U.S. patent application number 10/272914 was filed with the patent office on 2003-04-10 for mitochondrially targeted antioxidants.
This patent application is currently assigned to University of Otago. Invention is credited to Murphy, Michael P., Smith, Robin A.J..
Application Number | 20030069208 10/272914 |
Document ID | / |
Family ID | 27353842 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030069208 |
Kind Code |
A1 |
Murphy, Michael P. ; et
al. |
April 10, 2003 |
Mitochondrially targeted antioxidants
Abstract
The invention provides mitochondrially targeted antioxidant
compounds. A compound of the invention comprises a lipophilic
cation covalently coupled to an antioxidant moiety. In preferred
embodiments, the lipophilic cation is the triphenyl phosphonium
cation, and the compound is of the formula
P(Ph.sub.3).sup.+XR.multidot.Z.sup.- where X is a linking group, Z
is an anion and R is an antioxidant moiety. Also provided are
pharmaceutical compositions containing the mitochondrially targeted
antioxidant compounds, and methods of therapy or prophylaxis of
patients who would benefit from reduced oxidative stress, which
comprise the step of administering the compounds of the
invention.
Inventors: |
Murphy, Michael P.;
(Dunedin, NZ) ; Smith, Robin A.J.; (Dunedin,
NZ) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Assignee: |
University of Otago
|
Family ID: |
27353842 |
Appl. No.: |
10/272914 |
Filed: |
October 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10272914 |
Oct 18, 2002 |
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09968838 |
Oct 3, 2001 |
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09968838 |
Oct 3, 2001 |
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09577877 |
May 25, 2000 |
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6331532 |
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Current U.S.
Class: |
514/75 ;
568/9 |
Current CPC
Class: |
C07F 9/5456 20130101;
C09B 69/001 20130101; A61K 47/543 20170801; C07F 9/65522 20130101;
C07F 9/54 20130101; C07F 9/572 20130101; A61K 31/66 20130101 |
Class at
Publication: |
514/75 ;
568/9 |
International
Class: |
C07F 009/02; A61K
031/66 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 25, 1997 |
NZ |
329255 |
Claims
1. A mitochondrially-targeted antioxidant compound comprising a
lipophilic cation covalently coupled to an antioxidant moiety,
wherein the antioxidant moiety is capable of being transported
through the mitochondrial membrane and accumulated within the
mitochondria of intact cells, with the proviso that the compound is
not thiobutyltriphenylphosph- onium bromide.
2. A compound as claimed in claim 1 wherein the lipophilic cation
is the triphenylphosphonium cation.
3. A mitochondrially-targeted antioxidant compound as claimed in
claim 1, wherein said compound has the formula 8wherein X is a
lining group, Z is an anion, and R is an antioxidant moiety.
4. A compound as claimed in claim 3, wherein X is a C.sub.1 to
C.sub.30 carbon chain, optionally including one or more double or
triple bonds, and optionally including one or more substituents
and/or unsubstituted or substituted alkyl, alkenyl or alkynyl side
chains.
5. A compound as claimed in claim 4, wherein X is (CH.sub.2).sub.n
where n is an integer of from 1 to 20.
6. A compound as claimed in claim 5 wherein X is an ethylene,
propylene, butylene, pentylene or decylene group.
7. A compound as claimed in claim 3 wherein said compound has the
formula 9including all stereoisomers thereof.
8. A compound as claimed in claim 7 wherein Z is Br.
9. A compound as claimed in claim 3, having the formula 10wherein:
Z is a pharmaceutically acceptable anion, m is an integer of from 0
to 3, each Y is independently selected from groups, chains and
aliphatic and aromatic rings having electron donating and accepting
properties, (C).sub.n represents a carbon chain optionally
including one or more double or triple bonds, and optionally
including one or more substituents and/or unsubstituted or
substituted alkyl, alkenyl or alkynyl side chains; and n is an
integer of from 1 to 20.
10. A compound as claimed in claim 9, wherein (C).sub.n is an alkyl
chain of the formula (CH.sub.2).sub.n wherein n is an integer of
from 1 to 20.
11. A compound as claimed in claim 10, wherein each Y is
independently selected from the group consisting of alkoxy,
thioalkyl, alkyl, haloalkyl, halo, amino, nitro and optionally
substituted aryl, or when m is 2 or 3, two Y groups, together with
the carbon atoms to which they are attached, form an aliphatic or
aromatic carbocyclic or heterocyclic ring fused to the aryl
ring.
12. A compound as claim in claim 11 wherein each Y is independently
selected from methoxy and methyl.
13. A compound as claimed in claim 9 wherein said compound has the
formula 11
14. A compound as claimed in claim 13 wherein Z is Br.
15. A pharmaceutical composition suitable for the treatment of a
patient who would benefit from reduced oxidative stress, which
comprises an effective amount of a mitochondrially-targeted
antioxidant as defined in claim 1 in combination with one or more
pharmaceutically acceptable carriers or diluents.
16. A pharmaceutical composition as claimed in claim 15 wherein the
mitochondrially-targeted antioxidant has the formula I 12wherein X
is a linking group, Z is an anion and R is an antioxidant
moiety.
17. A pharmaceutical composition as claimed in claim 16 wherein the
mitochondrially targeted antioxidant compound is 13
18. A pharmaceutical composition as claimed in claim 15 wherein the
mitochondrially targeted antioxidant compound has the formula
14wherein: Z is a pharmaceutically acceptable anion, m is an
integer of from 0 to 3, each Y is independently selected from
groups, chains and aliphatic and aromatic rings having electron
donating and accepting properties, (C).sub.n represents a carbon
chain optionally including one or more double or triple bonds, and
optionally including one or more substituents and/or
unsubstituted-or substituted alkyl, alkenyl or alkynyl side chains;
and n is an integer of from 1 to 20.
19. A pharmaceutical composition as claimed in claim 18, wherein
(C).sub.n is an alkyl chain of the formula (CH.sub.2).sub.n wherein
n is an integer of from 1 to 20.
20. A pharmaceutical composition as claimed in claim 18, wherein
each Y is independently selected from the group consisting of
alkoxy, thioalkyl, alkyl, haloalkyl, halo, amino, nitro and
optionally substituted aryl, or, when m is 2 or 3, two Y groups,
together with the carbon atoms to which they are attached, form an
aliphatic or aromatic carbocyclic or heterocyclic ring fused to the
aryl ring.
21. A pharmaceutically composition as claimed in claim 20, wherein
each Y is independently selected from methoxy and methyl.
22. A pharmaceutical composition as claimed in claim 17 wherein the
mitochondrially targeted antioxidant compound is: 15
23. A method of therapy or prophylaxis of a patient who would
benefit from reduced oxidative stress, which comprises the step of
administering to the patient a mitochondrially-targeted antioxidant
as defined in claim 1.
24. A method of reducing oxidative stress in a cell which comprises
the step of administering to the cell a mitochondrially-targeted
antioxidant as defined in claim 1.
Description
TECHNICAL FIELD
[0001] The invention relates to antioxidants having a lipophilic
cationic group and to uses of these antioxidants, for example, as
pharmaceuticals.
BACKGROUND OF THE INVENTION
[0002] Oxidative stress contributes to a number of human
degenerative diseases associated with ageing, such as Parkinson's
disease, and Alzheimer's disease, as well as to Huntington's
Chorea, diabetes and Friedreich's Ataxia, and to non-specific
damage that accumulates with aging. It also contributes to
inflammation and ischaemic-reperfusion tissue injury in stroke and
heart attack, and also during organ transplantation and surgery. To
prevent the damage caused by oxidative stress a number of
antioxidant therapies have been developed. However, most of these
are not targeted within cells and are therefore less than optimally
effective.
[0003] Mitochondria are intracellular organelles responsible for
energy metabolism. Consequently, mitochondrial defects are
damaging, particularly to neural and muscle tissues which have high
energy demands. They are also the major source of the free radicals
and reactive oxygen species that cause oxidative stress inside most
cells. Therefore, the applicants believe delivering antioxidants
selectively to mitochondria will be more effective than using
non-targeted antioxidants. Accordingly, it is towards the provision
of antioxidants which may be targeted to mitochondria that the
present invention is directed.
[0004] Lipophilic cations may be accumulated in the mitochondrial
matrix because of their positive charge (Rottenberg, (1979) Methods
Enzymol, 55, 547-560; Chen, (1988) Annu Rev Cell Biol 4, 155-181).
Such ions are accumulated provided they are sufficiently lipophilic
to screen the positive charge or delocalise it over a large surface
area, also provided that there is no active efflux pathway and the
cation is not metabolised or immediately toxic to a cell.
[0005] The focus of the invention is therefore on an approach by
which it is possible to use the ability of mitochondria to
concentrate specific lipophilic cations to take up linked
antioxidants so as to target the antioxidant to the major source of
free radicals and reactive oxygen species causing the oxidative
stress.
SUMMARY OF THE INVENTION
[0006] In its broadest aspect, the invention provides a
mitochondrially-targeted antioxidant which comprises a lipophilic
cation covalently coupled to an antioxidant moiety, wherein the
antioxidant moiety is capable of being transported through the
mitochondrial membrane and accumulated within the mitochondria of
intact cells, with the proviso that the compound is not
thiobutyltriphenylphosphonium bromide.
[0007] Preferably, the lipophilic cation is the
triphenylphosphonium cation.
[0008] Preferably, the mitochondrially-targeted antioxidant has the
formula 1
[0009] wherein Z is an anion, X is a linking group and R is an
antioxidant moiety.
[0010] Preferably, X is a C.sub.1-C.sub.30, more preferably
C.sub.1-C.sub.20, carbon chain, optionally including one or more
double or triple bonds, and optionally including one or more
substituents (such as hydroxyl, carboxylic acid or amide groups)
and/or unsubstituted or substituted alkyl, alkenyl or alkynyl side
chains.
[0011] Preferably, X is (CH.sub.2).sub.n where n is an integer of
from 1 to 20, more preferably of from about 1 to 15.
[0012] More preferably, X is an ethylene, propylene, butylene,
pentylene or decylene group.
[0013] Preferably, Z is a pharmaceutically acceptable anion.
[0014] In one particularly preferred embodiment, the
mitochondrially-targeted anti-oxidant of the invention has the
formula 2
[0015] including all stereoisomers thereof.
[0016] Preferably, Z is Br. The above compound is referred to
herein as "compound 1".
[0017] In another preferred embodiment, the
mitochondrially-targeted antioxidant has the general formula: 3
[0018] wherein:
[0019] Z is a pharmaceutically acceptable anion, preferably a
halogen,
[0020] m is an integer from 0 to 3,
[0021] each Y is independently selected from groups, chains and
aliphatic and aromatic rings having electron donating and accepting
properties,
[0022] (C).sub.n represents a carbon chain optionally including one
or more double or triple bonds, and optionally including one or
more substituents and/or unsubstituted or substituted alkyl,
alkenyl or alkynyl side chains, and
[0023] n is an integer of from 1 to 20.
[0024] Preferably, each Y is independently selected from the group
consisting of alkoxy, thioalkyl, alkyl, haloalkyl, halo, amino,
nitro, optionally substituted aryl, or, when m is 2 or 3, two Y
groups, together with the carbon atoms to which they are attached,
form an aliphatic or aromatic carbocyclic or heterocyclic ring
fused to the aryl ring. More preferably, each Y is independently
selected from methoxy and methyl.
[0025] Preferably, (C).sub.n is an alkyl chain of the formula
(CH.sub.2).sub.n.
[0026] In a particularly preferred embodiment, the
mitochondrially-targete- d antioxidant of the invention has the
formula 4
[0027] Preferably, Z is Br. The above compound is referred to
herein as "mitoquinol". The oxidised form of the compound is
referred to as "mitoquinone".
[0028] In a further aspect, the present invention provides a
pharmaceutical composition suitable for treatment of a patient who
would benefit from reduced oxidative stress which comprises an
effective amount of a mitochondrially-targeted antioxidant of the
present invention in combination with one or more pharmaceutically
acceptable carriers or diluents.
[0029] In a further aspect, the invention provides a method of
reducing oxidative stress in a cell which comprises the step of
administering to said cell a mitochondrially targeted antioxidant
as defined above.
[0030] In still a further aspect, the invention provides a method
of therapy or prophylaxis of a patient who would benefit from
reduced oxidative stress which comprises the step of administering
to said patient a mitochondrially-targeted antioxidant as defined
above.
[0031] Although broadly as defined above, the invention is not
limited thereto but also consists of embodiments of which the
following description provides examples.
DESCRIPTION OF DRAWINGS
[0032] In particular, a better understanding of the invention will
be gained with reference to the accompanying drawings, in
which:
[0033] FIG. 1 is a graph which shows the uptake by isolated
mitochondria of compound 1, a mitochondrially-targeted antioxidant
according to the present invention;
[0034] FIG. 2 is a graph which shows the accumulation of compound 1
by isolated mitochondria;
[0035] FIG. 3 is a graph which shows a comparison of a compound 1
uptake with that of the triphenylphosphonium cation (TPMP);
[0036] FIG. 4 is a graph which shows that compound 1 protects
mitochondria against oxidative damage;
[0037] FIG. 5 is a graph which compares compound 1 with vitamin E
and the effect of uncoupler and other lipophilic cations;
[0038] FIG. 6 is a graph which shows that compound 1 protects
mitochondrial function from oxidative damage;
[0039] FIG. 7 is a graph which shows the effect of compound 1 on
mitochondrial function;
[0040] FIG. 8 is a graph which shows the uptake of compound 1 by
cells;
[0041] FIG. 9 is a graph which shows the energisation-sensitive
uptake of compound 1 by cells; and
[0042] FIG. 10 is a graph which shows the effect of compound 1 on
cell viability.
[0043] FIG. 11 shows the UV-absorption spectrum of
[10-(6'-ubiquinonyl)dec- yltriphenyl-phosphonium bromide] (herein
referred to as "mitoquinone") and of the reduced form of the
compound [10-(6'-ubiquinonyl)decyltriphenylpho- sphonium bromide]
(herein referred to as "mitoquinol").
[0044] FIGS. 12A to 12D show reactions of
[10-(6'-ubiquinonyl)decyltriphen- ylphosphonium bromide]
("mitoquinone") and the reduced form of the compound ("mitoquinol")
with mitochondrial membranes. Beef heart mitochondrial membranes
(20 .mu.g/ml) were suspended in 50 mM sodium phosphate, pH 7.2 at
20.degree. C. In panel A rotenone and antimycin were present and
for the t=0 scan, then succinate (5 mM) was added and scans
repeated at 5 minute intervals as indicated. In panel B A.sub.275
was monitored in the presence of rotenone and antimycin and then
mitoquinone (50 .mu.M) was added, followed by succinate (5 mM) and
malonate (20 mM) where indicated. In Panel C rotenone,
ferricytochrome c (50 .mu.M) and malonate (20 mM) were present,
A.sub.275 was monitored and mitoquinol (50 .mu.M) and myxathiazol
(10 .mu.M) were added where indicated. In panel D A.sub.550 was
monitored and the experiment in Panel C was repeated in the
presence of KCN. Addition of myxathiazol inhibited this rate by
about 60-70%. There was no reaction between mitoquinone and
succinate or NADH in the absence of mitochondrial membranes,
however mixing 50 .mu.M mitoquinone, but not mitoquinol, with 50
.mu.M ferricytochrome c led to some reduction of A.sub.550;
[0045] FIG. 13 shows reactions of mitoquinol and mitoquinone with
pentane-extracted mitochondrial membranes. Pentane extracted beef
heart mitochondria (100 .mu.g protein/ml) were suspended in 50 mM
sodium phosphate, pH 7.2 at 20.degree. C. In Panel A NADH (125
.mu.M) was added and A.sub.340 was monitored and ubiquinone-1
(UQ-1; 50 .mu.M) added where indicated. This was repeated in Panel
b, except that mitoubiquinone (50 .mu.M) was added. In Panel C
pentane extracted mitochondria were incubated with mitoquinone (50
.mu.M), A.sub.275 was monitored and succinate (5 mM) and malonate
(20 mM) added where indicated. In Panel D pentane-extracted
mitochondria were incubated with NADH (125 .mu.M), ferricytochrome
c (50 .mu.M) and A.sub.550 was monitored and mitoquinone (50 .mu.M)
was added where indicated. Addition of myxathiazol inhibited the
rate of reduction by about 60-70%;
[0046] FIG. 14 shows reduction of mitoquinone by intact
mitochondria. Rat liver mitochondria (100 .mu.g/ml) were incubated
in 120 mM KCl, 10 mM HEPES, 1 mM EGTA, pH 7.2 at 20.degree. C. and
A.sub.275 monitored. In panel A rotenone and succinate (5 mM) were
present and mitoquinone (50 .mu.M) was added where indicated. This
experiment was repeated in the presence of malonate (20 mM) or FCCP
(333 nM). In panel B glutamate and malate (5 mM of each) were
present from the start and and mitoquinone (50 .mu.M) was added
where indicated. This experiment was repeated in the presence of
FCCP or with rotenone and FCCP. Addition of TPMP (50 .mu.M) instead
of mitoquinone did not lead to changes in A.sub.275;
[0047] FIG. 15 shows uptake of radiolabelled mitoquinol by
energised rat liver mitochondria and its release on addition of the
uncoupler FCCP; and
[0048] FIG. 16 shows the effect of mitoquinol on isolated rat liver
mitochondria. In A rat liver mitochondria energised with succinate
were incubated with various concentrations of mitoquinol and the
membrane potential determined as a percentage of control
incubations. In B the respiration rate of succinate energised
mitochondria under state 4 (black), state 3 (white) and uncoupled
(stippled) conditions, as a percentage of control incubations.
DESCRIPTION OF THE INVENTION
[0049] As stated above, the focus of this invention is on the
mitochondrial targeting of compounds, primarily for the purpose of
therapy and/or prophylaxis to reduce oxidative stress.
[0050] Mitochondria have a substantial membrane potential of up to
180 mV across their inner membrane (negative inside). Because of
this potential, membrane permeant, lipophilic cations accumulate
several-hundred fold within the mitochondrial matrix.
[0051] The applicants have now found that by covalently coupling
lipophilic cations (preferably the lipophilic triphenylphosphonium
cation) to an antioxidant the compound can be delivered to the
mitochondrial matrix within intact cells. The antioxidant is then
targeted to a primary production site of free radicals and reactive
oxygen species within the cell, rather than being randomly
dispersed.
[0052] In principle, any lipophilic cation and any antioxidant
capable of being transported through the mitochondrial membrane and
accumulated within the mitochondria of intact cells, can be
employed in forming the compounds of the invention. It is however
preferred that the lipophilic cation be the triphenylphosphonium
cation herein exemplified, and that the lipophilic cation is linked
to the antioxidant moiety by a carbon chain having 1 to 30 carbon
atoms, preferably 1 to 20 carbon atoms.
[0053] While it is generally preferred that the carbon chain is an
alkylene group (preferably C.sub.1-C.sub.20, more preferably
C.sub.1-C.sub.15), carbon chains which optionally include one or
more double or triple bonds are also within the scope of the
invention. Also included are carbon chains which include one or
more substituents (such as hydroxyl, carboxylic acid or amide
groups), and/or include one or more side chains or branches,
selected from unsubstituted or substituted alkyl alkenyl or alkynyl
groups.
[0054] In some particularly preferred embodiments, the linking
group is an ethylene, propylene, butylene, pentylene or decylene
group.
[0055] Other lipophilic cations which may covalently be coupled to
antioxidants in accordance with the present invention include the
tribenzyl ammonium and phosphonium cations.
[0056] Preferred antioxidant compounds of the invention, including
those of general formulae I and II as defined above, can be readily
prepared, for example, by the following reaction: 5
[0057] The general synthesis strategy is to heat a halogenated
precursor, preferably a brominated or iodinated precursor (RBr or
RI) in an appropriate solvent with 2-3 equivalents of
triphenylphosphine under argon for several days. The phosphonium
compound is then isolated as its bromide or iodide salt. To do this
the solvent is removed, the product is then triturated repeatedly
with diethyl ether until an off-white solid remains. This is then
dissolved in chloroform and precipitated with diethyl ether to
remove the excess triphenylphosphine. This is repeated until the
solid no longer dissolves in chloroform. At this point the product
is recrystallised several times from methylene chloride/diethyl
ether.
[0058] It will also be appreciated that the anion of the
antioxidant compound thus prepared, which will be a halogen when
this synthetic procedure is used, can readily be exchanged with
another pharmaceutically or pharmacologically acceptable anion, if
this is desirable or necessary, using ion exchange chromatography
or other techniques known in the art.
[0059] The same general procedure can be used to make a wide range
of mitochondrially targeted compounds with different antioxidant
moieties R attached to the triphenylphosphonium (or other
lipophilic cationic) salt. These will include a series of vitamin E
derivatives, in which the length of the bridge linking the
Vitamin-E function with the triphenylphosphonium salt is varied.
Other antioxidants which can be used as R include chain breaking
antioxidants, such as butylated hydroxyanisole, butylated
hydroxytoluene, quinols (including those of formula II as defined
above) and general radical scavengers such as derivatised
fullerenes. In addition, spin traps, which react with free radicals
to generate stable free radicals can also be synthesized. These
will include derivatives of 5,5-dimethylpyrroline-N-oxide,
tert-butylnitrosobenzene, tert-nitrosobenzene,
.alpha.-phenyl-tert-butyln- itrone and related compounds.
[0060] In some preferred embodiments of the invention, the
antioxidant compound is a quinol derivative of the formula II
defined above. A particularly preferred quinol derivative of the
invention is the compound mitoquinol as defined above. Another
preferred compound of the invention is a compound of formula II in
which (C).sub.n is (CH.sub.2).sub.5, and the quinol moiety is the
same as that of mitoquinol.
[0061] Once prepared, the antioxidant compound of the invention, in
any pharmaceutically appropriate form and optionally including
pharmaceutically-acceptable carriers or additives, will be
administered to the patient requiring therapy and/or prophylaxis.
Once administered, the compound will target the mitochondria within
the cell.
[0062] Set out below are synthetic schemes which may be used to
prepare some other specific mitochondrially targeted antioxidant
compounds of the present invention, namely (1) a mitochondrially
targeted version of buckminsterfullerene; (2) a mitochondrially
targeted spin trap compound; and (3) a further synthetic route for
a mitochondrially targeted spin trap compound. 6
[0063] The invention will now be described in more detail with
reference to the following non-limiting examples.
EXAMPLES
Example 1
Experimental
[0064] 1. Synthesis of a Mitochondrially-Targeted Vitamin-E
Derivative (Compound 1)
[0065] The synthesis strategy for a mitochondrially-targeted
vitamin-E derivative (compound 1) is as follows. The brominated
precursor (compound 2)
2-(2-bromoethyl)-3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzop-
yran was synthesized by bromination of the corresponding alcohol as
described by Grisar et al, (1995) (J Med Chem 38, 2880-2886). The
alcohol was synthesized by reduction of the corresponding
carboxylic acid as described by Cohen et al., (1979) (J. Amer Chem
Soc 101, 6710-6716). The carboxylic acid derivative was synthesized
as described by Cohen et al. (1982) (Syn Commun 12, 57-65) from
2,6-dihydroxy-2,5,7,8-tetramethylchrom- an, synthesized as
described by Scott et al., (1974) (J. Amer. Oil Chem. Soc.
101,6710-6716). 7
[0066] For the synthesis of compound 1, 1 g of compound 2 was added
to 8 ml butanone containing 2.5 molar equivalents of
triphenylphosphine and heated at 100.degree. C. in a sealed Kimax
tube under argon for 7-8 days. The solvent was removed under vacuum
at room temperature, the yellow oil triturated with diethyl ether
until an off-white solid remained. This was then dissolved in
chloroform and precipitated with diethyl ether. This was repeated
until the solid was insoluble in chloroform and it was then
recrystallised several times from methylene chloride/diethyl ether
and dried under vacuum to give a white hygroscopic powder.
[0067] 2. Mitochondrial Uptake of Compound 1
[0068] To demonstrate that this targeting is effective, the
exemplary vitamin E compound 1 was tested in relation to both
isolated mitochondria and isolated cells. To do this a
[.sup.3H]-version of compound 1 was synthesized using
[.sup.3H]-triphenylphosphine and the mitochondrial accumulation of
compound 1 quantitated by scintillation counting (FIG. 1) (Burns et
al., 1995, Arch Biochem Biophys 332, 60-68; Burns and Murphy, 1997,
Arch Biochem Biophys 339, 33-39). To do this rat liver mitochondria
were incubated under conditions known to generate a mitochondrial
membrane potential of about 180 mV (Burns et al., 1995; Burns and
Murphy, 1997). Under these conditions compound 1 was rapidly
(<10 s) taken up into mitochondria with an accumulation ratio of
about 6,000. This accumulation of compound 1 into mitochondria was
blocked by addition of the uncoupler FCCP (carbonyl
cyanide-p-trifluoromethoxyphenylhydrazone) which prevents
mitochondria establishing a membrane potential (FIGS. 1 and 2)
(Burns et al., 1995). Therefore compound 1 is rapidly and
selectively accumulated into mitochondria driven by the
mitochondrial membrane potential and this accumulation results in a
concentration of the compound within mitochondria several thousand
fold higher than in the external medium. This accumulation is
rapidly (<10 s) reversed by addition of the uncoupler FCCP to
dissipate the mitochondrial membrane potential after accumulation
of compound 1 within the mitochondria. Therefore the mitochondrial
specific accumulation is solely due to the mitochondrial membrane
potential and is not due to specific binding or covalent
interaction.
[0069] The mitochondrial specific accumulation of compound 1 also
occurs in intact cells. This was measured as described by Burns and
Murphy, 1997 and the accumulation was prevented by dissipating both
the mitochondrial and plasma membrane potentials. In addition,
compound 1 was not accumulated by cells containing defective
mitochondria, which consequently do not have a mitochondrial
membrane potential. Therefore the accumulation of compound 1 into
cells is driven by the mitochondrial membrane potential.
[0070] The accumulation ratio was similar across a range of
concentrations of compound 1 and the amount of compound 1 taken
inside the mitochondria corresponds to an intramitochondrial
concentration of 4-8 mM (FIG. 2). This uptake was entirely due to
the membrane potential and paralleled that of the simple
triphenylphosphonium cation TPMP over a range of membrane
potentials (FIG. 3). From comparison of the uptake of TPMP and
compound 1 at the same membrane potential we infer that within
mitochondria about 84% of compound 1 is membrane-bound (cf. About
60% for the less hydrophobic compound TPMP).
[0071] Further details of the experimental procedures and results
are given below.
[0072] FIG. 1 shows the uptake of 10 .mu.M [.sup.3H] compound 1 by
energised rat liver mitochondria (continuous line and filled
symbols). The dotted line and open symbols show the effect of
addition of 333 nM FCCP at 3 min. Incubation with FCCP from the
start of the incubation led to the same uptake as for adding FCCP
at 3 min (data not shown). Liver mitochondria were prepared from
female Wistar rats by homogenisation followed by differential
centrifugation in medium containing 250 mM sucrose, 10 mM Tris-HCL
(pH 7.4) and 1 mM EGTA and the protein concentration determined by
the biuret assay using BSA as a standard. To measure [.sup.3H]
compound 1 uptake mitochondria (2 mg protein/ml) were suspended at
25.degree. C. in 0.5-1 ml 110 mM KCl, 40 mM Hepes-KOH, pH 7.2, 0.1
mM EDTA supplemented with nigericin (1 .mu.g/ml), 10 mM succinate,
rotenone 1.33 .mu.g/ml and 60 nCi/ml [.sup.3H] compound 1 and 10
.mu.M compound 1. After the incubation mitochondria were pelleted
by centrifugation and the [3H] compound 1 in the supernatant and
pellet quantitated by scintilation counting.
[0073] FIG. 2 shows the mitochondrial accumulation ratios
[(compound 1/mg protein)/(compound 1/.mu.l)] obtained following 3
min incubation of energised rat liver mitochondria with different
concentrations of compound 1 (filled bars) and the effect of 333 nM
FCCP on these (open bars). The dotted line and open circles show
compound 1 uptake by mitochondria, corrected for FCCP-insensitive
binding To measure [.sup.3H] compound 1 accumulation ratio
mitochondria (2 mg protein/ml) were suspended at 25.degree. C. in
0.5-1 ml 110 mM KCl, 40 mM Hepes-KOH, pH 7.2, 0.1 mM EDTA
supplemented with nigericin (1 .mu.g/ml), 10 mM succinate, rotenone
1.33 .mu.g/ml and 6-60 nCi/ml [.sup.3H] compound 1 and 1-50 .mu.M
compound 1. After the incubation mitochondria were pelleted by
centrifugation and the [.sup.3H] compound 1 in the supernatant and
pellet quantitated by scintillation counting.
[0074] FIG. 3 shows a comparison of compound 1 uptake with that of
TPMP at a range of mitochondrial membrane potentials. Energised rat
liver mitochondria were incubated for 3 min with 10 .mu.M compound
1 and 1 .mu.M TPMP and different membrane potentials established
with 0-8 mM malonate or 333 nM FCCP. The accumulation ratios of
parallel incubations with either 60 nCi/ml [.sup.3H] compound 1 or
50 nCi/ml [.sup.3H] TPMP were determined, and the accumulation
ratio for compound 1 is plotted relative to that of TPMP at the
same membrane potential (slope=2.472, y intercept=319, r=0.97).
Mitochondria (2 mg protein/ml) were suspended at 25.degree. C. in
0.5-1 ml 110 mM KCl, 40 mM Hepes-KOH, pH 7.2, 0.1 mM EDTA
supplemented with nigericin (1 .mu.g/ml), 10 mM succinate, rotenone
1.33 .mu.g/ml.
[0075] 3. Anti-Oxidant Efficacy of Compound 1
[0076] The compounds of the invention are also highly effective
against oxidative stress. To demonstrate this, exemplary compound 1
was further tested using rat brain homogenates. The rat brain
homogenates were incubated with or without various concentrations
of the test compounds (compound 1; native Vitamin E
(.alpha.-tocopherol), bromobutyl triphenylphosphonium bromide,
Trolox (a water soluble form of Vitamin E) and compound 2,
ie2-(2-bromoethyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-b-
enzopyran-6-ol, the precursor of compound 1 ("Brom Vit E")) and the
oxidative damage occurring over the incubation was quantitated
using the TBARS assay (Stocks et al., 1974, Clin Sci Mol Med
47,215-222). From this the concentration of compound required to
inhibit oxidative damage by 50% was determined. In this system 210
nM compound 1 inhibited oxidative stress by 50% while the
corresponding value for native Vitamin E was 36 nM. The value for
bromobutyltriphenylphosphonium bromide, which contains the
triphenylphosphonium moiety but not the antioxidant Vitamin E
moiety was 47 .mu.M. These data show that compound 1 is an
extremely effective antioxidant, within an order of magnitude as
effective as Vitamin E. Comparison with
bromobutyltriphenylphosphonium bromide shows that the antioxidant
capability is due to the Vitamin E function and not to the
phosphonium salt. Further details of the experimental procedures
and results are set out below.
[0077] The IC.sub.50 values for inhibition of lipid peroxidation
were determined in rat brain homogenates, and are means .+-. SEM or
range of determinations on 2-3 brain preparations. Octan-1-ol/PBS
partition coefficients are means .+-. SEM for three independent
determinations. N.D. not determined. Partition coefficients were
determined by mixing 200 .mu.M of the compound in 2 ml
water-saturated octanol-1-ol with 2 ml octanol-saturated-PBS at
room temperature for 1 h, then the two layers were separated by
brief centrifugation and their concentrations determined
spectrophotometrically from standard curves prepared in PBS or
octanol. To measure antioxidant efficacy four rat brains were
homogenised in 15 ml 40 mM potassium phosphate (pH 7.4), 140 mM
NaCl at 4.degree. C., particulate matter was pelleted
(1,000.times.g at 4.degree. C. for 15 min) and washed once and the
combined supernatants stored frozen. Aliquots were rapidly thawed
and 5 mg protein suspended in 800 .mu.l PBS containing antioxidant
or ethanol carrier and incubated at 37.degree. C. for 30 min.
Thiobarbituric acid reactive species (TBARS) were quantitated at
532 nm by adding 200 .mu.l conc. HClO.sub.4 and 200 .mu.l 1%
thiobarbituric acid to the incubation, heating at 100.degree. C.
for 15 min and then cooling and clarification by centrifugation
(10,000.times.g for 2 min). The results are shown in Table 1
below.
1TABLE 1 Partition coefficients and antioxidant efficacy of
compound 1 and related compounds IC.sub.50 for inhibition of lipid
Octanol:PBS partition Compound peroxidation (nM) coefficient
Compound 1 210 .+-. 58 7.37 .+-. 1.56 Bromo Vit E 45 .+-. 26 33.1
.+-. 4.4 .alpha.-Tocopherol 36 .+-. 22 27.4 .+-. 1.0 Trolox 18500
.+-. 5900 N.D. BrBTP 47000 .+-. 13000 3.83 .+-. 0.22
[0078] When mitochondria were exposed to oxidative stress compound
1 protected them against oxidative damage, measured by lipid
peroxidation and protein carbonyl formation (FIG. 4). This
antioxidant protection was prevented by incubating mitochondria
with the uncoupler FCCP to prevent uptake of compound 1, and
lipophilic cations alone did not protect mitochondria (FIG. 5).
Most importantly, the uptake of compound 1 protected mitochondrial
function, measured by the ability to generate a membrane potential,
far more effectively than Vitamin E itself (FIG. 6). This shows
that the accumulation of compound 1 into mitochondria selectively
protects their function from oxidative damage. In addition, we
showed that compound 1 is not damaging to mitochondria at the
concentrations that afford protection (FIG. 7).
[0079] The next step was to determine whether compound 1 was
accumulated by intact cells. Compound 1 was rapidly accumulated by
intact 143B cells, and the amount accumulated was greater than that
by .rho..degree. cells derived from 143B cells. This is important
because the .rho..degree. cells lack mitochondrial DNA and
consequently have far lower mitochondrial membrane potential than
the 143B cells, but are identical in every other way, including
plasma membrane potential, cell volume and protein content (FIG.
8); this suggests that most of the compound 1 within cells is
mitochondrial. A proportion of this uptake of compound 1 into cells
was inhibited by blocking the plasma and mitochondrial membrane
potentials (FIG. 9). This energisation-sensitive uptake corresponds
to an intra mitochondrial concentration of compound 1 of about 2-4
mM, which is sufficient to protect mitochondria from oxidative
damage. These concentrations of compound 1 are not toxic to cells
(FIG. 10).
[0080] Further details of the experimental procedures and results
are discussed below.
[0081] FIG. 4 shows the protection of mitochondria against
oxidative damage by compound 1. Mitochondria were exposed to
oxidative stress by incubation with iron/ascorbate and the effect
of compound 1 on oxidative damage assessed by measuring TBARS
(filled bars) and protein carbonyls (open bars). Rat liver
mitochondria (10 mg protein) were incubated at 25.degree. C. in a
shaking water bath in 2 ml medium containing 100 mM KCl, 10 mM
Tris, pH 7.7, supplemented with rotenone (1.33 .mu.g/ml), 10 mM
succinate, 500 .mu.M ascorbate and other additions. After
preincubation for 5 min, 100 .mu.M FeSO.sub.4 was added and 45-55
min later duplicate samples were removed and assayed for TBARS or
protein carbonyls.
[0082] FIG. 5 shows a comparison of compound 1 with vitamin E and
the effect of uncoupler and other lipophilic cations. Energised rat
liver mitochondria were exposed to tert-butylhydroperoxide and the
effect of compound 1 (filled bars), .alpha.-tocopherol (open bars),
compound 1+333 nM FCCP (stippled bars) or the simple lipophilic
cation bromobutyl triphenylphosphonium (cross hatched bars) on
TBARS formation determined. Rat liver mitochondria (4 mg protein)
were incubated in 2 ml medium containing 120 mM KCl, 10 mM
Hepes-HCl pH 7.2; 1 mM EGTA at 37.degree. C. in a shaking water
bath for 5 min with various additions, then tert butyl
hydroperoxide (5 mM) was added, and the mitochondria incubated for
a further 45 min and then TBARS determined.
[0083] FIG. 6 shows how compound 1 protects mitochondrial function
from oxidative damage. Energised rat liver mitochondria were
incubated with iron/ascorbate with no additions (stippled bars), 5
.mu.M compound 1 (filled bars), 5 .mu.M .alpha.-tocopherol (open
bars) or 5 .mu.M TPMP (cross hatched bars), and then isolated and
the membrane potential generated by respiratory substrates measured
relative to control incubations in the absence of iron/ascorbate.
Rat liver mitochondria were incubated at 25.degree. C. in a shaking
water bath in 2 ml medium containing 100 mM KCl, 10 mM Tris, pH
7.7, supplemented with rotenone (1.33 .mu.g/ml), 10 mM succinate,
500 .mu.M ascorbate and other additions. After preincubation for 5
min, 100 .mu.M FeSO.sub.4 was added and after 30 min the incubation
was diluted with 6 ml ice-cold STE 250 mM sucrose, 10 mM Tris-HCL
(pH 7.4) and 1 mM EGTA, pelleted by centrifugation (5 min at
5,000.times.g) and the pellet resuspended in 200 .mu.l STE and 20
.mu.l (=1 mg protein) suspended in 1 ml 110 mM KCl, 40 mM HEPES,
0.1 M EDTA pH 7.2 containing 1 .mu.M TPMP and 50 nCi/ml [3H] TPMP
either 10 mM glutamate and malate, 10 mM succinate and rotenone, or
5 mM ascorbate/100 .mu.M TMPD with rotenone and myxothiazol (2
.mu.g/ml), incubated at 25.degree. C. for 3 min then pelleted and
the membrane potential determined as above and compared with an
incubation that had not been exposed to oxidative stress.
[0084] FIG. 7 shows the effect of compound 1 on the membrane
potential (filled bars) and respiration rate of coupled (open
bars), phosphorylating (stippled bars) and uncoupled mitochondria
(cross hatched bars), as a percentage of values in the absence of
compound 1. The effect of various concentrations of compound 1 on
the membrane potential of isolated mitochondria was determined from
the distribution of [.sup.3H] TPMP by incubating rat liver
mitochondria (2 mg protein/ml) in 0.5 ml medium as above containing
1 .mu.M TPMP and 50 nCi/ml [.sup.3H] TPMP at 25.degree. C. for 3
min. After the incubation mitochondria were pelleted by
centrifugation and the [.sup.3H] TPMP in the supernatant and pellet
quantitated by scintilation counting and the membrane potential
calculated assuming a volume of 0.5 .mu.l/mg proteins and that 60%
of intramitochondrial TPMP is membrane bound. To measure the effect
of compound 1 on coupled, phosphorylating and uncoupled respiration
rates, mitochondria (2 mg protein/ml) were suspended in 120 mM KCl,
10 mM Hepes-HCl pH 7.2, 1 mM EGTA, 10 mM K Pi in a 3 ml Clark
oxygen electrode then respiratory substrate, ADP (200 .mu.M) and
FCCP (333 nM) were added sequentially to the electrode and
respiration rates measured.
[0085] FIG. 8 shows the uptake of compound 1 by cells. Here
10.sup.6 143B cells (closed symbols) or .rho..degree. cells (open
symbols) were incubated with 1 .mu.M [3H] compound 1 and the
compound 1 accumulation ratio determined. Human osteosarcoma 143B
cells and a derived .rho..degree. cell line lacking mitochondrial
DNA were cultured in DMEM/10% FCS (foetal calf serum) supplemented
with uridine and pyruvate under an atmosphere of 5% CO.sub.2/95%
air at 37.degree. C., grown to confluence and harvested for
experiments by treatment with trypsin. To measure [.sup.3H]
compound 1 accumulation cells (10.sup.6) were incubated in 1 ml
HEPES-buffered DMEM. At the end of the incubation, cells were
pelleted by centrifugation, the cell pellet and the supernatant
prepared for scintillation counting and the accumulation ratio
[compound 1/mg protein)/(compound 1/.mu.l)] calculated.
[0086] FIG. 9 shows the amount of compound 1 taken up by 10.sup.6
143B cells over 1 h incubation, corrected for inhibitor-insensitive
binding. Human osteosarcoma 143B cells were incubated in 1 ml
HEPES-buffered DMEM with 1-50 .mu.M compound 1 supplemented with
6-60 nCi/ml [.sup.3H] compound 1. To determine the
energistration-dependent uptake, parallel incubations with 12.5
.mu.M oligomycin, 20 .mu.M FCCP, 10 .mu.M myxathiazol, 100 nM
valinomycin and 1 mM ouabain were carried out. At the end of the
incubation, cells were pelleted by centrifugation and prepared for
scintillation counting and the energisation-sensitive uptake
determined.
[0087] FIG. 10 shows the effect of compound 1 on cell viability.
Here, confluent 143B cells in 24 well tissue culture dishes were
incubated with various concentrations of compound 1 for 24 h and
cell viability measured by lactate dehydrogenase release.
Example 2
Synthesis of [10-(6'-ubiquinolyl)decyltriphenylphosphonium Bromide]
(Herein Referred to as "Mitoquinol")
[0088] Synthesis of Precursors
[0089] To synthesise 11-bromoundecanoic peroxide 11-bromoundecanoic
acid (4.00 g, 15.1 mmol) and SOCl.sub.2 (1.6 mL, 21.5 mmol) were
heated, with stirring, at 90.degree. C. for 15 min. Excess
SOCl.sub.2 was removed by distillation under reduced pressure (15
mm Hg, 90.degree. C.) and the residue (IR, 1799 cm.sup.-1) was
dissolved in diethyl ether (20 mL) and the solution cooled to
0.degree. C. Hydrogen peroxide (30%, 1.8 mL) was added, followed by
dropwise addition of pyridine (1.4 mL) over 45 min. Diethyl ether
(10 mL) was added and the mixture was stirred for 1 h at room
temperature then diluted with diethyl ether (150 mL) and washed
with H.sub.2O (2.times.70 mL), 1.2 M HCl (2.times.70 mL), H.sub.2O
(70 mL), 0.5 M NaHCO.sub.3 (2.times.70 mL) and H.sub.2O (70 mL).
The organic phase was dried over MgSO.sub.4 and the solvent removed
at room temperature under reduced pressure, giving a white solid
(3.51 g). IR (nujol mull) 1810, 1782.
[0090] 6-(10-bromodecyl)ubiquinone was synthesised by mixing crude
material above (3.51 g, 12.5 mmol max), (ubiquinone.sub.0, 1.31 g,
7.19 mmol, Aldrich) and acetic acid (60 mL) and stirring the
mixture for 20 h at 100.degree. C. The mixture was diluted with
diethyl ether (600 mL) and washed with H.sub.2O (2.times.400 mL),
1M HCl (2.times.450 mL), 0.50 M NaHCO.sub.3 (2.times.450 mL) and
H.sub.2O (2.times.400 mL). The organic phase was dried over
MgSO.sub.4. The solvent was removed under reduced pressure, giving
a reddish solid (4.31 g). Column chromatography of the crude solid
on silica gel (packed in CH.sub.2Cl.sub.2) and elution with
CH.sub.2Cl.sub.2 gave the product as a red oil (809 mg, 28%) and
unreacted ubiquinone as a red solid (300 mg, 1.6 mmol, 13%). TLC:
R.sub.f (CH.sub.2Cl.sub.2, diethyl ether 20:1) 0.46; IR (neat)
2928, 2854, 1650, 1611, 1456, 1288; .lambda.max (ethanol): 278 nm;
.sup.1H NMR (299.9 MHz) 3.99 (s, 6H, 2.times.--OCH.sub.3), 3.41 (t,
J=6.8 Hz, 3H, --CH.sub.2--Br), 2.45 (t, J=7.7 Hz, 2H,
ubquinone-CH.sub.2-), 2.02, (s, 3H, --CH.sub.3). 1.89 (quin, J=7.4
Hz, 3H, --CH.sub.2--CH.sub.2--Br), 1.42-1.28 (m, 20H,
--(CH.sub.2).sub.7-); .sup.13C NMR (125.7 MHz) 184.7 (carbonyl),
184.2 (carbonyl), 144.3 (2C, ring), 143.1 (ring), 138.7 (ring),
61.2 (2.times.--OCH.sub.3), 34.0 (--CH.sub.2-), 32.8 (--CH.sub.2-),
29.8 (--CH.sub.2-), 29.4 (2.times.--CH.sub.2-), 29.3
(--CH.sub.2--), 28.7 (2.times.--CH.sub.2-), 28.2 (--CH.sub.2-),
26.4 (--CH.sub.2-), 11.9 (--CH.sub.3). Anal. Calcd. For
C.sub.19H.sub.29O.sub.2Br:C, 56.86; H, 7.28; Found: C, 56.49, H,
7.34; LREI mass spectrum: calcd. For C.sub.19H.sub.29O.sub.2Br
400/402; Found 400/402.
[0091] To form the quinol, 6-(10-bromodecyl)-ubiquinol, NaBH.sub.4
(295 mg, 7.80 mmol) was added to a solution of the quinone (649 mg,
1.62 mmol) in methanol (6 mL) and stirred under argon for 10 min.
Excess NaBH.sub.4 was quenched with 5% HCl (2 mL) and the mixture
diluted with diethyl ether (40 mL). The organic phase was washed
with 1.2 M HCl (40 mL) and saturated NaCl (2.times.40 mL), and
dried over MgSO.sub.4. The solvent was removed under reduced
pressure, giving a yellow oily solid (541 mg, 83%). .sup.1H NMR
(299.9 MHz) 5.31 (s, 1H, --OH), 5.26 (s, 1H, --OH), 3.89 (s, 6H,
2.times.--OCH.sub.3), 3.41 (t, J=6.8 Hz, 2H, --CH.sub.2--Br), 2.59
(t, J=7.7 Hz, 2H ubquinol-CH.sub.2-), 2.15 (s, 3H, CH.sub.3) 1.85
(quin, J=7.4 Hz, 2H, --CH.sub.2--CH.sub.2--Br), 1.44-1.21 (m, 19H,
--CH.sub.2).sub.7-).
[0092] Synthesis of 10-(6'-ubiquinolyl)decyltriphenylphosphonium
Bromide (`Mitoquinol`)
[0093] To synthesise 10-(6'-ubiquinolyl)decyltriphenylphosphonium
bromide. To a 15 mL Kimax tube were added
6-(10-bromodecyl)ubiquinol (541 mg, 1.34 mmol), PPH.sub.3 (387 mg,
1.48 mmol), ethanol (95%, 2.5 mL) and a stirring bar. The tube was
purged with argon, sealed and the mixture stirred in the dark for
88 h at 85.degree. C. The solvent was removed under reduced
pressure, giving an oily orange residue. The residue was dissolved
in CH.sub.2Cl.sub.2 (2 mL) followed by addition of pentane (20 mL).
The resultant suspension was refluxed for 5 min at 50.degree. C.
and the supernatant decanted. The residue was dissolved in
CH.sub.2Cl.sub.2 (2 mL) followed by addition of diethyl either (20
mL). The resultant suspension was refluxed for 5 min at 40.degree.
C. and the supernatant decanted. The CH.sub.2Cl.sub.2/diethyl ether
reflux was repeated twice more. Residual solvent was removed under
reduced pressure, giving crude product as a cream solid (507 mg).
.sub.1H NMR (299.9 MHz) 7.9-7.6 (m, 20H, --P.sup.+ Ph.sub.3), 3.89
(s, 6H, 2 .times.--OCH.sub.3), 3.91-3.77 (m, 2H,
--CH.sub.2--P.sup.+Ph.sub.3), 2.57 (t, J=7.8 Hz, 2H
ubquinol-CH2--), 2.14 (s, 3H, CH.sub.3), 1.6-1.2 (m, 23H,
--(CH.sub.2).sub.8-). .sup.31P NMR (121.4 MHz) 25.1.
[0094] The crude product (200 mg) was oxidized to
10-(6'-ubiquinonyl)decyl- triphenylphosphonium bromide (the
oxidised form) by stirring in CDC 1.sub.3 under an oxygen
atmosphere for 13 days. The oxidation was monitored by .sup.1H NMR
and was complete after 13 days. The solvent was removed under
reduced pressure and the resultant residue dissolved in
CH.sub.2Cl.sub.2 (5 mL). Excess diethyl ether (15 mL) was added and
the resultant suspension stirred for 5 min. The supernatant was
decanted and the CH.sub.2Cl.sub.2/diethyl ether precipitation
repeated twice more. Residual solvent was removed under reduced
pressure, giving crude product as a brown sticky solid (173
mg).
[0095] The quinone was reduced to the quinol by taking a mixture of
crude quinone and quinol (73 mg, ca. 3:1 by 1H NMR) in methanol (1
mL) was added NaBH.sub.4 (21 mg, 0.55 mmol). The mixture was
stirred slowly under an argon atmosphere for 10 min. Excess
NaBH.sub.4 was quenched with 5% HBr (0.2 mL) and the mixture
extracted with CH.sub.2Cl.sub.2. The organic extract was washed
with H.sub.2O (3.times.5 mL). Solvent was removed under reduced
pressure, giving a mixture of quinone and quinol (ca 1:5 by .sup.1H
NMR) as a pale yellow solid (55 mg).For routine preparation of the
quinol form the ethanolic solution, dissolve in 5 vols of water,
(=1 ml) add a pinch of NaBH.sub.4 leave on ice in the dark for 5
min, then extract 3.times.0.5 ml dichloromethane, Wash with
water/HCl etc blow off in nitrogen, dissolve in same vol of etoh
and take spectrum and store at -80 under argon. Yield about 70-80%.
Oxidises rapidly in air so should be prepared fresh. vortex with 1
ml 2M NaCl. Collect the upper organic phase and evaporate to
dryness under a stream of N.sub.2 and dissolve in 1 ml ethanol
acidified to pH 2 .
[0096] Synthesis of [.sup.3H]-10-(640
-ubiquinonyl)decyltriphenylphosphoni- um Bromide
[0097] To a Kimax tube was added 6-(10-bromodecyl)ubiquinol ( 6.3
mg; 15.6 .mu.mol) triphenylphosphine (4.09 mg; 15.6 .mu.mol) and
100 .mu.l ethanol containing [.sup.3H] triphenylphosphine (74
.mu.Ci custom synthesis by Moravek Biochemicals, Brea, CA, U.S.A.,
Spec Ac 1 Ci/mmol) and 150 .mu.l ethanol added. The mixture was
stirred in the dark under argon for 55 h at 80.degree. C. Then it
was cooled and precipitated by addition of 5 ml diethyl ether. The
orange solid was dissolved in few drops of dichloromethane and then
precipitated with diethyl ether and the solid was washed (x4) with
-2 ml diethyl ether. Then dissolved in ethanol to give a stock
solution of 404 .mu.M which was stored at -20.degree. C. The UV
absorption spectrum and TLC were identical to those of the
unlabelled 10-(6'-ubiquinonyl)decyltriphenylphosphonium bromide and
the specific activity of the stock solution was 2.6 mCi/mmol.
[0098] Extinction Coefficients
[0099] Stock solutions of the quinone in ethanol were stored at
-80.degree. C. in the dark and their concentrations confirmed by
31P nmr. The compound was converted to the fully oxidised form by
incubation in basic 95% ethanol over an hour on ice or by
incubation with beef heart mitochondrial membrane at room
temperature, either procedure leading to the same extinction
coefficient of 10,400 M.sup.-1 cm.sup.-1 at the local maximum of
275 nm, with shoulders at 263 and 268 nm corresponding to the
absorption maxima of the triphenylphosphonium moiety (Smith et al,
Eur. J Biochem., 263, 709-716, 1999; Burns et al, Archives of
Biochemistry and Biophysics, 322, 60-68, 1995) and a broad shoulder
at 290 nm due to the quinol (Crane et al, Meth. Enzymol., 18C,
137-165, 1971). Reduction by addition of NaBH4 gave the spectrum of
the quinol which had the expected peak at 290 nm with an extinction
coefficient of 1800 M.sup.-1 cm.sup.-1 and the extinction
coefficient for at 268 nm was 3,000 M.sup.-1 cm.sup.-1 the same as
that for the phosphonium moiety alone (Burns, 1995 above). The
extinction coefficient of 10,400 M.sup.-1 cm.sup.-1 at 275 nm was
lower than that for other quinones which have values of 14,600
M.sup.-1 cm.sup.-1 in ethanol (Crane, 1971 above) and 12,250
M.sup.-1 cm.sup.-1 in aqueous buffer (Cabrini et al, Arch Biochem
Biophys, 208, 11-19, 1981). While the absorbance of the quinone was
about 10% lower in buffer than in ethanol, the discrepancy was not
due to an interaction between the phosphonium and the quinone as
the absorbance of the precursor quinone before linking to the
phosphonium and that of the simple phosphonium
methyltriphenylphosphonium were additive when 50 .mu.M of each were
mixed together in either ethanol or aqueous buffer. The
.DELTA..epsilon..sub.ox- -red was 7,000 M.sup.-1 cm.sup.-1 .
[0100] The spectrum of fully oxidised mitoquinone (50 .mu.M) in 50
mM sodium phosphate, pH 7.2 is shown in FIG. 11. Addition of
NaBH.sub.4 gave the fully reduced compound, mitoquinol. The UV
absorption spectrum of the reduced (quinol) and oxidised (quinone)
mitoquinone/ol are shown in FIG. 11. To determine whether the
mitochondrial respiratory chain could also oxidise or reduce the
compound mitoquinone was incubated with beef heart mitochondrial
membranes (FIG. 12). In panel A the spectrum of fully oxidised
mitoquinone in the presence of antimycin inhibited membranes is
shown (t=0; FIG. 12A). Addition of succinate led to the gradual
reduction of the mitoquinol as measured by repeating the
measurement every five minutes and showing that the peak at 275 nm
gradually disappeared, the presence of antimycin prevented the
oxidation of the quinol by mitochondrial complex III. Succinate did
not lead to the complete reduction of mitoquinone to mitoquinol, as
can be seen by comparing the complete reduction brought about by
borohydride (FIG. 11), instead it reduced about 23% of the added
ubiquinone (FIG. 12A). This is presumably due to equilibration of
the quinol/quinone couple with the succinate/fumarate couple (Em
Q=40 mV at pH 7, Em Suc=30 mV), hence this proportion corresponds
to an Eh of about +8 mV.
[0101] The reduction of mitoquinone can be followed continuously at
A.sub.275 nm (FIG. 12B). On addition to rotenone inhibited
mitochondrial membranes the small amount of mitoquinol remaining
was oxidised leading to a slight increase in A275, but on addition
of the Complex II substrate succinate mitoquinone was rapidly
reduced and this reduction was blocked by malonate, an inhibitor of
Complex II (FIG. 12B). The rate of reduction of mitoquinone was
51.+-.9.9 nmol /min/mg protein, which compares with the rate of
reduction of cytochrome c by succinate in the presence of KCN of
359 nmol/min/mg. Allowing for the 2 electrons required for
mitoquinone reduction compared with 1 for cytochrome c the rate of
electron flux into the mitoquinone pool is of similar order to the
electron flux through the respiratory chain.
[0102] To determine whether mitoquinol was oxidised by Complex III
of the respiratory chain, mitoquinol was added to beef heart
membranes which had been inhibited with rotenone and malonate (FIG.
12C). The mitoquinol was oxidised rapidly by membranes at an
initial rate of about 89.+-.9 nmol mitoquinol/min/mg protein (mean
of 2+/-range) and this oxidation was blocked by myxathiazol an
inhibitor of complex III (FIG. 12C). To confirm that these
electrons were being passed on to cytochrome c, mitoquinol was then
added to membrane supplemented with ferricytochrome c and the rate
of reduction of cytochrome c monitored (FIG. 12D). Addition of
mitoquinol led to reduction of cytochrome c at an initial rate of
about 93 +/-13 nmol/min/mg (mean +/-range). This rate was largely
blocked by myxathiazol, although a small amount of cytochrome c
reduction (about 30-40%) was not blocked by myxathiazol.
[0103] Mitoquinone/ol may be picking up and donating electrons
directly from the active sites of the respiratory complexes, or it
could be equilibrating with the endogenous mitochondrial ubiquinone
pool. To address this question the endogenous ubiquinone pool was
removed from beef heart mitochondria by pentane extraction. In the
absence of endogenous ubiquinone as an electron acceptor the
pentane extracted beef heart mitochondria could not oxidise added
NADH, but addition of ubiquinone-1, a ubiquinone analogue that can
pick up electrons from the active site of complex I, the oxidation
of NADH is partially restored (FIG. 13A). Similarly, addition of
mitoquinone also restored NADH oxidation indicating that
mitoquinone can pick up electrons from the complex I active site
(FIG. 13B). Succinate could also donate electrons to mitoquinone in
pentane extracted beef heart mitochondrial in a malonate sensitive
manner, suggesting that mitoquinone could also pick up electrons
from the active site of Complex II (FIG. 13C). Finally, the effect
of the quinone on the flux of electrons to cytochrome c was
detemined and it was shown that there was no NADH-ferricytochrome c
activity until mitoquinone was added (FIG. 13D), and this was
partially inhibited by myxathizol (60-70%).
[0104] The next step was to see if mitoquinone also accepted
electrons within intact mitochondria (FIG. 14). When mitoquinone
was added to intact energised mitochondria it was rapidly reduced
(FIG. 14A). In the presence of the uncoupler FCCP to dissipate the
membrane potential the rate was decreased about 2-3 fold,
presumably due to the prevention of the uptake of the compound in
to the mitochondria (FIG. 14A). The complex II inhibitor malonate
also decreased the rate of reduction of mitoquinone (FIG. 14A). Use
of the NADH-linked substrates glutamate/malate also led to the
rapid reduction of mitoquinone by intact mitochondria which again
was decreased by addition of the uncoupler FCCP (FIG. 14B). The
Complex I inhibitor rotenone also decreased the rate of reduction
of mitoquinone (FIG. 14B).
[0105] The next step was to see if mitoquinol was accumulated by
energised mitochondria. To do this a tritiated version of the
compound was made, incubated with energised mitochondria and the
amount taken up into the mitochondria determined. It can be seen
that the compound is accumulated rapidly and that this accumulation
is reversed by addition of the uncoupler FCCP (FIG. 15).
[0106] The next assays were to determine the toxicity of these
compounds to mitochondria and cells. To determine the toxicity to
isolated mitochondria the effect on membrane potential and
respiration rate were measured (FIG. 16). It can be seen from FIG.
16 that 10 .mu.M mitoquinol had little effect on mitochondrial
function and at 25 .mu.M and above there was some uncoupling and
inhibition of respiration.
[0107] Industrial Application
[0108] The compounds of the invention have application in selective
antioxidant therapies for human patients to prevent mitochondrial
damage. This can be to prevent the elevated mitochondrial oxidative
stress associated with particular diseases, such as Parkinson's
disease, diabetes or diseases associated with mitochondrial DNA
mutations. They could also be used in conjunction with cell
transplant therapies for neurodegenerative diseases, to increase
the survival rate of implanted cells.
[0109] In addition, these compounds could be used as prophylactics
to protect organs during transplantation, or ameliorate the
ischaemia-reperfusion injury that occurs during surgery. The
compounds of the invention could also be used to reduce cell damage
following stroke and heart attack or be given prophylactically to
premature babies, which are susceptible to brain ischemia. The
methods of the invention have a major advantage over current
antioxidant therapies--they will enable antioxidants to accumulate
selectively in mitochondria, the part of the cell under greatest
oxidative stress. This will greatly increase the efficacy of
antioxidant therapies. Related lipophilic cations are being trialed
as potential anticancer drugs and are known to be relatively
non-toxic to whole animals, therefore these
mitochondrially-targeted antioxidants are unlikely to have harmful
side effects.
[0110] Those persons skilled in the art will appreciate that the
above description is provided by way of example only, and that
different lipophilic cation/antioxidant combinations can be
employed without departing from the scope of the invention.
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