HK1148951A - Use of novel compound having affinity for amyloid, and process for production of the same - Google Patents

Description

Application and preparation method of novel compound with affinity to amyloid

Technical Field

The present invention relates to the use of a novel compound having affinity for amyloid and a method for producing the same, and particularly to a reagent for detecting amyloid in a biological tissue, which can be used for detecting amyloid in a focal site in the diagnosis of systemic amyloidosis and other diseases associated with amyloid accumulation.

Background

Diseases caused by deposition of fibrillar proteins called amyloid in various organs or tissues in the body are collectively called amyloidosis. A common feature of amyloidosis is that fibrillar proteins called amyloid proteins, rich in β -sheet structures, are deposited systemically or locally at sites in various organs such that dysfunction is triggered in the organ or tissue. Amyloid generally refers to a protein aggregate formed by aggregation of various amyloid precursor proteins in an organism, such as amyloid-beta, mutant transthyretin, and beta 2-microglobulin. Amyloid proteins, although formed from any amyloid precursor protein, have a characteristic structure that is rich in β -sheet. Thus, compounds that bind to the β -sheet structure, such as congo red and thioflavin T, are characterized by their affinity for amyloid.

Amyloidosis is classified into systemic amyloidosis and localized amyloidosis, depending on the deposition pattern of amyloid.

Systemic amyloidosis is a disease in which amyloid deposition occurs in various parts of the body. Examples of systemic amyloidosis include: familial amyloidosis (in which amyloid produced in the liver is deposited in various organs throughout the body to cause disease), senile TTR amyloidosis (in which amyloid is deposited in the heart and large joints such as hand joints), dialysis amyloidosis (occurring in the bones and joints of patients undergoing chronic dialysis), reactive AA amyloidosis (secondary amyloidosis, resulting from the deposition of amyloid produced by serum amyloid a, which is an acute phase protein secondary to chronic inflammatory diseases such as chronic rheumatism), and immunocytotic amyloidosis (in which amyloid produced by immunoglobulin is deposited in various organs throughout the body).

Localized amyloidosis is a disease in which amyloid deposition occurs only in certain organs. Examples of localized amyloidosis include: cerebral amyloidosis such as alzheimer's disease (where amyloid is deposited in the brain), cerebrovascular amyloidosis and creutzfeldt-jakob disease, endocrine amyloidosis (where amyloid is deposited in the islets and insulinomas associated with I I type diabetes mellitus, or in the atria), cutaneous amyloidosis (where amyloid is deposited in the skin), and localized nodular amyloidosis (where nodular amyloid is deposited in the skin and lungs).

In the case of systemic amyloidosis, the diagnosis of amyloidosis is carried out as follows: first, tissue is harvested from skin, kidney, and gastrointestinal tract, etc. biopsy sites; and stained with congo red or thioflavin T. Congo red is a fluorescent compound with a high affinity for the beta-sheet structure of amyloid, and exhibits birefringence under a polarizing microscope due to its directionality, so it can selectively stain amyloid deposits in tissues. Similarly, thioflavin T is also a fluorescent compound having affinity for amyloid, and can be used similarly to congo red. After the tissue staining was found to be positive, confirmation of diagnosis was performed by combining immunostaining using an antibody. However, it is often difficult to determine a positive result by congo red or thioflavin T staining even when the staining is observed under a polarizing microscope.

On the other hand, it is considered to perform image diagnosis of systemic amyloidosis using recently widely developed image diagnosis apparatuses such as PET, SPECT and MRI.

However, when congo red and thioflavin T are labeled and used as probes for image diagnosis, they have a problem that the binding specificity to amyloid is poor so that good detection sensitivity cannot be obtained.

In addition, since congo red has carcinogenicity, it is not suitable for diagnosis of human body.

Therefore, it has been proposed that bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene (BSB) and derivatives thereof, which are congo red derivatives, are used as a fluorescent reagent for detecting amyloid, have high affinity and detection sensitivity for systemic amyloid, and can be used for in vivo detection (non-patent document 14, patent document 7). It is reported that BSB has high affinity with amyloid protein produced by brain amyloidosis and systemic amyloidosis, and has no benzidine structure in its structure, so that it has little carcinogenic problem and can be radiolabeled for use as a probe for image diagnosis.

On the other hand, for alzheimer's disease (hereinafter referred to as AD), which is a typical cerebral amyloidosis, attempts have been made to detect AD in vivo using a compound having high affinity for amyloid as a marker because a biopsy specimen cannot be collected.

Such probes for image diagnosis of amyloid in brain are mostly hydrophobic low molecular weight compounds, which have high affinity with amyloid and high brain transferability, and are used with various radionuclides such as¹¹C、¹⁸F and¹²³and I, marking. For example, there is a report that,¹¹c or radiohalogen-labelled compounds include the respective 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]Benzoxazoles (hereinafter IBOX) and 6- [2- (fluoro) ethoxy]-2- [2- (2-dimethylaminothiazol-5-yl) ethenyl]Benzoxazoles (non-patent document 6, non-patent document 7); DDNP derivatives 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 imidazoleAnd pyridine 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). Further, as for some probes for image diagnosis, human body image studies have been conducted, and it has been reported that there is a 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).

International publication WO2007/002540 discloses a series of compounds having a group having affinity for amyloid, which is linked to a site labeled with a radionuclide via ethylene glycol or polyethylene glycol (patent document 5).

International publication WO2007/063946 pamphlet discloses a series of compounds that are linked to a five-membered aromatic heterocyclic group to prevent their metabolism in the brain (patent document 6).

[ patent document 1] JP-T-2004-506723

[ patent document 2] JP-T-2005-504055

[ patent document 3] JP-T-2005-512945

[ patent document 4] JP-T-2002-

[ patent document 5] International publication WO2007/002540 pamphlet

[ patent document 6] International publication WO2007/063946 pamphlet

[ patent document 7] International publication WO2005/016888 pamphlet

[ non-patent document 1] J.A.Hardy & G.A.Higgins, "Alzheimer's Disease: the Amyloid Cascade Hypothesis. ", Science, 1992, 256, p.184-185

[ non-patent document 2] G.McKhann et al, "Clinical diagnostics of Alzheimer's disease: report of the NINCDS-ADRDA word Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's disease ", Neurology, 1984, 34, p.939-944

[ non-patent document 3] Z. -P.Zhuang et al, "Radiodinated Styrylbenzenes and Thioflavins as Probes for analog Aggregates.", J.Med.chem., 2001, 44, p.1905-1914

[ non-patent document 4] Masahiro Ono et al, "11C-labeled stilbene derivatives as A β -aggregate-specific PET imaging agents for Alzheimer's disease", nucleic Medicine and Biology, 2003, 30, p.565-571

[ non-patent document 5] H.F.Kung et al, "Novel Stilbenes as Probes for analog devices", J.American Chemical Society, 2001, 123, p.12740-12741

[ non-patent document 6] Zhi-Ping Zhuang et al, "IBOX (2- (4' -dimethyl aminophenyl) -6-iodobendoxazole): a ligand for imaging particulate compositions in the broad. ", nucleic acids and Biology, 2001, 28, p.887-894

[ non-patent document 7]Furumoto Y et al, "A" and "B", "¹¹C]BF-227：A New ¹¹C-Labeled2-Ethenylbenzoxazole Derivative for Amyloid-β PlaquesImaging.”，European Journal of Nuclear Medicine and MolecularImaging，2005，32，Sup.1，P759

[ non-patent document 8] Eric D.Agdeppa et al, "2-Dialkylamino-6-acryloylinnonitrile treated naphthanes (DDNP Analogs): novel Diagnostic and Therapeutic Tools in Alzheimer's disease ", Molecular Imaging and Biology, 2003, 5, p.404-417

[ non-patent document 9] Zhi-Ping Zhuang et al, "Structure-Activity Relationship of Imidazo [1, 2-a ] Pyridines as Ligands for Detecting β -Amyloid plants in the brain", J.Med.chem, 2003, 46, p.237-243

[ non-patent document 10] W.E.Kluk et al, "Imaging woven analog in Alzheimer's disease with Pittsburgh Compound-B.", Ann.Neurol., 2004, 55, p.306-319

[ non-patent document 11] Nicolaas P.L.G.Verhoeff et al, "In-Vivo Imaging of Alzheimer Disease β -Amyloid With [11C ] SB-13 PET.", American Journal of Geriatric Psychiatry, 2004, 12, p.584-595

[ non-patent document 12] Hiroyuki Arai et al, "[ 11C ] -BF-227 AND PET to Visualize Amyloid analog in Alzheimer's diseases Patients", Alzheimer's & Dementia: the Journal of The Alzheimer' S Association, 2006, 2, Sup.1, S312

[ non-patent document 13] Christopher M.Clark et al, "Imaging Amyloidwith I123 IMPY SPECT", Alzheimer's & Dementia: the Journal of Alzheimer' S Association, 2006, 2, Sup.1, S342

[ non-patent document 14] D.M.Skovronsky et al, Proc.Natl.Acad.Sci., 2000, 97, 7609

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-like proteins, and clinical applications thereof have been sought.

The results of the experiment in the normal mouse show that the use of [ alpha ], [ alpha ]¹²⁵I]Labeled TZDM, IBOX and m-I-SB will all be transferred into the brain 2 minutes after administration. However, clearance of these compounds from normal tissues is inadequate and gradually accumulates in the brain over time after administration (JP-T-2005-512945; Zhui-Ping Zhuang et al, Nuclear Medicine and Biology, 2001, 28, p.887-894; H.F.Kung et al, J.Am.chem.Soc., 2001, 123, p.12740-12741). A problem arises when clearance from normal tissue is inadequate: sufficient contrast cannot be obtained at the site of amyloid accumulation. Use 2¹¹C]The labeled SB-13 was shown in rat experiments to have clearance from normal tissue, but the clearance rate was not said to be sufficiently rapid (Masahiro Ono et al, Nuclear Medicine and Biology, 2003, 30, p.565-571).

Also, it is disclosed that¹²⁵I]Experimental results of labeled compounds show that a compound having an imidazopyridine skeleton such as IMPY has a property of transferring to the brain and accumulating on amyloid after administration, and also has an excellent property of being rapidly eliminated from normal tissues, which is different from the above-mentioned compounds. However, IMPY is a compound that is positive in the back-mutation assay. In order to use such a compound as a probe for image diagnosis, the dosage and administration mode must be sufficiently considered (pamphlet of International publication WO 03/106439).

FDDNP was also reported to be positive in the back-mutation assay. (WO 03/106439).

The amyloid-targeting image diagnostic probe is preferably a compound which has excellent affinity for amyloid and is cleared from normal tissues sufficiently rapidly like IMPY, but can suppress toxicity such as mutagenicity. However, no compounds having such properties have been disclosed.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a compound having affinity for amyloid and inhibiting toxicity such as mutagenicity, which enables highly sensitive detection of amyloid in vivo or in vitro.

Means for solving the problems

The present inventors found that a specific novel compound having an imidazopyridine-phenyl skeleton having a carbon atom bonded to a hydroxyl group has affinity with amyloid and low toxicity such as mutagenicity, and that amyloid is detected with high sensitivity in vivo or in vitro by using a series of such compounds as a probe. Thus, the present invention has been completed.

That is, according to one aspect of the present invention, there is provided a reagent for detecting amyloid deposited on a biological tissue, comprising a compound represented by the following formula (1):

or a salt thereof.

The biological tissue may be various tissues in which amyloid is known to be deposited in amyloidosis. Typical examples of these biological tissues include brain, heart, lung, pancreas, bone, and joint, and brain is given as the most typical biological tissue. In the case of a brain subject, typical amyloidosis includes alzheimer's disease and dementia with Lewy bodies.

In the formula (1), R³As shown in the following formula:

R¹is a radioactive halogen substituent, m is an integer from 0 to 4, and n is 0 or 1. As the radioactive halogen, various nuclides can be used, and preferably, a compound selected from the group consisting of¹⁸F、⁷⁵Br、⁷⁶Br、¹²³I、¹²⁴I、¹²⁵I and¹³¹halogen of I, more preferably selected from¹⁸F、¹²³I and¹²⁵halogen of I. In formula (1), when n is 0, m is preferably 0 to 4, and when n is 1, m is preferably 1 to 4.

A¹，A²，A³And A⁴Independently represent carbon or nitrogen, and it is necessary that at least 1 of them represents carbon. Preferably, A¹，A²，A³And A⁴More preferably, all of them represent carbon. In the formula (1), R³And A¹，A²，A³Or A⁴The indicated carbon bonds. When A is¹，A²，A³And A⁴Respectively represent not R³Bonded carbon, the hydrogen atom bonded thereto is unsubstituted. The hydroxyl group shown in formula (1) may be bonded to any carbon constituting the phenyl skeleton, but it is preferable that the hydroxyl group is bonded to a carbon at the 4' -position of the phenyl skeleton. R³May be A¹，A²，A³Or A⁴Any one of them as long as it is carbon, but it is preferably A³The carbon indicated, i.e., is the 6-position carbon.

The compound of the above formula (1) is a novel compound, and according to another aspect of the present invention, there is provided a method for preparing a radioactive halogen-labeled organic compound, which comprises:

a step of preparing a reaction solution containing a compound represented by the following formula (2) or a salt thereof and a radioactive halide ion:

wherein A is¹，A²，A³And A⁴Independently represent carbon or nitrogen, R⁴Is a group represented by the formula:

m is an integer of 0 to 4,

n is an integer of 0 or 1, and

when m is 0, R²Non-radioactive halogen substituents, nitro substituents, trialkylammonium substituents having 3 to 12 carbon atomsA substituent of a trialkyl stannyl or triphenylstannyl having 3 to 12 carbon atoms, R is not equal to 0 and/or n is not equal to 0²A non-radioactive halogen substituent, a mesyloxy substituent, a trifluromesyloxy substituent or an aromatic sulfonyloxy substituent,

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, R⁴And A¹，A²，A³Or A⁴The carbon indicated is bonded to the carbon to which,

and

a step of providing reaction conditions to the reaction solution to synthesize a compound represented by the following formula (1) or a salt thereof:

wherein A is¹，A²，A³And A⁴As in the case of the formula (2),

R³is a group represented by the formula:

R¹is a substituent of a radioactive halogen, and is,

m and n are the same as in formula (2),

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, R³And A¹，A²，A³Or A⁴The carbon indicated is bonded to the carbon to which,

in the formula (2), A¹，A²，A³And A⁴Independently represents carbon or nitrogen, if necessary, itAt least 1 of them represents carbon. Preferably, A¹，A²，A³And A⁴More preferably, all of them represent carbon. In the formula (2), R⁴And A¹，A²，A³Or A⁴The indicated carbon bonds. When A is¹，A²，A³And A⁴Respectively represent not R⁴Bonded carbon, the hydrogen atom bonded thereto is unsubstituted. The hydroxyl group shown in formula (2) may be bonded to any carbon constituting the phenyl skeleton, but it is preferable that the hydroxyl group is bonded to a carbon at the 4' -position of the phenyl skeleton. R⁴The bonding position of (A) is not particularly limited as long as it is A¹，A²，A³Or A⁴Carbon represented by, but preferably A³The carbon indicated, i.e., is the 6-position carbon.

The step of preparing a reaction solution containing the precursor compound represented by the above formula (2) or a salt thereof and radioactive halogen ions is carried out, for example, by dissolving the precursor compound or a salt thereof in an inert organic solvent and adding thereto a solution containing radioactive halogen ions obtained by a known method.

As the inert organic solvent, various solvents which are not reactive with the precursor compound or a salt thereof and the radioactive halogen ion can be used, and for example, when the radioactive halogen ion used is radioactive iodine, methanol can be preferably used, and when the radioactive halogen ion used is radioactive fluorine, acetonitrile can be preferably used.

In the step of synthesizing the compound represented by the above formula (1) or a salt thereof, the reaction conditions to be given to the reaction solution are not particularly limited as long as it is permissible wherein the substituent R of the compound of the formula (2)²The conditions for the substitution reaction with the radioactive halogen ion added to the reaction solution may be any conditions, and known reaction conditions suitable for the kind of radioactive halogen ion may be used.

In the method for producing a radioactive halogen-labeled organic compound of the present invention, as the radioactive halide ion, for example, a radioactive halide ion can be used¹⁸F、⁷⁵Br、⁷⁶Br、¹²³I、¹²⁴I、¹²⁵I or¹³¹I. When preparing a compound in which R is¹The radioactive halogen substituent is¹²³I、¹²⁴I、¹²⁵I or¹³¹When the compound of formula (1) of I is used, each¹²³I ions,¹²⁴I ions,¹²⁵I ion or¹³¹The I ion acts as a radioactive halide. As the compound of formula (2), preferably used are: wherein when m ═ n ═ 0, R²Is a compound of iodine, bromine, a trialkylstannyl substituent having 3 to 12 carbon atoms or a triphenylstannyl substituent, wherein R is not equal to 0 and/or n is not equal to 0²A compound that is iodine; particularly preferred is where R is when m ═ n ═ 0²Is a compound of iodine, trimethylstannyl substituent, tributylstannyl substituent and triphenylstannyl substituent.

When preparing a compound in which R is¹The radioactive halogen substituent is¹⁸When F is a compound of the formula (1), use is made of¹⁸As the radioactive halide ion, as the compound of formula (2), F ion is preferably used, wherein R is preferably used when m ═ n ═ 0²A compound which is a nitro substituent or a trialkylammonium substituent having 3 to 12 carbon atoms, and wherein R is not equal to 0 and/or n is not equal to 0²Compounds which are mesyloxy, trifluoromethanesulfonyloxy or aromatic sulfonyloxy substituents, it being particularly preferred if R.noteq.0 and/or n.noteq.0²Is a compound having a trifluoromethanesulfonyloxy substituent or a tosyloxy substituent. When preparing a compound in which R is¹The radioactive halogen substituent is⁷⁵Br or⁷⁶When Br is used, the compounds of formula (1) are used⁷⁵Br ion or⁷⁶Br ion as radioactive halide ion, as the compound of formula (2), preferably used is one in which R²A compound that is bromine.

Further, according to another aspect of the present invention, there is provided a precursor compound for producing a radioactive halogen-labeled organic compound, which is represented by the following formula (2), or a salt thereof:

wherein A is¹，A²，A³And A⁴Independently represent carbon or nitrogen, or a salt thereof,

R⁴is a group represented by the formula:

m is an integer of 0 to 4,

n is an integer of 0 or 1,

when m is 0, R²A non-radioactive halogen substituent, a nitro substituent, a trialkylammonium substituent having 3 to 12 carbon atoms, a trialkylstannyl substituent having 3 to 12 carbon atoms or a triphenylstannyl substituent, R being equal to 0 when m and/or n is equal to 0²A non-radioactive halogen substituent, a mesyloxy substituent, a trifluromesyloxy substituent or an aromatic sulfonyloxy substituent,

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, R⁴And A¹，A²，A³Or A⁴The indicated carbon bonds.

As R in formula (2)²The non-radioactive halogen substituent may be a halogen capable of targeting in a nucleophilic substitution reaction using radioactive fluorine or a halogen capable of targeting in an isotope exchange reaction with radioactive iodine, and preferably iodine, bromine, or chlorine may be used. As the trialkylstannyl substituent, various substituents can be used, and preferably, a trimethylstannyl substituent and a tributylstannyl substituent can be used. In the formula (2), R is preferred²Selected from iodine, bromine, trimethylStannyl substituents, tributylstannyl substituents, trifluoromethanesulfonyloxy substituents, and triphenylstannyl substituents. Meanwhile, in formula (2), when n is 0, m is preferably 0 to 4; when n is 1, m is preferably 1 to 4.

In the formula (2), A¹，A²，A³And A⁴Independently represent carbon or nitrogen, and it is necessary that at least 1 of them represents carbon. Preferably, A¹，A²，A³And A⁴More preferably, all of them represent carbon. In the formula (2), R⁴And A¹，A²，A³Or A⁴The indicated carbon bonds. When A is¹，A²，A³And A⁴Respectively represent not R⁴Bonded carbon, the hydrogen atom bonded thereto is unsubstituted. The hydroxyl group shown in formula (2) may be bonded to any carbon constituting the phenyl skeleton, but it is preferable that the hydroxyl group is bonded to a carbon at the 4' -position of the phenyl skeleton. R⁴Is a bonding position of¹，A²，A³Or A⁴The carbon is not particularly limited. But is preferably A³The carbon indicated, i.e., the carbon at the 6-position.

Effects of the invention

According to the present invention, an amyloid detection reagent having affinity with amyloid and capable of suppressing toxicity such as mutagenicity, and thus useful for in vitro and in vivo detection of amyloid associated with a wide range of amyloidosis, a method for producing the same, and an intermediate for production thereof can be obtained.

Drawings

FIG. 1 is a scheme for the synthesis of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine.

FIG. 2 is a synthetic route for 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine.

FIG. 3 is a synthetic route for 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyridine.

FIG. 4 is a synthetic route for 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrimidine.

FIG. 5 is a synthetic route for 6- (3 '-fluoropropoxy) -2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine.

FIG. 6 is a synthetic route for 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine.

FIG. 7 is a synthetic route for 6-fluoro-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine.

FIG. 8 is a synthetic route for 2- (4' -hydroxyphenyl) -6-nitroimidazo [1, 2-a ] pyridine.

Fig. 9(a) is an autoradiogram of brain sections after injection of compound 6, and fig. 9(b) is a fluorescence micrograph of thioflavin T-stained samples (magnified of the site of injection of amyloid suspension).

FIG. 10 is a scheme for the synthesis of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine.

FIG. 11 is a synthetic route for 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine.

FIG. 12 is a synthetic route for 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrazine.

FIG. 13 is a synthetic route for 8-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine.

Fig. 14(a) is an autoradiogram of brain sections after injection of compound 60, and fig. 14(b) is a fluorescence micrograph of thioflavin T-stained samples (magnified of the site of injection of amyloid suspension).

Fig. 15(a) is an autoradiogram of a brain section after injection of compound 61, and fig. 15(b) is a fluorescence micrograph of a thioflavin T-stained sample (magnified image of the site of injection of amyloid suspension).

Fig. 16 is a graph showing the ability of compound 6 to bind to amyloid.

Detailed Description

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

Hereinafter, a synthesis method of a precursor compound for preparing a radiohalogen-labeled organic compound according to one aspect of the present invention will be described by taking 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine as an example.

First, 4 '-hydroxyacetophenone is reacted with copper bromide to prepare 2-bromo-4' -hydroxyacetophenone (fig. 1, step 1). In this case, the reaction can be carried out according to a conventional method, for example, a method described in King, L.Carroll and Ostrum, G.Kenneth, Journal of Organic Chemistry, 1964, 29(12), p.3459-3461.

Then, 2-bromo-4 '-hydroxyacetophenone prepared as described above was reacted with 2-amino-5-bromopyridine to prepare 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (fig. 1, step 2). This step can be performed according to the following method.

First, 2-bromo-4 '-hydroxyacetophenone and 2-amino-5-bromopyridine are dissolved in an inert solvent such as acetonitrile, and then they are reacted with each other at a reflux temperature for 2 to 6 hours to prepare a hydrobromide salt of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine as a white precipitate. The solvent used at this time may be acetonitrile or other solvents commonly used for similar reactions, such as methanol and acetone. The reaction temperature may be a temperature that allows reflux, for example 90 ℃ when the solvent is acetonitrile. The amount of the solvent used may be an amount sufficient to effect the reaction, but it should be noted that if the solvent is too much, precipitation of the reaction product is difficult to obtain. For example, when 2-bromo-4' -hydroxyacetophenone equivalent to an amount of 10mmol is used in the reaction, the amount of the solvent used may be about 40 to 50 mL.

Subsequently, the reaction solution was filtered to recover the precipitate. The white precipitate was suspended in a methanol/water (1: 1) mixture. Then, a saturated aqueous sodium bicarbonate solution was added thereto in a very large excess amount with respect to the precipitate to release 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine as a precipitate. The newly generated precipitate was filtered to recover 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 1, step 2) as the target compound in this step. The amount of the water/methanol mixed liquid is not particularly limited as long as it is sufficient to effect the reaction. However, it should be noted that if the amount of the mixed liquid is too large, precipitation of the product is disturbed. For example, when 2-bromo-4' -hydroxyacetophenone is used in an amount corresponding to 10mmol, a mixed solution of water/methanol may be used in an amount of about 40 to 100 mL. In addition, the amount of sodium hydrogencarbonate is also not particularly limited as long as it is in a very large excess relative to the above-mentioned precipitate as a reaction substrate. For example, when the reaction is carried out under the above-mentioned conditions, the amount of the saturated aqueous sodium bicarbonate solution added to the reaction solution may be about 25 mL.

Then, the 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine prepared above was dried well and dissolved in dioxane, and after addition of triethylamine, bis (tributyltin) and a catalytic amount of tetrakis (triphenylphosphine) palladium were added. The reaction mixture was heated at about 90 ℃ and reacted for about 24 hours, and then the solvent was distilled off and purified by chromatography to obtain 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine as a target compound (fig. 1, step 3). The amount of bis (tributyltin) used at this time may be an amount satisfying an excess condition with respect to the reaction substrate, and specifically, it is preferable that its molar ratio is about 1.5 times with respect to the reaction substrate 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine.

When a compound is obtained in which the substituent at the 6-position is a trialkylstannyl substituent other than the tributylstannyl substituent, various bis (trialkyltin) suitable for the purpose may be used in step 3 in place of bis (tributyltin). For example, when a compound having a trimethylstannyl substituent as a substituent at the 6-position is synthesized, the same reaction as described above may be performed with bis (trimethyltin) in step 3.

In order to obtain a compound in which the substituent at the 6-position is a non-radioactive halogen substituent, the compound obtained in step 2 may be used as it is as a compound containing bromine as a halogen substituent, and for compounds in which the halogen substituent at the 6-position is fluorine, chlorine and iodine, the same reaction as in step 2 is carried out except that 2-amino-5-fluoropyridine, 2-amino-5-chloropyridine and 2-amino-5-iodopyridine are used in place of 2-amino-5-bromopyridine in step 2, respectively.

In order to obtain a compound having a substituent attached to the 6-position via an oxygen atom, in step 2, 2-amino-5-hydroxypyridine may be used in place of 2-amino-5-bromopyridine to synthesize 2- (4' -hydroxyphenyl) -6-hydroxyimidazo [1, 2-a ] pyridine, and a bromide compound having a substituent desired to be introduced may be reacted therewith in the presence of a base. For example, to obtain a compound having a 3-fluoropropoxy substituent at the 6-position, 2- (4' -hydroxyphenyl) -6-hydroxyimidazo [1, 2-a ] pyridine may be reacted with 1-bromo-3-fluoropropane in the presence of potassium carbonate.

In addition, in order to obtain a compound having a substituent attached to the 6-position via an alkyl chain, the following operation may be performed. For example, for compounds in which the substituent at the 6-position is 3 ' -bromopropyl, the 2- (4 ' -hydroxyphenyl) -6-bromoimidazo [1, 2-a ] pyridine obtained in step 2 can be converted to 6-allyl-2- (4 ' -hydroxyphenyl) imidazo [1, 2-a ] pyridine by reaction with allyltributyltin. Hydroboration and oxidation reactions are then carried out to convert to 2- (4 '-hydroxyphenyl) -6- (3' -hydroxypropyl) imidazo [1, 2-a ] pyridine. In addition, bromination of the hydroxyl group can be carried out by tetrabromomethane in the presence of triphenylphosphine.

In accordance with the above process, a compound wherein a is a can be prepared except that 2-amino-5-bromopyrimidine is used in place of 2-amino-5-bromopyridine in step 2 of fig. 1¹，A²，A³And A⁴A in (A)¹A compound represented by the above formula (1) which is nitrogen, and wherein A⁵，A⁶，A⁷And A⁸A in (A)⁵A compound represented by the above formula (2) which is nitrogen.

In accordance with the above method, except that 6-amino-3-bromo-1, 2, 4-triazine is used in place of 2-amino-5-bromopyridine in step 2 of FIG. 1, a compound wherein A is¹，A²，A³And A⁴A in (A)²And A⁴A compound represented by the above formula (1) which is nitrogen, and wherein A⁵，A⁶，A⁷And A⁸A in (A)⁶And A⁸A compound represented by the above formula (2) which is nitrogen.

(method for synthesizing radiohalogen-labeled organic Compound)

Then, 2- (4' -hydroxyphenyl) -6-, is used¹²³I]Iodoimidazo [1, 2-a]Pyridine is an example, and a method of preparing a radiohalogen-labeled organic compound according to another aspect of the present invention is described.

In the range of 2- (4' -hydroxyphenyl) -6-, [ solution ]¹²³I]Iodoimidazo [1, 2-a]In the preparation of pyridine, first, radioactive halide ions for labeling are obtained¹²³I]Sodium iodide solution. The [ 2] can be obtained by a known method using, for example, xenon as a target and exposing to proton bombardment¹²³I]Radioactive iodine. The [ alpha ], [ alpha ]¹²³I]Radioactive iodine is prepared into¹²³I]Sodium iodide solution, and used for labeling.

Then, the labeling precursor 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]Pyridine is dissolved in an inert organic solvent, and [ 2], [ solution ] is added thereto¹²³I]Sodium iodide solution, an acid and an oxidizing agent, and reacting them to obtain 2- (4' -hydroxyphenyl) -6-, [2- ], a target compound¹²³I]Iodoimidazo [1, 2-a]Pyridine. As the inert organic solvent for dissolving the precursor compound, the precursor of the label and¹²³I]various solvents having no reactivity with sodium iodide, and methanol can be preferably used.

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

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

A compound labeled with a radioactive halogen other than iodine can be synthesized by labeling a labeled precursor suitable for the purpose of synthesis with a radioactive halogen suitable for the purpose. For example, to synthesize 6-, [ 2]¹⁸F]Fluoropropoxy-2- (4' -hydroxyphenyl) imidazo [1, 2-a]Pyridine, the labeling precursor 2- (4 '-hydroxyphenyl) -6- (3' -p-toluenesulfonyloxypropoxy) imidazo [1, 2-a ] may be reacted in the presence of a phase transfer catalyst and potassium carbonate]Pyridine and [ alpha ], [ alpha¹⁸F]Fluorine ion reaction.

(method of preparing and Using the detection reagent of the present invention)

Amyloid has a variety of different structures depending on the kind of precursor protein, but they have in common a β -sheet structure. Many staining agents that target amyloid proteins, such as thioflavin T and congo red, are known to target the β -sheet structure and have equal staining abilities against different classes of amyloid proteins.

The compound of formula (1) of the present invention has affinity for amyloid having amyloid β -protein (hereinafter referred to as a β) as a precursor compound, and also has activity of inhibiting the binding of thioflavin T, which is known to bind to a wide range of amyloid, to amyloid.

Therefore, the compound of formula (1) of the present invention is considered to have affinity for the β -sheet structure of amyloid, as with thioflavin T. This suggests that the compounds of formula (1) according to the invention have the same affinity for various amyloid proteins.

That is, the amyloid detection reagent of the present invention can be used for diagnosing systemic amyloidosis and localized amyloidosis, similarly to thioflavin T and congo red. Systemic amyloidosis includes immunoglobulin amyloidosis, reactive AA amyloidosis, familial amyloidosis, dialysis amyloidosis, senile amyloidosis, etc. The localized amyloidosis includes cerebral amyloidosis, endocrine amyloidosis, cutaneous amyloidosis, localized nodular amyloidosis, and the like.

The amyloid detection reagent of the present invention can be used not only as a reagent for in vitro biopsy but also as a radiodiagnostic agent for in vivo use.

The amyloid detection reagent of the present invention can be prepared as a solution by mixing the radioactive halogen-labeled compound of the formula (1) with water, physiological saline, ringer's solution, or the like, which is optionally adjusted to an appropriate pH, as desired, in the same manner as other generally known radioactive diagnostic agents. At this time, the concentration of the compound of the present invention should be adjusted not to exceed a concentration at which stability of the compound of the present invention is ensured. There is no particular limitation on the dosage of the compound of the present invention as long as it is sufficient to obtain a distribution image of the administered agent. For example, iodine-123 (¹²³I) Labeled compound and fluorine-18: (¹⁸F) For labeled compounds, about 50-600MBq/60kg of adult body weight can be administered intravenously or topically. The distribution of the administered agent can be visualized by known methods. For example, iodine-123 (can be made by the column PECT apparatus¹²³I) Visualization of the labeled compound, alternatively fluorine-18 (by means of a PET apparatus¹⁸F) The labeled compound is imaged.

By administering the amyloid detection reagent of the present invention to an organism, amyloid deposited in biological tissues such as brain, heart, lung, digestive tract, blood vessel, liver, pancreas, kidney, joint and bone can be imaged, and used for imaging amyloid deposition in biological tissues such as brain, heart, lung, pancreas, bone and joint, which are difficult to biopsy and collect.

Examples

Hereinafter, the present invention will be described in more detail by examples, comparative examples and reference examples. However, the following examples in no way limit the scope of the present invention.

In the following examples, the names of the respective compounds used in the experiments are as defined in Table 1-1.

Table 1: compound names of evaluation compounds used in examples

Name of Compound	Common name
		Compound 1	6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a]Pyridine compound
Compound 2	2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine compound
		Compound 3	6- (3 '-Fluoropropoxy) -2- (4' -hydroxyphenyl) imidazo [1, 2-a]Pyridine compound
Compound 4	2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyrimidines
		Compound 5	[125I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine compound
Compound 6	[123I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine compound
		Compound 7	6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a]Pyrazine esters
Compound 8	2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyrazine esters

Compound 9	[123I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyrimidines
		Compound 10	[123I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyrazine esters
Compound 11	[123I]-2- (4' -hydroxyphenyl) -8-iodoimidazo [1, 2-a]Pyridine compound

Example I-1 Synthesis of 6-Tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of copper bromide to obtain a suspension, and a solution of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in a mixture of 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 1, step 1).

2.15g (corresponding to 10.0mmol) of 2-bromo-4' -hydroxyacetophenone and 1.74g (corresponding to 10.0mmol) of 2-amino-5-bromopyridine were dissolved in 50mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 105 ℃ for 6 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 20mL of water and 20mL of methanol. Then, about 25mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 2.41g (equivalent to 8.32mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 1, step 2).

138mg (equivalent to 0.476mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine was dissolved in 20mL of dioxane, and 2mL of triethylamine was added thereto. Then, 360. mu.L (equivalent to 0.713mmol) of bis (tributyltin) and 20mg (catalytic amount) of tetrakis (triphenylphosphine) palladium were added thereto. After the reaction mixture was stirred at 90 ℃ for 22 hours, the solvent was distilled off under reduced pressure. The residue was purified by preparative TLC (elution solvent: hexane/ethyl acetate 1/4), and the obtained crude product was purified by preparative HPLC (HPLC apparatus: LC-908 (trade name; manufactured by Nippon analytical industries, Ltd.), column: 2 JAIGEL 2H (trade name; manufactured by Nippon analytical industries, Ltd.) in series, mobile phase: chloroform) to obtain 47mg (equivalent to 94.9. mu. mol) of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 1, step 3).

The results of NMR measurement of the obtained 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (internal standard: tetramethylsilane) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 8.01-7.94(m, 1H), 7.71-7.67(m, 2H), 7.70-7.67(m, 1H), 7.64-7.60(m, 1H), 7.20-7.11(m, 1H), 6.89-6.85(m, 2H), 1.62-1.46(m, 6H), 1.34(sext, J ═ 7.3Hz, 6H), 1.18-1.03(m, 6H), 0.90(t, J ═ 7.3Hz, 9H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ 157.85, 145.11, 144.72, 131.90, 129.93, 127.62, 124.02, 122.59, 116.14, 116.09, 106.19, 28.96, 27.27, 13.62, 9.81.

(example I-2)¹²⁵I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Synthesis of pyridine

To 53. mu.L of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]To a methanol solution of pyridine (concentration: 1mg/mL), 75. mu.L of 1mol/L hydrochloric acid, 136MBq [ alpha ], [ solution ]¹²⁵I]Sodium iodide (40. mu.L volume) and 10. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was allowed to stand at 50 ℃ for 10 minutes, the solution was subjected to HPLC under the following conditions to obtain [ 2], [ solution ]¹²⁵I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine fraction.

HPLC conditions:

a chromatographic column: phenomenex Luna C18 (trade name; manufactured by Phenomenex Co., Ltd.; specification: 4.6X150mm)

Mobile phase: water with 0.1% trifluoroacetic acid/acetonitrile with 0.1% trifluoroacetic acid 80/20 → 0/100(17 min, linear gradient)

Flow rate: 1.0 mL/min

A detector: an ultraviolet visible absorption photometer (detection wavelength: 282nm) and a radioactivity counter (model: STEFFI, manufactured by Raytest Co., Ltd.)

To this fraction was added 10mL of water. The resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters Corp.; filling amount of filler: 130mg) so that the column was adsorbed and collected [, [ 2]¹²⁵I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine. The column was rinsed with 1mL of water, and then 1mL of ethanol was passed therethrough to elute¹²⁵I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine. The radioactivity of the resulting compound immediately after the synthesis was 37.5 MBq. Further, as a result of TLC analysis under the following conditions, the radiochemical purity of this compound was 96.5%.

TLC analysis conditions:

TLC plate: RP-¹⁸F254 (trade name; manufactured by Merck)

Mobile phase: 20/1% methanol/water

A detector: bio-image analyzer BAS-2500 (model: BAS-2500; manufactured by Fuji film Co., Ltd.)

(example I-3)¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Synthesis of pyridine

To 70. mu.L of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]To a methanol solution of pyridine (concentration: 1mg/mL), 75 to 100. mu.L of 1mol/L hydrochloric acid, 236-¹²³I]Sodium iodide (volume 15-120. mu.L), 7.5-10. mu.L of 1mmol/L sodium iodide solution and 10-15. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was heated at 50 ℃ for 10 minutes, HPLC was performed under the same conditions as in example I-2 to obtain [ alpha ], [ beta ] -an¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine fraction.

To this fraction was added 10mL of water. LetThe resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters Co., Ltd.; filling amount of a filler: 130mg) so that the column was adsorbed and collected [, [ 2]¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine. The column was rinsed with 1mL of water, and then 1mL of diethyl ether was passed therethrough to elute¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine. The radioactivity of the resulting compound immediately after synthesis was 21-180 MBq. Further, as a result of TLC analysis under the same conditions as in example I-2, the radiochemical purity of this compound was 99.5%.

Example I-4 Synthesis of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 2, step 1).

2.15g (corresponding to 10.0mmol) of 2-bromo-4' -hydroxyacetophenone and 1.74g (corresponding to 10.0mmol) of 2-amino-5-bromopyridine were dissolved in 50mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 105 ℃ for 6 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 20mL of water and 20mL of methanol. Then, about 25mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 2.41g (equivalent to 8.32mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 2, step 2).

The results of NMR measurement of the obtained 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (internal standard: dimethyl sulfoxide) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylsulfoxide-d 6, resonance frequency: 500 MHz): δ 9.54(br.s, 1H), 8.83-8.81(m, 1H), 8.17(s, 1H), 7.79-7.74(m, 2H), 7.51(d, J ═ 9.6Hz, 1H), 7.30(dd, J ═ 9.6, 1.8Hz, 1H), 6.86-6.81(m, 2H).

¹³C-NMR (solvent: dimethyl sulfoxide-d 6, resonance frequency: 125 MHz): δ 158.15, 146.40, 143.79, 127.82, 127.67, 127.14, 125.01, 117.87, 116.15, 108.60, 106.05.

Example I-5 Synthesis of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyridine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 3, step 1).

441mg (equivalent to 2.0mmol) of 2-bromo-4' -hydroxyacetophenone and 449mg (equivalent to 2.0mmol) of 2-amino-5-iodopyridine were dissolved in 15mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 5 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 10mL of water and 10mL of methanol. Then, about 10mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 526mg (equivalent to 1.56mmol) of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyridine (FIG. 3, step 2).

The results of NMR measurement of the obtained 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyridine (internal standard substance: dimethyl sulfoxide) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylsulfoxide-d 6, resonance frequency: 500 MHz): delta 8.86-8.84(m, 1H), 8.14(s, 1H), 7.78-7.74(m, 2H), 7.40-7.35(m, 2H), 6.86-6.82(m, 2H).

¹³C-NMR (solvent: dimethyl sulfoxide-d 6, resonance frequency: 125 MHz): δ 158.08, 145.87, 143.87, 132.48, 131.72, 127.67, 124.99, 118.14, 116.14, 108.02, 75.85.

Example I-6 Synthesis of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrimidine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 4, step 1).

646mg (corresponding to 3.0mmol) of 2-bromo-4' -hydroxyacetophenone and 668mg (corresponding to 3.0mmol) of 2-amino-5-iodopyrimidine are dissolved in 20mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 8 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 10mL of water and 10mL of methanol. Then, about 15mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 3 minutes. The precipitate was collected by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 737mg (equivalent to 2.19mmol) of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrimidine (FIG. 4, step 2).

The results of NMR measurement of the obtained 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrimidine (internal standard substance: dimethylformamide) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylformamide-d 7, resonance frequency: 500 MHz): δ 9.80(br.s, 1H), 9.35(d, J ═ 2.3Hz, 1H), 8.60(d, J ═ 2.3Hz, 1H), 8.23(s, 1H), 7.94-7.90(m, 2H), 6.98-6.94(m, 2H).

¹³C-NMR (solvent: dimethylformamide-d 7, resonance frequency: 125 MHz): δ 158.87, 154.00, 147.18, 146.77, 139.07, 127.68, 124.50, 115.85, 106.10, 73.46.

Example I-7 Synthesis of 6- (3 '-Fluoropropoxy) -2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine

31.11g (corresponding to 178.88mmol) of 2-bromo-3-hydroxypyridine was dissolved in 95.8mL of dimethyl sulfoxide, and 89.9mL (corresponding to 89.9mmol) of a 1mol/L sodium methoxide-methanol solution was added thereto. The reaction solution was then heated to 90 ℃ to distill off methanol. After the reaction solution was cooled to 5 ℃ or lower, 29.2g (corresponding to 205.62mmol) of methyl iodide was added, followed by stirring at room temperature for 17 hours. After completion of the reaction, the reaction solution was poured into ice water, and extracted with chloroform 2 times. The combined chloroform layers were washed with 1mol/L sodium hydroxide solution, and then with saturated sodium chloride solution 2 times, and dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, 20.74g (corresponding to 110.31mmol) of 2-bromo-3-methoxypyridine was obtained (FIG. 5, step 1).

83mL of concentrated sulfuric acid was cooled to-5 ℃ and 83mL of 90% nitric acid was carefully added thereto. Subsequently, 20.69g (corresponding to 110.04mmol) of 2-bromo-3-methoxypyridine were carefully added thereto. After the reaction mixture was stirred in the ice bath for 5 minutes, the mixture was stirred at room temperature for 10 minutes, then heated to 55 ℃, and stirred for an additional 1 hour. After the reaction solution was cooled to room temperature, the reaction solution was poured little by little into crushed ice to cause precipitation. The precipitate was filtered and washed with water and then dried over phosphorus pentoxide under reduced pressure to give 17.41g (corresponding to 74.71mmol) of 2-bromo-3-methoxy-6-nitropyridine (FIG. 5, step 2).

17.36g (corresponding to 74.50mmol) of 2-bromo-3-methoxy-6-nitropyridine were dissolved in 520mL of ethanol, and 11.63g (50% humidity) of 10% palladium on carbon was added thereto under an argon flow. 88.4mL of hydrazine monohydrate was then added dropwise to the mixture. After the reaction mixture was heated under reflux for 45 minutes, the reaction solution was cooled to room temperature. Then, after filtering off the palladium on carbon, the residue was washed with ethanol, and the washings and the filtrate were combined. The combined solution was concentrated under reduced pressure. To the concentrate were then added 402mL of water and 38mL of concentrated aqueous ammonia, and the resulting mixture was extracted 8 times with chloroform. The combined chloroform layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude product was distilled off under reduced pressure to give 8.14g (equivalent to 65.57mmol) of 2-amino-5-methoxypyridine (FIG. 5, step 3).

13.50g (corresponding to 59.66mmol) of 4' -benzoyloxyacetophenone were dissolved in 1100mL of methanol, and 34.52g (corresponding to 71.59mmol) of tetra-n-butylammonium tribromide was added thereto. The mixture was stirred at room temperature overnight and distilled under reduced pressure to remove the solvent. The residue was dissolved in ethyl acetate, washed 2 times with water and then with saturated sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting crude product was purified by flash silica gel column chromatography (elution solvent: hexane/dichloromethane ═ 1/1) to obtain 13.38g (equivalent to 43.84mmol) of 4' -benzoyloxy-2-bromoacetophenone (fig. 5, step 4).

13.33g (equivalent to 43.68mmol) of 4' -benzoyloxy-2-bromoacetophenone and 5.67g (equivalent to 45.67mmol) of 2-amino-5-methoxypyridine were dissolved in 481mL of ethanol. The resulting solution was heated to reflux for 2 hours. After the reaction solution was cooled, 6.64g (corresponding to 79.09mmol) of sodium hydrogencarbonate was added thereto. The resulting reaction mixture was heated to reflux for 4 hours. After the reaction was completed, the solvent was concentrated under reduced pressure. The resulting residue was dissolved in chloroform and then washed with water. After the chloroform layer was dried over anhydrous sodium sulfate, the solvent was distilled off. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/ethyl acetate 20/1) to obtain 10.20g (equivalent to 30.87mmol) of 2- (4' -benzoyloxyphenyl) -6-methoxyimidazo [1, 2-a ] pyridine (fig. 5, step 5).

4.90g (corresponding to 14.83mmol) of 2- (4' -benzoyloxyphenyl) -6-methoxyimidazo [1, 2-a ] pyridine, which has been dried sufficiently to remove water, are dissolved in 245mL of chloroform and cooled to-15 ℃. To this solution was added dropwise a solution prepared by dissolving 12.62mL (corresponding to 133.48mmol) of boron tribromide in 134mL of dichloromethane. After the temperature of the resulting solution was raised to room temperature, the resulting solution was stirred for 17 hours. After completion of the reaction, the reaction solution was cooled with ice and 668mL of methanol was added thereto, followed by stirring at room temperature for 3 hours. The reaction mixture was then concentrated under reduced pressure. The resulting crude product was supplemented with 290mL of chloroform and 29mL of methanol to obtain a slurry, and then the precipitate was recovered by filtration. The recovered precipitate was washed with chloroform and then dried under reduced pressure to obtain 3.00g (equivalent to 13.28mmol) of 2- (4' -hydroxyphenyl) -6-hydroxyimidazo [1, 2-a ] pyridine (FIG. 5, step 6).

560mg (equivalent to 2.48mmol) of 2- (4' -hydroxyphenyl) -6-hydroxyimidazo [1, 2-a ] pyridine is dissolved in 21mL of dimethylformamide, and 1.37g (equivalent to 9.90mmol) of potassium carbonate and 349mg (equivalent to 2.48mmol) of 1-bromo-3-fluoropropane are added thereto. The solution was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure, and then 10mL of chloroform and 10mL of methanol were supplemented to obtain a slurry. The slurry was filtered and the filtrate was concentrated. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1) to obtain 151mg (equivalent to 0.527. mu. mol) of 6- (3 '-fluoropropoxy) -2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 5, step 7).

The results of NMR measurement of the obtained 6- (3 '-fluoropropoxy) -2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (internal standard substance: dimethyl sulfoxide) are shown below.

NMR apparatus used: JNM-GSX-270 (manufactured by JEOL, Japan electronic Co., Ltd.)

¹H-NMR (solvent: dimethylsulfoxide-d 6; resonance frequency: 270 MHz): δ 9.52(s, 1H), 8.22(d, J ═ 2.2Hz, 1H), 8.08(s, 1H), 7.75-7.65(m, 2H), 7.44(d, J ═ 9.6Hz, 1H), 6.99(dd, J ═ 9.6, 2.2Hz, 1H), 6.85-6.75(m, 2H), 4.62(dt,²J_HF ＝47.0Hz，J ＝6.0Hz，2H)，4.05(t，J＝6.0Hz，2H)，2.13(dquint，³J_HF＝25.9Hz，J ＝6.0Hz，2H)。

example I-8 Synthesis of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and then recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 6, step 1).

748mg (equivalent to 3.5mmol) of 2-bromo-4' -hydroxyacetophenone and 605mg (equivalent to 3.5mmol) of 2-amino-5-bromopyrimidine are dissolved in 30mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 5 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 10mL of water and 15mL of methanol. Then, about 10mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, dried under reduced pressure, and the resulting crude product was recrystallized from N, N-dimethylformamide to give 289mg (equivalent to 0.997mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine (FIG. 6, step 2).

The results of NMR measurement (internal standard: dimethyl sulfoxide) of the obtained 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylsulfoxide-d 6, resonance frequency: 500 MHz): δ 9.56(br.s, 1H), 9.21(d, J ═ 2.5Hz, 1H), 8.46(d, J ═ 2.5Hz, 1H), 8.09(s, 1H), 7.79-7.75(m, 2H), 6.83-6.79(m, 2H).

¹³C-NMR (solvent: dimethyl sulfoxide-d 6, resonance frequency: 125 MHz): δ 158.63, 149.99, 147.68, 146.88, 134.78, 127.93, 124.52, 116.23, 106.83, 103.94.

Example I-9 Synthesis of 6-fluoro-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine

70mL of ethyl acetate was added to 40.0g (equivalent to 179mmol) of copper bromide to obtain a suspension, and a solution of 11.6g (equivalent to 85.3mmol) of 4' -hydroxyacetophenone in a mixture of 70mL of ethyl acetate and 70mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5.5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 10.2g (equivalent to 47.3mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 7, step 1).

439mg (equivalent to 2.0mmol) of 2-bromo-4' -hydroxyacetophenone and 224mg (equivalent to 2.0mmol) of 2-amino-5-fluoropyridine were dissolved in 20mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 5 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 8mL of water and 8mL of methanol. Then, about 10mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 302mg (equivalent to 1.32mmol) of 6-fluoro-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 7, step 2).

The results of NMR measurement of the obtained 6-fluoro-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (internal standard substance: dimethyl sulfoxide) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylsulfoxide-d 6, resonance frequency: 500 MHz): δ 9.45(br.s, 1H), 8.65(ddd,³J_HF＝4.6Hz，J＝2.5，0.7Hz，1H)，8.16-8.15(m，1H)，7.75-7.69(m，2H)，7.56-7.51(m，1H)，7.23(ddd，³J_HF＝8.4Hz，J＝9.9，2.5Hz，1H)，6.82-6.76(m，2H)。

¹³C-NMR (solvent: dimethyl sulfoxide-d 6, resonance frequency: 125 MHz): δ 157.82, 152.81(d,¹J_CF＝232.3Hz)，146.58，142.92，127.35，124.99，117.19(d，³J_CF＝9.6Hz)，116.40(d，²J_CF＝25.9Hz)，115.89，113.66(d，²J_CF＝41.8Hz)，109.48。

¹⁹F-NMR (solvent: dimethylsulfoxide-d 6, resonance frequency: 470 MHz): δ 141.93 (br.s).

Example I-10 Synthesis of 2- (4' -hydroxyphenyl) -6-nitroimidazo [1, 2-a ] pyridine

70mL of ethyl acetate was added to 40.0g (equivalent to 179mmol) of copper bromide to obtain a suspension, and a solution of 11.6g (equivalent to 85.3mmol) of 4' -hydroxyacetophenone in a mixture of 70mL of ethyl acetate and 70mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5.5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 10.2g (equivalent to 47.3mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 8, step 1).

432mg (equivalent to 2.0mmol) of 2-bromo-4' -hydroxyacetophenone and 279mg (equivalent to 2.0mmol) of 2-amino-5-nitropyridine are dissolved in 20mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 6 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 8mL of water and 8mL of methanol. Then, about 8mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 3 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 148mg (equivalent to 0.580mmol) of 2- (4' -hydroxyphenyl) -6-nitroimidazo [1, 2-a ] pyridine (FIG. 8, step 2).

The results of NMR measurement of the obtained 2- (4' -hydroxyphenyl) -6-nitroimidazo [1, 2-a ] pyridine (internal standard substance: dimethyl sulfoxide) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylsulfoxide-d 6, resonance frequency: 500 MHz): δ 9.74-9.72(m, 1H), 9.59(br.s, 1H), 8.39(s, 1H), 7.87(dd, J ═ 9.9, 2.3Hz, 1H), 7.79-7.74(m, 2H), 7.65-7.61(m, 1H), 6.84-6.80(m, 2H).

¹³C-NMR (solvent: dimethyl sulfoxide-d 6, resonance frequency: 125 MHz): δ 158.47, 148.51, 145.25, 136.63, 127.93, 127.81, 124.06, 118.92, 116.09, 115.92, 110.37.

Example II-1 Synthesis of 6-Tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 10, step 1).

748mg (equivalent to 3.48mmol) of 2-bromo-4' -hydroxyacetophenone and 605mg (equivalent to 3.48mmol) of 5-bromo-2-aminopyrimidine were dissolved in 30mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 5 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 10mL of water and 15mL of methanol. Then, about 10mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure. The resulting solid was recrystallized from DMF to give 289mg (equivalent to 1.00mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine (FIG. 10, step 2).

75.4mg (equivalent to 0.260mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine was dissolved in 10.0mL of dioxane, and 2.0mL of triethylamine was added thereto. Then, 0.20mL (equivalent to 0.39mmol) of bis (tributyltin) and 20.1mg (catalytic amount) of tetrakis (triphenylphosphine) palladium were added thereto. After the reaction mixture was stirred at 90 ℃ for 10 hours, the solvent was distilled off under reduced pressure. The residue was purified by flash silica gel column chromatography (elution solvent: hexane/ethyl acetate ═ 1/1) to give 24.0mg (equivalent to 0.048mmol) of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine (fig. 10, step 3).

The results of NMR measurement of the obtained 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrimidine (internal standard: tetramethylsilane) are shown below

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 8.41(s, 1H), 8.23(s, 1H), 7.80(d, J ═ 8.7Hz, 2H), 7.63(s, 1H), 6.93(d, J ═ 8.7Hz, 2H), 1.57-1.51(m, 6H), 1.37-1.23(m, 6H), 1.16-1.12(m, 6H), 0.88(d, J ═ 7.3Hz, 9H)

Example II-2 Synthesis of 6-Tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 11, step 1).

4.66g (corresponding to 21.6mmol) of 2-bromo-4' -hydroxyacetophenone and 2.53g (corresponding to 14.5mmol) of 5-bromo-2-aminopyrazine were dissolved in 100mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 3.5 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 10mL of water and 10mL of methanol. Then, about 20mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 10 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 1.32g (equivalent to 4.55mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine (fig. 11, step 2).

1.00g (equivalent to 3.45mmol) of 6-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine was dissolved in 50.0mL of dioxane, and 20.0mL of triethylamine was added thereto. Then, 4.5mL (equivalent to 5.18mmol) of bis (tributyltin) and 239mg (catalytic amount) of tetrakis (triphenylphosphine) palladium were added thereto. After the reaction mixture was stirred at 90 ℃ for 24 hours, the solvent was distilled off under reduced pressure. The residue was purified by flash silica gel column chromatography (elution solvent: hexane/ethyl acetate 1/1) to give 314mg (equivalent to 0.628mmol) of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine (fig. 11, step 3).

The results of NMR measurement of the obtained 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine (internal standard: tetramethylsilane) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 9.21(s, 1H), 7.95(s, 1H), 7.79(d, J ═ 8.7Hz, 2H), 7.77(s, 1H), 6.91(d, J ═ 8.7Hz, 2H), 1.70-1.55(m, 6H), 1.38-1.31(m，6H)，1.18-1.15(m，6H)，0.89(d，J＝7.3Hz，9H)

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ 157.3, 146.4, 143.5, 140.3128.0, 124.9, 123.6, 116.1, 106.9, 29.0, 27.3, 13.7, 10.0.

Example II-3 Synthesis of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrazine

314mg (corresponding to 0.628mmol) of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyrazine obtained in example II-2 are dissolved in 5.0mL of dichloromethane, to which 114mg (corresponding to 0.942mmol) of iodine dissolved in 5.0mL of dichloromethane are added. The reaction mixture was stirred at a temperature of 0 ℃ for 10 minutes and at room temperature for 30 hours. Then, a saturated aqueous sodium hydrogencarbonate solution and a saturated aqueous sodium thiosulfate solution were added thereto. The precipitate was collected by filtration, washed with water and ethyl acetate in this order, and dried under reduced pressure to obtain 131mg (equivalent to 0.389mmol) of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrazine (FIG. 12, step 1).

The results of NMR measurement of the obtained 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrazine (internal standard: tetramethylsilane) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: dimethylformamide-d 7, resonance frequency: 500 MHz): δ 9.89(s, 1H), 9.01(s, 1H), 8.82(s, 1H), 8.42(s, 1H), 7.92(d, J ═ 8.7Hz, 2H), 6.93(d, J ═ 8.7Hz, 2H).

Example II-4 Synthesis of 8-Tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine

50mL of ethyl acetate was added to 28.17g (equivalent to 126mmol) of cupric bromide to obtain a suspension, and a mixture of 8.18g (equivalent to 60.0mmol) of 4' -hydroxyacetophenone in 50mL of ethyl acetate and 50mL of chloroform was added thereto. The resulting mixture was then heated to reflux. After 5 hours, the reaction mixture was cooled to room temperature and filtered. The resulting filtrate was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and decolorized by adding activated carbon. Then, the resulting solution was concentrated by filtration. The obtained crude product was purified by flash silica gel column chromatography (elution solvent: chloroform/methanol-20/1), and recrystallized from ethyl acetate/petroleum ether to obtain 7.25g (equivalent to 33.7mmol) of 2-bromo-4' -hydroxyacetophenone (fig. 13, step 1).

432mg (equivalent to 2.01mmol) of 2-bromo-4' -hydroxyacetophenone and 348mg (equivalent to 2.01mmol) of 3-bromo-2-aminopyridine were dissolved in 20mL of acetonitrile. The resulting solution was heated to reflux in an oil bath at 110 ℃ for 6 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, and the precipitate was recovered by filtration. The precipitate was washed with acetonitrile and dried under reduced pressure. The resulting crude crystals were suspended in a mixture of 8mL of water and 8mL of methanol. Then, about 8mL of a saturated sodium bicarbonate solution was added thereto, and the mixture was sonicated with a sonicator for 5 minutes. The precipitate was recovered by filtration from the resulting mixture, washed well with water, and dried under reduced pressure to obtain 368mg (equivalent to 1.27mmol) of 8-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (FIG. 13, step 2).

75.2mg (equivalent to 0.260mmol) of 8-bromo-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine was dissolved in 10.0mL of dioxane, and 2.0mL of triethylamine was added thereto. Then, 0.20mL (equivalent to 0.39mmol) of bis (tributyltin) and 20.1mg (catalytic amount) of tetrakis (triphenylphosphine) palladium were added thereto. After the reaction mixture was stirred at 90 ℃ for 11 hours, the solvent was distilled off under reduced pressure. The residue was purified by flash silica gel column chromatography (elution solvent: hexane/ethyl acetate 1/1) to give 62.5mg (equivalent to 0.125mmol) of 8-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (fig. 13, step 3).

The results of NMR measurement of the obtained 8-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a ] pyridine (internal standard: tetramethylsilane) are shown below.

NMR apparatus used: JNM-ECP-500 (manufactured by JEOL Ltd.)

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 8.01(d, J ═ 6.4Hz, 1H), 7.87(d, J ═ 8.7Hz, 2H), 7.70(s, 1H), 7.17(d, J ═ 6.4Hz, 1H), 6.87(d, J ═ 8.7Hz, 2H), 6.68-6.66(m, 1H), 1.69-1.56(m, 6H), 1.38-1.30(m, 6H), 1.28-1.16(m, 6H), 0.88(t, J ═ 7.3Hz, 9H)

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ 145.2, 141.0, 139.2, 132.4, 131.8, 127.7, 127.3, 125.0, 115.4, 112.2, 106.4, 29.2, 27.4, 13.7, 10.2.

(example II-5)¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Synthesis of pyrimidines

To 100. mu.L of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]To a methanol solution of pyrimidine (concentration: 1mg/mL), 100. mu.L of 2mol/L hydrochloric acid, 621MBq of¹²³I]Sodium iodide (volume 150. mu.L), 20. mu.L of a 1mmol/L sodium iodide solution and 20. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was left to stand at 50 ℃ for 10 minutes, the solution was subjected to HPLC under the same conditions as in example I-2 to obtain [ 2], [¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]A pyrimidine fraction.

Performing the same operation as described in the above paragraph to obtain [ 2]¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyrimidine (amount of reagent added: 150. mu.L of 6-tributylstannyl-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a)]A methanol solution of pyrimidine (concentration: 1mg/mL), 75. mu.L of 2mol/L hydrochloric acid, 487MBq of¹²³I]Sodium iodide (volume 150. mu.L), 20. mu.L of a 1mmol/L sodium iodide solution and 30. mu.L of 10% (W/V) hydrogen peroxide).

The two fractions obtained in the two stages above were mixed and 10mL of water was added. The resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters; filling amount of filler: 145mg) so thatThe column is adsorbed and collected¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]A pyrimidine. The column was rinsed with 1mL of water, and then 1mL of diethyl ether was passed therethrough to elute¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]A pyrimidine. The radioactivity of the resulting compound immediately after the synthesis was 67 MBq. Further, as a result of TLC analysis under the following conditions, the radiochemical purity of this compound was 92.5%.

TLC analysis conditions:

TLC plate: silica gel 60F₂₅₄(trade name; manufactured by Merck Co., Ltd.)

Mobile phase: chloroform/methanol/triethylamine 100/1/2

A detector: rita Star (trade name; manufactured by raytest)

(example II-6)¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Synthesis of pyrazines

To 100. mu.L of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]To a methanol solution of pyrazine (concentration: 1mg/mL), 75. mu.L of 2mol/L hydrochloric acid, 469MBq [ sic ], [ solution ]¹²³I]Sodium iodide (100. mu.L in volume), 20. mu.L of a 1mmol/L sodium iodide solution and 20. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was left to stand at 50 ℃ for 10 minutes, the solution was subjected to HPLC under the same conditions as in example I-2 to obtain [ 2], [¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]A pyrazine fraction.

To this fraction was added 10mL of water. The resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters Co.; filling amount of filler: 145mg) so that the column was adsorbed and collected [, [ 2]¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]A pyrazine. The column was rinsed with 1mL of water, and then 1mL of diethyl ether was passed therethrough to elute¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]A pyrazine. The radioactivity of the resulting compound immediately after the synthesis was 133 MBq. Further, under the same conditions as in example II-5TLC analysis was carried out, and as a result, the radiochemical purity of this compound was 99.0%.

(example II-7)¹²³I]-2- (4' -hydroxyphenyl) -8-iodoimidazo [1, 2-a]Synthesis of pyridine

To 70. mu.L of 8-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]To a methanol solution of pyrazine (concentration: 1mg/mL), 50. mu.L of 2mol/L hydrochloric acid, 454 MBq's [ alpha ], [ beta ]¹²³I]Sodium iodide (100. mu.L in volume), 20. mu.L of a 1mmol/L sodium iodide solution and 20. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was left to stand at 50 ℃ for 10 minutes, the solution was subjected to HPLC under the same conditions as in example I-2 to obtain [ 2], [¹²³I]-2- (4' -hydroxyphenyl) -8-iodoimidazo [1, 2-a]Pyridine fraction.

To this fraction was added 10mL of water. The resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters Corp.; filling amount of filler: 130mg) so that the column was adsorbed and collected [, [ 2]¹²³I]-2- (4' -hydroxyphenyl) -8-iodoimidazo [1, 2-a]Pyridine. The column was rinsed with 1mL of water, and then 1mL of diethyl ether was passed therethrough to elute¹²³I]-2- (4' -hydroxyphenyl) -8-iodoimidazo [1, 2-a]Pyridine. The radioactivity of the resulting compound immediately after the synthesis was 185 MBq. Further, as a result of TLC analysis under the same conditions as in example II-5, the radiochemical purity of this compound was 91.7%.

(reference example 12 [ ]¹²⁵I]Synthesis of IMPY

Synthesized for evaluation of logP according to the following procedure_{Octanol (I)}The [ 2], [ used in the comparative example (comparative example I-6)¹²⁵I]-IMPY。

6-Tributylstannyl-2- [ 4' - (N, N-dimethylamino) phenyl ] was synthesized according to the method described in literature (Zhi-Ping Zhuang et al, J.Med.chem., 2003, 46, p.237-243)]Imidazo [1, 2-a ]]Pyridine, and dissolved in methanol (concentration: 1 mg/mL). To 53. mu.L of the resulting solution, 75. mu.L of 1mol/L hydrochloric acid, 20. mu.L of 13.5MBq [ alpha ], [ solution ]¹²⁵I]Sodium iodide and 10. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was allowed to stand at 50 ℃ for 10 minutes, the solution was subjected to HPLC under the same conditions as in example I-2 to obtain [ 2], [¹²⁵I]-an IMPY fraction.

To this fraction was added 10mL of water. The resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters Corp.; filling amount of filler: 130mg) so that the column was adsorbed and collected [, [ 2]¹²⁵I]-IMPY. The column was rinsed with 1mL of water, and then 1mL of ethanol was passed therethrough to elute¹²⁵I]-IMPY. The radioactivity of the resulting compound immediately after the synthesis was 2.6 MBq. Further, as a result of TLC analysis under the same conditions as in example I-2, the radiochemical purity of this compound was 98.0%.

(reference example 2 [ ]¹²³I]Synthesis of IMPY

The [ 2], [ comparative example I-7 ] used in the comparative example for evaluation of accumulation in the brain was synthesized according to the following procedure¹²³I]-IMPY。

6-Tributylstannyl-2- [ 4' - (N, N-dimethylamino) phenyl ] was synthesized according to the method described in literature (Zhi-Ping Zhuang et al, J.Med.chem., 2003, 46, p.237-243)]Imidazo [1, 2-a ]]Pyridine, and dissolved in methanol (concentration: 1 mg/mL). To 53. mu.L of the resulting solution, 100. mu.L of 1mol/L hydrochloric acid, 20 to 50. mu.L of 190-¹²³I]Sodium iodide, 10. mu.L of a 1mmol/L sodium iodide solution and 10. mu.L of 10% (W/V) hydrogen peroxide. After the mixture was allowed to stand at 50 ℃ for 10 minutes, the solution was subjected to HPLC under the same conditions as in example I-2 to obtain [ 2], [¹²³I]-an IMPY fraction.

To this fraction was added 10mL of water. The resulting solution was passed through a reverse phase column (trade name; Sep-Pak (registered trade name) Light C18 bridges, manufactured by Waters Corp.; filling amount of filler: 130mg) so that the column was adsorbed and collected [, [ 2]¹²³I]-IMPY. The column was rinsed with 1mL of water, and then 1mL of diethyl ether was passed therethrough to elute¹²³I]-IMPY. At the very end of the synthesis, the resulting compoundsThe radioactivity of the product is 47-56 MBq. Further, as a result of TLC analysis under the same conditions as in example I-2, the radiochemical purity of this compound was 98.0%.

(examples I-11 to I-14, comparative examples I-1 to I-5) measurement of amyloid-binding Properties

The compounds of the invention were tested for affinity for amyloid by the in vitro binding assay described below.

(1) Mixing A beta_1-40(manufactured by peptide research Co., Ltd.) was dissolved in a phosphate buffer (pH7.4) and shaken at 37 ℃ for 62 to 72 hours to obtain an aggregated A.beta.suspension (concentration: equivalent to 1mg/mL, hereinafter, referred to as amyloid suspension in this example).

(2) According to the literature (Naiki, H. et al, Labeled Investigation,74p.374-383(1996)) based on fluorescence spectrophotometry using thioflavin T (manufactured by Fluka corporation), to confirm that the agglutinated a β obtained in (1) is amyloid (measurement conditions: excitation wavelength 446nm, fluorescence wavelength 490 nm).

(3) According to the literature (Wang, Y. et al, J.Labeled Compound amplified Pharmaceut).44S239(2001)) from the labeling precursor 2- (4' -aminophenyl) benzothiazole¹²⁵I]2- (3 '-iodo-4' -aminophenyl) benzothiazole (hereinafter referred to as "[ solution ]¹²⁵I]3' -I-BTA-0) dissolved in ethanol. Use of a solution of 12 to 71MBq¹²⁵I]Sodium iodide (volume 10-30. mu.L) is prepared to obtain a sodium salt of 1-22MBq at the end of the synthesis¹²⁵I]3' -I-BTA-0. Congo red, thioflavin T and 6-methyl-2- [ 4' - (N, N-dimethylamino) phenyl]Benzothiazole (hereinafter referred to as 6-Me-BTA-2) was directly weighed in the form of a commercially available reagent and used.

(4) According to the literature (Wang, Y. et al, J.Labeled Compounds Radiopharmaceut, respectively.44S239(2001)) and literature (Zhuang, z.p. et al, j.med.chem.46Synthesis of 2- (3' -iodo) according to the method described in 237(2003)-4 '-aminophenyl) benzothiazole (hereinafter referred to as 3' -I-BTA-0) and IMPY.

(5) Preparation of¹²⁵I]3' -I-BTA-0, each evaluation compound and amyloid were dissolved in a phosphate buffer (pH7.4) containing 0.1% bovine serum albumin, and their final concentrations were as shown in Table 2. The resulting samples were filled in each well (volume about 0.3mL) of a 96-well microplate.

Table 2: final concentration of each compound in the sample solution

(6) The microplate filled with the sample solution was shaken at a specified speed (400rpm) at 22 ℃ for 3 hours. Each sample solution was then passed through a glass fiber filter (trade name; Mulutiscreen;)^TM-FC, manufactured by Millipore) filtration to separate from free [ solution ]¹²⁵I]Separating amyloid-binding protein from 3' -I-BTA-0¹²⁵I]3′-I-BTA-0。

(7) The glass fiber filter used for filtering each sample solution was washed (0.5 mL. times.5) with a phosphate buffer solution (pH7.4) containing 0.1% bovine serum albumin, and the radioactivity of the glass fiber filter was measured by the Autowell Gamma System (model: ARC-301B, manufactured by Aloka). This radioactivity was used as the radioactivity level of each sample solution bound to amyloid to calculate the inhibition rate (hereinafter, a represents the radioactivity level in the sample when the concentration of each evaluation compound was zero (0), and B represents the radioactivity level in the sample when the concentration of each evaluation compound was 0.001nmol/L or more).

(8) Further, a solution containing 15. mu. mol/L of 6-Me-BTA-2, 400pmol/L in a phosphate buffer solution (pH7.4) containing 0.1% bovine serum albumin was prepared¹²⁵I]3' -I-BTA-0 and 1. mu. mol/L of Abeta_1-40The radioactivity level was measured by the same procedures as described in (6) and (7) above. Will be determinedThe radioactivity level was defined as the background radioactivity level and used to calculate the inhibition rate (hereinafter referred to as BG).

(9) The inhibition rate was determined by the following formula (1) using the radioactivity levels measured in the above (7) and (8).

The value converted by probability unit (probit) from the obtained inhibition rate with respect to the logarithm of the evaluation compound concentration was plotted to obtain an approximate straight line by the least square method. Using this line, the concentration of each evaluation compound when the radioactivity level was half that of a sample not containing each evaluation compound was evaluated, and defined as the 50% inhibitory concentration (hereinafter referred to as IC) of each compound₅₀% value). Using the values as indices, evaluation was made for each of the evaluation compounds and amyloid (aggregated A.beta._1-40) The affinity of (a).

IC of each evaluation Compound_50％The values are shown in Table 3. Compounds 1 to 4 all showed IC's of less than 100_50％Value, compared to congo red and thioflavin T, to amyloid (agglutinated Abeta)_1-40) Has higher affinity. As a result, it was found that the compounds 1 to 4 were found to be associated with amyloid (agglutinated Abeta)_1-40) Has good affinity. In particular, Compound 1 compares to amyloid (aggregated A β) as compared to 3' -I-BTA-0 and 6-Me-BTA-2_1-40) Has higher affinity and the affinity is equivalent to that of IMPY.

Table 3: IC of the Compounds of the invention_50％

Experiment of	Evaluation of Compounds	IC50％Value (nmol/L)
			Comparative example I-1	3′-I-BTA-0	10.1
Comparative example I-2	Congo red	＞1000
			Comparative example I-3	Thioflavin T	＞1000
Comparative example I-4	6-Me-BTA-2	25.4
			Comparative example I-5	IMPY	4.0
Examples I to 11	Compound 1	4.4
			Examples I to 12	Compound 2	46.0
Examples I to 13	Compound 3	54.4
			Examples I to 14	Compound 4	54.1

(example I-15, examples II-8 to II-10, comparative example I-6) measurement of partition coefficient based on octanol extraction

The partition coefficient (hereinafter referred to as logP) was measured by octanol extraction which is generally known as an indicator of the penetration of a compound through the blood brain barrier (hereinafter referred to as BBB)_{Octanol (I)})。

An ether solution of the compound 5 prepared in example I-2 (example I-15), an ether solution of the compound 9 prepared in example II-5 (example II-8), an ether solution of the compound 10 prepared in example II-6 (example II-9), an ether solution of the compound 11 prepared in example II-7 (example II-10) and an ether solution prepared in reference example 1 were each diluted with a physiological saline solution containing 10mg/mL of ascorbic acid¹²³I]IMPY in ether (comparative example I-6), adjusted to a radioactive concentration of 20-30 MBq/ml. To 2mL of octanol were added 10. mu.L each of the prepared sample solutions, and 2mL of 10mmol/L phosphate buffer solution (pH7.4) was added, followed by stirring for 30 seconds. After the mixture was centrifuged (2000rpmx60 minutes) by a low-speed centrifuge, 1mL each of the octanol layer and the aqueous layer was sampled, and the respective radioactivity count was determined by using an Autowell Gamma system (model: ARC-301B, manufactured by Aloka). Using the obtained radioactivity counts, logP was calculated according to equation (2)_{Octanol (I)}。

The results are shown in Table 4. logP exhibited by Compound 5_{Octanol (I)}Value of 1.6¹²⁵I]logP indicated by IMPY_{Octanol (I)}The value was 2.1. logP of compounds known to permeate across the BBB_{Octanol (I)}Values ranged from 1 to 3 (Douglas D.Dischino et al, J.Nucl.Med., (1983), 24, p.1030-1038). Thus, this indicates that both compounds have BBB permeability equivalent to IMPY.

Table 4: logP of the Compounds of the invention_{Octanol (I)}Value of

Experiment of	Compound (I)	logPOctanol (I)Value of
			Comparative example I-6	[125I]-IMPY	2.1
Examples I to 15	Compound 5	1.6
			Examples II to 8	Compound 9	1.7
Examples II to 9	Compound 10	2.3
			Examples II to 10	Compound 11	3.0

(examples I-16, comparative example I-7) measurement of metastatic and clearance in brain

Using compound 6, the change with time of radioactive accumulation in the brain of male Wistar rats (7 weeks old) was measured.

Under thiopentan anesthesia, Compound 6 (example I-16) was dissolved in a 0.05mL solution (radioactive concentration of 20-30MBq/mL) in a physiological saline solution containing 10mg/mL ascorbic acid, and prepared in the above reference example 2¹²³I]IMPY (comparative example I-7) dissolved in a physiological saline solution containing 10mg/mL of ascorbic acid (radioactive concentration 20-30MBq/mL) was injected into the tail vein of each Wistar rat (7 weeks old). After 2, 5, 30 and 60 minutes from injection, these rats were sacrificed by exsanguination from the abdominal aorta, brains were removed, and radioactivity of the brains (hereinafter, referred to as a in this example) was measured using an autowell gamma system (model: ARC-301B, manufactured by Aloka), and further, the quality of the brains was measured 2, 5, 30 and 60 minutes after injection. In addition, the radioactivity of a 1000-fold diluted solution of 0.05mL of the injected solution (hereinafter, referred to as B in this example) was measured in the same manner as described above. Using these measurement results, the radioactivity distribution (% ID/g) per unit brain weight at each time point was calculated according to the following formula (3).

At each time point, two animals were used for example I-16 and comparative example I-7.

The results are shown in Table 5. As shown in Table 5, at the time point of 2 minutes after injection, Compound 6 showed accumulation and¹²³I-IMPY was comparable and subsequently showed a tendency to clear rapidly within 60 minutes. These resultsIn addition, compound 6 has the formula¹²³I-IMPY has the same excellent brain transferability and rapid clearance from the brain.

Table 5: accumulation of radioactivity in the brain following intravenous injection of Compound 6 (rat)

(example I-17) confirmation of amyloid in brain by imaging

The following experiment was performed to examine whether amyloid in the brain can be imaged by the compound of the present invention.

(1) Mixing A beta_1-40(manufactured by peptide research Co., Ltd.) was dissolved in a phosphate buffer (pH7.4) and shaken at 37 ℃ for 72 hours to obtain an aggregated A.beta.suspension (A.beta.concentration: equivalent to 1mg/mL, hereinafter, referred to as amyloid suspension in this example).

(2) mu.L (equivalent to 25. mu.g) of the amyloid suspension was injected into the amygdala on one side of male Wistar rats (7 weeks old). As a control, 25. mu.L of phosphate buffered saline solution (pH7.4) was injected into the amygdala on the other side of the rat. The rats were examined 1 day after injection of the amyloid suspension and phosphate buffered saline solution (pH 7.4).

(3) Compound 6 was dissolved in a physiological saline solution containing 10mg/mL of ascorbic acid to obtain a sample solution (radioactive concentration of 32 MBq/mL). The solution was injected into rats through the tail vein (dose: 0.5mL, radioactivity given: equivalent to 16 MBq).

(4) The brain was removed 60 minutes after the injection, and a brain section having a thickness of 10 μm was prepared with a microtome (model: CM3050S, manufactured by LEICA). The brain slice was exposed on an imaging plate for 20 hours, and then image analysis was performed by using a bioimage analyzer (model: BAS-2500; manufactured by Fuji film Co., Ltd.).

(5) After image analysis with a bioimaging analyzer, pathological staining was performed with thioflavin T, and imaging was performed by using a fluorescence microscope (model: TE 2000-U; manufactured by Nikon Co., Ltd.; excitation wavelength: 400-. Thus demonstrating amyloid deposition on the sections (fig. 9 b).

Figure 9 shows autoradiograms and thioflavine T stained images of brain sections from rats injected intracerebrally with amyloid. As shown in fig. 9, significant radioactive accumulation was observed in the amygdala on the side injected with the amyloid suspension. From the results of the staining of the radioactive accumulation site with thioflavin T, the presence of amyloid at the accumulation site was confirmed. On the other hand, no significant accumulation of radioactivity was observed in the amygdala on the side of the saline solution injection compared to other sites.

These results suggest that compound 6 has the property of accumulating on amyloid in the brain and has the ability to image amyloid in the brain.

Examples I-18 to I-20 Back-mutation assay

To test the gene mutagenicity of compounds 1, 2 and 4, a back-mutation test (hereinafter referred to as Ames test) was performed using Salmonella typhimurium TA98 and TA 100.

The test was carried out with and without the addition of S9 mix. Dimethylsulfoxide was used as a negative control. The positive control used 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide without the addition of S9mix and 2-aminoanthracene with the addition of S9 mix.

The amount of each sample added to the test plate was 7 doses (geometric ratio: 4), and the maximum dose was 5000. mu.g/plate. Each test sample solution and the test strain (TA98 or TA100), or the test sample, S9mix and the test strain were mixed together, and then the mixture was overlaid in layers on the medium of the test plate with soft agar, followed by incubation at 37 ℃ for 48 hours. The number of revertant colonies on the plate was counted after the culture, and when the number of revertant colonies was 2 times or more as large as that of the negative control and an increase in concentration-dependent behavior was exhibited, the mutagenicity was judged to be positive.

The results are shown in Table 6. The number of reversion colonies of each test strain in the groups treated with compounds 1, 2 and 4 was 2 times smaller than that of the group treated with the negative control, regardless of whether S9mix was added to each strain and the amount of the test sample added. From the above results, it was confirmed that compounds 1, 2 and 4 were negative in the Ames test and had no gene mutagenicity.

Table 6: results of Ames test

(examples II-11, II-12, comparative example II-1) measurement of metastatic property and clearance property in brain

Using compounds 10 and 11, the change with time of radioactive accumulation in the brain of male Wistar rats (7 weeks old) was measured.

Under thiopentone sodium anesthesia, Compound 10 (example II-11), Compound 11 (example II-12) and the [ prepared ] in reference example 2 above¹²³I]Each 0.05mL solution (radioactive concentration 20-31MBq/mL) of IMPY dissolved in a physiological saline solution containing 10mg/mL of ascorbic acid was injected into the tail vein of each Wistar rat. After 2, 5, 30 and 60 minutes of injection, these rats were sacrificed by exsanguination from the abdominal aorta, and the brains were removed and measured for brain mass, and then radioactivity of the brains (hereinafter, referred to as A in this example) was measured by a single channel analyzer (model of detector: SP-20, manufactured by Utility light research). In addition, the radioactivity of the remaining part of the whole body was measured in the same manner as described above (hereinafter, referred to as "B" in this example). Using these measurement results, the radioactivity accumulation (% ID/g) per unit brain weight at each anatomical time point was calculated from the following formula (4).

Experiments were performed using 3 animals at each time point.

The results are shown in Table 7. As shown in Table 7, at a time point of 2 minutes after the injection, the compounds 10 and 11 were used in combination with [ alpha ], [ beta¹²³I]IMPY, likewise, shows a marked accumulation of radioactivity, followed by a tendency to rapidly eliminate within 60 minutes. These results suggest that Compounds 10 and 11 and [, ]¹²³I]IMPY, as well, has excellent intracerebral transferability and rapid clearance from the brain.

Table 7: radioactive accumulation of the Compounds of the invention in the brain following intravenous injection (rats)

(examples II-13) Ex vivo autoradiographs of Compound 10 Using an amyloid-injected rat model

(1) Mixing A beta_1-42(manufactured by Peptist research Co., Ltd.) was dissolved in a phosphate buffer (pH7.4) and shaken at 37 ℃ for 72 hours to obtain a 1mg/mL aggregated A β suspension (hereinafter, referred to as an amyloid suspension in this example).

(2) 2.5 μ L (equivalent to 25 μ g) of the amyloid suspension was injected into the amygdala on one side of male Wistar rats (7 weeks old). As a control, 2.5. mu.L of phosphate buffered saline solution (pH7.4) was injected into the amygdala on the other side of the rat. The rats were examined 1 day after injection of the amyloid suspension and phosphate buffered saline solution (pH 7.4).

(3) Compound 10 was dissolved in a physiological saline solution containing 10mg/mL of ascorbic acid to obtain a sample solution (in which the radioactive concentration was 31 MBq/mL). The solution was injected into rats through the tail vein under thiopentan anesthesia (dose: 0.5mL, radioactivity given: equivalent to 15 MBq).

(4) The brain was removed 60 minutes after the injection, and a brain section having a thickness of 10 μm was prepared with a microtome (model: CM3050S, manufactured by LEICA). The brain slice was exposed on an imaging plate for 20 hours, and then image analysis was performed by using a bioimage analyzer (model: BAS-2500; manufactured by Fuji film Co., Ltd.).

(5) After image analysis with a bioimaging analyzer, pathological staining was performed with thioflavin T, and imaging was performed by using a fluorescence microscope (manufactured by Nikon Co., Ltd.; model: TE 2000-U; excitation wavelength: 400-. Thus demonstrating amyloid deposition on the section (fig. 14 b).

Figure 14 shows autoradiograms and thioflavine T stained images of brain sections from rats injected intracerebrally with amyloid. As shown in fig. 14, significant radioactive accumulation was observed in the amygdala on the side injected with the amyloid suspension. On the other hand, no significant accumulation of radioactivity was observed in the amygdala on the side of the saline solution injection compared to other sites. On the autoradiogram, almost no accumulation of radioactivity was observed at positions other than the site of amyloid injection. From the results of thioflavin T staining, the presence of amyloid at the accumulation site was confirmed (fig. 14 b). These results suggest that compound 10 has the property of accumulating on amyloid in the brain and has the ability to image amyloid in the brain.

(examples II-14) Ex vivo autoradiographs of Compound 11 using an amyloid-injected rat model

The same procedures as in examples II-13 were carried out, except that a solution obtained by dissolving Compound 11 in 10mg/mL ascorbic acid solution (the radioactive concentration in the sample solution was 30MBq/mL) was used as the sample solution.

Figure 15 shows autoradiograms and thioflavine T stained images of brain sections from rats injected intracerebrally with amyloid. As shown in fig. 15, significant radioactive accumulation was observed in the amygdala on the side injected with the amyloid suspension. From the results of thioflavin T staining, the presence of amyloid at the accumulation site was confirmed (fig. 15 b). On the other hand, no significant accumulation of radioactivity was observed in the amygdala on the side of the saline solution injection compared to other sites. These results suggest that compound 11 has the property of accumulating on amyloid in the brain and has the ability to image amyloid in the brain.

Example II-15 Back-mutation assay

To test the gene mutagenicity of compound 8, a back-mutation assay (hereinafter referred to as Ames assay) was performed using Salmonella typhimurium TA98 and TA 100.

The test was carried out without addition of S9mix and with addition of S9 mix. Dimethylsulfoxide was used as a negative control. The positive control used 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide without the addition of S9mix and 2-aminoanthracene with the addition of S9 mix.

The amount of compound 8 added to the test plates was 7 doses (geometric ratio: 3), and the maximum dose was 5000. mu.g/plate. After mixing the compound 8 and the test strain (TA98 or TA100), or the compound 8, S9mix and the test strain together, the mixture was overlaid in layers on the medium of the test plate with soft agar and then incubated at 37 ℃ for 48 hours. The number of revertant colonies on the plate after incubation was counted, and when the number of revertant colonies was not less than 2 times that of the negative control and showed a concentration-dependent increase, the mutagenicity was judged to be positive.

The results are shown in Table 8. The number of reversion colonies of each test strain in the group treated with compound 8 was 2 times smaller than that of the group treated with the negative control, regardless of whether S9mix was added to each strain and the amount of the test sample added. On the other hand, a significant increase in the number of revertant colonies was observed in the group treated with the positive control. From the above results, it was confirmed that Compound 8 was negative in the Ames test and had no gene mutagenicity.

Table 8: results of Ames test

(example III-1 to example III-2) confirmation of amyloid binding

To investigate the binding mechanism of the compounds of the present invention, experiments were performed to inhibit the binding to thioflavin T using amyloid protein produced with dextrin as a precursor compound. Dextrin is an amyloid protein that accumulates in the pancreas in I I type diabetes.

Method

(1) Dextrin (human) (manufactured by Wako corporation) was dissolved in a phosphate buffer (pH7.4) and shaken at 37 ℃ for 72 hours to obtain a coagulated dextrin suspension (hereinafter, referred to as an amyloid suspension in this example) of 1 mg/mL.

(2) According to the literature (Naiki, H. et al, Labeled Investigation,74p.374-383(1996)) based on fluorescence spectrophotometry using thioflavin T (manufactured by Fluka corporation), to confirm that the agglutinated dextrin obtained in (1) is an amyloid protein (measurement conditions: excitation wavelength 446nm, fluorescence wavelength 490 nm).

(3) The amyloid suspension was dissolved in 50mM phosphate buffer (pH7.4) at a dextrin concentration of 15. mu.M. Meanwhile, thioflavin T was dissolved in 50mM glycine-NaOH buffer (pH 8.5) at a concentration of 15. mu.M.

(4) Samples were prepared in which each evaluation compound and amyloid were dissolved in 50mM phosphate buffer (pH7.4) and the final concentrations were as shown in Table 9, respectively. Each 50. mu.L of the amyloid solution and thioflavin T solution prepared in (3), and 50. mu.L of the sample solution were filled in each well of a 96-well microplate (volume about 0.3 mL).

Table 9: final concentration of each compound in the sample solution

(5) A sample in which 100. mu.L of 50mM phosphate buffer (pH7.4) and 50. mu.L of 50mM glycine-NaOH buffer (pH 8.5) were mixed was prepared as a blank, and the same procedure as in (4) was carried out to calculate the inhibition ratio (hereinafter referred to as BG).

(6) The microplate filled with the sample solution was allowed to stand at room temperature for 30 hours. Then, the fluorescence intensity of each sample solution (measurement conditions: excitation wavelength 446nm and fluorescence wavelength 490nm) was measured with a microplate reader (model: SPECTRA MAX GEMINI XS, manufactured by Molecular Devices) (hereinafter, A represents the fluorescence intensity in a sample in which the concentration of each evaluation compound was zero (0), and B represents the fluorescence intensity in a sample in which the concentration of each evaluation compound was 1.5. mu. mol/L or more).

(7) Using the fluorescence intensity measured in the above (6), the inhibition ratio was obtained by the following formula (5):

the inhibition ratio of each evaluation compound is shown in table 10. Compounds 1 and 2 at any concentration inhibited thioflavin T binding. This result indicates that compounds 1 and 2 competitively inhibit the binding of thioflavin T. It is generally known that thioflavin T binds to amyloid by recognizing its beta-sheet structure. Thus, this suggests that compounds 1 and 2 have the same mechanism of amyloid binding as thioflavin T, i.e., recognize the β -sheet structure to bind to.

Table 10: inhibition ratio (%) of Thioflavin T binding to amyloid (dextrin) by the Compound of the present invention

Example III-3 determination of amyloid affinity

Amyloid affinity of the compounds of the invention was evaluated by the following in vitro binding assay.

Method

(1) Using the method described in examples III-1 and III-2, a 1mg/mL suspension of the agglutinated dextrin (hereinafter, in this example, referred to as an amyloid suspension) was prepared.

Insulin having a crosslinked β -sheet structure was prepared by the following operations (2) and (3), and operations (2) and (3) are known to the literature (Burke, m.j. et al, Biochemistry.11The method described in p.2435-2439(1972)) is slightly modified.

(2) 5mg of insulin (manufactured by Sigma Aldrich Japan) was dissolved in 1mL of deionized water adjusted to pH 2 with hydrochloric acid, and the reaction mixture was heated at 90 ℃ for 10 minutes and then rapidly cooled in dry ice/ethanol.

(3) The sample solution of (2) was heated at 90 ℃ for 5 minutes and then quenched in dry ice/ethanol. After repeating the same operation 10 times, it was visually confirmed that the sample solution became a gel form.

(4) The sample of (3) was centrifuged (16000g × 15 minutes), and then the supernatant was removed, and the precipitate was dissolved in 1mL of deionized water to obtain a suspension of aggregated insulin (hereinafter, in this example, referred to as an insulin amyloid suspension).

Beta 2-microglobulin having a cross-linked beta-sheet structure was prepared by the following procedures (5) and (6), procedures (5) and (6) being in the known literature (Ohhashi, y. et al, biochemistry. et al, Journal of Biological Chemistry.280The method described in p.32843-32848(2005), but with minor modifications.

(5) To a 1mg/mL solution of β 2-microglobulin (manufactured by Oriental Yeast Co., 1 td.), 50mM glycine-HCl buffer (pH 2.5) containing 100mM NaCl was added, followed by ultrasonic treatment at 37 ℃ for 1 minute in a hot ultrasonic water bath, and then heating at 37 ℃ for 9 minutes without ultrasonic treatment

(6) After repeating the operation of (5) 17 times, the sample solution was treated in the same manner as in (2) above. The sample solution was centrifuged (16000g × 15 minutes), the supernatant was removed, and the precipitate was dissolved in 0.5mL of 50mM glycine-HCl buffer solution (pH 2.5) containing 100mM NaCl to obtain a suspension of aggregated β 2-microglobulin (hereinafter, in this example, referred to as β 2-m-amyloid suspension).

(7) According to the qualitative experiments based on the fluorescence spectrophotometry using thioflavin T (manufactured by Fluka Co.) as described in example III-1 and example III-2, it was confirmed that the aggregated insulin and the aggregated β 2 microglobulin obtained in (4) and (6) were amyloid.

(7) A solution of Compound 6 synthesized by the method of example I-3 described above (radioactive concentration of 500MBq/mL) was prepared, and diluted with a phosphate buffer solution (pH7.4) containing 0.1% bovine serum albumin to prepare a solution of 2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a ] pyrimidine in a total amount of 1.0 to 101.0 pM.

(8) To each well of a 96-well microplate, 50. mu.L of the solution prepared in (7) (final concentration: 0.2 to 20.2pM) and 50. mu.L of a solution (final concentration: 0.8. mu.g/mL) prepared by diluting a dextrin amyloid suspension, an insulin amyloid suspension or a. beta.2-m-amyloid suspension with a phosphate buffer (pH7.4) containing 0.1% bovine serum albumin were added, followed by 150. mu.L of the same buffer.

(9) The microplate was shaken at a specified rate (400rpm) for 3 hours at 22 ℃. The mixture in each well was then passed through a glass fiber filter (trade name: Mulutiscreen:)^TM-FC, manufactured by Millipore) to separate amyloid-binding compound 6 from free compound 6.

(10) The glass fiber filter after the mixture was filtered was washed with a phosphate buffer containing 0.1% bovine serum albumin (0.2 mL. times.5 times), and then the radioactivity of the glass fiber filter was measured by an Autowell Gamma system (model: ARC-7001, manufactured by Aloka).

(11) The relationship between the amount of amyloid bound and the amount of amyloid added to compound 6 was evaluated from the measurement results of (10). From the sample to which no amyloid suspension was added in (8) above, non-specific binding was determined (example I-3).

The relationship between the concentration of compound 6 in the sample solution and the radioactive Count (CPM) of the glass fiber filter measured in (11) above is shown in fig. 16. In the group to which the amyloid suspension was added (fig. 16, the group to which dextrin amyloid was added, the group to which insulin amyloid was added, and the group to which β 2-m-amyloid was added), the radioactivity was all higher than that in the group to which the amyloid suspension was not added (fig. 16, the group to which amyloid was not added), and the radioactivity on the glass fiber filter increased in proportion to the addition concentration of compound 6. In the conditions of this experiment, all amyloid (and amyloid bound to compound 6) was larger than the pore size of the glass fibers. Thus, amyloid is retained on glass fibers, whose radioactive count is a value reflecting the amount of compound 6 bound to the amyloid. Since the value of the radioactivity count of the glass fiber increased with the increase in the concentration of compound 6 and the radioactivity thereof was higher than that of the group to which no amyloid protein was added, it was suggested that compound 6 was a compound having a property of specifically binding to amyloid protein.

Example IV measurement of radioactivity distribution Rate in Each organ

In order to demonstrate that the compound of the present invention can be distributed in the target organ and has a good property of being cleared to the outside of the body, the change with time of radioactive accumulation to each organ was measured in SD rat (8 weeks old) using compound 6.

To 400. mu.L of 6-tributylstannyl-2- (4' -hydroxyphenyl) imidazo [1, 2-a]To an acetonitrile solution of pyridine (concentration: 1mg/mL), 400. mu.L of 1mol/L sulfuric acid, 10. mu.L of 1mmol/L sodium iodide, and 270. mu.L of 1074MBq¹²³I]Sodium iodide and 10. mu.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 obtain [ 2], [¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine fraction.

HPLC conditions:

a chromatographic column: YMC-Pack Pro C8 (trade name; manufactured by YMC Co., Ltd.; specification: 4.6X150mm)

Mobile phase: 10mM formic acid (pH 3.0)/acetonitrile 80/20 → 80/20 → 10/90(0 min-20 min-30 min)

Flow rate: 1.0 mL/min

A detector: an ultraviolet visible absorption photometer (detection wavelength: 254nm) and a radioactivity counter (model: STEFFI manufactured by Raytest Co., Ltd.)

To this fraction was added 10mL of water. The resulting solution was passed through a Sep-Pak C18 column (trade name; Sep-Pak (registered trade name) Light C8 cards, manufactured by Waters Co., Ltd.; filling amount of the filler: 130mg) so that the column was adsorbed and collected [, [ 2] ]¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine. The column was rinsed with 1mL of water, and then 1mL of diethyl ether was passed therethrough to elute¹²³I]-2- (4' -hydroxyphenyl) -6-iodoimidazo [1, 2-a]Pyridine (compound 6). The radioactivity of the resulting compound immediately after the synthesis was 470 MBq. Further, TLC analysis was carried out under the following conditions, and as a result, the compound was obtainedThe radiochemical purity is 98%.

TLC analysis conditions:

TLC plate: silica gel 60F₂₅₄(trade name; manufactured by Merck Co., Ltd.)

Mobile phase: ethyl acetate/methanol/diethylamine-100/4/1

A detector: rita Star (trade name; manufactured by raytest Co., Ltd.)

The ether solution of Compound 6 was diluted with a physiological saline solution containing 10mg/mL of ascorbic acid, and the radioactive concentration was adjusted to 8-12 MBq/mL. 0.2mL of the prepared sample solution was administered to the tail vein of the above rat without anesthesia. These rats were sacrificed by exsanguination from the abdominal aorta and the organs shown in table 11 were removed 5, 30, 60 and 180 minutes after dosing. The mass and radioactivity of each removed organ were measured in the same manner as in example II-11. Meanwhile, radioactivity of the whole body of the rat (hereinafter referred to as the remaining part of the whole body) after organ removal was measured. Using these measurement results, the radioactivity distribution (% ID/g) per unit weight of each organ at each time point was calculated according to the following formula (6)

At each time point, experiments were performed using 3 animals.

R^T: radioactivity of target organ (cpm)

R^S: total radioactivity in all organs (cpm)

R^R: radioactivity (cpm) in the remainder of the body

M^T: quality of target organ (g)

The results are shown in Table 11. As shown in table 11, compound 6 was distributed in each organ at a time point of 5 minutes after administration, and then most of the radioactivity was distributed in the small and large intestines. In addition, its radioactivity distribution shifts from the small intestine to the large intestine. We have therefore found that compound 6 is rapidly excreted via the bile after administration and is well cleared to the outside of the body.

Meanwhile, observing the brain, heart, lung, pancreas and bone in which amyloid is considered to accumulate, significant radioactive accumulation was found in all these organs at a time point of 5 minutes after administration, thus confirming the distribution of compound 6. In addition, the ratio of the time point 5 minutes after the administration to the time point 180 minutes after the administration ((% ID/g at the time point 5 minutes after the administration)/(% ID/g at the time point 180 minutes after the administration)) showed high values, for example, 122 for the brain, 13 for the heart, 7 for the lung, 14 for the pancreas, and 4 for the bone. Thus, it was shown that rapid radioactivity distribution and rapid clearance were exhibited in organs where amyloid was thought to accumulate.

From the foregoing, it can be seen that the early radioactivity distribution after administration and the rapid clearance in vitro required for amyloid detection agents in biological tissues are achieved.

TABLE 11

Industrial applicability

The reagent for detecting amyloid in biological tissues of the present invention can be used as an in vitro and in vivo diagnostic agent for amyloid in amyloidosis such as systemic amyloidosis.

Claims (17)

1. A reagent for detecting amyloid deposited on a biological tissue, comprising a compound represented by the following formula (1) or a salt thereof:

wherein A is¹，A²，A³And A⁴Independently represent carbon or nitrogen, and

R³is a group represented by the formula:

wherein R is¹Is a radioactive halogen substituent;

m is an integer of 0 to 4; and is

n is an integer of 0 or 1,

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, and R³And A¹，A²，A³Or A⁴The indicated carbon bonds.

2. The reagent of claim 1, wherein A is¹，A²，A³And A⁴At least 3 of which represent carbon.

3. The reagent of claim 2, wherein A¹，A²，A³And A⁴All represent carbon.

4. The reagent of any one of claims 1-3, wherein R¹Is selected from¹⁸F、⁷⁵Br、⁷⁶Br、¹²³I、¹²⁴I、¹²⁵I and¹³¹I。

5. the agent of any one of claims 1 to 4, wherein the biological tissue is brain, heart, lung, pancreas, bone or joint.

6. A method for preparing a radiohalogen-labeled organic compound, comprising:

a step of preparing a reaction solution containing a compound represented by the following formula (2) or a salt thereof and a radioactive halide ion:

wherein A is¹，A²，A³And A⁴Independently represent carbon or nitrogen, and

R⁴is a group represented by the formula:

wherein m is an integer from 0 to 4;

n is an integer of 0 or 1; and is

When m is 0, R²A non-radioactive halogen substituent, a nitro substituent, a trialkylammonium substituent having 3 to 12 carbon atoms, a trialkylstannyl substituent having 3 to 12 carbon atoms or a triphenylstannyl substituent; when m ≠ 0 and/or n ≠ 0, R²A non-radioactive halogen substituent, a mesyloxy substituent, a trifluromesyloxy substituent or an aromatic sulfonyloxy substituent,

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, and R⁴And A¹，A²，A³Or A⁴The carbon bonds represented; and

a step of giving reaction conditions to the above reaction solution to synthesize a compound represented by the following formula (1):

wherein A is¹，A²，A³And A⁴Is the same as in formula (2), and

R³is a group represented by the formula:

wherein R is¹Is a radioactive halogen substituent; and is

m and n are the same as in formula (2),

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, R³And A¹，A²，A³Or A⁴The indicated carbon bonds.

7. The method for preparing a radiohalogen-labeled organic compound of claim 6, wherein A¹，A²，A³And A⁴At least 3 of which represent carbon.

8. The method for preparing a radiohalogen-labeled organic compound of claim 7 wherein A¹，A²，A³And A⁴All represent carbon.

9. A process for the preparation of a radiohalogen-labelled organic compound according to any one of claims 6 to 8, wherein R²Selected from iodine, bromine, trialkylstannyl substituents having 3 to 12 carbon atoms and triphenylstannyl substituents, and the radioactive halide is selected from¹²³I ions,¹²⁴I ions,¹²⁵I ion and¹³¹i ion, and R¹Is selected from¹²³I、¹²⁴I、¹²⁵I and¹³¹I。

10. the method for preparing a radiohalogen-labeled organic compound of claim 9, wherein R is²Selected from the group consisting of iodine, trimethylstannyl substituents, tributylstannyl substituents, and triphenylstannyl substituents.

11. A process for the preparation of a radiohalogen-labelled organic compound according to any one of claims 6 to 8, wherein R²Selected from nitro substituents, trialkylammonium substituents having 3 to 12 carbon atoms, mesyloxy substituents, trifluoromethanesulfonyloxy substituentsA substituent group and an aromatic sulfonyloxy substituent group, the radioactive halide ion being¹⁸F ion, R¹Is that¹⁸F。

12. The method for preparing a radiohalogen-labeled organic compound of claim 11, wherein R is²Is a trifluoromethanesulfonyloxy substituent or a tosyloxy substituent.

13. A process for the preparation of a radiohalogen-labelled organic compound according to any one of claims 6 to 8, wherein R²Is bromine and the radioactive halide ion is⁷⁵Br ion or⁷⁶Br ion, R¹Is that⁷⁵Br or⁷⁶Br。

14. A precursor compound for use in the preparation of a radioactive halogen-labeled organic compound, which is represented by the following formula (2):

wherein A is¹，A²，A³And A⁴Independently represent carbon or nitrogen, and

R⁴is a group represented by the formula:

wherein m is an integer from 0 to 4;

n is an integer of 0 or 1; and is

When m is 0, R²A non-radioactive halogen substituent, a nitro substituent, a trialkylammonium substituent having 3 to 12 carbon atoms, a trialkylstannyl substituent having 3 to 12 carbon atoms or a triphenylstannyl substituent, R being equal to 0 when m and/or n is equal to 0²Non-radioactive halogen substituent, mesyloxy substituent, trifluoromethylA sulfonyloxy substituent or an aromatic sulfonyloxy substituent,

with the proviso that A¹，A²，A³And A⁴At least 1 of (a) represents carbon, R⁴And A¹，A²，A³Or A⁴The indicated carbon bonds.

15. The precursor compound for the preparation of a radiohalogen-labeled organic compound or a salt thereof according to claim 14, wherein a¹，A²，A³And A⁴At least 3 of which represent carbon.

16. The precursor compound for the preparation of a radiohalogen-labeled organic compound or a salt thereof according to claim 15, wherein a¹，A²，A³And A⁴All represent carbon.

17. The precursor compound for the preparation of a radiohalogen-labeled organic compound or a salt thereof according to any one of claims 14 to 16, wherein R is²Selected from the group consisting of iodo, bromo, trimethylstannyl, tributylstannyl, trifluoromethanesulfonyloxy, and triphenylstannyl substituents.