KR20170110496A - New compounds or pharmaceutically acceptable salt thereof selective for amyloid-β plaque and method for imaging amyloid-β plaque in vivo using the same - Google Patents

Abstract

The present invention relates to a novel compound which is selective for beta amyloid plaques or a pharmaceutically acceptable salt thereof and a method for imaging beta amyloid plaques using the same, more specifically, And a method for synthesizing a novel compound or a pharmaceutically acceptable salt thereof, which can be used therefor, in a high-resolution three-dimensional imaging through a low-energy-source two-photon microscope.
A novel compound, or a pharmaceutically acceptable salt thereof, selective to beta amyloid plaques according to the present invention releases strong two-photon fluorescence and is selectively stained with amyloid. In addition, the compound or its pharmaceutically acceptable salt has a property of passing through the blood brain barrier of a rat efficiently because of its low molecular weight and electrically neutral nature. Based on these properties, amyloid deposition in the brain of transgenic mice can be imaged into a high-resolution three-dimensional image using a two-photon microscope.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel compound which is selective for beta amyloid plaques or a pharmaceutically acceptable salt thereof, and a method for imaging beta amyloid plaques in vivo using the same or a salt thereof for amyloid-β plaques and methods for imaging amyloid- in vivo using the same}

The present invention relates to a novel and novel method for the deposition of beta amyloid which can provide real-time two-photon microscopy (TPM) images of amyloid-β (Aβ) plaques in vivo Or a pharmaceutically acceptable salt thereof, and a method for imaging beta amyloid plaques in vivo using the same.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to chronic dementia and cognitive decline.

Pathological hallmarks of AD include misfolded protein aggregation and unbalanced reactive oxygen, metal ions, and acetylcholine levels.

Among them, the formation of beta amyloid plaques and neurofibrillary tangles within specific brain regions have been identified as the earliest inventions of AD.

At the same time as the additional effort for AD, we were devoted to AD drug development to reduce or inhibit A [beta] species.

However, little is known about the underlying mechanisms for the formation of A [beta] plaques and the effects of A [beta] plaques on AD.

Therefore, in order to solve the above problem, it is highly desirable to visualize a clear three-dimensional (3D) visualization of A [beta] plaques through real-time methods in vivo.

The TPM for performing the 3D visualization method is an attractive tool, and in particular the TPM contains deep tissue imaging and intrinsic section stability and utilizes two adjacent IR photons (> 700 nm).

The TPM can be performed in a noninvasive manner because it has the requisite high spatial resolution and can provide 3D visualization of the intact tissue deep inside.

In addition to advances in micro-endoscopic and video-rate scanning systems, TPM also has many potential for clinical use, including early diagnosis, monitoring therapy, and accurate diagnosis.

However, for practical application of the TPM, a sensitive, light-stable two-photon probe and a TP excited (two-photon excited) fluorescence are used to provide visualization through a high signal-to-noise fluorescence (TPEF) technology should be combined.

In order to visualize the Aβ plaques, a number of small molecule compounds have been developed and bis-styrylbenzene derivatives such as MeO-XO4 have been frequently used for in vivo TPM imaging, but small values of two- photon action cross section (hereinafter 'Φδ _TPA '), and low solubility limit the use of bis-styrylbenzene derivatives in TPM.

In addition, due to the photochemical instability such as photo-isomerization, the open chain system of the conjugating bridge (C = C) in the low molecular weight compound can induce rapid light discoloration It is impossible to image the door for a long time.

For this reason, there is a need for the synthesis and development of novel compounds for in vivo imaging of A [beta] plasmids having large values of [Phi] _TPA , good solubility, and light stability.

Korean Patent Publication No. 2016-0067208

It is an object of the present invention to provide a method for treating or preventing Alzheimer's disease by using a novel compound capable of selectively binding to beta amyloid (hereinafter referred to as 'Aβ') plaque or a pharmaceutically acceptable salt thereof, And to provide a method for imaging the distribution of A [beta] plaques.

In order to achieve the above object, the present invention provides a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1, R ₁ to R ₄ are the same or different from each other,

A halogen, a C ₁ to C ₄ alkoxy, a C ₁ to C ₄ alkyl, a C ₁ to C ₄ alcohol, or a group represented by the following formula (1-1)

[Formula 1-1]

m is an integer of 1 to 5,

R ₅ to R ₆ are the same as or different from each other,

Hydrogen, a hydroxyl group, a halogen, C ₁ to alkoxy, C ₁ to alkyl, C ₁ to alcohol, the formula 1-2 or formula 1-3, C ₄ of the C ₄ C _4,

[Formula 1-2]

[Formula 1-3]

n is an integer of 8 to 15,

R ₇ to R ₁₀ are the same or different from each other,

2 or more is fluorine (F) and the remainder is hydrogen.

The present invention also provides a two-photon fluorescence probe comprising the compound or a pharmaceutically acceptable salt thereof.

The present invention also provides a composition for detecting beta amyloid comprising the compound or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition for the treatment or prevention of Alzheimer's disease comprising the compound or a pharmaceutically acceptable salt thereof and a physiologically active substance.

The present invention also relates to a method for the preparation of a pharmaceutical composition comprising the steps of: injecting a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof into a sample separated from a living body; Combining said compound or a pharmaceutically acceptable salt thereof with a beta amyloid plaque in a sample isolated from a living body; Irradiating an excitation source to a sample separated from the living body; And observing fluorescence generated from a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof by a two-photon microscope.

The novel compounds or their pharmaceutically acceptable salts according to the present invention have a large two-photon operating cross-section (PHImax = 420 GM), which can be administered intravenously and then injected into the individual beta amyloid in vivo by using real-time TPM imaging. Or < RTI ID = 0.0 > 'A ') < / RTI > plaque.

It can also be used for dual-color TPM imaging for 3D visualization of A [beta] plaques with internal blood vessels and cerebral amyloid angiopathy (CAA), which is useful for early diagnosis and treatment of AD.

1 is a ¹ H-NMR spectrum (400 MHz) of Compound 1 in CDCl ₃ ;
Figure 2 shows the ¹³ C-NMR spectrum (100 MHz) of Compound 1 in CDCl ₃ containing 2 drops of d ₆ -DMSO;
Figure 3 is a high-resolution mass spectrometry (HRMS) spectrum of Compound 1;
4 is a ¹ H-NMR spectrum (400 MHz) of compound 2 (QAD1) in d ₆ -DMSO;
5 is a diagram showing the ¹³ C-NMR spectrum (100 MHz) of Compound 2 (QAD1) in d ₆ -DMSO;
6 is a graph showing the HRMS spectrum of Compound 2 (QAD1);
7 is a ¹ H-NMR spectrum (400 MHz) of Compound 1 in CDCl ₃ ;
Figure 8 shows ¹³ C-NMR spectra (100 MHz) of compound 2 in CDCl ₃ containing 2 drops of d ₆ -DMSO;
9 shows HRMS spectrum of compound 3;
Fig. 10 shows the one-photon absorption spectra (a, c, e) and the phosphate buffer (a) for compound 1 (a, b), compound 2 (QAD1) (c, d) 2, and 3 in PBS (saline; hereinafter 'PBS') buffer solution (10 mM, pH 7.4);
Figure 11 shows the effect of Compound 1 (a (1)) in 1,4-dioxane, ethanol (EtOH), EtOH: PBS (v / v, 1: 1) and PBS buffer (a, c, e) and emission spectra (b, d, f) of Compound 2 (QAD1) (c, d) and Compound 3 1;
Figure 12 shows the normal absorption spectrum (a) and luminescence spectrum (b) for 1 μM Compound 2 (QAD1) in 1,4-dioxane, EtOH, and PBS buffer (10 mM, pH 7.4); Twophoton excited fluorescence (hereinafter, referred to as 'TPEF') spectrum obtained from beta amyloid (hereinafter referred to as 'Aβ') plaques in brain slices labeled with Compound 2 (QAD1) Luminescence spectrum (c) for Compound 1, Compound 2 (QAD1), and Compound 3 obtained in ethanol; (TPA) spectrum (d) for Compound 1, Compound 2 (QAD1), and Compound 3 obtained from ethanol;
Changes in fluorescence intensity of the composite of Figure 13 is PBS buffer solution (10 mM, pH 7.4) Aβ 1 -42 oligomer (0 to 10 μM) Compound 2 (QAD1) (1 μM) with a in (a) and fluorescence titration (B) of the curve (the calculated value is represented by a solid line and the excitation wavelength is 407 nm);
14 is a diagram showing the molar absorptivity spectra of Compound 1, Compound 2 (QAD1), and Compound 3 obtained from ethanol;
Figure 15 shows the cis-isomer structure of Compound 2 (QAD1) (a), and Compound 3 (b), an optimized geometry by DFT in ethanol;
16 is a graph showing optimized geometrical structures and molecular orbital distribution of compounds 2 (QAD1) (a) and 3 (b) in ethanol by density functional theory (DFT);
17 is a PBS buffer solution (10 mM, pH 7.4) for _{1 -42} Aβ oligomer and compound 1 in the presence of BSA (a, excitation wavelength: 417 nm), compound 2 (QAD1) (b, 417 nm), and the compound 3 (c, 326 nm); Fig.
Figure 18 is the pH for the general purpose buffer (0.1 M citric acid (citric acid), 0.1 M KH 2 PO 4, 0.1 M Na 2 B 4 O 7, 0.1 M Tris, and 0.1 M KCl) Compound 2 (QAD1) in (Excitation wavelength: 407 nm);
Fig. 19 shows the results of the administration of 0 (a), 30 (b), 60 (c), and 150 min (d) after injecting intravenously (hereinafter 'iv') Compound 2 ) In vivo TPM images of the frontal lobe of transformed 5XFAD mice; To visualize the Aβ plaque distribution, an accumulated 230 TPM image (e) along the z-direction at a depth of about 300 μm from the surface of the envelope; 3D reconstructed TP images (f, g) from the surface of the envelope (at a depth of about 300 [mu] m) from the frontal lobe of transformed 5XFAD mice after intravenous Compound 2 (QAD1) (10 mg / kg) and dextran 40 kDa- about 270 TPM images accumulated along the z-direction, and the scale bars are 50 [mu] m (a) and 30 [mu] m (e);
Figure 20 shows the TPM image (a) of the envelope sections of transgenic 5XFAD mouse brains stained with 20 μM of Compound 1 (a), Compound 2 (QAD1) (c), and Compound 3 , c, e); Signal-to-noise (S / S) ratio measured by Compound 1, Compound 2, and Compound 3 and intensity of TP excited fluorescence (TPEF) N ') (b, d, f) (TPEF is collected at 450 to 520 nm at 750 nm excitation in a femtosecond pulse, and the scale bar is 48 μm);
Figure 21 shows the TPM image of the envelope sections of transformed 5XFAD rat brains co-labeled with Compound 2 (QAD1) (a) and Congo red (b) for 90 minutes at 37 & a, b, c); And 20 times magnified merged images (c); TPM image (d) of epidermal tissue section of transgenic 5XFAD rat brain treated with compound 2 (QAD1); As a function of time, the corresponding TPEF intensity e for the A, B, and C shown in (d) (the intensity digitized continuously at intervals of 2 seconds for 1 hour continuously using the xyt mode) was recorded continuously TPEF Is collected at 450 to 520 nm at 750 nm excitation in a femtosecond pulse, and the scale bar is (c) 72 μm and (d) 96 μm.

Hereinafter, a novel compound or a pharmaceutically acceptable salt thereof which is selective to beta amyloid plaques of the present invention and a method of imaging beta amyloid plaques using the same will be described in more detail.

The inventors of the present invention have found that when a novel compound or a pharmaceutically acceptable salt thereof is used, TPM can be imaged in real time by specifically binding to beta amyloid in vivo, thereby completing the present invention.

The present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1, R ₁ to R ₄ are the same or different from each other,

A halogen, a C ₁ to C ₄ alkoxy, a C ₁ to C ₄ alkyl, a C ₁ to C ₄ alcohol, or a group represented by the following formula (1-1)

[Formula 1-1]

m is an integer of 1 to 5,

R ₅ to R ₆ are the same as or different from each other,

Hydrogen, a hydroxyl group, a halogen, C ₁ to alkoxy, C ₁ to alkyl, C ₁ to alcohol, the formula 1-2 or formula 1-3, C ₄ of the C ₄ C _4,

[Formula 1-2]

[Formula 1-3]

n is an integer of 8 to 15,

R ₇ to R ₁₀ are the same or different from each other,

2 or more is fluorine (F) and the remainder is hydrogen.

The compound or a pharmaceutically acceptable salt thereof may be represented by the following formula (2).

(2)

In Formula 2, R ₁ to R ₄ are the same or different from each other,

Hydrogen, C ₁ to C ₄ alkyl, C ₁ to C ₄ alcohols, or the following formula (2-1)

[Formula 2-1]

R ₅ to R ₆ are the same as or different from each other,

Hydrogen, a C ₁ to C ₄ alcohol, the following Formula 2-2 or Formula 2-3.

[Formula 2-2]

[Formula 2-3]

The compound or a pharmaceutically acceptable salt thereof may be represented by any one of the following formulas (3-1) to (3-14).

[Formula 3-1]

[Formula 3-2]

[Formula 3-3]

[Chemical Formula 3-4]

[Formula 3-5]

[Chemical Formula 3-6]

[Chemical Formula 3-7]

[Chemical Formula 3-8]

[Chemical Formula 3-9]

[Chemical Formula 3-10]

[Formula 3-11]

(3-12)

[Chemical Formula 3-13]

[Chemical Formula 3-14]

A representative compound in the compound according to the present invention or a pharmaceutically acceptable salt thereof may be a compound (QAD1) represented by the following formula (3-2), wherein QAD1 is a cyclic conjugating bridge and a dissolution unit (DAD) structure with a solubilizing unit, especially QAD1, exhibits a high sensitivity and selectivity for bright phosphorescent excited fluorescence (hereinafter "TPEF"), considerable solubility, light stability and Aβ .

[Formula 3-2]

The present invention also provides a two-photon fluorescence probe comprising the compound or a pharmaceutically acceptable salt thereof.

The two-photon fluorescence probe can specifically bind to beta amyloid plaques.

The present invention also provides a composition for detecting beta amyloid comprising the compound or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition for the treatment or prevention of Alzheimer's disease comprising the compound or a pharmaceutically acceptable salt thereof and a physiologically active substance.

The physiologically active substance may be any one selected from the group consisting of drugs, peptides, proteins, and genes, but is not limited thereto.

The present invention also relates to a method for the preparation of a pharmaceutical composition comprising the steps of: injecting a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof into a sample separated from a living body; Combining said compound or a pharmaceutically acceptable salt thereof with a beta amyloid plaque in a sample isolated from a living body; Irradiating an excitation source to a sample separated from the living body; And observing fluorescence generated from a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof by a two-photon microscope.

The excitation source may have a wavelength in the range of 700 to 800 nm, but is not limited thereto.

Hereinafter, a novel compound or a pharmaceutically acceptable salt thereof and a method for imaging beta amyloid plaques using the same according to the present invention will be described in more detail. However, the present invention is not limited by these examples.

Compound 1, compound 2 (QAD1), and compound 3, which are novel quadrupole compounds having a cyclic conjugation bridge, were synthesized by the following Reaction Scheme 1 and the synthesis route was as follows.

[Reaction Scheme 1]

(a) Potassium tert-butoxide (KOtBu), THF, 0 to 20 占 폚;

(b) Triethyl phosphite (hereinafter referred to as 'P (OEt) ₃ '), 125 ° C;

(c) i: Compound 1, 2-bromoethylacetate, sodium hydride (NaH), 18-Crown-6, THF, 80 ° C; ii: Sodium metoxide (NaOMe '), methanol, 0-20 ° C.

Referring to Scheme 1 above, compounds 1 and 2 (QAD1) can be synthesized by reduction-induced cyclization of 4-dialkylamino-2-nitrobenzaldehyde and 4-dialkylamino- Was synthesized in a 65-75% yield via Horner-Wadsworth-Emmons coupling reaction between bisphosphonates substituted with tetrafluorobenzene.

Compound 3 was synthesized by hydrolysis at a yield of 58% by substitution reaction of compound 1 with 2-bromoethylacetate.

Specifically, Compound 1, Compound 2 (QAD1) and Compound 3 were synthesized according to Reaction Schemes 2 to 4, and the reagents and conditions according to Reaction Schemes 2 to 4 were as follows.

[Reaction Scheme 2]

Reagents and conditions: (i) Iodomethane, proton-sponge, CH ₃ CN, reflux, 16 h; (ii) Phosphorous oxychloride (POCl ₃ '), DMF, 90 ° C, 8 hours; (iii) Potassium tert-butoxide (hereinafter referred to as 'KOtBu'), THF, at 0 ° C, room temperature for 12 hours; (iv) Triethyl phosphite (P (OEt) ₃ ) at 125 占 폚 for 12 hours.

[Reaction Scheme 3]

Reagents and conditions: (i) 2-Bromoethanol, calcium carbonate, potassium iodide, H ₂ O, 100 ° C, 8 hours; (ii) acetyl chloride, triethylamine, 35 C, 12 h; _{(iii) POCl 3, DMF,} 90 ℃, 12 hours; (iv) potassium tert-butoxide THF, at 0 ° C, room temperature for 12 hours; (v) P (OEt) ₃ , 125 DEG C, 12 hours; (vi) Sodium methoxide (NaOMe '), methanol, at 0 ° C, room temperature for 12 hours.

[Reaction Scheme 4]

Reagents and conditions: (i) 2-bromoethylacetate, sodium hydride (NaH), 18-Crown-6, THF, room temperature at 80 占 폚; (ii) NaOMe, methanol.

≪ Example 1 >

Compound A

To a stirred solution of 3-nitroaniline (2.0 g, 14.5 mmol) and proton-sponge (6.83 g, 31.86 mmol) in anhydrous acetonitrile (anhydrous CH ₃ CN, 70 mL) Iodomethane (2.0 mL, 31.9 mmol) was added.

The total reaction was stirred at 80 < 0 > C for 8 hours under a nitrogen atmosphere. To obtain a residue was dissolved in chloroform (CHCl _3, 100 ㎖) The solvent was evaporated and washed with water after the (100 ㎖) and _{1 (N) H 2 SO 4} (50 ㎖).

The organic fraction (organic portion) of sodium sulfate (Na ₂ SO ₄₎ the dried crude product (crude product) the eluent (eluent) in EtOAc / hexane (ethylene acetate / hexane, 1: 1 v / v) using a column chromatography Purification by flash chromatography gave a red solid (Compound A).

Yield: 1.51 g (63%). ^{1 H-NMR (400MHz, CDCl} 3): δ (ppm) 7.52-7.47 (m, 2H), 7.32 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 8.8 Hz, J = 2.8 Hz, 1H), < / RTI > 3.03 (s, 6H). ¹³ C-NMR (100 MHz, CDCl ₃ ):? (Ppm) 150.4, 148.9, 129.3, 117.4, 110.2, 105.7, 40.2.

Compound B

A solution of A (1.3 g, 7.83 mmol) in anhydrous DMF (2.17 mL, 28.27 mmol) was cooled to 0 C in an ice bath and POCl ₃ (1.07 mL, 11.74 mmol) was added dropwise.

The reaction mixture was then heated to 80 < 0 > C within 1 h and stirred at 80 < 0 > C for an additional 6 h.

After the stirring, the dark solution was cooled to room temperature, cooling water was poured into the cooled cancer solution, and the resulting precipitate was filtered. The residue was recrystallized from acetone to give a brown solid (Compound B).

Yield: 1.2 g (79%). ¹ H-NMR (400 MHz, CDCl ₃ ):? (Ppm) 10.12 (s, 1H), 7.91 (d, J = 2.8 Hz, 1H), 7.09 dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 3.14 (s, 6H) ppm;

¹³ C-NMR (100 MHz, CDCl ₃ ):? (Ppm) 186.5, 154.2, 150.9, 131.5, 117.3, 114.5, 105.9, 40.3.

Compound D

NaOtBu (0.654 g, 5.83 mmol) was added at 0 < 0 > C under nitrogen atmosphere to a stirred solution of compound C (1.0 g, 2.33 mmol) dissolved in anhydrous TFH (80 mL).

After 30 min, a solution of compound B (0.95 g, 4.89 mmol) in THF (20 mL) was added to the reaction and stirred overnight at room temperature.

Water was added to terminate the reaction, followed by a red precipitate, a solid residue, which was washed with water and hexane and then with THF. The residue was dried in vacuo to give a red solid (Compound D).

Yield: 0.730 g (63%). ¹ H-NMR (400 MHz, CDCl ₃ ):? (Ppm) 7.86 (d, J = 16 Hz, 1H), 7.64 6.87 (m, 2H), 3.07 (s, 6 H).

Compound 1

A solution of Compound D (0.05 g, 0.094 mmol) in P (OEt) ₃ (5 mL) was heated at reflux overnight in a nitrogen atmosphere.

The solvent was removed in vacuo and the product was purified on a silica column using EtOAc / hexane (ethylene acetate / hexane, 3: 7 v / v) to give a brick red solid (Compound 1).

1 to 3 show the ¹³ C-NMR spectra (100 MHz) and the high-resolution mass spectrometry ( ¹ H-NMR spectrum) of CDCl ₃ containing 2 drops of d ₆ -DMSO, ; Hereinafter, 'HRMS') spectrum, and the data for FIGS. 1 to 3 are as follows.

Yield: 0.032 g (72%). ^{1 H-NMR (400 MHz,} CDCl 3): δ ppm 9.12 (s, 1H), 7.40 (d, J = 8.8 Hz, 1 H), 6.97 (s, 1H), 6.69-6.64 (m, 2H), 2.90 (s, 6 H).

^{13 C-NMR (100 MHz,} CDCl 3 contained 2-drops of d 6 -DMSO): δ (ppm) 148.5, 144.7, 142.3 (d, J = 10.7Hz), 138.2, 122.2, 121.1, 119.7, 110.1, 106.8 , 93.5, 41.4. HRMS (FAB ⁺ ): m / z calcd for [C ₂₆ H ₂₂ N ₄ F ₄ ]: 466.1775, found: 466.1776

Compound E

3-nitroaniline (10.0 g, 72.4 mmol), 2-bromoethanol (12.14 mL, 181.0 mmol) and calcium carbonate (CaCO _{3 ,} 18.11 g, 181.0 mmol) were added to water (150 mL) mmol) and potassium iodide (KI, 1.2 g, 7.2 mmol) were added and refluxed for 16 hours.

After filtration, the filtrate was extracted with EtOAc (50 mL x 3) and the combined organic layers were washed with saturated NaCl (50 mL x 2), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated to give the crude product .

The crude product was purified by silica column chromatography using eluant EtOAc / hexane (ethylene acetate / hexane, 3: 7 v / v) to give a yellow solid (Compound E).

Yield: 6.20 g (38%). ^{1 H-NMR (400MHz, CDCl} 3): δ (ppm) 7.36-7.35 (m, 2H), 7.16 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 8.4 Hz, J = 2.8 Hz, 1H), 4.68 (t, J = 4.8 Hz, 2H), 3.69-3.65 (m, 4H), 3.47 (t, J = 5.6 Hz, 4H).

¹³ C-NMR (100 MHz, CDCl ₃ ):? (Ppm) 148.9, 148.6, 129.3, 117.7, 110.2, 105.8, 59.4, 54.8.

Compound F

A solution of Compound E (5.4 g, 23.87 mmol) in triethylamine (7.33 mL, 52.52 mmol) was cooled to 0 < 0 > C.

Then, acetyl chloride (3.73 mL, 52.52 mmol) was added dropwise to the solution, and the mixture was stirred overnight at 35 ° C. After cooling to room temperature, water (25 mL) was added to hydrolyze the mixture.

After vigorous stirring for 1 hour after the hydrolysis, THF was evaporated and a saturated aqueous sodium bicarbonate solution was used to neutralize.

After extracting with chloroform (3 x 50 mL), the combined organic layers were dried over Na ₂ SO ₄ , filtered and the solvent removed in vacuo to give a yellow solid (Compound F).

Yield: 7.0 g (95%). ^{1 H-NMR (400 MHz,} CDCl 3): δ (ppm) 7.45 (t, J = 2.8 Hz, 1H), 7.32 (dd, J = 8.0 Hz, J = 2.0 Hz, 1 H), 7.17 (t, J = 8 Hz, 1H), 6.94 (dd, J = 8.4 Hz, J = 6.0 Hz, 1H), 4.12 ), 1.88 (s, 6H).

¹³ C-NMR (100 MHz, CDCl ₃ ):? (Ppm) 170.2, 148.9, 147.8, 129.5, 117.1, 110.7, 105.7, 60.8, 49.3, 20.4.

Compound G

Freshly distilled DMF (3.10 mL, 32.22 mmol) was cooled to 0 ° C in an ice bath and POCl ₃ (1.62 mL, 17.72 mmol) was added dropwise.

After stirring at room temperature for 1 hour, compound F (5.0 g, 16.11 mmol) was added to DMF and the reaction was stirred overnight at 70 < 0 > C. Thereafter, the cancer solution was cooled to room temperature and poured with cooling water.

Saturated aqueous sodium acetate solution was added slowly and the mixture was stirred overnight. After stirring overnight, a brown precipitate was obtained and washed with acetone to give a dark brown solid (Compound G).

Yield: 2.80 g (52%). ¹ H-NMR (400 MHz, CDCl ₃ ):? (Ppm) 10.09 (s, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.28 dd, J = 8.8, J = 2.8 Hz, 1H), 4.27 (t, J = 5.6 Hz, 4H), 3.74 (t, J = 5.6 Hz, 4H), 2.03 (s, 6H).

¹³ C-NMR (100 MHz, CDCl ₃ ):? (Ppm) 186.2, 170.6, 152.4, 151.8, 131.5, 118.4, 114.7, 106.5, 60.8, 49.8, 20.9.

Compound H

To a stirred solution of compound C (0.5 g, 1.11 mmol) in anhydrous THF (25 mL) was added NaOtBu (0.274 g, 2.44 mmol) at 0 <0> C under a nitrogen atmosphere.

After 30 minutes, a solution of Compound G (0.826 g, 2.44 mmol) dissolved in anhydrous THF (20 mL) was added to the reaction mixture and stirred overnight at room temperature.

Water was then added to terminate the reaction, and a precipitate was obtained, which was then washed sequentially with water and hexane to obtain a crude product.

The crude product was purified by silica column chromatography using eluant EtOAc / hexane (ethylene acetate / hexane, 7: 3 v / v) to yield a red solid (Compound H).

Yield: 0.205 g, 25%. ¹ H NMR (400 MHz, CDCl ₃ ):? (Ppm) 7.83 (d, J = 16.8 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H) J = 5.6 Hz, 1 H), 6.88 (d, J = 16.8 Hz, 1H), 4.28 (d, J = J = 5.6 Hz, 4H), 2.05 (s, 6 H) ppm.

¹³ C NMR (100 MHz, CDCl ₃ ):? (Ppm) 170.7, 149.4, 147.9, 145.9, 143.3, 131.7, 128.9, 120.2, 116.3, 115.2, 107.3, 61.2, 49.8, 21.1.

Compound I

A solution of compound H (0.08 g, 0.098 mmol) in P (OEt) ₃ (5 mL) was heated at 125 &lt; 0 &gt; C under refluxing reaction overnight.

The solvent was removed in vacuo and the product was purified by silica column chromatography using eluent EtOAc / hexane (ethylene acetate / hexane, 7: 3 v / v) to yield a red solid (Compound I).

Yield: 0.050 g (68%). ^{1 H-NMR (400 MHz,} CDCl 3 contained 2-drops of d 6 -DMSO): δ (ppm) 9.31 (s, 1H), 7.34 (d, J = 8.8 Hz, 1 H), 6.89 (s, 1H J = 6.0 Hz, 4H), 6.59 (s, 1H), 6.58 (dd, J = 8.8 Hz, , &Lt; / RTI &gt; 1.93 (s, 6H).

¹³ C-NMR (100 MHz, CDCl ₃ contained 2-drops of d ₆ -DMSO):? (ppm) 170.5, 144.6, 142.2, 138.5, 138.3, 122.3, 121.5, 119.9, 108.9, 106.5, 93.3, 61.4, 50.3, 20.8.

Compound 2 (QAD1)

A solution of compound I (0.05 g, 0.026 mmol) in methanol (10 mL) was added to a solution of sodium methoxide (NaOMe, 20 μL) and the reaction was stirred at 0 ° C. for 60 minutes.

The solvent was then evaporated and the residue was dissolved in CHCl ₃ (25 mL) with 2.5 mL methanol and washed with saturated aqueous ammonium chloride (NH ₄ Cl) solution (25 mL).

The organic layer was dried over anhydrous magnesium sulfate (MgSO ₄₎ and concentrated in vacuo.

The crude product eluent of MeOH / CHCl ₃ (methanol / chloroform, 1: 20 v / v) for further purification by column chromatography of silica fatigue dark brown solid (compound 2) was obtained by using a substantial amount.

4 to 6 is a compound 2 (QAD1) d ₆ -DMSO from ¹ H-NMR spectrum (400 MHz), d ₆ -DMSO at ¹³ C-NMR spectrum (100 MHz), and HRMS will showing the spectrum of FIG. 4 to 6 are as follows.

¹ H-NMR (300 MHz, d ₆ -DMSO):? (Ppm) 10.89 (s, 1H), 7.41 (d, J = 9.2 Hz, 1H) H), 6.57 (d, J = 8.0 Hz, 1H), 4.78 (bs, 2H), 3.59 (t, J = 6.0 Hz, 4H), 3.48 (t, J = 6.4 Hz, 4H).

¹³ C-NMR (100 MHz, d ₆ -DMSO):? (Ppm) 145.3, 139.1, 127.8, 124.5, 122.7, 120.8, 120.4, 118.7, 109.6, 108.6, 58.3, 53.9. ^{HRMS (FAB +): m /} z calcd for [C 30 H 30 N 4 O 4 F 4]: 586.2198, found: 586.2198

Compound J

Sodium hydride (NaH, 0.016 g, 0.68 mmol) and 18-Crown-6 (0.011 g, 0.045 mmol) were dissolved in anhydrous THF (10 mL) under nitrogen atmosphere and stirred for 10 min. And refluxed at 80 ° C for 1 hour.

After 1 hour, the reaction mixture was stirred at room temperature, and a solution of 2-bromoethylacetate (36 μL, 0.33 mmol) in THF was added dropwise at 0 ° C. and stirred overnight at room temperature.

After the reaction was complete, THF was evaporated in vacuo and the residue was dissolved in ethylacetate (50 mL) and washed with water (2 x 25 mL). The organic layer was dried with Na ₂ SO ₄ and evaporated the organic solvents.

The product was purified by column chromatography using eluent EtOAc / hexane (ethylene acetate / hexane, 3: 7 v / v) to give a yellow solid (Compound I).

Yield: 0.067 g (71%). ^{1 H-NMR (400MHz, CDCl} 3): δ (ppm) 7.55 (d, J = 8.8 Hz, 1H), 6.84 (dd, J = 8.8 Hz, J = 2.4 Hz, 1 H), 6.70 (d, J = 1.6 Hz, 1H), 6.50 (s, 1H), 4.30-4.27 (m, 4H), 3.04 (s, 6H), 1.92 (s, 3H).

¹³ C-NMR (100 MHz, CDCl ₃ ):? (Ppm) 170.6, 148.5, 145.9, 143.3, 139.1, 122.2, 121.7, 120.3, 113.2, 110.2, 106.7, 93.1, 64.4, 41.9, 29.9, 20.8.

Compound 3

A solution of compound J (0.05 g, 0.078 mmol) in methanol (10 mL) was added to NaOMe (20 [mu] L) and the reaction mixture was stirred at 0 [deg.] C for 60 min.

The solvent was then evaporated and the residue was dissolved in CHCl ₃ (25 mL) with 2.5 mL methanol and washed with saturated aqueous ammonium chloride (NH ₄ Cl) solution (25 mL).

The organic layer was dried over anhydrous magnesium sulfate (MgSO ₄₎ and concentrated in vacuo.

The crude product was purified by silica column chromatography using eluent EtOAc / hexane (ethylene acetate / hexane, 7: 3 v / v) to give a yellow solid (Compound 3).

7 to 9 show the ¹ H-NMR spectrum (400 MHz) of Compound 3 in CDCl ₃ of Compound ₃ , the ¹³ C-NMR spectrum (100 MHz) in CDCl ₃ containing 2 drops of d ₆ -DMSO, and the HRMS spectrum The data for Figs. 7 to 9 are as follows.

Yield: 0.034 g (81%). ^{1 H-NMR (300 MHz,} CDCl 3): δ (ppm) 7.55 (d, J = 8.4 Hz, 1 H), 6.83 (dd, J = 8.8 Hz, J = 2.0 Hz, 1 H), 6.68 (d J = 2.0 Hz, 1H), 6.63 (s, 1H), 4.21 (t, J = 5.6 Hz, 2H), 4.84 (t, J = 5.2 Hz, 2H).

^{13 C-NMR (100 MHz,} CDCl 3 contained 2-drops of d 6 -DMSO): δ (ppm) 145.5, 138.9, 132.6, 122.6, 121.4, 120.2, 113.4, 109.9, 106.3, 93.4, 57.8, 42.8, 29.8 . ^{HRMS (FAB +): m /} z calcd for [C 30 H 30 N 4 O 2 F 4]: 554.2299, found: 554.2297.

Compound A1

^{1 H-NMR (400MHz, d} 6 -DMSO): δ (ppm) 10.89 (s, 1H), 7.32 (d, J = 8.4Hz, 1H), 6.50-6.48 (m, 2H), 5.66 (s, 1H ), 2.72 (s, 3 H).

Compound A2

^{1 H-NMR (400MHz, CDCl} 3): δ (ppm) 7.53 (d, J = 8.8Hz, 1H), 6.82 (dd, J = 8.8 Hz, J = 2.4 Hz, 1 H), 6.70 (d, J J = 5.6 Hz, 1H), 3.52-3.48 (m, 4H), 3.46-3.43 (m, 2H) (m, 4 H), 3.33 (s, 3 H), 3.02 (s, 6 H).

Compound A3

^{1 H-NMR (400MHz, CDCl} 3): δ (ppm) 7.52 (d, J = 8.8 Hz, 1H), 6.81 (dd, J = 8.8 Hz, J = 2.0 Hz, 1 H), 6.70 (s, 1H ), 6.60 (s, 1H), 3.65-3.60 (m, 31H), 3.54-3.52 (m, 9H), 3.48-3.42 ).

Compound A4

¹ H-NMR (400 MHz, CDCl ₃ contained 2-drops of d ₆ -DMSO):? (Ppm) 8.83 (s, 1H), 7.47 (d, J = 9.2 Hz, 1H) ), 6.72-6.70 (m, 1H), 3.74 (t, J = 6.0 Hz, 4H), 3.68-3.60 (m, 34H).

Compound B1

Compound B2

Compound B3

Compound B4

Compound B5

Compound B6

Compound B7

Experimental Example 1 Characterization of Novel Compounds or Their Pharmaceutically Acceptable Salts

1. Solubility measurement

To, (hereinafter 'DMSO' dimethyl sulfoxide) stock solution (1.0 x 10 ^-2 M) to prepare a novel compound, a compound of the quadrupole type 1, compound 2 (QAD1), and Compound 3 dimethylsulfoxide .

The stock solution was diluted to 1.0 x 10 ^-5 to 1.0 x 10 ^-7 and added to a cuvette containing 3.0 ml of PBS buffer (10 mM, pH 7.4) using a micro syringe. The concentration of DMSO in PBS buffer was maintained at 0.1%.

Plots of absorbance intensity were linear at low concentrations relative to the total amount of dyes injected into the cuvette and exhibited curvature as the concentration increased. The maximum point of the linear section means the solubility.

2. Octanol-water partition coefficient (logP _oct )

A solution of a small aliquot (10 μL) of the compound solution (20 mM) in DMSO was added to the vial containing n-octanol (5 mL) using a microsyringe. To the vial was added PBS buffer (5 ml) and the mixture was vigorously stirred and maintained in dark for 1 hour.

The concentration of the compound in each layer was measured, and the log P _oct value was calculated by the following equation (1) after measuring the molar extinction coefficient of each dye.

[Equation 1]

log P _oct = log [probe] _oct -log [probe] _PBS

In the above equation (1), [probe] _oct and [probe] _PBS represent concentrations of compound in n-octanol and PBS.

3. Spectroscopic measurement

The absorption spectrum was measured with a UV-Vis spectrophotometer (S-3100) and the fluorescence spectrum was measured with a fluorescence spectrophotometer (FluoroMate FS-2) in a 1 cm standard quartz cell (1 cm standard quartz cell). The fluorescence quantum yield was determined by literature method using coumarin 307 (0.95 in 陸 = methanol) as a reference.

4. Measurement of two-photon cross section

The two-photon cross-section (δ) was measured using femtosecond ('fs') fluorescence measurements. Compound 2 (QAD1) (1.0 x ^10-6 M) was dissolved in ethanol and the two-photon induced fluorescence intensity was measured using rhodamine 6G at 720-880 nm.

The intensities of the two-photon-sensitive fluorescent spectrum of the reference and the sample were determined at the same excitation wavelength. The cross-sectional area of the two-photon fluorescence was calculated using the following equation.

&Quot; (2) &quot;

In Equation 2 s and r represent sample and reference molecules, S is a charge coupled device; represents the signal intensity collected from (Charge Coupled Device hereinafter 'CCD') detector, Φ represents the fluorescence quantum yields, Φ is Represents the total fluorescence collection efficiency of the experimental instrument, c represents the molecular density in solution, and [delta] _r represents the two-photon fluorescence cross-sectional area of the reference molecule.

5. Animal (Animal)

Five familial Alzheimer's disease ("5XFAD") transgenic mice (The Jackson Laboratory, Bar Harbor, ME) were used to study in vivo and in vitro brain imaging.

5XFAD mice express three mutations of human amyloid precursor protein (APP) and two human presenilin-1 (PS-1) mutations through a neuron-specific promoter A large amount of beta amyloid ("Aβ") plaques was found in the brains of 10 months of age due to betrayal.

Therefore, neuronal cell death and memory loss occur due to a large amount of A [beta] plaques.

The mice used in the experiment were raised in a 12/12 hour light-dark cycle in a facility without a specific pathogen. All animal experiments were approved by Seoul National University Animal Experimental Ethics Committee.

6. Animal Surgery

In order to observe the two-photon image in vivo, skull cranial surgery was performed.

The mice were anesthetized by injecting a mixture of anesthetics into 5XFAD transgenic mouse muscle (Zoletil 50, 2 ml / kg; Virbac, Carros, France) - Rompun (Rompun, 25%; Bayer Korea, Seoul, South Korea) . The mice were then fixed on a heating plate and the body temperature of the mice (37 ° C) was maintained during surgery and imaging.

After removal of the scalp and periosteum of the rats, a skull area of between 0.5 mm and 3.5 mm on the basis of bregma and between 0.5 mm and -3.5 mm on the basis of sagittal suture (sagittal suture) And the exposed area was covered with a cover glass of about 3 mm.

Compound 2 (QAD1) was injected intravenously (i.v.) (10 mg / kg) prior to imaging.

7. Two-Photon Fluorescence Microscopy (TPFM) analysis

Two-photon fluorescence microscope images of the compounds labeled Compound 1, Compound 2 (QAD1), and Compound 3 synthesized according to an embodiment of the present invention had numerical aperture (NA) of 0.30, 1.30, and 1.30, , × 40 oil lens and × 100 oil objectives lenses through spectral confocal and multiphoton microscopes (Leica TCS SP8 MP).

The two-photon fluorescence microscope images were prepared by inserting compound 1, compound 2 (QAD1), and compound 3 into a model-locked titanium-sapphire laser light source device (Mai Tai HP; Spectra Physics, 80 MHz per second, 100 fs pulse width) And excited with an output of 2680 mW (corresponding to an average power of approximately 3.0 mW at the focal plane) and observed with a DMI 6000B microscope (Leica).

Real-time imaging using a Chamlide IC (Live Cell Instrument) was performed by maintaining appropriate temperature, humidity, and pH values for a long time for a stable image environment.

To obtain images in the 450 to 520 nm range, internal PMTs were used to acquire 8-bit signals of 512 × 512 and 1024 × 1024 pixels, respectively, at a scan rate of 400 Hz.

Two-photon laser scanning microscopy (LSM 7 MP, Carl Zeiss Inc., Oberkochen, Germany) was used to observe Aβ plaque staining using Compound 1, Compound 2 (QAD1), and Compound 3 synthesized according to an embodiment of the present invention .

In addition, Compound 1, Compound 2 (QAD1), and Compound 3 of the present invention were observed in vivo in two-photon brain using a titanium-sapphire femtosecond laser (Chameleon Ultra®, Coherent, Santa Clara, CA).

To observe brain images, laser power was set to 30 to 50 mW and 780 nm wavelength was used.

In order to obtain 4D images, z-stack imaging technique of deep tissue was used. Specifically, 1-μm intervals were adjusted and time-lapse z-stack images were used. Image processing was performed with Volocity software (PerkinElmer, Boston, Mass.).

8. Cortical Slice Preparation for ex vivo Two Photon Imaging

Transepithelial sections were prepared from transgenic 5XFAD mice.

Specifically, 5XFAD mice were sacrificed and the whole brain was rapidly extracted and NaCl (124 mM), KCl (3 mM), NaH ₂ PO ₄ (1.25 mM), MgCl ₂ (1 mM) and NaHCO ₃ , D-glucose (10 mM) , and CaCl bubbling (bubbled) a cold artificial cerebrospinal fluid ₂ comprises a (2 mM) and, 95% O _2, and _{5% CO 2 (artificial cerebrospinal fluid} ; hereinafter 'aCSF') Lt; / RTI &gt; for 30 seconds.

The tissue sections were then cut into 400 μm thickness using a vibrating-blade microtome in aCSF.

With 95% O _2, and 5% CO ₂ in han aCSF within the bubbling (bubbled) compound 1, compound 2 (QAD1), and Compound 3 (a total of 20 μM) to cut to 2 μL at 37 ℃ After preparing the fragments Lt; / RTI &gt; for 1 hour and 30 minutes.

The sections were then washed three times with aCSF and transferred to glass-bottomed dishes (NEST) and observed with confocal microscopy.

9. Experimental results

10 shows the one-photon absorption spectra (a, c, e) and the PBS buffer solution (10 mM) for Compound 1 (a, b), Compound 2 (QAD1) (c, d) , pH 7.4) with respect to Compound 1, Compound 2 (QAD1), and Compound 3. FIG.

10, the solubilities of Compound 2 (QAD1) and Compound 3 in phosphate buffered saline (PBS) (pH 7.4) determined by the UV-vis titration method were 4 μM and 3 μM On the other hand, the solubility of Compound 1 was 1.0 μM. It can be seen that Compound 2 (QAD1) has high solubility in PBS buffer solution.

Next, sensitivity to solvent polarity was analyzed.

Figure 11 shows the effect of Compound 1 (a (1)) in 1,4-dioxane, ethanol (EtOH), EtOH: PBS (v / v, 1: 1) and PBS buffer (a, c, e) and emission spectra (b, d, f) of Compound 2 (QAD1) (c, d) and Compound 3 Table 1 and Table 2 show that Compound 1, Compound 2 (QAD1), and Compound 2 (Compound 1) are dissolved in various solvents (1,4-dioxane, ethanol, ethanol: PBS buffer solution (1: 1, v / ) And Compound 3, and the photophysical data on the compounds A1 to A7, which are the novel compounds.

Referring to FIG. 11 and Table 1, in the case of the compound 1, the emission maximum (emission maximum) (hereinafter referred to as &quot;? _Fl '') increases as the solvent polarity is increased from 1,4-dioxane to ethanol And gradually shifted from the 489 nm to the 515 nm to the red region. On the other hand, since the solubility of the compound 1 in the PBS buffer solution was very small, no luminescence was observed in the PBS buffer solution.

[Table 1]

[Table 2]

a: Numbers in parentheses are average parameters of solvent polarity.

b: One-photon absorption spectrum maximum wavelength (λ _max ) (unit: nm).

c: Photon emission spectrum maximum wavelength (λ _max ) (unit: nm).

d: Fluorescence quantum yield (error range: ± 10%).

e: Two-photon excitation spectrum maximum wavelength (? _max ) (unit: nm).

f: Peak cross-sectional area per photon at 10 ^-50 cm ⁴ s / photon (unit: GM).

g The peak two-photon action cross-section in 10 ^-50 cm ⁴ s / photon.

h: PBS buffer solution (10 mM, pH 7.4).

E ^N _T values were based on water.

i: Not measured.

One-photon and two-photon excitation fluorescence signals were too small to be assigned values.

12 (a) and 12 (b) show the normal absorption spectrum (a) and the emission spectrum of compound 1 (QAD1) at 1 μM in 1,4-dioxane, EtOH and PBS buffer solution (10 mM, pH 7.4) Fig. 5 shows the spectrum (b).

Referring to Figs. 12 (a) and 12 (b), Compound 2 (QAD1) exhibited similar behavior in the PBS buffer solution of Compound 1 except for the lambda _fl portion. Specifically, the ethanol of Compound 1 migrated to the red region (546 nm) more than lambda _fl . This confirmed that Compound 2 (QAD1) is a more polar sensitive compound than Compound 1.

13 (a) shows the change in fluorescence intensity of a complex of Compound 2 (QAD1) (1 μM) having Aβ _{1 -42} oligomer (0 to 10 μM) in PBS buffer solution (10 mM, pH 7.4) Fig.

Referring to Figure 13 (a), Aβ _{1 -42} oligomers (Aβ _{1 -42} oligomer; hereinafter 'AβO') λ _fl of compound 2 (QAD1) in the presence of the λ (a blue region in a PBS buffer solution _fl = 510 nm ).

Referring to Figure 12 (b), a similar result (508 nm) was obtained from the Aβ plaque labeled with compound 2 (QAD1) in transgenic mouse brain slices. This indicates that the ethanol solvent appropriately represents the polarity to the microenvironment of the A [beta] plaque.

The photophysical properties of Compound 1, Compound 2 (QAD1), and Compound 3 in ethanol were analyzed and summarized in Table 3.

[Table 3]

12 (c) is a graph showing the luminescence spectra of Compound 1, Compound 2 (QAD1) and Compound 3 obtained in ethanol.

Referring to FIG. 12 (c), the brightness (hereinafter referred to as '?') Of Compound 2 (QAD1) was significantly larger than that of Compound 1.

Referring to FIG. 11 and Table 1, although Compound 3 exhibited a solvatochromic shift, Compound 3 showed a rapidly decreased brightness in the absorption spectrum shifted to blue .

To identify the major differences between compound 2 (QAD1) and compound 3, the structure of compound 2 (QAD1) and compound 3 was determined by density functional theory (DFT) at the B3LYP / 6-31G * Respectively.

Table 4 is a table showing the calculated values of Compound 2 (QAD1) and Compound 3 obtained from ethanol.

Referring to Table 4, first, the energy differences between the compound 2 (QAD1) and the cis isomer and the trans isomer of the compound 3 were almost the same.

[Table 4]

15 is a diagram showing the cis-isomer structure of Compound 2 (QAD1) (a) and Compound 3 (b) which are optimized geometries by DFT in ethanol.

Referring to FIG. 15, the compound 3 has a twisted structure between the central? -Core and the bridge heterocycle, while the optimized geometry in the ground state of the compound 2 (QAD1) is a planar structure.

16 is a diagram showing optimized geometrical structures and molecular orbital distribution by density functional theory (DFT) of compound 2 (QAD1) (a) and compound 3 (b) in ethanol .

16, the HOMO-LUMO energy and oscillator strength of Compound 3 were 2.67 eV and 0.98, respectively, while Compound 2 (QAD1) showed HOMO-LUMO energy of 2.56 eV and oscillator strength of 1.91 Bar, Compound 2 (QAD1) had lower HOMO-LUMO energy than compound 3 and exhibited twice the vibrator intensity value.

Therefore, the stronger brightness of compound 2 (QAD1) is attributable to the planar structure, which may enable efficient ICT. However, the weaker brightness of compound 3 results from steric repulsion between bis-ethanol and? -Core due to the twisted structure.

In addition, the selective binding profiles of Compound 1, Compound 2 (QAD1), and Compound 3 against AβO and bovine serum albumin (BSA) were analyzed.

13 (a) is a graph showing the change in fluorescence intensity for a complex of Compound 2 (QAD1) (1 μM) having Aβ _{1 -42} oligomer (0 to 10 μM) in PBS buffer solution (10 mM, pH 7.4) to be.

The dissociation constant (K _d ) for compound 2 (QAD1) / AβO was determined by fluorescence titration.

Specifically, K _d for Compound 2 (QAD1) / AβO The value was 21.5 nM. This means that the binding affinity of Compound 2 (QAD1) to AβO is higher than the K _d value for Compound 1 and Compound 3.

Figure 17 (b) is a view showing the change of the luminescence intensity to the PBS buffer solution, compound 2 (QAD1) (b, 417 nm) in the presence of _{1 -42} Aβ oligomers, and to BSA (10 mM, pH 7.4).

Referring to Figure 17 (b), Compound 2 (QAD1) showed negligible interaction with BSA when performed under similar experimental conditions.

17 (a) is a graph showing the change in luminescence intensity for Compound 1 (a, excitation wavelength: 417 nm) in the presence of Aβ _{1 -42} oligomer and BSA in PBS buffer solution (10 mM, pH 7.4).

Referring to FIG. 17 (a), the increase in the luminescence intensity of Compound 1 in the presence of BSA was higher than that of AβO, suggesting that Compound 1 has high binding affinity for BSA.

17 is a view showing a change of the emission intensity of the compound 3 (c, 326 nm) in the presence of _{1 -42} Aβ oligomers and BSA in PBS buffer (10 mM, pH 7.4).

Referring to FIG. 17, similar results were observed in Compound 3, except for a slight luminosity.

Thus, due to improved solubility and planar morphology, Compound 2 (QAD1) is found to bind specifically to AβO than BSA.

[Table 5]

a: a letter about the chemical structure in Scheme 1;

b: All measurements were carried out in _{1 -42} Aβ oligomer (10 μM) under the presence PBS buffer solution (10 mM, pH 7.4) member.

c is the maximum wavelength (λ _max ) (unit: nm) of a single photon absorption / absorption spectrum.

d is the maximum wavelength (λ _max ) of a single photon emission spectrum in nm.

e: Fluorescence quantum yield (error range: ± 10%).

f: _{1 -42} Aβ oligomer (0 to 10 μM) the dissociation constants measured by the one-photon process in the presence (Dissociation constants, error range: ± 10%)

g: Fluorescence enhancement factor (FF _min ) / F _min )

Figure 18 shows the effect of Compound 2 ((R)) at 407 nm excitation wavelength in a universal buffer (0.1 M citric acid, 0.1 M KH ₂ PO ₄ , 0.1 M Na ₂ B ₄ O ₇ , 0.1 M Tris, Lt; RTI ID = 0.0 &gt; QAD1) &lt; / RTI &gt;

Referring to FIG. 18, the luminescence intensity of compound 2 (QAD1) did not respond sensitively to pH changes in the biologically relevant pH range.

From the above results, it can be seen that compound 2 (QAD1) undergoes minimal interference by BSA and pH, and can bind to AβO sensitively.

The 陸_TPA value of Compound 2 (QAD1) determined by TPEF was analyzed.

FIG. 12 (d) is a diagram showing the two-photon action (TPA) spectrum (d) for Compound 1, Compound 2 (QAD1) and Compound 3 obtained in ethanol.

Table 3 shows the results of analyzing the photophysical properties of Compound 1, Compound 2 (QAD1), and Compound 3 obtained from ethanol.

Referring to FIG. 12 (d) and Table 3, the compound 2 (QAD1) Φδ _TPA value showed 420 GM at 750 nm, which was higher than that of the non-polar compound having a Φδ _TPA value of about 100 GM.

Referring to Table 3, the reason that Compound 1 exhibited a smaller value than Compound 2 (QAD1) was attributed to the smaller? Value of Compound 1. In addition, the minimum? 隆_max value of compound 3 inhibits efficient intramolecular charge transfer (ICT) due to the twisted structure.

Specifically, with respect to the value of the two-photon operating cross-section (PHA _TPA ), the size of the two-photon absorption cross-section (? _TPA ) of the molecule is proportional to the range of the ICT characteristics, including the donating and accepting ability, , Form suggestion, and symmetry.

Among various types of two-photon absorption dyes, electron donor (D) -substituted acceptor (A) and center symmetric quadrupole (DAD) have proven to be promising motifs for the δ _TPA value.

In addition, multi-fluorinated centers, including bis-styrylbenzene derivatives, exhibit selective binding affinities for A [beta] plaques.

Therefore, the DAD type for Aβ plaques including tetrafluorobenzene as an amino group and an electron acceptor as a strong electron donor is preferable, and an indolyl ring which can expect higher light stability than an open chain system It is preferable to use compound 2 (QAD1), since it is desirable to encapsulate a C = C bond within the compound.

Compound 2 (QAD1) showed Φδ _TPA values greater than 420 GM to resist photobleaching, and also showed high binding affinity for Aβ plaques and ability to penetrate BBB. Thereby enabling real-time and providing a high spatial resolution 3D image for the in vivo Aβ plaque.

Table 6 shows the log P _oct values for Compound 1, Compound 2 (QAD1), and Compound 3.

[Table 6]

Referring to Table 6, the lipophilicity value (logP _oct ) of Compound 2 (QAD1) obtained by cleavage between n-octanol and PBS buffer solution was 3.42, indicating that the blood brain barrier (BBB) (Log P = 2.0 to 3.5) for penetration of the water.

[Table 7]

Fig. 19 shows the results of the administration of 0 (a), 30 (b), 60 (c), and 150 minutes (d) after injecting intravenously (hereinafter 'iv') Compound 2 ) In vivo TPM images for the frontal lobe of transformed 5XFAD mice; To visualize the Aβ plaque distribution, an accumulated 230 TPM image (e) along the z-direction at a depth of about 300 μm from the surface of the envelope; 3D reconstructed TP images (f, g) from the surface of the envelope (at a depth of about 300 [mu] m) from the frontal lobe of transformed 5XFAD mice after intravenous Compound 2 (QAD1) (10 mg / kg) and dextran 40 kDa- about 270 TPM images accumulated along the z-direction, and the scale bars are 50 m (a) and 30 m (e)).

Referring to FIG. 19, these results suggest that compound 2 (QAD1) is a sensitive compound that brightens TPM in vivo in Aβ plaques.

The ability of compound 2 (QAD1) on A [beta] plaques in brain tissue was monitored.

Transepithelial sections were obtained from transgenic 5XFAD mice, which were AD model mice in which brain Aβ plaques were formed.

Figure 20 shows the TPM image (a) of the envelope sections of transgenic 5XFAD mouse brains stained with 20 μM of Compound 1 (a), Compound 2 (QAD1) (c), and Compound 3 , c, e).

20, the fragments labeled with Compound 1 and Compound 3 showed a faint TPEF with an important background signal, while the fragments labeled with Compound 2 (QAD1) with the highest S / N ratio showed the TP of the TPM image Bright spots) were observed.

This result is due to the high? 隆_TPA value and high sensitivity to A? Plaque of compound 2 (QAD1).

To confirm that compound 2 (QAD1) can be correctly located in the A [beta] plaque, a co-localization experiment was performed between Congo red, a well known fluorescent marker for A [beta] plaques, and the compound.

Figures 21 (a) to 21 (c) show the results of transgenic 5XFAD mice co-labeled with Compound 2 (QAD1) (a) and Congo red (b) (A, b, c) and 20-fold magnified merged images (c) of the epidermal tissue section of the brain.

Referring to Figures 21 (a) to 21 (c), the bright TPEF region of compound 2 (QAD1) was merged with the signal of Congo red with Pearson's colocalization coefficient (0.85). This confirms that the bright spot of the TPM image reflects the Aβ plaque.

The TPEF spectrum of the Aβ plaques in the tissues was symmetrical, and with reference to FIG. 12 (b), it matched the emission spectrum obtained from ethanol.

16 (d) and 16 (e), the TPEF intensity remained almost the same at 750 nm excitation in the femtosecond pulse after 60 minutes.

Showed high light stability due to the structural motif of Compound 2 (QAD1) upon encapsulation of the C = C bond in the ring.

Thus, the usefulness of in vivo Compound 2 (QAD1) was analyzed.

Transgenic 5XFAD mice were anesthetized and a cranial window was surgically installed for direct TPM imaging.

Compound 2 (QAD1) (10 mg / kg) was injected intravenously and the TPM image was directly collected at 780 nm excitation.

Referring to FIG. 19 (a), the initial image shows a bright TPEF through the blood vessels in the outer skin region. Referring to the white arrows in Figs. 19 (b) to 19 (d), it rapidly moved to a plaque near the blood vessel with time.

Through the infiltration of the BBB, the plaque could be visualized clearly by the QAD dyeing process.

Also, referring to Fig. 19 (e), when TPM images were obtained in the deeper regions, the labeled individual plies were clearly visualized with a high S / N ratio.

Because 3D visualization of Aβ plaques along the blood vessels is important for clinical application, dextran 40 kDa-Texas red, a well-known blood marker, was injected.

Subsequently, a dual-color TPM image was obtained immediately along the z-direction deeper than 300 μm from the shell surface and about 270 3D TPM images accumulated along the z-direction were obtained.

Referring to Figures 19 (f) and 19 (g), individual Aβ plaques of varying sizes in specific regions were clearly visualized according to the blood vessels.

In addition, cerebral amyloid angiopathy (CAA) was deposited on Aβ plaques formed around the central nervous wall of the central nervous system and observed directly.

These results demonstrate the usefulness of Compound 2 (QAD1) for 3D visualization of in vivo Aβ plaques in real time, and for the clinical diagnosis of Aβ plaques for diagnosis and precise treatment.

The novel compounds synthesized for in vivo imaging of A [beta] plaques or their pharmaceutically acceptable salts exhibit a large two-photon operating cross-section ([phi] _max = 420 GM), substantial solubility, high sensitivity, and selectivity for such A [ , As well as the ability to load easily into Aβ plaques in living rat brains.

These quadrupole-like compound features not only allow direct 3D visualization of individual plaques, but also demonstrate that quadrupole-form compounds can be useful in biomedical applications, including easy diagnosis and treatment of AD.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (10)

1. A compound represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

In Formula 1, R ₁ to R ₄ are the same or different from each other,
A halogen, a C ₁ to C ₄ alkoxy, a C ₁ to C ₄ alkyl, a C ₁ to C ₄ alcohol, or a group represented by the following formula (1-1)
[Formula 1-1]

m is an integer of 1 to 5,
R ₅ to R ₆ are the same as or different from each other,
Hydrogen, a hydroxyl group, a halogen, C ₁ to alkoxy, C ₁ to alkyl, C ₁ to alcohol, the formula 1-2 or formula 1-3, C ₄ of the C ₄ C _4,
[Formula 1-2]

[Formula 1-3]

n is an integer of 8 to 15,
R ₇ to R ₁₀ are the same or different from each other,
2 or more is fluorine (F) and the remainder is hydrogen. The method according to claim 1,
The compound, or a pharmaceutically acceptable salt thereof,
A compound or a pharmaceutically acceptable salt thereof, which is represented by the following formula (2):
(2)

In Formula 2, R ₁ to R ₄ are the same or different from each other,
Hydrogen, C ₁ to C ₄ alkyl, C ₁ to C ₄ alcohols, or the following formula (2-1)
[Formula 2-1]

R ₅ to R ₆ are the same as or different from each other,
Hydrogen, a C ₁ to C ₄ alcohol, the following Formula 2-2 or Formula 2-3.
[Formula 2-2]

[Formula 2-3]

The method according to claim 1,
The compound, or a pharmaceutically acceptable salt thereof,
A compound or a pharmaceutically acceptable salt thereof, which is represented by any one of the following formulas (3-1) to (3-14):
[Formula 3-1]

[Formula 3-2]

[Formula 3-3]

[Chemical Formula 3-4]

[Formula 3-5]

[Chemical Formula 3-6]

[Chemical Formula 3-7]

[Chemical Formula 3-8]

[Chemical Formula 3-9]

[Chemical Formula 3-10]

[Formula 3-11]

(3-12)

[Chemical Formula 3-13]

[Chemical Formula 3-14]

A two-photon fluorescent probe comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof. The method of claim 4,
In the two-photon fluorescence probe,
Beta-amyloid plaques, which specifically bind to beta-amyloid plaques. A composition for detecting beta amyloid, comprising a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for the treatment or prevention of Alzheimer's disease comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a physiologically active substance. The method of claim 7,
The physiologically active substance may be,
A pharmaceutical composition for the treatment or prevention of Alzheimer's disease, wherein the composition is any one selected from the group consisting of drugs, peptides, proteins, and genes. 4. A method for the treatment of cancer, comprising: injecting a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof into a sample separated from a living body;
Combining said compound or a pharmaceutically acceptable salt thereof with a beta amyloid plaque in a sample isolated from a living body;
Irradiating an excitation source to a sample separated from the living body; And
2. A method of imaging a beta amyloid plaque, comprising observing fluorescence generated from a compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof by a two-photon microscope. The method of claim 9,
The excitation source,
Characterized in that it has a wavelength in the range of 700 to 800 nm.

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