JPWO2014132919A1 - Diagnostic composition - Google Patents

Abstract

For the purpose of providing a clinically practical β-amyloid protein imaging probe for SPECT and an imaging probe for diagnosing diabetes, a compound represented by the general formula (I): wherein R1, R2, R3, And R4, any one represents a radioactive iodine atom, the other represents a hydrogen atom, and R5, R6, R7, and R8 are independently of each other a hydrogen atom, an amino group, a methylamino group, a dimethylamino group, A methoxy group or a hydroxy group is represented, and X represents CH or N. Or a pharmaceutically acceptable salt of these compounds, a composition for diagnosing conformation disease is provided.

Description

  The present invention relates to a composition for diagnosing a conformational disease such as Alzheimer's disease, and particularly to a composition used for single photon tomography.

  In recent years, the increase in patients with dementia in developed countries has become a major social problem. Alzheimer's disease (AD) accounts for the largest proportion of dementia, with an estimated 26.6 million AD worldwide worldwide in 2006. In the future, with the rapid aging of the population, the number of AD patients is expected to reach 106.8 million in 2050, and there is a strong demand for the development of early diagnosis methods and fundamental treatments. .

  AD has clinical symptoms such as memory and cognitive decline and brain atrophy at the onset and mild cognitive impairment (MCI). On the other hand, neurofibrillary tangles consisting of senile plaques and β-amyloid protein (Aβ) with a sheet structure, which are characteristic pathological changes in the brain of AD, and hyperphosphorylated tau protein It is known that the accumulation of is recognized from the normal stage of cognitive function and has reached almost constant at the time when clinical symptoms are observed. Senile plaque deposits are formed by aggregation and fibrosis of Aβ (1-40) and Aβ (1-42), which are sequentially excised from amyloid precursor protein by β-secretase and γ-secretase, which are degrading enzymes. . Neurofibrillary tangles occur when tau protein is phosphorylated excessively and loses its ability to bind to microtubules, and free tau proteins aggregate together. In particular, the deposition of senile plaques with Aβ aggregates as the main component begins at the earliest stage of the onset of AD, and because of its high disease specificity, Aβ is important in the early diagnosis of AD and the development of therapeutic agents Target molecules.

The nuclear medicine molecular imaging method is considered to be a method suitable for early clinical diagnosis of AD because it can perform quantitative evaluation with high accuracy and high sensitivity by making use of the permeability of radiation. Many Aβ imaging probes for positron tomography (PET) have been developed so far. Among them, (E) -4- (2- (6- (2- (2- (2- [ ¹⁸ F] fluoroethoxy ) Ethoxy) ethoxy) pyridin-3-yl) vinyl l) -N-methylaniline (AV-45) was approved by the US Food and Drug Administration (FDA) in April 2012 and is now in clinical use in the United States. (Cselenyi Z, Jonhagen ME, Forsberg A, Halldin C, Julin P, et al. (2012) J Nucl Med 53: 415-424). However, problems with PET probes include the lack of facilities with PET devices and the short physical half-life of the radionuclide used.

In the future, in order to respond to the diagnosis of AD, which is expected to further increase in the number of patients, single photons are superior in versatility in that they can use radionuclides with a larger number of facilities and a longer physical half-life than PET. The use of tomography (SPECT) is expected. So far, compounds containing radioactive iodine and compounds labeled with ^99m Tc have been developed as Aβ imaging probes for SPECT. [ ¹²³ I] 6-Iodo-2- (4′-dimethylamino) phenyl-imidazo [1,2-a] pyridine (IMPY) is the first Aβ imaging probe for SPECT tested in humans (Kung MP , Hou C, Zhuang ZP, Zhang B, Skovronsky D, et al. (2002) Brain Res 956: 202-210, Kung MP, Hou C, Zhuang ZP, Cross AJ, Maier DL, et al. (2004) Eur J Nucl Med Mol Imaging 31: 1136-1145, Newberg AB, Wintering NA, Plossl K, Hochold J, Stabin MG, et al. (2006) J Nucl Med 47: 748-754). This compound showed affinity for the synthesized aggregates of Aβ peptide and selectively bound to amyloid plaques in brain sections of AD patients, but there was a significant difference in biodistribution between AD patients and healthy individuals. Not shown (Newberg AB, Wintering NA, Plossl K, Hochold J, Stabin MG, et al. (2006) J Nucl Med 47: 748-754). This is thought to be due to the high lipophilicity of IMPY and low in vivo stability. As described above, several Aβ imaging probes for SPECT have already been developed, but at present there are no clinically practical ones.

Recently, the present inventors have reported that a pyridylbenzofuran derivative (Non-patent Document 1) or a pyridylbenzoxazole derivative (Non-patent Document 2) labeled with ¹⁸ F can be used as an Aβ imaging probe for PET. However, there is no report about using these derivatives as Aβ imaging probes for SPECT.

  As described above, Aβ is a typical amyloid associated with a disease, but many other amyloids associated with a disease are known besides Aβ. For example, it is known that amyloid is also deposited on pancreatic islets of Langerhans in patients with type 2 diabetes (Opie E, J. Exp. Med., 5: 527-541, 1991). The main component of this amyloid is a peptide called amylin, and human amylin has a β-sheet structure that is important for deposition as amyloid (Glenner GG, Eanes ED, Wiley CA, Biochem). Biophys. Res. Comm., 155: 608-614, 1988).

  Attempts have been made to diagnose not only AD but also diabetes by PET and SPECT. For example, an imaging probe was created based on a peptide that binds to GLP-1R (glucagon-like peptide 1 receptor) distributed in pancreatic β cells, and a method for diagnosing diabetes by measuring the amount of islets using this probe was reported. (Patent Document 1). However, imaging probes targeting amylin and diagnostic methods for diabetes have not been reported so far.

International Publication WO2010 / 032509

Ono M, Cheng Y, Kimura H, Cui M, Kagawa S, et al. (2011) J Med Chem 54: 2971-2979 Cui, M .; Wang, X .; Yu, P .; Zhang, J .; Li, Z .; Zhang, X .; Yang, Y .; Ono, M .; Jia, H .; Saji, H .; Liu, BJ Med. Chem. 2012, 55, 9283-96

  An object of the present invention is to provide an Aβ imaging probe for SPECT that is clinically practical. Another object of the present invention is to provide an imaging probe for diagnosing diabetes.

  As a result of intensive studies to solve the above problems, the present inventor administered a pyridylbenzofuran derivative or a pyridylbenzoxazole derivative containing radioactive iodine to AD model mice and wild type mice, and performed SPECT imaging. It was found that there is a clear difference in radioactivity accumulation in the mouse brain, that is, the derivative can be a clinically practical Aβ imaging probe for SPECT.

As described above, it is already known to use a pyridylbenzofuran derivative or a pyridylbenzoxazole derivative as an Aβ imaging probe for PET. However, for the following reasons, at the time of filing the present application, it was not predicted that these derivatives could be used as an Aβ imaging probe for SPECT.
1) Since iodine has a higher molecular weight than fluorine, it changes the properties of the substance to be labeled. For this reason, pyridyl benzofuran derivatives labeled with radioactive iodine may not exhibit the same properties as pyridyl benzofuran derivatives labeled with radioactive fluorine.
2) As mentioned above, the biodistribution of IMPY showed no significant difference between AD patients and healthy individuals. A pyridylbenzofuran derivative or a pyridylbenzoxazole derivative containing iodine is similar in structure to IMPY (both contain iodine, and are composed of a condensed ring of 6-membered ring and 6-membered ring and a 6-membered ring). Similarly, it was expected that there would be no difference in biodistribution between AD patients and healthy individuals.

  The present inventor has also found that the above-mentioned pyridylbenzofuran derivative containing radioactive iodine also binds to amylin, which is a main component of amyloid found in patients with type 2 diabetes.

  The present invention has been completed based on the above findings.

That is, the present invention provides the following (1) to (9).
(1) General formula (I)
[In the formula, any one of R ¹ , R ² , R ³ , and R ⁴ represents a radioactive iodine atom, the other represents a hydrogen atom, and R ⁵ , R ⁶ , R ⁷ , and R ⁸ are Independently, it represents a hydrogen atom, an amino group, a methylamino group, a dimethylamino group, a methoxy group, or a hydroxy group, and X represents CH or N. ]
Or a pharmaceutically acceptable salt of these compounds. A composition for diagnosing a conformational disease, comprising:
(2) The composition for diagnosis of conformation disease according to (1), wherein R ³ in the general formula (I) is a radioactive iodine atom, and R ¹ , R ² , and R ⁴ are hydrogen atoms. .
(3) ^{R 7} in the general formula (I) is an amino group, methylamino group, or dimethylamino ^group, R 5, ^{R 6,} and ^{R 8} is characterized in that it is a hydrogen atom (1) or ( The composition for diagnosis of conformation disease according to 2).
(4) The composition for diagnosis of conformation disease according to any one of (1) to (3), wherein the radioactive iodine atom is ¹²³ I or ¹²⁵ I.
(5) The composition for diagnosing conformation disease according to any one of (1) to (4), which is used for single photon tomography.
(6) The composition for diagnosing conformation disease according to any one of (1) to (5), wherein the conformation disease is Alzheimer's disease.
(7) The composition for diagnosing conformation disease according to any one of (1) to (5), wherein the conformation disease is type 2 diabetes.
(8) The composition for diagnosis of conformation disease according to any one of (1) to (4) is administered to an animal, an image of a brain of the animal is taken, and the image is represented by the general formula (I) A method for diagnosing Alzheimer's disease, comprising diagnosing Alzheimer's disease based on the state of a compound.
(9) The composition for diagnosis of conformation disease according to any one of (1) to (4) is administered to an animal, an image of the pancreas of the animal is taken, and the image is represented by the general formula (I) A method for diagnosing type 2 diabetes, comprising diagnosing type 2 diabetes based on the state of the compound.

  The present invention provides a clinically practical Aβ imaging probe for SPECT. The present invention also provides an imaging probe for diagnosing diabetes.

The figure which shows the synthetic pathway of the pyridyl benzofuran derivative containing an iodine. The numbers in the figure indicate the compound numbers. The figure which shows the synthetic pathway of the pyridyl benzofuran derivative containing a radioactive iodine. The numbers in the figure indicate the compound numbers. The figure which shows the change of the radioactivity in each organ of the mouse | mouth which administered the pyridyl benzofuran derivative containing a radioactive iodine. Brain SPECT images of Tg2576 mice (A) and wild type mice (B) after administration of Compound [ ¹²³ I] 8. Ex vivo autoradiograms of brain sections of Tg2576 mice (A) and wild-type mice (B) after administration of Compound [ ¹²³ I] 8. Images of fluorescent staining with thioflavin S of the same brain section as A and B are shown in C and D, respectively. In vitro autoradiogram of AD brain sections incubated with Compound [ ¹²⁵ I] 7 (A), Compound [ ¹²⁵ I] 8 (B), and Compound [ ¹²⁵ I] 9 (C). Aβ plaques were confirmed by immunostaining (D). The figure which shows the synthetic pathway of the pyridyl benzoxazole derivative containing an iodine. The numbers in the figure indicate the compound numbers. The figure which shows the synthetic pathway of the pyridyl benzoxazole derivative containing a radioactive iodine. The numbers in the figure indicate the compound numbers. The figure which shows the change of the radioactivity in each organ of the mouse | mouth which administered the pyridyl benzoxazole derivative containing a radioactive iodine. The figure which shows the change of the radioactivity in the brain of the normal mouse | mouth which administered compound [ ^125I ] 29 ((circle)), compound [ ^125I ] 30 ((triangle | delta)), or compound [ ^125I ] 31 ((circle)). In vitro autoradiograms of AD brain sections incubated with compound [ ¹²⁵ I] 29 (A), compound [ ¹²⁵ I] 30 (B), and compound [ ¹²⁵ I] 31 (C). Aβ plaques were confirmed by immunostaining (D). Brain sections of 16-month-old Tg2576 mice (A), 25-month-old Tg2576 mice (D), 16-month-old wild-type mice (C), and 25-month-old wild-type mice (F) administered with the compound [ ¹²³ I] 31 Ex vivo autoradiogram. The same brain sections as those of Tg2576 mice were stained with thioflavin S (B and E). Brain SPECT images of 25-month-old Tg2576 mice (A) and 25-month-old wild-type mice (B) after administration of Compound [ ¹²³ I] 31. The figure which shows the result of the binding saturation experiment to the amylin aggregate of compound [ ^125I ] 9. In vitro autoradiogram of pancreas sections incubated with compound [ ¹²⁵ I] 9.

  Hereinafter, the present invention will be described in detail.

R ¹ , R ² , and R ⁴ in the general formula (I) are preferably hydrogen atoms.

R ³ in the general formula (I) is preferably a radioactive iodine atom.

R ⁵ , R ⁶ and R ⁸ in the general formula (I) are preferably hydrogen atoms.

R ⁷ in the general formula (I) is preferably an amino group, a methylamino group, or a dimethylamino group, more preferably a methylamino group when X is CH, and when X is N. Is a dimethylamino group.

The radioactive iodine atom is preferably ¹²³ I or ¹²⁵ I, and more preferably ¹²³ I.

  Representative compounds among the compounds represented by formula (I) are shown in the following table. In the table, “Me” represents a methyl group, and “I” represents a radioactive iodine atom.

  Among the above compounds, preferred compounds are I-12 (Compound 7), I-13 (Compound 8), I-14 (Compound 9), I-33 (Compound 29), I-34 (Compound 30), I -35 (Compound 31) can be mentioned, and more preferred compounds include I-13 and I-35.

  The compound represented by the general formula (I) can be synthesized according to the method described in the Examples or according to a method in which those methods are appropriately modified or modified with reference to the description.

  In the composition for diagnosing conformation disease of the present invention, a pharmaceutically acceptable salt of the compound represented by the general formula (I) may be used instead of the compound represented by the general formula (I). . Examples of such salts include alkali metal salts (sodium salt, potassium salt, lithium salt), alkaline earth metal salts (calcium salt, magnesium salt), sulfate, hydrochloride, nitrate, phosphate, and the like.

  In the present invention, “conformation disease” means a disease group caused by a protein abnormalized by conformational transformation such as Aβ, tau protein, prion, amylin, etc. In addition to AD, genetics associated with Down's syndrome and Dutch amyloidosis Hereditary cerebral hemorrhage with amyloidosis (Dutch type: HCHWA-D), Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and type 2 diabetes. In addition, diseases to be diagnosed include precursor symptoms of diseases that are generally not recognized as “diseases”. Examples of prodromal symptoms of such diseases include mild cognitive impairment (MCI) seen before the onset of AD.

  The diagnosis of conformation disease using the above composition is usually performed by administering this composition to an animal, and then taking an image of the organ of the animal, and the state (quantity) of the compound represented by the general formula (I) in the image. , Distribution, etc.). Specifically, it can be diagnosed by SPECT or the like. The target animal may be a human (diagnostic subject) or a non-human animal (such as a laboratory animal such as a mouse, rat, or rabbit). The organ to be imaged may be determined according to the conformation disease to be diagnosed. For example, if AD is diagnosed, a brain image is taken, and if type 2 diabetes is diagnosed, a pancreas image is taken. .

The method of administration of the composition is not particularly limited and can be appropriately determined according to the type of compound, the type of labeling substance, etc., but is usually intradermal, intraperitoneal, intravenous, arterial or spinal fluid injection or Administer by infusion. The dose of the composition is not particularly limited, and can be appropriately determined according to the type of compound, the type of labeling substance, etc. In the case of an adult, the compound represented by the general formula (I) is 10 ⁻¹⁰ per day. It is preferable to administer ~ 10 ^-3 mg, and more preferably 10 ^-8 to 10 ^-5 mg.

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

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
[Example 1] Synthesis of pyridylbenzofuran derivative and its application [Experimental method]
(1) Reagents, instruments, etc. All reagents used in the synthesis were purchased from Nacalai Tesque, Wako Pure Chemical Industries, or Aldrich, and used without further purification. ¹ H-NMR spectra (400 MHz) were obtained on a JEOL JNM-AL400 NMR spectrometer at room temperature with TMS as an internal standard. Chemical shifts are reported as δ values (ppm) compared to internal TMS. Coupling constants are reported in hertz. Multiplicity is defined by s (single line), d (double line), t (triple line), and m (multiple line). Mass spectra were obtained with Shimadzu GC-MS-QP2010 Plus (ESI). Fluorescence observation was performed with a microscope using a BV-2A filter set (Nikon, Eclipse 80i) (excitation, 400-440 nm; dichroic mirror, 455 nm; long pass filter, 470 nm). Reversed phase HPLC and analysis of radiolabeled and non-radioactive analogues were performed using a Cosmosil C18 column (Nacalai Tesque, 5C ₁₈ −) with a mobile phase (acetonitrile: water = 7: 3) supplied at a flow rate of 1.0 mL / min. AR-II, 4.6 mm x 150 mm) and Shimadzu system (LC-10AT pump with SPD-10A UV detector (λ = 254 nm)). ddY mice (5 weeks old, 22-25 g, male) were purchased from Shimizu Experimental Materials Co., Ltd. Tg2576 transgenic mice and wild type mice were purchased from Taconic Farms. Micro-SPECT / CT for small animals (FX3300, Gamma Medica Ideas) was performed at the Radioisotope Research Center (Kyoto University). All animal experiments were conducted according to the inventor's organizational guidelines and were approved by the Kyoto University Animal Care Committee.

(2) Synthesis of pyridylbenzofuran derivatives
Synthesis of 5- (5-bromobenzofuran-2-yl) pyridin-2-amine (Compound 1)
2-Bromobenzofuran-2-boronic acid (722 mg, 3.0 mmol), 2-amino-5-iodopyridine (660) in 2 M Na ₂ CO ₃ (aq) / dioxane (150 ml, 1: 1) as solvent mg, 3.0 mmol), and a solution of Pd (Ph ₃ P) ₄ (366 mg, 0.3 mmol) were stirred at reflux overnight. The mixture was cooled to room temperature and 1M NaOH (20 mL) was added. After extraction with ethyl acetate, the organic phase was dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated and the residue was purified by silica gel chromatography (hexane: ethyl acetate = 1: 6) to give 273 mg of compound 1 (31.5%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 4.66 (s, 2H), 6.57 (d, 1H, J = 8.4 Hz), 6.79 (s, 1H), 7.32 (d, 1H, J = 2.4 Hz), 7.33 (d, 1H, J = 2.0 Hz), 7.66 (d, 1H, J = 1.6 Hz), 7.86 (dd, 1H, J ₁ = 8.8 Hz, J ₂ = 2.4 Hz), 8.58 (d, 1H, J = 2.4 Hz) .MS: m / z 290 (M ⁺ + H).

Synthesis of 5- (5-bromobenzofuran-2-yl) -N-methylpyridin-2-amine (Compound 2)
5-Bromobenzofuran-2-boronic acid (120 mg, 0.5 mmol), 5-iodo-N-methylpyridine with 2 M Na ₂ CO ₃ (aq) / dioxane (20 ml, 1: 1) as solvent A solution of 2-amine (117 mg, 0.5 mmol) and Pd (PH ₃ P) ₄ (61 mg, 0.05 mmol) was stirred at reflux overnight. The mixture was cooled to room temperature and 1 M NaOH (20 mL) was added. After extraction with ethyl acetate, the organic phase was dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give 57.6 mg of compound 2 (38.0%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 2.98 (d, 3H, J = 5.2 Hz), 4.80 (s, 1H), 6.45 (d, 1H, J = 8.8 Hz), 6.75 (s, 1H), 7.32 (d, 1H, J = 7.2 Hz), 7.52 (d, 1H, J = 8.4 Hz), 7.64 (d, 1H, J = 2.0 Hz), 7.87 (dd, 1H, J ₁ = 8.8 Hz, J ₂ = 2.4 Hz), 8.60 (d, 1H, J = 2.8 Hz). MS: m / z 304 (M ⁺ + H).

Synthesis of 5- (5-bromobenzofuran-2-yl) -N, N-dimethylpyridin-2-amine (compound 3)
5-Bromobenzofuran-2-boronic acid (120 mg, 0.5 mmol), 5-iodo-N, N-dimethyl in 2 M Na ₂ CO ₃ (aq) / dioxane (20 ml, 1: 1) as solvent A solution of pyridin-2-amine (124 mg, 0.5 mmol) and Pd (Ph ₃ P) ₄ (61 mg, 0.05 mmol) was stirred at reflux overnight. The mixture was cooled to room temperature and 1M NaOH (20 mL) was added. After extraction with ethyl acetate, the organic phase was dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 7: 3) to obtain 59.0 mg of compound 3 (37.2%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.14 (s, 6H), 6.55 (d, 1H, J = 8.8 Hz), 6.71 (s, 1H), 7.17 (dd, 1H, J ₁ = 8.8 Hz, J ₂ = 2.4 Hz), 7.32 (d, 1H, J = 2.4 Hz), 7.53 (d, 1H, J = 8.8 Hz), 7.88 (dd, 1H, J ₁ = 8.8 Hz, J ₂ = 2.4 Hz), 8.65 (d, 1H, J = 2.4 Hz). MS: m / z 318 (M ⁺ + H).

Synthesis of 5- (5- (tributylstannyl) benzofuran-2-yl) pyridin-2-amine (compound 4) Compound 1 (273 mg, 0.95 in a mixed solvent (40 mL, 3: 1 dioxane / triethylamine mixture) mmol), bis (tributyltin) (0.8 mL), and (Ph ₃ P) ₄ PD (100 mg) were stirred at 90 ° C. overnight. After extraction with ethyl acetate, the organic phase was dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give 115.6 mg of compound 4 (24.6%). NMR (400 MHz, CDCl ₃ ): δ 0.87-0.91 (m, 9H), 1.06-1.10 (m, 6H), 1.32-1.39 (m, 6H), 1.54-1.62 (m, 6H), 4.75 (s, 2H), 6.54 (d, 1H, J = 8.0 Hz), 6.82 (s, 1H), 7.31 (d, 1H, J = 8.0 Hz), 7.47 (d, 1H, J = 8.4 Hz), 7.63 (s, 1H), 7.85 (dd, 1H, J ₁ = 8.4 Hz, J ₂ = 2.4 Hz), 8.59 (d, 1H, J = 2.4 Hz) .HRMS (EI): m / z calcd for C ₂₅ H ₃₆ N ₂ OSn (M ⁺ ) 500.1849, found 500.1847.

Synthesis of N-methyl-5- (5- (tributylstannyl) benzofuran-2-yl) pyridin-2-amine (Compound 5) 27.7 mg of compound using a reaction similar to that described above for the preparation of Compound 4 5 was obtained from compound 2 in 28.4% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 0.89 (t, 9H, J = 7.2 Hz), 1.32-1.35 (m, 12H), 1.62-1.66 (m, 6H), 3.04 (s, 3H), 6.75 (d, 1H, J = 8.8 Hz), 6.87 (s, 1H), 7.32 (d, 1H, J = 7.2 Hz), 7.46 (d, 1H, J = 8.4 Hz), 7.64 (d, 1H, J = 2.0 Hz), 8.05 (d, 1H, J = 8.4 Hz), 8.40 (d, 1H, J = 3.2 Hz) .HRMS (EI): m / z calcd for C ₂₆ H ₃₈ N ₂ OSn (M ⁺ ) 514.2006 , found 514.1998.

Synthesis of N, N-dimethyl-5- (5- (tributylstannyl) benzofuran-2-yl) pyridin-2-amine (Compound 6) Using a reaction similar to that described above for the preparation of Compound 4, 88.9 mg Compound 6 was obtained from Compound 3 in 25.1% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 0.89 (t, 9H, J = 7.2 Hz), 1.04-1.12 (m, 6H), 1.30-1.39 (m, 6H), 1.53-1.60 (m, 6H) , 3.14 (s, 6H), 6.57 (d, 1H, J = 9.6 Hz), 6.79 (s, 1H), 7.29 (d, 1H, J = 8.0 Hz), 7.47 (d, 1H, J = 8.4 Hz) , 7.62 (s, 1H), 7.88 (dd, 1H, J ₁ = 9.6 Hz, J ₂ = 2.4 Hz), 8.67 (d, 1H, J = 2.4 Hz). HRMS (EI): m / z calcd for C ₂₇ H ₄₀ N ₂ OSn (M ⁺ ) 528.2162, found 528.2158.

5- (5-Iodobenzofuran-2-yl) pyridin-2-amine (Compound 7)
CHCl ₃ (5 mL) Compound 4 (100 mg, 0.20 mmol) in a solution of was added iodine solution in _{CHCl 3 (0.5 mL, 1 M} ) at room temperature. The mixture was stirred for 10 minutes at room temperature. NaHSO ₃ solution (3 ml, 5% in water) was added sequentially. After the mixture was stirred for 5 minutes, the organic phase was separated. The aqueous phase was extracted with CHCl ₃ and the combined organic phases were dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give 34.9 mg of compound 7 (52.0%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 4.66 (s, 2H), 6.57 (d, 1H, J = 8.4 Hz), 6.77 (s, 1H), 7.27 (s, 1H), 7.51 (d, 1H , J = 8.0 Hz), 7.84 (s, 1H), 7.85 (d, 1H, J = 2.4 Hz), 8.58 (d, 1H, J = 2.4 Hz) .HRMS (EI): m / z calcd for C ₁₃ H ₉ IN ₂ O (M ⁺ ) 335.9760, found 335.9752.

5- (5-Iodobenzofuran-2-yl) -N-methylpyridin-2-amine (Compound 8)
Using a reaction similar to that described above for the preparation of compound 7, 50.2 mg of compound 8 was obtained from compound 5 in 65.1% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 2.98 (d, 3H, J = 4.8 Hz), 4.79 (s, 1H), 6.45 (d, 1H, J = 8.8 Hz), 6.74 (s, 1H), 7.30 (s, 1H), 7.50 (d, 1H, J = 8.4 Hz), 7.85 (s, 1H), 7.87 (d, 1H, J = 2.4 Hz), 8.60 (d, 1H, J = 2.4 Hz). HRMS (EI): m / z calcd for C ₁₄ H ₁₁ IN ₂ O (M ⁺ ) 349.9916, found 349.9923.

5- (5-Iodobenzofuran-2-yl) -N, N-dimethylpyridin-2-amine (Compound 9)
Using a reaction similar to that described above for the preparation of compound 7, 32.8 mg of compound 9 was obtained from compound 6 in 45.6% yield. ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.14 (s, 6H), 6.55 (d, 1H, J = 8.8 Hz), 6.70 (s, 1H), 7.25 (d, 1H, J = 4.0 Hz), 7.47 (d, 1H, J = 8.4 Hz), 7.82 (s, 1H), 7.84 (d, 1H, J = 2.4 Hz), 8.65 (d, 1H, J = 2.4 Hz). HRMS (EI): m / z calcd for C ₁₅ H ₁₃ IN ₂ O (M ⁺ ) 364.0073, found 364.0065.

(3) Radioactive synthesis The radioactive iodine labeled body of the pyridyl benzofuran derivative was synthesize | combined from the corresponding tributyltin derivative. Briefly, to initiate the reaction, 100 μL H ₂ O ₂ (3%) was added to a tributyltin derivative (0.5 mg / 100 μL EtOH), 7.4 MBq [ ¹²³ I] or 0.74 MBq in a sealed vial. Of [ ¹²⁵ I], and 1 N of 100 μL HCl. The reaction was allowed to proceed for 10 minutes at room temperature and was terminated by the addition of NaHSO ₃ . After neutralizing with sodium bicarbonate, the reaction mixture was extracted with ethyl acetate. The extract was dried by passing through an anhydrous Na ₂ SO ₄ column and dried with a stream of nitrogen gas. Radiolabeled ligand was purified by HPLC.

(4) In vitro binding analysis using aggregated Aβ peptide in solution
Aβ (1-42) was purchased from Peptide Institute (Osaka, Japan). Aggregation was performed by gently dissolving the peptide (0.25 mg / ml) in a buffer (pH 7.4) containing 10 mM sodium phosphate and 1 mM EDTA. The solution was incubated for 42 hours at 37 ° C. with gentle constant shaking. Contains 50 μL iodinated pyridylbenzofuran derivative (0.008 pM-400 μM in 10% EtOH), 50 μL [ ¹²⁵ I] IMPY (0.02 nM), 50 μL Aβ (1-42) aggregates and 850 μL 10% EtOH The mixture was incubated for 3 hours at room temperature. The mixture was then filtered through Whatman GF / B filters using a Brandel M-24 cell harvester and the radioactivity of the filter containing bound ¹²⁵ I ligand was measured with a γ counter. The half-maximal inhibitory concentration (IC ₅₀ ) values were determined from the displacement curves of three independent experiments using GraphPad Prism 5.0, and for inhibition constants (Ki), the Cheng-Prusoff equation ( Cheng Y, Prusoff WH (1973) Biochem Pharmacol 22: 3099-3108): calculated using K _i = IC ₅₀ / (1+ [L] / K _d ), where [L] is used in the assay Concentration of [ ¹²⁵ I] IMPY, and Kd is the dissociation constant of IMPY (4.2 nM).

(5) Biodistribution in normal mice
Under 2% isoflurane anesthesia, 10% ethanol (100 μL) containing ¹²⁵ I-labeled pyridylbenzofuran derivative (37 kBq) was directly injected into the tail vein of ddY mice. Mice (n = 5 at each time point) were sacrificed at 2, 10, 30, and 60 minutes after injection. The target organ was removed and weighed, and the radioactivity was measured with an automatic gamma counter (COBRAII, Packard). The dose ratio for each organ was calculated by comparing the tissue values to appropriately diluted aliquots of the injection. The ratio of dose per gram of sample was calculated by comparing the sample value with the diluted initial dose value.

(6) SPECT / CT for small animals
SPECT and CT data were acquired and processed using a preclinical imaging system FX3300 (Gamma Medica) equipped with a multi-modality preclinical imaging platform FLEX Triumph (Gamma Medica). Tg2576 transgenic mice (28 months old, female) and wild type mice (28 months old, female) were used as models for Alzheimer's disease and age-matched controls, respectively. Compound [ ¹²³ I] 8 (20.5-26.5 MBq) in 10% EtOH aqueous solution containing 0.1% Tween80 was injected into the tail vein. Mice were anesthetized with isoflurane (2.0% isoflurane) 10 minutes after dosing and a 4-head detector camera was used to perform a tomographic spiral SPECT scan of the anesthetized mice. Acquisition parameters were as follows: rotation angle, 360 °; projection time, 90 seconds; number of projections, 32; collimator, multi-pinhole collimator (N5F75A10); turning radius, 35mm. Immediately after obtaining SPECT, CT of anesthetized mice was obtained. Acquisition parameters were as follows: gantry rotation in continuous flying mode; tube voltage, 61 kV; tube current, 305 μA; spot size, 50 μm; 2 × 2 binning and 1184 × 1120 projection matrix size, 360 ° With a complete scan, a total of 512 images / frame was acquired.

A modified 3D cone-beam felt kamp algorithm obtained with a voxel size of 0.177 x 0.177 x 0.177 mm (512 x 512 x 512 image volume) was used to reconstruct the CT projection. SPECT projection determination was processed for the generation of quantitative images. A 20% energy window centered at 159 keV was used for ¹²³ I acquisition. To reconstruct the SPECT projection, a 3D ordered subset expectation maximization algorithm (5 iterations, 8 subsets) that yields an 80x80x80 image matrix with a voxel size of 0.835x0.826x0.598 mm used. Amira's 3D data analysis and visualization software (Mercury Computer Systems) was used to perform image analysis.

(7) Ex vivo autoradiography of transgenic mouse brain Animals were killed by decapitation after SPECT analysis. The brain was immediately removed and frozen in a dry ice / hexane bath. A 20 μm section was cut out and exposed to a BAS imaging plate (Fuji Film, Tokyo, Japan) overnight. Ex vivo film autoradiogram was obtained. After autoradiographic examination, the same sections were stained with thioflavin-S to confirm the presence of Aβ plaques. For staining of thioflavin-S, sections were immersed in a 0.125% thioflavin-S solution containing 50% EtOH for 5 minutes and washed with 50% ethanol. After drying, the sections were examined using a microscope (Nikon Eclipse 80i) equipped with a B-2A filter set (excitation, 450-490 nm; dichroic mirror, 505 nm; long pass filter, 520 nm).

(8) In vitro autoradiography using human AD brain slices
Postmortem brain tissue from an AD autopsy confirmed case (93 years old, female) was obtained from Kyoto University Graduate School of Medicine. Serial sections of 6 micrometer thickness of paraffin embedded blocks were used for in vitro autoradiography. Sections were incubated with ¹²⁵ I-labeled tracer (444 kBq / 50 μL) for 1 hour at room temperature, then immersed in saturated Li ₂ CO ₃ in 40% EtOH (2 × 2 min wash), with 40% EtOH Washed (washed once for 2 minutes) and washed with water for 30 seconds. After drying, ¹²⁵ I-labeled sections were exposed to BAS imaging plates (Fuji Film, Tokyo, Japan) overnight. Autoradiographic images were obtained using a BAS5000 scanner system (Fuji Film). After autoradiographic examination, the same section was immunostained using an amyloid β protein immunohistochemical staining kit (Wako Pure Chemical Industries, Ltd.).

〔Experimental result〕
(1) Synthesis of pyridylbenzofuran derivatives An important step in the formation of pyridylbenzofuran skeleton was achieved by the Suzuki coupling reaction (Fig. 1) (Miyaura N, Yamada K, Suzuki A (1979) Tetrahedron Lett 36: 3437-3440 ). Suzuki coupling gave the desired compound 1-3 in 31.5%, 38.0%, and 37.2% yields, respectively. The bromo compound (1-3) is reacted with bis (tributyltin) using Pd (0) as a catalyst to give the corresponding tributyltin derivative (4-6) of 24.6%, 28.4%, and 25.1%, respectively. Obtained in yield. These tributyltin derivatives were immediately reacted with iodine in chloroform at room temperature to give iodine derivatives (7-9) in yields of 52.0%, 65.1%, and 45.6%, respectively. Furthermore, these tributyltin derivatives can also be used as starting materials for radioiodine labeling in the preparation of compound [ ^125I ] 7, compound [ ^123I / ^125I ] 8, and compound [ ^125I ] 9. A novel radioiodine labeled pyridylbenzofuran derivative was obtained by iodination destannyl reaction using hydrogen peroxide as an oxidant (FIG. 2). It was expected that carrier-free synthesis would be the final product with theoretical specific activity similar to ¹²⁵ I (81.4 TBq / mmol). The radiochemical identity of the radioiodine labeled ligands was confirmed from their HPLC profiles by co-injection with non-radioactive compounds by HPLC profile. Compound [ ¹²⁵ I] 7, Compound [ ¹²³ I / ¹²⁵ I] 8, and Compound [ ¹²⁵ I] 9 were> 99% radiochemical purity in 61-89% radiochemical yield after HPLC purification Was obtained.

(2) In vitro binding analysis to Aβ (1-42) aggregates Initial screening for the affinity of iodinated pyridylbenzofuran derivatives uses [ ¹²⁵ I] IMPY as a competitive radioligand and uses Aβ (1- 42) Aggregates (Cheng Y, Ono M, Kimura H, Kagawa S, Nishii R, et al. (2010) ACS Med Chem Lett 1: 321-325, Ono M, Haratake M, Saji H, Nakayama M (2008) Bioorg Med Chem Lett 16: 6867-6872).
These derivatives inhibited [ ¹²⁵ I] binding with Ki values in the nanomolar range (Ki = 2.4-10.3 nM). This indicates that they had excellent affinity for Aβ (1-42) aggregates (Table 2). The Ki values of IMPY and PIB as controls were 10.5 nM and 9.0 nM, respectively.

(3) In vivo biodistribution in normal mice The inventor of the present invention used radioactivity after intravenous injection of compound [ ^125I ] 7, compound [ ^125I ] 8, and compound [ ^125I ] 9 in normal mice. The in vivo distribution was determined (FIG. 3). All of these ¹²⁵ I-labeled derivatives passed through the blood-brain barrier (BBB) immediately after injection (4.14-4.67% ID / g at 2 minutes after injection) and radioactivity was removed from the brain over time (post-injection 1.30-3.69% ID / g in 60 minutes). Compound [ ¹²⁵ I] 8 reached a peak at 2 minutes after injection, and Compound [ ¹²⁵ I] 7 and Compound [ ¹²⁵ I] 9 reached a peak at 10 minutes after injection. Generally, the brain _2min / brain _60min ratio as an index is used to compare the outflow ratio from the brain. The ratio of brain _{2 min} / brain _{60 min of} compound [ ¹²⁵ I] 7, compound [ ¹²⁵ I] 8, and compound [ ¹²⁵ I] 9 was 1.30, 3.21 and 1.12. In addition to accumulation in the brain, radioactivity was also distributed to other organs such as the liver, kidney, stomach and intestine. The stomach and intestine showed radioactivity accumulation over time, while the liver and kidney showed high initial uptake and efflux over time (Figure 3).

(4) SPECT / CT for small animals
To demonstrate the feasibility of Aβ imaging in vivo, SPECT / CT was performed in Tg2576 transgenic and wild type mice after intravenous injection of compound [ ¹²³ I] 8. Due to significant Aβ deposition in the brain, Tg2576 transgenic mice have been widely used for evaluation of Aβ imaging agents in vitro and in vivo (Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, et al (1996) Science 274). A clear difference in radioactivity accumulation in the brain was observed between the Tg2576 mouse and the wild-type mouse in the SPECT image of the cross section (FIG. 4).

(5) Ex vivo autoradiography of transgenic mouse brain SPECT / CT for ex vivo autoradiography to further characterize the specific binding of compound [ ¹²³ I] 8 to Aβ plaques in the brain Immediately after the experiment, the brain was removed, frozen and sectioned (FIG. 5). Autoradiographic images showed extensive labeling of Aβ plaques in the brains of Tg2576 mice (FIG. 5A), but not in wild type controls (FIG. 5B). Aβ plaque labeling was confirmed by co-staining of the same section with thioflavin-S (a pathological dye commonly used for amyloid staining) (FIG. 5C).

(6) Autoradiography of postmortem AD brain sections
When AD brain sections were incubated with ¹²⁵ I-labeled pyridylbenzofuran, autoradiographic images revealed excellent visualization of Aβ plaques with minimal background noise (FIG. 6). No significant difference was observed in the accumulation of radioactivity in brain sections of AD between compound [ ¹²⁵ I] 8 and compound [ ¹²⁵ I] 9 (FIGS. 6B and 6C). However, compound [ ¹²⁵ I] 7 labeled Aβ plaques, but showed non-specific binding radioactivity in brain white matter (FIG. 6A). The radioactive spot derived from compound [ ¹²⁵ I] 8 in the cortex was confirmed by immunostaining of the same brain section with Aβ antibody (FIG. 6D).

[Example 2] Synthesis of pyridylbenzoxazole derivative and its application [Experimental method]
(1) Reagents and equipment
[ ¹²⁵ I] NaI used was Iodine-125 manufactured by MP Biomedical, Inc, and [ ¹²³ I] NH ₄ I used was manufactured by Nippon Mediphysics. Aβ (Human, 1-42) [TFA form] was purchased from Peptide Institute. Transgenic (Tg2576) mice and wild type mice were purchased from Taconic and used after breeding. As other reagents, commercially available general reagents or special grade reagents were used.

The medium pressure preparative liquid chromatography apparatus used was a pump PUMP 580D purchased from Yamazen Co., Ltd., an ultraviolet detector prep UV-254D, and a fraction collector Parallel Frac FR-260. A liquid chromatograph LC-20AD equipped with an ultraviolet-visible detector SPD-20A manufactured by Shimadzu Corporation was used for UV-visible spectroscopic analysis using high performance liquid chromatography (HPLC). In addition, NDW-101 manufactured by Aloka was used for radioactivity analysis using HPLC. As the column, a Cosmosil 5C ₁₈ -AR-II column (4.6 × 150 mm) manufactured by Nacalai Tesque was used.

  A microtome CM1900 manufactured by Leica was used for preparing frozen mouse brain sections, and BZ-9000 manufactured by KEYENCE was used for the fluorescence microscope.

¹ H NMR was measured using JEOL JNM-LM400 with tetramethylsilane as an internal standard substance. Mass spectrometry was measured using SHIMADZU GCMS-QP2010. For measurement of radioactivity, Wizard ² 1470 manufactured by PerkinElmer was used. For analysis of autoradiography, a BAS imaging plate and a BAS5000 scanner system manufactured by FujiFilm were used. Image collection by SPECT / CT was performed using GMI FX-3300 Pre-Clinical Imaging System, and OSEM was used for data analysis. Brandel M-24R cell harvester and Whatman GF / Bfilter were used for suction filtration in binding experiments using Aβ aggregates.

(2) Synthesis of pyridylbenzoxazole derivatives
Synthesis of 5- (5-bromobenzo [d] oxazol-2-yl) pyridin-2-amine (Compound 23)
2-Amino-4-bromophenol (376 mg, 2 mmol) and 5- (trifluoromethyl) pyridin-2-amine (486 mg, 3 mmol) are dissolved in 1 M aqueous sodium hydroxide solution (10 mL). The mixture was stirred for 3 hours while heating at 90 ° C. The precipitated solid was collected by suction to obtain 412 mg (yield: 71.2%) of Compound 23. ¹ H NMR (400 MHz, CDCl ₃ ) δ4.87 (s, 2H), 6.60 (d, J = 9.4 Hz, 1H), 7.42 (s, 1H), 7.84 (t, J = 1.1 Hz, 1H), 8.22 (dd, J = 8.7, 2.3 Hz, 1H), 8.94 (d, J = 1.8 Hz, 1H).

Synthesis of 5- (5-bromobenzo [d] oxazol-2-yl) -N-methylpyridin-2-amine (Compound 24) Compound 23 (184 mg, 0.64 mmol) and paraformaldehyde (115 mg, 3.84 mmol) It melt | dissolved in methanol (10 mL), the methanol solution (0.512 mL, 5 M) of sodium methoxide was added, and it recirculate | refluxed for 1 hour. After returning to room temperature, sodium borohydride (121 mg, 3.2 mmol) was slowly added with stirring and refluxed for 2 hours. After returning to room temperature and adding a saturated aqueous sodium hydrogen carbonate solution, the mixture was extracted with chloroform and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 1 as an elution solvent to obtain 150 mg (yield: 77.5%) of Compound 24. ¹ H NMR (400 MHz, CDCl ₃ ) δ3.03 (d, J = 5.3 Hz, 3H), 5.03 (s, 1H), 6.49 (d, J = 9.4 Hz, 1H), 7.41 (s, 2H), 7.83 (s, 1H), 8.21 (dd, J = 8.9, 2.5 Hz, 1H), 8.94 (d, J = 2.3 Hz, 1H).

Synthesis of 5- (5-bromobenzo [d] oxazol-2-yl) -N, N-dimethylpyridin-2-amine (Compound 25) Compound 23 (350 mg, 1.21 mmol) and paraformaldehyde (436 mg, 14.5 mmol ) Was dissolved in acetic acid (7 mL), sodium cyanoborohydride (608 mg, 9.68 mmol) was slowly added with stirring, and the mixture was stirred at room temperature for 21 hours. A saturated aqueous sodium hydrogen carbonate solution was added, the mixture was extracted with chloroform, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 1 as an elution solvent to obtain 109 mg (yield: 28.4%) of Compound 25. ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.87-1.66 (m, 27H), 3.20 (s, 6H), 6.60 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H) , 7.53 (d, J = 7.8 Hz, 1H), 7.81 (s, 1H), 8.23 (dd, J = 8.9, 2.5 Hz, 1H), 9.01 (d, J = 2.3 Hz, 1H).

Synthesis of 5- (5- (tributylstannyl) benzo [d] oxazol-2-yl) pyridin-2-amine (Compound 26) Compound 23 (320 mg, 1.11 mmol) was converted to 1,4-dioxane (10 mL) Bis (tributyltin) (1.1 mL), tetrakistriphenylphosphine palladium (509 mg, 0.44 mmol) and triethylamine (5 mL) were added and refluxed for 45 minutes. After evaporating the reaction solvent under reduced pressure, the residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 1 as an elution solvent to obtain 196 mg (yield: 35.3%) of Compound 26. ¹ H NMR (400 MHz, CDCl ₃ ) δ0.87-1.64 (m, 27H), 4.85 (s, 2H), 6.60 (d, J = 9.2 Hz, 1H), 7.38 (dd, J = 7.8, 0.9 Hz , 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.83 (s, 1H), 8.24 (dd, J = 8.7, 2.3 Hz, 1H), 8.96 (d, J = 1.6 Hz, 1H).

Synthesis of N-methyl-5- (5- (tributylstannyl) benzo [d] oxazol-2-yl) pyridin-2-amine (Compound 27) Compound 24 (345 mg, 1.14 mmol) was converted to 1,4-dioxane Dissolved in (10 mL), bis (tributyltin) (1.1 mL), tetrakistriphenylphosphine palladium (527 mg, 0.46 mmol), and triethylamine (5 mL) were added and refluxed for 45 minutes. After evaporating the reaction solvent under reduced pressure, the residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 1 as an elution solvent to obtain 161 mg (yield: 27.4%) of Compound 27. ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.87-1.60 (m, 27H), 3.02 (d, J = 5.3 Hz, 3H), 5.06 (s, 1H), 6.49 (d, J = 8.7 Hz, 1H) , 7.36 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.82 (s, 1H), 8.24 (dd, J = 8.7, 2.3 Hz, 1H), 8.97 (d, J = 2.1 Hz, 1H).

Synthesis of N, N-dimethyl-5- (5- (tributylstannyl) benzo [d] oxazol-2-yl) pyridin-2-amine (Compound 28) Compound 25 (55 mg, 0.17 mmol) was converted to 1,4 -Dissolved in dioxane (10 mL), bis (tributyltin) (0.17 mL), tetrakistriphenylphosphine palladium (78.6 mg, 0.07 mmol), and triethylamine (5 mL) were added and refluxed for 45 minutes. After evaporating the reaction solvent under reduced pressure, the residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 3 as an elution solvent to obtain 20.7 mg (yield: 23.0%) of Compound 28. ¹ H NMR (400 MHz, CDCl ₃ ) δ 0.87-1.66 (m, 27H), 3.20 (s, 6H), 6.60 (d, J = 9.2 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H) , 7.53 (d, J = 7.8 Hz, 1H), 7.81 (s, 1H), 8.23 (dd, J = 8.9, 2.5 Hz, 1H), 9.01 (d, J = 2.3 Hz, 1H).

Synthesis of 5- (5-iodobenzo [d] oxazol-2-yl) pyridin-2-amine (Compound 29) Compound 26 (195 mg, 0.39 mmol) was dissolved in chloroform (5 mL) and iodine in chloroform ( 1.5 mL, 0.2 M) was added, and the mixture was stirred at room temperature for 15 minutes. A saturated aqueous sodium hydrogen sulfite solution was added to stop the reaction, the chloroform layer was separated, dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 1 as an elution solvent to obtain 24.8 mg (yield: 18.9%) of Compound 29. ¹ H NMR (400 MHz, CDCl ₃ ) δ4.87 (s, 2H), 6.60 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 7.61 (dd, J = 10.9 , 1.8 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 8.22 (dd, J = 8.7, 2.1 Hz, 1H), 8.93 (d, J = 1.8 Hz, 1H) .MS m / z 337 (M ⁺ ).

Synthesis of 5- (5-iodobenzo [d] oxazol-2-yl) -N-methylpyridin-2-amine (Compound 30) Compound 27 (160 mg, 0.31 mmol) was dissolved in chloroform (5 mL) and iodine Of chloroform (1.5 mL, 0.2 M) was added, and the mixture was stirred at room temperature for 15 minutes. A saturated aqueous sodium hydrogen sulfite solution was added to stop the reaction, the chloroform layer was separated, dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 1 as an elution solvent to obtain 61.2 mg (yield: 56.2%) of Compound 30. ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.03 (d, J = 5.3 Hz, 3H), 5.01 (s, 1H), 6.49 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.2 Hz , 1H), 7.59 (dd, J = 8.5, 1.6 Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H), 8.21 (dd, J = 8.9, 2.3 Hz, 1H), 8.94 (d, J = 2.3 Hz, 1H). MS m / z 351 (M ⁺ ).

Synthesis of 5- (5-iodobenzo [d] oxazol-2-yl) -N, N-dimethylpyridin-2-amine (Compound 31: PBOX-3) Compound 28 (20 mg, 0.04 mmol) in chloroform (5 mL ), A solution of iodine in chloroform (1.5 mL, 0.2 M) was added, and the mixture was stirred at room temperature for 15 minutes. A saturated aqueous sodium hydrogen sulfite solution was added to stop the reaction, the chloroform layer was separated, dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative column chromatography using ethyl acetate: hexane = 1: 3 as an elution solvent to obtain 13.9 mg (yield: 100%) of Compound 31. ¹ H NMR (400 MHz, CDCl ₃ ) δ 3.20 (s, 6H), 6.60 (d, J = 8.9 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.57 (dd, J = 8.5, 1.4 Hz, 1H), 8.01 (d, J = 1.6 Hz, 1H), 8.19 (dd, J = 8.9, 2.5 Hz, 1H), 8.98 (d, J = 2.3 Hz, 1H) .MS m / z 365 ( M ⁺ ).

(3) Synthesis of ¹²⁵ I-labeled pyridylbenzoxazole derivatives [ ¹²⁵ I] NaI (3.7-7.4 MBq), 1 M HCl in ethanol solution (50 μL, 1 mg / mL) of the tributyltin compound, the labeling precursor of each compound (50 μL) and 3% (w / v) H ₂ O ₂ (50 μL) were added and reacted at room temperature for 5 minutes. Subsequently, a saturated sodium sulfite aqueous solution (100 μL) was added to stop the reaction, and a saturated sodium hydrogen carbonate aqueous solution (100 μL) was added to neutralize the reaction solution. The mixture was extracted by adding ethyl acetate, passed through a Pasteur pipette filled with anhydrous sodium sulfate and dehydrated, and then the solvent was distilled off by nitrogen stream. The radioactive iodine-labeled compounds were separated and purified by reversed-phase HPLC (water: acetonitrile = 4: 6-1: 1) using the corresponding non-radioactive compounds as standards.

(4) Synthesis of ¹²³ I-labeled PBOX-3 To an ethanol solution of compound 30 (50 mL, 1 mg / mL), [ ¹²³ I] NH ₄ I (148 MBq), 1 M HCl (50 mL), 3% (w / v) H ₂ O ₂ (50 mL) was added and allowed to react at room temperature for 15 minutes. Subsequently, a saturated sodium sulfite aqueous solution (100 μL) was added to stop the reaction, and a saturated sodium hydrogen carbonate aqueous solution (100 μL) was added to neutralize the reaction solution. The mixture was extracted by adding ethyl acetate, passed through a Pasteur pipette filled with anhydrous sodium sulfate and dehydrated, and then the solvent was distilled off by nitrogen stream. Radioactive iodine-labeled compounds were separated and purified by reversed-phase HPLC (water: acetonitrile = 4: 6) using the corresponding non-radioactive compounds as standards.

(5) Preparation of Aβ (1-42) aggregate
PBS (pH 7.4) was used to prepare Aβ (1-42) at a concentration of 0.25 mg / mL. An Aβ (1-42) aggregate solution was prepared by incubating at 37 ° C. for 42 hours. The aggregate solution was stored at −80 ° C. until used for experiments.

(6) calculating a K _i of by competitive inhibition experiments using Aβ aggregates
K _i was calculated in the same manner as in Example 1.

(7) In vitro autoradiography using brain slices from AD patients
Brain slices from AD patients were provided by Kyoto University Graduate School of Medicine (6 μm, 78 years old, female). Wash with xylene (10 minutes x 2), 100% EtOH (1 minute x 2), 90% EtOH, 80% EtOH, 70% EtOH wash (1 minute x 1) and purified water wash (30 seconds x 1) The deparaffinization process was performed. The compound was diluted to 15 kBq / mL with 10% EtOH solution. The prepared radioligand was dropped on the section and incubated for 1 hour. Then, it was washed twice with 50% EtOH for 1 minute. After fixing on a BAS imaging plate for 24 hours, analysis was performed using a BAS5000 scanner system.

(8) Immunostaining using anti-Aβ antibody An anti-Aβ (1-42) monoclonal antibody (clone No. BC05) was used as an antibody for immunostaining. After treatment with formic acid (90%), it was washed with purified water for 5 minutes. Further, the plate was washed with PBS-Tween 20 for 2 minutes and then incubated with a 0.05% trypsin solution for 15 minutes. The plate was washed twice with PBS-Tween 20 for 5 minutes and then incubated with blocking serum for 30 minutes. Thereafter, the cells were incubated overnight with an anti-Aβ (1-42) antibody at room temperature. The plate was washed with PBS-Tween 20 for 3 minutes for 2 minutes and incubated with a streptavidin biotin peroxidase complex solution for 3 hours. The plate was washed with PBS-Tween 20 for 3 minutes for 2 minutes and then incubated with a diaminobenzidine solution at room temperature for 10 minutes. It was washed with distilled water for 1 minute, and observed with a microscope after enclosing.

(9) In vivo radioactivity distribution experiment of ¹²⁵ I-labeled pyridylbenzoxazole derivatives in normal mice
[ ¹²⁵ I] -labeled product was diluted with 10% EtOH-containing physiological saline or 0.1% Tween 80 and 10% EtOH-containing physiological saline so as to be 19-37 kBq / mL. One group of five 5-week-old ddY male mice (20-25 g) was prepared by adding Compound [ ¹²⁵ I] 29, Compound [ ¹²⁵ I] 30 and Compound [ ¹²⁵ I] 30 prepared from 1.9-3.7 kBq (100 μL) from the tail vein. ¹²⁵ I] 31 (PBOX-3) was administered, sacrificed at 2, 10, 30, and 60 minutes, blood was collected, and the major organs were removed. Next, after immediately measuring the weight of blood and organs, the radioactivity was measured with a gamma counter.

(10) SPECT imaging and ex vivo autoradiography using Tg2576 mice
[ ¹²³ I] PBOX-3 solution (13-15 or 35-53 MBq) dissolved in physiological saline containing 0.1% Tween 80 and 10% EtOH was mixed with Tg2576 mice (female, 16 or 25 months old) and wild type mice ( Female, 16 or 25 months old) were administered via the tail vein. Five minutes after administration, the patient was placed under anesthesia by inhalation of 3% isoflurane. SPECT imaging was performed for 50 minutes from 10 minutes after administration, and then CT imaging (tube current: 310 mA, tube voltage: 60 kV) was performed. 100 minutes after administration, the mice were sacrificed, the brains were removed, embedded in SCEM, and frozen in a hexane bath. Serial sections with a thickness of 20 mM were prepared using a microtome, fixed on a BAS imaging plate for 12 hours, and then analyzed using a BAS5000 scanner system. The same section was stained with thioflavin S solution and observed with a fluorescence microscope.

〔Experimental result〕
(1) Synthesis of Pyridylbenzoxazole Derivative FIG. 7 shows a synthesis route of a pyridylbenzoxazole derivative. The synthesis of the pyridylbenzoxazole skeleton was performed according to a previously reported method (Qiao, JX; Wang, TC; Hu, C .; Li, J .; Wexler, RR; Lam, PY Org. Lett. 2011, 13 , 1804-7). 2-Amino-4-bromophenol and 5- (trifluoromethyl) pyridin-2-amine were reacted in a 1 M aqueous sodium hydroxide solution to obtain Compound 23 in a yield of 71%. Compound 23, in which monomethyl group is introduced by methylation of compound 23 using sodium borohydride, sodium methoxide and paraformaldehyde, and dimethylamino group is introduced by using sodium cyanoborohydride and paraformaldehyde. Compound 25 was obtained in 78% and 28% yields, respectively. Tributyltin derivative 26-28 was obtained from the corresponding bromo compound by Pd (0) -catalyzed bromo-tin exchange reaction (yield 23-35%). The synthesized tributyltin compound 26-28 and iodine were reacted at room temperature in a chloroform solvent to obtain the corresponding iodine compound 29-31 (yield 19-100%).

(2) Radioiodine labeling of pyridylbenzoxazole derivatives FIG. 8 shows ¹²⁵ I labeling and ¹²³ I labeling routes. The target compound [ ¹²⁵ I] 29, compound [ ¹²⁵ I] 30, compound [ ¹²⁵ I] 31 (PBOX-3) and compound [ ¹²³ I] 31 (PBOX-3) use H ₂ O ₂ as an oxidizing agent. And obtained with a radiochemical yield of 30-50% and a radiochemical purity of 95% or more. Identification of ¹²⁵ I-labeled substance and ¹²³ I-labeled substance was performed by reversed-phase HPLC using a corresponding non-radioactive compound.

(3) Examination of binding affinity of pyridylbenzoxazole derivatives to Aβ (1-42) aggregates In order to investigate the binding properties of various synthesized pyridylbenzoxazole derivatives to Aβ (1-42) aggregates, [ ¹²⁵ I] In vitro competitive inhibition experiments using IMPY as a competitive ligand were performed, inhibition experiments were prepared using GraphPad Prism 4.0, and IC ₅₀ was calculated (Table 3). The inhibition constant was calculated using the formula K _i = IC ₅₀ / (1+ [L] / K _d ).

As a result of the competitive inhibition experiment, Compound 29, Compound 30, and Compound 31 (PBOX-3) inhibited the binding of [ ¹²⁵ I] IMPY in a concentration-dependent manner. Therefore, any of the pyridylbenzoxazole derivatives was Aβ (1-42 ) It was found to have binding affinity for aggregates. In addition, there was a difference in binding properties depending on the type of substituent introduced. The binding properties improved in the order of primary, secondary and tertiary amines, and PBOX-3 showed the strongest binding affinity.

(4) Evaluation of brain migration, disappearance from the brain, and in vivo radioactivity distribution In vivo radioactivity distribution experiments using normal mice examined the uptake of pyridylbenzoxazole derivatives into the brain and subsequent clearance. . FIG. 9 shows the results of in vivo radioactivity distribution after administration of pyridylbenzoxazole derivatives to normal mice, and FIG. 10 shows the radioactivity distribution in the brain. Compound [ ¹²⁵ I] 29, Compound [ ¹²⁵ I] 30 and Compound [ ¹²⁵ I] 31 (PBOX-3) all showed high brain migration of 4.7-6.6% ID / g 2 minutes after administration. Further, the value in the brain at 60 minutes after administration was 0.4-1.4% ID / g, indicating that the pyridylbenzoxazole derivative is rapidly cleared after transfer into the brain. From this result, it was revealed that the pyridylbenzoxazole derivative improved the radioactivity behavior in the brain, which was a problem of the 2-phenylindole derivative.

(5) Examination of binding to senile plaques on AD patient brain slices
In vitro autoradiography using AD patient brain sections was performed to examine the binding to senile plaques accumulated on AD patient brain sections (FIG. 11). As a result, Compound 31 (PBOX-3) was found to accumulate a large amount of radioactivity on brain sections of AD patients. This site was consistent with the site stained by immunostaining with anti-Aβ antibody, indicating that PBOX-3 has binding to senile plaques accumulated on brain sections of AD patients. On the other hand, in the study using Compound 29, no radioactivity accumulation was observed on the brain section, and the radioactivity accumulation of Compound 30 was unclear. These results reflected the _Ki values calculated in in vitro competitive inhibition experiments.

(6) Examination of binding to amyloid plaques ex vivo
The compound 31 (PBOX-3) considered to have the most suitable properties as an Aβ imaging probe for SPECT was subjected to ex vivo autoradiography using Tg2576 mice and wild type mice (FIG. 12). Accumulation of amyloid plaques in the brain of Tg2576 mice begins at 12 months and is known to be noticeable at 23 months (Kawarabayashi, T .; Younkin, LH; Saido, TC; Shoji, M .; Ashe, KH; Younkin, SGJ Neurosci. 2001, 21, 372-81). In this study, 16 and 25 month old Tg2576 mice and wild type mice were used. On the brain section of 16-month-old Tg2576 mice, almost no staining site with thioflavin S was observed, and no difference in radioactivity accumulation from wild-type mice was observed even in ex vivo autoradiography. On the other hand, on the 25-month-old Tg2576 mouse brain section, a large number of radioactivity accumulation not observed on the wild-type mouse brain section was observed, and the accumulation area coincided with the fluorescent staining area of thioflavin S. From the above results, it was revealed that PBOX-3 has a binding property to amyloid plaques in the living body of the mouse although non-specific binding was observed in the brain.

(7) SPECT imaging
SPECT / CT imaging using 25-month-old Tg2576 mice and wild-type mice was performed, and the usefulness of 31 (PBOX-3) as an Aβ imaging probe for SPECT was evaluated. An arrow cross-section (Sagittal) and a transverse cross-section (transverse) image are shown in FIG. In the SPECT image of the brain of a 25-month-old Tg2576 mouse, higher radioactivity accumulation was observed compared to the brain of a wild-type mouse of the same age. From this result, it was revealed that amyloid plaques accumulated in the mouse brain can be imaged by SPECT imaging using PBOX-3.

[Example 3] Examination of binding properties of radioactive iodine-labeled pyridylbenzofuran derivative to amylin aggregate [Experimental method]
(1) Preparation of amylin aggregates The amylin aggregates were prepared with reference to Langmuir 2010, 26, 3453-3461.
Amylin (Human) (0.5 mg, Peptide Institute, 4219-v) was dissolved in a mixed solution of DMSO (100 μL), 20 mM Tris-HCl, 100 mM NaCl (pH 7.5) (900 μL). Further, it was diluted to 100 μM with 20 mM Tris-HCl and 100 mM NaCl (pH 7.5) to obtain a stock solution.

(2) Binding saturation experiment Compound [ ¹²⁵ I] 9 (EtOH solution) (500,000 cpm) and non-radioactive compound 9 (DMSO solution) (250 nM) were each diluted in order, 16,000-500,000 cpm, 7.8-250 nM To the solution. Glass tubes contain various concentrations of [compound [ ¹²⁵ I] 9 (50 μL), various concentrations of non-radioactive compound 9 solution (50 μL), amylin aggregates (final concentrations 25 nM, 50 μL), 10% DMSO An aqueous solution (850 μL) was added, and the mixture was allowed to stand at room temperature for 3 hours. In addition, non-specific binding was carried out in DMSO solution (50 μL) of non-radioactive compound 9 at a final concentration of 50 nM and 10% DMSO-containing aqueous solution (850 μL) in which thioflavin T (Wako) was dissolved to a final concentration of 10 mM. ) Was added. After standing, the reaction solution was permeated through a GF / B filter using an M-24 cell harvester. The radioactivity remaining on the filter was measured with a γ counter, and the obtained data was analyzed with GraphPad Prism to calculate the dissociation constant K _d .

(3) In vitro autoradiography using pancreas slices
10 μCi / 1 mL of compound [ ¹²⁵ I] 9 containing 10% EtOH in aqueous solution of pancreas from Type 2 diabetes (BioChain, Female, 69 years old) and pancreas from healthy subjects (BioChain, male, 71 years old) Each was added and allowed to stand at room temperature for 1 hour. Then, it was washed with an aqueous solution containing 50% EtOH (1 min x 2) and water (30 sec), exposed to an imaging plate (BAS-SR, Fuji Photo Film Co., Ltd.) for 3 hours, and then subjected to an image analyzer (Bio Imaging Analyzer BAS5000, Each autoradiogram was obtained using Fuji Film Co., Ltd. The same section was stained with thioflavin S (100 μM), washed, and observed with a fluorescence microscope.

〔Experimental result〕
(1) Bond saturation experiment Scatchard analysis was performed from the obtained saturation curve (Fig. 14), and the bond dissociation constant (Kd) was calculated. As a result, Kd = 8.11 nM, and the compound [ ¹²⁵ I] 9 was converted into amylin aggregates. It became clear that high binding property was shown.

(2) In vitro autoradiography using a pancreas section Compared with a healthy person's pancreas tissue section (FIG. 15 right), remarkable radioactivity accumulation was confirmed on the diabetic patient section (FIG. 15 left). This radioactivity accumulation coincided with the fluorescence image of thioflavin S, an amyloid stain, suggesting that compound [ ¹²⁵ I] 9 has binding properties to amyloid deposited in diabetic tissue.

  It can be used for the development of imaging reagents for Aβ for the purpose of AD diagnosis, support for the development of therapeutic agents targeting Aβ, and the determination of medical conditions using Aβ accumulation in AD patients as an index.

  This specification includes the contents described in the specification and / or drawings of the Japanese Patent Application (Japanese Patent Application No. 2013-38304) which is the basis of the priority of the present application. In addition, all publications, patents, and patent applications cited in this specification are incorporated herein by reference as they are.

Claims (9)

Formula (I)
[In the formula, any one of R ¹ , R ² , R ³ , and R ⁴ represents a radioactive iodine atom, the other represents a hydrogen atom, and R ⁵ , R ⁶ , R ⁷ , and R ⁸ are Independently, it represents a hydrogen atom, an amino group, a methylamino group, a dimethylamino group, a methoxy group, or a hydroxy group, and X represents CH or N. ]
Or a pharmaceutically acceptable salt of these compounds. A composition for diagnosing a conformational disease, comprising: The composition for diagnosing conformation disease according to claim 1, wherein R ³ in the general formula (I) is a radioactive iodine atom, and R ¹ , R ² , and R ⁴ are hydrogen atoms. The R ⁷ in the general formula (I) is an amino group, a methylamino group, or a dimethylamino group, and R ⁵ , R ⁶ , and R ⁸ are hydrogen atoms. Composition for diagnosis of conformational disease. The composition for diagnosing conformation disease according to any one of claims 1 to 3, wherein the radioactive iodine atom is ^123I or ^125I .   The composition for diagnosing conformation disease according to any one of claims 1 to 4, wherein the composition is used for single photon tomography.   The composition for diagnosing conformation disease according to any one of claims 1 to 5, wherein the conformation disease is Alzheimer's disease.   The conformation disease diagnosis composition according to any one of claims 1 to 5, wherein the conformation disease is type 2 diabetes.   The composition for diagnosis of conformation disease according to any one of claims 1 to 4 is administered to an animal, an image of a brain of the animal is taken, and the state of the compound represented by the general formula (I) in the image A method for diagnosing Alzheimer's disease, comprising diagnosing Alzheimer's disease based on the above.   A composition for diagnosis of conformation disease according to any one of claims 1 to 4 is administered to an animal, an image of the pancreas of the animal is taken, and the compound represented by the general formula (I) in the image is obtained. A method for diagnosing type 2 diabetes, comprising diagnosing type 2 diabetes based on a state.