JP7809338B2 - Diagnostic imaging agent for amyloid oligomers - Google Patents

Description

The present invention relates to an imaging diagnostic agent used to detect amyloid beta oligomers, and a curcumin derivative or a salt thereof.

The number of dementia patients in Japan exceeds 5 million and is estimated to reach 7 million by 2025, making its resolution an urgent and important issue. Alzheimer's disease (AD) is the most common type of dementia, and its clinical symptoms include memory impairment and higher brain dysfunction (aphasia, apraxia, agnosia, and constructional apraxia). These symptoms are often shared with other dementias, making it extremely difficult to definitively diagnose Alzheimer's disease based on clinical symptoms alone.

Furthermore, efforts to develop a cure for AD have all failed. Brain pathology in Alzheimer's disease progresses 20 to 30 years before the onset of symptoms, highlighting the importance of preemptive medicine, which diagnoses and treats the condition before symptoms appear.

Characteristic histopathological findings of Alzheimer's disease include senile plaques and neurofibrillary tangles. The main component of the former is amyloid beta protein with a beta-sheet structure, while the main component of the latter is hyperphosphorylated tau protein. It is known that in Alzheimer's disease, the above-mentioned pathological tissue changes, such as the accumulation of aggregated amyloid beta protein, begin in the brain well before the onset of clinical symptoms. Therefore, detecting aggregated amyloid beta protein as a marker is one method for early diagnosis of diseases involving amyloid accumulation, particularly Alzheimer's disease.

Amyloid beta peptide (Aβ) oligomers play an important role in the early stages of AD before its onset. Aβ aggregates then become Aβ oligomers and then Aβ fibrils. Because treatment of AD after its onset is difficult, it is important to establish accurate diagnostic methods and develop treatments for people in their 40s and 50s, when toxic oligomers begin to accumulate.

With this in mind, research has been progressing in recent years into radioactive contrast agents for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) that selectively bind to amyloid beta protein in the brain. Classic compounds with high affinity for amyloid include Congo Red, thioflavin S, and thioflavin T, which are used for the definitive pathological diagnosis of Alzheimer's disease. Many of these compounds have difficulty crossing the blood-brain barrier and barely penetrate into the brain even when administered intravenously.

Furthermore, nuclear magnetic resonance imaging (MRI) is one diagnostic method that does not use radionuclides. The inventors have previously reported successful imaging of senile plaques using fluorine nuclear magnetic resonance imaging (fluorine MR imaging) with curcumin derivatives (Patent Documents 1 and 2). Furthermore, Patent Document 3 reports a curcumin derivative that interacts with Aβ fibrils and can be used to detect Aβ fibrils.

However, to date, there have been no reports of small molecule compounds that specifically bind to Aβ oligomers.

International Publication No. 2010/098502 International Publication No. 2014/109296 Japanese Patent Application Publication No. 2018-138528

The object of the present invention is to provide an imaging diagnostic agent used to detect amyloid beta oligomers, and a substance that specifically binds to amyloid beta oligomers.

The inventors have previously demonstrated that curcumin and curcumin derivatives bind not only to Aβ fibrils but also to Aβ oligomers.

As a result of extensive research to achieve the above-mentioned objective, the inventors analyzed the chemical mechanism of curcumin derivatives such as those described in Patent Documents 1 and 2. They concluded that the keto-enol tautomerism of curcumin plays an important role, and synthesized a curcumin derivative that exclusively adopts the keto structure. As a result, they discovered that such a curcumin derivative that exclusively adopts the keto structure specifically binds to Aβ oligomers.

The present invention was completed based on these findings and through further research, and provides the following diagnostic imaging agents, curcumin derivatives or salts thereof, etc., used to detect amyloid beta oligomers:

Item 1. A diagnostic imaging agent used to detect amyloid β oligomers, comprising, as an active ingredient, a curcumin derivative or a salt thereof having only a keto structure.
Item 2. The curcumin derivative is represented by formula (I):

Item 2. An imaging diagnostic agent according to Item 1, which is a curcumin derivative represented by the formula: (wherein ^R1 's are each independently a hydrogen atom, a fluorine atom, _CH3- , _CH2F- , _CHF2- , _CF3- , _CH3O- , _CH2FO- , _CHF2O- or _CF3O- , R2 ^'s are each independently a hydrogen atom or a fluorine atom, A is alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- , ^R3 is hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 , ^R4 and ^R5 are each independently a hydrogen atom or alkyl, ^and m is an integer from 1 to 5).
Item 3. The diagnostic imaging agent according to Item 2, wherein each ^R1 independently represents a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- , or _CF3O- .
Item 4. The diagnostic imaging agent according to any one of Items 1 to 3, which is used for diagnosing a disease in which amyloid β protein accumulates.
Item 5. The diagnostic imaging agent according to Item 4, wherein the disease associated with accumulation of amyloid β protein is Alzheimer's disease.
Item 6. The diagnostic imaging agent according to any one of Items 1 to 5, wherein the diagnostic imaging is MRI.
Item 7. A method for detecting amyloid β oligomers, which uses the diagnostic imaging agent according to any one of Items 1 to 3.
Item 8. A method for diagnosing a disease associated with accumulation of amyloid β protein, using the diagnostic imaging agent according to any one of items 1 to 3.
Item 9. Formula (IA):

A curcumin derivative or salt thereof represented by the formula: (wherein ^R1's are each independently a hydrogen atom, a fluorine atom, _CH3- , _CH2F- , _CHF2- , _CF3- , _CH3O- , _CH2FO- , _CHF2O- or _CF3O- , ^R2 's are each independently a hydrogen atom or a fluorine atom, A is alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- , ^R3 is hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 , ^R4 and ^R5 are each independently a hydrogen atom or ^alkyl , and m is an integer of 1 to 5 (however, when ^R1 's are both _CH3O- or _CF3O- and ^R2 's are both hydrogen atoms, A must not be _CH3- ).
Item 10. The curcumin derivative or a salt thereof according to Item 9, wherein each ^R1 independently represents a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- , or _CF3O- .

The diagnostic imaging agent of the present invention contains as an active ingredient a compound that has high binding specificity for Aβ oligomers and can be used to detect Aβ oligomers. Furthermore, the curcumin derivative or a salt thereof of the present invention has high binding specificity for Aβ oligomers and is useful as an active ingredient in a diagnostic imaging agent used to detect Aβ oligomers.

According to the present invention, Aβ oligomers play an important role in the early stages of AD before its onset, making it possible to diagnose diseases in which amyloid β protein accumulates at an early pre-symptomatic stage.

1 is a graph showing the results of an analysis of binding to Aβ oligomers and Aβ fibers using a quartz crystal microbalance (QCM) measurement device performed in Test Example 1. The upper row shows the frequency change for Aβ fibers, and the lower row shows the frequency change for Aβ oligomers. *p<0.05 vs vehicle (after 20 minutes, Mann Whitney test), Aβ oligomers and Aβ fibers n=5, vehicle n=3 1 is a graph showing the results of the analysis of binding to Aβ oligomers and Aβ fibers using a QCM device carried out in Test Example 1. The upper row shows the total amount of compound bound to Aβ fibers or the solvent, and the lower row shows the total amount (pmol/1 nmol Aβ) of compound bound to Aβ oligomers or the solvent. *p<0.05 vs vehicle (Mann Whitney test), Aβ oligomers and Aβ fibers n=5, vehicle n=3 1 is a graph showing the time course of the peak area of the ^19F -NMR signal measured by MRI after administering Compound 1 to APP/PS1 mice and wild-type mice via the tail vein in Test Example 2. The solid line shows the results using 16-month-old APP/PS1 mice, and the dashed line shows the results using 12-month-old wild-type mice. n=3 MRI brain images obtained by administering Compound 1 to APP/PS1 mice and wild-type mice via the tail vein in Test Example 2. The left image shows the results from 16-month-old APP/PS1 mice, and the right image shows the results from 12-month-old wild-type mice.

The following describes in detail the embodiments of the present invention.

The imaging diagnostic agent of the present invention used to detect amyloid beta oligomers is characterized by having as its active ingredient a curcumin derivative or its salt that contains only a keto structure.

In this invention, amyloid beta protein refers to a protein consisting of 38 to 43 amino acids, produced from amyloid precursor protein by the action of protease, and Aβ oligomers are aggregates of 2 to several tens of Aβ molecules (particularly 2 to 30 molecules).

The curcumin derivatives used in the present invention are compounds that have only a keto structure. The keto structure refers to a curcumin derivative shown below in which both central parts are ketones, and the enol structure refers to a curcumin derivative in which one of the central parts is enolized.

The curcumin derivative or its salt of the present invention has the property of high binding specificity to Aβ oligomers, which play an important role in the early stages before the onset of AD.

The curcumin derivatives or salts thereof of the present invention are not particularly limited as long as they are compounds that have the structure of curcumin and only take the keto structure. When the curcumin derivatives or salts thereof of the present invention contain an asymmetric carbon, both optical isomers separated by conventional methods and the racemate are included in the compounds of the present invention.

The curcumin derivatives of the present invention may be in the form of a salt, and such salts may be any pharmaceutically acceptable salt, including, for example, alkali metal salts such as potassium salts and sodium salts; alkaline earth metal salts such as calcium salts; and organic amine salts such as triethanolamine salts and tris(hydroxymethyl)aminomethane salts. Some of these salts contain water of crystallization.

A preferred curcumin derivative of the present invention is a curcumin derivative represented by the following formula (I) or a salt thereof.
Formula (I):

(In the formula, ^{each R1} is independently a hydrogen atom, a fluorine atom, _CH3- , _CH2F- , _CHF2- , _CF3- , _CH3O- , _CH2FO- , _CHF2O- or _CF3O- ; each ^R2 is independently a hydrogen atom or a fluorine atom; A is alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- ; ^R3 is hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 ; ^R4 and ^R5 are independently a hydrogen atom or an alkyl; and m is an integer of 1 to ^5. )

In addition, the curcumin derivative represented by the following formula (IA) or a salt thereof is a novel compound that has not been described in the literature and is included in the curcumin derivative represented by formula (I) or a salt thereof.
Formula (IA):

wherein R1 ^'s _are each independently a hydrogen atom, a fluorine atom, _CH3- , CH2F-, _CHF2- , _CF3- , _CH3O- , _CH2FO- , _CHF2O- or _CF3O- ; ^R2 's are each independently a hydrogen atom or a fluorine atom; A is alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- ; ^R3 is hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 ; ^R4 and ^R5 are each independently a hydrogen atom or an alkyl; and m is an integer of 1 to ⁵ , preferably 1 to 3 (provided that when both ^R1 's are _CH3O- or _CF3O- and both ^R2 's are hydrogen atoms, A must not be _CH3- ).

Preferably, each ^R1 is independently a fluorine atom, CH ₂ F—, CHF ₂ —, CF ₃ —, CH ₂ FO—, CHF ₂ O— or CF ₃ O—.

The alkyl of A, ^R4 , and ^R5 may be a straight or branched _C1-6 alkyl, preferably a straight or branched _C1-3 alkyl, which also applies to the alkyl of alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, and hydroxyalkoxy in the compound of formula (I).

Specific examples of C _1-6 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and hexyl.

Specific examples of C _1-3 alkyl include methyl, ethyl, n-propyl, and isopropyl.

The curcumin derivative of formula (I) or formula (IA) or its salt can be prepared by the method described below.

The compound of formula (I) can be prepared by hydrolyzing the compound of formula (II).

(wherein R ¹ , R ² and A are as defined above.)

Solvents for this reaction include alcohols such as methanol, ethanol, n-propanol, iso-propanol, and butanol; ethers such as aqueous tetrahydrofuran and aqueous dioxane; acid amides such as aqueous dimethylformamide and aqueous dimethylacetamide; sulfoxides such as aqueous dimethyl sulfoxide, and mixed solvents of these.

It is desirable to add a mineral acid to promote this reaction. Examples of such mineral acids include hydrochloric acid, sulfuric acid, nitric acid, and perchloric acid. The mineral acid can be used in an amount of 3 to 10 times, preferably 4 to 6 times, the molar amount of the compound of formula (II).

This reaction can usually be carried out at 0 to 150°C, preferably 30 to 100°C, and the reaction time is usually about 1 to 150 hours.

Compound (II) can be prepared by reacting compound (III) with methyl iodide.

(wherein R ¹ , R ² and A are as defined above.)

Solvents for this reaction include aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic hydrocarbons such as pentane, hexane, petroleum ether, and ligroin; ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and dioxane; ketones such as acetone and 2-butanone; nitriles such as acetonitrile and propionitrile; acid amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide, and mixed solvents of these.

To promote this reaction, it is desirable to add a base. Examples of such bases include organic bases such as triethylamine, pyridine, N-methylmorpholine, 1,8-diazabicyclo[5,4,0]-7-undecene, and N,N-dimethylaniline; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate; alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; and alkaline earth metal hydroxides such as barium hydroxide and calcium hydroxide. The base can be used in an amount of 3 to 20 times the molar amount of the compound of formula (III), preferably 5 to 10 times the molar amount.

This reaction can usually be carried out at 0 to 70°C, and the reaction time is approximately 1 to 48 hours.

It is also desirable to use methyl iodide in an amount of 2 to 20 times the molar amount of the compound of formula (III).

The compound of formula (III) can be produced by condensing the compound of formula (IV) with the compound of formula (V). However, the compound of formula (IV) must be reacted in a molar amount twice that of the compound of formula (V). The compound of formula (V) can be produced by known methods.

(wherein R ¹ , R ² and A are as defined above.)

To ensure efficient reaction, it is desirable to conduct the reaction in a solvent in the presence of a boron compound and a base. Examples of boron compounds that can be used in this reaction include boric acid, diboron trioxide, trimethyl borate, triethyl borate, tripropyl borate, tri-n-butyl borate, tri-tert-butyl borate, and mixtures of diboron trioxide with various boric acid esters. It is desirable to use 0.5 to 6 times the molar amount of the boric acid compound relative to the compound of formula (IV).

Examples of the base include primary amines such as n-butylamine, sec-butylamine, tert-butylamine, n-propylamine, n-hexylamine, and cyclohexylamine; and secondary amines such as morpholine, piperidine, and 1,2,3,4-tetrahydroquinoline. It is desirable to use a 1-fold molar amount of base relative to the compound of formula (V).

Examples of solvents that can be used include aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic hydrocarbons such as pentane, hexane, heptane, petroleum ether, and ligroin; ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and dioxane; esters such as methyl acetate, ethyl acetate, and methyl propionate; acid amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide; phosphoric acid amides such as hexamethylphosphortriamide, and mixed solvents thereof.

The reaction temperature is usually 0 to 150°C, preferably 0 to 100°C, and the reaction time is usually about 0.5 to 24 hours.

After the reaction, the reaction mixture must be treated with an acid to decompose the resulting boron complex of the compound of formula (III). Examples of acids that can be used include mineral acids such as hydrochloric acid and sulfuric acid, and organic acids such as acetic acid and propionic acid.

The compound of formula (IV) can be produced by reacting the compound of formula (VI) with chlorodimethyl ether. The compound of formula (VI) can be produced by known methods.

(wherein ^R1 and ^R2 are as defined above.)

Examples of solvents include aromatic hydrocarbons such as benzene, toluene, and xylene; aliphatic hydrocarbons such as pentane, hexane, heptane, petroleum ether, and ligroin; ethers such as diethyl ether, dipropyl ether, dibutyl ether, tetrahydrofuran, and dioxane; ketones such as acetone and 2-butanone; nitriles such as acetonitrile and propionitrile; acid amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide; halogenated hydrocarbons such as dichloromethane, carbon tetrachloride, and 1,2-dichloroethane, and mixed solvents thereof.

To promote this reaction, it is desirable to add a base. Examples of such bases include organic bases such as triethylamine, pyridine, N-methylmorpholine, N-methylpiperidine, 1,8-diazabicyclo[5,4,0]-7-undecene, and N,N-dimethylaniline; alkali metal carbonates such as sodium carbonate and potassium carbonate; alkali metal bicarbonates such as sodium bicarbonate and potassium bicarbonate; and alkali metal hydroxides such as sodium hydroxide and potassium hydroxide. The base can be used in an amount of 1 to 3 times the molar amount of the compound of formula (VI), preferably 1.4 to 1.8 times the molar amount.

This reaction can usually be carried out at 0 to 50°C, and the reaction time is usually around 1 to 48 hours.

The compound of formula (I) obtained by the above-mentioned production method and its associated methods can be isolated and purified by known means, such as concentration, vacuum concentration, distillation, fractional distillation, solvent transfer, solvent extraction, crystallization, recrystallization, chromatography, etc.

When the compound of formula (I) is obtained in its free form, a salt can be formed by conventional methods.

Specific examples of compounds of formula (I) are shown in Table 1 below.

Many curcumin derivatives of formula (I) are hydrophobic compounds with low solubility in water. For compounds to be administered to living organisms, high water solubility is desirable, and among compounds of formula (I), salts are more desirable.

Curcumin derivatives or salts thereof containing only the keto structure can be used as diagnostic imaging agents for detecting Aβ oligomers. MRI is preferred as the diagnostic imaging method.

When a curcumin derivative or a salt thereof having only the keto structure is used as an imaging diagnostic agent, the compound can specifically detect Aβ oligomers in the brain. In particular, when Aβ oligomers are detected noninvasively using ^19F -MRI, the detection sensitivity depends on the number of fluorine atoms, so among the compounds of formula (I), compounds containing more fluorine atoms are more desirable.

Curcumin derivatives or salts thereof that contain only the keto structure specifically bind to Aβ oligomers, which play an important role in the early stages of AD before the onset of the disease, making it possible to detect the locations where Aβ oligomers are present. Therefore, curcumin derivatives or salts thereof that contain only the keto structure can be used as diagnostic imaging agents for diseases in which amyloid beta protein accumulates, enabling early diagnosis of diseases in which amyloid beta accumulates at a pre-symptomatic stage.

When a curcumin derivative or a salt thereof containing only the keto structure is used as a diagnostic imaging agent, it may be administered locally or systemically. There are no particular restrictions on the administration method, and it may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intraperitoneal, intravenous, arterial, or spinal fluid injection, and infusion.

Diagnostic imaging agents containing curcumin derivatives or salts thereof containing only the keto structure are in a pharmaceutically acceptable form suitable for human administration and contain physiologically acceptable additives. Such compositions may contain, as appropriate, pharmaceutically acceptable diluents, buffers, solubilizers (e.g., surfactants such as cyclodextrin, polyethylene glycol, Pluronic™, Tween™, Cremophor™, or phospholipids), soothing agents, etc., and may further contain, as necessary, components such as pharmaceutically acceptable solvents, stabilizers, or antioxidants (e.g., ascorbic acid, etc.). The dosage of the curcumin derivative or salt thereof containing only the keto structure is selected appropriately depending on the dosage method, the patient's age, sex, and other conditions, and the severity of the disease.

Diseases in which amyloid beta protein accumulates include Alzheimer's disease and Down syndrome.

Next, synthesis examples and test examples related to the present invention are described, but the present invention is not limited to these.

Synthesis Example 1 Synthesis of 1,7-bis(4'-hydroxy-3'-trifluoromethoxy)phenyl-4-ethyl-4-methyl-1,6-heptadiene-3,5-dione (Compound 1) (1) A solution of 4-hydroxy-3-(trifluoromethoxy)benzaldehyde (2.06 g; 10 mmol) in acetone (18 mL) was added with potassium carbonate (2.21 g; 16 mmol) and cooled on ice. To this mixture, chloromethyl methyl ether (1.07 mL; 14 mmol) was added in small portions with stirring. The reaction mixture was stirred at room temperature for 10 hours, and the solvent was evaporated under reduced pressure. The resulting residue was extracted with ethyl acetate. The extract was washed with water, saturated aqueous sodium bicarbonate, and saturated brine, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:3) to give 2.48 g (99%) of 4-methoxymethoxy-3-(trifluoromethoxy)benzaldehyde as a colorless oil.

(2) To a solution of 3-ethyl-2,4-pentanedione (256 mg; 2.0 mmol) in ethyl acetate (4.0 mL), boron trioxide (140 mg; 2.0 mmol) was added and the mixture was heated at 70°C for 30 minutes. Tributyl borate (1.08 mL; 4.0 mmol) and 4-methoxymethoxy-3-(trifluoromethoxy)benzaldehyde (1.00 g; 4.0 mmol) obtained in step (1) were then added and the mixture was heated at the same temperature for 30 minutes. Butylamine (0.20 mL; 2.0 mmol) was then added and the mixture was heated at the same temperature for 5 hours. After cooling to room temperature, 2M hydrochloric acid (10 mL) was added and the mixture was stirred vigorously at room temperature for 30 minutes, followed by extraction with ethyl acetate. The extract was washed with water, then with saturated brine, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate: n-hexane = 1:3) to give 1,7-bis(4'-methoxymethoxy-3'-trifluoromethoxy)phenyl-4-ethyl-1,6-heptadiene-3,5-dione (282 mg; 24%).
¹⁹ FNMR (CDCl ₃ ): δ -59.50 (s), ¹ HNMR (CDCl ₃ ): δ1.20 (3H, t, J=7.2Hz), δ2.58 (2H, q, J=7.2Hz), δ3.50 (6H, s), δ5.26 (4H, s), δ6.96 (2H, d, J=15.6Hz), δ7.2-7.3 (2H), δ7.4-7.5 (4H), δ7.67 (2H, d, J=15.6Hz), δ17.38 (1H, s)

(3) To a solution of 1,7-bis(4'-methoxymethoxy-3'-trifluoromethoxy)phenyl-4-ethyl-1,6-heptadiene-3,5-dione (237 mg; 0.40 mmol) obtained in the above step (2) and methyl iodide (0.25 mL; 4.0 mmol) in acetone (10 mL), powdered potassium carbonate (550 mg; 4.0 mmol) was added and heated at 55°C for 3 hours. The solvent was distilled off under reduced pressure, and the resulting residue was extracted with ethyl acetate. The extract was washed with water, then with saturated brine, and then dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:3) to obtain 230 mg (95.0%) of 1,7-bis(4'-methoxymethoxy-3'-trifluoromethoxy)phenyl-4-ethyl-4-methyl-1,6-heptadiene-3,5-dione as a pale yellow oil.
¹⁹ FNMR (CDCl ₃ ): δ -59.59 (s), ¹ HNMR (CDCl ₃ ): δ0.82 (3H, t, J=7.2Hz), δ1.42 (3H, s), δ2.05 (2H, q, J=7.2Hz), δ3.46 (6H, s), δ5.23 (4H, s), δ6.65 (2H, d, J=15.6Hz), δ7.17 (2H, d, J=8.4Hz), δ7.38 (2H, br.s), δ7.40 (2H, dd, J=2Hz, 8.4Hz), δ7.62 (2H, d, J=15.6Hz)

(4) To a solution of 1,7-bis(4'-methoxymethoxy-3'-trifluoromethoxy)phenyl-4-ethyl-4-methyl-1,6-heptadiene-3,5-dione (182 mg; 0.30 mmol) obtained in step (3) in ethanol (6.0 mL) was added 1 M hydrochloric acid (1.80 mL; 1.80 mmol) and the mixture was heated to 60-65°C for 4 hours. The solvent was evaporated under reduced pressure, and the resulting residue was extracted with ethyl acetate. The extract was washed with water, then with saturated brine, and dried over magnesium sulfate. The solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:3) to yield 101 mg (65%) of colorless crystals of 1,7-bis(4'-hydroxy-3'-trifluoromethoxy)phenyl-4-ethyl-4-methyl-1,6-heptadiene-3,5-dione. Mp89-91℃ ¹⁹ FNMR (CDCl ₃ ): δ -59.22 (s), ¹ HNMR (CDCl ₃ ): δ0.82 (3H, t, J=7.2Hz), δ1.42 (3H,s), δ2.05 (2H, q, J=7.2Hz), δ5.69 (2H, s), δ6.64 (2H, d, J=15.6Hz), δ7.01 (2H, d, J=8.4Hz), δ7.35 (2H, br.s), δ7.39 (2H, dd, J=2Hz, 8.4Hz), δ7.61 (2H, d, J=15.6Hz)

Compound 3 used in the following test examples was synthesized according to the description in Synthesis Example 1 of JP 2014-105196 A. Compound 3 has the following structure. Compound 3 is a compound that exhibits keto-enol tautomerism.

Test Example 1: Analysis of Binding to Aβ Oligomers and Aβ Fibers Using a Quartz Crystal Microbalance (QCM) Apparatus. The AffinitxQ (Initium, Tokyo) quartz crystal microbalance (QCM) measurement apparatus used in this study was a 27-MHz QCM analyzer. This apparatus utilizes the decrease in vibration frequency upon binding between a compound immobilized on the quartz crystal sensor and the test substance, enabling highly sensitive detection of trace substance binding. In this study, Aβ fibers with a β-sheet structure or Aβ aggregates such as Aβ oligomers were immobilized on the sensor of the QCM apparatus and reacted with the respective reagents. Aβ fibers (fibrils) and Aβ oligomers (globulomer-type) were prepared as follows.

To prepare Aβ aggregates, Aβ _1-42 peptide (Peptide Institute, Inc.) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 1 mM, cooled at 37°C for 1 hour, the HFIP was removed by aspiration, and the aggregates were stored at -30°C. Before use, the peptide was dissolved in dimethyl sulfoxide (DMSO, dehydrated) to a concentration of 5 mM. Aβ fibrils were prepared by diluting the 5 mM Aβ _1-42 DMSO solution with Dulbecco's phosphate-buffered saline (D-PBS, Ca- and Mg-free, Nacalai Tesque, Inc.) to a 100 μM solution and incubating at 37°C for 48 hours. Aβ oligomers were prepared by diluting a 5 mM Aβ _1-42 DMSO solution to 400 μM with 0.2% sodium dodecyl sulfate (SDS) in D-PBS and incubating at 37°C for 6 hours, adding pure water to a final concentration of 100 μM, and further incubating at 37°C for 18 hours. This solution was centrifuged at 14,000 g for 10 minutes, and the supernatant was used.

Next, in binding tests using a QCM device, 2 μL of Aβ oligomers, Aβ fibrils, or solvent was dried and immobilized on the sensor (Aβ oligomers were left overnight in a humid state at 4°C and then dried for 24 hours at 4°C). The sensor was immersed in PBS in a glass cell container to stabilize it, and then compounds 1 and 3 were added to a final concentration of 10 μM, and thioflavin T (Anaspec) was added to a final concentration of 30 μM. Immediately after this, the change in frequency was recorded.

The results are shown in Figures 1 and 2 and Table 2. The upper panel of Figure 1 shows the frequency change for Aβ fibrils, and the lower panel shows the frequency change for Aβ oligomers. When compound 3 was added, a significant decrease in frequency was observed for both the sensor immobilized with Aβ fibrils (fibril) and the sensor immobilized with Aβ oligomers (oligomer) compared to the control sensor (vehicle) immobilized with solvent alone (fibril n=5, oligomer n=5, vehicle n=3). On the other hand, when compound 1 was added, a decrease in frequency was observed for the sensor immobilized with Aβ oligomers, but there was almost no difference between the sensor immobilized with Aβ fibrils and the control. Thioflavin T, which is known to bind specifically to Aβ fibrils, decreased the frequency for the sensor immobilized with Aβ fibrils, but there was no difference between the sensor immobilized with Aβ oligomers and the control without Aβ.

The upper panel of Figure 2 shows the total amount of compound bound to Aβ fibers within 20 minutes of compound addition, and the lower panel shows the total amount of compound bound to Aβ oligomers. Compound 3 bound significantly more to both Aβ fibers and Aβ oligomers, compound 1 bound only to Aβ oligomers, and thioflavin T bound only to Aβ fibers.

Furthermore, Table 2 shows the molar ratio of the amount of Aβ oligomers bound to Aβ fibers. This value increases when binding is more readily to Aβ oligomers than to Aβ fibers. Table 2 also shows that compound 1 has higher binding selectivity for Aβ oligomers than compound 3 and thioflavin T.

These results indicate that compound 3 has the ability to bind to both Aβ oligomers and Aβ fibrils, while compound 1 binds to Aβ oligomers but has low binding affinity to Aβ fibrils.

Test Example 2: ^19F -MR Imaging of Compound 1 10 mg of Compound 1 was weighed out, and 0.125 ml of 80% Cremophor-EL was added and dissolved by warming. Then, 0.875 ml of saline was added to prepare a dosing solution (10 mg/ml). This dosing solution was administered via the tail vein at 0.5 ml/kg/min to wild-type mice (12 months old, 1 male, 2 females) or APP/PS1 mice (16 months old, 2 males, 1 female) anesthetized with pentobarbital sodium, for a total dose of 200 mg/kg.

The ^19F -MR signals of the mouse head were measured using an MR system. First, a 10-minute single-pulse measurement was performed to acquire the NMR spectrum, followed by a 50-minute measurement for imaging using the Chemical Shift Imaging (CSI) method. After the measurement, the data were summed to create an image.

The results are shown in Figures 3 and 4. Figure 3 shows the time course of the ^19F -NMR signal peak area in wild-type mice (dashed line) and APP/PS1 mice (solid line) administered Compound 1. The peak area in APP/PS1 mice was larger than that in wild-type mice at all time points, indicating that Compound 1 accumulated in the brains of APP/PS1 mice.

In Figure 4, the left image shows a ¹⁹ F-MR image of an APP/PS1 mouse, and the right image shows a ¹⁹ F-MR image of a wild-type mouse. In the ¹⁹ F-MR image, signals (arrows) presumed to be Aβ oligomers were detected in the brain.

Test Example 3: Analysis of Brain Delivery of Compound 1 10 mg of Compound 1 was weighed out, and 0.125 ml of 80% Cremophor-EL was added and dissolved by warming. Then, 0.875 ml of saline was added to prepare a dosing solution (10 mg/ml). This dosing solution (200 mg/kg) and a dosing solution containing only the vehicle were administered via the tail vein to wild-type mice (6.5 months old, one female each). Thirty minutes after administration, the mice were euthanized by an overdose of sodium pentobarbital, and the brains were removed and homogenized in four volumes of _CD3OD to prepare a methanol extract solution.

Next, analysis was performed using a liquid chromatograph mass spectrometer (LC/MS). The LC system used was an Agilent 1290 binary pump. The analytical conditions were: analytical column: ZORBAX Eclipse Plus C18 1.8 μm 2.1 × 50 mm, A: ₄ mM HCOONH4 (pH 6.4), B: CAN, 0 min A90%, 1 min A90%, 6 min A20%, 7 min A20%. The MS system used was a QTRAP5500 in negative ion mode.

Table 3 below shows the concentration of compound 1 in the mouse brain sample solution determined from the ion intensity observed by LS/MS.

Claims (7)

An imaging diagnostic agent used to detect amyloid β oligomers, comprising, as an active ingredient, a curcumin derivative or a salt thereof having only a keto structure, wherein the curcumin derivative is represented by formula (I):
An imaging diagnostic agent which is a curcumin derivative represented by the formula: (wherein ^R1's are each independently a hydrogen atom, a fluorine atom, _CH3- , _CH2F- , _CHF2- , _CF3- , _CH3O- , _CH2FO- , _CHF2O- or _CF3O- , ^R2 's are each independently a hydrogen atom or a fluorine atom, A is alkyl, cyano, carboxy, alkoxycarbonyl or ^R3- ( _CH2 ) _m- , ^R3 is hydroxy, carboxy, cyano, alkylcarbonyloxy, alkoxycarbonyl, alkoxyalkoxy, hydroxyalkoxy or ^CONR4R5 , ^R4 and ^R5 are each independently a hydrogen atom or ^alkyl , and m is an integer from 1 to 5). 2. The diagnostic imaging agent according to claim 1, wherein each ^R1 independently represents a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- , or _CF3O- . The diagnostic imaging agent according to claim 1 or 2, which is used to diagnose diseases in which amyloid β protein accumulates. The diagnostic imaging agent according to claim 3, wherein the disease in which amyloid beta protein accumulates is Alzheimer's disease. The diagnostic imaging agent according to any one of claims 1 to 4, wherein the diagnostic imaging is MRI. Formula (IA):
A curcumin derivative or salt thereof represented by the formula: (wherein ^R1's are each independently a hydrogen atom, a fluorine atom, _CH3- , _CH2F- , _CHF2- , _CF3- , _CH3O- , _CH2FO- , _CHF2O- or _CF3O- , ^R2 's are each independently a hydrogen atom or a fluorine atom, and A is methyl or ethyl (provided that when R1's are both hydrogen atoms, CH3O- or CF3O-, or a hydrogen atom and CH3O-, and ^R2 's _{are both} hydrogen _atoms ^, A must not be _CH3- )). Formula (IA):
A curcumin derivative or salt thereof represented by the formula: (wherein R1 ^'s are each independently a fluorine atom, _CH2F- , _CHF2- , _CF3- , _CH2FO- , _CHF2O- or _CF3O- , R2 ^'s are each independently a hydrogen atom or a fluorine atom, and A is methyl or ethyl (however, when R1 ^'s are both _CF3O- and ^R2's are both hydrogen atoms, A must not be CH3-) _.