JPWO2007063950A1 - Long wavelength fluorescent substances and their use for conformation disease diagnosis and treatment - Google Patents

Abstract

A compound represented by formula: (I) or a salt or solvate thereof, a composition and kit for diagnosing conformation disease containing the same, a pharmaceutical composition for treating and / or preventing conformation disease, and the above compound or a compound thereof A method for diagnosing conformation disease using a salt or solvate, and a method for treating and / or preventing conformation disease.

Description

  The present invention uses a long-wavelength fluorescent substance for diagnosing and treating conformation disease, and a composition and kit for diagnosing conformation disease including the same, a pharmaceutical composition for treating and / or preventing conformation disease, and the aforementioned substance The present invention relates to a method for diagnosing conformation disease and a method for treating and / or preventing conformation disease.

  Diseases in which proteins having a β-sheet structure peculiar to conformation diseases accumulate include various diseases characterized by deposition of insoluble fibrillar proteins in various organs and tissues in the body. These diseases include Alzheimer's disease, prion disease, Lewy body disease, Parkinson's disease, Huntington's disease, bulbar spinal muscular atrophy, dentate nuclei / palliary bulbous atrophy, spinal cord / cerebellar degeneration, Machado- Examples include Joseph Disease, Amyophic Lateral Sclerosis (ALS), Down's Syndrome, Pick's Disease, FTDP-17 (Frontotemporal Dementia and Parkinsonism linked to Chromosome 17), LNTD (Limbic Neurofibrillary Tangle Demetia), Sudanophilic Leukodystrophy, Amyloidosis, and the like.

  Among them, Alzheimer's disease (AD) is currently regarded as one of the most difficult diseases to treat, and an accurate early diagnosis is desired. Alzheimer's disease is a disease characterized by progressive intellectual dysfunction that occurs mainly from presenescence to old age. Pathologically, it is characterized by global atrophy of the cerebrum, marked degeneration and loss of nerve cells, neurofibrillary tangles and appearance of senile plaques. It is known that the greatest risk factor for Alzheimer's disease is aging. Therefore, the increase in the number of patients accompanying the increase in the number of elderly people is particularly remarkable in Japan, the United States, and European countries, which are aging societies. ing.

  In Japan, the number of Alzheimer's disease patients is estimated to be about 1 million, and it is expected that the number of patients will increase as the population ages. The cost of Alzheimer's disease patients, including care costs, is considered to be between 1 million and 3 million yen per patient per year, so we have already paid socio-economic costs between 1 trillion and 3 trillion yen in Japan. It will be. It is now common knowledge in the world that treatment in Alzheimer's disease before or as soon as symptoms become apparent has a great medical economic effect.

  In recent years, even in Alzheimer's disease, immunotherapy such as vaccines and fundamental treatment methods represented by β or γ secretase inhibitors are being developed. Non-patent documents 1 to 4 are included in immunotherapy-related papers such as vaccines, and non-patent documents 5 to 7 are included in papers related to β and γ-secretase inhibitors. This suggests that by diagnosing as early as possible and adding these fundamental therapies, an era in which people can live their lives without suffering from Alzheimer's disease as long as it is before the onset at the time of diagnosis is coming.

  Therefore, the key is to detect Alzheimer's disease before onset. There are various current methods for diagnosing Alzheimer's disease. In Japan, neuropsychological evaluation methods such as Hasegawa, ADAS, MMSE, etc. that quantitatively evaluate the decline in cognitive function of individuals suspected of Alzheimer's disease are common. In rare cases, diagnostic imaging methods (MRI, CT, etc.) are used as an auxiliary.

  In Alzheimer's disease, it is known that the pathological image in the brain has already progressed to an irreversible state when the family or clinician surrounding the patient notices the first clinical symptoms. Many studies have shown that neurodegeneration with Alzheimer's disease has already begun long before the first clinical symptoms appear (approximately 40 years in the long case). Considering the progression characteristics of the disease states and the rapid increase in the number of patients as described above, the necessity and significance of an accurate early diagnosis of Alzheimer's disease are extremely great.

  The histopathology of Alzheimer's disease is represented by two main features. Senile plaques and neurofibrillary tangles. The former main component is amyloid β (Aβ) protein having a β-sheet structure, and the latter is hyperphosphorylated tau protein. A definitive diagnosis of Alzheimer's disease relies on the appearance of these pathological features in the patient's brain.

  Amyloid β protein is characteristic of diseases in which amyloid accumulates, including Alzheimer's disease, and is closely related. Therefore, detection of amyloid β protein having a β sheet structure in the body, particularly in the brain, as a marker is one of important diagnostic methods for diseases in which amyloid accumulates, particularly Alzheimer's disease.

  In recent years, Alzheimer's disease diagnostic methods using a positron-labeled probe (PET probe) that specifically and selectively recognizes amyloid β protein are being realized. Exploratory clinical trials have already been carried out with three probes of FDDNP (Patent Document 1), (6-OH BTA-1 (= PIB) (Patent Document 2), and SB-13 (Patent Document 3) [FDDNP For non-patent document 8; for non-patent document 9 for 6-OH BTA-1 (= PIB); see non-patent document 10 for SB-13].

  However, when MRI or PET is used for diagnosis of conformation diseases such as Alzheimer's disease, the equipment becomes large, the energy consumption is extremely large, and the price of the equipment becomes very expensive. In addition, the equipment is not portable and a dedicated building is required. Furthermore, MRI and PET have a long diagnosis time of about 2 hours per patient and are not suitable for mass screening because the number of patient treatments per day is small. For this reason, diagnosis using these devices can be performed only in a very limited facility.

  In recent years, a compound that binds to amyloid β protein and emits long-wavelength fluorescence is systemically administered, and the binding of amyloid β protein and the compound in the brain is quantified in vivo using a long-wavelength fluorescence measurement device and its space. There is an attempt to diagnose amyloid-accumulating diseases from a spatial distribution. Such compounds include AOI-987 [see Non-Patent Document 11], NIAD-4 [see Non-Patent Document 12], and the like.

In general, long-wavelength light is known to have high biological permeability. However, since absorption of hemoglobin is present below 600 nm and absorption of water occurs above 1000 nm, it is limited to compounds having a wavelength of 600 nm to 1000 nm. Is done. This region of 600 nm to 1000 nm is called a “biological spectroscopic window” (see FIG. 1). Therefore, in order to use a compound that binds to amyloid β protein and emits long-wavelength fluorescence as a diagnostic compound, both its excitation wavelength and emission wavelength must be within the range of the “spectroscopic window of the living body”. .
PCT / US99 / 18966 (WO2000 / 10614) PCT / US2001 / 026427 (WO2002 / 016333) PCT / US2002 / 27201 (WO2003 / 018070) Schenk et al., Nature 400, 173-177, 1999 Bard et al., Nature Medcine, Vol. 6, pages 916-919, 2000 Hock et al., Nature Medcine, Vol. 8, pp. 1270-1275, 2002 Fox et al., Neurology, (Neurology) 64, 1563-1572, 2005 John et al., Journal of Medicinal Chemistry, 46, 4625-4630, 2003 Tian et al., Journal of Biological Chemistry, 277, 31499-31505, 2002 Kimura et al., Bioorganic and Medicinal Chemstry Letters, 15: 211-215, 2005 Shoghi-Jadid et al., American Journal of Geriatric Psychiatry, 10, 24-35, 2005 Klunk et al., Anals of Neurology, 55, 306-319, 2004 Verhoeff et al., American Journal of Geriatric Psychiatry, 12, 584-589, 2004 Hintersteiner et al., Nature Biotechnology, 23, 577-583, 2005 Nesterov et al., Angew Chem int. Ed, 44-2-5, 2005

  It was an object of the present invention that the equipment used is not large, consumes little energy, is inexpensive and portable, and allows easy diagnosis of conformation disease. It was also an object of the present invention to enable a diagnosis of conformation disease suitable for mass screening with a short diagnosis time per patient and hence a large number of patient treatments. Furthermore, the development of a medicament for effectively treating or preventing conformation disease was also an object of the present invention.

  The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and specifically bind to amyloid β protein, and the excitation and emission wavelength fall within the range of “spectroscopic window of living body”. I came to find. Such a fluorescent compound is a compound having a deep color effect, that is, the fluorescence intensity is remarkably enhanced by binding to amyloid β protein. By using such compounds, non-specific binding and non-binding can be ignored. Therefore, since an image of only the binding of amyloid β protein and the compound can be obtained immediately after administration of such a compound, it becomes possible to diagnose an amyloid accumulating disease in a very short time. Long-wavelength fluorescence obtained by administering such a compound to a living body by intravenous administration or the like and irradiating a living brain with an appropriate excitation wavelength from the outside is measured using a long-wavelength fluorescence measuring device, and in vivo, particularly in the brain In addition to quantifying the accumulation of amyloid β protein, conformational diseases such as Alzheimer's disease, Down's syndrome, prion disease, and the like, which are caused by the accumulation of protein having a β sheet structure, can be diagnosed.

  According to the present invention, a pharmaceutical composition for diagnosis and treatment / prevention of conformation disease is provided. Conformation disease diagnosis using the composition is not large-scale equipment, consumes little energy, is inexpensive, and is portable. Furthermore, such a diagnosis has a short diagnosis time per patient, and therefore has a large number of patient treatments and is suitable for mass screening. In addition, conformation disease can be effectively treated or prevented using the composition.

FIG. 1 is a diagram showing an outline of light absorption by a living body. Yukio Yamada: Light and Biology -Invitation to Ecological Spectroscopy-, "Medical Diagnosis by Light"; Editing Mamoru Tamura: pp19-36, quoted from Kyoritsu Shuppansha, 2001 (Tokyo) FIG. 2-a is a contour map showing the three-dimensional fluorescence spectrum of the buffer solution. For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 2-b is a contour map showing the three-dimensional fluorescence spectrum of 1 μM THK-565 alone. The vertical axis represents the excitation wavelength (Ex. Wavelength), and the horizontal axis represents the emission wavelength (Em. Wavelength) in nanometers (nm). For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 2-c is a contour map showing a three-dimensional fluorescence spectrum of 1 μM THK-565 bound to 5 μM amyloid β having a β sheet structure. The vertical axis represents the excitation wavelength (Ex. Wavelength), and the horizontal axis represents the emission wavelength (Em. Wavelength) in nanometers (nm). For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 3-a is a diagram showing a fluorescence spectrum of THK-565 bound to amyloid β protein when the excitation wavelength is 640 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 3B is a diagram showing a fluorescence spectrum of THK-565 bound to amyloid β protein when the excitation wavelength is 650 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 3-c shows the fluorescence spectrum of THK-565 bound to amyloid β protein when the excitation wavelength is 660 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 3D is a diagram showing a fluorescence spectrum of THK-565 bound to amyloid β protein when the excitation wavelength is 670 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 3-e is a diagram showing a fluorescence spectrum of THK-565 bound to amyloid β protein when the excitation wavelength is 680 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 3-f is a diagram showing a fluorescence spectrum of THK-565 bound to amyloid β protein when the excitation wavelength is 690 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 4-a is a contour diagram showing the three-dimensional fluorescence spectrum of the buffer solution. For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 4-b is a contour map showing the three-dimensional fluorescence spectrum of 1 μM THK-585 alone. The vertical axis represents the excitation wavelength (Ex. Wavelength), and the horizontal axis represents the emission wavelength (Em. Wavelength) in nanometers (nm). For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 4-c is a contour map showing a three-dimensional fluorescence spectrum of 1 μM THK-585 bound to 5 μM amyloid β having a β sheet structure. The vertical axis represents the excitation wavelength (Ex. Wavelength), and the horizontal axis represents the emission wavelength (Em. Wavelength) in nanometers (nm). For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 5-a is a diagram showing a fluorescence spectrum of THK-585 bound to amyloid β protein when the excitation wavelength is 640 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 5-b shows the fluorescence spectrum of THK-585 bound to amyloid β protein when the excitation wavelength is 650 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 5-c shows the fluorescence spectrum of THK-585 bound to amyloid β protein when the excitation wavelength is 660 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 5-d is a diagram showing a fluorescence spectrum of THK-585 bound to amyloid β protein when the excitation wavelength is 670 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 5-e shows the fluorescence spectrum of THK-585 bound to amyloid β protein when the excitation wavelength was 680 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 5-f is a diagram showing a fluorescence spectrum of THK-585 bound to amyloid β protein when the excitation wavelength is 690 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 6A is a contour map showing the three-dimensional fluorescence spectrum of the buffer solution. For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 6-b is a contour map showing the three-dimensional fluorescence spectrum of 1 μM AOI-987 alone. The vertical axis represents the excitation wavelength (Ex. Wavelength), and the horizontal axis represents the emission wavelength (Em. Wavelength) in nanometers (nm). For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 6-c is a contour map showing a three-dimensional fluorescence spectrum of 1 μM AOI-987 bound to 5 μM amyloid β having a β sheet structure. The vertical axis represents the excitation wavelength (Ex. Wavelength), and the horizontal axis represents the emission wavelength (Em. Wavelength) in nanometers (nm). For the alternate long and short dash line ellipse and the broken line ellipse, see the description of the first embodiment. FIG. 7A is a diagram showing a fluorescence spectrum of AOI-987 bound to amyloid β protein when the excitation wavelength is 640 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of AOI-987 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 7-b is a diagram showing a fluorescence spectrum of AOI-987 bound to amyloid β protein when the excitation wavelength is 650 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of AOI-987 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 7-c is a diagram showing a fluorescence spectrum of AOI-987 bound to amyloid β protein when the excitation wavelength is 660 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of AOI-987 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 7-d is a diagram showing a fluorescence spectrum of AOI-987 bound to amyloid β protein when the excitation wavelength is 670 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of AOI-987 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 7-e shows the fluorescence spectrum of AOI-987 bound to amyloid β protein when the excitation wavelength was 680 nm. The vertical axis represents fluorescence intensity (Int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves, the upper curve shows the fluorescence spectrum of AOI-987 bound to amyloid β protein, and the lower curve shows the spectrum of the buffer solution. FIG. 8 shows a fluorescent microscope image (ex vivo) when THK-265 was intravenously administered to a Tg mouse (Tg2576) in which amyloid β protein accumulates. FIG. 9 shows a lumino image analyzer image (in vivo) when THK-265 was intravenously administered to a rat model in which amyloid β protein was injected into the brain. The solid circle represents the amyloid β protein administration site, and the broken circle represents the buffer administration site. FIG. 10 shows THK-265 stained images (left panel) and anti-amyloid β (Aβ) antibody stained images (adjacent sections of the left panel) in brain sections of Alzheimer's disease patients. Open arrows indicate amyloid β protein. FIG. 11 is a view showing a THK-265 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein, and open arrowheads indicate neurofibrillary tangles. FIG. 12 is a view showing the strongly enlarged image of FIG. FIG. It is a figure which shows the THK-449 dyeing | staining image in an Alzheimer's disease patient brain section. Open arrows indicate amyloid β protein. FIG. 14 is a view showing a THK-539 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 15 shows a THK-556 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 16 is a view showing a THK-558 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 17 is a view showing a THK-559 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 18 is a view showing a THK-561 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 19 is a view showing a THK-562 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 20 is a view showing a THK-563 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 21 shows THK-565 stained images (left panel) and anti-amyloid β (Aβ) antibody stained images (adjacent sections of the left panel) in brain sections of Alzheimer's disease patients. Open arrows indicate amyloid β protein. FIG. 22 is a view showing a THK-585 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 23 is a diagram showing a THK-533-stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 24 is a view showing a THK-535 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 25 is a diagram showing a THK-557 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 26 is a diagram showing a THK-577-stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 27 is a view showing a THK-653-stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 28 is a view showing a THK-700 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 29 is a view showing a THK-705 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 30 is a view showing a THK-706-stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 31 is a view showing a THK-715-stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 32 is a view showing a THK-716 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 33 is a view showing a THK-717-stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein. FIG. 34 is a view showing a THK-753 stained image in a brain section of an Alzheimer's disease patient. Open arrows indicate amyloid β protein.

(I)
［式中、Ｒ_１は水素、Ｃ_１−６アルキル、ハロゲン、またはＣ_１−６アルキル−ハロゲンであり、
Ｒ_２およびＲ_３はそれぞれ独立して水素またはＣ_１−６アルキルであり、
Ｒ_４は水素またはＣ_１−６アルキルであり、
ＥはＣＨ_２または不存在であり、
Ａは５員環または６員環で、下記の構造：

または

または

を有し；
ＸおよびＹは独立してＮまたはＣＨであり；
ＺはＯ、Ｓ、ＣＨ_２またはＮ−Ｃ_ｐＨ_２ｐ＋１であり；
ＧはＮまたはＣＨであり；
ＪはＯ、Ｓ、ＣＨ_２またはＮ−Ｃ_ｑＨ_２ｑ＋１であり；
Ｒ_５は水素、Ｃ_１−６アルキル、ピロール、ピラゾール、イミダゾール、トリアゾール、またはＮＲ^ＩＲ^ＩＩであり、
Ｒ_６は水素、Ｃ_１−６アルキル、またはハロゲンであり、
Ｒ^Ｉ、Ｒ^ＩＩはそれぞれ独立して水素またはＣ_１−６アルキルであるか、あるいは一緒になってピロリジン環、モルホリン環、ピペリジン環または窒素原子がＣ_１−３アルキルで置換されていてもよいピペラジン環を形成し、
ｎ_１は１ないし４の整数であり；
ｎ_１'は１ないし３の整数であり；
ｎは１ないし４の整数であり；
ｐは１ないし４の整数であり；
ｑは１ないし４の整数であり；
２つの環部分をつなぐ二重結合まわりの配置はシス型、トランス型のいずれであってもよい］
で示される化合物、またはその塩もしくは溶媒和物を提供する。In a first aspect, the present invention provides a compound of formula (I) for use in diagnosis, treatment and / or prevention of conformation disease:

(I)
[Wherein R ₁ is hydrogen, C _1-6 alkyl, halogen, or C _1-6 alkyl-halogen;
R ₂ and R ₃ are each independently hydrogen or C _1-6 alkyl;
R ₄ is hydrogen or C _1-6 alkyl;
E is CH ₂ or absent,
A is a 5-membered or 6-membered ring and has the following structure:

Or

Or

Having
X and Y are independently N or CH;
Z is O, S, CH ₂ or N—C _p H _{2p + 1} ;
G is N or CH;
J is O, S, CH ₂ or N—C _q H _{2q + 1} ;
R ₅ is hydrogen, C _1-6 alkyl, pyrrole, pyrazole, imidazole, triazole, or NR ¹ R ^II
R ₆ is hydrogen, C _1-6 alkyl, or halogen;
R ^I and R ^II are each independently hydrogen or C _1-6 alkyl, or together, the pyrrolidine ring, morpholine ring, piperidine ring or nitrogen atom may be substituted with C _1-3 alkyl. Forming a piperazine ring,
n ₁ is an integer from 1 to 4;
n ₁ ′ is an integer from 1 to 3;
n is an integer from 1 to 4;
p is an integer from 1 to 4;
q is an integer from 1 to 4;
The arrangement around the double bond connecting the two ring parts may be either cis or trans]
Or a salt or solvate thereof.

Preferred substituents of formula (I) are as follows:
R ₁ is preferably hydrogen, C _1-6 alkyl, halogen, particularly bromine or fluorine.
R ₂ and R ₃ are preferably methyl.
R ₄ is preferably hydrogen or methyl.
Examples of R ₅ include hydrogen, C _1-6 alkyl, and NR ^I R ^II . When ring A is phenyl, NR ^I R ^II is preferable.
R ^I and R ^II are preferably methyl, and it is also preferred that they together form a morpholine ring.
R ₆ is preferably hydrogen.
When R ₆ is hydrogen, preferred n is 4 (ie, an unsubstituted phenyl ring).
Preferred n ₁ is 1 to 3, and 2 is particularly preferred.
The arrangement around the double bond connecting the two ring portions may be either a cis type or a trans type, but is preferably a trans type.

  Considering the results of dark color effect, optimal excitation wavelength and optimal emission wavelength, brain barrier permeability, recognition of amyloid protein with β-sheet structure, acute toxicity test, and ability to bind to amyloid β protein, which are described later, are preferable Compounds of formula (I) are THK-449, THK-533, THK-539, THK-556, THK-558, THK-559, THK-561, THK-562, THK-563, THK-564, THK- 565, THK-573, THK-585, THK-653, THK-700, THK-705, THK-715, THK-716, THK-717, among which the preferred compound of formula (I) is THK-565. THK-585, THK-700, THK-705.

(II)
［式中、Ｒ_７−Ｒ_１４は独立して水素、ハロゲン、ＯＨ、ＣＯＯＨ、ＳＯ_３Ｈ、ＮＲ’Ｒ’’、ＮＯ_２、Ｃ_１−６アルキル、Ｃ_１−６アルキル−ハロゲン、Ｏ−Ｃ_１−６アルキル、Ｏ−Ｃ_１−６アルキル−ハロゲン、ＳＨ、ＣＮ、またはケトであり、
Ｒ’およびＲ’’は独立して水素またはＣ_１−４アルキルであり、
Ｘは窒素または炭素であり、
Ｙは窒素または炭素であり、
ｎ_２は１ないし４の整数であり、
２つの環部分をつなぐ二重結合まわりの配置はシス型、トランス型のいずれであってもよい］
で示される化合物、またはその塩もしくは溶媒和物を提供する。In a second aspect, the present invention relates to formula (II) used for diagnosis, treatment and / or prevention of conformation disease:

(II)
[Wherein R _{7 to} R ₁₄ are independently hydrogen, halogen, OH, COOH, SO ₃ H, NR′R ″, NO ₂ , C _1-6 alkyl, C _1-6 alkyl-halogen, O— C _1-6 alkyl, O—C _1-6 alkyl-halogen, SH, CN, or keto,
R ′ and R ″ are independently hydrogen or C _1-4 alkyl;
X is nitrogen or carbon;
Y is nitrogen or carbon;
n ₂ is an integer of 1 to 4,
The arrangement around the double bond connecting the two ring parts may be either cis or trans]
Or a salt or solvate thereof.

  The bond indicated by the dotted line in the formula (II) forms a conjugated double bond. Moreover, the compound of a formula (II) includes those, when it has a keto-enol tautomer.

Preferred substituents in formula (II) are as follows.
R ₇ is preferably hydrogen or C _1-4 alkyl,
R ₈ is preferably OH, SH, methyl or keto,
R ₉ is preferably hydrogen, C _1-4 alkyl, or CN,
R ₁₀ is preferably methyl, OH or keto,
R ₁₁ is preferably methyl, OH or keto,
R ₁₂ is preferably hydrogen, C _1-4 alkyl, or CN,
R ₁₃ is preferably OH, SH, methyl or keto,
R ₁₄ is preferably hydrogen or C _1-4 alkyl.
As X ₂ and Y ₂ , nitrogen is preferable.
n ₂ may be 1, ₂ , 3, or 4.
The arrangement around the double bond connecting the two ring portions may be either a cis type or a trans type, but is preferably a trans type.

  Among the compounds of formula (II), those having high recognition specificity of amyloid protein having a β sheet structure are THK-265, THK-535, THK-577, THK-380, THK-536, THK-537, THK. -706, THK753. Considering the results of dark color effect, optimal excitation wavelength and optimal emission wavelength, brain barrier permeability, recognition of amyloid protein with β-sheet structure, acute toxicity test, and ability to bind to amyloid β protein, which are described later, are preferable The compounds of the formula (II) are THK-265, THK-380, THK-540 (see Table 1), and the preferred compounds of the formula (II) are THK-265 and THK-540.

(III)
［式中、Ｒ_１５およびＲ_１６は独立して水素またはＣ_１−６アルキルであるか、あるいは一緒になってピロリジン環、モルホリン環、ピペリジン環または窒素原子がＣ_１−３アルキルで置換されていてもよいピペラジン環を形成し、ベンゼン環はＣ_１−４アルキルまたはハロゲンにより置換されていてもよい］
で示される化合物、またはその塩もしくは溶媒和物を提供する。In a third aspect, the present invention relates to formula (III) used for diagnosis, treatment and / or prevention of conformation disease:

(III)
[Wherein R ₁₅ and R ₁₆ are independently hydrogen or C _1-6 alkyl, or together, the pyrrolidine ring, morpholine ring, piperidine ring or nitrogen atom is substituted with C _1-3 alkyl. An optionally piperazine ring is formed, and the benzene ring may be substituted with C _1-4 alkyl or halogen]
Or a salt or solvate thereof.

Preferred R ₁₅ and R ₁₆ of the compound of formula (III) are when both are ethyl or together form a morpholine ring.

  Considering the results of dark color effect, optimal excitation wavelength and optimal emission wavelength, brain barrier permeability, recognition of amyloid protein with β-sheet structure, acute toxicity test, and ability to bind to amyloid β protein, which are described later, are preferable Compounds of formula (III) are THK-449 and THK-527 (see Table 1).

(IV)
［式中、Ｒ_１７およびＲ_１８は独立して水素、Ｃ_１−６アルキル、ハロゲン、Ｃ_１−６アルキル−ハロゲン、またはベンゼン環であり、
ｎ_４は１ないし４の整数であり、
２つの環部分をつなぐ二重結合まわりの配置はシス型、トランス型のいずれであってもよい］
で示される化合物、またはその塩もしくは溶媒和物を提供する。In a fourth aspect, the present invention relates to formula (IV) used for diagnosis, treatment and / or prevention of conformation disease:

(IV)
[Wherein R ₁₇ and R ₁₈ are independently hydrogen, C _1-6 alkyl, halogen, C _1-6 alkyl-halogen, or a benzene ring;
n ₄ is an integer of 1 to 4,
The arrangement around the double bond connecting the two ring parts may be either cis or trans]
Or a salt or solvate thereof.

Preferred R ₁₇ and R _{18 in} formula (IV) are C _1-3 alkyl or a benzene ring.
The arrangement around the double bond connecting the two ring portions may be either a cis type or a trans type, but is preferably a trans type.

  Considering the results of dark color effect, optimal excitation wavelength and optimal emission wavelength, brain barrier permeability, recognition of amyloid protein with β-sheet structure, acute toxicity test, and ability to bind to amyloid β protein, which are described later, are preferable Compounds of formula (IV) are THK-534, THK-539, THK-557 (see Table 1), and particularly preferred compounds of formula (IV) are THK-534 and THK-539.

（Ｖａ）
または

（Ｖｂ）
［式中、Ｒ_１９、Ｒ_２０、Ｒ_２１、Ｒ_２２は独立して水素、ハロゲンまたはＣ_１−４アルキルであり；
ｎ_５、ｎ_５'、ｎ_５''、ｎ_５'''は独立して１〜４の整数であり；
ａ_５、ｂ_５は独立して１〜６の整数であり；
２つの環部分をつなぐ二重結合まわりの配置はシス型、トランス型のいずれであってもよい］
で示される化合物を提供する。
２つの環部分をつなぐ二重結合まわりの配置はシス型、トランス型のいずれであってもよいが、好ましくはトランス型である。好ましいＲ_１９、Ｒ_２０、Ｒ_２１、Ｒ_２２は水素であり、好ましいａ_５は３、好ましいｂ_５は４である。In a fifth aspect, the present invention relates to formula (V) used for diagnosis, treatment and / or prevention of conformation disease:

(Va)
Or

(Vb)
[Wherein R ₁₉ , R ₂₀ , R ₂₁ , R ₂₂ are independently hydrogen, halogen or C _1-4 alkyl;
n ₅ , n ₅ ′, n ₅ ″, n ₅ ′ ″ are independently an integer of 1 to 4;
a ₅ and b ₅ are each independently an integer of 1 to 6;
The arrangement around the double bond connecting the two ring parts may be either cis or trans]
Is provided.
The arrangement around the double bond connecting the two ring portions may be either a cis type or a trans type, but is preferably a trans type. Preferred R ₁₉ , R ₂₀ , R ₂₁ , R ₂₂ are hydrogen, preferred a ₅ is 3, and preferred b ₅ is 4.

Typical examples of the above-mentioned compounds (I) to (V) of the present invention are shown in Table 1.

In particular, the present invention provides a compound represented by formula (I) or a salt or solvate thereof used as a diagnostic probe for conformation disease. The present invention also provides a composition for diagnosing conformation disease and a composition for treating and / or preventing conformation disease comprising a compound represented by formula (I), or a salt or solvate thereof. Furthermore, the present invention provides a method for diagnosing, treating and / or preventing a conformational disease characterized by administering the compound of the present invention, particularly the compound of formula (I), to a subject. In particular, the present invention
(1) A compound represented by the formula (I) or a salt or solvate thereof used as a diagnostic probe, therapeutic agent and / or prophylactic agent for conformation disease;
(2) THK-449, THK-533, THK-539, THK-556, THK-558, THK-559, THK-561, THK-562, THK-563, THK-564, THK-565, THK-573 A compound according to (1) selected from the group consisting of THK-585, THK-653, THK-700, THK-705, THK-715, THK-716 and THK-717;
(3) The compound according to (2), selected from the group consisting of THK-565, THK-585, THK-700, and THK-705;
(4) In the case of binding to an amyloid protein having a β-sheet structure, fluorescence with a wavelength of about 600 nm to about 1000 nm is generated when irradiated with light having a wavelength of about 600 nm to about 1000 nm. Compound;
(5) In the case of binding to an amyloid protein having a β sheet structure, fluorescence with a wavelength of about 700 nm to about 710 nm is generated when irradiated with light having a wavelength of about 650 nm to about 680 nm. Compound;
(6) A composition for diagnosing conformation disease, comprising the compound according to any one of (1) to (5) or a salt or solvate thereof;
(7) A kit for diagnosing conformation disease comprising the compound according to any one of (1) to (5) or a salt or solvate thereof as a constituent component;
(8) A pharmaceutical composition for treatment and / or prevention of conformation disease, comprising the compound according to any one of (1) to (5) or a salt or solvate thereof;
(9) A compound according to any one of (1) to (5) or a salt or solvate thereof, or a composition according to (6) is administered to a subject and irradiated with light having an excitation wavelength from outside the subject. A method for diagnosing conformation disease in a subject, characterized by measuring long-wavelength fluorescence obtained by the method;
(10) A compound disease according to any one of (1) to (5), a salt or solvate thereof, or a composition according to (8), which is administered to the subject. Methods of treatment and / or prevention;
(11) Use of the compound according to any one of (1) to (5) or a salt or solvate thereof for producing a composition or kit for diagnosing conformation disease in a subject;
(12) Use of the compound according to any one of (1) to (5) or a salt or solvate thereof for producing a pharmaceutical composition for the treatment and / or prevention of conformation disease in a subject. It is to provide.

The terms “C _1-6 alkyl”, “C _1-4 alkyl” and “C _1-3 alkyl” in the present specification mean an alkyl group having 6, 4 or 3 carbon atoms, respectively. These terms also include structural isomers. For example, a saturated alkyl having 3 carbon atoms includes an n-propyl group and an i-propyl group. As used herein, the term “halogen” includes fluorine, chlorine, bromine and iodine.

  In the present specification, “compound of the present invention”, “compound of formula (I)”, “compound of formula (II)”, “compound of formula (III)”, “compound of formula (IV)”, References to “a compound of formula (V)” and “a compound of formula (I)-(V)”, when present, include salts and solvates thereof. In addition, when referring to “a compound of the present invention” or “a compound of formula (I)-(V)”, formula (I), formula (II), formula (III), formula (IV) and formula (V) These compounds are intended to be included.

Salts of the compounds of formula (I)-(V) are also included in the present invention, if present. Salts may be formed with nitrogen atoms or any functional groups in compounds of formula (I)-(V). For example, when a carboxyl group or a sulfonic acid group is present in the compound, a salt may be formed between this and a metal. Examples of such salts include salts with alkali metals such as lithium, sodium and potassium, and salts with alkaline earth metals such as magnesium, calcium and barium. When the compound of the formula (I)-(V) contains a hydroxyl group, a compound in which the hydrogen is a metal such as sodium or potassium is also included in the present invention. Further, when a complex formed with a compound of the formula (I)-(V) and a metal salt (for example, a complex formed with a metal salt such as magnesium chloride or iron chloride) can be present, the present specification In the book, it is included in the salt of the compound of formula (I)-(V). The compound of the present invention, salt or solvate thereof can be used as it is or as a composition or kit for diagnosis, treatment and / or prevention of conformation disease. When the compound of the present invention is in the form of a salt, it is preferably a pharmaceutically acceptable salt. Also, Formulas (I) - is by the compounds on the kind of substituents R ₁ to R ₁₈ of (V) may form an onium salt with anions, halide ions as such an anion, organic acid ion, a sulfonate An acid ion, a perchlorate ion, etc. are mentioned. Such onium salts are also preferably pharmaceutically acceptable. Pharmaceutically acceptable salts of the compounds of formula (I)-(V) include, for example, salts with halide ions such as chlorine, bromine and iodine, or salts with metals such as sodium, potassium and calcium. . Such salts are also encompassed by the present invention. Further, the compound of the formula (I)-(V) may form a complex with a metal salt such as iron chloride or cobalt chloride, and such a complex is also included in the present invention. Also, solvates of compounds of formula (I)-(V), if present, are included in the present invention. Examples of the solvate include hydrate, methanol solvate, ethanol solvate, ammonia solvate and the like. When used in the composition or kit for the diagnosis of conformation disease, the therapeutic and / or prophylactic composition of the present invention, a pharmaceutically acceptable one is also preferable, and a pharmaceutically acceptable solvate is hydrated. Products, ethanol hydrates and the like.

  The invention relates in a further aspect to a composition for the diagnosis of conformation diseases comprising a compound of the invention, in particular a compound of formula (I) or a salt or solvate thereof.

  In order to diagnose conformation disease, a compound of the invention, particularly a compound of formula (I), is administered to a subject. The administration subject is preferably a living mammal, more preferably a primate, and most preferably a human. Administration can be local or systemic. At this time, the compound of the present invention may be administered alone, but it is preferably administered as a composition containing the compound of the present invention, particularly the compound of formula (I). The administration route includes intradermal, intraperitoneal, intravenous, arterial, or spinal fluid injection or infusion, and can be selected depending on factors such as the type of disease, the compound used, the condition of the subject, and the examination site.

  The diagnostic composition of the present invention may be in any form, but for that purpose, it is preferably in a form that can be injected or infused. Therefore, the carrier is preferably a pharmaceutically acceptable liquid, and is an aqueous solvent such as potassium phosphate buffer, physiological saline, Ringer's solution, or distilled water, or polyethylene glycol, vegetable oils, ethanol, glycerin, dimethyl sulfone. There are non-aqueous solvents such as, but not limited to, xylene and propylene glycol. The mixing ratio of the carrier and the compound of the present invention can be appropriately selected depending on the application site, detection means, etc., but is usually a ratio of about 100,000 to 1 to about 10 to 1, preferably about 10,000 to 1 to 1. The ratio is about 100 to 1. In addition, the composition of the present invention further contains known antibacterial agents (eg, antibiotics), local anesthetics (eg, procaine hydrochloride, dibucaine hydrochloride, etc.), buffers (eg, tris-HCl buffer, hepes buffer, etc.), penetration A pressure regulator (for example, glucose, sorbitol, sodium chloride, etc.) may be contained. The dosage of the compound of the present invention as the diagnostic composition of the present invention is about 0.01 mg to about 1000 mg, preferably about 1 mg to about 50 mg per diagnosis.

  The compound of the present invention is administered to a subject as it is, preferably in the form of a diagnostic composition containing the compound, and after the time for binding to β-structured amyloid β protein has elapsed (usually about 5 Conformation disease can be diagnosed by irradiating the examination site with long-wavelength light and detecting and analyzing the longer-wavelength fluorescence generated. When the compound of the present invention or a diagnostic composition containing the compound is used for the diagnosis of conformation disease, the wavelength of the long wavelength light irradiated to the target site (in this specification, “excitation wavelength”, “Ex. Wavelength”) Is also from about 550 nm to about 1200 nm, preferably from about 600 nm to about 1000 nm, such as from about 630 nm to about 700 nm, such as from about 630 nm to about 700 nm, within the “biological spectroscopic window” range. is there. The wavelength of fluorescence generated when the compound of the present invention is irradiated with light having such a wavelength (also referred to as “emission wavelength” or “Em. Wavelength” in the present specification) is about 550 nm to about 1200 nm. About 600 nm to about 1000 nm, which is within the range of the “spectroscopic window of”. Preferably Em. The wavelength is longer than the excitation wavelength, more preferably at least about 20 nm longer than the excitation wavelength, and even more preferably at least about 30 nm longer. When the compound of formula (I) of the present invention or a diagnostic composition containing the compound is used for diagnosis of conformation disease, the preferred excitation wavelength of light irradiated to the target site is about 640 nm to about 690 nm, Preferably, it is about 650 nm to 680 nm. The wavelength of the fluorescence obtained is preferably at least about 20 nm longer than the excitation wavelength of the irradiated light, more preferably at least about 30 nm longer. One preferred example of using the compound of formula (I) or a diagnostic composition containing the compound is that the target site is irradiated with light having a wavelength of about 650 nm to about 680 nm, and a wavelength of about 700 nm to about 710 nm is used. Is to detect fluorescence. When the compound of the present invention or a diagnostic composition containing the compound is used, the time required for diagnosis is, for example, about 5 minutes to about 30 minutes when an adult human brain is used as a test site. , Less burden on the subject.

  In order to generate light having a long wavelength, known means such as a lamp that generates light in the visible to infrared region, a spectroscopic system for irradiating a specific wavelength, and the like can be used. Focusing means for efficiently irradiating the specimen with the generated long wavelength light, for example, known means such as a lens and a slit may be used. The intensity and irradiation time of the long wavelength light emitted from the apparatus can be appropriately changed depending on the type and state of the specimen.

  Known means can be used to detect and analyze long wavelength fluorescence arising from the subject. Analysis of long wavelength fluorescence includes measuring its intensity, peak wavelength, and the like. As means for detecting and analyzing long-wavelength fluorescence, for example, as an excitation system, a semiconductor laser irradiation device having characteristics in the excitation wavelength region, a mercury or xenon lamp irradiation device combined with a filter having characteristics in the excitation wavelength region, etc. Equipped with a photomultiplier tube (photomultiplier tube) having a high quantum yield in the measurement long wavelength fluorescent region as a measurement system, or a CCD camera having a high quantum yield in the measurement long wavelength fluorescent region Can be used. These means can be appropriately selected depending on factors such as the type of disease, the compound used, the condition of the subject, and the site to be examined.

  As described above, the diagnosis / test of conformation disease using the compound of the present invention or the composition containing the compound in the case of living mammals has been described. However, the subject is not only a living mammal but also dead. It may be a mammal. In addition, the compound of the present invention or a composition containing the same can be used not only for in vivo diagnosis but also for in vitro conformation disease. For example, a sample obtained from any part of a living or dead mammal is brought into contact with the compound of the present invention or a diagnostic composition containing the same, and the sample is irradiated with light having a long wavelength as described above. In addition, the generated long wavelength fluorescence can be detected and analyzed.

  As described above, it has been clarified by the present inventor that the fluorescence intensity of amyloid β protein greatly increases by taking a β structure, that is, exhibits a deep color effect. Therefore, when using the compound of the present invention or a diagnostic composition containing the same, it is preferable to confirm the dark color effect of the generated long wavelength fluorescence using amyloid β protein not taking β sheet as a control. If the dark color effect is confirmed, it can be confirmed that the amyloid β protein having the β structure is present in the target or the sample. The confirmation of the dark color effect is the intensity of long-wavelength fluorescence obtained using a target or sample that may contain amyloid β protein that does not have β sheet structure and a control (amyloid β protein that does not have β sheet structure). This can be done by comparing For example, by drawing and comparing a contour map and a wavelength versus intensity graph as shown in the embodiment, the dark color effect can be confirmed, and the diagnosis and examination of conformation disease can be ensured.

  In a further aspect, the present invention provides a kit for diagnosing conformation disease comprising the compound of the present invention as a constituent. Usually, the kit contains each component of the compound of the present invention, a solvent for dissolving it, a buffer, an osmotic pressure adjusting agent, an antibacterial agent, a local anesthetic agent, etc. separately or together in each container. It is a collection of what you put in. The compound of the present invention may be provided as a solid such as a lyophilized powder or may be provided by dissolving in a suitable solvent. The solvent may be the same as the carrier used in the above-described composition of the present invention. In addition, each component such as a buffer, an osmotic pressure regulator, an antibacterial agent, and a local anesthetic may be the same as that used in the above-described composition of the present invention. Various containers can be selected as appropriate, but the container can have a shape suitable for label introduction into the compound of the present invention, and can be made of a light-shielding material depending on the properties of the compound, or a patient For convenience in administration, it may be shaped like a vial or a syringe. The kit may appropriately include instruments necessary for diagnosis, such as a syringe, an infusion set, or a device that emits light of long wavelength, a device that detects and / or analyzes long wavelength fluorescence, and the like. Usually, instructions are attached to the kit.

  Further, as described above, since the compound of the present invention specifically binds to amyloid protein having a β structure, the action / function of the amyloid protein having the β structure is suppressed or eliminated, or the β structure by the cell is taken. It is thought to suppress the production of amyloid protein.

  Accordingly, the present invention relates to the fact that accumulation of β-structured amyloid protein containing a compound of the present invention or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier is an etiology or a part of the pathogenesis. Conformational diseases such as Alzheimer's disease, prion disease, Lewy body disease, Parkinson's disease, Huntington's disease, spinal and spinal muscular atrophy, dentate nuclei / pallidulitis atrophy, spinal cord / cerebellar degeneration, Machado -Prevention and / or treatment of Joseph Disease, Amyophic Lateral Sclerosis (ALS), Down syndrome, Pick disease, FTDP-17 (Frontotemporal Dementia and Parkinsonism linked to Chromosome 17), LNTD (Limbic Neurofibrillary Tangle Demetia), Sudanophilic Leukodystrophy, Amyloidosis, etc. Pharmaceutical compositions for use are provided.

  The formulation forms of such pharmaceutical compositions are various, but liquid formulations are preferred, and injectable formulations are particularly preferred. Such injectable formulations can be injected directly into the brain, or, as will be shown later in the examples, the compounds of the present invention have high blood / brain barrier permeability, so the pharmaceutical composition can be injected intravenously or intravenously. It can also be formulated and administered for infusion. Such a liquid formulation can be prepared by a method known in the art. For the preparation of the solution, the compound of the present invention is dissolved in an appropriate carrier, water for injection, physiological saline, Ringer's solution, etc., sterilized with a filter or the like, and then filled into an appropriate container such as a vial or an ampoule. It is also possible to freeze-dry the solution and prepare the solution again with an appropriate carrier at the time of use. The suspensions can be prepared by sterilizing the compounds of the invention, for example, by exposure to ethylene oxide and then suspending in a sterile liquid carrier. Methods for preparing such formulations and other methods of preparation are known in the art. The dose of the compound of the present invention as the pharmaceutical composition of the present invention depends on the disease state, sex, age, weight, etc. of the patient, but generally, it is about 0 per day for an adult weighing 70 kg. .1 mg to about 5000 mg, preferably about 1 mg to about 100 mg. Treatment can be carried out with such a dose for a certain period, and the dose can be increased or decreased depending on the result.

  The compound of the present invention used in the above-mentioned diagnosis of conformation disease, compositions and kits therefor, and pharmaceutical compositions for prevention and / or treatment of conformation disease may be any, but preferably Compounds of formula (I), of which THK-449, THK-533, THK-539, THK-556, THK-558, THK-559, THK-561, THK-562, THK-563, THK-564, THK-565, THK-573, THK-585, THK-653, THK-700, THK-705, THK-715, THK-716, THK-717 are preferred, THK-565, THK-585, THK-700, THK-705 is particularly preferred.

  In another aspect, the present invention provides a compound of the present invention, particularly a compound of formula (I), a salt or solvate thereof, or a diagnostic composition containing the same, administered to a subject, and an excitation wavelength from outside the subject. There is provided a method for diagnosing conformation disease in a subject, characterized by measuring long-wavelength fluorescence obtained by irradiating the light.

  In yet another aspect, the present invention provides administration of a compound of the present invention, particularly a compound of formula (I), a salt or solvate thereof, or a therapeutic and / or prophylactic composition containing it to a subject. A method of treating and / or preventing conformational disease in a subject is provided.

  The preferred compounds and their dosages used in these methods of the invention are the same as those described above.

  In yet another aspect, the invention provides the use of a compound of the invention, in particular a compound of formula (I), a salt or solvate thereof, for the manufacture of a composition or kit for the diagnosis of conformation disease in a subject. provide.

  In yet another aspect, the present invention provides a compound of the invention, in particular a compound of formula (I), a salt or solvent thereof, for the manufacture of a pharmaceutical composition for the treatment and / or prevention of conformation disease in a subject. Provide the use of Japanese products.

  These preferred compounds for use in the present invention and their dosages are similar to those described above.

  The present invention will be described in more detail and specifically with reference to the following examples, but the examples are merely illustrative and do not limit the present invention.

Experimental Method for Examination of Dark Color Effect Excitation Wavelength and Emission Wavelength of Compound of the Present Invention Dissolved in Potassium Phosphate Buffer Solution, and Excitation Wavelength of Compound of the Present Invention Bound to Amyloid β Protein Taking β Sheet Structure The emission wavelength was measured and compared, and the presence or absence of the hyperchromic effect of the compound of the present invention was examined.
Amyloid β protein 1-40 (Peptide Laboratories 4307-v) was dissolved in potassium phosphate buffer (pH 7.4) to a concentration of 20 μM and allowed to stand at 37 ° C. for 4 days to give a β sheet structure. The amyloid β protein was diluted with the same buffer solution, and the compound of the present invention dissolved in the same buffer solution was added to the amyloid β protein. The emission wavelength was measured (final concentration of amyloid β protein: 5 μM, final concentration of the compound of the present invention: 1 μM).
In the optimal wavelength measurement of the compound of the present invention alone, the compound of the present invention is dissolved in a potassium phosphate buffer (pH 7.4), and is measured with a fluorescence spectrophotometer (JASCO FP-6300-WRE-362). Excitation and emission wavelengths were measured (final concentration of the compound of the invention: 1 μM).
AOI-987 [Hintersteiner et al., Nature Biotechnology, Vol. 23, 577-583, 2005] was used as a control compound.
The measurement items are as follows.
1) THK-565 three-dimensional fluorescence spectrum (contour map)
2) THK-565 fluorescence spectrum obtained with an excitation wavelength of 640 to 690 nm 3) THK-585 three-dimensional fluorescence spectrum (contour map)
4) THK-585 fluorescence spectrum obtained with an excitation wavelength of 640 to 690 nm 5) Three-dimensional fluorescence spectrum (contour map) of the control compound AOI-987
6) Fluorescence spectrum of the control compound AOI-987 obtained with an excitation wavelength of 640 to 680 nm

Experimental results 1) THK-565 three-dimensional fluorescence spectrum (contour map)
Fig. 2-a shows the buffer solution, Fig. 2-b shows the one-dimensional THK-565 alone, and Fig. 2-c shows the three-dimensional fluorescence of 1 µM THK-565 bound to the 5 µM amyloid β protein having the β-sheet structure. The spectrum (contour map) is shown. The vertical axis in FIG. 2 indicates the excitation wavelength (EX. Wavelength), and the horizontal axis indicates the emission wavelength (Em. Wavelength) in nanometers (nm).
With THK-565 alone, new contour lines that could not be seen in the contour map of the buffer were observed (parts indicated by the dashed-dotted ellipses). THK-565 bound to 5 μM of amyloid β protein having a β-sheet structure gave new contour lines that were not found in the buffer on the same coordinate axis as THK-565 alone, but took the β-sheet structure. The contour line when bound to amyloid β protein was clearly nectar (the part indicated by the dashed ellipse).
This indicates that THK-565 is enhanced in fluorescence intensity by binding to amyloid β protein having a β sheet structure. Such a phenomenon is generally called a deep color effect (Hyperchromic effct).

Δ蛍光強度＝（βシート構造をとったアミロイドβ蛋白に結合した化合物の蛍光強度）−（化合物単独の蛍光強度）2) THK-565 fluorescence spectrum obtained with an excitation wavelength of 640 to 690 nm As a result of three-dimensional fluorescence spectrum analysis, THK-565 exhibits a dark color effect by binding to amyloid β protein having a β sheet structure. Therefore, further detailed analysis was performed by changing the excitation wavelength.
FIG. 3A to FIG. 3F show the fluorescence spectra of THK-565 bound to amyloid β protein when the excitation wavelength was moved from 640 nm to 690 m. In FIG. 3, the vertical axis represents fluorescence intensity (int.), And the horizontal axis represents emission wavelength in nanometers (nm). Of the two curves in FIG. 3, the upper curve shows the fluorescence spectrum of THK-565 bound to amyloid β protein, and the lower curve shows the fluorescence spectrum of THK-565 alone. Table 1 shows the dark color effect of THK-565 bound to amyloid β protein having a β sheet structure. As shown in FIG. 3 and Table 2, THK-565 binds to amyloid β protein having a β sheet structure. By doing so, the fluorescence intensity was remarkably enhanced, and a typical dark color effect was exhibited. The optimum excitation wavelength at that time was 650 to 680 nm, and the optimum emission wavelength was 700 to 710 nm.

Δfluorescence intensity = (fluorescence intensity of compound bound to amyloid β protein having β sheet structure) − (fluorescence intensity of compound alone)

3) Three-dimensional fluorescence spectrum of THK-585 (contour map)
Fig. 4-a shows three-dimensional fluorescence of buffer, Fig. 4-b shows 1 µM THK-585 alone, and Fig. 4-c shows 1 µM THK-585 bound to 5 µM amyloid β protein having a β-sheet structure. The spectrum (contour map) is shown. The vertical axis in FIG. 4 indicates the excitation wavelength (EX. Wavelength), and the horizontal axis indicates the emission wavelength (Em. Wavelength) in nanometers (nm).
With THK-585 alone, new contour lines that could not be seen in the contour map of the buffer solution were observed (the part indicated by the dashed-dotted ellipse). THK-585 bound to 5 μM amyloid β protein with β-sheet structure gave new contours that were not found in the buffer on the same coordinate axis as THK-585 alone, but took β-sheet structure. The contour line bound to amyloid β protein was clearly denser (the part indicated by the dashed ellipse).
This indicates that THK-585 has enhanced fluorescence intensity by binding to amyloid β protein having a β sheet structure, that is, THK-585 has a dark color effect.

Δ蛍光強度＝（βシート構造をとったアミロイドβ蛋白に結合した化合物の蛍光強度）−（化合物単独の蛍光強度）4) Fluorescence spectrum of THK-585 obtained with an excitation wavelength of 640 to 690 nm As a result of three-dimensional fluorescence spectrum analysis, THK-585 exhibits a deep color effect by binding to amyloid β protein having a β sheet structure. Therefore, further detailed analysis was performed by changing the excitation wavelength.
FIG. 5-a to FIG. 5-f show the fluorescence spectra of THK-585 bound to amyloid β protein when the excitation wavelength was moved from 640 nm to 690 m. The vertical axis in FIG. 5 indicates the fluorescence intensity (int.), And the horizontal axis indicates the emission wavelength in nanometers (nm). Of the two curves in FIG. 5, the upper curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein, and the lower curve shows the fluorescence spectrum of THK-585 alone.
Table 3 shows the dark color effect of THK-585 bound to amyloid β protein having a β sheet structure. As shown in FIG. 5 and Table 3, THK-585 binds to amyloid β protein having a β sheet structure. By doing so, the fluorescence intensity was remarkably enhanced, and a typical dark color effect was exhibited. The optimum excitation wavelength at that time was 650 to 680 nm, and the optimum emission wavelength was 700 to 710 nm.

Δfluorescence intensity = (fluorescence intensity of compound bound to amyloid β protein having β sheet structure) − (fluorescence intensity of compound alone)

Δ蛍光強度＝（βシート構造をとったアミロイドβ蛋白に結合した化合物の蛍光強度）−（化合物単独の蛍光強度）5) Other representative compounds showing dark color effect Table 4 shows typical compounds showing dark color effect in addition to THK-565 and THK-585. The optimum excitation wavelength for each was 650 to 680 nm, and the optimum emission wavelength was 696 to 710 nm.

Δfluorescence intensity = (fluorescence intensity of compound bound to amyloid β protein having β sheet structure) − (fluorescence intensity of compound alone)

6) Three-dimensional fluorescence spectrum (contour map) of the control compound AOI-987
FIG. 6-a shows the three-dimensional fluorescence spectrum (contour map) of 1 μM AOI-987 alone, and FIG. 6-b shows 1 μM AOI-987 bound to 5 μM amyloid β protein having a β-sheet structure. The vertical axis in FIG. 6 indicates the excitation wavelength (EX. Wavelength), and the horizontal axis indicates the emission wavelength (Em. Wavelength) in nanometers (nm).
The coordinates of the contour map of AOI-987 bound to amyloid β protein having a β sheet structure and the density thereof were almost the same as those of AOI-987 alone. This indicates that AOI-987 does not have a dark color effect even when bound to amyloid β protein having a β sheet structure.

7) Fluorescence spectrum of the control compound AOI-987 obtained by an excitation wavelength of 640 to 680 nm As a result of the three-dimensional fluorescence spectrum analysis, AOI-987 is also a dark color effect by binding to amyloid β protein having a β sheet structure. However, in order to confirm this, further detailed analysis was performed by moving the excitation wavelength.
FIGS. 7A to 7E show the fluorescence spectra of AOI-987 bound to amyloid β protein when the excitation wavelength was moved from 640 nm to 680 m. The vertical axis in FIG. 7 indicates the fluorescence intensity (int.), And the horizontal axis indicates the emission wavelength in nanometers (nm). Of the two curves in FIG. 7, the upper curve shows the fluorescence spectrum of AOI-987 alone, and the lower curve shows the fluorescence spectrum of THK-585 bound to amyloid β protein.
As shown in FIG. 7, the fluorescence intensity of AOI-987 bound to amyloid β protein having a β sheet structure is almost the same as that of AOI-987 alone, but rather weakly bound to amyloid β protein. It was.

As described above, the compounds of the present invention represented by THK-565, THK-585, THK-533, THK-564, THK-700, THK-705, THK-717, etc. showed a dark color effect. The positive control compound AOI-987 did not show a dark color effect. A compound exhibiting a dark color effect has a very low fluorescence intensity in a state where it does not bind to amyloid β protein, but in a state where it binds, the fluorescence intensity is dramatically increased. When this is used for actual clinical diagnosis such as Alzheimer's disease, the nonspecific binding and non-conjugated fluorescence are almost negligible, so the nonspecificity as in the conventional probe for PET (positron emission tomography) There is no need to wait until the bound and unbound bodies are cleared from the brain. Therefore, since an image of only the binding of amyloid β protein and the compound can be obtained immediately after administration of such a compound, it becomes possible to diagnose an amyloid accumulating disease in a very short time.
On the other hand, in the diagnosis using the positive control compound AOI-987 which does not show a dark color effect, it is necessary to wait until the non-specific binding and non-binding body are cleared from the brain.

Measurement of the optimum excitation wavelength and optimum emission wavelength of the inventive compound in methyl alcohol solution The inventive compound is dissolved in methyl alcohol, adjusted to a concentration of 1 μM, and optimal with a fluorescence spectrophotometer (FP-6300-WRE-362) Excitation and emission wavelengths were measured. The results are shown in Table 5.

いずれの化合物のエキサイテーション波長およびエミッション波長も、βシート構造をとったアミロイドβ蛋白に結合すると長波長側にシフトした。試験した化合物の多くはβシート構造をとったアミロイドβ蛋白に結合した場合、エキサイテーション波長、エミッション波長ともに「生体の分光学的窓」の範囲内にあった。このうちエミッション波長がエキサイテーション波長よりも約３０ｎｍ以上長波長であるものは、分析が容易であり好ましい。Measurement of Optimal Excitation Wavelength and Optimum Emission Wavelength of Inventive Compound Bound to Amyloid β Protein Having β Sheet Structure Amyloid β protein 1-40 (Peptide Institute) is 20 μM in potassium phosphate buffer (pH 7.4). And was allowed to stand at 37 ° C. for 4 days to give a β sheet structure. The amyloid β protein is diluted with the same buffer solution, and the inventive compound dissolved in the buffer solution is added thereto. Optimal excitation and emission wavelength with a fluorescence spectrophotometer (JASCO FP-6300-WRE-362) (Final concentration of amyloid β protein: 5 μM, final concentration of the inventive compound: 1 μM). The results are shown in Table 6.

The excitation wavelength and emission wavelength of both compounds were shifted to the longer wavelength side when bound to amyloid β protein having a β sheet structure. Many of the compounds tested were within the range of the “biological spectroscopic window” when both the excitation wavelength and emission wavelength were bound to amyloid β protein having a β sheet structure. Among these, those whose emission wavelength is longer than the excitation wavelength by about 30 nm or more are preferable because they are easy to analyze.

Study method for amyloid β protein brain accumulation model 1) Study using transgenic (Tg) mice in which amyloid accumulates in the brain Tg mice (Tg2576) were used. A test compound was administered from the tail vein of a Tg mouse, and 1 hour later, the chest was opened under pentobarbital Na deep anesthesia, and 10% neutral buffered formalin was percutaneously fixed. Thereafter, the brain was cleaved and the brain was removed and immersed in 30% sucrose for 12 hours or more. Next, the extracted brain is quickly frozen in finely pulverized dry ice, and the cryosection (Bright, Model-OT) is used to place the frozen section on a slide glass coated with poly-L-lysine. Produced. In a state where the prepared brain section was not encapsulated, it was microscopically examined with a fluorescence microscope (Nikon Eclipse 80i) and photographed with a digital camera (Nikon Dxm1200F or Photometrics Cool SNAP ES).

2) Examination of amyloid β protein intracerebral injection model
Slc: Wistar male rats were used. Amyloid β protein 1-40 (Peptide Institute 4307-v)) was dissolved in 50 mM potassium phosphate buffer (pH 7.4) to a concentration of 500 μM and incubated at 37 ° C. for 4 days or longer to form a β sheet structure. Sonicated and VORTEX treated and used for intracerebral administration. The rats were anesthetized by intraperitoneal administration of 50 mg / kg, pentobarbital Na, and fixed to a stereotaxic apparatus (Narige Scientific Instruments SR-5N). One side amygdala of Paxinos and Watson's brain coordinates (Bregma as the origin, anteroposterior, -3.0mm; mediolateral, ± 4.9mm; dorsoventral, -8.8mm) cannulated with 500μM amyloid β protein 1-40 170 [mu] m (made of glass of 170 to 250 [mu] m) was injected into 9 locations while shifting 0.5 [mu] l to the back side by 0.5 mm. The test compound was administered into the caudal vein 3 days after the injection of amyloid β protein, in which the buffer solution was injected in the same procedure on the opposite side. Twenty minutes after administration, the rat brain was photographed using a lumino image analyzer (Fuji Photo Film LAS-300) under pentobarbital Na anesthesia. The peak excitation wavelength was 630 nm, and the peak emission wavelength was 670 nm.

Experimental results 1) Examination using transgenic (Tg) mice in which amyloid accumulates in the brain FIG. 8 shows that 2.5 mg / kg THK-265 was intravenously administered to Tg mice, and the brain was taken out after 1 hour and frozen sections. The example which produced this and observed with the fluorescence microscope was shown.
As shown in FIG. 8, THK-265 administered intravenously penetrated the blood-brain barrier and selectively bound to amyloid plaques of Tg mice.

2) Examination in the amyloid β protein intracerebral injection model Figure 9 shows the rat brain in vivo using 1 mg / kg THK-265 intravenously administered to the amyloid β protein intracerebral injection model rat, and 20 minutes later using a lumino image analyzer. The images taken are shown.
As shown in FIG. 9, THK-265 administered intravenously permeated the blood-brain barrier and selectively bound to amyloid β protein injected into the unilateral brain (solid line circle). On the other hand, a THK-265 binding image was not observed on the opposite side to which the buffer solution was administered (inside the broken line circle).
As can be seen from FIG. 9, these phenomena could be imaged in vivo with a lumino image analyzer, so that by using the compound of the present invention, a protein having a β-sheet structure peculiar to conformation disease, For example, amyloid β protein in Alzheimer's disease and abnormal prion protein accumulation in prion disease can be imaged by using the compound of the present invention, in other words, various conformation diseases based on images of brain accumulation and spatial distribution of these proteins. It can be diagnosed.

測定した本発明の化合物のうち、ＴＨＫ−５３３、ＴＨＫ−５５８、ＴＨＫ−５６５、ＴＨＫ−５８５、ＴＨＫ−６５１、ＴＨＫ−７００、ＴＨＫ−７０５、ＴＨＫ−７１５、ＴＨＫ−７１６はβシート構造をとったアミロイドβ蛋白の認識度が高かった。Measurement of the β sheet structure recognition degree of the amyloid β protein of the compound of the present invention The thioflavin-T method was used. The measurement method will be described below.
Several methods have already been reported for quantifying the degree of binding between β-sheet-structured amyloid β protein and a compound in vitro. In this study, the method of LeVine (Protein Science, Vol. 2, 404- 410, 1993) and the method of Woods et al. (Journal of Molecular Biology, Vol. 256, pages 870-877 1996) were modified for use in measurement. That is, amyloid β protein 1-40 (manufactured by Peptide Institute) was dissolved in potassium phosphate buffer (pH 7.4) and allowed to stand at 37 ° C. for 4 days. The compound of the present invention dissolved in the same buffer solution was dispensed into a 96-well microplate in a volume of 25 μl, then 25 μl of amyloid β protein solution (final concentration 5 μM) left for 4 days was added and stirred for 15 minutes. Subsequently, 50 μl of thioflavin T (fluorescing depending on the degree of β structure of amyloid β protein) dissolved in glycine-NaOH buffer solution (pH 8.5) was added in units of 50 μl each (final concentration 3 μM), and after stirring for 15 minutes, fluorescence micro The fluorescence was measured with a plate reader (Spectra Max Gemini XS type, manufactured by Molecular Devices) at an excitation wavelength of 442 nm and a measurement wavelength of 485 nm. When the fluorescence intensity of thioflavine alone is A, B in the presence of amyloid β protein and thioflavine is B, and C in the presence of amyloid β protein, thioflavin T and the compound of the present invention is C, the β sheet structure of each compound of the present invention The degree of recognition of amyloid β protein was calculated by the following formula (unless otherwise specified, the concentration of the compound of the present invention was 1 μM).

Amyloid β protein recognition degree (%) having a β sheet structure of the compound of the present invention
= {(BC) / (BA)} X100)

It can be said that the higher the β structure recognition degree, the higher the binding specificity of the compound of the present invention for amyloid β protein. The results are shown in Table 7.

Among the measured compounds of the present invention, THK-533, THK-558, THK-565, THK-585, THK-651, THK-700, THK-705, THK-715, THK-716 have a β sheet structure. The degree of recognition of amyloid β protein was high.

試験した本発明の化合物は動物体内に投与した場合、安全性が高いことがわかった。Acute toxicity test The acute toxicity of the compound of the present invention was examined by intravenous administration using mice. CRJ: CD1 (ICR) male mice were used as 3 mice per group (average weight of each group was 30-40 g. Each compound was dissolved in a mixture of DMSO, 1N HCl, polyethylene glycol 400, distilled water, Alternatively, it was dissolved in DMSO, diluted with distilled water, administered through the tail vein, and observed thereafter until day 7. Examples of test results are shown in Table 8.

The tested compounds of the present invention were found to be highly safe when administered into animals.

Method of staining the compounds of the present invention on brain sections of Alzheimer's disease patients (i) Brain specimens in temporal lobes or hippocampus of patients and normal elderly who were pathologically confirmed as Alzheimer's disease were used. The specimen was provided by the Longevity Medical Research Institute, a welfare village hospital, a joint research partner, and was approved by the bereaved family for use for research purposes (Permission No. 20 of the welfare village hospital ethics committee).
(Ii) Paraffin-embedded brain tissue was sliced at a thickness of 6 μm or 8 μm, spread on a slide glass, and dried. The paraffin brain sections were deparaffinized in the order of 10 minutes × 2 with xylene, 5 minutes × 2 with 100% ethanol, 5 minutes with 90% ethanol and 10 minutes with running water.
(Iii) As a pretreatment for staining with the compound of the present invention, a treatment for removing autofluorescence by lipofuscin was performed. First, the deparaffinized section was immersed in a 0.25% KMnO ₄ solution for 20 minutes. After washing with PBS for 2 minutes × 2 times, it was immersed in a 0.1% K ₂ S ₂ O ₅ / oxalic acid solution for about 5 seconds, and further washed with PBS for 2 minutes × 3 times.
(Iv) About 150 μl of 100 μM compound solution of the present invention dissolved in 50% ethanol was dropped and reacted for 10 minutes. After being soaked in tap water five times, it was sealed with Fluor Save Reagent (Calbiochem) and microscopically examined using a fluorescence microscope (Nikon, Eclipse 80i). Images were taken with a digital camera (Nikon Dxm1200F or Photometrics Cool SNAP ES).

Immunostaining method (a) Immunostaining method of amyloid β protein After deparaffinization, wash in distilled water for 2 minutes × 2, surround the tissue with immunopen, drop about 150 μl of formic acid and leave at room temperature for 5 minutes. did. After washing with tap water for 5 minutes, it was immersed in cold PBS-Tween 20 for 2 minutes, and then approximately 150 μl of 0.05% trypsin solution was dropped and reacted at 37 ° C. for 15 minutes.
After washing with cold PBS-Tween20 for 5 minutes × 2 times in an ice bath, drop 2 drops of blocking serum and react at 37 ° C. for 30 minutes to remove excess water, then amyloid β protein specific antibody About 150 μl of 6F / 3D (DAKO, diluted 1:50) was added and reacted at 37 ° C. for 1 hour.
Further, after washing with cold PBS-Tween 20 for 2 minutes × 5 times, 2 drops of anti-mouse IgG (H + L), goat, and biotin binding solution were dropped and reacted at 37 ° C. for 1 hour, and then with cold PBS-Tween 20 for 2 minutes × 3 After washing twice, 2 drops of ABC solution (streptavidin-biotin-peroxidase complex solution) was added dropwise and allowed to stand for 30 minutes. Again, after washing with cold PBS-Tween 20 for 2 minutes × 3 times, DAB solution (10 mg of DAB dissolved in 20 ml of 0.05 mol / l Tris-HCl buffer and 100 μl of 3% hydrogen peroxide solution added immediately before use) About 150 μl of the solution was dropped to obtain a sufficient color, and then washed with distilled water for 1 minute to stop the reaction.
(B) Immunostaining method for neurofibrillary tangles After deparaffinization, the plate was washed twice with chilled PBS-Tween 20 for 5 minutes, and then 2 drops of blocking serum were dropped and reacted at 37 ° C for 30 minutes. After removing excess water, two drops of AT-8 (Mia Nobels, diluted 1: 100), which is a tau specific antibody, was added dropwise and reacted at 4 ° C. overnight.
The following day, after washing with cold PBS-Tween 20 for 2 minutes x 5 times, drop 2 drops of anti-rabbit IgG, goat, biotin binding solution at 37 ° C for 1 hour, and wash with cold PBS-Tween 20 for 2 minutes x 3 times Then, 2 drops of ABC solution (streptavidin-biotin-peroxidase complex solution) was dropped and allowed to stand for 30 minutes.
Again, after washing with cold PBS-Tween 20 for 2 minutes × 3 times, DAB solution (10 mg of DAB dissolved in 20 ml of 0.05 mol / l Tris-HCl buffer and 100 μl of 3% hydrogen peroxide solution added immediately before use) About 150 μl of the solution was dropped to obtain a sufficient color, and then washed with distilled water for 1 minute to stop the reaction. The blocking serum, anti-rabbit IgG, goat, biotin binding solution, and ABC solution were those included in the Phosphorylated Tau Immunohistostain Kit (Wako 299-57301).

Experimental Results THK-265 specifically bound to amyloid β protein (FIG. 10) and neurofibrillary tangles (FIGS. 11 and 12) in Alzheimer's disease brain sections. This is clear from THK-265 that the staining position corresponds to the staining position of immunostaining (amyloid β protein and neurofibrillary tangles) in adjacent sections.
Similarly, THK-449, THK-539, THK-556, THK-558, THK-559, THK-561, THK-562, THK-563, THK-565, THK-585, THK-533, THK-535 , THK-557, THK-577, THK-653, THK-700, THK-705, THK-706, THK-715, THK-716, THK-717, THK-753 are also amyloid β protein in brain sections of Alzheimer's disease patients It was found to specifically bind to (Figs. 13 to 34, respectively).
Similar results were obtained for other compounds of the present invention.

  The present invention can be used in the fields of manufacturing drugs and kits for testing / diagnosis of conformation disease, manufacturing of treatment and / or prevention of conformation disease, testing / diagnosis of conformation disease, treatment or prevention, etc. . The present invention can also be used in fields such as the production of conformational research reagents.

Claims (12)

Formula (I) for use as a diagnostic probe, therapeutic agent and / or prophylactic agent for conformation disease:

(I)
[Wherein R ₁ is hydrogen, C _1-6 alkyl, halogen, or C _1-6 alkyl-halogen;
R ₂ and R ₃ are each independently hydrogen or C _1-6 alkyl;
R ₄ is hydrogen or C _1-6 alkyl;
E is CH ₂ or absent,
A is a 5-membered or 6-membered ring and has the following structure:

Or

Or

Having
X and Y are independently N or CH;
Z is O, S, CH ₂ or N—C _p H _{2p + 1} ;
G is N or CH;
J is O, S, CH ₂ or N—C _q H _{2q + 1} ;
R ₅ is hydrogen, C _1-6 alkyl, pyrrole, pyrazole, imidazole, triazole, or NR ¹ R ^II
R ₆ is hydrogen, C _1-6 alkyl, or halogen;
R ^I and R ^II are each independently hydrogen or C _1-6 alkyl, or together, the pyrrolidine ring, the morpholine ring, the piperidine ring, or the nitrogen atom may be substituted with C _1-3 alkyl. Forming a good piperazine ring,
n ₁ is an integer from 1 to 4;
n ₁ ′ is an integer from 1 to 3;
n is an integer from 1 to 4;
p is an integer from 1 to 4;
q is an integer from 1 to 4;
The arrangement around the double bond connecting the two ring parts may be either cis or trans]
Or a salt or solvate thereof.   THK-449, THK-533, THK-539, THK-556, THK-558, THK-559, THK-561, THK-562, THK-563, THK-564, THK-565, THK-573, THK- The compound of claim 1 selected from the group consisting of 585, THK-653, THK-700, THK-705, THK-715, THK-716 and THK-717.   The compound of claim 2 selected from the group consisting of THK-565, THK-585, THK-700 and THK-705.   The compound according to claim 1, wherein the compound emits fluorescence having a wavelength of about 600 nm to about 1000 nm when irradiated with light having a wavelength of about 600 nm to about 1000 nm when bound to an amyloid protein having a β sheet structure.   The compound according to claim 1, wherein the compound emits fluorescence having a wavelength of about 700 nm to about 710 nm when irradiated with light having a wavelength of about 650 nm to about 680 nm when bound to an amyloid protein having a β sheet structure.   A composition for diagnosing conformation disease, comprising the compound according to any one of claims 1 to 5, or a salt or solvate thereof.   A diagnostic kit for conformation disease comprising the compound according to any one of claims 1 to 5 or a salt or solvate thereof as a constituent component.   A pharmaceutical composition for the treatment and / or prevention of conformation disease, comprising the compound according to any one of claims 1 to 5, or a salt or solvate thereof.   The compound according to any one of claims 1 to 5 or a salt or solvate thereof, or the composition according to claim 6 is administered to a subject, and is obtained by irradiating light of an excitation wavelength from outside the subject. A method for diagnosing conformation disease in a subject, characterized by measuring long-wavelength fluorescence.   Treatment of conformation disease in a subject and / or administration of the compound according to any one of claims 1 to 5 or a salt or solvate thereof, or a composition according to claim 8 to the subject. Prevention method.   Use of the compound according to any one of claims 1 to 5, or a salt or solvate thereof, for producing a composition or kit for diagnosing conformation disease in a subject.   Use of a compound according to any one of claims 1 to 5 or a salt or solvate thereof for the manufacture of a pharmaceutical composition for the treatment and / or prevention of conformation disease in a subject.

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