U.S. patent application number 16/410776 was filed with the patent office on 2019-10-31 for enantiomers of 8-hydroxyquinoline derivatives and the synthesis thereof.
This patent application is currently assigned to AVIDIN Co. Ltd. The applicant listed for this patent is AVIDIN Co. Ltd., SONEAS Research Co. Ltd., Synaging SAS. Invention is credited to Mario GYURIS, Laszlo HACKLER, Istvan KANIZSAI, Thierry PILLOT, Laszlo PUSK S, Andras SZABO, Ferenc TAK CS.
Application Number | 20190330188 16/410776 |
Document ID | / |
Family ID | 56289533 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330188 |
Kind Code |
A1 |
PUSK S; Laszlo ; et
al. |
October 31, 2019 |
ENANTIOMERS OF 8-HYDROXYQUINOLINE DERIVATIVES AND THE SYNTHESIS
THEREOF
Abstract
Enantiomer derivatives of 8-hydroxyquinoline derivatives with
general formula (I) and (II) and the synthesis thereof and
pharmaceutically acceptable salts and metal complexes thereof, and
the medicinal and/or pharmaceutical compositions comprise these
compounds: ##STR00001## The compounds according to the invention
can be used preferably as cytoprotective, neuroprotective,
cardioprotective, anxiolytic and antidepressant agent for treatment
of neuropsychiatric and neurologic diseases and diseases in
connections with transplantations and with ischemia and reperfusion
injuries thereof, and inhibition of organ, advantageously skin
graft rejection.
Inventors: |
PUSK S; Laszlo; (Szeged,
HU) ; KANIZSAI; Istvan; (Szeged, HU) ; PILLOT;
Thierry; (Crevic, FR) ; GYURIS; Mario;
(Szeged, HU) ; SZABO; Andras; (Budapest, HU)
; TAK CS; Ferenc; (Monor, HU) ; HACKLER;
Laszlo; (Szeged, HU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AVIDIN Co. Ltd.
SONEAS Research Co. Ltd.
Synaging SAS |
Szeged
Budapest
Nancy |
|
HU
HU
FR |
|
|
Assignee: |
AVIDIN Co. Ltd
Szeged
HU
SONEAS Research Co. Ltd.
Budapest
HU
Synaging SAS
Nancy
FR
|
Family ID: |
56289533 |
Appl. No.: |
16/410776 |
Filed: |
May 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15065385 |
Mar 9, 2016 |
10287265 |
|
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16410776 |
|
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|
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PCT/HU2016/000012 |
Mar 7, 2016 |
|
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15065385 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
41/00 20180101; A61P 25/18 20180101; A61P 37/06 20180101; A61P
25/28 20180101; A61P 25/00 20180101; A61P 25/08 20180101; A61P
21/02 20180101; A61P 37/00 20180101; A61P 25/14 20180101; C07D
401/12 20130101; A61P 9/00 20180101; A61P 25/24 20180101; A61P
25/16 20180101; A61P 25/22 20180101 |
International
Class: |
C07D 401/12 20060101
C07D401/12 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 9, 2015 |
HU |
HU/P1500098 |
Claims
1. Novel R-enantiomeric derivatives of 8-hydroxyquinoline
derivatives of general formula (I) and novel S-enantiomeric
derivatives of 8-hydroxyquinoline derivatives of general formula
(II), and pharmaceutically acceptable salts and complexes with
divalent or polyvalent metals thereof, ##STR00009## wherein in
general formulas (I) and (II) R.sub.1 is a lower alkyl group, lower
cycloalkyl group, aryl group, aralkyl group or six membered
heteroaryl or heteroaralkyl group wherein said cyclic groups are
optionally substituted at the ortho, meta or para positions with
one, two, three or four electron withdrawing groups or electron
donating groups or optionally substituted five membered heteroaryl
or heteroaralkyl group wherein the five or six membered heteroaryl
or heteroaralkyl groups comprising one, two or three nitrogen;
oxygen or sulfur atoms or combinations thereof; R.sub.2 is a
hydrogen atom; aryl group; or six membered heteroaryl group wherein
said cyclic groups are optionally substituted at the ortho; meta or
para positions with one, two, three or four electron withdrawing
groups or electron donating groups or optionally substituted five
membered heteroaryl group wherein the five or six membered
heteroaryl groups comprising one, two or three nitrogen; oxygen or
sulfur atoms or combinations thereof; R.sup.3 represents a hydrogen
atom; lower alkyl group; --CH.sub.2F; CHF.sub.2; --CF.sub.3;
--CH.sub.2CH.sub.2F; --CH.sub.2CHF.sub.2; --CH.sub.2CF.sub.3;
--CH.sub.2OR.sup.5; --CH.sub.2CH.sub.2OR.sup.6; or
--CH.sub.2--NR.sup.7R.sup.8 group; R.sup.4 represents a hydrogen
atom; halogen atom; methylthio group; methylsulfinyl group;
methylsulfonyl group; or azido group; R.sup.5 represents a hydrogen
atom; or lower alkyl group; R.sup.6 represents a hydrogen atom; or
lower alkyl group; R.sup.7 represents a hydrogen atom; or lower
alkyl group; R.sup.8 represents a hydrogen atom; or lower alkyl
group; R.sup.7 and R.sup.8 represents jointly --(CH.sub.2).sub.n--
group; or --CH.sub.2CH.sub.2OCH.sub.2CH.sub.2-- group or
--CH.sub.2CH.sub.2SCH.sub.2CH.sub.2-- group or
--CH.sub.2CH.sub.2NR.sup.9CH.sub.2CH.sub.2-- group, wherein n is 4,
5 or 6; R.sup.9 represents a lower alkyl group; or --COR.sup.10
group, R.sup.10 represents a hydrogen atom; lower alkyl group;
methoxy group; or ethoxy group; in the general formula (I) X
represents a hydrogen substituted C atom with "R" configuration; in
the general formula (II) Y represents a hydrogen substituted C atom
with "S" configuration; with the proviso that R.sup.1 cannot
represent non-substituted phenyl group, in case R.sup.2 represents
a non-substituted phenyl group; or non-substituted 2-pyridyl group;
or 4-carboxyphenyl group; or 2-carboxyphenyl group; R.sup.3
represents a hydrogen atom or methyl group; R.sup.4 represents
hydrogen atom or chlorine substituent; and R.sup.1 cannot represent
3,4-dimethylphenyl group, in case R.sup.2 represents a
non-substituted 2-pyridyl group; R.sup.3 represents methyl group
and R.sup.4 a hydrogen atom; and R.sup.1 cannot represent 2-furyl
group, in case R.sup.2 represents a non-substituted 2-pyridyl
group; R.sup.3 represents a hydrogen atom and R.sup.4 represents a
chlorine substituent, and R.sup.1 cannot represent a
non-substituted 2-pyridyl group, in case R.sup.2 represents
5-methylisoxazol-3-yl group; R.sup.3 represents a hydrogen atom; R4
represents a hydrogen atom.
2. Novel R-enantiomeric derivatives of 8-hydroxyquinoline
derivatives of general formula (I') according to claim 1 and
pharmaceutically acceptable salts and complexes with divalent or
polyvalent metals thereof, ##STR00010## wherein in general formula
(I') R.sup.1' represents an aryl group substituted with an electron
withdrawing group in meta or para position, or an aryl group
substituted with an electron donating group in ortho, meta or para
position; or a double-substituted aryl group with electron
withdrawing groups in meta and para positions; or an aryl group
double-substituted with electron withdrawing groups in ortho and
para positions; or a substituted or unsubstituted heteroaryl group;
R.sup.2' represents an aryl group substituted with an electron
withdrawing group in para position, or an aryl group substituted
with an electron donating group in ortho, meta or para position; or
an unsubstituted heteroaromatic group or a heteroaromatic or aryl
group substituted with alkyl group and/or with electron withdrawing
groups in ortho, meta or para positions; R.sup.3' represents a
hydrogen atom R.sup.4' represents a hydrogen atom and in general
formula (I') X represents a hydrogen substituted C atom with "R"
configuration.
3. Novel S-enantiomeric derivatives of 8-hydroxyquinoline
derivatives of general formula (II') according to claim 1 and
pharmaceutically acceptable salts and complexes with divalent or
polyvalent metals thereof, ##STR00011## wherein in general formula
(II') R.sup.1' represents an aryl group substituted with an
electron withdrawing group in meta or para position, or an aryl
group substituted with an electron donating group in ortho, meta or
para position; or a double-substituted aryl group with electron
withdrawing groups in meta and para positions; or an aryl group
double-substituted with electron withdrawing groups in ortho and
para positions; or a substituted or unsubstituted heteroaryl group;
R.sup.2' represents an aryl group substituted with an electron
withdrawing group in para position, or an aryl group substituted
with an electron donating group in ortho, meta or para position; or
an unsubstituted heteroaromatic group or a heteroaromatic or aryl
group substituted with alkyl group and/or with electron withdrawing
groups in ortho, meta or para positions; R.sup.3' represents a
hydrogen atom; R.sup.4' represents a hydrogen atom and in general
formula (I') Y represents a hydrogen substituted C atom with "S"
configuration.
4. Novel R-enantiomeric derivatives of 8-hydroxyquinoline
derivatives of general formula (I'') according to claim 1 and
pharmaceutically acceptable salts and complexes with divalent or
polyvalent metals thereof, ##STR00012## wherein in general formula
(I'') R.sup.1'' represents a phenyl or pyridyl group optionally
single or double substituted with a trifluoromethyl group, hydroxy
group, fluorine atom or isopropoxy group; R.sup.2'' represents a
phenyl group optionally single or double substituted with a
trifluoromethyl group or methoxy-carbonyl group; or a pyridyl,
pyrimidyl, pyrrolidinyl, oxazolidinyl group optionally single or
double substituted with a methyl group or fluorine atom; R.sup.3''
represents hydrogen atom; R.sup.4'' represents hydrogen atom; and
in general formula (I'') X represents a hydrogen substituted C atom
with "R" configuration;
5. Novel S-enantiomeric derivatives of 8-hydroxyquinoline
derivatives of general formula (II'') according to claim 1 and
pharmaceutically acceptable salts and complexes with divalent or
polyvalent metals thereof, ##STR00013## wherein in general formula
(II'') R.sup.1'' represents a phenyl or pyridyl group optionally
single or double substituted with a trifluoromethyl group, hydroxy
group, fluorine atom or isopropoxy group; R.sup.2'' represents a
phenyl group optionally single or double with a trifluoromethyl
group or methoxy-carbonyl group; or a pyridyl, pyrimidyl,
pyrrolidinyl, oxazolidinyl group optionally single or double
substituted with a methyl group or fluorine atom; R.sup.3''
represents hydrogen atom; R.sup.4'' represents hydrogen atom and in
general formula (II'') Y represents a hydrogen substituted C atom
with "S" configuration.
6. Novel enantiomeric derivatives of 8-hydroxyquinoline derivatives
according to claim 1 and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals thereof characterized
in listed as follows:
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]-methyl]quinolin-8-ol),
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol, Potassium
7-[(R)-[(4-methylpirimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-olate, Potassium
7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-olate, Natrium
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-olate,
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-ol fumarate,
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-ol fumarate,
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol zinc complex,
7-[(R)-[(6-Methylpyridin-2-yl)amino]4-nitrophenyl)methyl]-quinolin-8-ol,
7-[(S)-[(6-Methylpyridin-2-yl)amino]4-nitrophenyl)methyl]-quinoline-8-ol,
7-[(R)-[(6-Methylpyridin-2-yl)amino]3-hydroxyphenyl)methyl]-quinoline-8-o-
l,
7-[(S)-[(6-Methylpyridin-2-yl)amino]3-hydroxyphenyl)methyl]-quinoline-8-
-ol,
7-[(R)-[(6-Methylpyridin-2-yl)amino](4-hydroxy-3-methoxiphenyl)methyl-
]-quinoline-8-ol,
7-[(S)-[(6-Methylpyridin-2-yl)amino](4-hydroxy-3-methoxiphenyl)methyl]-qu-
inoline-8-ol,
7-[(R)-[(6-Methylpyridin-2-yl)amino](5-bromopyridin-2-yl)methyl]-quinolin-
e-8-ol,
7-[(S)-[(6-Methylpyridin-2-yl)amino](5-bromopyridin-2-yl)methyl]-q-
uinoline-8-ol,
7-[(R)-[(6-Methylpyridin-2-yl)amino]2-hydroxyphenylmethyl]-quinoline-8-ol-
,
7-[(S)-[(6-Methylpyridin-2-yl)amino]2-hydroxyphenylmethyl]-quinoline-8-o-
l,
5-Chloro-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phen-
yl]methyl]quinolin-8-ol,
5-Chloro-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinoline-8-ol,
5-Chloro-7-[(R)-[(6-methylpyridin-2-yl)amino][4-(trifluoromethyl)phenyl]m-
ethyl]quinoline-8-ol,
2-Methyl-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinoline-8-ol,
2-Methyl-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinoline-8-ol,
2-[(Dimethylamino)methyl]-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifl-
uoromethyl)phenyl]methyl]quinoline-8-ol,
2-[(Dimethylamino)methyl]-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifl-
uoromethyl)phenyl]methyl]quinoline-8-ol,
2-[(Dimethylamino)methyl]-7-[(R)-[(4-methylpyridin-2-yl)amino][4-(trifluo-
romethyl)phenyl]methyl]quinoline-8-ol,
2-[(Dimethylamino)methyl]-7-[(S)-[(4-methylpyridin-2-yl)amino][4-(trifluo-
romethyl)phenyl]methyl]quinoline-8-ol,
5-Nitro-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-
methyl]quinoline-8-ol,
5-Nitro-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-
methyl]quinoline-8-ol,
7-[(R)-[(Pyridin-2-yl)[4-(trifluoromethyl)phenylamino]methyl]quinoline-8--
ol,
7-[(S)-[(Pyridin-2-yl)[4-(trifluoromethyl)phenylamino]methyl]quinoline-
-8-ol
2-(Hydroxymethyl)-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluor-
omethyl)phenyl]methyl]quinoline-8-ol,
2-(Hydroxymethyl)-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluorometh-
yl)phenyl]methyl]quinoline-8-ol.
7. Novel enantiomeric derivatives of 8-hydroxyquinoline derivatives
according to claim 1 and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals thereof wherein the
metal complexes are iron, zinc or copper complexes.
8. Novel enantiomeric derivatives of 8-hydroxyquinoline derivatives
according to claim 1 and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals thereof wherein the
metal complex is zinc complex.
9. Novel enantiomeric derivatives of 8-hydroxyquinoline derivatives
according to claim 6 and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals thereof, wherein the
enantiomeric derivative is
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol.
10. Novel enantiomeric derivatives of 8-hydroxyquinoline
derivatives according to claim 6 and pharmaceutically acceptable
salts and complexes with divalent or polyvalent metals thereof,
wherein the enantiomeric derivative is
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol.
11. Novel enantiomeric derivatives of 8-hydroxyquinoline
derivatives according to claim 6 and pharmaceutically acceptable
salts and complexes with divalent or polyvalent metals thereof,
wherein the salts and the complex are listed as follows: Potassium
7-[(R)-[(4-methylpirimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-olate, Potassium
7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-olate, Natrium
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-olate,
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-ol fumarate,
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-ol fumarate,
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol zinc complex.
12. Medicinal and/or pharmaceutical compositions comprising the
novel enantiomeric derivatives of 8-hydroxyquinoline derivatives
according to claim 1 and/or pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals thereof.
13. Medicinal and/or pharmaceutical compositions according to claim
12, further comprising the novel enantiomeric derivatives of
8-hydroxyquinoline derivatives according to claim 1 and/or
pharmaceutically acceptable salts and metal complexes thereof and
inert, pharmaceutically acceptable, solid or liquid carrier and/or
excipient.
14. Medicinal and/or pharmaceutical compositions according to claim
12, further comprising medicinal acceptable carriers and/or
excipients as following listed: starch, gelatinized starch,
cellulose, microcrystalline cellulose or cellulose-derivatives,
lactose, lactose monohydrate, talcum, mannitol, sodium chloride,
sodium carbonate, saccharose, maltose, calcium carbonate, colloidal
anhydrous silicon dioxide, stearic acid, magnesium stearate and/or
isomalt.
15. Medicinal and/or pharmaceutical compositions according to claim
12, wherein the composition is solid, semi-solid or liquid.
16. Medicinal and/or pharmaceutical compositions according to claim
12, wherein the composition is tablet, inhalation powder, capsule,
suppository or solution for injection.
17. Medicinal and/or pharmaceutical compositions according to claim
12, wherein the composition is tablet.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/065,385, filed Mar. 9, 2016, which claims priority from
Hungarian Patent Application No. HU/P1500098, filed Mar. 9, 2015,
and is also a continuation of PCT International Application No.
PCT/HU2016/00012, filed Mar. 7, 2016, which claims priority from
Hungarian Patent Application No. HU/P1500098, filed Mar. 9, 2015,
all of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] Our invention relates to novel R-enantiomeric derivatives of
8-hydroxyquinoline derivatives of general formula (I) and novel
S-enantiomeric derivatives of 8-hydroxyquinoline derivatives of
general formula (II), and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals, advantageously iron,
copper or zinc complexes thereof,
##STR00002##
[0003] wherein
[0004] in general formulas (I) and (II) [0005] R.sub.1 is a lower
alkyl group, lower cycloalkyl group, aryl group, aralkyl group or
six membered heteroaryl or heteroaralkyl group wherein said cyclic
groups are optionally substituted at the ortho, meta or para
positions with one, two, three or four electron withdrawing groups
or electron donating groups or optionally substituted five membered
heteroaryl or heteroaralkyl group wherein the five or six membered
heteroaryl or heteroaralkyl groups comprising one, two or three
nitrogen; oxygen or sulfur atoms or combinations thereof; [0006]
R.sub.2 is a hydrogen atom; aryl group; or six membered heteroaryl
group wherein said cyclic groups are optionally substituted at the
ortho; meta or para positions with one, two, three or four electron
withdrawing groups or electron donating groups or optionally
substituted five membered heteroaryl group wherein the five or six
membered heteroaryl groups comprising one, two or three nitrogen;
oxygen or sulfur atoms or combinations thereof; [0007] R.sup.3
represents a hydrogen atom; lower alkyl group; --CH.sub.2F;
--CHF.sub.2; --CF.sub.3; --CH.sub.2CH.sub.2F; --CH.sub.2CHF.sub.2;
--CH.sub.2CF; --CH.sub.2OR.sup.5; --CH.sub.2CH.sub.2OR.sup.6; or
--CH.sub.2--NR.sup.7R.sup.8 group; [0008] R.sup.4 represents a
hydrogen atom; halogen atom; methylthio group; methylsulfinyl
group; methylsulfonyl group; or azido group; [0009] R.sup.5
represents a hydrogen atom; or lower alkyl group; [0010] R.sup.6
represents a hydrogen atom; or lower alkyl group; [0011] R.sup.7
represents a hydrogen atom; or lower alkyl group; [0012] R.sup.8
represents a hydrogen atom; or lower alkyl group; [0013] R.sup.7
and R.sup.8 represents jointly --(CH.sub.2).sub.n-- group; or
--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2-- group or
--CH.sub.2CH.sub.2SCH.sub.2CH.sub.2-- group or
--CH.sub.2CH.sub.2NR.sup.9CH.sub.2CH.sub.2-- group, [0014] wherein
[0015] n is 4, 5 or 6; [0016] R.sup.9 represents a lower alkyl
group; or --COR.sup.10 group, [0017] R.sup.10 represents a hydrogen
atom; lower alkyl group; methoxy group; or ethoxy group;
[0018] in the general formula (I) [0019] X represents a hydrogen
substituted C atom with "R" configuration;
[0020] in the general formula (II) [0021] Y represents a hydrogen
substituted C atom with "S" configuration;
[0022] with the proviso that [0023] R.sup.1 cannot represent
non-substituted phenyl group, in case [0024] R.sup.2 represents a
non-substituted phenyl group; or non-substituted 2-pyridyl group;
or 4-carboxyphenyl group; or 2-carboxyphenyl group; [0025] R.sup.3
represents a hydrogen atom or methyl group; [0026] R.sup.4
represents hydrogen atom or chlorine substituent; and [0027]
R.sup.1 cannot represent 3,4-dimethylphenyl group, in case [0028]
R.sup.2 represents a non-substituted 2-pyridyl group; [0029]
R.sup.3 represents methyl group and [0030] R.sup.4 a hydrogen
atom;
[0031] and [0032] R.sup.1 cannot represent 2-furyl group, in case
[0033] R.sup.2 represents a non-substituted 2-pyridyl group; [0034]
R.sup.3 represents a hydrogen atom and [0035] R.sup.4 represents a
chlorine substituent
[0036] and [0037] R.sup.1 cannot represent a non-substituted
2-pyridyl group, in case [0038] R.sup.2 represents
5-methylisoxazol-3-yl group [0039] R.sup.3 represents a hydrogen
atom [0040] R4 represents a hydrogen atom
[0041] The subject matter of the invention furthermore relates
advantageously to novel R-enantiomeric derivatives of
8-hydroxyquinoline derivatives of general formula (I') and novel
S-enantiomeric derivatives of 8-hydroxyquinoline derivatives of
general formula (II'), and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals, advantageously iron,
copper or zinc complexes thereof,
##STR00003##
[0042] wherein
[0043] in general formulas (I') and (II') [0044] R.sup.1'
represents an aryl group substituted with an electron withdrawing
group in meta or para position, or an aryl group substituted with
an electron donating group in ortho, meta or para position; or a
double-substituted aryl group with electron withdrawing groups in
meta and para positions; or an aryl group double substituted with
electron withdrawing groups in ortho and para positions; or a
substituted or unsubstituted heteroaryl group; [0045] R.sup.2'
represents an aryl group substituted with an electron withdrawing
group in para position, or an aryl group substituted with an
electron donating group in ortho, meta or para position; or an
unsubstituted heteroaromatic group or a heteroaromatic or aryl
group substituted with alkyl group and/or with electron withdrawing
groups in ortho, meta or para positions; [0046] R.sup.3' represents
advantageously a hydrogen atom [0047] R.sup.4' represents
advantageously a hydrogen atom and
[0048] in general formula (I') [0049] X represents a hydrogen
substituted C atom with "R" configuration;
[0050] In general formula (II') [0051] Y represents a hydrogen
substituted C atom with "S" configuration.
[0052] The subject matter of the invention furthermore relates
advantageously to novel R-enantiomeric derivatives of
8-hydroxyquinoline derivatives of general formula (I'') and novel
S-enantiomeric derivatives of 8-hydroxyquinoline derivatives of
general formula (II'') and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals, advantageously iron,
copper or zinc complexes thereof,
##STR00004##
[0053] wherein
[0054] in general formulas (I'') and (II'') [0055] R.sup.1''
represents advantageously a phenyl or pyridyl group optionally
single or double substituted with a trifluoromethyl group, hydroxy
group, fluorine atom or isopropoxy group; [0056] R.sup.2''
represents advantageously a phenyl group optionally single or
double substituted with a trifluoromethyl group or methoxy-carbonyl
group; or a pyridyl, pyrimidyl, pyrrolidinyl, oxazolidinyl group
optionally single or double substituted with a methyl group or
fluorine atom; [0057] R.sup.3'' represents advantageously hydrogen
atom; [0058] R.sup.4'' represents advantageously hydrogen atom;
[0059] In general formula (I'') [0060] X represents a hydrogen
substituted C atom with "R" configuration;
[0061] In general formula (II'') [0062] Y represents a hydrogen
substituted C atom with "S" configuration.
[0063] The subject matter of the invention furthermore relates
advantageously to novel enantiomeric derivatives of
8-hydroxyquinoline derivatives, and pharmaceutically acceptable
salts and complexes with divalent or polyvalent metals,
advantageously iron, copper or zinc complexes thereof, especially
advantageously zinc complexes thereof as listed detailed as
follows: [0064]
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol), [0065]
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol, [0066] Potassium
7-[(R)-[(4-methylpirimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-olate, [0067] Potassium
7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-olate, [0068] Natrium
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-olate, [0069]
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-ol fumarate, [0070]
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinolin-8-ol fumarate, [0071]
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinolin-8-ol zinc complex, [0072]
7-[(R)-[(6-Methylpyridin-2-yl)amino]4-nitrophenyl)methyl]-quinolin-8-ol,
[0073]
7-[(S)-[(6-Methylpyridin-2-yl)amino]4-nitrophenyl)methyl]-quinolin-
e-8-ol, [0074]
7-[(R)-[(6-Methylpyridin-2-yl)amino]3-hydroxyphenyl)methyl]-quinoline-8-o-
l, [0075]
7-[(S)-[(6-Methylpyridin-2-yl)amino]3-hydroxyphenyl)methyl]-quin-
oline-8-ol, [0076]
7-[(R)-[(6-Methylpyridin-2-yl)amino](4-hydroxy-3-methoxiphenyl)methyl]-qu-
inoline-8-ol, [0077]
7-[(S)-[(6-Methylpyridin-2-yl)amino](4-hydroxy-3-methoxiphenyl)methyl]-qu-
inoline-8-ol, [0078]
7-[(R)-[(6-Methylpyridin-2-yl)amino](5-bromopyridin-2-yl)methyl]-quinolin-
e-8-ol, [0079]
7-[(S)-[(6-Methylpyridin-2-yl)amino](5-bromopyridin-2-yl)methyl]-quinolin-
e-8-ol, [0080]
7-[(R)-[(6-Methylpyridin-2-yl)amino]2-hydroxyphenylmethyl]-quinoline-8-ol-
, [0081]
7-[(S)-[(6-Methylpyridin-2-yl)amino]2-hydroxyphenylmethyl]-quinol-
ine-8-ol, [0082]
5-Chloro-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinolin-8-ol, [0083]
5-Chloro-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinoline-8-ol, [0084]
5-Chloro-7-[(R)-[(6-methylpyridin-2-yl)amino][4-(trifluoromethyl)phenyl]m-
ethyl]quinoline-8-ol, [0085]
2-Methyl-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinoline-8-ol, [0086]
2-Methyl-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinoline-8-ol, [0087]
2-[(Dimethylamino)methyl]-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifl-
uoromethyl)phenyl]methyl]quinoline-8-ol, [0088]
2-[(Dimethylamino)methyl]-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifl-
uoromethyl)phenyl]methyl]quinoline-8-ol, [0089]
2-[(Dimethylamino)methyl]-7-[(R)-[(4-methylpyridin-2-yl)amino][4-(trifluo-
romethyl)phenyl]methyl]quinoline-8-ol, [0090]
2-[(Dimethylamino)methyl]-7-[(S)-[(4-methylpyridin-2-yl)amino][4-(trifluo-
romethyl)phenyl]methyl]quinoline-8-ol, [0091]
5-Nitro-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-
methyl]quinoline-8-ol, [0092]
5-Nitro-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-
methyl]quinoline-8-ol, [0093]
7-[(R)-[(Pyridin-2-yl)[4-(trifluoromethyl)phenylamino]methyl]quinoline-8--
ol, [0094]
7-[(S)-[(Pyridin-2-yl)[4-(trifluoromethyl)phenylamino]methyl]qu-
inoline-8-ol [0095]
2-(Hydroxymethyl)-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluorometh-
yl)phenyl]methyl]quinoline-8-ol, [0096]
2-(Hydroxymethyl)-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluorometh-
yl)phenyl]methyl]quinoline-8-ol.
[0097] The subject matter of the invention furthermore relates
especially advantageously to the novel enantiomeric derivatives of
8-hydroxyquinoline derivatives, and pharmaceutically acceptable
salts and complexes with divalent or polyvalent metals,
advantageously iron, copper or zinc complexes thereof, especially
advantageously zinc complexes thereof as listed detailed as
follows: [0098]
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinoline-8-ol, [0099]
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinoline-8-ol.
[0100] The subject matter of the invention furthermore relates
especially advantageously to the pharmaceutically acceptable salts
and zinc complex of novel enantiomeric derivatives of
8-hydroxyquinoline derivatives, as listed detailed as follows:
[0101] Potassium
7-[(R)-[(4-methylpirimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinoline-8-olate, [0102] Potassium
7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinoline-8-olate, [0103] Natrium
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinoline-8-olate, [0104]
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinoline-8-ol fumarate, [0105]
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)-phenyl]methyl]-
quinoline-8-ol fumarate, [0106]
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-methyl]-
quinoline-8-ol zinc complex.
[0107] The subject matter of the invention furthermore relates to
medicinal and/or pharmaceutical compositions comprising novel
enantiomeric derivatives of 8-hydroxyquinoline derivatives
disclosed by general formulas (I) and (II), advantageously (I') and
(II'), advantageously (I'') and (II'')--henceforth General Formulas
according to the Invention--and advantageously named specifically
as above, and/or pharmaceutically acceptable salts and/or complexes
with divalent or polyvalent metals, advantageously iron, copper or
zinc complexes thereof, as active agent, which compositions are
containing inert, pharmaceutically acceptable, solid or liquid
carrier and/or excipient, advantageously starch, gelatinized
starch, cellulose, microcrystalline cellulose or
cellulose-derivatives, lactose, lactose monohydrate, talcum,
mannitol, sodium chloride, sodium carbonate, saccharose, maltose,
calcium carbonate, colloidal anhydrous silicon dioxide, stearic
acid, magnesium stearate or isomalt.
[0108] The subject matter of the invention furthermore relates to
medicinal and/or pharmaceutical composition, advantageously solid
composition, especially advantageously tablet, inhalation powder or
capsule, advantageously semi-solid composition, especially
advantageously suppository, or advantageously liquid composition
especially advantageously solution for injection.
[0109] The subject matter of the invention furthermore relates to
process for the preparation of medicinal and/or pharmaceutical
compositions comprising novel enantiomeric derivatives of
8-hydroxyquinoline derivatives described by General Formulas
according to the Invention and advantageously named specifically as
above, and pharmaceutically acceptable salts and/or complexes with
divalent or polyvalent metals, advantageously iron, copper or zinc
complexes thereof by mixing the enantiomeric derivatives and/or
pharmaceutically acceptable salts and/or complexes thereof,
according to the invention with pharmaceutically applicable
carriers and/or excipients disclosed as above, then by formulating
the mixture to a medicinal and/or pharmaceutical composition
advantageously to tablet, inhalation powder or capsule, suppository
or solution especially advantageously to tablet, using the usual,
standard formulation technics.
[0110] The subject matter of the invention furthermore relates to
novel, stereoselective process for the preparation of novel
enantiomeric derivatives of 8-hydroxyquinoline derivatives
described by General Formulas according to the Invention and
advantageously named specifically as above, and pharmaceutically
acceptable salts and/or complexes with divalent or polyvalent
metals, advantageously iron, copper or zinc complexes thereof,
specifically concerning the enantiomeric derivatives of general
formula (I) and (II) by reacting an 8-hydroxyquinoline derivative
of general formula III
##STR00005##
[0111] with an amine of general formula (IV),
R.sup.2--NH.sub.2 IV
[0112] and with an oxo-compound of general formula (V)
##STR00006##
[0113] using quinidine (obtaining R-enantiomer) or quinine
(obtaining S-enantiomer) as catalyst.
##STR00007##
[0114] By this method pure R-enantiomeric derivative of general
formula (I) or pure S-enantiomeric derivative can be obtained, and
can be used as active agent or can be converted to pharmaceutically
acceptable salts or to complexes with divalent or polyvalent
metals, advantageously iron, copper or zinc complexes specifically
advantageously to zinc complexes thereof or can be released from
the salt or complex thereof.
[0115] The terms quinidine or quinine catalysts are interpreted to
have the following chemical structures with the well-known meaning
in the art:
##STR00008##
[0116] See, for example, X. Liu et al., 7 Organic Letters, 167-169
(2005).
[0117] Concerning the further advantageous versions of the
compounds of general formulas according to the invention, the
process according to the invention is described as above, using the
proper R' and R'' substituents indexed properly in the general
formulas (III'), (III'), (IV') (IV'') and (V'') and (V'') of the
starting materials and reagents according to the different versions
of General Formulas according to the Invention and compounds
advantageously specifically named as above.
[0118] The subject matter of the invention furthermore relates to
novel, stereoselective process for the preparation of novel
R-enantiomeric derivatives of 8-hydroxyquinoline derivatives of
general formulas (I), (I') and (I''), and advantageously named
specifically as above, and pharmaceutically acceptable salts and/or
complexes with divalent or polyvalent metals, advantageously iron,
copper or zinc complexes thereof, specifically concerning the
enantiomeric derivatives of general formula (I) and (II) by
reacting an 8-hydroxyquinoline derivative of general formula (III),
advantageously (III'), further advantageously (III'') with an amine
of general formulas (IV), advantageously (IV'), further
advantageously (IV'') and with an oxo-compound of general formulas
(V), advantageously (V'), further advantageously (IV''),
advantageously using quinidine as catalyst and obtaining by this
method pure R-enantiomeric derivative.
[0119] The subject matter of the invention furthermore relates to
novel, stereoselective process for the preparation of novel
S-enantiomeric derivatives of 8-hydroxyquinoline derivatives of
general formulas (I), (I') and (I''), and advantageously named
specifically as above, and pharmaceutically acceptable salts and/or
complexes with divalent or polyvalent metals, advantageously iron,
copper or zinc complexes thereof, specifically concerning the
enantiomeric derivatives of general formula (I) and (II) by
reacting an 8-hydroxyquinoline derivative of general formula (III),
advantageously (III'), further advantageously (III'') with an amine
of general formulas (IV), advantageously (IV'), further
advantageously (IV'') and with an oxo-compound of general formulas
(V), advantageously (V'), further advantageously (IV''),
advantageously using quinine as catalyst and obtaining by this
method pure S-enantiomeric derivative.
[0120] For the process according to the invention solvent,
advantageously organic solvent or water, especially advantageously
acetonitrile was used as reaction medium.
[0121] The reaction can be implemented by using advantageously
acidic catalyst, especially advantageously formic acid.
[0122] For the preparation of the compounds according to the
invention the following novel, general stereoselective processes
have been applied according to the invention, depending on the
starting materials:
[0123] For the process according to the invention solvent,
advantageously organic solvent or water, especially advantageously
acetonitrile was used as reaction medium.
[0124] The reaction can be implemented by using advantageously
acidic catalyst, especially advantageously formic acid.
[0125] For the preparation of the compounds according to the
invention the following novel, general stereoselective processes
have been applied according to the invention, depending on the
starting materials:
[0126] Process "R" for Preparation of Enantiomers with "R"
Configuration:
[0127] To a solution of 50 mmol of quinidine in the proper solvent
(180 ml), formic acid (0.84 equivalent) was added in inert
atmosphere, then amine derivative (1 equivalent), aldehyde compound
(1 equivalent) and 8-hydroxyquinoline derivative (1.2 equivalent)
were added.
[0128] The mixture was stirred at a proper temperature until the
product was formed in a desired quantity.
[0129] The reaction mixture was concentrated in vacuum to its third
volume, the residue was dissolved in dichloromethane. The solution
was washed
with 1 M NaOH and extracted further by 1M NaOH 6 times.
[0130] A non-polar solvent was added to the organic phase then the
dichloromethane was evaporated off. The solution obtained was added
to 100 ml of 3 M HCl. The phases were separated and the organic
layer was extracted with 3 M HCl solution.
[0131] To the combined HCl phase methyl-t-butyl-ether was added
then the pH of the biphasic system was adjusted with 40% NaOH
solution to four.
[0132] The precipitated quinidine was filtered off, the biphasic
filtrate was separated, then the water layer was washed twice by
methyl-t-butyl-ether, the combined organic phase was dried on
sodium sulfate and filtered.
[0133] A proper solvent was added to the filtrate, the
methyl-t-butyl-ether was evaporated off and the residue was stirred
at room temperature for 16 hours. The precipitated racemic crystals
were filtered.
[0134] The mother liquor was concentrated in reduced pressure and
the remaining pure R-enantiomer was dissolved in 40 ml of
isopropanol, stirred at room temperature for 48 hours, then the
precipitated crystals were filtered off, to give pure crystalline
R-enantiomer.
[0135] Process "S" for Preparation of Enantiomers with
S-Configuration
[0136] To a solution of quinine (55 mmol) in a proper solvent (300
ml) in inert atmosphere, formic acid (0.8 equivalent), amine (2.5
equivalent), aldehyde derivative (3.7 equivalent), and finally
8-hydroxyquinoline derivative (1.0 equivalent) were added.
[0137] The mixture was stirred at a proper temperature until the
product was formed in a desired quantity.
[0138] The solvent was evaporated off in vacuum, the residue was
dissolved in dichloromethane and chromatographed on silica gel. The
fractions containing the product were collected and the solvent was
evaporated off.
[0139] The raw product obtained was purified by normal phase Flash
chromatography using hexane-ethyl-acetate gradient, then the
fractions containing the product were collected and
concentrated.
[0140] The residue was dissolved in 2-propanol. After 2 hours
stirring the precipitated racemic crystals were filtered off. The
mother liquor was concentrated in vacuum to get the S-enantiomer.
The raw product was isolated by the usual methods (e.g. filtration,
centrifugation) and was purified by known methods if needed (e.g.
recrystallization or chromatography).
[0141] The subject matter of the invention furthermore relates to
the use of novel enantiomeric derivatives of 8-hydroxyquinoline
described by General Formulas according to the Invention and
advantageously named specifically as above and pharmaceutically
acceptable salts and complexes with divalent or polyvalent metals,
advantageously iron, copper or zinc complexes thereof, for the
manufacture of a medicinal and/or pharmaceutical composition
suitable for the treatment and/or prevention of neuropsychiatric
diseases advantageously anxiety disorders, schizophrenia,
depression, bipolar disorder, especially advantageously bipolar
disorder, depression.
[0142] The subject matter of the invention furthermore relates to
the use of novel enantiomeric derivatives, advantageously the
R-enantiomeric derivatives of 8-hydroxyquinoline described by
General Formulas according to the Invention and advantageously
named specifically as above and pharmaceutically acceptable salts
and complexes with divalent or polyvalent metals, advantageously
iron, copper or zinc complexes thereof, for the manufacture of a
medicinal and/or pharmaceutical composition suitable for the
treatment and/or prevention of neurologic diseases, advantageously
epilepsy, amnesia, different memory disorders, cognitive functional
problems, neurodegenerative diseases especially advantageously
memory disorders, epilepsy, amnesia, cognitive functional problems,
Alzheimer's disease, Huntington disease, Parkinson disease, Wilson
disease, amyotrophic lateral sclerosis (ALS).
[0143] The subject matter of the invention furthermore relates to
the use of novel enantiomeric derivatives of 8-hydroxyquinoline
described by General Formulas according to the Invention and
advantageously named specifically as above and pharmaceutically
acceptable salts and complexes with divalent or polyvalent metals,
advantageously iron, copper or zinc complexes thereof, for the
manufacture of a medicinal and/or pharmaceutical composition
suitable for the treatment and/or prevention of ischemia and
reperfusion injuries thereof, advantageously of cardiovascular
disorders, blood vessel catastrophes, traumatic injuries,
neurodegenerative traumas, diseases in connection with
transplantations and in connection with ischemia and reperfusion
injuries thereof, advantageously of impairments of the brain,
heart, liver, kidney or lung especially advantageously of traumatic
brain injuries and for inhibition of organ, advantageously of skin
graft rejection, especially advantageously for inhibition of skin
graft rejection.
[0144] The subject matter of the invention furthermore relates to
the use of novel enantiomeric derivatives of 8-hydroxyquinoline
described by General Formulas according to the Invention and
advantageously named specifically as above and pharmaceutically
acceptable salts and complexes with divalent or polyvalent metals,
advantageously iron, copper or zinc complexes thereof, for the
manufacture of metal chelate forming, cyto-protective,
neuroprotective and/or cardio-protective medicinal and/or
pharmaceutical composition, showing favourable binding to the
target protein, suitable for the treatment and/or prevention of
neuropsychiatric, neurologic diseases and of diseases in connection
with ischemia.
[0145] The subject matter of the invention furthermore relates to
the use of novel enantiomeric derivatives of 8-hydroxyquinoline
described by General Formulas according to the Invention and
advantageously named specifically as above and pharmaceutically
acceptable salts and complexes with divalent or polyvalent metals,
advantageously iron, copper or zinc complexes thereof, for the
manufacture of a cyto-protective, neuroprotective and/or
cardio-protective medicinal and/or pharmaceutical composition
suitable for protecting the cells from the cytotoxic attacks, and
for the treatment of patients suffering in diseases combined with
"cell-death" and/or prevention of their diseases.
[0146] The subject matter of the invention furthermore relates to
the use of medicinal and/or pharmaceutical compositions comprising
novel enantiomeric derivatives of 8-hydroxyquinoline described by
General Formulas according to the Invention and advantageously
named specifically as above and pharmaceutically acceptable salts
and complexes with divalent or polyvalent metals, advantageously
iron, copper or zinc complexes thereof for treatment and prevention
of the above described diseases, which compositions can be
administered by a usual method in medicine, advantageously by oral,
buccal, sublingual, parenteral or intravenous, rectal or inhalation
method especially advantageously administered by oral method.
[0147] The subject matter of the invention furthermore relates to a
cytoprotective, neuroprotective or cardioprotective process for
treatment and/or prevention of cardiovascular diseases, blood
vessel catastrophes, traumatic injuries, neurodegenerative traumas,
diseases in connection with transplantations and in connection with
ischemia and reperfusion injuries thereof, advantageously of
impairments of the brain, heart, liver, kidney or lung especially
advantageously of traumatic brain injuries and for inhibition of
organ, advantageously for skin graft rejection, especially
advantageously for inhibition of skin graft rejection, by
administering a medicinal and/or pharmaceutical composition by a
usual method in medicine, advantageously by oral, buccal,
sublingual, parenteral or intravenous, rectal or inhalation method
especially advantageously administered by oral method, which
composition comprises novel enantiomeric derivatives of
8-hydroxyquinoline described by General Formulas according to the
Invention and advantageously named specifically as above and
pharmaceutically acceptable salts and complexes with divalent or
polyvalent metals, advantageously iron, copper or zinc complexes
thereof.
[0148] The subject matter of the invention furthermore relates to a
cytoprotective, neuroprotective process for treatment and/or
prevention of neuropsychiatric diseases advantageously anxiety
disorders, schizophrenia, depression, bipolar disorder, especially
advantageously bipolar disorder, and depression and further of
neurologic diseases, advantageously epilepsy, amnesia, different
memory disorders, cognitive functional problems, neurodegenerative
diseases especially advantageously memory disorders, epilepsy,
amnesia, cognitive functional problems, Alzheimer's disease,
Huntington disease, Parkinson disease, Wilson disease, amyotrophic
lateral sclerosis (ALS) by administering a medicinal and/or
pharmaceutical composition by a usual method in medicine,
advantageously by oral, buccal, sublingual, parenteral or
intravenous, rectal or inhalation method especially advantageously
administered by oral method, which composition comprises novel
enantiomeric derivatives of 8-hydroxyquinoline described by General
Formulas according to the Invention and advantageously named
specifically as above and pharmaceutically acceptable salts and
complexes with divalent or polyvalent metals, advantageously iron,
copper or zinc complexes thereof.
HISTORY, THE STATE OF THE ART, MECHANISM OF ACTION
[0149] 1) The specification of the history, state of the art,
mechanism of action of the novel enantiomeric derivatives of
8-hydroxyquinoline according to the invention, described by General
Formulas according to the Invention and advantageously named
specifically as above and pharmaceutically acceptable salts and
metal complexes thereof and medicines suitable for treatment and/or
prevention of different diseases and comprising the subject
compounds.
[0150] The prior art and patents referred and cited in the present
specification hereinafter are all part of the state of the art.
[0151] The various etiological cell injuries and cell deaths are
the main characteristics of many cardiovascular, neurological and
inflammatory disorders. Cell injuries may occur as the results of
cellular hypoxia or ischemia, formation of various kinds of
oxidants or free radicals and/or overproduction of various
biological mediators (cytokines, chemokines, lipid mediators) and
overproduction and/or aggregation of different toxic peptides (e.g.
.beta.-amyloid peptides) or of proteins (synuclein, huntigtin,
prion) in case of neurodegenerative diseases (comprehensive
literature: Orrenius, 2007; Mattson, 2006).
[0152] These processes are often interdependent; so those occur as
parts of self-amplifying ("suicidal") intracellular cycles and form
the determining basis of many human diseases.
[0153] Though cell death is typically referred as apoptosis or
necrosis, these two forms only represent the two ends of the range
of the forms of cell injuries.
[0154] The intercellular mechanisms taking part in the above cell
death processes are complex, but often activate the cell death
effector family called caspases and mitochondrial dysfunction,
mitochondrial depolarisation, generation of reactive oxygen species
and release of mitochondrial components into the cytosol
(comprehensive literature: Szabo, 2005; Duprez et al., 2009;
Degterev and Yuan, 2008; Wang et al., 2009; Stefanis, 2005).
[0155] In the case of Alzheimer's disease, neuronal cell death
results mainly from the direct cytotoxic effects of aggregated
.beta.-amyloid peptides which, in part are generated by
caspase-triggered apoptosis.
[0156] The other reason of the deterioration of the cognitive
functions is the quantitative decreasing of different synaptic
proteins playing role in neurotransmission and the reduced
reactivity of different receptors like acetylcholine and muscarine
(Machova et al., 2008; Bartus, 2000).
[0157] There are more and more evidences, that in the course of
.beta.-amyloid peptide aggregation not the fibrillary but the
oligomer form has a correlation with neuropathological changes in
connection with mortality and dementia, like development of protein
tangles and neuritic disorders (e.g. reduced dentrit-spike and
reduced numbers of active synapses) (comprehensive literature: Lue
et al., 1999; McLean et al., 1999; Wang et al., 1999).
[0158] However, besides the oligomer forms of .beta.-amyloid
peptide aggregates the fibrillary forms results also neuronal
destruction and reduced functionality partly through LDI receptors
(Janciauskiene et al., 1999).
[0159] The compounds preventing cell injury and cell death are
usually called "cytoprotective" compounds. Cytoprotection may be
achieved by many pharmacological and biochemical methods. The
following examples of them are mentioned here: scavengers of
oxidants and free radicals, inhibitors of certain "death effector
pathways", stabilisation of cell membranes, etc. In the course of
ischemia or several related disease processes, iron and copper
cations are released from the tissues which catalyse hydroxy-free
radical formation in the Haber-Weiss pathway in a known manner
causing cell injuries. Inactivation or chelate formation of these
metal cations may result in a cytoprotective effect. Thus
experiments were conducted to mitigate the catalytic efficiency of
iron and copper cations in such a way that iron-chelate forming
siderophores (e.g. deferoxamine) were administered (Lewen et al.,
2000; Britton et al., 2002).
[0160] It is known that glutamate is released along with zinc
cations from the synaptosomes of the nervous system cells using
glutamate as a chemical messenger. Usually, the zinc released in
the nerve synapse is quickly built again in the synaptosomes. As a
result of ischemia, lasting attacks and cerebral lesion, the zinc
released from the synaptosomes is accumulated in the extracellular
liquid surrounding the neurons. When an excessive amount of zinc
enters the cell body, zinc may trigger cell death via apoptosis and
necrosis. Zinc-chelate forming through that mechanism may result in
neuroprotection and influence the outcome of various
neuropsychiatric diseases. (Regland et al., 2001; Koh et al.,
1996).
[0161] Therefore, the zinc-chelating agents may also be useful in
treatment of the Alzheimer's disease by binding zinc occurring in
the plaques thus weakening the structure of the plaques
(Frederickson et al., 2005; Schafer et al., 2007). The
zinc-chelating agents may also be useful in the treatment of
Huntington's disease (Nguyen et al., 2005).
[0162] According to another way of cytoprotection, the
intracellular pathways mediating protective effects are induced. A
prototype of this approach is the so-called "ischemic
preconditioning" where the cells or organs are subjected to
ischemia for a short time in order to induce over-regulation of the
cytoprotective genes (e.g. genes of antioxidant enzymes, heat shock
proteins and others). Induction of heme oxygenase enzyme (HMOX-1)
has demonstrated cytoprotection in several experimental systems
(e.g. Li et al., 2007; Idris et al., 2008).
[0163] Compounds used for inhibition of endoplasmatic reticulum
stress have been described in the American patent application
publication no. US 2008/293699.
[0164] Part of the state of the art is our own earlier patent
application also, where recently new racemic 8-hydroxyquinoline
derivatives and indications thereof have been described and where
some members of this compound family have been identified by
cell-based screening tests for systematic identification of
cytoprotective compounds (WO2011148208).
[0165] In this test a certain form of cell injury was simulated and
a chemical library was screened in order to identify compounds
preventing or retarding cell injury (e.g. Gero et al., 2007).
[0166] By means of the cell-based screening method, we have found
and identified novel racemic 8-hydroxyquinoline derivatives.
[0167] These compounds protect the cells from injuries induced by
oxidative stress therefore these can potentially be used in the
treatment of many diseases.
[0168] The racemic compounds exert various cellular effects e.g.
iron-copper and zinc chelating, inhibition of PARP-activation,
inhibition of mitochondrial dysfunction, or activation of heme
oxygenase enzyme.
[0169] However, according to our earlier invention, only the
racemic compounds have been described, the enantiomerically pure
derivatives thereof have not been prepared at all, and neither the
chemical characters thereof have been disclosed.
[0170] Moreover concerning the earlier cytoprotection tests, no
direct evidence has been provided, that neuronal destruction
triggered by the different aggregated forms of .beta.-amyloid
peptides (oligomer, and high molecular weight fibrillary complexes)
can be prevented by the racemic compounds and whether they can be
used for increasing cognitive functions.
[0171] Earlier it was also not known, through which molecular
mechanisms the positive effects of each enantiomer can be achieved
on neurons, cardiac sells and in neurodegenerative animal models
induced by beta oligomers.
[0172] It was shown that, the pure R-enantiomeric derivative has
effect on several targets that can be related to the
neuroprotective effect and inhibiting effect concerning
neuroinflammation of the clinical candidate.
[0173] The R-enantiomer compound of the Example 2 according to the
invention inhibits in micromolar concentration the caspase-3 and
5-lipoxygenase enzymes and the transcriptional induction regulated
by the activated T-cell nuclear factor (NF-AT).
[0174] The activation of caspase-3 is strongly related to several
degenerative processes of the aging brain (Lynch et al., 2002) and
to the pathogenesis of neurodegenerative diseases occurred in old
age (Eckert et al., 2003).
[0175] The activation of caspase-3 is a common connection point of
many toxic stimulations, included the oxidative injury and the
toxicosis induced by the aggregated .beta.-amyloid peptides.
[0176] The activated caspase-3 protease induces degradation of
several key intracellular proteins, resulting apoptosis at the end
(Hengartner, 2000).
[0177] However, the caspase-3 plays important role not only in the
late phase of neurodegeneration inducing neuronal destruction but
also encourages the development of the pathological changes in the
early stage, because it is responsible also for the proteolytic
cleavage of both of amyloid precursor protein (APP) and the GGA3
adaptor which are transforming to toxic amyloid peptide aggregates
at the end.
[0178] The axonal microtubule-associated tau protein forming
neurofibrillary tangles is also substrate of the caspase-3 enzyme,
so increased function of the caspase-3 enzyme reduces the binding
of the tau with total length to microtubule, leading to a neuritic
degeneration at the end.
[0179] Moreover, it has been shown that the fibrillary tau protein
deposits in neurons are not causes but consequences of the
degenerative processes within the cell induced by the activation of
caspase (Calignon et al., 2010).
[0180] Coming from all these, the neuronal destruction related to
the neurodegenerative processes for different reasons (oxidative
stress, effects of .beta.-amyloid peptide aggregates) and the
developed deterioration of the cognitive processes can be inhibited
inter alia by using the pure R-enantiomeric derivative according to
the invention and to the Example 2 for inhibition the caspase
enzyme in the brain.
[0181] Disturbance in calcium ion homeostasis and the following
activation of calcineurin can induce pathological changes related
to the Alzheimer's disease (Liu et al., 2005.
[0182] The activated T-cell nuclear factor (NFAT) is
dephosphorylated by calcineurin, translocated in the cell nucleus
by dephosphorylation, triggering expressions of several genes which
are inducing neuronal destruction, activation of astrocyte and
neuroinflammatory processes. Among these the induction of
interleukin-1.beta. (IL-1.beta.) is of primary importance, being
one of the causes of the increased level of extracellular glutamic
acid, and are involved in hyperexcitable synaptic activity,
generally in induction and maintenance of neuroinflammatory
processes related to several neurodegenerative diseases (Sama et
al., 2008).
[0183] Recently it was shown that the neurotoxicity triggered by
.beta.-amyloid could be reduced by pharmacological inhibition of
NFAT in transgenic mouse (Hudry et al., 2012).
[0184] Furthermore different levels of each NAFT isoforms have been
stated in brain samples of patients of Alzheimer's disease
suffering from dementia with different severity (Abdul et al.,
2009).
[0185] It was published by the same authors also, that the protein
level of excitatory amino acid transporter (EAAT-2) was NFAT
depending reduced by .beta.-amyloid oligomers in astrocytes and the
level of glutamic acid resulting neuronal destruction was
increased.
[0186] Using general NFAT inhibitors all these effects could be
reversed proving that NFAT transcriptional activation has important
role in pathological processes related to the central nervous
system and with the inhibition thereof it was proved that the NFAT
system could be an important target concerning the therapy of
neurodegenerative diseases.
[0187] It is well known that the inhibition of calcineurin/NFAT
system reduces the organ graft rejection, so the compounds
according to the invention could also be suitable for inhibition of
organ graft rejection.
[0188] It is well known that the inhibition of calcineurin/NFAT
system reduces the organ graft rejection, so the compounds
according to the invention could also be suitable for inhibition of
organ graft rejection. (Lee es Park, 2006): Lee M, Park J.
Regulation of NFAT activation: a potential therapeutic target for
immunosuppression. Mol Cells. 2006 Aug. 31; 22(1):1-7.)
[0189] NFAT transcriptional induction triggered by A23187 ionophore
and PMA inductive agent was inhibited by the compound according to
the Example 2 and the invention in micromolar concentration.
Therefore, it can be concluded that the enantiomerically pure
R-enantiomeric derivative according to Example 2 is able to inhibit
the increased calcineurin activity induced by .beta.-amyloid and by
the increased level of intracellular calcium and the consequent
calcineurin activity, and therefore it is able to inhibit the
apoptotic and neuroinflammatory processes, and positive effect may
have generated in different neurodegenerative diseases and by
transplantations.
[0190] The calcineurin/NFAT system and the caspase-3 apoptotic
activation system are related to each other.
[0191] The calcineurin over function induces caspase-3 activation
and apoptosis (Asai et al., 1999).
[0192] As the compound according to Example 2 inhibits both NFAT
transcriptional activity and caspase-3 enzyme activity the compound
according to the invention reduces the neuronal destruction by
double effects, but also has positive effect on the
neuroinflammatory processes induced by NFAT.
[0193] Therefore, it is assumed that pure R-enantiomeric derivative
has more efficiency than the solely on NAFT system active or solely
the caspase-3 enzyme inhibiting compounds.
[0194] The third target of the pure R-enantiomeric derivative
according to the invention and to Example 2 is the enzyme
5-lipoxygenase (5-LOX) having an important role in inflammatory and
traumatic (excitotoxic and ischemic) processes related to central
nervous system (Zhou et al., 2006) and in neurodegenerative
diseases related to aging (Uz et al., 1998). It was shown in
transgenic mice that inhibition of 5-LOX reduced .beta.-amyloid
aggregates and improved the cognitive functions (Firuzi et al.,
2008).
[0195] Increased activity of 5-LOX reduced the level of PSD-95,
synaptophysin and MAP2 proteins being important in synaptic
integrity (Chu et al., 2013).
[0196] It is concluded from all these that, the R-enantiomeric
derivative according to the Example 2 having micromole activity
validly, reduces the neuroinflammatory processes by inhibition of
5-LOX enzyme thereof and has positive influence on pathological
changes related to .beta.-amyloid peptides and tau proteins
[0197] The cell injury caused by hypoxia is a common characteristic
of different diseases including brain infarct, brain stroke and
infarct of heart muscle.
[0198] An important and central element of cell's accommodation to
hypoxia is the hypoxia-inducible factor (HIF), a transcription
factor activating expressions of several genes inter alia including
the genes playing important role in glucose metabolism, in the
antioxidant system, in angiogenesis or in blood cell formation
(comprehensive article: Chowdhury et al., 2008).
[0199] It was shown that, impacts causing rise of the level of HIF,
have advantageous effect on ischemic diseases and on reproduction
of stem cells (Zhang et al., 2006).
[0200] Using a screening system described earlier, HIF system
activating small molecules, including several 8-hydroxyguinoline
type compounds with furcate structure could be identified (Smirnova
et al., 2010). However, among the molecules listed in the examples
there was not any pure enantiomer, only racemic mixtures have been
worked with.
[0201] The subject racemic compounds activated the expression of
genes with a value between 2-10 .mu.M IC.sub.50 by 2-7.times.
maximal value. According to our invention, it has been found that,
the R-enantiomeric derivatives according to Example 2 and
S-enantiomeric derivatives according to Example 3 were capable to
increase the gene activity of phosphoglycerate kinase 1 (PGK-1),
the vascular endothelial growth factor (VEGF), the hem-oxygenase 1
(HMOX-1) and the erythropoietin (EPO) in the cells, all regulated
by HIF.
[0202] According to our invention the highest activation was found
in case of EPO gene, in submicromolar concentration with over
20.times. maximal activation value.
[0203] The PGK1, VEGF, EPO activations have been disclosed in the
subject document (Smirnova et al., 2010), but it was not shown any
activity of 8-hydroxyquinoline type compounds
[0204] Furthermore, recently it has been shown that, the
hem-oxygenase 1 (HMOX-1), an antioxidant enzyme induced by HIF has
strong cytoprotective effect in neurons (Chen et al., 2000) and in
endothelial cells of brain capillaries (Bresgen et al., 2003).
[0205] As oxidative stress plays an important role in several
neurodegenerative and neuroinflammatory processes, the
pharmacological induction of HMOX-1 is judged as a logic and
desirable therapeutic solution (Calabrese et al., 2003; Jazwa and
Cuadrado., 2010).
[0206] The induction of HMOX-1 influenced also the outcome of heart
transplantation advantageously (Bach 2006).
[0207] In one of our earlier publication it has been shown that, a
racemic 8-hydroxyquinoline causes increase of HMOX-1 gene activity
in cardiac sells, explaining partly the cardioprotective
characteristics thereof in cell system and protective effect
[0208] thereof in case of heart muscle injury caused by
ischemia/reperfusion in rat (Korkmaz et al., 2013).
[0209] On the other hand, none of the enantiomerically pure form of
the compound group described in the present patent application of
general formula has been examined yet.
[0210] It was shown that, the R-enantiomeric derivative according
to Example 2 and S-enantiomeric derivative according to Example 3
caused the induction of HMOX-1 in astrocytes, concluding that, both
pure enantiomer described by us can have cytoprotective effect
concerning the diseases with oxidative stress.
[0211] The highest inductive effect of R-enantiomeric derivative
according to Example 2 and S-enantiomeric derivative according to
Example 3 on expression of gene was stated in case of EPO gene.
[0212] Moreover, it has been shown that, by oral administration,
the R-enantiomeric derivative according to Example 2 resulted
increased EPO gene activity in cerebral cortex and hippocampus of
elderly mice chronically.
[0213] Recently it was shown that, the EPO in brain has outstanding
role in neuroprotection and advantageous characteristics thereof
revealed itself in several psychiatric diseases like depression,
schizophrenia, bipolar disorder, epilepsy (Newton et al., 2013) in
neurodegenerative diseases like Alzheimer's disease, Parkinson
disease (Arabpoor et al., 2012; Xue et al., 2007), traumatic brain
injury, blood vessel catastrophes in brain (Mammis et al., 2009),
diseases characterized by neuroinflammation, like sclerosis
multiplex (Hagemeyer et al., 2012), and had positive effect in
animal models and several clinical tests concerning other diseases
related to central nervous system, like amyotrophic lateral
sclerosis (ALS) (Nob at al., 2014; Merelli et al., 2013; Chong et
al., 2013). Because of this there were several methods examined
targeting the ingestion of the EPO directly into the brain.
[0214] The intravenous EPO injection could cause system side
effects, therefore there were experiments also for ingesting EPO
through the nose (Merelli et al., 2011).
[0215] However, these kinds of applications could have several
disadvantages inter alia the costly production, the involved
administration, stability, dosage and the side effect on possible
blood cell forming organs of EPO.
[0216] Therefore, according to an advantageous solution an EPO
synthesis could be induced locally in the brain by a small
molecule.
[0217] This goal has been achieved by the enantiomeric derivatives
according to the invention, described in the present patent
application and concrete examples has been given concerning the EPO
inducing effect of R-enantiomeric derivative according to Example 2
and S-derivative according to Example 3 in cell system.
[0218] Concerning the R-enantiomeric derivative according to the
Example 2 and the invention it was shown that, it can increase the
EPO gene activity in different regions of brain also in living
animal.
[0219] By these characteristics of the compounds according to the
invention and disclosed in the present patent application it is
obvious to use them in case of different psychiatric diseases,
diseases related to ischemia and central nervous system,
neurodegenerative disorders, and inflammatory processes related to
the brain.
[0220] 2) History of the process according to the invention
[0221] The process is actually a modification according to the
invention of the known Betti-reaction, which makes possible to
prepare novel enantiomeric derivatives by novel, stereoselective
synthesis.
[0222] In the course of the stereoselective process according to
our invention the R- or S-enantiomeric derivatives have been
prepared by using cinchona stereoisomer (quinidine or quinine) in a
novel enantioselective version of Betti reaction (Betti, 1900;
Betti, 1903; Phillips et al., 1954; Phillips et al., 1956;
Phillips, 1956) modified and optimised by us.
[0223] The advantage of our process is that the proper enantiomers
were obtained by a simple recrystallization over 99% enantiomeric
excess, without using any separation technique (e.g. chiral
preparative HPLC) using low-cost cinchona organocatalysts:
catalysed by quinidine R-enantiomer, catalysed by quinine
S-enantiomer could be obtained.
[0224] The known processes disclosed in the background literature
of stereoselective Betti-reaction mainly enantiomerically pure
reactant (e.g. aralkyl amine) was used, then the proper enantiomers
were isolated by separation of the chiral support (Palmieri,
2000).
[0225] Organocatalysed asymmetric reactions using cinchona
alkaloids or modified derivatives thereof are known in case of
Mannich reactions (Verkade at al., 2008).
SUMMARY OF THE INVENTION
[0226] The essence and advantages of the invention can be
summarised as follows.
[0227] The enantiomerically pure R-enantiomeric derivative prevents
the destruction of neurons, the neuroinflammatory processes induced
by astrocytes and microglia and furthermore advantageously reduces
the pathological reactions related to amyloid peptides and to tau
proteins which are destroying the neurotoxic and synaptic
systems,
[0228] In consequence the compounds according to the invention
advantageously to Example 2 reduces the deterioration of the
cognitive functions related to neurodegenerative processes and
therefore they could be therapeutically effective in treatment of
several degenerative diseases related to the central nervous
system.
[0229] As the of enantiomerically pure compounds according to the
invention advantageously to Example 2 influence all the three
identified targets (caspase-3, NAFT, 5-LOX), effectively, they are
effective not only in neurodegenerative processes, but also in
different ischemic processes like ischemic and inflammatory
processes related to heart vascular system, therefore the compound
could be also effective in treatment of diseases related to these
processes.
[0230] The active ingredient of the medicinal and/or pharmaceutical
compositions according to our invention is a molecule on one side
not complexed with iron, copper or zinc cations, on the other side
forms complex with iron, copper or zinc cations. The essence of the
subject matter of the invention and base of novelty is that all
prior art documents and patents being part of the state of the art
disclosed only the racemic products and characteristics, biological
effect thereof, the pure enantiomers according to the invention and
characteristics, biological effect thereof are disclosed in our
present patent application for the first time.
[0231] The essence of our invention is furthermore that the novel
enantiomers according to the subject matter of invention are
prepared by the stereoselective synthesis also according to the
subject matter of the invention therefore the novel pharmaceutical
compositions are well applicable for prevention and/or treatment of
the diseases listed in present specification, which applications
are also part of the subject matter of the invention.
[0232] The essence of the stereoselective process according to the
invention is that using quinidine catalyst enantiomerically pure
R-enantiomer, using quinine catalyst enantiomerically pure
S-enantiomer is formed.
[0233] The medicinal and/or pharmaceutical compositions according
to our invention can be used for prevention and/or treatment of the
diseases listed in present specification as cytoprotective,
neuroprotective and cardioprotective agent.
[0234] So the subject matter of our invention furthermore relates
to a cytoprotective, neuroprotective and cardioprotective process
for prevention and/or treatment of the diseases listed in present
specification by administering the medicinal and/or pharmaceutical
compositions according to the invention by a usual method in
medicine, advantageously administered by oral method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0235] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the U.S.
Patent and Trademark Office upon request and payment of the
necessary fee.
[0236] FIG. 1
[0237] The calculated configuration of the conformer of the
S-enantiomer compound according to the invention (1) with the
lowest energy.
[0238] FIG. 2
[0239] The FTIR (Fourier transform infrared spectroscopy) and VCD
(Vibrational Circular Dichroism) spectrum of the sample of
R-enantiomer compound according to the invention measured in
CDCl.sub.3 (deuterated chloroform) solvent.
[0240] FIG. 3
[0241] The calculated VCD spectrum of the conformers of the
S-enantiomer compound according to the invention with greater
population than 1%.
[0242] FIG. 4
[0243] The theoretical VCD spectrum of S- and R-enantiomer
compounds according to the invention and the conformers thereof
measured in CDCl.sub.3 calculated as with the population weighted
sum.
[0244] FIG. 5
[0245] The inhibiting effect of the R- and S-enantiomer compounds
according to the invention on different matrix metalloprotease
enzymes is shown.
[0246] FIGS. 6A and 6B
[0247] The effect of R-enantiomer compound according to the
invention on cell death in vitro caused by hydrogen-peroxide on
cardiac sells is shown.
[0248] FIGS. 7A and 7B
[0249] The effect of R-enantiomer compound according to the
invention on cell death in vitro caused by hydrogen-peroxide on
SH5Y neuron cells is shown.
[0250] FIGS. 8A and 8B
[0251] The effect of S-enantiomer compound according to the
invention on cell death in vitro caused by hydrogen-peroxide on
cardiac sells is shown.
[0252] FIGS. 9A and 9B
[0253] The effect of S-enantiomer compound according to the
invention on cell death in vitro caused by hydrogen-peroxide on
SH5Y neuron cells is shown.
[0254] FIG. 10
[0255] The effect of sodium salt of R-enantiomer compound according
to the invention on cell death in vitro caused by fibrillar
aggregates of .beta.-amyloid peptides on primer cortical neuron
cells is shown.
[0256] FIG. 11
[0257] The effect of zinc complex of R-enantiomer compound
according to the invention on cell death in vitro caused by
fibrillar aggregates of .beta.-amyloid peptides on primer cortical
neuron cells is shown.
[0258] FIG. 12
[0259] The effect of R-enantiomer and S-enantiomer compounds
according to the invention and according to Example 2 and 3 on
short-term memory disorders in vivo caused by oligomer aggregate of
.beta.-amyloid peptides is shown.
[0260] FIG. 13
[0261] The effect of different concentration of R-enantiomer
compound according to the invention and according to Example 2 on
short-term memory disorders in vivo caused by oligomer aggregate of
.beta.-amyloid peptides is shown.
[0262] FIG. 14
[0263] The effect of R-enantiomer and S-enantiomer compounds
according to the invention and according to Example 2 and 3 on
short-term memory disorders in vivo caused by scopolamine is
shown.
[0264] FIG. 15
[0265] The diagram of inhibition effect of R-enantiomeric
derivative according to the invention and according to Example 2 on
NFAT protein transcriptional activity
[0266] FIG. 16
[0267] The effect of R-enantiomeric derivative according to the
invention and according to Example 2 influencing the gene
expression in primer astrocytes
[0268] FIG. 17
[0269] The effect of R-enantiomeric derivative according to the
invention and according to Example 2 influencing erythropoietin
gene expression in hippocampus and cerebral cortex of elderly
animals in course of chronical treatment.
[0270] FIG. 18
[0271] The effect of R-enantiomeric derivative according to the
invention and according to Example 2 influencing the survival of
the skin graft in course of chronical treatment after skin
grafting.
DETAILED DESCRIPTION OF THE INVENTION
Explanation of Expressions, Abbreviations Used in the
Specification
[0272] Expression "enantiomerically pure":
[0273] Characteristic property of a certain enantiomeric form
containing the other enantiomeric form to a specified percentage:
so e.g. in case of 100% purity there is no other enantiomeric form
at all, in case of 98% purity the other enantiomeric form is in 2%
present etc.
[0274] Expression "lower alkyl group":
[0275] Straight or branched chained alky groups with 1-4 carbon
atoms like e.g. methyl, ethyl, isopropyl groups etc.
[0276] Expression "cyclo alkyl group"
[0277] Cyclic groups comprising 3-8 carbon atoms like cyclopropyl,
cyclobutyl, cyclohexyl group etc.
[0278] Expression "aryl group":
[0279] Monocyclic or bicyclic aromatic hydrocarbon groups like e.g.
phenyl group, naphthyl group
[0280] Expression "aralkyl group":
[0281] Alkyl group described above, substituted by one or two aryl
groups described above, like e.g. benzyl, beta phenyl-ethyl group
etc.
[0282] Expression "heteroaryl group":
[0283] 5 or 6 membered aryl groups containing one or more oxygen,
nitrogen and/or sulfur atoms like pyridyl, pyrimidyl, pyrroryl,
oxazolyl groups etc.
[0284] Expression "halogen atom":
[0285] Fluorine, chlorine, bromine or iodine atoms
[0286] Expression "Electron withdrawing substitutes":
[0287] The indicator of electron withdrawing groups, represented as
substituents, advantageously halogen atoms, nitro groups,
trifluoromethyl, methysulfinyl or methylsulfonyl groups.
[0288] Expression "Electron donating group":
[0289] The indicator of electron donating groups represented as
substituents advantageously lower alkyl groups with 1-4 carbon
atoms like e.g. methyl group.
[0290] Expression "General Formulas according to the Invention" The
common meaning of the general formulas (I) and (II), advantageously
(I') and (II'), especially advantageously (I'' and II''), where the
general formulas signed by comma are meaning the advantageous
properly substituted versions.
[0291] Pharmaceutically acceptable salts:
[0292] The novel enantiomers of 8-hydroxyquinoline derivatives
according to the invention described by the General Formulas
according to the Invention as given above and named specifically
can form salts with bases on the hydroxy group and with acids on
nitrogen atom in the substituent attached to position 7 of the
quinoline ring.
[0293] For the salt formation pharmaceutically suitable bases
advantageously alkaline metal hydroxydes like sodium-hydroxyde
potassium-hydroxyde or organic acids advantageously hydrogen
bromide, acetic acid, fumaric acid, maleic acid, malic acid,
succinic acid, tartaric acid, benzenesulfonic acid,
p-toluenesulfonic acid or methane sulfonic acid can be used.
[0294] Pharmaceutically acceptable complexes:
[0295] The novel enantiomers of 8-hydroxyquinoline derivatives
according to the invention described by the General Formulas
according to the Invention as given above and named specifically
can coordinatively bind divalent or polyvalent metals (like e.g.
iron, zinc, copper etc.) and forming complexes with them.
[0296] In such complexes the free electron pairs of the oxygen atom
of hydroxyl group and of nitrogen atom of the quinolone are
participating.
[0297] Chelate forming effect:
[0298] The novel enantiomers of 8-hydroxyquinoline derivatives
according to the invention described by the General Formulas
according to the Invention as given above and named specifically
are capable to form chelates with the free electron pairs described
as above the same way as complexes are formed. Thank to this
chelate forming feature the unbeneficial processes caused by metal
ions can be inhibited in different diseases, and also the
functioning of proteins containing metal ions can be influenced
advantageously.
[0299] Indications, Explanations of Sets of Diseases
[0300] The following statement of sets and collective nouns of
indications and diseases gives help to interpret and to understand
the specification of the application and the use claims containing
the indications. [0301] 1) Neuropsychiatric diseases [0302] anxiety
disorders, schizophrenia, depression, bipolar disorder [0303] 2)
Neurologic diseases: [0304] epilepsy, amnesia, different memory
disorders, cognitive functional problems and neurodegenerative
diseases including memory disorders, epilepsy, amnesia, cognitive
functional problems, Alzheimer's disease, Huntington disease,
Parkinson disease, Wilson disease, amyotrophic lateral sclerosis
(ALS). [0305] 3) Ischemia and reperfusion injuries thereof in
connection mainly but not solely with cardiovascular disorders,
blood vessel catastrophes, traumatic injuries and neurodegenerative
traumas, diseases in connection with transplantations: [0306]
impairments of the brain, including traumatic brain injuries, and
impairments of heart, liver, kidney or lung and organ,
advantageously skin graft rejections.
CHEMICAL EXAMPLES
[0307] The subject matter of our invention is supported by the
following examples without limiting the scope of protection to the
examples.
Example 1
[0308] The Determination of the Absolute Configuration
[0309] The racemic mixture of molecules according to Example 2 and
Example 3 comprises one chiral center, but they have significant
conformation freedom so to determine the absolute configuration
also detailed analysis of conformation was needed.
[0310] The essence of the determination of the absolute
conformation based on VCD spectroscopy is the ab initio (DFT)
levelled calculation of the VCD spectrum of one or the other
enantiomeric form, and then the comparison of the calculated and
the measured spectrum thereof. As the VCD spectrum comprises high
number of bands with alternating signals the comparison is
generally extremely reliable.
[0311] In case the position and signal-pattern of the bands of
calculated and measured spectrum correspond, the absolute
configuration corresponds with the configuration of the chosen
enantiomeric form, if mirrored, the configuration is the
opposite.
[0312] In presence of more conformers the measured spectrum should
be compared with the theoretic spectrum obtained as
population-weighted average of calculated spectrum of each of
enantiomeric compounds.
[0313] The FTIR and VCD spectra of the sample were recorded by a
Bruker PMA-37 typed VCD-module connected with a Bruker Equinox55
FTIR spectrometer with resolution of 4 cm.sup.-1, in a BaF.sub.2
cuvette with 0.05 mm layer thickness, in CDCl.sub.3 solvent, in 100
mg/ml concentration, using MCT detector cooled by liquid N.sub.2.
The spectrometer was optimized on fingerprint-range therefore an
optical filter transparent in 1800-800 cm.sup.-1 range was used and
the photoelastic modulator of the device was set on 1300 cm.sup.-1
wave number.
[0314] The calibration of the device was implemented with the help
of a Cds standard double refracted crystal (CdS multiple
wave-plate, MPW) and a polarisation filter as analyser with metal
grid and with KRS-5 carrier.
[0315] In case of the sample and the reference approximately 42000
interferograms were averaged that corresponds to a recording time
if 12 hours to optimize the bad signal-to-noise ratio caused by the
low signal-intensity of VCD spectra.
[0316] To correct the baseline of the VCD spectrum, the solvent
spectrum recorded under same condition was extracted from the raw
spectrum.
[0317] The infrared spectrum can be obtained together with the VCG
spectrum from the ratio of one channelled DC spectrum of the sample
and that of the reference (solvent) (this is a global infrared
signal not containing any chiral information).
[0318] The quantum chemical calculations were performed on a
Supermicro server (2.times. Intel Xeon.TM. X5680 3.3 GHz 6-corn
processor, 72 GB RAM) using a Gaussian 09 software package (Frisch
et al., 2010.). The quantum chemical calculations were performed on
the S-enantiomeric form on B3LYP/6-31G** DFT theoretical level,
under vacuum condition, the mapping of the conformers was performed
along the five torsion angle (.theta..sub.1-.theta..sub.5) (FIG. 1)
describing the flexibility of the molecule with partly systematic
partly heuristic searching.
[0319] The calculation of the IR- and VCD-spectra of the structures
with optimized geometry was performed also on B3LYP/6-31G** DFT
level.
[0320] The calculated frequency (wave number) values were corrected
by 0.97 scale factor usable in case of B3LYP/6-31G** theoretical
level.
[0321] For the generation of the theoretical spectra were assumed a
signal-shape of Lorentz diagram and half-width measured at 6
cm.sup.-1 mid-height.
[0322] For visualization of the vibrational spectrum GaussView 5.0
program package has been used.
[0323] For estimating the population formed on temperature T=298K,
Boltzmann-distribution was assumed. According to the Boltzmann
distribution the relative population of the ith conformer compared
to the conformer having the lowest free enthalpy (i=0) is:
N i N 0 = e - .DELTA. G i RT ##EQU00001## .DELTA. G i = G i - G 0
##EQU00001.2##
[0324] The mole fraction of ith conformer (the population measured
to the whole conformer mixture) is:
x i = N i i N i ##EQU00002##
[0325] In that case the calculated IR- or VCD spectrum is the
weighted sum of the spectra of each conformers:
S ( v ~ ) = i x i S i ( v ~ ) ( IR , VCD ) ##EQU00003##
[0326] It can be stated after implementation of the population
analysis of the 32 calculated conformer results that there are very
small energy differences between the individual conformers so no
dominant conformer can be determined.
[0327] Among of these eleven have higher population than 1%.
Summarizing these eleven conformers they take 99.6%, so the
spectral contribution of conformers with higher energy was
neglected.
[0328] The geometrical data, relative free enthalpies and
population of the eleven conformers with higher population than 1%
are summarized in Table 1.
TABLE-US-00001 TABLE 1 The characterising torsion angle data,
relative free enthalpy and estimated population of the S-enantiomer
at 298 K temperature, on the basis of quantum chemical calculations
carried out on B3LYP/6-31G** levelled vacuum status. Con- .DELTA.G
Popula- former .theta..sub.1 (.degree.) .theta..sub.2 (.degree.)
.theta..sub.3 (.degree.) .theta..sub.4 (.degree.) .theta..sub.5
(.degree.) (kJ/mol) tion (%) 1 142.0 -173.5 -95.5 -74.4 -179.2 0.0
20.3 1 136.3 -14.7 162.1 70.6 179.9 0.6 15.9 3 82.7 -172.8 65.5
87.1 178.9 1.0 13.6 4 137.3 166.7 160.5 71.0 179.8 1.3 12.2 5 83.3
-172.8 -109.4 87.4 178.9 1.4 11.5 6 88.3 -10.8 13.7 -136.6 179.4
3.1 5.8 7 88.4 -10.8 -166.6 -136.9 179.4 3.1 5.7 8.sup.a 80.9 8.2
62.2 87.0 178.7 3.3 5.4 9.sup.a 81.2 8.2 119.0 87.4 178.6 3.8 4.4
10 143.8 6.7 -93.0 -73.7 -179.5 4.2 3.7 11 89.6 169.4 12.7 -136.4
179.3 7.5 1.0 .sup.aDiffering only in spacing of CF.sub.3 group
[0329] It is characteristic for all these conformers that they have
H-bound between the hydrogen atom of the hydroxyl group and the
nitrogen of the quinolone ring. In some conformers not mentioned
here this is missing, respectively the conformers with H-bound with
the N-atom of the aminopyrimidine part are energetically also less
favourable. The spatial structure of the conformers with the lowest
energy is shown on Scheme 1.
[0330] Infrared and VCD Spectra, the Determination of Absolute
Configuration
[0331] The FTIR and VCD spectrum of compound according to Example
2, is shown in FIG. 2 in the spectral range of 1700-1000
cm.sup.-1.
[0332] This approximately corresponds to the spectral region
limited by the characteristics of optics of VCD spectrometer and
the transmission of the solvent (CDCl.sub.3) used in 0.05 mm
cuvette.
[0333] It can be stated that there are more bands in the VCD
spectrum than in the FTIR spectrum referring to the fact that in
several cases there is a band overlap in the infrared spectrum so
the corresponding bands of VCD spectrum can be identified either
only as a "shoulder" or cannot be identified at all.
[0334] The extreme values (maximums or minimums) in VCD and in
infrared spectrum should appear at identical wavelength in
principle; small differences can be explained also by band overlap.
It can generally be stated that the vibrations of aromatic and
heteroaromatic rings couple strongly with the functional groups
attached to them hence their assignation is fairly difficult due to
their delocalization.
[0335] The most intensive band of the IR spectrum at 1326 cm.sup.-1
derives from the coupling of the symmetric deformation vibration of
CF.sub.3 group and the skeletal vibration of benzene ring, but the
corresponding VCD band has a very low intensity.
[0336] One of the most indicative part of VCD the spectrum is the
negative-positive band-pair at 1502 cm.sup.-1 and 1512 cm.sup.-1
that are not separated in the IR spectrum but they merge into a
broader band at 1507 cm.sup.-1. According to the calculations the
positive band at 1512 cm.sup.-1 has a special diagnostic value as
it derives from coupling of the deformation of the C--H bond
attached to the chiral center and the in-plane deformation
vibration of C--N--H part of 2-aminopyrimidine group. The negative
band at 1507-cm.sup.-1 derives from the skeletal vibration of
quinoline ring and the in-plane OH deformation vibration of its OH
substituent, to a certain extent coupled with the deformation of
the C--H bond of the chirality center. The intensive band at 1583
cm.sup.-1 derives from coupling of the skeletal vibration of
pyrimidine ring and the in-plane N--H deformation vibration of the
seconder amino group attached to it.
[0337] The calculated VCD spectrum of the relevant conformers of
the S-enantiomer is shown in FIG. 3. It can be seen from the
spectrum that the pattern of the VCD bands depends strongly from
the conformers, resulting high averaging and due to to coupling of
vibrations it is not possible to abstract from the conformation
conditions of the achiral aromatic and heterocyclic parts. In the
case of the bands at the area of 1500 cm.sup.-1 mentioned above
having high diagnostic value, there is luckily almost no change at
least concerning the conformers with higher population. The
analysis of all calculated spectra of the conformers (32 pieces)
resulted in that the sign of these bands with diagnostic value
turns to the opposite only in case of conformers where the N--H
bond and the bond of chiral center have the same direction. These
conformers are energetically negligible, very unfavourable forms
with a population far below 0.1%. It is obvious that for the
determination of absolute configuration both the experimental
spectrum and the calculated spectrum weighted with population are
needed (FIG. 4), but according to FIG. 3 it is also shown that the
signs of most of the bands (included also the bands at the area of
1500 cm.sup.-1) are opposite from the signs of experimental VCD
spectrum.
[0338] Considering that the VCD spectra of enantiomers are
mirrored, based on the wave number and sing-pattern of VCD bands of
the measured spectrum and the theoretical, calculated spectrum
weighted with the population (FIG. 4) it has been concluded that
the compound according to Example 2 is an R-enantiomer (Scheme 2).
The measured VCD spectrum is in good agreement with its theoretical
spectrum and it has basically opposite spectrum compared to the
molecule with S-configuration used in the course of molecular
modelling.
Example 2
Preparation of Enantiomerically-Pure
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-ol
[0339] In inert atmosphere 180 ml of acetonitrile, 16.22 g (50
mmol, 0.5 eq) of quinidine, 3.87 g (84 mmol, 0.84 eq) of formic
acid, 10.91 g (100 mmol, 1.0 eq) of 2-amino-4-methylpyrimidine,
17.41 g (100 mmol, 1.0 eq) of 4-trifluoromethylbenzaldehyde, and
finally 17.42 g (120 mmol, 1.2 eq) of 8-hydroxyquinoline were added
into a 500 ml round bottom flask. The mixture was stirred for 16
hours at acetonitrile reflux temperature.
[0340] The acetonitrile solution was concentrated in reduced
pressure to its third volume; the residue was dissolved in 100 ml
of dichloromethane. The solution was washed twice with 100 ml of 1M
NaOH solution and extracted further six times by 50 ml of 1M NaOH
solution. 100 ml of toluene was added to the organic phase then the
dichloromethane was evaporated off. The solution obtained was added
to 100 ml of 3 M HCl. The phases were separated and the toluene
phase was extracted with 30 ml of 3 M HCl solution. To the combined
HCl phases methyl-t-butyl-ether was added then the pH of the
biphasic system was adjusted with 40% NaOH solution to 4.
[0341] The precipitated quinidine was filtered off, the biphasic
filtrate was separated, then the water layer was washed twice by 20
ml of methyl-t-butyl-ether, the combined ether phase was dried on
sodium sulphate and filtered. 30 ml acetonitrile was added to the
filtrate, the methyl-t-butyl-ether was evaporated off and the
acetonitrile residue was stirred at room temperature for 16 hours.
The precipitated crystals were filtered to get 7.61 g (18.5 mmol,
HPLC: 95%) of racemic crystalline product.
[0342] The mother liquor was concentrated in reduced pressure to
give 14.3 g of crude enantiomerically-pure R-isomer (HPLC:
80%).
[0343] The crude R-enantiomer was then dissolved in 40 ml of
isopropanol, stirred at room temperature for 48 hours, then the
precipitated crystals were filtered off, to give 5.67 g (13.8 mmol,
HPLC: 98.9%, ee: 99%) of pure crystalline R-enantiomer.
[0344] .sup.1H-NMR (ppm): 10.1, 1H, s; 8.85, 1H, dd; 8.30, 1H, dd;
8.15, 1H, d; 8.07, 1H, d; 7.75, 1H, d; 7.65, 2H, d; 7.60, 2H, d;
7.53, 1H, dd; 7.41, 1H, d; 7.09, 1H, d; 6.51, 1H, d; 2.25, 3H,
s
Example 3
Preparation of Enantiomerically-Pure
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-ol
[0345] In inert atmosphere 300 ml of acetonitrile, 18 g (55 mmol,
0.5 eq) of quinine, 3.05 g (66 mmol, 0.80 eq) of formic acid, 22.00
g (200 mmol, 2.5 eq) of 2-amino-4-methylpyrimidine, 51.00 g (290
mmol, 3.7 eq) of 4-trifluoromethylbenzaldehyde, and finally 11.5 g
(79 mmol, 1.0 eq) of 8-hydroxyquinoline were added in a 1 l, four
necked round bottom flask.
[0346] The mixture was stirred at 73.degree. C. for six days.
[0347] The solvent was evaporated off in reduced pressure. The
residue (93.4 g) was dissolved in 200 ml of dichloromethane and
chromatographed on 100 g silica gel.
[0348] The fractions containing the product were collected and the
solvent was evaporated off (60.4 g). The raw product obtained was
purified by normal phase Flash chromatography using
hexane-ethyl-acetate gradient, then the fractions containing the
product were collected and concentrated (19.81 g).
[0349] The residue was dissolved in 190 ml of 2-propanol. After 2
hours stirring the precipitated racemic crystals were filtered off.
The mother liquor was concentrated in vacuum obtaining 13.44 g of
pure product (HPLC: 96.2%, ee: +99%).
[0350] .sup.1H-NMR (ppm): 10.1, 1H, s; 8.85, 1H, dd; 8.30, 1H, dd;
8.15, 1H, d; 8.07, 1H, d; 7.75, 1H, d; 7.65, 2H, d; 7.60, 2H, d;
7.53, 1H, dd; 7.41, 1H, d; 7.09, 1H, d; 6.51, 1H, d; 2.25, 3H,
s.
Example 4
Preparation of Enantiomerically-Pure
Potassium-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)pheny-
l]methyl]quinolin-8-olate
[0351] 10 g (24.4 mmol) of neutral compound according to Example 2
was dissolved in 60 ml of ethanol, then a solution of 2.73 g (24.4
mmol) of potassium tert-butoxide in ethanol (60 ml) was added
drop-wise. After 1 hour stirring 360 ml of methylcyclohexane was
added and the alcohol was evaporated off in reduced pressure. After
16 hours stirring the precipitated crystals were filtered to get
6.09 g of pure product (13.6 mmol, HPLC: 98.1%, ee: +99%).
[0352] .sup.1H-NMR (ppm): 9.97, 1H, s; 8.43, 1H, dd; 8.07, 1H, d;
7.87, 1H, dd; 7.76, 2H, d; 7.51, 2H, d; 7.24, 1H, d; 7.14, 1H, dd;
6.39, 1H, d; 6.38, 1H, d; 6.15, 1H, d; 3.38, 1H, s; 2.21, 3H,
s.
[0353] .sup.13C-NMR (ppm): 161.5, 160.3, 150.0, 145.3, 139.8,
138.0, 129.6, 128.5, 127.3, 124.6, 122.7, 121.0, 109.8, 107.6,
67.0, 56.6, 25.1, 23.7
Example 5
Preparation of Enantiomerically-Pure
Potassium-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)pheny-
l]methyl]quinolin-8-olate
[0354] 6.12 g (13.9 mmol) of neutral compound according to Example
3 was dissolved in 17 ml of ethanol, then a solution of 1.56 g
(13.9 mmol) of potassium tert-butoxide in ethanol (9 ml) was added
drop-wise. After 1 hour stirring 55 ml of methylcyclohexane was
added, then the alcohol was evaporated off in reduced pressure and
after 16 hours stirring the precipitated crystals were filtered to
get 5.08 g of pure product (13.6 mmol, HPLC: 98.1%, ee: +99%).
[0355] .sup.1H-NMR (ppm): 9.97, 1H, s; 8.43, 1H, dd; 8.07, 1H, d;
7.87, 1H, dd; 7.76, 2H, d; 7.51, 2H, d; 7.24, 1H, d; 7.14, 1H, dd;
6.39, 1H, d; 6.38, 1H, d; 6.15, 1H, d; 3.38, 1H, s; 2.21, 3H,
s.
[0356] .sup.13C-NMR (ppm): 161.5, 160.3, 150.0, 145.3, 139.8,
138.0, 129.6, 128.5, 127.3, 124.6, 122.7, 121.0, 109.8, 107.6,
67.0, 56.6, 25.1, 23.7
Example 6
Preparation of Enantiomerically-Pure Sodium
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-olate
[0357] 0.50 g (1.22 mmol) of neutral compound according to Example
2 was dissolved in 5 ml of ethanol, then this solution was added
drop-wise to 1 ml (1.22 mmol) of ethanol solution of sodium
ethoxide (28 mg Na+1 ml ethanol). After 1 hour stirring 360 ml of
methylcyclohexane was added, then the alcohol was evaporated off in
reduced pressure and after 16 hours stirring the precipitated
crystals were filtered, and 408 mg (0.94 mmol) pure product was
obtained.
Example 7
Preparation of Enantiomer-Pure
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-ol fumarate
[0358] 22.6 g (55.0 mmol) of neutral compound according to Example
2 was dissolved in 30 ml of ethanol, then a solution of 3.2 g (27.5
mmol) of fumaric acid in ethanol (90 ml) was added drop-wise. After
16 hours stirring the precipitated crystals were filtered to get
16.8 g of pure product (35.9 mmol, HPLC: 98.1%, ee: 99.4%).
[0359] .sup.1H-NMR (ppm): 8.86, 1H, d; 8.30, 1H, dd; 8.15, 1H, d;
8.06, 1H, d; 7.74, 1H, d; 7.65, 2H, d; 7.59, 2H, d; 7.55, 1H, dd;
7.41, 1H, d; 7.07, 1H, d; 6.63, 1H, s; 6.51, 1H, d; 2.25, 3H,
s.
[0360] .sup.13C-NMR (ppm): 167.4, 165.9, 161.5, 149.5, 148.3,
148.1, 138.0, 136.0, 133.9, 127.6, 127.3, 127.1, 126.7, 125.1,
124.5, 121.8, 117.5, 110.3, 51.6
Example 8
Preparation of Enantiomerically-Pure
7-[(S)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uiolin-8-ol fumarate
[0361] 9.21 g (22.4 mmol) of neutral compound according to Example
3 was dissolved in 15 ml of ethanol, then a solution of 1.30 g
(11.2 mmol) of fumaric acid in ethanol (40 ml) was added drop-wise.
After 16 hours stirring the precipitated crystals were filtered to
get 8.34 g of pure product (17.8 mmol, HPLC: 98.5%, ee: 99%).
[0362] .sup.1H-NMR (ppm): 8.86, 1H, d; 8.30, 1H, dd; 8.15, 1H, d;
8.07, 1H, d; 7.74, 1H, d; 7.65, 2H, d; 7.59, 2H, d; 7.55, 1H, dd;
7.41, 1H, d; 7.07, 1H, d; 6.63, 1H, s; 6.51, 1H, d; 2.25, 3H,
s.
[0363] .sup.13C-NMR (ppm): 167.4, 165.9, 161.5, 149.5, 148.3,
148.1, 138.0, 136.0, 133.9, 127.6, 127.3, 127.1, 126.7, 125.1,
124.5, 121.8, 117.5, 110.3, 51.6
Example 9
Preparation of Enantiomerically-Pure
7-[(R)-[(4-Methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-ol zinc complex
[0364] 1.098 g (5.0 mmol) of zinc acetate dihydrate was dissolved
in 150 ml of tetrahydrofuran, then a solution of 4.104 g (10 mmol)
of neutral compound according to Example 2 in tetrahydrofuran (150
ml) was added drop-wise. To the mixture obtained 300 ml of hexane
was added drop-wise.
[0365] After 16 hours stirring the precipitated crystals were
filtered to get 2.73 g (3.09 mmol) of pure product.
[0366] .sup.1H-NMR (ppm): 8.80, 1H, d; 8.75, 1H, s; 8.32, 1H, d;
8.16, 1H, d; 7.75, 2H, d; 7.58, 2H, d; 7.50, 2H, d; 6.85, 1H, d;
6.58, 1H, d; 6.48, 1H, d; 3.60, 1H, t; 2.28, 3H, s; 1.75, 1H,
s.
[0367] .sup.13C-NMR (ppm): 161.5, 160.3, 150.0, 145.3, 139.8,
138.0, 129.6, 128.5, 127.3, 124.6, 122.7, 121.0, 109.8, 107.6,
67.0, 56.6, 25.1, 23.7.
Example 10
Preparation of Enantiomerically-Pure
7-[(R)-[(6-Methylpyridin-2-yl)amino]4-nitrophenyl)methyl]-quinolin-8-ol
[0368] In inert atmosphere into 18 ml of acetonitrile 1.622 g (5.0
mmol, 0.5 eq) of quinidine, 0.387 g (8.4 mmol, 0.84 eq) of formic
acid, 1.091 g (10 mmol, 1.0 eq) of 2-amino-6-methyl-pyridine, 4.379
g (29 mmol, 3.7 eq) of 4-nitrobenzaldehyde, and finally 1.742 g (12
mmol, 1.2 eq) of 8-hydroxyquinoline were added.
[0369] The mixture was stirred at room temperature for four days.
The reaction mixture was processing up as given above to get the
product as pure R-enantiomer (HPLC: 98.9%, ee: 99%).
[0370] C.sub.22H.sub.18N.sub.4O.sub.3 (MS: 386.1); HPLC (Chiralpak
ADn-Hexane/IPA/TEA=90/10/0.1): Tr=10.30 min. .sup.1H NMR
(DMSO-d.sub.6) .delta. 2.2 (3H, s, CH.sub.3), 6.37 (1H, d, J=7.0
Hz), 6.49 (1H, d, J=7.9 Hz), 6.98 (1H, d, J=8.8 Hz, NHCH), 7.28
(1H, t, J=7.9 Hz), 7.40 (2H, t, J=7.9 es 8.8 Hz), 7.50-7.55 (1H,
m), 7.59-7.66 (3H, m), 8.16 (2H, d, J=8.8 Hz), 8.28 (1H, d, J=7.9
Hz), 10.1 (1H, wide s, OH); .sup.13C NMR (DMSO-d.sub.6) .delta.
24.2 (CH.sub.3), 51.5 (CH), 105.6 (CH), 111.6 (CH), 117.7 (CH),
121.9 (CH), 123.5 (2.times.CH), 124.5 (Cq), 126.7 (CH), 127.7 (Cq),
128.2 (2.times.CH), 136.1 (CH), 137.3 (CH), 138.2 (Cq), 146.2 (Cq),
148.4 (CH), 149.8 (Cq), 152.0, 155.7 and 157.3 (Cq)
Example 11
Preparation of Enantiomerically-Pure
7-[(S)-[(6-Methylpyridin-2-yl)amino]4-nitrophenyl)methyl]-quinolin-8-ol
[0371] In inert atmosphere into 30 ml of acetonitrile 1.8 g (5.5
mmol, 0.7 eq) of quinine, 0,305 g (6.6 mmol, 0.8 eq) of formic
acid, 2.20 g (20 mmol, 2.0 eq) of 2-amino-6-methylpyridine, 4.38 g
(29 mmol, 3.7 eq) of 4-nitrobenzaldehyde, and finally 1.15 g (7.9
mmol, 1.0 eq) of 8-hydroxyquinoline were added.
[0372] The mixture was stirred at room temperature for six days.
The reaction mixture was processing up as given above to get the
product as pure S-enantiomer (HPLC: 98.9%, ee: 99%).
[0373] C.sub.22H.sub.19N.sub.4O.sub.3(MS: 386.1); HPLC (Chiralpak
ADn-Hexane/IPA/TEA=90/10/0.1): Tr=10.30 min. .sup.1H NMR
(DMSO-d.sub.6) .delta. 2.2 (3H, s, CH.sub.3), 6.37 (1H, d, J=7.0
Hz), 6.49 (1H, d, J=7.9 Hz), 6.98 (1H, d, J=8.8 Hz, NHCH), 7.28
(1H, t, J=7.9 Hz), 7.40 (2H, t, J=7.9 es 8.8 Hz), 7.50-7.55 (1H,
m), 7.59-7.66 (3H, m), 8.16 (2H, d, J=8.8 Hz), 8.28 (1H, d, J=7.9
Hz), 10.1 (1H, wide s, OH); .sup.13C NMR (DMSO-d.sub.6) .delta.
24.2 (CH.sub.3), 51.5 (CH), 105.6 (CH), 111.6 (CH), 117.7 (CH),
121.9 (CH), 123.5 (2.times.CH), 124.5 (Cq), 126.7 (CH), 127.7 (Cq),
128.2 (2.times.CH), 136.1 (CH), 137.3 (CH), 138.2 (Cq), 146.2 (Cq),
148.4 (CH), 149.8 (Cq), 152.0, 155.7 and 157.3 (Cq)
Example 12
Preparation of Enantiomerically-Pure
7-[(R)-[(6-Methylpyridin-2-yl)amino]3-hydroxyphenyl)-methyl]quinolin-8-ol
[0374] In inert atmosphere into 18 ml of acetonitrile 1.622 g (50
mmol, 0.5 eq) of quinidine, 0.387 g (8.4 mmol, 0.84 eq) of formic
acid, 1.091 g (10 mmol, 1.0 eq) of 2-amino-6-methyl-pyridine, 3.538
g (29 mmol, 3.7 eq) of 3-hydroxybenzaldehyde, and finally 1.742 g
(12 mmol, 1.2 eq) of 8-hydroxyquinoline were added.
[0375] The mixture was stirred at reflux temperature of the solvent
for 16 hours. The reaction mixture was processing up as given above
to get the product as pure R-enantiomer (HPLC: 98.9%, ee: 99%).
[0376] C.sub.22H.sub.18N.sub.4O.sub.3 (MS: 357); HPLC (Chiralpak AD
n-Hexane/IPA/TEA=90/10/0.1): Tr=8.90 min.
Example 13
Preparation of Enantiomerically-Pure
7-[(S)-[(6-Methylpyridin-2-yl)amino]3-hydroxyphenyl)-methyl]quinolin-8-ol
[0377] In inert atmosphere into 30 ml of acetonitrile 1.8 g (5.5
mmol, 0.7 eq) of quinine, 0.305 g (6.6 mmol, 0.8 eq) of formic
acid, 2.2 g (20 mmol, 2.0 eq) of 2-amino-6-methyl-pyridine, 3.538 g
(29 mmol, 3.7 eq) of 3-hydroxybenzaldehyde, and finally 1.15 g (7.9
mmol, 1.0 eq) of 8-hydroxyquinoline were added.
[0378] The mixture was stirred at reflux temperature of the solvent
for six days. The reaction mixture was processing up as given above
to get the product as pure S-enantiomer (HPLC: 98.9%, ee: 99%).
[0379] C.sub.22H.sub.18N.sub.4O.sub.3 (MS: 357); HPLC (Chiralpak AD
n-Hexane/IPA/TEA=90/10/0.1): Tr=17.18 min.
Example 14
Preparation of Enantiomerically-Pure
7-[(R)-[(6-Methylpyridin-2-yl)amino]3-methoxyphenyl)-methyl]quinolin-8-ol
[0380] In inert atmosphere into 18 ml of acetonitrile 1.622 g (50
mmol, 0.5eq) of quinidine, 0.387 g (8.4 mmol, 0.84 eq) of formic
acid, 1.091 g (10 mmol, 1.0 eq) of 2-amino-6-methyl-pyridine, 1.52
g (10 mmol, 1.0 eq) of 4-hydroxy-3-methoxy-benzaldehyde, and
finally 1.742 g (12 mmol, 1.2 eq) of 8-hydroxyquinoline were
added.
[0381] The mixture was stirred at reflux temperature of the solvent
for 16 hours. The reaction mixture was processing up as given above
to get the product as pure R-enantiomer (HPLC: 95%, ee: 99%).
[0382] C.sub.22H.sub.18N.sub.4O.sub.3 (MS=387); HPLC (Chiralpak AD
n-Hexane/IPA/TEA=90/10/0.1): Tr=8.61 min.
Example 15
Preparation of Enantiomerically-Pure
7-[(S)-[(6-Methylpyridin-2-yl)amino]3-methoxyphenyl)-methyl]quinolin-8-ol
[0383] In inert atmosphere into 30 ml of acetonitrile 1.8 g (5.5
mmol, 0.7 eq) of quinine, 0.305 g (6.6 mmol, 0.8 eq) of formic
acid, 2.2 g (20 mmol, 2.0 eq) of 2-amino-6-methyl-pyridine, 4.408 g
(29 mmol, 3.7 eq) of 4-hydroxy-3-methoxy-benzaldehyde, and finally
1.15 g (7.9 mmol, 1.0 eq) of 8-hydroxyquinoline were added.
[0384] The mixture was stirred at reflux temperature of the solvent
for six days. The reaction mixture was processing up as given above
to get the product as pure S-enantiomer (HPLC: 95%, ee: 99%).
[0385] C.sub.22H.sub.18N.sub.4O.sub.3(MS=387); HPLC (Chiralpak AD
n-Hexane/IPA/TEA=90/10/0.1): Tr=11.14 min.
Example 16
Preparation of Enantiomerically-Pure
7-[(R)-[(6-methylpyridin-2-yl)amino](5-bromopyridin-2-yl)methyl]quinolin--
8-ol
[0386] Using 8-hydroxyquinoline, 5-bromopyridine-2-carbaldehyde,
2-amino-6-methylpyridine and quinidine, pure R-enantiomer was
obtained according to the general R-process (HPLC: +99%, ee:
+99%).
[0387] C.sub.21H.sub.17BrN.sub.4O (MS=421); HPLC (Lux 5u
Cellulose-4, 100*4.6 n-Hexane/IPA/TEA=90/10/0.1): Tr=5.20 min.
Example 17
Preparation of Enantiomerically-Pure
7-[(S)-[(6-Methylpyridin-2-yl)amino](5-bromopyridin-2-yl)methyl]quinolin--
8-ol
[0388] Using 8-hydroxyquinoline, 5-bromopyridine-2-carbaldehyde,
2-amino-6-methlypyridine and quinine, pure R-enantiomer was
obtained according to the general S-process (HPLC: +99%, ee:
+99%).
[0389] C.sub.22H.sub.17BrN.sub.4O (MS=421); HPLC (Lux 5u
Cellulose-4, 100*4.6 n-Hexane/IPA/TEA=90/10/0.1): Tr=6.56 min.
Example 18
Preparation of Enantiomerically-Pure
7-[(R)-[(6-Methylpyridin-2-yl)amino]2-hydroxyphenyl)-methyl]quinolin-8-ol
[0390] Using 8-hydroxyquinoline, 2-hydroxybenzaldehyde,
2-amino-6-methylpyridine and quinidine, pure R-enantiomer was
obtained according to the general R-process (HPLC: +99%, ee:
+99%).
[0391] C.sub.22H.sub.19N.sub.3O.sub.2 (MS=357); HPLC (Lux 5u
Cellulose-4, 100*4.6 n-Hexane/IPA/TEA=90/10/0.1): Tr=2.39 min.
Example 19
Preparation of Enantiomerically-Pure
7-[(S)-[(6-Methylpyridin-2-yl)amino]2-hydroxyphenyl)-methyl]quinolin-8-ol
[0392] Using 8-hydroxyquinoline, 2-hydroxybenzaldehyde,
2-amino-6-methlypyridine and quinine, pure S-enantiomer was
obtained according to the general S-process (HPLC: +99%, ee:
+99%).
[0393] C.sub.22H.sub.19N.sub.3O.sub.2 (MS=357); HPLC (Lux 5u
Cellulose-4, 100*4.6 n-Hexane/IPA/TEA=90/10/0.1): Tr=7.04 min.
Example 20
Preparation of Enantiomerically Pure
5-Chloro-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinolin-8-ol
[0394] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinidine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-methylpirimidine then 580 mg
(3.33 mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally
716 mg (4 mmol, 1.2 eq) 5-chloro-8-hydroxyquinoline were added into
a round bottom flask.
[0395] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0396] The reaction mixture was processing up as usual to get the
pure product.
[0397] C.sub.22H.sub.16ClF.sub.3N.sub.4O; mass (ESI positive mode):
445 (444+H.sup.+). HPLC (Lux4; Hexane:Isopropanol 95:5) Tr=22.8
minute.
[0398] 1H NMR (500 MHz, D6MSO) .delta. 10.43 (wide s, 1H),
8.95-8.91 (m, 1H), 8.47-8.42 (m, 1H), 8.20 (d, J=9.6 Hz, 1H), 8.15
(d, J=4.9 Hz, 1H), 7.71-7.66 (m, 1H), 7.65 (d, J=8.2 Hz, 2H), 7.58
(d, J=8.1 Hz, 2H), 7.09 (1H, d, J=9.5 Hz), 6.51 (1H, d, J=4.95 Hz),
2.24 (3H, s); 13C-NMR (125 MHz, D6MSO): 25.5, 110.5, 118.8, 123.0,
125.0, 125.3, 125.4, 126.6, 127.8, 132.5, 138.7, 147.5, 149.2,
149.4, 161.5.
Example 21
Preparation of Enantiomerically Pure
5-Chloro-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinolin-8-ol
[0399] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-methylpirimidine then 580 mg
(3.33 mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally
716 mg (4 mmol, 1.2 eq) 5-chloro-8-hydroxyquinoline were added into
a round bottom flask.
[0400] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0401] The reaction mixture was processing up as usual to get the
pure product.
[0402] C.sub.22H.sub.16ClF.sub.3N.sub.4O; mass (ESI positive mode):
445 (444+H.sup.+). HPLC (Lux4; Hexane:Isopropanol 95:5) Tr=22.8
minute.
[0403] 1H NMR (500 MHz, D6MSO) .delta. 10.43 (wide s, 1H),
8.95-8.91 (m, 1H), 8.47-8.42 (m, 1H), 8.20 (d, J=9.6 Hz, 1H), 8.15
(d, J=4.9 Hz, 1H), 7.71-7.66 (m, 1H), 7.65 (d, J=8.2 Hz, 2H), 7.58
(d, J=8.1 Hz, 2H), 7.09 (1H, d, J=9.5 Hz), 6.51 (1H, d, J=4.95 Hz),
2.24 (3H, s); 13C-NMR (125 MHz, D6MSO): 25.5, 110.5, 118.8, 123.0,
125.0, 125.3, 125.4, 126.6, 127.8, 132.5, 138.7, 147.5, 149.2,
149.4, 161.5.
Example 22
Preparation of Enantiomerically Pure
5-Chloro-7-[(R)-[(6-methylpyridin-2-yl)amino][4-(trifluoromethyl)phenyl]m-
ethyl]quinolin-8-ol
[0404] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinidine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-6-picoline then 580 mg (3.33
mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally 716
mg (4 mmol, 1.2 eq) 5-chloro-8-hydroxyquinoline were added into a
round bottom flask.
[0405] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0406] The reaction mixture was processing up as usual to get the
pure product.
[0407] C.sub.23H.sub.17ClF.sub.3N.sub.3O; mass (ESI positive mode):
444 (443+H.sup.+).
Example 23
Preparation of Enantiomerically Pure
2-Methyl-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinolin-8-ol
[0408] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinidine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-methylpirimidine then 580 mg
(3.33 mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally
636 mg (4 mmol, 1.2 eq) 8-hydroxyquinaldine were added into a round
bottom flask.
[0409] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0410] The reaction mixture was processing up as usual to get the
pure product.
[0411] C.sub.23H.sub.19F.sub.3N.sub.4O, mass (ESI positive mode):
425 (424+H.sup.+)
Example 24
Preparation of Enantiomerically Pure
2-Methyl-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl-
]methyl]quinolin-8-ol
[0412] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-methylpirimidine then 580 mg
(3.33 mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally
636 mg (4 mmol, 1.2 eq) 8-hydroxyquinaldine were added into a round
bottom flask.
[0413] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0414] The reaction mixture was processing up as usual to get the
pure product.
[0415] C.sub.23H.sub.19F.sub.3N.sub.4O, mass (ESI positive mode):
425 (424+H.sup.+)
Example 25
Preparation of Enantiomerically Pure
2-[(Dimethylamino)methyl]-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifl-
uoromethyl)phenyl]methyl]quinolin-8-ol
[0416] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinidine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-methylpirimidine then 580 mg
(3.33 mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally
808 mg (4 mmol, 1.2 eq) 2-((dimethylamino)methyl)quinolin-8-ol were
added into a round bottom flask.
[0417] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0418] The reaction mixture was processing up as usual to get the
pure product.
[0419] C.sub.2H.sub.24F.sub.3N.sub.5O; mass (ESI positive mode):
468 (467+H.sup.+)
Example 26
Preparation of Enantiomerically Pure
2-[(Dimethylamino)methyl]-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifl-
uoromethyl)phenyl]methyl]quinolin-8-ol
[0420] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-methylpirimidine then 580 mg
(3.33 mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally
808 mg (4 mmol, 1.2 eq) 2-[(dimethylamino)methyl]quinolin-8-ol were
added into a round bottom flask.
[0421] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0422] The reaction mixture was processing up as usual to get the
pure product.
[0423] C.sub.25H.sub.24F.sub.3N.sub.5O; mass (ESI positive mode):
468 (467+H.sup.+)
Example 27
Preparation of Enantiomerically Pure
2-[(Dimethylamino)methyl]-7-[(R)-[(4-methylpyridin-2-yl)amino][4-(trifluo-
romethyl)phenyl]methyl]quinolin-8-ol
[0424] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinidine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-picoline then 580 mg (3.33
mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally 808
mg (4 mmol, 1.2 eq) 2-[(dimethylamino)methyl]quinolin-8-ol were
added into a round bottom flask.
[0425] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0426] The reaction mixture was processing up as usual to get the
pure product.
[0427] C.sub.26H.sub.25F.sub.3N.sub.4O; tomeg (ESI positive mode):
467 (466+H.sup.+)
Example 28
Preparation of Enantiomerically Pure
2-[(Dimethylamino)methyl]-7-[(S)-[(4-methylpyridin-2-yl)amino][4-(trifluo-
romethyl)phenyl]methyl]quinolin-8-ol
[0428] In inert atmosphere 3 ml of acetonitrile, 540 mg (1.67 mmol,
0.5eq) of quinine, 129 mg (2.8 mmol, 0.84 eq) of formic acid, 364
mg (3.33 mmol, 1.0 eq) of 2-amino-4-picoline then 580 mg (3.33
mmol, 1.0 eq) of 4-(trifluoromethyl)benzaldehyde, and finally 808
mg (4 mmol, 1.2 eq) 2-[(dimethylamino)methyl]quinolin-8-ol were
added into a round bottom flask.
[0429] The mixture was stirred for 6 days at 75.degree. C.
temperature.
[0430] The reaction mixture was processing up as usual to get the
pure product.
[0431] C.sub.26H.sub.25F.sub.3N.sub.4O; mass (ESI positive mode):
467 (466+H.sup.+)
Example 29
Preparation of Enantiomerically Pure
5-Nitro-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-
methyl]quinolin-8-ol
[0432] Using 5-nitro-8-hydroxyquinoline,
4-(trifluoromethyl)benzaldehyde, 2-amino-4-methlypyrimidine and
quinidine, pure R-enantiomer was obtained according to the general
R-process (HPLC: +99%, ee: +99%).
[0433] C.sub.22H.sub.16F.sub.3N.sub.5O.sub.3; mass (ESI positive
mode): 456 (455+H.sup.+)
Example 30
Preparation of Enantiomerically Pure
5-Nitro-7-[(S)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]-
methyl]quinolin-8-ol
[0434] Using 5-nitro-8-hydroxyquinoline,
4-(trifluoromethyl)benzaldehyde, 2-amino-4-methlypyrimidine and
quinine, pure S-enantiomer was obtained according to the general
S-process (HPLC: +99%, ee: +99%).
[0435] C.sub.22H.sub.16F.sub.3N.sub.5O.sub.3; mass (ESI positive
mode): 456 (455+H.sup.+)
Example 31
Preparation of Enantiomerically Pure 7-[(R)-[(Pyridin-2-yl)
[4-(trifluoromethyl)phenylamino]methyl]quinolin-8-ol
[0436] Using 8-hydroxyquinoline, 4-(trifluoromethyl)aniline,
2-pyridine-carbaldehyde and quinidine, pure R-enantiomer was
obtained according to the general R-process.
[0437] C.sub.2H.sub.16F.sub.3N.sub.3O.sub.3; mass (ESI positive
mode): 396 (395+H.sup.+)
Example 32
Preparation of Enantiomerically Pure 7-[(S)-[(Pyridin-2-yl)
[4-(trifluoromethyl)phenylamino]methyl]quinolin-8-ol
[0438] Using 8-hydroxyquinoline, 4-(trifluoromethyl)aniline,
2-pyridine-carbaldehyde and quinine, pure S-enantiomer was obtained
according to the general S-process.
[0439] C.sub.22H.sub.16F.sub.3N.sub.30; mass (ESI positive mode):
396 (395+H.sup.+)
Example 33
Preparation of Enantiomerically Pure
2-(Hydroxymethyl)-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluorometh-
yl)phenyl]methyl]quinolin-8-ol
[0440] Using 2-hydroxymethyl-8-hydroxyquinoline,
4-(trifluoromethyl)benzaldehyde, 2-amino-4-methylpyrimidine and
quinidine, pure R-enantiomer was obtained according to the general
R-process.
[0441] C.sub.23H.sub.19F.sub.3N.sub.4O.sub.2; mass (ESI positive
mode) 441 (440+H.sup.+)
Example 34
Preparation of Enantiomerically Pure
2-(Hydroxymethyl)-7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluorometh-
yl)phenyl]methyl]quinolin-8-ol
[0442] Using 2-hydroxymethyl-8-hydroxyquinoline,
4-(trifluoromethyl)benzaldehyde, 2-amino-4-methylpyrimidine and
quinine, pure S-enantiomer was obtained according to the general
S-process.
[0443] C.sub.23H.sub.19F.sub.3N.sub.4O.sub.2; mass (ESI positive
mode) 441 (440+H.sup.+)
VII. Biological Examples: In Vitro Procedures
Example 35
Inhibition of Matrix Metalloproteinase 2 (MMP-2, 72 kDa
Gelatinase), Matrix Metalloproteinase 8 (MMP-8), Matrix
Metalloproteinase 10 (MMP-10), Matrix Metalloproteinase 12
(MMP-12), Matrix Metalloproteinase 13 (MMP-13), and Matrix
Metalloproteinase 14 (MMP-14) Activity with Compounds
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phen-
yl]methyl]quinoline-8-ol and
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Examples 2 and 3
[0444] Different matrix metalloproteinase activities were
investigated in a set of fluorophotometric, biochemical
measurements. Recombinant matrix metalloproteinase enzymes were
pre-incubated at 37.degree. C. with different concentrations (25
.mu.M-195 nM) of compounds
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol and
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol according to Examples 2 and 3.
[0445] Cleavage of the applied substrate resulted in a
fluorescently active molecule which was measured one hour after
reaction start with a Wallac Victor microtiter plate reader by
using 355 nm extinction and 460 nm emission filters. Enzyme
activities were compared to controls which were not treated with
the inhibitor, and are given as percentages. FIG. 5 shows the
activity of the six investigated MMP enzymes being dependent on
concentrations of compounds according to Examples 2 and 3.
According to the IC.sub.50 values (half maximal inhibitory
concentrations) shown in the figure, the R- and S-enantiomers
inhibited activities of the investigated enzymes to a similar
extent.
Example 36
Treatment with the Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2 for Inhibition of
H.sub.2O.sub.2-Induced Cell Death of Cardiac Muscle Cells In
Vitro
[0446] Cell line H9c2 (ATCC, Rockville, 10MD, USA), derived from
embryonic rat heart, was cultured in Dulbecco's Modified Eagle
Medium containing 10% bovine serum, 4 mM L-glutamine
(Sigma-Aldrich, Hungary), 100 U/ml penicillin, and 100 .mu.g/ml
streptomycin. Cells were cultured in 100 mm TC-treated culture
dishes (Orange Scientific, Belgium) in an incubator set at
37.degree. C. and 5% CO.sub.2 with humid air. For real-time
monitoring of cell viability, a Roche xCELLigence SP and DP
(ACEA-Roche, Hungary) was used, which gives us information about
cell viability on the basis of changes in cell conductivity. Prior
to plating, the special 96-well e-plate was covered with 0.2% type
I collagen, and then placed into the incubator for 30 min.
Cell-free baseline impedance was measured once in a minute for 10
min. After plating (6000 cells/well), measuring began. Treatment of
cells was always carried out the morning after plating. Measurement
proceeded for 72 h.
[0447] Results are shown in FIG. 6. Curve 1 refers to untreated
control, curve 2. to control treated with H.sub.2O.sub.2, curve 3,
4, 5, 6 refer to different concentrations (3: 0.11 .mu.M compound
according to Example 2+900 .mu.M of H.sub.2O.sub.2, 4: 0.33 .mu.M
compound according to Example 2+900 .mu.M of H.sub.2O.sub.2, 5: 1
.mu.M compound according to Example 2+900 .mu.M of H.sub.2O.sub.2,
6: 3 .mu.M compound according to Example 2+900 .mu.M of
H.sub.2O.sub.2) of the compound according to Example 2
(7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phe-
nyl]methyl]quinoline-8-ol). B: refers to percentage of viability at
6 and 24 h after treatment, compared to control cells.
Example 37
Treatment with the Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2 for Inhibition of
H.sub.2O.sub.2-Induced Cell Death of SH-SY5Y Human Neuroblastoma
Cells In Vitro
[0448] SH-SY5Y cells (ATCC, Rockville, 10MD, USA) were cultured in
100 mm TC-treated culture dishes (Orange Scientific, Belgium) in an
incubator set at 37.degree. C. and 5% CO.sub.2 with humid air. For
real-time monitoring of cell viability, a Roche xCELLigence SP and
DP (ACEA-Roche, Hungary) was used, which gives us information about
cell viability on the basis of changes in cell conductivity. For
plating, DMEM (Dulbecco's Modified Eagle Medium) supplemented with
10% FBS was used. Prior to plating, the special 96-well e-plate was
covered with 0.2% type I collagen, and then placed into the
incubator for 30 min. Cell-free baseline impedance was measured
once in a minute for 10 min. After plating (20.000 cells/well),
measuring began. Treatment of cells was always carried out the
morning after plating. Measurement proceeded for 72 h.
[0449] Results are shown in FIG. 7 Curve 1 refers to untreated
control, curve 2 to control treated with H.sub.2O.sub.2, curve 3,
4, 5, 6 refer to different concentrations (3: 0.11 .mu.M compound
according to Example 2+500 .mu.M of H.sub.2O.sub.2, 4: 0.33 .mu.M
compound according to Example 2+500 .mu.M of H.sub.2O.sub.2, 5: 1
.mu.M compound according to Example 2+500 .mu.M of H.sub.2O.sub.2,
6: 3 .mu.M compound according to Example 2+500 .mu.M of
H.sub.2O.sub.2) of the compound according to Example 2
(7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phe-
nyl]methyl]quinoline-8-ol). B: refers to percentage of viability at
6 and 24 h after treatment, compared to control cells.
Example 38
Treatment with the Compound
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 3 for Inhibition of
H.sub.2O.sub.2-Induced Cell Death of Cardiac Muscle Cells In
Vitro
[0450] Cell line H9c2 (ATCC, Rockville, 10MD, USA), derived from
embryonic rat heart, was cultured in Dulbecco's Modified Eagle
Medium containing 10% bovine serum, 4 mM L-glutamine
(Sigma-Aldrich, Hungary), 100 U/ml penicillin, and 100 .mu.g/ml
streptomycin. Cells were cultured in 100 mm TC-treated culture
dishes (Orange Scientific, Belgium) in an incubator set at
37.degree. C. and 5% CO.sub.2 with humid air. For real-time
monitoring of cell viability, a Roche xCELLigence SP and DP
(ACEA-Roche, Hungary) was used, which gives us information about
cell viability on the basis of changes in cell conductivity. Prior
to plating, the special 96-well e-plate was covered with 0.2% type
I collagen, and then placed into the incubator for 30 min.
Cell-free baseline impedance was measured once in a minute for 10
min. After plating (6000 cells/well), measuring began. Treatment of
cells was always carried out the morning after plating. Measurement
proceeded for 72 h.
[0451] Results are shown in FIG. 8. Curve 1. refers to untreated
control, curve 2. to control treated with H.sub.2O.sub.2, curve 3,
4, 5, 6 refer to different concentrations (3: 0.11 .mu.M compound
according to Example 3+900 .mu.M of H.sub.2O.sub.2, 4: 0.33 .mu.M
compound according to Example 3+900 .mu.M of H.sub.2O.sub.2, 5: 1
.mu.M compound according to Example 3+900 .mu.M of H.sub.2O.sub.2,
6: 3 .mu.M compound according to Example 3+900 .mu.M of
H.sub.2O.sub.2) of the compound according to Example 3.
(7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl-
]quinoline-8-ol). B: refers to percentage of viability at 6 and 24
h after treatment, compared to control cells.
Example 39
Treatment with the Compound
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 3 for Inhibition of
H.sub.2O.sub.2-Induced Cell Death of SH-SY5Y Human Neuroblastoma
Cells In Vitro
[0452] SH-SY5Y cells (ATCC, Rockville, 10MD, USA) were cultured in
100 mm TC-treated culture dishes (Orange Scientific, Belgium) in an
incubator set at 37.degree. C. and 5% CO.sub.2 with humid air. For
real-time monitoring of cell viability, a Roche xCELLigence SP and
DP (ACEA-Roche, Hungary) was used, which gives us information about
cell viability on the basis of changes in cell conductivity. For
plating, DMEM (Dulbecco's Modified Eagle Medium) supplemented with
10% FBS was used. Prior to plating, the special 96-well e-plate was
covered with 0.2% type I collagen, and then placed into the
incubator for 30 min. Cell-free baseline impedance was measured
once in a minute for 10 min. After plating (20.000 cells/well),
measuring began. Treatment of cells was always carried out the
morning after plating. Measurement proceeded for 72 h.
[0453] Results are shown in FIG. 9 Curve 1. refers to untreated
control, curve 2 to control treated with H.sub.2O.sub.2, curve 3,
4, 5, 6 refer to different concentrations (3: 0.11 .mu.M compound
according to Example 3+500 .mu.M of H.sub.2O.sub.2, 4: 0.33 .mu.M
compound according to Example 3+500 .mu.M of H.sub.2O.sub.2, 5: 1
.mu.M compound according to Example 3+500 .mu.M of H.sub.2O.sub.2,
6: 3 .mu.M compound according to Example 3+500 .mu.M of
H.sub.2O.sub.2) of the compound according to Example 3
(7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phe-
nyl]methyl]quinoline-8-ol). B: refers to percentage of viability at
6 and 24 h after treatment, compared to control cells.
Example 40
Treatment with the Compound
Potassium-7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phen-
yl]methyl]quinoline-8-olate According to Example 4 for Inhibition
of Cell Death Caused by Fibrillar Aggregates of Amyloid Peptides in
Primary Cortical Neurons In Vitro
[0454] Cortical neurons from embryonic day 16-17 Wistar rat fetuses
were maintained as previously described [1]. Peptide used: A 1-42
(Bachem, ref. H1368, batch #1025459). Oligomer preparation: the
preparation of A oligomers was performed according to SynAging
standard operating procedures. The oligomeric preparation contains
a mixture of stable dimers, trimers and tetramers of A 1-42
peptide, as well as monomeric forms of the peptide. The same
preparation of oligomers was used for all experimental set-ups and
has been previously characterized in terms of oligomer composition,
neurotoxicity in vitro as well as induction of cognitive
impairment.
[0455] Primary cortical neurons were incubated with increasing
concentrations of the compound according to Example 2 in the
presence or absence of 1 .quadrature.M A 1-42 oligomers. Following
24 h incubation, neuronal viability was monitored using the
calcein-AM assay as previously described [1, 2]. Data shown in FIG.
10 are obtained from 3-4 separate experiments (mean.+-.SEM).
Student's t-test (**, p<0.05, ***, p<0.001) as well as ANOVA
followed by a Scheffe's post hoc test were used to test statistical
significance.
Example 41
Treatment with the Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol zinc Complex According to Example 9 for Inhibition
of Cell Death Caused by Fibrillar Aggregates of Amyloid Peptides in
Primary Cortical Neurons In Vitro
[0456] Cortical neurons from embryonic day 16-17 Wistar rat fetuses
were maintained as previously described [1]. Peptide used: A 1-42
(Bachem, ref. H1368, batch #1025459). Oligomer preparation: the
preparation of A oligomers was performed according to SynAging
standard operating procedures. The oligomeric preparation contains
a mixture of stable dimers, trimers and tetramers of A 1-42
peptide, as well as monomeric forms of the peptide. The same
preparation of oligomers was used for all experimental set-ups and
has been previously characterized in terms of oligomer composition,
neurotoxicity in vitro as well as induction of cognitive
impairment.
[0457] Primary cortical neurons were incubated with increasing
concentrations of the compound according to Example 2 in the
presence or absence of 1 .quadrature.M A 1-42 oligomers. Following
24 h incubation, neuronal viability was monitored using the
calcein-AM assay as previously described [1, 2]. Data shown in FIG.
11. are obtained from 3-4 separate experiments (mean.+-.SEM).
Student's t-test (**, p<0.05, ***, p<0.001) as well as ANOVA
followed by a Scheffe's post hoc test were used to test statistical
significance.
Biological Examples, In Vivo Procedures
[0458] In vivo experiments were carried out according to the
National Institute of Health guidelines for the care and use of
laboratory animals, approved by the French Ministry for Research
and Technology. In the course of experiments, animals were housed
at a standard temperature (22.+-.1.degree. C.) and in a
light-controlled environment (lights on from 7 a.m. to 8 p.m.) with
ad libitum access to food and water. Male C57BL/6 mice (C57BL/6J
Rj, ref. SC-CJ-12w-M, Janvier, France) were housed 5 animals per
cage. From one week before the start of the experiment until the
end of the experiment, mice were housed individually. Animals were
monitored twice-a-day by laboratory personnel (8 a.m. and 4 p.m.).
In this project, young (14 weeks old) as well as aged (15-16 months
old) mice were used. Experiments were carried out using 12 mice per
experimental group.
[0459] Investigation of Short-Term Working Memory--Y Maze Test
[0460] Y maze tests were carried out as previously described [1,
3]. Prior to testing, mice were brought to the experimental room
for at least 30 min to acclimatize to experimental room conditions.
The Y maze is made of opaque Plexiglas and each arm is 40 cm long,
16 cm high, 9 cm wide and positioned at equal angles. The apparatus
was placed in the test room in such a way that it was brightened
homogeneously with 12-15 lux in the arms as well as in the central
zone. One mouse at a time was placed at the end of one arm and was
allowed to move in the maze freely during a 5 min examination. Each
arm entry was recorded with those entries being counted where the
mouse completely placed its hind paws in the chosen arm.
Alternation was defined as successive entry into the 3 arms on
overlapping triplet sets. Results were calculated as the percentage
of the ratio of actual detected alternations to possible
alternations (defined as the number of total arm entries minus
2).
Example 42
Effects of Treatment with Compounds
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol and
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2 and 3 on Short-Term Memory
Impairment Caused by Fibrillar Aggregates of Amyloid Peptides in In
Vivo C57BL6/J Mice
[0461] For the examination of memory impairment caused by A
oligomers, a mouse model developed and validated by SynAging was
used [1-3]. Mice receiving icy injection of A oligomers develop
memory deficits associated with a decrease of hippocampal synaptic
protein levels. Under anesthetization, 1 .mu.l of soluble AB
oligomers (50 pmol) or vehicle (saline) was injected into the right
ventricle. The stereotaxic coordinates from the bregma are as
follows (in mm): AP-0.22, L-1.0 and D-2.5. For treatments, a 10 ml
Hamilton microsyringe fitted with a 26-gauge needle was used.
Treatment with molecules described in Example 2. and 3. was carried
out once daily for four days with the use of a per os probe (100
.mu.l/animal). Four experimental groups were established: Group
A--vehicle/A oligomer, Group B--compound according to Example 2 (4
mg/kg body weight) and A oligomer, Group C--compound according to
Example 3 (4 mg/kg body weight) and A oligomer, Group D--compounds
from both Example 2. and 3. (4 mg/kg body weight) and A oligomer.
At day 4 post A treatment, short-term memory was assessed with the
Y maze test.
[0462] Treatment with compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol according to Example 2 inhibited while treatment
with compound
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)pheny-
l]methyl]quinoline-8-ol) according to Example 3 did not inhibit
short-term memory impairment caused by fibrillar aggregates of
amyloid peptides in in vivo C57BL6/J mice.
Example 43
Effects of Treatment with Different Concentrations (Related to Body
Weight) of Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2 on Short-Term Memory
Impairment Caused by Fibrillar Aggregates of Amyloid Peptides in In
Vivo C57BL6/J Mice
[0463] Treatment with A oligomers was carried out as previously
described in Example 16. Treatment with the examined compound
according to Example 2 was carried out once daily for four days
with the use of a per os probe (100 .mu.l/animal). Four
experimental groups were established: Group A--vehicle/A oligomer,
Group B--compound according to Example 2. (0.5 mg/kg body weight)
and A oligomer, Group C--compound according to Example 2 (2 mg/kg
body weight) and A oligomer, Group D--compound according to Example
2 (4 mg/kg body weight) and A oligomer.
[0464] At day 4 post A treatment, short-term memory was assessed
with the Y maze test. Results are summarized in FIG. 13.
[0465] Treatment with compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol according to Example 2 inhibited short-term memory
impairment caused by fibrillar aggregates of amyloid peptides in in
vivo C57BL6/J mice in a dose-dependent manner.
Example 44
Effect of Compounds
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol and
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2 and 3 on Scopolamine-Induced
Short-Term Memory Impairment in In Vivo C57BL6/J Mice
[0466] Scopolamine has been widely used to induce cognitive
deficits (typically short-term memory impairment) in animals [FR4].
Test protocol: 30 min prior to behavioral test, scopolamine (0.6
mg/kg) or vehicle was administered i.p. in control and treated mice
(12 mice per experimental groups). Mice were returned to their cage
until the start of the test. Administration of drug-candidates
described in Example 2. and 3. was carried out with a per os gavage
(administration time based on PK data). Y-maze tests were carried
out as previously described. Results are summarized in FIG. 14.
[0467] Treatment with compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol according to Example 2 inhibited, while treatment
with compound
7-[(S)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)pheny-
l]methyl]quinoline-8-ol according to Example 3 did not inhibit
scopolamine-induced short-term memory impairment in in vivo
C57BL6/J mice.
Example 45
Inhibition of the Activity of Caspase 3 Peptidase with Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2
[0468] Throughout the experiments, human caspase 3 enzyme produced
in E. coli was used as previously described (Mittl PRE, Marco S D,
Krebs J F, Karanewsky D S, Priestle J P, Tomaselli K J and Grutter
M G (1997) Structure of Recombinant Human CPP32 in Complex with the
Tetrapeptide Acetyl-Asp-Val-Ala-Asp Fluoromethyl Ketone. J Biol
Chem. 272: 6539-6547). In brief: the enzyme (150 U/ml) was
pre-incubated together with the compound according to Example 2 (10
.mu.M) in modified HEPES buffer (50 mM HEPES, pH=7.4, 100 mM NaCl,
0.1% CHAPS, 1 mM EDTA, 10% glycerin, 10 mM DTT) for 15 min at
37.degree. C. After the addition of 50 mM Ac-DEVD-AMC substrate,
the reaction ran for 60 min. The amount of generated AMC
(7-amino-4-methylcoumarine), which gives information about the
enzyme activity, was detected by fluorescence at 360/465 nm. At the
applied concentration of 10 mM, the compound according to Example 2
reduced the activity of caspase 3 enzyme by 74%.
Example 46
Transcriptional Inhibition of NF-AT (Nuclear Factor of Activated T
Cells) Protein with the Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2
[0469] Experiments were carried out on human T Jurkat cells
transfected with a reporter construct expressing 1-galactosidase
and containing the binding site of NFAT1 transcription factor based
on the previously described method (Emmel E A, Verweij C L, Durand
D B, Higgins K M, Lacy E and Crabtree G R (1989) Cyclosporin A
specifically inhibits function of the nuclear proteins involved in
T cell activation. Science. 246:1617-1620; Karttunen J and Shastri
N (1991) Measurement of ligand-induced activation in single viable
T cells using the lacZ reporter gene. Proc Natl Acad Sci USA.
88:3972). Cells (3.times.10.sup.6 cells/ml) were incubated with 10
.mu.M compound according to Example 2 in RPMI-1640 medium (pH=7.4)
for 20 min at 37.degree. C. Following this, incubation lasted for
an additional 4 h in the presence of 0.5 .mu.M A23187 and 50 ng/ml
PMA (12-O-tetradecanoylphorbol-13-acetate). Induced effects can be
determined by the -galactosidase activity of cells treated with the
compound according to Example 2 and untreated cells as it catalyzes
the transformation of FDG (fluorescein di- -D-galactopyranoside)
into fluorescein. Reading of results was carried out with a
SpectraFluor Plus plate reader. Decrease of -galactosidase activity
was compared to the effect of 1 .mu.M cyclosporine A which was
served as a positive control. The compound according to Example 2
Reduced the transcriptional activity of NF-AT protein by 2.2 .mu.M
half-value concentration. Results are summarized in FIG. 15.
Example 47
Inhibition of 5-Lipoxygenase Enzyme Activity with the Compound
7-[(R)-[(4-Methylpirimidine-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]-
quinoline-8-ol According to Example 2
[0470] Human 5-lipoxygenase produced by Sf9 cells was used
throughout the experiments. The enzyme was pre-incubated with 10
.mu.M compound according to Example 2 at room temperature,
then--after the addition of 25 .mu.M arachidonic acid
substrate--the amount of generated rhodamine 123 was determined by
fluorescence measurement. Percentage of inhibition rate was
calculated by comparison to untreated control reactions. In 10
.mu.M concentration the compound according to Example 2 inhibited
5-lipoxygenase activity by 54%.
Example 48
Gene Expression Influencing Effect of the R-Enantiomeric Derivative
According to Example 2 on Primary Astrocytes
[0471] Pure astrocyte culture was obtained from neonatal rats
according to the protocol found in literature (Protocols for Neural
Cell Culture 2001, pp 117-127. ch 9. Ruth Cole, Jean de Vellis.
Preparation of Astrocyte, Oligodendrocyte, and Microglia Cultures
from Primary Rat Cerebral Cultures). Astrocytes (1 million) were
cultured in 100 mm TC-treated culture dishes (Orange Scientific,
Belgium) in an incubator set at 37.degree. C. and 5% CO.sub.2 with
humid air. The R-enantiomeric derivative according to Example 2 was
added to cells in two final concentrations: 330 nM and 1000 nM.
Cells were also treated with 1000 nM PBT2 which bears the basic
structure of the R-enantiomer according to Example 2
(8-hydroxy-quinoline ring) but contains conformational differences
and was developed for the treatment of neurodegenerative disorders
(Pub. No.: WO/2004/007461). Treatments were carried out with a 100
.mu.M DMSO-based stock solution. Control cells were treated with
the same amount of DMSO without the agent. Each treatment was
conducted in three separate culture dishes. After treatment, cells
were incubated at 37.degree. C. for 3 h, then the medium was
removed, cells were washed with PBS and total RNA was extracted
from cells with the Accuzol.TM. Total RNA Extraction Solution
(Bioneer, Daejeon, South Korea), according to the manufacturer's
protocol. cDNA transcription was carried out with the High Capacity
cDNA Reverse Transcription Kit (Life Technologies, Foster City,
Calif., USA). During the test, the expression of four genes (PGK1,
EPO, HMOX-1, VEGF) controlled by HIF1a was determined by qRT-PCR,
with HPRT gene serving as a control. Relative expression of genes
in treated samples was compared to untreated controls. The PCR was
conducted with a LightCyclerD Nano Instrument (Roche, Budapest,
Hungary) in the presence of UPL (Universal Probe Library) probes
(HPRT: 95, HMOX1: 4,EPO: 16, VEGF: 1,PGK1: 66), specific primers
(HPRT1: 5'-gaccggttctgtcatgtcg-3', HPRT2:
5'-acctggttcatcatcactaatcac-3'; HMOX1-1:
5'-gtcaagcacagggtgacaga-3', HMOX1-2: 5'-ctgcagctcctcaaacagc-3';
EPO1: 5'-agtcgcgttctggagaggta-3', EPO2: 5'-ccttctgcacagcccatt-3';
VEGF1: 5'-aaaaacgaaagcgcaagaaa-3', VEGF2:
5'-tttctccgctctgaacaagg-3'; PGK1-1: 5'-ccagataacgaataaccaaagga-3',
PGK1-2: 5'-gacttggctccattgtcca-3') in 20 .mu.l PCR reaction volume
with 20 ng cDNA template and 10 .mu.l Lightcycler DNA Probes Master
(5.times.) Reagent Kit (Roche) according to the following protocol:
activation of enzyme at 95.degree. C. for 10 min, 50 cycles:
denaturation at 95.degree. C. for 15 sec, hybridization and
polymerization, then detection at 60.degree. C. for 30 sec. Results
are shown in FIG. 16. While the compound according to Example 2
caused dose-dependent and robust activation of EPO and HMOX-1
genes, even 1000 nM of PBT2 did not induced differences in gene
activity.
Example 49
Effect of Chronic Treatment with the R-Enantiomeric Derivative
According to Example 2 on Erythropoietin Gene Expression in the
Hippocampus and Cortex of Aged Animals
[0472] Twenty-four 18-month-old C57BL/6 female mice (Innovo Ltd.,
Budapest, Hungary) were divided into two groups randomly: Group
1--untreated control, Group 2--treated with the R-enantiomer
according to Example 2, administered in drinking water (20 mg/l)
for four months. From both groups, 10-10 mice were sacrificed by
CO.sub.2 inhalation. After the withdrawal reflex ceased, the whole
animal was subjected to perfusion with 1.times. phosphate buffer
according to the described protocol (Whole Animal Perfusion
Fixation for Rodents; Gregory J. Gage, Daryl R. Kipke, William
Shain; J. Vis. Exp. (65), e3564, doi:10.3791/3564 (2012). After
perfusion, brain tissue was removed from the animals and
hippocampal and cortical regions were separated. Half of the brain
was processed per animal. Half of the samples was used for gene
expression studies, the other half was used for protein expression
(Example 35.) studies.
[0473] To study gene expression, total RNA was extracted from the
brain tissues with Accuzol.TM. Total RNA Extraction Solution
(Bioneer, Daejeon, South Korea), according to the manufacturer's
protocol. cDNA transcription was carried out with the High Capacity
cDNA Reverse Transcription Kit (Life Technologies, Foster City,
Calif., USA). Expression of the gene coding for erythropoietin, the
EPO gene was determined by qRT-PCR (with HPRT gene serving as a
control) with the method and instrument described in Example 33.
but here, mouse-specific UPL probes (HPRT: 95, EPO: 16) and primer
pairs were applied (mouse HPRT1: 5'-tcctcctcagaccgctttt-3', mouse
HPRT2: 5'-cctggttcatcatcgctaatc-3'; mouse EPO1:
5'-tctgcgacagtcgagttctg-3', mouse EPO2: 5'-cttctgcacaacccatcgt-3').
One hippocampal control, one cortical control and one treated
cortical sample did not give appreciable results. Results are shown
in FIG. 17. Chronic treatment with the R-enantiomeric derivative
according to Example 2 induced both hippocampal and cortical EPO
expression compared to samples of untreated control mice.
Example 50
Effect of Chronic Treatment with the R-Enantiomeric Derivative
According to Example 2 on Erythropoietin Protein Expression in the
Hippocampus and Cortex of Aged Animals
[0474] Twenty-four 18-month-old female C57BL/6 mice (Innovo Ltd.,
Budapest, Hungary) were treated according to the experimental setup
described in Example 34. in two groups: Group 1--control group
given normal drinking water, Group 2--treated with the R-enantiomer
according to Example 2, administered in drinking water (20 mg/l)
for four months. Brain preparation was carried out under conditions
described in Example 34. Brain samples were washed with 1.times.PBS
to remove blood, then tissues were homogenized in 1.times.PBS and
for 3 days, they were held at -20.degree. C. overnight and
25.degree. C. at daytime every day. After the third round of
thawing, samples were centrifuged (5 min, 5000 rpm) and the
supernatant was collected for further protein expression studies.
Determination of erythropoietin levels of samples was done with
Quantikine.RTM. ELISA Mouse Erythropoetin (R&D Systems, cat.
no.: MEPOOB) according to the manufacturer's protocol. At the end
of the test, the developed colour was measured with a
THERMO-LABSYSTEMS Multiskan FC Absorbance Plate Reader at 450 nm.
Results obtained through ELISA and measured at 450 nm were compared
to standard dilution series of purified mouse EPO. Values (pg/ml)
obtained based on the dilution series were compared to actual
protein content of tissues. Protein content of tissue homogenates
was measured with Nanodrop-1000 adjusted for BSA protein, at 280
nm. Two hippocampal and two cortical controls as well as one
treated hippocampal and cortical sample did not give appreciable
results. Results are shown in FIG. 18. Chronic treatment with the
R-enantiomeric derivative according to Example 2 induced increased
EPO protein levels in both hippocampal and cortical samples
compared to samples of untreated control mice.
Example 51
Inhibition of Skin Graft Rejection with the Compound
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4
(trifluoromethyl)phenyl]methyl]quinoline-8-ol According to Example
2
[0475] Mouse skin transplantation is a standard method to assay
host T cell responses to MHC-disparate donor antigens, which
protocol was followed in the present example (Garrod and Cahalan;
2008). Briefly, WT C57BL/6 recipient mice (8-12 weeks old) and WT
BALB/c donor mice (8-12 weeks old) were anesthetized with a
combination of 10 mg/kg Xylazine and 100 mg/kg Ketamine
administered by i.p. injection and 0.05 mg/kg Buprenorphine was
administered by subcutaneous injection for analgesia. Ear skin (1.0
cm2) from the WT BALB/c donor mouse was grafted onto the flank of
the WT C57BL/6 recipient. The implanted skin (graft) was covered
with a sterile bandage, which was removed on day 7 post-transplant.
Recipients received either no treatment (controls), or treated with
compound according to Example 2 orally at 5 mg/kg dose, once every
day until the end of the experiment from the day of
transplantation. As positive control we administered tacrolimus at
5 mg/kg dose, once every day until the end of the experiment from
the day of transplantation via intraperitoneal administration.
[0476] Different scores were used to define the quality of the
grafts: intact grafts (Score 0), early stages of rejection (Score
1-4, corresponding to first clear signals of graft rejection: 1;
>25% rejection rate: 2; >50% rejection rate: 3; >75%
rejection rate: 4) up to complete graft rejection (Score 5). The
different scores describe rejection by the area that was destroyed
by the immune system of the host. Scoring was done at day 9 and day
10 post-transplant. Each group contained 6 animals. Average scores
with SEM were calculated for each group.
[0477] We can see from the results (FIG. 18) that both the positive
control, tacrolimus compound as well as the compound according to
Example 2 resulted in smaller score values both on day 9 and on day
10, from which we can conclude that both compounds prevented skin
graft rejection.
FORMULATION EXAMPLES
Example 52
[0478] 20 mg of active ingredient, enantiomerically pure
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-ol was mixed with microcrystalline cellulose, mannitol,
colloidal anhydrous silicon dioxide and magnesium stearate and the
mixture was formulated under pressure, with the usual formulation
technic to tablet.
Example 53
[0479] 20 mg of active ingredient, enantiomerically pure
7-[(R)-[(4-methylpyrimidin-2-yl)amino][4-(trifluoromethyl)phenyl]methyl]q-
uinolin-8-ol was mixed with microcrystalline cellulose, mannitol,
colloidal anhydrous silicon dioxide and magnesium stearate and the
homogenised mixture was filled into gelatin capsules as solid
powder.
REFERENCES
[0480] Abdul et al., 2009: Abdul H M, Sama M A, Furman J L, Mathis
D M, Beckett T L, Weidner A M, Patel E S, Baig I, Murphy M P,
LeVine H 3rd, Kraner S D, Norris C M. Cognitive decline in
Alzheimer's disease is associated with selective changes in
calcineurin/NFAT signaling. J Neurosci. 2009, 29:12957-12969.
[0481] Araboor et al., 2012: Arabpoor Z, Hamidi G, Rashidi B,
Shabrang M, Alaei H, Sharifi M R, Salami M, Dolatabadi H R, Reisi
P. Erythropoietin improves neuronal proliferation in dentate gyrus
of hippocampal formation in an animal model of Alzheimer's disease.
Adv Biomed Res. 2012, 1:50. [0482] Asai et al., 1999: Asai A, Qiu
Jh, Narita Y, Chi S, Saito N, Shinoura N, Hamada H, Kuchino Y,
Kirino T. High level calcineurin activity predisposes neuronal
cells to apoptosis. J Biol Chem. 1999, 274:34450-34458. [0483]
Bartus, 2000: Bartus, R. T. On neurodegenerative diseases, models,
and treatment strategies: lessons learned and lessons forgotten a
generation following the cholinergic hypothesis. Exp. Neurol. 2000,
163:495e529. [0484] Bach, 2006: Bach F H. Heme Oxygenase-1 and
Transplantation Tolerance Human Immunology 2006, 67:430-432. [0485]
Betti, 1900: Betti, M. Gazz. Chim. Ital. 1900, 30 II, 301; [0486]
Betti, 1903: Betti, M. Gazz. Chim. Ital. 1903, 33 II, 2; [0487]
Bresgen et al., 2003: Bresgen N, Karlhuber G, Krizbai I, Bauer H,
et al. Oxidative stress in cultured cerebral endothelial cells
induces chromosomal aberrations, micronuclei, and apoptosis, J.
Neurosci. Res. 2003 72:327-333. [0488] Britton et al., 2002:
Britton R S, Leicester K L, Bacon B R. Iron toxicity and chelation
therapy. Int J Hematol. 2002, 76:219-228. [0489] Calabrese et al.,
2010: Calabrese V, Butterfield D A, Stella A M, Nutritional
antioxidants and the heme oxygenase pathway of stress tolerance:
novel targets for neuroprotection in Alzheimer's disease, Ital. J.
Biochem. 2003, 52:177-181; Targeting heme oxygenase-1 for
neuroprotection and neuroinflammation in neurodegenerative
diseases. Jazwa A, Cuadrado A. Curr Drug Targets. 2010,
11:1517-1531. [0490] Chen et al., 2003: Chen K, Gunter K, Maines M
D. Neurons overexpressing heme oxygenase-1 resist oxidative
stress-mediated cell death, J. Neurochem. 2000, 75:304-313.) [0491]
Chong et al., 2013: Chong Z Z, Shang Y C, Mu Y, Cui S, Yao Q,
Maiese K. Targeting erythropoietin for chronic neurodegenerative
diseases. Expert Opin Ther Targets. 2013, 17:707-720. [0492]
Chowdhury et al., 2008: The human oxygen sensing machinery and its
manipulation. Chowdhury R, Hardy A, Schofield C J. Chem Soc Rev.
2008, 37:1308-1319. [0493] Chu et al., 2013: Chu J, Li J G,
Ceballos-Diaz C, Golde T, Pratic D. The influence of 5-lipoxygenase
on Alzheimer's disease-related tau pathology: in vivo and in vitro
evidence. Biol Psychiatry. 2013, 74:321-328; [0494] Cole and de
Vellis, 2001: Ruth Cole, Jean de Vellis. Preparation of Astrocyte,
Oligodendrocyte, and Microglia Cultures from Primary Rat Cerebral
Cultures, Protocols for Neural Cell Culture konyv 2001, 9. fejezet,
pp 117-127; [0495] De Calignon et al., 2010: de Calignon A, Fox L
M, Pitstick R, Carlson G A, Bacskai B J, Spires-Jones T L, Hyman B
T. Caspase activation precedes and leads to tangles. Nature. 2010,
464: 1201-1204. [0496] Degterev and Yuan, 2008: Degterev A, Yuan J.
Expansion and evolution of cell death programmes. Nat Rev Mol Cell
Biol. 2008, 9:378-390. [0497] Eckert et al., 2003: Eckert A,
Marques C A, Keil U, Schussel K, Muller W E. Increased apoptotic
cell death in sporadic and genetic Alzheimer's disease. Ann N Y
Acad Sci. 2003, 1010:604-609. [0498] Emmel et al., 1991: Emmel E A,
Verweij C L, Durand D B, Higgins K M, Lacy E and Crabtree G R
(1989) Cyclosporin A specifically inhibits function of the nuclear
proteins involved in T cell activation. Science. 246:1617-1620.
[0499] Firuzi et al. 2008: Firuzi O, Zhuo J, Chinnici C M,
Wisniewski T, Pratic D. 5-Lipoxygenase gene disruption reduces
amyloid-beta pathology in a mouse model of Alzheimer's disease.
FASEB J. 2008, 22:1169-1178. [0500] Frederickson et al., 2005:
Frederickson C J, Koh J Y, Bush A I. The neurobiology of zinc in
health and disease. Nat Rev Neurosci. 2005, 6:449-462. [0501] Gage
et al., 1999: Gage G J, Kipke D R, Shain W. Whole Animal Perfusion
Fixation for Rodents J. Vis. Exp. 2012, (65: e3564; [0502] Frisch M
J, et al., 2010: Frisch M J, Trucks G W, Schlegel H B, Scuseria G E
et al., Gaussian 09 rev. B01, Gaussian, Inc., Wallingford Conn.,
2010. [0503] Hagemeyer et al., 2012: Hagemeyer N, Boretius S, Ott
C, Von Streitberg A, Welpinghus H, Sperling S, Frahm J, Simons M,
Ghezzi P, Ehrenreich H. Erythropoietin attenuates neurological and
histological consequences of toxic demyelination in mice. Mol Med.
2012, 18:628-635. [0504] Hengartner, 2000: Hengartner M O. The
biochemistry of apoptosis. Nature 2000, 407:770-776; [0505] Hudri
et al., 2012: Hudry E, Wu H Y, Arbel-Ornath M, Hashimoto T,
Matsouaka R, Fan Z, Spires-Jones T L, Betensky R A, Bacskai B J,
Hyman B T. Inhibition of the NFAT pathway alleviates amyloid .beta.
neurotoxicity in a mouse model of Alzheimer's disease. J Neurosci.
2012, 32:3176-3192.). [0506] Idris et al., 2008: Idriss N K, Blann
A D, Lip G Y. Hemoxygenase-1 in cardiovascular disease. J Am Coll
Cardiol. 2008, 52:971-978. [0507] Janciauskiene et al., 1999:
Janciauskiene S, Wright H T, Lindgren S. Fibrillar Alzheimer's
amyloid peptide Abeta(1-42) stimulates low density lipoprotein
binding and cell association, free radical production and cell
cytotoxicity in PC12 cells. Neuropeptides. 1999, 33:510-516. [0508]
Jazwa and Cuadrado, 2010: Jazwa A, Cuadrado A. Targeting heme
oxygenase-1 for neuroprotection and neuroinflammation in
neurodegenerative diseases. Curr Drug Targets. 2010, 11:1517-1531.
[0509] Karttunen et al. 1991: Karttunen J and Shastri N.
Measurement of ligand-induced activation in single viable T cells
using the lacZ reporter gene. Proc Natl Acad Sci USA. 1991,
88:3972. [0510] Koh et al., 1996: Koh J Y, Suh S W, Gwag B J, He Y
Y, Hsu C Y, Choi D W. The role of zinc in selective neuronal death
after transient global cerebral ischemia. Science. 1996,
272:1013-1016. [0511] Korkmaz et al., 2013: Korkmaz S, Barnucz E,
Loganathan S, Li S, Radovits T, Hegedus P, Zubarevich A, Hirschberg
K, Weymann A, Puskas L G, Ozsvari B, Farago N, Kanizsai I, Fabian
G, Gyuris M, Merkely B, Karck M, Szabo C, Szabo G. Q50, an
iron-chelating and zinc-complexing agent, improves cardiac function
in rat models of ischemia/reperfusion-induced myocardial injury.
Circ J. 2013; 77:1817-1826. [0512] Lewen et al., 2000: Lewen A,
Matz P, Chan P H. Free radical pathways in CNS injury. J
Neurotrauma. 2000, 17:871-890; [0513] Li et al., 2007: Li C,
Hossieny P, Wu B J, Qawasmeh A, Beck K, Stocker R. Pharmacologic
induction of heme oxygenase-1. Antioxid Redox Signal. 2007,
9:2227-2239. [0514] Liu et al., 2005: Liu F, Grundke-Iqbal I, Iqbal
K, Oda Y, Tomizawa K, Gong C X. Truncation and activation of
calcineurin A by calpain I in Alzheimer disease brain. J Biol Chem.
2005, 280:37755-37762. [0515] Lue, et al., 1999: Lue L F1, Kuo Y M,
Roher A E, Brachova L, Shen Y, Sue L, Beach T, Kurth J H, Rydel R
E, Rogers J. Soluble amyloid beta peptide concentration as a
predictor of synaptic change in Alzheimer's disease, Am J Pathol
1999, 155:853-862. [0516] Lynch et al., 2002: Lynch A M, Lynch M A.
The age-related increase in IL-1 type I receptor in rat hippocampus
is coupled with an increase in caspase-3 activation. Eur J Neurosci
2002, 15:1779-1788. [0517] Machova et al., 2008: Machova, E.,
Jakubik, J., Michal, P., Oksman, M., Iivonen, H., Tanila, H.,
Dolezal, V. Impairment of muscarinic transmission in transgenic
APPswe/PS1dE9 mice. Neurobiol. Aging 2008, 29:368e378. [0518]
Mammis et al., 2009: Mammis A, McIntosh T K, Maniker A H.
Erythropoietin as a neuroprotective agent in traumatic brain
injury. Surg Neurol. 2009, 71:527-531. [0519] Mattson, 2006:
Mattson M P. Neuronal life-and-death signaling, apoptosis, and
neurodegenerative disorders. Antioxid Redox Signal. 2006,
8:1997-2006. [0520] McLean et al., 1999: C. McLean, R. Cherny, F.
Fraser, S. Fuller, M. Smith, K. Beyreuther, A. Bush and C. Masters,
Soluble pool of A.beta. amyloid as a determinant of severity of
neurodegeneration in Alzheimer's Disease, Ann Neurol 1999,
46:860-866 [0521] Merelli et al., 2011: Merelli A, Caltana L,
Lazarowski A, Brusco A. Experimental evidence of the potential use
of erythropoietin by intranasal administration as a neuroprotective
agent in cerebral hypoxia. Drug Metabol Drug Interact. 2011,
26:65-69. [0522] Merelli et al., 2013: Merelli A, Czornyj L,
Lazarowski A. Erythropoietin: a neuroprotective agent in cerebral
hypoxia, neurodegeneration, and epilepsy. Curr Pharm Des. 2013,
19:6791-801. [0523] Mittl et al., 1997: Mittl PRE, Marco S D, Krebs
J F, Karanewsky D S, Priestle J P, Tomaselli K J and Grutter M G
Structure of Recombinant Human CPP32 in Complex with the
Tetrapeptide Acetyl-Asp-Val-Ala-Asp Fluoromethyl Ketone. J Biol
Chem. 1997, 272: 6539-6547. [0524] Newton et al. 2013: Newton S S,
Fournier N M, Duman R S. Vascular growth factors in
neuropsychiatry. Cell Mol Life Sci. 2013, 70:1739-1752. [0525]
Nguyen et al., 2005: Nguyen T, Hamby A, Massa S M. Clioquinol
down-regulates mutant huntingtin expression in vitro and mitigates
pathology in a Huntington's disease mouse model. Proc Natl Acad Sci
USA. 2005, 102:11840-11845. [0526] Noh et al., 2014: Noh M Y, Cho K
A, Kim H, Kim S M, Kim S H. Erythropoietin modulates the
immune-inflammatory response of a SOD1(G93A) transgenic mouse model
of amyotrophic lateral sclerosis (ALS). Neurosci Lett. 2014,
574:53-58. [0527] Orrenius, 2007: Orrenius S. Reactive oxygen
species in mitochondria-mediated cell death. Drug Metab Rev. 2007,
39:443-455. [0528] Palmieri, 2000: Palmieri, G. A Practical
o-Hydroxybenzylamines Promoted Enantioselective Addition of
Dialkylzincs to Aldehydes with Asymmetric Amplification Tetrahedron
Asymmetry 2000, 11:3361. [0529] Phillips et al., 1954: J. P.
Phillips, R. W. Keown, Q. Fernando; The reaction of anils with
8-quinolinol., J. Org. Chem. 1954, 19:907. [0530] Phillips and
Barrall, 1956: J. P. Phillips, E. M. Barrall. Notes--Betti
Reactions of Some Phenols. J. Org. Chem. 1956, 21:692. [0531]
Phillips, 1956: J. P. Phillips, The Reactions Of 8-Quinolinol,
Chem. Rev. 1956, 56:286. [0532] Regland et al., 2001: Regland B,
Lehmann W, Abedini I, Blennow K, Jonsson M, Karlsson I, Sjogren M,
Wallin A, Xilinas M, Gottfries C G. Treatment of Alzheimer's
disease with clioquinol. Dement Geriatr Cogn Disord. 2001,
12:408-414. [0533] Sama et al., 2008: Sama M A, Mathis D M, Furman
J L, Abdul H M, Artiushin I A, Kraner S D, Norris C M.
Interleukin-1beta-dependent signaling between astrocytes and
neurons depends critically on astrocytic calcineurin/NFAT activity.
J Biol Chem. 2008, 283:21953-21964. [0534] Schafer et al., 2007:
Schafer S, Pajonk F G, Multhaup G, Bayer T A. Copper and clioquinol
treatment in young APP transgenic and wild-type mice: effects on
life expectancy, body weight, and metal-ion levels. J Mol Med
(Berl). 2007, 85:405-413. [0535] Smirnova et al., 2010: Smirnova N
A, Rakhman I, Moroz N, Basso M, Payappilly J, Kazakov S,
Hernandez-Guzman F, Gaisina I N, Kozikowski A P, Ratan R R,
Gazaryan I G. Utilization of an in vivo reporter for high
throughput identification of branched small molecule regulators of
hypoxic adaptation. Chem Biol. 2010, 17:380-391. [0536] Stefanis,
2005: Stefanis L. Caspase-dependent and -independent neuronal
death: two distinct pathways to neuronal injury. Neuroscientist.
2005, 11:50-62. [0537] Szabo, 2005: Szabo C. Mechanisms of cell
necrosis. Crit Care Med. 2005, 33:S530-534. [0538] Uz et al., 1998:
Uz, T., Pesold, C., Longone, P., and Manev, H. Aging associated
up-regulation of neuronal 5-lipoxygenase expression: putative role
in neuronal vulnerability. FASEB J. 1998, 12:439-449. [0539]
Verkade et al., 2008: Verkade J M M, van Hemert L J C, Quaedflieg P
J L M, Rutjes F P J T. Organocatalysed asymmetric Mannich
reactions; Chem. Soc. Rev., 2008, 37:29. [0540] Wang et al., 1999:
J. Wang, D. W. Dickson, J. Q. Trojanowski and V. M. Lee, The levels
of soluble versus insoluble brain Abeta distinguish Alzheimer's
disease from normal and pathologic aging, Exp Neurol. 1999,
158:328-337. [0541] Xue et al., 2007: Xue Y Q, Zhao L R, Guo W P,
Duan W M. Intrastriatal administration of erythropoietin protects
dopaminergic neurons and improves neurobehavioral outcome in a rat
model of Parkinson's disease. Neuroscience. 2007, 146:1245-1258.
[0542] Zhang et al., 2006: Zhang, C. P., Zhu, L. L., Zhao, T.,
Zhao, H., Huang, X., Ma, X., Wang, H., and Fan, M. Characteristics
of neural stem cells expanded in lowered oxygen and the potential
role of hypoxia-inducible factor-1Alpha. Neurosignals 2006,
15:259-265. [0543] Zhou et al., 2006: Zhou, Y., Wei, E. Q., Fang,
S. H., Chu, L. S., Wang, M. L., Zhang, W. P., Yu, G. L., Ye, Y. L.,
Lin, S. C., and Chen, Z. Spatio-temporal properties of
5-lipoxygenase expression and activation in the brain after focal
cerebral ischemia in rats. Life Sci. 2006, 79:1645-1656. [0544]
Garrod K R, Cahalan M D (2008) Murine skin transplantation. J Vis
Exp 11:634.
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