EA043204B1 - ADAMANTANE DERIVATIVE AND ITS APPLICATIONS - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to an adamantane derivative and a pharmaceutically acceptable salt thereof. The present invention also relates to a pharmaceutical composition containing such a compound and a method for treating or preventing a disease using such a compound.

Prerequisites for the creation of the invention

The ATP-sensitive K+ channel ( _KATP channel) is an internal rectifying K+ channel that connects intracellular metabolism and cell membrane excitation and is known to have a heterologous octamer structure consisting of a sulfonylurea receptor (SUR), belonging to the ABC family of proteins, and two transmembrane subunits Kir6.1 and Kir6.2. K _atp channel activity is controlled by various types of openers, K+ channel inhibitors, or intracellular nucleotides. All of them have active sites in SUR subunits. Their responses have been reported to differ depending on the subtype of SUR (NPL 1).

Some of the adamantane derivatives having a cell type structure are used as medicines. Amantadine is used as an antiviral drug and therapeutic agent for Parkinson's disease. Memantine hydrochloride is approved as a therapeutic agent for moderate/severe dementia of the Alzheimer's type even in Japan. Memantine is a non-competitive NMDA receptor inhibitor and has been reported to have a mechanism of action that prevents nerve cell death due to ischemia-induced excessive release of glutamic acid (NPL 2).

Adamantane derivatives having activity as a medicine have been described in various literatures (Patent Literature 1-3).

List of reference documents

Patent Literature.

Patent Literature 1: National Publication of International Patent Application No. 2011-529057.

Patent Literature 2: Japanese Patent Application Laid-Open No. 2010-522203.

Patent Literature 3: National Publication of International Patent Application No. 2009-508956.

non-patent literature.

Non-Patent Literature 1: Folia pharmacologica Japonica, 126, 311 to 316 (2005).

Non-Patent Literature 2: Folia pharmacologica Japonica, 124, 145 to 151 (2004).

The essence of the invention

Technical task.

A therapeutic and prophylactic method that provides a sufficient effect on a cognitive disease or disorder such as Alzheimer's disease has not yet been developed, and thus a new therapeutic and prophylactic agent with a different mechanism of action from existing medicines needs to be developed. In addition, the development of a new therapeutic and prophylactic agent for diabetes is highly desirable.

In one aspect, it is an object of the present invention to provide a pharmaceutical composition for use in the treatment or prevention of a cognitive disease or disorder. Yet another object of the present invention is to provide a method for treating or preventing a cognitive disease or disorder using a predetermined adamantane derivative.

In one aspect, it is an object of the present invention to provide a pharmaceutical composition for use in the treatment or prevention of diabetes or a diabetic complication. Yet another object of the present invention is to provide a method for treating or preventing diabetes or a diabetic complication using a predetermined adamantane derivative.

The ATP-sensitive K+ channel (K _atp channel) contains Kir6.1 and Kir6.2 subunits and is known to serve as an active site for, for example, an antidiabetic drug.

In one aspect, it is an object of the present invention to provide an anti-Kir6.1 channel inhibitor or an anti-Kir6.2 channel inhibitor of the K _atp channel. Yet another object of the present invention is to provide a pharmaceutical composition for use in the treatment or prevention of a disease associated with the Kir6.1 channel or the Kir6.2 channel of the K _atp channel. Another object of the present invention is to provide a method for treating or preventing a disease associated with the Kir6.1 channel or the Kir6.2 channel of the K _atp channel using a predetermined adamantane derivative.

The solution of the problem.

The inventors of the present invention have made intensive studies in order to achieve the above objectives. As a result, they found that the adamantane derivative has a Kir6.2 channel inhibitory activity, a Kir6.1 channel inhibitory activity, a therapeutic effect on a cognitive disease or disorder, and a hypoglycemic effect. Based on this, the present invention was created. The description discloses the following inventions presented in [1-1]-[1-20].

- 1 043204

[1-1] The compound represented by formula (I)

(I)

Chemical formula 1 where R ¹ is a hydrogen atom or (C ₁ . ₆ alkyl)carbonyl optionally substituted with one or more halogen atoms;

R ² represents a hydrogen atom or (C ₁ . ₆ alkyl)carbonyl optionally substituted with one or more halogen atoms;

X is O or NR ⁵ ;

R ³ is phenyl optionally substituted with one or more substituents selected from X ¹ , 5- or 6-membered heteroaryl optionally substituted with one or more substituents selected from X ¹ or COOR ⁶ ;

R ⁴ represents a hydrogen atom, a halogen atom, azido, -OR ⁷ or -NHR ⁸ ;

R ⁵ represents a hydrogen atom or C _1-6 alkyl;

R ⁶ represents a hydrogen atom or C _1-6 alkyl;

R ⁷ represents a hydrogen atom, C _1-6 alkyl, C _1-6 alkoxy-C _1-6 alkyl or (C _1-6 alkyl) carbonyl optionally substituted with one or more halogen atoms;

R ⁸ represents a hydrogen atom, C _1-6 alkyl or (€ _1-6 alkyl)carbonyl optionally substituted with one or more halogen atoms; And

X ¹ represents C _1-6 alkyl, halogen atom, C _1-6 alkoxy, nitro or cyano, its enantiomer, its diastereomer or its pharmaceutically acceptable salt.

[1-2] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [1-1], wherein R ⁴ is a chlorine atom or an azido.

[1-3] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [1-1] or [1-2], wherein R ¹ is trifluoroacetyl.

[1-4] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [1-1]-[1-3], wherein R ² is (C _1-6 alkyl)carbonyl, optionally substituted one or more halogen atoms.

[1-5] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [1-4], wherein R ² is trifluoroacetyl.

[1-6] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of [1-1]-[1-5], wherein R ³ is phenyl optionally substituted with one or more substituents selected from X ¹ , or pyridyl optionally substituted with one or more substituents selected from X ¹ .

[1-7] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [1-1] selected from (R)-(( 1 R,2S,3R,5R,7S)-5-azido -1-hydroxyadamantan-2-yl)(phenyl)methyl acetate;

ethyl (S)-2-acetamido-2-(( 1 R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate;

ethyl (R)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate;

(1R,2S,3R,5R,7R)-5-chloro-2-((S)-2-methoxy-2-oxo-1-(2,2,2-trifluoroacetamido)ethyl)adamantan-1-yl 2 ,2,2-trifluoroacetate;

(1S,2R,3S,5S,7S)-5-chloro-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate;

(S)-2-amino-2-((1R,2S,3R,5R,7S)-1,5-dihydroxyadamantan-2-yl)acetic acid;

N-((R)-((1S,2R,3S,5S,7S)-5-chloro-1-hydroxyadamantan-2-yl)(phenyl)methyl)-2,2,2-trifluoroacetamide;

(1S,2R,3S,5R,7S)-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate;

(1 S,2R,3S,5S,7R)-5-(2-methoxyethoxy)-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1 -yl 2,2,2 -trifluoroacetate;

N-((R)-((1S,2R,3S,5S,7S)-5-chloro-1-hydroxyadamantan-2-yl)(pyridin-3-yl)methyl)-2,2,2trifluoroacetamide;

2,2,2-trifluoro-N-((R)-((1S,2R,3S,5R,7S)-1-hydroxyadamantan-2-yl)(phenyl)methyl)acetamide; and (1S,2R,3S,5S,7R)-5-methoxy-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate; or an enantiomer, diastereomer or pharmaceutically acceptable salt of such a compound.

[1-8] A pharmaceutical composition containing a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [1-1]-[1-7].

[1-9] A pharmaceutical composition according to [1-8] for use in the treatment or prevention of a cognitive disease or disorder.

- 2 043204

[1-10] The pharmaceutical composition according to [1-9], wherein the cognitive disease or disorder is selected from Alzheimer's type dementia, cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, mental illness, and neurodegenerative disease.

[1-11] A pharmaceutical composition according to [1-8] for use in the treatment or prevention of diabetes or a diabetic complication.

[1-12] A Kir6.2 channel inhibitor comprising a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [1-1]-[1-7].

[1-13] A Kir6.1 channel inhibitor, comprising a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [1-1]-[1-7].

[1-14] A method for treating or preventing a cognitive disease or disorder, comprising administering to a subject a therapeutically effective amount of a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of [1-1]-[1-7].

[1-15] The method according to [1-14], wherein the cognitive disease or disorder is selected from Alzheimer's type dementia, cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, mental illness, and neurodegenerative disease.

[1-16] A method for treating or preventing diabetes or a diabetic complication, comprising administering to a subject a therapeutically effective amount of a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of [1-1]-[1-7].

[1-17] A method for treating or preventing a disease associated with a Kir6.1 channel or a Kir6.2 channel, comprising administering to a subject a therapeutically effective amount of a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of [1-1 ]-[1-7] as a Kir6.1 channel inhibitor or a Kir6.2 channel inhibitor.

[1-18] The method according to [1-17], wherein the disease associated with the Kir6.1 channel or the Kir6.2 channel is a cognitive disease or disorder or diabetes or a diabetic complication.

[1-19] The method according to [1-17] or [1-18], wherein the disorder associated with the Kir6.1 channel or the Kir6.2 channel is a cognitive disease or a disorder selected from Alzheimer's type dementia, cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, mental illness and neurodegenerative disease.

[1-20] The method according to [1-17] or [1-18], wherein the disease associated with the Kir6.1 channel or the Kir6.2 channel is diabetes or a diabetic complication.

The description discloses the following inventions presented in [2-1]-[2-12].

[2-1] The compound represented by formula (I)

(I)

Chemical formula 2 where R ¹ represents a hydrogen atom or (C ₁ . ₆ alkyl)carbonyl optionally substituted with one or more halogen atoms;

R ² represents a hydrogen atom or (C ₁ . ₆ alkyl)carbonyl optionally substituted with one or more halogen atoms;

X is O or NR ⁵ ;

R ³ is phenyl optionally substituted with one or more substituents selected from X ¹ or COOR ⁶ ;

R ⁴ represents a hydrogen atom, a halogen atom, azido, -OR ⁷ or -NHR ⁸ ;

R ⁵ represents a hydrogen atom or C ₁ . ₆ alkyl;

R ⁶ represents a hydrogen atom or C ₁ . ₆ alkyl;

R ⁷ is a hydrogen atom, C ₁ . ₆ alkyl or (C ₁ . ₆ alkyl)carbonyl optionally substituted with one or more halogen atoms;

R ⁸ represents a hydrogen atom, C ₁ . ₆ alkyl or (C ₁ . ₆ alkyl)carbonyl optionally substituted with one or more halogen atoms; And

X ¹ is C ₁ . ₆ alkyl, halogen atom, C ₁ . ₆ alkoxy, nitro or cyano, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof.

[2-2] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [2-1], wherein R ⁴ is a chlorine atom or an azido.

[2-3] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [2-1] or [2-2], wherein R ¹ is trifluoroacetyl.

[2-4] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof in

- 3 043204 in accordance with any of [2-1]-[2-3], where R ² represents trifluoroacetyl.

[2-5] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of [2-1]-[2-4], wherein R ³ is phenyl optionally substituted with one or more substituents selected from ^X1 .

[2-6] A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to [2-1] selected from (R)-((1R,2S,3R,5R,7S)-5-azido-1 -hydroxyadamantan-2-yl)(phenyl)methyl acetate;

ethyl (S)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate;

ethyl (R)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate;

(1R,2S,3R,5R,7R)-5-chloro-2-((S)-2-methoxy-2-oxo-1-(2,2,2-trifluoroacetamido)ethyl)adamantan-1-yl 2 ,2,2-trifluoroacetate;

(1S,2R,3S,5S,7S)-5-chloro-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate; and (S)-2-αmino-2-((1R,2S,3R,5R,7S)-1,5-dihydroxuadαmαmαnαn-2-yl)acetic acid, or an enantiomer, diastereomer, or pharmaceutically acceptable salt of such a compound.

[2-7] A pharmaceutical composition containing a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [2-1]-[2-6].

[2-8] A pharmaceutical composition according to [2-7] for use in the treatment or prevention of a cognitive disease or disorder.

[2-9] The pharmaceutical composition according to [2-8], wherein the cognitive disease or disorder is selected from Alzheimer's type dementia, cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, mental illness, and neurodegenerative disease.

[2-10] A pharmaceutical composition according to [2-7] for use in the treatment or prevention of diabetes or a diabetic complication.

[2-11] A Kir6.2 channel inhibitor, comprising a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [2-1]-[2-6].

[2-12] A Kir6.1 channel inhibitor, comprising a compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof according to any one of [2-1]-[2-6].

Useful effects of the invention.

In one aspect, the present invention provides a pharmaceutical composition for use in the treatment or prevention of a cognitive disease or disorder. In another aspect, the present invention provides an inhibitor of the Kir6.1 channel or Kir6.2 channel of the K _atp channel.

Brief description of the figures

Fig. 1 is a graph showing CaMKII activity enhanced by the compound of the present invention in cells (Neuro2A cells) overexpressing the Kir6.2 channel. All indicators related to significant difference are relative to control (C: Kir6.2 expressing non-drug-treated cells). In the drawings in this application, a significant difference, marked as ** or ++, means P<0.01; or significant difference marked as + or * means P<0.05.

Fig. 2a shows (results) the expression of the Kir6.2 channel in N2A cells, which was monitored using immunoblotting using an anti-Kir6.2 channel antibody to Kir6.2 channel overexpressing cells. The case having a significant difference from the non-drug treated group (-) is indicated as **.

Fig. 2b shows the results of whole cell patch clamp analysis showing that TP-014 suppresses potassium outflow in Kir6.2 channel-overexpressing cells. The results show that TP-014 inhibits the Kir6.2 channel and reduces potassium current.

Fig. 3a shows the results of calcium imaging analysis showing that the intracellular calcium concentration in Kir6.2 channel-overexpressing cells is increased by administration of TP-014. The change in the concentration-dependent amount of calcium over time (4 min) was monitored in cases of treatment with the compound of the present invention and memantine. The results show that TP-014 inhibits the Kir6.2 channel and increases the intracellular calcium concentration.

Fig. 3b shows the results of calcium imaging analysis showing that the intracellular calcium concentration in Kir6.2 channel-overexpressing cells is increased by administration of TP-014. The amount of calcium was measured 4 minutes after treatment with memantine and the compound of the present invention. The group using Kir6.2-expressing cells (Neuro2A cells) was confirmed to have a significant difference compared to the non-drug group (-). The results show that TP-014 inhibits the Kir6.2 channel and increases the intracellular calcium concentration.

Fig. 4a shows the results of an experiment in which TP-014 was administered to model mice with more

- 4 043204 Alzheimer's disease (APP23 mice) (age 12 months) for two months and the analysis was performed by the Y-maze method, showing that cognitive function was effectively improved.

Fig. 4b shows the results of an experiment in which TP-014 was administered to model mice with Alzheimer's disease (APP23 mice) (age 12 months) for two months and analysis was performed by the Y-maze method, showing that cognitive function was effectively improved. The case having a significant difference between wild-type (WT) and APP23 mice in the percentage of correct answers (change) in memory learning is indicated as **; and the case having a significant difference between APP mice and controls (untreated group) is indicated as ++.

Fig. 4c is a graph showing the results of an experiment in which TP-014 was administered to model mice with Alzheimer's disease (APP23 mice) (age 12 months) for two months and analyzed by a novel object recognition test, showing that cognitive function was effectively improved. . The case of significant difference between New (new object) and Familiar (same object) in each group of mice is indicated as **

Fig. 4d shows the results of an experiment in which TP-014 was administered to model mice with Alzheimer's disease (APP23 mice) (age 12 months) for two months and analysis was performed by a conditioned freezing test, showing that cognitive function was effectively improved. In memory ability tests, the case having a significant difference from WT is listed as **; and the case having a significant difference with APP23 mice is indicated as +.

Fig. 4e shows the results of an experiment in which TP-014 was administered to model mice with Alzheimer's disease (APP23 mice) (age 12 months) for two months and analysis was carried out for long-term potentiation phenomenon (LTP) as an indicator of memory formation by electrophysiological method.

Fig. 4f shows the results of an experiment in which TP-014 was administered to model mice with Alzheimer's disease (APP23 mice) (age 12 months) for two months and analyzed for long-term potentiation phenomenon (LTP) as an indicator of memory formation by electrophysiological method.

Fig. 4g shows the results of an experiment in which TP-014 was administered to model mice with Alzheimer's disease (APP23 mice) (age 12 months) for two months and analyzed for long-term potentiation phenomenon (LTP) as an indicator of memory formation by electrophysiological method. The case having a significant difference with WT is listed as **; and the case having a significant difference with APP23 mice is listed as ++ or +.

Fig. 5a shows bands (image) obtained by immunoblot electrophoresis showing the results of protein phosphorylation, which were analyzed by immunoblotting using antibodies against CaMKII, CaMKIV and ERK.

Fig. 5b shows the results of quantitative analysis of the signal intensity of the bands (in Fig. 5a) obtained by immunoblot electrophoresis. The case having a significant difference with WT(-) (non-treated group) is indicated as **; and the case having a significant difference with the APP23 group of mice not treated with drug (-) is indicated as +.

Fig. 5c shows bands (image) obtained by immunoblot electrophoresis showing the results of protein phosphorylation, which were analyzed by immunoblotting using antibodies against CaMKII, CaMKIV and ERK.

Fig. 5d shows the results of quantitative analysis of the signal intensity of the bands (in Fig. 5c) obtained by immunoblot electrophoresis. The case having a significant difference with WT(-) (non-treated group) is indicated as **; and the case having a significant difference with the APP23 group of mice not treated with drug (-) is indicated as ++.

Fig. 6a shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. olfactory bulb removed mice (OBX mice) for two months and Y-maze analysis was performed, showing that cognitive function was effectively improved.

Fig. 6b shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. olfactory bulb removed mice (OBX mice) for two months and Y-maze analysis was performed, showing that cognitive function was effectively improved. In OBH mice, the case having a significant difference with WT in percent correct (change) in memory learning is indicated as **; and the case having a significant difference between control (untreated group) and OBH mice is indicated as ++.

Fig. 6c shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. olfactory bulb removed mice (OBX mice) for two months, and analysis was carried out with a novel object recognition test, showing that cognitive function was effectively improved. The case of significant difference between Novel (new object) and Familiar (familiar object) in each group of mice, indicated as **

Fig. 6d shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. mice with removed olfactory bulbs (OBX mice), in

- 5 043204 for two months and the analysis was carried out using the conditioned freezing test, showing that the cognitive function is effectively improved. In memory ability tests, the case having a significant difference from WT is listed as **; and the case having a significant difference with OBX mice is indicated as +.

Fig. 6e shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. olfactory bulb ablated mice (OBX mice) for two months and analyzed for long-term potentiation phenomenon (LTP), which is an indicator of memory formation, by the electrophysiological method.

Fig. 6f shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. olfactory bulb ablated mice (OBX mice) for two months and analyzed for long-term potentiation phenomenon (LTP), which is an indicator of memory formation, by the electrophysiological method.

Fig. 6g shows the results of an experiment in which TP-014 was administered to model mice with a neurodegenerative disease, ie. olfactory bulb ablated mice (OBX mice) for two months and analyzed for long-term potentiation phenomenon (LTP), which is an indicator of memory formation, by the electrophysiological method. The case having a significant difference from the simulation is indicated as **; and a case having a significant difference with OBX mice as ++ or +.

Fig. 7a shows bands (image) obtained by immunoblot electrophoresis showing the results of phosphorylation of proteins that were analyzed by immunoblotting using antibodies against CaMKII, CaMKIV and ERK.

Fig. 7b shows the results of quantitative analysis of the signal intensity of the bands (in Fig. 7a) obtained by immunoblot electrophoresis.

Fig. 7c shows bands (image) obtained by immunoblot electrophoresis showing the results of protein phosphorylation, which were analyzed by immunoblotting using antibodies against CaMKII, CaMKIV and ERK.

Fig. 7d shows the results of quantitative analysis of the signal intensity of the bands (in Fig. 7c) obtained by immunoblot electrophoresis. The case having a significant difference with WT(-) (non-treated group) is indicated as **; and the case having a significant difference with the OBX group of mice not treated with drug (-) is indicated as ++.

Fig. 8a is a graph showing the results of an experiment in which TP-014 was administered to Kir6.2 channel-deficient mice for two months and analyzed by the Y-maze method, showing that cognitive function was effectively improved.

Fig. 8b shows the results of an experiment in which TP-014 was administered to Kir6.2 channel-deficient mice for two months and analyzed by the Y-maze method, showing that cognitive function was effectively improved. The case having a significant difference between Kir6.2-deficient mice and WT in percent correct (change) in memory learning is indicated as * or **.

Fig. 8c is a graph showing the results of an experiment in which TP-014 was administered to Kir6.2 channel-deficient mice for two months and analysis was performed by a novel object recognition test, showing that cognitive function was effectively improved. The case of significant difference between Novel (new object) and Familiar (familiar object) in each group of mice is indicated as **.

Fig. 8d is a graph showing the results of an experiment in which TP-014 was administered to Kir6.2 channel-deficient mice for two months and analysis was performed by a conditioned freezing test, showing that cognitive function was effectively improved. In memory ability tests, the case having a significant difference from WT is shown as *.

Fig. 8e shows the results of an experiment in which TP-014 was administered to Kir6.2 channel-deficient mice for two months and analyzed for long-term potentiation phenomenon (LTP) as an indicator of memory formation by electrophysiological method.

Fig. 8f shows the results of an experiment in which TP-014 was administered to Kir6.2 channel-deficient mice for two months and analyzed for long-term potentiation phenomenon (LTP) as an indicator of memory formation by electrophysiological method.

Fig. 8g shows the results of an experiment in which TP-014 was injected with Kir6.2 channel-deficient mice for two months and analyzed for long-term potentiation (LTP) phenomenon, which is an indicator of memory formation, by electrophysiological method. A case that has a significant difference from WT is listed as ** or *.

Fig. 9a shows bands (image) obtained by immunoblot electrophoresis showing the results of protein phosphorylation, which were analyzed by immunoblotting using antibodies against CaMKII, CaMKIV and ERK.

Fig. 9b shows the results of quantitative analysis of the signal intensity of the bands (in Fig. 9a) obtained by immunoblot electrophoresis. Case having significant difference with WT(-) (group, not

- 6 043204 drug-treated), is indicated as ** or *.

Fig. 10 shows the results of staining sections of mouse brain sections with APP23 showing the effect of the compound of the present invention on Ae aggregation.

Fig. 11a shows the results of a test that investigates whether or not the compound of the present invention has the effect of improving depression-like symptom in OVH mice. The case with a significant difference from sham (control group) is indicated as **; and the case having a significant difference with OBX mice is indicated as +.

Fig. 11b shows the results of a test that investigates whether or not the compound of the present invention has the effect of improving depression-like symptom in OBH mice. The case with a significant difference with the sham (control group) is indicated as **, and with a significant difference with OBX mice is indicated as +.

Fig. 12a shows the results of a test that examines whether or not the compound of the present invention has an improvement effect on depression by inhibitory action on the Kir6.1 channel. The case having a significant difference with WT (control group) is indicated as **.

Fig. 12b shows the results of a test that examines whether or not the compound of the present invention has an ameliorating effect on depression by inhibitory action on the Kir6.1 channel. The case having a significant difference with WT (control group) is indicated as **.

Fig. 13a shows the results of a test that investigates whether the compound of the present invention actually inhibits the Kir6.1 channel and activates CaMKIV, thus having an improving effect on depression. The case having a significant difference with WT (control group) is indicated as **.

Fig. 13b shows the results of a test that investigates whether the compound of the present invention actually inhibits the Kir6.1 channel and activates CaMKIV, thus having an improving effect on a depression-like symptom. The case having a significant difference with WT (control group) is indicated as **.

Fig. 14 shows the results of a test that examines whether or not the compound of the present invention has a hypoglycemic effect. The term weeks refers to the time period of long-term administration, the case having a significant difference between ob/ob (saline solution) in each week is indicated as *.

Fig. 15 is an illustration showing the mechanism of action of TP-014. When the Kir6.2 channel located in the spinal cord is inhibited, the potassium present in the cells cannot flow out of them, raising the cell membrane threshold. As a result, calcium outside the cell enters the cell faster, activates CaMKII, activates GluA1 (Ser-831) (AMPA acceptor) downstream of CaMKII, and presumably improves cognitive function. TP-014 similarly inhibits the Kir6.1 channel located in the nerve cell body. As a result, calcium enters the cells by a similar mechanism. Calcium influx activates CaMKIV, activates CREB (Ser-133), and presumably induces neurogenesis, improving depression. TP-014 is a novel cognition-enhancing drug having both a cognition-enhancing effect (Alzheimer's main symptom) based on an inhibitory Kir6.2 channel of action, and a depression-improving effect (Alzheimer's peripheral symptom) based on an inhibitory Kir6.1 actions.

Fig. 16 shows the structure of the plasmid vector: pcDNA3.1-Kir6.2.

Fig. 17-1 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.2.

Fig. 17-2 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.2.

Fig. 17-3 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.2.

Fig. 17-4 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.2.

Fig. 17-5 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.2.

Fig. 18a is a graph showing CaMKIV activity enhanced by the compound of the present invention in cells (Neuro2A cells) overexpressing the Kir6.1 channel. All cases with a significant difference from the control (C: Kir6.1 expressing non-drug-treated cells) were noted.

Fig. 18b shows the results of Kir6.1 channel expression in N2A cells, which was monitored using immunoblotting using an anti-Kir6.1 channel antibody to Kir6.1 channel overexpressing cells. The case having a significant difference from the non-drug treated group (-) is indicated as **.

Fig. 18c shows the results of determining the current of potassium flowing out of the cells, checked using Kir6.1 channel-overexpressing cells and measured by the conventional patch clamp method.

Fig. 19a shows the results of sensitivity analysis of groups of mice (used in the test) to anxiety using the elevated plus maze method. With regard to the time spent by the mouse in the open arm of the maze, the case having a significant difference from WT(-) is indicated as ** or *; and the case having a significant difference with WT (CORT) is listed as ++.

- 7 043204

Fig. 19b shows a photograph of the apparatus used in the elevated plus maze method.

Fig. 19c shows the results of the light/dark test method. The case having a significant difference with WT(-) is indicated as **; and the case having a significant difference with WT (CORT) is listed as ++.

Fig. 19d shows a photograph of the device used in the light/dark test method.

Fig. 19e shows the results of the bead burying test. The case having a significant difference with WT(-) is indicated as **; and the case having a significant difference with WT (CORT) is listed as +.

Fig. 19f shows a photograph of the device used in the beading method.

Fig. 19g shows the results of the open field test. The case having a significant difference with WT(-) is indicated as **; and the case having a significant difference with WT (CORT) is listed as ++.

Fig. 19h shows a photograph of the device used in the open field method.

Fig. 19i shows the results of the conditioned fading test. The case that is significantly different from WT(-) is listed as ** or *; and the case having a significant difference with WT (CORT) is listed as ++.

Fig. 20 shows the structure of the plasmid vector: pcDNA3.1-Kir6.1.

Fig. 21-1 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.1.

Fig. 21-2 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.1.

Fig. 21-3 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.1.

Fig. 21-4 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.1.

Fig. 21-5 shows the sequence of the plasmid vector: pcDNA3.1-Kir6.1.

Description of Embodiments

The present invention is more specifically described below.

According to one aspect of the present invention, there is provided a pharmaceutical composition for the treatment or prevention of a cognitive disease or disorder, comprising a compound represented by formula (I), an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof. More specifically, the compound of the present invention includes a compound represented by the following formulas (I) and (II).

Chemical formula 3

In the description of the invention C _1-6 alkyl refers to a linear, branched, cyclic or partially cyclic alkyl group containing 1-6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, 3-methylbutyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, n-hexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-ethylbutyl and 2-ethylbutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cyclopropylmethyl. For example, C _1-4 alkyl and C _1-3 alkyl are also included.

In the description of the invention, C _1-6 alkoxy refers to an alkyloxy group [-O-(C _1-6 alkyl)] containing an alkyl group with 1-6 carbon atoms as defined above. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, t-butoxy, n-pentoxy, 3-methylbutoxy, 2-methylbutoxy, 1-methylbutoxy, 1-ethylpropoxy, n-hexyloxy, 4-methylpentoxy, 3-methylpentoxy, 2-methylpentoxy, 1-methylpentoxy, 3-ethylbutoxy, cyclopentyloxy, cyclohexyloxy and cyclopropylmethyloxy. For example, C _1-4 alkoxy and C _1-3 alkoxy are also included. In the description of the invention, C _1-4 alkoxy includes, for example, C _1-3 alkoxy.

In the description of the invention, azido refers to -N ₃ .

In the specification, (C _1-6 alkyl)carbonyl refers to an alkylcarbonyl group containing a C _1-6 alkyl group as defined above. Examples include methylcarbonyl(acetyl), ethylcarbonyl, t-butylcarbonyl and (C _1-3 alkyl)carbonyl.

In the description of the invention, 5- or 6-membered heteroaryl is not specifically limited, provided that it is a heteroaryl consisting of a 5-membered ring or a 6-membered ring containing at least one heteroatom selected from an oxygen atom, a nitrogen atom, and sulfur atom. Examples include pyridyl, pyrimidyl, pyridazinyl, pyrazyl, furanyl (furyl), thiophenyl (thienyl), oxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl and tetrazolyl.

In the specification, C _1-6 alkoxy-C _1-6 alkyl refers to C _1-6 alkyl containing a C _1-6 alkoxy substituent as defined above, and the C _1-6 alkyl alkyl portion is as defined above. Examples include methoxymethyl, ethoxymethyl, 2-methoxyethyl, 1-methoxyethyl, 3-methoxypropyl, 2-methoxypropyl and 1-methoxypropyl.

Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

In the description of the invention, examples of (C _1-6 alkyl)carbonyl optionally substituted with one or more halogen atoms include trifluoroacetyl, difluoroacetyl, 2,2,2-trifluoroethylcarbonyl and

- 8 043204 perfluoroethylcarbonyl.

If the compound represented by formula (I) forms a solvate such as a hydrate, the present invention can be carried out using the solvate. In addition, the compound of the present invention can be appropriately carried out using the compound in a mixture or solution state or crystal polymorphism.

In the specification, substituted with one or more substituents includes, for example, substitution with 1-3 substituents.

The present invention relating to the compound represented by formula (I) includes a tautomer, a geometric isomer, various stereoisomers such as an optical isomer, and a diastereomer, and a mixture thereof. Examples of the compound represented by formula (I) include the compound represented by the following formulas (Ia) to (Ih).

Chemical formula 4

As the compound of the present invention, for example, the compound described in the examples in the present specification can be used. More specifically, the following connections can be used:

(R)-((1R,2S,3R,5R,7S)-5-azido-1-hydroxyadamantan-2-yl)(phenyl)methyl acetate (TP-009);

ethyl (S)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate (TP-010);

ethyl (R)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate (TP-011);

(1 R,2S,3R,5R,7R)-5-chloro-2-((S)-2-methoxy-2-oxo-1-(2,2,2-trifluoroacetamido)ethyl)adamantan-1-yl 2,2,2-trifluoroacetate (TP-012);

(1 S,2R,3S,5 S,7 S)-5-chloro-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1 -yl 2,2,2trifluoroacetate (TP -014);

(S)-2-amino-2-(( 1 R,2S,3R,5R,7S)-1,5-dihydroxyadamantan-2-yl)acetic acid (TP-015);

N-((R)-(( 1 S,2R,3S,5 S,7 S)-5-chloro-1-hydroxyadamantan-2-yl)(phenyl)methyl)-2,2,2-trifluoroacetamide (TP -048);

(1 S,2R,3S,5R,7 S)-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1 -yl 2,2,2-trifluoroacetate (TP-049) ;

(1 S,2R,3S,5 S,7R)-5-(2-methoxyethoxy)-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1 -yl 2,2, 2-trifluoroacetate (TP-050) ;

N-((R)-(( 1 S,2R,3S,5 S,7 S)-5-chloro-1-hydroxyadamantan-2-yl)(pyridin-3-yl)methyl)-2,2,2trifluoroacetamide (TR-051);

2,2,2-trifluoro-N-((R)-(( 1 S,2R,3S,5R,7S)-1-hydroxyadamantan-2-yl)(phenyl)methyl)acetamide (TP-052); and (1 S,2R,3S,5 S,7R)-5-methoxy-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1 -yl 2,2,2trifluoroacetate (TP -053).

A pharmaceutically acceptable salt of the compound represented by formula (I) is not particularly limited, as long as it is a salt that can be used as a pharmaceutical product.

Examples of the salt formed by the compound of the present invention and a base include a salt with an inorganic base such as sodium, potassium, magnesium, calcium and aluminum; and a salt with an organic base such as methylamine, ethylamine and ethanolamine. The salt may be an acid addition salt. Examples of the acid addition salt include a salt with a mineral acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid; and an acid addition salt with an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid.

Atoms (for example, a hydrogen atom, a carbon atom, an oxygen atom, a nitrogen atom, and a sulfur atom), containing

- 9 043204 huddled in the compound represented by formula (I) may be atoms of isotopes other than those most commonly encountered. Isotope atoms may be radioactive isotopes. More specifically, according to one aspect of the present invention, there is provided a compound represented by formula (I) already defined in the specification and labeled with an isotope atom, or a salt thereof. Isotope labeling can be, for example, radioactive isotope labeling (eg ³ H, ¹⁴ C, ³² P). In order to easily obtain a compound, labeling with ³ H is preferred.

In one embodiment of the present invention, a compound represented by formula (I), its enantiomer, its diastereomer, or a pharmaceutically acceptable salt thereof, is administered as a prodrug to be converted to the active compound in vivo.

In the present invention, examples of treating a cognitive disease or disorder include the treatment of Alzheimer's type dementia, cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, mental illness, and neurodegenerative disease. In the present invention, the pharmaceutical composition can be used to improve brain dysfunctions, for example, brain dysfunction caused by cardiovascular disease, traumatic brain injury, brain tumor, viral encephalitis, hypoxic encephalopathy, and alcohol intoxication. The present invention can be applied, in particular, to cognitive dysfunctions such as memory impairment, attention deficit disorder, executive function disorder and social behavior disorder. Examples of cognitive dysfunction include neurodegenerative disease (Alzheimer's disease, Parkinson's disease, Pick's disease and Huntington's disease, etc.), mental illness (schizophrenia, bipolar disorder, depression, phobia, sleep disorder, drug addiction, etc.) and general disorder development (autism, Asperger's syndrome, mental retardation, hyperactivity disorder, tic disorder, etc.).

In the present invention, examples of the diabetic complication include hyperglycemia, diabetic coma, ketone coma, nonketotic hyperosmolar coma, lactic acidosis, hypoglycemic coma, acute infection, microangiopathy, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, macroangiopathy, cerebral vascular disease, coronary heart disease, diabetic gangrene , hyperlipidemia, chronic infection, cholelithiasis and cataracts.

In one embodiment of the present invention, the compound represented by formula (I), its enantiomer, its diastereomer or its pharmaceutically acceptable salt is used as a Kir6.2 channel inhibitor or a Kir6.1 channel inhibitor. More specifically, the compound represented by formula (I), its enantiomer, its diastereomer, or its pharmaceutically acceptable salt can be used for the treatment or prevention of a disease in which the Kir6.2 channel is involved, such as cognitive disease or disorder, hyperglycemia, diabetes, and diabetic complication. ; and for the treatment or prevention of a disease in which the Kir6.1 channel is involved, such as cognitive disease or disorder, hyperglycemia, diabetes, diabetic complication, and mental illness.

The pharmaceutical composition of the present invention may take various dosage forms. Examples of dosage forms for oral administration include tablet, capsule, powder preparation, granule, pill, liquid preparation, emulsion, suspension, solution, spray, syrup, extract, and elixir. Examples of dosage forms for parenteral administration include injection such as subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection; transdermal injection or patch, as well as ointment or lotion. Examples of dosage forms for intraoral administration include sublingual form and oral patch. Examples of nasal administration include an aerosol. However, dosage forms are not limited to this. These drugs can be obtained by methods known in this field and commonly used for the formulation of drugs.

The pharmaceutical composition may contain various commonly used components; for example, may contain at least one type of pharmacologically acceptable excipient, disintegrant, diluent, lubricant, flavor, color, sweetener, flavor, suspending agent, wetting agent, emulsifier, dispersing agent, adjuvant, preservative, buffer, binder, stabilizer, and agent coatings. The pharmaceutical composition of the present invention may be in a sustained release form or a sustained release form.

The dose of the therapeutic agent, prophylactic agent or pharmaceutical composition of the present invention can be appropriately selected depending on, for example, the route of administration, body size, age, physical condition of the patient, severe or mild symptom of the disease, and the period of the disease after the onset of the disease. The pharmaceutical composition of the present invention may contain a therapeutically effective amount and/or a prophylactically effective amount of a compound represented by formula (I). In the present invention, the compound represented by formula (I) can be used generally at a dose of 1-1000 mg/day for an adult patient or 0.01 to 20 mg/day/kg of body weight. Introduction of pharmaceutical composition

- 10 043204 may be single or multiple doses.

The pharmaceutical composition of the present invention may contain, if necessary, components known in the art, such as a colorant, a preservative, a chemical flavor, a flavor, a coating agent, an antioxidant, a vitamin, an amino acid, a peptide, a protein, and a mineral (iron, zinc, magnesium, iodine, etc.). The therapeutic agent or prophylactic agent of the present invention may have dosage forms suitable for, for example, pharmaceutical composition, functional food, health food, beverage and supplement, for example, solid preparations such as granule (including dry syrup), capsule (soft capsule, hard capsule), tablet (including chewable dosage form), powdered dosage form (powder) and pill, or liquid preparations such as oral solution (including liquid drug, suspension, syrup). The therapeutic agent or prophylactic agent of the present invention can be used directly, for example, as a pharmaceutical composition, functional food, health food and supplement.

Examples of additives for formulating a drug product include an excipient, a lubricant, a binder, a disintegrant, a thinning agent, a dispersing agent, a wetting agent, a preservative, a thickener, a pH modifier, a coloring agent, a flavoring agent, a surfactant, and a solubilizing agent. To formulate a liquid drug, a thickening agent such as pectin, xanthan gum, and guar gum can be added to the mixture. In addition, coated tablets can be prepared using a coating agent and a pasty adhesive can be formed. In the case of other dosage forms, drug products can be obtained in accordance with the usual method.

Examples

The present invention will be more specifically described using examples; however, the present invention is not limited to these examples.

Example 1

O

H ₃ C

^ph and ^pt LiCI, THF;

TMSCI

O

X

N CH _}

EYOS

TMSOTf

CH ₂ CI ₂

TiCI ₄

CH ₂ C1 ₂ o

n ₃ s

C1

H ₃ C

TR-010

CI

Chemical formula 5

To a solution of bis((R)-1-phenylethyl)amine (1.8 g, 18 mmol) in THF (30 ml) was added dropwise a solution of n-BuLi in hexane (1.56 M, 4.8 ml, 7. 33 mmol) under ice cooling. The reaction solution was stirred for 30 minutes at the same temperature, the reaction solution was cooled to -78°C. Trimethylsilyl chloride (TMSCl, 1.7 mL, 13.3 mmol) was then added followed by a solution of 7-methylenebicyclo[3.3.1]nonan-3-one (1.0 g, 16.6 mmol) in THF (5 mL ) through the cannula. After stirring for 1 hour, water was added to the reaction solution, and the solution was extracted with diethyl ether. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure. The residue was subjected to silica gel chromatography (hexane:ethyl acetate=9:1) to give the title compound, TMS enol ester (1.24 g, 84%) as a colorless oil.

TMS-prepared enol ester (400 mg, 1.80 mmol) and ethyl (E)-2-(acetylimino)acetate (5.4 mmol) prepared according to the method disclosed in the literature (Kobayashi S et al., J. Combi. Chem., 2001, 3, 401) was dissolved in dichloromethane (9 ml). The reaction solution was cooled to 0°C. To this solution was added trimethylsilyl trifluoromethanesulfonate (TMSOTf, 240 mL, 900 mmol). The solution was stirred at the same temperature for 1 hour and then saturated aqueous NaHCO ₃ was added to stop the reaction. The reaction solution was extracted with ethyl acetate. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure to give a crude product (410 mg, oil) of the Mannich reaction. The crude product (400 mg) was dissolved in dichloromethane (7 ml) and cooled to -30°C. TiCl ₄ (120 ml, 1.09 mmol) was added to the solution. After stirring the solution for 1 hour at the same temperature, water was added to stop the reaction. The solution was extracted with diethyl ether. The resulting organic layer was washed with brine and then dried over MgSO4, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=10:1 to 9:1) to give TP010 (148 mg, 25%) and

- 11 043204

TP-011 (228 mg, 52%).

TP-010 (84% ee): amorphous;

[a]D ²⁹ =3.4 (c=1.832, CHCl ₃ ); (1H-NMR (400 MHz, CDCl ₃ ): δ 6.15 (d, J=8.8 Hz, 1H), 4.90 (t, J=9.6 Hz, 1H), 4.22-4 .15 (m, 2H), 3.00 (s, 1H), 2.29 (br.s, 1H), 2.15-1.95 (m, 8H), 2.04 (s, 3H), 1.85-1.78 (m, 2H), 1.50 (brd, J=12.7 Hz, 1H), 1.37 (brd, J=13.4 Hz, 1H), 1 .28 (t, J=7.3 Hz, 3H);

¹³ C-NMR (100 MHz, CDCl ₃ ): δ 173.1, 170.3, 70.8, 66.6, 61.8, 56.7, 51.8, 51.7, 47.9, 46 .6, 37.7, 33.3, 31.8, 29.4, 23.4, 14.0;

IR (solvent free, cm ^-1 ): 3336, 1725, 1654;

MS (EI): m/z 329 (M ⁺ ), 256 (100%);

HRMS (EI): calculated for C ₁₆ H ₂₄ NO ₄ Cl (M+) 329.1394, found 329.1399.

TP-011 (84% ee): m.p. 65-68°C (Et ₂ O-n-hexane);

[a]D ²⁹ = -2.4 (c=1.72, CHCl3);

1H-NMR (400 MHz, CDCl ₃ ): δ 7.64 (br s, 1H), 4.42 (d, J=10.1, 3.9 Hz, 1H), 4.20 (q, J=7.1Hz, 2H), 3.70(s, 1H), 2.30(brs, 1H), 2.20-1.88(m, 9H), 1.98(s, 3H ), 1.88 (brd, J=13.9 Hz, 1H), 1.57 (brd, J=12.5 Hz, 1H), 1.38 (brd, J=12, 5 Hz, 1n), 1.28 (t, J=7.1 Hz, 3H);

13C-NMR (100 MHz, CDCl ₃ ): δ 171.7, 170.4, 72.6, 66.0, 61.1, 56.6, 55.1, 48.9, 47.3, 46, 2, 38.3, 34.3, 31.8, 29.0, 22.9, 14.1;

IR (solvent free, cm ^-1 ): 3377, 1739, 1650;

MS (EI): m/z 329 (M ⁺ ), 256 (100%);

HRMS (EI) : calculated for C ₁₆ H ₂₄ NO ₄ Cl (M+) 329.1394, found 329.1415.

Example 2

TR-013 o o cat. RuCI ₃ -3H ₂ O sash D. H ₂ N mon

NYU ₄ -2H ₂ O ^F 3 ^C 00CF ₃ 2MNaOH

MaCN SP Η o- ^G MeO ₂ CrA0 THF Lil

Μ6ϋΐΜ-υυΐ4-ΓΊ2νΛ f I ch ₂ n ₂ ZkA ^cl

_Et2O

TP-012 TP-015

Chemical formula 6

To a solution of (1R,2S,5S)-2-((R)-hydroxy(phenyl)methyl)-7-methylenebicyclo[3.3.1]nonan-3-one (750 mg,

2.9 mmol), which was obtained in accordance with the method described in J. Am. Chem. Soc., 2014, 136, 17591-17600, diphenylphosphatazide (DPPA, 820 mL, 3.81 mmol) and triphenylphosphine (1.20 g, 4.4 mmol) in THF (15 mL) was added with ice-cooling diisopropyl azodicarboxylate (DIAD, 2.2 ml, 4.4 mmol).

After stirring for 1 hour at the same temperature, the solvent was distilled off under reduced pressure. Dichloromethane (15 ml) was added to the residue and TiCl ₄ (820 ml, 2.3 mmol) was added under ice-cooling. After the reaction solution was stirred at room temperature for 4 hours, a saturated NaHCO ₃ aqueous solution was added under ice-cooling. The reaction solution was filtered through Celite (registered trademark). The filtrate was extracted with diethyl ether. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane:ethyl acetate=10:1) to obtain (1 R,2S,3R,5R,7R)-2-((S)-azido(phenyl)methyl)- 5-chloroadamantan-1-ol (756 mg, 92%) as a white solid.

To a solution of the obtained azide compound (750 mg, 2.67 mmol) in THF (14 ml) was added LiAlH ₄ (300 mg, 8.00 mmol) under ice-cooling. After stirring for 1 hour at the same temperature, an aqueous ammonia solution was added to the reaction solution. The reaction solution was filtered through Celite (registered trademark), the solvent was distilled off under reduced pressure. Dichloromethane (15 ml) was added to the residue, and then triethylamine (2.2 ml, 16.0 mmol) and anhydrous trifluoroacetic acid (TFAA, 1.2 ml, 8.0 mmol) were added under ice-cooling. After the reaction solution was stirred at room temperature overnight, a saturated aqueous NaHCO ₃ solution was added. The reaction solution was extracted with dichloromethane. The resulting organic layer was dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to a

- 12 043204 overnight silica gel chromatography (hexane:ethyl acetate=15:1) to give TP-013 (871 mg, 56%) as a white solid.

T. pl. 83-85°C (colorless needles, n-hexane-Et ₂ O);

[α% ³¹ = -84.1 (c=1.08, CHCI3);

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.35-7.27 (m, 5H), 6.63 (d, J=11.1 Hz, 1H), 5.44 (t, J= 10.4 Hz, 1H), 3.26 (d, J=11.1 Hz, 1H), 2.99 (d, J=11.1 Hz, 1H), 2.45-2.41 (m, 3H), 2.26-2.13 (m, 5H), 1.96 (brd, J=12.4 Hz, 2H), 1.47 (brd, J=14.0 Hz, 1H );

¹³ C-NMR (100 MHz, CDCl ₃ ): δ 156.2 (q. J=37.4 Hz), 154.9 (q. J=42.3 Hz), 139.1, 129.2 , 128.7, 127.1, 115.8 (Q, J=288.1 Hz), 113.3 (Q, J=287.3 Hz), 86.6, 65.1, 53.4 , 50.2, 48.0, 46.9, 46.1, 35.6, 34.6, 31.7, 28.5;

IR (solvent free, cm ⁻¹ ): 3296, 2945, 1775, 1698;

MS (EI): m/z 483 (M ⁺ ), 202 (100%);

HRMS (EI): calculated for C ₂₁ H ₂₀ ClF ₆ NO ₃ (M ⁺ ) 483.1036, found 483.1046.

To a solution containing TP-013 (550 mg, 1.14 mmol) in acetonitrile (1.8 ml)-carbon tetrachloride (1.8 ml)-water (1.8 ml) was added under ice-cooling RuCl ₃ x3H ₂ O (114 mmol) and H1O ₄ x2H ₂ O (3.6 g, 16.0 mmol). The reaction solution was vigorously stirred at the same temperature for 8 hours. Water was added to the reaction solution. The reaction solution was extracted with dichloromethane. A solution of diazomethane in diethyl ether was added to the resulting organic layer under ice-cooling until the solution turned yellow. After 30 minutes, the solution was purged with nitrogen to remove diazomethane, and then the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=15:1) to give TP-012 (235 mg, 44%) as a white solid.

[a]D ²⁴ = -22.7 (c=1.84, CHCI3);

¹ H-NMR (400 MHz, CDC13): δ 7.10 (d, J=9.8 Hz, 1H), 5.02 (d, J=10.1 Hz, 1H), 3.76 (s, 3H), 2.92 (dd, J=10.3, 2.0 Hz, 1H), 2.74 (d, J=11.5 Hz, 1H), 2.69 (dd, J=11.5 , 1.7 Hz, 1H), 2.43 (br.s, 1H), 2.30-2.10 (m, 7H), 1.86 (d, J=14.1 Hz, 1H), 1 .46 (d, J=14.1 Hz, 1H);

¹³ C-NMR (100 MHz, CDC13): δ 171.1, 157.1 (q. J=37.9 Hz), 155.2 (q. J=41.2 Hz), 115.5 ( sq. J=285.4 Hz), 113.8 (q. J=284.6 Hz), 87.0, 64.8, 53.0, 51.3, 49.8, 47.6, 46.5, 45.9, 34.2, 33.5, 31.7, 28.8;

IR (solvent free, cm- ¹ ): 3319, 1780, 1714;

MS (EI): m/z 406 (M-CO2CH3);

HRMS (EI): calculated for C ₁₅ H ₁₅ NO ₃ F ₆ Cl (M+) 406.0645, found 406.0651.

To a solution of TP-012 (256 mg, 550 mmol) in THF (2.0 ml) was added an aqueous solution of NaOH (2 M, 2.0 ml) under ice-cooling. The reaction solution was stirred for 2 hours. THF was distilled off, then the reaction solution was neutralized with 10% HCl aqueous solution, subjected to ion exchange chromatography (DOWEX50), eluted with 0.23 N ammonium chloride aqueous solution, and lyophilized to obtain TP-015 (48.5 mg , 34%) as a white solid.

[a] _D ²⁸ = -46.3 (c=0.78, MeOH);

¹ H-NMR (600 MHz, CD3OD): δ 3.87 (d, J=9.6 Hz, 1H), 2.26 (br s, 1H), 2.19 (br s, 1H), 2.12 (d, J=10.9 Hz, 1H), 2.08 (d, J=12.4 Hz, 1H), 1.80-1.62 (m, 5H), 1.66 (br .s, 2H), 1.50 (brd, J=12.4 Hz, 1H), 1.37 (brd, J=13.1 Hz, 1H);

¹³ C-NMR (150 MHz, CD3OD): δ 174.6, 70.3, 68, 6, 54.7, 53.8, 49.4, 44.3, 43.4, 38.7, 32, 9, 30.9, 29.2;

IR (solvent free, cm- ¹ ): 3336, 1730;

MS (FAB): m/z 242 (M+1);

HRMS (FAB): calculated for C ₁₂ H ₂₀ NO ₄ (M+1) 242.1387, found 252.1383.

Example 3

ⁿ 9 n o ^Ac 2° ^Ac 9 n o ^tmsn 3 ^Ac 9 n 9 ⁿ

Et ₃ N, DMAP BF ₃ OEt ₂ O sn ₂ C1 ₂ CH ₂ ci ₂

TP-009

Chemical formula 7

To a solution of (1R,2S,5S)-2-((R)-hydroxy(phenyl)methyl)-7-methylenebicyclo[3.3.1]nonan-3-one (256 mg, 1.00 mmol) in dichloromethane (5 ml) trimethylamine (0.42 ml, 3 mmol), dimethylaminopyridine (DMAP, 12 mg, 0.1 mmol) and anhydrous acetic acid (0.14 ml, 1.5 mmol) were added under ice cooling. After the reaction solution was stirred at room temperature for 20 minutes, a saturated NaHCO ₃ aqueous solution was added under ice-cooling, and extracted with dichloromethane. The resulting organic layer was dried over MgSO ₄ and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=4:1) to give (1R,2S,5S)-2-((R)-acetoxy(phenyl)methyl)-7-methylenebicyclo[3.3.1]nonane-3 -one (289 mg, 97%) as a white solid.

To a solution of the resulting product (75.2 mg, 0.252 mmol) in dichloromethane (2.5 ml) was added trimethylsilyl azide (TMSN3, 0.10 ml, 0.76 mmol) and BF ₃ xOEt ₃ (0.04 ml, 0.30 mmol) at -20°C. Temperature

- 13 043204 the reaction solution was gradually raised to room temperature, and the reaction solution was stirred for 3 hours. Thereafter, a saturated NaHCO ₃ aqueous solution was added to the reaction solution under ice-cooling. The resulting reaction solution was extracted with dichloromethane. The resulting organic layer was dried over MgSO ₄ and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=2:1) to give TP-009 (33.5 mg, 39%) as a white solid.

T. pl. 114°C (colorless crystals, H-hexane-Et ₂ O);

[a]D ¹⁹ =+56.0 (c=0.67, CHCl ₃ );

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.37-7.31 (m, 5H), 6.01 (d, J=10.6 Hz, 1H), 2.95 (br s, 1H), 2.27 (br.s, 1H), 2.25 (d, J=10.6 Hz, 1H), 2.15 (d, J=13.0 Hz, 1H), 2.01 ( s, 3H), 1.89-1.83 (m, 2H), 1.76-1.61 (m, 7H), 1.18 (d, J=13.0 Hz, 1H);

¹³ C-NMR (100 MHz, CDCl ₃ ): δ 169.6, 138.9, 128.7, 128.5, 127.3, 77.0, 71.3, 60.0, 51.9, 50 .7, 41.6, 40.4, 38.8, 32.5, 30.6, 29.3, 21.6;

IR (solvent free, cm ⁻¹ ): 3460, 2931, 2091, 1732;

MS (EI): m/z 323 (M+-H2O), 107 (100%);

HRMS (EI): calculated for C ₁₉ H ₂₁ N ₃ O ₂ (M+-H2O) 323.1634, found: 323.1613.

Example 4

TP-014

Chemical formula 8

A solution of n-butyllithium in hexane ( 1.56 M, 28.2 ml, 44 mmol) under ice-cooling. The reaction solution was stirred at the same temperature for 30 minutes and then cooled to -78°C. A solution of 7-methylenebicyclo[3.3.1]nonan-3-one (6.00 g, 40 mmol) in THF (60 mL) was added to the reaction mixture via cannula. After stirring the reaction solution for 1 h, a solution of benzaldehyde (6.1 ml, 60 mmol) in THF (40 ml) was added via cannula. After the reaction solution was stirred for 2 hours, acetic acid and a saturated ammonium chloride aqueous solution were successively added to the reaction solution. The reaction solution was extracted with diethyl ether. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane:ethyl acetate=4:1) to obtain (1S,2R,5R)-2-((S)-hydroxy(phenyl)methyl)7-methylenebicyclo[3.3. 1]nonan-3-one (8.3 g, 81%) as a white solid. This material was recrystallized from diethyl ether to give colorless needles.

T. pl. 122°C;

[a] _D ²¹ = -17.9 (c=0.32, CHCl ₃ );

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.38-7.25 (m, 5H), 4.79 (d, J=1.8 Hz, 1H), 4.76 (d, J= 1.8 Hz, 1H), 4.71 (d, J=6.8 Hz, 1H), 2.90 (s, 1H), 2.64 (dd, J=15.7, 6.8 Hz, 1H), 2.48-2.18 (m, 6H), 2.01 (brd, J=14.3 Hz, 1H);

¹³ C-NMR (100 MHz, CDCl ₃ ): δ 211.0, 141.6, 128.8, 127.6, 114.8, 74.6, 62.7, 45.7, 42.2, 41 .3, 32.4, 31.9, 28.4;

IR (solvent free, cm ⁻¹ ): 3390, 1711;

MS (EI): m/z 256 (M ⁺ ), 95 (100%);

HRMS (EI): calculated for C ₁₇ H ₂₀ O ₂ (M+) 256.1463, found 256.1450.

To a solution of (1S,2R,5R)-2-((S)-hydroxy(phenyl)methyl)-7-methylenebicyclo[3.3.1]nonan-3-one (2.00 g, 7.5 mmol), DPPA (2.3 ml, 11 mmol) and triphenylphosphine (3.0 g, 11 mmol) in THF (38 ml) were added DIAD (2.2 ml, 11 mmol) under ice cooling. After stirring for 1 hour at the same temperature, the solvent was distilled off under reduced pressure. Dichloromethane (38 ml) was added to the residue and TiCl ₄ (0.8 ml, 7.5 mmol) was added under ice-cooling. After the reaction solution was stirred at room temperature for 4 hours, a saturated NaHCO ₃ aqueous solution was added under ice-cooling. The reaction solution was filtered through Celite (registered trademark) and the filtrate was extracted with diethyl ether. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and tetrahydropyran (THP, 40 ml) was added to the residue. LiAlH ₄ (430 mg, 11 mmol) was added to the mixture while cooling

- 14 043204 ice. The reaction mixture was stirred at the same temperature for 30 minutes, and an aqueous ammonia solution was added to the reaction solution. The reaction solution was filtered through Celite (registered trademark), and the solvent was distilled off under reduced pressure. Dichloromethane (40 ml) was added to the residue, and then triethylamine (6.3 ml, 45 mmol) and TFAA (3.2 ml, 23 mmol) were added under ice-cooling. The resulting reaction solution was stirred at room temperature overnight, and a saturated NaHCO ₃ aqueous solution was added, followed by extraction with dichloromethane. The resulting organic layer was dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane:ethyl acetate=15:1) to give the crude product as a white solid. This material was recrystallized from diethyl ether-hexane to give TP-014 (1.27 g, 35%) as a white solid.

T. pl. 89°C;

[α] d ²¹ =+89.1 (c=0.31, CHCl ₃ );

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.35-7.27 (m, 5H), 6.63 (d, J=11.1 Hz, 1H), 5.44 (t, J= 10.4 Hz, 1H), 3.26 (d, J=11.1 Hz, 1H), 2.99 (d, J=11.1 Hz, 1H), 2.45-2.41 (m, 3H), 2.26-2.13 (m, 5H), 1.96 (brd, J=12.4 Hz, 2H), 1.47 (brd, J=14.0 Hz, 1H );

¹³ C-NMR (100 MHz, CDCl ₃ ): δ 156.2 (q. J=37.4 Hz), 154.9 (q. J=42.3 Hz), 139.1, 129.2 , 128.7, 127.1, 115.8 (Q, J=288.1 Hz), 113.3 (Q, J=287.3 Hz), 86.6, 65.1, 53.4 , 50.2, 48.0, 46.9, 46.1, 35.6, 34.6, 31.7, 28.5;

IR (solvent free, cm- ¹ ): 3296, 2945, 1775, 1698;

MS (EI): m/z 483 (M ⁺ ), 202 (100%);

HRMS (EI): calculated for C ₂₁ H ₂₀ ClF ₆ NO ₃ (M ⁺ ) 483.1036, found 483.1046;

Anal.: calculated for C ₂₁ H ₂₀ ClF ₆ NO ₃ : C, 52.13; H, 4.17;

N, 2.89; found C, 52.27; H, 4.18; N, 2.88.

Example 5

TR-014 TR-048

Chemical formula 9

To a solution of TP-014 (84.7 mg, 0.175 mmol) in THF (2 ml) was added 0.5 M NaOH aqueous solution (1 ml) under ice-cooling. The reaction solution was stirred at the same temperature for 15 minutes, and a saturated aqueous NH4Cl solution was added and extracted with ethyl acetate. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane:ethyl acetate=8:1 to 4:1) to give TP-048 (65.5 mg, 96%) as a white solid.

[a]D ²⁶ =+109.2 (c=0.772, CHCI3);

¹ H-NMR (400 MHz, CDCI3): δ 7.41-7.32 (m, 5H), 6.98 (br, 1H), 5.34 (t, J=9.7 Hz, 1H) , 2.36-2.29 (m, 3H), 2.19-2.00 (m, 7H), 1.77 (brd, J=11.6 Hz, 1H), 1.41-1 .33 (m, 2H);

¹³ C-NMR (100 MHz, CDCI3): δ 156.2 (q., J=37.1), 140.5, 129.4, 128.6, 127.4, 115.8 (q., J =288.1Hz), 72.3, 66.1, 56.7, 54.2, 52.4, 47.7, 46.3, 38.6, 34.4, 31.8, 28.8 ;

IR (solvent free, cm- ¹ ): 3553, 3297, 2940, 1698, 1552, 1208, 1183, 1165;

MS (EI): m/z 387 (M ⁺ ), 202 (100%);

HRMS (EI): calculated for C ₁₉ H ₂₁ C1F ₃ NO ₂ (M+) 387.1213, found 387.1196.

Example 6

o 9 o o o

X A y A A

F ₃ C^OH ^H N ^CF 3 f3Ao hhn CF3 ho h ^h n cf3 rvA ^Ph AIBN, < ^Me 3 ^s ')3 ^SiH А-far ° ^{5 M Na0H aq} gAt Ph toluene, vJA ^THF AjA Reflux ^TP ' ⁰¹⁴ TP-049 TP-052

Chemical formula 10

To a solution of TP-014 (30.0 mg, 0.062 mmol) in toluene (2 mL) was added tris(trimethylsilyl)silane (29 mL, 0.095 mmol) and azobisisobutyronitrile (AIBN, 2.0 mg, 0.012 mmol) at room temperature. The reaction solution was heated under reflux overnight, then the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=15:1) to give TP-049 (23.0 mg, 83%) as a white solid.

[a]D ²⁹ =+106.4 (c=0.385, CHCI3);

¹ H-NMR (400 MHz, CDCI3): δ 7.33-7.27 (m, 5H), 6.31 (brd, J=10.1 Hz, 1H), 5.50 (dd, J =10.9, 10.1 Hz, 1H), 3.20 (brd, J=10.9 Hz, 1H), 2.60 (brd, J=11.6 Hz, 1H), 2 .45 (broad.d, J=12.1 Hz, 1H), 2.28-2.27 (m,

- 15 043204

3H), 2.04-1.80 (m, 6H), 1.72 (br s, 2H);

¹³ C-NMR (100 MHz, CDCI3): δ 156.0 (q. J=37.1 Hz), 155.1 (q. J=41.8 Hz), 139.8, 129.0, 128.4, 127.2, 115.8 (Q, J=288.1 Hz), 113.5 (Q, J=287.3 Hz), 87.5, 53.6, 49.4, 41.3, 37.2, 36.1, 33.0, 30.6, 30.4, 30.2;

IR (solvent free, cm ⁻¹ ): 3335, 2927, 1775, 1700, 1556, 1218, 1169;

MS (EI): m/z 449 (M ⁺ ), 202 (100%);

HRMS (EI): calculated for C ₂₁ H21F ₃ NO ₃ (M ⁺ ) 449.1426, found 449.1447.

To a solution of TP-049 (61.5 mg, 0.137 mmol) in THF (1.4 ml) was added an aqueous solution of NaOH (0.5 M, 0.5 ml) under ice-cooling. After stirring the reaction solution at the same temperature for 5 minutes, 2 M hydrochloric acid was added to the reaction solution. The reaction solution was extracted with ethyl acetate. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane:ethyl acetate=4:1 to 2:1) to give TP-052 (49.4 mg, quantitative) as a white solid.

TP-052: [α] d ¹⁴ =+130.7 (c=0.243, CHCl3);

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.39-7.31 (m, 5H), 6.77 (brd, J=8.9 Hz, 1H), 5.40 (dd, J=9.7, 8.9 Hz, 1H), 2.32 (brd, J=9.7 Hz, 1H), 2.31-2.07 (m, 4H), 1.85-1 .79 (m, 2H), 1.72-1.57 (m, 5H), 1.52-1.44 (m, 2H), 1.29 (br., 1H);

¹³ C-NMR (100 MHz, CDCl3): δ 156.1 (q. 37.1 Hz), 140.7, 129.4, 128.5, 127.5, 115.9 (q. 288 Hz) ), 77.2, 54.3, 53.0, 50.5, 48.5, 41.4, 39.6, 39.4, 33.2, 30.6, 29.6;

IR (solvent free, cm ⁻¹ ): 3566, 3291.2919, 1698, 1183;

MS (EI): m/z 353 (M ⁺ ), 151 (100%);

HRMS (EI): calculated for C ₁₉ H ₂ 2F ₃ NO2 (M ⁺ ) 353.1603, found 353.1604.

Example 7

Chemical formula 11

To a solution of (1S,2R,5R)-2-(R-azido(phenyl)methyl)-7-methylenebicyclo[3.3.1.]nonan-3-one (57.4 mg, 0.204 mmol) in dichloromethane (2 ml ) were added successively 2-methoxyethanol (78 ml, 1.0 mmol) and scandium trifluoromethanesulfonate (5.0 mg, 0.01 mmol) under ice-cooling. After the reaction solution was stirred at room temperature for two days, a saturated NaHCO ₃ aqueous solution was added under ice-cooling. The reaction solution was extracted with dichloromethane. The resulting organic layer was dried over MgSO ₄ and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=1:2 to 1:1) to obtain (1S,2R,3S,5S,7S)-2-((R)-azido(phenyl)methyl)-5-( 2-methoxyethoxy)adamantan-1-ol (41.2 mg, 56%) as a colorless oil.

To a solution of the resulting azide compound (39.6 mg, 0.111 mmol) in THF (1 ml) was added LiAlH ₄ (8.0 mg, 0.21 mmol) under ice-cooling. The temperature of the reaction solution was gradually raised to room temperature, and the reaction solution was stirred for 1 hour. The reaction solution was ice-cooled, and then LiAlH4 (8.0 mg, 0.21 mmol) was added. After the reaction solution was stirred at room temperature for 1 hour, an aqueous ammonia solution was added to the reaction solution under ice-cooling. The reaction solution was filtered through Celite (registered trademark). The filtrate was dried over Na ₂ SO ₄ and the solvent was distilled off under reduced pressure. Dichloromethane (1 ml) was added to the residue, then triethylamine (77 ml, 0.56 mmol) and anhydrous trifluoroacetic acid (TFAA, 47 ml, 0.33 mmol) were added under ice-cooling. After the reaction solution was stirred at room temperature for 5 hours, a saturated NaHCO3 aqueous solution was added under ice-cooling. The reaction solution was extracted with dichloromethane. The resulting organic layer was dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane:ethyl acetate=1:4 to 1:2) to obtain TP-050 (31.6 mg, 54%) as a colorless oil.

[α]π ²⁵ =+72.1 (c=0.965, CHC1h);

¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.34-7.23 (m, 5H), 6.33 (brd, J=9.9 Hz, 1H), 5.44 (dd, J=10.9, 9.9 Hz, 1H), 3.59-3.56 (m, 2H), 3.51-3.48 (m, 2H), 3.37 (s, 3H), 3 .17 (brd, J=10.9 Hz, 1H), 2.65 (brd, J=10.6 Hz, 1H), 2.43-2.37 (m, 3H), 1, 95-1.81 (m, 7H), 1.38 (brd, J=11.6 Hz, 1H);

¹³ C-NMR (100 MHz, CDCl3): δ 156.1 (q. J=37.4 Hz), 154.9 (q. J=42.1 Hz), 139.4, 129.1, 128.5, 127.2, 115.8 (Q, J=288.1 Hz), 113.4 (Q, J=287.3 Hz), 87.6, 73.7, 72.3, 60.2, 59.1, 53.5, 48.5, 45.0, 41.1, 39.9, 36.3, 30.5, 29.2;

IR (solvent free, cm ⁻¹ ): 3303, 2936, 1775, 1698, 1554, 1221, 1172;

- 16 043204

MS (EI): m/z 523 (M+), 202 (100%);

HRMS (EI): calculated for C ₂₄ H ₂₇ F ₆ NO ₅ (M+) 523.1793, found 523.1797.

Example 8

TP-053

Chemical formula 12

To a solution of (1S,2R,5R)-2-(R-azido(phenyl)methyl)-7-methylenebicyclo[3.3.1.]nonan-3-one (238 mg, 0.848 mmol) in methanol (8.5 ml ) scandium trifluoromethanesulfonate (20 mg, 0.04 mmol) was added under ice-cooling. After the reaction solution was stirred at room temperature for 18 hours, a saturated NaHCO ₃ aqueous solution was added to the reaction solution under ice-cooling. The reaction solution was extracted with ethyl acetate. The resulting organic layer was washed with saturated saline and dried over MgSO ₄ and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=1:4 to 1:2) to obtain (1S,2R,3S,5S,7S)-2-((R)-azido(phenyl)methyl)-5-methoxyadamantane -1-ol (225 mg, 85%) as a colorless oil.

To a solution of the obtained azide compound (225 mg, 0.716 mmol) in THF (4 ml) was added LiAlH ₄ (41 mg, 1.1 mmol) under ice-cooling. After stirring for 1 hour at the same temperature, an aqueous ammonia solution was added to the reaction solution. The reaction solution was filtered through Celite (registered trademark), and the solvent was distilled off under reduced pressure. Dichloromethane (4 ml) was added to the residue and triethylamine (497 ml, 3.86 mmol) and anhydrous trifluoroacetic acid (TFAA, 299 ml, 2.15 mmol) were added under ice-cooling. After the reaction solution was stirred at room temperature for 40 h, a saturated NaHCO ₃ aqueous solution was added under ice-cooling. The reaction solution was extracted with dichloromethane. The resulting organic layer was dried over MgSO ₄ and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=1:8 to 1:2) to give TP-053 (262 mg, 75%) as a white solid.

[a]D ¹⁴ =+97.2 (c=0.179, CHCl ₃ );

¹ H-NMR (400 MHz, CDCl3): δ 7.33 (m, 5H), 6.35 (brd, J=9.9 Hz, 1H), 5.45 (dd, J=10.6 , 9.9 Hz, 1H), 3.25 (s, 3H), 3.17 (brd, J=10.6 Hz, 1H), 2.61 (br d J=10.6 Hz, 1H ), 2.45-2.37 (m, 3H), 1.97-1.73 (m, 7H), 1.39 (brd, J=13.5 Hz, 1H);

¹³ C-NMR (100 MHz, CDCl3): δ 156.0 (q. J=37.4 Hz), 155.0 (q. J=41.8 Hz), 139.4, 129.1, 128.6, 127.1, 115.8 (Q, 288.1 Hz), 113.4 (Q, 287.0 Hz), 87.7, 75.5, 53.5, 48.7, 48.6, 44.5, 40.8, 39.5, 36.3, 33.3, 30.4, 29.3;

IR (solvent free, cm ⁻¹ ): 3299, 2941, 1776, 1697, 1221, 1172;

MS (EI): m/z 479 (M ⁺ ), 202 (100%);

HRMS (EI): calculated for C ₂₂ H ₂₃ F ₆ NO ₄ (M ⁺ ) 479.1531, found 479.1486.

Example 9

O

TR-051

Chemical formula 13

To a solution of bis((S)-1-phenylethyl)amine (2.5 ml, 11 mmol) and lithium chloride (850 mg, 20 mmol) in THF (25 ml) was added dropwise a solution of n-butyl lithium in hexane (1 56 M, 7.1 ml, 11 mmol) under ice-cooling. After stirring the reaction solution at the same temperature for 30 minutes, the reaction solution was cooled to -78°C. A solution of 7-methylenebicyclo[3.3.1]nonan3-one (1.52 g, 10 mmol) in THF (15 mL) was added to the reaction mixture via cannula. After stirring the reaction solution for 30 min, a solution of nicotinaldehyde (1.1 ml, 12 mmol) in THF (10 ml) was added via cannula. After the reaction solution was stirred for 40 minutes, acetic acid and a saturated ammonium chloride aqueous solution were successively added to the reaction solution. The reaction solution was extracted with ethyl acetate. The resulting organic layer was washed with brine and dried over K2CO3. The solvent was distilled off under reduced pressure. The remainder was subjected

- 17 043204 silica gel column chromatography (hexane-acetone=3:2 to 1:2) to obtain (1S,2R,5R)-2-((S)hydroxy(pyridin-3-yl)methyl)-7-methylenebicyclo [3.3.1]nonan-3-one (2.7 g, 81%) as a white solid. This material was recrystallized from ethyl acetate to give colorless crystals (99% ee).

Diisopropyl azodicarboxylate (DIAD, 214 ml , 1.1 mmol) under ice-cooling. The temperature of the reaction solution was gradually raised to room temperature. After the reaction solution was stirred for 5 hours, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=4:1 to 2:1) to obtain (1S,2R,5R)-2-((R)-azido(pyridin-3-yl)methyl)-7methylenebicyclo[3.3 .1] nonan-3-one (187 mg, 66%) as a colorless oil.

To a solution of the resulting azide compound (187 mg, 0.66 mmol) in dichloromethane (7 ml) was added TiCl ₄ (300 ml, 0.27 mmol) under ice-cooling. After the reaction solution was stirred at room temperature for 3 hours, a saturated NaHCO ₃ aqueous solution was added under ice-cooling. The reaction solution was extracted with diethyl ether. The resulting organic layer was washed with brine and dried over MgSO ₄ . The solvent was distilled off under reduced pressure, and the resulting solid was washed with cold diethyl ether to give (1S,2R,3S,5S,7S)-2-((R)-azido(pyridin-3-yl)methyl)-5-chloroadamantan-1 -ola (98.5 mg, 92%).

To a solution of the resulting compound (75.4 mg, 0.257 mmol) in THF (2 ml) was added LiAlH ₄ (23 mg, 0.61 mmol) under ice-cooling. After stirring the reaction solution for 1 hour at the same temperature, an aqueous ammonia solution was added to the reaction solution under ice-cooling. The reaction solution was filtered through Celite (registered trademark), and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (CHCl ₃ :methanol=1:0 to 4:1) to give a crude amine.

Dichloromethane (2 ml) was added to the resulting crude amine, and then triethylamine (178 ml, 1.28 mmol) and anhydrous trifluoroacetic acid (TFAA, 107 ml, 0.76 mmol) were added under ice-cooling. The temperature of the reaction solution was raised to room temperature. After stirring the reaction solution for 4 hours, a saturated NaHCO ₃ aqueous solution was added under ice-cooling. The reaction solution was extracted with dichloromethane. The resulting organic layer was dried over Na ₂ SO ₄ . The solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (hexane:ethyl acetate=2:1 to 1:4) to give TP-051 (48.8 mg, 49%) as a white solid.

[a]D ²⁰ =+53.9 (c=0.379, CHCI3);

1H-NMR (400 MHz, CDCl ₃ ): δ 8.57 (d, J=1.0 Hz, 1H), 8.50 (dd, J=4.9, 1.5 Hz, 1H), 7, 72 (brd, J=7.8 Hz, 1H), 7.41 (brd, J=9.8 Hz, 1H), 7.32 (dd, J=7.8, 4.9 Hz , 1H), 5.35 (dd, J=9.8, 9.3 Hz, 1H), 2.40-2.38 (m, 2H), 2.29 (br.s, 1H), 2, 22-1.99 (m, 7H), 1.75 (br., 1H), 1.68 (b.l, J=13.7 Hz, 1H), 1.48 (b.l, J=13 .2 Hz, 1H), 1.42 (br.d, J=13.2 Hz, 1H);

¹³ C-NMR (100 MHz, CDCl ₃ ): δ 156.4 (q. J=37.1 Hz), 148.2, 147.7, 138.3, 136.5, 124.0, 115, 8 (Q, J=287.8 Hz), 71.9, 66.1, 57.3, 52.6, 51.7, 47.6, 46.3, 38.3, 34.3, 31 .6, 28.6;

IR (solvent free, cm ⁻¹ ): 3292, 2938, 1700, 1558, 1212, 1184, 1161, 759;

MS (EI): mz 388 (M ⁺ ), 203 (100%);

HRMS (EI) : calculated for C ₁₈ H ₂₀ ClF ₃ N ₂ O2 (M+) 388.1165, found 388.1177.

Test example 1.

A plasmid vector containing the cDNA of the Kir6.2 channel inserted into it: pcDNA3.1-Kir6.2 was obtained from Dr. Toru Ishizuka (Graduate School of Life Sciences, Tohoku University). The plasmid vector, pcDNA3,1-Kir6.2, was conditioned according to the instructions supplied with the GenElute HP Plasmid Maxiprep Kit (manufactured by Sigma-Aldrich). Culture solution (composition: 450 ml DMEM culture solution containing 50 ml bovine serum and 100 units penicillin/streptomycin) of Neuro2A cells (N2A cells, National Instituted of Biomedical Innovation) cultured in DMEM culture solution (Gibco) was exchanged with Opti-Mem (Gibco) (contains Lipofectamine R2000 (1 mg/1 ml)) containing pcDNA3.1-Kir6.2 (1 mg/ml) conditioned as above and cultured for 5 h to obtain N2A cells overexpressing Kir6 .2 channel. The culture solution was again exchanged with DMEM and cultured for two days. Then memantine (manufactured by Sigma-Aldrich) and a compound of the present invention (n=4 per group) were added to the culture solutions (DMEM, Gibco) to give a concentration of 10 nM per culture solution, and the culture solutions were allowed to stand for 1 hour. Then, the Kir6.2 channel overexpressing N2A cells were harvested, and SDS sample buffer was added to the N2A cells to prepare a suspension. The suspension was immunoblotted using an anti-phosphorylated CaMKII antibody (Fukunaga K. et al., J. Biol. Chem., 1992, 267, 22527-22533) as the primary antibody and an anti-rabbit IgG antibody (manufactured by Southern Biotech) as the secondary antibody ( other conditions, except that the above antibodies were the same

- 18 043204 which are used in conventional immunoblotting), to study the activation of CaMKII. As a result, TR-009, TR-010, TR-011, TR-012, TR-014, TR-015, TR-048, TR-049, TR-050, TR-051, TR-052, TR- 053, bands were obtained showing a reaction with an antibody against phosphorylated CaMKII. It has been confirmed that CaMKII activation is enhanced. The results are shown in FIG. 1. In FIG. 1 result for the case (control: c) where no test compound was added is considered 100%. Activation of CaMKII in cases containing memantine, TP-009, TP-010, TP-011, TP-012, TP-014, TP-015, TP-048, TP-049, TP-050, TP-051, TP- 052, TP-053 (corresponding to 9, 10, 11, 12,12, 15, 48, 49, 50, 51, 52 and 53 M respectively) is shown in FIG. 1.

Test example 2.

Using the Kir6.2 channel-overexpressing cells obtained in Test Example 1, the potassium current flowing out of the cells was measured by the conventional patch clamp method. The results are shown in FIG. 2. An ATP-sensitive potassium channel (Kir6.2 channel) was localized in the cell membrane of nerve cells. If the channel is inhibited and closed, the threshold of the nerve cell membrane rises, leading to a state that is similar to that where an action potential is temporarily generated, resulting in potassium current flowing out of the cell and instead calcium current flowing into the cell from outside. Fig. 2a shows that the Kir6.2 channel is overexpressed in N2A cells (upper FIG. shows immunoblot stained images; while lower FIG. quantifies the signal intensity of the bands). This was confirmed using immunoblotting (using the same conditions as in Test Example 1, except for anti-Kir6.2 channel antibody, n=5) with anti-Kir6.2 channel antibody (prepared in the usual way) to Kir6.2 channel-overexpressing cells (obtained as described above). No change was observed in the constitutive gene product, ie. β tubulin (anti-β tubulin antibody was obtained from Sigma-Aldrich and other conditions are the same as in Kir6.2 detection). Fig. 2b (confirmed results) shows that if Kir6.2 channel-overexpressing cells are allowed to stand in an electrophysiological experimental buffer containing TP-014 to obtain a concentration of 10 nM, the potassium current that flows out when the membrane potential of nerve cells changes towards plus, suppressed (n=5 per group). The results show that TP-014 inhibits the Kir6.2 channel and inhibits potassium current flowing out of cells.

Test example 3.

Using the same Kir6.2 channel-overexpressing cells as in Test Example 1, the amount of calcium that flows into the cells from the outside when treated with TP-014 was measured by calcium imaging. The results are shown in FIG. 3. The calcium imaging method is the measurement of the amount of calcium based on the fluorescence intensity of cultured nerve cells that were treated in a culture solution containing a calcium fluorescent dye (Fura2, manufactured by Dojindo Laboratories) at a concentration of 4 µM. Imaging was performed with an imaging device (LAMBDA10-2, manufacturer of SUTTER INSTRUMENT) according to the instructions supplied with the device. Fig. 3a shows the change in TP-014 concentration-dependent amount of calcium over time (over 4 min) when treated with TP-014 (1-100 nM) and memantine (100 nM). Fig. 3b shows the results of calcium measurements when the measurement was taken 4 minutes after treatment with memantine (100 nM) and TP-014 (1-100 nM) (n=5 per group). TP-014 has a greater calcium-increasing effect than memantine. It was confirmed that the amount of calcium in the cells was significantly increased by treatment with TP-014 as a result of the inhibition of potassium leakage from the cells, confirmed in Test Example 2.

Test example 4.

Model mice with Alzheimer's disease (APP23 mice, Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 1997, 94, 13287-13292) (age 12 months) were orally administered TP-014 (1 mg/kg) times per day for two months (long-term treatment). As a result, a significant effect of improving cognitive function was confirmed. The results are shown in FIG. 4. FIG. 4a-4d show the results of the behavior analysis. fig. 4a and 4b show attention function analyzed in WT mice (C57BL/6J, Japan SLC) and APP23 mice (n=5 per group) by the conventional Y-maze method. As a result, it was confirmed that the effect of significantly improving the attention function is exhibited by processing TP-014. The Y-maze method is a method that allows the mouse to move freely in the three arms of the maze for 8 minutes. The sleeves will be conditionally designated as A, B, C, respectively. The mouse located in A will move to B or C (sleeve). The case where the mouse moves to B and then moves to C, in short the case of sequential movement A-B-C, is considered the correct answer; whereas the case where the mouse moves A-B-A and avoids choosing a new sleeve is considered an incorrect answer. The arms into which the mouse entered were recorded in chronological order and the number of times the mouse entered each of the arms during a certain time was counted and determined as the total number of entries into the arms. Of these, the number of cases of a correct response (the case where the mouse consecutively chooses three different arms) was counted and defined as the number of alternating behaviors (number of alternations). The ratio of the number of alternations to the number, gender

- 19 043204 calculated by subtracting 2 from the total number of visits to the arms, expressed as alternation (%) and used as an index of normal alternation behavior (percentage of correct responses of spatial working memory).

Mice prefer a new (unfamiliar) object. Normal mice give the correct answer 70% of the time; however, APP2 3 mice give the correct answer only in about 50% of cases. Based on these percentages, the attention function (cognitive function) is analyzed.

Fig. 4c shows the results of novel (unfamiliar) object memory for WT mice and APP23 mice (both groups, n=5) assessed by the conventional novel object recognition method. The test for recognition of new objects was carried out as follows. Two structural elements identical in shape are placed in a cage and the mice are allowed to play with them for 10 minutes (this is called a learning test). One hour later, one of the structural elements is replaced by a structural element of a different shape. Since normal mice show interest in a new object, they play with a structural element with a different shape for longer. Mice with Alzheimer's disease (model) appear to have a memory impairment because they cannot recognize a new object (structural element). The mice are allowed to play freely for another 5 minutes with two structural elements of different shapes (this is called the memory retention test). In the learning test and the memory retention test, the number of times the mouse contacted each of the two objects was counted. The ratio (%) of the number of contacts with a differently shaped structural element to the total number of contacts in the memory retention test was calculated and used as a discrimination index.

Fig. 4d shows the results of memory of the feeling of fear, which was analyzed by the usual method of conditioned freezing (n=5 per group). The conditioned freezing method is an assay that takes advantage of the mouse's predisposition to a dark spot rather than a light spot. Day 1: The mouse is placed in a bright place. Since the mouse likes a dark place, the mouse goes into a dark place (dark box). When the mouse is electrically stimulated at this time, the mouse is surprised and returns to the bright place and never enters the dark place. Day 2: The mouse is placed in a bright place (the same place as on day 1), and then whether the mouse enters the dark place or not is observed for 5 minutes. If the mouse immediately enters a dark place, this is defined as a decrease in the mouse's memory of the feeling of fear. Latency means the period of time (seconds) until a mouse placed in a bright place enters a dark place (dark room) on day 2. Since the APP23 mice immediately entered the dark place, this confirmed that the fear memory was reduced; however, in mice treated with TP-014 for two months, the improvement effect was confirmed.

Fig. 4e-4g show the results of the analysis of the phenomenon of long-term potentiation (LTP) (serving as an index of memory formation) by the electrophysiological method. The hippocampus in the brain plays an important role in memory. The hippocampus was cut into sections (400 microns thick). Sections were placed in artificial cerebrospinal fluid (126 mM NaCl, 5 mM KCl, 26 mM NaHCO ₃ , 1.3 mM MgSO ₄ -7H ₂ O, 1.26 mM KH2PO4, 2.4 mM CaCl ₂ -2H ₂ O, 10 mM glucose) saturated with 95% O ₂ /5% CO ₂ gas at 34° C. for 2 hours and reduced. Hippocampal slices were transferred to the measurement chamber and perfused with artificial cerebrospinal fluid containing TP-014. The activity of nerve cells was recorded upon application of electrical stimulation and the excitatory postsynaptic potential (fEPSP) was measured. Based on this, the degree of improvement in LTP was evaluated. The recorded waveforms are shown in FIG. 4e. After that, electrical stimulation (100 Hz) was applied to the hippocampus to induce a selective change (it is believed that memory is formed by a selective change in the hippocampus). It was confirmed that the growth rate of neuronal excitation is reduced in APP23 mice; while the rate improves in mice treated with long-term treatment with TP-014. Memory learning has been shown to be effectively improved by improving LTP.

Test example 5.

The APP23 mouse hippocampus was excised. SDS sample buffer was added to hippocampal sections to prepare a suspension which was immunoblotted with antibodies against CaMKII, CaMKIV and ERK (CaMKII: Fukunaga et al., J. Biol., Chem., 1992, 267, 22527-22533, CaMKIV: Kasahara et al., J. Biol. Chem., 2001, 276, 24044-24050, ERK: manufactured by Sigma-Aldrich). In this way, the phosphorylation of individual proteins was analyzed. The results are shown in FIG. 5a and 5b. CaMKII, CaMKIV and ERK are molecules thought to play an important role in memory formation. As a result, CaMKII phosphorylation was found to be reduced in normal APP23 mice; while in APP23 mice receiving long-term treatment with TP-014 (treatment conditions are the same as in Test Example 4), CaMKII phosphorylation is accelerated. Based on this result, CaMKII activation was shown to be essential for effective memory enhancement in APP23 mice when treated with TP-014.

GluA1 (Ser-831), synapsin I (Ser-603) and CREB (Ser-133) are molecules known to be activated if CaMKII is activated. These molecules were analyzed by immunoblotting of hippocampal sections suspended in SDS sample buffer. Antibodies against individual mo

- 20,043,204 molecules were obtained from Millipore. The results are shown in FIG. 5c and 5d. It has been shown that activation of GluAl (Ser-831) and CREB (Ser-133) is induced by CaMKII activation. In FIG. 5a and 5c show bands (image) actually obtained by immunoblot electrophoresis. Fig. 5b and 5d show analysis results quantifying the signal intensities of the bands shown in FIG. 5a and 5s.

Test example 6.

The same experiment as shown in Fig. 4 was performed using mouse models of neurodegenerative diseases, ie olfactory bulb ablation mice (OBX mice). The results are shown in FIG. 6a-6g. Cognitive dysfunction in OBH mice improved significantly with long-term administration (2 weeks) of TP-014. 10 week old male DDY mice (Nippon SLC, Hamamatsu, Japan) were used to generate OBX mice. The surgery to remove the olfactory bulb was performed under anesthesia with sodium pentobarbital (50 mg/kg, ip; Dainippon, Osaka, Japan). The mouse was fixed on a fixation device for operations on the brain and the skull above the olfactory bulb was drilled to obtain a hole with a diameter of 1 mm. The olfactory bulb was removed with a suction pump so as not to damage the prefrontal cortex. A sham group was prepared by subjecting mice to the same operation as in the OBX group, except for removal of the olfactory bulb by suction.

Test example 7.

The intracellular mechanism of cognitive dysfunction in OBX mice was examined in the same manner as in FIG. 5. The results are shown in FIG. 7a-7d. The activation of CaMKII and CaMKIV has been found to be important in the hippocampus, which plays an important role in memory formation. In addition, activation of GluA1 (Ser-831) and CREB (Ser-133), which are downstream molecules of CaMKII and CaMKIV upon activation, was also confirmed to be important. Anti-GluA1 (Ser-831) and CREB (Ser-133) antibodies were obtained from Millipore. From the results shown in FIG. 47, it was found important to accelerate the activation of CaMKII and CaMKIV for the cognitive improvement effect of TP-014. Because cognitive dysfunction is not observed in CaMKIV gene-defective mice, CaMKII plays an important role in improving cognitive function.

Test example 8.

To confirm that the action of TP-014 is a Kir6.2 inhibitory action, the site of action of TP-014 was identified using the same behavioral experiment as in FIG. 4 (Figs. 8a and 8b: Y-maze method, Fig. 8c: novel object recognition method, Fig. 8d: conditioned fading method, Figs. 8e-8g: LTP improvement score, n=5 per group) using Kir6. 2 channel-deficient mice. The results are shown in FIG. 8. Cognitive dysfunction was confirmed to be induced in Kir6.2 channel-deficient mice. The results show that the Kir6.2 channel is essential for memory formation. It has also been shown that memory impairment and LTP attenuation in Kir6.2-deficient mice do not improve with long-term treatment with TP-014 (two months). The result shows that the site of action of TP-014 is the Kir6.2 channel. Analytical methods are the same as in Test Examples 4-7. Of note, Kir6.2-defective mice were obtained from Professor Susumu Seino, School of Medicine of Kobe University (Miki T. et al., Proc. Natl. Acad. Sci., USA, 1998, 95, 10402-10406).

Test example 9.

CaMKII activation was examined in the same manner as in FIG. 4-7. In addition, GluA1 (Ser-831), which is known as a molecule activated by CaMKII activation, was investigated. Hippocampal sections were suspended in SDS sample buffer to obtain a suspension which was immunoblotted. Thus, the intracellular mechanism of cognitive dysfunction in Kir6.2-defective mice was analyzed. The results are shown in FIG. 9 (Fig. 9a: images of the bands obtained by immunoblotting, Fig. 9b: results of quantification of the signal intensity of the bands). In the hippocampus of Kir6.2-defective mice, CaMKII activation was accelerated. Even in the case of long-term treatment with TP-014, no effect was observed. An abnormality was observed in intracellular and extracellular calcium homeostasis (balance) in Kir6.2 channel defect, and CaMKII phosphorylation was accelerated. TP-014 has been shown to have no effect on CaMKII activation, suggesting that the site of action of TP-014 is the Kir6.2 channel.

Test example 10.

The hypothesis that amyloid-β (Ab) is the cause of Alzheimer's disease remains important. Av aggregation that occurs in APP23 mice (14 months of age) was confirmed by immunostaining. The brains of WT (control mice) and APP23 mice were cut into 50 micron sections. Sections were stained with 6E10 (Ab antibody, manufactured by Abcam) and thioflavin. The results (aggregate score index) are shown in FIG. 10. Conditions other than the above were the same as in the conventional immunostaining method. Ae aggregation was found to be accelerated in mouse APP23, in particular, many aggregates were observed in the cerebral cortex (PFC). On the contrary, aggregation was practically not observed in the hippocampus (CA1). Av aggregation was inhibited by long-term treatment with TP-014. The result shows that TP-014 has an inhibitory effect against Av

- 21 043204 aggregation.

Test example 11.

The improving effect of TP-014 on depression-like symptom was tested using OBX mice as a mouse model of depression. The results are shown in FIG. 11. OBH mice were originally established as a mouse model of depression, although deterioration in cognitive function was observed. Depression was analyzed by the tail suspension method (a) and the forced swimming method (b). The tail hanging method is a way of pinching the tail of the mouse and hanging the mouse upside down. If the suspended mouse has depression, the immobility time is long. Because a normal mouse moves even if it is hanging, the time of immobility is short. In the forced swim method, the mouse is forced to swim in water in a glass. A mouse with depression does not swim or move (just floats). Thus, the time of immobility is measured. The immobility time was long in OBX mice in both the tail suspension method (a) and the forced swimming method. The immobility time of the TP-014 long-term administration group (2 weeks, same administration method as above) improved. Based on the results, it was demonstrated that TP-014 has the effect of improving depression-like symptom in CVD mice (n=5 per group).

Test example 12.

Immobility time in Kir6.1-deficient mice (heterotype, n=5 per group) was measured by the tail suspension method (a) and the forced swimming method (b) in the same manner as in FIG. 11. Heterotypic mice are mice in which the expression level of the Kir6.1 channel is half (homotypic mice develop arrhythmia after birth and die) in contrast to homotypic mice (completely defective mice). The results are shown in FIG. 12. Kir6. 1-defective mice observed a hyperactivated depression-like symptom. The results showed that Kir6.1 is an important molecule for depression. Long-term treatment with TP-014 is ineffective. It was confirmed that TP-014 has the effect of improving depression through the inhibitory Kir6.1 channel action. Kir6.2 1 defective mice were obtained from Professor Susumu Seino, School of Medicine of Kobe University (Miki T. et al., Nature Medicine, 2002, 8, 466-472).

Test example 13.

CaMKIV induced by the Kir6.1 channel was analyzed using CaMKIV-deficient mice (n=5, per group*) in the same manner as in FIG. 12. The results are shown in FIG. 13. Accelerated development of a depression-like symptom was also observed in CaMKIV-deficient mice. Based on this result, CaMKIV was found to be important in the mechanism of development of depression. TP-014 had no effect on CaMKIV depression-like symptom (increased immobility time). TP-014 was found to inhibit the Kir6.1 channel and have the effect of improving depression through activation of CaMKIV. CaMKIV-deficient mice were obtained from professor, Hiroyuki Sakagami, Kitasato University School of Medicine (Takao K. et al., PLoS One, 2010, 5, e9460).

Test example 14.

The hypoglycemic effect of TP-014 was tested by measuring blood glucose levels with an assay kit (manufactured by Technicon International co). The results are shown in FIG. 14. Measurements were carried out for 4 weeks. As a result of long-term treatment with TP-014 (1 mg/kg) for 4 weeks, blood glucose levels significantly decreased even after 3 weeks. Tolbutamide was used as a control. The Kir6.2 channel binds to SUR1 (urea receptor) on the cell membrane to form a channel. The mechanism of action is considered in connection with the Kir6.2 channel inhibitory action. Tolbutamide binds to SUR1, inhibiting the Kir6.2 channel.

Test example 15.

A plasmid vector containing the cDNA of the Kir6.1 channel inserted into it: pcDNA3.1-Kir6.1 was obtained from professor Toru Ishizuka of Graduate School of Life Sciences, Tohoku University. N2A cells overexpressing the Kir6.1 channel were obtained in the same manner as in the production of N2A cells overexpressing the Kir6.2 channel of Test Example 1, except that the above plasmid was used.

CaMKIV activation was analyzed (measured) using the obtained Kir6.1 channel-expressing cells. The same immunoblotting as in Test Example 1 was used as the analysis method. Anti-phosphorylated CaMKIV antibody (Kasahara J. et al., J. Biol., 2001, 276, 24044-50) was used as the primary antibody. An anti-rabbit IgG antibody (manufactured by SouthernBiotech) was used as a secondary antibody.

Using the obtained Kir6.1 channel-overexpressing cells, the potassium current flowing out of the cells was measured by the conventional patch clamp method. The results are shown in FIG. 18. An ATP-sensitive potassium channel (Kir6.1 channel) was localized in the cell membrane of nerve cells. If the channel is inhibited and closed, the threshold of the nerve cell membrane rises, leading to a state that is similar to that where an action potential is temporarily generated, resulting in potassium current flowing out of the cell and instead calcium current flowing into the cell from outside. Fig. 18a shows that the Kir6.1 channel is overexpressed in N2A cells (top figure shows stained images, half

- 22 043204 immunoblotted; while the bottom figure quantifies the signal intensity of the fringes). This was confirmed using immunoblotting (using the same conditions as in test example 1, except for anti-Kir6.1 channel antibody, n=5) with anti-Kir6.1 channel antibody (prepared in the usual way) to Kir6.1 channel -overexpressing cells (obtained as described above). No change was observed in the constitutive gene product, ie. β tubulin (anti-β tubulin antibody was obtained from Sigma-Aldrich and other conditions are the same as in Kir6.1 detection). The figure shows that if Kir6.2 channel-overexpressing cells are allowed to soak in an electrophysiological experimental buffer containing TP-014 to obtain a concentration of 10 nM, the potassium current that flows out when the membrane potential of nerve cells changes towards plus is suppressed ( n=5 per group). The results show that TP-014 inhibits the Kir6.1 channel and inhibits potassium current flowing out of cells.

Test Example 16.

Using WT mice (C57BL/6J, Japan SLC, two months old) injected with corticosterone (5 mg/kg dose administered once a day for 2 weeks); and Kir6.1-defective mice injected with corticosterone as a mouse model of anxiety symptomatic disease were subjected to five behavioral tests regarding anxiety-related behavior. Of note, Kir6.1-defective mice were obtained from Professor Susumu Seino, School of Medicine of Kobe University (Miki T. et al., Nature Medicine, 2002, 8, 466-472).

In WT mice and Kir6.1-defective mice injected with corticosterone, TP-014 (1 mg/kg) was administered orally once a day for 2 weeks (long-term treatment). As a result, it was confirmed that a significant improving effect of accelerating anxiety symptoms was obtained. The results are shown in FIG. 19.

In FIG. 19a shows the results of the anxiety analysis of each group (n=5 per group) using the elevated plus maze method (Fig. 19b). In the device used in this test, the cruciform device with sleeves is provided in a high position and the individual sleeves are either open (visible) or closed. Anxious mice remain in the closed arm for a long time; whereas the anxiety resistant mice remain in the open arm (visible). The retention time in the open sleeve is indicated on the vertical axis of FIG. 19a.

Fig. 19c shows the results of the light/dark test (FIG. 19d) (n=5 per group). The time was measured until the moment when the mouse, placed in a black box (dark place), feels the desire for light and leaves the box to a lighted place. The time until the mouse exits (entering the open compartment) is indicated on the vertical axis of FIG. 19s.

Fig. 19e shows the results of the beading test (FIG. 19f) (n=5 per group). Wood shavings were placed in the mouse cage and 20 balls were spread out so that the mouse could see them. The mice were allowed to move freely for 30 min. The number of pellets buried and hidden in a wood chip mat was counted. Since the mouse does not like a luminous object, anxiety-resistant mice come into contact with many balls. The number of balls buried is indicated on the vertical axis of FIG. 19th.

Fig. 19g shows the results of the open field test (FIG. 19h) (n=5 per group). The mouse is placed in a square box and allowed to move for 30 min inside the box. Usually, a mouse that is very anxious tends to move around the edge of the box; whereas a mouse with strong anxiety tolerance often runs around in the central part of the box. This trend is used as a benchmark. The residence time in the central part of the box is indicated in Fig. 19h.

Fig. 19i shows the results of the conditioned freeze test (n=5 per group). Using the same device as in the light/dark method, the mouse is placed in a dark place, and then sound (upper) is generated for 30 seconds, followed by electrical stimulation for 3 seconds. After sound, electrical stimulation is performed three times. In this case, the mouse remembers that electrical stimulation will be given after the sound. The next day, the sound continues for 5 minutes. The mouse, if it feels fear/anxiety, does not move. The time of mouse immobility is measured. The immobility time is indicated on the vertical axis of FIG. 19i.

All of the above test results confirmed that long-term administration (2 weeks) of TP-014 improved the acceleration of the anxiety-like symptom. Kir6.1-defective mice injected with corticosterone show anxiety-like symptoms; however, when TP-014 was administered, no improvement effect was obtained. The results confirmed that the improving effect of the compound of the present invention on the acceleration of anxiety-like symptoms is manifested by Kir6.1.

It should be noted that in the drawings in this application, indications of a significant difference ** or ++ mean P<0.01; while indications of a significant difference + or * mean P<0.05.

Claims (14)

1. Compound represented by formula (I) (I) Chemical formula 1 where R ¹ represents a hydrogen atom or (C _1-6 alkyl)carbonyl optionally substituted with one or more halogen atoms; R ² represents a hydrogen atom or (C _1-6 alkyl) carbonyl optionally substituted with one or more halogen atoms; X is O or NR ⁵ ; R ³ is phenyl optionally substituted with one or more substituents selected from X ¹ , 5- or 6-membered heteroaryl containing at least one heteroatom selected from an oxygen atom, a nitrogen atom and a sulfur atom, optionally substituted with one or more substituents selected from X ¹ , or COOR ⁶ ; R ⁴ represents a hydrogen atom, a halogen atom, azido or -OR ⁷ ; R ⁵ represents a hydrogen atom; R ⁶ represents a hydrogen atom or C _1-6 alkyl; R ⁷ represents a hydrogen atom, C _1-6 alkyl or C _1-6 alkoxy-C _1-6 alkyl; X ¹ is C _1-6 alkyl or a halogen atom, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof. 2. The compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to claim 1, where R ⁴ represents a chlorine or azido atom. 3. The compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to claim 1 or 2, wherein R ¹ is trifluoroacetyl. 4. A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1-3, wherein R ² is (C _1-6 alkyl)carbonyl optionally substituted with one or more halogen atoms. 5. The compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to claim 4, wherein R ² is trifluoroacetyl. 6. A compound, an enantiomer thereof, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 5, wherein R ³ is phenyl optionally substituted with one or more substituents selected from X ¹ , or pyridyl optionally substituted with one or more substituents selected from X ¹ . 7. The compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to claim 1, selected from (R)-(( 1 R,2S,3R,5R,7S)-5-azido-1-hydroxyadamantane-2- yl)(phenyl)methyl acetate; ethyl (S)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate; ethyl (R)-2-acetamido-2-((1R,2S,3R,5R,7R)-5-chloro-1-hydroxyadamantan-2-yl)acetate; (1 R,2S,3R,5R,7R)-5-chloro-2-((S)-2-methoxy-2-oxo-1-(2,2,2-trifluoroacetamido)ethyl)adamantan-1-yl 2,2,2-trifluoroacetate; (1 S,2R,3S,5S,7S)-5-chloro-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate; (S)-2-amino-2-(( 1 R,2S,3R,5R,7S)-1,5-dihydroxyadamantan-2-yl)acetic acid; N-((R)-(( 1 S,2R,3S,5S,7S)-5-chloro-1-hydroxyadamantan-2-yl)(phenyl)methyl)-2,2,2-trifluoroacetamide; (1S,2R,3S,5R,7S)-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate; (1 S,2R,3S,5S,7R)-5-(2-methoxyethoxy)-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1 -yl 2,2,2 -trifluoroacetate; N-((R)-(( 1 S,2R,3S,5S,7S)-5-chloro-1-hydroxyadamantan-2-yl)(pyridin-3-yl)methyl)-2,2,2trifluoroacetamide; 2,2,2-trifluoro-N-((R)-(( 1 S,2R,3S,5R,7S)-1-hydroxyadamantan-2-yl)(phenyl)methyl)acetamide; (1 S,2R,3S,5S,7R)-5-methoxy-2-((R)-phenyl(2,2,2-trifluoroacetamido)methyl)adamantan-1-yl 2,2,2trifluoroacetate; or an enantiomer, diastereomer or pharmaceutically acceptable salt of such a compound. 8. A pharmaceutical composition having inhibitory activity on the Kir6.2 channel and Kir6.1 channel of the K _atp channel, containing a therapeutically effective amount of a compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to any one of claims 1 to 7, and pharmaceutically acceptable additives. - 24 043204 9. The use of a pharmaceutical composition according to claim 8 for the treatment or prevention of a disease associated with the Kir6.1 channel or Kir6.2 channel of the K _atp channel. 10. Use according to claim 9, wherein the disorder associated with the Kir6.1 channel or the Kir6.2 channel of the K _atp channel is a cognitive disease or disorder. 11. The use of claim 10 wherein the cognitive disease or disorder is selected from Alzheimer's type dementia, cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, mental illness, and neurodegenerative disease. 12. Use according to claim 9, wherein the disease associated with the Kir6.1 channel or the Kir6.2 channel of the K _ATP channel is diabetes or a diabetic complication. 13. Kir6.2 inhibitor of the K _atp channel channel, which is a compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to any one of claims 1-7. 14. Kir6.1 inhibitor of the K _atp channel, which is a compound, its enantiomer, its diastereomer or its pharmaceutically acceptable salt according to any one of claims 1-7.