subsequent binding experiments, α-Syn aggregates prepared in 30 mM Tris-HCl buffer (200 mM NaCl, pH 6.0) were used.

2.3. Evaluation of binding affinity for α-Syn aggregates To evaluate the binding affinity of BFC derivatives to α-Syn aggregates (250 nM), adsorption experiments of BFC derivatives were performed (Fig. 15). As a result, among the BFC derivatives, [ ¹²⁵ I] 30 had an adsorption rate of about 10% with respect to the α-Syn aggregate, indicating that the adsorbability with respect to the α-Syn aggregate was particularly high. Regarding the BFC derivative having an amino group, [ ¹²⁵ I] 39 showed an adsorption rate of about 4%, while [ ¹²⁵ I] 33 and [ ¹²⁵ I] 36 showed almost no adsorptivity.

As a result of the adsorption experiment, it was found that [ ¹²⁵ I] 30 has high adsorptivity to α-Syn aggregates among the BFC derivatives evaluated this time. Therefore, alpha-Syn for aggregates with respect to binding affinity of ^[125 I] 30, in order to study in more detail, it was binding saturation experiments ^[125 I] 30 for alpha-Syn aggregates. The value obtained by subtracting non-specific binding from the concentration of the compound bound to the α-Syn aggregate at each concentration was plotted, and further the Scatchard analysis was performed to calculate the dissociation constant K _d value and the maximum binding amount B _max value. . As a result, the binding of [ ¹²⁵ I] 30 to α-Syn aggregates is represented by a binding saturation curve, showing a Scatchard plot that fits the single-binding site model, K _d = 141 nM, B _max = 332 mmol / Calculated as mol protein (FIG. 16). This indicated that [ ¹²⁵ I] 30 has binding affinity for α-Syn aggregates. The BFC derivative evaluated this time has a weak affinity for α-Syn, so it can be used as a PrP ^Sc selective amyloid imaging probe.

2.4. Evaluation of binding affinity for Aβ _1-42 aggregates To evaluate the binding affinity of BFC derivatives to Aβ _1-42 aggregates, binding saturation experiments were performed. Plot the value obtained by subtracting non-specific binding from the concentration of the compound bound to the Aβ _1-42 aggregate at each concentration, and further calculate the dissociation constant K _d value and maximum binding amount B _max value by performing Scatchard analysis. (Table 4). As a result, the binding of the BFC derivative to the Aβ _1-42 aggregate was a binding saturation curve, which showed a Scatchard plot that fits the single-binding site model. The BFC derivative has a K _d value of 102-238 nM for the Aβ _1-42 aggregate and a B _max value of 82.7-972 mmol / mol protein, and has a binding affinity for the Aβ _1-42 aggregate. Indicated. However, its binding affinity was shown to be much lower than [ ¹⁸ F] Florbetapir (K _d = 2.9 nM), which is approved by the FDA as a PET drug.

Here, regarding the K _d values of the BFC derivatives for rMoPrP aggregates, Aβ _1-42 aggregates and α-Syn aggregates, the investigations of the present inventors and the results obtained in this study are summarized in Table 5. It was. Overall, it is suggested that BFC derivatives have a high affinity for rMoPrP aggregates, but do not have a high affinity for other Aβ aggregates or α-Syn aggregates. When K _d (Aβ) / K _d (rMoPrP) of the BFC derivative is calculated, compounds 30, 33, 36 and 39 are calculated as 3.0, 3.9, 7.2 and 11, respectively, and compound 39 is most selective for rMoPrP aggregates. Was shown to be high.

2.5. Evaluation of binding to amyloid on mouse brain slices
2.5.1. Evaluation of binding to PrP ^Sc on mBSE-infected mouse brain sections To evaluate the binding to PrP ^Sc deposited on mouse brain sections, we performed fluorescence staining of mBSE-infected mouse brain sections using BFC derivatives. (FIG. 17). As a control group, fluorescent staining of brain sections obtained from uninfected mice of the same week was performed. As a result, in the fluorescence staining of the non-infected mouse brain section, a clear fluorescence image of each BFC derivative was not observed, but only background fluorescence was observed (FIGS. 17a, e, i, m), and also in PrP antibody staining. No PrP ^Sc deposition was observed (Fig. 17b, f, j, n). In mBSE-infected mouse brain sections, compound-derived fluorescence images were observed for each BFC derivative (FIGS. 17c, g, k, o). Further, when immunostaining was performed on the brain section adjacent to the brain section used in the fluorescence staining to confirm the site where PrP ^Sc was present, a stained image with the antibody was observed in the mBSE-infected mouse brain section (FIG. 17d). , h, l, p). Therefore, it was suggested that the BFC derivative recognizes the PrP ^Sc deposition site in the brain.

2.5.2. Evaluation of binding to Aβ aggregates on Tg2576 mouse brain slices To evaluate the binding to Aβ aggregates deposited on mouse brain slices, Tg2576 was overexpressed with APP using a BFC derivative. Mouse brain sections were fluorescently stained (FIG. 18). Further, adjacent sections of Tg2576 mouse brain sections were stained with ThT, which is a fluorescent staining reagent for amyloid. As a result, among the BFC derivatives, compounds 33 and 36 matched the ThT positive site of the Tg2576 mouse brain section with the compound-derived staining site. However, the fluorescence from the stained compound was weak overall, suggesting that the binding affinity of BFC derivatives to Aβ aggregates on Tg2576 mouse brain sections was low. Moreover, as shown in FIGS. 9 and 17, strong fluorescence derived from the compound at the PrP ^Sc deposition site was observed in mBSE-infected mouse brain sections. Therefore, it was suggested that the BFC derivative may function as a selective imaging probe for PrP ^Sc .

From the above results, it was suggested that BFC derivatives have lower binding properties to amyloid other than PrP ^Sc compared to existing compounds. Furthermore, since these compounds are excellent in brain migration for deployment as an in vivo imaging probe, the possibility of clinical application as a PrP ^Sc selective imaging probe was shown.

References
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2. Ono, M .; Maya, Y .; Haratake, M .; Nakayama, M. Synthesis and characterization of styrylchromone derivatives as β-amyloid imaging agents. Bioorg. Med. Chem. 2007, 15, 444-450.
3. Atarashi, R .; Moore, RA; Sim, VL; Hughson, AG; Dorward, DW; Onwubiko, HA; Priola SA; Caughey, B. Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Methods. 2007, 4, 645-650.
4. Atarashi, R .; Satoh, K .; Sano, K .; Fuse, T .; Yamaguchi, N .; Ishibashi, D .; Matsubara, T .; Nakagaki, T .; Yamanaka, H .; Shirabe, S .; Yamada, M .; Mizusawa, H .; Kitamoto, T .; Klug, G .; McGlade, A .; Collins SJ; Nishida, N. Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat. Med. 2011, 17, 175-178.
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The compound of the present invention exhibits high binding specificity for amyloid protein, high blood brain barrier permeability, and rapid disappearance from normal tissues, and is useful for diagnosis of amyloid-related diseases. In addition, the compounds of the present invention can be useful as imaging probes that are selective for abnormal prion protein aggregates (PrP ^Sc ).

This application is based on Japanese Patent Application No. 2018-037948 filed in Japan, the contents of which are incorporated herein by reference in their entirety.

Claims (15)

1. Formula (I):
   

   

   (Where
   X ¹ represents O, S, or NH;
   X ² represents CH or N;
   R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen atom; halogen atom; hydroxy group; carboxy group; sulfo group; nitro group; amino group; C _1-6 alkyl group; An optionally substituted C _1-6 alkoxy group; a mono (C _1-6 alkyl) amino group; or a di (C _1-6 alkyl) amino group, and R ⁵ , R ⁶ , R ⁷ and R ⁸ are , each independently, a hydrogen atom; a halogen atom; hydroxy group; a carboxy group; a sulfo group; a nitro group; an amino group; C _1-6 alkyl group; a halogen atom or a hydroxy which may be substituted by a group C _1-6 An alkoxy group; a mono (C _1-6 alkyl) amino group; or a di (C _1-6 alkyl) amino group. )
   Or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X ¹ in formula (I) is O and X ² is CH.
3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein any one of R ¹ , R ² , R ³ and R ⁴ in formula (I) is a halogen atom.
4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein any one of R ¹ , R ² , R ³ and R ⁴ in formula (I) is an iodine atom.
5. X ¹ in the formula (I) is O, X ² is CH, R ¹ , R ² , R ⁴ , R ⁵ and R ⁸ are hydrogen atoms, R ³ is an iodine atom R ⁶ is a hydrogen atom; a C _1-4 alkyl group; a C _1-4 alkoxy group optionally substituted by a hydroxy group; a mono (C _1-4 alkyl) amino group; or a di (C _1-4) Alkyl) amino group and R ⁷ is hydroxy group; C _1-4 alkoxy group optionally substituted by hydroxy group; amino group; mono (C _1-4 alkyl) amino group; or di (C ₁ The compound according to claim 1, which is a _-4 alkyl) amino group, or a pharmaceutically acceptable salt thereof.
6. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, which is labeled with a radionuclide.
7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is a positron emitting nuclide.
8. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the radionuclide is a γ-ray emitting nuclide.
9. The radionuclide is ¹²⁵ I, or a compound or a pharmaceutically acceptable salt thereof according to claim 6.
10. A composition for diagnosing amyloid-related diseases, comprising the compound according to any one of claims 6 to 9 or a pharmaceutically acceptable salt thereof.
11. Amyloid-related diseases include Creutzfeldt-Jakob disease, Alzheimer's disease, Lewy body dementia, Mediterranean fever, Maccle-Wells syndrome, idiopathic myeloma, amyloid polyneuropathy, amyloid cardiomyopathy, systemic senile amyloidosis, amyloidosis Hereditary cerebral hemorrhage, Down syndrome, scrapie, Kruo disease, Gerstman-Stroisler-Scheinker syndrome, medullary thyroid cancer, isolated atrial amyloid, β ₂ -microglobulin amyloid in dialysis patients, inclusion body myositis, muscle wasting disease A composition according to claim 10, selected from the group consisting of β ₂ -amyloid deposits in and Langerhans type II diabetes insulinoma.
12. An imaging agent for amyloid deposits comprising the compound according to any one of claims 6 to 9 or a pharmaceutically acceptable salt thereof.
13. A mammal into which a detectable amount of the compound according to any one of claims 6 to 9 or a pharmaceutically acceptable salt thereof is introduced is computed tomography (SPECT) or positron tomography (PET) A method for imaging amyloid deposits, comprising the step of imaging.
14. A step of administering a test substance to a model animal of amyloid-related disease, a step of administering a composition for diagnosing amyloid-related disease according to claim 10 or 11 to the model animal, and a formula contained in the brain of the model animal ( A screening method for a therapeutic or prophylactic agent for amyloid-related diseases, which comprises the step of examining the distribution or amount of the compound represented by I).
15. A step of administering a therapeutic or prophylactic agent for amyloid-related disease to a model animal of amyloid-related disease, a step of administering to the model animal the composition for diagnosing amyloid-related disease according to claim 10 or 11, and A method for evaluating a therapeutic or prophylactic agent for amyloid-related diseases, comprising a step of examining the distribution or amount of a compound represented by formula (I) contained in the brain.

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