U.S. patent application number 13/588383 was filed with the patent office on 2013-02-21 for pharmaceutical compositions for preventing or treating degenerative brain disease and method of screening the same.
This patent application is currently assigned to KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY. The applicant listed for this patent is Hyunah Choo, Seonmi Jo, Daesoo Kim, Ji Yoon Kim, Changjoon Justin LEE, Boeun Yoon. Invention is credited to Hyunah Choo, Seonmi Jo, Daesoo Kim, Ji Yoon Kim, Changjoon Justin LEE, Boeun Yoon.
Application Number | 20130046093 13/588383 |
Document ID | / |
Family ID | 46650451 |
Filed Date | 2013-02-21 |
United States Patent
Application |
20130046093 |
Kind Code |
A1 |
LEE; Changjoon Justin ; et
al. |
February 21, 2013 |
PHARMACEUTICAL COMPOSITIONS FOR PREVENTING OR TREATING DEGENERATIVE
BRAIN DISEASE AND METHOD OF SCREENING THE SAME
Abstract
A pharmaceutical composition for preventing or treating a
degenerative brain disease, and a method of screening a material
for preventing or treating a degenerative brain disease. The method
may effectively screen a prophylactic or therapeutic candidate
material for preventing or treating a degenerative brain disease. A
variety of degenerative brain diseases may be effectively prevented
or treated using the pharmaceutical composition including a
screened material for preventing or treating a degenerative brain
disease.
Inventors: |
LEE; Changjoon Justin;
(Seoul, KR) ; Jo; Seonmi; (Suncheon-si, KR)
; Yoon; Boeun; (Seoul, KR) ; Choo; Hyunah;
(Seoul, KR) ; Kim; Ji Yoon; (Seoul, KR) ;
Kim; Daesoo; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LEE; Changjoon Justin
Jo; Seonmi
Yoon; Boeun
Choo; Hyunah
Kim; Ji Yoon
Kim; Daesoo |
Seoul
Suncheon-si
Seoul
Seoul
Seoul
Daejeon |
|
KR
KR
KR
KR
KR
KR |
|
|
Assignee: |
KOREA INSTITUTE OF SCIENCE AND
TECHNOLOGY
Seoul
KR
|
Family ID: |
46650451 |
Appl. No.: |
13/588383 |
Filed: |
August 17, 2012 |
Current U.S.
Class: |
540/597 ;
435/7.1; 544/127; 546/114; 546/115 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 27/04 20180101; A61P 25/00 20180101; A61P 25/28 20180101; G01N
33/5058 20130101; A61P 21/04 20180101; G01N 2800/2814 20130101;
C07D 513/04 20130101; A61P 9/00 20180101; A61P 43/00 20180101; G01N
33/6896 20130101; C12N 2503/02 20130101; G01N 2800/302 20130101;
A61P 25/16 20180101; C07D 498/04 20130101; A61P 25/24 20180101;
G01N 33/9426 20130101; A61P 25/08 20180101; A61P 25/22 20180101;
A61P 31/04 20180101; A61P 31/12 20180101 |
Class at
Publication: |
540/597 ;
435/7.1; 546/115; 546/114; 544/127 |
International
Class: |
C07D 498/04 20060101
C07D498/04; C07D 513/04 20060101 C07D513/04; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 18, 2011 |
KR |
10-2011-0082345 |
Aug 16, 2012 |
KR |
10-2012-0089402 |
Claims
1. A method of screening candidate materials for preventing or
treating a degenerative brain disease, the method comprising: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
determining if the target assay sample reduces a concentration of
.gamma.-aminobutylic acid (GABA) in the reactive astrocyte or
reduce release of GABA from the reactive astrocyte, wherein the
target assay sample is determined as a candidate material for
diagnosing or treating a degenerative brain disease if the target
assay sample is determined to reduce the concentration of GABA in
the reactive astrocyte or to reduce the release of GABA from the
reactive astrocyte.
2. A method of screening candidate materials for preventing or
treating a degenerative brain disease, the method comprising: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
measuring an expression amount of a gene encoding a monoamine
oxidase B (MAO-B) in the reactive astrocyte, or an amount or
activity of an MAO-B protein, wherein the target assay sample is
determined as a candidate material for diagnosing or treating a
degenerative brain disease if the expression amount of the gene
encoding the MAO-B, or the amount or activity of the MAO-B protein
is found to be down-regulated.
3. A method of screening candidate materials for preventing or
treating a degenerative brain disease, the method comprising: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
determining a subcellular localization pattern of a bestrophine 1
channel in the reactive astrocyte, wherein the target assay sample
is determined as a candidate material for diagnosing or treating a
degenerative brain disease if the subcellular localization pattern
of the bestrophine 1 channel is determined to be changed from a
cell body and a main process Into a microdomain direction.
4. A method of screening candidate materials for preventing or
treating a degenerative brain disease, the method comprising: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
measuring an expression amount of a gene encoding Best1 in the
reactive astrocyte or an amount or activity of Best1 protein,
wherein the target assay sample is determined as a candidate
material for preventing or treating the degenerative brain disease
if the expression amount of the gene encoding Best 1, or the amount
or activity of the Best1 protein is found to be down-regulated.
5. A method of screening candidate materials for preventing or
treating a degenerative brain disease, the method comprising: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
measuring an expression amount of a gene encoding a
.gamma.-aminobutylic acid (GABA) transaminase in the reactive
astrocyte, or an amount or activity of a GABA transaminase protein,
wherein the target assay sample is determined as a candidate
material for preventing or treating the degenerative brain disease
if the expression amount of the gene encoding the GABA
transaminase, or the amount or activity of the GABA transaminase
protein is found to be up-regulated.
6. The method of claim 1, wherein the reactive astrocyte originates
from a brain tissue of an animal model with brain injury, a brain
tissue of a virus-infected animal, a brain tissue of a Parkinson's
disease animal model, or a brain tissue of an Alzheimer's disease
animal model.
7. The method of claim 6, wherein the brain tissue is selected from
the group consisting of the hippocampus, corpus striatum,
substantia nigra pars compacta, and thalamic nuclei.
8. The method of claim 1, wherein the degenerative brain disease is
selected from among Alzheimer's disease, mild cognitive impairment,
vascular dementia, frontotemporal dementia, lewy body dementia,
Creutzfeld-Jakob disease, traumatic head injuries, syphilis,
acquired immune deficiency syndrome (AIDS) and other viral
infections, brain abscess, brain tumor, multiple sclerosis,
dementia in metabolic disease, hypoxia, Parkinson's disease,
Huntington's disease, Pick's disease, amyotrophic lateral sclerosis
(ALS), epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
post-traumatic stress disorder, spinal cord injury, and
myelitis.
9. A pharmaceutical composition for preventing or treating a
degenerative brain disease, the pharmaceutical composition
comprising an effective component that is a material reducing a
concentration of .gamma.-aminobutylic acid (GABA) in a reactive
astrocyte.
10. A pharmaceutical composition for preventing or treating a
degenerative brain disease, the pharmaceutical composition
comprising an effective component that is a material suppressing an
expression of a gene encoding a monoamine oxidase B (MAO-B) in a
reactive astrocyte, or a material reducing an activity of an MAO-B
protein.
11. The pharmaceutical composition of claim 10, wherein the
material reducing the activity of the MAO-B protein is a compound
selected from the group consisting of a compound represented by
Formula I below, a pharmaceutically acceptable salt, an isomer, a
solvate, a hydrate, and a combination thereof. ##STR00082##
wherein, in Formula I above, R1 and R2 are each independently a
hydrogen atom, a halogen atom, a nitro group, a cyano group, a
carboxyl group, a hydroxyl group, a substituted or unsubstituted
C1-C12 alkyl group, a substituted or unsubstituted C2-C12 alkenyl
group, a substituted or unsubstituted C2-C12 alkynyl group, a
substituted or unsubstituted C3-C15 cycloalkyl group, a substituted
or unsubstituted C3-C40 heterocycloalkyl group, (a substituted or
unsubstituted C6-C20 aryl) C1-C12 alkyl group, a substituted or
unsubstituted C1-C12 alkoxy group, a substituted or unsubstituted
C6-C20 arylamine group, a substituted or unsubstituted
C.sub.6-C.sub.30 diarylamine group, a substituted or unsubstituted
C6-C20 aryloxy group, a substituted or unsubstituted C6-C20 aryl
group, or a substituted or unsubstituted C5-C20 heteroaryl group;
and X is --O--, --S--, or --N(H)--.
12. The pharmaceutical composition of claim 10, wherein the
material reducing the activity of the MAO-B protein is a compound
selected from the group consisting of a compound represented by
Formula I below, a pharmaceuetically acceptable salt, an isomer, a
solvate, a hydrate, and a combination thereof. ##STR00083##
wherein, in Formula I above, R1 and R2 are each independently a
hydrogen atom, a halogen atom, a nitro group, a cyano group, a
substituted or unsubstituted C1-C12 alkyl group, or a substituted
or unsubstituted C1-C12 alkoxy group; and X is --O--, or --S--.
13. The pharmaceutical composition of claim 10, wherein the
material reducing the activity of the MAO-B protein is
N-cyclohexyl-2-phenyloxazolo[5,4-b]pyridine,
2-phenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine,
2-phenyl-5-(pyrrolidine-1-yl)thiazolo[5,4-b]pyridine,
2-(2-chlorophenyl)-5-(pyrrolidine-1-yDoxazolo[5,4-b]pyridine,
2-(3-chlorophenyl)-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine,
2-(4-fluorophenyl)-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine,
2-phenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
2-phenyl-5-(piperidine-1-yl)thiazolo[5,4-b]pyridine,
2-(2-chlorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
2-(3-fluorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
2-(3-chlorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
3-(5-(piperidine-1-yl)oxazolo[5,4-b]pyridine-2-yl)benzonitrile,
2-(4-fluorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
2-(4-bromophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
2-(4-methoxyphenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
2-(3-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine, or
2-(4-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine.
14. A pharmaceutical composition for preventing or treating a
degenerative brain disease, the pharmaceutical composition
comprising an effective component that is a material changing a
subcellular localization pattern of a bestrophine 1 channel in a
reactive astrocyte from a cell body and a main process into a
microdomain direction.
15. A pharmaceutical composition for preventing or treating a
degenerative brain disease, the pharmaceutical composition
comprising an effective component that is a material inhibiting an
expression of a gene encoding bestrophin1 channel in a reactive
astrocyte, or a material reducing an activity of a bestrophin1
protein.
16. A pharmaceutical composition for preventing or treating a
degenerative brain disease, the pharmaceutical composition
comprising an effective component that is a material inducing an
expression of a gene encoding a .gamma.-aminobutylic acid (GABA)
transaminase in a reactive astrocyte, or a material increasing an
activity of a GABA transaminase protein.
17. The pharmaceutical composition of claim 9, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
18. The method of claim 2, wherein the reactive astrocyte
originates from a brain tissue of an animal model with brain
injury, a brain tissue of a virus-infected animal, a brain tissue
of a Parkinson's disease animal model, or a brain tissue of
Alzheimer's disease animal model.
19. The method of claim 3, wherein the reactive astrocyte
originates from a brain tissue of an animal model with brain
injury, a brain tissue of a virus-infected animal, a brain tissue
of a Parkinson's disease animal model, or a brain tissue of
Alzheimer's disease animal model.
20. The method of claim 4, wherein the reactive astrocyte
originates from a brain tissue of an animal model with brain
injury, a brain tissue of a virus-infected animal, a brain tissue
of a Parkinson's disease animal model, or a brain tissue of
Alzheimer's disease animal model.
21. The method of claim 5, wherein the reactive astrocyte
originates from a brain tissue of an animal model with brain
injury, a brain tissue of a virus-infected animal, a brain tissue
of a Parkinson's disease animal model, or a brain tissue of
Alzheimer's disease animal model.
22. The method of claim 2, wherein the degenerative brain disease
is selected from among Alzheimer's disease, mild cognitive
impairment, vascular dementia, frontotemporal dementia, lewy body
dementia, Creutzfeld-Jakob disease, traumatic head injuries,
syphilis, acquired immune deficiency syndrome (AIDS) and other
viral infections, brain abscess, brain tumor, multiple sclerosis,
dementia in metabolic disease, hypoxia, Parkinson's disease,
Huntington's disease, Pick's disease, amyotrophic lateral sclerosis
(ALS), epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
post-traumatic stress disorder, spinal cord injury, and
myelitis.
23. The method of claim 3, wherein the degenerative brain disease
is selected from among Alzheimer's disease, mild cognitive
impairment, vascular dementia, frontotemporal dementia, lewy body
dementia, Creutzfeld-Jakob disease, traumatic head injuries,
syphilis, acquired immune deficiency syndrome (AIDS) and other
viral infections, brain abscess, brain tumor, multiple sclerosis,
dementia in metabolic disease, hypoxia, Parkinson's disease,
Huntington's disease, Pick's disease, amyotrophic lateral sclerosis
(ALS), epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
post-traumatic stress disorder, spinal cord injury, and
myelitis.
24. The method of claim 4, wherein the degenerative brain disease
is selected from among Alzheimer's disease, mild cognitive
impairment, vascular dementia, frontotemporal dementia, lewy body
dementia, Creutzfeld-Jakob disease, traumatic head injuries,
syphilis, acquired immune deficiency syndrome (AIDS) and other
viral infections, brain abscess, brain tumor, multiple sclerosis,
dementia in metabolic disease, hypoxia, Parkinson's disease,
Huntington's disease, Pick's disease, amyotrophic lateral sclerosis
(ALS), epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
post-traumatic stress disorder, spinal cord injury, and
myelitis.
25. The method of claim 5, wherein the degenerative brain disease
is selected from among Alzheimer's disease, mild cognitive
impairment, vascular dementia, frontotemporal dementia, lewy body
dementia, Creutzfeld-Jakob disease, traumatic head injuries,
syphilis, acquired immune deficiency syndrome (AIDS) and other
viral infections, brain abscess, brain tumor, multiple sclerosis,
dementia in metabolic disease, hypoxia, Parkinson's disease,
Huntington's disease, Pick's disease, amyotrophic lateral sclerosis
(ALS), epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
post-traumatic stress disorder, spinal cord injury, and
myelitis.
26. The pharmaceutical composition of claim 10, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
27. The pharmaceutical composition of claim 11, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
28. The pharmaceutical composition of claim 12, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
29. The pharmaceutical composition of claim 13, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
30. The pharmaceutical composition of claim 14, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
31. The pharmaceutical composition of claim 15, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
32. The pharmaceutical composition of claim 16, wherein the
degenerative brain disease is selected from the group consisting of
Alzheimer's disease, mild cognitive impairment, vascular dementia,
frontotemporal dementia, Louis corpuscle dementia, Creutzfeld-Jakob
disease, traumatic head injuries, syphilis, acquired immune
deficiency syndrome (AIDS), viral infection, brain abscess, brain
tumor, sclerosis, dementia in metabolic disease, hypoxia,
Parkinson's disease, Huntington's disease, Pick's disease,
amyotrophic lateral sclerosis (ALS), epilepsy, ischemia, stroke,
attention deficit hyperactivity disorder (ADHD), schizophrenia,
depression, manic-depression, stress disorder, spinal cord injury,
and myelitis.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATION
[0001] This application claims the benefit of Korean Patent
Application No. 10-2011-0082345, filed on Aug. 18, 2011, and Korean
Patent Application No. 10-2012-0089402, filed on Aug. 16, 2012, in
the Korean Intellectual Property Office, the disclosures of which
are incorporated herein in their entirety by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present disclosure relates to a pharmaceutical
composition for diagnosing or treating a degenerative brain
disease, and a method of screening the same.
[0004] 2. Description of the Related Art
[0005] Degenerative brain diseases, including Alzheimer's disease,
mostly cause memory and cognitive dysfunction and behavioral
problems. In particular, Alzheimer's disease is a chronic disease
gradually worsening over several years, which causes severe
emotional distress to the patient and family, and huge medical
expenses. Drugs developed so far for treatment of Alzheimer's
disease temporarily relieve symptoms only, and thus, there is a
high demand for fundamental treatment or progress suppression of
the disease.
[0006] For example, a main target in developing a therapeutic agent
for Alzheimer's disease has been neurotransmitters found in
Alzheimer's disease, such as cholinergic neurons, and thus,
cholinesterase inhibitors (such as Aricept, Exelon, Reminyl, or the
like) are commercially available. A recently FDA-approved
medication called memantine, which is a glutamate receptor
antagonist, is also developed with a representative
neurotransmitter glutamate as a target. However, these drugs
basically can not block the progression of the disease itself, and
in recent years, there has been active research into the
development of drugs targeting .beta.-amyloid (A.beta.), which
forms amyloid plaques as a key component found in Alzheimer's
disease, to suppress a .beta.- or .gamma.-secretase that are
important in the generation of the .beta.-amyloid (A.beta.), or to
decompose the generated .beta.-amyloid (A.beta.). However, this
targeting on a normal protein present in the human body may inhibit
a normal function of the protein, leading to a side effect.
[0007] Therefore, there is a demand for a prophylactic or
therapeutic pharmaceutical composition targeting on new proteins
associated with a variety of degenerative brain diseases, based on
existing technologies.
SUMMARY OF THE INVENTION
[0008] The present disclosure provides a method of screening a
prophylactic or therapeutic candidate material for preventing or
treating a degenerative brain disease.
[0009] The present disclosure also provides a prophylactic or
therapeutic pharmaceutical composition for preventing or treating a
degenerative brain disease.
[0010] According to an aspect of the present disclosure, there is
provided a method of screening candidate materials for preventing
or treating a degenerative brain disease, the method including: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
determining if the target assay sample reduces a concentration of
.gamma.-aminobutylic acid (GABA) in the reactive astrocyte or
reduce release of GABA from the reactive astrocyte, wherein the
target assay sample is determined as a candidate material for
diagnosing or treating a degenerative brain disease if the target
assay sample is determined to reduce the concentration of GABA in
the reactive astrocyte or to reduce the release of GABA from the
reactive astrocyte.
[0011] According to an aspect of the present disclosure, there is
provided a method of screening candidate materials for preventing
or treating a degenerative brain disease, the method including: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
measuring an expression amount of a gene encoding a monoamine
oxidase B (MAO-B) in the reactive astrocyte, or an amount or
activity of an MAO-B protein, wherein the target assay sample is
determined as a candidate material for diagnosing or treating a
degenerative brain disease if the expression amount of the gene
encoding the MAO-B, or the amount or activity of the MAO-B protein
is found to be down-regulated.
[0012] According to an aspect of the present disclosure, there is
provided a method of screening candidate materials for preventing
or treating a degenerative brain disease, the method including: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
determining a subcellular localization pattern of a bestrophine 1
channel in the reactive astrocyte, wherein the target assay sample
is determined as a candidate material for diagnosing or treating a
degenerative brain disease if the subcellular localization pattern
of the bestrophine 1 channel is determined to be changed from a
cell body and a main process Into a microdomain direction.
[0013] According to an aspect of the present disclosure, there is
provided a method of screening candidate materials for preventing
or treating a degenerative brain disease, the method including: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
measuring an expression amount of a gene encoding Best1 in the
reactive astrocyte or an amount or activity of Best1 protein,
wherein the target assay sample is determined as a candidate
material for preventing or treating the degenerative brain disease
if the expression amount of the gene encoding Best 1, or the amount
or activity of the Best1 protein is found to be down-regulated.
[0014] According to an aspect of the present disclosure, there is
provided a method of screening candidate materials for preventing
or treating a degenerative brain disease, the method including: (a)
contacting a target assay sample to a reactive astrocyte; and (b)
measuring an expression amount of a gene encoding a
.gamma.-aminobutylic acid (GABA) transaminase in the reactive
astrocyte, or an amount or activity of a GABA transaminase protein,
wherein the target assay sample is determined as a candidate
material for preventing or treating the degenerative brain disease
if the expression amount of the gene encoding the GABA
transaminase, or the amount or activity of the GABA transaminase
protein is found to be up-regulated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The above and other features and advantages of the present
disclosure will become more apparent by describing in detail
exemplary embodiments thereof with reference to the attached
drawings in which:
[0016] FIG. 1 shows confocal fluorescent images of hippocampal
reactive astrocytes of a mouse model of Alzheimer's disease,
illustrating .gamma.-aminobutylic acid (GABA) in the hippocampal
reactive astrocyte;
[0017] FIG. 2 shows confocal fluorescent images illustrating an
amount of expression of monoamine oxidase B (MAO-B) in the
hippocampal reactive astrocytes of the mouse model of Alzheimer's
disease;
[0018] FIG. 3 shows confocal fluorescent images illustrating a
subcellular localization pattern of bestrophine 1 channel in the
hippocampal reactive astrocytes of the mouse model of Alzheimer's
disease;
[0019] FIG. 4 shows confocal fluorescent images illustrating an
amount of expression of GABA transaminase in the hippocampal
reactive astrocytes of the mouse model of Alzheimer's disease;
[0020] FIG. 5 shows confocal fluorescent images illustrating
distribution of GABA in the hippocampal tissue of the mouse model
of Alzheimer's disease;
[0021] FIG. 6 shows confocal fluorescent images illustrating a
subcellular localization pattern of bestrophine 1 channel in the
hippocampal reactive astrocytes of a mouse model with a brain
injury;
[0022] FIG. 7 shows confocal fluorescent images illustrating an
amount of expression of MAO-B in the hippocampal reactive
astrocytes of the mouse model with a brain injury;
[0023] FIG. 8 shows confocal fluorescent images illustrating an
amount of expression of a GABA transaminase in the hippocampal
reactive astrocytes of the mouse model with a brain injury;
[0024] FIG. 9 shows microscopic images illustrating GABA reactive
astrocytes of postmortem cerebral tissues from a human Alzheimer
patient and a normal subject;
[0025] FIG. 10 shows confocal fluorescent images illustrating
expression levels of MAO-B and GABA in the reactive astrocytes of
the postmortem cerebral tissues from the human Alzheimer patient,
and a plot of correlation between expression levels of GFAP and
MAO-B;
[0026] FIG. 11 shows confocal fluorescent images illustrating
reactive astrocytes and GABA in reactive astrocytes in the thalamic
nuclei domains of a virus-infected model mouse;
[0027] FIG. 12 shows confocal fluorescent images illustrating
reactive astrocytes and MAO-B and GABA in the reactive astrocytes
in the substantia nigra pars compacta of a Parkinson's model
rat;
[0028] FIG. 13 shows confocal fluorescent images illustrating
reactive astrocytes and Best1 and GABA in the reactive astrocytes
in substantia nigra pars compacta of a Parkinson's model mouse;
[0029] FIG. 14 is a graph illustrating activity of an MAO-B enzyme
in a hippocampal extract of an Alzheimer model mouse;
[0030] FIG. 15 is a figure and a graph illustrating concentration
of GABA in hippocampal tissues of the Alzheimer model mouse;
[0031] FIG. 16 is a figure and a graph showing inhibitory current
levels affected by tonic GABA in hippocampal dentate gyrus granular
cells of the Alzheimer model mouse.
DETAILED DESCRIPTION OF THE INVENTION
[0032] As used herein, the term "and/or" includes any and all
combinations of one or more of the associated listed items.
Expressions such as "at least one of," when preceding a list of
elements, modify the entire list of elements and do not modify the
individual elements of the list.
[0033] The inventors of the present disclosure made efforts to
research and develop drugs for preventing an onset of degenerative
brain diseases, including Alzheimer's disease, or treating the
same, and found an increase in concentration of gamma-aminobutyric
acid (GABA) as a neurotransmitter in hippocampal reactive
astrocytes using an animal model of Alzheimer's disease, along with
a cause thereof, such as increased expression of MAO-B as an
essential enzyme involved in the production of GABA or the reduced
expression of GABA transaminase. The inventors also found a change
in subcellular localization pattern of bestrophine 1 channel
through which GABA can pass or an increased expression of
bestrophine 1 in the reactive astrocytes.
[0034] The present disclosure relates to a method of screening a
prophylactic or therapeutic material for preventing or treating a
degenerative brain disease, the material reducing a concentration
of GABA in reactive astocytes, and in particular, the method
targeting MAO-B, bestrophine 1 channel, or a GABA transaminases in
association with degenerative brain diseases. Thus, this novel use
of the proteins as a target in preventing or treating
neuroegenerative diseases is based on the inventors' findings that
hippocampal reactive astrocytes in a mouse model of Alzheimer's
disease have increased expression of MAO-B, and a reduced
expression of GABA transaminases, relative to a normal mouse model,
so that a concentration of GABA in the hippocampal reactive
astrocytes is increased, a subcellular localization pattern of
bestrophine 1 channel is changed, and a degree of expression of
bestrophine 1 is increased, leading to secretion of GABA out of the
reactive astrocytes, and thus, generates tonic GABA outside the
reactive astrocytes.
[0035] Tonic GABA, in the form of being bound to a tonic GABA
receptor in nerve cells, allows chloride ions (Cl--) to enter
neurons. This interferes with normal neuronal signaling by lowering
a resting membrane potential of nerve cells. Absence epilepsy is
known as a disease in which neuronal signaling is inhibited by
tonic GABA (David W. Cope, Giuseppe Di Giovanni, Sarah J. Fyson,
Gergely Orban, Adam C. Errington, Magor L. Larincz, Timothy M.
Gould, David A. Carter, and Vincenzo Crunelli, Nat. Med., 2009,
15(12); 1392-1398). In a stroke, inhibiting tonic GABA is known to
facilitate nerve recovery (Andrew N. Clarkson, Ben S. Huang, Sarah
E. MacIsaac, Istvan Mody, and S. Thomas Carmichael, Nature, 2010,
468; 305-309). Furthermore, reportedly, inhibiting a
hippocampus-specific receptor of tonic GABA may improve the memory
and cognitive abilities (G. R. Dawson, K. A. Maubach, N. Collinson,
M. Cobain, B. J. Everitt, A. M. MacLeod, H. I. Choudhury, L. M.
McDonald, G. Pillai, W. Rycroft, A. J. Smith, F. Sternfeld, F. D.
Tattersall, K. A. Wafford, D. S. Reynolds, G. R. Seabrook, and J.
R. Atack, JPET, 2006, 316(3):1335-1345; H. Lal, B. Kumar, and M. J.
Forster, The FASEB Journal, 1988, 2(11: 2707-2711).
[0036] A degenerative brain disease collectively refers to any
brain disease occurring from a degenerative change in nerve cells
of the central nervous system.
[0037] The causes of degenerative brain diseases are mostly
unknown, and the disease has slow onset and continuously progresses
via selective intrusion into associated nerve systems. In some
embodiments, for example, the degenerative brain disease is
selected from the group consisting of Alzheimer's disease, mild
cognitive impairment, vascular dementia, frontotemporal dementia,
Louis body dementia, Creutzfeld-Jakob disease, traumatic head
injuries, syphilis, acquired immune deficiency syndrome (AIDS) and
other viral infections, brain abscess, brain tumor, sclerosis,
dementia in metabolic disease, hypoxia, Parkinson's disease,
Huntington's disease, Pick's disease, amyotrophic lateral sclerosis
(ALS), epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
stress disorder, spinal cord injury, and myelitis.
[0038] According to the method of the present disclosure, a method
of screening a pharmaceutical composition for preventing or
treating a degenerative brain disease includes contacting a target
sample to be assayed and a reactive astrocyte. In some embodiments,
the reactive astrocyte may originate from a brain tissue of an
animal model with a brain injury, a brain tissue of a
virus-infected animal, a brain tissue of an animal model with
Parkinson's disease, or a brain tissue of an animal model with
Alzheimer's disease. The animal model may originate from mammals,
and in some embodiments, may originate from rodents such as mice or
rats, or primates such as monkeys, but is not limited thereto. In
some embodiments, the brain tissue may be the hippocampus, corpus
striatum, substantia nigra pars compacta, or thalamic nuclei, but
is not limited thereto.
[0039] The term "sample" used with regard to the screening method
refers to a to unidentified candidate material to be screened that
is tested to determine if it may reduce a concentration of GABA in
reactive astrocytes or may reduce release of GABA from the reactive
astrocytes. A mechanism to reduce the concentration of GABA or the
release of GABA may be, for example, as follows: i) increased
expression of a gene encoding MAO-B in the reactive astrocytes,
increased amount of the MAO-B protein, or increased activity of the
MAO-B protein, .quadrature.) increased expression of a gene
encoding Best1, increased amount or activity of the Best1 protein
in the reactive astrocytes, or a shift in subcellular localization
pattern of the bestrophine 1 channel in the reactive astrocytes
from a microdomain direction into a cell body and a main process,
or .quadrature.) a reduced expression of a gene encoding a GABA
transaminase in the reactive astrocytes, reduced amount of the GABA
transaminase protein, or reduced activity of the GABA transaminase
protein. Non-limiting examples of the sample are chemical
materials, nucleotides, anti-sense-RNA, short hairpin RNA (shRNA),
small interfering RNA (siRNA), and natural extracts.
[0040] Subsequently, it is determined if a sample to be assayed may
reduce the concentration of GABA in treated reactive astrocytes
using the following three methods:
[0041] i) An expression amount of a gene encoding MAO-B in the
reactive astrocytes, an amount of the MAO-B protein, or activity of
the MAO-B protein is determined. As a result, if the expression
amount of the gene encoding the MAO-B, or the amount or activity of
the MAO-B protein is found to be down-regulated, the target assay
sample may be determined as a candidate prophylactic or therapeutic
material for a degenerative brain disease.
[0042] ii) An expression amount of a gene encoding Best1 in the
reactive astrocytes or an amount or activity of the Best1 protein
is measured. As a result, if the expression amount of the gene
encoding Best 1, or the amount or activity of the Best1 protein is
found to be down-regulated, the target assay sample may be
determined as a candidate material for preventing or treating a
degenerative brain disease.
[0043] iii) A subcellular localization pattern of the bestrophine 1
channel in the reactive astrocytes is determined. As a result, the
target assay sample may be determined as a candidate prophylactic
or therapeutic material for preventing or treating a degenerative
brain disease if the subcellular localization pattern of the
bestrophine 1 channel is determined to be changed from a cell body
and a main process into a microdomain direction.
[0044] iv) An expression amount of a gene encoding a GABA
transaminase in the reactive astrocyte, or an amount or activity of
a GABA transaminase protein may be determined. As a result, if the
expression amount of the gene encoding the GABA transaminase, or
the amount or activity of the GABA transaminase protein is found to
be up-regulated, the target assay sample may be determined as a
candidate prophylactic or therapeutic material for preventing or
treating a degenerative brain disease.
[0045] The change in expression amount of the gene encoding the
protein (MAO-B, Best1, or GABA transaminase) may be measured by any
of a variety of methods, for example, via hybridization reaction
using RT-PCR, northern blotting or a cDNA microarray, or via in
situ hybridization reaction. For example, when using an RT-PCR
protocol, after separation of a total RNA from treated cells of a
sample, a first chain cDNA is prepared using an oligo dT primer and
a reverse transcriptase. Subsequently, PCR is performed using the
first chain cDNA as a template and a set of primers specific to the
genes encoding the proteins. Afterward, a PCR amplification product
is subjected to electrophoresis to analyze bands from the
electrophoresis, thereby measuring a change in the expression
amount of the specific protein-encoding gene.
[0046] The change in amount of the proteins (MAO-B, bestrophine 1
channel, or GABA transaminase) or in subcellular localization
pattern may be measured using any of a variety of methods. For
example, the change in amount of MAO-B, Best1, or GABA transaminase
may be identified using immunohistochemistry, radioactive
immunoassay, radioactive immunoprecipitation, immunoprecipitation,
western blotting, ELISA, capture-ELISA, or sandwich assay, but is
not limited thereto. The shift in subcellular localization pattern
of the protein may be identified using, but not limited to,
immunohistochemistry and transmission electron microscopy (TEM).
The change in activity of the proteins may be measured using an in
vitro enzyme activity assay, which is known in the art.
[0047] According to another aspect of the present disclosure, a
pharmaceutical composition for preventing or treating a
degenerative brain disease includes an effective component that is
a material reducing a concentration of GABA in a reactive
astrocyte.
[0048] According to still another aspect of the present disclosure,
a pharmaceutical composition for preventing or treating a
degenerative brain disease includes an effective component that is
a material suppressing an expression of a gene encoding a monoamine
oxidase B (MAO-B) in a reactive astrocyte, or a material reducing
an activity of an MAO-B protein.
[0049] In one embodiment, the material reducing the activity of the
MAO-B protein may be a compound selected from the group consisting
of a compound represented by Formula I below, a pharmaceuetically
acceptable salt thereof, an isomer thereof, a solvate thereof, a
hydrate thereof, and a combination thereof.
##STR00001##
[0050] wherein, in Formula I above, R.sub.1 and R.sub.2 are each
independently a hydrogen atom, a halogen atom, a nitro group, a
cyano group, a carboxyl group, a hydroxyl group, a substituted or
unsubstituted C.sub.1-C.sub.12 alkyl group, a substituted or
unsubstituted C.sub.2-C.sub.12 alkenyl group, a substituted or
unsubstituted C.sub.2-C.sub.12 alkynyl group, a substituted or
unsubstituted C.sub.3-C.sub.15 cycloalkyl group, a substituted or
unsubstituted C.sub.3-C.sub.40 heterocycloalkyl group, (a
substituted or unsubstituted C.sub.6-C.sub.20 aryl)
C.sub.1-C.sub.12 alkyl group, a substituted or unsubstituted
C.sub.1-C.sub.12 alkoxy group, a substituted or unsubstituted
C.sub.6-C.sub.20 arylamine group, a substituted or unsubstituted
C.sub.6-C.sub.30 diarylamine group, a substituted or unsubstituted
O.sub.6--O.sub.20 aryloxy group, a substituted or unsubstituted
O.sub.6--O.sub.20 aryl group, or a substituted or unsubstituted
C.sub.5--O.sub.20 heteroaryl group; and X is --O--, --S--, or
--N(H)--.
[0051] As used herein, the term "alkyl" refers to a monovalent
saturated, branched, or straight hydrocarbon group derived by
removing one hydrogen atom from a single carbon atom of a parent
alkane. Non-limiting examples of the alkyl group are methyl, ethyl,
propyl, such as propane-1-yl, propane-2-yl, and cyclopropane-1-yl,
and butyl, such as butane-1-yl, butane-2-yl, 2-methyl-propane-1-yl,
2-methyl-propane-2-yl, cyclobutane-1-yl, and tert-butyl. In some
other embodiments, the alkyl group may include from 1 to 12 carbon
atoms. At least one hydrogen atom in the alkyl group may be
substituted with a halogen atom, a hydroxyl group, a lower alkyl
group, or the like. The term "lower alkyl" refers to an alkyl group
including from 1 to 6 carbon atoms.
[0052] The term "alkenyl" refers to an unsaturated branched,
straight, or cyclic alkyl group with at least one carbon-carbon
double bond derived by removing one hydrogen atom from a single
carbon atom of a parent alkene. The alkenyl group may be in Z- or
E-form (or a cis or trans form), near the double bond. Non-limiting
examples of the alkenyl group are ethenyl; propenyl, such as
prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl(allyl),
prop-2-ene-2-yl, and cycloprop-1-ene-1-yl; cycloprop-2-ene-1-yl;
and butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl,
2-methyl-prop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl,
buta-1,3-diene-1-yl, buta-1,3-diene-2-yl, cyclobut-1-ene-1-yl,
cyclobut-1-ene-3-yl, and cyclobuta-1,3-diene-1-yl. In some
embodiments, the alkenyl group may include from 2 to 12 carbon
atoms, and in some other embodiments, may be a "lower alkenyl"
group having from 2 to 6 carbon atoms.
[0053] The term "alkynyl" refers to an unsaturated branched or
straight hydrocarbon group with at least one carbon-carbon triple
bond derived by removing one hydrogen atom from a single carbon
atom of a parent alkyne. Non-limiting examples of the alkynyl
groups are ethinyl, propynyl, butynyl, 2-pentynyl, 3-pentynyl,
2-hexynyl, and 3-hexynyl. In some embodiments, the alkynyl group
may have from 2 to 12 carbon atoms, and in some other embodiments,
may be a "lower alkynyl" group having from 2 to 6 carbon atoms.
[0054] The term "alkoxy" refers to a radical --OR, wherein R is an
alkyl group. Non-limiting examples of the alkoxy group are methoxy,
ethoxy, propoxy, butoxy, and cyclohexyloxy.
[0055] The term "aryl" refers to a monovalent aromatic hydrocarbon
group derived by removing one hydrogen atom from a single carbon
atom of a parent aromatic ring system. The aryl group may include a
5- and 5-membered carbocyclic aromatic ring, for example, benzene;
a bicyclic system of which at least one ring may be a carbocyclic
and aromatic group, such as naphthalene, indane, and tetraline; a
tricyclic system of which at least one ring may be a carbocyclic
and aromatic group, such as fluorene. For example, the aryl group
may include a 5- and 5-membered carbocyclic aromatic ring fused to
a 5- to 7-membered heterocycloalkyl group including at least one
heteroatom selected from among N, O, and S. In some embodiments the
aryl group may include from 6 to 10 carbon atoms. However, the aryl
does not include or overlap with heteroaryl groups independently
defined below. Therefore, if at least one carbocyclic aromatic ring
is fused with a heterocycloalkyl aromatic ring, a resulting cyclic
system is a heteroaryl defined herein, not an aryl group.
[0056] The term "carboxy" refers to a radical --C(O)OH.
[0057] The term "cyano" refers to a radical --CN.
[0058] The term "cycloalkyl" refers to a saturated or unsaturated
nonaromatic cyclic alkyl group. To define a certain level of
saturation, the term "cycloalkaneyl" or "cycloalkenyl" is used.
Non-limiting examples of the cycloalkyl are groups derived from
cyclopropane, cyclobutane, cyclopentane, and cyclohexane. In some
other embodiments, the cycloalkyl group may be a C3-10 cycloalkyl
group, for example, a C3-6 cycloalkyl group.
[0059] The term "heterocycloalkyl" refers to a saturated or
unsaturated non-aromatic cyclic alkyl group, wherein at least one
carbon atom (and relevant hydrogen atom) may be independently
appropriately substituted with the same or a different heteroatom
and a relevant hydrogen atom thereof. Non-limiting examples of the
heteroatoms are N, P, O, S, and Si. Non-limiting examples of the
heterocycloalkyl group are groups derived from epoxide,
imidazolidine, morpholine, piperazine, piperidine, pyrazolidine,
pyrrolidine, quinuclidine, tetrahydrofuran, tetrahydropyran, or the
like. Examples of the substituted heterocycloalkyl are cyclic
systems substituted with at least one oxo(.dbd.O) or oxide (--O--)
group, such as piperidineyl N-oxide, morpholinyl-N-oxide,
1-oxo-1-thiomorpholinyl, and 1,1-dioxo-1-thiomorpholinyl.
[0060] The term "halo" refers to a fluoro group, a chloro group, a
bromo group, or an iodo group.
[0061] The term "heteroaryl" refers to a monovalent heteroaromatic
group derived by removing one hydrogen atom from a single atom of a
parent heteroaromatic ring system. The heteroaryl group may include
a 5- to 7-membered aromatic, monocyclic ring including at least one
heteroatom, for example, 1 to 4 heteroatoms, and in some
embodiments, 1 to 3 heteroatoms, selected from among N, O, and S,
and carbon atom in the rest of the rings; a polycyclic
heterocycloalkyl ring including at least one heteroatom, for
example, 1 to 4 heteroatoms, and in some embodiments, 1 to 3
heteroatoms, selected from among N, O, and S, and carbon atom in
the rest of the rings; and a polycyclic heterocycloalkyl ring
including at least one heteroatom in an aromatic ring. In some
embodiments, the heteroaryl group may include a 5- to 7-membered
heteroaromatic ring fused with a 5- to 7-membered cycloalkyl ring;
and a 5- to 7-membered heteroaromatic ring fused with a 5- to
7-membered heterocycloalkyl ring. In these fused bicyclic
heteroaryl rings in which only one ring contains at least one
heteroatom, the heteroaromatic ring or cycloalkyl ring may be a
fusing site. If a total number of S and O atoms in the heteroaryl
group is greater than 1, the heteroatoms may not be adjacent to
each other. In some embodiments, the total number of S and O atoms
in the heteroaryl group may be 2 or less, and in some other
embodiments, may be 1 or less. The heteroaryl group does not
include or overlap with the aryl groups as defined above.
Non-limiting examples of the heteroaryl group are groups derived
from acridine, arsindole, carbazole, .beta.-carboline, chromane,
chromene, cinnoline, furan, imidazole, indazole, indole, indulines,
idolizing, isobenzofuran, isochromene, isoindole, isoindoline,
isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,
oxazole, perinidine, phenanthridine, phenanthroline, phenazine,
phthalazine, pterdine, purine, pyran, pyrazine, pyrazole,
pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine,
quinazoline, quinoline, quinolizine, quinoxaline, tetrazole,
thiadiazole, thiazole, thiophene, triazole, xanthenes, or the like.
In some embodiments, the heteroaryl group may be a 5- to
20-membered heteroaryl group, for example, a 5- to 10-membered
heteroaryl group. Non-limiting examples of the heteroaryl group are
groups derived from thiophene, pyrrole, benzothiophene, benzofuran,
indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.
[0062] In some embodiments, in the compound of Formula I above,
R.sub.1 and R.sub.2 may be each independently a hydrogen atom, a
halogen atom, a nitro group, a cyano group, a substituted or
unsubstituted C.sub.1-C.sub.12 alkyl group, or a substituted or
unsubstituted alkoxy group, wherein X may be --O--, or --S--.
[0063] In some embodiments, the material reducing the activity of
the MAO-B protein may be
N-cyclohexyl-2-phenyloxazolo[5,4-b]pyridine, [0064]
2-phenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine, [0065]
2-phenyl-5-(pyrrolidine-1-yl)thiazolo[5,4-b]pyridine, [0066]
2-(2-chlorophenyl)-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine,
[0067]
2-(3-chlorophenyl)-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine,
[0068]
2-(4-fluorophenyl)-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine,
[0069] 2-phenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine, [0070]
2-phenyl-5-(piperidine-1-yl)thiazolo[5,4-b]pyridine, [0071]
2-(2-chlorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
[0072]
2-(3-fluorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
[0073]
2-(3-chlorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
[0074]
3-(5-(piperidine-1-yl)oxazolo[5,4-b]pyridine-2-yl)benzonitrile,
[0075]
2-(4-fluorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
[0076] 2-(4-bromophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
[0077]
2-(4-methoxyphenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine,
[0078] 2-(3-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine,
or [0079]
2-(4-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine.
[0080] According to another aspect of the present disclosure, a
pharmaceutical composition for preventing or treating a
degenerative brain disease includes an effective component that is
a material changing a subcellular localization pattern of
bestrophine 1 channel in a reactive astrocyte from a cell body and
a main process into a microdomain direction.
[0081] According to another aspect of the present disclosure, a
pharmaceutical composition for preventing or treating a
degenerative brain disease includes an effective component that is
a material inducing an expression of a gene encoding a GABA
transaminase in a reactive astrocyte, or a material increasing an
activity of a GABA transaminase protein.
[0082] In some embodiments, the pharmaceutical composition may
include nucleotides, antisense, shRNA, siRNA oligonucleotides, or
natural extracts as an effective component.
[0083] In some embodiments, the degenerative brain disease may be
selected from the group consisting of Alzheimer's disease, mild
cognitive impairment, vascular dementia, frontotemporal dementia,
Louis corpuscle dementia, Creutzfeld-Jakob disease, traumatic head
injuries, syphilis, acquired immune deficiency syndrome (AIDS),
viral infection, brain abscess, brain tumor, sclerosis, dementia in
metabolic disease, hypoxia, Parkinson's disease, Huntington's
disease, Pick's disease, amyotrophic lateral sclerosis (ALS),
epilepsy, ischemia, stroke, attention deficit hyperactivity
disorder (ADHD), schizophrenia, depression, manic-depression,
stress disorder, spinal cord injury, and myelitis, but are not
limited thereto.
[0084] The pharmaceutical composition may include a
pharmaceuetically acceptable carrier. Non-limiting examples of the
pharmaceutically acceptable carrier that may be used in the
pharmaceutical field include commonly-used lactose, dextrose,
sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate,
alginates, gelatin, calcium silicate, micro-crystalline cellulose,
polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose,
methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium
stearate, and mineral oil, but is not limited thereto. The
pharmaceutical composition may further include, for example, a
lubricant, a wetting agent, a sweetener, a flavor enhancer, an
emulsifying agent, a suspending agent, a preservative, or the like.
Other suitable pharmaceutically acceptable carriers and drugs are
described in Remington's Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition may be orally or parenterally
administered (for example, via intravenous injection,
intraperitoneal injection, intramuscular injection, subcutaneous
injection, or local administration)
[0085] A suitable dosage of the pharmaceutical composition may
depend on a variety of factors, including formulation methods,
administration methods, ages of patients, body weight, gender,
pathologic conditions, diet, administration time, administration
route, excretion speed, and reaction sensitivity. In some
embodiments, a dosage of the pharmaceutical composition may be from
about 0.001 to about 100 mg/kg (of body weight) a day.
[0086] The pharmaceutical composition may be formulated with a
pharmaceutically acceptable carrier and/or an excipient in the form
of a unit or multiple dosage(s) by a well-known method in the art.
In this regard, the formulation may be a solution in oil or an
aqueous medium, a suspension, an emulsified solution, an extract,
powder, granules, a tablet, or a capsule, and may further include a
dispersing or a stabilizing agent.
[0087] One or more embodiments of the present disclosure will now
be described in detail with reference to the following examples.
However, these examples are for illustrative purposes only and are
not intended to limit the scope of the one or more embodiments of
the present disclosure.
Example 1-1
Verification of Increased GABA in Reactive Astrocytes of a Mouse
Model with Alzheimer's Disease
[0088] To identify whether an amount of GABA was increased in
reactive astrocytes changed from normal astrocytes by Alzheimer's
disease, an immunohistochemical analysis of a well-known
APPswe/PSEN1 transgenic mouse model of Alzheimer's disease
(purchased from The Jackson Laboratory, http://www.jax.org) was
performed. After immunostaining a sample tissue, further staining
with thioflavin-S was performed to observe amyloid plaque as a
feature of Alzheimer's disease.
[0089] After deeply anesthetizing an about 8- to 9-month aged
APPswe/PSEN 1 mouse with Avertin, perfusion fixation was followed
using 4% paraformaldehyde. After isolating the brain from the
mouse, a coronal cryostat section having a thickness of about 30
.mu.m from the hippocampus was washed with phosphate buffered
saline (PBS) three times, and was then reacted with a blocking
solution (0.3% Triton-X, 2% normal serum in 0.1M PBS, available
from Sigma) for about 1 hour. The resultant was shaking-cultured
together with Chicken anti-GFAP antibody (1:500, Chemicon) and
guinea pig anti-GABA antibody (1:1000, available from Chemicon) at
4.quadrature. overnight. The resulting cultured product was washed
with PBS three times, was subsequently reacted with anti-chicken
Alexa 488 (1:200, Invitrogen) and anti-guinea pig Alexa 647 (1:200,
Invitrogen) that were conjugated with corresponding secondary
antibodies, for about 3 hours, and then was washed again with PBS
three times. A resulting product was reacted with a solution of 1
mM thioflavin-S dissolved in an aqueous solution of 50% ethanol for
about 8 minutes, and then was washed twice with 80% ethanol each
for 10 seconds, and then three times with tertiary distilled water
each for 10 seconds.
[0090] The tissue stained was moved into PBS and then onto a slide
glass, and then mounted onto a mounting medium (Dako) to be
observed using an FV1000 confocal microscope (Olympus) to obtain a
series of confocal fluorescence images, which were then processed
with Olympus FLUOVIEW software ver.2.1.
[0091] High-magnification (.times.40) confocal immunohistochemical
images of the resulting hippocampus of the transgenic APPswe/PSEN1
mouse were obtained using antibodies against GFAP and GABA. The
results are shown in FIG. 1. Referring to FIG. 1, amyloid plaques
(blue), which are not found in normal mice, were observed in the
hippocampus of the mouse model of Alzheimer's disease. Reactive
astrocytes (green) in the Alzheimer's disease model were found to
be stained in astrocyte marker GFAP (green) and to have increased
cell body size and increased thickness of the main process, as
compared with normal astrocytes. Zero or almost zero GABA (red)
appeared in the normal astrocytes, while the amount of
intracellular GABA was dramatically increased. With regard to
interneurons stained with anti-GABA antibody, but not stained with
anti-GFAP antibody, there was found no difference in size or degree
of GABA staining between the normal mouse and the Alzheimer mouse
model.
Example 1-2
Verification of Increased GABA in Alzheimer Patient's Cerebral
Reactive Astrocytes
[0092] To identify whether an amount of GABA was increased in
Alzheimer patient's reactive astrocytes with a change of normal
astrocytes into the reactive astrocytes, an immunohistochemical
analysis was performed using postmortem cerebral tissues (from
School of Medicine in Boston University) from a normal subject and
an Alzheimer patient. 30 .mu.m-thick coronal cryostat sections were
obtained from the fixed postmortem brain tissues, and were then
reacted in a hydrogen peroxide solution to suppress activity of the
peroxidase remaining in the tissue, followed by washing them with
PBS three times, and reacting them in a blocking solution (0.3%
Triton-X, 2% normal serum in 0.1M PBS, available from Sigma) for
about 1 hour. Next, a mixture of the sample tissues with a guinea
pig anti-GABA antibody (1:1000, Chemicon) was incubated while
shaking at about 4.quadrature. overnight. The resulting product was
washed with PBS three times, and was then reacted with a
corresponding secondary antibody-conjugated anti-guinea pig
horseradish peroxidase (HRP) (1:200, Invitrogen) for about 3 hours,
followed by washing with PBS three times. Next, the resulting
product was reacted with a DAB solution until it was stained brown
by the activity of the HRP, and was then transferred into PBS,
mounted onto a slide glass, and then onto a mounting medium
(available from Dako) for optical microscopic observation.
High-magnification (.times.40) immunohistochemical images of the
postmortem cerebral tissues from the normal subject and the
Alzheimer patient were obtained. The results are shown in FIG. 9.
Referring to FIG. 9, no or nearly no GABA (brown color) was not
found in the cerebral astrocytes from the normal subject, and a
sharp increase in the amount of subcellular GABA was found in the
Alzheimer patient's cerebral astrocytes. These results indicate
that GABA-accumulating reactive astrcytes are found both in the
Alzheimer mouse model and human patients, and thus supporting that
the present disclosure may be applicable in preventing or treating
Alzheimer disease.
Example 2-1
Verification of Expression Changes in MAO-B, Bestrophine 1 and GABA
Transaminase in Reactive Astrocytes
[0093] To investigate changes in MAO-B, bestrophine 1, and GABA
transaminase in reactive astrocytes, immunohistochemical staining
was performed in the same manner as in Example 1, except that
different types of antibodies were added.
[0094] As primary antibodies, further to the chicken anti-GFAP
antibody (1:5001 Chemicon) and guinea pig anti-GABA antibody
(1:1000, Chemicon), a rabbit anti-MAO-B antibody (1:50, Sigma),
rabbit anti-Best1 antibody (1:100, Soria et al. 2006), or a rabbit
anti-GABA transaminases antibody (1:100 Epitomics) was added. As
secondary antibodies, further to the anti-chicken DyLight 488
(1:200, JacksonIR) and anti-guinea pig Alexa 647 (1:200,
Invitrogen), an anti-rabbit Alexa 555 (1:200 Invitrogen) was added
for reaction
[0095] As a result, as seen in FIG. 2, an increase in MAO-B was
found in the reactive astrocytes, and referring to FIG. 3, a
reduced expression of the GABA transaminase was found in the
reactive astrocytes. The increased expression of the MAO-B and the
reduced expression of the GABA transaminase lead to increased GABA
in the reactive astrocytes. Referring to FIG. 4, as compared with
the wild type, a change in subcellular localization of bestrophine
1 was found in the reactive astrocytes, indicating that a changed
secretion pattern of GABA resulting from the changed subcellular
localization of bestrophine 1 leads to Alzheimer's disease.
Example 2-2
Verification of Increased Cerebral MAO-B Protein Activity in
Alzheimer Mouse Model
[0096] A stock solution was prepared using a human MAO-B enzyme
(purchased from Aldrich) and a Amplex.RTM. Red monoamine oxidase
assay kit according to a preparation manual. The kit includes a
5.times. reaction buffer, an Amplex.RTM. red reagent (1 mg), HRP
(horseradish peroxidase), DMSO, H.sub.2O.sub.2, p-tyramine
(substrate of MAO-A, B), benzylamine (substrate of MAO-B),
clorgiline (inhibitor of MAO-A), and pargyline (inhibitor of
MAO-B). Among these reagents in the kit, benzylamine was used as a
substrate for MAO-B, and pargyline was used as an MAO-B inhibitor A
solution as overall substrates was prepared as follows.
[0097] 200 ul of a solution of 1 mg of Amplex.RTM. red sufficiently
dissolved in 200 ul of DMSO, 100 ul of a mixed solution of HRP and
1 ml of a 1.times. buffer, 200 ul of a solution of benzylamine
dissolved in 1.2 ml of dH.sub.2O were added to 9.5 ml of a 1.times.
buffer to reach a total volume of 10 mL, which is sufficient for
100 wells. 0.5 ul of a mixture of MAO-B inhibitor pargyline and 1
ml of dH.sub.2O was put into each well.
[0098] First, a hippocampal extract from the Alzheimer mouse was
put into 1.sup.st and 2.sup.nd rows of the 96 wells, while a
hippocampal extract from the normal mouse was put into 3.sup.rd and
4.sup.th rows of the wells. After anesthetizing the mice, the
hippocampus was isolated from each mouse, followed by separating a
CA1 region and a DG region from the hippocampus. Immediately after
being separated from the hippocampus, the fresh tissues from each
mouse were homogenized in a homogenization solution. A relatively
large tissue lump was removed by weak centrifugation, and a
supernatant was collected and centrifuged at a high speed (13000
rpm for 20 minutes) to obtain a mitocondria-rich precipitate. The
precipitate obtained from the hippocampus was used by about 50
micrograms per each well to determine the activity of the MAO-B
enzyme. 0.5 ul of a pargyline, the MAO-B inhibitor was further
added into the 2.sup.nd and 4.sup.th row of the wells. To reduce an
experimental error for accuracy, the test was repeated three times
for each compound. After 30 minutes, 100 ul of the substrate
solution was added into each well in a darkroom. The test was
performed in the darkroom due to light sensitivity of the
Amplex.RTM. reagent. Finally, a total volume of the reaction
solution per well reached 200 ul. After about 2 to 3 hours,
chromophoric degrees of the samples were measured. A variation in
data values for the 1.sup.st and 2.sup.nd rows of the wells
indicates the pure reaction activity of the MAO-B enzyme with the
substrate in the hippocampus of the Alzheimer mouse. A variation in
data values for the 3.sup.rd and 4.sup.th rows of the wells
indicates the pure reaction activity of the MAO-B enzyme with the
substrate in the hippocampus of the normal mouse.
[0099] Referring to FIG. 14, the Alzheimer mouse was found to have
a higher hippocampal MAO-B activity in the hippocampal extract as
compared with the normal mouse, and in particular, in the
hippocampal DG region by about 25%, indicating that an increased
MAO-B activity causes Alzheimer's disease.
Example 2-3
Verification of Increased MAO-B Expression in Alzheimer Patient's
Cerebral Reactive Astrocytes
[0100] To investigate changes in expression of MAO-B in reactive
astrocytes, immunohistochemical staining was performed using
postmortem cerebral tissues of the Alzheimer patient in the same
manner as in Example 2, except that additional types of antibodies
were used.
[0101] A chicken anti-GFAP antibody (1:500, Chemmicon), guinea pig
anti-GABA antibody (1:1000, Chemicon), and rabbit anti-MAO-B
antibody (1:50, Sigma) were used as primary antibodies. As
secondary antibodies, further to the anti-chicken DyLight 488
(1:200, JacksonIR) and anti-guinea pig Alexa 647 (1:200,
Invitrogen), an anti-rabbit Alexa 555 (1:200 Invitrogen) was added
for reaction.
[0102] As a result, as shown in FIG. 10A, the expression of the
MAO-B was found to be increased in the reactive astrocytes. This
increased expression of the MAO-B led to an increased in GABA in
the reactive astrocytes. This indicates that an increased
expression of MAO-B in human reactive astrocytes of the Alzheimer
patient causes an increase in GABA and consequently Alzheimer
disease.
[0103] An increased expression level of cerebral MAO-B mRNA in the
Alzheimer patient was identified using quantitative RT-PCR. Total
RNA was prepared from the postmortem cerebral tissues of the
Alzheimer patient. First-strand cDNA was prepared from the total
RNA using an oligo (dT) primer and a reverse transferase
Subsequently, PCR was performed using the first-strand cDNA as a
template and a gene-specific primer set encoding a MAO-3 protein
and a GFAP protein. The resulting amplified PCR product was
subjected to electrophoresis, and subsequently the resulting bands
were analyzed to measure changes in expression of the genes
encoding the proteins were measured by band analysis.
[0104] As a result, an expression level of GFAP as an astrocyte
marker in the Alzheimer patient's brain was increased as shown in
FIG. 10B, and an increased expression level of the MAO-B enzyme
producing GABA was also found as shown in FIG. 100. FIG. 10D is a
plot of correlation between the expression levels of GFAP and MAO-B
in each patient sample. This indicates that expression levels of
both MAO-B protein and MAO-B mRNA were increased in the Alzheimer
patient's brain.
Example 3-1
Verification of Increase in Hippocampal Tonic GABA from Production
of GABA in Reactive Astrocytes of Alzheimer Mouse Model
[0105] Low-magnification (.times.10) confocal fluorescent images
were obtained from the slide sample manufactured in Example 1 using
an FV1000 confocal microscope (Olympus). As seen in FIG. 5, a
GABA-stained area was increased in most of the hippocampal dentate
gyrus molecular layer of the Alzheimer mouse model (denoted by
white arrows), meaning that the amount of GABA was increased both
in and outside the cells. As in Example 2, an increased expression
of MAO-B in the reactive astrocytes led to subcellular accumulation
of GABA, and a change in subcellular localization of bestrophine 1
led to exocytic secretion of GABA to become tonic GABA. This change
in secretional pattern of the GABA is considered to cause
Alzheimer's disease.
Example 3-2
Verification of Increased Hippocampal Extracellular GABA Resulting
from GABA Production in Reactive Astrocytes of Alzheimer Model
Mouse
[0106] An increased level of hippocampal extracellular GABA in the
Alzheimer model mouse was identified using microdialysis and
high-performance liquid chromatography (HPLC). After being
anesthetized with isoflurane, the Alzheimer model mouse was fixed
in a stereotaxic instrument, and a guide cannula and a
microdialysis probe was implanted into an amyloid plaque-rich
region of the hippocampus, as shown in FIGS. 15a and 15b. After
completely waking up from the anesthesia, an artificial
cerebrospinal fluid (ASCF) was flowed through the probe during
microdialysis, and then liquid from the microdialysis was collected
at 20-minute intervals and was then analyzed using HPLC to measure
a GABA level in each liquid sample.
[0107] Referring to FIG. 15, the Alzheimer model mouse was found to
be higher in hippocampal extracellular GABA level by about 2 times,
as compared with the normal mouse. This indicates that GABA
produced and accumulated in the reactive astrocytes was released
out of the cells, becoming tonic GABA, and the increased release of
GABA led to Alzheimer disease.
Example 3-3
Verification of Increased Hippocampal Neuro-Inhibitory Signal
Resulting from GABA Production in Reactive Astrocytes of Alzheimer
Model Mouse
[0108] The degree in which the hippocampal neurons of the Alzheimer
model mouse were affected by a GABA-mediated inhibitory signal was
measured using whole-cell patch clamp recording.
[0109] After being anesthetized with halothane, the Alzheimer model
mouse was decapitated, followed by quick extraction of the brain
from the skull, and soaking it in an ice-cold cutting solution
including: 250 mM sucrose, 26 mM NaHCO.sub.3, 10 mM D(+)-glucose, 4
mM MgCl.sub.2, 3 mM myo-inositol, 2.5 mM KCl, 2 mM Sodium pyruvate,
1.25 mM NaH.sub.2PO.sub.4, 0.5 mM Ascorbic acid, 0.1 mM CaCl.sub.2,
and 1 mM Kynurenic acid (pH 7.4) All solutions were gas-treated
with 95% O.sub.2-5% CO.sub.2. After trimming the forehead and
cerebellum parts using a knife, and 300-micrometer-thick slices
containing the hippocampus were cut using a microtome (Leica VT
1000), and transferred to artificial cerebrospinal fluid (ASCF)
solution including; 126 mM NaCl, 24 mM NaHCO.sub.3, 1 mM
NaH.sub.2PO.sub.4, 2.5 mM KCl, 2.5 mM CaCl.sub.2, 2 mM MgCl.sub.2,
and 10 mM D(+)-Glucose (pH 7.4). Slices were incubated for about 40
minutes at least at room temperature. For whole-cell patch clamp
recording, hippocampal slices were transferred to an
electrophysiological recording chamber (RC-26G, Warner Instruments)
which is continuously superfused with artificial cerebrospinal
fluid (ASCF) solution (flow rate; 2 ml/min) and controlled by a
flow controller (Synaptosoft) and a vacuum pump (Charles Austen,
model Capex 8C). Slice chamber was mounted on the stage of an
upright microscope (Olympus, Japan) and viewed with an X60 water
immersion objective with differential interference contrast and
infrared optics. Cellular morphologies were visually identified by
Imaging Workbench 6.0 (INDEC Systems, Inc), camera controller
(Hamamatsu, C4742-95), and light microscope controller (Olympus,
TH4-200). Whole cell voltage-clamp recording was made from granular
cell stomas mostly located in the hippocampal dentate gyrus.
[0110] For granular cell recording patch pipettes (8-12 M.OMEGA.)
were constructed from thick-walled borosilicate glass capillaries
(SC150F-10, Warner instrument Corp), and pipette was filled with an
internal solution containing; 135 mM CsCl, 4 mM NaCl, 0.5 mM
CaCl.sub.2, 10 mM HEPES, 5 mM EGTA, 2 mM Mg-ATP, 0.5 mM
Na.sub.2-GTP, and 10 mM QX-314 (pH adjusted to 7.2 with CsOH)
(278-285 mOsmol) (Rossi, et al., 2003). With this internal
solution, E.sub.cl=0 mV with voltage clamp and holding potential of
-70 mV, inward current was elicited. During whole-cell patch clamp
recording using patch pipettes with granular cells, an artificial
cerebrospinal fluid (ASCF) solution containing AP-5 and CNQX was
flowed for 5 minutes or longer to suppress neurotransmission
facilitated by excitatory neurotransmitter stimulator, and record
selectively only a neurosignal induced by the inhibitory material
GABA. After 5 minutes, an artificial cerebrospinal fluid (ASCF)
solution containing bicuculline that inhibits GABAergic
neurotransmission was flowed for about 3 minutes to suppress GABA
signals. The amplitude of a tonic GABA signal transferred to the
granular cells may be understood by comparing baselines before and
after the inhibition with bicuculilline.
[0111] The signals were digitized and sampled at 50 .mu.s intervals
with Digidata 1440A (Molecular Devices) and Multiclamp 700B
amplifier (Molecular Devices) using pCLAMP 10.2 software (Molecular
Devices). Off-line analysis was carried out using Clampfit 10.2
(Molecular Devices), SigmaPlot 10.0 (SPSS), and Excel 2003
(Microsoft).
[0112] All the drugs and chemicals used in this example were
purchased from SIGMA-Aldrich if not mentioned otherwise; QX-314
(Tocris), AP-5 (Tocris), CNQX (Tocris), and bicuculline
methobromide (Tocris).
[0113] Numerical data was presented as means.+-.S.E.M. The
significance of data for comparison was assessed by Student's
two-tailed unpaired t test, and significance level was represented
as *(p<0.05), **(p<0.01), ***(p<0.001). Data was filtered
at 2 kHz.
[0114] As a result, the hippocampal granular cells in the dentate
gyrus of the Alzheimer model mouse were found to be more influenced
by tonic GABAergic inhibition as compared with the normal mouse
(FIG. 16). A current level representing the intracellular influx of
chloride ions (Cl--) induced by GABA was about 12 pA for the
Alzheimer model mouse and about 8 pA in the normal mouse, both on
average. Tonic current density based on the cell area was also
higher in the Alzheimer mouse twice then the normal mouse. This
indicates that the extracellular efflux of GABA generated in the
hippocampal reactive astrocytes of the Alzheimer mouse may inhibit
nerve cells Finally, this may inhibit smooth signal transfer
between neurons, impairing normal brain functions, which leads to
memory impairment as a main symptom from the Alzheimer disease.
Example 4-1
Verification of Increased GABA in Reactive Astrocytes of
Virus-Infected Model Mouse
[0115] A wildtype C57BL/6 mouse (purchased from The Jackson
Laboratory), instead of the Alzheimer model mouse, was used. The
wildtype C57BL/6 mouse under anesthesia was fixed to a stereotaxic
instrument, and adenovirus expressing a fluorescent FGP protein was
injected into a thalamic nuclei domain to induce viral infection.
An immunochemical test was performed on the virus-infected mouse in
the same manner as in Example 1.
[0116] Referring to FIG. 11, nearly no GABA was in the astrocytes
(green) of the normal thalamic nuclei with no viral infection,
whereas GABA-rich astrocytes (yellow) were found in the
adenovirus-infected thalamic nucleic as in the Alzheimer model
mouse.
[0117] These results indicate that GABA-accumulating reactive
astrocytes may be generated in Alzheimer disease and other
degenerative brain diseases, including viral infection and brain
impairment.
Example 4-2
Verification of Increased GABA and Expression Changes of MAO-B and
Bestrophine 1 in Reactive Astrocytes in Rat and Mouse Models with
Parkinson's Disease
[0118] A wildtype rat with intracerebrally injected 6-OHDA and a
wildtype C57BL/6 mouse with intraperitoneally injected MPTP
(purchased from The Jackson Laboratory) were used as Parkinson's
disease model rat and mouse, respectively. Dopamine neurons in the
substantia nigra pars compacta of the two models were killed to
induce Parkinson's symptoms, followed by an immunochemical test in
the same manner in Example 1.
[0119] Referring to FIG. 12, expression levels of both MAO-B and
GABA were found to be increased in the Parkinson's model rat, as in
the Alzheimer model described above. As shown in FIG. 13, a gradual
shift in subcellular localization pattern of bestrophine 1 channel
in the reactive astrocytes from a microdomain direction into a cell
body and a main process, and an increased expression level of the
bestrophine 1 channel were identified in the Parkinson's model
mouse. The amount of GABA was also increased in the reactive
astrocytes.
[0120] These results indicate that GABA-accumulating reactive
astrocytes may be generated in Alzheimer disease and other
degenerative brain diseases, including Parkinson's disease and
brain impairment.
Example 4-3
Verification of Increased GABA and Expression Changes of MAO-B,
Bestrophine 1, and GABA Transaminase in Hippocampal Reactive
Astrocytes of a Mouse Model with a Hippocampal Injury
[0121] A transgenic GFAP-EGFP mouse (available from The Jackson
Laboratory), instead of the Alzheimer mouse model, was used, in
which after anesthetization in the same manner as in Example 1,
hippocampal damage was induced by inserting a sharp pin into the
hippocampus of the anesthetized mouse. An immunohistochemical test
was performed on the mouse on the 5th and 14th days after the
hippocampal damage.
[0122] As seen in FIG. 6, similar to an Alzheimer model, the
transgenic GFAP-EGFP was found to have an intracellular
distribution pattern of bestrophine 1 channel in the reactive
astrocytes that gradually changed from micro-domains into cell
bodies and main process, and to have an increased expression of
MAO-B in the reactive astrocytes 5 days after the hippocampal
damage. In addition, on the 5th day after the hippocampal damage,
the astrocytic body and main process were found to be larger in
size, while an expression of the GABA transaminase per cell area
was reduced.
[0123] These results indicate that GABA-accumulating reactive
astrocytes may be generated with other degenerative brain diseases
and brain damage, further to with Alzheimer disease.
Example 5
Preparation of Compound for Screening MAO-B Activity Inhibitory
Material
[0124] To screen compounds inhibiting the activity of MAO-B
associated with the treatment of degenerative brain diseases in the
present disclosure as described above in the examples, a total of
79 kinds of compounds were obtained using the following
methods:
Example 5-1
Synthesis of N-(2,6-dichloropyridine-3-yl)benzamide
##STR00002##
[0126] 0.038 ml (0.33 mmol) of benzoyl chloride was dissolved in 4
ml of acetonitrile, and 50 mg (0.30 mmol) of
2,6-dichloropyridine-3-amine was added thereto to obtain a mixture,
which was then refluxed at about 70.quadrature. for about 6 hours.
Termination of the reaction was confirmed by thin layer
chromatography (TLC), and the reaction product was cooled to room
temperature, followed by distillation under reduced pressure. A
small amount of methanol was added to the resulting brown solid so
that the color of the brown solid was changed to white. This white
solid was filtrated and dried to obtain 58.7 mg (0.22 mmol) of a
target compound with a yield of about 73% to 90%.
[0127] .sup.1H NMR (400 MHz, MeLD) .delta. 8.29 (d, J=8.4 Hz, 1H),
8.00-7.98 (m, 2H), 7.62 (t, J=6.1 Hz, 1H), 7.57-7.50 (m, 3H)
Example 5-2
Synthesis of 2-chloro-N-(2,6-dichloropyridine-3-yl)benzamide
##STR00003##
[0129] 65 mg (0.19 mmol) of a target compound was obtained with a
yield of about 75% in the same manner as in Example 5-1, except
that 0.04 ml (0.33 mmol) of 2-chlorobenzoyl chloride was used as a
starting material.
[0130] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.98 (dd=8.6 Hz,
1H), 8.68 (s, 1H), 7.80 (d, J=6.67 Hz, 1H), 7.56-7.52 (m, 2H),
7.49-7.45 (m, 1H), 7.39 (d, J=8.6 Hz, 1H)
Example 5-3
Synthesis of N-(2,6-dichloropyridine-3-yl)-3-fluorobenzamide
##STR00004##
[0132] 45 mg (0.18 mmol) of a target compound was obtained with a
yield of about 60% to 100% in the same manner as in Example 5-1,
except that 0.04 ml (0.33 mmol) of 3-fluorobenzoyl chloride was
used as a starting material.
[0133] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.93 (d, J=8.5 Hz,
1H), 8.37 (s, 1H), 7.68 (t, J=7.5 Hz, 2H), 7.57 (q, J=8.0 Hz, 1H),
7.41-7.34 (m, 2H)
Example 5-4
Synthesis of 3-chloro-N-(2,6-dichloropyridine-3-yl)benzamide
##STR00005##
[0135] 58.8 mg (0.19 mmol) of a target compound was obtained with a
yield of about 59% in the same manner as in Example 5-1, except
that 0.04 ml (0.33 mmol) of 3-chlorobenzoyl chloride was used as a
starting material.
[0136] .sup.1H NMR (400 MHz, MeOD) .delta. 8.22 (d, J=8.3 Hz, 1H),
7.97 (t, J=1.9 Hz, 1H), 7.90-7.88 (m, 1H), 7.62-7.61 (m, 1H), 7.5
(dd, J=7.9, 11. 6 Hz, 2H)
Example 5-5
Synthesis of N-(2,6-dichloropyridine-3-yl)-3-methylbenzamide
##STR00006##
[0138] 1 g (3.6 mmol) of a target compound was obtained with a
yield of about 83% in the same manner as in Example 5-1, except
that 0.63 ml (4.7 mmol) of 3-methylbenzoyl chloride was used as a
starting material.
[0139] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.92 (d, J=8.4 Hz,
1H), 8.36 (s, 1H), 7.72 (s, 1H), 7.70-7.67 (m, 1H), 7.43 (d, J=4.2
Hz, 1H), 7.35 (d, J=3.3 Hz, 1H), 2.47 (s, 3H)
Example 5-6
Synthesis of N-(2,6-dichloropyridine-3-yl)-3-nitrobenzamide
##STR00007##
[0141] 960 mg (3.07 mmol) of a target compound was obtained with a
yield of about 99% in the same manner as in Example 5-1, except
that 626 mg (3.4 mmol) of 3-nitrobenzoyl chloride was used as a
starting material.
[0142] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.87 (d, J=8.4 Hz,
1H), 8.77 (s, 1H), 8.49 (dd, J=2.4, 6.0 Hz, 1H), 8.37 (s, 1H), 8.24
(d, J=7.8 Hz, 1H), 7.89 (t, J=8.1 Hz, 1H), 7.58 (d, J=3.6 Hz,
1H)
Example 5-7
Synthesis of 3-cyano-N-(2,6-dichloropyridine-3-yl)benzamide
##STR00008##
[0144] 1.5 g (5.1 mmol) of a target compound was obtained with a
yield of about 83% in the same manner as in Example 5-1, except
that 1 g (6.1 mmol) of 3-cyanobenzoyl chloride was used as a
starting material.
[0145] .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.8.41 (s, 1H),
8.29 (d, J=10.5 Hz, 1H), 8.18-8.09 (m, 2H), 7.78 (t, J=7.8 Hz, 1H),
7.6 5 (dd, J=0.9, 7.2 Hz, 1H)
Example 5-8
Synthesis of N-(2,6-dichloropyridine-3-yl)-4-fluorobenzamide
##STR00009##
[0147] 70 mg (0.28 mmol) of a target compound was obtained with a
yield of about 94% in the same manner as in Example 5-1, except
that 0.04 ml (0.33 mmol) of 4-fluorobenzoyl chloride was used as a
starting material.
[0148] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.72 (d, J=4.5 Hz,
1H), 8.24 (s, 1H), 7.72 (d, J=8.0 Hz, 2H), 7.46 (d, J=8.1 Hz, 2H),
7.28 (d, J=8.0 z, 1H)
Example 5-9
Synthesis of 4-chloro-N-(2,6-dichloropyridine-3-yl)benzamide
##STR00010##
[0150] 90 mg (0.29 mmol) of a target compound was obtained with a
yield of about 99% in the same manner as in Example 5-1, except
that 0.04 ml (0.33 mmol) of 4-chlorobenzoyl chloride was used as a
starting material.
[0151] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.93 (d, J=4.7 Hz,
1H), 8.34 (s, 1H), 7.89 (d, J=8.3 Hz, 2H), 7.56 (d, J=8.3 Hz, 2H),
7.39 (d, J=8.6 Hz, 1H)
Example 5-10
Synthesis of 4-bromo-N-(2,6-dichloropyridine-3-yl)benzamide
##STR00011##
[0153] 610 mg (1.77 mmol) of a target compound was obtained with a
yield of about 56% to 95% in the same manner as in Example 5-1,
except that 739 mg (3.37 mmol) of 4-bromobenzoyl chloride was used
as a starting material.
[0154] .sup.1H NMR (300 MHz, MeOD) .delta. 8.27 (d, J=8.3 Hz, 1H),
7.92-7.89 (m, 2H), 7.76-7.73 (m, 2H), 7.51 (d, J=8.4 Hz, 1H)
Example 5-11
Synthesis of N-(2,6-dichloropyridine-3-yl)-4-methylbenzamide
##STR00012##
[0156] 970 mg (3.6 mmol) of a target compound was obtained with a
yield of about 80% in the same manner as in Example 5-1, except
that 0.63 ml (4.7 mmol) of 4-methylbenzoyl chloride was used as a
starting material.
[0157] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.97 (d, J=8.6 Hz,
1H), 8.38 (s, 1H), 7.84 (d, J=8.1 Hz, 2H), 7.38 (d, J=8.3 Hz, 3H),
2.49 (s, 3H)
Example 5-12
Synthesis of N-(2,6-dichloropyridine-3-yl)-4-methoxybenzamide
##STR00013##
[0159] 1.8 g (6.0 mmol) of a target compound was obtained with a
yield of about 98% in the same manner as in Example 5-1, except
that 0.95 ml (6.7 mmol) of 4-methoxybenzoyl chloride was used as a
starting material.
[0160] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.96 (d, J=8.6 Hz,
1H), 8.33 (s, 1H), 7.94-7.90 (m, 2H), 7.38 (d, J=8.5 Hz, 1H),
7.09-7.04 (m, 2H), 3.94 (s, 3H)
Example 5-13
Synthesis of N-(2,6-dichloropyridine-3-yl)-4-nitrobenzamide
##STR00014##
[0162] 1.6 g (5.2 mmol) of a target compound was obtained with a
yield of about 84% in the same manner as in Example 5-1, except
that 620 mg (6.7 mmol) of 4-nitrobenzoyl chloride was used as a
starting material.
[0163] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.93 (d, J=8.6 Hz,
1H), 8.45 (d, J=8.6 Hz, 2H), 8.41 (s, 1H), 8.13 (d, J=8.9 Hz, 2H),
7.43 (d, J=8.6 Hz, 1H)
Example 5-14
Synthesis of 4-cyano-N-(2,6-dichloropyridine-3-yl)benzamide
##STR00015##
[0165] 1.7 g (5.9 mmol) of a target compound was obtained with a
yield of about 96% in the same manner as in Example 5-1, except
that 1.13 ml (6.8 mmol) of 4-cyanobenzoyl chloride was used as a
starting material.
[0166] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.92 (d, J=8.6 Hz,
1H), 8.38 (s, 1H), 8.05 (d, J=8.3 Hz, 2H), 7.90 (d, J=8.2 Hz, 2H),
7.42 (d, J=8.6 Hz, 1H)
Example 5-15
Synthesis of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
##STR00016##
[0168] 58 mg (0.22 mmol) of N-(2,6-dichloropyridine-3-yl)benzamide
and 25 mg of sodium carbonate were added to 2.2 ml of
dimethylformamide to obtain a mixture, which was then refluxed at
about 160.quadrature. for about 24 hours. Termination of the
reaction was confirmed by TLC, and the reaction product was cooled
to room temperature and then concentrated under reduced pressure. A
small amount of distilled water was added into a resulting
concentrated solution, and an aqueous phase was treated with
methylene chloride to get an organic phase. The extracted organic
phase was dried using anhydrous MgSO.sub.4, filtrated, and then
concentrated under reduced pressure to obtain a concentrated
solution, which was then separated using column chromatography
(hexane:ethylacetate=20:1) to obtain 48 mg (0.21 mmol) of a target
compound with a yield of about 95%.
[0169] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.30 (d, J=6.4 Hz,
2H), 8.05 (d, J=8.2 Hz, 1H), 7.67-7.57 (m, 3H), 7.43 (d, J=8.2 Hz,
1H)
Example 5-16
Synthesis of 5-chloro-2-(2-chlorophenyl)oxazolo[5,4-b]pyridine
##STR00017##
[0171] 400 mg (1.5 mmol) of a target compound was obtained with a
yield of about 75% in the same manner as in Example 5-15, except
that 600 mg (2.0 mmol) of
2-chloro-N-(2,6-dichloropyridine-3-yl)benzamide was used as a
starting material.
[0172] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.20 (d, J=8.6 Hz,
1H), 7.99 (d, J=8.2 Hz, 1H), 7.65 (d, J=8.1 Hz, 1H), 7.45-7.33 (m,
3H)
Example 5-17
Synthesis of 5-chloro-2-(3-fluorophenyl)oxazolo[5,4-b]pyridine
##STR00018##
[0174] 130 mg (0.52 mmol) of a target compound was obtained with a
yield of about 51% to about 80% in the same manner as in Example
5-15, except that 300 mg (1.0 mmol) of
N-(2,6-dichloropyridine-3-yl)-3-fluorobenzamide was used as a
starting material.
[0175] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.07 (d, J=8.1 Hz,
2H), 7.97 (d, J=9.1 Hz, 1H), 7.57 (q, J=8.0 Hz, 1H), 7.44 (d, J=8.2
Hz, 1H), 7.36-7.30 (m, 1H)
Example 5-18
Synthesis of 5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine
##STR00019##
[0177] 275 mg (1.03 mmol) of a target compound was obtained with a
yield of about 63% in the same manner as in Example 5-15, except
that 500 mg (1.65 mmol) of
3-chloro-N-(2,6-dichloropyridine-3-yl)benzamide was used as a
starting material.
[0178] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.29 (s, 1H), 8.50
(dd, J=2.9, 4.6 Hz, 1H), 8.06 (d, J=8.2 Hz, 1H), 7.63-7.51 (m, 2H),
7.45 (d, J=8.2 Hz, 1H)
Example 5-19
Synthesis of 5-chloro-2-m-tolyloxazolo[5,4-b]pyridine
##STR00020##
[0180] 130 mg (0.53 mmol) of a target compound was obtained with a
yield of about 76% in the same manner as in Example 5-15, except
that 200 mg (0.7 mmol) of
N-(2,6-dichloropyridine-3-yl)-3-methylbenzamide was used as a
starting material.
[0181] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.07-7.97 (m, 3H),
7.46-7.32 (m, 3H), 2.46 (s, 3H)
Example 5-20
Synthesis of 5-chloro-2-(3-nitrophenyl)oxazolo[5,4-b]pyridine
##STR00021##
[0183] 103 mg (0.37 mmol) of a target compound was obtained with a
yield of about 50% in the same manner as in Example 5-15, except
that 230 mg (0.74 mmol) of
N-(2,6-dichloropyridine-3-yl)-3-nitrobenzamide was used as a
starting material.
[0184] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.9.11 (s, 1H), 8.56
(d, J=5.4 Hz, 1H), 8.44 (d, J=5.1 Hz, 1H), 8.08 (d, J=8.4 Hz, 1H),
7.78 (t, J=8.1 Hz, 1H), 7.45 (d, J=8.4 Hz, 1H)
Example 5-21
Synthesis of
3-(5-chlorooxazolo[5,4-b]pyridine-2-yl)benzonitrile
##STR00022##
[0186] 165 mg (0.65 mmol) of a target compound was obtained with a
yield of about 38% in the same manner as in Example 5-15, except
that 500 mg (1.0 mmol) of
3-cyano-N-(2,6-dichloropyridine-3-yl)benzamide was used as a
starting material.
[0187] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.8.55 (s, 1H), 8.47
(d, J=5.2 Hz, 1H), 8.06 (d, J=8.2 Hz, 1H), 7.87 (d, J=5.1 Hz, 1H),
7.70 (t, J=7.8 Hz, 1H), 7.44 (d, J=8.2 Hz, 1H)
Example 5-22
Synthesis of 5-chloro-2-(4-fluorophenyl)oxazolo[5,4-b]pyridine
##STR00023##
[0189] 250 mg (1.01 mmol) of a target compound was obtained with a
yield of about 73% in the same manner as in Example 5-15, except
that 400 mg (1.37 mmol) of
N-(2,6-dichloropyridine-3-yl)-4-fluorobenzamide was used as a
starting material.
[0190] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.32-8.26 (m, 2H),
8.04 (d, J=8.2 Hz, 1H), 7.43 (d, J=8.2 Hz, 1H), 7.32-7.21 (m,
2H)
Example 5-23
Synthesis of 5-chloro-2-(4-chlorophenyl)oxazolo[5,4-b]pyridine
##STR00024##
[0192] 255 mg (0.96 mmol) of a target compound was obtained with a
yield of about 60% in the same manner as in Example 5-15, except
that 500 mg (1.65 mmol) of
4-chloro-N-(2,6-dichloropyridine-3-yl)benzamide was used as a
starting material.
[0193] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.8.51 (dd, J=1.8,
5.1 Hz, 2H), 8.01 (d, J=8.2 Hz, 1H), 7.54 (dd, J=1.8, 5.2 Hz, 2H),
7.40 (d, J=8.2 Hz, 1H)
Example 5-24
Synthesis of 2-(4-bromophenyl)-5-chlorooxazolo[5,4-b]pyridine
##STR00025##
[0195] 300 mg (0.97 mmol) of a target compound was obtained with a
yield of about 57% in the same manner as in Example 5-15, except
that 600 mg (1.7 mmol) of
4-bromo-N-(2,6-dichloropyridine-3-yl)benzamide was used as a
starting material.
[0196] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.15 (d, J=8.6 Hz,
2H), 8.05 (d, J=8.2 Hz, 1H), 7.74 (d, J=8.6 Hz, 2H), 7.44 (d, J=8.2
Hz, 1H)
Example 5-25
Synthesis of 5-chloro-2-p-tolyloxazolo[5,4-b]pyridine
##STR00026##
[0198] 346 mg (1.4 mmol) of a target compound was obtained with a
yield of about 88% in the same manner as in Example 5-15, except
that 450 mg (1.6 mmol) of
N-(2,6-dichloropyridine-3-yl)-4-methylbenzamide was used as a
starting material.
[0199] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.18 (d, J=8.3 Hz,
2H), 8.02 (d, J=8.2 Hz, 1H), 7.42-7.37 (m, 3H), 2.50 (s, 4H)
Example 5-26
Synthesis of 5-chloro-2-(4-methoxyphenyl)oxazolo[5,4-b]pyridine
##STR00027##
[0201] 434 mg (1.6 mmol) of a target compound was obtained with a
yield of about 72% in the same manner as in Example 5-15, except
that 700 mg (2.3 mmol) of
N-(2,6-dichloropyridine-3-yl)-4-methoxybenzamide was used as a
starting material.
[0202] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.23 (dd, J=2.0,
4.9 Hz, 2H), 7.99 (d, J=8.2 Hz, 1H), 7.39 (d, J=8.2 Hz, 1H), 7.09
(dd, J=2.0, 4.9 Hz, 2H), 3.95 (s, 3H)
Example 5-27
Synthesis of 5-chloro-2-(4-nitrophenyl)oxazolo[5,4-b]pyridine
##STR00028##
[0204] 103 mg (0.4 mmol) of a target compound was obtained with a
yield of about 51% in the same manner as in Example 5-15, except
that 230 mg (0.7 mmol) of
N-(2,6-dichloropyridine-3-yl)-4-nitrobenzamide was used as a
starting material.
[0205] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.47 (s, 4H), 8.13
(d, J=8.3 Hz, 1H), 7.50 (d, J=8.3 Hz, 1H)
Example 5-28
Synthesis of
4-(5-chlorooxazolo[5,4-b]pyridine-2-yl)benzonitrile
##STR00029##
[0207] 197 mg (0.8 mmol) of a target compound was obtained with a
yield of about 45% in the same manner as in Example 5-15, except
that 500 mg (1.7 mmol) of
4-cyano-N-(2,6-dichloropyridine-3-yl)benzamide was used as a
starting material.
[0208] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.41 (d, J=8.3 Hz,
2H), 8.11 (d, J=8.3 Hz, 1H), 7.90 (d, J=8.3 Hz, 2H), 7.49 (d, J=8.3
Hz, 1H)
Example 5-29
Synthesis of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
##STR00030##
[0210] 50 mg (0.2 mmol) of N-(2,6-dichloropyridine-3-yl)benzamide
and 117 mg (0.3 mmol) of Lawesson's reagent were added to 3 ml of
toluene to obtain a mixture, which was then refluxed at about
110.quadrature. for about 5 hours. Termination of the reaction was
confirmed by TLC, and the reaction product was cooled to room
temperature, and then concentrated under reduced pressure. 80 mg
(0.6 mmol) of K2CO3 and 3 ml of dimethylformamide were added into a
resulting concentrated solution to obtain a mixture, which was then
refluxed at about 160.quadrature. for about 3 hours. Termination of
the reaction was confirmed by TLC, and the reaction product was
cooled to room temperature. After a small amount of distilled water
was added into the reaction product, and an acqueous phase was
treated with methylene chloride to get an organic phase. The
organic phase was dried using anhydrous Mg2SO4, filtrated, and then
concentrated under reduced pressure to obtain a concentrated
solution, which was then separated using column chromatography
(hexane:ethylacetate=20:1) to obtain 22 mg (0.1 mmol) of a target
compound with a yield of about 50% to about 70%.
[0211] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.8.22 (d, J=8.5 Hz,
1H), 8.09-8.07 (m, 2H), 7.55-7.50 (m, 3H), 7.45 (d, J=8.5 Hz,
1H)
Example 5-30
Synthesis of
2-phenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine
##STR00031##
[0213] 200 mg (0.9 mmol) of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
and 10 equivalents of pyrrolidine were added to 1 ml of
dimethylformamide to obtain a mixture, which was then refluxed at
about 135.quadrature. for about 4 hours. Termination of the
reaction was confirmed by TLC, and the reaction product was cooled
to room temperature, followed by distillation under reduced
pressure to remove pyrrolidine.
[0214] A saturated NaHCO3 solution was added to the concentrated
solution, and an aqueous phase was treated with methylene chloride
to obtain an organic phase. The organic phase was dried using
anhydrous Na2SO4, filtrated, and concentrated under reduced
pressure to obtain a concentrated solution, which was then
separated using column chromatography (hexane:ethylacetate=20:1) to
obtain 33 mg (0.1 mmol) of a target compound with a yield of about
15%.
[0215] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.23-8.20 (m, 2H),
7.82 (d, J=8.7 Hz, 1H), 7.53-7.49 (m, 3H), 6.40 (d, J=8.7 Hz, 1H),
3.56 (t, J=6.7 Hz, 4H), 2.10-2.06 (m, 4H)
Example 5-31
Synthesis of
2-phenyl-5-(pyrrolidine-1-yl)thiazolo[5,4-b]pyridine
##STR00032##
[0217] 74 mg (0.3 mmol) of a target compound was obtained with a
yield of about 65% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
was used as a starting material.
[0218] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.03 (d, J=8.9 Hz,
3H), 7.50-7.45 (m, 3H), 6.54 (d, J=9.0 Hz, 1H), 3.58 (t, J=6.7 Hz,
4H), 2.10-2.05 (m, 4H)
Example 5-32
Synthesis of
2-chlorophenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine
##STR00033##
[0220] 38 mg (0.13 mmol) of a target compound was obtained with a
yield of about 32% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(2-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0221] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.21-8.16 (m, 1H),
7.91 (d, J=8.7 Hz, 1H), 7.60-7.54 (m, 1H), 7.44-7.38 (m, 2H), 6.4 3
(d, J=8.8 Hz, 1H), 3.58 (s, 4H), 2.08 (s, 4H)
Example 5-33
Synthesis of
2-chlorophenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine
##STR00034##
[0223] 30 mg (0.1 mmol) of a target compound was obtained with a
yield of about 26% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0224] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.22 (s, 1H),
8.09-8.06 (m, 1H), 7.82 (d, J=8.7 Hz, 1H), 7.46-7.44 (m, 2H), 6.42
(d, J=8.7 Hz, 1H), 157 (t, J=6.6 Hz, 4H), 2.11-2.07 (m, 4H)
Example 5-34
Synthesis of
2-fluorophenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine
##STR00035##
[0226] 25 mg (0.1 mmol) of a target compound was obtained with a
yield of about 22% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(4-fluorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0227] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.11 (d, J=8.9 Hz,
2H), 7.90 (d, J=8.1 Hz, 1H), 7.34-7.32 (m, 1H), 6.67 (d, J=8.9 Hz,
2H), 3.44 (t, J=6.6 Hz, 4H), 2.12-2.09 (m, 4H)
Example 5-35
Synthesis of
2-chlorophenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine
##STR00036##
[0229] 40 mg (0.13 mmol) of a target compound was obtained with a
yield of about 34% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(4-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0230] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.16-8.12 (m, 2H),
7.84-7.80 (m, 1H), 7.51-7.47 (m, 2H), 7.30 (s, 1H), 6.44 (m, 1H),
3.57 (d, J=4.4 Hz, 4H), 2.09 (d, J=4.7 Hz, 4H)
Example 5-36
Synthesis of
2-bromophenyl-5-(pyrrolidine-1-yl)oxazolo[5,4-b]pyridine
##STR00037##
[0232] 20 mg (0.06 mmol) of a target compound was obtained with a
yield of about 15% in the same manner as in Example 2-30, except
that 100 mg (0.4 mmol) of
2-(4-bromophenyl)-5-chlorooxazolo[5,4-b]pyridine was used as a
starting material.
[0233] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.06 (d, J=8.6 Hz,
2H), 7.81 (d, J=8.7 Hz, 1H), 7.64 (d, J=8.6 Hz, 2H), 6.40 (d, J=8.7
Hz, 1H), 3.56 (t, J=6.5 Hz, 4H), 2.11-2.06 (m, 4H)
Example 5-37
Synthesis of 2-phenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00038##
[0235] 28 mg (0.1 mmol) of a target compound was obtained with a
yield of about 26% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
and 10 equivalents of piperidine were used as starting
materials.
[0236] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.22-8.16 (m, 2H),
7.80 (d, J=8.7 Hz, 1H), 7.83-7.47 (m, 3H), 6.70 (d, J=8.7 Hz, 1H),
3.63 (s, 4H), 1.68 (s, 6H)
Example 5-38
Synthesis of
2-phenyl-5-(piperidine-1-yl)thiazolo[5,4-b]pyridine
##STR00039##
[0238] 83 mg (0.3 mmol) of a target compound was obtained with a
yield of about 73% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
was used as a starting material.
[0239] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.04 (d, J=9.2 Hz,
3H), 7.51-7.48 (m, 3H), 6.84 (d, J=9.2 Hz, 1H), 3.67 (s, 4H), 1.71
(s, 6H)
Example 5-39
Synthesis of
2-chlorophenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00040##
[0241] 21 mg (0.06 mmol) of a target compound was obtained with a
yield of about 18% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(2-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0242] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.19-8.16 (m, 1H),
7.91 (d, J=8.9 Hz, 1H), 7.59-7.56 (m, 1H), 7.44-7.41 (m, 2H), 6.7 3
(d, J=8.9 Hz, 1H), 3.67 (s, 4H), 1.72 (s, 6H)
Example 5-40
Synthesis of
2-(3-fluorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00041##
[0244] 23 mg (0.1 mmol) of a target compound was obtained with a
yield of about 19% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(3-fluorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0245] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.00 (d, J=7.8 Hz,
1H), 7.90 (d, J=8.0 Hz, 1H), 7.84 (d, J=8.9 Hz, 1H), 7.53-7.18 (m,
1 H0, 7.21 (t, J=5.9 Hz, 1H), 6.73 (d, J=8.9 Hz, 1H), 3.67 (s, 4H),
1.72 (s, 6H)
Example 5-41
Synthesis of
2-chlorophenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00042##
[0247] 67 mg (0.2 mmol) of a target compound was obtained with a
yield of about 56% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0248] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.22 (s, 1H), 8.09
(d, J=6.5 Hz, 1H), 7.84 (d, J=8.8 Hz, 1H), 7.47 (s, 2H), 6.72 (d,
J=8.9 Hz, 1H), 3.67 (s, 4H), 1.72 (s, 6H)
Example 5-42
Synthesis of
5-(piperidine-1-yl)-2-m-tolyloxazolo[5,4-b]pyridine
##STR00043##
[0250] 61 mg (0.2 mmol) of a target compound was obtained with a
yield of about 52% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of 5-chloro-2-m-tolyloxazolo[5,4-b]pyridine
was used as a starting material.
[0251] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.07 (s, 1H), 8.02
(d, J=7.7 Hz, 1H), 7.41 (t, J=7.6 Hz, 1H), 7.32 (d, J=7.6 Hz, 1H),
3.66 (s, 4H), 2.47 (s, 3H), 1.72 (s, 6H)
Example 5-43
Synthesis of
2-nitrophenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00044##
[0253] 39 mg (0.12 mmol) of a target compound was obtained with a
yield of about 30% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(3-nitrophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0254] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.9.06 (s, 1H), 8.51
(d, J=7.8 Hz, 1H), 8.35 (d, J=5.0 Hz, 1H), 7.87 (d, J=8.9 Hz, 1H),
7.71 (t, J=8.0 Hz, 1H), 6.75 (d, J=8.9 Hz, 1H), 3.70 (s, 4H), 1.73
(s, 6H)
Example 5-44
Synthesis of
3-(5-(piperidine-1-yl)oxazolo[5,4-b]pyridine-2-yl)benzonitrile
##STR00045##
[0256] 26 mg (0.09 mmol) of a target compound was obtained with a
yield of about 22% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
3-(5-chlorooxazolo[5,4-b]pyridine-2-yl)benzonitrile was used as a
starting material.
[0257] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.50 (s, 1H), 8.42
(d, J=5.3 Hz, 1H), 7.85 (d, J=8.9 Hz, 1H), 7.78 (d, J=3.56 Hz, 1H),
7.6 5 (t, J=7.9 Hz, 1H), 6.75 (d, J=8.9 Hz, 1H), 3.71 (s, 4H), 1.73
(s, 6H)
Example 5-45
Synthesis of
2-(4-fluorophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00046##
[0259] 20 mg (0.1 mmol) of a target compound was obtained with a
yield of about 11% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(4-fluorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0260] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.11 (d, J=9.1 Hz,
2H), 7.92 (d, J=8.2 Hz, 1H), 7.33 (t, J=8.1 Hz, 1H), 7.36 (d, J=9.1
Hz, 2H), 3.43 (s, 4H), 1.72 (s, 6H)
Example 5-46
Synthesis of
2-chlorophenyl-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00047##
[0262] 30 mg (0.1 mmol) of a target compound was obtained with a
yield of about 17% in the same manner as in Example 5-30, except
that 150 mg (0.6 mmol) of
5-chloro-2-(4-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0263] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.14 (d, J=8.7 Hz,
2H), 7.83 (d, J=8.9 Hz, 1H), 7.50 (d, J=8.7 Hz, 2H), 6.72 (d, J=8.9
Hz, 1H), 3.67 (s, 4H), 1.72 (s, 6H)
Example 5-47
Synthesis of
2-(4-bromophenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00048##
[0265] 40 mg (0.1 mmol) of a target compound was obtained with a
yield of about 23% in the same manner as in Example 2-30, except
that 150 mg (0.5 mmol) of
2-(4-bromophenyl)-5-chlorooxazolo[5,4-b]pyridine was used as a
starting material.
[0266] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.11 (d, J=9.1 Hz,
2H), 7.92 (d, J=8.2 Hz, 1H), 7.33 (t, J=8.1 Hz, 1H), 7.35 (d, J=9.1
Hz, 2H), 3.43 (s, 4H), 1.72 (s, 6H)
Example 5-48
Synthesis of
5-(piperidine-1-yl)-2-p-tolyloxazolo[5,4-b]pyridine
##STR00049##
[0268] 30 mg (0.1 mmol) of a target compound was obtained with a
yield of about 25% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of 5-chloro-2-p-tolyloxazolo[5,4-b]pyridine
was used as a starting material.
[0269] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.11 (d, J=8.2 Hz,
2H), 7.83 (d, J=8.8 Hz, 1H), 7.34 (d, J=8.0 Hz, 2H), 6.70 (d, J=8.8
Hz, 1H), 3.66 (s, 4H), 2.46 (s, 3H), 1.72 (s, 6H)
Example 5-49
Synthesis of
2-(4-methoxyphenyl)-5-(piperidine-1-yl)oxazolo[5,4-b]pyridine
##STR00050##
[0271] 20 mg (0.06 mmol) of a target compound was obtained with a
yield of about 17% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(4-methoxyphenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0272] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.15 (d, J=9.0 Hz,
2H), 7.80 (d, J=8.8 Hz, 1H), 7.04 (dd, J=2.8, 6.9 Hz, 2H), 6.69 (d,
J=8.8 Hz, 1H), 3.91 (s, 3H), 3.64 (s, 4H), 1.71 (s, 6H)
Example 5-50
Synthesis of
4-(5-(piperidine-1-yl)oxazolo[5,4-b]pyridine-2-yl)benzonitrile
##STR00051##
[0274] 18 mg (0.06 mmol) of a target compound was obtained with a
yield of about 15% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
4-(5-chlorooxazolo[5,4-b]pyridine-2-yl)benzonitrile was used as a
starting material.
[0275] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.30 (d, J=8.5 Hz,
2H), 7.83 (q, J=8.9 Hz, 3H), 6.75 (d, J=8.9 Hz, 1H), 3.70 (s, 4H),
1.73 (s, 6H)
Example 5-51
Synthesis of
2-(3-chlorophenyl)-N-cyclopentyloxazolo[5,4-b]pyridine-5-amine
##STR00052##
[0277] 18 mg (0.06 mmol) of a target compound was obtained with a
yield of about 15% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine and 10
equivalents of cyclopentylamine were used as starting
materials.
[0278] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.21 (s, 1H),
8.10-8.06 (m, 1H), 7.80 (d, J=8.6 Hz, 1H), 7.50-7.45 (m, 2H), 6.44
(d, J=8.6 Hz, 1H), 4.75 (d, J=6.2 Hz, 1H), 4.25-4.19 (m, 1H),
2.21-2.11 (m, 2H), 1.83-1.68 (m, 4H), 1.58-1.50 (m, 2H)
Example 5-52
Synthesis of
N-cyclohexyl-2-phenylthiazolo[5,4-b]pyridine-5-amine
##STR00053##
[0280] 10 mg (0.03 mmol) of a target compound was obtained with a
yield of about 9% in the same manner as in Example 5-30, except
that 99 mg (0.4 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
and 10 equivalents of cyclopentylamine were used as starting
materials.
[0281] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.03-7.99 (m, 3H),
7.48 (s, 3H), 6.53 (d, J=8.9 Hz, 1H), 4.68 (d, J=6.4 Hz, 1H), 3.75
(t, J=3.4 Hz, 1H), 2.13 (d, J=12.1 Hz, 2H), 1.84-1.80 (m, 2H),
1.73-1.69 (m, 1H), 1.54-1.42 (m, 2 H), 1.33-1.22 (m, 3H)
Example 5-53
Synthesis of
2-(3-chlorophenyl)-N-cyclohexyloxazolo[5,4-b]pyridine-5-amine
##STR00054##
[0283] 24 mg (0.07 mmol) of a target compound was obtained with a
yield of about 19% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0284] .sup.1H NMR (300 MHz, MeOD) .delta. 8.07 (s, 1H), 8.03-8.00
(m, 1H0, 7.68 (d, J=8.7 Hz, 1H), 7.54-7.51 (m, 2H), 6.53 (d, J=8.7
Hz, 1H), 3.79-3.75 (m, 1H), 2.06 (d, J=9.7 Hz, 2H), 1.82 (d, J=6.2
Hz, 2H), 1.70 (d, J=3.6 Hz, 1H), 1.54-1.40 (m, 2H), 1.33-1.25 (m,
3H)
Example 5-54
Synthesis of 5-(azepane-1-yl)-2-phenyloxazolo[5,4-b]pyridine
##STR00055##
[0286] 70 mg (0.2 mmol) of a target compound was obtained with a
yield of about 57% in the same manner as in Example 5-30, except
that 97 mg (0.4 mmol) of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
and 10 equivalents of hexamethyleneimine were used as starting
materials.
[0287] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.24-8.20 (m, 2H),
7.82 (d, J=8.8 Hzm1H), 7.54-7.50 (m, 3H), 6.54 (d, J=8.9 Hz, 1H),
3.74 (t, J=5.9 Hz, 4H), 1.88 (s, 4H), 1.63-1.59 (m, 4H)
Example 5-55
Synthesis of 5-(azepane-1-yl)-2-phenylthiazolo[5,4-b]pyridine
##STR00056##
[0289] 51 mg (0.2 mmol) of a target compound was obtained with a
yield of about 47% in the same manner as in Example 5-30, except
that 85 mg (0.35 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
was used as a starting material.
[0290] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.05-8.00 (m, 3H),
7.50-7.48 (m, 3H), 6.68 (d, J=9.2 Hz, 1H), 3.75 (t, J=5.9 Hz, 4H),
1.87 (s, 4H), 1.63-1.59 (m, 4H)
Example 5-56
Synthesis of
5-(azepane-1-yl)-2-(4-fluorophenyl)oxazolo[5,4-b]pyridine
##STR00057##
[0292] 29 mg (0.1 mmol) of a target compound was obtained with a
yield of about 23% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(4-fluorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0293] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.23-8.18 (m, 2H),
7.80 (d, J=8.8 Hz, 1H), 7.20 (t, J=8.7 Hz, 2H), 6.54 (d, J=8.9 Hz,
1H 0, 3.73 (t, J=5.9 Hz, 4H), 1.88 (s, 4H), 1.63-1.60 (m, 4H)
Example 5-57
Synthesis of N-benzyl-2-phenyloxazolo[5,4-b]pyridine-5-amine
##STR00058##
[0295] 30 mg (0.1 mmol) of a target compound was obtained with a
yield of about 25% in the same manner as in Example 5-30, except
that 97 mg (0.4 mmol) of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
and 10 equivalents of benzylamine were used as starting
materials.
[0296] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.24-8.21 (m, 2H),
7.82 (d, J=8.6 Hz, 1H), 7.55-7.51 (m, 3H), 7.46-7.31 (m, 5H), 6.4 6
(d, J=8.6 Hz, 1H), 5.04 (s, 1H), 4.67 (d, J=5.7 Hz, 2H)
Example 5-58
Synthesis of N-benzyl-2-phenylthiazolo[5,4-b]pyridine-5-amine
##STR00059##
[0298] 49 mg (0.2 mmol) of a target compound was obtained with a
yield of about 65% in the same manner as in Example 5-30, except
that 60 mg (0.24 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
was used as a starting material.
[0299] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.06-8.00 (m, 3H),
7.54-7.31 (m, 8H), 6.57 (d, J=8.9 Hz, 1H), 5.10 (s, 1H), 4.67 (d,
J=5.7 Hz, 2H)
Example 5-59
Synthesis of
N-benzyl-2-(3-fluorophenyl)oxazolo[5,4-b]pyridine-5-amine
##STR00060##
[0301] 30 mg (0.1 mmol) of a target compound was obtained with a
yield of about 35% in the same manner as in Example 5-30, except
that 70 mg (0.3 mmol) of
5-chloro-2-(3-fluorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0302] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.00 (d, J=7.8 Hz,
1H), 7.93-7.88 (m, 1H), 7.82 (d, J=8.6 Hz, 1H), 7.54-7.32 (m, 6H),
7.26-7.19 (m, 1H), 6.48 (d, J=8.6 Hz, 1H), 5.07 (s, 1H), 4.67 (d,
J=5.8 Hz, 2H)
Example 5-60
Synthesis of
N-benzyl-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine-5-amine
##STR00061##
[0304] 20 mg (0.1 mmol) of a target compound was obtained with a
yield of about 23% in the same manner as in Example 5-30, except
that 70 mg (0.3 mmol) of
5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0305] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.22 (s, 1H),
8.10-8.08 (m, 1H), 7.81 (d, J=8.6 Hz, 1H), 7.51-7.34 (m, 7H), 6.48
(d, J=8.6 Hz, 1H), 5.08 (s, 1H), 4.67 (d, J=5.6 Hz, 2H)
Example 5-61
Synthesis of
N-benzyl-2-(4-chlorophenyl)oxazolo[5,4-b]pyridine-5-amine
##STR00062##
[0307] 29 mg (0.09 mmol) of a target compound was obtained with a
yield of about 32% in the same manner as in Example 5-30, except
that 70 mg (0.3 mmol) of
5-chloro-2-(4-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0308] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.15 (d, J=8.8 Hz,
2H), 7.81 (d, J=8.6 Hz, 1H), 7.51 (d, J=8.8 Hz, 2H), 7.46-7.30 (m,
5H), 6.47 (d, J=8.6 Hz, 1H), 5.06 (s, 1H0, 4.67 (d, J=5.7 Hz,
2H)
Example 5-62
Synthesis of
N-(2-morpholinoethyl)-2-phenylthiazolo[5,4-b]pyridine-5-amine
##STR00063##
[0310] 31 mg (0.1 mmol) of a target compound was obtained with a
yield of about 50% in the same manner as in Example 5-30, except
that 50 mg (0.2 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
and 3 equivalents of 2-morpholinoethanamine were used as starting
materials.
[0311] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.05-8.00 (m, 3H),
7.54-7.47 (m, 3H), 6.59 (d, J=8.9 Hz, 1H), 5.38 (s, 1H), 3.78 (t,
J=4.6 Hz, 4H), 3.52 (q, J=5.1 Hz, 2H), 2.69 (t, J=6.0 Hz, 2H), 2.55
(t, J=4.4 Hz, 4H)
Example 5-63
Synthesis of
N-benzyl-N-methyl-2-phenyloxazolo[5,4-b]pyridine-5-amine
##STR00064##
[0313] 218 mg (0.7 mmol) of a target compound was obtained with a
yield of about 80% in the same manner as in Example 5-30, except
that 200 mg (0.9 mmol) of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
and 5 equivalents of N-benzylmethylamine were used as starting
materials.
[0314] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.25-8.21 (m, 2H),
7.85 (d, J=8.8 Hz, 1H), 7.54-7.50 (m, 3H), 7.39-7.27 (m, 5H), 6.5 8
(d, J=8.8 Hz, 1H), 4.91 (s, 2H), 3.21 (s, 3H)
Example 5-64
Synthesis of
N-benzyl-N-methyl-2-phenylthiazole[5,4-b]pyridine-5-amine
##STR00065##
[0316] 100 mg (0.3 mmol) of a target compound was obtained with a
yield of about 72% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of 5-chloro-2-phenylthiazolo[5,4-b]pyridine
was used as a starting material.
[0317] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.05 (d, J=9.1 Hz,
3H), 7.54-7.47 (m, 3H), 7.40-7.29 (m, 5H), 6.71 (d, J=9.1 Hz, 1H),
4.93 (s, 2H), 3.21 (s, 3H)
Example 5-65
Synthesis of
N-benzyl-2-(2-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00066##
[0319] 70 mg (0.2 mmol) of a target compound was obtained with a
yield of about 67% in the same manner as in Example 5-30, except
that 80 mg (0.3 mmol) of
5-chloro-2-(2-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0320] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.19-8.16 (m, 1H),
7.92 (d, J=8.8 Hz, 1H), 7.60-7.57 (m, 1H), 7.46-7.28 (m, 7H), 6.6 0
(d, J=8.9 Hz, 1H), 4.92 (s, 2H), 3.22 (s, 3H)
Example 5-66
Synthesis of
N-benzyl-2-(3-fluorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00067##
[0322] 118 mg (0.4 mmol) of a target compound was obtained with a
yield of about 98% in the same manner as in Example 5-30, except
that 90 mg (0.4 mmol) of
5-chloro-2-(3-fluorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0323] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.01 (d, J=7.8 Hz,
1H), 7.93 (d, J=5.6 Hz, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.53-7.46 (m,
1 H), 7.40-7.28 (m, 5H), 7.24-7.18 (m, 1H), 6.59 (d, J=8.8 Hz, 1H),
4.91 (s, 2H), 3.21 (s, 3H)
Example 5-67
Synthesis of
N-benzyl-2-(3-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00068##
[0325] 57 mg (0.2 mmol) of a target compound was obtained with a
yield of about 65% in the same manner as in Example 5-30, except
that 80 mg (0.3 mmol) of
5-chloro-2-(3-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0326] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.24-8.22 (m, 1H),
8.11-8.08 (m, 1H), 7.85 (d, J=8.8 Hz, 1H), 7.48-7.46 (m, 2H),
7.40-7.35 (m, 2H), 7.35-7.28 (m, 4H), 6.59 (d, J=8.9 Hz, 1H), 4.91
(s, 2H), 3.22 (s, 3H)
Example 5-68
Synthesis of
N-benzyl-2-(4-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00069##
[0328] 121 mg (0.3 mmol) of a target compound was obtained with a
yield of about 92% in the same manner as in Example 5-30, except
that 100 mg (0.4 mmol) of
5-chloro-2-(4-chlorophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0329] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.15 (d, J=8.6 Hz,
2H), 7.83 (d, J=8.8 Hz, 1H), 7.50 (d, J=8.6 Hz, 2H), 7.40-7.28 (m,
5H), 6.58 (d, J=8.8 Hz, 1H), 4.90 (s, 2H), 3.21 (s, 3H)
Example 5-69
Synthesis of
N-benzyl-N-methyl-2-p-tolyloxazolo[5,4-b]pyridine-5-amine
##STR00070##
[0331] 130 mg (60%) of a target compound was obtained with a yield
of about 60% in the same manner as in Example 5-30, except that 172
mg (0.7 mmol) of 5-chloro-2-p-tolyloxazolo[5,4-b]pyridine was used
as a starting material.
[0332] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.12 (d, J=8.2 Hz,
2H), 7.83 (d, J=8.7 Hz, 1H), 7.39-7.28 (m, 7H), 6.56 (d, J=8.8 Hz,
1H), 4.90 (s, 2H), 3.21 (s, 3H), 2.46 (s, 3H)
Example 5-70
Synthesis of
N-benzyl-2-(4-methoxyphenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00071##
[0334] 67 mg (0.2 mmol) of a target compound was obtained with a
yield of about 57% in the same manner as in Example 5-30, except
that 88 mg (0.3 mmol) of
5-chloro-2-(4-methoxyphenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0335] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.17 (d, J=9.0 Hz,
2H0, 7.81 (d, J=8.7 Hz, 1H), 7.37-7.28 (m, 5H), 7.04 (d, J=6.9 Hz,
2 H), 6.55 (d, J=8.8 Hz, 1H), 4.90 (s, 2H), 3.92 (s, 3H), 3.20 (s,
3H)
Example 5-71
Synthesis of
N-benzyl-N-methyl-2-(4-nitrophenyl)oxazolo[5,4-b]pyridine-5-amine
##STR00072##
[0337] 65 mg (0.2 mmol) of a target compound was obtained with a
yield of about 99% in the same manner as in Example 5-30, except
that 45 mg (0.2 mmol) of
5-chloro-2-(4-nitrophenyl)oxazolo[5,4-b]pyridine was used as a
starting material.
[0338] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.36 (s, 4H), 7.88
(d, J=8.9 Hz, 1H), 7.40-7.26 (m, 5H), 6.63 (d, J=8.9 Hz, 1H), 4.92
(s, 2H), 3.24 (s, 3H)
Example 5-72
Synthesis of
4-(5-benzyl(methyl)amino)oxazolo[5,4-b]pyridine-2-yl)benzonitrile
##STR00073##
[0340] 61 mg (0.2 mmol) of a target compound was obtained with a
yield of about 71% in the same manner as in Example 5-30, except
that 63 mg (0.3 mmol) of
4-(5-chlorooxazolo[5,4-b]pyridine-2-yl)benzonitrile was used as a
starting material.
[0341] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.30 (d, J=8.4 Hz,
2H0, 7.88-7.28 (m, 3H), 7.40-7.28 (m, 5H), 6.62 (d, J=8.9 Hz, 1H),
4.92 (s, 2H), 3.23 (s, 3H)
Example 5-73
Synthesis of N-methyl-2-phenyloxazolo[5,4-b]pyridine-5-amine
##STR00074##
[0343] 50 mg (0.16 mmol) of
N-benzyl-N-methyl-2-phenyloxazolo[5,4-b]pyridine-5-amine and 0.25
ml of 95% H.sub.2SO.sub.4 were mixed together by stirring for about
12 hours. Termination of the reaction was confirmed by TLC, and the
reaction product was cooled to room temperature, followed by
dropwise addition of 1.25 ml of H2O. The reaction product was
titrated using a 15% NaOH solution to a pH 4. After a resulting
solid was filtrated, a resulting filtrate was titrated to a pH 10.
An aqueous phase was separated with methylene chloride, followed by
distillation under reduced pressure to obtain 25 mg (0.1 mmol) of a
target compound with a yield of about 69%.
[0344] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.24-8.20 (m, 2H0,
7.83 (d, J=8.6 Hz, 1H), 7.55-7.51 (m, 3H), 6.46 (d, J=8.6 Hz, 1H0,
4.75 (s, 1H), 3.07 (d, J=5.1 Hz, 3H)
Example 5-74
Synthesis of N-methyl-2-phenylthiazolo[5,4-b]pyridine-5-amine
##STR00075##
[0346] 47 mg (0.2 mmol) of a target compound was obtained with a
yield of about 92% in the same manner as in Example 5-73, except
that 70 mg (0.2 mmol) of
N-benzyl-N-methyl-2-phenylthiazolo[5,4-b]pyridine-5-amine was used
as a starting material.
[0347] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.05-8.01 (m, 3H),
7.52-7.48 (m, 3H), 6.56 (d, J=8.9 Hz, 1H), 4.87 (s, 1H), 3.06 (d,
J=5.1 Hz, 3H)
Example 5-75
Synthesis of
2-(3-fluorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00076##
[0349] 31 mg (0.1 mmol) of a target compound was obtained with a
yield of about 58% in the same manner as in Example 5-73, except
that 70 mg (0.2 mmol) of
N-benzyl-2-(3-fluorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-ami-
ne was used as a starting material.
[0350] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.00 (d, J=5.4 Hz,
1H), 7.90 (d, J=5.6 Hz, 1H), 7.83 (d, J=8.6 Hz, 1H), 7.54-7.47 (m,
1H), 7.25-7.18 (m, 1H), 6.47 (d, J=8.6 Hz, 1H), 4.79 (s, 1H), 3.07
(d, J=5.1 Hz, 3H)
Example 5-76
Synthesis of
2-(3-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00077##
[0352] 30 mg (0.1 mmol) of a target compound was obtained with a
yield of about 72% in the same manner as in Example 5-73, except
that 57 mg (0.2 mmol) of
N-benzyl-2-(3-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-ami-
ne was used as a starting material.
[0353] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.22 (s, 1H),
8.10-8.07 (m, 1H), 7.82 (d, J=8.6 Hz, 1H), 7.50-7.43 (m, 2H), 6.47
(d, J=8.6 Hz, 1H), 4.79 (s, 1H), 3.08 (d, J=5.1 Hz, 3H)
Example 5-77
Synthesis of
2-(4-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00078##
[0355] 43 mg (0.2 mmol) of a target compound was obtained with a
yield of about 83% in the same manner as in Example 5-73, except
that 70 mg (0.2 mmol) of
N-benzyl-2-(4-chlorophenyl)-N-methyloxazolo[5,4-b]pyridine-5-ami-
ne was used as a starting material.
[0356] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.13 (d, J=8.6 Hz,
2H), 7.80 (d, J=8.6 Hz, 1H), 7.50 (d, J=8.6 Hz, 2H), 6.45 (d, J=8.6
Hz, 1H), 4.78 (s, 1H), 3.06 (d, J=5.1 Hz, 3H)
Example 5-78
Synthesis of N-methyl-2-p-tolyloxazolo[5,4-b]pyridine-5-amine
##STR00079##
[0358] 21 mg (0.09 mmol) of a target compound was obtained with a
yield of about 75% in the same manner as in Example 5-73, except
that 40 mg (0.12 mmol) of
N-benzyl-N-methyl-2-p-tolyloxazolo[5,4-b]pyridine-5-amine was used
as a starting material.
[0359] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.09 (d, J=7.6 Hz,
2H), 7.79 (d, J=8.5 Hz, 1H), 7.32 (d, J=7.8 Hz, 2H), 6.42 (d, J=8.4
Hz, 1H), 3.04 (s, 3H), 2.44 (s, 3H)
Example 5-79
Synthesis of
2-(4-methoxyphenyl)-N-methyloxazolo[5,4-b]pyridine-5-amine
##STR00080##
[0361] 18 mg (0.07 mmol) of a target compound was obtained with a
yield of about 60% in the same manner as in Example 5-73, except
that 40 mg (0.12 mmol) of
N-benzyl-2-(4-methoxyphenyl)-N-methyloxazolo[5,4-b]pyridine-5-am-
ine was used as a starting material.
[0362] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.8.14 (d, J=9.0 Hz,
2H), 7.77 (d, J=8.5 Hz, 1H), 7.03 (d, J=9.0 Hz, 2H), 6.42 (d, J=8.6
Hz, 1H), 3.90 (s, 3H), 3.04 (d, J=5.1 Hz, 3H)
Example 5-80
Synthesis of
N-cyclohexyl-2-phenyloxazolo[5,6-b]pyridine-5-amine
##STR00081##
[0364] 20 mg (0.07 mmol) of a target compound was obtained with a
yield of about 34% in the same manner as in Example 5-30, except
that 50 mg (0.2 mmol) of 5-chloro-2-phenyloxazolo[5,4-b]pyridine
and 10 equivalents of cyclopentylamine were used as starting
materials.
[0365] .sup.1H NMR (300 MHz, MeOD) .delta. 8.12-8.11 (m, 2H), 7.70
(d, J=8.7 Hz, 1H), 7.57-7.53 (m, 3H), 6.54 (d, J=8.7 Hz, 1H), 3.79
(s, 1H), 2.08 (d, J=12.2 Hz, 2H), 1.83 (dd, J=3.9, 5.7 Hz, 2H),
1.71 (d, J=13.2 Hz, 1H), 1.52-1.42 (m, 2H), 1.30 (t, J=12.0 Hz,
3H)
Example 6
Screening of MAO-B's activity inhibitory material
[0366] Whether the compounds of Example 5 inhibit activity of MAO-B
was tested.
[0367] A stock solution was prepared using a human MAO-B enzyme
(purchased from Aldrich) and a Amplex.RTM. Red monoamine oxidase
assay kit according to a preparation manual. The kit includes a
5.times. reaction buffer, an Amplex.RTM. red reagent (1 mg), HRP,
DMSO, H.sub.2O.sub.2, p-tyramine (substrate of MAO-A, B),
benzylamine (substrate of MAO-B), clorgiline (inhibitor of MAO-A),
and pargyline (inhibitor of MAO-B). Among these reagents in the
kit, benzylamine was used as a substrate for MAO-B, and pargyline
was used as an MAO-B inhibitor. A solution as overall substrates
was prepared as follows. 200 ul of a solution of 1 mg of
Amplex.RTM. red sufficiently dissolved in 200 ul of DMSO, 100 ul of
a mixed solution of HRP and 1 ml of a 1.times. buffer, 200 ul of a
solution of benzylamine dissolved in 1.2 ml of dH.sub.2O were added
to 9.5 ml of a 1.times. buffer to reach a total volume of 10 mL,
which is sufficient for 100 wells. 0.5 ul of a mixture of MAO-B
inhibitor pargyline and 1 ml of dH.sub.2O was put into each well.
First, the activity of MAO-B was determined using 10 uM of the
synthesized compound.
[0368] 96 wells were injected with positive and negative types, and
the wile type. The positive type included only substrate and
hydrogen peroxide, and the negative type included only substrate.
For the wild type, corresponding wells were injected with the
enzyme, substrate, and MAO-B inhibitor, but with no synthesized
compound. Afterward, 2 ul of the synthesized compound (1 mM) was
added into each well, and the human MAO-B enzyme was put only into
the 1.sup.st row of wells. 0.5 ug of the human MAO-B was put into
each well along with 100 ul of a 1.times. buffer. The human MAO-B
enzyme was put into the 2.sup.nd row of the wells along with 0.5 ul
of a pargyline, the MAO-B inhibitor. To reduce an experimental
error for accuracy, the test was repeated three times for each
compound. After 30 minutes, 100 ul of the substrate solution was
added into each well in a darkroom. The test was performed in the
darkroom due to light sensitivity of the Amplex.RTM. reagent.
Finally, a total volume of the reaction solution per well reached
200 ul. After about 2 to 3 hours, chromophoric degrees of the
samples were measured. A variation in data values for the 1.sup.st
and 2.sup.nd rows of the wells indicates the pure reaction activity
of the MAO-B enzyme with the substrate. Using the samples with the
synthesized compound the remaining activity of MAO-B after
inhibited by the synthesized compound may be determined. This is
because the activities of the other enzymes excluding the MAO-B
enzyme may be excluded through this method. Compounds with high
inhibitory activity at a concentration of 10 uM were screened from
among the synthesized compounds at a compound concentration of 10
uM. Afterward, concentration-dependent IC.sub.50 values of these
compounds may be obtained through an activity assay at different
concentrations of 0.001 uM, 0.01 uM, 0.1 uM, 1 uM, and 10 uM
[0369] The results of the test on whether the compounds of Example
5 inhibited the activity of MAO-B are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Compound Remaining activity of MAO-B (%)
IC.sub.50(.mu.M) Example 5-30 2.31 0.41 Example 5-31 1.7 7.3
Example 5-32 7.7 111.9 Example 5-33 14.2 16.3 Example 5-34 8.4 8.7
Example 5-35 42.4 -- Example 5-36 33.5 -- Example 5-37 3.09 2
Example 5-38 1.8 8.3 Example 5-39 0.6 13.2 Example 5-40 13.8 0.27
Example 5-41 17 38.4 Example 5-42 -7.8 -- Example 5-43 -21.5 --
Example 5-44 10.1 -- Example 5-45 26.5 10.1 Example 5-46 56.0 --
Example 5-47 28.6 0.096 Example 5-48 46.2 -- Example 5-49 15.8 1.52
Example 5-50 71.2 -- Example 5-51 37.2 -- Example 5-52 43 --
Example 5-53 39.3 -- Example 5-54 57.5 -- Example 5-55 -1.8 --
Example 5-56 61.4 -- Example 5-57 63.1 -- Example 5-58 60.8 1.9
.times. 10.sup.3 Example 5-59 51.9 -- Example 5-60 55.4 -- Example
5-61 46.4 -- Example 5-62 42.7 -- Example 5-63 53.2 5.2 .times.
10.sup.4 Example 5-64 47.8 -- Example 5-65 84.1 -- Example 5-66 43
-- Example 5-67 47.3 68.2 Example 5-68 24.1 5.08 Example 5-69 84.1
-- Example 5-70 52.4 -- Example 5-71 101.2 -- Example 5-72 108.8 --
Example 5-73 51 -- Example 5-74 27.2 15.7 Example 5-75 22 9.86
Example 5-76 4.4 2.99 Example 5-77 4.14 3.02 Example 5-78 45.8 --
Example 5-79 84.5 -- Example 5-80 11.1 8.72 Selegiline 0.1 9.7
[0370] As described above, a method according to the one or more
embodiments of the present disclosure may effectively screen a
prophylactic or therapeutic candidate material for preventing or
treating a degenerative brain disease. A variety of degenerative
brain diseases may be effectively prevented or treated with
pharmaceutical composition including the screened prophylactic or
therapeutic candidate material.
[0371] While the present disclosure has been particularly shown and
described with reference to exemplary embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the present disclosure as defined by
the following claims.
* * * * *
References