Compounds inhibiting the aggregation of superoxide dismutase-1

• US 20060194821A1
• Filed: 02/16/2006
• Published: 08/31/2006
• Est. Priority Date: 02/18/2005
• Status: Abandoned Application

• Current Assignee: Brigham and Women's Hospital Incorporated
• Original Assignee: Brigham and Women's Hospital Incorporated
• Inventors: Choi, Andrew, Nowak, Richard, Lansbury, Peter, Ray, Soumya
• Application Number:
  US11/354,896
• Publication Number:
  US 20060194821A1
• Time in Patent Office: Days
• US Class Current: 514/263.23
• CPC Class Codes: A61K 31/165 having aromatic rings, e.g. colchicine, atenolol, progabide
• CPC Class Codes: A61K 31/52 Purines, e.g. adenine
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

The invention is directed to methods of inhibiting the rate at which superoxide dismutse-1 (SOD) aggregates using compounds that stabilize SOD dimers. The methods are useful in the study and therapy of amyotrophic lateral sclerosis. The invention also includes assays that can be used to identify compounds that stabilize dimers and SOD molecules that have been modified for use in these assays.

Claims

1. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula I:

2. The method of claim 1, wherein in said compound of formula I:

a, b, c and d are each independently selected from C and N, with the proviso that a, b, c and d are not all N;

at least one of R₁, R₂ and R₆ is —

(CH₂)_n-Z-(CH₂)_m—

R₇,

where Z is C, N or S,

R₇ is H, CH₃ or a phenyl optionally substituted at one or more positions with CH₃ halogen, OH or NH₂,

n is an integer from 0 to 3 inclusive,

m is an integer from 0 to 3 inclusive;

when R₁ is —

(CH₂)_n-Z-(CH₂)_m—

R₇, R₂ and R₆ may also each independently be selected from;

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂;

when R₂ is —

(CH₂)_n-Z—

(CH₂)_m—

R₇, R₁ and R₆ may also each independently be selected from;

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂;

when R₆ is —

(CH₂)_n-Z-(CH₂)_m—

R₇, R₁ and R₂ may also each independently be selected from;

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂;

R₃ and R₅ are each independently either O or S; and

R₄ is H, a (C₁-C₃)alkyl, OH or NH₂.

3. The method of claim 2, wherein, in said compound of formula I, a, b, c and d are either C or N, with the proviso that no more than two of a, b, c and d may be N.

4. The method of any one of claims 1-3, wherein, in said compound of formula I, R₁ is H, a halogen, a (C₁-C₃)alkyl, OH or NH₂.

5. The method of any one of claims 1-3, wherein, in said compound of formula I, R₂ is H, a halogen, a (C₁-C₃)alkyl, OH or NH₂.

6. The method of any one of claims 1-3, wherein, in said compound of formula I, R₆ is H, a halogen, a (C₁-C₃)alkyl, OH or NH₂.

7. The method of any one of claims 1-3, wherein, in said compound of formula I, R₁ and R₂ are each independently selected from:

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂.

8. The method of any one of claims 1-3, wherein, in said compound of formula I, R₁ and R₆ are each independently selected from:

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂.

9. The method of any one of claims 1-3, wherein, in said compound of formula I, R₂ and R₆ are each independently selected from:

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂.

10. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula II:

11. The method of claim 10, wherein R₈ is group a), b) or c);

and wherein R₉ and R₁₃ are each independently selected from H, a halogen, a (C₁-C₃)alkyl, OH and NH₂.

12. The method of claim 10, wherein R₉ is group a), b) or c);

and wherein R₈ and R₁₃ are each independently selected from;

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂.

13. The method of claim 10, wherein R₁₃ is group a), b) or c);

and wherein R₈ and R₉ are each independently selected from;

H, a halogen, a (C₁-C₃)alkyl, OH and NH₂.

14. The method of any one of claims 11-13, wherein R₁₀, R₁₁ and R₁₂ are each independently selected from:

a halogen, a (C₁-C₃)alkyl, OH and NH₂.

15. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula III:

16. The method of claim 15, wherein:

a) no more than three of a, b, c, d, e and f are N;

b) no more than one of R₁₇, R₁₈ and R₂₂ is

c) no more than one of R₁₉, R₂₀ and R₂₁ is

17. The method of claim 16, wherein:

R₁₇ is

R₂₀ is

18. The method of any one of claims 15-17, wherein no more than two of a, b, c, d, e and f are N.

19. The method of any one of claims 15-18 wherein no more than one of a, b, c, d, e and f are N.

20. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula IV:

21. The method of claim 20, wherein R₂₈ is NH₂ or NH—

NH₂.

22. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula V:

23. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula VI:

24. The method of claim 23, wherein:

n is 1 or 2;

Z is N; and

R₃₂ is NH₂.

25. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula VII:

26. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound selected from the group consisting of:

a) N-nitroso-5-(phenylsulfinyl)pyridin-2-amine;

b) 6-[(4-chlorophenyl)amino]pyrimidine-2,4(1H,4H)-dione;

c) 6-[(4-chlorobenzyl)thio]1,2,4-triazine-3,5(2H,4H)-dione;

d) 4-bromo-2-{(E)-[(4-fluorophenyl)imino]methyl}phenol;

e) 6-(ethylthio)-thioxo-4,5-dihydro-1,2,4-triazin-3 (2H)-one;

f) 2-[2-(2-amino-4-methylphenyl)ethyl]-5-methylaniline;

g) 6-[(4-fluorobenzyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

h) 2-(3-fluorophenyl)hydrazinecarboxamide;

i) 3-benzyl-2-hydroxylbenzohydrazide;

j) 4-hydroxybenzaldehyde semicarbazone;

k) 4-(1,3-benzothiazol-2-yl)butanamide;

l) 2-(1H-benzimidazol-2-yl)acetohydrazide;

m) N-[(1R,4R)-4-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl]urea;

n) trimacicolone; and

o) 6-amino-methy-adenosine.

27. A mutated form of SOD having the sequence of SEQ ID NO:

1 but wherein at least one amino acid is replaced with a substitute amino acid that can undergo fluorescent labeling, said at least one amino acid being selected from the group consisting of;

Gly51;

Asp52;

Thr54;

Aala55;

Ser59;

Ala1;

Thr2;

Ala4;

Val5;

Val7;

Lys9;

Gly10;

Asp11;

Gly12;

Gln15;

Ser107;

Gly108;

Asp109;

Cys111;

Ile113;

Gly114;

Arg115;

Thr116; and

Leu117.

29. The method of claim 27, wherein said substitute amino acid is cys.

30. The mutated SOD of claim 27, wherein lysine at position 9 is changed to cys.

31. A dimer of the SOD of any one of claims 27-30.

28. An SOD enzyme having the sequence shown in FIG. 4 wherein said SOD is fluorescently labeled at a position selected from the group consisting of amino acid:

51;

52;

54;

55;

59;

1;

4;

5;

7;

9;

10;

11;

12;

15;

107;

108;

109;

111;

113;

114;

115;

116; and

117; and

wherein the amino acid that is shown in each of these positions in FIG. 4 may be replaced with a substitute amino acid to facilitate fluorescent labeling.

32. An assay for measuring the ability of a test compound to stabilize SOD dimers, comprising:

a) incubating a labeled SOD homodimer or labeled SOD heterodimer in which both SOD molecules in said homodimer or heterodimer are fluorescently labeled with an unlabeled SOD homodimer in which neither SOD is fluorescently labeled, wherein the incubation is performed in the presence of said test compound;

b) measuring the rate of mixed heterodimer formation in the incubation of step a) by determining the rate of loss of fluorescence attributable to said labeled SOD homodimer or labeled SOD heterodimer over time, wherein said mixed heterodimer has one SOD molecule that is labeled and one SOD molecule that is unlabeled;

c) comparing the rate of mixed heterodimer formation in step b) with the rate determined for a similar incubation carried out in the absence of said test compound; and

d) concluding that said test compound stabilizes the dimerized form of SOD if the rate of mixed heterodimer formation determined in the presence of said test compound is slower than the rate determined in the absence of said test compound.

33. An assay for measuring the ability of a test compound to stabilize SOD dimers, comprising:

a) incubating a first labeled SOD homodimer with a second labeled SOD homodimer in the presence of said test compound, wherein the SOD in said first homodimer is labeled with a donor fluorophore and the SOD in said second homodimer is labeled with an acceptor fluorophore;

b) measuring the rate of mixed donor/acceptor heterodimer formation in the incubation of step a) by measuring the change in fluorescence over time, wherein said mixed donor/acceptor heterodimer has one SOD labeled with said donor fluorophore and one SOD labeled with said acceptor fluorophore;

c) comparing the rate of mixed donor/acceptor heterodimer formation in step b) with the rate determined for a similar incubation carried out in the absence of said test compound; and

d) concluding that said test compound stabilizes the dimerized form of SOD if the rate of mixed donor/acceptor heterodimer formation determined in the presence of said test compound is slower than the rate determined in the absence of said test compound.

34. An assay for measuring the ability of a test compound to stabilize SOD dimers, comprising:

a) incubating a labeled SOD heterodimer with an unlabeled SOD homodimer in the presence of said test compound, wherein said labeled SOD heterodimer has one SOD molecule labeled with a donor fluorophore and a second SOD molecule labeled with an acceptor fluorophore and wherein neither SOD in said SOD homodimer is fluorescently labeled;

b) determining the rate of dimer dissociation by measuring the loss in fluorescence attributable to said labeled SOD heterodimer over time,

c) comparing the rate of fluorescence loss in step b) with the rate determined for a similar incubation carried out in the absence of said test compound; and

d) concluding that said test compound stabilizes the dimerized form of SOD if the rate of fluorescence loss in the presence of said test compound is slower than the rate of fluorescence loss in the absence of said test compound.

35. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula VIII:

36. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound of formula IX:

37. A method of inhibiting the aggregation of superoxide dismutase, comprising contacting superoxide dismutase dimers with an effective amount of a compound selected from the group consisting of:

a) methyl3-(3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoate;

b) 6-[(3,5-dimethyl-1H-pyrazol-4-yl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

c) 3-methyl-6-[methyl(phenyl)amino]pyrimidine-2,4(1H,3H)-dione;

d) 2-(2-methyl-1H-benzimidazol-1-yl)acetamide;

e) hydroxy(oxo) {4-[(2-oxo-1,2,3,6-tetrahydopyrimidin-4-yl)amino]phenyl}ammonium;

f) 6-[(2-chlorophenyl)amino]pyrimidine-2,4(1H,3H)-dione;

g) 6-[(4-pyrrolidin-1-ylphenyl)amino]pyrimidine-2,4(1H,3H)-dione;

h) 6-[(4-methylphenyl)amino]pyrimidine-2,4(1H,3H)-dione;

i) 5-ethylpyrimidine-2,4(1H,3H)-dione;

j) 6-anilino-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5carbonitrile;

k) 6-anilino-1-methylpyrimidine-2,4(1H,3H)-dione;

l) 3-methyl-6-[(4-methylphenyl)amino]pyrimidine-2,4(1H,3H)-dione;

m) 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione;

n) 6-[(4-chlorophenyl)amino]-3-methylpyrimidine-2,4(1H,3H)-dione;

o) 6-(allylthio)-1,2,4-triazine-3,5(2H,4H)-dione;

p) ethyl[(3,5-dioxo-2-tetrahydrofuran-2-yl-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)thio]acetate;

q) 6-[(imidazo[1,2-a]pyridin-2-ylmethyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

r) 6-[(1-naphthylmethyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

s) 6-[(2-morpholin-4-yl-2-oxoethyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

t) 6-{[2-(4-methoxyphenyl)-2-oxoethyl]thio}-1,2,4-triazine-3,5(2H,4H)-dione;

u) 6-{[2-(2-chlorophenyl)-2-oxoethyl]thio}-1,2,4-triazine-3,5(2H,4H)-dione;

v) 6-{[(2-phenyl-1,3-thiazol-4-yl)methyl]thio}-1,2,4-triazine-3,5(2H,4H)-dione;

w) 6-[(2-chloro-6-flourobenzyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

x) 4-(1,3-benzothiazol-2-yl)-N-phenylbutanamide;

y) N-(aminocarbonyl)-2-(1,3-benzothiazol-2-yl)acetamide;

z) 4-(1,3-benzothiazol-2-yl)butanoic acid;

aa) 3-(1,3-benzothiazol-2-yl)-N-1,3-thiazol-2-ylpropanamide;

bb) 3-(1,3-benzothiazol-2-yl-NN-dimethylpropanamide;

cc) methyl-4-(1,3-benzothiazol-2-yl)butanoate;

dd) 3-(1,3-benzothiazol-2-yl-N-phenylpropanamide;

ee) 3-(1,3-benzothiazol-2-yl)-N-methyl-N-phenylpropanamide;

ff) 2-[4-(1,3-benzothiazol-2-yl)piperidin-1-yl]acetamide;

gg) 6-(1-naphthylamino)pyrimidine-2,4(1H,3H)-dione;

hh) methyl-4-[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)amino]benzoate;

ii) 6-{[(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl]thio}-1,2,4-triazine-3,5(2H,4H)-dione;

jj) 1,2-di-[6-Mercapto-2H-[1,2,4]triazine-3,5-dione]ethane; and

kk) di-{5-[1H-pyrimidine-2,4-dione]methyl}thioether.

38. A compound selected from the group consisting of:

1,2-di-[6-Mercapto-2H-[1,2,4]triazine-3,5-dione]ethane; and

di-{5-[1H-pyrimidine-2,4-dione]methyl}thioether.

39. A pharmaceutical composition in unit dosage form comprising a therapeutically effective amount of a compound selected from the group consisting of:

a) N-nitroso-5-(phenylsulfinyl)pyridin-2-amine;

b) 6-[(4-chlorophenyl)amino]pyrimidine-2,4(1H,4H)-dione;

c) 6-[(4-chlorobenzyl)thio]1,2,4-triazine-3,5(2H,4H)-dione;

d) 4-bromo-2-{(E)-[(4-fluorophenyl)imino]methyl}phenol;

e) 6-(ethylthio)-thioxo-4,5-dihydro-1,2,4-triazin-3(2H)-one;

f) 2-[2-(2-amino-4-methylphenyl)ethyl]-5-methylaniline;

g) 6-[(4-fluorobenzyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

h) 2-(3-fluorophenyl)hydrazinecarboxamide;

i) 3-benzyl-2-hydroxylbenzohydrazide;

j) 4-hydroxybenzaldehyde semicarbazone;

k) 4-(1,3-benzothiazol-2-yl)butanamide;

l) 2-(1H-benzimidazol-2-yl)acetohydrazide;

m) N-[(1R,4R)-4-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl]urea;

n) trimacicolone;

o) 6-amino-methy-adenosine;

p) methyl3-(3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoate;

q) 6-[(3,5-dimethyl-1H-pyrazol-4-yl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

r) 3-methyl-6-[methyl(phenyl)amino]pyrimidine-2,4(1H,3H)-dione;

s) 2-(2-methyl-1H-benzimidazol-1-yl)acetamide;

t) hydroxy(oxo) {4-[(2-oxo-1,2,3,6-tetrahydopyrimidin-4-yl)amino]phenyl}ammonium;

u) 6-[(2-chlorophenyl)amino]pyrimidine-2,4(1H,3H)-dione;

v) 6-[(4-pyrrolidin-1-ylphenyl)amino]pyrimidine-2,4(1H,3H)-dione;

w) 6-[(4-methylphenyl)amino]pyrimidine-2,4(1H,3H)-dione;

x) 5-ethylpyrimidine-2,4(1H,3H)-dione;

y) 6-anilino-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5carbonitrile;

z) 6-anilino-1-methylpyrimidine-2,4(1H,3H)-dione;

aa) 3-methyl-6-[(4-methylphenyl)amino]pyrimidine-2,4(1H,3H)-dione;

bb) 1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione;

cc) 6-[(4-chlorophenyl)amino]-3-methylpyrimidine-2,4(1H,3H)-dione;

dd) 6-(allylthio)-1,2,4-triazine-3,5(2H,4H)-dione;

ee) ethyl[(3,5-dioxo-2-tetrahydrofuran-2-yl-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)thio]acetate;

ff) 6-[(imidazo[1,2-a]pyridin-2-ylmethyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

gg) 6-[(1-naphthylmethyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

hh) 6-[(2-morpholin-4-yl-2-oxoethyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

ii) 6-{[2-(4-methoxyphenyl)-2-oxoethyl]thio}-1,2,4-triazine-3,5(2H,4H)-dione;

jj) 6-{[2-(2-chlorophenyl)-2-oxoethyl]thio}-1,2,4-triazine-3,5 (2H,4H)-dione;

kk) 6-{[(2-phenyl-1,3-thiazol-4-yl)methyl]thio}-1,2,4-triazine-3,5 (2H,4H)-dione;

ll) 6-[(2-chloro-6-flourobenzyl)thio]-1,2,4-triazine-3,5(2H,4H)-dione;

mm) 4-(1,3-benzothiazol-2-yl)-N-phenylbutanamide;

nn) N-(aminocarbonyl)-2-(1,3-benzothiazol-2-yl)acetamide;

oo) 4-(1,3-benzothiazol-2-yl)butanoic acid;

pp) 3-(1,3-benzothiazol-2-yl)-N-1,3-thiazol-2-ylpropanamide;

qq) 3-(1,3-benzothiazol-2-yl-N,N-dimethylpropanamide;

rr) methyl-4-(1,3-benzothiazol-2-yl)butanoate;

ss) 3-(1,3-benzothiazol-2-yl-N-phenylpropanamide;

tt) 3-(1,3-benzothiazol-2-yl)-N-methyl-N-phenylpropanamide;

uu) 2-[4-(1,3-benzothiazol-2-yl)piperidin-1-yl]acetamide;

vv) 6-(1-naphthylamino)pyrimidine-2,4(1H,3H)-dione;

ww) methyl-4-[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)amino]benzoate;

xx) 6-{[(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl]thio}-1,2,4-triazine-3,5(2H,4H)-dione;

yy) 1,2-di-[6-Mercapto-2H-[1,2,4]triazine-3,5-dione]ethane;

zz) di-{5-[1H-pyrimidine-2,4-dione]methyl}thioether.