U.S. patent application number 11/337664 was filed with the patent office on 2006-08-10 for aminophenoxyacetamide derivatives and pharmaceutical composition containing thereof.
This patent application is currently assigned to DAIICHI ASUBIO PHARMA CO., LTD.. Invention is credited to Hirokazu Annoura, Norihito Murayama, Naohiro Takemoto.
Application Number | 20060178401 11/337664 |
Document ID | / |
Family ID | 18624324 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060178401 |
Kind Code |
A1 |
Takemoto; Naohiro ; et
al. |
August 10, 2006 |
Aminophenoxyacetamide derivatives and pharmaceutical composition
containing thereof
Abstract
The present invention relates to an amiophenoxyacetamide
derivative of the formula (I): ##STR1## wherein R.sup.1 to R.sup.4
are, independent from each other, a hydrogen atom or an optionally
substituted alkyl group; E.sup.1 is --NR.sup.4--; and E.sup.2 is an
oxygen atom or --NR.sup.10--; Q is the group --X--Y-Q', wherein X
and Y are connecting bonds or X is an alkylene or alkenylene group
and Y is selected from a group comprising C.dbd.O, NHC(.dbd.O), and
C(.dbd.O)NH, and Q' is a hydrogen atom or a phenyl or pyridyl group
which may be substituted; and pharmaceutically acceptable salts
thereof. The present invention further relates to compositions
comprising compounds of the formula (I) and methods of using said
compounds for treating cerebral functional disorders and cerebral
organic disorders.
Inventors: |
Takemoto; Naohiro; (Osaka,
JP) ; Annoura; Hirokazu; (Kyoto, JP) ;
Murayama; Norihito; (Osaka, JP) |
Correspondence
Address: |
DRINKER BIDDLE & REATH (DC)
1500 K STREET, N.W.
SUITE 1100
WASHINGTON
DC
20005-1209
US
|
Assignee: |
DAIICHI ASUBIO PHARMA CO.,
LTD.
|
Family ID: |
18624324 |
Appl. No.: |
11/337664 |
Filed: |
January 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10009566 |
Dec 12, 2001 |
7067533 |
|
|
PCT/JP01/03198 |
Apr 13, 2001 |
|
|
|
11337664 |
Jan 24, 2006 |
|
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|
Current U.S.
Class: |
514/317 ;
514/326; 546/208; 546/223 |
Current CPC
Class: |
A61P 25/16 20180101;
A61P 25/28 20180101; A61P 21/02 20180101; C07D 417/04 20130101;
A61P 43/00 20180101; A61P 25/00 20180101; A61P 9/00 20180101; A61P
9/10 20180101; C07D 211/58 20130101 |
Class at
Publication: |
514/317 ;
514/326; 546/208; 546/223 |
International
Class: |
A61K 31/454 20060101
A61K031/454; A61K 31/445 20060101 A61K031/445; C07D 211/56 20060101
C07D211/56; C07D 401/02 20060101 C07D401/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 13, 2000 |
JP |
2000-112100 |
Claims
1. An aminophenoxyacetamide derivative represented by the following
formula (I): ##STR48## wherein: R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 are, independent from each other, hydrogen atom or lower
alkyl group; R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are, independent
from each other, hydrogen atom or lower alkyl group; E.sup.1 is
group --NR.sup.9-- (in which, R.sup.9 is hydrogen atom or lower
alkyl group); E.sup.2 is oxygen atom or group --NR.sup.10-- (in
which, R.sup.10 is hydrogen atom or lower alkyl group which may be
substituted); Q is a group of --X--Y-Q', wherein X is a connecting
bond, lower alkyl group, lower alkenyl group, or lower alkynyl
group; Y is a connecting bond, or a group selected from the groups
consisting of C.dbd.O, C(.dbd.O)NH, NHC(.dbd.O), --O--, --S--,
CH(OH), --O--CH(OH)-- and --O--CH.sub.2--CH(OH)--, in which
hydrogen atom of amido group may be substituted with lower alkyl
group; and Q' is hydrogen atom or a cyclic group selected from the
groups consisting of aryl group, heteroaryl group, saturated or
unsaturated cyclic hydrocarbon group, and saturated or unsaturated
heterocyclic group, wherein one or more of the hydrogen atoms in
the cyclic group of Q' may be substituted; either in the case that
X and Y are both connecting bond then Q' is not hydrogen atom; or
in the case that one of X and Y is other than connecting bond then
E.sup.2 is the group --O-- and all of the groups of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are not hydrogen atom; or a
pharmaceutically acceptable salt thereof.
2. The aminophenoxyacetamide derivative of formula (I) claimed in
claim 1, wherein X and Y are both connecting bond; or
pharmaceutically acceptable salts thereof.
3. The aminophenoxyacetamide derivative of formula (I) claimed in
claim 1, wherein, one of X and Y is other than connecting bond and
E.sup.2 is the group --O-- and all of the groups of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 are other than hydrogen atom, wherein
X, Y, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are the same as defined
above in claim 1; or pharmaceutically acceptable salts thereof.
4. A composition comprising an aminophenoxyacetamide derivative or
a pharmaceutically acceptable salt thereof represented by the
formula (I) in claim 1 as an active ingredient.
5. A composition comprising an aminophenoxyacetamide derivative or
a pharmaceutically acceptable salt thereof represented by the
formula (I) in claim 2 as an active ingredient.
6. A composition comprising an aminophenoxyacetamide derivative or
a pharmaceutically acceptable salt thereof represented by the
formula (I) in claim 3 as an active ingredient.
7. A method for inducing the production of CalbindinD-28K wherein
said method comprises administering to a patient a composition
according to claim 4.
8. A method for inducing the production of CalbindinD-28K wherein
said method comprises administering to a patient a composition
according to claim 5.
9. A method for inducing the production of CalbindinD-28K wherein
said method comprises administering to a patient a composition
according to claim 6.
10. A method for improving or treating cerebral function and/or
organic function disorders wherein said method comprises
administering to a patient a composition according to claim 4.
11. The method of claim 10, wherein said cerebral function
disorders are caused by ischemic disorders.
12. The method of claim 11, wherein said ischemic disorders are
selected from the group consisting of cerebral infarction,
intracerebral hemorrhage and cerebral arteriosclerosis.
13. The method of claim 10, wherein said organic function disorders
are selected from the group consisting of senile dementia, cerebral
injury, cerebral operation, Alzheimer's disease, Parkinson's
disease, and amyotrophic lateral sclerosis.
14. A method for improving or treating cerebral function and/or
organic function disorders wherein said method comprises
administering to a patient a composition according to claim 5.
15. The method of claim 14, wherein said cerebral function
disorders are caused by ischemic disorders.
16. The method of claim 15, wherein said ischemic disorders are
selected from the group consisting of cerebral infarction,
intracerebral hemorrhage and cerebral arteriosclerosis.
17. The method of claim 14, wherein said organic function disorders
are selected from the group consisting of senile dementia, cerebral
injury, cerebral operation, Alzheimer's disease, Parkinson's
disease, and amyotrophic lateral sclerosis.
18. A method for improving or treating cerebral function and/or
organic function disorders wherein said method comprises
administering to a patient a composition according to claim 6.
19. The method of claim 18, wherein said cerebral function
disorders are caused by ischemic disorders.
20. The method of claim 19, wherein said ischemic disorders are
selected from the group consisting of cerebral infarction,
intracerebral hemorrhage and cerebral arteriosclerosis.
21. The method of claim 18, wherein said organic function disorders
are selected from the group consisting of senile dementia, cerebral
injury, cerebral operation, Alzheimer's disease, Parkinson's
disease, and amyotrophic lateral sclerosis.
22. A method for selecting a neuroprotective compound, wherein said
method comprises evaluating the activation of receptors for various
kinds of physiologically active substances and the phosphorylation
of the FGF receptor caused by the induction of CalbindinD-28k
production.
23. The method for selecting a neuroprotective compound according
to claim 22, wherein said method comprises evaluating the
autophosphorylation of the FGF receptor.
24. The method for selecting a neuroprotective compound according
to claim 22, wherein said method is performed by evaluating for
neuroprotective effect of the physiologically active substance
against glutamate-induced neurodegeneration together with one or
more of the following tests (i)-(iii): (i) evaluating for
antagonism against the neuroprotective effect of the
physiologically active substance by treatment with MTA
(5-deoxy-5-methyl-thioadenosine), which inhibits
autophosphorylation of the FGF receptor, and by treatment with
inhibitors of various physiologically active substance receptors,
to determine if the neuroprotective effect is due to
autophosphorylation of receptors of the FGF receptor; (ii)
evaluating the CalbindinD-28k inducing effect of the
physiologically active substance; or (iii) confirming that the
neuroprotective effect of the physiologically active substance is
due to its inducing CalbindinD-28k production, by treating with the
antisense oligonucleotide of CalbindinD-28k and determining if
CalbindinD-28k production is antagonized.
25. The method according to claim 22, wherein said physiological
active substance receptors are selected from the group consisting
of receptors for neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5),
brain-derived neurotrophic factor (BDNF), insulin-like growth
factor-I/II (IGF-I/II), platelet-derived growth factor (PDGF), and
estrogen.
26. A neuroprotective compound selected by the method according to
claim 22.
27. A composition comprising a neuroprotective compound according
to claim 26.
28. A method of treating or improving cerebral functional disorders
and/or cerebral organic disorders, wherein said method comprises
administering the composition according to claim 27 to a patient in
need thereof.
29. The method according to claim 28, wherein said cerebral
functional disorders are caused by ischemic disorders.
30. The method according to claim 29, wherein said ischemic
disorders are selected from the group consisting of cerebral
infarction, intracerebral hemorrhage and cerebral
arteriosclerosis.
31. The method according to claim 28, wherein said cerebral organic
disorders are selected from the group consisting of senile
dementia, cerebral injury, cerebral operation, Alzheimer's disease,
Parkinson's disease, and amyotrophic lateral sclerosis.
Description
RELATED APPLICATION DATA
[0001] This application is a continuation application of U.S.
patent application Ser. No. 10/009,566, filed Dec. 12, 2001, which
is a .sctn.371 application of PCT/JP01/03198 filed Apr. 13, 2001,
which claims priority to JP 2000-112100 filed Apr. 13, 2000. The
entire contents of each of these applications is incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to cerebral functional or
organic disorders improving and treating agents containing
aminophenoxyacetamide derivatives and pharmaceutically acceptable
salt thereof as an active ingredient, having neuroprotective effect
by inducing the production of CalbindinD-28k, one of
Ca.sup.2+-binding proteins, and to the methods for selecting these
neuroprotective aminophenoxyacetamide derivatives. More
specifically, the present invention relates to the therapeutic and
improving agents for various cerebral dysfunction due to various
ischemic disorders such as cerebral infarction, intracerebral
hemorrhage and cerebral arteriosclerosis. Furthermore, the present
invention relates to therapeutic and improving agents for various
cerebral organic disorders due to senile dementia, sequelae of
cerebral injury, or surgical operation, Alzheimer's disease,
Parkinson's disease, and amyotrophic lateral sclerosis, and
Huntington's disease, etc.
BACKGROUND ART
[0003] It is considered that the progressive and delayed death of
nerve cells, observed in cerebral injury and cerebrovascular
disease such as intracerebral hemorrhage, transient cerebral
ischemia, and cerebral infarction, is mainly caused by the increase
of the intracellular Ca.sup.2+ concentration, the various factors
of which are related to signal transduction to cause, for example,
the abnormal activation of receptors by over releasing glutamate
which is internal excitability, the activation of ion channels, and
the induction of reactive oxygen species/free radicals. [F. B.
Meyer, Brain Res. Rev., 14, 227 (1989); E. Boddeke et al., Trends
Pharmacol. Sci., 10, 397 (1989); J. M. McCall et al., Ann. Rep.
Med. Chem., 27, 31 (1992)].
[0004] From these points of view, antagonists for glutamate
receptors, calcium channel blockers, antioxidants and so on have
been applied for medicaments of preventing or suppressing the
neurodegeneration. However, these clinically used medicaments
suppress only a few pathways relating to the increase of the
cellular Ca.sup.2+ concentration, and therefore are not yet
sufficient enough for preventing or suppressing the
neurodegeneration.
[0005] On the contrary, the internal production of CalbindinD-28k
is induced by activation of receptors for many physiologically
active substance's such as FGF, NT-3, NT-4/5, BDNF, IGF-I/II, PDGF,
estrogen and so on, and as well as by activation of FGF receptor,
which is one of nerve growth factor receptors [C. V.-Abejon et el.,
Neuron, 15, 105 (1995); A. Silva et al., Brain Res. Bull., 1, 35
(2000)]. And CalbindinD-28k, one of Ca.sup.2+-binding proteins and
mainly distributed in vulnerable site against ischemic disorders in
the central nervous system, which is known to show buffer action
against the increase of intracellular Ca.sup.2+ concentration. [A.
M. Lacopino et al., Neurodegeneration, 3, 1 (1994); M. P. Mattson
et al., Neuron, 6, 41 (1991)]
[0006] Accordingly, it is expected to achieve sufficient
neuroprotective effects against the increase of intracellular
Ca.sup.2+ concentration caused by any kinds of pathways if
CalbindinD-28k, one of the Ca.sup.2+-binding proteins per se, can
be supplied in a cell. Namely, it is expected that medicaments
containing CalbindinD-28k would be extremely effective therapeutic
and improving agents against cerebral functional and due to various
ischemic disorders such as cerebral infarction, intracerebral
hemorrhage and cerebral arteriosclerosis. It is also expected to be
effective against cerebral dysfunction due to cerebral ischemic
disorders due to sequelae of senile dementia, cerebral injury and
surgical operation, Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis and so on.
[0007] However, it is very difficult and therefore it is not likely
to administer the CalbindinD-28k protein directly into the
desirable site in the central nervous system of a body in view of
the limitations existing in the pharmacological and pharmaceutical
methodology because CalbindinD-28k itself is an unstable macro
molecular weight protein having 28 Kd (Kilo Dalton) of molecular
weight.
[0008] On the other hand, the lower molecular weight compounds
capable of including the production of CalbindinD-28k protein can
be easily prepared into the various kinds of pharmaceutical
compositions by the conventional technique. Therefore, these lower
molecular weight compounds would induce the production of the
neuroprotective CalbindinD-28k protein once easily administered
into a body, showing the buffering action against the increase of
the intracellular Ca.sup.2+ concentration. That is, these lower
molecular weight compounds can be effective pharmaceutical
compounds for improving and treating cerebral functional and
organic disorders.
[0009] Under these circumstances, one objective of the present
invention is to select and to provide the lower molecular-weight
neuroprotective compounds capable of inducing the production of
CalbindinD-28k, one kind of Ca.sup.2+-binding proteins, via
phosphorylation of receptors of various physiologically active
substances, as well as to provide the pharmaceutical compositions
of low toxicity in suitable preparations such as intravenous
injectable solution.
[0010] The other objective of the present invention is to provide
the therapeutic and improving agents for cerebral functional
disorders due to various ischemic disorders such as cerebral
infarction, intracerebral hemorrhage and cerebral arteriosclerosis,
as well as cerebral organic disorders such as sequelae of senile
dementia, cerebral injury, or surgical operation, Alzheimer's
disease, Parkinson's disease and amyotrophic lateral sclerosis.
DISCLOSURE OF THE INVENTION
[0011] As one aspect of the present invention, it is provided
aminophenoxyacetamide derivatives represented by the following
formula (I): ##STR2## wherein,
[0012] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are, independent from
each other, hydrogen atom or lower alkyl group which may be
substituted;
[0013] R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are, independent from
each other, hydrogen atom or lower alkyl group which may be
substituted;
[0014] E.sup.1 is group --NR.sup.9 (in which, R.sup.9 is hydrogen
atom or alkyl group which may be substituted);
[0015] E.sup.2 is oxygen atom or group --NR.sup.10 (in which,
R.sup.10 is hydrogen atom; alkyl group which may be substituted;
aryl group which may be substituted or aralkyl group which may be
substituted); Q is a group of --X--Y-Q', in which X is a connecting
bond, lower alkyl group, lower alkenyl group or lower alkynyl
group; Y is a connecting bond, or a group selected from the groups
consisting of C.dbd.O, C(.dbd.O)NH, NHC(.dbd.O), --O--, --S--,
CH(OH), --O--CH(OH), and --O--CH.sub.2--CH(OH), in which hydrogen
atom of amido group may be substituted with lower alkyl group; and
Q' is hydrogen atom or a cyclic group selected from the groups
consisting of aryl group, heteroaryl group, saturated or
unsaturated cyclic hydrocarbon group, and saturated or unsaturated
heterocyclic group, wherein one or more of the hydrogen atom in the
cyclic group of Q' may be substituted;
[0016] provided that X and Y are both connecting bond then Q' is
not hydrogen atom; or provided that one of X and Y is other than
connecting bond then E.sup.2 is the group --O-- and all of the
groups of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are not hydrogen
atom;
or a pharmaceutically acceptable salt thereof.
[0017] In the description of a lower alkyl group may be
specifically a straight or branched alkyl group of the number of
carbon atoms from C.sub.1 to C.sub.6, for example, methyl, ethyl,
n-propyl, isopropyl and so on and more preferably, methyl or ethyl.
In the description of lower alkenyl group may be specifically
C.sub.1 to C.sub.6 alkenyl group, and lower alkynyl may be
specifically C.sub.1 to C.sub.6 alkynyl group.
[0018] Furthermore, the present invention provides the
aminophenoxyacetamide derivatives of the formula (I), in which;
[0019] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 all are methyl
group;
[0020] when E.sup.1 is oxygen atom; E.sup.2 is the group --NR.sup.9
(in which, R.sup.9 is hydrogen atom; alkyl group which may be
substituted; aryl group which may be substituted or aralkyl group
which may be substituted); or when E.sup.1 is group --NR.sup.10 (in
which, R.sup.10 is hydrogen atom; alkyl group which may be
substituted; aryl group which may be substituted; or aralkyl group
which may be substituted); E.sup.2 is oxygen atom;
[0021] R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are, independent from
each other, hydrogen atom or lower alkyl group;
[0022] Q is group --X--Y-Q' (in which, Q' is hydrogen atom, phenyl
group which may be substituted, pyridyl group which may be
substituted, quinolyl group which may be substituted, isoquinolyl
group which may be substituted; benzothiazole group which may be
substituted or benzimidazole group which may be substituted; or
pharmaceutically acceptable salts thereof.
[0023] More specifically, the following compound groups (1) to (4)
are the specific embodiments of the aminophenoxyacetamide
derivatives of the formula (I) of the present invention having the
excellent effect. [0024] (1) The aminophenoxyacetamide derivatives
claimed in claim 1, wherein;
[0025] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are, independent from
each other, hydrogen atom; or alkyl group which may be
substituted;
[0026] R.sup.5 is hydrogen atom or alkyl group which may be
substituted;
[0027] E.sup.1 is --NH--;
[0028] E.sup.2 is oxygen atom;
or pharmaceutically acceptable salts thereof.
[0029] (2) The aminophenoxyacetamide derivatives claimed in claim
1, wherein;
[0030] R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are, independent from
each other, hydrogen atom; or alkyl group which may be
substituted;
[0031] R.sup.5 is hydrogen atom or alkyl group which may be
substituted;
[0032] E.sup.1 and E.sup.2 are --NH--;
or pharmaceutically acceptable salts thereof.
[0033] (3) The aminophenoxyacetamide derivatives claimed in claim
2, wherein;
[0034] R.sup.5 is hydrogen atom or alkyl group which may be
substituted;
[0035] E.sup.1 is --NH--;
[0036] E.sup.2 is oxygen atom;
[0037] when X is connecting bond, Y is --CONH--; or when X is
--CONH--, Y is connecting bond;
[0038] Q' is phenyl group which may be substituted;
or pharmaceutically acceptable salts thereof.
[0039] (4) The aminophenoxyacetamide derivatives claimed in claim
2, wherein;
[0040] R.sup.5 is hydrogen atom or alkyl group which may be
substituted;
[0041] E.sup.1 is --NH--;
[0042] E.sup.2 is oxygen atom;
[0043] X is connecting bound or alkylene group and Y is one of the
groups consisting of --CH(OH)--, --O--CH(OH)--, and
--O--CH.sub.2--CH(OH)--;
[0044] Q' is phenyl group which may be substituted;
or pharmaceutically acceptable salts thereof.
[0045] According to the present inventor's investigations, it is
confirmed that the aminophenoxyacetamide derivatives represented by
the formula (I) effectively induced the production of
CalbindinD-28K in low concentration and possessed excellent
neuroprotective effect. Further, these compounds are also confirmed
to have high safety margin, and are suitable for preparation of
various kinds of pharmaceutical compositions.
[0046] Therefore, as a further embodiment, the present invention
provides an improving and therapeutic agent for the cerebral
functional and organic disorders containing aminophenoxyacetamide
derivatives represented by the formula (I) or pharmaceutically
acceptable salt thereof, as an active ingredient.
[0047] As another embodiment, the present invention provides
effective and simple method of selecting (screening) lower
molecular weight compounds capable of inducing the production of
the CalbindinD-28k, one of Ca.sup.2+-binding proteins.
[0048] The method of selecting low molecular weight compounds
consists of several evaluation tests mentioned below;
[0049] (1) Evaluation test to compare the neuroprotective effect of
the test compounds against glutamate-induced neurodegeneration,
between the administration thereof prior to the glutamate addition
and the simultaneous administration thereof.
[0050] (2) The test to confirm whether or not the aforementioned
neuroprotective effect is neuroprotective through phosphorylation
of receptors for various physiologically active substances. These
tests are conducted by the antagonistic effect of the inhibitors
for each of the receptors such as FGF NT-3, NT-4/5, BDNF, IGF-I/II,
PDGF, or estrogen, and MTA (5-Deoxy-5-Methylthioadenosine), which
specifically inhibits autophosphorylation of FGF receptor.
[0051] (3) Evaluation test of inducing capability for each test
compounds to produce CalbindinD-28k.
[0052] (4) Confirmation test for neuroprotective effect of
CalbindinD-28k by the inhibition using its antisense
oligonucleotide.
[0053] By the above stated evaluation tests, effective compounds
having the following features can be selected.
Evaluation Test (1):
[0054] This test is to evaluate whether the test compounds have
neuroprotective effect against glutamate induced neurodegeneration,
by administrating such testing compounds before or simultaneously
along with the glutamate to induce the neuronal cell injury.
[0055] If the test compound shows greater neuroprotective effect
against neurodegeneration induced by glutamate administration in
case of pre-treatment than that in case of simultaneously
treatment, then the compound may possess effect of inducing protein
like substance, which shows neuroprotective effect. Therefore, the
compound possessing neuroprotective effect based on the protein
like substance induced, including CalbindinD-28k, one of
Ca.sup.2+-binding proteins, is selected by this evaluation
test.
Evaluation Test (2):
[0056] In the case where neuroprotective effect disappears by the
administration of inhibitors to receptors such as FGF, NT-3,
NT-4/5, BDNF, IGF-I/II, PDGF and estrogen, then it is confirmed
that such neuroprotective effect is caused by the activation of
these receptors. Furthermore, in the living cell, MTA
(5-Deoxy-5-Methylthioadenosine) specifically inhibits
autophosphorylation of FGF receptors. Inhibition of neuroprotective
activity by the treatment with MTA (specific inhibitor for
self-physpholylation of FGF receptors) confirms that such
neuroprotective effect involves phosphorylation of FGF receptors.
Therefore, this evaluation test would select the compounds which
neuroprotective effect is expressed by the activation of receptors
of various physiologically active substances and through
phosphorylation of FGF receptor.
Evaluation Test (3):
[0057] The compound having effect of inducing CalbindinD-28k
production would be selected by this evaluation test.
Evaluation Test (4):
[0058] It is necessary for the protective protein to be produced
via the signal transduction of cells through the phosphorylation of
receptors of various physiologically active substances to provide
the neuroprotective effect of the compounds, and the CalbindinD-28k
is one of that protective proteins. Therefore, with this evaluation
test, the compound which has neuroprotective activity due to the
CalbindinD-28k production, is inhibited by using CalbindinD-28k
antisense. In this test, the compound having neuroprotective effect
is confirmed based on the CalbindinD-28k produced.
[0059] The present invention provides effective and simple
selecting method of lower molecular weight neuroprotective
compounds based on CalbindinD-28k production induced, by using all
of the evaluation tests, or using the combination of evaluation
tests (1) and (2), evaluation tests (1), (2) and (3), evaluation
tests (1) and (3) or evaluation tests (1), (3) and (4).
[0060] FIG. 1 shows the flow chart of the selecting methods of the
present invention to show the overview of selecting method of lower
molecular weight compounds possessing neuroprotective effect based
on CalbindinD-28k production induced, by combining aforementioned
evaluation tests.
[0061] In accordance with the selecting methods of the present
invention, the compounds specifically described in the description
of the present invention is selected as lower molecular weight
compounds possessing the inducing effect on the production of
CalbindinD-28k, one of Ca.sup.2+-binding protein. However, these
selecting methods can be applied to selecting various compounds
possessing neuroprotective effect based on activation of
physiologically active substance's receptors and CalbindinD-28k
production inducing effect involving autophosphorylation of FGF
receptor, and are not limited to the selection of the compounds
described in this specification.
BRIEF DESCRIPTION OF DRAWING
[0062] FIG. 1 shows the flow chart of the selecting methods of
lower molecular weight compounds possessing neuroprotective effect
based on production of CalbindinD-28k induced of the present
invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0063] The aminophenoxyacetamide derivatives of the present
invention include aminophenoxyacetamides, aminoanilinoacetamides,
aminothiophenoxyacetamides, oxyanilinoacetamides and
thioanilinoacetamides. Therefore, "aminophenoxyacetamide
derivatives" in this specification include all the derivatives
stated above as long as not stated otherwise.
[0064] In the aminophenoxyacetamide derivatives of the formula (I)
provided by the present invention with reference to various
substitution group of R.sup.1 to R.sup.10, "halogen atom" includes
fluorine atom, chlorine atom and bromine atom.
[0065] The term "alkoxy group" stands for a straight-chained or
branched-chained C.sub.1-C.sub.5 alkoxy group, and may include, for
example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,
sec-butoxy, tert-butoxy and the like.
[0066] The term "alkyl group which may be substituted" stands for a
straight-chained or branched-chained C.sub.1-C.sub.5 alkyl group
which may be halogen-substituted, and may include, for example,
methyl, ethyl, propyl, trifluoromethyl group, and the like.
[0067] The "aryl", a part of the term "aryl group which may be
substituted", stands for C.sub.4-C.sub.14 aryl group containing at
least one hetero atom(s) such as nitrogen and oxygen atom(s).
Examples of the preferred aryl group include phenyl, pyridyl and
naphthyl. The suitable substituents of said aryl group include
halogen atom such as fluorine atom, chlorine atom and bromine atom;
hydroxy group; a straight-chained or branched-chained
C.sub.1-C.sub.5 alkoxy group having 1 to 5 carbon atoms such as
methoxy group and ethoxy group; and a straight-chained or
branched-chained C.sub.1-C.sub.5 alkyl group which can be
substituted by halogen atom such as methyl, ethyl and
trifluoromethyl.
[0068] The "aralkyl", a part of the term "aralkyl group which may
be substituted", stands for C.sub.5-C.sub.12 aralkyl group
containing at least one hetero ring atom(s) such as nitrogen and
oxygen atom(s). The examples include benzyl, phenethyl,
pyridylmethyl, and pyridylethyl.
[0069] The suitable substituents of said aralkyl group include
halogen atoms such as fluorine atom, chlorine atom and bromine
atom; hydroxy group; a straight-chained or branched-chained
C.sub.1-C.sub.5 alkoxy group such as methoxy group and ethoxy
group; and a straight-chained or branched-chained C.sub.1-C.sub.5
alkyl group which can be substituted by halogen atom such as
methyl, ethyl and trifluoromethyl.
[0070] The "aryl", a part of the term "aryl group which may be
substituted" represented as "Q", stands for C.sub.4-C.sub.14 aryl
group which may contain at least one hetero atom(s) such as
nitrogen and oxygen atom(s). The examples include phenyl, pyridyl
and naphthyl. The suitable substituents of said aryl group include
halogen atom such as fluorine atom, chlorine atom and bromine atom;
hydroxy group; a straight-chained or branched-chained
C.sub.1-C.sub.5 alkoxy group having 1 to 5 carbon atoms such as
methoxy group and ethoxy group; and a straight-chained or
branched-chained C.sub.1-C.sub.5 alkyl group which can be
substituted by halogen atom such as methyl, ethyl and
trifluoromethyl. Furthermore, these substituents may also include a
straight-chained or branched-chained C.sub.1-C.sub.5 alkyl group
which may be substituted by halogen atom such as fluorine atom,
chlorine atom and bromine atom.
[0071] The "alkylene", a part of the term "alkylene group which may
be substituted by hydroxyl group", refers to the substituets "X"
and "Y", and preferably represents a straight-chained or
branched-chained C.sub.1-C.sub.6 alkylene group such as methylene,
methylmethylene, ethylene, trimethylene, tetramethylene,
cyclopropylmethylene and the like.
[0072] The term "cycloalkylene" preferably stands for
C.sub.3-C.sub.6 cycloalkylene and may include 1,1-cyclopropylene,
1,2-cyclopropylene, 1,1-cyclobutylene, 1,1-cyclopentylene,
1,1-cyclohexylene and the like. Among them, 1,1-cyclopropylene and
1,2-cyclopropylene are more preferable.
[0073] The "alkenylene", a part of the term "alkenylene group which
may be substituted by lower alkyl group", may include
C.sub.2-C.sub.4 alkenylene such as vinylene, and butadiene, and
vinylene is preferably used. The lower alkyl group, which is
substituent of alkenylene group, may be methyl, ethyl, propyl,
isopropyl and the like.
[0074] The term "connected bond" with reference to "X" and "Y"
means direct bond. Therefore, if "X" and/or "Y" are connected bond,
two adjacent substituents of "X" and/or "Y" are connected directly,
and these substituents do not exist as "X" and/or "Y".
[0075] The suitable substituents represented as "Q" for "phenyl
group which may be substituted", "phenoxy group which may be
substituted", "benzoyl group which may be substituted", "pyridyl
group which may be substituted", "quinolyl group which may be
substituted", "isoquinolyl group which may be substituted",
"benzothiazole group which may be substituted" and "benzimidazolyl
group which may be substituted", may include halogen atom such as
fluorine atom, chlorine atom and bromine atom; hydroxy group; a
straight-chained or branched-chained C.sub.1-C.sub.5 alkoxy group
such as methoxy, ethoxy group and so on. Furthermore, these
substituents may also include a straight-chained or
branched-chained C.sub.1-C.sub.5 alkyl group which may be
substituted by halogen atom such as methyl, ethyl, trifluoromethyl
and the like.
[0076] It is understood that when the aminophenoxyacetamide
derivatives of the formula (I) of the present invention exist in
the isomer forms, each isomers per se, as well as the isomeric
mixture, shall be included in the compounds of the present
invention. Namely, the structural isomers may exist due to the
substituents on the benzene ring. Furthermore, optical isomers may
exist due to the asymmetric carbon atom of the hydroxy substituted
"X" or "Y" of alkylene group. These isomers shall be included
within the scope of the compounds of the present invention.
[0077] The aminophenoxyacetamide derivatives of the formula (I)
include the compounds (Ia), (Ib), (Ic) and (Id) obtained by the
synthetic process mentioned latter. For example, these compounds
may be prepared by the following.
[0078] The compound (IV), obtained by the reaction of the compound
(II) with the ester derivative (III), is hydrolyzed to convert into
the carboxylic acid derivative (V). Furthermore, the compound
(VIII) is obtained by the reaction of the amine derivative (VI)
with the compound (VII), and the protecting group of the compound
(VIII) is removed to obtain the amine derivative (IX). Then, the
obtained compound (V) is converted into amide compound (X) by the
condensation reaction with the compound (IX). Further, the
protecting group in the compound (X) thus obtained is removed to
obtain compound (Ia), the compound of formula (I) in the claim 1 of
the present invention (Process 1).
[0079] The compound (Ib), the aminophenoxyacetamide derivative of
formula (I) in the claim 2 of the present invention, can be
obtained by the following. The amide compound (XII) is obtained by
condensation reaction of the carboxylic acid derivative (V'), which
is obtained in the Process 1, with compound (XI), and the
protecting group of the resultant was removed (Process 2).
[0080] The compound (Ib), obtained in the Process 2, can be
converted to the compound (Ic) by the reaction with the compound
(XIII) (Process 3).
[0081] Furthermore, the compound (Id) can be obtained by reacting
the compound (Ib) with the compound (XIV) (Process 4).
[0082] Each process will be further illustrated by the following
reaction scheme. ##STR3## wherein, R.sup.1 to R .sup.8, E.sup.1 and
E .sup.2 have the same definitions as above; Q has the same meaning
as defined in claim 1; and R.sup.11 is alkyl group which may be
substituted, aryl group which may be substituted; aralkyl group
which may be substituted; tert-butoxycarbonyl group; ethoxycarbonyl
group; acetyl group; benzyloxycarbonyl group;
p-methoxybenzyloxycarbonyl group; R.sup.12 is a straight-chained or
branched-chained C.sub.1-C.sub.5 alkyl group; L.sup.1 is leaving
group which can easily be replaced with amino, hydroxy and mercapto
group; L.sup.2 is leaving group which can be easily replaced with
amino, and boric acid; P.sup.1 is tert-butoxycarbonyl group,
ethoxycarbonyl group, acetyl group, benzyloxycarbonyl group,
p-methoxybenzyloxycarbonyl group, benzyl group or trifluoroacetyl
group.
[0083] According to this process 1, the compound (Ia) can be
obtained from the known starting compound (II).
[0084] Namely, for the first step, the compound (II) is reacted
with 1.0 to 1.5 mole equivalent of ester compound (III) in the
inert solvent, and if necessary in the presence of the base, under
stirring at -20.degree. C. to 150.degree. C., preferably at
0.degree. C. to 100.degree. C.
[0085] The inert solvent to be used in the reaction may be benzene,
toluene, tetrahydrofuran, dioxane, dimethyformamide, dimethyl
sulfoxide, acetonitrile, acetone, methanol, ethanol, isopropyl
alcohol, tert-butyl alcohol, ethylene glycol, diethyl ether and the
like.
[0086] The base to be used in the above reaction may be an organic
base such as triethylamine, diisopropylethylamine, pyridine and the
like, or an inorganic base such as sodium, sodium hydride,
potassium, potassium hydride, sodium methoxide, potassium
tert-butoxide, sodium carbonate, potassium carbonate, cesium
carbonate, cesium fluoride, sodium bicarbonate, potassium
bicarbonate and the like. These organic base and inorganic base may
be used in combination, and sodium iodide, potassium iodide or
tetrabutylammonium iodide can be added in the reaction mixture.
[0087] The substituent "L.sup.1" in the ester derivative (III) may
be the leaving group which can easily be replaced with amino,
hydroxy or mercapto group, and examples include halogen atom such
as chlorine atom, bromine atom, iodide atom; alkylsulfonyloxy group
such as methanesulfonyloxy group; arylsulfonyloxy group such as
p-toluenesulfonyloxy group, 3-nitrobenzenesulfonyloxy group and the
like.
[0088] The compound (II) and compound (III) to be used in this
reaction can be commercially available and known compounds, or can
be easily prepared from known compounds by using common
methods.
[0089] Examples of the compound (II) include
4-(tert-butoxy-carbonylamino)phenol,
4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylphenol,
2-(tert-butoxycarbonylamino)-3,4,5,6-tetramethylphenol,
3-(tert-butoxycarbonylamino)-2,4,5,6-tetramethylphenol,
4-(tert-butoxycarbonylamino)-2,3,5-trimethylphenol,
4-(tert-butoxycarbonylamino)-2-chloro-3,5,6-trimethylphenol,
4-(tert-butoxycarbonylamino)-2,3,6-trimethylphenol,
4-(tert-butoxycarbonylamino)-2,3-dimethylphenol,
4-(tert-butoxycarbonylamino)-2,5-dimethylphenol,
2-(tert-butoxycarbonylamino)-4,6-dimethylphenol,
5-(tert-butoxycarbonylamino)-2-methoxyphenol,
5-(tert-butoxycarbonylamino)-4-chloro-2-methoxyphenol,
4-(tert-butoxy-carbonylamino)-2,6-dichlorophenol,
4-(tert-butoxycarbonylamino)-2,3,4,6-tetramethylaniline,
4-methoxy-2-methylaniline,
4-(tert-butoxycarbonylamino)-2,5-dimethylaniline,
2-(tert-butoxycarbonylamino)-4,5-dimethylaniline,
3-(tert-butoxycarbonylamino)-2,4,6-trimethylaniline,
2-(tert-butoxycarbonylamino)-4,5-dimethylaniline,
4-(tert-butoxycarbonylamino)-2,5-dichloroaniline,
4-(tert-butoxycarbonylamino)-2,6-dichloroaniline,
2-(tert-butoxycarbonylamino)-4,5-dichloroaniline,
4-(tert-butoxycarbonylamino)-2-methoxy-5-methylaniline,
4-(tert-butoxycarbonylamino)-2,5-dimethoxyaniline,
4-(benzyloxycarbonylamino)phenol,
4-(benzyloxycarbonylamino)-2,3,5,6-tetramethylphenol,
2-(benzyloxycarbonylamino)-3,4,5,6-tetramethylphenol,
3-(benzyloxycarbonylamino)-2,4,5,6-tetramethylphenol,
4-(benzyloxycarbonylamino)-2,3,5-trimethylphenol,
4-(benzyloxycarbonylamino)-2-chloro-3,5,6-trimethylphenol,
4-(benzyloxycarbonylamino)-2,3,6-trimethylphenol,
4-(benzyloxycarbonylamino)-2,3-dimethylphenol,
4-(benzyloxycarbonylamino)-2,5-dimethylphenol,
2-(benzyloxycarbonylamino)-4,6-dimethylphenol,
5-(benzyloxycarbonylamino)-2-methoxyphenol,
5-(benzyloxycarbonylamino)-4-chloro-2-methoxyphenol,
4-(benzyloxycarbonylamino)-2,6-dichlorophenol,
4-(benzyloxycarbonylamino)-2,3,4,6-tetramethylaniline,
4-methoxy-2-methylaniline,
4-(benzyloxycarbonylamino)-2,5-dimethylaniline,
2-(benzyloxycarbonylamino)-4,5-dimethylaniline,
3-(benzyloxycarbonylamino)-2,4,6-trimethylaniline,
2-(benzyloxycarbonylamino)-4,5-dimethylaniline,
4-(benzyloxycarbonylamino)-2,5-dichloroaniline,
4-(benzyloxycarbonylamino)-2,6-dichloroaniline,
2-(benzyloxycarbonylamino)-4,5-dichloroaniline,
4-(benzyloxycarbonylamino)-2-methoxy-5-methylaniline,
4-(benzyloxycarbonylamino)-2,5-dimethoxyaniline and so on.
[0090] The ester compound of the formula (III) includes, for
example, ethyl bromoacetate, ethyl 2-bromopropionate, ethyl
2-bromo-2-methylpropionate, and so on.
[0091] Then, the obtained compound (IV) is hydrogenated to convert
into carboxylic acid derivative (V) by the common methods.
[0092] The compound (IX) to be used for the condensation reaction
with the above-obtained carboxylic acid derivative (V) can be
obtained by the following manner.
[0093] Namely, for the first step, the amine derivative (VI) is
conducted by the condensation reaction with the compound (VII) in
the inert solvent, and if necessary in the presence of the base,
under stirring at the room temperature to 180.degree. C., to obtain
the compound (VIII).
[0094] The inert solvent to be used in the reaction may be benzene,
toluene, xylene, diethylaniline, tetrahydrofuran, diethylether,
dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform,
methanol, ethanol, propane-2-ol, butyl alcohol and the like.
[0095] The base to be used in the above reaction may be an organic
base such as triethylamine, diisopropylamine, and the like, or an
inorganic base such as sodium hydride, potassium hydride, sodium
tert-butoxide, potassium, tert-butoxide, sodium ethoxide, sodium
carbonate, sodium bicarbonate, cesium carbonate and the like.
[0096] The reaction of the amine compound (VI) with the compound
(VII) can also be conducted in the inert solvent such as benzene,
toluene, xylene and tetrahydrofuran, and in the presence of
palladium catalyst such as tris(dibenzylidene-acetone)dipalladium,
diacetoxypalladium, palladium chloride and the like, phosphine
coordination compound such as tir-n-butylphosphine,
tri-tert-butylphosphine, tri-o-tolylphosphine, BINAP and the like,
and the base such as sodium tert-butoxide and cesium carbonate
under stirring at 50.degree. C. to 150.degree. C.
[0097] Furthermore, the reaction of the compound (VII), in which
the substitute "L.sup.2" is boronic acid residue, with the amine
compound (VI) can be conducted in the inert solvent, and in the
presence of the base and 1.0 to 2.0 mole equivalent of copper
acetate (CuOAc.sub.2), under stirring at the room temperature to
100.degree. C. [D. M. T. Chan et al., Tetrahedron Letters, 39, 2933
(1998)].
[0098] The inert solvent to be used in this reaction may be
dichloromethane, chloroform and the like, and the base may be
triethylamine, pyridine and the like.
[0099] The compound (VI) to be used for the reaction with the
compound (VII) is known compound (cf. R. H. Mach et al., J. Med.
Chem., 36, 3707 (1993)), or can be easily prepared by the methods
described in EP 0184257 A1 [R. A. Stokbroekx, et al.].
[0100] Then, the protecting group at nitrogen atom of the compound
(VIII) thus obtained is removed to obtain the amine derivative
(IX).
[0101] This reaction may vary depending on the protecting group on
the nitrogen atom of the compound (VIII). For example, the compound
(VIII) is treated with acids such as acetic acid, trifluoroacetic
acid, methanesulfonic acid, trifluoromethane-sulfonic acid,
hydrochloric acid, sulfuric acid, or nitric acid in an inert
solvent such as benzene, toluene, acetonitrile, tetrahydrofuran,
dioxane, dichloromethane, chloroform, carbon tetrachloride, water,
methanol, ethanol, and the like.
[0102] Furthermore, the removal of the protecting group may also be
carried out by hydrogenolysis of the compound (VIII) under 1 to 5
atom of hydrogen, in the presence of a catalyst such as
palladium-carbon, palladium hydroxide, platinum, or platinum oxide,
in an inert solvent such as methanol, ethanol, isopropyl alcohol,
ethyl acetate or acetic acid.
[0103] Then, the carboxylic acid derivative of the formula (V) is
converted into amide derivative (X) by reaction with the compound
(IX).
[0104] The reaction conditions of this amidation reaction may vary
according to the methods described in "Compendium for Organic
Synthesis" (wiley-Interscience: A Division of John Wiley & Sons
Ltd.).
[0105] For example, the compound (V) is treated, optionally in the
presence of an organic or an inorganic base, with diethyl
cyanophosphonate (DEPC), diphenylphosphoryl azide (DPPA),
dicyclohexylcarbodiimide (DCC),
1-ethyl-3-(dimethylamino-propyl)carbodiimide hydrochloride or
2-iodo-1-methyl-pyridinium iodide, and then reacted with compound
(IX) to obtain the amide compound (X). Furthermore, the compound
(V) is converted into the activated ester compound such as acid
halide, symmetric acid anhydride, or mixture acid anhydride, and
then, reacted with the compound (IX) to obtain the amide compound
(X).
[0106] The compound (X) thus obtained is converted into the
aminophenoxyacetamide derivatives of the formula (Ia), the compound
of the present invention, by the removal reaction of the protecting
group on the nitrogen atom of the amide compound (X).
[0107] Although each compounds obtained in the above process 1 may
be used for the next reaction without further purification, it can
also be used after further purification if necessary in
conventional manner such as recrystallization or column
chromatography and so on. ##STR4## wherein, R.sup.5 to R.sup.8 and
R.sup.11 have the same definitions as above, E.sup.1 and E.sup.2
have the same meanings as defined in claim 2, and P.sup.2 is
tert-butoxycarbonyl group, ethoxycarbonyl group, acetyl group,
benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, benzyl
group or trifluoroacetyl group.
[0108] According to this process 2, the aminophenoxyacetamide
derivative of the formula (Ib) can be synthesized from the compound
(V') [wherein, R.sup.1 to R.sup.4 are methyl groups, E.sup.1 is
oxygen atom and E.sup.2 is --NR.sup.9; or E.sup.1 is --NR.sup.10
and E.sup.2 is oxygen atom] obtained in the process 1 mentioned
above.
[0109] Namely, the compound (V') [wherein, R.sup.1 to R.sup.4 are
methyl groups, E.sup.1 is oxygen atom and E.sup.2 is --NR.sup.9; or
E.sup.1 is --NR.sup.10 and E.sup.2 is oxygen atom] is reacted with
the compound (XI) to obtain the amide compound (XII), and then, the
protecting group of the resultant compound (XII) is removed off to
give the aminophenoxyacetamide derivative (Ib).
[0110] This reaction may be carried out by the same manner as
described in the Process 1. ##STR5## wherein, R.sup.5 to R.sup.8
and have the same definitions as above, n, X, Y, Q', E.sup.1 and
E.sup.2 have the same meanings as defined in claim 2.
[0111] According to this process 3, the aminophenoxyacetamide
derivative of the formula (Ic) can be obtained from the compound
(Ib) by reacting with the compound (XIII).
[0112] Namely, the compound (Ib) is reacted with 1.0 to 1.5 mole
equivalent of the compound (XIII) in the inert solvent such as
benzene, toluene, tetrahydrofuran, dioxan, dimethylformamide,
dimethyl sulfoxide, acetonitrile, acetone, ether, dichloromethane,
chloroform and carbon tetrachloride in the presence of the base, at
-50.degree. C. to 120.degree. C., preferably at -20.degree. C. to
80.degree. C.
[0113] The base to be used in the reaction may be an organic base
such as triethylamine, diisopropylethylamine, pyridine and the
like, or an inorganic base such as sodium, sodium hydride,
potassium, potassium hydride, sodium ethoxide, sodium
tert-butoxide, sodium carbonate, potassium carbonate, cesium
carbonate, cesium fluoride, sodium bicarbonate, potassium
bicarbonate and the like. Sodium iodide, potassium iodide or
tetrabutylammonium iodide can be added in the reaction mixture.
[0114] The substituent "L.sup.3" in the compound (XIII) is the
leaving group, which can easily be replaced by amino group, and
examples include halogen atom such as chlorine atom, bromine atom,
iodine atom; alkylsulfonyloxy group such as methanesulfonyloxy
group; arylsulfonyloxy group such as p-toluenesulfonyloxy group and
the like.
[0115] In this process 3, the aminophenoxyacetamide derivative of
the formula (Ic) can be produced as well. ##STR6## wherein, R.sup.5
to R.sup.8 and L.sup.3 have the same definitions as previously
mentioned, Q', E.sup.1 and E.sup.2 are the same meanings as defined
in the claim 2, and m is integer 0 to 3.
[0116] According to this process 4, the aminophenoxyacetamide
derivative of the formula (Id) of the present invention can be
obtained from the reaction of the compound (Ib), obtained in the
process 2 mentioned above, with the compound (XIVa) or the compound
(XIVb).
[0117] For example, the compound (Ib) is reacted with 0.9 to 1.5
moles equivalent of the compound (XIVa) or (XIVb) in an inert
solvent at from room temperature to about 200.degree. C.,
preferably at about 50.degree. C. to about 150.degree. C., to
produce the aminophenoxyacetamide of the formula (Id).
[0118] The inert solvent to be used in the reaction may be benzene,
toluene, tetrahydrofuran, diethyl ether, ethylene glycol dimethyl
ether, dioxane, dimethyformamide, dimethyl sulfoxide, acetonitrile,
methanol, ethanol, isopropyl alcohol, tert-butyl alcohol, ethylene
glycol and the like.
[0119] Examples of the compound (XIVa) include epibromohydrin,
epichlorohydrin, (R)-epichlorohydrin, (S)-epichlorohydrin and the
like, and examples of the compound (XIVb) include glycidyl
tosylate, (R)-glycidyl tosylate, (S)-glycidyl tosylate,
(R)-glycidyl 3-nitro-benzensulfonate, (S)-glycidyl
3-nitrobenzesulfonate, (R)-glycidyl 4-nitro-benzoate, (S)-glycidyl
4-nitrobenzoate, gylcidyltrimethylammonium chloride and the
like.
[0120] In this process 4, the aminophenoxyacetamide derivative of
the formula (Id) can be produced as well.
[0121] The aminophenoxyacetamide derivatives of the formula (I)
thus obtained may be isolated and purified in conventional manner,
such as recrystallization, column chromatography and the like.
[0122] Further, each isomers contained in the compounds of the
formula (I) of the present invention can be obtained by resolution
of the isomeric mixture of these compounds by the conventional
methods, such as recrystallization, column chromatography, HPLC,
and the like, or by using optically active reagents.
[0123] The aminophenoxyacetamide derivatives of the present
invention represented by the formula (I) may be used in the form of
free bases or suitable pharmaceutically acceptable acid addition
salts thereof. The pharmaceutically acceptable salts can be
obtained by treating the compound (I) with an inorganic acid or an
organic acid in suitable organic solvent such as ether,
tetrahydrofuran, dichloromethane, chloroform, benzene, toluene,
methanol, isopropanol, ethanol and the like.
[0124] Examples of the inorganic acid include hydrochloric acid,
sulfuric acid, hydrobromic acid, phosphoric acid, periodic acid and
the like. Further, examples of the organic acid include formic
acid, acetic acid, butyric acid, oxalic acid, malonic acid,
propionic acid, valeric acid, succinic acid, fumaric acid, maleic
acid, tartaric acid, citric acid, malic acid, benzoic acid,
p-toluenesulfonic acid, methanesulfonic acid and the like.
[0125] The aminophenoxyacetamide derivatives of the present
invention represented by the formula (I) or pharmaceutically
acceptable salts thereof shows low toxicity and may be administered
per se. However, it may be converted in the form of
pharmaceutically acceptable composition with the conventional
pharmaceutically acceptable carriers for improvement or treatment
of various kinds of diseases due to cerebral functional or organic
disorder.
[0126] The dosage forms may include oral formulations such as
capsules, tablets or parenteral formulations such as injection
solution containing the compound of the formula (I) per se, or
using the conventional excipients. For example, the capsules can be
prepared by mixing the compound of the formula (I) in powder form
with a suitable excipient such as lactose, starch or derivatives
thereof or cellulose derivatives, and then filled in gelatin
capsules.
[0127] Also, the tablets can be prepared by mixing the active
ingredients with the above-mentioned excipients, binders such as
sodium carboxymethylcellulose, alginic acid or gum arabic and
water, then if necessary, making the resultant mixture into
granules. Then, it may be further mixed with lubricant such as talc
or stearic acid, and compressed into tablet by mean of common
tableting machine.
[0128] Injectable formulations for parenteral route also can be
prepared by dissolving the compound of the formula (I) or salts
thereof in sterile distilled solution or sterile physiological
saline solution with solution adjuvant, and filling it into ample.
A stabilizer or buffer can be used in the injectable solution, and
the injectable formulation may be administered intravenously or by
dripping.
[0129] In administration of the compound of the formula (I), which
possesses neuroprotective effect by induction of CalbindinD-28k,
one of Ca.sup.2+-bindind proteins, the therapeutically effective
dosage for improving cerebral functional and organic disorders is
not particularly limited and may vary depending on the various
kinds of factors. These factors may be the patient's condition, the
severity of the disease, age, existence of a complication,
administration route, formulation, as well as number of times for
administration.
[0130] A usual recommended daily dose for oral administration is
within the range of 0.1-1,000 mg/day/person, preferably 1-500
mg/day/person, while a usual recommended daily dose for parenteral
administration is within the range of 1/100 to 1/2 based on dose of
the oral administration. These doses also may vary depending on
age, as well as the patient's condition.
EXAMPLES
[0131] The present invention is illustrated in more detail by way
of the following examples, but it is to be noted that the present
invention is not limited by these Examples in any way.
[0132] The compound numbers in the following examples are identical
to those of the Table's mentioned later.
Example 1
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-(4-piperidinyl)acetamide
(1)
[0133] A solution of 457 mg of
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylphenoxy]acetic
acid, 363 mg of
1-(tert-butoxy-carbonylamino)-4-methylaminopiperidine, 2.16 g of
25% propane phosphonic acid anhydride [Japanese Patent Kokai Showa
55-100346] in ethyl acetate solution and 985 .mu.l of triethylamine
in 5 ml of dichloromethane was stirred over night under room
temperature. After the reaction, saturated sodium hydrogen
carbonate aqueous solution was added to the reaction mixture and
the mixture was extracted with dichloromethane. The extract was
washed with saline, dried and concentrated under reduced pressure
to give the residue. The obtained residue was dissolved in 8 ml of
dichloromethane, and to this solution was added 2 ml of
trifluoroacetic acid under ice-cooling, then the mixture was
stirred for 1 hour at the room temperature. After removal of the
solvent, the resultant residue was purified by amine-coated silica
gel (Fuji Silysia Chemical Ltd.) column chromatography
(dichloromethane:methanol=30:1) to give 192 mg (42%) of the
above-mentioned compound (1).
Example 2
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-(4-piperidinyl)propanamide
(2)
[0134] The title compound (2) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylphenoxy)propionic
acid and 1-(tert-butoxycarbonylamino)-4-methylaminopiperidine by
the same manner as the Example 1.
Example 3
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-2-methyl-N-methyl-N-(4-piperidinyl)-
propanamide (3)
[0135] The title compound (3) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylphenoxy]-2-methyl
propionic acid and
1-(tert-butoxycarbonylamino)-4-methylaminopiperidine by the same
manner as the Example 1.
Example 4
2-(2-Amino-3,4,5,6-tetramethylphenoxy)-N-methyl-N-(4-piperidinyl)acetamide
(4)
[0136] The title compound (4) was obtained from
2-[2-(tert-butoxycarbonylamino)-3,4,5,6-tetramethylphenoxy]acetic
acid and 1-(tert-butoxylcarbonylamino)-4-methylaminopiperidine by
the same manner as the Example 1.
Example 5
2-(3-Amino-2,4,5,6-tetramethylphenoxy)-N-methyl-N-(4-piperidinyl)acetamide
(5)
[0137] The title compound (5) was obtained from
2-[3-(tert-butoxycarbonylamino)-2,4,5,6-tetramethylphenoxy]acetic,
acid and 1-(tert-butoxycarbonylamino)-4-methylaminopiperidine by
the same manner as the Example 1.
Example 6
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-(1-phenethyl-4-piperidin-
yl)acetamide (6)
[0138] To a mixture solution of 99 mg of the compound (1) obtained
in the Example 1 and 42.3 .mu.l of phenethyl bromide in 2 ml of
acetonitrile was added 65 .mu.l of triethylamine, and the mixture
was stirred for 5 hours at 60.degree. C. After the reaction,
saturated sodium hydrogen carbonate aqueous solution was added to
the reaction mixture and the mixture was extracted with ethyl
acetate. The extract was washed with saline, dried and concentrated
under reduced pressure to give the residue. The obtained residue
was purified by amine-coated silica gel (Fuji Silysia Chemical
Ltd.) column chromatography (dichloromethane:ether=1:1) to give 86
mg (65%) of the above-mentioned compound (6).
Example 7
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-[1-(2-anilino-2-oxoethyl)-4-piper-
idinyl]-N-methylacetamide (7)
[0139] The title compound (7) was obtained from the compound (1)
obtained in the Example 1 and N-phenyl-2-bromoacetamide by the same
manner as the Example 6.
Example 8
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-(1-benzoyl-4-piperidinyl)-N-methy-
lacetamide (8)
[0140] The title compound (8) was obtained from the compound (1)
obtained in the Example 1 and benzoyl chloride by the same manner
as the Example 6.
Example 9
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-(1-butyl-4-piperidinyl)-N-methyla-
cetamide (9)
[0141] The title compound (9) was obtained from the compound (1)
obtained in the Example 1 and butyl bromide by the same manner as
the Example 6.
Example 10
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-[1-(2-phenyl-2-oxoethyl)-4-piperi-
dinyl]-N-methylacetamide (10)
[0142] The title compound (10) was obtained from the compound (1)
obtained in the Example 1 and phenacyl bromide by the same manner
as the Example 6.
Example 11
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-[1-(2-hydroxy-2-phenylethyl)-4-pi-
peridinyl]-N-methylacetamide (11)
[0143] To a mixture solution of the compound (10) obtained in the
Example 10 in methanol was added 1.0 equivalent of sodium
borohydride at 0.degree. C., and the mixture was stirred for 1.5
hours at the room temperature. After the reaction, the solvent was
removed under reduced pressure, and the resulting residue was
purified by amine-coated silica gel (Fuji Silysia Chemical Ltd.)
column chromatography (dichloromethane:methanol=20:1) to give the
above-mentioned compound (11) in the yield of 58%.
Example 12
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-(1-cyclopropylmethyl-4-piperidiny-
l)-N-methylacetamide (12)
[0144] The title compound (12) was obtained from the compound (1)
obtained in the Example 1 and cyclopropylmethyl bromide by the same
manner as the Example 6.
Example 13
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-[1-(trans-2-phenyl-1-cyc-
lopropylmethyl)-4-piperidinyl]acetamide (13)
[0145] The title compound (13) was obtained from the compound (1)
obtained in the Example 1 and trans-2-phenyl-1-cyclopropylmethyl
bromide by the same manner as the Example 6.
Example 14
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-[1-(2-phenoxyethyl)-4-pi-
peridinyl]acetamide (14)
[0146] The title compound (14) was obtained from the compound (1)
obtained in the Example 1 and 2-phenoxyethyl bromide by the same
manner as the Example 6.
Example 15
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-{1-[2-(N-methylanilino)--
2-oxoethyl]-4-piperidinyl}acetamide (15)
[0147] The title compound (15) was obtained from the compound (1)
obtained in the Example 1 and N-methyl-N-phenyl-2-bromoacetamide by
the same manner as the Example 6.
Example 16
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-{1-[2-(4-morpholinyl)eth-
yl]-4-piperidinyl}acetamide (16)
[0148] The title compound (16) was obtained from the compound (1)
obtained in the Example 1 and N-(2-bromoethyl)morpholine
hydrochloride by the same manner as the Example 6.
Example 17
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-{1-[2-(2-hydroxy-2-phenylethoxy)e-
thyl]-4-piperidinyl}-N-methylacetamide (17)
[0149] The title compound (17) was obtained from the compound (1)
obtained in the Example 1 and 2-(chloroethoxy)-1-phenylethanol by
the same manner as the Example 6.
Example 18
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-[1-(4-cyanobenzyl)-4-piperidinyl]-
-N-methylacetamide (18)
[0150] The title compound (18) was obtained from the compound (1)
obtained in the Example 1 and 4-cyanobenzyl bromide by the same
manner as the Example 6.
Example 19
4-({4-[[(4-Amino-2,3,5,6-tetramethylphenoxy)acetyl]-(methyl)amino]-1-piper-
idinyl}methyl)benzamide (19)
[0151] To a mixture solution of 82 mg of the compound (18) obtained
in the Example 18 in methanol were added 58 .mu.l of 30% hydrogen
peroxide aqueous solution and 150 .mu.l of 3N-sodium hydroxide
aqueous solution under ice-cooling, and the reaction mixture was
stirred for 6 hours at the room the temperature. After the
reaction, saturated sodium hydrogen carbonate aqueous solution was
added to the reaction mixture, and extracted with dichloromethane.
The organic layer was washed with saturated saline, dried and the
solvent was removed under reduced pressure. The resulting residue
was purified by amine-coated silica gel (Fuji Silysia Chemical
Ltd.) column chromatography (dichloromethane:methanol=10:1) to give
66 mg (77%) of the above-mentioned compound (19).
Example 20
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-{1-[2-(phenylthio)ethyl]-
-4-piperidinyl}acetamide (20)
[0152] The title compound (20) was obtained from the compound (1)
obtained in the Example 1 and 2-(chloroethyl)phenyl sulfide by the
same manner as the Example 6.
Example 21
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-[1-(2-propynyl)-4-piperi-
dinyl]acetamide (21)
[0153] The title compound (21) was obtained from the compound (1)
obtained in the Example 1 and propargyl bromide by the same manner
as the Example 6.
Example 22
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-[1-(1-methyl-2-phenyleth-
yl)-4-piperidinyl]acetamide (22)
[0154] The title compound (22) was obtained from the compound (1)
obtained in the Example 1 and 2-bromo-1-phenylpropane by the same
manner as the Example 6.
Example 23
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-(1-cyclopropylmethyl-4-piperidiny-
l)-N-methylpropanamide (23)
[0155] The title compound (23) was obtained from the compound (2)
obtained in the Example 2 and cyclopropylmethyl bromide by the same
manner as the Example 6.
Example 24
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-(1-butyl-4-piperidinyl)-N-methylp-
ropanamide (24)
[0156] The title compound (24) was obtained from the compound (2)
obtained in the Example 2 and 1-bromobutane by the same manner as
the Example 6.
Example 25
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-{1-[2-(4-morpholinyl)eth-
yl]-4-piperidinyl}propanamide (25)
[0157] The title compound (25) was obtained from the compound (2)
obtained in the Example 2 and N-(2-bromoethyl)morpholine
hydrochloride by the same manner as the Example 6.
Example 26
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-[1-(trans-2-phenyl-1-cyc-
lopropylmethyl)-4-piperidinyl]propanamide (26)
[0158] The title compound (26) was obtained from the compound (2)
obtained in the Example 2 and trans-2-phenyl-1-cyclopropylmethyl
bromide by the same manner as the Example 6.
Example 27
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N-methyl-N-{1-[2-(N-methylanilino)--
2-oxoethyl]-4-piperidinyl}propanamide (27)
[0159] The title compound (27) was obtained from the compound (2)
obtained in the Example 2 and N-methyl-N-phenyl-2-bromoacetamide by
the same manner as the Example 6.
Example 28
2-(4-Amino-2,3,5,6-tetramethylphenoxy)-N,2-dimethyl-N-[1-(2-phenylethyl)-4-
-piperidinyl]propanamide (28)
[0160] The title compound (28) was obtained from the compound (3)
obtained in the Example 3 and phenethyl bromide by the same manner
as the Example 6.
Example 29
2-(2-Amino-3,4,5,6-tetramethylphenoxy)-N-methyl-N-[1-(2-phenylethyl)-4-pip-
eridinyl]acetamide (29)
[0161] The title compound (29) was obtained from the compound (4)
obtained in the Example 4 and phenethyl bromide by the same manner
as the Example 6.
Example 30
2-(2-Amino-3,4,5,6-tetramethylphenoxy)-N-[1-(2-phenyl-2-oxoethyl)-4-piperi-
dinyl]-N-methylacetamide (30)
[0162] The title compound (30) was obtained from the compound (4)
obtained in the Example 4 and phenacyl bromide by the same manner
as the Example 6.
Example 31
2-(2-Amino-3,4,5,6-tetramethylanilino)-N-methyl-N-[1-(4-phenoxyphenyl)-4-p-
iperidinyl]acetamide (31)
[0163] The title compound (31) was obtained from
2-[2-(tert-butoxycarbonylamino)-3,4,5,6-tetramethylanilino]acetic
acid and 1-(4-phenoxyphenyl)-4-methyaminopiperidine by the same
manner as the Example 1.
Example 32
2-(2-Amino-3,4,5,6-tetramethylanilino)-N-{1-[4-(4-fluorobenzyl)phenyl]-4-p-
iperidinyl}-N-methylacetamide (32)
[0164] The title compound (32) was obtained from
2-[2-(tert-butoxycarbonylamino)-3,4,5,6-tetramethylanilino]acetic
acid and 1-[4-(4-fluorobenzyl)phenyl]-4-methyaminopiperidine by the
same manner as the Example 1.
Example 33
2-(3-Amino-2,4,5,6-tetramethylphenoxy)-N-(1-benzoyl-4-piperidinyl)-N-methy-
lacetamide (33)
[0165] The title compound (33) was obtained from the compound (5)
obtained in the Example 5 and benzoyl chloride by the same manner
as the Example 6.
Example 34
2-(3-Amino-2,4,5,6-tetramethylphenoxy)-N-[1-(4-cyanobenzyl)-4-piperidinyl]-
-N-methylacetamide (34)
[0166] The title compound (34) was obtained from the compound (5)
obtained in the Example 5 and 4-cyanobenzyl bromide by the same
manner as the Example 6.
Example 35
2-(3-Amino-2,4,5,6-tetramethylphenoxy)-N-methyl-N-[1-(2-phenylethyl)-4-pip-
eridinyl]acetamide (35)
[0167] The title compound (35) was obtained from the compound (5)
obtained in the Example 5 and phenethyl bromide by the same manner
as the Example 6.
Example 36
2-(4-Amino-2,3,5-trimethylphenoxy)-N-[1-(4-fluorophenyl)-4-piperidinyl]-N--
methylacetamide (36)
[0168] The title compound (36) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5-trimethylphenoxy]acetic acid
and 1-(4-fluorophenyl)-4-methyaminopiperidine by the same manner as
the Example 1.
Example 37
2-(4-Amino-2,3,5,6-tetramethylanilino)-N-[1-(1,3-benzothiazol-2-yl)-4-pipe-
ridinyl]-N-methylacetamide (37)
[0169] The title compound (37) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylanilino]acetic
acid and 1-(1,3-benzothiazol-2-yl)-4-methyaminopiperidine by the
same manner as the Example 1.
Example 38
2-(4-Amino-2,3,5,6-tetramethylanilino)-N-[1-(4-fluorophenyl)-4-piperidinyl-
]-N-methylacetamide (38)
[0170] The title compound (38) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylanilino]acetic
acid and 1-(4-fluorophenyl)-4-methyaminopiperidine by the same
manner as the Example 1.
Example 39
2-(3-Amino-2,4,6-trimethylanilino)-N-[1-(4-fluoro-phenyl)-4-piperidinyl]-N-
-methylacetamide (39)
[0171] The title compound (39) was obtained from
2-[3-(tert-butoxycarbonylamino)-2,4,6-trimethylanilino]acetic acid
and 1-(4-fluorophenyl)-4-methyaminopiperidine by the same manner as
the Example 1.
Example 40
2-(4-Amino-2,3,5,6-tetramethylanilino)-N-methyl-N-[1-(4-phenoxyphenyl)-4-p-
iperidinyl]propanamide (40)
[0172] The title compound (40) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylanilino]propionic
acid and 1-(4-phenoxyphenyl)-4-methyaminopiperidine by the same
manner as the Example 1.
Example 41
2-(4-Amino-2,3,5,6-tetramethylanilino)-N-(1-[1,1'-biphenyl]-4-yl-4-piperid-
inyl)-N-methylpropanamide (41)
[0173] The title compound (41) was obtained from
2-[4-(tert-butoxycarbonylamino)-2,3,5,6-tetramethylanilino]propionic
acid and 1-[1,1'-biphenyl]-4-yl-N-methyl-4-piperidine amine by the
same manner as the Example 1.
[0174] The physiochemical datum of the compounds obtained by the
above-mentioned examples is summarized in the following tables 1 to
7. TABLE-US-00001 TABLE 1 IR (KBr) No. Chemical Structure
Properties cm.sup.-1 .sup.1H-NMR (CDCl.sub.3) 1 ##STR7## white
powder (CHCl.sub.3) 2950, 2399, 1734, 1652, 1558, 1472, 1418, 1319,
1083 1.53-1.85(4H, m), 2.08(3H, s), 2.09(3H, s), 2.23(3H, s),
2.24(3H, s), 2.63(1H, m), 2.76(1H, m), 2.88 & 2.92(3H, each s),
3.15(2H, m), 3.48(2H, brs), 3.73 & 4.63(1H, each m), 4.31 &
4.36(2H, each s) 2 ##STR8## white powder 3378, 2938, 1638 1472,
1416, 1370 1286, 1257, 1077 1.42 & 1.43 (3H, d, J=6.5 Hz),
1.54-1.72(4H, m), 2.08(6H, s), 2.19(6H, s), 2.35 & 2.53(1H,
each m), 2.74(1H, m), 2.80 & 2.84(3H, each s), 3.08(2H, m),
3.44(2H, brs), 3.80(0.5H, m), 4.58(1.5H, m) 3 ##STR9## pale yellow
powder (CHCl.sub.3) 2401, 1624, 1474 1412, 1384, 1256 1145, 1076
1.41(6H, s), 1.61-1.80(4H, m), 2.04 & 2.07 & 2.08 &
2.11 & 2.13(12H, each s), 2.65(1H, m), 2.75(1H, m), 2.89 &
3.34(3H, each s), 3.14(2H, m), 3.45(2H, brs), 4.60 & 4.90(1H,
m) 4 ##STR10## white powder (CHCl.sub.3) 2401, 1654, 1474 1238,
1077, 1044 928 1.49-1.73(4H, m), 2.10(3H, s), 2.13(3H, s), 2.17(3H,
s), 2.22(3H, s), 2.53-2.81(2H, m), 2.76 & 2.91(3H, each s),
3.14(2H, m), 3.40 & 4.61(1H, m), 3.96-4.33(2H, brs), 4.47 &
4.51(2H, each s) 5 ##STR11## yellow oil (CHCl.sub.3) 2932, 2402,
1654 1451, 1320, 1122 1084, 1050 1.61-1.81(4H, m), 2.09 & 2.13
& 2.18 & 2.22(12H, each s), 2.63(2H, m), 2.77(2H, m), 2.88
& 2.92(3H, each s), 3.16 & 3.18(2H, each m), 3.52(2H, brs),
3.72 & 4.63(1H, each m), 4.34 & 4.38(2H, each s)
[0175] TABLE-US-00002 IR (KBr) No. Chemical Structure Properties
cm.sup.-1 .sup.1H-NMR (CDCl.sub.3) 6 ##STR12## white powder (2.HCl
salt) (Et.sub.2O/MeOH) 261-263.degree. C. (2.HCl salt) 3432, 2926,
2345, 1646, 1534, 1478, 1455, 1304, 1248, 1098 1.56-2.29(6H, m),
2.09(6H, s), 2.23(3H, s), 2.24(3H, s), 2.61(2H, m) 2.75-2.85(2H, #
m), 2.88 & 2.92(3H, each s), 3.09(2H, m), 3.47(2H, brs 3.69
& 4.58(1H, each m), 4.32 & 4.35(2H, each s), 7.20(3H, m),
7.24-7.33(2H, m) 7 ##STR13## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 196-200.degree. C. (2.HCl salt) 3425, 2950, 1692,
1636, 1556, 1498, 1447, 1314, 1248, 1097 1.68-2.04(4H, m), 2.09(6H,
s), 2.23(6H, s), 2.29-2.55(2H, m), 2.94 & # 2.96(3H, each s),
3.00(2H, m), 3.14 & 3.16(2H, each s), 3.49(2H, brs), 3.81 &
4.57(1H, each m), 4.33 & 4.36(2H, each s), 7.12(1H, m),
7.35(2H, m), 7.57(2H, d), 8.96 & 9.06(1H, each brs) 8 ##STR14##
white powder (HCl salt) (Et.sub.2O/MeOH) 202-203.degree. C. (2.HCl
salt) 3457, 2922, 2586 1633, 1530, 1448, 1318, 1250, 1108 1043, 713
1.78(4H, m), 2.08(6H, s), 2.22(6H, s), 2.89(3H, s), 2.89-3.06 (2H,
# brs), 3.47(2H, brs), 3.87(1H, brs), 4.06 & 4.79 (1H, each m),
4.32 & 4.37 (2H, each s), 4.82(1H, brs), 7.41(5H, m) 9
##STR15## white powder (2.HCl salt) (Et.sub.2O/MeOH)
229-230.degree. C. (2.HCl salt) 3428, 2956, 1648, 1522, 1463, 1419,
1309, 1248, 1106, 1091, 1046 0.92(3H, m), 1.33 & 1.45(4H, each
m), 1.74-2.32(8H, m), 2.08 (6H, # s), 2.22(6H, s), 2.88 & 2.90
(3H, each s), 2.99 & 3.01 (2H, each m), 3.46(2H, brs), 3.68
& 4.11 & 4.54(1H, each m), 4.31 & 4.34(2H, each s), 10
##STR16## white powder (2.HCl salt) (Et.sub.2O/MeOH)
201-203.degree. C. (2.HCl salt) 3417, 2938, 1694, 1646, 1598, 1450,
1417, 1247, 1101, 1.69(2H, m), 1.89(1H, m), 2.08(6H, s), 2.22(6H,
s), 2.30(1H, m), 2.89(3H, # s), 2.91 & 3.09(4H, each m), 3.74
& 4.59 & 4.81 (1H, each m), 3.82 & 3.85(2H, each s),
4.32 & 4.35(2H, each s), 7.46 & 7.57(3H, each m), 7.97(2H,
m) 11 ##STR17## white powder (2.HCl salt) (Et.sub.2O/MeOH)
229-230.degree. C. (2.HCl salt) 3375, 2945, 1646, 1460, 1248, 1100,
700 1.73-2.56(8H, m), 2.09(6H, s), 2.23(6H, s), 2.90 &
3.252(2H, each m), # 2.90 & 2.92(3H, each s), 3.47(2H, brs),
3.76 & 4.58(1H, each m), 4.33 & 4.35(2H, each s), 4.71
& 4.73(1H, each m), 7.36(5H, m)
[0176] TABLE-US-00003 IR (KBr) No. Chemical Structure Properties
cm.sup.-1 .sup.1H-NMR (CDCl.sub.3) 12 ##STR18## white powder (2.HCl
salt) (Et.sub.2O/MeOH) 223-225.degree. C. (2.HCl salt) 3425, 2932,
1648, 1460, 1414, 1306, 1248, 1095, 1032, 0.11(2H, m), 0.41(2H, m),
0.75 & 0.95(1H, each m), 1.65-2.18 (8H, m), # 1.99 (6H, s),
2.10 & 2.13(6H, each s), 2.78 & 2.81(3H, each s), 3.05(2H,
m), 3.37(2H, brs), 3.58 & 4.44 (1H, each m), 4.21 & 4.24
& 4.27(2H, each s) 13 ##STR19## white powder (2HCl salt)
(Et.sub.2O/MeOH) 216-217.degree. C. (2.HCl salt) 3431, 2941, 1637,
1498, 1460, 1417, 1248, 1096, 1032, 0.82(1H, m), 0.97(1H, m),
1.23(1H, m), 1.66-2.22(7H, m), 2.08 (6H, # s), 2.22(6H, s),
2.37(1H, m), 2.53(1H, m), 2.87 & 2.90(3H, each s), 3.12(2H, m),
3.46(2H, brs), 3.67 & 4.54(1H, each m), 4.31 & 4.33(2H,
each s), 7.04(2H, m), 7.14(1H, m), 7.25(2H, m) 14 ##STR20## white
powder (2.HCl salt) (Et.sub.2O/MeOH) 216-219.degree. C. (2.HCl
salt) 3417, 2950, 1638, 1598, 1494, 1413, 1305, 1246, 1099, 1044,
756 1.61-2.38(6H, m), 2.09(6H, s), 2.22 (6H, s), 2.82(2H, m), 2.88
& 2.91(3H, # each s), 3.08(2H, m), 3.46(2H, brs), 3.70 &
4.56(1H, each m), 4.09(2H, m), 4.31 & 4.34(2H, each s),
6.90(3H, m), 7.28(2H, m) 15 ##STR21## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 210-212.degree. C. (2.HCl salt) 3414, 2938, 1663,
1495, 1452, 1368, 1248, 1098, 703 1.79-2.20(6H, m), 2.07(6H, s),
2.17 & 2.21(6H, each s), 2.84 & 2.88(3H, # each s),
2.92(4H, m), 3.27(3H, s), 3.47(2H, m), 3.57 & 4.47(1H, m), 4.29
& 4.30(2H, each s), 7.18(2H, d=7.3 Hz, J=Hz), 7.36-7.45(3H, m)
16 ##STR22## white powder (3.HCl salt) (Et.sub.2O/MeOH)
290-293.degree. C. (3.HCl salt) 3442, 2945, 2402, 1668, 1452, 1306,
1294, 1248, 1121, 1102, 1060, 876 1.75-2.23(6H, m), 2.09(6H, s),
2.22(6H, s), 2.50(8H, m), 2.87 & 2.90(3H, # each s), 3.01(2H,
m), 3.47(2H, m), 3.47 & 4.54(1H, each m), 3.71(4H, m), 4.30
& 4.34(2H, each s) 17 ##STR23## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 228-231.degree. C. (2.HCl salt) 3427, 2950, 1651,
1452, 1415, 1306 1248, 1124, 1101, 1045, 700 1.67-2.24(6H, m),
2.09(6H, s), 2.22 & 2.23(6H, each s), 2.61 (2H, # m), 2.79(2H,
m), 2.89 & 2.92(3H, each m), 3.08(2H, m), 3.43 (2H, m),
3.47(2H, brs), 3.70 & 4.57(1H, each m), 3.75(2H, m), 4.32 &
4.35(2H, each s), 4.87(1H, m), 7.23(5H, m)
[0177] TABLE-US-00004 IR (KBr) No. Chemical Structure Properties
cm.sup.-1 .sup.1H-NMR (CDCl.sub.3) 18 ##STR24## white powder (2.HCl
salt) (Et.sub.2O/MeOH) 251-253.degree. C. (2.HCl salt) 3416, 2918,
2487, 1658, 1631, 1461, 1305, 1248, 1116, 1097 1.66-2.22 (6H, m),
2.08 (6H, s), 2.22(6H, s), 2.89 & 2.92(3H, each s), # 2.91 (2H,
m), 3.47 (2H, brs), 3.55(2H, s), 3.70 & 4.55 (1H, each m), 4.31
& 4.34 (2H, each s), 7.44(2H, m), 7.60(2H, m), 19 ##STR25##
white powder (2.HCl salt) (Et.sub.2O/MeOH) 226-229.degree. C.
(2.HCl salt) 3404, 2940, 1667, 1462, 1421, 1248, 1097 1.65-2.22
(6H, m), 2.08 (6H, s), 2.21(6H, s), 2.88 & 2.91(3H, each # s),
2.93 (2H, m), 3.46 (2H, brs), 3.53(2H, s), 3.66 & 4.54 (1H,
each m), 4.31 & 4.33 (2H, each s), 4.39(2H, m), 4.77(2H, m),
5.74 & 6.12 (2H, brs) 20 ##STR26## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 231-233.degree. C. (2.HCl salt) 3427, 2936, 1640,
1459, 1419, 1308, 1247, 1096, 1026, 746, 693 1.67-2.22(6H, m),
2.08(6H, s), 2.22(6H, s), 2.63(2H, m), 2.87 & 2.90(3H, # each
s), 3.03-3.07(4H, m), 3.67 & 4.53(1H, each m), 4.11 &
4.13(2H, each s), 7.18(1H, m), 7.27(2H, m), 7.34(2H, m) 21
##STR27## white powder (2.HCl salt) (Et.sub.2O/MeOH)
172-174.degree. C. (2.HCl salt) 3406, 2926, 2565 1638, 1459, 1420
1249, 1099 1.65-2.43(7H, m), 2.09 & 2.22 & 2.24 & 2.26
(12H, each # s), 2.88 & 2.91(3H, each s), 2.97(2H, m), 3.31(2H,
s), 3.47(2H, brs), 3.68 & 4.56(1H, m), 4.31 & 4.35(2H, each
s) 22 ##STR28## white powder (2.HCl salt) (Et.sub.2O/MeOH)
212-214.degree. C. (2.HCl salt) 3411, 2936, 1638 1454, 1420, 1249
1098, 1028 0.94(3H, t), 1.46-2.26(4H, m), 2.09 & 2.23 &
2.24(12H, each # s), 2.30-2.55(2H, m), 2.80-3.03(4H, m), 2.89 &
2.92(3H, each s), 3.47(3H, m), 3.63 & 4.54(1H, m), 4.32 &
4.35(2H, each s), 7.13-7.33(5H, m) 23 ##STR29## white powder (2.HCl
salt) (Et.sub.2O/MeOH) 220-221.degree. C. (2.HCl salt) 3408, 2939,
2592, 1657, 1463, 1415, 1251, 1107, 1079, 1024 0.01 & 0.43(4H,
each m), 0.76(1H, m), 1.34 & 1.35(3H, d, # J=6.2 Hz),
1.50-1.83(6H, m), 1.99(6H, s), 2.06-2.23(2H, m), 2.11 (6H, s), 2.72
& 2.76(3H, each s), 3.02(2H, m), 3.36(2H, brs), 3.60 &
4.43(1H, each m), 4.47(1H, m)
[0178] TABLE-US-00005 IR (KBr) No. Chemical Structure Properties
cm.sup.-1 .sup.1H-NMR (CDCl.sub.3) 24 ##STR30## white powder (2.HCl
salt) (Et.sub.2O/MeOH) 228-231.degree. C. (2.HCl salt) 3428, 2931,
1630, 1464, 1374, 1249, 1080, 1024 (2.HCl salt): # CD.sub.3OD 1.02
(3H, m), 1.42 (3H, m), 1.46 (2H, m), 1.71-1.97(4H, m), 2.19(2H, m),
2.24(6H, s), 2.29(6H, s), 2.87(3H, s), 3.06(4H, m), 3.66(2H, m),
4.12 & 4.60 (1H, each m), 4.79(1H, m) 25 ##STR31## white powder
(3.HCl salt) (Et.sub.2O/MeOH) 266-268.degree. C. (3.HCl salt) 3417,
2942, 2537, 1630, 1454, 1417, 1249, 1101 1.43(3H, m), 1.54-2.19(6H,
m), 2.07(6H, s), 2.19(6H, s), 2.46 & 2.52 8H, each m), 2.98(2H,
m), 3.45(2H, brs), 3.71(4H, m), 3.71 & 4.52 (1H, each m),
4.55(1H, m) 26 ##STR32## white powder (2.HCl salt) (Et.sub.2O/MeOH)
195-197.degree. C. (2.HCl salt) 3430, 2938, 1636, 1499, 1463, 1414,
1249, 1096, 700 0.81(1H, m), 0.96(1H, m), 1.22(1H, m), 1.43(3H, m),
1.55-2.39(8H, m), 2.07(6H, # s), 2.16 & 2.18(6H, each s),
2.52(1H, m), 2.78 & 2.83 (3H, each s), 3.06(2H, m), 3.44(2H,
brs), 3.71 & 4.53(1H, each m), 4.54(1H, m), 7.03(2H, m),
7.23(1H, m), 7.26(2H, m) 27 ##STR33## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 205-208.degree. C. (2.HCl salt) 3436, 2938, 1653,
1496, 1462, 1414, 1368, 1248, 1071, 700 1.40(3H, d, J=6.5 Hz),
1.41-2.17(6H, m), 2.08 & 2.17 & # 2.21(12H, each s), 2.84
& 2.88(3H, each s), 2.92(4H, m), 3.28(3H, s), 3.46(2H, brs),
3.59 & 4.45(1H, each m), 4.54(1H, m), 7.18(2H, d, J=7.6 Hz,
7.31-7.44(3H, m), 28 ##STR34## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 214-216.degree. C. (2.HCl salt) 3405, 2940, 2554,
1625, 1463, 1402, 1250, 1145, 1092, 752, 702 1.41(6H, s),
1.67-2.30(4H, m), 2.07 & 2.08 & 2.11 & # 2.13(12H, each
s), 2.19(2H, m), 2.58(2H, m), 2.80(2H, m), 2.90 & 3.35(3H, each
s), 3.09(2H, m), 3.46(2H, brs), 4.54 & 4.86(1H, each m),
7.17-7.31(5H, m). 29 ##STR35## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 245-247.degree. C. (2.HCl salt) 3410, 2928, 2634
1638, 1603, 1496 1462, 1415, 1323 1250, 1102, 703 (2.HCl salt: #
CD.sub.3OD) 1.89-2.27(4H, m), 2.23 & 2.25 & 2.31 &
2.34(12H, each s), 2.87 & 2.92(3H, each s), 3.06-3.40(6H, m),
3.69-3.79(2H, m), 3.85 & 4.66(1H, m), 4.88 & 5.00(2H, each
s), 7.31(5H, m)
[0179] TABLE-US-00006 TABLE 6 IR(KBr) No. Chemical Structure
Properties cm.sup.-1 .sup.1H-NMR(CDCl.sub.3) 30 ##STR36## pale
yellow powder (2.HCl salt) (Et.sub.2O/MeOH) 169-171.degree. C.
(2.HCl salt) 3435, 2934, 2618 1694, 1639, 1598 1451, 1232, 1102 757
1.44-1.70(2H, m), 1.81-2.34(4H, m), 2.10(3H, s), 2.13(3H, # s),
2.17(3H, s), 2.22 & 2.23(3H, each s), 2.77 & 2.92(3H, each
s), 3.10 (2H, m), 3.37 & 4.58(1H, m), 3.82 & 3.85(2H, each
s), 4.16(2H, brs), 4.47 & 4.51(2H, each s), 7.46(2H, t),
7.58(1H, t), 7.98(2H, d) 31 ##STR37## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 164-166.degree. C. (2.HCl salt) 3396, 2888, 2614
1641, 1588, 1508 1490, 1247, 1173 1098 1.72-2.09(4H, m), 2.11(3H,
s), 2.14(3H, s), 2.18(3H, s), # 2.23 (3H, s), 2.70 & 2.85(2H,
m), 2.79 & 2.95(3H, each s), 3.49 & 4.69(1H, m), 3.66(2H,
m), 4.19(2H, brs), 4.50 & 4.54(2H, each s), 6.88-7.08 (6H, m),
7.24-7.33(3H, m) 32 ##STR38## pale yellow powder (2.HCl salt)
(Et.sub.2O/MeOH) 165-167.degree. C. (2.HCl salt) 3410, 2916, 2614
1639, 1508, 1414 1323, 1221, 1158 820 1.69-2.07(4H, m), 2.10(3H,
s), 2.14(3H, s), 2.17(3H, s), # 2.23 (3H, s), 2.68 & 2.83(2H,
m), 2.77 & 2.92(3H, each s), 3.49 & 4.68(1H, m), 3.70(2H,
m), 3.87(2H, s), 4.20(2H, brs), 4.49 & 4.53(2H, each s),
6.82-6.92(2H, m), 6.95(2H, t), 7.05(2H, d), 7.08-7.16(2H, m) 33
##STR39## white powder (HCl salt) (Et.sub.2O/MeOH) 171-174.degree.
C. (HCl salt) 3423, 2934, 2577, 1626, 1518, 1447, 1318, 1279, 1250,
1107, 1025, 711 (2.HCl salt): # CD.sub.3OD 1.81(4H, m), 2.26 &
2.29 & 2.32(12H, each s), 2.90 & 2.92(3H, each s), 2.91
& 3.25 & 3.82(4H, each m), 4.50 & 4.59(2H, each s),
4.75(1H, m), 7.46(5H, m). 34 ##STR40## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 203-205.degree. C. (2.HCl salt) 3406, 2936, 1637,
1460, 1418, 1322, 1305, 1249, 1096, 1031, 944, 827 1.59-2.22(6H,
m), 2.09 & 2.13 & 2.18(12H, each s), # 2.90(2H, s), 2.88
& 2.92(3H, each m), 3.54(4H, brs), 3.67 & 4.55(1H, each m),
4.34 & 4.37(2H, each s), 7.43(2H, m), 7.60(2H, m), 35 ##STR41##
white powder (2.HCl salt) (Et.sub.2O/MeOH) 157-158.degree. C.
(2.HCl salt) 3421, 2940, 1638, 1492, 1457, 1420, 1312, 1248, 1099,
1032, 703 1.69-2.23(4H, m), 2.09 & 2.14 & 2.18(12H, each
s), # 2.60(2H, m), 2.79(2H, m), 2.87 & 2.92(3H, each s),
3.08(2H, m), 3.49(2H, brs), 3.66 & 4.58 (1H, each m), 4.35
& 4.38(2H, each s), 7.18-7.31(5H, m)
[0180] TABLE-US-00007 TABLE 7 IR (KBr) No. Chemical Structure
Properties cm.sup.-1 .sup.1H-NMR(CDCl.sub.3) 36 ##STR42## white
powder (2.HCl salt) (Et.sub.2O/MeOH) 198-200.degree. C. (2.HCl
salt) 3454, 2950, 2359 1655 1514, 1492 1414, 1291, 1238 1134, 1101
1.70-2.09(4H, m), 2.11(3H, s), 2.16(3H, s), 2.17 & 2.21(3H, #
each s), 2.68-2.87(2H, m), 2.89 & 2.96(3H, each s), 3.37(2H,
brs), 3.60 (2H, m), 3.98 & 4.54-4.68(1H, m), 4.59 &
4.64(2H, each s), 6.56 & 6.61(1H, each s), 6.83-7.00(4H, m) 37
##STR43## white powder (2.HCl salt) (Et.sub.2O/MeOH)
190-194.degree. C. (2.HCl salt) 3424, 2929, 1624, 1540, 1468, 1412,
1262, 1126, 1095, 1018, 762 (2.HCl salt): # DMSO 1.65-1.87(4H, m),
2.17(6H, s), 2.22(3H, s), 2.24(3H, s), 2.77 & 2.80(3H, each s),
3.30(2H, m), 3.96(2H, s), 4.13(2H, m), 4.59 (1H, m), 7.10(1H, dd),
7.30(1H, dd), 7.47(1H, d), 7.78(1H, d) 38 ##STR44## pale yellow
foam (2.HCl salt) (Et.sub.2O/MeOH) 231-235.degree. C. (2.HCl salt)
3420, 1651, 1511, 1451, 1407, 1237, 1166, 1101, 1012, 844
1.67-2.04(4H, m), 2.13(6H, s), 2.29(6H, s), 2.65 & # 2.82(2H,
each m), 2.77 & 2.92(3H, each s), 3.46(2H, m), 3.61 &
3.66(2H, each s), 4.70(1H, m), 6.88-6.98(4H, m) 39 ##STR45## white
powder (2.HCl salt) (Et.sub.2O/MeOH) 193-195.degree. C. (2.HCl
salt) 3418, 2846, 2590 1638, 1638, 1575, 1513 1488, 1409, 1358
1238, 1104 1.63-2.07(4H, m), 2.10 & 2.12 & 2.19 & 2.25
& 2.26(9H, # each s), 2.58-2.86(2H, m), 2.78 & 2.92(3H,
each s), 3.38-3.55 & 4.65(1H, m), 3.48(2H, brs), 3.60(2H, m),
3.72 & 3.76(2H, each s), 4.53(1H, brs), 6.68 & 6.75(1H,
each s), 6.84-7.00(4H, m) 40 ##STR46## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 197-200.degree. C. (2.HCl salt) 3365, 2928, 2470
1638, 1588, 1507 1488, 1248, 1200 1112, 1074 1.23 & 1.32(3H,
each d), 1.41-1.98(4H, m), 2.10(3H, s), # 2.11(3H, s), 2.24(3H, s),
2.27(3H, 5), 2.55-2.88(2H, m), 2.71 & 2.79(3H, each s),
3.29-3.68(4H, m), 3.45 & 4.62(1H, m), 3.97(1H, m),
6.84-7.33(9H, m) 40 ##STR47## white powder (2.HCl salt)
(Et.sub.2O/MeOH) 175-177.degree. C. (2.HCl salt) 3384, 2922, 2478,
1638, 1573, 1486, 1452, 1393, 1364, 1335, 1075 (2.HCl salt):
CD.sub.3OD 1.23 & 1.33(3H, # each d), 1.45-1.99(4H, m),
2.10(3H, s), 2.11(3H, s) 2.24(3H, s), 2.27(3H, s), 2.33-2.95(2H,
m), 2.70 & 2.79(3H, each s), 3.30-3.85(4H, m), 3.47 &
4.66(1H, m), 3.97(1H, m), 6.92-7.03(2H, m), 7.24-7.34(3H, m),
7.40(1H, m), 7.47-7.59(3H, m)
[0181] The effect of aminophenoxyacetamide derivatives of the
present invention represented by the formula (I) was evaluated by
the following biological testing methods. [0182] Test 1: Evaluation
for neuroprotective effect against glutamate induced
neurodegeneration, by comparing the administration of the test
compound prior to the glutamate addition with the simultaneous
administration of the test compound along with the glutamate.
[0183] Test 2: Evaluation for antagonism against cell death by
treatment of various kinds of receptor inhibitor and MTA
[5-deoxy-5-methyl-thioadenosine]. [0184] Test 3: Evaluation for
CalbindinD-28k production increasing effect. [0185] Test 4:
Evaluation for neuroprotective inhibiting effect by antisense
oligonucleotide. [0186] Test 5: Evaluation for cerebral edema
suppressing effect.
[0187] By using of the above-mentioned biological tests, the
selection of the compounds having neuroprotective effect by
activating the receptor of FGF, due to the introduction of the
CalbindinD-28k, one of Ca.sup.2+-binding proteins, was performed by
combining all the Test 1 to 4, by combining Test 1 and 2, by
combining Test 1, 2 and 3, or by combining Test 1, 3, and 4,
respectively.
[0188] The following are the detailed description of the test
methods.
Biological Test 1: Evaluation for Neuroprotective Effect Against
Glutamate Induced Neuronal Cell Death
[0189] Primary cultures were prepared from cerebral cortices of
fetal Wistar rats (E18) according to the modified method of Mattson
and Kater [M. P. Mattson, Brain Res. Rev., 13, 179 (1988)]. After
papain-dissociation, neurons were seeded on poly-L-lysine coated 96
wells plates (Sumitomo Bakelite Co., Ltd.) at density of
5.times.10.sup.4 cells/well and cultivated in 100 .mu.l of DMEM
medium (Dulbecco's modified Eagle medium (Gibco) supplemented with
10 mM NaHCO.sub.3, 15 mM KCl, 1 mM sodium pyruvate, and 10%
(vol/vol) horse serum). Cultures were maintained at 37.degree. C.
in a 90% air/10% CO.sub.2 humidified incubator. Glutamate was added
to the culture at day 4 to the final concentration of 1 mM. Cell
survival was then determined 1 day later by using
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide
(MTT). MTT was dissolved in phosphate buffered saline (PBS, pH 7.4)
at 5 mg/ml and filtered to sterilize and remove a small amount of
insoluble residue present in some batches of MTT, and added to the
cultures at a final concentration of 0.5 mg/ml. Six hours after
incubation, culture medium was discarded to stop the reaction.
Dimethyl sulfoxide was added to all wells and mixed thoroughly to
dissolve the dark blue crystals. After a few minutes at room
temperature, the plates were read by using Micro ELISA Reader at a
test wavelength of 570 nm and a reference wavelength of 650 nm.
[0190] The test compounds (1 .mu.M) was added 24 hrs prior to
glutamate treatment and simultaneously with glutamate.
[0191] The effect of the test compounds was determined as the
survival rate of living cells (%) according to the following
equation: Survival rate of living cells (%)=[(test compound
group-glutamic acid treated group)/(control group-glutamic acid
treated group)].times.100
[0192] That is, the survival rate of living cells after incubation
of the control group was converted to 100%, and the survival rate
of living cells of the tested compounds was calculated.
Biological Test 2: Evaluation for Antagonism Against Neuronal Cell
Death by Treatment of Various Kinds of Receptor Inhibitor of
Physiological Active Substances and MTA
[0193] This biological test is performed to determine whether the
neuroprotective effect of the test compounds is due to the
activation of receptors of physiological active substances or not,
by using antagonistic test for neutralizing antibody and inhibitor
for FGF, NT-3, NT-4/5, BDNF, IGF-I/II, NGF, PDGF and estrogen,
respectively.
[0194] MTA (5-deoxy-5-methylthioadenosine) specifically inhibits
the autophosphorylation of FGF receptor in the living cells [P. A.
Mather, J. Bio. Chem., 268, 4244 (1993)]. Therefore, the
neuroprotective effect of the test compounds is inhibited by
treatment of MTA, this effect is depended by the signal transfer
effect through the phosphorylation of FGF receptor.
[0195] The inhibitors of the various kinds of receptor were
dissolved in the optimum concentration, and MTA was dissolved in
the concentration of 7.5 mM, just before the using. 30 minutes
before the treatment of the test compounds, the optimum
concentration of each inhibitors or 0.75 mM of MTA was added, and
the neuroprotective effect of the test compounds was determined by
mean of MTT method.
[0196] The results of each Biological Test 1 and 2 were shown in
the following Table 8. TABLE-US-00008 TABLE 8 Survival Rate (%)
Compound Survival Rate (%) (Compound: 1 .mu.l & No. (Compound:
1 .mu.l) MTA treatment) 6 54 25 7 76 32 8 78 24 9 104 5 10 86 31 11
74 56 12 54 33 13 65 13 16 101 33 17 85 55 18 91 57 19 96 28 22 79
32 24 61 22 25 87 16 28 103 46 32 73 34 33 120 46 34 74 21 36 97 25
37 95 20 38 124 19 39 94 28
Biological Test 3: Evaluation for CalbindinD-28k Inducing
Effect
[0197] Primary cultures were prepared from cerebral cortices of
fetal Wistar rats (E18) according to the modified method of Mattson
and Kater (M. P. Mattson, Brain Res. Rev., 13, 179 (1988)]. After
papain-dissociation, neurons were seeded on poly-L-lysine coated 6
wells plates (Falcon) at density of 5500 cells/mm.sup.2 and
cultivated in 2 ml of DMEM medium.
[0198] Test compounds were added 5 days after initiation of the
incubation, and after 7 days of incubation, the protein was
extracted with homogenized buffer solution [containing 20 mM of
Tris-HCl (pH=7.4), 1 mM of EDTA, and 0.1 mM of
phenylmethyl-sulfonyl fluoride].
[0199] The effect of the test compounds was determined by the
western blot technique using polyclonal anti CalbindinD-28k Swant
(Swant Co., Ltd.) as antibody.
[0200] Table 9 shows the test results. In the table, the amount of
induced CalbindinD-28k of the control group (none-treated group)
was indicated as 100 percents. TABLE-US-00009 TABLE 9 Amount of
induced CalbindineD-28k Compound (% vs. control) No. (Compound: 1
.mu.M) 6 163 Control 100
Biological Test 4: Evaluation for Neuroprotective Inhibiting Effect
by Antisense Oligonucleotide
[0201] It is necessary to produce the protective protein for the
signal transfer action of cells through the phosphorylation of FGF
receptor for the neuroprotective effect of the test compounds, and
the CalbindinD-28k is one of that protective proteins having
Ca.sup.2+ buffering function. Therefore, the following test
determined whether CalbindinD-28k is concerned in the
neuroprotective effect of the test compounds by using an antisense
oligonucleotide.
[0202] Primary cultures were prepared from cerebral cortices of
fetal Wistar rats (E18). After papain-dissociation, neurons were
seeded on poly-L-lysine coated 96 wells plates (Sumitomo Bakelite
Co., Ltd.) at density of 4.times.10.sup.4 cells/well and cultivated
in 100 .mu.l of DMEM medium.
[0203] From 2 days after the incubation to 8 days, 100 .mu.l of
Neurobasal medium/2% B-27 Supplement and 5 .mu.M of three kinds of
antisense oligonucleotides were added respectively, on every 24
hours. 7 days after incubation, 1 .mu.M and 10 .mu.M of the test
compounds were added, and 8 days after incubation, 300 .mu.M of
glutamic acid was added. Cell survival was then determined 1 day
later by using MTT. Six hours after incubation, culture medium was
discarded to stop the reaction. Dimethyl sulfoxide was added to all
wells and mixed thoroughly to dissolve the dark blue crystals.
After a few minutes at room temperature, the plates were read by
using Micro ELISA Reader at a test wavelength of 570 nm and a
reference wavelength of 650 nm.
[0204] The effect of the test compounds was determined as the
survival rate of living cells (%) according to the equation as
indicated in the Biological Test 1: That is, the survival rate of
living cells after incubation of the control group was converted to
100%, and the survival rate of living cells of the tested compounds
was calculated.
[0205] The sequences of the antisense oligonucleotides to be used
in this test are following. TABLE-US-00010 calbindin antisense 1:
5-TGA CTG CAG GTG GGA TTC TGC-3 calbindin antisense 2: 5-ACC GTC
GAA ATG AAG CCA GA-3 calbindin antisense 3: 5-CGT ATC ATC CAC GGT
CTT GTT-3
[0206] Table 10 shows the test results. TABLE-US-00011 TABLE 10
Test Survival Rate (%) Survival Rate (%) Compound [antisense (-)]
[antisense (+): 5 .mu.M] BFGF (1 ng) 71 8 BFGF (10 ng) 101 35 No. 6
(0.1 .mu.M) 103 30 No. 6 (1 .mu.M) 108 34 No. 38 (0.1 .mu.M) 100 28
No. 38 (1 .mu.M) 106 33
Biological Test 5: Evaluation for Cerebral Edema Suppressing
Effect
[0207] Adult male Wistar rats weighing 210-230 g were used in the
present study and housed in an artificially controlled environment
with 12-hour light/dark cycle. Animals were anesthetized with
pentobarbital sodium (50 mg/kg of body weight, i.p., Nembutal,
Dinabbott, Japan) and placed in a stereotaxic apparatus. The skull
was exposed and a hole was made on the parietal bone; 1.5 mm
posterior and 0.8 mm lateral to the bregma. All procedures were
performed under a surgery microscope to avoid excess irritation to
the meninges and the underlying brain. A brass screw with a blunt
tip (1.0 mm in diameter and 3.0 mm in length, Biomedica, Japan) was
inset in the hole. The screw tip protruding intracranially was
approximately 2.5 mm, which stuck into the parietal part of the
right cerebrum through the meninges. In sham-operated rats, scalp
was closed without attaching a screw to the hole. After surgery,
all rats were kept in their original dens until sacrifice. Rats
were allowed to feed chow and water ad libitum.
[0208] Under anesthesia the rat cerebral hemispheres of the target
were processed for water content on post-TBI day 6 (n=6). The
cerebral hemispheres dissected out were weighed, dried for 24 hours
at 110 degree (Celsius) and then weighed again. The subtracted
amount of the wet and dry weights was regarded as tissue water of
the target, and utilized for the analysis of the cerebral
edema.
[0209] The water content was calculated by using the following
formula: Water content (%)=[(wet weight of hemisphere-dry weight of
hemisphere)/wet weight of hemisphere].times.100
[0210] The test compounds were intravenously administered just
after the operation via tail vein of the rats.
[0211] Table 11 shows the test results. TABLE-US-00012 TABLE 11
Compound No. Cerebral edema suppressing (administration amount)
rate (%) 6 (3 mg/kg) 24.9 8 (1 mg/kg) 14.0 12 (3 mg/kg) 14.7 13 (3
mg/kg) 13.1 14 (1 mg/kg) 18.7 15 (1 mg/kg) 14.7 18 (1 mg/kg) 22.1
24 (1 mg/kg) 10.6 25 (1 mg/kg) 19.1 26 (3 mg/kg) 11.9 27 (3 mg/kg)
11.8 29 (1 mg/kg) 13.8
INDUSTRIAL APPLICABILITY
[0212] As described above, the present invention provides lower
molecular compounds, especially aminophenoxyacetamide derivatives
of the formula (I), which induce the CalbindinD-28k, one of
Ca.sup.2+-binding proteins, and can be easily administrated. Since
the induction of CalbindinD-28k caused by the administration of the
compound provided by the present invention cause neuroprotective
effect and cerebral functional and organic disorder improving and
treating effect, it can be understood that the agent of the present
invention is highly applicable in pharmaceutical field.
Sequence CWU 1
1
3 1 21 DNA Artificial Sequence Antisense DNA to act as a blocker
for production of calbindin-D 28K 1 tgactgcagg tgggattctg c 21 2 20
DNA Artificial Sequence Antisense DNA to act as a blocker for
production of calbindin-D 28K 2 accgtcgaaa tgaagccaga 20 3 21 DNA
Artificial Sequence Antisense DNA to act as a blocker for
production of calbindin-D 28K 3 cgtatcatcc acggtcttgt t 21
* * * * *