JP7652425B2 - Photooxygenation catalyst compound having a phenothiazine skeleton and medicine containing the same - Google Patents

Description

The present invention relates to a pharmaceutical for preventing or treating diseases involving various pathogenic amyloids.

In general, proteins fold to form specific native structures and perform vital functions, but on the other hand, they can misfold and aggregate (amyloidize) into fibers rich in β-sheet structures. The aggregates (oligomers, protofibrils, and fibers) produced during this amyloidization process are known to cause various functional disorders (such diseases are collectively known as "amyloid diseases"), and more than 35 types of proteins have been identified as causative agents of amyloid diseases. Examples of such amyloids include tau protein, α-synuclein in Parkinson's disease, amylin in diabetes, transthyretin in systemic amyloidosis, and huntingtin in Huntington's disease.

On the other hand, Alzheimer's disease is a progressive neurodegenerative disease that causes brain atrophy and cognitive decline, and the number of patients is increasing year by year. Alzheimer's disease is also a type of amyloid disease, and its onset is thought to be related to neurotoxicity caused by aggregates formed by amyloid beta peptides (Aβ), and treatments targeting Aβ have been actively researched so far. Known therapeutic drugs and treatments targeting Aβ include, for example, inhibitors of enzymes that produce Aβ from precursor proteins, Aβ decomposition enzyme promoters, immunotherapy, and Aβ aggregation inhibitors. However, these conventional treatments have problems such as side effects and low pharmacological effects, and have not yet been put to practical use. For this reason, the development of new methods that lead to safe and effective treatment of Alzheimer's disease is desired.

In response to this, the present inventors have focused on the characteristic of Aβ aggregating through hydrophobic interactions and have developed photocatalytic compounds that suppress aggregability and toxicity by selectively adding hydrophilic oxygen atoms to Aβ (Non-Patent Documents 1, 2, etc.). However, in order to apply these conventional photocatalytic compounds to actual disease treatment, there was room for improvement in terms of oxygen activation under near-infrared light irradiation, water solubility, pharmacokinetics including brain transferability, etc. In addition, conventional photocatalytic compounds only have a mechanism for expressing oxygen activation by interaction with aggregated Aβ (Aggregated Aβ) that forms a cross-β sheet structure as shown in "Path (I)" of Figure 1, while it was difficult to efficiently oxygenate Aβ (Low Aggregated Aβ) that is more toxic, such as oligomers, as shown in "Path (II)" of Figure 1.

Taniguchi, A. et al., Nat. Chem. 2016, 8, 974-982 Ni, J. et al., M. Chem 2018, 4, 807-820

In view of the problems of the conventional techniques, the present invention aims to develop a photocatalytic compound that can efficiently oxygenate amyloids in a low-aggregation state and at low concentrations, which was difficult to achieve with conventional methods, and that can reduce cytotoxicity under light irradiation. Another object of the present invention is to provide a drug for preventing and treating amyloid-related diseases using such a novel photocatalytic compound.

The inventors of the present invention have conducted intensive research to solve the above problems, and have found that by using a photocatalytic compound having a mother skeleton in which a phenothiazine structure and a morpholine structure are linked, and a side chain having a specific structure is introduced, stacking of catalyst molecules can be suppressed, cytotoxicity can be reduced, and selective oxygenation of amyloids by light irradiation can be achieved. In addition, the inventors have also found that the compound can efficiently oxygenate amyloids in a low-aggregation state and at low concentrations. These findings have led to the completion of the present invention. Note that the term "oxygenation" is used here to broadly mean the reaction of oxidation, particularly the reaction of bonding oxygen atoms.

That is, in one aspect, the present invention relates to a photooxygenation catalyst compound for amyloids, more specifically,
<1> A compound represented by the following formula (I) or a salt thereof:
(wherein X is an optionally substituted aryl group or an optionally substituted heteroaryl group; Y is a carboxyl group or a biological equivalent thereof; ^L1 and ^L2 are each a spacer group; and R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms);
<2> The compound or salt thereof according to the above <1>, wherein L ¹ and L ² each independently represent an alkylene chain or an ether chain;
<3> The compound or salt thereof according to the above <1> or <2>, wherein X is an optionally substituted phenyl group;
<4> The compound or salt thereof according to any one of the above <1> to <3>, wherein Y is a carboxyl group, an aliphatic carboxyl group, a nitrogen-containing heteroaryl group, a sulfonamide group, or a phosphate ester group;
<5> The compound according to the above <1>, represented by the following formula (II), or a salt thereof:
(In the formula, R ^a is a hydrogen atom or 1 to 5 substituents which may be the same or different and are independently selected from an alkyl group, an amino group, a hydroxyl group, an alkoxy group, a halogen, a nitro group, or a carbonyl group; R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; m is a natural number from 1 to 10; n is a natural number from 1 to 10; and p is a natural number from 1 to 5);
<6> A catalyst for oxygenating pathogenic amyloid, comprising the compound according to any one of the above items <1> to <5> or a salt thereof; and <7> an agent for inhibiting the aggregation of pathogenic amyloid, comprising the compound according to any one of the above items <1> to <5> or a salt thereof.

In another aspect, the present invention also relates to medicaments and methods of treatment comprising the above compounds, more particularly
<8> A pharmaceutical composition comprising the compound or salt thereof according to any one of the above <1> to <5> and a pharma- ceutically acceptable carrier;
<9> The pharmaceutical composition according to the above <8>, which is a preventive or therapeutic drug for a disease associated with pathogenic amyloid;
<10> The pharmaceutical composition according to the above-mentioned <9>, wherein the disease associated with pathogenic amyloid is Alzheimer's disease;
<11> Use of the compound or a salt thereof according to any one of the above <1> to <5> for the manufacture of a prophylactic or therapeutic agent for a disease associated with pathogenic amyloid;
<12> A method for preventing or treating a disease associated with pathogenic amyloid, comprising administering an effective amount of the compound or salt thereof according to any one of the above <1> to <5>;
<13> The method according to <12> above, which comprises administering the compound or salt thereof according to any one of <1> to <5> above, and then irradiating an affected area of a patient with light from outside the body; and <14> The method according to <12> or <13> above, wherein the disease associated with pathogenic amyloid is Alzheimer's disease.

According to the present invention, amyloids in a low aggregated state and at low concentrations can be selectively and efficiently photooxygenated while reducing cytotoxicity. This has the effect of suppressing or reducing the aggregation and toxicity of pathogenic amyloids such as Aβ peptides in the body (e.g., in the brain) by a non-invasive method of administering the compound intravenously or the like and then irradiating it with light from outside the body. Therefore, the present invention is extremely beneficial in that it enables the prevention and treatment of diseases involving pathogenic amyloids by a novel, minimally invasive method.

FIG. 1 is a schematic diagram showing a scheme of a photooxidation reaction of amyloid β peptide (Aβ). FIG. 2 shows UV-vis absorption spectra of the compound of the present invention and a comparative compound. FIG. 3 is a graph of fluorescence intensity showing the generation of superoxide anion for the compounds of the present invention and comparative compounds. FIG. 4 is a graph showing the cytotoxicity due to light irradiation for the compounds of the present invention and the comparative compounds. FIG. 5 is a graph showing the amount of oxygenated Aβ produced in the photooxygenation reaction of Aβ using the compound of the present invention and a comparative compound (CRANAD catalyst) (Aβ concentration: 20 μM). FIG. 6 is a graph showing the amount of oxygenated Aβ produced in the photooxygenation reaction of Aβ using the compound of the present invention and the comparative compound (CRANAD catalyst) (Aβ concentration: 2 μM). FIG. 7 is a graph showing peptide selectivity in photooxygenation reactions by the compounds of the present invention.

The following describes an embodiment of the present invention. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be modified and implemented as appropriate without departing from the spirit of the present invention.

1. Definitions
In this specification, the term "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

In the present specification, the term "alkyl or alkyl group" may be any of linear, branched, cyclic, and aliphatic hydrocarbon groups consisting of a combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, and may be, for example, 1 to 20 carbon atoms (C _1-20 ), 1 to 15 carbon atoms (C _1-15 ), or 1 to 10 carbon atoms (C _1-10 ). In the present specification, the alkyl group may have one or more optional substituents. For example, C _1-8 alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. The substituent may be, for example, an alkoxy group, a halogen atom (which may be any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group, but is not limited thereto. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl portion of other substituents containing an alkyl portion (e.g., an alkoxy group, an arylalkyl group, etc.).

In this specification, "alkylene" refers to a divalent group consisting of a linear or branched saturated hydrocarbon, such as methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, 1,2 -dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyltrimethylene, and the like.

In this specification, "aryl or aryl group" may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, or may be an aromatic heterocycle containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms) as ring constituent atoms. In this case, it is called a "heteroaryl group" or a "heteroaromatic group". Whether the aryl is a monocyclic or condensed ring, it may be bonded at any possible position. Non-limiting examples of monocyclic aryl include a phenyl group (Phe), a thienyl group (2- or 3-thienyl group), a pyridyl group, a furyl group, a thiazolyl group, an oxazolyl group, a pyrazolyl group, a 2-pyrazinyl group, a pyrimidinyl group, a pyrrolyl group, an imidazolyl group, a pyridazinyl group, a 3-isothiazolyl group, a 3-isoxazolyl group, a 1,2,4-oxadiazol-5-yl group, or a 1,2,4-oxadiazol-3-yl group. Non-limiting examples of fused polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthryl, indazolyl, quinolyl, isoquinolyl, 1,2-dihydroisoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2,3-dihydrobenzofuranyl, and 2,3-dihydrobenzofuranyl. Examples of the aryl group include 1-yl group, 2,3-dihydrobenzofuran-2-yl group, naphthyridinyl, dihydronaphthyridinyl, tetrahydronaphthyridinyl, imidazopyridinyl, pteridinyl, purinyl, quinolizinyl, indolizinyl, tetrahydroquinolizinyl, and tetrahydroindolizinyl, 2,3-dihydrobenzothiophen-1-yl group, 2,3-dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazolyl group, fluorenyl group, and thioxanthenyl group. In the present specification, the aryl group may have one or more optional substituents on the ring. Examples of the substituents include, but are not limited to, alkyl groups, alkoxy groups, halogen atoms, amino groups, mono- or di-substituted amino groups, substituted silyl groups, and acyl. When the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing an aryl moiety (e.g., aryloxy group, arylalkyl group, etc.). When an aryl group has two or more substituents, they may be the same or different. In this specification, "aromatic ring" means a monocyclic or condensed polycyclic conjugated unsaturated hydrocarbon ring structure, which may contain one or more heteroatoms (e.g., oxygen atom, nitrogen atom, sulfur atom, etc.) as ring-constituting atoms.

As used herein, the term "ring structure" refers to a heterocyclic or carbocyclic group when formed by the combination of two substituents, and such groups can be saturated, unsaturated, or aromatic. It thus includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl, as defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperdinyl, imidazolyl, pyrrolidinyl, and pyridyl. As used herein, a substituent can form a ring structure with another substituent, and when such substituents are bonded together, one skilled in the art will understand that a particular substitution, such as a bond to hydrogen, is formed. Thus, when certain substituents are described as forming a ring structure together, one skilled in the art will understand that the ring structure can be formed and is readily produced by conventional chemical reactions. Both such ring structures and the process of their formation are within the purview of one skilled in the art.

As used herein, "ester" includes both ROCO--(when R=alkyl, alkoxycarbonyl-) and RCOO--(when R=alkyl, alkylcarbonyloxy-). As used herein, "alkylamino" and "arylamino" refer to an amino group in which the hydrogen atom of the _-NH2 group is substituted with one or two of the above alkyl or aryl groups. Examples include methylamino, dimethylamino, ethylamino, diethylamino, ethylmethylamino, benzylamino, and the like.

In the present specification, when a functional group is defined as "optionally substituted," the type of the substituent, the substitution position, and the number of the substituent are not particularly limited, and when there are two or more substituents, they may be the same or different. Examples of the substituent include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may further have a substituent. Examples of such substituents include, but are not limited to, halogenated alkyl groups.

2. Photooxygenation Catalyst Compound of the Present Invention The compound of the present invention is characterized by having a mother skeleton in which a phenothiazine structure and a morpholine structure are linked together, a side chain of a specific structure, and a structure represented by the following formula (I).

In formula (I), X is an electron-donating substituent, specifically an optionally substituted aryl group or an optionally substituted heteroaryl group. Typically, X can be a monocyclic 5- or 6-membered ring, and examples of such monocyclic aryl or heteroaryl include a phenyl group, a pyridyl group, an oxazolyl group, a pyrazolyl group, a 2-pyrazinyl group, a pyrimidinyl group, a pyrrolyl group, an imidazolyl group, a pyridazinyl group, a 3-isothiazolyl group, a 3-isoxazolyl group, a 1,2,4-oxadiazol-5-yl group, or a 1,2,4-oxadiazol-3-yl group. X may also be a condensed polycyclic aryl or heteroaryl, and examples of such groups include a 1-naphthyl group, a 2-naphthyl group, or the like. The aryl or heteroaryl group may have one or more optional substituents on the ring, such as an alkyl group, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group. When the aryl or heteroaryl group has two or more substituents, they may be the same or different. Preferably, X is a phenyl group.

In formula (I), ^L1 is a spacer group for linking the phenothiazine skeleton portion and X. Preferably, ^L1 can be an alkylene or ether chain. More preferably, L1 is a linear or branched alkylene having 2 to 10 carbon atoms or an ether chain having 2 to 10 carbon atoms. The length of the spacer group ^L1 can be appropriately adjusted for selective binding to amyloids.

In formula (I), R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Preferably, R is a hydrogen atom or a methyl group.

The left side chain "X-N(R)-L ¹ -N(R)-" in formula (I) can cause quenching upon light irradiation by forming intramolecular stacking with the phenothiazine skeleton moiety and/or by photoinduced electron transfer (PET) to the phenothiazine skeleton moiety. This results in an effect that, when amyloids are not present, catalytic activity is suppressed (OFF), but when the photooxidation catalyst compound of formula (I) binds to amyloid, these quenching operations are released and catalytic activity is selectively turned ON. In addition, such side chains suppress stacking between catalyst molecules, thereby suppressing the generation of undesirable superoxide anions and reducing cytotoxicity upon light irradiation.

The left chain "X--N(R)-L ¹ -N(R)--" is preferably linked to the meta or para position, more preferably the meta position, relative to the S atom in the phenothiazine skeleton.

In formula (I), Y is a substituent for suppressing cytotoxicity by introducing an anionic structure into the compound of formula (I), and is a carboxyl group or a biological equivalent thereof having an acidic proton (a group that is likely to become anionic like carboxylic acid). That is, Y can be a group derived from a heteroaromatic ring having an acidic proton, such as a sulfonamide, a phosphate ester, or a tetrazole, which is known as a biological equivalent thereof. Representative examples of Y are a carboxyl group, an aliphatic carboxyl group, a nitrogen-containing heteroaryl, a sulfonamide group, or a phosphate ester group. Preferably, Y is a tetrazolyl group, an imidazolyl group, a triazolyl group, a pentazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, or an oxazolyl group. More preferably, Y is a tetrazolyl group. By using such a highly hydrophobic tetrazolyl group, the amphiphilicity of the entire molecule is eliminated, thereby further reducing toxicity.

In formula (I), ^L2 is a spacer group for linking the morpholine skeleton portion and Y. Preferably, ^L1 can be an alkylene or ether chain. More preferably, L1 is a linear or branched alkylene having 2 to 10 carbon atoms or an ether chain having 2 to 10 carbon atoms. ^L1 and ^L2 may be the same or different, and each independently can be an alkylene or an ether chain.

The right side chain "-L ² -Y" in formula (I) is a group capable of reducing the cytotoxicity derived from the catalyst itself. In addition, as described below, by making the right side chain link to the phenothiazine skeleton via a morpholine moiety, the morpholine structure acts as a steric hindrance, suppressing stacking between catalyst molecules, further suppressing the generation of undesirable superoxide anions, and reducing cytotoxicity upon light irradiation.

In a typical embodiment, X in formula (I) can be an optionally substituted phenyl group, ^L1 can be an ether chain, ^L2 can be an alkylene, and Y can be a tetrazolyl group. In this case, the compound of the present invention has a structure represented by the following formula (II).

In formula (II), R ^a can be any substituent on the phenyl group as described above. More specifically, R ^a can be a hydrogen atom or 1 to 5 substituents, which may be the same or different, selected from an alkyl group, an amino group, a hydroxyl group, an alkoxy group, a halogen, a nitro group, and a carbonyl group.

In formula (II), R is, as in formula (I) above, a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and preferably, R is a hydrogen atom or a methyl group.

In formula (II), m, n, and p all define the length of the spacer portion. m is a natural number from 1 to 10, preferably from 1 to 5. n is a natural number from 1 to 10, preferably from 1 to 5. Furthermore, p is a natural number from 1 to 5, preferably from 1 to 3. These m, n, and p may be the same or different, but preferably m and n are the same.

The compounds of the present invention represented by the above formulas (I) and (II) may exist as salts. Examples of such salts include base addition salts, acid addition salts, and amino acid salts. Examples of base addition salts include metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, ammonium salts, and organic amine salts such as triethylamine salts, piperidine salts, and morpholine salts. Examples of acid addition salts include mineral acid salts such as hydrochlorides, sulfates, and nitrates, and organic acid salts such as carboxylates, methanesulfonates, paratoluenesulfonates, citrates, and oxalates. Examples of amino acid salts include glycine salts. However, the salts are not limited to these salts.

The compounds of the present invention may have one or more asymmetric carbons depending on the type of substituent, and may exist as stereoisomers such as optical isomers or diastereoisomers. Pure stereoisomers, any mixtures of stereoisomers, racemates, etc. are all included within the scope of the present invention.

In addition, the compounds of the present invention or salts thereof may exist as hydrates or solvates, and all of these substances are included in the scope of the present invention. The type of solvent that forms the solvate is not particularly limited, but examples include solvents such as water, ethanol, acetone, and isopropanol.

The examples in this specification specifically show production methods for representative compounds included in the compounds of the present invention, so that a person skilled in the art can appropriately produce any compound included in formulas (I) and (II) by referring to the disclosure in this specification and, if necessary, by appropriately selecting starting materials, reagents, reaction conditions, etc.

2. Pharmaceutical compositions containing the compounds of the present invention The compounds of the present invention can catalyze the oxygenation reaction of pathogenic amyloid. The oxygenation reaction proceeds by exciting the compounds of the present invention by light irradiation, thereby adding oxygen atoms to amino acid residues in amyloid, and oxidizing them. This makes it possible to inhibit or reduce the aggregation of pathogenic amyloid.

Therefore, in another aspect, the present invention relates to a pathogenic amyloid oxygenation catalyst or a pathogenic amyloid aggregation inhibitor, comprising the above-mentioned compound or a salt thereof. Furthermore, the present invention also relates to a pharmaceutical composition containing the above-mentioned compound or a salt thereof and a pharma- ceutically acceptable carrier.

The compound of the present invention has a feature that it can selectively and efficiently photo-oxygenate amyloids in a low aggregated state and at a low concentration while reducing cytotoxicity. Without necessarily being bound by theory, the left side chain "X-N(R)-L ¹ -N(R)-" in formula (I) can cause quenching upon light irradiation by forming intramolecular stacking with the phenothiazine skeleton moiety and/or by photoinduced electron transfer (PET) to the phenothiazine skeleton moiety. As a result, the catalytic activity is suppressed (OFF) when amyloids are not present, but when the photooxidation catalyst compound of the present invention binds to amyloids, the quenching action is released and the catalytic activity is selectively turned ON, allowing the amyloids to be selectively oxygenated.

As shown in the above formulas (I) and (II), the compound of the present invention has a structure in which a morpholine portion is linked to a phenothiazine skeleton, and the morpholine structure acts as a steric hindrance to inhibit intermolecular stacking between catalyst molecules, thereby making it possible to inhibit the generation of undesirable superoxide anions that cause cytotoxicity upon light irradiation. In addition, as described above, the substituent X of "X-N(R)-L ¹ -N(R)-" on the left chain forms an intramolecular stack with the phenothiazine skeleton portion, thereby inhibiting intermolecular stacking between catalyst molecules, thereby further suppressing the generation of undesirable superoxide anions and reducing cytotoxicity upon light irradiation. Thus, according to the present invention, it is possible to prevent and treat diseases involving pathogenic amyloids by a novel, minimally invasive technique.

"Pathogenic amyloids" include, but are not limited to, amyloids such as amyloid beta (Aβ) peptide, amylin, transthyretin, α-synuclein, tau protein, and huntingtin, which are known to be involved in Alzheimer's disease, Parkinson's disease, diabetes, Huntington's disease, and systemic amyloidosis in animals, including humans.

For example, when Aβ peptide is oxidized by the compound of the present invention, it is sufficient that one or more of the 40 or 42 amino acid residues constituting the Aβ peptide are oxidized, but it is preferable that one or more amino acid residues selected from His and Met are oxidized. It is more preferable that the oxidation is in the form of a hydroxyl group or an oxo group (oxide) being added to each amino acid residue. In the case of His, the oxidized form of the amino acid residue is presumed to have a structure in which the imidazole ring of the histidine residue is oxidized, that is, a dehydroimidazolone ring or a hydroxyimidazolone ring. In addition, in the case of Met, it is presumed that oxygen is added to the sulfur atom in the methionine residue.

The compound of the present invention preferably has a maximum absorption wavelength (λmax) in the range of 650 to 800 nm, and is preferably capable of being excited at that wavelength. By having an absorption band on the long wavelength side, it can be excited by long wavelength light that has high biological permeability.

Pharmaceutical compositions containing the compound of the present invention or a salt thereof can be prepared by various preparation methods using pharma- ceutical acceptable carriers and by selecting an appropriate formulation according to the method of administration. Examples of dosage forms of pharmaceutical compositions containing the compound of the present invention as a main ingredient include tablets, powders, granules, capsules, liquids, syrups, elixirs, oily or aqueous suspensions, etc., as oral preparations.

Injectables may contain stabilizers, preservatives, and dissolution aids, and the solution, which may contain these aids, may be stored in a container and then freeze-dried or otherwise processed into a solid preparation to be prepared just before use. A single dose may be stored in one container, or multiple doses may be stored in one container.

Examples of topical preparations include solutions, suspensions, emulsions, ointments, gels, creams, lotions, sprays, patches, etc.

Solid preparations contain the compound of the present invention and pharma- ceutically acceptable additives, such as fillers, extenders, binders, disintegrants, dissolution enhancers, wetting agents, lubricants, etc., which can be selected and mixed as necessary to produce a formulation. Liquid preparations include solutions, suspensions, emulsions, etc., and may contain suspending agents, emulsifiers, etc. as additives.

When the compound of the present invention is used as a medicine for human use, the daily dosage for an adult is preferably in the range of 1 mg to 1 g, and more preferably 1 mg to 300 mg.

In a further aspect, the present invention also relates to a method for preventing or treating a disease associated with pathogenic amyloid, comprising administering an effective amount of the compound or a salt thereof. The method preferably includes irradiating the affected area of the patient with light from outside the body after administration of the compound or a salt thereof. As described above, the compound of the present invention can be excited by long-wavelength light that has high biological permeability, and therefore, the aggregation and toxicity of pathogenic amyloid in the body (e.g., in the brain) can be suppressed or reduced by a non-invasive method of irradiating the patient with light from outside the body after administration by intravenous administration or the like.

Specifically, the compound of the present invention or a salt thereof is introduced into a living body or into a cell, and light is irradiated when the compound has been transferred to the desired site. Examples of means of administration into the living body include intramuscular injection, intravenous injection, topical administration, and oral administration.

Diseases associated with pathogenic amyloid include Alzheimer's disease, Parkinson's disease, diabetes, Huntington's disease, systemic amyloidosis, etc. in animals, including humans. Typically, the disease associated with pathogenic amyloid is Alzheimer's disease.

The present invention will be described in further detail below with reference to examples, but the present invention is not limited to these.

1. Synthesis of Compounds of the Present Invention Compounds of the present invention having the following structures were synthesized via compounds (1) to (7) described below (in the formula, Me means a methyl group).

[Synthesis of 2,3-dihydro-4H-1,4-benzoxazine-4-butnenitrile (1)]
_To a solution of 3,4-dihydro-2H-1,4-benzoxazine (1.17 mL, 10.0 mmol) in MeCN (56 mL) was added TBAI (1.85 g, 5 mmol), NaI (5.99 g, 40 mmol), _K2CO3 (2.76 g, 20 mmol), and 4-bromobutanenitrile (1.99 mL, 20 mmol). The resulting mixture was refluxed overnight under argon atmosphere and cooled to room temperature. The reaction mixture was then filtered through a pad of Celite containing AcOEt. The filtrate was concentrated and the residue was purified by column chromatography on silica gel (eluent: Hex/AcOEt = 4/1 to 3/1) to give compound (1) (1.66 g, 82%) as a beige solid.
¹ H NMR (500 MHz, CDCl ₃ ): δ= 6.84-6.77 (m, 2H), 6.65-6.62 (m, 2H), 4.24 (t, J = 4.5 Hz, 2H),3.38 (t, J = 6.9 Hz, 2H), 3.33 (t, J = 4.5 Hz, ¹³ C NMR (125 MHz, CDCl ₃ ): δ = 144.2, 134.6, 121.6, 119.2, 118.2, 116.6, 112.1, 64.3, 49.7, 47.6, 22.6, 14.7.

[Synthesis of 2,3-dihydro-4H-1,4-benzoxazine-4-(3-(1H-tetrazol-5-yl)propyl) (2)]
To a solution of 2,3-dihydro-4H-1,4-benzoxazine-4-butonitrile (1) (1.66 g, 8.2 mmol) and trimethylsilyl azide (2.18 mL, 24.6 mmol, 3.0 equiv.) in dry toluene (13.5 mL) was added dibutyltin oxide (816.5 mg, 3.3 mmol, 0.40 equiv.) and the mixture was heated for 24-72 h until the nitrile was consumed (TLC analysis). The reaction mixture was concentrated in vacuo. The residue was dissolved in methanol and reconcentrated. The residue was partitioned between ethyl acetate and saturated sodium bicarbonate solution. The organic phase was extracted with an additional portion of saturated sodium bicarbonate solution. The combined aqueous extracts were acidified to pH 2 with concentrated hydrochloric acid solution and then extracted with AcOEt (×2). The combined organic extracts were dried over Na ₂ SO ⁴ , filtered, and concentrated to give compound (2) (1.86 g, 92%) as a clear, colorless oil. No further purification was necessary.
¹ H NMR (500 MHz, CDCl ₃ ): δ = 6.72 (ddd, J = 7.8 Hz, 7.7 Hz, 1.1 Hz, 1H), 6.65-6.62 (m, 2H),6.51 (ddd, J = 7.8 Hz, 7.7 Hz, 1.1 Hz 1H), 8 = 4.24 (t, J = 4.0 Hz, 2H), 3.34-3.28 (m, 4H), 3.00(t, J = 7.4 Hz, 2H), 2.08 (tt, J = 7.4 Hz, 7.4 Hz, 2H).
^13C NMR (125 MHz, CD ₃ OD): δ= 145.5,136.3, 122.5, 118.6, 117.2, 113.4, 65.5, 51.0, 49.8, 48.1, 25.2, 21.6

[Synthesis of (5-(3-(2,3-dihydro-4H-1,4-benzoxazine-4-yl)propyl)-1H-tetrazol-1-yl)methyl acetate (3)]
To a flask containing compound (2) (1.72 g, 7.0 mmol) in THF (14 mL), bromomethyl acetate (854.1 μL, 9.1 mmol, 1.3 equiv.) and iPr ₂ NEt (1.59 mL, 9.1 mmol, 1.3 equiv.) were added at room temperature. The resulting mixture was stirred at room temperature overnight and quenched with water. The resulting mixture was extracted with AcOEt (×2), washed with water and brine, then dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a crude mixture. The crude mixture contained two regioisomers, which can be separated by silica gel column chromatography (DCM/MeOH=50/1 to 20/1). The desired isomer, compound (3), was obtained as a clear oil (937.2 mg, 43%) and the other isomer was obtained as a clear colorless oil (693.6 mg, 31%).
1H NMR (400 MHz, CDCl ₃ ): δ = 6.79-6.74 (m, 2H), 6.61-6.55 (m, 2H), 6.08 (s, 1H), 4.19 (t, J= 4.4 Hz, 2H), 3.38 (t, J = 7.1 Hz, 2H), ¹³ C NMR (125 MHz, CDCl ₃ ): δ = 168.8, 167.1, 144.0,135.0, 121.6, 117.5, 116.4, 112.1, 71.0, 64.4, 50.2, 47.2, 24.4, 22.9, 20.4.

[Synthesis of 2-(2-(methyl(4-(2-(4-bromophenyl)diazenyl)phenyl)amino)ethoxy)-ethan-1-ol (4)]
To a cooled solution (0°C) of 4-bromobenzenediazonium-tetrafluoroborate (877 mg, 4.2 mmol) in water (14.7 mL) was slowly added a solution of NaOAc (121.2 mg, 1.5 mmol) and 2-[2-(methylphenylamino)ethoxy]-ethan-1-ol (823.8 mg, 4.2 mmol) in EtOH ( _7.2 mL). The reaction mixture immediately turned dark red. The mixture was stirred at room temperature for 10 h and diluted with AcOEt (x2). The organic phase was washed with water, brine, dried over _Na2SO4 , filtered and concentrated under reduced pressure to give a crude mixture. The crude mixture was purified by silica gel column chromatography (Hex/AcOEt = 2/1 to 1/2). A yellow-red solid, compound (4), was obtained as the product, yield (991 mg, 62%).
¹ H NMR (400 MHz, CDCl ₃ ): δ = 7.85 (d, J = 9.0 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 7.57 (d, J =8.5 Hz, 2H), 6.77 (d, J = 9.0 Hz, 2H), 3.70-3.63 (m, 6H), 3.55 (t, J = 4.9 Hz, 2H), 3.10 (s, 3H).
^13C NMR (125 MHz, CDCl ₃ ): δ = 151.9, 151.6, 143.5, 132.0, 125.2, 123.7, 123.3, 111.5, 72.4, 68.6,61.8, 52.2, 39.2.

[Synthesis of 2-(2-((4-aminophenyl)methylamino)ethoxy)-ethanol (5)]
Compound (4) (113.5 mg, 0.30 mmol) was dissolved in methanol (3.6 mL). Hydrogenation was carried out using palladium on charcoal as catalyst (10 wt% Pd, 31.9 mg, 0.03 mmol, p( _H2 ) = 1 atm). The reaction mixture was stirred at 50°C under hydrogen atmosphere until the starting material was consumed. The catalyst was removed by filtration through a Celite pad and the filtrate was evaporated. The crude product was quickly purified by silica gel column chromatography (DCM/MeOH = 10/1 to 3/1). Compound (5) was obtained as a beige solid (59.1 mg, 94%).

[Synthesis of 4-(3-(1-acetoxymethyl)-1H-tetrazol-5-yl)propyl)-8-((2-(2- hydroxyethoxy) ethyl)(methyl)amino)-3,4-dihydro-2H-1,4-oxazino[2,3-b]phenothiazine-6-ium (7)]
Compound (5) (124.5 mg, 0.59 mmol) was added to a round bottom flask, deionized water (3.7 mL) and methanol (7.3 mL) were added under an argon atmosphere, and the solution was cooled to 0° C. To the solution was added sodium thiosulfate (103.0 mg, 0.65 mmol, 1.1 equiv) in water (1.0 mL) in one portion. Sodium persulfate (140.9 mg, 0.59 mmol, 1.0 equiv) in water (2.74 mL) was added dropwise over 15 min. The reaction mixture was stirred at 0° C. for 3 h and then warmed to room temperature over 1 h. Compound (6) was obtained crude as a black solution.

To a reaction mixture of compound (6) (S-(2-amino-5-((2-(2-hydroxyethoxy)ethyl)(methyl)amino)phenyl)O-hydrogen sulfurothioate) (0.59 mmol) was added a solution of compound (3) (187.9 mg, 0.59 mmol) in MeOH. The mixture was heated to 40° C. and Fetizon reagent (Ag ₂ CO ₃ on Celite; 489.7 mg, 1.78 mmol, 3.0 equiv.) was added in portions over 15 min. The mixture was then stirred at 80° C. for 3 h. After removing the solids by filtration, the filtrate was concentrated in vacuo. The crude mixture was purified by reverse phase HPLC to give compound (7) (39.9 mg, 10%) as a red-blue solid.
¹ H NMR (400 MHz, CD ₃ CN): δ = 7.93 (d, J = 9.9 Hz, 1H), 7.53 (s, 1H), 7.50-7.47 (dd, J = 9.9Hz, 3.1 Hz, 1H), 7.42 (s, 1H), 7.34 (d, J = 3.1 Hz, 1H), 6.17 (s, 2H), 4.35 (m, 2H), 3.86 (t, J =4.9 Hz, 2H), 3.81-3.74 (m, 8H), 3.53 (t, J = 4.5 Hz, 2H), 3.49-3.47 (m, 2H), 3.31 (s, 3H), 3.08
(t, J = 7.1 Hz, 2H) 2.03 (s, 3H).
^13C NMR (125 MHz, Acetone-d ₆ ): δ = 170.2, 156.6, 154.5,148.3, 145.6, 138.3, 137.0, 134.3, 131.7, 120.6, 119.6, 106.9, 106.0, 73.8, 69.2, 67.7, 64.4, 61.8,54.6, 53.9, 52.0, 48.3, 40.7, 23.8, 20.4 (One peak is missing due to the overlap in the aromatic region).
HRMS (ESI(+)): m/z calcd for C26H32N7O6S ⁺ [M] ⁺ 554.2180, found 554.2175.

[Synthesis of 4-(3-(1H-tetrazol-5-yl)propyl)-8-(methyl(2-(2-(methyl(phenyl) amino) ethoxy)ethl)amino)-3,4-dihydro-2H-1,4-oxazino[2,3-b]phenothiazine-6-ium (the compound of the present invention)]
Prior to the reaction, compound (7) (12.0 mg, 0.018 mmol) was azeotropically dried with benzene. To a solution of compound (7) in MeCN (0.13 mL), DMSO (35.9 μL, 0.50 mmol, 28.5 equiv.) and _iPr2NEt (16.8 μL, 0.15 mmol, 8.3 equiv.) were added under argon atmosphere and stirred until dissolved. To the reaction mixture was added _SO3 ·Py (34.1 mg, 0.214 mmol) in one portion at room temperature under argon flow conditions. After stirring overnight at room temperature, the resulting mixture was concentrated in vacuo and purified by reverse phase HPLC to give the product (5.3 mg) as a blue-green solid.

To a solution in MeOH (0.1 mL) was added N-methylaniline (0.78 μL, 7.20 μmol, 0.9 equiv) and decaborane (0.29 mg, 2.39 μmol, 0.3 equiv) and the reaction mixture was stirred at room temperature under argon overnight. The reaction mixture was concentrated under reduced pressure and dissolved in 0.1 M phosphate buffer/DMSO (v/v = 10/1). Pig liver esterase (18 units per mg of starting material) was added to the resulting solution and the reaction mixture was stirred at 37°C overnight. The crude product was evaporated to dryness and purified by reverse phase HPLC to give the product (Catalyst 3) (1.3 mg, 24% for two steps), a compound of the present invention, as a red-blue solid.
HRMS (ESI(+)): m/z calcd for C30H35N8O2S ⁺ [M] ⁺ 571.2598, found 571.2599.

In addition, the comparative compound (MB1) was synthesized by the following procedure.

[Synthesis of 8-(dimethylamino)-4-methyl-3,4-dihydro-2H-1,4-oxazino[2,3-b]phenothiazine-6-ium (MB1)]
S-(2-amino-5-(dimethylamino)phenyl)-O-hydrogen sulfurothioate (12.4 mg, 0.05 mmol) and 4-methyl-3,4-dihydro-2H-1,4-benzoxazine (7.46 mg, 0.05 mmol, 1.0 equiv.) were dissolved in MeOH- _H2O mixed solvent (v/v = 5/2, 1.51 mL). The mixture was heated to 40°C, and Fetizon _reagent ( _Ag2CO3 on Celite; 27.4 mg, 0.1 mmol, 2.0 equiv.) was added portionwise over 15 min. The mixture was then stirred at 80°C for 3 h. After the solids were removed by filtration, the filtrate was concentrated in vacuo. The crude mixture was purified by reversed-phase HPLC to give the product (MB1) (1.8 mg, 8%) as a red-blue solid.

2. Cytotoxicity Study To verify the cytotoxicity of the photooxygenation catalyst, the amounts of singlet oxygen ( ¹ O ₂ ) and superoxide (O ₂ ⁻ .) generated under light irradiation conditions in the absence of amyloids were examined.

As comparative examples, methylene blue derivative compounds not having a morpholine structure ("Catalyst 1" and "Catalyst 2"), methylene blue (MB), and the compound "MB1" in which a morpholine structure is linked to the methylene blue synthesized above were used.

First, the UV spectrum of Comparative Example 1 (Catalyst 1), which does not have a morpholine structure, was measured, and it was found that the amount of singlet oxygen generated in this comparative example was significantly lower than that of MB, while a large amount of undesirable superoxide was generated (Figure 3). When the UV-vis absorption spectrum of Comparative Example 1 in phosphate buffer was measured, a peak at 610 nm derived from the dimer was observed in addition to a peak at 651 nm derived from the monomer, suggesting that the catalyst molecules form dimers and associate with each other. From this, it was found that in the catalyst molecules of Comparative Example 1, stacking occurs between molecules in the ground state, and a mechanism involving one electron transfer proceeds, resulting in the generation of superoxide anions.

Next, the UV-vis absorption spectrum and fluorescence spectrum were measured for the compound of the present invention and other comparative compounds, and the results are shown in Figures 2 and 3, respectively. As a result, as shown in Figure 2, the compound of the present invention in which a morpholine structure is linked to a phenothiazine structure ("Catalyst 3" in the figure) does not have an absorption peak near 610 nm derived from a dimer, and it was found that the formation of intermolecular stacking can be suppressed. Note that even in Comparative Example 2 (Catalyst 2), in which the spacer group in the side chain of Comparative Example 1 was changed from an alkyl chain to an ether chain, the absorption peak near 610 nm was reduced due to improved hydrophilicity, and dimer formation was reduced to some extent.

From the results in Figure 3, it was confirmed that the compound of the present invention (Catalyst 3) significantly reduced the amount of superoxide anion generated compared to Comparative Examples 1 and 2.

Furthermore, in the presence of living cells, the catalyst compound was added to evaluate the cytotoxicity caused by light irradiation. The cell culture medium containing the compound of the present invention (Catalyst 3) or Comparative Example 1 (Catalyst 1) was incubated at 37°C under 5% _CO2 for 48 hours, and the cell viability was analyzed by WST-8 (n=2, mean ± SEM). The compound concentrations were 0.1, 0.6, 1.5, 3.0, 5.0, and 10 μM. As a result, as shown in Figure 4, the compound of the present invention (Catalyst 3) maintained a higher cell viability than Comparative Example 1 (Catalyst 1) (Figure 4).

These results demonstrate that the compound of the present invention (Catalyst 3) can significantly reduce the generation of superoxide anions, which are a cause of cytotoxicity, by inhibiting stacking of catalyst molecules through derivatization of methylene blue molecules.

3. Oxidation of Amyloid β (Aβ) Next, an experiment on selective photooxygenation of Aβ, a pathogenic amyloid, was carried out using the compound of the present invention. As a comparative example, the CRANAD catalyst (Ni, J. et al. Chem. 2018, 4, 807-820), which is known as a conventional catalyst, was used. The CRANAD catalyst oxygenates amyloid through binding to the cross-β sheet, as shown in "Path (1)" in Figure 1.

First, Aβ1-42 was prepared in situ from 26-O-acylisopeptide (commercially available from Peptide Institute, Inc.) according to the references (Taniguchi, A.; Sasaki, D.; Shiohara, A.; Iwatsubo, T.; Tomita, T.; Sohma, Y.; Kanai, M. Angew. Chem. Int. Ed. 2014, 53, 1382).

As described in the above references, Aβ1-42 isopeptide (200 μM in 0.1% aqueous trifluoroacetic acid), angiotensin IV (200 μM in water), [Tyr ⁸ ]-Substance P (200 μM in water), leuprorelin acetate (200 μM in water), and somatostatin (200 μM in water) were diluted with 100 mM phosphate buffer or phosphate-buffered saline (PBS; pH 7.4) to a final peptide concentration of 20 μM (pH 7.4). For Aβ1-42, the solutions were incubated at 37°C for 30 min to 6 h.

To each solution, a compound of the present invention (Catalyst 3) or a comparative CRANAD catalyst (2 mM in dimethylsulfoxide) was added. The mixtures containing the compounds at a final concentration of 10 μM were irradiated with a light-emitting diode (LED) (λ = 655 nm) at 37 °C. For the CRANAD catalyst, an LED with λ = 780 nm was used. The power of the 655 nm LED source was 14 mW, and the light irradiation was performed at a distance of approximately 5 to 10 cm from the sample. A corresponding reaction sample without light irradiation was also prepared as a control. The reactions were monitored and analyzed using MALDI-TOF MS. If necessary, the reaction solutions were desalted with ZipTip U-C18 (Millipore Corporation) before MS analysis. The degree of oxygenation is expressed as the oxygenation intensity ratio (%) = (sum of MS peak intensities of n[O] adducts) / (sum of MS peak intensities of remaining starting material and n[O] adducts) x 100.

As shown in Figure 5, the compound of the present invention (Catalyst 3) was confirmed to interact with Aβ (20 μM) in a low aggregated state and to exhibit excellent oxygenation activity. On the other hand, the conventional CRANAD catalyst, which has the function of oxygenating amyloid through binding to cross-β sheets, was unable to oxygenate Aβ in a low aggregated state.

In addition, the oxygenation activity was also evaluated for Aβ at a low concentration (2 μM) close to physiological conditions (Figure 6). As a result, the compound of the present invention (Catalyst 3) showed efficient oxygenation activity even at such low concentration conditions.

In other words, it has been demonstrated that the compound of the present invention (Catalyst 3) has high affinity and strong oxygenation activity even for Aβ with a low amount of cross-β sheets, and therefore can maintain a high oxygenation yield even for Aβ in a low aggregated state, for which a decrease in oxygenation yield was confirmed with conventional catalysts.

On the other hand, it was also confirmed that the compound of the present invention (Catalyst 3) showed low oxygenation yields when other peptides containing tryptophan, tyrosine, histidine, methionine, etc. as off-target substrates were used (Figure 7). This result indicates that the compound of the present invention has high selectivity for Aβ. In other words, it suggests that the compound of the present invention shows high oxygenation activity only when it interacts with Aβ, as the PET mechanism and intramolecular stacking in the ground state are eliminated.

Claims (7)

A compound represented by the following formula ( II ) or a salt thereof:

(In the formula, R ^a is a hydrogen atom or 1 to 5 substituents which may be the same or different and are independently selected from an alkyl group, an amino group, a hydroxyl group, an alkoxy group, a halogen, or a nitro group; R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; m is a natural number from 1 to 10; n is a natural number from 1 to 10; and p is a natural number from 1 to 5.) A catalyst for the oxygenation of pathogenic amyloid, comprising the compound according to claim 1 or a salt thereof. An agent for inhibiting pathogenic amyloid aggregation, comprising the compound according to claim 1 or a salt thereof. 10. A pharmaceutical composition comprising the compound of claim 1 or a salt thereof and a pharma- ceutically acceptable carrier. The pharmaceutical composition according to claim 4 , which is a preventive or therapeutic drug for a disease associated with pathogenic amyloid. The pharmaceutical composition of claim 5 , wherein the pathogenic amyloid-associated disease is Alzheimer's disease. Use of the compound or a salt thereof according to claim 1 for the manufacture of a drug for the prevention or treatment of a disease involving pathogenic amyloid.

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