Note: Descriptions are shown in the official language in which they were submitted.
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NITRATE ESTERS AND THEIR USE FOR
MITIGATING CELLULAR DAMAGE
BACKGROUND OF THE INVENTION
This invention relates to nitrate esters and use thereof in mitigating
cellular damage. Particularly, this invention relates to selected organic
nitrates,
most particularly those bearing a sulfur or phosphorus atom R or y to the
nitrate
group, having therapeutic utility as agents that protect tissues from
oxidative
injury.
The nitrate ester glyceryl trinitrate (GTN) or nitroglycerin, has been used
as a vasodilator in the treatment of angina pectoris for over a hundred years,
and the dominant contemporary belief is that GTN exerts its therapeutic effect
through in vivo release of nitric oxide (NO). Other organic nitrates, such as
isosorbide dinitrate, have also been identified as effective and clinically
important vasodilators. NO itself has been identified as Endothelium Derived
Relaxing Factor (EDRF) and several classes of compounds, for example
nitrosothiols, in addition to organic nitrates, have been proposed as NO
donors
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or NO prodrugs. Endogenous stimulation or exogenous administration of NO
have been shown to inhibit production of reactive oxygen species (ROS) and
expression of oxidant-mediated molecular or tissue injury. Well-known
examples of these classes of compounds and one nitrate, GTN itself, have been
suggested to demonstrate neurotoxic or neuroprotective effects by dint of
interactions with the redox modulatory site of the N-methyl-D-aspartate
(NMDA) excitatory amino acid receptor. Thus GTN is firstly a potent
vasodilator and secondly possesses potential neuroprotective properties. In
addition, GTN has been found to suppress renal oxidant damage caused by
potassium bromate (Rahman et al., Redox Rep. 4: 263-9, 1999). Several
attempts have been made to increase the efficacy or potency of alternative
organic nitrates as vasodilators relative to GTN, for example, by
incorporation
of propanolamine or cysteine functionalities. However, no attempt has been
made to separately regulate the vasodilatory and cytoprotective effects of
GTN.
Indeed, postural hypotension and weakness are signs of cerebral ischemia, and
are adverse effects associated with the vasodilatory effects of GTN. Observed
in treatment, these effects are highly contraindicative of GTN itself, and by
extrapolation GTN derivatives (1,2,3-trinitratopropane derivatives), as
clinically useful protective therapeutic agents.
SUMMARY OF THE INVENTION
In as much as the potent vasodilatory effects of organic nitrates may
prove (a) deleterious to, or alternatively (b) synergistic with the protective
effects of GTN, it is postulated herein that regulation of these two effects
is
required for development of new and useful protective therapeutic agents.
Further, it is postulated that such regulation may be achieved through use of
an
appropriate organic nitrate, such as, for example, nitrate esters
incorporating
sulfur-containing or phosphorus-containing functionalities into the structure
of
the nitrate esters.
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Accordingly, a first aspect the invention features nitrate esters having the
Y
X
R3-C R4
Rn
R2- ONO2
I
R
formula: m , or a pharmaceutically acceptable salt
thereof, wherein:
each of m and n is, independently, an integer from 0 to 10;
each of R3, R4, R17 is, independently, hydrogen, a nitrate group, or A;
R' is hydrogen or A;
where A is selected from: a substituted or unsubstituted aliphatic group
having from 1 to 24 carbon atoms in the chain, which optionally contains 1 to
4
0, S, NR6, and/or unsaturations in the chain, optionally bearing from 1 to 4
hydroxy, nitrate, amino, aryl, or heterocyclic groups; an unsubstituted or
substituted cyclic aliphatic moiety having from 3 to 7 carbon atoms in the
aliphatic ring, which optionally contains 1 to 2 0, S, NR6, and/or
unsaturations
in the ring, optionally bearing from 1 to 4 hydroxy, nitrate, amino, aryl, or
heterocyclic groups; an unsubstituted or substituted aliphatic moiety
comprising
a linkage from 0 to 5 carbon atoms between R' and R3 and/or between R17 and
R4, which optionally contains 1 to 2 0, S, NR6, and/or unsaturations in the
linkage, optionally bearing from 1 to 4 hydroxy, nitrate, amino, aryl, or
heterocyclic groups; a substituted or unsubstituted aliphatic group having
from
1 to 24 carbon atoms in the chain, containing linkages selected from C=O, C=S,
and C=NOH, which optionally contains 1 to 4 O, S, NR6, and/or unsaturations
in the chain, optionally bearing from 1 to 4 hydroxy, nitrate, amino, aryl, or
heterocyclic groups; a substituted or unsubstituted aryl group; a heterocyclic
group; an amino group selected from alkylamino, dialkylamino, cyclic amino,
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cyclic diamino, cyclic triamino, arylamino, diarylamino, and alkyarylamino; a
hydroxy group; an alkoxy group; and a substituted or unsubstituted aryloxy
group;
each of Rz, Rs, R' 8 is, independently, hydrogen, A, or X-Y;
where X is F, Br, Cl, NO2 CH2, CF2, 0, NH, NMe, CN, NHOH, N2H3,
NZH2R13, N2HR13R14 , N3, S, SCN, SC(=NH)N(R's)2, SC(=NH)NHR's,
SC(O)N(R's)2, SC(O)NHR's, SO3M, SH, SR7, SO2M, S(O)Rg, S(O)2R9,
S(O)ORg, S(O)20R9, PO2HM, PO3HM, P03M2, P(O)(OR's)(OR16),
P(O)(OR16)(OM), P(O)(R's)(ORg), P(O)(OM)R's, CO2M, CO2H, CO2R",
C(O), C(O)R'Z, C(O)(OR13), POZH, PO2M, P(O)(OR'a), P(O)(R13), SO, SO2,
C(O)(SR13), SRs, SSR7, or SSRs;
Y is F, Br, Cl, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7, CN, NHOH,
N2H3, N2H2R13, N2HR13R'4 , N3, S, SCN, SC(=NH)N(R's)2, SC(=NH)NHR's,
SC(O)N(R's)2, SC(O)NHR's, SO3M, SH, SR7, SOZM, S(O)Rg, S(O)2R9,
S(O)ORB, S(O)20R9, PO2HM, P03M2, P(O)(ORIS)(OR16), P(O)(OR16)(OM),
P(O)(R's)(ORg), P(O)(OM)R's, CO2M, CO2H, CO2R", C(O)R12, C(O)(OR13),
C(O)(SR13), SRs, SSR7, or SSR 5, or does not exist;
each of R6, R7 > R8> R9, R" , R' 2 , R' 3 > R' a > R's , R' 6 is,
independently, an
alkyl or acyl group containing 1-24 carbon atoms, which may contain 1-4
ONO2 substituents; a C1-C6 connection to R'-Ra in a cyclic derivative; a
hydrogen, a nitrate group, or A; and
M is H, Na+, K+, NHa+, or N+HkR"(a_k) where k is 0 to 3, or other
pharmaceutically acceptable counterion;
with the proviso that:
when m= 0; n= 1;
each of R18 and R3 is, independently, H, a nitrate group, or a C1-C4 alkyl
chain, which may include one 0 linking R' 8 and R3 to form a pentosyl,
hexosyl,
cyclopentyl, or cyclohexyl ring, which ring optionally bears a hydroxyl
substituent;
each of R" and R4 is, independently H, a nitrate group, a C,-Ca alkyl,
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optionally bearing 1 to 3 nitrate groups, or an acyl group (-C(O)RS);
each of RS , R6 , R8 , R9, R' z, R' 3, R' a , R's , R' 6 is, independently, an
alkyl
group containing 1 to 12 carbon atoms, which may contain 1 to 4 ON02
substituents; or a C 1 or C2 connection to R' g, R' 7, or R3 in a cyclic
derivative;
each of R7 and R" is, independently, a C1 to C8 alkyl or acyl group;
M is H, Na+, K+, NHa+, or N+HkR"(a_k) where k is 0 to 3; and
X is CH2, 0, NH, NMe, CN, NHOH, NZH3, N2H2R13, N2HR13R'4, N3, S,
SCN, SC(=NH)N(R15)2, SC(=NH)NHR15, SC(O)N(R15)2, SC(O)NHR15, SO3M,
SH, SR7, SO2M, S(O)R8, S(O)2R9, S(O)ORg, S(O)ZOR9, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R15, CO2M,
CO2H, C02R", C(O), C(O)R12, C(O)(OR13), PO2M, P(O)(OR'a), P(O)(R13),
SO, SOz, C(O)(SR13), or SSRa;
then Y is not CN, N2H2R13, N2HR13R14, N3, SCN, SC(=NH)N(R15)2,
SC(=NH)NHR15, SC(O)N(R15)Z, SC(O)NHR15, SO3M, SH, SO2M, P03M2,
PO3HM, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(OM)R15, COZM, CO2H,
C02R", C(O)R12, C(O)(SR13), SR4, SR5, or SSR5, or Y does not exist.
In accordance with the present invention, one skilled in the recognizes
that one of m, n, or p must be equal to at least one.
In one embodiment, the compound of the invention is:
0
02NO S-S 0
P 02N0' ~ S CI ON02 S
'i\ONOz CI H3C N
> >
OEt
O O
02NO S-S OEt O2N0 S\ - O2NO S-S N-
r S \ / / \ /
ON02 ON02 ON02 -
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O _ _
H
F '
2
ON02 ; ONO2 FON02 or
F S-S Q
ON02
G3 2
ONO2 , wherein G' is Me, OMe, Cl, NO2, Br, or H; G is CO2Et,
COZH, CO2Me, CONH2, or CO(CH2)2NEt2; and G3 is Cl, OMe, or CONH2.
In another embodiment, the compound is:
HO2C
NH2
OzNO S-S NH2 O2NO S-S HN-C~l
O
OH ~NH
[Vk] ON02 0 ; ON02 0 \-COzH
H3
02NO S-S HN-~ 02NO S-S N
O
~
OEt
ON02 0 = ON02 0
0
02NO S-S O2NO S4 HO S-S OH
-j \- - CH3
ONO2 0 ON02 ONO2 O2NO
O
O
S-S O2NO S-S - O2NO S-S
r
Et02C
ONO2 O2NO ; ONO2 ONO2
02NO S-S OH O2NO S-S
_j \-~ -j S-)__~ON02
ONOZ O2NO or ONO2 O2NO
In another embodiment, the compound is:
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02NO 02NO S O 'O
02NO ON02 ~S203Na
N(CH3)2
NaO3S2 S2O3Na ; ON02 ON02 ~ ~
02NO S-S1O
CH3
2
O-'~) ON02 O-") ONO
ONO2 ~N~~SO3H. ~,N,,'~,~SO3Na.
> > >
O\ ~O
02NO HN-S
L OZNO OS ON02 HO SSO3Na
NN
~02NO ON02
ON02 CF3 . ONO2 or
, > >
H
02S' N ON02
n
N(CH3)2 , wherein n is 2 or 3.
In another embodiment, the compound is:
0 CH3
O 02NO S
02NO SH 02NO S4 SCH3
CH3 ON02 'O'
ON02 . ON02 F
> > >
O
O 02NO S
O H3C
02NO S ONO2 O
H3C
ONO2
O O
02NO S O2NO S ~
iBu
H3C OMe H3C -
- ~ ONO2
ON02
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F
4FF
O F 02N0 S O2NO S H3C ON02 ONO2 HO
0
CI O2NO
-
02NO S O HN ON02 HN CI
CI CH3 CI
ONO2
O
02NO S HN O2NO S=
H3C CH3
ON02 ONO2 ; or
02NO S
CH3
ON02 0
In another embodiment, the compound is:
ON02
ON02 -
ON02 S ~ / ONO2
SS 0 S / ~
O CH3 S ONO2 O CH3
ONO2
ON02 . ~ . ONOZ 0
> > > >
ON02 -
S ON02
\ /
ON02 S O g
SS--\,-O 01\// ON02 CH3 ONO2 0 H3C
O /
/ \ OI ~
ON02 O S~N or S
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In yet another embodiment, the compound is:
02NO g
OZNO S HO2C S'S 02NO S p
ON02 O2NOJ-S; O2NO ON02
> > >
S-S N ~ ON02
_ O2NO S
S 02NO O
ON02 [IIIam]; 0 ; or HO OH.
In a second aspect, the invention features a compound having the
formula:
Y
X
R3-C Ra
Rn
R2-C ONO2
R'
m , or a pharmaceutically acceptable salt thereof,
containing from 1 to 3 nitrate groups and an S atom in proximity to a nitrate
group,
wherein
each of m and n is, independently, an integer from 0 to 10;
R' is a hydrogen or A;
each of R2, R5, and R18 is, independently, hydrogen or A;
each of R3, R4, and R' 7, is independently, a hydrogen, a nitrate group, or
A;
each of R6 R7 Rg R9 R11, Rlz R13 R1a Rls and R16 is, inde endentl
> > , , , , > > p Y,
A, a hydrogen, a nitrate group, or a C1-C24 alkyl or acyl group, optionally
containing 1-4 ON02 substituents or a CI -C6 linkage to Rl, R2, R3, or R4 in
cyclic derivatives;
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each of R' and R" is, independently, a substituted or unsubstituted C1-
C8 alkyl or acyl group;
A is selected from:
a C1-C24 alkyl group, which optionally contains 1 to 4 0, S, NR6, and/or
unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, Cl, F,
amino,
unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or
unsubstituted or substituted heterocyclic groups, or 1-2 nitrate groups;
a C3-C24 alkyl group, containing 1-5 C=O, C=S, or C=NOR7 linkages,
which optionally contains 1 to 4 0, S, NR6, and/or unsaturations in the carbon
chain, optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino,
unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or
unsubstituted or substituted heterocyclic groups;
a C3-C7 linkage to any of R', R2, R3, R4, or R'7, forming an aliphatic
ring, which optionally contains 1 to 2 0, S, NR6, and/or unsaturations in the
linkage, optionally bearing from 1 to 6 substituents, independently selected
from unsubstituted or substituted alkyl, unsubstituted or substituted aryl,
unsubstituted or substituted CI -C4 alkaryl, unsubstituted or substituted
heteroaryl, unsubstituted or substituted C1-C4 alkheteroaryl, unsubstituted or
substituted heterocyclic, unsubstituted or substituted C1-C4 alkheterocyclic,
hydroxy, nitrate, Cl, F, and amino groups;
a Co-C5 linkage to or between any of R', R3, R4, or R17, which optionally
contains 1 to 2 0, S, NR6, and/or unsaturations in the linkage, bearing two or
more substituents, independently selected from unsubstituted or substituted
alkyl, unsubstituted or substituted aryl, unsubstituted or substituted C1-C4
alkaryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted
CI -
C4 alkheteroaryl, unsubstituted or substituted heterocyclic, unsubstituted or
substituted C1-C4 alkheterocyclic, hydroxy, nitrate, Cl, F, and amino groups;
an unsubstituted Co-C5 linkage to or between any of R', R3, and R4,
which optionally contains 1 to 2 non-adjacent 0, S, NR6, and/or unsaturations
in the linkage;
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a CI -C5 linkage to or between any of R', R3, R4, and R17 containing 1 to
2 C=O, C=S, or C=NOR 7 linkages, which optionally contains 0, S, NR6, and/or
unsaturations in the linkage, optionally bearing from 1 to 4 substituents,
independently selected from unsubstituted or substituted alkyl, unsubstituted
or
substituted aryl, unsubstituted or substituted C1-C4 alkaryl, unsubstituted or
substituted heteroaryl, unsubstituted or substituted CI -C4 alkheteroaryl,
unsubstituted or substituted heterocyclic, unsubstituted or substituted C1-C4
alkheterocyclic, hydroxy, nitrate, Cl, F, and amino groups;
a substituted or unsubstituted aryl group;
a substituted or unsubstituted heteroaryl group;
a substituted or unsubstituted heterocyclic group;
an amino, cyclic amino, diamino, triamino, alkylamino, dialkylamino,
arylamino, diarylamino, or alkylarylamino group;
a hydroxy group;
an alkoxy group; and
a substituted or unsubstituted aryloxy group;
X is F, Br, NO2, CH2 CF2, 0, NH, NMe, NHOH, N2H3, N2H2R13,
N2HR13R14, N3, S, SC(=NH)N(Rl5)2, SC(=NH)NHR15, SC(O)N(R's)2 ,
SC(O)NHR15, SH, SRS, SR7, S(O)R8, S(O)R5, PO2HM, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR), P(O)(OM)R15, COZIVI,
COZH, CO2R", C(O), C(O)(OR13), PO2H, PO2M, P(O)(OR14), P(O)(R13), SO,
SO2, C(O)(SR13), SR5, SR7, or does not exist;
Y is F, Br, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7, NHOH, NZH3,
N2H2R13, N2HR'3R'4, N3, SC(=NH)N(R15)2, SC(=NH)NHR15, SC(O)N(R15)2 ,
SC(O)NHR15, SH, SR7, SO2M, S(O)R8, S(O)R5, PO2HM, PO3M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R'5, CO2M,
CO2H, CO2R5, C(O)(OR13), C(O)(SR13), C(S)R5, C(S)R'Z, C(S)OR12, or does
not exist; and
M is H, Na+, K+, NH4+, N+HkR"(4_k) where k is 0-3, or other
pharmaceutically acceptable counterion;
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and with the proviso that,
when m=0 and n= l;
each of R' g and R3 is, independently, a hydrogen, a nitrate group, or a
C1-C4 alkyl chain, which may include one 0, linking R18 and R3 together to
forrn a pentosyl, a hexosyl, a cyclopentyl, or a cyclohexyl ring, said ring
optionally bearing from 1-4 hydroxyl substituents;
each of R17 and R4 is, independently, a hydrogen, a nitrate group, a C1-
C4 alkyl group, optionally bearing from 1-3 nitrate groups, or an acyl group (-
C(O)Rs);
each of Rs, R6, R8, R9, R12> R13 > R14 > R's > and R16 is, independently, a C1-
C12 alkyl group, optionally bearing from 1-4 ONOZ substituents, or a C1-C2
alkyl linkage to R'8, R17, or R3;
X is F, Br, NO2, CH2 CFz, 0, NH, NMe, NHOH, N2H3, N2H2R13,
N2HR13R14, N3, S, SC(=NH)N(R's)2, SC(=NH)NHR's, SC(O)N(R's)Z ,
SC(O)NHR's, SH, SRs, SR7, S(O)R8, S(O)R5, POZHM, PO3HM, P03M2,
P(O)(OR's)(OR16), P(O)(OR16)(OM), P(O)(R's)(ORg), P(O)(OM)R's, CO2M,
CO2H, COZR", C(O), C(O)(OR13), POZH, POZM, P(O)(OR14), P(O)(R13), SO,
SO2, C(O)(SR13), SRs, SR7; and
Y is not CN, N2H2R13, N2HR13R14, N3, SCN, SC(=NH)N(R's)2,
SC(=NH)NHR's, SC(O)N(R's)2, SC(O)NHR's, SO3M, SH, SO2M, PO3M2,
PO3HM, P(O)(ORIS)(OR16), P(O)(OR16)(OM), P(O)(OM)R's, CO2M, CO2H,
CO2Rs, C(O)R12, C(O)(SR13), SR4, SR5, or SSR5, or Y does not exist.
In one embodiment of the second aspect, the compound is:
02NO S-S
O CH3
02NO S-S ~--N O
>- -~N / Ni~CH3 ON02 O
H3C )~
ON02 0 N H3C
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CH3
02NO S-S CH3
p
I H3C OH
H3C G
CH
ONO2 G_ Br CH3 02NO S-S 3
G = OH \-\ O CH3
G = NH2 O CH3
G = NHCHO ONO2 0
02NO S-S CH3
02NO S-S Y ~N
~CH3
ONO2 0
ONO2 S,,/' N 0 OMe
, or
0
02NO S-S HN-~=O
1-0- NH
ONO2
CI CH3
In another embodiment, the compound is:
p H3C /
OzNO S ~ I
p HN
02NO S -
N / _
ON02 CH3 O
p ON02 H3C
0
O 02NO S CH3
N
02NO S O
/ ONOZ
ON02 H3C- N - ~ CH3
/ -
3
O or CH3O CI .
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In another embodiment, the compound is:
H3C OH
CH3 OzNO S-S CH3
02NO S-S
O CH3 02NO 0
O CH3
02NO 0 or O OMe,
In another embodiment the compound is:
02NO
S 02N0 S H3C S\ 02N0 S\go \ I S S
H3C I S 02NO H3C
02NO ON02 . S= 02NO S O=
> > >
H3C
02NO S X
s ~S S
H3C S
; or 02NO
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In another embodiment, the compound is:
H3C
N H3C
~ \ CH3 N
O2NO S I\ O NO SCH3
02N0 2 I
S ON02
H3C
H3C N
/ N 02N0 /S-~/CF3
~ i
02N0' S ON02. ON02 .
> >
H3C
// N H3C H3C
02NO~/~S ONO2 N N
S ~
02NOts~ CH3 CF3,
ON02 . ON02 .
s
> > >
H3C
S OH H3C CF3
N
N 02N0 S
HO 02NO/ S\ CH3
02NO O S
> > >
H3C / HO \ OH
N I /
~ CF3
02NO S> -
O ~ O2NO ONO2
H3C CH3
t/s N N~CH3 N
O2NO CH3
" S ~ 0,
~~
0 02NO S CH3.
H3C CH3
CH3 N N H3C
~ t N
O NO N~ H3C S S CH3 ~O CH3
2 ~S CH3 02NO CH3 H3C S~-y
CF3 H3C . ON02
> > >
H3C
~ CF3
H3C S~
ON02 .
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O NO N OZNON azz Z s N
O2NO
ON02 , S~
02NO N O2NO N
S~OH S
~ CI
F3C
O2NO N O2NO N
S
S I CF3.
02NO N 02NO N
S I~ CF S I~ CF
3 3
CI
~ /
O2NO / N
O2NO S CH3 S
N
02N0
S
ON02
~ CI ~ I S
CH3 CH3
OZNO S O2NO S NO2
;or
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In another embodiment, the compound is:
H3C CH3
H3C H3C
O OOH $ O I CH3 HgC $ I~ CH3
O 3C CH3 / OH 02NO OH
02NO O2NO H3C H3C
or
S-S
O2NO
O
O OMe
In yet another embodiment of the second aspect, the compound is:
N N
N\ (CF3 ().yCH3 CF3 H3C N\
~ ~~
OzNO" $= ON02 . ON02 . ON02 . 02NO $=
> > > > >
N H3C
C 3
H3C N H3C /~ N
/ H3 S
O2N0 S~ = O2NO 02NO
> > >
/ \ H3C N
O2NO S
or 02NO $"
;
In a third aspect, the invention features a compound having the formula:
/ \ -
02NO H3C N N
$ N_N\ / 3/ \ /
- - CF3 (
$~ S s
~ ~ ON02 02N0
H3C H3C F3C
N \ ~
02NO $; 02NOS N02 ; 02N0~ $
ONOz 02NO ON02 02NO
S-S S-S
O2NO ONO2 02NO ON02
N N
- - ; - - ;
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2 cssIII N S S /S
02N0 S-S S-S ON02; 02NO ONO2
S / I
~
0 OuCH3 0 OuCH3 02N0~- -SOgH
II IOI N N
02NO 0 ~ 02N0
02NO S, S S,
S
02N ONO2 O2NO ON02
02NO or 02NO ONOz
;
In a fourth aspect, the invention features a composition that includes a
pharmaceutically acceptable carrier and a compound having the general
formula:
Y
X
R3 1118]
n
R2-C ON02
R'
m
or a pharmaceutically acceptable salt thereof, containing 1 to 3 nitrate
groups and an S atom in proximity to a nitrate group,
wherein
each of m and n is, independently, an integer from 0 to 10;
R' is a hydrogen or A;
each of RZ, R5, and R18 is, independently, hydrogen or A;
each of R3, R4, and R17, is independently, a hydrogen, a nitrate group, or
A;
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CA 02580627 2007-03-15
WO 2006/029532 PCT/CA2005/001417
each of R6, R7 , R8, R9, R11, R12, R13, RI4, R15, and R16 is, independently,
A, a hydrogen, a nitrate group, or a C1-C24 alkyl or acyl group, optionally
containing 1-4 ON02 substituents or a Cl-C6linkage to Rl, R2, R3, or R4 in
cyclic derivatives;
each of R7 and R" is, independently, a substituted or unsubstituted Cl-
C8 alkyl or acyl group;
A is selected from:
a C1-C24 alkyl group, which optionally contains 1 to 4 0, S, NR6, and/or
unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, Cl, F,
amino,
unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or
unsubstituted or substituted heterocyclic groups, or 1-2 nitrate groups;
a C3-C24 alkyl group, containing 1-5 C=O, C=S, or C=NOR' linkages,
which optionally contains 1 to 4 0, S, NR6, and/or unsaturations in the carbon
chain, optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino,
unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, or
unsubstituted or substituted heterocyclic groups;
a C3-C7 linkage to any of R', R2, R3, R4, or R17, forming an aliphatic
ring, which optionally contains 1 to 2 0, S, NR6, and/or unsaturations in the
linkage, optionally bearing from 1 to 6 substituents, independently selected
from unsubstituted or substituted alkyl, unsubstituted or substituted aryl,
unsubstituted or substituted C1-C4 alkaryl, unsubstituted or substituted
heteroaryl, unsubstituted or substituted C1-C4 alkheteroaryl, unsubstituted or
substituted heterocyclic, unsubstituted or substituted C1-C4 alkheterocyclic,
hydroxy, nitrate, Cl, F, and amino groups;
a Co-C5linkage to or between any of R1, R3, R4, or R17, which optionally
contains 1 to 2 0, S, NR6, and/or unsaturations in the linkage, bearing two or
more substituents, independently selected from unsubstituted or substituted
alkyl, unsubstituted or substituted aryl, unsubstituted or substituted C1-C4
alkaryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted
CI -
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C4 alkheteroaryl, unsubstituted or substituted heterocyclic, unsubstituted or
substituted C1-C4 alkheterocyclic, hydroxy, nitrate, Cl, F, and amino groups;
an unsubstituted Co-C5 linkage to or between any of R', R3, and R4,
which optionally contains 1 to 2 non-adjacent 0, S, NR6, and/or unsaturations
in the linkage;
a CI-C5linkage to or between any of R', R3, R4, and R" containing 1 to
2 C=O, C=S, or C=NOR7 linkages, which optionally contains 0, S, NR6, and/or
unsaturations in the linkage, optionally bearing from 1 to 4 substituents,
independently selected from unsubstituted or substituted alkyl, unsubstituted
or
substituted aryl, unsubstituted or substituted Ct-C4 alkaryl, unsubstituted or
substituted heteroaryl, unsubstituted or substituted CI -C4 alkheteroaryl,
unsubstituted or substituted heterocyclic, unsubstituted or substituted C i-C4
alkheterocyclic, hydroxy, nitrate, Cl, F, and amino groups;
a substituted or unsubstituted aryl group;
a substituted or unsubstituted heteroaryl group;
a substituted or unsubstituted heterocyclic group;
an amino, cyclic amino, diamino, triamino, alkylamino, dialkylamino,
arylamino, diarylamino, or alkylarylamino group;
a hydroxy group;
an alkoxy group; and
a substituted or unsubstituted aryloxy group;
X is F, Br, NOz, CH2 CF2, 0, NH, NMe, NHOH, N2H3, NZH2R' 3,
N2HR13R14, N3, S, SC(=NH)N(R'S)Z, SC(=NH)NHR'S, SC(O)N(R's)2,
SC(O)NHR15, SH, SRS, SR7, S(O)Rg, S(O)R5, PO2HM, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R15, CO21VI,
COZH, CO2R", C(O), C(O)(OR13), PO2H, POZM, P(O)(OR14), P(O)(R13), SO,
SOZ, C(O)(SR13), SR5, SR7, or does not exist;
Y is F, Br, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7 , NHOH, N2H3,
N2H2R13, N2HR13R14, N3, SC(=NH)N(R'S)2, SC(=NH)NHR15, SC(O)N(R15)2 ,
SC(O)NHR15, SH, SR7, SOZM, S(O)R8, S(O)R5, PO2HM, P03M2,
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P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R'S, CO2M,
CO2H, C02R5, C(O)(OR13), C(O)(SR13), C(S)R5, C(S)R'Z, C(S)OR12, or does
not exist; and
M is H, Na+, K+, NH4+, N+HkR"(4_k) where k is 0-3, or other
pharmaceutically acceptable counterion;
and with the proviso that,
when m=0 and n=1;
each of R18 and R3 is, independently, a hydrogen, a nitrate group, or a
C1-C4 alkyl chain, which may include one 0, linking R'g and R3 together to
form a pentosyl, a hexosyl, a cyclopentyl, or a cyclohexyl ring, said ring
optionally bearing from 1-4 hydroxyl substituents;
each of R17 and R4 is, independently, a hydrogen, a nitrate group, a C1-
C4 alkyl group, optionally bearing from 1-3 nitrate, groups, or an acyl group
(-
C(O)RS);
each of RS> R6> Rg, R9, R12, R13 , R1a > R's > and R16 is, independently, a Ci-
C1Z alkyl group, optionally bearing from 1-4 ONOZ substituents, or a C1-C2
alkyl linkage to R' g, R' 7, or R3;
X is F, Br, NO2, CH2 CF2, 0, NH, NMe, NHOH, N2H3, N2H2R' 3,
N2HR13R14, N3, S, SC(=NH)N(R'S)Z, SC(=NH)NHR'S, SC(O)N(R's)2,
SC(O)NHR15, SH, SRS, SR7, S(O)R8, S(O)R5, PO2HM, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(Rls)(ORg), P(O)(OM)R'S, CO2M,
CO2H, CO2R", C(O), C(O)(OR13), PO2H, PO2M, P(O)(OR14), P(O)(R13), SO,
SO2, C(O)(SR13), SR5, SR7; and
Y is not CN, N2H2R13, N2HR13R14, N3, SCN,SC(=NH)N(R")2,
SC(=NH)NHR15, SC(O)N(R15)2 , SC(O)NHR15, SO3M, SH, SO2M, PO3M2,
PO3HM, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(OM)R15, COzM, CO2H,
CO2R5, C(O)R12, C(O)(SR13), SR4, SR5, or SSR5, or Y does not exist.
In a fifth aspect, the invention features a composition that includes a
pharmaceutically acceptable carrier and one of the following compounds:
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O2NO
H3C S N'N ~ 3 N N N
CF3 (
\ S~ S /S
ON02 02NO
> > >
H3C H3C F3C
N ~
0N0 /S 02N0 S N02 02N0~ S
ON02 02NO ON02 02NO
S-S S-S
02N0 ON02 02N0 ON02
N N
- - ; - - ;
F3C
ON02 02N0
/ ~ / /
- - ~ s s s
02N0 S-S S-S ON02; 02NO ON02
S p
I/ 0 O CH 0 OuCH3 y $ II O2NO--_ /--\ __-SO3H
02NO 0 ~ 02N0 O ~ NN
02NO 02NO S,
O2NO S\ ONO O2NO
2
ON02
ON02 ; or 02NO
In a sixth aspect, the invention features a method for preventing or
mitigating tissue and/or cellular damage in a subject by modulating
intercellular
and/or intracellular free radical concentration in the subject by
administering to
the subject an effective amount of a compound containing at least one
aliphatic
nitrate group and at least one sulfur atom in proximity to the nitrate group.
By
"sulfur atom in proximity" or "proximal functional group" is meant a sulfur
atom or functional group that is connected through bonds in a(3, y, or S
relationship to a nitrate ester group (i.e., the atom connectivity is 1,2, or
1,3, or
1,4). The functional group may also be referred to as "proximally located" or
"situated in proximity." Proximal functional groups also include those groups
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WO 2006/029532 PCT/CA2005/001417
that have a through-space intramolecular juxtaposition with a nitrate group
that
is within 3A.
In one embodiment, the tissue and/or cellular damage can be associated
with aging, septic shock, ischemia/reperfusion injury, ulcers, gastritis,
ulcerative colitis, Crohn's disease, diabetes, rheumatoid arthritis, asthma,
cirrhosis of the liver, allograft rejection, encephalomyelitis, meningitis,
pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis,
glomerulonephritis, uveitis, glaucoma, blepharitis, chalazion, allergic eye
disease, comeal ulcer, keratitis, cataracts, age-related macular degeneration,
optic neuritis, ileitis, hemorrhagic shock, anaphylactic shock, bacterial
infection, viral infection, fungal infection, parasitic infection,
hemodialysis,
chronic fatigue syndrome, stroke, toxic shock syndrome, adult respiratory
distress syndrome, cachexia, myocarditis, eczema, psoriasis, dermatitis,
urticaria, cerebral ischemia, systemic lupus erythematosis, chronic
neurodegenerative disease, priapism, cystic fibrosis, schizophrenia,
depression,
premenstrual syndrome, anxiety, addiction, migraine, gastrointestinal motility
disorders, obesity, hyperphagia, hematologic cancers, myelofibrosis, graft-
versus-host disease, CNS trauma, hepatitis, renal failure, chronic hepatitis
C,
drug-induced lung injury (e.g., paraquat), bacterial translocation,
circulatory
shock, traumatic shock, vascular aneurysm, metastatic cancer, or myocardial
infarction.
In another embodiment, the tissue and/or cellular damage can associated
with neurological diseases such as, for example, Parkinson's disease;
Alzheimer's disease; Huntington's disease; multiple sclerosis; amylotrophic
lateral sclerosis; AIDS-induced dementia; epilepsy; alcoholism; alcohol
withdrawal; drug-induced seizures; viral/bacterial/fever-induced seizures;
trauma to the head; hypoglycemia; hypoxia due to myocardial infarction;
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WO 2006/029532 PCT/CA2005/001417
cerebral vascular occlusion; cerebral vascular hemorrhage; hemorrhage; or
environmental excitotoxins of plant, animal, or marine origin.
In yet another embodiment, the tissue and/or cellular damage can be
associated with cytokine therapy, wherein a nitrate ester of the invention is
administered to the subject before, during, and/or after the administration of
the
therapeutic cytokine.
In another embodiment of the sixth aspect, the compound has the
formula:
Y
X
R3-C R4
Rn
R2-C ON02
R
J m , containing from 1 to 3 nitrate groups and an S
atom in proximity to a nitrate group, wherein
m is an integer from 0 to 10;
n is an integer from 0 to 10;
each of R3 4 17 is, independently, hydrogen, a nitrate group, or A;
Rl is hydrogen or A;
A is selected from:
a substituted or unsubstituted C1-C24 alkyl group, optionally containing
1 to 4 0, S, NR6, and/or unsaturations in the chain, optionally bearing from 1
to
4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or
unsubstituted or substituted heterocyclic groups;
an unsubstituted or substituted cyclic moiety having from 3 to 7 carbon
atoms in the ring, which optionally containing 1 to 2 0, S, NR6, and/or
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CA 02580627 2007-03-15
WO 2006/029532 PCT/CA2005/001417
unsaturations in the ring, optionally bearing from 1 to 4 hydroxy, nitrate,
Cl, F,
amino or unsubstituted or substituted aryl, or unsubstituted or substituted
heterocyclic groups;
an unsubstituted or substituted moiety constituting a linkage from 0 to 5
carbons, to or between any of R1, R3, R17 and R4, which optionally contains 1
to
4 0, S, NR6, and/or unsaturations in the linkage, and optionally bearing from
1
to 4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or
unsubstituted or substituted heterocyclic groups;
a substituted or unsubstituted CI-C24 alkyl group, containing 1-4
linkages selected from C=O, C=S, and C=NOH, which optionally contains 0,
S, NR6, and/or unsaturations in the chain, optionally bearing from 1 to 4
hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or
unsubstituted or substituted heterocyclic groups;
a substituted or unsubstituted aryl group;
a substituted or unsubstituted heterocyclic group;
an amino group selected from alkylamino, dialkylamino, cyclic amino,
cyclic diamino, cyclic triamino, arylamino, diarylamino, and alkyarylamino;
a hydroxy group;
an alkoxy group; and
a substituted or unsubstituted aryloxy group;
R2, R5, R' g, are optionally hydrogen, A, or X-Y;
X is F, Br, Cl, NO2, CH2, CF2, 0, NH, NMe, CN, NHOH, N2H3,
N2H2R13, N2HR13R14, N3, S, SCN, SC(=NH)N(R'S)Z, SC(=NH)NHR's,
SC(O)N(R15)2 , SC(O)NHR15, SO3M, SH, SR', SO2M, S(O)R8, S(O)ZR9,
S(O)R5, S(O)2R5, S(O)ORB, S(O)20R9, PO2HM, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R15, CO2M,
COZH, COzR", C(O), C(O)R'Z, C(O)(OR13), PO2H, PO2M, P(O)(OR'a),
P(O)(R13), SO, SO2, C(O)(SR'3), SRS, SSR' or SSRS, SS, or does not exist;
Y is F, Br, Cl, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7, CN, NHOH,
N2H3, NZH2R'3, N2HR'3R14, N3, S, SCN, SC(=NH)N(R15)2, SC(=NH)NHR's,
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SC(O)N(R's)2, SC(O)NHR's, SO3M, SH, SR7, SO2M, S(O)R8, S(O)2R9,
S(O)ORg, S(O)Rs, S(O)2Rs, S(O)2OR9, PO2HM, P03M2, P(O)(OR's)(OR16),
P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R's, CO2M, COZH, C02Rs,
C(O)R'Z, C(O)(OR13), C(O)(SR13), SRs, SSR7 or SSRs, C(S)Rs, C(S)R12,
C(S)OR'Z, or does not exist;
each of R6 , R7 , Rg, R9, R" , R12 , R' 3 , R' a , R's , and R' 6 is,
independently, a
C1-C24 alkyl group, optionally containing 1-4 ON02 substituents, a C1-C24 acyl
group, optionally containing 1-4 ON02 substituents, a CI -C6 ring-forming
connection to any of R1-R4, a hydrogen, a nitrate group, or A; and
M is H, Na+, K+, NHa+, N+HkRI'(a_k) where k is 0-3, or other
pharmaceutically acceptable counterion;
and with the proviso that,
when m=0; n=1;
each of R18 and R3 is, individually, H, a nitrate group,or a C1- C4 alkyl
group, which may include one 0, linking R'g and R3 to form pentosyl, hexosyl,
cyclopentyl, or cyclohexyl rings, which optionally bears hydroxyl
substituents;
each of R17 and Ra is, individually, H, a nitrate group, a C1-C4 alkyl
group, optionally bearing from 1-3 nitrate groups, or -C(O)Rs;
each of Rs R6 Rg R9 R12 R13 R'a R's R16 is, individually, a C-C
~ , , > > > > > i ia
alkyl group, optionally containing 1-4 ON02 substituents or a C1-C2 ring-
forming connections to R18,R17, or R 3 ;
each of R7 and R" is, independently, a CI-C8 alkyl group or a C1-C8
acyl group;
M is H, Na+, K+, NHa+, N+HkR"(a_k) where k is 0-3;
X is CH2, 0, NH, NMe, CN, NHOH, N2H3, NZHZR13, N2HR13R14, N3, S,
SCN, SC(=NH)N(R's)2, SC(=NH)NHR's, SC(O)N(R's)2 , SC(O)NHR's, SO3M,
SH, SR7, SO2M, S(O)R8, S(O)ZR9, S(O)ORB, S(O)20R9, PO3HM, P03M2,
P(O)(OR's)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)R's, CO2M,
CO2H, CO2R", C(O), C(O)R12, C(O)(OR13), PO2M, P(O)(OR'a), P(O)(R13),
SO, SO2, C(O)(SR13), SR 5, or SSRa; and
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WO 2006/029532 PCT/CA2005/001417
Y is not CN, N2H2R13, N2HR13R14, N3, SCN, SC(=NH)N(R's)Z,
SC(=NH)NHR15, SC(O)N(R15)2 , SC(O)NHR15, SO3M, SH, SO2M, P03M2,
PO3HM, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(OM)R15, CO2M, CO2H,
C02R5, C(O)R'Z, C(O)(SR13), SR4, SR5, or SSR5, or Y does not exist.
In another embodiment, the compound contains at least 2 nitrate groups.
In another embodiment, the nitrate is beta or gamma to said sulfur atom.
In another embodiment of the sixth aspect, the compound can be any of
the compounds that were cited individually as an embodiment of either the
first
or second aspect of the invention.
In another embodiment, the compound is:
02NO 02NO 02NO
SCN S02Ph SS03M
ON02 ON02 ON02
02N0 02N0 S_S 0S 0
NCSH2C CH2SCN NCSH2C CH2Br ~
ON02 ON02 O2NO ON02> = 02N0 ON02 .
> > >
SCN
02NO 02NO NCSH2C---/ 02NO
\-__/SO3Na V__/SSO3Na ON02 V~--/SCOCH3
00 0,0 0\0 0
S S-S S p S P.
~ u ~
02N0 = 02N0 = 02NO Br = 02N0 = 02N0
> > > > >
NH2
N I N 02NO ON02
O2NO O~ N \S) OZNO S-S ON02
~ O ~ O2NO ON02 ON02 02N0
S ,
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WO 2006/029532 PCT/CA2005/001417
H
N
ON02 \
O N OZNO S
03S '-~ ON02 ~ ~ =
02NO ONOZ
H3C
tN CFg S S \ / / H3C
S H3C N
/
0NO S 02N0 /S\N02
ON02 02NO >
> > >
ON02 02NO
F3C S-T
02
~N 02NO ~ ~ ON
02N0 S-
ON02 02NO S
ON02 02NO
02N0 \S-S ONO2 - - ~
N N
- 02NO S-S S-S ON02;
/
S ~
I
F3C C
~/ N /O O CH
S S S ~ a
02NO ONOZ ~ 02NO 0
S p
0 OuCH3
02NO--\_ __/-SO3H
02N0 0
02NO 02NO
02NO S\ ONO 02NO S
2
ONO2
ON02 ~ or 02NO
In yet another embodiment of the sixth aspect, the compound is:
02NO ON02
N S-S
a'~
02NO S or ON02 02NO
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As used herein, the terms "alkyl" and the prefix "alk-" are inclusive of
both straight chain and branched chain saturated or unsaturated groups, and of
cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unless otherwise
specified, acyclic alkyl groups are from 1 to 6 carbons and contain at least
one
C-H bond. The number of carbons in an alkyl group refers to the total number
of carbons contained in the group. Cyclic groups can be monocyclic or
polycyclic and preferably have from 3 to 8 ring carbon atoms. Exemplary
cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl
groups. Unless otherwise indicated, alkyl groups may be substituted or
unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl,
alkylthio, arylthio, halogen, hydroxyl, fluoroalkyl, perfluoralkyl, amino,
aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl,
and carboxyl groups.
By "aryl" is meant a carbocyclic aromatic ring or ring system. Unless
otherwise specified, aryl groups are from 6 to 18 carbons. . Examples of aryl
groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl groups.
By "heterocycle" is meant an aromatic or non-aromatic ring system that
contains at least one ring heteroatom (e.g., 0, S, N). The term "heteroaryl"
refers to an aromatic heterocyclic ring or ring system that contains at least
one
ring heteroatom (e.g., 0, S, N). Unless otherwise specified, heteroaryl rings
contain from 1 to 9 carbons. Exemplary heteroaryl groups include furanyl,
thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl,
isothiazolyl, triazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl,
pyrimidyl,
pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole,
indazolyl,
indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl,
quinazolinyl,
naphtyridinyl, phthalazinyl, phenanthrolinyl, purinyl, and carbazolyl rings or
ring systems.
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Unless otherwise specified, non-aromatic heterocyclic groups are from 2
to 9 carbons and can include, for example, dihydropyrrolyl,
tetrahydropyrrolyl,
piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl,
tetrahydrofuranyl, dihydrothiophene, tetrahydrothiophene, and morpholinyl
groups. As defined in the present application, the definition of heterocycle
specifically excludes R-lactam rings.
Unless otherwise specified, aryl, heteroaryl, or heterocyclic groups may
be unsubstituted or substituted by one or more substituents selected from the
group consisting of C 1_6 alkyl, hydroxy, halo, nitro, C 1_6 alkoxy, C1_6
alkylthio,
trifluoromethyl, C1_6 acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, C1_6
alkoxycarbonyl, arylalkyl (wherein the alkyl group has from 1 to 6 carbon
atoms) and heteroarylalkyl (wherein the alkyl group has from 1 to 6 carbon
atoms).
By "halide" or "halogen" or "halo" is meant bromine, chlorine, iodine,
or fluorine. As used herein, the terms "alkyl" and the prefix "alk-" are
inclusive
of both straight chain and branched chain saturated or unsaturated groups, and
of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unless otherwise
specified, acyclic alkyl groups are from 1 to 6 carbons. Cyclic groups can be
monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms.
Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and
adamantyl groups. Alkyl groups may be substituted or unsubstituted.
Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio,
arylthio,
halogen, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl,
disubstituted
amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
By "alkoxy" is meant a chemical substituent of the formula -OR, where
R is an alkyl group. By "aryloxy" is meant a chemical substituent of the
formula -OR', where R' is an aryl group. By "alkaryl" is meant a chemical
CA 02580627 2007-03-15
WO 2006/029532 PCT/CA2005/001417
substituent of the formula -RR', where R is an alkyl group and R' is an aryl
group, By "alkheteraryl" is meant a chemical substituent of the formula RR",
where R is an alkyl group and R" is a heteroaryl group.
What is meant by "aliphatic nitrate" is a nitrate group that is connected
to a non-aromatic carbon.
What is meant by "antioxidant" is a compound that attenuates or
prevents oxidation of a target biomolecule through the inhibition of the
initiation or propagation steps that constitute oxidative and peroxidative
processes.
"Mitigating neurodegeneration" as used herein involves affecting
neuroprotection, inhibiting or preventing neurodegeneration, and/or
ameliorating the manifestations or impact of neurodegeneration. Such
amelioration includes effecting cognition enhancement, as is quantified by
tests
known in the art (e.g., Venault et al., 1992, incorporated herein by
reference).
"Modulating" a biological process as used herein (for example, modulating
activity of the non-glutamate neuroreceptors), encompasses both increasing
(positively modulating) and decreasing (negatively modulating) such activity,
and thus inhibition, potentiation, agonism, and antagonism of the biological
process.
It is an object of the present invention to provide provide novel organic
nitrates, including aliphatic nitrate esters bearing a sulfur or phosphorus
moiety
P or y to a nitrate group, or congeners thereof. Another object of the present
invention is to provide methods for making such novel organic nitrates.
Another object of the invention is to provide methods for affecting
neuroprotection, mitigating neurodegeneration, affecting cognition
enhancement, and/or protecting tissues from oxidative injury employing
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selected organic nitrates. Another object of the present invention is to
provide
novel drugs as neuroprotective agents. Another another object of the present
invention is to provide novel drugs for use in cognition enhancement. Another
object of the invention is to provide novel drugs for use in protecting
tissues
from oxidative injury.
It will be understood by those skilled in the art, that when organic
radicals, such as R", X, or Y, are represented as "does not exist," that the
valency of the carbon bound to the radical is adjusted accordingly (i.e., by a
point of unsaturation).
Compounds may be constructed according to formulas of the invention
in which an S atom is appropriately proximally placed with respect to a
nitrate
functional group, but in which a carbonyl group intervenes to form a thioester
linkage. Such compounds do not form part of the invention if cleavage of this
thioester bond produces two entirely separate molecules, one containing the
nitrate funtionality, and another the S-functionality. An example of such a
compound is one containing the motif, O2NOCH2C(CH3)2C(O)S-(organic
radical), which would liberate on thioester cleavage an aliphatic nitrate that
does not contain a S-functionality. It is understood by one skilled in the art
that
facile cleavage of a thioester in an aqueous biological milieu will render an
aliphatic nitrate that does not contain a proximal S, and therefore, such a
thioester does not comprise part of the current invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure I is a synthetic scheme showing the use of Bunte salt IVd in the
preparation of compounds Va, IVr, Vbb, and Vbc.
Figure 2 is a graph showing a comparison of 50 M GTN (stippled
bars), 10 M Va (cross-hatched bars), 30 M Va (upward hatched bars) and 50
M Va (downward hatched bars) on lactate dehydrogenase (LDH) release from
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isolated perfused rat heart after left coronary artery occlusion (LCAO) for 45
min followed by reperfusion for 90 min. Hearts were perfused with drugs for
ten min prior to and throughout the period of LCAO. Data are the mean ~
standard errors (n= 3-6).
Figure 3 is a graph showing a comparison of 50 M GTN (stippled
bars), 50 M Va (hatched bars) and 50 M compound IIIam (cross-hatched
bars) on lactate dehydrogenase (LDH) release from isolated perfused rat heart
after left coronary artery occlusion (LCAO) for 45 min followed by reperfusion
for 90 min. Hearts were perfused with drugs for ten min prior to and
throughout the period of reperfusion. Data are the mean standard errors (n=
4-6).
Figure 4 is a graph showing a comparison of 50 gM GTN (open bar) and
50 M Va (hatched bar) on infarct size in isolated perfused rat heart after
left
coronary artery occlusion (LCAO) for 45 min followed by reperfusion for 90
min. Hearts were perfused with drugs for ten min prior to and throughout the
period of LCAO. Data are the mean standard errors (n= 5-6).
Figure 5 is a graph showing a comparison of 50 M GTN (open bar), 50
M Va (hatched bar) and 50 M compound IIIam (cross-hatched bar) on infarct
size in isolated perfused rat heart after left coronary artery occlusion
(LCAO)
for 45 min followed by reperfusion for 90 min. Hearts were perfused with
drugs for ten min prior to and throughout the period of reperfusion. Data are
the mean standard errors (n = 3-6).
Figure 6 is a graph showing a comparison of 50 M GTN (stippled bars)
and 50 M Va (hatched bars) on coronary perfusion pressure in isolated
perfused rat heart after left coronary artery occlusion (LCAO) for 45 min
followed by reperfusion for 90 min. Hearts were perfused with drugs for ten
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min prior to and throughout the period of LCAO. Coronary perfusion pressure
was recorded just prior to LCAO (baseline), at the beginning of reperfusion (0
min reperfusion) and at the end of the 90 min reperfusion period (90 min
reperfusion). Data are the mean standard errors (n = 6).
Figure 7 is an immunocytochemical analysis of tyrosine hydroxylase
immunoreactivity in the substantia nigra of rats. Figure 7A is an analysis of
a
brain section in which the animal was administered the neurotoxic compound 6-
hydroxydopamine (6-OHDA). Figure 7B is an analysis of a brain section in
which the animal was administered only vehicle control (dimethyl sulfoxide).
Figure 7C is an analysis of a brain section in which the animal was treated
with
compound Va before and after administration of 6-OHDA. Figure 7D is an
analysis of a brain section in which the animal was administered only vehicle
control. .
Figure 8 is a graph showing the inhibition and potentiation of lipid
peroxidation induced by FeSO4 (50 M) as assessed by TBARS (thiobarbituric
acid reactive substances) determination, on incubation of rat brain
synaptosomes with ascorbic acid or (x-tocopherol (n=3). Percentage TBARS
detected is given relative to TBARS in presence of FeSO4 (100%). "Control"
experiment is in the presence of vehicle and absence of FeSO4. Error bars show
S.E.M.
Figure 9 is a graph showing the inhibition of lipid peroxidation induced
by FeSO4 (50 M) as assessed by TBARS determination, on incubation of rat
brain synaptosomes with Trolox (n=3). Percentage inhibition is given relative
to: control TBARS in presence of vehicle and absence of FeSO4 (100%);
TBARS in presence of vehicle and FeSO4 (0%). Error bars show S.E.M; data is
fitted to a sigmoidal curve (EC50 = 6.8 x 10-5 M).
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Figure 10 is a graph showing the inhibition of lipid peroxidation induced
by FeSO4 (50 M) as assessed by TBARS determination, on incubation of rat
brain synaptosomes with lipoic acid (LA, dashed line) or dihydrolipoic acid
(LAH2, solid line) (n=3). Percentage inhibition relative to: control TBARS in
presence of vehicle and absence of FeSO4 (100%); TBARS in presence of
vehicle and FeSO4 (0%). Error bars show S.E.M.
Figure 11 is a graph showing the inhibition of lipid peroxidation induced
by FeSO4 (50 M) as assessed by TBARS determination, on incubation of rat
brain synaptosomes with: GTN (varied) + LAH2 (1 mM) (~, solid line); Va (V,
dashed line); or Va (varied) + LAH2 (1 mM) (A, dashed line), (n=3).
Percentage inhibition relative to: control TBARS in presence of vehicle and
absence of FeSO4 (100%); TBARS in presence of vehicle, adjuvants and FeSO4
(0%). In experiments with adjuvant LAH2, 0% inhibition corresponds to
TBARS in presence of vehicle containing LAH2. Error bars show S.E.M.
Figure 12 is a graph showing the inhibition of lipid peroxidation induced
by FeSO4 (50 M) as assessed by TBARS determination, on incubation of rat
brain synaptosomes with: Va (varied) + cysteine (1mM) (~, solid line); or Va +
PhSH (1 mM) (A, solid), (n=3). For comparison, inhibition curve with
NONOate, DETA/NO (in the absence of adjuvants; =, dashed line) is shown.
Percentage inhibition relative to controls :TBARS in presence of vehicle and
absence of FeSO4 (100%); TBARS in presence of vehicle, adjuvant and FeSO4
(0%). In both experiments with adjuvant thiols, 0% inhibition corresponds to
TBARS in presence of vehicle containing thiol (1mM). Error bars show S.E.M.
Figure 13 is a graph showing the inhibition of lipid peroxidation induced
by FeSO4 (50 M) as assessed by TBARS detennination, on incubation of rat
brain synaptosomes with: IVr (varied) (~); or Va + PhSH (1 mM) (A), (n=3).
Percentage inhibition relative to: control TBARS in presence of vehicle and
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absence of FeSO4 (100%); TBARS in presence of vehicle, adjuvant, and FeSO4
(0%). In experiments with thiol, 0% inhibition corresponds to TBARS in
presence of vehicle containing adjuvant PhSH. Error bars show S.E.M.
Figure 14 is a graph showing the effects of NO-donors. Inhibition of
lipid peroxidation induced by FeSO4 (50 M) as assessed by TBARS
determination, on incubation of rat brain synaptosomes with: (a) DEA/NO
(diethylamine NONOate, ~, solid line); and Sper/NO (spermine NONOate, ~,
dashed lines), (n=3). Percentage inhibition relative to: control TBARS in
presence of vehicle, adjuvant, and absence of FeSO4 (100%); TBARS in
presence of vehicle and FeSO4 (0%). Error bars show S.E.M. Data is fitted to
sigmoidal curves (EC50: DEA/NO = 7 x 10-5 M; Sper/NO = 2 x 10-4 M).
Figure 15 is a graph showing the inhibition of lipid peroxidation induced
by FeSO4 (50 M) as assessed by TBARS determination, on incubation of rat
brain synaptosomes with i-amyl nitrite (IAN, ~ solid line) (n=3). Percentage
inhibition relative to: control TBARS in presence of vehicle and absence of
FeSO4 (100%); TBARS in presence of FeSO4 (0%). Data is fitted to a sigmoidal
curve: EC50 = 1.6 x 10-4 M.
Figure 16 is a graph showing the comparison of inhibition by nitrate IVs
in: (1) FeSO4 (50 M) induced lipid peroxidation in rat brain synaptosomes
assessed by TBARS determination (0 dashed line); and, (2) ABAP induced
lipid peroxidation in liposomes assessed by BODIPY peroxidation (= solid
line) (n=3). Percentage inhibition relative to: control response in presence
of
vehicle and absence of initiator (100%); response in presence of vehicle and
initiator (0%). Data is fitted to a sigmoidal curve: EC50 (synaptosomes) = 1.0
x
10"3 M, (liposomes) = 1.3 x 10"4 M.
Figure 17 is a graph showing the comparison of inhibition of lipid
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peroxidation induced by FeSO4 (50 M) as assessed by TBARS determination,
on incubation of rat brain synaptosomes with nitrate Vbb (V solid line) and
nitrate Vbc (~ solid line), compared to Trolox (o dashed line) (n=3).
Percentage inhibition relative to: control TBARS in presence of vehicle and
absence of FeSO4 (100%); TBARS in presence of FeSO4 (0%). Data is fitted to
a sigmoidal curve: ECso Vbb = 2.0 x 10"5 M; ECso Vbc = 7 x 10-7 M and 7 x 10"
s
M).
DETAILED DESCRIPTION OF THE INVENTION
This invention pertains to methods and compositions useful for treating
neurodegeneration or preventing or mitigating tissue and/or cellular damage by
administering to a subject a therapeutic nitrate ester. Neuroprotection and/or
cognition enhancement can be affected, for example, by modulating an
interaction with guanylyl cyclase (GCase, the enzyme responsible for cGMP
production in various areas of the brain), modulating a glutamate or non-
glutamate neuroreceptor or attenuating free radical damage. The attenuation of
free radical concentration by a nitrate ester of the invention can also be
useful
for preventing or mitigating tissue and/or cellular damage.
According to certain aspects of the invention, neurodegeneration is
mitigated by stimulating cerebral GCase. One of the major targets for the
novel
organic nitrates of the invention is GCase activation, resulting in the
production
of cGMP. Experimental evidence obtained in a number of in vitro model
systems supports the notion that elevated levels of cGMP help to prevent
apoptotic (programmed) cell death. Thus, a cGMP-dependent mechanism
significantly increases the survival of trophic factor-deprived PC 12 cells
and rat
sympathetic neurons (Farinelli et al., 1996), and of primary cultures of rat
embryonic motor neurons (Estevez et al., 1998). The mechanism of action for
selected organic nitrates in preventing apoptotic cell death may be inhibition
of
caspase-3 activation indirectly through elevations in cGMP levels or directly
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via protein S-nitrosylation of the enzyme by an NO-intermediate (Kim et al.,
1997). Caspase-3 is a member of the cysteine protease family of enzymes that
are essential for the execution step in apoptosis (Cohen, 1997; Nicholson and
Thornberry, 1997). Activation of caspase-3 is required for apoptotic cell
death
in trophic factor-deprived PC 12 cells (Haviv et al., 1997) and in glutamate-
mediated apoptotic cell death of cultured cerebellar granule neurons (Du et
al.,
1997). In animal models of cerebral ischemia, caspase-3 activity is induced
and
may be responsible for the apoptotic component of delayed neuronal cell death
(Chen et al., 1998; Namura et al., 1998; Ni et al., 1998). Inhibitors of
caspase-3
significantly decrease the apoptotic component of delayed neuronal cell death
in response to ischemic injury both in vitro (Gottron et al., 1997) and in
vivo
(Endres et al., 1998). A secreted region of the Alzheimer's disease (3-amyloid
precursor protein lowers intracellular calcium levels and provides
neuroprotective effects on target cells through increases in cGMP levels and
activation of protein kinase G (Barger et al., 1995; Furukawa et al., 1996).
In
preferred embodiments of the methods of the invention, nitrated molecules that
have the capacity to activate GCase directly or via release of an NO-
containing
intermediate are used to modulate GCase activity.
According to certain other aspects of the invention, cognition
enhancement (e.g., improved memory performance) is achieved by stimulating
cerebral GCase. Several lines of experimental evidence support the notion that
GCase and cGMP are involved in the formation and retention of new
information. cGMP has been directly implicated in both long-term potentiation
(LTP) and long-term depression (LTD), which are proposed cellular models for
learning and memory (Arancio et al., 1995; Wu et al., 1998). In animal models,
elevation of hippocampal cGMP levels leading to increased protein kinase G
activity has been shown to be important for retention and consolidation of new
learning (Bernabeu et al., 1996, 1997). Thus, stimulation of cerebral GCase
activity is expected to improve learning and memory performance in
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individuals in whom cognitive abilities are impaired by injury, disease, or
aging.
We have shown that novel organic nitrate esters have differential effects
to activate soluble GCase and to cause cGMP accumulation in vascular and
brain tissue. There is a clear dissociation between the vascular relaxation
effects of organic nitrate esters and ability to affect neuroprotection.
Activation
of GCase and accumulation of cGMP have been shown to be important in the
neuroprotection of hippocampal brain slices subjected to a period of in vitro
ischemia.
Cerebral ischemia results in marked increases in the release of the
excitatory amino acid glutamate in the affected brain region (Bullock et al.,
1998; Huang et al., 1998; Yang et al., 1998). In both humans (Bullock et al.,
1998) and experimental animals (Huang et al., 1998; Goda et al., 1998; Yang et
al., 1998), the amount of glutamate released during ischemia is positively
correlated with the extent of brain injury. In experimental animal models of
cerebral ischemia, decreased release of glutamate during ischemia (Goda et
al.,
1998) or blockade of glutamate receptors with antagonists (Ibarrola et al.,
1998;
O'Neill et al., 1998; Umemura et al., 1997) significantly reduces the extent
of
brain injury. However, these interventions are only effective when given prior
to or during the ischemic insult. To be broadly useful, a therapeutic
intervention is preferably effective when administered after the period of
ischemia. We have designed a class of novel organic nitrate esters having high
efficacy in effecting neuroprotection in vivo in models of transient global
and
focal cerebral ischemia when given after the ischemic insult. It will be
appreciated, therefore, that these selected organic nitrates can be used for
treatment of conditions including but not limited to: stroke; Parkinson's
disease; Alzheimer's disease; Huntington's disease; multiple sclerosis;
amylotrophic lateral sclerosis; AIDS-induced dementia; epilepsy; alcoholism;
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alcohol withdrawal; drug-induced seizures; viral/bacterial/fever-induced
seizures; trauma to the head; hypoglycemia; hypoxia; myocardial infarction;
cerebral vascular occlusion; cerebral vascular hemorrhage; hemorrhage;
environmental excitotoxins of plant, animal, or marine origin; and the like.
The direct effects of selected organic nitrates on amino acid
neurotransmitter receptors has been tested using the Xenopus oocyte expression
system and two-electrode voltage-clamp recording methods. Selected organic
nitrates were found to have direct, modulatory effects on GABAA receptor
function (see Working Examples below). These allosteric modulatory effects
of selected organic nitrates were not shared by direct NO-generating
compounds, indicating a novel mechanism of action for selected organic
nitrates to interact with GABAA receptors. In behavioural models of learning
and memory, drugs which decrease GABAA receptor function improve
performance on learning and memory tasks (Venault et al., 1992). Thus, the
behavioural effect of selected organic nitrates, developed to act as
modulators
of GABAA receptor function, will be to improve memory performance and
cognition in patient populations. It will be appreciated, therefore, that
these
selected organic nitrates can be used for treatment of conditions including
but
not limited to: stroke; dementias of all type; trauma; drug-induced brain
damage; and aging.
According to certain aspects of the invention, neurodegeneration is
mitigated by inhibition of free radical damage. Reoxygenation and reperfusion
after a period of ischemia contributes significantly to the development of
brain
injury. Oxygen radicals, especially superoxide and peroxynitrite, formed in
the
period after an ischemic event may initiate processes such as breakdown of
membrane lipids (lipid peroxidation), leading to loss of cell membrane
integrity
and inhibition of mitochondrial function (Macdonald and Stoodley, 1998;
Gaetani et al, 1998). Oxidative stress is also believed to be one factor
involved
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in initiation of apoptotic neuronal cell death (Tagami et al., 1998). In
experimental animal models of ischemic brain injury, free radical scavengers
and enhanced activity of superoxide dismutase have been found to reduce the
extent of neuronal injury and cell death (Chan et al., 1998; Mizuno et al.,
1998;
Tagami et al., 1998).
It has been shown that 2,3-dinitrooxy-(2,3-bis-nitrooxy-propyldisulfanyl)-
propane, compound Va, improves task acquisition in cognitively impaired
animals (see Smith et al., NeuroReport 11: 3883, 2000), suggesting to us that
novel nitrates administered to an intact animal can have direct effects on the
brain. These observations also lead us to postulate that the nitrates of the
present invention could protect tissues against oxidative injury. Accordingly,
we tested the effects of compound Va in the 6-hydroxydopamine (6-OHDA)
model of Parkinson's disease. 6-OHDA is a neurotoxin selectively taken up
into dopaminergic neurons, resulting in a selective killing of these neurons,
via
a mechanism involving oxidative stress that is evident by 4 days after
injection
of the toxin. Previous studies have demonstrated that the monoamine oxidase
type B (MAO-B) inhibitor, deprenyl, can prevent 6-OHDA-induced killing of
dopaminergic neurons. We therefore employed deprenyl as a positive control
in this study. Loss of dopaminergic innervation to the striatum results in an
upregulation of postsynaptic dopamine receptors, and the development of
dopamine receptor supersensitivity 2-3 weeks after 6-OHDA lesioning of the
substantia nigra. This supersensitivity to dopamine can be unmasked by
challenging the animal with a dopamine receptor agonist, such as apomorphine.
Since the substantia nigra pars compacta is a bilateral structure, unilateral
destruction of the substantia nigra will induce a dopamine receptor imbalance,
which can manifest behaviourally as apomorphine-induced turning in the
direction contralateral to the lesion. This behavioural manifestation of 6-
OHDA toxicity provides a convenient, quantifiable index of neuronal injury
and neuroprotection. Immunocytochemical analysis confirmed that compound
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Va preserved TH-positive neurons in the substantia nigra of 6-OHDA-injected
rats (see Example 3 and Figure 7). These data demonstrate that compound Va
is a very effective neuroprotective agent against 6-OHDA-induced killing of
dopaminergic neurons in the rat substantia nigra pars compacta.
Accordingly, in certain aspects and embodiments of the invention, the
invention features nitrated molecules which have the capacity to inhibit
production of free radicals and/or which act as free radical scavengers.
Free radical overproduction is associated with a wide range of disease
states and/or indications, such as, for example, aging, septic shock,
ischemia,
overexpression of cytokines, ulcers, inflammatory bowel disease (e.g.,
gastritis,
ulcerative colitis or Crohn's disease), diabetes, arthritis (e.g., rheumatoid
arthritis), asthma, cirrhosis, allograft rejection (e.g., transplant
rejection),
encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis,
lymphocytic
choriomeningitis, glomerulonephritis, ophthalmologic diseases (e.g., uveitis,
glaucoma, blepharitis, chalazion, allergic eye disease, corneal ulcer,
keratitis,
cataract, retinal disorders, age-related macular degeneration, optic neuritis,
and
the like), ileitis, inflammation induced by overproduction of inflammatory
cytokines (e.g., liver inflammation, renal inflammation, airway inflammation,
and the like), hemorrhagic shock, anaphylactic shock, burn, infection leading
to
the overproduction of inflammatory cytokines (including bacterial (e.g., E.
coli
infection), viral (e.g., HIV), fungal (e.g., Candidiosis and histoplasmosis)
and
parasitic (e.g., Leishmaniasis and Schistosomiasis) infections), hemodialysis,
chronic fatigue syndrome, stroke, cancers, including metastatic cancers (e.g.,
breast cancer, bladder cancer, lung cancer, colon cancer, or cancer of the
other
organs, or skin or other noncutaneous portions of the body), cardiovascular
diseases associated with overproduction of inflammatory cytokines (e.g., heart
disease, cardiopulmonary bypass, ischemic/reperfusion injury, and the like),
ischemic/reperfusion associated with overproduction of inflammatory
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cytokines, toxic shock syndrome, adult respiratory distress syndrome,
cachexia,
myocarditis, autoimmune disorders, eczema, psoriasis, heart failure,
dermatitis,
urticaria, cerebral ischemia, systemic lupus erythematosis, AIDS,
neurodegenerative disorders (e.g., chronic neurodegenerative disease), chronic
pain, priapism, cystic fibrosis, schizophrenia, depression, premenstrual
syndrome, anxiety, addiction, migraine, gastrointestinal motility disorders,
obesity, hyperphagia, solid tumors (e.g., neuroblastoma), malaria, hematologic
cancers, myelofibrosis, lung injury, graft-versus-host disease, head injury,
CNS
trauma, hepatitis, renal failure, liver disease (e.g., chronic hepatitis C),
drug-
induced lung injury (e.g., paraquat), transplant rejection and preservation,
fertility enhancement, bacterial translocation, circulatory shock, traumatic
shock, and vascular aneurysm (e.g., aortic aneurysm), ileus, or myocardial
infarction.
In addition, the compounds or methods of the present invention may find
use in cytokine therapy (with consequent induction of free radical
overproduction) which, for example, is commonly used in the treatment of
cancers, including metastatic cancers (e.g., breast cancer, bladder cancer,
lung
cancer, colon cancer, or cancer of the other organs, or skin or other
noncutaneous portions of the body), autoimmune disease, and in AIDS patients.
Systemic hypotension due to the induction of free radical overproduction is a
dose-limiting side effect of cytokine therapy. Thus, a large patient
population
exists which will benefit from the invention methods.
It will also be appreciated by those skilled in the art, that any organic
nitrate in which vasodilatory potency is reduced and neuroprotective potency
increased, represents a new and useful therapeutic agent for use in
neuroprotection, particularly in treatment of conditions including but not
limited to: stroke; Parkinson's disease; Alzheimer's disease; Huntington's
disease; multiple sclerosis; amylotrophic lateral sclerosis; AIDS-induced
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dementia; epilepsy; alcoholism; alcohol withdrawal; drug-induced seizures;
viraUbacterial/fever-induced seizures; trauma to the head; hypoglycemia;
hypoxia; myocardial infarction; cerebral vascular occlusion; cerebral vascular
hemorrhage; hemorrhage; environmental excitotoxins of plant, animal, or
marine origin. GTN itself, proposed as a neuroprotective agent, has no
clinical
utility as a neuroprotective agent in therapy owing to its extraordinarily
high
vasodilatory potency. Similarly, by extrapolation, 1,2,3-trinitratopropane
(GTN) derivatives are not expected to have clinical utility as neuroprotective
agents in therapy owing to their especially high vasodilatory potency.
It will additionally be appreciated by those skilled in the art, that the use
in therapy of any organic nitrate in cognition enhancment, represents a new
and
useful treatment for cognition enhancement, particularly in treatment of
conditions including but not limited to: stroke; dementias of all type,
trauma,
drug-induced brain damage, and aging.
In particular, the therapeutic compounds of the invention comprise at
least one nitrate group. The nitrate groups(s) can optionally be covalently
bound to a carrier moiety or molecule (e.g., an aromatic group, an aliphatic
group, peptide, steroid, nucleoside, peptidomimetic, steroidomimetic, or
nucleoside analogue, or the like). In addition to functioning as a carrier for
the
nitrate functionality, the carrier moiety or molecule can enable the compound
to
traverse biological membranes and to be biodistributed preferentially, without
excessive or premature metabolism. Further, in addition to functioning as a
carrier for the nitrate functionality, the carrier moiety or molecule can
enable
the compound to exert amplified neuroprotective effects and/or cognition
enhancement through synergism with the nitrate functionality.
In one aspect, the invention provides a method of treating a neurological
condition and/or preventing an undesirable mental condition (e.g., memory
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loss) including the step of administering to a subject an effective amount of
a
therapeutic compound capable of mitigating neurodegeneration which has at
least one nitrate group. In one embodiment, the therapeutic compound is
capable of effecting neuroprotection. In another embodiment of the invention,
the therapeutic compound is capable of effecting cognition enhancement. The
therapeutic compound has the formula (Formula I):
\ /E-F-G
N O
/ +
O-
wherein E, F, G are organic radicals which may contain inorganic
counterions; so that a neurological condition is treated.
In another aspect, the invention provides a pharmaceutical composition
including a physiologically acceptable carrier and a compound having the
formula (Formula I):
\ /E-F-G
N O
/ +
O-
wherein: E, F, G are organic radicals which may contain inorganic
counterions. The composition is employed for mitigating neurodegeneration,
effecting neuroprotection and /or effecting cognition enhancement. The
composition may also be employed for preventing or mitigating tissue and/or
cellular damage in a subject by modulating intercellular and/or intracellular
free
radical concentration in the subject.
In another aspect, therapeutic compounds of the invention that effect
neuroprotection and/or effect cognition enhancement in a subject to which the
therapeutic compound is administered have the formula (Formula II):
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R19
R3 R4 ~ p
R17 R18
Il Jn
R2 ON02
R1
m
in which: m, n, p are integers from 0 to 10; R3,17 are each, independently,
hydrogen, a nitrate group, or A; Rl' 4 are each, independently, hydrogen or A,
where A is selected from: a substituted or unsubstituted aliphatic group
(preferably a branched, or straight-chain aliphatic moiety having from 1 to 24
carbon atoms in the chain, which optionally contains 1 to 4 0, S, NR6, and/or
unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, nitrate,
amino or aryl, or heterocyclic groups; an unsubstituted or substituted cyclic
aliphatic moiety having from 3 to 7 carbon atoms in the aliphatic ring, which
optionally contains 1 to 2 0, S, NR6, and/or unsaturations in the ring,
optionally
bearing from 1 to 4 hydroxy, nitrate, amino or aryl, or heterocyclic groups;
an
unsubstituted or substituted aliphatic moiety constituting a linkage from 0 to
5
carbons, between R' and R3 and/or between R17 and R4, which optionally
contains 1 to 2 0, S, NR6, and/or unsaturations in the linkage, and optionally
bearing from 1 to 4 hydroxy, nitrate, amino or aryl, or heterocyclic groups);
a
substituted or unsubstituted aliphatic group (preferably a branched, cyclic or
straight-chain aliphatic moiety having from 1 to 24 carbon atoms in the
chain),
containing linkages selected from C=O, C=S, and C=NOH, which optionally
contains 1 to 4 0, S, NR6, and/or unsaturations in the chain, optionally
bearing
from 1 to 4 hydroxy, nitrate, amino or aryl, or heterocyclic groups; a
substituted
or unsubstituted aryl group; a heterocyclic group; an amino group selected
from
alkylamino, dialkylamino, cyclic amino, cyclic diamino, cyclic triamino,
arylamino, diarylamino, and alkyarylamino; a hydroxy group; an alkoxy group;
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and a substituted or unsubstituted aryloxy group; R2, R5, R18, R19 are
optionally
hydrogen, A, or X-Y; where X is F, Br, Cl, NO2, CH2, CF2, 0, NH, NMe, CN,
NHOH, N2H3, N2H2R13, N2HR13R14, N3, S, SCN, SC(=NH)N(R'5)2,
SC(=NH)NHR15, SC(O)N(R15)2 , SC(O)NHR15, SO3M, SH, SR7, SO2M,
S(O)R8, S(O)2R9, S(O)OR8, S(O)ZOR9, PO2HM, PO3HM, P03MZ,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(Rls)(OR), P(O)(OM)R15, CO2M,
COZH, C02R", C(O), C(O)R12, C(O)(OR13), PO2H, PO2M, P(O)(OR14),
P(O)(R13), SO, SO2, C(O)(SR'3), SR5, SSR7 or SSR5; Y is F, Br, Cl, CH3,
CF2H, CF3, OH, NH2, NHR6, NR6R7, CN, NHOH, N2H3, N2H2R13, N2HR13R14,
N3, S, SCN, SC(=NH)N(Rls)2, SC(=NH)NHR15, SC(O)N(R15)2 , SC(O)NHR15,
SO3M, SH, SR', SO2M, S(O)R8, S(O)ZR9, S(O)ORB, S(O)20R9, PO2HM,
P03M2, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R15)(OR8), P(O)(OM)R15,
,
CO2M, CO2H, CO2R", C(O)R12, C(O)(OR13), C(O)(SR13), SRS, SSR7 or SSRS
or does not exist; R6, R7, R8, R9, R", R12, R13, R14, R15, R16 are the same or
different alkyl or acyl groups containing 1-24 carbon atoms which may contain
1-4 ONO2 substituents; or C1 - C6 connections to R' - R4 in cyclic
derivatives;
or are each independently hydrogen, a nitrate group, or A; M is H, Na+, K+,
NH4+, N+HkR"(4_k) where k is 0-3, or other pharmaceutically acceptable
counterion.
Pharmaceutical compostions comprising a compound of Formula II in
admixture with a pharmaceutically acceptable carrier therefor are provided by
the invention. The invention further provides methods of mitigating
neurodegeneration, effecting neuroprotection and/or effecting cognition
enhancement in a subject comprising the step of administering a compound of
Formula II to a subject such that said mitigation and /or said neuroprotection
an/or cognition enhancement occurs.
According to this aspect of the invention, preferred therapeutic
compounds for effecting neuroprotection and/or cognition enhancement in a
47
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subject to which the compound is administered include compounds in which
R19 is X-Y. In some preferred embodiments: R'9 is X-Y and R5, R6, R8, R9, R12,
R' 3, R' 4, R's, R' 6 are the same or different alkyl groups containing 1-24
carbon
atoms which may contain 1-4 ON02 substituents, or C, or C2 connections to R'
- R3 in cyclic derivatives; R' and R3 are the same or different and selected
from H, C1-C4, alkyl chains, which may inlude one 0, linking R' and R3 to form
pentosyl, hexosyl, cyclopentyl, or cycohexyl rings, which rings optionally
bear
hydroxyl substituents; R2 and R4, are the same or different and selected from
H,
a nitrate group, Cl-C4 alkyl optionally bearing 1-3 nitrate group, and acyl
groups (-C(O)R5); and R7, R" are the same or different C1- C8, alkyl or acyl.
In certain embodiments in which R19 is X-Y, m, p = 1, and n = 0. In
other embodiments in which R19 is X-Y, X is selected from: CH2, 0, NH,
NMe, CN, NHOH, N2H3, N2H2R13, N2HR13R14, N3, S, SCN, SC(=NH)N(R'S)2,
SC(=NH)NHR15, SC(O)N(R15)Z , SC(O)NHR15, SO3M, SH, SR', SOZM,
S(O)R8, S(O)2R9, S(O)ORg, S(0)20R9, PO3HM, P03M2, P(O)(OR15)(OR16),
P(O)(OR16)(OM), P(O)(R15)(OR8), P(O)(OM)R15, CO2M, CO2H, CO2R",
C(O), C(O)R12, C(O)(OR13), P02M, P(O)(OR14), P(O)(R13), SO, SO2,
C(O)(SR13), SSR4. In certain other embodiments in which R19 is X-Y, Y is
selected from CN, N2H2R13, N2HR13R14, N3, SCN, SC(=NH)N(R'S)2,
SC(O)N(R15)2 , SC(O)NHR15, SO3M, SR4, SO2M, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(ORg), P(O)(OM)R15, CO2M,
CO2H, CO2R", C(O)R'Z, C(O)(SR13), SR5, SSR5, or does not exist. In some
embodiments, X and/or Y contains a sulfur-containing functional group. In
certain embodiments, the compound of the invention comprises a heterocyclic
functionality, more preferably, a nucleoside or nucleobase. In other
embodiments, the compound of the invention comprises a carbocyclic
functionality, more preferably, a steroidal or carbohydrate moiety.
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In another aspect of the invention, a therapeutic compound of the
invention is represented by the formula (Formula III):
Y
X
R3 - C R4
R17- I R~s
n
R2-C ONO2
Rl
m
in which: m, n are 1-10; R1-9 , RI1"ls, X, and Y have the meaning as
defined above. In certain preferred embodiments, R6 - R9 and R" - R16 are the
same or different alkyl or acyl groups containing 1-24 carbon atoms which may
contain 1-4 ON02 substituents, or C, - C6 connections to R' - R4 in cyclic
derivatives. In certain preferred embodiments, R18 is A and m = n = 1.
Examples and preferred embodiments of this aspect include:
02NO 02NO
F CO2H OCF2CF2H
ON02 (IIIa); ON02 (IIIb); ON02 (IIIc);
02NO
NO2
ON02 (IIId);
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H
N
ONOZ
= O
H + N~
H H 03S~ONO2
02N0l" H "ON02 (IIIg);
02NO ONO2
S-S
02N0 ONO 2
ON02 02NO (IIIh);
0S O 02N0 02NO
BrH2C CH2Br NCSH2C CH2CCN
02NOf (IIIi); ON02 (IIIj); ON02 (IIIk);
02N0 0S0
02N0
NCSH2C CH26r BrH2C CH2Br
ON02 (IIII); 02NO ON02 (IIIm); OH (IIIo);
O2NO NCSH2C--r CH2CCN O2NO ONO2
BrH2CJ-CH2Br (IIIp); ON02 (III(4); NaO3S2 S2O3Na
[IIIr];
HO SSO3Na
O~ ONOz O'-j ONO2 ~
~N--~SO3H [Ills]; ~N,-,'L,,~SO3Na [IIIu]; ONO2 [IIIag];
OEt
O
S~ -
02NO 02NO
S203Na g
ON02 [IIIai]; ON02 [IIIaj]; and s ON02
[IIIam].
CA 02580627 2007-03-15
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In another aspect of the invention, a therapeutic compound of the
invention is represented by the formula III in which the compound contains
from 1 to 3 nitrate groups and an S atom in proximity to a nitrate group,
where
each of m and n is, independently, an integer from 0 to 10; R' is a hydrogen
or
A; each of R2, R5, and R18 is, independently, hydrogen or A; each of R3, R4,
and
Rl7
, is independently, a hydrogen, a nitrate group, or A; each of R6, R7, Rg, R9,
R11 R1Z R13 Ri4 Rls and R16 is, inde endentl A, a h dro en a nitrate rou
, > > , , p Y> Y g ~ g P,
or a C1-C24 alkyl or acyl group, optionally containing 1-4 ON02 substituents
or
a C1-C6 linkage to R', R2, R3, or R4 in cyclic derivatives; each of R7 and Rl'
is,
independently, a substituted or unsubstituted Cl-C8 alkyl or acyl group;
A is a CI -C24 alkyl group, which optionally contains 1 to 4 0, S, NR6,
and/or unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, Cl,
F,
amino, unsubstituted or substituted aryl, unsubstituted or substituted
heteroaryl,
or unsubstituted or substituted heterocyclic groups, or 1-2 nitrate groups; a
C3-
C24 alkyl group, containing 1-5 C=O, C=S, or C=NOR7 linkages, which
optionally contains 1 to 4 0, S, NR6, and/or unsaturations in the carbon
chain,
optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino, unsubstituted
or
substituted aryl, unsubstituted or substituted heteroaryl, or unsubstituted or
substituted heterocyclic groups; a C3-C7 linkage to any of R1, RZ, R3, R4, or
R17,
forming an aliphatic ring, which optionally contains 0, S, NR6, and/or
unsaturations in the linkage, optionally bearing from 1 to 6 substituents,
independently selected from unsubstituted or substituted alkyl, unsubstituted
or
substituted aryl, unsubstituted or substituted C1-C4 alkaryl, unsubstituted or
substituted heteroaryl, unsubstituted or substituted C,-C4 alkheteroaryl,
unsubstituted or substituted heterocyclic, unsubstituted or substituted C1-C4
alkheterocyclic, hydroxy, nitrate, Cl, F, and amino groups; a Co-C5linkage to
or
between any of R', R3, R4, or R17, which optionally contains 0, S, NR6, and/or
unsaturations in the linkage, bearing two or more substituents, independently
selected from unsubstituted or substituted alkyl, unsubstituted or substituted
aryl, unsubstituted or substituted C1-C4 alkaryl, unsubstituted or substituted
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heteroaryl, unsubstituted or substituted C1-C4 alkheteroaryl, unsubstituted or
substituted heterocyclic, unsubstituted or substituted CI -C4 alkheterocyclic,
hydroxy, nitrate, Cl, F, and amino groups; an unsubstituted Co-C5 linkage to
or
between any of R', R3, and R4, which optionally contains 1 to 2 non-adjacent
0,
S, NR6, and/or unsaturations in the linkage; a CI -C5 linkage to or between
any
of R', R3, R4, and R17 containing 1 to 2 C=O, C=S, or C=NOR7 linkages, which
optionally contains 1 to 2 0, S, NR6, and/or unsaturations in the linkage,
optionally bearing from 1 to 4 substituents, independently selected from
unsubstituted or substituted alkyl, unsubstituted or substituted aryl,
unsubstituted or substituted CI-C4 alkaryl, unsubstituted or substituted
heteroaryl, unsubstituted or substituted C1-C4 alkheteroaryl, unsubstituted or
substituted heterocyclic, unsubstituted or substituted CI -C4 alkheterocyclic,
hydroxy, nitrate, Cl, F, and amino groups; a substituted or unsubstituted aryl
group; a substituted or unsubstituted heteroaryl group; a substituted or
unsubstituted heterocyclic group; an amino, cyclic amino, diamino, triamino,
alkylamino, dialkylamino, arylamino, diarylamino, or alkylarylamino group; a
hydroxy group; an alkoxy group; or a substituted or unsubstituted aryloxy
group;
X is F, Br, NO2, CH2 CF2, 0, NH, NMe, NHOH, N2H3, N2H2R13,
N2HR13R'4 , N3, S, SC(=NH)N(R'S)2, SC(=NH)NHR15, SC(O)N(R15)2 ,
SC(O)NHR15, SH, SR5, SR7, S(O)R8, S(O)R5, PO2HM, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(OR8), P(O)(OM)Rls, CO2M,
CO2H, CO2R", C(O), C(O)(OR13), PO2H, PO2M, P(O)(OR14), P(O)(R13), SO,
SO2, C(O)(SR13), SR5, SR7, or does not exist;
Y is F, Br, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7 , NHOH, N2H3,
N2H2R13, N2HR13R'4 , N3, SC(=NH)N(RIS)2, SC(=NH)NHR15, SC(O)N(R15)2 ,
SC(O)NHR15, SH, SR7, SO2M, S(O)R8, S(O)R5, PO2HM, PO3M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R's)(ORg), P(O)(OM)R15, CO2M,
CO2H, C02R 5, C(O)(OR13), C(O)(SR13), C(S)R5, C(S)R12, C(S)OR12, or does
not exist; and
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M is H, Na+, K+, NH4+, N+HkR"(4_k) where k is 0-3, or other
pharmaceutically acceptable counterion;
and with the proviso that, when m=0 and n=1, each of R18 and R3 is,
independently, a hydrogen, a nitrate group, or a C1-C4 alkyl chain, which may
include one 0, linking R18 and R3 together to form a pentosyl, a hexosyl, a
cyclopentyl, or a cyclohexyl ring, said ring optionally bearing from 1-4
hydroxyl substituents; each of R" and R4 is, independently, a hydrogen, a
nitrate group, a C1-C4 alkyl group, optionally bearing from 1-3 nitrate
groups,
or an ac Y1 group (-C(O)RS); each of R5, R6, R8, R9, R' , R' 2 > R' 3 > R' a
, R' S, and
R' 6 is, independently, a C 1-C 12 alkyl group, optionally bearing from 1-4
ON02
substituents, or a C1-C2 alkyl linkage to R18, R17, or R3; X is F, Br, NOZ,
CH2
CF2, 0, NH, NMe, NHOH, N2H3, N2H2R13, N2HR13R14, N3, S,
SC(=NH)N(R15)Z, SC(=NH)NHR15, SC(O)N(R15)2 , SC(O)NHR15, SH, SRS,
SR7, S(O)Rg, S(O)R5, PO2HM, PO3HM, P03M2, P(O)(OR15)(OR16),
P(O)(OR16)(OM), P(O)(R15)(ORg), P(O)(OM)R15, COzM, CO2H, COzR",
C(O), C(O)(OR13), PO2H, PO2M, P(O)(OR14), P(O)(R13), SO, SO2, C(O)(SR'3),
SRS, SR7 ; and Y is not CN, NzH2R13, N2HR13R14, N3, SCN, SC(=NH)N(R'S)2,
SC(=NH)NHR15, SC(O)N(R15)2 , SC(O)NHR15, SO3M, SH, SOZM, P03M2,
PO3HM, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(OM)R15, CO2M, CO2H,
COZRS, C(O)R12, C(O)(SR13), SR4, SR5, or SSR5, or Y does not exist.
Examples and preferred embodiments of this aspect include:
0 CH3
02NO S-S ~ -N
N / Ni~CH3
~ H3C ~)~
ON0Z 0 N
02NO S-S
ON02 JO
L 0
H3~ [Vt];
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CH3
02NO S-S O ~ CH3
~ I H3C OH
H3C ~ G -
CH
ON02 G_ Br CH3 02NO S-S 3
G = OH \,-N ~OCH3
G = NH2 O CH3
G = NHCHO . ONO2 0
02NO S-S CH3
Y
02NO S-S H3 '
ON02 0
\-~-
ON02 S,,// N [VY]; 0 OMe [Vx];
0 02NO S-S HN~ 02NO S
O
CI ~ \ NH ONO
N
2
ON02 - CH3
CI CH3 O [Vay];
H3C ~ O
~ I O2NO S
O HN
O2NO S - -
/
O ONOz H3C N
CH3
ON02 H3C [Vaw]; O \ ~ [Vat];
OZNO 0 CH3 H3C OH
S
O
N CH3
OZNO S-S
ON02 O CH3
- \~ 0 CH3
CH3O CI [Van]; O2NO 0 ~
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02NO S-S CH3 O2NO
S O 02NO S
02N0 0 Cz H3C I S
O J
OMe [Vx]= 02NO ON02 ~ S
~
H3C S
S
~
O2NO
02NO S =
H3C
CH3
02NO S 02NO S H3C X 02N0
~ S ~~S S OzNO~/S
H3C H3C S 02NO S
=
0 I S
> > > >
H3C
~N H3C
02N0 S~CH3 ,/t N
ON02 . O2NO S ONO2.
> >
H3C
N
02N0 a
S)I/CF3
iON02
H3C
,// N H3C
02NO '~/~S ONO2
02N0 t/N
S>T __~ /CH3
S ON02 .
> >
H3C
~N CF3
OZNO SI-Y
ON02
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H3C
s OH H3C aN CF3
HO N OzNO S 02NO CH3
~ \.
02NO ~ O ~ S
H3C S OH
~ N HO
~ CF3
O2NO S> 0 O2NO ON02
H3C CH3
N N~CH3
02N0 $
0
H3C CH3
CH3 ~N Ni
CH3 N
NJ~O, 02N0 ~(/ ~ CH H3C S S CFi3
T~S 3 02N0 CH3
02N0 SCH3. CF3 H3C
> > >
H3C CH3
H3C H3C O O OH
S
,O / N CH3 ~N CF3 O CH3
H3C $ H3C $~ H3C
ION02 . ON02 . 02NO
> > >
S-S
H3C H3C
S CH3 H3C $ CH3
02N0 N
S OH 02NO OH
02NO H3C H3C O OMe =
, > >
-
02N0 S =
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N &yCF3 (yCF3 ~~ CH3 S H3C N3\
ON02 . ON02 ON02 . O2NO S.
> > > >
H3C N
O2NO /S
H CH3 N- H3C H3C
H3C S s N ~~
02NO . 02NO O2NO S' .
, > >
[Ve] G=Me
[VfJ G=OMe
FS-S G [Vg] G=CI
ONO2 [Vh] G=N02
ONO2 [Vi] G=Br
[Vj] G=H
[Vu] G=F
S-S [Vm] G=CO2Et
~ ~ [Vs] G=CO2H
ON02 [Vaa] G=CO2Me
ONO2 G [Vae] G=CONH2
[Vag] G=CO(CH2)2NEt2
s-s Q U W~ G G COMe 02N0\ ~S~S ~~ CI
rONO2 G=CONH2 '~ \ -
ONO2 G [Vaf] ON02 CI [Vp];
0
O2NO S-S O
02NO S-S OEt
ON02 - ~ S
H3C N [Vr]; ON02
02NO S-S N-
/_\ /
ON02 [Vn];
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-
0
HO S-S OEt 02NO S= 02NO S
CH3
ON02 ONOZ ONO2 0 HO2C
-i/-NH2
02NO S-S NH2 O2NO S-S HN4
O
OH
~-j
ONOZ 0 [Vk]; ON02 0 \--CO2H
CH3
02NO S-S HN--~ 02NO SH 02NO S-S N
0
OEt ~ 0
ON02 0 ONOz [IVs]> ' ON02 0
0
O2NO S-S 02N0 S-~ HO S-S OH
CH3
ON02 0 ; ON02 [IVt]; ONO2 02NO [Vz];
O
O
S-S OzNO S-S
~ ~tO2C
ONO2 O2NO [Vac]; ONO2 [VI];
O2NO S-S
ON02 [Vab];
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OzNO S-S OH 02NO S-S'
S-)__~ONOz
ON02 O2NO ON02 OZNO [IVr];
02N0 S 8'O
~ \-'\N(CH3)2
ON02
O
02NO HN \S~
O
02NO S-S~ O2NO S ON02
CH3 ~--~ N
-N
~02N0 ONO2 ~ ~ ~ I
ON02 ON02 [IVp]; CF3
[Vam];
O
H
02S 02NO S ~ ~
' N ONO2 O -
'tn H3C
O2NO S4
CH3 ON02 0
nis2or3
N(CH3)2 ONO2 [IVt]; [Var];
0 CH3
O2NO S
S~CH3
\ON02 0
F [Vax];
0 O
02NO S
H3C
ON02 [Vba];
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O O
02NO S 02NO S-
~iBu
H3C OMe H3C
ON02 [Vap]; ON02 [Vao];
F
4FF
O F 02N0 S O2N0 S H3C ON02 [Val]; ON02 HO [Vaj];
0
CI 02NO S
- -
02N0 S O HN \/ ONO2 HN CI
~-j ci CH3 CI
ON02 [Vas]; [Vaz];
ONO2 -
0 ON02 S \ /
02NO S HN SS S
0 CH3
H3C CH3 ON02 -/
ON02 [Vak]; ON02 ; O
ON02
S / \
ON02
ON02 ON02
S
S \ / ONO2 0
ON02 O CH3 SS--\\.-O N ONO2 CH3
O
ONO O \ / . S N
O 2
> > >
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ON02 -
S S~
0 H3C 02NO S.
ON02 O~~ J 02N0 /~ S~SHO2C S
~--~S ONOZ ~
02NO S,S
O2NO-~
S-S N ON02
S ~4 ~ _\ O2NO S
p 02NO O
02NO ON02 ; O ; or HO OH.
Pharmaceutical compostions comprising a compound of Formula III in
admixture with a pharmaceutically acceptable carrier therefor are provided by
the invention. The invention further provides methods of mitigating
neurodegeneration, effecting neuroprotection and/or effecting cognition
enhancement in a subject comprising the step of administering a compound of
Formula III to a subject such that said mitigation and /or said
neuroprotection
and/or cognition enhancement occurs.
In another aspect of the invention, a therapeutic compound of the
invention can be represented by the formula (Formula IV):
Y
X
R3-C R4
Rn
R2- i ON02
R'
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in which n = 0, X is CH2 or does not exist, and Y is selected from F, Br,
Cl, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7, CN, NHOH, N2H3, N2H2R13,
N2HR13R14, N3, S, SCN, SC(=NH)N(R15)2, SC(=NH)NHR15, SC(O)N(Rl5)2,
SC(O)NHR15, SO3M, SH, SR7, SO2M, S(O)R8, S(O)2R9, S(O)ORg, S(O)2OR9,
PO2HM, P03M2, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R]s)(ORs),
P(O)(OM)R15, CO2M, CO2H, C02R,1, C(O)RI2, C(O)(OR13), C(O)(SR13), SR5,
SSR7 or SSR5. R2, R4, R5, R6, R7, R8, R9, Ri1, R12, R13, R14, R15, and R16 are
as
defined above. In certain preferred embodiments, R2 and R4 are optionally H, a
nitrate group or a connection to R5-R9 and RI I -R16in cyclic derivatives.
Pharmaceutical compositions comprising a compound of Formula IV in
admixture with a pharmaceutically acceptable carrier therefor are provided by
the invention.
The invention further provides methods of mitigating neurodegeneration,
effecting neuroprotection and/or effecting cognition enhancement in a subject
comprising the step of administering a compound of Formula IV to a subject
such that said mitigation and /or said neuroprotection and/or cognition
enhancement occurs.
Examples and preferred embodiments of compounds of the invention
according to Formula IV are as follows:
0
11,OCH2CH3
CI SCN P~OCH2CH3 S203Na
ON02 EONO2 ONO2 EONO2
E
ON02 (IVa); ON02 (IVb); ONOz (IVc), ON02
(IVd);
o o 0 0
S~o S 0
S2O3Na ~/ p CON0.2 (IVe); 02NO (IVf); 02N0 (IVg); 02NO (IVh);
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CN Br P~ O O:Ba S02Ph
ON02 FON02 L0N02 EON02
L
ON02 (IVi); ON02 (I'Ij); ON02 (IVl), and ON02
In yet another aspect of the invention, a compound of the invention can '
be represented by the formula (Formula V):
SSR5
R3 - C R4
Rn
R2-C ON02
R'
m
in which R2 is optionally H or a connection to R5 in cyclic derivatives,
R4 is H or a nitrate group, and R5 is as described above.
Pharmaceutical compositions comprising a compound of Formula V in
admixture with a pharmaceutically acceptable carrier therefor are provided by
the invention.
The invention further provides methods of mitigating neurodegeneration,
effecting neuroprotection and/or effecting cognition enhancement in a subject
comprising the step of administering a compound of Formula V to a subject
such that said mitigation and /or said neuroprotection and/or cognition
enhancement occurs.
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Examples and preferred embodiments of compounds of the invention according
to formula V (Formulae Va-c) are as follows:
OZNO ONO2 S-S S-S
S-S ~
~
ONO2 O2NO (Va); 02N0 ON02 (Vb); or 02N0 OH
(Vc).
In another aspect, the invention features one of the following nitrate
esters:
O2NO S H3C N cI~)
N
N'N - - ~ CF3 / S~ 3 S
~ [IIIad]~ ON02 ; 02NO ~
H3C H3C F3C
/ \\
O2NO S~ O2NO S N02 [IIIah]; O2NO' \/ ~
S;
ON02 02NO ONO2 O2NO
S-S S-S
O2NO ~ ~ ~ ~ ONO2 02NO ON02
N N
- - ; - - ;
S F3C
ON02 OO O2N0 N
/ ~ / / S S S
O2NO S-S S-S ONO2; O2NO ONOz
02NO S,
02NO S ONO O S O I N
2 < > S
/~(\ 02NO
ON02 Br ONO2 (IIIn); ONO2 (IIIf);
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02NO S\ S \ I \ S
O2NO O O CH O OuCH3
ON02 ~ 3 I I
02N0 ~ O2NO 0 ; O2N0 O ;
NH2
N~
O~ N
OZNO
O2NO--\__N l N ~ __-SO3H V O
; or S (Ille).
In another aspect, the invention features a method for preventing or
mitigating tissue and/or cellular damage in a subject by modulating
intercellular and/or intracellular free radical concentration in the subject.
The
method includes administering to the subject an effective amount of a
compound containing at least one aliphatic nitrate group and at least one
sulfur
atom in proximity to said nitrate, such as, for example, a compound of formula
1. In one embodiment, the nitrate of this method contains at least 2 nitrate
groups. In another embodiment, the nitrate is beta or gamma to a sulfur atom.
Yet another embodiment features compounds of formula III for
preventing or mitigating tissue and/or cellular damage where m is an integer
from 0 to 10; n is an integer from 0 to 10; each of R3 4 17 is, independently,
hydrogen, a nitrate group, or A; R' is hydrogen or A, with A is selected from:
a
substituted or unsubstituted C1-C24 alkyl group, optionally containing 1 to 4
0,
S, NR6, and/or unsaturations in the chain, optionally bearing from 1 to 4
hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or
unsubstituted or substituted heterocyclic groups; an unsubstituted or
substituted
cyclic moiety having from 3 to 7 carbon atoms in the ring, which optionally
contains O, S, NR6, and/or unsaturations in the ring, optionally bearing from
1
to 4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or
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unsubstituted or substituted heterocyclic groups; an unsubstituted or
substituted
moiety constituting a linkage from 0 to 5 carbons, to or between any of R1,
R3,
R'7 and R4, which optionally contains 0, S, NR6, and/or unsaturations in the
linkage, and optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino or
unsubstituted or substituted aryl, or unsubstituted or substituted
heterocyclic
groups; a substituted or unsubstituted C1-C24 alkyl group, containing 1-4
linkages selected from C=O, C=S, and C=NOH, which optionally contains 0,
S, NR6, and/or unsaturations in the chain, optionally bearing from 1 to 4
hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or
unsubstituted or substituted heterocyclic groups; a substituted or
unsubstituted
aryl group; a substituted or unsubstituted heterocyclic group; an amino group
selected from alkylamino, dialkylamino, cyclic amino, cyclic diamino, cyclic
triamino, arylamino, diarylamino, and alkyarylamino;a hydroxy group; an
alkoxy group; and a substituted or unsubstituted aryloxy group;
R2, R5, R'g, are optionally hydrogen, A, or X-Y;
X is F, Br, Cl, NO2, CH2, CF2, 0, NH, NMe, CN, NHOH, N2H3,
N2H2R13, N2HR13R14, N3, S, SCN, SC(=NH)N(R'S)2, SC(=NH)NHR's,
SC(O)N(R15)2 , SC(O)NHR15, SO3M, SH, SR7, SO2M, S(O)Rg, S(O)2R9,
S(O)R5, S(O)2R5, S(O)ORB, S(O)20R9, POZHM, PO3HM, P03M2,
P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(Rls)(OR), P(O)(OM)R'S, CO2M,
CO2H, CO2R", C(O), C(O)R12, C(O)(OR13), PO2H, PO2M, P(O)(OR'a),
P(O)(R13), SO, SO2, C(O)(SR'3), SRS, SSR' or SSRS, SS, or does not exist;
Y is F, Br, Cl, CH3, CF2H, CF3, OH, NH2, NHR6, NR6R7, CN, NHOH,
N2H3, N2H2R13, N2HR13R14, N3, S, SCN, SC(=NH)N(R'S)Z, SC(=NH)NHR'S,
SC(O)N(R15)Z , SC(O)NHR15, SO3M, SH, SR7, SO2M, S(O)R8, S(O)2R9,
S(O)ORB, S(O)R5, S(O)2R5, S(O)20R9, PO2HM, P03M2, P(O)(OR15)(OR16),
P(O)(OR16)(OM), P(O)(R15)(OR8), P(O)(OM)R15, CO2M, CO2H, CO2R5,
C(O)R12, C(O)(OR13), C(O)(SR13), SRS, SSR7 or SSR5, C(S)R5, C(S)R12,
C(S)OR12, or does not exist;
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each of R6 R7 R8, Rlo R' i Rlz R13 Rla Rls and R16 is,
> > , > > , ~ , > > independently, a C1-C24 alkyl group, optionally containing
1-4 ONOz
substituents, a C1-C24 acyl group, optionally containing 1-4 ONOz
substituents,
a C1-C6 ring-forming connection to any of R'-Ra, a hydrogen, a nitrate group,
or A; and
M is H, Na+, K+, NHa+, N+HkR"(a_k) where k is 0-3, or other
pharmaceutically acceptable counterion;
and with the proviso that, when m=0 and n=1, each of R18 and R3 is,
individually, H, a nitrate group,or a C, - C4 alkyl group, which may include
one
0, linking R18 and R3 to form pentosyl, hexosyl, cyclopentyl, or cyclohexyl
rings, which optionally bears hydroxyl substituents; each of R" and R 4 is,
individually, H, a nitrate group, a C 1-Ca alkyl group, optionally bearing
from 1-
3 nitrate rou s, ~ or -C(O)Rs= each of R 5, R6, R8 R9, R1z , R13 , R14 , R's >
R'6 is,
g p , individually, a C1-C12 alkyl group, optionally containing 1-4 ONOz
substituents
or a CI -Cz ring-forming connections to R'g,R", or R3; each of R7 and R" is,
independently, a C1-C8 alkyl group or a C1-C8 acyl group; M is H, Na+, K+,
NHa+, N+HkR"(a_k) where k is 0-3;
X is CH2, 0, NH, NMe, CN, NHOH, N2H3, N2H2R13, N2HR13R14, N3, S,
SCN, SC(=NH)N(R15)2, SC(=NH)NHR's, SC(O)N(R's)z, SC(O)NHR's, SO3M,
SH, SR7, SO2M, S(O)R8, S(O)2R9, S(O)ORg, S(O)20R9, PO3HM, P03M2,
P(O)(ORIS)(OR16), P(O)(OR16)(OM), P(O)(Rls)(OR8), P(O)(OM)R's, CO2M,
COzH, CO2R", C(O), C(O)R1z, C(O)(OR13), PO2M, P(O)(OR'a), P(O)(R13),
SO, SOz, C(O)(SR13), SR 5, or SSR4; and
Y is not CN, N2H2R13, N2HR13R14 , N3, SCN, SC(=NH)N(R15)2,
SC(=NH)NHR's, SC(O)N(R's)z, SC(O)NHR's, SO3M, SH, SO2M, P03Mz,
PO3HM, P(O)(OR's)(OR16), P(O)(OR16)(OM), P(O)(OM)R's, CO2M, COZH,
C02R 5, C(O)R1z, C(O)(SR13), SR4, SR 5, or SSR 5, or Y does not exist.
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Preferred compounds include any of the compounds of formulas II, III,
IV, and V that have been individually described herein. Other preferred
compounds include:
02NO OZNO 02NO
SCN S02Ph SSO3M
ON02 ON02 ON02
02N0 02N0 S_S 0g 0
NCSH2C CH2SCN NCSH2C CH2Br ~
ON02 ON02 = O2NO ON02> = 02N0 ON02 .
> > >
SCN
02NO 02NO NCSH2C~ 02NO
V-_./SO3Na V__/SSO3Na ON02 V---/SCOCH3
> > > >
0,0 0 0 Q\ O 0
S~. S-S S~ S;O p
02N0 = OzNO O2NO Br = 02N0 O2NO
> > > > >
NH2
N/ N 02NO ON02
O2NO O N O2N0 \S, OZNO S-S ON02
ON02= ON02 02N0
, , ,
s
H
N
ONO2 / ~
O N 02NO g
I
,N
03S ONO2 N \ ~ .
02N0 ON02 ~ H3C
cs NCF3 ( H3C H3C N
~ S S / \\ / ~
ON02 ~ 02NO 02N0 g 02N0 S NO2 ~
ON02 02NO
F3C S-S
~N 02N0 ON02
O2NO-S; - -
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ONOz 02NO S
S-S ONOz / ~ / ~ 02NO
02N0 ~ ~ ~ ONOz ' _ -
/~
_ N N_ ; 02NO S-S S-S ONOz;
c~SCH3
S 02NO ONOz ~ O2NO IOI
S p
0 OuCH3
y 02NO--\_ __/-SO3H
02NO O
02NO 02NO S,
O2NO S\S ONO O2NO S
z
ONOz
ON02 ~ or 02N0
Particularly preferred compounds for preventing or mitigating tissue
and/or cellular damage are:
02NO ONOz
N S-S
\
O2NO S(IVk) and ONOz OzNO (Va).
It will be noted that the structure of some of the compounds of this
invention includes asymmetric carbon atoms. It is to be understood accordingly
that the isomers (e.g., enantiomers, diastereomers) arising from such
asymmetry
are included within the scope of this invention. Such isomers can be obtained
in substantially pure form by classical separation techniques and by
asymmetric synthesis. For the purposes of this application, unless expressly
noted to the contrary, a compound shall be construed to include both the R and
S stereoisomers at each stereogenic center.
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In certain embodiments, a therapeutic compound of the invention
comprises a cation (i.e., in certain embodiments, one of X or Y includes a
cation, e.g., in the compound of formula IVd). If the cationic group is a
proton,
then the compound is considered an acid. If the proton is replaced by a metal
ion or its equivalent, the compound is a salt. Pharmaceutically acceptable
salts
of the therapeutic compound are within the scope of the invention. For
example, M can be a pharmaceutically acceptable alkali metal (e.g. Li, Na, K),
ammonium, alkaline earth metal (e.g. Ca, Ba, Mg), higher valency cation, or
polycationic counter ion (e.g., polyammonium cation) (see e.g., Berge et al.,
1977). It will be appreciated that the stoichiometry of an anionic portion of
the
compound to a salt-forming cation will vary depending on the charge of the
anionic portion of the compound and the charge of the counterion. Preferred
pharmaceutically acceptable salts include a sodium, potassium, or calcium
salt,
but other salts are also contemplated within their pharmaceutically acceptable
range.
A therapeutic compound of the invention.can be administered in a
pharmaceutically acceptable vehicle. As used herein "pharmaceutically
acceptable vehicle" includes any and all solvents, excipients, dispersion
media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying
agents, and the like which are compatible with the activity of the compound
and
are physiologically acceptable to the subject. An example of a
pharmaceutically acceptable vehicle is buffered normal saline (0.15 M NaCI).
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 therapeutic compound, use thereof in the compositions
suitable for pharmaceutical administration is contemplated. Supplementary
active compounds can also be incorporated into the compositions.
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Carrier or substituent moieties useful in the present invention may also
include moieties which allow the therapeutic compound to be selectively
delivered to a target organ. For example, delivery of the therapeutic compound
to the brain may be enhanced by a carrier moiety using either active or
passive
transport (a "targeting moiety"). Illustratively, the carrier molecule may be
a
redox moiety, as described in, for example, U.S. Patents 4,540,654 and
5,389,623, both to Bodor. These patents disclose drugs linked to
dihydropyridine moieties which can enter the brain, where they are oxidized to
a charged pyridinium species which is trapped in the brain. Thus drugs
accumulate in the brain. Other carrier moieties include compounds, such as
amino acids or thyroxine, which can be passively or actively transported in
vivo. Such a carrier moiety can be metabolically removed in vivo, or can
remain intact as part of an active compound. Structural mimics of amino acids
(and other actively transported moieties) including peptidomimetics, are also
useful in the invention. As used herein, the term "peptidomimetic" is intended
to include peptide analogues which serve as appropriate substitutes for
peptides
in interactions with, for example, receptors and enzymes. The peptodomimetic
must possess not only affinity, but also efficacy and substrate function. That
is,
a peptidomimetic exhibits functions of a peptide, without restriction of
structure
to amino acid constituents. Peptidomimetics and methods for their preparation
and use are described in Morgan et al. (1989), the contents of which are
incorporated herein by reference. Many targeting moieties are known, and
include, for example, asialoglycoproteins (see e.g., Wu, U.S. Patent
5,166,320)
and other ligands which are transported into cells via receptor-mediated
endocytosis (see below for further examples of targeting moieties which may be
covalently or non-covalently bound to a target molecule).
In the methods of the invention, neurodegeneration in a subject is
mitigated, and/or neuroprotection and/or cognition enhancement is effected, by
administering a therapeutic compound of the invention to the subject. The
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invention also features methods for preventing or mitigating tissue and/or
cellular damage in a subject by administering a therapeutic compound of the
invention to the subject, thereby modulating intercellular and/or
intracellular
free radical concentration. The term "subject" is intended to include living
organisms in which the particular neurological condition to be treated can
occur. Examples of subjects include humans, apes, monkeys, cows, sheep,
goats, dogs, cats, mice, rats, and transgenic species thereof. As would be
apparent to a person of skill in the art, the animal subjects employed in the
working examples set forth below are reasonable models for human subjects
with respect to the tissues and biochemical pathways in question, and
consequently the methods, therapeutic compounds and pharmaceutical
compositions directed to same. As evidenced by Mordenti (1986) and similar
articles, dosage forms for animals such as, for example, rats can be and are
widely used directly to establish dosage levels in therapeutic applications in
higher mammals, including humans.
In particular, the biochemical cascade initiated by cerebral ischemia is
generally accepted to be identical in mammalian species (Mattson and Scheff,
1994; Higashi et al., 1995). In light of this, pharmacological agents that are
neuroprotective in animal models such as those described herein are believed
to
be predictive of clinical efficacy in humans, after appropriate adjustment of
dosage. Specifically, there are comparable memory-deficit patterns between
brain-damaged rats and humans, which indicates that the rat can serve as an
excellent animal model to evaluate the efficacy of pharmacological treatments
or brain damage upon memory (Kesner, 1990). The only approved drug for the
clinical treatment of occlusive stroke in humans is tissue plasminogen
activator,
which is administered at a dose of 0.9 mg/kg by intravenous injection
(Wittkowsky, 1998). This drug is also effective in protecting the rat brain
subjected to cerebral ischemia by occlusion of the middle cerebral artery,
when
administered at a dose of 10 mg/kg intravenously (Jiang et al., 1998). Thus,
the
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rat model of focal cerebral ischemia used in the development of the novel
organic nitrate esters described herein has been shown to be shown to be
predictive of clinical efficacy with at least one other class of
pharmacological
agents.
As would also be apparent to a person skilled in the art, the invention
further encompasses methods of the invention employed ex vivo or in vitro. For
example, the Examples describe studies utilizing tissue homogenates according
to the invention. Furthermore, diagnostic tests or studies of efficacy of
selected
compounds may conveniently be performed ex vivo or in vitro, including in
animal models. Such tests, studies and assays are within the scope of the
invention.
The pharmaceutical compositions may be formulated according to
conventional pharmaceutical practice (see, e.g., Remington: The Science and
Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams &
Wilkins, 2000, Philadelphia, and Encyclopedia of Pharmaceutical Technology,
eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Administration of the compositions of the present invention to a subject to be
treated can be carried out using known procedures, at dosages and for periods
of time effective to mitigate neurodegeneration, to effect neuroprotection, to
effect cognition enhancement, and/or to prevent or mitigate tissue and/or
cellular damage in the subject. An effective amount of the therapeutic
compound necessary to achieve a therapeutic effect may vary according to
factors such as the amount of neurodegeneration that has already occurred at
the clinical site in the subject, the age, sex, and weight of the subject, and
the
ability of the therapeutic compound to mitigate neurodegeneration, to effect
neuroprotection, to effect cognition enhancement, and/or to prevent or
mitigate
tissue and/or cellular damage in the subject. Dosage regimens can be adjusted
to provide the optimum therapeutic response. For example, several divided
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doses may be administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation. A non-limiting
example of an effective dose range for a therapeutic compound of the invention
(e.g., Va) is between 0.5 and 500 mg/kg of body weight per day. In an aqueous
composition, preferred concentrations for the active compound (i.e., the
therapeutic compound that can mitigate neurodegeneration, effect
neuroprotection, effect cognition enhancement, and/or prevent or mitigate
tissue and/or cellular damage) are between 5 and 500 mM, more preferably
between 10 and 100 mM, and still more preferably between 20 and 50 mM.
The therapeutic compounds of the invention can be effective when
administered orally. Accordingly, a preferred route of administration is oral
administration. Alternatively, the active compound may be administered by
other suitable routes such as transdermal, subcutaneous, intraocular,
intravenous, intramuscular or intraperitoneal administration, and the like
(e.g.,
by injection). Depending on the route of administration, the active compound
may be coated in a material to protect the compound from the action of acids,
enzymes and other natural conditions which may inactivate the compound.
The compounds of the invention can be formulated to ensure proper
distribution in vivo. For example, the blood-brain barrier (BBB) excludes many
highly hydrophilic compounds. To ensure that the therapeutic compounds of
the invention cross the BBB, they can be formulated, for example, in
liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents
4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or
more moieties which are selectively transported into specific cells or organs
("targeting moieties"), thus providing targeted drug delivery (see, e.g.,
Ranade
et al., 1989). Exemplary targeting moieties include folate and biotin (see,
e.g.,
U.S. Patent 5,416,016 to Low et al.); mannosides (Umezawa et al., 1988);
antibodies (Bloeman et al., 1995; Owais et al., 1995); and surfactant protein
A
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receptor (Briscoe et al., 1995). In a preferred embodiment, the therapeutic
compounds of the invention are formulated in liposomes; in a more preferred
embodiment, the liposomes include a targeting moiety.
Delivery and in vivo distribution can also be affected by alteration of an
anionic group of compounds of the invention. For example, anionic groups
such as phosphonate or carboxylate can be esterified to provide compounds
with desirable pharmocokinetic, pharmacodynamic, biodistributive, or other
properties. Exemplary compounds include IVl and pharmaceutically acceptable
salts or esters thereof.
To administer the therapeutic compound by other than parenteral
administration, it may be necessary to coat the compound with, or co-
administer
the compound with, a material to prevent its inactivation. For example, the
therapeutic compound may be administered to a subject in an appropriate
carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable
diluents include saline and aqueous buffer solutions. Liposomes include water-
in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et
al.,
1984).
The therapeutic compound may also be administered parenterally (e.g.,
intramuscularly, intravenously, intraperitoneally, intraspinally, or
intracerebrally). Dispersions can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary conditions of
storage
and use, these preparations may contain a preservative to prevent the growth
of
microorganisms. Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the composition must be sterile and
must
be fluid to the extent that easy syringability exists. It must be stable under
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conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi. The
vehicle can be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable oils. The
proper
fluidity can be maintained, for example, by the use of a coating such as
lecithin,
by the maintenance of the required particle size in the case of dispersion,
and by
the use of surfactants.
Prevention of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In some cases, it will be
preferable to include isotonic agents, for example, sugars, sodium chloride,
or
polyalcohols such as mannitol and sorbitol, in the composition. Prolonged
absorption of the injectable compositions can be brought about by including in
the composition an agent which delays absorption, for example, aluminum
monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the
therapeutic compound in the required amount in an appropriate solvent with
one or a combination of ingredients enumerated above, as required, followed by
filter sterilization. Generally, dispersions are prepared by incorporating the
therapeutic compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile injectable
solutions, the
preferred methods of preparation are vacuum drying and freeze-drying which
yield a powder of the active ingredient (i.e., the therapeutic compound)
optionally plus any additional desired ingredient from a previously sterile-
filtered solution thereof.
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The therapeutic compound can be orally administered, for example, with
an inert diluent or an assimilable edible carrier. The therapeutic compound
and
other ingredients may also be enclosed in a hard or soft shell gelatin
capsule,
compressed into tablets, or incorporated directly into the subject's diet. For
oral
therapeutic administration, the therapeutic compound may be incorporated with
excipients and used in the form of ingestible tablets, buccal tablets,
troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage
of
the therapeutic compound in the compositions and preparations may, of course,
be varied. The amount of the therapeutic compound in such therapeutically
useful compositions is such that a suitable dosage will be obtained.
It is especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited as unitary
dosages for the subjects to be treated; each unit containing a predetermined
quantity of therapeutic compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical vehicle. The
specification for the dosage unit forms of the invention are dictated by and
directly dependent on (a) the unique characteristics of the therapeutic
compound and the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such a therapeutic compound for
the treatment of neurological conditions in subjects.
Therapeutic compositions can be administered in time-release or depot
form, to obtain sustained release of the therapeutic compounds over time. The
therapeutic compounds of the invention can also be administered transdermally
(e.g., by providing the therapeutic compound, with a suitable carrier, in
patch
form).
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Active compounds are administered at a therapeutically effective dosage
sufficient to mitigate neurodegeneration, to effect neuroprotection, to effect
cognition enhancement, and/or to prevent or mitigate tissue and/or cellular
damage in a subject. A "therapeutically effective dosage" preferably mitigates
neurodegeneration by about 20%, more preferably by about 40%, even more
preferably by about 60%, and still more preferably by about 80% relative to
untreated subjects. The ability of a compound to mitigate neurodegeneration
can be evaluated in model systems that may be predictive of efficacy in
mitigating neurodegeneration in human diseases, such as animal model systems
known in the art (including, e.g., the method of transient middle cerebral
artery
occlusion in the rat) or by in vitro methods, (including, e.g., the assays
described herein).
It will be appreciated that the ability of a compound of the invention to
mitigate neurodegeneration will, in certain embodiments, be evaluated by
observation of one or more symptoms or signs associated with
neurodegeneration in vivo. Thus, for example, the ability of a compound to
mitigate neurodegeneration may be associated with an observable improvement
in a clinical manifestation of the underlying neurodegeneration-related
disease
state or condition, or a slowing or delay in progression of symptoms of the
condition. Thus, monitoring of clinical manifestations of disease can be
useful
in evaluating the neurodegeneration-mitigating efficacy of a compound of the
invention.
The method of the invention is useful for treating neurodegeneration
associated with any disease in which neurodegeneration occurs. Clinically,
neurodegeneration can be associated with conditions including but not limited
to: stroke; Parkinson's disease; Alzheimer's disease; Huntington's disease;
multiple sclerosis; amylotrophic lateral sclerosis; AIDS-induced dementia;
epilepsy; alcoholism; alcohol withdrawal; drug-induced seizures;
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viral/bacterial/fever-induced seizures; trauma to the head; hypoglycemia;
hypoxia; myocardial infarction; cerebral vascular occlusion; cerebral vascular
hemorrhage; hemorrhage; environmental excitotoxins of plant; animal, or
marine origin; dementias of all type; trauma; drug-induced brain damage; and
aging; or result from surgical procedures such as cardiac bypass.
Novel compounds according to the invention can be synthesized by
methods set forth herein or in U.S. Patent Nos. 5,807,847; 5,883,122;
6,310,052; and 6,365,579, for example. Various compounds for use in the
methods of the invention are commercially available and/or can be synthesized
by standard techniques. In general, nitrate esters can be prepared from the
corresponding alcohol, oxirane or alkene by standard methods, that include:
nitration of alcohols and oxiranes, mixed aqueous/organic solvents using
mixtures of nitric and sulfuric acid and/or their salts, with temperature
control
(see Yang et al., 1996); nitration of alcohols and oxiranes in acetic
anhydride
using nitric acid or its salts with or without added acid catalyst, with
temperature control (see, e.g., Louw et al., 1976); nitration of an alcohol
with a
nitronium salt, e.g. a tetrafluoroborate; nitration of an alkene with thallium
nitrate in an appropriate solvent (see Ouellette et al., 1976).
The following Examples further illustrate the present invention and are
not intended to be limiting in any respect. Those skilled in the art will
recognize, or be able to ascertain using no more than routine experimentation,
numerous equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention and are
covered by the claims.
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Example 1. Preparation of Nitrate Esters
Synthesis of Compound IVr
As shown in Figure 1, the synthesis of compound IVr proceeded from
the Bunte salt, 2,3-dinitrooxypropane-l-thiosulfonate (compound IVd), which
was prepared from 1,2-dinitrooxy-3-bromopropane as follows: 3-
bromopropane-1,2-diol was added dropwise into a cold mixture of HNO3 (68-
70%, 4.Oeq) and H2SO4 (95%, 4.0 eq) in CH2Cl2 (50 mL) at room temperature
over 30 min. The organic layer was separated, washed, dried and concentrated
to yield a yellow oil which was purified by flash chromatography on Si02 to
give 3-bromopropane-1,2-diol dinitrate in 45% yield (29). The Bunte salt was
prepared by reacting 3 -bromopropane- 1,2-diol dinitrate with an equimolar
portion of Na2S203 in 3:1 MeOH/H20 at 50 C for 10 hours and subsequently
purifying by flash chromatography on Si02 (29). The Bunte salt was oxidized
with a small molar excess of H202 (30%) in EtOH:H20 mixture (1:1) with a
catalytic amount of HZSO4 for 2 days. Extraction with CH2C12, concentration,
and purification by flash chromatography on Si02 yielded compound IVr as a
yellow oil (Rf = 0.65; CH2ClZ:hexane = 65:35; 5%). 'H-NMR (CDC13, 400
MHz): 5.55-5.65 (m, 1H), 4.87-4.94 (dd, 1H, J 12.94, 2.94), 4.62-4.70 (m, 1H,
J 12.88), 3.13-3.30 (m, 2H). 13C-NMR: (CDC13, 100 MHz): 76.74, 69.46/69.42,
36.65/36.63. Mass spec. (m/z, EI+): 380.1 (M-NO2)+ 290%; 426.1 (M)+ 100%;
427.1 (M+1)+ 10%; 428.1 (M+2)+ 17%; 429.2 (M+3)+ 1.5%; 430.3 (M+4)+
1.3%; calculated for C6H10N4012S3 426Ø Elemental analysis: calculated for
C6HION4014S2: C, 16.90; H, 2.36; S, 15.04; calculated for C6H,oN4012S3 : C,
16.90; H, 2.36; S, 22.56; found: C, 17.27; H, 2.38; S, 21.68.
Synthesis of Compound Va, Vbb, and Vbc
As shown in Figure 1, the syntheses of compounds Va, Vbb, and Vbc
proceeded from the Bunte salt, compound IVd. A round-bottomed flask
equipped with a dropping funnel, a thermometer, and a mechanical stirrer was
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charged with a solution of compound IVd and cooled to 0 with the aid of an
ice-salt bath. A cold solution of the thiol precursor of compounds Va, Vbb, or
Vbc was added rapidly, with vigorous stirring for 3 minutes, followed by the
addition of aqueous saturated NaCI. The mixture was warmed to 5 C and
stirring stopped after 10 minutes. The crude disulfides Va, Vbb, or Vbc, were
extracted 3x with diethyl ether. The extracts were combined, dried over
calcium sulfate, and filtered through a glass-wool plug. Removal of the
solvent
leaves disulfide product, which can be further purified by silica gel
chromatography.
Synthesis of Compounds Ve, Vf, Vg, Vh, Vi, Vj, and Vu
Alkyl bromides or alkyl mercaptans were obtained commercially or by
adaptation of literature procedures. Bunte salts were obtained from the
appropriate alkyl bromide by reaction with sodium thiosulfate, as described
above for compound IVd. Bunte salts (9.67 mmoles) were dissolved in
distilled water (IOmL). To this solution, a solution of mercaptan (6.46
mmoles) in 1 M NaOH (7mL) was added dropwise. The resulting emulsion was
stirred for 1 to 15 minutes and then extracted with dichlorometane or
ethylacetate. The combined organic extracts were washed with H20, dried over
MgSO4 and concentrated under vacuum. The residue was purified by flash
colunm chromatography on silica gel to give the following: Compound Vj
(53% yield),13C NMR (75Mhz, CDC13) 36.9, 69.8, 77.6, 128.6, 129.5, 129.8,
136.0; Compound Vi (43% yield),13C NMR (75Mhz, CDC13) 36.9, 69.8, 77.4,
122.7, 130.9, 132.9, 135.1; Compound Vh (9% yield),13C NMR (75Mhz,
CDC13) 36.9, 69.6, 77.6, 124.5, 126.9, 144.7, 147.0; Compound Vg (81%
yield),13C NMR (75Mhz, CDC13) 36.8, 69.8, 77.5, 129.9, 130.8, 134.5, 134.8;
Compound Vf (56% yield),13C NMR (75Mhz, CDC13) 36.5, 55.8, 69.7, 77.8,
115.4, 126.7, 133.7, 160.9; Compound Ve (52% yield), 13C NMR (75Mhz,
CDC13) 21.5, 36.8, 69.8, 77.7, 130.5, 130.6, 132.5, 139.2; and Compound Vuj
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(61% yield),13C NMR (75Mhz, CDC13) 36.7, 69.9, 77.6, 116.8-117.1(d),
131.3- 131.3(d), 132.5-132.6(d), 161.6-164.9(d).
Synthesis of Compound Vb
1,4-dibromo-2,3-dinitrobutanediol (8.88 mmol) and Na2SzO3.5H20
(2.81 g; 18 mmol) were dissolved in a mixture of 100 mL of MeOH and 45 mL
of H20. The resulting solution was heated during 4 days at 40-45 . After this
time the reaction mixture was partially evaporated to reduce the volume of
solvents. The resulting mixture was extracted with 4x50 mL of Et20. The
extracts were combined, washed (H20), dried (MgSO4) and concentrated. The
residue was purified by flash colunm chromatography on silica gel
(hexane:EtOAc 85:15), yielding the title compound (10%) 13C (CDC13): 83.9,
31.7.
Tables 1 and 21ist characterization data determined for other compounds
of the invention.
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Table 1
I 'H NMR 13C NMR
IIla (CDC13): 5.34-5.57 (1H, dm, 3JHF 20.6), 4.53-4.87 (CDC13): 79.47 (d, IJCF
177), 76.73
(411, sup
2 4 erposition several multiplets, OZNO-CH +(d, ZJcF 20.6), 67.84 (d, 3JCF
6.87)
CH F, JHF 46.7, JHF 0.66
IIIb (CDC13):8 (CDC13): 6
IIIc (CDC13): S 5.7 (1H, t, zJHF 54), 5.45 (1H, m), 4.5- (CDC13): 8 75.55,
68.05, 60.76
4.9 2H,m,4.15-4.35 1H,m
IIId (CDC13): S 5.46 (1H, m), 4.80-4.87 (1H, dd, J 3.5, (CDC13): S 77.24,
68.57, 39.86
12.9), 4.65-4.72 (1H, dd, J 6.2, 12.9), 3.7-3.8 (2H,
m
IIIf (CDC13) 6 8.72 (s, 1H), 5.38 (t, 1H), 4.6 (d, 2H), -
2.45 (s, 3H
IlIg (DMSOd6) CHONOz only: 6 4.8-5.8 (DMSOd6) CONOz only: 6 85.68,
84.17, 82.47, 76.50
IIIh CD3OD S 4.85 3H, m), 3.5 1H, m) CD3OD S 70.61, 36.74
IIIi (CDC13): 8 6.95 (dd, 1H), 6.71 (dd, 1H), 6.09 (m, (CDC13): 6 137.9,
132.5, 76.6, 52.9
1H), 3.80 (dd, 1H , 3.32 (dd, 1H
IIlj (CDC13): S 5.62 (2H, m), 3.60 (4H, m) (CDC13): S 77.87, 25.22
IIIk CD3C : S 3.45 (m, 2H , 5.72 (m, 2H) CD3CN : S 79.98, 28.87
IIIl - CD3C : 8 79.48, 33.45, 28.47
IIIm DMSOd6 : 8 5.97 (m, 2H), 3.80 (m, 4H) DMSOd6 : S 78.84, 52.60
IIIn (CDC13): S 5.73 (m, 1H), 4.62 (m, 1H), 3.96-3.77 (CDC13): 6 81.47, 57.85,
53.50,
(m, 1H), 3.58-3.32 (m, 1H 38.75
IIIo - (CDC13): 8 81.24, 69.79, 33.26,
27.24
IIIp (CDC13): S 5.36 (m, 1H), 3.11-3.60 (m, 4H), 2.33 (CDC13): S 78.92, 33.66,
30.64,
(m, 2H 27.36
IIlq (CDC13): S 5.47 (m, 1H), 3.53-3.05 (m, 4H), 2.29 (CDC13): 6 81.32, 37.12,
32.97,
m, 211) 30.98
Ni (CDC13): S 5.45 (1H, m), 4.83 (1H, dd), 4.65 (1H, (CD3OD): 8 116.44, 75.37,
71.20,
dd), 2.9 (2H, m) 19.19
IVk (CDC13) S 8.55 (s, 1H), 4.55 (t, 2H), 3.15 (t, 2H), (CDC13) 6 150.9,
150.7, 125.3,
2.37 (s, 31172.53, 24.47, 15.18
Vb (CDC13) S 5.56 (m, 2H), 3.38-2.95 (m, 4H) (CD3OD) S 85.93, 32.77
Vc (CDC13): 8 5.85-5.91 (1H, m), 4.50-4.58 (1H, m), (CDC13): 6 87.6, 74.96,
36.20, 31.54
3.22-3.29 (1H, dd, J 5.47, 12.78), 2.97-3.05 (1H,
dd, J 4.6, 11.88), 2.82-2.90 (1H, dd, J 2.87, 12.78),
2.74-2.83 1H, dd, J 3.15, 11.9)
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Table 2
1H NMR 13C NMR
ozNO (CDC13): 8 2.43 (s, 3H), 2.89- _
N 3.05 (4H, superposition of a
o2NO~-ss--\ 41,
Js
triplet with multiplets), 3.18
(t, 2H), 4.8 (dd, 2H), 5.51 (m,
1H), 8.62 (s, 1H)
o2No-\~/ N (CDC13): S 0.95 (d, 3H, _
S J=6.9), 1(d, 3H, J=6.9), 2.15
ONO2 (m, 1H), 2.36 (s, 3H), 3.13 (t,
2H, J= 6.8), 4.6 (t, 2H,
J=6.8), 4.7 (d, 1H, J=5.1).
OZNO-\~/ " (CDC13) : 8 2.44 (s, 3H), 3.21 (CDC13): S 15.27,
S ~
(t, J=6.7, 2H), 4.63 (t, J=6.7, 24.67, 72.37, 125.24,
2H), 7.4 (m, 3H), 7.9 (m, 2H) 126.43, 129.05,
130.04, 133.7, 151.1,
166.
(CDC13) : 8 2.33 (s, 3H), 2.63 (CDC13): 6 163.58,
OzNO / N
(s, 3H), 3.11 (t, 2H), 4.57 (t, 149.29, 124.36,
2H) 72.42, 24.43, 19.26,
14.94
O2NO~ N (CDC13): S 2.57 (s, 3H), 3.32 (CDC13): S 15.45,
o (t, J=6.43, 2H), 4.68 (t, 25.19, 71.31, 114.34,
F3C J=6.43, 2H). 118.18, 138.18,
155.29, 156.31
02NO (CDC13): 6 5.21 (q, 1H), 4.62 (CDC13): S 160.64,
/ N
S%~/oH (t, 2H), 3.2 (t, 2H), 2.4 (s, 149.8, 128.3, 125.11,
F3e~ 3H) 121.36, 71.9, 24.53,
14.75.
oZNO-\_~/-" (CDC13): S 7.82 (d, 2H, (CDC13) 164.13,
~
S J=8.4), 7.4 (d, 2H, J=8.4), 151.35, 136.02,
4.64 (t, 2H), 3.21 (t, 2H), 132.24, 129.34,
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2.43 (s, 3H) 127.66, 125.71,
72.28, 24.69, 15.25
~N (CDC13): S 7.91 (m, 2H), -
oZrvo S I~ 7.44 (m, 3H), 4.61 (s, 2H),
~ 2.54 (s, 3H)
~N (CDCl3): 6 8.03 (d, 2H), 7.7 (CDC13): 6 166.73,
oZNO S (d, 2H), 5.62 (t, 2H), 2.56 (s, 155.86, 132.5,
CF3 3H) 132.06, 126.98,
126.25 (q), 122.87,
65.99, 15.56
(CDC13): 8 8.0 (d, 2H), 7.68 (CDC13): 6 163.52,
N
oZNO S (d, 2H), 4.65 (t, 2H), 3.23 (t, 151.78, 136.8,
cF' 2H), 2.46 (s, 3H) 126.75, 126.64,
126.13 (q), 125.91,
122.3, 72.19, 24.7,
15.25
(CDC13): 6 7.78 (d, 2H), 7.23 (CDC13): 8 165.74,
N
ozNO S (d, 2H), 4.63 (t, 2H), 3.19 (t, 150.88, 140.36,
2H), 2.39 (s, 3H) 131.05, 129.79,
126.4, 124.67, 72.4,
24.65, 21.6, 15.24
(CDC13): 6 6.62(dd, 1 H), -
02N0 S CH3 6.56(dd, 1H), 4.56(t, 2H),
3.09(t, 2H), 2.49(s, 3H)
(CDC13): 6 7.20 (dd, 1 H), _
O2NO S 6.95(dd, 1 H), 6.89(dd, 1H),
4.64(t, 2H), 3.23(t, 2H)
c' (CDC13): 6 7.96 (m, 2H), (CDC13): 8 166.09,
~i
7.59 (m, 2H), 7.45 (m, 5H) 152.92, 134.52,
OZNO / N
s 4.68 (t, 2H), 3.37 (t, 2H). 133.44, 133.15,
120.49, 130.31,
129.18, 129.01,
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126.67, 72.27, 25.37.
~N (CDC13): 6 7.85 (d, 2H), 7.41 (CDC13): 6 167.38,
02NO S (d, 2H),5.60 (t, 2H), 2.53 (s, 155.53, 136.76,
ci 3H) 131.78, 129.46,
127.94, 121.91,
65.12, 15.5.
I\ ON02 (CDC13): 8 7.78(m, 1H), -
S 7.76(m, 1 H), 7.31(m, 2H),
7.11(s, 1 H), 4.71(t, 2H),
3.29(t, 2H)
CH3 (CDC13): s 6.70(s, 1 H), _
6.56(s, 1H), 3.78(t, 2H),
02NO S
2.09(t, 2H), 2.19(s, 3H)
CH3 (CDC13): S 6.74(s, 1H), _
4.67(t, 2H), 3.12(t, 2H),
OzNO S NO2
2.59(s, 3H)
Example 2
Characterization of Cardioprotection in Isolated, Perfused Heart
In order to test for potential cardioprotective properties, the effects of Va
and compound IIIam were tested in an in vitro model of cardiac ischemia, in
which isolated, perfused rat hearts were subjected to transient left coronary
artery occlusion (LCAO) followed by reperfusion. Drug treatments [DMSO
(drug vehicle), GTN, compound Va or compound IIIam were initiated at two
distinct time points: (i) prior to and throughout the 45 minute period of LCAO
(protection) or (ii) prior to and throughout the 90 minute reperfusion period
(salvage). Drug-induced reduction of lactate dehydrogenase (LDH) release and
reduction of infarct size were assessed as measures of cardioprotection. Rat
hearts were excised and mounted for retrograde aortic perfusion at a constant
flow rate of 6-8 mL/min/g heart weight. The coronary perfusion pressure was
monitored by a pressure transducer connected to the perfusion line. To induce
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regional ischemia, the left coronary artery was occluded for 45 minutes, after
which the occlusion was released and the heart reperfused for 90 minutes. At
the end of the reperfusion period, the LCA was re-occluded and 0.5 mL of 1%
Evan's Blue dye was slowly infused into the heart, via the aotic cannula, to
stain the area of myocardium perfused by the patent right coronary artery.
Thus
the area-at-risk (AAR) for infarction was determined by negative staining.
Acute ischemic damage was assessed by measuring the release of the cytosolic
enzyme, LDH, into the perfusate, and by quantitation of infarct size by
staining
for viable tissue using 2,3,5-triphenyltetrazolium chloride (TTC) followed by
computerized planimmetry. Infarct size was expressed as infarct area (negative
staining after TTC staining) as a percent of the area-at-risk (negative
staining
after Evan's Blue dye). Left coronary artery occlusion was associated with a 5-
fold increase in LDH release (Figures 2 and 4, solid bars), whereas LDH
release was not increased in non-occluded hearts (Figures 2 and 3, open bars).
When administered prior to and during the period of LCA occlusion (protection
protocol), compound Va, in a concentration-dependent manner, significantly
reduced LDH release during the subsequent period of reperfusion (Figure 2, * P
< 0.01 vs. LCAO + DMSO, one-way ANOVA, Tukey-Kramer post-hoc test),
whereas GTN had no effect. Using the same drug infusion protocol, compound
Va significantly reduced the size of the myocardial infarct after LCAO and
reperfusion (Figure 4, * P < 0.01 vs DMSO-treated hearts, one-way ANOVA,
Tukey-Kramer post-hoc test) whereas GTN had no effect. As a measure of the
functional recovery of the heart after ischemia/reperfusion injury, the
increase
in perfusion pressure during the reperfusion period was monitored (Figure 6).
In hearts subjected to LCAO, perfusion pressure was increased by about 2-fold
by the end of the reperfusion period. This increase was markedly reduced in
hearts treated with compound Va, whereas GTN had no effect. When GTN,
compound Va and compound IIlam were infused prior to and throughout the
reperfusion period (salvage protocol), all three drugs decreased LDH release
during the reperfusion period (Figure 3, * P < 0.01 vs. LCAO + DMSO, one-
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way ANOVA, Tukey-Kramer post-hoc test). However, only compound Va and
compound IIlam reduced infarct size when administered prior to and
throughout the reperfusion period (Figure 5). These results indicate that
compound Va reduces the severity of ischemia-reperfusion injury when
administered prior to and throughout an acute ischemic insult, and that
compounds Va and compound IIIam reduce the severity of ischemia-
reperfusion injury when administered just prior to and throughout reperfusion,
after a prolonged ischemic insult. However, the prototypical nitrate, GTN, is
ineffective at protecting the heart from acute ischemic insult.
Example 3
Neuroprotection against 6-hydroxydopamine-induced killing of dopaminergic
neurons in the rat substantia nigra pars compacta
Male Long-Evans rats were anesthetized with sodium pentobarbital, and
received stereotaxic, unilateral injections of 6-OHDA (6 g in 2 l) into the
right substantia nigra pars compacta. Vehicle (dimethylsulfoxide, DMSO) or
compound Va were given by subcutaneous injection every hour for 6 hours,
beginning 30 minutes before 6-OHDA. Each dose of compound Va was 200
mol/kg. Two weeks after the administration of 6-OHDA or vehicle, the rats
received a single injection of apomorphine (1 mg/kg, s.c.), and contralateral
rotations were counted at 15 minute intervals for 60 minutes. In some animals,
the brains were fixed, and frozen sections cut for immunocytochemical analysis
of tyrosine hydroxylase (TH).
In vehicle-treated animals, apomorphine induced rotations contralateral
to the lesion that persisted for the entire 60 minute observation period. In
contrast, animals that received compound Va exhibited essentially no rotations
over 60 minutes in response to apomorphine injection, suggesting virtually
complete protection against the neurotoxic effects of 6-OHDA (Table 3).
Immunocytochemical analysis confirmed that compound Va preserved TH-
positive neurons in the substantia nigra of 6-OHDA-injected rats (Figure 7).
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These data demonstrate that compound Va is a very effective neuroprotective
agent against 6-OHDA-induced killing of dopaminergic neurons in the rat
substantia nigra pars compacta.
Table 3: Apomorphine-Induced Rotations after Unilateral 6-Hydroxydopamine
Lesion
Treatment Rotations/15 minutes
Vehicle 32 6 (n = 8)
Compound Va 0.2 0.1 (n = 10)*
Deprenyl 0.1 0.1 (n = 4)*
p < 0.001 compared to vehicle control, One-way analysis of variance with
Bonferroni's Multiple Comparison's test.
Synaptosome Assay The methodology for measurement of thiobarbituric
acid reactive substances (TBARS) from synaptosomes was adapted from that of
Keller et al. Neuroscience 80: 685-696, 1997. Adult Sprague-Dawley rats
(250-300g) were anesthetized with halothane for 20 sec. and decapitated. The
brain was removed and the cerebral cortex separated from white matter. The
cerebral cortex was homogenized in a solution containing sucrose 0.32 M,
EDTA 2mM and TRIS=HCl 10 mM, pH 7.2, using a Teflon pestle. The tissue
was 5% w/v in the homogenizing buffer. The homogenate was centrifuged for
minutes at 310 g at 4 C. The supematant was then centrifuged for 10
minutes at 20,000 g at 4 C. The pellet was collected, resuspended in Locke's
buffer (154 mM NaCI, 5.6 mM KCI, 2.3 mM CaCl2=2H20, 1.0 mM
MgCl2=6H2O, 3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES, pH 7.2) and
centrifuged for 10 minutes at 20,000 g at 4 C. The procedure of washing the
pellet was repeated 2-3 times in order to reduce transition metal ion
concentrations. Finally, the pellet was resuspended in Locke's buffer for use
in
the lipid peroxidation assay. All assays were performed in triplicate and on
three separate synaptosome preparations from different animals.
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Potential antioxidants and prooxidants were freshly prepared: in Locke's
buffer [FeSO4; ascorbic acid (AA); Trolox; cysteine]; in 10 mM NaOH
[NONOates]; or in organic solvent, such as methanol or DMSO, [a-tocopherol
(ctTH); nitrates; nitrites; PhSH; lipoic acid (LA); dihydrolipoic acid
(LAHZ)];
final dilution 52.5% (v/v) organic component). Pro/antioxidants (or solvent
vehicle in control experiments) were added to the synaptosome preparation,
followed immediately by freshly prepared aqueous FeSO4 (or buffer in control
experiments) and incubated for 30 minutes at 37 C (air equilibrated). After
incubation, TBA reagent (TBA 0.375% w/v, C13CCO2H 15% w/v, HCl 1M
25% v/v) was added to the homogenate (homogenate: TBA reagent, 1:2 v/v),
and the sealed samples were boiled for 15 minutes at approx. 96-100 C. The
cooled samples were then centrifuged for 10 minutes at 9,000 g at room
temperature. The pink supematant was transferred into microplates and the
absorbance was measured at 530 nm on a Dynex MRX microplate reader. TBA
reagent solutions were freshly made and calibrated using solutions of
authentic
malondialdehyde.
Lipid peroxidation: synaptosome experimental results. Preliminary lipid
peroxidation experiments explored the time course of synaptosome lipid
peroxidation, incubating homogenate with FeSO4 (lO M - 150 M) in Locke's
buffer, for time intervals from 15 minutes to 180 minutes (data not shown).
Under these experimental conditions, the level of peroxidation, as measured by
TBARS, was seen to be below saturation at 30 minutes using 50 M FeSO4.
The ability of this system to provide concentration dependent lipid
peroxidation
data was demonstrated using the antioxidant a-tocopherol (aTH), and ascorbic
acid (AH), which is known to act as a prooxidant in Fe(II)-induced lipid
peroxidation systems (see Figure 8). These conditions thus were chosen for all
further synaptosome experiments.
The Fe/synaptosome/TBARS system was designed to provide
concentration-response curves for inhibition of lipid peroxidation, which
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be quantified by EC50 values. Absolute EC50 values measured in such systems
are highly dependent on experimental conditions, and therefore must be
benchmarked against well-studied antioxidants, such as Trolox, a water soluble
chroman carboxylate derivative of aTH (see Figure 9).
Thiols can display mixed pro- and antioxidant activity towards lipid
peroxidation. In particular, in the presence of transition metals, either
added to
lipid preparations, or adventitious metal ions present in tissue homogenates,
thiols may act as prooxidants. The vic-dithiol, dihydrolipoic acid (LAH2, ~,
solid line in Figure 10) yielded a concentration dependent prooxidant effect,
akin to ascorbic acid, whereas the oxidized disulfide lipoic acid (LA, =,
dashed line in Figure 10) showed very modest inhibition of lipid peroxidation
at
the highest concentration applied. Cysteine (1 mM) was a prooxidant in the
presence of FeSO4 giving 117% of the lipid peroxidation seen in the presence
of FeSO4 alone, whereas PhSH (1 mM) in the presence of FeSO4, gave 110%
of the lipid peroxidation seen in the presence of FeSO4 alone (data not
shown).
Of further consideration in analysis of lipid peroxidation data is the
requirement
for added thiols in experiments with nitrates. Clearly, any antioxidant effect
of
nitrates may be masked by the prooxidant effect of the adjuvant thiol. Thus,
it
was chosen to present the data as "percentage inhibition of lipid
peroxidation",
by normalizing TBARS measurements to: (i) 100% inhibition of lipid
peroxidation (corresponding to TBARS in the presence of vehicle and absence
of FeSO4); and, (ii) 0% inhibition of lipid peroxidation (corresponding to
TBARS in the presence of vehicle, FeSO4, and any added thiol). This
methodology and protocol was applied uniformly to all experiments graphed in
Figures 9-17.
The antioxidant trolox yielded a potent concentration dependent
reduction in TBARS products with EC50 = 6.8 x 10-5 M (Figure 9), which can
be contrasted to the effect of lipoic acid (LA) or dihydrolipoic acid (LAH2,
see
Figure 10).
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GTN alone had no effect on Fe-induced lipid peroxidation (data not
shown), nor did varied concentrations of GTN have any significant effect in
the
presence of added LAH2 (1 mM) (Figure 11). Indeed, GTN produced no
significant effect on lipid peroxidation with any thiol used (e.g. cysteine,
PhSH), over the effect of the thiol itself (see GTN with LAH2, ~, solid line
in
Figure 11). However, compound Va (A, dashed line, Figure 11) did inhibit
lipid peroxidation at higher concentrations in the presence of LAH2. Further,
in
contrast to GTN, compound Va inhibited TBARS formation with the water
soluble thiol, cysteine, and the more lipophilic thiophenol (PhSH) (see Va +
cysteine, ~, solid line or Va + PhSH, =, solid line in Figure 12). Addition of
PhSH (1mM) yielded a concentration dependent inhibition curve for lipid
peroxidation: EC50 = 1.4 x 10-5 M. At high millimolar concentrations,
compound Va showed some prooxidant activity in the presence of cysteine.
Data for inhibition of iron-induced lipid peroxidation by compound IVr
can be fit to a curve leading to 100% efficacy with an EC50 of 1.2 x 10-4 M
(or
fitted without constraint on efficacy to EC50 = 2.7 x 10-4 M; efficacy = 78%).
Moreover, compound Va, in the absence of thiol, showed a modest inhibition of
lipid peroxidation, more pronounced at lower concentrations (see Figure 13).
This antioxidant effect is clearly not an ubiquitous property of disulfides,
since
lipoic acid (LA) does not show such properties in the identical assay (Figure
10).
Concentration response curves were derived from TBARS data for the
NO donor NONOate, Sper/NO (spermine NONOate, dashed line), and for
DEA/NO (diethylamine NONOate, solid line) (see Figure 14). The potency
and efficacy of inhibition of lipid peroxidation by Sper/NO and by IAN (see
Figure 15) were both observed to be approximately identical (EC50 IAN = 1.6 x
10-4 M, Sper/NO = 2 x 10"4 M). TBARS measured for the compound IVs
(dashed lines in Figure 16), in the absence of any adjuvants, revealed similar
efficacy for this compound compared to Sper/NO and IAN, but a potency lower
by an order of magnitud (EC50 (synaptosomes) = 1.0 x 10-3 M, (liposomes) _
92
CA 02580627 2007-03-15
WO 2006/029532 PCT/CA2005/001417
1.3 x 10"4 M). The observed behavior of compounds IVs and IVr was similar,
but measurements on compound IVr could not be extended to higher
concentrations because of solubility. The potency and efficacy of inhibition
of
lipid peroxidation by compounds Vbb (V, solid line in Figure 17) and Vbc (~,
solid line in Figure 17) were also measured and found to be 2.0 x 10-5 M for
Vbb and and 7 x 10-7 M and 7 x 10-5 M for Vbc.
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All publications and patents cited in this specification are hereby
incorporated by reference herein as if each individual publication or patent
were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding, it will be
readily apparent to those of ordinary skill in the art in light of the
teachings of
this invention that certain changes and modifications may be made thereto
without departing from the spirit or scope of the appended claims.
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