U.S. patent application number 16/122116 was filed with the patent office on 2019-03-07 for pentacyclic compound.
This patent application is currently assigned to Eisai R&D Management Co., Ltd.. The applicant listed for this patent is Eisai R&D Management Co., Ltd.. Invention is credited to Koji Hagiwara, Tamaki Hoshikawa, Yoshihisa Kobayashi, Yoshihiko Norimine, Yoshiaki Ohashi, Nobuhiro Sato, Yu Yoshida.
Application Number | 20190071452 16/122116 |
Document ID | / |
Family ID | 65517037 |
Filed Date | 2019-03-07 |
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United States Patent
Application |
20190071452 |
Kind Code |
A1 |
Ohashi; Yoshiaki ; et
al. |
March 7, 2019 |
PENTACYCLIC COMPOUND
Abstract
The present invention provides compounds represented by formulas
(I) to (VI) or pharmaceutically acceptable salts thereof.
##STR00001##
Inventors: |
Ohashi; Yoshiaki; (Tsukuba,
JP) ; Norimine; Yoshihiko; (Tsukuba, JP) ;
Hoshikawa; Tamaki; (Tsukuba, JP) ; Yoshida; Yu;
(Tsukuba, JP) ; Kobayashi; Yoshihisa; (Tokyo,
JP) ; Sato; Nobuhiro; (Tsukuba, JP) ;
Hagiwara; Koji; (Tsukuba, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Eisai R&D Management Co., Ltd. |
Tokyo |
|
JP |
|
|
Assignee: |
Eisai R&D Management Co.,
Ltd.
Tokyo
JP
|
Family ID: |
65517037 |
Appl. No.: |
16/122116 |
Filed: |
September 5, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 495/22 20130101;
A61P 25/16 20180101; A61P 25/28 20180101 |
International
Class: |
C07D 495/22 20060101
C07D495/22; A61P 25/28 20060101 A61P025/28; A61P 25/16 20060101
A61P025/16 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 7, 2017 |
JP |
2017-172169 |
Claims
1. A compound selected from the group consisting of:
3-fluoro-6,11-dimethyl-6,7,10,11,12,13-hexahydrobenzo[f]pyrido[4'',3'':4'-
,5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-5,14-dione:
##STR00027##
5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4-
,5]pyrimido[1,2-a]thieno[2,3-f][1,4]diazepine-4,13-dione:
##STR00028##
5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4-
,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione:
##STR00029##
(3aS,14aR)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,-
14aH)-dione: ##STR00030##
(3aR,14aR)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,-
14aH)-dione: ##STR00031## and
(3aS,14aS)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,
14aH)-dione: ##STR00032## or a pharmaceutically acceptable salt
thereof.
2.
3-Fluoro-6,11-dimethyl-6,7,10,11,12,13-hexahydrobenzo[t]pyrido[4'',3''-
:4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-5,14-dione or
a pharmaceutically acceptable salt thereof according to claim 1:
##STR00033##
3.
5,10-Dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3-
':4,5]pyrimido[,2-a]thieno[2,3-f][1,4]diazepine-4,13-dione or a
pharmaceutically acceptable salt thereof according to claim 1:
##STR00034##
4.
5,10-Dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3-
':4,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione or a
pharmaceutically acceptable salt thereof according to claim 1:
##STR00035##
5.
(3aS,14aR)-5,10-Dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f-
]pyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(-
2H,14aH)-dione or a pharmaceutically acceptable salt thereof
according to claim 1: ##STR00036##
6.
(3aR,14aR)-5,10-Dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f-
]pyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(-
2H,14aH)-dione or a pharmaceutically acceptable salt thereof
according to claim 1: ##STR00037##
7.
(3aS,14aS)-5,10-Dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f-
]pyrido[44'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13-
(2H,14aH)-dione or a pharmaceutically acceptable salt thereof
according to claim 1: ##STR00038##
8. A pharmaceutical composition comprising the compound or
pharmaceutically acceptable salt thereof according to claim 1 and
one or more pharmaceutically acceptable additives.
9. A method of treating a disease selected from a group consisting
of Alzheimer's disease, Dementia with Lewy bodies and Parkinson
disease with dementia comprising administering the compound or
pharmaceutically acceptable salt thereof according to claim 1 to a
patient in need thereof.
10. The method according to claim 9 wherein the disease is
Alzheimer's disease.
11. The method according to claim 9 wherein the disease is Dementia
with Lewy bodies.
12. The method according to claim 9 wherein the disease is
Parkinson disease with dementia.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of Japanese patent
application No. 2017-172169 filed on Sep. 7, 2017, the disclosure
of which is herein incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates to a pentacyclic compound or a
pharmaceutically acceptable salt thereof having cholinergic neuron
activation and/or neuroprotective effect, and pharmaceutical use
thereof. The present invention also relates to pharmaceutical
compositions comprising the above compound as an active
ingredient.
BACKGROUND
[0003] Cholinergic neurons which release acetylcholine as a
transmitter are widely projected in the forebrain from the nucleus
basalis of Meynert and the septal nucleus of the basal forebrain to
the hippocampus, amygdala, and cerebral cortex, and are involved in
the modulation of memory, learning, cognition, and attention
(Non-Patent Literature 1). Moreover, cholinergic neurons in the
pedunculopontine tegmental nucleus and laterodorsal tegmental
nucleus of the brain stem are projected in the striatum, accumbens
nucleus, substantia nigra, and thalamus, and are considered to be
involved in the control of motivation and vigilance (Non-Patent
Literatures 2 to 4).
[0004] In particular, the role of cholinergic neurons in the basal
forebrain has been more clarified by analysis using many animal
models such as lesion model. Especially, the correlation between
functional disorder of cholinergic neurons and decreased memory and
learning has been shown in the animal models (Non-Patent
Literatures 5 to 7), and it has been shown that cognitive
performance is improved by increasing the amount of acetylcholine
using a cholinesterase inhibitor, and enhancing the function of
cholinergic neurons (Non-Patent Literatures 8 to 12).
[0005] It has been reported that Nerve Growth Factor (NGF) shows
the neuroprotective effect on cholinergic neurons in the animal
model indicating loss of cholinergic neurons. (Non-Patent
Literature 13 to 15).
[0006] Particularly for Alzheimer's disease (AD), loss of
cholinergic neurons is found from early stage of AD and is one of
the pathological features of AD. Accumulation of senile plaques by
deposits of amyloid beta and neurofibrillary tangles by tau protein
aggregation are also pathological features of AD, and particularly
neurofibrillary tangles are known to increase with the progress of
the disease status and bring neuronal death. Neurofibrillary
tangles are found in nucleus basalis of Meynert and entorhinal
cortex from the early stage of AD. Among them, it is reported that
loss of cholinergic neurons in nucleus basalis of Meynert by tau
protein aggregation is found at earlier stage and that there is a
correlation between the loss and a decrease in cognitive function
score (Non-Patent Literatures 16 and 17). Similarly to AD,
hyperphosphorylation and abnormal accumulation of tau protein is
found in genetically modified mice having a P301S mutation which
has been found in familial frontotemporal dementia (human tau P301S
transgenic mice). Consequently, neurofibrillary tangles, a
pathological feature of AD, are formed (Non-Patent Literature 18)
and bring cognitive dysfunction by synaptic impairment,
neurodegeneration and loss of neurons. Based on these findings,
human tau P301S transgenic mice are widely used as AD-like animal
models (Non-Patent Literatures 19-22), and improvement of cognitive
decline and suppression of disease status progress in Alzheimer's
disease can be expected by suppressing AD-like pathological changes
in human tau P301S transgenic mice.
[0007] Furthermore, multiple analyses using genetically modified
mice and animal models of disorders suggest that axonal transport
deficit is one of the causes of loss of cholinergic neurons
(Non-Patent Literatures 23-25).
[0008] Among them, the axon of cholinergic neurons which projects
from septal area to hippocampus is impaired in a fimbria-fornix
lesioned model and impairment of retrograde transport of molecules
involved with survival and function brings loss of neurons
(Non-Patent Literatures 26-28). The impairment of retrograde
transport is found also in genetically modified mice (Non-Patent
Literatures 23 and 24) and loss of cholinergic neurons by
fimbria-fornix lesion reflects one aspect of the disease status.
Accordingly, improvement of cognitive decline and suppression of
disease status progress in Alzheimer's disease can be expected by
suppression or improvement of loss of cholinergic neurons in this
model of the disorder.
[0009] Dementia with Lewy bodies (DLB) and Parkinson disease (PD)
are progressive neurodegenerative disorders in which abnormal
inclusion bodies (Lewy bodies) mainly composed of alpha synuclein
appear in neurons and bring degeneration and loss of neurons.
Cognitive dysfunction develops if Lewy bodies are mainly
distributed in cerebral cortex and Parkinsonism develops if Lewy
bodies are mainly distributed in brain stem. In addition to that,
psychiatric symptoms such as visual hallucination, hallucination
and delusion, sleep disorder and autonomic symptoms also develop.
The diagnosis is dementia with Lewy bodies if dementia appears
before or within one year from the onset of Parkinsonism and the
diagnosis is Parkinson disease with dementia (PDD) if Parkinsonism
has appeared before one year or more from the onset of dementia.
Dementia with Lewy bodies, Parkinson disease with dementia and
Parkinson disease are pathologically same diseases and
comprehensively referred to as Lewy body disease (LBD) though these
are different in cognitive dysfunction and appearance order and
degree of Parkinsonism. In dementia with Lewy bodies and Parkinson
disease with dementia, similarly to Alzheimer's disease, neurons of
nucleus basalis of Meynert, a nuclei of origin of cholinergic
nerve, are degenerated and lost and it is reported that severe
cholinergic neuron disorder appears in hippocampus and cortex
(Non-Patent Literatures 29-31). Furthermore, there is a correlation
between progress of cholinergic neuron disorder and cognitive
dysfunction (Non-Patent Literature 29), and cholinesterase
inhibitors have been demonstrated to improve cognitive function.
Based on these findings, cognitive function improves by the
improvement of function of cholinergic neurons, and similarly to
Alzheimer's disease, improvement of cognitive decline and
suppression of disease status progress in dementia with Lewy bodies
and Parkinson disease with dementia can be expected by suppression
or improvement of loss of cholinergic neurons in several models of
the disorder.
[0010] Therefore, based on these findings, an improvement in
reduced cognitive performance caused by the dysfunction of
cholinergic neurons can be expected by achieving functional
activation and/or neuroprotective effect on cholinergic neurons in
clinical practice.
[0011] In addition to the above diseases, examples of diseases for
which association between decrease in cognitive function and the
dysfunction of cholinergic neurons has been reported include
Huntington's chorea, Down's syndrome, amyotrophic lateral sclerosis
(ALS), major depression, schizophrenia, and the like.
CITATION LIST
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"Coordinate expression of the vesicular acetylcholine transporter
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SUMMARY
[0043] An object of the present invention is to provide a compound
or a pharmaceutically acceptable salt thereof having cholinergic
neuron activation and/or neuroprotective effect and having a
potential use of a therapeutic agent for Alzheimer's disease,
dementia with Lewy bodies and Parkinson disease with dementia.
[0044] As a result of extensive studies to solve the above
problems, the present inventors found a pentacyclic compound or
pharmaceutically acceptable salts thereof having cholinergic neuron
activation and or neuroprotective effect.
[0045] That is, the present invention relates to the following
<1> to <30>.
<1> A compound selected from the group consisting of: [0046]
3-fluoro-6,11-dimethyl-6,7,10,11,12,13-hexahydrobenzo[f]pyrido[4'',3'':4'-
,5']thieno[2',3' 1,2-a]
[0046] ##STR00002## [0047]
5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4-
,5]pyrimido[1,2-a]thieno[2,3-f][1,4]diazepine-4,13-dione:
[0047] ##STR00003## [0048]
5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4-
,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione:
[0048] ##STR00004## [0049]
(3aS,14aR)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,-
14aH)-dione:
[0049] ##STR00005## [0050]
(3aR,14aR)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,
14aH)-dione:
##STR00006##
[0050] and [0051]
(3aS,14aS)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,-
14aH)-dione:
##STR00007##
[0051] or a pharmaceutically acceptable salt thereof.
<2>3-Fluoro-6,11-dimethyl-6,7,10,11,12,13-hexahydrobenzo[f]pyrido[4-
'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-5,14-dione
or a pharmaceutically acceptable salt thereof:
##STR00008##
<3>5,10-Dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thien-
o[2',3':4,5]pyrimido[1,2-a]thieno[2,3-f][1,4]diazepine-4,13-dione
or a pharmaceutically acceptable salt thereof:
##STR00009##
<4>5,10-Dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thien-
o[2',3':4,5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione
or a pharmaceutically acceptable salt thereof:
##STR00010##
<5>(3aS,14aR)-5,10-Dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclop-
enta[f]pyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-
-4,13(2H,14aH)-dione or a pharmaceutically acceptable salt
thereof:
##STR00011##
<6>(3aR,14aR)-5,10-Dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclop-
enta[f]pyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-
-4,13(2H,14aH)-dione or a pharmaceutically acceptable salt
thereof:
##STR00012##
<7>(3aS,14aS)-5,10-Dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclop-
enta[f]pyrido[4'',3'':4',5']thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-
-4,13(2H,14aH)-dione or a pharmaceutically acceptable salt
thereof:
##STR00013##
<8> A pharmaceutical composition comprising the compound or
pharmaceutically acceptable salt thereof according to any one of
<1> to <7> and one or more pharmaceutically acceptable
additives. <9-1> The pharmaceutical composition according to
<8>, which is a neuron activating agent. <9-2> The
pharmaceutical composition according to <8>, which is a
neuron protecting agent. <10> The pharmaceutical composition
according to <8> for the treatment of cognitive dysfunction.
<11> A therapeutic agent for cognitive dysfunction comprising
the compound or pharmaceutically acceptable salt thereof according
to any one of <1> to <7>. <12> A method of
treating cognitive dysfunction, comprising administering the
compound or pharmaceutically acceptable salt thereof according to
any one of <1> to <7> to a patient in need thereof.
<13> The compound or pharmaceutically acceptable salt thereof
according to any one of <1> to <7> for use in the
treatment of cognitive dysfunction. <14> Use of the compound
or pharmaceutically acceptable salt thereof according to any one of
<1> to <7> for the manufacture of a therapeutic agent
for cognitive dysfunction. <15> A therapeutic agent for
Alzheimer's disease comprising the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7>. <16> A method of treating Alzheimer's disease,
comprising administering the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7> to a patient in need thereof. <17> The compound or
pharmaceutically acceptable salt thereof according to any one of
<1> to <7> for use in the treatment of Alzheimer's
disease. <18> Use of the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7> for the manufacture of a therapeutic agent for
Alzheimer's disease. <19> A therapeutic agent for Dementia
with Lewy bodies comprising the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7>. <20> A method of treating Dementia with Lewy
bodies, comprising administering the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7> to a patient in need thereof. <21> The compound or
pharmaceutically acceptable salt thereof according to any one of
<1> to <7> for use in the treatment of Dementia with
Lewy bodies. <22> Use of the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7> for the manufacture of a therapeutic agent for Dementia
with Lewy bodies. <23> A therapeutic agent for Parkinson
disease with dementia comprising the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7>. <24> A method of treating Parkinson disease with
dementia, comprising administering the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7> to a patient in need thereof. <25> The compound or
pharmaceutically acceptable salt thereof according to any one of
<1> to <7> for use in the treatment of Parkinson
disease with dementia. <26> Use of the compound or
pharmaceutically acceptable salt thereof according to any one of
<1> to <7> for the manufacture of a therapeutic agent
for Parkinson disease with dementia. <27> A method of
treating a disease selected from a group consisting of Alzheimer's
disease, Dementia with Lewy bodies and Parkinson disease with
dementia comprising administering the compound or pharmaceutically
acceptable salt thereof according to any one of <1> to
<7> to a patient in need thereof. <28> The method
according to <27> wherein the disease is Alzheimer's disease.
<29> The method according to <27> wherein the disease
is Dementia with Lewy bodies. <30> The method according to
<27> wherein the disease is Parkinson disease with
dementia.
[0052] The pentacyclic compounds represented by formulas (I) to
(VT) (hereinafter referred to as "the compounds (I) to (VI)") or
pharmaceutically acceptable salts thereof according to the present
invention have neuron activation and/or neuroprotective effect, as
shown in activity data in pharmacological test examples provided
later. The compounds (I) to (VT) of the present invention lead to
an improvement of cognitive performance due to their neuron
activation and/or neuroprotective effect, and thus have a potential
use as therapeutic agents for Alzheimer's disease, Dementia with
Lewy bodies and Parkinson disease with dementia.
DETAILED DESCRIPTION
[0053] Hereinafter, the contents of the present invention will be
described in detail.
[0054] In the present specification, the structural formulas of the
compounds may represent specific isomers for convenience; however,
the present invention may include rotational isomers and tautomers,
as well as isomeric mixtures, is not limited to the formulas
described for convenience, and may be any of the isomers or a
mixture containing the isomers in any proportion.
[0055] Further, polymorphic crystals may also exist; however, the
present invention is also not limited to any of them and may be a
singly crystal form or a mixture thereof. Moreover, the present
invention also includes amorphous forms, and the compounds
according to the present invention include anhydrates and solvates
(particularly hydrates).
[0056] The present invention also includes isotope-labeled
compounds of the compounds (I) to (VI). The isotope-labeled
compounds are the same as the compounds (I) to (VI), except that
one or more atoms are replaced by one or more atoms having an
atomic mass or mass number different from those generally found in
nature. Examples of isotopes that can be incorporated into the
compounds of the present invention include isotopes of hydrogen,
carbon, nitrogen, oxygen, fluorine, phosphorus, sulfur, iodine, and
chlorine, and specifically include .sup.2H, .sup.3H, .sup.11C,
.sup.14C, .sup.15N, .sup.18O, .sup.18F, .sup.32P, .sup.35S,
.sup.123I, .sup.125I, and the like.
[0057] The above isotope-labeled compounds, for example, compounds
into which radioactive isotopes, such as .sup.3H and/or .sup.14C,
are incorporated, are useful for the tissue distribution assay of
medicines and/or substrates. .sup.3H and .sup.14C are considered to
be useful because of the ease of the preparation and detection
thereof. Isotopes .sup.11C and .sup.18F are considered to be useful
for PET (positron emission tomography), isotope .sup.125I is
considered to be useful for SPECT (single-photon emission computed
tomography), and all of them are useful for brain imaging.
Replacement by heavier isotopes, such as .sup.2H, results in some
types of therapeutic advantages, including an increase in the in
vivo half-life period or a decrease in the required dose due to
higher metabolic stability, and is therefore considered to be
useful under certain situations. The above isotope-labeled
compounds can be similarly prepared by carrying out the procedures
disclosed in the following Examples using easily usable reagents
labeled with isotopes in place of reagents not labeled with
isotopes.
[0058] The "pharmaceutically acceptable salts" in the present
specification are not particularly limited as long as they are
salts formed with the compounds according to the present invention,
and specific examples include acid addition salts, such as
inorganic acid salts, organic acid salts, and acidic amino acid
salts.
[0059] The "pharmaceutically acceptable salt" in the present
specification is any salt formed in a suitable ratio unless there
is any especially limiting description, and the number of acid
molecules per molecule of the compound in the formed salt is not
particularly limited; however, it is preferable that the number of
acid molecules per molecule of the compound be about 0.5 to about
2, and it is more preferable that the number of acid molecules per
molecule of the compound be about 0.5, about 1, or about 2.
[0060] Preferable examples of the inorganic acid salts include
hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and
preferable examples of organic acid salts include acetate,
succinate, fumarate, maleate, tartrate, citrate, lactate, stearate,
benzoate, methanesulfonate, p-toluenesulfonate, and
benzenesulfonate.
[0061] Preferable examples of the acidic amino acid salts include
aspartate and glutamate.
[0062] When the compounds (I) to (VT) according to the present
invention are obtained in a free form, they can be converted into
salts that may be formed by the compounds (I) to (VI) or hydrates
thereof in accordance with a conventional method.
[0063] When the compounds (I) to (VI) according to the present
invention are obtained as salts of the compounds (I) to (VI) or
hydrates of the compounds (I) to (VI), they can be converted into
free forms of the compounds (I) to (VI) in accordance with a
conventional method.
[0064] Moreover, various isomers (e.g., optical isomers, rotational
isomers, stereoisomers, etc.) obtained from the compounds (I) to
(VI) according to the present invention can be purified and
isolated by general separation means, such as recrystallization,
diastereomeric salt method, enzymatic resolution method, and
various chromatographic techniques (e.g., thin-layer
chromatography, column chromatography, gas chromatography,
etc.).
[0065] [Pharmaceutical Preparation]
[0066] The pharmaceutical composition according to the present
invention can be produced by mixing pharmaceutically acceptable
additives with a compound selected from the group of compounds (I)
to (VI) or pharmaceutically acceptable salts thereof. The
pharmaceutical composition according to the present invention can
be produced by a known method, for example, the method described in
the General Rules for Preparations of The Japanese Pharmacopoeia
Seventeenth Edition.
[0067] The pharmaceutical composition according to the present
invention can be appropriately administered to a patient depending
on the dosage form thereof.
[0068] The dose of the compounds (I) to (VI) according to the
present invention or pharmaceutically acceptable salts thereof
varies depending on the severity of symptoms, age, sex, body
weight, dosage form, type of salt, specific type of disease, and
other conditions; however; in general, the dose for an adult per
day by oral administration is about 30 .mu.g to 10 g, preferably
100 .mu.g to 5 g, and more preferably 100 .mu.g to 1 g; the dose
for an adult per day by injection administration is about 30 .mu.g
to 1 g, preferably 100 .mu.g to 500 mg, and more preferably 100
.mu.g to 300 mg; and the above dose is administered once or several
times.
[0069] The compounds of the present invention can be used as
chemical probes for capturing the target proteins of bioactive
low-molecular-weight compounds. That is, the compounds of the
present invention can be converted into affinity chromatography
probes, photoaffinity probes, etc., by introducing labeling groups,
linkers, or the like into a moiety different from a structural
moiety essential for the development of the activity of the
compounds using a method described, for example, in J. Mass
Spectrum. Soc. Jpn. Vol. 51, No. 5, 2003, pp. 492-498,
WO2007/139149, or the like.
[0070] Examples of labeling groups, linkers, etc., used in chemical
probes include groups shown in the group consisting of the
following (1) to (5):
(1) protein-labeling groups, such as photoaffinity-labeling groups
(e.g., a benzoyl group, a benzophenone group, an azide group, a
carbonylazide group, a diaziridine group, an enone group, a diazo
group, a nitro group, etc.) and chemical affinity groups (e.g., a
ketone group in which the alpha carbon atom is replaced by a
halogen atom, a carbamoyl group, an ester group, an alkylthio
group, a Michael acceptor such as .alpha.,.beta.-unsaturated ketone
or ester, and an oxirane group); (2) cleavable linkers, such as
--S--S--, --O--Si--O--, monosaccharides (a glucose group, a
galactose group, etc.), or disaccharides (lactose, etc.); and
oligopeptide linkers cleavable by enzyme reaction; (3) fishing tag
groups, such as biotin and a
3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propion-
yl group; (4) radioactive labeling groups, such as .sup.125I,
.sup.32P, .sup.3H, and .sup.14C; fluorescent labeling groups, such
as fluorescein, rhodamine, dansyl, umbelliferone, 7-nitrofurazanyl,
and a
3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacen-3-yl)propion-
yl group; chemiluminescent groups, such as luciferin and luminol;
and markers capable of detecting heavy metal ions, such as
lanthanoid metal ions and radium ions; or (5) groups to be attached
to solid carriers, such as glass beads, glass beds, microtiter
plates, agarose beads, agarose beds, polystyrene beads, polystyrene
beds, nylon beads, and nylon beds.
[0071] Probes prepared by introducing labeling groups, etc.,
selected from the group consisting of the above (1) to (5) into the
compounds of the present invention by the methods described in the
above documents or the like can be used as chemical probes for
identifying labeled proteins useful to search novel drug design
targets, etc.
EXAMPLES
[0072] The compounds (I) to (VI) of the present invention can be
produced by, for example, the methods described in the following
Examples, and the effects of the compounds can be confirmed by the
methods described in the following Test Examples. However, these
are just examples, and the present invention is not limited to the
following specific examples in any case and may be modified within
a range that does not depart from the scope of the present
invention.
[0073] Compounds described with document names, etc., indicate that
the compounds were produced according to the documents, etc.
[0074] Moreover, the abbreviations used in the present
specification are welt-known and common to a person skilled in the
art. In the present specification, the following abbreviations are
used.
DCE: 1,2-dichloroethane DCM: dichloromethane
DIPEA: N,N-diisopropylethylamine
[0075] DMT-MM:
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
DMSO: dimethylsulfoxide EDC:
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride HATU:
O-(7-ambenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate HOBT: 1-hydroxybenzotriazole n-: normal
NMM: N-methylmorpholine
[0076] t-: tertiary TBD:
1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine TBME: tertiary
butyl methyl ether TEA: triethylamine THF: tetrahydrofuran
.sup.1H-NMR: proton nuclear magnetic resonance spectrometry MS:
mass spectrometry HPLC: high-performance liquid chromatography
[0077] The term "room temperature" in the following Examples and
Production Examples generally refers to about 10.degree. C. to
about 35.degree. C. % refers to weight percent unless otherwise
specified.
[0078] Chemical shifts of proton nuclear magnetic resonance spectra
are denoted in .delta.-unit (ppm) relative to tetramethylsilane,
and coupling constants are recorded in Hertz (Hz). Patterns are
designated as s: singlet, d: doublet, t: triplet, q: quartet, m:
multiplet, br: broad, br.s: broad singlet.
[0079] For the optical resolution of the compound, Parallex
Flex.TM. produced by Biotage (column: one of CHRALPAK.RTM. AD-H,
IA, IB, and IC produced by DAICEL; and CHIRALCEL.RTM. OD-H and OJ-H
produced by DAICEL) was used.
[0080] In the reactions using a microwave reactor in the Production
Examples, Reference Examples, and Examples, Initiator.TM. or
Initiator+.TM. produced by Biotage was used.
[0081] Regarding chromatography, as silica gel, Silica Gel60
produced by Merck (70-230 mesh or 230-400 mesh ASTM) or PSQ60B
produced by Fuji Silysia Chemical Ltd. was used, or a pre-packed
column {column: Hi-Flash.TM. Column (Silicagel) produced by
YAMAZEN, size: one of S (16.times.60 mm), M (20.times.75 mm), L
(26.times.100 mm), 2 L (26.times.150 mm), and 3 L (46.times.130
mm); or Biotage.TM. SNAP Ultra Silica Cartridge produced by
Biotage, size: one of 10 g, 25 g, and 50 g} was used.
[0082] As NH silica gel, CHROMATOREX NH-DM2035 produced by Fuji
Silysia Chemical Ltd. was used, or a pre-packed column {column:
Hi-Flash.TM. Column (Amino) produced YAMAZEN, size: one of S
(16.times.60 mm), M (20.times.75 mm), L (26.times.100 mm), 2 L
(26.times.150 mm), and 3 L (46.times.130 mm); or Presep.TM. (Luer
Lock) NH2(HC) produced by Wako Pure Chemical Industries, Ltd.,
size: one of type M (14 g/25 mL), type L (34 g/70 mL), type 2 L (50
g/100 mL), and type 3 L (110 g/200 mL)} was used.
[0083] As names of the compounds shown below, except for generally
used reagents, those shown in the "E-Notebook" Version 12
(PerkinElmer) were used.
Production Example 1
Synthesis of ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
##STR00014##
[0085] TEA (61.6 mL, 442 mmol) was added at room temperature to a
mixture of 1-methyl-4-piperidone (CAS No. 1445-73-4) (51.5 mL, 442
mmol), ethyl cyanoacetate (CAS No. 105-56-6) (47.2 mL, 442 mmol),
sulfur (CAS No. 7704-34-9) (14.2 g, 442 mmol), and ethanol (800
mL). The reaction mixture was stirred at 40.degree. C. for 15
hours, and then concentrated under reduced pressure. The residue
was purified by column chromatography (NH silica gel, ethyl
acetate). The obtained concentrated residue was triturated with
ethyl acetate. The precipitates were collected by filtration,
washed with ethyl acetate, and dried under reduced pressure to
yield the title compound (58.4 g).
[0086] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.33 (t,
J=7.0 Hz, 3H), 2.44 (s, 3H), 2.62-2.70 (m, 2H), 2.79-2.88 (m, 2H),
3.37 (t, J=2.0 Hz, 2H), 4.26 (q, J=7.3 Hz, 2H), 5.97 (br. s,
2H).
[0087] MS (ESI) m/z: 241 [M+H].sup.+
Production Example 2
Synthesis of
7-fluoro-4-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione
##STR00015##
[0089] Sarcosine (CAS No. 107-97-1) (5.16 g, 58.0 mmol) was added
at room temperature to a solution of
6-fluoro-1H-benzo[d][1,3]oxazine-2,4-dione (CAS No. 321-69-7) (10.0
g, 55.2 mmol) in pyridine (100 mL), and the reaction mixture was
stirred at 100.degree. C. for 8 hours. The reaction mixture was
cooled to room temperature. The precipitates were collected by
filtration and washed with diethyl ether. The obtained solid was
dried under reduced pressure to yield the title compound (5.34
g).
[0090] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 3.30 (s,
3H), 3.90 (s, 2H), 6.97 (dd, J=8.8, 4.5 Hz, 1H), 7.20 (ddd, J=8.6,
7.6, 2.9 Hz, 1H), 7.67 (dd, J=9.0, 3.1 Hz, 1H), 7.99 (br. s,
1H).
[0091] MS (ESI) m/z: 209 [M+H].sup.+
Production Example 3
Synthesis of
4-methyl-3,4-dihydro-1H-thieno[3,2-e][1,4]diazepine-2,5-dione
##STR00016##
[0093] A mixture of 1H,2H,4H-thieno[3,2-d][1,3]oxazine-2,4-dione
(CAS No. 78756-28-2) (300 mg, 1.77 mmol), sarcosine (395 mg, 4.43
mmol), and water (10 mL) was heated under reflux for 2 hours. The
reaction mixture was cooled to 0.degree. C. The precipitates was
collected by filtration, and washed sequentially with water and
diethyl ether. The obtained solid was dried under reduced pressure
to yield the title compound (165 mg).
[0094] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 3.24 (s,
3H), 4.00 (s, 2H), 6.72 (d, J=5.3 Hz, 1H), 7.52 (d, J=5.3 Hz, 1H),
7.96 (br. s, 1H).
[0095] MS (ESI) m/z: 197 [M+H].sup.+
Production Example 4
Synthesis of
4-methyl-1,4-dihydro-1H-thieno[2,3-e][1,4]diazepine-2,5-dione
##STR00017##
[0097] 1H,2H,4H-thieno[2,3-d][1,3]oxazine-2,4-dione (CAS No.
103979-54-0) (600 mg, 3.55 mmol) was added to a solution of
sarcosine (790 mg, 8.87 mmol) in water (12 mL). The reaction
mixture was heated under reflux for 1.5 hours. The reaction mixture
was cooled to room temperature. Chloroform was added to the
reaction mixture, and the organic layer was separated. The aqueous
layer was extracted with chloroform (twice) and ethyl acetate (3
times). The combined organic layer was dried over anhydrous sodium
sulfate and filtered, and the filtrate was concentrated under
reduced pressure. The obtained solid was dried to yield the title
compound (430 mg).
[0098] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 3.23 (s,
3H), 3.99 (s, 2H), 6.90 (d, J=5.9 Hz, 1H), 7.29 (d, J=5.7 Hz, 1H),
8.39 (br. s, 1H).
[0099] MS (ESI) m/z: 197 [M+H].sup.+
Production Example 5
Synthesis of
(5aS,8aR)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
##STR00018##
[0100] (1) Synthesis of methyl
2-((1S,2R)-2-((t-butoxycarbonyl)amino)-N-methylcyclopentanecarboxamide)ac-
etate
[0101] TEA (22.2 mL, 159 mmol), HOBT/monohydrate (11.7 g, 76.3
mmol), and EDC (14.6 g, 76.3 mmol) were sequentially added under
ice cooling to a mixture of
(1S,2R)-2-((t-butoxycarbonyl)amino)cyclopentane-1-carboxylic acid
(CAS No. 137170-89-9) (14.6 g, 63.6 mmol), sarcosine methyl ester
hydrochloride (CAS No. 13515-93-0) (10.7 g, 76.3 mmol), and THF
(150 mL). After the reaction mixture was stirred at room
temperature for 15 hours, ethyl acetate and water were added, and
the organic layer was separated. The aqueous layer was extracted
with ethyl acetate. The combined organic layer was washed
sequentially with a saturated sodium hydrogen carbonate aqueous
solution and a saturated sodium chloride solution, dried over
anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure. The obtained residue was purified twice by column
chromatography (silica gel, 25-30% ethyl acetate/n-heptane) to
yield the title compound (16.1 g).
[0102] MS (ESI) m/z: 337 [M+Na].sup.+
(2) Synthesis of
(5aS,8aR)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
[0103] A 4 N hydrogen chloride/1,4-dioxane solution (160 mL, 640
mmol) was added under ice cooling to methyl
241S,2R)-2-((t-butoxycarbonyl)amino)-N-methylcyclopentanecarboxamide)acet-
ate (16.1 g, 51.3 mmol). The reaction mixture was stirred at the
same temperature for 30 minutes, then stirred at room temperature
for 45 minutes, and concentrated under reduced pressure. TBD (8.57
g, 61.6 mmol) was added under water cooling to a solution of the
residue in methanol (130 ml). The reaction mixture was stirred
under water cooling for 3 hours, and then cooled to 0.degree. C.
The resulting solid was collected by filtration, washed 3 times
with ice-cooled methanol, and dried under reduced pressure to yield
the title compound (5.22 g).
[0104] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.41-1.59
(m, 2H), 1.78-1.98 (m, 2H), 2.00-2.15 (m, 1H), 2.36-2.53 (m, 1H),
3.08 (s, 3H), 3.18-3.32 (m, 1H), 3.49 (dd, J=15.5, 1.7 Hz, 1H),
3.91-4.04 (m, 1H), 4.51 (d, J=15.4 Hz, 1H), 5.54 (br. s, 1H).
[0105] MS (ESI) m/z: 183 [M+H].sup.+
Production Example 6
Synthesis of (5aR,8
aR)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
##STR00019##
[0106] (1) Synthesis of t-butyl
2-((1R,2R)-2-((t-butoxycarbonyl)amino)-N-methylcyclopentanecarboxamide)ac-
etate
[0107] DIPEA (1.81 mL, 10.5 mmol) and HATU (1.99 g, 5.23 mmol) were
sequentially added at room temperature to a mixture of
(1R,2R)-t-butoxycarbonyl-2-aminocyclopentanecarboxylic acid (CAS
No. 245115-25-7) (1.00 g, 4.36 mmol), sarcosine t-butyl ester
hydrochloride (CAS No. 136088-69-2) (872 mg, 4.80 mmol), and DCM
(10 mL). The reaction mixture was stirred at room temperature for 1
hour, and then directly purified by column chromatography (silica
gel, 30-50% ethyl acetate/n-heptane) to yield the title compound
(1.61 g).
[0108] MS (ESI) m/z: 357 [M+H].sup.+
(2) Synthesis of
(5aR,8aR)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
[0109] A 4 N hydrogen chloride/1,4-dioxane solution (16 mL, 64
mmol) was added at room temperature to t-butyl
2-((1R,2R)-2-((t-butoxycarbonyl)amino)-N-methylcyclopentanecarboxamide)ac-
etate (1.61 g, 4.52 mmol), and the mixture was stirred for 20
hours. The reaction mixture was concentrated under reduced
pressure. Sodium hydrogen carbonate (0.911 g, 10.8 mmol), methanol
(24 mL), NMM (0.099 mL, 0.90 mmol), and DMT-MM (12.3% H.sub.2O,
1.80 g, 5.70 mmol) were sequentially added to the residue at room
temperature, and the mixture was stirred for 20 hours. The reaction
mixture was concentrated under reduced pressure, and the residue
was washed with DCM. The washed liquid was concentrated under
reduced pressure, and the residue was purified by column
chromatography (silica gel, 5-20% methanol/ethyl acetate) to yield
the title compound (745 mg).
[0110] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.56-1.88
(m, 3H), 1.91-2.02 (m, 1H), 2.13-2.23 (m, 1H), 2.26-2.39 (m, 1H),
3.07 (s, 3H), 3.08-3.16 (m, 1H), 3.51-3.62 (m, 1H), 3.79 (d, J=18.0
Hz, 1H), 4.58 (d, J=18.0 Hz, 1H), 6.76 (br. s, 1H).
[0111] MS (ESI) m/z: 183 [M+H].sup.+
Production Example 7
Synthesis of
(5aS,8aS)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
##STR00020##
[0112] (1) Synthesis of t-butyl
2-((1S,2S)-2-((t-butoxycarbonyl)amino)-N-methylcyclopentanecarboxamide)ac-
etate
[0113] HATU (1.99 g 5.23 mmol) was added at room temperature to a
mixture of (1S,2S)-t-butoxycarbonyl-2-aminocyclopentanecarboxylic
acid (CAS No. 143679-80-5) (1.00 g, 4.36 mmol), sarcosine t-butyl
ester hydrochloride (872 mg, 4.80 mmol), DIPEA (1.81 mL, 10.5
mmol), and DCM (10 mL). The reaction mixture was stirred at room
temperature overnight, and then directly purified by column
chromatography (silica gel, 30-50% ethyl acetate/n-heptane) to
yield the title compound (1.55 g).
[0114] MS (ESI) m/z: 357 [M+H].sup.+
(2) Synthesis of
(5aS,8aS)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
[0115] A 4 N hydrogen chloride/1,4-dioxane solution (16 mL, 64
mmol) was added at room temperature to t-butyl
2-((1S,2S)-2-((t-butoxycarbonyl)amino)-N-methylcyclopentanecarboxamide)ac-
etate (1.55 g, 4.35 mmol), and the mixture was stirred for 16
hours. The reaction mixture was concentrated under reduced
pressure. Sodium hydrogen carbonate (0.877 g, 10.4 mmol), methanol
(24 mL), NMM (0.096 mL, 0.87 mmol), and DMT-MM (12.3% H.sub.2O,
1.73 g, 5.48 mmol) were sequentially added to the residue at room
temperature, and the mixture was stirred for 3 hours. The reaction
mixture was concentrated under reduced pressure, and the residue
was washed with DCM. The washed liquid was concentrated under
reduced pressure, and the residue was purified by column
chromatography (silica gel, 0-20% methanol/ethyl acetate) to yield
the title compound (753 mg).
[0116] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.55-1.88
(m, 3H), 1.91-2.02 (m, 1H), 2.11-2.22 (m, 1H), 2.25-2.40 (m, 1H),
3.07 (s, 3H), 3.07-3.16 (m, 1H), 3.51-3.62 (m, 1H), 3.78 (d, J=18.0
Hz, 1H), 4.57 (d, J=18.0 Hz, 1H), 6.54 (br. s, 1H).
[0117] MS (ESI) m/z: 183 [M+H].sup.+
Example 1
Synthesis of
3-fluoro-6,11-dimethyl-6,7,10,11,12,13-hexahydrobenzo[f]pyrido[4'',3'':4'-
,5]thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-5,14-dione
##STR00021##
[0119] Phosphorus oxychloride (4.65 mL, 49.9 mmol) was added at
room temperature to a mixture of ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
(6.00 g, 25.0 mmol) obtained in Production Example 1,
7-fluoro-4-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione
(5.20 g, 25.0 mmol) obtained in Production Example 2, and DCE (300
mL). The reaction mixture was stirred at 80.degree. C. for 20
hours. While stirring under ice-cooling, sodium ethoxide (a 20%
solution in ethanol, 80 mL, 207 mmol) was added to the reaction
mixture. The reaction mixture was stirred at room temperature for
20 minutes. A saturated sodium hydrogen carbonate aqueous solution
and ethyl acetate were added to the reaction mixture, and the
organic layer was separated. The aqueous layer was extracted with
ethyl acetate. The combined organic layer was dried over magnesium
sulfate and filtered, and the filtrate was concentrated under
reduced pressure. The residue was sequentially purified by column
chromatography (NH silica gel, 50-100% ethyl acetate/n-heptane) and
column chromatography (silica gel, 0-50% methanol/ethyl acetate).
The obtained solid was triturated with TBME, and the precipitates
were collected by filtration. The obtained solid was washed with
TBME and dried under reduced pressure to yield the title compound
(4.56 g).
[0120] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 2.51 (s,
3H), 2.66-2.76 (m, 1H), 2.77-2.88 (m, 1H), 3.04-3.18 (m, 2H), 3.25
(s, 3H), 3.57-3.75 (m, 2H), 4.09 (d, J 15.2 Hz, 1H), 4.47 (d,
J=14.8 Hz, 1H), 7.25-7.31 (m, 1H), 7.60-7.64 (m, 1H), 7.67 (dd,
J=9.0, 4.7 Hz, 1H).
[0121] MS (ESI) m/z: 385 [M+H].sup.+
Example 2
Synthesis of
5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2',3':4-
,5]pyrimido[1,2-a]thieno[2,3-f][1,4]diazepine-4,13-dione
##STR00022##
[0123] Phosphorus oxychloride (0.157 mL, 1.68 mmol) was added at
room temperature to a mixture of ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
(303 mg, 1.26 mmol) obtained in Production Example 1,
4-methyl-3,4-dihydro-1H-thieno[3,2-e][1,4]diazepine-2,5-dione (165
mg, 0.841 mmol) obtained in Production Example 3, and 1,4-dioxane
(10 mL). The reaction mixture was stirred at 70.degree. C. for 2
hours, and then stirred at 90.degree. C. for 5 hours. Sodium
ethoxide (a 20% solution in ethanol, 2.60 mL, 6.73 mmol) was added
to the reaction mixture cooled to room temperature. The reaction
mixture was stirred at room temperature for 40 minutes. Ethyl
acetate and a saturated sodium hydrogen carbonate aqueous solution
were added to the reaction mixture, and the organic layer was
separated. The aqueous layer was extracted with ethyl acetate. The
combined organic layer was dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, 50% methanol/ethyl
acetate) to yield the title compound (90.0 mg).
[0124] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 2.51 (s,
3H), 2.66-2.87 (m, 2H), 3.07-3.20 (m, 2H), 3.26 (s, 3H), 3.56-3.74
(m, 2H), 4.21 (d, J=15.0 Hz, 1H), 4.56 (d, J=15.0 Hz, 1H), 7.54 (d,
J=5.3 Hz, 1H), 7.59 (d, J=5.3 Hz, 1H).
[0125] MS (ESI) m/z: 373 [M+H].sup.+
Example 3
Synthesis of
5,10-dimethyl-5,6,9,10,11,12-hexahydropyrido[4'',3'':4',5']thieno[2'3':4,-
5]pyrimido[1,2-a]thieno[3,2-f][1,4]diazepine-4,13-dione
##STR00023##
[0127] Phosphorus oxychloride (1.43 mL, 15.3 mmol) was added at
room temperature to a mixture of
4-methyl-3,4-dihydro-1H-thieno[2,3-e][1,4]diazepine-2,5-dione (1.00
g, 5.10 mmol) obtained in Production Example 4, ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
(1.84 g, 7.64 mmol) obtained in Production Example 1, and
1,4-dioxane (30 mL) The reaction mixture was stirred at room
temperature for 5 minutes, and stirred at 90.degree. C. for 2
hours. Sodium ethoxide (a 20% solution in ethanol, 21.7 mL, 56.1
mmol) was added over 5 minutes to the reaction mixture cooled to
room temperature. The reaction mixture was stirred at room
temperature for 1.5 hours. Ethyl acetate, a saturated sodium
hydrogen carbonate aqueous solution, and water were sequentially
added to the reaction mixture, and the organic layer was separated.
The aqueous layer was extracted with ethyl acetate. The combined
organic layer was dried over anhydrous magnesium sulfate and
filtered, and the filtrate was concentrated under reduced pressure.
The residue was purified by column chromatography (silica gel,
20%-50% methanol/ethyl acetate). The obtained solid was triturated
with ethanol, and the precipitates were collected by filtration.
The obtained solid was washed with ethanol, and dried under reduced
pressure to yield the title compound (712 mg).
[0128] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 2.52 (s,
3H), 2.71-2.87 (m, 2H), 3.05-3.30 (m, 5H), 3.59-3.75 (m, 2H), 4.23
(d, J=14.8 Hz, 1H), 4.57 (d, J=14.8 Hz, 1H), 7.35 (d, I=6.2 Hz,
1H), 7.39 (d, J=5.9 Hz, 1H).
[0129] MS (ESI) m/z: 373 [M+H].sup.+
Example 4
Synthesis of (3
aS,14aR)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]pyri-
do[4'',3'':4',5]thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,14a-
H)-dione
##STR00024##
[0131] Phosphorus oxychloride (7.93 mL, 85.1 mmol) was added at
room temperature to a mixture of
(5aS,8aR)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
(3.10 g, 17.0 mmol) obtained in Production Example 5-(2), ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
(8.18 g, 34.0 mmol) obtained in Production Example 1, and DCE (300
mL). The reaction mixture was stirred at 80.degree. C. for 14.5
hours. A saturated sodium hydrogen carbonate aqueous solution was
added to the reaction mixture at 0.degree. C., the mixture was
stirred at room temperature for 3.5 hours, and then the organic
layer was separated. The aqueous layer was extracted with ethyl
acetate. The combined organic layer was washed sequentially with a
saturated sodium hydrogen carbonate aqueous solution and a
saturated sodium chloride solution, dried over anhydrous magnesium
sulfate, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography (NH silica gel,
30-60% ethyl acetate/n-heptane). The obtained concentrated residue
was triturated with TBME, and the precipitates were collected by
filtration. The obtained solid was washed 3 times with TBME, and
dried under reduced pressure to yield the title compound (3.70
g).
[0132] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.51-1.73
(m, 2H), 1.94-2.18 (m, 2H), 2.30-2.41 (m, 1H), 2.44-2.59 (m, 4H),
2.71-2.82 (m, 2H), 3.04-3.19 (m, 5H), 3.42-3.54 (m, 1H), 3.64 (s,
2H), 4.17 (d, J=15.6 Hz, 1H), 4.75 (d, J=15.6 Hz, 1H), 5.69-5.82
(m, 1H).
[0133] MS (ESI) m/z: 359 [M+H].sup.+
[0134] Specific rotation: [.alpha.].sub.D.sup.20-146.0 (c 0.50,
CHCl.sub.3)
[0135] Analysis by HPLC:
[0136] (Analysis conditions) Column: CHIRALPAK IB (produced by
Daicel Chemical Industries, Ltd.) (0.46 cm .phi..times.15 cm),
40.degree. C., eluent: ethanol/hexane=20/80 (v/v), flow rate: 1
ml/min., detection: UV (254 nm).
[0137] (Analysis results) When the title compound was analyzed
under the above analysis conditions, the retention time was 10.38
minutes, the optical purity was >98% ee, and the optical
rotation was (-). The retention time of the enantiomer was
confirmed by the product synthesized similarly using a racemic
mixture as a starting material.
Example 5
Synthesis of (3
aR,14aR)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]pyri-
do[4'',3'':4',5]thieno[2',3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,14a-
H)-dione
##STR00025##
[0139] Phosphorus oxychloride (0.793 mL, 8.51 mmol) was added at
room temperature to a mixture of
(5aR,8aR)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
(310 mg, 1.70 mmol) obtained in Production Example 6-(2), ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
(613 mg, 2.55 mmol) obtained in Production Example 1, and DCE (16
mL). The reaction mixture was stirred at 70.degree. C. for 2.5
hours and then returned to room temperature, and ethyl acetate (15
mL) and a saturated sodium hydrogen carbonate aqueous solution (30
mL) were added. The reaction mixture was stirred at room
temperature for 5 days, ethyl acetate was added, and the organic
layer was separated. The aqueous layer was extracted with ethyl
acetate. The combined organic layer was dried over anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography (NH silica gel,
50-70% ethyl acetate/n-heptane). The obtained product was washed 3
times with diethyl ether, then washed with TBME, and dried under
reduced pressure to yield the title compound (143 mg).
[0140] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.29-1.49
(m, 1H), 1.68-1.83 (m, 1H), 1.82-2.21 (m, 3H), 2.50 (s, 3H), 2.76
(t, J=5.7 Hz, 2H), 2.98-3.23 (m, 6H), 3.40-3.54 (m, 1H), 3.57-3.68
(m, 2H), 4.17-4.34 (m, 2H), 5.30 (d, J=17.4 Hz, 1H).
[0141] MS (ESI) m/z: 359 [M+H].sup.+
[0142] Analysis by HPLC:
[0143] (Analysis conditions) Column: CHIRALPAK IC (produced by
Daicel Chemical Industries, Ltd.) (0.46 cm .phi..times.15 cm),
40.degree. C., eluent: ethanol, flow rate: 1 mL/min., detection: UV
(254 nm)
[0144] (Analysis results) The retention time of the title compound
was 6.64 minutes, the optical purity was >99% ee, and the
optical rotation was (-).
Example 6
Synthesis of
(3aS,14aS)-5,10-dimethyl-3,3a,5,6,9,10,11,12-octahydro-1H-cyclopenta[f]py-
rido[4'',3'':4',5']thieno[2%3':4,5]pyrimido[1,2-a][1,4]diazepine-4,13(2H,1-
4aH)-dione
##STR00026##
[0146] Phosphorus oxychloride (0.859 mL, 9.22 mmol) was added at
room temperature to a mixture of
(5aS,8aS)-4-methyloctahydrocyclopenta[e][1,4]diazepine-2,5-dione
(336 mg, 1.84 mmol) obtained in Production Example 7-(2), ethyl
2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate
(665 mg, 2.77 mmol) obtained in Production Example 1, and DCE (17
mL). The reaction mixture was stirred at 60.degree. C. for 3.5
hours, and then returned to room temperature. Ethyl acetate (15 mL)
and a saturated sodium hydrogen carbonate aqueous solution (30 mL)
were added to the reaction mixture. After the reaction mixture was
stirred at room temperature for 5 days, ethyl acetate was added,
and the organic layer was separated. The aqueous layer was
extracted with ethyl acetate. The combined organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatography
(NH silica gel, 40-80% ethyl acetate/n-heptane). The obtained
product was washed 3 times with diethyl ether and dried under
reduced pressure to yield the title compound (166 mg).
[0147] .sup.1H-NMR (400 MHz, CDCl.sub.3) .delta. (ppm): 1.31-1.50
(m, 1H), 1.69-1.83 (m, 1H), 1.84-1.97 (m, 1H), 1.97-2.20 (m, 2H),
2.50 (s, 3H), 2.73-2.80 (m, 2H), 3.02-3.23 (m, 6H), 3.41-3.55 (m,
1H), 3.57-3.69 (m, 2H), 4.19-4.34 (m, 2H), 5.30 (d, J=17.2 Hz,
1H).
[0148] MS (ESI) m/z: 359 [M+H].sup.+
[0149] (Analysis conditions) Column: CHIRALPAK IC (produced by
Daicel Chemical Industries, Ltd.) (0.46 cm .phi..times.15 cm),
40.degree. C., eluent: ethanol, flow rate: 1 mL/min., detection: UV
(254 nm)
[0150] (Analysis results) The retention time of the title compound
was 8.34 minutes, the optical purity was >99% ee, and the
optical rotation was (+).
Pharmacological Test Examples
[0151] The following pharmacological tests were conducted using the
compounds of Examples 1 to 6.
[0152] Measurement of Acetylcholine (ACh) Release in the Rat
Primary Septal Neuron Culture System in the Presence of NGF
[0153] (1) Rat Primary Septal Neuron Culture
[0154] The septal area was isolated from Sprague-Dawley (SD) rats
(Charles River Laboratories Japan, Inc.) at a fetal age of 18 days,
and cultured. Specifically, fetuses were aseptically removed from
pregnant rats under isoflurane anesthesia. The brain was extracted
from each fetus, and immersed in ice-cooled L-15 medium (11415-064,
Thermo Fisher Scientific). The septal area was dissected from the
extracted brain under a stereoscopic microscope. The dissected
septal area was subjected to enzyme treatment in an enzyme solution
containing 0.25% trypsin (15050-065, Thermo Fisher Scientific) and
0.01% DNase (D5025-150KU, Sigma) at 37.degree. C. for 30 minutes,
thereby dispersing the cells. In this case, the enzyme reaction was
terminated by adding inactivated horse serum (26050-088, Thermo
Fisher Scientific). The enzyme-treated solution was centrifuged at
1000 rpm for 3 minutes, and the supernatant was removed. A medium
in an amount of 10 mL was added to the obtained cell mass. The
medium used was Dulbecco's Modified Eagle's Medium (044-29765,
WAKO) supplemented with N2 supplement (17502-048, Thermo Fisher
Scientific), 1 mM sodium pyruvate (11360-070, Thermo Fisher
Scientific), and Penicillin-Streptomycin (15140-1221, Thermo Fisher
Scientific). The cells of the cell mass to which the medium was
added were redispersed by gentle pipetting, and then centrifuged
again at 1000 rpm for 3 minutes, and the supernatant was removed.
The medium in an amount of 10 mL was added to the obtained cell
mass, and the cell dispersion was filtered through a 40-.mu.m nylon
mesh (Cell Strainer) to remove the cell mass, thereby obtaining a
neuronal cell suspension. The neuronal cell suspension was diluted
with the medium, and 10% inactivated bovine serum (26140-079,
Thermo Fisher Scientific) and 10% inactivated horse serum were
added. Thereafter, 100 .mu.L/well of the suspension was seeded in a
96-well plate (354461, CORNING) pre-coated with poly-D-lysine so
that the initial culture density was 1.4.times.10.sup.5
cells/cm.sup.2. After the seeded cells were cultured under 5%
CO.sub.2-95% air in a 37.degree. C. incubator for 2 days, the
entire medium was replaced with 120 .mu.L of fresh medium, and the
cells were subsequently cultured for 5 days.
(2) Compound Addition
[0155] On the 7th day of culture, compound was added in the
following manner. A solution of the test compound in DMSO was
diluted with the medium so that the concentration was 10 times
higher than the final concentration. NGF (450-01, PEPRO TECH, INC.)
was prepared at 0.3 ng/mL. These two solutions were added each in
an amount of 15 .mu.L/well, and the mixture was mixed well. The
final DMSO concentration was 0.1% or less. Moreover, only DMSO and
NGF were added to the control group.
(3) ACh Release Measurement
[0156] One day after compound addition, an amount of ACh release
was measured by HPLC in the following manner. A warmed buffer was
added at 100 .mu.L/well to the well after the medium was
eliminated, and the buffer was immediately removed. Thereafter, a
buffer to which 10 .mu.m choline, 10 .mu.m physostigmine, and 6 mM
KCl were added was added at 120 .mu.L/well. The buffer was prepared
by adding 125 mM NaCl, 25 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1.2 mM
KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, 2.2 mM CaCl.sub.2 (2H.sub.2O),
and 10 mM glucose to sterilized water, and the final pH of the
solution was set to 7.4. After the 96-well plate to which the
buffer was added was incubated under 5% CO.sub.2-95% air in a
37.degree. C. incubator for 40 minutes, 80 .mu.L of buffer was
collected. An internal standard solution IPHC (5.times.10.sup.-7 M)
was added in an amount of 6 .mu.L to the collected buffer, and the
buffer was transferred to a tube for HPLC measurement and subjected
to HPLC measurement. The results are represented by the effect of
each compound as the percentage (% of control) of the ACh
concentration in the buffer of the control group, and the compound
concentrations showing a 20% increase from the ACh concentration in
the buffer of the control group are shown in the following Table
1.
TABLE-US-00001 TABLE 1 Concentration (.mu.M) showing a 20% or more
increase from Example the amount of ACh in the control group 1 0.1
2 0.1 3 0.1 4 0.1 5 0.1 6 0.03
[0157] Measurement of Choline Acetyltransferase (ChAT) mRNA
Expression Levels in the Rat Septal Area
(1) Compound Administration
[0158] In this study, SD male rats (Charles River Laboratories
Japan, Inc.) with a body weight of about 250 to 350 g were used.
The compound was dissolved in 0.01 mol/L hydrochloric acid, and
orally administered.
(2) Sampling
[0159] At 24 hours after the administration of the compound, the
whole brain tissue was collected under pentobarbital anesthesia.
The medial septum was isolated from whole brain on ice and frozen
with liquid nitrogen, and then stored at -80.degree. C.
(3) Measurement of ChAT mRNA Expression Levels
[0160] For RNA purification, RNeasy Plus Mini Kit (#74136: QIAGEN)
was used in this study. RNA purification was performed by the
method described in the kit. After RNA purification, the total RNA
concentration was measured by using QIAxpert Instrument (QIAGEN).
cDNA was synthesized using SuperScript.RTM. VILO.TM. cDNA Synthesis
Kit (#11754: Thermo Fisher Scientific). The synthesis of cDNA was
performed by the method described in the kit. The synthesized cDNA
was diluted 4 times with RNase free water, and the diluted cDNA
solution was used as a sample. Taqman Universal PCR Master Mix
(#4304437: Thermo Fisher Scientific), Taqman.RTM. Gene Expression
Assays, INVENTORIED (#4331182: Thermo Fisher Scientific), RNase
free water, and the cDNA solution were mixed in amounts of 10
.mu.l, 1 .mu.l, 4 .mu.l, and 5 respectively, and the resulting
mixture was used as a measurement sample solution. Quantitative
polymerase chain reaction (qPCR) was conducted using ABI PRISM.RTM.
7900HT (Thermo Fisher Scientific) by a fluorescence probe method.
Analysis was performed by SDS 2.4 (Thermo Fisher Scientific). The
results were calculated by the percentage of the amount of ChAT
mRNA expression levels in the compound administration group
increased from the amount of ChAT mRNA expression levels in the
vehicle administration group. The results are shown in the
following Table 2.
TABLE-US-00002 TABLE 2 Amount (%) increased from the amount of ChAT
mRNA expression levels in the Example Dose vehicle administration
group 1 10 mg/kg 73.3 2 3 mg/kg 38.0 3 10 mg/kg 56.4 4 10 mg/kg
42.4 5 3 mg/kg 33.6 6 10 mg/kg 32.0
[0161] Measurement of Acetylcholine (ACh) in Rat Cerebrospinal
Fluid (CSF)
(1) Background
[0162] Correlation between increase and decrease of intracerebral
neurotransmitters and those in cerebrospinal fluid (CSF) was
revealed by studies on rodents and the correlation was also seen in
human (Lowe S et al. Psychopharmacology 219 (2012) 959-970). Thus,
the changes in acetylcholine in CSF were measured in order to
determine the changes in intracerebral acetylcholine by the test
compounds.
(2) Compound Administration
[0163] In this study, Fischer344 male rats (Charles River
Laboratories Japan, Inc.) with a body weight of about 150 to 250 g
were used. The test compounds were orally administered to the rats
once a day at 10 mg/kg for three days. The vehicle used was 0.01
mol/L hydrochloric acid.
(3) Sampling
[0164] At 24 hours after the administration of the vehicle and the
test compounds, the CSF was collected from cisterna magna in a tube
containing AchE inhibitors under pentobarbital anesthesia. The
collected CSF was centrifuged at 3500.times.g at 4.degree. C. for
10 minutes and the supernatant was collected. The collected
supernatant was frozen with liquid nitrogen, and then stored at
-80.degree. C.
(4) Measurement of Ach by LC-MS
[0165] To 10 .mu.L of the CSF was added 50 .mu.L of
acetylcholine-d9 chloride (ACh-d9) at a final concentration of 0.34
nmol/L as an internal standard. The mixture was pipetted and
centrifuged at 1500.times.g at 4.degree. C. for 10 minutes. The
supernatant was collected and subjected to LC/MS (NexeraX2 (MS),
TSQ Altis (HPLC)), and Ach was detected as precursor ion at m/z
146.050 and as product ion at m/z 87.071 and ACh-d9 as an internal
standard was detected as precursor ion at m/z 155.088 and as
product ion at m/z 87.000. The results were shown as calculations
of a percentage of increase in ACh concentration in CSF in the test
compound administration group with respect to that in vehicle
administration group (% of control). The results were shown in
Table 3.
TABLE-US-00003 TABLE 3 Amount (%) increased with respect to from
the amount of Example ACh in CSF the vehicle administration group 1
160.0 3 156.8
[0166] Evaluation in Human Tau P301S Transgenic Mouse
(1) Compound Administration
[0167] In this study, the test compounds were orally administered
to human tau P301S transgenic mice once a day for three months from
four-month-old to seven-month-old. The vehicle used was 0.01 mol/L
hydrochloric acid.
(2) Sampling
[0168] At the initial day of the administration (four-month-old)
and at the next day of the final administration, mice of vehicle
administration group and test compound administration group were
anesthetized under pentobarbital (50 mg/kg, i.p.) and perfused with
PBS. After the perfusion, the forebrain including the medial septal
area was collected and fixed with 4% paraformaldehyde.
(3) Preparation of Brain Coronal Frozen Section
[0169] The collected forebrain including the medial septal area was
immersed and shaken overnight in 4% paraformaldehyde. The immersion
solution was replaced with 7.5% sucrose solution. It was immersed
and shaken overnight in 7.5% sucrose solution, and the immersion
solution was replaced with 15% sucrose solution and it was immersed
and shaken overnight. The immersion solution was replaced with 30%
sucrose solution and it was immersed and shaken overnight. Brain
coronal frozen sections with 30 .mu.m thickness were prepared from
the forebrain including the medial septal area by using a microtome
(Leica, SM2000R).
(4) Immunohistochemistry of Choline Acetyltransferase (ChAT)
Positive Cells
[0170] The prepared brain coronal frozen sections were stained with
DAB (DAB PEROXIDASE SUBSTRATE KIT (Vector, SK-4100)) using a ChAT
antibody (Santa Cruz, SC-20672) as a primary antibody. The section
image including the medial septal area as shown in "The mouse Brain
in stereotaxic coordinates" (COMPACT THIRD EDITION, Keith B. J.
Franklin & George Paxinos) was taken by an all-in-one
fluorescence microscope (KEYENCE, BZ-X710) and ChAT positive cells
around the major axis of the medial septal area were counted by BZ
analysis software (KEYENCE). The results were shown as a percentage
of the number of ChAT positive cells in the vehicle administration
group and the test compound administration group with respect to
the number of ChAT positive cells at the time of initial
administration (four-month-old). Data are expressed as the
mean.+-.SEM. The differences between the group at the time of
initial administration and the vehicle-treated group (significant:
*) was analyzed by an unpaired t-test, and also the differences
between the vehicle-treated group and compound-treated group
(significant: .sup.#) was analyzed by unpaired t-test. A value of
P<0.05 was considered statistically significant. Statistical
analyses were performed using the GraphPad Prism version 7.02. The
results were shown in Table 4.
TABLE-US-00004 TABLE 4 Ratio (%) of number of ChAT positive cells
Treatment Group compared to that in the initial administration
Group at the time of 100.0 .+-. 4.5 initial administration Vehicle
administration 83.0 .+-. 5.8* group Example 1 105.0 .+-. 4.0.sup.#
administration group (Dose: 10 mg/kg) Example 3 105.3 .+-.
4.3.sup.# administration group (Dose: 5 mg/kg)
[0171] Neuroprotective and Restorative Effect on Cholinergic
Neurons Using Fimbria-Fornix Lesioned Rat Model
(1) Preparation of Fimbria-Fornix Lesioned Rat Model
[0172] In this study, Sprague-Dawley male rats (Charles River
Laboratories Japan, Inc.) with a body weight of about 250 to 350 g
were used. The rat was anesthetized under the combination of three
drugs: midazolam (2 mg/kg s.c.), medetomidine hydrochloride (0.15
mg/kg s.c.) and butorphanol tartrate (2.5 mg/kg s.c.) and fixed
with a brain stereotaxis apparatus (Narishige Co., Ltd.). The
cranial was exposed and a hole with 5 mm width was drilled in the
skull from the median line 2 mm posterior to Bregma. A razor with 4
mm width was pierced into the Bregma in 5.5 mm depth to cut
fimbria-fornix. After hemostasis, the scalp was sutured. After the
operation, the rat was brought back to the cage and recovered from
the anesthesia. In the sham-operated group, a hole with 5 mm width
was drilled in the skull from the median line 2 mm posterior to
Bregma, but no razor was pierced.
(2) Compound Administration
[0173] The test compounds were orally administered to the rats once
a day from five days to nine days after the operation (Example 1:
10 mg/kg) or from seven days to fourteen days after the operation
(Example 3: 3 mg/kg). The vehicle used was 0.01 mol/L hydrochloric
acid. In the sham-operated group, the vehicle was orally
administered once a day similarly to the test compound
administration group.
(3) Sampling
[0174] The rats were anesthetized under pentobarbital and
transcardially perfused with ice-cold PBS. After the perfusion, the
forebrain including the medial septal area was collected and
immersed and shaken overnight with 4% paraformaldehyde. The
immersion solution was replaced with 7.5% sucrose solution. It was
immersed and shaken overnight in 7.5% sucrose solution, and the
immersion solution was replaced with 15% sucrose solution and it
was immersed and shaken overnight. The immersion solution was
replaced with 30% sucrose solution and it was immersed and shaken
overnight. Brain coronal frozen sections with 30 .mu.m thickness
were prepared from the forebrain including the medial septal area
by using a microtome (Leica, SM2000R).
(4) Immunohistochemistry of Choline Acetyltransferase (ChAT)
Positive Cells and Vesicular Acetylcholine Transporter (VAChT)
[0175] The prepared brain coronal frozen sections were stained with
DAB (DAB PEROXIDASE SUBSTRATE KIT (Vector, SK-4100)) using a ChAT
antibody (Santa Cruz, SC-20672) or a VAChT antibody (Merck
Millipore, ABN100) as a primary antibody. The section image
including the medial septal area or hippocampus as shown in "The
mouse Brain in stereotaxic coordinates" (COMPACT THIRD EDITION,
Keith B. J. Franklin & George Paxinos) was taken by an
all-in-one fluorescence microscope (KEYENCE, BZ-X710) and ChAT
positive cells of the medial septal area or optical density (OD) in
hippocampal VAChT were measured by BZ analysis software (KEYENCE).
The results were shown as a percentage of the number of ChAT
positive cells of the medial septal area or OD in hippocampal VAChT
in the vehicle administration group and the test compound
administration group with respect to the number of ChAT positive
cells of the medial septal area or OD in hippocampal VAChT in the
sham-operated group. Data are expressed as the mean.+-.SEM. The
differences between the vehicle-treated group and compound-treated
(significant: #) was analyzed by unpaired t-test. A value of
P<0.05 was considered statistically significant. Statistical
analyses were performed using the GraphPad Prism version 7.02. The
results were shown in Tables 5 and 6.
TABLE-US-00005 TABLE 5 Number of ChAT Number of ChAT Number of ChAT
positive cells (%) positive cells (%) in positive cells (%) at in
vehicle test compound initial administration administration Example
administration group group 1 59.9 .+-. 6.0 43.3 .+-. 12.3 79.1 .+-.
15.7 3 57.0 .+-. 7.5 38.4 .+-. 5.0 74.1 .+-. 9.3.sup.#
TABLE-US-00006 TABLE 6 OD in OD in hippocampal hippocampal OD in
hippocampal VAChT (%) in VAChT (%) in VAChT (%) at vehicle test
compound initial administration administration Example
administration group group 1 35.4 .+-. 4.4 22.8 .+-. 9.5 77.2 .+-.
14.6.sup.# 3 51.7 .+-. 13.1 19.5 .+-. 6.4 66.1 .+-. 14.2.sup.#
* * * * *