HK1193815B - Compound having affinity for amyloid - Google Patents

Description

Compounds having affinity for amyloid

Technical Field

The present invention relates to compounds for diagnosing cerebral degenerative diseases. More specifically, the present invention relates to compounds useful for detecting amyloid at a focal site in the diagnosis of alzheimer's disease and other diseases associated with amyloid accumulation.

Background

Diseases caused by deposition of fibrous proteins called amyloid in various organs or tissues in the body are collectively called amyloidosis. A common feature of amyloidosis is that a fibrous protein called amyloid, rich in β -sheet structures, deposits systemically or locally at sites in various organs such that dysfunction is triggered in the organ or tissue.

Alzheimer's disease (hereinafter referred to as AD) is a typical amyloidosis disease, which is known to be a disease causing dementia. As deposits of amyloid accumulate in the brain, the disease is fatal and, therefore, is said to be a disease of greater social interest than other amyloidosis disorders. In recent years, the number of AD patients in advanced countries of aging society has increased dramatically, leading to social problems.

From a histopathological point of view, AD is characterized by three pathological findings in the brain, namely the development of senile plaques (senileplaques), the formation of neurofibrillary tangles (neurofibrillary neurons) and extensive neuronal loss. Senile plaques have a structure mainly composed of amyloid, which is said to be evident in the initial stages of the onset of AD, and thus, this pathological phenomenon is found in the brain about 10 years or more before clinical symptoms appear.

Diagnosis of AD involves the assessment of various cognitive functions (e.g., the hisaka dementia scale (hasegawa scale), ADAS-JCog, and MMSE) with the aid of a combination of image diagnoses, such as CT and MRI. However, the methods based on the evaluation of cognitive functions have a problem that the diagnostic sensitivity is low in the early stage of onset of disease, and the diagnostic result is susceptible to the innate cognitive function of an individual. Currently, when AD patients are still present, it is practically impossible to perform definitive diagnosis of AD because definitive diagnosis requires biopsy of the diseased part (non-patent document 1).

Meanwhile, a report indicates that amyloid constituting senile plaques is an aggregate of amyloid β protein (hereinafter referred to as a β). Meanwhile, many research reports indicate that a β aggregates form β -sheet structures that lead to neurocytotoxicity. Based on these findings, a so-called "amyloid interlock reaction hypothesis" has been proposed, which suggests that brain precipitation of a β causes downstream phenomena, i.e., formation of neurofibrillary tangles and neuronal loss (non-patent document 2).

Based on these facts, in recent years, in vivo detection of AD using a compound having a high affinity for amyloid as a marker has been attempted.

The probe used for image diagnosis of such intracerebral amyloid is mostly a hydrophobic low molecular weight compound which has high affinity with amyloid and high brain transferability, and uses various radionuclides such as¹¹C、¹⁸F and¹²³i, and the like. For example, the following compounds have been reported,¹¹c or radiohalogen labelled compounds include various thioflavine derivatives such as 6-iodo-2- [4' - (N, N-dimethylamino) phenyl]Benzothiazole (hereinafter referred to as TZDM) and 6-hydroxy-2- [4' - (N-methylamino) phenyl]Benzothiazole (hereinafter referred to as 6-OH-BTA-1) (patent document 1, non-patent document 3), stilbene compounds such as (E) -4-methylamino-4 ' -hydroxystilbene (hereinafter referred to as SB-13) and (E) -4-dimethylamino-4 ' -iodostilbene (hereinafter referred to as m-I-SB) (patent document 2, non-patent document 4, non-patent document 5), benzoxazole derivatives such as 6-iodo-2- [4' - (N, N-dimethylamino) phenyl ] stilbene]Benzoxazoles (hereinafter IBOX) and 6- [2- (fluoro) ethoxy]-2- [2- (2-dimethylaminothiazol-5-yl) ethenyl]Benzoxazole (non-patent document 6, non-patent document 7), DDNP derivative such as 2- (1- {6- [ (2-fluoroethyl) (methyl) amino group]-2-naphthyl } ethylene) malononitrile (hereinafter referred to as FDDNP) (patent document 4, non-patent document 8), and imidazopyridine derivatives such as 6-iodo-2- [4' - (N, N-dimethylamino) phenyl]Imidazo [1,2-a ]]Pyridine (hereinafter referred to as IMPY) (patent document 3, non-patent document 9), and the like; and a compound obtained by bonding a nitrogen-containing 5-membered aromatic heterocyclic group to an imidazopyridine-phenyl group via a carbon, with a radioactive halogenAnd the like (patent document 5, patent document 6). In addition, human body image studies have been conducted on some of these probes for image diagnosis, and it has been reported that there is significant accumulation of radioactivity in the brain of AD patients as compared with normal persons (non-patent document 10, non-patent document 11, non-patent document 12, non-patent document 13).

Documents of the prior art

Patent document

Patent document 1 Japanese Kokai publication Hei-2004-506723

Patent document 2 Japanese Kokai publication Hei-2005-504055

Patent document 3 Japanese Kokai publication No. 2005-512945

Patent document 4 Japanese Kohyo publication No. 2002-523383

Patent document 5 pamphlet of International publication No. 2007/063946

Patent document 6 pamphlet of International publication No. 2010/128595

Non-patent document

Nonpatent document 1 J.A.Hardy & G.A.Higgins, "Alzheimer's disease, the Amyloi dCascado Hypohesis", Science, 1992, 256, p.184-185

Non-patent document 2 G.McKhann et al, "Clinicaldianosis of Alzheimer's disease," report of VernonincDS-ADRDAWark Grouputhei Auguspericeps of department of Healthand HumanservicesTaskForceon Alzheimer's disease ", Neurology, 1984, 34, p.939-944

Non-patent document 3, Z. -P.Zhuang et al, "Radiodinated styrylbenzenzenesand thioflavinsasprobes for Amyloid doggergates", J.Med.chem., 2001, 44, p.1905-1914

Non-patent document 4 Masahirono et al, "11C-labeldstibenederivavisaas β -aggregate-specific PEstimagingmessorzaorer's disease", NuclearMedicineandbiology, 2003, 30, p.565-571

Non-patent document 5 "NovelStilbenesa Probesesforylidpalaques", H.F.Kung et al, J.American chemical society, 2001, 123, p.12740-12741

Non-patent document 6, Zhi-PingZHuang et al, "IBOX (2- (4' -methylenestyryl) -6-iodobensoxazole): aligndmegamydidplayerthybridain", NuclearMedicineandbiology, 2001, 28, p.887-894

Non-patent document 7, "11C ] BF-227," [11C ] ANew11C-Labeled 2-Ethylenebenzoxazoledirivativefor Amyloid-beta-platques imaging ", European journal of nucleic acids and molecular imaging, 2005, 32, Sup.1, P759

Non-patent document 8, "2-Dialkylamino-6-AcylmalononitrilSubstistitedNaphhalenes" (DDNPanalogs), "NovelDiagnostitalcutaneuttolToolszheimer" in EricD.Agdppa et al, molecular imaging and dBiology, 2003, 5, p.404-417

Non-patent document 9, Zhi-PingZHuang et al, "Structure-Activity relationship of Imidazo [1,2-a ] pyridine ligand for detecting. beta. -Amyloid Plaquesitin brain., J.Med.Chem, 2003, 46, p.237-243

Non-patent document 10 "imagingbrainamyloidin alzheimer's disease with pittsburgh co-extruded-b.", ann.neurol., 2004, 55, p.306-319

Nonpatent document 11 Nicolaasp.L.G.Verhoeff et al, "In-vivo imaging of Alzheimer disease. beta. -amyloid with [11C ] SB-13 PET.", American journal of GeriatroatricPsychiatry, 2004, 12, p.584-595

Non-patent document 12 HiroyukiArai et al, "[ 11C ] -BF-227ANDPETtoVisualizeAmyloidinAlzheimer ' S diseases Patients", Alzheimer ' S & Dementia, the journal of Alzheimer ' S Association, 2006, 2, Sup.1, S312

Non-patent document 13 ChristopherM Clark et al, "imagingAmyloidWithI 123IMPYSPECT," Alzheimer 'S & Dementia: the Journaroftheimer' S Assocination, 2006, 2, Sup.1, S342

Disclosure of Invention

Problems to be solved by the invention

As described above, various compounds have been disclosed as probes for image diagnosis of amyloid-based subjects, and clinical applications thereof have been sought. However, no compounds identified to date have the ability to tolerate clinical use. In addition, in view of wide clinical applications, it is desired to develop compounds having sufficient diagnostic performance not only with PET nuclides but also with SPECT nuclides.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel compound effective as an image diagnostic probe targeting amyloid, and an alzheimer's disease diagnostic agent containing the same.

Means for solving the problems

The present inventors have conducted extensive studies and, as a result, have found that a diagnostic agent for amyloidosis having sufficient diagnostic performance can be obtained by using a compound in which the 5-membered nitrogen-containing heterocycle is bonded to the carbon at the 4' -position of the phenyl group of the imidazopyridine-phenyl skeleton via the nitrogen atom of the nitrogen-containing heterocycle, and have completed the present invention.

According to one aspect of the present invention, there are provided a compound represented by the following formula (1) or a salt thereof and an Alzheimer's disease diagnostic agent containing the compound represented by the following formula (1) or a salt thereof,

in the formula (1), R¹Is a radioactive halogen substituent. As R¹Various radioactive halogens can be used, and those selected from the group consisting of¹⁸F、⁷⁶Br、¹²³I、¹²⁴I、¹²⁵I and¹³¹the radioactive halogen in I can be more preferably used¹⁸F or¹²³I。

A¹And A²Each independently is CH or N.

Therefore, according to a preferred embodiment of the present invention, there can be provided a compound represented by the following formula (3), (4) or (5) or a salt thereof and an alzheimer's disease diagnostic agent containing the compound represented by the formula (3), (4) or (5) or a salt thereof.

According to another aspect of the present invention, there is provided a compound represented by the following formula (2) or a salt thereof,

in the formula (2), R²Is a group selected from a non-radioactive halogen substituent, a nitro group, a trialkylammonium group having an alkyl chain of 1 to 4 carbon atoms, a trialkylstannyl group having an alkyl chain of 1 to 4 carbon atoms or a triphenylstannyl group, A³And A⁴Each independently is CH or N.

The compound represented by the formula (2) can be preferably used as a labeling precursor of the compound represented by the above formula (1).

As non-radioactive halogen substituents, it is possible to use: halogen that can be a target in nucleophilic substitution reaction using radioactive fluorine or halogen that can be a target in isotope exchange reaction with radioactive iodine, and chlorine, iodine, or bromine can be preferably used. As the trialkylstannyl substituent, various substituents can be used, and trimethylstannyl and tributylstannyl substituents can be preferably used.

Therefore, according to a preferred embodiment of the present invention, there is provided a compound represented by the following formula (6), (7) or (8).

Effects of the invention

According to the present invention, a novel compound having affinity for amyloid and having good amyloid developability in vivo, and a diagnostic agent for alzheimer's disease can be obtained.

Drawings

FIG. 1 is a synthetic route for 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine.

FIG. 2 is a scheme for the synthesis of 2- [4- (1H-pyrrol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine.

FIG. 3 is the use [ alpha ]¹²³I]-6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl]Imidazo [1,2-a ]]Autoradiograms of brain sections of AD patients with pyridine.

FIG. 4 is the use [ alpha ], [ beta ], [ alpha ]¹²³I]-6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl]Imidazo [1,2-a ]]Autoradiograms of brain sections of AD patients with pyridine.

FIG. 5 is the use [ alpha ], [ beta ], [ alpha ]¹²³I]Autoradiograms of brain sections of AD patients from IMPY.

Figure 6 is immunostaining of brain sections of AD patients using anti-amyloid antibodies.

Detailed Description

(method for synthesizing precursor Compound of Radioactive halogen-labeled Compound)

The method for synthesizing a precursor compound of a radiohalogen-labeled compound according to one embodiment of the present invention will be described below with reference to 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine as an example. This compound is a compound preferably used as a precursor compound of the radioiodinated compound of the present invention.

A synthetic route for 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine is shown in FIG. 1. In the synthesis of 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine, 2- (4-azidophenyl) -6-iodoimidazo [1,2-a ] pyridine is synthesized by first reacting 4-azidophenacyl bromide with 2-amino-5-iodopyridine (FIG. 1, step 1). The reaction at this time can be carried out according to a conventional method, for example, a method described in the literature (Zhi-PingZhuang et al, J.Med.chem, 2003, 46, p.237-243).

Then, the 6-iodo-2- (4-azidophenyl) imidazo [1,2-a ] pyridine synthesized as described above is reacted with trimethylsilylacetylene by a known method (for example, the method described in James T. Fletcher et al, tetrahedron Lett, 2008, 49, p.7030-7032) to give 6-iodo-2- [4- (4-trimethylsilyl-1H-1, 2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine (FIG. 1, step 2), then, the trimethylsilyl group was removed (FIG. 1, step 3) to give 6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine.

Then, the obtained 6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine is reacted with bistributyltin by a known method (for example, the method described in the document Zhi-PingZHuang et al, J.Med.Chem, 2003, 46, p.237-243) (FIG. 1, step 4) and purified to obtain the desired 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine.

In the case of obtaining a compound in which the substituent at the 6-position in the imidazopyridine ring is a trialkylstannyl substituent other than the tributylstannyl substituent, various kinds of bistrialkyltin may be used depending on the purpose, instead of bistriabutyltin in step 4 of fig. 1. For example, when a compound in which the substituent at the 6-position is a trimethylstannyl substituent is synthesized, the same reaction as described above may be carried out using bistrimethyltin in step 4 of fig. 1.

In addition, other precursor compounds of the present invention can also be synthesized by using commonly available starting materials and combining reactions well known to those skilled in the art. For example, a compound in which the substituent at the 6-position in the imidazopyridine ring is a nitro group can be synthesized in a known manner using 2-amino-5-nitropyridine instead of 2-amino-5-iodopyridine in step 1 of fig. 1. In addition, A in the above formula (2)³And A⁴Compounds that are both CH can be synthesized according to the procedure of FIG. 1 above by using 4- (1H-pyrrol-1-yl) phenacyl bromide in place of 4-azidophenacyl bromide in step 1 of FIG. 1 and omitting step 3 of FIG. 1. In addition, A in the above formula (2)³Is CH and A⁴The compound that is N can be synthesized according to the procedure of fig. 1 described above by using 4- (1H-imidazol-1-yl) phenacyl bromide in place of 4-azidophenacyl bromide in step 1 of fig. 1 and omitting step 3 of fig. 1.

(method for synthesizing a radiohalogen-labeled Compound)

Next, a method for producing a radioactive halogen-labeled compound according to another aspect of the present invention will be described with reference to a radioactive iodine-labeled compound as an example.

The synthesis of the radioiodinated compound can be carried out by: the labeling precursor compound synthesized in the above-mentioned manner is dissolved in an inert organic solvent, and [2 ] obtained by a known method is added thereto¹²³I]Sodium iodide solution, etc., and an acid and an oxidizing agent are added to react them. As dissolution-labelled precursor compoundsThe inert organic solvent used in (1), and the labeling precursor and [2 ]¹²³I]Sodium iodide and the like, and acetonitrile can be preferably used.

As the acid, various acids can be used, and hydrochloric acid is preferable.

The oxidizing agent is not particularly limited as long as it can oxidize iodine in the reaction solution, and hydrogen peroxide or peracetic acid can be preferably used. The amount of the oxidizing agent to be added may be an amount sufficient for oxidizing iodine in the reaction solution.

A radioactive halogen label other than iodine can be synthesized by labeling a labeled precursor according to the purpose of synthesis with a radioactive halogen according to the purpose. For example, in Synthesis [ alpha ], [¹⁹F]-6-fluoro-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl]Imidazo [1,2-a ]]In the case of pyridine, 6-nitro-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl group as a labeling precursor]Imidazo [1,2-a ]]Pyridine with [2 ] in the presence of a phase transfer catalyst and potassium carbonate¹⁸F]And (3) reacting fluorine ions.

(method of preparing and Using the diagnostic agent of the present invention)

The diagnostic agent of the present invention can be prepared in the same manner as other conventionally known radioactive diagnostic agents as a liquid in which the radioactive halogen-labeled compound of the present invention is mixed with water or physiological saline adjusted to an appropriate pH as desired, or a green's solution or the like. The concentration of the compound in this case is required to be set to a concentration at which stability of the compound contained therein can be obtained or less. The amount of the compound to be used is not particularly limited as long as it is a concentration sufficient for imaging the distribution of the administered drug. For example, in use¹²³I labeled compounds and¹⁸in the case of the F-labeled compound, the compound can be administered intravenously or topically at a rate of about 50 to 600MBq per adult weighing 60 kg. The distribution of the administered agent can be visualized by known methods, e.g. in use¹²³In the case of I-labeled compounds, imaging can be performed using a SPECT apparatus, and¹⁸in the case of F-labeled compounds, PET devices may be usedImaging is performed.

The present invention will be described in more detail by way of examples, comparative examples and reference examples, but the present invention is not limited to these. Note that, in the following examples, the names of the respective compounds used in the experiments are as defined in table 1.

Table 1 compound names of evaluation compounds used in examples

Example 1 Synthesis of 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine (FIG. 1)

218mg (equivalent to 0.909 mmol) of 4-azidophenacyl bromide and 200mg (equivalent to 0.909 mmol) of 2-amino-5-iodopyridine were dissolved in 1.0mL of acetonitrile and heated in an oil bath at 80 ℃ for 3 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and the precipitate was filtered off, then, washed with acetonitrile and dried under reduced pressure. The obtained crude crystal was suspended in a mixture of 3mL of water and 3mL of methanol, and then, about 4mL of a saturated sodium bicarbonate solution was added thereto and shaken in an ultrasonic washer for 5 minutes. The precipitate was filtered off from the resulting mixture, washed well with water and dried under reduced pressure to give 214mg (equivalent to 0.593 mmol) of 2- (4-azidophenyl) -6-iodoimidazo [1,2-a ] pyridine (FIG. 1, step 1).

Using an NMR apparatus: JNM-ECP-500 (manufactured by Japan electronic Co., Ltd.)

¹H-NMR (solvent: deuterated chloroform, resonance frequency: 500 MHz): 8.89 (s, 1H), 8.31 (s, 1H), 7.99 (d, J =8.7Hz, 2H), 7.42 (s, 1H), 7.19 (d, J =8.7Hz, 2H).

214mg (equivalent to 0.593 mmol) of the obtained 2- (4-azidophenyl) -6-iodoimidazo [1,2-a ] pyridine was dissolved in 3.0mL of dimethylformamide, 0.164mL (equivalent to 1.18 mmol) of trimethylsilylacetylene was added, and then 29.6mg (equivalent to 0.118 mmol) of copper sulfate pentahydrate was added, and the mixture was stirred with heating at 80 ℃ for 3 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, 5mL of water was added thereto, and the precipitated solid was filtered and washed with water thoroughly. The resulting solid material was dried under reduced pressure to obtain 137mg of crude crystals of 6-iodo-2- [4- (4-trimethylsilyl-1H-1, 2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine (FIG. 2, step 2).

137mg of the obtained crude crystals of 6-iodo-2- [4- (4-trimethylsilyl-1H-1, 2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine were suspended in 3.0mL of tetrahydrofuran, and 0.3mL of a 1.0mol/L tetrahydrofuran solution of tetrabutylammonium fluoride was added. After stirring under heating reflux for 4 hours, the reaction was cooled to room temperature, and the precipitate was filtered off, washed with tetrahydrofuran and diethyl ether and dried under reduced pressure. The obtained crude crystal was suspended in a mixture of 2.0mL of methanol, and then, about 3.0mL of a saturated sodium bicarbonate solution was added thereto, followed by shaking in an ultrasonic washer for 15 minutes. The precipitate was filtered off from the resulting mixture, washed well with water and dried under reduced pressure to give 97.4mg (equivalent to 0.252 mmol) of 6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine (4) (FIG. 1, step 3).

Using an NMR apparatus: JNM-ECP-500 (manufactured by Japan electronic Co., Ltd.)

¹H-NMR (solvent: deuterated chloroform, resonance frequency: 500 MHz): 8.92 (s, 1H), 8.84 (d, J =0.9Hz, 1H), 8.41 (s, 1H), 8.15 (d, J =8.7Hz, 2H), 7.98 (d, J =8.7Hz, 2H), 7.96 (d, J =0.9Hz, 1H), 7.45 (s, 1H).

50mg (equivalent to 0.129 mmol) of 6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] imidazo [1,2-a ] pyridine are dissolved in 2.0mL of dioxane, 0.5mL of triethylamine is added, followed by 0.129mL (equivalent to 0.258 mmol) of bistributyltin and 14.9mg (catalytic amount) of tetrakis (triphenylphosphine) palladium. The reaction mixture was stirred at 100 ℃ for 16 hours, the solvent was then distilled off under reduced pressure, and the residue was purified by flash silica gel column chromatography (eluent: hexane/ethyl acetate 2/1) to give the desired 2- [4- (1H-1,2, 3-triazol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine in an amount of 43mg (equivalent to 0.078 mmol) (fig. 1, step 4).

Using an NMR apparatus: JNM-ECP-500 (manufactured by Japan electronic Co., Ltd.)

¹H-NMR (solvent: deuterated chloroform, resonance frequency: 500 MHz): 8.12 (d, J =8.7Hz, 2H), 8.04 (d, J =1.2Hz, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.87 (d, J =1.2Hz, 1H), 7.82 (d, J =8.7Hz, 2H), 7.61 (d, J =8.7Hz, 1H), 7.20 (d, J =8.7Hz, 1H), 1.64-1.49 (m, 6H), 1.36 (tt, J =7.3, 7.3Hz, 6H), 1.20-1.06 (m, 6H), 0.91 (t, J =7.3Hz, 9H).

(example 2 [ ]¹²³I]-6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl]Imidazo [1,2-a ]]Synthesis of pyridine (Compound 1)

2- [4- (1H-1,2, 3-triazol-1-yl) phenyl synthesized in example 1 at 90. mu.L]-6-tributylstannyl imidazo [1, 2-a)]170. mu.L of hydrochloric acid 1mol/L and 674MBq [ alpha ], [ beta ] -pyridine/1 mol/L of acetonitrile¹²³I]60 μ L of sodium iodide and 10 μ L of 30% (W/V) hydrogen peroxide. The mixture was allowed to stand at 40 ℃ for 10 minutes, and then subjected to HPLC under the following conditions to thereby obtain a product of "HPLC¹²³I]-6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl]Imidazo [1,2-a ]]Pyridine fraction.

HPLC conditions:

column: YMCPackProC8 (trade name, manufactured by YMC Co., Ltd., size: 4.6X 150 mm)

Mobile phase: 0.1% trifluoroacetic acid/acetonitrile 20/80 → 0/100 (20 min)

Flow rate: 1.0 mL/min

A detector: ultraviolet visible absorption photometer (detection wavelength: 260 nm) and radiation detector (Raytest, STEFFI model)

10mL of water was added to this fraction, and the resulting liquid was passed through a Sep-PakC18 column (trade name: Sep-Pak (registered trade name) LightC18Cartridges, manufactured by Waters Corp., filler filling amount: 130 mg) to be adsorbed and collected¹²³I]-6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl]Imidazo [1,2-a ]]Pyridine. The column was washed with 1mL of water, and then 1mL of diethyl ether was introduced to elute¹²³I]-6-iodo-2- [4- (1H-1,2, 3-triazol-1-yl) phenyl]Imidazo [1,2-a ]]Pyridine. The radioactivity obtained was 134.5MBq immediately after the synthesis. Further, as a result of TLC analysis under the following conditions, the radiochemical purity was 99.5%.

TLC analysis conditions:

TLC plate: TLC plate: silica gel 60F254 (trade name, manufactured by Merck)

Developing phase: chloroform/methanol/diethylamine-100/1/2

A detector: ritasar (trade name, Raytest Co., Ltd.)

Example 3 Synthesis of 2- [4- (1H-pyrrol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine

110mg (equivalent to 0.594 mmol) of 4- (1H-pyrrol-1-yl) acetophenone were dissolved in 3mL of dichloromethane and 248. mu.L of triethylamine, and then 154. mu.L (equivalent to 1.19 mmol) of trimethylbromosilane was added dropwise under ice cooling. After stirring overnight at room temperature under an argon atmosphere, the reaction mixture was washed with water and saturated brine, and dried over magnesium sulfate. The solvent was distilled off, and the resulting residue was dissolved in 3.0mL of tetrahydrofuran, and 106mg (equivalent to 0.594 mmol) of N-bromosuccinimide was added thereto and stirred at room temperature for 30 minutes. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by flash silica gel column chromatography (eluent: hexane/ethyl acetate 7/1) to give 120mg (equivalent to 0.454 mmol) of 4- (1H-pyrrol-1-yl) phenacyl bromide (fig. 2, step 1).

Using an NMR apparatus: JNM-ECP-500 (manufactured by Japan electronic Co., Ltd.)

¹H-NMR (solvent: deuterated chloroform, resonance frequency: 500 MHz): 8.07 (d, J =8.5Hz, 2H), 7.70 (d, J =8.5Hz, 2H), 7.19-7.18 (m, 2H), 6.41-6.40 (m, 1H), 4.44 (s, 2H).

120mg (equivalent to 0.454 mmol) of 4- (1H-pyrrol-1-yl) phenacyl bromide and 99.9mg (equivalent to 0.454 mmol) of 2-amino-5-iodopyridine were dissolved in 2.0mL of acetonitrile and dry-distilled under heating for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and the precipitate was filtered off, then, washed with acetonitrile and dried under reduced pressure. The obtained crude crystal was suspended in a mixture of 10mL of water and 10mL of methanol, and then about 20mL of a saturated sodium bicarbonate solution was added thereto, followed by shaking in an ultrasonic washer for 20 minutes. The precipitate was filtered off from the resulting mixture, washed well with water and dried under reduced pressure to give 120mg (equivalent to 0.312 mmol) of 6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl ] imidazo [1,2-a ] pyridine (FIG. 2, step 2).

Using an NMR apparatus: JNM-ECP-500 (manufactured by Japan electronic Co., Ltd.)

¹H-NMR (solvent: deuterated chloroform, resonance frequency: 500 MHz): 8.90 (s, 1H), 8.34 (s, 1H), 8.02 (d, J =8.7Hz, 2H), 7.65 (d, J =8.7Hz, 2H), 7.45-7.41 (m, 3H), 6.19 (brs, 1H).

50mg (equivalent to 0.129 mmol) of 6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl ] imidazo [1,2-a ] pyridine are dissolved in 2.0mL of dioxane, after addition of 0.5mL of triethylamine, 0.130mL (equivalent to 0.258 mmol) of bistributyltin and 15.0mg (catalytic amount) of tetrakis (triphenylphosphine) palladium are added. The reaction mixture was stirred at 100 ℃ for 16 hours, the solvent was then distilled off under reduced pressure, and the residue was purified by flash silica gel column chromatography (eluent: hexane/ethyl acetate: 2/1) to give 52mg (equivalent to 0.095 mmol) of 2- [4- (1H-pyrrol-1-yl) phenyl ] -6-tributylstannyl imidazo [1,2-a ] pyridine (fig. 2, step 3).

Using an NMR apparatus: JNM-ECP-500 (manufactured by Japan electronic Co., Ltd.)

¹H-NMR (solvent: deuterated chloroform, resonance frequency: 500 MHz): 8.02-8.00 (m, 3H), 7.83 (s, 1H), 7.60 (d, J =8.7Hz, 1H), 7.46 (d, J =8.7Hz, 2H), 7.18-7.14 (m, 3H), 6.37-6.36 (m, 2H), 1.62-1.50 (m, 6H), 1.36 (tt, J =7.3, 7.3Hz, 6H), 1.19-1.06 (m, 6H), 0.91 (t, J =7.3Hz, 9H).

(example 42 [ ]¹²³I]-6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl]Imidazo [1,2-a ]]Synthesis of pyridine (Compound 2)

2- [4- (1H-pyrrol-1-yl) phenyl synthesized in example 3]-6-tributylstannyl imidazo [1, 2-a)]To 200. mu.L of an acetonitrile solution of pyridine (concentration: 1 mg/mL) were added 200. mu.L of 1mol/L sulfuric acid, 12. mu.L of 1mmol/L aqueous sodium iodide solution, and 1243MBq¹²³I]170 μ L of sodium iodide, 20 μ L of 30% (W/V) hydrogen peroxide. The mixture was allowed to stand at 40 ℃ for 10 minutes, and then subjected to HPLC fractionation under the following conditions¹²³I]-6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl]Imidazo [1,2-a ]]Pyridine fraction.

HPLC conditions:

column: YMCPackProC8 (trade name, manufactured by YMC Co., Ltd.; Standard: 4.6X 150 mm)

Mobile phase: 0.1% trifluoroacetic acid/acetonitrile 20/80 → 0/100 (20 min)

Flow rate: 1.0 mL/min

A detector: ultraviolet visible absorption photometer (detection wavelength: 260 nm) and radiation detector (Raytest, STEFFI model)

10mL of water was added to this fraction, and the resulting liquid was passed through a Sep-PakC18 column (trade name: Sep-Pak (registered trade name) LightC18Cartridges, manufactured by Waters Corp., filler filling amount: 130 mg) to be adsorbed and collected¹²³I]-6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl]Imidazo [1,2-a ]]Pyridine. The column was washed with 1mL of water, and then 1mL of diethyl ether was introduced to elute¹²³I]-6-iodo-2- [4- (1H-pyrrol-1-yl) phenyl]Imidazo [1,2-a ]]Pyridine. The radioactivity obtained was 235MBq immediately after the synthesis. Further, as a result of TLC analysis under the following conditions, the radiochemical purity was 98.4%.

TLC analysis conditions:

TLC plate: TLC plate: silica gel 60F₂₅₄(trade name, manufactured by Merck)

Developing phase: chloroform/methanol/diethylamine-100/1/2

A detector: ritasar (trade name, Raytest Co., Ltd.)

(reference example 12 [ ]¹²³I]Synthesis of IMPY

Synthesized for determining logP according to the following procedure_{Octanol (I)}And [2 ] used in comparative example for evaluating accumulation in the brain¹²³I]-IMPY。

Synthesis of 2- [4' - (N, N-dimethylamino) phenyl ] according to the method described in the literature (Zhi-PingZhuang et al, J.Med.chem., 2003, 46, p.237-243)]-6-tributylstannyl imidazo [1, 2-a)]Pyridine, dissolved in acetonitrile (concentration: 1 mg/mL). To 50. mu.L of the solution was added 2mol/L of hydrochloric acid (50. mu.L, 1075 MBq)¹²³I]80 μ L of sodium iodide, 23 μ L of 1mmol/L sodium iodide solution, and 15 μ L of 30% (W/V) hydrogen peroxide. The mixture was allowed to stand at 40 ℃ for 10 minutes and then subjected to HPLC under the same conditions as in example 2 to give [2 ], ]¹²³I]-an IMPY fraction.

10mL of water was added to this fraction, and the resulting liquid was passed through a Sep-PakC18 column (trade name: Sep-Pak (registered trade name) LightC18Cartridges, manufactured by Waters Corp., filler filling amount: 130 mg) to be adsorbed and collected¹²³I]-IMPY. The column is washed with 1mL of water, and then eluted with 1mL of diethyl ether¹²³I]-IMPY. The radioactivity obtained was 170MBq immediately after the synthesis. Further, TLC analysis was carried out under the same conditions as in example 2,as a result, the radiochemical purity was 98.5%.

Example 5 determination of partition coefficient based on octanol extraction

Measurement of partition coefficient by octanol extraction (hereinafter referred to as logP)_{Octanol (I)}) It is generally known as an index of the permeability of a compound through the blood-brain barrier (hereinafter referred to as BBB). .

(method)

Preparing Compound 1 and Compound 2 to about 1MBq/mL with a water-saturated 1-octanol solution, adding 30. mu.L to an equilibration vessel, adding water-saturated 1-octanol and 1-octanol saturated water to each equilibration vessel to 200. mu.L, 400. mu.L, or 800. mu.L, respectively, stirring the equilibration vessels, shaking for 5 minutes (20 to 25. + -. 2 ℃ C., 20 revolutions/minute), centrifuging each mixture (23 ℃ C., 3000g × 20 minutes) with a centrifuge (model: T2-MC, manufactured by BECKMAN), then, sampling 50. mu.L each of water-saturated 1-octanol and 1-octanol saturated water, measuring a radioactivity count with an AutowellGamma system (model: ARC-7001, manufactured by Aloka), calculating a logP according to the following formula (1) using the measured radioactivity count_{Octanol (I)}。

(results)

The results are shown in Table 2. logP of Compound 1 and Compound 2_{Octanol (I)}The values were 1.98 and 2.45, respectively. The best logP of compounds in BBB permeability is known_{Octanol (I)}The values ranged between 1-3 (Douglas D.Dischino et al, J.Nucl.Med., (1983), 24, p.1030-1038). The above results show that compound 1 and compound 2 have BBB permeability.

TABLE 2 logP of the Compounds of the invention_{Octanol (I)}Value of

Compound (I)	logPOctanol (I)Value of
		Compound 1	1.98
Compound 2	2.45

Example 6 calculation of dissociation constant (hereinafter referred to as K) obtained from binding experiment using brain tissue of Alzheimer's disease (hereinafter referred to as AD) patient_d) And maximum number of bonds (hereinafter referred to as B)_max）

This was performed using a homogenate of gray matter of brains of AD patients prepared from brain tissues (frontal tablets) of AD patients sold by analytical biologicalcalcalcalevices, Inc. (USA).

(method)

A mixed solution of compound 1 (about 35 kBq/100. mu.L) and compound 3 (62.5 nmol/L) was diluted with 5mmol/L phosphate-buffered saline containing 0.1% bovine serum albumin (hereinafter referred to as BSA) to prepare 0.2nmol/L to 25nmol/L in the reaction solution, 100. mu.L of 5mmol/L phosphate-buffered saline containing 0.1% BSA, 100. mu.L of the prepared mixed solution of compound 1 and compound 3 were added to each well of a 96-well microplate, and then, 50. mu.L of AD patient gray homogenate was added to start the reaction.the reaction solution was shaken for 3 hours (22 ℃ C., 400 rpm), the reaction solution was filtered using a glass fiber filter (Multiscreen SFB, manufactured by Millipore corporation). the filtered filter was washed with 5mmol/L phosphate-buffered saline containing 0.1% BSA (200. mu.L × 3 times), and then, Gaowema ARC: (7001: 7001 model: 7001) was used to wash the filtered filter with 5mmol/L phosphate-buffered saline containing 0.1% BSA (model: 7003)Manufactured by stew) the radioactivity remaining in the filter was measured. For non-specific binding, 6-OH-BTA-1 (synthesized according to the method described in "C.A. Mathis et al, J.Med.Cem., (2003), 46, p.2740") was added to the reaction solution to 1. mu. mol/L, and the reaction solution was counted and expressed in the same manner. The obtained counts were analyzed by GraphPadPrismVer.5 (GraphPad software Co., Ltd.) to calculate binding parameters (K)_d，B_max）。

(results)

Compound 1 exhibits K_d：4.94nmol/L、B_max: 2242fmol/mg protein. This result suggests that compound 1 has high binding activity to amyloid aggregates in the brain of AD patients.

Example 7 measurement of transferability and washability in brain

The change with time of radioactive accumulation in the brain of male Wistar rats (8 weeks old) was measured using compound 1 and compound 2.

(method)

Each of the compounds 1 and 2 was dissolved in a physiological saline solution containing 50mmol/L of L-cysteine hydrochloride to prepare a sample solution (each radioactive concentration was 37 MBq/mL). The sample solution was injected into the tail vein of male Wistar rats (8 weeks old) without anesthesia (dose: 0.2mL, radioactivity of administration: equivalent to 7.4 MBq). Blood and brain were collected 2 minutes, 5 minutes, 15 minutes, 30 minutes, and 60 minutes after the administration, with the head broken without anesthesia. The brain mass was measured, and the radioactivity of the brain (hereinafter referred to as "A" in this example) was measured using a single channel analyzer (model: SP-20, manufactured by Utility industries, Ltd.). In addition, the radioactivity of the remaining part of the whole body including blood was measured in the same manner as described above (hereinafter, referred to as "B" in this example). Using these measurement results, the amount of radioactivity accumulated (% ID/g) per unit brain weight at each anatomical time point was calculated from the following formula (2).

Further, the preparation of [2 ]¹²³I]IMPY was dissolved in a liquid (radioactivity concentration: 37 MBq/mL) containing 50mmol/L of L-cysteine hydrochloride in physiological saline, and the amount of radioactivity accumulated per unit brain weight (% ID/g) at each dissection time point was calculated in the same manner as described above.

In this example, experiments were carried out using 3 animals at each time point.

(results)

The results are shown in Table 3. As shown in table 3, compound 1 and compound 2 showed the following tendency: confirmation and administration 2 min after administration¹²³I-IMPY accumulates with the same high radioactivity and then rapidly disappears over 60 minutes. This result suggests that Compound 1 and Compound 2 are used in combination with [ alpha ], [ alpha¹²³I]IMPY, as well, has excellent brain transferability and rapid clearance from the brain.

TABLE 3 intracerebral radioactivity accumulation of the Compounds of the invention after intravenous injection (in rats)

Example 8 confirmation of binding of Compound to brain slice of AD patient Using autoradiogram

To evaluate whether the compounds of the present invention can map amyloid in the brain of AD patients, the following experiments were performed.

(method)

(1) AD patient brain sections of 5 μm in thickness were prepared using AD patient brain tissue sold by analytical biologicalcalcalcalevices, Inc. (USA).

(2) The brain sections were immersed in PBS for 15 minutes, 5 minutes and 5 minutes, respectively, and then immersed in PBS containing 1% BSA for 30 minutes, to prepare solutions containing Compound 1, Compound 2 and¹²³I]IMPY in PBS with 1% BSA (radioactive concentration 10 kBq/mL), brain sections were immersed for 30 min at room temperature. Then, the brain sections were washed by immersing the brain sections in 1% BSA-containing PBS, and PBS for 5 minutes. The washed brain sections were thoroughly dried, exposed to light for 16 hours on an imaging plate, and subjected to autoradiography image analysis using a bioimage analyzer (model: BAS-2500, manufactured by Fuji photo film Co., Ltd.) (FIGS. 3, 4, and 5).

(3) Immunostaining of amyloid deposition sites was performed by anti-amyloid antibody using adjacent sections. Anti-human amyloid β (N) (82E1) mouse IgGMoAb (shinkansen ) was used as an anti-amyloid antibody, and anti-mouse IgG (H + L) goat IgGFab' -HRP (shinkansen ) was used as a secondary antibody. Amyloid deposition sites were detected by applying DAB + (3, 3' -diaminobenzidine hydrochloride) · substrate kit (Dako) to HRP bound to secondary antibody (fig. 6).

(results)

Immersing in a solution containing compound 1, compound 2 and [2 ]¹²³I]Autoradiograms of sections of the solution of-IMPY are shown in FIGS. 3, 4 and 5, respectively. The gray matter portion of the frozen brain section of the AD patient used in this experiment was confirmed by immunostaining for amyloid deposition (fig. 6), and the binding of the immunostained compound to the amyloid deposition site was confirmed in any autoradiogram. The above results show that Compound 1 and Compound 2 of the present invention are used in combination with [ [ alpha ], ]¹²³I]IMPY likewise images amyloid deposits in the brain.

Industrial applicability

The compounds of the invention can be used in the field of diagnostic agents.

Claims (5)

1. A compound represented by the following formula (1) or a salt thereof,

in the formula, R¹Is radioactive iodine, A¹And A²Each independently is CH or N.

2. The compound or salt according to claim 1, wherein R is¹Is selected from¹²³I、¹²⁴I、¹²⁵I and¹³¹I。

3. a compound represented by the following formula (2) or a salt thereof,

in the formula, R²Is a group selected from a trialkylstannyl substituent having an alkyl chain with 1-4 carbon atoms or a triphenylstannyl group, A³And A⁴Each independently is CH or N.

4. An Alzheimer's disease diagnostic agent comprising a compound represented by the following formula (1),

in the formula, R¹Is radioactive iodine, A¹And A²Each independently is CH or N.

5. The diagnostic agent for Alzheimer's disease according to claim 4, wherein R is¹Is selected from¹²³I、¹²⁴I、¹²⁵I and¹³¹I。