U.S. patent application number 10/941126 was filed with the patent office on 2005-03-24 for treatment of mitochondrial diseases.
Invention is credited to Bodupalli, Sekhar, Miller, Guy, Walkinshaw, Gail, Wang, Bing.
Application Number | 20050065099 10/941126 |
Document ID | / |
Family ID | 34425947 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050065099 |
Kind Code |
A1 |
Walkinshaw, Gail ; et
al. |
March 24, 2005 |
Treatment of mitochondrial diseases
Abstract
The invention relates the method of treatment or amelioration of
mitochondrial disorders such as Alzheimer's disease, Parkinson's
disease, Friedreich's ataxia (FRDA), cerebellar ataxias, Leber's
hereditary optic neuropathy (LHON), mitochondrial myopathy,
encephalopathy, lactacidosis, stroke (MELAS), Myoclonic Epilepsy
with Ragged Red Fibers (MERFF), amyotrophic lateral sclerosis
(ALS), motor neuron diseases, Huntington's disease, macular
degeneration, and epilepsy, with chroman derivatives of Formula I
or Formula II as described herein.
Inventors: |
Walkinshaw, Gail; (Mountain
View, CA) ; Bodupalli, Sekhar; (San Jose, CA)
; Wang, Bing; (Cupertino, CA) ; Miller, Guy;
(San Jose, CA) |
Correspondence
Address: |
GALILEO PHARMACEUTICALS, INC.
(PREVIOUSLY GALILEO LABORATORIES, INC.)
5301 PATRICK HENRY DRIVE
SANTA CLARA
CA
95954
US
|
Family ID: |
34425947 |
Appl. No.: |
10/941126 |
Filed: |
September 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60504391 |
Sep 19, 2003 |
|
|
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60541737 |
Feb 4, 2004 |
|
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Current U.S.
Class: |
514/27 ; 514/456;
514/80 |
Current CPC
Class: |
A61P 3/10 20180101; A61K
31/496 20130101; A61K 31/7048 20130101; C07D 311/94 20130101; C07D
405/12 20130101; C07D 405/06 20130101; A61P 11/06 20180101; A61P
21/00 20180101; A61P 43/00 20180101; C07D 311/74 20130101; A61P
25/00 20180101; C07D 405/14 20130101; A61P 29/00 20180101; A61P
37/00 20180101; C07D 295/096 20130101; A61K 31/357 20130101; A61P
9/10 20180101; A61P 41/00 20180101; A61P 13/12 20180101; A61P 27/02
20180101; C07D 319/08 20130101; A61P 35/00 20180101; A61P 19/02
20180101; C07D 417/06 20130101; A61P 25/16 20180101; C07D 311/58
20130101; A61P 1/00 20180101; A61P 11/00 20180101; A61P 9/00
20180101; C07D 409/04 20130101; C07D 417/12 20130101; A61P 17/06
20180101; A61K 31/353 20130101; A61P 25/14 20180101; A61P 17/02
20180101; A61P 17/10 20180101; A61P 17/00 20180101; A61K 31/352
20130101; C07D 311/70 20130101; A61P 25/08 20180101; A61P 15/00
20180101; A61P 25/28 20180101; A61K 31/675 20130101 |
Class at
Publication: |
514/027 ;
514/080; 514/456 |
International
Class: |
A61K 031/7048; A61K
031/675; A61K 031/353 |
Claims
What is claimed is:
1. A method of treating or ameliorating a subject suffering from a
mitochondrial disorder comprising administering to said subject a
therapeutically effective amount of a compound of Formula I or
Formula II: 11wherein: -A-B-- is --CH.sub.2--CH.sub.2--;
--CH.dbd.CH-- or --CH.sub.2--O--; n is 0; R.sup.1 is C.sub.1-.sub.4
alkyl; R.sup.2 is C.sub.1-20 alkyl or C.sub.2-20 alkenyl, halogen,
cyano, or R.sup.2 and A together with the atoms to which they are
attached form a ring; R.sup.3 is hydrogen; halogen; aralkyl,
optionally substituted with one or more substituents selected from
alkyl, haloalkyl, hydroxy, alkoxy, halogen, oxo, cyano, nitro,
amino, --SO.sub.2NR.sub.2 or --C(O)OR; heteroaralkyl optionally
substituted with one or more substituents selected from alkyl,
haloalkyl, hydroxy, alkoxy, halogen, oxo, cyano, nitro, amino,
--SO.sub.2NR.sub.2 or --C(O)OR; C.sub.1-.sub.6 alkyl;
C.sub.2-.sub.20 alkenyl;
--(CR.sub.2).sub.1-3S(O).sub.0-2(CR.sub.2).sub.1-3C(O)OR;
--(CR.sub.2).sub.1-3OR.sup.a; --(CR.sub.2).sub.1-3NR.sup.bR.sup.c
--C(O)R.sup.a; or nitro; R.sup.4 is hydrogen; optionally
substituted C.sub.1-.sub.6 alkyl; C.sub.2-.sub.12 alkenyl;
hydroxyalkyl; acyl; glucoside; phosphoryl; phosphoryloxyalkyl;
carboxyalkylcarbonyl; or aminoalkylcarbonyl; R.sup.5 and R.sup.6
are independently of each other hydrogen, halogen, haloalkyl,
nitro, acyl, C.sub.1-.sub.6 alkyl or C.sub.2-.sub.12alkenyl; or
R.sup.5 and R.sup.6 taken together with the carbon to which they
are attached form a 5-6 membered aliphatic, unsaturated or aromatic
ring, optionally substituted with C.sub.1-6 alkyl, C.sub.1-6
alkoxy, hydroxy, carboxy, carboxyalkyl, alkoxycarbonyl,
alkoxycarbonylalkyl, aminocarbonyl, aminocarbonylalkyl, or
hydroxyalkyl; R is hydrogen or C.sub.1-6 alkyl; R.sup.a is
hydrogen; optionally substituted C.sub.1-.sub.6 alkyl; optionally
substituted C.sub.2-.sub.12 alkenyl; optionally substituted aryl;
optionally substituted cycloalkyl; or optionally substituted
saturated, partially unsaturated or unsaturated heterocyclyl;
R.sup.b and R.sup.c are independently of each other hydrogen;
C.sub.1-.sub.6 alkyl; hydroxyalkyl; aminoalkyl; optionally
substituted aryl; optionally substituted benzyl; or optionally
substituted heterocyclyl; or R.sup.b and R.sup.c taken together
with the atom to which they are attached form a 5 to 8 membered
aromatic, saturated or unsaturated ring, optionally incorporating
one additional atom chosen from N, O, or S and optionally
substituted with a substituent selected from the group consisting
of lower alkyl, halo, cyano, alkylthio, lower alkoxy, oxo, phenyl,
benzyl and carboxy; with the proviso that the compound is not
alpha-tocopherol; or 12wherein: G is --O--, --S--, --SO--,
--SO.sub.2--, a secondary or tertiary amine group, a phosphate
group, a phosphoester group, or an unsubstituted or substituted
methylene group, R.sup.7 and R.sup.8 independently are hydrogen,
hydroxy, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester,
unsubstituted or substituted amine, amide, halogen or unsubstituted
or substituted sulfonyl, or jointly complete a 5- or 6-member
aliphatic or aromatic ring, R.sup.9 and R.sup.10 independently are
hydrogen, hydroxy, alkyl, aryl, alkenyl, alkynyl, aromatic, ether,
ester, unsubstituted or substituted amine, amide, halogen or
unsubstituted or substituted sulfonyl, or jointly complete a 5- or
6-member aliphatic, aromatic or heterocyclic ring, R.sup.11 is
hydrogen, hydroxy, alkyl, aryl, alkenyl, alkynyl, aromatic, ester
or unsubstituted or substituted amine, R.sup.12 is --COOH,
--COOR.sup.13, --CONH.sub.2, --CONHR.sup.13, --CONR.sup.13R.sup.14,
--NH.sub.2, --NHR.sup.13, --NR.sup.13R.sup.14, or a carboxylate
salt, R.sup.13 and R.sup.14 independently are unsubstituted or
substituted alkyl, aryl, alkaryl, aralkyl, alkenyl or alkynyl, p is
0 to 3, and m is 0 to 5; or single stereoisomers, mixtures of
stereoisomers, or pharmaceutically acceptable salts thereof.
2. The method of claim 1, comprising administering to said subject
a therapeutically effective amount of a compound of Formula I.
3. The method of claim 2, comprising administering the compound of
Formula I wherein R.sup.3 is hydrogen, C.sub.1-6 alkyl, or
C.sub.2-20 alkenyl.
4. The method of claim 2, comprising administering the compound of
Formula I wherein R.sup.5 and R.sup.6 are independently of each
other C.sub.1-4 alkyl or halogen.
5. The method of claim 4, comprising administering the compound of
Formula I wherein R.sup.1 is C.sub.1-6 alkyl and R.sup.2 is
C.sub.1-6 alkyl.
6. The method of claim 2, comprising administering the compound of
Formula I wherein R.sup.1 is C.sub.1-6 alkyl, and R.sup.2 is
C.sub.16 alkyl or C.sub.16 alkenyl.
7. The method of claim 2, comprising administering the compound of
Formula I wherein R.sup.5 and R.sup.6 taken together with the
carbon to which they are attached form a 5-6 membered carbocyclic
ring, optionally substituted with C.sub.1-6 alkyl, C.sub.1-6
alkoxy, hydroxy, carboxy, carboxyalkyl, alkoxycarbonyl,
alkoxycarbonylalkyl, aminocarbonyl, aminocarbonylalkyl, or
hydroxyalkyl.
8. The method of claim 1, comprising administering to said subject
a therapeutically effective amount of a compound of Formula II.
9. The method of claim 8, comprising administering the compound of
Formula II, wherein R.sup.12 is --COOH, or --COOR.sup.13 and
R.sup.13 is C.sub.1-6 alkyl.
10. The method of claim 1, comprising administering a compound
selected from:
8-Chloro-2-(3-chloro-propyl)-2,5-dimethyl-chroman-6-ol;
5-Chloro-2-(3-chloro-propyl)-2,8-dimethyl-chroman-6-ol;
7-tert-Butyl-2-(3-chloro-propyl)-2-methyl-chroman-6-ol; Acetic acid
2,2,5-trimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl ester;
2,2,5,7-Tetramethyl-8-trifluoromethyl-chroman-6-ol;
5-Methoxy-2,2,7,8-tetramethyl-chroman-6-ol;
8-Fluoro-2,2,5,7-tetramethyl-- chroman-6-ol;
2,2,5,7-Tetramethyl-8-(3-methyl-butyl-chroman-6-ol;
6-Hydroxy-2,2,5,7-tetramethyl-chroman-8-carbaldehyde;
2,2,5,7-Tetramethyl-8-nitro-chroman-6-ol;
5-(1-Hydroxy-ethyl)-2,2,7,8-tet- ramethyl-chroman-6-ol;
5-Hydroxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
5-(2-Hydroxy-ethyl)-2,2,7,8-tetramethyl-chroman-6-ol;
10-Methoxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ol;
2-Ethynyl-2,5,7,8-tetramethyl-chroman-6-ol;
2-(6-Hydroxy-2,5,7,8-tetramet-
hyl-chroman-2-ylmethoxy)-N-(3-trifluoromethyl-phenyl)-acetamide;
(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-piperazin-1-yl-methanone;
6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxamidine;
N-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethyl)-N-methyl-acetamide;
2,2,5,7,8-Pentamethyl-2H-chromen-6-ol;
2-(6-Hydroxy-2,2,7,8-tetramethyl-c- hroman-5-ylmethyl
)-propane-1,3-diol; 2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-
-(3-methyl-but-2-enyl)-chroman-6-ol;
2,2,5,8-Tetramethyl-chroman-6-ol;
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol;
2-(3-Chloro-propyl)-2,7,8-trimethyl-chroman-6-ol;
2-(2-Chloro-ethyl)-2,7,- 8-trimethyl-chroman-6-ol;
2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-acetic
acid methyl ester;
5,6,9-Trimethyl-8-oxa-tricyclo[7.3.1.0.sup.2,7]trideca-2,4,-
6-trien4-ol; 2,2,7,8-Tetramethyl-5-(3-methyl-butyl)-chroman-6-ol;
2,2,7,8-Tetramethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol;
3-(6-Hydroxy-2,8-dimethyl-chroman-2-yl)-propionic acid;
2,2,7,8-Tetramethyl-chroman-6-ol;
3-[6-Hydroxy-2,7,8-trimethyl-5-(3-methy-
l-but-2-enyl)-chroman-2-yl]-propionic acid;
3-(5-Bromo-6-hydroxy-2,7,8-tri- methyl-chroman-2-yl)-propionic
acid; 3-(6-Hydroxy-2,7,8-trimethyl-5-nitro--
chroman-2-yl)-propionic acid;
3-(2,5,7,8-Tetramethyl-chroman-2-yl)-propion- ic acid;
3-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-propionic acid;
3-(6-Hydroxy-2-methyl-chroman-2-yl)-propionic acid;
3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic acid methyl
ester; 3-(6-Hydroxy-2-methyl-chroman-2-yl)-propionic acid methyl
ester; 3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic acid;
2-Hydroxymethyl-2,5,7,8-tetramethyl-chroman-6-ol;
5-[3-(6-Hydroxy-2,7,8-t-
rimethyl-chroman-2-yl)-propyl]-thiazolidine-2,4-dione;
2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[6-hydroxy-2,7,8-trimethyl-2-(4,-
8,12-trimethyl-tridecyl)-chroman-5-ylmethylsulfanyl]-ethylcarbamoyl}-butyr-
ic acid;
5-(4-Benzyl-piperazin-1-ylmethyl)-2,7,8-trimethyl-2-(4,8,12-trime-
thyl-tridecyl)-chroman-6-ol;
3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-- 2-yl)-propionic acid
methyl ester; 3-(6-Hydroxy-2-methyl-3,4-dihydro-2H-be-
nzo[h]chroman-2-yl)-propionic acid;
4-[1-(Carboxymethyl-carbamoyl)-2-merca-
pto-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propiony-
lamino]-butyric acid); 2,2,7,8-Tetramethyl-chroman-6-ol; Delta
tocopherol; Delta tocotrienol; Gamma-tocopherol; Gamma-tocotrienol;
and Alpha tocotrienol; or single stereoisomers, mixtures of
stereoisomers, or pharmaceutically acceptable salts thereof.
11. The method of claim 1, comprising administering a
therapeutically effective amount of a compound of Formula I or of
Formula II to a subject suffering from a disease involving
mitochondrial dysfunctions selected from Alzheimer's disease,
Parkinson's disease, Friedreich's ataxia (FRDA), cerebellar
ataxias, Leber's hereditary optic neuropathy (LHON), mitochondrial
myopathy, encephalopathy, lactacidosis, stroke (MELAS), Myoclonic
Epilepsy with Ragged Red Fibers (MERFF), amyotrophic lateral
sclerosis (ALS), motor neuron diseases, Huntington's disease,
macular degeneration, and epilepsy.
12. The method of claim 1, comprising administering a
therapeutically effective amount of a compound of Formula I or of
Formula II to a subject suffering from Friedreich's ataxia (FRDA),
MELAS, or MERFF.
13. The method of claim 12, comprising administering a
therapeutically effective amount of a compound selected from:
8-Chloro-2-(3-chloro-propyl- )-2,5-dimethyl-chroman-6-ol;
5-Chloro-2-(3-chloro-propyl)-2,8-dimethyl-chr- oman-6-ol;
7-tert-Butyl-2-(3-chloro-propyl)-2-methyl-chroman-6-ol; Acetic acid
2,2,5-trimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl ester;
2,2,5,7-Tetramethyl-8-trifluoromethyl-chroman-6-ol;
5-Methoxy-2,2,7,8-tetramethyl-chroman-6-ol;
8-Fluoro-2,2,5,7-tetramethyl-- chroman-6-ol;
2,2,5,7-Tetramethyl-8-(3-methyl-butyl-chroman-6-ol;
6-Hydroxy-2,2,5,7-tetramethyl-chroman-8-carbaldehyde;
2,2,5,7-Tetramethyl-8-nitro-chroman-6-ol;
5-(1-Hydroxy-ethyl)-2,2,7,8-tet- ramethyl-chroman-6-ol;
5-Hydroxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
5-(2-Hydroxy-ethyl)-2,2,7,8-tetramethyl-chroman-6-ol;
10-Methoxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ol;
2-Ethynyl-2,5,7,8-tetramethyl-chroman-6-ol;
2-(6-Hydroxy-2,5,7,8-tetramet-
hyl-chroman-2-ylmethoxy)-N-(3-trifluoromethyl-phenyl)-acetamide;
(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-piperazin-1-yl-methanone;
6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxamidine;
N-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethyl)-N-methyl-acetamide;
2,2,5,7,8-Pentamethyl-2H-chromen-6-ol;
2-(6-Hydroxy-2,2,7,8-tetramethyl-c-
hroman-5-ylmethyl)-propane-1,3-diol;
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5--
(3-methyl-but-2-enyl)-chroman-6-ol;
2,2,5,8-Tetramethyl-chroman-6-ol;
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol;
2-(3-Chloro-propyl)-2,7,8-trimethyl-chroman-6-ol;
2-(2-Chloro-ethyl)-2,7,- 8-trimethyl-chroman-6-ol;
2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-acetic
acid methyl ester;
5,6,9-Trimethyl-8-oxa-tricyclo[7.3.1.0.sup.2,7]trideca-2,4,-
6-trien4-ol; 2,2,7,8-Tetramethyl-5-(3-methyl-butyl)-chroman-6-ol;
2,2,7,8-Tetramethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol;
3-(6-Hydroxy-2,8-dimethyl-chroman-2-yl)-propionic acid;
2,2,7,8-Tetramethyl-chroman-6-ol;
3-[6-Hydroxy-2,7,8-trimethyl-5-(3-methy-
l-but-2-enyl)-chroman-2-yl]-propionic acid;
3-(5-Bromo-6-hydroxy-2,7,8-tri- methyl-chroman-2-yl)-propionic
acid; 3-(6-Hydroxy-2,7,8-trimethyl-5-nitro--
chroman-2-yl)-propionic acid;
3-(2,5,7,8-Tetramethyl-chroman-2-yl)-propion- ic acid;
3-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-propionic acid;
3-(6-Hydroxy-2-methyl-chroman-2-yl)-propionic acid;
3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic acid methyl
ester; 3-(6-Hydroxy-2-methyl-chroman-2-yl)-propionic acid methyl
ester; 3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic acid;
2-Hydroxymethyl-2,5,7,8-tetramethyl-chroman-6-ol;
5-[3-(6-Hydroxy-2,7,8-t-
rimethyl-chroman-2-yl)-propyl]-thiazolidine-2,4-dione;
2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[6-hydroxy-2,7,8-trimethyl-2-(4,-
8,12-trimethyl-tridecyl)-chroman-5-ylmethylsulfanyl]-ethylcarbamoyl}-butyr-
ic acid;
5-(4-Benzyl-piperazin-1-ylmethyl)-2,7,8-trimethyl-2-(4,8,12-trime-
thyl-tridecyl)-chroman-6-ol;
3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-- 2-yl)-propionic acid
methyl ester; 3-(6-Hydroxy-2-methyl-3,4-dihydro-2H-be-
nzo[h]chroman-2-yl)-propionic acid;
4-[1-(Carboxymethyl-carbamoyl)-2-merca-
pto-ethylcarbamoyl]-2-[3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propiony-
lamino]-butyric acid); 2,2,7,8-Tetramethyl-chroman-6-ol; Delta
tocopherol; Delta tocotrienol; Gamma-tocopherol; Gamma-tocotrienol;
and Alpha tocotrienol; or single stereoisomers, mixtures of
stereoisomers, or pharmaceutically acceptable salts thereof.
14. The method of claim 12, comprising treating or ameliorating a
subject in need of protection of Friedreich's ataxia comprising
administering a therapeutically effective amount of a tocopherol
selected from beta-tocopherol, delta-tocopherol, gamma-tocopherol,
alpha-tocotrienol, beta-tocotrienol, delta-tocotrienol, and
gamma-tocotrienol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This U.S. patent application claims priority under 35 U.S.C.
119(e) to U.S. Provisional Applications Ser. No. 60/504,391, filed
Sep. 19, 2003, and Ser. No. 60/541,737, filed Feb. 4, 2004
incorporated herein by reference in their entirety.
BACKGROUND INVENTION
[0002] The present invention relates to a method of treatment,
amelioration, or prevention of mitochondrial diseases with certain
pharmaceutical formulations containing chroman derivatives.
[0003] Mitochondrial dysfunction leads to impaired calcium
buffering, generation of free radicals that may participate in
intracellular and extracellular processes, changes in mitochondrial
permeability and oxidative damage which is observed in several
neurodegenerative diseases. Some disorders involved in
mitochondrial dysfunction affecting cellular processes include but
are not limited to as Alzheimer's disease, Parkinson's disease,
Friedreich's ataxia and other ataxias, Leber's hereditary optic
neuropathy (LHON), mitochondrial myopathy, encephalopathy,
lactacidosis, stroke (MELAS), Myoclonic Epilepsy with Ragged Red
Fibers (MERFF), amyotrophic lateral sclerosis (ALS) and other motor
neuron diseases, Huntington's disease, macular degeneration, and
epilepsy.
[0004] Friedreich's ataxia ("FRDA") is an autosomal recessive
multi-system degenerative disorder that results in progressive
damage to the nervous system and causes symptoms ranging from
muscle weakness and speech problems to heart disease. Ataxia
results from the degeneration of nerve tissue in the spinal cord
and of nerves that control muscle movement in the arms and legs.
Symptoms usually begin between the ages of 5 and 15 but can appear
as early as 18 months or as late as 30 years of age. The first
symptom is usually difficulty in walking. The ataxia gradually
worsens and slowly spreads to the arms and then the trunk. Foot
deformities such as clubfoot, flexion (involuntary bending) of the
toes, hammer toes, or foot inversion (turning in) may be early
signs. Rapid, rhythmic, involuntary movements of the eyeball are
common. Most people with Friedreich's ataxia develop scoliosis (a
curving of the spine to one side), which, if severe, may impair
breathing. Other symptoms include chest pain, shortness of breath,
and heart palpitations.
[0005] Mitochondrial myopathy, encephalopathy, lactacidosis, stroke
("MELAS") is a progressive neurodegenerative disorder. The typical
presentation of patients with MELAS syndrome includes features that
comprise the name of the disorder such as mitochondrial
encephalomyopathy, lactic acidosis, and stroke like episodes. Other
features, such as diabetes mellitus and hearing loss, clearly are
part of the disorder. MELAS is characterized by stroke-like
episodes and a mitochondrial myopathy.
[0006] Alzheimer's disease ("AD") is a progressive disease of the
human central nervous system. It is manifested by dementia in the
elderly, by disorientation, loss of memory, difficulty with
language, calculation, or visual-spatial skills, and by psychiatric
manifestations. It is associated with degenerating neurons in
several regions of the brain. Alzheimer's disease is reviewed by
Price, D. L. et al. (Clin. Neuropharm. 14:S9-S14 (1991)); Pollwein,
P. et al. (Nucl. Acids Res. 20:63-68 (1992)); Regland, B. et al.
(Med. Hypoth. 38:11-19 (1992)) and Johnson, S. A. (In: Review of
Biological Research in Aging, Vol. 4., Rothstein, M. (Ed.),
Wiley-Liss, NY, 163-170 (1990)).
[0007] Parkinson's disease ("PD") is a neurodegenerative disease
especially affecting the dopaminergic neurons of the substantia
nigra--pars compacta--and its nigrostriatal projections in nearly
one million Americans. The symptomatic manifestations are motor
disorders such as tremor, muscular rigidity, bradykinesia, poor
balance, and walking problems. Secondary symptoms include
depression, sleep disturbances, dizziness, stooped posture,
constipation, dementia, and problems with speech, breathing,
swallowing, and sexual function symptoms.
[0008] Amyotrophic lateral sclerosis ("ALS"), also known as Lou
Gehrig's disease or Charcot syndrome, is a progressive motor neuron
disease characterized by both upper and lower motor neuron damage.
The disease is characterized by degeneration of motor neurons in
the cortex, brainstem and spinal cord (Principles of Internal
Medicine, 1991 McGraw-Hill, Inc., New York; Tandan et al. Ann.
Neurol., 18:271-280, 419-431, 1985). Generally, the onset is
between the third and sixth decade, typically in the sixth decade.
Symptoms may include tripping, stumbling and falling, loss of
muscle control and strength in hands and arms, difficulty speaking,
swallowing and/or breathing, chronic fatigue, and muscle twitching
and/or cramping. Symptoms of upper motor neuron damage include
stiffness (spasticity), muscle twitching (fasciculations), and
muscle shaking (clonus). Symptoms of lower motor neuron damage
include muscle weakness and muscle shrinking (atrophy).
[0009] The use of certain chroman-ylmethylamino derivatives for the
treatment of Parkinson's disease and epilepsy has been disclosed in
U.S. Pat. Nos. 5,663,294; 5,541,199; 5,670,667; 5,684,039;
5,756,521; 6,235,774; and 6,331,561. The use of chromans for
treating mitochondria associated diseases including Alzheimer's
disease, diabetes mellitus, Parkinson's disease, neuronal and
cardiac ischemia, Huntington's disease, and stroke is disclosed in
U.S. Pat. Nos. 6,498,191 and 6,511,966 and U.S. patent application
US 2003/0176448. Triphenyl phosphonium tocopherol analogs having
cardioprotective or mitochondrially targeted antioxidant properties
have been described by Gisar, J M in EP 545,283 and by Murphy, M.
in Annals of the New York Academy of Sciences (2002), 959, 263-274
and in U.S. Pat. No. 6,331,532, US 2202/00523242 and US
2003/0069208.
[0010] The use of antioxidants targeted to mitochondria shown to be
effective at slowing disease progression has been reported by
Jauslin, M L in FASEB Journal, express article
10.1096/fj.03-0240fje. Therapeutic benefit of administering
.gamma.-tocopherol derivatives and metabolites as antioxidants and
nitrogen oxide scavengers which treat high blood pressure,
thromboembolic diseases, cardiovascular disease, cancer,
natriuretic disease, formation of neuropathological lesion and
reduced immune system response are disclosed in U.S. Pat. Nos.
6,555,575; 6,242,479; 6,150,402; and 6,410,589. The use of certain
chroman derivatives in cosmetic and dermatological preparations is
disclosed in US 2002/0127252. Beneficial effects of Vitamin E in
the progression of a number of major degenerative diseases of the
nervous system is examined in Fryer, Nutritional Neuroscience,
(1998) Vol.1, 327-351.
[0011] The present invention addresses the desire to provide new
therapies for conditions characterized by mitochondrial
dysfunction. The present invention particularly addresses the
method of treating or ameliorating the symptoms of Friedreich's
ataxia, a condition affecting young children, for which there is no
effective cure or treatment. Most children afflicted by this
debilitating condition, Friedreich's ataxia, die in or before early
adulthood.
SUMMARY OF THE INVENTION
[0012] It has surprisingly been found that certain compounds limit
or prevent damage to organelles, cells, and tissues caused by
mitochondrial dysfunction, oxidative stress or neuroinflammation,
as demonstrated by providing protection in standard experimental
models of mitochondrial dysfunction caused by MPP.sup.+/MPTP
(1-methyl-4-phenylpyridinium/1-methy-
l-4-phenyl-1,2,3,4-tetrahydropyridine) or of oxidative stress
caused by beta amyloid or high glutamate. These compounds show
protection in the experimental model using FRDA fibroplasts and may
be used for the treatment of Friedreich's disease and other
ataxias, Leber's hereditary optic neuropathy (LHON), mitochondrial
myopathy, encephalopathy, lactacidosis, stroke (MELAS), Myoclonic
Epilepsy with Ragged Red Fibers (MERFF), macular degeneration,
Down's syndrome, Creutzfeldt-Jakob syndrome.
[0013] In a first aspect, the present invention relates to the
method of treating or ameliorating a subject suffering from a
mitochondrial disorder comprising administering to said subject a
therapeutically effective amount of a compound of Formula I or
Formula II: 1
[0014] wherein:
[0015] -A-B-- is --CH.sub.2--CH.sub.2--; --CH.dbd.CH-- or
--CH.sub.2--O--;
[0016] n is 0;
[0017] R.sup.1 is C.sub.1-.sub.4 alkyl;
[0018] R.sup.2 is C.sub.1-20 alkyl or C.sub.2-20 alkenyl, halogen,
cyano, or R.sup.2 and A together with the atoms to which they are
attached form a ring;
[0019] R.sup.3 is
[0020] hydrogen;
[0021] halogen;
[0022] aralkyl, optionally substituted with one or more
substituents selected from alkyl, haloalkyl, hydroxy, alkoxy,
halogen, oxo, cyano, nitro, amino, --SO.sub.2NR.sub.2 or
--C(O)OR;
[0023] heteroaralkyl optionally substituted with one or more
substituents selected from alkyl, haloalkyl, hydroxy, alkoxy,
halogen, oxo, cyano, nitro, amino, --SO.sub.2NR.sub.2 or
--C(O)OR;
[0024] C.sub.1-.sub.6 alkyl;
[0025] C.sub.2-.sub.20 alkenyl;
[0026]
--(CR.sub.2).sub.1-3S(O).sub.0-2(CR.sub.2).sub.1-3C(O)OR;
[0027] --(CR.sub.2).sub.1-3OR.sup.a;
[0028] --(CR.sub.2).sub.1-3NR.sup.bR.sup.c
[0029] --C(O)R.sup.a; or
[0030] nitro;
[0031] R.sup.4 is hydrogen; optionally substituted C.sub.1-.sub.6
alkyl; C.sub.2-.sub.12 alkenyl; hydroxyalkyl; acyl; glucoside;
phosphoryl; phosphoryloxyalkyl; carboxyalkylcarbonyl; or
aminoalkylcarbonyl;
[0032] R.sup.5 and R.sup.6 are independently of each other
hydrogen, halogen, haloalkyl, nitro, acyl, C.sub.1-.sub.6 alkyl or
C.sub.2-.sup.12alkenyl; or R.sup.5 and R.sup.6 taken together with
the carbon to which they are attached form a 5-6 membered
aliphatic, unsaturated or aromatic ring, optionally substituted
with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy, carboxy,
carboxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, aminocarbonyl,
aminocarbonylalkyl, or hydroxyalkyl;
[0033] R is hydrogen or C.sub.1-.sub.6 alkyl;
[0034] R.sup.a is hydrogen; optionally substituted C.sub.1-.sub.6
alkyl; optionally substituted C.sub.2-.sub.12 alkenyl; optionally
substituted aryl; optionally substituted cycloalkyl; or optionally
substituted saturated, partially unsaturated or unsaturated
heterocyclyl;
[0035] R.sup.band R.sup.c are independently of each other hydrogen;
C.sub.1-.sub.6 alkyl; hydroxyalkyl; aminoalkyl; optionally
substituted aryl; optionally substituted benzyl; or optionally
substituted heterocyclyl; or R.sup.b and R.sup.c taken together
with the atom to which they are attached may form a 5 to 8 membered
aromatic, saturated or unsaturated ring, optionally incorporating
one additional atom chosen from N, O, or S and optionally
substituted with a substituent selected from the group consisting
of lower alkyl, halo, cyano, alkylthio, lower alkoxy, oxo, phenyl,
benzyl and carboxy;
[0036] with the proviso that the compound is not
alpha-tocopherol;
[0037] or 2
[0038] wherein:
[0039] G is --O--, --S--, --SO--, --SO.sub.2--, a secondary or
tertiary amine group, a phosphate group, a phosphoester group, or
an unsubstituted or substituted methylene group,
[0040] R.sup.7 and R.sup.8 independently are hydrogen, hydroxy,
alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester,
unsubstituted or substituted amine, amide, halogen or unsubstituted
or substituted sulfonyl, or jointly complete a 5- or 6-member
aliphatic or aromatic ring,
[0041] R.sup.9 and R.sup.10 independently are hydrogen, hydroxy,
alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester,
unsubstituted or substituted amine, amide, halogen or unsubstituted
or substituted sulfonyl, or jointly complete a 5- or 6-member
aliphatic, aromatic or heterocyclic ring,
[0042] R.sup.11 is hydrogen, hydroxy, alkyl, aryl, alkenyl,
alkynyl, aromatic, ester or unsubstituted or substituted amine,
[0043] R.sup.12 is --COOH, --COOR.sup.13, --CONH.sub.2,
--CONHR.sup.13 , CONR.sup.13R.sup.14, --NH.sub.2, --NHR.sup.13,
--NR.sup.13R.sup.14, or a carboxylate salt,
[0044] R.sup.13 and R.sup.14 independently are unsubstituted or
substituted alkyl, aryl, alkaryl, aralkyl, alkenyl or alkynyl,
[0045] p is 0 to 3, and
[0046] m is 0 to 5;
[0047] or
[0048] single stereoisomers, mixtures of stereoisomers, or
pharmaceutically acceptable salts thereof.
[0049] In a preferred embodiment the method of treating or
ameliorating a subject suffering from a mitochondrial disorder
comprises administering to said subject a therapeutically effective
amount of a compound of Formula I; in another preferred embodiment
the method comprises administering a compound of Formula I wherein
R.sup.3 is hydrogen, C.sub.1-6 alkyl, or C.sub.2-20 alkenyl and in
another embodiment the the method comprises administering a
compound of Formula I wherein R.sup.5 and R.sup.6are C.sub.1-4
alkyl or halogen, preferably wherein R.sup.5 and R.sup.6 are
C.sub.1-4 alkyl or halogen and R.sup.1 is C.sub.1-6 alkyl and
R.sup.2 is C.sub.1-20 alkyl. In another embodiment, the method
comprises administering a compound of Formula I wherein R.sup.1 is
C.sub.1-6 alkyl and R.sub.2 is C.sub.16 alkyl or C.sub.16 alkenyl,
and in yet another embodiment the method comprises administering a
compound of Formula I wherein R.sup.5 and R.sup.6 taken together
with the carbon to which they are attached form a 5-6 membered
carbocyclic ring, which may be optionally substituted with
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy, carboxy, carboxyalkyl,
alkoxycarbonyl, alkoxycarbonylalkyl, aminocarbonyl,
aminocarbonylalkyl, or hydroxyalkyl.
[0050] In another preferred embodiment the method of treating or
ameliorating a subject suffering from a mitochondrial disorder
comprises administering to said subject a therapeutically effective
amount of a compound of Formula II; and in another preferred
embodiment the method comprises administering a compound of Formula
II wherein R.sup.12 is --COOH or --COOR.sup.13 and R.sup.13 is
C.sub.1-6 alkyl.
[0051] In a preferred embodiment, the invention relates to a method
of treating or ameliorating a subject suffering from disorders
including but not limited to a disease involving mitochondrial
disorders selected from Alzheimer's disease, Parkinson's disease,
Friedreich's ataxia (FRDA), cerebellar ataxias, Leber's hereditary
optic neuropathy (LHON), mitochondrial myopathy, encephalopathy,
lactacidosis, stroke (MELAS), Myoclonic Epilepsy with Ragged Red
Fibers (MERFF), amyotrophic lateral sclerosis (ALS), motor neuron
diseases, Huntington's disease, macular degeneration, and epilepsy
by administering to said subject a therapeutically effective amount
of a compound of Formula I or Formula II or a pharmaceutically
acceptable salt thereof. In a more preferred embodiment the
invention relates to a method of treating or ameliorating a subject
suffering from Friedreich's ataxia, MELAS, or MERFF by
administering to said subject a therapeutically effective amount of
a compound of Formula I or Formula II.
[0052] In another preferred embodiment the treatment comprises
protecting or ameliorating a subject in need of protection of
Friedreich's ataxia comprising administering a therapeutically
effective amount of a tocopherol selected from beta-tocopherol,
delta-tocopherol, gamma-tocopherol, alpha-tocotrienol,
beta-tocotrienol, delta-tocotrienol, or gamma-tocotrienol.
[0053] Particularly preferred are those methods of treatment and
uses in the manufacture of pharmaceutical compositions therefor,
wherein the compound of Formula I or Formula II is selected from
the preferred compounds, and particularly from the compounds
selected from:
[0054] 2,2,7,8-Tetramethyl-2H-chromen-6-ol;
[0055]
3-(6-Hydroxy-2-methyl-3,4-dihydro-2H-benzo[h]chromen-2-yl)-propioni-
c acid;
[0056] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid methyl ester;
[0057]
2,7,8-Trimethyl-5-morpholin4-ylmethyl-2-(4,8,12-trimethyl-tridecyl)-
-chroman-6-ol;
[0058]
5-(4-Benzyl-piperazin-1-ylmethyl)-2,7,8-trimethyl-2-(4,8,12-trimeth-
yl-tridecyl)-chroman-6-ol;
[0059] 3-(7,8-Dihydroxy-2-methyl-chroman-2-yl)-propionic acid;
[0060]
5-[3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propyl]-thiazolidine--
2,4-dione;
[0061] 2-Hydroxymethyl-2,5,7,8-tetramethyl-chroman-6-ol;
[0062]
3-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-2-meth-
yl-propionic acid;
[0063]
2-Hydroxymethyl-6-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethox-
y)-tetrahydro-pyran-3,4,5-triol;
[0064] 3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid;
[0065] 3-(6-Hydroxy-2-methyl-chroman-2-yl)-propionic acid methyl
ester;
[0066] 3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic acid
methyl ester;
[0067]
3-[8-(2-Methoxycarbonyl-ethyl)-3,5,6,8-tetramethyl-1,2,3,8,9,10-hex-
ahydro-pyrano[3,2-f]chromen-3-yl]-propionic acid methyl ester;
[0068]
3-[8-(2-Carboxy-ethyl)-3,5,6,8-tetramethyl-1,2,3,8,9,10-hexahydro-p-
yrano[3,2-f]chromen-3-yl]-propionic acid;
[0069] 3-(6-Hydroxy-2-methyl-chroman-2-yl)-propionic acid;
[0070] 3-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-propionic
acid;
[0071] 3-(2,5,7,8-Tetramethyl-chroman-2-yl)-propionic acid;
[0072] 3-(6-Hydroxy-2,7,8-trimethyl-5-nitro-chroman-2-yl)-propionic
acid;
[0073] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid;
[0074]
3-[6-Hydroxy-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman-2-yl]--
propionic acid;
[0075] 3-(6-Hydroxy-2,8-dimethyl-chroman-2-yl)-propionic acid;
[0076] 2,2,7,8-Tetramethyl-chroman-6-ol;
[0077]
2,2,7,8-Tetramethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol;
[0078] 2,2,7,8-Tetramethyl-5-(3-methyl-butyl)-chroman-6-ol;
[0079]
(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-acetic
acid methyl ester;
[0080] 2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
[0081] 2-(3-Chloro-propyl)-2,7,8-trimethyl-chroman-6-ol;
[0082] 2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
[0083]
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman--
6-ol;
[0084]
1-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethyl)-pyrrolidine-2--
carboxylic acid methyl ester;
[0085] 2,2,5,8-Tetramethyl-chroman-6-ol;
[0086] 7-Bromo-2,2,5,8-tetramethyl-chroman-6-ol;
[0087] 2,2,7,8-Tetramethyl-5-(3-methyl-pentyl)-chroman-6-ol;
[0088] 2,2,5,8-Tetramethyl-7-(3-methyl-butyl)-chroman-6-ol;
[0089] 5-Isopropoxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
[0090] 5-Hexyloxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
[0091] 5-tert-Butoxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
[0092]
3-(5-Bromo-6-hydroxy-2-methyl-3,4-dihydro-2H-benzo[h]chromen-2-yl)--
propionic acid
[0093] 2-(2-Hydroxy-ethyl)-2,7,8-trimethyl-chroman-6-ol;
[0094] Disodium salt of Phosphoric acid
mono-[2,2,7,8-tetramethyl-5-(3-met-
hyl-butyl)-chroman-6-yloxymethyl]ester;
[0095]
2-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethyl)-propane-1,3-di-
ol;
[0096] 6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carbonitrile;
[0097] 2,2,5,7,8-Pentamethyl-2H-chromen-6-ol;
[0098] 2-Ethynyl-2,5,7,8-tetramethyl-chroman-6-ol;
[0099]
10-Methoxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ol;
[0100] 5-(2-Hydroxy-ethyl)-2,2,7,8-tetramethyl-chroman-6-ol;
[0101]
(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-morpholin-4-yl-methano-
ne;
[0102] 5-(1-Hydroxy-ethyl)-2,2,7,8-tetramethyl-chroman-6-ol;
[0103] 5-Hydroxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
[0104] 2,2,5,7-Tetramethyl-8-nitro-chroman-6-ol;
[0105] 6-Hydroxy-2,2,5,7-tetramethyl-chroman-8-carbaldehyde;
[0106] 2,2,7,8-Tetramethyl-5-nitro-chroman-6-ol;
[0107]
2,2,5,7-Tetramethyl-8-(3-methyl-but-2-enyl)-chroman-6-ol;
[0108] 8-Fluoro-2,2,5,7-tetramethyl-chroman-6-ol;
[0109] 2,2,5,7-Tetramethyl-8-(3-methyl-butyl-chroman-6-ol;
[0110] 5-Methoxy-2,2,7,8-tetramethyl-chroman-6-ol;
[0111] 8-tert-Butyl-2,2,5-trimethyl-chroman-6-ol;
[0112] 2,2,5,7-Tetramethyl-8-trifluoromethyl-chroman-6-ol;
[0113] Acetic acid
2,2,5-trimethyl-3,4-dihydro-2H-benzo[h]chromen-6-yl ester;
[0114] 7-tert-Butyl-2-(3-chloro-propyl)-2-methyl-chroman-6-ol;
[0115]
2-(3-Chloro-propyl)-8-isopropyl-2,5-dimethyl-chroman-6-ol;
[0116] 5-Chloro-2-(3-chloro-propyl)-2,8-dimethyl-chroman-6-ol;
[0117] 8-Chloro-2-(3-chloro-propyl)-2,5-dimethyl-chroman-6-ol;
[0118] 3-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-propionic
acid methyl ester;
[0119]
2,5,8-trimethyl-2-(4,8,12-trimethyltridecyl)-6-chromanol;
[0120]
2,7,8-trimethyl-2-(4,8,12-trimethyltridecyl)-6-chromanol;
[0121] 2,8-Dimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol;
[0122]
2,5,7,8-Tetramethyl-2-(4,8,12-trimethyl-trideca-3,7,11-trienyl)-chr-
oman-6-ol;
[0123]
2,5,8-trimethyl-2-(4,8,12-trimethyl-trideca-3,7,11-trienyl)-chroman-
-6-ol;
[0124]
2,7,8-trimethyl-2-(4,8,12-trimethyl-trideca-3,7,11-trienyl)-chroman-
-6-ol;
[0125]
2,8-dimethyl-2-(4,8,12-trimethyl-trideca-3,7,11-trienyl)-chroman-6--
ol;
[0126] or single stereoisomers, mixtures of stereoisomers, or
pharmaceutically acceptable salts thereof.
[0127] Certain embodiments of the invention provide novel and
preferred combinations of substituent groups pendant from the
formulae of the different inventions
DETAILED DESCRIPTION OF THE INVENTION
[0128] Definitions
[0129] As used in the present specification, the following words
and phrases are generally intended to have the meanings as set
forth below, except to the extent that the context in which they
are used indicates otherwise.
[0130] The term "optional" or "optionally" means that the
subsequently described event or circumstance may or may not occur,
and that the description includes instances where said event or
circumstance occurs and instances in which it does not. For
example, "optionally substituted alkyl" means either "alkyl" or
"substituted alkyl," as defined below.
[0131] It will be understood by those skilled in the art with
respect to any group containing one or more substituents that such
groups are not intended to introduce any substitution or
substitution patterns that are sterically impractical and/or
physically non-feasible.
[0132] The term "acyl" refers to the groups --C(O)--H,
--C(O)-(optionally substituted alkyl), --C(O)-(optionally
substituted cycloalkyl), --C(O)-(optionally substituted alkenyl),
--C(O)-(optionally substituted cycloalkenyl), --C(O)-(optionally
substituted aryl), and --C(O)-(optionally substituted
heterocyclyl).
[0133] The term "alkenyl" refers to a monoradical branched or
unbranched, unsaturated or polyunsaturated hydrocarbon chain,
having from about 2 to 20 carbon atoms, more preferably about 2 to
10 carbon atoms. This term is exemplified by groups such as
ethenyl, but-2-enyl, 3-methyl-but-2-enyl (also referred to as
"prenyl", octa-2,6-dienyl, 3,7-dimethyl-octa-2,6-die- nyl (also
referred to as "geranyl"), and the like.
[0134] The term "alkoxy" refers to the groups --O-alkyl,
--O-alkenyl, --O-cycloalkyl, --O-cycloalkenyl, and --O-alkynyl.
Preferred alkoxy groups are --O-alkyl and include, by way of
example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,
tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy,
and the like.
[0135] The term "alkyl" refers to a monoradical branched or
unbranched saturated hydrocarbon chain preferably having from about
1 to 20 carbon atoms, more preferably about 1 to 10 carbon atoms,
and even more preferably about 1 to 6 carbon atoms. The term
"alkyl" also means a combination of linear or branched and cyclic
saturated hydrocarbon radical consisting solely of carbon and
hydrogen atoms. This term is exemplified by groups such as methyl,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl,
tetradecyl, and the like. The term "lower alkyl refers to a
monoradical branched or unbranched saturated hydrocarbon chain of 1
to 6 atoms.
[0136] The term "substituted alkyl" refers to an alkyl group in
which 1 or more (up to about 5, preferably up to about 3) hydrogen
atoms is replaced by a substituent independently selected from the
group: .dbd.O, .dbd.S, acyl, acyloxy, optionally substituted
alkoxy, optionally substituted amino (wherein the amino group may
be a cyclic amine), azido, carboxyl, (optionally substituted
alkoxy)carbonyl, (optionally substituted amino)carbonyl, cyano,
optionally substituted cycloalkyl, optionally substituted
cycloalkenyl, halogen, hydroxyl, nitro, sulfamoyl, sulfanyl,
sulfinyl, sulfonyl, and sulfonic acid. One of the preferred
optional substituents for alkyl is hydroxy, exemplified by
hydroxyalkyl groups, such as 2-hydroxyethyl, 3-hydroxypropyl,
3-hydroxybutyl, 4-hydroxybutyl, and the like; dihydroxyalkyl groups
(glycols), such as 2,3-dihydroxypropyl, 3,4-dihydroxybutyl,
2,4-dihydroxybutyl, and the like; aminoalkyl groups such as
dimethyl aminoalkyl, piperidinylalkyl, morpholinylalkyl, and those
compounds known as polyethylene glycols, polypropylene glycols and
polybutylene glycols, and the like. Another preferred optional
substituent for alkyl is sulfanyl exemplified by allylsulfanyl,
carboxypropylsulfanyl, 2-methyl-propionyl-pyrrolidine-2-ca-
rboxylic acid, 5-methyl-1-H-benzimidazol-2-yl-sulfanyl,
sulfoxyethylsulfanyl, 4,6-dimethyl-pyrimidin-2-ylsulfanyl, 4
carboxy-benzyl-sulfanyl, isobutylsulfanyl, and the like. Other
preferred optional substituents for alkyl are --N-hydroxyureidyl,
--N-hydroxythioureidyl or --N-hydroxyacetamide.
[0137] The term "amino" refers to the group --NH.sub.2 as well as
to the groups --NHR or --NRR where each R is independently selected
from the group: optionally substituted alkyl, optionally
substituted cycloalkyl, optionally substituted alkenyl, optionally
substituted cycloalkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted heterocyclyl,
acyl, optionally substituted alkoxy, carboxy and alkoxycarbonyl,
and where --NRR may be a cyclic amine.
[0138] The term "amino acid" or "natural amino acid" refers to any
of the twenty (20) common amino acids as generally accepted in the
peptide art.
[0139] The term "aralkyl" refers to the moiety "-alkylene-aryl"
each having the meaning as defined herein.
[0140] The term "aryl" refers to an aromatic cyclic hydrocarbon
group of from 6 to 20 carbon atoms having a single ring (e.g.,
phenyl) or multiple condensed (fused) rings (e.g., naphthyl or
anthryl). Preferred aryls include phenyl, naphthyl and the
like.
[0141] The term "substituted aryl" refers to an aryl group as
defined above, which unless otherwise constrained by the definition
for the aryl substituent, is substituted with from 1 to 5
substituents, and preferably 1 to 3 substituents, independently
selected from the group consisting of: hydroxy, thiol, acyl,
acyloxy, optionally substituted alkenyl, optionally substituted
alkoxy, optionally substituted alkyl (such as tri-halomethyl),
optionally substituted alkynyl, optionally substituted amino,
optionally substituted aryl, optionally substituted aryloxy, azido,
carboxyl, (optionally substituted alkoxy)carbonyl, (optionally
substituted amino)carbonyl, cyano, optionally substituted
cycloalkyl, optionally substituted cycloalkenyl, halogen,
optionally substituted heterocyclyl, optionally substituted
heterocyclooxy, hydroxyl, nitro, sulfanyl, sulfinyl, sulfanyl, and
sulfonic acid. Preferred aryl substituents include alkyl, alkenyl,
alkoxy, halo, cyano, nitro, trihalomethyl, carboxy, amino, amido,
sulfonamido, and sulfinyl.
[0142] The term "carbonyl" refers to the di-radical
"--C(.dbd.O)--", which is also illustrated as "--C(O)--".
[0143] The term "(optionally substituted amino)carbonyl" refers to
the group --C(O)-(optionally substituted amino). This moiety is
also referred to as a primary, secondary or tertiary
carboxamide.
[0144] The term "carboxy" or "carboxyl" refers to the moiety
"--C(O)OH", which is also illustrated as "--COOH".
[0145] The term "cycloalkyl" refers to non-aromatic cyclic
hydrocarbon groups of having about 3 to 40 (preferably about 4 to
15) carbon atoms having a single ring or multiple condensed or
bridged rings. Such cycloalkyl groups include, by way of example,
single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclooctyl, and the like, or multiple ring structures
such as adamantanyl, and the like. The term "cycloalkyl"
additionally encompasses spiro systems wherein the cycloalkyl ring
has a carbon ring atom in common with another ring.
[0146] The term "halo" or "halogen" refers to fluoro, chloro, bromo
and iodo.
[0147] The terms "heterocycle", "heterocyclic", "heterocyclo", and
"heterocyclyl" refer to a monovalent, saturated, partially
unsaturated or unsaturated (aromatic), carbocyclic radical having
one or more rings incorporating one, two, three or four heteroatoms
within the ring (chosen from nitrogen, oxygen, and/or sulfur).
Preferred heterocycles include morpholine, piperidine, piperazine,
thiazole, thiazolidine, isothiazole, oxazole, isoxazole, pyrazole,
pyrazolidine, pyrazoline, imidazole, imidazolidine, benzothiazole,
pyridine, pyrazine, pyrimidine, pyridazine, pyrrole, pyrrolidine,
quinoline, quinazoline, purine, carbazole, benzimidazole,
pyrimidine, thiophene, benzothiophene, pyran, tetrahydropyran,
benzopyran, furan, tetrahydrofuran, indole, indoline, indazole,
xanthene, thioxanthene, acridine, quinuclidine, and the like.
[0148] The terms "substituted heterocycle", "substituted
heterocyclic", "substituted heterocyclo" and "substituted
heterocyclyl" refer to a heterocycle group as defined above, which
unless otherwise constrained by the definition for the heterocycle,
is substituted with from 1 to 5 substituents, and preferably 1 to 3
substituents, independently selected from the group consisting of:
hydroxy, thiol, acyl, acyloxy, optionally substituted alkenyl,
optionally substituted alkoxy, optionally substituted alkyl (such
as tri-halomethyl), optionally substituted alkynyl, optionally
substituted amino, optionally substituted aryl, optionally
substituted aryloxy, azido, carboxyl, (optionally substituted
alkoxy)carbonyl, (optionally substituted amino)carbonyl, cyano,
optionally substituted cycloalkyl, optionally substituted
cycloalkenyl, halogen, optionally substituted heterocyclyl,
optionally substituted heterocyclooxy, hydroxyl, nitro, sulfanyl,
sulfinyl, and sulfonic acid. Preferred substituted heterocycles
include thiazolidine-2,4-dione and
3-methyl-5-oxo-4,5-dihydro-1H-pyrazol.
[0149] The term "heterocycloalkyl" refers to the moiety
"-alkylene-heterocycle" each having the meaning as defined
herein.
[0150] The term "substituted heterocycloalkyl" refers to the
moiety" -(optionally substituted alkylene)-(optionally substituted
heterocycle)", each having the meaning as defined herein.
[0151] The term "mitochondrial diseases or disorders" of which
hundreds of varieties have been identified--can cause a complex
variety of symptoms. These include muscle weakness, muscle cramps,
seizures, food reflux, learning disabilities, deafness, short
stature, paralysis of eye muscles, diabetes, cardiac problems and
stroke--like episodes, to name a few. The symptoms can range in
severity from life-threatening to almost unnoticeable, sometimes
taking both extremes in members of the same family. Because some
people have specific subsets of these symptoms, clinical
researchers have grouped those that occur together into
"syndromes," producing a bewildering array of descriptive acronyms
such as MELAS (mitochondrial encephalomyopathy with lactic acidosis
and stroke-like episodes) or MERFF (myoclonus epilepsy with ragged
red fibers). This term also includes disorders such as Kearns-Sayre
syndrome (KSS), Leigh's syndrome, maternally inherited Leigh's
syndrome (MILS), Myogastrointestinal encephalomyopathy (MNGIE),
Neuropathy, ataxia and retinitis pigmentosa (NARP), Friedreich's
ataxia (FRDA), amyotrophic lateral sclerosis (ALS) and other motor
neuron diseases, Huntington's disease, macular degeneration,
epilepsy, Alzheimer's, Leber's hereditary optic neuropathy (LHON),
Progressive external ophthalmoplegia (PEO), and Pearson
syndrome.
[0152] The term "neurodegenerative disorders" refers to disorders
characterized by a loss of neurons and may or may not include an
inflammatory process. Neurodegenerative disorders include stroke,
head trauma, cerebral hypoxia, spinal cord injury, senile dementia,
Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and other
motor neuron diseases, cerebral amyloid angiopathy, HIV-related
dementia, Parkinson's disease, Huntington's disease, prion
diseases, myasthenia gravis, Down's syndrome, Creutzfeldt-Jakob
disease, Friedreich's ataxia (FRDA), Fergusson and Critchley's
ataxia and other ataxias, Leber's hereditary optic neuropathy
diabetic neuropathy (LHON), neuropathic pain, encephalitis,
meningitis, and Duchenne's muscular dystrophy.
[0153] The term "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable excipient" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0154] The term "pharmaceutically acceptable salt" refers to salts
which retain the biological effectiveness and properties of the
compounds of this invention and which are not biologically or
otherwise undesirable. In many cases, the compounds of this
invention are capable of forming acid and/or base salts by virtue
of the presence of amino and/or carboxyl groups or groups similar
thereto. Pharmaceutically acceptable base addition salts can be
prepared from inorganic and organic bases. Salts derived from
inorganic bases, include by way of example only, sodium, potassium,
lithium, ammonium, calcium and magnesium salts. Salts derived from
organic bases include, but are not limited to, salts of primary,
secondary and tertiary amines, such as alkyl amines, dialkyl
amines, trialkyl amines, substituted alkyl amines, di(substituted
alkyl) amines, tri(substituted alkyl) amines, alkenyl amines,
dialkenyl amines, trialkenyl amines, substituted alkenyl amines,
di(substituted alkenyl) amines, tri(substituted alkenyl) amines,
cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,
substituted cycloalkyl amines, disubstituted cycloalkyl amine,
trisubstituted cycloalkyl amines, cycloalkenyl amines,
di(cycloalkenyl) amines, tri(cycloalkenyl) amines, substituted
cycloalkenyl amines, disubstituted cycloalkenyl amine,
trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,
triaryl amines, heterocyclic amines, diheterocyclic amines,
triheterocyclic amines, mixed di- and tri-amines where at least two
of the substituents on the amine are different and are selected
from the group consisting of alkyl, substituted alkyl, alkenyl,
substituted alkenyl, cycloalkyl, substituted cycloalkyl,
cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic, and the
like. Also included are amines where the two or three substituents,
together with the amino nitrogen, form a heterocyclic group.
[0155] Specific examples of suitable amines include, by way of
example only, isopropylamine, trimethyl amine, diethyl amine,
tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine,
2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine,
caffeine, procaine, choline, betaine, ethylenediamine, glucosamine,
N-alkylglucamines, theobromine, purines, piperazine, piperidine,
morpholine, N-ethylpiperidine, and the like.
[0156] Pharmaceutically acceptable acid addition salts may be
prepared from inorganic and organic acids. Salts derived from
inorganic acids include hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like. Salts
derived from organic acids include acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid,
and the like.
[0157] The term "seizure" is used herein as meaning the physical
affliction characterized by transient clouding of consciousness,
generally associated with a disturbance in the electrical activity
of the cortex of the brain. Illustrative of such seizures are those
associated with petit mal epilepsy.
[0158] The term "therapeutically effective amount" refers to that
amount of a compound of this invention that is sufficient to effect
treatment, as defined below, when administered to a mammal in need
of such treatment. The therapeutically effective amount will vary
depending upon the subject and disease condition being treated, the
weight and age of the subject, the severity of the disease
condition, the particular compound chosen, the dosing regimen to be
followed, timing of administration, the manner of administration
and the like, all of which can readily be determined by one of
ordinary skill in the art.
[0159] The term "tocopherol" means any of a family of molecules
(including both tocopherols and tocotrienols and derivatives
therof) which are characterized by a chroman-6-ol ring structure
and a side chain at the 2-position. Tocopherols possess a
4',8',12'-trimethyltridecyl phytyl side chain and the trocotrienols
differ by the presence of double bonds at 3',7',11" positions of
the side chain. The chemical names for the tocopherols are:
[0160] beta-tocopherol:
2,5,8-trimethyl-2-(4,8,12-trimethyltridecyl)-6-chr- omanol;
[0161] gamma-tocopherol:
2,7,8-trimethyl-2-(4,8,12-trimethyltridecyl)-6-ch- romanol;
[0162] delta-tocopherol:
2,8-dimethyl-2-(4,8,12-trimethyl-tridecyl)-chroma- n-6-ol;
[0163] alpha-tocotrienol:
2,5,7,8-tetramethyl-2-(4,8,12-trimethyl-trideca--
3,7,11-trienyl)-chroman-6-ol
[0164] beta-tocotrienol:
2,5,8-trimethyl-2-(4,8,12-trimethyl-trideca-3,7,1-
1-trienyl)-chroman-6-ol;
[0165] gamma tocotrienol:
2,7,8-trimethyl-2-(4,8,12-trimethyl-trideca-3,7,-
11-trienyl)-chroman-6-ol;
[0166] delta tocotrienol:
2,8-dimethyl-2-(4,8,12-trimethyl-trideca-3,7,11--
trienyl)-chroman-6-ol.
[0167] The term "treatment" or "treating" means any treatment of a
disease or disorder in a mammal, including:
[0168] preventing or protecting against the disease or disorder,
that is, causing the clinical symptoms not to develop;
[0169] inhibiting the disease or disorder, that is, arresting or
suppressing the development of clinical symptoms; and/or
[0170] relieving the disease or disorder that is, causing the
regression of clinical symptoms.
[0171] It will be understood by those skilled in the art that in
human medicine, it is not always possible to distinguish between
"preventing" and "suppressing" since the ultimate inductive event
or events may be unknown, latent, or the patient is not ascertained
until well after the occurrence of the event or events. Therefore,
as used herein the term "prophylaxis" is intended as an element of
"treatment" to encompass both "preventing" and "suppressing" as
defined herein. The term "protection," as used herein, is meant to
include "prophylaxis."
[0172] Nomenclature
[0173] In general, the nomenclature used in this Application was
generated using or with the help of version 2.2 of the AUTONOM.TM.
naming package within the ChemOffice.RTM. version 7.0.3 suite of
programs by CambridgeSoft Corp (Cambridge, Mass.).
Synthesis of the Compounds of the Invention
[0174] Synthetic Reaction Parameters
[0175] The terms "solvent", "inert organic solvent" or "inert
solvent" mean a solvent inert under the conditions of the reaction
being described in conjunction therewith. Solvents employed in
synthesis of the compounds of the invention include, for example,
methanol ("MeOH"), acetone, water, acetonitrile, 1,4-dioxane,
dimethylformamide ("DMF"), benzene, toluene, tetrahydrofuran
("THF"), chloroform, methylene chloride (also named
dichloromethane( ("DCM"), diethyl ether, ethyl acetate ("EtOAc"),
pyridine and the like, as well as mixtures thereof. Unless
specified to the contrary, the solvents used in the reactions of
the present invention are inert organic solvents.
[0176] The term "q.s." means adding a quantity sufficient to
achieve a stated function, e.g., to bring a solution to the desired
volume (i.e., 100%).
[0177] Unless specified to the contrary, the reactions described
herein take place at atmospheric pressure within a temperature
range from 0.degree. C. to 110.degree. C. (preferably from
0.degree. C. to 25.degree. C.; most preferably at "room" or
"ambient" temperature ("RT"), e.g., 20.degree. C.). Further, unless
otherwise specified, the reaction times and conditions are intended
to be approximate, e.g., taking place at about atmospheric pressure
within a temperature range of about 0.degree. C. to about
110.degree. C. (preferably from about 0.degree. C. to about
25.degree. C.; most preferably at about "room" or "ambient"
temperature, e.g., approximately 20.degree. C.) over a period of
about 1 to about 10 hours (preferably about 5 hours).
[0178] Isolation and purification of the compounds and
intermediates described herein can be effected, if desired, by any
suitable separation or purification procedure such as, for example,
filtration, extraction, crystallization, column chromatography,
thin-layer chromatography or thick-layer chromatography, or a
combination of these procedures. Specific illustrations of suitable
separation and isolation procedures can be had by reference to the
examples herein below. However, other equivalent separation or
isolation procedures can also be used.
[0179] Some compounds of the present invention are available
commercially or can be synthesized as known in the art. Some
exemplary syntheses of compounds of this invention are also
described in Examples.
Preferred Compounds
[0180] The compounds of Formula I or Formula II encompass the
chroman derivatives of the invention as disclosed, and/or the
pharmaceutically acceptable salts of such compounds. In addition,
the compounds of this invention include the individual
stereochemical isomers and mixtures thereof, arising from the
selection of substituent groups. It will be understood by those
skilled in the art with respect to any group containing one or more
substituents that such groups are not intended to introduce any
substitution or substitution patterns that are sterically
impractical and/or synthetically non-feasible.
[0181] Preferred for the compounds, pharmaceutical formulations,
methods of manufacture and use of the present invention are the
following combinations and permutations of substituent groups of
Formula I and Formula II.
Utility, Testing and Administration
[0182] General Utility
[0183] Compound, compositions, formulations, and methods of the
present invention are useful for the treatment of disorders
characterized by defective mitochondrial activity. In particular,
compounds of the present invention can be used in the treatment of
diseases such as degenerative diseases of the brain
((Wernicke-Korsakoff disease, Kreuzfeldt-Jakob disease (KJD),
Hallervorden-Spatz disease, Schilder's disease, Alzheimer's
disease, senile dementia, Down's syndrome in middle age,
Abercrombie's disease, Prion diseases, Zellweger syndrome, Alper's
Syndrome), spinocerebellar degenerations (spinal ataxia, cerebellar
cortical degenerations, Friedreich's ataxia (FRDA) and other
ataxias), multiple system degenerations (Menzel, Dejerine-Thomas,
Shy-Drager, and Machado Joseph), systemic disorders (Refsum
disease, ataxia telangiectasia), epilepsy, mitochondrial disorders
(MELAS, MERFF, KSS, Leigh's, MILS, MNGIE, NARP, PEO, Pearson),
demyelinating core disorders (multiple sclerosis, acute transverse
myelitis), muscular atrophies (amyotrophic lateral sclerosis (ALS),
multiple sclerosis (MS), infantile spinal muscular atrophy,
Huntington's disease, spinobulbar atrophy (SBA), juvenile spinal
muscular atrophy, myasthenia gravis and other motor neuron
diseases), movement disorder (drug-induced Parkinsonism or
Parkinson's disease), retinopathy (Leber's hereditary optic
neuropathy, age-related macular degeneration (AMD), cataracts),
cerebral ischemia ("stroke" most often caused by thrombosis,
vasoconstriction and embolism), myocardial ischemia (including
chronic stable angina, angina pectoris, unstable angina and
Prinzmetal's angina, silent ischemia, reinfarction, reocclusion,
restenosis, myocardial infarction and other forms of heart
disease), diabetes, renal disease, pre-menstrual syndrome (PMS),
asthma, cardiopulmonary inflammatory disorders, chronic heart
failure, rheumatoid arthritis, muscle fatigue, irritable bowel
syndrome, inflammatory bowel disease, intermittent claudication and
for the preservation of allograft tissue for transplantation.
Certain compounds of the present invention are also useful in
treating conditions falling with the group of dermatologic
conditions, in particular prevention and protecting skin tissue
against age-related damage or damage resulting from insults such as
harmful ultraviolet (UV) radiation, stress and fatigue, and in the
treatment of contact dermatitis, skin irritation, skin
pigmentation, psoriasis, or acne.
[0184] Testing
[0185] This section describes how compositions incorporating
compositions of the present invention are selected, using in vitro
and/or in vivo animal models, for example, and used as therapeutic
interventions in the exemplary indications, i.e., stroke, epilepsy,
Parkinson's disease, Friedreich's ataxia, MELAS, macular
degeneration, ALS, and Alzheimer's disease.
[0186] MPTP/MPP.sup.+-induced neurodegeneration of dopaminergic
neurons is a well characterized model which is therefore widely
used to understand the pathogenesis of Parkinson's disease. The
compounds were tested on MPTP/MPP.sup.+ induced neuronal death in
vitro and in vivo as shown in the following examples.
[0187] In vitro evaluation of protection against mitochondrial
dysfunction is carried out using substantia nigra-derived
dopaminergic progenitor cell line as described in Son J H, et al J
W. (1999) J Neurosci, 19: 10-20, exposed to
1-methyl-4-phenylpyridinium (MPP.sup.+)
[0188] In vivo evaluation is carried out using mice that have been
treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a
neurotoxin. MPTP is metabolized by astrocytes into
1-methyl-4-phenylpyridinium (MPP.sup.+), a substrate for the
dopamine transporter which then selectively inhibits complex 1 of
the mitochondrial electron transport chain. This results in
depletion of ATP, the production of reactive oxygen species and,
consequently cell death. In a number of species, including humans,
non-primates and rodents, MPTP produces an irreversible and severe
parkinsonian syndrome which includes virtually all the clinical
features of the disease. The striking pathologic and clinical
similarities between idiopathic Parkinson's disease and
MPTP-induced Parkinsonism suggest that the two disorders share
common pathogenic mechanism.
[0189] A cellular assay using FRDA-patient derived fibroblasts (as
described by Jauslin, M L et al, Human Molecular Genetics 11;
3055-3063 (2002)); is used to determine the cell protecting effect
of the test compounds by analyzing survival of dermal fibroblasts
taken from FRDA patients and unaffected normal donors under
conditions of partial GSH depletion. Exposure of FRDA fibroblasts
to BSO (L-buthionine (S,R)-sulfoximine) under conditions of
restricted selenium causes depletion of cellular glutathione (GSH)
and severe plasma membrane damage leading to cell death.
Preincubation with the test compounds before the addition of BSO is
used to determine if they can protect FRDA cells from BSO-mediated
cell death.
[0190] Protection against redox stress can be further evaluated in
cell culture using high glutamate induced oxidative stress (HGOS)
in mouse dopaminergic cell lines. The cytotoxic effect of glutamate
is not due to excitotoxicity, as this cell line is devoid of
inotropic glutamate receptors. Rather, the glutamate-induced
toxicity of dopaminergic cells is associated with an inhibition of
cystine transport which subsequently leads to depletion of
intracellular glutathione (GSH) levels (Murphy T. H., et al. Neuron
2, 1547-1558, 1989), activation of neuronal 12-lipoxygenase (Li, Y.
et al., Neuron 19,453-463, 1997), increased ROS production (Tan S.
et al., J. Cell Biol. 141, 1423-1432, 1998) and elevated
intracellular Ca.sup.2+ (Li, Y. et al., see supra). Some molecules
were measured for their ability to protect such cells against
glutamate-induced stress and the assay is detailed in Examples.
[0191] In addition IL-1 elevation has been associated with many
neurodegenerative diseases. There is increasing evidence for a role
of IL-1 in Alzheimer's Disease (AD) (Mrak R E et al. Neurobiol
Aging 22(6):903-908, 2001). Elevated levels of IL-1.beta. have been
shown to surround amyloid plaques in the disease and recent genetic
studies have indicated that a polymorphism in IL-1.alpha. is linked
to an increased risk of AD (3-6 fold increase) (Griffin W S et al.,
J Leukoc Biol 72(2):233-238, 2002). This polymorphism has also been
correlated with rate of cognitive decline in AD patients (Murphy G
M et al., Neurology, 56(11)1595-1597, 2001). The risk of AD is
increased even further when the polymorphism in IL-1.alpha. is
found in combination with another polymorphism in IL-1.beta. (see
Griffin W S, supra), providing convincing evidence that these
cytokines play an important role in the pathology of the
disease.
[0192] This assay measures the release of IL-1.beta. from a mouse
microglial cell line following an inflammatory challenge with LPS
and interferon-gamma. The ability of test articles to inhibit
microglial cell activation and IL-1.beta. release is determined by
co-incubation of the test article with the inflammatory challenge.
Cytokine release is measured using a mouse IL-1.beta. ELISA and
cell toxicity is determined using Cell Tracker Green (a fluorescent
dye that measures cell viability).
[0193] Cerebral ischemic insults are modeled in animals by
occluding vessels to, or within, the cranium (Molinari, G. F.,
1986, in H. J. M. Barnett, et al., (Eds) Stroke: Pathophysiology,
Diagnosis and Management, Vol. 1, Churchill Livingstone, N.Y.). The
rat middle cerebral artery occlusion (MCAO) model is one of the
most widely used techniques to induce transient focal cerebral
ischemia approximating cerebral ischemic damage in humans, e.g.,
those who suffer from a stroke. The middle cerebral artery used as
the ischemic trigger in this model is the most affected vessel in
human stroke. The model also entails a period of reperfusion, which
typically occurs in human stroke victims. MCAO involving a two-hour
occlusion has been found to produce the maximum size of cortical
infarction obtainable without increased mortality at twenty-four
hours.
[0194] Further validation of efficacy in neuroprotection can be
assessed in functional tests, such as the grip strength test or the
rotorod test. Animals treated with compounds that show
neuroprotection maintain their pre-MCAO grip strength values after
MCAO, as compared to untreated animals, which showed a significant
reduction in grip strength, indicating loss of sensorimotor
function. Likewise, animals treated with compounds that show
neuroprotection also maintained their pre-MCAO rotorod activity
scores after MCAO, as compared to untreated animals, which showed a
significant reduction in rotorod scores, indicating loss of
sensorimotor function at higher brain levels.
[0195] Administration
[0196] The compounds of this invention are administered at a
therapeutically effective dosage, e.g., a dosage sufficient to
provide treatment or amelioration for the disease states previously
described. Administration of the compounds of the invention or the
pharmaceutically acceptable salts thereof can be via any of the
accepted modes of administration for agents that serve similar
utilities.
[0197] While human dosage levels have yet to be optimized for the
compounds of the invention, generally, a daily dose is from about
0.01 to 10.0 mg/kg of body weight, preferably about 0.1 to 5. mg/kg
of body weight, and most preferably about 0.3 to 1.0 mg/kg of body
weight. The amount of active compound administered will, of course,
be dependent on the subject and disease state being treated, the
severity of the affliction, the manner and schedule of
administration and the judgment of the prescribing physician.
[0198] In employing the compounds of this invention for treatment
or amelioration of the above conditions, any pharmaceutically
acceptable mode of administration can be used. The compounds of
this invention can be administered either alone or in combination
with other pharmaceutically acceptable excipients, including solid,
semi-solid, liquid or aerosol dosage forms, such as, for example,
tablets, capsules, powders, liquids, suspensions, suppositories,
aerosols or the like. The compounds of this invention can also be
administered in sustained or controlled release dosage forms,
including depot injections, osmotic pumps, pills, transdermal
(including electrotransport) patches, and the like, for the
prolonged administration of the compound at a predetermined rate,
preferably in unit dosage forms suitable for single administration
of precise dosages. The compositions will typically include a
conventional pharmaceutical carrier or excipient and a compound of
this invention or a pharmaceutically acceptable salt thereof. In
addition, these compositions may include other medicinal agents,
pharmaceutical agents, carriers, adjuvants, and the like,
including, but not limited to anticoagulants, blood clot
dissolvers, permeability enhancers and slow release
formulations.
[0199] Generally, depending on the intended mode of administration,
the pharmaceutically acceptable composition will contain about 0.1%
to 90%, preferably about 0.5% to 50%, by weight of a compound or
salt of Formulae I or II, the remainder being suitable
pharmaceutical excipients, carriers, etc.
[0200] One preferred manner of administration for the conditions
detailed above is oral, using a convenient daily dosage regimen
which can be adjusted according to the degree of affliction. For
such oral administration, a pharmaceutically acceptable, non-toxic
composition is formed by the incorporation of any of the normally
employed excipients, such as, for example, mannitol, lactose,
starch, magnesium stearate, sodium saccharine, talcum, cellulose,
sodium crosscarmellose, glucose, gelatin, sucrose, magnesium
carbonate, and the like. Such compositions take the form of
solutions, suspensions, tablets, dispersible tablets, pills,
capsules, powders, sustained release formulations and the like.
[0201] Preferably the compositions will take the form of a pill or
tablet and thus the composition will contain, along with the active
ingredient, a diluent such as lactose, sucrose, dicalcium
phosphate, or the like; a lubricant such as magnesium stearate or
the like; and a binder such as starch, gum acacia,
polyvinylpyrrolidine, gelatin, cellulose and derivatives thereof,
and the like.
[0202] Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc. an active
compound as defined above and optional pharmaceutical adjuvants in
a carrier, such as, for example, water, saline, aqueous dextrose,
glycerol, glycols, ethanol, and the like, to thereby form a
solution or suspension. If desired, the pharmaceutical composition
to be administered may also contain minor amounts of nontoxic
auxiliary substances such as wetting agents, emulsifying agents, or
solubilizing agents, pH buffering agents and the like, for example,
sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan
monolaurate, triethanolamine acetate, triethanolamine oleate, etc.
Actual methods of preparing such dosage forms are known, or will be
apparent, to those skilled in this art; for example, see
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 15th Edition, 1975. The composition or formulation to
be administered will, in any event, contain a quantity of the
active compound in an amount effective to alleviate the symptoms of
the subject being treated.
[0203] Dosage forms or compositions containing active ingredient in
the range of 0.005% to 95% with the balance made up from non-toxic
carrier may be prepared.
[0204] For oral administration, a pharmaceutically acceptable
non-toxic composition is formed by the incorporation of any of the
normally employed excipients, such as, for example pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, talcum,
cellulose derivatives, sodium crosscarmellose, glucose, sucrose,
magnesium carbonate, sodium saccharin, talcum and the like. Such
compositions take the form of solutions, suspensions, tablets,
capsules, powders, sustained release formulations and the like.
Such compositions may contain 0.01%-95% active ingredient,
preferably 0.1-50%.
[0205] For a solid dosage form, the solution or suspension, in for
example propylene carbonate, vegetable oils or triglycerides, is
preferably encapsulated in a gelatin capsule. Such diester
solutions, and the preparation and encapsulation thereof, are
disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545.
For a liquid dosage form, the solution, e.g. in a polyethylene
glycol, may be diluted with a sufficient quantity of a
pharmaceutically acceptable liquid carrier, e.g. water, to be
easily measured for administration.
[0206] Alternatively, liquid or semi-solid oral formulations may be
prepared by dissolving or dispersing the active compound or salt in
vegetable oils, glycols, triglycerides, propylene glycol esters
(e.g. propylene carbonate) and the like, and encapsulating these
solutions or suspensions in hard or soft gelatin capsule
shells.
[0207] Other useful formulations include those set forth in U.S.
Pat. No. Re. 28,819 and U.S. Pat. No. 4,358,603.
[0208] The formulation can be administered in a single unit dosage
form for continuous treatment or in a single unit dosage form ad
libitum when relief of symptoms is specifically required. For
example, the formulation may be administered as a bolus or as a
continuous intravenous infusion after onset of symptoms of stroke,
myocardial infarction or chronic heart failure.
[0209] Another preferred manner of administration is the topical
administration. "Topical administration" refers to application of
the present compositions by spreading, spraying, etc. onto the
surface of the skin. The typical amount applied may vary from about
0.1 mg of composition per square centimeter of skin to about 25 mg
of composition per square centimeter of skin. The compounds of the
present invention may be formulated for topical administration to
the epidermis as ointments, creams or lotions or as transdermal
patch. Formulations suitable for topical administration in the
mouth include lozenges, pastilles and mouthwashes.
[0210] Parenteral administration is generally characterized by
injection, either subcutaneously, intramuscularly or intravenously.
Injectables can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for solution or
suspension in liquid prior to injection, or as emulsions. Suitable
excipients are, for example, water, saline, dextrose, glycerol,
ethanol or the like. In addition, if desired, the pharmaceutical
compositions to be administered may also contain minor amounts of
non-toxic auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, solubility enhancers, and the like,
such as for example, sodium acetate, sorbitan monolaurate,
triethanolamine oleate, cyclodextrins, etc.
[0211] A more recently devised approach for parenteral
administration employs the implantation of a slow-release or
sustained-release system, such that a constant level of dosage is
maintained; see, e.g., U.S. Pat. No. 3,710,795. The percentage of
active compound contained in such parenteral compositions is highly
dependent on the specific nature thereof, as well as the activity
of the compound and the needs of the subject. However, percentages
of active ingredient of 0.01% to 10% in solution are employable,
and will be higher if the composition is a solid which will be
subsequently diluted to the above percentages. Preferably the
composition will comprise 0.2-2% of the active agent in
solution.
[0212] Nasal solutions of the active compound alone or in
combination with other pharmaceutically acceptable excipients can
also be administered.
[0213] Formulations of the active compound or a salt may also be
administered to the respiratory tract as an aerosol or solution for
a nebulizer, or as a microfine powder for insufflation, alone or in
combination with an inert carrier such as lactose. In such a case,
the particles of the formulation have diameters of less than 50
microns, preferably less than 10 microns.
EXAMPLES
[0214] The following preparations and examples are given to enable
those skilled in the art to more clearly understand and to practice
the present invention. They should not be considered as limiting
the scope of the invention, but merely as being illustrative and
representative thereof.
[0215] General Characterization Methods
[0216] As reported in the following examples, Nuclear Magnetic
Resonance (NMR) spectra were recorded on a Bruker DTX 300
spectrometer using, in most cases, tetramethyl silane (TMS) as the
internal reference. Mass spectra were obtained on an Agilent 1100
LC/MSD instrument using either electrospray ionization (positive or
negative mode) (ESI) or atmospheric pressure chemical ionization
(positive or negative mode) (APCI).
Example 1
Beta-Amyloid Cell Death Assay
[0217] Media Composition
[0218] Neurobasal/B27i: Neurobasal medium plus 1.times.B27
supplement, 0.5 mM L-glutamine, 25 .mu.M L-glutamic acid, and
0.5.times. Penicillin/Streptomycin
[0219] Neurobasal/B27m: Neurobasal medium plus 1.times.B27
supplement and 0.5 mM L-glutamine
[0220] BSS (Ca/Mg free): HBSS (calcium/magnesium free) plus 10 mM
Hepes (pH 7.25), 1.times. Penicillin/Streptomycin, and 1 mM Sodium
Pyruvate
[0221] Glucose-free BSS.sub.0: 143.6 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl.sub.2, 0.8 mM MgSO.sub.4, 1 mM NaH.sub.2PO.sub.4, 26.2 mM
NaHCO.sub.3, 10 mg/l phenol red, 0.25.times.
Penicillin/Streptomycin, and 10 mM Hepes (pH 7.4)
[0222] Papain Quench solution: Neurobasal medium plus 1.times.B27
supplement, 1.times. Penicillin/Streptomycin and 0.5 mg/ml
DNase1
[0223] Assay media: Neurobasal medium plus 1.times.B27 (minus AO)
supplement, 0.5 mM L-glutamine, and 0.25.times.
Penicillin/Streptomycin.
[0224] Experimental Procedure
[0225] Hippocampal Cell Culture
[0226] Hippocampal neurons were isolated from E18 rat embryos as
follows. Embryos were decapitated and the heads immersed in cold
BSS (Ca/Mg free). Using a dissecting microscope the hippocampi were
dissected out and placed in cold BSS (Ca/Mg free). The isolated
hippocampi were then centrifuged at 1000 rpm for 2 min, the BSS
aspirated off and 2 ml of 2 mg/ml Papain in Neurobasal media added
per 10 embryos. After mixing on a rotational shaker for 10 min at
37.degree. C., 5 ml Papain Quench solution was added. Cells were
then centrifuged at 1000 rpm for 2 min, the supernatant was
aspirated and 2 ml of Neurobasal/B27i was added. The cells were
triturated 6 times with siliconized pipettes (decreasing bore size)
after which an additional 5 ml Neurobasal/B27i was added. The cell
suspension was then centrifuged at 1000 rpm for 2 min, the
supernatant was aspirated and 2 ml of Neurobasal/B27i was added.
Cells were triturated again as described above and the volume of
Neurobasal/B27i was adjusted to 1 ml/embryo. Cells were then
counted and seeded at a density of 75,000 cells per well in a
poly-D-lysine coated 24-well plate. After four days media was
removed from the cells and replaced with Neurobasal/B27m media+5
.mu.M Ara-C (cytosine arabinoside). Seven days after isolation the
media was removed again and replaced with fresh Neurobasal/B27m
media. Ten days after isolation the hippocampal cultures were used
in the assay described below.
[0227] Preparation of Oligomeric Beta-Amyloid (A.beta.) Peptide
[0228] Aggregation of A.beta.(1-42) (American Peptide Co,
Sunnyvale, Calif.) into oligomers was carried out according to the
method of Dahlgren et al, (2002) Oligomeric and fibrillar species
of amyloid-beta peptides differentially affect neuronal viability.
J Biol Chem 277: 32046-32053. The A.beta. peptide was dissolved to
1 mM in hexafluoroisopropanol (HFIP) and aliquoted into sterile
microcentrifuge tubes. The HFIP was removed under vacuum and the
peptide film stored at -20.degree. C. The day before the assay, the
peptide film was resuspended in dry DMSO to a concentration of 5
mM. Ham's F-12 media was then added to bring the peptide to a final
concentration of 100 .mu.M, and this solution was incubated at
4.degree. C. for 24 hours to allow formation of oligomers.
[0229] Treatment of HippocamDal Neurons with Oligomeric A.beta.
[0230] The existing growth medium was aspirated from the
hippocampal cultures and the monolayer was washed once with 500
.mu.l glucose free-BSS.sub.0. Test articles were diluted to 2-fold
the desired testing concentration in assay media and 250 .mu.L was
added to the cells. From the 100 .mu.M oligomeric beta-amyloid
peptide solution described above, a working solution of 6 .mu.M was
made in assay media. 250 .mu.L of this working solution was also
added to the cells. The final volume for each well was 500 .mu.L
and the final concentration of A.beta. peptide was 3 .mu.M. As a
negative control, cells were incubated with 500 .mu.L assay media
with no additions.
[0231] Cells were incubated in a 39.degree. C. incubator (5%
CO.sub.2) for 24 hours. After this time, the number of live neurons
remaining in each well was determined using a fluorescent vital
cell stain, Cell Tracker Green (Molecular Probes, Eugene, Oreg.).
Assay media was aspirated from the cells and 400 .mu.L of 2.5 .mu.M
Cell Tracker Green was added to each well. Cells were placed in a
37.degree. C. incubator for 5 minutes after which time the cell
stain was aspirated off and 500 .mu.l of HBSS (Invitrogen, Life
Technologies, Carlsbad, Calif.) was added to each well. The number
of live cells in each well was then quantitated using an automated
fluorescent microscope/imaging system (Universal Imaging,
Downingtown Pa.).
[0232] Certain compounds of the present invention such as
[0233] 2,2,7,8-Tetramethyl-chroman-6-ol; and
[0234] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid methyl ester;
[0235] when tested as described above exhibited assay between 10%
and 40% protection in the .beta. amyloid cell assay
Example 2
MPP.sup.+Cell Death Assay
[0236] Media Composition
[0237] RF media: DMEM-No glucose, glucose (29.1 mM), L-glutamine
(1.4 mM), 10% heat-inactivated FBS, and 1.times.
penicillin/streptomycin (P/S)
[0238] Wash media: DMEM-No glucose and 1.times.P/S
[0239] Low serum media: DMEM-No glucose, glucose (29.1 mM),
L-glutamine (1.4 mM), 0.5% FBS, and 1.times.P/S
[0240] Assay Media: DMEM-No glucose, L-glutamine (1.4 mM), 0.5%
FBS, and 1.times.P/S
[0241] Experimental Procedure
[0242] A substantia nigra-derived dopaminergic progenitor cell line
was seeded in poly-D-lysine-coated 24-well plates at a density of
4500 cells per well in RF media. The cells were left to attach for
16 hours in a 33.degree. C. incubator (5% CO.sub.2) after which
time they were washed once with 500 .mu.L wash media and then
differentiated into a neuronal phenotype by incubating in low serum
media for 24 hours in a 39.degree. C. incubator (5% CO.sub.2).
[0243] After 24 hours the low serum medium was aspirated from the
cells and the monolayer was washed once with 500 .mu.L wash media.
Test articles were diluted to 2-fold the desired testing
concentration in assay media and 250 .mu.L was added to the cells.
From a 10 mM stock, a working solution of 140 .mu.M
1-methyl-4-phenylpyridinium (MPP.sup.+) (Sigma, St. Louis, Mo.) was
made in assay media and 250 .mu.L of this working solution was also
added to the cells. The final volume in each well was 500 .mu.L and
the final concentration of MPP.sup.+was 70 .mu.M. As a negative
control, cells were incubated with 500 .mu.L assay media with no
additions.
[0244] Cells were incubated in a 39.degree. C. incubator (5%
CO.sub.2) for 24 hours. After this time, the number of live neurons
remaining in each well was determined using a fluorescent vital
cell stain, Cell Tracker Green (Molecular Probes, Eugene, Oreg.).
Assay media was aspirated from the cells and 400 .mu.L of 2.5 .mu.M
Cell Tracker Green was added to each well. Cells were placed in a
37.degree. C. incubator for 5 minutes after which time the cell
stain was aspirated off and 500 .mu.L of HBSS (Invitrogen Life
Technologies, Carlsbad, Calif.) was added to each well. The number
of live cells in each well was then quantitated using an automated
fluorescent microscope/imaging system (University Imaging,
Downingtown Pa.)
[0245] Results:
[0246] Certain compounds of the present invention such as
[0247] 2,2,7,8-Tetramethyl-chroman-6-ol;
[0248]
4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-[3-(6-h-
ydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyric
acid;
[0249] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid methyl ester;
[0250]
2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chrome-
n-6-ol;
[0251] Tetramethyl-5-(3-methyl-butyl)-chroman-6-ol;
[0252]
5,6,9-Trimethyl-8-oxa-tricyclo[7.3.1.0.sup.2,7]trideca-2,4,6-trien4-
-ol;
[0253]
(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-acetic
acid methyl ester;
[0254]
2-Methyl-2-thiophen-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-ben-
zo[h]chromen-6-ol;
[0255]
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman--
6-ol; and
[0256]
2,2-Dimethyl-5-(3-methyl-but-2-enyl)-3,4,7,10-tetrahydro-7,10-ethan-
o-2H-benzo[h]chromen-6ol
[0257] when tested as described above provided protection in at
least 30%, preferably in at least 50% of the cells tested at
concentrations ranging from 1 to 25 .mu.M.
Example 3
MPTP Animal Model
[0258] Male C57/BL6 mice (Harlan, Ind.), weight 25-30 g, were used
in all studies. MPTP-HCl (Sigma) was administered i.p. according to
one of the following protocols. The maximum volume which was given
per injection is 200 .mu.L. In all studies, animals were euthanized
with carbon dioxide and, brains were removed for subsequent
determination of dopamine depletion where appropriate.
[0259] Subacute Model
[0260] Animals received 25 mg/kg of MPTP once a day for 5
consecutive days. The end point was 2 days after the final
dose.
[0261] Acute Model
[0262] Animals received 4.times.20 mg/kg of MPTP at 2 hour
intervals. The end point was at either 7 or 14 days.
[0263] Subchronic Model
[0264] Animals received 2.times.40 mg/kg of MPTP with this repeated
16 hours later. The end point was at either 14 or 28 days.
[0265] Chronic Model
[0266] Animals received 25 mg/kg of MPTP, given twice weekly for 5
weeks. The end point was either 1, 3 or 24 weeks after the final
dose.
[0267] Neurobehavioral Outcome Measures
[0268] Compound efficacy was examined with the use of
neurobehavioral models. These models allowed the determination of a
given compound's ability to reverse the motor deficits seen with
MPTP treatment.
[0269] All animals received pre-training for the individual model
and a baseline reading was obtained one day prior to the
commencement of MPTP treatment.
[0270] Open Field Test
[0271] The open field test measures spontaneous activity. Deficits
can be observed with hippocampal and basal ganglia lesions, and
with hindlimb dysfunction. It is sensitive to moderate dopamine
(DA) depletion.
[0272] The open field test was carried out in a clear, acrylic open
box 60 cm.times.60 cm. The base of the box was marked into a
5.times.5 grid of 12 cm.times.2 cm squares. Animals were
individually placed in the box and allowed to roam free. The number
of squares an animal crossed in a 90 second time period was
recorded. In order to be scored, the animal must either have had
all four limbs within a given square or, all four limbs must have
left a given square.
[0273] Tactile Adhesion Model
[0274] The tactile adhesion model measures an animal's ability to
complete a complex sensorimotor task. It is sensitive to moderate
to severe DA depletion.
[0275] A tactile stimulus (0.5.times.0.5 cm square of "sticky
tape") was applied to each side of the animal's face and the time
taken to remove it is recorded. The following measurements were
recorded:
[0276] a. Latency to contact left side tape
[0277] b. Latency to contact right side tape
[0278] c. Order of side contacted (left vs. right)
[0279] d. Latency to remove left side tape
[0280] e. Latency to remove right side tape
[0281] Pole Test
[0282] The pole test evaluates motor co-ordination.
[0283] A rough surfaced metal pole (diameter 8 mm, height 50 cm)
protruding from a cage filled with animal bedding was used for this
test. Animals were placed head upwards at the top of the pole. They
were required to turn and descend face downwards and the time taken
for this was recorded (latency to reach bedding). Timing was
started when the individual animal gripped the top of the pole and
was stopped when all four limbs contacted the bedding.
[0284] Compound Administration
[0285] All test compounds were administered ip. The maximum volume
which was given per injection was 200 .mu.L. Compounds were dosed
up to twice daily, two days prior and 7 days post MPTP
treatment.
[0286] Data Analysis
[0287] A baseline reading was taken for each animal one day prior
to MPTP treatment. All subsequent readings were normalized to the
individual animal's baseline. Values were expressed as a percent
baseline.
[0288] Data was percent baseline and expressed as mean.+-.std
dev:
[0289] Summary
[0290] Compound and vehicle given for 9 days in total, 2 days prior
to MPTP and 7 days after MPTP treatment. L-DOPA given 1 hour prior
to the pole test, each day.
[0291] Results
[0292] Certain compounds of the present invention such as
[0293] 2,2,7,8-Tetramethyl-2H-chromen-6-ol;
[0294] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid methyl ester;
[0295]
3-[6-Hydroxy-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman-2-yl]--
propionic acid;
[0296] 3-(6-Hydroxy-2,8-dimethyl-chroman-2-yl)-propionic acid;
[0297]
2,2,7,8-Tetramethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol;
[0298]
(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-acetic
acid methyl ester.
[0299] at a dose of 30 mg/kg/day significantly reduced the
functional deficit produced by MPTP on each of days 1-3. Functional
outcome was significantly better on days 1 and 2 than in vehicle
treated animals and in both cases were comparable to that of the
negative control group.
[0300] Doses of 10 mg/kg/day and 3 mg/kg/day reduced the MPTP
induced deficit on days 2 and 3 but this may not be significantly
different from vehicle treated animals.
Example 4
FRDA Fibroblast Assay for Protection from Oxidative Stress
[0301] A. Cell Culture and Reagents
[0302] Primary fibroblasts were derived from donors with a
molecular diagnosis of FRDA and control donors with no
mitochondrial disease. Lines F2, C2 and C3 were obtained from
Coriell Cell Repositories (Camden, N.J., USA; catalog #'s GM04078,
GM 08402 and GM08399, respectively). All cell types were diagnosed
at the molecular level for intronic GAA triplet repeat length in
the trataxin gene using a PCR-based method, according to methods
known in the art. FRDA-fibroblasts types had .about.400-450 repeats
(F2 line) or more (F1 and F3), whereas control cell lines displayed
PCR products of normal length. The cells were seeded in microtiter
plates at a density of 4000 cells per 100 .mu.l in growth medium
consisting of 25% (v/v) M199 EBS and 64% (v/v) MEM EBS without
phenol red (Bioconcept, Allschwil, Switzerland) supplemented with
10% (v/v) fetal calf serum (PAA Laboratories, Linz, Austria), 100
U/ml penicillin, 100 .mu.g/ml streptomycin (PAA Laboratories, Linz,
Austria), 10 .mu.g/ml insulin (Sigma, Buchs, Switzerland), 10
.mu.g/ml EGF (Sigma, Buchs, Switzerland), 10 .mu.g/ml bFGF
(PreproTech, Rocky Hill, N.J., USA) and 2 mM glutamine (Sigma,
Buchs, Switzerland). The cells were incubated in the presence of
the various test compounds for 24 h before addition of 1 mM BSO
(L-buthionine (S,R)-sulfoximine).
[0303] B. Cell Viability Measurements
[0304] Cell viability was measured after the first signs of
toxicity appeared in the BSO-treated controls (typically after
16-48 h). The cells were stained for 60 min at room temperature in
PBS with 1.2 .mu.m calceinAM and 4 .mu.m ethidium homodimer
(Live/Dead assay, Molecular Probes, Eugene, Oreg., USA).
Fluorescence intensity was measured with a Gemini Spectramax XS
spectrofluorimeter (Molecular Devices, Sunnyvale, Calif., USA)
using excitation and emission wavelengths of 485 and 525 nm,
respectively.
[0305] C. Data and Statistics
[0306] In experiments carried out in support of the present
invention, certain compounds such as
[0307] 2,2,7,8-Tetramethyl-2H-chromen-6-ol;
[0308]
4-[1-(Carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-2-[3-(6-h-
ydroxy-2,7,8-trimethyl-chroman-2-yl)-propionylamino]-butyric
acid);
[0309] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid methyl ester;
[0310]
2-Amino-4-{1-(carboxymethyl-carbamoyl)-2-[6-hydroxy-2,7,8-trimethyl-
-2-(4,8,12-trimethyl-tridecyl)-chroman-5-ylmethylsulfanyl]-ethylcarbamoyl}-
-butyric acid;
[0311]
5-[3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-y)-propyl]-thiazolidine-2-
,4-dione;
[0312] Delta tocopherol;
[0313] Delta tocotrienol;
[0314] Gamma-tocopherol;
[0315] Gamma-tocotrienol;
[0316] 3-(6-Hydroxy-2,8-dimethyl-chroman-2-yl)-propionic acid;
[0317] 2,2,7,8-Tetramethyl-5-(3-methyl-butyl)-chroman-6-ol;
[0318] 2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
[0319]
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman--
6-ol; and
[0320] significantly reduced cell death in FRDA fibroblasts
compared to untreated FRDA fibroblasts with an EC.sub.50 of between
0.01 .mu.M and 6 .mu.M.
Example 5
High Glutamate-Induced Oxidative Stress Assay (HGOS)
[0321] This procedure was used to induce high glutamate-induced
oxidative stress (HGOS) in a dopaminergic neuronal cell line. Using
this assay the potency and efficacy of test articles against HGOS
neuronal cell injury and cell death was established in a high
throughput manner.
[0322] Materials
[0323] Dopaminergic neuronal cell lines
[0324] DMEM-No Glucose (Life Technologies Cat #11966-025)
[0325] L-glutamine (Life Technologies Cat #25030-081)
[0326] L-glutamic acid, monosodium salt (Sigma Cat #G5889)
[0327] D-glucose (Sigma Cat #G-6151)
[0328] 10.times.HBSS buffer(pH 7.4) (950 ml Pyrogen-free water,
2.44 g/L MgCl2.6H20, 3.73 g/L KCl, 59.58 g/L Hepes, 58.44 g/L NaCl,
1.36 g/L KH2PO4, 1.91 g/L CaCl2.2H2O and pH to 4.5 with HCl)
[0329] Cell Tracker Green fluorescent dye (Molecular Probes, Cat
#2925). Prepare a 5 .mu.M solution in pre-warmed HBSS just prior to
use.
[0330] Sterile 96-well plates precoated with poly-D-lysine (Corning
Catalog #3665)
[0331] 96-well deep well mother plate, DyNA Block 1000 (VWR Catalog
#40002-008)
[0332] Neuronal Cells
[0333] The cells were seeded into 96-well plates at a density of
2000 per well and left to grow for 72 hours in a 33.degree. C.
incubator with 5% CO.sub.2 in air atmosphere. The passage number of
the cells for each assay experiment were no later than p11 in order
to minimize experimental variation.
[0334] Compound PreDaration in Deep-Well Mother Plates
[0335] VWRBrand DyNA Block 1000, deep well mother plates (VWR Cat.
#40002-008) were used for the preparation of the test
compounds.
[0336] All compounds were dissolved in DMEM-No Glu containing 1 mM
glucose, 30 mM glutamate and 1.times. Pen/Strep. DMEM-No Glu with 1
mM glucose and 1.times.P/S was used as the negative control,
DMEM-No Glucose with 1 mM glucose, 100 M glutamate was used as a
positive control and 100 .mu.M Glutathione was added to the
positive control as a standard. All of the procedures for this
involving the making and dilution of compounds were performed using
aseptic conditions and with minimal light.
[0337] Cell Preparation
[0338] The plates were removed from the incubator and examined
under the microscope for morphological appearance and density.
Using an aseptic technique and an 8-channel aspirator the media was
carefully removed from the cells and replaced with 200 .mu.l of
1.times.HBSS. This was done as quickly as possible to prevent the
cells drying out. The plates were then placed in the humidified
37.degree. C. incubators of the Biomek 2000 Side Loader. Four
plates were washed at a time so as to minimize the time that the
cells were sitting in 1.times.HBSS prior to addition of the
compound test solution.
[0339] Experimental Setup
[0340] The Beckman Biomek workstations were used to load the
compounds and controls from the mother plates onto the cell plates
that were prewashed with HBSS under sterile conditions. The plates
were incubated in the upper HTS incubator at 37.degree. C. in 5%
CO.sub.2 for exactly 16 hrs. The following day, using the Beckman
Biomek workstations, the plates were removed from the incubator.
Using Cell Tracker Addition, the compounds were removed from the
plates, washed once with 200 .mu.M of pre-warmed 1.times.HBSS and
then 100 .mu.L of 5 .mu.M Cell Tracker Green was added to each
well. The plates were incubated at 37.degree. C. for 30 min to
allow the dye to enter the cell and be cleaved by the esterases.
After washing the cells twice with prewarmed 1.times.HBSS, the
plates were read with the 485 excitation; 538 emission filter pair
on a Fluoroskan.
[0341] Certain compounds of the present invention such as:
[0342] 6-Hydroxy-2,2,5,7-tetramethyl-chroman-8-carbaldehyde;
[0343] 2,2,5,7-Tetramethyl-8-nitro-chroman-6-ol;
[0344] 5-(1-Hydroxy-ethyl)-2,2,7,8-tetramethyl-chroman-6-ol;
[0345] 5-Hydroxymethyl-2,2,7,8-tetramethyl-chroman-6-ol;
[0346] 5-(2-Hydroxy-ethyl)-2,2,7,8-tetramethyl-chroman-6-ol;
[0347]
10-Methoxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromen-6-ol;
[0348] 2-Ethynyl-2,5,7,8-tetramethyl-chroman-6-ol;
[0349]
2-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethoxy)-N-(3-trifluor-
omethyl-phenyl)-acetamide;
[0350]
(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)-piperazin-1-yl-methano-
ne;
[0351] 6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxamidine;
[0352]
N-(6-Hydroxy-2,5,7,8-tetramethyl-chroman-2-ylmethyl)-N-methyl-aceta-
mide;
[0353] 2,2,5,7,8-Pentamethyl-2H-chromen-6-ol;
[0354]
2-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethyl)-propane-1,3-di-
ol;
[0355]
2-(2-Chloro-ethyl)-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman--
6-ol;
[0356] 2,2,5,8-Tetramethyl-chroman-6-ol;
[0357] 2-(3-Chloro-propyl)-2,7,8-trimethyl-chroman-6-ol;
[0358] 2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
[0359] 2-(2-Chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol;
[0360]
(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylsulfanyl)-acetic
acid methyl ester;
[0361]
5,6,9-Trimethyl-8-oxa-tricyclo[7.3.1.0.sup.2,7]trideca-2,4,6-trien--
4-ol;
[0362] 2,2,7,8-Tetramethyl-5-(3-methyl-butyl)-chroman-6-ol;
[0363] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid;
[0364] 2-Hydroxymethyl-2,5,7,8-tetramethyl-chroman-6-ol;
[0365]
5-[3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propyl]-thiazolidine--
2,4-dione;
[0366]
5-(4-Benzyl-piperazin-1-ylmethyl)-2,7,8-trimethyl-2-(4,8,12-trimeth-
yl-tridecyl)-chroman-6-ol;
[0367] 3-(5-Bromo-6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic
acid methyl ester;
[0368]
3-(6-Hydroxy-2-methyl-3,4-dihydro-2H-benzo[h]chroman-2-yl)-propioni-
c acid; and
[0369] 2,2,7,8-Tetramethyl-chroman-6-ol;
[0370] were considered to be active when they exhibited protection
against HGOS cell injury and cell death with an EC.sub.50 in a
range of 5 .mu.M or less.
Example 6
2-(2-chloro-ethyl)-2,7,8-trimethyl-chroman-6-ol
[0371] 3
[0372] Step 1:
[0373] To a solution of 4-chloro-butanone-2 (2.65 g, 25 mmol) in 25
ml of THF at -20.degree. C. was added dropwise a solution of 1M
vinyl magnesium bromide (40 ml, 40 mmol). After the completion of
addition the mixture was stirred for one more hours at room
temperature. To the mixture was added 10 ml of water at 0.degree.
C. with stirring. The mixture was dried over MgSO.sub.4. After the
removal of the solvent, the residue was purified via flash column
purification with 1:3 ethyl acetate and hexane as elute to afford
800 mg of 5-chloro-3-methyl-pent-1-en-3-ol. .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. (ppm): 5.86-5.90 (dd, 1H); 5.29 (d, 1H), 5.13
(d, 1H), 3.50-3.59 (t, 3H); 1.87-1.83 2.18 (m, 2H); 1.63-1.69 (m,
2H), 1.31 (s, 3H)
[0374] Step 2:
[0375] A mixture of, 2,3-dimethyl-dihydroquinone (794 mmol), 1.0 ml
(3.97 mmol) of BF.sub.3.Et.sub.2O and 25 ml of dioxane was heated
up to 110.degree. C. under a nitrogen atmosphere. A solution of
5-chloro-3-methyl-pent-1-en-3-ol (1.12 g, 8.6 mmol) in 24 ml of
dioxane was added slowly to the mixture. The addition was completed
after two hours and the mixture was continued to reflux for an
additional 3 hours. After cooling down to room temperature, the
mixture was poured to water, extracted with ethyl acetate, washed
with water and dried over MgSO.sub.4. After the removal of
solvents, the residue was mixed with methanol/HCl and let stirred
overnight at room temperature. After the removal of the solvents
the residue was purified via flash column chromatography (1:3
EtOAc/Hex) twice to give 2-(2-chloro-ethyl)-2,7,8-tri-
methyl-chroman-6-ol .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.
(ppm): 6.39 (s. 1H), 4.26 (S, 1H), 3.80-3.65 (m, 2H), 2.85-2.70 (m,
2H), 2.16 (s, 3H), 2.12 ( s, 3H), 2.20-2.05 (m, 2H), 1.85-1.75 (m,
2H) 1.31 (s, 3H). MS: 255 (M+H.sup.+, 100%)
Example 7
2,2,7,8-Tetramethyl-4H-benzo[1,3]dioxin-6-ol
[0376] 4
[0377] Step 1
[0378] To a mixture of 2,3-dimethylhydroquinone (1.38 g, 10 mmol),
K.sub.2CO.sub.3 (2.76 g, 20 mmol), potassium iodide (0.83 g, 5
mmol) in 50 mL dry acetone was added benzyl bromide (1.88 g, 11
mmol). The resulting suspension was vigorously stirred for 48 at
RT. The solid was filtered off and the liquid was concentrated. The
residue was chromatographed to afford the benzyl derivative,
4-benzyloxy-3-methyl-phe- nol, as a light brown solid (1.15 g).
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 7.49-7.35 (m, 5
H), 6.70 (d, J=8.7, 1 H), 6.61 (d, J=8.7, 1 H), 5.03 (s, 2 H), 4.43
(s, 1 H), 2.26 (s, 3 H), 2.22 (s, 3 H); MS (ESI) m/z 229
(M+H.sup.+, 100%).
[0379] Step 2:
[0380] To 684 mg (3 mmol) of 4-benzyloxy-3-methyl-phenol in 10 mL
toluene and 1.5 mL DME (dimethoxyethane) in a sealable tube was
added paraformaldehyde (1.8 g, 60 mmol). The tube was flushed with
argon and sealed. It was heated to 130.degree. C. for 48 h under
stirring. After cooling to room temperature, the solid was filtered
off and washed with 1:1 hexane/EtOAc and the liquid was
concentrated. The residue was chromatographed to afford
4-benzyloxy-6-hydroxymethyl-2,3-dimethyl-phenol- , as a light brown
solid (640 mg). .sup.1H-NMR (300 MHz, CDCl.sub.3/CD.sub.3OD))
.delta. (ppm): 7.43-7.29 (m, 5 H), 6.55 (s, 1 H), 4.95 (s, 2 H),
4.70 (s, 2 H), 2.18 (s, 3 H), 2.16 (s, 3 H); MS (ESI) m/z 241
(M-OH.sup.-, 100%).
[0381] Step 3:
[0382] A solution of
4-benzyloxy-6-hydroxymethyl-2,3-dimethyl-phenol (86 mg, 0.33 mmol)
in dimethoxypropane (10 mL) in the presence of toluene sulfonic
acid (7 mg) was stirred at RT for 15 h. It was added 30 mg of
anion-exchange resin and stirring was continued for 20 more min.
The resin was then filtered off and the solution was concentrated.
The crude product was purified by chromatography on a silicagel
column to afford
6-benzyloxy-2,2,7,8-tetramethyl-4H-benzo[1,3]dioxine as a white
sticky solid (86 mg). .sup.1H-NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 7.51-7.35 (m, 5 H), 6.45 (s, 1 H), 5.02 (s, 2 H), 4.85 (s, 2
H), 2.26 (s, 3 H), 2.19 (s, 3 H), 1.59 (s, 6 H); .sup.13C-NMR
.delta. (ppm): 150.6, 143.3, 137.8, 128.5, 127.8, 126.3, 125.8,
115.9, 105.4, 99.1, 70.9, 61.1, 24.9, 12.2, 11.5.
[0383] Step 4
[0384] To a solution of
6-benzyloxy-2,2,7,8-tetramethyl-4H-benzo[1,3]dioxi- ne (86 mg, 0.29
mmol) in 10 mL EtOH was added Pd/C (15 mg, 10%). It was stirred in
a hydrogen atmosphere for 1.5 h and filtered. The solution was
concentrated and the crude product was purified by chromatography
on silicagel to afford 2,2,7,8-tetramethyl-4H-benzo[1,3]dioxin-6-ol
as a white solid (54 mg). .sup.1H-NMR (CDCl.sub.3, 300 MHz) .delta.
(ppm): 6.28 (s, 1 H), 4.77 (s, 1 H), 4.76 (s, 2 H), 2.17 (s, 3 H),
2.13 (s, 1 H) 1.55 (s, 3 H), 1.54 (s, 3 H); .sup.13C-NMR .delta.
(ppm): 147.1, 142.9, 126.1, 122.7, 116.6, 107.5, 99.1, 60.9, 24.8,
11.9, 11.5.
Example 8
2,2,7,8-Tetramethyl-chroman-6-ol
[0385] 5
[0386] Step 1
[0387] To a solution of 2,3-dimethylhydroquinone (2 g, 14.5 mmol)
and BF.sub.3.Et.sub.2O (3.3 g, 23.2 mmol) in 30 mL of dioxane was
slowly added a dilute solution of 2-methyl-but-3-en-2-ol (1.0 g,
11.6 mmol) in 10 mL of dioxane over a period of 30 min. Upon the
completion of the alcohol addition, it was allowed to stir for 4 h.
The reaction was quenched by pouring over ice (70 g) and the
mixture was extracted with EtOAc (3.times.50 mL). The combined
organic layers were dried over Na.sub.2SO.sub.4 and concentrated.
The crude product was purified by chromatography (hexane/EtOAc=9:1)
to afford 2,3-dimethyl-5-(3-methyl-but-- 2-enyl)-benzene-1,4-diol
as a light brown oil (2 g). MS (ESI) m/z: 207 (M+H+, 100%).
[0388] Step 2
[0389] A solution of
2,3-dimethyl-5-(3-methyl-but-2-enyl)-benzene-1,4-diol (2.54 g, 12.3
mmol) and BF.sub.3.Et.sub.2O (2.8 mL, 22.4 mmol) in 50 mL dioxane
was heated to reflux for 5 h. It was cooled to RT and quenched onto
ice (100 g). The mixture was extracted with EtOAc (3.times.60 mL)
and dried over Na.sub.2SO.sub.4. The residue was purified by
chromatography (Hexane/EtOAc=5:1) to afford
2,2,7,8-Tetramethyl-chroman-6- -ol as a brown oil (1.5 g).
.sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 6.28 (s, 1 H),
5.30 (s, 1 H), 2.6 (t, J=6.8 2 H), 2.10 (s, 3 H), 2.09 (s, 3 H),
1.70 (t, J=6.8, 2 H), 1.26 (s, 6 H); .sup.13C-NMR .delta. (ppm):
145.4, 145.9, 125.8, 122.2, 118.1, 112.6, 73.6, 33.0, 27.0, 22.7,
12.1; (ESI) m/z: 207 (M+H.sup.+, 100%).
Example 9
2,2,7,8-Tetramethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol
[0390] 6
[0391] To a solution of 2,2,7,8-tetramethyl-chroman-6-ol (305 mg,
1.39 mmol), prepared as described above, in 5 mL dry dioxane was
added boron trifluoride (296 mg, 2.1 mmol). It was stirred for 3
min followed by dropwise addition of 2-methyl-but-3-en-2-ol
solution (143 mg, 1.66 mmol, in 3 mL of dioxane). The reaction was
allowed to stir for 5 h at RT before quenching on to ice (80 g).
The mixture was extracted with DCM (3.times.50 mL) and the combined
organic layers were dried over Na.sub.2SO.sub.4 and concentrated
under reduced pressure. The crude product was purified by
chromatography (hexane) to afford
2,2,7,8-tetramethyl-5-(3-methyl-but-2-enyl)-chroman-6-ol as a light
brown oil (229 mg). .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta.
(ppm): 5.16 (m, 1 H), 4.70 (s, 1 H), 3.34 (d, J=6.8, 2.18 (s, 3 H),
2.14 (s, 3 H), 1.86 (s, 3 H), 1.81 (t, J=13.8, 2 H), 1.77 (s, 3 H),
1.32 (s, 6 H); .sup.13C-NMR .delta. (ppm): 145.7, 145.4, 134.1,
123.5, 122.2, 122.0, 121.8, 116.3, 72.5, 33.1, 26.7, 25.8, 20.8,
17.9, 12.1, 11.9; (ESI) m/z: 275 (M+H.sup.+, 100%).
Example 10
3-[6-Hydroxy-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman-2-yl]-propion-
ic acid
[0392] 7
[0393] A solution of
3-(6-hydroxy-2,7,8-trimethyl-chroman-2-yl)-propionic acid (150 mg,
0.557 mmol) in 1M aq. NaOH (1.11 mL, 1.11 mmol) was cooled to
0.degree. C., and treated with prenyl bromide (0.064 mL, 0.557
mmol). Following stirring for 2 h at ambient temperature the
reaction mixture was made slightly acidic with 0.5M HCl and shaken
with EtOAc. The organic phase was evaporated yielding a brown
residue which was subjected to column chromatography (SiO.sub.2:
hexane:EtOAc, 8:2 v/v) yielded
3-[6-hydroxy-2,7,8-trimethyl-5-(3-methyl-but-2-enyl)-chroman-2-yl]-propio-
nic acid as a pale brown solid (50 mg). .sup.1H-NMR (300 MHz,
CDCl.sub.3) .delta. (ppm): 5.15 (t, J=7, 1H), 3.33 (d, J=7, 2H),
2.73 (t, J=7, 2H), 2.59 (t, J=7, 2H), 2.17 (s, 3H), 2.12 (s, 3H),
1.86-2.10 (m, 2H), 1.85 (s, 3H), 1.80-1.85 (m, 2H), 1.76 (s, 3H),
1.26 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3,) .delta. (ppm):
180.2, 145.7, 145.2, 134.3, 123.7, 122.5, 122.0, 121.9, 116.2,
73.4, 34.4, 31.8, 28.7, 25.9, 25.6, 23.3, 20.4, 18.0, 12.2, 11.9.
MS ESI-Pos m/z 333.2 (M+H.sup.+).
Example 11
2-Hydroxymethyl-2,5,7,8-tetramethyl-chroman-6-ol
[0394] 8
[0395] To a solution of
6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (1.0 g, 4
mmol) in 50 mL dry THF at RT under argon atmosphere was added small
portions of LiAlH.sub.4 over a period of 30 min. The reaction was
stirred for 2 h and quenched on to ice water (100 mL). It was
extracted with EtOAc (3.times.50 mL) and the combined organic
layers were washed with water (100 mL). The organic solution was
dried over Na.sub.2SO.sub.4 and concentrated. The residue was
chromatographed with hexane/EtOAc (3:1) and the desired compound
2-hydroxymethyl-2,5,7,8-tetra- methyl-chroman-6-ol was isolated as
a cream-colored solid (655 mg). .sup.1H-NMR (300 MHz, CDCl.sub.3,)
.delta. (ppm): 4.90 (s, 1 H), 3.68 (m, 2 H), 2.70 (m, 2 H), 2.46
(m, 1 H), 2.20 (s, 3 H), 2.16 (s, 3 H), 2.15 (s, 3 H), 2.03 (m, 1
H), 1.76 (m, 1 H), 1.26 (s, 3 H); .sup.13C-NMR .delta. (ppm):
145.2, 144.98, 122.6, 121.9, 119.3, 117.4, 75.2, 69.4, 27.9, 20.5,
20.4, 12.4, 12.0, 11.5; MS (ESI) m/z: 259 (M+Na.sup.+, 100%), 237
(M+H.sup.+, 45%).
Example 12
5-(4-Benzyl-piperazin-1-ylmethyl)-2,7,8-trimethyl-2-(4,8,12-trimethyl-trid-
ecyl)-chroman-6-ol
[0396] 9
[0397] To a solution of bromo-tocopherol prepared as described
above (1.00 g) in 25 ml of methylene chloride was added
1-benzylpiperazine (0.8 g) at room temperature. The solution was
stirred at room temperature for 2 hours. More methylene chloride
was added, washed with water, and dried over magnesium sulfate.
Evaporation and chromatography (silica gel, methylene chloride)
gave 250 mg of 5-(4-benzyl-piperazin-1-ylmethyl)-2,7,-
8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol. .sup.1H-NMR
(300 MHz, CDCl.sub.3) .delta. (ppm) 7.35-7.20 (m, 5H), 3.63 (s,
2H), 3.51 (s, 2H), 2.14 (s, 3H), 2.10 (s, 3H). MS (PAl-ES) m/z 605
(M+H.sup.+, 100%).
[0398] Similarly replacing benzylpiperazine with other amines the
following compounds were produced:
[0399]
2,7,8-trimethyl-5-piperazin-1-ylmethyl-2-(4,8,12-trimethyl-tridecyl-
)-chroman-6-ol. .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. (ppm)
3.62 (s, 2H), 2.14 (s, 3H), 2.10 (s, 3H). MS (PAI-ES) m/z 515
(M+H.sup.+, 100%).
[0400]
2,7,8-trimethyl-5-morpholin-4-ylmethyl-2-(4,8,12-trimethyl-tridecyl-
)-chroman-6-ol .sup.1H-NMR (300 MHz, CDCl.sub.3) .delta. (ppm) 3.74
(br. s, 4H), 3.64 (s, 2H), 2.65-2.50 (m, 6H), 2.14 (s, 3H), 2.10
(s, 3H).
[0401]
2,7,8-trimethyl-5-pyrrolidin-1-ylmethyl-2-(4,8,12-trimethyl-tridecy-
l)-chroman-6-ol; MS (ESI) m/z: 500 (M+H.sup.+, 100%), and
[0402]
2,7,8-trimethyl-5-piperidin-1-ylmethyl-2-(4,8,12-trimethyl-tridecyl-
)-chroman-6-ol; MS (ESI) m/z: 514 (M+H.sup.+, 100%)
Example 13
2,2,7,8-Tetramethyl-2H-chromen-6-ol
[0403] 10
[0404] Step 1:
[0405] A mixture of 2,3-dimethyl-1,4-dihydroquinone (5.0 g, mmol),
acetone (80 mL), anhydrous potassium carbonate (18 g, ) and
dimethyl sulfate (14 mL) was gently refluxed under nitrogen for 10
hours. The suspension was cooled to room temperature and poured
into 300 mL of water. The precipitate was collected and washed with
water, then dried in air to give 5.5 g of
1,4-dimethoxy-2,3-dimethyl-benzene as a purple solid.
[0406] Step 2:
[0407] A solution of 1,4-dimethoxy-2,3-dimethyl-benzene (1 g) in
dichloromethane was stirred and cooled in an ice-water bath, and
TiCl.sub.4 (1.1 mL) was slowly added, followed by the addition of
CHCl.sub.2OCH.sub.3 (0.58 g). The solution was stirred for another
15 min with ice-water bath, 30 min at RT, then 15 min at 35.degree.
C. Then the red solution was poured into ice and extract with
dichloromethane. Workup and purification by chromatography
(silicagel column, eluting with EtOAc/:hexane 1:4) gave 973 mg of
2,5-dimethoxy-3,4-dimethyl-benzaldehyde- .
[0408] Step 3:
[0409] A solution of 2,5-dimethoxy-3,4-dimethyl-benzaldehyde (155
mg) in dichloromethane was stirred and cooled in an ice-water bath.
BBr.sub.3 (1M in CH.sub.2Cl.sub.2, 1.80 mL) was slowly added and
the solution was stirred for another hour, then the red solution
was poured into ice and extracted with dichloromethane. Workup and
purification by chromatography (silicagel column, eluting with
EtOAc/:hexane 1:4) gave 110 mg of
2,5-dihydroxy-3,4-dimethyl-benzaldehyde.
[0410] Step 4:
[0411] To a solution of 2,5-dihydroxy-3,4-dimethyl-benzaldehyde
(2.0 g) in dichloromethane (50 mL) was added 3,4-dihydro-2H-pyran
(DHP)(1.5 g), followed by p-toluenesulfonic acid monohydrate (200
mg). The solution was stirred at RT for 1 hour and quenched by
adding 1 mL of a sodium bicarbonate solution. Then dichloromethane
was dried over MgSO4 and concentrated. The residue was purified by
silica gel column chromatography eluting with hexane and ethyl
acetate (8:2) to give 1.5 g of
2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-benzaldehyde.
[0412] Step 5:
[0413] A mixture of
2-hydroxy-3,4-dimethyl-5-(tetrahydro-pyran-2-yloxy)-be- nzaldehyde
(300 mg), methyl 3,3-dimethylacrylate (300 mg), and K.sub.2CO.sub.3
(300 mg) in 10 mL of DMF was stirred at 160.degree. C. for 5 h.
After letting it cool down to room temperature, it was worked up
and purified by silica gel column chromatography eluting with 5%
ethyl acetate in hexane, to give the THP-protected
2,2,7,8-tetramethyl-2H-chrom- en-6-ol intermediate. Removal of the
protection group was accomplished as described above with
toluenesulfonic acid in MeOH for 5 h. Purification by
chromatography (silica gel column, eluting with 15% EtOAc in
hexane) gave 83 mg of colorless solid of
2,2,7,8-tetramethyl-2H-chromen-6-ol. .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. (ppm) d: 6.30 (s, 1H), 6.20 (d, J=9.6 Hz,1H),
5.58 (d, J=9.6 Hz, 1H), 4.50 (s, 1H, OH), 2.13, 2.12 (2s, 6H), 1.39
(s, 6H) ppm. .sup.13C NMR (CDCl.sub.3, 75 MHz) d: 147.02, 144.56,
130.88, 125.55, 123.59, 122.47, 119.03, 109.80, 75.49, 27.57,
12.18, 11.74. MS (m/z): 205 (M+H.sup.+).
[0414] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto. All patents and publications cited above are
hereby incorporated by reference
* * * * *