KR20160049819A - Compositions and methods for the detection of amyloid plaques and the diagnosis of alzheimer′s disease - Google Patents

Abstract

The purpose of the present invention is to provide a composition for diagnosing Alzheimer′s disease through detection of beta-amyloid plaques, and a manufacturing method thereof. The composition for diagnosing Alzheimer′s disease is a diagnostic composition for detecting beta-amyloid plaques, having low molecular weight, manufactured by a simple method, having excellent phosphorescent properties, having high selectivity for beta-amyloid plaques, having excellent binding affinity with a single beta-amyloid as well as beta-amyloid plaques, enabling a clinical image diagnosis, and to a manufacturing method thereof.

Description

[0001] The present invention relates to a composition for detecting beta amyloid plaques and for diagnosing or treating Alzheimer's disease,

The present invention relates to beta amyloid plaque detection and compositions for diagnosing Alzheimer's disease.

In Korea, the prevalence of dementia is rapidly increasing due to the rapid aging of the population. According to the Ministry of Health and Welfare 's survey on the prevalence of dementia, the population of demented elderly people aged 65 and over is expected to reach 522,000 in 2012 and 750,000 in 2020. However, Korea has a high awareness of the risk of dementia, but the perception of dementia symptoms is lower than the global average.

In particular, dementia is a symptom that gradually develops, so if you are diagnosed early and have a good diagnosis, there is a possibility of improvement. However, many people miss the treatment period and develop into severe dementia symptom in a short time. The early diagnosis of dementia is a very important factor as it has a very large impact and is developing into a social problem.

About 70% of patients with dementia are Alzheimer's patients, and a number of studies have identified proteins involved in Alzheimer's disease, but the cause of the disease is still unclear. However, neurofibrillary tangles formed by amyloid plaques and hyperphosphorylated tau protein filaments in nerve cells, which were formed by accumulation of beta-amyloid peptides between nerve cells, Have been reported.

Beta amyloid is composed of 36-43 amino acids of peptide. Accumulation of beta amyloid peptide is an early pathogenesis of brain damage and development of nerve fiber masses.

In recent years, methods for inhibiting or eliminating beta amyloid production have been studied as a treatment for Alzheimer's disease, and new ligands for beta amyloid plaque detection have been developed by specifically binding to beta amyloid plaques.

Conventionally developed beta amyloid plaque detecting fluorescent ligands are considered to be very complicated to manufacture and that the fluorescent ligands bind also to phosphorylated tau protein fibers and thus have low selectivity for beta amyloid plaques. The beta amyloid detection ligands, which have virtually no clinically proven effects because they do not show a significant change in their properties, are the biggest challenges of development.

No. 10-2012-0115538

It is an object of the present invention to provide a diagnostic composition for detecting beta amyloid plaques, which has a small molecular weight, a simple manufacturing method, a high fluorescence property, a high selectivity to a beta amyloid plaque, And a composition for diagnosing or treating Alzheimer's disease by detecting beta amyloid plaques which can be clinically diagnosed by imaging.

The present invention provides a composition for diagnosing degenerative brain disease comprising a dimethylbenzene amine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(Wherein Z is a hetero atom selected from nitrogen or boron,

Wherein R ₁ to R ₂ may be independently of each other hydrogen, (C ₁ -C ₁₀ ) alkyl, hydroxy or carboxy.

The Z of the present invention is a heteroatom selected from nitrogen or boron, and wherein R ₁ to R ₂ are hydrogen, (C ₁ -C ₅ ) alkyl or hydroxy.

The composition of the present invention is characterized in that R ₁ to R ₂ are hydrogen, hydroxy, methyl or ethyl.

The formula 1 of the present invention is exemplified by a composition characterized in that it is any one selected from the group consisting of the following compounds.

The composition of the present invention provides a composition characterized by binding to beta amyloid oligomers and beta amyloid plaques.

The degenerative brain disease of the present invention may be a disease characterized by a disease selected from the group consisting of Alzheimer's disease, senile dementia, neurodegenerative disorder or amyloidosis.

There is provided a diagnostic imaging agent for a disease selected from the group consisting of Alzheimer's disease, senile dementia, neurodegenerative disorder or amyloidosis, which comprises the composition of the present invention.

There is provided a kit for diagnosing or treating a disease selected from the group consisting of Alzheimer's disease, senile dementia, neurodegenerative disorder or amyloidosis, which comprises the diagnostic imaging agent of the present invention.

The composition for diagnosing Alzheimer's disease according to the present invention can be used for the diagnosis of Alzheimer's disease by preparing a beta-amyloid fluorescent diagnostic composition capable of binding to not only amyloid plaques but also single beta amyloid in existing beta amyloid plaque diagnoses limited to selectivity for amyloid plaques only, It is possible to diagnose Alzheimer's disease as an early diagnosis by providing a diagnostic composition containing a fluorescent ligand compound that has a remarkable increase in fluorescence after binding to beta amyloid as a diagnostic imaging agent. And the diagnostic accuracy can be improved.

Brief Description of the Drawings Figure 1 is a comparative graph of changes in fluorescence intensity before and after binding of single beta amyloid and complex beta amyloid of the compound of the present invention.
Figure 2 shows the results of binding affinity measurement of the compound of the present invention against beta amyloid in mouse brain tissue.
Figure 3 shows in-vivo (in vivo) measurements of beta amyloid plaque-selective binding of the compounds of the invention in brain and tissue sections of dementia-induced mice.

The present invention provides a diagnostic composition comprising a dimethylbenzene amine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(Wherein Z may be substituted with a hetero atom selected from nitrogen, boron, oxygen or sulfur,

R ₁ to R ₂ may be independently selected from hydrogen, (C ₁ -C ₁₀ ) alkyl, hydroxyl or carboxyl group.

The dimethylbenzenamine derivative compound of Formula 1, which is a dimethylbenzene amine derivative of the present invention, can be easily used as a beta amyloid detecting fluorescent ligand by binding to beta amyloid.

Preferably, in formula (1), Z is a heteroatom selected from nitrogen or boron, and R ₁ to R ₂ may be a structure having at least one of (C ₁ -C ₅ ) alkyl and a hydroxyl group have.

As used herein, the term " alkyl " means a straight or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isobutyl, pentyl or hexyl.

The formula 1 according to an embodiment of the present invention may be selected from the following compounds, but is not limited thereto.

The present invention also relates to a diagnostic composition comprising a dimethylbenzene amine derivative represented by Formula 1 and a pharmaceutically acceptable salt thereof, wherein the dimethylbenzene amine derivative represented by Formula 1 is a beta amyloid detecting fluorescent ligand, And may be an imaging composition capable of selectively detecting beta amyloid by selectively binding to amyloid or beta-amyloid plaques.

More preferably, the compound of Formula 1 is a compound wherein R ₁ to R ₂ are substituted with boronic acid.

The present inventors have conducted studies to develop a diagnostic agent having selectivity for beta amyloid oligomer (single beta amyloid) as well as beta amyloid plaque, which is a diagnostic agent having selectivity limited to beta amyloid plaques, and found that the combination of a beta amyloid oligomer and a fluorescent ligand A boronic acid was introduced into the fluorescent ligand compound represented by the above formula (1) to prepare a dimethylbenzene amine derivative compound having an excellent binding affinity for beta amyloid.

Alzheimer's Dementia occurs when beta amyloid oligomers form plaques, induce tau protein, and cause various vascular degeneration. Accordingly, the present invention provides a dimethylbenzene amine derivative compound as a fluorescent ligand having selectivity not only to a beta amyloid plaque but also to a beta amyloid oligomer (single beta amyloid) for forming a plaque.

In another embodiment of the present invention, the diagnostic composition comprising the dimethylbenzenamine derivative compound or a pharmaceutically acceptable salt thereof is administered to a patient in need of such treatment by administering to a patient a disease associated with the accumulation and deposition of beta amyloid peptide selectively binding to a single beta-amyloid or beta amyloid plaque Of the present invention.

As a composition for diagnosing or treating a disease associated with the accumulation and deposition of a beta amyloid peptide comprising the derivative compound or a pharmaceutically acceptable salt thereof, the composition has high binding affinity for beta amyloid, high initial brain uptake and excellent pharmacokinetics It can have an excellent effect as an imaging method for early diagnosis of a disease associated with amyloid accumulation and deposition, and as a therapeutic use for delaying the treatment, prevention or onset of the disease.

The composition may further include additives such as pharmaceutical substances for therapeutic use in early diagnosis of the composition, treatment, prophylaxis or delayed onset of the disease, but the present invention is not limited thereto.

The above additives may be formulated together with carriers, excipients or diluents in the form of preparations for parenteral administration such as tablets, hard or soft capsules, granules, pills, powders, etc., or injections, depending on the purpose of administration .

Preferred formulations for this purpose can be administered parenterally, e. G. Subcutaneously, intravenously, intramuscularly or intraperitoneally. Parenteral administration preparations such as injectable solutions or suspensions, injectable preparations such as ready-to-use injectable dry powders which can be used as distilled water for syringes, or topical preparations such as ointments are preferable.

At this time, generally used filler; extender; Binders such as gum arabic, corn starch, microcrystalline cellulose or gelatin; Wetting agents; Disintegrants such as alginic acid, corn starch or potato starch; A diluent such as a surfactant or an excipient such as calcium phosphate or lactose may be used together.

In addition, the dimethylbenzene amine derivative compound of the present invention can be used as a diagnostic agent for diagnosing diseases related to the accumulation and deposition of degenerative brain diseases or beta amyloid peptides as a beta amyloid detecting fluorescent ligand.

Previously reported beta amyloid imaging agents have a complexity in their manufacturing process and also have a limitation in that they lack beta amyloid plaque because they have the property of binding to phosphorylated tau protein fibers that are not only beta amyloid plaques.

Accordingly, the present invention has developed a diagnostic imaging agent using the dimethylbenzene amine derivative compound of the present invention, so that it has not only a beta amyloid plaque but also a specific selectivity for a single beta amyloid, and the fluorescence intensity after beta amyloid is remarkably increased, Can have an excellent effect as a diagnostic imaging agent for diseases associated with brain diseases or beta amyloid accumulation and deposition.

Diseases related to degenerative brain disease or beta amyloid accumulation and deposition include, for example, Alzheimer's disease, Down's syndrome, mild cognitive impairment, cerebral amyloid angiopathy, vascular dementia, It may be, but is not limited to, a multi-infarct dementia.

In addition, since the fluorescence intensity of the fluorescence intensity is increased after binding with the beta amyloid aggregates, the imaging agent can be detected even in the wavelength range of 400 to 700 nm. Therefore, Can detect beta amyloid plaques, and thus has excellent ability to image beta amyloid plaques in vivo (ex vivo / in vivo).

More specifically, the following compound having boronic acid introduced into the dimethylbenzene amine derivative represented by the above formula (1) has a high binding affinity with beta amyloid

And it is able to detect beta amyloid specifically in deep biological cells or tissues from the surface by increasing more than 90 times as compared with that before binding of fluorescence intensity after binding with beta amyloid, and it is possible to detect single beta amyloid and have high accuracy And can be used as a diagnostic imaging agent with excellent imaging ability in vivo (ex vivo / in vivo).

In yet another embodiment of the present invention, a kit for diagnosing or treating a disease associated with degenerative brain disease or beta amyloid accumulation and deposition comprising the diagnostic imaging agent may be provided.

The kit may be an RT-PCR kit, a DNA chip kit, a microarray, or a protein chip kit, but is not limited thereto. The kit for diagnosis or treatment may be prepared by a conventional method used in the art using a diagnostic imaging agent or a composition for diagnosing or treating a disease associated with the degenerative brain disease or beta amyloid accumulation and deposition.

Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples. It is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is possible.

Accordingly, the spirit of the present invention should not be construed as being limited to the embodiments described, and all of the equivalents or equivalents of the claims, as well as the following claims, belong to the scope of the present invention .

[Example 1-3] Synthesis of dimethylbenzeneamine derivative compound

Synthesis of 1- (5- (4- (dimethylamino) phenyl) furan-2-yl) ethanone (Compound b)

4- (dimethylamino) benzeneboronic acid (Sigma-Aldrich) was added to a DME solution in which 5-bromo-2-acetylfuran (compound a) (1.6 g, 8.5 mmol) Pd (PPh ₃ ) ₄ (0.98 g, 0.85 mmol) was dissolved in 2M Na ₂ CO ₃ (10 mL), and the solution was further added to the solution. was refluxed. the resulting solution was dried over and then was extracted with ethyl acetate, washing with water, MgSO _4. After evaporation of the solvent in vacuo, the residue was analyzed by chromatography (chromatographed) to afford the _{(SiO 2, EtOAc / n -hexane} = 4/1 (v / v)), the compound b (1.3g, 71 %).

Synthesis of Dimethylbenzenamine Derivatives

(Compound b) and the corresponding 4-benzaldehyde (Sigma-Aldrich, St. Louis, Mo.) were added to the above prepared 1- (5- (4- (dimethylamino) , MO, USA) in DMF was added 5 N NaOH (50 mL) at 0 DEG C. The reaction mixture was stirred at room temperature for 8 hours until the starting materials disappeared. the insert into cold ice water, which was then adjusted to pH 6 with 1 n HCl. the resulting precipitate was filtered, and the residue was chromatographed _{(SiO 2, EtOAc / n -hexane} = 1/4 (v / v)), Dimethylbenzene amine derivative compound of the following Table 1 was obtained.

Dimethylbenzene amine derivative compound Example 1

¹H NMR (500 MHz, DMSO-d₆,d, ppm): 2.96 (6H, s), 6.77 (2H, d, J = 9.2 Hz), 6.96 (1H, d, J = 3.7 Hz), 7.68 (2H, m), 7.71 (2H, m), 7.77 (2H, m), 7.84 (2H, m), 7.88 (1H, d, J = 3.7 Hz), 8.11 (OH, s); ¹³CNMR(125MHz,DMSO-d₆, d, ppm): 39.8, 105.4, 111.9, 116.3, 122.6, 122.8, 126.2, 127.5, 134.5, 135.9, 141.5, 150.9, 151.2, 159.4, 162.2, 175.1; ESI-MS (M + H) m/z = 362.0.((E) -3- (5- (4- (dimethylamino) phenyl) furan-2-yl) prop- 1-enyl) phenylbromonic acid)
(4 - ((E) -3- (5- (4- (dimethylamino) phenyl) furan-2-yl) prop-

^{1 H NMR (500 MHz, DMSO} -d 6, d, ppm): 2.96 (6H, s), 6.77 (2H, d, J = 9.2 Hz), 6.96 (1H, d, J = 3.7 Hz), 7.68 ( 2H, m), 7.71 (2H, m), 7.77 (2H, m), 7.84 (2H, m), 7.88 (1H, d, J = 3.7 Hz), 8.11 (OH, s); ¹³ CNMR (125 MHz, DMSO-d ₆ , d, ppm): 39.8, 105.4, 111.9, 116.3, 122.6, 122.8, 126.2, 127.5, 134.5, 135.9, 141.5, 150.9, 151.2, 159.4, 162.2, 175.1; ESI-MS (M + H) m / z = 362.0. Example 2
(Dimethylamino) phenyl) furan-2-yl) prop-2-en-1-one)
(E) -3- (4- (diethylamino) phenyl) -1- (5- (4- (dimethylamino) phenyl) furan-

^{1 H NMR (500 MHz, CDCl} 3, d, ppm): 1.20 (6H, t, J = 7.1 Hz), 3.03 (6H, s), 3.41 (4H, q, J = 7.1 Hz), 6.59 (1H, d, J = 3.7 Hz), 6.66 (2H, d, J = 8.8 Hz), 6.75 (2H, d, J = 8.8 Hz), 7.28 (1H, d, J = 15.4 Hz) ,, 7.33 (1H, d J = 3.7 Hz), 7.54 (2H, d, J = 8.9 Hz), 7.71 (2H, d, J = 8.9 Hz), 7.82 (H, d, J = 15.4 Hz); ¹³ CNMR (125 MHz, CDCl ₃ , d, ppm): 12.6, 40.2, 44.5, 104.9, 111.3, 112.0, 116.2, 117.9, 119.4, 122.2, 126.3, 130.6, 143.6, 149.5, 150.8, 152.5, 158.7, 177.4; ESI-MS (M + H) m / z = 389.1. Example 3
Furan-2-yl) prop-2-en-1-one)
(E) -3- (4- (dimethylamino) phenyl) -1- (5- (4- (dimethylamino) phenyl) furan-

^{1 H NMR (500 MHz, CDCl} 3, d, ppm): 3.03 (6H, s), 3.04 (6H, s), 6.61 (1H, d, J = 3.7 Hz), 6.71 (2H, d, J = 8.8 Hz), 6.74 (2H, d , J = 8.8 Hz), 7.31 (1H, d, J = 15.4 Hz) ,, 7.34 (1H, d, J = 3.7 Hz), 7.57 (2H, d, J = 8.9 Hz ), 7.72 (2H, d, J = 8.9 Hz), 7.84 (H, d, J = 15.4 Hz); ¹³ CNMR (125 MHz, CDCl ₃ , d, ppm): 40.1, 40.2, 104.9, 111.8, 112.0, 116.7, 117.8, 119.6, 123.0, 126.3, 130.3, 143.6, 150.1, 151.9, 152.5, 158.8, 177.3; ESI-MS (M + H) m / z = 361.1.

[Example 4] Evaluation of fluorescence property of dimethylbenzene amine derivative compound against beta amyloid.

Experiments were conducted to measure fluorescence intensities before and after binding of the dimethylbenzene amine derivatives of Examples 1 to 3 with single beta amyloid and complex beta amyloid.

The compounds of Table 1 were mixed with single beta amyloid and complex beta amyloid for 15 minutes at room temperature, respectively, and then the fluorescence intensities of the compounds before binding and the fluorescence intensities after binding were compared. Respectively.

(1) Preparation of A? 42 fibril

Aβ peptide was dissolved in pH 7.4 PBS buffer solution to a final concentration of 100 μM Aβ42 (rPeptide (Bogart, GA, 30622)) and then stirred at room temperature for 3 days using a magnetic bar at 1200 rpm. The formation of A [beta] fibrils was confirmed by the ThT assay.

(2) Fluorescence spectra measurement of the compounds of the present invention in vitro

The maximum excitation and emission wavelength? _Max of the three dimethylbenzene amine derivative compounds of Examples 1 to 3 according to the present invention ≪ / RTI > SpectraMax M2 (Molecular Devices). To the PBS buffer solution, 10 mM of Ab42 fibril prepared by the above method, 5 mu M of the compounds of Examples 1 to 3 was used as a final concentration. In general, here λ _max is the fixed first Are determined by scanning the emission, emission λ _max is Lt; / RTI > is determined by scanning the excitation spectrum with a fixed? _Em .

As a result, the FAβ / F0 values of Examples 1 to 3 were 93.62, 58.9 and 48.9, respectively, with respect to fluorescence reactivity with the aggregated Aβ peptide (Table 2). Also in FIG. 1, it is suggested that the compound according to the present invention has a marked difference in the emission wavelengths before and after binding with the amyloid plaques.

(3) ement of binding affinity (Kd).

The dissociation constants (Kd) for the three dimethylbenzene amine derivative compounds of Examples 1 to 3 according to the present invention were measured using 10 μM of the aggregated Aβ42 peptide. Aggregated Aβ42 (10 μM final concentration) was mixed with various concentrations of each compound in PBS buffer (10, 5, 2.5, 1.25, 0.5 μM). Nonlinear regression analysis was performed using GraphPad Prism. The dissociation constant (Kd) was obtained from the best fitted curve.

As can be seen from the following Table 2, the Kd values of the compounds of Examples 1 to 3 were found to have a high binding affinity for A? 42 of not more than 2.5. In order to be a preferred compound for detecting amyloid aggregates, the binding affinity for beta amyloid should be high. When the compounds according to the present invention have high binding affinity, have.

The fluorescence intensities of the compounds of Examples 1 to 3 were increased when the compounds of Examples 1 to 3 were bound to the beta amyloid. In the case of Example 1, beta amyloid and beta amyloid Indicating that the fluorescence intensity after conjugation is increased about 90 times or more.

[Example 5] In vivo imaging experiment of a brain tissue fragment of Alzheimer's disease model mouse using the dimethylbenzene amine derivative compound of the present invention.

(1) Comparison test for detection of beta amyloid in general mouse and dementia-induced mouse using the compound of Example 1.

The compound of Example 1 was injected into the tail vein of 13-month-old dementia-inducing mice and normal mice, and the brains and organs of each mouse were harvested 30 minutes later. The mice were then subjected to fluorescence imaging (Maestro 2.0 in vivo imaging system The results are shown in FIG. 2. As shown in FIG. 2, fluorescence was not detected in brain tissues obtained after intravenous injection into general mice, but brain tissue obtained after intravenous injection into dementia- , It was confirmed that the compound of Example 1 binds to the beta amyloid plaques which are present in the brain tissues and exhibits a high fluorescence intensity.

(2) Fluorescence imaging test of brain tissue sections of dementia-induced mice using the compounds of Examples 1 to 3.

Experiments were conducted to confirm the binding affinities of the compounds of Examples 1 to 3 as in-vivo diagnostic imaging agents using disease model mice.

The compounds of Examples 1 to 3 were injected into the tail vein of the 13-month old dementia-induced mouse, and the brains and organs of each mouse were extracted 30 minutes later. A fluorescence image (laser confocal scanning microscope) was taken to identify beta amyloid plaques associated with the compounds of Examples 1-3. The results are shown in Fig. The extracted brain tissue was photographed with a fluorescence microscope. As a result, it was found that the compounds of Examples 1 to 3 correctly detected the lesion in which beta amyloid was accumulated.

Claims (8)

A composition for diagnosing degenerative brain disease comprising a dimethylbenzeneamine derivative compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

(Wherein Z is a hetero atom selected from nitrogen or boron,
Wherein R ₁ to R ₂ may be independently of each other hydrogen, (C ₁ -C ₁₀ ) alkyl, hydroxy or carboxy.
The method according to claim 1,
Wherein Z is one heteroatom selected from nitrogen or boron, and R ₁ to R ₂ are hydrogen, (C ₁ -C ₅ ) alkyl or hydroxy.
3. The method of claim 2,
Wherein R ₁ to R ₂ are hydrogen, hydroxy, methyl or ethyl.
3. The method of claim 2,
Wherein the formula (1) is any one selected from the group consisting of the following compounds.

The method according to claim 1,
Wherein said composition binds to beta amyloid oligomers and beta amyloid plaques.
The method according to claim 1,
Wherein the degenerative brain disease is a disease selected from the group consisting of Alzheimer's disease, senile dementia, neurodegenerative disorders or amyloidosis.
A diagnostic imaging agent for a disease selected from the group consisting of Alzheimer's disease, senile dementia, neurodegenerative disorders or amyloidosis, comprising the composition according to any one of claims 1 to 6.
A kit for diagnosing or treating a disease selected from the group consisting of Alzheimer's disease, senile dementia, neurodegenerative disorders or amyloidosis, comprising the diagnostic imaging agent according to claim 7.

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